key: cord- -blbaqbo authors: wu, dongdong; pan, pinhua; su, xiaoli; zhang, lemeng; qin, qingwu; tan, hongyi; huang, li; li, yuanyuan title: interferon regulatory factor- mediates alveolar macrophage pyroptosis during lps-induced acute lung injury in mice date: - - journal: shock doi: . /shk. sha: doc_id: cord_uid: blbaqbo previously, we demonstrated that pyroptosis in alveolar macrophages (ams) plays an essential role in lipopolysaccharide (lps)-induced acute lung injury. however, the underlying mechanism remains largely unclear. here, we show that the absence of interferon regulatory factor (irf- ) in genetic knock-out mice strongly abrogates pyroptosis in ams and alleviates the lps-induced lung injury and systemic inflammation. our study demonstrates that irf- contributes to caspase- activation and apoptosis-associated speck-like protein containing a caspase activation and recruitment domain pyroptosome formation in ams and leads to downstream inflammatory cytokine release, including that of il- β, il- , and hmgb . the nuclear translocation of irf- is linked to the presence of toll-like receptor (tlr ). our findings suggest that pyroptosis and the downstream inflammatory response in ams induced by lps is a process that is dependent on tlr -mediated up-regulation of irf- . in summary, irf- plays a key role in controlling caspase- -dependent pyroptosis and inflammation. acute lung injury and acute respiratory distress syndrome (ali/ards) are serious clinical disorders of the lung. the mortality rates resulting from ali/ards in intensive care units (icus) remain high at % to % ( ) . sepsis is one of the main risk factors for ards, and many animal models of sepsis have been developed to study ali ( ) . lipopolysaccharide (lps) models of inflammation have not reproduced the complex physiology of sepsis and our study focuses mainly on sepsisrelated ards. so identification of novel and effective therapeutic targets and approaches is vital if the outcomes in ards cases are to be improved. alveolar macrophages (ams) account for the % of the cells in bronchoalveolar lavage fluid (balf) ( ) . in view of the role ams as guardians for the alveolar-blood interface against airborne particles and microbes, their pivotal role in the pathogenesis of ali/ards has recently come under close scrutiny. pyroptosis is a recently identified caspase- dependent form of cell death, which features rapid plasma membrane rupture, dna fragmentation, and production of pro-inflammatory cytokines ( , ) . following oligomerization of inflammasome components, the nod-like receptor protein (nlrp ), interacts with the adaptor molecule apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (asc) through an n-terminal pyrindomain (pyd) domain. the caspase-recruitment domain of asc then assembles to nucleate the polymerization and filament formation of pro-caspase- , leading to its self-activation and proinflammatory cytokines release (il- b and il- ) ( ) . inhibitors of cysteine protease caspase- have long been sought as a therapeutic tool because mice lacking caspase- are resistant to lps-induced endotoxic shock ( ) . a previous study conducted by our group observed caspase- activation in ams during the development of ali/ ards, and further, that treatment with a specific caspase- inhibitor has been shown to reduce lps-induced lung injury in mice ( ) . caspase- activation in am is thus closely related to the occurrence of ali/ards. however, the mechanism modulating caspase- activation during pyroptosis remains unclear. interferon regulatory factor (irf- ) has recently been recognized as an interferon-induced transcription factor with pro-inflammatory and pro-injury functions ( ) . irf- promotes cell death with caspase- activation, which has been associated with oligodendrocyte pyroptosis in multiple sclerosis and encephalomyelitis lomyelitis ( ) ( ) ( ) ( ) . while the role of irf- in ams' susceptibility to injury is largely unknown. inflammatory injury to lung tissue of irf- -knockout mice is reduced significantly during sepsis ( ) ( ) ( ) . it has been found by our research group that ams in mice with lps-induced ali/ards present with activation of caspase- , which is a characteristic of pyroptosis. therefore, we hypothesize that irf- may be involved in the pathogenesis of ali/ ards by regulating alveolar macrophage pyroptosis and the release of inflammatory mediators. in the present study, we showed that the absence of irf- in genetic knockout mice strongly alleviates lps-induced lung injury and abrogates its pyroptotic effects in ams. these findings suggest that irf- plays a key role in controlling caspase- -dependent pyroptosis and inflammation, which is, in turn, a tlr -dependent process. our study provides a novel perspective on the pathogenesis of ali/ards. lps (e coli :b ) and adenosine triphosphate (atp) were obtained from sigma-aldrich (st. louis, mo). rabbit polyclonal caspase- p (m- ) antibody was sourced from santa cruz, ca. rabbit polyclonal tlr , irf- , il- b, and glyceraldehyde- -phosphate dehydrogenase (gapdh) antibody were all from cell signaling technology (boston, ma). rabbit polyclonal histone h antibody and rabbit polyclonal asc antibody was obtained from immunoway biotechnology co (newark, de). alexa -conjugated secondary antibody was obtained from molecular probes inc (eugene, or). male irf- ko, tlr ko mice, and the control mice (c bl/ j) were purchased from the jackson laboratory (bar harbor, me). animals were maintained in a specific pathogen-free, laminar-flow atmosphere under controlled temperature, humidity, and light. all animal protocols were approved by the animal care and use committee of the central south university and were performed in accordance with the national institutes of health guidelines for the care and use of laboratory animals. male irf- ko, tlr ko, and matched c bl/ j ( - -week old) mice were given intraperitoneal injections of a lethal dose of lps ( mg/kg). control mice received injections of sterilized phosphate buffered saline (pbs). in some experiments group survival rates of h were observed. in other experiments, mice were sacrificed h post-lps. following euthanasia, the lungs (n ¼ per group) were excised from the mice via a median sternotomy. the wet weight (w) of the left lung was measured using an electronic scale and then desiccated in an oven at c for h to determine the dry weight (d). the water content was measured by calculating w/d weight ratio. the right lung was removed and fixed in % paraformaldehyde for h. lungs were instilled with ml pbs each time through an intratracheal catheter as descried previously ( ) , and lavaged > times by slowing withdrawing more than . ml lavage fluids each time. a total of ml bronchoalveolar lavage (bal) was withdrawn from each mouse. the ml lavage fluid was centrifuged at  g for min to pellet ams. another ml balf was harvested immediately following sacrifice from other mice to detect the total protein level and cytokine level in the balf. cytokine (il- b, il- , and hmgb ) concentrations from ml balf and serum were detected using relevant elisa kits. formalin-fixed, paraffin-embedded lung tissue was cut into sections of -um thickness and stained with hematoxylin-eosin. histopathological changes were viewed under a microscope by a pathologist who was blinded to the treatment. each histological characteristic was evaluated on a scale of to ( ¼ normal; ¼ mild; ¼ moderate; ¼ severe). total aggregate scores of lung injury were calculated according to the sum of the score for alveolar edema and hemorrhage, numbers of infiltrating of leukocytes, and thickness of alveolar walls and epithelium, as described previously ( ) . to determine the expression of cleaved caspase- in the lung tissue of mice, immunohistochemistry analyses were carried out. sections were routinely stained with antimouse caspase- p (m- ) at : (santa cruz, ca). the reaction products were visualized by treating the slide with , -diaminobenzidine and counterstaining with hematoxylin. for negative controls, the specific primary antibody was replaced by an equivalent volume of isotype control antibody. we observed the results under a light microscope (olympus, tokyo, japan). ams were isolated by bal as previously described ( ) . then, ams harvested from mice by lavage were cultured in rpmi (gibico, carlsbad, ca) supplemented with % fbs, mmol/l glutamine, mmol/l -( hydroxyethyl)- -piperazine ethan esulfonic acid, u/ml penicillin, and ug/ml streptomycin. after h of adherence, the cells were washed twice, after which time whole cell lysates were harvested and subjected to western blot. as previously described ( ) , the viability of the am isolate was > %, as evaluated by trypan blue exclusion. the purity was > %, as determined using fluorescently labeled abs (mabs) that specifically recognize proteins expressed by mouse macrophages (surface ags f / and cd b). the mouse monocyte/ macrophage cell line j .a was purchased from american type culture collection and maintained in dmem supplemented with % fbs, ug/ml streptomycin, and u/ml penicillin. lentivirial constructs expressing shrna directed against mouse irf- mrna were manufactured by hanyin (shanghai, china). the shrna sequences were as follows: irf- shrna: '-gcactaaatgagtcctattcc- ', and nonspecific shrna: -ttctccgaacgtgtcacgt- . the constructs expressing irf- shrna were infused with green fluorescent protein. the lentivirial constructs expressing mouse irf- infused with puromycin resistance genes were manufactured by the same vendor (hanyin). the irf- cdna was obtained from a commercially available source (national center for biotechnology information, bethesda, md, usa). all lentivirial vectors were purified to a titer of  tu/ml. at a multiplicity of infection of : h after transfection, the medium was exchanged for fresh medium, and the cells were cultured for an additional h. the transfection efficiency of irf- shrna was observed by fluorescence microscope. for irf- expressing group, the medium was replaced and ug/ml puromycin was applied for selecting for days after h of transfection, then irf- expression of stable transfection is obtained. the effects of irf- knockdown and overexpression were determined using qpcr and western blot analysis and we were able to achieve more than % transduction efficiency using the lentivirus delivery system (data not shown). total rna was isolated from ams using trizol reagent from invitrogen (carlsbad, ca, usa). the reverse transcript (cdna) was synthesized from ug of total rna using the all-in-one first-stand cdna synthesis kit (cenecopoeia). quantitative real-time pcr was carried out using all-in-one qpcr mix from cenecopoeia. the reaction mix of a total volume of ul was programmed as follows: c for min, cycles at c for s, c for s, and c for s. gapdh was used as the reference gene. the sequence of primers used for quantitative pcr was as follows: tlr forward: collected cells were centrifuged at g for min. these pellets were then resuspended on ice with cytoplasmic extraction reagent (vazyme, china) combined with protease inhibitor mix for min, and centrifuged at g for min at c to extract the cellular protein. for nuclear protein isolation, the cell pellet was lysed with nuclear extraction reagent (nanjing, vazyme, china) combined with a protease inhibitor mix. protein concentration was determined by the bca method, and ug of protein per sample was mixed with sample loading buffer and boiled for min. protein samples were electrophoresed in % to % sodium dodecyl sulfatepolyacrylamide (sds-page) gels and transferred onto polyvinylidene fluoride (pvdf) membranes (bio-rad laboratories, berkeley, ca). after blocking with % nonfat milk in tbs-t for h at room temperature, membranes were incubated overnight at c with primary antibodies against caspase- p ( : , ), gapdh ( : , ), histone h ( : ), irf- ( : , ), and il- b ( : ). after three washes, membranes were incubated with the secondary antibody conjugated with horseradish peroxidase at room temperature for h. the blots were developed with super signal chemiluminescent substrate (pierce chemical co, rockford, il) and exposed to film. the relative quantities of the proteins were determined with a densitometer and expressed in absorbance units (au). j .a cells were placed onto glass cover slides, fixed with % paraformaldehyde for min and permeabilized with . % tritonx- for min at room temperature. cells were then blocked with % bovine serum albumin (bsa) in pbs for h at room temperature, and then incubated with rabbit polyclonal asc antibody ( : in % bsa in pbs) overnight at c. after washing three times with pbs, the cells were stained with alexa -conjugated secondary antibody (diluted to : , ) for min at room temperature. the cells were washed three times in pbs and stained with dapi. slides were visualized with an olympus fluoroview confocal microscope. il- b, il- , and hmgb evels in the serum, balf, and cell culture supernatant were determined using commercially available mouse il- b and il- elisa kits. respectively (ebioscience, san diego, ca) according to the manufacturer's instructions. hmgb levels in the samples were determined by an hmgb detection kit (chondrex inc, redmond, wa) according to the manufacturer's instructions. caspase- activity was measured using colorimetric assay (biovision, mountain view, ca). the assay is based on spectrophotometric detection of the chromophore p-nitroanilide (pna) after cleavage by caspase- from the labeled substrate yvad-pna. the pna light emission can be quantified using lactate dehydrogenase (ldh) activity in cell-culture supernatants following lps/atp stimulation was determined to enable evaluation of cell death. ldh activity was quantified by spectrophotometric analysis using the cytotoxicity detection kit (roche, basel, switzerland), according to the manufacturer's instructions. ldh activity in % triton x- -lysed cells was determined relative to total activity at %. the released ldh activity was expressed as a percentage of total cellular ldh activity. data are expressed as mean ae sd. statistical comparisons were performed by one-way anova or two-tailed student t test. survival rates were analyzed with the kaplan-meier test. spss . was used for statistical analyses. a p value < . was considered to be statistically significant. previously, we demonstrated that pyroptosis occurs in ams during lps-induced ali in mice ( ) . here, we set out to determine the role of irf- during lps-induced ali in mice, and the association between tlr and caspase- . it has already been established that caspase- is a biomarker of pyroptosis. we isolated the ams from the ali mouse model. as shown in figure a , western blot analysis demonstrated that the protein levels of tlr , irf- (p < . ), and caspase- increased in ams after lps administration. we also found that mrna expression coding for tlr (fig. b, p < . ), irf- (fig. b, p < . ) , and caspase- (fig. b, p < . ) were significantly higher when compared with the control group, a result that was consistent with our western blot analysis. to determine the levels of caspase- in the lung tissue, caspase- was detected in lung sections by immunohistochemistry staining. higher expression levels of caspase- were observed in lung tissue from the ali mouse model (fig. c) . these results suggest that lps does indeed induce tlr and irf- expression and pyroptosis in alveolar macrophages in ali. irf- ko mice were used to investigate whether irf- mediates lps-induced acute lung injury and cytokine release. to determine whether irf- contributes to mortality following lps administration, -h survival rates were noted. significantly, lps-induced mortality was % in the wt mice at h, whereas all irf- ko mice survived for h postadministration ( fig. a) . irf- ko mice demonstrated significantly improved -h survival rates compared with the control wt mice (p < . ). in a further set of experiments, four animal groups were created: wt/pbs group; wt/lps group; irf- ko/pbs group; and irf- ko/lps group. an examination of the pathology of the lung tissue showed that the wt/lps group developed exacerbated lung inflammation, hemorrhaging and alveolar septal thickening, while lung lesions were fewer in the irf- ko mice (fig. b) . the level of total proteins in balf (fig. c , p < . ), lung injury score (fig. d , p < . ), and pulmonary w/d weight ratio (fig. e , p < . ) significantly reduced in the irf- ko/lps group when compared with the wt/lps group, which indicated that the lung injury in irf- ko mice was alleviated. for cytokine levels both in serum and balf, we discovered that irf- deletion significantly blocked lps-induced il- ( well as il- b (fig. c, p < . ; fig. d , p < . ), and hmgb (fig. e, p < . ; fig. f , p < . ) compared with the wt/lps group. these pyroptosis-associated cytokines have been shown to be important mediators of tissue damage. these data indicate that irf- plays an important role in the pathogenesis of lps-induced ali. having shown that lps-induced lung injury is associated with the pyroptosis of ams ( ), we set out to analyze whether irf- deletion enhances protection by inhibiting pyroptosis in ams. as shown in figure a , lps challenge resulted in a dramatic activation of caspase- (p < . ) and il- b in ams in wt mice. however, the levels of caspase- p and il- b were strongly attenuated in the ams of irf- ko mice. moreover, caspase- was also detected in lung sections by immunohistochemical staining. indeed, reduced expression of caspase- was observed in lung tissue in irf- ko mice, as well as highly positive ams, compared with the control group (fig. b) . to determine whether irf- nuclear expression and caspase- activation are modulated by tlr in lps-induced acute lung injury, tlr ko mice were challenged with lps by ip injection for h. ams were isolated from mice to perform western blotting and quantitative pcr. as shown in figure , both protein level (fig. a) , and mrna level (fig. b ) of irf- and caspase- were increased in ams during lps-induced acute lung injury, while they were both dramatically more inhibited in the tlr ko/lps group than the model group (p < . for both). furthermore, immunohistochemistry gave a negative result in the lung sections of lps-treated tlr ko mice, but a positive result in the lps-treated wt mice (fig. c) . together, these findings demonstrate that irf- expression and caspase- activation are dependent on intact tlr signaling. lps/atp-induced pyroptosis and cytokine release in j .a cells are irf- dependent we also studied the kinetics of irf- expression in j .a cell responses to lps. the results showed that the nuclear lpsinduced irf- protein expression was time dependent and reached a peak at h (fig. a, p < . ) and that lps induced nuclear irf- expression in a dose-dependent manner in a certain range, reaching a peak at a dose of ng/ml. continued increases of the dosage did not increase irf- expression (fig. b, p < . ) . the involvement of irf- signaling in macrophages was examined directly in protection experiments in which irf- expression was knocked down using lentivirus-delivered shrna. j .a cells were transduced with irf- shrna lentiviral vectors and a control lentiviral vector (carrying nonspecific shrna). due to the fact that lps combined with atp is a powerful inducer of pyroptosis, the cells were stimulated with lps ( ng/ml) for h and atp ( mm) for h. following stimulation, western blottings tests were performed to assess the irf- nuclear translocation and caspase- activation in cytoplasm. in addition, caspase- activity and ldh release were also carried out post-treatment. asc pyroptosomes were also examined by immunofluorescence. western blotting analysis of the protein samples confirmed that transduction of irf- shrna of j .a cells with irf- shrna vector resulted in significant reduction of irf- expression in both lps/atp-treated and untreated groups compared with controls (fig. a) . as a result of the reduced irf- expression, the activation of caspase- in response to lps/atp was significantly suppressed compared with the controls, both in western blot analysis (fig. a ) and colorimetric assay (fig. c, p < . ) . asc pyoptosomes were also not up-regulated in j .a cells following lps/atp stimulation in the presence of irf- shrna (fig. g) . in an effort to examine the role of irf- further, we induced irf- overexpression. j .a cells were transduced with an irf- -expression lentiviral vector and a control lentiviral vector. once this was done, the cells were stimulated with lps ( ng/ml) for h and atp ( mm) for h. nuclear and cytoplasmic protein in j .a cells was confirmed by western blotting (fig. b) . transduction of j .a cells with irf- lentivirus vector resulted in significant caspase- activation (fig. b , p < . ; fig. d , p < . ) compared with the negative control in both lps/atp-treated and untreated groups. ldh release (fig. f , p < . ) also rose progressively compared with the controls. thus, lps/atp-induced macrophage pyroptosis was shown to be dependent on irf- . supernatants were also collected from the above-mentioned lps/atp treated j .a cells in the presence of irf- shrna and irf- lentivirus. as suspected, il- , il- b, and hmgb were heavily suppressed in the presence of irf- shrna even in the presence of lps/atp stimulation (fig. a , p < . ; fig. c , p < . ; fig. e , p < . ). in the irf- over expression j .a cells however, these cytokines were significantly higher in supernatants compared with negative controls (fig. b , p < . ; fig. d , p < . ; fig. f , p < . ). these findings suggest that overexpression of irf- may have caused higher inflammation and that downregulation of irf- can counteract severe inflammation. in sum, irf- plays a key role in controlling caspase- -dependent pyroptosis and inflammation. ali/ards is a complex and devastating disorder of the lung associated with excessive inflammation. pyroptosis is a form of programed cell death dependent on caspase- activation and cytokine release. however, the exact link between the am pyroptosis and lung injury in ali/ards remains to be elucidated. in the present study, we identify the critical role of irf- in mediating lung injury during sepsis-related ali/ards induced by lps. indeed, as a gene noted for its involvement in the interferon (ifn) pathway, irf- is often characterized as elevated in ards patients ( ) . additionally, disruption of irf- in the mouse model alleviated lung injury and protected against lps insult. these studies indicate that irf- plays an important role in mediating lung injury in ali/ards. we, therefore, have investigated the role of irf- in mediating am pyroptosis during the development of ali/ards. we showed that tissue-derived alveolar macrophages from lps-stimulated irf- ko mice exhibit less pyroptosis than their wt counterparts (fig. a) . furthermore, we found that overexpression of irf- in j .a cells alone is a sufficient stimulation to induce pyroptosis. consequently, our data provides evidence that irf- activation induces pyroptosis by modulating asc pyroptosome formation and caspase- activation (fig. ) . indeed, it has been shown that irf- is an essential transcription factor involved in pyroptotic cell death. previous studies have proved that the caspase- gene contains an irf- -binding element (ire), and that caspase- expression cannot be up-regulated in oligodendrocyte progenitor cells stimulated by ifn without the presence of irf- ( , ) . irf- can also induce the pyroptosis of oligodendrocyte in the development of multiple sclerosis, promote the inflammatory reaction, and lead to inflammatory demyelination ( ) . the presence of ire as a gene promoter of caspase- makes it possible that irf- directly regulates caspase- activity and alveolar macrophage pyroptosis from the structure. irf- may be also the actor controlling the release of inflammatory mediators. the present study demonstrates that il- , il- b, and hmgb are significantly reduced both in serum and balf in irf- ko mice during lps-induced acute lung injury (fig. ) . we also validated that overexpression of irf- serves as a strong promoter of the abovementioned inflammatory cytokine release (fig. ) . currently, caspase- -mediated tissue damage is thought to require the pro-inflammatory caspase- substrates il- b and il- , while the existence of another caspase- -dependent mediator of endotoxemia, hmgb , has been described. reduced hmgb levels in caspase- -deficient mice correlated with their resistance to lps, while the neutralization of hmgb with abs protected mice deficient in il- b and il- from succumbing to a lethal dose of lps ( ). furthermore, irf- fig. . irf- mediates lps/atp-induced cytokine release in j .a cells. transduced j .a cells were stimulated with or without lps ( ng/ml) for h and atp ( mm) was added during the last hour of culture in the presence of irf- shrna or irf- lentivirus. two milliliters of the supernatants were harvested and tested for levels of pro-inflammatory cytokines. knockdown of irf- in j .a cells inhibited the expression of il- (a), il- b (c), and hmgb (e) in the supernatants. overexpression of irf- in j .a cells boosted the expression of il- (b), il- b (d), and hmgb (f) in the supernatants. * p < . versus the nc group; # p < . versus the ncþlps/atp group; þ p < . versus the irf- shrna group. results are representative of three independent experiments. irf- indicates interferon regulatory factor ; lps, lipopolysaccharide. mediates the release of hmgb by post-translational modification through acetylation ( ) . these results imply that irf- could play a key role in pyroptosis-associated cytokines. toll-like receptor (tlr ) functions in pathogen-associated molecular pattern recognition and initiation of immune responses and inflammatory processes. it has been confirmed that administration of lps up-regulates lung tlr expression and gene silencing of tlr attenuates inflammatory response and lung injury ( ) . collectively, these data support an emerging concept that the quantity of tlr expressed on ams will modulate irf- activation to modify pyroptotic inflammatory signaling and elicit cytokine expression. we also identified tlr as necessary for the activation of irf- (fig. ) . however, the mechanism by which tlr mediates irf- expression in ams compromised by lps remains to be elucidated. the myeloid differentiation factor (myd ) adaptor protein has been shown to recruit members of the irf- family of transcription factors to evoke tlr target genes. indeed, irf- has been shown to be a participant in myd signaling and migrates to the nucleus more effectively after stimulation with various tlr ligands ( ) . our experiments demonstrate that irf- serves as a link between the initial upstream lps-induced tlr signal and downstream pathological mechanisms by activating genes with propyroptotic functions. based on these observations, we conclude that that irf- plays a key role in controlling caspase- -dependent pyroptosis and inflammation. lps-induced pyroptosis in ams is a process that is dependent on tlr -mediated upregulation of irf- . by identifying the molecular mechanisms of irf- -mediated caspase- signaling in macrophage pyroptosis and the associated inflammation, our study provides a novel therapeutic approach to management of ali/ards. incidence and outcome of acute lung injury and acute respiratory distress syndrome in the surgical intensive care unit animal models of acute lung injury diverse macrophage populations mediate acute lung inflammation and resolution caspase- -induced pyroptotic cell death pyroptosis: host cell death and inflammation the adaptor asc has extracellular and 'prionoid' activities that propagate inflammation inflammasome-dependent release of the alarmin hmgb in endotoxemia inhibition of alveolar macrophage pyroptosis reduces lipopolysaccharide-induced acute lung injury in mice interferon regulatory factor regulation of oligodendrocyte injury and inflammatory demyelination irf- as a negative regulator of cell proliferation interferon-regulatory factor- is critical for tamoxifen-mediated apoptosis in human mammary epithelial cells apoptosis of oligodendrocytes via fas and tnf-r is a key event in the induction of experimental autoimmune encephalomyelitis irf- signaling in central nervous system glial cells regulates inflammatory demyelination interferon regulatory factor- regulates the autophagic response in lps-stimulated macrophages through nitric oxide interferon regulatory factor- mediates the release of high mobility group box- in endotoxemia in mice splenocyte apoptosis and autophagy is mediated by interferon regulatory factor during murine endotoxemia tolllike receptor mediates alveolar macrophage response to pneumocystis murina acute lung injury using oleic acid in the laboratory rat: establishment of a working model and evidence against free radicals in the acute phase isolation and partial characterization of subpopulations of alveolar macrophages, granulocytes, and highly enriched interstitial macrophages from rat lung neutrophil il- beta processing induced by pneumolysin is mediated by the nlrp /asc inflammasome and caspase- activation and is dependent on kþ efflux sars-cov virus-host interactions and comparative etiologies of acute respiratory distress syndrome as determined by transcriptional and cytokine profiling of formalinfixed paraffin-embedded tissues overexpression of the dominant-negative form of interferon regulatory factor in oligodendrocytes protects against experimental autoimmune encephalomyelitis stat /irf- signaling pathway mediates the injurious effect of interferon-gamma on oligodendrocyte progenitor cells burn-induced acute lung injury requires a functional toll-like receptor evidence for licensing of ifn-gammainduced ifn regulatory factor transcription factor by myd in toll-like receptor-dependent gene induction program key: cord- -h owwjao authors: xiong, jing; miller, virginia m.; hunter, larry w.; li, yunman; jayachandran, muthuvel title: leukocyte- and platelet-derived microvesicle interactions following in vitro and in vivo activation of toll-like receptor by lipopolysaccharide date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: h owwjao background: pro-coagulant membrane microvesicles (mv) derived from platelets and leukocytes are shed into the circulation following receptor-mediated activation, cell-cell interaction, and apoptosis. platelets are sentinel markers of toll-like receptor (tlr ) activation. experiments were designed to evaluate the time course and mechanism of direct interactions between platelets and leukocytes following acute activation of tlr by bacterial lipopolysaccharide (lps). methodology/principal findings: blood from age-matched male and female wild type (wt) and tlr gene deleted (dtlr ) mice was incubated with ultra-pure e. coli lps ( ng/ml) for up to one hour. at designated periods, leukocyte antigen positive platelets, platelet antigen positive leukocytes and cell-derived mv were quantified by flow cytometry. numbers of platelet- or leukocyte-derived mv did not increase within one hour following in vitro exposure of blood to lps. however, with lps stimulation numbers of platelets staining positive for both platelet- and leukocyte-specific antigens increased in blood derived from wt but not dtlr mice. this effect was blocked by inhibition of tlr signaling mediated by my and trif. seven days after a single intravenous injection of lps ( ng/mouse or ng/gm body wt) to wt mice, none of the platelets stained for leukocyte antigen. however, granulocytes, monocytes and apoptotic bodies stained positive for platelet antigens. conclusions/significance: within one hour of exposure to lps, leukocytes exchange surface antigens with platelets through tlr activation. in vivo, leukocyte expression of platelet antigen is retained after a single exposure to lps following turn over of the platelet pool. acute expression of leukocyte antigen on platelets within one hour of exposure to lps and the sustained expression of platelet antigen on leukocytes following a single acute exposure to lps in vivo explains, in part, associations of platelets and leukocytes in response to bacterial infection and changes in thrombotic propensity of the blood. acute and chronic infection, especially that induced by gramnegative bacteria is associated with increased risk of thrombosis and atherosclerotic disease [ , , , , ] . little is known about the underlying cellular mechanisms responsible for these risks. lipopolysaccharide (lps), a component of the cell wall of gram-negative bacteria, is an antigen which initiates inflammation and innate immune responses by interacting with toll-like receptor (tlr ). tlr is expressed on the surface of cells, including leukocytes and platelets [ , , ] . under physiological conditions, platelets and leukocytes circulate in quiescent state and do not interact with each other. however, once activated under pathophysiological conditions such as those associated with infection, platelets change shape, secrete prothrombogenic inflammatory and cellular adhesion molecules from alpha-and densegranules which cause the platelets to adhere to each other or to leukocytes and/or vascular endothelium [ , , , ] . the physiological consequences of stimuli associated with infection, like lps stimulation, are acute but can be sustained. for example, half-life of platelets was shortened and the activation state of newly formed platelets from bone marrow megakaryocytes increased within seven days following a single acute intravenous injection of lps in mice [ , ] . however, cellular events, specifically those occurring among blood elements, contributing to the shortened half-life and increased activation state of platelets remains to be clarified. one mechanism offered to explain how infection contributes to the onset and progression of cardiovascular diseases is through increased production of proinflammatory cytokines [ , ] . however, this explanation does not address how the production of inflammatory cytokines might proceed nor does it identity the cell types which are targets for the lps stimulation. platelets may represent one of the first blood borne elements to react to lps stimulation as changes in platelet reactivity via tlr seems to occur prior to sustained changes in circulating levels of cytokines [ ] . alternatively, comparable activation of leukocyte as well as platelet result in formation of cell-derived microvesicles (mv) which may contribute to increased thrombogenic propensity of the blood, pro-inflammatory immune processes and thus cardiovascular risk [ , , , , , , , ] . clarifying the interactions of these blood elements (platelets and leukocytes) in the setting of tlr activation might provide insight into how infection initiates or facilitates progression of cardiovascular disease. mv are cell membrane-derived vesicles ranging in size from . to micron in diameter which are shed in response to cellular activation, cell-cell interaction and apoptosis [ , , , , ] . these cell-derived vesicles are an interface of activation between cellular components of the blood with the vascular wall and between soluble components of the blood associated with immunity including response to infection [ , , ] . for example, phosphatidylserine (ps) on the surface of mv provides catalytic sites for prothrombinase complex to generate thrombin needed for the conversion of fibrinogen to fibrin in formation of clots [ , , ] . furthermore, exposure of diluted blood to lps in vitro increased production of platelet-derived as well as tissue factor positive mv within to hours [ , , ] . while those experiments provide evidence that lps modulates platelet activation, they do not provide any insight about the interactions of platelet with other blood elements within the earliest stages of activation especially at time points prior to the period when measurable changes in circulating cytokines are observed in vivo [ ] . therefore, the present study was designed to test the hypothesis that acute exposure to a sentinel dose of lps would induce mv production and exchange of specific proteins/ receptors between platelets and leukocytes via tlr activation. mv transport of biologically active cell contents including cell surface receptors among cells were identified using antibodies specific for cell antigens (i.e. cd or cd , which is platelet-or leukocyte-specific antigens respectively) [ ] ; mv derived from platelets and/or leukocytes were distinguished by cell specific fluorescein conjugated antigen staining using calibrated flow cytometry. four to eight month old, male and female c bl snj mice (wild type, wt) and c bl scn mice homozygous for deletion of tlr (dtlr ) were obtained from the jackson laboratory, bar harbor, maine. these mice do not express the il- rb mutation that was originally described for this strain [ ] . mice of each sex and age were used randomly in each of the various protocols. mice were housed in a temperature-controlled environment ( uc; % relative humidity), / light/dark cycle, and fed standard chow. experiments were approved by the institutional animal care and use committee, mayo clinic, rochester, mn. ultra-pure e.coli lipopolysaccharide (lps, :b strain-tlr ligand, product number tlrl-pelps), pepinh-myd (product number tlrl-pimyd) or pepinh-trif (product number tlrl-pitrif) inhibitory peptide (invivogen, san diego, ca) were prepared as suggested by the supplier. mouse thrombin and bovine serum albumin were purchased from sigma chemical co., st. louis, mo, usa. cellular origin of antigens was determined using platelet (rat anti-mouse cd antibody) and total leukocyte (rat anti-mouse cd antibody) membrane specific fluorescein conjugated {phycoerythrin (pe)-or fluorescein isothiocyanate (fitc)-} antibodies by flow cytometry. pe-or fitc-conjugated annexin-v and matched isotype control antibodies were purchased from bd pharmingen international, san diego, ca. all other reagents and solvents used in this study were of analytical/ reagent grade. blood was collected from the retro-orbital sinus plexus ( - ml/mouse) of wild type and dtlr mice through siliconized capillary tubes coated with hirudin (thrombin inhibitor) and soybean trypsin inhibitor (sti, factor xa inhibitor) into . ml polypropylene tubes containing ml of mm hirudin and mm sti [ ] . for in vitro experiments, anticoagulated blood was aliquoted into pairs of tubes within min after collection so that measurements from a vehicle-treated, control tube and lpstreated tube could be analyzed from each mouse at each time point. vehicle (saline) or lps ( ng/ml) was added to one of each paired aliquots of blood. at designated time points ( , , and minutes) after addition of vehicle or lps, ml whole blood from each aliquot was diluted into ml ( : ) hepes/ hanks' buffer ( mm nacl, . mm kcl, . mm cacl , . mm mgso , . mm na hpo , mm hepes, ph . ) with mg/ml albumin and mm sti for staining of platelets positive for leukocyte antigen and leukocyte positive for platelet antigen. the remaining blood was used to prepare platelet free plasma (pfp) for mv analysis. for in vivo experiments, male mice ( month of age) received a single injection of lps ( ng/mouse or ng/gm body weight) into the tail vein. seven days after the injection, blood was collected as described above. for analysis of platelet antigen positive leukocyte sub-populations, flow cytometry was triggered with total leukocyte marker cd -conjugated with allophycocyanin (cd -apc). all cd positive events containing granulocytes, monocytes, lymphocytes and apoptotic bodies were separated by light forward and side scatter blot analysis and gated each sub-type of leukocytes based on their size. each gate was verified using cell specific antibodies (cd b for granulocytes, cd for monocytes and cd for t-lymphocytes and cd for b-lymphocytes. differential leukocyte populations were then subjected to two-color analysis to differentiate leukocytes positive for phosphatidylserine and platelet antigen (annexin-v-fitc vs cd -pe) from leukocytes negative for phosphatidylserine and platelet antigen. percentages of positive events were calculated above the set threshold from isotype control antibody. a blood sample from each mouse was divided into two ml aliquots; one was diluted with ml saline (control), the other with ml saline containing lps ( ng/ml final concentration). samples were incubated at uc. at time-points (prior to lps or vehicle), and min, a ml aliquot was removed from each sample, diluted in ml % paraformaldehyde and incubated for an additional min. each sample was then diluted in ml pbs ( . mm pore membrane-filtered) and centrifuged at g for min. the supernate, which contained platelets, was removed, placed into a new vial and used for imaging analysis. an aliquot of each platelet suspension was diluted : in pbs, and a ml drop was placed on a glass slide, cover-slipped and sealed with glue. platelets were then imaged in dark-field mode using a light microscope (olympus bx with a oil-immersion lens) coupled to a cytoviva illumination system (cytoviva, inc., auburn, al). scanning from a corner of each cover-slip, platelets in each field were counted until were totaled. platelets were categorized according to shape morphology (discoid, irregular, flattened, pseudopodia) or whether exhibiting membrane granules or in aggregates (each aggregate was counted as ). the number of platelets in each category was expressed as percentage of total platelets counted. diluted ( : ) whole blood ( ml) was incubated with peconjugated platelet-and leukocyte-specific antibodies (cd and cd , respectively), or separately with annexin v-fitc (binding to phosphatidylserine) for min, after which % paraformaldehyde ( ml) was added. matched fluorescein conjugated isotype control antibodies were used simultaneously staining to set the threshold and exclude nonspecific binding. interactions between cell (platelets or leukocytes) and cell-derived mv and ps expression on both platelets and leukocytes were analyzed by flow cytometry (facscalibur tm and facscanto tm , bd biosciences, san jose, ca). platelets were identified by forward and side scatter and with fluorescein conjugated cd antibody until , events gated for each sample, respectively ( figure a ). all buffers and antibodies were filtered twice through . mm membrane (millipore) filters for mv analysis. platelet free plasma (pfp) was prepared by double centrifugation at g for min. pfp was diluted ( ml pfp+ ml hepes/hanks' buffer) and incubated with ml fitc-conjugated annexin-v and pe-conjugated cd or cd antibodies for min in dark, at which time ml hanks' balanced salts buffer (ph . ) with . mm cacl was added. matched isotype control antibodies were used to set threshold and exclude nonspecific binding. mv were quantified based on counts of calibration beads (trucount tm beads) added immediately to the samples prior to analysis by flow cytometry (facscanto tm , bd biosciences, san jose, ca) as previously described [ , , ] . in this study, mv were defined as events of , mm in diameter using size calibration beads and positive for annexin-v and platelet-and leukocyte-specific markers. analysis of tlr intracellular signaling pathway lps stimulated tlr signaling was analyzed by introducing myd or trif inhibitory peptides as described in other studies [ , ] . anticoagulated blood from wt mice was aliquoted into six tubes and treated with pepinh-control (vehicle), pepinh-myd ( mm, myd inhibitory peptide which binds to myd to block tlr stimulated myd signaling) and/or pepinh-trif ( mm which binds to trif to block tlr stimulated trif signaling) alone or in combination for min after which either saline or lps ( ng/ml) was added for one hour. at this time, an aliquot was diluted for analysis by flow cytometry to measure cellular origin of mv and platelet positive leukocyte antigen and leukocyte positive platelet antigen. data are presented as percentage or fold increase from the paired vehicle and lps treated blood samples at each time point. all data are presented as mean sem; n = number of animals used in each experiment. statistical significance was evaluated by paired or unpaired two-tailed student's t-test. statistically significance was accepted at p, . . prior to addition of lps, platelets from wt mice were discoid, formed few aggregates and did not have extended psuedopodia (figure ). surface expression of ps on platelets or leukocytes was similar in wt and dtlr mice. within each group of mice, expression of ps was significantly lower on leukocytes than on platelets (table ) . during the first hour of incubation of the blood with lps, platelets from wt mice underwent a shape change, extended pseudopodia and formed aggregates (figure ). within this first hour, neither surface expression of ps on either platelets or leukocytes or numbers of platelet-or leukocyte-derived mv changed significantly in wt mice or dtlr mice (table ) . however, the number of platelets positive for leukocyte-specific antigen (defined by the platelet-gate on the flow cytometer, figure ) increased significantly in a time-dependent manner in blood of wt mice, but not dtlr mice (figures ) . using the total leukocyte specific antibody (cd -apc) to define total leukocytes, numbers of leukocytes positive for platelet-specific antigen did not increase significantly in either wt or dtlr mice ( figure ) . in blood from wt mice, the percentage of platelets positive for leukocyte antigen (cd ) was reduced significantly and to the same extent by myd and trif (figure ). the combination of myd with trif did not reduce the percentage of aggregates to a greater extent than either alone (from . . to . . ). seven days following a single intravenous injection of a sentinel dose of lps ( ng/gm body weight which is ng/mouse), none of the platelets were positive for leukocyte antigen. on the contrary, compared to leukocytes obtained from animals treated with vehicle or a week before lps injection in the same mouse, granulocytes and monocytes were significantly positive for platelet antigen seven days after the single lps injection (figure ) . apoptotic bodies also stained positive for platelet antigen (figure ). understanding how infection alters cell-cell interactions and release of mv from specific blood borne elements may help to identify new targets for reducing cardiovascular/thrombotic risk with infection. this study demonstrates the acute, immediate interaction of platelets and leukocytes after incubation of whole blood with a sentinel dose of lps through tlr signaling. exchange of antigens and associations of specific cell-derived mv among cells is a mechanism for the transfers signaling molecules to specific cells. for example, mv derived from neutrophils induced platelet activation by binding to platelets gp ba (glycoprotein b a) via activated a m b on mv [ ] , while platelet-derived mv mediate leukocyte-leukocyte aggregation, activate leukocyte phagocytic properties and amplify leukocyte-mediated tissue injury in thrombotic and inflammatory disorders [ ] . results from the present study demonstrate that the number of platelets positive for leukocyte antigen increased within min of exposure to lps. this increase was not accompanied by increased expression of ps on cell or mv surface. because expression of the leukocyte antigen on platelets was defined using the platelet size gate and platelet specific marker cd on the flow cytometry, larger leukocytes would be excluded. therefore, these leukocyte antigens may represent a membrane exchange during platelet-leukocyte adhesion or adhesion of leukocyte-derived mv to the platelets. the half-life of whole platelet-leukocyte aggregates may be shorter and therefore, we did not determine whole platelet-leukocyte aggregates in this study. with agonist binding, the tlr dimerizes and undergoes a conformational change required for the recruitment of signaling molecules [ ] , such as the adaptor molecules myeloid differentiation protein (myd ) and toll-interleukin- (il- ) receptor figure . representative dark-field micrographs of platelets from a wt mouse taken immediately before or after and min. incubation of the blood with ng/ml lps. prior to lps application, platelet aggregates (of more than platelets) were absent, and most platelets were discoid shaped (left panels). at and minutes incubation with saline, platelet morphology did not change (top panels); whereas, large aggregates of irregularly-shaped platelets were observed in those incubated with lps (bottom panels, inset shows platelets with pseudopodia). each bar represents mm. similar results were observed with blood from two additional animals (n = ). doi: . /journal.pone. .g domain containing adaptor inducing interferon-b (trif), which mediate myd dependent or independent pathway respectively [ , ] . lps activates both myd and trif pathways, which are important in the tlr mediated intracellular signaling [ ] . the acute effects of lps on platelet and leukocyte activation were most likely mediated through activation of tlr as platelet positive leukocyte antigen was not observed in blood from dtlr mice. furthermore, leukocyte antigen expression on platelet was reduced by inhibition of myd -and trif-dependent pathways alone or in combination. these signaling pathways may be potential molecular targets to inhibit infection/inflammation induced interactions among formed elements in the blood. a single tlr sentinel dose injection of lps, such as used in this study, shortened platelet half-life and increased platelet production without increases in cytokine production within hours of stimulation [ ] . although in vivo interaction of plateletand leukocyte-aggregates with the vascular wall could stimulate or exacerbate proinflammatory immune responses [ , ] , the half-life of these cell aggregates is not known. a significant finding of the present in vivo study is that leukocytes sustain or retain platelet antigen seven days after an in vivo injection of lps. this time point corresponds to turnover of the platelet pool and is consistent with observations that changes in platelet half-life and increases in platelet turnover are dependent on the concentration of agonist, i.e. lps, activation [ , ] . quantification of dual-positive events is usually not considered in studies of cellular or mv quantification with lps stimulation. results from the present study, therefore, suggest that platelet-leukocyte antigen could be used as an additional biomarker of cellular activation for diagnostic or prognostic purposes in settings of sub-clinical or asymptomatic exposures to infective agents. monocytes showed the greatest expression of platelet antigen following lps injection. since monocytes are considered to be the primary leukocytic cell involved with development of atherosclerotic lesions [ , , ] , the present results provide insight into a mechanism linking low to moderate levels of infection to progression of cardiovascular disease. to our knowledge, results of the present study represent the first to examine time-dependent changes in production of mv/ exchange of cell-specific antigens in cells derived from whole blood incubated with lps. in diluted blood, addition of lps ( ng or mg/ml) increased the number of mv from platelets and those positive for tissue factor but only after four hours of incubation [ ] . because the blood was diluted, unlike the present study, cell-cell interactions were most likely attenuated and no evaluation was performed relative to interaction with leukocytes which could have indirectly affected platelet activation and production of cytokines. unexpectedly, ps expression on platelets (or leukocytes) did not increase significantly with acute exposure to lps. ps is expressed in the inner leaflet of plasma membrane but rapidly inverts to outer surface following activating stimuli [ , , ] . exposure of ps to the outer membrane surface also occurs with release of mv [ ] . results of the present study are consistent with reports that production of mv is not accompanied by ps expression on cell of origin but might be restricted to that portion of the membrane undergoing mv blebbing [ ] . within one hour of exposure of whole blood to a concentration of lps that has threshold effect on cytokine production in vivo, platelets become positive for leukocyte antigen. platelet-leukocyte interactions require tlr signaling as the dual antigen positivity of platelets was observed in blood derived from wild type but not dtlr mice. furthermore, peptide inhibitors of tlr signaling molecules blocked the interaction. these events occur within hour after the initial exposure to lps. in addition, effects of lps stimulation are sustained at least up to days past the initial lps exposure, as leukocytes express platelet antigen at this time point. collectively, these results identify an acute and rapid signaling mechanism by which sentinel-grade acute infection through tlr alters blood hemostasis and sustained leukocyte activation which may contribute to progression of cardiovascular diseases. chlamydia pneumoniae binds to platelets and triggers p-selectin expression and aggregation: a causal role in cardiovascular disease? a major outbreak of severe acute respiratory syndrome in hong kong virulent strains of helicobacter pylori and vascular diseases: a meta-analysis association between chronic dental infection and acute myocardial infarction risk of myocardial infarction and stroke after acute infection or vaccination lipopolysaccharide stimulates platelet secretion and potentiates platelet aggregation via 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production through toll-like receptor activation in platelets from recently menopausal women platelet-derived microparticles stimulate proliferation, survival, adhesion, and chemotaxis of hematopoietic cells role of platelet-derived microparticles in angiogenesis and tumor progression megakaryocyte-derived microvesicles loss of estrogen receptor ß decreases mitochondrial energetic potential and increases thrombogenicity of platelets in aged female mice increased platelet microvesicle formation is associated with mortality in a porcine model of endotoxemia levels of microparticle tissue factor activity correlate with coagulation activation in endotoxemic mice shiga toxin and lipopolysaccharide induce platelet-leukocyte aggregates and tissue factor release, a thrombotic mechanism in hemolytic uremic syndrome a point mutation in the il- rbeta gene underlies the il- unresponsiveness of lpsdefective c bl/ sccr mice peptide-mediated interference of tir domain dimerization in myd inhibits interleukin- -dependent activation of nf-{kappa}b differential involvement of bb loops of toll-il- resistance (tir) domain-containing adapter proteins in tlr -versus tlr -mediated signal transduction expression, activation, and function of integrin alphambeta (mac- ) on neutrophil-derived microparticles platelet-derived microparticles and the potential of glycoprotein iib/iiia antagonists in treating acute coronary syndrome toll-like receptor signalling myd -dependent and -independent activation of trem- via specific tlr ligands myd -dependent and independent pathways of toll-like receptors are engaged in biological activity of triptolide in ligand-stimulated macrophages exovesicles from human activated dendritic cells fuse with resting dendritic cells, allowing them to present alloantigens in vitro study of pro-inflammatory and antitumour properties of microvesicles from bacterial cell wall of pantoea agglomerans evolving concepts in the triad of atherosclerosis, inflammation and thrombosis nanoparticle pet-ct imaging of macrophages in inflammatory atherosclerosis functional cd ligand is expressed on human vascular endothelial cells, smooth muscle cells, and macrophages: implications for cd -cd ligand signaling in atherosclerosis back and forth: the regulation and function of transbilayer phospholipid movement in eukaryotic cells reconstitution of phospholipid scramblase activity from human blood platelets calcium involvement in aminophospholipid exposure and microparticle formation during platelet activation: a study using ca +-atpase inhibitors phospholipid composition of cell-derived microparticles determined by one-dimensional high-performance thin-layer chromatography microparticles in cardiovascular diseases key: cord- - rg f i authors: legband, nathan; buesing, keely; borden, mark; terry, benjamin title: the treatment of acute respiratory distress syndrome in rats with a peritoneal dosing system date: - - journal: j med device doi: . / . sha: doc_id: cord_uid: rg f i nan current medical treatments for conditions involving an impaired respiratory system provide inefficient methods of delivering oxygen to the patient. patients with these respiratory dysfunctions are often unable to get the required amount of oxygen to the brain and body. this can lead to delayed recovery from acute illness, multisystem organ failure, and ultimately death. the mortality rate for cases of respiratory dysfunctions has been recently reported in the range of - % [ ] . this is especially troubling considering there are many causes of respiratory failure leading to acute respiratory distress syndrome (ards), including pneumonia, sepsis, trauma, chemical inhalation, and bacterial or viral infection. methods of oxygenation and ventilation that bypass the lungs have been explored in an effort to promote lung recovery. so far, only extracorporeal membrane oxygenation (ecmo) has been approved for medical use. however, ecmo is an unsatisfactory treatment as it possesses many of its own risks, including hemorrhage, thrombosis, and cannula malfunction. these risks eliminate ecmo as a viable treatment alternative for many patients. therefore, we have researched an alternative technology for delivering oxygen to the body through the peritoneal cavity. we have developed a peritoneal dosing system (pds) which delivers an infusate solution to the peritoneal cavity. in order to provide a patient with supplemental oxygenation, phospholipid shelled oxygen microbubbles (ombs) would be administered to the patient. previously, we have shown that peritoneal membrane oxygenation (pmo) with ombs is a possible treatment method in acute lung injury models [ ] . we are now extending the validity of pmo treatment by testing in long term disease models, which more accurately reflect clinical scenarios. ards is a medical condition that has been well established in animal models. rat models of ards have been well established by tracheal delivery of the endotoxin, lipopolysaccharide (lps) [ ] . our study involves two phases: ( ) development and characterization of the ards disease model in rats; ( ) design and validation of ambulatory infusion device. in all phases, male wistar rats are housed and cared for according to the university of nebraska iacuc guidelines. animals are allowed to acclimate for days prior to the experiments. in phase , the model for ards is evaluated and characterized. rats are sedated with ketamine-xylazine ( - mg/kg). healthy baseline measurements of weight, chest radiographs (prx , bowie), pulse oximetry (physiosuite, kent scientific corp.), and venous blood analysis are then done. analysis of venous blood from the tail vein was performed with a handheld blood analyzer (vetscan istat , abaxis) to determine the animal's blood gases, ph, chemistry, and hematocrit. intratracheal administration of . ml saline with lps ( mg/kg, sigma-aldrich) was performed with the microsprayer v r aerosolizer (model ia- b-r, penncentury). an additional negative control (nc) group was not administered with any intratracheal solution. observation and collection of daily blood samples and chest radiographs of each animal were performed until death or at days post lps administration. upon completion of the observation period, living animals are then euthanized with sodium pentobarbital. lung tissue was hematoxylin and eosin stained and sent to an independent pathologist for lung injury scoring [ ] . phase is the development of an ambulatory delivery system that will infuse fluid to the rat's peritoneal cavity for treatment. the pds must fulfill several design criteria: ( ) continuously treat four rats simultaneously with the same or different infusates; ( ) infusate needs to be stored at a temperature range of - c; ( ) warm the infusate to c immediately before delivery to the animal; ( ) gently agitate solution to prevent dissociation of microbubbles; ( ) prevent tubing from restraining the rat or being harmed by the rat; ( ) be compatible with current facility housing and cages. the pds will be validated by constant observation in trials with healthy animals to ensure the system fulfills the animal's safety and tubing requirements. for phase , trials have been completed for day lps and nc groups. eight rats (m ¼ g) were successfully administered aerosolized lps and all developed ards while two rats (m ¼ g) were selected for the nc group. the overall mortality rate was . % for lps trials and % for nc trials. all deaths occurred within hr after administration of lps. the remaining lps rats began recovering over the course of the day observation period. chest radiographs taken of lps rats show clear indications of bilateral infiltrates and interstitial edema in the lungs (fig. ) . the lps group showed a dramatic loss in lung function from the observed spo levels when compared to ncs (fig. ) . table shows lung injury scores from lps deaths at day , and nc and lps trials euthanized on day . in phase , final design of the pds has been completed (fig. ) . the pds is comprised of a peristaltic pump, warm water bath, a spring pullbox for dynamic tubing restraint, and a labview control system. the pds is compatible with current housing arrangements of the rats and will not restrict their mobility. once the prototype of the pds has been constructed, tests will be completed with healthy rats to validate the system and provide the rat with full mobility. we expect to be able to continuously dose saline, inert gas microbubbles (imbs), and ombs into the peritoneal cavity of four animals with the current pds design. based upon our previous animal experiments with lung injuries, we anticipate improved pulse oximetry, blood gases, and survivability in rats provided ombs compared to those administered saline or imbs. we have developed an alternative extrapulmonary oxygenation method by delivering ombs to the peritoneal cavity. in verifying the clinical benefits of pmo treatment we have devised the current study. trials of phase show that we are able to repeatedly induce ards in rats with our current methodology. analysis of bal specimens is pending, as we recently identified a lab able to conduct the analysis. the final design of the pds has been completed and build of the prototype has begun. upon completion of phase and , pmo treatment will be evaluated. the future direction of this work after validation will focus on reproducing results in large-animal models of moderate to severe ards, and commercializing the technology. we will begin development of large scale manufacture of ombs. we believe pmo treatment will prove to be a safe and reliable lung bypass therapy for patients with severe ards who cannot tolerate the significant risk profile inherent to ecmo. as we move toward clinical translation, we foresee the implementation of pmo in intensive care units, military combat settings, and even space exploration vehicles. scale ranges from no ( ) and severe ( ) injury. a one rat suffered pulmonary injury during euthanasia, which artificially increased their score. extracorporeal carbon dioxide removal for patients with acute respiratory failure secondary to the acute respiratory distress syndrome: a systematic review systemic oxygen delivery by peritoneal perfusion of oxygen microbubbles intratracheal aerosolization of endotoxin (lps) in the rat: a comprehensive animal model to study adult (acute) respiratory distress syndrome the effects of ketamine, midazolam and ketamine/xylazine on acute lung injury induced by a-naphthylthiourea in rats we wish to thank unl's institutional animal care staff who helped us in performing the procedures and for caring for the animals used in this study. funding was provided by uno nasa space grant no. . key: cord- - q g authors: imai, kenji; kotani, tomomi; tsuda, hiroyuki; nakano, tomoko; ushida, takafumi; iwase, akira; nagai, taku; toyokuni, shinya; suzumura, akio; kikkawa, fumitaka title: administration of molecular hydrogen during pregnancy improves behavioral abnormalities of offspring in a maternal immune activation model date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: q g the aim of the present study was to investigate long-term outcomes of the offspring in a lipopolysaccharide (lps)-induced maternal immune activation (mia) model and the effect of maternal molecular hydrogen (h( )) administration. we have previously demonstrated in the mia mouse model that maternal administration of h( ) attenuates oxidative damage and neuroinflammation, including induced pro-inflammatory cytokines and microglial activation, in the fetal brain. short-term memory, sociability and social novelty, and sensorimotor gating were evaluated using the y-maze, three-chamber, and prepulse inhibition (ppi) tests, respectively, at postnatal or weeks. the number of neurons and oligodendrocytes was also analyzed at postnatal weeks by immunohistochemical analysis. offspring of the lps-exposed dams showed deficits in short-term memory and social interaction, following neuronal and oligodendrocytic loss in the amygdala and cortex. maternal h( ) administration markedly attenuated these lps-induced abnormalities. moreover, we evaluated the effect of h( ) on lps-induced astrocytic activation, both in vivo and in vitro. the number of activated astrocytes with hypertrophic morphology was increased in lps-exposed offspring, but decreased in the offspring of h( )-administered dams. in primary cultured astrocytes, lps-induced pro-inflammatory cytokines were attenuated by h( ) administration. overall, these findings indicate that maternal h( ) administration exerts neuroprotective effects and ameliorates mia-induced neurodevelopmental deficits of offspring later in life. the association between maternal immune activation (mia) and subsequent neurodevelopmental disorders in offspring has become increasingly recognized, with both epidemiological evidence and research findings in various animal models [ ] [ ] [ ] [ ] . mia against viral or bacterial infections influences the developing fetal central nervous system (cns), and increases the risk of schizophrenia and autism spectrum disorder (asd) later in life [ ] [ ] [ ] . it has been suggested that mia has a strong impact on microglial development, and microglial disturbance disrupts neurogenesis, neuronal migration, and myelination, thus leading to consequent impairments in the brain function of the offspring . based on these findings, mia is believed to be a disease primer . it has also been reported that mia and mia-induced fetal neuroinflammation stimulate the generation of reactive oxygen species (ros) and disturbances in pro-inflammatory cytokine production in the fetal brain , . these changes are reported to maternal h administration attenuated the behavioral deficits induced by lps exposure. since mia has been considered to be a cause of behavioral abnormalities in offspring, including asd/ schizophrenia-like behavior, we subsequently evaluated the effect of lps and maternal administration of hw on short-term memory, sociability, social novelty, and sensorimotor gating. as shown in fig. a , spontaneous alternation was markedly reduced in the lps group than in the control group (p < . ) in the y-maze test, which is indicative of impaired short-term memory. treatment with h significantly attenuated the lps-induced impairment of short-term memory (p < . ). there was no difference in the total number of arm entries among the three groups (control, . ± . ; lps, . ± . ; hw + lps, . ± . ). in order to investigate social behaviors, we evaluated sociability and preference for social novelty in the three-chambered social test. during the habituation phase, the mice in each group spent equal amounts of time exploring both compartments, with no biased preference for either of the two empty cylinders (data not shown). during the sociability phase, the mice in each group demonstrated a preference for spending more time in the chamber containing an unfamiliar mouse (stranger ) relative to the opposite, empty chamber. however, the lps-treated offspring approached the chamber containing stranger significantly less frequently than the control and hw + lps groups (p < . and p < . , respectively; fig. c ). during the social novelty preference phase, the mice in each group showed a significant preference for the new, unfamiliar mouse (stranger ) over the previous, now familiar, mouse (stranger ). however, the lps-treated offspring approached the chamber containing the stranger significantly less frequently than the control group (p < . ; fig. d ). there was no difference between the lps and the hw + lps groups, although a trend toward attenuation of the lps-induced reduction in the preference for social novelty was detected in the hw + lps group. the prepulse inhibition (ppi) test was performed to evaluate sensorimotor integration by measuring the startle response to administered acoustic pulses, however, no significant differences were detected among the groups (fig. b ). maternal h administration attenuated the lps-induced neuronal loss. in our previous investigation, activated microglia was most accumulated in mainly temporal association area, h after the lps insult. neurons in this area were reported to be related to sociability deficits . in addition, the association between autism and amygdala, especially in lateral amygdala , , is well known. moreover, the amygdala interacts with the hippocampus in relation to memory . based on those findings, we subsequently performed nissl staining and evaluated the number of neurons in the amygdala, cerebral cortex, and hippocampus to investigate the brain regions related to the results of the behavioral deficits induced by lps exposure. the quantitative results revealed significant reductions in the number of neurons in the amygdala and cerebral cortex in the lps-treated offspring than in the control group (p < . and p < . , respectively; fig. a , panels a,b,d,e and b), but not in the hippocampus (fig. s ). the lps-induced neuronal loss in the amygdala and cerebral cortex was improved in the hw + lps group than in the lps group (p < . and p < . , respectively; fig. a , panels c,f and b). maternal h administration attenuated lps-induced oligodendrocytic loss and suppressed astrocytic activation. the pathophysiologic mechanisms of the brain injury due to mia are incompletely understood; however, under prenatal inflammation, activated astrocyte was reported to have a potential cause an adverse effect on neurons and oligodendrocyte, which would result in the abnormal behaviors in later life . the number of oligodendrocytes was evaluated by using olig , a transcription factor involved in the differentiation of cells in the oligodendroglial lineage, and a mature oligodendrocyte marker , . compared with the control group, the number of olig -positive cells was clearly reduced in the white matter, amygdala, and cerebral cortex in the lps group (p < . , p < . , and p < . , respectively; fig. a , panels a,b,d,e,g,h and c). h treatment completely blocked the lps-induced decrease of olig -positive cells in all three regions (p < . , p < . , and p < . , respectively; fig. a , panels c,f,i and c). the hallmarks of astrocytic activation include the development of a hypertrophic morphology with fewer processes and upregulation of intermediate filament proteins, particularly glial fibrillary acidic protein (gfap) , . in the control group, some gfap-positive cells were detected, most of which were in the resting state (fig. b , panels a,d). compared with the control group, a significantly increased number of activated astrocytes, characterized by a large soma and fewer processes (thus obtaining a more rounded shape), was observed in the white matter and amygdala of the lps-treated offspring (p < . and p < . , respectively; fig. b , panel b,e and d). to evaluate the hypertrophic soma, the cellular size of the astrocytes was also quantified by calculating the ratio of gfap immunostained area to the number of gfap-positive cells. larger soma were observed in the white matter of lps-exposed mouse brains (p < . ; fig. b , panel b and e). although the number of gfap-positive cells was unchanged following h treatment, the size of the gfap-positive cells was significantly reduced in the white matter (p < . ; fig. b , panel c and e), indicating a reduced number of reactive astrocytes by h treatment. in the amygdala, the number of gfap positive astrocytes in the hw + lps group was significantly decreased (p < . ; fig. b , panel f and d). h treatment suppressed lps-induced activation in primary cultured astrocyte. because the over-activation of immune cells, including astrocytes, could have an adverse effect on neurons with the overproduction of pro-inflammatory cytokines , we investigated the anti-inflammatory effect of h on astrocyte using primary cultured astrocyte. as shown in fig. , stimulation of astrocytes with lps for three hours led to marked increases in tumor necrosis factor α (tnf-α) (p < . ), interleukin (il)- β (p < . ), and il- (p < . ) mrna expression. these increases in expression of tnf-α (p < . ), il- β (p < . ) and il- (p < . ) were significantly attenuated by h treatment. altered gene expression patterns in primary cultured astrocytes from the control group, the lps group, and the hw + lps group. as we previously described the gene expression profile of microglia , differential gene expression patterns of astrocytes from newborn mice in the control group, the lps group, and the hw + lps group were also observed (tables s -s , supplementary information). these results suggest that h might also influence the function not only of microglia but also of astrocytes via changes in gene expression. for the first time, we have demonstrated that maternal h administration improved mia-induced neurological impairments of offspring, including short-term memory and social interaction at postnatal - weeks. previously, we reported that maternal h administration suppresses microglial activation in the same model. in the present study, h also suppressed the activation of astrocytes, both in vitro and in vivo. taken together, these results suggest that h exerts a protective role against mia-induced neuroinflammation through the suppression of microglia and astrocytes. as a result, mature oligodendrocytes were restored and neurons were protected from damage, thus leading to improved long-term neurological outcomes following maternal h administration. prenatal exposure to lps also resulted in a transient reduction in body weight of the offspring between p -p , which was attenuated by maternal h administration. this might be due to a change in the activity of the hypothalamic-pituitary axis . deficient post-neonatal growth has been reported to contribute to poor neurologic outcome , thus, the transient decrease of weight observed in this model might be related to the neurological impairments in the offspring. on the basis of the epidemiological evidence, several translational rodent models have been established to investigate a potential causal relationship between mia and asd-like behavioral abnormalities . following the discovery, in the s, of the increased risk of schizophrenia after maternal influenza infection , models of viral-like immune activation by polyriboinosinic-polyribocytidilic acid (poly(i:c)) have been widely studied , , . in addition, models of bacterial-like immune activation by lps are also well known to precipitate inflammatory responses and behavioral abnormalities in offspring, similar to the poly(i:c) models . however, with respect to neurotoxicity, lps exposure may have a stronger effect on the fetal brain than poly(i:c) exposure . thus, the type of pathogen is important for the pattern of neurodevelopmental abnormalities of the offspring, and the timing and dose of the pathogen's administration, in addition to the strain or species, may also have an influence. the present model of prenatal exposure to lps led to impairments in short-term memory and social interaction at to weeks after birth. alterations in short-term memory were demonstrated by the marked reduction in alternation behavior in the y-maze test, which is consistent with previous reports , and is a persistent finding in poly(i:c) models . the y-maze test is well known as a hippocampal-dependent short-term learning task. in our mia model, a remarkable loss of neurons was observed in the amygdala and cerebral cortex of the offspring, but not in the hippocampus. the formation of memory is encoded in a broad network of cortical/subcortical regions including the hippocampus, cingulate cortex, and amygdala . moreover, the amygdala is directly connected to and dynamically interacts with the hippocampus in relation to memory . therefore, a decreased number of neurons in the amygdala and cerebral cortex could consequently result in short-term memory impairments. in this model, increased activation of astrocytes was observed in the white matter and amygdala, which be related to impaired short-term memory , . previous reports have demonstrated a causal relationship between prenatal exposure to lps and deficits in social behavior and sensorimotor gating in offspring , [ ] [ ] [ ] [ ] . in this model, the social behavior including sociability and social novelty preference was impaired. the number of oligodendrocytes in the amygdala, cerebral cortex, and white matter in the offspring was decreased, which is consistent with previous findings ; and a reduced number of neurons in the amygdala and cortex was also observed. strong evidence suggests a crucial involvement of the amygdala in social processing and social cognition in humans [ ] [ ] [ ] , as well as in social behavior in animals [ ] [ ] [ ] . a recent report also suggested an association between the function of the cortex and the amygdala, and social behavior . therefore, the social behavior impairment observed in the present study is compatible with the neuronal and oligodendrocytic loss in the amygdala and cortex. a locomotor test was not performed in this study, although we observed no apparent differences in speed of movement among all groups in the three-chamber social test. interestingly, there were no remarkable differences in the ppi test in our mia model, and sensorimotor gating was also normal in comparison with the control group. a possible explanation for these results is that ppi abnormalities might occur following exposure to a pathogen at mid-gestation , while impairments in short-term memory and social behavior might be independent of the timing of exposure to a pathogen . thus, the negative results of the ppi test might be due to the fact that in the present model, lps exposure occurs at late gestation, on embryonic day (e ). impairments in short-term memory and social behavior are well described in schizophrenia and asd , . ppi impairments manifest primarily in adults with asd , and are not observed in children with asd . a recent study further suggested that sensorimotor gating is only impaired in certain asd subgroups , although it may be a globally common feature in schizophrenia . therefore, the present model may reflect asd-like rather than schizophrenia-like behavioral impairments. several characteristics that are reported in human asd brains were also observed in the present model, including neuronal loss in the amygdala and cerebral cortex , and astrocytic activation in the white matter . we and others have also previously demonstrated microglial activation, enhanced pro-inflammatory cytokine levels, including il- , and elevated oxidative damage in fetal brains , [ ] [ ] [ ] . in the mia model, microglia play a major role, along with astrocytes, in synaptic pruning and neural circuit formation . in addition, a single injection of il- is known to lead to the same behavioral abnormalities as those observed in the lps models (il- is thought to be a key cytokine in the link between mia and the behavioral deficits in the offspring ). in our previous report, maternal h administration reduced both the excessive microglial activation and the increased il- levels in the mia fetal brains , which could ameliorate the behavioral deficits later in life. previous studies in adult rodents reported that h treatment could mitigate the behavioral dysfunctions, including short-term memory deficits, cognitive impairments, and depressive-like behaviors, by regulating inflammation and ros production , , which is in line with our current results. moreover, the present study demonstrated that prenatal h administration restored the mia-induced neuronal and oligodendrocytic loss, and suppressed the excessive activation of astrocytes at postnatal weeks. it was also shown that h directly attenuated the lps-induced expression of pro-inflammatory cytokines, including tnf-α, il- β, and il- , in primary cultured astrocytes. these cytokines are known to induce apoptosis in oligodendrocytes and hypomyelination in the neonatal rodent . thus, the suppressive effect of h on both microglial and astrocytic activation would protect neurons and oligodendrocytes against lps exposure. the pathological mechanism by which mia causes psychiatric diseases in offspring is completely unknown, however we speculate that the following steps may be involved , . intrauterine inflammation activates fetal microglia during the acute phase, and subsequently, microglia also activate astrocytes. in the chronic phase, microglial activation is attenuated, while astrocytes are continuously activated . both microglia and astrocytes are important for normal neurodevelopment, however under prenatal inflammation, they are activated, their function is altered, and they have an adverse effect on neurons and axons, with the overproduction of pro-inflammatory cytokines . inflammation also prevents the maturation of oligodendroglial progenitor cells, followed by a reduction of the oligodendrocyte lineage , . this reduction leads to axonal loss. the neuronal and axonal damage caused by microglia or astrocytes in the developing brain would result in the abnormal behavioral features in later life. in our model, we have previously reported over-activation of fetal microglia h after the lps insult , and the present study demonstrated that activation of astrocytes continued even ~ weeks after the insult. moreover, the number of neurons and oligodendrocytes were reduced in the amygdala and white matter, respectively. we have previously reported that h has a suppressive effect on microglial activation , and the present study demonstrated that h had a similar effect on astrocytes, as described above. from these findings, we speculate that h would protect neurons and oligodendrocytes indirectly via suppression of the over-activation of immune cells, including microglia and astrocytes, which would result in a reduction of the behavioral abnormalities present in mia offspring. however, in this study, a direct effect of h on neurons and oligodendrocytes was not evaluated in vitro, thus, this possibility cannot be excluded. several limitations of this study should be noted. in the current mia model, additional core features of asd, including restricted and repetitive behavioral patterns, were not investigated . thus, the effect of maternal h administration on these symptoms remains unknown. the sensorimotor gating abnormality may be evaluated at an older age than was described in the present model, however, this might require changing other factors including the timing of exposure. since the mouse brain is not considered to be fully mature until weeks of age , the interpretation of the results of behavioral tests in the present study requires considerable caution. in addition, we could not avoid the detrimental effects of maternal separation required for the measurement of body weight on offspring development. however, the separation was performed similarly for the offspring in all three groups, thus any effect should be minimal, and consistent across groups. in humans, it is currently thought that the occurrence of multiple risk factors, including genetic mutations and postnatal exposure to inflammation or other environmental triggers, may be required for the onset of asd , . before advancing the clinical application of h , it should be investigated whether h could exert similar effects in so called 'multi-hit' models. finally, in the previous and present studies, maternal h administration was performed prior to the lps exposure; thus, the therapeutic potential of h remains unclear. therefore, it might be relevant to evaluate the therapeutic effects of h by administering h to dams after exposure to lps and in offspring. in conclusion, we have provided evidence that maternal h administration reduced deficits in short-term memory and social interaction in the lps-induced mia model, and also demonstrated an attenuation of the lps-induced neuronal and oligodendrocytic cell loss. remarkably, the neuroprotective effect of h appeared in the amygdala and cortex in this mouse model. the neuroprotection is thought to be due to the suppression of neuroinflammation, including excessive activation of microglia and astrocytes and the increased level of pro-inflammatory cytokines, as reported previously . although further investigation is required, our current and previous results point to the potential efficacy of maternal h administration on the long-term neurological outcomes of offspring exposed to inflammation in utero. reagents. lps (serotype o :b ) was obtained from sigma-aldrich (st. louis, mo, usa). hw and hydrogen medium (hm) were prepared by dissolving h gas as described previously , . both hw and hm had a concentration of > . mm h . animals and treatments. the experimental protocols in this study were approved by the animal experiment committee of nagoya university (approval number: ), and were carried out in accordance with the regulations on animal experiments in nagoya university. pregnant icr (cd- ) mice ( - weeks) were purchased from charles river laboratories (kanagawa, japan). all mice were allowed free access to food and water and were maintained on a -h light/dark cycle (lights on at : am). the pregnant mice were assigned randomly to three groups of five dams each: control group, lps group, and hw + lps group (fig. ) , as in our previous report . all pregnant mice, except for those in the control group, received an intraperitoneal injection (i.p.) of μg of lps dissolved in sterile saline on e . in the control group, the pregnant mice were injected with an equal volume of sterile saline. in the hw + lps group, the pregnant mice were administered hw, beginning h before lps injection and continuing until parturition. hw was aliquoted in glass drinking bottles to prevent h degassing as well as air refilling. offspring growth evaluation and behavioral testing. as shown in fig. , after birth, all offspring were housed with their mother until p , when they were weaned, as previously reported , . we only used male offspring in all of the experiments because of previously reported sex effects . all animals were left undisturbed except for measuring body weights and weekly cage changes. body weight was recorded at p , , , , , , and . the order of the behavioral tests was as follows: ( ) y-maze test at postnatal weeks ; and ( ) three-chamber sociability and social novelty test and ppi test at postnatal weeks, according to previous reports (fig. ). all behavioral tests were conducted between pm and pm. y-maze test. the y-maze test was carried out as described previously . briefly, the test was performed in a y-shaped maze with three opaque plastic arms situated at ° angles from each other. each mouse was placed individually in the center of the apparatus and allowed to explore the maze freely during an -min session. the series of arm entries were recorded visually. alternation was defined as successive entries into the three arms, on overlapping triplet sets. the percent alternation was calculated as the ratio of actual to possible alternations (defined as the total number of arm entries minus two) multiplied by . spontaneous alternation (%) was used to quantify short-term memory. three-chamber social test. the three-chamber social test was performed as described previously . briefly, the apparatus consisted of a black plexiglas rectangular box and two identical clear plexiglas cylinders. there were three interconnected chambers in the box. the light was conditioned at lux in an experimental room. during the habituation phase, empty cylinders were placed in each of the end chambers. a mouse was introduced to the center chamber and its behavioral approach to the end chambers was monitored for min. during the sociability test, an unfamiliar male icr (cd- ) mouse (stranger ) that had no prior contact with the test mouse was placed in one of the empty chambers, and the behavioral approach of the test mouse to the empty chamber and stranger was monitored for min. during the social novelty test, a new unfamiliar male icr (cd- ) mouse (stranger ) was placed in the third chamber, and the behavioral approach of the test mouse to stranger and stranger was monitored for min. the time spent in each zone was calculated using the ethovision automated tracking program (noldus, wageningen, the netherlands). prepulse inhibition test. the ppi test was carried out as described previously with the sr-lab system (san diego instruments, san diego, california). briefly, after habituation for min, the animals received startle trials, no-stimulus trials, and ppi trials. each startle trial consisted of a single db white noise burst lasting msec. the ppi trials consisted of a prepulse ( msec burst of white noise at , , , or db intensity) followed, msec later, by the startle stimulus ( db, msec white noise). the total session lasted min. the resulting movement of the animal in the startle chamber was measured for msec after the startle stimulus onset (sampling frequency khz), rectified, amplified, and input into a computer, which calculated the maximal response over the -msec period. the basal startle amplitude was determined as the mean amplitude of the startle trials. the ppi was calculated according to the following formula: × [ − (pp×/p )] %, in which ppx was the mean of the ppi trials (pp , pp , pp , or pp ) and p was the basal startle amplitude. after the behavioral analysis, offspring in each group were selected randomly and sacrificed by decapitation at postnatal weeks (fig. ) . the offspring were perfused via the heart with % paraformaldehyde (pfa) in pbs. the brains were then fixed in % pfa for at least h, embedded in paraffin, and cut in coronal planes (approximately . mm posterior to bregma). immunofluorescence staining was performed using the following primary antibodies: rabbit monoclonal anti-olig antibody (ab , : ; abcam, tokyo, japan) or rabbit polyclonal anti-gfap antibody ( - -ap, : ; proteintech, chicago, il). the sections were incubated with the primary antibodies at °c overnight and were further incubated with secondary antibodies (alexa fluor , : or alexa fluor , : ; invitrogen, carlsbad, ca) for min at room temperature. nissl staining was also performed in order to evaluate the number of neuronal cells. following deparaffinization, the tissue sections were incubated in . % cresyl violet (muto pure chemicals co.), and subsequently dehydrated in graded ethanol, and permanently mounted. images were acquired with a bz- microscope (keyence corporation, osaka, japan). the bz image measurement software program (keyence) was used to quantify the number of neuronal cells and olig -positive cells, as well as the number and the soma size of the gfap-positive cells. astrocytic cultures. mouse primary astrocytes were isolated from primary mixed glial-cell cultures of newborn icr mice, as described previously , . the purity of the astrocytes was > %, as determined by immunostaining with the anti-gfap antibody. astrocytes were plated at a density of × cells/well in -well dishes. cells were maintained in dulbecco's modified eagle's minimum essential medium (sigma-aldrich) supplemented with % fetal bovine serum, mg/ml bovine insulin, and . % glucose. to assess the expression of pro-inflammatory cytokines, confluent astrocytes were treated with μg/ml lps and/or hm. h treatment was performed by replacing the astroglial medium with hm, and dehydrogenated hm was used as a control medium. the time course of h concentration in hm was described previously . replacement of the astroglial medium with hm or control medium was performed h before the addition of lps. quantitative reverse transcription-polymerase chain reaction (rt-pcr). total rna from the astrocyte cultures was extracted using the rneasy mini kit (qiagen inc., tokyo, japan). the rt reaction with ng of total rna was carried out with a first strand cdna synthesis kit (revertra ace; toyobo co., ltd, osaka, japan). the expression levels of mrnas encoding tnf-α, il- β, and il- were evaluated by quantitative pcr (qpcr) using the thermal cycler dice (takara bio inc., tokyo, japan) and sybrii premix ex taq (takara bio inc.) reagents. primers for qrt-pcr are listed in table . statistical analysis. the data are presented as means ± standard error of the mean (sem). to analyze the neonatal body weights, two-way repeated measures analysis of variance (anova) was used, followed by a bonferroni post-hoc test. the mrna expression levels in astrocyte cultures were also analyzed by two-way anova, followed by a bonferroni post-hoc test. all other data were analyzed using one-way anova followed by tukey's test. the statistical analyses were performed using prism for windows (graphpad software, san diego, ca). values of p < . were considered to be statistically significant. tnf-α ′-gtagcccacgtcgtagcaaac- ′ ′-ctggcaccactagttggttgtc- ′ il- ′-acaaccacggccttccctac- ′ ′-tccacgatttcccagagaaca- ′ il- β ′-catccagcttcaaatctcgcag- ′ ′-cacacaccagcaggttatcatc- ′ β-actin ′-cgtgggccgccctaggcacca- ′ ′-acacgcagctcattgta- ′ table . list of primers for qrt-pcr. maternal immune activation: implications for neuropsychiatric disorders maternal immune activation and abnormal brain development across cns disorders beyond infection -maternal immune activation by environmental factors, microglial development, and relevance for autism spectrum disorders prenatal maternal immune activation and brain development with relevance to psychiatric disorders prenatal maternal infection, neurodevelopment and adult schizophrenia: a systematic review of population-based studies maternal infection and immune involvement in autism epidemiologic studies of exposure to prenatal infection and risk of schizophrenia and autism brain injury in premature infants: a complex 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labile cognitive impairment seizure-dependent mtor activation in -ht neurons promotes autism-like behaviors in mice combined effect of neonatal immune activation and mutant disc on phenotypic changes in adulthood heterozygous disruption of autism susceptibility candidate causes impaired emotional control and cognitive memory excitatory amino acid transporter expression by astrocytes is neuroprotective against microglial excitotoxicity coronavirus infection induces h- antigen expression on oligodendrocytes and astrocytes the author would like to thank dr. j. kawanokuchi (institute of traditional chinese medicine, suzuka university of medical science) for technical support for primary cultured astrocytes. we would like to acknowledge mrs sachiko morisaki for her laboratory work. we would like to thank editage (www.editage.jp) for english language editing. this work was supported by jsps kakenhi grant number h and k and msd life science foundation, public interest incorporated foundation. the authors have no conflicts of interest to declare in association with this study. supplementary information accompanies this paper at https://doi.org/ . /s - - - . the authors declare no competing interests.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- -chd ezba authors: anas, adam; poll, tom van der; de vos, alex f title: role of cd in lung inflammation and infection date: - - journal: crit care doi: . /cc sha: doc_id: cord_uid: chd ezba this article is one of ten reviews selected from the yearbook of intensive care and emergency medicine (springer verlag) and co-published as a series in critical care. other articles in the series can be found online at http://ccforum.com/series/yearbook. further information about the yearbook of intensive care and emergency medicine is available from http://www.springer.com/series/ . toll-like receptors (tlr) on the surface of cells of the respiratory tract play an essential role in sensing the presence of microorganisms in the airways and lungs. th ese receptors trigger infl ammatory responses, activate innate immune responses, and prime adaptive immune responses to eradicate invading microbes [ ] . tlr are members of a family of pattern-recognition receptors, which recognize molecular structures of bacteria, viruses, fungi and protozoa (pathogen-associated molecular patterns or pamps), as well as endogenous structures and proteins released during infl ammation (damage/ danger-associated molecular patterns or damps). to date, ten diff erent tlr have been identifi ed in humans and twelve in mice. tlr are expressed on all cells of the immune system, but also on parenchymal cells of many organs and tissues. th e binding of a pamp to a tlr results in cellular activation and initiates a variety of eff ector functions, including cytokine secretion, prolifera tion, co-stimulation or phagocyte maturation. to facilitate microbial recognition and to amplify cellular responses, certain tlr require additional proteins, such as lipopolysaccharide (lps) binding protein (lbp), cd , cd and high mobility group box- protein (hmgb- ). in this chapter, the role of cd as an accessory receptor for tlr in lung infl ammation and infection is discussed. th e central role of cd in the recognition of various pamps and amplifi cation of immune and infl ammatory responses in the lung is depicted in figure . cd was characterized as a receptor for bacterial endotoxin (lps) in , almost a decade before the discovery and characterization of tlr, and can be regarded as the fi rst described pattern-recognition receptor [ ] . th e protein was fi rst identifi ed as a diff erentiation marker on the surface of monocytes and macrophages and was designated cd at the fi rst leukocyte typing workshop in paris in . th e genomic dna of human cd was cloned in and the gene was later mapped to chromo some q - . several polymorphisms have been found in the cd gene, of which nucleotide polymorphisms at position - and - correlated with decreased lung function in endotoxin-exposed farmers [ ] . th e cd gene consists of two exons which code for a single mrna that is translated into a protein of amino acids. th e cd protein is composed of eleven leucin-rich repeats, which are also found in tlr and which are important in pamp binding. moreover, the crystal structure of cd revealed that the protein has a `horseshoe' shape, similar to tlr , and that lps is bound within the pocket [ ] . in contrast to tlr, however, cd lacks a transmembrane domain, and thus cannot initiate intracellular signal transduction by itself. th e cd protein is processed in the endoplasmatic reticu lum and expressed as a kda glycoprotein on the cell surface via a glycosylphosphatidyl (gpi) anchor [ ] . like other gpianchored proteins, cd accumulates on the cell surface in microdomains known as lipid rafts, which are fairly rich in cholesterol and accumulate several kinases at the intracellular site. cd is expressed pre dominantly on the surface of `myeloid' cells, such as mono cytes, macrophages and neutrophils, but at lower levels also on epithelial cells, endothelial cells and fi broblasts. in addition to being expressed as a gpi-anchored membrane protein, cd is also expressed in a soluble form (scd ) [ ] . scd may result from secretion of the protein before coupling to the gpi anchor or from shedding or cleavage from the surface of monocytes. scd is present in the circulation and other body fl uids and levels of scd in plasma increase during infl ammation and infection. since interleukin (il)- induces scd expression in liver cells it is regarded as an acute phase protein. in bronchoalveolar lavage (bal) fl uid from patients with acute respiratory distress syndrome (ards), scd levels were strongly increased and correlated with total protein levels and neutrophil numbers in the bal fl uid [ ] , suggesting that scd contributes to the infl ammatory process in the lung. cd is a molecule with a wide range of functions. in addition to functioning as a pattern recognition receptor for a variety of microbial ligands, cd also acts as a receptor for endogenous molecules like intercellular adhesion molecule (icam)- on the surface of apoptotic cells, amyloid peptid, ceramide, and urate crystals. ligation of cd by these ligands, except for apoptotic cells, mediates activation of infl ammatory responses. lps is the major constituent of the outer membrane of gram-negative bacteria and is one of the most potent tlr ligands. cd together with lbp plays an essential role in binding of lps to the tlr /md- complex [ ] . lbp, which, among others, is present in the bloodstream and bal fl uid [ ] , binds to lps aggregates and transfers lps monomers to cd . cd associates with tlr / md- and transfers the lps monomer to this complex [ ] . likewise, scd is able to mediate lps-activation of cells with low membrane cd expression, such as epithelial and endothelial cells [ ] . however, at high con cen trations, lbp and scd are also able to downregulate lps-induced responses by transfer of lps to lipoproteins for subsequent removal [ ] . recent data indicate that lps is bound by md- within the tlr / md- complex [ ] and that subsequent conformational changes in tlr lead to reorganization of its cytoplasmic domain, enabling the recruitment of the adaptor proteins, myeloid diff erentiation primary-response protein (myd ) and tir-domain-containing-adaptorprotein-inducing-inter feron (ifn)-β (trif) [ ] . th ese adaptors initiate signal transduction to the nucleus by activation of nuclear factor (nf)-κb and ifn regulatory transcription factor (irf)- , leading to the production of cytokines that regulate infl ammatory cells [ ] . in macrophages, trif-dependent signaling is essential for the expression of the majority of lps-induced genes, including ifn-α/β. cd , which lacks an intracellular domain for signal transduction, is expressed on the surface of alveolar macrophages, infi ltrating monocytes and neutrophils, and at lower levels also on epithelial and endothelial cells in the lung. cd recognizes and binds various structures from invading microbes, such as lipopolysaccharide (lps) from gram-negative bacteria, lipoteichoic acid (lta) from gram-positive bacteria, lipoarabinomannan (lam) from mycobacteria, viral double stranded (ds) rna and f glycoprotein (f-gp) from respiratory syncytial virus (rsv). cd subsequently transfers these bound components to toll-like receptors (tlr) which than trigger cell activation. binding of lps to cd is regulated by additional accessory receptors in the lung, including lps-binding protein (lbp) and a number of surfactant proteins (sp). furthermore, soluble cd (scd ) enhances lps-induced activation of cells with low cd expression. depending on the microbe and the pamps it expresses, cd -amplifi ed responses can either be benefi cial to the host by induction of an adequate infl ammatory and immune response to eradicate the invading microbe, or detrimental to the host by excessive infl ammation and/or dissemination of the pathogen. recently, it was reported that, in the absence of cd , the tlr /md- complex can distinguish between diff erent chemotypes of lps [ ] . smooth lps is synthesized by most gram-negative bacteria and consists of three modules: th e lipid a moiety, a core poly saccharide, and an o-polysaccharide of variable length (made up of to over monosaccharide units) [ ] . gram-negative bacteria that fail to add the core polysaccharide or the o-polysaccharide chain to the lipid a moiety produce `rough' lps, named after the rough morphology of the colonies these bacteria form. lipid a, the bioactive part of both smooth and rough lps, is responsible for most of the pathogenic eff ects in gram-negative bacterial infections [ , ] . murine macrophages lacking cd secreted equal amounts of tumor necrosis factor-α (tnf) to macrophages expressing cd upon stimulation with rough lps, but failed to secrete tnf in response to smooth lps, an eff ect which was reversed by addition of scd [ ] . moreover, macrophages lacking cd failed to secrete ifn-α/β in response to either rough or smooth lps. th ese fi ndings indicate that cd is required for activation of the tlr /trif pathway by either smooth or rough lps, and required for the activation of tlr / myd pathway by smooth but not by rough lps [ ] . in addition to lps, cd also facilitates tlr activation by other pamps including certain viral components [ , ] . in the lung, binding of lps to tlr is infl uenced by a number of surfactant proteins (sp), including sp-a, sp-c and sp-d [ ] . th ese surfactants are able to infl uence the interaction between tlr and lps by direct binding to lps; i.e., sp-a binds to rough lps and lipid a, but not to smooth lps, sp-c also binds to rough lps, and sp-d binds to both rough and smooth lps. sp-a and sp-c binding to lps inhibits tnf secretion by alveolar macrophages, whereas sp-d binding to lps moderately enhances tnf secretion by alveolar macrophages. in addition, sp-a, sp-c and sp-d also bind to cd at the site which recognizes lps. strikingly, binding of sp-a to cd enhanced the binding of rough lps and binding of sp-c to cd augmented binding of smooth lps [ ] , whereas binding of sp-a to cd reduced binding of smooth lps and binding of sp-d to cd decreased binding of both smooth and rough lps. furthermore, sp-d infl uences lps-induced tnf secretion by alveolar macrophages by regulating matrix metalloproteinasemediated cleavage of cd from the surface of these cells [ ] . together, these fi ndings suggest that lps recognition in the lung and subsequent induction of infl ammatory immune response is a complexly regulated process. in addition to lps-induced activation of tlr , cd also amplifi es a number of tlr-dependent responses triggered by other bacterial pamps, including peptidoglycan, lipoteichoic acid (lta) and lipoarabinomannan (lam) [ ] [ ] [ ] . peptidoglycan is an essential cell wall component of virtually all bacteria. peptidoglycan is a polymer of nacetylglucosamine and n-acetylmuramic acid, crosslinked by short peptides. breakdown products of peptido glycan are recognized by diff erent classes of pattern-recognition receptors [ ] . polymeric soluble peptidoglycan is recognized by tlr on the surface of cells, and the interaction of peptidoglycan with tlr triggers myd -dependent activation and nuclear translocation of nf-κb, and subsequently the transcription and secretion of cytokines. muramyl dipeptide and γ-dglutamyl-meso-diaminopimelic acid, which are lowmolecular weight breakdown fragments of peptidoglycan, are recognized by intracellular pathogen recognition receptors, nucleotide-binding oligomerization domain containing (nod) and nod , respectively [ ] . ligand binding to these receptors triggers interaction with the receptor-interacting protein kinase, rip , which activates nf-κb. of these peptidoglycan breakdown products, only polymeric peptidoglycan binds to cd , and cd enhances polymeric peptidoglycan-induced tlr activation. th e low molecular weight fragments of peptidoglycan, like muramyl dipeptide, do not bind to cd , do not induce cell activation through cd and also do not interfere with the binding of polymeric peptidoglycan to cd [ ] . furthermore, unlike lps, peptidoglycan bound to scd is not able to activate epithelial and endothelial cells with low membrane cd expression. lta is a constituent of the cell wall of gram-positive bacteria, anchored on the outer face of the cytoplasmic membrane and commonly released during growth and antibiotic therapy. like polymeric peptidoglycan, lta induces nf-κb activation and cytokine secretion in a tlr -dependent manner. lta is recognized by lbp and cd , and these accessory receptors both enhance ltainduced cell activation [ ] . presumably in a similar manner, cd also enhances tlr -dependent cellular activation by lam derived from the cell-wall of mycobacteria. lam derived from slowly growing virulent mycobacteria like mycobacterium tuberculosis and m. leprae is capped with mannose (manlam), whereas lam from avirulent and fast growing mycobacterial species is uncapped (aralam). strikingly, aralam from avirulent mycobacteria is much more potent in inducing tnf secretion by macrophages than manlam from virulent mycobacterial strains [ ] . aralam-, but not manlam-induced tnf secretion by monocytes and macrophages was largely cd -, tlr -and myd dependent [ ] . recently cd was also found to enhance the innate immune response triggered by the tlr ligand poly(i:c), a synthetic mimic of double stranded rna [ ] . tlr together with tlr and tlr are regarded as sensors for viral infection, since these receptors recognize viral nucleic acids, like single and double stranded rna. th e potentiating eff ect of cd on tlr activation resulted from increased uptake of poly(i:c) and intracellular delivery to the compartment where tlr resides [ ] . taken together, these fi ndings suggest that cd plays an important role in the induction and amplifi cation of infl ammatory responses evoked by a wide variety of pathogens. th e contribution of cd to tlr ligand-induced lung infl ammation has been investigated in several animal studies (table ) . intratracheal administration of lps did not signifi cantly induce tnf release and neutrophil accumulation in the lungs of rabbits, unless lps was complexed with lbp [ ] or the animals were subjected to mechanical ventilation [ ] . intratracheal instillation of anti-cd antibodies together with lps/lbp or intravenous pretreatment with anti-cd or anti-tlr antibodies before mechanical ventilation markedly reduced these infl ammatory responses [ , ] . despite a reduction in lung neutrophil number, intravenous anti-cd treatment of rabbits exposed to lps and subjected to ventilation did not cause a decrease in lung chemokines, including cxcl (il- ), growth related oncogene (gro) and monocyte chemoattractant protein (mcp)- , whereas anti-tlr treatment did lower the level of gro moderately and of cxcl signifi cantly [ ] . th ese fi ndings reveal that lps alone does not cause signifi cant lung infl ammation in rabbits and suggest that additional accessory signals are required. whether mechanical ventilation induces increased release of lbp or release of (endogenous) damps which potentiate the lps-induced response remains to be determined. in contrast to rabbits, administration of lps alone to lungs of naive mice induced severe pneumonitis, irrespective of the manner of lps delivery (inhalation or intra tracheal or intranasal instillation) or the source of lps (escherichia coli or acinetobacter baumannii). using antibody-treated and gene-defi cient mice, cd was found to be critically involved in the development of lps-induced lung infl ammation [ ] [ ] [ ] [ ] . a study with cd -defi cient mice and tlr mutant mice (lacking a functional tlr ) showed that lps-induced vascular leakage, neutrophil infi ltration, nuclear translocation of nf-κb. th e release of cytokines (tnf and il- ) and chemo kines (cxcl and cxcl ) in the lung was completely dependent on these pattern recognition receptors [ ] . similar observations were made by others using mice treated intravenously with anti-cd infl uenza a mouse cd -/-/~clearance, ~lymphocyte recruitment and activation, ~neutrophil infl ux, ~cytokines antibodies [ ] and by our group using cd -defi cient and tlr -defi cient mice [ ] . furthermore, intratracheal treatment of cd -defi cient mice with scd restored the infl ammatory response to the level present in wildtype mice, whereas treatment with wild-type alveolar macrophages restored the neutrophil infi ltration of the lung but not pulmonary tnf release [ ] . moreover, treatment with wild-type alveolar macrophages also restored neutrophil infi ltration in the lung of lpsexposed tlr -defi cient mice [ ] . th ese fi ndings indicate that scd , and cd and tlr on the surface of alveolar macrophages contribute to the development of lps-induced lung infl ammation. however, when a high dose of lps was administered to the lungs of mice, acute lung infl ammation was absent in mice lacking functional tlr , but only partially reduced in cd defi cient mice [ ] . th us, lps-induced lung infl am mation is entirely dependent on tlr and, depending on the dose of lps, also on the presence of cd in the lung. our group determined whether cd also contributes to the development of lung infl ammation induced by lta, a tlr ligand from the cell wall of gram-positive bacteria [ , ] . lung infl ammation induced by staphylo coccus aureus lta was completely dependent on tlr , but independent of lbp and only moderately dependent on cd expression. as compared to wildtype mice, s. aureus lta-induced neutrophil infl ux was unchanged in cd -defi cient mice, whereas tnf and cxcl release in the lung were partially reduced [ ] . strikingly, however, pulmonary infl ammation was also greatly diminished in tlr -defi cient mice, as well as in mice defi cient for platelet activating factor receptor (pafr), a known receptor for lta on epithelial cells. similarly, lung infl ammation induced by streptococcus pneumoniae lta, which is less potent compared s. aureus lta, was also completely dependent on tlr expression. however, in contrast to s. aureus lta, neutrophil infi ltration of the lung was moderately reduced in cd -defi cent mice treated with pneumococcal lta, whereas tnf and cxcl release in the lung was unchanged [ ] . moreover, pneumococcal ltainduced lung infl ammation was moderately diminished in tlr -defi cient mice. th us, despite the amplifying eff ect on lta-induced tlr -mediated responses in vitro, cd contributes minimally to lung infl ammation induced by lta. th e unexpected contribution of tlr to lta-induced lung infl ammation may result from damps generated during the infl ammatory process in the respiratory tract. in line with the fi ndings that cd contributes to lpsinduced lung infl ammation in mice, a number of studies have shown that cd is essential for the host defense response in the lung against gram-negative bacteria, such as nontypeable haemophilus infl uenzae, a possible cause of community acquired pneumonia, and a. baumannii and e. coli, which are frequent inducers of nosocomial pneumonia (table ) . nontypeable h. infl uenzae expresses the tlr ligands lps and lipooligosaccharide on its cell wall, as well as several tlr ligands, including lipoproteins and porins. previously, we found that activa tion of alveolar macrophages by nontypeable h. infl uenzae depended on expression of tlr , tlr , and cd [ ] . moreover, bacterial clearance after intranasal infection with nontypeable h. infl uenzae was markedly reduced in cd -defi cient and tlr -defi cient mice, as well as in tlr -defi cient mice at later stages of the disease [ ] . interestingly, despite impaired bacterial clearance in cd -defi cient and tlr -defi cient mice, the infl ammatory response in the lung was strongly reduced in tlr defi cient mice, but elevated in cd defi cient mice. similar observations were made with encapsulated h. infl uenzae in tlr -mutant mice [ ] . furthermore, clearance of nontypeable h. infl uenzae was also significantly impaired in myd -defi cient mice, but not in mice lacking functional trif [ ] . in a similar manner, cd was involved in the host defense response against a. baumanii [ ] . cd -defi cient mice, like tlr defi cient mice, suff ered from impaired bacterial clearance in the lungs and enhanced bacterial dissemination after intranasal infection with a. baumannii. however, unlike tlr -defi cient mice, cd -defi cient mice developed similar infl ammatory responses compared to wild-type mice. th ese fi ndings suggest a role for cd in antibacterial responses against nontypeable h. infl uenzae and a. baumannii. although the role of tlr (and tlr ) in phagocytic killing is controversial, it is unknown whether cd is involved in such processes. th e role of cd in e. coli-induced pneumonia was determined in anti-cd antibody treated rabbits. intravenous anti-cd antibody treatment of rabbits inoculated with e. coli by bronchial instillation, resulted in decreased bacterial clearance from the lungs, but had no eff ect on neutrophil infi ltration or cytokine release in the lungs [ ] . however, anti-cd treatment protected against sustained hypotension and reduced the levels of nitrate and nitrite in the blood. th e contribution of cd to e. coli-induced pneumonia has not been investigated in mice, whereas the role of the other components of the lps receptor complex (tlr , md- , myd , trif) has been determined using gene-defi cient or mutant mice. although analysis of bacterial clearance after intranasal infection of tlr -mutant mice with e. coli produced inconsistent results [ ] , lack of md- or trif resulted in impaired bacterial clearance after e. coli instillation in the lungs [ , ] . moreover, e. coli-induced neutrophil accumulation and cytokine release was signifi cantly reduced in mice devoid of functional tlr , md- , myd or trif [ ] [ ] [ ] . th ese fi ndings indicate that signaling through the tlr receptor complex is essential in the host defense response against e. coli, and suggests that cd may contribute to these e. coli-induced responses. to our knowledge, it is unclear whether cd contributes to host defense against pseudomonas aeruginosa, a frequent cause of nosocomial pneumonia, and burkholderia cepacia, a prevalent gram-negative bacterium, together with p. aeruginosa, in patients with cystic fi brosis. recently, it was found that both tlr and tlr are critical in the host response to p. aeruginosa and that tlr -defi cient mice were not susceptible to intratracheal p. aeruginosa infection unless a bacterial mutant devoid of fl agellin production was used [ ] . a similar approach is required to determine a role for cd in pseudomonas-induced pneumonia. it is plausible that cd also contributes to the host response against b. cepacia, since lps from this bacterium signals through tlr and anti-cd antibodies dramatically inhibited b. cepacia-induced chemokine secretion by lung epithelial cells [ ] . whether cd contributes to host defense response against klebsiella pneumoniae, a known cause of nosocomial pneumonia, also remains to be determined, but data from our study with tlr -mutant mice indicate that signaling through tlr is essential for successful clearance of this bacterium [ ] . in contrast to the essential role of pulmonary tlr and cd in the host defense response against most gramnegative bacteria, we found that tlr was not involved and cd played a remarkable detrimental role in the host response to b. pseudomallei, the causative organism of melioidosis (the most common cause of communityacquired sepsis in southeast asia) [ , ] . cd defi cient mice infected intranasally with b. pseudomallei were protected from mortality, accompanied by enhanced bacterial clearance in the lung, blood and liver, and reduced cellular infi ltration in the lung [ ] , whereas the course of disease in tlr -defi cient mice was indistinguishable from wild-type mice [ ] . moreover, intranasal administration of scd to cd -defi cient mice partially reversed the phenotype into that of wild-type mice [ ] . interestingly, these fi ndings in b. pseudo mallei-infected cd -defi cient mice strongly resemble our previous results found with tlr -defi cient mice, and are in line with the observation that b. pseudomallei expresses an atypical lps which signals through tlr [ ] . whether cd interacts with tlr in b. pseudo mallei-induced responses, and by which mechanism these receptors facilitate the growth and dissemination of b. pseudomallei after intranasal infection remains to be determined. in the model for s. pneumoniae-induced pneumonia, we observed an unexpected detrimental role for cd in the innate host defense response. s. pneumoniae, a gram-positive bacterium and the single most frequent pathogen causing community-acquired pneumonia, induces severe lung infl ammation and sepsis in wild-type mice after intranasal instillation. strikingly, cd defi cient mice were protected against pneumococcal pneumonia, presumably as a result of reduced bacterial spread to the circulation and reduced lung infl ammation [ ] . in contrast, tlr -defi cient and tlr -mutant mice were not protected against pneumococcal pneumonia [ , ] , but in fact tlr seemed redundant for effi cient bacterial clearance and tlr -mutant mice were more susceptible to pneumonia, accompanied by impaired bacterial clearance. however, as in cd -defi cient mice, lung infl ammation was also reduced in pneumococciinfected tlr -defi cient mice [ ] . since intrapulmonary treatment with scd rendered cd -defi cient mice equally susceptible to s. pneumoniae as wild-type mice [ ] , these results suggest that s. pneumoniae abuses (s) cd in the lung to cause invasive respiratory tract infection. interestingly, the phenotype of cd defi cient mice strongly resembled the phenotype of mice defi cient for pafr [ ] , a receptor for phosphoryl choline from the pneumococcal cell wall which facilitates pneumococcal invasion of cells. further studies are required to determine whether cd serves as a chaperone in the presentation of s. pneumoniae to the pafr so that the phosphoryl-pafr-mediated invasion is facilitated. since m. tuberculosis expresses a number of molecules, such as lipoproteins, which activate immune cells in a cd -dependent manner, we and others investigated whether cd also contributed to the host immune response in mice with lung tuberculosis [ ] . although initially after intranasal infection of wild-type and cd defi cient mice no diff erences in bacterial loads, cell infi ltration and release of most cytokines in the lung were found [ , ] , at later time points (> weeks after infection) cd -defi cient mice were protected from mortality presumably as a result of a reduced infl ammatory response in the lungs [ ] . th ese fi ndings are completely opposite to the results from m. tuberculosisinfected tlr -defi cient and tlr -mutant mice, which suff ered from reduced bacterial clearance, chronic infl ammation, increased cellular infi ltration of the lungs and reduced survival [ ] [ ] [ ] . th e mechanism underlying the detrimental eff ect of cd in the host response against m. tuberculosis remains to be established. in addition to its role in (myco)bacterial infections, cd may also play a role in the pulmonary host response against respiratory syncytial virus (rsv), the most common cause of lower respiratory tract disease in infants and young children worldwide, and infl uenza a virus, a cause of pneumonia in very young children, the elderly and immunocompromised patients. th e envelop f glycoprotein from rsv and certain infl uenza a virus components activate macrophages in a cd -dependent manner [ , ] . experiments with wild-type and tlr mutant mice infected intranasally with rsv showed that viral clearance was reduced in the absence of functional tlr [ ] , due to impaired natural killer (nk) cell migration and function and impaired cytokine secretion. recently, it was found that tlr and tlr are also involved in recognition of rsv [ ] . whether cd contributes to these tlr-mediated immune responses against rsv remains to be determined. using cd defi cient mice, we demonstrated that cd played a minimal role in infl uenza a virus-induced pneumonia [ ] . during the entire course of disease, viral loads were slightly reduced in cd -defi cient mice, but this did not result from improved lymphocyte recruitment or lympho cyte activation, or consistent changes in pulmonary cytokines [ ] . th us, despite the fact that infl uenza a expresses ligands that require cd for immune cell activation [ ] , cd seems redundant in the host defense response against infl uenza a virus. cd plays a central role in the lung in the recognition and binding of a variety of (myco)bacterial and viral components, and in the amplifi cation of subsequent host responses. th e studies discussed in this chapter indicate that the contribution of cd to the pulmonary host defense responses may range from benefi cial to detrimental, depending on the microbe and the pamps it expresses. interfering with cd -lps or cd -lta inter actions reduced lung infl ammation. interference with cd -pathogen interactions, however, did not have a signifi cant eff ect on m. tuberculosis or infl uenza a virus infection, resulted in reduced clearance of nontypeable h. infl uenzae, e. coli or a. baumannii in the lung, but enhanced clearance (and reduced dissemination) of b. pseudomallei or s. pneumoniae. th e latter observation indicates that certain pathogens may abuse cd in the lung to cause invasive disease. whether cd is a suitable target for intervention in these latter infectious diseases and/or in aberrant infl ammatory responses during pneumonia requires further study. abbreviations ards = acute respiratory distress syndrome, bal -broncoalveolar lavage, damp = damage/danger-associated molecular pattern, f-gp = f glycoprotein, gpi = glycosylphosphatidyl, gro = growth related oncogene, hmgb- = high mobility group box- protein, icam = intracellular adhesion molecule, ifn = interferon, il = interleukin, irf = ifn regulatory transcription factor, lam = lipoarabinomannan, lbp = lipopolysaccharide binding protein, lps = lipopolysaccharide, lta = lipoteichoic acid, mcp = monocyte chemoattractant protein, myd = myeloid diff erentiation primary-response protein , nf = nuclear factor, nk = natural killer, nod = nucleotide-binding oligomerization domain containing, pafr = platelet activating factor resceptor, pamp = pathogen-associated molecular pattern, rip = receptorinteracting protein kinase, rsv = respiratory syncytial virus, sp = surfactant protein, tlr = toll-like receptors, tnf = tumour necrosis factor, trif = tir-domain-containing-adaptor-protein-inducing-interferon-β toll-like receptors: function and roles in lung disease cd and innate recognition of bacteria polymorphisms in the cd gene associated with pulmonary function in farmers crystal structure of cd and its implications for lipopolysaccharide signaling cd , a receptor for complexes of lipopolysaccharide (lps) and lps binding protein relationship between soluble cd , lipopolysaccharide binding protein, and the alveolar infl ammatory response in patients with acute respiratory distress syndrome innate immune sensing and its roots: the story of endotoxin van der poll t: pulmonary lipopolysaccharide (lps)-binding protein inhibits the lps-induced lung infl ammation in vivo lipopolysaccharide activation of human endothelial and epithelial cells is mediated by lipopolysaccharidebinding protein and soluble cd modulatory eff ects of scd and lbp on lps-host cell interactions the structural basis of lipopolysaccharide recognition by the tlr -md- complex pathogen recognition and innate immunity cd is required for myd -independent lps signaling pattern recognition receptors tlr and cd mediate response to respiratory syncytial virus augusto: la interactions between lps and lung surfactant proteins surfactant protein-d regulates soluble cd through matrix metalloproteinase- cd is a pattern recognition receptor lipoteichoic acid (lta) of streptococcus pneumoniae and staphylococcus aureus activates immune cells via toll-like receptor (tlr)- , lipopolysaccharide-binding protein (lbp), and cd , whereas tlr- and md- are not involved peptidoglycan recognition in innate immunity double-stranded rna-mediated tlr activation is enhanced by cd lipopolysaccharide binding protein and cd interaction induces tumor necrosis factor-alpha generation and neutrophil sequestration in lungs after intratracheal endotoxin eff ect of toll-like receptor blockade on pulmonary infl ammation caused by mechanical ventilation and bacterial endotoxin eff ect of cd blockade on endotoxin-induced acute lung injury in mice distinct roles of pattern recognition receptors cd and toll-like receptor in acute lung anas et al. critical care diff erential roles of cd and tolllike receptors and in murine acinetobacter pneumonia cd is an essential mediator of lps induced airway disease the role of toll-like receptor in environmental airway injury in mice lipoteichoic acid-induced lung infl ammation depends on tlr and the concerted action of tlr and the platelet-activating factor receptor role played by toll-like receptors and in lipoteichoic acid-induced lung infl ammation and coagulation the myd -dependent, but not the myd -independent, pathway of tlr signaling is important in clearing nontypeable haemophilus infl uenzae from the mouse lung toll-like receptor mediates innate immune responses to haemophilus infl uenzae infection in mouse lung eff ect of cd blockade in rabbits with escherichia coli pneumonia and sepsis tlr- pathway mediates the infl ammatory response but not bacterial elimination in e. coli pneumonia toll/il- receptor domaincontaining adaptor inducing ifn-beta (trif)-mediated signaling contributes to innate immune responses in the lung during escherichia coli pneumonia myeloid diff erentiation protein- -dependent and -independent neutrophil accumulation during escherichia coli pneumonia control of pseudomonas aeruginosa in the lung requires the recognition of either lipopolysaccharide or fl agellin burkholderia cepaciainduced il- gene expression in an alveolar epithelial cell line: signaling through cd and mitogen-activated protein kinase role of toll-like receptor in gram-positive and gram-negative pneumonia in mice toll-like receptor impairs host defense in gram-negative sepsis caused by burkholderia pseudomallei (melioidosis) cd impairs host defense against gram-negative sepsis caused by burkholderia pseudomallei in mice van der poll t: cd facilitates invasive respiratory tract infection by streptococcus pneumoniae toll-like receptor plays a role in the early infl ammatory response to murine pneumococcal pneumonia but does not contribute to antibacterial defense improved host defense against pneumococcal pneumonia in platelet-activating factor receptor-defi cient mice van der poll t: cd contributes to pulmonary infl ammation and mortality during murine tuberculosis toll-like receptor (tlr) -and tlr -mediated pathogen recognition in resistance to airborne infection with mycobacterium tuberculosis toll-like receptor expression is required to control chronic mycobacterium tuberculosis infection in mice toll-like receptor -defi cient mice succumb to mycobacterium tuberculosis infection toll-like receptor plays a protective role in pulmonary tuberculosis in mice respiratory syncytial virus activates innate immunity through toll-like receptor van der poll t: cd plays a limited role during infl uenza a virus infection in vivo the authors declare that they have no competing interests. key: cord- -a acr o authors: koch, r. m.; diavatopoulos, d. a.; ferwerda, g.; pickkers, p.; de jonge, m. i.; kox, m. title: the endotoxin-induced pulmonary inflammatory response is enhanced during the acute phase of influenza infection date: - - journal: intensive care med exp doi: . /s - - - sha: doc_id: cord_uid: a acr o background: influenza infections are often complicated by secondary infections, which are associated with high morbidity and mortality, suggesting that influenza profoundly influences the immune response towards a subsequent pathogenic challenge. however, data on the immunological interplay between influenza and secondary infections are equivocal, with some studies reporting influenza-induced augmentation of the immune response, whereas others demonstrate that influenza suppresses the immune response towards a subsequent challenge. these contrasting results may be due to the use of various types of live bacteria as secondary challenges, which impedes clear interpretation of causal relations, and to differences in timing of the secondary challenge relative to influenza infection. herein, we investigated whether influenza infection results in an enhanced or suppressed innate immune response upon a secondary challenge with bacterial lipopolysaccharide (lps) in either the acute or the recovery phase of infection. methods: male c bl/ j mice were intranasally inoculated with × ( ) pfu influenza virus (ph n , strain a/netherlands/ / ) or mock treated. after (acute phase) or (recovery phase) days, mg/kg lps or saline was administered intravenously, and mice were sacrificed min later. cytokine levels in plasma and lung tissue, and lung myeloperoxidase (mpo) content were determined. results: lps administration days after influenza infection resulted in a synergistic increase in tnf-α, il- β, and il- concentrations in lung tissue, but not in plasma. this effect was also observed days after influenza infection, albeit to a lesser extent. lps-induced plasma levels of the anti-inflammatory cytokine il- were enhanced days after influenza infection, whereas a trend towards increased pulmonary il- concentrations was found. lps-induced increases in pulmonary mpo content tended to be enhanced as well, but only at days post-infection. conclusions: an lps challenge in the acute phase of influenza infection results in an enhanced pulmonary pro-inflammatory innate immune response. these data increase our insight on influenza-bacterial interplay. combing data of the present study with previous findings, it appears that this enhanced response is not beneficial in terms of protection against secondary infections, but rather damaging by increasing immunopathology. patients with influenza infection often suffer from severe secondary bacterial infections, which are associated with high morbidity and mortality rates [ , ] . a striking example of this relationship was provided by bacteriological and histopathological analysis of infected lung tissue obtained from people who died of influenza during the - "spanish flu" pandemic, in whom bacterial pneumonia was found to be the predominant cause of death [ ] . these data suggest that an influenza infection profoundly influences the immune response upon a secondary bacterial infection. several studies have evaluated immunological interactions between influenza and bacterial infections, including infections with gram-negative bacteria [ ] . in vitro studies in which influenza-infected alveolar macrophages were subsequently stimulated with bacterial lipopolysaccharide (lps), a bacterial compound that induces a profound innate immune response, revealed increased levels of pro-inflammatory cytokines tumor necrosis factor (tnf) α, interleukin (il)- β, and il- [ ] [ ] [ ] [ ] [ ] , indicative of a priming effect on these cells by influenza. data from in vivo animal studies are ambiguous. similar to the in vitro data, some report enhanced responses. for instance, influenza infection in mice was shown to enhance the inflammatory response and neuropathogenicity resulting from lps administration on days and after influenza inoculation [ ] . likewise, murine influenza infection resulted in increased levels of pro-inflammatory cytokines in both plasma and lungs, and enhanced pulmonary neutrophil influx upon pneumococcal infection days later [ ] . similar results were observed in mice days after influenza infection [ ] . however, two otherwise largely comparable studies demonstrated reduced pulmonary pro-inflammatory cytokine concentrations upon streptococcus pneumoniae and staphylococcus aureus infections in mice infected with influenza days before, indicative of influenza-induced immunosuppression [ , ] . these equivocal results may be due to differences in the severity or kinetics of the influenza infection or the use of different bacteria as secondary challenges, thereby targeting various complex multi-receptor signaling pathways. also, the use of live bacteria could have contributed to these ambiguous results. for instance, if influenza would induce immunosuppression and thereby facilitate outgrowth of bacteria upon a secondary live infectious challenge, the increased bacterial burden can eventually result in fulminant inflammation, which would wrongfully suggest influenza-induced augmentation of the immune response. in the present study, we investigated whether influenza infection results in an enhanced or suppressed innate immune response upon a secondary challenge with lps. furthermore, we assessed the kinetics of these influenza-induced effects by performing the lps challenges in either the acute or the recovery phase of influenza infection. all procedures described were in accordance with the requirements of the dutch experiments on animals act, the ec directive / , and approved by the animal ethics committee of the radboud university nijmegen medical center (ru-dec - ). forty-eight male c bl/ j mice (charles river, sutzfield, germany) aged - weeks and weighing - g were used. mice were housed in individually ventilated cages, with five mice per cage at the central animal facility of the radboud university. at day , six groups of eight mice (total n = ) were anesthetized by isoflurane and intranasally inoculated with a sublethal dose of influenza virus (ph n , strain a/ netherlands/ / , × pfu) or mock treated (nacl . %) in a volume of μl. following infection, all mice were monitored and weighed daily. the temperature was recorded with an infrared thermometer on the skin, and physical condition was scored using a scoring and weight sheet (weight, body temperature, ruffled coat, hunched back, reduced mobility, and moribund). at either day (acute phase) or day (recovery phase), mice were placed in a temperature-controlled chamber to receive lps (e coli, serotype :b , mg/kg) or nacl . % by intravenous injection in the tail vein. ninety minutes after lps or nacl administration, mice were deeply anesthetized with isoflurane and exsanguinated through orbital extraction, followed by cervical dislocation after which organs were collected. ethylenediaminetetraacetic acid (edta)-anticoagulated blood was centrifuged at ×g for min at room temperature after which plasma was stored at − °c until analysis. subsequently, perfusion of the lungs was performed by intracardiac injection with phosphate-buffered saline (pbs), after which lung lobes were harvested and snap frozen in liquid nitrogen and stored at − °c until homogenization. lung tissue was placed in ml lysis buffer containing pbs, . % triton x- , and a protease inhibitor cocktail (complete edta-free tablets, roche, woerden, the netherlands, tablet per ml lysis buffer). subsequently, lung lobes were homogenized at hz, using a polytron homogenizer, and subjected to two rapid freeze-thaw cycles using liquid nitrogen. finally, homogenates were centrifuged ( min, , ×g, °c), and the supernatant was stored at − °c until cytokine analysis. concentrations of tnf-α, il- β, il- , and il- in plasma and lung homogenates were measured using a luminex assay (milliplex, millipore, billerica, ma) according to the manufacturer's instructions. the lower detection limit of the assay was pg/ml for all cytokines. plasma il- β levels were below the detection limit in the majority of animals. lung homogenate cytokine concentrations were normalized to total protein content determined by bicinchoninic acid assay (bca protein assay; thermo fisher scientific). myeloperoxidase (mpo) content was measured in lung homogenates using an enzyme-linked immunosorbent assay (hycult biotech, uden, the netherlands) according to the manufacturer's instructions. concentrations were normalized to total protein content as described above. all data were normally distributed according to the shapiro-wilk test. the grubbs test (extreme studentized deviate method) was used to exclude significant outliers from analysis (maximum of one exclusion per dataset). to determine the number of animals required per group, we performed a power calculation based on a minimal detectable difference of % in lps-induced plasma tnf-α levels between influenza-infected and non-influenza-infected mice. mean ± sd ( ± pg/ml) tnf-α plasma levels were obtained from previous work from our group, in which male c bl/ j mice were also injected intravenously with mg/kg lps and sacrificed min later [ ] . using a two-sided α of . and a power of % (β of . ) in an unpaired t test design, six animals per group were required. to account for potential loss of animals due to influenza infection, eight animals per group were used. the effect size was based on previous work [ ] , in which influenza infection modulated the plasma cytokine response to lps administration by at least %. comparisons were analyzed using unpaired student's t tests and repeated measures one-way analysis of variance (anova) as indicated in the figure legends. statistical analyses were performed in graphpad prism . for windows (graphpad software, san diego, ca). two-tailed p values < . were considered statistically significant. all influenza-inoculated mice showed clinical signs of infection, including weight loss, lethargy, and pyrexia. four influenza-infected mice were prematurely taken out of the experiment because of signs of severe infection. body weight decreased in all influenza-infected mice in the acute phase of infection, whereas it remained stable in mock-inoculated mice (fig. ) . from day onwards, body weight started to increase, marking the recovery phase of influenza infection (fig. ) . influenza infection by itself did not result in increased plasma levels of any of the cytokines measured at both and days post-infection (fig. ) . expectedly, lps fig. body weight of influenza-or mock-inoculated mice. data are presented as mean with sem. dagger indicates the two time points at which mice in the respective groups were sacrificed administration led to profoundly increased plasma concentrations of tnf-α, il- , and il- . although tnf-α and il- plasma levels appeared to be somewhat higher in mice challenged with lps days after influenza infection compared with mock-inoculated mice, this did not reach statistical significance (p = . and p = . , respectively). plasma concentrations of the anti-inflammatory cytokine il- were however significantly enhanced in mice challenged with lps days after influenza infection. no differences in any of the plasma cytokine levels were measured between influenza-infected and mock-inoculated mice at days. in lung homogenates, influenza by itself caused mildly elevated levels of tnf-α, il- β, il- , and il- at days post-infection and to a lesser extent at days after infection (fig. ) . similar to what was found in plasma, lps challenge also led to increased concentrations of all measured cytokines in lung tissue. a synergistic increase of all pro-inflammatory cytokines in the lungs was found in influenza-infected mice challenged with lps days later and, to a lesser extent, in mice challenged with lps days post-influenza infection. for il- , the potentiating effect was additive rather fig. plasma levels of tnf-α, il- , and il- in mice that received influenza/mock followed by lps/nacl or days later. data are presented as scatter-dot plots with horizontal lines indicating the mean value. *p < . , **p < . , ***p < . (calculated by unpaired student's t tests) fig. levels of tnf-α, il- β, il- , and il- in lung homogenates of mice that received influenza/mock followed by lps/nacl or days later. data are presented as scatter-dot plots with horizontal lines indicating the mean value. *p < . , **p < . , ***p < . , # p = . - . (calculated by unpaired student's t tests) than synergistic, only observed at days post-influenza infection, and reached a trend towards statistical significance. in accordance with pulmonary cytokine levels, influenza infection by itself led to increased mpo content in the lungs days after infection and tended to result in increased mpo content days post-infection (fig. ) . again, lps administration also resulted in increased mpo levels in lung tissue, and there was a trend towards enhanced mpo content in influenza-infected mice challenged with lps days after infection. in the present study, we demonstrate that a systemic lps challenge in the acute phase of influenza infection ( days post-infection) results in an enhanced pulmonary, but not systemic pro-inflammatory cytokine response. this effect was synergistic rather than additive, indicating that influenza infection actually modulates the immune response to a subsequent challenge with lps. furthermore, this effect remained present, although less pronounced, in the recovery phase of influenza infection ( days post-infection). the lps-induced increase in mpo content in lung homogenates, reflecting pulmonary neutrophil influx or sequestration, tended to be enhanced in the acute phase of influenza infection as well. these results suggest that influenza infection, especially in the acute phase, may cause a more pronounced pulmonary pro-inflammatory immune response upon a secondary bacterial infection. our results are in accordance with in vitro data reporting a cellular priming effect of influenza observed upon secondary stimulation with lps [ ] [ ] [ ] [ ] [ ] , as well as with other murine in vivo studies that report increased inflammation and pulmonary neutrophil influx or sequestration upon a secondary bacterial infection or lps challenge in the acute phase of influenza infection [ , ] . for example, a preceding influenza infection in mice gravely enhanced lung injury induced by a secondary infection with streptococcus pneumoniae days later, resulting in a severe necrotic pneumonia accompanied by increased mortality [ ] . also, fig. mpo content in lung homogenates of mice that received mock/influenza followed by nacl/lps or days later. data are presented as scatter-dot plots with horizontal lines indicating the mean value. *p < . , **p < . , ***p < . , # p = . - . (calculated by unpaired student's t tests) the increased mpo content observed in our study is an important hallmark of acute respiratory distress syndrome (ards) [ , ] , a severe complication of influenza infection caused by excessive pulmonary inflammation. these and our study reveal that the enhancing effect on the pro-inflammatory innate immune response is most evident in the lungs, probably because the influenza-induced damage and consequent inflammatory effects are most pronounced at this site. in this context, our data are in line with the recommendation to use corticosteroids in patients with severe influenza infections in the intensive care unit to counteract the pulmonary hyperinflammatory response causing ards. several underlying mechanisms may contribute to the observed effects. at the cellular level, studies have shown that influenza and certain bacterial pathogens, such as haemophilus influenzae and streptococcus pneumoniae, utilize similar immunological pathways and that the overlap in the inflammatory mediators produced thereby creates augmentation of the immune response during sequential infection, in turn causing immunopathology [ , ] . furthermore, it has been hypothesized that influenza stimulates tnf-α gene transcription activators or may interfere with labile transcription repressor proteins and stabilizes tnf-α mrna by delaying its degradation [ ] . alternatively, the increased lung mpo levels observed do not necessarily reflect pmn infiltration into the lungs, but may (also) result from pmns trapped in the vasculature, as circulating activated neutrophils become rigid and can be trapped within the small capillaries of the lung [ ] . as such, increased entrapment of leukocytes in the pulmonary vasculature during influenza infection could also contribute to the enhanced inflammatory cytokine levels upon lps challenge. we can only speculate on this, because no histological data are available, which represents a limitation of this work. it may be argued that the enhanced pro-inflammatory immune response induced by influenza serves as a means to efficiently eliminate the primary pathogen and to enhance host defense towards a secondary infection. for instance, pro-inflammatory cytokines are induced in influenza-infected cells to limit viral replication and to initiate downstream immune responses [ ] . however, this is not supported by previous work, where an increased bacterial burden was observed irrespective of an enhanced or suppressed response [ ] [ ] [ ] . several explanations for this observation may be put forward. first, next to potentiating pro-inflammatory cytokine responses, the present study and work by others [ ] have shown that influenza infection also potentiates production of the key anti-inflammatory cytokine il- , which was demonstrated to be crucial in facilitating bacterial outgrowth upon secondary challenge with streptococcus pneumoniae [ ] . second, influenza may on the one hand prime for production of innate cytokines produced by myeloid cells, but impair t cell-derived cytokines that are instrumental for the adaptive immune response. this was elegantly demonstrated by kudva et al., who showed that, in line with our results, infection with staphylococcus aureus days after influenza resulted in increased pulmonary levels of innate cytokines such as il- and mcp- , and increased neutrophil influx to the lungs, but decreased concentrations of t-cell-derived il- and il- , which were demonstrated to play a pivotal role in fending off the staphylococcal infection [ ] . whereas the enhancing effects of influenza on pro-inflammatory innate immune parameters were less pronounced at days post-infection, a suppressed response was neither evident. this could be partly biased by the exclusion of two mice in both recovery groups due to severe influenza infection. however, it might also be argued that days post-infection is too soon for these effects to manifest. for example, profound desensitization towards lps and flagellin, another toll-like receptor (tlr) ligand, was observed in alveolar macrophages obtained from mice up to weeks after influenza infection [ ] . furthermore, the direction of the response upon a secondary challenge is probably highly dependent on the pathogen or stimulus used, each using distinct intracellular signaling pathways. with regard to this, it is well-known that influenza virus particularly predisposes to aspergillus fumigatus, which is present in % of all influenza patients [ , ] , causing infections such as invasive pulmonary aspergillosis that are associated with very high mortality rates. as different mechanisms may be important in host defense towards various pathogens, the specific response towards aspergillus fumigatus could be suppressed by a preceding influenza infection. the use of corticosteroids may be another important factor in the observed vulnerability towards particular secondary infections, as steroid use was shown to be independently associated with the presence of aspergillus fumigatus in sputum of cystic fibrosis patients [ ] and with a substantially increased risk of community-acquired staphylococcus aureus bacteremia [ ] . furthermore, a meta-analysis revealed that the use of corticosteroids was significantly associated with nosocomial infections [ ] . to the best of our knowledge, these putative detrimental effects of corticosteroid treatment during influenza on secondary infections have yet to be studied systematically in animal models. in any case, it remains to be determined whether the overall effects of corticosteroid treatment are beneficial or not, as they may lead to increased susceptibility in a subset of influenza virus-infected patients but may also provide health benefits in another subset of influenza virus-infected patients. an lps challenge in the acute phase of influenza infection results in an enhanced pulmonary pro-inflammatory innate immune response. these data increases our insight concerning viral-bacterial interplay. combined with previous findings, it appears that this enhanced pro-inflammatory response does not lead to protection against secondary infections but rather causes immunopathology leading to damage, and thereby to organ failure. predominant role of bacterial pneumonia as a cause of death in pandemic influenza: implications for pandemic influenza preparedness secondary bacterial infections associated with influenza pandemics influenza-induced type i interferon enhances susceptibility to gram-negative and gram-positive bacterial pneumonia in mice infection of macrophages by influenza a virus: characteristics of tumour necrosis factor-alpha (tnf alpha) gene expression the potentiating effect of lps on tumor necrosis factor-alpha production by influenza a virus-infected macrophages cytokine release from human peripheral blood leucocytes incubated with endotoxin with and without prior infection with influenza virus: relevance to the sudden 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during endotoxemia cxcl -cxcr enhances the development of neutrophil-mediated fulminant lung injury of viral and nonviral origin contribution of neutrophil-derived myeloperoxidase in the early phase of fulminant acute respiratory distress syndrome induced by influenza virus infection insights into the interaction between influenza virus and pneumococcus patterns in bacterial-and viralinduced immunosuppression and secondary infections in the icu the neutrophil in vascular inflammation new fronts emerge in the influenza cytokine storm influenza a inhibits th -mediated host defense against bacterial pneumonia in mice sustained desensitization to bacterial toll-like receptor ligands after resolution of respiratory influenza infection invasive pulmonary aspergillosis is a frequent complication of critically ill h n patients: a retrospective study influenzaassociated aspergillosis in critically ill patients inhaled corticosteroids and aspergillus fumigatus isolation in cystic fibrosis use of glucocorticoids and risk of community-acquired staphylococcus aureus bacteremia. a population-based case-control study mayo corticosteroids for the treatment of human infection with influenza virus: a systematic review and meta-analysis. clinical microbiology and infection: the official publication of the european society of clinical microbiology and infectious diseases the authors thank fred van opzeeland, elles simonetti, francine van der poll, ilona van de brink, and jelle gerretsen for their help with the mouse experiments and laboratory analyses. this work was supported by an efro (dutch: "europees fonds voor regionale ontwikkeling," english: "european regional development fund") grant ( - ). efro had no role in the design of the study; in the collection, analysis, and interpretation of data; and in writing the manuscript. data sharing is not applicable to this article as no reusable datasets were generated or analyzed during the current study.authors' contributions rk designed and conducted the study, analyzed and interpreted the data, and drafted the manuscript. dd and gf aided in the study design and conduct, interpreted the data, and critically revised the manuscript. pp and mdj supervised the study, interpreted the data, and critically revised the manuscript. mk designed and supervised the study, interpreted the data, and critically revised the manuscript. all authors read and approved the final manuscript. all procedures described were in accordance with the requirements of the dutch experiments on animals act, the ec directive / , and approved by the animal ethics committee of the radboud university nijmegen medical center (ru-dec - ). not applicable. the authors declare that they have no competing interests. key: cord- -isw jeir authors: flori, laurence; gao, yu; laloë, denis; lemonnier, gaëtan; leplat, jean-jacques; teillaud, angélique; cossalter, anne-marie; laffitte, joëlle; pinton, philippe; de vaureix, christiane; bouffaud, marcel; mercat, marie-josé; lefèvre, françois; oswald, isabelle p.; bidanel, jean-pierre; rogel-gaillard, claire title: immunity traits in pigs: substantial genetic variation and limited covariation date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: isw jeir background: increasing robustness via improvement of resistance to pathogens is a major selection objective in livestock breeding. as resistance traits are difficult or impossible to measure directly, potential indirect criteria are measures of immune traits (its). our underlying hypothesis is that levels of its with no focus on specific pathogens define an individual's immunocompetence and thus predict response to pathogens in general. since variation in its depends on genetic, environmental and probably epigenetic factors, our aim was to estimate the relative importance of genetics. in this report, we present a large genetic survey of innate and adaptive its in pig families bred in the same environment. methodology/principal findings: fifty four its were studied on large white pigs vaccinated against mycoplasma hyopneumoniae and analyzed by combining a principal component analysis (pca) and genetic parameter estimation. its include specific and non specific antibodies, seric inflammatory proteins, cell subsets by hemogram and flow cytometry, ex vivo production of cytokines (ifnα, tnfα, il , il , il , ifnγ, il , il , il ), phagocytosis and lymphocyte proliferation. while six its had heritabilities that were weak or not significantly different from zero, and its had moderate ( . <h ≤ . ) or high (h > . ) heritability values, respectively. phenotypic and genetic correlations between its were weak except for a few traits that mostly include cell subsets. pca revealed no cluster of innate or adaptive its. conclusions/significance: our results demonstrate that variation in many innate and adaptive its is genetically controlled in swine, as already reported for a smaller number of traits by other laboratories. a limited redundancy of the traits was also observed confirming the high degree of complementarity between innate and adaptive its. our data provide a genetic framework for choosing its to be included as selection criteria in multitrait selection programmes that aim to improve both production and health traits. increasing robustness by improving resistance/tolerance to pathogens is an important selection objective in most livestock species, particularly in pigs. in the past years, selection for growth, carcass leanness, meat quality and prolificacy, combined with stringent sanitary rules, vaccination and use of antibiotics, has been highly effective in pigs [ ] . since the early 's, prophylactic use of antibiotics as growth promoters has been forbidden by european legislation. as a result, the health status of numerous farms has deteriorated, leading to an increase in the therapeutic use of antibiotics. indeed, animals highly selected for production traits may be more susceptible to pathogens or less able to maintain performance after infection. deterioration of the global health status may also be due to environmental trends. in this context, including health traits in existing breeding schemes using direct and/or indirect strategies is an emerging trend in pig breeding. direct strategies target animal resistance/tolerance to specific pathogens but may result in increased susceptibility to other diseases [ , ] . alternatively, an indirect and putatively more global approach focuses on immune traits (its) providing a measure of immune capacity (i.e. immunocompetence) and hopefully predicting the responses to pathogens in general [ ] . the choice of relevant its is further based on knowledge of the immune system. this highly interactive and cooperative system is classically separated into two arms referred to as innate and adaptive, which produce a combined response. innate immunity is the first line of defence. its activation is non pathogen-specific and depends on the recognition of evolutionarily conserved pathogenassociated molecular patterns such as lipopolysaccharides constituting bacterial cell walls [ ] . innate immunity involves physical barriers, innate immune cells such as dendritic cells (dcs), monocytes, natural killers (nk cells) or cd t lymphocytes, and inflammatory cytokines such as il b, il and tnf. adaptive immunity is antigen-specific and requires the recognition of specific ''non-self'' antigens via a process of antigen presentation and results in an immunological memory. adaptive immunity is divided into cell-and humoral-mediated immunity with different effector functions [ ] . in order to include its in a breeding plan to improve pig immunocompetence, the genetic and phenotypic parameters of the different its need first to be estimated. several studies in swine, mice, poultry and cattle demonstrated the possibility of selecting animals with high or low immune response (ir) as characterized by one or a few its [ , , , , ] . a study on yorkshire pigs selected for eight generations for high and low adaptive ir (hir and lir, respectively) on an index combining four standardized measures of specific antibodies and cellmediated ir, after stimulation with specific antigens (bacillus calmette-guérin and hen egg white lysozyme), has revealed that hir and lir animals differ in response to immunization and infection [ , , , , ] . other studies have also shown that various innate and adaptive its are genetically controlled. for example, variation in innate its, such as nk cells, monocytes, interferon a (ifna) production or phagocytosis [ , , ] is heritable and several adaptive its have moderate to high heritability values including total white blood cells (wbc), cd + t lymphocyte, cd a + t lymphocyte and b lymphocyte subsets [ , , ] , delayed-type hypersensitivity reaction [ , ] , lymphocyte proliferative response [ ] , interleukin- (il ) production by lymphocytes [ ] and antibody response [ , , , ] . clapperton and colleagues have also reported that variation in acute phase protein levels is heritable [ , ] . finally, several significant qtls for total leukocyte count ( [ , ] ; animal-qtldb, http://www.animalgenome.org/cgi-bin/qtldb/index), mitogen-induced proliferation [ ] , antibody response [ , ] , cytokine production (il and ifnc) [ ] , complement activity [ ] , and acute phase protein serum concentration [ ] have been detected and mapped to different pig chromosomes. taken together these data demonstrate that variation in some its is under genetic control. however, most of the results reported so far have targeted a limited number of traits and very few studies have combined innate and adaptive its. our global goal is to identify immunocompetence traits for inclusion in selection schemes aiming to improve both zootechnical performances and health traits in pigs. for this purpose, we have launched a genetic and genomic study of numerous its covering innate and adaptive ir [ ] . in this report, we present the results of a global genetic study, combining principal component analysis (pca), and genetic parameter estimation applied to a large number of innate and adaptive its in a pig population vaccinated against mycoplasma hyopneumoniae (m. hyopneumoniae). a set of its was measured on a population of pigs three weeks after vaccination against m. hyopneumoniae (tables and ; table s ; figure ). these its comprise either traits related to ir (phagocytosis, lymphocyte proliferation, cytokine production after in vitro stimulations, levels of total and specific antibodies, levels of acute phase proteins) or traits related to total leukocyte and leukocyte subpopulation counts. the various characteristics and descriptive statistics of the traits measured on each animal of the studied population (n = to ) are summarized in tables and . among the cell-mediated its evaluated after diverse stimulations, higher responses in cytokine levels were observed after phorbol myristate acetate (pma)-ionomycin (pmaiono) stimulation compared to lipopolysaccharide (lps) and concanavalin a (cona) stimulations, except for il production. conversely, a higher lymphocyte proliferation was detected after cona and pmaiono stimulations than after lps stimulation. ample phenotypic variation was observed for most traits. the coefficient of variation (cv) was equal to . on average and ranged from . to . (tables and ). limited dispersion (cv# . ) was observed for traits derived from hemograms, cell subsets characterized by fluorescence-activated cell sorting (facs), phagocytosis capacity and non-specific immunoglobulins. moderate dispersion ( . ,cv# . ) was observed for most cytokines produced in vitro except for tumour necrosis factor a (tnfa) and mitogen proliferation-related traits. finally, the cv for seric c reactive protein (crp) and haptoglobin (hapt) levels were close to . and , respectively. the highest cv ( . ) was obtained for the specific iggs directed against m. hyopneumoniae. these data clearly indicate that the seric inflammatory protein levels and the specific iggs had the greatest phenotypic variance in our study. in order to analyse the factors causing the variation, we performed a normed pca with traits (tables and ). for cellmediated adaptive ir, we included only those traits related to cytokine production and lymphocyte proliferation after pmaionomycin stimulation. for innate its, we included facscharacterized cell subtypes including the percentages of b lymphocytes (igm + ), cd t lymphocytes (tcrcd + ), three subsets of ab t lymphocytes (cd + cd + , cd -cd + and cd + cd -), nk cells (cd + cd + ) and three monocyte subsets (cd + cd a + , cd + mhcii + , mhcii + cd a + ). we excluded from the analysis four haematological traits not directly involved in immunity: red blood cell count (rbc), hematocrit (ht), red blood cell distribution width (rdw) and platelet count (plt). the percentage of variance (inertia) explained by the first five components was over % (figure a ). each of these five components explained more than % of the total variance and the first two components accounted for . and . % of the total variance, respectively. taking into account the components from pca, multivariate normal mixture modelling and modelbased clustering (see materials and methods) were used to identify clusters of its. the highest bayesian information criterion (bic) was obtained using the diagonal model with variable shape and variable variance (vvi in pink on figure b ) and k = (first factorial plan on figure c ). no parameter was located near the correlation circle indicating that the phenotypic correlations between its are globally weak. a first cluster (k in blue on figure c ) groups together four hemogram-derived cell counts: white blood cell count (wbc), lymphocyte count (lym), monocyte count (mon) and neutrophil count (neu). this cluster is representative of total cell number traits despite the eosinophil count (eos) not being included. a second cluster (k in green on figure c ) groups together all the traits related to facscharacterized leukocyte subpopulations (expressed as the percentage of cells with one or two surface antigens) except cd t lymphocytes (tcrcd + ), with a cell response parameter (il -pmaiono) and the seric level of haptoglobin (hapt). this cluster can be considered as representative of the leukocyte subsets. a third cluster (k in red on figure c ) includes all other cell response traits, one hemogram-derived cell count (eos) and one facs-characterized leukocyte subpopulation. note that k and k related traits, which mainly correspond to cell subsets and explain around % of the phenotypic variance, show clear clustering ( figure c ). these traits are grouped on the first pca axis and separated on the second axis. traits belonging to k , representative of cell activity (cytokine production, phagocytosis and antibody production), are more spread out on the other axes (data not shown). interestingly, cluster analysis did not highlight any cluster of innate or adaptive its. the estimation of phenotypic correlations (r r p ) with wombat confirmed that the its are weakly correlated, except i) among a few cell count traits (wbc, lym, mon, neu), and ii) between cell count traits and a few leukocyte subsets (wbc, lym, cd + cd + and cd -cd + ) for whichr r p greater than . were estimated (table s ; figure s ). weakr r p were mainly positive ( ) with negative. no strongly negativer r p (# . ) were found. taken together, pca and estimations of phenotypic correlations showed that the level of redundancy between the different immune parameters was limited. heritability estimates of the analyzed its was equal to . on average (se = . ; table for the large set of adaptive its, the mean heritability was . (se = . ). no significant difference in average heritability values was detected either between group of its qualifying the innate and adaptive immunity or the humoral and cellular adaptive immunity. in addition, an equivalent proportion of innate and adaptive its had significant heritability values. indeed, % ( / ), % ( / ) and among the traits involved in cell-mediated immunity, variation in ab t lymphocyte (cd -cd + , cd + cd + and cd + cd cells) counts was highly heritable. heritability estimates of the three cytokine (il , il , ifng) levels were moderate to high after pmaiono and cona stimulations and weak to moderate after lps stimulation. for those cytokines induced by cona or lps stimulation, confidence interval ( ci) for the heritabilities overlapped zero, except for il -cona. il production after pmaiono, cona and lps stimulations gave high estimates of heritability significantly different from zero for il -pmaiono and il -lps. proliferation measurements after various stimulations (prolif-cona, prolif-pmaiono, prolif-lps) provided moderate estimates of heritability not significantly different from zero. among the traits involved in humoralmediated adaptive immunity, heritabilities for total igg and iga antibody levels were higher than for total igm and specific antibodies, and weak h values were obtained for b lymphocyte count (igm + cells). heritability for innate its such as i) total cell number (eos and neu), ii) leukocyte subsets (cd + cd + cells, cd + cd acells, cd + mhcii + cells and tcrcd + lymphocytes), iii) cytokine production (ifna and il ), and iv) phagocytosis were high and significantly different from zero. in addition, several innate its showed weak to moderate heritability, including proinflammatory cytokines (il b, il , tnf and il ), mon, cd -cd + cells, cd + cd cells, mhcii -cd a + cells, mhcii + cd acells, cd -cd a + cells and cd + cd a + cells. variation in acute phase proteins was moderately (crp) to highly (hapt) heritable. among the four traits, which measured the total number (mon) or proportions (mhcii + cd a + , cd + cd a + and cd + mhcii + ) of monocytes, moderate to high h , but not significantly different from zero, were estimated, except for cd + mhcii + cells. other haematological traits (rbc, ht, rdw and plt) gave high h estimates, of which ht and plt were significant. pairwise genetic correlations are presented in table s and illustrated in figure . genetic correlation estimates among most its were generally weak but a few high genetic correlations were observed. the number of positive genetic correlations ( ) was higher than that of negative correlations ( ), as already observed for phenotypic correlations. positive genetic correlations were higher (in absolute values) than negative ones (table s , figures and s ). only ( . % of the total number of correlations) and five r ĝ ( . % of the total number of correlation estimates) were higher than . or lower than - . , respectively. the unsupervised hierarchical clustering distinguished two main clusters of traits ( figure ). the first cluster of traits (cluster a) could be divided into two groups: i) a group of four traits including one innate immunity cytokine (ifna), two antibody levels (total igg and specific igg-mh), and one facscharacterized leukocyte subpopulation (cd + cd a -) and ii) a group of traits with nine hemogram-based cell counts or facscharacterized leukocyte subpopulations (wbc, lym, mon, neu, eos, cd + mhcii + , cd + cd + , cd -cd + cells) and two cell activity traits (igm and il -pmaiono). the second cluster of traits (cluster b) is also subdivided into two groups of traits: i) a group of three different cell response traits (il , iga and ifng-pmaiono) and one facs-characterized leukocyte subpopulation (igm + cells), and ii) a group of nine cell response traits (il , il -pmaiono, il -pmaiono, tnf, prolif-pma, hapt, crp, il b, il and phag) and four facs-characterized leukocyte subpopulations (cd + cd -, cd + cd -, tcrcd + and mhcii + cd a + cells). in cluster a, the first group of traits showed moderately to highly positive genetic correlations with each other. indeed, r ĝ values greater than . were estimated between wbc, lym, mon, neu, eos, cd -cd + and cd + cd + (nk) cells ( figure , table s ). in cluster b, r ĝ values greater than . were estimated between i) tnf, il and phag, and ii) crp and hapt ( figure , table s ). strong negative r ĝ values (, . ) were found between a few traits from both clusters: i) tnf and three cell number traits (wbc, lym, mon), ii) il and cd + mhcii + , and iii) crp and iga. the measured its globally cover innate and adaptive immunity the large-scale study reported here allowed us to estimate the genetic and phenotypic parameters of numerous its measured on pigs bred in the same environment. innate immunity is represented by traits, humoral-mediated immunity by five traits and cell-mediated adaptive immunity by traits. we have also considered the total number of white blood cells and lymphocytes, and four other haematological traits (rbc, ht, rdw and plt). within cell-mediated immunity, we explored both th and th responses by measuring cytokine production. figure summarizes the traits that we selected to cover immunity globally. these traits include in vivo measures on blood such as quantification of cell populations by hemogram, dosage of circulating immunoglobulins and acute phase proteins, as well as ex vivo measures obtained after in vitro tests such as lymphocyte proliferation, phagocytosis capacity and cytokine production after blood stimulation. all these its have been widely studied in humans [ , ] . the trait typology we have used (table ) follows a model based on a clear distinction between innate and adaptive immunity, which may be over-simplistic since both immune systems are closely interconnected [ , ] . monocytes are involved in innate immunity but are also antigen-presenting cells required for adaptive immunity, and cytokines such as il are at the interface between innate and adaptive immunity. similarly, the cell-mediated and humoral adaptive immunity subdivision is artificial. for example, il is a cytokine produced by th lymphocytes that is usually classified as part of adaptive cellmediated immunity, whereas it is also involved in antibody production and thus adaptive humoral immunity. in addition, the conventional paradigm that cd + ab t lymphocytes differentiate into th and th lineages expressing specific cytokines is collapsing. indeed, recent studies have revealed that cytokine production by the different cd + t cell subsets (th , th , th, th and itreg) is highly flexible, providing new insight into the th cell plasticity [ ] . nevertheless, although schematic, the approach used in our report provides a comprehensive overview of genetic variation and co-variation across the entire immune spectrum in pigs (figure ). table illustrates various ranges of phenotypic variation in the measured immune traits, with most having a cv over . . such variability has already been reported in large panels of healthy humans [ ] , and in previous studies on pigs [ , , , ] . for instance, we substantiated the high level of variation of cytokine production previously reported for il production and virusinduced ifna production in a swedish yorkshire population [ ] . for innate immunity-related cytokines and il , stimulation was performed with a mixture of lps, pma and ionomycin. il was the cytokine produced with the highest levels followed by il b, tnf, il and lastly il . these four cytokines are not expected to be expressed at similar levels at all time points after stimulation and a kinetic study would help to improve comparison of cytokine production levels. weaker levels of adaptive ir cytokines are observed after lps stimulation compared to cona or pmaiono. these differences could be related to the distinct modes of action of these molecules. indeed, pma, a plant-derived functional analog of diacylglycerol, in conjunction with ionomycin, a calcium ionophore produced by streptomyces conglobatus, and cona, a lectin originally extracted from the jack-bean canavalia ensiformis, are known to be potent mitogens of blood lymphocytes [ , ] . lps, a major structural component of the outer membrane of gram-negative bacteria, which binds the cd / the five first components, which explain more than % of the total variance, are in red. b. plot of the bayesian information criterion (bic) calculated with different models according to number of clusters. six models are compared: eii (spherical with equal volume and equal shape), vii (spherical with variable volume and equal shape), eei (diagonal with equal shape and equal volume), vei (diagonal with variable shape and equal volume), evi (diagonal with equal shape and variable volume), vvi (diagonal with variable shape and variable volume). c. first factorial plan ( : first component, : second component) with three clusters identified by multivariate normal mixture modelling and model-based clustering taking into account the components (clusters k , k and k are in blue, green and red, respectively). doi: . /journal.pone. .g tlr /md receptor complex and promotes the secretion of proinflammatory cytokines, has been extensively used to study innate immune response [ ] . we have already shown that transcriptome modifications in peripheral blood mononuclear cells (pbmcs) differ between pmaiono and lps stimulation and that pmaiono and lps target different cells and cellular pathways [ ] . the combination of pma and ionomycin induces a stronger stimulation that may be related to a higher production of cytokines as detected in the present study. in addition, the lymphocyte proliferation induced by lps is weaker than that observed with other stimulants, as expected. our study provides the first heritability estimates for innate and adaptive cytokine production and for lymphocyte proliferation after pma-ionomycin and lps stimulations in pig. pro-inflammatory cytokines appear to show less heritable variation than adaptive system-related cytokines. among the adaptive system-related cytokines, estimated heritability was weakest for cytokines produced after lps stimulation, except for il production. heritability estimates for lymphocyte proliferation after cona, pma and lps stimulations were moderate and that of lymphocyte proliferation after cona stimulation was comparable to the value obtained by edfors-lilja and colleagues [ ] . moderate to high heritability estimates for cell count traits from hemogram or facs also confirmed those previously obtained for wbc, total lymphocytes, neutrophils, eosinophils and some leukocyte subsets (for cd + and cd + t lymphocytes, cd t lymphocytes, cd r + , cd r + cd a -, cd r + cd a + and cd + mhcii + ) [ , , , ] . likewise, our results confirmed the high heritability estimate for phagocytosis [ ] . conversly, a lower heritability estimate for crp than previously reported was observed [ , ] . overall, the heritability estimates for these traits appear robust regardless of populations, environments and protocols. some discrepancies exist between our heritability estimates and previous results for humoral-mediated adaptive its and some innate its. indeed, in our study, b lymphocyte levels (igm + cells) are not significantly heritable contrary to other results [ , ] and specific iggs (igg-mh) have lower heritability estimates ( . , se = . ) than previously reported (range from . to . ) for specific antibodies directed against other antigens [ ] . our estimated heritability for total igg is higher ( . , se = . ) than in published reports [ , , , ] . in addition, our estimated h for ifna production is moderate to high ( . , se = . ), contrary to previous results (range from to . ) [ ] , and for haptoglobin ( . , se = . ) is higher than previously published (range from . to . ) [ , ] . these discrepancies could be due to differences in the pig breeds and in environment factors but also to the absence of common standardised protocols between laboratories. in order to better qualify the phenotypes, protocol standardisation is needed. overall, we show that variation in both innate and adaptive its is under substantial genetic control (figure ; tables and ) . similar heritability estimates for innate and adaptive its and also between cell number and cell response parameters were observed. further, heritability estimates do not differ consistently between in vivo and ex vivo measures with no apparent bias due to phenotyping methods. these data also suggest candidate its for qtl mapping. indeed, mapping studies have already started for total leukocyte count ( [ , ] ; animalqtldb, http://www.animalgenome.org/cgi-bin/qtldb/index), mitogen-induced proliferation [ ] , antibody response [ , ] , cytokine production (il , il and ifnc) [ , ] , complement activity [ ] , and acute phase protein serum concentration [ ] . compared to the previously limited data on genetic correlations between its in pigs, our study provides a large-scale estimation of phenotypic and genetic correlations among its. pca results and correlation estimations highlight the weak phenotypic and genetic correlations between the different its, except mainly for cell subsets. no cluster of innate and adaptive its is revealed. these results illustrate that many of the its included in our study provide more or less independent potential clues for selecting for improved immunocompetence. such complementarity is expected since innate immunity is in place or ready for activation prior to infection or antigenic stimulation and collaborates with adaptive immunity, which is induced by infection or antigenic stimulation. nevertheless, a few highly positive genetic correlations have been detected between total number of white blood cells and some leukocytes subsets, and between some leukocyte subsets such as total number of lymphocytes, cd -cd + lymphocytes (which contains ab cd -cd + lymphocytes and nk), and nk cells (cd + cd + cells). phagocytosis, production of il and tnf, two pro-inflammatory cytokines produced by monocytes and macrophages, were positively correlated with acute inflammatory phase proteins produced by hepatocytes i.e. crp and hapt. a high correlation was also found between crp and hapt. clapperton and colleagues [ ] have shown that phenotypic correlations between leukocyte subsets and acute phase proteins are weak (, . ) and not significantly different from zero in agreement with our results. in contrast to our study, clapperton and collaborators have not detected any significant genetic and phenotypic correlations between different leukocyte subsets except when one subset was nested in another [ ] . our results provide a framework for including its in multitrait selection for immunocompetence in pigs. criteria for inclusion should take into account heritability, biological relevance, biological sensitivity and feasibility of measurement [ ] . the weak genetic correlations between most its suggest that it will be difficult to choose only a few its to select for immunocompetence, and that a combination of many traits may be required [ ] . the moderate to high heritabilities estimated for many traits together with the selection study on pigs carried out by wilkie and colleagues a decade ago support the feasibility of selecting for immunocompetence [ , ] . chickens have also been successfully divergently selected for carbon clearance (phagocytic activity), high antibody response to newcastle disease virus three weeks after vaccination (adaptive humoral ir) and wing web response to pha (high cell-mediated immune response) for more than twelve generations [ , ] . in order to include immunocompetence in selection for improved health, a major challenge will be to correlate variation in heritable its in healthy animals with inter-individual variability in response to various pathogens. testing this hypothesis will be a key point for further use of its as indirect selection criteria in multitrait selection to improve resistance to disease. some results on genetic and phenotypic relationships between immunocompetence and susceptibility to specific pathogens have already been reported in the literature for pigs. among pigs selected for eight generations for high (hir) or low (lir) response based on an index of four cell and humoral-mediated immunity traits, an increased specific antibody response and lower polyserositis were observed in the hir pigs compared to the lir pigs after challenge with a novel pathogen, mycoplasma hyorhinis [ , ] . thus, animals with a high ir level to unrelated challenges, as defined by wilkie and colleagues [ , ] , have a better response to infection with mycoplasma hyorhinis. however, hir pigs develop more severe arthritis than lir pigs. indeed, the levels of humoral and cell-mediated adaptive its included in the wilkie et al index induce the formation of immune complexes and/or the development of inflammatory responses, central to the pathogenesis of mycoplasma hyorhinis-induced arthritis. other correlation tests between its and response to various infections are needed. however, it is important to remain cautious with high responder animals, which could develop autoimmune pathologies or pathological iummne responses. all the studies on immunocompetence and resistance to disease will have to be completed by estimation of genetic correlations with economically important traits already under selection. negative genetic correlations have been reported between some its (monocytes, cd r + cells) and average daily gain [ ] . however a larger correlation study considering a higher number of its and pig performances is needed and is ongoing in our population. in the future, a more sustainable production system may require a compromise with a slight decrease in performance traded off for a gain in animal robustness. more studies are required to better understand the correlations between its and production traits and it is not established which levels of its would be good predictors for resistance to pathogens if any. in conclusion, our results show that variation in many its is under significant genetic control in pigs and these findings may provide insights in other species. moreover, based on heritability and correlation estimations, some of the its that we have studied might be incorporated into selection schemes, provided they are associated with improved global health and do not exhibit strong antagonisms with other economically important traits. our experiment was conducted in accordance with the french national regulations for humane care and use of animals in research. no ethics approval was required for the vaccination and the collection of blood samples under the then current regulations. experiments were performed under the individual license numbers - assigned to marcel bouffaud who was responsible for experiments in the test farm, and - assigned to a veterinarian, dr silvia vincent-naulleau. the experimentation agreement number for the test farm at le rheu was a - - . a total of large white pigs (castrated males, dam line) tested for performance traits in a pig test station (ue , inra, le rheu, france) was included in the study. the pigs were distributed in seven contemporary groups and belonged to nuclear families obtained from boars, with an average of . (+/ . ) piglets per boar. animals were born and weaned in different selection herds and arrived in the test station at five weeks of age with no prior vaccination. they were placed into pens of piglets in a post weaning unit and vaccinated against m. hyopneumoniae (stellamune, pfizer, one injection) one day after their arrival in the test station, when . days old in average. all pigs were apparently healthy with no clinical sign of infection. all animals were sampled three weeks after vaccination. blood samples were collected via the external jugular vein into tubes with or without anti-coagulants, according to further use. blood collected in ml tubes with no anti-coagulant was centrifuged at g for min at uc. the serum was collected and stored at uc until use. plasma was collected from blood sampled in heparinised tubes and stored at uc before use. hemograms were measured with an ms - counter (eli-techgroup, france) with blood sampled in edta tubes. among a set of traits, nine were included in the genetic analyses: total number of leukocytes, lymphocytes, monocytes, neutrophils, eosinophils, erythrocytes, platelets and hematocrit (tables and ). total concentrations of immunoglobulin subsets were measured by elisa as previously described [ ] . plasma samples were diluted : , : and : , to detect igm, iga and igg, respectively, in tris-buffered saline and added to plates coated with immunoglobulin class specific pig antibody (bethyl laboratories inc., interchim, france). the different subsets were detected with the appropriate peroxidase anti-pig igm, iga or igg (bethyl laboratories inc.) and were quantified by reference to standard curves constructed with known amounts of pig immunoglobulin subsets. anti-m. hyopneumoniae igg titers were also measured by elisa using a commercial kit (elisa id screenh m. hyopneumoniae indirect, idvet, france). absorbance was read at nm using an elisa plate reader (spectra thermo, tecan, nc, usa) and the biolise . data management software. haptoglobin and c reactive protein levels were measured in pig serum by colorimetric tests (phase haptoglobin assay, abcys biologie, france) and elisa assays (porcine c reactive protein assay, abcys biologie, france), respectively. absorbance was read at nm using an elisa plate reader (mrx revelation, dynex). pbmcs were purified by density gradient centrifugation. a volume of ml heparinised blood was added to leucosept tubes (greiner bio-one, france) pre-filled with ml ficoll (lymphocytes separation medium, eurobio, france) and centrifuged at rpm for min. pbmcs were collected at the ficoll interface and washed in ml d-pbs without mgcl and cacl (gibco, invitrogen, france). cells were then incubated in ml bd pharmlyse x (bd biosciences, france) at room temperature. purified pbmcs were washed in ml d-pbs without mgcl and cacl , incubated with ml pig serum at uc for min, washed again in ml d-pbs without mgcl and cacl and then washed in ml s/w buffer ( g/l nan , g/l bovine serum albumin in pbs, ph . ) at a final concentration of . cells/ml. cells were used for each antibody labelling. single, double or triple staining was performed using monoclonal antibodies (mabs) directed against i) cd (msa , isotype igg a, vmrd) and cd (mca , isotype igg , serotec), ii) cd (pt a, isotype igg a, vmrd) and cd a (pt b, isotype igg b, vmrd) iii) tcrcd (mac , isotype igg a, bd biosciences pharmingen), iv) igm (pig a, isotype igg b, vmrd) and v) mhcii (msa , isotype igg a, vmrd), cd (mca , isotype igg , serotec) and cd a ( - - a, isotype igg b, bd biosciences pharmingen). briefly, pbmcs were stained with primary mabs for min at uc, washed in s/w buffer and stained with allophycocyanin-conjugated anti-mouse igg (bd biosciences, france), phycoerythrin-conjugated antimouse igg a (southern biotech, france), fitc-conjugated antimouse igg b (southern biotech, france), apc-conjugated antimouse igg (bd biosciences, france), phycoerythrin-conjugated anti-rat igg a (bd pharmingen, france), or phycoerythrinconjugated anti-mouse igg b (southern biotech, france). after washing in s/w buffer, cells were fixed in bd cellfix solution (becton dickinson, germany). data acquisition and analysis were carried out with the facscan and cellquest software (becton dickinson, uk). synthesis of ifna by leukocytes of pigs was tested in vitro by incubating diluted total blood in the presence of pseudorabies virus (prv, suid herpesvirus )-infected, glutaraldehyde-fixed, pk cell monolayers, according to a protocol previously described for transmissible gastroenteritis virus [ ] . confluent pk cell monolayers grown in well plates were infected by prv at a multiplicity of infection of , fixed with . % glutaraldehyde h post-infection and washed with d-pbs and rpmi before the addition of blood samples. for each animal, monolayers were incubated with diluted heparinized blood ( ml of blood diluted : in dmem supplemented with antibiotics) for h at uc. plates were then centrifuged at x g for min at uc, and supernatants were collected and stored at - uc. ifna was assayed in the supernatants using a classical sandwich elisa test as previously described [ ] . production and dosage of il b, il , il , tnfa and il heparinized blood samples ( ml) were fivefold diluted in well plates in . ml rpmi medium (biowhittaker, belgium) supplemented with % heat-inactivated fetal bovine serum (qb perbio, uk), mmol/l l-glutamine, u/ml penicillin and mg/ml streptomycin. for stimulation, a mixture of ng/ml pma (sigma, france), mg/ml ionomycin (sigma, france) and mg/ml lps from escherichia coli o :b (sigma, france) was added to the diluted blood. for mock stimulation, a volume of pbs equal to the volume of stimulation reagents was added to the diluted blood. after incubation at uc for h, culture supernatants were collected by centrifugation at g for min and stored at uc before use. the cytokines il b, il , il , tnf and il were quantified using commercial elisa tests (duoset elisa development kits, r&d systems, usa). for quantification of basal levels of cytokines in supernatants from mock-stimulated cells, the samples were not diluted for quantification. supernatants collected from stimulated cells were diluted ( : for il b and il , : for il , : for tnf and : for il ). all samples were tested in duplicates. absorbance was read at nm using an elisa plate reader (mrx revelation, dynex). results were expressed as pg of cytokine/ml. heparinized blood diluted : in complete culture medium consisting of dmem (dulbecco's modified eagle medium, eurobio, france) supplemented with % fetal calf serum (hyclone, perbio, france), mm l-glutamine, u/ml penicillin and mg/ml streptomycin (eurobio, france) was stimulated with mg/ml cona (sigma, france), or with ng/ml of pma (sigma, france) and mg/ml of ionomycin (sigma, france) or mg/ml lps from escherichia coli (sigma, france). cytokine content was measured in supernatants using elisa tests as already described [ ] . briefly, purified fractions of anti-swine il- , il- , ifnc (clones a d f , a b f and a d b respectively, biosource, france) and il (clone , r and d system, france) were used as capture antibodies, in conjunction with the biotinylated anti-swine il- , il- , il and ifnc monoclonal antibodies (clones a d h , a b c and a d c , respectively, biosource, clinisciences, france) or anti-swine polyclonal antibody (goat anti-porcine il- , r and d system, france). streptavidin-horseradish peroxidase (biosource) and tmb (fermentas, md, usa) were used for detection. absorbance was read at nm using an elisa plate reader (spectra thermo, tecan, nc, usa) and the biolise . data management software. recombinant pig il- , il- , il and ifnc were used as standards. the detection limits were pg/ ml, pg/ml, pg/ml and pg/ml for il- , il- , il and ifnc, respectively. results were expressed as pg of cytokine/ ml. lymphocyte proliferation was performed in well plates as already described [ ] . briefly, heparinized blood samples were diluted : in complete culture medium consisting of dmem (dulbecco's modified eagle medium, eurobio, france) supplemented with % fetal calf serum (hyclone, perbio, france), mm l-glutamine, u/ml penicillin and mg/ml streptomycin (eurobio, france). for detection of unspecific lymphocyte proliferation, the diluted blood samples were seeded in well plates ( ml/well) and mock-stimulated for h by incubation in culture medium (control wells), or stimulated for h by incubation with the culture medium supplemented with either mg/ml cona (sigma, france), or ng/ml of pma (sigma, france) and mg/ml of ionomycin (sigma, france), or mg/ml lps (sigma, france). control wells remained unstimulated. after h of incubation at uc, . mci of h-methylthymidine (icn, france) was added to each well. after another h incubation period, the cells were harvested through glassfiber filters (whatman, united kingdom) by means of an automatic harvester (titerteck-skatron, molecular devices, france). incorporation of tritiated thymidine was measured with a liquid scintillation beta counter (kontron instruments, france). results were expressed as a stimulation index of lymphocyte proliferation calculated as mean counts per min (cpm) of the triplicate cultures in stimulated culture/mean cpm in control non-stimulated culture. preliminary statistical analyses were performed using r software [ ] . in order to test if trait distributions deviated from gaussian, a d'agostino normality test was used (p = . ). since most traits were not sampled from a gaussian distribution, they were all normalized using a box-cox transformation except for phenotypes reaching the value zero, which were normalized using ln( +x) transformation. significant effects of age at the time of vaccination, of time of vaccination, of breeding unit and of time of experiment were detected for most traits by variance analysis taking into account these effects. normed principal component analysis (pca, [ ] , dudi.pca function, ade package [ ] , r software [ ] ) on a subset of its adjusted for age at the time of vaccination, time of vaccination, breeding unit and experiment (table ) were performed using a linear model. clusters of its were detected using the r package mclust for normal mixture modelling and model-based clustering [ ] . it combines model-based agglomerative hierarchical classification, based on the classification likelihood, and the expectation-maximization (e-m) algorithm for maximum likelihood estimation of multivariate mixture models. variance components, genetic parameters and their standard errors were estimated by the restricted maximum likelihood (reml) method [ ] , using the wombat software [ ] . this is the reference method to estimate genetic parameters with a mixed model. univariate and bivariate mixed linear animal models were employed to estimate heritability and genetic correlations, respectively. for the univariate analyses, the fixed part of the model included experiment time, age at the time of vaccination, vaccination time and herd of origin effects and the random part included a common litter environmental effect and direct genetic effects. in matrix notation y~x b zw a azw c cze where y = the vector of observations; x b , w a and w c are known incidence matrix relating observations to fixed and random effects; ß = a vector of fixed effects and covariates; a = the vector of direct genetic effects; c = the vector of common litter environmental effects; and e = the vector of random residual effects. all random effects were assumed to follow a normal distribution with zero mean. for bivariate analyses, the same effects as for univariate analyses were taken into account in the fixed part of the model and a direct genetic effect was included in the random part of the model. % confidence intervals ( ci) were calculated for heritability (h ) estimates (h ). heritability estimates have been classified: high (h . . ), moderate ( . ,h , . ) or weak (h # . ). a graphical representation of the genetic correlations combined with a hierarchical clustering (euclidian distance, average link) was obtained with the heatmap function from the bioconductor software [ ] . comparisons of heritability average between subsets of traits were tested using the wilcoxon mann-whitney test (significance threshold p-value = . ). figure s heatmap of the phenotypic correlations between its. the correspondence between colour scale and genetic correlation levels are presented on the right-hand side of the heatmap. 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tang, lu; feng, jian; wang, yi; han, zhihai; meng, jiguang title: downregulation of paralemmin- ameliorates lipopolysaccharide-induced acute lung injury in rats by regulating inflammatory response and inhibiting formation of tlr /myd and tlr /trif complexes date: - - journal: inflammation doi: . /s - - - sha: doc_id: cord_uid: qnxyhmw previous studies have demonstrated paralemmin- (palm ) participates in toll-like receptor (tlr) signaling. this study investigated the effect of palm knockdown on lipopolysaccharide (lps)-induced acute lung injury (ali) and its underlying mechanisms. we constructed a recombinant adenoviral vector containing short hairpin rna for palm to knockdown palm expression. a transgene-free adenoviral vector was used as a negative control. the ali rat model was established by lps peritoneal injection at -h post-transfection. results showed that downregulation of palm improved the survival rate, attenuated lung pathological changes, alleviated pulmonary edema, lung vascular leakage and neutrophil infiltration, inhibited the production of proinflammatory cytokines and activation of nuclear factor κb and interferon β regulatory factor , and promoted the secretion of anti-inflammatory cytokine interleukin- and expression of suppressor of cytokine signaling- in the ali rat model. however, palm knockdown had no effect on tlr , myeloid differentiation factor (myd ), and toll-interleukin- receptor domain-containing adaptor inducing interferon β (trif) expression. moreover, palm knockdown reduced the interaction of tlr with myd or trif induced by lps in rat lungs. therefore, the downregulation of palm protected rats from lps-induced ali and its mechanisms were partially associated with the modulation of inflammatory responses and inhibition of tlr /myd and tlr /trif complex formation. acute lung injury (ali) and its advanced stage, acute respiratory distress syndrome (ards), are clinical syndromes characterized by acute hypoxemic respiratory failure, decreased pulmonary compliance, and severe imbalance of ventilation and blood flow ratio resulting from excessive pulmonary inflammation, non-cardiogenic pulmonary edema, and diffuse alveolar damage [ , ] . because of the lack of specific and effective treatments for ali/ ards, the mortality rate among these patients remains as high as - % [ ] . the pathogenesis of ali/ards is complex, and one of the most crucial factors is an uncontrolled inflammatory response [ , ] . controlling the excessive inflammatory response during acute respiratory failure is an important therapeutic target for treating ali/ards. the toll-like receptor (tlr) signaling pathway plays a key role in host defense, innate immunity, and inflammation [ , , ] . prototypic signaling in inflammation occurs through the lipopolysaccharide (lps)-tlr pathway. lps induces the dimerization of tlr that initiates signal transduction involving multiple adaptors, leading to the activation of inflammatory transcription factors including nuclear factor kappa beta (nf-κb) and interferon β regulatory factor (irf ), which mediates the upregulation of inflammatory cytokine gene expression leading to an inflammatory response [ , ] . however, unchecked, excessive or inappropriate tlr signaling activation can lead to severe inflammation and immunity-related tissue damage [ , ] . single immunoglobulin interleukin (ig il)- receptor-related molecule (sigirr) is an important negative regulator of tlr signaling [ ] . through the negative regulation of tlr signaling, sigirr can depress the inflammatory response to lps, block the transduction cascade, inhibit nf-κb activation, decrease the production of inflammatory mediators, and ultimately attenuate organ injury [ ] . trapping key components of the tlr signaling pathway is the mechanism by which sigirr inhibits tlr signaling transduction [ , ] . the toll-interleukin- receptor (tir)-domain of sigirr is necessary for sigirr to interact with tir-domain containing adaptors [ ] . paralemmin (palm)- belongs to the palm protein family and was first described in xenopus laevis as xlgv / xlcaax- by cornish et al. [ ] . in our previous study, we used the c-terminal part of sigirr containing the tir domain as bait to screen sigirr-binding proteins in a human lung cdna library and found that palm- is a novel interactive partner of sigirr [ ] . we also showed that silencing palm expression inhibited the release of inflammatory cytokines induced by lps in vitro [ ] . moreover, previous research demonstrated that the downregulation of palm was protective against lps-induced ali in mice [ ] . although the downregulation of palm has been shown to benefit lps-induced ali in mice, the explicit underlying mechanisms against ali, including whether the downregulation of palm has a protective effect against lps-induced ali in other animal models, a negative effect on the expression of adaptors in the lps-tlr pathway, blocks lps-tlr signal transduction, and regulates the inflammatory transcription factors nf-κb and irf , remain unclear. indeed, species diversity usually leads to different research results, even under the same experimental conditions. for example, comprehensive physiologic studies of lung injury are better in rats than in mice [ ] . therefore, the present study investigated the effect of the downregulated expression of palm on lps-induced ali using a rat model. to elucidate the potential mechanisms involved, the production of pro-and anti-inflammatory cytokines in bronchoalveolar lavage fluid (balf) and serum, and the expression of tlr , myeloid differentiation factor (myd ), tir domain-containing adaptor inducing interferon β (trif), and nf-κb and irf activities in the lungs were examined. additionally, the interaction of tlr with myd or trif in rat lung was also detected by coimmunoprecipitation. cells in the exponential growth phase were used in the experiments described below. hek cells (microbix, toronto, canada) were grown in dulbecco's modified eagle's medium (gibco/brl, grand island, ny), supplemented with % fetal bovine serum (gibco/ brl), u/ml penicillin, u/ml streptomycin, and mmol/l l-glutamine (gibco/brl) at °c in a humidified chamber with % co . hek cells were seeded in m flasks or -well plates ( × /well) prior to transfection. in accordance with the principles of rna interference sequence design, the sequence ′-agatcttgatggag ggttt- ′ was chosen as the target sequence. the primers for this target sequence were ′-ccggggagatcttg atggagggtttctcgagaaaccctccatca agatctcctttttg- ′ (sense) and ′ -a att c a a a a a g g a g at c t t g at g g a g g g t t t c tcgagaaaccctccatcaagatctcc- ′ (antisense) (genechem co. ltd., shanghai, china). the short hairpin oligonucleotide and complementary strand targeting the consensus sequences of palm were designed and inserted into the shuttle plasmid pgv (genechem) to generate the plasmid pgv -palm -shrna. the pgv -palm -shrna plasmid was identified by pcr and dna sequencing. recombinant viruses were generated using packaging cells cotransfected with the shuttle plasmid and the framing plasmid (pbhglox) via the admax™ system (microbix). viral stocks were purified using cscl gradients. the recombinant virus was named ad.palm -shrna. a control vector containing no transgene (ad.v) was constructed using the same method. titers of the recombinant adenoviruses were determined by means of % tissue culture infectious dose, and for ad.palm -shrna and ad.v, this was × plaqueforming units (pfu)/ml and × pfu/ml, respectively. adult wistar rats ( weeks old) were purchased from the laboratory animal center of the academy of military medical sciences (certificate number scxk-army- - , beijing, china). all rats used in this research were raised and used in accordance with the arrive (animal research: reporting of in vivo experiments) guidelines on the use of laboratory animals [ ] and the care and housing of rats were approved by the animal care and use committee of navy general hospital (approval number: scxk-army- - ). animals were housed in specific pathogen-free conditions at a regular temperature ( ± °c) and humidity ( ± °c) with a standard diet and clean water provided ad libitum. after acclimatization for week in the animal housing facility, rats were anesthetized by ether (fuyu chemical co., ltd., tianjin, china) inhalation as follows: cotton wool containing ether was placed in an animal anesthesia bottle and the rats were placed into this bottle for min. after the rats were lightly anesthetized, they were removed and inoculated intranasally with a total of × pfu (in μl) of ad.palm -shrna (ad.palm -shrna group) or ad.v (ad.v group) using a -ml blunt head syringe as previously described [ , ] . forty-eight hours later, lps (escherichia coli : b , sigma-aldrich, st. louis, mo, usa) at mg/kg (dissolved in saline) was injected into the peritoneal cavity of conscious rats to establish the a l i r a t m o d e l . r a t s w e r e s a c r i f i c e d w i t h a supraphysiological dose of pentobarbital sodium ( mg/kg) at , , , , , or h after lps challenge (n = per time point in each group). after each rat was sacrificed, an -gauge sterile catheter (carelife co. ltd., shanghai, china) was inserted into the left bronchus to collect balf as previously described [ ] . the right lungs were rapidly removed from all rats and washed in rnasefree ice-cold saline. the upper lobe of the right lung was frozen in ml trizol reagent (invitrogen, shanghai, china) and stored in liquid nitrogen for rna extraction. the middle lobe of the right lung was frozen in ml cell lysis buffer (beyotime chemical co., jiangsu, china) and stored in liquid nitrogen for protein isolation and tissue homogenization. the lower lobe of the right lung was immediately fixed with % paraformaldehyde phosphate buffer solution (bosterbio, wuhan, china) for histological evaluation. blood samples were obtained from the right side of the heart ( - ml per rat), allowed to clot for . h on ice, and then centrifuged at ×g for min ( °c). the sera were stored in small aliquots at − °c until the cytokine levels were measured. total rna was extracted from the lung tissue using trizol reagent (invitrogen) according to the manufacturer's instructions. the rna was further purified by an rna cleanup kit (cwbio co., beijing, china). reverse transcription (rt) of the lung samples was performed using the takara primescript rt reagent kit (takara, dalian, china). then, quantitative real-time polymerase chain reaction (qrt-pcr) was performed using itaq universal sybr green supermix (takara) according to the manufacturer's instructions. the primers for rat palm were ′-gaggcagggatcttgatgtc- ′ (sense) and ′-gcccaacaccctcaagacta- ′ (antisense) (takara). the primers for rat β-actin were ′-ggag attactgccctggctccta- ′ (sense) and ′-gact catcgtactcctgcttgctg- ′ (antisense) (takara). β-actin was selected as an internal standard. the pcr parameters were set as °c for s, followed by cycles of °c for s and °c for s. all reactions were performed in triplicate, and reports were generated by rotor-gene real-time analysis software . (corbett research, australia). the relative expression of the target genes (palm and β-actin) was calculated by the −△△ct method. protein was extracted from the lung tissue as previously described [ , ] . protein concentrations were measured by the bicinchoninic acid (bca) method (sigma-aldrich) at nm with a μquant microplate spectrophotometer (biotek inc., winooski, vt, usa). protein samples were analyzed by western blotting. equal amounts of protein were separated by % sodium dodecyl sulfate (sds)/polyacrylamide gel electrophoresis (page) and transferred onto a polyvinylidene fluoride filter membrane in a semidry blotting apparatus (bio-rad co. ltd., shanghai, china). to reduce non-specific binding, the membrane was blocked in a % (w/v) non-fat milk (beyotime) solution in tris-buffered saline-tween (tbst: mm nacl, mm tris/hcl, ph . and . % tween ) (beyotime) at °c for h. the membranes were then incubated with anti-palm- (cat. no. sc- , ; polyclonal goat anti-rat; santa cruz) ( : in tbst) and anti-β-actin antibodies (cat. no. af ; monoclonal mouse anti-rat; beyotime) ( : in tbst) overnight at °c. after extensive washing with tbst, the membranes were incubated with horseradish peroxidase (hrp)-conjugated secondary antibody ( : in tbst) (beijing golden bridge biotech, beijing, china) at room temperature for h. the blots were washed twice in tbst buffer and the signals were detected by enhanced chemiluminescence following the manufacturer's instructions (beyotime). the membrane was photographed using the gel documentation and analysis system (gbox-hr, syngene, usa), and band intensities were measured with adobe photoshop version . . software (adobe systems, usa) and normalized to the expression of β-actin for quantitative analysis. a second cohort of rats (n = ) was used to evaluate the effect of ad.palm -shrna on the outcome of lpsinduced ali. rats were divided into two groups (n = rats per group) and given the same treatment as previously described. seven-day survival rates were recorded and analyzed. lung tissues were treated with a % paraformaldehydephosphate buffer solution (bosterbio) (ph = . ) overnight at °c, dehydrated, embedded, and sectioned to . -μm thickness. tissue slides were then stained with hematoxylin and eosin (he) (bosterbio) and analyzed under identical light microscope conditions (olympus co., ltd., tokyo, japan). lung injury was scored by a blinded observer according to the following three criteria: ( ) alveolar and interstitial edema, ( ) alveolar hemorrhage, and ( ) infiltration or aggregation of neutrophils. each criterion was graded according to a four-point scale: = normal, = mild change, = moderate change, and = severe change. the scores for criteria to were summed to represent the lung damage score (total score: - ) [ , ] . the left lungs were lavaged five times with pbs ( ml each time) and approximately % of the instilled volume was retrieved [ , ] . balfs were then centrifuged at ×g for min ( °c), and the clear supernatant was transferred to sterile eppendorf tubes and stored at − °c for the analysis of total protein concentration [ , ] by the bca method (sigma-aldrich). adult wistar rats (n = per group) were treated as described in the bmaterials and methods^section bestablishment of ali rat model and experimental design.^at h after lps challenge, the right lungs were harvested and stored in liquid nitrogen for the analysis of nf-κb phospho-p , phospho-irf , tlr , myd , trif, suppressor of cytokine signaling (socs ) protein levels and coimmunoprecipitation assay, and the left lungs were harvested for the analysis of lung wet/dry weight ratio. the lung tissues were weighed immediately then subjected to desiccation in a °c oven until a stable dry weight was achieved after h. the scales and oven were purchased from whaisp scientific instrument co., ltd. (wuhan, china). the wet/dry weight ratio was calculated by dividing the wet weight with the dry weight to quantify the magnitude of pulmonary edema [ ] . balf was prepared as described above, and balf pellets were resuspended in cold pbs ( °c). balf ( μl) was mounted on a glass slide by centrifugation and the glass slide was stained with he (bosterbio). for analysis of the neutrophil cell count, we counted cells per slide in randomly selected high-powered fields (× ) under identical light microscope conditions (olympus). balf neutrophil cell counts were expressed as cell count/ml of balf [ ] . to measure tissue myeloperoxidase (mpo) activity, frozen lungs were thawed and mpo was extracted following the homogenization and sonication procedure described previously [ ] . mpo activity in the supernatant was measured at an absorbance of nm, and changes in mpo activity were determined following the decomposition of h o in the presence of o-dianisidine. the specific activity of mpo in the lung was expressed as u/g of the wet lung tissue. serum and balf were prepared as described above. levels of tumor necrosis factor-α (tnf-α), interleukin- β (il- β), il- (r&d systems, inc., minneapolis, mn), and interferon β (ifn-β) (cusabio, wuhan, china) in balf and serum were measured by commercially available enzyme-linked immunosorbent assay (elisa) kits for rat, following the manufacturer's instructions. in brief, diluted standards or samples were added to -well plates precoated with affinity purified polyclonal antibodies specific for tnf-α, il- β, il- , and ifn-β, respectively. enzyme-linked polyclonal antibodies were then added to the wells prior to incubation at °c for min, followed by five final washes. the intensities detected at nm were measured after the addition of substrate solutions for min. each sample was assayed in triplicate. levels of tnf-α, il- β, il- , and ifn-β were calculated according to standard curves. nuclear extracts were prepared using the ne-per nuclear-cytoplasmic extraction reagents kit (thermo fisher scientific, usa) according to the manufacturer's protocol. the extracts were stored at − °c, and the protein concentration of the nuclear extracts was quantified by the bca method (sigma-aldrich). nf-κb p dna binding activity was assessed on isolated nuclear extracts by elisa using the transam™ nf-κb transcription factor assay kit according to the manufacturer's protocol (active motif, carlsbad, ca) [ ] . each sample was assayed in triplicate. equal amounts of raji nuclear extract were used as positive controls. western blot analysis of nf-κb phosho-p , phospho-irf , tlr , myd , trif, and socs lung tissues samples were obtained as described in the bmaterials and methods^section blung wet/dry ratios.^the nuclear extract and total protein were prepared, and the procedure of western blot analysis was performed as described above. expressions of nf-κb phospho-p and phospho-irf in the nuclear extracts were analyzed by immunoblotting with phospho-specific anti-nf-κb p (cat. no. an ; monoclonal rabbit antirat; beyotime) ( : in tbst) antibody or phosphospecific anti-irf (cat. no. ab ; polyclonal rabbit anti-rat; abcam, cambridge, uk) ( : in tbst) antibody, and histone h (cat. no. sc- , ; monoclonal mouse anti-rat; santa cruz) ( : in tbst) was used as a lysate control. expressions of tlr , myd , trif, and socs in total protein were analyzed by immunoblotting with anti-tlr (cat. no. sc- , ; monoclonal mouse anti-rat; santa cruz) ( : in tbst), anti-myd (cat. no. ; monoclonal rabbit anti-rat; cell signaling technology) ( : in tbst), rabbit polyclonal anti-trif (cat. no. ab ; polyclonal rabbit anti-rat; abcam, cambridge, uk) ( : in tbst), or anti-socs (cat. no. sc- , ; monoclonal mouse anti-rat; santa cruz) ( : in tbst). β-actin (beyotime) ( : in tbst) was used as a lysate control. corresponding hrpconjugated secondary antibodies (beijing golden bridge) were used to display the protein signal. to examine endogenous protein-protein interactions, coimmunoprecipitation assays were performed. lung tissue samples were obtained as described in the bmaterials and methods^section blung wet/dry ratios.^protein was extracted from the lung tissue as previously described [ ] . the rat lung tissue was homogenized and lysed in tne buffer ( mm tris, ph . , mm nacl, mm edta, % np- , % glycerol with protease inhibitor cocktail [sigma-aldrich]) ( ml of tne/g lung lysate) and clarified by centrifugation at , ×g for min at °c. after centrifugation, proteins were quantified using the bca method (sigma-aldrich). approximately mg samples of rat lung lysates were incubated with a primary rat-specific anti-tlr ( ) antibody ( : in tbst) (cat. no. sc- , ; monoclonal mouse anti-rat; santa cruz), and the same amount of lysate was incubated with normal rat igg (santa cruz) as a negative control at °c for h and then with μl of protein a/g-agarose (beyotime) at °c with rocking overnight. the pellets obtained after centrifugation were washed five times with washing buffer ( mm tris (ph . ), mm mgcl , mm edta, and mm pmsf). the pellets were resolved by sds-page and boiled for min. after centrifugation, the supernatants were obtained as immunoprecipitates for western blotting analysis by using anti-tlr antibody (santa cruz), anti-myd antibody (cell signaling technology), or anti-trif antibody (abcam). data are expressed as the means ± standard error of the mean (sem). statistical significance was estimated by one-way analysis of variance (anova) followed by the student-newman-keuls test. kaplan-meier curves and the log-rank test were used to assess survival data. statistical significance was determined for p values less than . . lung tissues were obtained at , , , , , and h after lps injection to detect palm expression using quantitative real-time polymerase chain reaction (qrt-pcr) and western blot analysis. in the ad.v group, palm gene and protein expression in the lung tissues increased significantly, peaking at h after lps challenge (p < . vs. -h time point; p > . -h vs -h time point) (fig. ) , indicating that lps enhanced palm expression in a time-dependent manner. this result was consistent with our previous report in vitro [ ] . the administration of ad.v did not influence palm expression before lps stimulation (p > . vs. normal rats) (fig. ) . compared with the ad.v group and normal rats, palm mrna and protein expression in the ad.palm -shrna group were significantly decreased, although increases were noted after lps stimulation at h (p < . vs. ad.v group at each time point) (fig. ) . moreover, the palm expression levels in the ad. palm group decreased by about % compared with those in the ad.v group at each time point. to determine the effect of the downregulation of palm expression on the outcome of lps-induced ali, survival rates of the ad.v and ad.palm -shrna groups were compared using the log-rank test. compared with the ad.v group, the survival rate of the ad.palm -shrna group was significantly increased. rats receiving the ad.palm -shrna and control adenoviral vectors had an overall survival rate of and %, respectively, over days (p < . ) (fig. ) . histopathological staining showed that lung sections from the ad.v group displayed typical histological features of ali after lps challenge, including the infiltration of numerous neutrophils, alveolar congestion and hemorrhage, and marked swelling of the alveolar walls. moreover, the magnitude of lung injury was increased over time ( fig. a (a-e) ). ad.palm -shrna pretreatment improved these lps-induced histological changes of lung tissue compared with the ad.v group (fig. a (a-j) ). in addition, we used a pathological scoring system to quantify the severity of lung tissue damage in both groups. as shown in fig. b , the lung injury scores of both groups were very low before lps stimulation, demonstrating that the adenovirus vector had no impact on lung injury (fig. b) . furthermore, the lung injury scores in the ad.palm -shrna group were significantly decreased at each time point after lps challenge compared with those in the ad.v group (p < . vs. ad.v group at each time point) (fig. b) . because protein concentrations in the balf and lung water content are usually used to quantify the increase of vascular permeability in ali, we measured total protein levels in balf and lung wet/dry ratios in both groups. the concentration of total protein in the balf of both groups increased after lps-stimulation (p < . vs. -h time point) (fig. a) . however, total protein levels in the balf of the ad.palm -shrna group were significantly lower than those in the ad.v group after lps stimulation (p < . vs. ad.v group at each time point) (fig. a) . this result also demonstrated that the total protein level in the balf of both groups reached a peak at h after lps stimulation (fig. a) , indicating that lung vascular leakage was most severe at the -h time point. consequently, the lung wet/dry ratios at this time point were measured to confirm changes of lung vascular permeability. we found that lps led to increased lung wet/dry ratios in both ad.v and ad.palm -shrna groups (p < . vs. normal rats) (fig. b) , and ad.palm -shrna pretreatment markedly attenuated lps-induced pulmonary edema (p < . vs. lps-treated and lps + ad.v-treated rats) (fig. b) . balf neutrophil counts commonly indicate the severity of inflammation in ali. as shown in fig. a , the balf neutrophil count was significantly increased in the ad.v group after lps injection (p < . vs. -h time point) (fig. a) . however, in the ad.palm -shrna group, the balf neutrophil count was significantly decreased at each time point after lps exposure (p < . vs. group ad.v at each time point) (fig. a) . myeloperoxidase (mpo) activity is an index of neutrophil sequestration. thus, the mpo activity in lung tissue was detected to determine the extent of neutrophil infiltration. mpo activity was normalized by the weight of wet lung tissue. in unstimulated rats, mpo levels in the lung tissues were very low ( . ± . u/g wet lung tissue in the ad.v group and . ± . u/g wet lung tissue in the ad.palm -shrna group) (fig. b) . mpo activities in lung tissues increased sharply after lps injection in both groups (p < . vs. -h time point) (fig. b) . nevertheless, increases of mpo activity in the lung were significantly decreased in the ad.palm -shrna group at each time point after lps exposure compared with that in the ad.v group (p < . vs. group ad.v at each time point) (fig. b) . increased levels of proinflammatory cytokines are an indication of local pulmonary injury/systemic inflammation in ali, as reported previously [ , , ] . thus, we evaluated levels of the proinflammatory cytokines tnf-α, il- β, and ifn-β in the balf and serum. when ad.palm -shrna and ad.v were administered without lps treatment, there were no significant differences in cytokine levels in balf and serum between the two groups (p > . ) (fig. ) . after lps stimulation, the levels of tnf-α (fig. a, b) , il- β (fig. c, d) , and ifn-β (fig. e, f) in balf and serum were significantly increased in the ad.v group. in contrast, ad.palm -shrna pretreatment reduced the elevated levels of tnf-α (fig. a, b) , il- β (fig. c, d) , and ifn-β (fig. e, f) in balf and serum (p < . vs. ad.v group at each time point) (fig. ) . il- is an anti-inflammatory cytokine with negative immune regulatory effects in the tlr inflammatory signal transduction pathway [ ] . thus, we evaluated the levels of anti-inflammatory cytokine il- in the balf and serum. lps exposure increased the levels of il- in the balf (fig. a) and serum (fig. b) . in the ad.palm -shrnatreated group, the lps-induced increase of il- was further enhanced compared with the ad.v-treated group (p < . vs. ad.v group at each time point) (fig. ) . socs is an important negative regulator of inflammation and plays a protective role in lps-induced lung inflammatory responses [ , ] . previous studies have shown that il- induced socs expression [ , ] . thus, we evaluated the protein levels of socs in lung tissues using western blot analysis. the results showed that lps induced the expression of socs in all lps-treated rats (p < . vs. normal rat) (fig. c) . however, ad.rpalm pretreatment further increased the elevated socs protein levels in lung tissues (p < . vs. lps-treated and lps + adv-treated rats) (fig. c) . nf-κb is considered a key transcription factor in lps-tlr signaling that mediates lps-induced ali [ ] . thus, the nf-κb-dna-binding activity and nf-κb phospho-p protein level in rat lungs was determined as described above. the elisa result showed that all lps-injected rats exhibited an increase in nf-κb-dna-binding in comparison with lps-free rats (p < . vs. -h time point) (fig. a) . ad.palm -shrna pretreatment significantly attenuated the lps-induced increase in nf-κb activation compared with the ad.v group (p < . vs. ad.v group at each time point) (fig. a) . elisa demonstrated that the activity of nf-κb reached a peak at h after lps stimulation (fig. a) . therefore, we also examined the nf-κb phospho-p protein level in nuclear extracts at this time point using western blot analysis to confirm the inhibitory action of ad.palm -shrna on lps-induced nf-κb activation. as shown in fig. b , at h after lps stimulation, the nf-κb and phospho-p protein levels in the ad.palm -shrna group were reduced by about % compared with those in lps-treated and lps + ad.v-treated rats (p < . ) (fig. b) . irf is also an important transcription factor in cells and activates the ifn-β gene to regulate the release of proinflammatory cytokines induced by lps [ ] . we therefore examined the phospho-irf protein levels in nuclear extracts using western blot analysis to detect the influence of palm on lps-induced irf activation. at h after lps stimulation, phospho-irf protein levels were significantly increased in all rats. however, ad.rpalm pretreatment reduced the elevated phospho-irf protein levels (p < . vs. lps-treated and lps + ad.vtreated rats) (fig. c) . effects of downregulation of palm on lps-induced tlr , myd , and trif protein expression tlr , myd , and trif are important adaptors in the tlr signaling pathway [ ] . thus, we examined tlr , myd , and trif expression levels in lung tissues using western blot analysis to detect the influence of palm on their expression in the ali rat model. as shown in fig. , lps significantly induced the expression of tlr , myd , and trif in lung tissues (p < . vs. normal rats). however, palm shrna and control adenovirus did not have significant effects on tlr , myd , and trif protein expression in lps-induced ali rats (p > . vs. lps-treated and lps + ad.v-treated rats) (fig. ) . myd and trif are recruited to the tlr intracellular domain after stimulation by lps, which triggers the formation of tlr /myd and tlr / trif complexes [ ] . as shown in fig. , the formation of tlr /myd and tlr /trif complexes was significantly increased in lung tissues after lps stimulation (p < . vs. normal rats). pre-treatment with ad.palm -shrna reduced the i n t e n s i t y o f t h e m y d a n d t r i f b a n d coimmunoprecipitated using anti-tlr antibody (p < . vs. lps-treated and lps + ad.v-treated rats). however, the control adenovirus (ad.v) pre-treatment did not have significant effects on lpsinduced tlr /myd and tlr /trif complex formation in comparison with lps-treated rats (p > . vs. lps-treated rats) (fig. ) . fig. . the downregulation of palm has no effect on lps-induced tlr , myd , and trif protein expression. rats were instilled with μl of ad.palm -shrna or ad.v intranasally. at h, lung injury was induced by a peritoneal injection of lps ( mg/kg). normal and ali rats were used as control groups, and lung tissues were collected at h after lps challenge. a representative immunoblots of tlr , myd , and trif. β-actin served as an internal control. b quantification of densitometric measurement as a ratio of tlr , myd , and trif relative to β-actin. data are expressed as means ± sem (n = ). *p < . vs. normal rats. fig. . effects of downregulation of palm on lps-induced tlr /myd and tlr /trif complex formation in lungs. rats were instilled with μl of ad.palm -shrna or ad.v intranasally. at h, lung injury was induced by a peritoneal injection of lps ( mg/kg). normal and ali rats were used as control groups, and lung tissues were collected at h after lps challenge. a representative coimmunoprecipitated bands of myd and trif by anti-tlr antibody. β-actin served as an internal control. b quantification of densitometric measurement of palm knockdown on interactions of tlr with myd and trif. data are expressed as means ± sem (n = ). *p < . vs. normal rats and #p < . vs. lps + ad.palm -shrna-treated rats. the present study revealed that palm expression in rat lung was upregulated in a time-dependent manner in response to lps. furthermore, the downregulation of palm improved the survival rate of rats, ameliorated the severity of lung injury, alleviated pulmonary edema, lung vascular leakage and neutrophil infiltration, inhibited the production of proinflammatory cytokines (tnf-α, il- β, and ifn-β) and activation of nf-κb and irf , and promoted the secretion of the antiinflammatory cytokine il- and the expression of socs in the lps-induced ali rat model. however, the downregulation of palm did impact the upregulation of tlr , myd , and trif protein expression induced by lps in rat lung tissues. moreover, the downregulation of palm impeded the interaction of tlr with myd or trif induced by lps in rat lungs. these results suggest that the downregulation of palm exerts a potential protective effect against lpsinduced ali in rats and may be a promising potential treatment for ali. its protective effects were partially associated with the downregulation of proinflammatory cytokines, upregulation of the anti-inflammatory cytokine (il- ) and negative regulator of inflammation (socs ), and the inhibition of nf-κb and irf activities. its mechanisms may be attributed to the modulation of inflammatory responses and inhibition of tlr / myd and tlr /trif complex formation. palms are a small protein family that includes palm , palm , palm , and palmdelphin (palmd) [ ] , which are highly expressed in the brain, kidney, adrenal gland, mammary gland, and breast cancers [ ] [ ] [ ] . previous studies have verified that palm is implicated in controlling cell shape, plasma membrane dynamics, cell motility, invasiveness and metastatic potential of cancer cells, modulation of cell migration and maturation, and tumor lymphangiogenesis [ , , ] . however, little is known about the biological functions of the other palm isoforms. hultqvist and colleagues [ ] considered that there is functional diversification and specific biological roles among palm isoforms. palm was first reported in by cornish et al. [ ] and was speculated to act as an adaptor to link intrinsic membrane proteins to each other, to the cytoskeleton, or to motor proteins [ , ] . we reported for the first time in that palm is a novel interactive partner of sigirr, and we also found that lps upregulated the expression of palm [ ] . our present study confirms the findings of our previous studies, which demonstrated the modulation of palm expression in response to lps [ , ] . our results revealed that palm expression was detected in the lung tissue of normal rats, and that the control adenovirus ad.v had no effect on palm expression. after lps stimulation, palm protein and mrna expression levels in the lungs of the ad.v group were significantly upregulated in a time-dependent manner, which was in line with our previous result in vitro [ ] . moreover, the expression pattern of palm was similar with those of the adaptors in tlr signaling, including myd and trif [ ] [ ] [ ] [ ] . the pattern of palm upregulation might be related to its functional involvement in lps-tlr signal transduction, similar to the adaptors [ ] . in our present study, palm gene and protein expression in lung tissues was downregulated by administering a recombinant adenovirus expressing shrna for rat palm . although palm expression in the ad.palm -shrna group increased after lps stimulation for h, this expression level remained lower than that of the normal and ad.v group rats during the experiment. this result demonstrated that the palm gene transcript in lung tissue was silenced successfully using the interference adenoviral vector-mediated rna through intranasal instillation. increased palm expression levels in the lungs of the ad.palm -shrna group may also be caused by enhanced palm gene transcription during the process of inflammatory signal transduction. an uncontrolled inflammatory response is one of the most crucial factors in the pathogenesis of ali [ , ] . during the early stage of ali, inflammatory and immune responses involve a vast of array of mediators; therefore, inhibition of the inflammatory cascade response is critical for the treatment of this disorder [ ] . downregulation of palm gene transcription to impede tlr signal transduction at the early stage may inhibit the excessive inflammatory responses and improve the outcome of inflammatory diseases, including ali/ ards. thus, we determined the survival rate, lung histological changes, pulmonary edema, lung vascular leakage, neutrophil infiltration, and mpo activity in the lung to assess the effect of downregulation of palm on lps-induced ali in rats. our findings showed that the downregulation of palm expression significantly improved the aforementioned assessment indexes, similar to previous studies [ , ] . however, in these previous studies, the underlying mechanisms have not been explored. lps induces the dimerization of tlr , resulting in conformational changes of the tlr homodimer that induces the recruitment of adaptor proteins containing tir domains. tir domains of tlr recruit tir domaincontaining adaptor proteins, myd -adaptor-like (mal) and myd (myd -dependent pathway), or trif and toll-il- receptor-containing adapter molecule (ticam- ) (myd -independent pathway) [ ] . activation of the myd -dependent pathway induces the activation and translocation of transcription factors such as nf-κb and activator protein- (ap- ) in the nucleus and induces the production of proinflammatory cytokines [ ] . activation of the myd independent pathway leads to the activation and translocation of the transcription factor irf in the nucleus to induce the production of type i interferon [ ] . the myd -independent pathway also plays a key role in the late-phase activation of nf-κb [ ] . in the present study, we found that the downregulation of palm suppressed nf-κb-dna-binding activity and decreased nf-κb phospho-p and phospho-irf protein levels in the nucleus of ali rat lung tissues. these findings suggested that the downregulation of palm inhibited the activation of nf-κb and irf in the ali rat model. the increase of proinflammatory cytokines in the balf are thought to be indices of local pulmonary inflammation in ali [ , , ] , and the levels of inflammatory mediators in serum generally reflect the extent of systemic inflammatory response and the prognosis of inflammatory diseases [ , , , ] . in our present study, a significantly increased survival rate was observed in the ad.palm -shrna group, accompanied with a decrease in inflammatory mediator levels in the balf and serum. these results suggest that this therapy directed to the lung through intranasal instillation inhibited local inflammation in the lungs and suppressed systemic inflammation to improve the outcome of ali. mitigation of pulmonary inflammation and reduction of alveolar capillary permeability decreased the translocation of inflammatory mediators from lung tissues into the systemic circulation ameliorating severity of the systemic inflammatory response [ , ] . the negative effect of the downregulated expression of palm on the production of proinflammatory cytokines and the infiltration of neutrophils might be ascribed to its negative regulatory function on transcription factors nf-κb and irf . alleviation of inflammatory responses can finally lead to the amelioration of lung tissue damage. our results also revealed that the downregulation of palm did not impact the upregulation of tlr , myd , and trif protein expression after ali in lung tissues. previous studies have shown that palms are associated with lipid rafts and have been proposed to function as adaptors between membrane proteins or with the cortical cytoskeleton [ , ] . with this in mind, we speculated that the inhibition of palm expression to impair its function with adaptors in the lps-tlr signaling pathway may be a potential mechanism for the downregulation of palm protection against lpsinduced inflammatory responses and lung injury. downregulating the palm suppression of transcription factors nf-κb and irf might not be mediated by inhibiting the expression of the adaptors tlr , myd , and trif in lps-induced inflammation. therefore, we further investigated the effect of the downregulation of palm on the interaction of tlr with myd or trif in lung tissues of ali rats. the result of coimmunoprecipitation analysis showed that palm knockdown inhibited the interaction of tlr with myd or trif. our previous study demonstrated that palm interacted with the tir domain of sigirr [ ] . moreover, our in vitro study reveals that palm interacts with myd , irak- , traf- , and ticam- , important adaptors in the tlr signaling pathway, in a ligand-dependent manner (accepted but unpublished data). therefore, we presumed that palm might act as a bbridge^to link tlr to the adaptor proteins (myd , irak- , traf- , and tiacm- ) in lps-tlr signal transduction. downregulation of palm expression might diminish the bridging efficacy of palm during lps-tlr signal transduction, which might reduce further the interaction of tlr with myd or trif. inhibition of tlr /myd and tlr /trif complex formation may be a potential mechanism of the suppression of nf-κb and irf activation in lps-induced inflammation. further investigation is still needed to illuminate the detailed molecular mechanisms of the regulatory effect of palm on lps-tlr signaling. il- is a negative feedback regulatory molecule in the tlr signaling pathway that degrades the nf-κb p subunit and inactivates mitogen-activated protein kinase (mapk) signaling [ ] . in this study, we also found that lps-induced il- expression in the balf and serum was significantly increased after palm gene silencing. this finding suggests that palm inhibits anti-inflammatory cytokine il- secretion in lps-induced ali. however, the downregulation of palm decreased the inhibitory effect of palm on il- secretion. promotion of anti-inflammatory cytokine il- secretion may also be a possible mechanism of downregulation of palm protection against lps-induced lung injury. the detailed mechanisms of palm inhibition against lpsinduced il- secretion warrant further research. previous studies have shown that socs proteins have pivotal roles in attenuating cytokine and tlr signaling in myeloid cells [ ] . in mice deficient for socs in hematopoietic and endothelial cells, injection with granulocytecolony stimulating factor (g-csf) induced a pronounced inflammatory response involving enhanced neutrophilia and neutrophil infiltration of multiple tissues [ ] . furthermore, the exogenous delivery of socs significantly enhanced the survival of mice challenged with lps-induced endotoxic shock [ ] . in the present study, we demonstrated that the downregulation of palm promoted the expression of socs in the lung tissues of ali rats. it was reported that il- induced socs expression to inhibit the expression of inflammatory cytokines in lpsstimulated macrophages [ ] . thus, downregulating the palm promotion of socs expression might be indirectly mediated by enhancing the expression of il- in lps-induced inflammation. the detailed molecular mechanisms of the regulatory effect of palm on socs expression require further investigation. the lps model of lung injury displays the key features of clinical ards, including inflammation, pulmonary edema, and mortality, and is still widely used to investigate the pathogenesis and treatment of ali and ards [ , , , ] . therefore, in the present study, we used the lps-induced ali rat model to investigate the effect of the downregulation of palm on ali. the rat ali model may be superior to the mouse ali model based on the following reasons. first, the availability of the rat genome will be extremely useful, as opportunities for comprehensive physiologic studies of lung injury are better in rats than in mice [ , ] . second, rats have a larger blood volume in contrast to mice, which benefits the analysis of cytokine levels in serum, to further illustrate the relationship between the local and systemic inflammation and the mechanisms of ali. the palm gene family is an early gene family that has been maintained throughout vertebrate evolution and may be important for the development and plasticity of complex nervous systems [ ] . at present, there are no relevant literature reports on palm knockouts; therefore, whether palm knockout possesses potentially fatal consequences remains unclear. the effect of palm knockout on the entire animal and the pathophysiological changes of ali in palm knockout animals will be investigated in future studies by constructing palm gene knockout rat models. with respect to ali/ards, transgene expression does not need to last a lifetime and a single administration of adenoviral vector could cover the critical period of ali/ ards [ ] . we therefore chose an adenoviral vector as a transient gene transfer tool in our study. our results showed that although palm expression increased in the palm knockdown group after lps stimulation for h, this expression level did not exceed that of normal rats at all time points. this finding indicates that the rna interference activity of ad.palm -shrna lasted for the duration of the experiment. moreover, in this study, we did not determine the effect of palm on inflammatory cells and immune cells, such as dendritic cells and macrophages, as it was not clear whether palm was expressed in these cells; however, this was not the main emphasis of the present study. future research will focus on this emerging issue using in vitro experiments. in summary, our results demonstrated that palm expression was induced by lps in a time-dependent manner, and that downregulation of palm in the lung, via interfering adenoviral vector-mediated rna, improved the survival rate in an lps-induced rat model. this protective effect of palm -knockdown on ali might be mediated by attenuating pulmonary pathological injury, alleviating alveolar capillary permeability, and decreasing neutrophil infiltration. downregulation of proinflammatory cytokine secretion, upregulation of anti-inflammatory cytokine secretion and socs expression, and inhibition of nf-κb and irf activities likely represent the mechanisms for the downregulation of palm protection against lpsinduced inflammatory responses and lung tissue injury. downregulating palm suppression of lps-tlr signaling might not be mediated by modulating the expression of tlr , myd , and trif, but rather through inhibiting the interaction of tlr with myd or trif. these results suggest that modulating palm expression may be a promising potential treatment for ali/ards. future research will focus on the emerging issues from this study and on the detailed molecular mechanisms of the palm regulation of lps-tlr signaling. programmed cell death receptor ligand modulates the regulatory t cells' capacity to repress shock/sepsis-induced indirect acute lung injury by recruiting phosphatase src 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lps-induced inflammatory response in human renal proximal tubular epithelial cells by regulating tlr -myd -tak -mediated nf-κb and mapk pathway molecular characterization and immunohistochemical localization of palmdelphin, a cytosolic isoform of the paralemmin protein family implicated in membrane dynamics paralemmin, a prenyl-palmitoyl-anchored phosphoprotein abundant in neurons and implicated in plasma membrane dynamics and cell process formation paralemmin- is expressed in lymphatic endothelial cells and modulates cell migration, cell maturation and tumor lymphangiogenesis paralemmin- is over-expressed in estrogen-receptor positive breast cancers cellular and subcellular localization of paralemmin- , a protein involved in cell shape control, in the rat brain, adrenal gland and kidney evolution of the vertebrate paralemmin gene family: ancient origin of gene duplicates suggests distinct functions inhibiting toll-like receptor signaling ameliorates pulmonary fibrosis during acute lung injury induced by lipopolysaccharide: an experimental study down-regulation of endothelial tlr signalling after apo a-i gene transfer contributes to improved survival in an experimental model of lipopolysaccharide-induced inflammation expression of tlr -myd and nf-κb in the iris during endotoxin-induced uveitis sulphoraphane inhibited the expressions of intercellular adhesion molecule- and vascular cell adhesion molecule- through myd -dependent toll-like receptor- pathway in cultured endothelial cells hydroxysafflor yellow a ameliorates lipopolysaccharide-induced acute lung injury in mice via modulating toll-like receptor signaling pathways silencing angiopoietin-like protein (angptl ) protects against lipopolysaccharide-induced acute lung injury via regulating sirt /nf-kb pathway mesenchymal stem cellbased developmental endothelial locus- gene therapy for acute lung injury induced by lipopolysaccharide in mice keratinocyte growth factor gene transduction ameliorates acute lung injury and mortality in mice intratracheal instillation of keratinocyte growth factor decreases hyperoxia-induced mortality in rats socs is a critical physiological negative regulator of g-csf signaling and emergency granulopoiesis gene delivery of socs protects mice from lethal endotoxic shock rat genetics: attaching physiology and pharmacology to the genome the authors thank the national natural science foundation of china (chen, x.x., no. ; feng, j., no. ) and the innovative cultivation foundation of navy general hospital of the pla (chen, x.x., no. cxpy ) for their great support in financing these researches. conflict of interest. the authors declare that they have no conflict of interests.funding. this study was funded by the national natural science foundation of china (chen, x.x., no. ; feng, j., no. ) and the innovative cultivation foundation of navy general hospital of the pla (chen, x.x., no. cxpy ). key: cord- - eblcke authors: gallego, carolina; middleton, andrew m.; martínez, nhora; romero, stefany; iregui, carlos title: interaction of bordetella bronchiseptica and its lipopolysaccharide with in vitro culture of respiratory nasal epithelium date: - - journal: vet med int doi: . / / sha: doc_id: cord_uid: eblcke the nasal septa of fetal rabbits at days of gestation were harvested by cesarean section of the does while under anesthesia and then exposed to bordetella bronchiseptica or its lipopolysaccharide (lps) for periods of and hours. a total of explants were used. the tissues were examined using the hematoxylin & eosin technique. then, semithin sections ( . μm) were stained with toluidine blue and examined with indirect immunoperoxidase (ipi) and lectin histochemistry. the most frequent and statistically significant findings were as follows: ( ) cell death and increased goblet cell activity when exposed to bacteria and ( ) cell death, cytoplasmic vacuolation and infiltration of polymorphonuclear leukocytes when exposed to lps. the lesions induced by the bacterium were more severe than with lps alone, except for the cytoplasmic vacuolation in epithelial cells. ipi stained the ciliated border of the epithelium with the bacterium more intensely, while lps lectin histochemistry preferentially labeled the cytoplasm of goblet cell. these data indicate that b. bronchiseptica and its lps may have an affinity for specific glycoproteins that would act as adhesion receptors in both locations. bordetella bronchiseptica is a gram-negative bacterium capable of colonizing the respiratory tract of a large range of mammalian hosts, including mice, rats, guinea pigs, rabbits, cats, dogs, pigs, sheep, horses, and bears [ ] . b. bronchiseptica is responsible for a wide spectrum of overt respiratory diseases such as kennel cough in dogs, atrophic rhinitis in pigs, and snuffles and pneumonia in rabbits [ ] [ ] [ ] . b. bronchiseptica can also lead to permanent asymptomatic colonization of the respiratory tract [ , ] . in dogs, b. bronchiseptica causes tracheobronchitis with two patterns of histological lesions. one pattern consists of focal areas of epithelial degeneration, necrosis, and cellular disorganization with vacuolation and pyknosis represented by congestion in the lamina propria, infiltrated with macrophages and lymphocytes. the second pattern consists of mucopurulent exudates that accumulate in the lumen of the airway with edema in the lamina propria, marked infiltration of polymorphonuclear leukocytes and clumps of bacteria located between the cilia of the tracheobronchial epithelium [ ] . in piglets, b. bronchiseptica can cause upper respiratory illness, leading to nonprogressive atrophic rhinitis; histologically, other common lesions include hyperplasia of the epithelium with metaplasia and deciliated cells. in rabbits, b. bronchiseptica causes a suppurative bronchopneumonia with interstitial pneumonia; histologically, peribronchial lymphocytic cuffing has been described in [ , ] . in species other than piglets [ ] [ ] [ ] , detailed descriptions of the initial changes in the respiratory epithelium of the nasal cavity have not been reported. numerous b. bronchiseptica virulence factors have been implicated as being responsible for damaging the host cells. these factors include toxins such as adenylate cyclase toxin, dermonecrotic toxin, the type iii secretion system, and adhesins such as filamentous hemagglutinin, pertactin, and fimbriae [ , ] . most of the effects of these factors have been described from in vitro studies working with isolated cell cultures exposed to the respective virulence factor. despite the importance of b. bronchiseptica infection in rabbits [ , ] , as well as of the lipopolysaccharide (lps) of b. bronchiseptica as a virulence factor, no reports on the effects of the whole microorganism or its lps using a nasal septum culture have been documented in this species. recent work from our group with another respiratory pathogen of rabbits, pasteurella multocida, showed damage caused by the lps of this pathogen to the respiratory epithelium of rabbit fetuses using the same model presented in this study [ ] . in addition, the author found that the lps of p. multocida significantly increased the number of bacteria adhering to the epithelium when this molecule was applied minutes before or simultaneously with exposure to the microorganism. a similar effect has been described for the lps of other gram negative bacteria, such as salmonella enterica [ ] and helicobacter pylori [ ] . the goal of this work was to detail the changes caused by b. bronchiseptica in the respiratory epithelium of the nasal septum of rabbits during the first hours of infection. additionally, we sought to examine whether the lps of this pathogen by itself could cause lesions that would complicate the damages caused by the bacterium. to test this, a novel experimental approach more similar to natural conditions was used, namely, a tissue culture from the nasal septum of fetal rabbits. . . bacteria. b. bronchiseptica was isolated from rabbits having clinical symptoms of rhinitis or pneumonia; the animals originated from commercial farms located on the flat plain near bogotá, colombia ( , m.a.s.l.). samples were collected from nares or trachea, cultured on brain heart infusion agar (bhi), and were stored in % glycerol at − ∘ c until use. large-scale virulent b. bronchiseptica cultures were collected in bhi agar and its biomass was harvested. the bacterium was suspended in distilled water (dw) with . % thimerosal at ∘ c for inactivation and conservation. the cells were centrifuged at , g for min in sterile dw. westphal and jann's [ ] phenol-hot water method was used. inactivated bacteria were suspended in dw, treated with % phenol v/v for min at ∘ c, and stored at ∘ c for h. when phase separation (aqueous, phenolic interphase, and precipitate) was evident, the samples were centrifuged at , g for min at ∘ c [ ] . the aqueous phase was separated and treated with : volume of % ethanol that was then stored for h at − ∘ c and then centrifuged at , g for min to obtain crude lps. lps precipitate was suspended in physiological saline solution (pss), centrifuged at , g for min, and then dialyzed against sterile dw. the lps was quantified by the lee and tsai's colorimetric method [ ] . lps ( g in l pss) was intraperitoneally inoculated into five mice to evaluate its biological activity. an additional five mice were injected with sterile pss. the first five mice were euthanized when clinical signs appeared, and their tissues were processed by routine histopathological technique. hyperimmune antisera production. one mature sheep was used for hyperimmune antiserum production against b. bronchiseptica . next, days after the first inoculation of killed b. bronchiseptica with complete freund adjuvant was administered, one repetition with incomplete freund adjuvant and one repetition with only the bacterium, the animal was bled. the antiserum was immunoadsorbed with fetal rabbit turbinate macerates to eliminate cross-reaction with rabbit tissues, and they were washed with pss and diluted : in sterile buffer (tris saline, ph . ). one ml of antiserum was admixed with ml macerate, washed with pss, and centrifuged at g for h at room temperature. the supernatant contained the immunoadsorbed antiserum and was kept at - ∘ c until use. the antiserum titer was calculated by indirect immunodot at : - : dilutions. culture. twenty-six day pregnant does ( = , explants) were subjected to cesarean section under aseptic conditions; they had been previously anesthetized with mg/kg xylazine, mg/kg ketamine. fetuses were immediately euthanized by medullar sectioning. three sequential -mm-thick transversal sections were obtained from the nasal cavity. the sections were then freed of skin, bone and turbinate, retaining only the nasal septum. the septa were washed tree times with dulbecco's modified eagle medium (mem) high in glucose and were treated as shown in tables and . the septa were placed in veterinary medicine international changes to the respiratory epithelium exposed to whole b. bronchiseptica and to b. bronchiseptica lps were quantitatively and semiquantitatively evaluated. changes such as the number of vacuolated cells, number of dead cells, desquamated cells (for descriptive morphological purposes the following were accepted as cell death criteria: pyknosis and karyorrhexis, desquamated cells in the lumen with such nuclear changes and cell detritus in the lumen), and polymorphonuclear neutrophils (pmn) infiltrating the epithelium were quantified over epithelial cells with a x objective. a semiquantitative analysis of the activity of the goblet cells, detritus and mucus in the lumen was also performed. four fields were examined for each tissue section. the presence and localization of bacteria on the respiratory epithelium were evaluated using walker and mayer's [ ] iip technique, with some modifications. the immunoadsorbed anti-b. bronchiseptica polyclonal antiserum described in section . was used as a primary antibody; as a secondary conjugate, protein g (sigma biochemicals, st. louis, mo, usa) labeled with peroxidase was used. septa not exposed to bacteria and septa in which the primary antiserum was replaced with preimmune ovine sera were included as negative controls. septa of rabbits suffering from a natural disease and from which b. bronchiseptica had been isolated and diagnosed by iip and histopathology were included as positive controls. a final : dilution of the immunoadsorbed antiserum was used. to detect the lps of b. bronchiseptica in the respiratory epithelium of the nasal septum a lectin histochemical technique was implemented [ ] . briefly, nasal septa were incubated with a lectin-enzyme conjugate which consisted of lectin from limulus polyphemus (lpa) conjugated to the alkaline phosphatase enzyme (alkaline phosphatase conjugated limulus polyphemus lectin horseshoe crab, ey laboratories inc., usa), which binds specifically to the sugar -keto- -deoxyoctonate (kdo) in the core of lps [ ] . experimental error was controlled for as follows: random application of treatments, blind evaluation of tissue, independence with the durbin-watson test and normality with shapiro-wilk's test. a test for comparing means was used for the proposed hypotheses, which proved to be significant. mice intraperitoneally inoculated with lps showed signs compatible with endotoxemia after h, including depression, nasal and ocular secretion, bristled hair, and watery feces. macroscopically, the lungs were congested and had petechial hemorrhages in their serous membranes. histopathologically, microcirculatory changes in the lung consisting of edema, congestion, alveolar hemorrhages and infiltration of pmn in the alveolar septa and the alveolar space were observed. in the liver, mild perivascular mononuclear inflammatory infiltrate and moderate congestion were the main findings. respiratory epithelial cells of the nasal septa experimentally incubated with b. bronchiseptica for h or h showed no statistically significant changes; in consequence, for statistical purposes the results of both experimental time points were grouped. however, the changes in the epithelium of exposed septa were significantly different than those at , , and h that had not been exposed to the microorganism. respiratory epithelial cell death (figure (a) ) and pmn infiltration into the respiratory epithelium of the septa exposed to b. bronchiseptica were significantly different ( < . ) than negative controls. cellular degeneration, such as cytoplasmic vacuolation and reactive changes, such as increased goblet cell activity (defined by a larger cytoplasm of the gc, protrusion above the ciliated cells and the gc liberating their content into the lumen (figure (b) )), were also significantly different ( < . ) than the control groups (figure (c) ). cytoplasmic vacuolation of respiratory epithelial cells was present in some of the control tissues at hours but was minimal (figure (d) ). (e) fetal rabbit nasal septum respiratory epithelium exposed to b. bronchiseptica. positive staining of ciliated border is indicated (arrow). a x magnified iip is shown. (f) negative control fetal rabbit nasal septum respiratory epithelium not exposed to b. bronchiseptica. a x magnified iip is shown. of pmn into the epithelium, and increased goblet cell activity (figure (a) ). all of these lesions, including death of the epithelial cells, were statistically significant when compared to control tissues ( < . ) (figure (b) ). . . nasal septa exposed to b. bronchiseptica: iip. b. bronchiseptica was observed attaching to the ciliated border over the course of hours (figure (e) ). conversely, no specific staining was found in control tissues (figure (f) ). histochemistry. the specific reaction of b. bronchiseptica lps with lectin histochemistry was found mostly within goblet cell cytoplasm (figure (c) ); controls did not show a similar reaction. while descriptions of the respiratory nasal epithelial lesions caused by b. bronchiseptica in piglets are well documented [ ] [ ] [ ] , they are less so for dogs, cats, and rabbits. additionally, a detailed study of the cellular changes caused by some of the virulence factors of this pathogen in cell cultures has been reported [ ] [ ] [ ] . however, to the best of our knowledge, a thorough study of the cellular changes during the initial stages of the infection and the distribution of b. bronchiseptica or its lps in an isolated manner by using a model that more closely reflects the in vivo conditions (i.e., reconstructing the architecture and cell relationships of the respiratory epithelium in a natural host of this microorganism) has not been documented. in this work, we successfully developed a nasal septa tissue culture from fetal rabbits to study the first steps of infection with b. bronchiseptica and its lps. tissue culture models from human trachea, lung, nasopharynx, and nasal turbinates for studying the colonization, invasion, and pathogenic effects of other microorganisms, such as streptococcus pneumoniae, neisseria meningitides, and mycobacterium tuberculosis, have been previously developed [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . here, we cultured the nasal septum from fetal rabbits at -day of gestation for h without major morphological changes in the epithelial cells. importantly, we also examined the earliest changes during the interaction between b. bronchiseptica or its lps with that epithelium, as that is the time during which the first steps of adhesion and colonization take place. as early as hours postexposure to b. bronchiseptica or its lps, several degenerative, reactive, and deadly changes were present in the respiratory epithelial cells. these consisted of cytoplasmic vacuolation, activation of gc, infiltration of pmn and dead cells within the epithelial layer or desquamated into the lumen. vacuolation is considered a sign of cellular degeneration and cell death [ , ] . a similar finding has been described in vivo in rabbits experimentally infected with pasteurella multocida and in rabbits with natural respiratory disease [ , ] . nevertheless, no previous publications have documented this lesion within the respiratory epithelium of rabbits or other species during the first hours of infection induced by b. bronchiseptica or its lps, using similar experimental conditions as those employed in this study. goblet cell activity was observed after challenge with either b. bronchiseptica or its lps; the cells were enlarged due to an apparent accumulation and enlargement of their cytoplasmic content, and a higher number of gc were observed protruding above the normal apical limit of the respiratory epithelium or were found extruding their content, which led to mucus accumulation in the luminal septa. tesfaigzi et al. [ ] stated that lps induces an increase in the number of epithelial cells in the respiratory mucosa by increasing the quantity of gc (i.e., hyperplasia). this contention has been contradicted by other authors who have proposed that the synthesis and secretion of mucus are synchronic and do not alter the number or size of gc [ , ] . our study was not intended to determine whether there was gc hyperplasia, metaplasia, or hypertrophy; however, the enlargement of these cells observed in the epithelia exposed to b. bronchiseptica or its lps could not be explained without any increase in the size of their cytoplasm. this increase need not be due to a higher synthesis of mucin, but it is more likely due to a looser arrangement (due to hygroscopy) of the granula contents or the fusion of their membranes prior to expulsion. the mechanism by which the lps induces mucus production is not completely understood. however, it is known that lps increases mrna expression of muc ac, muc b, and il- and that it stimulates the secretion of both mucins (muc ac up to %, muc b up to %) and the inflammatory cytokine il [ ] . in vivo, increased expression of genes coding for mucin and inducing mucous metaplasia due to lps have been demonstrated [ ] ; the lps stimulates inflammatory mediator synthesis by the pmn, which in turn drives the expression of the aforementioned genes. given the short experimental time in our study, it would be quite difficult for a cell to perform some of these elaborated processes, during which the activation of several genes must take place. typical morphological changes of cell death in human tracheal cell line cultures induced by b. bronchiseptica have been described [ ] . studies using b. bronchisepticainfected macrophages and various epithelial cell lineages have also documented cytotoxicity in a nonapoptotic mechanism that is type iii secretory system-dependent. nogawa et al. [ ] and kuwae et al. [ ] reported that the mechanism by which b. bronchiseptica induces necrosis is mediated by the complex bopc and bopb-bopd proteins, with tyrosine phosphatase activity translocated through the secretory type iii system. the activity apparently lies in the ability of bopd and bopb to induce the formation of pores in the cytoplasmic membrane of eukaryotic cells through which other effector proteins are introduced into the cytoplasm. morphologically, the dying cells seem necrotic rather than apoptotic, showing cytoplasmic swelling and extensive membrane blebbing without nuclear changes [ ] . qualitatively, the changes induced by the b. bronchiseptica lps were similar to those caused by the bacterium. quantitatively, however, the two differed in that, with the exception of pmn infiltration into the epithelium, the lesions were less severe with the lps than with the bacterium. it has been previously indicated that other pathogen-associated molecular patterns (pamps) expressed by the bacterium, in addition to the lps, can induce genomic expression in host cells mediated by tlrs other than tlr . this is further supported by the observation that live b. bronchiseptica induces a small subset of genes that are not activated by the lps [ , ] . in this work, lps induced a higher infiltration of pmn in the respiratory epithelium and the propria of nasal septa than did the bacterium. multiple studies have demonstrated that the main inflammatory cells in tracheobronchial lavages of rats that were intranasally and intratracheally exposed to different doses ( to g) of pseudomonas aeruginosa lps were neutrophils, with macrophages, lymphocytes, and eosinophils being absent. such lps doses are enough to initiate neutrophilic inflammation in the lungs [ , ] . the dose used in our work was g/ml ( g final concentration); this dose closely resembles those reported by others [ ] [ ] [ ] be explained by selective activation induced by the lps of the pmn through its pamp recognition receptors, more specifically through tlr . mann et al. [ , , ] and kirimanjeswara [ ] documented tlr activation by b. bronchiseptica lps and they noted its importance for pmn recruitment and cytokine production. several proteins are required for the migration of a pmn from the venular lumen to its final destination in the epithelium, be it in the interstitial tissues or the respiratory track; all of these proteins were present and most likely expressed at high levels in the experimental tissue cultures employed in this study. however, the question of where the increased numbers of pmn observed infiltrating the epithelium of the septum came from still remains. this pmn infiltration cannot be easily explained in this study as the nasal septa were completely separated from the vascular bed of the fetuses. one hypothesis could be that these cells are in an inactive state within the remaining blood vessels or in the interstitial matrix of the nasal septum and that they are not recognizable until activated by the bacteria or their products. additional studies are required to address this issue. however, the importance of pmn in lps-mediated processes cannot be ignored. considering the similarity of the lesions caused by b. bronchiseptica and its lps and the fact that the differences were only in quantitative terms, it is tempting to hypothesize that the lesions caused by the bacterium could simply be the result of the lps still attached to its outer membrane. this hypothesis should be addressed in future studies. b. bronchiseptica and its lps distribution on the nasal septum respiratory epithelium were evaluated by the indirect immunoperoxidase and lectin histochemistry techniques. b. bronchiseptica immunostaining indicated that bacteria were distributed mainly on the ciliated border of the epithelium and less frequently in detritus and mucus accumulated in the lumen. in both cases, the staining was granular in nature. on the other hand, b. bronchiseptica lps was visualized mainly within gc cytoplasm. these results suggest that b. bronchiseptica, as well as its lps, adhered to receptors containing glycoconjugates, be it in the glycocalyx of the interciliary space or in those produced by the gc. filamentous hemagglutinin (fha) of b. bronchiseptica is necessary and sufficient to mediate in vitro rat lung epithelial cell adherence [ ] . the fha of b. pertussis possesses a carbohydrate recognition domain (crd) that mediates its attachment to the ciliated respiratory epithelial cells and macrophages in vitro. a lectinlike activity for heparin and other sulfated carbohydrates has also been identified in b. bronchiseptica and b. pertussis, which can mediate adherence to nonciliated epithelial cell lines [ ] [ ] [ ] [ ] . regarding the lps, it is possible that a short duration of time is needed for it to bind to its receptors, as our experiments found lps within gc cytoplasm in the early stages of exposure. these findings could be explained by alexander and rietschel's [ ] results, which indicated that lbp and cd mediated rapid cellular internalization of high molecular weight lps aggregates. furthermore, huber et al. [ ] proposed that the lps could bind soluble cd receptors, which would permit cd -negative cells to respond to the lps. in this research, we successfully established a nasal septum culture from rabbit fetuses for studying the first steps of the relationship between b. bronchiseptica or its lps with the respiratory epithelium. models such as these, which use a complete tissue culture instead of isolated cells, more closely reflect the natural processes of an infection and allow for an improved understanding of the initial phases of the pathogen-host interaction and further examine new preventive avenues. we also demonstrated that b. bronchiseptica and its lps induce very similar lesions on the septal respiratory epithelium after h of exposure. the lesions in the epithelial cells were mainly degenerative, early reactive, and deadly in nature and the changes induced by the whole bacterium were even more severe. the labeling of the bacterium and its gc cytoplasm mucosubstance-associated lps suggests that b. bronchiseptica and its lps have an affinity for interciliar and gc glycoproteins, representing their respective initial adhesion locations. bacterial and parasitic diseases of rabbits the pathogenesis of turbinate atrophy in pigs caused by bordetella bronchiseptica pasteurella multocida and bordetella bronchiseptica infections in rabbits role of bordetella bronchiseptica in infectious tracheobronchitis in dogs rabbit respiratory system: clinical anatomy, physiology and disease adherence of bordetella bronchiseptica to porcine trachea maintained in organ culture adherence of pasteurella multocida or bordetella bronchiseptica to the swine nasal epithelial cell in vitro bordetella bronchiseptica persists in the nasal cavities of mice and triggers early delivery of dendritic cells in the lymph nodes draining the lower and upper respiratory tract prior infection with bordetella bronchiseptica increases nasal colonization by haemophilus parasuis in swine an improved 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within human macrophages adhesion of bacteria to mucosal surfaces combining sites of bacterial fimbriae r-form lps, the master key to the activation of tlr /md- -positive cells key: cord- -h ordzm authors: felts, paul a.; woolston, anne-marie; fernando, himali b.; asquith, stephen; gregson, norman a.; mizzi, oliver j.; smith, kenneth j. title: inflammation and primary demyelination induced by the intraspinal injection of lipopolysaccharide date: - - journal: brain doi: . /brain/awh sha: doc_id: cord_uid: h ordzm inflammation is a prominent feature of several disorders characterized by primary demyelination, but it is not clear whether a relationship exists between inflammation and myelin damage. we have found that substantial demyelination results from the focal inflammatory lesion caused by the injection of lipopolysaccharide (lps; ng) directly into the rat dorsal funiculus. within h, such injections caused a focal inflammatory response consisting of a substantial number of polymorphonuclear cells and ed -positive and inducible nitric oxide synthase (inos)-positive macrophages/microglia. the number of inflammatory cells was substantially reduced by day . ox- -positive t-cells were less frequently observed but were present in the meninges at h, reached a maximum in the dorsal funiculus at days, and were rare at days. the inflammation was followed by the appearance of a large lesion of primary demyelination that encompassed up to ∼ % of the cross-sectional area of the dorsal funiculus. treatment with dexamethasone significantly reduced the number of cells expressing inos, but did not prevent the demyelination. by days the lesions were largely remyelinated, usually by schwann cells. these changes were not observed in control, saline-injected animals. we conclude that the intraspinal injection of lps results in inflammation and subsequently in prominent demyelination. the mechanisms underlying the demyelination are not clear, but it is notable that it typically begins with disruption of the adaxonal myelin. indeed, there is an early loss of myelin-associated glycoprotein within the lesion, despite the persistence of proteolipid protein. this combination is a feature of the pattern iii lesion recently described in multiple sclerosis (lucchinetti et al., ), and we therefore suggest that lps-induced demyelination may serve as the first experimental model available for the study of this type of multiple sclerosis lesion. inflammation within the cns is a common feature of neurological disorders and infection, but whether it contributes directly to neuronal or glial damage in vivo can be difficult to determine. for example, in a disease such as multiple sclerosis, and its animal model experimental autoimmune encephalomyelitis (eae), immune-mediated inflammation can be prominent but it is difficult to disentangle the indirect consequences of the inflammation from other ongoing immune-mediated disease processes. experimentally, inflammation can be induced in most tissues by the local injection of lipopolysaccharide (lps), and typically large numbers of neutrophils and monocytes invade the tissue from the bloodstream within minutes, macrophages remaining at the site for a number of days (issekutz et al., ; cybulsky et al., ) . however, lps injection into the parenchyma of the adult brain results in an altered and attenuated cellular response. thus, a quantity of lps which elicits a typical inflammatory response in the skin ( ng) does not result # the author ( ) . published by oxford university press on behalf of the guarantors of brain. all rights reserved. for permissions, please email: journals.permissions@oupjournals.org in the recruitment of either neutrophils or monocytes when examined - days after injection into the hippocampus (andersson et al., a) . cellular influx is attenuated despite the fact that - ng of lps is effective in rapidly upregulating the expression of endothelial intercellular adhesion molecule- (icam- ) and vascular cell-adhesion molecule (vcam) (bell and perry, ) , and of proinflammatory cytokines such as interleukin b (il- b) and tumour necrosis factor a (tnf-a; stern et al., ) . increasing the quantity of lps injected to - ng, which results in a florid extravasation of neutrophils in the skin, still does not result in significant neutrophil recruitment in the brain parenchyma, although it does cause activation of microglia and recruitment of monocytes (andersson et al., a; montero-menei et al., ) . studies of the consequences of lps-induced inflammation in the brain have so far tended to focus on relatively acute effects, and they have not used high-resolution histological techniques to assess changes in tissue architecture. moreover, studies have so far neglected the injection of lps into the spinal cord despite the observation that the inflammatory response can differ between the brain and the spinal cord following traumatic lesions (schnell et al., ) . we have therefore examined in detail the short-and long-term consequences of injecting lps into spinal white matter. our study used light and electron microscopy and immunohistochemistry to examine tissue up to days after injection in order to determine the time course of cellular recruitment, and changes in local tissue structure. we report that, following the recruitment of neutrophils and macrophages, a large primary demyelinating lesion formed in the dorsal funiculus after a delay of up to week following injection. it is possible that the inflammation-associated demyelination observed in this study may illuminate the mechanisms underlying the inflammation-associated demyelination in multiple sclerosis, particularly in pattern iii lesions. using sterile technique and under deep halothane anaesthesia, a quarter laminectomy was performed at the t vertebral level in adult male sprague-dawley rats ( g, mean sd). two small holes, mm apart, were made in the dura over the left dorsal column and a drawn glass micropipette (typical external tip diameter mm) was inserted into the dorsal column and . ml of lps ( ng/ml in saline) was injected at each of the two sites at depths of . and . mm ( ml in total). the injection sites were marked by placing a small amount of sterile charcoal on the adjacent dura. control animals received injections of saline alone, and the lesion site was marked in the same way. lps from salmonella abortus equi, s. typhimurium or escherichia coli (serotype :b ) (sigma, gillingham, dorset, uk; catalogue numbers l- , l- and l- respectively) was used without further purification. the endotoxin activity of the commercially obtained lps was estimated by a semiquantitative dilution method using the limulus amoebocyte lysate assay (e-toxate kit; sigma). in one set of experiments, highly purified lps from s. abortus equi (alexis biochemicals, nottingham, uk; catalogue number alx- - ) was used to determine if the lesions were the result of injecting contaminants known to be present in many less highly purified commercial preparations of lps. animals were perfused via the left ventricle under deep halothane anaesthesia at the following times. lps-injected animals were perfused at h (n = ), day (n = ), days (n = ), days (n = ), days (n = ), days (n = with unpurified lps and n = with purified lps), days (n = ) and days (n = ). saline-injected animals were perfused at h (n = ), h (n = ), days (n = ), days (n = ) and days (n = ). the vasculature was rinsed with approximately ml of normal saline containing u/l heparin, . % lignocaine, . % nano and . m n- -hydroxyethylpiperazine-n - -ethanesulphonic acid (hepes; ph . ) followed by approximately ml of % glutaraldehyde in . m phosphate buffer (ph . ; pressure head cm). the tissue was cut transversely into . mm thick blocks, which were postfixed in . % osmium tetroxide in . m phosphate buffer, dehydrated in graded alcohols, passed through propylene oxide and embedded in taab resin (taab laboratories, aldermaston, uk). for light microscopy, . mm thick sections were cut on an ultramicrotome, stained with toluidine blue and examined and photographed using a zeiss axiophot microscope equipped with a jvc tk-f frame capture camera. for electron microscopy, nm thick sections were stained with uranyl acetate and lead citrate and examined in a hitachi h transmission electron microscope. animals whose tissues were to be examined by immunohistochemistry were perfused at postinjection times of h, , , , and days (at each time point, n ! for lps-injected animals and n ! for saline-injected animals). six additional animals (two lps-injected and one saline-injected at both and h after injection) were perfused specifically to examine the expression of the inducible form of nitric oxide synthase (inos) and icam- in short-term lesions. in addition, one naive animal was also perfused to obtain normal tissue. the method was as above, except that the fixative consisted of freshly prepared % paraformaldehyde in . m phosphate buffer. following perfusion, the spinal cords were either cryoprotected by immersion in % sucrose for h, embedded in oct compound (bdh, poole, uk), frozen, and stored until use at À c, or processed into polyester wax using standard techniques (kent, ) . frozen sections ( mm) were cut using a cryostat and floated in phosphate-buffered saline (pbs) for immunohistochemical labelling. polyester wax sections ( mm) were cut on a microtome equipped with a peltier-cooled chuck and floated on % gelatine solution and attached to slides. apart from anatomical landmarks and the dural scar, the presence of charcoal on the surface of the spinal cord confirmed the injection site. immunohistochemical labelling was used to identify cell types and to examine the expression of adhesion molecules, inflammatory factors and myelin proteins. both peroxidase and fluorescent reporters were used. in experiments using peroxidase, sections were incubated in pbs containing . % azide and . % h o for min, followed by non-immune rat serum ( %; sigma) for min. tissues were then incubated in primary antibody (dilution : except where noted, and including pbs, % bovine serum albumin, % rat serum and . % triton-x ) overnight at c, followed by thorough washing and incubation at room temperature for h in horseradish peroxidase-conjugated rat anti-mouse (f(ab') fragment) secondary antibody (dilution : ; jackson immunoresearch laboratories, west grove, pa). following thorough washing, immunolabelling was visualized with , -diaminobenzidine (dab substrate kit; vector laboratories, peterborough, uk) and the sections were mounted on slides, dehydrated in graded ethanols, cleared in xylene and mounted in styrolite (bdh, poole, uk). in experiments using fluorescent labelling, sections were incubated with primary antibody solution (including % normal goat serum) overnight at c. after thorough washing, sections were incubated h at room temperature with appropriate goat anti-rabbit or goat antimouse (f(ab ) fragment) secondary antibodies conjugated to fluorescein or rhodamine (diluted : ; chemicon, harrow, uk). inflammatory cells were identified using the monoclonal primary antibodies mrc ox- , ed and rln. d (all from serotec, kidlington, uk; : dilution), which recognize t lymphocytes, macrophages/activated microglia, and b lymphocytes respectively. oligodendrocytes were labelled with a monoclonal antibody (clone cc- ) raised against a fragment of the adenomatous polyposis coli (adpc) protein (bhat et al., ) (polyester wax sections; oncogene research products, san diego, ca, usa; dilution : ). monoclonal antibodies were also used to demonstrate the presence of inos (dilution : ; clone ; affiniti research product, exeter, uk), icam- (dilution : ; clone h : a gift from prof. david male, open university, uk) and il- b (serotec; clone silk ). a polyclonal antibody recognizing il- b (serotec) was used in some doublelabelling experiments. the cell types expressing il- b were investigated using fluorescence double-labelling and monoclonal primary antibodies against macrophages (ed ), resting microglia (ox- ; serotec) and astrocytes [glial fibrillary acidic protein (gfap); sigma, : ]. expression of the myelin components proteolipid protein (plp) and myelin-associated glycoprotein (mag) within the lps lesion was examined in polyester wax sections double-labelled with a rabbit polyclonal anti-mag antibody [raised against a peptide located at the carboxy terminal of l-mag (butt et al., ) ; dilution : ; rhodamine conjugated secondary as above] and a monoclonal anti-plp (serotec; dilution : ; fluorescein conjugated secondary antibody as above). cells labelling with ox- , ed or rln. d were counted using a · objective in transverse sections of spinal cord at or near the centre of the lesion. a single value for each animal was derived for several regions of the spinal cord (e.g. dorsal funiculus, lateral columns; table ) by averaging the counts from three non-adjacent transverse sections. polymorphonuclear cells (pmns) were identified in resin sections by their multilobular nuclear morphology. for all cell types examined, only extravasated cells were included; thus, labelled cells within the vascular lumen were excluded. fluorescent adpc-positive cells were counted in the dorsal funiculus of animals injected with saline (n = ) or lps (n = ) days after injection. at this interval the lesion was clearly discernible using differential interference contrast optics as a region of tissue disruption in the dorsal funiculus, allowing adpc-positive cells to be counted in both demyelinated and apparently normal areas of the dorsal funiculus of lps-injected animals. adpcpositive cells were counted in montages of digital images captured using a · objective. in order to examine the effect of a broad-spectrum anti-inflammatory agent on lps-induced demyelination, daily intraperitoneal injections of either dexamethasone ( . mg/kg, n = ) or saline (n = ) were given to animals beginning days prior to the injection of lps into the dorsal funiculus. to determine the extent of suppression of the inflammatory response, three animals from each treatment group were reanaesthetized and perfused with % paraformaldehyde in . m phosphate buffer a day after lps injection. the spinal cords at the injection site were frozen and examined for inos expression by immunohistochemistry as above, using a polyclonal rabbit ant-inos primary antibody (transduction laboratories, oxford, uk; dilution : ). sections were subsequently photographed using a digital camera (spot rt) and labelled cells within the dorsal funiculus were counted. to determine the consequences of dexamethasone treatment on the extent of demyelination, the remaining four animals from each treatment group were perfused days after lps injection with % glutaraldehyde in . m phosphate buffer and the tissues were processed for light and electron microscopy as above. the cross-sectional area of the lesion within the dorsal funiculus was measured from digital photographs using an image analysis program (sigma scan). as lps has both lipid and carbohydrate moieties, it is likely to exhibit some detergent-like properties that could solubilize cell membranes and lead to demyelination. to assess whether this property might confuse the findings, the ability of lps directly to attack cell membranes was assessed in an erythrocyte lysis assay using the detergents sodium dodecyl sulphate (sds) and lysophosphatidylcholine for comparison. erythrocytes, suspended in isotonic saline, were exposed to lps or detergent for min at room temperature; the suspension was centrifuged and haemoglobin release assessed by measuring absorbance at and nm. salmonella typhimurium lps stock solution was diluted to mg/ml in carbonate buffer and ml was added to the wells of immulon Ò b elisa plates and left overnight at c. after washing once, they were blocked with ml of % fish gelatine (sigma) in pbs for h at c. rat sera were collected at (n = ), (n = ), (n = ), (n = ) and (n = ) days after lps injection. sera were diluted in % bovine serum albumin (bsa)-pbs, from : to : , and ml was added to the wells overnight at c. second antibody, alkaline phosphatase-conjugated anti-rat (sigma) at : , was added for h at c and substrate development was for h at c before optical density was read at nm. linbro enzyme immunoassay (eia) plates were coated with ml of rat cns myelin, mg protein per ml in . % methylglyoxal, ph . , for h at c. the plates were carefully washed with pbs- . % tween three times and then blocked with ml of % fish gelatine-pbs for h at c. rat sera, collected at (n = ), (n = ) and (n = ) days after lps injection, were diluted from : to : in % bsa-pbs, ml was added to the wells, and the wells were incubated overnight at c. the plates were washed carefully three times with pbs-tween before adding alkaline phosphatase conjugated anti-rat immunoglobulin g ( : ) for h at c. after washing three times, the plates were developed with pnitrophenol and the optical density was read at nm. prior exposure to pathogens could influence the reaction of animals to lps injection, and so in one group of experiments we monitored animals for a number of infectious agents. surveillance animals, housed in the same room as the experimental animals, were returned to the supplier (harlan uk, loughborough, uk) for routine pathogen screening. injection of lps resulted in an early invasion of inflammatory cells into the parenchyma of the spinal cord. the invasion was not limited to the dorsal funiculus and was not initially associated with apparent damage to the nerve fibres of the injected dorsal funiculus. however by - days after lps injection a large, focal demyelinating lesion had formed within the dorsal funiculus. repair by remyelination commenced by days, and by days large numbers of remyelinated axons were present; most of the remyelination was by schwann cells. assay of the injected lps solutions using the semiquantitative limulus amoebocyte lysate method yielded the following endotoxin activities: s. abortus equi - , endotoxin units/ml, s. typhimurium - endotoxin units/ml and e.coli - , endotoxin units/ml. lps derived from each of the three bacteria was found to produce similar lesions and will therefore be considered together. spinal cords injected with saline ( fig. ) showed damage restricted to a very small number of axons undergoing either table populations of inflammatory cells present in the spinal cord at various times following the injection of lps or saline into the dorsal funiculus wallerian degeneration or demyelination. such damage was present at time periods greater than days after injection and occurred at the pial surface in the region of the insertion of the injection pipette, or along the line of the injection pipette deep within the dorsal funiculus. remyelinated axons were not observed in the saline-injected animals. eight hours after lps injection. a number of inflammatory cells were present within the spinal cord (see below, cell populations within the spinal cord), but the cord otherwise appeared grossly normal and the myelin sheaths in the dorsal funiculus appeared intact. one day after lps injection. by day after the lps injection into the left dorsal column there were signs of an intense localized response, with tissue damage in the grey matter adjacent to the deep portions of the left dorsal column. in the tissue immediately surrounding the injection site within the dorsal column the appearance was grossly normal ( fig. a) . examination in the electron microscope revealed a mild increase in extracellular space within the white matter which was not, however, very widespread. occasional demyelinated axons were seen but this may have been associated with needle trauma. infrequent debris-containing monocytic cells were present. in contrast, pmns were numerous, both close to blood vessels and within the parenchyma of the dorsal column, and particularly in the grey matter adjacent to the deep portions of the left dorsal column. three days after lps injection. under the light microscope the spinal cord had a similar appearance to that observed day after injection, including obvious cellular infiltration (fig. b) . although white matter damage was not prominent, very occasional demyelinated axons (< per section) were observed. large numbers of inflammatory cells were evident in the grey matter adjacent to the deep portion of the left dorsal column, with cuffs consisting of multiple layers of mononuclear and polymorphonuclear cells surrounding some vessels in this region (fig. c ). electron microscopy revealed obvious oedema (fig. a) , which extended into the otherwise normal adjacent white matter. the expanded extracellular space contained many small process profiles as well as fluid-filled spaces that appeared to arise from damaged axons. electron microscopy confirmed the presence of infrequent demyelinated axons (fig. a ) as well as demonstrating many myelinated fibres in which the internal mesaxon was obvious. the endothelium in small vessels showed a low density of microvilli but did not show obvious signs of activation (i.e. hypertrophy). phagocytes containing myelin and possibly axonal debris were found throughout the lesion. five days after lps injection. light microscopy indicated that cellular infiltration was reduced, while oedema was more obvious (fig. d ). this expanded extracellular space was confirmed under the electron microscope and was found to contain floccular material, presumably precipitated protein. (fig. b ). debris-containing macrophages were prominent, particularly in perivascular spaces, which were also enlarged, with some withdrawal of astrocyte processes. all of these changes were indicative of a breakdown of the blood-brain barrier. small areas of non-myelinated axons, usually less than mm in calibre, were seen, but whether these were the result of demyelination was difficult to determine. seven days after lps injection. large lesions were present within the dorsal funiculus ( fig. a and b), consisting of demyelinating axons, and most commonly occupying the ventral half or ventral two-thirds of both right and left dorsal funiculus, including, but not restricted to, the corticospinal tract. characteristically, the breakdown of the myelin sheath appeared to progress from the adaxonal region outwards, or from the cytoplasmic regions of schmidt-lanterman incisures or paranodal regions. the myelin breakdown appeared in transverse section to progress from a small vesicular appearance to a larger disorganized mesh of membrane ( fig. c-e) . in more oblique sections the small vesicles were seen as tubes, an appearance reminiscent of that seen in the early stages of lysophosphatidylcholine-induced demyelination (hall and gregson, ) . the demyelination in most instances appeared to be taking place without the close apposition of a myelomonocytic cell, and apoptotic cells were seen; however, because of the dissolution of nuclear and cytoplasmic components it was difficult to identify such cells on morphological criteria. many phagocytic cells containing myelin debris were seen, but the extracellular space also contained considerable amounts of membranous myelin debris. despite the prominence of the demyelination, axon loss, as indicated by the number of fibres undergoing wallerian degeneration, was small, in both the sensory, ascending, portion of the dorsal column mm rostral to the lesion (fig. a ) and in the descending corticospinal tract mm caudal to the lesion (fig. b) . fourteen days after lps injection. light microscopy revealed large, mixed lesions containing demyelinated axons, debris-filled macrophages, and axons associated with cells ( fig. c and d) . these axon-cell associations were most often on a one-to-one basis, suggesting that the cells were schwann cells. an enlarged extracellular space remained, and under the electron microscope this was found to contain membrane debris. examination in the electron microscope also provided convincing evidence of the presence of naked axons, other cell processes (most often not identifiable) and many debris-filled phagocytes. demyelinated axons encircled by cell processes were common and the cell body of the ensheathing cell was often present in the plane of section. in many cases these cells appeared to be initiating remyelination. twenty-eight days after lps injection. lesion repair and remyelination were well advanced ( fig. e and f) and oedema was reduced. there were many fewer debriscontaining phagocytes. remyelination was accomplished by a mixture of schwann cells and oligodendrocytes, typically with schwann cell remyelination predominant in the core of the lesion. small amounts of myelin membrane debris were present in the extracellular compartment, and scattered cystic vacuoles, which appeared to be derived from dead axons, were present. in larger lesions the area of the dorsal funiculus appeared enlarged (fig. e ) and the density of myelinated fibres appeared lower than normal (fig. f) . inflammatory cells are present, particularly surrounding blood vessels in the grey matter immediately adjacent to the dorsal funiculus. one such blood vessel, outlined by the box in b, is shown at higher magnification in c. both mononuclear cells and pmns can be seen in the wall of this vessel. little or no demyelination, is present at days (d), and this can be seen more clearly in the inset, which shows the region within the box at higher magnification. scale bar (shown in d) = mm in a, b and d (main image), mm in c, mm in inset. no more than one pmn was present in any of the various regions within the spinal cord examined h after saline injection, the interval producing the greatest number of cells in the animals injected with lps (table ). the meninges surrounding the spinal cord had pmns. at days, the interval producing the greatest number of ox- -positive cells in the lps-injected spinal cord, only one labelled cell was observed in the saline-injected cord. an additional two cells were present in the meninges. also at this time, small numbers of ed -positive cells were present in the salineinjected spinal cord; however, most were found either in the dorsal funiculus or meninges. the ed -positive cells in the dorsal funiculus were found extending in a line into the left dorsal column from the pial surface, and were presumed to result from minor damage associated with the injection track. most of the ed -positive cells found in the meninges were associated with the few particles of charcoal used to mark the injection site. following lps injection, invasion of the spinal cord by inflammatory cells was apparent by h, the earliest time point examined. over the following days the overall number of inflammatory cells present, and the cell types comprising this population, progressed through a characteristic pattern, as described in the following paragraphs. although for the large majority of axons, myelin is not grossly disrupted at this time, very small numbers of demyelinated axons (arrows in inset) are present. the lack of myelin debris in the extracellular space surrounding these demyelinated axons suggests that the demyelination occurred soon after the injection. the morphology was similar days after lps injection (b), with the myelin sheaths of most fibres grossly normal but with small regions of cleanly demyelinated axons also present (e.g. arrows in inset). however, days after injection (c, d and e), sheaths exhibiting enlarged adaxonal compartments containing various amounts of lamellar debris were common. scale bar (shown in e) = mm in a (inset = mm), mm in b (inset = mm), mm in c and d, and . mm in e. fig. light micrographs of dorsal funiculi days (a and b), days (c and d) and days (e and f) after the injection of lps. for each pair of micrographs, the image on the left shows the dorsal funiculus and adjacent grey matter at low magnification and the image on the right shows a region of the lesion at high magnification. in the low-magnification micrographs (a, c and e) the margins of the dorsal funiculus are outlined (arrowheads). at days after injection, most of the deep dorsal funiculus, including all of the corticospinal tract, is undergoing demyelination. b shows a number of axons with disintegrating myelin (arrows), with the lamellae separating either at the adaxonal surface or in midsheath. several debris-filled macrophages (e.g. arrowheads in b) are present. at days after injection a similar region of lesion is present (c); however, in d it is apparent that the lesion now consists mainly of either apparently naked demyelinated axons (e.g. arrows) or demyelinated axons in the early stages of association with cells (e.g. arrowheads). a number of debris-filled phagocytes are obvious. at days after injection a very large lesion is present, occupying nearly all of the dorsal funiculus. at this time, axons with the signet ring appearance characteristic of schwann cell remyelination predominate (arrows in f). scale bar (shown in f) = mm in a, c and e, and mm in b, d and f. pmns. eight hours after injection, pmns were far more numerous than either ed -positive macrophages or ox- positive t lymphocytes (table ) . given the differences in the thickness of sections used to count pmns versus those used for macrophages and t lymphocytes, direct comparisons cannot be made. however, it is worth noting that on average at h there were more pmns observed in . mm thick resin sections than there were ed -positive macrophages observed in mm thick cryostat sections. at h the greatest number of pmns was present in the dorsal funiculus and meninges, although substantial numbers were found in most regions, in particular in the grey matter. by h the number of pmns had fallen by approximately three-quarters, with most cells concentrated in the dorsal funiculus and the grey matter. very few pmns were observed days after injection, or later. macrophages/ microglia. ed -positive cells (table , fig. a ) were present in the spinal cord at h after lps injection, and there was an approximately -fold increase in these cells between and h. at h the dorsal funiculus accounted for more than % of the total ed -positive cells. a substantial increase in the number of ed -positive cells present in the dorsal funiculus and grey matter occurred between and h. by days the total number of ed -positive cells had fallen, and this decline continued between and days. at , and days the number of ed -positive cells was relatively stable, and these cells were concentrated within the dorsal funiculus. t lymphocytes. the total number of ox- -positive t lymphocytes within the sections was always fewer than the ed -positive cells, and exhibited two peaks (table ). the first, smaller, peak occurred h after injection; all of the cells were associated with the meninges and not in the neural parenchyma. indeed, prior to day no t lymphocytes were observed within the dorsal funiculus. the second peak in cell numbers occurred days after injection (fig. b) and was mostly the result of cells within the dorsal funiculus. by days the number of t lymphocytes had fallen to near zero and this remained the case at days. b lymphocytes. rln. d -positive cells were not observed in the spinal cord at any time after lps injection. oligodendrocytes. significantly fewer adpc-positive cells were present within the lesioned region of the dorsal funiculus days after lps injection ( . . cells/mm , mean sd) than in either the adjacent, unlesioned region of the dorsal funiculus from the same animals ( . . cells/mm ; one way anova with student-newman-keuls post-test, p = . ) or the dorsal funiculus of saline-injected control animals ( . . cells/mm ; p = . ). astrocytes. gfap-positive stellate, process-bearing cells and cell processes were present in the lps-injected dorsal funiculus h and , and days after injection. an increase in labelling intensity and process thickness was present days after injection, as was an increase in the gfap immunoreactivity within the grey matter directly adjacent to the lps-injected dorsal funiculus (fig. ) . this increased labelling in the adjacent grey matter persisted at days, and at this time nearly the entire lesioned area was a fine meshwork of moderately gfap-immunoreactive processes, with occasional thicker, more intensely labelled processes present. the entire lesioned area remained moderately gfappositive days after lps injection, at this time exhibiting a cobbled appearance. the occasional intensely labelled processes present appeared to be confined to the periphery of the lesion. this lesion remyelinates predominately by schwann cells, which, prior to myelination, can express gfap (jessen et al., ) . thus, it seems likely that the moderate labelling present across the lesion at and days represents labelling of gfap within schwann cells prior to or immediately after myelination. any disappearance of astrocytes from this schwann cell territory would be predicted from previous studies of regions of schwann cell remyelination in the cns (blakemore, ; felts and smith, ) . small numbers of inos-immunolabelled cells were observed within the dorsal funiculus and adjacent grey matter as early as h after lps injection. however, the most prominent inos immunolabelling was observed h after lps injection and thereafter it declined until only small numbers of inospositive cells were observed at days, and none were present days after injection. both naive spinal cord and spinal cord taken . cm rostral to the site of lps injection exhibited light icam- labelling in some larger blood vessels (fig. d) . lps injection markedly increased such labelling, and by h after lps injection labelling had increased in both the number of labelled vessels and the apparent intensity of labelling (fig. c) . increased labelling was also observed , and days after lps injection. in addition to labelled blood vessels, we also observed two populations of icam- -positive cells in the lps-injected dorsal funiculus. one was a group of rounded cells that were most often closely associated with a blood vessel, and the (table ) . up-regulation of icam- on blood vessels is evident at the site of lps injection within the dorsal funiculus h after injection (c), compared with the very light labelling present in the dorsal funiculus of the same animal . cm rostral to the site of injection (d). the intensity of blood vessel labelling in naive spinal cord is similar to that shown in d (data not shown). scale bar (shown in d) = mm. second was a number of cells with ramified processes scattered throughout the parenchyma of the dorsal funiculus. both populations were present in relatively small numbers; the former was observed up to days after injection, and the second up to days after injection, when only a few labelled cells were present. since a variety of cells likely to be present in the lps lesion have been reported to up-regulate expression of icam- in response to inflammatory mediators, including microglia (zielasek et al., ) , macrophages (goebeler et al., ) , astrocytes and oligodendrocytes (satoh et al., ) , it is likely that several cell types contributed to the observed icam- labelling. substantial numbers of il- b-positive cells were present across the whole cross-section of the spinal cord h after lps injection (fig. ) . compared with il- b expression h after injection (examined in a parallel study, data not shown), where positive cells were concentrated in or adjacent to the dorsal funiculus, labelled cells appeared both more numerous and more widespread at h. in addition, the morphology of the il- b-positive cells differed somewhat at and h, with both ramified, ox- -positive cells and rounded, ed -positive cells common at h, but with ramified, ox- -positive cells predominating at h. two populations of il- b-positive cells were also present h after lps injection. rounded il- b-positive cells were located throughout the dorsal funiculus and adjacent grey matter, whereas il- bpositive cells with short, stubby processes were found at a distance from the dorsal funiculus. at h, fewer il- bpositive cells were present compared with h, and most of these exhibited a round morphology. by week only a very small number of il- b-positive cells were present, and these were ox- -negative but ed -positive. the cells were often adjacent to large blood vessels, and they formed only a small subset of the substantial population of ed -positive fig. light micrographs showing gfap immunoreactivity at the interface between the dorsal funiculus (df) and the grey matter (gm) in an animal days after the injection of lps into the spinal cord. a transverse section cm rostral to the injection site is shown in a, and a similar region at the site of lps injection is shown in b. note the fine labelling of astrocyte processes present at the site distant from the lesion in both the dorsal funiculus (arrows in a) and grey matter (arrowheads in a). labelling of astrocytes is increased at the injection site, with thickened astrocyte processes in both the dorsal funiculus (arrows in b) and the grey matter (arrowheads in b), and an overall increase in labelling density in the grey matter. scale bar = mm. plp and mag immunoreactivity was examined in doublelabelled sections of lps lesions induced , and days earlier. three days after injection there was no obvious loss of either of these myelin proteins within the injected dorsal funiculus, compared with other white-matter regions in the same sections. however, by days after injection, immunoreactivity for mag, but not plp, was reduced in the deep dorsal funiculus, the most typical site for development of the lps-induced demyelinating lesion ( fig. a and b). this differential loss of mag at the site of lps injection was even more pronounced days after injection ( fig. c and d) ; at this time only very faint mag labelling was observed within the deep dorsal funiculus (fig. h ) compared with the immunoreactivity present in the superficial dorsal columns (fig. f) . plp immunoreactivity was not decreased in the lesion (compare figs. e and g) , and indeed plp labelling appeared slightly more intense in this region, possibly due to increased antigen availability produced by myelin disruption. treatment with dexamethasone was effective in reducing the magnitude of the inflammatory response ( fig. a and b) , as indicated by a significant reduction in the average number of cells expressing inos in transverse sections of the dorsal funiculus at the site of lps injection [ (mean sd) in dexamethasone-treated animals day after lps injection versus cells in saline-treated animals; p = . , student's t-test]. however, dexamethasone treatment did not reduce the extent of lps-induced demyelination in the spinal cord ( fig. c-f) . when examined days after lps injection, the average cross-sectional area of demyelination in the dorsal funiculus of animals treated with dexamethasone ( . . mm ) was actually greater than that in animals treated with saline ( . . mm ), although this difference was not statistically significant (p > . , student's t-test). at days after lps injection, the axons in lesions from dexamethasonetreated animals tended to be surrounded by whorls of myelin debris, whereas naked demyelinated axons, or demyelinated axons associated with cell processes, were particularly common in lps lesions from saline-treated animals. in the dexamethasone-treated animals, the seemingly increased size of the lesions might have been due to expansion of their areas due to the physical bulk of increased amounts of myelin debris. lesions were examined days after the injection of a highly purified lps preparation into the dorsal funiculus. in the three animals examined, this lps preparation produced lesions (fig. ) that were similar to those seen with the unpurified lps preparation, causing lesions occupying , and % of the cross-sectional area of the dorsal funiculus. of the sera examined, only one appeared to have titratable anti-lps antibody. this sample was from a single animal days after the lps injection; otherwise there was no indication of an immune response developing against the lps. all sera examined showed some reactivity with myelin at a low dilution (< : ), but there was no indication of an increase in reactivity following lps injection. erythrocyte lysis was not increased by a min exposure to lps, even at a concentration approximately % higher than the concentration injected into the spinal cord in this study (data not shown). in contrast, lysophosphatidylcholine or sds caused substantial erythrocyte lysis at the same concentration. examination of surveillance animals by the supplier revealed that no animals were positive for coronavirus, rat parvovirus, hantavirus, sendai virus, theiler's murine encephalomyelitis virus, kilham rat virus, toolan's h- virus, pneumonia virus of mice, reovirus or lymphocyte choriomeningitis virus. animals were also free of bordetella bronchiseptica, clostridium piliforme, corynebacterium kutscheri, mycoplasma species, salmonella species, streptobacillus moniliformis, bhaemolytic streptococci and streptococcus pneumoniae. two-thirds of animals tested were positive for pasteurella pneumotropica. the response of animals to lps injection into the dorsal funiculus in this series (n = ) was similar to that observed in other experiments. we have described an inflammatory and demyelinating lesion that arises following the injection of lps into rat spinal white matter. substantial numbers of pmns and ed -positive cells appear within the white matter and adjacent spinal regions within h of lps injection, reach a peak within the first day, and decline thereafter. during this early phase of the lesion, which is centred at the grey-white matter border, the myelin sheaths in the dorsal funiculus are intact. however, after a delay of - days a large demyelinating lesion develops, persisting between and days and often affecting more than % of the cross-sectional area of the dorsal funiculus at the injection site. the demyelination is primary, and there is relatively little wallerian degeneration rostral (in the sensory . c-f show light and electron micrographs of lesions in dexamethasone-treated (c and e respectively) and saline-treated (d and f respectively) animals days after lps injection. saline-treated animals exhibit a lesion similar to that of untreated animals, with nests of packed demyelinated axons (e.g. arrows in d and f) and cell-associated demyelinated axons (e.g. arrowheads in d). relatively small amounts of extracellular myelin debris are present at this time (e.g. upper left corner in f). dexamethasone treatment does not prevent the demyelination, but does prolong the presence of debris, and the axons appear surrounded by whorls of disaggregated myelin (e.g. arrows in c and e). comparing d with c, it is clear that debris-filled macrophages (e.g. m in d) are more prominent in lesions from animals treated with saline than in those treated with dexamethasone. scale bar (shown in f) = mm in a and b, mm in c and d, and mm in e and f. portion of the dorsal funiculus) or caudal (in the corticospinal tract) to the injection site. oligodendrocyte numbers appear to be reduced in the region of demyelination; however, the possibility that these cells survive but lose the antigen reacting with the adpc antibody cannot be discounted. a number of studies have examined the effects of injecting inflammogens into the cns, including lps (andersson et al., a; bell and perry, ; stern et al., ) , il- b (andersson et al., b; minghetti et al., ; schnell et al., ; bernardes-silva et al., ) , tnf-a (andersson et al., b; minghetti et al., ; schnell et al., ; hall et al., ) and interferon g (minghetti et al., ) . typically, an inflammatory reaction is evoked in the cns, but it is substantially muted when compared with similar injections in other tissues (andersson et al., a) . in general, little or no demyelination has been reported, although demyelination was not specifically examined in most of the studies. in two studies where myelin was examined, some limited myelin loss was reported. matyszak and perry ( ) found that the injection of killed bacillus calmette-guérin (bcg) directly into the hippocampus produced an acute inflammatory response but did not result in myelin loss. however, when animals were subsequently given bcg peripherally, a delayed-type hypersensitivity developed which was associated with myelin loss, as assessed by immunohistochemistry. in a subsequent study (matyszak et al., ) it was demonstrated that, at least in small pockets, this myelin loss resulted from primary demyelination. lehnardt and colleagues found a reduction in immunolabelling with the oligodendrocyte marker rip following large doses of lps ( mg) injected into the pericallosal white matter of neonatal rats (lehnardt et al., ) . however, such doses of lps produced destructive, cystic lesions in many animals, and so although the nature of the myelin loss was not examined in detail, it seems likely to have been secondary to axonal degeneration. in summary, the present study is the first report of substantial primary demyelination resulting directly from the injection of an inflammogen into the cns. the mechanism of the demyelination following injection of lps into the dorsal funiculus is not known, but several possibilities can be considered. lps is an amphiphilic compound and could display detergent-like properties, so it might directly solubilize oligodendrocyte membranes. however, two lines of evidence argue against this mechanism. first, using an erythrocyte lysis assay we found no evidence of membrane damage. secondly, and perhaps most compellingly, a detergent effect would be expected to occur relatively promptly [cf. lysophosphatidylcholine-induced demyelination (hall and gregson, ) ]; however, there was a delay of nearly a week between the injection of lps and the appearance of significant demyelination. it is also possible that the lps-induced inflammation activated a latent viral infection, leading to destruction of oligodendrocytes and demyelination. certain viruses, for example the neurotropic coronaviruses and theiler's virus, can cause demyelination within the cns (for review see fazakerly and walker, ) . however, serological screening of surveillance animals within the animal holding facility did not reveal antibodies to a range of pathogens, including coronavirus and theiler's virus. we consider it unlikely that either detergent action or viral activation is responsible for the demyelination. it is possible that lps may be directly toxic to oligodendrocytes, perhaps acting via plasmalemmal receptors. such direct cell damage is suggested by studies demonstrating a detrimental effect of lps on the survival of oligodendrocyte progenitors in vitro (molina-holgado et al., ) . however, mrna for lps receptors is either absent [toll-like receptor (tlr )] or found only at very low levels (cd ) in oligodendrocyte precursors (lehnardt et al., ) . on the other hand, many commercial preparations of lps have been shown to contain contaminants that are highly bioactive, and can activate receptors other than tlr , including toll-like receptor (tlr ) (hirschfeld et al., ) , a receptor that has been reported to be expressed by oligodendrocytes (bsibsi et al., ) . this contamination is perhaps best illustrated by the ability of these preparations to activate leucocytes from c h/hej mice, which lack functional tlr (morrison et al., ) . to examine the possibility that our typical lps preparation was indeed acting via other receptors, we also examined the effect of an lps preparation that does not activate cells from c h/hej mice. the lesions resulting from such injections (fig. ) appeared similar to those produced by the less pure lps preparation, indicating that the lesions are the result of the lps, as expected, and probably mediated by tlr activation. thus, if tlr is absent in adult oligodendrocytes they would be unlikely to respond directly to lps since this receptor is thought to play a key role in signal transduction following lps binding (poltorak et al., ; hoshino et al., ) . these considerations suggest that lps may be acting via other, indirect, mechanisms. the apparent conflict between the findings of molina-holgado et al. ( ) , indicating that oligodendrocyte progenitors are sensitive to lps, and the seeming absence of appropriate receptors, as demonstrated by lehnardt et al. ( ) , is probably due to the degree of cellular purity of the examined cultures. lehnardt and colleagues found that the deleterious effect of lps on oligodendrocyte progenitors was dependent on the presence of microglia (lehnardt et al., ) , and a role for these cells is also supported by the observation that conditioned media from lps-exposed microglia (or astrocytes) injured oligodendrocyte progenitors (pang et al., ) . the factor(s) responsible are not clear, but a role for tnf-a is possible since this cytokine has been shown to damage rodent (selmaj and raine, ; cammer, ) and human (jurewicz et al., ) oligodendrocytes in vitro. however, injection of tnf-a into white matter in vivo does not result in demyelination (hall et al., ) , and so if this agent is involved it may act in concert with other factors; or perhaps demyelination can result from chronic, rather than acute, exposure to tnf-a. recent evidence suggests that peroxynitrite (formed from nitric oxide and superoxide anion) plays a key role in lps-induced, microglial-mediated oligodendrocyte damage in vitro (li et al., ) . thus, although lps might not be directly toxic to oligodendrocytes, it could cause demyelination indirectly via factors derived from activated microglia, astrocytes or macrophages. arguing against this possibility is the fact that dexamethasone treatment reduced the inflammatory response, as indicated by the reduction in cells expressing inos, yet demyelination was at least as extensive as that seen in control animals. dexamethasone is known to inhibit the lps-induced activation of the transcription factors nuclear factor-kb and activated protein- in monocytic cells, leading to decreased production of inflammatory mediators such as il- b (jeon et al., ) . there are several possible explanations for why dexamethasone did not reduce the extent of demyelination. first, the dexamethasone treatment may not have adequately suppressed the inflammation. this may be the case, since inos activity was suppressed, but not eliminated, in the lesion. secondly, the cellular actions of both lps and dexamethasone are complex, and activation of cells by lps via pathways unaffected by dexamethasone may also have occurred, perhaps via the myd -independent cascade following tlr activation (for review see akira and takeda, ) . certainly, dexamethasone has recently been shown actually to enhance expression of tissue factor (reddy et al., ) , a transmembrane glycoprotein present on monocytic cells which activates coagulation cascades. local activation of tissue factor might have resulted in ischaemia, thereby augmenting the lesions. in summary, although the mechanism of lps-induced demyelination is not known, we favour the idea that oligodendrocytes are killed by a factor or factors produced by activated inflammatory cells, particularly in the light of the finding that lps-activated microglia are capable of inducing oligodendrocyte damage in culture (lehnardt et al., ) . could the current observations on lps-induced demyelination illuminate the mechanisms involved in the demyelination observed in inflammatory demyelinating disease? cns demyelination occurs in association with inflammation in a number of disorders, including multiple sclerosis (lassmann, ), devic's neuromyelitis optica (baudoin et al., ) , recurrent transverse myelitis (pandit and rao, ) , htlv- associated tropical spastic paraparesis (ijichi et al., ) and acute disseminated encephalomyelitis (stuve and zamvil, ) , including the form of the latter disorder which is associated with infection with the gram-negative bacterium chlamydia pneumoniae (heick and skriver, ) . however, our understanding of the mechanism of demyelination in such disorders is often incomplete. it is possible that the mechanism involved in one or more of these disorders may be common to that operating in the current experimental lesion; if so, the current lesion could act as a useful model with which to explore the mechanism for potential therapeutic targets. we note in particular that in the current lesion mag is lost before other myelin proteins, and this loss appears to occur just prior to the initiation of demyelination. such preferential loss of mag also occurs in some inflammatory demyelinating lesions in multiple sclerosis, designated 'pattern iii' by lucchinetti and colleagues (lucchinetti et al., ; aboul-enein et al., ) . the unusual initiation of demyelination in the periaxonal myelin may be another manifestation of this early mag loss, as mag is normally found in the periaxonal myelin in the cns (trapp et al., ) . this pattern of demyelination is not reproduced by the usual experimental model for multiple sclerosis, namely eae (lucchinetti et al., ) . it is notable that in both multiple sclerosis (h. lassmann, personal communication) and the current lps model this unusual pattern of demyelination occurs in conjunction with microglial activation and the prominent expression of inos. the current findings suggest that lps-induced demyelination might be a useful experimental model for demyelinating lesions exhibiting the pattern iii phenotype. it will also be interesting to determine whether the demyelination that we have observed occurs by a mechanism similar to that responsible for producing the lesions recently described in early multiple sclerosis lesions by barnett and prineas ( ) . certainly, both lesions exhibit prominent oligodendrocyte loss and vesicular demyelination, which occur in the absence of a local, noticeable infiltration of lymphocytes. furthermore, in neither lesion does the demyelination appear to depend upon myelin stripping by phagocytes. preferential loss of myelin-associated glycoprotein reflects hypoxialike white matter damage in stroke and inflammatory brain diseases toll-like receptor signalling the acute inflammatory response to lipopolysaccharide in cns parenchyma differs from that in other body tissues intracerebral injection of proinflammatory cytokines or leukocyte chemotaxins induces minimal myelomonocytic cell recruitment to the parenchyma of the central nervous system devic's neuromyelitis optica: a clinicopathological review of the literature in connection with a case showing fatal dysautonomia relapsing and remitting multiple sclerosis: pathology of the newly forming lesion adhesion molecule expression on murine cerebral endothelium following the injection of a proinflammagen or during acute neuronal degeneration recruitment of neutrophils across the blood-brain barrier: the role of e-and p-selectins expression of the apc tumor suppressor protein in oligodendroglia remyelination by schwann cells of axons demyelinated by intraspinal injection of -aminonicotinamide in the rat broad expression of toll-like receptors in the human nervous system differential expression of the l-and s-isoforms of myelin associated glycoprotein (mag) in oligodendrocyte unit phenotypes in the adult rat anterior medullary velum apoptosis of 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evidence for tlr as the lps gene product recent perspectives in japan lymphocyte traffic through chronic inflammatory lesions: differential migration versus differential retention dexamethasone inhibits il- b gene expression in lps-stimulated raw . cells by blocking nf-kb/rel and ap- activation three markers of adult non-myelin-forming schwann cells, c(ran- ), a e and gfap: development and regulation by neuron-schwann cell interactions tnf-induced death of adult human oligodendrocytes is mediated by c-jun nh -terminal kinase- immunohistochemistry using polyester wax the pathology of multiple sclerosis and its evolution the toll-like receptor tlr is necessary for lipopolysaccharideinduced oligodendrocyte injury in the cns lipopolysaccharide-activated microglia kill developing oligodendrocytes by generating peroxynitrite heterogeneity of multiple sclerosis lesions: implications for the pathogenesis of demyelination demyelination in the central nervous system following a delayed-type hypersensitivity response to bacillus calmette-guerin ultrastructural studies of an immune-mediated inflammatory response in the cns parenchyma directed against a non-cns antigen in vivo expression of cyclooxygenase- in rat brain following intraparenchymal injection of bacterial endotoxin and inflammatory cytokines lps/ifngamma cytotoxicity in oligodendroglial cells: role of nitric oxide and protection by the anti-inflammatory cytokine il- lipopolysaccharide intracerebral administration induces minimal inflammatory reaction in rat brain isolation of a lipid a bound polypeptide responsible for 'lps-initiated' mitogenesis of c h/hej spleen cells recurrent myelitis effects of lipopolysaccharide on oligodendrocyte progenitor cells are mediated by astrocytes and microglia defective lps signaling in c h/hej and c bl/ sccr mice: mutations in tlr gene dexamethasone enhances lps induction of tissue factor expression in human monocytic cells by increasing tissue factor mrna stability cytokine-induced expression of intercellular adhesion molecule- (icam- ) in cultured human oligodendrocytes and astrocytes acute inflammatory responses to mechanical lesions in the cns: differences between brain and spinal cord tumor necrosis factor mediates myelin and oligodendrocyte damage in vitro spatiotemporal induction patterns of cytokine and related immune signal molecule mrnas in response to intrastriatal injection of lipopolysaccharide pathogenesis, diagnosis, and treatment of acute disseminated encephalomyelitis the myelin-associated glycoprotein is enriched in multivesicular bodies and periaxonal membranes of actively myelinating oligodendrocytes expression of intercellular adhesion molecule- on rat microglial cells we wish to thank mr meirion davies, ms sally gavin and mr matthew purcell for their excellent technical assistance, professor david male for the gift of the anti-icam- antibody, and mr robert stevenson for performing the endotoxin assay. the work was supported by grants from the multiple sclerosis society of great britain and northern ireland, the wellcome trust and the charitable fund for guy's and st thomas' hospitals. key: cord- -wsjob p authors: wang, yan-hang; lv, hai-ning; cui, qing-hua; tu, peng-fei; jiang, yong; zeng, ke-wu title: isosibiricin inhibits microglial activation by targeting the dopamine d /d receptor-dependent nlrp /caspase- inflammasome pathway date: - - journal: acta pharmacol sin doi: . /s - - - sha: doc_id: cord_uid: wsjob p microglia-mediated neuroinflammation is a crucial risk factor for neurological disorders. recently, dopamine receptors have been found to be involved in multiple immunopathological processes and considered as valuable therapeutic targets for inflammation-associated neurologic diseases. in this study we investigated the anti-neuroinflammation effect of isosibiricin, a natural coumarin compound isolated from medicinal plant murraya exotica. we showed that isosibiricin ( − μm) dose-dependently inhibited lipopolysaccharide (lps)-induced bv- microglia activation, evidenced by the decreased expression of inflammatory mediators, including nitrite oxide (no), tumour necrosis factor-α (tnf-α), interleukin- (il- ), interleukin- β (il- β) and interleukin- (il- ). by using transcriptomics coupled with bioinformatics analysis, we revealed that isosibiricin treatment mainly affect dopamine receptor signalling pathway. we further demonstrated that isosibiricin upregulated the expression of dopamine d / receptors in lps-treated bv- cells, resulting in inhibitory effect on nucleotide binding domain-like receptor protein (nlrp )/caspase- inflammasome pathway. treatment with dopamine d / receptor antagonists sch ( μm) or sultopride ( μm) could reverse the inhibitory effects of isosibiricin on nlrp expression as well as the cleavages of caspase- and il- β. collectively, this study demonstrates a promising therapeutic strategy for neuroinflammation by targeting dopamine d / receptors. dopamine, as a crucial neurotransmitter, can transmit neuronal signals to regulate neurological function by acting on specific dopamine receptors [ , ] . currently, the dopamine receptor family is considered to play a key role in various advanced neural functions, including emotion, reward and autonomic movement [ ] . in addition, increasing attention has been paid to the role of the dopamine signal system in immunomodulation and inflammation control [ , ] . previous studies have shown that the genetic and pharmacologic regulation of dopamine receptors can have potential anti-inflammatory effects. for instance, dopamine d receptor (drd ) activation is widely involved in the alleviation of inflammatory bowel diseases [ , ] , pancreatic inflammation [ ] and arthritis [ ] . specifically, drd /d have been found to be widely expressed not only in neurons and t cells [ ] but also in microglia [ ] , indicating that drd /d might also be potential therapeutic targets for neuroinflammation in the central nervous system (cns). recently, the pharmacologic activation of drd was shown to exert an obvious inhibitory effect on neuroinflammation induced by intracerebral haemorrhage, by regulating the nuclear factor-κb (nf-κb) signalling pathway [ ] . moreover, new therapies based on drd /d agonists have been developed for experimental autoimmune encephalomyelitis [ ] . therefore, treating the microglia-derived inflammatory response by targeting dopamine receptors appears to be a promising therapeutic strategy for neuroinflammation. in particular, dopamine receptors have been found to be highly associated with the inflammasome signalling pathway. some previous research has suggested that spinal cord injury induces inflammatory cytokine production by activating the nucleotide-binding domain-like receptor protein inflammasome pathway, which is significantly suppressed by drd agonists [ ] . moreover, drd can also block nlrp inflammasome activation in a β-arrestin-dependent manner [ ] . collectively, these findings provide a potential therapeutic strategy for treating the neuroinflammatory response by targeting the drd-mediated inflammasome pathway. murraya exotica l. (rutaceae) has traditionally been used in china for the treatment of infectious diseases and pains [ ] . modern pharmacological studies have suggested that murraya koenigii and m. exotica possess a wide range of biological activities, including anti-inflammatory, antinociceptive, antioxidative stress, anti-mutagenic stress, and chondroprotective and anticancer metastasis effects [ , ] . isosibiricin is a natural bioactive coumarin compound isolated from m. exotica. several studies have indicated that the coumarin derivatives isofraxidin, osthole and urolithin can exert obvious anti-inflammatory activities both in vitro and in vivo [ ] [ ] [ ] . therefore, in this study, we explored the mechanism of the anti-neuroinflammatory effects of isosibiricin in a bv- microglial model and highlighted that isosibiricin can significantly inhibit the production of multiple inflammatory mediators induced by bacterial lipopolysaccharide stimulation via targeting the drd /d -dependent inflammasome pathway, providing a potential therapeutic strategy for inflammation-related neurological disorders. mouse strains balb/c mice ( - weeks old) were purchased from the vital river laboratories (beijing, china). all mice were raised in a specific pathogen-free environment. the bv- cell line was purchased from the cell bank of peking union medical college in china. the cells were cultured in dulbecco's modified eagle's medium containing % fetal bovine serum, u/ml penicillin and mg/ml streptomycin. the cell culture conditions were maintained at °c in a % co incubator under % absolute humidity. nitrite oxide analysis bv- cells were treated with isosibiricin ( , , and μm) with or without lps for h. then, the supernatants were used to analyse the production of nitrite oxide (no) with a nitric oxide analysis kit (jiancheng bioengineering institute, nanjing, china) enzyme-linked immunosorbent assay bv- cells were treated with isosibiricin ( , , and μm) with or without lps. after treatment for h, μl of the cell supernatants was centrifuged at × g for min to evaluate the accumulation of tumour necrosis factor-α (tnf-α). in addition, μl of the cell supernatants was also centrifuged at × g for min to evaluate il- levels h after treatment. the two experiments were carried out with enzyme-linked immunosorbent assay (elisa) kits (excell bio company, shanghai, china) following the manufacturer's protocol. the cells were collected and rna was extracted by the rnaprep pure cell/bacteria kit (tiangen biotech, beijing, china). target analyses were carried out by the yuanquan yike company (beijing, china). amino allyl-labelled antisense rna was prepared and purified prior to hybridization. purified, coupled antisense rna was quantified using nanodrop nd- to explore the targets affected by isosibiricin. transcriptome analysis pathway analysis [ ] on isosibiricin was also performed by the yuanquan yike company. after the targets of isosibiricin were determined by amino allyl-labelled antisense rna hybridization, kyoto encyclopedia of genes and genomes (kegg) pathway enrichment was used to perform the pathway analysis. signal pathways significantly influenced by isosibiricin compared with lps were selected. western blotting after treatment, the cells were lysed in ice-cold ripa buffer containing % protease inhibitors (macgene, beijing, china). whole-cell proteins were collected by centrifugation at × g for min. the concentration of the total proteins was detected by a protein assay kit (transgen, beijing, china). protein solutions of equal concentration were separated by %- % sds-polyacrylamide gel electrophoresis and transferred to pvdf membranes. next, the pvdf membranes were blocked with % skim milk at °c for min. subsequently, the membranes were incubated with primary antibodies at °c for h. after incubation with a second antibody for h, the membranes were developed with supersignal west femto maximum sensitivity substrate. the protein bands were imaged by a tanon imaging analysis system (tanon, shanghai, china). relative densitometry analysis was carried out by imagej software. reverse-transcription pcr the cells were collected and total rna was extracted using the rnaprep pure cell/bacteria kit (tiangen biotech, beijing, china). the mrna was reverse transcribed using a cdna synthesis kit (transgen, beijing, china). reverse-transcription pcr (rt-pcr) was performed using a rt-pcr superkit (transgen, beijing, china) on an agilent technologies stratagene mx p system. a single cycle was carried out at °c for s, °c for s and °c for s, and this cycle was repeated times. the relative transcriptional levels of drd and drd normalized to those of gapdh was calculated by the comparative −ΔΔct method [ ] . dopamine receptor agonist analysis hek cells stably expressing drd /gα were cultured in -well plates for h. after the supernatant was removed, fluo- / am, a calcium fluorescence probe, was added at °c for min in an incubator under % absolute humidity. the fluo- /am was removed and a calcium buffer solution was added. flexstation ii was used to measure the levels of gα protein activation induced by isosibiricin ( , . nm, . nm, nm, nm, nm, μm and μm) at nm. dopamine was used as a positive control. the ec was analysed by graphpad prism. all experiments were carried out by the national center for drug screening (shanghai, china). nine balb/c mice were randomly divided into the control, lps and isosibiricin groups. the isosibiricin group was treated with isosibiricin ( mg/kg body weight). after h, both the lps group and the isosibiricin group were injected intraperitoneally with lps ( mg/kg body weight). then, the brain tissues were sectioned and stained with specific antibodies. cycle adenosine monophosphate analysis bv- cells were treated with μm isosibiricin with or without lps for h. then, the cell lysates were used to analyse the level of cycle adenosine monophosphate (camp) with a camp direct immunoassay kit (bioway, beijing, china) all experiments were carried out at least three times in triplicate. statistical analyses were performed by using one-way analysis of variance with graphpad prism . software. multiple comparisons were performed by comparing the mean of each column with the mean of every other column. student's t-test was used without correcting for multiple comparisons. all data were expressed as the means ± s.d. a p-value < . was considered to be significant. in this study, the anti-inflammatory effect of isosibiricin (fig. a) was measured by using an no release assay. as shown in fig. b , lps obviously stimulated no release, which was significantly inhibited by isosibiricin ( , and μm) in a concentrationdependent manner. to evaluate the effect of isosibiricin on other inflammatory factors, the levels of tnf-α and il- were further analysed by elisa. as shown in fig. c , d, tnf-α and il- expression was significantly increased upon lps treatment; however, isosibiricin ( , and μm) inhibited tnf-α and il- production in a concentration-dependent manner. moreover, cox- and inos expression was markedly upregulated in lpstreated bv- cells, and isosibiricin reduced cox- and inos expression in a concentration-dependent manner (fig. e) . taken together, these findings indicated that isosibiricin can attenuate neuroinflammation by inhibiting the expression of multiple inflammatory mediators in lps-treated bv- cells. isosibiricin upregulates drd /d expression in lps-treated bv- cells and balb/c mice to investigate the potential mechanism of the anti-inflammatory effect of isosibiricin, transcriptomics coupled with bioinformatics analysis was performed to explore the anti-inflammatory signalling pathway of isosibiricin in lps-treated bv- cells. the results suggested that genes, including upregulated genes and downregulated genes ( genes related to inflammation and genes related to the synthesis, storage, release, termination of dopamine and the expression of drds) were significantly changed by isosibiricin compared with lps. notably, dopamine receptors were most strongly related to isosibiricin treatment (fig. a) . moreover, kegg pathway analysis was carried out to explore the pathway affected by isosibiricin. as shown in fig. b , dopamine activation of the neurological reward system was strongly influenced by isosibiricin. therefore, we speculated that isosibiricin might exert an anti-inflammatory effect by targeting the dopamine receptor signalling pathway. nevertheless, isosibiricin did not directly target or regulate the functions of dopamine receptors (supplementary table ). thus, we hypothesized that isosibiricin can regulate the expression of dopamine receptors. as shown in fig. c , d, lps downregulated the mrna expression of drd and drd , and this was significantly reversed by isosibiricin. in addition, isosibiricin also upregulated drd /d expression in the ca region and cortex of lps-treated balb/c mice (supplementary fig. s a) . collectively, these observations revealed that isosibiricin can effectively upregulate drd and drd expression in vitro and in vivo, which might represent a crucial antiinflammatory mechanism. isosibiricin inhibits the nlrp /caspase- inflammasome pathway in lps-or nigericin-treated bv- cells and lps-treated balb/c mice it has been reported that the expression of the pro-inflammatory mediator il- β significantly increases in drd -null mice compared with wild-type mice [ ] . in addition, drd agonists are able to inactivate the nlrp inflammasome pathway [ ] . thus, we detected the effect of isosibiricin on the nlrp inflammasome pathway. as shown in fig. a , isosibiricin significantly reduced nlrp , caspase- , il- β and il- expression in a concentrationdependent manner, indicating that isosibiricin exerts an inhibitory effect on the nlrp /caspase- inflammasome pathway. moreover, the nlrp inflammasome inducer nigericin was used to activate bv- cells and we found that nigericin significantly increased nlrp , caspase- , il- β and il- expression, which was the predicted pathways (a) and targets (b) of isosibiricin inferred by the differential expressed gene-based method. c, d isosibiricin upregulated dopamine d receptors (c) and dopamine d receptor (d) expressions in lps-induced bv- cells. ## p < . vs. control group. **p < . vs. lps group effectively inhibited by isosibiricin (fig. b) [ ] . animal experiments were also carried out to evaluate the anti-inflammatory effect of isosibiricin. as shown in fig. c , isosibiricin significantly inhibited inflammasome-related il- β and il- induced by lps in the ca region and cortex of balb/c mice. these results suggested that isosibiricin exerts an anti-inflammatory effect via the inhibition of the nlrp inflammasome pathway. drd /d inhibitors reverse the isosibiricin-mediated inactivation of nlrp /caspase- inflammasome pathway considering the effect of isosibiricin on dopamine receptors (fig. c, d) and the inflammasome pathway (fig. a, b) , we hypothesized that isosibiricin might inhibit the inflammasome pathway through dopamine receptors. therefore, drd and drd inhibitors, sch and sultopride were used to investigate whether drd /d are involved in isosibiricinmediated anti-inflammatory effects. we found that the anti-inflammatory effect of isosibiricin was significantly reversed upon treatment with sch and sultopride. nlrp , caspase- , il- β and il- expression was markedly increased by sch and sultopride (fig. a) . furthermore, sch and sultopride were also used in nigericin-treated bv- cells. similar to what was observed in the lps-induced experiment, nlrp , caspase- , il- β and il- expression was reversed in the sch -and sultopride-treated groups compared with the isosibiricin-treated group (fig. d) . taken together, the results suggested that drd and drd inhibitors are able to reverse the isosibiricin-mediated inhibition of the nlrp /caspase- inflammasome pathway. dopamine agonists (das), such as dopamine, act on dopamine receptors to regulate a great diversity of biological functions [ ] . fig. isosibiricin inhibits nlrp /caspase- inflammasome pathway in vitro and in vivo. a isosibiricin inhibited inflammasome-related nlrp , pro-caspase- , caspase- , pro-il- β, il- β, pro-il- and il- expressions in lps-induced bv- cells. quantification for caspase- , il- β and il- expressions were relative to gapdh in lps-induced bv- cells. b isosibiricin inhibited inflammasome-related nlrp , pro-caspase- , caspase- , pro-il- β, il- β, pro-il- and il- expressions in nigericin-induced bv- cells. quantification for caspase- , il- β and il- expressions were relative to gapdh in nigericin-induced bv- cells. c isosibiricin inhibited inflammasome-related il- β and il- in the ca region and cortex stained with the specific antibodies il- β and il- . arrows indicate the activated inflammatory factors. ## p < . vs. control group. **p < . vs. lps group or nigericin group in the past decade, das have been mainly used clinically for the treatment of parkinson's disease and, recently, the development and clinical application of das for various other neurological diseases have begun to draw attention from researchers [ , ] . neuroinflammation is an inflammatory response in the cns characterized by increased microglial over-activation [ , ] . thus, the development of anti-neuroinflammatory agents is of great importance for the treatment of inflammation-related neurological disorders, such as motor paralysis, amyotrophic lateral sclerosis and dementia [ ] [ ] [ ] . in the current study, we reported that isosibiricin exerts a significant anti-neuroinflammatory effect on lps-treated bv- cells. lps is a common inflammation inducer that is used to investigate different types of inflammatory pathology, such as tissue inflammation [ ] , vascular inflammation [ ] and myocarditis [ ] . dopaminergic neurons play an important role in the pathological process of parkinson's disease [ ] , and lps is widely used to induce neuroinflammation to establish models of parkinson's disease. in addition, some references have indicated that dopamine receptors are also involved in the regulation of the classical lps signalling pathway [ ] [ ] [ ] . therefore, lps was used to induce neuroinflammation and damage dopamine receptors in the substantia nigra [ ] . the potential mechanism of isosibiricin may involve the upregulation of drd /d expression and the further inactivation of the nlrp /caspase- inflammasome pathway, which results in the blockage of the production of multiple inflammatory mediators. in addition, we used pipeline pilot to measure the solubility, intestinal absorption and blood-brain barrier (bbb) level of isosibiricin. isosibiricin exhibited good solubility in water at °c and was easily absorbed by the intestine. the bbb level was , which means that isosibiricin can pass through the bbb. thus, our study provides an attractive therapeutic methodology for inflammation-associated neurological disorders, as well as an anti-neuroinflammatory lead compound that may become a clinical candidate. dopamine receptors were recently discovered as key therapeutic targets involved in immunoregulation and the inflammatory response [ ] . previously reported active compounds were shown to mainly activate or inhibit dopamine receptors [ , ] ; however, we found that isosibiricin did not directly activate drd /d but significantly upregulated the expression of drd /d mrna, further promoting drd /d -dependent inflammasome signalling pathway inactivation and blocking inflammatory mediator production. in addition, the level of camp was regulated by drd /d , but we detected that isosibiricin did not affect the level of camp ( supplementary fig. s b ). these mechanisms are quite different from those of current small molecular regulators, which directly activate drd /d [ ] . however, the molecular mechanism by which drd mrna expression is regulated still needs to be explored. we speculate that some transcription factors, such as nf-κb, nrf and creb, might be involved in this process. dopamine receptors, including five subtypes from drd to drd , are seven-transmembrane-spanning g-protein-coupled receptors [ , ] . based on their structures and pharmacological characteristics, dopamine receptors are divided into two major categories: dopamine d -like receptors (drd and drd ) and d like receptors (drd , drd , and drd ) [ ] . notably, the specific drd inhibitors sch (for drd ) and sultopride (for drd ) significantly reversed the inhibitory effect of isosibiricin on the nlrp signalling pathway, further supporting the observation that isosibiricin suppresses neuroinflammation by blocking the nlrp / caspase- inflammasome pathway. it is worth mentioning that the regulatory effect of drd inhibitors on the isosibiricin-dependent inhibition of il- β production was more significant in lps-induced bv- cells than in nigericin-induced bv- cells. a possible explanation may be that the mechanism by which lps induces inflammasome signal activation differs from that of nigericin. fig. dopamine d / receptor inhibitors reverse isosibiricin-mediated inhibition of nlrp /caspase- inflammasome pathway. a sch and sultopride reversed isosibiricin-mediated inhibition of nlrp , pro-caspase- , caspase- , pro-il- β, il- β, pro-il- and il- expressions in lps-induced bv- cells. quantification for caspase- , il- β and il- expressions was relative to gapdh in lps-induced bv- cells. b sch and sultopride reversed isosibiricin-mediated inhibition of nlrp , pro-caspase- , caspase- , pro-il- β, il- β, pro-il- and il- expressions in nigericin-induced bv- cells. quantification for caspase- , il- β and il- expressions was relative to gapdh in nigericin-induced bv- cells. ## p < . vs. control group. $$ p < . vs. lps group or nigericin group. **p < . vs. the isosibiricin group isosibiricin regulates drd / to inhibit neuroinflammation yh wang et al. meanwhile, isosibiricin-mediated anti-neuroinflammation 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with three-dimensional biologically relevant spectrum characterization of an invertebrate-type dopamine receptor of the american cockroach, periplaneta americana this work was financially supported by the national natural science foundation of china (numbers , and ) and the drug innovation major project (number zx - - ). kwz, pft, and yj designed the research. yhw and hnl conducted majority of the experiments. qhc coordinated the experiments. kwz and yhw wrote the manuscript. kwz provided suggestions for the experimental design and manuscript. the online version of this article (https://doi.org/ . /s - - - ) contains supplementary material, which is available to authorized users.competing interests: the authors declare no competing interests. key: cord- - k t authors: yuan, qing; jiang, yan-wen; ma, ting-ting; fang, qiu-hong; pan, lei title: attenuating effect of ginsenoside rb on lps-induced lung injury in rats date: - - journal: j inflamm (lond) doi: . /s - - - sha: doc_id: cord_uid: k t background: sepsis causes neutrophil sequestration in the lung which leads to acute lung injury (ali). radix ginseng (rg), a traditional herb used as herbal remedy in eastern asia for thousands of years, which has been traditionally used in china to improve blood circulation and ameliorate pathological hemostasis. this study investigated whether ginsenoside rb , the main components of rg, can attenuate ali induced by lps. methods: in vivo, male wistar rats were divided into three groups (n = each groups) on the basis of the reagent used, which were subjected to lps injection with or without ginsenoside rb ( mg/kg) treatments to induce ali model. lung injury was assessed by pulmonary histology, lung wet-weight to dry-weight (w/d) ratio, the number of myeloperoxidase (mpo) positive cells, immunohistochemical analysis of intercellular adhesion molecule- (icam- ), gene expression of icam- , ultrastructure changes of pulmonary microvasculature, concentration of inflammatory markers and in plasma. in vitro, pulmonary microvascular endothelial cells (pmvecs) were stimulated with lps in the presence and absence of ginsenoside rb ( mm), nuclear factor-κb (nf-κb) p was measured by immunocytochemistry staining and western blotting. results: infusion of lps induced lung injury, in vivo, as demonstrated by pulmonary edema with infiltration of neutrophils and hemorrhage, the increase in lung w/d ratio, the number of mpo positive cells, the level of inflammatory markers such as tnf-α, mcp- and il- , enhanced expression of icam- and icam- gene. moreover, resulted in the changes of intercellular junctions in the endothelial cells of pulmonary microvasculature. in vitro, the significant increased release of nf-κb p and its subsequent translocation into the nucleus in pmvecs were observed. in contrast, ginsenoside rb treatment significantly ameliorated the lps-induced lung injury, as judged by the marked improvement in all these indices. conclusions: these results indicate that ginsenoside rb attenuated lps-induced lung injury through an inhibition of the inflammatory signaling pathway, besides the direct inhibitory effect on proinflammatory molecules. acute lung injury (ali) and acute respiratory distress syndrome (ards) in their most severe forms are still major challenges in modern intensive care medicine that significantly contribute to morbidity and mortality of critically ill patients. a recent epidemiological study indicate that ali leads to , deaths annually in the united states [ ] . respiratory failure is caused by an excessive inflammatory response to both pulmonary and extrapulmonary stimuli, including pneumonia, acid aspiration, ischemia-reperfusion and sepsis [ ] . inflammatory mediators can disrupt the pulmonary capillary barrier, leading to the influx of a proteinrich edema with severe consequences for gas exchange and the functional integrity of remote organ systems [ ] . excessive infiltration of polymorphonuclear leukocytes (pmns) into the lungs has been identified as a pivotal event in the early development of ali. pulmonary microvascular endothelial cells(pmvecs) are critically involved in the pathogenesis of acute lung injury. pmvecs can be stimulated by pro-inflammatory cytokines including tnf-α to express adhesion molecules such as intercellular cell adhesion molecule- (icam- ) for leukocytes and other inflammatory cells. increased expression of adhesion molecules on pmvecs leads to leukocyte recruitment via interactions with their cognate ligands on leukocytes at the sites of atherosclerosis. pmvecs play an important role in initiation and development of pulmonary inflammation procedure as well as early target cells [ ] . radix ginseng (rg), a traditional used as a herbal remedy in eastern asia for thousands of years, which has been traditionally used in china to improve blood circulation and ameliorate pathological hemostasis and has also recently become popular in western countries. recently, it was reported that there are some active compounds in rg which could scavenge radical, inhibit the leukocytes adhesion to venular wall or protect lipopolysaccharide (lps)-induced microcirculatory injury. as so far, among identified ginsenosides, ginsenoside-rb , −ro, −rg , −rc, and -re are highly abundant. in particular, ginsenoside rb makes up . - . % of ginseng extracts [ ] . cell culture studies have shown that ginsenoside rb can inhibit lps-induced expression of the proinflammatory cytokine tnf-α. we previously identified that ginsenoside rb , which is isolated from notoginseng and ginseng in chinese herbal medicine efficiently can attenuate lpsinduced intestinal injury by inhibiting nf-κb activation [ ] . however, the effect of ginsenoside rb on lung microcirculatory injury has not been reported thus far. therefore, in the present study, we developed a rat model of ali induced by lps, in vivo. meanwhile, an in vitro model of pmvecs was established to observe the inflammatory injury induced by lps. the goal of the present study was to clarify the effects of ginsenoside rb on lps-induced rat lung injury and analyzed the detailed molecular mechanisms in vivo and in vitro. ginsenoside rb was purchased from the national institute for the control of pharmaceutical and biological products. the saponin was chromatographically pure, and the chemical structure was shown in figure . lps (e.coli lps serotype : b ), endothelial cell growth supplement (ecgs), fetal bovine serum (fbs) were obtained from sigma (st. louis, mo, usa), mouse anti-intercellular adhesion molecule- (icam- ) was purchased from bd pharmingen (san diego, ca), rabbit antimyeloperoxidase (mpo) was purchased from neomarkers (fremont, ca, usa). moloney murine leukemia virus (m-mlv) reverse transcriptase and dulbecco's modification of eagle's medium dulbecco (dmem) were obtained from invitrogen (carlsbad, ca, usa), rabbit anti-factor viii-related antigen, β-actin, anti-β-actin, anti-nf-κb p and abc kit were obtained from santa cruz biotechnology inc ( santa cruz, ca, usa). all animals were handled according to the procedures approved by the committee on the ethics of animal experiments of capital medical university. this investigation was carried out in strict accordance with the recommendations in the guide for the care and use of laboratory animals of the national institutes of health. all surgery was performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering. male wistar rats weighing to g were obtained from the animal center of capital medical university. the rats were fed a standard laboratory chow diet and maintained at ± °c, relative humidity of % ± % with a -h- -h light-dark cycle. the animals were fasted for h before the experiment, allowing free access to water. according to the report, ali was induced by intravenous injection of lps [ ] . the rats were divided randomly into three groups: control group, lps group and lps + ginsenoside rb group. in the control group, the ml normal saline was administered intravenously in min, followed by continuous intravenous injection of normal saline ml/kg/h for mins continuously (n = ). in the lps group, lps ( ug/kg) dissolved in ml saline was administered intravenously in min, the animals were observed for mins, then followed by intravenous injection of normal saline at ml/kg/h for mins continuously (n = ). in the lps + ginsenoside rb group, lps ( ug/kg) dissolved in ml saline was administered intravenously in min, the animals were observed for mins, then followed by intravenous injection of ginsenoside rb at mg/kg/h for mins continuously (n = ). the dose of ginsenoside rb was determined from the reports of sun etal [ ] . the blood-free w/d ratio was determined as described by xie etal [ ] . briefly, the lung was homogenized with an identical weight of distilled water. homogenate and blood samples were weighed and dried at °c for hours. dry weights were measured, and the w/d ratios of the homogenate and blood were calculated. a sample of the homogenate was centrifuged at , rpm for hour, and blood samples were diluted with an equal volume of distilled water. to determine the hb levels in the homogenate and blood, μl of the homogenate supernatant or diluted blood was tested with a blood cell analyzer (cell-dyn , dainabot, tokyo, japan). the weight of blood in the wet lung was then calculated. the blood-free w/d ratio was then determined. the same rats were used for histological evaluation. after the infusion of the reagents, the right upper lung of each mouse was removed and fixed with % buffered formalin. the samples were further processed as routine, and mounted sections were stained with hematoxylin and eosin for light microscopy. for immunohistochemistry, frozen sections of lung were acetone-fixed and then incubated with % normal rabbit serum in pbs ( minutes, °c) to block nonspecific staining. sections were then incubated with mouse anti-intercellular adhesion molecule- (icam- ) or rabbit anti-myeloperoxidase (mpo) overnight at °c, followed by incubation with biotinylated donkey anti-rabbit or donkey anti-mouse igg ( : ) for min. positive staining was revealed by diaminobenzidine, according to the manufacture's instruction of the abc kit. the number of mpo positive cells was counted within a field of view under the microscope with a × objective lens and fields were selected randomly in each section with the imaging-pro plus . . real-time rt-pcr analysis was performed as described previously with some modifications [ , ] . briefly, total rna was extracted from lung tissue as described above and then poly(a) + rna was isolated using oligotex™ dt (takara, tokyo, japan). the first-strand cdna was generated by reverse transcription reaction using mmlv reverse transcriptase and the poly(a) + rna preparations as templates. the cdna was amplified by rt-pcr by using a light cycler (roche diagnostics, indianapolis, in) with the specific upstream and downstream primers for icam- mrna analysis under the following reaction conditions: denaturation at °c for mins, followed by cycles of denaturation at °c for seconds, annealing at °c for seconds, and extension at °c for seconds. the sequences of the upstream and downstream primers for icam- and β-actin were as follows: ′-caaacgggagatgaatggta- ′ and ′-aataggtgtaaatggacgcc- ′ for icam- ; and ′-cctgtatgcctctggtcgta- ′ and ′-ccatc tcttgctcgaagtct- ′ for β-actin, respectively. the product sizes were bp for icam- and bp for βactin, respectively. the amplified products were analyzed by melting curve analysis and stained using sybr greeni. the icam- mrna level was normalized with the βactin mrna level in each poly(a) + rna preparation. relative gene expressions were calculated by using the −ΔΔct method, in which ct indicates cycle threshold, the fractional cycle number where the fluorescent signal reaches detection threshold [ ] . the normalized Δct value of each sample is calculated using up to an endogenous control gene (β-actin). fold change values are presented as average fold change = −(averageΔΔct) for genes in treated relative to control samples. the fresh left upper lung tissues ( × × mm) were taken for electron microscopy. the specimen was fixed in . % glutaraldehyde and phosphate buffer. the specimen was then rinsed in phosphate buffer, postfixed with % osmic tetroxide in phosphate buffer. after graded dehydration in ethyl alcohol and propylene oxide, specimen was embedded in spurr resin. then, the embedded tissues were thin-sectioned, mounted on copper grids, and stained with uranyl acetate and lead citrate. the images were taken by electron microscope (h- iv, hitachi, tokyo, japan). peripheral blood tnf-α, mcp- and il- assay after the infusion, blood was collected via abdominal aorta of each animal and anticoagulated with heparin ( unit/ml blood). the plasma was isolated by centrifugation. fifty microliters of plasma or standard was incubated with μl capture beads for h at room temperature, and then mixed with μl phycoerythrobilin (pe)-labeled tnf-α, mcp- and il- detection antibodies and incubated for h at room temperature to form a sandwich complex. following incubations, ml of washing buffer (bd, biosciences pharmingen, usa) was added to each tube. the mean fluorescence intensities of tnf-α, mcp- and il- were measured by flow cytometry (facs calibur, b.d. co., usa) and the data were analyzed by bd cytometric bead array analysis software. rat pmvecs were isolated from peripheral parts of the lung of wistar rats, purified, and cultured as described in kim [ ] . briefly, wistar rats were maintained under specific pathogen-free and controlled light conditions ( °c, % humidity, and -hour day/night rhythm). after animals were died, the thoracic cavity was opened and the lung was exposed fully, lung tissue was cut from peripheral parts of the lung, pasted in culture flasks and cultured in dmem with % fbs, lg/ml ecgs. behind cells emigrated from the pasting tissue and were grown to confluence, cells were purified by different speed adherence method. the cell morphology was observed in inverted optical microscope. primary rat pulmonary microvascular endothelial cells (pmvecs) were grown in a humidified atmosphere with % co . experimental data were obtained from cells in their third to fifth generation. the immunocytochemical staining of factor viii-related antigen was to performed to identify the pmvecs. the cells were fixed with . % paraformaldehyde and permeabilized with . % triton x- . after rehydrating with % bovine serum albumin, the cells were exposed to rabbit anti-factor viii-related antigen. on washing times with pbs, they were exposed to goat anti-rabbit igg. the cells were then stained with , ′-diaminobenzidine and the negative control group received only the secondary antibody [ ] . the cells were analyzed and identified under inverted microscope. the protocol was approved by the committee on the ethics of animal experiments of capital medical university. all surgeries were performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering. cultured rat pmvecs were randomly divided into two groups: control group and lps group. cells in control group were received vehicle ( . % dmso) only. in lps group the cells were stimulated with ng/ml lps previously for h. at the end of the stimulation, the cells were treated with mm ginsenoside rb or vehicle ( . % dmso) for h, respectively. to observe the expression of nf-κb p on pmvecs, immunocytochemistry staining was carried out as described previously. the cells were fixed with . % paraformaldehyde and permeabilized with . % triton x- . after rehydrating with % bovine serum albumin, the cells were exposed to rabbit anti-nf-κb p . on washing times with pbs, they were exposed to goat anti-rabbit igg. the cells were then stained with , ′-diaminobenzidine and the negative control group received only the secondary antibody. the cells were analyzed under inverted microscope. total and nuclear proteins were extracted from cells to measure the nf-κb p at the end of observation. the cells were washed twice with cold pbs. then, cells were lysed with sds sample buffer containing mm tris (ph . ), % sds (wt/vol), % -mercaptoethanol and % glycerol. cell homogenates were centrifuged at , rpm at °c for min. supernatants of cells were collected, and protein concentration of each sample was measured with a bicinchoninic acid assay kit using bsa as standard. an equal amount of protein from each sample ( mg) was resolved in % tris-glycine sds polyacrylamide gel. protein band was blotted to nitrocellulose membrane. after incubation for h in blocking solution ( % dry milk in tris buffered saline with tween ) at room temperature (rt), the membrane was incubated for h with anti-β-actin ( : ), anti-nf-κb p ( : ) at °c, respectively. the secondary antibody (horseradish peroxidase-conjugated donkey anti-rabbit immunoglobulin) was added at : dilution and incubated at room temperature for h. peroxidase labeling was detected with the enhanced chemiluminescence western blotting detection system (amersham pharmacia biotech, piscataway, nj) and analyzed by a densitometry system. the relative protein level was normalized to β-actin. all values were presented as mean ± se. for the remaining parameters, the means of different groups were compared by anova and f-test. a value of p < . was designed as significant. we examined the effect of ginsenoside rb treatment on lung injury after lps infusion. lung injury was assessed by morphological examination, with the w/d ratio as an index of pulmonary edema [ ] . sections of lung excised from control animals were essentially normal (figure a top) . in contrast, lps animals exhibited serious alveolar collapse, severe thickening of interalveolar septa with marked infiltration of neutrophil (figure a, middle) . ginsenoside rb treatment greatly suppressed lung injury, and reduced histological damage (figure a, bottom) . the w/d ratio of lung are shown in figure b . the w/d ratio was significantly higher in the lps group than in the control (p < . ) and lps (p < . ) groups. there was no significant difference in the w/d ratio between the control and ginsenoside rb groups. the marked decrease in lung w/d ratio, which reached the same level as the control animals ( figure b ). as an indicative enzyme of neutrophil granulocytes, mpo was revealed by immunohistochemical staining after lps infusion on lung to evaluate the leukocyte infiltration. the representative images and the quantifications of the mpopositive cells in the three groups are presented in figure . the mpo-positive cells was hardly detectable in con group ( figure a top) but significantly increased by lps, indicative of leukocyte infiltration in this situation ( figure a middle) . noticeably, the lps elicited increase in mpo-positive cells was significantly inhibited by administration of ginsenoside rb ( figure a, bottom) . since cell adhesion molecules play pivotal roles in pulmonary inflammation by the recruitment of leukocytes into the lung [ , ] , immunohistochemical analysis of icam- was carried out in the lung mins after lps infusion. as noticed from figure rare expression of icam- was detected on microvascular endothelium of lung in con group ( figure a, top) . in contrast, the immunochemical staining for the expression of icam- was prominent in lps group ( figure a, middle) , which was attenuated significantly by treatment with ginsenoside rb (figure a, bottom) . this result was confirmed by a quantitative evaluation of the icam- mrna level, which was almost similar with immunohistochemical analysis. the icam- mrna level was detectable in con group whereas it was markedly increased in the lps group. in contrast, in lps plus ginsenoside rb group the level was significantly decreased to almost the same as that of the control group ( figure b ). ultrastructure changes of pulmonary microvasculature figure presents the electron micrographs of rat pulmonary microvasculature in each group. in the con group ( figure a ) microvasculature was lined by a layer of endothelial cells, which exhibited a rather smooth inner face with occasionally occurring vesicles in the cytoplasm. at min after lps infusion, an apparent alteration in the ultrastructure of the endothelial cell occurred, characterized by the increase in the gap of intercellular junctions ( figure b ). lps-induced alterations in the ultrastructures of endothelial cell were abated by treatment with ginsenoside rb ( figure c ). the concentrations of the cytokines tnf-α and mcp- in plasma are presented in figure . in the con group, the concentrations of tnf-α, mcp- and il- were . ± . , . ± . and . ± . ng/l, respectively. the concentrations of the two cytokines were enhanced dramatically by lps stimulation to . ± . , . ± . and . ± . ng/l, respectively. treatment with ginsenoside rb inhibited the lps-induced production of tnf-α, mcp- and il- markedly ( figure ). activation of nf-κb pathway leads to phophorylation of p and p . our investigation found a noticeable increased expression of nf-κb p and its subsequent translocation into the nucleus mins after lps stimulation in pmvecs ( figure a, middle) , which was ameliorated by ginsenoside rb , indicating that ginsenoside rb inhibited the activation of nf-κb p ( figure a , bottom). the expression of nf-κb p was quantified as a parameter for nf-κb activation by western blotting. the results showed that the expression of nf-κb p increased in the lps group, similarly, this increase was significantly inhibited by treatment with ginsenoside rb (figure b -c). the present study demonstrated lps infusion caused severe lung injury with the characteristic features of ali, as revealed by the evidence of alveolar septal thickening due to interstitial edema with infiltration of inflammatory cells [ ] . we also confirmed that ginsenoside rb , a main component of traditional chinese herb rg, on the lps-induced lung injury and inflammation. the results in the present study demonstrated that ginsenoside rb significantly attenuated the lps-induced lung injury, as shown by the lesser tissue injury compared with that observed in the lps animals, the w/d ratio, the expression of icam- along the vascular endothelium, the mrna level of icam- , inflammatory factors, and nf-κb p expression in the lung. these results suggest that, ginsenoside rb may protect the lung from lpsinduced tissue injury. lps is responsible for the initiation of the septic cascade in gram-negative bacterial infections, which involves upregulation of icam- and production of large amount of cytokines [ , ] , the two processes that are both mediated by nf-κb [ , ] . because nf-κb activation can lead to enhanced expression of proinflammatory cytokines (mcp- , tnf-α), chemokines and adhesion molecules, modulation of nf-κb activation may provide a direct way of inhibiting inflammatory mediators. lps caused severe lung injury with the characteristic features of ali, as revealed by the evidence of alveolar septal thickening due to interstitial edema with infiltration of inflammatory cells [ ] (figure a , middle), which was confirmed by the increase in lung w/d ratio. we have previously reported that treatment with yiqifumai, whose main ingredient is rg, significantly attenuates lpsinduced microcirculatory disturbance in rat mesentery suggesting that yiqifumai is a promising regime for treatment of lps-evoked sepsis thanks to its multiple targeting potential for the initial steps of the process. the present experiment demonstrate that ginsenoside rb , the main ingredient of rg, may be specifically capable of attenuating the lps-induced lung injury. it is known that the number of mpo-positive cells in tissues is markedly relevant to tissue neutrophils accumulation and served as an index of inflammation, because mpo is an enzyme that is released mainly from neutrophils [ ] . the endothelial permeability thus increases due to the structure damage of the endothelial cells as well as to the enzymatic cleavage of adherent junction proteins [ ] , which eventually results in the transmigration of leukocytes across the endothelial lining into the surrounding tissues and the loss of plasma into extravascular space leading to severe hypoxemia and life-threatening edema in the lung [ , ] . the result of the present research demonstrated that treatment with ginsenoside rb reduced the accumulation of neutrophils in rat lung tissue exposed to lps. histological examination and mpo assay in lung tissue showed more extensive neutrophil infiltration in the ali group. meanwhile, lps is known to cause pulmonary inflammation in association with neutrophil sequestration in the pulmonary microvasculature, followed by adhesion and activation [ , ] . we therefore studied the effects of ginsenoside rb on expression of the adhesion molecule required for neutrophil recruitment in lung tissue. expression of icam- , which is an important adhesion molecule for neutrophil activation, markedly increased in the vascular endothelium after lps infusion [ ] . the sepsis-associated organ injury is further complicated by the implication of overproduction of cytokines such as tnf-α, mcp- and il- , which are believed to be pro-inflammatory factors, are produced by activated monocyte/macrophages, and acts mainly to attract neutrophils and monocytes [ ] . these chemokines are all associated with the influx, accumulation and activation of highly destructive cells involved in figure the effect of ginsenoside rb on the activation of nf-κb p in pmvecs. a. representative images for the effect of ginsenoside rb treatment on activation of nf-κb p in pmvecs. b. representative images of western blotting for the effect of ginsenoside rb treatment on activation of nf-κb p in pmvecs. c. effect of ginsenoside rb treatment on expression of nf-κb p in pmvecs. con: control group; lps: lps group; lps + rb : lps plus ginsenoside rb group. data were expressed as mean ± s.e of animals. * p < . vs. con group, # p < . vs. lps group. local inflammatory processes. as wang has reported ginsenoside rb significantly reduced excessive accumulation of inflammatory cytokines in the peripheral blood [ ] . we have acquired the similar outcomes in this study, ginsenoside rb alleviated remarkably the overproduction of inflammatory markers (tnf-α, mcp- and il- ). in contrast, ginsenoside rb treatment significantly attenuated the expression of these proinflammatory molecules compared with lps infusion alone. thus, these findings suggest that ginsenoside rb possesses potent anti-inflammatory properties, and hence is able to suppress lps-induced pulmonary tissue inflammation and injury. in line with these findings, the result of electron microscopy in the present study showed the gap of intercellular junctions apparently increased at min after lps infusion, whereas the endothelial cells themselves remained intact with ginsenoside rb treatment, implying that lps-induced plasma leakage observed in the present situation was mainly inhibited by ginsenoside rb . however, it has been reported that lps acts to upregulate the gene expression of il- , a proinflammatory chemokine, through the il- receptor-associated kinase intracellular signaling pathway, resulting in the activation of nf-κb [ ] . in animal model, our results showed that lps injection markedly promoted nf-κb p phosphorylation [ ] . in this study, lps-induced over phosphorylated nf-κb p was notably suppressed by ginsenoside rb , in vitro. these results demonstrated that the antiinflammatory property of ginsenoside rb was very likely mediated by inactivation of nf-κb phosphorylation. in this study, the beneficial effects found for ginsenoside rb may be attributed at least in part to decrease of nf-κb activity, which plays a crucial role in regulating the expression of proinflammatory molecules including tnf-α and mcp- . the molecular mechanism behind the protective effect of ginsenoside rb on the lps-induced lung injury is unclear and requires further identification, although the advantage of using ginsenoside rb is evident. role of the pulmonary epithelium and inflammatory signals in acute lung injury french congenital diaphragmatic hernia study group: pathophysiology of persistent pulmonary hypertension of the newborn: impact of the perinatal environment protection of vascular barrier integrity by activated protein c in murine models depends on protease-activated receptor- protection from lipopolysaccharide-induced pulmonary microvascular endothelial cell injury by activation of hedgehog signaling pathway protective effects of ginsenoside rb on septic rats and its mechanism attenuating effect of pretreatment with yiqifumai on lipopolysaccharide-induced intestine injury and survival rate in rat penehyclidine hydrochloride attenuates lps-induced acute lung injury 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improving effect of pretreatment with yiqifumai on lps-induced microcirculatory disturbance in rat mesentery attenuating effect of ginsenoside rb on lps-induced lung injury in rats the study was supported by beijing municipal health bureau youth research foundation (qn - ), beijing, china. the funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. study on railway workers chronic disease management network information platform no. z -c. the authors declare that they have no competing interests.authors' contributions qy carried out the lung histologic study, cytokines detection, immunohistochemical study and drafted the manuscript, y-wj participated in the molecular biology studies and cell culture. t-tm carried out the ultrastructure assessment. q-hf participated in the design of the study and performed the statistical analysis. lp conceived of the study, and participated in its design and coordination and helped to draft the manuscript. all authors read and approved the final manuscript. key: cord- -kkxdmzk authors: smirnova, s. s.; pisareva, m. m.; smirnova, t. d.; sivak, k. v.; vorobiev, k. v. title: long-term maintenance of the functional changes induced by influenza a virus and/or lps in human endothelial ecv- cell sublines date: - - journal: cell tissue biol doi: . /s x sha: doc_id: cord_uid: kkxdmzk influenza a virus and secondary bacterial infection may have remote effects in the form of cardiovascular complications or fibrosis in different organs. however, the mechanisms governing the development of complications remain poorly studied. the present work reports the comparative assessment of the functional changes which take place in human ecv- endothelial cell sublines obtained previously by the long-term culturing of cells after exposure to varying infectious doses (ids) of influenza a virus, and/or bacterial lipopolysaccharide (lps). it has been demonstrated that, in the course of long-term culturing (six passages) after exposure to pathogenic agents (influenza virus and/or lps), endothelial cells maintain changes in their migratory activity, permeability, and expression of mrna for cytokines tnfα and tgfβ (along with the changes in their proliferation activity, which has been demonstrated earlier). the pattern of changes depended on the type of the agent (agents) to which the cells were exposed. the differences in migratory activity (which was at its maximum h after wounding) between the cell sublines at the sixth passage correlated with the differences in their proliferation activity at the first passage (proliferation data were obtained previously). in particular, an increase in migration and proliferation was observed in the sublines exposed to low virus doses (ecv- id), as well as exposed to lps (ecv-lps), while the suppression of migration and proliferation was observed in the subline exposed to high virus doses (ecv- id). in the ecv- id, ecv-lps, and most notably in ecv- id + lps sublines, we detected an increase in the expression of mrna for cytokines tnfα and tgfβ, which, however, didn’t lead to the induction of apoptosis. we have also demonstrated an increase in cell permeability in the analyzed sublines, which was indicated by a decrease in the expression of the mrnas for the genes encoding occludin and zo- , the tight junctions proteins . this paper also reports an evaluation of the effects of the antiviral preparations rimantadine and alpisarin on the functional state of cell sublines. as a result, it has been demonstrated that these drugs may be able to prevent the development of the pathological changes caused by influenza a virus and/or lps in endothelial cells. the results obtained in the present work may be of use when studying the mechanisms of development of the influenza a virus and secondary bacterial infection complications. endothelial cells form a semipermeable barrier between circulating liquid and the surrounding tissues. changes in the endothelial barrier function contribute significantly to the development of different pathological states including inflammation and impaired wound sealing. moreover, they may contribute to the development of acute encephalopathies and organ dysfunctions (as a result of cardiovascular system involvement), organ fibrosis, increased angiogenesis, and oncogenesis (di pietro, ; you and stallcup, ) . disruption of the endothelial barrier in the lungs (in particular, as a result of influenza virus or bacterial infection) may cause liquid to penetrate into alveoli, resulting in pulmonary edema and development of acute respiratory distress syndrome (ards) (kwok et al., ) . impaired migration of endotheliocytes and changes in their proliferation and apoptotic activity, as well as altered cell permeability, are critical for the development of pathological states. abbreviations: ros-reactive oxygen species, id-infective dose, lps-lipopolysaccharide, moi-multiplicity of infection, fbs-fetal bovine serum, aj-adherence junctions, cldn -claudin- , ocld-occludin, tgfβ-transforming growth factor beta, tj-tight junctions, tnfα-tumor necrosis factor alpha, vegf-vascular endothelial growth factor. the ability of endothelial cells to regenerate a damaged intravascular layer relies on the activation of migration of the cells most closely located to the wound, as well as on strengthening of their capacity to proliferate. the process of cell migration includes cell polarization, formation of protrusions in the direction of movement, and detachment and contraction of the posterior cell margin, which are associated with dynamic reorganization of the actin cytoskeleton and microtubules involving signaling molecules from the rho family of small gtpases (kasa et al., ) . key physiological regulators of intracellular signaling pathways in endothelial cells are represented by reactive oxygen and nitrogen species (breton-romero, lamas, ) . endogenous hydrogen peroxide (h o ) accumulates in actively migrating endothelial cells at the site of injury caused by the inflammation inducers of different type, in particular, of bacterial or viral nature. the main source of reactive oxygen species (ros) are nox nad(p)h oxidases. endothelial cells are rich in nox and its homolog nox (basuroy et al., ) . a particularly important role in the stimulation of the signaling systems associated with endothelial cell migration is played by the vascular endothelium growth factor (vegf) which interacts with ros produced not only in the cytosol by nox and nox , but also in mitochondria (kim et al., ) . endothelial barrier is controlled by the locally produced vasoactive agents, such as histamine, prostaglandin, thrombin, and vegf. they modulate cell membrane permeability by promoting dynamic changes in adherens (aj) and tight junctions (tj), and regulate the centripetal cell tension as a result of the cortical actin layer reorganization and formation of actomyosin stress fibers (dejana et al., ; kasa et al., ) . an increase in endothelial cell migration in the presence of bacterial lipopolysaccharide (lps) is used as an indicator of the conversion of endothelial cells into activated fibroblasts via the endothelial to mesenchymal transition (sarmiento et al., ) . similarly to lps, influenza virus is also able to trigger ros production in epithelial and endothelial cells by means of nox and nox induction, which may also facilitate the reproduction of the virus itself (armatore et al., ) . it has been demonstrated that the infection of endothelial cells with influenza virus leads to the disruption of intercellular contacts and a decrease in the content of claudin- and claudin- (cldn ) proteins that form part of tjs (armstrong et al., ; short et al., ) . inflammatory response in the cells, induction of apoptosis, increase in cell permeability, and reorganization of cytoskeleton proteins are all performed by influenza a virus through the same cellular mechanisms and signaling pathways (rho/rock, p , and jnk), which form part of the mechanism of action of lps (zhang et al., ; . the data presented above were obtained for in vivo acute viral infection or during the early period ( - h) after the exposure to lps or high infectious doses (ids) of influenza a virus in vitro. however, we haven't found any data on the effects of simultaneous exposure of cell cultures to influenza virus and lps. earlier, we have for the first time demonstrated an increase in proliferation of the influenza virusinfected human ecv- endothelial cells, which are nonpermissive for the influenza virus, with the decrease in the virus id for - days after the infection, which was accompanied by an increase in cell apoptosis. normal reproduction of influenza virus is impaired in nonpermissive cells, and the presence of virus is detected only at the level of the viral surface protein mrna during no more than one or two passages (danilenko et al., ) . the change in the physiological state of the cells at the first passage ( - days) was the first stage in the changes induced in the cells by influenza virus, with further cell culturing (passages to ) resulting in the stably increased proliferation at low apoptosis levels (smirnova et al., ) . the aim of the current work was to find a link between the primary exposure of ecv- cells to the agents (high and low influenza virus ids and/or lps) and the pattern of remote functional changes in the ecv- cell sublines at the sixth passage after the exposure. we have analyzed the changes in the migratory activity of the cells and gene expression at mrna level, including the genes encoding tumor necrosis factor alpha (tnfα), transforming growth factor beta (tgfβ), and nox , as well as the proteins forming part of tj and controlling the permeability of cell membranes, cldn , occludin (ocld), and zo- . additionally, we assessed the correlation between the functional changes and the proliferation activity and apoptosis in cells of sublines that were analyzed previously (smirnova et al., ) , as well as evaluating the ability of rimantadine and alpisarin antiviral preparations to restore the normal functioning of endothelial cells. sublines of the passaged human endothelium ecv- cell line obtained after the infection with the influenza a virus brisbane/ / (h n ) were used in the work. cells were infected with the low virus dose ( id) corresponding to the multiplicity of infection (moi) equal to . or high virus dose ( id, moi = . ) and additionally (or exclusively) exposed to ng/ml of the escherichia coli lps (sigma, united states). cell were passaged on the sixth to seventh day after infection by splitting them in a ratio of : using the alpha-mem medium (biolot, russia) containing % fetal bovine serum (fbs) (hyclone, united states) without antibiotics. to detach cells from the surface, a solution containing vercene and chymopsin was used. both control (intact) and experimental cells were at the sixth passage after the exposure to the agents (smirnova et al., ) . depending on the agent (virus and corresponding id, lps, or virus and lps together), the cell sublines were given the following names: ecv- id, ecv- id, ecv-lps, ecv- id + lps, and ecv- id + lps. assessment of migratory activity. cell suspensions of the corresponding sublines were inoculated into the wells of six-well plates (nunc, denmark) in a concentration of × cells/ml ( ml per well) in the alpha-mem medium with % fbs, and the plates were placed into the co incubator ( °c and % co ). the next day, the cell monolayer was scratched with a -μl tip, cells were washed with serum-free medium, and alpha-mem medium with % fbs was added to the cells. an amount of . ml ( / medium volume) of tenfold solutions of each of the two analyzed antiviral preparation were applied into the wells. immediately after the application of the antiviral preparations (point zero) and then , , , and h after the application, the selected region of the wound surface was imaged using the axio vert a microscope (carl zeiss, germany) with × magnification. with the aid of the axiovision rel. . software, the wound surface area was calculated in the obtained images, and the change in the surface area with time, i.e., wound sealing, was determined. the end result was expressed as a percentage relative to the change in the wound surface area in the control samples (which were not subjected to infection and treated with antiviral drugs) at each time point. analysis of gene expression. on the fourth day after wounding (when the wound was almost completely sealed), cells were removed from the well surface, counted, washed with the serum-free medium, centrifuged, and used to assess the changes in the expression of the genes encoding cytokines and other cellular factors by real-time pcr. rna was isolated from the analyzed samples using the amplipraim ribo-prep kit (central research institute of epidemiology, russia) according to the manufacturer's instructions. to assess gene expression, cdna was synthesized on the rna template in reverse transcription reaction using the reverta-l kit (central research institute of epidemiology, russia). amplification reaction was carried out in the rotor-gene thermal cycler (corbett research, australia) according to the following program: denaturation at °c for min, cycles including °c for s, then °c for s, and °c for s, each. the reaction mixture contained μl of dna, an amount of μl of ready-made . × pcr buffer (sintol, russia), . μl of syntaq dna polymerase ( u/μl), and . μl of each primer and probe ( pmol/μl) (dna-sintez, russia) in μl. primer for the actin gene (actb) were used as an internal standard. the nucleotide sequences of the oligonucleotide primers and probes for tnfα, tgfβ, nox , cldn , ocld, zo- , and actb are provided in table . expression levels were determined for two or three times, and the end result was expressed in relative units, indicating a change in gene expression relative to the control, and calculated according to the for- table . nucleotide sequences of primers and taqman probes used to analyze mrna expression for the genes encoding cytokines, tight junction proteins, and other cellular factors forward primer '- ' probe '- ' reverse primer '- ' mula -ΔΔct , where c t is the cycle threshold value for the analyzed sample (wong and medrano, ) . plants, russia) solutions were prepared using serumfree alpha-mem medium and were used in final concentrations of μg/ml and μg/ml, respectively. at each time point, two wells were analyzed. experiments were performed not less than in triplicates. statistical data analysis was performed using the mann-whitney u test for the comparison of the two groups of nonparametric samplings. differences were considered significant with p < . . analysis of cell migration was performed using the previously obtained ecv- cell sublines (smirnova et al., ) at the sixth passage after the exposure to influenza virus and/or lps treatment. migratory activity was assessed by the change in wound surface area with time (wound sealing). as a result, we have demonstrated that the maximum migratory activity is observed in all cell sublines (compared to the control intact cells) during the first h (here and in what follows, time is given in hours after wounding). cell migration grew slower by the th- th hours and remained almost at the -h and control levels up to the th hours (fig. a) . the comparison of different sublines showed that highest migratory activity was exhibited by the endothelial cells after the contact with the minimum infective dose of influenza a virus (ecv- id subline, migratory activity % of the control activity h after wounding). the migration exhibited by the cells infected with the high virus dose (ecv- id) was the minimum ( % of the control h after wounding). the cells after the contact with lps (both in the case of individual exposure and together with the virus) showed slightly increased migratory activity during the first hours after wounding ( - h), while later migration remained at the control level (fig. a) . to assess the effects of the antiviral preparations (rimantadine and alpisarin) on the migratory activity of the cells, the preparations were applied to the wells containing cells immediately after wounding. as a result, it was demonstrated that they significantly affect cell migration. rimantadine decreased the migratory activity of the control cells by % h after wounding, while alpisarin decreased it by %; however, migration was restored to the control level h after wounding. rimantadine had almost no effect on the migration of the ecv- id subline cells ( % in h), while the level of ecv- id subline cell migration in the presence of this drug grew higher than the control level (up to % in h). alpisarin caused even higher increase in the migratory activity of these subline cells in h (up to % in ecv- id subline and up to % in the ecv- id subline). the highest increase in cell migration was caused by both preparations in the sublines treated with lps, namely, ecv-lps (up to % in the case of rimantadine and % in the case of alpisarin), ecv- id + lps ( and %, respectively), and ecv- id + lps ( and %, respectively) . in all the sublines, maximum migration level in the presence of the antivirals was also observed h after wounding, together with a decrease in the level of migration by eighth hour; however, in this case, migratory activity remained higher than in the control (fig. a) . we have also revealed that the pattern of differences in the migratory activity between the cell sublines at the sixth passage at the maximum activity point ( h after wounding) correlated with the pattern of changes in proliferation activity of the cells at the first passage (proliferation data were obtained previously (smirnova et al., ) ). in particular, the enhancement of cell migration and proliferation was observed in the ecv- id and ecv-lps sublines, while enhancement of suppression was observed in the ecv- id subline (figs. b, c) . comparison of the migration and proliferation patterns for the cells at the sixth passage showed that the similarity was retained for most sublines, except for the ecv-lps subline, in which proliferation activity decreased at the sixth passage, and the ecv- id subline, in which, by contrast, an increase in proliferation was detected (figs. b, c) . therefore, the differences in the migratory activity of the cells in the analyzed sublines at the sixth passage are determined by the initial (at the first passage) effects of the agents (high or low doses of the virus and/or lps). on the fourth day after wounding (when the wound was almost completely sealed), the analyzed subline cells were detached from the surface of the six-well plates and used to determine the expression levels of mrnas for cytokines (tnfα and tgfβ), nox oxidase, and tj proteins (ocld, zo- , and cldn ) (ecv- id and ecv- id + lps sublines were not used). pcr has revealed an increased expression of mrnas for the cytokines tnfα and tgfβ in the cells that were exposed to the virus and/or lps, with the highest level of expression for these cytokines having been observed in the ecv- id + lps subline cells ( and times higher, respectively, than in the control). treatment with the antiviral preparations rimantadine and alpisarin considerably decreased the expression of the mrnas for these cytokines enhanced by the exposure to virus and/or lps, although, rimantadine on its own stimulated the expression of tnfα mrna ( times higher than in the control) (fig. ) . a slight increase in the expression level of nox mrna was detected only in the ecv- id subline ( . times higher than in the control), while antiviral preparations reversed it to the control levels. the expression of the mrna for the cldn gene encoding the tj protein was at the control level in all cell sublines except for ecv- id + lps, in which cldn mrna expression was . times higher than in the control. the treatment with antiviral preparations increased mrna expression level for the gene encoding this protein in all sublines, and especially so in the case of alpisarin (with the maximum increase-by . times-being in the ecv-lps subline), which may indicate that cell permeability returns to its normal level (fig. ) . in a separate experiment, we assessed the expression levels of mrnas for the genes encoding other proteins within the tj family, namely, ocld and zo- . the obtained results revealed that the expression level of mrnas for these genes gradually decreased in ecv- cells from the first passage to the fifth passage after a single exposure to different doses of influenza a virus and/or lps, this effect being clearer in the case of ocld than in the case of zo- (fig. ) . these data provide evidence that the exposure to influenza virus and/or lps leads to an increase in the permeability of cell membranes, which is further maintained in the analyzed cell sublines for a long period of time. angiogenesis plays an important role in many physiological and pathological processes, while apoptosis serves as one of the regulatory factors of angiogenesis. the ability of the highly virulent avian influ- enza virus strains to cause a lethal disease in chicken as a result of apoptosis of the endothelial cells in different organs is well-known (swayne, ) . however, the pathological mechanisms of influenza infection, in particular, the link between the infection and apoptosis and barrier function impairment, are not always clear. it has been demonstrated in vitro that human influenza a viruses are able to trigger tnf-induced apoptosis of endothelial cells (sumikoshi et al., ) . at the same time, increased cell permeability in human microvascular endothelium (hmvec) and association with apoptosis has been demonstrated only for live (infectious) influenza a virus; uv-inactivated avirulent influenza virus also caused an increase in cell permeability, but in this case not as a result of apoptosis, but rather as a result of the degradation of the tj protein cldn- (armstrong et al., ) . the role of the cytokines tnfα and tgfβ in both apoptosis induction and cell barrier function impairment is well-known, although these processes often develop independently of each other (petrache et al., ; meeteren and ten dijke, ). in our previous work, we have demonstrated very low levels of apoptosis, just as in control cells, in all the obtained endothelium cell sublines cultured during more than three passages (six passages, in particular). at the same time, the antiviral preparations rimanta-dine and alpisarin reduced the increased proliferation of the ecv- id subline cells at the sixth passage and simultaneously stimulated apoptosis in all the analyzed sublines (smirnova et al., ) . the comparative study of the human endothelial ecv- cell sublines carried out in the present work and in our previous work (smirnova et al., ) has demonstrated that the infection of nonpermissive cells with influenza a virus (both in high and in very low doses) and exposure to lps can change migratory, proliferation, and apoptotic activity of cells and impair cell barrier function. we have shown for the first time that these functional changes may be observed during a long period of cell culturing after the exposure. in the analyzed cell sublines, the expression of mrna for ocld and zo- encoding the proteins that form part of the tj complex gradually decreased with an increase in the passage number, which indicated an increase in cell permeability (kasa et al., ) . the results obtained in the present work, as well as in the previous one (smirnova et al., ) , show that all sublines of endothelial cells acquire resistance to apoptosis in the process of culturing (after the third passage), notwithstanding the high level of expression of mrna for both tnfα and tgfβ, especially in the ecv- id + lps subline. this indicates a link between the cytokines tnfα and tgfβ and the increase in cell permeability, but not the induction of apoptosis in this case. this conclusion is supported also by the fact that, in the presence of the antivirals rimantadine and alpisarin, expression of tnfα and tgfβ genes is inhibited and cell permeability is restored (due to the increase in the cldr mrna expression) with a simultaneous increase in the apoptosis level, which also indicates that these drugs are capable of restoring the functional state of the cells. it is characteristic that the level of nox mrna expression appeared to be only slightly increased and only in a single subline, which was obtained after the infection with the low dose of influenza virus (ecv- id) and differed from all others in its increased proliferation level. this allows it to be suggested that the dysfunction of cells in the analyzed sublines that develops over the period of their long-term culturing may be caused by the increase in ros content not only in the cytosol (produced by nox and nox oxidases), but, apparently, to a greater extent in mitochondria (harel et al., ) . the multifunctional cytokine tgfβ can enhance cell proliferation and apoptosis resistance through the alk smad / /nf-κb signaling pathway, which induces the transformation of endothelial cells into miofibroblasts (van meeteren and ten dijke, ; montorfano et al., ) . tgfβ is considered to be the key cytokine participating in fibrosis. reduced sensitivity of lung fibroblasts to fas-induced apoptosis (resistance to apoptosis) with increased alveolar epithelial cell apoptosis level is a characteristic feature of pulmonary fibrosis, in which the regions of a disrupted epithelial cell layer with reduced reparation capacity are filled with apoptosis-resistant fibroblasts (maher et al., ; liu and desai, ) . our findings (increased expression level for tgfβ mrna along with the development of apoptosis resistance) provide support for the idea that there is a link between influenza virus infection and, especially, lps treatment with myofibroblast formation and the possibility of pulmonary fibrosis development. thus, the addition of lps at the time of ecv- cell infection with influenza a virus leads to a more serious dysfunction of endothelial cells, in the same way as a secondary bacterial infection, even in the case of mild influenza infection leads to serious and, in many cases, complications that occur much later (mccullers, ) . our data also indicate that endothelial cells infected only with influenza a virus may become atypical (demonstrate increased proliferation activity and an extremely low apoptosis level), which may facilitate the expansion of these cells and hinder the reparation of damaged blood vessel regions with normal endothelial cells. this may lead to enhanced angiogenesis, which in turn promotes oncogenesis, and may possibly cause intravascular lesions of unknown origin with the characteristics of pseudotumor hyperplasia (diaz-flores et al., ) . to summarize, the results of the present work have revealed that under our experimental conditions (the use of varying virus doses and/or lps with subsequent long cell culturing), changes in endothelial cell functioning can be observed for a long period of time after the exposure. we have also demonstrated that there is a connection between the agent used (virus and/or lps) and the direction of functional changes (increase or decrease) in migratory and proliferation activity and cell permeability. the antiviral preparations rimantadine and alpisarin have a substantial effect on all the analyzed live processes in the cell sublines, which indicates that these preparations may pre- the authors declare that they have no conflict of interest. this article does not contain any studies involving animals or human participants performed by any of the authors. influenza virus replication in lung epithelial cells depends on redox-sensitive pathways activated by nox 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ros-induced ros release orchestrated by nox , nox , and mitochondria in vegf signaling and angiogenesis anti-inflammatory effects in indirubin derivatives on influenza a virusinfected human pulmonary microvascular endothelial cells reciprocal regulation of tgf-b and oxygen species: a perverse cycle of fibrosis molecular mechanisms in lipopolysaccharide-induced pulmonary endothelial barrier dysfunction insights into interaction between influenza virus and pneumococcus oxidative stress mediates the conversion of endothelial cells into myofibroblasts via a tgf-b and tgf-b -dependent pathway differential effect of mlc kinase in tnfa-induced endothelial cell apoptosis and barrier dysfunction endotoxin-induced vascular endothelial cell migration is dependent on tlr /nf-kb pathway, nad(p)h oxidase activation, and transient receptor potential melastatin calcium channel activity change in the functional state of cells of the human endothelial cell line ecv- under the influence of influenza a virus, lipopolysaccharide from e. coli, and some drugs human influenza virus infection and apoptosis induction in human vascular endothelial cells understanding the complex pathobiology of high pathogenicity avian influenza viruses in birds real-time pcr for mrna quantitation location of vegf to vascular ecm is an important aspect of tumor angiogenesis, cancers p mapk, rho/rock and pkc pathway are involved in influenza-induced cytoskeletal rearrangement and hyperpermeability in pmvec via phosphorylating erm key: cord- -qfnukav authors: nan title: irish thoracic society annual scientific meeting, ramada hotel, belfast: th– th november date: - - journal: ir j med sci doi: . /s - - -y sha: doc_id: cord_uid: qfnukav nan welcome to the irish thoracic society annual scientific meeting . we are delighted that the meeting has made a return to belfast this year and in honour of this we've put together a programme that we feel sure will make for a highly interesting and worthwhile experience. a central feature will be the presentation of original research in both oral and poster form, showcasing the wide range of important and innovative work being carried out throughout the island. the focus of this year's symposium is lung cancer and we are delighted to welcome a panel of leading international speakers who will share their knowledge and insights on this important topic. additional guest lectures on ciliary dyskinesia and asthma by distinguished specialists in both fields complete a varied and, we hope, highly stimulating programme. it is my pleasure to welcome you to the irish thoracic society annual scientific meeting . on behalf of the irish thoracic society i wish to thank dr. jackie rendall for her outstanding work in conjunction with the its office in organising this year's programme. thanks to her sterling efforts we look forward to a rewarding and stimulating meeting. has been a busy year for the irish thoracic society and i would like to take this opportunity to reflect on some of the highlights. february saw the publication of the inhale report, nd edition (ireland needs healthier airways and lungs -the evidence). compiled by the its in conjunction with dr neil brennan and dr terry o'connor, the report underlines the serious resource deficits that still exist in respiratory health-care. clearly a lot more work is needed in this area and the society will continue advocating for a respiratory strategy to tackle the imbalance. throughout the year the society has made representations on a broad range of issues including copd, tuberculosis, critical care services and lung cancer. with respect to the latter, the irish thoracic society lung cancer sub-committee has been working with the national cancer control programme towards the development of improved services for lung cancer care. the society has also been represented on the national copd strategy group and the national tb advisory committee and we look forward to the respective reports on this work. significant headway has been made in the area of education and research. the irish thoracic society boehringer ingelheim research fellowship, launched last year, has recently been awarded for a second time, promising valuable contributions to respiratory research from two very worthy projects in the coming years. our ability to communicate with members has improved dramatically thanks to a radical upgrade of the irish thoracic society website -www.irishthoracicsociety.com. this provides information on the society's activities and other relevant issues. many delegates will have become familiar with its facilities for on-line registration and submission of abstracts in the lead-up to the meeting and we trust they have proven convenient and user-friendly. password protected members areas are designed for more specialist interest content and we encourage members to help us develop these areas further as a resource for sharing information and discussion of issues. we recognise the important role our members continue to play in all these activities. in order to sustain our efforts, the continued support of members and the expansion of our membership base is vital. i would also like to take this opportunity to thank our partners in the pharmaceutical and medical equipment sectors. their support over the years has been central to the society's development and is now more important than ever -we look forward to continued collaboration in and beyond. dr. jj gilmartin, president, the irish thoracic society patient's with copd are known to have decreased levels of activity. this study looks at free-living activities as measured by the sense-ware armband to determine if there was a relationship with standard exercise field tests for this patient population. thirty one patients with copd were recruited: men (n = ), female (n = ). ethical approval and written consent was obtained. the senseware Ò armband was worn for seven consecutive days and distance on the shuttle walk test was measured. pearson's correlations were undertaken using spss version . the mean age of the study population was . yrs (± . ) with a mean fev % (± ). the shuttle walk test (swt) was m (± ). free-living activities as measured by; physical activity level (pal) of . patients low levels of activities in daily life as measured by the senseware Ò armband parameters correlates with the shuttle walk test making it a reliable outcome measure. the shuttle walk test can provide us with valuable insight into the patient's free-living activities and sleep pattern. international literature points to inequity in the provision of palliation in favour of patients with cancer despite the high mortality associated with copd. chronicity is associated with biographical disruption, loss and shifting relationships with healthcare professionals all of which may influence the nature of palliative care in advanced copd. a three phased project is underway aimed at developing palliative care for patients with copd. the purpose of phase one is to identify palliative care needs of these patients. a mixed method research design was employed for phase one involving health status measurement and qualitative interviews. inclusion criteria were those patients who were hospitalised for exacerbation of copd. the patient sample was mainly derived from one hospital over a one year period with a primary diagnosis of copd. gatekeepers were in place to protect confidentiality. data was collected using the st george's respiratory questionnaire(sgrq), hospital anxiety and depression scale (hads), and the medical research council (mrc) dyspnoea scale. in a nd round of interviews, the questions are open and semi-structured. interviews were undertaken in patients' homes. an integrated analysis is underway of data represented in different forms using spss and nvivo software. twenty six patients were interviewed. ages ranged from - yrs (mean = ). anxiety and depression scores averaged . and . respectively. scores of [ indicating moderate and severe levels were found in cases for anxiety and for depression. average sgrq scores = . indicating significant impact on quality of life. themes from qualitative data include a catastrophic diagnostic event along the illness trajectory, ambivalence towards opd visits and rigid daily routine to control breathlessness. issues emerged regarding recruitment, the construing of palliative care in copd and articulation of experiences of quality of care. conclusion: preliminary findings suggest significant disability and lay expertise; isolation; anxiety; impact on relationships and poorly articulated fears of the future. unmet palliative care needs are evident and challenge nursing to find appropriate ways of construing palliative care in copd. patients with severe copd are likely to have repeated exacerbations and early mortality. in ventilated patients herpes simplex virus- (hsv- ) is frequently identified and is associated with an increased mortality. we determined the frequency of hsv- in copd patients (stable and exacerbated) and if it was associated with disease severity and mortality. methods: stable and exacerbated copd patients were recruited. spirometry was performed. sputum was obtained and lysed by ddt. nucleic acids were extracted and specimens were tested for hsv- and gapdh using real-time pcr. results: one hundred and thirty six patients with exacerbations of copd and stable patients were recruited. hsv- was detected in % of copd patients during an exacerbation and % of stable copd patients. no significant differences in hsv- copy numbers were seen on comparison of these groups. detection of hsv- was associated with increasing copd disease severity, p \ . . the presence of hsv- during exacerbations was associated with increased mortality, p \ . , predominantly from respiratory causes, p = . . conclusion: hsv- is frequently detected in the sputum of copd patients. it is more commonly found in patients with severe airways disease and its presence during exacerbations is associated with increased mortality. pulmonary rehabilitation (pr) is associated with symptomatic and physiologic improvements in patients with copd. however, biologic effects on systemic inflammatory and profibrotic cytokines are unproven. thirty two patients with moderate or severe copd (age . ± . y, fev ± . % predicted) were recruited to a pr programme. cardiopulmonary exercise testing was performed before and after the programme. serum c-reactive protein (crp), tumour necrosis factor-alpha (tnf-a), interleukin- (il- ), transforming growth factor-beta (tgf-b) and oxidative burst were measured before exercise, at peak exercise and at recovery. there were statistically significant improvements in all domains of the st georges respiratory questionnaire, chronic respiratory disease questionnaire and hospital anxiety and depression questionnaire. there were no significant changes in crp or tnf-a associated with exercise or pulmonary rehabilitation. exercise was associated with a surge in oxidative burst. endurance exercise was associated with an increase in il- (p = . ) that was attenuated by pulmonary rehabilitation (p = . ). incremental exercise was associated with an increase in tgf-b (p = . ) that was attenuated by pulmonary rehabilitation (p = . ). demonstrating biological effects of pr has proved elusive to date. this is the first study to demonstrate modulation of both circulating inflammatory and profibrotic cytokines by pr in patients with copd. queen's university, royal victoria hospital, belfast city hospital, n. ireland beta cryptoxanthin is a pro-vitamin a carotenoid which is reported to be a good biomarker of fruit and vegetable intake. we hypothesised that levels of serum beta cryptoxanthin would be related to fev . in , men aged to years were recruited into the belfast arm of the prospective epidemiological study of myocardial infarction (prime). we describe the cross-sectional analysis of the men who had a valid spirometry trace and plasma sample at year follow-up. beta cryptoxanthin levels were measured using hplc analysis. fev values at years were modelled using simple linear regression, and adjusted for covariates. serum beta cryptoxanthin levels were positively correlated with fev (r = . , p \ . ). for each nanomole per litre increment in serum beta cryptoxanthin levels, fev was . mls greater. following adjustment for the covariates, for each nanomole per litre increment in serum beta cryptoxanthin levels, fev was . mls greater ( %ci . to . , p \ . ). serum beta cryptoxanthin levels are positively correlated with fev . this suggests that in this population a moderate increase in serum beta cryptoxanthin levels (achievable by a modest increase in dietary intake of fruit and vegetables) may have a protective effect on lung function. the study was undertaken to evaluate organ utilization in the republic of ireland. a retrospective review of potential donors from january to august was performed. donors organ were selected according to criteria set by the international society of heart and lung transplantation (ishlt), abo group compatibility and predicted tlc. this included a donor age \ years old, a satisfactory history, a normal chest radiogram, arterial blood gases (abg) of [ kpa ( mmhg) (fio of % and a peep of ). potential offers for organ donation occurred. the median donor age was years (range - ), the mean period of mechanical ventilation was days (range [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . sixty percent ( offers) were declined on the basis of ishlt criteria, % were declined because of age, % due to poor blood gases, % due to abnormal chest x-ray and % because of chest trauma. sixty-three offers were evaluated at the donor site. five percent ( offers) were excluded because of size and hla crossmatch constraints, these were offered to uk transplant. lungs ( . %) were successfully transplanted. organ donation in republic of ireland is high ( per million population), however lung utilisation is low. the pathway to increase number of lung transplantation may include a framework for optimising donor physiology. the use of endoscopic ultrasound with fine-needle aspiration (eus-fna) is well established in diagnosing and staging non-small cell lung cancer with positron emission tomography (pet) positive posterior mediastinal lymph nodes. the sensitivity of eus-fna ranges between and %. it is less invasive and has lower complication rates when compared to surgical staging of mediastinal nodes. this study aims to describe our initial experience of eus-fna in lung cancer. eus-fna was used prospectively for the assessment of pet positive mediastinal lymph nodes between january and july . when eus-fna did not show malignant invasion, a confirmatory mediastinoscopy was done. endpoints were performance of eus-fna, morbidity and length of hospital stay. patients underwent eus-fna during the study period for both diagnosis and staging. patients had positive lymph node invasion and had no evidence of malignant invasion on eus-fna. negative cytology on the latter was confirmed on mediastinoscopy giving eus-fna a sensitivity of % for the study period. it upstaged the disease in patients. eus-fna is reliable, non-surgical tool for mediastinal staging. it reduces the need for surgical staging procedures in lung cancer patients with suspected mediastinal involvement. the limitation of this study is the poor documentation of the lymph node stations that were sampled. the role played by the innate immune system in determining survival in non-small-cell lung cancer (nsclc) is unclear. the aim of this study was to investigate the prognostic significance of cytotoxic t-lymphoctye infiltration in nsclc. immunohistochemistry was used to detect cd + t-lymphocytes in the tumor islets and tumor stroma in patients with surgically resected nsclc. quantification of immune infiltration was performed using a novel automated image analysis algorithm. univariate cox regression analysis, kaplan-meier analysis and the log-rank test were used to illustrate differences in overall survival according to the expression of tumour to stroma lymphocyte infiltration ratio. lymphocytes were detected in both the tumour islets and stroma in all patients. we used the median of tumor:stroma cd + infiltration ratio as a threshold to dichotomise patients to either high or low infiltration rate. results showed that those with a higher intratumoral lymphocyte infiltration had a significantly better survival compared to those with a low tumour/stroma infiltration ratio (p \ . ). microlocalization of infiltrating cytotoxic t-lymphocytes is a powerful predictor of outcome from surgically resected nsclc. the biologic explanation for this and its implications for the use of adjunctive treatment require further evaluation. . ebus-tbna for lung cancer -initial irish experience accurate mediastinal staging is essential in lung cancer patients under consideration for surgical resection. compared to mediastinoscopy or l anterior mediastinotomy (the gold standard), ebus-tbna is less invasive and may be carried out during diagnostic bronchoscopy, offering the potential for one-stop diagnosis and staging. ebus commenced in sjh in mid . we retrospectively analysed the first cases, ( %) of whom had tbna. patients with known or suspected lung cancer had ebus-tbna of n glands. cytology was positive for malignancy in / ( %). in the patients where cytology was negative, had no further follow up. of the other , only were proven node positive, but were confirmed node negative at mediastinoscopy/surgery and had resolution of nodes at repeat ct. therefore, in these patients ebus-tbna had a negative predictive value of %, sensitivity % and overall accuracy %. in patients with primary paratracheal mass, ebus-tbna was positive in all . of a total lung cancer cases where ebus-tbna was positive, this was the only positive sample in ( %). in % of patients ebus-tbna obviated the need for more invasive sampling of the mediastinum-this is one-stop diagnosis and staging. the national patient safety agency (npsa) (may ) highlights the role of ultrasound guidance for pleural drain procedures. we review our practice from a district general hospital. pleural studies were identified from the hospital pas and the radiology database. case notes of those who had their effusions drained by catheter were studied. patients (m:f : ; age - [mean . ]) had drains placed in the period august through july . were in patients; electively admitted for the procedure and remained an outpatient. ( %) drains were sited under ultrasound guidance, ( %) placed on ward ( of these marked in ultrasound) and ( %) sited in ct. patients had single drain placement, had drains; had drains and patient had drains. ( %) drains were placed by career radiology staff, ( %) by ward based staff. consent was documented on ( %) occasions. ( %) drains were flushed regularly as instructed by radiologist. average time in situ was days (range - days). ( %) patients were discharged home with drains in situ. patients died with drains in place. drain removal was performed by hospital ward staff. we conclude that most drain placements conform to npsa recommendations. consent is poorly documented. this will be addressed by implementation of a chest drain management chart. protocol for the care of pleural-sited catheters has been written. ultrasound is more sensitive than clinical examination or chest x-ray for determining the presence of pleural fluid and helps guide thoracentesis. we report our initial experience with chest ultrasound examinations performed at the bedside by the respiratory consult service for assessment of pleural effusion. ( %) of referrals were from the oncology/haematology service, ( %) from other medical teams and patients from general surgery. on ( %) of ultrasound examinations pleural fluid was detected and drainage was performed. in / ( %) fluid was drained by aspiration alone and in the remaining ( %) a seldinger chest drain was placed. patients ( %) had malignant or paramalignant exudative effusions ( with lung primary; with metastatic cancer from other sites and with lymphoma), ( %) had parapneumonic effusions and there were transudates, hemothorax and unexplained exudates. the procedure was well tolerated by all patients. one patient had a small post-aspiration pneumothorax on chest x-ray that required no further intervention and there was one drain misplacement. ultrasound is safe, portable and sensitive for detection of pleural fluid. it can be used at the bedside by respiratory physicians to guide management of pleural effusion. in an effort to standardise treatment of primary spontaneous pneumothorax (psp) and secondary spontaneous pneumothorax (ssp), the british thoracic society (bts), in , published the first evidence based guidelines for management of this condition. the objective of this audit was to assess compliance with the bts guidelines in amnch. retrospective hipe data, chart and radiology review of all spontaneous pneumothoraces admitted to amnch during . there were spontaneous pneumothoraces admitted to hospital in the year studied ( psp's and ssp's). of the psp's: mean age was . years; male:female ratio was : ; were classified as large and as small; attempted aspiration; intercostal drains were inserted with an average drain time in situ of days; mean drain calibre was fr. of the ssp's: mean age was . years; male:female ratio was : ; underlying pulmonary disease was copd in and cystic fibrosis in ( patients); were classified as large and as small; none were aspirated; intercostal drains were inserted with an average drain time in situ of days; mean drain calibre was fr. conclusion: bts guidelines are not being adhered to in amnch. in particular, aspiration, which is the recommended first line treatment in psp, is an underutilised therapeutic procedure. on average, the calibre of intercostals drain used was too large and not in keeping with the guidelines which recommend initial placement of small calibre ( - fr) drains. it is important to note that our data reflects patients admitted to hospital, and does not include patients managed and discharged from the emergency department. penetrated chest trauma is potentially fatal if not promptly addressed. we aim to review the incidence, demographics and the outcome of patients with napct who attended a single level-iii trauma centre. we conducted a retrospective study of all napct's presented to cork university hospital between - .our definition of napct is non accidental injury of chest involving sharp objects penetrating skin and muscular layers with or without injury to deep structure above the level of the diaphragm requiring hospital admission. patients with napct wounds were admitted to c.u.h from january to december . there were females ( . %) and males ( . %) with a median age of years (range - ). the average length of stay in hospital was days.the highest incidence was . / population (n = ), which was in . there was a decreasing trend in the following nine years. the incidence in was . injuries / population (n = patients). the incidence of napct's was low and the annual incidence is decreasing over the last years. young men were the commonest victims. pleural infection is a difficult management issue with approximately % patients requiring surgical intervention and a % mortality rate. early diagnosis and appropriate therapy is vital to reducing morbidity, mortality and health care costs. this is an audit of the management of pleural infection in the ulster hospital from february to april as per bts guidelines. pleural infection was diagnosed as pleural fluid with ph \ . , ldh [ iu/l, glucose \ . mmol/l. eight patients were identified, ( . %) male with a mean (sd) age (± ) years. all patients had pleural fluid sampling within hours of suspected infection. all patients had chest drains inserted with median (iq range) duration of drainage ( - ) days. positive bacterial culture from pleural fluid was obtained in ( %) patients (haemophilus influenzae, enterococcus) and from sputum in ( . %) patients (h.influenzae, coliforms, klebsiella). antibiotic therapy was as per bts guidelines. median (iq range) hospital stay was ( - ) days while surgical intervention was required in ( . %) patients. one patient died at day from a non-respiratory complication. this audit demonstrates appropriate management of pleural infection but shows that pleural infection remains a difficult management problem with significant morbidity and mortality. twenty-two patients died awaiting lung transplantation. the highest mortality rate was among those patients with idiopathic pulmonary fibrosis n = . the mode of death was acute exacerbations of ipf. cf patients, emphysema patients and sarcoid patient, patient with bronchiectasis died awaiting lung transplantation. these data indicate that the mortality rate of patients awaiting lung transplantation is highest amongst patients with idiopathic pulmonary fibrosis suggesting that early referral on the basis of a gas transfer less that % predicted as per international guidelines should be considered. approximately lung cancers are diagnosed annually in northern ireland, however despite recent advances in the management of this disease little impact has been made on survival rates . the short interval from diagnosis to death and the complex problems that are a reality for many of these patients make it imperative that health care professionals gain a greater understanding of the experiences of those living with this disease. this qualitative study aimed to explore the experiences of patients and carers living with a lung cancer diagnosis within a northern ireland context. a secondary aim was to describe participants' experiences of service delivery in order to identify any areas for improvement. a semi-structured in depth interview process was used and a purposive sample of participants was identified, comprising patients with advanced lung cancer and carers. the central emerging theme of ''living with dying'' highlighted the struggle of dealing with a poor prognosis and the pervasive uncertainty associated with an unpredictable disease trajectory, whilst trying to maintain some quality of life and positivity. the findings confirm the need for effective support and information pathways that provide timely and responsive services which embrace the holistic needs of patients and carers. the total number of patients was and complete data was available on ( %) patients. the mean age was ( - ). there were ( %) male and ( %) female patients. the mean duration from first radiological investigation suspicious for lung cancer to first procedure, first diagnostic procedure and first treatment regardless of modality were , and days respectively. the mean interval from first procedure to first diagnostic procedure, and mean interval from first diagnostic procedure to first treatment were and days respectively. there were ( %) patients with confirmed cancer diagnosis and ( %) of them went for curative surgery. in conclusion, our practice in management of lung cancer is consistent with the bts guidelines. transbronchial needle aspiration (tbna) is a bronchoscopic technique that enhances the diagnosis and staging of patients with thoracic cancers and other diseases. while endobronchial ultrasound guided-tbna (eus-tbna) is the gold standard, many units do not have access to the technology and expertise required. this study explored the clinical utility of tbna in the diagnosis and staging of patients with thoracic cancer in a university hospital. we studied the yield of tbna in patients diagnosed with thoracic cancers in our institution from september st to august st . from patients with thoracic cancer, patients had tbna performed. eleven specimens ( %) were diagnostic ( from hilar nodes and from subcarinal nodes) and of the ( %) were the only positive diagnostic specimen, avoiding the need for further diagnostic procedures such as transthoracic needle biopsy and mediastinoscopy in these patients. three of the eleven cases were small cell lung carcinoma, were squamous cell carcinoma and were adenocarcinoma. while eus-tbna is the gold standard for the diagnosis and staging of patients with thoracic cancer, tbna provides a clinically useful alternative in units that are not equipped to provide this service. however, dedicated training in the use of tbna should preclude the routine use of this procedure in bronchoscopy units. types of cancers in our study are as follow: poorly differentiated small cell cancer (all smokers), squamouss cell cancer (all smokers), nonsmall cell ca ( nonsmokers), adenocarcinoma (all were smokers), nonclassified , wide spread metastasis , carcinoid tumour .and one patient presented with mesothelioma transformed to sarcomatous changes. among nonsmokers had nonsmall cell cancer, mesthelioma, metastatic lung cancer, was unclassified. patients underwent bronchoscopy, bronchoscopic biopsies, bronchial washings sent, patientst under went ct guided biopsies, had mediastinoscopy and lymph node biopsy to confirm diagnosis. patients were given chemotherapy and got radiotherapy. at the time of diagnosis patient were referred for surgery, among them had lobectomies, pneumonectomies, wedge resection, deemed unresectable perioperatively. patients were given chemotherapy and got radiotherapy. patients deemed unsuitable for treatment. majority of these patients referred by general practitioners and the early diagnosis determined the final out come of the patients. there needs to be high index suspicion for early patients refrall in the presence of risk factors for lung cancer. . audit of lung mass/nodule service regional hospital department of respiratory medicine, midland regional hospital mullingar a lung mass/nodule service was established in . service was provided by consultant respiratory physician and respiratory nurse specialist. we performed a retrospective audit on this service and held regular weekly mdt meetings involved oncology service in tullamore and st james's lung cancer service to monitor the progress of all possible lung cancers. aims of study: aim of the service is to provide prompt access to the patients, their investigations, management and proper follow up. in last five years patients were reviewed. ( %) were male. average age was (range - ). number of patients evaluated each year was in , in , in , in , and in (jan-aug). more detailed analysis was performed of a subgroup this population; all patients who presented from jan-june . total number of patients was . ( %) presented with lung mass, ( %) with pulmonary nodule, ( %) had associated pulmonary infiltrates, ( %) haemoptysis, ( %) enlarged mediastinal lymph nodes. ( %) had bronchoscopy, ( %) ct/us guided biopsy, ( %) thoracentesis, deemed unsuitable for further evaluation. ( %) were diagnosed with bronchogenic carcinoma ( non small cell cancer %, ( squamous cell cancer . %, adenocarcinoma . %, undifferentiated . %), small cell cancer . %, sarcoidosis ( . %), and had either an initial nondiagnostic evaluation or assigned to follow-up ct scan protocol (as per fleischner society guidelines ). of the lung cancer patients, ( %) were referred for chemotherapy/radiotherapy, ( %) for surgery, ( %) for palliative management. of the ( %) assigned to the follow-up protocol, was subsequently diagnosed with cancer. lung mass/nodule service facilitates early diagnosis of lung cancer and subsequent assignment to appropriate therapy; it also provides a coordinated careful follow-up of lung nodules. many patients with lung cancer are symptomatic from diagnosis, and quality of life (qol) may be maximised through use of specialist palliative care in parallel with other treatments. patients are at increased risk of psychological disorders such as depression and anxiety. this study explored anxiety, depression and qol of a small group of patients (n = ), predominantly male ( . %), mean age years, using the marie curie ''breathing space'' outpatient clinic over a four week period. ''breathing space'' is a nurse led multidisciplinary clinic using integrative, person-centred care to maximise qol of patients with lung cancer through weekly assessments and interventions to enhance breathing, stamina, relaxation, mood, independence and well-being. a prospective survey design incorporated qualitative and quantitative approaches using semi structured interviews at baseline and after four weeks. qualitative data explored patient expectations and experiences of clinic attendance. most reported preconceived fears about the clinic due to poor information which were later dispelled. anxiety, depression and quality of life scores improved for this small sample. the core elements of ''breathing space'' may contribute to improve qol for patients with lung cancer. further work is needed on a larger sample to confirm the effect on anxiety and depression. we performed a retrospective analysis of procedures done in the endoscopy/bronchoscopy unit in a dublin teaching hospital from to . since , all data in the unit has been entered live to an electronic patient record, replacing hand written reports. this excludes procedures carried out in areas other than the endoscopy unit, such as icu, hdu, bone marrow transplant unit etc. a total of procedures were carried out, which consisted of upper gi endoscopy , ( %), colonoscopy , ( %), bronchoscopy , ( . %), cystoscopy %, ercp %, sigmoidoscopy . %, trus biopsy . %, ileoscopy . %. during bronchoscopy, the following procedures were performed; tbbx ( %), endobronchial bx ( . %), tbna of mediastinal glands or peripheral lesions ( %). indications for bronchoscopy were; mass on the chest radiograph %, haemoptysis %, consolidation %, persistent symptoms with normal chest radiograph . %, pleural effusion %, recurrent infections %, hoarseness . %, stridor . %, others %. focal endobronchial tumours were present in ( %) cases, the vast majority of which were cancer. excessive haemorrhage occurred in ( . %), which was controlled in all cases with standard measures. the incidence of pneumothorax post transbronchial biopsy was \ %. mortality was zero. an electronic database provides a useful tool for audit of clinical practice. bronchoscopy is the third most common procedure performed in an endoscopy unit of a major teaching hospital in dublin. the most common pathological diagnosis in this unit is lung cancer. the workplace smoking ban protects adults from secondhand smoke (shs/ets) but there is now concern about the protection of children from (shs) in the home or in cars. we evaluated the levels of shs/ ets that might be experienced by a child in the back of a car, where the driver is smoking. a particle was located in the back of a car at the height of a child's car seat. measurements were recorded prior, during, and minutes after smoking had stopped. this was repeated with the drivers window open, and with it closed. these results show that particulate levels rise significantly with active smoking. the levels are significantly higher when the driver's window is closed during smoking, however even with the window open the levels are significantly elevated. post smoking levels are higher than the levels during smoking with the window open. these results are comparable with those reported from the us. children in the back seat of cars with the driver smoking are subject to very high levels of ets, which persist even after the smoking has stopped. having the driver's window open reduces the exposure, but it is still significantly higher than background levels. cells (gift from dieter c. gruenert, usf) were pretreated ± % cse and then stimulated with lps; cytomix (tnf-a, il- b, lps) or cytomix (tnf-a, il- b, ifn-c) and il- release measured. exposure to cse significantly reduced the stimulated il- release in all cases in the cfbe o-cells (cytomix : . ± . pg/ml, +cse . ± . pg/ml; cytomix : ± . pg/ml, +cse ± . pg/ml; lps-pa lg/ml . ± . pg/ml, +cse . ± . pg/ml; all p \ . ). however, il- release from hbe o-cells was only significantly inhibited basally and after stimulation with cytomix (cytomix ; ± . pg/ml, +cse ± . pg/ml; p \ . ). these data indicate that the response of the cf cell line to cse differs from that of the normal cell line. cse inhibits via tlr in a cells causing a reduction in both basal and lps stimulated il- release. this would provide a rationale for reduced responses to cytomix or lps. further studies will examine the effect of cse on tlr in our cell lines. previous studies have demonstrated improved lung health in bar workers, and reduction in cardiac morbidity following such bans. we sought to evaluate medical admissions before & following implementation of the ban, to assess for any impact on respiratory and cardiovascular admissions. data were obtained for all medical emergency admissions to our institution in the -month periods from january -december , and january -december using the hospital in-patient enquiry system. data were examined for trends in respiratory and cardiovascular disease between the study periods. medical admissions increased over the study period ( / n = ; / n = ). however, there was a decrease in the proportion of admissions due to pneumonia (rr . ), asthma (rr . ), spontaneous pneumothorax (rr . ), stroke (rr . ), and unstable angina (rr . ). admissions with copd increased (rr . ). these changes were seen in smokers & non-smokers. no significant mortality impact was observed. the proportion of medical admissions for respiratory and cardiovascular disease decreased in the years following the implementation of the smoking ban. any impact on chronic diseases may take many more years to become apparent. the ban on smoking in public places came into force in northern ireland on the th april . the objective was to look at the impact of the smoking ban on smoking prevalence in diabetic patients a year before and a year after the smoking ban. a retrospective analysis of smoking habit data held on a computerised data base (the diamond system) a year before and a year after the introduction of the smoking ban. the data of diabetic patients was analysed. there were % male and % female. type diabetics were % and % of the patients had type diabetes mellitus. a year before the smoking ban, ( %) were smokers- % male, % female. the prevalence of smoking in type male diabetic was % and in type female diabetic %. the prevalence of smoking in type male diabetics was % and in females %. one year after the smoking ban ( %) were smoking- % male and % female (p [ . ). to date, there has been no statistical difference in the number of patients with diabetes mellitus who smoke since the introduction of the smoking ban. the ban on smoking in public places came into force in northern ireland on the th april . our objective was to look at the impact of the smoking ban on blood pressure control in a group of patients with diabetes mellitus. a retrospective analysis of smoking habit data held on a computerised data base (the diamond system) was performed, one year before and one year after the introduction of smoking ban. the data of diabetic patients was analysed, % male and % female. type diabetics were % and % of the patients had type diabetes mellitus. mean blood pressure of the non-smokers was / mmhg before the smoking ban and / mmhg one year after the introduction of smoking ban. the mean blood pressure of smokers was / mmhg before and / mmhg a year after the ban (p [ . ). there was a small improvement in the blood pressure control of nonsmoking diabetic patients a year after the ban, however it was not statistically significant. overall, blood pressure control was at target. the law prohibiting workplace smoking improved the respiratory health of dublin bar workers. however, non-smoking bar workers living with a smoker are exposed to cigarette smoke at home. ( ) . for this study barworkers were studied. current smokers (n = ), and asthmatics (n = ) were excluded from analysis. ( %) lived with a smoker, and ( %) did not. all barworkers had similar baseline co levels, but those without exposure at home had a more significant reduction yr post ban. breathing symptoms in those without home exposure were lower at baseline, while ent symptoms were similar in both groups. fev remained constant in those not exposed to cigarette smoke at home, while it declined (not significantly) in those with home exposure. exposure to cigarette smoke in the home continues to put nonsmokers at increased risk of significant respiratory symptoms. smokefree homes would bring improved health. smoking adversely affects the health of patients with cf. study aims: to determine active and passive smoking exposure among adult irish cf patients. methods: cf patients attending cuh completed a questionnaire relating to personal smoking and second-hand smoke (shs) exposure, correlated with pulmonary function and exacerbation-rate data. results: patients ( male) completed the questionnaire (table ) . . % were currently smokers. . % admitted to having tried smoking at some time. in the never-smoked group (n = ), % were currently exposed to shs; mean duration of exposure was . pack-years ( %ci: . - . ), while % had previous shs exposure; mean duration of exposure was . pack-years. % were never exposed to shs. in those currently exposed, the source was from a parent in . % and a sibling in . %. anthropometric data and exacerbation rates were similar between groups, but smokers showed a trend towards better lung function. a large level of exposure to shs exists among irish cf patients, with a clinically relevant proportion actively smoking. this study identifies a need for more aggressive smoking cessation strategies for both patients and caregivers. supported by cfai. a non-probability sampling of self-identified glc was recruited using electronic and print media advertisements between december and march . , respondents completed the questionnaires. otc data for the same period was analysed (n = , respondents). appropriate statistical analyses were performed to compare the mean differences in smoking rates between these two surveyed populations across age, gender and socio-economic groups (ses). adjusted current rates in glc were % and . % in general population (p = . ) and ''heavy'' smoking prevalence was . % in glc and . % in general population (p = . ). upper ses glcs are ''heavy'' smokers compared to general population of similar ses group (p = . ). glcs (\ years) were ''heavy'' smokers compared to general population of same age-groups (p = . ). no significant gender differences were observed. more glc were ''heavy'' smokers than the general irish population, but current smoking rates among the glc in ireland were not significantly different from the general population. a sampling frame of post-primary schools was used to randomly select schools for the issac study. , children ( - years) completed the isaac questionnaire. smoking prevalence was based on children's self-reported answer to the question ''if you travel by car does anyone smoke cigarettes in the car [yes/no]? for part , we used ''upgreen counters'' for vantage points: a shopping car park (saturday - pm); near a school (monday - pm); a busy sub-traffic junction (monday - pm), and ''moving cameras'' for the th vantage point, the civic offices (monday - am). smoking prevalence in cars was . % in ireland. for the four vantage locations, the prevalence rates were: . % (n = / ), . % (n = / ), . % (n = / ), and . % (n = / ), respectively. smoking in cars is a public-health policy issue. our findings show that children are regularly exposed to second-hand-smoke in cars in ireland and this demands legislation. in march we performed a pilot study to determine the feasibility of a pulmonary outreach programme in the midlands area. there are currently two similar programmes in ireland, but this was the first in a rural setting. our programme has two pathways of care: patients discharged \ h received home visits on consecutive days and were followed up for days (early discharge programme, edp); if discharged [ h due to unsuitability for the edp, they were reviewed for the first two weeks by specialist respiratory support within the community (outreach programme op). all patients were subsequently enrolled into pulmonary rehab. to date, patients have been enrolled to pop. % were male; mean age years; majority had severe disease ( . % stage ii, . iii, . % iv). . % required ltot, % home-nppv. . % were active smokers. . % were readmitted within the first weeks. the mean length of inpatient stay was . d for the edp, and . d for op; the average los nationally is . days. overall there were substantial financial savings. thus, rural pulmonary outreach programmes are feasible as they lead to a reduction in hospital los, improved patient knowledge of their disease, and are cost effective. the physical and psychological symptom burden associated with patients with advanced copd has been compared to that of patients dying with cancer. traditionally palliative care services have focussed on people with cancer, however recently the american thoracic society have endorsed the concept that palliative care should be available to patients at all stages of their illness [ ] endorsing the who palliative care definition [ ] . the irish hospice foundation and hse undertook a study in examining how all levels of palliative care can be extended to people with copd. challenges identified for introducing palliative care for people with copd include the uncertainty of the copd disease trajectory, the tension between delivering hope and planning for the inevitable, the lack of comprehensive respiratory services and the need for further education and research. the development of a model of care for patients with stage iii / iv copd providing a clear pathway of access to all levels of palliative care, the production of information and educational material, the requirement that all palliative care services are accessible to copd patients as required and the need for further collaboration between respiratory and palliative care services are key recommendations in the report of the study. school of pharmacy, queen's university , and mater hospital , belfast introduction: self-management plans for copd is derived from success in asthma. patients may benefit from the early intervention following selfmanagement plan . methods: patients ( y; % females) with mod-severe copd, were randomly assigned to an intervention group ( ) and usual care ( ). a pharmacist delivered an education program on disease state, medications, home exercise and breathing techniques. a booklet and a customised action plan for acute exacerbations (antibiotics and steroids) were given. follow up was at three months by telephone and a six months scheduled visit. the eq- d health status and sgrq were administered to all patients. outcomes included admissions, a/e visits and quality of life. results: at months the intervention group had reduction in both admissions [ ( %) vs ( %); p = . ], and a/e visits [ ( %) vs s ( %); p \ . ]. on the sgrq there was improvement in the symptom (- . ; p = . ), impact (- . ; p = ). and total score (- . ; p = . ). physical activity scores did not improve. the difference in the eq- d scores improved both vas scale [ . vs . ; p = . ], and utility scale [ . vs . ; p = . ]. this ongoing study indicates that a clinical pharmacy led management programme can reduce the need for hospital care in patients with moderate-to-severe copd and improve aspects of their health related quality of life. copd is a leading cause of morbidity and mortality worldwide. exacerbations of copd result in frequent hospitalisation and account for % of the costs associated with the disease. our objective was to identify risk factors which predict relapse requiring readmission following an exacerbation of copd. from to , consecutive exacerbations of copd admitted to hospital were prospectively studied. baseline demographics, number of hospitalisations in the previous year, oxygen use and smoking history were assessed. breathlessness and quality of life scores were recorded and oxygen saturations and spirometry measured. rehospitalisation data was collected at day , weeks and months. during the follow up period, patients ( %) were readmitted by day , ( %) were admitted by six weeks and ( %) were admitted by three months. logistic regression analysis identified hospitalisation in previous months (p = . , or . , ci . - . ) and borg score or higher (p = . , or . , ci . - . ) predicted readmission in % of patients at day . home oxygen use (p = . , or . , ci . - . ), pack year [ (p = . , or . , ci . - . ) and borg score [ (p = . , or . , ci . - . ) predicted week admission in . %. admission in the previous year and borg score of c predict early relapse, while home oxygen use, pack-year history c and borg score of c predict later relapse following an acute exacerbation of moderate copd. aecopd is an inflammatory lung disease associated with systemic consequences. a systematic analysis was undertaken of alpha- antitrypsin (a at), c-reactive protein (crp) and procalcitonin (pct) in the serum of patients with aecopd and matched inflammatory controls (cellulitis), pre-and post-antibiotic therapy. a at and crp are acute phase proteins. pct, a serum calcitonin precursor, is also raised in bacterial infections. venous samples from patients were analysed in this prospective study. amongst the aecopd and controls (cellulitis), were males and females (aged to ). crp(mg/l) levels were elevated in cellulitis (mean ± std error, . ± . ) and aecopd ( . ± . ) patients prior to treatment. a at(lmol/l) levels were also significantly elevated in cellulitis ( . ± . ) and aecopd ( . ± . ) patients. following intravenous antibiotic therapy, crp levels fell in cellulitis ( . ± . ) and copd ( . ± . ) patients. similarly, a at values fell in cellulitis ( . ± . ) and in aecopd ( . ± . ) patients. pct (ng/ml) levels were not elevated in all individuals with either cellulitis ( / ) or aecopd ( / ), but did decrease significantly in those that were elevated following antibiotic therapy. crp and a at levels are significantly elevated during aecopd and cellulitis. both levels fell significantly post antibiotic treatment (p = . for crp and p = . for a at). pct levels, when elevated, were reduced post antibiotic therapy. these data suggest that aecopd elicits a systemic response similar to a non-respiratory infection and this response to treatment can be monitored using biomarkers. in copd there is a cytotoxic t-cell infiltrate in the airway mucosa. it has been suggested that a virus may be a co-factor. we have recently shown high levels of ebv in severe disease . we wanted to establish if it was present in early disease. we recruited smoking ( pack y) subjects ( y) with early copd with mean fev . ( %) and smoking ( pack y) unobstructed smokers ( y) with mean fev . ( %). none of the subjects had used inhaled or oral steroids. nose and throat swabs were taken. induced sputum was obtained using hypertonic saline. total nucleic acids were extracted, and ebv dna was detected using taqman quantitative pcr. results: ebv was detected more often in the copd ( / swabs and / sputum) than the control ( / swabs and / sputum). p = . and . for swabs and sputum respectively (fisher's exact test). there was a wide range of copy numbers which were not different among those who were positive. conclusion: ebv is present more frequently in early copd than in unobstructed smoking controls. ebv is known to be a cyclical herpes virus which comes and goes. it may have a role in the pathogenesis of copd. background and method: niv is a valuable treatment for hypercapnic respiratory failure. [ ] transcutaneous co monitors(tosca) has provided novel approach to monitor these patients.. we assessed niv service and the use of transcutaneous co monitors in our tertiary care center. charts of patients attended from july to dec were retrospectively evaluated. data was retrievable on from total of patients. there mean age was ± yrs. were female. copd was in %, decompensated obesity/hypoventilation was % and neuromuscular/chest wall deformity was %. fev (mean) was . % - . %. % had type and ( %) had type respiratory failure. their average-ph was ( . - . ). mean pco was ( . - . ). only % of patients had repeat blood gas analysis at - hour, tosca was used to monitor non-invasive ventilation in ( %) of patients. the average number of use per patient was . - . . there length of stay(avg) was . days. their mean ipap and epap were . and respectively. ( %) patients were commenced niv in hdu. ( %) died due to respiratory failure. conclusion: % of patients were successfully monitored with tosca. we conclude that tosca is a valuable tool for monitoring patients at ward level. respiratory assessment unit, crest directorate, st. james's hospital, dublin as part of its transformation programme, the hse established a group in september to develop a national strategy for the management of copd. the aim of this survey was to capture information regarding the availability and range of physiotherapy services for persons with copd in ireland and thus inform the report of the national copd strategy group. the survey was emailed to physiotherapy managers across care settings in november . the survey sought information relating to the range of physiotherapy services for persons with copd provided at each site, as well as perceived service deficits and existing/potential innovations in practice. data were analysed using descriptive statistics. fifty-seven sites responded to the survey. no formal joint services between acute hospitals and pccc were reported. pulmonary rehabilitation programmes (prps) were available in sites only. service deficits reported related to lack of appropriate treatment space, lack of specialised respiratory staff, the absence of prps, and lack of interaction between acute hospitals and community services. physiotherapy services for persons with copd vary greatly across sites and settings. prps are not widely available and are primarily hospital based. there is a need for wider availability of joint hospital/ community based initiatives such as copd outreach and prps. pulmonary rehabilitation has established efficacy, but patients often require follow-up care or maintenance. there are few studies that explore the patients' experience of pulmonary rehabilitation and maintenance. also, there are no guidelines for health professionals as to what constitutes effective maintenance for clients who complete pulmonary rehabilitation. the study aim was to explore patients' perceptions of pulmonary rehabilitation and the maintenance options provided to them. a qualitative, exploratory descriptive design used focus groups to collect data. the purposive sample (n = ), had a diagnosis of either copd or bronchiectasis, and had attended a pulmonary rehabilitation programme within the last year. a focus group schedule using open ended questions and prompts was designed. discussions were transcribed verbatim and burnard's ( ) thematic content analysis was used to guide data analysis. the dynamics of group participation and peer support were identified as important incentives for patients. increased confidence and personal achievement were described as outcomes. the reasons for non-participation in maintenance were also elucidated by patients. this study provides an important contribution in relation to the experience of patients and the findings enhance current quantitative studies. patients' experience of outcomes and expectations has the potential to influence future services. following recommendations from the hse transformation programme , a multidisciplinary committee was established to develop a national strategy for the management of copd. as a member of the committee representing anail, the author conducted a survey to establish the range of inpatient and outpatient services provided by respiratory nurses for persons with copd. a questionnaire was devised to gather information regarding the provision of services such as inhaler technique, oxygen assessments, copd outreach programmes, pulmonary rehabilitation programmes (prp's) and palliative care services for persons with end stage copd. thirty-five members of anail were surveyed in october . data were analysed using descriptive statistics. a response rate of % was achieved. inpatient and outpatient services such as respiratory nurse reviews, oxygen assessments, self management plans were provided by more than % of respondents. of those who replied % provided inhaler technique education, % can refer persons with copd for prp, % run an outreach programme and % providing limited palliative care services. a number of current innovations and deficits within the services provided by respiratory nurses were highlighted. the contribution made by specialist nurses to the acute and chronic respiratory service is reflected by this survey. the inspiratory fraction-inspiratory-to-total-lung capacity (ic/ tlc) is an independent risk factor for mortality in chronic obstructive pulmonary disease (copd) . ic/tlc b %predicted is associated with significantly shorter survival. little data exists about the effect of pulmonary rehabilitation (pr) on survival in copd . the purpose of this study is to examine the effect of pr on ic/tlc. patients (mean age . ± . ), with clinical evidence of copd (mean fev . ± %predicted, mean ic/tlc . ± . %predicted) were enrolled in an week pr programme, consisting of twice-weekly sessions of exercise and education. assessments/re-assessments consisted of lung function (spirometry, diffusion, sniff nasal inspiratory pressure, capacity), exercise tests (shuttle, treadmill) and quality-of life-questionnaires (qol). patients, re-assessed at months, demonstrated improvements in exercise and qol compared to baseline (p \ . ). these patients were divided into groups-group ic/tlc b % predicted (n = ), group ic/tlc [ % predicted (n = ) at baseline. there were no between-group differences in improvements in exercise or qol at months. group ic/tlc improved at / from baseline . % predicted to . % predicted, and group from . to . % predicted (not significant). although the results are not significant there appears to be a trend in improved ic/tlc following pr. this study should be repeated with a larger sample size. department of medicine, midland regional hospital, mullingar, ireland primary objective was to assess the appropriateness of our hospital admissions for copd exacerbations as per nice guidelines. we also assessed the quality of their outpatient copd medical care. all copd related admissions mar-may were prospectively reviewed. variables as per nice guidelines were considered with one point for each variable. a score of zero was considered an inappropriate admission while c was appropriate. patients were included. mean age was ( - ), ( %) were male. ( %) patients were admitted as per guidelines, ( %) patient met no criteria. commonest variables present were: poor level of activity ( %), significant co morbidities ( %), inability to cope at home ( %). least common variables were: impaired level of consciousness ( %), cyanosis ( %), acute confusion ( %). ( %) received antibiotics. ( %) had spirometry performed for diagnosis. out of smokers ( %) were offered cessation advice. ( %) were appropriately on inhaled steroids. out of an eligible ( %) were enrolled in pulmonary rehab. mean los was . days, and there was linear relationship between length of stay and guideline score. there was excellent compliance with nice guidelines for copd admissions. quality of outpatient care was good in the domains evaluated. up to % of copd hospital inpatients will be readmitted within weeks of discharged. specific predictive markers of readmission are not currently in routine clinical practice. in this study, we assessed a remote monitoring system for continuous readout of patients' pulse rate and o saturation (biancamed, ireland). a cohort of normal volunteers (n = ) and copd patients (n = ) were enrolled and full remote monitoring and psychological profiling (via a modified hospital anxiety and depression score (hads)) of patients' well being was performed. in controls and patients, mean percentage of recording time was % (range: %- %) and % (range: %- %) respectively. principal reasons for loss of recording were a) patients moving out of range of monitor, b) non-compliance due to impracticality and/or discomfort while wearing the device, and c) accidental slippage of the oximeter probe. analysis of time of o saturation below %, %, % and % per hour revealed significant improvement over time in % of patients. one patient subsequently readmitted showed a significant deterioration prior to admission. in conclusion, this system shows potential in the early identification of copd patients who clinically deteriorate at home. in , % of respiratory inpatient discharges related to copd. information on hospital services for copd patients was required for the development of a national strategy. a survey on relevant staffing, wards and diagnostic units, policies and practice and access to specialist services was distributed to acute hse hospitals via hospital networks. hospitals responded ( %). written policies are in place for management of copd ( %), non invasive ventilation (niv) ( %) and long term oxygen therapy ( %). niv is provided in the emergency department (ed) ( . %), medical assessment unit (mau) ( . %), icu ( . %), hdu ( . %), respiratory ward ( . %), all medical wards ( . %). in almost two thirds of hospitals, all inpatients with copd can access respiratory nurse specialists, smoking cessation officers and palliative care services. access by ed/mau patients is possible in %, % and % of hospitals respectively and by gp referral in %, % and %. ten hospitals have pulmonary rehabilitation programmes ( %), five have onward referral mechanisms and four were planning a programme. the waiting time for programmes is up to one year. three hospitals have outreach programmes in place. this survey highlights the variation in hospital based services for copd patients and opportunities for service development. in the health services executive established a steering group to develop a national strategy for the management of copd. this study aims to describe the range of services available to copd patients and ease of access from the primary care perspective. a postal survey was distributed to a random sample of gps by the icgp. data was analysed using excel. valid questionnaires were returned (response rate . %) from practices in counties. . % have access to spirometry within their own practice. a practice nurse usually conducts the test ( %) and a gp interprets the results ( %). patients are unable to access patient support groups ( . %), pulmonary rehabilitation ( . %), rapid access respiratory clinics ( . %) or community options for management of an exacerbation-home based ( . %) or local community unit/district hospital ( . %). waiting times are up to six months for physiotherapy and pulmonary function testing and up to one year for respiratory consultant review, long term oxygen therapy assessment and pulmonary rehabilitation. this survey highlights geographical variation and gaps to be addressed for a shift to occur towards a community-based, responsive, flexible service for copd patients. oxygen therapy is an important treatment option for patients with severe copd, as long term continuous therapy (ltot). information on relevant community resources for ltot was required for the development of a national copd strategy. a survey was distributed by e-mail via each local health office (lho) manager ( ), covering activity and costs of aids and appliances, policy and procedure. data was analysed using excel. twenty two responses were received from local health areas ( %). there were wide population differences in the rate of ltot between areas, from - / , . home oxygen can be prescribed by hospital consultant, gp, respiratory nurse specialist or physiotherapist. arrangements for follow-up of patients on long term home oxygen vary considerably. half of respondents have difficulty with the level of detail provided on home oxygen prescriptions. % have a policy on provision of portable oxygen cylinders. % are aware of arrangements for ongoing maintenance of oxygen appliances. cost of ltot in was estimated to be in excess of € million. long term oxygen is an important copd therapy but is costly and has potential for harm. standardised practices are required for its use in the community. spirometry is the gold standard for diagnosis of copd. additional pulmonary function tests (pfts) can also assist in management. details of respiratory diagnostic resources in ireland were required to inform the national copd strategy. a questionnaire was developed in conjunction with the irish association of respiratory scientists and circulated to members via email. questions focused on staffing, workload, waiting times, tests available, referral sources and educational activities. ten laboratories responded ( %). pft activity ranged from , to , . minimum waiting times ranged from days to weeks and maximum from four days to eight weeks. laboratories accepted referrals for basic pfts from respiratory consultants (all), other hospital consultants (all), respiratory nurse specialists ( %), emergency departments ( %), medical assessments units ( %) and gps ( %). tests confined to respiratory team/other consultant referrals included bronchial provocation, minute walk, long term oxygen therapy and fitness to fly assessments. five hospitals participated in training relevant to copd in the hospital and two in the community. additional copd services included participation in pulmonary rehabilitation and outreach programmes. respiratory diagnostic laboratories are predominantly resourced for hospital referrals. examples are provided where scientists also provide a service to the community, for diagnostic tests and education. patients with copd have higher blood levels of markers of inflammation such as tumour necorsis factor (tnf-a), interleukin (il- ) interleukin (il- ) and c-reactive protein (crp). these are independent risk factors for decreased lung function and are associated with increased symptoms such as shortness of breath and respiratory rate. recently, tools to measure activity have been developed which continuously record patient free living activity and sleep. we hypothesized that there may be a relationship between levels of systemic inflammation and measures of free-living activities. thirty one patients were recruited: men (n = ), female (n = ). ethical approval and written consent was obtained. venous blood samples were taken (il- , il- , tnf-a and crp which were logged for normal distribution). a senseware Ò activity monitor was worn for consecutive days and the st.george's respiratory questionnaire were measured. pearson's correlations were undertaken using spss version s mean age of . yrs (+/- . ) with an fev or (+/- ) and a mean smoke pack history of (+/- ). a medium negative correlation was found between lgcrp and physical activity duration [r = - . , n = , p = . ]. a large correlation was found between the lgcrp and the st. georges respiratory questionnaire [r = . , n , p = . ]. this was also reflected in the impact section of the questionnaire [r = . , n = , p = . ]. c-reactive protein blood levels appear to be inversely correlated to free-living activities and quality of life. these data suggest that the measure of crp may be an important factor to include in the assessment of the severity of copd. during the months of july and august , in a prospective study, patients were transferred from the adelaide and meath hospital within days of their acute admission with copd, to peamount hospital for airc. patients were enrolled: males with a mean age of . yrs and a mean fev of . l ( %). the mean length of stay (los) in the acute hospital was . days and the mean los in peamount hospital was . days, with a total mean hospital stay of days. we hypothesise that this extended hospital stay and targeted respiratory care will improve patients overall quality of life, breathlessness, and exercise capacity, and reduce their dependency on the acute hospital service and re-admission rates. these patients will be followed up over the next year as a continuation of this study. the miners' disability score (mds), developed during the compensation process for uk miners, utilises a ten-point scale. the medical research council(mrc) dyspnoea scale, previously validated using the incremental shuttle walking test(iswt), utilises a five-point scale which may be less discriminating. we aimed to validate the mds as a score of respiratory disability in copd patients. patient data (mds/iswt/endurance shuttle walking test(eswt)) from our pulmonary rehabilitation programme were initially analysed (n = ; median fev = . l; mean age = yrs). subsequently, inpatients (median fev = . l; mean age = . yrs) had baseline mrc dyspnoea grade, mds, and manchester respiratory activities of daily living score (mradl) determined. degree of association between variables was assessed using the spearman rank correlation. mds correlated well with iswt (rs = - . , %ci - . to - . ), but not with eswt. fev was not associated with mds grade. mds correlated well with mrc dyspnoea grade (rs = . , %ci . to . ). mrc grade and mds correlated well with mradl (mrc rs = .- . , %ci - . to - . ; mds rs = - . , %ci - . to - . ) score. the mds showed a more favourable association. the mds is a valid measure of respiratory disability that could be used to complement fev and may provide an accurate reflection of performance status and disability in patients with copd. copd is an unremitting disease that impacts negatively on quality of life. the aim of this study was to compare functional capacity (fc); a measure of weight distance over six minutes, with standard tools used in the assessment of patients with stable copd. forty one patients with severe copd: fev % ± % predicted were recruited: men (n = ), women (n = ). senseware Ò armbands were worn for seven days to quantify their average daily steps. ethical approval and written consent were obtained. pearson's and spearman's correlations were performed using spss version . functional capacity was significantly associated with mean daily steps: men (r = . , p = . ) women (r = . , p = . ), shuttle walk test: men (r = . , p = . ) women (r = . , p = . ) and fev in men only (r = . , p = . ). there was no relationship between fc and borg: men (r = . , p = . ) women (r = . , p = . ) or the saint-george respiratory questionnaire: men (r = - . , p = . ) women (r = . , p = . ). we found that quantifying ''free-living'' measures is an important dimension of functional status not ordinarily captured and that functional capacity is a reliable outcome measure for assessing stable copd. the only gender difference identified was in male fev . patients (n = , mean age ) admitted to castle hill hospital with acute exacerbation of copd were studied. patients were either treated with standard therapy plus mg erdosteine bd (n = ) or standard therapy alone. (n = ) and followed up at day five and day ten. there was no significant improvement in subjective measures of breathlessness. at day subjective cough frequency was reduced by % in the +erd group as compared with deterioration in the -erd group. fev increased by ml -erd group and ml in the +erd group. hacc hour recordings on nine patients revealed coughs on day one falling to coughs by day five. there was a % reduction in cough frequency on the +erd group and % reduction in the -erd group. cough counting may be a useful objective marker to judge the success or failure of treatment strategies in acute exacerbation. chronic obstructive pulmonary disease is a lung disease characterized by chronic airflow obstruction that is not fully reversible measured using spirometry. the aim of this audit was to assess use of spirometry in diagnosis of copd in primary care. two hundred questionnaires were sent to primary care practices, seventy nine were completed. questionnaires identified which practices used spirometry. information was obtained on who performed spirometry within the practice, what training had been received, what criteria for screening for copd was utilised and general information on management of copd. we found % of practices had a spirometer. the most common reasons for not were cost involved ( %) and lack of confidence in interpreting results ( %). spirometry was performed most commonly by practice nurses ( %), interpretation of results was largely done by general practitioners ( %). only % had received recognised training in spirometry. the largest group of patients screened were symptomatic smokers over years old, however only % of patients screened had spirometry performed. these data indicate that we need to promote training in the use of spirometry for the diagnosis and management of copd in primary care in ireland. physiological responses to pulmonary rehabilitation (pr) are measured using a variety of clinical exercise tests. we compared incremental with endurance cardiopulmonary exercise testing (cpet) in a series of patients attending a pr programme. thirty two patients with moderate or severe copd (age . ± . y, fev ± . % predicted) were recruited to an -week pr programme. exercise capacity was assessed using incremental cpet before and after pr in patients and endurance cpet (at % of the peak incremental cpet workload) before and after pr in patients. among the incremental exercise group, there were no significant differences in vo max (mls/min) (p = . ), vo max (mls/kg/min) (p = . ), vco max (mls/min) (p = . ) or maximum workload achieved (watts) (p = . ) before and after pr. among the endurance exercise group, there was a significant difference (p = . ) in exercise duration ( vs seconds), but no differences in vo max (mls/min) (p = . ), vo max (mls/kg/min) (p = . ) or vco max (p = . ) (mls/min) before and after pr. incremental cpet is a poor tool to measure physiological changes in exercise capacity associated with pr. endurance cpet is the more ideal test, demonstrating significant increases in endurance time associated with pr despite unchanged peak oxygen consumption and carbon dioxide production. cardiopulmonary exercise testing (cpet) provides a global assessment of the integrative exercise responses involving the pulmonary, cardiovascular, haematopoietic, neuropsychological, and skeletal muscle systems, which are not adequately reflected through the measurement of individual organ system function. this case report looks at how cpet makes the initial diagnosis of mcardle's syndrome. a year old man initially presented to the cardiologists complaining of muscle fatigue after a short period of sustained exertion. all his cardiac investigations were normal. deconditioning would have explained the young mans symptoms adequately. as such, he was sent for cardiopulmonary exercise testing (cpet) to differentiate between poor aerobic conditioning and a possible pathological aetiology. the patient managed to exercise for six minutes and the test was limited by muscle fatigue. there was early failure in the aerobic metabolic pathway with a significantly reduced vo max (oxygen uptake-aerobic metabolism) and the absence of a corresponding rise in the vco signalling a concurrent failure of the anaerobic pathway. these results pointed towards a rare muscle enzyme deficiency. diagnosis was confirmed in the conventional way using a muscle biopsy. this case represents a unique and non invasive way of diagnosing a rare and often under diagnosed enzyme deficiency and underlines the versatility and diagnostic value of cpet. non-invasive ventilation (niv) is increasingly provided at ward level with implications for skills and practice development, support and inter-professional decision-making. despite recommendations by the british thoracic society ( ) that niv can be provided outside of the intensive care unit, use of niv at ward level remains problematic, presenting particular contextual challenges to care. a qualitative research study was undertaken, involving focus group interviews with nursing staff (n = ) and individual semistructured interviews with doctors (n = ) from specialised (respiratory) and non-specialised units in a regional teaching hospital. a number of support issues were identified. niv was considered a time-consuming procedure, with a perception of inadequate staffing levels at ward level. access to experienced medical and nursing support was viewed as an integral part of niv service provision. knowledge gaps exist at local level specifically in relation to inadequate education and training. clinical practice guidelines for niv were recommended to guide practice. this research study sought to inform practice development, specifically the greater acceptance and use of niv at ward level, through examining care issues. the themes expressed in the findings point s towards the need for review of present service provision, particularly in the areas of education, training and guideline development. pulmonary function laboratories interface with all medical disciplines. there is anecdotal evidence that pulmonary function tests (pfts) are often requested inappropriately. an audit was undertaken in the pulmonary function laboratory, belfast city hospital to determine how many referrals were appropriate, the origin of each referral and the designation of the referrer. the audit randomly considered requests over a six-month period. requests were reviewed by a clinical scientist and a consultant chest physician. a request was deemed inappropriate if tests unlikely to contribute to the patient's management were sought, or if tests were omitted that should have been requested. the requests originated from the following main specialities: respiratory medicine ( %), general surgery ( %), general medicine ( %) and haematology ( %). sixty-seven percent of referrals were made by junior doctors (junior or senior house officers) and % of these were appropriate. thirteen percent of requests were made by consultants of which % were appropriate. only % of respiratory referrals were appropriate. overall % of requests were considered appropriate, however there was significant variability among disciplines ( %- %). the results indicate that many pft requests are inappropriate. additionally, the quality of respiratory referrals is not better than nonrespiratory referrals. consultant requesting does not guarantee correct referral. the findings have both resource and educational implications. a phenomenological approach enabled the researcher to gain an insight into the participants lived experiences and uncover their stories. the researcher is a respiratory nurse specialist and therefore has a particular interest in this area. a husserlian phenomenological approach with bracketing of preconceived ideas underpinned the chosen methodology. a total of seven interviews were transcribed by the researcher in this study. the participants were patients on long term non invasive ventilation. data was generated using unstructured interviews, which were tape-recorded. data was analysed using colaizzi's framework. beginning the therapy, process of adjustment to the therapy and gaining a new independence were the major themes identified within the study. this study is small however; the findings have implications for nursing practice, education and management locally and highlighted areas that require further research. dysregulation of pulmonary inflammation has been proposed as contributing to airways disease in cystic fibrosis (cf). the aim of this project was to compare two t helper- cytokines (interleukin (il)- and il- ) for their relative stability, activity and interaction with glycosaminoglycans (gags) which are highly abundant in the cf lung. bronchoalveolar lavage fluid (balf), serum and sputum pre-and post-nebulised hypertonic saline (hts) were collected from cf patients and compared to balf and serum from non-cf controls. western blots and elisas were used to visualize and quantify cytokine levels respectively. il- was undetectable within cf balf and was shown to be degraded by neutrophil elastase. as a biological consequence significantly reduced levels of il- were secreted by jurkat t lymphocytes (p = . ). il- was competitively displaced from gags by il- , which binds gags via electrostatic interactions. exposure of cf balf to hts or treatment of cf patients with hts displaced il- from gag matrices rendering the chemokine susceptible to proteolytic cleavage and reducing the chemoattractant capacity of cf sputum. in conclusion, gags possess the ability to influence the cytokine profile of the cf lung promoting a neutrophil dominated immune response and hts treatment may improve resolution of this inflammation. human cathelicidin, ll- , a amino acid antimicrobial peptide produced by neutrophils and respiratory epithelium has been shown to have antimicrobial activity as well as possess immunomodulatory properties. we have investigated this potential immunomodulatory effect of ll- using lps stimulated thp- monocytes. effects of ll- on the lps signalling pathway were investigated using western blot and elisa. ll- was shown to inhibit the degradation of ijba and ijbb during lps stimulation, whilst preventing the phosphorylation of ijba, ikk, stat- , akt, c-jun and atf- . cytokine data showed a partial reduction in lps induced il- and tnf-a with lg/ml ll- . further investigation revealed that lps induced cytokine production could be reduced to control levels when ng and ng of lps was used to challenge cells in the presence of lg/ml of ll- . washing of cells following pretreatment with ll- abolished ll- 's inhibitory effects on lps-induced il- production when compared to unwashed samples. results suggest that ll- is exerting its anti-inflammatory effect primarily by neutralising lps activity as nearly all these effects can be inhibited by higher ll- :lps ratios. exposure of bacteria such as p. aeruginosa, growing within a biofilm in the lungs of cf patients, to antibiotics during treatment of recurring pulmonary exacerbations, may result in the development of antibiotic resistance. the aim of this study was to compare biofilm formation and antibiotic susceptibility of matched p. aeruginosa isolates cultured from cf sputum before and after antibiotic treatment of an acute exacerbation of pulmonary infection. biofilm formation ( hours) by matched pairs of p. aeruginosa isolates, cultured from sputum samples prior to commencing and at the end of antibiotic treatment, was assessed by total viable count using the calgary biofilm device. all isolates formed biofilms with no differences in biofilm formation apparent between any of the matched pairs of isolates. prior to commencing antibiotic treatment, p. aeruginosa isolates from (caz), (tob), (pip/taz) and (mer) patients were susceptible. following antibiotic treatment, the susceptibility status of isolates changed from sensitive to resistant for (caz), (tob), (pip/taz) and (mer) patients. these results indicate that antibiotic treatment had no effect on the ability of p. aeruginosa isolates to form bacterial biofilms but in some patients resulted in the development of antibiotic resistance. cause of death. paradoxically, neutrophils are recruited into the lungs but fail to clear infections. the question that this project will address is; are cf neutrophils intrinsically abnormal? within this study we shall focus on neutrophil membrane proteins and present the first proteome study on normal and cf membranes. a pure neutrophil membrane fraction was prepared by sucrosedensity ultracentrifugation. the solubilizing power of nonionic and zwitterionic detergents as membrane protein solubilizers for twodimensional electrophoresis was investigated. ief was performed with immobilized ph gradients. optimized solubilization of membrane proteins was achieved by combining the zwitterionic detergent chaps ( %) or sb - ( %) with the nonionic detergent triton x- ( %). excellent reproducibility of protein-spots was observed on ph linear gradient strips ( - and - ), allowing for comparative studies. with our now optimized protocol we propose to screen circulating neutrophils from cf patients during periods of exacerbation, and to look for quantitative changes in membrane protein expression (up-regulation, down-regulation or post-translational changes) using a stable-isotope labeling approach. data arising from this project will identify candidate proteins that could be used as biomarkers and/or contribute to a better understanding of disease progression in cf. neutrophil dominated inflammation characterises acute lung injury, pneumonia, copd, cystic fibrosis and bronchiectasis. factors modulating neutrophil mediated inflammation may have important therapeutic potential in these conditions. the anti-inflammatory protein, secretory leukoprotease inhibitor (slpi), is a non-glycosylated molecule produced by epithelial cells, macrophages and neutrophils. this study aims to enhance our knowledge of the anti-inflammatory effects of slpi and to investigate the relationship between slpi and the human neutrophil. neutrophils were purified from whole blood and subcellular fractionation performed employing sucrose gradients and ultracentrifugation techniques. translocation of slpi to the outside of the cell post pma( ng/ml) or fmlp( - m) activation was assessed by western blot analysis. our experimental results confirm the findings of sallenave et al [ ] and demonstrate that slpi resides within the neutrophil cytosol. however, contrary to previously published data we have found that slpi does not co-localise with lactoferrin in the secondary granules [ ] (figure ). cytosolic spli migrated as a dimer on sds-page and upon cell activation translocated to the outside of the cell in predominantly monomeric form. our results may support the concept that slpi orchestrates diverse effects within the neutrophil, with monomer and dimer forms of the molecule possessing distinct anti-inflammatory modes of action. the primary cause of morbidity and mortality is infection by gramnegative bacteria such as pseudomonas aeruginosa, resulting in chronic airway inflammation characterized by release of interleukin (il)- . to avoid the innate immune system, p. aeruginosa can undergo genetic changes [ ] , such as modification of the lipid a component of the lipopolysaccharide (lps) structure [ ] . the aim of this study was to compare the pro-inflammatory response of various types of purified lps isolated from cf patients with that of commercially available lps. human (hte) and cf (cfte) tracheal epithelial cells at * % confluency were serum starved ( h), then stimulated with lps from sigma (laboratory stain) or isolates that differed in their lipid a structure: pak (mild cf), se (severe cf), se (infant cf), bronc (bronchiectasis) and il- release measured. in order to achieve similar il- release, sigma lps was required at -fold higher concentrations than cf lps isolates (ug/ml vs. ng/ml). there was a differential response to lps between hte and cfte cells: se and pak strains induce a higher response in cfte cells when compared to hte. in conclusion, the inflammatory response to p. aeruginosa is dependent upon strain and environment, which may be due to changing lipid a structures. we investigated the ability of secreted bacterial proteinases from three pathogens (burkholderia multivorans, burkholderia cenocepacia, and pseudomonas aeruginosa) involved in chronic bacterial infections in cystic fibrosis to degrade various host defence-related molecules. these included secretory leukocyte proteinase inhibitor (rhslpi), alpha- antitrypsin (aat), secretory iga (siga), igg, lactoferrin and lysozyme. host defence-related molecules were co-incubated with cell-free bacterial supernatants from hour biofilm cultures from all three pathogens under investigation. no degradation of aat, siga, igg, and lactoferrin was observed for any of the organisms. only one out of isolates tested demonstrated the ability to degrade lysozyme. all isolates of b. multivorans (n = ) and p. aeruginosa (n = ) were able to degrade rhslpi however, out of five bacterial isolates tested for b. cenocepacia only two demonstrated a limited ability to degrade the molecule with [ % of the protein band still remaining intact at the end of the experiment. this study demonstrates that the majority of the host defence molecules investigated are resistant to degradation by bacterial proteinases from b. multivorans, b.cenocepacia and p. aeruginosa when grown as a biofilm. however, rhslpi was vulnerable to significant degradation which could result in aberrant serine proteolysis in regions of the lungs containing biofilm growth. children are ten times more sensitive to radiation-induced cancer than adults. we aimed to determine the cumulative radiation exposure associated with imaging in a paediatric population with cf, to identify contributing factors and to suggest ways of reducing their lifetime radiation exposure. medical and radiology records were reviewed. effective radiation dose (msv) and cumulative lifetime radiation doses were calculated for each patient using national radiological protection board (uk)data files. patients, mean age . ( - . ) years with a total follow up time of person years, had chest radiographs, abdominal radiographs and computerized tomography (ct) scans, including thoracic ct scans. average cumulative radiation exposure per patient was . ( . - ) msv. radiation exposure increased with age (p = . ) and with increasing numbers of cts (p = . ). radiation dose was significantly increased in the subgroup who presented with meconium ileus (p = . , independent of age).radiation dose was not significantly related to lung disease severity (measured as forced expiratory volume in second (fev ). radiation exposure in our cf population compares favourably with other tertiary centres worldwide. radiation dose can be minimised by reducing frequency of scans and altering scanning technique. a number of mirna expression profiling studies have shown mir- to be highly expressed in rat and human lung. tom a predicted target of mir- has been shown to interact with tollip and proposed as a negative regulator of il- b and tnf-a signalling pathways. the aim of this study was to validate tom as a target of mir- and elucidate its role in tlr and il- signalling pathways in cystic fibrosis (cf) versus non-cf airway epithelial cells. expression of mir- and tom were evaluated by qpcr. overexpression of premir- was performed by reverse transfection and tom was subsequently detected by western blot. mir- was found to be down-regulated (p = . ) and tom mrna significantly up-regulated (p = . ) in cf bronchial cells when compared to their non-cf counterparts. overexpression of mir in cf cells led to a decrease in tom protein production. this data shows that mirna is differentially regulated in cf airway epithelial cells and that tom is a target of mir- and may have an important role in regulating innate immune responses in the cf lung. case : y.o. male with recurrent infective exacerbations was noted to experience more severe and longer exacerbations compared to other similar patients. common variable immunodeficiency (cvid) was diagnosed based on low iga, igm, igg , igg and lack of antibody response to pneumovax. treatment with ivig has commenced. case : y.o. male who experienced severe anxiety during transition to adult cf care. obsessive compulsive disorder was recognized as exemplified by patient using alcohol wipes weekly to clean himself. he has responded well to cognitive behavioural psychotherapy. we conclude that physicians should appreciate the spectrum of coexisting conditions that are separate to a diagnosis of cf and can contribute to morbidity and mortality. cfrd adversely affects pulmonary function however diabetic control did not significantly impact function any further. this finding warrants larger prospective studies to confirm that a diagnosis of cfrd impacts pulmonary function but that diabetic control may not. with improving cf survival, fertility issues emerge. this descriptive study assesses knowledge & approaches to fertility information provision in cf care. prospective anonymous questionnaires were mailed to a male cf cohort (n = ). sections included demographics, fertility knowledge, investigation & personal relationships. response rate was % (n = ). mean age years (range - , sd . ). all knew that cf affected fertility but only . % (n = ) were able to provide explanations. of this group, . % (n = ) provided the correct explanation. % (n = ) have discussed fertility with a healthcare professional and half (n = ) selfinitiated this. mean discussion age was . years (range - , sd . ). one third stated preference for earlier discussion. . % (n = ) who had discussions were satisfied with information provided. commonest first source where patients heard of infertility was written material ( . %, n = ). three-quarters of respondents (n = ) requested further fertility information. the preferred source was written material ( . %, n = ). . % (n = ) have had semen analysis & all remaining (n = ) would accept an opportunity for this if offered. all respondents were aware of infertility however most unaware of explanation. few have formally discussed fertility. the majority want further information (preferred method written material) & an opportunity for semen analysis. this study identifies significant gaps existing in sex education during provision of cf care. a. sahadevan, s.h. chotirmall, a.k. mann, p. branagan, c. gunaratnam, n.g. mcelvaney discovering predictors of mortality in cf within ireland has therapeutic implications. we aim to determine factors predicting mortality in an irish cf cohort. a retrospective analysis of clinical, microbiological and radiological parameters in deceased cf patients over an -year period ( - ) was conducted (n = ). this was age matched to a living cf cohort. spss version . was used-chi-squared and independent student t-testing applied. mean age . years (sd +/- . , range - ) [deceased group] and . years (sd +/- . , range - ) [living group]. % (n = ) and . % (n = ) were female in the deceased and living groups respectively. within the deceased cohort, . % (n = ) had abnormal liver function (p = . ), . % (n = ) grew pseudomonas (p = . ) and . % (n = ) had candida in sputum (p = . ). correspondingly, in the living cohort . % (n = ) had abnormal liver tests, . % (n = ) and . % (n = ) respectively grew sputum pseudomonas and candida species. the deceased had poorer lung function (p \ . ), weight (p \ . ) and bmi (p \ . ). mean fev was . litres and mean weight . kilograms less than that of the living cohort. poor pulmonary function (fev , fvc), abnormal liver function, sputum culture of pseudomonas and candida spp and suboptimal nutrition (weight, bmi) were all predictors of mortality in our cohort. abnormal lfts are common in cf. we aim to determine any relationship between abnormal lfts & pulmonary function in a cf cohort. cf patients were included during the -month study ( - ). serum bilirubin, alanine aminotransferase (alt), alkaline phosphatase (alkp) & international normalised ratio (inr) were obtained in the outpatient clinic when exacerbation free. lung function (fev ) was concurrently determined. spearman (nonparametric) correlation was applied where appropriate. mean bilirubin was . umol/l (range . - umol/l) and mean alt . iu/l (range - iu/l). . % (n = ) had both above average bilirubin and alt of which ( . %) and ( . %) respectively in the bilirubin and alt groups had fev abnormal liver function did not impact fev however inr showed negative correlation. this may relate to malabsorption of fat soluble vitamins or be explained by a residual coagulopathic state following recurrent pulmonary exacerbations. osteoporosis & vitamin malabsorption contribute to poor nutritional status in cf. we aim to determine the effect of vitamin d deficiency and bone fragility on pulmonary function. systematic random sampling of an outpatient cf cohort was studied. pulmonary function (fev ), vitamin d status (serum) and bone fragility (z-score on dexa) was determined. chi squared analysis was applied to results (spss version . ). patients were included in the study (age - ). . % (n = ) exhibited vitamin d deficiency. of these, a single patient had normal lung function (fev [ % predicted), reduced function (fev - % predicted), markedly reduced function (fev - % predicted) and patient fev \ % predicted (p = . ). vitamin d deficiency was commoner in males (n = ). within the cohort, patients had normal bmd (z-score [ - ), had osteopenia (z-score - - . ) & had osteoporosis (all male) (z-score [ - . ) (n = ). in those with vitamin d deficiency (n = ), patient had osteoporosis & a further osteopenia ( . %) (p = . ). vitamin d deficiency is characterized by lower fev & bmd (osteopenia) however osteoporosis was present in cases of normal vitamin d levels. our study suggests the role bone health plays in the cf ''gender gap'' may be overestimated. biopsy showed nodular glomerulosclerosis (ngs) occurring in the absence of diabetes mellitus, amyloidosis and any other known cause of ngs. a recent paper has suggested that the pathogenesis of ngs in normoglycaemic, non-diabetic cf patients is similar to that of classic diabetes induced ngs and may be mediated by the development of advanced glycosylation end products (age). in cf, chronic pulmonary infection/inflammation, in combination with reduced glutathione levels contribute to an oxidative state and increased levels of age and to s /calgranulin. it is postulated these ligands interacting with rage resulting in the formation of nodular glomerulosclerosis in patients with cystic fibrosis. we conclude that increasing life spans of cf patients may lead to an increased identification of proteinuric renal disease, the aetiology of which may include this newly described pathological process. this is the first case described in a european cystic fibrosis population and the fourth case worldwide. nebulised hypertonic saline is an effective and safe therapy for cf lung disease. however reports show over % of patients cannot tolerate this treatment, and up to % of patients are totally noncompliant when using standard nebuliser units. positive expiratory pressure nebulizer devices splint open the airways and have a more controlled rate of nebulisation. we tested if patients who had failed hypertonic saline via standard nebuliser units could tolerate this therapy via a pep nebulizer. we prospectively recruited adult cf patients over a month period, who had previously failed hypertonic saline trials and commenced them on hypertonic saline via a pep nebulizer. patients completed a questionnaire on tolerability of the new device. notes were examined retrospectively on mean time to intravenous antibiotic usage pre and post therapy and mean time to next exacerbations. there was a subjective reduction of over [ % noted in coughing, chest tightness and bad taste using the pep nebulizer, with all patients tolerating this form of treatment. in this small study we found an absolute reduction in antibiotic usage of % post hypertonic saline usage and a fold increase in time to next exacerbation post nebulized pep treatment. hypertonic saline administered via a pep nebulizer may be a novel therapeutic strategy for patients who cannot tolerate hypertonic saline through a standard nebulizer. chronic lung infection with p. aeruginosa is responsible for most of the morbidity and mortality in patients with cf. cross infection involving the epidemic strains liverpool (les), manchester (mes), midlands (mid ) and clone c has been documented. regular genotyping is recommended to assess distribution of genotypes. genotyping is not currently performed in the belfast trust. the aim of this study is to perform molecular typing of isolates. the results will inform future strategies for laboratory screening, and infection control. p.aeruginosa isolates collected during routine clinics were typed using pulsed field gel electrophoresis (pfge), restriction patterns were analysed using bionumerics. a multiplex pcr ( ) was used to type isolates. samples were typed by both methods and results compared. % of isolates were defined by pfge as the les genotype. . % of adult isolates were defined as clone c. no mes or mid isolates were reported. there was . % correlation between pfge and pcr results. this study reports prevalence of the les strain in the ni cf population. it is recommended that patients with cf infected by p.aeruginosa have all isolates of varying phenotypes genotyped. pcr detection of les isolates will be a useful tool for screening. having successfully derived a method to culture nasal epithelial cells (necs) from cystic fibrosis (cf) and non-cf subjects, the aim of this study was to characterise the electrophysiological responses of these cells. cells from f del/f del patients and non-cf controls were used for patch-clamp investigation. cultured cells were separated and plated on glass coverslips chambers. culture medium was replaced with standard external solution (ses) before establishing whole-cell configuration. membrane ion currents were recorded using patchclamp. once a stable current was achieved, amiloride and forskolin were applied to the cells to elicit their responses. the f del/ f del cells responded to amiloride by rapid reduction in wholecell current, when forskolin was added to the bath it had little or no effect on the cell current amplitude in the cf cells (n = ). in the non-cf cells however, there was a response to the addition of forskolin. these responses to both amiloride and forskolin were as expected and demonstrate that this cell model of nasal epithelial cells obtained from nasal brushings proves to be a very feasible model for future studies of cf and can be used as an ideal model for research into the nature of action of numerous cf drugs. it has been shown that females with cystic fibrosis (cf) have worse lung function compared with cf males. gender and bmi have been correlated with lung function in adults. we aimed to show a similar trend in a paediatric population. we studied children aged years and older attending our cf unit. fev , height and weight were measured at the clinic when patients were at their baseline. of patients, were males ( %) and were females ( %). of the males had fev [ % ( %), had fev - % ( %) and with fev \ % ( %). within the female subgroup, had fev [ % ( . %), had fev - % ( . %) and had fev \ % ( . %) (p = . ). bmi was divided into groups; \ th percentile indicating poor nutrition that may affect lung function and [ th percentile (table ) . no statistical difference was found (p = . ). there was no statistical difference between genders with regards to lung function although there was a trend in favour of males. in our group of well nourished patients, there was no correlation between bmi and fev . this is in contrast to adult data in a similar study. piperacillin-tazobactam induced fever is well documented in patients with cystic fibrosis. here, we report adverse reactions which occurred in three patients treated with piperacillin-tazobactam over a three month period. setting: tertiary care, academic medical centre (beaumont hospital, dublin, ireland). patients and methods: three patients were evaluated retrospectively for piperacillin-tazobactam induced fever and evidence of bone marrow suppression. results: two of our series had evidence of drug induced fever (using criteria by young et al.). both patients had a mean duration of piperacillin-tazobactam exposure of . days with an average temperature of . c. fever resolved in both patients within hours of discontinuation of the antibiotic. neither had evidence of a septic focus (determined by cxr, blood cultures, ivc tip analysis or msu). two of our series developed bone marrow suppression, one becoming pancytopenic requiring bone marrow biopsy, the other becoming transiently neutropenic. both recovered within hours of cessation of offending agent. components of the new iv piperacillin-tazobactam preparation (ph buffers or stabilising agents) may be involved in the development of these late reactions. objectives: cystic fibrosis patients suffer from chronic bacterial colonisation and repeated exacerbations. one of the most challenging elements of treating these patients is that they develop antibiotic resistance. the aim of this study was to assess if changes in antibiotic sensitivity were related to the number of exacerbations (noe). we compared the noe requiring intravenous antibiotics with respiratory cultures at the time. the sensitivities of pseudomonas to antibiotics were analysed from to . we correlated the changes in sensitivities with the noe they had in this period. a univarious analysis was performed using a non-parametric test. noe was used as a dependent variable. thirty-two patients were included. in the piperacillin/tazobactam group, % became resistant with a mean noe of . . (p = . ) no resistance developed with colomycin. % of patients developed resistance to gentamicin. % of patients developed resistance to ceftazidime with average noe of . . (p = . ) only % of patients developed resistance to ciprofloxacin. the findings of this study showed considerable variations with antibiotic sensitivities. twenty-one patients demonstrated some change in sensitivities, and eleven with none. those with antibiotics resistance had a higher noe compared to those with constant sensitivities. slpi is an anti-inflammatory antiprotease that negatively regulates tlr , , and . we examined the effect of the viral rna mimic polyic on cytokine production by evaluating responses it induced in airway epithelial cells; we then evaluated slpi's effect on these responses. we performed selective inhibition studies to identify the receptor by which polyic induces its effects. rna was isolated from cystic fibrosis (cf) and non-cf bronchial epithelial cells and used in quantitative rtpcr reactions with gene- fev \ % ( %) ( %) s specific primers to each receptor. cells were stimulated with polyic in time course and dose response experiments and il- and interferon (ifn)-beta production quantified by elisa. the effect of pre-treatment with slpi or receptor inhibitors was evaluated. polyic induced il- but not ifn beta production. il- production was inhibited by pretreatment with slpi but not by inhibitors to pkr, rig , or mda . further rtpcr experiments demonstrated deficiency of phosphatase shp- , important for interferon production. polyic induces expression of the proinflammatory cytokine il- via a mechanism involving tlr . slpi inhibits this effect. polyic does not induce ifn beta production in airway cells. shp- deficiency demonstrated is a proposed mechanism for this effect. objective: in cystic fibrosis the mechanisms that lead to initial bacterial colonization, the development of a sustained and predominantly neutrophilic inflammatory response, and the ultimate destruction of the lung over decades remains unclear. here we investigate whether humoral autoimmunity could play a role in pathogenesis of cystic fibrosis. circulating autoantibodies in plasma of cystic fibrosis patients (n = ) and of healthy controls (n = ) were studied using immunofluorescense using hep cells as a substrate. selected samples were studied using immunofluorescense on primary bronchial epithelial cells. eight out of cf patients presented igg autoantibodies against hep epithelial cell line and against primary bronchial epithelial cells. there was no apparent correlation between fluorescence intensity and autoantibody titers and the disease intensity. the fluorescence pattern was in all cases mixed (speckled and nucleolar). igg autoantibodies with avidity for bronchial epithelium are present in some patients with cystic fibrosis. this suggest that autoreactive adaptive responses directed against bronchial epithelium may be important in aetiology of the disease and warrant further investigations. the role of pulmonary rehabilitation (pr) has not been widely investigated in patients with diagnoses other than copd. the aim of this study was to investigate the effects of an week pr programme in patients with bronchiectasis. seventeen patients with a diagnosis of bronchiectasis were recruited from respiratory consultant clinics. all patients underwent an -week programme ( supervised sessions) of exercise training and education. subjects were assessed at baseline and on programme completion on measures of exercise capacity and quality of life. data were analysed using minitab version . thirteen patients ( female, male) completed the programmemean age . (sd = . ) years, mean %predicted fev . % (sd = . ). after eight weeks, there was a significant increase (p = . ) in the incremental shuttle walk test distance of . metres ( % ci: . to . m). there was a trend toward a statistically significant improvement (p = . ) in the st george's respiratory questionnaire impacts subscale although there was no statistically significant improvement in the overall score (p = . ). results of this observational study support the potential role of pr in patients with bronchiectasis. robust trials are necessary to assess the effect of such programmes on a range of outcomes, as well as the efficacy of individual programme components. pkcd genetically depleted mice had baseline capillary filtration coefficient (kfc) compared to their wild type counterparts. there was no difference between these groups.similarly, there was no difference between wet-to-dry ratio's at baseline or in response to hydrostatic challenge.however, pkcd knockout mice experienced a significant protective effect when challenged with lps for hours injected intraperitoneally compared to their wild type. this correlated with reduction in neutrophil recruitment to the lung as well as significant attenuation of histological evidence of lung injury. however, there was no significant difference in the respective cytokine profiles. pkcd plays a central role the development of acute lung injury following exposure to lps and may represent a potential therapeutic target in attenuating acute lung injury. the precise mechanisms remains to be elucidated, however it is likely mediated through impaired neutrophil response. mycobacterium tuberculosis (mtb) is responsible for almost million deaths annually (who). the success of the bacillus is largely due to its ability to evade the host immune response. mycobacteria survive s within macrophages by blocking fusion of phagosomes and lysosomes, thus avoiding exposure to antimicrobial peptides and enzymes present in lysosomes. il- inhibits the progression of phagosome maturation in murine macrophages, however little is known about the role of il- on phagosome maturation in human macrophages. pma-differentiated thp- cells were seeded at . x cells/ml on glass coverslips. monocyte derived macrophages (mdms) were isolated from buffy coats obtained from the irish blood transfusion board. cells were treated with anti-il- monoclonal or isotype control antibody, infected with live or killed gfp-bcg and pkh green-labeled m. tuberculosis h ra, and incubated with lamp- antibody and alexa fluorescent stain. the colocalisation of mycobacteria-containing phagosomes with lysosomes, as identified by lamp- , was determined by confocal microscopy. colocalisation of mycobacteria-containing phagosomes with acidified lysosomes was infrequent in untreated thp- cells, % ± . . however colocalisation increased when killed mycobacteria were internalised ( . % ± . ). similarly in cells treated with anti-il- colocalisation increased to % ± . . mdms treated with anti-il- and infected with gfp-bcg showed a significant increase in phagosome maturation compared to untreated mdms. this data suggests il- suppresses the ability of macrophages to proceed with phagosome maturation, favouring survival of mycobacteria within the host. the prevalence of tuberculosis (tb) in ireland is not decreasing and management is becoming more complex with a multi-cultural population and increased drug resistance. there are new presentations (e.g. tb associated with biological agents). shorter rotations for junior doctors may be associated with less familiarity with tb management. we reviewed the appropriateness of our practice by examining the management of randomly selected patients attending our clinic. there was an equal gender balance and % were non-nationals. forty-two percent had pulmonary tb. significant deficiencies in management and documentation were identified. bcg status was not recorded in cases. incomplete data was available on the patients' hiv status. while all patients received pyridoxine prophylaxis, % of patients receiving ethambutol did not have an ophthalmology review. only % of patients received ethambutol. one patient (with multi-drug resistant tb) was transferred to another centre for negative pressure isolation. of the patients who were treated, one did not complete their treatment. this review indicated a need for improved documentation of bcg, hiv status and attention to issues such as ophthalmology review. to achieve this we have developed a clinical care pathway for management of tb in our clinic. background: northern ireland has consistently had a low tb prevalence ( . / , ). however, there has been an increase in cases of mdr-tb admitted to the rvh since . retrospective chart review of mdrtb cases. six patients were admitted between - . five were suspected to have mdr-tb on admission based on epidemiological risks; two had prior tb treatment; one was a contact of mdrtb; two were from countries of high mdr prevalence. four patients had primary mdrtb. the rifampicin resistance probe was positive in all cases. susceptibility testing showed isolates to be resistant to a median of drugs. all were susceptible to second line injectable agents, and / were susceptible to quinolones. one patient was hiv co-infected. patients converted their sputum to culture negative in a median of weeks (range - ) and were considered for discharge when they had negative cultures month apart. hospital admissions were prolonged due to drug toxicities and/or social issues (median hospital stay months, range - ). discussion: due to globalisation, even countries with low tb prevalence need to manage mdrtb. management of these patients is complex, and significant toxicities were seen. prolonged isolation also has significant resource implications. a year old male was admitted from the local psychiatric unit with apparent pneumonia. a chronic schizophrenic and heavy smoker, he had spent some years in institutional care in various facilities. he was treated with high flow humidified oxygen and nebulised bronchodilators. hours later he was found to be sputum smear positive for aafb. he was isolated and the infection control team mobilised. he turned out to have a fully sensitive m.tb organism and responded well to treatment. bts tb guidelines were followed and all staff and patients in the same ward bay as the patient were informed and letters sent to all gps. one patient contact had oesophageal carcinoma and was a chronic alcoholic. he developed post operative pleural effusion (tb culture positive) some months post exposure. a second contact (also alcoholic) was investigated as possible lung cancer some months after exposure and was smear positive for aafb. both patients died on treatment for their tb. all tb isolates were identical. the index case is alive and well. this highlights the danger of even short delays in diagnosis and appropriate isolation of tb patients. streptococcus pneumonia is a leading cause of invasive diseases which will pose an important health problem in ireland. efficacy of pneumovax is between and % (ref ). the vaccination would prevent severe pneumococcal infections (ref ). the purpose of the audit was to determine the rate of update of pneumovax among the chronic respiratory group and to identify reasons why the vaccination may not have been administered. subsequently, address ways of improving current policy. patients who attended the respiratory outpatient clinic in st. james's hospital from july till jan were questioned whether they have the pneumovax given at any stage of the life and ever receive booster dose; and reasons why if they had not been vaccinated. a total of patients were interviewed. . % were male and . % were female. mean age of male was . and mean age of female was . . approx % of patient has obstructive airflow diseases. % has received pneumovax at some stage while % never received pneumovax. the main reason ( . %) is poor awareness of the importance of the vaccine. the main implication from the audit is to raise awareness of pneumovax via campaign, reminding gp; and the funded pneumovax clinic. nursing home acquired pneumonia (nhap) is an important subset of healthcare associated pneumonia. we have prospectively compared three different pneumonia severity scores: pneumonia severity index (psi), modified bts score and naughton score and several inflammatory markers including procalcitonin levels to determine their ability to predict fatality in nhap. this study was carried out on fifty patients presenting to our hospital over a two year period, july to june inclusive. the case fatality rate was %. the modified bts score most accurately identified fatal outcome ( n = ; n = ; n = ). psi and naughton scores were poor predictors with % of deaths characterized as psi category or . procalcitonin levels co-related well with disease severity as measured by the psi and modified bts scores. none of the inflammatory markers accurately predicted patients at low risk of death from nhap. we conclude that the different pneumonia severity scores and several commonly used inflammatory markers fail to accurately predict patients at low risk of death from nhap. the adelaide and meath hospital, tallaght opened in and celebrates its th anniversary this year. we sought to assess changes in the incidence, demographic characteristics, and microbiological profile of tb infection in our institution over this period. patients diagnosed with active tb in the years & were included. patients were identified using laboratory, public health, and hipe records. demographic, clinical, and microbiological data were obtained by retrospective chart review. there was marked growth in the incidence of tb over this period our data emphasizes the re-emergence of tb as a major public health issue in ireland, and highlights immigration as a major contributing factor. the emergence of resistant infection has not to date been seen in our institution, but can be anticipated in the near future. we report a case of a -year-old white male who presented with one month history of pleuritic chest pain. chest radiograph demonstrated left upper lobe cavitation. bronchoalveolar lavage was smear positive for acid-fast bacilli and culture grew pan-sensitive mycobacterium tuberculosis complex. standard anti-tuberculous treatment with rifater and ethambutol was instituted; however the patient developed a severe cutaneous reaction after weeks. skin biopsy demonstrated findings consistent with allergic dermatitis. the rash resolved after discontinuation of therapy; however it recurred after sequential re-exposure to rifampicin, ethambutol, isoniazid and moxifloxacin. the patient underwent desensitization to rifampicin; ethambutol and isoniazid using modified penicillin protocols. titration to target dosing with each individual drug was achieved allowing reinstitution of standard therapy with rifater and ethambutol. the sequential desensitisation processes were each complicated by the reoccurrence of a mild rash, which was controlled by oral prednisolone. to our knowledge this is the first reported case of hypersensitivity to four anti-tuberculosis agents with a favourable response to a strategy of sequential rapid oral desensitisation. introduction: tb is a major infectious disease. in n.ireland the incidence remains low but is increasing. in cases were notified, from the southern trust. a tb nurse was appointed in september . this audit was undertaken to review the tb clinic and optimise the service provided. a retrospective month case note audit. all aspects of the service were reviewed; nationality of those attending, use of interpreter services, the problem of missed appointments and the reason for failure to attend. contact tracing and mantoux testing were assessed as this is a new role for the tb nurse. compliance with treatment in the light of mdrtb cases. results: patients attended the tb clinic in the month period. ( . %) were not native of n.ireland. there have been cases of mdrtb in the trust in the last year which has posed particular operational problems. tb remains an active problem in the southern trust. patient attendance at the clinic may be optimized by having appointment letters in the patients own language and introducing services at peripheral sites. lack of negative pressure ventilation facilities is an ongoing problem. pharmacy dispensing of full treatment course has improved compliance. we sought to analyze differences between patients with latent, pleural and pulmonary tuberculosis (tb) attending a dedicated tb clinic in the mercy university hospital, cork from july to june . two hundred and sixty nine patients were referred to the clinic. one hundred twenty eight ( %) had active tuberculosis, ( %) had latent tuberculosis infection (ltbi), ( %) had an alternative diagnosis and ( %) had atypical mycobacterial infection. among those with active tuberculosis, ( %) had pulmonary tb and had extra pulmonary tb, of whom had pleural tb ( %). in this group of patients with tb infection, female sex and foreignborn status were more frequent in those with ltbi compared with those with pulmonary tb. in contrast, smoking was more prevalent in patients with pulmonary tb. % of patients with pulmonary tb were asymptomatic. mantoux induration was less in those with pleural tb compared with those with pulmonary tb and ltbi. directly observed therapy was implemented predominantly in patients with pulmonary tb and at a rate considerably short of world health organisation recommendations across all groups. the purpose of this study was to determine if the lysophospholipids sphingosine -phosphate (s p) and lysophosphatidic acid (lpa), which have been implicated in allergy, induce up-regulation of adhesion molecules and eosinophil chemoattractants in an in vitro cholinergic nerve cell model, imr- cells. s p and lpa act mainly via g-protein coupled receptors s p - and lpa - respectively. eosinophils accumulate at innervating cholinergic nerves in fatal asthma and in animal models of asthma and adhere to nerve cells in culture via intercellular adhesion molecule- (icam- ). the methods used were real-time pcr and western blotting. s p , s p , lpa , lpa and lpa were expressed on imr- cells. both s p and lpa induced erk phosphorylation and erkand g i -dependent up-regulation of icam- expression in imr- cells, with differing time courses. lpa also induced erk-and g i -dependent up-regulation of the eosinophil chemoattractant, ccl- . the eosinophil granule protein eosinophil peroxidase (epo) induced erk-dependent up-regulation of transcription of s p , lpa , lpa and lpa . thus s p and lpa, acting via g i -coupled nerve cell receptors, induce up-regulation of adhesion molecules and chemoattractants which stimulate eosinophil accumulation and adhesion to cholinergic nerve cells. in turn, epo induces up-regulation of s p and lpa receptors, potentially perpetuating s p-and lpa-induced effects. asthma and rhinitis are characterised by eosinophilic inflammation. however, recent studies indicate that eosinophils are not essential for clinical symptoms, but instead exert a remodelling effect on the local tissues. we propose that neural remodelling involving the bmp pathway, enhancing a cholinergic phenotype, is a potential mechanism of airway remodelling in asthma. imr cells behave like cholinergic neurons when cultured with sodium butyrate. we exposed imr cells to eosinophil granule proteins and to bone morphogenetic proteins & and harvested the cells. proteins were separated into fractions and western blot analysis was performed. rna was isolated, converted to copy dna, and analysed using quantitative pcr. we found that major basic protein, but not eosinophil peroxidase, produced a down-regulation of bmpreceptor a (bmpr a) gene expression ( % reduction at hrs, p = . ). mbp decreased bmpr a in membrane protein and increased bmpr a within the nuclear protein. co-incubation of bmp with mbp attenuated expression of the bmp pathway transcription target id and of choline acetyltransferase. co-incubation with bmp & mbp produced increased expression of id and choline acetyltransferase. these results indicate that eosinophil granule proteins change bmp receptor balance, producing a downstream effect on cholinergic gene expression and therefore on the cholinergic phenotype of cells. exercise-induced bronchoconstriction (eib) has a reported prevalence of - %. this study aimed to measure for the first time, the prevalence of eib and asthma in professional rugby players and to demonstrate the utility of a sport specific field-test as a screening tool in field-sport. prospectively a cohort of senior international rugby players underwent spirometry before and after a rugby-specific exercise challenge. exercise intensity levels were also assessed. a fall in forced expiratory volume in one second (fev ) c % from baseline after exercise challenge was considered diagnostic of eib. during analysis, players were divided into two groups: those with airflow obstruction (ao) and those without (nao). ao airflow obstruction, nao no airflow obstruction forty-two players were tested. table summarises the spirometric results. twelve players ( %) had a history of, or were diagnosed with, airflow obstruction (ao group). seven players had a previous diagnosis of asthma and were on inhaled treatment, of these; % (n = ) had eib after exercise despite regular inhaled therapy. five players ( %) were newly diagnosed with eib. eib is more prevalent in professional rugby players than in the general population. a pre-existing diagnosis of asthma with regular inhaled therapy does not preclude eib. sports-specific field-testing is a useful method of screening in players. ulster hospital, dundonald, regional immunology service, royal hospitals, belfast. asthma is a major risk factor of anaphylactic deaths in children with peanut allergy. peanut allergy is a lifelong condition but some children outgrow their coexistent asthma. it is currently not known whether children who have outgrown their asthma symptoms have ongoing eosinophilic airways inflammation. exhaled nitric oxide is recognised as a non-invasive marker of eosinophillic airways inflammation. the aim of our project was to examine the levels of exhaled nitric oxide in peanut allergic children. children with peanut allergy were recruited at the ulster hospital and royal belfast hospital for sick children, northern ireland. exhaled nitric oxide levels (eno) were measured using the niox mino in all children. results: children were enrolled over a month period, age range to years (median years). ( %) had no history of wheeze, ( %) had outgrown asthma, ( %) had current active asthma and ( %) had occasional wheeze within the last year but were not taking any regular asthma medication. levels of eno were significantly elevated in those with outgrown asthma and those with occasional wheeze but no regular asthma medication (p \ . ). exhaled nitric oxide levels were elevated in children with a history of asthma outgrown and those with current 'untreated' asthma. this would suggest ongoing allergic airways inflammation. our study gives a rationale for checking eno in children with peanut allergy. consideration should then be given to starting inhaled corticosteroid therapy in peanut allergic children with elevated exhaled nitric oxide levels. guideline-defined asthma control, physical activity and bmi omalizumab has been shown to be effective in severe persistent allergic. this study describes our local experience in a group of carefully selected asthmatic sufferers. a retrospective audit was performed on patients with severe persistent allergic asthma who fulfilled the criteria for omalizumab therapy. only those who were regarded as omalizumab-responders were included in this analysis (n = ). the primary outcome measures for the study were acute hospital admissions, exacerbation rates, reliever usage and change in fev . these data were analysed for the six month period prior to commencement of omalizumab and for the six months following. the results showed that as a group acute hospital admissions reduced by %, with a corresponding reduction in exacerbation rates of %. mean fev increased by ml and reliever usage was reduced by %.this is consistent with that reported in the literature. omalizumab has proven effective in our local population of carefully selected severe asthmatics. introduction: difficult to treat asthma (dta) is associated with frequent symptoms despite therapy with high dose inhaled corticosteroids (ics). adolescent asthma presents special difficulties given the associated development issues. we sought to determine objective features of dta in this group. methods: all patients ( - yrs) attending the adolescent asthma clinic were reviewed. clinical data was collected at referral. dta was defined as requiring high dose ics (bdp) of [ mcg/day (\ yr) or [ mcg/day ( - yrs). the dta group was compared with remainder on low dose ics. of a total of ( f: m) patients, ( %) had dta with a mean ics of mcg/day. there were no significant differences in steroid rescue, bmi, fev /fvc, serum ig or eosinophil level, dust mite responsiveness, eczema, rhinitis, or smoking. dta patients were more likely to have elevated eno ( vs. ; p = . ) and lower ige level ( vs. ; p = . ). co-existing conditions more likely in dta included grass allergy (p = . ), any food allergy (p = . ), gord (p = . ) and vcd (p = . ). conclusions: dta in adolescents was associated with higher eno and lower ige levels, grass and food allergy, gord and vcd. identification of these features early can potentially facilitate more comprehensive and effective management. omalizumab is a monoclonal ige antibody which reduces asthma exacerbations. it is only cost effective in severe asthmatics with recurrent exacerbations. to assess the outcome of omalizumab treatment in severe asthmatic patients in the respiratory department of connolly hospital. a retrospective chart review of asthmatic patients treated with omalizumab. baseline demographics, omalizumab dose and frequency, other asthma medications, fev , exacerbation rates, and side effects were reviewed. male to female ratio was : . mean age was . years. mean ige level prior to omalizumab was . u/ml. mean fev prior to treatment was . %. mean fev post treatment was . %. prior to omalizumab five patients were on step of gina treatment guidelines, patients were on step . four patients reduced their shortacting beta agonist requirements, one patient was weaned to a lower dose of steroid and patient increased their inhaled steroid dose during the treatment period. patients had recurrent exacerbations prior to treatment. mean number of exacerbations during year of treatment was . . the most common side effect experienced was joint pains. treatment with omalizumab reduced exacerbation rates in these severe poorly controlled asthmatics. airway sensory hyperreactivity (shr) is an important clinical feature in chronic cough and is characterised by bouts of coughing triggered by relatively innocuous stimuli including exposure to aerosols, scents and changes in air temperature. these abnormal sensory responses are often what distress a patient most about their condition . the aim of this study was to determine the prevalence and clinical features of shr in patients with chronic cough. we undertook a retrospective case review of sequential referrals to the belfast city hospital cough clinic. we defined shr + as those individuals reporting cough provoked by one or more of the following; ) change in air temperature (thermoactivation), ) exposure to aerosols, scents, odours (chemoactivation), ) talking, laughing or singing (mechanoactivation). we compared shr + with shr-patients across a range of variables using chi-square and analysis of variance as appropriate. charts were available for review. we identified shr + in ( %) with significantly more females in the shr + ( % versus %, p = . ). no other features including age, cough duration, cough aetiology, atopic status, preceding urti or pc reliably distinguished shr + from shr-patients. these preliminary results suggest shr is a common problem especially among females with chronic cough. omalizumab is a humanized monoclonal antibody to ige which prevents binding to fceri receptor. it is known to increase eosinophilic apoptosis and inhibit the th immune response culminating in a reduction in eosinophil recruitment, activation and tissue migration. omalizumab use in the management of css however has been reported in one case to ameliorate the asthmatic component of the disease and to significantly lower plasma eosinophil counts at three months. our patient, mb, a year old man diagnosed with css years ago manifesting with uncontrolled asthma, severe pan-sinusitis with recurrent nasal polyposis, eosinophilic gastoenteritis histologically confirmed with duodenal biopsy and subacute bowel obstruction. initial eosinophil count was . /l., with an elevated ige level of u/ml. autoantibodies were negative. mb's extrapulmonary symptoms were well contolled with oral steroids and azathioprine. efforts to minimize oral steroid doses were hampered by persistent asthma and rhinosinusitis as well as a relapse of eosinophilic gastroenteritis in . omalizumab was commenced in june . after weeks, apreciable improvements in asthmatic and sinusitis symptomes were noted. average peak flow improved from to l/m and a reduction in peripheral eosinophil count from . to . /l. a paediatric asthma telephone clinic (patc) was set up in our hospital to provide follow up for children with mild asthma. structured telephone interviews with children aged between and and their carers who received detailed asthma education, was conducted and subsequent appropriate clinical action initiated. the aim of this study is to assess the effectiveness of the patc for medical surveillance by the asthma nurse. a retrospective review of case notes of the children referred to the patc was conducted. unscheduled use of health care & use of antibiotics or steroids between time of the patc and the next out patient clinic were recorded. pulmonary function was compared pre and post the patc. descriptive statistics were carried out and a paired t test was used to detect any significant change in lung function. forty one patients were referred to the patc with asthma. only ( %) had an unscheduled visit to the gp and no patient presented to s the emergency department. there was no significant change in pulmonary function. routine follow up of children with mild asthma and their carers by an asthma nurse via a structured telephone consultation can be considered an alternative to face to face follow up. further evaluation comparing the patc to an outpatient clinic and assessment of child/ carer satisfaction could confirm this. approximately , ( . %) patients received inhaled shortacting beta agonists in combination with a regular standard-dose inhaled corticosteroid.. a further , ( . %) patients were also prescribed a regular inhaled long-acting beta agonist (salmeterol or formoterol). patients ( . %) on combination therapy were coprescribed four different asthmatic treatments inclusive of oral prednisolone. approximately ( . %) of the patients prescribed a respiratory drug were co-prescribed nicotine replacement therapy. in total there were , patients prescribed a mucolytic drug in combination with a respiratory drug b) there were significant levels of coprescribing of salbutamol with beta blocking agents at . % ( %ci: , . ). in addition . % ( %ci: . , . ) of the patients prescribed theophylline in were also prescribed ciprofloxacin. c) levels of co-prescribing with antibiotics was %. the antibiotics coprescribed were augmentin, clarithromycin, cephalosporins and ciprofloxacin. assessing changes in asthma control is difficult. peak flow diaries are not completed and history is subject to recall bias. with a view to developing an electronic asthma management system, we attempted to use breath acoustics to assess changes in respiratory status. spirometry and breath sounds were simultaneously recorded in asthmatic subjects during histamine challenge. breath sounds were recorded by a microphone over the trachea. data was collected from seven male and four female subjects with a mean age . yrs. acoustic features were extracted from the breath sounds and evaluated for a correlation with the percentage change in fev . the highest correlation occurred between the duration of exhalation and percentage change in fev (r = - . ). fev showed a correlation with number of wheezing episodes (r = - . ), median wheeze frequency (r = - . ), maximum wheeze duration (r = . ), frequency of maximum duration wheezing group (r = - . ) and mean frequency of the wheezes(r = - . ). using these features together (combined in a linear discriminant classifier) a % drop in fev was detected with a sensitivity of % and specificity of %. the results of this study suggest a strong relationship between duration of exhalation and fev .the study also showed that combining acoustic features can be beneficial in detecting a decrease in fev . the liver disease of alpha- antitrypsin deficiency (aatd) is associated with endoplasmic reticulum(er) stress. seps is a selenoprotein that through a chaperone activity decreases er stress. we aimed to determine the effect of seps on er stress in this condition by measuring activity of the grp promoter and levels of active atf as markers of the unfolded protein response in hepg cells transfected with zaat transgene. we investigated levels of nfjb activity, a marker of the er overload response. to determine the effect of selenium supplementation on the function of seps we investigated glutathione peroxidase activity, grp promoter and nfjb activity. we also investigated the anti-inflammatory effect of selenium through the -deoxy-d , -prostaglandin j pathway( d-pgj ) and checked selenium levels in a population of zz and mm phenotypes for aatd. seps reduced levels of active atf . overexpression of seps also inhibited grp promoter and nfjb activity and this effect was enhanced in the presence of selenium supplementation. increased d-pgj concentrations were found in selenium supplemented cells. we demonstrated serum selenium levels to be in the low normal range in the patients tested. this data demonstrates a role for seps in this conformational disease and suggests a possible therapeutic potential for selenium supplementation. alpha- antitrypsin (a at) is a glycoprotein synthesised chiefly in the liver and functions as the most important antiprotease in the lung and also demonstrates anti inflammatory properties. it has previously been demonstrated that a at is packaged along with neutrophil elastase within the primary granules of these cells [ ] . thus there remains a paradox as to why an enzyme and cognate inhibitor would simultaneously compartmentalize, potentially impeding protease antimicrobial activity. this aim of this study was to reevaluate the localisation of a at within the neutrophil. compartmentalisation of a at within the neutrophil was established by sub-cellular fractionation, western blot analysis and confocal immunofluorescence. our data clearly show that a at is a genuine outer membrane protein of neutrophils associated with cholesterol-and sphingolipidenriched membrane domains called lipid rafts. we have observed that treatment of neutrophil membranes with phosphatidylinositol-specific phospholipase c (piplc) or high nacl concentrations removed a at from the neutrophil membrane indicating that localization of a at in lipid rafts is mediated by electrostatic interactions to a glycosylphosphatidyl-inositol (gpi) linked membrane protein. further studies will address the relevance of neutrophil associated a at and may support the theory that the anti inflammatory effects of a at are not simply related to modulation of serine proteases activity. aat deficiency (aatd) is a hereditary disorder, resulting from mutations in the serpina gene, classically presenting with earlyonset emphysema and liver disease. the most common mutation associated with aat deficiency is the z mutation, with the s mutation also associated with lung disease. aat deficiency is under-diagnosed and prolonged delays in diagnosis are common. world health organisation guidelines advocate screening patients with copd, asthma, cryptogenic liver disease and first degree relatives of known aatd patients. zz aatd patients on the national alpha- registry (n = , . +/- . years, male, female) were compared to a cohort of mm copd patients (n = , . +/- . years, male, female). mean aat levels in the zz group were . +/- . g/l compared to . +/- . g/l in the mm copd cohort. the mean fev for all zz patients was . +/- . % compared to . +/- . % for mm copd patients. however, when zz cases identified by family screening were removed, the mean fev of the zz cohort was lower than the mm group ( +/- . %, p = . , compared to mm group). when mm and zz groups were stratified by smoking status, zz smokers had mean fev of . +/- . % compared to . +/- . % for never smokers, while mm smokers had mean fev of . +/- . % compared to . +/- . % for never smokers. these findings underline the clinical significance of the zz phenotype and smoking in the development of copd. ). in the present study we examined the immunomodulatory activity of aat and investigated whether nadph-oxidase activation via the g-protein coupled n-formyl-methionyl-leucyl-phenylalanine (fmlp) receptor was inhibited by aat. oxygen (o ) consumption was quantified using a clark-type oxygen electrode and o production was determined by superoxide dismutase (sod)-inhibitable reduction of cytochrome c. both the rate of o consumption and o production elicited by fmlp ( - m) was significantly inhibited in the presence of aat ( lm). in addition, inhibition of o production was dose dependent and almost completely inhibited by . lm aat. mechanisms of inhibition were investigated and found to be mediated through a decrease in intracellular camp. levels of camp at seconds post fmlp stimulation were elevated to . ± . pmol/ neutrophils, whilst co-treatment with aat ( . lm) reduced camp levels to . ± . pmol/ cells. in conclusion, the observed inhibition of neutrophil nadphoxidase activity by aat, is further evidence supporting a role for this molecule as an anti-inflammatory mediator. alpha- antitrypsin (aat) is a serum glycoprotein that inhibits proteases, and is produced mainly by hepatocytes. it is particularly important in dampening the action of neutrophil elastase, which can damage the lungs. aat deficiency results from both a qualitative and a quantitative deficiency of the protein which predisposes to the development of emphysema, chronic bronchitis, bronchiectasis, and liver disease. we investigated patients registered on the irish alpha- database as aat deficiency mz phenotype. the information gathered from the database was supplemented with chart reviews for clinical information and pulmonary function tests. mz patients had a mean fev % predicted of . +/- . %. we note that there is a negative correlation between cigarette pack years and fev % predicted (r = . ). the serum level of aat does not necessarily correlate negatively with fev (r = . ). nearly % of mz patients were detected by family screening of known aat deficient patients. it remains uncertain whether mz patients are predisposed to aat deficiency sequelae when compared to the general population. we aim to settle this uncertainty by comprehensively describing the characteristics of this cohort of patients. this may represent a change in the way mz patients are managed and could implicate earlier preventative measures to decrease the likelihood of developing emphysema. alpha- antitrypsin (aat) is produced by hepatocytes, and is the most important antiprotease in the lung. aat deficiency (aatd) is a hereditary disorder resulting from mutations in the aat gene, presenting with emphysema in adults and liver disease in childhood. who guidelines advocate a targeted strategy in screening copd, non-responsive asthma, and cryptogenic liver disease patients and also relatives of known aatd patients. the most common phenotype associated with disease is zz followed by sz. a chart review of aatd patients on the national alpha- registry was performed on zz (n = ) and sz (n = ) patients. the mean age at diagnosis for zz patients was . +/- . years for males and . +/- . years for females. we demonstrate that zz individuals identified as a result of family screening have significantly increased fev ( . +/- . %, . +/- . years) when compared to zz patients identified by targeted symptomatic screening ( . +/- . %, . +/- . , p = . ). zz and sz patients who smoked had significantly decreased lung function compared to nonsmoking zz and sz and that a positive correlation between pack years and fev exists. our results emphasize the need for increased awareness and early detection of asymptomatic aatd. identification of patients from a targeted detection programme should include aggressive family screening and allow the initiation of preventative measures before significant lung disease has occurred. thirty-three females age - mean and males, age - , mean underwent cpx. mean bmi in females was . ( . ) and . ( . ) in males. most tests were done because of unexplained dyspnoea ( ). other reasons for requesting cpx were: assessment for fitness for lung cancer surgery ( ); assessment of fitness in patients with sarcoid ( ); cardiac transplant assessment ( ); pre surgical assessment for other major surgical procedures ( ). the data on the patients who underwent cpx for unexplained dyspnoea was analysed. mean bmi was . . thirty-two of these patients had normal lung function. as a group these patients were extensively investigated before coming for cpx, had ct scans performed, had lung perfusion scanning, had echocardiography. forty-one of the patients had sub maximal tests with no evidence of cardiac or respiratory disease, the test being limited by reconditioning. in conclusion, cpx is a valuable tool in this district general hospital with various reasons for requesting the test. in patients with unexplained dyspnoea, it may be prudent to request cpx at an earlier stage in the investigative journey. sarcoidosis is a multisystemic disease of unknown aetiology. prognostic biomarkers are not part of routine clinical practice. our hypothesis is that enhanced activity for myofibroblast differentiation in sarcoidosis at the initial diagnosis and is associated with an adverse prognosis and the development of pulmonary fibrosis. in addition we investigate the role of tgf-b in this process. fifty patients with biopsy proven sarcoidosis (stage i n = , stageii n = , stage iii n = ) were enrolled. bronchoalveolar lavage (bal) samples were obtained at initial evaluation. primary lung fibroblasts (ccd- lu) were incubated with bal samples and a-smooth muscle actin (asma) mrna expression as a marker of myofibroblast differentiation was assessed via rt-pcr. asma mrna was significantly elevated up to % (above control) in patient's samples. we also found a significant correlation between asma mrna expression and progression of pulmonary disease in sarcoidosis, (p \ . ). to investigate the role of tgf-b contributing to this enhanced myofibroblast differentiation, we coincubated bal samples with saturating concentrations of anti-tgf-b antibody and found a % reduction in this biological activity, (p \ . ). in conclusion we demonstrate firstly enhanced capacity of bal samples to induce myofibroblast differentiation, secondly it is of prognostic significance and finally tgf-b is a significant contributor to this biological activity (fig. ). we present a case of cs seen recently at our hospital and the management undertaken for this yr old, the son of a tuberculosis specialist in south africa. his main symptom was exertional syncope with evidence of conduction abnormality on ecg, poor cardiac function on echocardiogram with normal coronary angiogram. an exercise stress test induced ventricular tachycardia. a dual chamber pacemaker and a defibrillator were placed. he had several hospital admissions over a year with ventricular tachycardia/ ventricular fibrillation. cardiac ablation followed electrophysiological studies. cardiac biopsies confirmed cs. there was no evidence of pulmonary or eye involvement. we discuss diagnostic tests and criteria and also treatment options. corticosteroids are believed to control the inflammation and fibrosis, preventing cardiac dysfunction. we closely follow his progress to assess long term effect of steroid treatment especially on his arrhythmia. idiopathic pulmonary fibrosis (ipf) is a progressive disease frequently associated with terminal respiratory failure. the insight patients with ipf have into their disease process and prognosis is unknown. this may lead to delayed communication regarding end-of-life issues. we studied insight into disease and attitudes towards invasive ventilation (iv) amongst a group of ipf patients and compared these outcomes with copd patients with a comparable severity of disease. ten patients with ipf and eight patients with copd were studied. spirometry, the minute walk test and arterial blood gas were recorded as markers of severity. patient insight into disease and attitudes towards iv were surveyed using a newly developed questionnaire. % of patients with ipf felt they had sufficient information about their illness compared to % of copd patients. % of ipf patients had discussed prognosis with their doctor, versus % with copd. if the need arose, % of ipf patients wanted iv compared with % of copd patients. only one patient felt that the issue of iv should not be discussed. there is a deficiency in knowledge regarding disease process and prognosis among ipf patients. end of life issues and iv should be carefully addressed with these patients. coeliac disease is a gluten sensitive enteropathy that results in a chronic malabsorptive disorder. the disorder is rarely associated with pulmonary conditions though the link between the gut and lung remains obscure. conditions include the rare association with pulmonary haemosiderosis (hamilton lane syndrome) and for the first time to the best of our knowledge, pulmonary alveolar microlithiasis(pam). we also describe a literature review of the pulmonary associations of coeliac disease. pulmonary haemosiderosis is a form of pulmonary haemorrhage syndrome progressing to pulmonary fibrosis. we describe pulmonary haemosiderosis and capillaritis in a year old female coeliac, an association known as hamilton lane syndrome. treatment involved corticosteroids and a gluten free diet with resolution of her symptoms. pam is a rare disease where minute calculi are found in alveoli resulting in progressive pulmonary fibrosis. no association with coeliac disease has previously been published. we describe a case of a year old female coeliac who over years developed pulmonary fibrosis secondary to pam. there is no effective treatment and individuals may progress to transplantation. coeliac disease should be considered in patients presenting with pulmonary haemorrhage/ haemoptysis and in patients with interstitial lung disease where histological findings are consistent with pam. the vdi at and months with pulmonary involvement were significantly higher (p = . , p \ . respectively), but not at month. a vdi [ has been associated with a fold increase in mortality. ( %) with pulmonary involvement had a vdi [ at initial presentation compared to ( %) (p \ . ) without pulmonary involvement. the percentage with vdi [ in the pulmonary patients reduced at and months ( % and % respectively, p = . ). ( %) of patients with pulmonary involvement at presentation had eu-vas criteria for generalised and severe subgroups compared to ( %) without pulmonary involvement (p \ . ). of patients died had pulmonary involvement; one was attributed to vasculitis. we conclude that pulmonary involvement at presentation is highly predictive of severe organ damage (vdi [ ) at initial presentation and year and more extensive disease activity (bvas (all stages) and euvas) in anca-associated vasculitis. background: levels of adenosine can be rapidly increased during settings of inflammation and acute lung injury. these increases in adenosine have been implicated in pathological progression of lung diseases, as well as, protection against acute lung injury. we tested the effects of elevated adenosine levels on endothelial barrier function in vivo and in vitro. methods: intracellular levels of adenosine were elevated in rodents through inhibition of adenosine deaminase with pentostatin, and lung edema was measured in models of acute lung injury caused by a-naphthylthiourea (antu). the degree of antu-induced lung edema, as measured by lung wet to dry weight ratios and filtration coefficients (k f ), was significantly diminished in the rodents given pentostatin either before or after the antu injury. in vitro analyses using pulmonary artery endothelial cells plated on a monolayer demonstrated that adenosine receptor a a and a b agonist, n-ethylcarboxamidoadenosine (neca), improved permeability. while no significant effects were noted with adenosine receptor a , a a , a b , or a inhibitors or adenosine transporter inhibitors alone, we noted a significant attenuation of the adenosineinduced barrier function enhancement in the presence of adenosine receptor a a and a b antagonists, and adenosine transporter inhibitor, nitrobenzylthioinosine (nbti). adenosine enhances the pulmonary endothelial barrier function in acute lung injury through interaction with a a and a b receptors. childhood interstitial lung disease (child) comprises a spectrum of heterogenous disorders characterised by tachypnoea, radiological diffuse pulmonary infiltrates and abnormal histology. pathologies largely unique to children presenting in the first two years of life are now appreciated and up to % of cases may be inborn errors of surfactant metabolism leading to surfactant dysfunction . we report our experience of child in infants presenting to the royal belfast hospital for sick children over years and describe the presentation, investigations and clinical outcome in this group. all children presented with chronic tachypnoea, hypoxaemia, crackles, indrawing and failure to thrive. differential diagnoses of cystic fibrosis, aspiration, immunodeficiency and cardiac disease were considered and excluded in all cases. nine children had a hrct scan, the commonest finding being a 'mosaic' or diffuse interstitial pattern and four underwent lung biopsy, none of which resulted in a definitive diagnosis. eleven children were treated with inhaled corticosteroids and three with additional systemic steroids and all have experienced clinical resolution over time. the recent identification of the genes responsible for surfactant dysfunction disorders may, in future, obviate the need for a lung biopsy and be diagnostic in approximately % of children. screening of patients with contrast echocardiography, thoracic computerised tomography (ct) and cerebral magnetic resonance imaging (mri) has identified patients with definite hht, ( %) of whom had epistaxis, ( %) had telangiectasia and ( %) had a first-degree relative with hht. contrast echocardiography and/or ct were performed in patients, identifying patients ( %) with pulmonary arteriovenous malformations (pavms). nineteen patients with single or multiple pavms had embolization procedures performed, with - pavms embolized per procedure. mri was performed in ( %) patients but no cerebral arteriovenous malformations (cavms) were diagnosed. hht incidence in ireland is thought to be in - , , suggesting that there are many more undiagnosed cases nationally. internationally published data suggest a prevalence of - % for pavms and - % for cavms in patients with hht. while the prevalence of pavms in our group is consistent with these data, the prevalence of cavms is not, suggesting that irish patients with hht may differ genotypically and phenotypically from those in other countries. atrial and c-type natriuretic peptides (anp, cnp) are known vasodilators in many vascular beds. the role of npr-c in natriuretic peptide (np) mediated pulmonary vasodilation remains unknown. furthermore the role of endothelium in mediating vasoactive effects of np is controversial. using isolated ventilated-perfused lungs to monitor pulmonary artery (pa) pressures upon exposure to increasing doses of angiotensin ii in the presence of vehicle or anp, cnp, or the selective npr-c ligand, canf. additionally, using isolated pa rings constricted with phenylephrine, concentration dependent relaxations were measured in response to nps in endothelium intact/denuded vessels. results: anp and cnp but not canf significantly attenuated the vasoconstrictive properties of angiotensin ii in isolated perfused lung. similarly, canf had no vasodilatory effect in constricted pa rings. anp and cnp both vasodilated the pa rings. however, only cnp had endothelium dependent vasodilation at doses higher than - m. the endothelium dependent vasodilation was completely abolished by pretreatment with l-name, no synthase inhibitor, and iberiotoxin, a k + channel blocker. pretreatment with a-glycyrrhetinic acid ( a-ga), a myoendothelial gap junction inhibitor, and indomethacin, a cyclo-oxygenase inhibitor, had no effect on endothelium dependent vasodilation. conclusion: npr-c plays limited role in np mediated pulmonary vasodilation. the endothelium dependent effect of cnp is mediated by no and bkca channels. pulmonary embolism with potential fatal consequences is common amongst acutely ill medical patients. it is recognised that venous thromboembolism (vte) prophylaxis is underutilised in this population. we postulated that an education campaign could increase its usage. we prospectively studied consecutive medical admissions for one month before and after an educational intervention to see if the rate of thromboprophylaxis prescribed increased in patients who warranted such prophylaxis as recommended by the american college of chest physicians (accp) guidelines. prior to an educational intervention, we studied ninety-nine consecutive medical admissions. thirty-six patients in this group met criteria for prophylaxis and had no contraindication to prophylaxis. nineteen ( . %) of these patients received prophylaxis and seventeen ( . %) did not receive prophylaxis. the data were presented to the medical staff and the guidelines were discussed (educational intervention). subsequently, of consecutive medical admissions, met the criteria for prophylaxis. thirty-six ( . %) of these patients received thromboprophylaxis. compliance with the accp guidelines therefore rose from . % to . % (p = . ; fisher's exact test) of those eligible following the educational intervention. our data supports the use of education to increase adherence to guidelines which may improve outcome in acutely ill medical patients. venous thromboembolic disease is a major cause of morbidity and mortality amongst medical inpatients. prophylactic therapy with low molecular weight heparin, unfractionated heparin, and graded compression stockings has been shown to significantly reduce risk of pe & dvt. we sought to assess compliance with current international guidelines in a medical inpatient population, as well as the impact of a simple, prominent educational poster at ward and emergency department level. data was collected using a proforma derived from current accp guidelines. collection was performed on dates, before and one month after intervention. patients were assessed on each date. at baseline, prophylaxis was indicated in % (n = ) of patients. of these % (n = ) received appropriate therapy. compliance was best among specialist disciplines such as stroke medicine. acute s medical teams fared less well. among the post-survey cohort, prophylaxis was indicated in % (n = ), and prescribed in (n = %) (p = . for comparison) of these. vted prophylaxis is under prescribed. our results suggest that simple educational measures can improve compliance with established international guidelines. however, more interactive methods may yield greater benefits. pulmonary arteriorovenous malformation (pavm), a rare cause of hypoxia, are familial in % manifesting as osler weber rendu (owr) syndrome and are associated with embolic stokes in % ( , ) . this report highlights a case of hypoxia secondary to pavm with a family history of embolic strokes not associated with owr. a year old man was admitted with dyspepsia. admission chest x-ray identified a left mid zone mass. he had no medical history. his mother died from a stroke aged and had an ''abnormality in her lung''. his brother died at age from an embolic stroke. shortly after admission, he complained of dyspnoea. physical exam revealed oxygen saturations of %, tachypnea and tachycardia. oxygen saturations improved to % on % oxygen. he had no telangectasia or other signs of owr syndrome. ct pulmonary angiogram displayed a large pavm in left lower lobe. a contrast echo identified a large extracardiac right to left shunt. endoscopy confirmed oesophagitis secondary to excessive alcohol intake. he was discharged on aspirin pending review by cardiothoracic surgery for definitive treatment of his pavm to prevent worsening of his symptomatic hypoxia and reduce his risk of an embolic stroke. it has been hypothesised that fatigue of the genioglossus muscle contributes to collapse of the upper airway in osas. in this study, fatigability of the genioglossus was compared in healthy control subjects (aged - ) and osas patients (aged - ; apnoea/ hypopnoea frequency - /hr) using surface electromyographic (emg) signals recorded with a novel intra-oral electrode. emg signals were recorded during sustained isometric tongue protrusion at % of maximum voluntary contraction. endurance time was the point at which force fell % below the target level. muscle fibre conduction velocity (cv), an index of fatigue, was estimated from two adjacent emg signals and the rate of muscle fibre cv decrease during each contraction was calculated. the mean endurance time was lower in patients ( . ± . secs) than controls ( . ± . secs). in addition, the mean rate of decrease of muscle fibre cv, when normalised to an initial value of one, was greater in patients ( . ± . percent per second) than in controls ( . ± . percent per second). together, these results suggest increased susceptibility to genioglossus fatigue in untreated osas patients. furthermore, this approach provides a means of examining the role of fatigue in the pathophysiology of osas. obstructive sleep aponea (osa) is characterised by recurrent collapse of the airway during sleep leading to reduced or complete cessation of airflow despite respiratory effort, causing fragmented sleep and excessive daytime sleepiness. sleep deprivation is thought to be responsible for up to % of road traffic accidents. we examined data on the occupation of consecutive patients treated for osa with continous positive airway pressure (cpap) in our institution. specifically the numbers driving vehicles as the primary part of their occupation. drivers made up % ( ) of the total. this included taxi drivers ( ), bus drivers ( ) and train drivers, delivery persons ( ) and drivers of other vehicles ( ) . this data is of major importance given that the rate of osa in the populaiton is estimated at - %, whilst drivers making up % of our diagnosed osa polulation. it has major implications for service provision, the safety of all road users and those legislating in this regard. to avoid expensive sleep laboratory diagnosis of osa, we provide a home-based monitoring service with overnight oximetry and/or five channel limited sleep study. we wished to review these studies to assess their usefulness in reaching a diagnosis and to look at practical difficulties that may arise. we reviewed all oximetries and sleep studies, in patients with suspected osa, requested by one consultant in months period in st. john's hospital limerick and the mid-west regional hospital. of the oximetries, . % were suggestive of mild, . % moderate and . % severe osa. % were normal, . % borderline and . % malfunctioned. patients did not attend their first appointment. % of patients were waiting less than weeks. of the limited sleep studies, . % were suggestive of mild, . % moderate and . % severe osa. . % were normal. patients ( . %) had prior non-diagnostic oximetries. there were a large number ( ) of non-attendants for sleep study; we attribute this to longer waiting time and lack of secretarial back-up. there was minimal damage to the equipment. patients were started on long term cpap, to date nearly % are persisting with this. this confirms that home diagnosis of osa is both feasible and practical. both conditions represent a significant burden to the health service in terms of diagnosis, treatment and management. volunteers agreed to undergo a home limited cardiopulmonary sleep study and to interview with questionnaires including the epworth score. studies were manually scored to determine the apnoea hypopnoea index. results: volunteers were recruited, were excluded due to incomplete studies and withdrew consent. subjects ( female) were analysed, mean age: yrs (range - ), mean bmi: (± . ). significant osahs (ahi [ ) was found in females ( %) and male ( %) subjects, % overall. conclusion: osahs is very common in this group of patients without being the primary reason for attendance. this would suggest that osahs remains under-diagnosed particularly in the context of cardiovascular disease. we suggest that there should be routine screening for osahs in this patient group. to assess newly diagnosed patients with osahs for cardiac dysfunction by echocardiography. background: osahs is a common condition occurring in approximately % of men and % of women. it is associated with an increased morbidity and mortality from cardiovascular disease. newly diagnosed patients (ahi [ ) attending the sleep clinic in st. james's hospital were sent for echo to determine evidence of early heart changes in this group. results: patients were scanned ( female). average age: years, (range - yrs) with average weight of kg (+/- ) evidence of diastolic dysfunction was found in female ( %) and of the male subjects ( % the nf-jb inflammatory pathway is selectively activated by intermittent hypoxia in vitro and in patients with obstructive sleep apnoea syndrome (osas). nitric oxide (no) acts as an important signalling molecule in several biological processes including inflammation. we hypothesise that no plays a critical role in regulating the microenvironment of intermittent hypoxia and in modulation of the associated nf-jb response. serum nitrite and nitrate levels were measured in osas patients with no other medical disorder and healthy controls (body mass index-and age-matched). levels in osas patients were remeasured following continuous positive airway pressure (cpap) therapy. we also investigated the effect of no on transcriptional events initiated by intermittent hypoxia in an in vitro model. serum nitrite and nitrate levels did not differ between osas patients and controls (p = . and p = . respectively). nitrite levels in osas patients increased significantly following cpap therapy ( . lm compared to . lm (p = . )), indicating increased endothelial nitric oxide synthase (nos) activity. in our translational in vitro model no increases oxygen bioavailability in hypoxia through mitochondrial inhibition and decreases intermittent hypoxia-induced nf-jb activity. conversely, nos inhibition increases nf-jb activity. the findings support a role for no in regulating the inflammatory response to intermittent hypoxia. continous positive pressure (cpap) is an effective therapy for obstructive sleep aponea (osa). it's efficacy is limited by several factors including variable compliance with therapy. compliance is defined as cpap usage of greater than hours more than % of nights. studies of predictors of compliance have shown conflicting results. data on patients with osa treated with cpap in our institution was examined including aponea-hyponea index (ahi), oxygen desaturation index (odi), minimum oxygen saturation, epworth sleepiness score (ess) and compliance rates. the overall compliance rate was found to be %, with overall compliance at weeks following initiation of therapy . %. this rate compared with compliance of . % at months. this suggests that week compliance rates are a good marker of longer-term compliance. obstructive sleep aponea (osa) is characterised by recurrent collapse of the airway during sleep leading to reduced or complete cessation of airflow despite respiratory effort. this leads to sleep fragmentation through multiple arousals with poor sleep and has been associated with major co-morbidities including impaired cognition, poor quality of life, and increased risk of accidents. evidence is emerging that osa is an independent risk factor for adverse cardiovascular outcomes. we treat over one hunderd patients for osa with continous positive airway pressure (cpap) in our institution. patient data including body mass index, neck circumference, aponea-hyponea index (ahi), oxygen desaturation index (odi), lowest oxygen (o ) saturation, epworth sleepiness score (ess) was collected. neck circumference was correlated with markers of disease severity and found to be a predictor of more severe disease as defined by higher ahi and lowest o saturation. no correlation was found with odi or ess. we propose that this is a useful and easily obtained marker of severity of osa. prompt management of exacerbations is a cornerstone of effective treatment of cystic fibrosis. it is therefore imperative that patients promptly report changes in symptoms to their physician/cf team. we sought to clarify which symptoms patients would report to the clinical team and when they would report them. an anonymous questionnaire was sent to a random sample of stable adult patients with cystic fibrosis ( male and female). predictors of early response were evaluated using logistic regression. % returned questionnaires that were suitable for analysis. for all symptoms a significant number of patients would not contact the medical team before their next routine out-patient appointment or even report it at all (table ). there was a wide variation in the time to alerting the clinical team between and within symptoms. females were significantly more likely to report a change in small volume haemoptysis or cough before their next out-patient appointment. younger patients were more likely to report small and large volume haemoptysis. delayed patient responsiveness to changes in respiratory symptoms is a barrier to prompt management of exacerbations in cf. supported by: health research board, cf association of ireland. bronchoalveolar lavage can be used to investigate pulmonary aspiration in children by measuring pepsin concentration, however, it is invasive. sputum induction is a potential non-invasive way of obtaining samples. we aimed to: ( ) assess safety and feasible of measuring pepsin in inducted sputum in children and ( ) determine whether the sputum induction procedure caused gastro-oesophageal reflux with refluxed pepsin contaminating samples. children with no respiratory or gastroesophageal symptoms were recruited (n = , range - years). following spirometry, sputum induction was carried out and pepsin concentration measured by an 'in house' elisa. spirometry was repeated and any complications noted. only one child (aged ) produced no sample, however two other year olds did complete sputum induction. no adverse effects were reported. one child required a sabutamol nebuliser following a % decrease in fev . of the sputum samples ( %) were positive for pepsin. sputum induction appeared a safe procedure in children. it is well tolerated by children and can be successfully carried out in children as young years of age. however, the analysis of pepsin in sputum obtained by induction is not useful in the investigation of respiratory associated gastroesophageal reflux disease as % asymptomatic children have positive samples. immunodeficiency may result in recurrent pulmonary infection leading to chronic lung damage and bronchiectasis. the aim of our study was to identify immunodeficiency in idiopathic bronchiectasis and the parameters that correlate with disease severity. patients with idiopathic bronchiectasis were assessed. patients were recruited over a one year period from the respiratory out-patient and in-patient service. serum immunoglobulins, igg subclasses and pneumococcal antibody levels were measured. clinical, physiological, microbiologic and radiologic data was obtained. patients with igg subclass deficiency (iggsd) were significantly more likely to be hospitalised with a respiratory exacerbation as compared with the immunocompetent group (p \ . ). iggsd was associated with lower fev (p \ . ), more severe obstructive airways disease (p \ . ) and persistent sputum purulence (p \ . ). iggsd correlated with low pneumococcal antibody levels. there was no significant difference in bronchiectasis severity or lobar involvement on high resolution ct. our findings support the hypothesis that patients with iggsd and documented bronchiectasis suffer more severe exacerbations even in the contaxt of radiologically mild disease. patients with evidence of bronchiectasis radiologically should have serum immunoglobulins and igg subclasses performed. this cohort is high risk for progressive lung damage in the setting of recurrent severe exacerbations and should be identified early to ensure optimal management. macrophages undergo apoptosis after infection with m. tuberculosis (mtb). this macrophage response deprives the bacillus of its niche cell, and supports the host response through better antigen presentation.virulent strains of mtb do not cause apoptosis at low multiplicities of infection (moi) which may contribute to this pathogen's ability to survive long-term in macrophages. we investigated the ability of mtb to cause macrophage cell death at a high moi ( - ) which is likely to represent the high bacillary burden seen in the later stages of infection. the mechanism of cell death was analysed by fluorescent microscopy and nucleosome elisa. macrophages infected with virulent (h rv) mtb displayed several features typical of apoptosis including dna fragmentation and exposure of phosphatidylserine. however, mitochondrial cytochrome c release and nuclear fragmentation were not observed, suggesting that mtb-induced cell death differs from classical apoptosis. cell death was significantly reduced (p \ . ) following treatment with serine protease inhibitors (aebsf and tpck) but was not effected by the caspase inhibitor zvad-fmk. in summary, mtb triggers a novel serine protease-dependent macrophage cell death pathway which may facilitate dissemination in the later stages of infection. a better understanding of this macrophage response may direct new vaccine and treatment options. patients had microbiological confirmation of tb, were culture positive and patients were pcr positive. the mean (sd) age was ( ). male:female ratio : . of the pulmonary cases, bronchoscopy (bronchial washings/ biopsy) was required to make a microbiological diagnosis in cases ( %). of the cases where sensitivity data was available, there were cases ( . %) of drug resistant tb. of these, ( %) were mono-resistant, ( %) were poly-resistant and ( %) were multidrug-resistant tb. various factors including the presence of a resistant organism determined duration of treatment and site of care. six patients had disease in multiple sites the high rate of drug resistant tb in this population has implications not only for the management of index cases but also for the management of contacts and the management of latent tb in the population as a whole. resource limitations have raised interest in portable monitoring systems that can be used to improve access to the diagnosis of obstructive sleep apnoea syndrome (osas). this prospective study compares a combined electrocardiogram and oximetry recorder (holter-oximeter) in an unattended home setting against attended polysomnography for detection of osas. subjects ( male: female) with suspected osas underwent attended polysomnography (psg) in hospital and subsequent unattended evaluation at home with a holter-oximeter (nemon dr +). an algorithm for estimation of the (apnoea-hypopnoea index) ahi using only a single lead of electrocardiograph (ecg) and the oximetry trace had previously been developed using a database of psg recordings. osas was defined as ahi c , non-osas as ahi \ , and b ahi \ as indeterminate. evaluation methodology was in accordance with the recommended american academy of sleep medicine guidelines (flemons et al. ) . sensitivity and specificity were %. positive and negative likelihood ratios were [ and respectively. estimated ahi agreed closely with psg ahi (r = . ; p \ . ). combined holter-oximeter monitoring compares well against polysomnography for identifying osas and is potentially a suitable device for home screening of sleep apnea in a population suspected of having osas. current international guidelines suggest the combined use of pre test probability scores, d dimers and ct pulmonary angiography (ctpa) for the management of suspected pulmonary embolism (pe). our aim was to audit adherance to international guidelines for the management of suspected pe in a teaching hospital. a retrospective audit was performed on all ctpa's carried out in merlin park hospital from the / / to the / / . patients had clinical notes available for analysis. only % of patients had a documented well's or geneva score. ctpa confirmed pe in % of cases overall. when a well's score was calculated in these patients, % were high risk, % were intermediate risk and % were low risk. a third of patients in the low and intermediate risk groups had no d dimers measured. half of patients in the high risk group had an inappropiate d dimers. no patient with a confirmed pulmonary embolism had a normal d-dimers. % of patients had d dimers measured and . % of these of were normal. all ctpa's in this subgroup were negative for pulmonary embolism. when a well's score was calculated, retrospectively, from clinical notes patients were in an low or intermediate risk group where ctpa was not indicated. we conclude that pre test probability scores are underutilised. it confirms that patients with low or intermediate risk of pe and negative ddimers should not require ctpa. it also highllights that d-dimers should be used appropriately in low or intermediate risk groups. common variable immunodeficiency (cvid) is a primary immunodeficiency syndrome characterised by diminished serum immunoglobulin levels and impaired antibody responses. cvid may present as granulomatous disease and masquerade as ''sarcoidosis''. we have previously described four patients referred to us with a diagnosis of sarcoidosis with granulomatous disease on biopsy who in fact had cvid. sarcoidosis is more typically associated with hypergammaglobulinaemia. we have evaluated serum immunoglobulin levels in patients with granulomatous disease on biopsy with clinical and radiological pattern compatible with sarcoidosis. thirty seven ( %) were male. mean age of presentation was . (sd +/- , range - ). % of patients demonstrated hypergammaglobulinaemia (n = ). % (n = ) demonstrated low igm levels. % (n = ) demonstrated an isolated low igg level. a single patient had low igg and igm levels and has been further evaluated for cvid. % of patients had low/ normal iga levels (n = ). in conclusion, hypergammaglobulinaemia was less common than anticipated in this cohort of patients with granulomatous disease believed to be secondary to sarcoidosis. patients with a putative diagnosis of sarcoidosis should have immunoglobulin levels determined to exclude cvid. in chronic asthma, goblet cell hyperplasia and decreased ciliogenesis are characteristic features which may be influenced by th cytokines (eg il- and il- ). in vitro basal mucociliary differentiation and differences in paediatric epithelial cells (normal & asthmatics) exposed to il- were studied. blind non-bronchoscopic bronchial brushings obtained from children were differentiated at air liquid interface for days. cells s were treated with ng/ml il- and ng/ml il- . transepithelial resistance (ter), number of ciliated (anti a -acetylated tubulin antibody) and goblet cells (muc ac + ) were assessed as a measure of tissue differentiation. both asthmatics and normal cell cultures formed well differentiated pseudostratified epithelium (ter [ x/cm ). asthmatic cultures expressed significantly more goblet cells ( . %, sd = . ) when compared with non-asthmatic cultures ( . %, sd = . ) under basal culture conditions (p = . ). significant more goblet cells are seen in asthmatic cultures when chronically exposed to il ( ng/ml and ng/ml) when compared with identically treated nonasthmatic cultures (p \ . ). asthmatic cultures expressed significantly less ciliated cells ( . %, sd = . ) when compared with nonasthmatic cultures ( . %, sd = . ) under basal culture conditions (p = . ). asthmatic cells differentiate at basal conditions with higher proportion of goblet cells and decreased number of ciliated cells when chronically exposed to il- . combining inhaled corticosteroids with long-acting b -agonists results in improved asthma symptom control and fewer asthma exacerbations compared to inhaled corticosteroids alone. however, there are limited data as to whether these beneficial effects are due to enhanced antiinflammatory actions, or whether such combination therapies impact on airway remodeling in asthma. we sought to determine the effects of inhaled budesonide/formoterol combination therapy, versus inhaled budesonide alone or inhaled placebo, on allergen-induced airway responses, airway inflammation and airway remodeling. fourteen asthmatic subjects with dual responses after allergen inhalation were included in this prospective randomized, double-blind, -period cross-over study. outcomes included asthmatic responses, changes in airway responsiveness and sputum eosinophilia, measured before and after allergen challenge, and numbers of airway submucosal myofibroblasts measured before and after study treatment. combination treatment attenuated the maximal early asthmatic responses and also resulted in a -doubling dose attenuation of allergen-induced airway hyperresponsiveness compared to budesonide or placebo treatment (p \ . ). allergen-induced increases in sputum eosinophils and in submucosal tissue myofibroblasts were significantly reduced by combination treatment (p \ . ), but not by budesonide alone when compared to placebo. inhaled budesonide/formoterol combination therapy attenuated allergen-induced airway responses and provided greater anti-inflammatory effects than either budesonide alone or placebo. combination therapy also attenuated the allergen-induced increases in submucosal myofibroblast numbers, suggesting that clinical benefits associated with such combination treatment may relate to effects on airway remodeling. the purpose of this study was to determine the effects of sub-cytotoxic concentrations of eosinophil peroxidase (epo) on expression and sub-cellular localisation of the linked cell growth and cell cycle mediators, focal adhesion kinase (fak), cyclin-dependent kinase inhibitor p kip and the epidermal growth factor receptors egfr and erbb . eosinophils exert many of their inflammatory effects in allergic disorders by degranulation and release of cationic granule proteins including epo. in sub-cytotoxic concentrations, eosinophil granule proteins increase transcriptional expression of various growth factors in airway cells. the methods used were real-time pcr, western blotting of membrane, nuclear and cytoplasmic cell fractions and immunoprecipitation. epo induced a concomitant time-dependent egress of fak and p kip from the cell nucleus to the cytoplasm. immunoprecipitation indicated a physical association between fak and p kip , implying that fak acts as a nuclear-cytoplasmic shuttle for p kip . epo also induced up-regulation of expression of egfr and erbb . our results imply that epo potentially induces cell proliferation, by up-regulating egfr and erbb and cell cycle, by driving p kip from the nucleus. this has implication for the role of eosinophils in tissue remodelling and turnover in conditions from asthma to cancer. alpha- antitrypsin (a at) deficiency predisposes individuals to early onset emphysema and is a debilitating disease in which neutrophils play a central role. it is becoming more evident that a at possess key anti inflammatory properties and the aim of this project was to examine the possible role of a at in modulating neutrophil chemotaxis. western blot and facs analysis was utilised to examine the effect of a at on release of cd b, a key molecule in chemotaxis and adhesion, from the neutrophil membrane. the effect of a at on neutrophil migration using il- ( - ng/ . cells) was quantified employing a multiwall chemotaxis chamber. our experimental results revealed that a at ( . lm) prevented the release of cd b from the neutrophil membrane. inhibition of il- chemotaxis was dose dependent and almost completely inhibited by . lm aat. in addition, neutrophils of a at deficient (zz) individuals displayed decreased levels of cd b. this study highlights the importance of serum levels of a at for modulating neutrophil activity and aims to evaluate whether infused a at possess the ability to bind and govern the activity of circulating neutrophils in vivo. aat deficiency (aatd) is a hereditary disorder, resulting from mutations in the serpina gene, and classically presents with earlyonset emphysema and liver disease. the most common mutation causing aatd is the z mutation, with the s mutation also associated with lung disease. aat deficiency is under-diagnosed and prolonged delays in diagnosis are common. the world health organisation advocates screening copd, poorly-controlled asthma, cryptogenic liver disease patients and first degree relatives of known aatd patients. , individuals with copd, asthma, or cryptogenic liver disease were screened in the national targeted detection programme. , healthy individuals from the tcd biobank were genotyped for s and z alleles. targeted screening identified zz, sz, ss, mz, ms, and mi individuals, yielding gene frequencies of . and . for s and z respectively. biobank screening of , healthy individuals identified ms, mz, sz and a single ss case, yielding gene frequencies of . and . for s and z. the allele frequencies for s and z in ireland were previously estimated at between . - . and . - . ( ). our pilot study shows s and z alleles occur at higher frequencies, suggesting , zz individuals and over , carriers on the island of ireland. the z mutation is more clinically significant with a higher penetrance than s in the groups we have evaluated. inspiratory-to-total lung capacity ratio predicts mortality in patients with chronic obstructive pulmonary disease joint accp/aacvpr evidence-based clinical practice guidelines sarcoidosis: clinical update hereditary hemorrhagic telangiectasia pulmonary arteriovenous malformations: techniques and long-term outcome of embolotherapy obstructive sleep apnoea in a patient with bilateral carotid body tumours, a unique case references obstructive sleep apnea caused by carotid body tumor: case report obstructive sleep apnea due to a carotid body paraganglioma continuous positive airway pressure therapy compliance rates across three sleep centres we present a unique case of a patient who presented with obstructed sleep apnoea (osa) caused by bilateral carotid body tumours.the patient was treated during a year period, during which time he required non-invasive ventilation at night via continuous positive airways pressure (cpap). he also underwent surgical resection of his right and left carotid body tumours sequentially while on cpap. during family screening his daughter was discovered to have a carotid body tumour but without osa.at presentation, his apnoea-hypopnoea index (ahi) was . , and his epworth score was out of possible . after resection of both carotid body tumours, his ahi score fell to . , and he was symptomatically improved and he was able to return to work.this unusual case highlights the need to investigate patients with osa for an underlying treatable cause. to our knowledge, this is the first report of bilateral familial carotid body tumours causing osa; to date osa has only been reported with unilateral carotid body tumours. , to determine long-term compliance rates for patients prescribed continuous positive airway pressure (cpap) therapy from three sleep centres. having 'limited study' devices now in use for - years now, we have the opportunity to compare cpap compliance rates between sleep centres using these, and established centres using full polysomnography (psg).a population-based analysis of patients prescribed cpap therapy over a -year period - , with compliant vs. non-compliant status determined in june .it is possible to achieve long-term compliance rates of [ % with limited study systems and correct follow-up care. however, factors such as patient education and follow up, along with a high level of home service, are vital. . is brain natriuretic peptide a good marker for obstructive sleep apnea and the efficacy of continuous positive airway pressure therapy in obstructive sleep apnea patients? the aim to assess natriuretic peptide levels n-terminal pro b-type and b natriuretic peptide in congestive heart failure (chf) patients with/without obstructive sleep apnea (sa). the effect of cpap on natriuretic peptides in a sleep apnea population was assessed.a blind study in a known population, sleep apnea status was unknown. biosyn triage measured bnp, roche measured nt bnp. both were measured in congestive heart failure patients ( female, male) with obstructive sleep apnea ( female, male) and normal patients ( female, male).bnp is a good marker for chf. nt probnp is a good marker for chf, it could distinguish chf patients from chf osa patients. cpap didn't reduce natriuretic peptide concentrations. aim: to show that osahs is a common feature in patients with metabolic syndrome. background: metabolic syndrome has been described as a constellation of risk factors for cardiovascular disease. the who places the incidence at % of the population. osahs occurs in - % of males and females. key: cord- -jne jqf authors: macparland, sonya a.; ma, xue-zhong; chen, limin; khattar, ramzi; cherepanov, vera; selzner, markus; feld, jordan j.; selzner, nazia; mcgilvray, ian d. title: lipopolysaccharide and tumor necrosis factor alpha inhibit interferon signaling in hepatocytes by increasing ubiquitin-like protease (usp ) expression date: - - journal: j virol doi: . /jvi. - sha: doc_id: cord_uid: jne jqf inflammation may be maladaptive to the control of viral infection when it impairs interferon (ifn) responses, enhancing viral replication and spread. dysregulated immunity as a result of inappropriate innate inflammatory responses is a hallmark of chronic viral infections such as, hepatitis b virus and hepatitis c virus (hcv). previous studies from our laboratory have shown that expression of an ifn-stimulated gene (isg), ubiquitin-like protease (usp) is upregulated in chronic hcv infection, leading to impaired hepatocyte responses to ifn-α. we examined the ability of inflammatory stimuli, including tumor necrosis factor alpha (tnf-α), lipopolysaccharide (lps), interleukin- (il- ) and il- to upregulate hepatocyte usp expression and blunt the ifn-α response. human hepatoma cells and primary murine hepatocytes were treated with tnf-α/lps/il- /il- and usp , phosphorylated (p)-stat and myxovirus (influenza virus) resistance (mx ) expression was determined. treatment of huh . cells and primary murine hepatocytes with lps and tnf-α, but not il- or il- , led to upregulated usp expression and induced an ifn-α refractory state, which was reversed by usp knockdown. liver inflammation was induced in vivo using a murine model of hepatic ischemia/reperfusion injury. hepatic ischemia/reperfusion injury led to an induction of usp expression in liver tissue and promotion of lymphocytic choriomeningitis replication. these data demonstrate that certain inflammatory stimuli (tnf-α and lps) but not others (il- and il- ) target usp expression and thus inhibit ifn signaling. these findings represent a new paradigm for how inflammation alters hepatic innate immune responses, with usp representing a potential target for intervention in various inflammatory states. importance inflammation may prevent the control of viral infection when it impairs the innate immune response, enhancing viral replication and spread. blunted immunity as a result of inappropriate innate inflammatory responses is a common characteristic of chronic viral infections. previous studies have shown that expression of certain interferon-stimulated genes is upregulated in chronic hcv infection, leading to impaired hepatocyte responses. in this study, we show that multiple inflammatory stimuli can modulate interferon stimulated gene expression and thus inhibit hepatocyte interferon signaling via usp induction. these findings represent a new paradigm for how inflammation alters hepatic innate immune responses, with the induction of usp representing a potential target for intervention in various inflammatory states. i nterferon (ifn) is a key endogenous mediator of viral clearance by the innate immune response. one excellent example of this is hepatitis c virus (hcv) infection of the liver ( ) . interferon signaling drives the expression of multiple interferon-stimulated genes (isgs), which mediate viral clearance. isgs, however, can also be induced by other factors, including inflammatory stimuli such as tumor necrosis factor alpha (tnf-␣) ( ) . hcv infection, which induces chronic inflammation of the liver, is associated with high serum tnf-␣ ( , ) . interestingly, anti-tnf-␣ treatment has been shown to lead to an improved virologic response to ifn-␣/ribavirin antiviral therapy for hcv ( ) , while other data show safety of combined treatment but no effect on hcv viral loads of hcv treatment-naive individuals ( ) . the purpose of the present study is to define an important causal link between inflammation and the host hepatic innate immune response, with broad viral relevance. our recent work in the host innate immune response to chronic hcv infection suggested an association between hepatocytes and the liver resident immune cells that drive liver inflam-mation ( ) ( ) ( ) . there is a dichotomous response to chronic hcv infection in the liver, and this response predicts who will and who will not respond to exogenous ifn-␣ treatment. in patients that do not respond to therapy with ifn/ribavirin, hepatocytes have strong preactivation of the ifn-response, with high expression of a subset of isgs ( ) ( ) ( ) . in patients exhibiting a sustained virologic response on therapy with ifn/ribavirin, tissue macrophages show a high expression of isgs ( , ) . the expression of isgs in macrophages or hepatocytes is more predictive of treatment outcome than il b polymorphisms ( ) ; this finding suggests a link between hepatic inflammatory cell activation and viral clearance. liver kupffer cell inflammatory responses are induced by and play a role in the control and clearance of multiple viral infections of the liver ( , ) . although the mechanisms underlying these patterns are undoubtedly complex, the association between liver macrophages and hepatocytes raises the question of how the hepatocytes react to the cytokines produced by the macrophages (and other inflammatory cells). if isg expression in hepatocytes reflects a response to inflammatory stimuli, then how the liver responds to a viral infection will be modulated by the inflammatory milieu. in hcv infection, we have found that the isg /usp pathway is an important regulatory pathway. both isg and usp are isgs that are strongly upregulated in the livers of ifn treatment-resistant patients ( ) ( ) ( ) . isg is a ubiquitin-like protein that is strongly upregulated by ifn and conjugates to multiple cellular proteins ( , ) . usp is an isg -specific protease that is also upregulated by ifn-␣ ( , ) . both isg and usp have been suggested to blunt type ifn signaling ( , ( ) ( ) ( ) . in addition to its ability to remove isg from its conjugated proteins, usp has been shown to bind to the type ifn receptor and blunt ifn signaling ( ) . upregulation of usp may represent a negative-feedback loop counteracting the effects of type ifn. furthermore, knockdown of usp increases both isg induction and anti-hcv activity of ifn-␣ ( ) , and data have shown that ifn-␣ treatment given to mice in vivo increases hepatic usp and blunts the effect of a subsequent dose of ifn-␣ ( ) . the mechanisms controlling usp expression in the liver are poorly understood but will have relevance to understanding innate immune mechanisms in chronic viral infection. although the majority of work on usp has centered on its upregulation by type ifn, interferon stimulated genes have multiple upregulatory stimuli other than ifn-␣ ( , ) . for example, inflammatory stimuli, e.g., endotoxin and lipopolysaccharide (lps), have been shown to upregulate usp in peritoneal exudate macrophages ( ) . if similar stimuli lead to increased usp expression in hepatocytes, then this pathway could represent a novel link between inflammatory and innate immune responses in the liver. the present study is based on the hypothesis that liver inflammation will directly impact hepatocyte expression of usp and therefore will impact ifn signaling. this link will have relevance to multiple diseases, since inflammatory stimuli such as tnf-␣ and lps have been shown to play roles in many liver diseases, including viral hepatitis ( ) ( ) ( ) . the link may also help to explain our observations in chronic hcv infection, since chronic hcv infection is characterized by increased serum tnf-␣ ( , ), increased hepatocyte usp , and impaired ifn responsiveness ( ) . quite apart from hcv, the importance of the link between hepatic inflammation and innate immune response lies in the downstream effects of the impairment of innate immunity. we speculate that by blocking ifn-␣ signaling, usp expression may lead to an enhanced susceptibility to infection with interferon-sensitive viruses and enhanced viral proliferation. support for this notion is found in a mouse study in which expression of usp in macrophages led to lower ifn responsiveness, leading to locally restricted replication of vsv ( ) . in the present study, treatment of hepatic cells with lps and tnf-␣, but not il- or il- , led to upregulated usp expression in hepatocytes. the enhanced usp expression was associated with decreased ifn-␣-stimulated expression of p-stat and isgs, a phenomenon reversed by usp knockdown. as an in vivo correlate of our in vitro findings, experimentally induced hepatic ischemia/reperfusion injury induced usp mrna expression in liver and enhanced lymphocytic choriomeningitis (lcmv) replication, an effect not seen in usp Ϫ/Ϫ mice. these data demonstrate that certain inflammatory stimuli (tnf-␣ and lps), as well as ischemic injury, but not other cytokines (il- and il- ) can lead to enhanced hepatocyte usp expression and thereby inhibit ifn signaling. these findings lend new knowledge to our understanding of how inflammation can modulate hepatic innate immune responses, with usp representing a potential target for intervention to reverse any proviral effect of inflammation. phorylated-stat (p-stat- ; cell signaling technology, boston, ma), rabbit polyclonal anti-stat- (santa cruz biotechnology), goat polyclonal anti-ube antibody (santa cruz biotechnology), or mouse monoclonal anti-actin (sigma) antibodies, followed by anti-mouse or anti-rabbit igg conjugated to horseradish peroxidase (calbiochem, billerica, ma). an enhanced chemiluminescence detection kit (amersham pharmacia biotech, uppsala, sweden) was used to determine the levels of protein expression. flow cytometric quantification of usp expression in huh . cells. usp expression on huh . cells treated with lps or tnf was quantified by using flow cytometry as previously described ( ) . briefly, cells were fixed, permeabilized, and stained with mouse anti-human usp polyclonal antibody (abnova) or a relevant isotype-matched control antibody. staining was detected with a secondary goat anti-mouse igg labeled with fluorescein isothiocyanate (fitc; santa cruz biotechnology). the cells were acquired using a facscalibur cytometer (bd biosciences, san jose, ca). a minimum of , events were collected. the resulting data were analyzed using flowjo software (tree star, inc.). the experiments were repeated four times. sirna targeting murine usp and ube l. usp small interfering rna (sirna) is a pool of three target-specific -to -nucleotide (nt) sirnas designed to knock down usp gene expression that was obtained from santa cruz biotechnology. the ube l sirna used was a pool of three to five target-specific -to -nt sirnas designed to knockdown mouse ube l gene expression and was obtained from santa cruz. usp and ube l sirna was transfected into ϫ primary murine hepatocytes according to the manufacturer's instructions using the santa cruz sirna reagent system sc- (santa cruz) and as previously described ( ) . inhibition of inflammatory signaling in primary mouse hepatocytes. to inhibit lps-or tnf-␣-stimulated nf-b activation, primary mouse hepatocytes were incubated with the following inhibitors for min prior to h of stimulation with lps or tnf-␣: p , a protein kinase a inhibitor ( ); nsc , a jak inhibitor ( ); p , a protein kinase c inhibitor ( ); pd , a mitogen-activated protein kinase kinase inhibitor ( ); silibinin, an inhibitor of ikk␣ ( ) ; and wortmannin, a phosphatidylinositide -kinase inhibitor ( ) . the concentrations, sources, and main targets of these inhibitors are described in table . after lps or tnf-␣ stimulation, hepatocytes were harvested and usp and il- ␤ (a surrogate of nf-b activation) mrna expression was evaluated as described below. rna isolation and quantitative real-time pcr analysis. huh . cells and primary murine hepatocytes were treated with ifn-␣ ( u/ml), tnf-␣ ( ng/ml), lps ( ng/ml), il- ( ng/ml), or il- ( ng/ ml) over a -h time course, and usp expression was determined by quantitative pcr (qpcr) as previously described ( , ) . briefly, total rna was prepared from cells using the trizol reagent (invitrogen) according to the manufacturer's instructions. the reverse transcription reactions of the extracted rna were performed with a first-strand cdna synthesis kit (amersham pharmacia biotech) according to the manufacturer's directions. first, g of extracted rna was added in a total volume of l of combined cdna reaction reagents with random hexamer oligonucleotides as the first-strand primer in a . -ml reaction tube. samples were heated to °c for min, chilled on ice for min, and incubated at °c for h, followed by a -min incubation at °c. the specific primers for all of the detected genes for the pcrs were based on genbank-published sequences: mus musculus ubiquitin-specific peptidase (usp ; nm_ ) forward primer ( =-tacagcagagagcagcagga) and reverse primer ( =-cacatgtcggagcttgctaa); mus musculus myxovirus (influenza virus) resistance (mx ; nr_ ) forward primer ( =-tctgaggagagccagacaat- =) and reverse primer ( =-actctggtccccaatgacag); mouse hypoxanthine guanine phosphoribosyltransferase (hprt; nm_ ) forward primer ( =-tcagt caacgggggacataaa) and reverse primer ( =-ggggctgtac tgcttaaccag); mus musculus il- ␤ (nm_ ) forward primer ( =-gaaatgccaccttttgacagtg) and reverse primer ( =-tgg atgctctcatcaggacag); homo sapiens ifn-␣ b (ifna b; ay ) forward primer ( =-gcttgggatgagaccctccta) and reverse primer ( =-cccaccccctgtatcacac); homo sapiens ifn-␥ (ifng; nm_ ) forward primer ( =-tcggtaactgacttgaatg tcca) and reverse primer ( =-tcgcttccctgttttagctgc); homo sapiens actin, beta-like (actbl ; nm_ ) forward primer ( =-gtctgccttggtagtggataatg) and reverse primer ( =-tcgagg acgccctatcatgg); homo sapiens ubiquitin specific peptidase (usp ; nm_ ) forward primer ( =-aggagaagcgtccctt tcca) and reverse primer ( =-tggtccttaatcaggttccagag); and homo sapiens myxovirus (influenza virus) resistance- (mx ; nm_ ) forward primer ( =-ggtggtccccagtaatgtgg) and reverse primer ( =-cgtcaagattccgatggtcct). quantitative real-time pcr was performed on an abi prism ht machine (applied biosystems, foster city, ca) with sybr green realtime pcr master mix (applied biosystems) according to the directions provided by the manufacturer. all of the rna samples and controls were assayed in duplicate. real-time pcr conditions were as follows: min at °c, followed by cycles of s at °c and s at °c monitor fluorescence in sybr channel during a °c annealing/extension step. the results were analyzed by using applied biosystems sds . software (applied biosystems). experimental hepatic ischemia/reperfusion injury model. hepatic ischemia/reperfusion injury (hiri) was simulated as before ( ) . partial ( %) hepatic ischemia was induced for min in mice, after which the surgical clamps were removed. control (sham) animals underwent anesthesia and laparotomy alone. animals were euthanized or h after ischemia/reperfusion, the affected liver segments were removed, and target gene expression was determined by qpcr in whole liver tissue. lcmv strain we was propagated in l cells (atcc ccl- ) as previously described ( ) . in additional experiments, after min hiri or sham laparotomy, mice were infected with ϫ pfu of lcmv we by intravenous tail vein injection. the ischemic liver lobes were harvested at day postinfection. snap-frozen liver tissue samples were homogenized in ␣-mem (multicell, usa) supplemented with % fbs, l-glutamine, and u of penicillin/ml plus g of streptomycin/ml using the tissuelyser lt system (qiagen, netherlands). viral titers were examined on mc cells (atcc crl- ) using a focus-forming assay as previously described ( ) . statistical analysis. all data were analyzed using prism version . software (graphpad software, san diego, ca). statistically significant differences in fig. e were calculated by using a two-tailed student t test (prism software). the impact of hiri on lcmv replication (see fig. a and b) was evaluated by one-way analysis of variance with the tukey's post hoc test. usp is induced in hepatocytes by lps and tnf-␣ but not by il- and il- . we have previously shown that usp can modulate the type ifn response ( ) . we sought to determine whether inflammatory stimuli could increase usp expression in hepatocytes. liver tissue inflammation is mediated by both proand anti-inflammatory stimuli, acting through diverse pathways. we therefore compared the effects of three proinflammatory stimuli (tnf-␣, lps, and il- ) and one anti-inflammatory stimulus (il- ), all four signaling through independent receptors and signaling cascades ( , ) . in a -h time course experiment, usp mrna expression was measured after stimulation with lps ( ng/ml), tnf-␣ ( ng/ml), il- ( ng/ml), or il- ( ng/ml). usp mrna expression was induced by tnf-␣ and lps but not by il- or il- ( fig. a) . meanwhile, neither tnf-␣ nor lps treatments had a marked effect on mx mrna expression (fig. b) , indicating that the effects we observe are not due to a generalized upregulation of isgs or to type ifn signaling. tnf-␣ and lps treatment also led to augmented usp protein expression by western blotting (fig. c ) and by intracellular staining (fig. d to f). treatment of huh . cells with tnf-␣ or lps induced expression of usp in . % Ϯ . % and . % Ϯ . %, respectively, compared to untreated controls in which usp expression was . % Ϯ . % (fig. fi) . usp induction was also demonstrated with a significant shift in the mean fluorescence intensity of usp staining after tnf-␣ or lps simulations (fig. fii ). in these assays, the degree of protein expression did not correlate completely with mrna expression, a finding that is consistent with many genes in the liver ( ) in that the degree of mrna induction was higher than the observed usp protein expression. however, the functional effects we observed in terms of ifn-␣ responses were robust. tnf-␣ and lps block ifn-␣ signaling in huh . hepatoma cells. to determine whether inflammatory stimuli such as tnf-␣ and lps can interfere with type ifn signaling, we sought to determine whether pretreatment of hepatoma cells with tnf-␣ or lps blocked ifn-␣ signaling. huh . cells were treated with tnf-␣ ( ng/ml) or lps ( ng/ml) for h. the cells were subsequently treated with ifn-␣ for h. we chose h to measure mx expression based on previous descriptions of ifn-stimulated mx induction in the literature ( ) and based on our observations with primary mouse hepatocytes. control cells were left untreated, followed by a -h ifn-␣ treatment at h. after the -h ifn-␣ treatment, the cells were harvested, and mx expression was measured by qpcr as a measure of ifn-inducible gene expression. as expected, a -h exposure to ifn-␣ strongly induced mx expression, whereas a -h exposure to tnf-␣ and lps did not (fig. , columns , , and ). both tnf-␣ and lps pretreatment inhibited the effect of h of exposure to ifn-␣ (fig. , columns , , and ), indicating that ifn-␣ signaling was impaired in the presence of these inflammatory stimuli. having shown that certain inflammatory stimuli both increase hepatocyte usp and blunt ifn-␣ signaling, we next sought to determine whether the blunting of ifn-␣ signaling is mediated via increased usp . we addressed this question by selective knockdown of usp mrna. thus, huh . cells transfected with usp sirna or control sirna were treated with lps ( ng/ml), tnf-␣ ( ng/ml), and/or ifn-␣ ( iu/ml) for h or pretreated with lps ( ng/ml) or tnf-␣ ( ng/ml) for h and then either left untreated or treated with ifn-␣ ( iu/ml) for h (a -h time point was selected for optimal expression of pstat in huh . cells). the expression of pstat , stat , and isg conjugates (as a marker of usp knockdown), usp , and actin was detected by western blotting. as seen in fig. a , usp expression was largely knocked down in cells transfected with usp sirna. ifn-␣-induced phosphorylation of stat- was inhibited by pretreatment (for h) with tnf-␣ or lps (fig. a , sirna control lanes f and h versus lane d), but this effect is reversed by knockdown of usp (fig. a , sirna usp , lanes f and h versus lane d). of note, usp knockdown did not increase pstat in response to ifn-␣ before h; these findings are consistent with previous observations ( ) . we next wanted to confirm whether our findings from hepatoma cells would hold true in primary mouse hepatocytes. with this in mind, primary murine hepatocytes from usp ϩ/ϩ mice were isolated, transfected with usp sirna or control sirna, and then exposed to ifn-␣ ( iu/ml), lps ( ng/ml), or tnf-␣ ( ng/ml) for h. after a washing step, the cells were treated with ifn-␣ for an additional h. mx isg mrna expression was measured by qpcr (normalized to expression of the hprt housekeeping gene) as an index of downstream ifn-␣ effect. ifn-␣, lps, and tnf-␣ treatment blocked the effect of the final dose of ifn-␣ (fig. b, columns , , , and ) . however, knockdown of usp reversed this effect and augmented the effect of ifn-␣ (fig. b, columns , , , and ) . the data from this experiment are consistent with those obtained with human huh . cells (data not shown). thus, the ability of tnf-␣ and lps to block ifn signaling is seen in both primary and immortalized hepatocytes. as noted earlier, usp has dual roles: it both strips isg from its target proteins and impairs type ifn signaling independent of its protease activity ( ) . to determine whether the usp -dependent blunting of hepatocyte ifn signaling was due to its ability to strip isg from its conjugates, we blocked the process of isgylation by knockdown of the isg e enzyme, ube l. murine hepatocyte ube l was knocked down via transfection with sirna specific for ube l. usp ϩ/ϩ murine hepatocytes transfected with ube l sirna or control sirna were stimulated with ifn-␣ ( iu/ml), lps ( ng/ml), or tnf-␣ ( ng/ml) for h and then treated with an additional dose of ifn-␣ for h. knockdown was measured in the hepatocytes transfected with ube l sirna by western blotting probing for ube l expression and isg conjugate formation (fig. a) . next, to examine the effect of this knockdown on isg induction, wild-type murine hepatocytes transfected with ube l sirna or control sirna were stimulated with tnf-␣ ( ng/ml) and lps ( ng/ml) for h and then restimulated with ifn-␣ ( iu/ ml) for h, at which time mx isg mrna expression was measured by qpcr. as seen in fig. a , ube l knockdown was achieved, resulting in a decrease in isg conjugates. however, as seen in fig. b , we observed that neither the presence nor the relative absence of isg conjugation impacted the ability of tnf-␣ and lps to block ifn-␣ signaling since there was no change in isg expression after h of ifn-␣ treatment when usp ϩ/ϩ hepatocytes transfected with anti-ube l sirna were compared to hepatocytes transfected with irrelevant sirna (fig. b , sirna control, columns , , and , and ube l sirna, columns , , and ). these results are consistent with there being no role for isgylation (and, thus, for the ability of usp to strip isg from its protein conjugates) in the ability of tnf-␣ and lps to block type ifn signaling in hepatocytes, and this finding is in agreement with previous data showing that usp blocks ifn signaling independent of its enzymatic activity ( ) . we next sought to determine whether tnf-␣ and lps could induce usp expression in hepatocytes via the induction of ifn-␣. as observed in fig. a , stat phosphorylation shows distinct activation profiles with ifn-␣ stimulation compared to tnf-␣ or lps stimulation (fig. a , sirna control and sirna usp , lanes b and c compared to lane d). although these data strongly suggest that the effect of lps and tnf-␣ stimulation are a result of direct induction of isgs and not secondary to the induction of ifn-␣, we wanted to confirm whether lps and tnf-␣ could induce type or type ifn (ifn-␣ or ifn-␥) in primary murine hepatocyte. thus, primary murine hepatocytes were treated with ifn-␣ ( u/ml), lps ( ng/ml), or tnf-␣ ( ng/ml) for , , , or h; they were then lysed and assessed for ifn-␣, ifn-␥, and usp expression by qpcr. we observed that neither ifn-␣, lps, nor tnf-␣ induce much, if any, hepatocyte expression of ifn-␣ or ifn-␥, although the same doses induce strong expression of usp (fig. ) . thus, the induction of isgs by lps and tnf-␣ is very unlikely to reflect the induction of hepatocyte type or type ifn, which suggests that the observed usp induction is not due to type or type ifn secretion and autocrine stimulation of the ifn-␣ receptor. experimental hepatic ischemia/reperfusion induces usp expression and enhances lcmv replication. we then assessed whether tissue-wide hepatic inflammatory stress increases liver usp expression, as an in vivo confirmation of our in vitro findings. this was approached by inducing partial ( %) hepatic ischemia/reperfusion injury (hiri) for min in mice, euthanizing the animals at or h (time points relevant to our in vitro time course) and measuring induction of usp by qpcr. we chose hiri as a very well-characterized inflammatory stress, known to be driven both by tnf-␣ and lps ( ) . as seen in fig. a , hiri alone induced usp mrna expression in whole livers by Ͼ fold that of untreated animals. thus, in vivo liver inflammation leads to increased usp expression at the organ level. after determining that hiri induces usp expression, we next wanted to examine the impact of hiri on viral control. thus, we induced % hiri for min prior to lcmv we infection of usp ϩ/ϩ and usp Ϫ/Ϫ mice. as seen in fig. b , hiri led to a significant increase in lcmv viral titers in usp ϩ/ϩ mice, an effect that was not observed in usp Ϫ/Ϫ mice. having shown that lps/ tnf-␣ stimulation increases hepatocyte usp expression, we then sought to determine whether we could pharmacologically inhibit the induction of usp by lps and tnf-␣. we focused on small-molecule agents that have been linked to the nf-b signaling pathway, because of the central role of this pathway in inflammatory activation in response to a large number of inflammatory mediators, including lps and tnf-␣ ( ) . we were particularly interested in inhibitors, such as silibinin, that in addition have been linked to clinical suppression of hepatic viral production (in the case of silibinin and hcv) ( , ) . thus, we preincubated primary mouse hepatocytes for min with various inhibitors of lps and tnf-␣ signaling and measured their impact on usp induction, while simultaneously measuring expression of proinflammatory cytokine il- ␤ mrna, since nf-b is also known to promote il- ␤ transcription ( , ) . as seen in fig. a and as table , tnf-␣ induced expression of usp was potently downregulated by all inhibitors tested, and this inhibition coincided with impaired il- ␤ mrna expression (fig. b) . meanwhile, only two inhibitors potently (Ͼ %) inhibited usp expression as well as il- ␤ mrna expression in response to lps stimulation; nsc (an inhibitor of jak ) and silibinin (an inhibitor of ikk␣) (fig. a and b and table ). these data suggest that the induction of usp by tnf-␣ and lps, and pos-sibly other inflammatory stimuli, is promoted by nf-〉 signaling and that hepatocyte usp expression in particular-compared to il- ␤-may be an attractive target for pharmacologic manipulation in the setting of liver inflammation. in this study we examined the role of various inflammatory stimuli in the induction of usp and the downstream establishment inflammatory stimuli, we examined the induction of stat- phosphorylation in huh . cells with or without usp knockdown (a) and the expression of isg mrna (mx ) in primary mouse hepatocytes and the ability of usp knockdown to restore isg induction after lps and tnf-␣ stimulation (b). (a) huh . cells were transfected with anti-usp sirna or control irrelevant sirna. huh . cells were then pretreated with lps ( ng/ml), tnf-␣ ( ng/ml), or ifn-␣ ( u/ml) or left untreated for h and then exposed to ifn-␣ or left untreated for an additional h. as controls, huh . cells were treated with lps, tnf-␣, and ifn-␣ for h only. the expression of usp , pstat , stat , and isg conjugates and actin proteins was measured by western blotting. (b) primary murine hepatocytes from usp ϩ/ϩ mice were isolated, transfected with anti-usp sirna or control irrelevant sirna, and pretreated with ifn-␣ ( iu/ml), lps ( ng/ml), or tnf-␣ ( ng/ml) for h (ifn-␣ pre, lps pre, or tnf-␣ pre, respectively). after being washed, the cells were cultured in the presence or absence of ifn-␣ for h (ifn-␣ final). mx isg mrna expression was measured by qpcr and normalized to the expression of the hprt housekeeping gene. the data were generated from pooled triplicate experiments analyzed in duplicate. error bars represent the sem for duplicate pcrs. of an ifn-␣ refractory state. we used multiple methods to show that certain inflammatory stimuli, including lps and tnf-␣ are able to induce the expression of usp , which results in downstream downregulation of ifn-␣-induced isg expression. the role of usp in the ifn-␣ refractory state has been previously demonstrated by human and mouse usp knockdown studies ( , ( ) ( ) ( ) . other inflammatory stimuli, including il- and il- , did not induce usp or impair expression of isgs, including usp . in vivo, hepatic inflammatory stress (ischemia/reperfusion injury) led to increased hepatic usp gene expression that was associated with poor control of lcmv infection. thus, the hepatic inflammatory milieu, contributed to by individual inflammatory cytokines and stimuli, modulates usp expression. these results demonstrate one mechanism by which liver inflammation directly impacts the hepatocellular innate immune response. usp is known to be induced by multiple inflammatory stimuli in macrophages ( ) and lymphocytes ( ) . our findings in hepatocytes are consistent with work done by other groups in immune cells. for example, lps treatment of a murine macro- phage cell line upregulates usp in an irf -dependent manner ( ) . the cytokine specificity of our results is also consistent with finding that il- alone is not able to induce usp in murine t cells ( ) . only when t cells were treated with il- and another proinflammatory cytokine, such as transforming growth factor ␤, il- ␤, and il- , was usp expression induced ( ) . these data point out that usp can be induced by inflammatory stimuli in multiple cell types in the absence of ifn. usp is therefore well positioned to act as the mediator of "cross talk" between innate immunity and inflammatory responses. in the present study, we show that increased usp expression following exposure of hepatocytes by inflammatory stimuli blunts the ifn response. the binding of usp with the ifn-␣ receptor has been shown to inhibit the interaction of stat- with the ifn-␣ receptor and thus block downstream ifn signaling ( , ) . our data are consistent with this mechanism, since the blunting of hepatocyte ifn signaling after exposure to inflammatory stimuli is independent of usp -mediated removal of isg from its target proteins. the degree of tnf-␣ and lps-induced ifn-␣ refractoriness was not changed by ube l knockdown, although isgylation was considerably reduced. however, other mechanisms may also be at play. recent work has demonstrated that usp has the ability to deubiquitinate the transforming growth factor-activated kinase (tak ) complexes required for nf-b activation in t cells and that overexpression of usp leads to decreased nuclear activation and impaired formation of tak complexes ( ) . usp -mediated nf-b inhibition may be of importance not only for t cell adaptive immunity but also for liver inflammation. the role of tak in innate and adaptive immunity has been previously demonstrated ( ) , and studies have also shown that tak deletion interferes with hepatocyte homeostasis and leads to hepatic injury ( ) . thus, increased hepatocyte usp in response to inflammatory stimuli may constitute a negative-feedback cycle with relevance to multiple aspects of the liver's response to infection. our in vivo experiments demonstrated that inflammation resulting from ischemia/reperfusion injury induces usp expression, which coincides with diminished lcmv control in mouse livers. the general finding that liver inflammation promotes lcmv production is in agreement with work showing that polymicrobial sepsis, characterized by high tnf-␣ and inflammation, leads to increased susceptibility to lcmv infection ( ) . in our present study, hepatic ischemia/reperfusion injury did not enhance lcmv production in usp knockout mice. these data are intended as proof-of-concept but do suggest that the role of usp in hepatic viral infection and inflammation deserves further investigation. our in vitro and in vivo findings suggest a new model for how inflammation alters hepatic innate immune responses. in this model, liver inflammation leads to increased hepatocyte usp , which in turn makes the liver more susceptible to infections targeting the hepatocyte, such as hcv. this mechanism may help to explain the clinical observation that hcv infection of a transplanted, hcv-naive liver graft (that has gone through an ischemia/reperfusion cycle) is more aggressive than the original infection and that the severity of the reinfection correlates with the severity of the ischemia/reperfusion injury suffered during the transplant ( , ) . however, we have also found that usp is necessary but not sufficient on its own to induce an ifn-␣ refractory state ( ) . with this in mind, we hypothesize that the innate immune response and its ability to control ifn-sensitive viruses will depend on cumulative effect of multiple intrahepatic signals, including the induction of usp . if these results are to be translated into a clinical setting, then one approach is to target usp induction pharmacologically. using multiple inhibitors of tnf-␣/lps signaling, we found that two inhibitors, silibinin (an inhibitor of ikk␣) and nsc (a jak inhibitor) possess the ability to inhibit tnf-␣ and lps-induced usp expression and proinflammatory effects. these findings raise the possibility of pharmacologically targeting usp expression during inflammatory events to prevent the establishment of an ifn-␣ refractory state. previously, inhibition of jak signaling led to a protection of mouse livers from ischemic insult ( ) . as well, silibinin has been shown to reduce of hcv liver graft reinfection ( ) and enhance hcv clearance in ifn-␣ nonresponders ( ) via multiple mechanisms, including by direct inhibition of hcv ns b rna polymerase ( ) . usp modulation may be one mechanism by which silibinin exerts its anti-hcv effects. the present study focuses on a relatively small number of inflammatory stimuli; while tnf-␣ and lps are important to a large number of liver diseases, they are far from being the only drivers of the hepatic inflammatory response and tissue levels of usp are likely influenced by other stimuli as well. for example, we previously demonstrated that increased usp expression in the liver is and enhances lcmv replication. to examine the in vivo effect of an inflammatory stimulus on usp expression and viral replication, a hepatic ischemia/reperfusion model was used. (a) partial ( %) hepatic ischemia was induced for min, after which the portal vascular clamp was removed. control animals (sham) underwent anesthesia and a laparotomy alone. animals were euthanized and h after ischemia/reperfusion (i/r), and the affected liver segments were removed. usp mrna expression was determined by qpcr in whole liver tissue and normalized to hypoxanthine-guanine phosphoribosyltransferase (hprt) expression. data are expressed as means Ϯ the sem with n ϭ to mice/group. a p value of Ͻ . was considered significant. (b) after min of hiri or sham laparotomy, mice were infected with ϫ pfu of lcmv we. liver lobes were harvested at day postinfection. viral titers were examined on mc cells using a focus-forming assay. data are expressed as means Ϯ the sem with n ϭ to mice/group. a p value of Ͻ . was considered significant. inhibition of nf-b activation impairs lps-and tnf-␣-stimulated usp induction. to investigate the link between lps/tnf-␣ stimulation and usp induction, inhibitors of nf-b activation were added to primary mouse hepatocytes for min prior to stimulation with ng of tnf-␣/ml or ng of lps/ml for h (the inhibitor names, targets, and concentrations used are given in table ). inhibitor-treated cells were also left unstimulated as a control. as a control for usp induction, hepatocytes were stimulated with u of ifn-␣/ml for h. usp (a) and il- ␤ (b) mrna expression levels were evaluated by pcr and normalized to the hprt housekeeping gene expression prior to determining the fold increase versus mock-treated samples. the data were generated from pooled triplicate experiments analyzed in duplicate. error bars represent the sem for duplicate pcrs. predictive of patients with chronic hcv infection who will not respond to ifn-based anti-hcv therapy ( ) . although we found that tnf-␣ hepatic mrna expression was increased in treatment nonresponders, it was relatively more increased in treatment responders ( ) . we suspect that whereas increased tnf-␣ does contribute to usp expression, there are other stimuli, for example, lps and perhaps the recently described interferon-lambda ( ) , that also modulate hepatic usp expression. we propose that the ultimate usp expression is due to the liver's coordinate response to multiple stimuli. the finding that hepatic usp expression is modulated by inflammatory stimuli is a new paradigm for the interaction of the liver inflammatory microenvironment and viral infection. taken together, these data may suggest that usp represents a good target for intervention in numerous inflammatory states and in the clinical setting of hcv-related liver transplantation. hiri-induced upregulation of usp could lead to worsened outcomes posttransplant, including a quicker, more aggressive reinfection of the new organ with higher hcv rna titers. we suggest that the finding that ifn responses and usp expression are tightly linked to the hepatic inflammatory response helps to explain the finding that tnf-␣ antibody treatment improves treatment outcomes of chronic hcv with treatment combinations, including ifn-␣ ( ). mechanism of action of interferon and ribavirin in treatment of hepatitis c interferon, mx, and viral countermeasures hepatitis c virus infection induces inflammatory cytokines and chemokines mediated by the cross talk between hepatocytes and stellate cells tumor necrosis factor alpha gene expression and the response to interferon in chronic hepatitis c etanercept as an adjuvant to interferon and ribavirin in treatment-naive patients with chronic hepatitis c virus infection: a phase randomized, double-blind, placebocontrolled study safety of anti-tumor necrosis factor-alpha therapy in patients with rheumatoid arthritis and chronic hepatitis c virus infection hepatic celltype specific gene expression better predicts hcv treatment outcome than il b genotype hepatic gene expression discriminates responders and nonresponders in treatment of chronic hepatitis c viral infection cell-type-specific gene expression signature in liver underlies response to interferon therapy in chronic hepatitis c infection tissue macrophages suppress viral replication and prevent severe immunopathology in an interferon-idependent manner in mice the role of kupffer cells in hepatitis b and hepatitis c virus infections alpha interferon induces long-lasting refractoriness of jak-stat signaling in the mouse liver through induction of usp /ubp the isg /usp ubiquitin-like pathway (isgylation system) in hepatitis c virus infection and resistance to interferon therapy silencing of usp potentiates the antiviral activity of interferon against hepatitis c virus infection ubp is a novel regulator of interferon signaling independent of its isg isopeptidase activity usp -based negative feedback control is induced by type i and type iii interferons and specifically inactivates interferon alpha response gene induction pathways mediated by distinct irfs during viral infection lipopolysaccharide activates the expression of isg -specific protease ubp via interferon regulatory factor tumor necrosis factor-alpha in liver ischemia/reperfusion injury the role of intestinal endotoxin in liver injury: a long and evolving history viral and host factors induce macrophage activation and loss of toll-like receptor tolerance in chronic hcv infection enforced viral replication activates adaptive immunity and is essential for the control of a cytopathic virus complete replication of hepatitis c virus in cell culture tnf-alpha-induced sphingosine -phosphate inhibits apoptosis through a phosphatidylinositol -kinase/ akt pathway in human hepatocytes oncostatin m is a potent inducer of hepcidin, the iron regulatory hormone lipopolysaccharide, immune activation, and liver abnormalities in hiv/hepatitis b virus (hbv)-coinfected individuals receiving hbv-active combination antiretroviral therapy protein interferon-stimulated gene conjugation delays but does not overcome coronavirus proliferation in a model of fulminant hepatitis hepatitis c virus persisting after clinically apparent sustained virological response to antiviral therapy retains infectivity in vitro protein-kinase inhibitor-( - )-amide peptide analogs with standard and nonstandard amino-acid substitutions for phenylalanine- : inhibition of campdependent protein-kinase bone marrow-derived myofibroblasts promote colon tumorigenesis through the il- /jak /stat pathway nonmuscle myosin is regulated during smooth muscle contraction pd- is a specific inhibitor of the activation of mitogen-activated protein-kinase kinase in-vitro and in-vivo silibinin inhibits constitutive and tnf alpha-induced activation of nf-b and sensitizes human prostate carcinoma du cells to tnf alpha-induced apoptosis wortmannin is a potent phosphatidylinositol -kinase inhibitor: the role of phosphatidylinositol , , -trisphosphate in neutrophil responses protective strategies against ischemic injury of the liver interleukin- determines viral clearance or persistence in vivo quantification of lymphocytic choriomeningitis virus with an immunological focus assay in -well or -well plates dubbing down innate immunity malignant pirates of the immune system a comparison of selected mrna and protein abundances in human liver kinetic differences in the induction of interferon stimulated genes by interferon-alpha and interleukin b are altered by infection with hepatitis c virus nf-b in the liver-linking injury, fibrosis, and hepatocellular carcinoma successful prevention of hepatitis c virus (hcv) liver graft reinfection by silibinin mono-therapy differential in vitro effects of intravenous versus oral formulations of silibinin on the hcv life cycle and inflammation dexamethasone inhibits il- ␤ gene expression in lps-stimulated raw . cells by blocking nf-b/rel and ap- activation nf-b regulates il- ␤ transcription through a consensus nf-b binding site and a nonconsensus cre-like site usp inhibits nf-b and nfat activation during th differentiation by deubiquitinating the tak -tab complex essential function for the kinase tak in innate and adaptive immune responses disruption of tak in hepatocytes causes hepatic injury, inflammation, fibrosis, and carcinogenesis polymicrobial sepsis increases susceptibility to chronic viral infection and exacerbates cd ϩ t cell exhaustion recipient age affects long-term outcome and hepatitis c recurrence in old donor livers following transplantation prolonged rewarming time during allograft implantation predisposes to recurrent hepatitis c infection after liver transplantation the ubiquitin specific protease usp is necessary but not sufficient for a hepatocyte ifn refractory state: variable roles in type i and type iii ifn responsiveness blockade of janus kinase- signaling ameliorates mouse liver damage due to ischemia and reperfusion silibinin is a potent antiviral agent in patients with chronic hepatitis c not responding to pegylated interferon/ribavirin therapy a variant upstream of ifnl (il b) creating a new interferon gene ifnl is associated with impaired clearance of hepatitis c virus we thank dong er zhang for the gift of the usp Ϫ/Ϫ mice. s.a.m. thanks the casl/cihr hepatology fellowship program and the national cihr research training program in hepatitis c for financial support. key: cord- - q qxsr authors: maru, yoshiro title: explanation of metastasis by homeostatic inflammation date: - - journal: inflammation and metastasis doi: . / - - - - _ sha: doc_id: cord_uid: q qxsr if inflammation caused by either non-self or self molecules can disseminate throughout the body and inflammatory sites actively allow entry of circulating tumor cells and assist regrowth, then circulating tumor cells metastasize to the sites of inflammation. however, disrupted sites of homeostatic inflammation do not necessarily guarantee metastatic spread and subsequent regrowth. if inflammation caused by either non-self or self molecules can disseminate throughout the body and inflammatory sites actively allow entry of circulating tumor cells and assist their regrowth, then circulating tumor cells metastasize to the sites of inflammation. however, disrupted sites of homeostatic inflammation do not necessarily guarantee metastatic spread and subsequent regrowth. before explaining how tumor cells regrow in premetastatic organs with inflammatory properties, there is a fundamental issue of whether or not extrinsic factorinduced inflammation, systemic or localized, facilitates metastasis in the inflammatory lesion. although a rare case of metastasis of lung cancer to a minor traumatic lesion has been reported, mechanical injury seldom allows metastatic growth of tumor, otherwise surgeons lose their job. radiation to the thorax or pelvis of tumor-non-bearing normal mice increased serum tgfβ levels by twofold for at least days [ ] . mmtv/pymt transgenic model of metastatic mammary tumor showed that thoracic irradiation at gy by shielding other portions of the body enhanced lung surface metastasis by threefold at weeks after irradiation. this took place in a manner dependent on tgfβ in the tumor cells, because conditional depletion of tgfβrii using cre/lox technology reversed the effect. tgfβ , which is induced by irradiation in a tumor-independent manner, is thought to facilitate directly the survival of the tumor cells. in this case, the lungs were not irradiated to cause inflammation but tumor burden by itself induce premetastatic conditions in the lungs and irradiation-induced serum tgfβ activates ctc to reach the lungs. uv-irradiated epidermal keratinocytes secrete hmgb , which activates tlr to recruit neutrophils in the irradiated skin in which cd b + ly c + ly g + cells dominate [ ] . in transgenic mouse model of melanoma that overexpressed hgf and an oncogenic cdk , repeatedly irradiated mice by uv for weeks exhibited some melanoma cells expanding along the endothelial cells, often observed in human melanoma. this angiotropism enhanced lung metastasis, both of which were abrogated in ko mice of either tlr or myd . also in this experiment, lungs were not treated by uv, but the generated melanoma by uv prepared premetastatic soil in the lungs. in both experiments, irradiation of tumor induced host cell responses, which provide favorable conditions for tumor cells to achieve metastasis, such as potentials to intravasate and survive. meta-analysis of patients who suffered from chronic liver diseases, including fatty liver, chronic hepatitis virus b or c infection, and cirrhosis, revealed significantly lower rates of liver metastasis in colorectal cancer (crc) [ ] . readers should remember that the organ first encountered by circulating tumor cells from the primary tumor is liver by crc, lungs by any tumors that do not drain into the portal vein, and any organs by lung cancer. a more focused analysis of chronic viral hepatitis (cvh) b and c, liver metastasis took place in . % ( of cases) compared with . % ( of ) in non-cvh. in c bl/ mice with cirrhosis, which was induced by carbon tetrachloride gastrogavage for months, b f melanoma cell injection via the portal vein caused seven-to ninefold increase in frequency of tumor in the liver by histological analysis in cirrhosis over the untreated group [ ] . no surface metastatic nodules were found in the lungs. intravital kinetic analysis showed that the tumor cell movement was slower in the cirrhotic group. given the presence of regeneration, fiber generation and inflammatory cells in both clinical autopsy and experimental mouse settings, i assume that inflammatory nature in the liver and its matching with tumor cell property may make a difference. treatment of b melanoma cells with lipopolysaccharide (lps) or lipid a at μg/ ml each for h, which induced ccl expression, followed by extensive washing and subcutaneous implantation, reduced the tumor growth compared with untreated b cells in both wild-type and tlr -ko mice. however, lps promotes tumor cell migration in vivo [ ] . supportive evidence has been provided by experiments in which direct intratracheal administration of either escherichia coli or lps before injection of tumor cells, such as b f melanoma or rm- cells, through the tail vein enhanced lung metastasis at days after tumor challenge [ ] . tumor cell injection at any time points earlier than h, but not later than days, of lps was effective for the enhancement. bal fluid h after lps had increased amounts of il- , g-csf, kc, sdf- , and extracellular ubiquitin. the major chemokine receptor of neutrophils in acute lung inflammation is cxcr , which is activated by mip- and kc (remember that kc is a rodent version of il- in humans). as with tlr , cxcr is expressed in both blood and non-blood cells, both of which contribute to neutrophil accumulation and pulmonary permeability in the lungs. both ubiquitin and sfd- are known to bind cxcr , and cxcr inhibitor amd blocked the enhancement of metastasis. the reasonable but important point is that they found no metastasis in the extrapulmonary organs. in our laboratory, lps administration through a catheter only to the left bronchus with microspheres to make sure of the stimulation sites induced vascular permeability at h and accumulation of intravenously injected labeled tumor cells in the left but not right lung. therefore, a local tlr activation of a given pulmonary segment causes a limited inflammatory space to which tumor cells are allowed to metastasize. if the lung tlr is activated by an endocrine manner, the whole lungs should be transformed to be premetastatic soil. mmtv-pyv mt mice display no primary mammary tumor at weeks, but during the period of - weeks tumors with spontaneous metastasis appear. rheumatoid arthritis mouse model by type ii collagen injection was performed intradermally at weeks of mmtv-pymt mice to induce arthritis in - weeks of tumor development [ ] . the combined systems at early developmental stages of mammary tumor are assumed to see the systemic inflammatory effect at the premetastatic phase. bone metastasis took place only in the arthritis mice, and more than twofold increase in lung metastasis was observed in the same group. the arthritis induction caused leukocyte infiltration in the lungs, which was enhanced by roughly fourfold in the background of mammary tumor. elevation of factors in the lungs and circulation was detected, including il- , il- , and pge . anti-il- antibody and cox- inhibitor both reduced the metastasis. the same group demonstrated that arthritis induced mast cell recruitment in the lungs in a tumorindependent manner, but tumor cell engagement enhanced the mast cell activation underpinning the enhanced metastasis [ ] . anti-c-kit antibody also was effective to reduce metastasis. importantly, the detailed experiments tell us that premetastatic soil can be cultivated by inflammation of other organs. the experimental method to induce allergic inflammation in lungs has been established. intraperitoneal injections of chicken ovalbumin (ova) followed by repeated aerosol challenges can provoke mucus accumulation in respiratory tracts and airway hyperresponsiveness, serving as an asthma model [ ] . the james j. lee group has demonstrated that cd + t-cell-dependent recruitment of eosinophils in both bronchoalveolar lavage fluid (bal) and interstitial peribronchial and perivascular areas are required for the asthmatic phenotypes by engineering eosinophil-lacking phil mice in which expression of cytocidal diphtheria toxin is congenitally driven by the eosinophil peroxidase gene promoter. gαi -dependent ccr signaling promotes transendothelial migration of eosinophil [ ] . when b f melanoma cells were injected through the tail veil after aerosol challenges, numbers of metastatic foci on the pulmonary surface were increased by threefold [ ] . t cell depletion by anti-cd antibody (gk . ), corticosteroid inhalation and gαi -ko condition, but not the phil background, abrogated the enhanced metastasis. therefore, it is not eosinophils by themselves but cd + tcell-dependent soil preparation to allow transendothelial migration of cells that appears to play a critical role for allergy-facilitated lung metastasis. the same group provided data of breast cancer patients with lung metastasis. approximately % suffered from asthma but with no corticosteroid treatment, which is about twice the frequency of asthma in a random population of american females. cc , alternatively called club cell secretory protein (ccsp) or uteroglobin (ug), is secreted by club cells in a steroid-inducible manner. cc -ko mice are susceptible to lps inhalation with increased leukocytes in bal h after lps [ ] . the concentration gradient of s a was detected in the lungs down to blood in cc -ko mice [ ] . cc -ko enhanced lung metastasis after injection of b f melanoma cells through the tail vein. although s a was not expressed in b f cells, they expressed rage whose ligand includes age and s a / s a . an anti-rage sirna-mediated knockdown of rage in b f prior to the tail veil injection suppressed lung metastasis. i assume that cc -ko lungs are already cultivated by constant assaults of bacteria with lps for the expression of s a and s a before tumor cell injection. the calcineurin-nf-atc pathway has been known as located downstream of antigen-presenting cell engagement to t cells but also is activated individually by the vegf-vegfr and thrombin-par- systems [ ] . both exert vascular permeability as i stated earlier. from cdna microarray experiments of huvec stimulated by vegf and thrombin, down syndrome critical region- (dscr- , also called calcipressin , mcip- , and adapt ) was found dramatically induced by . -fold and . -fold, respectively at h [ ] . it turned out that dscr- inhibited nuclear localization of nf-atc in huvec, i.e., inhibited calcineurin activity by direct interaction. the nf-atc-mediated transcription induces expression of cytokines, such as il- , but simultaneous induction of the inhibitory molecule dscr- forms a negative feedback loop to brake hyperactivation of the cytokine storm. the expression of icam- , e-selectin, and vcam- were negatively affected by more than % in thrombin-stimulated huvec infected with dscr- adenovirus [ ] . not only the mrna expression level but also number of genes was down-regulated by dscr- . the same author stated that of thrombinactivated genes in huvec were down-regulated by dscr- overexpression. given that both vegfr and par- provoke inflammation, the autoinhibitory mechanism serves to avoid an overshoot and maintain the oscillating physiological levels of calcineurin-nf-atc activity. therefore, this also can be called homeostatic inflammation. adenovirus-mediated gene transfer of dscr- in b melanoma xenograft at approximately mm tumor size, in which angiogenesis was actively taking place, inhibited the tumor growth. however, syngrafts of murine renal carcinoma (renca) and colon cancer (mc ) in dscr- À ko mice displayed diminished tumor growth with significant reduction in sma + cd + vessels [ ] . mechanistically, constitutive activation of calcineurin not only induced precocious nf-atc nuclear localization in a cyclosporine-inhibitable manner but also dephosphorylated bad to activate the apoptosis pathway in endothelial cells. cerebral hemorrhage was detected in % of dscr- -ko at embryonic days and (e and e ). those experiments tell us important logics in homeostasis. there are several modes in negative feedback by molecules downstream, including catalytic inhibition of the molecule upstream by keeping the cells alive and cancellation of the biological outcome, such as growth, by eliminating the cells by apoptosis. it depends on the developmental stage of embryos and tumors. another example of endothelial homeostasis is vegf. anti-vegf antibody also resulted in side effects, such as brain hemorrhage [ ] . vegf-ko mice are embryonic lethal [ ] . in ectopic syngraft model with egfp-labeled b f melanoma cells, premetastatic lungs were assumed by days after subcutaneous implantation [ ] . they found fibrinogen-fibrin deposit generated by vascular instability that is caused by expression of angiopoietin (ang ), mmp , and mmp . they could hardly detect up-regulation of vegf in the lungs and claimed that both tgfβ and tnfα were responsible for up-regulation of those molecules, sirna-mediated knockdown of which resulted in suppressed infiltration of both myeloid cells and tumor cells. ang is usually expressed in vascular remodeling sites in which vessels are more plastic with loosened endothelial junctions. our experiments showed that blocking tgfβ and tnfα individually suppressed up-regulation of s a and s a in premetastatic lungs of tumor-bearing mice. when b f melanoma cells were injected via the tail vein to dscr -lacz mice, in which lacz expression was engineered to manifest the calcineurin-nf-atc activity, tyrosinase mrnas were not detected until day after tumor cell injection. because it could be detected as early as days - before tumor cell arrival, they assumed the period as the premetastatic period. interestingly, b f injection in dscr- -ko mice significantly enhanced lung metastasis as judged by day . orthotopic renca tumor model and llc footpad model also aggravated lung metastasis in dscr- -ko mice, while growth of the primary tumor was suppressed as just described. the difference between the primary sites and metastatic lungs turned out to be ang expression in the lungs without endothelial apoptosis. up-regulation of both vegf and activated vegfr was recognized in the tumor-injected dscr- -ko mice. i have repeatedly stated the importance of vascular permeability in inflammation ( fig. . ; see also chap. ). what will happen when endothelial cells are systemically injured? it has been intensively studied from the standpoint of hypertension and vascular tone. vascular tone is how much a vessel is constricted compared with maximum dilatation with largest lumen diameters. for example, angiotensin ii (atii) (amino acids - ) increases vascular tone, while by atrial natriuretic peptide (anp) decreases it. an early study in showed that atii induced vascular permeability with interstitial edema in rabbit aorta and dermis in min with evans blue as an indicator, which was accompanied by widening of inter-endothelial gaps by endothelial contraction [ ] . atii-stimulated vessels secrete pge, which further increases permeability and induces vasodilatation. atii infusion for days in mice displayed hypertension, reduced acetylcholine-mediated relaxation, increased maximum response to norepinephrine, hypertrophic remodeling with prominent macrophage infiltration, nox up-regulation, and nfκb activation [ ] . those are attenuated in op/op mice that are deficient in m-csf showing monocytopenia and macrophage deficiency. in the same vessel injury model under the background of ldl-r-ko to induce hypercholesterolemia, development of atherosclerosis and aortic aneurysm was reduced by whole-body genetic deletion of atii type a receptor (at a) [ ] . the atii-induced inflammation in vessels is accompanied by leukocyte migration through activation of endothelial at a of both endothelial cells and vascular smooth muscle cells (vsmc) and the stimulated vsmc secretes the potent permeability factor vegf and stimulated endothelial cells secrete chemokines, including ccl , rantes, mip- α, and ip- [ ] . conversely, vegf-induced vascular permeability is blocked by pharmacological inhibitors against atii receptor. here the vasoconstrictor induces inflammation-mediated increase of vascular tone. in the renin-angiotensin system (ras), angiotensinogen is processed to ati (amino acids - ) by renin, which is further cleaved by ace to atii. both ati and atii are inactivated by ace , a homolog of ace, to at (amino acids - ) and at (amino acids - ), respectively. ace negatively regulates ras. although the baseline levels of pulmonary atii were not changed in ace -ko mice, lung injury by acid (see chap. ) significantly augmented its levels in ace -ko mice, which was accompanied by severe lung edema due to enhanced pulmonary vascular permeability as judged by evans blue but not to changes in pulmonary perfusion pressures. the high permeability was abrogated in at a-ko mice or pharmacological inhibitor for at receptor [ , ] . when b f melanoma cells were intravenously injected, lung metastasis was reduced in at a-ko mice [ , ] . conversely, oral administration of atii enhanced the metastasis. the authors showed an engagement between b f cells with atii-induced expression of p-selectin glycoprotein ligand- (psgl- ) and platelets expressing p-selectin prompted vegf release from the platelets. it is likely that atii not only stimulates pulmonary endothelial cells to induce inflammation but also b f cells to induce psgl- expression to facilitate the engagement. the important point is that at a-expressing tumor cells may reach the premetastatic lungs and wait for an angiogenic switch, which could be mediated by atii that plays a homeostatic role in the lung microenvironment. macrophages also express ace to generate atii [ ] . tnfα can induce vascular permeability as stated before. transgenic mice of tnfα exhibited lung emphysema and severe pulmonary hypertension (ph) with vascular remodeling, such as interstitial thickening and perivascular fibrosis [ ] . because nitric oxide (no) at ppm failed to restore the right ventricular pressure, ph was not due to sustained vasoconstriction. in a skeletal muscle, tnfα infusion induced increased capillary permeability but only a minor effect on vascular tone [ ] . the vascular permeability factor eventually increases vascular tone after sustained tnfα-induced inflammation. knockout mice of npr-a, one of the receptors for anp, displayed systemic hypertension [ ] . however, lps-induced vascular permeability in the lungs could be partially attenuated by anp infusion even in npr-a-ko mice, suggesting that anp also may use other receptors, such as npr-c, for pulmonary barrier protection [ ] . therefore, anp not only decreases vascular permeability but also decreases vascular tone, i.e., causes vasodilatation. lung metastasis induced by intravenous infusion of b melanoma cells after pretreatment with lps was reduced by % by anp infusion prior to the tumor cell challenge [ ] . pretreatment with atii also enhanced lung metastasis, which was inhibited by candesartan, an aii receptor blocker [ ] . now i state on arachidonic acid (aa) metabolites in vascular tone. in endothelial cells, aa is metabolized to ( ) pgi by cyclooxygenase (cox), ( ) -or -hete (hydroxyeicosatetraenoic acid) by lipoxygenase (lox) and , -or , -eet (epoxyeicosatrienoic acid) by cytochrome p epoxygenases like cyp c and cyp j . the eets are subsequently metabolized into dhet (dihydroxyeicosatrienoic acid) with reduced biopotency by soluble epoxide hydrolase (seh) (see fig. . in chap. ). endothelial cells decrease their vascular tone individually by pgi , no, and endothelium-derived hyperpolarizing factor (edhf), such as eet [ ] . independence of those factors can be understood by non-no and non-pgi -mediated relaxation even in the presence of pharmacological inhibitors against nos and cox. to complicate the circumstances, many factors participate in their release. for example, histamine induces release of pgi and eet. eet also is released by acetylcholine and bradykinin. eet opens ca +-activated k-channel and hyperpolarizes the membrane. syngeneic xenograft tumor models, including b f melanoma, t fibrosarcoma, and llc in the background of tie promoter-driven cyp c , cyp j transgenic (tg) and seh-ko mice to achieve high levels of eet in endothelial cells exhibited dramatic increase in the tumor growth with enhanced tumor angiogenesis as judged by cd -positive cells [ ] . transgenic mice of tie -seh gave opposite effects. an enhanced corneal tumor angiogenesis was observed after systemic administration of , -eet. in addition, stimulation of metastasis after removal of llc primary tumor was further augmented in the background of those eet-high mice. i underline that they observed that the affected metastatic organs were multiple, including lymph nodes, liver, and lungs even without removal of the primary tumor. the eet-high tumor-bearing mice after tumor removal showed high levels of plasma vegf and the resected tumor expressed high vegf mrnas. most importantly and intriguingly, the authors performed parabiosis experiments to share circulation between, for example, a tumor-bearing tie -seh-ko mouse as a donor with high eet and a tie -seh-tg mouse as a recipient with low eet. this parabiosis gave enhanced tumor growth but the opposite condition, i.e., tumor in eet-low mice failed to enhance it. the shared high levels of eet were not sufficient to enhance tumor growth and endothelial eet levels were critical. it also was the case in metastasis. a tumor-bearing tie -cyp c -tg mouse with high eet failed to enhance metastasis in the recipient tie -seh-tg mouse, demonstrating that endothelial eet levels control the metastatic microenvironment. eet is produced in endothelial cells and therefore it appears to circulate and act on endothelial cells of the whole body. however, even in conditions where eet could work in an endocrine manner, local actions in each endothelial cells appear to control its bioactivity. both transgenic and ko engineering just provides artificial circumstances in which the endothelial eet simultaneously affects all endothelial cells of the whole body. benign fat tumors are functionally malignant ( fig. . ; see also chap. ). a similarity can be recognized between cancer and adipose tissue. ccr -and tlr promoted myeloid cells are mobilized in the primary tumor sites, premetastatic sites, and adipose tissues in obesity. the accumulated macrophages facilitate the tumor proliferation in the primary sites and hypertrophy of the adipocytes. therefore, adipose tissues can be assumed to be benign tumors, because adipocytes do not make metastatic progression to different locations of adipose tissues nor circulating bone marrow-derived cells differentiate into adipocytes. analyses of adipose tissue of mice that underwent bmt with gfp + bone marrow revealed that there were no gfp + cells that also were positive perilipin, an adipocyte marker [ ] . a situation in which tumor cells utilize host homeostatic systems was proposed by peter bannasch in [ ] (fig. . ) . the glycogen storage system in liver is homeostatically regulated by glycogenolysis by epinephrine and glucagon and glycogenosis by insulin. as i stated in chaps. and , glycogenolysis in liver and active glycolysis are metabolic hallmarks in cancer. however, an early preneoplastic event common to both chronic liver disease patients with a high risk for hepatocellular carcinoma and n-nitrosomorpholine-treated chemical hepatocarcinogenesis model in rats is glycogenotic activity that mimics the insulin effect [ ] . this also can be recognized as a homeostatic response to a driving force for glycogenolytic activity that still is latent. a similar phenomenon in angiogenesis is observed. semaphorin a (sema a) is one of the endogenous angiogenesis inhibitors by signaling through cellular cytoskeleton (see chap. ). in biopsy samples of human uterine cervical cancer patients, sema a was highly expressed in the epithelial cells and some endothelial cells in high-grade dysplasia called cin- , which was totally absent in cervical squamous cell carcinomas (scc) [ ] . this was supported by the hpv/e mouse model in which cervical tumor progression could be time-dependently observed from low-grade (cin- / ), cin- lesions, and scc. sema a was highly expressed in endothelial cells in cin- lesions. it is likely that the up-regulation of sema a is a homeostatic response against the angiogenic switch by the tumor before full tumor angiogenesis. the idea of lps mimicry is based on the demonstration (see below) by us and other group that md- , the co-receptor of tlr (see fig. . in chap. ), binds not only lps but also s a and saa . in biochemical levels, we used full-length s a and saa proteins expressed in and purified from mammalian cells, and synthetic peptides of various lengths theoretically to avoid contamination of lps. surface plasmon resonance analyses revealed that kd is nm for mammalian s a and . nm for mammalian saa when tested with baculovirus-purified tlr /md- proteins [ ] . the apparent kd between tlr /md- and lipid a was nm (see chap. ). the synthetic peptide of - amino acids of s a binds md- purified from baculovirus with kd . μm and stimulated cell migration in vitro in a manner dependent on tlr , md- , and myd . the binding domain is localized in the c-terminal amino acids s a , which is supported by the results of docking simulation [ ] . overlapping peptide scanning of amino acids of saa revealed that amino acids - bind the md- with kd μm and stimulated cell migration dependently on tlr , md- , and myd . injection of the bioactive -mer to achieve a serum concentration around the kd value in tumor-non-bearing mice could mobilize bmdc to the lungs in an md- -dependent manner [ ] . when human peripheral mononuclear cells were stimulated by either μg/ml of e. coli-derived lps or purified s a with endotoxin < . pg/ml for h and the culture supernatants were subjected to cytokine array, both induced for example ccl but ip- and cxcl were induced only by lps. cytokines specifically induced by s a were not found [ ] . the up-regulation of s a and s a in premetastatic lungs is dependent on vegf and tnfα, both of which are produced by the primary tumor [ ] . the lung metastasis is blocked by anti-s a antibody or tlr -ko. let us consider pneumonia. lps in the alveolar space promotes permeability in epithelial barrier. anti-s a antibody can suppress transepithelial migration of leukocytes from the interstitial to alveolar space. given the bacteriocidal roles of leukocytes in the alveolar space, it is reasonable for bacteria to promote epithelial permeability for invasion and for leukocytes to counter-migrate. because anti-s a antibody can block transendothelial migration of leukocytes from circulation to interstitial space, s a is likely to contribute to extravasation of leukocytes in physiological and of tumor cells in pathological condition. exogenously borne microbes through the airway induce mobilization of bone marrowderived myeloid cells (bmdc) to the lungs. the triggering mechanism may involve microbial lps that activates tlr in club cells in terminal bronchioles. the paracrine signaling goes in the direction to the circulation side. expression of endogenous ligands, such as s a and saa , in endothelial cells in sterile premetastatic lungs is induced by primary tumor-derived growth factors, such as ccl , from the circulation side and the paracrine signaling goes in an opposite direction from the circulation to airway side to result in amplification of saa in club cells. bone marrow may misrecognize those endogenous ligands as lps derived from lung infection and mobilize the myeloid cells there to battle against the phantom microbes leukocyte extravasation requires their initial attachment with endothelial cells. bone marrow leukocytes are known to up-regulate mac- (cd b-cd ) expression in response to not only lps but also other inflammatory factors including tnfα, ltb , and c a. in neutrophils, in which roughly % of cytosolic protein is s a and a , native s a -s a heterodimers purified from neutrophils failed to enhance adhesion to fibrinogen whose receptor is mac- at concentrations up to μm, whereas e. coli-expressed s a could do so by changing cd (¼β integrin) into an active form that can be recognized by monoclonal antibody mab [ ] . if the recombinant s a protein was contaminated by lps, it should be the lps activity via tlr . if not, the activity should have been mediated by a receptor that binds s a but not s a ; the most likely one is cd (see below) whose activation results in expression of tnfα that may work in a paracrine fashion. s a and s a were claimed to be in vivo substrate of mmp whose knockout reduced lung metastasis [ ] . the peter angel group nicely engineered hepatocyte-specific conditional double transgenic mice of s a and s a , which displayed no disease phenotypes including inflammatory lesions [ ] . both proteins were detected weakly in the liver but not increased in the serum from the baseline. interestingly, they showed roughly twofold up-regulation of serum concentration of cxcl with systemic enrichment of gr + s a + monocytic cells in circulation and passively in liver. given that s a was up-regulated in the serum of tumor-bearing mice in our experiments, s a induction of cxcl is sufficient for leukocyte mobilization from the bone marrow. i am eager to see if portal injection of tlr or rageexpressing cells can augment liver metastasis. for cxcl , also refer to chaps. and . plasminogen activator inhibitor- (pai- ) (see chaps. and ), which is induced by lps or atf activation, plays a defensive role in pneumonia by facilitating transepithelial migration of leukocytes into the air space. both s a and saa can potentially activate atf (see below). therefore, the defensive mechanism against bacteria by mobilizing leukocytes is <hijacked> by tumor cells. just like leukocytes moving toward lps, tumor cells move toward those endogenous tlr ligands in the lungs resulting in metastatic dissemination. pai- inhibits plasminogen activator responsible for generating plasmin, which can activate mmp required for metastatic microenvironment. this seems paradoxical. however, s a and saa cause vascular permeability and fibrin-fibrinogen deposition, which as a next step activates fibrinolytic events. . . . s a and the eph-ephrin system s a induces ephrin-a expression [ ] . the original finding was that lps induces tnfα, which then promotes expression of ephrin-a in huvec. the prototype of the eph (the cdna was originally isolated from a nude mouse transplantable tumor named erythropoietin-producing hepatoma) family of receptor tyrosine kinases, now called epha , was discovered by me in , and currently the family is grouped into epha with ten members and ephb with six members. their ligands are either gpi-anchored ephrin-a through a or transmembrane ephrin-b to b proteins, respectively. the system forms a counter-receptor system giving both forward (ephrin to eph) and reverse (eph to ephrin) signaling. the family members play important roles in many biological settings, including ephb ephrin-b in arteriovenous differentiation, ephrin-b in vegfr activity, and epha /a -ephrin-a /a in neuronal pathfinding. systemic administration of lps mimics bacterial assaults from the most exposed organs, i.e., lungs sensing air-borne lps and liver responsible for perception of gut-derived lps via portal vein. the misrecognition by those organs was reflected in the time-dependent up-and down-regulation of both epha /a and ephrin-a expressions, which is presumably involved in vascular permeability to allow leukocyte extravasation there [ ] . a small elevation of lps from whatever tissues attacked by bacteria is homeostatically controlled by this way. we have shown that the epha and epha can bind the membrane-bound ephrin-a in lung endothelial cells serving as adhesion molecules in a manner independent of its tyrosine kinase activity [ ] . however, a soluble form of ephrin-a , which is released from the primary tumor by adam -mediated shedding, reaches in an endocrine fashion and disrupts the pulmonary vascular barrier disorganizing ve-cadherin. we also have shown that the ephrin-a -fc recombinant soluble protein causes contraction of endothelial cells in a tyrosine kinasedependent manner through the sam domain binding to integrin-linked kinase and the subsequent rhoa-rock pathway [ ] . in both epha -ko and epha -ko mice, vascular permeability in the lungs was actually increased. the information indicates the disruption of molecules that usually participate in lung homeostasis as adhesion machinery causes inflammation in the lungs, i.e., vascular permeability and cell migration. the critical mechanism is likely switching between on and off state of the epha tyrosine kinase activity. therefore, the mechanisms of premetastasis cannot be explained without putting into consideration of molecules directly involved in lung homeostasis. the classical danger, such as invading bacteria or tumor cells and necrotic cells, are absent in the premetastatic lungs, but both vascular permeability and cell migration actively take place as in the case of physiological conditions. however, their levels are higher than those in physiological circumstances (see below). after arrival of tumor cells, which is the true danger, danger hypothesis-based events continue as found in the primary tumor. what is the mechanism by which to explain the sustained expression of s a and saa in the lungs while their serum levels decrease - days after implantation of tumor cells? detailed analysis of stimulation and expression pattern of s a , saa , and tnfα revealed that the triggering mechanism is primary tumor-secreted ccl that activates ccr in the hyperpermeable regions in the lungs to induce s a expression in the endothelial cells. s a from endothelial cells is secreted into the interstitium where macrophages are located and stimulated to secrete saa . the interstitial saa then stimulates tlr expressed in club cells in the terminal bronchiole regions, a preferential site of lung metastasis. club cells then express saa , which stimulates their own tlr resulting in autoamplification of saa [ ] (fig. . ) . at this stage, the paracrine cascade starting from ccl may be dispensable for the establishment of premetastatic milieu even if the primary tumor is removed. it is assumed that tlr inhibition may be one of the reasonable ways to put an end to the saa autoamplification. an initial lps exposure is known to induce tolerance against subsequent lps challenge. the mechanisms involve tlr -induced activation of aryl hydrocarbon receptor (ahr) whose ligands include dioxin of environmental origin and endogenous kynurenine, a product of tryptophan catabolism by indoleamine , -dioxygenase (ido ) that also is induced by tlr activation, as manifested by deceased serum levels of tnfα and il- [ ] . ahr-ko mice were more sensitive to endotoxemia by impaired mitigation of the tlr -nfκb signaling by ahr. ahr is expressed in club cells that metabolize xenobiotics and can autoamplify saa . by utilizing ahr-expressing primary hepatocytes derived from ahr-ko mice, it was shown that a potent ahr agonist tcdd ( , , , tetrachlorodibenzo-p-dioxin) repressed saa expression induced by il- β and il- [ ] . the authors showed that similar effects were observed with different ahr agonists, such as benzopyrene and naphthoflavone, and that the mechanisms involved inhibited recruitment of p rela and c/ebpβ to the saa promoter. however, ongoing studies in our laboratory have shown that neither s a nor saa lack this feedback (tsukahara and maru, unpublished results). readers should remember that ccl is also involved homeostatic inflammation in alveolar recruitment of monocytes (see chap. ). we have shown that both s a and ccl can induce vascular permeability in the lungs. i will review interesting experiments by jeffrey pollard group [ ] . they defined inflammatory ly c + (therefore gr + ) (see chap. . . . ) and resident ly c -(gr -) monocytes sharing cd + cd b + cd + phenotypes (cd is csf -r and almost all cd + cells are ly g -), which were sorted from csf -r-gfp transgenic mice to track down after adoptive transfer into mmtv-pymt mice. while gr monocytes were recruited in the primary tumor, gr monocytes were mobilized to the late-stage lung metastatic lesions and forced pulmonary metastatic nodules by intravenous injection of a pymt-induced mouse mammary tumor cell line (met- ), and intriguingly lungs h after intravenous injection of met- cells before extravasation in the lungs, but not -week-old pymt mice with yet premalignant mammary tumors presumably without expression of ccl even if they make an entry into circulation. high levels of ccr expression were observed in the recruited gr monocytes. notably, ccl expression levels in tumor cells were homogenous in the metastatic lung nodules but heterogenous in the primary tumor. therefore, ccl expression appears to confer a metastatic trait on the tumor cells. given that even ctc expressing ccl alone without primary tumor induced gr + monocytes in the lung, augmented levels of ccl in circulation may be misrecognized by gr + monocytes in the bone marrow as a danger producing ccl in the lungs. the recruited monocytes are destined to prepare premetastatic soil by producing vegf in contact with ccl -secreting tumor cells and vegf works in concert with ccl to facilitate tumor cell extravasation into the lungs. they showed that myeloid cell-specific conditional knockout of vegf by the tamoxifen-inducible system linked to the csf -r promoter abrogated the tumor cell seeding in the lungs. vegf by itself was not required for the recruitment of gr monocytes since vegf-null monocytes were recruited at similar levels to the control. tlr is a double-edged sword but seemingly defensive. the tlr polymorphism d g in the extracellular domain showed reduced lps response with a greater risk of sepsis and decreased ability for binding hmgb , an endogenous tlr ligand (see part ii). the frequency of metastasis by years after breast cancer surgery was % in d g patients compared with . % in those without polymorphism, suggesting the polymorphism is loss of defensive function [ ] . autosomal recessive mutations in myd within the irak -interacting domain, such as l p result in failed interaction with irak leading to severe infection with pyrogenic bacteria during the early infancy [ ] . compared with reactions in the initial exposures to exogenous bacteria in neonates, those in early adolescence get less and less severe. conversely, a gain of function mutation l p in myd , which results in constitutive myd signaling with production of cytokines, such as il- , can facilitate diffuse large b-cell lymphoma progression [ ] . thus, the tlr -myd pathway appears to be defensive against bacteria and tumor progression, but too much defensive reaction favors tumor progression. lps reduces the expression of ccl through the tlr -socs pathway in high endothelial venules in lymph nodes, which results in less mobilization of ccr -expressing lymphocytes as attackers to the lymph nodes [ ] . in this case, tlr weakens the defense via ccl . when tumor cells express ccr , ccl promotes their mobilization through afferent lymphatic ducts that are dilated by vegf-c. here, ccl promotes the attack. in both cases, it is favorable for attack by tumor cells. this apparent discrepancy can be easily understood by thinking of homeostatic mimicry and the hijacking idea. lps mobilizes defensive bmdc. s a also mobilizes defensive bmdc [ ] . mobilized bmdc participates in more s a production but find no enemy or attackers to battle against in the premetastatic lungs. overexpressed s a mobilizes aggressive tumor cells expressing tlr in postmetastatic lungs. an sirna-mediated knockdown of tlr in tumor cells abrogated their lung metastasis [ ] . at this stage, defenders are substituted with attackers. tlr activation inhibited growth of breast cancer cells with wild-type p by increasing growth-suppressing ifnγ secretion but promotes growth of those with p mutations by expression progrowth cytokines, including il- and cxcl [ ] . as i just stated, both plasma s a and saa elevate in tumor-bearing mice and days, respectively, after subcutaneous implantation of tumor cells with recruitment cd b + myeloid cells in the premetastatic lungs. there are some differences between intravenously injected lps and the endogenous tlr ligands produced in tumor burden. reciprocal bmts between wild-type and tlr -ko mice can theoretically generate ( ) bm tlr -ko with most of the pulmonary population of cells, such as epithelial and endothelial cells are of wild-type, and ( ) lung tlr -ko with recruitment of a small population of wild-type cells from the bone marrow. when systemic lps was administered at . mg/kg, neutrophil recruitment in the lungs was observed within h as monitored by myeloperoxidase activity and chloroacetate esterase staining in the lungs, which was abrogated in tlr -ko and lung tlr -ko, but not bm tlr -ko, e-and p-selectin double ko and cd -ko [ ] . lps-activated tlr was reflected in the p-selectin expression in the lungs in totally wild-type and bm tlr -ko mice but not tlr -ko and lung tlr -ko mice. collectively, lps stimulates tlr expression in the lung resident cells to induce neutrophil recruitment. when similar experiments were performed by tumor challenge, which increases plasma levels of both s a and saa , pulmonary recruitment of mac + myeloid cells was impaired in tlr -ko and bm tlr -ko mice but not lung tlr -ko mice, indicating that blood cell tlr is important, but the experiments failed to reduce the significance of lung tlr . because club cells, which express tlr , are derived from bm, a mixture of tlr + and tlr club cells was present in lung tlr -ko mice. more than members are known in the s family proteins [ ] . they are homologous to each other by - % in amino acid levels. information of the members with determined crystal structure revealed that they have an ef-hand motif and form antiparallel homo-or hetero-dimers. they can further associate to form high-order multimers. high-resolution nmr spectroscopy revealed that binding of ca + opens up the hydrophobic pocket allowing their interaction with the c-type immunoglobulin domain of rage, i.e., ca + molecularly switches the binding. this ca + sensor is engaged in a variety of biological events by functioning in intracellular and/or extracellular space. s a can preferably form a hetero-dimer with s a . in peripheral blood cells from s a -ko mice, s a proteins were under detection, although s a mrnas were present, suggesting that s a proteins are responsible for stabilization of s a proteins in mice [ ] . an opposite situation was reported in humans. human s a proteins were unstable in the absence of s a proteins [ ] . both s a and s a proteins were initially identified in the synovial fluid of rheumatoid arthritis patients [ ] . corpora amylacea in prostate glands of prostate cancer patients mainly consists of amyloid (see chap. ) of s a and s a [ ] . s a /s a proteins purified from granulocytes showed a propensity to aggregate in vitro forming fibrillar structures in weeks in a manner dependent on zn + and ca + as judged by atomic force microscopy and transmission electron microscopy [ ] . liquid chromatography-electrospray ionization mass spectrometry of corpora amylacea revealed the presence of hemoglobin subunits and myeloperoxidase of host origin as well as bacterial dna sequences, strongly indicating the involvement of bacterial infection-induced inflammation in the formation of this crystal. s a expression is up-regulated not only in pre-metastatic lungs but also in synovial fluid in rheumatoid arthritis patients, serum from sle and intestinal epithelial cells of crohn's inflammatory bowel disease and most importantly in tumors [ , ] . in contrast to most secretory proteins exported by the er-golgi pathway, unconventional, i.e., still unknown mechanisms underline the release of il- β, il- , il- , proil- α, fgf, and s a , all of which lack the leader sequence [ ] . it is known that il- β secretion depends on narlp inflammasome and caspase- activation [ ] . in addition to soluble factors, such as tnfα and lps, hif- induces expression of both s a and s a by binding to their hypoxia response element (hre) in the promoters in benign prostate epithelial hyperplasia bph- cells and prostate cancer pc- and du- cells [ ] . analysis of patients who were subjected to radical prostatectomy revealed similar expression patterns of s a and hif- α in immunostaining and a negative correlation between the expression levels of s a and the time required for recurrence. although dexamethasone alone failed to induce s a expression in h but enhanced lps-stimulated expression in thioglycolate-elicited murine macrophages, it directly induced s a expression in human blood monocytes. synovial knee joint biopsy of ra patients revealed that s a -positive cell numbers increased h after intravenous administration of methylprednisolone [ ] . up-regulation after steroid may imply that s a is at least seemingly antiinflammatory. however, the story is not so simple. for example, increased expression of s a in acute lymphoblastic leukemia with mll gene translocation confers glucocorticoid resistance on the patients. in this case, abundant intracellular s a is assumed to interfere with glucocorticoid-elicited ca + fluxes from the er to mitochondria [ ] . two receptors are known for s a : tlr and rage [ , ] (fig. . ). more precisely, we showed that s a binds the md- coreceptor of the tlr -md- complex. only n-glycosylated rage, which constitutes only % of total rage, can bind s a [ ] . from the rage side, it can bind the authentic ligand age, s a , and s a [ , ] . one of the unique features of s proteins is that they lack signal peptides usually essential for secretory proteins, but they are actually secreted actively and passively into the extracellular space by an as yet unknown mechanism. members that participate in tumor biology include s a , s a , s a , s a , s a , s a , and s b [ ] . clinical correlation between the expression and invasive levels has been reported in s a (also called metastasin) in particular. this is supported by two logical experiments, i.e., metastasis in mmtv-neu mice was enhanced when crossed with s a transgenic mice, while that in mmtv-pymt with s a -ko was suppressed, indicating that s a is necessary and sufficient for the tumor metastasis although the mechanism is still unclear [ ] . both stromal cells and tumor cells are influenced in terms of expression levels of s a in those tumor models. similar to s a , it is abundant in the synovial fluid of rheumatoid arthritis patients and in the psoriatic skin. intraperitoneal administration of anti-s a antibody in the human psoriasis xenograft in scid mice was effective by reducing the epidermal thickness and ki- + proliferating cells [ ] . while expression of s a in tumor cells induces their metastatic potentials, various types of cells, including t cells, dc, macrophages, fibroblasts, and myofibroblasts, are s a -positive in psoriatic skin. secreted forms of s a are thought to bind rage. s a and s a can form homo-dimers and hetero-dimers, but their partial synthetic peptides do not necessarily do so (fig. . ) . i mean either monomer or homo-dimer by s a and heterodimer by s a /s a . in , marion a. hofmann initially showed evidence of a linkage of s family with inflammation by s a binding to rage whose authentic ligand is age (see chap. ) pathologically essential in diabetes mellitus [ ] . mice lack the s a gene of which s a may function in place. in a skin carcinogenesis model induced by chemicals dmba/tpa, s a , s a , mip- α, mip- β, and mip- were up-regulated [ ] . those are target genes of nfκb that is activated by rage or tnfα. knockout mice of either tnfα or rage were resistant to this chemical carcinogenesis, suggesting sustained nfκb activation by a positive feed-forward loop of s a /s a to rage to nfκb to s a /s a . reciprocal bmt experiments with wild-type and rage-ko mice revealed that rage in bone marrow cells is responsible for carcinogenesis and dermal infiltration of neutrophils and macrophages. now rage has at least five ligands, i.e., age, s a , s a (only human), s a /s a , and hmgb . conversely, s a has at least three receptors: rage, tlr , and emmprin. tomas leanderson group has shown that human s a and dimeric s a -s a bound human rage with kd of nm and . nm, respectively [ ] . it is of note that s a bound rage with kd at nm. they also bound the human tlr -md- complex with kd of . nm and . nm, respectively. because the binding was not inhibited by simultaneous application of md- proteins, it is likely that s a binds the tlr side. affinity isolation as spectrometry in search of binding proteins for s a identified emmprin also called cd [ ] . s a stimulation induced expression of cxcl , tnfα, ephrin-a , and mmp in the emmprin signaling presumably via traf . rage, another receptor for s a , failed to form a heterodimer with emmprin. intravenously injected emmprin-expressing melanoma cells metastasized to the skin of s a but not s a transgenic mice with the epidermis-specific involucrin promoter. furthermore, pull-down experiments of human monocyte-derived dendritic cells after hiv- infection with fc-fused cd j, which belongs to the human leukocyte immunoglobulin-like receptor family, revealed binding of s a [ ] . they reported that stimulation of purified nk cells by recombinant tetrameric but not monomeric s a proteins expressed and purified from e. coli in endotoxin-free conditions as they claimed increased production of tnfα and enhanced their anti-hiv- activity. if homeostatic, lps should have a feedback loop. see fig. . in chap. for the fundamental tlr signaling cascade. negative regulation by a was stated in chap. . in addition, upon tlr activation, nfκb-mediated atf transcription takes place [ ] . atf directly associates with hdac to inhibit tlr -driven gene expression [ ] . although lps stimulation induced expression of neutrophil chemoattractant cxcl in atf -ko mice, neutrophil recruitment in the atf -ko lungs was not observed [ ] . gene expression profile analysis revealed that tiam was down-regulated in atf -ko, which was responsible for the migration defect. in the ova-induced model of bronchial asthma, atf is up-regulated in the lungs. atf -ko increased the hyperresponsiveness, pulmonary eosinophilia, and th cytokine production [ ] . when you remember this model combined with lung metastasis by b f melanoma cells (see above), you would expect aggravation of metastasis in the lungs. however, myeloid specific atf -ko or general atf -ko both reduced lung metastasis in the background of syngeneic orthotopic mmtv-pymt mammary cancer model [ ] . interestingly, there was no difference in the primary tumor growth. transcription factor atf is activated in a variety of cells essentially by stress, such as hypoxia, il- , and tnfα. atf -activated macrophages, for example, switch the balance to m and directly activate mmp expression. this may be one reason to explain the metastasis-suppressing activity in the atf -ko mice. activated tlr also induces pi k-and rapl-mediated activation of cd b, i.e., changing its conformation from an inactive to active state. as you remember that cd b is an integrin αmβ , this is an inside-out signal. this elicits a sequential activation of src and syk associated with itam [ ] . then syk phosphorylates myd and trif, to which an e ligase cbl-b binds and ubiquitinates for proteasomal degradation. this eventually weakens the lps-induced signaling by cd b-mediated negative feedback. calcineurin inhibitor, such as fk , or an sirna-mediated knockdown of calcineurin in raw cells augmented the tlr -nfκb cascade-mediated expression of tnfα [ ] . conversely, a constitutively active calcineurin by deleting the autoinhibitory and calmodulin-binding regions reduced lps-stimulated activation of nfκb. given that tnfα production by fk was reduced in macrophages deficient in myd , trif, tlr , and tlr and that calcineurin co-immunoprecipitates with them, the mechanisms may involve interaction, direct or indirect, between calcineurin and those molecules. lps activates akt. while traf located downstream of tlr signaling can serve as a direct e ubiquitin ligase for akt important for its membrane translocation and activation in growth factor-activated tumor cells, akt controls regulatory micrornas [ ] . for example, akt up-regulates mirna let- e that is capable of repressing tlr expression within a few hours, which is restored to the baseline in h. akt -ko macrophages displayed exaggerated responses to lps. the deduced amino acid sequence portion between mouse and human saa are highly homologous to each other, and a single nucleotide insertion in the human saa exon results in a frameshift to generate a shorter saa with a unique c-terminal saa sequence of amino acids. interestingly, this type of evolutionary genetic alteration occurs in primates, including chimpanzees ( fig. . ) . because no expression of human saa has been reported so far, it has been assumed to a pseudogene. although we could not find human saa transcripts in the database of the expressed pseudogenes in tumor cells [ ] , elaborate qpcp analysis of human lung cancer samples in our laboratory revealed that there exists a chimeric saa -saa fusion mrnas [ ] . the human saa exon is connected to the human saa exon with roughly kb between the exons to yield the saa -saa fusion protein that could be recognized by a monoclonal antibody against the unique sequence of human saa . the corresponding gene product activates erk by binding and stimulating lox- , an endothelial cell-specific scavenger for oxidized ldl. the binding affinity was nm as judged by elisa between c-terminal synthetic -mer human saa peptide and soluble lox- . given that neither the saa -saa purified from mammalian cells nor synthetic peptide of human saa bound the tlr -md- complex, human saa signaling is diverged from that of mouse. exogenous lps of initial encounter induced expression of ccr , which in turn bound endogenous ccl in germfree mice, suggesting that innate stimulation by exogenous microbes induce receptors for endogenous mediators (hiratsuka and maru, unpublished data). we have observed up-regulation of individually ccl + and ccr + cells in the lungs of lps-treated but not pbs-treated germ-free neonatal mice. however, neonatal mice housed in the standard spf conditions, in which they were exposed to lps-containing bacteria, increased numbers of ccl + and ccr + cells were recognized even pbs-treated mice. in the spf neonates lps administration followed by llc injection induced the tumor cell recruitment in the lungs, which was totally abrogated in ccr -ko mice (hiratsuka and maru, unpublished data). thus, neonatal exposure to exogenous lps activates the ccl -ccr system, which leads to the endogenous tlr cascade of the s a -saa paracrine system. a similar situation is known in saa expression in adipose tissue through gut [ , ] . levels of saa mrna in epididymal adipose tissue and colon of germfree swiss-webster mice were approximately ten-and sevenfold, respectively, higher than those raised conventionally. myd -ko reduced the saa mrna levels by sixfold compared with the wild type. lps simulates tlr in the colon epithelial cells as well as macrophages to induce saa expression. serum levels of saa in conventionally raised mice were higher than those in germ-free mice and lps administration induced saa expression in adipose tissue. given that both authentic ligand lps and endogenous one saa bind tlr in adipose tissue macrophages to secrete saa by a feed-forward mechanism. thus lps-triggered and autoamplified saa production stimulates myeloid cell mobilization from bone marrow resulting in accumulation of macrophages in both tissues. the inflammatory response in both colon and adipose tissue is stabilized at homeostatic levels. we should pay attention to the mechanism by which lps-induced inflammation in colon can spread to other tissue(s) if lps can induce expression of endogenous tlr ligands, because they can function as lps in bacteria-free tissues. what are the roles in premetastatic lungs of mdsc that i stated in the chap. ? in our initial report of premetastatic lung establishment by s a , we monitored mac- + cells. this population of cells includes cd b + gr + cells, i.e., mdsc. in breast cancer ectopic xenograft model with t cells labeled by gfp, which causes spontaneous metastasis to the lungs, flow cytometric analysis and qpcr analysis of gfp of the lungs showed that mice did not have tumor cells in the lungs until days after tumor implantation. coculture of sorted mdsc with normal lung cells revealed that the number of ifnγ-producing macrophages was significantly decreased [ ] . histological analysis of the premetastatic lungs showed that costaining of cd b + gr + and mmp + cells. in addition, huvec cocultured with mdsc producing mmp disrupted ve-cadherin of huvec. it is likely that mdsc cultivates the soil by reducing antitumor effect and by promoting pulmonary vascular permeability. mdsc accumulate not only in the premetastatic lungs but also in other parts of the body, such as the spleen, by more than fivefold in weeks after tumor cell transplantation. analysis of the splenic population of mdsc revealed several interesting features: ( ) mdsc failed to accumulate in tumors transplanted in s a -ko mice but instead cd + and cd + t cells infiltrated the tumor and of mice rejected tumor; ( ) s a overexpression induced mdsc accumulation by blocking myeloid differentiation via the nox complex-mediated production of ros. is s a -saa expression necessary and/or sufficient for premetastatic soil establishment? both anti-s a and anti-saa antibodies individually blocked lung metastasis as we reported previously [ , ] . saa -ko mice showed no prominent phenotypes in metastasis experiments in our hands (tomita t and maru y, unpublished results). because s a -ko mice were embryonic lethal, we are currently trying to establish conditional s a -ko mice to uncover the homeostatic role of s a . we have discussed on inflammatory conditions, including lps administration and acute pneumonia, which promote lung metastasis. knockout mice of uteroglobin, which is an anti-inflammatory protein also called ccsp or cc (see chaps. and ), a specific marker of lung club cells, displayed up-regulated expression of lung s a and enhanced lung metastasis when injected with b f melanoma cells through the tail vein in a tumor nonbearing condition [ ] . in mice with keratin -directed expression of iκbα to the epidermis (k -iκbα), inflammatory hyperplasia with s a + neutrophil that dominated over f / + macrophages took place followed by the development of well-differentiated squamous cell carcinomas at months of age. up-regulation of tnfα, s a , and saa was found in the skin lesions, which were sensitive to uvb at j/m to induce apoptosis [ ] . the dermatitis and skin tumor formation were abrogated when k -iκbα mice were crossed with tnfr -ko but not ccl -ko mice [ , ] . murine mammary cancer met cells were subcutaneously implanted in k -iκbα mice, and the tumor was resected to promote lung metastasis. in premetastatic lungs, cd b + gr + cell numbers were elevated but reduced after the resection. however, the resection induced development of macroscopic metastatic nodules in the lungs but not in the skin where premetastatic factors that we proposed, such as s a and tnfα, were constantly up-regulated (tomita, toftgard and maru, unpublished data). lung metastasis after resection can be explained by elimination of negative angiogenic switch on the tumor cells that reached the lungs before resection. however, the tumor cells could hardly reach the inflammatory skin, suggesting the presence of organotropic factors that control the lung metastasis. binding of receptor activator of nuclear factor kb (rank) in osteoclast precursors and rank ligand (rankl) in osteoblasts induces osteoclast differentiation in the presence of m-csf [ ] (fig. . ) . the osteoclast precursors are derived from bone marrow hematopoietic stem cells and c-kit + m-csfr + cd b low. this homeostasis in bone is hijacked by tumor cells to achieve bone metastasis by ectopically expressing both rankl and rank [ ] . rank stimulation elicits signaling through akt and erk, inducing cell migration but not cell proliferation or cell death. rank is frequently expressed in breast and renal cell cancer [ ] . rank is also expressed in normal mammary gland epithelial cells. a report of clear cell carcinoma of kidney showed that rank, rankl, and the soluble form of decoy receptor for rankl called osteoprotegerin (opg) were expressed in %, %, and %, respectively, as judged by immunostaining. although roughly % of renal cell cancer patients develop bone metastasis, opg-high patients were free of bone metastasis. patients with strong expression of both rank and rankl had extramedullary metastasis to skin and liver. rank-expressing tumor cells migrate towards rankl-expressing osteoblasts in bone. on arrival, rankl in tumor cells and induced rankl in osteoblasts by tumor cell-derived pthrp stimulate rank to induce active osteoclastogenesis and subsequent osteolytic bone metastasis. given that rankl is induced by lps in osteoblasts, endogenous tlr ligands, such as s a , derived from either myeloid cells or tumor cells might play a role in osteoclastogenesis [ ] . intracardiac injection of b f melanoma cells that express high levels of rank resulted in metastasis in bones, ovaries, adrenal glands, and brain. opg not only inhibited rankl-dependent cell migration of tumor cells in vitro but also suppressed bone metastasis in vivo. intriguingly, however, metastasis to other organs was not affected. this information indicates that hijacking the rank-rankl system participates not only in the mobilization of tumor cells but also in organ specificity in metastasis. in addition to tumor cells, lps-activated tlr -expressing neutrophils express rankl. this also is the case in neutrophils from the synovial fluid of exacerbated rheumatoid arthritis patients in which endogenous tlr ligands s a and s a are abundant [ ] . therefore, rankl not only in tumor cells but also in neutrophils participates in osteoclastic bone resorption. cox- expression (see chap. ) is observed not only in a variety of tumor cells but also stromal cells. lps induces pge production in macrophages. in postmetastatic bone experiments with melanoma cells, we have shown by using coculture systems and gene knockout mice that b melanoma cell-osteoblasts contacts resulted in osteoblastic expression of rankl in a membrane-bound (m) pges- (see chap. )-dependent manner, leading to osteoclast formation [ ] . bone metastasis was abrogated in genetic background of mpges- -ko or in the presence of an ep antagonist ae - when melanoma cells were injected intravenously through the tail vein. thus, in this case the rank-rankl system is hijacked by melanoma cell-stimulated activation of mpges-pge -ep signaling in osteoblasts. another way to hijack homeostasis is found in one of the mechanisms of brain metastasis by breast cancer and melanoma. connexin mediates homocellular communications in endothelial cells (see chap. ). endothelial cell-specific knockout of connexin resulted in dramatic no elevation and hypotension suggesting that vascular gap junctions contributes to vascular homeostasis [ ] . intraperitoneal injection of lps into mice also induced bladder connexin expression in bladder smooth muscle cells, which was largely blocked by an inos inhibitor. this suggests complicated mechanisms of connexin expression in a variety of cells in which no generation systems are involved [ ] . an emt regulator twist up-regulates connexin expression in breast cancer cells, which mediates heterocellular communication between the cancer cells and brain endothelial cells [ ] . the heterocellular connection appears to be functional as judged by the passage of calcein orange dye from the tumor cells to the brain endothelial cells, which might exchange other bioactive molecules. importantly, using t- breast cancer cells that were labeled with pkh membrane dye, which is retained when labeled cells are not dividing but lost after three cell divisions, the authors showed that the metastatic tumor cells that initiated heterocellular communication with brain endothelial cells may be dormant cells as exemplified by cancer stem cells. autacoids function in a paracrine fashion in physiological conditions. however, when overexpressed in pathological conditions they appear in the blood stream and affect the distant target cells in an endocrine manner. autocrine secretion of vegf in endothelial homeostasis, that of s a in myeloid cells as exemplified in immune suppressive functions of mdsc and paracrine secretion of tnfα within a variety of tissues are known. plasma levels of those factors increase in tumor patients in general, which allows their action in tissues distant from the sites of original overproduction, i.e., primary tumors. for example, s a -a protein levels in plasma were elevated in gastric cancer patients [ ] . although the s a -a producing primary tumor is fixed in stomach, the plasma s a -a works in an endocrine manner to stimulate bone marrow-derived mdsc, which secretes s a -a forming an autocrine loop. mdsc circulate and migrate to a tissue including the primary tumor and distant ones apart from it, in which the original functions of s a -a are transmitted. in the danger theory, matzinger proposed that the immune system is activated by damaged cells (fig. . ) [ ] . this led to the molecular identification of damage signals by so-called alarmins or danger-associated molecular patterns (damps), including uric acids, hmgb (see chap. ), and s a (chap. ). although matzinger described by mistake that the immune system fails to be activated by tumors, because they usually do not become necrotic to give damps [ ] , current understanding is that tumors cause tissue damages and therefore premetastasis, which is devoid of tissue damages, precedes the arrival of tumor cells as a true danger. hijacking of homeostatic roles played by endogenous mediators by tumor cells gives inflammatory conditions in premetastatic tissues. tumor cells inhibition of tgf-β with neutralizing antibodies prevents radiation-induced acceleration of metastatic cancer progression ultraviolet-radiation-induced inflammation promotes angiotropism and metastasis in melanoma lower incidence of hepatic metastases of colorectal cancer in patients with chronic liver diseases: meta-analysis impact of cirrhosis on the development of experimental hepatic metastases by b f melanoma cells in c bl/ mice activation of toll-like receptor on tumor cells in vitro inhibits subsequent tumor growth in vivo the ubiquitin-cxcr axis plays an important role in acute lung infection-enhanced lung tumor metastasis collagen induced arthritis increases secondary metastasis in mmtv-pyv mt mouse model of mammary cancer arthritis augments breast cancer metastasis: role of mast cells and scf/c-kit signaling influence of the route of allergen administration and genetic background on the murine allergic pulmonary response defining a link with asthma in mice congenitally deficient in eosinophils allergic pulmonary inflammation promotes the recruitment of circulating tumor cells to the lung clara cells attenuate the inflammatory response through regulation of macrophage behavior lack of an endogenous anti-inflammatory protein in mice enhances colonization of b f melanoma cells in the lungs vascular endothelial growth factor-and thrombininduced termination factor, down syndrome critical region- , attenuates endothelial cell proliferation and angiogenesis a protein encoded within the down syndrome critical region is enriched in striated muscles and inhibits calcineurin signaling thrombin-induced autoinhibitory factor, down syndrome critical region- , attenuates nfat-dependent vascular cell adhesion molecule- expression and inflammation in the endothelium targeted deletion of the calcineurin inhibitor dscr suppresses tumor growth bevacizumab safety in patients with central nervous system metastases heterozygous embryonic lethality induced by targeted inactivation of the vegf gene the calcineurin-nfat-angiopoietin- signaling axis in lung endothelium is critical for the establishment of lung metastases effects of angiotensin ii and some analogues on vascular permeability in the rabbit reduced vascular remodeling, endothelial dysfunction, and oxidative stress in resistance arteries of angiotensin ii-infused macrophage colonystimulating factor-deficient mice: evidence for a role in inflammation in angiotensin-induced vascular injury hypercholesterolemia stimulates angiotensin peptide synthesis and contributes to atherosclerosis through the at a receptor angiotensin ii increases expression of ip- and the renin-angiotensin system in endothelial cells negative regulation of vegf-induced vascular leakage by blockade of angiotensin ii type receptor angiotensin-converting enzyme protects from severe acute lung failure angiotensin ii type a receptor signaling facilitates tumor metastasis formation through p-selectin-mediated interaction of tumor cells with platelets and endothelial cells the renin-angiotensin system and malignancy adipocyte-derived lipids increase angiotensin-converting enzyme (ace) expression and modulate macrophage phenotype pulmonary hypertension in tnf-alpha-overexpressing mice is associated with decreased vegf gene expression in vivo effects of tumor necrosis factor-alpha on capillary permeability and vascular tone in a skeletal muscle hypertension, cardiac hypertrophy, and sudden death in mice lacking natriuretic peptide receptor a atrial natriuretic peptide attenuates agonist-induced pulmonary edema in mice with targeted disruption of the gene for natriuretic peptide receptor-a anp/gc-a signaling attenuates pulmonary metastasis of b melanoma enhanced by lipopolysaccharide or angiotensin-ii angiotensin ii type i antagonist prevents pulmonary metastasis of murine renal cancer by inhibiting tumor angiogenesis arachidonic acid metabolites as endothelium-derived hyperpolarizing factors epoxyeicosanoids stimulate multiorgan metastasis and tumor dormancy escape in mice bone marrow-derived circulating progenitor cells fail to transdifferentiate into adipocytes in adult adipose tissues in mice early bioenergetic changes in hepatocarcinogenesis: preneoplastic phenotypes mimic responses to insulin and thyroid hormone enhancement and phenotypic modulation of nnitrosomorpholine-induced hepatocarcinogenesis by dehydroepiandrosterone mouse colon carcinoma cells established for high incidence of experimental hepatic metastasis exhibit accelerated and anchorageindependent growth the s a -serum amyloid a -tlr paracrine cascade establishes a pre-metastatic phase eritoran inhibits s a -mediated tlr /md- activation and tumor growth by changing the immune microenvironment serum amyloid a binds md- to activate p and nf-kappab pathways in a myd -dependent manner s a and s a induce cytokine expression and regulate the nlrp inflammasome via ros-dependent activation of nf-kappab( .) tumour-mediated upregulation of chemoattractants and recruitment of myeloid cells predetermines lung metastasis s a mediates neutrophil adhesion to fibronectin through activation of beta integrins effect of ablation or inhibition of stromal matrix metalloproteinase- on lung metastasis in a breast cancer model is dependent on genetic background hepatocyte-specific s a and s a transgene expression in mice causes cxcl induction and systemic neutrophil enrichment ephrin-a expression induced by s a is mediated by the toll-like receptor endothelial epha receptor stimulation increases lung vascular permeability adam -cleaved ephrin-a contributes to lung metastasis epha interacts with integrin-linked kinase and regulates cell morphology and motility imbalance of clara cell-mediated homeostatic inflammation is involved in lung metastasis aryl hydrocarbon receptor control of a disease tolerance defence pathway ah receptor represses acute-phase response gene expression without binding to its cognate response element ccl recruits inflammatory monocytes to facilitate breasttumour metastasis toll-like receptor -dependent contribution of the immune system to anticancer chemotherapy and radiotherapy pyogenic bacterial infections in humans with myd deficiency oncogenically active myd mutations in human lymphoma salmonella disrupts lymph node architecture by tlr -mediated suppression of homeostatic chemokines bone marrow-derived progenitor cells are important for lung repair after lipopolysaccharide-induced lung injury tlr has a tp -dependent dual role in regulating breast cancer cell growth endothelium-derived toll-like receptor- is the key molecule in lps-induced neutrophil sequestration into lungs s proteins in cancer loss of s a (mrp ) results in reduced interleukin- -induced cd b surface expression, a polarized microfilament system, and diminished responsiveness to chemoattractants in vitro s a as a pharmacological target molecule in inflammation and cancer two calcium-binding proteins in infiltrate macrophages of rheumatoid arthritis amyloid formation by the pro-inflammatory s a /a proteins in the ageing prostate biophysical characterization of s a and s a in the absence and presence of bivalent cations increased serum levels of s a /a and s a are associated with cardiovascular disease in patients with inactive systemic lupus erythematosus phagocyte-specific s proteins are released from affected mucosa and promote immune responses during inflammatory bowel disease active caspase- is a regulator of unconventional protein secretion activation of the nlrp inflammasome in dendritic cells induces il- beta-dependent adaptive immunity against tumors hypoxia and hif- increase s a and s a expression in prostate cancer regulation of s a by glucocorticoids elevated s a /s a expression causes glucocorticoid resistance in mll-rearranged infant acute lymphoblastic leukemia mrp and mrp are endogenous activators of toll-like receptor , promoting lethal, endotoxin-induced shock rage, carboxylated glycans and s a /a play essential roles in colitis-associated carcinogenesis n(epsilon)-(carboxymethyl)lysine adducts of proteins are ligands for receptor for advanced glycation end products that activate cell signaling pathways and modulate gene expression hexameric calgranulin c (s a ) binds to the receptor for advanced glycated end products (rage) using symmetric hydrophobic target-binding patches rage mediates a novel proinflammatory axis: a central cell surface receptor for s /calgranulin polypeptides s b and s a differentially modulate cell survival by interacting with distinct rage (receptor for advanced glycation end products) immunoglobulin domains s protein family in human cancer significance of the s a protein in psoriasis rage signaling sustains inflammation and promotes tumor development identification of human s a as a novel target for treatment of autoimmune disease via binding to quinoline- -carboxamides s a is a novel ligand of emmprin that promotes melanoma metastasis s a protein is a novel ligand for the cd j receptor and its interaction is implicated in the control of hiv- replication by nk cells systems biology approaches identify atf as a negative regulator of toll-like receptor atf is a novel regulator of mouse neutrophil migration activating transcription factor is a negative regulator of allergic pulmonary inflammation transcription factor atf links host adaptive response to breast cancer metastasis integrin cd b negatively regulates tlr-triggered inflammatory responses by activating syk and promoting degradation of myd and trif via cbl-b calcineurin negatively regulates tlr-mediated activation pathways the kinase akt controls macrophage response to lipopolysaccharide by regulating micrornas expressed pseudogenes in the transcriptional landscape of human cancers human serum amyloid a (saa ) protein, expressed as a fusion protein with saa , binds the oxidized low density lipoprotein receptor adipocyte-derived serum amyloid a and hyaluronan play a role in monocyte recruitment and adhesion regulation of serum amyloid a (saa ) in mouse colonic epithelium and adipose tissue by the intestinal microbiota gr- +cd b+ myeloid cells tip the balance of immune protection to tumor promotion in the premetastatic lung squamous cell carcinomas and increased apoptosis in skin with inhibited rel/nuclear factor-kappab signaling tumor necrosis factor receptor -mediated signaling is required for skin cancer development induced by nf-kappab inhibition timed nf-kappab inhibition in skin reveals dual independent effects on development of hed/eda and chronic inflammation the molecular understanding of osteoclast differentiation regulation of cancer cell migration and bone metastasis by rankl increased rankl expression is related to tumour migration and metastasis of renal cell carcinomas gene expression of osteoclast differentiation factor is induced by lipopolysaccharide in mouse osteoblasts via toll-like receptors surface rankl of toll-like receptor -stimulated human neutrophils activates osteoclastic bone resorption direct melanoma cell contact induces stromal cell autocrine prostaglandin e -ep receptor signaling that drives tumor growth, angiogenesis and metastasis endothelial cell-specific knockout of connexin causes hypotension and bradycardia in mice reciprocal regulation between proinflammatory cytokine-induced inducible no synthase (inos) and connexin in bladder smooth muscle cells role of connexins in metastatic breast cancer and melanoma brain colonization increased myeloid-derived suppressor cells in gastric cancer correlate with cancer stage and plasma s a /a proinflammatory proteins tolerance, danger, and the extended family the danger theory: years later key: cord- -tsk pakb authors: jesmin, subrina; gando, satoshi; zaedi, sohel; sakuraya, fumika title: differential expression, time course and distribution of four pars in rats with endotoxin-induced acute lung injury date: - - journal: inflammation doi: . /s - - - sha: doc_id: cord_uid: tsk pakb the hypothesis that the expression of protease-activated receptors (pars) protein is regulated at the level of transcription and that par isoforms, par- , par- , par- , and par- , in lung tissue show different patterns of expression in lipopolysaccharide (lps)-induced acute lung injury (ali) was tested. male wistar rats were rendered endotoxemic by intra-peritoneal injection of lps ( mg/kg body weight). we examined the expression of protein and mrna and the immunohistochemical localization of par isoforms in lung tissues , , , and h after lps administration. induction of ali by lps was confirmed based on histopathological changes. lps administration induced significant increases in the expression of par isoforms (protein) at the level of transcription in ali. while the time course of par- and - expressions were different, those of par- and - were almost similar. an immunohistochemical analysis showed localization of par isoforms in the vascular endothelium, alveolar epithelium, and alveolar macrophages. however, the cellular distribution patterns of par isoforms were different. we conclude that lps induces increase in protein expression of par isoforms at the level of transcription in rats with ali. the differential expression patterns (over a time course) and distribution of par isoforms suggests a distinct role for each isoform in the pathogenesis of lps-induced ali. intra-alveolar and -vascular fibrin deposition is common in acute lung injury (ali) and acute respiratory distress syndrome (ards) [ , ] . the up-regulation of procoagulant pathways, impairment of physiological anticoagulant systems, and depression of the fibrinolytic pathway, collectively lead to florid alveolar fibrin deposition [ ] . fibrin deposits, in turn, enhance the inflammatory response by increasing vascular permeability, and by activating neutrophils and endothelial cells to produce proinflammatory cytokines [ , ] . these processes are the hallmarks of ali/ards and contribute to its pathogenesis. recent evidence suggests that progressive ali/ards is closely linked to the activation of inflammation and coagulation [ , ] , and that protease-activated receptors (pars) are important candidates in this interaction [ ] . to date, four distinct par isoforms, namely par- , - , - , and - have been described. thrombin activates par- , - , and - , whereas, trypsin and mast cell tryptase activate par- [ y ]. recent evidence suggests that tissue factor/factor viia (fviia) and ternary tissue factor/fviia/fxa complexes activate par- , and par- and - , respectively [ , ] . although the distribution patterns of some of the par isoforms have been determined in several tissues, it still remains unclear in the lungs of ali/ards. we have previously demonstrated the pulmonary expression of proinflammatory cytokine tumor necrosis factor-! (tnf) and key procoagulant molecules of tissue factor, plasminogen activator inhibitor- (pai- ), and fibrin in rabbits with endotoxin-induced ali [ ] . in addition, we were able to observe increased levels of pars protein, and to co-localize par- and procoagulant molecules in the alveolar epithelium and vascular endothelium. collectively, these results suggest that increase in expression of pars, together with proinflammatory cytokine and procoagulant molecules may underlie the development of ali during endotoxemia. the aims of the present study were to complement the data of a previous study by testing the hypothesis that: ( ) the pulmonary expression of par isoforms (protein) is regulated at the level of transcription and that ( ) the distribution pattern of each of the four par isoforms is distinct in the lung. to test this hypothesis, we performed experiments using a rat model of lipopolysaccharide (lps)-induced acute lung injury. male wistar rats ( y g, weeks old) were used in all experiments. endotoxemia was induced by administration of bacterial lps from escherichia coli :b ( mg/kg), dissolved in sterile saline, via i.p injection. at this dose, lps induces lung injury, as well as the expression of inflammatory cytokines. groups of animals (n= ) were killed using sodium pentobarbital ( mg/kg bw, i.p.) at different time-points after lps administration ( , , , and h). the control group received an equal volume of sterile saline ( ml/body), without lps. at the indicated time, the blood samples were collected by cardiac puncture for blood gas analysis, and lung tissue specimens were harvested with care, frozen immediately in liquid nitrogen, and then stored at j -c. for paraffin sections, lung tissue specimens were postfixed in % paraformaldehyde overnight and then embedded in paraffin. all the experimental procedures were approved by the animal care and use committee of hokkaido university graduate school of medicine animal care and use committee. in order to determine the arterial blood pressure and heart rate of rats, a microtip pressure transducer catheter (spc- , millar instruments, houston, tx, usa) was inserted into the left carotid artery of anaesthetized animals (sodium pentobarbital ( mg/ kg body weight, i.p.)). then, the arterial blood pressure (bp) and heart rate (hr) were monitored using a pressure transducer (model sck- , gould, ohio, usa) and recorded (bp and hr) using a polygraph system (amplifier, ap- g, nihon kohden, tokyo, japan; tachometer, at- g, nihon kohden; thermalpen recorder, wt- g, nihon kohden). lung tissues were harvested, blotted dry and weighed in order to determine the weight of the lung in the wet state and calculate the wet-to-dry weight ratio, as follows: the lung tissues were weighed; wrapped loosely in aluminum foil; placed in a drying oven overnight; and weighed again. immediately after harvest, the specimens were fixed in % buffered formalin solution, dehydrated, embedded in paraffin, and then sliced into -mm-thick sections. after deparaffinization, tissue sections were stained using standard hematoxylin and eosin (he) staining method. morphological injury in lung was semi-quantified by two pathologists blinded to the experimental design by analyzing, from each section, about randomly selected images. the average score was then determined or calculated (n= rat per each group). for determining the cellular distribution of proteins of interest, tissue specimens were fixed in % buffered formalin solution, dehydrated, embedded in paraffin, and then sliced into -mm-thick sections. the sections were then deparaffinized and treated for min with citrate buffer ( mm citric acid, ph . ) in a microwave oven ( w) before immunostaining. in some cases, frozen sections were fixed in acetone and air dried. endogenous peroxidase activity was quenched by incubation in % hydrogen peroxide for min. one percent bovine albumin in tris was used for min at room temperature to block non-specific staining caused by secondary antibodies. the sections were then incubated with primary antibodies overnight at -c, rinsed in phosphate buffer solution and then exposed to the fluorescence secondary antibody, rhodamine-conjugated affinipure or fluorescein-conjugated affinipure anti-sheep, anti-rabbit, anti-goat or anti-mouse igg (jackson immuno research laboratories), for h according to the manufacturer_s instructions. the specificity of immunoreactivity was confirmed by negative controls where nonimmune igg was used instead of primary antibodies. the coverslips were mounted with immunon (thermo shandon). immunofluorescent images were observed using a laser scanning confocal imaging system (mrc- , bio-rad laboratories). immunofluorescence staining was semi-quantitated using a scale (+ to ++++) and the average score of randomly selected images was calculated. two pathologists blinded to the experimental design evaluated each slide. the same set of experiments was repeated at least three times. the immunoblotting procedure used in the present study has already been described in our previous report [ ] . briefly, ice-cold lung tissues were minced with scissors, homogenized, and centrifuged, followed by determination of protein concentration of the supernatant using the bicinchoninic acid protein assay (pierce biotechnology). samples were then boiled in reducing sds sample buffer for min, loaded onto an sdsypage ( y % polyarylamide) gel under reduced conditions, subjected to electrophoresis, and electrophoretically transferred to polyvinylidine difluoride filter membrane. to reduce non-specific binding, the membrane was blocked for h at room temperature with % non-fat milk in pbs ( mm nacl, . mm kcl, . mm na hpo , . mm kh po ) containing . % tween , incubated overnight at -c with primary antibodies in pbsytween buffer, washed three times with pbsytween buffer, and then the membrane was incubated with a suitable secondary antibody coupled to horseradish peroxidase for min at room temperature. the blots were washed five times in pbsytween buffer and subsequently visualized with an enhanced chemiluminescence detection system (amersham), exposed to x-ray film (fuji photo film). intensity of total protein bands per lane was evaluated by densitometry. negligible loading/transfer variation was observed between samples. in each experiment, ßactin was used as the loading control. for immunological-based detections, the following antibodies were used: anti-human par- rabbit polyclonal antibody, anti-human par- goat polyclonal antibody, anti-mouse par- goat polyclonal antibody and anti-mouse par- goat polyclonal antibody (santa cruz biotechnology); anti-rabbit fibrinogen sheep polyclonal antibody (cedarlane laboratories); anti-human fibrin mouse monoclonal antibody (chemicon international), anti-rabbit inducible nitric oxide (no) synthase (inos) mouse monoclonal antibody (affinity bioreagents, golden, co, usa), and anti-xenopus laevis ßactin mouse monoclonal antibody (abcam). in most cases, the specificity of each antibody was initially confirmed by blocking its expression using a competing peptide against which the antibody was raised. it should also be noted that no positive immunoreactivity was observed when non-immune igg was used instead of the primary antibodies. for par- and - , rat liver tissue was used as positive control for both immunofluorescence staining and the immunoblot analysis, according to the manufacturer_s instructions (santa cruz biotechnology) [ ] , whereas, for par- and - immunoreactivities, rat liver tissue and rat uterus were used as positive controls, respectively [ ] . please note that each of the anti-par antibodies showed no cross-reactivity with other par isoforms. anti-human fibrin mouse monoclonal antibody and anti-rabbit fibrinogen sheep polyclonal antibody recognize fibrin and fibrinogen, respectively, but can also detect each other_s target peptide. the immunogen of the anti-fibrin antibody is fibrin-like ß-peptide gly-his-arg-pro-leu-asp-lys-cys. total rna samples were prepared from lung tissue specimens using the guanidinium thiocyanatephenol-chloroform single-step extraction method with isogen (nippon gene, toyama, japan), which is routinely used in our laboratory [ ] . after isolation, the rna was processed as follows: treated with dnase i; quantified and then reverse transcribed to cdna by omniscript reverse transcriptase using a first-strand cdna synthesis kit (qiagen). the reverse transcription reaction was performed at -c for min. the expression of par isoform mrnas were analyzed by real-time quantitative pcr with taqman probe using an abi prism sequence detector (perkinyelmer applied biosystems, foster, ca, usa), as previously described [ ] . gene-specific primers and taqman probes were synthesized from primer express v. . software (perkinyelmer applied biosystems), according to published cdna sequences of each gene. the sequences of the oligonucleotides were as follows: tnf levels in the plasma and lung tissues were detected using an enzyme-linked immunosorbent assay (elisa) kit for screening rat tnf (pierce biotechnology, rockford, il). for inos, we used a human inos immunoassay kit (r and d systems, minneapolis, mn). the results were expressed as meantsd (n=total number of animals in each group). the means were compared by a one-way factorial analysis of variance, followed by scheffé_s test for multiple comparisons. differences were considered to be significant at a value of p< . . as shown in table , levels of both systolic and diastolic blood pressure decreased significantly after lps administration, in comparison to control rats. the peak levels of plasma tnf and its (tnf) concentration in the lung were significantly elevated at h after lps administration. similarly, levels of plasma inos and its (inos) protein and mrna expression in the lung increased after lps administration. table summarizes the values for blood gases and lactate concentrations in rats before and after lps was given. arterial pao was significantly reduced from control animals at all time points after lps administration. as a quantitative measure of fluid clearance in lungs, wet-to-dry weight ratios were evaluated in lungs removed from rats killed at specified times after lps administration. the effect of lps on the ratios (wet-to-dry weight ratios) occurred in a time-dependent manner, leading to a significant (p< . ) increase from baseline ( . t . ) to peak value ( . t . ) after lps administration. the lungs from control rats (untreated) showed no detectable injury, based on the histological analysis. in contrast, h after lps administration, the lungs of treated animals showed congestion (+), neutrophil infiltration (+) and thickening of alveolar septum (+). these changes were also observed at and h after lps administration. at h following the administra- tion of lps, the features of lung injury became more evident. the lungs showed congestion (+), infiltration of inflammatory cells in the alveoli (++) and a thickening of alveolar septum (++), which together are called glanulomatous changes. these changes are shown in fig. . the relative amounts of immunodetectable fibrinogen/fibrin increased steadily following induction of sepsis with lps. relative levels of fibrinogen/fibrin vs. control ( . ) at , , , and h after lps administration were . t . / . t . , . t . / . t . , . t . / . t . , and . t . / . t . , respectively (p< . ). fibrin was poorly detectable in control lungs (data not shown). at h after lps administration, fibrin deposition was evident in the intra-and extra-vascular spaces, in the alveoli and in the bronchial epithelium (data not shown). the par- specific antibody reacted with one band of õ kda that is consistent with the predicted molecular mass of this receptor [ , ] . its expression increased in a time-dependent manner (fig. ) , whereas, that of par- was observed at all time points after lps administration, peaking ( . -fold) h after lps administration (fig. ) . peak expressions of par- and - proteins were seen h after lps administration (fig. ) . par- was detected as a band of õ kda, consistent with the manufacturer_s information. in addition, the molecular weights of the bands believed to be those of par- and - were comparable to those of the genbank sequences. the mrna expression of the par isoforms, as determined by real-time pcr, corresponded to levels of their respective proteins in the lung (fig. ) . immunohistochemical staining of par- (fig. ) in the control lung were essentially nil. however, h after lps administration, par- was predominantly expressed in the endothelium of small-to micro-sized blood vessel, and vascular smooth muscle cells of medium-to large-sized blood vessel, while only a modest expression was observed in vascular smooth muscle cells of small-micro-sized blood vessels. the endothelium of medium-to large-sized blood vessels only exhibited moderate staining of par- . the alveolar epithelium and bronchial epithelium exhibited strong positive staining of par- , with only modest staining observed in the alveolar macrophages. similar to the pattern of par- immunoreactivity, par- (fig. ) immunoreactivity was essentially nil in the control lungs. however, h after lps administration, it (par- ) was localized in the endothelium and vascular smooth muscle cells of small to micro size blood vessels, at comparable intensities. in the vascular endothelium of medium-to large-sized blood vessels, par- immunoreactivity was strong, but was weakly stained in the vascular smooth muscle cells. the intensity of par- immunoreactivity in the alveolar and bronchial epithelia and alveolar macrophages were similar. unlike par- and - , there was some slight immunostaining observed for par- in the control lung (fig. ) , and by h after lps administration, it (par ) was intensively expressed in both the endothelium and vascular smooth muscle cells of small-to medium-sized blood vessels. in contrast, only moderate immunoreactivity was localized in the vascular smooth muscle cells of large-sized blood vessel, but nonetheless, strong staining in the endothelium. the intensity of par- immunoreactivity in the alveolar and bronchial epithelia, and alveolar macrophage, was similar to par- and - . immuno-expression of par- in control lungs was similar to par- and - (fig. ) , and h after lps administration, strong signals were localized in the vascular smooth muscle cells of small-to medium-sized blood vessels, and moderately expressed in the vascular endothelium. this was in contrast to the vascular endothelium of large-sized blood vessels, which exhibited strong par- immunoreactivity, with modest staining in vascular smooth muscle cells. strong positive staining of par- is localized in alveolar epithelium, while moderate staining is observed in alveolar macrophages and the bronchial epithelium. these results are summarized on table . here, we test the hypothesis that levels of par isoforms in lungs are regulated at the level of transcription, but each (isoform) with a distinct pattern of tissue distribution. following lps administration, we observed a significant reduction in blood pressure and an increased expression of plasma inos, which occurred in parallel to inos immunoreactivity in the lung [ ] . in addition, he staining demonstrated a potent infiltration of inflammatory cells and thickening of alveolar septum called glanuloma [ ] . collectively, these findings support the notion that lps (via i.p.) induces sepsis and the development of ali in a rat model. the present study observed increased expression of tnf protein in both lung tissue and plasma immediately after lps administration. tnf is known to induce the expression of tissue factor, which leads to the activation of the extrinsic coagulation pathway, and increases in levels of fxa and thrombin, and ultimately, enhancement of coagulation [ ] . tnf is also known to increase levels of pai- , which, in turn, inhibit fibrinolysis [ ] . following increase in tnf levels, we found a marked elevation of fibrin/fibrinogen protein levels in the lung tissues. this observation was confirmed by immunohistochemical analysis that demonstrated clear fibrin deposition in both intra-vascular and -alveolar spaces. although, in the present study, we did not measure levels of tissue factor or the pai- , in our previous investigation these molecules were highly expressed in the lung of lps-treated rats, as revealed by western blot and immunohistochemical analyses [ ] . collectively, these data suggest that enhanced inflammation, coagulation activation, and the inhibition of fibrinolysis lead to fibrin deposition in acutely injured lung, following lps administration. these results are consistent with the clinical and experimental evidence for ali and ards [ y ] . here, we also found that lps induces increases in the protein expression of pars isoforms to in the lung of rats. the parallel changes observed in levels of both protein and mrna of par isoforms suggests that lps may exert its influence at the transcription level. the disparity in the timing of pars expression between our previous and the present studies may be due to species differences [ ] . indeed, nysted et al. [ ] demonstrated that stimulation with the cytokines tnf and interleukin- (il- ), as well as bacterial lps resulted in a five to ten fold elevation of par- gene expression in a dose-dependent manner using huvec. in addition, the exogenous treatment of human skeletal muscle cell and monocyte with proinflammatory cytokines increased expression of par- and - [ , ] . lan et al. [ ] demonstrated that inflammatory stimuli induced by influenza a virus expressed par- , - , - , and - mrna in the lung of intact mice. it is interesting to note that the occurrence of peak levels for plasma and lung tnf ( h after lps administration), coincided with the point at which significant elevations of pars- ,- , and- were observed, compared to control. these results suggest that tnf, together with, or as well as, lps rapidly induce expression of pars gene expression through transcription factors in the injured rat lungs. nuclear factor-kappab (nf-kb) is a transcriptional factor that plays a critical role in sepsis by regulating gene expression of many inflammatory cellular mediators and receptors involved in immune recognition [ ] . the nf-kb activators include an extensive list of grampositive and -negative bacteriae and their products such as lps; viruses and their components; protozoan parasites; cytokines (e.g., tnf, il- ); free radical; and oxidants. although determination of factors likely to regulate the transcription of pars is important, it goes beyond the aims of the present study. we believe that nf-kb may be one of the most important transcriptional factors regulating pars expression in lps-induced ali and, as such, plan study it in our future projects. although little is known about the cellular distribution of par- and - in lung tissue, pars- and - have been demonstrated in the airway epithelial and smooth muscle cells within the respiratory tract, as well as endothelial and vascular smooth muscle cells [ ] . par- and - are also present in the terminal bronchial epithelium and type ii epithelial cells of the peripheral lung, and lung macrophages, mast cells, granulocytes, and lymphocytes [ ] . while our previous study demonstrated the immunolocalization of par- in these cells and tissues in lps-treated rabbits, the present study showed strong immunoreactivities for all isoforms of pars in the endothelium, alveolar epithelium, and lung macrophages using a rat model of ali [ ] . in addition to increased immunoreactivities of tissue factor and pai- in the endothelium and alveolar epithelium, as reported in the previous study, we also confirmed here a strong fibrin deposition in the alveolar epithelium, and intra-alveolar and -vascular spaces. both the endothelium and alveolar epithelium are known to play important roles in the pathogenesis of ali. the co-localization of the key procoagulant molecules and the four isoforms of par in these tissues imply that these molecules may also play a pivotal role in inducing permeability in endothelial and epithelial cells, as observed in lps-induced ali. in contrast to the distinct patterns in onset and course of expression, and localizations of par- and - , par- and - had almost a similar pattern. it has been suggested that, in certain instances, binding of protease to one receptor can facilitate cleavage of another receptor. this appears to be the case for par- and - on mouse platelets [ ] . other studies suggest that par- facilitates activation of par- in the presence of low thrombin concentration [ , ] . some data indicate that par- is a cofactor for par- in mouse platelets: the hirudin-like site of par- binds and concentrates thrombin at the cell surface, and, thereby, promote cleavage of thrombin to par- [ ] . while the pathological significance of these phenomena has not been elucidated, the co-localization and co-expression of par and - , as observed in the present study, may indicate a similar cofactor relationship. a recent study provides strong evidence that neutrophil elastase-mediated apoptosis plays a pivotal role in the pathogenesis of ali, through the par- dependent pathway [ ] . vogel et al. [ ] demonstrated abrogation of thrombin-induced increase in pulmonary microvascular permeability in par- knockout mice. furthermore, the absence of par- signaling appears to attenuate bleomycin-induced lung inflammation and fibrosis [ ] . indeed, using par- knockout mouse, su et al. [ ] demonstrated that par- activation induces lung inflammation and pulmonary edema. in addition to these studies, we found expression of par- and - and their localization in endothelium, alveolar epithelium and macrophages in the lung. taken together, these data suggest that par- and - may play a critical role in the pathogenesis of ali. however, the physiological and or pathological significance of par- and - expressions in the injured lung is currently unclear. the limitations of the present study include: ( ) a narrow window of time after administration of lps was investigated to study the pulmonary expression of pars; ( ) the single moderate dose of lps (and not higher dose), although able to induce endotoxemia in rat, could not induce severe endotoxemia. thus, the current investigation could not provide insights on pars expression patterns associated with severe endotoxemia. for this reason, future studies should investigate a possible correlation between the degree of severity of induced endotoxemia and the pattern of pars expression. in addition, a more extended and prolonged time course study should be undertaken that will enable examination of the pulmonary pars expression possible. in summary, we demonstrate expression of par- , - , - , and - proteins, which occurred at the level of mrnas for up to h after lps administration in a rat model of ali. the timing in expression, i.e., in onset and over the course of time, for the four isoforms of pars were distinct. they were also localized in diverse cells, including vascular endothelium, alveolar epithelium, and alveolar macrophages, a similar localization pattern as tissue factor and pai- , observed in our previous study. in addition, we also found tnf expression, as well as intra-alveolar and -vascular fibrin deposition in the lung. collectively, these results suggest that each par isoform plays a distinct important role in the pathogenesis of lps-induced ali. coagulation, fibrinolysis, and fibrin deposition in acute lung injury coagulation abnormalities in acute lung injury and sepsis bronchoalveolar coagulation and fibrinolysis in endotoxemia and pneumonia coagulation and inflammation in acute lung injury thrombin signaling and protease-activated receptors proteinase-activated receptors: novel mechanisms of signaling by serine proteases proteinase-activated receptors nonhemostatic activity of coagulation factor xa: potent implications for various diseases tissue factorand factor x-dependent activation of protease-activated receptor by factor viia temporal changes in pulmonary expression of key procoagulant molecules in rabbits with endotoxin-induced acute lung injury: elevated expression levels of protease-activated receptors diminished penile expression of vascular endothelial growth factor and its receptors at the insulin-resistant stage of a type ii diabetic rat model: a possible cause for erectile dysfunction in diabetes protease-activated receptor isoform expression in pregnant and nonpregnant rat myometrial tissue effects of exercise training on expression of endothelin- mrna in the aorta of aged rats thrombin and protease-activated receptor- agonists promote lipopolysaccharide-induced hepatocellular injury in perfused liver role of expression of thrombin receptor par- in muscle cells and neuromuscular junctions during the synapse elimination period in the neonatal rat immunochemical localization of inducible nitric oxide synthase in endotoxin-treated rats acute respiratory distress syndrome. a comprehensive clinical approach pathogenesis of disseminated intravascular coagulation in sepsis the proteinase-activated receptor is induced by inflammatory mediators in human endothelial cells. comparison with the thrombin receptor thrombin receptor induction by injury-related factors in human skeletal muscle cells thrombin receptor expression and responsiveness of human monocytic cells to thrombin is linked to interferon-induced cellular differentiation altered expression and in vivo lung function of protease-activated receptors during influenza a virus infection in mice nuclear factor-kb role of proteaseactivated receptors in airway function: a target for therapeutic intervention? par is a cofactor for par activation by thrombin protease-activated receptors in hemostasis, thrombosis and vascular biology proteaseactivated receptor- mediates elastase-induced apoptosis of human lung epithelial cells abrogation of thrombin-induced increase in pulmonary microvascular permeability in par- knockout mice absence of protease-activated receptor- signaling affords protection from bleomycin-induced lung inflammation and fibrosis protease-activated receptor- activation induces acute lung inflammation by neuropeptide-dependent mechanisms key: cord- - fa hk n authors: ge, xin; meng, xianglin; fei, dongsheng; kang, kai; wang, qiubo; zhao, mingyan title: lycorine attenuates lipopolysaccharide-induced acute lung injury through the hmgb /tlrs/nf-κb pathway date: - - journal: biotech doi: . /s - - - sha: doc_id: cord_uid: fa hk n lung injury associated with systemic inflammatory response is a common problem affecting human health. previous studies have shown that lycorine exerts a anti-inflammatory effect. however, whether lycorine alleviates lung injury remains unclear. to explore this issue, balb/c mice and mle- cells were treated with lipopolysaccharide (lps) to establish lung injury mouse model and cell model, respectively. glycyrrhizic acid, known as an inhibitor of ali, was also used to study the effects of lycorine in vitro. our results showed that after lps treatment, the lung injury score, lung wet-to-dry weight ratio, and malondialdehyde (mda) production in the lung tissues and the expression levels of tumor necrosis factor-α, interleukin- β, and interleukin- in bronchoalveolar lavage fluid were significantly increased, whereas their levels were decreased by lycorine. additionally, lps injection activated the high-mobility group box (hmgb )/toll-like receptors (tlrs)/nf-κb pathway. however, lycorine treatment attenuated the activity of the hmgb /tlrs/nf-κb pathway in the lung tissues. in vitro studies showed that lycorine administration significantly decreased the levels of inflammatory cytokines and mda and attenuated the activity of the hmgb /tlrs/nf-κb pathway in lps-treated cells. moreover, the inhibitory effects of lycorine on the inflammatory response and oxidative stress in lps-treated lung cells were similar with that of glycyrrhizic acid, and this inhibition was intensified by both lycorine and glycyrrhizic acid treatment. we suggest that lycorine could alleviate lps-induced lung injury of inflammation and oxidative stress by blocking the hmgb /tlrs/nf-κb pathway, which gives a new perspective for ali therapy to treat lycorine as a potential treatment clinically. acute lung injury (ali) is a clinical syndrome results from various direct or indirect injury factors, characterized by pulmonary edema, inflammatory responses, and surfactant dysfunction . ali usually leads to further clinical outcomes, including acute respiratory distress syndrome (ards) and hypoxemia . therefore, it is necessary to explore new therapeutic approaches and therapeutic targets for the treatment of ali. imbalance in a variety of complex reactions, including inflammatory responses and oxidative stress, is the main cause of lung injury. inflammatory responses involve the activation of inflammatory cells and the release of inflammatory cytokines (park et al. ) . in response to injury stimulation, neutrophils, macrophages, endothelial cells, and mast cells are activated and produce various cytotoxic substances, including pro-inflammatory factors, leading to lung injury and further acute hypoxic respiratory insufficiency or respiratory failure (butt et al. ; herold et al. ) . additionally, when the organisms are adversely stimulated, the oxidation and antioxidant processes become out of balance, resulting in the accumulation of peroxidation products, including reactive oxygen species (ros) and malondialdehyde (mda), and further cell death (guo and ward ) . moreover, the accumulation of peroxidation products is also associated with the activation of inflammatory responses. specifically, ros could regulate the expression levels of pro-inflammatory factors through the nuclear factor-κb (nf-κb) pathway and chromatin remodeling (rahman et al. ). it has been reported that the inhibition of inflammation response and oxidative stress effectively attenuated lipopolysaccharide (lps)-induced ali (jiang et al. ; lei et al. ) . mechanism studies have shown that this inhibition was achieved by blocking the toll-like receptor (tlr )/nf-κb pathway and nuclear factor e -related factor (nrf )/heme oxygenase- (ho- ) pathway (deng et al. ; lei et al. ) . collectively, inflammatory response and oxidative stress may be therapeutic targets for the treatment of lung injury. lycorine is a natural alkaloid isolated from lycoris and displays a variety of biological efficacy, including antitumor, anti-virus, and anti-malaria (guo et al. ; nair and van staden ; ying et al. ) . early studies showed that lycorine also had anti-inflammatory efficacy and improved the survival rate of mice with lps-induced septic shock (citoglu et al. ; kang et al. ) . however, whether lycorine alleviates lps-mediated ali needs further investigation. high-mobility group box (hmgb ) is a key regulator of ali. hmgb mediated the activation of the tlr /nf-κb pathway and the blockade of tlr / nf-κb pathway relieved lps-induced ali . moreover, the evidence revealed that lycorine could inhibit hmgb expression in cancer progression (roy et al. ) . based on the above, we hypothesized that lycorine could alleviate lps-induced ali via blocking the hmgb / tlr /nf-κb pathway. in the present study, we found that lps treatment triggered significant lung injury and induced inflammatory response and oxidative stress in the lung, which was attenuated by lycorine. mechanism studies showed that the activation of hmgb /tlrs/nf-κb pathway induced by lps increased inflammatory responses and oxidative stress, while its blockade by lycorine effectively suppressed pulmonary inflammatory response and oxidative stress, and further alleviated lung injury. male balb/c mice aged weeks were adaptively fed for week. all animals had free access to water and were housed at a temperature of ± °c with a h: h light-dark cycle. the animals were randomly divided into four groups: control group, lps group, lps + lycorine ( mg/kg) group, and lps + lycorine ( mg/kg) group. for the establishment of ali model, mice were instilled with lps intratracheally ( mg/kg, dissolved in μl saline), while the control group was given the same amount of saline. for the treatment group, mice were received lycorine intraperitoneally ( mg/kg or mg/kg) h before lps treatment. after h of lps treatment, all animals were sacrificed. bronchoalveolar lavage fluid (balf) and lung tissues were collected for subsequent experiments. lps and lycorine were purchased from aladdin biochemical technology co., ltd. (shanghai, china). the lung tissue samples were fixed with % formaldehyde, embedded with paraffin, and then sectioned. lung sections ( μm) were stained with hematoxylin and eosin according to the manufacturer's instructions. subsequently, the sections stained with hematoxylin and eosin were observed and photographed under a microscope (olympus, tokyo, japan) (magnification, ×). blind lung injury scoring system was used to calculate the lung injury score. in short, alveolar congestion, alveolar wall thickening and edema, and alveolar cell infiltration scored - ( : none, : mild, : moderate, : severe), with a maximum score of (toba et al. ). fresh lung tissues (left lung) were collected. after removing other tissues, the wet weight of lung tissues was measured. then, the lungs were placed in an oven at °c for h to remove all moisture, and the dry weight was determined. finally, the lung wet/dry weight ratio was calculated. the expression levels of tumor necrosis factor-α (tnf-α), interleukin- β (il- β), and interleukin- (il- ) in balf and cells were detected using elisa kits (boster, wuhan, china). the optical density (od) values at nm and nm were detected with a microplate reader (biotek, winooski, vt, usa). the lung tissues were homogenized and centrifuged to obtain the supernatant. radioimmunoprecipitation assay lysis buffer (solarbio, beijing, china) was used to lyse cells. the lysate was then centrifuged to obtain the supernatant. the protein concentration was measured using bicinchoninic acid (bca) protein concentration kit (beyotime, shanghai, china) and then diluted to mg/ml with saline. subsequently, the mda detection kit (jiancheng, nanjing, page of china) was used to measure the mda level according to the manufacturer's instructions. the lung sections were incubated with % h o for min at room temperature to eliminate endogenous peroxidase activity. after treated with % goat serum for min at room temperature, the sections were incubated with anti-hmgb antibody ( : , abclonal, wuhan, china) at °c overnight. then, sections were washed with pbs for three times ( min/time) and incubated with horseradish peroxidase (hrp)-labeled secondary antibodies ( : , thermo fisher, waltham, ma, usa) for h at °c. after washing three times with pbs ( min/time), the sections were treated with , n-diaminobenzidine tetrahydrochloride reagent ( μl) (solarbio). subsequently, hematoxylin (solarbio) was counterstained for min. finally, the slices were observed and photographed under a microscope (olympus) (magnification, ×). total proteins were extracted from lung tissues and cells. bca protein concentration kit (beyotime) was used to detect protein concentration. equal amount ( µg, μl per lane, μg/ μl) of protein was separately by % sds-page. after transferred onto polyvinylidene difluoride membranes, the proteins were incubated with primary antibodies at °c overnight. subsequently, the membranes were incubated with hrpconjugated anti-rabbit/mouse secondary antibodies ( : , beyotime) for h at room temperature. then, the membranes were visualized with chemiluminescence reagent and the optical density values were analyzed by gel-pro-analyzer software. β-actin was used as an internal control to standardize the target protein levels. the primary antibodies used in the present study were: anti-hmgb ( : , proteintech, wuhan, china), anti-tlr ( : , proteintech), anti-tlr ( : , proteintech), anti-myd ( : , affinity, cincinnati, oh, usa), anti-p /p-p (ser ) ( : , affinity), anti-iκbα/ p-iκbα (ser /ser ) ( : , affinity), and anti-β-actin ( : , santa cruz biotechnology, santa cruz, ca, usa). total rna was extracted from lung tissues and cells. with the help of super m-mlv reverse transcriptase and rnase inhibitor, reverse transcription pcr was performed to obtain complementary dna (cdna). subsequently, rt-qpcr was performed using cdna, primers, × power taq pcr mas-termix, and sybr green. the expression levels of the target genes were normalized with β-actin. the primers (genscrip, nanjing, china) for rt-qpcr were as follows: lung sections were fixed in % paraformaldehyde for min and then incubated with . % triton x- for min. after washing three times with pbs ( min/time), the sections were blocked with goat serum for min. subsequently, sections were incubated with anti-hmgb antibody ( : , abclonal, wuhan, china) at °c overnight, followed by cy labeled goat anti-rabbit secondary antibody ( : , beyotime). after washing three times ( min/time) with pbs, the sections were treated with ′, -diamidino- -phenylindole (aladdin) for min. finally, sections were observed and photographed (olympus) (magnification, ×). all data were mean ± standard deviation. graphpad prism . was used for data analysis. differences between multiple groups were analyzed by one-way anova. p < . was considered statistically significant. we first investigated the effects of lycorine on lps-induced lung injury. the results showed that compared with control group, lps treatment resulted in pathological damage, including alveolar congestion, alveolar wall thickening, and edema (fig. a) . the lung injury score in lps group was significantly elevated as compared with control group (fig. a , p < . ). however, compared with lps group, the severity of alveolar congestion, alveolar wall thickening, and edema in lps + lycorine groups was decreased (fig. a) . the lung injury score in lps + lycorine groups was lower than that in lps group (fig. a , p < . ). as shown in fig. b , compared with control group, the lung wet-to-dry weight ratio was significantly increased after lps challenged (p < . ). however, the lung wet-to-dry weight ratio in lps + lycorine ( mg/kg) group was significantly decreased compared with lps group (fig. b, p < . ) . lps treatment resulted in the accumulation fig. lycorine alleviated lps-induced lung injury, inflammatory response, and oxidative stress in mice. one hour before challenged with lps ( mg/kg, dissolved in μl saline) for h, balb/c mice were treated with or without lycorine ( mg/kg or mg/kg). , control group; , lps group; , lps + lycorine ( mg/kg) group; , lps + lycorine ( mg/kg) group. a representative histological images of lung lesions and the lung injury score were obtained by hematoxylin-eosin staining, scale bar = μm. b, c the lung wet/dry weight ratio and the levels of mda in the lung tissues were measured in each group. d the expression levels of tnf-α, il- β, and il- in each group were measured by enzyme-linked immunosorbent assay. n = . data are means ± sd. a superscript with the same letter (such as a and ab) means that the difference is not significant, and a superscript with different letter (such as a and b) means that the difference is significant (p < . ). lps lipopolysaccharide, mda malondialdehyde, tnf-α tumor necrosis factor-α, il- β interleukin- β, il- interleukin- of mda in the lung issues, which was decreased by lycorine ( fig. c , p < . ). additionally, the expression levels of pro-inflammatory factors in balf were detected by elisa assay. as shown in fig. d , the expression levels of tnf-α, il- β, and il- in lps group were significantly up-regulated as compared with control group, whereas lycorine treatment inhibited lps-induced tnf-α, il- β, and il- elevation in a dose-dependent manner in balf (p < . ). therefore, the results suggested that lps treatment successfully induced severe lung injury in the lungs accompanied by pulmonary edema, inflammatory response, and oxidative stress, which was ameliorated by lycorine. we further explored the mechanism of lycorine in alleviating lps-induced lung injury. as shown in fig. a , the expression level of hmgb measured by immunohistochemistry assay in lps group was higher than that in control group, while lycorine treatment inhibited the production of hmgb induced by lps. furthermore, compared to the control group, the protein levels of hmgb , tlr , tlr , myd , p-p , and p-iκbα in lps group were observably increased and iκbα protein level in lps group was significantly decreased (p < . ). however, compared with lps group, the protein levels of hmgb , tlr , tlr , myd , p-p , and p-iκbα in lps + lycorine groups were markedly decreased and iκbα level was increased (fig. b , p < . ). consistently, the mrna expression levels of hmgb , tlr , and tlr were markedly increased in lps group as compared with control, whereas their levels were significantly decreased in lps + lycorine groups as compared with lps group (fig. c , p < . ). therefore, these results indicated that lycorine inhibited lps-induced activation of the hmgb /tlrs/nf-κb pathway. we also established a cell model with lps. the results showed that compared with control group, the tnf-α, il- β, il- , and mda levels in lps group were significantly increased, while their levels in lps + lycorine and lps + glycyrrhizic acid groups were decreased as compared with lps group, respectively ( fig. a -d, p < . ). moreover, the inhibitory effect of lycorine on the expression levels of pro-inflammatory factors was enhanced by glycyrrhizic acid (fig. a -d, p < . ). overall, these results indicated that lycorine inhibits lps-induced inflammatory response and oxidative stress, which is consistent with in vivo studies. moreover, the combined effect of lycorine and glycyrrhizic acid is more conducive to relieving lps-induced lung injury. as presented in fig. a , lps treatment increased hmgb expression level as compared with control group. however, lycorine treatment decreased hmgb expression level as compared with the lps group, and the decrease was further enhanced by glycyrrhizic acid (fig. a) . moreover, compared with control group, lps triggered hmgb , tlr , tlr , myd , p-p , and p-iκbα elevation, and decreased iκbα level (p < . ). however, lycorine and glycyrrhizic acid administration significantly inhibited the accumulation of hmgb , tlr , tlr , myd , p-p , and p-iκbα in lps-treated cells (fig. b , p < . ). the trend of hmgb , tlr , and tlr mrna levels in each group was consistent with their protein expression (fig. c , p < . ). furthermore, the inhibitory effect of lycorine on the hmgb / tlrs/nf-κb pathway was enhanced by glycyrrhizic acid (fig. a -c, p < . ). overall, lycorine and glycyrrhizic acid can inhibit the activation of the lps-induced hmgb / tlrs/nf-κb pathway. co-treatment of lycorine and glycyrrhizic acid enhances this inhibitory effect. in the present study, we revealed the important role of lycorine in alleviating lps-induced ali. increasing evidence suggested that inflammatory response is a vital cause of lung injury. severe inflammatory response and overexpression of inflammatory factors il- β, tnf-α, il- , and il- were observed in both lung injury animal models and patients with ards (dong and yuan ; meduri et al. ) . additionally, studies showed that highly expressed inflammatory factors were also associated with poor prognosis of patients with ards, including il- ( bouros et al. ; meduri et al. ) . the present study showed that lpstreated mice exhibited inflammatory response and overexpression of tnf-α, il- β, and il- , which is consistent with the previous results (deng et al. ). in addition, oxidative stress is considered to be another important cause of lung damage. it can not only directly induce tissue damage, but also regulate pathways involved in pulmonary inflammatory response (crapo ; guo and ward ) . severe oxidative stress was observed in both lung injury patients and animal models, and the mda level was significantly elevated (lei et al. ; winterbourn et al. ) . consistent with the previous studies, the present study showed that lps treatment resulted in the accumulation of oxidative stress and mda in the lungs and cells, indicating that inflammatory response and oxidative stress play a crucial role in the induction of lung injury. it has been reported that drugs that inhibit inflammation and oxidation effectively attenuated lung damage, including astaxanthin, cordycepin, and linarin (bi et al. ; han et al. ; lei et al. ). lycorine has been reported to be an effective inhibitor of tumor development and also had many other benefits, including reducing lps-induced bone loss in mice and inhibiting intervertebral disc degeneration (park et al. ; wang et al. ). here, we found that lycorine alleviated lung injury and inhibited pulmonary edema, inflammatory response, and oxidative stress. the results suggested that lycorine may be a potential treatment for ali. hmgb is an inflammatory regulator related to sepsis and immune diseases, which is highly expressed in inflammatory diseases (qu et al. a) . hmgb is the upstream regulator of tlr (di candia et al. ) . in lps-induced fig. lycorine blocked hmgb /tlrs/nf-κb pathway in lpstreated mice. one hour before challenged with lps ( mg/kg, dissolved in μl saline) for h, balb/c mice were treated with or without lycorine ( mg/kg or mg/kg). , control group; , lps group; , lps + lycorine ( mg/kg) group; , lps + lycorine ( mg/ kg) group. a the expression of hmgb in the lung tissues was measured by immunohistochemistry assay, scale bar = μm. b relative protein expression levels of hmgb , tlr , tlr , myc , p , p-p , iκbα, and p-iκbα in the lung tissues were determined by western blot analysis. β-actin as an internal control. c the mrna expression levels of hmgb , tlr , and tlr in each group were measured by rt-qpcr. n = . data are means ± sd. a superscript with the same letter (such as a and ab) means that the difference is not significant, and a superscript with different letter (such as a and b) means that the difference is significant (p < . ). lps lipopolysaccharide, hmgb the high-mobility group box , tlr toll-like receptor, rt-qpcr quantitative real-time pcr lung injury, hmgb mediated activation of the tlr / nf-κb signaling pathway, while hmgb down-regulation significantly blocked the tlr /nf-κb pathway . mechanism studies have shown that upon stimulation, phosphorylated iκbα dissociates from nf-κb dimer (p :p ), which is subsequently degraded by proteasomes. activated nf-κb is transferred from the cytoplasm to the nucleus to bind to the κb site in the target gene promoter or enhancer, and then, p is phosphorylated by protein kinase to initiate the transcription of inflammatory factors and oxidative stress-related factors (ghosh and hayden ; giridharan and srinivasan ) . thus, the nf-κb pathway is critical for the induction of inflammatory response and oxidative stress. additionally, the early studies showed that the blockade of tlr /nf-κb pathway effectively inhibited oxidative stress and inflammatory responses in the lungs (deng et al. ; zhang et al. ) . therefore, the hmgb / tlr /nf-κb pathway is an important pathway to induce inflammatory response and oxidative stress. in addition, tlr has been reported to be highly expressed in inflammatory diseases and tissues, and tlr knockdown significantly decreased the release of inflammatory cytokines (gao et al. ; ito et al. ) . moreover, tlr could activate nf-κb pathway (yan et al. ) . here, we found that lps treatment up-regulated hmgb , tlr , tlr , myd , p-p , and p-iκbα expression levels, whereas the up-regulation was suppressed by lycorine. the results suggested that lycorine may inhibit lps-induced inflammatory response and oxidative stress via hmgb /tlrs/nf-κb pathway. glycyrrhizic acid is the main component of licorice. it is well known that glycyrrhizic acid has a positive effect on anti-inflammatory treatment (yang et al. ) . glycyrrhizic acid is identified as an inhibitor of hmgb . studies showed that glycyrrhizic acid inhibited oxidative stress and inflammatory response in lung injury by inhibiting hmgb expression (qu et al. b; zhao et al. ) . here, we found that lycorine has similar regulatory mechanisms with glycyrrhizic acid in alleviating lung injury. in addition, the synergistic effect of glycyrrhizic acid and lycorine is more conducive to the improvement of lung injury, which may provide a new idea for the treatment of lung injury. fig. lycorine alleviated lps-induced inflammatory response and oxidative stress in mle- cells. mle- cells were pretreated with lycorine ( μm) or/and glycyrrhizic acid ( mmol/l) for h, and then treated with lps ( μg/ml) for h. , control group; , lps group; , lps + lycorine group; , lps + glycyrrhizic acid group; , lps + lycorine + glycyrrhizic acid group. a-c the expression levels of tnf-α, il- β, and il- were assessed by enzyme-linked immuno-sorbent assay in mle- cells in each group. d the mda levels were measured in all the groups. n = , data are means ± sd. a superscript with the same letter (such as a and ab) means that the difference is not significant, and a superscript with different letter (such as a and b) means that the difference is significant (p < . ). lps lipopolysaccharide, mda malondialdehyde, tnf-α tumor necrosis factor-α, il- β interleukin- β, il- interleukin- fig. lycorine blocked hmgb /tlrs/nf-κb pathway in lpstreated mle- cells. mle- cells were pretreated with lycorine ( μm) or/and glycyrrhizic acid ( mmol/l) for h, and then treated with lps ( μg/ml) for h. , control group; , lps group; , lps + lycorine group; , lps + glycyrrhizic acid group; , lps + lycorine + glycyrrhizic acid group. a the expression of hmgb detected by immunofluorescence staining in each group. scale bar = μm. red represents hmgb staining and blue for nuclei identification. b the protein expression levels of hmgb , tlr , tlr , myc , p , p-p , iκbα, and p-iκbα in all groups were normalized to the level of β-actin. c the mrna expression levels of hmgb , tlr , and tlr in each group were measured by rt-qpcr. n = , data are means ± sd. a superscript with the same letter (such as a and ab) means that the difference is not significant, and a superscript with different letter (such as a and b) means that the difference is significant (p < . ). lps lipopolysaccharide, hmgb the high-mobility group box , tlr toll-like receptor, rt-qpcr quantitative real-time pcr taken together, our results provide evidence that lps can trigger lung injury accompanied by increased pulmonary edema, inflammatory response, and oxidative stress, which is attenuated by lycorine. moreover, we further illustrate that lycorine may alleviate lps-induced lung injury via blocking hmgb /tlrs/nf-κb pathway. astaxanthin alleviated acute lung injury by inhibiting oxidative/nitrative stress and the inflammatory response in mice the clinical significance of serum and bronchoalveolar lavage inflammatory cytokines in patients at risk for acute respiratory distress syndrome acute lung injury: a clinical and molecular review evaluation of analgesic, anti-inflammatory and hepatoprotective effects of lycorine from sternbergia fisheriana (herbert) rupr oxidative stress as an initiator of cytokine release and cell damage lianqinjiedu decoction attenuates lps-induced inflammation and acute lung injury in rats via tlr /nf-kap-pab pathway hmgb is upregulated in the airways in asthma and potentiates airway smooth muscle contraction via tlr accelerated inflammation and oxidative stress induced by lps in acute lung injury: iotanhibition by st acute respiratory distress syndrome: advances in diagnosis and treatment regulation of alveolar macrophage death in acute lung inflammation long noncoding rna malat regulates sepsis in patients with burns by modulating mir with tlr new regulators of nf-kappab in inflammation mechanisms of nf-kappab p and strategies for therapeutic manipulation role of oxidants in lung injury during sepsis a conserved inhibitory mechanism of a lycorine derivative against enterovirus and hepatitis c virus linarin prevents lpsinduced acute lung injury by suppressing oxidative stress and inflammation via inhibition of txnip/nlrp and nfkappab pathways novel concepts of acute lung injury and alveolar-capillary barrier dysfunction role of tlr in inflammation and tissue damage after intestinal ischemia-reperfusion injury the protective effect of trillin lps-induced acute lung injury by the regulations of inflammation and oxidative state lycorine inhibits lipopolysaccharide-induced inos and cox- up-regulation in raw . cells through suppressing p and stats activation and increases the survival rate of mice after lps challenge cordycepin inhibits lps-induced acute lung injury by inhibiting inflammation and oxidative stress persistent elevation of inflammatory cytokines predicts a poor outcome in ards. plasma il- beta and il- levels are consistent and efficient predictors of outcome over time the protective effect of dexmedetomidine on lpsinduced acute lung injury through the hmgb -mediated tlr / nf-kappab and pi k/akt/mtor pathways antiplasmodial lycorane alkaloid principles of the plant family amaryllidaceae lycorine attenuates autophagy in osteoclasts via an axis of mros/trpml / tfeb to reduce lps-induced bone loss cytokine balance in the lungs of patients with acute respiratory distress syndrome high-mobility group box (hmgb ) and autophagy in acute lung injury (ali): a review glycyrrhizic acid ameliorates lps-induced acute lung injury by regulating autophagy through the pi k/akt/ mtor pathway redox modulation of chromatin remodeling: impact on histone acetylation and deacetylation nf-kappab and pro-inflammatory gene expression lycorine downregulates hmgb to inhibit autophagy and enhances bortezomib activity in multiple myeloma xb deficiency enhances lipopolysaccharide-induced septic response and acute lung injury lycorine suppresses endplate-chondrocyte degeneration and prevents intervertebral disc degeneration by inhibiting nf-kappab signalling pathway protein carbonyl measurements show evidence of early oxidative stress in critically ill patients tlr silencing reduced hyperammonaemia-induced liver injury by inhibiting oxidative stress and inflammation responses via inactivating nf-kappab and mapk signals the anti-inflammatory activity of licorice, a widely used chinese herb lycorine inhibits breast cancer growth and metastasis via inducing apoptosis and blocking src/fak-involved pathway protective effects of the suppressed nf-kappab/tlr signaling pathway on oxidative stress of lung tissue in rat with acute lung injury glycyrrhizic acid prevents sepsis-induced acute lung injury and mortality in rats acknowledgements this study was supported by grants from the national natural scientific foundation of china (no. ). conflict of interest the authors declare that they have no conflicts of interest.ethics approval all animal experiments were performed according to the national institutes of health guide for the care and use of laboratory animals and with the approval of the ethic committee of harbin medical university. key: cord- -v as gde authors: sukhotnik, igor; shehadeh, naim; rothem, lilah; lurie, michael; mogilner, jorge; shiloni, eitan; shamir, raanan title: oral insulin up-regulates toll-like receptor expression and enhances intestinal recovery following lipopolysaccharide-induced gut injury in a rat date: - - journal: dig dis sci doi: . /s - - - sha: doc_id: cord_uid: v as gde in the present study, we evaluated the protective effect of oral insulin (oi) on intestinal mucosa following lipopolysaccharide-induced intestinal damage in a rat. male sprague-dawley rats were divided into three experimental groups: sham rats, lps-rats that were treated with lipopolysaccharide (lps), and lps-ins rats that were treated with oi given in drinking water h before and following injection of lps. intestinal structural changes, enterocyte proliferation, enterocyte apoptosis, and mucosal expression of toll-like receptor (tlr ) were determined h after the last lps injection. lps-ins animals showed a significantly greater bowel and mucosal weight in jejunum and ileum, mucosal dna and protein in jejunum and ileum, villus height in ileum, crypt depth in jejunum and ileum, cell proliferation rates in jejunum, and significantly lower apoptotic index in ileum compared to lps- animals. lps rats demonstrated % increase in tlr expression in jejunum compared to sham animals. treatment with oi resulted in a three-fold increase in tlr expression in jejunum, compared to lps animals. in conclusion, oi improves intestinal recovery after lps endotoxemia in a rat. sepsis and its consequences, adult respiratory distress syndrome (ards) and multiple organ failure (mof), are major causes of mortality in severely sick and injured patients. the exact mechanisms of sepsis and mof are still an enigma, but it has been suggested that the gut is the ''motor'' of such septic states [ ] and the first step of a ''gutliver-lung axis'' [ ] . a number of factors have been shown to predispose to the passage of bacteria or bacteria products such as endotoxin from the lumen of the gut into the bloodstream (bacterial translocation). these include shock, parenteral nutrition, intestinal epithelial damage, and antibiotic therapy [ ] . translocation of bacteria and endotoxin may lead to activation of the immune inflammatory system and the production of cytokines and other immune inflammatory mediators that can promote systemic inflammatory response, multiple organ failure, and death [ ] . sepsis by itself may lead to the alteration of the gut flora, impaired host immune defenses, or direct gut mucosal injury and may result in gut barrier failure. all of the above derangements, acting in concert, may ultimately lead to multiple organ failure and death [ ] . the mechanisms by which sepsis damages intestinal mucosa are poorly understood. the innate immune recognition of bacterial and viral products is mediated by a family of transmembrane receptors known as toll-like receptors (tlrs). since tlrs were initially found as mammalian homologues of the drosophila membrane protein toll [ ] , ten mammalian homologues have been identified and are designated tlr - , respectively [ ] . several experimental studies have shown that lipopolysaccharide (lps), a major cell wall constituent of gram-negative bacteria, is usually recognized by tlr receptor whereas tlr recognizes gram-positive bacterial cell wall components [ ] . in enterocytes, exposure to lps results in endothelial activation through a tlr receptor. a complex of several immunologic and nonimmunologic processes maintains the barrier function of the gastrointestinal tract. recent evidence suggests that the combination of an intact intestinal mucosa and a normally functioning immune system provides adequate barrier function. in this context, identification of factors that promote growth and regeneration of the intestinal epithelium may suggest new therapeutic strategies for maintaining gut integrity in septic patients. although the intestine is not a classic target tissue for insulin, extensive experimental studies have established that insulin supplementation has important physiological effects in the context of intestinal growth, cell maturation, and differentiation of the small intestine [ ] [ ] [ ] . the purpose of this study was to evaluate the effects of oral insulin in preventing mucosal damage caused by lps endotoxemia in a rat model, including its effect on enterocyte proliferation and death via apoptosis, and to determine whether alterations in toll-like receptor -mediated signaling may occur during lps endotoxemia and insulin administration. the experimental protocol was approved by the ''guide for the care and use of laboratory animals'', rappaport faculty of medicine, technion (haifa, israel). male sprague-dawley rats weighing - g were kept in individual stainless steel cages at constant temperature ( °c) and humidity, and a -h light-dark cycle was maintained. the rats were acclimatized for a minimum of week before experimentation. the rats had free access to water and were pair fed with standard chow. animals were randomly assigned to one of three experimental groups of rats each: ( ) sham rats (group a, n = ) underwent i.p. injection of sterile saline once a day; ( ) lipopolysaccharide (lps) rats (group b, n = ) were treated with lps and given i.p. once a day at a dose of mg/kg for h (two doses); and ( ) lps-ins rats (group c, n = ) were pretreated with oral insulin given in drinking water ( %) h before and following injection of lps. twenty-four hours after the last lps injection, animals were anesthetized with i.p. sodium pentobarbital ( mg/ kg) and were sacrificed by inducing open pneumothorax. the small bowel was excised quickly, washed with cold isotonic saline and divided into two segments: proximal jejunum immediately after treitz ligament and terminal ileum proximal to ileo-cecal junction. each segment was weighed, cut longitudinally, and the diameter was measured at three equidistant places. mucosa was scraped using a glass slide, collected, and weighed. dna and protein were extracted from the mucosa of jejunum and ileum using trizol reagent as described by chomczynski [ ] . dna concentration was recorded spectrophotometrically and calculated per cm of bowel length/ g body weight. final protein concentration was measured spectrophotometrically using a commercially available kit (bio-rad, protein assay) and was calculated per cm of bowel length. histological sections were prepared from the proximal jejunum and distal ileum. the samples of intestinal tissues were fixed in a % formaldehyde solution ( - % methanol), then were embedded in paraffin wax using standard techniques. sections ( lm each) were cut and stained with hematoxylin and eosin. the mucosal damage of the small bowel or intestinal injury was graded using the park score [ ] : normal mucosa, subepithelial space at villus tip, more extended subepithelial space, epithelial lifting along villus sides, denuded villi, loss of villus tissue, crypt layer infarction, transmucosal infarction, and transmural infarction. the villus height and crypt depth for each specimen were measured using an objective mounted micrometer ( · magnification) and an optical microscope ( · magnification). villus height and crypt depth data were derived from eight rats in each group, and each measurement consisted of the mean of ten villi and crypts. standard -bromodeoxyuridine ( -brdu) labeling reagent (zymed laboratories, inc., san francisco, ca) was injected intraperitoneally at a dose of ml/ g body weight h before sacrifice. crypt cell proliferation was assessed using the biotinylated monoclonal anti-brdu antibody system provided in a kit form (zymed laboratories, inc, san francisco, ca). an index of proliferation was determined as the ratio of crypt cells staining positively for brdu per five crypts. immunohistochemistry for caspase- (caspase- cleaved concentrated polyclonal antibody; dilution : ; biocare medical, walnut greek, ca) was performed for identification of apoptotic cells using a combination of the streptovidin-biotin-peroxidase method and microwave antigen retrieval on formalin-fixed, paraffin-embedded tissues according to manufacturers' protocols. briefly, lm sections were deparaffinized, rehydrated in graded alcohol, and microwave-pretreated in edta buffer for min. after washing in phosphate-buffered saline (pbs), the specimens were incubated in peroxidase quenching solution ( % h o in methanol) for min, then were treated with serum blocking solution for min, and thereafter were stained with primary caspase- cleaved polyclonal antibodies (diluted : ) for min in a moist chamber. after washing, the slides were incubated with human-absorbed, biotinylated, affinity-purified antibody to react with primary antibodies. enhanced horseradish peroxidase conjugated streptavidin was then bound to the biotinylated secondary antibodies. the dab was then used to create an intense brown deposit around the antigenantibody-enzyme complex in the sample. for each group, the number of stained cells was counted in at least ten villi in areas without necrosis. the apoptotic index (ai) was defined as the number of apoptotic cells per ten villi. all measurements were performed by a qualified pathologist blinded as to the source of intestinal tissue. intestinal segments (proximal jejunum and distal ileum) were quickly isolated and frozen after sacrifice and conserved at - °c. for total rna extractions, approximately mg of tissue was homogenized with a glass homogenizer. total rna was isolated using trizol reagent (gibco brl, usa), as described by chomczynski [ ] , and then was serially diluted with water pretreated with diethylpyrocarbonate. a portion of total rna ( lg in a total volume of ll) was reverse transcribed using moloney murine leukemia virus (mmlv) first strand cdna synthesis kit (gene choice, inc., md, usa). the reaction buffer contains random hexamer primers and dntps. portions of cdna (* ng) synthesized from cells were amplified using pmol of each primer in · reddy mix pcr master mic reaction buffer according to the manufacturer's instructions (abgene, epson, surrey, uk). the pcr reaction was performed as follows: initial melting at °c for min, followed by cycles each of min at °c, annealing at °c for rtlr and rb-actin for s, elongation at °c for min, followed by a -min extension at °c. after pcr, the amplified product ( ml) was run on a % agarose gel stained with ethidium bromide and photographed. the level of gene expression was expressed as the ratio of the gray density of the objective gene over the gray density of b-actin at densometry. the sequences for the gene-specific tlr and b-actin primers are depicted in table (all primers were purchased from sigma). the data are expressed as the mean ± sem. a nonparametric kruskal-wallis anova test was used for statistical analysis with p \ . considered statistically significant. all animals in groups a survived. injection of lps (group b) resulted in a % mortality rate ( of rats died). pretreatment with insulin resulted in a % mortality ( of rats died). the remaining rats recovered from the surgery uneventfully. about % of lps-animals (groups b and c) suffered from appetite loss, diarrhea, and weight loss. lps endotoxemia intestinal mucosal parameters lps endotoxemia (group b) resulted in a significant decrease in bowel weight in jejunum ( %, p \ . ) and ileum ( %, p \ . ), mucosal weight in jejunum ( %, p \ . ) and ileum ( %, p \ . ), mucosal dna in jejunum ( %, p \ . ) and ileum ( %, p \ . ), and mucosal protein in jejunum ( %, p \ . ) and ileum ( %, p \ . ) ( table ) compared to control animals (group a). following oral insulin administration, lps-rats (group c) demonstrated a significant increase in jejunal ( %, p \ . ) and ileal ( %, p \ . ) bowel weight, jejunal ( %, p \ . ) and ileal ( %, p \ . ) mucosal weight, jejunal ( %, p \ . ) and ileal ( %, p \ . ) mucosal dna, and jejunal ( %, p \ . ) and ileal ( %, p \ . ) mucosal protein compared to lps-animals (group b). a significant decrease in villus height in jejunum ( %, p \ . ) and ileum ( %, p \ . ) and crypt depth in ileum ( %, p \ . ) was seen in rats following lps endotoxemia (group b) compared to sham animals (group a) ( table ) . following oral insulin administration (group c), lps animals demonstrated a significant increase in ileal ( %, p \ . ) villus height as well as jejunal ( %, p \ . ) and ileal ( %, p \ . ) crypt depth compared to lps-animals (group b). park's injury score (table ) increased significantly following lps endotoxemia (group a) in jejunum (fivefold, p \ . ) and ileum (two-fold, p \ . ) compared to sham animals (group a). pretreatment with oral insulin did not change intestinal injury score significantly compared to lps-animals (group b). enterocyte proliferation and apoptosis lps rats (group b) showed a significant decrease in the proliferation index in ileum ( ± vs. ± brdu positive cells per crypts, p \ . ) compared to sham animals (group a) and a trend toward a decrease in cell proliferation in jejunum ( ± vs. ± brdu positive cells per crypts, p = . ); however, this trend did (figs. and ). following pretreatment with oral insulin (group c), lps rats showed a significant decrease in apoptotic index in ileum ( . ± . vs. . ± . apoptotic cells per villi, p \ . ) compared to lps-animals (group b), and tendency toward decrease in cell apoptosis in jejunum; however, this trend did not achieve statistical significance. lps rats (group b) demonstrated a % increase in tlr expression in jejunum (fig. ) compared to sham animals (group a). pretreatment with oral insulin (group c) resulted in a three-fold increase in tlr expression in jejunum compared to lps-animals (group b). there was no significant change in the tlr expression in ileum between the three experimental groups. extensive studies in various experimental models have established that insulin plays an important role in normal intestinal physiology [ , , [ ] [ ] [ ] . a fact that insulin is present in human milk and that breast milk stimulates maturation and proliferation of intestinal mucosa, led to the hypothesis that insulin might have a trophic gut effect [ ] . shulman et al. [ ] have demonstrated that administration of oral insulin causes an increase in ileal mass and disaccharidase activity in the newborn miniature pig without apparent concomitant changes in serum glucose, insulin, or cortisol levels. in another experiment, the same authors observed an increase in ileal lactase activity following oral insulin administration in the newborn miniature pig [ ] . in a recent clinical trial, these authors has shown that enteral administration of insulin to preterm infants ( - weeks of gestational age) leads to a higher lactase activity and less feeding intolerance [ ] . buts et al. [ ] analyzed the distribution, ontogeny, and molecular properties of insulin receptors in immature and mature enterocytes using radioligand binding assays. they have shown that iinsulin binding to the insulin receptor was five times higher in crypt cells than in villus cells and two times higher in the ileum than in the jejunum. these authors concluded that increased responsiveness of rat immature enterocytes to insulin could be related to high membrane concentrations of insulin receptor and that normal rat enterocytes express a -kda phosphotyrosine protein identified as a direct substrate of the insulin receptor tyrosine kinase. in another study, these researchers have demonstrated that insulin activates the brush border membrane hydrolases in rat immature intestine by binding the hormone to its intestinal receptor and indirect triggering of the transcription of hydrolases genes and possibly by changes in intracellular polyamine concentrations [ ] . we have recently shown that oral insulin supplementation exerts intestinal trophic effects, as well as systemic effects in rats when administered up to weeks beyond the suckling period [ ] . since insulin has trophic gut effects, we hypothesized that insulin may have a beneficial effect on gut growth and recovery in different gastrointestinal disorders. we have recently demonstrated that oral insulin exerts a strong stimulating effect on bowel re-growth following massive small bowel resection in a rat [ ] . in a different experiment, we have shown that oral insulin does not prevent ischemic damage but accelerates intestinal recovery, enhances enterocyte proliferation, and decreases cell death via apoptosis following intestinal ischemia-reperfusion in jury in rats [ ] . in the present experiment, we investigated the effects of oral insulin in preventing mucosal damage caused by lipopolysaccharide endotoxemia in a rat. in a recent study, we have demonstrated that lps endotoxemia impairs the integrity of the gastrointestinal mucosa in a rat. decreased cell proliferation and increased apoptosis were the main mechanisms responsible for decreased cell mass in this model [ ] . our results show that lps endotoxemia causes a direct intestinal mucosal injury (this is evident from increased park's intestinal injury score) and leads to mucosal hypoplasia. the observed decreased bowel and mucosal weight, decreased mucosal dna and protein, and decreased villus height and crypt depth in this model support this conclusion. the changes in body weight and small bowel morphology in the current report were similar to those observed in our previous experiment [ ] . the data from the previous study [ ] and the current one suggest that intestinal hypoplasia is the predominant feature of mucosal damage. although the sham-insulin group was not included in the current study, we have recently shown that oral insulin exerts a trophic effect on normal intestine. in rats, oral insulin given in the postweaning period resulted in a significant increase in mucosal weight as well as intestinal dna content and villus height. the maximal changes were observed in the first week of insulin treatment, and most significant changes were observed in the proximal intestine [ ] . the present data clearly indicate that oral insulin had a strong stimulating effect on intestinal recovery from intestinal mucosal injury caused by lps similar to that seen previously in suckling rats. increased bowel and mucosal weight as well as mucosal dna and protein support this conclusion. parallel increases in mucosal dna and protein indicate that the greater mucosal mass of animals treated with oral insulin can be attributed to cellular hyperplasia. because the dna content is directly proportional to mucosal cell number, these measurements exclude such factors as edema or vascular engorgement as responsible for differences in mucosal mass. increased villus height may be the result of increased proliferation and accelerated migration along the villus, and is a marker for the increased absorptive surface area and increased numbers of epithelial cells. increased mucosal proliferation in functioning intestine as demonstrated by the increased cell proliferation index following oral insulin administration together with decreased cell death via apoptosis, suggests an activated enterocyte turnover and may be considered a main mechanism of mucosal hyperplasia in recovering bowel. our data indicate that oral insulin did not prevent the damaging effect of lps on gut. this is evident from unchanged injury score scale following insulin administration. the intestinal mucosal surfaces are continuously exposed to large amounts of bacterial products such as lps. lps, a well-known bacterial pyrogen, is recognized by several receptors, including the toll-like receptor (tlr) , on various cells including enterocytes. tlr recognizes lps and transduces a proinflammatory signal through the adapter molecule myeloid differentiation marker myd [ ] . many studies in various experimental models have established that tlr expression in different organs and tissues is up-regulated following lps administration. janardhan et al. [ ] have shown that tlr expression in lung is sustained up to h after intra-tracheal instillation of lps. lps-induced suppression of cardiac myocyte contractility is tlr -dependent [ ] . in accordance with other tissues, several experimental studies have shown that tlr mrnas is upregulated in the small intestine after lps administration [ ] . the fact that the intestinal epithelium seems to be unresponsive to intestinal luminal lps was explained by low or absent expression of tlr , md- , and the coreceptor molecule cd in cells constituting the intestinal mucosal surface [ ] . in the present study, we observed a % increase in the tlr receptor expression in jejunum following lps administration; however, in ileum tlr expression remained unchanged. therefore, we suggest that the signaling pathways leading from lps to tlr activation in jejunum and ileum may be different. because the bacterial contamination in ileum is significantly greater than that in jejunum, the mechanism of the ubiquitous activation of tlr in the presence of lps is limited in the ileum to protect the mucosa against massive amounts of bacteria and bacterial lps present in the intestinal lumen and do not initiate acute inflammatory responses as would be elicited along mucosal surface in jejunum. our observations are consistent with data of other investigators who note that the human intestine exhibits specific mechanisms for lps tolerance, given its proximity to the enteric flora and their otherwise strong innate response to lps [ ] [ ] [ ] . exposure to oral insulin resulted in three-fold increase in tlr expression in jejunum compared to lps-nontreated rats. this increase of tlr expression together with increased lps did not lead to mucosal damage in this group compared to lps-animals. in contrast, lps-ins rats showed a significantly enhanced cell turnover. it should be emphasized that parallel increase in lps exposure and upregulation of tlr expression has less significant damaging effect on intestinal mucosa compared to other organs and tissues. indeed, insulin fragments themselves exert their stimulated effect on cell turnover through binding to the other receptors. it is likely that other specific growthrelated genes are also up-regulated by insulin in the gastrointestinal tract. similar to lps, oral insulin exerted minimal effect on tlr expression in the ileum. this may be explained by the presence of bacteria in the ileum that may lead to a temporary insensitivity of cells toward subsequent lps and insulin stimulation. the mechanism by which insulin increases tlr are unknown. since one would assume that increased tlr is part of the lps induced injury, it is reasonable to suggest that the adaptive response of the intestine to oral insulin is stronger than its effects on tlr . in conclusion, oral insulin does not prevent intestinal mucosal injury caused by lps endotoxemia in the rat, but it enhances intestinal mucosal recovery following lps administration. increased enterocyte proliferation and decreased enterocyte apoptosis may be responsible for this positive effect. oral insulin increases the stimulating effect of lps on toll-like receptor expression in the proximal intestine, suggesting that tlr is not the mediator of injury in this model. the role of intestinal barrier failure and bacterial translocation in the development of systemic infection and multiple organ failure the intestine-liver-lung axis in septic syndrome nutrition and the gut mucosal barrier. in: daly j (ed) current opinions in general surgery role of the gut in multiple organ failure: bacterial translocation and permeability changes nutritional support of the gut: how and why the dorsoventral regulatory gene cassette spätzle/toll/ cactus controls the potent antifungal response in drosophila adults a toll-like receptor recognizes bacterial dna cutting edge: recognition of gram-positive bacterial cell wall components by the innate immune system occurs via toll-like receptor trophic factors for the gastrointestinal tract oral insulin increases small intestinal mass and disaccharidase activity in the newborn miniature pig effect of oral insulin on lactase activity, mrna, and posttranscriptional processing in the newborn pig a reagent for the single-step simultaneous isolation of rna, dna and proteins from cell and tissue samples the sequence of development of intestinal tissue injury after strangulation ischemia and reperfusion effect of enteral administration of insulin on intestinal development and feeding tolerance in preterm infants: a pilot study expression of insulin receptors and of -kda receptor substrate in rat mature and immature enterocytes premature stimulation of rat sucraseisomaltase (si) by exogenous insulin and the analog b-asp is regulated by a receptor-mediated signal triggering si gene transcription intestinal and systemic effects of oral insulin supplementation in rats after weaning oral insulin enhances intestinal re-growth following massive small bowel resection in rat beneficial effects of oral insulin on intestinal recovery following ischemia-reperfusion injury in rat oral arginine reduces gut mucosal injury caused by lipopolysaccharide endotoxemia in a rat toll-like receptor- is required for intestinal response to epithelial injury and limiting bacterial translocation in a murine model of acute colitis toll like receptor- expression in lipopolysaccharide induced lung inflammation toll-like receptor , nitric oxide, and myocardial depression in endotoxemia obstructive jaundice increases sensitivity to lipopolysaccharide via tlr upregulation: possible involvement in gut-derived hepatocyte growth factorprotection of hepatocytes decreased expression of toll-like receptor- and md- correlates with intestinal epithelial cell protection against dysregulated proinflammatory gene expression in response to bacterial lipopolysaccaride physical contact between lipopolysaccharide and toll-like receptor revealed by genetic complementation mechanisms of endotoxin tolerance in human intestinal microvascular endothelial cells key: cord- -idffrnac authors: xiang, meng; fan, jie title: pattern recognition receptor–dependent mechanisms of acute lung injury date: - - journal: mol med doi: . /molmed. . sha: doc_id: cord_uid: idffrnac acute lung injury (ali) that clinically manifests as acute respiratory distress syndrome is caused by an uncontrolled systemic inflammatory response resulting from clinical events including sepsis, major surgery and trauma. innate immunity activation plays a central role in the development of ali. innate immunity is activated through families of related pattern recognition receptors (prrs), which recognize conserved microbial motifs or pathogen-associated molecular patterns (pamps). toll-like receptors were the first major family of prrs discovered in mammals. recently, nacht–leucine-rich repeat (lrr) receptors and retinoic acid–inducible gene–like receptors have been added to the list. it is now understood that in addition to recognizing infectious stimuli, both toll-like receptors and nacht-lrr receptors can also respond to endogenous molecules released in response to stress, trauma and cell damage. these molecules have been termed damage-associated molecular patterns (damps). it has been clinically observed for a long time that infectious and noninfectious insults initiate inflammation, so confirmation of overlapping receptor-signal pathways of activation between pamps and damps is no surprise. this review provides an overview of the prr-dependent mechanisms of ali and clinical implication. modification of prr pathways is likely to be a logical therapeutic target for ali/acute respiratory distress syndrome. (tlrs - and tlrs - ) in mice have been defined ( ) . tlrs , , and are expressed intracellularly, whereas tlrs , , , , and are expressed on the cell surface. tlrs are expressed on a range of immune cells including macrophages, dendritic cells, b cells and certain types of t cells, as well as on certain nonimmune cells, such as endothelial cells, smooth muscle cells and epithelial cells that lie at potential sites of entry, including the skin and the respiratory, intestinal and genitourinary tracts. the expression of tlrs is modulated by activation, matu-ration or differentiation of the different cell types ( , ) . tlr proteins are a family of type i transmembrane receptors characterized by an nh -terminal extracellular leucine-rich repeat (lrr) domain, which mediate the recognition of their respective pamps, and a cooh-terminal intracellular tail containing a conserved region called the toll/interleukin (il- ) receptor (tir) homology domain. the tir domain is the defining motif of the tlr/il- superfamily, and it is likely to be one of the earliest signaling domains to have evolved ( ) . tlrs can recognize a diverse range of pamps, generate inflammatory signals to coordinate innate immune responses and modulate adaptive immune responses. the list of tlr ligands is growing. however, the ligand for tlr and mouse tlr remains unknown at present. activation of tlrs initiates two major pathways: the myd -dependent pathway, which is used by all tlrs except tlr , resulting in the activation of nuclear factor (nf)-κb and activator protein- (ap- ); and the trif-dependent pathway, which is initiated by tlr and tlr , resulting in the activation of type i interferons (ifns) ( , , ) . expression of numerous proinflammatory cytokines, such as tumor necrosis factor (tnf)-α, il- , il- and ifns, is one of the major outcomes of the activation of the pathways ( ) . tlr signaling is summarized and shown in figure . rlrs as dexd/h-containing rna helicases are expressed in the cytoplasm in a variety of cells, including immune and nonimmune cells. unlike membranebond tlr , tlr and tlr , which are localized on the endosome and recognize viral double stranded rna, singlestranded rna and dna, respectively, rlrs are cytoplasmic proteins that recognize viral rna produced as a consequence of viral replication ( , ) . rlrs consist of three family members: rig-i, melanoma differentiation-associated gene (mda ) and laboratory of genetics and physiology (lgp ) ( , ) . role of prrs in mediating inflammation and organ injury. infection causes pamp release, but also causes tissue and cell damage and subsequent damp release. similarly, injury caused by trauma or various other factors not only leads to damp release but also renders the patient more susceptible to infection and therefore pamp release. in turn, the pamps and damps act through prrs, which include tlrs, nlrs and rlrs, to activate the innate immune system, yet they can also contribute to persistent and deleterious systemic inflammation and organ injury, including ali. structurally, rig-i and mda contain a dexd/h box rna helicase domain and two caspase-recruiting domain (card)like domains required for eliciting downstream signaling pathways ( , ) . the c-terminal region of rig-i contains a repressor domain (rd), which inhibits downstream signaling. the mda c-terminal region is similar to the rd of rig-i; however, its function is not clear. lgp contains a dexd/h helicase domain and an rd, but lacks the card-like region. lgp was suggested to play an inhibitory role in virus-induced response, because the lgp rd binds the rig-i rd and suppresses signaling as a consequence of interfering with the self-association of rig-i ( , , ) . rig-i is essential for the recognition of a series of rna viruses, which include sendai virus, newcastle disease virus, influenza virus, vesicular stomatitis virus and japanese encephalitis virus ( ) . mda is required for the recognition of other rna viruses, including picornaviruses such as encephalomyocarditis virus, mengo virus and theiler virus ( , ) . thus, rig-i and mda have specificities in their detection of rna viruses, presumably through recognition of distinct structures of viral rna ( , ) . recent studies revealed a pathway of rlr regulation of nf-κb. rig-i/mda card domains, through a card-containing adaptor protein, ifnβ promoter stimulator , also known as mitochondrial antiviral signaling protein, and card adaptor inducing ifnβ, ultimately activate irf and nf-κb ( , ) . the role of rlrs in the mechanism of ali has not been elucidated. the nlr family is a group of recently identified cytoplasmic prrs that contain more than members in humans ( ) . the major role of nlrs is to recognize cytoplasmic microbial pamps and/or endogenous danger signals and initiate immunological responses, although the physiological function of most nlrs is poorly understood at present ( ) . members of the nlr family are categorized into at least five subfamilies according to their n-terminal structure, including nods (nucleotidebinding oligomerization domain- ), nalps (nacht-, lrr-and pyrindomain-containing proteins), ipaf (ice-protease activating factor), naips (neuronal apoptosis inhibitor factors) and class ii transactivator (ciita) ( ) . the nlr family shares a domain organization consisting of a c-terminal lrr domain, a central nucleotide-binding nacht domain, and an n-terminal protein-protein interaction domain composed of a card, pyrin domain (pyd) or baculovirus inhibitor of apoptosis repeat (bir) domain ( ) . nods and ipaf contain card effector domains, whereas nalps have pyd domains, and naips possess bir domains. the functions of nod , nod , ipaf and nalp are more studied. nod and nod are the first nlrs that are reported (tlr in association with tlr or tlr ), tlr , tlr and tlr are localized on the cell surface for ligand recognition. tlr , tlr and tlr are localized in the endosome for ligand recognition in the lumen of endosome. all tlrs, except tlr , recruit myd , and tlr , tlr , tlr and tlr recruit the additional adaptor tirap, which links the tir domain with myd . tlr and tlr recruit trif. tlr requires the additional linker adaptor tram, which links the tir domain of tlr with trif. stimulation of the cells with tlr , tlr , tlr , tlr and tlr ligands initiates the myd -dependent pathway whereas tlr ligands initiate the trif-dependent pathway. tlr activates both myd -dependent and trif-dependent pathways. in the myd dependent pathway, myd recruits the irak family of proteins and traf . in turn, traf activates tak . the activated tak activates the ikk complex, which activates nf-κb subunits. the activated tak also activates the mapk pathway. in the trif-dependent pathway, trif interacts with rip and traf . activated traf and rip activate nf-κb and mapks. trif also interacts with traf and activates tbk /ikki, which activate irf and irf . cells stimulated with tlr and tlr ligands activate nf-κb and mapks via the myd -dependent pathway. to induce type i ifns, myd associates with the irak family of proteins. irak and ikkα activate irf . irak also interacts with traf and activates irf . the activated nf-κb subunits and irfs are translocated to the nucleus. nf-κb and mapks initiate the transcription of inflammatory cytokine genes whereas irfs initiate the transcription of type i interferons. (the figure is adapted from [ ] and used with permission from elsevier.) to have a direct function as prrs in the recognition of peptidoglycan (pgn)derived peptides. nod senses γ-d-glutamyl-meso-diaminopimelic acid (that is,-dap) that is derived from gramnegative bacteria ( ) , whereas nod senses muramyl dipeptide, which is from both gram-positive and gram-negative bacteria ( , ) . when nods bind with pgn-derived peptides, they rapidly form oligomers, which lead to the recruitment of the receptor-interacting protein (rip ) kinase through card-card interactions ( ) . this complex of nod -rip or nod -rip then recruits the inhibitor of nk-κb kinase complex (ikk), leading to the activation of nf-κb. activation nod and nod can also initiate an mapk pathway that leads to the activation of p and erk. in addition, nod signaling can activate jnk as well ( ) . nalp proteins are characterized by the presence of an n-terminal pyrin effector domain ( ) . several nlrs, namely nalp , nalp and nalp , have an important role in activation of proinflammatory caspases through formation of inflammasome ( , ) . the inflammasome is a multiprotein complex of more than kda that is responsible for the activation of caspases and , leading to the processing and secretion of the proinflammatory cytokines il- β and il- ( , ) . martinon et al. have presented different caspase activation platforms in which different components constitute the various inflammasomes ( ) . two types of nalp inflammasome are better studied: the nalp inflammasome that is composed of nalp , the adaptor protein asc, caspase- and caspase- ( ) , and the nalp /nalp inflammasome that contains nalp or nalp cardi-nal, asc and caspase- ( ) . tlrs - are expressed in lung tissue ( ) , and individual tlrs are differentially regulated in specific lung cell populations in response to microbial stimula-tion. tlr , tlr , tlr and tlr are the most likely to be involved in recognition of bacteria in the lungs ( ) ( ) ( ) . a study by bernard et al. has shown that ali/ ards induced by lipopolysaccharide (lps) is a major cause of mortality among humans ( ) . the lps membrane receptor complex is composed of several accessory molecules, which include phosphatidylinositol-anchored cd , tlr , md and md ( ) . the lpsbinding protein (lbp) enhances the binding of lps to its receptor ( ) . absence of cd , md or lbp abrogates most lps responses ( , ) . expression of functional tlr has been found in many cell types in the lung ( ) , and lps-induced lethal shock and ali have been shown to be tlr dependent ( ) ( ) ( ) . thus, tlr plays a critical role in the mechanism of infection-related ali. a recent study has shown that respiratory infections in the human lung initiated by tlr agonist lipoteichoic acid (lta, a component of gram-positive bacteria) and tlr agonist lps (a component of gram-negative bacteria) exhibit different inflammatory responses ( ) . the study was performed on healthy subjects with lps or lta instillation into the contralateral lung. alveolar macrophages (amφ) isolated from bronchoalveolar lavage fluid were analyzed by multiplex ligation-dependent probe amplification. the results show that whereas both lps and lta elicited neutrophil recruitment, only lps instillation was associated with activation of neutrophils (pmn) (cd b surface expression and degranulation) and consistent rises of chemokine/cytokine levels. moreover, lps but not lta activated am, as reflected by enhanced expression of proinflammatory mediators and increased spontaneous cytokine release upon incubation ex vivo. remarkably, only lta induced c a release. these data suggest that stimulation of tlr or tlr results in differential pulmonary inflammation, which may be of relevance for understanding the differences during gram-positive and gram-negative respiratory tract infection ( ) . pulmonary endothelium is a major component of the alveolar-capillary unit, and is susceptible to injury from noxious agents that are either inhaled or delivered to the lung through the pulmonary circulation ( ) . in ali, pulmonary endothelium plays a major role by (a) altering metabolic activity to affect pulmonary and systemic homeostasis, (b) mediating polymorphonuclear pmn adhesion to promote pmn infiltration, (c) changing pmn barrier permeability to cause pulmonary edema and (d) secreting cytokines and chemokines to induce lung inflammation ( ) . a recent study by andonegui and colleagues has shown that endothelial cells (ecs) are the key sentinel cells for detecting infection by gram-negative bacteria and recruiting pmn to peripheral tissues ( , ) . indeed, previous studies have shown that direct activation of circulating pmn with lps is not sufficient to induce their sequestration within the lung ( ) . interactions of pmn with ecs seems important for the process of pmn sequestration into the lung ( , ) . lps stimulates the cd and tlr complex, which in turn activates nf-κb ( ) and increases the expression of adhesion molecule e-selection, intercellular adhesion molecule- (icam- ) and vascular cell adhesion molecule ( , ) . the tlr signaling also leads to production and release of various bioactive molecules, including il- β, il- , tnf-α, chemokines and nitric oxide ( ) , all of which are actively involved in the development of ali. more importantly, tlr signaling can also upregulate other tlrs, such as tlr , and thus amplify the inflammatory responses. although tlr is predominantly expressed in the first-line host defense cells (monocytes, macrophages, dendritic cells and pmn) ( , ) , its expression is low in ecs and epithelial cells ( ) . studies from our laboratory showed that lps through tlr -and myd -dependent signaling induced tlr upregulation in ecs ( ) . we have demonstrated that tlr signaling, through activating nf-κb, upregulates tlr expression in ecs, and this process is enhanced by oxidant signaling generated by pmn nad(p)h oxidase. the functional relevance of nad(p)h oxidase in mediating tlr -induced tlr expression in ecs is evident by markedly elevated and stable icam- expression as well as augmented pmn migration in response to sequential challenge with lps and peptidoglycan ( ) . thus, tlr activation, signaled by tlr and as regulated by pmn nad(p)h oxidase, is an important mechanism responsible for amplifying pmn transmigration to sites of infection ( figure ). the tlr -tlr interaction suggests a highly coordinated, oxidant-mediated upregulation of tlr in response to lps. when one considers the interactions of the innate immune system as microbes are first encountered, the value of such temporal organization is significant. for example, gram-negative bacteria persist in tissues and, if they are not immediately killed through the activation of pmn, complement and other antimicrobial factors, they may spill out systemically and result in septic shock. survival in the face of such infections depends on the innate immune system, which must be able to monitor and respond to pathogens over a prolonged period of time. given the need for a prolonged response to bacterial infection, it has always seemed somewhat surprising that response to lps is temporally finite. this endotoxin tolerance means that within hours after exposure to lps, innate immune cells are incapable of responding again to a rechallenge ( ) . but it is now clear that as lps sensitivity wanes, the immune system has at its disposal the capability of marshaling responses via oxidative metabolites and their ability to upregulate other tlrs ( ) . the subsequent means of responding to bacteria depend on the ability of the innate immune system to destroy microbes and enhance the release of alternative immune stimuli. the tlrs that are utilized are the ones that bind the constituents of degrading bacteria, such as lipopeptides, pgn, heat shock proteins and cpg dna. it seems plausible that activated pmn may even alter the phenomenon of lps tolerance, at least in a localized context, by setting into action a positive feedback loop at sites to which pmn are chemoattracted ( ) . this would enhance inflammatory responses locally and help fight infection. however, in a setting of posttrauma sirs or ali, the primed pmn activation serves as an amplifier to cause enhanced pmn infiltration and organ injury. extensive pmn influx into the lungs is one of the characteristics of ali. studies have shown that excessive induction of proinflammatory cytokines in pmn and delayed pmn apoptosis are associated with higher mortality and more severe organ dysfunction in sepsis patients ( , ) . human pmn express all tlr mrna except tlr . tlr are more abundant than tlr on pmn ( , ) . pmn recruitment to the lung after lps inhalation is primarily dependent on tlr -nf-κb signaling ( , ) . the pi k/akt pathway was also reported to be involved in tlr -induced expression of il- β, tnf-α and chemokine macro phage inflammatory protein (mip)- ( ) . we have reported that tlr , through regulating g-protein-coupled receptor kinases (grks), promotes pmn migration. we demonstrated that mip- induces grk and grk expression in pmns through phosphoinositide- -kinase (pi k)-γ signaling, and lps-activated signaling through the tlr pathway transcriptionally downregulates the expression of grk and grk in response to mip- . the reduced expression of grks lowers chemokine receptor desensitization and markedly augments the pmn migratory response. these data indicate that tlr modulation of pmn surface chemokine receptor expression after the downregulation of grk and grk expression is a critical determinant of pmn migration ( ) (figure ) . a recent study explored a novel role of mtor complex (mtorc ) in tlr and tlr -induced pmn activation ( ) . administration of rapamycin, an inhibitor of mtorc , decreased the severity of lung injury after intratracheal lps or pam (a tlr ligand) administration, as determined by diminished neutrophil accumulation in the lungs, reduced interstitial pulmonary edema, and diminished levels of tnf-α and il- in bronchoalveolar lavage fluid. these results indicate that mtorc activation is essential in tlr -and tlr -induced pmn activation, as well as in the development and severity of ali. the e ubiquitin ligase cblb has a crucial role in the prevention of chronic inflammation and autoimmunity. however, a recent study showed that cblb also has an unexpected function in acute lung inflammation ( ) . cblb attenuates the sequestration of pmn in the lungs after administration of lps. in a model of polymicrobial sepsis in which acute lung inflammation depends on tlr , the loss of cblb expression accentuates acute lung inflammation and reduces survival. cblb controls the association between tlr and the intracellular adaptor myd . expression of wt cblb, but not expression of a cblb mutant that lacks e ubiquitin ligase function, prevents the activity of a reporter gene for nf-κb in monocytes that have been challenged with lps. the downregulation of tlr expression on the cell surface of pmn is impaired in the absence of cblb. these data reveal that cblb regulates the tlr mediated acute inflammatory response that is induced by sepsis ( ) . pmn apoptosis is a crucial injurylimiting mechanism of inflammatory resolution. circulating pmn undergo constitutive apoptosis that results in the shutdown of secretary capacity and allows pmn recognition and removal by macrophages ( , ) . several inflammatory agents, such as lps, tnf, il- , il- , il- and granulocyte colony-stimulating factor (g-csf), can delay apoptotic response, providing pmn with a longer life span, which in turn allows the pmn to accumulate at local tissue sites of inflammation/infection ( , ) . protein (p ) is a transcription factor that is important in multicellular organisms, where it regulates the cell cycle and promotes apoptosis. modulation of p by nutlin- α diminished the response of pmn and macrophages to stimulation through tlr or tlr as well as attenuated lps-induced ali. nf-κb has been reported as a modulator of apoptosis in inflammatory cells ( ) . p can negatively regulate nf-κb activity by decreasing binding of nf-κb to the promoters of genes for proinflammatory cytokines. in p -/mice, the inflammatory process and severity of ali in response to lps are enhanced ( ) . amφ account for approximately % of airspace leukocytes ( ) . tissue damage induced by lps is mediated mainly by inflammatory products released from amφ ( , ) , thus activated amφ play a critical role in the development of ali ( ) . lps inhalation induces amφ to produce and release inflammatory mediators tnf-α, il- β and mip- in a tlr dependent manner ( ) , which further result in the recruitment of pmn into the lower respiratory tract and activate other cell types, including epithelia and endothelia ( ) . tlr is constitutively expressed in amφ, and tlr can be induced in response to lps or proinflammatory cytokines. the inducible tlr expression might be important in responding to other bacterial components from grampositive bacteria ( ) . the role of cd in the regulation of lps-tlr signaling in macrophages has recently been reported ( ) . cd is a transmembrane adhesion molecule and hemopoietic cd has an essential role in hyaluronan clearance and resolution of noninfectious lung injury. following intratracheal lps treatment, cd -/mice demonstrated an exaggerated inflammatory response characterized by increased inflammatory cell recruitment, elevated chemokine expression in bronchoalveolar lavage fluid and a marked increase in nf-κb dna-binding activity in lung tissue in vivo and in macrophages in vitro. furthermore, cd -/mice were more susceptible to lps-induced shock. the study further found that the induction of the negative regulators of tlr signaling il- r-associated kinase-m, toll-interacting protein and a by intratracheal lps in vivo and in macrophages in vitro was significantly reduced in cd -/mice. collectively, these data suggest that cd plays a role in preventing exaggerated inflammatory responses to lps by promoting the expression of negative regulators of tlr- signaling ( ). impairment of the alveolar epithelial barrier is important in the development of ali. under physiologic conditions the epithelial barrier is less permeable than the endothelial barrier; thus, destruction of epithelial barrier integrity prompts a progressive influx of protein-rich fluid into the alveoli ( ) . on the other hand, the loss of epithelial integrity represents an impairment of physiologic transepithelial fluid transport and further inhibits the reabsorption of alveolar edema ( ) . tlrs are critical for airway epithe-lial cell recognition of inhaled pathogens and for innate immune signaling. in cultured human lung epithelial cells, mrna of all tlrs has been detected ( , ) . tlr , tlr , tlr and tlr have the highest expression, and the ligands for these tlrs increased il- and vascular endothelial growth factor (vegf) production in normal human bronchial epithelial cells ( ) . tlr is a heterodimer with tlr and tlr , and each of these is present on the airway epithelial surface. tlr , which recognizes dsrna or poly(i:c), is located in endosomes in unstimulated human bronchial epithelial cells ( ) . tlr is also present on the airway epithelial surface where it can interact with epidermal growth factor receptor (egfr). studies have shown that tlr ligands stimulated il- and vegf production via egfr and the downstream signaling that might include map kinases and nf-κb ( , ) . interestingly, heat shock proteins (hsp), such as hsp and hsp , appear to be intimately involved in the recognition of lps. extracellular hsp released from virally infected airway epithelial cells induces il- express in human bronchial epithelial cells, resulting in the recruitment and activation of pmn via tlr ( , ) . type ii alveolar epithelial cells can also be activated by lps mediated through tlr signaling and in turn promote pulmonary inflammatory processes ( , ) . a study by togbe et al. of whether tlr gene dosage contributes to infection has demonstrated that overexpression of tlr augmented an lps-induced bronchoconstrictive effect, as well as tnf-α and cxc chemokine ligand (keratinocyte-derived chemokine) production ( ) . the study further showed that pmn recruitment, microvascular and alveolar epithelial injury with protein leak in the airways, and damage of the lung microarchitecture were dependent on tlr gene dose. therefore, the tlr expression level determines the extent of acute pulmonary response to inhaled lps, and tlr may thus be a valuable target for immunointervention in acute lung inflammation as a result of infection. in addition to the tlrs, nlrs are critically involved in the sensing of bacterial pathogens. nod senses diaminopimelic acid-containing peptidoglycan present in gram-negative bacteria, whereas nod senses the muramyl dipeptide present in most organisms. because of the apparent lack of direct effects on cell signaling induced by activators of nlrs, it is suggested that their role in pathogen sensing is one of cooperation with the tlrs ( ) . however, studies suggested that the actions of nod vary between cell types and, unlike those seen with lps, the in vivo effects may be independent of leukocyte activation. for instance, cartwright and colleagues have shown that although selective activation of nod in macrophages has no apparent effect, in vascular cells nod activation results in the profound induction of nosii and shock in vivo ( ) . nod is thought to be important in the maintenance of a healthy gut barrier because individuals who carry a defective nod have an increased risk of crohn disease and other intestinal disorders ( ) . although the importance of tlr family in sensing pathogens is well recognized, it is also plausible that they may function in noninfectious diseases because tlr expression is also regulated in conditions other than infection. indeed, growing evidence has shown that prrs play a central role in the mechanisms of noninfectious ali. several tlrs not only have the ability to recognize more than one ligand, but often recognize ligands with completely different chemical structures. such tlr activation does not occur under normal circumstances but only when there is a change in the environment that either leads to the release of endogenous ligands from a cellular compartment, or leads to the modification of endogenous mediator that gives them the ability to activate tlrs ( , ) . because of the association of many endogenous ligands with tissue injury, the nomenclature of damps has been suggested. the best characterized damps include those products released from cells in response to stress or undergoing abnormal death, including hsp , hsp , the extra domain a of fibronectin, oligosaccharides of hyaluronic acid and high-mobility group box (hmgb ). most of these ligands act as agonists of tlr or tlr , or both receptors ( ) . resuscitated hemorrhagic shock (hs) often promotes the development of lung injury by priming the immune system for an exaggerated inflammatory response to a second, often trivial, stimulus, the so-called "two hit hypothesis" ( ) . we used a simplified animal model of the two-hit paradigm to address the mechanisms of hs-primed pmn migration and lung inflammation ( ) . in this model, animals are subjected to a nonsevere resuscitated hs (hypotension at mmhg for hour, followed by a small dose of intratracheal lps. although neither shock nor lps alone induces injury, the combination caused lung pmn accumulation and increased i-albumin transpulmonary flux. findings from this model have suggested that the mechanisms underlying the priming of pmn and inflammation involve a complicated receptor cross-talk process and interaction between pmn and amφ, which is described below. we demonstrated that lps-tlr signaling upregulates tlr expression in amφ, and hs-activated pmn play a critical role in the mechanism of tlr upregulation ( ) . this cross-talk between tlr and tlr in amφ results in the amplification of expression of cytokines and chemokines in response to the bacterial products lps and pgn, and subsequently leads to enhanced pmn sequestration in the lung. these findings reveal a novel mechanism underlying hsprimed lung injury, namely that hs-activated pmn that were initially sequestered into the alveoli can instruct amφ to upregulate tlr , thereby sensitizing amφ to tlr ligands and promoting enhanced lung inflammation. how does hs-activated pmn enhance tlr upregulation of tlr in amφ? we found that reactive oxygen species (ros) derived from pmn nad(p)h oxidase play an important role in amplifying the tlr upregulation ( ) . studies have also shown that lack of endogenous nad(p)h oxidase in the amφ caused a decrease in tlr expression in response to lps stimulation; however, the decrease was restored when the amφ was coincubated with pmn isolated from wild-type mice subjected to hs. these results indicate that although the endogenous nad(p)h oxidase in amφ is also involved in the signaling, the exogenous oxidants from pmn nad(p)h oxidase are essential for inducing amplified tlr expression in amφ in response to lps. the tlr gene promoter contains multiple binding sites for transcriptional factors, which include nf-κb, ccaat/enhancer binding protein, camp response element-binding protein and stat (signal transducer and activator of transcription) ( ) . of these, nf-κb has been reported to regulate tlr expression in response to cytokines and mycobacterial infection ( , ) . it has been demonstrated that lps-tlr -induced tlr upregulation in amφ is largely mediated through the nf-κb signaling pathway, because the nf-κb inhibitor ikk-nbd significantly decreased lps-induced tlr expression in amφ ( ) . although oxidants are involved in the nf-κb signal transduction pathway ( ) ( ) ( ) , their molecular targets have not yet been defined. the contribution of redox regulation and location of potential redox-sensitive sites within the nf-κb activation pathway are the subjects of controversy ( ) . recently we reported that hmgb /tlr signaling mediates the hs-induced increase in tlr surface expression and decrease in tlr surface expression in the lung as well as in mouse lung vascular ecs (mlvec) ( ) . these alterations in tlr and tlr expression result in hmgb -mediated activation of nad(p)h oxidase and expression of icam- in mlvec that is tlr dependent in the early phase and switches to being tlr dependent in the late phase following hs. more importantly, the hs-induced surface expression of tlr contributes to an enhanced activation of mlvec and augmented pulmonary pmn infiltration in response to the tlr agonist pgn. thus, the study demonstrates a novel mechanism underlying hs-augmented lung inflammation, namely that induction of increased tlr surface expression in lung endothelial cells, which is induced by hs/r and mediated by hmgb activation of tlr signaling, is an important mechanism responsible for ec-mediated inflammation and organ injury following hs ( ) . we have shown that hs-induced pmn nad(p)h oxidase activation is mediated by hmgb -tlr signaling. hmgb was originally defined as a nuclear protein that functions to stabilize nucleosome formation, and it also acts as a transcription factor that regulates the expression of several genes ( ) . hmgb can be secreted by innate immune cells in response to microbial products or other inflammatory stimuli ( , ) . hmgb is also released by injured cells and is known as one of the main prototypes of the emerging damps ( ) ( ) ( ) . hmgb was initially identified as an inflammatory cytokine that is a late mediator of lethality in sepsis ( , ) . however, recent studies suggest that hmgb acts as an early mediator of inflammation, contributing to the development of ali after hemorrhage ( ) , and hepatic injury after liver ischemia-reperfusion ( ) . we found in our study that hs/r activates the tlr -myd -irak signaling pathway through hmgb , and further activates p mapk and akt pathways to initiate pmn nad(p)h oxidase activation. pmn nad(p)h oxidase-derived oxidants, in turn, mediate tlr -tlr cross-talk in amφ and sensitize amφ response to tlr ligands, which act in a positive feedback manner to amplify pulmonary pmn infiltration and inflammation ( ) . we have also addressed a fundamental question regarding how hs globally regulates pmn infiltration in the lungs. we have shown that hs, through alarmin hmgb , induced il- secretion from macrophages in an autocrine and tlr signaling-dependent manner. in turn, il- , through an il- -g-csf-mediated mechanism, induced pmn egress from bone marrow. therefore a sustained and hs-primed migration of pmn was maintained. we have also shown that β-adrenergic-receptor activation by catecholamine of macrophages mediated the hs-induced release of hmgb . these data indicate that hs, a global ischemia/ reperfusion stimulus, regulates pmn mobilization through a series of interacting pathways that include neuroendocrine and both innate and acquired immune systems ( ) . hyaluronan (ha) is a massive sugar polymer in the extracellular matrix. under physiologic conditions, ha exists as a high-molecular-weight polymer (> d) and undergoes dynamic regulation resulting in accumulation of lower molecular weight species ( - kd) after tissue injury. ha fragments can trigger innate immune responses in a manner that overlaps with both grampositive and gram-negative organism recognition pathways ( ) . it has been demonstrated that fragmented ha accumulates during tissue injury ( ) ( ) ( ) . cd is required to clear ha during tissue injury, and impaired clearance of ha results in unremitting inflammation. additionally, fragmented ha stimulates the expression of inflammatory genes by inflammatory cells at the injury site ( ) . recently, jiang et al. demonstrated that ha fragments require both tlr and tlr to stimulate mouse macrophages to produce inflammatory chemokines and cytokines. in a noninfectious lung injury model, mice deficient in both tlr and tlr showed an impaired transepithelial migration of inflammatory cells, increased tissue injury, elevated lung epithelial cell apoptosis and decreased survival ( ) . lung epithelial cell overexpression of high molecular mass ha protected mice against ali and apoptosis, in part through tlr-dependent basal activation of nf-κb. the exaggerated injury in tlr -and tlr -deficient mice appears to be due to impaired ha-tlr interactions on epithelial cells. these studies demonstrate that host-matrix component ha and tlr interactions provide signals that initiate inflammatory responses, maintain epithelial cell integrity and promote recovery from ali ( ) . mechanical ventilation (mv) provides life-saving support for many patients with respiratory failure ( ) . however, mechanical stresses produced by mv can induce lung injury, termed ventilator-induced lung injury ( ) . evidence from animal experimental studies has demonstrated that mv per se can induce inflammatory responses ( ) ( ) ( ) . a recent report has demonstrated that tlr , but not tlr , played a role in development of the inflammatory response after shorttime mv ( ) . mv not only causes ventilator-induced lung injury in healthy animals, but also exacerbates damage in the injured lung ( ) . tlr blockade reduces pulmonary inflammation caused by the combination of lps and mv ( ) . in the mechanism of hyperoxia-induced ali, tlr expression and activation seems impotent. exposure of human epithelial cells to hyperoxia in the absence of an exogenous viral pathogen significantly increased tlr expression. in vivo studies showed that both the absence of tlr via gene deletion in mice and the presence of an anti-tlr antibody in wild-type mice conferred significant protection in a hyperoxia-mediated lung injury model ( ) . the inflammasome is a multiprotein complex that mediates the activation of caspase- , which promotes secretion of the proinflammatory cytokines il- β and il- , as well as pyroptosis, a form of cell death induced by bacterial pathogens. members of the nlr family, including nlrp , nlrp and nlrc , and the adaptor asc are critical components of the inflammasome that link microbial and endogenous danger signals to caspase- activation. the role of nalp in mediating noninfectious ali has been revealed by two recent studies. gasse and colleagues reported that uric acid locally produced in the lung upon bleomycin (blm)-induced dna damage and degradation triggers nalp inflammasome activation, and in turn causes lung injury ( ) . reduction of uric acid levels using the inhibitor of uric acid synthesis allopurinol or uricase leads to a decrease in blm-induced il- β production, lung inflammation, repair and fibrosis. local administration of exogenous uric acid crystals recapitulates lung inflammation and repair, which depend on the nalp inflammasome, myd and il- r pathways and tlr and tlr for optimal inflammation but are independent of the il- receptor ( ) . babelova et al., however, reported the role of biglycan, a ubiquitous lrr proteoglycan of the extracellular matrix, in mediating ali through interacting with tlr and tlr on macrophages ( ) . the study showed that in macrophages soluble biglycan induces the nlrp /asc inflammasome and subsequent activation of caspase- and release of mature il- β without need for additional costimulatory factors. this is caused by the interaction of biglycan with tlr / and purinergic p × /p × receptors, which induces receptor cooperativity. furthermore, ros formation is involved in biglycan-mediated activation of the inflammasome. by signaling through tlr / biglycan stimulates the expression of nlrp and pro-il- β mrna. these results provide evidence for direct activation of the nlrp inflammasome by biglycan and suggest a fundamental paradigm of how tissue stress and injury are monitored by innate immune receptors detecting the release of the extracellular matrix components and turning such a signal into a robust inflammatory response ( ) . susceptibility and response to infectious disease is, in part, heritable. potential associations between clinical outcome from sepsis and many inflammatory cytokine gene polymorphisms, innate immunity pathway gene polymorphisms and coagulation cascade polymorphisms have been observed. we may yet be able to tease out the complex influence of genetic variation on susceptibility and response to infectious disease ( ) . in , arbour and colleagues reported that two polymorphisms of the tlr gene were present in a higher proportion of individuals who are hyporesponsive to inhaled lps ( ) . this finding led to a number of studies investigating the potential impact of these tlr polymorphisms on the course of infectious diseases and the development of septic shock and tlr polymorphisms ( ) ( ) ( ) . these polymorphisms do not seem to confer sus-ceptibility to all gram-negative infections, because other groups ( , ) have shown no correlation in other infectious diseases such as meningococcal disease, and again one should be mindful that very significant hypofunctioning tlr alleles are likely to have a very strong negative selection pressure across the generations ( ) . tlr- polymorphisms have also been linked to susceptibility to staphylococcal infection ( ) and lepromatous leprosy ( , ) . although some of these studies are still relatively small in scale, they reinforce the important role of tlrs in pathogen recognition and immune response in humans. cd polymorphisms, key accessory molecules for tlr signaling, have been associated with increased prevalence of positive bacterial culture findings and sepsis attributed to gramnegative infections in a critically ill population ( ) , as well as the susceptibility to chronic chlamydia pneumoniae infection in patients with coronary artery disease ( ) . the discovery of the importance of prrs in the pathogenesis of sirs and organ injury, including ali, has led to a therapeutic strategy targeting prrs. tlrs are the most extensively studied family of prrs, and thus recently developed new drugs mainly target tlrs and are either agonists of tlrs to enhance immune responses against infectious agents or antagonists designed to reduce inflammation due to infection or autoimmune responses ( ) . the approaches to modulating tlr activity have focused on the following aspects: (a) ligands or analogues such as eritoran (e ) from eisai (woodcliff lake, nj, usa), a synthetic analogue of bacterial lipid a that inhibits lps from activating cells through the tlr /cd /md complex ( ) ( ) ( ) ; (b) monoclonal antibodies, soluble receptors and other accessory proteins, such as a natural soluble form of tlr , found in mouse plasma and breast milk, which acts to block tlr ligand stimulation ( ) , and a member of the tlr/il- receptor family (tir or sigirr) that inhibits nf-κb signaling and may be an endogenous inhibitor of the tlr system ( ); (c) signal transduction blockers; many of the key molecules in the signaling pathways for each tlr have been identified and are considered potential drug targets ( ) ( ) ( ) . the structural bases of tir domain interactions between tlrs and adapters such as myd , mal, tram and trif have been modeled, and small peptidic sequences based on the tir domain bb loop or peptidomimetics of this region have been made that can block the interactions ( , , ) . (d) sirna and antisense; studies with knockout mice suggest that deficiency in individual tlrs has limited consequences for animals under normal conditions, but exhibits impact under conditions of specific infectious challenge ( , , ) . however, sirna sequences themselves may be ligands for intracellular tlrs ( ) ; thus attention to the design of appropriate sequences is necessary. drugs targeting tlrs have not yet been clinically applied to the treatment of ali, but because of the critical role of prrs in the development of ali, targeting of prrs has opened up a productive area for the therapy of ali. current pharmacotherapy has not been highly successful in increasing patient survival in cases of ali/ards. since prrs were recognized, their significance in the mechanisms of ali has been quickly identified. the combined activation of these different receptors may result in complementary, synergistic or antagonistic effects that modulate the process of ali. therefore, modification of prr pathways is likely to be a logical therapeutic target for ali/ards. however, a complete understanding of the role of prrs in the mechanism of ali requires further "decoding" of these multiple receptor interactions. this work was supported by the national institutes of health grant r -hl- , national institutes of health center grant p -gm- , and a va merit award. the authors declare that they have no competing interests as defined by molecular medicine, or other interests that might be perceived to influence the results and discussion reported in this paper. neutrophils and adult respiratory distress syndrome: two interlocking perspectives in early predictors of postinjury multiple organ failure acute respiratory distress in adults multiple organ failure in polytrauma patients adult respiratory distress syndrome: risk with common predispositions the formation of a structure with the features of synovial lining by subcutaneous injection of air: an in vivo tissue culture system recent 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inflammation and fibrosis biglycan: a danger signal that activates the nlrp inflammasome via toll-like and p x receptors bench-tobedside review: association of genetic variation with sepsis tlr mutations are associated with endotoxin hyporesponsiveness in humans relevance of mutations in the tlr receptor in patients with gram-negative septic shock human toll-like receptor mutations but not cd polymorphisms are associated with an increased risk of gram-negative infections polymorphisms in tolllike receptor (tlr ) are associated with protection against leprosy a functional polymorphism of toll-like receptor is not associated with likelihood or severity of meningococcal disease variation in toll-like receptor and susceptibility to group a meningococcal meningitis in gambian children reducing the toll of inflammatory lung disease a novel polymorphism in the toll-like receptor gene and its potential association with staphylococcal infection cutting edge: a toll-like receptor polymorphism that is associated with lepromatous leprosy is unable to mediate mycobacterial signaling toll-like receptor (tlr ) polymorphisms are associated with reversal reaction in leprosy polymorphisms in cd , mannose-binding lectin, and toll-like receptor- are associated with increased prevalence of infection in critically ill adults cd promoter polymorphism - c>t is associated with susceptibility to chronic chlamydia pneumoniae infection in peripheral blood monocytes targeting toll-like receptors for drug development: a summary of commercial approaches lipoprotein distribution of a novel endotoxin antagonist, e , in plasma from human subjects with various lipid levels continuous pharmacodynamic activity of eritoran tetrasodium, a tlr antagonist, during intermittent intravenous infusion into normal volunteers tlr antagonists for endotoxemia and beyond soluble forms of tolllike receptor (tlr) capable of modulating tlr signaling are present in human plasma and breast milk si-girr inhibits interleukin- receptor-and tolllike receptor -mediated signaling through different mechanisms identification of critical residues of the myd death domain involved in the recruitment of downstream kinases pivotal advance: inhibition of myd dimerization and recruitment of irak and irak by a novel peptidomimetic compound peptide-mediated interference of tir domain dimerization in myd inhibits interleukin- -dependent activation of nf-{kappa}b the family of five: tir-domain-containing adaptors in tolllike receptor signalling cutting edge: role of toll-like receptor in mediating immune response to microbial lipoproteins cutting edge: tlr -deficient and myd -deficient mice are highly susceptible to staphylococcus aureus infection activation of the mammalian immune system by sirnas key: cord- - tq p z authors: wang, ting; yegambaram, manivannan; gross, christine; sun, xutong; lu, qing; wang, hui; wu, xiaomin; kangath, archana; tang, haiyang; aggarwal, saurabh; black, stephen m. title: rac nitration at y is involved in the endothelial barrier disruption associated with lipopolysaccharide-mediated acute lung injury date: - - journal: nan doi: . /j.redox. . sha: doc_id: cord_uid: tq p z acute lung injury (ali), a devastating illness induced by systemic inflammation e.g., sepsis or local lung inflammation e.g., covid- mediated severe pneumonia, has an unacceptably high mortality and has no effective therapy. ali is associated with increased pulmonary microvascular hyperpermeability and alveolar flooding. the small rho gtpases, rhoa and rac are central regulators of vascular permeability through cytoskeleton rearrangements. rhoa and rac have opposing functional outcome: rhoa induces an endothelial contractile phenotype and barrier disruption, while rac stabilizes endothelial junctions and increases barrier integrity. in ali, rhoa activity is increased while rac activity is reduced. we have shown that the activation of rhoa in lipopolysaccharide (lps)-mediated ali, is dependent, at least in part, on a single nitration event at tyrosine (y) . thus, the purpose of this study was to determine if the inhibition of rac is also dependent on its nitration. our data show that rac inhibition by lps is associated with its nitration that mass spectrometry identified as y , within the switch i region adjacent to the nucleotide-binding site. using a molecular modeling approach, we designed a nitration shielding peptide for rac , designated nipr (nitration inhibitor peptide for the rho gtpases ), which attenuated the lps-induced nitration of rac at y , preserves rac activity and attenuates the lps-mediated disruption of the endothelial barrier in human lung microvascular endothelial cells (hlmvec). using a murine model of ali induced by intratracheal installation of lps we found that nipr successfully prevented rac nitration and rac inhibition, and more importantly attenuated pulmonary inflammation, reduced lung injury and prevented the loss of lung function. together, our data identify a new post-translational mechanism of rac inhibition through its nitration at y . as nipr also reduces sepsis induced ali in the mouse lung, we conclude that rac nitration is a therapeutic target in ali. the current pandemic of covid- has caused over a hundred and sixty thousand deaths in usa, mainly due to a severe form of lung inflammation and edema, called acute lung injury (ali) or its severe form acute respiratory distress syndrome (ards) [ ] . ali and ards represent the same disease spectrum characterized by hypoxemic respiratory insufficiency from pulmonary edema [ ] . the acute lung inflammation and increased vascular hyperpermeability associated with ali can be caused by systemic inflammation such as sepsis, or lung inflammation such as severe pneumonia induced by covid- or other bacteria or viruses [ ] . besides supportive care with low tidal volume mechanical ventilation, general anti-inflammatory steroids, and extracorporeal membrane oxygenation (ecmo) in the intensive care unit (icu), there are no specific drug therapies for ali/ards, which still has an unacceptably high mortality of ~ % [ ] . thus, a devastating demand for effective therapies for ali/ards exists. respective primary antibody in blocking buffer, and the secondary antibody in blocking buffer after three time of wash in tbst. reactive bands were visualized using chemiluminescence (pierce) using either a kodak cf image station or a li-cor biosciences odyssey imaging system. band intensity was quantified using either kodak d image processing software or image studio software version . hlmvec were homogenized in pierce ip lysis buffer (thermo-fisher). after centrifugation, the supernatant was collected and mixed with a/g agarose beads (emd/calbiochem) with the pull-down antibody for h at ºc. the beads were collected after centrifugation at , rpm for min and washed times with ip wash buffer (thermo-fisher). the beads were then boiled with x laemmli buffer for min. proteins were separated on % gels and transferred to pvdf membrane and analyzed using western blot methods described above. in another experiment, immunoprecipitated proteins in gel stained with coomassie blue dye were digested with chymotrypsin and used for mass spectrometry. rac activity was measured using the rac pull-down activation assay biochem kit (cytoskeleton inc.). briefly, hlmvec and mouse lung tissue were lysed using the cell lysis buffer and the lysate with pak-pbd beads was incubated at °c on a rotator for h. the pak-pbd beads were centrifuged at , g and washed with wash buffer. equal j o u r n a l p r e -p r o o f volume of x laemmli sample buffer was added to each tube and boiled for min. samples were analyzed by sds-page and western blot analysis as described above. all spectra were taken on an ab sciex maldi-tof-tof mass spectrometer in positive reflector mode ( kv) with a matrix of chca. masses were calibrated to sciex nitration, we exposed nipr to authentic peroxynitrite ( μm) for min. samples were desalted using a c- ziptip, mixed with chca matrix and were spotted directly onto a maldi plate for further ms and ms/ms analysis. the ability of nipr or nipr f peptides to bind to rac was also analyzed by mixing ng/ml of peptide with ng/ml of human recombinant rac protein for min at rt and using maldi ms linear mode acquisition with sinapinic acid as a matrix. a nitro-y rac specific antibody (rac -y -no ) was raised against a synthetic peptide antigen (llisyttnafpgey no iptvfd), where y-no represents -nitrotyrosine as previously described [ ] . the peptide was used to immunize rabbits. tyrosine nitration-reactive rabbit antiserum was first purified by affinity chromatography. further purification was carried out using immunodepletion using non-nitrated peptide (llisyttnafpgeyiptvfd) resin chromatography, after which the resulting eluate was tested for antibody specificity by immunoblotting and immunoprecipitation followed by mass spectrometry. the rhoa-y -no antibody was generated as previously described [ ] . j o u r n a l p r e -p r o o f his-tagged rac recombinant protein was expressed in e. coli as previously described [ ] . briefly, isopropyl-beta-d-thiogalactopyranoside (iptg, mm) was added and the cells were incubated for - h at °c. bacteria were then harvested by centrifugation and the pellet was immediately lysed in mm tris-hcl, % glycerol, mg/ml lysozyme, mm nacl, protease inhibitor cocktail, ribonuclease a (sigma), and deoxyribonuclease i (sigma). the pellet was gently rocked for minutes, sonicated and subjected to ultracentrifugation. the supernatant was loaded onto a hisprep ff / column (sigma) using binding buffer ( mm tris-hcl, mm nacl, % glycerol, mm imidazole) at . ml/min flow. the column was washed with mm tris-hcl, mm nacl, % glycerol, mm imidazole using a flow rate of . ml/min. elution of the histidine-tagged protein was accomplished using elution buffer ( mm tris-hcl, mm nacl, % glycerol, mm imidazole) at . ml/min flow. collected fractions were loaded for size-exclusion gel filtration on a hiload / superdex column (sigma) using gel filtration buffer ( mm tris-hcl, mm nacl, % glycerol) at . ml/min flow. fractions were collected and analyzed by coomassie blue staining and western blot. all purification steps were performed at °c, and purified protein was stored at - °c. recombinant human rhoa protein was also purified using a bacterial expression system as previously described [ ] . j o u r n a l p r e -p r o o f pulmonary arterial endothelial cells (paec) were cultured as described [ ] . ovine paec, isolated as previously described [ ] , were transduced with a his-tagged rac adenovirus (moi = ) and incubated for h at °c. cells were lysed and the histagged rac protein was purified using hispur ni-nta columns (thermo scientific) and stored at - °c. fractions were analyzed by coomassie blue staining and western blot analysis. peptides containing amino acids (fpgeyiptvf) from aa - of rac fused with the cell permeable tat sequence were synthesized by peptide . inc (chantilly, va). this peptide was designated nipr (nitration inhibitory peptide for the rho gtpases ) and designed to interact with the flap region of the rac protein. a similar phenylalanine-substituted peptide (nipr f) was also synthesized harboring a y f mutation. the electrical resistance of the endothelial cell monolayer was measured with the electrical cell impedance sensor (ecis) technique [ ] . hlmvec were cultured on gold plated electrodes ( e +) until % confluence. the change in electric resistance across the monolayer was measured continuously. the data was normalized to the initial values of basal resistance. in the transwell permeability assay, hlmvec were seeded on a semi-permeable membrane in the upper chamber of the transwell ( μm pore size, bd biosciences, san jose, ca). after appropriate treatments, , mw fitc-dextran j o u r n a l p r e -p r o o f ( μg/μl) (sigma-aldrich) was added to the upper chamber. the amount of fitc-dextran infiltrating into the lower chamber was determined using a fluoroskan ascent fluorometer. the committee on animal research at georgia regents university and university of arizona approved all animal protocols and procedures. in the pre-injury model, male c bi/ mice ( weeks) received vehicle (saline) or mntmpyp ( mg/kg body weight) via an intraperitoneal injection min before intratracheal installation of e. coli :b lps, . x endotoxin units/g body weight, sigma-aldrich). mice were examined h after lps challenge. at the end of the treatment, animals were anesthetized, and the lungs were flushed with ml of ice-cold pbs (to remove blood) gently, and excised. a portion of the lung was quickly snap-frozen in liquid nitrogen and stored at - ºc. in the post-injury model, male c bi/ mice ( - weeks) received vehicle ( . % saline) or e. coli :b lps ( . mg/kg body wt, sigma-aldrich) prepared in . % saline via an intratracheal injection days before administration of nipr or nipr f peptide (intraperitoneal injection, mg/kg). on day (post lps) mice were euthanized, lungs were flushed with ml of ice-cold pbs (to remove blood) gently, and excised. a portion of the lung was quickly snap-frozen in liquid nitrogen and stored at - ºc. the remaining lung tissue was fixed in pbs buffered formalin for histology analysis. j o u r n a l p r e -p r o o f balf was obtained by instilling and withdrawing ml of ice-cold hanks buffer (sigma) via a tracheal cannula. bal fluid was then centrifuged g for min to collect pelleted cells. bal cells were then resuspended in red cell lysis buffer (rclb) for s, centrifuged g for min to remove rclb, and resuspended in µl of pbs. total cell count was then determined using a haemocytometer. cell suspension ( µl) was also separated by cytospin centrifuge (thermo-fisher) g for min onto glass slides. after air dry, the slides were stained with dif-quik staining (vwr) for differential cell count. lungs were inflated with % formalin under cm h o pressure and immersed in the same solution before tissue processing into paraffin-embedded blocks and μm sections were then cut stained with h&e. histopathological assessment was conducted by two researchers who were masked to treatment group. h&e stained sections were scored for the presence of neutrophil in the alveolar space, neutrophils in the interstitial space, the existence of hyaline membranes, proteinaceous debris filling the airspaces and alveolar septal thickening as described previously [ ] . mice were remaining anesthetized with isoflurane ( % with oxygen), tracheostomized with a metal . mm (internal diameter) cannula and connected to a flexi vent (scireq inc) ventilator. ventilation was initiated at a tidal volume of ml/kg and a rate of /min. a tlc maneuver was performed, followed by sec later, by a sinusoidal hz j o u r n a l p r e -p r o o f oscillation. subsequently, an sec forced oscillatory signal ( . - . hz) was applied, the mechanical input impedance of the respiratory system was calculated, and a model containing a constant phase tissue compartment was fit to input impedance in order to evaluate tissue elastance. dynamic pressure-volume maneuvers were performed by stepwise increasing the airway pressure to cm h o and then reversing the process. transcutaneous oxygen saturation were monitored via a small animal pulse oximeter (mouseox plus, starr life sciences corporation, oakmont, pa, usa) by placing the non-invasive sensor on the neck, as previously described [ ] . statistical analysis was performed using graphpad prism version . (graphpad). the mean ± sd or sem was calculated for all samples, and the significance was determined either by the unpaired t-test (for groups) and anova (for > groups). for the anova analyses, newman-kuels post-hoc testing was employed. a value of p < . was considered significant. in cultured hlmvec, lps challenge ( eu/ml) induced significant decrease in rac gtpase activity and this was attenuated by the peroxynitrite scavenger, mntmpyp ( figure a) . utilizing immunoprecipitation analyses we demonstrated that rac is subject to protein nitration and again this is attenuated by mntmpyp ( figure b&c ). these findings suggest that lps-mediated rac nitration is associated with reduced rac gtpase activity. to further confirm the nitration on rac peptide and identify the j o u r n a l p r e -p r o o f nitration site, we first utilized recombinant human rac protein purified using an expression purification system based in e.coli [ ] . purity of rac was determined to be > % using both sds page with coomassie staining (figure a ) and western blot analysis ( figure b ). the purified recombinant human rac protein was exposed to authentic peroxynitrite ( µm, min) and then digested using chymotrypsin endoproteinase. the resulting peptide fragments were subjected to maldi-tof ms and ms/ms analysis. a nitrated peptide ( . da) with sequence ttnafpgey was identified with a single nitration site present on the tyrosine residue. a ms peak with + da in molecular weight difference (equal to nitro group addition) was observed with the nitrated rac peptide fragment, within the region of aa - ( figure c ). further ms-ms analysis confirmed the peptide sequence and the position of the nitro-group as y ( figure c ). we next investigated the nitration of rac in cultured pulmonary arterial endothelial cells (paec). in order to achieve a quantifiable intracellular level of rac , paec were transduced with an adenovirus construct containing a his-tagged human wildtype rac . we confirmed that human rac protein could be purified utilizing a hispur ni-nta column ( figure d ). rac purification was confirmed by sds page with coomassie staining ( figure d ) and the identification of a single band by western blot analysis ( figure e ). transduced paec were then exposed to the peroxynitrite donor, sin- ( µm for h) and western blot analysis utilized to confirm that peroxynitrite did not alter total rac levels in the cells ( figure f ). however, utilizing a nitrotyrosine antibody which specifically binds nitrated tyrosine we were able to see a strong band in western blot at . kda indicating a nitrated rac protein in transduced paec exposed to sin- ( figure g ). the nitrated rac protein was then purified using hispur j o u r n a l p r e -p r o o f ni-nta column and the peptide fragments generated by overnight chymotrypsin digestion were subjected to maldi-tof ms and ms/ms analysis. after exposure to sin- , the same nitration site y was detected in the rac protein isolated from paec ( figure h ). these results indicate that peroxynitrite is capable of inducing rac nitration at a specific site, y both in vitro and in vivo. next, using the known rac protein structure and a previously established computational modeling method [ ] , we simulated the structural impact of y nitration on rac . this modeling predicts that the y containing region (the "flap" next to the switch i region of the catalytic domain) leads to a "close" state in the flap region ( figure i ), compared to the "open" state induced by nitration of y in the flap region of rhoa [ ] ( figure j ). these computational modeling data, coupled with our rac -gtpase activity measurements, ( figure a ) clearly demonstrate that lps-mediated peroxynitrite stress induces rac nitration at y and that this is inhibitory for rac gtpase activity. in order to increase the detection sensitivity for y nitrated rac , we next generated a polyclonal antibody (rac -y -no ) that specifically recognizes y nitrated rac following the protocol from the previous study which used an antibody (rhoa-y -no ) that recognized y nitrated rhoa [ ] . we confirmed that the rac -y -no antibody selectively detects y nitrated rac in recombinant protein exposed to authentic peroxynitrite in-vitro ( figure a ) and in-vivo hlmvec exposed to sin- ( figure b ). further, we validated that rac -y -no and rhoa-y -no antibodies are preferential for nitrated rac and nitrated rhoa respectively ( figure c ). using rac -y -no j o u r n a l p r e -p r o o f antibody we were next able to confirm that there was significant increase in rac nitration in the mouse lung h after i.t. lps challenge, and that again rac nitration was significantly suppressed by mntmpyp ( figure d ). these changes in rac nitration correlated with change in rac gtpase activity ( figure e ). these data demonstrate that nitration of rac at y , is intimately involved in the loss of rac activity in the mouse lung during sepsis-mediated ali. we next aimed to design a peptide which specifically targets the flap region of the rac protein to prevent its nitration at y . docking simulations ( figure a ) were used to design a nitration shielding approach that would bind to the flaps region of rac similar to that used earlier for rhoa [ ] . this resulted in the design of a peptide with sequence hrkkrrqrrrqfpgeyiptvf designated nitration inhibitory peptide for rho gtpases (nipr ). the molecular mass of the nipr peptide of . da was confirmed by maldi ms ( figure b ) and peptide sequence was confirmed by ms/ms ( figure c ). the nipr peptide was then exposed to authentic peroxynitrite ( µm, figure e ). as expected, when the nipr f peptide was exposed to peroxynitrite ( µm, min) there was no mass difference observed in ms and ms/ms spectrum because of absence of the target tyrosine residue required for nitration ( figures d and e) . using maldi ms linear mode acquisition, with sinapinic acid as a matrix, we also confirmed that both nipr and nipr f peptides we able to bind efficiently to intact human recombinant rac ( figure f ). to evaluate the therapeutic potential of nipr , we utilized a mouse model of ali figure a ). as expected, nipr , but not nipr f, significantly attenuated lps-mediated rac nitration ( figure b ) and restored rac gtpase activity ( figure c ). mice were also used to perform additional physiological, biochemical and morphological studies to evaluate the efficacy of the nipr peptide in attenuating symptoms of ali. cell infiltration into the balf was significantly increased in lps treated animals ( figure d ). nipr , but not nipr f, reduced this increase ( figure d ). we also assessed histopathological changes in the lungs. lps induces severe alveolar damage that includes the presence of large numbers of neutrophils and red blood cells in the alveolar and interstitial space, formation of hyaline membranes, septal thickening and debris accumulation in the j o u r n a l p r e -p r o o f alveoli ( figure e ). again, nipr , but not nipr f, reduced these pathological changes ( figure e &f) . we next assessed the effect of nipr on lung function in lps challenged mice. nipr restored oxygen saturation from ~ % to ~ % ( figure a ). using flexivent technology, we also performed respiratory pressure-volume loop (pv loop) measurement and analysis. lps induced a characteristic downward shift of the pv loop, compared to control mice ( figure b ). consistent with the oxygen saturation findings, nipr , but not nipr f, restored the pv loop, indicative of an improvement in respiratory mechanical function. specifically, nipr increased the respiratory maximal volume at highest pressure ( figure c ). nipr also prevented the lps-mediated attenuation of total respiratory compliance ( figure d ) and the increase in respiratory elastance ( figure e ). together these data demonstrate that nipr significantly improved lung function in this murine model of acute lung injury. ali is a complex syndrome with an unacceptable mortality. ali pathogenesis involves disruption of the epithelial and endothelial barriers leading to an increased permeability and decreased edema fluid clearance [ ] . lung barrier permeability is tightly regulated by adherens junctions, tight junctions, and gap junctions. junctional integrity is vital for cell-cell adhesion, actin cytoskeleton remodeling, intercellular signaling, and transcriptional regulation [ ] . rac regulates lung endothelial integrity via cytoskeleton j o u r n a l p r e -p r o o f rearrangements that determine cell shape and junctional integrity of the lumen [ ] . interestingly, within the rho-family small gtpases, rac and rhoa are antagomirs [ ] . rac is involved in the maintenance and stabilization of microvascular endothelial barrier functions (maintains junctional formation and cell relaxation), whereas rhoa primarily acts antagonistically to impair barrier properties [ ] . post-translational modification can directly influence the structure, function and stability of the rho gtpases [ ] . this particular study confirms, for the first time, that rac can be nitrated at y , which leads to a persistent inhibition of gtpase activity. crystal structure analysis of rac indicates that y nmr and x-ray crystal structures for rho family gtpases propose that the cys thiol in the gxxxxgk(s/t)c motif is accessible for solvent and suggest reactive oxygen species and reactive nitrogen species possibly target the cys thiol [ ] [ ] [ ] . further, we have shown that s-nitrosylation of rhoa attenuates the activity of rhoa [ ] . the effect of s-nitrosylation on rac is unresolved but we speculate that as nitration activates while s-nitrosylation inhibits rhoa that s-nitrosylation may stimulate rac activity. however, further studies will be required to test this possibility. besides nitration a number of other post translational modifications have been reported that have the potential to alter its activity/function. these include ubiquitination [ ] [ ] [ ] , phosphorylation [ , ] , and adenylylation (ampylation) [ ] , all of which have all been shown to play important roles in the regulation of rac . interestingly, it has been reported that y in rac is a target for ampylation [ ] . therefore, we speculate that nitration might inhibit phosphorylation and/or ampylation of y in rac which could permanently alter its activity. thus, it is likely that the competition among these various potential modifications likely has both physiological and pathological implications. again, further studies will be required to investigate this. during sepsis-like conditions, lps exposure stimulates the production of peroxynitrite which leads to protein nitration of tyrosine residues, a nonreversible event which often affects protein structure and function. previously we had reported that peroxynitrite j o u r n a l p r e -p r o o f stress induced by lps challenge causes rhoa nitration at y which facilitates its persistent activation [ ] . rac and rhoa, as a balanced control mechanism of lung endothelial integrity, is disrupted by peroxynitrite stress. even though rhoa and rac share structural homology, rhoa is involved in endothelial barrier disruption during the development of ali, while rac is involved in endothelial barrier recovery during the resolution phase of ali. the fact that each protein acts in a different phase of ali could provide a therapeutic window to target rac , instead of rhoa, in the patients in icu, most of whom have passed the earlier phase of endothelial disruption and are most likely in the slow recovery phase of this devastating disease. this possibility is supported by our animal studies in which we have utilized our nipr peptide to target rac nitration in a "reversal"-, instead of "prevention"-, strategy to effectively "treat" experimental ali. our study reveals a new mechanism by which endothelial integrity loss is maintained through nitration-mediated rac inhibition during sepsis-associated ali. importantly, we developed two new research tools in this study, a polyclonal antibody which specifically detects y -nitrated rac , and nipr , a synthetic peptide which prevents rac nitration at y . since rac is wildly expressed in multiple tissues with multidisciplinary function, our nitration specific rac antibody has significant potential as a research tool in a number of pathologic states. further, we validated the nitration shielding potency nipr in both cultured cells and a murine model of sepsis induced ali. thus, nipr , or its next generation, may have clinical utility given the fact that ali still does not have a selective drug therapy. however, this possibility should be tempered by the fact that previous ali drug clinical trials have all failed. we propose that this is due to the fact that ali is a syndrome that results from different insults (e.g., j o u r n a l p r e -p r o o f covid- induced ali is clearly different from trauma induced ali), and the involvement of multiple systems failure such as endothelial barrier disruption, macrophage activation, and leukocyte infiltration. in this study, we report that nipr selectively targets rac nitration and effectively blocks murine lung injury, but the therapeutic efficacy in ali patients is hard to predict. we anticipate that a successful clinical efficacy of nipr or similar product might require: ) precision medicine approach to identify patients in the sub-group with satisfactory responsiveness of rac nitration blockade, as not all triggers of ali (e.g., trauma) will lead to endothelial oxidative stress and peroxynitrite generation; ) combination therapy with other effective reagents, including suppressor of the cytokine storm and/or neutrophil eliminators; ) selective tissue delivery vehicle, to increase therapeutic index. further, we propose that conditional genetically modified in-vivo models will be needed to fully elucidate the biological functions of the rho gtpases in different physiological environments and pathological conditions, and this information will likely be required to develop precise pharmacological inhibitors. lps is a known activator of endothelial ros generation [ , ] . interestingly the generation of ros might be "designed" a result of detoxification of oxidative species in oxygen-rich environment, and sometimes as a signaling cascade, since it can be generated and removed quickly. however, these protective or regulatory signaling pathways can be overwhelmed, leading to "oxidative stress". in the lungs, multiple cell types, including endothelial cells, macrophages, epithelial cells, and infiltrated inflammatory leukocytes, are good ros generators. lps can activate several j o u r n a l p r e -p r o o f endogenous machineries to generate ros including nadph oxidase (nox), uncoupled nos, dysfunctional mitochondria, and xanthine oxidase, all of which have been reported to be involved with the oxidative stress associated with ali [ ] [ ] [ ] [ ] [ ] . the link between increased oxidative stress and lung injury, as well as the success of antioxidant therapy in preclinical models makes ros generation an attractive target in ali. however, the complex roles of ros in ali development and recovery have restricted the efficacy of antioxidant therapy. ros is involved in tissue injury [ ] , barrier disruption [ ] , proinflammatory cytokine production [ ] . while it is also involved in endothelial barrier recovery [ ] , and endothelial adherens junction re-assembly [ ] . endothelial cells have abundant no which is mainly produced by enos, which, upon lps-challenge mediated uncoupling, also starts to generate ros. the downstream effector of nos uncoupling is likely peroxynitrite, formed from the interaction of no with superoxide generated from different sources. we propose that targeting adverse events downstream of ros, such as rac nitration, will be more selective and effective to terminate ros associated adverse outcome, and reduce ali. nitration unlike phosphorylation, which is selectively mediated by kinases and phosphatases, and modulated by signaling cascades regulating these enzymes, is not dependent on enzymes to facilitate the post-translational modification. peroxynitrite mediated tyrosine nitration is a covalent modification that adds a nitro group (-no ) to one ortho carbon of tyrosine's phenolic ring to form -nitrotyrosine. protein tyrosine nitration introduces a net negative charge to the nitrated tyrosine at physiological ph, thus altering structural properties. previously, we had reported that like rac , its antagomir rhoa can also be nitrated at y , a similar location in the flexible flap region, leading to its persistent j o u r n a l p r e -p r o o f activation. we speculate that nitration will only occur at accessible tyrosine residues that are already sites for other post-translational modifications such as phosphorylation. indeed, nitration is very similar to phosphorylation in terms of charge, and thus could lead to a similar structural/functional outcome to that obtained by phosphorylation. alternatively, the presence of a -nitrotyrosine modification could potentially prevent the tyrosine residue being available for phosphorylation events. since no enzyme has been identified as being responsible for the removal of nitration sites, mimicking the counter part of phosphatase for phosphorylation, the structure/function effect is persistent and thus deleterious as it is not balanced and countered. this might also explain the opposite functional outcome between rac and rhoa nitration at a similar domain. in conclusion, for the first time, we have used mass spectrometry as well as cell and molecular biology methodologies to identify rac as being susceptible to nitration. this single nitration site located at y leads to rac gtpase inhibition and enhances lpsmediated barrier disruption. we have also demonstrated that rac nitration plays an important role in sepsis-mediated ali and that blocking rac nitration using a shielding peptide approach has therapeutic potential to reverse lps-induced lung injury. further studies will be required but this approach has the potential to produce a treatment for ali that has so far proven to be intractable to all the pharmacologic approaches tried to date. acute respiratory failure in covid- : is it "typical" ards? prevalence, etiologies and outcome of the acute respiratory distress syndrome among hypoxemic ventilated patients. srlf collaborative group on mechanical ventilation. société de réanimation de langue française spontaneous breathing during mechanical ventilation. risks, mechanisms, and management update on ards: beyond the low tidal volume endotoxemia and endotoxin tolerance in patients with ards revisiting pharmacology of oxidative stress and endothelial dysfunction in cardiovascular disease: evidence for redox-based therapies production of nitric oxide and peroxynitrite in the lung during acute endotoxemia mechanisms of nitric oxide synthase uncoupling in endotoxin-induced acute lung injury: role of asymmetric dimethylarginine peroxynitrite-mediated tyrosine nitration catalyzed by superoxide dismutase protein nitration in parkinson's disease inhibition of arginase activity enhances inflammation in mice with allergic airway disease, in association with increases in protein s-nitrosylation and tyrosine nitration increased calcification and protein nitration in arteries of chronic kidney disease patients quantitative assessment of proteinbound tyrosine nitration in airway secretions from patients with inflammatory airway disease cytoskeletal regulation of pulmonary vascular permeability rhoa, rac , and cdc exert distinct effects on epithelial barrier via selective structural and biochemical modulation of junctional proteins and f-actin rho and rac but not cdc regulate endothelial cell permeability lysophosphatidylcholine increases endothelial permeability: role of pkcalpha and rhoa cross talk myosin phosphatase and cofilin mediate camp/camp-dependent protein kinase-induced decline in endothelial cell isometric tension and myosin ii regulatory light chain phosphorylation lipopolysaccharide-induced lung injury involves the nitrationmediated activation of rhoa harvesting, identification and barrier function of human lung microvascular endothelial cells nitric oxide and superoxide generation from endothelial nos: modulation by hsp altered carnitine homeostasis is associated with decreased mitochondrial function and altered nitric oxide signaling in lambs with pulmonary hypertension nitration of tyrosine inhibits protein kinase g- α activity by attenuating cyclic guanosine monophosphate binding endothelial nitric oxide synthase deficient mice are protected from lipopolysaccharide induced acute lung injury particulate matter disrupts human lung endothelial barrier integrity via ros-and p mapk-dependent pathways an official american thoracic society workshop report: features and measurements of experimental acute lung injury in animals dimethylarginine dimethylaminohydrolase ii overexpression attenuates lps-mediated lung leak in acute lung injury altered expression of tight junction molecules in alveolar septa in lung injury and fibrosis ve-cadherin: at the front, center, and sides of endothelial cell organization and function role of gtpases in control of microvascular permeability rho gtpases and the regulation of endothelial permeability rac and rhoa: networks, loops and bistability rho gtpases, their post-translational modifications, diseaseassociated mutations and pharmacological inhibitors crystal structure of human rhoa in a dominantly active form complexed with a gtp analogue the crystal structure of human rac , a member of the rho-family complexed with a gtp analogue nitric oxide-induced inhibition of smooth muscle cell proliferation involves s-nitrosation and inactivation of rhoa rhoa s-nitrosylation as a regulatory mechanism influencing endothelial barrier function in response to g(+)-bacterial toxins the tumour suppressor hace controls cell migration by regulating rac degradation iaps regulate the plasticity of cell migration by directly targeting rac for degradation scf e ligase f-box protein complex scf(fbxl ) regulates cell migration by mediating rac ubiquitination and degradation tyrosine phosphorylation of rac : a role in regulation of cell spreading akt protein kinase inhibits rac -gtp binding through phosphorylation at serine of rac the fic domain: regulation of cell signaling by adenylylation irak- contributes to lipopolysaccharide-induced reactive oxygen species generation in macrophages by inducing nox- transcription and rac activation and suppressing the expression of antioxidative enzymes in vivo lipid-derived free radical formation by nadph oxidase in acute lung injury induced by lipopolysaccharide: a model for ards oxidative stress contributes to lung injury and barrier dysfunction via microtubule destabilization role of nadph oxidase in lipopolysaccharide-induced proinflammatory responses by human aortic endothelial cells mechanisms of nitric oxide synthase uncoupling in endotoxin-induced acute lung injury: role of asymmetric dimethylarginine lipopolysaccharide induces mitochondrial dysfunction in rat cardiac microvascular endothelial cells upregulation of xanthine oxidase by lipopolysaccharide, interleukin- , and hypoxia. role in acute lung injury signaling in the pathogenesis of acute lung injury (ali) and acute respiratory distress syndrome (ards) autophagy maintains the integrity of endothelial barrier in lps-induced lung injury superoxide potentiates nf-kappab activation and modulates endotoxin-induced cytokine production in alveolar macrophages regulation of endothelial barrier function by reactive oxygen and nitrogen species reactive oxygen species mediate rac-induced loss of cell-cell adhesion in primary human endothelial cells • endotoxin exposure induces site specific nitration of rac at y via peroxynitrite stress.• rac nitration at y leads to persistent rac gtpase inhibition and endothelial barrier disruption. • novel rac nitration shielding peptide, nipr blocks rac nitration and rescues endotoxin induced lung inflammation. • nipr is potentially an effective therapy for sepsis induced lung injury by targeting rac y nitration. the authors have no conflict of interest to declare related to this research article . key: cord- - yiuuye authors: mims, cedric a.; dimmock, nigel j.; nash, anthony; stephen, john title: mechanisms of cell and tissue damage date: - - journal: mims' pathogenesis of infectious disease doi: . /b - - - - . - sha: doc_id: cord_uid: yiuuye nan the impact on the host of microbial damage depends very much on the tissue involved. damage to muscle in the shoulder or stomach wall, for instance, may not be serious, but in the heart the very existence of the host depends on a strong muscle contraction continuing to occur every second or so, and here the effect of minor functional changes may be catastrophic. the central nervous system is particularly vulnerable to slight damage. the passage of nerve impulses requires normal function in the neuronal cell membrane, and viruses especially have important effects on cell membranes. also a degree of cellular or tissue oedema that is tolerable in most tissues may have serious consequences if it occurs in the brain, enclosed in that more or less rigid box, the skull. therefore, encephalitis and meningitis tend to cause more severe illness than might be expected from the histological changes themselves. oedema is a serious matter also in the lung. oedema fluid or inflammatory cell exudates appear first in the space between the alveolar capillary and the alveolar wall, decreasing the efficiency of gaseous exchanges. respiratory function is more drastically impaired when fluid or cells accumulate in the alveolar air space.* the effect of tissue damage is much less in the case of organs such as the liver, pancreas or kidney, which have considerable functional reserves. more than two-thirds of the liver must be removed before there are signs of liver dysfunction. cell damage has profound effects if it is the endothelial cells of small blood vessels that are involved. the resulting circulatory changes may lead to anoxia or necrosis in the tissues supplied by these vessels. here too, the site of vascular lesions may be critical, effects on organs such as the brain or heart having a greater impact on the host, as discussed above. rickettsiae characteristically grow in vascular endothelium, and this is an important mechanism of disease production. by a combination of direct and immunopathological factors there is endothelial swelling, thrombosis, infarcts, haemorrhage and tissue anoxia. this is especially notable in the skin, and forms the basis for the striking rashes in typhus and the spotted fevers. these skin rashes, although important for the physician are less important for the patient than similar lesions in the central nervous system or heart. it is damage to cerebral vessels that accounts for the cerebral disturbances in typhus; involvement of pulmonary vessels causes pneumonitis, and involvement of myocardial vessels causes myocardial oedema. in q fever, rickettsiae sometimes localise in the endocardium, and this causes serious complications. sometimes an infectious agent damages an organ, and loss of function in this organ leads to a series of secondary disease features. the signs of liver dysfunction are an accepted result of infections of the liver, just as paralysis or coma is an accepted result of infection of the central nervous system. diabetes may turn out to be caused by infection of the islets of langerhans in the pancreas. coxsackie and other virus infections of the islets of langerhans can certainly cause diabetes in experimental animals, and coxsackieviruses have been associated with juvenile diabetes in man. there are many diseases of unknown aetiology for which an infectious origin has been suggested. sometimes it is fairly well established that an infectious agent can at least be one of the causes of the disease, but in most instances it is no more than a hypothesis, with little or no good evidence. for conditions as common and as serious as multiple sclerosis, cancer and rheumatoid arthritis it would be of immense importance if a microorganism were incriminated, since this would give the opportunity to prevent the disease by vaccination. accordingly, there is a temptation to accept or publicise new reports even though the evidence is weak or the observations poorly controlled. as if to warn us about this and remind us of possibilities from environmental toxins, parkinson's disease, a chronic neurological condition in which there is a loss of neurons in a sharply defined region of the brain (substantia nigra), can be caused by exposure to the chemical mptp. one example which raises the possibility that subtle cns disturbances may be caused by viruses is experimental infection with borna disease virus. this virus was used to infect tree shrews (tupaia glis) which are primitive primates. there is little overt disease, but afterwards the male is no longer able to enact the ritual courtship behaviour which (as students well know), is an essential preliminary to mating in all primates, and the frustrated male usually ends up bitten by the female. thus it can be said that infection with borna disease virus renders the male psychologically sterile. presumably the virus in some way alters the functioning of neurons concerned in this particular pathway. all other behavioural and physiological aspects appear normal. borna disease virus is not known to occur in man, but speculation about an analogous human situation is fuelled by the finding of borna disease virus-specific antibodies in patients with psychiatric/ behavioural disorders. in an entirely different clinical context, infection of a particular strain of rats with borna disease virus causes immense obesity, the underlying physiological basis of which is not understood. since the aetiology of such diseases raises interesting problems in pathogenesis, the present state of affairs is summarised in table . , which includes some of the human diseases whose infectious origin is probable, possible, conceivable, or inconceivable. causal connections between infection and disease states are particularly difficult to establish when the disease appears a long time after infection. it was not too difficult to prove and accept that the encephalitis that occasionally occurs during or immediately after measles was due to measles virus. but it was hard to accept that a very rare type of encephalitis (subacute sclerosing panencephalitis or sspe), occurring up to years after apparently complete recovery from measles, was also due to measles virus and this was only established after careful studies and the eventual difficult isolation of a mutant form of measles virus from brain cells. 'slow' infections, in which the first signs of disease appear a long time after infection are now an accepted part of our outlook. the disease kuru occurred in new guinea and was transmitted from person to person by cannibalism. the incubation period of the disease in man appears to be - years, and was caused by a nonisolatable infectious agent that grew in the brain. this was established when the same disease appeared in monkeys several years after the injection of material from the brain of kuru patients. a similar agent called scrapie (see ch. ) infects sheep, mice and other animals and also has an incubation period representing a large portion of the life span of the host. in both kuru and sspe the agent was eventually shown to be present in the brains of patients. so far this has only been demonstrated indirectly as, despite strenuous efforts, the causative agent has yet to be isolated. if in a slow infection the microorganism that initiated the pathological process is no longer present by the time the disease becomes manifest, then the problem of establishing a causal relationship will be much greater. this may possibly turn out to be true for diseases like multiple sclerosis and rheumatoid arthritis. liver cancer in humans and leukaemia in mice, cats, humans and cattle can be caused by slow type virus infections. cancer or leukaemia appears as a late and occasional sequel to infection. the virus, its antigens or fragments of its nucleic acid are detectable in malignant cells. one important factor that often controls the speed of an infectious process and the type of host response, is the rate of multiplication of a microorganism.* different infectious agents show doubling times * every infection is a race between the spread and multiplication of the microbe and the generation of an antimicrobial response by the host. a day or two's delay in this response may let the microbe reach the critical levels of growth that give tissue damage and disease. varying from min to weeks, and some of these are listed in table . . often the rate of multiplication in the infected host, in the presence of antimicrobial and other limiting factors and when many bacteria are obliged to multipy inside phagocytic cells, is much less than the optimal rate in artificial culture. clearly a microorganism with a doubling time of a day or two will tend to cause a more slowly evolving infection and disease than one that doubles in an hour or less. it is uncommon for an infectious agent to cause exactly the same disease in all those infected. its nature and severity will depend on infecting dose and route, and on the host's age, sex, nutritional status, genetic background, and so on. many infections are asymptomatic in more than % of individuals, clinically characterised disease occurring in only an occasional unfortunate host, as 'the tip of the iceberg'.* asymptomatically infected individuals are important because they are not identified, move normally in the community, and play an important part in transmission. this chapter deals with demonstrable cell and tissue damage or dysfunction in infectious diseases. but one of the earliest indications of illness is malaise, 'not feeling very well'. this is distinct from fever or a specific complaint such as a sore throat and although it is difficult to define and impossible to measure, we all know the feeling. it can precede the onset of more specific signs and symptoms, or accompany them. sometimes it is the only indication that an infection is taking place. almost nothing is known of the basis for this feeling. toxins', of course, have been invoked and the earliest response to pyrogens (see pp. - ) before body temperature has actually risen, may play a part. interferons may have something to do with it because pure preparations of human a or ß interferons cause malaise and often headaches and muscle aches after injection into normal individuals. if interferon is eventually recognised as an important antimicrobial force we may have to regard these side effects as unfortunate but acceptable. soluble mediators of immune and inflammatory responses, such as interleukin- (see glossary) or other cytokines may also play a part. in some infectious diseases weakness and debility are prominent during convalescence. this can be especially notable following influenza and hepatitis, but its basis is as mysterious as in the case of malaise. the infections that matter are those causing pathological changes and disease. before giving an account of the mechanisms by which these changes are produced, it is important to remember that many infectious agents cause little or no damage in the host. indeed, it is of some advantage to the microorganism to cause minimal host damage, as discussed in ch. . virus infections as often as not fall into this category. thus, although infection with rabies or measles viruses nearly always causes disease, there are many enterovirus, reovirus and myxovirus infections that are regularly asymptomatic. even viruses that are named for their association with disease (poliomyelitis, influenza, japanese b encephalitis) often give an antibody response as the only sign of infection in the host. tissue damage is too slight to cause detectable illness. there is also a tendency for persistent viruses to cause no more than minor or delayed cellular damage during their persistence in the body, even if the same virus has a more cytopathic effect during an acute infection, e.g. adenoviruses, herpes simplex (see ch. ). a few viruses are remarkable because they cause no pathological changes at all in the cell, even during a productive infection in which infectious virus particles are produced. for instance, mouse cells infected with lcm or leukaemia virus show no pathological changes. a mouse congenially infected with lcm virus shows a high degree of immune tolerance, and all tissues in the body are infected. throughout the life of the animal, virus and viral antigens are produced in the cerebellum, liver, retina etc. without discernible effect on cell function. but sometimes there are important functional changes in infected cells which lead to a pathological result. for example, the virus infects growth hormoneproducing cells in the anterior pituitary. although the cells appear perfectly healthy, the output of growth hormone is reduced, and as a result of this, suckling mice fail to gain weight normally and are runted. when bacteria invade tissues, they almost inevitably cause some damage, and this is also true for fungi and protozoa. the extent of direct damage, however, is sometimes slight. this is true for treponema pallidum, perhaps because the lipopolysaccharide-protein components that might have induced inflammatory responses, are not exposed on the surface of the bacteria. it produces no toxins, does not cause fever, and attaches to cells in vitro without harmful effects. leprosy and tubercle bacilli eventually damage and kill the macrophages in which they replicate, but pathological changes are to a large extent caused by indirect mechanisms (see below). in patients with untreated lepromatous leprosy, the bacteria in the skin invade blood vessels, and large numbers of bacteria, many of them free, may be found in the blood. in spite of the continued presence of up to bacteria m l - of blood there are no signs or symptoms of septicaemia or toxaemia. mycobacterium leprae can be regarded as a very successful parasite that induces very little host response in these patients, even when the bloodstream is invaded. the resident bacteria inhabiting the skin and intestines of man and animals do not invade tissues and are normally harmless; indeed, as discussed in ch. , they may benefit the host. bacteria such as meningococci and pneumococci, whose names imply pathogenicity, spend most of their time as harmless inhabitants of the normal human nasopharynx: only occasionally do they have the opportunity to invade tissues and give rise to meningitis or pneumonia. cell and tissue damage are sometimes due to the direct local action of the microorganism. however, it is not at all clear how viruses cause the death of cells. many virus infections result in a shutdown of rna synthesis (transcription), protein synthesis (translation) and dna synthesis in the host cell, but usually these are too slow to account for the death of the cell. after all, cells like neurons never synthesise dna, and the half life of most proteins and even rnas is at least several hours. a possible alternative mechanism is the alteration of the differential permeability of the plasma membrane. this is important as the cell has a high internal k + concentration and low n a + concentration, while the reverse is true of body fluids. viruses do alter membrane permeability, but the unresolved question is whether or not this is responsible for the death of the cell or is merely an after-effect. it now appears that at least some virus infections (hiv, adenovirus, influenza virus, sindbis virus) cause the cells to commit suicide by a mechanism called 'programmed cell death' or 'apoptosis'. this is the natural process by which the body controls cell numbers and rids itself of superfluous or redundant cells during development. a familiar example is a tadpole 'losing' its tail. cells do not disintegrate but round up, and are then removed by phagocytes. a possible example of apoptosis is seen in the common cold when caused by the rhino virus group of picorna viruses. rhinoviruses infect nasal epithelial cells, and at an early stage the cells round up, fall off the mucosal surface and are carried away, often with their cilia still beating, in a stream of fluid induced by the infection.* this leaves areas of raw mucosa, with the exposed underlying tissues inflamed, oedematous, and susceptible to infection by the normally harmless resident bacteria. the gross pathology of cells infected with viruses appears generally nonspecific, very like that induced in cells by the toxins of diphtheria bacilli and streptococci, or by physical and chemical agents. the most common and potentially reversible change, the oedema seen as 'cloudy swelling' by routine histology, is associated with membrane permeability changes. changes in the endoplasmic reticulum, mitochondria and polyribosomes are seen by electron microscopy at this stage. later the nuclear chromatin moves to the edge of the nucleus ('margination' of chromatin) and becomes condensed (pycnosis), but the cell has already died by this time. there are two more characteristic types of morphological change produced by certain viruses, and these were recognised by histologists more than years ago. the first are inclusion bodies, parts of the cell with altered staining behaviour which develop during infection. they often represent either cell organelles or virus factories in which viral proteins and/or nucleic acids are being synthesised and assembled. herpes group viruses form intranuclear inclusions, rabies and poxviruses intracytoplasmic inclusions, and measles virus both intranuclear and intracytoplasmic inclusions. the second characteristic morphological change caused by viruses is the formation of multinucleate giant cells. this occurs, for instance when hiv 'fusion' proteins (gpl -gp ) present in nascent virus particles budding from an infected cell attach to cd receptors in the plasma membranes of neighbouring cells; membranes then fuse and multinucleate cells are formed. it also happens in measles and certain herpes virus infections. before leaving the subject of direct damage by viruses, one supreme example will be given. here the direct damage is of such a magnitude that the susceptible host dies a mere six hours after infection. if rift valley fever virus, an arthropod-borne virus infecting cattle, sheep and man in africa, is injected in very large doses intravenously into mice, the injected virus passes straight through the kupffer cells and endothelial cells lining liver sinusoids (see ch. ) and infects nearly all hepatic cells. hepatic cells show nuclear inclusions within an hour, and necrosis by four hours. as the single cycle of growth in hepatic cells is completed, massive liver necrosis takes place, and mice die only six hours after initial infection. the host defences in the form of local lymph nodes, local tissue phagocytes etc. are completely overcome by the intravenous route of injection, and by the inability of kupffer cells to prevent infection of hepatic cells. direct damage by the replicating virus destroys hepatic cells long before immune or interferon responses have an opportunity to control the infection. this is the summit of virulence. the experimental situation is artificial but it illustrates direct and lethal damage to host tissues after all host defence mechanisms have been overwhelmed. most viruses, rickettsiae and chlamydia damage the cells in which they replicate, and it is possible that some of this damage is due to the action of toxic microbial products. this action, however, is confined to the infected cell, and toxic microbial products are not liberated to damage other cells. mycoplasma (see table a . ) can grow in special cell-free media, but in the infected individual they generally multiply while attached to the surface of host cells. as studied in culture and on the respiratory epithelium they 'burrow' down between cells, inhibit the beat of cilia and cause cell necrosis and detachment. the mechanism is not clear. if a complete lawn of mycoplasma covers the surface of the host cell, some effect on the health of the cell is to be expected, but it is possible that toxic materials are produced or are present on the surface of the mycoplasma. dental caries provides an interesting example of direct pathological action. colonisation of the tooth surface by streptococcus mutans leads to plaque formation, and the bacteria held in the plaque utilise dietary sugar and produce acid (see p. ). locally produced acid decalcifies the tooth to give caries. caries, arguably the commonest infectious disease of western man, might logically be controlled by removing plaque, withholding dietary sugar, or vaccinating against streptococcus mutans. however, fluoride in the water supply or in toothpaste has been the method of choice, and has been very successful. it acts by making teeth more resistant to acid. bacteria generally damage the cells in which they replicate, and these are mostly phagocytic cells (see ch. ). listeria, brucella and mycobacteria are specialists at intracellular growth, and the infected phagocyte is slowly destroyed as increasing numbers of bacteria are produced in it. bacteria such as staphylococci and streptococci grow primarily in extracellular fluids, but they are ingested by phagocytic cells, and virulent strains of bacteria in particular have the ability to destroy the phagocyte in which they find themselves, even growing in the phagocytes, as described in ch. . many bacteria cause extensive tissue damage by the liberation of toxins into extracellular fluids. various toxins have been identified and characterised. most act locally, but a few cause pathological changes after spreading systemically through the body. this is a huge and growing part of our subject and we need to define the term toxin, a task which is more difficult than one might think. an attempt was made by bonventre who in defined toxins as a 'special class' of poisons which differ from, for example, cyanide or mercury by virtue of their microbial origin, protein structure, high molecular weight, and antigenicity. this view is too embracing, because it includes proteins of doubtful significance in disease, and also too restrictive, because it excludes nonprotein toxic complexes such as endotoxin. another suggestion is that toxin must include all naturally occurring substances (of plant, animal, bacterial or whatever origin) which when introduced into a foreign host are adverse to the well-being or life of the victim. this, too, is unsatisfactory because some substances-potent toxins within the scope of this definition-are being used in some contexts as therapeutic agents! perhaps it is pointless to strive for an all-embracing definition, although the obvious differences between bacterial and fungal toxins warrant the continued use of the appropriate prefix. for example, bacterial toxins are usually of high molecular weight and hence antigenic, whereas fungal toxins tend to be low molecular weight and not antigenic. the problem of definition is compounded because there are substances (aggressins) which help to establish an infective focus as well as those whose action is uniquely or largely responsible for the disease syndrome. also there are substances known to be produced by bacteria in vitro, whose properties on a priori grounds make them potential determinants of disease, but which have not been shown to play a role in vivo. we will concentrate on those toxins known to be, or likely to be, involved in some aspect of disease causation. it is not difficult to demonstrate the production of'toxins' by bacteria in culture, as judged by some bioassays. primary consideration will be given to those substances which are produced under ecologically significant conditions (i.e. in the natural host or relevant animal model) and cause (also in biologically relevant systems) damage to cells or tissues thereby contributing to disease. exotoxins are produced and then either secreted by, or released upon lysis from, both gram-positive and gram-negative bacteria. they are proteins, some of which are enzymes. when liberated locally they can cause local cell and tissue damage. those that damage phagocytic cells and are therefore particularly useful to the microorganism have been described in ch. . those that promote the spread of bacteria in tissues have been referred to in ch. . a description of some of the more interesting exotoxins follows. proteases and hyaluronidases, which help the spread of bacteria through tissues have already been mentioned in ch. . here we consider toxins which act on extracellular substances and are responsible for many of the main features of the diseases caused by the infecting organism. pseudomonas aerwgmosa-elastase, and one of at least six proteases of legionella pneumophila, both induce fibrinopurulent exudation in the rat lung (a model for p. aeragmosa-induced pneumonia in human cystic fibrosis) and the guinea-pig lung (a model for legionnaires' disease) respectively. these characteristics almost certainly arise from the release of oligopeptides from extracellular matrix components of the host which are chemotactic for leucocytes and fibroblasts. the l. pneumophila protease is the same major secretory protein (the m r zinc metalloprotease) already considered in ch. in relation to survival within macrophages. staphylococcal exfoliatin is important in staphylococcal 'scalded skin syndrome' (ssss), a disease of newborn babies. the disease is characterised by a region of erythema which usually begins around the mouth and, in - days, extends over the whole body. during this period, small yellowish exudative lesions often appear. the most striking feature of the disease, however, is that the epidermis, although apparently healthy, can be displaced and wrinkled like the skin of a ripe peach by the slightest pressure. soon large areas of the epidermis become lifted by a layer of serous fluid and peel at the slightest touch. large areas of the body rapidly become denuded in this way and the symptoms resemble those of massive scalding. while the toxin causes cleavage of desmosomes (specialised cell membrane thickenings through which cells are attached to each other) in the stratum granulosum, it may also act intracellularly. despite numerous attempts to characterise the biological activity of exfoliatin, the genetically predicted serine protease and/or lipase activity has never been demonstrated. some enterotoxigenic e. coli elaborate families of low molecular weight heat stable (st) peptides as well as heat labile (lt; cholera-like) toxin. sts bind to a receptor which then activates a tightly coupled membranebound guanylate cyclase in gut cells, resulting in the transmission of a signal to the inside of the cell, thereby elevating cgmp, or some other second message. as described later in the section on diarrhoea this gives rise to efflux of ions, and hence water, from enterocytes. we know that these toxins are able to traverse membranes since their targets are intracellular; we still know very little about the details of how they achieve this. these proteins have common features: one component (usually described as the b fragment or subunit) binds to and interacts with a cell receptor and promotes the uptake of an active component (a fragment or subunit) in which resides the biological activity which confers toxicity. there are several ways in which one can classify these toxins. ( ) biologically-some kill cells (cytotoxic toxins) whereas others (sometimes called cytotonic toxins) 'deregulate' cells. ( ) biochemically-some belong to a well defined biochemical group recognised by their ability to cleave nad + into nicotinamide and adp-ribose moieties. they are designated adp-ribosyl (adpr) transferases since they transfer adpr to different target proteins; this group straddles groups and . ( ) genetically-the third possibility reflects the genetic origin of these toxins: (i) production from a single gene of a single peptide which undergoes post-translational modification into a and b fragments which are covalently linked; (ii) production from separate genes of a and b subunits which noncovalently associate into stable complexes; (iii) production from separate genes of different proteins which do not associate into stable complexes but which must act in concert to express toxicity. these are known as binary toxins; again, group straddles group . examples of each of these groups will be given illustrating how they work, their importance in disease, and surprisingly (!) how some of the most toxic substances known to man are being used or may be used for therapeutic purposes. diphtheria toxin (dt). corynebacterium diphtheriae organisms multiply on the epithelial surfaces of the body (nose, throat, skin) but do not penetrate deeply into underlying tissues. the infection on the body surface causes necrosis of mucosal cells with an inflammatory exudate and the formation of a thick 'membrane' (hence the name c. diphtheriae: gr., diphthera = membrane) and if the infection spreads into the larynx there may be respiratory obstruction. the toxin probably assists colonisation of the throat or skin by killing epithelial cells and polymorphs. active immunisation with diphtheria toxoid has made diphtheria a clinical rarity in developed countries. dt is disseminated from the infection site and has important actions, especially on the heart and nervous system. dt is encoded by lysogenic phage ß but its expression is controlled by the host bacterium under conditions of iron stress. it is synthesised and processed as shown in fig. . . there is intense interest in seeking to understand the mechanisms of uptake of the active moieties of diphtheria and other toxins whose target is intracellular. this is driven by the desire to understand fundamental mechanisms in cell biology and to develop selective cytotoxic therapies' in clinical medicine. the mechanism of internalisation of the active a under the acidic conditions of the endosome, some dt-a is released by reduction, dt-b undergoes conformational change and interacts with the endosomal membrane resulting in the opening of cation channels. the latter are not absolutely necessary, but their formation is associated with times greater uptake of dt-a. in contrast, most other toxins which enter the cytosol appear to do so by more complicated routes which initially involving endocytosis. shiga toxin (and ricin, a toxic plant lectin believed to have been located in the tip of an umbrella and used to poison a bulgarian spy!) are not translocated across the endosomal membrane but are transported to the trans-golgi network and all the way back to the endoplasmic reticulum. somewhere in the exocytotic pathway conditions exist for the translocation of shiga toxin (sht) into the cytoplasm. sht acts by modifying s ribosomal rrna. for cholera toxin, it used to be thought that a l ( fig. . ) moved down through the central hole of the ring-shaped complex of ct-b which was anchored to the membrane by five ganglioside gm binding sites. structural work on this family of proteins shows that this is not physically possible. the finding that the c-terminal sequence lysine-aspartateglutamate-leucine (the kdel* motif) in the a subunit of cholera toxin and related sequences in e. coli heat labile toxin and pseudomonas exotoxin a raises the possibility that transport to the endoplasmic reticulum may also be necessary for these a subunits to reach the cytoplasm. a new and exciting development is beginning. for decades, attempts have been made to make immunotoxins by coupling native toxins to antibodies specific to some surface antigen on tumour cells, with little practical success, as yet. however, scientists have now genetically engineered dt by substituting that portion of the dt structural gene encoding the native toxin receptor-binding domain with modified cdna encoding one of a variety of cytokines and growth factors including il- , il- , il- , egf and α-msh. the resultant fusion toxin bears a new 'cellular address', but retains all of the other biological properties of the native dt molecule as well as a three-dimensional structure nearly identical with native dt. the il- receptor-binding domain fusion toxin has undergone phase i/ii clinical trials for the treatment of il- receptor positive haematological malignancies (and other il- related conditions) and has been shown to be safe and well tolerated. such constructs are potent against a murine model of il- receptor positive lymphoma, and the hope is that a durable remission of disease will be achieved in humans. (an era of new 'magic bullets'?) p. aeruginosa exotoxin a. p. aeruginosa, already alluded to above, is common in soil and water and can occasionally be isolated from the faeces of normal, healthy individuals. it is virtually harmless for healthy adults, but its ability to multiply in almost any moist environment and its resistance to many antibiotics have made the bacterium a major cause of * the kdel motif is normally found in proteins which having been processed in the golgi are trapped by the endoplasmic reticulum which recognises the kdel motif. this prevents the protein being lost to the cell via exocytotic trafficking and results in its translocation into the cytoplasm. the schema shows a round of peptide elongation and illustrates the key role played by two enzymes, ef and ef . dta and pea each adp-ribosylates diphthamide (a modified histidine) in ef -gtp which can no longer translocate the newly elongated peptide from the a site to the p site. sht a fragment is a specific n-glycosidase which cleaves an adenine residue from near the '-prime end of the s ribosomal rna. this depurination results in failure of ef -dependent binding of aminoacyl-trna to site a and hence inhibits protein synthesis. poliovirus achieves selective inhibition of host protein synthesis at an earlier stage than is depicted here. host mrna is first modified (capped) then bound to the small ribosomal subunit; poliovirus mrna is not capped. the function of a cap-binding protein, which recognises and binds host mrna to the ribosome, is inhibited by a poliovirus virion protein thereby allowing differential translation of virus messenger rna. ef-Ια: nucleotide-binding protein. hospital-acquired infection, particularly among patients with impaired host defence mechanisms such as those with chronic illness, genetic immunodeficiencies, those under treatment with immunosuppressive drugs, or patients suffering from extensive burns. p. aeruginosa causes localised infection in the urinary tract, respiratory tract, burns and wound infections. in severely debilitated patients these localised infections may develop into general septicaemia, with mortality in such cases approaching %. the mechanisms of pathogenicity of p. aeruginosa are complicated and unclear because (unlike c. diphtheriae) the organism elaborates several potentially toxic extracellular products, including a phospholipase, several proteases, lipase, haemolysin, enterotoxin, lipopolysaccharide endotoxin, elastase, exoenzyme s (pes), and exotoxin a (pea); evidence definitely implicates the last three. elastase has already been mentioned above (p. ). pes is without question a virulence determinant, but at present its biological mode of action is not wholly understood. pea is in many ways like dt. it inhibits protein synthesis by cleavage of nad + and subsequent adp-ribosylation of ef at the same diphthamide residue, is synthesised predominantly during the decline phase of cell growth, and its production appears to be dependent upon the concentration of iron in the medium. its m r is , and it can be activated in cell-free systems by proteolysis, or by reduction with thiols in the presence of urea and other chaotropic agents. all the enzymic activity of the toxin resides in a proteolytically derived fragment (m r approximately ). however, dissimilarities exist between the two toxins. firstly, the active a fragment resides in the cooh-terminal portion of the molecule, whereas the diphtheria a fragment is at the nh -terminal end. secondly, the b fragment of pea recognises a different receptor to that of dt, and there are no sequence homologies or serological crossreactivity between the two toxins. shiga toxin. this toxin is a subunit protein with an ab type structure ( a subunit; b, an oligomer of subunits). certain biotypes of e. coli make vero toxins (vts) or shiga-like toxins (slts) which are identical (slt ) or near identical (slt ) with shiga toxin. the role of shiga toxin in bacillary dysentery is discussed below. slt-producing strains of e. coli are responsible for haemorrhagic colitis and haemolytic urea syndrome. very recent crystallographic studies have shown a remarkable similarity in organisational structure of the b subunits of shiga toxin and those of the cholera toxin family discussed below, despite the near complete absence of sequence homology. the b subunits attach to a ganglioside structure gb or gb (cholera toxin attaches to gm ) on susceptible cell surfaces. sht-a fragment is a specific n-glycosidase which cleaves an adenine residue from near the '-prime end of s ribosomal rna. this depurination results in failure of efl-dependent binding of aminoacyl-trna to site a and hence inhibits protein synthesis ( fig. . ). this is based on studies on cultured cells, and how such cytotoxic activity gives rise to the enterotoxic activity-watery diarrhoea seen in cases of dysentery preceding the bloody mode of action of actin-adp-ribosylating toxins. c. botulinum c toxin component binds to the cell membrane followed by c i. the latter is internalised and upsets the equilibrium between polymerisation and depolymerisation of actin. adp-ribosylation of actin inhibits its polymerisation and turns g-actin into a capping protein which binds to the fast growing (concave) ends of actin filaments. capping of the concave ends increases the critical concentration for actin polymerisation. since the slow-growing (pointed) ends of actin filaments are free, depolymerisation of actin occurs at these ends. released actin is substrate for the toxin and will be withdrawn from the treadmilling pool of actin by adpribosylation, i.e. trapped. both reactions will finally induce the breakdown of the microfilament network. (taken with permission of authors (k. aktorieseia/.) and publisher (springer-verlag gmbh & co. kg, heidelberg, germany) from botulinum c toxin. clostridium botulinum c toxin is not a neurotoxin but belongs to a family of cytotoxins {clostridium perfringens iota toxin, clostridium spiroforme toxin, clostridium difficile adp ribosyltransferase) whose common features are that they are binary toxins and that they target cytoskeletal actin. of these c. botulinum c is the best studied at present. component c i is the equivalent of the a subunit and c ii, the b subunit. they are produced in parallel although different strains produce different ratios. more c is produced during sporulation than in the vegetative phase of growth. c ii is activated by proteolysis to produce a m r protein which binds to cell surfaces and thereby exposes a binding site for c i (m r ca. ) either on the cell surface or on attached c ii. once inside, c i adp-ribosylates monomeric actin and not polymerised f actin. the substrate specificity is high. the toxin will modify a specific arginine residue present in certain isoforms of actin. neither subunit is toxic on its own, but together the ld dose for a mouse is ca. - ng. (other examples of such 'binary' activity include anthrax toxin and staphylococcal leucocidin). the two components may be injected into different sites or by different routes but toxic effects always occur at the site of injection of c ii. this mode of action, summarised in fig. . , is believed to underlie the biological manifestations of toxicity which, in rats, include hypotension, haemorrhaging in and fluid accumulation around the lungs as a result of vascular permeability changes. the toxin is currently being used as a tool in cell biology to study cytoskeletal systems and dissect mechanisms of cell signal transduction. cholera and related toxins. there is a growing number of toxins in this category. easily the most studied is cholera toxin (ct), a major research area for three decades, which was originally stimulated by the desire to make a more effective vaccine against cholera. ironically, effective immunity is best mediated by antibacterial antibodies which prevent colonisation of the gut with v. cholerae rather than antitoxin-neutralising antibodies. cholera toxin is an ab subunit toxin ( fig. . ). enterotoxigenic e. coli (etec) strains produce two heat labile toxins: lti and ltii. ltii exists as two minor variants, ltiia and ltiib, the latter requiring trypsin treatment for full activation in the y- adrenal cell assay. the b subunits of ct, ltiia and ltiib recognise and are bound to cell surface gangliosides gm , gdlb and gdi a respectively and mediate the translocation of a l subunits into the cytoplasm; lti binds to either gm or a glycoprotein. the same reaction occurs as was described above for dt: nad + is cleaved into nicotinamide and adp-ribose (adpr) moieties, with two important differences: ( ) adpr transferase is greatly enhanced by a group of proteins (both membrane bound and soluble) of ca. m r designated adpribosylation factors (arfs; of as yet unknown physiological significance); ( ) adpr is transferred to the a s subunit of the g s regulator of adenylate cyclase as illustrated in fig. . . this results in the elevation of camp levels in the cell with the consequences described below in the section on diarrhoea. bordetella pertussis toxin (pertussigen) and adenylate cyclasehaemolysin (ac-hly). whooping cough (pertussis) is a severe respiratory tract infection characterised by prolonged paroxysmal coughing, attacks of which continue long after infection has cleared. the disease is capable of striking all ages but is particularly prevalent and severe in young children, where hospitalisation is required in about % of cases. the causative agent of pertussis, bordetella pertussis, is transmitted aerially from the respiratory tract of an infected individual to that of a susceptible host. the organism attaches (probably via its filamentous haemagglutinin, pili, and a m r outer membrane protein) to, and colonises the mucosal surface between the cilia, and multiplies there during the incubation period of the disease which is commonly around seven days. the infection then manifests as a slight fever and catarrh which is often indistinguishable from a common cold. however, one to two weeks later bouts of uncontrollable coughing begin. it is this paroxysmal coughing, along with the notorious 'whoop' as the child attempts to draw breath, which characterises the disease. the paroxysmal coughing stage often lasts for several weeks and no treatment fully effective in controlling the symptoms. the only proven means of controlling whooping cough is vaccination but, in the uk at least, sporadic reports of vaccine-induced brain damage in infants has diminished public acceptance of the vaccine. it should be noted that permanent encephalopathy (brain damage) is a recognised though rare consequence of whooping cough infection. much current work is being devoted to producing immunogenic, completely nontoxic preparations of pertussis toxin by genetic manipulation of t h e gene encoding t h e s i s u b u n i t ( fig. . ). in clinical trials in italy, such engineered vaccines h a v e been shown to be both safe a n d effective a s j u d g e d by antibody titres to pertussis toxin. several toxic substances h a v e been isolated from bordetella pertussis including a h e a t labile toxin, tracheal cytotoxin, endotoxin, adenylate cyclase-haemolysin (ac-hly), a n d pertussis toxin. t h e latter h a s m a n y biological activities-histamine sensitisation, leucocytosis promotion, islet (a) the production of cyclic amp by adenyl cyclase. cyclic amp is an important second messenger invoked in the intracellular amplification of many cellular responses to external signals including hormones. the nature of the physiological response reflects the biochemical differentiation of the cell responding to the stimulus. for example, in gut cells the response would be altered ion transport and hence fluid secretion; in muscle cells it would be glycogen breakdown in response to the call for more energy. the production of camp is controlled both positively and negatively at two different levels. the central cycle represents a normal membranebound hormone receptor and the heterotrimeric (aß ) g protein regulator complex which is activated upon binding of hormone to the receptor. there are two receptors, each with regulatory g proteins: one system responds to stimulatory and the other responds to inhibitory stimuli; only one system is shown. in gut cells these receptors would be on the basolateral (nonluminal) side of enterocytes enabling responses to stimuli from the circulation. (b) the second level of control involves endogenous gtpase properties of both stimulatory (a s ) and the inhibitory (a a ) subunits of the g protein regulator which may be outlined as follows. stimulation: agonist stimulation of its receptor results in the dissociation of the a s ß g protein from the receptor and of the subunits from each other, and the binding of gtp to a s . a s -gtp will productively interact with the catalytic unit of adenyl cyclase (not shown) and stimulate the production of camp from atp. endogenous gtpase activity in a s results in a delayed conversion of a s -gtp to inactive a s -gdp which can no longer stimulate the cyclase but allows the reassociation of the trimeric g protein and loss of gdp. inhibition: antagonist stimulation of its receptor results in an analogous situation for inhibiting cyclase activity via c^: inhibitory cti-gtp inactivates the cyclase; cti-gdp does not inactivate the cyclase. (c) the level of camp may be affected by physiological stimuli (which stimulate removal and modification of the g-protein complex) or by perturbation of the normal regulatory cycle as illustrated, by cholera toxin (ct) or pertussis toxin (ptx). (d) cholera toxin acts first by interacting with its receptor resulting in the internalisation of the active a l subunit. a l adp-ribosylates a s -gtp which promotes continued dissociation of the heterotrimer; also the endogenous gtpase is no longer functional hence the stimulation of the cyclase continues. lt toxins act in a similar manner. (e) pertussigen acts first by interacting with its receptor resulting in the internalisation of the active si subunit. si adp-ribosylates the ai-gdpß -heterotrimer which can no longer associate with the receptor (or lose gdp) to undergo another cycle of gtp activation; activated cyclase can no longer be turned off. (adapted, with kind permission of author (gierschik, p.) and publisher (springer-verlag gmbh & co. kg, heidelberg, germany) from figure in 'adp-ribosylation of signal-transducing guanine nucleotide-binding proteins by pertussis toxin', current topics in microbiology andlmmunology ( ) , , edited by klaus aktories. activation and adjuvanticity-and is believed to be the most important but by no means the only virulence determinant of b. pertussis: it is called pertussigen and is the basis of the vaccine against whooping cough. pertussigen is a complex subunit toxin whose biochemical mode of action is identical to that of ct except that the target is the aj subunit of the gj regulator of adenylate cyclase (see fig. . ). it is not yet clear how such biochemical activity relates to the clinical syndrome. ac-hly has two functional domains. one confers adenylate cyclase activity and another haemolytic activity. its haemolytic activity arises from its pore-forming properties in cell membranes. pore formation is probably responsible for the translocation of the adenylate cyclase portion of the complex. ac-hly is also known as cyclolysin and belongs to the group of toxins whose common feature is the presence of an array of a nine amino acid repeats; they have been designated rtx (repeats in toxin) toxins. the prototype is the haemolysin of e. coli which is important in extraintestinal infections caused by this organism. other toxins of this group include the haemolysin from proteus vulgaris, and the leukotoxin from pasteurella haemolytica. anthrax toxin. anthrax is a disease of animals, particularly sheep and cattle, and to a lesser extent man, caused by infection with bacillus anthracis. infection takes place following the ingestion of spores, the inhalation of spores, or by the entry of spores through abraded skin. the spores germinate and then the bacteria form a toxin that increases vascular permeability and gives rise to local oedema and haemorrhage. infection of the skin in man leads to the formation of a lesion (malignant pustule; a black eschar, hence b. anthracis; gr. anthrakos = coal) consisting of a necrotic centre surrounded by vesicles, blood-stained fluid and a zone of oedema and induration. in severe infections there is septicaemia with toxic signs, loss of fluid into tissues, with widespread oedema and eventually death. anthrax toxin, the discovery of which by smith and keppie in the early s was a major milestone in our subject, consists of three components, none of which are toxic by themselves: factor i (oedema factor or ef), factor ii (protective antigen or pa) and factor iii (lethal factor or lf). the toxin is largely responsible for local lesions, kills phagocytes, enters the circulation and accounts for the toxicity, oedema and death caused by b. anthracis. virulent strains also have a capsular polypeptide composed of d-glutamic acid, which inhibits opsonisation and phagocytosis (see ch. ). anthrax in man occurs mainly in those whose work brings them into contact with infected animals. it is not a common disease in the uk, and the usual source of infection is imported bones, hides, skins, bristles, wool and hair, or imported fertilisers made from the blood and bones of infected animals. most of what has just been described was discovered more than three decades ago but more recently new insights have been obtained as to the biochemical basis of the mode of action of anthrax toxin. ef was shown to be an inactive form of adenylate cyclase which is activated by calmodulin; pa and lf were not active. however combinations of pa and ef (but not pa and lf) caused an elevation of camp. thus anthrax toxin (at least the pa/ef combination) is a binary toxin. the response observed is more rapid and greater than that induced by cholera toxin. in contrast to cholera toxin, the anthrax complex is the cyclase, acts without a lag phase and its effects are instantly reversible upon washing toxin-treated cells. this suggests that the enzyme is either rapidly degraded after internalisation, or that it is not completely internalised but wsfhiw ii camp atp fig. . mode of action of anthrax toxin. factor ii (pa) interacts with the cell membrane. after proteolytic cleavage sites are exposed which bind factor i (ef) and facilitate internalisation of factor i (ef). this internalisation takes place via an endocytic step, with factor i being released into the cytosol. factor i must be rapidly inactivated since washing toxin-treated cells results in a rapid loss of adenylate cyclase activity. factor i interacts with calmodulin (cal) to become an active adenylate cyclase enzyme. interaction of factor i with factor ii and subsequent internalisation is blocked by prior binding of factor iii (lf) to factor ii. associated with the cell membrane in a manner allowing it to be readily removed. these facts together with the observations that lf would block the activity of ef are summarised in fig. . . this explains how lf blocks the effect of ef. they either compete for the same sites on pa, in which case the explanation is self-evident, or they bind to separate sites on pa which are situated such that occupancy of one occludes access to the other. one can extend these observations made in vitro to the in vivo situation. the model provides a basis for understanding the protective function of pa. antibodies to pa could either block the initial attachment to the cell membrane of target organs in vivo, or prevent the binding of ef to pa, or both. the fact that camp is known to be a potent secretagogue could explain how the oedematous reaction occurs when pa and ef are injected into test animals, i.e. ef is, after activation, an oedema factor. moreover, in biological tests, addition of lf (factor iii) depresses oedema production as would be predicted by this model. the observation that pa was probably fixed first may now be placed on a firmer basis since binding of pa is a prerequisite for the expression of ef (and by implication) lf activities. the case for the view that, in experimental anthrax in guinea-pigs, animals died (as do humans) of secondary shock, is also placed on a firmer basis with perhaps one important reservation. smith and coworkers claimed that factor iii (lf) decreased oedema but increased lethality; the former but not the latter observation is explicable by the model which is therefore too simplistic to explain the effects of the holotoxin. it is likely that lf exerts its activity together with pa on other cells in vivo. it has in fact been shown that lf in combination with pa is cytolytic for macrophages and macrophage-like cell lines-the probable basis of the earlier observation that anthrax toxin kills phagocytes and perhaps justification for talking about a plurality of anthrax binary toxins. clostridium tetani spores germinate in an infected wound and produce their toxin. spores are ubiquitous in faeces and soil, require the reduced oxygen tension for germination provided locally in the wound by foreign bodies (splinters, fragments of earth or clothing) or by tissue necrosis as seen in most wounds, the uterus after septic abortion, or the umbilical stump of the newborn.* the site of infection may be a contaminated splinter just as well as an automobile or battle injury. all strains of clostridium tetani produce the same toxin. it also reaches the central nervous system by travelling up other peripheral nerves following bloodborne dissemination of the toxin through the body. the motor nerves in the brain stem are short and therefore the cranial nerves are among the first to be affected, causing spasms of eye muscles and jaw (lockjaw). there is also an increase in tonus of muscles round the site of infection, followed by tonic spasms. in generalised tetanus there is interference with respiratory movements, and without skilled treatment the mortality rate is about %. botulism* caused by clostridium botulinum, a widespread saprophyte present in soil and vegetable materials. c. botulinum contaminates food, particularly inadequately preserved meat or vegetables, and produces a powerful neurotoxin. the toxin is destroyed at °c after min-of great importance to the canning industry-and there are at least seven antigenically distinct serotypes (a-g) produced by different strains of bacteria but which have a pharmacologically similar mode of action. it is absorbed from the intestine and acts on the peripheral nervous system, interfering with the release of acetylcholine at cholinergic synapses of neuromuscular junctions. somewhere between and hours after ingestion there are clinical signs suggesting an acute neurological disorder, with vertigo, cranial nerve palsies and finally death a few days later with respiratory failure. * botulus (latin) = sausage. in a large sausage was eaten by people in wildbad in germany; all became ill, and six died. the disease was subsequently referred to as botulism. t in recent years a less typical form of botulism has been described in small infants. the spores, present in honey applied to rubber teats, appear to colonise the gut, so that the toxin is produced in vivo after ingestion. tetanus and botulinum toxins are two of the most toxic substances known to man: g of botulinum toxin will kill mice. a great deal is now known about the genetics of both these toxins. tetanus toxin and botulinum toxin type g are encoded by plasmids, botulinum toxins types c and d are encoded by phages that infect clostridia, and botulinum type a is encoded in the bacterial genome; the genes have been sequenced. both toxins are synthesised as single peptides and are released from bacteria upon lysis. they are proteolytically activated by endogenous proteases to yield dichain derivatives consisting of -s -s -linked l (m r ) and h (m r ) chains. the recently adopted nomenclature reflects our current understanding of their peptide structures ( fig. . ). very recently, huge strides have been made in our knowledge of the mode of action of these toxins ( fig. . ). there is very little overall sequence homology between bonts and tetx; this could account for their immunological characteristics and the animal types normally affected. however, there is one structural feature common to all these neurotoxins located in the middle of the light chain and that is a zincbinding motif. in fact these neurotoxins are zinc endopeptidases. their targets are protein constituents of the synaptic fusion machinery responsible for the exocytotic release of neurotransmitters. the intracellular target cleaved by bonts b, d, and f and tetx is synaptobrevin (vamp, vesicle-associated membrane protein) present in small synaptic vesicles. bonts a and e cleave snap- (synaptosomal associated protein of m r ) a highly conserved protein known to be involved in exocytosis. bont c acts on syntaxin, a synaptic membrane protein also involved in exocytosis. however, some puzzles remain to be resolved. tetanus and botulinum toxins enter neuronal tissue preferentially at motoneuronal endplates, but the nature of the 'physiologically relevant ectoacceptors' is still not known. it remains unclear why botulinum toxin acts directly at the site of uptake and not, as observed with tetanus toxin, in the central nervous system, although a considerable amount of botulinum toxin (like tetanus toxin) is retrogradely transported. botulinum toxin blocks the release of acetyl choline at neuromuscular junctions to cause flaccid paralysis. likewise, one might ask why tetanus toxin fails to act at the motoneuronal junction at concentrations which would completely block release of neurotransmitter from gabaergic synapses. to reach inhibitory interneurons-its principal site of action-tetanus toxin must leave α-motoneurons after the primary uptake step, traverse the synaptic cleft to interneurons, leave those again in order to become finally internalised again from presynaptic membranes-a route identical to that of several neurotropic viruses. it acts by blocking the release of inhibitory transmitters (glycine or gab a) resulting in a failure to relax the affected muscle-pathophysiological 'tetanus'. only in rare cases does it act peripherally like botulinum toxin. the most toxic substances known to man are now being used as therapeutic agents to treat focal dystonias such as neck twists and eye squints, or eyelid closure and, more recently, some childhood palsies. the preparations are made from bont a and consist of toxin-haemagglutinin complex-the form in which the toxin is usually produced by the organism. this complex is less toxic than purified neurotoxin when administered parenterally but relatively more toxic when given orally; the haemagglutinin apparently protects the neurotoxin from proteolytic degradation in the gut. the effects of bont in relieving muscle spasms are not permanent but last for several months. treatment has to be repeated. to date the successes significantly outweigh the failures and no long-term adverse effects have been reported. antibody to the toxin has not so far been detected in the sera of the majority of patients. some toxins destroy membranes by virtue of their proteolytic activities, and some by their ability to degrade lipid components, while others are pore-forming or detergent-like in their mode of action. in addition to their action on protein components of lung connective tissue referred to above, pseudomonas aeragmosa-elastase and the zinc metalloprotease of legionella pneumophila are believed to destroy cell membranes by their proteolytic activity. this is the probable reason for the haemorrhage associated with lung infections caused by these pathogens, i.e. effects on type i alveolar epithelial and endothelial cells. clostridium perfringens α-toxin. a large number of bacterial enzymes are phospholipases some of which, but by no means all, are important toxins. the best example is the α-toxin of clostridium perfringens, the organism most commonly associated with gas gangrene. it is strictly anaerobic and occurs as a normal inhabitant in the large intestines of man and animals; its spores are ubiquitous in soil, dust and air. c. perfringens does not multiply in healthy tissues, but grows rapidly when it reaches devitalised and therefore anaerobic tissues. this could be after contamination of a natural wound with soil or dust, particularly on battlefields or in automobile accidents, or after contamination of a surgical operation site with clostridia from the patient's own bowels or skin. after abortions, particularly in the old days before antibiotics, intestinal clostridia often gained access to necrotic or devitalised tissues in the uterus and set up life-threatening infections. invasion of the blood was common and soon resulted in death, the clostridia localising and growing in internal organs such as the liver after death. c. perfringens has various enzymes that enable it to break down connective tissue materials, including collagen and hyaluronidase, thereby facilitating spread of the infection along tissue planes. most of the enzymes are toxic (a) reflex arc (top). mechanism for inhibiting the antagonists to a muscle contracting in response to stretch. muscles are reciprocally innervated with sensory and motor neurons, although for clarity this is shown only for the protagonist muscle. on stretch, the stretch receptors generate an impulse which is transmitted along the afferent sensory (s) neuron of the protagonist (p) muscle. this sp neuron enters the spinal cord by the dorsal root and synapses with the motor neuron supplying the protagonist muscle (mp) and with an interneuron (i) which in turn synapses with the motor neuron supplying the antagonist muscle (ma); the efferent motor neurons leave the spinal cord by the ventral root. at the sp/mp synapse an excitatory transmitter is released which induces an impulse in mp which leads to contraction of protagonist muscle. however, excitation of i causes release of an inhibitory transmitter at the i/ma synapse which leads to relaxation of the antagonist muscle. note that the basic reflex arc has been shown for simplicity but tetx acts mainly on voluntary muscles. (b) a simplified version of the biochemical events occurring in synapses (lower left). excitatory and inhibitory synapses, neurotransmitter release and action. gly, glycine; r, receptors of neuro transmitters; x, hitherto uncharacterised (candidates include glutamate, dopamine, atp, substance p, and somatostatin). to host cells and tissues, but α-toxin is easily the most important one. it is dermonecrotic, haemolytic (a feature seen mainly in tissues close to the focus of infection but sometimes responsible for large-scale intravascular haemolysis in infected patients), causes turbidity in lipoprotein-rich solutions and is lethal. while it is still true that these activities are all due to one molecular species, they are not different expressions of the one enzymic activity. historically, c. perfringens α-toxin was the first bacterial toxin to be characterised as an enzyme: it is a phospholipase c (plc) which removes the head group, phosphoryl choline, from phosphatidyl choline and from sphingomyelin. it is of undoubted importance in gas gangrene. toxoid prepared by formalin-treated toxin will protect sheep against infection caused by c. perfringens. however, one might ask why all enzymes with such biochemical specificity are not equally toxic or important in determining virulence; there are several reasons which can be put forward. by the combined use of monoclonal antibodies and molecular genetic analyses we now know that there are at least two functional domains in this c. perfringens α-toxin. if one compares c. perfringens α-toxin with the phosphatidyl choline-preferring, nontoxic phospholipase of bacillus cereus, one finds that two-thirds of the n-terminal sequence shows homology with the entire sequence of b. cereus plc. this portion of c. perfringens α-toxin retains its plc activity but not its haemolytic and lethal activities. the c-terminal part is not haemolytic, not enzymatically active and not cytotoxic for mouse lymphocytes, but is necessary for conferring toxicity on the n-terminal part of the protein. in fact, the c-terminus is a potent immunogen that will solidly protect mice -and hopefully man-against experimental infection with c. perfringens. surprisingly, the c-terminal domain of the nontoxic c. bifermentans enzyme shows sequence similarity with that of its c. perfringens α-toxin counterpart. the nontoxicity of this enzyme is ascribed to its comparatively much lower turnover rate, i.e. it is a much less efficient enzyme. recently, attention has been drawn to the distinct possibility that while haemolysis may be initially induced by the plc activity of c. perfringens α-toxin, it is possible that actual disruption of the membrane is due to the activation by the toxin of other phospholipases (phosphatidylinosotol- , -biphosphate (pip ) specific phospholipase and phospholipase d) present in the erythrocyte membrane. some now believe (c) sites of neurotoxin action (lower right). the predominant site of action of tetx is the intermotor neuron synapse; the exocytotic machine is interfered with by the endopeptidase action of tetx on vamp. bont acts at the neuromuscular junction inhibiting the release of acetyl choline (ach) by its proteolytic action on vamp (types b, d and f), or snap (types a and e), or syntaxin (type c). (amplified from figs and , 'bacterial toxins', nd edition, by j. stephen and r. a. pietrowski ( ), pp. and , van nostrand reinhold (uk).) that the basis of toxicity is not simply a cytolytic one, but rather a consequence of its ability, in sublytic doses, to release inosotol triphosphate (ip ) and activate the arachidonic acid cascade. there are other pathogenic clostridia that cause gas gangrene and produce similar toxins. infected tissues show inflammation, oedema and necrosis, not necessarily with the formation of gas, and the illness can be mild or very severe according to the extent of bacterial spread and the nature and quantity of toxins that are formed and absorbed. since the bacteria grow and produce their toxins only in devitalised tissues, the most important form of treatment is to remove such tissues. clostridia are strictly anaerobic, and exposure of the patient to hyperbaric oxygen (pure oxygen at - atmospheres in a pressure chamber) has been found useful in addition to chemotherapy. staphylococcal ß-toxin. staphylococcal ß-haemolysin is known to be produced in vivo. in ch. studies with isogenic mutants were described which indicate that it is important in killing neutrophils. it probably has the narrowest substrate specificity among the phospholipases, and is a hotr-cold haemolysin: lysis of erythrocytes occurs only on cooling after incubation at °c. the phenomenon, though of doubtful significance in vivo, has attracted attention and generated speculation about its mechanism. perhaps the most likely explanation is that, when cooled below their phase-transition temperature, the remaining phospholipids undergo quasi-crystalline formation, thereby generating intramembranous stresses incompatible with structural integrity. these proteins (made by some species, not all of which are pathogens) have been called oxygen-labile haemolysins because they are reversibly inactivated on standing in air, and their most studied property is their ability to lyse red cells. sh-activation of crude preparations is necessary for the expression of haemolytic activity but they are also active towards a variety of cell types. thiol-activated cytolysins share the following properties: they are cross-neutralised by hyperimmune sera, lyse a wide range of species of erythrocyte, exhibit similar ph and temperature optima for cytolytic activity, are lethal and cardiotoxic, and lose activity on incubation with erythrocyte ghosts. they are inactivated irreversibly by small amounts of cholesterol. interaction with cholesterol is the key primary event in their interaction with susceptible membranes which leads to the impairment of the latter; cholesterol plays no further part in the subsequent damage process. a schematic outline of how they damage cell membranes is shown in fig. . . several facts have recently reopened considerable interest in this group. first, the recognition of what these substances do to host defence cells when presented in sublethal doses. for example, pneumolysin from s. pneumoniae inhibits the respiratory burst in neutrophils, inhibits antibody synthesis in b cells and activates the classical complement pathway in the absence of antibodies. since complement is an important defence mechanism against the pneumococcus in vivo this could lead to depletion of complement levels and abrogate protection. in fact, immunisation of mice against pneumolysin affords some protection against challenge infection. second, in addition to its role in promoting invasive bacteraemia, pneumolysin has also been implicated in causing sensorineural deafness associated with meningitis caused by the pneumococcus; this perception is based on very recent work with a guinea-pig other pore-forming toxins t h e r t x toxins h a v e a l r e a d y b e e n referred to above in connection w i t h b. pertussis a c -h l y . b u t t h e r e a r e o t h e r pore-forming toxins. staphylococcal δ-toxin. this toxin acts in a manner similar to that of the sh-activated cytolysins, with an important difference: there is no initial specific binding. δ-toxin initially forms small pores and then islands of membrane or large micelles; this gives rise to its perceived detergent-like properties. there is a family of closely related δ-toxins which inhibit the growth of gonococci. it is not often that one can ascribe a positive function to a toxin which is beneficial for the organism producing it. in this case -toxin(s) could have important ecological significance in the mixed culture situation that is characteristic of the real microbial world. of great interest is the synergy that δ-toxin displays. sublytic amounts of δ-toxin causes release of cell constituents without lysis. but, only . haemolytic units of staphylococcal ß-toxin will cause lysis of cells in the presence of . lytic units of δ-toxin. this synergistic interaction could be the way in which staphylococcal toxins, which rarely exert their lethal effects in the majority of infections, exercise important cytolytic effects. of less obvious significance is the fact that δ-toxin is a poor antigen; for a long time its antigenicity was controversial. if δ-toxin were to prove of crucial importance as a cytolytic potentiator, then this could also partly explain why natural acquired immunity to staphylococcal infection is either non-existent or sufficiently low as to be easily overcome. staphylococcal a-and -toxins. staphylococci produce a range of toxins several of which we have already met. the α-toxin is considered as the main cytolysin produced by s. aureus. like streptolysin- and staphylococcal δ-toxin, it is secreted as a water-soluble protein and undergoes self-induced oligomerisation on cell membranes to form pores. it has recently been suggested that transmembrane channels formed by the hexameric form allow the penetration of the monomeric form which interferes with certain steps in translation. in systemic staphylococcal infections death is most probably due to the potent α-toxin but in localised pyogenic infections-such as mastitis in cattle, goats, rabbits and mice-its role is most likely one of killing phagocytes or conferring survivability on intracellular bacteria. -toxin is a binary toxin with a haemolytic action on rabbit erythrocytes. there is evidence that α, β and haemolysins and leucocidin are produced in vivo during natural and experimental infections. this is based on the presence of antibodies to these haemolysins in subjects undergoing staphylococcal infection and the extraction of toxin from peritoneal abscesses in mice. however, the precise roles that these or any other staphylococcal toxins play in localised infections is not clear. atrophie rhinitis is a disease of pigs in which the principal clinical sign is atrophy of the nasal turbinate bones and shortening of the snout. scientists have isolated and purified a toxin believed to be important in the causation of this disease. this is a remarkable toxin on at least two counts. it shows an incredible predilection for the target tissue in that it causes turbinate atrophy in gnotobiotic pigs, even when injected intraperitoneally; it also causes necrosis of spleen and thymus. perhaps even more surprising is the fact that it is a potent mitogen, hardly the kind of activity that one would associate with atrophy! one can only assume that it upsets the dynamic balance between the laying down and resorption of mesenchymal bone tissue by osteoblasts and osteoclasts respectively. the remainder of this section deals with other important (or potentially important) toxins which have not already been referred to in earlier chapters or in the section above. several staphylococcal toxins have already been referred to above. staphylococci are ubiquitous and responsible for a large group of diseases which range from the relatively harmless skin pimple through abscesses, impetigo, food poisoning, osteomyelitis, mastitis, primary pneumonia, staphylococcal scalded skin syndrome (ssss) and toxic shock syndrome (tss) to fatal septicaemias. in the case of ssss a toxin is known and is described on p. . toxic shock syndrome toxin (tsst- ). tss is seen characteristically in menstruating women whose tampons harbour multiplying staphylococci. it is due to a toxin called toxic shock syndrome toxin (tsst- ; one of the so-called staphylococcal enterotoxins, see next paragraph). toxic shock syndrome is characterised by sudden onset of fever, vomiting, diarrhoea, an erythematous rash followed by peeling of the skin, hypotensive shock, impairment of renal and hepatic functions and occasionally death. the main symptoms of the disease have been reproduced in rabbits by implanting chamber-enclosed tss-strains in the rabbit uterus or peritoneum or by injection of tsst- into rabbits. complex changes are observed including: haemorrhage in kidney and liver; congestion and haematomas in the lungs; leakage of blood into the thymus; and fluid in the pericardial sac and in the gut lumen. these effects in rabbits are very similar to those seen in humans and would certainly explain the shock and diarrhoeal syndrome so characteristic of the disease. all of this indicates that the primary effect of tsst- is on the integrity of capillary walls. the lethal effect of tsst- is enhanced considerably by endotoxin (see below). such synergy is also known for streptococcal erythrogenic toxin and lps, but the mechanism(s) involved in this complex synergy is not clear. although relatively uncommon in the uk, staphylococcal food poisoning accounts for over % of all cases of food poisoning in the united states. the disease is characterised by vomiting and diarrhoea commencing - h after consumption of contaminated food, especially dairy produce. symptoms usually last no longer than h. like botulism, staphylococcal food poisoning is commonly caused by the ingestion of food containing preformed toxins, known collectively as the staphylococcal enterotoxins. they are serotypically heterogeneous single-chain globular proteins, of molecular weight between and kda, secreted by certain strains of s. aureus. one of these, serotype f is identical to tsst- and staphylococcal pyrogenic exotoxin c. these toxins are not classical enterotoxins acting on intestinal cells but 'neurotoxins' activating receptors on the abdominal viscera, the stimulus reaching the vomiting centre via the vagus nerve. several of the staphylococcal toxins referred to above (tss- and enterotoxins), and also the erythrogenic toxins a and c of streptococcus pyogenes (see below) have an additional important action. they are among the most powerful t cell mitogens known, acting at picomolar concentrations. they bind to mhc class ii molecules on antigenpresenting cells and interact with the vß chain of the t cell receptor (see chs and ). this causes proliferation and cytokine release by the entire subset of t cells bearing that particular vß chain- - % of all t cells are affected, whereas only . - . % of t cells respond to a given regular antigen. this represents an important interference with a coordinated immune response, and the widespread polyclonal activation and cytokine release can be regarded as a microbial strategy, a 'diversion' of host immune defences. in addition, if the superantigen reacts with developing t cells (with the correct vß chain) in the thymus, these cells are deleted. it seems probable, therefore, that this is a more important biological function of these toxins than the one responsible for the characteristic disease, which may be no more than an 'accidental' phenomenon. it turns out that similar molecules are formed by mycoplasma and by certain retroviruses (e.g. the mis antigen of mouse mammary tumour virus). scarlet fever is one of the many conditions caused by streptococcal infection and may accompany a streptococcal sore throat or occasionally a streptococcal wound infection. there is a generalised erythematous rash together with fever and a sore throat. the rash is due to an erythrogenic toxin produced by certain strains of streptococcus pyogenes. erythrogenic toxin is a low molecular weight protein produced in a complex form with hyaluronic acid, which acts as a carrier. the protein consists of two parts. one is heat labile, carries the determinants of immunological specificity which give rise to three serotypes, a, b and c, and is responsible for primary toxicity manifestations, including pyrogenicity, low lethality, cytotoxic effects on cultured spleen macrophages, and suppression of the reticuloendothelial and immune systems. the second part is heat stable, antigenically common to types a, b and c, and is responsible for secondary toxicity; that is, hypersensitivity effects, including skin hyperreactivity (the basis of the rash), myocardial necrosis, enhancement of pyrogenicity and lethality, and enhanced host response to other injurious agents. thus, it is not possible to define the biological activity of 'erythrogenic' toxins without considering the immunological state of the host. an individual may suffer no reddening of the skin upon intradermal injection of erythrogenic toxin (negative dick test) because a high neutralising titre of antibody blocks the primary toxiphore or because of a lack of hypersensitivity to the common antigen. streptolysins o and s are dealt with in ch. . there is a plethora of toxins produced by numerous clostridial pathogens important in both human and veterinary medicine. the clostridial genus comprises a large number of toxigenic species, some of which are known to produce several toxins, extracellular enzymes, and other factors which are as yet recognised only by a letter of the greek alphabet. all the α-toxins are dermonecrotic and the others have haemolytic, enzymatic properties. toxin production in vitro is used as the basis of typing clostridia, and the picture is complex. no attempt will be made to describe every disease in man or animals associated with clostridia; only those are selected which best serve to illustrate the involvement of some recognisable toxins. in the case of sheep diseases-lamb dysentery, struck, enterotoxaemia, black disease, braxy, black quarter-some of the best evidence for implicating relevant toxins comes from field studies using multivalent vaccines (based on toxoids of these toxins). clostridium perfringens type c causes pig bei in man, essentially due to the production of ß-toxin. this is a rare disease in developed societies but a public health hazard in papua new guinea. four factors are responsible: the ubiquity of c. perfringens type c in the soil and faeces of man and pigs; the relatively low immunogenicity of ß-toxoids in young children, the group most at risk; the high carbohydrate, low protein nature of the staple diet; and the sporadic consumption of large quantities of pork on occasions of celebration. the latter dietary change promotes a proliferation of clostridia in the intestine which may lead to intestinal gangrene and death. ß-toxin damages the mucosa, reduces mobility of villi and causes more bacteria to become attached to the villi. more toxin is absorbed and the mucosa and underlying the intestinal wall become necrotic, leading to death in many cases. the influence of diet is additionally important in t h a t low protein diets cause decreased secretion of pancreatic proteolytic enzymes and sweet potato contains a trypsin inhibitor. these conditions promote the survival of ß-toxin which is highly sensitive to proteolytic inactivation. immunisation with ß-toxoid preparations has dramatically lowered the incidence of this fatal disease in children. gas gangrene in man may be caused by several bacterial species separately or in concert. these include clostridium perfringens type a, c. novyi types a and b, and c. septicum; c. perfringens and its a-toxin have already been discussed. far less is known about c. novyi and c. septicum and their toxins in gas gangrene in man. much more is known about the role these organisms play in diseases of animals, and multivalent vaccines confer a very high degree of immunity (particularly to sheep) against several clinically identifiable but separate diseases. a few of these diseases are described below. clostridium perfringens type b causes lamb dysentery. this is an acute, fatal disease of young lambs occurring during the first week of life and caused by absorption of toxin(s) generated by c. perfringens type b in the small intestine. clostridium perfringens type c causes struck in sheep, a disease occurring in the romney marshes of kent, but rare in other areas in the world. the pathological changes observed differ markedly from other enterotoxaemias and include enteritis. clostridium perfringens type d enterotoxaemia in sheep is another acute fatal disease. the most constant lesion is subendocardial haemorrhage around the mitral valve. c. perfringens type d e-toxin or its protoxin are recoverable from intestinal contents. clostridium novyi type b causes black disease of sheep or infectious necrotic hepatitis. this is an acute infectious disease of sheep (occasionally cattle) caused by the absorption of the α-toxin elaborated by the organism in necrotic foci in the liver, and is nearly always associated with invasion of the liver by immature liver flukes. how c novyi gets to the liver in the first place is not known but it is readily demonstrable in livers of normal sheep in areas where the disease is prevalent. experimental reproduction in guinea-pigs of a similar disease is possible by the combined action of c. novyi spores and liver fluke infestation. clostridium novyi type d causes a rapidly fatal disease in cattle (redwater disease) similar to, and regarded by some as an atypical manifestation of, black disease. the characteristic lesions include jaundice, various haemorrhagic manifestations, and anaemic infarcts in the liver; active liver fluke infestation may or may not be present. in culture this organism produces ß-toxin, which explains the haemoglobinuria, but no a-toxin. clostridium septicum causes braxy in sheep. the role of c. septicum in this acute, fatal disease is assumed because of its association with the characteristic haemorrhagic inflammatory lesion in the abomasum. the disease has not been reproduced experimentally with c. septicum but can be prevented by immunisation with sterile toxoids derived from this organism. clostridium chauvoei causes black quarter in sheep and cattle. this is a gas gangrene type infection of muscles and associated connective tissues in cattle and sheep; c. chauvoei is also the causative agent of parturient gas gangrene in sheep. the initial stimulus which activates the infection in cattle is not known, since the disease is hardly ever associated with any overt wounding. washed spores alone do not cause disease when injected, but do in conjunction with a tissue-necrotising agent. in sheep, wounding caused by parturition, castration, tailing, shearing, vaccination, as well as accidental damage will create a focus within which c. chauvoei can multiply. there are numerous examples now known of synergistic reactions between toxins of the same or different species. bacillus cereus makes a phosphatidyl choline-preferring phospholipase and a sphingomyelinase which are separately nontoxic; in concert they are haemolytic and termed cereolysin a-b. the examples of staphylococcal a-and δ-toxins and of staphylococcal tsst- and endotoxin, have already been alluded to above. streptococcal erythrogenic toxins increase sensitivity to other streptococcal factors (for example, streptolysin-o) and also to gramnegative endotoxin. the susceptibility of rabbits to endotoxin is increased times, and adult cynomolgus monkeys die within h, when injection of low levels of steptococcal toxin is followed h later by an otherwise sublethal dose of endotoxin. this may be because streptococcal toxin creates a state of hypersusceptibility to a wide variety of stressful agents. perhaps, in view of the high incidence of exposure of man and his domestic animals to streptococci, we should actively investigate possible toxin-mediated synergy in mixed infections involving streptococci. another entirely different type of example is that of the increase in toxicities of staphylococcal a-and -toxins, diphtheria toxin and endotoxin for neonatal ferrets preinfected with influenza virus. increases were -, -, -and -fold respectively. no increase in viral replication was observed. neonates died suddenly without clinical symptoms as in human babies dying from sudden infant death syndrome (sids). pathological examination showed inflammation of the upper respiratory tract, lung oedema and collapse, and early bronchopneumonia in animals receiving the dual challenge but not those receiving either toxin or virus on their own. thus, some bacterial toxins in conjunction with influenza virus could be one of the several causes of sids. many fungi contain substances that are harmful when taken by mouth, and there are two diseases that result from the ingestion of food containing preformed fungal toxins. as with c. botulinum, the disease is caused without the need for infection. aspergillus flavus infects ground nuts (monkey nuts) and produces a very powerful toxin (aflatoxin). contaminated (badly stored) ground nuts used to prepare animal feeds caused the death of thousands of turkeys and pigs in the uk in and the survivors of intoxication nearly all developed liver cancer. human disease has not yet been associated with this toxin. clavicepspurpurae is a rust fungus affecting rye, and it produces toxins (ergotamine especially) that give rise to ergot poisoning when contaminated grain is eaten. mushrooms and toadstools have long been recognised as sources of poisons and hallucinogens. unlike the toxins already discussed in this chapter, there is a group of toxins which are distinct structural components and are not released into the surrounding medium in any quantity except upon death and lysis of the bacteria. several protein toxins (e.g. clostridium difficile toxin a, tetanus toxin) are released during the decline phase of batch culture-probably on autolysis-and these are classified as exotoxins. here we deal with toxins which are known to comprise well-recognised structural entities which on a priori grounds must have key functions in the organism: they are found in the outer membranes of gram-negative organisms. there are two chemically distinct types of toxin considered: lipopolysaccharide (endotoxin; lps) and protein. the bulk of this section is taken up with endotoxin. many pathogenic organisms, however, are pathogenic by virtue of possessing various types of surface structure important in conferring virulence. these include, for example, adhesins which are important in colonising body surfaces or a variety of surface molecules (which may or may not be inside capsules) which render them resistant to phagocytosis. but the majority of adhesins and anti-phagocytic determinants are themselves nontoxic. the gram-negative bacterial cell wall is subject to considerable variations in both the composition of lps and in the number and nature of the proteins found in the outer cell membrane. apart from the examples given in ch. in relation to the gonococcus, such phenotypic variation in lps has rarely been examined in the context of pathogenicity. however, the examination of cell-bound proteins of yersinia pestis from organisms grown in vivo led to the discovery of a toxin lethal for mice and guinea-pigs. plague is one of the most deadly diseases of man and has, over several thousands of years, claimed millions of lives. in the fourteenth century, 'the black death' wiped out a quarter of the population of europe before spreading through the middle east and asia. fortunately, however, the last years or so have seen a drastic decrease in outbreaks of plague, though the threat of another epidemic is still with us. the causative organism of plague, yersinia pestis, is primarily a parasite of rodents in which it is endemic in many areas of the world. only when man comes into close proximity with infected rodents do outbreaks of human plague occur. the disease is spread from rat to rat and from rat to man by fleas. rodents in the terminal stages of infection with y. pestis suffer massive bacteraemia and so when a flea sucks the blood of an infected animal it swallows large numbers of bacteria. when the rodent eventually dies, the flea leaves the corpse and awaits a new host. in the meantime, however, y. pestis multiplies rapidly in the alimentary tract of the insect, often completely blocking the proventriculus. the flea becomes voraciously hungry and feeds on any suitable host, which sooner or later is man. however, because the flea's alimentary canal is blocked by bacteria it cannot feed efficiently and usually succeeds in imbibing blood only to regurgitate it, now contaminated with y. pestis, back into the wound. during the next - days, organisms become localised and multiply in the regional lymph nodes, causing considerable swelling. these swellings, which are often in the axillary and inguinal regions because the most common sites for flea-bites are the arms or legs, are referred to as primary buboes (greek boubon = groin); hence the name bubonic plague. secondary buboes may develop, followed by necrosis of the lymphoid tissue and vascular damage giving rise to haemorrhaging in many organs and tissues. these pathological changes are accompanied by prostration, high fever and delirium, followed in the terminal stages of the disease by shock and death. it is also possible for the disease to be transmitted from human to human by the aerosol route: exhaled organisms from heavily infected patients are capable of infecting others giving rise to pneumonic plague. the principal features of human plague can be reproduced in guineapigs and mice. monkeys show shock-like signs only during the terminal period of - h, when they become quiet, progressively weak, prostrate and hypothermic; for the previous - days infected animals are lively and vigorous. in the terminal stages blood pressure drops rapidly but there is no evidence of oligaemia (low blood volume) caused by haemorrhage, or of oedema, suggesting that vascular collapse must be associated with a vasodilatory factor(s), resulting in pooling of blood. in this respect monkeys differ from humans and guinea-pigs. the symptoms of plague-high fever and vascular damage -are characteristic of intoxication with endotoxin. however, it is extremely unlikely that endotoxin alone is the main toxin involved in plague. it is much more likely to act in conjunction with one or more other potentially toxic fractions from y. pestis. plague murine toxin is a protein which, although highly lethal for mice and rats, is relatively nontoxic for guinea-pigs, rabbits, dogs and monkeys. a completely separate guinea-pig toxin complex exists comprising at least two cell wall/membrane protein components, one of which will kill mice, although both are needed to kill guinea-pigs. however, the nature of the toxin or toxins of y. pestis and their role in the human disease syndrome are still far from clear. the classical approach to such a problem is to prepare a specific toxoid and to determine whether injection of this confers immunity to the disease. needless to say, the severity of human plague renders such experiments impossible. until such questions can be answered, we are left to argue whether, in the context of plague, man resembles a mouse, a monkey or a guinea-pig. endoxotins are part of the outer membrane of gram-negative bacteria. it has been known for many years that the cells (alive or dead) or cell extracts of a wide variety of gram-negative bacteria are toxic to man and animals. the literature on this subject is vast, sometimes confusing and often controversial; here we can give no more than a brief outline. some of the diseases in which endotoxin may play an important role include typhoid fever, tularaemia, plague and brucellosis, and a variety of hospital-acquired infections caused by opportunistic gram-negative pathogens which include escherichia coli, proteus, pseudomonas aeruginosa, enterobacter, serratia and klebsiella. in addition, endotoxin has been intensively studied as a possible causative agent of shock arising from post-operative sepsis or other forms of traumatic injury in which the normal flora of the gut is often the source of endotoxin. the toxins we have considered so far have been protein (or at least part protein) in nature but, in contrast, endotoxin is a complex lipopolysaccharide. it is also much more heat stable than protein toxins and much less easily toxoided. in addition to lethality, endotoxin displays a bewildering array of biological effects. the complex nature of the multi-layered gram-negative bacterial envelope is shown in fig. . (see also fig. . ) . the outer membrane is composed of a bimolecular leaflet arrangement as are other membranes but has a different composition from the cytoplasmic membrane. the lipopolysaccharide (lps) is unique in nature, only found in gramnegative bacteria, and is, or contains within it, what we designate endotoxin. immunoelectron microscopy indicates that lps exists in the outer leaflet of the membrane and extends outward up to nm; it is on, rather than in, the cell. thus it is evident that the term endotoxin is a misnomer which derives from the era when toxins were considered to be either exotoxins, which were synthesised and secreted by the viable organism, or endotoxins, which were intracellular and released only upon lysis. moreover, extraction with edta shows that approximately % of lps is held noncovalently linked in the membrane. extraction with a variety of different solvents yields material which is highly heterogenous and of apparent molecular weight - x . however, treatment with pyridine or addition of detergents reduces the polydispersity. the endotoxic glycolipid from the rough mutant of salmonella minnesota, r , has an m r of for the basic unit, from which complex aggregate structures are derived. lipopolysaccharide consists of three regions: polysaccharide side chains, core polysaccharide, and lipid a which consists of a di-glucosamine backbone to which long chain fatty acids are linked (fig. . ). the relationship of this type of molecule to the outer membrane is also shown in fig. . . the long chain fatty acids interdigitate between the phospholipids in the outer leaflet and may also be linked (or interact) with lipoproteins, which in turn may or may not be covalently anchored to the rigid peptidoglycan (pg). the polysaccharide side chains project outwards. this structure is not invariant. for example, many organisms when first isolated give rise to colonies with a smooth appearance on agar but on subculture produce colonies with a rough appearance. in general, 'smooth' strains of pathogenic species are more virulent than rough strains. this s->r conversion is accompanied by a loss of region i side chains, which contain the deoxy and dideoxy sugars found in these lps complexes. in addition to these somewhat drastic changes involving loss of side chains, it is possible to induce major compositional changes by manipulating the growth rate of these organisms in a chemostat. thus the lps of salmonella enteriditis, when grown with a mean generation time of min is nearly totally deficient in tyvelose (a dideoxy sugar), possesses % of the galactose and % of the glucose contents of lps obtained when the generation time is min. these genotypic s organisms exhibit an r-phenotype in terms of their vastly reduced o-agglutinability (see below); such observations are potentially very important in the context of the in vivo phenotype and pathogenicity, since it is well known that the growth rate of salmonella typhimurium in mice is - times lower than in vitro. examples have already been given in ch. of changes in lps structures in vivo in relation to neisseria gonorrheae and neisseria meningitidis. the extent to which lipid a is common between different genera is uncertain, but it is not likely to vary tremendously. the core polysaccharide structure is the same or very similar within groups of the enterobacteriaciae: thus polysaccharides from salmonellae are similar to each other, but differ from those of e. coli strains. however, within a group such as the salmonellae, there is a wide variation in the composition and detailed structures of the side chains, a fact which is exploited in the kauffman-white scheme for classifying salmonellae, giving rise to several thousand serotypes. the side chains carry the o-somatic antigen specificities of which there are far more than can readily be accounted for on the basis of the known number of sugars involved in the basic repeating units. in the side chains are found a range of depxy and dideoxy sugars. the general principles governing the relationship between the various chemotypes and serotypes are now well understood; the multiplicity of antibody specificities evoked may be explained in terms of antibodies which can recognise different aspects of one three-dimensional structure. lipid a is the primary toxiphore, but the polysaccharide plays an important part in conferring solubility upon, and optimising the size of micellar aggregates of lps, hence affecting biological activity. however, the immune status of the test animal may affect toxicity: as normal animals produce antibodies to the antigenic determinants on the surface of normal gut organisms (including o-somatic antigens), some of the biological effects of endotoxin may be mediated by hypersensitivity mechanisms. the most powerful evidence that lipid a is the primary toxiphore comes from studies on smooth (s) and rough (r) mutants whose biosynthetic capabilities are blocked at various points. this established that neither the o-side chains, nor the core polysaccharide are necessary for endotoxicity. pure lipid a can be made toxic by complexing it to a hydrophilic carrier like bovine serum albumin. one can make synthetic lipid a preparations which are toxic and assume space-filling configurations, which make it easy to see how they could fit into and be part of a bimolecular leaflet arrangement in the outer membrane. the range of biological properties of endotoxin is quite bewildering and the mode(s) of action very complicated. included among those effects which might play a role in gram-negative bacterial infections, are abortion, pyrogenicity, tolerance (not immune tolerance), the schwartzmann phenomenon, hypotension and shock, and lethality, but the precise part played by lps in these phenomena in gram-negative infections is far from clear. lps causes the release of vasoactive substances, activates the alternative pathway of the complement cascade, and also activates factor xii (hageman factor), the first step of the coagulation cascade, which sometimes results in disseminated intravascular coagulation (p. ). many, perhaps nearly all, the actions of lps are due to the stimulation of cytokine release from macrophages and other cells. there is an effect on the circulation, leading ultimately to vascular collapse. the vascular regions most affected differ from species to species; in man and sheep the main changes are found in the lungs. lps has powerful immunological actions, which is surely no accident; as well as activating the complement system, it induces il- production and is a potent b cell mitogen. man is one of the most sensitive of all species to the pyrogenic action of endotoxin. a dose of n g per kg of body weight injected intravenously into man causes the release of an endogenous pyrogen (interleukin- , see glossary) and tumour necrosis factor (tnf) from macrophages, which act on the hypothalamus to give an elevation of body temperature within an hour. it is possible that the pyrogenic action of lps helps to generate fever in gram-negative bacterial infections, but lps is not the only bacterial factor capable of inducing a febrile response. in spite of all these toxic actions, there have been suggestions that some of the responses to lps (by macrophages, polymorphs) could be advantageous to the host, possibly assisting in the recognition and destruction of bacteria. could it be that host responses to lps are, like the complement or the clotting systems, useful in moderation but harmful in excess? there are reports that when animals with less vigorous responses to lps are infected they suffer fewer symptoms, but permit greater growth of bacteria. very large numbers of gram-negative bacteria are normally present in the intestines (see ch. ), their continued death and exit in the faeces being balanced by multiplication in the lumen. there is a continuous, inevitable low-grade absorption of endotoxin from the intestine.* absorbed (endogenous) endotoxin enters the portal circulation and is taken * in addition, various antigens are absorbed in small quantities from the intestine, and in normal individuals antibodies are formed against various food proteins and to some extent against resident intestinal bacteria (see ch. ). kupffer cells remove any antigen-antibody complexes formed locally in the intestine and prevent them from entering the systemic circulation. up and degraded by reticuloendothelial cells, mainly kupffer cells in the liver. continuous exposure to endotoxin probably has profound effects on the immune system and on the histology of the intestinal mucosa, stimulating development of the immune system in the immature individual, but there are no obvious pathogenic consequences. normal people have low levels of antibody to endotoxin as a result of this continuous exposure. the sick individual may be much more susceptible to endogenous endotoxin, perhaps because of defects in removal by kupffer cells. after trauma or after genito-urinary instrumentation endotoxin is detectable in peripheral blood by the limulus test,* but this leads to no particular signs or symptoms. when large amounts of endotoxin enter the blood there are profound effects on blood vessels with peripheral vascular pooling, a drastic fall in blood pressure, collapse and sometimes death. thus, if enough endotoxin enters the blood during massive gramnegative bacterial sepsis, the vasomotor action of endotoxin becomes important and shock intervenes.! in experimental animals endotoxin also causes vasodilation and haemorrhage into the intestinal mucosa, and sometimes haemorrhage into the placenta with abortion, but these actions do not appear to be important in all gram-negative bacterial infections. to summarise, endotoxin, although studied so carefully and for so long, has not yet been shown to play a definitive role as a toxin in the pathogenesis of any infectious disease. however, there is a growing body of opinion that seeks to implicate endotoxin in the inflammatory response induced in meningitis caused by gram-negative bacteria. but, in spite of its effects on various host defence systems including polymorphs, lymphocytes, macrophages, complement, and on endothelial cells and platelets, its overall role in infection is still not clear. it can, however, cause shock when gram-negative bacteria invade the blood. it is for this reason that considerable effort in recent years has gone into the development of antilipid a antibodies for use as therapeutic agents to combat shock in such situations; the success rate is only partial and the expense enormous. for that reason several groups are seeking to exploit the wealth of chemical and biophysical information available on lps in attempts to develop synthetic derivatives which would neutralise the biological activity of lipid a. we await the outcome of such research. however, the characteristics of the o-antigen polysaccharide are sometimes important in determining virulence (pp. - ). * a sensitive test for endotoxin based on the ability of endotoxin to induce gelation of a lysate obtained from the blood cells of the horseshoe crab, limulus polyphemus. considerable space has been given to toxins because they are being intensively investigated as possible virulence determinants. the account illustrates the complexity of host-microbe interactions when analysed at the molecular level. most toxins are liberated from the microbial cell and can be studied with greater facility than many of the more elusive determinants of pathogenicity. but remember that microbes that replicate inside host cells are less likely to form powerful toxins because they cannot afford to damage at too early a stage the cell in which they are multiplying. thus, toxins are not prominent products in intracellular infections due to mycobacteria, brucella, rickettsiae, or chlamydia, and viruses do not form toxins. although a single molecule of a toxin like diphtheria toxin is enough to kill a cell, other toxins may do no more than impair cell function when present in sublethal concentrations. this can lead, for instance, to defective function in immune or phagocytic cells. low concentrations of the streptococcal streptolysins* will inhibit leucocyte chemotaxis. the ability to form toxins, whether encoded by plasmids or the microbial genome, is subject to selective forces. if toxin production puts a microorganism at a serious disadvantage it will tend to disappear. if it is advantageous it will be maintained, and will spread through the microbial population, just as the genetic changes that confer resistance to antimicrobial drugs are selected for when these drugs are widely used. it is therefore not unreasonable to ask how many of the well-known toxins are actually useful to the microbe as well as being important in causing disease in the host (table . ). however, microbes that multiply extracellularly must produce a variety of enzymes and other molecules involved in nutrition, adherence to substrate, and so on. in the case of free-living microbes these substances, as well as substances that damage or interfere with competing organisms, are of major importance. probably they cannot all be discarded when the parasitic mode of life is adopted. many will have a toxic action. yet, for the infecting microbe, these substances remain as unfortunate necessities, of no particular advantage and perhaps a disadvantage, in the parasitic way of life. in infectious diseases there is nearly always a certain amount of direct microbial damage to host tissues, as discussed above. host cells are destroyed or blood vessels injured as a direct result of the action of * the streptolysins are responsible for ß-haemolysis. most haemolysins will kill phagocytes. ) . inflammatory materials are liberated from necrotic cells, whatever the cause of the necrosis. also many bacteria themselves liberate inflammatory products and certain viruses cause living infected cells to release inflammatory mediators. therefore it is not always clear how much of the inflammation is directly microbial rather than host in origin.* but inevitably the host (see ch. ) generates inflammatory and other tissue responses, and these responses sometimes account for the greater part of the tissue changes. pathological changes can then be regarded as occurring indirectly as a result of these responses to the infection. inflammation causes redness, swelling, pain and sometimes loss of function of the affected part (see ch. ) and is generally a major cause of the signs and symptoms of disease. indirect damage attributable to the host immune response is discussed separately below. in most diseases direct and indirect types of damage both make a contribution to pathological changes, but in a given disease one or the other may be the most important. in a staphylococcal abscess the bacteria produce inflammatory materials, but they also kill infiltrating polymorphs whose lysosomal enzymes are thereby liberated and induce further inflammation. this type of indirect nonimmunological damage is sometimes important in streptococcal infections. virulent streptococci produce various toxins that damage phagocytes, and also bear on their surfaces substances that impede phagocytosis (see ch. ). nevertheless, with the help of antibody, all streptococci are eventually phagocytosed and killed and the infection terminated. unlike the staphylococci, however, killed group a streptococci pose a digestive problem for phagocytic cells. the peptidoglycan component of the streptococcal cell wall is very resistant to digestion by lysosomal enzymes. when streptococci are injected into the skin of a rabbit, for instance, streptococcal peptidoglycans persist in macrophages for as long as days. hence macrophages laden with indigestible streptococcal cell walls tend to accumulate in sites of infection. lysosomal enzymes, including collagenase, leak from these macrophages, causing local destruction of collagen fibres and the connective tissue matrix. macrophages secrete many other substances some of which may contribute to cell and tissue damage (see also p. ). many macrophages eventually die or form giant cells, sometimes giving rise to granulomatous lesions (see p. ). in this way persistent streptococcal materials sometimes cause chronic inflammatory lesions in the infected host. an additional immunopathological contribution to the lesions is to be expected if the host is sensitised to peptidoglycan components. other pathogenic microorganisms that are digested with difficulty by phagocytes include listeria, shigella, candida albicans and, of course, mycobacteria, but the importance of this in the pathogenesis of disease is not generally clear. the expression of the immune response necessarily involves a certain amount of inflammation, cell infiltration, lymph node swelling, even tissue destruction, as described in ch. . such changes caused by the immune response are classed as immunopathological. sometimes they are very severe, leading to serious disease or death, but at other times they play a minimal part in the pathogenesis of disease. with the possible exception of certain vertically transmitted virus infections and the transmissible 'prion' dementias (see ch. ), there are signs of an immune response in all infections. therefore it is to be expected that there will nearly always be some contribution of the immune response to pathological changes.* often the immunological contribution is small, but sometimes it forms a major part of the disease. for instance, in tuberculosis the pathological picture is dominated by the operation of a strong and persistent cmi response to the invading bacillus. in the classical tubercle a central zone of bacilli with large mononuclear and giant cells, often with some necrosis, is surrounded by fibroblasts and lymphocytes. mononuclear infiltrations, giant cells and granulomatous lesions (see p. ) are characteristic pathological features of tuberculosis. there are no recognised toxins formed by tubercle bacilli, and there seems to be no single antigen or other component that accounts for virulence. bacterial glycolipids (e.g. 'cord factor'), resistance to h (see pp. - ) and ability to utilise host fe (see p. ) have been correlated with pathogenicity, and inhibition of phagosome-lysosome fusion in macrophages (see pp. - ) by release of unidentified bacterial components would also contribute to pathogenicity. however, none of these factors is by itself absolutely necessary for virulence, which in such a complex, ancient parasite is likely to be multifactorial. as a gene library for m. tuberculosis is slowly built up there will be opportunities for clearer definition of virulence determinants. when macrophages are killed by intracellular mycobacteria the lysosomal enzymes and other materials released from the degenerating cell contribute to chronic inflammation as in the case of the streptococcal lesions referred to above. * a number of different microbial antigens are produced during most infections (see ch. ) and the possible immunological reactions are therefore numerous. for instance, at least types of circulating malarial antigen are found in heavily infected individuals. the mere enlargement of lymphoid organs during infectious diseases is a morphological change that can often be regarded as pathological. the lymph node swelling seen in glandular fever, for instance, is an immunopathological feature of the disease, and the same can be said of the striking enlargement of the spleen caused by chronic malaria and other infections in the condition known as tropical splenomegaly. as often as not the relative importance of direct microbial damage as opposed to immune and nonimmune inflammatory reactions have not yet been determined, but the picture is clearer in most of the examples given below. in one important human disease, pathological changes are certainly immunopathological in nature, but not enough is known about it to classify the type of reaction (see table . ). this disease is rheumatic fever, which follows group a streptococcal infections of the throat. it is the commonest form of heart disease in many developing countries. antibodies formed against a streptococcal cell wall or membrane component also react with the patient's heart muscle or valves, and myocarditis develops a few weeks later. many strains of streptococci have antigens that cross-react with the heart, and repeated infections with different streptococci cause recurrent attacks of rheumatic fever. there is genetic predisposition to the disease, based either on a particular antigen present in the heart of the patient or on a particular type of antibody response. chorea, a disease of the central nervous system, is a rare complication of streptococcal infection and antistreptococcal antibodies have been shown to react with neurons in the caudate and subthalamic nuclei of the brain. a number of microorganisms have antigens similar to host tissue components (p. ) so that in the course of responding immunologically to such infections the host is vulnerable to autoimmune damage (see ankylosing spondylitis, p. ). the antibodies to host components such as dna, igg, myofibrils, erythrocytes etc. that are seen in trypanosomiasis, mycoplasma pneumoniae, and eb virus infections appear to result from polyclonal activation of b cells (see p. ). it is not clear how important these autoimmune responses are in pathogenesis, but they reflect fundamental disturbances in immunoregulation. four types of immunopathology can be distinguished according to the classification of allergic reactions by coombs and gell, and microbial immunopathology will be described under these headings (see table . ). these depend on the reactions of antigens with reaginic (ige) antibodies attached to mast cells, resulting in the release of histamine, leukotrienes (see p. ) and heparin from mast cells, and the activation of serotonin and plasma kinins. if the antigen-antibody interaction takes place on a large enough scale in the tissue, the histamine that is released can give rise to anaphylactic shock, the exact features depending on the sensitivity and particular reaction of the species of animal to histamine. guinea-pigs suffer from bronchospasm and asphyxia, and in man there are similar symptoms, sometimes with a fall in blood pressure and shock. this type of immunopathology, although accounting for anaphylactic reactions to horse serum or to penicillin, is not important in infectious diseases. when the antigen-ige antibody interaction takes place at the body surface there are local inflammatory events, giving rise to urticaria in the skin, and hayfever or asthma in the respiratory tract. this local type of anaphylaxis may play a part in the pathogenesis of virus infections of the upper respiratory tract (e.g. common cold, respiratory syncytial virus infections of infants), or in skin rashes in infectious diseases. type reactions are common in helminth infections perhaps because ige antibodies have an important role in protection against these parasites. a dramatic type reaction can follow rupture of a hydatid cyst of echinococcus granulosus (the dog tapeworm). slow leakage of worm antigens means that mast cells are sensitised with specific ige antibody, and the sudden release of antigen can cause life-threatening anaphylaxis. when the larvae of ascaris lumbricoides pass through the lung on their journey from blood to intestine, they can give rise to igemediated respiratory symptoms, with infiltration of eosinophils. reactions of this type occur when antibody combines with antigen on the surface of a tissue cell, activates the complement sequence or triggers cytotoxicity by k cells (nk cells or phagocytes with fc receptors). k (killer) cell cytolysis is referred to as antibody-dependent cellular cytotoxicity (adcc). the antibody-coated cell is destroyed. as discussed in ch. , the same reaction on the surface of a microorganism (e.g. enveloped virus) constitutes an important part of antimicrobial defences, often leading to the destruction of the microorganism. cells infected with viruses and bearing viral antigens on their surface are destroyed in a similar way. clearly the antibody-mediated destruction of infected cells means tissue damage, and it perhaps accounts for some of the liver necrosis in hepatitis b, for instance, and probably in yellow fever. infected cells can also be destroyed by sensitised lymphocytes or nk cells independently of antibody (see below). in certain infections antibodies are formed against host erythrocytes and these cells are particularly sensitive to lysis. the haemolysis in malaria is caused by antibodies to parasite-derived antigens that have attached to red cells, rather than by autoantibodies to red cells themselves. in pneumonia due to mycoplasma pneumoniae (atypical pneumonia), antibodies (cold agglutinins) are formed against normal human group o erythrocytes. haemolytic anaemia is occasionally seen, and there is reticulocytosis (see glossary) in % of patients. the lesions in the lungs are perhaps based on cell-mediated immunopathological reactions. the combination of antibody with antigen is an important event, initiating inflammatory phenomena that are inevitably involved in the expression of the immune response. in the infected host, these inflammatory phenomena are most of the time of great antimicrobial value (see ch. ). but there are nevertheless immunopathological features of the infection, and immune complex reactions sometimes do a great deal of damage in the infected individual. the mechanisms by which antigenantibody reactions cause inflammation and tissue damage are outlined in fig. . . iga immune complexes are less harmful. antigens absorbed from the intestine can combine locally with iga antibody and the complex then enters the blood, to be filtered out in the liver and excreted harmlessly in bile (see p. ). when the antigen-antibody reaction takes place in extravascular tissues, there is inflammation and oedema with infiltration of polymorphs. if soluble antigen is injected intradermally into an individual with large amounts of circulating igg antibody, the antigen-antibody reaction takes place in the walls of skin blood vessels, and causes an inflammatory response. the extravasating poly morphs degenerate and their lysosomal enzymes cause extensive vascular damage. this is the classical arthus response. antigen-antibody reactions in tissues are not usually as serious as this, and milder inflammatory sequelae are more common as in the case of allergic alveolitis (see below), or the red zone seen round the borders of a smallpox vaccination site after seven or eight days. in the latter example circulating antibodies pass through vessel walls, meet vaccinia virus antigen in the dermal tissues and an inflammatory response is generated. a similar mild response can be induced experimentally to cause a reaction known as cutaneous anaphylaxis (see glossary), the test antigen being injected into the skin and reacting with blood-borne antibody. the resulting inflammation is detected by the visible local leakage of plasma proteins from blood vessels, circulating plasma proteins having been coloured by the intravenous injection of evans' blue. when the antigen-antibody reaction takes place in the blood to give circulating immune complexes, the sequelae depend to a large extent on size and on the relative proportions of antigen and antibody. if there is a large excess of antibody, each antigen molecule is covered with antibody and is removed rapidly by reticuloendothelial cells, which have receptors for the fc portion of the antibody molecule (see ch. ). when equal amounts of antigen and antibody combine, lattice structures are produced, and these form large aggregates whose size ensures that they are also rapidly removed by reticuloendothelial cells. if, however, complexes are formed in antigen excess, the poorly coated antigen molecules are not removed by reticuloendothelial cells. they continue to circulate in the blood and have the opportunity to localise in small blood vessels elsewhere in the body. the mechanism is not clear, but complexes are deposited in the glomeruli of the kidneys, the choroid plexuses, joints and ciliary body of the eye. factors may include local high blood pressure and turbulent flow (glomeruli), or filtering function of vessels involved (choroid plexus, ciliary body). in the glomeruli the complexes pass through the endothelial windows (fig. . ) and come to lie beneath the basement membrane. the smallest-sized complexes pass through the basement membrane and seem to enter the urine. this is probably the normal mechanism of disposal of such complexes from the body. immune complexes are formed in many, perhaps most, acute infectious diseases. microbial antigens commonly circulate in the blood in viral, bacterial, fungal, protozoal, rickettsial etc. infections. when the immune response has been generated and the first trickle of specific antibody enters the blood, immune complexes are formed in antigen excess. this is generally a transitional stage soon giving rise to antibody excess, as more and more antibody enters the blood and the infection is terminated. sometimes the localisation of immune complexes and complement in kidney glomeruli is associated with a local inflammatory response.* there is an infiltration of polymorphs, swelling of the glomerular basement membrane, loss of albumin, even red blood cells, in the urine and the patient has acute glomerulonephritis. this is seen following streptococcal infections, mainly in children (see below). as complexes cease to be formed the changes are reversed, and complete recovery is the rule. repeated attacks or persistent deposition of complexes leads to irreversible damage, often with proliferation of epithelial cells following the seepage of fibrin into the urinary space. under certain circumstances complexes continue to be formed in the blood and deposited subendothelially for long periods. this happens in certain persistent microbial infections in which microbial antigens are continuously released into the blood but antibody responses are only minimal or of poor quality (see below). complexes are deposited in glomeruli over the course of weeks, months or even years. the normal mechanisms for removal are inadequate. the deposits, particularly larger complexes containing high molecular weight antigens or antibodies (igm) are held up at the basement membrane and accumulate in the subendothelial space together with the complement components. as deposition continues, they gradually move through to the mesangial space ( fig. . ) where they form larger aggregates. mesangial cells, * see also footnote p. ; cells in kidney glomeruli, in joint synovium and in choroid plexuses bear fc or c b receptors. this would favour localisation in these tissues. of cell and tissue damage one of whose functions is to deal with such materials, enlarge, multiply and extend into the subepithelial space. if these changes are gradual there are no inflammatory changes, but the structure of the basement membrane alters, allowing proteins to leak through into the urine. later the filtering function of the glomerulus becomes progressively impaired. in the first place the glomerular capillary is narrowed by the mesangial cell intrusion. also, the filtering area is itself blocked by the mesangial cell intrusion, by the accumulation of complexes (fig. . ) , and by alterations in the structure of the basement membrane. the foot processes of epithelial cells tend to fuse and further interfere with filtration. the pathological processes continue, some glomeruli ceasing to produce urine, and the individual has chronic glomerulonephritis. circulating immune complex deposition in joints leads to joint swelling and inflammation but in choroid plexuses there are no apparent pathological sequelae. circulating immune complexes are also deposited in the walls of small blood vessels in the skin and elsewhere, where they may induce inflammatory changes.* the prodromal rashes seen in exanthematous virus infections and in hepatitis b are probably caused in this way. if the vascular changes are more marked they give rise to the condition called erythema nodosum, in which there are tender red nodules in the skin, with deposits of antigen, antibody and complement in vessel walls. erythema nodosum is seen following streptococcal infections and during the treatment of patients with leprosy. when small arteries are severely affected, for instance in some patients with hepatitis b, this gives rise to periarteritis nodosa. immune complex glomerulonephritis occurs as an indirect immunopathological sequel to a variety of infections. first there are certain virus infections of animals. the antibodies formed in virus infections generally neutralise any free virus particles, thus terminating the infection (see ch. ), but the infection must persist if antigen is to continue to be released into the blood and immune complexes formed over long periods. non-neutralising antibodies help promote virus persistence because they combine specifically with virus particles, fail to render them noninfectious, and at the same time block the action of any good neutralising antibodies that may be present. immune complexes in antigen excess are formed in the blood when the persistent virus or its antigens circulates in the plasma and reacts with antibody which is present in relatively small amounts. virus infections with these characteristics are * it is not clear how inflammation is caused. complement activation would presumably take place while complexes were circulating in the blood. perhaps the complexes bind more antibody after they have localised, or alternatively it is possible that free antigen circulates in the first place, localises, and later binds antibody to generate mediators of inflammation. included in table . . in each instance complexes are deposited in kidney glomeruli and sometimes in other blood vessels as described above. in some there are few if any pathological changes (ldv and leukaemia viruses in mice) probably because there is a slow rate of immune complex deposition, whereas in others glomerulonephritis (lcm virus in mice, adv in mink) or vasculitis (adv in mink) is severe. a persistent virus infection that induces a feeble immune response forms an ideal background for the development of immune complex glomerulonephritis, but there are no known viral examples in man. there are one or two other microorganisms that occasionally cause this type of glomerulonephritis and it is seen, for instance, in chronic quartan malaria and sometimes in infective endocarditis. in both these examples microbial antigens circulate in the blood for long periods. but immune complex deposition does not necessarily lead to the development of glomerulonephritis, and immune complexes are detectable in the glomeruli of most normal mice and monkeys. even in persistent virus infections the rate of deposition may be too slow to cause pathological changes as with ldv and leukaemia virus infections of mice (see table . ). during the acute stage of hepatitis b in man, when antibodies are first formed against excess circulating viral antigen (hepatitis b surface antigen), immune complexes are formed and deposited in glomeruli. but the deposition is short-lived and there is no glomerulonephritis. persistent carriers of the antigen do not generally develop glomerulonephritis, because their antibody is usually directed against the 'core' antigen (nephrotic syndrome in secondary syphilis) unknown causative agents man + + + + of chronic glomerulonephritis of the virus particle, rather than against the large amounts of circulating hepatitis b surface antigen. kidney failure in man is commonly due to chronic glomerulonephritis, and this is known to be mostly of the immune complex type, but the antigens, if they are microbial, have not yet been identified. immune complex glomerulonephritis occurs in man as an important complication of streptococcal infection, but this is usually acute in nature with inflammation of glomeruli, as referred to above. antibodies formed against an unknown component of the streptococcus react with circulating streptococcal antigen, perhaps also with a circulating host antigen, and immune complexes are deposited in glomeruli. streptococcal antibodies cross-reacting with the glomerular basement membrane may contribute to the picture. deposition of complexes continues after the infection is terminated, and glomerulonephritis develops a week or two later. the streptococcal infection may be of the throat or skin, and streptococcus pyogenes types and are frequently involved. when certain antigens are inhaled by sensitised individuals and the antigen reaches the terminal divisions of the lung, there is a local antigen-antibody reaction with formation of immune complexes. the resulting inflammation and cell infiltration causes wheezing and respiratory distress, and the condition is called allergic alveolitis. persistent inhalation of the specific antigen leads to chronic pathological changes with fibrosis and respiratory disease. exposure to the antigen must be by inhalation; when the same antigen is injected intradermally, there is an arthus type reaction (see p. ). there are a number of microorganisms that cause allergic alveolitis. most of these are fungi. a disease called farmer's lung occurs in farm workers repeatedly exposed to mouldy hay containing the actinomycete micromonospora faeni. cows suffer from the same condition. a fungus contaminating the bark of the maple tree causes a similar disease (maple bark stripper's disease) in workers in the usa employed in the extraction of maple syrup. the mild respiratory symptoms occasionally reported after respiratory exposure of sensitised individuals to tuberculosis doubtless have the same immunopathological basis. in addition to their local effects, antigen-antibody complexes generate systemic reactions. for instance, the fever that occurs at the end of the incubation period of many virus infections is probably attributable to a large-scale interaction of antibodies with viral antigen, although extensive cmi reactions can also cause fever. the febrile response is mediated by endogenous pyrogen (interleukin- ) and tumour necrosis factor (tnf) liberated from polymorphs and macrophages, as described on p. . probably the characteristic subjective sensations of illness and some of the 'toxic' features of virus diseases are also caused by immune reactions and liberation of cytokines. systemic immune complex reactions taking place during infectious diseases very occasionally give rise to a serious condition known as disseminated intravascular coagulation. this is seen sometimes in severe generalised infections such as gram-negative septicaemia, meningococcal septicaemia, plague, yellow fever and other haemorrhagic arthropod-borne virus diseases. immune complex reactions activate the enzymes of the coagulation cascade ( fig. . ), leading to histamine release and increased vascular permeability. fibrin is formed and is deposited in blood vessels in the kidneys, lungs, adrenals and pituitary. this causes multiple thromboses with infarcts, and there are also scattered haemorrhages because of the depletion of platelets, prothrombin, fibrinogen etc. systemic immune complex reactions were once thought to form the basis for dengue haemorrhagic fever. this disease is seen in parts of the world where dengue is endemic, individuals immune to one type of dengue becoming infected with a related strain of virus. they are not protected against the second virus, although it shows immunological cross-reactions with the first one. indeed the dengue-specific antibodies enhance infection of susceptible mononuclear cells, so that larger amounts of viral antigen are produced (see p. ). it was thought that after virus replication, viral antigens in the blood reacted massively with antibody to cause an often lethal disease with haemorrhages, shock and vascular collapse. however, it has proved difficult to demonstrate this pathophysiological sequence, and the role of circulating immune complexes and platelet depletion remains unclear. perhaps in this and in some of the other viral haemorrhagic fevers the virus multiplies in capillary endothelial cells. disease seems due to cytokines liberated from infected mononuclear cells. immune complex immunopathology is probable in various other infectious diseases. for instance, the occurrence of fever, polyarthritis, skin rashes and kidney damage (proteinuria) in meningococcal meningitis and gonococcal septicaemia indicates immune complex deposition. circulating immune complexes are present in these conditions. certain african arthropod-borne viruses with exotic names (chikungunya, o'nyong-nyong) cause illnesses characterised by fever, arthralgia and itchy rashes, and this too sounds as if it is immune complex in origin. immune complexes perhaps play a part in the oedema and vasculitis of trypanosomiasis and in the rashes of secondary syphilis. sensitive immunological techniques are available for the detection of circulating complexes and for the identification of the antigens and antibodies in deposited complexes. the full application of these techniques will perhaps solve the problem of the aetiology of chronic glomerulonephritis in man. the mere expression of a cmi response involves inflammation, lymphocyte infiltration, macrophage accumulation and macrophage activation as described in ch. , and can therefore by itself cause pathological changes. the cmi response to infection dominates the pathological picture in tuberculosis, with mononuclear infiltration, degeneration of parasitised macrophages, and the formation of giant cells as central features. these features of the tissue response result in the formation of granulomas (see glossary) which reflect chronic infection and accompanying inflammation. there is a ding-dong battle as the host attempts to contain and control infection with a microorganism that is hard to eliminate. the granulomas represent chronic cmi responses to antigens released locally. various other chronic microbial and parasitic diseases have granulomas as characteristic pathological features. these include chlamydial (lymphogranuloma inguinale), bacterial (syphilis, leprosy, actinomycosis), and fungal infections (coccidiomycosis). antigens that are disposed of with difficulty in the body are more likely to be important inducers of granulomas. thus, although mannan is the dominant antigen of candida albicans, glucan is more resistant to breakdown in macrophages and is responsible for chronic inflammatory responses. the lymphocytes and macrophages that accumulate in cmi responses also cause pathological changes by destroying host cells. cells infected with viruses and bearing viral antigens on their surface are targets for cmi responses as described in chs and . infected cells, even if they are perfectly healthy, are destroyed by the direct action of sensitised t lymphocytes, which are demonstrable in many viral infections. in spite of the fact that the in vitro test system so clearly displays the immunopathological potential of cytotoxic t cells, this is not easy to evaluate in the infected host. it may contribute to the tissue damage seen, for instance, in hepatitis b infection and in many herpes and pox virus infections. antigens from trypanosoma cruzi are known to be adsorbed to uninfected host cells, raising the possibility of autoimmune damage in chagas' disease, caused by this parasite.* it is also becoming * chagas' disease, common in brazil, affects million people, and is transmitted by blood-sucking bugs. after spreading through the body during the acute infection, the parasitaemia falls to a low level and there is no clinical disease. years later a poorly understood chronic disease appears, involving heart and intestinal tract, which contain only small numbers of the parasite but show a loss of autonomic ganglion cells. an autoimmune mechanism is possible (see p. ), because a monoclonal antibody to t. cruzi has been obtained that cross-reacts with mammalian neurons. clear that cells infected with certain protozoa (e.g. theileria parva in bovine lymphocytes) have parasite antigens on their surface and are susceptible to this type of destruction. little is known about intracellular bacteria. the most clearly worked out example of type (cmi) immunopathology is seen in lcm virus infection of adult mice. when virus is injected intracerebrally into adult mice it grows in the meninges, ependyma and choroid plexus epithelium, but the infected cells do not show the slightest sign of damage or dysfunction. after - days, however, the mouse develops severe meningitis with submeningeal and subependymal oedema, and dies. the illness can be completely prevented by adequate immunosuppression, and the lesions are attributable to the mouse's own vigorous cd + t cell response to infected cells. these cells present processed lcm viral peptides on their surface in conjunction with mhc i proteins, and sensitised cd + t cells, after entering the cerebrospinal fluid and encountering the infected cells, generate the inflammatory response and interference with normal neural function that cause the disease. the same cells destroy infected tissue cells in vitro, but tissue destruction is not a feature of the neurological disease. in this disease the cd + t cells probably act by liberating inflammatory cytokines. it may be noted that the brain is uniquely vulnerable to inflammation and oedema, as pointed out earlier in this chapter. the infected mouse shows the same type of lesions in scattered foci of infection in the liver and elsewhere, but they are not a cause of sickness or death. lcm infection of mice is a classical example of immunopathology in which death itself is entirely due to the cell-mediated immune response of the infected individual. this response, although apparently irrelevant and harmful, is nevertheless an 'attempt' to do the right thing. it has been shown that immune t cells effectively inhibit lcm viral growth in infected organs. however, a response that in most extraneural sites would be useful and appropriate turns out to be self-destructive when it takes place in the central nervous system. another type of t cell-mediated immune pathology is illustrated by influenza virus infection of the mouse. when inoculated intranasally, the virus infects the lungs and causes a fatal pneumonia in which the airspaces fill up with fluid and cells. the reaction is massive and the lungs almost double in weight. effectively the animal drowns. the cause is an influx of virus-specific cd + t cells. normally when an appropriate number of t cells had entered the lungs, the t cells would issue a feedback response to prevent such over-accumulation, but it is thought that influenza virus infects the t cells and inhibits this control process, so that the lungs are eventually overwhelmed. the action of the virus is subtle as it does not multiply in or kill the infected t cells, and it is presumed that it undergoes limited gene expression. one human virus infection in which a strong cmi contribution to pathology seems probable is measles. children with thymic aplasia show a general failure to develop t lymphocytes and cell-mediated immunity, but have normal antibody responses to most antigens. they suffer a fatal disease if they are infected with measles virus. instead of the limited extent of virus growth and disease seen in the respiratory tract in normal children, there is inexorable multiplication of virus in the lung, in spite of antibody formation, giving rise to giant cell pneumonia. this indicates that the cmi response is essential for the control of virus growth. in addition there is a total absence of the typical measles rash, and this further indicates that the cmi response is also essential for the production of the skin lesions. there is evidence that cell-mediated immune responses also make a contribution to the rashes in poxvirus infections. sometimes in infectious diseases there are prominent pathological changes which are not attributable to the direct action of microbes or their toxins, nor to inflammation or immunopathology. the stress changes mediated by adrenal cortical hormones come into this category. stress is a general term used to describe various noxious influences, and includes cold, heat, starvation, injury, psychological stress and infection. an infectious disease is an important stress, and corticosteroids are secreted in large amounts in severe infections (see also ch. ). they generally tend to inhibit the development of pathological changes, but also have pronounced effects on lymphoid tissues, causing thymic involution and lymphocyte destruction. these can be regarded as pathological changes caused by stress. it was the very small size of the thymus gland as seen in children dying with various diseases, especially infectious diseases, that for many years contributed to the neglect of this important organ, and delayed appreciation of its vital role in the development of the immune system. appreciation of the effects of stress on infectious diseases and the immune response in particular has led to the establishment of the science of neuroimmunology. properly controlled experiments are difficult to mount but there is a persuasive and growing body of evidence which shows that the nervous system affects the functioning of the immune system. the pathways of this communication are still poorly understood. work on mycobacterium bovis grew out of observations from the turn of the century that stress appears to increase the death rate in children with tb. in one type of experiment mice were stressed by being kept in a restraining device where movement was virtually impossible. this resulted in the reduction of expression of mhc class ii antigens on macrophages, which correlated with increased susceptibility to infection. similarly stressing mice infected with influenza virus caused several immunosuppressive events including reduction of inflammatory cells in the lung, and decreased production of interleukin- . suppression of antibody responses is found in people suffering a type of stress familiar to students-examinations! the best responses to hepatitis b vaccine in students immunised on the third day of their examinations were found in those who reported the least stress. finally, in a double blind trial at the common cold research unit in england with five different respiratory viruses, it was ascertained in human volunteers that stress gave a small but statistically significant increased likelihood of an individual developing clinical disease. pathological changes are sometimes caused in an even more indirect way as in the following example. yellow fever is a virus infection transmitted by mosquitoes and in its severest form is characterised by devastating liver lesions. there is massive mid-zonal liver necrosis following the extensive growth of virus in liver cells, resulting in the jaundice that gives the disease its name. destruction of the liver also leads to a decrease in the rate of formation of the blood coagulation factor, prothrombin, and infected human beings or monkeys show prolonged coagulation and bleeding times. haemorrhagic phenomena are therefore characteristic of severe yellow fever, including haemorrhage into the stomach and intestine. in the stomach the appearance of blood is altered by acid, and the vomiting of altered blood gave yellow fever another of its names, 'black vomit disease'. haemorrhagic phenomena in infectious diseases can be due to direct microbial damage to blood vessels, as in certain rickettsial infections (see p. ) or in the virus infection responsible for haemorrhagic disease of deer. they may also be due to immunological damage to vessels as in the arthus response or immune complex vasculitis, to any type of severe inflammation, and to the indirect mechanism illustrated above. finally there are a few infectious diseases in which platelets are depleted, sometimes as a result of their combination with immune complexes plus complement, giving thrombocytopenia and a haemorrhagic tendency (see also disseminated intravascular coagulation, p. ). thrombocytopenic purpura is occasionally seen in congenital rubella and in certain other severe generalised infections. infection during pregnancy can lead to foetal damage or death not just because the foetus is infected (pp. - ), but also because of infection and damage to the placenta. this is another type of indirect pathological action. placental damage may contribute to foetal death during rubella and cytomegalovirus infections in pregnant women. certain viruses undoubtedly cause tumours (leukaemia viruses, human papillomaviruses, several herpes viruses in animals) and this is to be regarded as a late pathological consequence of infection. as was discussed in ch. the tumour virus genome can be integrated into the host cell genome whether a tumour is produced or not, so that the virus becomes a part of the genetic constitution of the host. sometimes the host cell is transformed by the virus and converted into a tumour cell, the virus either introducing a transforming gene into the cell, activating expression of a pre-existing cellular gene, or inactivating the cell's own fail-safe tumour suppressor gene. the transforming genes of dna tumour viruses generally code for t antigens which are necessary for transformation, and the transforming genes of rna tumour viruses are known as one genes.* transformation has been extensively studied in vitro, and the features of the transformed cell described (changed surface and social activity, freedom from the usual growth restraints). simultaneous infection with two different microorganisms would be expected to occur at times, merely by chance, especially in children. on the other hand, a given infection generates antimicrobial responses such as as interferon production and macrophage activation which would make a second infection less likely. dual infections are commonest when local defences have been damaged by the first invader. the pathological results are made much more severe because there is a second infectious agent present. this can be considered as another mechanism of pathogenicity. classical instances involve the respiratory tract. the destruction of ciliated epithelium in the lung by viruses such as influenza or measles allows normally nonpathogenic resident bacteria of the nose and throat, such as the pneumococcus or haemophilus influenzae, to invade the lung and cause secondary pneumonia. if these bacteria enter the lung under normal circumstances, they are destroyed by alveolar macrophages or removed by the mucociliary escalator. in at least one instance the initial virus infection appears to act by interfering with the function of alveolar macrophages. mice infected with parainfluenza (sendai) virus show greatly increased susceptibility to infection with haemophilus influenzae, and this is largely due to the fact that alveolar macrophages infected with virus show a poor ability to phagocytose and kill the bacteria. specialised respiratory pathogens such as influenza, measles, parainfluenza or rhinoviruses damage the naso-* one genes (oncogenes) are also present in host cells, where they play a role in normal growth and differentiation, often coding for recognised growth factors (e.g. human platelet-derived growth factor). they can be activated and the cell transformed when tumour viruses with the necessary 'promoters' are brought into the cell. the one genes of the rna tumour viruses themselves originate from cellular oncogenes which were taken up into the genome of infecting viruses during their evolutionary history. pharyngeal mucosa and can lead in the same way to secondary bacterial infection, with nasal catarrh, sinusitis, otitis media or mastoiditis. the normal microbial flora of the mouth, nasopharynx or intestine are always ready to cause trouble if host resistance is lowered, but under normal circumstances they hinder rather than help other infecting microorganisms (see ch. ). one interesting example of exacerbation of infection occurs in mice dually infected with influenza virus and microorganisms such as streptococcus aureus or serratia marcescens. under these conditions animals suffer a more severe viral infection. this results from the need to proteolytically cleave the viral haemagglutinin protein which is done by a cellular enzyme. if the appropriate protease is in short supply or lacking completely, virions are formed but they are not infectious. under these circumstances the haemagglutinin can be cleaved extracellularly by microbial proteases with resulting increased amounts of infectious virus and disease. as a final example of dual infections, microorganisms that cause immunosuppression can activate certain pre-existing chronic infections. in measles, for instance, there is a temporary general depression of cmi; tuberculin-positive individuals become tuberculin negative, and in patients with tuberculosis the disease is exacerbated. in aids (see p. ) immunosuppression by hiv activates a variety of pre-existing persistent infections. diarrhoea deserves a separate section, since it is one of the commonest types of illness in developing countries and a major cause of death in childhood. particularly in infants, who have a very high turnover of water relative to their size, the loss of fluid and salt soon leads to life-threatening illness. it is estimated that on a global scale diarrhoea is responsible for - million deaths per year in children under five years old. in villages in west africa and guatemala the average - -year-old child has diarrhoea for about two months in each year.* diarrhoea also interacts with malnutrition and can cause stunted growth, defective immune responses and susceptibility to other infections (pp. - ). diarrhoea is also a common affliction of travellers from developed countries, * diarrhoea on a massive scale is not always confined to developing countries. there was a major outbreak of cryptosporidium infection in milwaukee, usa, in with more than cases; of these were diagnosed in the laboratory and they suffered watery diarrhoea (mean stools a day) for a mean of nine days. the small ( - μιη) oocysts, probably from cattle, had entered lake michigan, and then reached the community water supply because of inadequate filtration and coagulation treatment. and business deals, athletic successes and holiday pleasures can be forfeited on the toilet seats of foreign lands. the most reliable prophylaxis is to 'cook it, peel it, or forget it'. most attacks of diarrhoea are self-limiting. fluid and electrolyte replacement is a simple, highly effective, life-saving treatment that can be used without determining the cause of the diarrhoea. oral rehydration therapy (orf) means giving a suitable amount of salt and sugar in clean water and this is something that can be done by the mother. diarrhoea means the passage of liquid faeces,* or faeces that take the shape of the receptacle rather than have their own shape. this could arise because of increased rate of propulsion by intestinal muscles, giving less time for reabsorption of water in the large bowel, or because there was an increase in the amount of fluid held or produced in the intestine. in many types of infectious diarrhoea the exact mechanism is not known. diarrhoea, on the one hand, can be regarded as a microbial device for promoting the shedding and spreading of the infection in the community, or on the other hand as a host device to hasten expulsion of the infectious agent. diarrhoea is a superb mechanism for the dissemination of infected faeces (see p. ) and there is no doubt that strains of microbes are selected for their diarrhoea-producing powers. the advantages to the host of prompt expulsion of the infectious agent was illustrated when volunteers infected with shigella flexneri were given lomotil, a drug that inhibits peristalsis. they were more likely to develop fever and had more difficulty in eliminating the pathogen. in recent years significant strides have been made in our understanding of the pathophysiology of diarrhoeal disease. first, a quick resume of the normal structure and function of gut before attempting to understand the processes whereby it may be perturbed. the main function of the gut is the active inward transport of ions and nutrient solutes which is followed by the passive movement of water ( fig. . ). the driving force is the na + /k + atpase situated in the basolateral membrane of enterocytes on the villus (fig. . ) which maintains a low intracellular [na" "] thus creating the electrochemical gradient favourable for na + entry and, a high regional [na + ] in the intercellular spaces; cl~ follows na + . a similar situation exists in crypt cells: na + /k + atpase drives secretion. the key difference is the location of the carrier systems responsible for the facilitated entry of the actively transported species. in villus cells the carriers are present in the brush border, whereas in crypt cells they are located in the basal membrane: this is responsible for the vectorial aspects of ion/fluid traffic in villus/crypt assemblies. however, it is clear that several factors in addition to enterocytes are involved in * liquid faeces are not abnormal in all species. the domestic cow experiences life-long diarrhoea, but presumably does not suffer from it. (a) two methods of n a + cotransport are shown involving a glucose-linked symport and two coupled antiports; the latter results in the cotransport of cl~. the coupled antiports are functionally linked via h + and hc ~, the relative concentrations of which are a reflection of metabolic activity. these processes occur within the same cells but are shown separately for clarity. the driving force for n a + uptake is the low na + concentration maintained by the na + /k + pump (atpase) which creates the electrochemical gradient which promotes the inward movement of na + ; cl~ follows n a + by diffusion. water is drawn osmotically across the epithelium paracellularly (i.e. across tight junctions) and/or transcellularly, the former pathway accounting for approximately % of fluid movement. (b) secretion is the result of the coupled entry of n a + and cl~ across the basolateral membrane. n a + is recycled by the na + /k + pump and cl~ exits by diffusing down an electrochemical gradient and across the undifferentiated crypt cell apical membrane; n a + follows cl~ and water follows passively. if by whatever means, the level of nacl were to increase in such cells (as for example in rotavirus infection of neonatal mice, see below) then one could perceive how this could give rise to a cl~ driven hypersecretion of water. note, (i) the driving force results from the same mechanism that powers absorption i.e. the n a + / k + pump located in the basolateral membrane; it is the location of the 'port' 'diffusion' systems that determines the vectorial aspects of ion movement, (ii) the tight junctions are less tight in the crypts than villi. (iii) the apical membrane of the crypt cell is undifferentiated and only acquires micro villi during ascent into villous regions, φ : na + /k + pump; o: sym-, anti-port or diffusion channel. r e g u l a t i n g fluid t r a n s p o r t in t h e gut; t h e s e include t h e enteric n e r v o u s s y s t e m a n d t h e a n a t o m y of t h e microcirculation. t h e l a t t e r plays a profoundly i m p o r t a n t role in t h e u p t a k e of fluid. this is i l l u s t r a t e d in fig. . , which shows t h e existence of zones of g r a d e d osmotic fig. . schematic representation of a villus. note the central arterial vessel (av) which arborizes at the tip into a capillary bed drained by a subepithelial venous return (vr). movement of sodium into vr creates a concentration gradient between vr and av causing absorption of water from av and surrounding tissue. this results in a progressive increase in the osmolarity of incoming blood moving into the tip region through to vr. tip osmolarity is about three times higher than normal. this counter current system has been demonstrated in man and can be inferred in mice from the morphology of red blood cells which changes during ascent of the same vessel from base to tip regions of villi. the shaded areas indicate a vertical increase in osmolarity. left crypt: represents normal physiological secretion. right crypt: represents hyper secretion. ens, enteric nervous system, depicted schematically and not anatomically. potential. at the tips of villi in adult human gut, osmolalities range from to mosmkg _ h , which would generate huge osmotic forces. thus, current perceptions are that enterocytes are responsible for generating this gradient and the blood supply acts as a countercurrent multiplier which amplifies the gradient in a manner analogous to the loops of henle in the kidney. the hypertonic zone has been demonstrated directly in whole villi of infant mice in terms of the changing morphology of erythrocytes: in the lower regions of villi they show characteristic discoid morphology, whereas in the upper region they are crenated, indicating a hyperosmotic environment. the hypertonicity is dissipated if the blood flow is too slow and washed out if too fast. it is the villus unit rather than enterocytes by themselves which is responsible for fluid uptake. another consequence of the microcirculatory anatomy is that villus tip regions are relatively hypoxic. in addition, neonatal brush borders contain disaccharidases (principally lactase) which break down nonabsorbable disaccharides (e.g. lactose) into constituent absorbable monosaccharides. villus tips and crypts are regarded as the anatomical sites of physiological absorption and secretion respectively. fluid transport is a bidirectional process in the healthy animal with net absorption in health and net secretion in disease. the balance between absorption and secretion is poised at different points throughout the intestinal tract reflecting differences in both structure and function. proximal small intestine is relatively leaky; in contrast the colon is a powerfully absorptive organ. finally, crypts are the principal sites of cell regeneration, replacing cells which migrate up the epithelial escalator. the epithelium is renewed in approximately - days. at villus tips senescent cells are shed. diarrhoeal disease can result from interference with almost any one, or combination of these systems. diarrhoea-producing microbes are listed in table . . some examples are considered in detail below. rotaviruses are known to invade intestinal epithelial cells and cause diarrhoea in man, foals, dogs, pigs, mice etc. extensive multiplication takes place and very large amounts of virus ( particles g _ ) are shed in faeces. the conventional wisdom is that tips of villi especially are affected, leading to reduced absorption of fluid from the lumen. in addition destruction of enterocytes leads to a loss in lactase resulting in an accumulation of lactose in the gut causing an osmotic flux of fluid into the intestine. a major study of rota virus-induced diarrhoea in neonatal mice provides a different model of this important disease of children. oral infection of baby mice with a murine strain of rotavirus resulted in virus replication in enterocytes of the small intestine. before there had been detectable virus replication, villi became ischaemic, enterocytes severely damaged and villi shortened. in gut enterocytes this virus was demonstrably not cytopathic; the cell damage and villus responses were almost identical to that seen in experimentally induced ischaemia in physiological experiments. at the peak of virus replication, which was coincident with maximum shortening of villi, the gut was still absorptive, glucose transport intact and sufficient lactase activity remained to deal with the normal lactose load delivered to the stomach. after this the gut became secretory and diarrhoea clinically evident. villi were rapidly regenerated, but hyperaemia and diarrhoea persisted until the regeneration of the hypertonic zones as judged by the morphology of the red blood cells. peak diarrhoea coincided with the resynthesis of new villi (as judged by thymidine kinase levels, increased mitotic activity and morphometric analyses), and increased levels of intracellular nacl in the zone of cells where cell division was most active. transiently high levels of nacl have also been observed in dividing cells in culture. since ischaemic and hyperaemic villi were occasionally seen in control villi it may well be the case that the pathological changes reflect an exaggerated synchronised response of basic circulatory control mechanisms, which are part of the normal homeostatic mechanisms of villus physiology. thus, the pathophysiology of rotavirus-induced diarrhoea in neonatal mice may be summarised as follows: the reduction of red blood cells flowing through villi in the early stages of infection instigates hypoxia and hence atrophy of villi. the degree of atrophy in this infection is not associated with lack of absorption. the ensuing villus repair processes induce hypersecretion. the increase in blood flow throughout the remaining course of the infection reduces the hypertonicity of villi which impairs water absorption and thus prolongs diarrhoea. the preceding description of the self-limiting diarrhoea induced by rotavirus in neonatal mice is probably applicable to many diarrhoeas. however, the observed pathology may be different according to age, species, or the inducing pathogen. for example, in rotavirus-infected lambs, villus atrophy and crypt hypertrophy occur (the latter indicative of crypt cell division) but as in mice, infected lambs are not lactose intolerant. in rotavirus-infected piglets, crypt hypertrophy occurs but villus atrophy is severe, the animals are lactose intolerant and mortality high; a similar situation exists for the coronavirus, transmissible gastroenteritis (tge) virus. the latter has often been used as the model for infantile diarrhoea but the question is whether human infants are more like piglets or lambs. clinical studies have shown that recovery from mild, acute gastroenteritis of rota virus origin occurs within two weeks irrespective of the carbohydrate ingested. clearly, the severity of disease and the clinical outcome will depend on the extent of vertical' villus/ crypt involvement and the regions of intestine infected. when villus erosion is severe, then lactose may cause an 'osmotic' purge or be fermented by intestinal bacteria to short chain fatty acids which stimulate secretion in the colon. astroviruses, norwalk virus, certain coronaviruses, certain adenoviruses and probably toroviruses all cause gastroenteritic disease by infecting enterocytes. however, parvoviruses cause severe intestinal disease in dogs by virtue of their predilection for the mitotically active crypt cells; this causes the near complete erosion of villi similar to that seen after exposure to sublethal doses of irradiation. the classic paradigm for bacterial watery diarrhoea is cholera caused by vibrio cholerae in the small intestine. v. cholerae attaches to enterocytes of the proximal small bowel and is capable of producing at least three toxins: classical cholera toxin (ct); a toxin which disrupts the zonula occludens tight junction (designated zot); and another less well defined, auxilliary cholera enterotoxin (designated ace). as a result of toxic action, water and electrolytes are lost through the intact epithelial cells into the small intestine. as the multiplying bacteria increase in numbers and more and more epithelial cells are affected, the absorptive capacity of the colon is overwhelmed and there is profuse watery diarrhoea, as much as l l h - in severe cases.* the massive loss of isotonic fluid with excess of sodium bicarbonate and potassium leads to hypovolaemic shock, acidosis and haemoconcentration. anuria develops, and the collapsed, lethargic patient may die in - h. lives are saved by replacing the lost water and salts. the effect of toxin on an intestinal epithelial cell is long lasting, but the patient recovers as affected cells are shed and replaced in the normal fashion. the infection is particularly severe in children who easily develop low levels of plasma potassium. however, on a global scale this greatly feared disease, cholera, is only responsible for less than % of the total deaths due to diarrhoea. administration of as little as μg of purified ct alone to a healthy volunteer will reproduce the hugely dramatic, potentially lethal, purge of near isotonic fluid emphasising the central importance of ct in the causation of disease. the effect of zot has been demonstrated by electron microscopy in human biopsies, but the relative importance of zot and ace in human disease has not been quantified. how does ct work? as already outlined (pp. - ) the a subunit of this toxin is an adpribosyl transferase whose target is the a s subunit of the regulatory g protein which governs the expression of adenylate cyclase and hence the production of camp. this in turn results in the perturbation of the transport systems leading to a net efflux of ions and hence water. things are slightly more complicated, because elevation of camp occurs in villus tip cells (hence affecting absorption) but not in crypt cells. probably a signal is generated in intoxicated tip enterocytes which is transmitted to the crypt region by the ens causing release of neurotransmitters (e.g. serotonin, acetyl choline, and vasoactive peptide) which act on enterochromaffin (secretory) cells and on intestinal smooth muscle thereby augmenting crypt secretion and increasing peristalsis. studies have been made of sequential histological changes in human biopsies, taken from a series of patients from the onset of symptoms to recovery. it was clear that fluid secretion was not the result of massive desquamation of the epithelium. histological changes included 'engorgement of the capillaries' (i.e. interference with the blood supply), 'dilation of lacteals, vacuolation of enterocytes, exhaustion atrophy of enterocytes' (remember this is a noninvasive pathogen), 'accelerated shedding of cells, and increased mitotic activity'. most of these changes are similar to those described above for experimental murine rotavirus at post-peak diarrhoea and it seems that the range of histological reactions that can occur in the small intestine are limited so that qualitatively the picture is similar in different diarrhoeas. despite the undoubted importance of ct in the causation of the disease, and the potent antigenicity of ct, it is now recognised that protective immunity is very largely antibacterial. it is stopping effective colonisation which is important rather than neutralisation of the toxin. this has been partially achieved by using killed whole cell vaccines. several attempts have been made in the laboratory to genetically manipulate virulent strains (in practice this means deleting or inactivating the known toxin genes) such that the attenuated strain will colonise the gut and stimulate local immune responses and thereby prevent colonisation of the gut by virulent strains. to date, attenuated strains have been developed which fulfil these criteria but induce a mild transient diarrhoea which has prevented their adoption into vaccination programmes. however the recent emergence of v. cholerae strain (a new, third type) in india and bangladesh is a reminder that today's solution may not be adequate for tomorrow's problems. current vaccines are ineffective against this new strain. the picture withi£. coli is complicated since there are several biotypes of this pathogen which include: etec (enterotoxigenic e. coli, the principal cause of travellers' diarrhoea), epec (enteropathogenic e. coli), eiec (enteroinvasive e. coli), and ehec (enterohaemorrhagic e. coli) variants (see ch. ). the situation with etec is very similar to v. cholerae as far as heat labile toxin (lt, which is very closely related to ct) is concerned. it is additionally complicated by the fact that some strains of etec also make heat stable toxins (sts), which are nonantigenic low molecular weight peptides which activate membrane-bound guanylate cyclase which causes the production of cgmp which is the functional equivalent of camp. the 'classical' epec lesion is the formation of a characteristic pedestal-like lesion in the brush border of enterocytes with only limited invasion of the mucosa. just how it causes diarrhoea is not wholly clear. eiec is almost indistinguishable from shigella, and causes similar disease. ehec strains, which belong mainly (but not exclusively) to serogroup :h , cause a spectrum of intestinal disease ranging from asymptomatic carriage through mild diarrhoea to severe haemorrhagic colitis. they also cause extraintestinal infections such as haemolytic uraemic syndrome (hus). the initial lesion seen in the gut is similar to that caused by epec but ehec do not remain localised there: they multiply in the lamina propria and glandular crypts. while there is no conclusive proof of the involvement of slt in the pathogenesis of ehec diarrhoea there is a correlation between slt production and the severity of diarrhoeal disease or hus caused by this organism. salmonella spp. cause acute gastroenteritis and systemic typhoid disease in man, as well as other important infections in domestic animals. virulence in salmonella, especially in their natural hosts (see table . , footnote) isxomplex and mediated by numerous genes, some of which are present in otherwise nonpathogenic organisms such as e. coli. presumably all these bacteria share nutritional and environmental problems, whether inside or outside a host, that call for such genes. those restricted to salmonella are more likely to be critical in vivo. the clinical features of salmonella-induced diarrhoea (gastroenteritis) in humans and systemic typhoid infections are quite different. for example, gastroenteritis may follow - h after ingestion of contaminated food, whereas typhoid follows an incubation period of - days. diarrhoea, (which is usually watery, but may be severe, and sometimes bloody) is the predominating feature of gastroenteritis, whereas in adults, constipation is seen in the early clinical stages of typhoid; diarrhoea may occur much later. although fever may occur in gastroenteritis, in typhoid this may be so severe as to cause delirium. current perceptions are that gastroenteritis results from the initial interactions of salmonella (any one of many serotypes but frequently typhimurium or enteriditis, and/or their products) with the gut mucosa, whereas typhoid fever is produced by s. typhi organisms which translocate the mucosa, survive within macrophages, multiply and release endotoxin which triggers the highly complex endotoxin cascade. gastroenteritis is usually self-limiting, whereas in untreated typhoid mortality can be as high as %. the pathophysiology of fluid secretion caused by salmonellae is highly complex and as yet incompletely understood. the only two biological parameters which have been shown to correlate with induction of fluid secretion in a rabbit model (the best available small laboratory animal for human gastroenteritis) is the ability to invade the gut mucosa, and the induction of a leucocyte (mainly polymorphonuclear cell) influx into the mucosa and lumen of the gut. fluid secretion does not correlate per se with the ability to make an enterotoxin: avirulent as well as virulent strains make a cholera-like toxin though neither appear to release the toxin readily. the latter is therefore either not primarily involved, or is involved only after release from bacteria by means other than normal protein secretion mechanisms. these questions are as yet unresolved and the reader is referred to the reference list for a fuller discussion of these complex matters. currently, the major diarrhoeagenic pathogen of the small intestine in the developed world is campylobacter jejuni. the latter are bacteria present in wild birds, chickens and in the faeces of up to % of healthy cows in the uk. milk becomes contaminated and those who drink raw (unpasteurised) infected milk develop diarrhoea, sometimes with dysentery (blood and pus in the stools) and fever. the incubation period is - days and diarrhoea occurs after bacterial replication in the upper small intestine (jejunum). however, we are only at the beginning of understanding the detailed mechanisms and determinants responsible for disease causation by this pathogen. giardia lamblia is a noninvasive protozoan pathogen of the human small bowel which causes a spectrum of infection ranging from asymptomatic carriage through acute watery diarrhoea to chronic diarrhoea and malabsorption. giardia has a simple life cycle, existing in two forms: the multiplying trophozoite which infects mammalian hosts to cause disease and the environmentally resistant cyst. after ingestion, excystation is triggered by the ph in the stomach, and the trophozoite colonises the small bowel; the putative adherence factors have been described in ch. . the precise mechanisms involved in subsequent stages responsible for diarrhoea, malabsorption and cystation are less well understood. entamoeba histolytica causes lysis of target cells apparently by direct contact with the cell membrane. this pathogen produces under in vitro conditions a spectacular array of potential (but as yet unproven) virulence determinants including: proteases that round up cells, poreforming proteins, collagenases and oligosaccharidases and neurotransmitter-like compounds; the latter can induce intestinal fluid secretion. some of these factors have been implicated as the determinants responsible for liver abscess formation. not all diarrhoeal disease is the result of small bowel dysfunction. two important pathogens of the colon will be considered-clostridium difficile and shigella dysenteriae. c. difficile causes a spectrum of disease varied hepatitis a there are more than serotypes of salmonella, distinct from salmonella typhi and salmonella paratyphi. they are primarily parasites of animals, ranging from pythons to elephants, and their importance for man is their great tendency to colonise domestic animals. pigs and poultry are commonly affected, and human disease follows the consumption of contaminated meat or eggs. b other campylobacters cause sepsis, abortion and enteritis in animals. ranging from asymptomatic carriage through mild antibiotic-associated diarrhoea (aad) to fatal pseudomembranous colitis (pmc). normal flora play a major part-by means which are still not completely understood-in suppressing outgrowth of resident c. difficile spores. disruption of the normal flora, by antibiotic treatment for example, results in vegetative growth and production of several toxins of which c. difficile toxin a seems to be the most important in disease. this toxin is responsible for the secretory and inflammatory response in the intestine, and epithelial disruption. it probably acts by disrupting tight junctions between cells, and by causing chemotactic and other responses in polymorphs. diarrhoea could be due to the destruction of the 'absorptive epithelium' and failure to cope with the normal fluid load delivered by the small intestine, or an active type of secretion caused by replacement of the damaged epithelium, or both. the second example is that of dysentery caused by shigellae spp., in particular s. dysenteriae and s. flexneri. we have already dealt with the capacity of shigellae to invade (ch. ). invasiveness is a vital part of the virulence armoury of shigellae spp.; non-invasive mutants are a virulent. the pathogen invades the gut via m cells in peyer's patches and thence adjacent microvillus-bearing colonocytes. what is the role played by shiga toxin? mutants of s. dysenteriae devoid of the ability to make shiga toxin still retained the ability to cause lethal fulminant dysentery (low volume fluid production, pus cells, mucus) in macaque monkeys, and they invaded, multiplied within and rapidly killed hela cells. the major difference between the mutants and the toxin-positive wild type was that the latter caused a more severe disease with more haemorrhage, giving rise to blood in the stools, and, in some instances greater destruction of mucosal tissues. evidently the production of shiga toxin is not a sine qua non for causing bacillary dysentery: it exacerbates the disease. although much research has been focused on toxins, their mode of action, and their role in disease, it is useful to compare different types of intestinal infection and to refer to the concept of food poisoning. types of intestinal infection are set out in table . . food poisoning is a loosely used term, and usually refers to illnesses caused by preformed toxins in food, or sometimes to illnesses that come on within a day or so after eating contaminated food. food may be contaminated with plant poisons, fungal poisons (e.g. poisoning due to amanita phalloides), fish poisons,* heavy metals, as well as with bacterial toxins or bacteria. sourcebook of bacterial toxins the virology and immunology of lymphocytic choriomeningitis virus infection * ingestion of scombroid fish (mackerel etc.) containing large amounts of histamine or similar substances leads to headache, flushing, nausea and vomiting within an hour response of man to infection with vibrio cholerae. . clinical, serologic and bacteriologic responses to a known inoculum epitopes of streptococcal m proteins shared with cardiac myosin bacterial infections of respiratory and gastrointestinal mucosae pathogenesis and immunology of treponema pallidum t-lymphocyte stimulation by microbial antigens lessons from diarrhoeal diseases, demography to molecular pharmacology clostridium botulinum toxins: a general review of involvement in disease, structure, mode of action and preparation for clinical use disseminated intravascular coagulation: a review campylobacterpyloridis and gastritis: association with intercellular spaces and adaptation to an environment of mucus as important factors in colonization of the gastric epithelium typhoid fever: pathogenesis and immunologie control comparison of the alpha-toxin genes of clostridium perfringens type a and c strains: evidence for extragenic regulation of transcription pathogenic mechanism ofneisseriagonorrhoeae: observations on damage to human fallopian tubes in organ cultures by gonococci of colony type i or type rift valley fever virus in mice vi: histological changes in the liver in relation to virus multiplication viral aetiology of diseases of obscure origin campylobacter jejuni. current status and future trends cytolytic pore-forming proteins and peptides: is there a common structural motif? treatment of spasmodic torticollis with local injections of botulinum toxin pathogenesis of diseases caused by entamoeba histolytica: studies of adherence, secreted toxins and contactdependent cytolysis pituitary dwarfism in mice persistently infected with lymphocytic choriomeningitis virus tetanus and botulinum-b neurotoxins block neurotransmitter release by proteolytic cleavage of synaptobrevin tetanus toxin is a zinc protein and its inhibition of neurotransmitter release and protease activity depend on zinc experimental salmonella typhimurium induced gastroenteritis bacterial phospholipases c the development of respiratory syncytial virus-specific ige and the release of histamine in naso-pharyngeal secretions after infection immune complexes in human diseases key: cord- - p ma authors: duan, x.; nauwynck, h. j.; pensaert, m. b. title: effects of origin and state of differentiation and activation of monocytes/macrophages on their susceptibility to porcine reproductive and respiratory syndrome virus (prrsv) date: - - journal: arch virol doi: . /s sha: doc_id: cord_uid: p ma in this study, the susceptibility of porcine peripheral blood monocytes (bmo), peritoneal macrophages (pmφ) and alveolar macrophages (amφ) to prrsv was examined. to test the effect of differentiation and activation on their susceptibility, amφ and bmo were aged, cultivated in either adhesion or suspension and treated with bacterial lipopolysaccharide (lps) and phorbol myristate acetate (pma). it was found that freshly isolated pmφ and bmo were non-permissive to prrsv. pmφ remained refractory but a few bmo became susceptible after day cultivation. amφ were permissive with a significant increase of their susceptibility after one day cultivation. in a binding assay, it was demonstrated that the attachment of biotinylated prrsv to amf is much more efficient than to pmφ and bmo. two monoclonal antibodies (mabs) d and d which block prrsv infection of amφ and are directed against a candidate receptor for prrsv only reacted with the cell membrane of amφ. pma treatment of amφ blocked prrsv replication in the cells in a dose-dependent manner. the blocking effect of pma decreased after h continuous pre-treatment and diminished after h continuous pre-treatment. pma treatment did not affect the binding of prrsv and mab d and d to amφ. direct or indirect treatment of amφ and bmo with lps or cultivation in suspension did not significantly affect their susceptibility. these results provide clear evidence that prrsv has a strongly restricted tropism for only some sub-populations of porcine monocytes/macrophages and that some specific states of differentiation and activation of monocytes/macrophages considerably affect their susceptibility. porcine reproductive and respiratory syndrome virus (prrsv) resembles lactate dehydrogenase-elevating virus (ldv), equine arteritis virus (eav) and simian hemorrhagic fever virus (shfv), three other members of the arterivirus group with regard to morphology, genetic organisation and structural proteins [ , ] . one peculiar common characteristic of these viruses is that they have a strong tropism for monocytes/macrophages. of many procine cell systems tested, only porcine alveolar macrophages (amf) support replication of prrsv [ , , , ] . replication of prrsv in some cultivated porcine peripheral blood monocytes (bmo) has also been reported [ ] . unexpectedly it was also found that prrsv replicate in two non-porcine cell lines: an established cell line from monkey kidney, marc- [ ] , and a proprietary cell line, cl [ ] . similarly, the replication of ldv and shfv is also highly restricted to primary cultures of host macrophages in vitro. eav forms an exception as it replicates in many different cell types [ ] . prrsv also shows a strict cell speci®city``in vivo''. cells of the macrophage lineage have previously been identi®ed as the predominately and consistently infected cell type in prrsv infected pigs [ , , , ] . furthermore, several observations indicated that prrsv only replicates in some sub-populations of monocytes/macrophages. it was found that amf even during the period of the highest virus titres in the lungs after natural or experimental infection, only a low percentage of levaged amf carries prrsv antigens [ , ] . also, immature macrophages and macrophages progenitor stem cells are not or less susceptible to a prrsv infection. this was particularly evident for bone marrow cells, where replication of prrsv was not detected in experimentally prrsv infected pigs [ ] . similar evidence comes from``in vitro'' experiments in which different sub-populations of alveolar macrophages fractionated by density gradient centrifugation were shown to have different susceptibilities to a prrsv infection [ ] . such heterogeneity may be a re¯ection of the sate of differentiation and/or activation of the macrophages [ ] . a number of factors such as ageing, some cytokines and a few chemical products have been found to be able to differentiate and activate mononuclear phagocytes [ ] . bacterial lipopolysaccharide (lps) and phorbol myristate acetate (pma) are two frequently used stimulants. both lps and pma have a strong, rapid and easily reproducible activating capacity. their structure and sites of activation in the cells have been extensively studied [ , ] . lps is a structural component of the outer membrane of gram-negative bacteria. it activates monocytes and macrophages and stimulates them to produce certain factors, including tnf-a, il- , and prostaglandin e [ , ] . studying the effect of lps treatment on the susceptibility of amf to prrsv may give new insights in the pathogenesis of dual infection with prrsv and bacteria. pma has effects on monocyte/macrophages, including dramatic changes in cell shape, spreading, endocytosis, and release of lytic mediators and regulators [ , , ] . pma also stimulate premonocytic cells of some continuous cell lines to differentiate into a more mature monocyte/macrophage phenotype [ ] . because of those remarkable properties, pma has been used to investigate the relationship between cell activation/differentiation and viral replication with some viruses [ , ] . in this study, it was examined if porcine monocyte/macrophage lineage cells isolated from peritoneal cavity, lungs and peripheral blood are susceptible to prrsv and if their susceptibility was related to the degree of virus attachment. furthermore, the effect of differentiation and activation of monocytes/macrophages by ageing, adhesion and treatment with lps and pma on their susceptibility to prrsv was evaluated. two prrsv isolates were used: the lelystad strain of prrsv (kindly provided by dr. wensvoort) and a belgian isolate of prrsv designated v . a lelystad virus stock of the thirteenth passage grown in porcine amf with a titre of . tcid /ml was used in this study. the v was adapted to marc- cells, puri®ed and biotinylated as earlier described [ ] . brie¯y, a ®fth passage of v was ®rst clari®ed by centrifugation at  g for min, then precipitated at  g for h in a beckman t rotor at c. the pellets were resuspended in / of original volume in tne buffer ( mm nacl, mm edta, mm tris-hcl, ph . ) and centrifuged on a . to . m discontinuous sucrose gradient in a sw rotor at  g for h. after centrifugation, the virus band was harvested and its purity was determined with a sodium dodecyl sulphate-polyacrylamide gel electrophoresis (sds-page). the titre of the resulting virus preparation was to  tcid /mg as determined on marc- cells. the number of virus particles, as determined by negative staining electron microscopy, was to  /mg virus protein. puri®ed prrsv was labelled with biotin by using a protein biotinylation kit (amersham, buckinghamshire, uk). the virus was pelleted and re-suspended in biotinylation buffer ( mm na co , ph . ) at a protein concentration of mg/ml. after a brief sonication ml biotin reagent was added per mg of viral protein. the mixture was shaken for h at c and the reaction was terminated by addition of tris-hcl (ph . ) to a ®nal concentration of mm. biotinylated virus was collected after purifying on a sephadex g- column and diluted in pbs at a concentration of . mg/ml. after biotinylation, the titre of prrsv was reduced by %. biotinylated virions were stored at À c. a swine myeloid cell speci®c monoclonal antibody (mab), . . , was used to determine the percentage of porcine monocyte/macrophage cells [ ] . mabs against prrsv nucleocapsid protein, wbe and wbe ± were used for immuno¯uorescenece [ ] . two monoclonal antibodies (mab), d and d , which have been raised against amf and which are able to block prrsv infection of amf [ ] , were used for membrane immuno¯uorescence staining to test their reactivity with various cells. isotype matched irrelevant mabs e and g directed against suid herpesvirus type [ ] , were used as negative controls. porcine alveolar macrophages (amf) amf were obtained from -to -weeks old conventional belgian landrace pigs from a prrsv negative herd according to the method previously described by wensvoort et al. [ ] . brie¯y, the lungs were lavaged with ml cold phosphate buffered saline solution without calcium and magnesium (pbs). the lavaged cells were collected by centrifugation at  g for min at c. after two washings with cold pbs, a cell smear was made and stained with hemacolar reagents (diagnostica merck, darmstadt, germany) and the percentage of neutrophils was determined. the cells were stained with . . by membrane immuno¯uorescence. the percentage of cells from the monocytes/macrophages lineage was estimated by subtracting the percentage of neutrophils from the . . positive cells. by doing so, more than % of lung lavage cells were found to be monocyte/ macrophage lineage cells. pmf were isolated by lavaging the peritoneal cavity of four -to -weeks old conventional belgian landrace pigs originating from a prrsv negative herd with ml cold pbs. after centrifugation at  g for min at c and two washings with pbs, the cells were collected and the percentage of monocyte/macrophage lineage cells was determined with the technique described above. more than % of lavaged peritoneal cells were characterised as monocytes/macrophages. bmo were separated and cultivated as previously described [ ] . brie¯y, peripheral blood was obtained from four to weeks old conventional belgian landrace pigs originating from a prrsv negative herd and peripheral blood mononuclear cells (pbmc) were isolated from blood by ficoll-paque (pharmacia, uppsala, sweden) density gradient centrifugation according to the method recommended by the manufacturer.  pbmc were layered on a polystyrene cell culture dish (corning glass works, corning, ny, usa) which was coated with ml autologous plasma. after h incubation at c, non-adherent cells were removed by three washings with pbs. adherent cells representing enriched monocytes were harvested by gently¯ushing, the number of cells were counted and the percentage of monocytes was estimated with mab . . . more than % of cells were found to be monocyte/macrophage lineage cells. the viability of all cells used was b as assessed by % nigrosin staining. amf, pmf and bmo were brought in -well cell culture plates (nalge nunc international, roskilde, denmark) at a concentration of cells per well. the medium used for cultivation was rpmi supplemented with % of foetal calf serum. amf, bmo and pmf were inoculated by replacing the medium with ml stock solution containing . tcid prrsv. after incubation at c for h, three washings with pbs were performed. then, the cells were refed with medium and further incubated at c with % co . to evaluate the effect of maturation of monocytes/macrophages on their susceptibility to prrsv, freshly isolated amf, pmf and bmo from ®ve donors were seeded in -well tissue culture plates at a concentration of cells/ml/well and further incubated in rpmi medium plus % of foetal bovine serum at c with % co . after and h incubation, the cells were inoculated with prrsv. extracellular virus titre and the percentage of viral antigen positive cells were determined at , and h after inoculation. to test if the adhesion of bmo and amf affect their susceptibility to prrsv, freshly isolated amf and bmo from ®ve donors were cultivated either in suspension in te¯on inserts (poly labo, strasbourg, france) or attached to polystyrene in -well cell culture plates at a concentration of cells/well in rpmi medium plus % of foetal bovine serum at c with % co . after h cultivation, the cells were inoculated with prrsv. extracellular virus titre and the percentage of viral antigen positive cells were determined at , and hours after inoculation. the effect of pma treatment on prrsv replication in bmo was examined by treating one day cultivated bmo with ng/ml phorbol -myristate -acetate (pma) (sigma-aldrich vertriebs gmbh, deisenbofen, germany) for h before prrsv inoculation. after pma treatment, the cells were washed three times, then inoculated with prrsv. extracellular virus titre and the percentage of viral antigen positive cells were determined at , and h after inoculation. in order to examine the effect of duration of pma pre-treatment of amf on prrsv replication, amf were treated with ng/ml pma for different time periods prior to prrsv inoculation. after pma treatment, the cells were washed three times then inoculated with prrsv. intracellular virus titre was determined at h after inoculation. to test the effect of pma treatment during virus replication in amf, the amf were treated with ng/ml pma for h at different times after prrsv inoculation. intracellular virus titre was determined at h after inoculation. to estimate the dose dependent effect of pma treatment of amf on the prrsv replication, one day cultivated amf grown in -well-plates were treated with different concentrations of pma for h after h prrsv inoculation. the prrsv positive wells were determined by ®xing and staining the plate using an immunoperoxidase monolayer assay (impa) as earlier described [ ] after h inoculation. direct and indirect lps treatments were performed to evaluate the effect induced directly by lps or indirectly by lps-induced cytokines. for direct treatment, bacto lipopolysaccharide (lps) of e. coli :b (difco laboratories, detroit, michigen, usa) was used at a concentration of mg/ml medium. for indirect treatment, % of the supernatant from h lps treated porcine amf was used. after and h treatment, the cells were washed three times with pbs and inoculated with prrsv. extracellular virus titre and the percentage of viral antigen positive cells were determined at , and h after inoculation. pma and lps had no negative effect on the viability of the treated cells as assessed by nigrosin staining after each treatment. a cytotoxicity bioassay with pk cells for detecting tnf [ ] and a proliferation assay on d (n )m cells for determining il- concentrations [ ] were performed to asses the ef®cacy of the lps treatment. for determining the titre of extracellular virus, medium from prrsv infected cells was collected and centrifuged at  g for min. ml of tenfold dilutions of the supernatants were inoculated in -well microtiter plates (nalge nunc, roskilde, denmark) previously seeded with amf. for determining the titre of intracellular virus, amf, pmf and bmo were harvested by¯ushing, washing, washed twice with pbs and lysed by freeze-thawing. ml of tenfold dilutions were inoculated in -well microtiter plates previously seeded with cultivated amf. inoculated -well plates were ®xed h after inoculation and stained using an immunoperoxidase monolayer assay (ipma) as earlier described [ ] . macrophages and monocytes were detached by thorough¯ushing of the bottom of polystyrene dishes and washed once with pbs. cell smears were made using a cytocentrifuge at  g for min. the smears were ®xed in acetone for min at À c. a streptavidin-biotin immuno¯uorescence technique was performed. brie¯y, the smears were pre-incubated with : diluted sheep serum to block non-speci®c staining. the smears were ®rst incubated with a mixture of mabs wbe and wbe ± , then with biotinylated sheep-anti-mouse immunoglobulin and ®nally with streptavidin-¯uorescein isothiocyanate (fitc)-conjugate (amersham, buckinghamshire, uk). the smears were washed three times with pbs between each incubation. to con®rm the speci®city of the staining, two mouse ascites¯uids containing irrelevant mabs against suid herpesvirus type of the same isotype as wbe and wbe ± were used as negative controls [ ] . positive cells were counted using a leica dm rbe¯uorescence microscope. the membrane reactivity of biotinylated prrsv, mabs d and d to pbmc, bmo, pmf and pma-treated ( ng/ml pma for h) and -untreated amf were evaluated by ā ow cytometer facscalibur (becton dickinson)¯ow cytometer.  cells were washed three times with cold pbs and incubated with ml of % paraformaldehyde at room temperature for min. after washing once with cold pbs, the ®xed cells were ®rst incubated with biotinylated prrsv ( mg) or mabs ( ng/ml of d , d or e ), then : diluted streptavidin-¯uorescein isothiocyanate (fitc)-conjugate or goat antimouse igg fitc (amersham, buckinghamshire, uk) for h on ice. the cells were washed three times after each incubation. the mean¯uorescence intensity of each sample was measured on the¯ow cytometer. relative mean¯uorescence measurements were corrected for auto¯uorescence of control cells. all data were statistically analysed by the student's t-test. prrsv productively replicated in amf. table shows the virus titres and percentage of prrsv positive cells in freshly isolated and one day cultivated amf at , and h after prrsv inoculation. the virus titres and percentage of viral antigen positive cells in freshly isolated porcine amf at and h after inoculation were respectively to log tcid and to times lower than those of one day cultivated ones, which is signi®cantly different (p` . ). a productive replication of prrsv was not detected in freshly isolated bmo obtained from four pigs. however, when the bmo were inoculated after one day of cultivation, x to . tcid per cells virus and . to . % viral antigen positive cells were detected at hours and hours post inoculation, respectively ( table ) . productive replication of prrsv was not found in freshly isolated pmf obtained from ®ve pigs. after one day cultivation, pmf remained refractory to prrsv infection. as shown in table , no statistically signi®cant differences in both viral antigen expression and virus titres were observed between amf and bmo cultivated in suspension in te¯on inserts and those attached to polystyrene dishes. the ef®cacy of activation after lps treatment was shown by the presence of tnf-a (titre: biological units per ml for the supernatant of amf and biological units per ml for that of bmo) and il- (titre: biological units per ml for the supernatant of amf and biological units per ml for that of bmo) in the supernatant of amf and bmo after h treatment with lps. however, no signi®cant differences in both viral antigen expression and virus titres were found between untreated and the directly or indirectly lps treated amf (table ) . no virus replication was detected in the bmo directly or indirectly treated with lps (data not shown). no virus replication was detected in the bmo treated with pma. when amf were pre-treated with pma for a short time ( to h), they became resistant to prrsv infection (fig. ) . after h continuous exposure to pma, amf gradually regained their susceptibility to prrsv and after h continuous pre-treatment, virus production reached the level similar to that of untreated cultures. the effect of a h treatment of amf with pma at different time points after a prrsv inoculation on the virus replication is shown in fig. . simultaneous virus inoculation and pma treatment for h reduced the virus production to a level of . tcid per cells. replication of prrsv was completely blocked when amf had been inoculated for h before pma treatment. the longer the interval between inoculation and treatment, the higher the virus titre. the blocking effect was concentration dependent as shown in fig. . concentrations as low as  À mg/ml pma completely inhibited prrsv infection. the membrane reactivity of bmo, amf and pmf to biotinylated prrsv and anti-amf monoclonal antibodies d and d as shown in fig. , the binding of biotinylated prrsv to bmo, amf and pmf was demonstrated by¯ow cytometry with a signi®cantly higher uorescence intensity in amf. the binding of biotinylated prrsv to the amf was not affected by pma treatment. after staining with anti-porcine amf monoclonal antibodies d and d , the membrane¯uorescence was detected only on amf and not on pbmc, bmo, and pmf (fig. ) . the results of this study show that only some subsets of cells from the porcine monocyte/macrophage lineage are susceptible to prrsv and that the speci®c differentiation and activation state may considerably affect their susceptibility to prrsv infection in vitro. cells of the porcine monocyte/macrophage lineage from different anatomic locations are clearly heterogeneous in their permissiveness for prrsv infection. prrsv replication was detected in porcine amf, while freshly isolated bmo and pmf were completely resistant. these results are in agreement with earlier``in vivo'' experiments, in which the replication of prrsv was found in alveolar macrophages but not in peripheral blood mononuclear cells [ ] . in the present study, very low virus titres and few infected cells were detected in cultivated bmo. this is in contrast with the observations of voicu et al. [ ] , who found much higher virus titres in cultivated bmo. this variation may be due to differences in the genotype/phenotype of the donor animals, differences in prrsv isolates and differences in techniques for the isolation of bmo. genetic, antigenic and pathogenic variations among prrsv isolates in the usa and europe have been reported [ , ] . even with amf and cl , two commonly used cell systems for prrsv isolation, nearly one third of american prrsv isolates grown in one cell type failed to grow in the other one [ ] . the experiments also revealed that amf show some restriction to a prrsv infection when they were freshly isolated, even though they are relatively the most sensitive cell type for prrsv isolation [ , ] . the susceptibility clearly increases after one day cultivation. a similar increase of permissiveness to prrsv infection was observed in a very small number of cultivated bmo. these results suggest that the state of monocyte/macrophage differentiation plays an important role in determining their susceptibilty to prrsv. similar enhancing effects of differentiation on virus replication have been observed with other viruses such as the respiratory syncytial virus (rsv), parain¯uenza virus- , african swine fever virus, cytomegalovirus or herpes simplex virus (hsv), human immunode®ciency virus type (hiv- ), visna-maedi virus and pseudorabies virus in monocytes/macrophages [ , ] . since bmo are precursors of tissue macrophages and readily mature into macrophages when cultivated in vitro [ ] , it is logical to predict that some bmo may mature into susceptible macrophages similar to alveolar macrophages by cultivation in vitro. resident pmf, which represent another population of well-differentiated tissue macrophages from a restricted lineage, were completely resistant to a prrsv infection. in this study, treatment of amf with pma was associated with a clear reduction of the replication of prrsv and the blocking effect of pma was transitory. when amf were continuously pre-treated for to h, the virus replication was almost completely blocked. the cells regained full susceptibility after h of continuous treatment. this rather peculiar ®nding can be explained by the fact that, while the activating capacity of monocytes and macrophages by pma reaches a maximum after h exposure, a prolonged exposure to pma for more than h causes macrophages and monocytes to become refractory to pma stimulation [ ] . when monocyte/macrophage lineage cells are treated with pma, both enhanced or decreased viral replication has been observed with other viruses. for example, an increased virus replication with herpes simplex virus has been noticed upon cell differentiation [ ] whereas a down-regulation of viral replication was observed with feline immunode®ciency virus [ ] . the mechanism through which pma inhibits prrsv infection of amf is unclear. in contrast to the pma treatment, lps treatment of porcine amf and bmo did not in¯uence the susceptibility to prrsv infection. the biological properties of pma are generally considered to be due to the activation of protein kinase c, an enzyme involved in many cellular responses. although the activation of protein kinase c is also an essential process for lps induced activation of the macrophage, the signal pathways used by pma and lps in macrophages are quite different [ ] . therefore, it seems that pma modulated inhibition of prrsv infection might be mediated by a speci®c pma-signalling pathway in porcine amf. recent observations suggest that prrsv enters amf through a process of receptor-mediated endocytosis (unpubl. data). pma treatment does not change the binding activity of prrsv and monoclonal antibody against the putative prrsv receptor(s) to amf. therefore, the blocking effect of pma does not act through down-regulating the expression of the virus receptor(s) on the cells. mononuclear phagocyte differentiation is a process of normal cellular development and homeostasis and re¯ects a permanently altered expression of a cell's genetic potential, while macrophage activation is the process that causes reversible changes in macrophage phenotype and functions [ ] . the differentiation may induce some factors that are essential for virus replication, such as receptors or transcription factors, while activation may up-or downregulate the expression of these factors in cells. inef®cient binding of biotinylated prrsv to pmf, bmo and pbmc suggests that these cells lack the more speci®c binding sites that are expressed on the membrane of amf. different binding activity of mab d and d , which block prrsv infection of amf and recognise a candidate receptor on the cell, to different monocytes/macrophages provide further evidence that the restriction of prrsv replication in some subsets of monocytes/macrophages is due to a reduced expression of virus receptor(s). comparison of porcine alveolar macrophages and cl for the detection of porcine reproductive and respiratory syndrome (prrs) virus and anti-prrs antibody characterisation of swine infertility and respiratory syndrome (sirs) virus (isolate atcc vr- ) improved bioassay for the detection of porcine tumor necrosis factor using a homologous cell line: pk( ) infection of peritoneal macrophages in vitro and in vivo with feline immunode®ciency virus lipopolysaccharide receptors and signal transduction pathways in mononuclear phagocytes heterogeneity of porcine alveolar macrophages sub-population: immune functions and susceptibility to pears virus two distinct cytolytic mechanisms of macrophages and monocytes activated by phorbol myristate acetate isolation of swine infertility and respiratory syndrome virus (isolate atcc vr- ) in north america and experimental reproduction of the disease in gnotobiotic pigs molecular characterization of porcine reproductive and respiratory syndrome virus, a member of the arterivirus group production, characterization and reactivity of monoclonal antibodies to porcine reproductive and respiratory syndrome virus virus quanti®cation and identi®cation of cellular targets in the lungs and lymphoid tissues of pigs at different time intervals after inoculation with porcine reproductive and respiratory syndrome virus (prrsv) porcine reproductive and respiratory syndrome virus infection of alveolar macrophages can be blocked by monoclonal antibodies against cell surface surface antigens macrophages in viral infections immunohistochemical identi®cation of porcine reproductive and respiratory syndrome virus (prrsv) antigen in the heart and lymphoid system of three-week-old colostrumdeprived pigs simple, sensitive and speci®c bioassay of interleukin- enhanced replication of porcine reproductive syndrome (prrs) virus in a homogeneous subpopulation of ma- cell line phylogenetic analyses of the putative m (orf ) and n (orf ) genes of porcine reproductive and respiratory syndrome virus (prrsv): implication for the existence of two genotypes of prrsv in the u.s.a. and europe diagnosis of porcine reproductive and respiratory syndrome lelystad virus, the causative agent of porcine epidemic abortion and respiratory syndrome (pears), is related to ldv and eav in vitro induction of cytologic functional differentiation of the immature human monocyte cell line u- with phorbol myristate acetate replication of virulent aujeszky's disease virus (adv) in different blood mononuclear cell subpopulations virus production and viral antigen expression in porcine blood monocytes inoculated with pseudorabies virus effect of speci®c antibodies on the cell-associated spread of pseudorabies virus in monolayers of different cell types preparation and characterization of monoclonal antibodies reactive with porcine pbl lactate dehydrogenase-elevating virus, equine arteritis virus, and simian hemorrhagic fever virus: a new group of positive-strand rna viruses pathological, ultrastructural, and immunohistochemical changes by lelystad virus in experimentally induced infections of mystery swine disease (synonym: porcine epidemic abortion and respiratory syndrome (pears)) chronological immunohistochemical detection and localization of porcine reproductive and respiratory syndrome virus in gnotobiotic pigs mechanism generating functionally heterogeneous macrophages: chaos revisited lps induces selective translocation of protein kinase c-beta in lps-responsive mouse macrophages, but not in lps-nonresponsive mouse macrophages interaction of porcine reproductive and respiratory syndrome virus with swine monocytes mystery swine disease in the netherlands: the isolation of lelystad virus antigenic comparison of lelystad virus and swine infertility and respiratory syndrome (sirs) virus mechanism of intrinsic macrophage-virus interaction``in vitro isolation of a cytopathic virus from weak pigs on farms with a history of swine infertility and respiratory syndrome we thank c. bracke for technical assistance. we are grateful to dr. g. wensvoort for the supply of the lelystad isolate of prrsv and dr. t. w. drew for the gift of monoclonal antibodies wbe and wbe ± . received january , key: cord- - t lr z authors: moin, abu saleh md; sathyapalan, thozhukat; atkin, stephen l.; butler, alexandra e. title: pro-fibrotic m macrophage markers may increase the risk for covid in type diabetes with obesity()() date: - - journal: metabolism doi: . /j.metabol. . sha: doc_id: cord_uid: t lr z nan rate modified aptamer (soma)-scan plasma protein measurement [ ] was used to determine lps-binding protein (lbp), tgfβ- , pdgf-β, mmp and mmp protein concentration, expressed as relative fluorescent units (rfu). statistical analysis was performed using the student's t-test (graphpad prism . , san diego, ca). lps-binding (lbp) protein was reduced in plasma ( , . ± . vs , . ± . rfu, ot d vs control, p < . ). plasma lbp was also reduced in obese versus non-obese subjects regardless of diabetic status ( , . we report here that lps-related markers were associated with activated lung alveolar m macrophages in ot d, with a reduction in plasma lbp as a surrogate marker of circulatory lps elevation. previously increased lbp levels were reported in obesity [ , ] , a discrepancy compared to our observations that might be due to inclusion of obese subjects whom were smokers and alcoholic in those studies. lbp was increased in the bronchoalveolar lavage fluid (balf) of smokers [ ] . serum levels of lbp are also increased in heavy drinkers, probably reflecting high lps exposure due to alcohol-induced damage of the gastrointestinal barrier [ , ] . by contrast, in our study none of the ot d subjects were smokers or consumed alcohol, as those were exclusion criteria. moreover, lbp, the serum glycoprotein, plays a concentrationdependent dual role in determining lps-induced macrophage activation; low concentrations of lbp enhance the lps-induced activation of mononuclear cells (mnc), whereas the acute-phase rise in lbp concentration inhibits lps-induced cellular stimulation [ ] . furthermore, lbp is bound and internalized by host cells and colocalizes with lps in the cytoplasm [ ] . therefore, the significantly lower lbp levels reflect the elevated lps levels in ot d subjects in our study. the elevated tgf-β shown here may predispose to alveolar pre-fibrosis with their collapse following sars-cov- infection. tgf-β is detected in lung bronchoalveolar lavage fluid of % of patients with ards, major cellular sources of tgf-β in pulmonary fibrosis being alveolar macrophages and metaplastic type ii alveolar epithelial cells. activation of tgf-β is affected by mmp that was elevated here in ot d, contributing to enhancement of the pool of active tgf-β . mmp also weakens the airway epithelial barrier function by altering transepithelial electrical conductance and epithelial permeability to macromolecules [ ] . mmp , also elevated here, is increased in ards and associated with idiopathic pulmonary fibrosis (ipf), whilst pdgfβ, again elevated here, contributes to fibrosis development with tgf-β in ards. elevated plasma levels of tgf-β , pdgf-β, mmp and mmp determined early in the course of covid infection in a patient with ot d may indicate potential risk for more severe disease. the strengths of this study include the inclusion of a group of ot d subjects who were relatively treatment naïve and not on poly-pharmacy, and an age-matched healthy control group together with statehttps://doi.org/ . /j.metabol. . - /© elsevier inc. all rights reserved. of-the-art measurement of plasma proteins by soma-scan. limitations include the relatively small study subject numbers and that all participants were caucasian, so the results may not be generalizable to other ethnic groups. moreover, it is also possible that, apart from alveolar macrophages, other cellular sources, for example lung epithelial cells [ ] , arterial smooth muscle cells [ ] , epithelial cells of glandular tissues like prostate [ ] or bile duct epithelia [ ] , might contribute to lps-induced elevated plasma levels of those pro-fibrotic markers. in conclusion, in ot d the lung epithelial barrier integrity is likely destabilized in response to fibroproliferative activity of elevated tgf-β , pdgf-β, mmp or mmp derived from lung alveolar macrophages, increasing vulnerability to inhaled pathogens. this might lead to irreversible structural alterations and tissue stiffening in the lungs of ot d patients even prior to sars-cov- infection and thereby make these patients more vulnerable to covid -related infection with severe disease. the yorkshire and humber research ethics committee approved this study. all patients gave written informed consent. all authors gave their consent for publication. all the data for this study will be made available upon reasonable request to the corresponding author. no funding was received to perform this study. commentary: covid- and obesity: exploring biologic vulnerabilities, structural disparities, and weight stigma pulmonary postmortem findings in a series of covid- cases from northern italy: a two-centre descriptive study alveolar macrophage dysfunction and cytokine storm in the pathogenesis of two severe covid- patients pathological inflammation in patients with covid- : a key role for monocytes and macrophages the chemokine system in diverse forms of macrophage activation and polarization a vicious circle of alveolar macrophages and fibroblasts perpetuates pulmonary fibrosis via ccl effect of induced hypoglycemia on inflammation and oxidative stress in type diabetes and control subjects a marker of endotoxemia is associated with obesity and related metabolic disorders in apparently healthy chinese determinants of serum concentrations of lipopolysaccharide-binding protein (lbp) in the adult population: the role of obesity lipopolysaccharide-binding protein and cd are increased in the bronchoalveolar lavage fluid of smokers concentrations of lipopolysaccharide-binding protein, bactericidal/permeability-increasing protein, soluble cd and plasma lipids in relation to endotoxaemia in patients with alcoholic liver disease increased intestinal permeability to macromolecules and endotoxemia in patients with chronic alcohol abuse in different stages of alcohol-induced liver disease dual role of lipopolysaccharide (lps)-binding protein in neutralization of lps and enhancement of lps-induced activation of mononuclear cells lipopolysaccharide-binding protein is bound and internalized by host cells and colocalizes with lps in the cytoplasm: implications for a role of lbp in intracellular lps-signaling mmp modulates tight junction integrity and cell viability in human airway epithelia regulation of matrilysin expression in airway epithelial cells by pseudomonas aeruginosa flagellin lipopolysaccharide regulates mmp- expression through tlr /nf-κb signaling in human arterial smooth muscle cells lps/tlr signaling enhances tgf-β response through downregulating bambi during prostatic hyperplasia lipopolysaccharide enhances transforming growth factor β -induced platelet-derived growth factor-b expression in bile duct epithelial cells no authors have any conflict of interest or competing interests to declare. key: cord- -df ard o authors: müller-redetzky, holger c.; suttorp, norbert; witzenrath, martin title: dynamics of pulmonary endothelial barrier function in acute inflammation: mechanisms and therapeutic perspectives date: - - journal: cell tissue res doi: . /s - - - sha: doc_id: cord_uid: df ard o the lungs provide a large inner surface to guarantee respiration. in lung alveoli, a delicate membrane formed by endo- and epithelial cells with their fused basal lamina ensures rapid and effective gas exchange between alveolar and vascular compartments while concurrently forming a robust barrier against inhaled particles and microbes. however, upon infectious or sterile inflammatory stimulation, tightly regulated endothelial barrier leakiness is required for leukocyte transmigration. further, endothelial barrier disruption may result in uncontrolled extravasation of protein-rich fluids. this brief review summarizes some important mechanisms of pulmonary endothelial barrier regulation and disruption, focusing on the role of specific cell populations, coagulation and complement cascades and mediators including angiopoietins, specific sphingolipids, adrenomedullin and reactive oxygen and nitrogen species for the regulation of pulmonary endothelial barrier function. further, current therapeutic perspectives against development of lung injury are discussed. the inner walls of blood vessels are covered with a continuous endothelial cell monolayer, constituting a semi-permeable barrier between blood and interstitium. neighbouring endothelial cells (ecs) are closely connected to each other by interendothelial junctions. under physiologic conditions, the endothelial monolayer actively controls paracellular and transcellular extravasation of proteins, solutes and fluids, thereby adjusting interstitial fluid homeostasis (komarova and malik ) . in lung alveoli, endo-and epithelial cells and their merged basal laminas build a delicate membrane of less than μm thickness, which ensures rapid and effective gas exchange between alveolar and vascular lumens while at the same time forming a robust barrier against inhaled particles and microbes. moreover, this sophisticated structure importantly contributes to central metabolic and immunologic functions of the lung. however, upon infectious or sterile inflammatory stimulation via either the alveolar (e.g., in pneumonia and mechanical ventilation) or the vascular lumen (e.g., in bacteremia and sepsis), pulmonary endothelial barrier homeostasis may be disturbed, resulting in increased permeability, protein-rich fluid extravasation, lung oedema and finally acute respiratory distress syndrome (ards) with mortality rates ranging from to % depending on severity (ranieri, et al. ) (fig. ) . pneumonia is the most prevalent infectious disease worldwide and the third most frequent cause of death (world health organisation ) . pneumonia is also the most frequent cause of sepsis, a systemic inflammatory response of the organism, which may originate from infections at any other site of the body (abdominal, blood stream, urogenital, etc.) . in both pneumonia and sepsis, the initial innate immune response to invading bacteria, viruses or fungi is insufficient to avert the infection. despite subsequent antibiotic treatment, the interaction of pathogens and host defence culminates in complex inflammatory responses. liberation of inflammatory mediators, recruitment and activation of leukocytes to the lungs and activation of complement and coagulation cascades, are initiated, contributing to pulmonary endothelial hyperpermeability and ards development. for patients with ards, mechanical ventilatory support is an inevitable and life-saving treatment but may also perpetuate the inflammatory response and further enhance pulmonary endothelial barrier dysfunction (verbrugge et al. ) . specific pharmacologic therapies aiming at improvement of endothelial barrier function in patients with pneumonia, sepsis and/or ards are lacking. however, recent experimental studies enhanced our understanding of endothelial pathomechanisms contributing to the development of ards, potentially providing the basis for novel therapeutic strategies. therefore, we try here to give an overview on recent insights into the mechanisms of pulmonary endothelial barrier dysfunction in acute inflammation. inside endothelial cells, filaments of polymerised actin molecules together with polymerised tubulin molecules (microtubules) build the cytoskeleton, which is connected to glycocalyx (yoneda and couchman ) , focal adhesions and junctional proteins. whereas two types of endothelial junctions, adherens junctions (aj) and tight junctions (tj), are known, the current concept is that a sealing belt of tight junctions is present in ec of the blood-brain barrier but play a minor, if any, role in the barrier function of pulmonary endothelium. consisting in vascular endothelial (ve) cadherin and catenin, ajs maintain the tight connection of adjacent ecs and regulate the paracellular passage of fluids and solutes smaller than nm in radius across the endothelial monolayer (komarova and malik ) . in parallel, larger molecules including hormones, drugs, albumin and albumin-bound substances are transported across the endothelial barrier by transcellular trafficking. caveolar vesicles formed at the luminal side of ecs take up the molecules to be transported, cross the ecs and release the molecules at the abluminal surface by exocytosis (predescu et al. ). these two different transport mechanisms actively regulate endothelial permeability and thereby tissue fluid homeostasis. pathogens entering the alveolar compartment by inhalation or via the bloodstream are recognized by pathogen recognition receptors (prrs). the heterogeneous group of prrs comprises toll-like receptors (tlrs), cytosolic nod-like receptors (nlrs), rig-i-like receptors (rlrs) and dna sensors (opitz et al. ) . in alveoli, these receptors are expressed in epithelial cells, macrophages, dendritic cells, ecs and in subsequently recruited immune cells. prrs sense highly conserved microbial molecules called pathogen-associated molecular patterns (pamps) and specific endogenous molecules liberated by cell injury called danger-associated molecular patterns (damps). prr activation evokes cellular production of inflammatory cytokines, interferons and chemokines on transcriptional and post-translational levels (opitz et al ) , resulting in the activation of locally distributed cells and the recruitment of neutrophils and macrophages. thus, upon microbial infection and "sterile" tissue damage by various insults, prrs are central contributors to the inflammatory response. when being controlled, these inflammatory mechanisms are a prerequisite for pathogen clearance and thus for survival. however, control is frequently lost and once the inflammatory cascade is on track even effective antibiotic treatment is unable to stop it, which can (partly) be explained by ongoing pamp and damp release from dying bacteria and injured cells, respectively. inappropriate inflammation induces further unchecked synthesis of cytokines, chemokines and lipid mediators, accumulation and activation of leukocytes, uncontrolled activation of complement and coagulation cascades and last but not least endothelial barrier dysfunction. fig. a airspace-derived activation of the endothelium by mediators, bacterial toxins or physical stress due to mechanical ventilation starts a complex interplay of various inflammatory cascades resulting in vascular permeability. monocytes (m) are recruited to the endothelium (ec) and facilitate its further activation by secretion of tnfα, thereby augmenting the recruitment of neutrophils (pmn). activated platelets stimulate pmn. endothelium-pmn contact leads to permeability ( ). upon stimulation pmn undergo netosis, liberating neutrophil extracellular traps (nets) consisting in dna and histones that cause endothelial toxicity and barrier breakdown ( ). specific soluble mediators also increase permeability ( ). neutrophil-platelet complexes activate blood coagulation. central effector proteases like thrombin directly mediate vascular permeability. further, thrombin activates complement factor c to c a-a permeability increasing anaphylatoxin ( ). tnf tumor necrosis factor; il- β interleukin- β; ros/rns reactive oxygen and nitrogen species. b intracellular signalling regulates endothelial permeability. endothelial contraction results from actin myosin interaction after mlc-phosphorylation, which is regulated by myosin light chain kinase (mlck) and myosin light chain phosphatase (mlcp). mlcp is inhibited by rhoa-rock signalling while mlck is activated by c-src, rhoa and ca + /calmodulin. ca + enters the cytosol from endoplasmatic reticulum (er) or extracellular space. downstream of platelet activating factor (paf) and paf receptor (pafr), phospholipase c (plc) hydrolyses posphatidyl inositol bisphosphate (pip) into inositol , , -triphosphate (ip ) and diacylglycerol (dag). ip mediates ca + liberation from the er while dag opens transient receptor potential canonical (trpc) channels in the cellular membrane. the resulting increase of intracellular ca + leads to the activation of protein kinase c (pkc) α, to further rhoa activation and to ca + /calmodulin complexes, altogether finally leading to mlck activation. actin polymerisation forms stress fibres associated with endothelial contraction. various stimuli like il- β or mechanical force activate mitogen-activated protein kinase (mapk) p (p ), which activates mapk activated protein kinase (mk ), which phosphorylates heat shock protein (hsp ) leading to actin polymerisation. adherence junctions (aj) are mandatory for the sealing of intercellular contacts. ve-cadherin is anchored in peripheral cortical actin to the cytoskeleton. ve-cadherin phosphorylation leads to ve-cadherin internalisation and thereby to increased endothelial permeability. rhoa and c-src phosphorylate vecadherin. rac- and p rhoagap (p ) functionally antagonise rhoa activity. p rhoagap is recruited to the aj by p -catenin (p ), which itself inhibits ve-cadherin internalisation. rock inhibits p rhoagap and pkcα inactivates p -catenin thereby augmenting destabilisation of aj. iqgap recruits and stabilises rac- , protecting against ve-cadherin internalisation not only direct prr ligation by the pathogen but also liberated pathogenic factors may activate prr-dependent inflammatory cascades. for example, cell wall peptidoglycan of streptococcus pneumoniae activates tlr- (schroder et al. ) , while the pneumococcal exotoxin pneumolysin is recognized by tlr- and nlrp- (malley et al. ; witzenrath et al. ) . bacterial toxins rapidly compromise endothelial cell function (rubins et al. ; suttorp et al. suttorp et al. , suttorp et al. , . pneumolysin, for example, may rapidly induce ( ) ca + influx and ( ) liberation of platelet activating factor (paf) followed by thromboxane release (lucas et al. ; witzenrath et al. ). ca + increase and thromboxane receptor ligation both activate myosin light chain kinase (mlck) via pkcα and rho-kinase dependent signaling (hippenstiel et al. ; lucas et al. ; witzenrath et al. ). mlck phosphorylates mlc and subsequent actinmyosin-dependent cytoskeletal contraction evokes disruption of ajs, interendothelial gap formation and paracellular permeability (shen et al. ). in addition, pneumolysin is a cholesterol-dependent cytolysin that kills ecs by pore formation (tilley et al. ) . thus, pathogens may induce endothelial injury via host-dependent inflammatory and via direct mechanisms. upon acute inflammation, neutrophils and distinct monocyte subsets among other recruited leukocytes are involved in the pathophysiology of pulmonary vascular barrier failure. platelets also contribute to vascular injury by activating neutrophils and liberating soluble factors that directly interact with vascular barrier integrity. neutrophils are rapidly recruited to the lung upon different insults (grommes and soehnlein ; yoshida et al. ). in the lungs, the capillary compartment is the place of neutrophil transmigration, in contrast to other vascular beds where neutrophils pass the endothelial barrier in the venules. upon stimulation by various inflammatory agents, the cytoskeleton of the neutrophils changes by forming peripheral actin rims, which leads to neutrophil stiffening and trapping in the capillary bed (yoshida et al. ) . although the initial trapping is independent from expression of integrins and selectins on the cell surface (yoshida et al. ) , further recruitment may indeed depend on selectins and integrins in distinct scenarios (reviewed in grommes and soehnlein ) . however, endothelial leukocyte adhesion and alveolar recruitment of neutrophils does not induce significant vascular permeability per se (martin et al. ; rosengren et al. ) . although not yet shown for the pulmonary endothelium, studies performed in huvecs or cremaster vessel preparations reveal that during the process of neutrophil transmigration, endothelial disruption seems to be controlled by the formation of "endothelial domes / transmigratory cups" (carman and springer ; phillipson et al. ) that encapsulate the neutrophil and further by ring-like structures of neutrophil lfa- and endothelial icam- around the invading leukocyte, thereby potentially sealing the barrier through diapedesis (shaw et al. ) . however, activated neutrophils contribute to vascular permeability by ( ) secretion of soluble factors causing endothelial contraction, ( ) contact mediated mechanisms and ( ) generation of reactive oxygen species. amongst others, soluble factors of neutrophils include tnf-α, which binds to tnf-α receptor and and is known to induce vascular permeability. notably, although tnf-α leads to mlck and rho kinase (rock)-dependent actin stress fibre generation in endothelial cells, this is probably not the main mechanism of tnf-α induced endothelial permeability, as blocking rock or mlck did not ameliorate transcellular electric resistance (petrache et al. ). however, tnf-α also induced p mapk-dependent disarrangement of the microtubule system and thereby loss of intercellular ve-cadherin resulting in barrier disruption. blocking microtubule breakdown strongly protected against barrier failure induced by tnf-α (petrache et al. ) . further soluble factors include: ( ) thromboxane a , which is processed by endothelial cyclooxygenase- (cox ) from neutrophil-derived arachidonic acid, binds to the thromboxane receptor and may induce permeability in endothelial cells (kim et al. ); ( ) leukotriene a , which is processed by endothelial ltc synthetase and binds to the endothelial cysteinyl lt receptor subtype (cyslt r); and ( ) cxcl , - , - , - which bind to cxcr and are involved in endothelial barrier disruption (extensively reviewed in (distasi and ley )). further, neutrophil -endothelial contact via icam- and lfa- /mac- leads to ( ) rapid increase of intracellular ca + , which mediates actin polymerisation and endothelial contraction as well as disassembly of adherence junctions due to phosphorylation of ve-cadherin and ( ) to the secretion of heparin-binding protein, which is also secreted by neutrophils upon binding of ltb to the blt receptor, finally resulting in barrier failure by endothelial contraction (for detailed review of underlying mechanisms, refer to distasi and ley ). activation of neutrophils in the pulmonary microvasculature leads to endothelial hyperpermeability by generation of reactive oxygen species. gao et al. ( ) observed that ros generation upon tnf-α stimulation depends on class a phosphoinositide kinase and cd b/cd , resulting in nadph oxidase activation and finally generation of ros, causing pulmonary hyperpermeability (see below). platelets secrete various mediators upon activation, including thromboxane, thereby decreasing endothelial barrier integrity as observed in human umbilical vein endothelial cells (huvec) and in vivo (kim et al. ) . moreover, platelets mediate vascular permeability in infection and inflammation indirectly via activation of neutrophils (he et al. ; looney et al. ; zarbock et al. ). clark and colleagues have shown that platelets are activated by stimulation of tlr on their surface in murine sepsis (clark et al. ). upon activation, platelets secrete thromboxane, which is mandatory for the formation of permeability-mediating platelet-neutrophil complexes. in contrast, neutrophils solely attached to the endothelium after activation by tnf-α do not increase vascular permeability (he et al. ) . in mouse models of transfusion-related acute lung injury (trali) platelets are crucial for the development of permeability and pulmonary neutrophil sequestration (looney et al. ). further, platelets are involved in the generation of neutrophil extracellular traps (nets). neutrophils can undergo a process termed netosis in which the neutrophil expels its condensed dna, to which histones, antimicrobial peptides and enzymes like myeloperoxidase are bound. nets can bind and kill bacteria and thus contribute to the innate immune response against invading pathogens (brinkmann et al. ). on the other side, nets can be harmful. nets are involved in thrombus generation and cause endothelial permeability and sepsis related organ failure (caudrillier et al. ; clark et al. ; saffarzadeh et al. ) . in trali, platelets are mandatory for net formation in the lung and inhibition of platelet aggregation ameliorated net generation and consecutively pulmonary permeability (caudrillier et al. ). among peripheral blood monocytes a population of gr- high /ccr + /cxccr low monocytes can be defined, which are delivered from the bone marrow to sites of inflammation. this population rapidly homes in the pulmonary microvasculature upon lipopolysaccharide (lps) infusion or the onset of injurious mechanical ventilation and primes the lung for the development of pulmonary oedema formation when a second hit like lps, zymosan or ventilator-induced lung injury (vili) occurs wilson et al. ). the mechanism by which this damage is mediated is not fully clarified but the recruited monocytes secrete tnf-α and activate endothelial cells in a paracrine fashion, thereby directly or indirectly contributing to endothelial barrier dysfunction (o'dea et al. ) . further, they are involved in the process of neutrophil recruitment in ali (dhaliwal et al. ) . although the underlying mechanisms of leukocyte mediated barrier failure are of highest scientific interest, therapeutic interference to ameliorate acute lung injury by depletion or blocking of cell recruitment should raise concerns as neutrophils and monocytes are key players of pulmonary and systemic innate immune responses and therapeutic intervention at this level might leave the patient functionally immunosuppressed. elevated fibrin turnover is a hallmark of acute lung injury regardless of its genesis and may correlate with the severity of the diseases (glas et al. ; prabhakaran et al. ) . intrapulmonary fibrin deposition results from tissue factorfactor vii pathway activation, reduced pulmonary fibrinolytic capacity due to elevation of plasminogen activator inhibitor (pai- ) concentrations, diminished absolute and relative protein c activity due to reduced protein c production and shedding of thrombomodulin, an important activator of protein c on the cell surface, as well as reduced antithrombin iii levels (hofstra et al. ; prabhakaran et al. ; ware et al. ) . pulmonary coagulopathy occurs after alveolar flooding with protein-rich fluid due to high permeability oedema, resulting in alveolar fibrin deposition but coagulopathy also contributes to inflammation and vascular permeability itself, thereby aggravating the disease. thrombin, the central protease of the coagulation pathway activating fibrinogen, mediates proinflammatory effects by binding to protease activated receptors (par), thereby causing secretion of cytokines or leading to liberation of vascular endothelial growth factor (vegf), which contributes to vascular permeability (hippenstiel et al. ). furthermore, thrombin can directly cause endothelial cell contraction and processing of complement factor c a from c , a potent anaphylatoxin causing inflammation and vascular permeability (cirino et al. ; glas et al. ; huber-lang et al. ; khan et al. ; liu et al. ) . the complement system is part of the innate immune system and is also involved in functions of the adaptive immune response (mastellos et al. ) . the complement cascade can be activated by the classical, the lectin and the alternative pathways (markiewski and lambris ) . antigen-antibody complexes activate the classical pathway by binding c q, thereby processing c s, while in the lectin pathway mannose binding lectins (mbl) bind to pathogen associated molecular patterns on bacteria, assembling with mannose binding lectin proteases (mblp) and thereafter. c a and mbl/ mblp + subsequently interact with c and c , processing the c convertase c b a. the alternative pathway is activated after contact with, e.g., bacterial surfaces by spontaneous hydrolysis of c , which forms together with factor bb the alternative c convertase c bbb. both c convertases process c to c aan anaphylatoxinand c b, which is part of the c convertase. the c convertase cleaves c into c aa second anaphylatoxinand c b, the latter one being part of the membrane attack complex that leads to cell lysis, while c a and c a contribute to inflammation and vascular permeability. both c a and c a induce stress fibre generation in endothelial cells and thereby endothelial contraction. notably, the response was only of short duration after c a stimulation, while being prolonged after c a exposition (schraufstatter et al. ) . c a-induced permeability was more severe and phosphaditiyinositol- kinase-, src kinaseand epidermal growth factor (egf) receptor-dependent, while c a mediated its effects via rho kinase-controlled pathways (schraufstatteret al. ) . neutralising c a in rodent models of acute lung injury and systemic inflammatory responses reduced permeability in various organs including the lung (liu et al. ) . however, in c -deficient mice, immune complex-mediated lung injury including vascular permeability was not attenuated, while c a deficiency proved to be protective (huber-lang et al. ). this observation led to the understanding that c a can be alternatively processed by the protease thrombin defining another alternative pathway for complement activation downstream of c a. thus, targeting c a rather than c a to ameliorate vascular permeability seems to be reasonable. a study by kahn and colleagues also even observed aggravated microvascular injury in c -deficient mice suffering from acute rejection after trachea transplantation, while antagonisation of c a was highly protective. again, thrombin-mediated c a activation accounted for this observation (khan et al. ). toll-like receptor (tlr ) dependent signaling tlr is central for recognition of exogenous (e.g., lps) and endogenous (e.g., high mobility group box- , oxidised phospholipids) pro-inflammatory stimuli (imai et al. ; park et al. ) . systemic lps levels have been linked to severity of sepsis and related organ failure (marshall et al. ) . lps induced vascular permeability (mehta and malik ) and mice deficient for tlr were protected against lung injury due to different stimuli including lps, oleic acid, cecal ligation and puncture and gut or lung ischemia/reperfusion injury (ben et al. ; hilberath et al. ; imai et al. ; tauseef et al. ; zanotti et al. ). various signalling cascades have been linked to tlr -mediated pulmonary permeability. oxidised phospholipids induced tlr dependent activation of trif (tir domain-containing adapter-inducing interferon-β) and traf (tnf receptorassociated factor ) leading to nf-κb-dependent il- liberation, which contributed to lung oedema (imai et al. ) . after binding to the tlr /md receptor complex, lps induced nf-κb activation via myd , irak (interleukin- receptorassociated kinase) , irak and irak (kawagoe et al. ; medvedev et al. ) . further, recognition of lps by tlr increased intracellular diacylglycerol (dag) levels, activating transient receptor potential canonical (trpc) channels and leading to calcium influx, thereby activating mlck, which facilitates myosin light chain (mlc) phosphorylation inducing endothelial cell contraction. mlck activation further augmented lps-induced nf-κb-related inflammatory responses that contribute to vascular leakage (mehta and malik ; tauseef et al. ) . further, tlr activation evoked phosphorylation of src-kinase and consecutively of ve-cadherin and p , ultimately resulting in destabilisation of adherence junctions (gong et al. ) . tlr- is involved in the proinflammatory response to hmgb- in monocytes, which again was found to be myd -, irak , , -and nf-κb-dependent (park et al. ). moreover, hmgb- was linked to lung oedema formation in ventilator-induced lung injury (ogawa et al. ). however, hmgb- also induced endothelial permeability via the receptor for advanced glycation end products (rage) (wolfson et al. ) . in summary, tlr is often critically involved in the regulation of vascular barrier function during lung inflammation. thus, enthusiasm was aroused by the development of eritoran, an inhibitor of lps-binding to the tlr- adaptor molecule md- . eritoran reduced pulmonary inflammation in different animal models (mullarkey et al. ) as well as in humans exposed to lps bolus infusion (lynn et al. ) . in a phase ii clinical trial, patients with severe sepsis treated with eritoran tended to have reduced mortality as compared to placebotreated patients (tidswell et al. ). however, a recent multicentre phase iii study found no impact of eritoran on mortality or relevant secondary outcome parameters in sepsis (opal et al. ) , questioning the rationale of tlr inhibition for the treatment of sepsis and related organ failure including ards. although not proven by current data, it is tempting to speculate that targeting a single prr was unsuccessful because of the pleiotropic immune activation by various pamps and damps involving different prrs and downstream signaling pathways in sepsis. angiopoietin- (ang- ) to ang- are ligands of the receptor tyrosine kinase tie . ang- and - are well-known regulators of angiogenesis, inflammation and vascular leakage (reviewed in david et al. ; eklund and saharinen ) , whereas the role of ang- and its murine orthologue ang- has not been extensively investigated. tie is abundantly expressed in endothelium and also in pmns and a subpopulation of monocytes (lemieux et al. ; wong et al. ) . ang- is constitutively expressed in different cell types, including pericytes surrounding the vasculature, vascular smooth muscle cells, fibroblasts, thrombocytes and megakaryocytes (eklund and saharinen ) . steady tie activation by ang- importantly contributes to endothelial quiescence and barrier integrity. in contrast, ang- is expressed in endothelial cells, stored in weibel-palade bodies (fiedler et al. ) and rapidly released upon activation by inflammatory stimuli including tnf-α and thrombin (fiedler et al. (fiedler et al. , . ang- acts as an antagonist of ang- at the tie receptor, thus confirming endothelial quiescence and perpetuating proinflammatory, barrier-disintegrating mechanisms (fiedler et al. ; scharpfenecker et al. ) ang- mrna expression is increased upon stimulation by tnf-α, thrombin, hyperoxia, vegf, pdgf and many other factors (augustin et al. ). in , parikh and colleagues reported that ang- serum levels were generally increased in patients with sepsis, being even more increased when sepsis was accompanied by ards (parikh et al. ). in subjects with acute lung injury, plasma ang- had a prognostic value for mortality in non-infection-related but not in infection-related, acute lung injury (calfee et al. ) . in two experimental models of sepsis, ang- heterozygous mice had reduced ang- levels and were protected against lung injury, indicating that ang- plays a pathogenetic role besides being a marker of disease severity (david et al. ) . the perception of ang- being of central pathophysiologic importance in sepsis is being supported by the recent observation that ang- antibody treatment attenuated acute pericyte loss, permeability, hypotension and mortality in mice subsequent to intravenous lps injection (ziegler et al. ) . in vitro, ang- increased and ang- suppressed, endothelial adhesion molecule expression and pmn adhesion (fiedler et al. ; gamble et al. ) . ang- may also be able to directly recruit inflammatory cells, because the % monocytes expressing tie- have been shown to display chemotaxis towards ang- in vitro (murdoch et al. ). moreover, mice genetically overexpressing ang- or being treated with ang- showed reduced pulmonary cytokine and adhesion molecule expression, pmn infiltration and vascular leakage in endotoxin-or hydrogen peroxide-induced lung injury (mammoto et al. ; mccarter et al. ; witzenbichler et al. ; xu et al. ). ang- reduced pro-inflammatory gene expression and mediator production probably via interaction of the phosphorylated tie- receptor with currently unidentified inhibitors of nf-κb (hughes et al. ) . in addition to regulating inflammation, ang- and - directly alter endothelial integrity. in mice, ang- -induced tie- receptor phosphorylation stimulated the p rhogtpaseactivating protein (p rhogap) via pi -kinase and rac to inactivate rhoa, resulting in reduced f-actin stress fibre formation and diminished endothelial permeability (mammoto et al. ). for rac- activation by ang- , iq domain gtpase-activating protein- (iqgap- ) is required . in line, the ability of ang- to reduce endotoxemia-induced pulmonary vascular leakage was abolished by downregulation of p rhogap in mice (mammoto et al. ). further, ang- ( ) interfered with the inositol triphosphate (ip ) receptor, thereby blocking trpc -dependent ca + influx and reducing endothelial hyperpermeability in vitro (ahmmed et al. ; jho et al. ); ( ) increased the presence of junctional ve-cadherin protein via extracellular signal-regulated kinase (erk) / dependent activation of sphingosine kinase , thereby strengthening the tethering forces between adjacent endothelial cells ; and ( ) decreased basal and vegfinduced phosphorylation and subsequent internalisation of ve-cadherin (gavard et al. ) . adenoviral ang- gene transfer as well as administration of mesenchymal stem cells transfected with ang- almost completely abolished pulmonary hyperpermeability induced by subsequent lipopolysacharide injection witzenbichler et al. ). however, both approaches for ang- delivery were far from translation into effective clinical therapies. in this respect, the development of vasculotide, a pegylated mer peptide that activates tie- (tournaire et al. ) and the demonstration of vasculotide´s therapeutic potential in established abdominal sepsis in mice (kumpers et al. ) may represent important milestones on the long way from understanding the importance of tie- for endothelial barrier function to the clinical application of tie- activation. sphingolipids, a class of lipids containing sphingoid bases as a backbone, form a mechanically stable and chemically resistant outer leaflet of the plasma membrane lipid bilayer. some sphingolipids regulate biological processes, including sphingomyelin, ceramide, sphingosine and sphingosine- phosphate. the current understanding of the role of these four and other sphingoid bases in acute lung injury has been recently reviewed in detail (natarajan et al. ; uhlig and yang ) . ceramide is derived from palmitoyl-coa and serine in a multi-step process or from sphingomyelin by sphingomyelinase. ceramide is deacylated to sphingosine (sph) through the action of ceramidases (canals et al. ) and sph is rapidly phosphorylated by sphingosine kinase (sphk)- and - to sphingosine- -phoshate (s p). s p is either cleaved by s p lyase (s pl) to ethanol-amine phosphate and trans- -hexadecenal, or dephosphorylated to sphingosine by s p phosphatases and (s ppase) or by lipid phosphate phosphatases (lpp). ceramide deteriorates and s p improves, barrier integrity. of note, the gram-negative endotoxin lps and the pneumococcal exotoxin pneumolysin disrupt the pulmonary endothelial barrier in a platelet-activating factor (paf)-dependent manner (uhlig and yang ; witzenrath et al. ) , with paf increasing vascular permeability by an acid sphingomyelinase (asmase)-dependent process (goggel et al. ). in brief, asmase-produced ceramide recruits caveolin- , enos and trpc- channels into caveolae. no usually blocks trpc channels but caveolin- inhibits no production by enos, resulting in trpc activation followed by an increase of [ca + ] i , mlck activation, mlc phosphorylation and finally ec contraction and paracellular permeability (uhlig and yang ) . s p is produced by platelets, erythrocytes, hematopoietic and vascular endothelial cells (hanel et al. ; tani et al. ; venkataraman et al. ; yatomi et al. ) . coordinated biosynthesis and degradation maintain s p concentrations in plasma and tissues in the range required for most favourable physiologic functions, which include regulation of cell proliferation, differentiation, survival, migration, morphogenesis and barrier function (natarajan et al. ) . using mice that selectively lack s p in the plasma, camerer and colleagues noted that basal plasma levels of s p maintain endothelial barrier function. as compared to wild-type littermates, mice with a lack of plasma s p had increased pulmonary vascular leak and demonstrated enhanced susceptibility to paf stimulation, a phenotype reversed by s p transfusion (camerer et al. ) . s p acts as an intracellular messenger (le stunff et al. ) or as an extracellular ligand of five cell surface receptors (s p - ), which are differentially expressed and coupled to various g proteins (uhlig and yang ) . vascular endothelial cells primarily express s p , s p and s p . physiologic s p plasma concentrations ( . - μm) maintain microvascular barrier integrity via ligation of the g i -coupled s p and exogenous addition of s p to lung ecs increased monolayer integrity rapidly and dose-dependently through s p . s p binding to s p induces rac activation, peripheral mlc phosphorylation, adherens junction assembly and cortactin translocation, which protects endothelium from barrier-disruptive effects of thrombin . moreover, teijaro and colleagues recently observed that endothelial s p critically regulates innate immune responses in influenza pneumonia. activation of endothelial s p attenuated cytokine storm, immune cell recruitment and mortality during infection with human pathogenic influenza virus , suggesting that in this case endothelial cells are conducting the innate immunity orchestra (iwasaki and medzhitov ) . in addition to extracellular receptor-dependent effects of s p, intracellular s p enhanced barrier integrity independently from s p receptors requiring rac- and sphk -/mice were more susceptible to lps-induced lung injury compared with wild-type mice (wadgaonkar et al. ). along the same line, lps evoked increased expression and activity of the s p catabolising s pl, thereby reducing s p levels. constitutive reduction of s pl expression in vivo (s pl +/mice) or in ecs (by sirna) reduced lung injury and inflammation upon lps stimulation (zhao et al. ) . most importantly, infusion of s p reduced lung microvascular leakage and also cytokine release, leukocyte infiltration and histologic tissue changes in numerous different in vivo models of lung injury, including ischemia/reperfusion, pancreatitis and endotoxin challenge in mice and dogs mcverry et al. ; okazaki et al. ; peng et al. ). however, s p at supraphysiologic local concentrations (> μm) mediates rhoa-dependent barrier disruption through ligation of s p and s p , which couple to gi, gq and g / (sammani et al. ; siehler and manning ; wang and dudek ). moreover, s p stimulates contraction of human bronchial smooth muscle cells (rosenfeldt et al. ) , enhances murine airway hyperresponsiveness (roviezzo et al. ) and evokes bradycardia through s p (forrest et al. ). the latter findings suggest a rather small therapeutic window for s p, which may limit the therapeutic potential of s p and drugs that increase s p production or reduce s p catabolism. therefore, s p receptor agonists have gained considerable interest. for example, intratracheal as well as intravenous delivery of the s p agonist sew- reduced lung permeability after endotoxin injection (sammani et al. ) and the s p receptor and - ligand aal-r reduced lung permeability and mortality after influenza infection in mice ). closer to clinical application is a derivative of the fungal metabolite myriocin, fingolimod (fty ), which holds structural similarities with s p and has been approved as an immunosuppressive agent for the treatment of multiple sclerosis (brinkmann et al. ). in addition to its immunosuppressive effects, fty enhanced endothelial barrier function in vitro (sanchez et al. ) and in vivo (dudek et al. ) and ameliorated lps-evoked lung injury in mice natarajan et al. ). however, we recently observed that, although lower concentrations of fty enhanced barrier integrity in endothelial cell monolayers ( . - μm fty ) and in mechanically ventilated mice ( . mg/kg fty ), higher concentrations ( - μm fty ) evoked apoptosis and barrier dysfunction in vitro and in mechanically ventilated mice ( mg/kg) but not in spontaneously breathing mice (müller et al. ). if these experimental findings are translatable into the clinical setting, they suggest that, in fingolimod-treated ventilated patients with multiple organ dysfunction syndrome, in whom hepatic metabolism of fty is hampered, increased fty plasma concentrations could harm lungs that are sensitised by mechanical ventilation towards barrier-destabilising effects of the drug. despite recent studies providing valuable insights into possible mechanisms of barrier regulation by fty , the mode(s) of action remain unclear. fty is partly phosphorylated by sphk , thereby increasing its affinity to s p and s p (billich et al. ) . nevertheless, reduction of vegfinduced permeability by fty was independent from s p expression (sanchez et al. ) and endocytosis and degradation of s p by fty has been proposed (cyster ) . several further concepts may possibly explain fty -induced barrier enhancement and have recently been reviewed (natarajan et al. ) . notably, fty , like s p, induces bradycardia and dyspnea along with fev (forced expiratory volume in s) reductions (kappos et al. ). in conclusion, caution is warranted when considering fty for therapeutic lung barrier enhancement in critically ill patients. reactive oxygen species (ros) and reactive nitrogen species (rns) are crucial regulators of cellular function. ros and rns are tightly counterbalanced by antioxidant systems as superoxide dismutase or glutathione. however, excessive ros/rns production or critical reduction of their antioxidative counterparts leads to oxidative stress, which is involved in the pathogenesis of lung injury and particularly vascular permeability. among other molecules displaying oxidative properties, superoxide anions (o -), hydroxyl radical ( oh), hydrogen peroxide (h o ) and hypochloric acid (hocl) are summarised as ros, while metabolites of the nitric oxide ( no) metabolism like nitrite (no -) or peroxynitirite (onoo -) with oxidative power are termed rns. both ros and rns are physiological mediators of functional cell regulation. ros derived from mitochondrial oxidative phosphorylation can modulate the specific cellular pattern by reacting with redox-reactive cysteine residues, thereby altering enzyme activities and controlling cellular signalling (ray et al. ) . under inflammatory conditions, endothelial nadph oxidases, xanthine oxidase, cyclooxygenase and enos are involved in increased ros/rns production. neutrophils deliver even higher amounts of ros due to nadph oxydase activity, which are in part further processed to hocl by myeloperoxidase activity. in addition, neutrophils produce rns by inos . ros and rns contribute to acute lung injury upon different insults. perfusion of isolated rabbit lungs with h o evoked lung oedema (hippenstiel et al. ; seeger et al. ) . h o exposure resulted in a rapid and substantial decrease in endothelial camp content and the effects of h o on endothelial permeability were inhibited by adenylate cyclase activation (suttorp et al. b) . vili increased xanthine oxydoreductase (xor) activity and blocking xorprotected mice from pulmonary hyperpermeability (abdulnour et al. ) . ros signalling leads to mapk activation, which is involved in permeability generation in mice subjected to vili (dolinay et al. ; park et al. ) . underlying mechanisms are proinflammatory functions of this pathway and phosphorylation of heat shock protein (hsp ), which mediates stress fibre generation and endothelial contraction (abdulnour et al. ; damarla et al. ; dolinay et al. ) . further, mice deficient for the transcription factor nrf exhibited increased lung injury and permeability in vili due to significantly reduced antioxidative capacity and could be rescued from exacerbation of lung injury by supplementing the antioxidant n-acetyl-cysteine (papaiahgari et al. ) . no, the most prominent rns, is a highly diffusible and reactive free radical gas, produced from l-arginine in the lung by constitutively expressed endothelial no synthase (enos) in endothelial cells and by inducible nos (inos) in macrophages. expression of enos usually stays constant while enos activity can be rapidly increased, whereas inos expression is inducible but the activity is usually more or less constant. numerous inflammatory incidents induce no production and release, including endothelial stimulation by bacterial pore-forming toxins (suttorp et al. a ). the plethora of no´s biologic effects includes control of vascular tone and permeability, regulation of mitochondrial respiration and adhesion of platelets and leukocytes. no supports protection of cells against oxidant injury and microbial threats but can also have detrimental properties, e.g., activation of inflammatory processes, enzyme inhibition and dna damage. most probably, these cellular responses are differentially regulated by specific no concentrations (thomas et al. ) . the majority of no effects are mediated by ( ) nitrolysation of cysteine residues, ( ) reaction with transition metals like ion, zinc and copper and ( ) formation of onoothrough reaction with o -, which leads to nitration of proteins involved in the regulation of cellular function (korhonen et al. ) . inhaled nitric oxide (ino) is used as rescue therapy in individual cases of hypoxic respiratory failure in adults, children and newborns along with respiratory support and other appropriate treatments. the inhaled vasodilator reduces pulmonary arterial pressure without causing systemic vasodilation and selectively redistributes pulmonary blood flow towards ventilated lung regions, thereby reducing shunt flow and improving oxygenation (raoof et al. ) . nevertheless, although improvement of blood gases has been regularly noted during the first h of treatment, ino does not increase ventilator-free days or survival of ards patients (afshari et al. ) . in addition to its vasodilatory properties, no has endothelial barrier-regulating effects in the lungs but the published experimental studies paint a dichotomous picture. inhaled no was shown to protect against pulmonary barrier dysfunction in isolated perfused and ventilated rabbit lungs upon oxidative stress or ischemia/reperfusion (kavanagh et al. ; poss et al. ; schutte et al. b) . moreover, ino reduced pulmonary transvascular albumin flux in patients with acute lung injury (benzing et al. ). the precise mechanisms accounting for the stabilising effect of no remain to be elucidated but may involve increase of cyclic guanosine monophosphate (cgmp) through activation of guanylate cyclase (gc). no-induced barrier protection in rabbit lung ischemia/reperfusion was associated with increased cgmp production and could be further enhanced by inhibition of the cgmp-specific phosphodiesterase (pde) (schutte et al. ) . also, increase of cgmp by no (donors) and/or inhibition of cgmp-specific pde strengthened the endothelial barrier in pulmonary ecs upon h o treatment (seeger et al. ; suttorp et al. ) , in ecs and perfused mouse lungs stimulated with thrombin (seybold et al. ) and in mice with severe streptococcus pneumoniae pneumonia (witzenrath et al. ). the barrier-stabilising effects of no and cgmp may be partly explained by negative regulation of specific endothelial trp channels (yin et al. ) , some of which are central for [ca + ] i increase, pulmonary endothelial cell contraction and lung hyperpermeability in response to various stimuli (alvarez et al. ; boueiz and hassoun ; hamanaka et al. ; jian et al. ; kuebler et al. ; tiruppathi et al. ; yin et al. ) . on the other hand, endogenous no synthesis contributed to lung injury in hypoxic ischemia/reperfusion of isolated rabbit lungs (schutte et al. a) . moreover, inos expression was upregulated in response to mechanical ventilation in mice and ventilated inos -/mice as well as inos inhibitortreated mice had reduced lung inflammation and permeability compared with control wt mice . in line, pharmacologic inhibition of nos prevented the development of pulmonary hyperpermeability in rats subjected to vili (choi et al. ) . gain and loss of function studies have provided evidence for a contribution of soluble gc activation to ventilator-induced lung injury in mice (schmidt et al. ) . further, ino significantly increased endothelial permeability in rats with pseudomonas aeruginosa pneumonia independently from the inflammatory response (ader et al. ). thus, the individual effects of no on pulmonary vascular barrier function seem to depend on local no concentrations and the precise pathologic conditions. imatinib has been suggested for the treatment of increased vascular permeability. the tyrosine kinase inhibitor imatinib targets c-abl kinase, platelet-derived growth factor-derived receptors, c-kit, arg kinase and discoid domain receptors and and has been implemented into treatment of chronic myelogenous leukaemia. recently, imatinib was found to protect against endothelial barrier dysfunction evoked by thrombin in isolated endothelial cells, by vegf in a murine skin model and in the context of polymicrobial sepsis in mice. as the underlying mechanism, inhibition of arg kinase followed by augmented rac signalling and stabilised intercellular junctions and cell matrix adhesion has been identified (aman et al. ; chislock and pendergast ) . case reports have been published describing reduction of pulmonary oedema in the context of pulmonary venooclusive disease and resolution of bleomycin-induced pneumonitis (carnevale-schianca et al. ; overbeek et al. ) . with respect to clinical development, additional preclinical evidence for imatinib efficacy in ards is required. further, possible relevant undesirable effects have to be considered including cerebral haemorrhage particularly in patients with compromised coagulation, as malfunction of coagulation is also a major issue in sepsis patients (hoeper et al. ) . adrenomedullin (am) is an endogenous peptide with potent barrier protective properties that is expressed in various cells of the vascular system including endothelial and vascular smooth muscle cells and also in cardiomyocytes, epithelial cells and leukocytes. the am gene encodes for a preproadrenomedullin, which is processed to pro-am, from which am and proam n-terminal peptide (pamp) are generated. a m i d a t i o n b y p e p t i d o g l y c i n e a l p h a a m i d a t i n g monooxygenase (pam) is crucial for biologic function of the active am peptide (temmesfeld-wollbruck et al. b) . am binds to the calcitonin receptor like receptor (crlr), which assembles with receptor activity-modulating proteins (ramp) and . in endothelial cells, binding of am to the receptor results in intracellular accumulation of the second messenger camp and in activation of various kinases including protein kinase a (pka), pkc, map kinases and others (hippenstiel et al. ; temmesfeld-wollbruck et al. b) . mice deficient for am, crlr, pam or ramp die prematurely of hydrops fetalis, which highlights the role of am for vascular barrier integrity (bonder et al. ; caron and smithies ; cyster ; czyzyk et al. ; ichikawa-shindo et al. ) . am is up-regulated under inflammatory conditions like sepsis or experimental lung injury (agorreta et al. ; cheung et al. ; matheson et al. ) and mice heterozygous for am exhibit an aggravated inflammatory response and organ damage following lps challenge (dackor and caron ) . treatment with exogenous am protected against pulmonary hyperpermeability induced by various stimuli like staphylococcus aureus alpha toxin, hydrogen peroxide, lipopolysaccaride (lps) or hyperoxia and ventilator-induced lung injury (hippenstiel et al. ; itoh et al. ; müller et al. ; temmesfeld-wollbruck et al. a) . am also protected against barrier breakdown in the gut after challenge with staphylococcus aureus alpha toxin and in ischemia reperfusion injury and stabilised the blood-brain barrier (brell et al. a, b; higuchi et al. ; honda et al. ; kis et al. ; temmesfeld-wollbruck et al a ). at least two major mechanisms may contribute to the impressive function of am. first, am leads to the relaxation of the contractile apparatus of the endothelial cell by avoiding the generation of actin stress fibres and actin myosin interaction (temmesfeld-wollbruck et al. b) . we and others have observed a rise of intracellular camp upon am stimulation of endothelial cells, leading to the inhibition of mlc phosphorylation, thereby blocking actin-myosin interactionmediated cell contraction induced by thrombin or hydrogen peroxide in vitro, or evoked by mechanical ventilation in vivo (brell et al. b; hocke et al. ; müller et al. ). however, equally potent barrier protective effects of am are observed in gut epithelial cells that were not dependent on intracellular camp increase (temmesfeld-wollbruck et al. ). second, besides reducing cell contraction am increases intercellular adherence, thereby mediating barrier stabilisation. in rat intestine, staphylococcus alpha toxin infusion induced vascular hyperpermeability accompanied by loss of ve-cadherin in submucosal blood vessels, which was avoided by am treatment (hocke et al. ) . in endothelial cells, am protected against the loss of ve-cadherin and occludin derangement due to thrombin or staphylococcus alpha toxin stimulation and am enhanced the expression of claudin- in brain microvascular endothelial cells (hocke et al. ; honda et al. ) . immunomodulating effects of am have been described (gonzalez-rey et al. ); however, we observed that the strong barrier protection of am is not coupled to anti-inflammatory properties (müller et al. ) . although the underlying and obviously cellspecific mechanisms of am-mediated barrier protection partly remain elusive, the powerful properties observed in complex models regardless of the stimulus and independent from immunosuppressive effects indicate a high translational potential for am. acute inflammatory diseases including pneumonia and sepsis may result in ards, which is still associated with unacceptably high mortality. research has been successfully uncovering basic disease mechanisms, leading to improvements in therapy including ventilation and resuscitation strategies. nevertheless, although the pulmonary endothelium has long been noted to be central in the pathogenesis of ards and scientists have been elucidating innumerable important mechanisms of permeability increase, most therapeutic strategies to improve ards outcome based on the understanding of lung endothelial barrier dysfunction have so far been frustrating. these drawbacks should be understood as important sources of perception and it might be worth considering some general aspects when moving forward in this field. first, to regain endothelial barrier function once the endothelium is severely injured may be a barely achievable objective. interestingly, the only strategies so far decreasing mortality in ards, reduction of tidal volume and probably early prone positioning, short-term use of neuromuscular blockers and oesophageal pressure-guided positive endexspiratory pressure adjustment (guerin et al. ; network ards ; papazian et al. ; talmor et al. ) , are aimed at alleviation of further inflammatory stress by mechanical ventilation, thus being of a rather preventive nature. it may be promising to focus on strategies that decelerate the progress of "uncomplicated" pneumonia or sepsis to ards instead of trying to reverse severe parenchymal inflammation and injury. therefore, clinical and biological predictors of progress towards ards need to be identified and future therapies should be started before full-blown ards has developed. however, this notion should not encourage the performing of experimental studies in which the treatment of interest is commenced before onset of the initial disease (pneumonia or sepsis in this case), because such a preventive strategy can rarely be translated into clinics. second, the "real life aspect" needs to be respected. icu patients are frequently prone to ards due to multiple simultaneous incidents, unlike, e.g., lps-treated mice, which means that numerous redundant pathways may be differentially involved and should probably be addressed therapeutically at the same time. further, important inter-individual differences need to be considered. third, complexity is an important issue. as our understanding of central contributors to lung injury is growing, we are becoming aware of the differential effects one and the same pathomechanistic system may have. for example, s p seems to differentially affect endothelial integrity, depending on s p concentration, receptor expression and the exact local cellular setting, which implements a further dimension into the picture of barrier destructing mechanisms. probably, systems biology combined with mathematical multi-scale models that integrate knowledge from experimental studies (in vitro, in vivo and in silico), clinical trials and clinical and biological predictors of the individual patient will facilitate development of successful novel therapies and improvement of ards prevention. since the first description of ards in , researchers have made great efforts to unravel the mechanisms contributing to endothelial dysfunction in the lung in order to develop novel therapies. walking all the way to where we are standing today has sometimes been frustrating and possibly not even half of the whole distance has been accomplished. nevertheless, considering the high morbidity and mortality of ards, it is worth trying hard to 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carcinomas the leading causes of death in the world mesenchymal stem cell-based angiopoietin- gene therapy for acute lung injury induced by lipopolysaccharide in mice sphingosine- -phosphate: a platelet-activating sphingolipid released from agoniststimulated human platelets negative-feedback loop attenuates hydrostatic lung edema via a cgmp-dependent regulation of transient receptor potential vanilloid regulation of cytoskeletal organization by syndecan transmembrane proteoglycans neutrophil cytoskeletal rearrangements during capillary sequestration in bacterial pneumonia in rats novel critical role of toll-like receptor in lung ischemiareperfusion injury and edema complete reversal of acid-induced acute lung injury by blocking of platelet-neutrophil aggregation protection of lps-induced murine acute lung injury by sphingosine- -phosphate lyase suppression angiopoietin mediates microvascular and hemodynamic alterations in sepsis acknowledgement this work was funded by the deutsche forschungsgemeinschaft (sfb-tr b , z to n.s. and c , c to m.w.) key: cord- -fef qsik authors: kim, gwang-ho; yi, sun shin title: verification with the utility of an established rapid assessment of brain safety for newly developed vaccines date: - - journal: lab anim res doi: . /s - - - sha: doc_id: cord_uid: fef qsik in the twenty-first century, high contagious infectious diseases such as sars (severe acute respiratory syndrome), mers (middle east respiratory syndrome), fmd (foot-and-mouth disease) and ai (avian influenza) have become very prevalent, causing treat harm to humans and animals in aspect of public health, and economical issues. the critical problem is that newly-reported infectious diseases that humans firstly experience are expected to continue to emerge, and these diseases will be spreading out rapidly. therefore, rapid and safe supplies of effective vaccines are most pivotal to prevent the rapid prevalent of new infection, but international standards or assessing protocol the safety of urgent vaccines are not established well. in our previous study, since we established a module to assess the brain safety of urgent vaccines, therefore, it is necessary to verify that this established module for assessing brain safety could work effectively in commercially available two vaccines (one killed- and on live-vaccines). we compared the results of evans blue (eb) assay and qpcr analysis by injection of two kinds of vaccines, pbs and lipopolysaccharide (lps) under the condition of the module previously reported. we confirmed that the brain safety test module for urgent vaccine we established is very reproducible. therefore, it is believed that this vaccine safety testing method can be used to validate brain safety when prompt supply of a newly developed vaccines is needed. recently, a variety of infectious diseases with high rates of spread and mortality have been frequent. in addition, it has been reported that high contagious infectious diseases are spreading through various routes and carriers for recent two decades. it is important to prevent from spreading out a number of infectious diseases earlier in the outbreak of the causing agents because of our rapid transportation population density. therefore, prompt supplying of vaccines should be secured within a limit time, even at unexpected times and places. the speed of vaccine development is also a very critical issue. thus, safety of the vaccines also should be assured. nevertheless, it is difficult to obtain sufficient time to perform a complete safety evaluation for a rapid supply of urgent vaccines. it is very unfortunate that we have already mentioned in the previous report that there is not global standard for the safety assessment of these newly develop urgent vaccines. in our previous study, we established of minimal positive control conditions to ensure brain safety using experimental mice models during development of urgent vaccines before introducing to markets. we conducted several experiments to evaluate the safety of vaccines, and presented the ideal combination of the experiments: susceptible mice types according to the effective concentration of lipopolysaccharide (lps) destroying bloodbrain barrier (bbb), the appropriate lps exposure time destroying bbb, origin type of lps (salmonella enterica), and injection route of lps. in this study, we tried to evaluate whether the previously established assessment method could be available for evaluating brain safety rapidly with two commercial vaccines easily accessible vaccines (a killed-and a live-vaccine). we confirmed in the present study that the pre-established "vaccine safety module for the brain" was highly reproducible in the evaluation of the safety results by the commercially available vaccines. in this study, we described that this module can be useful in applying a commercially available vaccine to a rapid and effective positive-control for brain safety assessment of an urgent vaccine. this study will be of great help to ensure that vaccines should be urgently and promptly distributed to public with assured minimal safety of brain at an unexpected outbreak of certain infectious diseases in human and veterinary fields. most of experiments were performed based on our previous study. among the experiments, we primarily chose some combinations that we'd suggested in the previous reports by baek et al., [ ] . in brief, evidences for the bbb damage by commercially available vaccines (killed porcine genital respiratory syndrome vaccine vaccine and live canine parvovirus vaccine) were based on evans blue eb permeability into brain parenchyma and mrna expression of tight junctions (zo- and occludin) in the brain. experimental method for this is described in detail below. evans blue (eb) assay for brain permeability seven-weeks old icr mice and lps sources salmonella enterica were used. lps solutions concentration was . mg/ ml/kg, and i.p injection was performed. the lps from s.enterica serotype enteritidis (l ; sigma, usa) was dissolved in phosphate buffered saline (pbs; ph . ). two types of commercially available vaccines were used as experimental groups. one is a killed vaccine, a porcine genital respiratory syndrome vaccine (suishot prrs, choongang vaccine, korea). the other one is a live vaccine, a canine parvovirus vaccine (zoetis inc., usa). the concentration of the stock solution of these two vaccines is . × . tcid /ml. we set these concentrations to assess the effect of these vaccines on the bbb, depending on the concentration. through the previous studies we confirmed that the effective bbb destroyed concentration of these vaccines was -fold concentration of the stock solutions. therefore, the experiment was conducted under three conditions: -fold concentration of the stock solution, reference concentration of the stock solution and -fold diluted concentration of the stock solution. including negative control and positive control, the groups were divided groups according to vaccine types and concentrations: group , pbs/eb(n = ); group , lps/eb(n = ); group , -fold concentration of the porcine vaccine/eb (p , n = ); group , reference concentration of the porcine vaccine/eb (p , n = ); group , -fold diluted concentration of the porcine vaccine/eb (p , n = ); group , -fold concentration of the canine vaccine/eb (c , n = ); group , reference concentration of the canine vaccine/eb (c , n = ); group , fold diluted concentration of the canine vaccine/eb (c , n = ). we performed the eb assay according to the method of baek et al. [ ] , used in previous studies. for treatment, pbs, lps and the vaccines were intraperitoneally (i.p) injected into each mouse ( . mg/ ml/ kg body weight). at h post-injection (pi) of pbs/lps/ vaccines, eb dye ( % w/v in pbs, ml/kg, e ; sigma) was i.p injected into each mouse. all mice were sacrificed after h of eb exposure ( h pi). after sacrifice, blood was collected and serum was separated by using centrifugation. the brain was promptly removed after the mouse had been perfused with heparinized pbs ( . mg/l). serum and homogenized brain hemisphere of each mouse were dissolved in a % (w/v) trichloroacetic acid in pbs solution to eliminate proteins and then subjected to centrifugation at ×g for min. supernatant of each sample was diluted : with absolute ethanol. a spectrophotometer (infinite f ; tecan, switzerland) was used to determine the eb concentrations in the serum and brain hemisphere samples. the fluorescence intensity was measured at λ ex and em , and the eb concentration of each sample was calculated according to a standard curve that had been ordered to determine the penetration rate for eb in brain to eb in blood was calculated. following the eb assay, the safety of the vaccine was assessed by mrna expression of the molecules forming the tight junction of the brain. groups and experimental conditions were used same as eb assay. the lps solution was i.p. injected into all mice and sacrificed at the h pi time. after sacrifice, the brains were removed from each mouse and were prepared for next steps. rna was extracted by using trizol reagent (ambion; life technologies, usa) according to the manufacture's instruction. for rna extraction, the homogenized brain tissue was incubated in trizol and chloroform reagent and then subjected to centrifugation. the aqueous phase was removed and incubated with % isopropanol. after centrifugation, the obtained rna pellet was washed with % ice-cold ethanol and then dissolved in rnase-free water. the obtained rna extract solution was stored at − °c after undergoing °c heat incubation. quantification of mrna expression of zo- and occluding in the brain quantitative real-time polymerase chain reaction (qpcr) was performed with sybr green dye by using a step one plus real-time pcr system (life technologies). for relative quantitation of gene expression, we used the comparative cycle threshold (ct) method ( -△△ct ). results were normalized to that of the housekeeping/control gene glyceraldehyde -phosphate dehydrogenase (gapdh). the primer sequences for zonula occludens- (zo- ) and occludin have been used in our previous studies to establish positive control. these primer sequences were obtained from the national center for biotechnology information nucleotide database and are shown in table . data are presented as mean ± se or mean ± sem values for each experimental group. differences between means were analyzed by using student's t-test for single comparisons. p values< . were considered statistically significant. after i.p injection of pbs, lps ( . mg/ ml/kg) and two vaccines at three concentrations, the permeability of eb dye in brain parenchyma was measured by the ratio of the amount of eb dye in brain to the amount of eb dye in blood (fig. ) . in the first injection before eb injection, the result of the group injected pbs was negative control, and the result of the group injected lps was positive control. when compared to the pbs/eb group, the permeability of eb dye was significantly increased in mice of the group injected with lps/eb (figs. a and b) . the eb dye permeability of p group (; injected with fold concentration of porcine vaccine stock solution) and p group (; injected with reference concentration of porcine vaccine stock solution) was increased than pbs/ eb group, but not significantly, and eb dye penetrated through the brain parenchyma less than lps/eb group (fig. a) . however, the permeation rate of eb dye in c group (; injected with -fold concentration of canine vaccine stock solution) was not significant but higher than lps/eb group (fig. b) . the -fold diluted group of the two vaccines had similar or lower eb dye permeability than the pbs/eb group. the relative zo- and occludin mrna levels were plotted on the graph after h after i.p injection with pbs, lps, and two vaccines at three concentrations (fig. ) . zo- mrna levels were significantly higher in the lps group (positive control) than in the pbs group (negative control). in addition, zo- mrna levels were significantly increased compared to pbs group when -fold concentrations of two vaccine stock solutions were injected. at the -fold concentration of the stock solution, zo- mrna expression was lower in the p group (; porcine vaccine) but, higher in the c group (; canine vaccine) than the lps group, but not significantly. the zo- mrna expressions were similar or slightly higher in the p and c groups (; reference concentration of two vaccines stock solution) compared to the pbs group. and the zo- mrna expression was similar or slightly lower in the p and c groups (; -fold diluted concentration of two vaccines stock solution) compared to the pbs group. likewise, occludin mrna levels were significantly higher in the lps group (positive control) than in the pbs group (negative control). however, when -fold concentrations of two vaccines stock solutions were injected, occludin mrna expression was increased compared to the pbs group, but not significantly. in addition, the increase was smaller than zo- mrna level. in the p , c , p and c groups injected with reference concentrations and -fold diluted concentrations of the two vaccines, occludin expression was similar or slightly higher than the pbs group. humans and live stocks are easily exposed to the risk of infectious diseases caused by various micro-organisms such as bacteria and virus on a daily basis. this has become a very important issue as it affects health in our daily lives [ ] . once a high contagious infectious disease occurs, it can spread to other countries in an instant [ ] . thus, it is not limited to any region or country. rapid supply of vaccines that are effective in emergency situations is essential to prevent the spread of such infectious diseases [ ] . however, in urgent situation, both quick supply of vaccine and safe vaccine are important. all important biological actions of humans and live stocks are regulated and maintained primarily by the central nervous system (cns), especially the brain [ , , ] . brain is a complex network of numerous neurons. it is an important organ for regulating body homeostasis, behaviors, memory, and learning [ ] [ ] [ ] . this important organ is protected by a structure known as the bloodbrain barrier (bbb) [ ] . neurons in the brain and spinal cord are in directly contact with cerebrospinal fluid sequences of primers used for quantitative real-time pcr analysis. the sequence of the primers was used with reference to the previous description of baek et al. [ ] . zo- , zonula occludens- ; gapdh, glyceraldehyde -phosphate dehydrogenase (csf) [ ] . they are supplied with oxygen and nutrients. when blood and csf circulate, the material can selectively penetrate because the bbb limits mass transfer into brain parenchyma [ , ] . it is believed that the brain can examine components of blood and only let required substances selectively pass [ ] . if the bbb is exposed to any toxic substance, chronic high blood pressure, radiation, viruses, or bacteria, it can lead to brain problem [ , ] . if a newly developed vaccine or urgent vaccine fails to secure brain safety, brain parenchyma might become damaged and the health of the recipient may not be assured [ ] . this can become can be a serious public health hazard and result in serious economic damage to industrial animals. therefore, the safety assessment module we designed was focused on how to effectively measure effects of various vaccines on the stability of bbb in laboratory animals [ ] . the bbb is formed by a tight junction between endothelial cells of blood vessels distributed throughout the cns. it contains junctional molecules (jam), zonula occludens (zo), claudin, and occluding [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] . these structures are key components of tight junction of bbb [ , - , , ] . in our previous study, we have established a condition for positive control that can destroy the bbb structure by damage to the tight junction using lps mentioned earlier [ ] . these conditions include mouse species, age, type, concentration of lps, injection routes to mice, and exposure time after injection. eb assay, qpcr, and western blot analysis can be conducted under the condition of positive control to determine whether the bbb is destroyed [ , , , , , , ] . in the present study, we confirmed that these established conditions and modules of the positive control that destroyed the bbb could be used to assess brain safety of commercial vaccines [ ] . results of eb assay revealed that eb dye permeability of p group was higher than that of the pbs/eb group but lower than that of the lps/eb group. based on this, we could not conclude that the bbb was damaged in the vaccine concentration of the p group. on the other hand, the permeability of eb dye in the c group was similar or higher than that in the lps/eb group, meaning that bbb was damaged at vaccine concentration of c group. the same concentration of vaccine was injected. however, higher permeability of eb dye was seen in the c group. this result may be due to different vaccine types of porcine and canine vaccines. since the porcine vaccine is a killed-vaccine and the canine vaccine is a live-vaccine, the canine vaccine is more damaging to bbb. this was also confirmed by qpcr results of zo- mrna levels. as expected, at reference concentration and -fold diluted concentration of stock solution of two vaccines, eb dye permeability and relative zo- and occludin mrna levels were comparable to those of the negative control, meaning no significant damage to the bbb. occludin mrna expression was lower than zo- mrna expression level at -fold diluted concentration of the stock solution which was considered to be damaging to bbb [ ] . we have previously examined permeability of eb dye and changes in mrna and protein expression of zo- and occludin over time after lps injection. after h of lps injection, zo- mrna levels were dynamic and rapidly increased. the level of occludin mrna increased after h of injection, but increased rapidly after h. these results are highly correlated with our previous findings. however, in order amount of eb dye after canine parvovirus vaccine (live-vaccine) injection. the permeation rate of eb dye in c was higher, but not significantly higher than that in lps/eb group ( * , p < . ; ** , p < . ; *** , p < . ) to provide a rapid supply of urgent vaccines, rapid verification is required. considering this, h after injection of lps was established as a positive control condition of the module for evaluating bbb damage. consistent with results of previous studies, zo- levels, but not occludin levels, were increased rapidly after h of vaccine injection. therefore, when a commercially available vaccine is applied to our positive control and rapid safety assay modules, zo- tight junction primer is more suitable for qpcr. in addition, we have purposed three assays of eb assay, qpcr, and western blot as methods to determine damage to bbb in our previous study [ ] . however, since this module is aimed at assessing the safety of urgent vaccines, western blot analysis which requires a relatively long time might not be suitable. therefore, eb assay and qpcr would be more effective methods for rapid evaluation. it is recommended that we refer to an illustration of our research process on the safety assessment of vaccines (fig. ). in this study, we confirmed that the safety assessment module for urgent vaccines could be used to evaluate two commercial vaccines. however, in order for this module to be used in practice, it is necessary to apply more types of vaccines to the module. since there are many kinds of vaccines, they should be repeatedly applied and evaluated using this module. taken together, the usefulness of the rapid brain safety test module that we constructed previously was verified using vaccines fig. quantification of mrna levels of zonula occludens- (zo- ) and occludin in mouse brains. bbb breakdown and restoration were confirmed through expression level analysis of tight junctions in bbb. a zo- mrna expression after porcine genital respiratory syndrome vaccine (killedvaccine) injection. b zo- mrna expression after canine parvovirus vaccine (live-vaccine) injection. c occludin mrna expression after porcine genital respiratory syndrome vaccine (killed-vaccine) injection. d occludin mrna expression after canine parvovirus vaccine (live-vaccine) injection ( * , p < . ; ** , p < . ) commercially available in the veterinary market in korea. our results were highly reproducible. therefore, this evaluation module can be utilized in the future to assess the safety of various vaccines and adjuvants. fig. it is a safety evaluation method of vaccine against pre-established brain parenchyma. based on the results of our established experimental conditions according to baek et al. [ ] , lps (s. enterica-derived) and commercially available vaccines are injected intraperitoneally into -week-old icr mice ( . mg lps/ ml/kg) and brain hemispheres were removed after h post-injection. the mrna is extracted from the brain, and sequentially performed real time pcr (zo- and occluding). the tight junction mrna levels of the vaccine-treated group with those of the lpstreated group were compared and potential risk to the bbb of the vaccine is evaluated. (this conceptual diagram is shown by applying the method used by baek et al. in [ ] ) establishment of minimal positive-control conditions to ensure brain safety during rapid development of emergency vaccines the blood-brain barrier: an overview: structure, regulation, and clinical implications lipopolysaccharideinduced blood-brain barrier disruption: roles of cyclooxygenase, oxidative stress, neuroinflammation, and elements of the neurovascular unit infectious diseases epidemiology tight junction and polarity interaction in the transporting epithelial phenotype occludin: one protein, many forms the tight junction in inflammatory disease: communication breakdown the knockout of mir- and - alters smooth muscle cell maintenance and vascular homeostasis in mice: correlates with human disease the tight junction protein zo- establishes a link between the transmembrane protein occludin and the actin cytoskeleton experimental methods and transport models for drug delivery across the blood-brain barrier claudins and the modulation of tight junction permeability molecular physiology and pathophysiology of tight junctions in the blood-brain barrier lipopolysaccharide-induced blood brain barrier permeability is enhanced by alpha-synuclein expression neonatal influenza virus infection affects myelination in influenza-recovered mouse brain influenza and the work of the world health organization transient forebrain ischemia induces impairment in cognitive performance prior to extensive neuronal cell death in mongolian gerbil (meriones unguiculatus) lps induces occludin dysregulation in cerebral microvascular endothelial cells via mapk signaling and augmenting mmp- levels brain endothelial cell-cell junctions: how to "open" the blood brain barrier the system of cerebrospinal fluid-contacting neurons. its supposed role in the nonsynaptic signal transmission of the brain neuronal regulation of energy homeostasis: beyond the hypothalamus and feeding time-dependent changes of calbindin d- k and parvalbumin immunoreactivity in the hippocampus of rats with streptozotocin-induced type diabetes enhanced expressions of arginine vasopressin (avp) in the hypothalamic paraventricular and supraoptic nuclei of type diabetic rats all authors are appreciated the technical supports of mr. kyunghyun lim and mr. ji-kwang lee at department of biomedical laboratory science, soonchunhyang university. authors' contributions ghk; performing experiment, analysis of data and writing manuscript, ssy; designing of the study, analysis and interpretation of data and final approval of the version to be submitted. both authors read and approved the final manuscript. there were no more related information about the authors. there was some supporting data available for this work. the datasets used and/or analyzed in this study are available from the corresponding author on reasonable request. the authors declare that they have no competing interests. key: cord- - p wpav authors: huang, shang-hui; zhao, li-xiang; hong, chao; duo, cui-cui; guo, bing-nan; zhang, li-juan; gong, zheng; xiong, si-dong; gong, fang-yuan; gao, xiao-ming title: self-oligomerization is essential for enhanced immunological activities of soluble recombinant calreticulin date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: p wpav we have recently reported that calreticulin (crt), a luminal resident protein, can be found in the sera of patients with rheumatoid arthritis and also that recombinant crt (rcrt) exhibits extraordinarily strong immunological activities. we herein further demonstrate that rcrt fragments – (rcrt/ - ), rcrt/ - , rcrt/ - and rcrt/ - can self-oligomerize in solution and are – fold more potent than native crt (ncrt, isolated from mouse livers) in activating macrophages in vitro. we narrowed down the active site of crt to residues – , the activity of which also depends on dimerization. by contrast, rcrt/ - is almost completely inactive. when rcrt/ - is fractionated into oligomers and monomers by gel filtration, the oligomers maintain most of their immunological activities in terms of activating macrophages in vitro and inducing specific antibodies in vivo, while the monomers were much less active by comparison. additionally, rcrt/ - oligomers are much better than monomers in binding to, and uptake by, macrophages. inhibition of macrophage endocytosis partially blocks the stimulatory effect of rcrt/ - . we conclude that the immunologically active site of crt maps between residues – and that soluble crt could acquire potent immuno-pathological activities in microenvironments favoring its oligomerization. calreticulin (crt) is a kda ca + -binding glycoprotein in the endoplasmic reticulum of eukaryotic cells [ ] [ ] . it folds into domains including a lectin-like globular n domain (amino acid residues , a proline-rich p domain (residues - ) and a ca + -binding c domain (residues - ) [ ] [ ] [ ] . crt can also appear at the surface of various types of cells exhibiting multiple biological functions [ ] [ ] [ ] [ ] [ ] [ ] [ ] . recently it has been shown that soluble crt is present in the sera of patients with rheumatoid arthritis and with sle [ ] [ ] and that natural crt (ncrt), isolated from human or mouse tissues, can directly activate macrophages in vitro [ ] . additionally, rcrt/ - , a prokaryotically-expressed murine crt fragment covering amino acid residues - fused with an n-terminal his-tag, was extraordinarily potent in activating b cells and macrophages in vitro and also in eliciting specific ab production in mice [ ] . this recombinant polypeptide also exhibited potent adjuvanticity, effectively assisting igg production against conjugated target ags with or without t cell help [ ] [ ] . however, molecular mechanisms underlying this phenomenon are far from clear. recent x-ray crystographical studies by kozlov [ , ] . the sequence of rcrt/ - encompasses most of the globular n domain (aa residues , and we have previously shown that it possess lectin-like activity (selective binding with polysaccharides including carrageenan, alginic acids, and hyaluronic acids in elisas) [ ] , implying that the prokaryotically expressed recombinant polypeptide retained the lectin activity of crt. it is of interest to determine if destroying the carbohydrate binding and/or peptide-binding sites (by deleting first half of the n domain sequence) would also abolish the potent immunological activities observed in rcrt/ - . additionally, the fact that rcrt/ - is substantially more potent than ncrt in activating macrophages(see below) raised concerns regarding the possibility of lps contamination in the e. coli.-expressed recombinant product. the ''lps contamination'' hypothesis suggests tight binding between bacterial lps and rcrt and also that, due to a synergistic effect, the lps-crt complex is a more potent immune activator than free lps and rcrt alone. based upon the observation that interaction between the n and c domains of crt influences its structural stability as well as functional activity [ ] , a ''c-domain deletion'' hypothesis has also been postulated, suggesting that deletion of the c-domain (as in rcrt/ - ) leads to exposure of an immunologically active site (ias) in the n and/or p domains of crt. the present study compares the biochemical characteristics of ncrt and a series of truncated rcrt polypeptides and investigates the molecular mechanisms underlying the potent immunological activities of soluble rcrt. the results arising from this study have important implications for our understanding of the potential role of soluble crt in immunopathological conditions. native crt was extracted from mouse livers by (nh ) so precipitation followed by ion exchange chromatography on a deae-a column using a linear gradient of - mm nacl for elution. samples of the eluted fractions were assayed by sds-page (fig. a) , which showed that the eluent between - mm nacl contained protein bands of the expected molecular weight for ncrt ( kda), which showed approximately % homogeneity judging by density of the major band in coomassie blue (cbb)-stained gel (fig. c ). recombinant murine crt fragment with an n-terminal his-tag and without c-terminal kdel) was expressed in e. coli and affinity purified using a ni + -column. the resultant rcrt/ - product contained major protein bands at , and kda (figs. b & c); the two larger bands (namely rcrt- kda and rcrt- kda), but not the smaller one (cp ), were recognized by polyclonal rabbit-anti-crt antisera (crt-abs) in western blot (wb) (fig. d ). purified ncrt, but not bsa or recombinant enhanced green fluorescence protein (regfp, kda with a his-tag), was positively recognized by crt-abs. as evidenced by native page analysis, a substantial amount of rcrt/ - formed higher-molecular-weight oligomers, whilst ncrt existed mostly in monomeric form (figs. e & f). in our previous study, rcrt/ - could effectively activate mouse macrophages in vitro [ ] . interestingly, rcrt/ - was as effective as rcrt/ - in inducing no production by [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] were analyzed by a sds-page % gel followed by cbb staining. fractions - were combined, dialyzed against pbs, and then adjusted to mg/ml for use as ncrt. (b) a ni-column was employed for purification of rcrt/ - from a lysate of iptg-induced e. coli harboring the expression vector for rcrt/ - . samples of e. coli before and after iptg induction were loaded in lanes, flow through (binding buffer), wash through ( mm imidazole), resultant rcrt/ - ( mm imidazole), and stripping through were loaded, in the order of - into a sds-page % gel followed by cbb staining. samples of ncrt, rcrt/ - , bsa and regfp were compared in cbb-stained sds-page % gel (c) and native-page (e), followed by wb using rabbit anti-crt polyclonal antibody for detection (d, f). the secondary ab was hrplabeled goat-anti-rabbit igg, with opd as substrate. doi: . /journal.pone. .g mouse peritoneal macrophages in vitro, indicating that the presence, or deletion, of the c-domain did not affect the immunological activity of crt ( fig. a ). purified ncrt was also able to activate macrophages in vitro, but with a - -fold lower potency than rcrt/ - ( fig. a) . although rcrt/ - and rcrt/ - preparations had been repeatedly treated with polymyxin b (an efficient lps inhibitor) prior to functional assays, the possibility that bacterial lps might form a tight complex with rcrt, thereby resisting pmb treatment, was always a concern. figs. b & c demonstrate that ncrt was not able to bind lps in elisas, implying that formation of tight lps-crt complexes in the expressing e. coli cells is perhaps an unlikely event. moreover, lps and ncrt at sub-optimal concentrations did not show any synergistic effect in activating macrophages (fig. d) , providing circumstantial evidence against the ''lps contamination'' hypothesis. contaminating rpl does not contribute to rcrt activity when preparing rcrt/ - , cp was a relatively persistent contaminant protein (figs. & ) . to characterize this protein, the cp band was sliced out of sds-page gels and then (i) used to immunize c /bl mice for preparation of specific antisera; and (ii) subjected to q-tof mass spectrometry analysis for sequence identification. the resultant antisera (cp -abs) were able to recognize the immunizing protein band, but not the rcrt- kda and rcrt- kda bands, in wb (fig. b) . the ms result identified cp as bacterial s ribosomal protein l (rpl ), which was confirmed by positive recognition of recombinant rpl (rrpl , commercially available) by cp -abs (fig. b) . it is noteworthy that rrpl was unable to activate macrophages, as evidenced in no production assays (fig. c ), and also that rrpl was not recognized by crt-abs (figs. c & d). these results exclude the possibility that the contaminant cp could make a substantial contribution to the immunological activities of rcrt. in the culture supernatant was then determined using griess reagent and the results are expressed as mean no concentration (mm) sd. lps-based elisas were performed for detection of lps binding with crt (b) or lactoferrin (c). lf or ncrt ( mg/ml) were added to wells in polyvinyl plates pre-coated with lps ( mg/ml), with bsa as a negative control. combination of polyclonal rabbit abs against crt, or lactoferrin, and hrp-labeled goat-anti-rabbit igg was used for detection with opd as substrate. the results are expressed as absorbance at od nm sd. for sinergy analysis (d), freshly isolated mouse peritoneal macrophages were stimulated with ncrt ( . - mg/ml) in the presence, or absence, of lps ( . ng/ml) for h. cells in medium alone (medium) or stimulated with lps ( . ng/ml) alone (lps) were included as controls. tnf-a in the culture supernatant was then quantitated using an elisa kit and the results are expressed as mean concentration (pg/ml) sd. these are representatives of independent experiments. doi: . /journal.pone. .g since rcrt and ncrt differ in their ability to form oligomers in solution, we next asked if the potent immunological activities of rcrt polypeptides were the result of self-oligomerization. a sephadex g- column was employed for fractionation of rcrt/ - oligomers and monomers. fig. a shows that rcrt/ - was successfully separated into peaks, designated sequentially as orcrt (higher-molecular-weight rcrt/ - oligomers, major peak), mrcrt- kda (monomeric rcrt/ - , kda) and mrcrt- kda (monomeric rcrt- kda, aa - , see below). guided by the sds-page results ( , revealed their molecular mass as . kda and . kda, respectively. it can therefore be calculated that rcrt- kda is a degradation product of rcrt- kda less the c-terminal amino acid residues. as shown in fig. a , orcrts were modestly more effective than unfractionated rcrt/ - in eliciting tnf-a production by murine macrophages in vitro, while mrcrt- , mrcrt- and ncrt were - -fold less active than orcrts by comparison. a similar conclusion was also drawn when no production was taken as a measure for macrophage activation (fig. b) . moreover, s.c. immunization of balb/c mice with orcrts or unfractionated rcrt/ - (in the absence of adjuvant) elicited high titer serum igg capable of recognizing both orcrts and mrcrts in elisas (figs. c- f). by contrast, mrcrt- kda (monomeric rcrt/ - ) and mrcrt- kda (monomeric rcrt/ - ) were almost non-immunogenic in parallel experiments. in order to assess whether the ias is located in the n or p domain, the following fragments were designed: rcrt-n (residues - , full length n domain), rcrt/ - (partial n, half p domains), rcrt/ - (partial n, one third p domains), rcrt/ - (partial n, full length p domain), and rcrt/ - (full length p domain). all, but rcrt/ - (very low yield and poor solubility), were successfully expressed in e. coli and affinity-purified. both rcrt/ - and rcrt-n formed homodimers as well as higher-molecular-weight species (fig. a) . rcrt/ - was as potent as rcrt/ - and rcrt/ - in terms of eliciting no production by murine macrophages, while rcrt-n was almost completely inactive (fig. b ). similar to rcrt/ - , rcrt/ - also formed homodimers and higher-molecular-weight oligomers and showed strong macrophage-stimulatory activity in vitro (table ) . rcrt/ - , which contains a single cysteine residue (cys ) and could only form homodimers (fig. a) , was approximately fold less effective than rcrt/ - and rcrt/ - but nevertheless substantially more potent than mrcrts and ncrt (fig. b) . a mutant form of rcrt/ - (namely rcrt/ - -c a) was prepared by substituting the cysteine residue at position with alanine. the c a mutant was neither able to form homodimers/oligomers in solution nor activate macrophages in vitro (figs. c- e). these results map the ias of crt to residues - and suggest that the n-domain probably does not contribute to the immunological activity of the molecule. moreover, the importance of oligomerization for its immunological activities is further confirmed. in order to investigate the mechanisms for the relationship between rcrt oligomerization and its potent immunological activities, fractionated orcrts and mrcrt- kda (monomeric rcrt/ - ) were conjugated with fitc and then compared for ability to stain macrophages. after min incubation at uc, fitc-orcrts showed stronger binding to murine macrophages than fitc-mrcrts, as evidenced by flow cytometric analysis and confocal laser scanning microscopy (fig. a&b) . substantially more fitc-orcrts than fitc-mrcrts were endocytosed by macrophages after min incubation at uc (fig. b) . moreover, monodansylcadaverine (mdc), an endocytosis inhibitor [ ] , partially suppressed no production by macrophages under stimulation with orcrts, while lps-triggered macrophage activation was unaffected by the same treatment (fig. c ). in this study, we have examined different hypotheses (i.e. cdomain deletion hypothesis, lps contamination hypothesis, rpl hypothesis and oligomerization hypothesis) for explanation of the much stronger immunogenicity and immunostimulatory activities of rcrt than ncrt. our data strongly suggests that selfoligomerization of the rcrt polypeptides is a key factor for their strong immunological activities. as summarized in table , all four rcrt fragments containing residues - (including rcrt/ - , rcrt/ - , rcrt/ - and rcrt/ - ) self-oligomerized and exhibited potent macrophage activating ability in vitro and strong immunogenicity in vivo, while ncrt, which existed mainly in monomeric form, showed only modest stimulatory activities towards macrophages and was non-immunogenic in mice (figs. & ) . moreover, fractionated rcrt/ - oligomers were - folds more active than mrcrts in activating macrophages in vitro (fig. ) . the n domain polypep-tide (rcrt/ - ) also formed higher-molecular-weight oligomers (fig. a ), but it is almost completely inactive in terms of stimulating macrophages in vitro (fig. b ) and inducing ab responses in vivo (table ) . clearly, oligomerization is necessary but not sufficient to arm the rcrt polypeptides with potent immunological activities. in general, oligomerized (aggregated) proteins are more immunogenic than their monomeric counterparts. however, the immunogenicity of orcrts is by far the most impressive and not comparable by other protein aggregates. even in the absence of any adjuvant, minute amount ( ng/mouse) rcrt/ - or rcrt/ - can elicit strong igg responses in mice ( fig. ; ref. ). most, if not all, other protein antigens are unable to induce igg production in t-cell-deficient nude mice, yet rcrt/ - and rcrt/ - could do so relatively efficiently [ , ] . moreover, the potent adjuvanticity of the rcrt polypeptides is also quite phenomenal. for instance, rcrt/ - (mostly in oligomeric forms) is able to assist the production of igg abs against fused target proteins or conjugated polysaccharides in healthy mice or t-cell-deficient nude mice [ , ] . dimerization/oligomerization of soluble crt was also observed by previous investigators. for example, jorgensen et al documented that shielding of the free cys in the n domain is the main reason that ncrt exists mainly in monomeric form under physiological conditions. under partial unfolding conditions such as high temperature or low ph, however, the free cys could be exposed and subsequently help crt oligomerization [ ] . ncrt (isolated from human placenta) formed homodimers and higher-molecular-weight species through disulfide bonding as well as non-covalent association, and that oligomerized ncrt showed higher binding affinity to peptides and denatured proteins [ ] . in the case of prokaryotically expressed rcrt polypeptides (the folding of which may differ from ncrt), all cys residues in sequence could contribute to its olimerization, although it might not be absolutely necessary that all cys residues have to be available for inter-molecular cross-linking at the same time. mancino and colleagues illustrated that self-oligomerized rcrt could better assist hla folding in vitro [ ] . there are conserved cysteine residues in the amino acid sequence of crt. cys and cys form intramolecular disulfide bonds, while cys is free [ ] [ ] . it is likely that rcrt polypeptides are unable to form appropriate intra-molecular disulfide bonds like in ncrt, thereby allowing all cysteine residues to participate in self-oligomerization, although formation of higher-molecularweight oligomers could also occur through non-covalent association of crt [ , ] . crt is considered one of the heat shock proteins (hsps) that share many immunological and biochemical activities [ ] [ ] . interestingly, self-oligomerization also occurs to other hsps such as grp and hsp , which is likely associated with their chaperone function [ ] [ ] [ ] . koslov et al and chouquet et al have recently solved the crystal structure of the lectin site as well as a peptide-binding site in the crt n domain [ , ] , which apparently play important roles in the physiological function of crt. however, our data maps the ias of crt to a region of residues between aa - (fig. ). as rcrt-n was almost completely inactive in functional assays (fig. , table ), the ias may be narrowed down further to aa - in the p domain, although a series of truncated synthetic peptides covering this region would be needed for a concrete conclusion. interestingly, this sequence of amino acid residues contains the ra shared epitope (se)-binding site (residues - ) of crt recently mapped by ling et al. [ ] . such coinciding results from independent groups further emphasize the importance of the aa - region of the p domain to the immunological activities of crt. it has been documented that the crt p domain adopts a hairpin-shaped structure, its sequence is consisted of copies of a repeat motif (type : ixdpxxxk-pedwd) followed by copies of another repeat motif (type ) [ ] [ ] [ ] . interestingly, the se-binding site almost completely overlaps the type motif [ ] . it is reasonable to suggest that the type motif might also represent the core of ias of crt, responsible for direct biding with activation receptors on the surface of immune cells. functional comparison data showed that rcrt/ - and rcrt/ - (possessing type repeats, the latter without type repeats) are times more active than rcrt/ - (with type repeats, no type repeats) in activating macrophages (table , fig. b ), implying that (i) type repeats do not contribute to the immunological activity of the molecule; and (ii) the presence of copies of the type motif is of crucial importance for the potent immunological function of orcrts. it can be envisaged that oligomerization of crt multiplies its binding avidity to immune cells with receptors for the type motif, thereby enabling it to deliver stimulatory signals to the cells in a highly efficient manner. we further predict that the high-avidity binding of orcrts to macrophages may easily trigger their uptake process. indeed, orcrts can activate macrophages in an endocytosis-dependent pathway (fig. ) . perhaps orcrts could use certain intracellular sensors to deliver immunostimulatory activity to the responding immune cells. an example of an intracellular sensor for endocytosed polymeric proteins is the nod-like receptor (nlr) protein nlrp , a key component of the nlrp inflammasome and an important intracellular sensor for microbial ligands and endogenous danger signals [ ] . masters and colleagues demonstrated that soluble oligomers of islet amyloid polypeptide (iapp), a protein that forms amyloid deposits in the pancreas during type diabetes, could be endocytosed and trigger the nlrp inflammasome and generate mature il- b [ ] . finally, the region of aa - of crt has a % homology between mouse and human. it would be of interest to examine if there is a functional relationship between the se binding site and the ias of crt. irrespective of such a relationship, oligomerization might occur to extracellular crt released by tissue cells thereby converting crt into a highly active form, which may play important roles in the development and pathogenesis of autoimmune disorders in humans. purification of ncrt ncrt was purified from mouse livers using a modification of a previously described methods [ , ] . briefly, fresh mouse liver cells (erythrocytes depleted) were collected and centrifuged at rpm for min. the cell pellet was lysed in volumes of lysis buffer ( % triton-x , . mm pmsf in pbs) for min on ice, followed by centrifugation at , g for minutes. the supernatant was then precipitated using (nh ) so and the final precipitate dissolved in binding buffer ( mm nacl, mm tris, ph . ) followed by dialysis against this buffer. the sample was applied to a deae sephadex a column ( cm, ge healthcare, us) which was then sequentially washed with binding buffer and washing buffer ( mm nacl, mm tris, ph . ) at ml/min to remove contaminating proteins. the fractions were eluted with a linear salt ( - mm nacl) gradient. the preparation of rcrt/ - and regfp was as previously described [ ] and the other rcrt polypeptides used in this study were prepared using the same prokaryotic system. specific primers were as follows: -cccaagcttctaatctgtggggtcatc-gatcttg- . the rcrt/ - -c a mutant was constructed using takara mutan best kit (takara biotechnology co. ltd) following manufacturer's instruction. the mutant primer was as follows: sense: -attcacacacctataca-cactgatt- , antisense: -tcatcatccttagcccgga-tatcct- . all proteins were desalted by passing through pd columns (pierce, rockford, il, usa). protein concentration was determined using coomassie protein assay reagent (pierce, rockford, il, usa). all recombinant proteins were used at over % purity as judged by cbb-stained sds-page gels. the separated protein bands in sds-page gels were electroblotted onto pvdf membranes, at a constant current of ma in transbuffer ( mm tris, ph . , containing . m glycine and % methanol), using a bio-rad trans-blot cell. the strips were incubated for hr at room temperature in blocking buffer (tbs containing % nonfat milk), followed by a overnight incubation at uc with constant agitation in indicated antibody diluted in blocking buffer. after washes with tbs containing . % tween , strips were incubated for hr with hrpconjugated secondary antibody (southern biotechnology associates inc., usa) and visualized using the ecl detection system as recommended by the manufacturer (applygen technologies inc., beijing, china). lps-based and crt-based elisas were as previously described [ ] . briefly, elisa plates were coated at uc overnight with rcrt or lps and subsequently incubated with blocking solution ( % bsa in pbs) for hrs at uc. the wells were washed five times with pbs containing . % tween (pbs-t) prior to incubation at uc with ml of diluted mouse sera or with indicated protein (ncrt and lactoferrin) followed by corresponding antibody in triplicate. after washes with pbs-t, the plates were further incubated with hrp-labeled goat-antimouse or goat-anti-rabbit igg abs (southern biotechnology associates inc., al., usa) for hr at uc. the reaction was developed with ml of o-phenylenediamine (opd, sigma) for min and stopped with ml m h so . optical density (od) was measured at nm in an elisa spectrophotometer (titertek multiscan plus mk ii; icn flow laboratories, irvine, uk). all cells were cultured in complete r medium: rpmi- supplemented with % (v/v) fetal bovine serum (hyclone, usa), penicillin/streptomycin ( u/ml), l-glutamine ( mm), and b-me ( m). for preparation of mouse peritoneal macrophages, mice were injected i.p. with % thioglycollate ( ml/ mouse) and the macrophages retrieved from the peritoneum days later using a syringe. the resultant cells were. % positive for f / marker, as determined by facs analysis. relative macrophage stimulation activity (rmsa) of crt is estimated using ncrt as reference, which is able to induce tnf-a production by macrophages in vitro at a concentration of mg/ml or above (see fig. a ). the listed results are based on several batches of independent experiments including fig. a . b) ability to elicit specific igg responses in healthy balb/c mice after s.c. immunization, in the absence of adjuvant, with mg protein and a booster immunization with mg protein a fortnight later. the mice were monitored for up to days after the second immunization. nd: not detected; -: not immunogenic; +: strong humoral response; /+: weak response. freshly prepared c /bl mouse peritoneal macrophages ( . cells/well) were stimulated with rcrt fragments, or ncrt, or lps, in r medium in -well tissue culture plates for hrs in a % co incubator at uc. the concentration of tnf-a in the culture supernatant was determined using elisa kits (biolegend, san diego, usa) following the manufacturer's instructions. the concentration of no in the supernatant was determined by griess reagent. standard curves were established using nano . female c bl/ mice between the age of - weeks were purchased from the model animal research center, nanjing, china. all animals were maintained under specified-pathogen-free (spf) conditions and animal usage was conducted according to protocols approved by the soochow university institutional animal care and use committee. for immunization with rcrt, mice were immunized s.c. at the base of the tail with mg protein in total ml pbs. when booster immunization was needed, mg of protein in ml pbs was injected intraperitoneally (i.p.). for immunization with page gel slices containing cp , the band was cut out with a razorblade and then frozen in liquid nitrogen and emulsioned with cfa, which was then injected s.c. into female c /bl mice. serum samples were collected by tail bleeding, aliquoted and kept at uc until use. a sephadex g- (ge healthcare, us) column of cm was employed. ml of rcrt/ - at mg/ml was loaded into the column, followed by elution with . % nacl at ml/h and collected every ml. protein was labeled by fitc using fluorotag tm fitc conjugation kit (sigma, us) according to the manufacturer's instructions. in brief, mg of protein was dialyzed against . m na co , ph . at mg/ml. fluoresceine isothiocyanate (fitc, sigma) was dissolved in the same buffer with dmso at mg/ml and ml of this solution was added to the protein. the sample was gently mixed for h at rt. separation of labeled protein from unbound fitc was performed on a sephadex g- column. flow cytometric analysis freshly isolated peritoneal macrophages were stained with apc-anti-f / and then incubated with mg/ml of fitc-orcrt, fitc-mrcrt or fitc-ova for min at uc. the binding of crt by macrophages was determined by flow cytometry (bd facs canto ii, us). macrophages ( . /well) were plated onto poly-l-lysine coated glass slides and allowed to adhere. the cells were then incubated in a total volume of ml . % bsa in pbs with mg/ml fitc-orcrt, fitc-mrcrt or fitc-ova for min at uc or uc. after washing to remove unbound protein, macrophages were fixed in % paraformaldehyde and stored at uc until microscopic analysis. finally, % of glycerol was added and slides were counterstained with mg/ml dapi. the cells were imaged with a nikon confocal microscope system a . all experiments were repeated at least times and the results are expressed as mean standard deviation of the mean (sd). statistical analysis was performed using the independent-samples t test or two-side paired t test between groups using the spss . program (spss, chicago, il). differences were considered statistically significant at p, . . calreticulin: one protein, one gene, many functions definition of the lectin-like properties of the molecular chaperone, calreticulin, and demonstration of its copurification with endomannosidase from rat liver golgi delineation of the lectin site of the molecular chaperone calreticulin oligosaccharide binding characteristics of the molecular chaperones calnexin and calreticulin calreticulin is expressed on the cell surface of activated human peripheral blood t lymphocytes in association with major histocompatibility complex class i molecules cell surface calreticulin is a putative mannoside lectin which triggers mouse melanoma cell spreading thrombospondin mediates focal adhesion disassembly through interactions with cell surface calreticulin activation of human monocyte cell line u via cell surface calreticulin cell-surface calreticulin initiates clearance of viable or apoptotic cells through trans-activation of lrp on the phagocyte calreticulin in tlymphocytes. identification of calreticulin in t-lymphocytes and demonstration that activation of t cells correlates with increased levels of calreticulin mrna and protein regulation of peripheral t cell activation by calreticulin candidate autoantigens identified by mass spectrometry in early rheumatoid arthritis are chaperones and citrullinated glycolytic enzymes calreticulin is released from activated neutrophils and binds to c q and mannan-binding protein cd -dependent programming of t-helper cell responses following heat shock protein immunization functional analysis of recombinant calreticulin fragment - : implications for immunobiological activities of calreticulin in health and disease ajuvanticity of recombinant calreticulin calreticulin as a hydrophilic chimeric molecular adjuvant enhances igg responses to the spike protein of sars coronavirus structural basis of carbohydrate recognition by calreticulin x-ray structure of the human calreticulin globular domain reveals a peptide-binding area and suggests a multi-molecular mechanism calreticulin is a thermostable protein with distinct structural responses to different divalent cation environments amantadine and dansylcadaverine inhibit vesicular stomatitis virus uptake and receptor-mediated endocytosis of alpha -macroglobulin adjuvanticity of recombinant calreticulin fragment - in assisting anti-b-glucan igg responses in t celldeficient mice dimerization and oligomerization of the chaperone calreticulin calreticulin recognizes misfolded hla-a heavy chains calreticulin, a ca + -binding chaperone of the endoplasmic reticulum human placental calreticulin characterization of domain structure and post-translational modifications the metal ion binding properties of calreticulin modulate its conformational flexibility and thermal stability heatshock proteins as activators of the innate immune system heat shock proteins as regulators of the immune response heat-induced chaperone activity of hsp endoplasmic reticulum chaperone grp subunit assembly is regulated through a defined oligomerization domain substrate-binding characteristics of proteins in the kda heat shock protein family identification of the rheumatoid arthritis shared epitope binding site on calreticulin trosy-nmr reveals interaction between erp and the tip of the calreticulin pdomain nmr structure of the calreticulin p-domain nmr structures of and -residue fragments of the calreticulin p-domain nlrp : an immune sensor of cellular stress and infection activation of the nlrp inflammasome by islet amyloid polypeptide provides a mechanism for enhanced il- beta in type diabetes human placental calreticulin: purification, characterization and association with other proteins a single purification procedure for the major resident proteins of the er lumen: endoplasmin, bip, calreticulin and protein disulfide isomerase heat shock protein : specific binding of lipopolysaccharide key: cord- -flyx lr authors: hibbitts, alan j.; ramsey, joanne m.; barlow, james; macloughlin, ronan; cryan, sally-ann title: in vitro and in vivo assessment of pegylated pei for anti-il- /cxcl- sirna delivery to the lungs date: - - journal: nanomaterials (basel) doi: . /nano sha: doc_id: cord_uid: flyx lr inhalation offers a means of rapid, local delivery of sirna to treat a range of autoimmune or inflammatory respiratory conditions. this work investigated the potential of a linear kda poly(ethylene glycol) (peg)-modified kda branched polyethyleneimine (pei) (pei-lpeg) to effectively deliver sirna to airway epithelial cells. following optimization with anti- glyceraldehyde -phosphate dehydrogenase (gapdh) sirna, pei and pei-lpeg anti-il sirna nanoparticles were assessed for efficacy using polarised calu- human airway epithelial cells and a twin stage impinger (tsi) in vitro lung model. studies were then advanced to an in vivo lipopolysaccharide (lps)-stimulated rodent model of inflammation. in parallel, the suitability of the sirna-loaded nanoparticles for nebulization using a vibrating mesh nebuliser was assessed. the sirna nanoparticles were nebulised using an aerogen(®) pro vibrating mesh nebuliser and characterised for aerosol output, droplet size and fine particle fraction. only pei anti-il sirna nanoparticles were capable of significant levels of il- knockdown in vitro in non-nebulised samples. however, on nebulization through a tsi, only pei-peg sirna nanoparticles demonstrated significant decreases in gene and protein expression in polarised calu- cells. in vivo, both anti-cxcl- (rat il- homologue) nanoparticles demonstrated a decreased cxcl- gene expression in lung tissue, but this was non-significant. however, pei anti-cxcl- sirna-treated rats were found to have significantly less infiltrating macrophages in their bronchoalveolar lavage (bal) fluid. overall, the in vivo gene and protein inhibition findings indicated a result more reminiscent of the in vitro bolus delivery rather than the in vitro nebulization data. this work demonstrates the potential of nebulised pei-peg sirna nanoparticles in modulating pulmonary inflammation and highlights the need to move towards more relevant in vitro and in vivo models for respiratory drug development. oligonucleotide therapeutics offer a unique opportunity for accurate and specific disease treatment at a genetic level. to date, these have been investigated in a variety of endogenous and infectious conditions [ ] [ ] [ ] . of these, sirna has gained renewed prominence following the successful clinical approval of patisiran (onpattro™) by alnylam pharmaceuticals for hereditary ttr-mediated amyloidosis (hattr) [ ] . in part, the approval was based on patisiran's ability to rapidly and accurately reach its target cells (hepatocytes) by utilising the most appropriate delivery method (i.v. perfusion) [ ] . when the i.v. route is not practical or effective, utilising local delivery routes offer a means of vastly increasing the targeting of oligonucleotides and thereby improving the specificity and efficacy [ ] . local delivery is particularly promising as an approach for oligonucleotide therapeutics targeting respiratory diseases. this is especially true for episodes of acute pulmonary inflammation, including acute asthmatic episodes and pathogenic respiratory conditions such as acute respiratory distress syndrome (ards) [ , ] . the urgent need to mitigate the effects of respiratory viruses has become especially relevant with the emergence of the covid- pandemic. covid- is clinically manifest in the lungs in~ % of patients and mortality is strongly linked to cytokine storm-induced ards [ ] [ ] [ ] . in these cases, a fast-acting specific treatment of short duration may be more favourable than a continuous, systemic depression of the innate immune system in an otherwise healthy individual. one potential target for cytokine-induced respiratory distress is the chemotactic pro-inflammatory cytokine il- . il- is known to be secreted from pulmonary epithelial cells and is elevated in covid- patients [ , ] . furthermore, anti-il- therapy is currently under investigation in phase ii clinical trials using i.v.-delivered anti-il- monoclonal antibodies in covid- patients [ ] . however, the effective delivery of sirna to the lungs has been hampered by poor delivery device performance. this is especially true in the case of the nebulised delivery of sirna. nebulisation offers the benefits of a non-invasive, easy to use device with a large level of flexibility in the dose delivered. it has long been established that air-jet and older ultrasonic nebulisers are unsuited to sirna delivery due the high cost of the therapeutic cargo and their known deficiencies in output efficiency [ ] . previous tests have indicated that air-jet and ultrasonic nebuliser devices have a considerable discrepancy in their ability to effectively form droplets of the desired size [ ] . to address this, there has been a relatively new " rd generation" of nebulisers developed known as vibrating mesh nebulisers (vmns) (air-jet and ultrasonic nebulisers being the st and nd generations, respectively). these nebulisers function in a manner modified from previous ultrasonic nebulisers, whereby a plate containing approximately tapered holes vibrates at a frequency of roughly khz, thereby causing the ejection of liquid droplets [ ] . vibrating mesh nebulisers are now becoming widespread and there is investigation into their use for delivery of sensitive therapeutic cargoes, such as proteins [ ] , mirna and anti-mirna target site-blocking oligonucleotides [ , ] , mrna [ ] and sirna [ , ] . furthermore, the bulk of recent publications indicate that vmns deliver more drug to the patient with significantly less wastage, than the more prevalent air-jet nebulisers [ ] [ ] [ ] . in parallel to effective device-borne delivery to the lungs, the successful development of a locally delivered sirna therapy for pulmonary conditions must contend with the challenges of the lung micro-environment. major barriers to local pulmonary delivery include the mucociliary clearance action of the ciliated epithelial cells and the presence of mucus and alveolar fluid in different parts of the airways [ , ] . particles that are deposited on the ciliated cells are rapidly removed by muco-ciliary clearance and are eventually coughed up or swallowed. this mucus lines the respiratory epithelium from the nasal cavity to the terminal bronchioles [ ] . efforts to overcome the mucus barrier and improve the efficacy of delivered drugs has led to the development of "mucus penetrating particles". previous approaches to modifying cationic polymers for sirna delivery have drawn inspiration from naturally occurring molecules such as viral proteins [ ] . of particular interest is the use of poly(ethylene glycol) (peg)-modified nanoparticles, which has demonstrated much potential in this regard [ ] . recent research within our group has focused on developing nanoparticle carriers to better deliver sirna to the pulmonary epithelium [ , , ] . specifically, we have previously developed a block co-polymer of kda branched polyethyleneimine (pei) conjugated to a high degree ( %) with kda linear poly(ethylene glycol) (peg). this pei-lpeg was capable of higher levels of gene knockdown in fully polarised calu- epithelial monolayers compared to the pei controls, with limited nanomaterials , , of cellular toxicity evident [ ] . in this study, this pei-lpeg polymer will be further investigated for its potential to deliver anti-il- sirna to the lungs via a vibrating mesh nebuliser. given its prominent role in modulating pulmonary inflammation, the successful nebulisation of anti-il- sirna would serve as a promising proof of concept in combatting pulmonary inflammation at the genetic level. to achieve this, pei-lpeg sirna and pei sirna nanoparticles were nebulised using a vibrating mesh nebuliser, and their post-nebulisation stability was assessed in terms of their physicochemical properties and their ability to facilitate gene knockdown. cell studies first focused on validating the sequence specificity of anti-il- sirna in non-nebulised transfections. following this, the post-nebulisation efficacy was investigated following passage through a twin-stage impinger (tsi). this was first validated at a genetic level using the glyceraldehyde -phosphate dehydrogenase (gapdh) house-keeping gene before assessing the changes in il- protein levels. finally, anti-cxcl- (rat il- homologue [ ] ) sirna nanoparticles were assessed in an in vivo pilot study using a rat model of acute pulmonary inflammation involving intra-tracheal intubation, sirna delivery and subsequent lipopolysaccharide (lps) challenge. all the cell culture reagents were obtained from invitrogen corporation/thermofisher scientific (ca, usa) unless otherwise stated. the calu- bronchial epithelial cell line was obtained from the american tissue type culture collection (atcc) and used at passages - . all the poly (ethylene glycol) molecules were obtained from iris biotech (marktredwitz, germany). the sirna sequences for human gapdh, β-actin and il- were obtained from qiagen uk and were diluted : in te buffer as directed. the specific sequences remained the proprietary knowledge of qiagen. the sirna sequences for rat cxcl- ( uaacgagauauuuaacgccccc ) were obtained from (riboxx gmbh, radebeul, germany), and the sigenome non-targeting sirna # ( uaaggcuaugaagagauac ) scrambled sequence controls were obtained from dharmacon (now horizon discovery, cambridge, uk). all the other general chemicals and reagents used were of the highest grade possible and were obtained from sigma-aldrich company ltd. (wicklow, ireland) unless otherwise stated. branched kda pei was modified with kda linear peg by the reaction of succinimidyl-activated peg (peg-ssa) with pei under slightly basic aqueous conditions as previously described [ ] . briefly, g of kda pei was dissolved in ml of phosphate buffered saline (ph ). following this, mg of kda linear peg-ssa was dissolved in ml of . % dimethyl sulfoxide (dmso). this was added dropwise with stirring to the pei solution. the reaction proceeded for h at room temperature before being stopped by the addition of ml of deionised water. the reaction mixture was transferred to cellu•sep h membranes ( kda mwco) (orange scientific, braine-l'alleud, belgium) and dialysed overnight in excess deionised water to remove unreacted components before being lyophilised. the final products were then analysed for form and purity using h and c nmr spectroscopy (bruker avance ) and gel permeation chromatography (gpc) (perkin elmer, dublin, ireland) with electronic light scattering detection (agilent technologies, cork, ireland). pei and pei-peg polymer-sirna complexes were formed using pei nitrogen to rna phosphate (n/p) in ratios of and . briefly, the appropriate amount of polymer was added to an sirna solution ( µm) to yield a final concentration of µm sirna, vortexed for s and incubated for polyplex formation for min. following this, the solutions were diluted using pbs. the sirna nanoparticles were nebulised and the droplet size distributions, described by a volumetric median diameter (vmd), were measured by a malvern spraytec particle size analyser for the droplets produced (malvern instruments ltd., malvern, worcestershire, uk) with the rt sizer software (version . ). a l/min vacuum flow was implemented through the system, ensuring a laminar flow and reducing the artificial droplet size growth through collision with other droplets. the vacuum also ensured that the droplets passed through the laser beam only once. the centre of the emitted aerosol plume was directed through the centre of the laser beam to increase the accuracy of data acquisition. data acquisition was performed as previously described [ ] , beginning when beam obscuration exceeded % and continued until the end of dosing. the data acquisition rate was set to hz, which is individual readings per second taken to characterise the droplet size distribution. the data reported for each individual measurement is an average of the individual readings recorded over the course of the dose. in order to verify the accuracy of the generated data, the spraytec analyser's laser diffraction apparatus was tested with a reference reticle (malvern instruments ltd., malvern, worcestershire, uk). the droplet size is described by volumetric median diameter (dv ) and the fine particle fraction (fpf) (percentage of droplets less than µm in size). the surface tension of the sirna nanoparticle solutions was also examined using a ring/plate tensiometer (lauda scientific gmbh, lauda-königshofen, germany) at room temperature according to the manufacturer's instructions. in all cases, the changes in output rates and fluid surface tension were compared to pbs controls. the pei and pei-peg sirna nanoparticles were formed as previously described using . µg of sirna and diluting with pbs to a final volume of ml polyplex. the sirna samples were nebulised into a glass impinger at l/min using an aerogen ® pro vibrating mesh nebuliser (aerogen, galway, ireland). before nebulisation, pbs was added to the upper (stage a, ml) and lower (stage b, ml) stages. washes were quantitatively collected using pbs with the device, throat, stage a and stage b made to , , and ml respectively. a size analysis of the sirna polyplexes was performed using a malvern nano-zs zetasizer. pre-nebulisation samples underwent a in dilution in dh o before addition of pbs. post-nebulisation, rinsed samples were concentrated by centrifuging out excess pbs using centrisart centrifugal uf units ( kda mwco, sartorius). despite this, due to the high level of dilution that occurred and the strong ionic character of pbs, it was not possible to accurately measure the zeta potentials of the post nebulisation samples. size analysis programmes consisted of five separate scans which contained a minimum of sub-scans. calu- cells were seeded in mm transwell chambers at × cells/well cultured in complete media of : dmem:ham's f- ( % fbs and % pen/strep) in at a liquid-liquid interface for h and for a further - days at an air/liquid interface. a trans-epithelial electrical resistance (teer) value of > Ωcm was taken as a sign of tight junction formation. on the day of transfection, . µg/well ( nm) of sirna nanoparticles in pbs were formed as previously described. the sirna nanoparticles were administered directly to each well and incubated for h at • c and % co . following this, the apical ( µl) and baso-lateral ( . ml) layers of the transwells were aspirated off and collected for il- elisa analysis (invitrogen via bio-sciences, dublin, ireland) according to manufacturer's instructions and later normalised to account for differences in dilution. sequence specificity was demonstrated by use of non-complexed pei or pei-lpeg and gapdh-sirna negative controls. in addition to this, the teer value fluctuations over the h of transfection were recorded using an evom voltohmmeter (world precision instruments, stevenage, uk) at various time points for analysis. briefly, a baseline reading was established prior to transfection by equilibrating the apical layer in pbs for min at • c prior to reading. following the addition of the sirna nanoparticle pbs, readings were taken at min and at , , and h and compared to the pbs-only treated controls. calu- cells were seeded in . mm transwell plates at a density of . × cells/well using the previously described methods for calu- culture in transwell plates at liquid-liquid and air-liquid interfaces. cell monolayers were used for the transfection studies once a teer of > Ωcm was established [ ] . a glass tsi was assembled as previously described by grainger et al. [ ] , apart from the absence of solution in the lower chamber. an aerogen ® pro vibrating mesh nebuliser was sealed into place with parafilm at the entrance of the tsi ( figure a (i)). the adapter piece ( figure a (ii)) was removed and parafilm was wrapped around the base of the connecting tube to produce an attachment surface for the transwell insert. the transwell insert was then pushed onto the connecting tube until the tapered internal walls of the insert fastened firmly onto the parafilm ( figure b) . thus, air flowing down the connecting tube was diverted through the lateral ports of the insert. this caused particles to exit the air stream through inertial impaction and deposit onto the cell layer. following this, preformed pei/pei-lpeg sirna nanoparticles at a concentration of nm were delivered using either . , . or . µg of sirna per well. the vacuum pump was run at l/min and allowed to run for s past complete sample nebulisation. the nebuliser was rinsed by nebulising times with µl of pbs between doses with no transwell attached to the lower section. furthermore, the tsi itself was disassembled and rinsed out with sterile deionised water when switching between the pei and pei-lpeg samples. following the nebulisation of all samples, the cells were incubated for h at • c and % co . gapdh sirna was first delivered to validate the system using gene expression prior to the delivery of the anti-il- sirna and the protein expression analysis. nanomaterials , , of in addition to this, the teer value fluctuations over the h of transfection were recorded using an evom voltohmmeter (world precision instruments, stevenage, uk) at various time points for analysis. briefly, a baseline reading was established prior to transfection by equilibrating the apical layer in pbs for min at °c prior to reading. following the addition of the sirna nanoparticle pbs, readings were taken at min and at , , and h and compared to the pbs-only treated controls. calu- cells were seeded in . mm transwell plates at a density of . × cells/well using the previously described methods for calu- culture in transwell plates at liquid-liquid and air-liquid interfaces. cell monolayers were used for the transfection studies once a teer of > Ωcm was established [ ] . a glass tsi was assembled as previously described by grainger et al. [ ] , apart from the absence of solution in the lower chamber. an aerogen ® pro vibrating mesh nebuliser was sealed into place with parafilm at the entrance of the tsi ( figure a (i)). the adapter piece ( figure a (ii)) was removed and parafilm was wrapped around the base of the connecting tube to produce an attachment surface for the transwell insert. the transwell insert was then pushed onto the connecting tube until the tapered internal walls of the insert fastened firmly onto the parafilm ( figure b) . thus, air flowing down the connecting tube was diverted through the lateral ports of the insert. this caused particles to exit the air stream through inertial impaction and deposit onto the cell layer. following this, preformed pei/pei-lpeg sirna nanoparticles at a concentration of nm were delivered using either . , . or . μg of sirna per well. the vacuum pump was run at l/min and allowed to run for s past complete sample nebulisation. the nebuliser was rinsed by nebulising times with μl of pbs between doses with no transwell attached to the lower section. furthermore, the tsi itself was disassembled and rinsed out with sterile deionised water when switching between the pei and pei-lpeg samples. following the nebulisation of all samples, the cells were incubated for h at °c and % co . gapdh sirna was first delivered to validate the system using gene expression prior to the delivery of the anti-il- sirna and the protein expression analysis. following h of incubation, the basal secretions were collected and the apical layer of cells was washed and collected with an equivalent amount of sterile pbs. the gapdh gene knockdown was then assessed using real-time rt-pcr. rna was extracted using the rneasy micro kit (qiagen, uk) following the manufacturer's instructions. cdna for the real-time pcr was synthesised from rna using the high capacity reverse transcription kit (applied bio-systems via bio-sciences, dublin, ireland) according to the manufacturer's instructions. a quantitative pcr was performed in µl tubes using a rotor-gene (corbett research, uk, now qiagen, manchester, uk) thermal cycler with the real-time detection of fluorescence. the pcr was conducted in a volume of µl using rotor-gene sybr green rt-pcr kit (qiagen, manchester, uk). the percentage knockdown of gapdh was then quantified using the comparative quantitation software supplied by corbett research for the rotorgene. in all the experiments, human β-actin was used as a housekeeping gene to normalise the expression. in the case of anti-il- sirna delivery, the protein levels were assessed as previously described using an il- elisa. ) administration of anaesthetic (xylazine mg/kg and ketamine mg/kg) and were returned to their home cage until the anaesthetic took effect. a tail and toe pinch were used to assess the depth of anaesthesia. the anaesthetised rats were positioned supine on a biolite rodent intubation stand (kent scientific, torrington, ct, usa) suspended from the front incisors. the tongue was extended and moved to one side using forceps. a small animal laryngoscope was used to illuminate the vocal cords, epiglottis and opening to the trachea as well as to hold the tongue in place. once the airway of the rat had been visualised, a penn-century ia- c microsprayer ® (penn-century inc, wyndmoor, pa, usa) with a flexible stainless steel delivery tube of . mm ( -gauge) attached was used to access the lungs for sirna nanoparticle or lps delivery. prior to endotracheal intubation, pbs, lps or µg of sirna nanoparticles ( µl) were loaded into the microsprayer. this was achieved by aspirating the entire pre-prepared µl volume into the microsprayer and attaching spacer clips corresponding to µl each attached to the handle of the microsprayer. the dead volume was subsequently expelled by pressing down on the microsprayer handle until only µl remained. once the rat had been successfully intubated, approximately µl of the sample was delivered by pressing down the handle of the microsprayer quickly and steadily to completion and holding for approximately s to ensure full release of the sample. the microsprayer was immediately removed and the rats were administered µl of mg/ml atipamezole hydrochloride (sedastop™) diluted to µl using sterile pbs via i.p. injection to aid recovery and were placed on their sides in a heated incubator until consciousness was regained. the rats were then moved to a room with constant temperature set at • c for h with access to food and water ad libitum. then, h after the initial treatment, the rats underwent the intratracheal administration of either µl of sterile pbs or mg/ml of lps (from e. coli :b ) by the same procedure. the animals were not administered sedastop™ on this occasion and were placed in the incubator for h. after h, the rats were sedated again if necessary and then sacrificed via cervical dislocation for tissue harvesting. once the rats were euthanised, the thoracic cavity was opened. the lungs and heart were excised en bloc. one bronchus was clamped to allow bal collection from one side of the lungs only. ml nanomaterials , , of of saline was added to one side of the lungs through the trachea using a -gauge catheter with the needle removed and a ml syringe. saline was collected and a further ml of fresh saline was added. this procedure was repeated until the lung had been washed with ml of saline. the remaining lobes were collected for rt-pcr in ml of rnalater™ (thermofisher via bio-sciences, dublin, ireland). the collected bal fluid was centrifuged at × g for min at • c. supernatants were collected as bal fluid and stored at − • c. the cell pellets were resuspended in ml of red cell lysis buffer and centrifuged at × g for min at • c. the supernatants were discarded and the cell pellets were resuspended in µl of saline. an amount of µl of cell suspension was mixed with µl of trypan blue and the total cell counts taken using a haemocytometer. the remainder of the cell suspension was spun for min at rpm onto shandon coated microscope slides (fisher scientific, dublin, ireland) using shandon filter cards and shandon cytospin ® . the cells were fixed and stained using speedy-diff cell stain kit (clin-tech ltd., guildford, uk). briefly, the slides were immersed for a few seconds in speedy-diff fixative (coloured methanol) five times then immersed for a few seconds in speedy-diff a (buffered eosin y) at least times followed by blotting and washing in pbs and finally immersed in speedy-diff b (buffered azur/methylene blue) at least times, blotted and washed. the slides were dried and the differential cell counts were obtained using a ceti light microscope (medline scientific, chalgrove oxon, uk) at × magnification under oil immersion. the macrophages and neutrophils were counted in random fields and the total differential cell counts were calculated. a rat demonstration -plex ultra-sensitive kit assay (mesoscale discovery, rockville, md, usa was used to simultaneously detect ifn-γ, il- β, il- , il- , il- , kc/gro/cxcl- and tfn-α cytokines. the assay was set up and run according to the manufacturer's instructions. briefly, µl of provided diluent was added to each well and the plate was sealed and incubated at room temperature with vigorous shaking ( - rpm) for min. an -point serial dilution of rat demonstration -plex calibrator blend ( - pg/ml) was prepared in % bsa in pbs (for bal fluid calibration). the bal fluid samples were mixed with % bsa to reduce protein adherence to the micro-tubes. standards and bal fluid ( µl) were added in duplicate to wells and the plate was sealed and incubated for h at room temperature with vigorous shaking. the plate was washed times with pbs-t followed by the addition of µl of × detection antibody solution. the plate was resealed and vigorously shaken at room temperature for h. the washing times in pbs-t was repeated. an amount of µl of × read buffer t ( × read buffer t diluted in dh o) was added to each well and the plate was immediately analysed on a sector imager. standard curves were produced and the concentrations of analytes for each sample determined using the msd discovery workbench ® software. the fixed lung tissue was embedded in paraffin and the sections were mounted onto slides. the paraffin was removed by immersing slides in histochoice ® clearing agent for at least min. the tissue sections were hydrated by immersing them for a few seconds in % ethanol followed by %, % and % ethanol and finally dh o. after hydration, the sections were stained with haematoxylin for min followed by rinsing in dh o. the sections were differentiated in % acid alcohol to remove excess haematoxylin by immersing for a few seconds in % ethanol, then % ethanol followed by acid alcohol ( % concentrated hydrochloric acid in % ethanol (v/v)). the sections were then immersed in % ethanol followed by rinsing in dh o. the sections were immersed in alkaline ammonia water ( . % potassium bicarbonate and % magnesium sulphate) and rinsed by running under tap water for min. following rinsing, the sections were stained with eosin ( min) nanomaterials , , of and then immersed for a few seconds in dh o. the sections were then dehydrated by immersing for a few seconds in %, then %, % and finally % ethanol followed by a short immersion in histochoice ® clearing agent. coverslips were mounted onto the slides with dpx mounting medium. the lung sections were examined for inflammation by light microscopy. the degree of neutrophil-rich inflammation was scored as − (absent), +/− (very mild), + (mild) or ++ (moderate). scores were then assigned per treatment group. photomicrographs were acquired for each "score" using a nikon eclipse e microscope at ×, × and ×. all the samples were run in triplicate and the experiment was repeated on independent occasions unless otherwise stated. statistical significance was determined for in vitro assays using the graphpad prism software and the one or two-way anova method of statistical analysis using bonferroni multiple comparison tests analysis in all cases. for the in vivo work, significance was calculated using the kruskal-wallis test and dunn's post-hoc test. the data was expressed ± the standard deviation at all times (sd). the results were deemed to be statistically significant (*) where the p values were found to be < . , very significant (**) at p < . and extremely significant (***) at p < . . following purification, the pei-lpeg was analysed via gpc, where it was found that the final product demonstrated a faster elution time than either the pei or lpeg starting materials ( figure s ). this indicated a larger molecular weight co-polymer had been effectively synthesised. confirmation of the successful conjugation was also carried out using c nmr, which highlighted the presence of the carboxylic bonding between the pei and lpeg at ppm ( figure s ). finally, the % grafting was estimated using h nmr and the integration of the pei present at . ppm and the peg present at . ppm ( figure s ). this was approximately % grafted, as was previously the case when the initial in vitro work was reported [ ] . in order to establish an effective inhaled sirna therapy, it is important to examine the effect of nebulisation on the sirna nanoparticle size and to establish that, on reaching their site of action, the sirna nanoparticles were of the desired size for cell endocytosis. the sirna nanoparticles underwent particle sizing before nebulisation. following nebulisation using a vibrating mesh nebuliser (vmn) and passage through the tsi, the sirna nanoparticles were collected from the throat and stage a and b of the tsi and resized. it was found that the vibrating mesh nebulisation of sirna nanoparticles did not result in significant changes to the nanoparticle size in the respirable fraction ( figure ). in the case of the pei sirna nanoparticles, the greatest increases in nanoparticle size and polydispersity were found in stage a, which corresponds to the non-respirable fraction. however, the nanoparticle size was found to be unchanged in samples collected from stage b of the tsi, corresponding to the respirable fraction. similarly, the pei-lpeg sirna nanoparticles collected in the throat and upper chamber were found to have significant increases in size and polydispersity compared to the un-nebulised samples. however, the nanoparticles found in stage b demonstrated no significant changes. on comparing the pei and pei-lpeg sirna nanoparticles, the pegylated particles were significantly larger than the unmodified pei in the lower stage (stage b) of the tsi. however, the polydispersity indices for both did not demonstrate any significant differences. the aerogen pro vibrating mesh nebuliser was used to nebulise the pei and pei-lpeg sirna nanoparticles, and the vmd and %fpf was determined and compared to the nebulisation of pbs using the same device (table ) . on the examination of the vmd and %fpf of each sirna nanoparticle suspension, it was found that there were no significant changes when compared to the pbs controls. the vmd of the droplets containing sirna nanoparticles remained around ~ μm in all cases, which is within the required size range for successful delivery to the deep lungs [ ] . in addition, the %fpf of each nebulised sirna nanoparticle sample remained between % and %. however, on examining the changes in output rate, the pei-lpeg sirna n/p = nanoparticle samples demonstrated a % drop in output efficiency compared to the pbs controls. the surface tension of the pei and pei-lpeg sirna nanoparticle samples at the concentrations tested was measured, as this has been found to impact on the nebulisation of liquids. the pei-lpeg-sirna nanoparticle system had a decreased surface tension, although this was found not to be statistically significant compared to the other groups. table . effect of pei and pei-lpeg sirna nanoparticles on the vibrating mesh nebuliser output rate (ml/min), % fine particle fraction (%fpf) and by volumetric median diameter (dv ( )). surface the aerogen pro vibrating mesh nebuliser was used to nebulise the pei and pei-lpeg sirna nanoparticles, and the vmd and %fpf was determined and compared to the nebulisation of pbs using the same device (table ) . on the examination of the vmd and %fpf of each sirna nanoparticle suspension, it was found that there were no significant changes when compared to the pbs controls. the vmd of the droplets containing sirna nanoparticles remained around~ µm in all cases, which is within the required size range for successful delivery to the deep lungs [ ] . in addition, the %fpf of each nebulised sirna nanoparticle sample remained between % and %. however, on examining the changes in output rate, the pei-lpeg sirna n/p = nanoparticle samples demonstrated a % drop in output efficiency compared to the pbs controls. the surface tension of the pei and pei-lpeg sirna nanoparticle samples at the concentrations tested was measured, as this has been found to impact on the nebulisation of liquids. the pei-lpeg-sirna nanoparticle system had a decreased surface tension, although this was found not to be statistically significant compared to the other groups. table . effect of pei and pei-lpeg sirna nanoparticles on the vibrating mesh nebuliser output rate (ml/min), % fine particle fraction (%fpf) and by volumetric median diameter (dv ( )). to examine the suitability of pei/pei-peg sirna nanoparticles for the delivery of a potentially therapeutic anti-inflammatory sequence, anti-il- sirna was directly delivered to fully polarised calu- monolayers (n/p = ). for a more meaningful assessment of the potential therapeutic effect, the il- expression in calu- cells was investigated at the protein level using an il- -specific elisa. this also allowed for the examination of protein expression in both the apical and basal secretions of the calu- cells grown on transwell™ inserts. on the analysis of the pei anti-il sirna nanoparticle-treated samples, it was found that there were no significant changes in il- secretion in any of the apical samples. however, there were significant changes in the il- secretion in the basal samples ( figure a ) detected for pei anti-il- sirna nanoparticles ( pg/ml ± . pg/ml) in comparison to the pbs samples ( pg/ml ± . pg/ml). the administration of the anti-gapdh sirna nanoparticles or an equivalent concentration of free pei (as controls) did not result in any significant changes in the il- secretion. on the analysis of the pei-lpeg sirna nanoparticle-treated cells, decreases in basal secretions of il- were also observed ( figure b ). however, these were not found to be statistically significant. similar to the pei data, the administration of pei-lpeg anti-gapdh sirna control nanoparticles did not cause any significant changes in the il- expression. finally, the administration of pei-lpeg in solution at concentrations equivalent to n/p = nanoparticles to calu- cells did not result in a significant increase in il- secretion, as was also seen with the equivalent pei treated samples. as a means of investigating the effect that the administered sirna nanoparticles had on the calu- cell monolayer tight junctions, the teer values of the treated cells were taken at regular intervals and compared against pbs-treated cells ( figure c ). compared to the pbs-treated samples, the administration of pei and pei-lpeg sirna nanoparticle solutions did not significantly alter the teer integrity for the first h post-treatment. however, a comparative treatment with pei-lpeg sirna nanoparticles resulted in a greater, but non-significant, decrease ( % decrease at h) in the teer values compared to the pbs ( % decrease at h) or pei sirna nanoparticle ( % decrease at h)-treated samples. interestingly, the greatest drop in teer in the pei sirna nanoparticle-treated samples occurred at min post-treatment, whereas this point was not reached until h post-treatment in the pei-lpeg sirna nanoparticle-treated groups. finally, the teer values of both the pei and pei-lpeg nanoparticles were also found to recover and were significantly higher than the pbs controls at h post treatment. this represents a recovery of tight junction integrity in all the samples and an ability to withstand the stresses involved in sirna nanoparticle delivery. in order to comprehensively validate the tsi system, pei and pei-lpeg sirna nanoparticles at n/p = containing . , . or . µg/well of anti-gapdh sirna were nebulised in five independent experiments. for comparison, . µg/well of sirna was the standard amount used in previous non-nebulised transfections in section . . following the real-time rt-pcr analysis of gapdh inhibition, there was a significant difference in transfection efficiencies between the pei and pei-lpeg-treated samples (figure ) . the pei sirna nanoparticle-treated samples displayed highly variable levels of gapdh knockdown. even at the highest doses, the nebulised pei-mediated gapdh expression was . -fold (± . ) that of the pbs-treated cells. in contrast, the nebulised pei-lpeg sirna nanoparticles demonstrated significantly greater levels of gapdh knockdown versus the pbs-treated controls at higher doses. in . µg/well sirna nanoparticle-treated samples, the gapdh expression was found to be . (± . ) that of pbs-treated cells. this was significantly lower than both the pbs controls and the corresponding pei sirna nanoparticle-treated sample (* p < . ) when analysed via a -way anova. following the initial validation of the anti-il- sirna sequence specificity in standard transfection experiments and the validation of the tsi set-up using anti-gapdh sirna, the il- sirna nanoparticles were administered via the tsi ( figure ). in the case of pei anti-il- sirna nanoparticle-treated cells, protein inhibition was again observed in the basal secretions ( figure a ). using . and . µg sirna doses, the il- expression was reduced by up to % compared to the pbs-treated controls ( pg/ml ± sd vs. pg/ml ± sd, respectively) but were not found to be statistically significant. in contrast, the nebulised doses of pei-lpeg sirna demonstrated il- reduction in both the apical and basal samples ( figure b ). significant decreases in il- secretion of up to % were seen in apical secretions as well as decreases of up to % in basal secretions (non-significant). this demonstrated that only the nebulised pei-lpeg anti-il- sirna particles were capable of eliciting a significant inhibitory effect at the protein level. . -fold (± . ) that of the pbs-treated cells. in contrast, the nebulised pei-lpeg sirna nanoparticles demonstrated significantly greater levels of gapdh knockdown versus the pbstreated controls at higher doses. in . μg/well sirna nanoparticle-treated samples, the gapdh expression was found to be . (± . ) that of pbs-treated cells. this was significantly lower than both the pbs controls and the corresponding pei sirna nanoparticle-treated sample (* p < . ) when analysed via a -way anova. following the initial validation of the anti-il- sirna sequence specificity in standard transfection experiments and the validation of the tsi set-up using anti-gapdh sirna, the il- sirna nanoparticles were administered via the tsi ( figure ). in the case of pei anti-il- sirna nanoparticle-treated cells, protein inhibition was again observed in the basal secretions ( figure a ). using . and . μg sirna doses, the il- expression was reduced by up to % compared to the pbs-treated controls ( pg/ml ± sd vs. pg/ml ± sd, respectively) but were not found to be statistically significant. in contrast, the nebulised doses of pei-lpeg sirna demonstrated il- reduction in both the apical and basal samples ( figure b ). significant decreases in il- secretion of up to % were seen in apical secretions as well as decreases of up to % in basal secretions (non-significant). this demonstrated that only the nebulised pei-lpeg anti-il- sirna particles were capable of eliciting a significant inhibitory effect at the protein level. the intra-tracheal instillation of sirna nanoparticles in a lps-stimulated rat model of acute inflammation was investigated in a pilot study. based on the promising data obtained from in vitro monolayer culture experiments, n/p = was chosen as the n/p ratio, with a sirna dose of μg extrapolated from a previous in-house study [ ] . however, the n/p ratio was reduced due to the intra-tracheal instillation of sirna nanoparticles in a lps-stimulated rat model of acute inflammation was investigated in a pilot study. based on the promising data obtained from in vitro monolayer culture experiments, n/p = was chosen as the n/p ratio, with a sirna dose of µg extrapolated from a previous in-house study [ ] . however, the n/p ratio was reduced due to practical considerations and n/p = was chosen, as it was more comparable with the prior literature [ ] [ ] [ ] . on analysis, it was found that there was a significant increase in the overall bal cell population following lps stimulation, going from . (± . ) × cells/ml to . (± . ) × cell/ml ( figure a ). for groups treated with the pei and pei-lpeg anti-il sirna nanoparticles, no significant decrease in the overall cell number was evident. a similar level of inflammation across all the lps-treated groups was also evident upon histological analysis ( figure s ). using the differential cell staining of bal samples with eosin y and azur/methylene blue, it was in the case of the pei-lpeg sirna nanoparticle-treated groups, both the non-targeting (nt) and anti-cxcl- sirna-treated groups demonstrated -fold decreases in the cxcl- gene expression compared to the pbs-lps samples ( -vs. -fold respectively). while the gene expression was significantly decreased versus the lps-treated controls, there were no significant differences between the targeted and non-targeted sirna-treated animals. this indicated that the effect was not solely related to sirna-mediated protein inhibition. this was unexpected, considering the pei-lpeg polymer has been previously validated in vitro as causing no off-target effects in previous work [ ] as well as through the results in this study. the cxcl- protein expression in the bal fluid of treated rats was then examined as part of the -plex multi-analyte array ( figure b ). the results found that, at the early timepoint of h post lps challenge, only the pei-lpeg nt sirna-treated groups demonstrated significantly higher levels of cxcl- secretion when compared to the pbs-pbs samples. however, there were no significant differences compared to the pbs-lps-treated control animals. the results for each remaining cytokine of the multi-plex array were analysed and the test samples were compared for cytokine expression against the pbs-lps-treated control animals ( figure s ). overall, it was found that the administration of sirna nanoparticles did not lead to statistically relevant changes in cytokine levels compared to the pbs-lps-treated animals. when compared with the pbs-pbs-treated animals, there were similarly few significant changes in cytokine expression. the pei-lpeg nt sirna-treated animals were found to have a significantly higher il- expression compared to the pbs-pbs control. the pei-lpeg nt and anti-cxcl- sirna-treated animals had a higher il- secretion compared to the pbs-pbs-treated group. finally, it was noted that the cxcl- levels were significantly higher compared to several other cytokines in the pbs-pbs-treated animals ( figure s ). on analysis, it was found that there was a significant increase in the overall bal cell population following lps stimulation, going from . (± . ) × cells/ml to . (± . ) × cell/ml ( figure a ). for groups treated with the pei and pei-lpeg anti-il sirna nanoparticles, no significant decrease in the overall cell number was evident. a similar level of inflammation across all the lps-treated groups was also evident upon histological analysis ( figure s ). using the differential cell staining of bal samples with eosin y and azur/methylene blue, it was also possible to identify the separate cell populations in each treatment group ( figure b-h) . overall, macrophages were the predominant cell types identified in bal. the effect of lps stimulation on immune cell infiltration was evident, with the macrophage levels increasing from . (± . ) macrophages per field of view in the pbs-pbs-treated group to . (± . ) in the pbs-lps-treated group. however, for the animals treated with the pei anti-cxcl- sirna nanoparticles, there was a significant (p < . ) decrease in macrophage infiltration compared to those treated with the pei nt sirna nanoparticles ( . ± . vs. . ± . macrophages/field, respectively). for the groups treated with pei-lpeg sirna nanoparticles, no significant differences in macrophage infiltration were seen between the nt and anti-cxcl- groups ( ± cells and ± cells/field, respectively) as well as when compared to the pbs-lps-treated control group. the successful development of any pulmonary sirna nanoparticle therapy is dependent on both the efficient delivery of these nanoparticles through a suitable device and the bioactivity of the delivered sirna nanoparticles. the effect of the vibrating mesh nebulisation on the sirna nanoparticle size was first examined. an analysis of the effect of nebulisation on the sirna nanoparticle size determined that the most significant changes in nanoparticle size occurred in the fractions of the tsi corresponding to the non-respirable fractions. in contrast, the nanoparticles collected in the lower (respirable) fraction of the tsi demonstrated no significant changes. while the nanoparticle size and polydispersity were high, this is most likely due to the use of pbs as a suspension buffer to facilitate nebulisation. similarly, the use of pbs in the subsequent recovery of the particles from the tsi may have affected the size of the particles. for instance, the high ionic strength of pbs can cause electrostatically formed nanoparticles to swell considerably [ ] . however, as the fractions were treated in the same manner, the relative changes in size and pdi are thus comparable. therefore, it was encouraging to note that the pei-lpeg particles maintained their integrity in an undiluted and physiologically relevant buffer. furthermore, the pei-lpeg sirna nanoparticles demonstrated higher particle sizes than the unmodified pei sirna nanoparticles. this was expected since grafting the peg molecules to pei can result in a loss of charge density [ ] and thus become more sensitive to the effects of pbs dilution. overall, these results indicated the relatively low impact of vibrating mesh nebulisers on the integrity of the sirna nanoparticles for pulmonary delivery. the vibrating mesh nebuliser (vmn) performance was similarly unaffected when used for the nebulisation of the pei/pei-lpeg sirna nanoparticles. the emitted droplets were all approximately µm, which is in the desired size range for delivery to the lower airways [ ] . however, there was a slight decrease in the output efficiency apparent when the vmn were used to nebulise the pei-lpeg sirna samples. this can be explained by the ability of peg to decrease the surface tension in a solution, which can cause a reduction in the output rate. this has previously been related to the fact that samples with a low surface tension more readily wet the aperture surface and therefore decrease the efficiency of droplet formation, a phenomenon known as the "loxy effect" [ , ] . prior to assessing the nebulised sirna delivery and efficacy, the il- knockdown using the nanoparticles was first assessed by direct delivery onto calu- monolayers to establish the sequence specificity as well as investigating any underlying inflammatory effects. when examined for changes in il- protein levels, it was found that both the pei and pei-lpeg anti-il sirna nanoparticles were capable of eliciting decreases in il- expression, but only pei anti-il sirna significantly so and only on the basal side. previous work with polarised epithelial cells has found that cells preferentially secrete apically [ , ] . therefore, it is likely that apical secretion is prioritised and kept at homeostatic levels as much as possible with basal secretion bearing the decrease in il- production following anti-il- sirna treatment. furthermore, while cationic polymers can stimulate il- secretion in their own right [ ] , the persistent presence of the pei/pei-lpeg free polymer in solution on the apical cell surface did not trigger any significant increases versus the pbs-treated wells. recent studies have also highlighted that the delivery of exogenous sirna can mediate inflammation [ , ] . however, using anti-gapdh as a non-relevant negative control, it was also found that there were no non-specific changes in il- production using either polymer. an analysis of fluctuations in the teer values following direct anti-il sirna nanoparticle administration revealed that treatment with both pei and pei-lpeg sirna nanoparticles resulted in slight, but non-significant, decreases in teer following transfection, with significant increases in teer values h post-transfection. this slightly greater teer decrease seen for the pegylated constructs has also been previously reported using pegylated chitosan. in that study, cassettari et al. postulated that the greater decrease in teer values mediated by pegylated constructs was due to the higher masses used for pegylated polymers to reach equivalent concentrations of cationic polymer [ ] . this is also in keeping with findings regarding a temporary disruption recovery in epithelial cell tight junctions following transfection [ ] . once the utility of the vibrating mesh nebuliser and sequence specificity of the anti-il- sirna nanoparticles were established, it was then possible to investigate the impact of nebulisation on the bioactivity of the sirna nanoparticles. previously, our lab had investigated post-nebulisation sirna transfection using simple non-polarized calu- cell cultures [ ] . in this study, to better recapitulate the clinical environment, a twin-stage impinger deposition instrument was coupled with a polarised cell monolayer grown on a transwell insert (figure ) for transfection studies. in the initial gapdh validation of the system, experiments found that there were significant differences in sirna nanoparticle behaviour going from non-nebulised to nebulised transfection. the pei sirna nanoparticle transfection efficiency became highly variable following nebulisation, whereas the pei-lpeg sirna nanoparticles were capable of consistent and significant decreases in gapdh gene expression. while dispersing sirna nanoparticles through a twin-stage impinger has been previously recorded [ , ] , to date this has not been applied to monitor the post-nebulisation transfection efficiency. the positive role of pegylation in nebulisation and mucus trafficking was further demonstrated in tsi anti-il- sirna transfections of polarised calu- monolayers. the pei-lpeg sirna-transfected cells displayed decreased il- cytokine levels both apically and basally, with significant decreases in the apical secretion. in relation to the strong post-nebulisation efficiency of the pei-lpeg samples, this is most likely as pegylation is known to enhance colloidal stability as well as enhance the mucus penetration and trafficking of delivered particles [ ] [ ] [ ] . in addition to an enhanced ability to traverse the mucus barrier, it has also been found that the aerosol administration of drugs can result in a faster trafficking time compared to solution administration. specifically, the lower the apical fluid volume, the faster the transport [ , ] . this would explain the more obvious apical and basal il- inhibition in nebulised sirna nanoparticle-treated samples compared to the un-nebulised samples, with the mucus trafficking abilities of pegylation becoming more quickly apparent. following these positive results, the sirna nanoparticles were tested in a rat model of acute lps-mediated inflammation. the effects of this were then examined at the genetic, protein and cellular levels. at a genetic level, the use of lps to drive cxcl- up-regulation was validated with significant increases observed in gene expression. furthermore, pei anti-cxcl- sirna nanoparticles were also found to demonstrate reduced gene expression compared to their non-targeting controls although this was not found to be significant due to the variability in the nt-sirna-treated animals. interestingly, it was seen that there was some disparity in the non-targeting sirna treatment groups. the cxcl- gene expression in pei nt-sirna-lps-treated rats was found to be strongly up-regulated, while the pei-lpeg nt-sirna-treated animals demonstrated cxcl- upregulation to a much lower degree. considering that the same non-targeting sequence was used in both test groups, it is unlikely that this was nucleic acid sequence dependent. the in vitro work described here using non-targeted sirna also demonstrated that neither pei nor pei-lpeg elicited any significant changes in the il- expression. however, it is also known that pegylation can modulate the inflammatory effect of nanoparticles. previous work has shown that pei caused higher levels of complement activation, whereas conjugation with high molecular weight peg reduced the inflammatory reactions in vivo [ ] [ ] [ ] . however, it is possible that the lps is interacting with the pei-lpeg to some degree. previous work has demonstrated that peg chains with cationic moieties can neutralise lps [ ] . combined with the increased residency time in the lungs of high molecular weight pegylated compounds [ ] , this could explain why the diminished effect of lps at a genetic level may be more pronounced in the pei-lpeg-treated animals. the multi-parameter cytokine screen revealed that there were few significant changes in protein expression in treated groups compared to pbs-pbs sham-treated animals. this was likely due to the very short time between lps challenge and euthanasia of the animals ( h). previous work has demonstrated that it can take between - h for cytokine levels to peak in pulmonary instilled animals [ , ] . this was the case for cxcl- expression in all animals, with the exception of the pei-lpeg nt sirna-treated animals. these rats demonstrated significantly higher levels of cxcl- secretion when compared to the pbs-pbs-treated animals. however, the cxcl- levels in all the experimental groups were not significantly different from the pbs-lps-treated animals. however, it was also noted that there were some very high levels of expression in two out of four animals treated with pei-lpeg sirna. while it was not possible to say definitively what is causing this spike in cxcl- secretion in these animals, it is possible that animals may be reacting to the peg moieties. there is a growing body of literature (reviewed in [ ] ) highlighting the risks of pegylation in stimulating the immune system. however, the complexities of this are far from certain, with previous studies demonstrating immune responses are highly dependent on peg chain length and grafting density. specifically, higher levels of peg-grafting resulted in less pro-inflammatory effects without depleting macrophages [ ] . thus, care must be taken in the future in developing low intensity dosing regimens and delivery routes such as nebulisation that minimise this outcome. finally, intratracheally delivered pbs-pbs negative controls were also found to elicit~ pg/ml of cxcl- secretion, which is roughly equivalent to levels recorded in asthma patients ( pg/ml) [ ] . this would indicate that the procedure itself may be immune-stimulatory. when the ifn-γ levels were examined, it was found that the administration of lps resulted in significant increases in secretion. interestingly, similar significant increases in ifn-γ protein expression in the pei or pei-lpeg-treated groups were not observed following lps administration, regardless of the sirna type. this was in line with studies which have found that pei has a lower stimulatory effect on ifn-γ expression than other polymers in mice [ , ] . in addition to cytokine content, the cellular composition in the extracted bal fluid was investigated. here, it was found that the administration of lps resulted in a rapid and significant influx of a variety of immune cells. from the total cell population counts of the extracted bal fluid, there were no significant differences between all the lps-treated sample groups. however, differential staining for macrophages revealed that the delivery of anti-cxcl- sirna could elicit a significant reduction using pei nanoparticles. this further validates the in vitro evidence from this study demonstrating the higher rate of transfection with pei when directly administered to cells. this may also be aided by the fact that pei has also been documented as depleting bal macrophages when administered to the lungs [ ] (although no evidence of this was observed in nt-sirna-treated rats). given the large influx of innate immune cells, an important consideration for future sirna-based inflammatory therapies in the lungs would be to enable targeting of both the epithelial cells and macrophages. this would allow for a more complete inhibition of the inflammatory response. this is currently being investigated in our own lab and elsewhere [ ] [ ] [ ] and must be partnered with advanced d cell culture models for enhanced in vitro assessment [ ] . considering the in vitro/in vivo disparity in efficacy of pei-lpeg nanoparticles in significantly decreasing il- /cxcl- at the genetic level and indications of high levels of cytokine secretion in vivo, it is important to examine the differences between the nebulised and intratracheal delivery of sirna nanoparticles. specifically, in terms of invasiveness and direct delivery, intratracheal delivery was deemed necessary due to the high levels of sirna that would be required for in vivo nebulisation (a previous study of nebulised mrna at . mg/ml [ ] ) combined with the technical challenges associated with nebulised delivery to small animals. as a result of this, the n/p ratios that were optimised for nebulisation and had demonstrated efficient knockdown were now delivered directly to the lungs in a much more concentrated fashion. this concentrated dose would further exacerbate any underlying inflammation associated with sirna nanoparticle delivery. this is in agreement with a recent study by ng et al. which reported no differences between the bolus and intra-tracheal delivery of sirna in mice [ ] . therefore, the benefits of pegylation observed in the study were very much dependent on their means of delivery and may have been obscured by intra-tracheal/bolus administration in vivo. there is growing evidence now demonstrating delivery-related inflammatory responses to the bolus delivery of sirna [ , ] (indications of which were present in bal fluid). complement activation related pseudo-allergy (carpa) has triggered a move towards lower density/more controlled delivery methods such as i.v. perfusion for continued clinical development [ ] . similarly, this study has demonstrated that nebulisation represents an analogous low-density, aerosol approach to achieving high levels of transfection efficiency while avoiding inflammatory events associated with bolus delivery. future work for pulmonary delivery of sirna (and all oligonucleotide therapeutics) must now focus on not reverting to bolus delivery methods when progressing to pre-clinical studies. the continued development and refinement of existing pre-clinical models of nebulisation is a priority in this case. in this study, convergent device-nanoparticle development was addressed through the in vitro testing of knockdown efficiency of nebulised sirna nanoparticles in successively more complex cell assays. in parallel, the effect of sirna nanoparticle cargo on the nebuliser output and performance was also assessed and vice versa. finally, these sirna nanoparticles were progressed to in vivo testing in an lps-induced rat model of acute pulmonary inflammation. through these experiments, it was determined that sirna nanoparticles are capable of being efficiently nebulised through a vibrating mesh nebuliser without any impact on the integrity of the nanoparticles or the device performance. twin-stage impinger cell studies highlighted the significantly enhanced ability of pei-peg sirna nanoparticles to be nebulised, traverse the mucus lining of airway epithelial cells and reduce gene and protein expression compared to pei sirna nanoparticles. to support in vivo testing, a rat model of acute pulmonary inflammation was established. the cxcl- gene expression could be reduced using some of the sirna nanoparticle treatments to a level comparable to negative (healthy) controls. furthermore, in the case of pei anti-cxcl- sirna nanoparticle-treated animals, significant reductions in macrophage infiltration were also observed. however, there was significant variability and inconsistency in the effects seen for the treatment groups on cxcl- protein expression. in this study we have noted initial evidence of a relationship between the efficacy of the pegylated pei and the means of delivery. specifically, issues were identified when switching to direct intubation and bolus delivery instead of nebulisation, as was used in the in vitro studies. the genetic knockdown elicited by some of the treatments may well be realised at a protein level with further refinement in dosing, dose timing and the application of a less invasive means of delivery in future studies. supplementary materials: the following are available online at http://www.mdpi.com/ - / / / /s , figure s : gpc size and purity analysis of synthesized pei-lpeg overlaid with its respective starting materials., figure s : pei-lpeg carboxylic bonding presence was shown in c nmr at ppm, figure s : h nmr of synthesized pei-lpeg polymer with pei present at . ppm and peg present at . ppm, figure s figure s : bal cytokine expression levels in pbs-pbs treated rats (minimum n = ±sd, kruskal-wallis test and dunn's post-hoc test, * p < . ), figure s : pulmonary histopathology semi quantitative scoring of neutrophil-rich inflammation. haemotoxylin and eosin stained lung sections from top pbs, pbs-lps and sirna nanoparticle treated rats were scored based on the degree of neutrophil-rich inflammation observed (− absent, + mild, ++ moderate and +++ highly inflamed). images were acquired at ×, × and × magnification with arrows indicating evidence of inflammation and of blood and protein in the alveoli and loss of the alveolar lining at higher levels of severity. therapeutic oligonucleotides: state of the art co-delivery of free vancomycin and transcription factor decoy-nanostructured lipid carriers can enhance inhibition of methicillin resistant staphylococcus aureus (mrsa) rna-based therapeutics: from antisense oligonucleotides to mirnas the onpattro story and the clinical translation of nanomedicines containing nucleic acid-based drugs technologies for controlled, local delivery of sirna delivering drugs to the lungs: the history of repurposing in the treatment of respiratory diseases smart' non-viral delivery systems for targeted delivery of rnai to the lungs rna interference strategies as therapy for respiratory viral infections covid- : what has been learned and to be learned about the novel coronavirus disease covid- : consider cytokine storm syndromes and immunosuppression clinical features of patients infected with novel coronavirus in pathophysiological roles of interleukin- /cxcl in pulmonary diseases anti-il- ) for patients with covid- performance characteristics of conventional and prototype humidifiers and nebulizers effect of nebulizer type and antibiotic concentration on device performance performance of the vibrating membrane aerosol generation device: aeroneb micropump nebulizer effective nebulization of interferon-γ using a novel vibrating mesh precise targeting of mirna sites restores cftr activity in cf bronchial epithelial cells nebulised lipid-polymer hybrid nanoparticles for the delivery of a therapeutic anti-inflammatory microrna to bronchial epithelial cells 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vitro in vitro and in vivo complement activation and related anaphylactic effects associated with polyethylenimine and polyethylenimine-graft-poly(ethylene glycol) block copolymers pegylated polyplex with optimized peg shielding enhances gene introduction in lungs by minimizing inflammatory responses fate of pegylated antibody fragments following delivery to the lungs: influence of delivery site, peg size and lung inflammation design and facile solid-phase synthesis of peptide-based lps-inhibitors containing peg-like functionalities pulmonary administration of pegylated polylysine dendrimers: absorption from the lung versus retention within the lung is highly size-dependent local inflammation alters the lung disposition of a drug loaded pegylated liposome after pulmonary dosing to rats the importance of poly(ethylene glycol) alternatives for overcoming peg immunogenicity in drug delivery and bioconjugation inflammatory responses to pulmonary application of pei-based sirna nanocarriers in mice lung defense through il- carries a cost of chronic lung remodeling and impaired function evaluation of proinflammatory cytokine production induced by linear and branched polyethylenimine/plasmid dna complexes in mice influence of the mannose receptor in host immune responses the mannose receptor: linking homeostasis and immunity through sugar recognition targeted liposomal drug delivery to monocytes and macrophages the development of a tissue-engineered tracheobronchial epithelial model using a bilayered collagen-hyaluronate scaffold complement activation related hypersensitivity reactions to pegylated liposomal doxorubicin-experimental and clinical evidence, mechanisms and approaches to inhibition. in handbook of immunological properties of engineered nanomaterials this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license we thank lorraine nolan and awadh yadav, school of pharmacy, rcsi for assistance in preliminary experiments and drafting of animal ethics application. sincere thanks to anita umerska and lidja tajber of trinity college dublin for kind use of the ring tensiometer.conflicts of interest: r.m. is an employee of aerogen. key: cord- -tox tuzz authors: capellini, francesca maria; vencia, walter; amadori, massimo; mignone, giulia; parisi, erica; masiello, lucia; vivaldi, barbara; ferrari, angelo; razzuoli, elisabetta title: characterization of mdck cells and evaluation of their ability to respond to infectious and non-infectious stressors date: - - journal: cytotechnology doi: . /s - - -z sha: doc_id: cord_uid: tox tuzz the madin-darby canine kidney (mdck) cell line is widely used as epithelial cell model in studies ranging from viral infection to environmental pollutants, and vaccines production. however, little is known about basal expression of genes involved in innate immunity, and the ability to respond to infectious and non-infectious stressors. therefore, the aims of our study were to evaluate the basal level of expression of pivotal genes in the innate immune response and cell cycle regulation, as well as to evaluate the ability of this cell line to respond to infectious or non-infectious stressors. as surmised in our working hypothesis, we demonstrated the constitutive expression of genes involved in the innate immune response and cell defense alike, including tlrs, interleukins, myd , p /nf-kb and p . moreover, we described the ability of this cell line to respond to lps and cadmium (cd +) in terms of gene expression and cytokine release. these data confirm the possibility of using this cell line as a model in studies of host/pathogen interaction and response to non-infectious stressors. the madin-darby canine kidney (mdck) cell line is widely used as epithelial cell model. indeed, its properties of apico-basolateral polarity, cell junctions and in vitro growth rate are well known (dukes et al. ) . mdck cells are also used in studies of viral infections, and more recently in vaccine production (gregersen et al. ). however, little is known about basal expression of genes involved in innate immunity, dna repair, cell cycle regulation, as well as in the secretion of cytokines, and the response to infectious and non-infectious stressors. moreover, no data are available about the effect of cell passages and aging of confluent monolayers. these gaps may hinder a correct use of these cells for vaccine production and studies on host/pathogen interaction. owing to the above, the aims of our study were: -to evaluate the basal level of expression of pivotal genes in the innate immune response and cell cycle regulation. -to evaluate the ability of this cell line to respond to infectious or non-infectious stressors. in our study we decided to consider cell monolayer aging, a concept differing from cellular senescence; indeed, whereas the phenomenon known as replicative senescence is due to the ability of cells obtained from primary cultures to reduce their number of divisions until a complete stop within few weeks of culture (ogrodnik et al. ) , an immortalized cell line can indefinitely replicate in the presence of satisfactory environmental conditions (e.g. nutrients, temperature). however, after monolayer confluence the concentration of damage-associated molecular patterns (damps, such as self dna) in supernatants can increase and cause changes of gene expression; indeed sensing such dnas leads to activation of interferon regulatory factor (irf ) and nuclear factor kappa b (nf-jb), respectively (sok et al. ) . in this study, we chose cadmium (cd ?) as a noninfectious stressor. this heavy metal is widely known for its toxic and carcinogenic effects. cd ? toxicity has now been shown in nearly every organ and tissue of the body and the kidney is a critical organ for cd ? accumulation and toxicity. indeed, exposure to cd ? has been implicated in renal dysfunction (bernard ) . many studies demonstrated the ability of cd ? to modulate the activity of cellular enzymes, to suppress mitochondrial functions, to initiate oxidative stress, and to disrupt calcium homeostasis (tchounwou et al. ; rusanov et al. ) . moreover, in our previous study we demonstrated the ability of this heavy metal to modulate innate immune responses in ipec-j cells as a function of both time and concentration (razzuoli et al. ) . furthermore, cd ? exposure has been reported to alter a variety of immune cell functions in terms of both protein expression patterns and immune cell functions (maret and moulis ) . the major mechanism behind cd ? toxicity is oxidative stress that induces damages in organs such as kidney, liver, lung, brain and testis. this is due to the inflammatory response caused by cd-driven tissue damage, including leukocyte activation and recruitment. in particular, cd ? induces expression of il- b, tnf-a, il- , il- , which underlies the amplification of the inflammatory response (milnerowicz et al. ) . moreover, low levels of cd ? may be present in environment contaminants like dust (tan et al. ) , plastic and glass (turner ) . to mimic infectious stressors, we chose lipopolysaccharide (lps), present in the external wall of gram-negative bacteria and largely responsible for their toxicity. lps is recognized by tlr -coreceptors md and cd , which recognize the lipid a core of lps. tlr transduces the signal via myd and trif adapters to activate nf-jb-and/or irf mediated transcription of genes encoding pivotal molecules of the immune system, including cytokines and chemokines, which mimics major inflammatory response models (poltorak et al. ; rathinam et al. ) . moreover, lps is of concern since it is a common contaminant of cell cultures. it is an amphipathic molecule which adheres to hydrophobic materials like plastics and glassware. therefore, crosscontamination between experiments might occur if lps molecules were not effectively removed. if lps is introduced into laboratory equipment, its persistence can determine a memory effect provoking inflammatory responses or endotoxin tolerance. the control of lps contamination is difficult due to its ubiquity in nature, toxicity and stability in solutions (stable depending on ph, ions and surfactants), and even sterilization at temperatures [ °c (gorbet and sefton ) . this can cause contamination or crosscontamination between experiments if lps is not effectively removed from the system (schwarz et al. ) . as for the relevance of our study, mdck cells are widely used in many laboratories for viral isolation, vaccine development and production; furthermore, as they derive from renal tubule cells, they can be used in vitro to evaluate host-pathogen and host-chemical interaction. in this study, for the first time the basal expression of genes involved both in the inflammatory response and in the cell cycle was evaluated; furthermore, we investigated the effects of passage level and aging on the expression of the same genes. these parameters can affect the expression of cytokines, as shown, e.g. in ipec-j cells (razzuoli et al. ). furthermore, we evaluated the ability of mdck cells to interact with stressors of various nature. cell culture mdck cells (madin-darby canine kidney, izsler biobank oie code bs cl ) were grown in minimum essential medium (mem, carlo erba reagents s.r.l., milano, cat fa wl ) enriched with % (v/ v) fetal calf serum (fcs, gibco tm , thermofisher scientific, milano, cat - ) and a mixture of antibiotics (penicillin and streptomycin, % v/v, carlo erba reagents s.r.l., milano, cat fa wl ). cells were seeded into -well tissue culture plates ( ml per well, . cells/ml) and incubated at °c in % co until confluence ( - h). cells were tested at the rd, th and th passages; then, cells at confluence ( th passage) were further incubated for h to evaluate the effects of aging. each experiment was repeated ten times. monolayer cells were treated with lm cadmium (carlo erba reagents srl, milano, cat ). the choice of the exposure level was based on a previous study that defined low and moderate cadmium exposure levels (luevano and damodaran ) . in our study, we treated mdck with lm cd ?, which was equivalent to . % of the -h dl ( mg/kg) value after oral administration in dogs (venugopal and luckey ) . cd ? was dissolved in mem, and cells were stimulated for or h at °c in % co . we set up biological replicates; cells treated with medium only were used as negative control ( biological replicates, as well). each experiment was performed trice. we tested the ability of mdck to adsorb cd ? at different times after exposure: , and h. cells were treated with lm cadmium ( lg/ cells); then, intracellular concentration of cd ? at different time of exposure was checked using a graphite furnace atomic absorption spectroscopy (model zeenit p, analytik-jena, germany) as previously described (razzuoli et al. ) . each cd ? concentration was checked in quintuplicate and intracellular cd ? was expressed as lg cd ?/ cells. monolayer cells were treated with lg/ml lps from e. coli o :b (sigma-aldrich, inc., milano, cat l ). lps was dissolved in mem, and cells were stimulated for or h at °c in % co . we set up biological replicates; cells treated with medium only were used as negative control ( repeats). each experiment was performed thrice. in this study, we evaluated the expression of the following genes in untreated cells: tnf-a, inos, stat- a, ifn-c, il -b, il- , il- , il- , il- , il- , il- , il- , il- , il- , il- , il- , il- , il- , myd , nf-kb/p , tlr , tlr , tlr , tlr , tlr , tlr , tlr , tlr , tlr , md , cd , cd , cxcr , rad , p , hprt , pten, erbb , b m, gapdh, actb. to this purpose, we used primer sets described in previous studies (table ) , as well as others (table ) designed in our laboratory using the blast function of pubmed (https://www. ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?link_ loc=blasthome). the expression of the above genes was assessed by rt-qpcr total rna was extracted from mdck cells using rneasy mini kit (qiagen, milano, cat no ) using dnase digestion protocol, rna concentration was evaluated by uv absorbance (biophotometer, eppendorf, milan) and ng of rna ( ng/ll) were added to the reaction mix for cdna synthesis as previously described (razzuoli et al. ) . eva green rt-qpcr amplification were performed a cfx tm real-time system (bio-rad, milano) as previously described (razzuoli et al. ). to evaluate the basal level of expression, pcrnegative samples were given a ct fictitious value, whereas the positive ones showed ct values \ . after lps or cd ? treatment we evaluated the expression of the following genes: il- , il- , il- b, tlr , tlr , tlr , tlr , inos, cd , myd , nf-kb/p , tlr , md , il- . to normalize the results, we tested b m, b-act, hprt and gapdh as possible housekeeping genes by normfinder algorithm (peletto et al. ). in each sample, the relative expression of the selected genes was calculated using the formula -ddct , where ct is short for cycle of threshold and dct = ct (target gene) -ct (housekeeping). dct values are the mean of three test reverse gtccaatttcattaaggccaag replicates ± standard deviation and ddct = dct (different passages, th and th) -dct (control, rd passage). canine il- was measured in mdck supernatants by an elisa kit (r&d system, cat no ca ) according to the manufacturer's directions. il- was revealed by adding ll/well of ortho-phenylenediamine (opd) and . % h o as substrate. the reaction was blocked after min of incubation by adding ll/well of n h so ; then, plates were read spectrophotometrically at nm. cytokine concentration was calculated from a standard curve, created using eight, twofold dilutions of canine recombinant il- . data were analyzed by software prism , (graph pad software); the loq (limits of quantification) corresponded to pg/ml. data were expressed as average ± standard deviation. data sets were submitted to a kolmogorov-smirnov test to check normality of distributions; significant differences within normal distributions were checked by one-way anova followed by a dunnett's test for gene expression, cytokine release and cd ? uptake. a student's t test was applied to investigate the effect of aging on gene expression. the significance threshold was set at p \ . (prism , graphpad software). first, gapdh, actb, hprt and b m were evaluated as possible housekeeping genes (brinkhof et al. table ) . our results showed modulation of gene expression at the th and th passages (fig. ). in particular, at passage we observed an increase of tlr and a decrease of b m gene expression. regarding the th passage, il- , il- , il- , il- , tlr , tlr , tlr , tlr , p , cxcr , md and hprt gene expression was up-regulated. on the contrary, il- , tlr and tlr were down-regulated. after h aged cells, at passage , showed decrease of nf-kb/ p and b-act gene expression. myd , il- and cxcr were not modulated (data not shown) while other genes under study were up-regulated ( fig. ; p \ . ). our data showed a significant increase of intracellular cd ? after h of exposure with respect to the levels at h. in particular, we observed . %, . % and . % cd ? absorption after , and h of exposure, respectively, using lm cd ? ( lg of cd ?/ cells; fig. ). lm cd ? after h of exposure caused up-regulation of il- b, il- , il- , inos, tlr , tlr , tlr and tlr gene expression. on the contrary, cd was down-regulated (fig. ) . after h of exposure we observed up-regulation of il- , nf-kb/p and myd ; at the same time, inos, il- b, md , tlr and tlr were down-regulated. other genes under study were not significantly modulated. regarding il- release, we observed a significant increase of this cytokine in cell supernatants after h ( ± . pg/ml) and h ( ± . pg/ml) of cd ? exposure with respect to untreated cells (\ pg/ml). treatment with lg/ml lps for h caused upregulation of myd gene expression; at the same (fig. ) . after of treatment no significant modulation of gene expression was observed (fig. ) . concerning il- release, all samples of the lps study tested negative (il- \ pg/ml; data not shown). madin-darby canine kidney (mdck) is a continuous cell line of distal tubules of canine kidney widely used for different purposes, such as isolation of influenza viruses, production of flu vaccines, pathogenicity studies of bacterial strains, cytotoxicity tests. although several studies showed satisfactory results in mdck cells for these purposes, none of them described their characteristics in terms of both gene expression and involvement in the innate immune response. therefore, our study focused on the basal levels of protein release and gene expression in this cell line under physiological conditions and after exposure to diverse stressors. accordingly, we developed a rt-qpcr to evaluate the expression of a selected group of genes coding for molecules of different typology and function. our results demonstrated the expression of all the genes under study with the exception of il- , il- , il- , il- , il- and ifng; this is in agreement with other studies that reported no expression of these genes in epithelial cells (bianchi ; groeger and meyle ) . we also investigated the basal expression level of tlrs, a family of pattern recognition receptors (prrs), expressed by many cell types, in order to recognize microorganisms (e.g. bacteria, viruses and fungi) and to induce the innate immune response (velloso et al. ) . the expression in mdck of these molecules is in line with the routine use of this cell line: evaluation of the virulence of some bacterial strains, including bacterial penetration, adhesion and cytotoxicity (chou et al. ; lin et al. ) . the expression in all samples of p , one of the major members of the nf-kb protein family, along with myd , il- b, il- , il- and il- , implies the ability of this cell line to mount an inflammatory response. this assumption is confirmed by our results. in particular, cd ? exposure caused an inflammatory response characterized by upregulation of genes expression and il- release; this was associated to cd ? uptake, in agreement with previous studies conducted on enterocytes (razzuoli et al. ) . mdck cells showed the ability to absorb low levels of cd ?, causing an innate immune response, although this cell line is more resistant to cd ? exposure, compared with other cell lines like llc-pk (bonham et al. ) . also, a previous study showed that cd ? treatments at concentrations (bonham et al. ) . the basal levels of il- and il- , involved in the immune response, suggest the possibility to use this cell line in interaction studies between microbial agents and host krishnamoorthy et al. ) . in our study, the exposure of mdck to cd ? caused up-regulation of inos, tlr , tlr , tlr and tlr gene expression after h of exposure. the genes that are expressed in response to the activation of tlrs encode important proteins at various levels of the innate immune response, including not only cytokines, but also proteins involved in the mechanisms of bacterial killing, such as the inducible nitric oxide synthetase inos (tsutsuki et al. ) . this response could ease the replication of viral agents in mdck cells; in this respect, checconi et al. ( ) ( ) modulation of gene expression *** fig. cd ? treatment. cd ? uptake into mdck cells was measured after , and h of exposure. cells were treated with lm cadmium ( lg/ cells); then, intracellular concentration of cd ? was measured at different times of exposure. data are shown as mean ± sd. asterisk indicate significant differences (p \ . ) with respect to h of exposure, determined by one-way anova. gene expression was measured after treatment of mdck cells with lm cadmium for and h at °c in % co . data are expressed as -ddct where dct = ct (target gene) -ct (housekeeping); values are the mean of three test replicates ± standard deviation and ddct = dct (cd treatment) -dct (untreated control). negative samples were given a ct fictitious value. asterisks indicate significant differences: *p \ . , **p \ . , ***p \ . and ****p \ . demonstrated that the exposure to low levels of cadmium ( - lm) increased influenza virus replication in a dose-dependent manner. moreover, the exposure to lps caused in this study a downregulation of inos, cd , tlr and tlr , which confirms the ability of cells to react to both infectious and non-infectious stressors. treatment with lps caused no significant effects in terms of tnf-a, il- b, and il- gene expression and protein release in agreement with our previous study (razzuoli et al. ). however, these results differ from data obtained on myeloid cells like human macrophages and monocytes, where lps causes early pro-inflammatory responses through tlr /nf-kb signaling (petes et al. ) . the absence of il- b and il- responses to lps in mdck cells could be accounted for by a form of constitutive endotoxin tolerance (lotz et al. ) in these epithelial cells. concerning the cd ? treatment, we found modulation of gene expression and il- protein release in mdck cells as a function of time. in particular, we observed at different times of incubation with lm cd ? the modulation of some important pro-inflammatory cytokines and chemokines (il- , il- , il- b, il- ), and transcription factor genes (nf-kb/p ). similar results were obtained in ipec-j (razzuoli et al. ) and caco- cells, in which hyun et al. 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transcriptomic analysis tlr at the crossroads of nutrients, gut microbiota, and metabolic inflammation toxicology of non-radioactive heavy metals and their salts publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations acknowledgements the authors want to thank mrs. c. citati for the skilful technical assistance; her work is gratefully acknowledged. this study was supported by the italian ministry of health, grant rc c . conflict of interest the authors declare that they have no conflict of interest. key: cord- -o ffxgnn authors: lorts, angela; cornell, timothy t.; shanley, thomas p. title: sepsis date: - - journal: pediatric critical care study guide doi: . / - - - - _ sha: doc_id: cord_uid: o ffxgnn the health care provider faced with the management of a child with septic shock relies on a comprehensive understanding of the numerous disciplines embodied in the practice of pediatric critical care medicine. the child with septic shock may have simultaneous derangements in the function of virtually every system of the body including: cardiovascular, respiratory, immune, renal, coagulation, hepatic, metabolic and neurologic. the degree to which physiologic alterations are manifest in a given patient is variable and influenced by multiple host and non-host factors including: the developmental stage, the presence of co-morbidities, pathogen-related factors, and genetic influences on both the host inflammatory response as well as the response to pharmacologic agents, all combining to have a profound influence on outcome. the clinician must possess a systematic and multifaceted approach to these critically ill patients. the goal of this chapter is to provide a comprehensive description of the epidemiology, biology and pathophysiology (at both the cellular and organ level) of sepsis, as well as outlining the current principles of managing septic shock. it will be apparent that optimal management requires a strong working knowledge of cardiovascular physiology, infectious diseases, multiple organ interactions, immunity, coagulation, pharmacology, and the molecular biology of inflammation. the health care provider faced with the management of a child with septic shock relies on a comprehensive understanding of the numerous disciplines embodied in the practice of pediatric critical care medicine. the child with septic shock may have simultaneous derangements in the function of virtually every system of the body including: cardiovascular, respiratory, immune, renal, coagulation, hepatic, metabolic and neurologic. the degree to which physiologic alterations are manifest in a given patient is variable and infl uenced by multiple host and non-host factors including: the developmental stage, the presence of comorbidities, pathogen-related factors, and genetic infl uences on both the host infl ammatory angela lorts , timothy t. cornell, and thomas p. shanley response as well as the response to pharmacologic agents, all combining to have a profound infl uence on outcome. the clinician must possess a systematic and multifaceted approach to these critically ill patients. the goal of this chapter is to provide a comprehensive description of the epidemiology, biology and pathophysiology (at both the cellular and organ level) of sepsis, as well as outlining the current principles of managing septic shock. it will be apparent that optimal management requires a strong working knowledge of cardiovascular physiology, infectious diseases, multiple organ interactions, immunity, coagulation, pharmacology, and the molecular biology of infl ammation. before reviewing the epidemiology of pediatric sepsis, it must be appreciated that the conclusions of prevalence studies have been obscured in the past by several factors including a lack of a reliable case defi nition. it has only been in the s that consensus defi nitions for sepsis and septic shock were achieved. it was hoped that the development of standard defi nitions would not only enable accurate characterization of the epidemiology of sepsis, but also serve to stratify patients early in the course of sepsis for the purpose of clinical studies aimed at testing novel therapies. the most widely used defi nition of pediatric sepsis/septic shock is based on the american college of chest physicians/society of critical care medicine (accp/sccm) consensus conference, with adaptations for the pediatric population. the following four defi nitions resulted from these discussions: sirs , sepsis , septic shock , and severe sepsis . although there is overlap between some of these terms (particularly between septic shock and severe sepsis), each is intended to defi ne a particular patient population. longstanding clinical observations have identifi ed the presence of tachycardia, tachypnea, hyperthermia and leukocytosis as signs of infection, though these responses may also be present in the absence of any apparent infectious source. as a result, this physiologic response was defi ned as the systemic infl ammatory response syndrome (sirs). sirs defi nes a state of infl ammation/immune activation in a child and is based on the presence of at least two of the four criteria listed in table - . thus, patients with diverse clinical conditions such as sepsis, pancreatitis, burns, or severe trauma can meet criteria for sirs. it has been argued that the sirs defi nition is non-specifi c and that too broad a range of patients are ultimately classifi ed as having sirs. nevertheless, the criteria have been widely used in both prescriptive and interventional studies to enhance the "capture" of all patients at risk for the subsequent development of severe sepsis or septic shock. sirs i s a state of infl ammatory/ immune activation and is based on the presence of at least two of the four following clinical criteria: temperature > °c or < °c, heart rate > th percentile for age, respiratory rate > th percentile for age, or hyperventilation to paco < mm hg. the defi nition attempts to "capture" all patients at risk for the subsequent development of severe sepsis or septic shock. sepsis is defi ned as a sirs response which is secondary to an infection, either documented by microbiology cultures or other clinical evidence of infection. severe sepsis is defi ned by sepsis criteria plus evidence of insuffi cient end organ perfusion (table - ) . finally, septic shock is defi ned by sepsis criteria plus hypotension (two distinct measurements < rd percentile for age) after the administration of at least ml/kg of crystalloid or colloid, in addition to the criteria listed for severe sepsis (table - ) . these criteria have been used extensively for conducting clinical investigations and have proven to be of value despite criticism for lack of both sensitivity and specifi city. the latest consensus conference was convened in to further refi ne the diagnostic criteria and therapeutic recommendations, with specifi c considerations for the pediatric population. published in , the surviving sepsis campaign aims to improve the outcome in sepsis worldwide. the refi nement of pediatric-specifi c criteria for septic shock is also intended to aid future clinical trials and epidemiologic investigations in pediatric sepsis. the few published pediatric-specifi c studies illustrate the importance of sepsis in this age range. proulx analyzed the incidence and outcome of sirs, sepsis, severe sepsis, and septic shock in a single institution. over , admissions were analyzed over a -year period. sirs was present in % of patients, while % had sepsis, % had severe sepsis, and % had septic shock. the overall mortality for this population was % with a majority of deaths occurring in patients with multiple organ dysfunction syndrome (mods). an epidemiologic study using discharge international classifi cation of disease, th revision (icd- ) codes reviewed hospital records from seven large states representing nearly one-quarter of the united states population. while the criteria used for inpatient coding at discharge are not identical to accp/sccm consensus conference criteria, the study estimated an incidence of , cases of severe sepsis in individuals less than years of age ( . cases/ , population). the highest incidence was in neonates ( . cases/ , population), compared to children ages - who had an incidence of . cases/ , population. the overall mortality rate was . % ( , deaths nationally) consistent with the frequent observation that the mortality rate remains lower than comparable adult data. the study estimated an annual national health care cost of $ . billion associated with severe sepsis in children. a follow-up study with the same methodology appeared to show a % increase in the absolute number of cases of severe sepsis from and with the majority of this increase accounted for by severe sepsis in children less than year of age. the mortality rate had decreased to . % during this time period. collectively, these data illustrate that sepsis is a major health problem on the basis of incidence, mortality, and health care costs. there remains a need for further, well-designed epidemiologic studies of pediatric sepsis. future studies will enhance our understanding of not only epidemiology, but also the impact of new diagnostic and therapeutic approaches resulting from improved design of interventional trials specifi c to the pediatric population. sepsis is a systemic disease and can impact the functioning of all organ systems. the most common clinical manifestations of sepsis include: fever or hypothermia, tachypnea, tachycardia, leukocytosis or leukopenia, thrombocytopenia, and change in mental status. one of the earliest signs of infection is fever which results from the pyrogenic effect of cytokines, particularly interleukin (il)- b and tumor necrosis factor (tnf)-a . presentation with hypothermia can also occur, but is more common in infants. one traditional classifi cation of shock states divides this clinical state into three broad categories: hypovolemic, cardiogenic and distributive shock. the shock associated with c hapter • s eps is sepsis is unique in that all three forms are likely to be present. hypovolemic shock results from capillary leak, increased insensible losses, and decreased effective blood volume secondary to venodilation. cardiogenic shock is related to direct myocardial depression, the cause(s) of which remains the focus of investigation. finally, distributive shock is often apparent as brisk capillary refi ll, widened pulse pressure and bounding peripheral pulses and is caused by abnormally decreased systemic vascular resistance from pathologic vasodilation. the particular pattern of these hemodynamic physiologic perturbations manifested by any individual patient can be variable. some children have increased cardiac output with diminished systemic vascular resistance characteristic of distributive shock or the so-called "warm" shock state. in stark contrast to adults, in which this hemodynamic profi le (increased cardiac output/decreased systemic vascular resistance) is most common, children more frequently present with depressed cardiac output and elevated systemic vascular resistance. these patients appear cool with diminished pulses and poor capillary refi ll that is characteristic of the "cold" shock state. while important to recognize that patients may transition from one state to another, the presence of hypotension is often a late and particularly ominous sign that requires prompt intervention as its presence is associated with increased mortality. patients with sepsis often present with alterations in their respiratory system, notably tachypnea that refl ects a compensatory respiratory alkalosis aimed at neutralizing a metabolic acidosis related to hypoperfusion and anaerobic metabolism. chest x-ray fi ndings can reveal a small heart in the presence of hypovolemia with few vascular markings. alternatively, the combination of capillary leak, decreased myocardial function and the result of fl uid resuscitation in some children with sepsis can result in pulmonary edema. rapid progression to acute respiratory failure from ards is not uncommon. all organ systems and ultimately cellular functions are affected by poor perfusion and decreased oxygen delivery related to depressed cardiac and respiratory function. in addition, there may be direct injurious effects of bacterial toxins and circulating cytokines such as triggering of programmed cell death or apoptosis. the neurologic state of a child with sepsis is frequently altered and can range from agitation or irritability to frank obtundation. this depressed mental status can be present even in the absence of meningitis as a manifestation of cerebral hypoperfusion. skin manifestations are not uncommon and can include petechiae and purpura that are ominous signs of disseminated intravascular coagulation (dic) and purpura fulminans secondary to meningococcemia. diffuse erythema secondary to toxic shock syndromes can be present. there is also an increasing appreciation of sepsis-induced microvascular angiopathy contributing to distal skin and organ ischemia. an initial thorough and detailed physical exam provides both important clues to the diagnostic possibilities of pediatric septic shock and the underlying hemodynamic profi le. however, serial exams are imperative to follow pathophysiologic changes and to gauge the impact of therapeutic interventions in reversing the manifestations of shock. data from both clinical and basic science studies have supported the hypothesis that pathogens and/or their products initiate a host immune response that triggers widespread infl ammation causing tissue injury and organ dysfunction. potential initiating pathogens include gram-negative and gram-positive bacteria, viruses, fungi and protozoa. in some cases, overwhelming spread of pathogens (e.g. bacteremia) with release of toxins (e.g. endo-or exotoxins) may directly injure the host resulting in organ dysfunction. higher order organisms have evolved an immune system to eradicate pathogens which has evolved to include two systems: the innate or natural immune system and the acquired or adaptive immune system. the innate immune system is responsible for the highly conserved function of recognizing pathogens and mounting an effector response. it includes a series of molecules located on the cell surface termed pattern-recognition receptors (prr) which are capable of recognizing a broad array of conserved structures on a variety of children with sepsis may have hemodynamic characteristics that transcend traditional classifi cation. they often have elements of hypovolemia, cardiac dysfunction and abnormal vascular tone. in the septic child, the combination of capillary leak, decreased myocardial function and the result of fl uid resuscitation may result in rapid progression to acute respiratory failure. pathogens (so-called pathogen-associated molecular patterns, or pamp's). examples of pamp's include: lipopolysaccharide (lps) on gram-negative bacteria, lipoteichoic acid on gram-positive bacteria, mannans on yeast, double-stranded rna of rna viruses and unmethylated, cpg dna from bacteria. the effector responses that are regulated by the innate immune system (e.g. phagocytes, complement) are activated immediately upon infection and are designed to rapidly inhibit the replication of microorganisms. these cell surface pattern-recognition receptors (prr) are expressed on most antigen presenting cells of the innate immune system and represent diverse families of proteins. one group of prrs, the toll-like receptors (tlr), has been identifi ed as perhaps the most critical pathogen recognition receptor family in the context of sepsis biology. other families of prr include the c-type coll agenous lectins (collectins) that bind to a variety of carbohydrate moieties on cells, bacteria and viruses. most members of this family share structural homology to the complement protein c q and can functionally substitute for c q in activating the complement cascade. another family of prr possesses leucine-rich regions critical for protein-protein interactions that are necessary for immune recognition. examples of these leucine rich receptors include cd , a receptor on the cell surface of macrophages that binds to lps and the macrophage scavenger receptor that binds to bacterial cell walls. unbound circulating prrs exist and include pentraxins, such as c-reactive protein, an acute phase reactant synthesized by the liver and lipopolysaccharide-binding protein (lbp) which binds to lps to optimize its binding to the cd /toll-like receptor cellular complex. another key component of innate immunity is the complement system. the complement system is a complex cascade of proteins that possesses a broad array of anti-pathogen activities including: opsonization (c ), neutrophil chemotaxis (c a), perforating cytotoxicity (c - , mac complex) and the ability to bind to and directly lyse viruses (c ). an in depth discussion of the role of complement in the response to infection is beyond the scope of this chapter, but has been recently summarized. in summary, the host possesses a ubiquitous and diverse set of pathogen recognition receptors which function to protect the host from infectious challenges, but at the expense of triggering powerful effector responses. paramount to effector responses of the innate immune system is a proinfl ammatory action of numerous cytokines and chemokines. these biologically active proteins are critical to the activation and recruitment of cellular components of the adaptive immune system. while necessary for pathogen clearance, this acute, proinfl ammatory immune response must also ultimately subside in order to reestablish homeostasis and avoid cellular and tissue damage. a key pathophysiologic feature of sepsis is that this immune response often appears to become unregulated resulting in an overwhelming proinfl ammatory response and host autodestruction. this characteristic systemic infl ammatory response seen frequently in response to infection can also be observed in association with non-infectious triggers (e.g. trauma, burns, pancreatitis, cardiopulmonary bypass). lps recognition: recent epidemiologic surveys of the causative agents of sepsis have indicated an increase in the incidence of gram-positive organisms such that there is a roughly equivalent prevalence between these and gram-negative organisms. historically, sepsis research has focused on the role of gram-negative bacteria in evoking a pathologic response. the structure of endotoxin shows three domains: an outer polysaccharide hydrophilic chain which determines the o-antigenicity, an acidic core region, and a lipid-rich region. gramnegative organisms possess endotoxins with variable repeats of mono-and heteropolysaccharides with complex side chain structure to provide a basis for distinct antigenicity. the o-region is linked via an acidic core to the lipid a region that is highly conserved and responsible for much of the toxicity attributed to intact lps. a series of seminal observations have determined the molecular mechanisms by which the classic pamp, lps, initiates a proinfl ammatory response. first, a strain of lps-resistant mice, the c h/hej strain, was identifi ed and its resistance was found to be attributed to a single genetic mutation. second, it was shown that the lethal effects of endotoxin could be conferred by transfer of hematopoietic cells. endotoxin tolerant mice could be rendered the cells of the innate immune system contain cell surface molecules termed patternrecognition receptors (prr). these receptors are capable of recognizing a broad array of conserved structures on a variety of pathogens (so-called pathogenassociated molecular patterns, or pamp's). examples of pamp's include: lipopolysaccharide, lipoteichoic acid, viral rna and bacterial dna. toll-like receptors (tlr) are pathogen recognition receptors that have a critical role in sepsis. tlr is active in recognition of lps on gram-negative bacteria whereas tlr is active in the recognition of lipotechoic acid on gram-positive bacteria. a hallmark of sepsis is an immune response that appears to become unregulated resulting in an overwhelming proinfl ammatory response and host autodestruction. this characteristic systemic infl ammatory response is seen frequently in response to infection, but can also be observed in association with non-infectious triggers (e.g. trauma, burns, pancreatitis, cardiopulmonary bypass). c hapter • s eps is lps-sensitive after reconstitution with hematopoietic cells derived from the monocyte/macrophage lineage from an lps-sensitive strain. third, stimulation of monocyte-derived cells with endotoxin resulted in production of several cytokines and chemokines critical to the systemic infl ammatory response. among these, tnf and il- were shown to be critical initiators of the septic response and could in fact mimic the endotoxin response. finally, the elucidation of the lps receptor assisted the identifi cation of those signal transduction pathways by which endotoxin triggers infl ammatory gene expression. lps receptor: membrane bound cd- was shown to be required for lps signaling. however, it lacked a transmembrane extension required for cytoplasmic signaling indicating the presence of additional components of the receptor complex. investigators working with drosophila had identifi ed a gene, toll, which was responsible for dorsoventral polarization in embryonic development. when toll was functionally mutated, it was demonstrated to play a key role in host defense against aspergillus fumigatus . homology between the toll-like receptors and the mammalian il- family of receptors was discovered and provided additional evidence that this family was crucial to the human innate immune response. finally, it was determined that the c h/hej mouse strain which is hyporesponsive to lps possessed a mutation in toll-like receptor (tlr ), providing further evidence that this receptor was necessary for lps signaling. tlr is one of ten mammalian toll-like receptors that have been cloned to date, each being activated by a specifi c set of ligands. since these discoveries, other members of the lps-receptor complex have been elucidated and include both md- and myd . it is also known that circulating lpsbinding protein (lbp) facilitates lps binding to the cell surface receptor complex. together these components are able to "sense" lps at the cell surface and transmit this signal via a series of complex pathways. similarly, the products of gram-positive organisms, notably the cell wall component lipotechoic acid, activate cell activation through the related toll-like receptor (tlr ). after engagement of cell surface receptors (e.g. tlr and tlr ), several important signal transduction pathways are activated that elicit a number of transcriptional factors responsible for infl ammatory gene expression. among these, the nuclear factor-k b (nf-k b ) and the mitogen activated protein kinase (mapk) pathways play a prominent role in regulating the expression of a number of infl ammatory gene products key to propagating the sepsis response. in the case of nf-k b, stimulation of the lps receptor causes phosphorylation of the inhibitor of k b kinases (i k k) which in turn phosphorylates the intracellular inhibitor of nf-k b, i kappa b (i k b). upon phosphorylation, i k b undergoes poly-ubiquination followed by proteosomal degradation. the removal of i k b effectively unmasks a nuclear translocation sequence on nf-k b enabling it to proceed into the nucleus to bind to nf-k b consensus sequences present on the promoter regions of many infl ammatory genes: cytokines including tnf, chemokines including il- , adhesion molecules including e-selectin and others such as inos (see fig. - ). the role of nf-k b in sepsis is supported by studies demonstrating that survivors and non-survivors of sepsis are distinguishable on the basis of nf-k b binding activity in peripheral blood mononuclear cells. in addition, in sepsis-induced ards, increased activation of nf-k b in macrophages obtained by bal is found in ards patients when compared to icu controls. to a similar degree, the mapk signaling pathways are important in mediating the septic response. three mapk pathways exist: p protein kinase, extracellular-regulated protein kinase (erk), and c-jun-terminal kinase (jnk). evidence exists for the role of each of these signaling pathways in sepsis. tnf production by neutrophils and macrophages is dependent on p activation. lps stimulation of monocytes activates jnk with downstream activation of activating protein- (ap- ) and subsequent il- b production. lps induction of tnf is in part dependent on erk pathway activation. together, these two pathways, nf-k b and mapk's, appear to be critical to the propagation of signals from the cell surface to the nucleus where expression of infl ammatory gene products occurs. as such, these pathways remain valid targets for future strategies in modulating the septic response. nuclear factork b (nfk b ) and the mitogen activated protein kinase (mapk) pathways play a prominent role in regulating the expression of a number of infl ammatory gene products key to propagating the sepsis response. while numerous proteins have been shown to play a role in the septic response, a full review of each protein's function is beyond the scope of this study guide. instead, we aim to highlight some known principle mediators in this cascade. evidence that tnf mediates the septic response stems from numerous observations: it is produced by hematopoietic cells, its expression is temporally related to the development of septic shock, recombinant tnf induces experimental septic shock in animals, and passive immunization against tnf attenuates endotoxin-mediated responses. tnf possesses numerous functions in infl ammation such as driving adhesion molecule and chemokine expression to facilitate leukocyte-endothelial cell adhesion; upregulating tissue factor and inhibition of protein c to create a pathologic procoagulant state in the vasculature; and inducing nitric oxide synthase (inos) which mediates pathologic vasodilation. in human studies, levels of tnf have been shown to correlate with mortality, with the development of shock and purpura fulminans and with the development of sepsis-induced ards and shock. the name, il- , is now used to describe the family of proteins including two agonists (il- a and il- b ) and one antagonist, the il- receptor antagonist protein (il- ra). il- b which is secreted, mediates much of the systemic effects attributed to il- release in sepsis. synthesized as a propeptide, il- b requires proteolytic cleavage by the il- converting enzyme (ice) to become bioactive. il- b utilizes the -kda type i receptor which is tnf possesses numerous functions in infl ammation such as inducing adhesion molecules and chemokines that facilitate leukocyte-endothelial cell adhesion and inducing nitric oxide synthase (inos) which mediates pathologic vasodilation. tnf also upregulates tissue factor and inhibits protein c to create a pathologic procoagulant state in the vasculature. clinically, levels of tnf correlate with mortality, the development of shock and purpura fulminans and with the development of sepsis-induced ards and shock. p p iκ κ κ κk associated with a number of adapter proteins (e.g. myd ,tnf receptor-associated factor (traf ) and interleukin- receptor-associated kinase (irak)) to propagate signals through both the nf-k b and ap- pathways. il- b infusion elicits fever, hypotension and leukocytic infi ltration to the lungs. in a manner similar to tnf, il- stimulates monocyte activation and phagocytosis, increases adhesion molecule expression, and increases tissue factor expression while inhibiting thrombomodulin secretion, thus creating a procoagulant state. when detected in the circulation of septic patients, il- levels also correlate with mortality. of note, the il- ra is a circulating inhibitor of il- b that binds to the il- receptor without initiating a signal. the expression of il- ra has been shown to follow peak expression of il- . it is speculated that il- ra is an endogenous regulator of il- effects. however, in clinical trials, il- ra infusion failed to improve mortality in sepsis. furthering our molecular understanding of sepsis-induced organ dysfunction was the identifi cation of the "leukocyte-endothelial cell adhesion cascade". this cascade is characterized by cytokine activation of the selectin family of adhesion molecules (e.g. e-selectin) on the endothelium which initiate a process of neutrophil "rolling" via interaction with sialyated moieties constitutively present on circulating neutrophils. activation of the "rolling" neutrophil results in both increased expression and activation of the integrins which in turn bind to intercellular adhesion molecule (icam)- that is upregulated on the endothelial cell surface by tnf and il- b . this integrin-icam- interaction mediates fi rm adhesion of the neutrophil to the endothelial cell surface. finally, in response to various chemotactic cytokines or chemokines, neutrophils migrate to the site of infl ammation. release of both oxygen-and nitrogen-based radical species and proteases by the neutrophils may ultimately contribute to cellular injury and organ dysfunction. nitric oxide (no) is responsible for endothelium-derived relaxation of blood vessels. three isoforms of nitric oxide synthase are responsible for production of no: type i, a neuronal isoform (nnos); type ii, an inducible isoform (inos) and type iii, a constitutive, endothelial isoform (enos). tnf and il- b are capable of inducing inos and increased levels of circulating stable byproducts of no are found in both septic adults and children who simultaneously display low systemic vascular tone. this supports the hypothesis that no plays a principal role in septic shock via pathologic vasodilation. it has also been suggested that tnf and il- b may be the so-called "myocardial depressant factors" by increasing circulating no through induction of inos ; however, it is not clear that no is the exclusive mediator of these effects. in light of the evidence supporting the role of no in septic shock, clinical trials employing no synthesis inhibitors in septic shock were initiated. though early clinical reports and small studies reported that nos inhibitors could signifi cantly improve blood pressure, this was at the expense of decreasing cardiac output secondary to increased afterload. as a not uncommon hemodynamic profi le in pediatric septic shock is decreased cardiac output and elevated systemic vascular resistance, it is not cleat that nos inhibitors will have a therapeutic role in pediatric (or adult) sepsis in the future. studies employing agents directed against the early mediators of the septic response have been mostly ineffectual. this has led to the hypothesis that additional molecules with delayed kinetics of expression may infl uence the outcome in sepsis. as an example, it was observed that lps-challenged mice often die long after peak expressions of tnf and il- b suggesting that late-acting proteins may contribute to endotoxin-induced mortality. investigators searching for late expressed proteins identifi ed a member of the high mobility group (hmg)- non-histone chromosomal protein family in conditioned media h after lps-stimulation of macrophages. this protein renamed hmgb is a known ligand for the receptor for advanced glycation end products (rage). the rage receptor is expressed on monocytes and vascular smooth muscle. binding by hmgb activates both the nf-k b and mapk pathways. increased expression of hmgb was found in endotoxemic mice and in critically ill patients with surgical sepsis where increased levels correlated with non-survival. in animal models, the blockade of hmgb can inhibit the infl ammation associated with endotoxemia, cecal ligation and puncture and lps-triggered acute lung injury. identifi cation of hmgb and similar "late" mediators may provide a broader therapeutic window for successful immune modulating therapy in sepsis. regulatory processes and mediators exist for the purpose of modulation and eventual resolution of infl ammation and the septic response. an absence in the decline of proinfl ammatory mediators such as tnf and il- over the course of sepsis is an associated risk factor for mortality. monocyte activation results not only in production of proinfl ammatory cytokines, but also expression of a number of endogenous cytokine antagonists including soluble tnf receptors, the il- ra and additional anti-infl ammatory cytokines, such as il- and transforming growth factor-β (tgf-b ). il- has multiple anti-infl ammatory properties including inhibition of cytokine production from activated monocytes. il- inhibits expression of those cytokines known to contribute to sepsis, as well as important chemokines, including il- . in addition, il- increases expression of other anti-infl ammatory molecules such as il- ra and soluble tnf receptors. exogenous administration of il- in various experimental models has been used in an attempt to decrease infl ammatory cytokines and diminish organ injury. human studies showed that patients who did not survive ards had lower levels of il- in their bal fl uid compared to survivors. furthermore, the inability to increase il- in response to meningococcal infection was associated with increased mortality. thus, il- and additional regulatory cytokines (e.g. tgf-b , il- ) possess a number of anti-infl ammatory properties and are important contributors to the endogenous regulation of the acute septic response. dysregulation of the coagulation cascade occurs in sepsis as refl ected by activation of procoagulant pathways, consumption of clotting factors, alterations in fi brinolysis, and reduced anticoagulant activity. a common hematologic alteration in sepsis is the development of disseminated intravascular coagulation (dic) which is an acquired state of activation of coagulation and intravascular fi brin formation resulting in vascular thrombosis. in addition to proinfl ammatory cytokines, tissue factor (tf) activation also plays a prominent role in activating the coagulation cascade, initiating fi brin formation and contributing to the development of dic. concurrent with enhanced production of fi brin, there is decreased fi brinolysis related to increased plasminogen activator inhibitor type (pai- ), as well as dysfunction and/or depletion of antithrombin iii, protein c, protein s and tissue factor pathway inhibitor (tfpi). at iii which inhibits thrombin by forming thrombin-antithrombin (tat) complexes is decreased in sepsis related to degradation by elastases from activated neutrophils, dilution secondary to volume resuscitation, and impaired hepatic synthesis. despite a correlation between low at iii levels and mortality in patients with sepsis, replacement trials of at iii have failed to show a signifi cant effect on improving mortality. protein c is also noted to be depleted among patients with sepsis and septic shock. regulation of activation of protein c to activated protein c (apc) in the coagulation cascade is mediated in a complex manner and will not be discussed here. apc, upon dissociation from its receptor, binds to its co-factor, protein s, to subsequently inactivate factors va or viiia, thus playing a key role in inhibiting coagulation. it is both antithrombotic and profibrinolytic. apc also possesses anti-infl ammatory activity. in models of endotoxemia, apc infusion decreases cytokine production and attenuates neutrophil activation. these antiinfl ammatory effects appear to be independent of apc's anticoagulant effect. following an absence in the decline of proinfl ammatory mediators such as tnf and il- over the course of sepsis is an associated risk factor for mortality. monocyte activation results not only in production of proinfl ammatory cytokines, but also expression of a number of endogenous anti-infl ammatory cytokines including soluble tnf receptors, the il- ra and additional anti-infl ammatory cytokines, such as il- and tgfb . important anti-infl ammatory molecules include il- , il- ra and soluble tnf receptors. il- inhibits expression of proinfl ammatory cytokines known to contribute to sepsis, as well as important chemokines, including il- . in addition, il- increases expression of other anti-infl ammatory molecules such as il- ra and soluble tnf receptors. at iii inhibits thrombin by forming thrombin-antithrombin (tat) complexes. at iii is decreased in sepsis due to degradation by elastases from activated neutrophils, dilution secondary to volume resuscitation, and impaired hepatic synthesis. despite a correlation between low at iii levels and mortality in patients with sepsis, replacement trials of at iii have failed to show a signifi cant effect on improving mortality. these encouraging pre-clinical studies, clinical trials examining the effect of apc on mortality from sepsis were commenced culminating in the protein c worldwide evaluation in severe sepsis (prowess) trial. in this study, apc was associated with a statistically signifi cant reduction in -day mortality in septic adults. however, a recent pediatric study was stopped after an interim analysis showed that apc administration was highly unlikely to show improvement in outcome ( fig. - ). it is not uncommon to observe that patients exposed to seemingly identical pathogen insults display strikingly different pathophysiology and outcomes. it is believed that genetic differences among hosts are at least in part responsible for this variability in sepsis responses. as mentioned previously, the insensitivity to lps in the c h/hej mouse line was mediated by a mutation in the coding sequence for tlr . similar fi ndings of an attenuated response to pathogen stimulation have now been reported in patients with mutations in both the tlr and tlr gene. the polymorphism in tlr appears to confer an increased predisposition to severe gram-positive bacterial infections. more recently, a polymorphism within the cd promoter gene (c to t transition at base pair - ) was identifi ed with a particular genotype over-represented among septic shock patients compared to healthy controls. among the septic patients, the presence of this genotype also was associated with a signifi cantly higher mortality ( % versus %). these studies support the concept that genetic alterations in those genes known to participate in the septic response affect the host immune response and likelihood of survival. for a more complete review of the numerous examples of genetic alterations in key infl ammatory genes, the reader is directed to the suggested readings. the administration of activated protein c was associated with a statistically signifi cant reduction in -day mortality in septic adults. however, a recent pediatric study was stopped after an interim analysis showed that apc administration was highly unlikely to show improvement in outcome. the coagulation cascade in sepsis (reprinted with permission from bernard ) as the cellular response to sepsis has become better understood, the approach to treatment of sepsis has become broader. the treatment of sepsis involves four important components: initial resuscitation, elimination of pathogen, maintenance of oxygen delivery, and carefully directed regulation of the infl ammatory response. as reviewed above, sepsis is an immunologically complex response to an invasive pathogen necessitating tight physiologic regulation in order to eradicate the organism while maintaining cellular and organ homeostasis. in cases where the immunologic and infl ammatory responses continue to escalate, numerous pathways are altered and may ultimately prove amenable to immune modulating therapy, but this approach has been unsuccessful to date. the initial priority in the treatment of the septic child is respiratory and cardiovascular stabilization. the primary goals of therapy in those initial hours following clinical presentation are to maintain oxygenation and ventilation, achieve normal perfusion and blood pressure, and re-establish appropriate urine output for age. children with sepsis may have altered mental status which, if profound, raises concern about the ability to protect the airway. tachypnea associated with a primary or compensatory respiratory alkalosis is commonly present. the combination of increased lung vascular permeability and aggressive fl uid resuscitation to restore intravascular volume and maintain blood pressure may contribute to the subsequent development of pulmonary edema. in children with lung edema, the related changes in lung compliance and loss of functional residual capacity can dramatically increase the work of breathing ultimately necessitating tracheal intubation and mechanical ventilatory support. arterial blood gas analysis may show hypoxemia and metabolic acidosis; however, the decision to provide mechanical ventilatory support should not be based solely on laboratory fi ndings. the presence of increased work of breathing, hypoventilation or obtundation are all indications for instituting mechanical ventilatory support which holds additional benefi t in decreasing the overall oxygen consumption, especially when combined with sedation and paralysis. it should be stated, however, that children with warm shock can commonly be managed without endotracheal intubation so long as they are not obtunded or fl uid overloaded. disorientation or lethargy with intact responsiveness does not require placement of an artifi cial airway as many institutions manage these patients without intubation. the work of breathing associated with hyperventilation in the absence of pulmonary edema is not clinically signifi cant. furthermore, there is no evidence that decreasing work of breathing in the presence of distributive shock will result in redistribution of nutrient fl ow to vital organs, the very nature of distributive shock. however, it is more common for infants to present with cardiac dysfunction and pulmonary edema or seriously altered mental status requiring endotracheal intubation and mechanical ventilation. correction of intravascular volume depletion should be made prior to the institution of positive pressure ventilation. the decrease in venous return after the initiation of positive pressure ventilation may lead to further hemodynamic compromise in the child with intravascular volume depletion. caution should also be taken in choosing sedative agents for intubation, using agents that have the least impact on tenuous hemodynamics (e.g. ketamine). controversy exists over the adrenal suppressive effect of a single dose of etomidate when used for intubation in septic children and adults. the pediatric critical care clinician should be aware of the concern for adrenal suppression following a single dose of etomidate used for the intubation of children with septic shock and the published guidelines which do not recommend its use in this setting. following intubation, attention must be paid to matching the mechanically provided minute ventilation to that which was present during spontaneous respiratory effort so that respiratory compensation of acidemia is preserved. if it is deemed that positive pressure ventilation is not needed, supplemental oxygen should be provided to maintain normal oxygen saturations. treatment of sepsis involves four important components: initial resuscitation, elimination of pathogen, maintenance of oxygen delivery, and carefully directed regulation of the infl ammatory response. with regard to fl uid status, septic children have decreased effective intravascular volume related to a number of causes. poor oral intake of fl uid for a period of time prior to clinical presentation is common. increased vascular permeability leads to intravascular volume loss due to extravasation of fl uid from the vascular space, so-called "third spacing". finally, the no-mediated vasodilation reviewed above increases vascular capacitance thereby decreasing the effective circulating volume. thus, when sepsis is suspected, it is imperative to expeditiously achieve vascular access and initiate fl uid resuscitation with ml/kg of isotonic fl uid as quickly as possible. while debate continues as to the most effective fl uid for resuscitation, no pediatric literature exists to support colloid over crystalloid, the latter of which was recently demonstrated to be equally effective in a large adult icu trial. there is some support for using colloid fl uid in patients with a narrow pulse pressure; however, this practice is not supported by any large, well-designed clinical studies. while following the clinical exam for signs of intravascular volume overload (new onset of rales, increased work of breathing, development of a gallop, or hepatomegaly), fl uid should be administered quickly with the goal of monitoring heart rate response, urine output, capillary refi ll time and level of consciousness. initial fl uid resuscitation of the child with septic shock commonly requires a volume of up to or greater than ml/kg in the fi rst hour and one retrospective study demonstrated an increased survival in children given fl uid volumes of ml/kg or more within the fi rst hour. the accm clinical practice parameters for hemodynamic support of pediatric and neonatal septic shock recommend that the child with septic shock be repeatedly examined for the development of "rales, gallop rhythm, hepatomegaly, and increased work of breathing" during volume loading, and that in the absence of such fi ndings, volumes up to ml/kg can be administered in the fi rst hour. these guidelines further state that "the rate of fl uid administration should be reduced substantially when there are clinical signs of adequate cardiac fi lling without hemodynamic improvement." despite on-going fl uid resuscitation, hypotension and inadequate organ perfusion may persist requiring the initiation of inotropes and/or vasopressors. in children, vasoactive medicines should only be given in addition to fl uid resuscitation. however, consensus guidelines recommend that vasoactive infusions may be necessary in some cases to sustain perfusion pressure even when hypovolemia is not yet resolved. dopamine is the most common fi rst choice agent selected for hemodynamic support in those patients with fl uid-refractory shock. dopamine provides inotropic support at lower concentrations; however, it is often necessary to increase it to higher doses that provide vasopressor activity (up to m g/kg/min) to maintain adequate tissue perfusion. the decision of which agent to add in the setting of dopamine-refractory shock should be based on the underlying cause of cardiovascular compromise. for example, if hemodynamic instability is related to low cardiac output from direct cardio-depressant effects then increased inotropy from dobutamine or low-dose epinephrine may be indicated. if hypotension persists secondary to decreased vascular tone, then agents such as epinephrine and norepinephrine dosed in the alpha agonist range should be considered. finally, in children who demonstrate low cardiac output and/or increased afterload from vasoconstriction (i.e. increased systemic vascular resistance), agents with primarily inotropic or vasodilator function including milrinone, dobutamine or short-acting nitrovasodilators can been considered in the fl uid-resuscitated, normotensive child. as in evaluating the adequacy of fl uid resuscitation, similar clinical parameters should be followed in titrating vasoactive medications. appropriate endpoints include: capillary refi ll time less than s, normal peripheral pulses, warm extremities, urine output greater than . ml/kg/h, improved mentation, resolving acidemia, decreasing serum lactate and when available, superior vena cava (svc) oxygen saturation greater than %. invasive monitoring can further assist the clinician with goal directed endpoints. although frequent beside examination remains integral to the care of the child in septic shock, an additional task in the initial resuscitation phase is placement of appropriate and necessary vascular access and monitors. central venous access is a necessity for the child with fl uid refractory shock to provide for delivery of vasoactive medicines and large volumes of fl uid. these catheters can be useful for following the central venous pressure (cvp) during fl uid adequate volume resuscitation of the child with septic shock commonly requires a volume of up to or greater than ml/kg in the fi rst hour. appropriate endpoints of sepsis resuscitation include: capillary refi ll time less than s, normal peripheral pulses, warm extremities, urine output greater than . ml/kg/h, improved mentation, resolving acidemia, decreasing serum lactate and when available, superior vena cava oxygen saturation greater than %. administration. finally, when the tip is located in the superior vena cava, blood sampling can provide an approximate measure of the mixed venous oxygen saturation which has been validated as a critical target in adult shock resuscitation. the decision regarding the access site for a central venous catheter is dictated by a number of mitigating factors such as the experience level of the operator and the presence of coagulopathy. femoral catheters, in the absence of abdominal pathology, can be used to estimate cvp with good correlation. the cvp measured in the abdominal inferior vena cava must be assessed carefully as a low cvp can be a reliable indicator of hypovolemia, however, a normal or high cvp in the presence of abdominal distention does not automatically exclude the presence of hypovolemia. multiple adult studies have demonstrated that even an accurate intra-thoracic cvp may be a poor approximation of left ventricular end diastolic pressure and volume. the cvp can be elevated despite the presence of hypovolemia if pulmonary hypertension, right ventricular dysfunction with poor diastolic compliance, tricuspid regurgitation, cardiac tamponade or an intracardiac left-to-right shunt exists. even though precise determination of the true mixed venous saturation requires the presence of a pulmonary artery catheter, the approximations derived from the svc saturation have proven a useful target in septic adults. in contrast, because of differences in oxygen extraction between the upper extremities, abdomen, and lower extremities, venous oxygen saturations from a low lying femoral line do not accurately correlate with those measured in the pulmonary artery. consensus guidelines recommend therapeutic endpoints of superior vena cava oxygen saturation > % or mixed venous (pulmonary artery) oxygen saturation > %. placement of an intra-arterial catheter provides continuous monitoring of systemic blood pressure, pulse pressure and hemodynamic variation with respiration, as well as a means for drawing arterial blood gases, lactate levels and additional laboratory studies. the arterial blood also provides the most accurate measure of arterial oxygen content and can be used to both assess the function of the lungs and to maximize oxygen delivery. in the ventilated patient, variation in the amplitude of the arterial waveform has been found to correlate closely with intravascular volume status (see also chapter ). systolic pressure variation (spv), also referred to as "reverse pulsus paradoxus," is the variation in beat-to-beat amplitude of the arterial pulse during positive pressure ventilation. a single positive pressure breath normally affects the arterial pressure in a biphasic manner. the initial response to a positive pressure breath is to "squeeze" pulmonary vascular blood into the left atrium (the opposite, "pooling" of blood, occurs with negative pressure inspiration) leading to a rise in systolic pressure. in addition, positive intrathoracic pressure reduces the afterload on the left ventricle by virtue of the pressure gradient from the thorax outward further augmenting this early rise in arterial pressure. these two effects produce an upward movement of the systolic blood pressure coincident with the positive pressure breath, referred to as the d up component of spv. following this d up, a fall in systolic pressure occurs a few beats later as the decreased venous return (preload) to the right ventricle that occurred during positive pressure inspiration is now evident as decreased preload to the left ventricle after a few cardiac cycles. the transient reduction in right ventricular volume and output leads to a smaller left ventricular stroke volume and a brief reduction in arterial pressure that occurs later in the ventilator cycle ( d down). an exaggerated spv (> mm hg) has been seen early in the setting of hypovolemia. this is due to a greater d down component. several studies have shown that an increase in the spv occurs prior to a fall in arterial pressure and may a better predictor of hypovolemia than a low pulmonary capillary wedge pressure (pcwp) (< mm hg). an increase in the spv due to a greater d down component can also occur due to high airway pressures causing decreased venous return. recently, pulse pressure variation (ppv) has also been found to be a sensitive indicator of preload, and more importantly, fl uid responsiveness. ppv is defi ned as the maximal pulse pressure (systolic minus diastolic blood pressure) less the minimum pulse pressure divided by the average of these two pressures. the use of systolic pressure variation and pulse pressure variation is limited to those patients on mechanical ventilation. these measurements should occur when there is no spontaneous breathing. in the presence of a consistent delivered tidal volume, systolic pressure variation can be used to track the adequacy of intravascular volume over time (fig. - ) . the decision to use a pulmonary catheter (pac) with the goals of optimizing left ventricular preload, monitoring cardiac index and measuring oxygen delivery remains controversial. caveats to interpretation of pac data include the presence of an intracardiac shunt and an abnormally functioning mitral valve or other obstructed left heart lesion as either shunting, regurgitation, or inaccurate pressure determinations will alter cardiac index and/or the pulmonary capillary wedge pressure measurements. as previous data in adults have shown no benefi t of pac use, a recent consensus statement regarding their use stated that the role of pac remains unclear. studies of children with septic shock have shown that the information obtained from a pac aided in identifying hemodynamic profi les different from those presumed by care givers and has directly infl uenced care decisions. it was concluded by a recent consensus panel to be of potential benefi t in improving the management of pediatric patients. pulmonary artery catheter placement should be considered for pediatric patients who remain in shock after resuscitation and initiation of the usual vasoactive agents in whom the fl uid status and cardiac function remains unclear. in this setting, therapeutic endpoints are a cardiac index of > . and < . l/min/m and systemic and pulmonary vascular resistances within the normal range. early identifi cation of a possible offending pathogen and aggressive source control represent a crucial component of septic shock therapy. prompt initiation of appropriate antimicrobial therapy against the causative pathogen has been shown to be one of the most important predictors of outcome. in a 's study of over , adults, providing appropriate systolic pressure variation (spv) and pulse pressure variation (ppv) can accurately predict which patients will be responsive to further fl uid resuscitation (gunn and pinsky ) . antimicrobial coverage at least day prior to identifi cation of the organism was associated with improved survival. the pathogen itself has prognostic signifi cance. fungal infections, while accounting for only a minority of sepsis cases, carry the lowest survival rate, followed by gram-positive and gram-negative bacteria. survival rate has been reported to be the highest in patients in whom no pathogen was identifi ed. because of the importance of appropriate antimicrobial therapy, the decision of which agents to empirically start must balance potential side effects versus maximizing coverage. in this respect, it is important to be familiar not only with the most common causative pathogens, but also the local icu nosocomial risks and pathogen resistance patterns. initially, broad antibiotic coverage is initiated. neonates are most frequently placed on ampicillin and an aminoglycoside (e.g. gentamicin) or a third generation cephalosporin such as cefotaxime. in infants and children over the age of - weeks, the decision to start vancomycin empirically should be considered in light of the increasing antibiotic resistance of streptococcus pneumoniae and rising incidence of community acquired methicillin resistant staphylococcus aureus (mrsa). in addition, a rd or th generation cephalosporin (e.g. ceftriaxone) should be used. suspicion of a gram-negative infection or nosocomial infection requires additional coverage, usually in the form of an aminoglycoside, for the possibility of pseudmonas species and other resistant gram-negative organisms. because of its broad coverage, including many anaerobic species, and low renal toxicity, piperacillin/tazobactam is empirically administered with increasing frequency. the antiviral agent, acyclovir, should be administered if there is suspicion of a herpes virus infection. in immunocompetent children, the decision to start empiric antifungal therapy remains controversial. in the child who is not improving over the initial days of empiric coverage or in whom there is a higher risk for fungal infection (e.g. presence of indwelling devices, immunosuppression or other signifi cant co-morbidities), antifungal coverage may be indicated. the development of agents equally as effective as amphotericin, but with substantially reduced nephrotoxicity such as fl uconazole and caspofungin, may ultimately sway the risk/benefi t analysis towards more aggressive, earlier initiation of empiric antifungal coverage in select, high risk populations. the ability to narrow the spectrum of treatment once the causative organism has been identifi ed will reduce the number of potential side effects and curtail the development of pathogen resistance related to imprudent use of broad spectrum antibiotics. the current mainstay of supportive care in sepsis remains the maintenance of adequate oxygen delivery in the face of myocardial depression, capillary leak, acidosis, and massive cytokine release. while some early adult studies have suggested improved outcomes when achieving supra-normal levels of oxygen delivery, this approach in pediatric sepsis remains unproven. this is likely due to the fact that septic patients may have a perturbed ability to extract oxygen in addition to suboptimal oxygen delivery. clinically, this impairment in cellular oxygen uptake may be refl ected by an inappropriately high central venous oxygen saturation (s cv o ) in the face of a progressive and therapy refractory acidosis. optimizing appropriate oxygen delivery remains a clinical goal and incorporates the need for maximizing oxygen carrying capacity. while there is no recommended hemoglobin level for children, the most recent nih consensus conference suggested a hemoglobin concentration of g/dl for adults with cardiopulmonary compromise as part of a protocol toward achieving the therapeutic goal of s cv o > %, with improved outcomes demonstrated when this goal was achieved during initial resuscitation. in the context of fl uid loading with blood transfusion, empiric administration of diuretics to eliminate extra fl uid should be avoided until hemodynamic stability has been achieved or if the child exhibits signs of intravascular volume overload defi ned earlier in this chapter. similar clinical parameters can be assessed in response to blood transfusion. perhaps the best assessments are determinations that provide indirect evidence of the balance between oxygen delivery and consumption such as lactate levels and mixed venous oxygen saturation. in the context of fl uid loading with blood transfusion, empiric administration of diuretics to eliminate extra fl uid should be avoided until hemodynamic stability has been achieved or if the child exhibits signs of intravascular volume overload. finally, the nutritional status of the septic child must be addressed. patients with sepsis often have poor nutrition prior to admission to the picu and often may not be fed in the fi rst few days of illness. this state combined with the increased metabolic rate associated with sepsis place the septic patient at risk for protein calorie malnutrition. intestinal hypoperfusion in combination with absence of local enterocyte nutrition can cause mucosal barrier dysfunction and may contribute to translocation of bacteria and endotoxin from the intestine into the blood stream. while the use of enteral feeding in critical illness has been shown to improve survival and decrease hospital stay, its use must be balanced with the risk of stressing intestinal function in the face of poor splanchnic perfusion, especially in the child requiring the use of vasopressors such as epinephrine and norepinephrine. regardless of which mode of nutrition is chosen, the goal of achieving nitrogen balance is important for allowing recovery and return to physiologic homeostasis. in the absence of enteral feedings, protection from stress-related gastrointestinal ulcer formation is advised. because poor outcome in sepsis has been attributed to a dysregulated proinfl ammatory state, anti-infl ammatory agents, such as corticosteroids, have long been proposed as a potential therapeutic strategy. anecdotally, it has been observed that some patients treated with antibiotics appear to acutely worsen in a time frame consistent with the onset of antibiotic activity. this observation has been attributed to massive release of bacterial products following the lysis of high numbers of bacteria. to this end, investigators had shown that animals treated with anti-infl ammatory drugs prior to receiving antibiotics demonstrated a less severe response to bacterial lysis. despite encouraging preclinical studies, two subsequent large adult trials using high dose steroids early in sepsis showed no improvement in mortality. more recently, studies using lower doses of steroids over a longer period of time have suggested a possible benefi t including a reduced time to cessation of vasopressor therapy. these more recent observations have stimulated a resurgence in the use of corticosteroids in sepsis. adrenal insuffi ciency is frequently unrecognized in children with septic shock and a low basal circulating cortisol level, especially in association with an abnormal corticotropin stimulation test, has been associated with higher mortality rates. therefore, identifying "at risk" patients with a corticotropin stimulation test and treating this group may improve outcome in sepsis. a study in which adult septic shock patients who were classifi ed as "nonresponders" based on corticotropin stimulation results were treated with hydrocortisone and fl udrocortisone for days and had a signifi cantly reduced risk of death. however, this initial observation was not observed in larger follow-up studies. thus, the use of hydrocortisone and the application of a corticotropin stimulation test in septic patients remains highly controversial. in pediatrics, it is currently recommended that any child with fl uid-and catecholamine-refractory shock (inadequate response to two or more vasoactive agents), has a known history of adrenal insuffi ciency, or has previously received exogenous steroids should be considered for steroid replacement with hydrocortisone (usual dose between and mg/m /day divided every h). because the host response to sepsis is mediated by circulating infl ammatory molecules, it has been hypothesized that extracorporeal removal of these mediators via hemofi ltration or exchange transfusion may affect outcome. case reports suggest that arterial oxygenation and hemodynamics can be improved with use of hemofi ltration during sepsis and multiple organ failure. however, there exist many mitigating factors in evaluating the pediatric experience and the effi cacy of hemofi ltration remains unproven. challenges with instituting extracorporeal hemofi ltration include diffi culty with vascular access in smaller children, potential fl uid and electrolyte imbalance, hypothermia, anticoagulation requirements and acutely compromised hemodynamics during initiation. in addition, it is not known whether benefi cial proteins such as albumin, immunoglobulins, clotting factors and counter-regulatory cytokines are removed during this process. while experience shows that hemofi ltration can be safely performed in children with sepsis, it remains unclear if it will improve outcome. part of the infl ammatory response involves cytokines that cause widespread activation of the coagulation cascade with suppression of fi brinolysis as reviewed above. it is encouraging adrenal insuffi ciency is frequently unrecognized in children with septic shock. a low basal circulating cortisol level, especially in association with an abnormal corticotropin stimulation test, has been associated with higher mortality rates. that administration of activated protein c in adults with septic shock was associated with a signifi cant decrease in -day mortality. though activated protein c should not be routinely used in pediatric sepsis, indications for its use may be determined from further trials in pediatric sepsis. in the meantime, many other potential immune modulating therapeutic agents have been identifi ed and are currently under investigation. unfortunately, many of the antiinfl ammatory agents tried to date (anti-il- , anti-bradykinin, anti-endotoxin, anti-tnf-a , soluble tnf receptor and anti-platelet activating factor) have not shown any benefi t in large, randomized clinical trials. it is hoped that improvements in study design which include thoughtful stratifi cation of patients, timely identifi cation of the presence or absence of a pathogen and consideration of genetic factors that infl uence outcome will eventually assist in discovering and targeting pharmacologic agents that ultimately improve the outcome of the pediatric patient with septic shock. vascular bed-specifi c hemostasis: role of endothelium in sepsis pathogenesis corticosteroids for severe sepsis and septic shock: a systematic review and meta-analysis immunological therapy of sepsis: experimental therapies effi cacy and safety of recombinant human activated protein c for severe sepsis signal transduction during innate and adaptive immunity role of nfkappab in the mortality of sepsis new insights into its pathogenesis and treatment the accp-sccm consensus conference on sepsis and organ failure stress doses of hydrocortisone reverse hyperdynamic septic shock: a prospective, randomized, double-blind, single-center study clinical practice parameters for hemodynamic support of pediatric and neonatal septic shock: update from the american college of critical care medicine toll-like receptors: molecular mechanisms of the mammalian immune response prognostic values of tumor necrosis factor/cachectin, interleukin- , interferon-alpha, and interferon-gamma in the serum of patients with septic shock. swiss-dutch j immunoglobulin study group hemodynamic support in fl uid-refractory pediatric septic shock surviving sepsis campaign guidelines for management of severe sepsis and septic shock surviving sepsis campaign: international guidelines for management of severe sepsis and septic shock organization and regulation of mitogenactivated protein kinase signaling pathways lps induction of gene expression in human monocytes implications of arterial pressure variation in patients in the intensive care unit signal transduction by the c-jun n-terminal kinase (jnk) -from infl ammation to development the i kappa b kinase (ikk) and nf-kappa b: key elements of proinfl ammatory signalling use of corticosteroid therapy in patients with sepsis and septic shock: an evidence-based review myocardial dysfunction in septic shock: part ii. role of cytokines and nitric oxide rationale for restoration of physiological anticoagulant pathways in patients with sepsis and disseminated intravascular coagulation genomic polymorphisms in sepsis the contrasting effects of dopamine and norepinephrine on systemic and splanchnic oxygen utilization in hyperdynamic sepsis early enteral nutrition in acutely ill patients: a systematic review source control in the management of severe sepsis and septic shock: an evidence-based review role of the coagulation system in the local and systemic infl ammatory response new rationale for glucocorticoid treatment in septic shock anti-infl ammatory cytokines pediatric considerations epidemiology of sepsis and multiple organ dysfunction syndrome in children pulmonary artery catheter consensus conference: consensus statement drotrecogin alfa continuous plasmafi ltration in sepsis syndrome. plasmafi ltration in sepsis study group early goal-directed therapy in the treatment of severe sepsis and septic shock innate immune mechanisms triggering lung injury effects of nitric oxide in septic shock signal transduction pathways in acute lung injury: nf-k b and ap- sepsis remains one of the most pressing clinical challenges for the pediatric intensivist. it is apparent that while a great deal is now understood about the biological and molecular mechanisms involved in sepsis, this knowledge has not yet had a dramatic impact on improving outcome. at present, therapeutic modalities for sepsis remain largely supportive and founded on the fundamental physiologic principle of providing adequate oxygen delivery. with this approach, mortality in pediatric sepsis improved modestly over the past decades. however, the fact that over , children per year continue to die in association with severe sepsis argues that further advances be made. realization of the goal of improving survival requires investigators committed to achieving further mechanistic insights into the physiologic, molecular, and genetic biology of sepsis, in concert with large pediatric-specifi c interventional trials. a year old female is admitted to the pediatric intensive care unit with septic shock. she is well oxygenated on a % oxygen face mask. she has already received ml/kg of . % normal saline and has been started on a dopamine infusion at a rate of mcg/kg/min. in monitoring her response to these interventions, which of the following should not be used as a therapeutic endpoint to monitor her progress? a. capillary refi ll time b. echocardiographically measured ejection fraction c. mental status d. serum bicarbonate level e. urine output ³ ml/kg/h . a year old male with acute lymphocytic leukemia is admitted to the pediatric intensive care unit with vancomycin resistant enterococcus bacteremia. his vital signs reveal a temperature of . °c, a heart rate of bpm, a respiratory rate of breaths/min, and a blood pressure of / mm hg. he is lethargic, but arousable. his pulses are bounding and his capillary refi ll is brisk. an arterial blood gas reveals a ph . , a paco mm hg, a pao mm hg, an oxygen saturation of %, and a base defi cit of (− ). the oxygen saturation of venous blood sampled from the superior vena cava is %. a. the young man is bacteremic, but not in shock as evidenced by his bounding pulses, brisk refi ll, and normal systolic blood pressure. b. the young man is in shock with inadequate oxygen extraction at the tissue level evidenced by the elevated superior vena cava saturation. c. the young man has a primary metabolic acidosis, but has a normal oxygen extraction as oxygen saturation in the superior vena cava is normally higher than elsewhere in the body. d. the young man has a primary metabolic acidosis, but is not in shock, evidenced by his high superior vena cava saturation. e. the young man has a primary respiratory alkalosis that would benefi t from supplemental oxygen therapy. . there is suffi cient data to justify the use of which of the following adjuvant therapies in pediatric sepsis? a. the administration of activated protein c to a child in septic shock without thrombocytopenia or coagulopathy b. the administration of anti-tnf-a monoclonal antibodies to a septic patient to decrease the proinfl ammatory response c. the administration of stress dose hydrocortisone to a septic patient whose serum cortisol level fails to increase sufficiently in response to a corticotropin stimulation test d. the early initiation of high volume, continuous veno-venous hemofi ltration to remove proinfl ammatory cytokines e. the transfusion of packed red blood cells to maintain a hemoglobin ³ g/dl in order to provide supranormal oxygen delivery . it has become clear that dysregulation of the coagulation cascade occurs in sepsis as refl ected by activation of procoagulant pathways, consumption of clotting factors, alterations in fi brinolysis, and reduced anticoagulant activity. which of the following components of coagulation is increased during sepsis? a. antithrombin iii (at iii) b. plasminogen activator inhibitor type (pai- ) c. protein c d. protein s e. tissue factor pathway inhibitor (tfpi) key: cord- -psjyjvbu authors: zhou, yile; yang, yajie; liang, tao; hu, yan; tang, haihong; song, dongli; fang, hao title: the regulatory effect of microrna- a- p on the promotion of telocyte angiogenesis mediated by pi k (p α)/akt/mtor in lps induced mice ards date: - - journal: j transl med doi: . /s - - -z sha: doc_id: cord_uid: psjyjvbu background: telocytes (tcs) are newly identified interstitial cells that participate in tissue protection and repair. the present study investigated the mechanisms underlying the protective effect of tcs in a mouse model of respiratory distress. methods: the mouse model of acute respiratory distress syndrome (ards) was established by intratracheal instillation of lipopolysaccharide (lps). after instillation of tcs culture medium, lung injury was assessed, and angiogenesis markers, including cd and endothelial nitric oxide synthase (enos), were detected by immunofluorescence. bioinformatics analysis was used to screen significantly differentially expressed micrornas (mirnas) in cultured tcs stimulated with lps, and the regulation of downstream angiogenesis genes by these mirnas was analysed and verified. pi k subunits and pathways were evaluated by using a pi k p α inhibitor to study the involved mechanisms. results: in ards mice, instillation of tcs culture medium ameliorated lps-induced inflammation and lung injury and increased the protein levels of cd and enos in the injured lungs. a total of mirnas and mrnas were differentially regulated in tcs stimulated with lps. functional prediction analysis showed that the differentially expressed mrnas were enriched in angiogenesis-related processes, which were highly correlated with mir- a- p. culture medium from tcs with mir- a- p inhibition failed to promote angiogenesis in mouse models of lps-induced ards. in cultured tcs, lps stimulation upregulated the expression of mir- a- p, which further targeted the transcription factor e f and decreased notch protein expression. tcs culture medium enhanced hemangioendothelioma endothelial cells (eoma cells) proliferation, which was blocked by the mir- a- p inhibitor. the pi k p α inhibitor decreased vascular endothelial growth factor levels in lps-stimulated tcs and reversed the enhancing effect of tcs culture medium on eoma cells proliferation. conclusions: tcs exerted protective effects under inflammatory conditions by promoting angiogenesis via mir- a- p. the pi k p α subunit and transcriptional factor e f could be involved in this process. acute respiratory distress syndrome (ards) is a clinical syndrome characterised by acute progression of respiratory failure. according to an international multicentre research, the prevalence of ards was . % of icu admissions [ ] . inflammatory responses destroy underlying vascular endothelial cells and respiratory epithelial cells and impair the lungs' ability to exchange oxygen and carbon dioxide [ ] . therefore, decreasing inflammation and accelerating blood vessel repair are two key factors in the prevention and treatment of ards. since its severity and lack of effective pharmacologic treatments [ ] , it is of great significance to explore novel therapeutic strategies for ards. recently, cell therapy have been shown to have promising therapeutic potential. mesenchymal stem cells ameliorated ards due to paracrine mechanism [ ] . telocytes (tcs) are newly identified mesenchymal cells that play a role in providing nutrition to surrounding cells by cell-cell communication and have post-injury repair and regeneration functions [ ] [ ] [ ] . tcs contribute to angiogenesis within the myocardium [ ] . transplantation of cardiac tcs promotes post ischaemic myocardial repair [ ] . pulmonary tcs also assist with angiogenesis since they participate in forming the structure of the air-blood barrier [ ] . intratracheal administration of activated tcs has been reported to alleviate ventilator-induced lung injury in a mouse model by releasing angiogenic factors [ ] . however, the underlying mechanism remains unclear. class i phosphoinositide- -kinases (pi ks) or the four subtypes of catalytic subunit-p α, p β, p γ and p δ-are expressed in all mammalian cells. the catalytic subunits bind to p regulatory subunits, activate receptor tyrosine kinases (rtks), and transmit a variety of cell surface receptor signals, such as those from the epidermal growth factor receptor (egfr) or fibroblast growth factor receptor (fgfr), to promote cell growth [ ] . the pi k subunits p α and p δ were demonstrated to be associated with tissue repair; however, this function is mediated by different mechanisms. the activity of pi k p α can be enhanced by tyrosine kinase ligands, such as vascular endothelial growth factor (vegf) a, and can induce angiogenesis and vascular remodelling [ ] . moreover, p α regulates endothelial cell migration through the small gtpase rhoa, mediated by pi kcg, a gene encoding a p γ subunit, which has a protective effect on hypoxic-reoxygenated cardiomyocytes mediated by activation of the pi k/akt signalling pathway and inhibition of apoptosis [ ] . pi k (p δ)/ akt/mammalian target of rapamycin (mtor) signalling pathway mediates interferon-γ (ifn-γ) induced airway epithelial cell growth and proliferation through interaction with ceacam [ ] . micrornas (mirnas) are small, non-coding rnas that regulate the expression of target genes via posttranscriptional degradation of mrna and/or translational inhibition of protein expression. mir- a can influence cell proliferation, migration, invasion, apoptosis and tumour angiogenesis through the igf- /pi k/ akt signalling pathway in non-small cell lung cancer (nsclc) [ ] . mature mir- a- p was found to be secreted by lipopolysaccharide (lps)-activated macrophages in small vesicles, which were endocytosed and internalised by renal fibroblasts, thereby promoting the expression of fibrosis and inflammation markers in a mouse model of chronic renal allograft dysfunction (cad) in allogeneic kidney transplantation [ ] . antagonism of mir- a- p ameliorated cad in mouse model following kidney transplantation [ ] . in patients with renal allograft, elevation of urinary [ ] and plasma [ ] mir- level was correlated with interstitial fibrosis and tubular atrophy. the tcs line was established by transfection with simian vacuolating virus (sv ) and identified to maintain tcs morphology and immune characteristics [ ] . tcs proliferation was demonstrated to be regulated by transforming growth factor-β (tgf-β) and mediated by the pi k p α subunit and the pi k/akt/mtor signalling pathway [ ] . the present study was designed to investigate the underlying protective effect of tcs in a mouse model of respiratory distress. bioinformatics approaches were applied to analyse gene expression profiles in tcs challenged with lps. particular attention was devoted to the angiogenesis-related process. the protective mechanisms mediated by the pi k subunit in tcs were further examined in hemangioendothelioma endothelial cells (eoma cells) in vitro. the current study presents the theoretical bases of an alternative new potential therapeutic strategy for ards. both the mir- a- p inhibitor and nc were purchased from china ribobio (ribobio, guangzhou, china). tcs were transfected with the mir- a- p inhibitor and nc at a final concentration of nmol/l using a lipofectamine rnaimax transfection system (thermofisher scientific, carlsbad, ca) according to the manufacturer's protocol. cells were incubated with sirna in serum-free and antibiotic-free medium for h and then in normal growth medium for another h before the experiments were performed. gene expression profiling analysis of both mirna and mrna were performed with agilent microarray scanner (cat # g ca, agilent technologies, santa clara, ca). the data were normalised with the agimicrorna package [ ] . the gene expression files were analysed with r- . . software. differentially expressed genes (degs) were defined as those with an adjusted p-value of less than . . degs were further analysed with the limma package [ ] . heat maps were generated with the ggplot package [ ] . the online databases mirwalk [ ] and targetscan [ ] were used to screen potential mirna target genes. overlapping genes in the two databases were selected for further analysis. the online database string [ ] and the database for annotation, visualization and integrated discovery (david) v . [ ] were used to analyse gene function. the relationship between degs and mir-nas was further visualised with cytoscape . . [ ] . total rna was extracted from cultured tcs with trizol (takara, shiga, japan) according to the provided instructions. mirnas were reverse transcribed with a bulge-loop mirna qrt-pcr starter kit (ribobio, guangzhou, china), and mrnas were reverse transcribed to complementary dna (cdna) with a primescript rt reagent kit with gdna eraser (takara, shiga, japan). the expression levels of mir- a- p, mir- - p and mrnas were measured by quantitative real-time polymerase chain reaction (qpcr) on a bio-rad iq real-time pcr instrument, with u and gapdh used as the housekeeping genes for mirnas and mrnas, respectively. mirna pcr was performed with the bulge-loop mirna qrt-pcr starter kit, bulge-loop mmu-mir- a- p primer set and bulge-loop mmu-mir- - p primer set (ribobio, guangzhou, china). mrna primers were synthesised by sangon (shanghai, china). the following mouse-specific primers were used: gapdh sense primer: ′-gtt caa cgg cac agt caa g- ′, antisense primer: ′-gcc agt aga ctc cac gac at- ′; e f sense primer: ′-ctg ttt gca cga aca ctt atcag- ′, antisense primer: ′-gta ccg cgc tag gaa ttt gtg- ′; acvrl sense primer: ′-tga ttc ctg ttg ccg gcc t- ′, antisense primer: ′-cag tgt ggg ctc tca caa gt- ′; rbpj sense primer: ′-tgg cga gag ttt gtg gaa ga- ′, antisense primer: ′-agc act gtt tga tcc cct cg- ′; notch sense primer: ′-tgt ggc ttc ctt cta ctg cg- ′, antisense primer: ′-ctt tgc cgt tga cag ggt tg- ′; flt sense primer: ′-gtg agc act gcg gca aaa ag- ′, antisense primer: ′-act cat ttt ggg agg agc gt - ′; efnb sense primer: ′-cga ggt ggc aac aac aat gg- ′, antisense primer: ′-ata gtc ccc gct gac ctt ct - ′; thbs sense primer: ′-ctg cca atc ata acc agc g- ′, antisense primer: ′-ttc gtt aaa ggc cga gtg ct- ′; epas sense primer: ′-ctg agg aag gag aaa tcc cgt- ′, antisense primer: ′-tgt gtc cga agg aag ctg atg- ′; hypoxia inducible factor- α (hif- α) sense primer: ′-acc ttc atc gga aac tcc aaag- ′, antisense primer: ′-ctg ttg gct ggg aaa agt tagg- ′; pik ca sense primer: ′-cca cga cca tct tcg ggt g- ′, antisense primer: ′-acg gag gca ttc taa agt cacta- ′; pik cb sense primer: ′-cta tgg cag aca acc ttg acat- ′, antisense primer: ′-ctt ccc gag gta ctt cca act- ′; pik cd sense primer: ′-gta aac gac ttc cgc act aaga- ′, antisense primer: ′-gct gac acg caa taa gcc g- ′; and vegf sense primer: ′-gta cct cca cca tgc caa gt- ′, antisense primer: ′-tcc tat gtg ctg gct ttg gt- ′ . total protein was extracted from cultured tcs with lysis buffer ( mmol/l nacl, mmol/l edta, mmol/l naf, mmol/l dithiothreitol, μg/μl aprotinin, μg/ μl leupeptin, . mmol/l na vo , mmol/l phenylmethylsulfonyl fluoride (pmsf), and . % np- ). protein extracts ( μg) were separated by % sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (merck millipore, darmstadt, germany). after blocking with % non-fat milk/tris-buffered saline containing . % tween at room temperature for one hour, membranes were incubated with primary antibodies [specific for gapdh ( - -ig, proteintech, wuhan, china), e f (ab , abcam, cambridge, uk), deltalike (dll )(ab , abcam, cambridge, uk), notch (sc- , santa cruz, dallas, tx), notch (sc- , santa cruz, dallas, tx), notch (sc- , santa cruz, dallas, tx), phosphatase and tensin homolog deleted on chromosome ten (pten)(ab , abcam, cambridge, uk), pi k ( t, cst, boston, ma), p-pi k ( t, cst, boston, ma), mtor ( t, cst, boston, ma), p-mtor ( t, cst, boston, ma), akt ( s, cst, boston, ma), p-akt ( s, cst, boston, ma), and p α ( t, cst, boston, ma)] overnight at °c. protein expression levels were normalised to those of gapdh with imagej (nih, bethesda, md). eoma cells proliferation was assessed with a colorimetric assay-cell counting kit- (cck , yeasen, shanghai, china)-following the manufacturer's protocol. approximately eoma cells/well were seeded in a -well plate. after adhesion, eoma cells were incubated for h with culture medium from tcs transfected with the mir- a- p inhibitor or nc in the presence of lps. the pgl reporter vector (promega, madison, wi) was used to generate the plasmids pgl -wt-e f - ′-utr and pgl -mut-e f - ′-utr. human embryonic kidney cells were co-transfected with pgl -e f - ′-utr (wt or mut) and the mir- a- p mimic or nc with lipofectamine reagent (thermofisher scientific, carlsbad, ca). after incubation for h, luciferase activity was assessed by the dual-luciferase reporter assay system (promega, madison, wi) according to the manufacturer's protocol. the concentration of vegf in the tcs culture medium was measured by a commercial vegf elisa kit (westang, shanghai, china) according to the manufacturer's protocol. live observation of eoma cells was performed with a cell-iq cell culture platform (chip-man technologies, tampere, finland) equipped with a phase contrast microscope (nikon cfi achromat phase contrast objective with magnification) and a camera (nikon, fukasawa, japan). the equipment was controlled by imagen software (chip-man technologies). each group contained replicates of visual fields. images were acquired at -h intervals for h. lung tissues were fixed with % formalin solution and embedded in paraffin. each tissue was sectioned at μm and stained with haematoxylin-eosin (he, beyotime, shanghai, china) according to the manufacturer's protocol. for immunofluorescence staining, an antigen retrieval protocol was carried out with incubation in . % h o for min and heating to boiling in a microwave in citrate buffer for min. after blocking with % goat serum in tris-buffered saline, sections were incubated with diluted primary antibodies [cd ( : , ab , abcam, cambridge, uk), endothelial nitric oxide synthase (enos) ( : , cat , bd biotechnology, san jose, ca)] overnight at °c and then with secondary antibodies and ′, -diamidino- -phenylindole (dapi), separately. data are expressed as the means ± sds and were analysed by one-way analysis of variance (anova) and tukey's multiple comparisons test. a p-value of < . was considered statistically significant. all statistical analyses were performed with graphpad prism . (graphpad, san diego, ca). the ability for tcs protection was first estimated in ards mouse models. lps stimulation caused inflammatory infiltration, alveolar wall widening, and vessel destruction (fig. a) . the production of inflammatory cytokines was elevated in ards mice (fig. b) . since substances, including molecules and exosomes, released by tcs could be important factors affecting adjacent cells, the effect of tcs culture medium was assessed. instillation of tcs culture medium reduced the inflammatory infiltration, reduced the alveolar interstitial width and decreased the levels of inflammatory cytokines. bio-behaviours of tcs were recorded by cell-iq to show the typical morphology of cultured cells (additional file : figure s ). as angiogenesis is essential in tissue repair, the induction of angiogenic factors in tcs after stimulation with lps was assessed. in ards mice, the expression of the angiogenesis-related marker cd and enos was downregulated. however, an increase in cd and enos expression was observed in the wt tcs treatment group but not in the group treated with medium from tcs with mir- a- p inhibition (fig. ) . to identify the critical mirnas in the regulation of angiogenesis by tcs, mirna and mrna profiles were generated, and the relationship of differentially expressed mirnas with downstream angiogenesis factor-associated mrnas were analysed in lps-stimulated tcs. in the mrna expression of inflammatory cytokines in mice lungs in the above four groups. *p < . vs control, # p < . vs lps, **p < . vs lps/tcs, n = . il- β interleukin- β, il- interleukin- , tnf-α tumour necrosis factor-α lps-stimulated tcs, six mirnas, including mir- - p, mir- a- p, mir- , mir- - p, mir- a- p and mir- a- p, were upregulated, and one mirna (mir- - p) was downregulated with an absolute fold change > ( fig. a and table ). by referring these results to two online databases (mirwalk and targetscan), target genes were predicted to be downstream targets of the differentially expressed mirnas. in total, mrnas- upregulated and downregulated-were differentially expressed in tcs after lps stimulation ( fig. b and table ). a total of genes overlapped with those from the online prediction (fig. c) . pulmonary tcs were reported to promote angiogenesis in a mouse model of ards [ ] ; thus, particular attention was devoted to angiogenesis in the gene ontology (go) functional analysis. according to the david online database, degs were enriched in the processes of blood vessel formation, angiogenesis, blood vessel morphogenesis, blood vessel remodelling, and sprouting angiogenesis. for further analysis, the degs were enriched in the string database. according to the string database, the degs were enriched in angiogenesis-related processes: angiogenesis, blood vessel morphogenesis, vasculature development, blood vessel remodelling, sprouting angiogenesis, venous blood vessel sprouting, venous blood vessel morphogenesis, regulation of angiogenesis, and positive regulation of angiogenesis (fig. d) . as most genes participated in at least biological processes, those involved in more than three processes-i.e. e f , notch , epas , rbpj, flt , acvrl , efnb and thbs -were selected for further research. mir- a- p, mir- - p, mir- a- p and mir- - p regulated these genes (fig. e) . we next assessed the mrna levels of angiogenesis factors. the mrna expression of e f , notch , epas , rbpj, flt , acvrl , efnb and thbs was measured in fig. differentially expressed genes in tcs with lps treatment. a heat map of differential expressed mirnas in cultured tcs stimulated with lps. b heat map of differential expressed genes in cultured tcs stimulated with lps. c relationship between differential expressed mirnas and their differential expressed target mrnas. yellow indicated mirnas, green indicated downregulated mrnas, and red indicated upregulated mrnas. d interaction of the angiogenesis-related proteins in string. different colours indicated the involvement of proteins in different processes. red indicated angiogenesis, pink indicated blood vessel morphogenesis, purple indicated vasculature development, brown indicated blood vessel remodelling, blue indicated sprouting angiogenesis, cyan indicated venous blood vessel sprouting, orange indicated venous blood vessel morphogenesis, green indicated regulation of angiogenesis, and yellow indicated positive regulation of angiogenesis. e angiogenesis related mirnas and their downstream genes. red indicated upregulated mirnas, green indicated downregulated mirnas, yellow indicated genes that were enriched in more than three processes in string, blue indicated other mrnas tcs after lps stimulation. after lps stimulation, e f , efnb , and epas were significantly downregulated, while flt was upregulated. given that mirnas usually negatively regulate downstream genes, e f , efnb , and epas were further studied. lps stimulation significantly increased mir- a- p and mir- - p expression in tcs compared with that in cells under control conditions. to clarify the relationship between mirnas and mrnas, mirna inhibitors were applied. mir- - p inhibition restored the expression of epas but not efnb , and mir- a- p inhibition restored the expression of e f but not epas . mir- had been reported to increase proliferation, migration and tube formation of human umbilical vein endothelial cells (huvecs) and induce angiogenesis by directly targeting pten [ , ] . moreover, mir- a- p and its downstream target e f were further studied. after h, the protein expression of e f was decreased in tcs challenged with lps and was restored by inhibition of mir- a- p. the dual luciferase reporter assay indicated that e f was the direct target of mir- a- p (fig. ). the transcription factors e f / were reported to regulate vessel branching via dll -notch approaches [ ] or hif- α/vegfa signalling [ ] . in the present study, lps stimulation reduced the protein expression of notch but not notch , notch or dll . inhibition of mir- a- p restored notch protein expression in tcs in the presence of lps. lps did not affect hif- α expression. however, lps increased the expression of vegfa at the mrna level, and this increase was reversed by mir- a- p inhibition in cultured tcs (fig. ). pi k, especially the class i catalytic isoforms, plays an important role in angiogenesis. to study the mechanisms underlying the effect of mir- a- p in tcs on angiogenesis induction, pi k subunit expression was first examined. the mrna levels of the class i pi k isoforms pik ca, pik cb, and pik cd did not significantly change with lps stimulation. however, the protein level of p α in tcs was significantly increased with lps stimulation and decreased with mir- a- p inhibitor co-treatment. the pi k signalling molecules akt, mtor, and pten were unaffected by either lps or mir- a- p. these results indicated that pi k signalling might participate in angiogenesis via the p α isoform (fig. ). the proliferation of eoma cells was then estimated after co-culture with tcs pre-treated with the mir- a- p or pi k p α inhibitor. culture medium from tcs stimulated with lps promoted eoma cells proliferation, as determined by the cck assay. compared with medium from nc tcs, culture medium from tcs with mir- a- p inhibition significantly reduced eoma cells proliferation (fig. a) . the effect of p α was examined by dynamic real-time cell observation. the proliferation assay indicated that eoma cells proliferation decreased with lps stimulation but was restored by co-culture with tcs. inhibition of mir- a- p or p α (with its inhibitor hs- ) weakened the protective effect of tcs (fig. b, d) . the scratch assay showed similar results (fig. c, e) . vegf protein expression was significantly elevated with lps stimulation, and this increase was reversed by inhibition of either mir- a- p or p α (fig. f ) . the results above indicated that vegf is regulated by both mir- a- p and p α. this study reports that tcs culture medium can alleviate ards in mice probably via angiogenesis-associated factors regulated by mir- a- p. tcs exposed to lps exhibited increased mir- a- p expression and vegf production, which further promoted vascular endothelial cell proliferation. the protective effects of tcs mediated by mir- a- p might be regulated through pi k (p α)/ akt/mtor signalling and the expression levels of the downstream targets e f and notch (fig. ) . endotoxin-induced ards has been reported to affect both respiratory epithelial cells and the underlying vascular endothelial cells [ ] . in the present study, lps stimulation induced severe vascular damage in the lungs, as shown by the reduced levels of cd and enos. tcs are distinct from mesenchymal stem cells and fibroblasts and have been reported to have specific roles in cell signalling, tissue remodelling and angiogenesis [ ] . in the present study, tcs culture medium exhibited great potential to reverse the angiogenic signalling that was reduced by lps-induced inflammation, supporting the observation that tcs alleviate lps-induced lung injury in mice by releasing angiogenic factors [ ] . non-coding mirnas are involved in several pathological processes, including angiogenesis [ , ] . mir- - p [ ] , mir- a- p [ ] , and mir- a- p [ ] [ ] [ ] are reported to be associated with the angiogenesis process. mir- a- p and mir- - p were demonstrated to be involved in the promotion of angiogenesis in tcs. as mir- a- p was more frequently reported on angiogenesis, it was further studied. mir- a- p knockdown fig. mir- a- p and p α mediated the promotion of tcs on eoma proliferation induced by lps. a cells proliferation rate of eoma treated with lps and/or tcs and mir- a- p inhibitor measured by cck assay. *p < . vs control, # p < . vs lps/tc. b, d cell proliferation of eoma treated with lps and/or tcs and mir- a- p inhibitor or p α inhibitor measured by cell-iq. *p < . vs control, # p < . vs lps, **p < . vs lps/ tc, n = . c, e cell movement of eoma treated with lps and/or tcs and mir- a- p inhibitor or p α inhibitor measured by cell-iq. f vegfa levels secreted by tcs treated with lps and/or mir- a- p inhibitor or p α inhibitor measured by elisa. *p < . vs control, # p < . vs lps, n = : in tcs reduced cd and enos expression in the lungs of ards mice in vivo. mir- a- p exerts its protective effects on injury repair by inducing angiogenesis-associated signalling pathways. for instance, mir- a- p activates the akt pathway and increases matrix metalloproteinase- (mmp- ) expression to reduce the extent of the infarcted region in heart ischaemia/reperfusion injury [ ] , inhibits pten and sprouty homolog (spry ) to heal soft tissue wounds [ ] , and upregulates vegf and activates the ang- /tie- axis in traumatic brain injury [ ] . in the current study, the p α isoform in pi k/akt/mtor signalling pathway was demonstrated to be involved in mir- a- p-mediated angiogenic factor induction in tcs. however, the alteration of other protein levels and hif- α in tcs treated with lps and the mir- a- p inhibitor indicated that more complex signalling pathways were involved in regulating the angiogenic function of tcs. culture medium from lps-induced tcs promoted eoma cells proliferation in vitro, accompanied by elevated levels of vegf mrna and secretion, which further demonstrated that the functional mir- a- p was generated by tcs. these data support the hypothesis that mir- a- p plays a role in angiogenesis and profoundly demonstrate the mechanisms mediated by pi k p α. the e f family was first reported to induce cell proliferation [ ] , and e f family members are essential transcriptional regulators of cell cycle progression [ ] , as well as apoptosis, metabolism and angiogenesis [ ] . e f is an atypical transcriptional repressor in the e f family since it contains domains that differ from the canonical domains [ ] . by forming homodimers or heterodimers with e f , e f reduces the excessive and destructive activation of e f [ ] . however, reports of e f in angiogenesis in the literature are controversial. e f / has been reported to positively regulate the formation of blood vessels during embryonic development via hif- α/vegfa signalling [ ] . on the other hand, e f / suppresses tumour angiogenesis via the induction of dll [ ] . in the present study, e f expression was reduced after lps stimulation in tcs and restored with mir- a- p inhibition, indicating that e f plays a negative role in angiogenesis under inflammatory conditions. the notch family, which contains several receptors and ligands, is fundamental in the regulation of blood vessel branching [ ] . dll , a notch ligand, has an inhibitory function in blood vessel branching [ ] that is compromised by jagged activation [ ] . notch positively regulates angiogenesis [ ] , while notch negatively regulates cell proliferation [ ] and angiogenesis [ ] . in the initial stage of angiogenesis, inhibition of notch promotes vascular endothelial cell proliferation, while activation of notch reduces endothelial cell responses to vegf [ , ] . in the present study, notch expression was mediated by mir- a- p. however, the relationship between the transcription factor e f and notch was not illustrated. further experiments should be conducted to confirm the signalling pathway of e f /notch in angiogenesis. tcs have been reported to be important in tissue repair and healing processes. via mouse models, bioinformatics approaches and molecular biological methods, the present study shows that activated tcs promote endothelial regeneration and angiogenesis through mir- a- p-pi k (p α)/akt/mtor signalling and further demonstrates the key roles of vegf in tcs. the e f /notch signalling might also participates in this process. these findings shed light on mir- a- p in tcs as a new therapeutic target for vessel protection. epidemiology, patterns of care, and mortality for patients with acute respiratory distress syndrome in 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alleviates lps-induced injury by targeting smad in c /i chondrocytic cells notch activation during endothelial cell network formation in vitro targets the basic hlh transcription factor hesr- and downregulates vegfr- /kdr expression publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the authors would like to thank dr. dongli song supplementary information accompanies this paper at https ://doi. org/ . /s - - -z.additional file : figure s . the morphology of tcs. the pictures were gathered by cell-iq every h. the white arrow showed the typical telopode. not applicable. this study was approved by the ethics committee of the zhongshan hospital biomedical research department. not applicable. the authors declare that they have no competing interests. key: cord- - r n authors: sideras, paschalis; apostolou, eirini; stavropoulos, athanasios; sountoulidis, alexandros; gavriil, arianna; apostolidou, anastasia; andreakos, evangelos title: activin, neutrophils, and inflammation: just coincidence? date: - - journal: semin immunopathol doi: . /s - - - sha: doc_id: cord_uid: r n during the years that have elapsed since its discovery, activin-a, a member of the transforming growth factor β super-family originally discovered from its capacity to stimulate follicle-stimulating hormone production by cultured pituitary gonadotropes, has been established as a key regulator of various fundamental biological processes, such as development, homeostasis, inflammation, and tissue remodeling. deregulated expression of activin-a has been observed in several human diseases characterized by an immuno-inflammatory and/or tissue remodeling component in their pathophysiology. various cell types have been recognized as sources of activin-a, and plentiful, occasionally contradicting, functions have been described mainly by in vitro studies. not surprisingly, both harmful and protective roles have been postulated for activin-a in the context of several disorders. recent findings have further expanded the functional repertoire of this molecule demonstrating that its ectopic overexpression in mouse airways can cause pathology that simulates faithfully human acute respiratory distress syndrome, a disorder characterized by strong involvement of neutrophils. this finding when considered together with the recent discovery that neutrophils constitute an important source of activin-a in vivo and earlier observations of upregulated activin-a expression in diseases characterized by strong activation of neutrophils may collectively imply a more intimate link between activin-a expression and neutrophil reactivity. in this review, we provide an outline of the functional repertoire of activin-a and suggest that this growth factor functions as a guardian of homeostasis, a modulator of immunity and an orchestrator of tissue repair activities. in this context, a relationship between activin-a and neutrophils may be anything but coincidental. the transforming growth factor β (tgf-β) super-family encompasses a large group of structurally related polypeptide growth factors including tgf-β isoforms, activins, bone morphogenetic proteins (bmps), growth differentiation factors (gdfs), nodal, and müllerian inhibiting substance [ ] . numerous studies utilizing genetically modified transgenic or knock-out mouse lines have demonstrated that whereas normal signaling by this system plays a pivotal role during early development, adult homeostasis, and tissue injury-repair, its deregulation can contribute to development of pathology. activins together with structurally related inhibins form a subgroup within the tgf-β-super-family. although activin-a, the prototype member of this subfamily, was initially identified on the basis of its ability to stimulate the release of follicle-stimulating hormone (fsh) from pituitary gonadotrophes in vitro [ ] , numerous studies that followed have demonstrated that it is actually a remarkable pluripotent growth factor essential for embryonic development [ ] [ ] [ ] [ ] [ ] [ ] and adult tissue homeostasis [ ] [ ] [ ] [ ] [ ] [ ] . activins and inhibins are homo-or heterodimeric proteins formed by the interaction of "α" and "β" subunit which are encoded by distinct genes [ ] . one gene encoding for the "α" subunit (inhibin-α) and four genes encoding for "β" subunits (inhibin-βa, inhibin-βb, inhibin-βc, and inhibin-βe) have been characterized in mammals [ , [ ] [ ] [ ] . activins are formed by homo-or heterodimerization of inhibin-β subunits. so far, the homodimeric βa/βa (activin-a) and βb/βb (activin-b) and the heterodimeric βa/βb (activin-ab) dimers have been purified from biological fluids and their activity assessed, with activin-a receiving the greatest attention till now. heterodimerization of the inhibin-α subunit with the inhibin-βa or -βb subunits results in the production of the naturally occurring activin antagonists inhibin-a and inhibin-b, respectively (reviewed by de kretser et al. [ ] ). the functions of activin-a, activin-b, and activin-ab appear to be very similar; however, the expression pattern of the βa and βb subunits differs [ ] and mice deficient for either of these subunits exhibit different phenotypes [ , ] suggesting that these activin forms may perform overlapping functions, without however being fully redundant. absence of any detectable phenotype in βc or βe knockout mice led initially to the conclusion that their function can be compensated [ ] . however, subsequent studies demonstrated that the βc subunit can form homodimers or heterodimers with the βa or βb subunits and it was postulated that these forms might compete for receptor occupancy providing thus an antagonistic regulatory mechanism [ , ] . consistently, it was shown that recombinant activin-c could antagonize activin-a in vitro and transgenic animals overexpressing inhibin-βc were found to develop numerous abnormalities in testis, liver, and prostate [ ] . tgf-β super-family ligands signal via ternary receptor complexes that are formed by two types ii and i receptor polypeptides [ ] . both types of receptors possess serinethreonine kinase activity. the type ii receptors are constitutively active and thus upon interaction with the ligand come in proximity with the type i receptors, phosphorylate them and activate their kinase activity. activated type i receptors phosphorylate and activate receptor-activated smads (r-smads), the intracellular effector molecules of this system. tgf-β super-family signaling can be classified into two branches: the tgf-β branch, represented by ligands, such as tgf-β, activin, nodal, or myostatin, which leads to activation of smad and smad ; and the bmp branch, represented by ligands, such as bmps and gdfs, which leads to activation of smad , smad , and smad . phosphorylated r-smads form complexes with the common mediator, smad and enter the nucleus where they bind to dna and interact with transcription factors to regulate gene expression [ ] . finally, the inhibitory smads (i-smads) smad and smad are induced upon tgf-β superfamily ligand-mediated signaling and exert a negative feedback effect by competing with r-smads for receptor interaction and by marking the receptors for degradation [ ] . tgf-β super-family ligands can also signal via smad-independent intracellular pathways that involve mitogen-activated protein kinases and are referred to as "non-canonical" [ ] . activins utilize for signaling the type i receptors activinlike kinases (alks), alk- , alk- , and alk- and the type ii receptors act-riia and act-riib [ ] [ ] [ ] [ ] . activin-a binds to the type ii receptor and cooperatively recruits the type i receptor [ ] . activins, like other tgf-β superfamily proteins, are synthesized as large precursor proteins that contain nterminal pro-regions and c-terminal mature domains [ ] . the pro-domains guide correct folding and dimerization and contribute to biological activity of mature activins [ ] [ ] [ ] . the dimeric precursors are proteolytically cleaved and activins are secreted from the cell noncovalently associated with their pro-peptides. outside the cell, the pro-domain of activin-a can target it to heparin sulfated proteoglycans [ ] thus forming gradients around the cells that produce it and also protecting it from proteolytic attack [ ] . interestingly, activin-b lacks the critical residues required for binding to heparin sulfated proteoglycans and may diffuse more easily away from the source of its production, affecting the relative local concentration of mature activin-a and activin-b in a tissue [ ] . whereas tgf-β isoforms bind their propeptides with very high affinity and therefore are secreted from cells as latent complexes [ ] , the affinity of the βa pro-peptide for mature activin-a is very low (kd nm). although this affinity can support sequestration of activin-a within tissues, the pro-peptide can be easily displaced by the much higher affinity of the mature peptide to activin type ii receptors (kd pm), and therefore, activin-a is secreted in the extracellular spaces "practically" in an active form [ ] . the function of activins is tightly regulated at several levels. inhibin-a and inhibin-b, sharing the same β-subunits as activin-a and activin-b are able to compete for the activin receptors acting thus as extracellular regulators of activin signaling [ ] . furthermore, follistatin and follistatin-like protein- (fstl- ) [ ] [ ] [ ] , are synthesized and secreted independently, can bind to activins with high affinity and induce their rapid endocytosis and proteolytic degradation modulating thus the amounts of bioavailable activins in the tissues [ ] . follistatin was initially identified due to its ability to suppress fsh release by the rat pituitary [ ] , however, it was later demonstrated that this was mediated via blocking activin's access to its receptors [ ] . two follistatin variants generated through alternative splicing of a primary mrna have been described, namely, follistatin- (fs- ), and follistatin- (fs- ) [ ] . fs- possesses a heparan binding sequence that enables association with heparan sulfate proteoglycans. it normally associates with activins at the cell surface and facilitates their internalization and degradation [ ] . however, it can be released into the circulation by heparin [ , ] . fs- on the other hand is the predominant follistatin form in the circulation. it can bind to heparin sulfate only after interaction with activins due to a conformational change that exposes its heparan sulphate binding segment [ , ] . a third isoform, fst- has been identified as a proteolytic by-product of fst- [ ] . fstl- lacks heparan binding segment and thus behaves like fs- [ ] . the crystal structures of activin-a in complex with fstl- [ ] and fst- [ ] have been solved. in both these models, two follistatin molecules encircle the activin dimer, such that one quarter of the activin surface is buried. both forms of follistatin contact two discontinuous surfaces of activin, shielding both the types i and ii receptor binding sites [ , ] . interestingly, follistatin binds to activin-b with an affinity -fold lower that to activin-a [ , ] indicating a possibility for differential regulation of these two activins. furthermore, follistatin can inhibit other tgf-β ligands such as myostatin and gdf- [ ] and likewise, fstl- can bind myostatin [ , ] and some members of the bmp subfamily [ , , ] . thus, follistatin variants and follistatin-like proteins provide a versatile mechanism for local or systemic regulation of the levels of bioavailable activins and some other members of the tgf-β superfamily. bioavailability of activin-a can be modulated by the action of additional molecules, such as α -macroglobulin, cerberus, and lipovitelin (reviewed by philips [ ] ). activin-a expression is upregulated in disorders characterized by immuno-inflammatory and/or tissue remodeling pathophysiology one of the biological processes with which deregulated activin-a expression and function were implicated early on as contributor of pathogenesis, was fibrosis. hedger et al. demonstrated that activin-a could stimulate mitosis of t fibroblasts in vitro [ ] . although, its capacity to stimulate fibroblasts was a fraction of that of tgf-β, this study provided an early link between activin-a and fibrosis. expression of activin-a protein was detected by immunofluorescence in bronchiolar epithelium and smooth muscle cells of veins in both control and bleomycin-treated mice and increased activin-a secretion was observed in cultures of alveolar macrophages isolated from bleomycin-treated animals [ ] . although the bleomycin model resembles more acute lung injury (ali) than bona fide fibrosis, a subsequent report describing increased activin-a immunostaining in metaplastic epithelium, hyperplastic smooth muscle cells, desquamated cells, alveolar macrophages, and smooth muscle cells, admittedly in a very small group of patients with different forms of interstitial lung disease, firmly-associated activin-a with the fibrotic process in the literature [ ] . this association was further strengthened by the observations that: (a) activin-a could increase fibroblast migration and their differentiation to myofibroblasts in vitro [ ] , (b) prophylactic administration of follistatin could ameliorate bleomycin-induced pathology in mice [ ] , (c) increased levels of activin-a produced by hepatic stellate cells upon carbon tetrachloride-induced liver injury were contributing to development of hepatic fibrosis, the severity of which could be diminished by the co-administration of follistatin [ ] , and (d) the detection of increased serum levels of activin-a, in patients with systemic sclerosis and the demonstration that activin-a could upregulate in vitro collagen production by fibroblasts isolated from patients with systemic sclerosis [ ] . detection of skin abnormalities in animals deficient for either activin-a or follistatin and development of hyper-keratotic skin in the later provided the first evidence pointing towards involvement of the activin-a/follistatin system in skin remodeling [ , ] . consistently, substantial increase of activin-a and activin-b expressions was detected after skin injury in mice [ ] . in situ hybridization studies revealed different spatiotemporal expression pattern for the two activins, with activin-a being expressed in the granulation tissue below the wound and activin-b in the hyper-proliferative epithelium. while both activins were upregulated during the first days post-injury, after days, activin-a expression was back to baseline levels while activin-b expression persisted [ ] . overexpression of activin-a in the epidermis of transgenic animals under the control of the keratin- promoter caused dermal fibrosis, epidermal hyper-thickening, and enhanced wound repair [ ] . conversely, transgenic animals where follistatin was overexpressed using the same promoter as above, had a mild dermal and epidermal atrophy and after injury exhibited a severe delay in wound healing. however, upon healing the wounds presented substantially reduced scar formation [ , ] . it should be noted that the aforementioned studies constitute the earliest direct demonstration of the detrimental effect of deregulated activin-a expression in vivo. interestingly, local upregulation of activin-a has been detected by immunohistochemistry during the inflammatory and repair phases that occur in burnt skin as well [ ] . although collectively these reports have implicated activin-a in the processes associated with tissue repair and fibrosis, its actual role in the context of these processes must be quite complex. activin-a levels were found to be upregulated by several profibrotic agents such as tgf-β , tumor necrosis factor-α (tnf-α), endothelin, interleukin (il)- , thrombin, and angiotensin, and conversely activin-a itself was found to be able to stimulate production of pro-fibrotic factors, including tnf-α, connective tissue growth factor (ctgf), endothelin, type collagen, tissue inhibitor of metalloproteinase- , and plasminogen activator inhibitor- (pai- ) (reviewed by de kretser et al. [ ] ). activin-a has been implicated in the development of allergic airway inflammatory conditions and thus in the pathophysiology of asthma. rosendahl et al. [ ] using an experimental model of ovalbumin (ova)-induced airway inflammation demonstrated a dramatic increase in the numbers of inflammatory and structural cells expressing nuclear phosphorylated smad within the allergenchallenged lungs that was accompanied by substantial increase in the mrna levels of inhibin-βa, inhibin-βb, inhibin-βc, inhibin-βe, and alk (the activin type i receptor) and thus linked for the first time activin-a with respiratory inflammation [ ] . the observation that the rat basophilic leukemia (rbl- h ) [ ] and human mast cells (mc) [ ] could produce activin-a upon stimulation with ionomycin or ionomycin plus pma respectively, the colocalization of activin-a with mc in biopsies from human asthmatics, and the defective production of activin-a upon allergen challenge in mc-deficient mice [ ] linked activin-a with mc function and suggested that these cells were an important source of activin-a in the inflamed airways. increased activin-a levels were detected in the serum of patients with severe asthma compared with levels of patients with moderate asthma and healthy control subjects and increased mrna levels were detected in t cells from patients with moderate asthma. expression of activin-a by infiltrating lymphocytes and structural cells of the lung was detected in tissue sections from ova challenged mice [ ] or human asthmatic patients by immunohistochemistry [ , ] and activin-a was found to promote proliferation of human airway smooth muscle cells, and regulate migration and differentiation of bone marrow-derived mc progenitors [ ] . concurrent release of activin-a and follistatin in the lungs of ova challenged mice with maximal bal concentrations coinciding with maximal airway eosinophilia and frequency of il- , il- and il- producing cells in mediastinal lymph nodes was reported by hardy et al. [ ] . immunohistochemical staining pointed towards airway epithelial cells as the source of activin-a and follistatin, and decline of immunostaining for both molecules that paralleled goblet cell metaplasia was interpreted to postulate that these molecules were pre-stored and released upon allergen stimulation [ ] . although upregulation of activin-a in the context of airway inflammation is well documented, conflicting views have been put forward regarding the role of this molecule in asthma pathophysiology based on studies in the mouse ova allergic airway inflammation model. hardy et al. [ ] observed that neutralization of the allergen-induced activin-a with exogenous follistatin had a beneficial effect [ ] , however, semitekolou et al. [ ] using the same animal model observed exacerbation of allergen-induced pathology upon neutralization of activin-a using specific antibodies and attenuation of pathology upon treatment of the allergen challenged animals with recombinant activin-a. an interesting twist in the interplay of activin-a with allergic airway inflammation is the resent report demonstrating that activin-a, like tgf-β, could drive in vitro generation of t(h) cells [ ] . importantly, in vivo inhibition of t(h) differentiation induced by allergen and reduction of airway hyper-reactivity and collagen deposition could be accomplished only by blocking both activin-a and tgf-β [ ] . thus, although activin-a levels are increased during airway inflammatory responses, the actual role of this molecule and consequently it's potential therapeutic utility for asthma therapy definitely needs further substantiation. serendipitously, while studying the effect of castration on the levels of circulating follistatin in rams, philips et al. [ ] observed a robust elevation in the levels of circulating follistatin both in castrated and sum-operated rams. this surprising finding demonstrated that tissues other than the gonads could produce follistatin and also associated upregulation of follistatin levels with the inflammatory response associated with the surgery [ ] . this notion was further substantiated by replicating similar follistatin release patterns in sheep injected with lipopolysaccharide (lps) [ ] , sheep injected with recombinant il- β or in lambs subjected to intra-thoracic injection of yeast [ ] . a few years later, the development of activin-a-specific immunoassays allowed jones et al. [ ] to monitor activin-a levels in the serum of lps-injected sheep and demonstrate that activin-a was released within min from lps injection, slightly earlier than tnf-α and significantly earlier than il- [ ] . the observation that serum activin-a levels exhibited a biphasic response provided the basis for the hypothesis that the initial release of activin-a was due to pre-stored material released from responding cells, whereas the latter release was due to newly synthesized material [ ] . similar results were obtained in the mouse system where upon lps injection a rapid initial rise of serum activin-a was followed by a secondary rise - h later [ ] . survival and other pathology-associated parameters in lps-treated mice improved upon prophylactic administration of follistatin [ ] or prophylactic and therapeutic ( -h post-lps injection) administration of an activin neutralizing fusion protein composed of the extracellular portion of actriib fused to the fc portion of the human igg molecule [ ] . detection of increased levels of activin-a in the serum of patients with sepsis [ ] and increased activin-a levels in cerebrospinal fluid of rabbits [ ] and human patients [ ] with bacterial meningitis raised the possibility that activin-a could be a crucial player in the pathophysiology of sepsis. detection of increased levels of activin-a mrna in mouse models of inflammatory bowel disease [ ] and in biopsies of crohn's disease or ulcerative colitis patients [ ] suggested that activin-a may play an important role in the pathophysiology of inflammatory bowel disease. the demonstration by dohi et al. [ ] that prophylactic and therapeutic treatment with follistatin could improve the survival rate of mice in three mouse colitis models, attenuate several pathology-associated parameters and upregulate proliferation of intestinal epithelial cells and tissue repair, improving thus the barrier function of the colonic mucosa, strongly supported the association between activin-a and colitis pathophysiology [ ] . it should be noted that the study of dohi et al. is among the very few activin-a related studies where activin-a upregulation within the diseased tissues was verified by in situ mrna hybridization and the beneficial effect of follistatin was demonstrated in a therapeutic protocol with activin-a neutralization commencing after the onset of pathology. several studies have implicated activin-a in the pathophysiology of inflammatory arthropathies. high levels of activin-a, in some reports exceeding ng/ml were detected in synovial fluid of patients with rheumatoid arthritis and gout [ ] [ ] [ ] . activin-a was detected in fibroblastoid synovial cells and cd + macrophage-lineage cells in the proliferative reactive synovial membranes obtained from rheumatoid arthritis patients, as well as in and the smooth muscle and the endothelial layer of the arteries in these vascularized proliferative tissues [ , ] . furthermore production of activin-a by synoviocytes and chondrocytes was found to be upregulated in vitro by il- β, tgf-β, interferon-γ (ifn-γ), and il- [ , ] . in patients with heart failure, serum levels of activin-a were found to be significantly elevated with magnitudes correlating with disease severity, and increased levels of inhibin-βa mrna were detected in t cells but not monocytes [ ] . furthermore, in a rat model of heart failure induction of activin-a expression by cardiomyocytes and concerted increase in activin types i and i receptor mrna was observed after myocardial infarction. moreover, treatment of neonatal rat cardiomyocytes with activin-a was found to induce expression of mediators involved in infarction healing and myocardial remodelling such as matrix metalloproteinase- , tissue inhibitor of metalloproteinase- , transforming growth factor-beta- , and monocyte chemo-attractant protein- . the above findings were interpreted to suggest involvement of activin-a in the pathogenesis of heart failure. collectively, overexpression of activin-a has been observed in experimental models and patients suffering from acute inflammatory disorders (sepsis, meningitis, and endotoxemia), autoimmune disorders (rheumatoid arthritis, inflammatory bowel disease, and systemic lupus erythematosous), traumatic injuries (skin burns and surgery) and diseases with a strong tissue remodeling element (skin, liver, lung, and kidney fibrosis). the beneficial effect of activin-a neutralization via prophylactic, and, in some cases, therapeutic administration of follistatin in disease relevant animal models strongly suggested that activin-a was not a simple bystander but rather a key component of the pathogenetic mechanisms [ ] , raising hope that the activin-a signaling pathway could constitute an attractive target molecular system for development of therapeutic interventions. given the strong association of activin-a to human pathology, special effort has been allocated for the identification of the cellular sources of this growth factor in the context of the different physiological and pathological conditions with which it has been associated with, the characterization of the cellular responses triggered by activin-a and more importantly, the identification of the activin-a-induced responses which, when deregulated, could cause pathology. hence, numerous studies have provided information regarding the secretion of protein or the synthesis of mrna for activin-a, in tissues and isolated cell subpopulations of immune and non-immune origin. thus, it was shown that human peripheral blood monocytes produce activin-a upon stimulation with lps [ ] , granulocyte-macrophage colonystimulating factor (gm-csf), ifn-γ [ ] , or trem- [ ] . expression of mrnas for activin types i and ii receptors (actri, actrib, actrii, and actriib) and secretion of activin-a by mouse peritoneal macrophages was demonstrated after stimulation with agonists of toll-like receptor (tlr) , , and [ , ] . production of activin-a by activated microglia and infiltrating macrophages was demonstrated in an animal model of experimental meningitis induced by inoculation of streptococcus pneumoniae in the csn [ ] . human monocyte-derived dentritic cells and cd c + and cd + peripheral blood dendritic cell (dc) populations were found to express both types i and ii activin receptors and secrete high levels of activin-a after exposure to bacteria, specific tlr agonists, or cd ligand (cd l) [ ] [ ] [ ] . interestingly, blood plasmacytoid dc did not synthesize activin-a upon stimulation with influenza virus [ ] . human mc upon stimulation with pma plus calcium ionophore (a ) and cultured murine bone marrow mc (bmmc) upon stimulation by cross-linking the ige receptors were found to produce activin-a as well [ ] . anti-cd stimulated cd pos cd neg t cells, and not cd pos cd pos regulatory t cells, especially when cultured under th polarizing condition were found to secrete substantial amounts of activin-a in the culture medium, including thus t cells among the putative sources of bioactive activin-a [ ] . finally, activated b cells were also found to express types i and ii activin receptors and to secrete activin-a [ ] . the studies discussed earlier by jones et al. in sheep [ ] demonstrated that upon lps administration activin-a levels in the serum increased displaying a biphasic response. the biphasic activin-a response was interpreted as providing to support for the hypothesis that the early response was due to the release of pre-stored activin-a, whereas the second delayed response was due to de-novo synthesis. activin-a and follistatin were detected by immunohistochemical staining in mouse airways [ ] . the reduction of this immunostaining upon challenge of ova sensitized animals with allergen correlated with increased levels of activin-a and follistatin in the bal and development of allergic airway inflammation in the airways. these findings were used to further support the notion that preformed activin-a was released in the early stages of an inflammatory response [ ] . analysis of freshly isolated human neutrophils demonstrated that, compared to blood mononuclear cells, they contained -fold more activin-a which they could release rapidly upon stimulation with tnfα, introducing thus neutrophils as an important source of activin-a. the fact that tnfα stimulated neutrophils in vitro released activin-a within h although corresponding mrna levels were not increased until h of culture supported the "pre-stored activin-a" concept [ ] . expression of activin-a by neutrophils and stimulation of its secretion by tnfα was also confirmed with murine bone marrow derived neutrophils [ ] . finally, in a comprehensive study, wu et al. demonstrated that basically all analyzed tissues from untreated or lps-treated mice contained detectable activin-a and follistatin mrna and protein [ ] . the levels of activin-a in the different tissues analyzed ranged substantially indicating that homeostatic levels of activin-a differ among different tissues. the highest mrna expression was found in the liver, and the highest concentration of activin-a protein in the bone marrow. upon lps treatment, activin-a and follistatin mrna levels did not change significantly within the first hour after lps treatment in any of the analyzed tissues, however, a % decrease and a -fold increase in activin-a protein level was observed in bone marrow and lung respectively. these changes were associated with migration of bone marrow-derived, activin-a-loaded, neutrophils into the lung parenchyma. again the authors interpreted these observations as suggesting that the rapid increase in circulating activin-a during lps-induced inflammation is regulated at the posttranscriptional level, from newly translated and/or stored protein. interestingly, despite the implication of neutrophils as important activin-a producers, so far little is known regarding the effect of activin-a on the functionality of these cells. we have isolated epithelial, endothelial, inflammatory, and mesenchymal cells from enzymatically digested control and ova-challenged animals and analyzed the mrna levels for activin-a and follistatin. activin-a mrna was detected in all cell types in the untreated lungs, however, follistatin mrna was found exclusively in the mesenchymal fraction, i.e., epcamneg /cd neg /cd neg cells (unpublished observations). upon allergen challenge, the increased activin-a mrna (> -fold) was mainly in the cd pos fraction and again upregulation of follistatin mrna was detected exclusively in the mesenchymal fraction (unpublished observations). although these observations do not exclude the possibility that some pre-stored activin-a or follistatin could be released in the airways, they are more compatible with the notion that the bulk of these molecules, at least in the ova-induced allergic airway inflammation model, is most likely newly synthesized. activin-a was not only found to be produced by different cells of the immune system, as outlined above, it was also shown to affects their function in various ways depending on the activation state of the target cells and the coexistence of other stimulatory agents. thus, activin-a could stimulate the migration of immature murine dcs [ ] , acting as a proinflammatory agent. however, human monocyte derived dcs and cd c + peripheral myeloid dcs, when stimulated in vitro with cd ligand (cd l) in the presence of follistatin to block the autocrine action of activin-a, produced higher levels of dc cytokines (il- , il- , il- p , and tnf-α) and chemokines (il- , ip- , rantes, and mcp- ) [ ] suggesting thus indirectly a pro-inflammatory effect of activin-a on dcs. moreover, in the same study, it was shown that neutralization of the dc-derived activin-a significantly enhanced the capacity of dcs pulsed with chemically inactivated whole flu particles to stimulate expansion of viralantigen-specific effector cd + t cells in vitro [ ] . these findings combined with the demonstration that activin-a could inhibit maturation of dcs [ , ] and the fact that interaction of t cells with immature dcs could produce an immunosuppressive or tolerogenic response [ ] , could collectively indicate a potential, activin-a-mediated, autocrine feedback attenuation of dc-mediated responses. equally complex appeared to be the effect of activin-a on macrophage functions. thus, it has been shown that activin-a can stimulate monocytes/macrophages to produce inflammatory mediators, such as il- β, tnf-α, il- , nitric oxide (no), prostaglandin e , and thromboxanes [ ] [ ] [ ] [ ] , enhance the phagocytic capacity of mouse and rat peritoneal macrophages in vivo and in vitro [ , ] , and promote human monocyte to langerhans cell (lc) differentiation and migration in vitro [ ] . in contrast, other studies demonstrated that activin-a could have anti-inflammatory effects on macrophages that were already activated or in a primed state. thus, activin-a was shown to attenuate no release, phagocytic and pinocytic capacity and expression of cd and mhc ii by lps activated mouse peritoneal macrophages [ ] . moreover, decreased secretion of il- β and no as well as il- β and inos mrna, and reduced expression of cd , cd , and tlr was observed upon lps stimulation in the mouse macrophage cell line raw . [ ] . likewise, using activated human monocytic cell lines thp- and u- it was shown that activin-a could inhibit the production of il- β and enhance at the same time the production of il- receptor antagonist, resulting thus in attenuation of il- β biological activity [ ] . using the murine microglial cell line mg it was shown that activin-a could mitigate its activation by lps and attenuate upregulation of pro-inflammatory mediators including il- , il- and inos [ ] . likewise, using primary rat microglia cell cultures, it was shown that activin-a was able to decrease ifn-γ-induced synthesis of no and lps-induced tnf-α, il- , inducible no synthase, and il- β [ ] . based on the observation that activin-a could stimulate peritoneal macrophages to express arginase- (arg- ), one of the markers of alternatively activated macrophages (m ), and could also inhibit the ifn-γ-induced expression of inducible no synthase, one of the markers of inflammatory (m ) macrophages, ogawa et al. proposed that activin-a promotes differentiation of macrophages toward the m phenotype [ ] . however, a recent study by sierra-filardi et al. demonstrated that activin-a was expressed preferentially by m macrophages and more importantly, promoted differentiation of macrophages toward the m phenotype [ ] . neutralization of activin-a with specific antibodies in cultures of macrophages that were treated with gm-csf (conditions that normally lead to m polarization) led to reduced expression of m markers (ccl , ecscr, and ccr ) and increased expression of m markers (mafb, ets , folr , il , igf , and serpinb ), further supporting the notion that activin-a is an inducer of m macrophages [ ] . although the above two studies appear to contradict each other, they might actually present a variation of the same theme, namely, that activin-a affects in different ways naïve and activated or primed cells. the peritoneal macrophages used by ogawa et al. [ ] were murine and were collected after intra-peritoneal injection of thioglycolate, certainly not a resting macrophage population; whereas the macrophage populations analyzed in the study of sierra-filardi et al. [ ] originated from human peripheral blood monocytes and were polarized using appropriate culture conditions in vitro. activin-a was also found to affect t cell function. early studies demonstrated that recombinant activin-a could inhibit phytohaemaglutinin (pha)-induced rat thymocyte proliferation in vitro [ ] . more importantly, activin-a was shown to enhance in vivo the conversion of mouse peripheral naive t cells to foxp + t regulatory cells (tregs) and amplify the capacity of tgf-β to promote in vitro development of foxp + tregs [ ] . induction of another il- producing, however, foxp -t regulatory population by activin-a has been described by semitekolou et al. [ ] . thus, activin-a appears to attenuate t cell responses in two ways, either by inhibiting directly t cell responsiveness or indirectly by promoting the generation of t regulatory cells. natural killer (nk) cells have also been listed among the inflammatory cells affected by activin-a. robson et al. [ ] demonstrated that nk cells express types i and ii activin receptors and that activin-a directly regulates nk cell functions by downregulating expression of the t-box transcription factor t-bet, secretion of ifn-γ, and expression of cd [ ] . finally, b lymphocytes have not been exempt from the regulatory influences of activin-a as it has been shown that through direct action on resting b cells, activin-a could enhance igg and ige production [ ] , induce iga production from mouse mesenteric lymph node cells [ ] and decrease proliferation and autoantibody production from b cells of individuals with pulmonary alveolar proteinosis [ ] . in accordance with the broad expression pattern of activin-a described in the study of wu et al. [ ] , other cell types of non-immune origin have been described earlier as sources of activin-a and follistatin. for example, it has been shown that bovine aortic endothelial cells upon lps stimulation upregulate activin-a and follistatin mrna synthesis and protein secretion [ ] . bone marrow-derived stromal fibroblasts were found to produce activin-a, and this was enhanced by il- β and lps and inhibited by ifn-γ. interestingly, the opposite was observed with follistatin secretion, namely, il- β and lps exhibiting an inhibitory and ifn-γ a stimulatory effect on follistatin secretion by the same type of cells [ ] . production of activin-a has also been demonstrated in cultures of synoviocytes and chondrocytes upon stimulation with il- β, tgf-β, ifn-γ, and il- [ ] , consistent with the detection of high activin-a levels in the synovial fluid of patients with rheumatoid arthritis and gout. moreover, production of activin-a was demonstrated upon serum stimulation in cultures of quiescent, skin derived keratinocytes and fibroblasts, and again activin-a production was found further enhanced by il- β and tnf-α [ ] . hepatocytes and hepatic stellate cells have also been described as activin-a producers [ ] . collectively, the existing information indicates that activin-a mrna and protein are synthesized continuously in healthy tissues. in the context of immuno-inflammatory responses, different types of inflammatory cells and injured or stressed tissue-resident cells, such as endothelial, epithelial, and mesenchymal cells can contribute to activin-a production. activin-a depending on its concentration, context, and the previous history of its cellular targets or the presence of other regulatory signals in the micro-environment can stimulate sometimes apparently counteracting responses. despite the long list of putative functions that have been attributed to activin-a primarily from in vitro studies, and the continuously expanding list of diseases with which activin-a has been associated, until recently, little direct in vivo demonstration of the pathogenic consequences of deregulated activin-a expression has been produced [ , [ ] [ ] [ ] . therefore, to validate directly the consequences of deregulated activin-a expression in vivo, we overexpressed activin-a in the mouse airways using adenovirus-mediated gene transfer and meticulously monitored functional and structural alterations in the respiratory system for up to months, the time period post-viral instillation required for the activin-a-induced response to unfold fully [ ] . these studies demonstrated that ectopic overexpression of activin-a in mouse airways could cause severe and chronic respiratory pathology, which even four months after virus instillation was not fully resolved. virus-derived activin-a was detected in the broncho-alveolar lavage (bal) fluids of the experimental animals reaching maximum after seven days, and persisting at increased levels up to day . follistatin mrna levels were upregulated with a relatively delayed kinetic reaching maximum levels around days - and remaining high until day . the sequence of events associated with the development of activin-a-induced pathology is illustrated in fig. . the first stage (days - ) was characterized by a dramatic and transient wave of cell death, substantial increase in high-mobility group box- (hmgb ) immunostaining, appearance of two waves of inflammatory cells in the bal (the first consisting primarily of neutrophils and macrophages and the second, more robust wave, of neutrophils, macrophages and lymphocytes), secretion of numerous th , th , and th cytokines (with pro-inflammatory cytokines dominating early on and antiinflammatory cytokines later), development of hyaline membranes, a gradual and eventually dramatic reduction of lung compliance accompanied by decline of surfactant protein-c (spc), spb, and spa expression levels, development of a hyper-coagulant state accompanied by substantial upregulation of tissue factor (tf) mrna levels and upregulation of ctgf mrna levels. the second stage (days - ) was characterized by persistence of hmgb immunostaining, dramatic thickening of the alveolar septa and development of honeycomb-like histopathology, upregulation of collagens i and iii mrna levels, and increased deposition of collagen in the tissues. the third and final stage (days - months) was characterized by upregulation of tissue matrix metaloproteases (mmp and mmp ), gradual removal of the collage that was deposited earlier and eventual development of emphysematous lesions. although until stage ii, the process appeared to move towards the development of an interstitial fibrosis-like phenotype, during stage iii the process shifted completely direction leading eventually to the development of emphysema like pathology. the sequence of events triggered by the ectopic activin-a expression bears striking similarity to the pathophysiology of human acute lung injury/acute respiratory distress syndrome (ali/ards) [ ] , and consistently, dramatically increased levels of activin-a were detected in bal fluids from ards patients, strongly supporting the notion that activin-a could play a key role in the pathophysiology of this disorder [ ] . the association of activin-a with the pathophysiology of ali/ards is consistent with and complementary to earlier studies that have implicated activin-a in the pathophysiology of sepsis [ , ] . ali/ards and sepsis are distinct disease entities; however, they are intimately related [ ] [ ] [ ] [ ] . they both represent typical examples of severe pathology triggered by overzealous and inappropriately controlled immune reactivity. ali/ards represent fulminate respiratory conditions in which injury of the lung epithelium and endothelium triggers an uncontrolled inflammatory response and leads to often fatal functional and structural deterioration of the lung [ ] . despite the wide variety of recognized precipitating causes such as pneumonia and aspiration of gastric contents, the leading cause of ards is severe sepsis [ , ] . this "cause-effect" relationship could indicate that common harmful effector mechanisms are mobilized during their development, with activin-a being one of them. the histopathology induced by ectopic expression of activin-a in murine lungs shares some features with that induced by overexpression of other molecules such as tgf-β, il- β, and tnf-α, although it is overall quite unique to activin-a. whereas the inflammatory response that consist of neutrophils, macrophages, and lymphocytes, resembles the one induced by il- β overexpression [ ] , the histopathology resembles the one described in transgenic animals overexpressing tnf-α in the lung [ ] . despite the similarities, however, the tnf-α-induced phenotype requires considerably longer time to develop compared with the ∼ days for the activin-a adenovirus treated animals. interestingly, the phenotype in the activin-a treated animals is different from the one induced by tgf-β overexpression in the lung using either adenovirus in rats or transgenemediated expression in mice [ , ] . both tgf-β and activin-a utilize the same smad proteins and in the genetic background of the c bl mouse they both induce transient waves of cell death, mobilize macrophages, induce egr- and ctgf expression [ , ] , and seem to affect surfactant homeostasis [ ] and the coagulation system [ ] . however, the final outcome in the case of tgf-β overexpression is a more robust fibrotic response whereas in the case of activin-a a transient fibrotic response followed by development of emphysema [ ] . remarkably, the respiratory pathology bearing the highest similarity to the one induced by activin-a overexpression is caused by transient overexpression of gremlin, an inhibitor of bmp-mediated signaling [ ] . this surprising finding strongly suggests that basal levels of bmp signaling are part of the mechanism that maintains homeostasis in the adult lung tissue, and thus suppression of basal bmp signaling may cause pathology. interestingly, treatment of healthy animals by intra-tracheal instillation of a follistatin expressing adenovirus leads to upregulation of spc and cc expression, improvement of lung compliance and extension of tail-bleeding time, strongly suggesting that basal levels of activin-a signaling as well is continuously regulating lung homeostasis (unpublished observation). the development of pathology upon stimulation of the activin-a or suppression of the corresponding bmp signaling pathway, and the improvement of lung function parameters upon suppression of basal activin-a levels by the ectopic expression of follistatin, could indicate that a constant and dynamic balance between basal activities of bmp and activin-a signaling maintains homeostasis in the healthy lung. it appears that these pathways are constantly hardwired in the physiology of the lung and for this reason most likely abrupt changes in their concentration precipitate immediate and robust responses. the presence of activin-a protein and mrna in all the tissues of healthy animals analyzed by wu et al. [ ] might indicate that activin-a may play a similar role in maintaining homeostasis in other tissues as well. the earliest change detected in the lungs upon activin-a overexpression was an acute and transient wave of alveolar cell death that reached maximum levels around h postviral instillation. interestingly, a similar transient wave of alveolar cell death, with maximum levels again h post transgene activation, has been observed in lung-specific inducible tgf-β transgenic animals [ ] suggesting that cell death might be a common characteristic of abrupt changes in the levels of tgf-β super-family ligands. importantly, the transient tgf-β-triggered wave of cell death was crucial since its prevention with different anti-apoptotic reagents could ameliorate development of pathology [ ] . although, apoptotic cells in the tgf-β transgenic animals exhibited typical tunel + morphology, the tunel + staining induced by activin-a overexpression involved fragmented cell-debris either attached to basement membranes of alveolar-septa or present in clumps in airspaces, indicating a rapid transition of cells undergoing apoptosis into secondary necrosis and cellular rupture (fig. a) . the exact mechanism of activin-a-induced cells death has not been clarified as yet, however, it is very likely that the combination of the acute recruitment of neutrophils in the lung and probably the release of powerful mediators they carry, the activation of the coagulation cascade directly by the effect of activin-a on epithelial cells [ ] or indirectly through the activity of neutrophils [ , ] , the suppression of surfactant expression and possibly other responses that have not been analyzed as yet, such as activation of the complement system, could collectively lead to the tissue damage and enhanced cell death (fig. ) . tlr signaling appears to be a major inducer of activin-a production [ , , ] . interestingly, high levels of activin-a induce sustained release of hmgb (fig. d) and pro-inflammatory cytokines and chemokines, such as il- , mcp- , kc, il- β, il- , il- , and tnf-α [ ] . hmgb /cytokine complexes that are formed under these conditions are known to activate tlrs [ ] establishing thus a feed-forward regulatory loop that can sustain activin-a production as long as the infectious agents or signs of tissue injury persist in the affected tissues (fig. a) . this feed-forward loop can be turned on not only by infection but also as a consequence of "sterile" disturbance of the homeostatic balance like in the case of sterile tissue injury or other forms of stress [ ] . it is evident that such a feed-forward system, if gone out of control, can deteriorate into a vicious cycle and cause pathology. surely, activin-a was not selected evolutionary to cause damage. the activities induced by the acute upregulation of activin-a are powerful protective mechanisms that are beneficial for the majority of individuals facing an infection or a severe injury and obviously the collateral tissue damage is an inevitable trade-off for efficient protection [ ] . abrupt deviations from the homeostatic levels of activin-a in a tissue could be interpreted as a strong "danger" signal mobilizing thus appropriate protective mechanisms. activin-a is a "morphogen", i.e., designed evolutionary to stimulate different spectra of biological responses depending on its concentration, context, the previous history of its cellular targets or the presence of other regulatory signals in the micro-environment, and thus ideally suited to tailor appropriate tissue responses to different stress situations [ ] . different thresholds of activin-a in a tissue may reflect different levels of deviation from the homeostatic balance and could act as "yellow" or "red-alert"-like signals mobilizing to different extends the defense and/or repair mechanisms in the affected tissues [ ] . evidently, aberrant activation or loss of appropriate control under the influence of other co-morbidities or genetic predisposition could unnecessarily activate a "red-alert" response causing senseless collateral tissue damage and pathology. correct balance between innate and adaptive immunity is a prerequisite for effective defense against infectious intruders. although, innate immunity provides a rapid and efficient first line of defense, many of its powerful components do not discriminate between self and non-self and fig. a activation of inflammatory cells through tlr stimulation or sterile injury of tissue resident cells can lead to abrupt increase in the levels of bioavailable activin-a within a tissue. the combination of aggravated innate immunity, activation of the coagulation system, and alterations in molecular systems crucial for homeostasis, such as the surfactants in the lung, can collectively lead to acute necroptotic cell death which leads to the release of highly pro-inflammatory complexes of damps and cytokines that can activate via a feed-forward like response further activation of the tlr signaling pathway and further production of activin-a. if gone out of control, this feed-forward system can deteriorate into a vicious-cycle and cause pathology. b confocal image of the necroptotic response caused -h post-activin-a adenovirus instillation in the mouse lung. staining for type i pneumonocytes is shown with green and tunel signal is shown with red. c, d hmgb immunostaining of lung tissue section analyzed -h post-instillation of a control (c) or an activin-a-producing adenovirus (d) consequently collateral tissue damage is inevitable, especially in the context of prolonged and robust innate responses [ , ] . therefore, at the right point, innate responses must be contained and give way to the more sophisticated and specific effector mechanisms of adaptive immunity. failure to regulate correctly the transition between the innate and adaptive responses can lead to chronic inflammation, a condition associated with numerous inflammatory and autoimmune disorders. upregulation of activin-a expression appears to favor innate immunity in two ways, namely, by acting as a generic amplifier of innate inflammatory responses and by exerting inhibitory effects on adaptive responses. as discussed earlier, activin-a induces in vitro expression of numerous pro-inflammatory mediators, and it seems from the studies of apostolou et al. [ ] that either directly or indirectly it can stimulate these activities in vivo as well. thus, its capacity to upregulate tnf-α, il- , il- β, and hmgb production, and thus establish a feed-forward amplification loop by further activating the tlr pathway might explain the overt activation of innate responses by activin-a. on the other hand, the capacity of activin-a to inhibit maturation of dcs [ , ] , proliferation of t cells, induction of cytotoxic t cells, induction of foxp negative [ ] and via a tgf-β synergism, foxp - fig. activin-a: guardian of homeostasis, coordinator of innate and adaptive immunity, and orchestrator of tissue repair activities. activin-a is produced by numerous resident cells to maintain homeostatic balance in a tissue (the lung tissue is used as an example in the current cartoon). upon disturbance of homeostasis due to infection, injury, or tissue malfunction, high levels of activin-a can be produced by cells of either the innate or the adaptive immune system. positive and negative effects on different immune cells contribute to the coordination of immune reactivities and balance the relative involvement of innate and/or adaptive immunity. potentially, both innate and adaptive responses can cause collateral tissue injury. activin-a may orchestrate the tissue repair activities needed for restitution of yttissue integrity and homeostasis. other members of the tgf-β super-family, such as tgfs and bmps may also regulate some of the outlined interactions. red lines indicate sources of activin-a; dotted lines indicate positive (->) or negative (-|) influences on the indicated populations; mf macrophages, b b lymphocytes, t t lymphocytes, dcs dendritic cells, nl natural killer cells, treg t regulatory cells, tlrs toll-like receptors positive regulatory t cells [ ] illustrates some of the mechanisms by which activin-a could exert its negative effect on adaptive immune function. the dramatically prolonged survival of adenovirus infected cells in animals treated with activin-a expressing adenoviruses [ ] may reflect an inhibitory effect on the adaptive mechanisms that normally eliminate virus infected cells. interestingly, we have observed that lymphopenic rag- -deficient animals are much more sensitive to activin-a overexpression than wild-type animals of the same genetic background (unpublished observation). this may indicate that the adaptive system is not just a passive receiver of negative signals from activin-a but rather raises a counter regulatory response in an attempt to tempers the innate activities. such a dynamic balance between innate and adaptive responses could minimize the risk for collateral damage by an overzealous early innate response [ , ] . in conclusion, as previously argued [ , ] , activin-a together with follistatin (and other activin inhibitors) occupy a key position in the inter-phase of innate and adaptive immunity. high levels of bioavailable activin-a may intensify innate immunity, whereas, upregulation of follistatin or other activin-a modulating molecules and containment of activin-a reactivity could catalyze a shift towards adaptive-immune and tissue-repair activities. activin-a orchestrates tissue repair activities in an attempt to restitute homeostasis like a molecular "swiss army knife," activin-a appears to play an important role regulating repair of tissue injury as well. activation of fibroblasts and modulation of the extracellular matrix turnover either by stimulating collagen mrna synthesis or affecting the expression of mmps and tissue inhibitors of metaloproteases (timps) [ , , , ] are among the tissue repair related activities that can be modulated by activin-a. in the adenovirus-mediated activin-a overexpression in the lung, commencement of tissuerepair-related activities appears to correlate with upregulation of follistatin [ ] and is characterized by a decline in cytokine production with high il- mrna levels being the only remnant of the previous "cytokine storm" response, the appearance of "alternatively activated" like macrophages, the upregulation of collagens i and iii mrna levels and transient increase in the collagen deposition in the lung parenchyma. indeed, alternatively activated macrophages [ ] , il- [ ] , and hmgb , in the absence of other cytokines [ , ] , have been suggested to support tissue repair, and thus the late activation of these processes by activin-a are consistent with the notion that this molecule not only turns on activities that compromise lung functionality, but also coordinates the ensuing necessary remediating activities. due to the specially delicate architecture of the lung parenchyma clearance of collagen deposits that correlate with a shift in the protease/anti-protease balance in favor of the former may under certain circumstances lead to another form of tissue remodeling, namely development of emphysematous lesions. neutrophils constitute the most abundant white blood cell type in circulation and are the first inflammatory cells that are recruited from the circulation into tissues where homeostasis has been compromised either by infection, injury or other forms of stress. they have at their disposal an impressive arsenal of armaments, which, due to their nondiscriminatory nature, can be as hazardous to the host cells as to their intended targets, namely, the microbial intruders. neutrophils can phagocytize, release the content of their granules in the environment, or utilize a remarkable strategy known as netosis, an active form of cells death that involves the controlled release of chromatic decorated with antimicrobial granular and cytoplasmic proteins into the extracellular space [ , ] . extensive coverage of the biology of these extraordinary cells is beyond the scope of this review. without any doubt, many of the articles in this issue of "seminars in immunopathology" are providing adequate coverage, as numerous reviews in the literature have done previously [ ] [ ] [ ] [ ] . however, steadily accumulating evidence may imply a special relationship between neutrophils and activin-a, and consequently, this facet of neutrophil biology certainly deserves highlighting herein. neutrophils have been implicated in the pathophysiology of sepsis, ards, rheumatoid arthritis, inflammatory bowel disease, systemic lupus erythematosus, pre-eclampsia, coronary syndrome, and different forms of tissue injury (reviewed in refs. [ ] [ ] [ ] [ ] [ ] ) and netosis has been demonstrated in the context of some of these diseases and has been implicated in their pathophysiology. thus, nets have been detected in biopsies from human asthmatics that were characterized by infiltration of neutrophils [ ] , in the lungs of experimental animals that were challenged with sublethal doses of influenza a virus h n [ ] or lps [ ] and developed ards-like pathology, in the context of transfusion-related acute lung injury in humans and mice [ ] , and evidence for development of nets has been produced in systemic lupus erythematosus [ ] . interestingly, the abovementioned disorders are also characterized by upregulated expression of activin-a [ , , [ ] [ ] [ ] [ ] [ ] and most probably this correlation is not coincidental. the studies by chen et al. and wu et al. [ ] [ ] [ ] suggest that this correlation could stem from the fact that neutrophils are major producers of activin-a and consequently diseases characterized by neutrophilia might also exhibit enhanced activin-a production. however, recent observations in our laboratory may indicate that the relationship between neutrophils and activin-a is more complex than that. in the ova model of allergic airway inflammation, the levels of activin-a in the bal fluid of the ova-challenged animals ranges from to ng/ml and the great majority of the inflammatory cells in the bal are eosinophils ( - %). we have observed that raising the levels of activin-a in the airways of the ova-challenged animals using adenoviruses to levels in the bal ∼ - ng/ml could completely shift the eosinophil-dominated airway inflammatory response into a neutrophil-dominated (< % eosinophils and % neutrophils) (unpublished observation). although it is not known for the moment whether this preferential recruitment of neutrophils is caused by a direct action of activin-a on neutrophils or is mediated indirectly via the induction of tnf-α, il- , ifn-γ, il- , or any other pro-inflammatory mediator, it is quite clear that a very special, complex and dynamic relationship must link neutrophils and activin-a. indeed, kambas et al. have recently demonstrated that neutrophils from patients with sepsis could release large amounts of net-borne tf that could generate thrombin and activate platelets [ ] . therefore, neutrophils could activate the coagulation cascade indirectly, via an activin-a-mediated stimulation of tf production by epithelial cells [ ] and directly by releasing tf-loaded nets in the tissues [ ] . furthermore, saffarzadeh et al. have recently demonstrated that nets can directly induce epithelial and endothelial cell death [ ] , raising thus the possibility of activin-a-mobilized, neutrophil-derived nets could contribute to the acute wave of alveolar cell death observed in mice upon intra-tracheal instillation of activin-a-producing adenoviruses. remarkably, very little is known so far regarding the effect of activin-a signaling in neutrophils. in fact, a search for activin-a and neutrophils in the current literature resulted only in a handful of publications, with a tiny fraction of them directly addressing the consequences of activin-a-mediated signaling on neutrophils function [ ] . thus, tantalizing questions regarding the possible involvement of activin-a in the attraction of neutrophils in a tissue and/or the involvement of activin-a in the modulation of neutrophil functions, such as the formation and release of nets, remain still unanswered. a more comprehensive scrutiny of the neglected relationship between neutrophils and activin-a is therefore highly warranted. activin-a has been known for almost years and still the list of putative functions for this cytokine is steadily growing. the functional repertoire of this remarkable molecule includes numerous and occasionally contradicting pro-inflammatory, anti-inflammatory, and tissue remodeling activities. although there is still a great deal to learn, some features of activin-a biology are becoming better understood and hence a sketchy map that positions activin-a in the context of immuno-inflammatory processes can be drawn (fig. ) . thus, it is now recognized that different levels of activin-a mrna and protein are continuously produced in almost any normal tissue analyzed [ ] . these "basal" activin-a levels of production/activity are most likely important for the fine tuning of essential homeostatic functions. abrupt changes in the levels of bioavailable activin-a in a tissue rapidly activate a number of biological responses which, although they are meant to be protective, if not managed properly, they can cause severe tissue injury and pave the way to pathology (fig. ) . the potentially tissue-damaging responses triggered by activin-a include among others exuberant activation of the innate immune system, with neutrophils and macrophages being among the first to be recruited in tissues upon increase in activin-a expression, activation of the coagulation system that could lead to disseminated micro-vascular thrombosis, and in the respiratory system dramatic reduction of surfactant biosynthesis. the rationale behind the trade-off between protection and collateral tissue damage has been very eloquently discussed in review articles in the literature [ , ] . one can envisage situations where the crisis is so critical that a robust innate response that will restrict the spread of the intruder by activating the coagulation and complement systems and recruiting a robust neutrophil response could provide an effective protection, although at the cost of collateral tissue damage. it seems that activin-a does not function only during the acute phase of an inflammatory response, but as the response unfolds it may function as a coordinator of innate and adaptive immune responses, amplifying the former and suppressing directly or indirectly through induction of regulatory t cells the later. consequently, aberrant activin-a function in this context can lead to improper balance between innate and adaptive responses with potentially detrimental consequences. finally, activin-a regulates activities that are related to remediation of the tissue injury caused either by infection or by collateral damage inflicted by an overzealous or protracted activation of the immune system. thus, it has become evident that activin-a plays key roles at the interface of homeostasis, innate and adaptive immunity and tissue repair (fig. ) . although the rich functional repertoire of activin-a might appear confusing at a first glance, it becomes more palpable if we recognize that the different and occasionally counteracting activities that have been described by in vitro studies for activin-a, constitute in vivo integral components of a dynamic continuum in which the time factor is of key importance. it is very clear that as an inflammatory response unfolds, both the cells and the microenvironment involved evolve continuously (fig. ) . the tissue resident and the recruited inflammatory cells go through different stages of activation, express new receptors and co-receptors and often desensitize some, and different cytokines are produced at each stage from the onset till the eventual resolution of the response [ , , ] . consequently, in vivo, activin-a signaling targets "different" cells in the context of "different" microenvironments as the response unfolds time-wise. thus, pro-inflammatory activities of activin-a predominate during the acute, early phase of the response targeting primarily naïve cells, whereas, anti-inflammatory and matrix remodeling activities are manifested later in the response and target primed and activated cells. it is very likely that early and late activin-a-induced responses are linked with a "cause-effect" relationship, interlink in the fabric of a master homeostasis restitution program. as mentioned earlier, similar cause-effect relationship has been described between the transient apoptotic response induced by ectopic overexpression of tgf-β in the lung and the fibrotic response that followed much later [ ] . although this review has focused primarily on activin-a and its role in immuno-inflammatory and tissue-remodelingassociated processes, if one considers the extensive functional overlap between activin-a, activin-b, activin-ab, and tgf-βs, and the apparently antagonistic action of activin-c, activin-e, and bmps, the need of studying the tgf-βsuperfamily signaling system in an integrated manner becomes an absolute necessity. the therapeutic action of activin-a neutralizers, such as follistatin or a fusion protein composed of the extracellular potion of actriib, the type ii receptor for activins, linked to the fc portion of the human igg antibody, in colitis animal models [ ] and in the exogenous activin-a or lps-driven ali/ards animal models [ ] clearly indicate that even after establishment of pathology, neutralization of activin-a can still attenuate pathology associated parameters. this raises expectations for the clinical application of activin-a inhibitors and highlight the need for further research to further unwind the principles that govern and modulate its in vivo actions. likewise, the intimate relation between neutrophil biology and activin signaling that is slowly emerging necessitates a more thorough investigation. given their direct involvement in safeguarding and remediating homeostasis, such a relationship is most likely anything but coincidental. mechanisms of tgf-beta signaling from cell membrane to the nucleus purification and characterization of an fsh releasing protein from porcine ovarian follicular fluid insertion of inhbb into the inhba locus rescues the inhbanull phenotype and reveals new activin functions activin is an essential early mesenchymal signal in tooth development that is required for patterning of the murine dentition activin/inhibin beta b subunit gene disruption leads to defects in eyelid development and female reproduction identification of a potent xenopus mesoderm-inducing factor as a homologue of activin a responses of embryonic xenopus cells to activin and fgf are separated by multiple dose thresholds and correspond to distinct axes of the mesoderm regulation of prostate branching morphogenesis by activin a and follistatin development of cancer cachexia-like syndrome and adrenal tumors in inhibin-deficient mice inhibins, activins and follistatin: actions on the testis prevention of cachexia-like syndrome development and reduction of tumor progression in inhibin-deficient mice following administration of a chimeric activin receptor type ii-murine fc protein reversal of cancer cachexia and muscle wasting by actriib antagonism leads to prolonged survival the transforming growth factor-beta family regulation of activin's access to the cell: why is mother nature such a control freak mapping of genes for inhibin subunits alpha, beta a, and beta b on human and mouse chromosomes and studies of jsd mice molecular cloning of the mouse activin beta e subunit gene cloning of a new member of the tgf-beta family: a putative new activin beta c chain activin betac and betae genes are not essential for mouse liver growth, differentiation, and regeneration expression of activin subunits, activin receptors and follistatin in postimplantation mouse embryos suggests specific developmental functions for different activins functional analysis of activins during mammalian development beta a versus beta b: is it merely a matter of expression? localization of activin beta(a)-, beta(b)-, and beta(c)-subunits in humanprostate and evidence for formation of new activin heterodimers of beta(c)-subunit activin betac-subunit heterodimers provide a new mechanism of regulating activin levels in the prostate activin c antagonizes activin a in vitro and overexpression leads to pathologies in vivo the bmp /actrii extracellular domain complex provides new insights into the cooperative nature of receptor assembly smad regulation in tgf-beta signal transduction non-smad tgf-beta signals both smad and smad mediate activinstimulated expression of the follicle-stimulating hormone beta subunit in mouse gonadotrope cells activin b can signal through both alk and alk in gonadotrope cells smad mediates activin-induced transcription of follicle-stimulating hormone beta-subunit gene the biology of activin: recent advances in structure, regulation and function identification of human activin and tgf beta type i receptors that form heteromeric kinase complexes with type ii receptors characterization and determination of the biological activities of noncleavable high molecular weight forms of inhibin a and activin a requirement for activin a and transforming growth factor-beta pro-regions in homodimer assembly monomeric activin a retains high receptor binding affinity but exhibits low biological activity a common biosynthetic pathway governs the dimerization and secretion of inhibin and related transforming growth factor beta (tgfbeta) ligands activin a binds to perlecan through its proregion that has heparin/heparan sulfate binding activity growth factor binding to the pericellular matrix and its importance in tissue engineering new insights into the mechanisms of activin action and inhibition the recombinant proregion of transforming growth factor beta (latency-associated peptide) inhibits active transforming growth factor beta in transgenic mice expression cloning of an activin receptor, a predicted transmembrane serine kinase flrg (follistatin-related gene), a new target of chromosomal rearrangement in malignant blood disorders identification and characterization of a novel follistatin-like protein as a binding protein for the tgf-beta family follistatin-related protein (fsrp): a new member of the follistatin gene family antagonists of activin signaling: mechanisms and potential biological applications activin-binding protein from rat ovary is follistatin structural basis for the inhibition of activin signalling by follistatin primary structure of the human follistatin precursor and its genomic organization a novel role of follistatin, an activin-binding protein, in the inhibition of activin action in rat pituitary cells. endocytotic degradation of activin and its acceleration by follistatin associated with cell-surface heparan sulfate release of activin and follistatin during cardiovascular procedures is largely due to heparin administration structural and biophysical coupling of heparin and activin binding to follistatin isoform functions follistatin: a multifunctional regulatory protein molecular heterogeneity of follistatin, an activin-binding protein. higher affinity of the carboxylterminal truncated forms for heparan sulfate proteoglycans on the ovarian granulosa cell follistatin-related protein and follistatin differentially neutralize endogenous vs. exogenous activin the structure of fstl .activin a complex. differential binding of n-terminal domains influences follistatintype antagonist specificity the structure of the follistatin:activin complex reveals antagonism of both type i and type ii receptor binding differential binding and neutralization of activins a and b by follistatin and follistatin like- (fstl- /fsrp/flrg) the myostatin propeptide and the follistatinrelated gene are inhibitory binding proteins of myostatin in normal serum regulation of myostatin activity and muscle growth follistatin inhibits the function of the oocyte-derived factor bmp- inhibin and activin regulate [ h]thymidine uptake by rat thymocytes and t cells in vitro expression of immunoreactive and bioactive activin a protein in adult murine lung after bleomycin treatment expression of immunoreactive activin a protein in remodeling lesions associated with interstitial pulmonary fibrosis effects of activin a on proliferation and differentiation of human lung fibroblasts attenuation of bleomycin-induced pulmonary fibrosis by follistatin follistatin attenuates early liver fibrosis: effects on hepatic stellate cell activation and hepatocyte apoptosis activation of the activin a-alk-smad pathway in systemic sclerosis multiple defects and perinatal death in mice deficient in follistatin strong induction of activin expression after injury suggests an important role of activin in wound repair overexpression of activin a in the skin of transgenic mice reveals new activities of activin in epidermal morphogenesis, dermal fibrosis and wound repair the activin binding proteins follistatin and follistatinrelated protein are differentially regulated in vitro and during cutaneous wound repair impaired wound healing in transgenic mice overexpressing the activin antagonist follistatin in the epidermis temporal expression of activin in acute burn wounds-from inflammatory cells to fibroblasts the roles of activin a and its binding protein, follistatin, in inflammation and tissue repair activation of the tgf-beta/activin-smad pathway during allergic airway inflammation calcium-regulated expression of activin a in rbl- h mast cells regulation of activin a expression in mast cells and asthma: its effect on the proliferation of human airway smooth muscle cells activin a is an acute allergen-responsive cytokine and provides a link to tgf-beta-mediated airway remodeling in asthma activin and transforming growth factor-beta signaling pathways are activated after allergen challenge in mild asthma role of activin a in murine mast cells: modulation of cell growth, differentiation, and migration follistatin is a candidate endogenous negative regulator of activin a in experimental allergic asthma activin-a induces regulatory t cells that suppress t helper cell immune responses and protect from allergic airway disease activin a and tgf-beta promote t(h) cell-mediated pulmonary allergic pathology follistatin concentrations in male sheep increase following sham castration/ castration or injection of interleukin- beta plasma follistatin concentrations increase following lipopolysaccharide administration in sheep characterisation of the rapid release of activin a following acute lipopolysaccharide challenge in the ewe activin a is a critical component of the inflammatory response, and its binding protein, follistatin, reduces mortality in endotoxemia activin-a overexpression in the murine lung causes pathology that simulates acute respiratory distress syndrome serum concentrations of activin and follistatin are elevated and run in parallel in patients with septicemia increased activin levels in cerebrospinal fluid of rabbits with bacterial meningitis are associated with activation of microglia activin a concentrations in human cerebrospinal fluid are age-dependent and elevated in meningitis therapeutic potential of follistatin for colonic inflammation in mice activin a: a novel player and inflammatory marker in inflammatory bowel disease? suppression of il- biological activities by activin a and implications for inflammatory arthropathies expression of activin a in inflammatory arthropathies activin a induces cell proliferation of fibroblast-like synoviocytes in rheumatoid arthritis elevated levels of activin a in heart failure: potential role in myocardial remodeling activin and related proteins in inflammation: not just interested bystanders activin a/ erythroid differentiation factor is induced during human monocyte activation regulation of production of activin a in human marrow stromal cells and monocytes innate immune responses to trem- activation: overlap, divergence, and positive and negative cross-talk with bacterial lipopolysaccharide microglial cells and peritoneal macrophages release activin a upon stimulation with toll-like receptor agonists activin a stimulates type iv collagenase (matrix metalloproteinase- ) production in mouse peritoneal macrophages activin-a: a novel dendritic cell-derived cytokine that potently attenuates cd ligand-specific cytokine and chemokine production production and function of activin a in human dendritic cells induced expression of the new cytokine, activin a, in human monocytes: inhibition by glucocorticoids and retinoic acid activin a functions as a th cytokine in the promotion of the alternative activation of macrophages a dual role of activin a in regulating immunoglobulin production of b cells tumour necrosis factor-alpha stimulates human neutrophils to release preformed activin a regulation of activin a release from murine bone marrowderived neutrophil precursors by tumour necrosis factor-alpha and insulin acute regulation of activin a and its binding protein, follistatin, in serum and tissues following lipopolysaccharide treatment of adult male mice activin a induces dendritic cell migration through the polarized release of cxc chemokine ligands and the glycoprotein-hormones activin a and inhibin a interfere with dendritic cell maturation induction of interleukin -producing, nonproliferating cd (+) t cells with regulatory properties by repetitive stimulation with allogeneic immature human dendritic cells induction of prostanoid, nitric oxide, and cytokine formation in rat bone marrow derived macrophages by activin a activin a and retinoic acid synergize in cyclooxygenase- and thromboxane synthase induction during differentiation of j . macrophages a critical role of activin a in maturation of mouse peritoneal macrophages in vitro and in vivo effects of activin a on ige synthesis and cytokine production by human peripheral mononuclear cells activin a induces langerhans cell differentiation in vitro and in human skin explants activin a down-regulates the phagocytosis of lipopolysaccharideactivated mouse peritoneal macrophages in vitro and in vivo inhibitory effect of activin a on activation of lipopolysaccharidestimulated mouse macrophage raw . cells activin a regulates the production of mature interleukin- beta and interleukin- receptor antagonist in human monocytic cells activin as an anti-inflammatory cytokine produced by microglia regulation of activin a synthesis in microglial cells: pathophysiological implications for bacterial meningitis activin a skews macrophage polarization by promoting a proinflammatory phenotype and inhibiting the acquisition of anti-inflammatory macrophage markers activin a promotes the tgf-beta-induced conversion of cd + cd − t cells into foxp + induced regulatory t cells activin-a attenuates several human natural killer cell functions further characterization of activin ainduced iga response in murine b lymphocytes suppression of activin a in autoimmune lung disease associated with anti-gm-csf stimulatory effects of lipopolysaccharide on endothelial cell activin and follistatin potent induction of activin a secretion from monocytes and bone 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reperfusion injury neutrophil function in inflammation and inflammatory diseases eosinophil and neutrophil extracellular dna traps in human allergic asthmatic airways excessive neutrophils and neutrophil extracellular traps contribute to acute lung injury of influenza pneumonitis neutrophil extracellular traps directly induce epithelial and endothelial cell death: a predominant role of histones extracellular dna traps are associated with the pathogenesis of trali in humans and mice apoptosis and net formation in the pathogenesis of sle the role of neutrophils during intestinal inflammation neutrophils and clinical outcomes in patients with acute coronary syndromes and/or cardiac revascularisation. a systematic review on more than , subjects neutrophil nets: a novel contributor to preeclampsia-associated placental hypoxia? lupus neutrophils: 'net' gain in understanding lupus pathogenesis sensing sterile injury: opportunities for pharmacological control the emerging role of neutrophils in thrombosis-the journey of tf through nets acknowledgments work in the sideras laboratory is supported by core funding from the hellenic ministries of health and education and by grant cofounded by the european regional development fund and the hellenic ministry of education, general secretariat of research and technology (synergasia " syn- - "). work in the andreakos laboratory is supported by grants from the framework programme of the european commission (atheroremo, pre-dicta, tacit, and nilthera) and the grants cofounded by the european regional development fund and the hellenic ministry of education, general secretariat of research and technology (re-solve-asthma and midas). key: cord- -b a hhw authors: bellingan, g title: leukocytes: friend or foe date: journal: intensive care med doi: . /s sha: doc_id: cord_uid: b a hhw leukocytes have a fundamental role in innate and adaptive immunity, wound healing, tumour surveillance and in tissue remodelling. it is their function in the inflammatory response however that is of most interest to us in the intensive care setting. over the last three decades we have gained significant insights into leukocyte activation, recruitment and mediator secretion and the contribution of these agents to both the onset and resolution of sepsis and inflammation.¶the body relies on the inflammatory response for protection. leukocytes occupy a pivotal position in this but to maintain these cells in a state of permanent activation would be unsustainable, with widespread microvascular plugging, uncontrolled free radical release and an excessive metabolic demand. leukocytes thus circulate in a quiescent state and are rapidly activated by invading pathogens and other stimuli. a direct consequence of this protective strategy is that the inflammatory response may be inadequate, with the risk of overwhelming sepsis, or excessive, leading to rampant systemic inflammation and consequent multiple organ damage.¶it is now becoming apparent however that in addition to leukocytes other cells have important roles both in defence against invading pathogens and in driving malignant inflammation. this review will focus on two new facets of the innate immune system, the toll family of proteins as the signal transduction element for endotoxin, and the antimicrobial peptides. these exemplify potential damaging and protective response elements but importantly neither are restricted to leukocytes. the capacity of cells and tissues other than the leukocytes to participate and even lead in the inflammatory responses will also be explored. leukocytes are essential for a functional immune response, for normal wound healing, tumour surveillance and during development and tissue remodelling, it is their role in the inflammatory response however that is of most interest to us in the intensive care setting [ ± ] . it is now well established that leukocyte activation can be a double edged sword; the body is critically dependent upon these cells for protection from pathogens and an ineffective inflammatory/immune response can be lethal. a balance needs to be struck however as an excessive inflammatory response can also kill through progressive inflammation and consequent multi-organ dysfunction [ ± ] . leukocytes normally circulate in a quiescent state but can be rapidly activated to repel invading pathogens. this system of leukocyte activation has evolved because it would be unsustainable to maintain these cells in a state of permanent activation. this would lead to widespread leukocyte plugging, uncontrolled free radical release and an unwarranted metabolic de- abstract leukocytes have a fundamental role in innate and adaptive immunity, wound healing, tumour surveillance and in tissue remodelling. it is their function in the inflammatory response however that is of most interest to us in the intensive care setting. over the last three decades we have gained significant insights into leukocyte activation, recruitment and mediator secretion and the contribution of these agents to both the onset and resolution of sepsis and inflammation. the body relies on the inflammatory response for protection. leukocytes occupy a pivotal position in this but to maintain these cells in a state of permanent activation would be unsustainable, with widespread microvascular plugging, uncontrolled free radical release and an excessive metabolic demand. leukocytes thus circulate in a quiescent state and are rapidly activated by invading pathogens and other stimuli. a direct consequence of this protective strategy is that the inflammatory response may be inadequate, with the risk of overwhelming sepsis, or excessive, leading to rampant systemic inflammation and consequent multiple organ damage. it is now becoming apparent however that in addition to leukocytes other cells have important roles both in defence against invading pathogens and in driving malignant inflammation. this review will focus on two new facets of the innate immune system, the toll family of proteins as the signal transduction element for endotoxin, and the antimicrobial peptides. these exemplify potential damaging and protective response elements but importantly neither are restricted to leukocytes. the capacity of cells and tissues other than the leukocytes to participate and even lead in the inflammatory responses will also be explored. mand with progressive tissue damage and multi-organ dysfunction syndrome (mods). a direct consequence of this protective strategy is that the inflammatory response may be either inadequate, with the risk of overwhelming sepsis, or excessive, leading to rampant systemic inflammation [ ] . recently further layers of complexity have been described including the endogenous compensatory anti-inflammatory response syndrome (cars) protecting against excessive inflammation [ ] . moreover the underlying insult can impair the immune response and aggressive or persisting inflammation can lead to a state of anergy and heightened risk of further infection [ ] . to address the role of leukocytes in the inflammatory process and the balance that needs to be struck between an ineffective and an excessive response we need to understand a little about the inflammatory response itself. this also needs to be put into context as the leukocytes are not the only players involved. the genetic regulation of these processes is also of increasing importance. serious infections are the most common cause for admission to intensive care and the clinical features are well described [ ] . it is now clear that other conditions such as multiple trauma and burns also result in a state of generalised inflammation in the absence of infection. this has led to the description of the systemic inflammatory response syndrome (sirs), a multi-system inflammatory state characterised by excessive immuno-inflammatory cascade activation with widespread reduction in cellular oxygen utilisation, atp depletion, cell injury and death [ ] . the most common agents initiating inflammation are bacterial cell wall components including teichoic acid and peptidoglycans from gram-positive bacteria and lipopolysaccharide (lps) from gram-negative bacteria [ ] . activation induces multiple mediator networks including the complement, kinin, coagulation and fibrinolytic cascades, synthesis of lipid mediators, chemokines, cytokines and release of soluble receptors, along with free radical synthesis and leukocyte degranulation with release of numerous enzymes, including elastase, myeloperoxidase, proteases, collagenase and plasminogen activator all occurring as part of an interrelated network [ , , , ] . importantly, many cells other than leukocytes are also activated during inflammation, including endothelial cells, mesothelial cells and fibroblasts [ ± ]. these cells are all able to elaborate multiple inflammatory agents and represent a huge reservoir for mediator synthesis that does not often receive due consideration. as most infections occur primarily in the tissue and not in the blood stream, extravasation of leukocytes is essential in bring inflammatory cells and foreign pathogens into contact. this requires both a chemotactic gradient and co-ordinated up-regulation of endothelial and inflammatory cell adhesion molecule expression. pro-inflammatory agents rapidly up regulate e and pselectins which, with l-selectin, mediate leukocyte rolling along the endothelium [ ]. the b integrins lfa- (cd a), mac- (cd b) and p / (cd c) are the main leukocyte adhesion molecules responsible for firm adhesion, their endothelial ligands are icam- (cd ), icam- (cd ) and vcam- (cd ). pe-cam and vla- are essential for transmigration. these adhesion molecules are also all closely regulated by activating signals [ ± ] . once initiated, the intensity and duration of the inflammatory response is closely regulated [ ] . numerous mechanisms are invoked in this including ªanti-inflammatoryº cytokines (e. g. il- ra, il- , il- ) and endogenous anti-endotoxin antibodies acting to damp down the inflammatory response while the leukocytes phagocytose and kill the offending pathogens. leukocytes have only a finite life-span at the inflamed site with neutrophils rapidly undergoing apoptosis to be cleared by inflammatory macrophages, which themselves emigrate from the inflamed site during the resolution phase [ ± ]. thus a successful inflammatory event requires not only appropriate activation of cells and mediators with phagocytosis and removal of the inciting stimulus but also consequent elimination of the inflammatory cells and debris to allow the tissues to return to normal architecture and function. it is quite clear that leukocytes are fundamental for survival as exemplified so devastatingly by neutropenia [ ] . in addition, defects in leukocyte function can be just as harmful as reduction in numbers, with clear examples ranging from leukocyte adhesion deficiency (lad) where the cells are functional but cannot get to the site of infection, to chronic granulomatous disease (cgd) where cells form collections at sites of infection but lack functional killing ability [ , ] . the list of such conditions is long and simply serves to demonstrate the relative importance of different functions on adhesion, phagocytosis, free radical generation, and killing. neutrophils and macrophages not only directly phagocytose and kill pathogens but are also key regulators of the inflammatory response as they have a powerful capacity to initiate and perpetuate the multiple inflammatory mediator cascades described above. although billions of dollars have been spent on the, as yet, fruitless search for immunomodulatory agents that will effectively damp this response it has become clear that an intact and robust inflammatory response is indeed crucial for survival. anti-tnf therapy provides an excellent example of how important the normal inflammatory response is as inhibition of tnf-a activity leads to increased not decreased mortality in a number of septic models including caecal ligation and puncture, listeria, and candida albicans infections [ ± ] . in other diseases such as malaria, tnf-a blockade may be beneficial or harmful depending upon the underlying susceptibility which may be under genetic regulation [ , ] . this highlights the importance of differentiating between live micro-organisms and non-infectious agents such as lps when interpreting results from inflammatory models [ ] . furthermore the method whereby neutralising antibodies are delivered may also be critical [ ] . this is even more neatly demonstrated by yersinia enterocolitica infection where this bacterium produces a protein called yopb which directly inhibits host tnf-a production [ ] . inhibition of yopb with an anti-yopb serum increases tnf-a production and reduces bacterial growth again demonstrating the key role of tnf-a in controlling infection. other yop proteins may also be involved [ ] . severe critical illness can depress the immune system which can increase mortality [ ] . for example, patients with blunt or penetrating trauma exhibit reduced responses to usual recall antigens, the greater the injury severity the longer the period of anergy [ ] . haemorrhage also depressed macrophage antigen presentation by % or more in a mouse model probably through reduced antigen catabolism rather than reduced presentation [ , ] . similarly prolonged critical illness leads to immune hypo-responsiveness, the cause of which is unclear but can be related to reduced leukocyte responsiveness and monocyte hla-dr expression has successfully been used as a surrogate marker of this [ ]. boosting leukocyte functional responses by interferon g have been associated with a significant improvement in outcome suggesting that a fully functional leukocyte response throughout the inflammatory response is vital to successful outcome. leukocyte stimulation with g-csf or gm-csf also demonstrates the therapeutic value of these cells both with leukopenia and also in the treatment of infection in the non-neutropenic patients [ ± ] . other leukocyte functions vital to recovery from critical illness include wound healing, tissue remodelling and generation of an adaptive immune response with memory. this is in addition to those functions not immediately relevant to the intensive care setting including tumour surveillance and their role in growth and development. it is thus abundantly clear that leukocytes are friends without which we would not have survived or evolved. problems such as transplant rejection, hypersensitivity and allergy and white cell malignancies will not be considered in this review, although these are leukocyte driven and can lead to the requirement for intensive care. instead the focus will be on the potential for leukocytes to damage the host as part of the systemic inflammatory response syndrome. the concept that leukocytes could directly damage the host is not new. as so eloquently described by lewis thomas ªit is the information carried by the bacteria that we cannot abide. the gramnegative bacteria are the best examples of this. they display lipopolysaccharide endotoxin in their walls, and these macromolecules are read by our tissues as the very worst of bad news. when we sense lipopolysaccharide, we are likely to turn on every defence at our disposalº [ ] . this is supported by many observations that leukocyte depletion could prevent host damage as shown in table . this emphasises however the essential balance between leukocytes as a powerful motor of tissue damage and their key role in prevention of infection. ards is a particularly important condition in which the neutrophils are believed to act as a prime driving force. these cells have the capacity to damage the host with the production of free radicals and release of enzymes into a protected local microenvironment. moreover the neutrophil can secrete the pro-inflammatory the leukocytes are not the only cells that can synthesise and release significant quantities of inflammatory agents. endothelial cells, mesothelial cells and fibroblasts are all highly metabolically active and are amongst many cells that can produce pro-inflammatory cytokines, free radicals and lipid mediators, some of these are listed in table [ ± ]. moreover endothelial cells and mesothelial cells can present antigen while fibroblasts can phagocytose apoptotic cells. thus consider the peritoneum in the absence of neutrophils and macrophages. infection can elicit a massive surge of pro-inflammatory cytokines and free radicals, the omentum can wall off and localise infection while mesothelial cells can present antigen to lymphocytes driving an adaptive immune response. similarly in the lung the endothelial and epithelial cells along with fibroblasts can reproduce much of the classic inflammatory response that we normally associate with the leukocytes. these include many cytokines such as il- and tgfb which are directly implicated in the pathogenesis of ards and consequent fibrosis [ ] . it is becoming clearer that a greater understanding of the involvement of stromal cells in the synthesis and release of pro-inflammatory mediators in response to infection is required especially in regard to inflammation limited to specific regions or organs. as noted earlier the response to lps is uniquely sensitive but it is only recently that the signal transduction pathway for this has been fully established. in an intricate pathway lps has been shown to complex with circulating lipopolysaccharide binding protein (lbp) which binds to the cd receptor leading to inflammatory cell activation [ ] . cd has no cytoplasmic tail hence the mechanism through which it led to activation of nfkb and pro-inflammatory cytokine synthesis was not clear [ ] . detailed experiments on drosophila melanogaster have led to a much greater understanding of the innate immune response. it is now recognised that there are multiple pattern recognition receptors expressed on leukocytes and also on other cells that respond to major groups of pathogens [ ] . through these receptors activation responses are elicited to membrane components such as teichoic acid, peptidoglycans and endotoxin. the toll gene was first described for its function in dorsoventral pattern formation in drosophila, but it also controls the fly's immune response to fungal infection [ ] . one of the other four drosophila toll-like receptor (tlr) proteins called w is also involved in immune responses as antibacterial responses are compromised if this protein is deleted [ ] . the cytoplasmic domain of toll was found to be closely homologous to the cytoplasmic domain of the il- receptor although the extracellular domains are unrelated. although the toll protein family was first identified in drosophila it has now been characterised as the signal transducing element for lps in man [ , ] . lps induced signalling though toll leads to activation of nfkb and pro-inflammatory cytokine synthesis. importantly, non-myeloid cells can be activated by lps in a cd /lbp dependent fashion and toll-like receptors are expressed on cells other than peripheral blood leukocytes. there are tlr described in man, it is unknown as yet whether other tlr recognise other major pattern recognition molecules such as peptidoglycan, teichoic acid. certainly drosophila, which do not have an adaptive immune response, can elicit relatively specific responses to the major classes of bacteria through innate pattern recognition molecules [ ] . furthermore the tlr are not restricted to the phagocytes, indeed with expression on intestinal cells for example, their distribution is quite broad. this provides multiple cells with the capacity to respond to lps (and possibly to other major classes of bacterial cell wall proteins). hence it may well be through innate immune defences such as toll that cells other than phagocytes participate in the inflammatory response. we should thus beware of focusing on the leukocytes as the effector cells of the immune system when it is clear that functional receptors may have a much wider distribution than previously thought. it is now known that the main lps detoxification systems are bactericidal permeability increasing protein (bpi), serum amyloid protein and the lipoprotein system, especially hdl [ ± ]. the main source of bpi are the phagocytes and bpi inhibits lps delivery to monocyte cd and appears to condense lps aggregates whilst lbp promotes lps delivery to monocyte cd and disaggregates lps [ ] . lbp does however promote the transfer of lps into phospholipid micelles and most endotoxin added to blood ends up in the lipoprotein fraction. moreover lipoprotein depletion increases tnf-a levels and mortality while increased lipoprotein levels improve mortality and this is due at least in part to reduced production of pro-inflammatory cytokines [ ] . hdl is the main lps binding lipoprotein, although some is found in ldl but very little in vldl/chylomicrons. it seems that with time hdl hands lps over to ldl which takes it to the liver where it is secreted in the bile. septic patients often have very low hdl and apo a- levels which may increase their sensitivity to lps. furthermore increasing hdl through the use of reconstituted rhdl discs decreases tnf-a levels [ ] . this is despite an increase in detectable lps due to retention in the circulation in relatively inaccessible aggregates in hdl. the binding of lps to cells is far faster however than hdl inactivation, with cell binding occurring within minutes while hdl inactivation occurring over to hours [ ] . reconstituted hdl is a powerful binder and neutraliser of lps but is similarly slow. thus the major method for lps detoxification is not through leukocytes but via the lipoprotein micelles. it is not just the leukocytes that limit the inflammatory response through direct antimicrobial inhibitory mechanisms. key components of the innate immune response are the antimicrobial peptides which are listed in table . these are small antimicrobial peptides with usually less than amino acid residues that commonly carry a positive changes and are widely distributed across body surfaces and in secretions. they are reviewed by lehrer and ganz [ ] . although many of these are produced by leukocytes their major site of production is by cells lining the respiratory, renal and reproductive tracts and the epithelial surfaces. the defensins are b pleated sheet peptides of to amino acids in size. there are six a-defensins, human defensins ± are restricted to neutrophils while defensins and are produced by epithelial cells and protect the intestinal and female reproductive tracts respectively. the b-defensins produced by epithelial cells protect the respiratory, renal and reproductive tracts. likewise the cathelicidins (hcap- and its c terminal domain active fragment ll- ) are found at surface epithelial cells and mucous glands of the respiratory tract. the histatins are salivary proteins with activity against fungi, including azole resistant organisms [ ] . secretory leukoprotease inhibitor is found in many secretions and on epithelial surfaces. it is a amino acid peptide with antiprotease activity at the carboxy-terminal domain and broad spectrum antimicrobial activity at the amino terminal end. it is clear that in the antimicrobial peptides we have a broad and effective system to protect the body against infection that is mainly dependent table the main classes of human antimicrobial peptides on non-leukocyte production, hence the body does not rely solely on leukocytes as the only safeguard against pathogens. it is clear that leukocytes are essential to surviving an infective challenge, but that they can also cause overwhelming damage to the host. moreover, inflammation is a continually evolving process and different aspects of leukocyte function will be paramount at different stages, while the invading pathogens themselves can further modulate the host's inflammatory response. it is also clear that the leukocytes are not the only cells involved in the inflammatory response. endothelial cells, mesothelial cells, fibroblasts and epithelial cells are also all involved not only with their capacity to drive the inflammatory response through mediator generation but also in innate immune defences including through the production of antimicrobial proteins. paracrine signals between these cells will contribute to ªregionsº of inflammation rather than total systemic inflammation. our ability to monitor local and regional inflammation is only in its infancy. furthermore, early in the inflammatory response it may be appropriate to block excessive cytokine production while during later states anergy can develop where it would be more appropriate to boost leukocyte function. we do not have clear methods as yet to determine which stage inflammation is at nor do have the necessary tools to selectively block or boost specific leukocyte functions. leukocytes are an essential component in a system involving nearly every cell and tissue in the body. while excessive leukocyte activation is a key feature of malignant inflammation, many other cells are capable of synthesising pro-inflammatory cytokines and driving a damaging response. the complexity, redundancy and plasticity of host defence mechanisms make it unlikely that a global panacea for the inflammatory response will be discovered. chemokine expression in endothelial cells and monocytes is differentially regulated neutrophilic differentiation induced by human upper airway fibroblast-derived granulocyte/macrophage colony-stimulating factor (gm-csf) elevated transforming growth factor-alpha levels in bronchoalveolar lavage fluid of patients with acute respiratory distress syndrome activation of the adhesive capacity of cr on neutrophils by endotoxin: dependence on lipopolysaccharide binding protein and cd recognition of gram-negative bacteria and endotoxin by the innate immune system innate immunity: the virtues of a nonclonal system of recognition the dorsoventral regulatory gene cassette spatzle/toll/cactus controls the potent antifungal response in drosophila adults the toll-receptor family and control of innate immunity a human homologue of the drosophila toll protein signals activation of adaptive immunity toll-like receptor- mediates lipopolysaccharide-induced cellular signalling interchangeable endotoxinbinding domains in proteins with opposite lipopolysaccharide-dependent activities lipopolysaccharide (lps)-binding synthetic peptides derived from serum amyloid p component neutralize lps effects of specific antibodies, hormones, and lipoproteins on bacterial lipopolysaccharides injected into the rat lipopolysaccharide (lps)-binding proteins bpi and lbp form different types of complexes with lps lowdensity lipoprotein receptor-deficient mice are protected against lethal endotoxemia and severe gram-negative infections reconstituted high-density lipoprotein neutralizes gram-negative bacterial lipopolysaccharides in human whole blood bacterial lipopolysaccharide binds and stimulates cytokine-producing cells before neutralization by endogenous lipoproteins can occur antimicrobial peptides in mammalian and insect host defence candidacidal activity of salivary histatins. identification of a histatin -binding protein on candida albicans key: cord- -p joajvn authors: liu, zhicheng; yang, zhengtao; fu, yunhe; li, fenyang; liang, dejie; zhou, ershun; song, xiaojing; zhang, wen; zhang, xichen; cao, yongguo; zhang, naisheng title: protective effect of gossypol on lipopolysaccharide-induced acute lung injury in mice date: - - journal: inflamm res doi: . /s - - - sha: doc_id: cord_uid: p joajvn objective: gossypol has been reported to have anti-inflammatory properties. the purpose of this study was to evaluate the effect of gossypol on acute lung injury (ali) induced by lipopolysaccharide (lps) in mice. methods: male balb/c mice were pretreated with gossypol h before intranasal instillation of lps. then, h after lps administration, the myeloperoxidase in histology of lungs, lung wet/dry ratio and inflammatory cells in the bronchoalveolar lavage fluid (balf) were determined. the levels of tumor necrosis factor-α (tnf-α), interleukin- (il- ) and interleukin- β (il- β) in the balf were measured by elisa. the extent of phosphorylation of iκb-α, p nf-κb, p –p jnk, p –p erk, and p were detected by western blot. results: gossypol markedly attenuated the lps-induced histological alterations in the lung and inhibited the production of tnf-α, il- β and il- . additionally, gossypol reduced the inflammatory cells in balf, decreased the wet/dry ratio of lungs and inhibited the phosphorylation of iκb-α, p nf-κb, p –p jnk, p –p erk, and p caused by lps. conclusion: the data suggest that anti-inflammatory effects of gossypol against the lps-induced ali may be due to its ability of inhibition of the nf-κb and mapks signaling pathways. gossypol may be a promising potential therapeutic reagent for ali treatment. acute lung injury (ali), the basis of acute respiratory distress syndrome (ards), is characterized by severe hypoxemia, pulmonary edema and neutrophil accumulation in the lung [ , ] . lipopolysaccharide (lps), a main component of outer membrane of gram-negative bacteria, has been referred to be an important risk factor of ali [ , ] . intratracheal administration of lps has gained wide acceptance as a clinically relevant model of severe lung injury. in the clinical cases, ali is a major problem that has a high mortality rate of - % and there are still few effective measures or specific medicines to treat it [ , ] . therefore, the development of novel therapies for ali is urgently needed. gossypol, a yellow polyphenolic compound extracted from cottonseed ( fig. ) , has long been used as a male contraceptive drug [ , ] . in recent years, gossypol has been shown to exhibit a variety of other pharmacological activities, including anti-tumor, anti-oxidant, anti-virus and anti-inflammatory activities [ ] [ ] [ ] . gossypol was found to inhibit the activation of human t-lymphocytes stimulated with polyclonal activators, to suppress nf-jb activity and nf-jb-related genes expression in human leukemia u cells [ ] . although a number of studies have addressed the therapeutic potential of gossypol, its ability to protect against bacterial endotoxin-induced ali remains poorly understood. in this study, we sought to assess the preventive effects of gossypol in a lps-induced mouse ali model and elucidated the potential anti-inflammatory mechanism. male balb/c mice, weighing approximately - g, were purchased from the center of experimental animals of jilin university (changchun, china). and this study was approved by the ethical committee on animal research at the university of jilin (approval id: - ). the mice were housed in a room maintained at ± °c with - % humidity. all animals received food and water ad libitum. all animal experiments were performed in accordance with the guide for the care and use of laboratory animals established by the us national institutes of health. gossypol (purity:[ %) was purchased from the national institute for the control of pharmaceutical and biological products (beijing, china), dexamethasone (dex, purity: [ . %) was purchased from changle pharmaceutical co. (xinxiang, henan, china). mouse tnf-a, il- and il- b enzyme-linked immunosorbent assay (elisa) kits were purchased from biolegend (ca, usa). the myeloperoxidase (mpo) determination kit was provided by the jiancheng bioengineering institute of nanjing (nanjing, jiangsu, china). mouse monoclonal phospho-specific p antibody, mouse monoclonal phospho-specific p -p erk antibody, mouse monoclonal phospho-specific p -p jnk antibody, mouse mab phospho-nf-jb p , mouse mab phospho-ijb-a and rabbit mab ijb-a were purchased from cell signaling technology inc (beverly, ma). hrp-conjugated goat anti-rabbit and goat-mouse antibodies were provided by ge healthcare (buckinghamshire, uk). all other chemicals were of reagent grade. mice were randomly divided into eight groups: blank control group, lps group, gossypol ( . , , , and mg/kg) ? lps group, dex ? lps group. before inducing acute lung inflammation, gossypol ( . , , , and mg/kg) was given by intraperitoneal injection (i.p.), while dex, . mg/kg, was administrated intra-gastrically as a positive control. blank control and lps group mice were given an equal volume of distilled water by i.p. h later, mice were slightly anesthetized with an inhalation of diethyl ether, lg of lps in ll pbs was instilled intranasal (i.n.) to induce lung injury. blank control group mice were given a ll pbs by i.n. without lps. all the mice were alive after h lps treatment. the mice were killed by exsanguination at h after the administration of lps. collection of bronchoalveolar lavage fluid (balf) was performed three times through a tracheal cannula with autoclaved pbs, instilled up to a total volume of . ml. mice were randomly divided into six groups: blank control group, gossypol ( . , , , and mg/kg). gossypol ( . , , , and mg/kg) was given by intraperitoneal injection (i.p.). h after injection of gossypol, ll of peripheral blood was collected and mixed with the anticoagulant na -edta. automated hematological analysis was performed using a mek- k automated hematology analyzer (nihon kohden, japan). the following blood components were determined: white blood cell (wbc) count, the number of neutrophils and lymphocytes. lung wet-to-dry weight (w/d) ratio after the mice were euthanized, the lungs were removed and the wet weight recorded. the lungs were then placed in an incubator at °c for h to obtain the 'dry' weight. the ratio of wet lung to dry lung was calculated to assess tissue edema. the balf samples were centrifuged ( °c, , rpm, min) to pellet the cells. the cell pellets were resuspended in pbs for the total cell counts using a hemacytometer, and cytospins were prepared for differential cell counts by staining with the wright-giemsa staining method. determination of tnf-a, il- b and il- levels inflammatory cytokines of tnf-a, il- b and il- in balf were measured using specific elisa kits according the accumulation of neutrophils in the lung tissue was assessed by mpo activity. briefly, h after lps administration, mice under diethyl ether anesthesia were killed, and the right lungs were excised. one hundred milligrams of lung were homogenized and fluidized in extraction buffer to obtain % of homogenate. the sample including . ml homogenate and . ml of reaction buffer was heated to °c in water for min, on which occasion, the enzymatic activity was determined by measuring the change in absorbance at nm using a -well plate reader. histopathologic examination was performed on mice that were not subjected to balf collection. lungs were fixed with % buffered formalin, imbedded in paraffin and sliced. after hematoxylin and eosin (h&e) stain, pathological changes of lung tissues were observed under a light microscope. the lung injury score was quantificated by a scoring system as described elsewhere [ ] . the lung injury score was assessed as follows: no oedema, mild oedema, moderate oedema, severe oedema. for leucocyte or other cell infiltration, the grading system was that used to determine the extend of oedema: no cellular infiltration, mild cellular infiltration, moderate cellular infiltration, and severe cellular infiltration. each one gave a score for each from to . at h after the injection of lps, lung tissues were harvested and frozen in liquid nitrogen immediately until homogenization. proteins were extracted from the lungs using t-per tissue protein extraction reagent kit (thermo) according to the manufacturer's instructions. protein concentrations were determined by bca protein assay kit and equal amounts of protein were loaded per well on a % sodium dodecyl sulphate polyacrylamide gel. subsequently, proteins were transferred onto polyvinylidene difluoride membrane. the resulting membrane was blocked with tris-buffered saline containing . % tween- (tbs-t), supplemented with % skim milk (sigma) at room temperature for h on a rotary shaker, and followed by tbs-t washing. the specific primary antibody, diluted in tbs-t, was incubated with the membrane at °c overnight. subsequently, the membrane was washed with tbs-t followed by incubation with the peroxidase-conjugated secondary antibody at room temperature for h. the immunoactive proteins were detected by using an enhanced chemiluminescence (ecl) western blotting detection kit. all values are expressed as mean ± sem. differences between mean values of normally distributed data were analyzed using one-way anova (dunnett's t test) and twotailed student's t test. statistical significance was accepted at p \ . . to test if gossypol treatment effected the blood leukocytes of mice, we detected the blood leukocytes by routine blood test. the results showed gossypol treatment did not effect the blood leukocytes (table ) . gossypol inhibited lps-induced lung w/d ratio lps caused a significant increase in lung w/d ratio (p # \ . ) compared to the control group (fig. ) . gossypol ( and mg/kg) and dex significantly decreased the lung w/d ratio (p* \ . ) compared to those in the lps group (fig. ) . gossypol inhibited the inflammatory cell count in the balf of lps-induced ali mice the number of inflammatory cells, such as neutrophils and macrophages, in balf were analyzed at h after lps challenge. as shown in fig. , lps challenge significantly increased the number of total cells, neutrophils and macrophages compared with the control group (p # \ . ). meanwhile, pretreatment with gossypol ( . , , , and mg/kg) and dex ( . mg/kg) was found to significantly decrease the number of total cells (p \ . ), neutrophils (p \ . ), and macrophages (p \ . ). gossypol suppressed the production of cytokines in the balf of lps-treated ali mice the effect of gossypol on tnf-a, il- b and il- production was analyzed at h after lps challenge by elisa. as shown in fig. , the concentrations of tnf-a, il- , and il- b in balf were significantly increased after lps administration. gossypol ( . , , , and mg/kg) and dex significantly reduced tnf-a (p* \ . ), il- (p* \ . ), and il- b (p* \ . ) production compared to those in the lps group. the mpo activity (fig. ) was determined to assess the neutrophil accumulation within pulmonary tissues. lps challenge resulted in significantly increased lung mpo activity compared with the control group (p \ . ). however, this increase was apparently reduced by gossypol ( . , , , and mg/kg) (p \ . ) or dex (p \ . ). lung tissues, harvested at h after injection of lps, were subjected to he staining. as shown in fig. , lung tissues from the control showed a normal structure and no histopathologic changes under a light microscope (fig. a) . lung sections obtained from mice in lps group showed characteristic histological changes, including areas of inflammatory infiltration, focal areas of fibrosis with collapse of air alveoli and emphysematous, as well as thickening of the alveolar wall and pulmonary congestion (fig. b) . however, lps-induced pathological changes were significantly attenuated by gossypol ( , , and mg/kg) (fig. e-h) and dex ( . mg/ kg) treatment (fig. c) . evaluation of the lung injury score revealed that gossypol significantly attenuated lps-induced ali ( table ) . western blot analysis showed that nf-jb and mitogenactivated protein kinase (mapk) signaling pathways were activated h after lps treatment. pretreatment with gossypol ( mg/kg) inhibited the phosphorylation of ijb-a, p nf-jb (fig. ) , p , jnk and erk (fig. ) in all, these results showed that gossypol ( mg/kg) could simultaneously inhibit nf-jb and mapk signaling pathways efficiently in a mouse model of ali. lps-induced ali was characterized by the disruption of endothelial and epithelial integrity, lung edema, the release of inflammatory mediators, and extensive neutrophil infiltration [ ] . though several candidate therapies have been applied to reduce lung injury, there are still few effective measures or specific medicines to treat it. gossypol is a yellow polyphenolic compound isolated from cottonseed and has been shown to exhibit anti-inflammatory effects recently. in the present study, we observed the effect of gossypol on ali induced by lps in mice. the results showed that pretreatment with gossypol attenuated lung damage induced by lps and decreased the w/d ratio, proinflammatory cytokine production, inflammatory cell migration into the lung, protein leakage, the activation of nf-jb and mapk. this suggests that gossypol may be a promising potential therapeutic reagent for ali treatment. pulmonary edema is one of the major characteristics of ali [ ] . in this study, we evaluated the w/d ratio of the lung to quantify the magnitude of pulmonary edema. our experiments showed that gossypol significantly inhibits edema of the lung, as shown by a w/d ratio in the gossypol group that was significantly lower than the lps group. mpo activity, reflecting the parenchymal infiltration of neutrophils and macrophages, lps-induced ali is characterized by the infiltration of neutrophils in the lung, exhibiting increased mpo activity [ , ] . in this study, we found that lps administration significantly increased the mpo activity and pretreatment with gossypol decreased lps-induced increases in mpo activity in the lungs. this indicated that the protective effect of gossypol in ali is related to attenuation of neutrophil influx into the lung tissue. pro-inflammatory cytokines including tnf-a, il- b and il- participated in the development of ali [ ] [ ] [ ] . some reports have shown that lps-induced ali can lead to the overproduction of these cytokines. tnf-a is the earliest and primary pro-inflammatory factor produced when infection [ ] . il- b is a crucial mediator in ali. it plays an important role in the progression multiple organ failure in lps-induced endotoxic shock [ , ] . il- is also a marker of the acute inflammatory response in lpsinduced ali mode [ , ] . in the present study, gossypol significantly inhibited the production of tnf-a, il- b and il- induced by lps. these results indicate that the protective effects of gossypol on ali induced by lps may be attributed to the compound's inhibition of inflammatory factors. lps induces its inflammatory reaction through the activation of both nf-jb and mapks signaling pathways to regulate the release of pro-inflammatory cytokines [ ] . nf-jb is normally present in the cytoplasm as a heterodimer and is linked to the inhibitory proteins ijbs. once activated, nf-jb units p dissociates from its inhibitory protein ijb-a and translocates from the cytoplasm to the nucleus where they may trigger the transcription of specific target genes such as tnf-a, il- b and il- [ ] . in this study, we tested the effects of gossypol on nf-jb activation and ijb-a degradation. the results showed that lps stimulation dramatically increased the phosphorylation of ijb-a and nf-jb p protein. however, lps-induced ijb-a degradation and nf-jb p activation were significantly blocked by pretreatment with gossypol. mapks also play an important role in inducing cytokine production [ , ] . the lps stimulation of murine macrophages has been known to induce phosphorylation and activation of erk / , jnk, and p mapks [ ] . therefore, we investigated the effect of gossypol on activation (phosphorylation) of three mapks induced by lps in the mice of ali. the results showed that gossypol ( . , , and mg/kg) inhibited the phosphorylation of p -p erk, p , and p -p jnk in lps-stimulated mice. taken together, these results indicate that gossypol may exert its anti-inflammatory action by inhibition of the nf-jb and mapks signaling pathways activation. in conclusion, the present study demonstrated that gossypol has a protective effect against lps-induced ali, which may be related to its suppression of nf-jb and mapks activation, and subsequently leads to the reduction the inflammatory cell infiltration and proinflammatory cytokine expression in lung tissues. these findings suggest that gossypol may be an agent for preventing and treating lps induced ali. epidemiology of acute lung injury protective effect of florfenicol on acute lung injury induced by lipopolysaccharide in mice the pulmonary physician in critical care. : acute lung injury and the acute respiratory distress syndrome: definitions and epidemiology incidence and outcomes of acute lung injury the acute respiratory distress syndrome neutrophil activation and acute lung injury gossypol: a contraceptive for men a combined regimen of gossypol plus methyltestosterone and ethinylestradiol as a contraceptive induces germ cell apoptosis and expression of its related genes in rats induction of apoptosis by (-)-gossypol-enriched cottonseed oil in human breast cancer cells gossypol inhibits phosphorylation of bcl- in human leukemia hl- cells induction of neutrophil mac- integrin expression and superoxide production by the medicinal plant extract gossypol gossypol suppresses nf-kappab activity and nf-kappab-related gene expression in human leukemia u cells inhibition of inducible nitric oxide synthase attenuates acute endotoxin-induced lung injury in rats acute lung injury induces cardiovascular dysfunction: effects of il- and budesonide/formoterol antiinflammatory effects of hydrogen peroxide in neutrophil activation and acute lung injury role of inflammatory mediators in the pathophysiology of acute respiratory distress syndrome stem cells in sepsis and acute lung injury cytokine-mediated inflammation in acute lung injury local stimulation of alpha cholinergic receptors inhibits lps-induced tnf-alpha release in the mouse lung the role of nuclear factor-kappa b in pulmonary diseases tnf: a key neuroinflammatory mediator of neurotoxicity and neurodegeneration in models of parkinson's disease il- cytoprotection in hyperoxic acute lung injury occurs via pi k/akt-mediated bax phosphorylation phosphoinositide-mediated adaptor recruitment controls toll-like receptor signaling introduction to nf-kappab: players, pathways, perspectives map kinase activation in macrophages kinetics of mitogenactivated protein kinase family in lipopolysaccharide-stimulated mouse kupffer cells and their role in cytokine production isoeugenol suppression of inducible nitric oxide synthase expression is mediated by down-regulation of nf-kappab, erk / , and p kinase key: cord- -l ufs k authors: tomita, kengo; saito, yuna; suzuki, tokiko; imbaby, samar; hattori, kohshi; matsuda, naoyuki; hattori, yuichi title: vascular endothelial growth factor contributes to lung vascular hyperpermeability in sepsis-associated acute lung injury date: - - journal: naunyn schmiedebergs arch pharmacol doi: . /s - - - sha: doc_id: cord_uid: l ufs k vascular endothelial growth factor (vegf) is a prime regulator of vascular permeability. acute lung injury (ali) is characterized by high-permeability pulmonary edema in addition to refractory hypoxemia and diffuse pulmonary infiltrates. in this study, we examined whether vegf can be implicated as a pulmonary vascular permeability factor in sepsis-associated ali. we found that a great increase in lung vascular leak occurred in mice instilled intranasally with lipopolysaccharide (lps), as assessed by igm levels in bronchoalveolar lavage fluid. treatment with the vegf-neutralizing monoclonal antibody bevacizumab significantly reduced this hyperpermeability response, suggesting active participation of vegf in non-cardiogenic lung edema associated with lps-induced ali. however, this was not solely attributable to excessive levels of intrapulmonary vegf. expression levels of vegf were significantly reduced in lung tissues from mice with both intranasal lps administration and cecal ligation and puncture (clp)-induced sepsis, which may stem from decreases in non-endothelial cells-dependent vegf production in the lungs. in support of this assumption, stimulation with lps and interferon-γ (ifn-γ) significantly increased vegf in human pulmonary microvascular endothelial cells (hpmecs) at mrna and protein levels. furthermore, a significant rise in plasma vegf levels was observed in clp-induced septic mice. the increase in vegf released from hpmecs after lps/ifn-γ challenge was completely blocked by either specific inhibitor of mitogen-activated protein kinase (mapk) subgroups. taken together, our results indicate that vegf can contribute to the development of non-cardiogenic lung edema in sepsis-associated ali due to increased vegf secretion from pulmonary vascular endothelial cells through multiple mapk-dependent pathways. sepsis is a potentially life-threatening medical emergency that is caused by the body's extreme response to an infection. the advent of the new definition of sepsis, which has been published recently, prompts a reappraisal of organ dysfunction as the hallmark of sepsis (singer et al. ). sepsis affects every major organ, including the lung, liver, and kidney, within the body, ultimately leading to the failure of one or more organs. the respiratory system is the most affected organ of the body, and lung dysfunction is the first step in the development of multiple organ failure in septic patients. acute lung injury (ali) and its most extreme form, acute respiratory distress syndrome (ards), are the manifestations of lethal and complex respiratory dysfunction and are characterized by explosive and diffuse pulmonary infiltrates, leading to noncardiogenic alveolar edema and, ultimately, refractory hypoxemia (tsushima et al. ; matuschak and lechner ) . vascular endothelial growth factor (vegf), also known as vegf-a, is a glycoprotein originally isolated as a tumor cellsecreted vascular permeability factor (plouet et al. ) and, since then, it has long been documented that vegf serves as the prime regulator of vascular permeability (bates ) . while vegf has been subsequently shown to have additional potent mitogenic and angiogenic properties (ferrara ; sharma et al., ) , significant amounts of vegf are known to exist in the normal human lung without significant mitogenesis or angiogenesis (barratt et al. ). in the normal lung, vegf may function as a survival factor for epithelial cells and endothelial cells in a paracrine manner (gerber et al. ; mura et al. ; roberts et al. ). in the meanwhile, vegf may contribute to the development of non-cardiogenic pulmonary edema associated with ali/ards. lung-targeted overexpression of human vegf , using an adenoviral gene vector, has been shown to result in pulmonary edema and increased vascular permeability in mice (kaner et al. ) . in addition, it has been revealed that the high ventilation-induced increase in pulmonary microvascular permeability in mice can be attenuated by knockdown of vegf by short-interfering rnas (li et al. ) . moreover, pretreatment with adenovirus-encoding soluble vegf receptor (vegfr) has been found to prevent ischemia-reperfusion-induced lung injury in rats (godzich et al. ). however, a role of vegf in pulmonary vascular hyperpermeability accompanied by sepsis-associated ali is not fully understood. in the present study, by conducting in vivo and in vitro experiments using rodent models of sepsis-associated ali and human pulmonary microvascular endothelial cell line, respectively, we attempted to test the hypothesis that vegf may contribute to non-cardiogenic high vascular permeability pulmonary edema in ali associated with sepsis. all animal experimental procedures were conducted in accordance with the national institute of health guidelines on the use of laboratory animal and with approval of the care and use committee of the university of toyama. in the first series of experiments, we used the cecal ligation and puncture (clp)-induced sepsis mouse model. clp-induced sepsis is regarded as a highly clinically relevant model of polymicrobial sepsis, because it reproduces many hallmarks of sepsis occurring in human patients (hubbard et al. ) . the surgical procedure to generate clp-induced sepsis was conducted according to our previous reports (tomita et al. ; kawakami et al. ; yamashita et al. ) . in brief, male balb/c mice (sankyo lab service, tokyo japan), - weeks old, were anesthetized with - % sevoflurane by inhalation, and a middle abdominal incision was made. the cecum was mobilized, tightly ligated ( cm from the cecum tip), punctured twice with a -gauge needle, and gently squeezed to expel small amounts of feces. then, the bowel was repositioned to the peritoneal cavity, and the laparotomy site was closed with sterile suture (the skin and muscle were sutured separately). sham-operated control underwent the same procedure except for ligation and puncture of the cecum. both groups of animals were fed the same diet and water ad libitum, and housed in an environment with controlled temperature, constant humidity, and a daily -h light-dark cycle ). all animals after clp surgery were lethargic, showed lack of interest in their environment, displayed piloerection, and had crusty exudates around their eyes, as opposed to sham-operated animals that were healthy, moving freely and eating (matsuda et al. ). all mice received subcutaneous injection of . ml sterile normal saline immediately after surgery. at h after surgery, blood samples were collected and inflation-fixed lungs were harvested from mice treated with - mg/kg ketamine and mg/kg xylazine hydrochloride. in the second series of experiments, mice, under light anesthesia, were instilled intranasally with μg of lipopolysaccharide (lps) (escherichia coli :b ; list biological laboratories, campbell, ca, usa) in μl of sterile . % nacl solution. control animals received equivalent volume of vehicle saline solution. when bevacizumab, which neutralizes vegf and blocks its signal transduction through vegf receptors (papadopoulos et al. ) , was used, it was intravenously given to mice at a dose of μg at min before administration of lps. animals were euthanized at h after lps challenge. all experimental data were analyzed in a blinded fashion. the immortalized human pulmonary microvascular endothelial cell line (hpmec-st . r), which was developed by means of co-transfection of a plasmid encoding the catalytic component of telomerase and a plasmid encoding the simian virus large t antigen (krump-konvalinkova et al. ; unger et al. ) , was kindly provided by drs. c. james kirkpatrick and ronald e. unger at johannes gutenberg university (mainz, germany). hpmec-st . r cells were routinely maintained on tissue culture plastic ware in m medium (sigma-aldrich, st. louis, mo, usa) supplemented with % (v/v) heat-inactivated fetal bovine serum, μg/ml endothelial cell growth supplements (sigma-aldrich), μg/ml sodium heparin (sigma-aldrich), % (v/v) penicillin/streptomycin (nacalai tesque, kyoto, japan), and μg/ml g (nacalai tesque). cells were cultured at °c under a humidified atmosphere containing % co and % air. in hpmec-st . r cells, the presence of interferon (ifn)-γ can highly amplify the inflammatory responses to lps ). thus, cells were stimulated with μg/ml lps and ng/ml ifn-γ (r&d systems, minneapolis, mn, usa). when mitogen-activated protein kinase (mapk) inhibitors, pd ( μm; cayman chemical, ann arbor, mi, usa), sb ( μm; adipogen life sciences, epalinges, switzerland), sp ( μm; cayman chemical), and jnk-in- ( μm; merck, darmstadt, germany) were used, they were added to the medium for min before challenge with lps and ifn-γ. lps/ ifn-γ stimulation was stopped at the indicated time points by aspirating off the culture medium and then adding ice-cold pss before harvesting. blood levels of tumor necrosis factor (tnf)-α, interleukin (il)- β, il- , and monocyte chemoattractant protein- (mcp- ) were measured by the use of commercially available enzyme-linked immunosorbent assay (elisa) kit (r&d systems, minneapolis, mn) according to the manufacturer's instructions. to measure the concentrations of vegf in serum and culture medium samples, mouse vegf quantikine elisa kit (r&d system) and human vegf duoset elisa (r&d system) were used, respectively. the plate was read on a microplate reader (nippon-intermed, tokyo, japan). assays were performed in duplicate. to estimate pulmonary microvascular permeability, igm was selected as a marker of permeability and its concentration in bronchoalveolar lavage (bal) fluid was determined immunologically by igm mouse uncoated elisa kits with plates (thermo fisher scientific, rockford, il, usa). a polyethylene catheter was inserted into the trachea, bal was performed by repeatedly infusing and removing ml of pbs, and the third drainage of effluent was kept as bal fluid. total rna was isolated from hpmec-st . r cells and lung tissues with the use of sepazol®-rna i super g (nacalai tesque) according to the manufacturer's manual. revertra ace qpcr rt master mix (toyobo, osaka, japan) was used for the reverse transcription reaction, and real-time pcr analyses were performed using powerup™ sybr® green master mix (thermo fisher scientific), as described in the manufacturers' instructions. values were normalized to the housekeeping gene gapdh according to the manufacturer's protocol (mx p real-time pcr system; agilent technologies inc., santa clara, ca, usa). additional details are described by our laboratory (kawakami et al. ; yamashita et al. ; suzuki et al. ). the pcr primers were designed as follows: forward '-tgcagattatgcggatcaaacc- ′ and reverse ′-tgcattcacatttgttgtgctgtag- ′ for vegf, forward ′-caggcccagtttctgccatt- ′ and reverse ′-ttccagctcagcgtggtcgta- ′ for vegfr , forward ′-ccagcaaaagcagggagtct gt- ′ and reverse ′-tgtctgtgtcatcggagtga tatcc- ′ for vegfr , and forward ′-tgtg tccgtcgtggatctga- ′ and reverse ′-ttgc tgttgaagtcgcaggag- ′ for glyceraldehyde- phosphate dehydrogenase (gapdh). hpmec-st . r cells were grown in -mm dish, harvested, and lysed in μl of ripa buffer ( mm tris-hcl, mm nacl, % np- , % sodium deoxycholate, . % sds, ph . ) containing protease inhibitor cocktail on ice. the lysates were centrifuged at , ×g for min at °c and the resulting supernatants were collected. the protein concentration in the remaining supernatant was measured using bca protein assay kit (nacalai tesque). blotting procedure, chemiluminescent detection, and densitometric analysis were carried out as described in our previous reports (kawakami et al. ; suzuki et al. ) . samples ( - μg of protein) were separated with % sds-page gel electrophoresis and then transferred to polyvinylidene difluoride filter membrane. the membrane was probed with the following primary antibodies: anti-human extracellular signal-regulated protein kinase (erk) / mouse monoclonal antibody ( : ; cell signaling, danvers, ma, usa), anti-human phospho-erk / (thr- /tyr- ) rabbit monoclonal antibody ( : ; cell signaling), anti-human c-jun nterminal kinase (jnk) rabbit monoclonal antibody ( : ; cell signaling), anti-human p rabbit monoclonal antibody ( : ; cell signaling), anti-human phospho-p (thr- /tyr- ) mouse monoclonal antibody ( : ; cell signaling), anti-human jnk (thr- /tyr- ) mouse monoclonal antibody ( : ; cell signaling), and anti-human gapdh chicken polyclonal antibody ( : ; emd millipore, billerica, ma, usa). irdye®-labeled secondary antibodies were purchased from li-cor bioscience (lincoln, ne, usa) and odyssey clx infrared imaging system (li-cor bioscience) was employed for primary antibody detection. gapdh was used as the loading control. results are presented as mean ± standard error. data were analyzed by the use of prism software (version ; graphpad software, san diego, ca, usa). statistical analysis was performed by student's t test or one-way analysis of variance (anova) followed by tukey's multiple-comparison test. differences were considered to be statistically significant when a p value was < . . the clp rodent model, which causes peritonitis and leads to a polymicrobial sepsis, represents an indirect insult similar to the pathogenesis of ali/ards (villar et al. ) . indeed, we have clearly demonstrated that mice - h after clp surgery display marked hypoxemia, increased lung vascular permeability, and histological damage in lungs, including wall thickening, inflammatory infiltrate, and hemorrhage (takano et al. ; oishi et al. ; imaizumi et al. ) . when blood levels of pro-inflammatory cytokines were measured using an elisa, the sham-operated control animals had extremely low levels of the cytokines examined here. the mice h after clp-induced sepsis exhibited marked elevations in blood levels of tnf-α, il- β, il- , and mcp- (c) the mrna levels of vegf, vegfr , and vegfr in lung tissues were quantified by real-time pcr. lung tissues were harvested h after surgery. values are normalized to gapdh (n = ). * p < . , ** p < . , and *** p < . vs. the sham-operated control group (fig. a) . sepsis induction by clp also resulted in a significant elevation in plasma vegf protein levels in mice (fig. b) . plasma vegf in clp mice was increased . -fold in comparison with sham-operated control animals. however, the vegf mrna level was significantly downregulated in lung tissues from mice with clp-induced sepsis (fig. c) . we further examined vegfr mrna levels in clp mouse lungs. vegf regulates vascular permeability by activating receptors, vegfr (flt- ) and vegfr (kdr/flk ) (shibuya ) . as shown in fig. c , vegfr mrna was increased and vegfr mrna was decreased in lung tissues of clp mice compared with sham-operated controls. to assess changes in pulmonary vascular permeability, bal supernatant was analyzed for igm using an elisa. intranasal challenge with lps resulted in a highly significant . -fold increase in pulmonary vascular permeability (fig. a) . treatment with bevacizumab, which neutralizes vegf and blocks vegfr signaling (papadopoulos et al. ) , significantly but partially attenuated lung vascular permeability as compared with that shown in mice challenged with lps alone. we also examined vegf mrna levels in lung tissues of mice intranasally instilled with lps. as seen in clp-induced septic mice, pulmonary mrna expression was significantly downregulated in mice challenged with lps (fig. b ). in the immortalized human pulmonary microvascular endothelial cell line hpmec-st . r, the time course of changes in gene expression of vegf and its receptors after coadministration of lps and ifn-γ were investigated. the mrna level of vegf was gradually increased, reached a maximum at - h, and then showed a return toward the baseline at h (fig. a) . the mrna level of vegfr showed a trend toward increasing throughout - h observation period (fig. b) . vegfr mrna remained virtually unchanged in a wide range of time after stimulation with lps and ifn-γ (fig. c) . when the amounts of vegf in culture media were measured by an elisa, lps/ifn-γ challenge resulted in a significant increase in the protein level of vegf (fig. b) . the mapk signaling cascades are generally thought to be important in the pathogenesis of ali/ards (newton and holden ; qian et al. ) . when activation of the three major subgroups of mapk family, erk / , p , and jnk, was assessed by increases in their phosphorylation levels, coadministration of lps and ifn-γ resulted in significant activation of all three families of mapks in hpmec-st . r cells (fig. a) . we thus examined whether expression of vegf in human pulmonary microvascular endothelial cells is regulated by mapks. when hpmec-st . r cells were treated with pd , an inhibitor of mapk kinase which is an erk / upstream activator, or sb , which is widely used as a specific inhibitor of p mapk, the lps/ifn-γinduced increase in vegf protein levels was strongly blocked (fig. b) . treatment with sp , an anthrapyrazolone inhibitor of jnk, also abrogated the vegf protein increase in hpmec-st . r cells stimulated with lps/ifn-γ (fig. b) . the striking inhibition of the lps/ifn-γ-induced vegf upregulation was similarly observed by treatment with another fig. involvement of vegf in lung vascular hyperpermeability in mice after lps administration. lps ( μg) was instilled intranasally and animals were euthanized at h after lps challenge. (a) lung vascular permeability was assessed by igm levels in bal fluid from mouse lungs (n = - ). bevacizumab ( μg) was intravenously injected to mice min before lps challenge. (b) the mrna levels of vegf in lung tissues were quantified by real-time pcr. values are normalized to gapdh (n = ). *** p < . vs. control. ## p < . vs. lps alone jnk inhibitor jnk-in- , which is far more selective for jnk than sp (brain et al. ) (fig. b ). we demonstrated in this study for the first time that vegf contributed to pulmonary vascular hyperpermeability when lps was intranasally administrated in mice. intranasal administration of lps has long been widely used as an appropriate model in which ali can be directly induced (gharib et al. ; bosmann et al. ; juschten et al. ) , and increased pulmonary vascular permeability is a critical and non-redundant pathological process involved in the ali development (herold et al., ) . we found that treatment with the vegf-neutralizing monoclonal antibody bevacizumab resulted in a significant inhibition of the increase in pulmonary vascular permeability caused by intranasal lps challenge, as evidenced by changes in igm levels in bal fluid from mouse lungs. previous reports have well established that the amounts of igm in bal fluid are related with alterations in alveolarcapillary barrier and lung vascular permeability (kantrow et al. ; matute-bello et al. ; johnston et al. ) . intriguingly, while bevacizumab is clinically used for treatment of advanced cancers of the lung, colon, brain, kidney, and others by counteracting the angiogenic effect of vegf, its ability to reduce the increase in vascular permeability associated with vegf expression may help relieve patients with the potentially serious morbidity and symptoms that accompany peritumoral edema (gil-gil et al. ) . our findings imply the active participation of vegf in non-cardiogenic high vascular permeability pulmonary edema associated with lpsinduced ali (fig. ) . however, the preventive effect of bevacizumab on lps-induced pulmonary vascular hyperpermeability was partial. our previous study has shown that treatment with n g -nitro-l-arginine, an inducible nitric oxide (no) synthase (inos) inhibitor, or diphenhydramine, a histamine h -receptor antagonist, significantly but incompletely inhibited lps-induced lung vascular permeability in mice (matsuda et al. ) , which suggests that no and histamine may also be partly responsible for mediating increased lung vascular leak following lps challenge. we thus assume that several vascular permeability molecules, including vegf, no, and histamine, can be excessively produced and thereby actually contribute to the development of pulmonary edema in sepsis-associated ali. indeed, great increases in gene and protein expression of inos and histidine decarboxylase, an enzyme that only forms histamine in mammals, have been observed in the lungs of mice after induction of sepsis with lps (matsuda et al. ) . unexpectedly, expression of vegf in lung tissues was significantly downregulated rather than upregulated in two murine models of ali, intranasal lps administration and clp-induced sepsis. this observation is consistent with a string of human studies showing a reduction in intrapulmonary vegf in the early stages of ali/ards (maitre et al. ; thickett et al. ; abadie et al. ) , although a conflicting result was reported in mice exposed to lps in a nebulization chamber (karmpaliotis et al. ) . the reduced levels of intrapulmonary vegf in ali/ards may be explained by direct injury to and clearance of epithelial type cells (medford and millar ) which are considered to be the main source of vegf in lungs (kaner and crystal ) . in this regard, caution would be required in the interpretation of the observed decrease in vegf levels in lung tissues, since the total amount of intrapulmonary vegf may be determined by the deduction of vegf released from different intrapulmonary components, such as epithelial cells and vascular endothelial cells, which can be variably affected by endotoxin. as reported in human studies (maitre et al. ; thickett et al. ; azamfirei et al. ), we found a significant rise in plasma levels of vegf in mice with clp- fig. involvement of mapk activation in vegf released from human pulmonary microvascular endothelial cells after challenge with lps and ifn-γ. hpmec-st . r cells were stimulated with μg/ml lps and ng/ml ifn-γ. (a) activation of mapks in hpmec-st . r after challenge with lps and ifn-γ. levels of phosphorylation and total expression of erk / , p , and jnk before and min after lps/ifn-γ challenge were determined by western blotting. in the top trace of each panel, typical western blots are shown. in the bottom trace, the summary of quantification of densitometric measurements as ratio of phospho-mapk relative to mapk is presented (n = - ). * p < . and ** p < . vs. unstimulated value. (b) effects of mapk inhibitors on vegf levels in hpmec-st . r cells stimulated with lps/ifn-γ for h. pd ( μm), sb ( μm), sp ( μm), or jnk-in- ( μm) was added h before lps/ifn-γ challenge. the vegf levels released from cells into the cell culture medium were measured by an elisa (n = ). ** p < . vs. control. ### p < . vs. lps/ ifn-γ alone induced sepsis. this rise in plasma vegf levels seems likely to be attributed to the release from vascular endothelial cells. we showed that stimulation with lps/ifn-γ resulted in a significant upregulation of vegf expression in human pulmonary microvascular endothelial cells at mrna and protein levels, implying that endotoxin positively regulates endothelial cell-derived vegf in a transcription manner. it should be added that neutrophils may also contribute to the increased vegf plasma levels in clp-induced septic mice, because these hemocytes have been shown to produce various vascular permeability molecules, including vegf, in certain circumstances such as inflammation (taichman et al. ; distasi and ley ) . in line with our recent report , stimulation with lps/ifn-γ significantly activated three major subgroups of mapk family, erk / , p , and jnk, in human pulmonary microvascular endothelial cells, as assessed by their phosphorylation levels. each specific inhibitor of these mapk subgroups completely blocked the increase in vegf protein levels caused by lps/ifn-γ challenge. this suggests that endotoxin upregulates vegf expression in pulmonary microvascular endothelial cells through multiple mapkdependent pathways. it is noteworthy that mapk signaling is linked to increased expression of growth factors, including vegf, in different cell types (li et al. ; pagès et al. ; rak et al. ; schrma et al. ) . vegf displays broad vascular functions, including vascular permeability, via binding to and activating its specific receptors, vegfr and vegfr (shibuya ) . in this study, in lung tissues from clp-induced septic mice, vegfr mrna was upregulated and vegfr mrna was downregulated compared with sham-operated controls. furthermore, lps/ifn-γ application showed a trend to increase vegfr mrna in human pulmonary microvascular fig. schematic diagram of the participation of vegf released from pulmonary vascular endothelial cells in noncardiogenic high vascular permeability pulmonary edema associated with lps-induced ali. see text for details endothelial cells. however, it is not clear at this time whether altered expression of vegfr can be involved as regulatory mechanisms to transduce vegf-mediated vascular hyperpermeability signals. several lines of evidence provide that vegfr may function as a decoy receptor and negatively regulate vegf functions (sato et al. ; cao ) . accordingly, what role, if any, is played by altered vegfr expression observed in this study in the pathophysiology of sepsis-associated ali awaits further study. in conclusion, the present results indicate that vegf can contribute to the development of 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sepsis-induced acute lung injury model cardioprotective and functional effects of levosimendan and milrinone in mice with cecal ligation and puncture-induced sepsis publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations acknowledgments we thank drs. takahiro imaizumi and takuya sakamoto for their expert help in the commencing time of this study.author contributions k.t., n.m., and y.h. conceived and designed the experiments. k.t.., y.s., t.s., and s.i. performed the experiments. k.t., y.s., and t.s. analyzed data. k.h. and y.h. wrote the article. all authors read and approved the manuscript. the authors declare that all data were generated in-house and that no paper mill was used.funding information this study was supported by grant-in-aid for young scientists ( k , k ) and for scientific research ( k , h ) from japan society for promotion of science. all animal studies were approved by the animal care and use committee of the university of toyama, which are based on the national institute of health guide for the care and use of laboratory animals and the arrive guidelines. the authors declare that they have no conflict of interest. key: cord- - ltb dpa authors: li, xiangru; hao, zhenhua; liu, xiaorong; li, wei title: deficiency of mouse fhr- homolog, fhr-e, accelerates sepsis, and acute kidney injury through enhancing the lps-induced alternative complement pathway date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: ltb dpa alternative complement pathway (ap) plays an important role in the development of sepsis, which is life threatening. deficiency of factor h-related protein (fhr- ), which is a regulator of ap, has been considered as a susceptible factor for atypical hemolytic uremic syndrome (ahus) and other types of nephropathy when an inducer such as infection exists. however, the underlying mechanism of the disease development is largely unknown. there is no report on cfhr gene knockout in any animal infection model and its function in vivo is still unclear. here, a cfhr knockout mouse was generated for investigating ap in sepsis and sepsis-induced acute kidney injury (aki). we found that murine fhr- homolog (fhr-e) deficiency enhanced lipopolysaccharide (lps)-induced ap activation both in vitro and in vivo and that cfhr knockout mice exhibited more severe sepsis and aki in response to lps challenge. these results indicated that fhr-e deficiency promoted lps-induced sepsis and aki through ap over-activation, providing a mouse model for studying ap regulation and sepsis. this study revealed the function of fhr-e in vivo, which may further provide hints to the pathogenesis of fhr- deficiency-related diseases by enhancing lps-induced ap activation. sepsis is a critical health condition with high mortality rate ( , ) and acute kidney injury (aki) is a common severe complication of sepsis ( ) . gram-negative bacteria infection is a predominant cause of severe infection-triggered sepsis ( ) . sepsis is an intricate process during which complement system is activated and proved to be double-edged with benefits and harms ( ) . the complement system is crucial in immune surveillance ( ) and has extensive cross-talk with coagulation system and inflammation for homeostasis ( ) . it is triggered through three pathways among which the alternative complement pathway (ap) activation is responsible for more than % of terminal complement activation ( , ) . ap plays an important role in endotoxin clearance during the process of sepsis ( ) . lack of ap activation in individuals predisposes to infection ( ) ( ) ( ) . however, it is also life-threatening when complement is excessively activated, and inhibition of its over-activation prevents organ injuries ( ) . therefore, uncovering the precise regulatory mechanism of ap during sepsis may shed light on sepsis intervention. however, the mechanism of ap regulation during sepsis remains elusive. factor h (fh) and its related proteins (fhrs) are regulators of ap ( ) and responsible for determining complement activating surfaces ( ) . fh, the major regulatory factor of ap ( ) , has been widely investigated, while functions of fhrs are unclear or controversial. fh and fhrs consist of different number of complement control protein modules (ccps). all fhrs contain homologous ccps to ccp , of fh which are responsible for ligand recognition ( ) . in general, fh functions as a cofactor of factor i (fi) to cleave c b and accelerate the decay of c convertase ( , ) , while fhrs act as the competitor of fh ( ) . however, different fhrs may have different functions. fhr- inhibits c convertase ( ) . fhr- displays the cofactor activity for fi ( ) . fhr- inhibits c convertase in fluid phase and displays cofactor activity for fi ( ) . nevertheless, it remains elusive for the physiological functions of fhrs. mutations in fh and fhrs are associated with various diseases ( ) . cfhr -cfhr deletion increased the risk of atypical hemolytic uremic syndrome (ahus) and systemic lupus erythematosus (sle) ( ) ( ) ( ) . cfhr -cfhr deletion was proved to protect against iga nephropathy (igan) ( ) and age-related macular degeneration (amd) ( ) . the precise mechanism of this contradictory effect is unclear. in vitro studies of factor h-related protein (fhr- ) have shown that fhr- interferes with the regulation of fh by competing with fh and inhibits the activity of c convertase and the formation of terminal complement complex (tcc) ( ) . no obvious c and c regulatory activity at physiological concentration has been found in fhr- ( ) , albeit it is the most abundant fhr protein ( , ) . healthy individuals with cfhr -cfhr deletion showed higher frequency of patrolling monocytes, plasmacytoid dendritic cells (dcs), and lower frequency of classical monocytes, myeloid dcs. monocytes with cfhr -cfhr deletion secreted higher level of il- β in response to lps challenge ( ) . necrotic cells bound to fhr- promotes the secretion of tnf-α, il- β, il- , and il- by monocytes ( ) . these reports suggest multiple effects of cfhr deletion and highlight its complexity. the existence of relatively high frequency of healthy individuals with cfhr -cfhr deletion ( ) indicates that other triggers are essential to amplify the effect of fhr- deficiency, such as infections ( , ) . however, there is no report on fhr- deficiency in any animal infection model. three genes, fhrb, fhrc, and fhre (alias cfhr ), and two pseudogenes, fhra and fhrd, were identified in mouse ( , ) . recombinant fhr-a, fhr-b, and fhr-c were reported to interact with human c d, suggesting that murine fhrs function as homologs of human fhrs ( ) . none of fhr-a, fhr-b, and fhr-c contain the critical dimerization domain that exists in human fhr- , fhr- , and fhr- ( ), while fhr-e is predicted to contain it ( ) . thus, fhr-e is more likely the murine homolog of human fhr- or fhr- ( ) . nomenclature of human and mouse fhrs differs and there exist discrepancies in literature and existing database ( ) . inferred from the homology analysis (figure ) , we use mouse fhr- homolog fhr-e (np_ ) for the protein encoded by cfhr (refseq# at ncbi: nm_ , and a locus at mgi database: ) . homology analysis has shown the difference between human and murine fhrs ( , ) , suggesting that murine fhrs may function differently from their human counterparts. fhr-b, fhr-c, and fhr-e are functional fhrs in mouse. fhr-b and fhr-c were detected in mouse plasma with anti-fh antibody ( ) . mouse fhr-b interacts with c b and pentraxin to enhance complement activation and necrotic cells promoted this activation ( ) . fhrc mrna was found completely absent in the liver of three lupus-prone mouse strains and one diabetic-prone mouse strain ( ) . fhr-c may function on specific surfaces ( ) . it is unknown whether fhr-e exists in murine plasma and how it functions in ap. its human homolog, fhr- , was reported to function as a competitor of fh ( , , ) and its deletion was associated with various autoimmune diseases. however, its function in vivo and whether it has a regulatory role alone in the activation of ap remain unclear. in this study, cfhr was deleted on c bl/ mouse to study the function of fhr-e on ap and the effect of fhr-e deficiency on lps-induced sepsis. we found that fhr-e deficiency increased the mortality rate of lps-induced sepsis and potentiated kidney injury through enhancing ap activation. our data demonstrated a protective role of fhr-e during lps-induced sepsis in vivo and highlighted its importance in ap regulation. cfhr was deleted on c bl/ mouse by using the crispr/cas system developed by biocytogen co. (beijing, china). the heterozygous mouse was backcrossed with wild-type c bl/ for more than six generations. all the mice were bred in the animal facility of the institute of genetics and developmental biology (igdb), chinese academy of science. all procedures were approved by the institutional animal care and use committee of igdb. the age-matched wild-type (wt) or heterozygous littermates were used as controls. all experiments were conducted on the offspring of mice backcrossed more than six generations. the polyclonal antibody anti-fhr-e was generated in new zealand white rabbit. the dna fragment of the last three ccp domains (amino acids to ) of fhr-e was inserted into the expression vector pgex- t- and expressed in e. coli bl host cells. the recombinant protein was expressed as insoluble protein and separated by sds-page. the separated recombinant protein was used as an antigen to immunize the new zealand white rabbit every other week. the rabbit was sacrificed week after the fourth immunization and the sera was used as the anti-fhr-e polyclonal antibody. the rat anti-c (ab ), rat anti-c (ab ), sheep anti-fh (ab ), and rabbit anti-fibrinogen (ab ) were obtained from abcam (cambridge, uk). purified the upper bands were amplified with primers f and r . these bands can only be amplified in wild-type and heterozygous mice and cannot be amplified in homozygous mice because of the absence of the sequence of primer r . the lower bands were amplified with primers f and r . these bands can only be amplified in heterozygous and homozygous mice. the sequence between primers f and r is too long to be amplified in wild-type mice. (c) verification of cfhr deletion from rna level. hepatic rna of mice of different genotypes was extracted and reversely transcribed. primers spanning the open reading frame (orf, , bp, nm_ ) of cfhr were used for rt-pcr. actb was used as a semi-quantitative control. (d) verification of fhr-e deficiency at the protein level. plasma proteins of mice with different genotypes were separated by % sds-page gel. proteins were transferred to the membrane and analyzed by western blotting using anti-serum to mouse fhr-e, which was generated in rabbit immunized with a recombinant peptide of ccp - of fhr-e. an about kda specific fhr-e band was detected in wt and heterozygous mice, but not in homozygous mice. "unknown" indicates an unknown protein recognized by anti-fhr-e antibody. western blotting of fh was regarded as an internal control. rat anti-mouse c a ( ) and biotin rat anti-mouse c a ( ) were obtained from bd pharmingen (california, usa). mice were marked by cutting different toes and the toes were lysed by µl of lysis buffer (tris-hcl ph . mm, edta ph . mm, nacl mm, . % sds, and . mg/ml proteinase k) at • c for h. after transient centrifugation, µl of m nacl was added and mixed thoroughly. the mixture was centrifuged at , rpm for min and the supernatant was collected by adding double volume of ethanol and mixed thoroughly. the mixture was centrifuged at , rpm for min and then the supernatant was discarded. the pellet was washed with % ethanol. after the liquid was evaporated, µl of ddh o was added to dissolve the precipitated dna. one forward primer located on the upstream of the deleted sequence, one reverse primer located on the deleted region, and the other reverse primer located on the downstream of the deleted sequence were used for genotyping pcr assays. the primer's sequences are ′ -cagtaagactgcaagagacatatg- ′ , ′ -ctaagagcaacaggcgacag- ′ , and ′ -cattttaa aagaaaaataagccagcca- ′ , respectively. the amplified products are depicted in figure a . mouse hepatic rna was extracted using rneasy mini kit (qiagen, germantown, germany) and reversely transcribed with bio-rad transcript cdna synthesis kit (bio-rad, california, usa). the cdna was used as templates to amplify cfhr and actb (gene for β-actin). primers of cfhr were ′ -atggggtt ctgtcgcctgttgc- ′ and ′ -tcaatgaataaacgtattg tga- ′ . primers of actb were ′ -tgatggtgggaatggg tcaga- ′ and ′ -ccgctcgttgccaatagtgat- ′ . immunoblotting equal volume of serum or plasma of mice was diluted : with pbs (hyclone, utah, usa). equal volumes of diluted samples were separated with % sds-page gel and transferred onto polyvinylidene difluoride (pvdf) membrane (millipore, massachusetts, usa). the membrane was blocked with % defatted milk (bd, california, usa) for h at room temperature and immunoblotted with specific antibody overnight at • c followed by incubation with hrp-conjugated secondary antibody (zsgb-bio, beijing, china) for h at room temperature. the membrane was visualized by ecl (thermo fisher scientific, massachusetts, usa or ge healthcare, usa) and exposed with chemiluminescence apparatus (beijing sage creation, beijing, china). protein bands' gray values were measured by nih image j. serum was diluted by ap buffer ( . mm barbital, . mm sodium barbital, mm nacl, mm mgcl , and mm egta, ph . - . ). ten-fold diluted serum was incubated with . mg/ml lps o :b (sigma-aldrich, missouri, usa) or equal volume of pbs as control at • c for min. the reactions were stopped by mm edta. the samples were analyzed with % sds-page under reducing condition and protein bands' gray values were measured by nih image j. lps-stimulated ap activity was performed as previously published by elisa ( ) . briefly, lps ( µg/well) was coated on plates. ten-fold diluted serum with ap buffer was incubated on plates at • c for h. serum diluted with edta buffer ( . mm barbital, . mm sodium barbital, mm nacl, and mm edta, ph . - . ) was used as a negative control. after washing, the plates were incubated with rat anti-mouse c antibody and followed by incubation with hrp-conjugated antirat igg. tetramethylbenzidine (tmb, solarbio, beijing, china) was used as substrate and m h so was used as a stop solution. the absorbance was measured with multiskan go microplate spectrophotometer (thermo scientific, massachusetts, usa). for the detection of lps-induced c a production, -fold diluted serum was incubated with . mg/ml lps or equal volume of pbs as control at • c for h and the reactions were stopped by mm edta. the reaction mixtures were added to the plate, which was coated with purified rat anti-mouse c a antibody ( µg/ml) and incubated at • c for h. the mixtures were discarded and biotin rat anti-mouse c a ( . µg/ml) was added into the according plates as detection antibody. after washing, streptavidin hrp ( : , ) (bd pharmingen, california, usa) was added and incubated at • c for h. tmb (solarbio, beijing, china) was used as substrate and m h so was used as a stop solution. the absorbance was measured with multiskan go microplate spectrophotometer (thermo scientific, massachusetts, usa). six-to eight-week-old male mice were injected intraperitoneally (i.p.) with mg/kg lps dissolved in pbs and the control group mice were injected i.p. with equal volume of pbs. the edta anticoagulant blood was extracted from the tail tip at different time points and the plasma was collected to analyze with western blotting. mice were sacrificed at different time points and the edta anticoagulant blood was harvested from heart puncture. partial whole blood was used for complete blood counting using tek-ii mini full-automatic animal blood analyzer (tecom science, jiangxi, china). the plasma was collected for c a, c a, il- β and tnf-α concentration detection using elisa kit as described below. plasma c a, c a, il- β, and tnf-α measurements lps challenged mice were anesthetized and edta anticoagulant blood was collected by heart puncture. the whole blood was centrifuged at , rpm • c for min and the supernatant was collected. plasma levels of c a and c a were determined using elisa kits (mybiosource, california, usa) according to the manufacturer's instructions. the dilution factor of c a and c a was : . plasma il- β and tnf-α were measured with elisa kits (r&d systems, minnesota, usa) according to the manufacturer's instructions. about -week-old mice were injected i.p. with mg/kg lps. sixteen hours later, these mice were observed every half hour and the death of mice was recorded. the data were analyzed by graphpad prism version . using the log-rank (mantel-cox) test. mice were sacrificed at h after lps challenge and the left kidneys were harvested. half of the kidney was fixed in % paraformaldehyde for h at • c, dehydrated using graded ethanol, cleared with xylene, and embedded in paraffin. paraffin blocks were sliced into -µm sections. hematoxylin and eosin (he) staining was performed on the sections using hematoxylin and eosin staining kit (solarbio, beijing, china). pictures were captured with nicon ci-l microscope (nicon, tokyo, japan) at × magnification. tubular damage was assessed by tubular dilation, necrosis, and apoptosis. tubular dilations were counted in every non-overlap field and averaged. cell necrosis and apoptosis were determined using in situ cell death detection kit (roche, basel, switzerland). the fluorescent pictures were captured with an lsm confocal fluorescence microscope (zeiss, oberkochen, germany) at × magnification. only dots overlapped with nuclei were counted and the whole cell number of each field was counted by image j. proportion of apoptotic and necrotic cells was computed. paraffin slides that were processed by xylene de-waxing and gradient ethanol hydrating were blocked by ready-to-use goat serum (boster, wuhan, china) at • c for h and incubated with rabbit anti-fibrinogen ( : ) overnight at • c. after washing three times with pbs, the slides were incubated with alexa fluor goat anti-rabbit igg ( : , ) (invitrogen, california, usa) at • c for h. the slides were washed three times and mounted with mounting medium with dapi (zsgb-bio, beijing, china). images were captured using leica tcs sp confocal laser scanning microscope (leica microsystems, wetzlar, germany) at × magnification. the fluorescence intensity and fluorescence positive area were analyzed by image j. the protein sequences were downloaded from ncbi-protein (https://www.ncbi.nlm.nih.gov/protein/). amino acid sequences were aligned by clustalw with mega . ( ). the alignment diagram was drawn with genedoc. the evolutionary tree was constructed using the neighbor-joining method with mega . ( , ) . the domain structures of fhrs were analyzed by smart (http://smart.embl-heidelberg.de/). domain similarities among different fhrs were computed with pblast. data were presented as mean ± sem. student's t-test was used to compare the two groups. time course of factors of different time points after lps challenge was analyzed with one-way anova by spss software. comparison of survival curves was analyzed by mantel-cox test. significant differences were considered when the p-value was less than . and extremely significant differences were considered when the p-value was less than . . to ascertain the mouse fhr-e homolog of human fhr proteins, homology analysis between mouse and human fhrs was conducted. the evolutionary tree of fhrs showed that fhr-e converged with human fhr- , fhr- , and fhr- ( figure a) . the overall homology between mouse fhr-e and human fhr- / / is about %, while fhrb and fhrc have about % homology to fhr- / / ( ) . structure analyses of fhr-e and human fhr- , fhr- , and fhr- by smart showed that they all consist of ccp modules. amino acid sequence of each ccp in fhr-e was compared with human fhr- , fhr- , and fhr- by pblast and amino acid sequence alignment was analyzed by mega . . results showed that fhr-e shared more similarity with human fhr- (figures b-e) . with this regard, we selected the mouse cfhr gene that encodes the fhr-e protein for the generation of a gene knockout colony. the exons from to of cfhr were deleted by using the crispr/cas system on c bl/ mouse (figure a) . verification of cfhr deletion was conducted at dna, mrna, and protein levels (figures b-d) . the results demonstrated that fhr-e was detectable in murine plasma and was depleted in the knockout mice. our homemade anti-fhr-e antibody recognized murine fh as well due to the high epitope identities to scrs and of fh (data not shown). this is the first report of fhr-e in murine plasma. fhr-b and fhr-c has been detected in mouse plasma by using fh-specific antiserum ( ) . the unrecognized fhr-e in their experiment may be because of the antibody specificity. both heterozygotes (cfhr −/+ ) and homozygotes (cfhr −/− ) were viable and healthy bred in spf (specific pathogen free) mouse facility. under physiological condition, ap keeps at a low level of activation by spontaneous hydrolysis of c ( ) . to study whether cfhr deletion can trigger complement dysregulation under physiological condition, the plasma levels of c , c , and fh, central molecules of complement and main soluble regulator of ap, were detected on -to -week-old male sibling mice. western blotting results showed no obvious changes among mice of different genotypes (figure a) . the relative gray values of mice were analyzed and results showed that there were no significant differences (figures b-d) . furthermore, the concentrations of c a and c a, which are small cleaved fragments generated by complement activation, and the key mediators of inflammation were measured. neither the level of c a nor c a had significant differences among different genotypes (figures e,f) . these results suggested that under physiological condition, the deletion of cfhr was not sufficient to impact the ap regulation. ap activation is enhanced in cfhr −/− mice in response to lps treatment in vitro lps, the principal component of gram-negative bacteria cell wall, is an activator of ap ( ) and responsible for gramnegative bacteria induced sepsis ( ) . to test the effect of fhr-e deficiency on lps-induced ap, in vitro lps-induced ap activation experiments were performed. lps was incubated with serum from mice of different genotypes and the mixtures were analyzed by western blotting. a higher amount of c activated fragment in cfhr −/− mice was observed compared to the wild-type group (figures a,b) . ap activity measured by elisa assay ( , ) was also conducted to verify this effect. as expected, cfhr −/− mice showed more deposited c b on lps ( figure c) . furthermore, lps-induced c a production, an ensuing event of ap activation, was determined and significantly higher concentration of c a was detected in cfhr −/− mice compared to wild-type mice ( figure d ). an increasing tendency was observed in the heterozygotes, but there was no significant difference between wt and heterozygotes, suggesting that dose effects of fhr-e in heterozygotes may exist. thus, fhr-e deficiency promoted lps-induced ap activation in vitro. to further study the regulatory function of fhr-e, in vivo challenge of lps was applied. edta anticoagulant blood of different time points was collected from the tail tip to monitor the content changes of main complement or coagulation factors using western blotting. our results showed that the level of c decreased over time and that the level of c increased at first and decreased from h after lps challenge. no significant difference was observed among different genotypes at different points. however, more significant decrease of c content over time was observed in cfhr −/− mice compared with wild-type mice (figures a-c) . the levels of fh and von willebrand factor (vwf), which is involved in hemostasis, were also determined. the content of vwf increased at first and decreased from h post lps challenge (figures a,e) while the content of fh did not have any observable change over time (figures a,d) . this demonstrated that the hemostasis was promptly triggered and gradually vanished as the exhaustion of clotting factor, and that fhr-e deficiency and lps stimulation did not affect the level of fh. the contents of fhr-e in wild-type and heterozygous mice were also measured. interestingly, we found that fhr-e increased significantly at h post-lps challenge (figures a,f) . the data in figure were obtained from the same batches of mice. furthermore, another group of mice were challenged with lps and edta anticoagulant blood of different time points was harvested from heart puncture. the contents of plasma c a and c a of cfhr −/− mice were significantly higher than wild-type controls at h post-challenge (figures a,b) . these results demonstrated that fhr-e deficiency accelerated lps-induced ap. the plasma il- β and tnf-α contents at h post-challenge were detected and (c) detection of ap activation by plate bounded lps in diluted serum with elisa method. equivalent lps was coated on the plate. ap buffer and edta buffer diluted serum was added into the plate and incubated for h at • c. edta buffer diluted serum was used as a negative control for background subtraction. after washing, the lps-bound c b was detected with c antibody. twelve mice in each group were used. (d) measurement of the concentration of c a produced at h after lps in vitro stimulation. ap buffer diluted serum was incubated with lps or pbs for h at • c. equal volume of pbs was used as a negative control for background subtraction. the quantity of c a was detected with sandwich enzyme-linked immunosorbent assay. ten pairs of mice were used. n.s., not significant. *p < . . elevated concentrations of il- β and tnf-α were found in cfhr −/− mice (figures c,d) . subsequently, we observed that the proportion of granulocytes significantly increased and lymphocytes mildly decreased at h after challenge, which suggests that cfhr −/− mice had more severe inflammation (figures e,f) . the quantity of red blood cells in cfhr −/− mice at h after lps challenge was dramatically lower than the wild-type mice, which indicates that much more congestion could happen in cfhr deletion mice (figure g) . in summary, the cfhr −/− mice had features of enhanced complement activation and inflammation, which may lead to organ injury. to explore the endpoint effect of fhr-e deficiency on lpsinduced sepsis, the mortality assay was performed. cfhr −/− and wild-type mice were administrated i.p. injection of mg/kg lps. the survival data were recorded and analyzed. compared to the wild-type mice, the average survival time of cfhr −/− mice was significantly shorter and the mortality rate of cfhr −/− mice was significantly higher (figure h human cfhr deletion was associated with nephropathy ( , ) and kidney injury can be induced by complement overactivation ( ) . urea and creatinine, which are indicators of renal function, were tested at h post-challenge and significant increases were observed in both wt and ko groups (figures a,b) . however, we did not observe an apparent increase in the ko group compared with the wt group after lps treatment. this suggests that renal function was not worsened dramatically in fhr-e deficiency at the time of measurement. we then performed histological examination of kidney. renal tissue sections of h after lps challenge were prepared and he staining was conducted. kidney injury degree was assessed by the tubular dilations, apoptosis, and necrosis. tubular dilations were quantified, and cell apoptosis and necrosis were determined through tunel assay. more tubular dilations (figures c,d) and more cell apoptosis and necrosis (figures e,f) were exhibited in cfhr −/− mice. fibrin, which is the marker of vascular injury, was also stained to testify tissue injury. we found that cfhr −/− mice had significantly more fibrin deposition than wild-type mice (figures a-c) . plasma fibrin was also analyzed by western blotting and higher concentration of plasma fibrin was found in cfhr −/− mice ( figure d ). twenty non-overlapped fields were counted and averaged. (c) comparison of fluorescence intensity between wild-type and cfhr −/− mice. fluorescence intensity of each field was calculated by image j. twenty non-overlapped fields were counted and averaged. (d) western blotting analysis of plasma fibrin of h after lps challenge. western blotting of fhr-e was performed to determine the genotypes. equal volumes of plasma were loaded. the western blotting of fh on the same blot was regarded as an internal control. *p < . . in this study, cfhr knockout mice were generated to further investigate its role in ap regulation. no obvious changes of c a and c a concentration in plasma were found in cfhr knockout mice. these results suggest that the basal level of fhr-e plays negligible roles in spontaneously activated ap pathway. this may explain why many individuals with cfhr homozygous deletion are healthy ( ) . surprisingly, with fhr-e deficiency, increased c and c cleavage were found after lps stimulation in vitro, suggesting that fhr-e itself inhibits lps-induced ap activation. this may be explained by the conformation switch model in which soluble fh exists in a low-affinity latent conformation and transits to high-affinity activated conformation by interacting with self-surface targets ( ) . based on this model, fh may play negligible inhibition on lps-induced ap and fhr-e may compete c b with positive regulators of ap, like properdin, which plays an essential role in lps induced ap activation ( ) . when fhr-e is absent, ap activity is enhanced by the positive regulators. the inhibitory effect of fhr- on c convertase has been reported ( ) and was regarded as a therapeutic target ( ) . the result that more c a production after lps challenge in cfhr −/− mice was observed supports the idea that fhr-e has functional homology to human fhr- . cfhr deletion has been reported to be associated with renal disease in an incomplete penetrance and a second hit such as infection is considered to be essential for disease development ( ) . it is unknown whether cfhr homozygous deletion alone would trigger the onset of these conditions upon infection. one reported case showed that shiga toxin was a potential trigger of cfhr deletion-related thrombotic microangiopathy ( ) . here, we chose lps, which is the main constituent of the wall of gram-negative bacteria as a stimulator to study the effect of cfhr deletion in mice for possible pathological changes. both in vitro and in vivo assays showed that fhr-e deficiency significantly promoted lps-induced ap activation. these results suggested that fhr-e may play a coordinated role with fh in determining complement activation. however, the mechanism of selective activation of ap remains enigmatic. in many ahus patients with cfhr deletion, fh autoantibody was detectable ( ) . it was believed that ahus may result from the blocking of fh function by fh auto-antibody. however, this cannot explain those ahus patients with cfhr deletion without fh auto-antibody ( ) . our results suggest that fhr-e deficiency promotes infection-induced damage through enhancing ap activation. this may provide a hint for fhr- deficiency-related nephropathy. nevertheless, how fhr-e functions in this complex process warrants further investigation. in addition, we did not see much difference of fh level in fhr-e deficiency mice. to the best of our knowledge, there has been no report of fhrs on any inflammatory disease mouse models. our results revealed that cfhr knockout mice challenged with lps served as a good model to study the intricate network of complement, inflammation, and coagulation in sepsis and aki. we found that cfhr −/− mice showed higher content of c a and c a, more severe inflammation, more blood coagulation, and more severe renal injury after lps challenge compared with the wild-type group. however, we did not see significantly higher levels of urea and creatinine in cfhr −/− mice compared to the wildtype mice upon lps challenge. c a and c a, which are two main products of complement activation, have been shown to have anti-inflammatory ( , ) and pro-inflammatory function ( ) , respectively. administration of c a receptor antagonist peptide in thrombotic glomerulonephritis model significantly reduced leucocyte accumulation and thrombus formation in glomeruli ( ) and improved survival of mice with sepsis ( ), while administration of c a receptor antagonist exacerbated mortality of mice with sepsis ( ) . the regulation mechanism of c a and c a on sepsis development, which seemed to have converse effect, is still unclear. in our study, the deficiency of fhr-e promoted complement activation with more c a and c a production, which in turn induced more inflammation and eventually accelerated coagulation and tissue injury. these results suggested that c a may play a more potent role on sepsis development. our cfhr −/− mouse model provides a unique tool to study the underlying mechanism in which ap is activated upon lps challenge to induce tissue injury. beyond the scope of this study, it is speculative that patients with fhr deficiency may be susceptible to severe conditions under sars-cov- infection when sepsis is induced in covid- patients. the datasets generated for this study are available on request to the corresponding author. the animal study was reviewed and approved by institutional animal care and use committee of igdb (institute of genetics and developmental biology, chinese academy of science). xli, zh, and wl designed the experiments, analyzed the data, and supervised the project. xliu contributed to the initiation and design of the project. xli and wl wrote the manuscript. xli and zh performed the assays. this study was partially supported by grants from the ministry of science and technology of china ( yfc ) and from national natural science foundation of china ( ; ). severe sepsis in pre-hospital emergency care: analysis of incidence, care, and outcome incidence and trends of sepsis in us hospitals using clinical vs claims data acute renal failure and sepsis epidemiology of sepsis and infection in icu patients from an international multicentre cohort study complexity of complement activation 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profile in atypical haemolytic-uraemic syndrome cutting edge: targeted disruption of the c a receptor gene demonstrates a novel protective anti-inflammatory role for c a in endotoxin-shock is the complement activation product c a a proinflammatory molecule? re-evaluating the evidence and the myth in vitro and in vivo dependency of chemokine generation on c a and tnf-alpha the role of c a in the development of thrombotic glomerulonephritis in rats carboxypeptidase b deficiency reveals opposite effects of complement c a and c a in a murine polymicrobial sepsis model the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © li, hao, liu and li. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -w t zz authors: wu, beibei; wang, liyin; jiang, lili; dong, lili; xu, fengli; lu, yili; jin, jiahui; wang, zhanyue; liang, guang; shan, xiaoou title: n-butanol extract from folium isatidis inhibits the lipopolysaccharide-induced downregulation of cxcr and cxcr on human neutrophils date: - - journal: mol med rep doi: . /mmr. . sha: doc_id: cord_uid: w t zz neutrophils, immune cells crucial for protecting against invading pathogens, are important in sepsis. neutrophil migration is regulated by chemokine receptors and their cognate ligands. our previous study investigated the effect of n-butanol extract from folium isatidis on lipopolysaccharide (lps)-induced septic shock. the present study stimulated neutrophils with lps to explore the influence of lps on cell. neutrophils were then pretreated with n-butanol extract from folium isatidis followed by lps to examine the effect of this extract on neutrophil chemotaxis. the results showed that lps decreased the expression levels of cxc-chemokine receptor (cxcr) , cxcr and l-selectin (cd l), and increased the expression of interleukin- (il- ) by neutrophils. the addition of n-butanol extract from folium isatidis inhibited this lps-induced downregulation of cxcr , cxcr and cd l, and decreased the expression of il- on neutrophils. in addition, n-butanol extract promoted myeloperoxidase activity in neutrophils. taken together, lps downregulated the expression of chemokine receptors, leading to the failure of neutrophils to migrate to sites of infection. the addition of n-butanol extract, which promoted the ability of neutrophils to migrate, is a natural product and potential therapeutic agent with which to target neutrophil chemotaxis during lps stimulation. sepsis is a systemic inflammatory response syndrome associated with a high rate of mortality ( ) . the incidence of sepsis has been increasing over the last two decades. this increase has been more marked in developed countries, where - % of patients in intensive care units suffer from sepsis ( ) . sepsis remains the leading cause of mortality in patients in intensive care units ( ) and the associated burden of care incurs financial burden. however, the pathogenesis of sepsis remains to be fully delineated and effective therapies are lacking. a state of immunosuppression induced by sepsis has been demonstrated in clinical and experimental sepsis ( ) , with the majority of patients who have succumbed to sepsis-associated mortality showing evidence of immunosuppression and unresolved septic foci ( ) . polymorphonuclear neutrophils (pmns) are an essential component of the innate immune system, involved in the clearance of extracellular pathogens ( ) . as the most abundant subset of leukocytes, the involvement of pmn in sepsis is significant ( , , ) . the migration of pmns is regulated by chemoattractants and chemokine gradients ( , , ) . cxc-chemokine receptor (cxcr) and cxcr are the major chemokine receptors on pmns, with interleukin (il)- acting as a ligand of these receptors ( ) . severe sepsis is associated with the failure of pmns to migrate ( ) . in a previous clinical study, patients with suppression of pmn receptors were predisposed to inflammatory response syndrome ( ) . previous in vitro investigations have demonstrated that cxcr is downregulated upon stimulation with tumor necrosis factor-α (tnf-α) ( ) . the activation of toll-like receptor (tlr ) also suppresses the expression of cxcr ( ) . in an experimental mouse model of sepsis, the failure of pmns to migrate was shown to result in a high rate of mortality ( ). tancevski et al and van zee et al reported that promoting the recruitment of pmns ameliorates sepsis and attenuates sepsis-related injury and infection, respectively ( , ) . therefore, in order to improve treatment of inflammatory disorders, including sepsis, the promotion of pmn chemotaxis is an attractive target ( ). as an ancient chinese herb, folium isatidis is considered to have detoxification properties in traditional chinese medicine. in addition to being used to cure febrile diseases, including dipsosis, fever, jaundice and hematemesis, f. isatidis is also deployed against diseases, including mumps, viral hepatitis, influenza and epidemic encephalitis b ( ) ( ) ( ) . it was reported to be effective during the severe acute respiratory syndrome flu outbreak ( ) . these antimicrobial and anti-endotoxic properties indicate its potential to be developed into a natural antibiotic ( , ) . in our previous study ( ) , the main chemical components of f. isatidis were identified using high-performance liquid chromatography. it was also demonstrated that f. isatidis increased the survival of septic mice, and ameliorated lung injury by inhibiting the production of inflammatory cytokines tnf-α and il- through the myeloid differentiation primary response gene and nuclear factor-κb pathways ( ) . although the beneficial immunomodulatory effects of f. isatidis have been the focus of previous investigations, whether this herb has an effect on chemokine receptors remains to be elucidated. the present study aimed to determine whether f. isatidis affects the migration of pmns. isolation of neutrophils. the present study was approved by the ethics committee of the second hospital of wenzhou medical university (wenzhou, china), and informed consent was obtained. peripheral blood was collected from four - -year-old female healthy volunteers between august and december , who had been referred to the second affiliated hospital and yuying children's hospital of wenzhou medical university and who provided informed, written consent. the blood was transferred into heparin lithium-containing tubes (bd bioscience, san jose, ca, usa). the samples were processed within h of collection. whole blood was incubated with % dextran t- in the dark at room temperature for min for sedimentation of red blood cells (rbcs). following sedimentation, the white blood cell-enriched upper phase was layered over a ficoll-paque solution (ge healthcare life science, uppsala, sweden). centrifugation was performed at x g at room temperature for min without breaks, resulting in four distinct layers. the first three layers were discarded, and the remaining layer containing granulocytes and remnant rbcs was diluted in ack lysis buffer (gibco; thermo fisher scientific, inc., waltham, ma, usa). the cells were then washed with pbs, and the freshly isolated neutrophils were resuspended in rpmi (gibco; thermo fisher scientific, inc.). the purity of the isolated neutrophils was > % based on wright's staining and differential counts. flow cytometry. the isolated neutrophils were diluted to x cells/ml with rpmi (gibco; thermo fisher scientific, inc.). the cells were then incubated with either vehicle ( . % dmso) or increasing concentrations of n-butanol extract from folium isatidis ( , and µg/ml) at ˚c and % co for h. the specific method to obtain n-butanol extract from f. isatidis was described in previous study ( ) . the cells were then stimulated with . µg/ml lipopolysaccharide (lps; sigma; merck millipore, darmstadt, germany) for h at ˚c. the cells were washed with ice-cold pbs and then resuspended in µl facs buffer. the cells were then incubated with a : dilution of the following antibodies (the concentrations suggested by the manufacturer) for min at ˚c in the dark: fitc-conjugated anti-human cd (cxcr ; cat. no - - ), pe-conjugated anti-human cd (cxcr ; cat. no - - ) and apc-conjugated anti-human cd b (cat no. - - ) or fitc-conjugated anti-human l-selectin (cd l; cat. no - - ), pe-conjugated anti-human tlr (cat no. - - ) and apc conjugated anti-human tlr (cat no. - - ) (all from ebioscience, san diego, ca, usa). antibodies of the same isotype were used as negative controls. the cells were analyzed using a facs calibur system (bd biosciences). the mean fluorescence intensity (mfi) for , cells in each sample was determined using cellquest software, version . (bd biosciences). chemotaxis assays. the isolated neutrophils were pretreated as described above for h and then stimulated with lps ( . µg/ml) for h. the cells were then washed twice with pbs, and resuspended in rpmi at x cells/ml. the chemotaxis assays were performed using transwell inserts ( wells, µm pore size; corning, inc., corning, ny, usa), where , cells in µl were loaded in the upper inserts and µl of il- ( ng/ml; ebioscience; thermo fisher scientific, inc.) containing rpmi was added to the lower wells. following co-incubation for h at ˚c and a humidified % co atmosphere, the inserts were removed, and the migrated neutrophils in the lower wells were collected and quantified according to myeloperoxidase (mpo) activity using a myeloperoxidase assay kit (nanjing jiancheng bioengineering institute, nanjing, china). reverse transcription-quantitative polymerase chain reaction (rt-qpcr) analysis. total rna was isolated from cells using trizol reagent (invitrogen; thermo fisher scientific, inc.). rt-qpcr analysis was performed using an m-mlv platinum sybr green qpcr supermix-udg kit (invitrogen; thermo fisher scientific, inc.) according to the manufacturer's protocol. a total of µg rna was used for the determination. qpcr was performed in a µl reaction volume containing µl cdna target, . µl sybr green (bio-rad laboratories, inc., hercules, ca, usa), . µl sense primers, . µl anti-sense primers and . µl ddh o. an eppendorf mastercycler realplex detection system (eppendorf, hamburg, germany) was used for rt-qpcr analysis: the reaction consisted of the following conditions: ˚c for min, followed by ˚c for sec, ˚c for sec, and ˚c for sec ( cycles). the primers of the genes were synthesized by invitrogen (thermo fisher scientific, inc.) and the sequences were as follows: human cxcr , the result of real-time pcr was expressed as the threshold cycle (ct). the ct represents the pcr cycle at which the reported fluorescence rises above a set baseline threshold when the dna amplicon is replicating exponentially ( ) . results were normalized to β-actin as plotted as relative expression to the average of con or dmso, which were set as . quantification of il- . the neutrophils were pretreated for h with the extract as described above, and then stimulated with lps ( . µg/ml) for or h. the supernatant and cells were collected, and the levels of il- in the supernatant were determined using enzyme-linked immunosorbent assay (elisa) kits (cat no. - - ; ebioscience; thermo fisher scientific, inc.) according to the manufacturer's protocol. the total quantity of il- in the supernatant was normalized to that of total protein in the viable cell pellets. statistical analysis. all the experiments were performed in triplicate. data are expressed as the mean ± standard error of the mean. all the data were statistically analyzed using student's t test. p< . was considered to indicate a statistically significant difference. graphpad prism . software (graphpad software, inc., la jolla, ca, usa) was used for statistical analysis. and cd l. tlr and tlr /myeloid differentiation factor are vital in the recognition of lps in the host, and the triggering of these receptors can lead to neutrophil recruitment and migration via cxcr and cxcr . cd b and cd l, which are expressed on the surface of neutrophils, are critical for the occurrence of sepsis. lps, also known as endotoxin, is a component of the outer membrane of gram-negative bacteria, and is known to induce septic shock when present in high quantities. therefore, the present study measured the expression of neutrophil chemokine receptors under lps stimulation. the isolated neutrophils were treated with either vehicle or increasing concentrations ( , and , ng/ml) of lps for h, and the expression levels of cxcr , cxcr , tlr , tlr , cd b and cd l were measured using flow cytometry. the results showed that lps treatment resulted in decreased expression levels of cxcr , cxcr and cd l in a dose-dependent manner (fig. a-c) , which was in accordance with the results of a previous study ( ) . however, no significant changes were observed in the expression levels of tlr , tlr or cd b in the lps-treated neutrophils, compared with the vehicle-treated neutrophils (fig. d-f) . therefore, the expression levels of tlr , tlr and cd b were not examined in the remainder of the experiments. n-butanol extract from folium isatidis prevents the lps-induced downregulation of cxcr , cxcr and cd l. cxcr , cxcr and cd l may be important in sepsis, however, whether n-butanol extract affects these chemokine receptors remains to be elucidated. therefore, the present study assessed the effect of the extract on the expression of cxcr , cxcr and cd l. the isolated neutrophils were pre-incubated with either vehicle or increasing concentrations of extract ( , and µg/ml) for h, and were subsequently treated with lps ( . µg/ml) for h. subsequent analysis of the cells using flow cytometry indicated that the extract inhibited the lps-induced downregulation of cxcr , cxcr and cd l ( fig. a-c) in a dose-dependent manner. effect of n-butanol extract from folium isatidis on the gene expression levels of cxcr , cxcr and cd l. as the n-butanol extract obtained from f. isatidis had a significant effect on chemokine receptor protein levels, the present study investigated the gene expression levels of these receptors. the isolated neutrophils were pretreated with extract and then treated with lps ( . µg/ml) for or h (fig. a-f) . the extract increased the gene expression levels of cxcr , cxcr and cd l in a dose-dependent manner. il- , as the ligand of cxcr and cxcr , is important in the regulation of neutrophil migration. following exposure of the neutrophils to lps for h, the mrna expression of il- was significantly increased (fig. a) . therefore, the present study examined whether n-butanol extract affected the production of il- . the isolated neutrophils were pretreated with extract and then incubated with lps ( . µg/ml) for h (fig. b ) or h (fig. c) . the expression levels of il- were quantified using elisa. a decrease in il- was observed following exposure to lps for and h when pretreated with the extract. specifically, following exposure to lps for h, the cytokine expression of il- was decreased > -fold when the cells were treated with the extract at a dose of µg/ml, compared with lps stimulation (fig. c) . mpo levels are a measure of neutrophil migration. therefore, the present study examined the effect of the extract on the activity of mpo. neutrophils were pretreated with n-butanol extract for h followed by stimulation with lps for h. the activity of mpo increased in a dose-dependent manner following treatment with extract, compared with the control (fig. d) , suggesting that n-butanol extract promoted the function of neutrophils. sepsis remains a threat to human health, primarily due to a lack of effective therapies ( ). with current treatment options, the mortality rate of patients with sepsis remains high at ~ %, and mortality rates increase directly with disease severity ( ) . a previous worldwide survey of relevant pathogens in intensive care units found that the majority of patients with sepsis had blood cultures positive for gram-negative bacteria ( ) . lps, a component of the outer leaflet of the outer membrane of gram-negative bacteria, is a key molecule in the induction of sepsis. therefore, the present study used lps to simulate sepsis induced by gram-negative bacteria infection. as previously reported, the present study observed that lps stimulation decreased the expression of cxcr , cxcr and cd l on neutrophils. in addition to being effector cells involved in the acute phase of the inflammatory response, neutrophils are also important in the resolution of inflammation. the failure of neutrophils to migrate can lead to an inability to control infection due to weakened neutrophil phagocytic and bactericidal abilities ( ) . cxcr and cxcr are the major chemokine receptors on neutrophils. chemokine receptors regulate the migration of neutrophils to the site of infection to assist in controlling invading pathogens and protecting the body. duerschmied et al ( ) demonstrated that survival following lps-induced endotoxic shock improved upon the promotion of neutrophil recruitment during acute inflammation. n-butanol extract was found to have significant antiseptic abilities in our previous study. however, whether the extract affected neutrophil migration remained unclear. in the present study, it was demonstrated that n-butanol extract prevented the downregulation of cxcr , cxcr and cd l. in our previous study, it was demonstrated that n-butanol extract from folium isatidis significantly inhibited the activation of tlr and its signaling pathways ( ) . it has been reported that the systemic activation of tlrs and high levels of tnf-α are involved in the reduction of neutrophil recruitment through the downregulation of cxcr in neutrophils ( ) . therefore, it was hypothesized that n-butanol extract from f. isatidis prevents the downregulation of cxcr and cxcr through the activation of tlr and secretion of tnf-α. the primary function of cd l, a vascular adhesion molecule, is directing neutrophil migration. in addition to cxcr and cxcr , neutrophil migration requires the assistance of cd l. in the present study, the expression of cd l decreased following incubation with lps for h (fig. c) , whereas n-butanol extract promoted the expression of cd l in a dose-dependent manner (fig. c ). this suggested that the extract promoted the migration of neutrophils by inhibiting the sepsis-induced downregulation of cd l. il- is a ligand of cxcr and cxcr , and is rapidly secreted upon cell stimulation. the mrna expression of il- significantly increased upon stimulation with lps (fig. a) . however, n-butanol extract decreased the expression of il- in a dose-dependent manner ( fig. b and c) , and prevented the decreases in the expression of cxcr and cxcr . this suggested that the extract increased the activity of neutrophils and upregulated the expression of chemokine receptors, including cxcr and cxcr , rendering neutrophils more sensitive to the ligand il- . the present study quantified mpo activity as a measure of neutrophil migration. it was found the extract promoted the activity of mpo. the extract also promoted the ability of neutrophils to migrate by increasing the expression of cxcr and cxcr on the surface of neutrophils. to date, the treatment of sepsis consists primarily of supportive measures and experimental therapeutic approaches ( ) . therefore, novel pharmacological strategies are urgently required to promote the treatment of sepsis ( ) . with the ability to promote the expression of cxcr , cxcr figure . n-butanol extract from folium isatidis inhibits the downregulated protein levels of cxcr , cxcr and cd l. following pretreatment with a vehicle control (dmso) or n-butanol extract at the indicated concentration for h, neutrophils were incubated with lps ( . µg/ml) for h. expression levels of (a) cxcr , (b) cxcr and (c) cd l were measured using flow cytometry. the corresponding mean fluorescence intensity was calculated. each bar represents the mean ± standard error of the mean of three independent experiments. statistical significance relative to the lps group was determined. ** p< . . lps, lipopolysaccharide; cxcr , cxc-chemokine receptor ; cxcr , cxc-chemokine receptor ; cd l, l-selectin. and cd l, n-butanol extract itself is a potential candidate for the treatment of lps-induced sepsis. however, due to the presence of multiple bioactive components in the extract and limited approaches for preparing extracts from f. isatidis, it is difficult to identify the precise agent inhibiting lps-induced chemokine receptor downregulation. although the main active chemical components have been identified, there are more than eight major compounds in n-butanol extract ( ) . further investigation is required to identify the active compound in f. isatidis. in conclusion, the present study demonstrated that n-butanol extract obtained from f. isatidis prevented the lps-induced downregulation of cxcr , cxcr and cd l on neutrophils. the extract promoted neutrophil migration in response to il- . these results indicated that n-butanol extract from f. isatidis, which targets neutrophil chemotaxis during lps-induced sepsis, is a potential candidate for the treatment of sepsis. phospholipase d drives mortality in sepsis by inhibiting neutrophil extracellular trap formation and down-regulating cxcr sepsis biomarkers innate immune functions of immature neutrophils in patients with sepsis and severe systemic inflammatory response syndrome evaluation of toll-like, chemokine, and integrin receptors on monocytes and neutrophils from peripheral blood of septic patients and their correlation with clinical outcomes inflection points in sepsis biology: from local defense to 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ligation and puncture-induced murine sepsis does not cause lung injury international study of the prevalence and outcomes of infection in intensive care units neutrophils in sepsis: battle of the bands platelet serotonin promotes the recruitment of neutrophils to sites of acute inflammation in mice targeting neutrophils in sepsis early goal-directed therapy collaborative group: early goal-directed therapy in the treatment of severe sepsis and septic shock gastrin-releasing peptide receptor (grpr) mediates chemotaxis in neutrophils this study was supported by the provincial natural science foundation of zhejiang (grant no. y ). key: cord- -lku g k authors: zhang, shidong; wang, dongsheng; wang, xurong; li, shihong; li, jingyu; li, hongsheng; yan, zuoting title: aqueous extract of bai-hu-tang, a classical chinese herb formula, prevents excessive immune response and liver injury induced by lps in rabbits date: - - journal: journal of ethnopharmacology doi: . /j.jep. . . sha: doc_id: cord_uid: lku g k abstract ethnopharmacological relevance bai-hu-tang (bht) was traditionally used to reduce fever heat and promote generation of body fluids. aim of the study to investigate the effect and mechanism of bht in the prevention of lipopolysaccharide (lps) fever in manners of immune modulation. materials and methods the model of fever syndrome of chinese medicine pattern was imitated by lps injection i.v. in rabbits, and bht was gavaged. the serum levels of tumor necrosis factor-α (tnf-α), interleukin (il- , ) and immunoglobulin (igg, iga, and igm) were determined by enzyme-linked immunosorbent assay (elisa); alanine aminotransferase (alt) and aspartate aminotransferase (ast) were tested by biochemical methods. liver tissue damage was detected by hematoxylin–eosin (h&e) stain. subpopulation of t cells was detected by fluorescence activated cell sorter (facs). genes expression of toll-like receptor (tlr ) and lipopolysaccharide binding protein (lbp) in liver tissue were assayed by real-time polymerase chain reaction (rt-pcr). result the results demonstrated that bht prevented sudden increase of il- , tnf-α, alt and ast, and liver damage induced by lps. bht also prevented significant decrease of the percentage of cd + t cells since lps injection. at the same time, bht did not affect the gene expression of tlr and serum concentration of three immunoglobulins, which were increased by lps, but made gene expression of lbp higher. conclusion the results of this study indicated that bht played an important role in immunity protection and anti-injury through preventing immunoinflammatory damage by lps. the achievement thereby scientifically provided mechanism of bht in the prevention of febrile disease, and supported its traditional use. fever is a reaction of homeothermic animals and humans when thermogenic substance entered the body and interacted with the cells of the immune system (riedel and maulik, ) . it was a common response to infection, inflammation and trauma. clinically, febrile response was characterized as a rise in body temperature above the normal range, and often treated with eliminating excess heat (lisa, ) . although has different clinical courses, many communicable and infectious diseases can be classified into epidemic febrile disease according to therapies of traditional chinese medical (tcm) such as severe acute respiratory syndrome (sars) (peng, ; shen, ; gu et al., ) , avian influenza a (h n ) (li, ; nie and lin, ) , and swine influenza a (h n ) (zhou, ; liu, ; zheng et al., ) . because of the common feature of high fever, these diseases could be treated with antipyretic prescription according to principle of febrile disease treatment in tcm. lipopolysaccharide (lps), a bacterial endotoxin, is a group of heat stable molecules present in the outer membrane of gramnegative bacteria that possesses toxic effect (burell, ) . the injection of lps was a reasonable model of bacterial infection, and literatures were replete with records that lps caused a typical fever with intravenous or intraperitoneal injection (shibata et al., ; steiner et al., ; ravanelli et al., ) , and researchers often used lps to induce reproducible febrile responses in animals (roth et al., ) . actually, chinese researchers believed that an injection of lps can induce a representative fever syndrome of chinese medicine pattern in rabbit, and the fever pattern can be used to do studies for antipyretic traditional herb medicine and ethnopharmacological research (yang, ; yu et al., ; ai et al., ; ni and wei, ) . the traditional chinese medicine had cured over years of sage advice, making it one of the oldest and most widely used systems of medicine in the world. herbal medicine is one of the most essential elements of tcm, and bai-hu-tang (or white tiger decoction) is a classical natural chinese herbal formula with a long history of use. the formula originated from the treatise on febrile disease of shang han lun (or treatise on cold-attack) compiled by zhong-jing zhang who lived in eastern han dynasty in china. it was formulated with four herbs of liquorice, anemarrhena rhizome, gypsum and rice, and mainly used to reduce fever and promote generation of body fluids (charles, ; zhu, ) . it was also useful in treating diabetes mellitus (chen et al., ) , eczema, pruritus, some anxiety and emotional disorders (herb toxicities and drug interactions: a formula approach, ) .the formula has caused wide public concern over the recent years and practiced in many countries (european herbal and traditional medicine practitioners association, ) , and even some international press also published works for user guide (herb toxicities and drug interactions: a formula approach, ). therefore, the bht is an important traditional chinese formula that has been extensively used to prevent febrile disease in the world. but there was little basic theory report on its mechanism and effect. in this study, a febrigenic dosage of lps ( mg/kg i.v.) was injected into rabbits to form an animal febrile model, then the model animals were gavaged with bht at the same time, and the formula's function of liver damage prevention and immunomodulation was researched in rabbits. the method of animal treatment and a representative dosage of bht ( ml/kg) was an adaptation of other tcm researchers (chen et al., ; sun, ; xiong and liu, ) , as well as our preliminary test, which indicated there was no significant difference in the dose range of - ml/kg (data not shown in manuscript). the research achievement of this study will scientifically explore the mechanism and effect of bht, and provide theory support in traditional use of bht. the herbs of liquorice (sliced root of glycyrrhiza glabra l. in leguminosae), anemarrhena rhizome (sliced root of anemarrhena asphodeloides bunge in liliaceae), gypsum (crystal of calcium sulfate), and rice (nonglutinous rice, polished seed of oryza sativa l. in gramineae) were purchased from antaitang pharmaceutical co., ltd., china, and identified by dr. zuoting yan from lanzhou institute of animal & veterinary pharmaceutics of chinese academy of agricultural sciences. as shown in fig. glycyrrhizae radix . g, anemarrhena rhizome . g, gypsum . g and rice . g were extracted together by refluxing with boiling water twice. they were boiling water ( : , w/v) for h, and water ( : , w/v) for h, respectively. two-time juice was blended together, filtered with three-layer gauzes, and concentrated to ml experimental decoction. the experimental juice was autoclaved at c for min, and kept in airtight containers at c until used. lps (escherichia coli o :b ) was purchased from sigma chemical co. (st. louis, mo, usa), and dissolved in saline ( . % nacl) at clean benches. both of il and ig serum active elisa kits were purchased from r&d techno co. (minneapolis, mn, usa). monoclonal antibodies of mouse anti-rabbit cd + -fitc and cd + -fitc were obtained from abd serotec (kidlington, ox ge, uk). both of one-step rt-pcr kit (drr a) and sybr premix taq kit (drr a) for real-time pcr were provided by takara biotech co. (dalian, china). rna fixer was purchased from aidlab biotechnologies co. (beijing, china). primers for target genes were synthesized by bejing adult new zealand white rabbits weighted between . and . kg were used in the experiments, and housed individually in rabbit stocks under controlled room humidity of % and temperature of c with a h light and h darkness cycle. animals were fed commercial stock diet and water ad libitum, and allowed to stabilize for at least d in new surroundings before any experiments. all experimental animals were obtained from the animal center of lanzhou institute of biological products (lanzhou, china). the rabbits were divided into three groups randomly. group i served as control, and received only physiological saline; group ii was fever model, which received only lps ( mg/kg body weight) by intravenous injection (i.v.) into ear vein; group iii was gavaged with bht (a representative dose of ml/kg body weight) at the same time of lps intravenous injection. rabbits then received standard diet and water ad libitum. at the time of h after lps injection and bht administration, full conscious rabbits were immobilized on anchor platform through binding limbs outside body, and . ml blood was taken from heart by cardiac puncture with disposable syringe. after blood collection, the rabbits were anesthetized by an intraperitoneal injection of sodium pentobarbital ( mg/kg) for min, and sacrificed immediately. ml blood was placed into aseptic tubes, and centrifuged ( c, rpm, min) to get serum. . ml blood was placed into edtak tubes, and mixed to form anticoagulant blood. the serum sample was used for biochemical assays of il- , il- , tnf-α, igg, iga, and igm. at the same time, anti-coagulated whole blood samples from edtak tubes were used to analyze t lymphocyte subpopulations of cd + and cd + . some liver tissues of rabbits were cut away, flushed in saline ( . % nacl), and immersed in formalin for pathology analysis. one more part of liver tissue was immersed in rna fixer, and stored at À c for total rna extract. cd + t cells and cd + t cells were identified using directly conjugated anti-rabbit monoclonal antibodies. briefly, μl peripheral blood sample anti-coagulated by edtak were incubated in the dark with μl of the mouse anti-rabbit cd + -fitc or mouse antirabbit cd + -fitc antibody for min at c, and washed with μl of facs (fluorescence activated cell sorter) buffer. according to manufacturer, red cells were lysed by using ml lysing solution (becton dickinson). the remaining leukocytes were washed with pbs (ph . ) twice and then fixed with . ml facs fix solution (becton dickinson). fixed leukocytes were re-suspended in ml pbs, and analyzed in a becton dickinson facscalibur flow cytometer (becton dickinson). fluorescence data were collected from  cells, and analyzed by using cellquest software (becton dickinson). the serum samples were kept at À c until ready for measurement. according to the manufacturer's protocol, levels of igg, iga, igm, il- , il- , and tnf-α were measured by using commercially available elisa kits. the levels of alt and ast in serum samples were determined using an automatic biochemistry analyzer and biochemical kits (mindray biomedical electronics co., shenzhen, china), following the manufacturer's instruction. liver tissue was fixed in % neutral-buffered formalin, and embedded in paraffin. sections of -mm thickness were affixed to slides, deparaffinized, stained with hematoxylin-eosin (h&e) for general histopathology examination under a light microscope (olympus, japan), and assessed by a pathologist blind to the treatment groups. a two-step process was employed to determine relative quantity of selected genes. briefly, total rna was isolated from the liver using the trizol reagent according to the manufacturer's instructions and then μg total rna was reverse transcribed to cdnas by use of an rt-pcr kit following the manufacturer's directions. the information of pcr primers were shown in table . pcr was performed for cycles using the following conditions: preincubation at c for s, denaturation at c for s, annealing at c for s, and elongation at c for s. the pcr products were also verified by ethidium bromide-stained agarose gel electrophoresis (data not shown). for each real-time pcr sample was performed for target gene and the housekeeping gene (β-actin). real-time pcr was performed for quantification of gene expression of tlr and lbp, and using the quantitative pcr super mix in a final reaction volume of μl in  sybr green (molecular probes) according to the manufacturer's protocol. the data were expressed as the number of threshold cycle (c t ). the relative quantification of the target genes was determined by calculating the ratio between concentration of target gene and that of housekeeping gene. for each real-time pcr analysis, the individual experiments were performed in triplicate. data were reported as the means standard deviation (s.d.), and analyzed by using t-test for comparisons of significance level (p) between the control and the treated values. p o . was considered to be statistically significant, and if it was not significant then the p value was not less than . . calculations were made with the commercially available software spss . . fig. highlights that lps significantly increased tnf-α, il- , and il- , especially a sudden il- signaling that seems to become acute and life-threatening. after treatment with bht, il- decreased completely, and tnf-α reduced partially. although il- was still higher than control, it was lower than lps group significantly. these data suggested that lps led to an increase of inflammatory cytokines in rabbits, but bht might prevent the sudden increase, and protect animals from the injury of the immoderate inflammatory response. as biochemical marker for liver function, the serum levels of hepatic enzymes ast and alt were elevated significantly (p o . ) by lps injection. due to the work of bht, the elevation of these marker enzymes was significantly prevented (fig. a and b) . fig. c showed that liver pathological change of animals injected with lps. it showed centrilobular necrosis, infiltration of immunity cells (such as macrophages and lymphocytes) into portal tract and sinusoid, hepatocytes ballooning, necrosis, and disintegration. diffused areas of necrotic lesion, especially in the perivenular region which extends to the central zone, with collections of inflammatory cells, were observed after lps injection when compared to control. because of the absence of cellular necrosis and inflammatory infiltrates in the liver, it was evident that bht prevented hepatic lesions produced by lps, which was almost comparable to the control. these data indicated that bht prevented liver injury and dysfunction induced by lps in rabbits. the t cells subpopulation of cd + (th cells) and cd + (tc cells) in peripheral blood were determined by flow cytometry. the results showed that there was no change of cd + cells percentage ( fig. a ), but the percentage of cd + cells decreased significantly. moreover, serum igg, igm, and iga concentrations soared up after lps injection. due to administration of bht, the percentage of cd + cells was not decreased by lps (fig. b ), but enhanced immunoglobulins were still kept at a high level (fig. c-e) . the rt-pcr semi-quantitation demonstrated that mrna of tlr and lbp were up-regulated significantly after lps injection. due to the role of bht, mrna of tlr was down-regulated, but not significantly (p . ). note that bht made mrna of lbp expresses at a higher level (fig. b) . these results indicated that bht might prevent the lps immunological response by inhibiting excessive expression of crucial factor tlr in pro-inflammatory signal transduction in hepatic tissue. cytokines play an essential role in mediating interactions between cells of the immune system (fletcher and starr, ) . a sprinkling of cytokine can be specific responses to apart from infections and get transition into recovery, but an excessive cytokine production may cause the immune system out of control, and cause the precipitation of pathological consequences (allison and rosenthal, ) . some diseases of fever, kidney dysfunction, and liver problem could be attributed to body's response to excessive cytokine production that storm physiology of the body, such as il- and tnf-α (clark, ) . this virulent immune response with production of large amounts of inflammatory cytokines often was named as immunoinflammatory response, and played the most important role in the pathogenesis of liver damage and dysfunction (seely and christou, ; wang and ma, ; aldridge et al., ) . the results demonstrated that liver pathological changes were observed significantly in lps-treated animals, and the serum concentrations of alt and ast, which were functional readout for liver damage, increased drastically in lps injection animals, but decreased to normal level with bht prevention (fig. ) . these changes were accompanied by changes of inflammatory cytokines, especially sudden change of il- signaling (fig. c ). there were no significant changes of other cytokines of il- , il- , and ifn-γ (data not shown). this raised the intriguing possibility that excessive production of tnf-α, il- , and il- (fig. ) , which also caused excessive immune response, may be the direct cause of liver damage, and bht was able to prevent the pathological process. because the activation of the innate immune response can be a prerequisite for triggering of acquired immunity (akira et al., ) , the adaptive immune system always works in synchronization with innate immunity (abdelsadik and trad, ) . depending on the combination of cytokines produced in response to the stimulus, innate and adaptive immune responses are initiated. as reported, lps-induced proliferation of lymphoid cells is thought to be primarily restricted to b cells in acquired immune (tough et al., ) , and nearly all b cells require the help of th cells before they can mature and differentiate into antibody-secreting plasma cells. the conventional t cells played a critical role in tempering the inflammatory response recognized by innate immune system (kim et al., ) . in this study, the results of immunoglobulins and t cell subpopulations showed that humoral immunity mediated by b cells was activated by lps without cd + cell (th cells) increase, but cell immunity carried out by cd + cells (tc cells) was inhibited by lps. due to the role of bht, the cd + cell decrease was prevented totally (fig. b ), but enhanced humoral immunity was still kept at a high level (fig. c-e) . therefore, bht did not affect the activated adaptive immunity, but prevented the cell immunity inhibition and excessive cytokines in innate immunity by lps at the same time. in addition to cytokines, an important role in innate immunity has been ascribed to tlr family (lenert, ) . different tlr recognizes the different pattern-recognition receptors (prrs) expressed on the effector cells of immunity system, such as tlr recognized lipoproteins, tlr recognized double-stranded rna, and tlr recognized lps (akira et al., ; kawai and akira., ) . actually, tlrs as main prrs of immunity system played an important role in linking between rapid defense of innate immunity and delayed eradication of adaptive immunity (werling and jungi, ; pasare and medzhitov, ) . it was reported that tlr played an indispensable role in triggering lps fever, and the phase of febrile response to lps depended entirely on the tlr (steiner et al., ) . because lps firstly arrived at liver that is the main lps-processing and clearance organs after intravenous administration (li and blatteis, , it was ponderable to investigate expression of functional genes in liver. the gene expressions of tlr studied herein were not affected by bht (fig. a ), but lbp expression was increased (fig. b) , which bind and transport lps to activate cells in the immunity system (knapp et al., ) , and inhibiting inflammatory response at higher concentration (hamann et al., ) . this indicated that bht may play an important role in avoiding excessive immunity, and the elevated expression of tlr may be beneficial for the initiation of the following adaptive immunity linked by tlr . in conclusion, bht prevented excessive cytokines increase, and protected animals from liver damage by lps. bht also defened cd + cell immunity against lps, but did not affect adaptive immunity mediated by b cell and linked by tlr . therefore, bht played an important role in immunomodulation and anti-injury in the treatment of febrile disease. toll-like receptors on the fork roads between innate and adaptive immunity experimental study on feasibility of wei-qi-ying-xue syndrome in rabbit with toll-like receptors: critical proteins linking innate and acquired immunity pathogen recognition and innate immunity tnf/inos-producing dendritic cells are the necessary evil of lethal influenza virus infection cytokine storms: systemic 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patients with mild case treatment discussion on pathogenesis of h n in tcm shang han lun is the footstone of dialectical cure in tcm this research was supported by the national key science and technology support project in the th five-year plan of china ( badb b - ) and the central scientific research institutes for basic research fund of china ( ). gratitude is also sent to the anonymous and editor whose suggestions greatly improved the manuscript. key: cord- -nk thkme authors: downer, eric j.; jones, raasay s.; mcdonald, claire l.; greco, eleonora; brennan, sabina; connor, thomas j.; robertson, ian h.; lynch, marina a. title: identifying early inflammatory changes in monocyte-derived macrophages from a population with iq-discrepant episodic memory date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: nk thkme background: cells of the innate immune system including monocytes and macrophages are the first line of defence against infections and are critical regulators of the inflammatory response. these cells express toll-like receptors (tlrs), innate immune receptors which govern tailored inflammatory gene expression patterns. monocytes, which produce pro-inflammatory mediators, are readily recruited to the central nervous system (cns) in neurodegenerative diseases. methods: this study explored the expression of receptors (cd b, tlr and tlr ) on circulating monocyte-derived macrophages (mdms) and peripheral blood mononuclear cells (pbmcs) isolated from healthy elderly adults who we classified as either iq memory-consistent (high-performing, hp) or iq memory-discrepant (low-performing, lp). results: the expression of cd b, tlr and tlr was increased in mdms from the lp group when compared to hp cohort. mdms from both groups responded robustly to treatment with the tlr activator, lipopolysaccharide (lps), in terms of cytokine production. significantly, mdms from the lp group displayed hypersensitivity to lps exposure. interpretation: overall these findings define differential receptor expression and cytokine profiles that occur in mdms derived from a cohort of iq memory-discrepant individuals. these changes are indicative of inflammation and may be involved in the prodromal processes leading to the development of neurodegenerative disease. neuroinflammatory changes develop with age and are a feature of most neurodegenerative diseases including alzheimer's disease (ad). epidemiological studies indicate that long-term nonsteroidal anti-inflammatory treatment reduces the risk of developing ad [ ] suggesting that inflammatory processes may contribute to very early changes in the pathogenesis of the disease. the inflammatory changes in the central nervous system (cns) in normal ageing and ad are characterised by microglial activation with resultant changes in microglial phagocytic activity and neurotoxic consequences [ ] [ ] [ ] ; the evidence indicates that some of these cns changes have systemic parallels. the number of blood-derived monocytes is increased in ad patients [ ] , and it has been reported that circulating monocytes express specific cell adhesion molecules (cams) that are altered with age, and in patients with ad and mild cognitive impairment (mci) [ , ] . interestingly, circulating monocytes readily infiltrate the brain in murine models of ad [ , ] and evidence indicates that bloodborne monocytes can act as amyloid phagocytes and may be involved in amyloid-b (ab) clearance [ ] . these findings, alongside others which show that monocytes, in addition to in vitro differentiated macrophages, express several ad-related genes [ ] , suggest that circulating monocytes may be a valuable model system in assessing the role of microglial cells in the pathogenesis of neurodegenerative conditions. human monocytes express an array of cams and receptors that are crucial for cell-cell and cell-matrix interactions, transmigration and innate immune responses. of these, cd b is a receptor for intercellular adhesion and is a phenotypic monocytic cell marker, the expression of which increases with age, indicating a more activated phenotype [ ] . monocytes/macrophages also express a diverse array of toll-like receptors (tlrs), a family of signalling pattern recognition receptors that mediate innate immunity by recognising conserved motifs of microbial origin known as pathogen-associated molecular patterns (pamps) and endogenous damage-associated molecular patterns (damps) that are released by injured tissue [ ] . human blood monocytes express tlr , tlr , tlr , tlr , tlr , tlr , and tlr mrna, with tlr and tlr being the most highly expressed [ ] . tlrs continue to emerge as key players in cns diseases and much data link tlrs with ad pathology, with polymorphism in the tlr and tlr gene linked with the disease [ ] . the classical monocyte marker, cd , a glycosylphosphatidyl inositol (gpi)anchored myeloid glycoprotein, acts as a co-receptor for tlrs and is responsible for the uptake of gram-negative bacterial lipopolysaccharide (lps) via macropinocytosis [ ] . indeed, tlr acts as the primary signalling receptor for lps [ ] , with tlr further required for lps-induced tlr signalling [ ] . in an attempt to quantify relative decline and explore discrepancies between targeted cognitive domains that are central to ageing (e.g. episodic memory) and a proxy measure of lifetime cognitive capacity, we have identified a group of otherwise healthy adults who we classify as low performers (lp) because their memory performance is discrepant with their estimated iq. the advantage of adopting such an approach is threefold. first, it allows quantification of true age-related changes in the strength or weakness of memory relative to an estimate of lifetime cognitive capacity. second, it allows one to dissociate domain-specific changes in memory function. third, it allows classification of individuals as high or low memory performers based on standardised measures of memory relative to iq that can be utilised, not only in research, but also in a clinical setting. the purpose of the study was to compare the expression of receptors (cd b, tlr and tlr ) on circulating monocyte-derived macrophages (mdms) and the response of these cells to lps in samples prepared from the lp cohort and a cohort which we classified as iq memory-consistent (high-performing, hp) individuals. we show that mdms isolated from the lp group display enhanced expression of cd b, tlr and tlr , compared to the hp group. we further show that treatment of mdms with lps promoted pro-inflammatory cytokine production in hp and lp groups, but that the effect was exacerbated in mdms prepared from the lp group. adults ( female, male) with a mean age of . years (range of to ) were recruited from the older adult participant panel of the trinity college institute of neuroscience, dublin, ireland. participants were assigned to low and high performing sub-groups (lp and hp respectively) based on their memory performance relative to an estimate of their intelligence. z-scores were used to relate their performance on the wechsler memory scale story recall test [wms iii uk; ] to their scores on the national adult reading test [nart ; ] . participants were defined as lp if they scored more than . standard deviations below their nart-estimated iq on the wms. all other participants were classified as hp. this approach yielded hps ( female, male) with a mean age of . (sd = . ) and lps ( female, male) with a mean age of . (sd = . ). no significant difference was observed in mini mental state exam (mmse) scores between the two groups (table ) . participants received reimbursement of travel expenses to the maximum value of j . this study was approved by the ethics committee of the school of psychology at trinity college dublin, and all participants provided informed consent. all participants completed a detailed questionnaire about their own health and current medications, as well as any relevant health issues in their family. participants who had a history of head injury, stroke, epilepsy, neurological conditions, major psychiatric disorder, heart attack or diabetes were excluded from the study. a battery of tests was administered in one session, and focused on memory and executive function. the mmse was used as a screening measure. the nart was used as a proxy measure of general intellectual status. memory was assessed using subtests of wechsler memory scale: logical memory i and ii verbal paired associates i and ii visual reproduction i and ii. human pbmcs were prepared from heparinised venous whole blood samples ( ml per donor) from hp and lp individuals by density separation over lymphoprep tm (axis-shield, norway). plasma samples were separated following centrifugation, aliquoted and stored at - uc. cd + monocytes were isolated from pbmcs using positive selection with macs microbeads (miltenyi biotec, germany) and an automacs cell sorting instrument using a method which results in up to % purity as estimated by flow cytometry [ ] . the mean percentage monocytes in pbmcs from hp and lp groups was . . and . . , respectively (p = . ; -tailed unpaired t-test). in this study, cd + cells, obtained from pbmcs following macs sorting, were seeded ( cells/ml) on -well plates and cultured in rpmi supplemented with fbs ( %), antibiotics and human granulocyte macrophage colony-stimulating factor ( ng/ml; r&d systems, uk) for days. at the end of this period, cells were assessed again by flow cytometry; . % of the cells were cd + ( figure s ), which is consistent with previous studies that have been shown to yield . - % cd + cells [ ] [ ] [ ] . we refer to these cells as mdms in the present study. freshly-isolated pbmcs and mdms were washed times in facs buffer ( % fbs, . % nan in pbs) and blocked for min at uc with purified human igg ( mg/ml in facs buffer; sigma, uk). cells were incubated with anti-human cd b-apc (clone icrf ), anti-human tlr -fitc (clone t . ) and anti-human tlr -pe-cy (clone hta ) (all at : in facs buffer; biosciences, uk). a minimum of , events were collected and the percentage positive staining for each cellular target was calculated. total viable pbmcs were initially gated via forward and side scatter (demonstrated in figure s ) and mdms were stimulated with lps ( ng/ml) and after h rna was extracted using a nucleospinh rnaii isolation kit (macherey-nagel inc., germany). the concentration of lps used is in line with those used in various inflammatory paradigms in human monocytes and macrophages [ ] [ ] [ ] . the concentration of rna was determined using a uv/vis spectrophotometer (beckman coulter inc., ireland). cdna synthesis was performed on mg rna using a high capacity cdna rt kit (applied biosystems, usa) according to the manufacturer's instructions. equal amounts of cdna were used for rt-pcr amplification. real-time pcr primers were delivered as ''taqmanh gene expression assays'' containing forward and reverse primers, and a fam-labeled mgb taqman probe for each gene (applied biosystems, usa). primers used were as follows: cd b, tlr and tlr (taqmanh gene expression assay no. hs _m , hs _m and hs _m , respectively). a in dilution of cdna was prepared and real-time pcr performed using an applied biosystems real-time pcr system. cdna was mixed with qpcr tm mastermix plus (applied biosystems, usa) and the respective gene assay in a ml volume ( ml of diluted cdna, . ml taqmanh universal pcr mastermix, . ml target primer and . ml gapdh). human gapdh was used as an endogenous control and expression was conducted using a gene expression assay containing forward and reverse primers and a vic-labeled mgb taqman probe (# e; applied biosystems, usa). samples were run in duplicate and cycles were run as follows: min at uc and for each cycle, s at uc and min at uc. gene expression was calculated relative to the endogenous control and analysis was performed using the ddct method. in all experiments no change in relative gapdh mrna expression between treatment groups was observed. mdms were stimulated with lps ( ng/ml; h) and supernatants were assessed for tumor necrosis factor (tnf)a, il- , il- and il- p production using a human pro-inflammatory- -plex assay (mesoscale discovery, usa) according to manufacturer's instructions. briefly, plates were blocked in diluent for min at room temperature and duplicate samples and standards ( - , pg/ml) added for h at room temperature with vigorous shaking. detection antibody solution was added for h at room temperature with vigorous shaking. plates were washed again and read buffer was added to the plate. the plate was read immediately using a mesoscale sector imager plate reader and pro-inflammatory cytokine concentrations in test samples were evaluated with reference to the standard curve prepared using the -plex calibrator blend. plasma samples from hp and lp groups were analysed for concentrations of crp by elisa (duoset, r&d systems, uk) according to manufacturer's instructions. data were analysed using a student's t-test for independent means or two-way analysis of variance (anova) as appropriate. when analysis by anova indicated significance (p, . ), the post hoc student newman-keuls test was used. data are expressed as means standard errors of the mean (sem). human mdms isolated from the lp group display enhanced expression of cd b, tlr and tlr , compared to hps since neurological symptoms are often exacerbated by infection [ ] , we initially assessed plasma levels of the inflammatory reactant crp in plasma from hp and lp groups. no significant difference in plasma crp concentration was found between groups ( figure a ). pbmcs predominantly constitute lymphocytes and monocytes, with lower numbers of natural killer (nk) and dendritic cells. cd b is an adhesion molecule primarily expressed on the surface of monocytes, but also on activated lymphocytes and a subset of nk cells, where it mediates leukocyte adhesion and migration to orchestrate an inflammatory response [ ] . indeed, cd b expression is upregulated by many inflammatory mediators including cytokines [ ] . cd b expression was unchanged on pbmcs isolated from the hp and lp groups ( figure b , c, p = . ) and no difference in mdm cd mrna was observed between groups ( figure d ) suggesting that differentiation of monocytes to macrophages was similar in lp and hp individuals. however, the percentage of cd b + cells in the mdm preparation was significantly greater in the lp group, compared with the hp group ( figure e , f, p, . ) and the cd b staining intensity was also higher on mdms from the lp group, compared with the hp group ( figure g , p, . ). although cd b mrna was also enhanced in mdms from the lp, compared with the hp, group the difference did not reach statistical significance ( figure h , p = . ). since macrophages and other cells of the innate immune system recognise distinct microbial products via tlrs, we next assessed the expression of tlr and tlr since they are specifically implicated in inflammatory changes associated with neurodegeneration [ , ] . tlr expression on cd b + cells was similar on pbmcs isolated from the hp and lp groups ( figure i , j) but expression in mdms from the lp group was higher than the hp group, although it did not reach statistical significance ( figure k , l, p = . ); mfi values for the hp and lp groups were . and . respectively. tlr mrna was significantly greater in mdms prepared from the lp group compared with the hp group (p, . ; figure m ). tlr expression on cd b + cells was similar on pbmcs isolated from the hp and lp groups ( figure n , o) but expression on mdms from the lp group was significantly greater than from the hp group (p, . ; figure p , q); mfi values for the hp and lp groups were . and . respectively. tlr mrna was significantly greater in mdms prepared from the lp group compared with the hp group (p, . ; figure r ). overall, this indicates that differentiated mdms, but not freshly prepared pbmcs, isolated from the lp cohort display elevated expression of cd b and tlr / . since tlr acts as the primary signalling receptor for gramnegative bacterial lps [ ] and because tlr expression was increased in mdms prepared from the lp cohort, we examined the effect of lps on cytokine production in mdms from hp and lp groups. production of the cytokines tnfa, il- , il- and il- p was similar in unstimulated mdms from hp and lp groups ( figure ). however, lps significantly increased cytokine production in mdms from both groups, and importantly the production of tnfa (figure a ), il- ( figure b ), il- ( figure c ) and il- p ( figure d ) was exacerbated in mdms prepared from the lp group, compared with the hp group (p, . ). this indicates that mdms prepared from the lp group are hyper-responsive to lps, at least in terms of cytokine production, and the increase in tlr expression provides a plausible mechanism for this effect. we investigated the expression of tlr and , and the impact of in vitro treatment with lps on the production of cytokines in mdms prepared from an iq memory-discrepant lp cohort, compared with an iq memory-consistent hp cohort. the significant finding is that mdms from the lp group expressed higher levels of tlr and tlr , as well as cd b, and exhibited a more robust response to lps, than mdms from the hp group. ageing is associated with defects in both the innate and adaptive arms of the immune system. indeed, elderly individuals display reduced b cell production [ ] and t cell memory [ ] , ensuring greater susceptibility than young individuals to bacterial and viral infection. much data also indicate that ageing affects cells of the innate immune system, particularly associated with alterations in neutrophil [ ] and monocyte/macrophage phagocytic capacity [ ] , and ability to produce cytokines and chemokines [ ] . overall, cumulative data indicates that defective innate immunity is associated with advancing age. intermediate stages between normal ageing and dementia are recognised by several classification systems; the best recognised are the several clinical subtypes of mci, a preclinical stage of ad [ ] . the lack of diagnostic tools which enable early detection of conditions like mci and/or ad is a major stumbling block for treatment of disease and a method of detecting early changes in cognitive function relative to previous levels would be a significant advance. structural changes which are indicative of conversion from normal ageing to mci and ad have been identified using image analysis [ ] . in addition, a number of peripheral blood-based biomarkers have been suggested, however the most compelling data have been obtained from analysis of cerebrospinal fluid (csf) in which the ratio of ab/ phosphorylated tau combined with total tau levels yielded a positive predictive value in terms of conversion from mci to ad [ ] . myeloid cells, constituting blood-borne monocytes, tissue resident macrophages and parenchymal microglia, have defined functions in neurodegenerative diseases. monocytes and macrophages not only share the same origin with microglia in the brain, but also have the same antigens, functions, and regulatory mechanisms [ ] . much research has focused on characterising the neurodegenerative role of microglial cells which accumulate at senile plaques in ad [ ] , secreting proteolytic enzymes that degrade ab [ ] and expressing receptors that promote the phagocytosis of ab [ ] . monocytes and macrophages are professional phagocytic cells and central players in orchestrating the innate immune response. monocytes/macrophages have proinflammatory and cytotoxic properties, with the proclivity to produce inflammatory molecules such as tnfa and inducible nitric oxide synthase [ ] . blood-borne monocytes are highly mobile and rapidly recruit to inflamed cns tissue during bacterial infection [ ] , autoimmune disorders [ ] and neurodegenerative disease [ ] . indeed, circulating monocytes can infiltrate the cns parenchyma and differentiate into microglia [ ] , where they are important for clearing amyloid and limiting its deposition in murine models of ad [ ] . furthermore, macrophages display an activated phenotype in neurodegenerative disease, typified by increased expression of cell surface markers such as cd b [ ] , cns infiltration [ ] and enhanced production of inflammatory cytokines [ ] . here we show that expression of tlr was greater in mdms obtained from the lp cohort compared with the hp cohort and, consistently, that incubation of cells from the lp cohort responded more robustly to lps with significantly greater production of tnf, il- and il- than that produced by mdms from the hp group. mdms are a type i or classically activated (m ) macrophage that display a pro-inflammatory phenotype [ ] . mdms are considered a peripheral counterpart of microglia, as they share the same progenitor and antigen markers, and they have similar biological behaviours that mirror microglial function in the brain [ , ] . previous evidence has shown that they produce inflammatory cytokines in response to lps treatment [ ] and that they express tlr [ ] , which is confirmed here. interestingly monocytes from older, compared with younger, individuals have higher intracellular levels of tnf both at baseline figure . enhanced cd b, tlr and tlr expression in mdms from lps. a) plasma isolated from hp and lp groups displayed comparable levels of crp. data are presented as mean sem and represent triplicate determinations from and samples per hp and lp groups, respectively. b) cd b expression on pbmcs was unchanged between hp and lp subjects. c) representative plots of cd b + cells in pbmc populations. numbers beside gated areas indicate the percentage positive cells in that area. d) cd expression in mdms was similar in lp and hp individuals following days in culture. e) percentage cd b expression was increased in mdms derived from the lp group, compared with the hp group (p, . ). f) representative dot plots of cd b + cells in mdms (with values beside gated areas indicating the percentage positive cells in that area) show marked differences between cohorts. g) cd b mean fluorescence intensity on mdms was increased in the lp group compared with the hp group (p, . ). h) cd b mrna in mdms derived from the lp group was slightly, though not significantly, greater than values from the hp group (p = . ). i) tlr expression on cd b + pbmcs was similar in hp and lp groups and this is also shown in the representative dot plots of tlr + cells (j) which indicate the percentage positive cells. k) tlr expression on cd b + mdms was increased in the lp group, compared to the hp group (p = . ) and this is also shown in the representative dot plots of tlr + cells (l) which indicate the percentage positive cells. m) tlr mrna expression was increased on mdms derived from the lp group compared with the hp group (p, . ). n) tlr expression on cd b + pbmcs was similar in hp and lp groups and this is also shown in the representative dot plots of tlr + cells (o) which indicate the percentage positive cells. p) tlr expression on cd b + mdms was increased in the lp group compared with the hp group (p, . ) and this is also shown in the representative dot plots of tlr + cells ( and following lps stimulation [ ] , while pro-inflammatory cytokine [ ] and chemokine [ , ] levels are elevated in peripheral blood monocytes isolated from the elderly after lps stimulation. in addition, pbmcs from ad patients have been reported to produce enhanced levels of il- following lps stimulation compared with controls [ ] . these findings suggest that monocyte function is dysregulated both with age and in ad, but the present study significantly extends these observations to show that similar changes can be picked up in mdms prepared from a group of otherwise healthy adults whose memory performance is discrepant with their estimated iq. cd b is a receptor for intercellular adhesion molecule family members cd , cd and cd , enabling leukocyte adhesion and migration to mediate the inflammatory response [ ] . cd b is also a typical monocyte/macrophage marker, the expression of which is increased following activation [ ] . indeed, cd b expression is increased in monocytes from aged individuals [ ] and on microglial cells in aged animals [ ] . furthermore the number of cd b + activated microglia is increased in mouse models of ad [ ] and ms [ ] . the present data indicate that cd b expression was enhanced in mdms, but not pbmcs, from the lp cohort, indicating that these cells displayed an enhanced activated phenotype. this cell type-specific effect indicates that altering the phenotype of human monocytes in vitro may provide a mechanism which identifies alterations of myeloid cell function in cohorts with the most subtle cognitive changes. it is of interest to note that modifications of macrophage phenotype have been suggested in conditions where clear pathology is identified, i.e. in chronic inflammatory conditions [ ] , autoimmune disorders [ ] and ad [ ] . human monocytes express an array of tlrs [ ] and our data indicate that both cd b + pbmcs and mdms express tlr and tlr . in the present study we focused on examining expression of tlr and tlr since much evidence indicates the role of these receptors in neurodegenerative conditions. monocytes derived from elderly individuals display defective tlr signalling, particularly tlr / , with further decreases in relative tlr and tlr expression determined on peripheral blood monocytes from older subjects [ , ] . tlr / / mice are protected from cognitive impairment following ab immunisation [ ] with polymorphism in the tlr and tlr gene linked with ad [ ] . knockout studies suggest differential effects of tlrs on ad progression; deletion of cd , a co-receptor for tlr / , reduces plaque burden in a murine ad model [ ] , but deletion of myd enhances memory deficits [ ] . importantly, evidence indicates that ab binds microglial tlr and [ ] suggesting that tlrs function as members of the ab receptor complex. our findings indicate that the expression of tlr and tlr is increased in mdms from the lp group, compared with the hp group. since increased tlr expression does not occur on monocytes in healthy elderly individuals [ ] , while increased expression of tlr and tlr has been reported in pbmcs from patients with late-onset ad [ ] , the data may suggest a role for increased tlr expression in progression from the mildest manifestation of episodic memory loss to more profound cognitive decline. our results identify changes in the expression of receptors cd b, tlr and tlr on mdms isolated from a unique cohort of iq memory-discrepant older adults. consistent with the increased expression of tlr , stimulation with lps promoted enhanced cytokine production in mdms from this lp group, compared with the hp iq memory-consistent group. it is important to note that bacterial and(or) viral infections are associated with reduced cognitive performance [ ] , but no differences in plasma concentrations of crp were identified between the cohorts in this study. these peripheral cell changes, which are indicative of inflammation, may provide a biological substrate indicative of the earliest discernible changes in episodic memory decline which may be a precursor to more profound changes that are associated with the development of neurodegenerative disease. figure s flow cytometric analysis of mdms. flow cytometric analysis of mdms and selection of cd + cells. cd + monocytes were separated from pbmcs using a macs sorter. the cd + monocytes were cultured for days with gm-csf ( ng/ml), and stained with a cd antibody to determine purity by flow cytometry. gates were made using the isotype controls. after one week in culture . % of cells were cd + . (tif) figure s flow cytometric analysis of pbmcs. a) flow cytometric analysis of pbmcs and selection of live cells. pbmc gate location determined via forward/side scatter analysis of pbmcs. b) using a logical gating strategy, live pbmcs gated for cd b. 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changing perspective on the role of neuroinflammation in alzheimer's disease age-associated defect in human tlr- / function deletion of cd attenuates alzheimer's disease pathology by influencing the brain's inflammatory milieu myd -adaptor protein acts as a preventive mechanism for memory deficits in a mouse model of alzheimer's disease cd and tolllike receptors and are required for fibrillar a{beta}-stimulated microglial activation increased expressions of tlr and tlr on peripheral blood mononuclear cells from patients with alzheimer's disease our co-author and colleague, tom connor, passed away on th february ; we would like to dedicate this publication to his memory.the authors gratefully acknowledge the support of science foundation ireland ( /in. /b ) and atlantic philanthropies (neuroenhancement for independence in elder lives (niel). the authors would also like to thank l. penny for her assistance with phlebotomy, and b. moran for technical help with facs. conceived and designed the experiments: ejd rsj tjc ihr mal. performed the experiments: ejd rsj eg sb. analyzed the data: ejd rsj cm eg sb. wrote the paper: ejd. key: cord- -zvc s authors: choudhary, ishita; vo, thao; bathula, chandra s.; lamichhane, richa; lewis, brandon w.; looper, jayme; jeyaseelan, samithamby; blackshear, perry j.; saini, yogesh; patial, sonika title: tristetraprolin overexpression in non-hematopoietic cells protects against acute lung injury in mice date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: zvc s tristetraprolin (ttp) is a mrna binding protein that binds to adenylate-uridylate-rich elements within the ′ untranslated regions of certain transcripts, such as tumor necrosis factor (tnf) mrna, and increases their rate of decay. modulation of ttp expression is implicated in inflammation; however, its role in acute lung inflammation remains unknown. accordingly, we tested the role of ttp in lipopolysaccharide (lps)-induced acute lung injury (ali) in mice. lps-challenged ttp-knockout (ttp(ko)) mice, as well as myeloid cell-specific ttp-deficient (ttp(myeko)) mice, exhibited significant increases in lung injury, although these responses were more robust in the ttp(ko). mice with systemic overexpression of ttp (ttp(Δare)) were protected from ali, as indicated by significantly reduced neutrophilic infiltration, reduced levels of neutrophil chemoattractants, and histological parameters of ali. interestingly, while irradiated wild-type (wt) mice reconstituted with ttp(ko) hematopoietic progenitor cells (hpcs) showed exaggerated ali, their reconstitution with the ttp(Δare) hpcs mitigated ali. the reconstitution of irradiated ttp(Δare) mice with hpcs from either wt or ttp(Δare) donors conferred significant protection against ali. in contrast, irradiated ttp(Δare) mice reconstituted with ttp(ko) hpcs had exaggerated ali, but the response was milder as compared to wt recipients that received ttp(ko) hpcs. finally, the reconstitution of irradiated ttp(ko) recipient mice with ttp(Δare) hpcs did not confer any protection to the ttp(ko) mice. these data together suggest that non-hpcs-specific overexpression of ttp within the lungs protects against ali via downregulation of neutrophil chemoattractants and reduction in neutrophilic infiltration. acute lung injury (ali) and its severe form, acute respiratory distress syndrome (ards), are serious health concerns due to a high rate of mortality ( ) . ali is characterized by elevated levels of proinflammatory mediators, exaggerated neutrophil recruitment, and compromised pulmonary epithelial-endothelial barrier, resulting in increased vascular permeability ( ) . non-cardiogenic pulmonary edema, characterized by excessive accumulation of protein-rich edematous fluid and inflammatory cells in the alveolar spaces, results in hypoxemia in ards that requires aggressive clinical management including mechanical ventilation ( ) . despite significant health burdens posed by these diseases, the identity of key cellular and molecular players of host defense against ali remains unclear. zinc finger protein (zfp ), commonly known as tristetraprolin (ttp), is an mrna binding protein that binds to adenylate-uridylate-rich elements (ares) within the untranslated regions ( utrs) of target mrnas and increases their rate of decay ( ) . germline ttp-knockout (ttp ko ) mice exhibit the spontaneous development of a systemic inflammatory syndrome characterized by cachexia, erosive arthritis, myeloid hyperplasia, dermatitis, conjunctivitis, and autoimmunity ( ) . these phenotypes were shown to be essentially completely prevented in ttp ko mice with either tnf receptor deficiency, or when ttp ko mice were treated with anti-tnf antibodies ( ) . biochemical studies demonstrated that ttp binds to ares within the tumor necrosis factor (tnf ) mrna utr and results in tnf mrna degradation under normal conditions ( , ) . subsequent reports have shown that a number of other proinflammatory mediators including cxcl ( , ) , cxcl ( ), il- ( ), il- ( ) , ccl ( ) , and il- ( ) are also regulated by ttp ( ) . recently, using a systemic ttp overexpression (ttp are ) mouse model, we demonstrated protective effects of enhanced ttp levels in chronic immune-mediated inflammatory diseases including mouse models of arthritis, psoriasis, and autoimmune encephalomyelitis ( ) . ttp are mice lack ares in the utr of the endogenous ttp gene (zfp ) that results in increased stability of ttp mrna and, in turn, moderately increased expression of ttp protein in essentially all the tissues ( ) . together, these studies have indicated that ttp may be an endogenous anti-inflammatory protein and that enhancing its levels may be beneficial against various chronic inflammatory diseases. in the present study, we investigated the role of ttp in regulating lung inflammation in a mouse model of ali. using an oropharyngeal aspiration approach, lipopolysaccharide (lps)induced ali was modeled in adult mice, and the animals were monitored for signs of ali. to identify the protective role of ttp in a cell-specific manner, we performed bone marrow irradiation and reconstitution experiments in wild-type (wt), ttp ko ( ) , and systemic ttp overexpression (ttp are ) mice ( ) . our findings elucidate cell-specific roles of ttp in protection against ali, and indicate that ttp is an important modulator of endotoxin-induced ali. zfp floxed mice (zfp flox/flox ) ( ) were crossed with lysmcre recombinase expressing mice ( ) to generate mice for experimental (cre +/+ /zfp flox/flox ; ttp myeko ) and control (cre −/− /zfp flox/flox ; cre +/+ /zfp wt/wt ) groups. genotype status of progeny was determined by polymerase chain reaction (pcr) as described previously ( ) . ttp knockout mice (ttp ko ) and ttp overexpression mice (ttp are ) have been described before ( , ) . all the animal experiments were performed in accordance with principles and procedures outlined in the national institute of health guide for the care and use of laboratory animals and were approved by the louisiana state university animal care and use committee. both male and female adult ( - -week-old) mice were used for experiments. mice were anesthetized with isoflurane/oxygen followed by administration of µg lipopolysaccharide (lps) from escherichia coli o :b (l , sigma-aldrich) per mouse dissolved in sterile endotoxin-free saline ( µl total volume), or an equivalent volume of sterile endotoxin-free saline as a vehicle control, via oropharyngeal aspiration ( ) . mice were observed for signs of distress including anorexia, weight loss, hunched posture, ruffled haircoat, labored breathing, and dehydration every - h post lps challenge. mice exhibiting at least four of these clinical signs were humanely euthanized before the end of the study. following saline or lps treatment, mice were anesthetized with , , -tribromoethanol (sigma-aldrich, st. louis, mo, united states) at the indicated time points, and mid-line laparotomy was performed. briefly, bronchoalveolar lavage fluid (balf) was harvested from the right lung. recovered balf was processed and analyzed for total and differential cell counts by routine methods ( ) . unlavaged left lung lobes were fixed in % neutral buffered formalin (nbf) and used for preparation of slides for histopathological evaluation. right lung lobes were snap-frozen and stored at − • c. bronchoalveolar lavage fluid was harvested and centrifuged at × g for min, and the supernatant was stored at − • c for further analyses. the cell pellet was resuspended in µl of pbs and total cell counts were determined using a hemocytometer (brightline, horsham, pa, united states). cytospins were prepared using µl of cell suspension (statspin cytofuge ; hemocue, brea, ca, united states) followed by differential staining (modified giemsa kit; newcomer supply, middleton, wi, united states). mouse cytokine and chemokine levels were assayed in cell-free balf supernatant using luminex-xmap-based assay (mcytomag- k), according to the manufacturer's instructions (emd millipore, billerica, ma, united states). five micrometer sections of lung were stained with hematoxylin and eosin (h&e) for routine histology. histology: a semiquantitative histopathological scoring system was used to analyze the sections as follows: ( ) consolidation (percent of total surface area of lung section affected); ( ) bronchiolitis ( , no bronchioles affected; , one bronchiole affected; , between - bronchioles affected; , more than bronchioles affected); ( ) perivascular edema ( , minimal; , mild; , moderate; , severe); ( ) perivascular inflammation/inflammatory cells ( , absent; , minimal; , mild; , moderate; , severe); ( ) airspace edema ( , minimal; , mild; , moderate; , severe); ( ) airspace hemorrhages ( , absent, , patchy, mild; , extensive, moderate; , extensive, severe). slides were graded in a blinded manner without knowledge of sex and treatment groups. bone marrow transplantation experiments were performed as described previously ( ) . briefly, - -week old recipient mice were irradiated with megavolt x-rays from a linear accelerator (varian clinac ex) with two (dorsal and ventral) -rad ( cgy) doses. to prepare bone marrow cells for transplantation, femur bones of donor mice were flushed to collect bone marrows, and single cell suspensions were prepared. a total of × cells were injected into the tail vein of lethally irradiated recipient mice. reconstituted recipient mice were given . % neomycin sulfate dissolved in acidified water for the first weeks post-transplantation. lps-challenge experiments were performed weeks post bone marrow reconstitution, which has been previously shown to be an optimal period for repopulation of resident alveolar macrophages with donor cells following total body irradiation ( ) . lung tissue was lysed using pierce tm ripa buffer (thermo fisher scientific, waltham, ma, united states) supplemented with pierce tm protease inhibitor mixture (thermo fisher scientific, waltham, ma, united states) and phosphatase inhibitors ( mm sodium fluoride and mm sodium orthovanadate). tissues were mechanically homogenized using a bead beater (thermo fisher scientific, waltham, ma, united states). tissue lysates were centrifuged ( , × g, min, • c) to remove insoluble material and protein concentration of the supernatants was measured through bradford assay (bio-rad laboratories, hercules, ca, united states). equivalent amounts of denatured protein was separated on a - % bis-tris plus precast gels (invitrogen, carlsbad, ca, united states), transferred on to pvdf membrane (invitrogen, carlsbad, ca, united states) and probed with a : dilution of rabbit antiserum raised against a recombinant mouse ttp-maltose binding protein fusion ( ) followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit igg (bio-rad). signal was determined using supersignal west pico chemiluminescent substrate (pierce) on x-ray film. significant differences among groups were determined by oneway analysis of variance (anova) followed by tukey's post hoc test for multiple comparisons except for cytokine assays where two-way anova was used. measurements from two groups were compared using student's t-test assuming unequal variance. all data were expressed as mean ± sem. a p-value < . was considered statistically significant. statistical analyses were performed using graphpad prism . (graphpad software, la jolla, ca, united states). in order to explore the role of ttp in ali, ttp ko and littermate control wt mice were subjected to ali through oropharyngeal aspiration of endotoxin (lps) (single dose; µg lps/mouse) for a period of h. while saline-treated ttp ko and wt groups had comparable numbers of total immune cells in the balf (saline-treated wt; × ± × , saline-treated ttp ko , × ± × ), lps administration resulted in increased infiltration of immune cells in both the ttp ko and the wt groups. the total number of recovered immune cells in lps-challenged ttp ko mice ( × ± × ) were ∼ fourfold higher as compared to lps-challenged wt ( × ± × ) mice ( figure a) . increases in total cell counts in lps-challenged ttp ko mice were attributed to a significant increase in neutrophil ( figure b) , macrophage ( figure c) , and lymphocyte counts ( figure d ). these increases were associated with an increased injury to the pulmonary vascular barrier, as depicted by the presence of red blood cells in the cytospins prepared from the balf fluid of ttp ko mice ( figure e ; right panel, black arrow) versus control lps-challenged wt mice ( figure e ; left panel). histologically, the lungs of lps-challenged wt mice were characterized by mild to moderate consolidation (∼ % of total area of lung section), two-to fourfold increase in alveolar septal thickening (broken green arrow), moderate perivascular and airspace edema, and perivascular inflammation (figures f-h) . in contrast, the lung injury in lps-challenged ttp ko mice was characterized by severe consolidation (> % of total area of lung section) (figures f,g) that included infiltration of neutrophils, edema, fibrin, and airspace hemorrhage within the airway and alveolar lumen, multifocal loss of bronchiolar epithelium with infiltration of neutrophils and red blood cells within the bronchiolar lumen, and moderate to severe perivascular edema and inflammation (figures f-h) . of note, ∼ % lps-challenged ttp ko mice succumbed to lps challenge before -h and these had to be excluded from the analysis. these data suggest that systemic loss of ttp results in extreme susceptibility of mice to lpsinduced ali. in order to explore the role of myeloid cell-specific ttp on inflammatory response in ali, myeloid cell-specific ttp deficient mice (ttp myeko ; cre +/+ /zfp flox/flox ) and control (cre control; cre +/+ /zfp wt/wt and flox control; cre −/− /zfp flox/flox ) mice were challenged with lps. similar to saline-treated wt and ttp ko groups, saline-treated control and ttp myeko groups had comparable numbers of total . data are represented as mean ± sem. statistical analysis was performed by one-way anova followed by tukey's correction for multiple comparison test. *p < . ; **p < . ; ***p < . . n = each for wt and ttp ko saline controls; n = , wt group; n = , ttp ko group for lps-challenge group. three lps-challenged ttp ko mice succumbed to lps-challenge before h and were not lavaged for further analyses. representative photomicrographs of wright-giemsa stained balf cytospins of lps-challenged wt (e; left panel) and lps-challenged ttp ko (e; right panel) mice. neutrophil (red arrow), macrophage (green arrow), lymphocyte (blue arrow), red blood cell (black arrow) (original magnification × ). representative photomicrographs (f) from h&e-stained left lung lobe sections from adult lps-challenged wt (f; left panel) and lps-challenged ttp ko (f; right panel) mice. septal thickening (green broken arrow), intra-alveolar neutrophilic infiltrates (green arrow), intra-alveolar red blood cells (red arrow), bronchiolar lumen neutrophilic accumulation (blue arrow), and perivascular cellular infiltration (black arrow). asterisk represents alveolar space that is minimally affected (f; left) or severely consolidated with blood and neutrophils (f; right) (original magnification × ). semiquantitative histopathological scoring for consolidation (g) is shown as a percent of total surface area of the lung section affected in lps-challenged wt and lps-challenged ttp ko mice. semiquantitative histopathological scoring of lung sections for bronchiolitis, perivascular edema, perivascular inflammation, airspace hemorrhage, and airspace edema in lps-challenged wt and lps-challenged ttp ko mice (h). statistical analysis in g and h was performed using student's t-test. *p < . ; **p < . ; ***p < . ; ****p < . . immune cells (flox control; × ± × , ttp myeko ; × ± × ). lps administration resulted in increased numbers of immune cells in ttp myeko as well as both the control groups. this increase in total cell counts was, however, significantly greater in ttp myeko ( × ± × ) compared to cre control ( × ± × ) mice. the increase in cellular recovery did not reach statistical significance in lps-challenged ttp myeko when compared to the lps-challenged flox control group (p = . ) (figure a ). this effect was comparable in both sexes (data not shown). of note, the increase in cellular infiltration was ∼ threefold less in lps-challenged ttp myeko ( × ± × ) (figure a ) when compared to lps-challenged ttp ko mice ( × ± × ) ( figure a) . while neutrophil counts were comparable between lps-challenged ttp myeko and lpschallenged control mice (figures b,e) , macrophage counts were significantly elevated in the balf obtained from lps-challenged ttp myeko mice compared to the two groups of control mice (figures c,e) . lymphocyte counts did not differ between the lps-challenged ttp myeko and the two groups of control mice (figures d,e) . histopathological analysis revealed comparable levels of lung consolidation with widespread inflammatory cellular infiltrates within the airspaces of both lps-challenged ttp myeko and flox control mice; however, perivascular edema, perivascular inflammation, and airspace edema were somewhat exaggerated in lps-challenged ttp myeko mice compared to the flox control group (figures f-h) . unlike lps-challenged ttp ko mice, airspace hemorrhage was not observed in any of the groups. all the lps-challenged ttp myeko mice survived lps challenge, as compared to the lps-challenged ttp ko mice, in which ∼ % mortality was observed. these data indicate that myeloid cell-specific ttp is essential for protection against ali. mitigates lps-induced ali in mice during acute and sub-acute course of lung injury next, we examined whether the systemically ttp overexpressing (ttp are ) mice exhibit protection against ali. in experimental ali, while time points earlier than h of lps-challenge represent acute phases of ali, later time points represent somewhat sub-acute to chronic or resolution phases of endotoxin-induced ali in mice ( ) . therefore, we examined both lps-challenged wt and lps-challenged ttp are mice over time points representing acute to subacute phases, i.e., ± . × , . × ± . × , and . × ± . × cells at h, h, h, days, and days, respectively) ( figure a) . lipopolysaccharide administration resulted in increased numbers of total cells in balf from both wt and ttp are mice when compared to saline administration ( figure a) . total cell counts in balf from lps-challenged wt mice were . × ± . × , . × ± . × , × ± × , × ± . × , and . × ± . × at h, h, h, days, and days time points, respectively ( figure a) . interestingly, significantly reduced numbers of immune cells were recovered from the lungs of lps-challenged ttp are mice compared to lungs of lps-challenged wt mice at all time points examined post lpschallenge ( . × ± . × , . × ± . × , . × ± . × , . × ± . × , and . × ± . × at h, h, h, days, and days, respectively) ( figure a) . the decrease in total cell counts in lps-challenged ttp are mice was contributed by significantly reduced numbers of neutrophils at , , and h (figures b,e) . interestingly, however, the total numbers of macrophages were only significantly different between lps-challenged wt and lps-challenged ttp are mice at h and days time points (figures c,e) . lymphocyte counts were not significantly different in lps-challenged ttp are mice compared to lps-challenged wt mice (figures d,e) . cellular counts followed the same trend in both the sexes (data not shown). reduced cellular infiltration was also evident in cytological slides prepared from lps-challenged wt and lps-challenged figure | myeloid-ttp deficiency exacerbates lps-induced ali in mice. total cell counts (a) in the harvested balf of adult saline-(white bars; n = each for flox control and ttp myeko ) or lps-challenged cre control (cre +/+ /zfp wt/wt , gray bar; n = ), flox control (cre -/-/zfp flox/flox , purple bar; n = ), and ttp myeko (cre +/+ /zfp flox/flox , green bar; n = ) mice. differential cell counts are shown for neutrophils (b), macrophages (c), and lymphocytes (d). data are represented as mean ± sem. statistical analysis was performed by one-way anova followed by tukey's correction for multiple comparisons. ns, non-significant; *p < . ; ***p < . ; ****p < . . representative photomicrographs of wright-giemsa stained balf cytospins from lps-challenged flox control (e; left) and ttp myeko (e; right) mice. neutrophil (red arrow), macrophage (green arrow), lymphocyte (blue arrow) (original magnification × ). representative photomicrographs from h&e-stained left lung lobe sections from post- h lps-challenged flox control (f; left) and ttp myeko (f; right) mice (original magnification × ). asterisk represents alveolar spaces minimally obliterated in flox control (f; left) and severely obliterated in lps-challenged ttp myeko mice (f; right). bronchiolar lumen neutrophilic infiltrates (blue arrow), absent in lps-challenged flox control (f; left) but present in lps-challenged ttp myeko (f; right). semiquantitative histopathological scoring for consolidation (g) is shown as a percent of total surface area of the lung section affected in lps-challenged flox control and lps-challenged ttp myeko mice. semiquantitative histopathological scoring of lung sections for bronchiolitis, perivascular edema, perivascular inflammation, and airspace edema in lps-challenged flox control and lps-challenged ttp myeko mice (h). n = (flox control); n = (ttp myeko ). statistical analysis in g and h was performed using student's t-test. *p < . . ttp are bal fluid, which showed the sparsely present neutrophils, macrophages, and lymphocytes in lps-challenged ttp are mice compared to dense presence of these cells in lps-challenged wt mice ( figure e) . histologically, lpschallenged wt lungs exhibited ∼ % lung consolidation with significantly increased infiltration of immune cells within the airspaces, bronchiolitis, perivascular edema, and inflammation (figures f-h) . in contrast, bronchiolitis and perivascular edema were significantly attenuated in lps-challenged ttp are mice. lung consolidation, perivascular inflammation, and airspace edema were not significantly different between the two groups, and airspace hemorrhage was not observed in any group (figures f-h) . we next analyzed the changes in the protein expression levels of ttp over the course of ali in wt and ttp are mice whole lung tissue. as shown in figure i , basal expression levels of ttp were higher in ttp are versus wt lungs. upon lps administration, the expression levels of ttp increased in both wt and ttp are mice lungs by h post lps administration. this increase was significant in ttp are mice lungs as compared to unchallenged wt mice lungs. the levels of ttp started reducing at subsequent time points in both wt and ttp are lungs. while the ttp expression decreased to basal levels in wt mice, the ttp expression remained at relatively higher levels in the ttp are mice lungs at all subsequent time points. these data show that the presence of higher levels of ttp under basal conditions, combined with a significant increase at h post-lps challenge and maintenance of higher expression levels at later time points post lps challenge, protects ttp are mice from ali. cellular recruitment within the lung in response to lps challenge is mediated by the production of chemoattractants. therefore, next we sought to examine the levels of inflammatory cytokines and chemokines within the balf of lps-challenged wt and lps-challenged ttp are mice. of the cytokines/chemokines analyzed, four cytokines/chemokines, including g-csf, kc, il- , and il- p , were found to be significantly different between the lps-challenged wt and the lpschallenged ttp are mice. g-csf was significantly reduced in lps-challenged ttp are compared to lps-challenged wt mice at and h; kc and il- were significantly reduced at h; and il- (p ) was significantly reduced at h post-lps challenge (figures a-d) . interestingly, the levels of tnfα ( figure e ) and mip (figure f) , two known ttp targets, were not significantly different between the two groups. to determine the cell-specific role of ttp levels in ali, we modulated ttp levels in hematopoietic progenitor cells (hpcs) and non-hpcs. in order to test whether donor hpcs repopulate the recipient mouse lungs, we first made bone marrow chimeras in which total body irradiated wt mice were transplanted with hpcs from a mouse expressing green fluorescent protein (gfp) in their somatic cells. we found that greater than % of the cells recovered from the balf of these mice were gfp positive (data not shown). next, we generated three bone marrow chimeras in which irradiated wt recipient mice received hpcs from either wt (wt→wt), ttp are (ttp are →wt), or ttp ko (ttp ko →wt) mice ( figure a) . while saline-treated ttp ko →wt chimeras had no signs of cellular infiltration that included total cells (figure b) , neutrophils (figure c) , macrophages (figure d) , and lymphocytes (figure e) , lps-challenged wt→wt, ttp are →wt, and ttp ko →wt chimera mice had significant cellular recruitment, as indicated by balf total cellular counts (figure b) , neutrophils (figure c) , and macrophages ( figure d) . while lpschallenged ttp are →wt mice had somewhat reduced cellular infiltration as compared to lps-challenged wt→wt chimeras (figures b-d) , the lps-challenged ttp ko →wt chimera mice had remarkably higher number of bal cellular counts (figures b-d) . lymphocyte counts did not differ significantly between the three groups ( figure e) . histologically, ∼ , , and % of the lung parenchyma was consolidated in lps-challenged wt→wt, lps-challenged ttp are →wt, and lps-challenged ttp ko →wt group, respectively (figures a top panel, c) . consolidated parenchyma was characterized by the presence of large infiltrates of neutrophils and macrophages within the airspaces (alveolar and airway). other histological findings included the presence of edema and immune cells within the perivascular spaces, bronchial lumen cellular infiltrates, airspace edema, and occasional bronchoalveolar lymphoid aggregates (figures d-g) . these data suggest that while baseline expression of ttp in hpcs is essential for protection against exaggerated ali, its overexpression in these cells does not confer significant additional advantage. next, we generated bone marrow chimeras in which irradiated ttp are recipient mice received hpcs from either wt (wt→ttp are ), ttp are (ttp are →ttp are ), and ttp ko (ttp ko →ttp are ) mice ( figure a) . as compared to wt recipient chimera counterparts, lps-challenged wt→ttp are , lps-challenged ttp are →ttp are , and lps-challenged ttp ko →ttp are chimeras had significantly lower degrees of cellular recruitment, that included total cells ( figure b , blue solid bar, orange solid bar, green solid bar), neutrophils ( figure c , blue solid bar, orange solid bar, green solid bar), and macrophages ( figure d , blue solid bar, orange solid bar, green solid bar). lymphocyte counts were significantly reduced in lps-challenged ttp are →ttp are compared to the lps-challenged wt→wt chimeras ( figure e ). although the lps-challenged ttp ko →ttp are chimeras had significantly higher cellular recruitment as compared to lps-challenged wt→ttp are and lps-challenged ttp are →ttp are chimeras, the extent of recruitment was much diminished in lps-challenged ttp ko →ttp are chimera as compared to lps-challenged ttp ko →wt chimera ( figure b) . histologically, ∼ , %, % of the lung parenchyma was consolidated in lps-challenged wt→ttp are , lpschallenged ttp are →ttp are , and lps-challenged ttp ko →ttp are , respectively (figures a bottom panel, figure c ). histologically, mild increases in septal thickening with cellular infiltration, mild bronchiolitis, perivascular edema, inflammation, and airspace edema, and occasional balts were evident in all the three groups (figures d-g, solid blue, orange, and green bars). these data suggest that enhanced expression of ttp in lung non-hpc populations conferred significant protection against ali. further, this protection is somewhat compromised in the absence of baseline levels of ttp in hpcs. a tabular summary of differential cellular and ali responses in various chimeric mice is included in supplementary table . finally, we generated bone marrow chimeras in which irradiated ttp ko recipient mice received hpcs from ttp are (ttp are →ttp ko ). as expected, the cellular counts were significantly higher than any of the other lps-challenged chimeras (figures b-e, red solid bar) . however, total cellular counts in lps-challenged ttp are →ttp ko chimera were ∼ twofold lower than the lps-challenged ttp ko mice (figure ) . additionally, none of the lps-challenged ttp are →ttp ko chimeras succumbed to ali, as compared to % mortality in lps-challenged ttp ko mice (figure ) . these data suggest that complete loss of ttp in lung non-hpc populations significantly exaggerates ali, and that overexpression of ttp in hpcs may provide partial protection in severe ali. histologically, this group exhibited the most severe lung injury, characterized by ∼ % lung consolidation with neutrophils, macrophages, fibrin, and edema, severe bronchiolitis, and moderate to severe bronchiolar and alveolar hemorrhages (figures b-g) . tristetraprolin knockout (ttp ko ) mice exhibit a systemic inflammatory syndrome that is characterized by cachexia, polyarticular synovial arthritis, dermatitis, and myeloid hyperplasia ( ). however, the lungs of ttp ko mice exhibit very little spontaneous inflammation, characterized by the presence of rare foci of leucocytic infiltrates within the pulmonary parenchyma ( ) . these rare leucocytic infiltrates are abrogated upon combined deletion of ttp and tnf receptors, indicating the role for tnf activity in leucocytic infiltration ( ) . myeloid cell-specific loss of ttp (ttp myeko ) does not recapitulate the ttp ko phenotype, indicating that non-myeloid cells are required for the overall manifestation of the ttp deficiency syndrome ( ) . however, ttp myeko mice were found to be hypersensitive to endotoxin-induced systemic inflammation, particularly through exaggerated tnf production, delineating the critical role of myeloid cell-specific ttp in protection against systemic injury and inflammation ( ) . the role of ttp in localized lung inflammation, however, has remained unexplored. therefore, in this study we sought to explore the role of ttp in ali. we hypothesized that ttp modulates acute lung inflammation and that cell-specific modulation of ttp levels will differentially affect the outcome of acute lung inflammation. the unchallenged ttp ko mice display minor degrees of leukocytic infiltration in lung parenchyma that were thought to be contributed by tnf activity ( ) . here, in lps-challenged ttp ko mice, immune cell infiltration was ∼ fourfold higher as compared to lps-challenged wt mice. in fact, the susceptibility of ttp ko mice to lps-induced ali was so severe that ∼ % ttp ko mice succumbed to lps-challenge within - h post-lps administration, while no mortality was observed in lpschallenged wt mice. the surviving ttp ko mice displayed severe pulmonary pathology with exaggerated airspace and interstitial neutrophilic infiltration, exaggerated edema, vascular congestion, and lung injury that included epithelial and endothelial damage. it is likely that the increased production of inflammatory mediators, that are otherwise regulated by ttp, in lpschallenged ttp ko mice, contribute to their worsened pulmonary pathology. one such mediator, tnf, is already established to be highly secreted in ttp ko mice ( , ) . macrophages are the primary source of tnf in ali ( , ) although alveolar epithelial cells have also been suggested to release tnf in lps-induced lung inflammation ( ) . accordingly, we reasoned that, if macrophages are the primary source of tnf, deletion of ttp in myeloid cells would enhance its activity, leading to worsening of pulmonary pathology. although the lps-challenged ttp myeko mice had exaggerated pulmonary pathology as compared to lps-challenged wt mice, the extent of tissue damage and neutrophilic infiltration was not as severe as in lps-challenged ttp ko mice. these differences in the sensitivity of systemic versus myeloid cell-specific ttpdeficient mice indicate that ttp in cells other than myeloid-cells may play critical roles in modulating endotoxin-induced ali. these data suggest that while loss of myeloid cell-specific ttp worsens the ali, ttp expression in non-myeloid cells confers significant protection. clinically, the numbers of neutrophils within the balf of patients with ards have been shown to correlate with the severity of disease and poor outcome ( , ) . despite being the first line of defense against pathogenic insults, excessive recruitment and activation of neutrophils leads to lung tissue damage and loss of lung function ( , ) . therefore, targeting neutrophilic recruitment through suppressed release of key neutrophil chemoattractants may be an attractive therapeutic strategy against ali. we observed correlations between ttp deficiency or ttp overexpression and neutrophilic inflammation. for example, balf counts and tissue infiltration of neutrophils were overwhelming in the lps-challenged ttp ko mice. these outcomes were also exaggerated, but to a lesser degree, in lps-challenged ttp myeko mice. on the other hand, these outcomes were significantly attenuated in the lps-challenged ttp are mice. our findings are in line . data are represented as mean ± sem. statistical analysis in c-g was performed by one-way anova followed by tukey's correction for multiple comparisons within the three recipient groups and student's t-test between the three recipient groups. *p < . ; **p < . ; ***p < . ; ****p < . . with previous reports in other tissues. for instance, massive infiltration of neutrophils has been shown to occur in the skin of ttp ko mice subjected to psoriasis-like inflammation ( ) , whereas reduced airway neutrophilic infiltration was shown in mice genetically modified to express constitutively active endogenous unphosphorylated ttp following challenge with cigarette smoke ( ) . a number of studies have shown that ttp is phosphorylated at multiple sites by p -regulated kinase mapk-activated protein kinase (mapkapk ) and that ttp activity is significantly affected by its phosphorylation status ( ) ( ) ( ) . ttp phosphorylation has been shown to result in reduced ttp activity/reduced binding of ttp to ares, thus resulting in stabilization of its target mrnas ( ) . regulation of ttp activity by p mapk was in fact shown to result in a biphasic response of tnfα-induced il- expression in human bronchial smooth muscle cells ( ) . conversely, dephosphorylation of ttp resulted in reduced expression of il- and il- in a lung epithelial cells ( ) . since lps is an inducer of both ttp and p mapk, ttp would be expected to undergo phosphorylation and inactivation shortly after lps challenge. however, the effect of ttp phosphorylation is transient and would be expected to be reversed upon dephosphorylation when p is turned off. although we did not track the phosphorylation status of ttp in various lung cells during ali, we speculate that the p -mediated phosphorylation of ttp occurs well before h post lps challenge and is reversed by h time point. consistent with our speculation, we found differential effect of ttp overexpression on late phase cytokines (kc) versus early phase cytokines (tnf) (figure ) . tristetraprolin is a known regulator of key neutrophil chemoattractants including cxcl /kc ( , ) and cxcl /mip ( , ) . cxcl /kc is a central chemokine in neutrophil recruitment into the airspace in ards ( ) ( ) ( ) . clinically, increased concentrations of il- (cxcl /kc homolog in human), disease severity, and neutrophil migration into airspaces have been shown to be correlated ( ) ( ) ( ) . cxcl mrna utr contains ttp-binding sites and has been previously demonstrated to be a direct target of ttp ( , ) . consistent with this, the lps-challenged ttp are mice had significantly lower cxcl /kc concentrations. in contrast, the two well characterized ttp targets, tnf and mip /cxcl , were not significantly different between lps-challenged wt and lpschallenged ttp are mice. these observations are in line with our previous report, in which no differences were observed in the levels of tnf and mip /cxcl in the serum of wt and ttp are mice systemically challenged with lps ( ). these two inflammatory mediators peak early (before h in the serum) ( ) , and we speculate that overexpressed ttp may be less effective at mrna decay due to its phosphorylation status at these early time points. g-csf, another neutrophil chemoattractant, is also consistently detected within the balf of ali and ards patients and has been suggested to be associated with the accumulation and activation of neutrophils in ards ( ) . plasma g-csf levels have also been shown to correlate with clinical outcome in patients with ali ( , ) . g-csf has also been shown to exacerbate bleomycin induced lung injury in rats through marked infiltration of activating neutrophils ( ) . g-csf levels have been found to be increased in ttp ko mouse plasma ( ) . although g-csf has not been shown to be a direct ttp target, g-csf mrna utr in mouse possesses two ttp binding sites, uauuuau. the balf g-csf levels were significantly reduced in lps-challenged ttp are mice. g-csf levels were also found to be significantly reduced in ttp are mice in a previous study where we showed ttp are mice to be significantly protected from exhibiting collagen-antibody induced arthritis ( ) . lipopolysaccharide-challenged ttp myeko mice exhibited milder ali as compared to lps-challenged ttp ko mice, indicating that total loss of ttp expression in non-myeloid as well as myeloid cells contributes to severe ali. on the other hand, systemic ttp overexpression conferred significant protection against lps-induced ali. here, we addressed two logical questions: ( ) whether enhanced levels of ttp in non-hpcs would ameliorate endotoxin-induced ali, ( ) whether enhanced levels of ttp in hpcs would confer protection against endotoxin-induced ali. towards this, employing various bone marrow chimeras, we investigated cell-specific roles of ttp in protection against ali. in these experiments, we modulated ttp levels in hpcs by using bone marrow donors that were either wt, ttp are , or ttp ko . to modulate ttp levels in non-hpcs, wt, ttp are , or ttp ko recipients were used. one limitation of this study was that since hpcs also include various non-myeloid populations, we were not able to specifically investigate the effect of ttp modulation in myeloid cells. the bone marrow irradiation and reconstitution experiments revealed somewhat unexpected but interesting findings. as compared to wt→wt chimera, the ali in ttp are →wt chimeras was somewhat attenuated, whereas the ali in ttp ko →wt chimeras had worsened significantly. these data suggest that, when the ttp expression in non-hpcs is genetically unaltered (wt ttp in the recipient's non-hpcs), the ttp overexpression in hpcs is partially protective against lps-induced ali. however, when the ttp expression was ablated in non-hpcs [ttp depleted in the recipient's non-hpcs (ttp are →ttp ko chimera)], the overexpression of ttp in the hpcs was not sufficient to confer protection against ali. as compared to ttp are →ttp ko chimeras, in ttp ko →ttp are chimeras, the mere overexpression of ttp in non-hpcs (ttp overexpression in the recipient's non-hpcs) provided significant protection, even though the hpcs were ttp-deficient. however, this protection was further enhanced when the normal levels of ttp were restored in the hpcs (wt→ttp are chimera). based on these data, we conclude here that the ttp levels in non-hpcs are critical in protection against ali. further, while the wt levels of ttp in hpcs are essential for additional protection, the overexpression of ttp in hpcs is not significantly advantageous. since the non-hpcs population in lungs consists of a multitude of cell types including epithelial cells, endothelial cells, and fibroblasts, the cell-specific role of ttp still remains elusive. contribution of non-hpc-specific ttp toward modulation of inflammatory responses has also been suggested in previous reports. for instance, ttp regulation of tnf in keratinocytes, but not in myeloid or dendritic cells, was shown to protect mice from exacerbation of psoriasis-like pathology, development of spontaneous systemic inflammation, and dactylitis ( ) . another study suggested a role of intestinal epithelial cell-specific ttp in exacerbation of acute colitis ( ) . along similar lines, ttp depletion in myeloid cells did not replicate the ttp-deficiency phenotype ( ) , and the spontaneous reactive granulopoiesis seen in ttp ko mice was suggested to be caused by a non-cell autonomous mechanism likely contributed by liver cells ( ) . all these studies indicated an essential role of ttp in non-hpcs in regulating inflammation. among various non-hpc cell types in the lung, alveolar epithelial cells produce pro-inflammatory cytokines and chemokines ( ) . in in vitro conditions, lpschallenged pulmonary type ii epithelial cells have been shown to produce greater levels of neutrophil chemoattractants [il- , a human homolog for cxcl (kc), cxcl (mip ), and cxcl (lix)] indicating that ttp in lung alveolar epithelial cells may play an important role in regulating ali ( ) . consistent with these reports, while our data indicate critical roles of non-hpcs in lungs in mediating neutrophil recruitment to the airspaces, the exact identity of these non-hpcs remain unknown. in conclusion, our results show that (a) enhancing the levels of ttp is protective against endotoxin-induced ali; (b) the protective effect seen in ttp are mice is attributable to reduced neutrophilic recruitment and, in turn, reduced lung damage; (c) reduced neutrophilic recruitment is attributed to reduced secretion of chemoattractants, particularly kc and g-csf; and that, (d) ttp in non-hpcs plays an essential role in protection against endotoxin-induced ali. based on these results, we propose a model in which endotoxin damages epithelial cells within the lung, which then initiates a cascade of events leading to exaggerated release of proinflammatory mediators and neutrophilic chemoattractants, resulting in further lung damage and neutrophilic infiltration (figure ) . in this process, ttp acts as an intracellular regulator for the expression of proinflammatory mediators and neutrophilic chemoattractants. ttp expression in the non-hpcs, i.e., most likely epithelial and endothelial cells, confers protection against endotoxin-induced ali via suppression of mrnas encoding proinflammatory mediators and neutrophilic chemoattractants. hence, strategies to increase ttp expression or activity in non-hpcs together with hpcs population may prove beneficial for patients with ali/ards. in future studies, ttp expression within the lung could also be investigated as a prognostic indicator for the severity of ali/ards. our studies could also have implications for the lung hyper-inflammation and potentially life-threatening cytokine storms in the severe coronavirus disease (covid- ). the raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. the animal study was reviewed and approved by lsu iacuc. sp conceived and designed the study, maintained the animal colony, and performed histopathological analyses. ic, tv, and bl conducted animal necropsies. ys, cb, and rl performed balf cellularity assays. rl performed the intravenous injections. ys, tv, ic, and cb performed cytokine assays. jl performed the irradiations. sj provided technical expertise on bone marrow transplantations and reviewed the manuscript. pb provided various transgenic and knockout mice and edited the manuscript. sp and ys wrote and reviewed the manuscript for intellectual contents. all authors contributed to the article and approved the submitted version. the acute respiratory distress syndrome: 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murine chemokine kc il- ) in the bronchoalveolar lavage fluid from patients with the adult respiratory distress syndrome (ards) and patients at risk for ards interleukin -related neutrophil elastase and the severity of the adult respiratory distress syndrome increased interleukin- concentrations in the pulmonary edema fluid of patients with acute respiratory distress syndrome from sepsis elevated levels of nap- /interleukin- are present in the airspaces of patients with the adult respiratory distress syndrome and are associated with increased mortality alveolar granulocyte colony-stimulating factor and alpha-chemokines in relation to serum levels, pulmonary neutrophilia, and severity of lung injury in ards plasma granulocyte colony-stimulating factor levels correlate with clinical outcomes in patients with acute lung injury effects of granulocyte colony-stimulating factor (g-csf) on bleomycin-induced lung injury of varying severity deletion of tristetraprolin caused spontaneous reactive granulopoiesis by a non-cellautonomous mechanism without disturbing long-term hematopoietic stem cell quiescence tristetraprolin targets nos expression in the colonic epithelium differential regulation of cytokine release and leukocyte migration by lipopolysaccharide-stimulated primary human lung alveolar type ii epithelial cells and macrophages we thank thaya stoufflet for assistance with multiplex cytokine assays, sherry ring for histological tissue processing, and deborah stumpo for helping with the ttp mutant mice and their genotyping. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/ . /fimmu. . /full#supplementary-material conflict of interest: the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © choudhary, vo, bathula, lamichhane, lewis, looper, jeyaseelan, blackshear, saini and patial. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -iiw w authors: ding, xibing; jin, shuqing; tong, yao; jiang, xi; chen, zhixia; mei, shuya; zhang, liming; billiar, timothy r.; li, quan title: tlr signaling induces tlr up-regulation in alveolar macrophages during acute lung injury date: - - journal: sci rep doi: . /srep sha: doc_id: cord_uid: iiw w acute lung injury is a life-threatening inflammatory response caused by severe infection. toll-like receptors in alveolar macrophages (amΦ) recognize the molecular constituents of pathogens and activate the host’s innate immune responses. numerous studies have documented the importance of tlr-tlr cross talk, but few studies have specifically addressed the relationship between tlr and tlr . we explored a novel mechanism of tlr up-regulation that is induced by lps-tlr signaling in a dose- and time-dependent manner in amΦ from c bl/ mice, while the lps-induced tlr expression was significantly reduced in tlr (−/−) and myd (−/−) mice and following pretreatment with a nf-κb inhibitor. the enhanced tlr up-regulation in amΦ augmented the expression of cytokines and chemokines in response to sequential challenges with lps and poly i:c, a tlr ligand, which was physiologically associated with amplified amΦ-induced pmn migration into lung alveoli. our study demonstrates that the synergistic effect between tlr and tlr in macrophages is an important determinant in acute lung injury and, more importantly, that tlr up-regulation is dependent on tlr -myd -nf-κb signaling. these results raise the possibility that bacterial infections can induce sensitivity to viral infections, which may have important implications for the therapeutic manipulation of the innate immune system. mouse amΦ were isolated from the balf of wild-type (wt) mice and stimulated with lps ( , . , . , , or μ g/ml) in dmem containing % fbs for h. the graphed values represent the mean ± sem. five mice were analyzed per group. (c,d) mouse amΦ were isolated from the balf of wt mice and stimulated with lps ( μ g/ml) in dmem containing % fbs for - h. the graphed values represent the mean ± sem. five mice were analyzed per group. (a,c) a western blot of tlr protein expression in amΦ . actin expression was identified to normalize the densitometry of tlr expression. (b,d) rt-pcr analysis was used to evaluate tlr mrna expression in amΦ . β -actin mrna was detected for normalizing the value of tlr mrna. (e,f) qrt-pcr analysis was used to evaluate tlr mrna expression in amΦ . *p < . compared with the other groups. immunity against some viral infections. numerous studies have documented the importance of tlr cross talk, and multiple signaling pathways contribute to synergistic tlr ligand-dependent cytokine expression. in , alexopoulou l. et al. reported in nature that following an intraperitoneal injection of lps into mice, dramatic up-regulation of the expression of tlr mrna was observed in all tissues except the thymus, suggesting that the nuclei are stained with dapi (blue). the images were acquired using evosfl fluorescence microscopy. (d) qrt-pcr analysis was used to evaluate tlr mrna expression in amΦ . *p < . compared with the other groups; **p < . compared with the other groups. the expression of tlr is inducible. of note, a particularly high expression level of tlr mrna was observed in lung tissue. these results raised the possibility that bacterial infection can induce sensitivity to viral infection. this observation prompted us to further investigate the mechanism of tlr -tlr cross talk in amΦ . in the present study, using both an in vivo ali mouse model and the in vitro culture of amΦ from tlr −/− and myd −/− mice, we demonstrate that lps up-regulation of tlr in amΦ is dependent on tlr -myd -nf-κ b signaling. the functional relevance of the amplification in tlr -induced tlr expression in amΦ was demonstrated by a marked increase in the expression of the chemokines and cytokines, including macrophage inflammatory protein- (mip- ), macrophage chemoattractant protein- (mcp- ), tumor necrosis factor-alpha (tnf-α ), and interleukin- (il- ), in the ali mouse model. furthermore, we observed alveolar-capillary permeability and the induction of polymorphonuclear leukocyte (pmn) migration in response to sequential challenges with lps and poly i:c. thus, tlr up-regulation in amΦ is dependent on tlr -myd -nf-κ b signaling, which may represent an important mechanism responsible for amplifying pmn migration. to detect the expression of tlr in lps-induced amΦ , which were isolated from the bronchoalveolar lavage fluid (balf) of wild-type (wt) mice, lps was administered at different dosages ( , . , . , , and μ g/ml). tlr expression increased with . μ g/ml but was markedly increased following treatment with μ g/ml lps, and the increase in tlr expression trended back to the basal level at μ g/ml lps (fig. a,b ). as shown in fig. c , a μ g/ml lps challenge of amΦ from wt mice was associated with an increase in tlr protein expression at h and a more marked increase at h, while the increase in tlr expression trended back to the basal level at h. the tlr mrna expression changes shown in fig. d paralleled the protein expression changes. in addition, we obtained the same results using quantitative pcr (fig. e,f) . these results suggest that lps, a tlr ligand, can induce tlr expression in amΦ . lps up-regulates tlr expression in amΦ through tlr -myd -nf-κb signaling. as shown in fig. a , lps ( μ g/ml) challenge of amΦ , which were isolated from wt mice, resulted in an increase in tlr protein expression at h and a marked increase at h. however, in amΦ from tlr −/− mice, lps failed to induce tlr expression ( fig. a) , indicating that tlr signaling mediates the lps-induced up-regulation of tlr . all tlrs, with the exception of tlr , utilize myd , and tlr /myd signaling pathway is the primary signaling pathway used to induce the expression of pro-inflammatory cytokines . however, tlr can signal through both myd -dependent and -independent pathways, and the myd -independent signaling for tlr results in the production of type i ifns . to address the role of myd in mediating the lps-tlr -induced up-regulation of tlr , lps ( μ g/ml) was administered to amΦ from myd −/− mice, and tlr expression was assessed. as shown in fig. a , myd deficiency prevented lps-induced up-regulation of tlr . the changes in the mrna expression of tlr are shown in fig. b , and these results corresponded to the observed changes in protein expression. as shown in fig. c , the localization of tlr was further investigated in amΦ by immunofluorescence. in the absence of lps, tlr was found within small vesicles throughout the cytoplasm. tlr -containing vesicles increased in a time-dependent manner in amΦ from wt mice, but not in tlr −/− or myd −/− mice, and similar results were observed using quantitative pcr (fig. d) . can occur in the presence of myd . nf-κ b p was detected using nuclear protein extracts from amΦ to determine whether lps-tlr up-regulation of tlr was the result of activation of nf-κ b. as shown in fig. a , in response to lps in wt mice, the expression of nf-κ b p increased at . h and reached a significant level at . h. however, in tlr −/− mice, lps failed to induce nuclear nf-κ b p expression. to address the role of nf-κ b in mediating the lps-induced up-regulation of tlr , we determined the effects of ikk-nbd, an nf-κ b inhibitor , , on lps-induced tlr expression in amΦ . as shown in fig. b ,c, ikk-nbd ( μ m) prevented the lps-induced up-regulation of tlr mrna and protein expression in amΦ isolated from wt mice. we observed similar results by quantitative pcr (fig. d) . these results demonstrate that nf-κ b signaling plays a role in mediating tlr -tlr cross talk. to address the physiological relevance of lps/tlr activation and tlr expression in amΦ , we assessed mip- expression in the lungs using sequential challenges of lps and poly i:c. injection of lps at time followed by a saline injection (group ) at h resulted in a marked increase in mip- expression by h after lps challenge, which was further increased at h and then returned to the basal level by h after lps challenge (fig. a) . saline injection at time and poly i:c injection ( μ g/g body weight, intratracheally administered) (group ) at h caused a very small increase in mip- expression. however, the sequential injection of lps at time and poly the results show the effects of sequential challenges with lps and poly i:c on mip- expression in the lungs. in wt mice, lps ( μ g/g) or saline (sal) was injected intratracheally at time h, and hours later, poly i:c ( μ g/g) or sal was injected. in tlr −/− and tlr −/− mice, lps was injected intratracheally ( μ g/g) at time h, and poly i:c ( μ g/g) was injected intratracheally at h. lung tissue was harvested at the indicated times, and mip- protein expression was detected using western blotting (a,c), with actin being used to normalize the densitometry of mip- expression. additionally, mip- mrna expression was detected using rt-pcr (b), and qrt-pcr was used to detect mip- mrna expression in amΦ (d). β -actin mrna was used to normalize the value of mip- mrna expression. the graphed values represent the mean ± sem. five mice were analyzed per group. **p < . compared with the other groups; ***p < . compared with the other groups. because amΦ production of chemokines, such as mip- , is an important determinant of pmn migration, we also addressed the role of lps/tlr -mediated up-regulation of tlr in regulating pmn migration in the lungs. pmns were counted in the balf of wt, tlr −/− , and tlr −/− mice. in the lps (group ) and poly i:c (group ) only injection groups, mip- caused a slight increase in pmn migration in wt mice when compared with the saline control (group ). sequential injection of poly i:c at h after the initial lps injection (group ) markedly increased pmn migration in wt mice compared with the other groups. however, sequential challenge with lps and poly i:c did not significantly increase pmn migration in tlr −/− mice compared with wt mice (fig. ) . moreover, there was a similar increase in amΦ after sequential challenge with lps and poly i:c in tlr −/− mice (group ) when compared to lps alone (group ). additionally, the observed mip- expression level in the lung was consistent with pmn migration. together, these data show the important role of tlr signaling and amΦ mip- expression in mediating pmn migration in response to the up-regulation of tlr expression. to address the effect of lps/tlr -mediated activation of tlr in amΦ on inflammatory cytokines, we assessed tnf-α and il- in the serum and balf, as well as the chemokines mip- and mcp- in the balf, following sequential intratracheal challenges with lps and poly i:c. in the ali animal model, lps was injected intratracheally at time , and poly i:c was injected intratracheally at h (the time point when tlr was up-regulated in amΦ as described above). enzyme-linked immunosorbent assays (elisas) were used to assess mip- and mcp- in the balf as well as il- and tnf-α in both the balf and serum at h. lps (group ) or poly i:c (group ) alone induced a slight increase in mip- , mcp- , il- , and tnf-α when compared with the saline control (group ) (fig. ) , whereas sequential challenge with lps and poly i:c (group ) stimulated a marked increase in mip- , mcp- , il- , and tnf-α expression. however, the sequential challenge with lps and poly i:c did not significantly increase mip- , mcp- , il- , and tnf-α in tlr −/− mice (group ) when compared to wt mice (group ). the sequential challenge with lps and poly i:c also led to a similar increase in tlr −/− mice (group ) when compared to the lps alone group (group ) (fig. ) . because chemokine-dependent pmn migration and inflammatory cytokine secretion are important determinants of ali, we next addressed the role of lps/tlr -mediated up-regulation of tlr in alveolar-capillary permeability using evan's blue, which binds albumin, in wt, tlr −/− , and tlr −/− mice. we assessed permeability at , , , and h after lps/poly i:c administration. as shown in the results show the effects of sequential challenges with lps and poly i:c on pmn migration into the airspaces. in wt mice, lps ( μ g/g) or saline (sal) was injected intratracheally at time h, and h later, poly i:c ( μ g/g) or sal was injected. in tlr −/− and tlr −/− mice, lps ( μ g/g) was injected at time h, and poly i:c ( μ g/g) was injected intratracheally at h. pmn numbers in balf were counted at the times indicated. the graphed values represent the mean ± sem. five mice were analyzed per group. **p < . ; ***p < . compared between the groups as indicated. scientific reports | : | doi: . /srep weight, intratracheally administered) at h resulted in minimal changes (group ). however, the sequential injection of lps at time and poly i:c injection at h (at a time when tlr was up-regulated) in wt mice (group ) led to augmented changes in histopathology, including diffuse interstitial edema and inflammatory cell infiltration, compared with the single lps or poly i:c challenge. in contrast, the sequential injection of poly i:c at h after lps failed to induce augmented histopathology changes in tlr −/− mice (group ) compared with wt mice. in addition, we did not observe augmented histopathology changes in tlr mice (group ) and instead observed a change similar to that observed for lps alone (group ). taken together, these data show the important role of lps/tlr signaling in the up-regulation of tlr expression in ali. amΦ , which are located in the alveolar compartment of the lungs, provide a key initiation signal for ali [ ] [ ] [ ] . previous studies have shown that the later phase of ali is neutrophil-dependent , while amΦ contribute to the acute phase of lung injury. once activated by bacteria or viruses, amΦ generate and release a multitude of mediators, such as cytokines (il- , tnf-α ) and chemokines (mcp- , mip- ) , . these mediators act as chemoattractants for the migration of large numbers of activated inflammatory cells, such as monocytes and neutrophils, into the airspaces . synergy between viral and bacterial tlr signaling, which leads to amplification of the inflammatory response, has been reported previously . additionally, tian x. et al. found that poly i:c enhanced susceptibility to secondary pulmonary infections by bacteria . co-stimulation with lps and poly i:c markedly enhances the immune response, although the mechanism for this combined effect remains poorly understood. alexopoulou l. et al. found that when lps was injected intraperitoneally, tlr expression in lung tissue was dramatically up-regulated, which suggests that the expression of tlr is inducible. pan et al. provided direct evidence that lps induces tlr expression via a tlr -myd -irak-traf -nf-κ b-dependent signaling pathway in human the results show the effects of sequential challenges with lps and poly i:c on alveolar-capillary permeability and histological changes in the lung. in wt mice, lps ( μ g/g) or saline (sal) was injected intratracheally at time h, and h later, poly i:c ( μ g/g) or sal was injected. in tlr −/− and tlr −/− mice, lps ( μ g/g) was injected at h, and poly i:c ( μ g/g) was injected intratracheally at h. lung tissue and serum were harvested at the times indicated, and alveolar-capillary permeability was detected by using evan's blue staining (a). lung tissue was collected at the time indicated and subjected to hematoxylin and eosin (h&e) staining (x magnification) (b). **p < . ; ***p < . compared between the groups as indicated. scientific reports | : | doi: . /srep peripheral blood monocytes and monocytic cell lines, such as thp- cells. furthermore, we observed that lps could induce tlr expression in a dose-and time-dependent manner in amΦ . the stimulation of tlr by lps induces the release of critical proinflammatory cytokines that are necessary to activate a potent immune response. indeed, lps/tlr signaling has been intensively studied in recent years [ ] [ ] [ ] . in our study, the role of tlr signaling in regulating tlr expression was clearly shown using tlr −/− mice. lps challenge in wt mice induced the up-regulation of tlr , whereas this response was impaired in tlr −/− mice. the major adaptor molecules that bind to the intracellular domain of tlr are myd and trif , , and our results showed that myd mediated the tlr -tlr cross talk, as lps challenge of myd −/− mice failed to induce tlr expression. furthermore, the activation of nf-κ b was associated with an increase in tlr expression after lps challenge and a reduced expression of tlr in amΦ in which nf-κ b was inhibited by ikk-nbd. therefore, these results demonstrate an important role for tlr -myd -nf-κ b in mediating tlr expression. ali is caused by an uncontrolled systemic inflammatory response that results from direct (aspiration, pneumonia, ventilation-induced lung injury, etc.) or indirect injury (sepsis, hemorrhagic shock, etc.), leading to the activation of amΦ and the sequestration of neutrophils . excessive recruitment of leukocytes is critical to the pathogenesis of ali. neutrophils are the first leukocytes to be recruited to sites of inflammation in response to chemokines released by activated amΦ . specifically, stimulation of amΦ leads to the release of chemokines, which induce neutrophils to migrate from the intravascular space across the endothelium and epithelium into the airspaces . according to the relative position of the cysteine residues, chemokines have been classified into four subfamilies (cxc, cc, c, and cx c). among these, mip- , which is also known as cxcl , is thought to play a major role in mediating neutrophil recruitment . belperio j.a. et al. found that lung expression of mip- was correlated with lung injury and neutrophil sequestration during the pathogenesis of ventilation-induced lung injury, and cxcr −/− mice show a marked reduction in neutrophil sequestration and lung injury. additionally, mip- was shown to be up-regulated in the lungs and balf of animals, which was associated with neutrophil accumulation in the lungs after lps administration [ ] [ ] [ ] . furthermore, villar j. et al. showed that a cxcl polymorphism is associated with better outcomes in patients with severe sepsis . in the present study, we observed that lps/tlr signaling up-regulated tlr expression in amΦ . this cross talk between tlr and tlr in amΦ resulted in the amplification of cytokine (il- , tnf-α ) and chemokine (mip- , mcp- ) expression in response to lps and poly i:c, which activate tlr and tlr , respectively, and subsequently led to enhanced pmn sequestration into the lung, which was found to be correlated with ali based on the assessment of alveolar-capillary permeability and histological sections of lung tissue. thus, the present study demonstrates a novel mechanism by which lps can induce amΦ to up-regulate tlr expression, through a tlr -myd -nf-κ b-dependent pathway, thereby sensitizing amΦ to tlr ligands and promoting enhanced lung inflammation (fig. ) . in clinical scenarios, a variety of pathogens are involved in lung infections. in particular, viral infections are a significant risk factor for acquiring bacterial infections , . tlr is thought to be a major mediator of the cellular response to viral infection, because it responds to dsrna, a common by-product of viral replication . tlr has been implicated in infections by mouse cytomegalovirus (mcmv), reoviruses, lymphocytic choriomeningitis virus (lcmv), and influenza a virus (iav) . tlr −/− mice are more resistant to lethal west nile virus, a mosquito-borne ssrna flavivirus, which causes human disease of variable severity , and show reduced inflammation and lethality upon iav infection , . thus, up-regulation of tlr may contribute to an increased lung immune response to viral infection following a bacterial infection. despite the relatively well-known role of viral infections in promoting bacterial infections, it is still not clear whether bacterial infections also promote viral infections. therefore, our results are the first to offer new insight into this topic. there is also a growing body of evidence showing that most patients have a mixture of bacterial and viral infection , , and the cross talk between tlr and tlr induces a synergistic inflammatory response in cases of mixed infection. cameron r.j. et al. found that bacterial and viral pathogens interact to cause additional increases in inflammatory markers and an exacerbation of disease severity in patients with chronic obstructive pulmonary disease (copd). tlr has been shown to respond to dsrna, a replication intermediary for many viruses, but the heterologous rna released from necrotic cells or that is generated by in vitro transcription, such as mrna, also stimulates tlr signaling and induces immune activation. indeed, cavassani ka observed tlr activation during experimental polymicrobial sepsis and ischemia gut injury in the absence of an exogenous viral stimulus, and tlr −/− mice were protected from the lethal effects of sustained inflammation. moreover, treatment with a tlr antibody could attenuate the tissue injury associated with gut ischemia and significantly decrease sepsis-induced mortality. therefore, tlr serves as a regulator of the amplification of the immune response as well as an endogenous sensor of necrosis, independent of viral activation. our study showed that up-regulation of tlr significantly amplified il- , tnf-α , mcp- , and mip- expression, which then enhanced pmn migration and finally led to ali. tlr cross talk has obvious advantages for the host in protecting against infectious agents, because it enhances the initial immune reaction to pathogen infection and better primes the host for mounting a more robust adaptive immune response. the concept that multiple tlr-ligand interactions are required for effective host resistance to pathogens has important implications for the design of vaccinations and immunotherapies against infectious diseases. several studies have convincingly shown the improved efficacy of treatment with multiple tlr ligands compared with single tlr ligands in stimulating cellular immune responses in vivo. for example, co-administration of poly i:c and cpg odns increased serum cytokine production in a mouse tumor model when compared to the administration of either of the ligands alone . bone-marrow-derived dcs exposed to both poly i:c and a tlr ligand more effectively stimulated cytotoxic t lymphocyte responses compared to dcs exposed to either tlr ligand alone . these studies provide an important conceptual foundation to examine the protective efficacy of multiple tlr-ligand combinations in vaccines against infectious diseases. however, synergistic amplification of the inflammation response may be detrimental, because it can result in immune over-activation, which hampers immune homeostasis. as a consequence of an overactive response, the function of various organ systems may be compromised, resulting in multiple organ dysfunction syndrome (mods) and death. cytokines (tnf-α and il- ) are important components of the immune system because they act as messengers between cells, but they are also involved in many pathological aspects of the cascade leading to systemic inflammatory response syndrome (sirs) and ultimately mods. according to the two-hit hypothesis , patients who survive the initial inflammatory insult may die following a relatively minor second event that would not normally be life-threatening. in this study, viral (poly i:c) stimulation as a relatively minor secondary insult, led to an exaggerated secondary inflammatory response. thus, understanding the mechanism of this two-hit phenomenon may help to devise novel therapeutic strategies to prevent overwhelming and life-threatening inflammatory conditions such as septic shock and trauma-induced sirs. animals. male c bl/ wild type mice were purchased from the laboratory animal research center of shanghai. tlr knockout (tlr −/− ) mice, myd knockout (myd −/− ) mice and tlr knockout (tlr −/− ) were obtained from dr. billiar's lab at the university of pittsburgh. all mice used are on a c bl/ background. all experimental protocols involving animals were approved by institution animals care and use committee of tongji and pittsburgh university. the experiments were performed in accordance with the national institutes of health guidelines for the use of laboratory animals. mice were - weeks of age at the time of experiments and were maintained on standard rodent chow and water ad libitum. animals were anesthetized with mg/ kg ketamine and mg/kg xylazine administered intraperitoneally. animals were intratracheally administered lps ( μ g/g body wt; escherichia coli o :b ; sigma, st. louis, mo) in μ l of saline (sal) or sal at first time, then after h intratracheally administered poly i:c ( μ g/g body wt; - - ; invivogen) in μ l of saline (sal) or sal alone at the second time using a microspray syringe. the animals were randomly in one of four groups: sal/sal, sal/poly i:c, lps/sal, and lps/poly i:c. at various time points, a g sterile catheter was inserted into the trachea to collect bronchoalveolar lavage fluid (balf) as previously described . blood samples were immediately obtained by cardiac puncture and transferred to the laboratory for analysis of cytokines (il- , tnf-α ), chemokines (mip- , mcp- ) in serum and balf by elisa. pmn counts in balf were determined on wright-giemsa-stained slides. briefly, total cell counts were determined on a grid hemocytometer. then a total of cells were counted in cross-section per sample, and the number of pmns was calculated as the total cell count times the percentage in the balf sample. the lungs were rapidly removed from all mice and washed in ice-cold saline. half of the lung tissues were stored at − °c prior to biochemical analyses including mip- expression in lung lysates was measured by western blotting and rt-pcr, while the other half of the lung tissue was fixed in % formalin solution in preparation for histopathological analyses. for histological analysis, lung tissue samples were fixed in % paraformaldehyde in pbs overnight at °c. the samples were then dehydrated, embedded in paraffin, and cut into μ m sections. after deparaffinization, the tissues were stained with hematoxylin and eosin (h&e) for histological analysis. lung sections were scored for lung injury, including the following: ( ) alveolar and capillary edema, ( ) intravascular and peri-bronchial influx of inflammatory cells, ( ) thickness of the alveolar wall, and ( ) hemorrhage. the items were semiquantitatively scored as none, minimal, light, moderate, or severe (score , scientific reports | : | doi: . /srep , , or , respectively) by a pathologist blinded to the experimental group. the lung injury score was obtained by averaging the score from the animals within each group. amΦ isolation. bal was performed as previously described . normally, the bal fluid contains ~ % of amΦ and ~ % of other cells, including pmn and lymphocytes. the immunomagnetic separation system was used to isolate amΦ . magnetic nanoparticle-conjugated antibody (cd b microbeads, miltenyi biotec) was chosen to label and remove pmn and lymphocytes. the resulting cells consisted of > % macrophages, and cell viability was > %. amΦ from wild type, tlr −/− , tlr −/− and myd −/− mice were cultured in dmem containing % fbs and μ l/ml penicillin/streptomycin for days, then they were washed three times with pbs and the medium was changed to low-serum medium ( % fbs). cells were stimulated with lps (nc, . , . , , μ g/ml) for - h in dmem containing % fbs at a concentration of × cells/ml of medium. tlr expression in the amΦ lysates was measured by western blotting and rt-pcr. evans blue albumin (eba) as previous described , . evans blue ( . % eb, sigma-aldrich, st louis, mo, usa) was dissolved in ca + /mg + -free phosphate-buffered saline (pbs; sigma-aldrich), and conjugated to albumin ( % eba) that was prepared by adding bovine serum albumin (sigma-aldrich). after thoroughly dissolving by gently stirring with a magnetic bar, the eba solution was filtered through a . μ m syringe filter and aliquots were stored at − °c until use. each aliquot was used only once for each experiment. to evaluate alveolar-capillary barrier function, eba ( mg/kg body weight) was injected into the internal jugular vein h before euthanasia and lung harvesting. blood samples were obtained from the right heart, and the pulmonary vasculature was subsequently infused with ml pbs. the right lung was ligated at the level of the right mainstem bronchus, excised, blotted dry, weighed and stored in liquid nitrogen until these samples were used for eba analysis. after freeze/thaw, the lung tissue was homogenized in ml pbs and incubated with an additional ml of formamide (sigma-aldrich) ( h; °c). formamide extracts were centrifuged ( , g × min; °c), and the centrifuged supernatants were collected to quantify lung eba content using a dual-wavelength ( nm and nm) spectrophotometric method. pulmonary eba absorbance at nm was corrected by a correction factor with eba absorbance at nm. the eba permeability index was calculated by dividing pulmonary eba absorbance at nm/g of lung tissue by plasma eba absorbance at nm. nuclear protein extraction. nuclear protein extracts were prepared from amΦ following the kit instructions of thermo scientific (ne-per nuclear and cytoplasmic extraction reagents, , thermo fisher). amΦ were harvested with trypsin-edta and then centrifuge at × g for minutes. after washing cells twice with pbs, pellet by centrifugation at × g for minutes and discarded the supernatant. adding cer i (protease inhibitors and pmsf) to the cell pellet, vortex the tube vigorously for s, then incubated the tube on ice for minutes. added cer ii to the tube and vortex for s on the highest setting, incubated the tube on ice for minute. the tube was centrifuged for minutes at , × g, then transferred the supernatant (cytoplasmic extract) to a clean pre-chilled tube. nuclei pellet was suspended in ner and vortex for s. placed the sample on ice and continued vortexing for s every minutes, for a total of minutes. after centrifuged at , × g for minutes, supernatants containing nuclear proteins were frozen in liquid nitrogen in small aliquots and store at − °c. protein quantification was performed using bca protein assay. western blot analysis. amΦ and lung tissues were lysated in radio-immunoprecipitation lysis buffer (ripa), protease inhibitors (roche, mannheim, germany) and phenylmethylsulfonyl fluoride (pmsf). protein concentrations were subsequently determined by standard bca assay. after addition of × sodium dodecyl sulfate (sds) loading buffer, equivalent amounts of protein were heated ( °c; min) and separated by gel electrophoresis using a % sds-polyacrylamide electrophoresis gel. resolved proteins were then transferred to a nitrocellulose membrane and blocked with tris-buffered saline containing tween- (tbst) and % nonfat milk ( h; °c). nitrocellulose membranes were incubated overnight at °c with primary antibody against tlr (ab ; abcam, hong kong, china), nf-κ b p (ab , abcam, hong kong, china), abcam, mip- (ab , hong kong, china), pcna (ab , abcam, hong kong, china) and β -actin (ab , abcam, hong kong, china). the membranes were washed in tbst three times, incubated with secondary antibody ( - irdye mouse-anti-rabbit secondary antibody; licor biosciences, lincoln, ne, usa) for h at °c and then washed in tbst three additional times. the membranes were determined by using an odyssey image analysis system (licor biosciences). western blots were quantitated using quantity one software (bio-rad, foster city, ca, usa) and normalized to β -actin and pcna signal. total rna was extracted from amΦ and lung tissues using the trizol reagent (sigma-aldrich) and following the manufacturer's instructions. total rna was then reverse transcribed using a primescript rt reagent kit (takara bio inc. shiga, japan). primers for tlr amplification ( bp) were: position forward ′ -gtgagatacaacgtagctgactg- ′ , position reverse ′ -tcctgcatccaagatagcaagt- ′ . primers for mip- amplification ( bp) were: position forward ′ -ccaaccaccaggctacagg- ′ , position reverse ′ -gcgtcacactcaagctctg- ′ . primers for β -actin amplification ( bp) were: position forward ′ -ggctgtattcccctccatcg- ′ , position reverse ′ -ccagttggtaacaatgccatgt- ′ . the product of reverse transcription was amplified by following the premix taq version . instructions (takara bio inc.). pcr products were separated using a % agarose gel and identified by sybr green staining. expression of mrna was quantitated using image lab software (bio-rad) and normalized to the β -actin signal. scientific reports | : | doi: . /srep quantitative real-time pcr. quantitative real-time pcr (qpcr) reactions were performed using fast sybr green master mix (thermo fisher) in applied biosystems ht fast real-time pcr system according to the manufacturer's instructions. the cycling conditions were °c for min followed by cycles of °c for s, °c for s. at the end of the last cycle, the temperature was increased from °c to °c ( . °c/s) to produce a melting curve. the specificity of amplification was assessed for each sample by melting curve analysis. each pcr product showed a single peak. the size of the amplicon was checked by electrophoresis. agarose gel electrophoresis revealed a single product of the expected size. the analysis of the expression of tlr relative to the β -actin was performed with software in relative quantification mode following the manufacturer's instruction. the following criteria of sequences of all primers used in this study were applied in the course of designing the primers: product size from to bp, primer size from to bp, and a mean melting temperature of °c. immunofluorescence staining of cells and florescence microscopy. amΦ were cultured for a defined time period, fixed in % paraformaldehye/ × pbs for min. cells were washed three timers with × pbs and permeabilized using . % triton x- in × pbs, and blocked with % bsa for min and sequentially administered primary antibody and secondary antibody (alexa- -conjugated donkey anti rabbit secondary antibody). nuclei were stained with dapi for immunofluoresence analysis. stained cells were examined and recorded using evosfl fluorescence microscopy. statistics. the data are presented as the means ± sem of the indicated number of experiments and analyzed using anova; post hoc testing was performed using the bonferroni modification of the t-test. the individual studies performed throughout this work represent at least five independent studies. power analyses were performed by using a type i error probability of . , with a power of . , to determine the sample size necessary to reject the null hypothesis. all statistical analyses were carried out using the graphpad prism program. has mortality from acute respiratory distress syndrome decreased over time? a systematic review the acute respiratory distress syndrome: pathogenesis and treatment role of chemokines in the pathogenesis of acute lung injury hemorrhagic shock-activated neutrophils augment tlr signalinginduced tlr upregulation in alveolar macrophages: role in hemorrhage-primed lung inflammation tlr-signaling networks: an integration of adaptor molecules, kinases, and cross-talk toll-like receptor signaling pathways the macrophage response towards lps and its control through the p (mapk)-stat axis toll-like receptors and innate immunity tlr : interferon induction by double-stranded rna including poly(i:c) the toll-like receptor :dsrna signaling complex beyond dsrna: toll-like receptor signalling in rna-induced immune responses establishment of monoclonal antibody against human toll-like receptor that blocks double-stranded rna-mediated signaling subcellular localization of toll-like receptor in human dendritic cells mrna is an endogenous ligand for toll-like receptor tlr is an endogenous sensor of tissue necrosis during acute inflammatory events recognition of double-stranded rna and activation of nf-kappab by toll-like receptor cytomegalovirus enhances macrophage tlr expression and myd -mediated signal transduction to potentiate inducible inflammatory responses type i interferon signaling contributes to the bias that tolllike receptor exhibits for signaling mediated by the adaptor protein trif selective inhibition of nf-kappab activation by a peptide that blocks the interaction of nemo with the ikappab kinase complex tlr signaling induces tlr expression in endothelial cells via neutrophil nadph oxidase alveolar macrophage activation is a key initiation signal for acute lung ischemia-reperfusion injury diverse macrophage populations mediate acute lung inflammation and resolution role of resident alveolar macrophages in leukocyte traffic into the alveolar air space of intact mice neutrophils and acute lung injury role of alveolar macrophages in the inflammatory response after trauma role of alveolar macrophage and migrating neutrophils in hemorrhage-induced priming for ali subsequent to septic challenge the chemokine system in diverse forms of macrophage activation and polarization synergy between viral and bacterial toll-like receptors leads to amplification of inflammatory responses and preterm labor in the mouse poly i:c enhances susceptibility to secondary pulmonary infections by gram-positive bacteria bacterial lps up-regulated tlr expression is critical for antiviral response in human monocytes: evidence for negative regulation by cyld lps/tlr signal transduction pathway exploring the lps/tlr signal pathway with small molecules recognition of lipopolysaccharide pattern by tlr complexes myd : a central player in innate immune signaling. f prime rep beyond myd and trif pathways in toll-like receptor signaling the mercurial nature of neutrophils: still an enigma in ards? tnfalpha and mip- : role in particle-induced inflammation and regulation by oxidative stress critical role for cxcr and cxcr ligands during the pathogenesis of ventilator-induced lung injury syndecan- shedding facilitates the resolution of neutrophilic inflammation by removing sequestered cxc chemokines darc on rbc limits lung injury by balancing compartmental distribution of cxc chemokines role for macrophage inflammatory protein- in lipopolysaccharideinduced lung injury in rats a cxcl polymorphism is associated with better outcomes in patients with severe sepsis respiratory syncytial virus and staphylococcus aureus coinfection in children hospitalized with pneumonia incidence and characteristics of viral community-acquired pneumonia in adults transcriptional signaling by double-stranded rna: role of tlr toll-like receptor mediates west nile virus entry into the brain causing lethal encephalitis involvement of toll-like receptor in the immune response of lung epithelial cells to double-stranded rna and influenza a virus detrimental contribution of the toll-like receptor (tlr) to influenza a virus-induced acute pneumonia virus infection in exacerbations of chronic obstructive pulmonary disease requiring ventilation mixed bacterial-viral infections in septic children with leukemia synergistic activation of innate immunity by double-stranded rna and cpg dna promotes enhanced antitumor activity synergistic activation of dendritic cells by combined toll-like receptor ligation induces superior ctl responses in vivo modeling the two-hit hypothesis for evaluating strategies to prevent organ injury after shock/resuscitation rgd peptides protects against acute lung injury in septic mice through wisp -integrin β pathway inhibition wnt -inducible signaling pathway protein contributes to ventilator-induced lung injury competing financial interests: the authors declare no competing financial interests. key: cord- -l n is authors: nan title: poster sessions - date: - - journal: intensive care med doi: . /s - - - sha: doc_id: cord_uid: l n is nan total protein concentration in bal increased significantly and led to peak at ± mg/ml hour after intubations . mucin concentration was highest at hour after ventilation ( . ± . mg/ml). bal sp-a concentration ratio increased about times after hour ventilation. compare to mg/ml total protein, the ratio was . ± . in hour later, and . ± . in hours after ventilation.the change of bal wbc level led to peak in after hour ventilation, but blood wbc level led to peak in hours later. for elastase level both peak were hours later in bal and blood.in the caller components of bal, the neutrophyl cells were dominant in hour after intubation, but hours after ventilation, mast cells with phagocyted mucine and dusts were dominant. just introduction. coronary disease is prevalent in diabetic patients resulting in a frequency of invasive cardiac procedures four times that of non-diabetics. after cardiac surgery diabetics have twice the mortality and morbidity in early and late phases after operation. the reasons for this increased risk are poorly understood. diabetics exhibit complex abnormalities of lung structure and of the control of the cardiorespiratory system. these include pulmonary micro-vascular disease, autonomic neuropathy associated with an increased cardiovascular instability, an increased incidence of central and obstructive sleep apnoea and a reduced response to hypercapnia. this study was undertaken to determine whether at risk diabetic patients could be identified pre-operatively. methods. patients awaiting urgent cardiac surgical re-vascularisation were studied with measurement of: spirometry; percentage increase in transfer factor from sitting to lying position (tf) as an indicator of micro-vascular lung disease; overnight oximetry on air; and hour holter monitoring at present arf is one of the most spread and serious complication of postoperation period. practically the experience of carring nimv on patients with arf on early stadies of mosf is absent. until now, the criteria of uneffectiveness of nimv and indications for cessation of mask ventilation and moving of patients to mechanical ventilation are not determined. methods. there were included patients with ali in the examination. the cause of this condition was the mosf, developed in postoperative period. diagnosis of ali/ards was stated on the criteria adopted the american european consensus conference on ards ( ). presence of organs failure was determined on multiple organ dysfunction score (table ) . nimv was carried out by seances from to hours. average duration of nimv consisted . ± . hours. results. improvement of gases change was determined on patients ( %) out of . though patients with mosf were reintubated, out of which patients ( %) died lately as the result of mosf progressing. the condition of gases changing functions before intubation is one of the determining factors of prognosis. the patients reintubated under satisfactory indices of gase blood composition and early symptoms of mosf survived. patients, who were reintubated on decreased indices of arterial oxygenation under mosf progressing died in % cases ( nimv is effective method in complex therapy of arf, developing in postoperative period after cardiac surgery, that leads to significant improvement of lungs biomechanics and gases change function. progressing of mosf and storage disturbance of lung oxygenation is absolute indication for intubation and applications of special regimes of mechanical ventilation. references. . bernard gr, artigas a, brigham kl. am j respir crit care med - : : introduction. several bioimpedance cardiac output systems have been developed in the past in order to measure cardiac output in a wide variety of clinical situations. however, open thorax surgery negatively influences the accuracy of the measurement of thoracic electrical bioimpedance cardiac output (teb-co) ( ). the purpose of the present study was to evaluate the performance of a new bioimpedance cardiograph hl- (vrije universiteit medical centre amsterdam and hemologic amersfoort, the netherlands), using a new algorithm and a new electrode configuration, during open and closed chest in cabg patients, comparing teb-co with transcardiopulmonary thermodilution (tcpco). methods. after hospital ethics committee approval and written informed consent, fourteen patients with preserved lv-function at cineangiography or echocardiography, scheduled for coronary artery bypass grafting were included. for the teb system two current injecting electrodes were placed on the forehead and the left thigh respectively and two voltage sensing electrodes were used: one above the left clavicle at the base of the neck and the other at the level of the xyphoid in the left midaxillary line. for tcpco, the picco-system (pulsion, munich, germany) was used. hemodynamic measurements were recorded at three time points: t before the operation, t after weaning from bypass before sternal closure and t after sternal closure. teb-co and tcpco data were compared with pearson's r correlation coeficient. p< . was considered significant. bland-altman analysis ( ) with bias and precision was carried out at each of the three time points. results. ten males and females with age ± yr, body weight ± kg and height ± cm were included. a total of matched data pairs were available for analysis. table shows the results of correlation, bias and precision of the measurements at the three different time points. teb consistently underestimated tcpco. at all time points, there was a good correlation between both techniques. introduction. isoflurane sedation of icu patients has previously been shown to be useful but has not come into wide clinical use for a number of reasons.a new device(the anesthetic conserving device,"acd") enables easy and safe administration of isoflurane in the icu setting.we conducted a randomised, controlled study to evaluate efficacy of sedation and environmental safety during administration of isoflurane with the acd. the acd is a modified heat and moisture exchanger connected to the breathing circuit at the endotracheal tube.isoflurane is administered via a syringe pump to a vaporiser rod in the acd.due to the physical properties of the acd most of the exhaled isoflurane is returned to the patient. mechanically ventilated patients were randomised to receive isoflurane via the acd. control patients received midazolam intravenously. all patients received morphine analgesia. quality of sedation was assessed hourly in all patients."adequate sedation" was pre-defined as a set interval on the bloomsbury sedation scale. additionally, the patient's nurse determined if sedation over the previous hour in general had been adequate or not. time from discontinuation of the sedative drug until the patient followed verbal command and to extubation was compared between groups. in the isoflurane group a gas evacuation system was used during isoflurane administration. athmospheric concentration of isoflurane was measured at . m from the acd. results. in the isoflurane group patients were adequately sedated by the bloomsbury scale for ± % of the study period, compared to ± % in the control group.nurse satisfaction in the isoflurane group was % of time and % of time in the control group.mean time to extubation after cessation of sedative administration was min in the isoflurane group and min in the control group, mean time to patient cooperation was min in the isoflurane group, and min in the control group. no significant hemodynamic changes were noted at initiation of the sedation in either of the groups. no serious complications related to sedation were noted in either group.opioid requirements in the isoflurane group were lower, with a mean rate of . . mg/hr, compared with a mean rate of . . mg/hr in the control group.mean isoflurane infusion rate was . ml/hr, with mean end-tidal isoflurane concentrations of . % ( . - . %).environmental levels of isoflurane were generally low,with a mean of . ± . ppm, well below the recommended long-term exposure limit of ppm. brief peaks (< min) between and ppm were noted during endotracheal suctioning, etc on an average of . times/hour of exposure. conclusion. isoflurane administered via the acd for sedation of icu patients is environmentally safe, requires small volumes of isoflurane and may provide better quality of sedation than midazolam. it appears to be more titratable with a shorter time from adequate sedation to extubation and ability to cooperate. references. millane ta, bennett ed,grounds rm,anaesthesia ; : - .spencer em,willatts sm,intensive care brudney c. s. , gosling p. , manji m. anaesthesia, biochemistry, anaesthesia, university of birmingham, birmingham, united kingdom increased capillary permeability has been implicated in the pathogenesis of ards and organ failures. surgery and ischaemia-reperfusion injury are both associated with stimulation of the acute inflammatory response, an early feature of which is an increase in systemic capillary permeability. the kidneys amplify small changes in systemic capillary permeability ( ).the aim of this study was to explore any association between acr during and after cardiopulmonary bypass (cpb) and subsequent pulmonary and renal function. methods. forty patients ( female) mean (range) age . ( - ) yrs undergoing coronary artery bypass grafting were enrolled. patients with severely impaired left ventricular function (< % ef) were excluded. ten ml of urine was collected at intervals from the start of surgery until hours post cpb. microalbuminuria was measured by automated immunoturbidimetry and expressed as the albumin creatinine ratio (acr: ref. range < . mg/mmol). acr was compared with po /fio ratio, hours on ippv, renal function and duration of inotropic support, using spearman's rank correlation procedure. results. two patients were excluded (death at hours and acute renal failure post cpb). the median (range) duration of ippv was ( - ) hours. patients required inotropic support for median (range) ( - ) hours. median (range) acr increased during surgery and was maximal minutes post cpb. (table) two hour acr was inversely correlated with the mean po /fio ratio up to hours (rs = - . p = . ). two and hour acrs were both positively associated with duration of ippv (rs = . p = . and . p < . respectively). acr at and hours were associated with serum creatinine hours post cpb, (rs = . p = . , rs = . p = . respectively). acr at , and hours post cpb were associated with serum creatinine hours post cpb (rs = . p = . , rs = . p = . and rs = . p = . respectively). there was no significant association between duration of inotropic support and acr at any time point up to hours. conclusion. cpb leads to a perioperative microvascular insult, causing increased capillary permeability which influences later pulmonary and renal function. these rapid changes in microvascular permeability can be monitored as the acr, and in the patient group studied, the magnitude of the acr as early as hrs post cpb is associated with later organ function. acr may provide a tool allowing early identification of patients at risk of developing organ dysfunction, who may benefit from early intervention aimed at modifying the inflammatory response. acute lung injury (ali) is a major complication of gram-negative bacterial sepsis. to date, bacterial lipopolysaccharide has been held responsible for triggering ali ( ). whether additional bacterial toxins play a role in the development of acute pulmonary inflammation during gram-negative sepsis remains an unresolved issue. flagellin, a principal component of bacterial flagella, has been recently shown to elicit immune responses via activation of the toll-like receptor ( ). we have newly found that flagellin induces an expression of icam- and a massive production of il- by human lung epithelial cells. in mice, flagellin produces a severe acute lung inflammation with local release of pro-inflammatory cytokines, accumulation of inflammatory cells and increased pulmonary permeability that was more pronounced than following endotoxin ( ). the purpose of the present investigation was to evaluate the influence of flagellin on lung fluid filtration in rats. wistar rats ( - g) were exposed either to intravenous injection of flagellin . - mg/kg or corresponding volume of normal saline (controls). after - h, the rats were anesthetized and the lungs were isolated. the isolated lungs were ventilated under a normoxic condition and perfused with homologous blood ( ºc) at a constant flow for h or until development of irreversible edema. airway pressure, pulmonary arterial pressure, pulmonary vascular resistance, and changes in the lung weight were assessed. the increments in outflow pressure of . kpa for min were used to determine the fluid filtration rate and filtration coefficient in the lungs every min ( ). flagellin induced a dose-and time-dependent increment in the lung fluid filtration rate. in parallel, flagellin markedly increased airway pressure, pulmonary arterial pressure, pulmonary vascular resistance, and filtration coefficient. in contrast to the control lungs, all the lung preparations from flagellin-treated animals developed irreversible edema within the first two hours of perfusion. in isolated blood-perfused rat lungs, flagellin enhances fluid filtration, most likely, through elevation both of pulmonary microvascular permeability and hydrostatic pressure. the present study provides further evidence that flagellin may contribute to the development of sepsis-associated ali. . whether protein c conversely affects eosinophil function has not yet been reported. we investigated the effects of protein c and activated protein c on chemotaxis of eosinophils. possible involvement of endothelial protein c receptor (epcr) in the regulation was studied by using specific epcr antibodies. for preparation of eosinophils we used macs cd+ microbeads according to the manufactor's protocol. chemotaxis assays were performed using a -well boyden microchemotaxis chamber in which a -micrometer pore sized cellulose nitrate filter separates the upper and the lower chamber. eosinophils were pretreated by various protein c preparations with or without epcr antibodies, followed by washing and assessment of their migratory responses toward eotaxin. protein c and activated protein c exerted no significant chemotactic effect on eosinophils. however, eosinophils pretreated with protein c or activated protein c showed a sigificantly reduced response to the specific chemoattractant, eotaxin. moreover, this effects of protein c and activated protein c were inhibited using an antibody against epcr. conclusion. protein c as well as activated protein c inhibit the chemotactic effect of eotaxin on eosinophils via mechanisms involving epcr. this result indicates that protein c as well as activated protein c may decrease the number of eosinophils in tissue and thereby inhibiting inflammation and coagulation. deleterious effect of severe sepsis may be related to an oxidative stress, particularly related to peroxynitrite. selenium (se) toxicity is supposed to be related to oxidative stress through reaction with thiols. we perform a study to compare these toxicities. methods. wistar rats were studied. after day quarantine lipopolysaccharide (lps) or se was administered intraperitoneally in ml saline water. lps and se were administered in groups of rats with increasing doses from to mg/kg for lps, and from . to . mg/kg for se. mortality was observed at hours. animals were sacrificed under halothane. blood samples were taken in surviving rats of each group. nitric oxide (no) and nitrotyrosine (nit), a marker of oxidative stress especially related to peroxynitrite, were measured by elisa techniques, and plasma se concentration using atomic flame absorption. results. septic rats were rapidly sick. they rolled up into a ball. their fur was dull, and stood on end. they were asthenic and had diarrhea. at autopsy, intestinal abnormalities, and in some rats echymotics dots and hemolytic plasma were observed. rats were dehydrated. se rats developed an encephalopathy the first day and later recovered. se rats were lively, and seemed to required higher level of halothane for induction. ( )however the mechanisms responsible for this alteration remains under investigation. depressed micochondial respiration has also been found in different tissues during sepsis. ( ) the objective of this work was to study diaphragmatic function in rats after peritoneal sepsis and to correlate these findings with diaphragmatic mitocondrial respiration. cecal ligation and perforation was done under general anesthesia in wistar rats (septic group, n= ) . after hours the animals were monitored for arterial blood gases, systemic hemodynamia and body temperature. then, they were sacrified and the diaphragm force-frequency curves were obtained in vitro before and after fatigue. contraction time and relaxation time were also measured. mitochondrias were isolated from the diaphragm and oxygen consumption and other respiratory indexes were studied in septic animals. the results were compared to sham operated animals (control group, n= ). the septic group showed significantly lower values of aortic blood flow, arterial oxygen partial pressure, body temperature and arterial bicarbonate (p< . ) when compared to the control group. the forces measured at the different frequencies of stimulation were lower in the septic diaphragms both before and after fatigue when compared to controls (p< . ). mitochondrial respiration evaluated by oxygen consumption and rcr indexes was found decreased in the septic animals (p< . ). diaphragmatic contractile failure along with hemodynamic, respiratory and metabolic dysfunctions was found in peritoneal sepsis in rats. diaphragmatic dysfunction could be explained by mitochondrial damage during sepsis. we speculate that mitochondrial injury and dysfunction could be related to oxidative stress in this animal model. introduction. protein c is activated by thrombin bound to thrombomodulin and this effect is enhanced in the presence of the endothelial protein c receptor (epcr). in vivo and in vitro studies have revealed that components of this pathway may also inhibit inflammatory responses. protein c was able to inhibit leukocyte adhesion to vascular endothelial cells and to reduce neutrophil accumulation in rat lungs [ ] . protein c inhibits proinflammatory cytokine release in monocytes [ ] that were shown to express epcr [ ] . soluble epcr binds to proteinase- and cd b/cd of activated neutrophils [ ] , which were previously shown to synthesize thrombomodulin but not to promote thrombin-dependent protein c activation [ ] . if protein c directly affects neutrophil functions has not jet been sufficiently demonstrated. we investigated the in vitro effects of protein c and activated protein c on chemotaxis of isolated human neutrophils and explored wether epcr may be involved. neutrophils were obtained from forearm venous blood by standard methods. leukocyte migration toward gradients of soluble attractants into cellulose nitrate micropore filters was measured using a -well microchemotaxis chamber. cells were either directly exposed to gradients of protein c or were pretreated with protein c followed by washing; then chemotaxis toward typical attractants was tested. neither protein c nor activated protein c induce chemotaxis of neutrophils. both inhibit neutrophil chemotaxis toward interleukin- , fmlp and c a and there is no significant difference in the effects of these two substances. a blocking antibody against the epcr is able to diminish the effects of protein c and activated protein c. conclusion. protein c as well as activated protein c is able to inhibit neutrophil chemotaxis. this indicates that an activation of protein c is not necessary for effects on neutrophils to occur or that neutrophils are able to activate protein c followed by migration. the reduction of the protein c effects by an antibody against the endothelial protein c receptor suggests that neutrophils express epcr capable to signal anti-migratory stimuli. during sepsis increased vascular permeability results in fluid extravasation and edema. lymphatics contribute in draining interstitial fluid from the abdomen to central circulation, but several factors (outflow venous pressure, pattern of mechanical ventilation) can act upon flow in the thoracic duct ( , ). we have tested if lymph flow is affected by endotoxin infusion under different ventilatory conditions. methods. anesthetized pigs ( . ± kg) were studied. septic damage was induced by continuous infusion of endotoxin (lipopolysaccharide e.coli, lps). abdominal lymph flow was continuously recorded by an ultrasound flow probe positioned on the thoracic duct at the diaphragm level; hemodynamics, respiratory system data, bga and intra-abdominal pressure (iap) were registered. during the first . hours of lps infusion animals were ventilated in volume controlled mode tv - ml/kg, rr bpm, peep , fio . ; during the next hours animals were divided in group (control, peep ), (peep ) and (spontaneous breathing, cpap peep ). during lps infusion lymph flow significantly increased from . to . ml/min (p< . ), cardiac output and compliance decreased from . to . l/min * and to ml/cmh o * respectively, while mean pulmonary artery pressure and iap increased from to mmhg * and to cmh o * (* p< . ). in all the pigs a positive correlation was found between iap and lymph flow (mean pearson´s coefficient . ). no correlation was found between lymph flow and central venous pressure and airway pressure (mean pearson´s coefficient . and . ). in group and lymph flow changes averaged - % and + % (versus value before randomization). cpap increased lymph flow by %. lymph flow from the abdomen increases during lps infusion: role of lymphatics in draining abdominal fluid could thus be significant during sepsis (~ ml/h are drained). these preliminary results suggest that spontaneous breathing could improve lymphatic flow from the abdomen. despite the following rise in intra-thoracic pressure, increase of peep is not associated with lymph flow reduction. animals in peep group have however shown different patterns of response, and more data are needed to clarify this aspect. introduction. : ischemia/reperfusion or sepsis is initially responsible of an acute activation of pro-inflammatory cytokines (e.g. tumour necrosis factor (tnf-)). it is followed by a rise of anti-inflammatory cytokines (e.g. interleukin- (il- )). in human umbilical vein endothelial cell (huvec) tnf-induces a mitochondrial release of reactive oxygen species (ros) in a dosedependent manner. the signalisation pathway which links tnf-at mitochondria involves ceramide pathway ( ).the goal of our study is to evaluate the action of il- on the oxidative stress induced by tnf-in huvec and to define the mechanism of this interaction. huvec were grown on plastic cover slides. at confluence they were placed in a perfusion chamber under a microscope equipped with a digital camera connected to acquisition software. cells were perfused with krebs solution containing two fluorescent probes: dichlorodihydrofluorescein diacetate (dcfh) to study the release of reactive oxygen species (ros) and propidium iodide (pi) to study cell mortality. three cell groups were studied: a reference group, a tnf-group where, after one hour stabilisation, tnf-was added ( ng/ml) in perfusion medium during one hour, · a group tnf-+ il- where il- was added to perfusion medium minutes before tnf-. variations in fluorescence were recorded each minutes for dcfh and each one hour for pi. for a non lethal concentration (pi remaining unchanged), il- reduces significantly the ros production induced by tnf-(anova for repeated measures). interleukin- has an inhibitory effect on the release of ros induced by tnfin huvec. this effect could be the result of an interaction with acid sphingomyelinase. ( ) am. j. cell. molecular biol. : - , . the immunosuppresive drug cyclosporine a (csa) is an inhibitor of mitochondrial permeability transition (mpt) which could afford protection against cell death [ ] .to test whether csa protects against endotoxin-induced myocardial apoptosis [ ], we produced i-annexin v [ ], a marker of apoptotic cells, and measured its myocardial uptake during endotoxaemia in csa-treated rats. the specificity of the signal has been previously verified with caspase inhibitors and i-human serum albumin. methods. ) i-annexin v was produced with a radiochemical purity higher than % as confirmed by hplc. ) young male sprague-dawley rats were either given iv : saline ( . ml) : control group, n= , or lipopolysaccharide (lps) from e coli ( mg/kg) ± csa ( mg/kg): lps group, n= and lps+csa group, n= . h later, all animals were given i-annexin v ( mbq, mg protein). after h, hearts were harvested and divided into apex, septum, right and left ventricle (rv, lv) for determination of i-annexin v myocardial uptake with a lkb gamma counter. results were expressed as a mean percentage ± sd of the injected dose per gram of tissue (%id/g). statistical analysis was performed by mann-withney test; a p value < . was considered as significant (*). i-annexin v myocardial uptake is significantly increased in the lps group compared to control group; there is no significant difference between the septic groups . control lps lps+csa mean + -sd . + - . . + - . * . + - . ns mortality % % % i-annexin v myocardial uptake conclusion. our results confirm that endotoxaemia is associated with significant myocardial apoptosis but fail to demonstrate that csa can reduce the cell death signal detected by i-annexin v . in spite of its action on mpt and its myocardial dysfunction reducing effect in septic rats [ ] , csa provides no myocardial protection in this model . a reducing effect of csa on endotoxin-induced mortality is not excluded but remains to be demonstrated. further investigations are needed to clarify the effect of csa on the inflammatory responses due to endotoxaemia. sepsis induced alterations in hemostasis with dysbalances in fibrinolysis may lead to capillary obstruction due to fibrin deposition. the aim was therefore to investigate regional net fluxes of the fibrinolytic enzyme tissue-type plasminogen activator, tpa, and its main inhibitor plasminogen activator inhibitor type- , pai- , in response to endotoxemia. methods. anesthetized pigs (n= ) were instrumented for registration of cardiac output (co, thermodilution) and portal (qpv), hepatic (qha) and renal (qra) blood flows (ultrasound flowmetry, transonic). blood samples were collected from the aorta and pulmonary artery as well as the portal, hepatic and renal veins. after baseline registrations, all animals were subjected to an e. coli endotoxin infusion for min, followed by a volume/norepinephrine resuscitation for min targeting baseline co levels. plasma concentrations of both total and active tpa and pai- were determined as described [ , ] and net organ fluxes (ng/min) were calculated based on in-/outflowing plasma concentrations and local plasma flow [ ]. results. endotoxemia induced a low co state and a decrease in qpv. total liver blood flow was preserved due to a concomitant increase in qha. during resuscitation co and qpv were restored to baseline values. systemic plasma levels of total tpa increased over time during endotoxemia, peaking at min, whereupon a decline occurred. however, plasma levels of total tpa had not returned to baseline values at the end of the registration period ( min). changes in systemic levels of active tpa mirrored changes in total tpa. a marked ( -fold) increase in mesenteric net release of total tpa was observed. this response was paralleled by a pronounced increase in hepatic uptake of tpa. pai- described a different response to endotoxemia. by the end of the experiment plasma levels of both active and total pai- increased. in contrast, no significant net fluxes of pai- were observed across any of the investigated vascular beds except for the hepatic vascular bed, where a net release of both total and active pai- occurred at approximately min. hepatic pai- release rates then increased progressively. conclusion. endotoxemia induced a marked increase in mesenteric release of tpa which however was not entirely responsible for the increase in systemic plasma level of tpa. the results indicate that this profribrinolytic response at later stages are counteracted by increased plasma levels of pai- and this increase is mainly derived from the hepatic vascular bed. thus, patients with altered regional endothelial functions or liver capacity prior to a septic challenge can be expected to demonstrate varying susceptibility to thrombotic events. antithrombin has been shown to reduce mesenteric venular leukocyte interactions and intestine injury in a leukocyte-dependent model of endotoxemia ( ). however, endothelial damage during early endotoxemia has been shown to be leukocyte-independent ( ). the role of antithrombin in this setting is still unknown. therefore, it was the aim of the study to investigate the effects of antithrombin on leukocyte-independent endothelial damage. in male wistar rats, microvascular permeability (mp) and leukocyte-endothelialinteraction (leukocyte rolling, lr) were determined in mesenteric postcapillary venules using intravital microscopy at baseline, and min after start of a continuous infusion of endotoxin (etx; mg/kg/hr, e.coli o :b ) (group a, n= ). therefore animals were laparotomized and the mesentery was exposed beneath an in-vivo videomicroscope. mp was measured using fluorescein isothiocyanate (fitc) labelled albumin. leukocyte-endothelial interaction was blocked in all groups by fucoidin ( mg/kg b.w.), a l-selectin-binding carbohydrate, min before laparotomy. animals in group b (n= ) received antithrombin (kybernin®, aventis-behring, germany; ie/kg b.w.) prior to baseline measurement and additionally to the procedure described above. animals in group c (n= ) received equivalent volumes of nacl . % instead of antithrombin and endotoxin. statistical analysis was performed using two-way repeated measures anova followed by the scheffé test. a p-value < . was considered significant. in groups a-c, fucoidin prevented lr during the entire experiment. however, in all groups mp increased significantly, starting at min. animals in group a were characterized by a stronger increase in mp and showed significantly higher values in mp in comparison to groups b and c at min. there were no significant differences in mp between groups b and c. leukocyte-independent endothelial damage during early endotoxemia is attenuated by antithrombin. endothelial damage during early endotoxemia has been shown to be leukocyte-independent ( ). paf (platelet-activating factor)-and serotonin-receptor antagonism has been shown to reduce leukocyte-independent macromolecular leakage significantly ( , ). nevertheless, the exact mechanisms involved in leukocyte-independent endothelial dysfunction are unknown. therefore, it was the aim of the study to investigate the effects of nitric oxide (no) on leukocyte-independent endothelial damage during endotoxemia methods. in male wistar rats, microvascular permeability (mp) and leukocyte rolling (lr) were determined in mesenteric postcapillary venules using intravital microscopy at baseline, and min after start of the experiment. in all groups, leukocyte-endothelial interaction was blocked by fucoidin. rats were randomized into groups, animals each. the experiments were divided into two parts. part i (no-inhibitor): in group a, the mesentery was superfused with a l-name superfusion ( mmol/l) combined with a continuous infusion of endotoxin (etx; mg/kg/hr) after baseline measurement. group b received a l-name superfusion of the mesentery combined with a continuous infusion of saline . %. groups c and d were treated like groups a and b but without l-name. part ii (no-donator): group x received sin- (initial bolus of mg/kg b.w. followed by . mg/kg b.w. after min-measurement) followed by a continuous infusion of endotoxin (etx; mg/kg/hr). group y was treated similar to group c and group z was treated similar to group d. statistical analysis was performed using two-way repeated measures anova followed by the scheffé test. a p-value < . was considered significant. fucoidin prevented leukocyte-endothelial-interaction in all groups. part i: pe increased in all groups, being significant in group d at min (p< . vs. baseline) and being significant in groups a-c starting at min. animals in group d were characterized by a slighter increase in mp and showed significantly lower values in mp in comparison to groups a and b at min, and to groups a-c at min. there were no significant differences in mp between groups a-c at min. part ii: pe increased in all groups being significant in group z at min (p< . vs. baseline) and being significant in groups x and y starting at min. animals in group y were characterized by a stronger increase in mp and showed significantly higher values in mp in comparison to groups x and z at min. there were no significant differences in mp between groups x and z. leukocyte-independent endothelial damage during early endotoxemia is a nitricoxide mediated event. overproduction of nitric oxide (no) is thought to be a principal cause of the hypotension of septic shock. two nitric oxide synthase (nos) enzymes have been described in blood vessels: endothelial nos (enos) and inducible nos (inos). constitutive activity of enos in the endothelium is a major determinant of blood vessel tone in health; however, in experimental sepsis it appears endothelial enos expression is reduced while smooth muscle inos expression is increased ( ). in contrast, another model of human sepsis found an increase in enos but not inos in the vessel wall ( ). to resolve this discrepancy, we studied enos and inos protein concentrations in arterial smooth muscle (asm) from patients with clinical sepsis. asm was isolated from mesenteric vessels from patients undergoing bowel resection for perforated viscus (who in the perioperative period met the accp/sccm criteria for septic shock), and from controls with bowel cancer. after mechanical removal of endothelium and adventitia, the tissue was homogenised in protease inhibitor and frozen until sufficient samples had been accumulated. western blotting was performed under reducing conditions, with membranes incubated in : (inos) or : (enos) primary antibody followed by : peroxidase labelled secondary antibody. protein bands were quantified by computer analysis of the chemiluminescence detection film, then normalised to the protein concentration of the sample prior to dilution. . enos protein was increased in arterial smooth muscle from patients with septic shock (control . . units/mg, septic . . units/mg; n= controls and septics; p = . , student's t test). in contrast, there was no increase in concentration of inos; indeed inos protein was only detectable in asm from control and septic patients. we suggest that overexpression of enos, rather than inos, in the arterial smooth muscle of patients with septic shock may be responsible for the hypotension observed in these patients. introduction. data published in the literature concerning the effect of sepsis on intestinal motility found a reduction as well as a stimulation of intestinal motility . the settings used are mostly in vivo settings, and therefore not usable to investigate intestinal motility independent from circulatory changes. the aim of our study was to evaluate the direct effect of endotoxinemia on guinea-pig small bowel motility in vitro, independent from circulatory changes, and in a second step to evaluate the effect of vasoactive drugs on motility of these septic animals. two groups of guinea-pigs received mg/kg e. coli lps intraperitoneally or hours before the experiments started. in the following hours the animals developed severe symptoms of sepsis. a control group did not receive lps before the experiments started. the small bowel of sacrificed guinea-pigs was excised, cleaned and kept in tyrode's solution. after a resting period segments of cm length were set up in parallel organ bathes containing oxygenated tyrode's solution. peristaltic contractions were elicited by perfusion of the segments with tyrode's solution at a rate of . ml/min, against an aboral resistance of pascal. the intraluminal pressure increased gradually until it reached a pressure threshold (pt) which triggered peristaltic contractions. these contractions were recorded via a pressure transducer at the aboral end of the segments. increasing concentrations of epinephrine, norepinephrine, dopamine, dobutamine, clonidine and dexmedetomidine were cumulatively added to the organ bath at min intervals. each drug was tested on different segments. statistics was performed using ncss for windows, one-way and two-way anova for repeated measures were used, p values < . were considered statistically significant. in the control group all tested vasoactive drugs had a dose-and substance-dependent inhibitory effect on peristalsis. higher concentrations of all tested substances led to a complete block of peristalsis. hours after lps application a pronounced reduction of the inhibitory effects of clonidine, epinephrine, norepinephrine and dopamine were found. the reduced inhibitory effect of dexmedetomidine was not significant. hours after lps application the inhibitory effect was reduced again, but for most substances this reduction was not statistically significant. dobutamine was the only tested substance with a more pronounced effect after hours than after hours. endotoxinemia per se did not affect small bowel motility in vitro. a possible explanation for the controversy to in vivo data demonstrating an inhibitory effect on peristalsis might be that intestinal ischemia is a common event during sepsis, and ischemia in turn might cause paralysis. a described reduced sensitivity of alpha-adrenoceptors during sepsis, or a central effect of lps additionally inhibiting peristalsis ( ), might also be responsible for our findings. high cytokine levels in patients admitted to the emergency department are associated with an increased incidence of sepsis/septic shock. patients with cardiogenic shock (cs) who often develop sepsis during icu-stay,have not been particularly studied. we studied whether plasma levels of cytokines are better predictors of sepsis/septic shock than routinely determined laboratory parameters. il- ,il- and il- plasma levels were determined in pts with cs(cardiac index < . l/min/m²,pcwp > , mean arterial pressure < mmhg or need for vasopressor therapy and signs of organ hypoperfusion) on admission to the icu (median hrs after shock onset). patients who were not surgically treated during icu stay were eligible for the study and evaluated for development of sepsis or septic shock within week after onset of cs. c-reactive protein (crp) levels and white blood cell (wbc)-counts were routinely evaluated once daily in all patients until discharge. data are given as median and interquartile range. all pts with cs were free of demonstrable infection at time of blood sampling. nevertheless % had a crp-level > mg/dl at time of enrollment. pts ( %) developed septic shock within week after onset of cs. pneumonia ( %, n= ) and catheter related infections ( %, n= ) were the leading causes of sepsis. sepsis after cs was not associated with a higher mortality rate ( % vs. %, p=ns) and sirs that was encountered in % of cs pts at the time of blood sampling did not predispose for development of sepsis ( vs. %, p=ns).crp levels,and wbc-counts as well as il- , il- and il- plasma levels on admission to the icu did not differ significantly between cs-pts who developed sepsis and cs-patients without sepsis ( in pts who survived for more than hrs (n= ) the absolute crp levels hrs after admission (crp hrs) and the increase in crp levels over hrs following icu admission (dcrp) were significantly higher in pts who developed sepsis as compared to pts without sepsis. (crp hrs: . mg/dl [ - . ] vs. . [ . - . ], p= . ; dcrp: . mg/dl [ . - . ] vs. . [ . - . ], p= . ). a dcrp > . mg/dl in hrs was more sensitive than an absolute crp level > mg/dl hrs after icu-admission for predicting sepsis ( vs. %), but both parameters had equal specificity ( %). conclusion. although many pts with cs exhibit elevated crp levels the increase in crp over hrs (dcrp hrs)is a valuable parameter to identify pts at risk for sepsis. single-point determination of cytokines on admission to the icu is not superior to follow-up determinations of crp for predicting sepsis. mitochondrial dysfunction may be implicated in sepsis-induced multi-organ failure. glycolytically-generated atp may thus be an important alternative energy source if aerobic respiration is compromised. little is known about glycolysis during sepsis, though both up-and down-regulation are reported , . we therefore examined changes in glycolytic activity in a longterm sepsis model. an instrumented, fluid-resuscitated, faecal peritonitis rat model was used. this has a -hour mortality rate of approx. %. septic (n= ) and sham (n= ) rats were sacrificed at various time points ( , , , h) and liver samples harvested and assayed for maximal activity of the rate-limiting glycolytic enzymes, hexokinase (hk), phosphofructokinase (pfk) pyruvate kinase (pk). we demonstrate an initial rise (albeit non-significant) then significant downregulation in two rate-limiting glycolytic enzymes during sepsis. the lack of difference at h may reflect prior demise of the severely ill animals. whether the degree of glycolytic down-regulation is related to subsequent death requires further study. we presume the interesting finding of upregulation seen in the sham animals to be a response to surgery and/or fluid loading. recent studies have shown that low-dose vasopressin infusion or terlipressin bolus (tp, its long acting analogue; o'brien, ) restores blood pressure and reduces norepinephrine (ne) requirements in septic shock. however they have no effect upon blood pressure in non-septic patients. exact mechanisms underlying this hyperreactive effect in sepsis patients remain unknown. we chose to investigate this using our established in vivo and in vitro models of endotoxic shock in rats. in vivo -spontaneously breathing anaesthetised male wistar rats was given either saline (sham) or endotoxin (lps) (klebsiella mg kg - ) over mins and then fluid resuscitated with colloid mls kg - hr - for mins. at mins either a bolus of tp ( . mg kg - ) or a bolus and infusion of ne ( . mg kg- and mg kg hr - ) was administered. measurement of flow and pressure (mean arterial pressure -map) were made from appropriately sited probes and transducers. in vitro -rings of rat mesenteric artery (rma) were harvested, cleaned and incubated for h with or without mg ml - lps (s. typhosa). they were then mounted in organ baths for measurement of isometric tension. cumulative concentration-response curves to phenylephrine (pe: - to - m) or vasopressin (vp: - to - m) were then constructed. statistical analysis was by anova. results. in vivo -while ne had a significantly greater effect upon map in shams compared with lps rats (p= . ), tp caused a greater increase in lps animals than shams. a bolus of tp lasted approximately mins. in vitro -lps significantly depressed contractile responses to pe compared to control tissues (max contraction controls - . ± . g, lps - . ± . g, p< . , anova). however there was virtually no contractile response to vp even in control tissues after h incubation. the cytokine cascade activated in response to injury consists of a complex biochemical network with diverse effects on the injured host. leukocyte activation after trauma is essential for inflammation. it is a multistep process in which chemokine -interleukin (il)- has pivotal role. in two-hit hypothesis, sepsis represent a second insult to a previously injured and primed host, converting a low-grade or regulated host response into an accelerated or dysregulated host response, triggering new or progressive organ dysfunction ( ). aim of this study was to assess pro-inflammatory response to trauma with or without sepsis as a second insult. twenty five patients with severe trauma (explosive and sclopetarious) who developed sepsis and patients with same kind of severe trauma without sepsis were enrolled in this study. in the trauma+sepsis group patients developed multiple organ dysfunction syndrome (mods) and died. in trauma group developed mods and died. blood was drown on the first, third and fifth day of trauma. concentrations of il- , il- , tumor necrosis factor (tnf)alpha and interferon (ifn)-gamma were determined in plasma using elisa assays. when compared trauma+sepsis group with trauma group we found statistically highly significant difference (p< . ) in il- and ifn-gamma and statistically significant difference (p< . ) in tnf-alpha concentrations; mean values of il- were -fold higher, ifn-gamma -fold higher and tnf-alpha -fold higher in patients with trauma with sepsis. il- was not statistically different (p> . ) between two groups. when compared mods group with group without mods, we found statistically highly significant difference (p< . ) in il- and tnfalpha concentrations; mean values of il- were -fold higher and tnf-alpha . -fold higher in patients with mods; il- and ifn-gamma were not statistically different (p> . ) between two groups. when compared non-survivors with survivors, we found statistically highly significant difference (p< . ) in il- and tnf-alpha and statistically significant difference (p< . ) in il- concentrations; mean values of il- were . -fold higher in non-survivors, mean values of tnfalpha were . -fold higher in survivors, il- was also higher in survivors. ifn-gamma was not statistically different (p> . ) between two groups. there is augmented pro-inflammatory response after trauma with secondary sepsis. high concentrations of il- and tnf-alpha indicated higher severity (mods). but, fatal outcome was predicted with high concentrations of il- only; survivors had higher concentrations of tnf-alpha and il- . therefore, pro-inflammatory response was partly beneficial and partly detrimental to the host. in patients with shock hypoxia is considered to be the most important cause of organ failure and death. the goal of treatment therefore is to restore tissue oxygen delivery (tdo ). due to impaired oxygen extraction in distributive (septic shock) the relation between tdo and tissue oxygenation is less conclusive. direct measurement of tissue oxygen pressure (pto ) could be of great importance in gaining a better insight in tissue oxygenation in these patients. previously published data concerning pto in patients with sepsis/septic shock are contradictory ( , ). furthermore the techniques used were not easily applicable at the bedside. in a prospective observational study we performed bedside pto measurements in patients with sepsis/septic shock to gain insight in pto values and their dynamic changes related to the course of the illness, as well as investigating the practical applicability of tissue oxygen measurement in the icu setting. pto was measured continuously during the course of the illness using polarographic clark-type o electrodes (licox catheter measurement system, gms), which were placed subcutaneous in the upper arm. disease progression over time was expressed as the daily calculated sequential organ failure assessment (sofa) score. results. five men and women with septic shock n= or sepsis n= were included. the median (range) age was years ( - ), median apache-score on the day of admission was ( - ), median duration of pto measurement per patient was , days ( - ). in none of the patients technical problems were encountered during the pto measurements. the first day of measurement the median pto of the eight patients was ( - ) mmhg. in the six surviving patients the sofa score decreased over time and this was associated with a concomitant decrease in pto to a median of ( - ) mm hg. in the nonsurvivors an increasing sofa score was associated with an increase in the mean pto to mmhg on the day of death. in seven patients linear regression analysis showed a positive correlation between the daily sofa scores and the daily mean pto : r= . , . , . , . , . , . , . . in one patient no correlation was found. conclusion. bedside pto measurements in the icu using the licox measurement system are easily performed. pto in septic patients is variable but changes with the clinical course reflected by the sofa score: clinical improvement was associated with a decrease in pto while deterioration was associated with an increase of pto . these findings suggest that in patients with septic shock decreased oxygen utilisation may play a more important role than tissue hypoxia as such. to precise the diagnostic value of macrophage migration inhibitory factor (mif) as a marker of severity in patients with sepsis and to determine relations between mif and interleukin (il- ), we conduced a prospective, observational, cohort study, in two general intensive care units. we analyzed patients with septic shock, patients with sepsis, and healthy volunteers. the median mif serum level was significantly higher in septic shock patients ( . ng/ml, range . - . ) than in sepsis patients ( . ng/ml, range . - . ) or in healthy volunteers ( . ng/ml, range . - . ). there was a direct correlation between mif and il- concentrations (r= . , p< . ). the area under the curve (auc) of the receiver-operating characteristic (roc) for prediction of septic shock was . (p< . ) for mif and . (p< . ) for il- . the auc under the roc curve for prediction mortality was . (p< . ) for mif and . (p< . ) for il- . in this trial we found significant elevated serum levels of mif in patients with septic shock and sepsis. moreover, mif levels were discriminative for septic shock and mortality, and had a direct correlation with levels of il- with a similar diagnostic accuracy. in conclusion, mif appear to be a promissory marker of severity in sepsis. high density lipoprotein (hdl) modulates the inflammatory response to injury and infection via several pathways. hdl also directly binds and neutralises lps. administration of reconstituted hdl reduces cytokine release and attenuates shock in experimental endotoxaemia ( ). the hdl associated enzymes paraoxonase (pon) and lecithin cholesterol acyl transferase (lcat), destroy oxidised lipids that induce inflammatory changes in vascular endothelium ( ). incorporation of serum amyloid a (saa), an acute phase protein, into the hdl particle during the inflammatory response, may displace these protective enzymes producing a particle with proinflammatory properties ( ). alterations in hdl composition may, therefore, be implicated in dysregulation of the inflammatory response and could influence outcome from septic shock. methods. patients with septic shock, not given tpn or propofol, were recruited. apache ii scores and icu mortality were recorded. plasma and serum samples were taken within hours of the onset of shock. hdl cholesterol was measured by microenzymatic colorimetric assay. apolipoprotein ai (apo ai) was quantified by liquid phase radioimmunoassay. pon activity was determined by measuring the rate of paraoxon hydrolysis and described as percent of a control serum pool. lcat activity was quantified by measuring the esterification rate of c labelled cholesterol. saa was measured by elisa. results were compared with those of a pool of healthy volunteers and between survivors and nonsurvivors. (mann whitney u test). results. patients were recruited. there were survivors (s) and nonsurvivors (ns). pon activity was significantly higher in s than ns: . ( . - . ) vs. . ( . - . ), p< . . saa concentration was significantly higher in s than ns: ( . - ) severe trauma and sepsis are the major sources of morbidity and mortality despite the rapid development of intensive therapy. studies have indicated that there are marked alterations in immune response in patients exposed to major trauma or prolonged surgical procedures, including altered pro-and anti-inflammatory mediator/cytokine release ( ). traumatic injury results in profound immunosuppression which predisposes the patients to sepsis and/or multiple organ dysfunction syndrome (mods). aim of this study was to assess the prognostic value of anti-inflammatory cytokines: interleukin (il)- receptor antagonist (il- ra) , il- , il- and transforming growth factor (tgf)-beta regarding severity and outcome in patients with trauma and sepsis, trauma only and sepsis only. twenty five patients with severe trauma (explosive and sclopetarious) who developed sepsis, patients with same kind of severe trauma without sepsis and patients with severe sepsis were enrolled in this study. twenty nine patients developed mods (of all patients), died. blood was drown on the first, third and fifth day of trauma or sepsis. concentrations of il- ra, il- , il- and tgf-beta were determined in plasma using elisa assays. when compared mods group (regardless of initiating insult -trauma or sepsis) with group without mods, we found statistically highly significant difference (p< . ) in il- ra and il- concentrations; mean values of il- ra were -fold higher and il- -fold higher in patients with mods; il- and tgf-beta were not statistically different (p> . ) between two groups. when compared non-survivors with survivors, we found statistically highly significant difference (p< . ) in il- ra and il- concentrations; mean values of il- ra were . -fold higher and il- . -fold higher in non-survivors; il- and tgf-beta were not statistically different (p> . ) between two groups. when compared trauma+sepsis group with trauma group, we found statistically highly significant difference (p< . ) in il- ra and il- concentrations, they were higher in trauma+sepsis group (il- ra . -fold, il- -fold). il- and tgf-beta were not statistically different (p> . ) between two groups. when compared trauma+sepsis group with sepsis group and trauma group with sepsis group, we found no statistically significant difference in either one of anti-inflammatory cytokines. our study shows that il- ra and il- are excellent predictors of severity and outcome of critical illness; higher concentrations were found in group with more severe clinical status (mods) and in non-survivors. il- and tgf-beta had no significance as predictors of severity and outcome what so ever. fifty-eight patients admitted to two medical intensive care units for reasons other than acute coronary syndrome were consecutively included and analyzed according to their troponin status. thirty-day mortality, left ventricular ejection fraction, the presence or absence of underlying coronary artery disease, and a panel of inflammatory cytokines were compared between troponin-positive and troponin-negative patients. thirty-two of critically ill patients ( %) without evidence for an acute coronary syndrome were troponin-positive. positive troponin levels were associated with higher mortality ( . % vs. . %, p < . ) and lower left ventricular ejection fraction (p = . ). troponinpositive patients had significantly higher median levels of tumor necrosis factor a, its soluble receptor and interleukin- . a subgroup of ten aplastic patients was troponin-negative at study entrance. three became troponin-positive during leukocyte recovery and subsequently died, whereas all the others stayed troponin-negative and survived. conclusion. elevated troponin is a mortality risk factor for medical intensive care patients admitted for reasons other than acute coronary syndromes. it is associated with decreased left ventricular function, and this may be mediated by tumor necrosis factor a and mediators produced by neutrophilic granulocytes. it is very interesting to notice the high correlation among protein c and atiii activity levels and sofa scores (p< . ) and the dramatic decrease of the protein c system is already firmly present hours before negative outcome (not survivors). we also register the significant alterations of c inhibitor specially in the group (ns) patients with severe candidemia and this marker assumes a significant role with an interesting clinical future . tumour necrosis factor-a (tnf) is an important pro-inflammatory mediator and high levels of this cytokine have been associated with a poor outcome from sepsis. recently, genetic polymorphisms of the tnf locus and its promoter region have been associated with the incidence and outcome of severe sepsis ; , although the results have been conflicting . we chose to investigate the association between a known functional single nucleotide polymorphism (snp) in the tnf gene promoter (- g/a, guanine to adenine substitution) and outcome in severe sepsis and septic shock. caucasian adult patients with a diagnosis of severe sepsis or septic shock on icus in the uk and from an icu in sydney, australia were recruited. whole blood was collected in edta, dna extracted and amplified by pcr using specific primers and digested with the restriction endonuclease nco . this enzyme cuts the wild type (allele g) but this cutting site is abolished by the polymorphism (allele a). the restriction fragments were then size separated, visualised and scored on agarose gels. fisher's exact test was used for statistical analysis. shedding of membrane bound tumour necrosis factor receptors to produce soluble molecules (stnfrsf a and b) is an important inflammatory control mechanism . we and others have previously demonstrated that increased levels of stnfrsf a and stnfrsf b are associated with decreased survival from sepsis. furthermore, there appears to be an association between polymorphisms of the tnfrsf b locus and plasma levels of stnfrsf b . we have therefore investigated whether polymorphisms of the tnfrsf b gene and its promoter region might influence outcome from severe sepsis and septic shock. caucasian adult patients with a diagnosis of severe sepsis or septic shock from icus in the uk and from an icu in sydney, australia were recruited. we analysed polymorphisms of the tnfrsf b gene. a single nucleotide polymorphism in exon (snp t/g) was studied by pcr-rflp, a microsatellite in intron (ms ) using an abi a sequencer and a base pair insertion/deletion in the promoter region (indel) by polyacrylamide gel electrophoresis. analyses of associations between genotype and allele frequencies and outcome were by fisher's exact test. . icu mortality was %. overall genotype and allele frequencies for each of the polymorphisms were similar to published population frequencies. there were no statistically significant differences in allele frequencies in any of the three polymorphisms between survivors and non-survivors (snp p= . , ms p= . , indel, p= . ). the mortality was lower in patients homozygous for the base pair repeat in the microsatellite polymorphism in intron (the genotype associated with high levels of stnfrsf b) (mortality % v . %) and was higher in those with the snp (t/g or g/g) (mortality . % v . %). these differences, however, did not reach conventional levels of significance (p= . and . respectively). larger studies will be required to confirm or refute associations between tnfrsf b gene polymorphisms, particularly ms homozygosity, and outcome from sepsis. when associated with end organ dysfunction, sirs is a major cause of morbidity and mortality in the intensive care unit (icu) population ( ). lps concentrations in the gastro-intestinal tracts of these patients are elevated as a consequence of bacterial overgrowth. lps processing in the mesenteric circulation may influence the systemic inflammatory response ( ). tlr is an integral part of the lps receptor complex. a tlr polymorphism (asp gly) is associated with hypo-responsiveness to lps in human bronchial epithelial cells. we examined the association of this polymorphism with clinical outcome in icu patients with severe sirs. methods. adult icu patients with evidence of severe sirs were studied. patient demographics, apache ii data, length of stay and outcome data were collected. genotype was determined using pcr amplification. statistical analysis was performed using spss . . results. patients have been genotyped of whom are still in icu. of the remaining patients, / ( %) died in icu and died in hospital after discharge from icu, giving an overall hospital mortality rate of / ( %). mean (sd) apache ii score was ( ).the tlr genotype frequencies were asp/asp . % ( / ), asp/gly . % ( / ) and gly/gly . % ( / ). the allele frequencies were asp % and gly %, similar to previously reported frequencies in caucasians. preliminary analysis revealed no significant differences between apache ii scores in patients with the asp/asp genotype (mean . , sd . ) and those with asp/gly or gly/gly genotypes (mean . , sd . ) (p= . , student's t-test). / ( %) of patients who died during the hospital episode carried the gly polymorphism, compared to / ( %) of those who survived the hospital episode (p= . , fisher's exact test, or . , % ci . - . ). conclusion. no associations with severity of illness on admission to icu, icu length of stay or hospital outcome were detected with the present sample size. recruitment is ongoing, to attain sufficient power. we aim to study genes coding for components of the lps receptor complex, which are biologically relevant to innate immunity and the development of sirs. detailed, prospective study of the role of polymorphisms in innate immunity has the potential to improve our understanding of the pathogenesis of sirs, and to influence risk stratification and management of this severe complication of life-threatening infection. the present study was designed to evaluate the effect of low dose albumin infusion vs. control on the local inflammatory response following abdominal surgery. albumin loss during surgery is a well described phenomenon. in previous experiments a loss of plasma proteins, resp. albumin was observed during abdominal surgery. intravital microscopy for five hours was used to evaluate the effect of low dose albumin on the mesenteric microcirculation. urethan-anesthetized sprague-dawley rats underwent median laparatomy and placement of a doppler flow probe around the abdominal aorta. an ileal loop was prepared for eventration onto a microscopic stage using a plastic foil technique and the mesentery was immersed with krebs-henseleit buffer( %co in n ). low dose albumin ( . g/(kg bw*h)) was given vs. control (nacl . %) during the experiment. heart rate, map, aortic blood flow were registered on a beat-to-beat basis. abg's were drawn hourly for analysis of metabolic(be), respiratory (po , pco ) and hct values. rolling and adherent leukocytes significantly increased in the control group until the end of the experiment, whereas they constantly remained on a low level in the albumin group. velocity and shear rate in the mesenteric microcirculation were significantly higher in the albumin group which was supported by increased abdominal flow and stroke volume vs. control. low dose albumin infusion significantly reduces the inflammatory response on the mesenteric microcirculation following abdominal surgery. beneficial effects on systemic hemodynamics, mesenteric microcirculation and attenuation of leukocyte rolling and adhesion in mesenteric venules could only be observed in the albumin group, whereas the inflammation progressed in the control group. iasonidou c. , pertsas e. , koletsos k. , kapravelos n. , tsagalof s. , riggos d. icu, g.papanikolaou, thessaloniki, greece optimizing patient's hemodynamics in the icu can be challenging.the pa catheter has been used to determine preload, afterload and myocardial performance. however,insertion of a catheter is not a risk-free procedure and the values obtained can in some circumstances be misleading.the use of tee in icu has been increasing.previous studies have examined the correlation between the pulmonary vein (pv) velocities and mitral valve (mv) velocities and pcwp. the purpose of our study was to evaluate the relationship between these variables during different loading conditions as assessed by tee in icu patients. ( ) patients,with a mean age of ± years, requiring mechanical ventilation were prospectively studied. in all patients a pa catheter was inserted and baseline measurements were obtained.the pv velocities and mv velocities were evaluated during three different loading conditions: ) in a control situation ) in a state of decreased preload by intravenous administration of nitroglycerin )in a state of increased preload by administration of fluids.in all patients we used the following indices from the pv velocity : s (systolic),d (diastolic), decelaration time(dt) of d wave, apv (atrial reversal) and from mv velocity: e,a wave and deceleration time of e wave. the decrease in preload resulted in a trend toward a lower amplidute of d wave peak velocity as compared with the control state and a significant prolongation of the deceleration time (p< , ).there was a decrease in height of the systolic (s) wave (p< , )and the apv (p< , ). the mv curve demonstrated a significant decrease in e velocity (p< , ) and prolongation of deceleration time (p< , ).the increase in preload resulted in a significant increase in systolic and diastolic wave in pv (p< . ) with a shortening of the dt of d wave.the apv became significantly higher (p< , ).the mv curves demonstrated a significant increase in e wave (p< , ) with a decrease in dt.there was a good correlation between d wave and pcwp (r: , ),apv wave and pcwp (r: , ) and e wave and pcwp (r: , ).a direct correlation was present between changes in e and d waves (r: , ) and changes in dt of e and dt of d velocities (r: . ). this study provides evidence that tee gives information additive to the pa catheter in the assessment of preload in an icu population. examination of pv velocities and mv velocities and their changes during different loading conditions provide additional information regarding diastolic function. this may prove useful in minimizing the use of invasive methods for hemodynamic monitoring in icu patients. further investigation is required to correlate these doppler measures with the invasive hemodynamic measurements. methods. patients with spe ( women and men), with a mean age of years (sd of ; range: to years), were studied prospectively. coloured doppler-echocardiography was performed in all cases at admission, confirming that diagnosis by perfusion gammagraphy and / or helycoidal ct scan. emboli were observed in six patients ( %): in right atrial chamber, in pulmonary artery and in output tract of right ventricle. in patients ( %) right ventricular dilatation with a mean value of . mm (sd ), and tricuspid insufficiency in ( %) with mean estimated systolic pulmonary arterial pressure of mmhg (sd ). pulmonary acceleration time was measured in patients and found shortened in all of them: milliseconds (sd ), and septal abnormal movement was detected in patients ( %). out of patients had more than one sign of severe pulmonary embolism (spe), had two sign and the other had three or more signs. echocardiography is a simple technique, which allows the diagnosis of spe by the detection of emboli in the right heart cavity and / or the objectivation of indirect signs of functional alteration of right ventricle. coagulopathy and systemic inflammatory response have been previously reported in patients after cpr ( ). the coagulopathy includes activation of coagulation and inhibition of fibrinolysis, alterations similar to those reported in sepsis where profound depletion of anticoagulation proteins have been evidenced, and had significant therapeutic consequences ( ). however, anticoagulation proteins: protein c and s (pc -ps), as well as antithrombin (at) levels were not reported after cpr. consequently, serial measurements of markers of coagulation (thrombin-at [tat], d-dimers), fibrinolysis (plasminogen-activator inhibitor : pai- ), inflammation (il- ) and endothelial injury (soluble thrombomodulin: stm) were performed in patients (age: ± years; saps: ± ) after successful cpr.analyses on biomarker levels by anova were performed. the aim was to evaluate the effect of thrombolytic therapy in massive and submassive pulmonary embolism methods. patients ( women and men), with a mean age of years (sd ), range: to , studied prospectively. diagnosis at admission was confirmed with spiral ct scan and/or ventilation-perfusion (v/p) gammagraphy. a study protocol was performed in all patients consisting of: complete analysis, electrocardiography, thorax radiography and echocardiography.one hundred milligrams of rt-pa was infused in hours due to severity of the clinical presentation: haemodynamic instability ( cases) and/or severe hypoxemia or echocardiographic abnormalities ( patients). clinical improvement was seen in the entire group. studied variables pre and postthrombolysis are shown in the table. mean arterial pressure (map); right ventricular diameter (rvd), systolic pulmonary arterial pressure (spap), acceleration pulmonary time (apt), oxygen saturation (os), fibrinogen, hematocrit and heart rate (hr). postthrombolytic changes in electrocardiogram were objectivated, showing that some abnormalities had disappeared, such as: right bundle heart block in out of patients ( %), s q t pattern in out of ( %), t wave alterations in out of ( %), and the pulmonary p in out of ( %). minor hemorrhagic complications were observed in cases; only one needed transfusion. one patient had hematuria, one other hemarthrosis, and another one suffered pericardial blood effusion (after coronary by-pass graft). we have previously shown that the measurement of gut intraluminal redox potential (eh) during progressive bleeding and reperfusion is useful to monitor changes in oxygen transport . eh could provide with a different type of information from other parameters of tissue oxygenation, such as lactate and intramucosal ph. our goal was to show the rate of decrease of eh after the occlusion of superior mesenteric artery blood flow (qintestinal). eight anesthetized and mechanically ventilated sheep were studied. eh was measured as a voltage difference using a milivoltmeter with a platinum electrode, against a reference electrode. qintestinal was measured with an electromagnetic flowmeter. after basal measurements, superior mesenteric artery was occluded and eh was continuously registered during minutes. data (mean ± sd) were analyzed with repeated measures of anova followed by dunnett's test. response time was defined as a decline greater than three sd from basal values. assessment of heart rate variability (hrv) has been used in risk stratification after acute myocardial infarction, in congestive heart failure, and in the early diagnosis of diabetic neuropathy. patients with end-stage renal disease (esrd) constitute a population of increased cardiovascular morbidity and mortality. hemodialysis patients often show signs of autonomic neuropathy. data on hrv are usually derived from -hour holter recordings. however, short term rr interval variation as measured on standard -lead ecg holds important prognostic implications in subjects with dilated cardiomyopathy. the purpose of this study was to look at short term rr variation in esrd patients, and its modification after dialysis. methods. ( male, female) patients were included in the study.all of them were in three times a week hospital hemodialysis. twenty control subjects, of similar age and gender distribution, with normal renal function and blood pressure, were recruited among ward stuff. the rr intervals were measured from a continuous -min recording of lead ii. rr variation was calculated as the standard deviation of the rr intervals (rrsd), and the coefficient of variance of the rr (rrcv), i.e. standard deviation divided by the mean rr and expressed as percentage. ecgs were also analysed for left ventricular hypertrophy (lvh). . rrsd and rrcv were significantly decreased in dialysis patients compared to controls. rrsd was . ± . vs . . p= . , and rrcv was . ± . vs . ± . , p= . . rrsd and rrcv were not affected by dialysis, but were significantly decreased in those with ecg evidence of lvh, compared to those without. rrsd was . ± . vs . ± . (p= . ), and rrcv was . ± . vs . ± . , (p= . ). rrcv was associated with mg and k postdialysis. rrsd and rrcv were inversely correlated with cornell voltage, an ecg index of lvh. hemodialysis patients present with low short term rr variation in comparison with control subjects. electrocardiographically detected lvh among esrd patients is also associated with depressed rr variability. increased intracranial pressure,as that was seen in patients with large cerebral tumors, reduces frequency of the pulse.prolapsus of the brain masses in tentorial incissura of foramen magnum and consequently bradycardia,respiratory arrest,coma and death might occur in these patients. the aim of this study is to examine the ecg changes in patients with brain tumors in regard to the kind of tumors and localisation . the study group was consisted of patients ( male and female) of average age , years (range to ). there were patients with temporal lobe tumor ( left and right), patients with frontal lobe tumor ( left and right), with parietal lobe tumor ( left and right) and with occipital lobe tumor ( left and right). ecg changes were evaluated during the first hours from receiving in the icu. large cerebral tumors confirmed with ct, and definitive diagnosis was made pathohystologicaly. the most common ecg abnormalities associated with central lesions that we found were: prolongation of the q-t interval in . % patients with right and % with left cerebral hemisphere tumor; elevated, peaked, or notched t waves in . % patients with right and . % with left cerebral hemisphere tumor; and increased p-wave amplitude in . % with right and . % patients with left cerebral hemisphere tumor. the most frequent ecg changes that we registered among rhythm and conduction disturbances were: narrow-qrs tachycardia with regular rhythm; sinus tachycardia in % with right and % patients with left cerebral hemisphere tumor, sinus bradycardia in . % with right and % with left cerebral hemisphere tumor, and incomplete/complete right bundle branch block (bbb) in % patients with left cerebral hemisphere tumor. we did not find any specific differences according to the pathohystologicaly type of the tumors. conclusion. ecg abnormalities associated with central lesions in the patients with brain tumors are not depend from the kind of tumors and side of the brain where tumor is located. in the patients with brain tumors on left side of the brain prevails incomplete and complete right bundle branch block (bbb). prolonged mechanical ventilation support (mv) is associated with increased morbidity and less cost -effective admissions in the postoperative period (po) of heart surgery (hs). study conducted to identify variables associated with prolonged mv in patients that underwent hs. methods. cohort study; consecutive patients enrolled from / to / of . inclusion criteria: patients submitted to hs and admitted to intensive care unit (icu) in use of mv. exclusion criteria: non-cardiac surgery, admission to icu in spontaneous ventilation or death during the first hours of the po. variables that could be associated with prolonged mv were pre-selected for analysis and grouped according to the period it represented. preoperative period: in-hospital stay duration, age, body mass index, gender, severity of left ventricular dysfunction (lvd), pulmonary hypertension, chronic obstructive pulmonary disease, redo, urgency for the procedure. peroperative period: surgery, extracorporeal circulation (ecc) and aortic clamping duration, fluid and blood input/output differences, type of surgical procedure, combined procedures, need for post-ecc intraaortic balloon counterpulsation (iab). admission to the icu: oxygen alveolar/arterial difference, blood oxygen partial pressure/oxygen inspired fraction ratio (p/f). first hours of po: dobutamine or norepinephrine (nor) use, blood drainage volume, lowest blood lactate measurement and prognostic scores sofa, tiss and mods. for dichotomyc variables, mann-whitney test was applied; for continuous variables we used kendall's tau non-parametric correlation test; cuzick tendency test was used to evaluate association with lvd. results. median mv duration: hours; mean duration time . hours ( to ). increased mv duration was associated with emergency surgery (p= . ), coronary artery bypass graft surgery (p= . ), need of iab counterpulsation after ecc (p= . ), use of nor (p< . ). increases in mv duration were associated with increasing values of some variables (positive correlation): age (p< . ), surgery duration (p= . ), blood input/output difference (p< . ) and sofa, tiss and mods scores (p< . ). negative correlation was presented for fluids input/output difference (p= . ) and p/f (p< . ). increased linear tendency for mv duration correlates with worsening of lvd (p= . ). conclusion. more sophisticated statistical analysis should be applied in order to determine the cause-effect correlation for these variables. interventional studies will be conducted in the following months. cerebral vasospasm is a, potentially, life threatening phenomenon after aneurysmal subarachnoid hemorrhage (sah). as part of "triple h" therapy, fenylephrine and norepinephrine in combination with dobutamine and dopamine, are used most frequently to elevate systemic arterial blood pressure (abp) in order to preserve optimal cerebral blood flow. in obtaining increased arterial blood pressure during episodes of vasospasm we altered medical therapy from fenylephrine to norepinephrine but experienced an increasing incidence of paralytic ileus. in a retrospective cohort study we evaluated clinical outcome and the incidence of paralytic ileus. methods. in - , a consecutive series of patients had surgery (aneurysmal clip ligation) within hours after sah. patients with clinical vasospasm, were subdivided into two groups with respect to medication used. group a (n= ) was treated with a combination of dobutamine, dopamine and fenylephrine (mean increase syst. abp . ± . mm hg). in group b (n= ) norepinephrine was used instead of fenylephrine (mean increase in syst. abp . ± . mm hg). we compared basic variables of the two treatment groups, and investigated the clinical outcome using the glasgow outcome scale (gos), one year after initial sah. complications were registered and compared between these treatment groups. results. the two treatment groups were evenly matched concerning age (p= . ), wfnsscore at admission (p= . ), amount of subarachnoid blood on ct-scan (fisher) (p= . ), and observed prognostic variables as hypertension (p= . ) and smoking (p= . ). the clinical outcome, was not influenced by the kind of medication used (p= . ). the incidence of paralytic ileus differed between the two groups (group a: / vs group b: / , p< , ). paralytic ileus occurred mainly in patients treated with norepinephrine ( / = . %, odds ratio= . ). no relationship was found in height of systolic abp or dosage of norepinephrine administered to these patients. we observed a significant difference in duration of administration of norepinephrine in patients, who did develop a paralytic ileus (see - . / - . p= . table: norepinephrine use in cerebral vasospasm; patients with or without paralyticileus. conclusion. -the use of norepinephrine in patients with cerebral vasospasm after sah did not influence clinical outcome, although higher blood pressure levels were reached. -norepinephrine administered during longer periods than days, increases the risk of paralytic ileus.-fenylephrine is recommended in the treatment of cerebral vasospasm after sah. amigues l. , klouche k. , massanet p. , canaud b. , béraud j. j. intensive care unit, lapeyronie university hospital, montpellier, france introduction. slow discontinuous ultrafiltration (sduf) is nown recognized as an effective complementary treatment for congestive refractory end stage cardiac failure(escf). however, an organic kidney disease is often associated with heart failure and may worsen the prognosis. this study was undertaken to compare the effects of sduf in escf patients with and without previous kidney disease. methods. patients fullfilling escf criteria with fluid overload and oliguria (grade iv nyha) were treated by sduf. sduf was performed with a double head pump monitor (bsm , hospal), blood flow rate: - ml/mn, ultrafltration rate: . to l/h. vascular access was provided by femoral silicone twin catheters. a renal replacement therapy was institued when indicated. age, sex, cardiopathy, and nephropathy were collected in each patient. patient follow up before and after sduf: systolic arterial pressure, heart rate, diuresis, total fluid volume removed, cardiothoracic index, creatinemia, blood urea nitrogen, natremia, natriuresis, mortality and average survival. datas were compared between two groups: group without and group with nephropathy. mean age was ± yo and sex ratio was / , . myocardiopathy origin was: ischemia ( ), hypertension ( ), valvulopathy ( ), primary non-obstructive cardiopathy ( ), multifactorial ( ). oliguric renal failure: fonctional in patients (group ) and associated with mild chronic nephropathy in patients prior to heart attack (group ). no significant differences in clinical and biological datas were observed between the two groups except for blood urea nitrogen: , ± in group vs , ± , mmol/l in group . during scud, hemodynamic stability was observed in both groups; diuresis and natriuresis significantly increased and remained stable at the end of the treatment despite significant decreased diuretic doses. mean sessions of sduf was , ± , in group and , ± , in group (ns). renal replacement therapy was institued in both groups but the number of sessions was significantly higher in group : , ± , vs , ± , . mortality during hospitalisation was % in group and %in group . from the surviving patients, / patients in group and / patients in group underwent a chronic hemodialysis treatment. average survival was higher but not significant in group ( , ± vs , ± months). our study sugests that sduf remains a long term beneficent treatment for patients with both escf and renal failure. paradoxically, prognosis is slightly better than in patients with isolated refractory congestive heart failure. organic renal failure could artificially worsens cardiac function by increasing diuretic resistance which may be improved by sduf. ( ). this study aims to evaluate the influence of factors affecting renal blood flow including map, cvp, pulmonary artery wedge pressure (pawp), cardiac index (ci), systemic vascular resistance (svr) and pulmonary vascular resistance (pvr) on immediate graft function. methods. consecutive patients undergoing live-related kidney transplant were included. prior to anaesthesia, a . f continuous cardiac output pulmonary artery catheter was placed via the right internal jugular vein. a continuous cardiac output monitor (baxter edwards px ) was used for haemodynamic monitoring. baseline values of the map, cvp, pawp, ci, svr and pvr were computed. data was collected at -minute intervals thereafter and immediately before and after release of the vascular clamps. the ischemia time, intravenous fluids, dopamine use and blood transfusion were noted. if warranted by low preoperative haemoglobin or increased surgical blood loss, blood was administered as ml packed cell units using leukocyte filters. the outcomes chosen for graft function were the urine output on table (uo-ot), -hour urine output (uo- ), fall of serum creatinine from the preoperative value on day (creat- ) and day (creat- ). using spss statistical software, multiple linear regression analysis was done to find the variables significantly affecting the outcome. results. the only variable found to have a statistically significant influence on uo-ot was the map. no variable had any effect on the uo- . blood transfusion had a negative influence on the fall of creatinine on day and day ( since clinically adopted during cardiac surgery, cardiopulmonary bypass (cpb) has been implicated in complement activation and postoperative acute phase reaction. corticosteroids are usually employed as an attempt to dampen these phenomena and related postoperative morbidity. methods. after informed consent previously approved by the local ethical committee, we included adult patients submitted to cardiac surgery under cpb at a non-emergency setting. preoperative risk stratification employed euroscore (es) and cleveland clinic score (ccs). methylprednisolone (mp) - mg/kg, was added to cpb priming solution for group (n= ) but not for group (n= ). blood samples were collected from all patients at anaesthesia induction (t ), (t ), (t ) and -hour postoperative (t ) for measurement of total c and c-reactive protein (crp) levels, by nephelometric technique. postoperative multiple organ score (mods) were daily registered. results. the groups were considered comparable concerning to preoperative risk stratification, length of cpb and postoperative organ dysfunction at h postoperative (mod , as well. starting from similar levels of c and crp, we did not observe significant differences between groups and concerning to postoperative levels of c . nevertheless, patients treated with mp (group ) exhibited higher crp levels at h postoperative, as shown bellow: . ± . . ± . . perioperative administration of mp failed to show evidences of beneficial effect over postoperative organ failure and complement activation. the acute phase response, expressed as crp systemic levels, instead of softened, was significantly enhanced among patients to whose cpb priming solutions was added mp. these results support a larger randomised trial to reassess routine use of corticosteroids during cpb. objective: to evaluate the influence of enteral application of an immunoglobulin enriched bovine milk preparation on endotoxin plasma levels, endotoxin neutralizing capacity of plasma (enc) and the acute phase response (il- , crp) during and after cardiac surgery in a pilot study. design, patients and methods. patients who were treated by coronary bypass operation were enrolled in a controlled randomized study. the patients were treated by enteral application of g of a bovine colostrum milk preparation per day for days preoperatively. endotoxin and enc were sequentially determined intra-and postoperatively by a chromogenic modification of the limulus amebocyte lysate test. interleukin- , crp, transferrin, alpha- macroglobulin, albumin, apo-a, apo-b, igg, iga, igm were determined by "elisa" and nephelometrically. the clinical course was followed up by daily evaluation of the apache-ii-score. main results: demographic data were comparable in both groups. no differences of the apache-ii-score ( . . verum group, and . , control group, on admission) were observed. endotoxin plasma levels and enc showed high levels at the end of the procedure and seemed to have a trigger function for the acute phase response but were not significantly reduced throughout the observation period in patients receiving the milk preparation as calculated by comparing the area under the curve. plasma levels of endotoxin binding proteins did not differ significantly. plasma levels of il- increased to maximal median values of pg/ml in the verum and pg/ml in the control group and h after surgery. a tendency to lowered il- levels was observed throughout the whole observation period for the verum group. crp-levels showed their maximum values h after the procedure and were significantly reduced in patients of the verum group (p = . ). conclusion. this study revealed that endotoxemia occurs early during an elective surgical intervention, which is followed by a subsequent increase in mediators of the acute phase reaction. the prophylactic enteral application of a bovine milk preparation for two days in cardiac patients did reduce postoperative crp-plasma levels but contrary to a former prospective double blind study in abdominal surgery did not reduce perioperative endotoxemia. one reason could be the too low application of the bovine colostrum milk preparation. to compare a possible effect of improved therapeutical approaches in head trauma, epidemiological data should be compared at certain time points. due to the legal obligation to document all in-patient treatments in germany and to forward these data in an anonymous form to the office for statistical affairs (statistisches bundesamt) it is possible to provide a nationwide epidemiological analysis of head trauma and to compare the yearly obtained data. the incidence, the mortality, and the duration of hospital stay for the treatment of all hospitalized patients suffering from head trauma were calculated and compared to the data from while considering the data obtained from the office for statistical affairs in bonn and wiesbaden. the data were investigated while separating them according to the international classification of diseases (icd- ; no. - and - ). to further elucidate the causes of altered mortality and duration of hospital stay the number of cts and mris in german hospitals in and were compared. in addition, data indicating the number of patients admitted to neurological rehabilitation centers were analyzed. the incidence of head trauma did not change between and and was calculated to be at / . . the mortality, however, decreased from . / . in to . / . in ( vs. patients). in addition, the duration of hospital stay declined in all icd- encoded subgroups including mild brain trauma. this could be due to the increased number of ct devices and mris in german hospitals (ct: vs / mri: vs ) while comparing and . the number of patients transferred from hospitals to neurological rehabilitation centers increased from in to in (+ %). it could be speculated that both improved knowledge on the field of brain trauma therapy and a higher number of technical devices allowing rapid diagnosis of brain injury or potential intracranial complications following head trauma accounted for the reduction in mortality due to brain trauma in germany from through . the decline of the duration of hospital stay especially in patients with more severe head injury could also be due to a more rapid transfer of patients with head trauma from hospitals to rehabilitation centers. therapeutic hypothermia may improve outcome in patients with severe head injury, but clinical studies have produced conflicting results. we hypothesised that severe side effects of artificial cooling might have masked positive effects in earlier studies, and treated a large group of patients with severe head injury with hypothermia, using a strict protocol to prevent the occurrence of cooling-induced side effects. methods. consecutive patients admitted to our hospital with severe head injury (glasgow coma scale (gcs) < ) in whom icp remained above mmhg in spite of therapy according to a step-up protocol described previously [ ] were included in our study. those who responded to the last step of our protocol (barbiturate coma) constituted the control group (n= ). those who did not respond to barbiturate coma (n= ) were treated with moderate hypothermia ( oc- oc). average apache ii scores were higher, and average gcs at admission slightly lower in the hypothermia group, indicating greater severity of illness and more severe neurologic injury. predicted mortality was % for the hypothermia group vs. % in controls. actual mortality rates were significantly lower: % vs. %, p< . . the difference in overall mortality between hypothermic patients and controls was statistically significant (p< . ). the number of patients with good neurologic outcome was also higher in the hypothermia group: . % vs. . % for hypothermic patients vs. controls, respectively (p< . ). these differences were explained almost entirely by the subgroup of patients with gcs of or at admission (mortality % vs. %, p< . ; good neurologic outcome % vs. %, p< . ). artificial cooling can significantly improve survival and neurologic outcome in patients with severe head injury, when used in a protocol with great attention for the prevention of side effects. these effects are especially clear in patients with gcs of or at admission. because there is likely to have been bias against the hypothermia group in this study, the positive effects of hypothermia might even have been underestimated. introduction. s b, a glial calcium-binding protein, is a serum marker of cerebral damage. posttraumatically, however, s b in increased in all patients suffereing from hemorrhagic-traumatic shock, regardless of whether trauma is cerebral or extra-cerebral. the aim of this experimental study was to determine whether the posttraumatic s b increase is caused by extra-cerebral trauma or by hemorrhagic shock and whether it is influenced by the severity of shock. hemorrhagic shock was achieved by bleeding anesthesized rats to a mean arterial pressure (map) of - mm hg through a femoral catheter and maintaining this map until incipient decompensation. subsequently, map was either increased immediately to - mm hg (moderate shock) or maintained at - mm hg until % of shed blood had been returned (severe shock), and then increased to - mm hg. resuscitation was provided after - mm hg map had been maintained for min. trauma was achieved by midline laparotomy. hemorrhagic-traumatic shock caused an early s b increase at the onset of decompensation. s b in serum was highest at the end of the min. period during which map was maintained at - mm hg and was significantly higher at all time points after severe shock than after moderate shock. in contrast, trauma (laparotomy)without hemorrhagic shock did not cause any increase of s b in serum. the posttraumatic s b increase in serum appears to be caused by hemorrhagic shock rather than by extra-cerebral trauma. regardless of whether the source of s b is cerebral, indicating cerebral damage linked to shock, or extra-cerebral, the main determinant in the clinical setting remains the severity of shock. romera m. a. , chamorro c. , silva j. a. , pardo c. , marquez j. , ortega a. intensive care unit, clínica puerta de hierro, madrid, spain in patients with non-traumatic subarachnoid hemorrhage (sah), the development of myocardial abnormalities has been widely described. however, the true incidence of myocardial injury in this group of patients is unknown yet. we analyze the incidence of myocardial injury, in this population, using cardiac troponin i (tn i) assay and also we assess if the increase in tn i concentration has prognostic value. prospective study, including all patients with non-traumatic sah admitted to our intensive care unit (icu), from december to december . serum tn i concentration was measured, at least once, within the first hours after onset of symptoms. inmunoassay based on the "sandwich" principle was employed. the chi-squared test and fisher exact test were used for statistical analysis. of the patients admitted, were excluded ( admission later than hours, absence of tni determination, previous cardiopathy or renal failure ). eighty-two patients were included in the study ( women ). mean age ± years. the tni concentration was increased in / patients ( % ). sixteen ( . % ) patients died in icu. twelve of the ( % ) with a high tni concentration and / ( % ) with a normal tni concentration died [ relative risk (rr) . ( . to . ; % confidence interval (ci); p< . ]. thirty-seven ( % ) patients had a hunt-hess (hh ) grade greater or equal to iii. poor grades of sah ( hh>or = iii ) were associated with a higher incidence of raised tni concentration ; ci %); p< . ]. among this group of poor grade patients, elevated tn i levels were associated with a higher mortality [ / ( %) with a raised tni compared with / ( % ) with a normal tni concentration; rr ( . - ; ci % ); p< . ]. however, mortality in every case was related to neurological problems. seven patients ( . %) suffered from pulmonary edema and all had elevated tni levels. echocardiography was performed in all patients, being abnormal in of them. conclusion. in our series, the incidence of myocardial injury in sah was %. this cardiac injury was more frequent among patients with severe grades of sah. elevations in tn i levels had prognostic value, being associated with a higher mortality. therefore, we should closely monitor those patients with sah who develop an increase in the tni levels. renaud e. , matéo . j. , benlolo . s. , payen . d. dept of anesthesiology and critical care, lariboisiere hospital, department anesthesiology intensive care, lariboisiere, paris, france respiratory failure is one of the major complication of acute stroke ( ). we have investigated the impact of the location stroke on respiratory failure incidence, cause of intubation and outcome. we reviewed consecutive patients with acute stroke admitted to icu from to . following data were collected, glasgow coma score (gcs), cause of icu admission, presence of acute respiratory failure (arf), reason for intubation, presence of aspiration, length of mechanical ventilation (lomv), severity of hypoxia, length of stay in icu (los) and mortality. continuous data were compared by paired t-test and nominal data by chi-test. explicative variables for arf were assessed by univariate analysis. . patients had a middle cerebral artery (mca) stroke and had brainstem stroke (bs). age (mca ± sd yrs vs bs ± sd for), gsg score (mca ± sd vs bs ± sd for), length of stay in icu ( ± sd days for mca vs ± sd) were not significantly different. % bs and % mca patients were admitted in icu for respiratory failure (p= . ). admission to icu with loss of consciousness was significantly higher in mca ( / , %) than in bs ( / ) (p= . ). indication for intubation was always for aspiration pneumonia that was the leading cause of arf ( . ) associated with swallowing paralysis in bs (p= . ) and to unconsciousness in mca (p= . ). there was no difference for the lomv, the severity of hypoxia between the groups. arf, intubation or reason for intubation were not associated with mortality in the groups (p= . ). the major cause of death was the presence of cerebral herniation in the groups (p= . ). pulmonary complication due to aspiration more predominant in bs than mca stroke, represents the major cause of intubation and arf for bs patients. in the contrary, loss of consciousness in mca stroke group predominates for icu admission. outcome in all patients (mca and bs) was not influenced by presence of respiratory failure or reason for intubation. the major cause of death for stroke's patients is the neurologic state, and especially the presence of herniation. stroke code (sc) is a guidelines of united actuatio between out of hospital enmergency services from barcelone and the most four important hospitals of the city; which aim is to optimize the sequence time for stroke treatment; this allows to increase the number of candidates for reperfusion therapy. the present study aim is to evalute differents times sequences in the acute strokes in which trombolysis has been practised according to the acute stroke code first priority; and secundary to describe findings in the ct scan of these patients pro-inflammatory cytokines, such as tnf and il- are released in the brain within hours after closed head injury (chi). they were shown to have deleterious effects, mainly when active in the early post-injury period. a variety of anti-inflammatory and anti-apoptotic modalities have been shown to ameliorate the outcome of chi. erythropoietin (epo) is a kidneyderived cytokine regulating haematopoiesis both by acting as a growth factor and by inhibiting apoptotic cell death. recently it has been shown to be produced in cultured neurons, brain astrocytes and neurons under hypoxic/ischemic conditions and in response to oxidative stress. other studies have shown that the erythropoietin receptor (epor) is present under normal conditions on neuronal and brain capillary endothelial cells. epo has been found to have newly discovered neuroprotective properties in different models. these models include neuronal cultures against glutamate toxicity, global glutamate toxicity and rodent models of cerebral ischemia. in addition it induces brain endothelial cell proliferation and stimulates neovascularization in vivo. the present study was designed to test the protective effects of epo in rats udergoing controlled chi. methods. chi in rats was induced using a weight-drop device. clinical status was evaluated by the neurological severity score (nss), which tests tasks including reflexes, behavior and motor activity. a point is awarded for failing to perform a task so a higher score corresponds to a more severe trauma. study animals were treated with doses of i.p. units/dose ( ml) of rhu-epo, h and h after chi (treatment group) or with ml of vehicle injected i.p. at the same time points (control group). nss was evaluated by an observer blinded to the different groups at , and days post chi. nss scores were compared using a two tailed student t-test. control and study rats were subjected to chi of similar severity, ( h nss . + . and . + . respectively, p= . ) and followed at d, d and d following chi. clinical recovery was facilitated in the treatment group starting at h after chi and reached statistical significance at days post chi. the treatment group's d nss was . (n= ) vs. . in control animals (n= ) p= . . the present findings point to a neuroprotective role of epo in traumatic brain injury. brain tissue of treated and control animals is currently being analyzed for parenchymal cytokine levels. we have examined the role of post trauma treatment with epo of rats undergoing chi. as has been shown in other models of brain injury (stroke, ischemia, glutamate toxicity) epo seems to have a neuroprotective effect in head trauma. the exact mechanism of this protection has yet to be elucidated. this is the first time, to our knowledge, that epo has been studied in an animal model of traumatic head injury. ( ) ( ) ( ) is . %. it is difficult to know how to apply these figures to individual patients. we have used the anaerobic threshold in a prospective observational study to try and identify patients with an increased risk of mortality. forty-five patients scheduled for elective aaa repair had their anaerobic thresholds measured pre-operatively. the anaerobic threshold is the patient's oxygen consumption in ml/kg/min when anaerobic metabolism occurs(reference ). it is calculated by using a bicycle ergometer and a metabolic cart. clinical presentation and evolution of valvular heart disease (vhd) patients have great significance in determining the best moment for surgical correction but lacks correlation with surgical outcome in most cases. this study tries to determine the preoperative variables associated with mortality in the course of surgical treatment of vhd. cohort study conducted from january to february . inclusion criteria: patients submitted to vhd surgery during the period of study. exclusion criteria: vhd surgery combined with non -vhd procedures. data were analyzed with chi -squared, fisher and mann -whitney tests. one hundred five patients met the inclusion criteria. the preoperative variables associated with surgical mortality were: systemic arterial hypertension (p= . ), peripheral vascular disease (p= . ), redo (p= . ), age (p= . ), blood creatinine level (p= . ), left ventricular dysfunction (p= . ). conclusion. based on these data, efforts will be held in order to develop a prognostic score index for mortality in vhd surgical patients. in our setting, the diffusion of institutional education in basic cardiopulmonary resuscitation (bcpr) is low. the number of patients admitted to our units after resuscitation following cardiac arrest is rising due to the population demand on the out-of-hospital emergency services, . the patients with neurological sequelae secondary to incorrect bcpr in the first minutes are common. through the association of ex-patients of the intensive care medicine department, and with the psycho-social support of voluntary helpers on patient discharge, relatives are offered bcpr as part of the quality care programme. every three months, professionals from the department organise this course for relatives in the form of a hour module. the concepts of the prevention of ischaemic heart disease are presented together with the content of the national plan for bcpr. practical sessions are undertaken in small groups of to persons, using dummies. a total of relatives in courses have received this training over the past years. the mean age of the students was . (sd ) years ( - ), % women, % with middle and higher education, % housewives, and % manual labourers. the evaluation of the scores obtained in the item test before and after the course is shown in the tables below. multiorgan system failure (mosf) is an infrequent but very serious complications after cardiac surgery, with high rates of mortality. this study was undertaken to determine the frequency, prognosis and risk factors for mosf this study was performed in a twelve-bed cardiac surgery intensive care unit over a -month period. all adult consecutive patients undergoing coronary, valvular and combined (valvular and coronary) surgery were prospectively studied (n = ). all patients were assessed by the "modified" parsonnet score results. mosf developed in ( . %) patients, of whom ( . %) died. this was the main cause of overall hospital mortality ( / , . %). in a logistic-regression anlysis, the development of sepsis, postoperative low cardiac output syndrome, mechanical ventilation more than hours, a "modified" parsonnet score more than and and preoperative ventilatory support were independantly associated with the development of mosf. an organ system failure index (osfi) of or more was most significantly associated with icu mortality (p< . ). conclusion. in our series mosf was a leading cause of mortality after open-heart surgery. the development of mosf with an osfi of or more was the main predictor of postoperative mortality. we studied patients who underwent cabg surgery. fifteen patients ( male and female) were younger than years and patients ( male and female) were older than years. perioperative death occured in one patient from the < years group and in patients from the > years group (p=ns). categorical data were compared using the chi-square test and numerical data were analysed using the student t-test. differences were considered significant at p< . . in a n investigation conducted in ours icu, % of patients hospitalised after elective cardiac surgery presented a pain score > ( min score ; max ) . these results were considered inadequate. a quality improvement initiative was undertake. the aim of the present study was to test if pain evaluation and treatement improved following pain guidelines implementation in a surgical icu. the design consisted in observing de pain evaluation both before and month after implementation of guidelines. these guideline are divided in two item : first introduction of a regular pain evaluation using a visual analogue scale (vas) and second in a proportional vasderived analgesic prescription protocol. recommendation were given during repetitive meetings, feedback sessions and regular poster information on the icu walls. pocket guideline and vas tool was distributed. pain intensity evaluation of the nursing team was checked by an independent observer and compared with the nurses-charted vas. improvement of pain control was tested based on the following criteria: utilisation of the algorithm at least twice per working shift; corresponding analgesic drug to observed vas; and follow up check of vas after analgesic administration. the independent observer measure vas at a.m. and at p.m. postoperative day and . proportion of algorithm adherence before and after introduction of the recommendation were tested using fisher's exact test. variance of median vas was tested using mann-whitney test. these preliminary results indicate that the implementation of an algorithm on pain intensity evaluation and treatment increases the number of pain evaluation and re-evaluation after drugs administration. although the administration of analgesic drugs increased, the number of patients with insufficient pain treatment stays still high. the prognosis of liver transplant has improved the last few years due to advance in surgical techniques and immunosuppressive regimes, but early complications show a high prevalence affecting morbi-mortality in these patients a beds icu in a teaching rd level hospital. prospective observational study on all patients with the mentioned condition treated in our centre from october to october . follow up during icu stay. we have collected data from patients ( grafts) with a mean age of . ± . years, . % women, mean apache ii on admission ± . , median child score , ( - ) and mean sofa score . ± . . surgical data were as follows: fluids balance ± , hours of graft ischemia . ± . , reperfusion syndrome in % and fibrinolysis in . %. at admission mean core temperature was . ± ºc. median icu stay . ( - , max. ) days and median hours under mechanical ventilation ( . - , max ). the prescribed immunosuppression was cyclosporine in % and tacrolimus in % of patients results. icu mortality was . % ( patients). complications were present in % ( . % of them more than two episodes). patients had to be reoperated, one because early graft dysfunction treated with mars and retransplantation (death because a new graft dysfunction), and the other because abdominal haemorrhage. one patient developed an early rejection. metabolic complic . % (high insulin requirements . %) -renal failure . % (renal replacement , %)-cardiac complic . % (chf . %, hbp . %) -respiratory complic . % ( . % sdra) -bleeding % -neurological complic . % (myelinolysis patient) -infection . %. patients who died had higher apache ii, child and sofa scores, lower serum albumin levels, longer graft ischemia, higher percentage of fibrinolysis and reperfusion syndrome during surgery and higher percentage of acute renal failure an need for renal replacement (not statistical analysis due to the low mortality rate we report the effects of substitution with a virus-inactivated protein c (pc) concentrate in disseminated intravascular coagulation (dic) in preterm infants and children with sepsis (meningococcal in the children and aldolecent; staphylococcal and enterobacter in the preterms) associated with purpura fulminans. this was a prospective open-label study. a total of patients, paediatric and adolescent patients age . to . years with dic associated with severe acquired pc deficiency (range . to . iu/ml; median, . iu/ml) in meningococcal septic shock and purpura fulminans; and preterm infants with severe acquired pc deficiency (range . to . iu/ml; median, . iu/ml) due to staphylococcal and enterobacter sepsis were studied. replacement therapy was initiated with a virus-inactivated pc concentrate with an initial intravenous bolus of to iu/kg followed by iu/kg up to six times per day as an adjunctive therapeutic regimen to otherwise optimal intensive care treatment. after initial pc administration, plasma pc levels rose to normal ranges and were maintained under pc replacement therapy. improving or even normalising global hemostatic parameters were assessed in all patients. markedly elevated plasminogen activator inhibitor type (pai- ) levels prior to treatment, reflecting a reduced fibrinolytic potential, decreased rapidly under pc substitution. concomitantly improving signs of purpura fulminans reflected by decreasing size of skin lesions, demonstrated a restoring microcirculation. seven of the nine paediatric and all of the neonatal patients survived. one patient (paediatric) required limb amputation; two patients died because of multiorgan failure. both presented with a severely low plasma pc activity of . iu/ml on admission to the hospital. no adverse effects were observed with the pc concentrate administration. ait can be concluded that the administration of pc concentrate had a marked benefit on the deranged coagulation status of patients with purpura fulminans and septicaemia. normalisation or even partial correction of haemostasis as well as improvement of microcirculation accompanied by improving signs of purpura fulminans were demonstrated in all patients the main purpose of this study is to report medical and surgical complications of spine surgery in a third level universitary pediatric hospital with a reference spine surgical program. methods. study design is a retrospective clinical series of pediatric spinal surgeries.all spine surgeries performed on children under years of age between january and january were included. patient were grouped in four diagnostic categories (idiopathic, neuromuscular, congenital scoliosis and miscellaneous) and procedure performed (posterior (p) fusion, anterior/posterior (ap) fusion, anterior (a) fusion, (iw) instrumental withdrawal). next data were recorded from clinical chart:age, gender, needs of transfusion products, volume demands during first postoperative day,days on mechanical ventilation,medical and surgical complications. results. study sample included patients, female and male. age ranged between and years with average of . years. characteristics were: idiopathic , neuromuscular ,congenital scoliosis ,miscellaneous . procedures performed were:p fusion ,ap fusion ,a fusion ,iw . .average lenght of stay in pediatric intensive care unit were . days (range - ).average days on ventilatory support . ( range - . ). no patient required intubation after weaning.major complications were: deep wound infection( ), respiratory distress( ), large intraoperative blood loss ( ),and paraplegia ( ).no deaths were observed.minor complications were: atelectasis( ), pleural effusion( ), pneumonia( ), pneumotorax( ), superficial wound infection( ), urinary tract infection( ) and electrolitical disturbances( ). postoperative transfusion needs were . ml/kg ( % confidence interval (ci) . - . ) for ap fusion, . ml/kg ( % ci . - . )for p fusion; a fusion and iw doesn't need postoperative blood replacement. total blood transfusion was . ml/kg ( % ci . - . )for ap fusion, . ml/kg ( % ci . - . )for p fusion; . ml/kg for a fusion and . ml/kg for iw.volume demands(no blood products)during first postoperative day were . ml/kg ( % ci . - . )for ap fusion, . ml/kg ( % ci - . ) for p fusion; ml/kg for a fusion and . ml/kg for iw. conclusion. spine surgery has few major complications rate in a reference spine surgery pediatric hospital. minor respiratory complications affect % of our patients without repercussion in outcome. total blood loss is greater in ap fusion than in other procedures, but postoperative blood replacement in picu didn't differ between procedures. background elevated intra-abdominal pressure (iap) adversely affects pulmonary, cardiovascular, renal, splanchnic and central nervous system physiology, and it determines the common clinical picture called "the abdominal compartment syndrome". nevertheless the direct monitoring of iap is not always practicable, because it requires an abdominal drainage. a lot of authors demonstrated in the adults that the bladder pressure is a reliable index of iap, but there are not studies on pediatric population. the aim of this study is to evaluate the level of significance of this index in a pediatric population. population: we enlisted a group of pediatric patients, sedated and paralysed ( oltx, abdominal surgery, cardiac surgery), age . ± , (range - ) months. methods. the bladder pressure was measured with the patient in supine position, with a trasduction circuit connected to the bladder catheter and to the abdominal drainage ( jpratt, pig tail, catheter for peritoneal dialysis). to obtain a good transduction of pressure, a volume of saline was pushed into the bladder. the volume of saline was variable according to the weigth and age: we obtained a scheme (table ) from our empirical evaluation of the pediatric bladder compliance and urodinamic data. table , there aren't significative differences between the level of pressure measured in the bladder and in the peritoneal cavity ( p= . ). mean: , ds: , from to , pediatric patients (age range . to years, mean . years) were treated using nppv during distinct episodes of acute respiratory failure (arf) of neuromuscular origin. in all patients immediate intubation for an acute, life-threatening presentation was avoided and respiratory status improvement was achieved. few data are available up to now about nppv application and indications in the acute setting in infants affected by neuromuscular disorders (nmd). a prospective observational study was carried out on non-consecutive neuromuscular patients admitted to picu because of arf and managed with nppv in the acute phase;remarkably, out of were < months aged. all the patients were treated by a flowtriggered intensive care mechanical ventilator (siemens servo ventilator, siemens-elema, sweden) through a tight fitting face mask. nppv was administered for at least hours postadmission. a pressure-control mode was adopted for better compensation of leaks around the mask. flow-sensitive trigger permitted a better synchronization of patient's spontaneous breathing, limiting the need for deeper patient sedation (low-dose midazolam drip). initially, a relatively low ventilator frequency delivery was set ( - b/min). peak inspiratory pressure was tritrated upward to obtain an exhaled tidal volume of - ml/kg maintaining a paco value < mmhg and a ph > . ; peep value was adjusted to maintain an oxygen saturation > - % with a required fio < . . results. all patients were referred to picu on spontaneous breathing: those admitted with et tube already positioned were not considered eligible for this study. an oxygenation improvement was obtained in all patients within hours from the onset of nppv . the pao /fio increased from . ± . to . ± . (p< . ) and . ± . (p< . ) on selected time points ( and hours after nppv introduction, respectively); conformly, alveolar-to-arterial oxygenation difference (a-ado ) decreased from a . ± . to . ± . (p< . ) and . ± . (p< . ) respectively. conclusion. nppv resulted a safe and effective therapeutic approach in both hypoxemic and hypercapneic arf episodes in this children group affected by nmd. even in cases of emergency presentation or when resuscitation is needed, it is of importance to identify nmd children with residual ventilator-free breathing ability thus performing a nppv trial. life-threatening respiratory distress and young age should not preclude nppv application in a picu setting. pulse oximeters are widely used in paediatric intensive care but they have some severe limitations. the technique relies on the presence of adequate peripheral arterial pulsations, which are detected as photoplethysmographic signals (ppg). when peripheral perfusion is poor as in states of hypovolaemia, hypothermia and vasoconstriction oxygenation readings become extremely unreliable. hence, pulse oximetry becomes unreliable in a significant group of children just at the time when accurate readings are most needed. to overcome this limitation, the oesophagus has been investigated as a potential measurement site on the hypothesis that perfusion may well be better preserved at this central site. studies on adult patients have shown that measurable ppg signals at red and infrared wavelengths can be detected within the whole depth of the oesophagus. a new system to investigate the quality of oesophageal ppg signals is being constructed with the aim of developing a neonatal and paediatric oesophageal pulse oximeter. a reflectance optical sensor has been constructed comprising miniature infrared and red emitters and a photodetector. the sensor was design to fit into a conventional disposable transparent stomach tube, french gauge. the oesophageal ppg sensor within the stomach tube was inserted through the nose into the oesophagus of a kg, day old neonate. the stomach tube was advanced into the oesophagus under direct vision until the probe was cm from the nose. ppg traces from the oesophagus were recorded for approximately minutes at this depth on a laptop computer. measurements were repeated at and cm from the nose. measurable ppg traces of good quality were obtained in the oesophagus at all three depths. the ppg signals in the mid to lower region of the oesophagus on average had larger amplitudes at both red and infrared wavelengths than the ppgs recorded in the upper oesophagus. artefacts on both wavelengths due to oscillations as a result of high frequency ventilation. filtering successfully eliminates the artefact. the new oesophageal reflectance optical sensor has allowed ppg measurements to be made within the whole length of the neonatal oesophagus. the red and infrared wavelengths used are suitable for pulse oximetry. these results are the first to demonstrate that pulse oximetry may be feasible in the neonatal or paediatric oesophagus. further studies are required to develop a neonatal/paediatric pulse oximeter. we used protein c (ceprotein; baxter -immuno) in two patients with moderate or severe, therapy-resistant vod. . patient (e.g, swiss, , y) suffered from an acute myelogenous leukemia (m with t( ; ) of early infancy after complete first remission by conventional chemotherapy an allogeneic stem cell transplantation with a matched unrelated donor was performed. conditioning comprised busulfan, vp and cyclophosphamide. patient (m.k, iranian, y) suffered from beta-thalassemia major with secondary moderate hemosiderosis, as well as chronic persisting hepatitis c infection with liver fibrosis. he received a matched related bone marrow transplantation, using i.v. busulfan , reduced cyclophosphamide dose and fludarabine. both patients received low dose heparin ( iu/kg) and antithrombin iii substitution. in addition, pat. got prophylactical defibrotide ( mg/kg) and n-acetylcystein. two weeks after transplant both patients developed vod (severe (pat ); moderate to severe (pat ))with weight gain, hepatomegaly, massive ascites and severe thrombocytopenia. maximal bilirubin was mg/dl (pat ) and mg/dl)(pat ). therapy with defibrotide ( mg/kg) was started immediately. in pat. the pulmonary situation deteriorated rapidly with massive aszites and oxygen need and a reversed portal venous flow. defibrotide was stopped after days. thrombolytic therapy using rtpa and a continuous pc substitution (pc level %; bolus iu/kg, followed by iu/kg every h)) were started. lysis therapy had to be abandoned due to respiratory tract bleeding global coagulation (pt %, aptt sec) and pc level normalized within hours after pc substitution. a normal centripetal portal flow could be achieved by high dose defibrotide ( mg/kg) and continued pc substitution after several weeks. pat. showed only a temporary improvement under defibrotide treatment. due to clinical deterioration (hepatic pain, increased ascites) and low pc level ( %) a continuous pc substitution ( iu/kg every h) was initiated. there was a prompt recovery after adding pc with a dramatic reduction of ascites, weight and abdominal pain within - days after start of pc infusion. elevated bilirubin levels returned to normal in both patients. in our patients neither prophylactic administration of at iii nor of defibrotide were able to prevent moderate to severe vod.our data indicate that pc substitution may be a useful adjunctive treatment in severe vod. until controlled studied will be initiated we recommend a stratified treatment in vod, starting with defibrotide, and adding pc in unresponsive cases. ( ) showed recently that restrictive strategy of red-cell transfusion could be at least as effective as and possibly superior to a liberal transfusion strategy in critically ill patients. the aim of this study was to assess the impact of local transfusion guidelines emphasizing restrictive strategy on patients undergoing heart surgery and the prognostic value of transfusion following those restrictive criteria. methods. two groups of heart surgery patients were compared before and after the introduction of local transfusion guidelines. these guidelines involved general information on blood transfusion risks and obligation for the physician to respect predetermined transfusion criteria (hb < g/dl or > g/dl associated to systolic arterial pressure < mmhg or age over yrs or hr > /min or ci < . l/mim/m² or other associated disease . . . * . . . . * < . mortality (%) * * < . conclusion. introduction of local restrictive transfusion guidelines was associated to a significant reduction in red cell transfusion during the postoperative period of heart surgery. the global morbidity and mortality rates in the whole group of patients were not affected. however patients who required blood transfusion following the restrictive strategy had a worse outcome. transfusion was probably more the consequence than the cause of this worse prognosis. if transfusion was the cause of the worse prognosis, then morbidity and mortality rates would have been higher among patients requiring transfusion during liberal period than in the whole group of patient. ( ) hebert pc and col. a multicenter, randomized, controlled clinical trial of transfusion requirements in critical care. n engl j med ; : - . risk factors and outcome in european cardiac surgery higgins tl. quantifying risk and assessing outcome in cardiac surgery intensive care medicine, hospital del mar, servicio de microbiología to analyze if morbid obesity (mo) is associated with critical pathology in relation to patients undergoing vertical banded gastroplasty (vbg). all critical patients (cp) suffering mo, with a mean body mass index = , , receiving programmed surgical treatment, and admitted in the icu during the next period: st oct. to th feb. , were prospectively included. · surgery procedures. *restrictive: vbg according to masson's technic. *derivative: vbg + gip according to salmon's technic. vbg + gip according to capella's technic. ·type of study: descriptive. -vbg in association to other surgical procedures: cp ( cholecystectomies, right inguinal herniorhaphy, and other procedures). · mortality: cp: -septic shock -multiorganic disfunction · readmission: cp (subphrenic abcess and ards). complications . hypoxemia: cp ( % of the total) . . . . not secondary to hypoventilation: cp ( . %). . . . . associated to hypoventilation: cp ( . %) . need for noninvasive mechanical ventilation: cp ( %) . high blood pressure: cp ( . %). . disturbances of cardiac rythm and conduction: cp ( %). . metabolic acidosis: cp ( %). . other complications: cp ( %). conclusion. -the mo patient undergoing vbg, with or without gip, rather than a patient bound to the reanimation or recovery room, is indeed a patient who requires admission in the icu for, at least, - hours. -hypertension of difficult management and hypoxemia not due to hypoventilation nor shunt are the most frequent complications. -an important percentage of cp requires also mechanical ventilation. -complications related to surgery are exceptional. karlicek a. , haveman j. w. , verhoeven e. , van den dungen j. j. a. m. , tielliu . i. f. n. , hulsebos r. g. , nijsten m. w. n. surgery, groningen university hospital, groningen, netherlands introduction. the mortality in acute abdominal aortic aneurysms remains high. recent series still report a hospital mortality rate of more than % ( , ). despite the large number of published studies on hospital outcome, long-term outcome after icu admission has hardly been studied. here we present hospital survival and long-term outcome in patients with an acute abdominal aortic aneurysm. the records of all patients operated for aneurysm surgery between and were retrospectively reviewed. in patients surgery was performed for an acute abdominal aortic aneurysm. all operation reports were analysed. for complete follow-up the general practitioner was contacted if necessary. after arrival in the emergency department and confirmation of the diagnosis by physical examination and/or ultrasound all patients were immediately brought to the operation room. in our hospital even patients with cardiac arrest on arrival in the operation room are treated without delay, and were thus included in our study. all surviving patients were admitted at the intensive care. in case of postoperative haemodynamic instability, multiple organ failure, sepsis or diarrhea a sigmoidoscopy was performed to assess the presence of ischemia or infarction. three hundred and eight patients were operated for an acute abdominal aortic aneurysm, men and women. operative mortality was % ( / ). calculated from the moment of icu admission, day survival was %. cumulative survival rates calculated with the kaplan meier method at , , and years were %, %, % and % respectively. in patients in whom sigmoid resection was performed, day survival was % compared to % in the other patients. mortality in ruptured abdominal aortic aneurysm remains high, day survival was % in our group. sigmoid resection was associated with lower survival but sigmoidoscopy should be augmented to exclude sigmoid necrosis. outcome in these patients is not invariably poor. long term follow-up shows that also after discharge from the hospital these patients have a high mortality. carvalho a. g. r. , gomes r. v. , santos jr. b. , barbosa o. n. , weksler a. , pontes a. p. , camara a. c. surgical intensive care unit, instituto nacional de cardiologia laranjeiras, rio de janeiro, brazil introduction. prognostic markers developed in europe and north america cannot be applied in latin america where life expectancy is % lower according to world health organization. the objective of this study is to analyze patients profile submitted to heart surgery (hs), type of surgery distribution and the impact of variables, previously reported in medical literature, in the mortality and duration of intensive care unit (icu) stay in a public tertiary hospital. cohort study of patients submitted to hs from january to april . patients profile, type of surgery distribution and many variables were analyzed. variables that were studied: age, gender, body mass index (bmi), body area (ba), preoperative in-hospital stay (preop), extracorporeal circulation (ecc) duration, ventricular function (vf), surgical indication, combined procedures (comb), urgency for the surgery, presence of diabetes mellitus (dm), systemic arterial hypertension (ah) and cigarette smoke (cs). the profile and patients variables were analyzed and compared in two different groups. group a (ga): patients discharged from icu or in-icu stay lower than or equal to days (median in-icu stay in this study). group b (gb): death during icu admission or in-icu stay longer than days. t-student, mann-whitney, chi-squared and fisher tests were used in the statistical analysis. conclusion. there is a rather singular distribution of surgeries in this group. many of the previously described variables showed correlation with mortality or longer admission in the icu. prospective studies will be held in order to adjust these variables and determine new ones more relevant to underdeveloped countries. pierce c. m. , fortune p. , petros a. j. picu, great ormond street hospital, london, united kingdom, picu, royal children's hospital, melbourne, australia there are anecdotal reports of sildenafil, a type phospodiesterase inhibitor, being used to reduce pulmonary artery pressure in children with mainly cardiac induced pulmonary hypertension (pht). we have given oral sildenafil to children on our paediatric intensive care units with pht from various causes. diaphragmatic hernia ( ), avsd ( ) vsd ( ) pda ( ), pphn ( ) pulmonary hypoplasia ( ). the median age of the group was m (iqr - m). were receiving inhaled nitric oxide during sildenafil. median dose was . mg/kg (iqr . - . mg/kg hrly) and duration was days (iqr - ). pulmonary artery pressures were directly measured in of the cardiac children and deduced from doppler echocardiographic measurements of the tr jet in children.results. pap decreased significantly (p< . ) following oral doses of sildenafil (n= ). mean pulmonary/system (p/s) ratios decreased from . to . (n= ) within hours of the oral dose. systemic pressure was unaltered in all children. in one child with pulmonary hypoplasia the p/s ratio was unaffected. oral sildenafil can significantly reduce raised pap in children when there is a reversible etiology. this may be particularly useful in children and neonate with pphn. central venous catheters (cvc) are an important means of securing intravascular access in pediatric intensive care unit patients. one of the major morbidity's in use of cvc is catheter-related infection (cri). the incidence of bacteremia with cvc use is approximately . / catheter days and mortality as high as %. one approach to reduce the incidence of cri has been to decrease catheter bacterial colonization (cbc). reduction in cbc is achieved by coating or impregnating antimicrobial substances into the catheter material. use of minocycline/rifampin treated catheters has been shown to reduce the rate of cri in critically ill patients. the concern in pediatric population is the use of minocycline. tetracycline and its derivatives (minocycline), when used in young children, carry the risk of dental and sceletal abnormalities. the problem of potential eluting of minocycline from minocycline-impregnated catheters may pose a risk for young children. our study examined whether detectable levels of minocycline and rifampin were present in the serum of the pediatric intensive care unit patients with indwelling minocycline/rifampin impregnated cvc. methods. patients admitted to pccu age - years and in need of cvc were eligible for study. six patients were enrolled. each patient had two samples of blood and . ml withdrawn for rifampin and minocycline assays respectively. collection times were at the time of catheter insertion and h thereafter for seven days or until catheter removal, whichever came first. rifampin serum samples were processed prospectively soon after colection by standard hplc. minocycline serum samples were stored frozen in - centigrade and assayed in one batch using reverse phase hplc. results. demographic data are in table. ranges with mean values in ( ). none of the minocycline samples had detectable level of antibiotic. the limit of sensitivity for minocycline was . mg/l. therapeutic levels are . - . mcg/ml. one patient had consecutive samples to with low therapeutic levels of rifampin ( - mcg/ml). therapeutic levels of rifampin are - mcg/ml. rifampin sensitivity was mcg/ml. rifampin has distinct peak time and no interfering substances were identified.sex ( ), the emergence of this consequences required fast and corrective treatment. an inotropic agents commonly used in vlbw infants such as dopamine and norepinephrine in some cases do not produce elevation in blood pressure despite using of very high dose. in this study i would like to exam the influence of hydrocortisone administration in vlbw infants with hypotension unresponsive to stndard catecholamine treatment. i have reviewed the cardiovascular response to hydrocortisone therapy in preterm infants. mean gestation age was . ( - ) weeks, postnatal age . ( - )days, mean birth weight g ( - ). eight of them suffered from respiratory distress syndrome and eight from sepsis. the first line of hypotension therapy was always volume administration (normal saline or albumine) and catecholamine infusion. hydrocortisone at the dose mg/kg was administered when dopamine at the dose mcg/kg/min ( patients or norepinephrine . mcg/kg/min ( patients)failed to normalized arterial blood pressure. pneumococcal meningitis is an important cause of morbility and mortality in children. we describe the epidemiological characteristics and clinical features of pneumococcal meinigitis in children admitted to a children's hospital in barcelona. medical records of children with a diagnosis of pneumococcal meningitis based on identification of s. pneumoniae in the blood or cerebrospinal fluid between january, , to april, , were retrospectively reviewed. results. cases of pneumococcal meningitis were diagnosed in patients. median age was months (range . m- . y). children were younger than years old ( %). male-female ratio was . : . none of the children had a previous immunological deficit. thirteen patients ( %) were pre-treated with antibiotics. the most frequent signs on admission were fever ( %), vomiting ( %), headache and irritability ( %), othalgia ( %) and shock ( %). neurological findings were lowered level of consciousness in patients ( %), signs of meningismus in patients ( %) and arreactive mydriasis in patients ( %). the mean leukocyte counts in blood were /mm and the mean c-reactive protein was mg/l. cerebral spine fluid indices on admission were: white blood cell= ( - ) /mm ; protein= ( - ) mg/dl; and glucose= ( - ) mg/dl. main serogroups were: ( %), ( %), ( %), ( %), ( %), ( %), ( %) and ( %). overall, % of the pneumococcal isolates were penicilin-nonsusceptible, % cefotaxim-nonsusceptible and % were vancomycinnonsusceptible. an initial abnormal cranial computed tomography was found in patients. the median duration of parenteral antibiotic therapy was days. all patients were empirically treated initially with cefotaxime (associated to vancomycin in of them). twenty-six patients ( %) received dexamethasone. the administration of mannitol was necessary in patients ( %) and anticonvulsants were administrated in patients ( %). only patients ( %) needed inotropic support (no longer than hours). mechanical ventilation was required in patients ( %) during a mean of . days (range - ). acute complications were: metabolic acidosis ( / ), disseminated intravascular coagulopathy ( / ), seizures ( / ), siadh ( / ) and diabetes insipidus ( / ). twelve patients ( %) suffered deafness, three patients ( %) hemiparesia and four ( %) were exitus. the mean hospital stay was . days and mean intensive care stay was . days. there is an increased prevalence of pneumococco with decreased susceptibility to penicillin and to cefotaxime. deafness is one of the most common and serious sequelae of pneumococcal meningitis. corticotherapy has reduced the incidence of hearing loss. the new, antipneumococcal conjugated vaccine will confer effective prevention from the age of two months and will reduce the incidence of this meningitis. aims : to analize sedation/anesthesy methods used in our hospital for painfull or unconfortable procedures in children in relation to : )patient confort, )sedation complications, )and efficacy of the procedure a prospective study was conducted from january to march in disconfortable procedures in children. mean age was m ; their asa score was in %, in %, in %, and in %. more frequent procedures were : lumbar punctures (lp), thoracentesis or drainages, central catheters insertion, endoscopys . we identified different groups in relation to methods of sedation/analgesy : -procedures done in the emergency department with local anesthesia; -procedures done with administration of intravenous midazolan+ketamine; -procedures done with anesthesic support. we used the ramsay scale to classify the degree of anesthesia and the serna behavioral scale to classify the reaction to the procedure.results. group (n= ): %patients fighted against the procedure (serna scale ) and in % of the patients, complications of the procedure were found to be related to inadequate sedation. group (n= ): in %, sedation was considered inadequate -serna level (n= ) and (n= )-and in case there were complications of the procedure related to unsufficient sedation; there were ( %)cases of minor complications sedation-related; group (n= ): patient confort and adequacy of the sedation were found in %, with ( %) complications of the anesthesic method.conclusion. sedation/anesthesia were needed for the confort of the patients; only minor complications of sedation/anesthesy were found ; efficacy of the procedure was best achieved with the anesthesic method. introduction. the goals of emergency airway management are to antecipate and recognize respiratory problems and support therapy. the endotracheal intubation ( et) is not a routine procedure and it requests planning and personnel qualified to reduce the complications associated to this technique . the purpose of this study is to evaluate early complications associated with endotracheal intubation methods. data were collected prospectively from february to january in tertiary teaching hospital. the variables were obtained in four age groups: group (> month); group (between month to months); group (between months to months)and group (> months). the data were collected as demographic data, reason for endotracheal (et) intubation, sedation administered, local of et, physician responsible for et, complications associated with airway management. the major complications were defined as technical problems that resulted increased morbidity. minor complications were incidents that should be avoided. the complications were compared between emergency or elective et intubation. statistical analysis by chi-square, fisher exact test . we evaluated ( % female and % male) no consecutive patients. indication for intubation were: respiratory failure ( %), coma or depressed sensorium ( . %), post-operative ( %) and shock ( %). sedation and/or analgesic were used in % of patients and . % did not receive a sedative or analgesic for et intubation. a total et emergency intubation ( we report an outbreak due to rsv in a bedded picu with an annual admission rate of approximately patients, cardiac and medical patients accounting each for % of the population and % surgical.methods. an outbreak is defined as an event in which minimally patients develop bronchiolitis due to rsv following transmission via hands of carers within a limited period of week.nasopharyngeal aspirates were obtained from children with symptoms of lower airway infection, all samples were tested for rsv using the enzyme immuno assay, followed by tissue culture when the assay was negative. rsv positive children were isolated in cubicles and strict standards of hygiene were implemented. introduction.the objective of the study was to investigate the validity of outcome prediction after severe head injury using serum levels of protein s- b and of neuron specific enolase nse. methods.fifteen patients with severe head injury were included in this prospective study ( men and women) mean age yrs ( - ). none of the above patients had spinal cord injury or any other neurological disease. venous blood samples were taken on admission and consecutively the , , , , and day. immunoluminometric assay was used for the specimens. we tried to correlate the s- b and nse serum concentrations with the ct scan intacerebral pathology as well with the age, gender and outcome. results. all patients had elevated s- b and nse serum concentrations, with a gradual reduction towards the th day of icu stay. the mean values of day , for s- b were . ìg/l and for nse were . ìg/l. of day , they were for s- b . ìg/l and for nse . ìg/l. patients who died had the first day mean values of s- b . ìg/l and nse . ìg/l, whereas the survivors had mean values of s- b . ìg/l and of nse . ìg/l (p < . ). there was no strong correlation between the ct scan findings, the initial serum s- b and nse values and the gcs, on admission. conclusion. the protein s- b and nse are biochemical markers that seems to be elevated during the first days of injury, in patients with severe head trauma and could be used as markers of he severity of the injury. if protein s- b and nse could be used as a prognostic factor of the patient outcome, needs more investigation. our study is continued. estimates such as -day survival may be grossly misleading for assessment of intensive care utility. late mortality and morbidity may severely affect overall outcome. we studied -day survival rate in addition to survivors´ general health evaluation and prevalence of signs indicating post-traumatic stress disorder (ptsd). the setting is a university general intensive care unit. during the study period all adult patients who had been intubated and mechanically ventilated for at least hours were included (patients who died before hours are excluded). three to six months after their critical illness, survival data were retrieved from hospital and national registers. all patients surviving at this time were sent a health survey questionnaire (sf- ) and the post-traumatic stress syndrome -questions inventory (ptss- ). results. patients fulfilled the inclusion criteria. the mean age was years, % were women. health questionnaires were returned by ( %) of the survivors at follow-up time. -day survival rate was %, at days survival rate had fallen to %. among the responding survivors the frequency of a response pattern compatible with ptsd was %. survivors without signs of ptsd had sf- mean scores more than standard deviation (sd) below the swedish norm in the domains of physical functioning, role-physical and social functioning. survivors with signs of ptsd scored below non-ptsd survivors in every domain, and were more than sd below the swedish norm in the domains of social functioning, roleemotional and mental health. in total, there were only five persons ( % of respondents) who scored at or above the swedish norm for both the physical and the mental health summary scales. assuming the same outcome in non-respondents this figure would correspond to about % of all the included patients. conclusion. in this cohort of severely ill patients -day mortality was in the expected range but much mortality (another %) occurs in the following weeks, indicating a number of patients who have been subjected to long-lasting care with very meagre benefits. at - months following onset of their disease, survivors show considerably reduced subjective rating of their general health and life quality. as much as % of the survivors show signs compatible with ptsd. it could be estimated that about % of all patients included will both survive and within - months reach a level of general health comparable to that of the general population. the aim of this study is to probe that critically ill patients gender is not associated with differences in severity of illness and related mortality. we had tested the premise in front of a controversial evidence offered by several years of our icu activity. observational study. retrospective analysis using data prospectively collected in a medical-surgical icu of beds, in a teaching reference hospital, from november to july . we analyzed consecutive admissions considering reason for admission, age, icu length of stay, severity of illness (mpm , mpm , saps ii and spanish version of apache iii) and related risk of death. cases were analyzed according to gender and age decades. therapeutic effort was analyzed according nems system. standarized mortality ratio (smr) and its % ci was determined. one thousand and twelve cases out of were women. mean age (sd) was ( ) years. significative differences were founded in mpm prognostic values ( . ± . for men and . ± . for women, p . ). the rest of epidemiological data do not offer significant differences. smr for men was . , and for women . , but % ci overlapped . - . vs. . - . , p ns. the same differences were found when different age intervals were analyzed. only admission diagnostic (ischemic cardiopathy, post cardiopulmonar arrest and multiple trauma with no head trauma) showed greater mortality rates in women, but these differences disappeared when age intervals were considered. in spite of certain confusing data about greater mortality ratios in women admitted to our icu, accurate analysis does not show significant differences in severity of illness, associated prognosis and mortality, and therapeutic effort between male and female. bacterial infection is one of the most frequent and most feared complications in patients with a hematologic malignancy (phm). in a retrospective study, we found that bacteremia precipitating icu admission in phm was associated with a better outcome [ ]. however, it remained unclear whether this finding could be extrapolated to all bacterial infections. the aim of this prospective study was to evaluate whether bacterial pneumonia (bp) and bacterial sepsis or other bacterial infections (bs) had a better outcome compared to non-bacterial or noninfectious complications (nbc) in critically ill phm.methods. consecutive phm admitted to the icu over a year period were categorized into bp (n= ), bs (n= ) or nbc (n= ) according to strict diagnostic criteria by an independent panel of physicians who were blinded for the outcome. the impact of bp and bs on the inhospital mortality was assessed by logistic regression after adjustment for severity of critical and underlying hematologic illness, duration of hospitalization before icu admission and other potentially important prognostic factors. two models were tested, the first using a classical severity of illness score (apache iii) and the second using a score system especially designed for cancer patients (groeger score) [ ] . bacterial infection is one of the more favourable complications precipitating icu admission in phm and is associated with comparable mortality rates as in general icu patients. therefore, reluctance to admit phm to the icu for advance support is unjustified, especially when a bacterial infection is suspected to be the cause of deterioration. ( , ) the cleveland clinic score is the only one, to compile intraoperative data until the timepoint of icu admission.( , ) we wanted to find out, whether the combination of pre and postoperative score, in alliance with additional parameters, improves the predictability of outcome. from from until adult cardiothoracic patients were examined. logistic regression was used for analyzing those variables, dealing with mortality. the selection of significant factors is based on a stepwise forward procedure(p< , ). the accuracy of multivariate analysis is shown as roc(receiver-operator characteristic) curve. . variables, pre as well as intraoperative parameters proved to be statistically significant in the analysis, in the multivariate analysis: both scores, operation and aox time, preop at iii, assessment of intraop course, hb at icu admission, blood loss h< ml and rethoracotomy for bleeding. the pre and the postoperative cleveland clinic risk score were both statistically significant in the uni and multivariate analysis, but their combination improved roc. additional parameters had only little further impact. pre and postoperative cleveland clinic score are reliable in predicting the risk of cardiothoracic patients. adding further intra and postoperative data, risk stratification becomes more precise. the appearance of unexpected intraoperative difficulties was highly significant for adverse outcome. the collection of data should be continued on the icu and therapy should be reevaluated and modified any time. objective: to describe the frequency, etiologies, forms of presentation, and foci of bacteremia identified in patients admitted to the icu. prospective epidemiological surveillance study carried out from april to march . bacteremia was defined as the isolation of a pathogenic microorganism in one or more blood samples. bacteremias were classified into contaminating or true according to clinical manifestations. a descriptive analysis of variables including mean values, ranges, and standard deviations is presented. a total of episodes of bacteremia were identified, of which were true bacteremias ( . episodes per patients). the characteristics of patients with true bacteremia were as follows: mean (sd) age ( . ) years; male sex . %; mean apache ii score on admission . ( . ); and mean length of previous hospitalization ( ) days. in ( . %) cases, bacteremias were acquired in the icu and in ( . %) episodes were polymicrobial. a total of pathogens were cultured. these included gram-positive cocci in ( . %) cases, gram-negative bacteria in ( . %), and fungi in ( . %). initial presentation included severe sepsis in ( . %) cases and septic shock in ( . %). the most frequent origin of intra-icu true bacteremias was unknown in . % of cases (primary bacteremia) followed by catheterrelated bacteremia. crude mortality was . % and bacteremia-related mortality . %. primary bacteremia and catheter-related bacteremia were the most common. a total of . % bacteremias were polymicrobial. gram-positive cocci were the predominant causative pathogens. gyurov e. g. , milanov m. s. , milanov s. g. , neichev p. g. general icu, emergency medicine hospital "pirogov", sofia, bulgaria intensive care units are unique because they house seriously ill patients in confined environments where antibiotic use is extremely common. since our last publication ( ) there is a substantial rise in emergence of nosocomial infection namely gram-positive as well as changes of pattern of emergence. to study the frequency of emergence of nosocomial infection (nci) in intensive care unit (icu) we studied retrospectively data from case records and flow sheets of postoperative patients in our icu during - and compared data with last period. results. of patients in our icu during two years, we include those ( . %) who stayed for more than hours. according to results from cultures we divided them to three groups. group one included ( . %) patients without bacterial growth. group included patients with proved nosocomial infections /nci/. we obtained samples: from urinary catheters ( positive- . %), from tracheal tube ( positive- . %), from blood ( positive- . %), intradermal segments from central venous lines ( positive- . %), and from sputum ( positive- . %). the most common place for emergence of nci in our icu is respiratory tract. on -th icu day the tract became infected in almost % of the patients. the major role among pathogens played acinetobacter spp. ( . %), citrobacter spp. ( . %), p.aeruginosa ( % and serratia spp. ( %). the second place for emergence of nci is "reserved" for blood-stream infections. almost the half of the cultures ( . %) showed bacterial growth. the isolated pathogens were the same: acinetobacter spp ( %), serratia spp. ( %), but there was substantial rise in frequency of emergence of s. epidermidis during the last years (see figure) . its frequency almost equalized that of acinetobacter spp. the other two main sources for nci were urine catheters and cv catheters. they remained on -rd and -th place. group included patients with endogenous surgical wound infections. in this group we obtained samples from surgical wounds and drainages. in . % of cultures showed bacterial growth. during next this figure rose nearly twice ( . %). the leading role played the same acinetobacter spp., citrobacter spp., p. aeruginosa, enterococcus spp. and e. coli. the role of s. epidermidis increased greatly during this period de waele j. j. h. c. , hoste e. , blot s. , colardyn f. icu, ghent university hospital, gent, belgium introduction. intra abdominal infections frequently complicate the postoperative course of patients with acute necrotizing pancreatitis. the objective of this study was to analyze the incidence of pancreatic surinfection after surgery for acute necrotizing pancreatitis, describe its characteristics and identify associated risk factors. we retrospectively ( ) ( ) ( ) ( ) ( ) ( ) ( ) analyzed patients treated surgically for acute pancreatitis. surgical treatment consisted of debridement and postoperative continuous lavage. we recorded demographic characteristics, incidence of organ failure, data on surgical and infectious complications, data on surgical and medical treatment and disease severity by ranson and apache ii score. surinfection of the pancreatic necrosis was present in out of patients ( %). the surinfection was polymicrobial in patients. most of the organisms were gram-negative ( %), the others were gram-positives ( %) or fungi ( %). patients with surinfected necrosis were younger ( y vs. , p< . ), had surgical complications more often ( % vs. . %, p= . ), needed retroperitoneal lavage for a longer time ( days vs. , p< . ), and had a longer hospital stay ( days vs. , p< . ) than patients without surinfection. multivariate analysis demonstrated that age (or . ; % ci: . - . , p< . ) and the occurrence of a surgical complication or ; % ci . - . , p< . ) were independently associated with pancreatic surinfection. the mortality in patients with infected necrosis was higher ( % vs. %, p= . ), although in multivariate analysis no association was found. pancreatic surinfection is high after debridement and retroperitoneal lavage, with mainly gram negative bacteria involved. surgical complications and younger age are significant risk factors for surinfection. the aim of this report is to describe the current status of sap in spanish icu's methods. sap cases are identified in accordance with generally accepted criteria in each icu, such as ranson, imrie, pcr and ct-dynamic criteria. sap was selected from the data base of the national study of spanish nosocomial infection monitoring (envin). this study covered the period from to . envin is an observational, prospective and multicentre study. sap patients hospitalized during more than hours in all the participating icu's have been included in the study. these patients were monitored until their discharge from the icu or up to a maximum of - days. secondary infections have also been monitored. severity is measured by means of apache ii. infections, mortality, epidemiological data and antibiotics used as a means of prevention are described. the statistical analysis used the chi x test for the association of qualitative variables, the student t for the comparison of averages and the % statistical significance level results. patients ( . %) of the , patients monitored by envin were found to have sap. the average apache ii was . and the average stay was . days. the base illness was medical ( . %) and surgical ( %). . % of the patients underwent emergency surgery. ni accumulated incidence was . % and density incidence was / hospitalization days. crude mortality was % and ni-related mortality was %. infections were detected: of abdominal origin ( . %), ventilator-associated pneumonias ( . %), secondary bacteremias related to abdominal infection ( . %), catheter-associated urinary tract infections . %; primary bacteremias ( . %); central venous catheter-associated bacteremia ( . %). a total of pathogens were isolated. bgn . %, cgp . % (mrsa . %), fungii % (principally candidas), enterococci % and anaerobes . %. . % of the sap patients received antibiotic treatment. the antibiotic most frequently used in prophylaxis was imipenem-cilastatine ( %) and piperaciline-tazobactam ( %). the antibiotics most frequently used in absolute indication were imipenem %, piperaciline/tazobactam . %, metronidazol %, vancomicina . %, ciprofloxacino %, amikacina in % and fluconazol . % conclusion. sap cases in spanish icu's account for little more than % of all hospital cases, but they result in high levels of severity, morbidity and mortality. crude mortality and sap septic complication-related mortality in spanish icu's are much higher than the average indicated in the literature ( . % and . %). imipenem is the antibiotic most frequently used in prophlaxis. the irruption of candidas has been detected. fluid /blood warmers help to prevent hypothermia by raising the temperature of intravenously administered fluids & blood. the hl- hotline fluid warmer is the model used in our hospital. it consists of disposable tubing set with a central channel through which the fluid is infused and outer tubing through which heated water circulates. the water is contained in a reservoir, which is heated by an electric element. the manufacturer's instructions recommend changing the water in the reservoir every days. this water is a potential source of infection and we therefore sampled the water in the reservoir for microbiological contamination. this study was conducted at royal london hospital during the month of december .there are fluid warmers, all hotline in our operating theatres. samples of water were taken from each of the reservoirs at the end of the working day. using aseptic techniques ml of water were added to a labeled blood culture bottle. each sample was cultured for hours. after one week we repeated procedure results. after hours of incubation, pseudomonas sp. grown in out of culture bottles. the results from the second sets also grew pseudomonas sp. in the same out of water reservoirs.conclusion. the water in the reservoir is heated to - degree celsius. this temperature does not inhibit the growth of pseudomonas. each time the disposable tube is disconnected from the reservoir approximately ml of water is spilled potentially spreading microorganisms. in addition there are case reports of cracks/splits in the inner tubing of the disposable tubing potentially exposing infused fluid or blood to heated water from the reservoir ( ). methods. ( . %) of consecutive cardiac surgery patients operated at onassis cardiac surgery center, from january st to june th , developed t> . c and leucocytosis, without evidence of specific site of infection. those patients were examined for possible catheter related infection, by removing central and arterial catheters and sending them along with blood specimens for culture. infections within the first postoperative h were defined as early, whereas those developed after the first h were defined as late. we examined the relation between the incidence of catheter related infection and the type of microorganism isolated, the type of operation performed, the icu stay and the hospital mortality. . coronary artery bypass grafting(cabg), valve or ascending aorta replacement(vr), combined(cabg+vr), acute dissecting aneurysm(ada) and other operations were carried out. positive blood or catheter cultures were found in patients ( . %). staphylococcus epidermidis was cultured from all patients with early(n= ) and % of those with late(n= ) infection, while candida was found in % of those with late infection. icu stay and hospital mortality was ten times higher in patients with positive blood or catheter cultures compared to the general icu population ( . vs . days and . % vs . %, respectively). finally, mortality was higher in patients with late compared to those with early infection( % vs %). (pts) who had suffered traumatic brain injury (tbi) as well as the immune response of these pts. pts with moderate to severe tbi (gcs =< ) and age > were enrolled under the presupposition they remained on mechanical ventilation (mv) > days. a total of tbi pts were followed-up; infected pts were identified and associated factors were studied. in addition, serum immunoglobulin (sig) levels and soluble interleukin- receptors (sil- r) were measured in infected pts. c. albicans species. candidemia in icu patients is associated with a high mortality rate. c. albicans was the most common yeast isolated from blood. non-c. albicans species have a frequent occurence among candidemic icu patients. the moderate susceptibility of azoles against non-c. albicans species indicates the usefulness of susceptibility testing for antifungal treatment. prospective, cohort, observational, and multicenter study. urine cultures were performed once a week to all patients admitted to the icu. samples were processed at the different clinical microbiology laboratories of the participating hospital using specific culture medium (sabouraud) and the bactec technique and the a c (biomerieux) system for the identification of species. candiduria was defined as < cfu of candida spp. in the urine. frequencies are expressed as cumulative incidence (%) and incidence density (episodes per days of urinary catheter). . results. a total of patients admitted > days to the participating icus between may to january were included in the study. of these patients, ( %) had a urinary catheter inserted, with , urinary catheter days. one or more candida spp. in the urine were detected in patients. the rate of candiduria was per patients/icu and the incidence density . per days of urinary catheter. in cases, candida spp. in association with different bacteria ( . %) were found, mostly gram-negative pathogens ( cases), in particular p. aeruginosa (n= ) and e. coli (n= ), and gram-positive pathogens ( cases) especially enterococcos (n= ). in respect to candida spp., c. albicans predominated ( . %) followed by c. glabrata ( . %), c. tropicalis ( . %), c. parapsilosis ( . %), and c. krusei ( . %), independently of the week in which isolation of pathogens was made. conclusions: candiduria was diagnosed in % of critically ill patients admitted for more than days in the icu. candida albicans was the pathogen most frequently recovered ( . %), although c. non-albicans was isolated in one out of each three cases. a retrospective study was done over the last year that included neonatal patients admitted at the intensive care unit. all patients had congenital anomalies ( patients with gastroschisis ( . %), patients with esophageal atresia ( . %), and others with intestinal obstruction, duodenal atresia and malrotation). . % of the patients were on total parenteral nutrition and mechanical ventilation. the average stay in the icu was . days. candida albicans was checked for in swabs of wound, in blood-culture, stool-culture, urine-culture, tracheal aspirate, gastric asp results candida albicans was identified in patients ( . %). it usually appeared - days after the introduction of the antibiotic therapy. it was most commonly found in gastric aspirate ( . %), stool-culture ( . %) etc. it would first appear in gastrointestinal tract (stool-culture and gastric aspirate after days). in respiratory and urinary tract candida was identified after days, and in blood-culture after days. . % of the patients received cephtriaxon or ampicillin, and . % amikacin or gentamycin and metronidazol. morbidity in patients with yeast infection is very high. the most common causative agent is candida, and the predilection organ is digestive tract. risk factors are: prematurity, mechanical ventilation, total parenteral nutrition, longer hospital stay and widespectrum antibiotics. due to unspecific clinical picture early diagnosis is usually made according to the results of taken cultures. there are still many dilemmas regarding systemic antimycotic profilaxys. key: cord- -lkngp u authors: bachmaier, kurt; stuart, andrew; hong, zhigang; tsukasaki, yoshikazu; singh, abhalaxmi; chakraborty, sreeparna; mukhopadhyay, amitabha; gao, xiaopei; maienschein-cline, mark; kanteti, prasad; rehman, jalees; malik, asrar b. title: selective nanotherapeutic targeting of the neutrophil subset mediating inflammatory injury date: - - journal: biorxiv doi: . / . . . sha: doc_id: cord_uid: lkngp u inflammatory tissue injury such as acute lung injury (ali) is a disorder that leads to respiratory failure, a major cause of morbidity and mortality worldwide. excessive neutrophil influx is a critical pathogenic factor in the development of ali. here, we identify the subset of neutrophils that is responsible for ali and lethality in polymicrobial sepsis. the pro-inflammatory neutrophil subpopulation was characterized by its unique ability to endocytose albumin nanoparticles (anp), upregulation of pro-inflammatory cytokines and chemokines as well as the excessive production of reactive oxygen species (ros) in models of endotoxemia and septicemia. anp delivery of the drug piceatannol, a spleen tyrosine kinase (syk) inhibitor, to the susceptible subset of neutrophils, prevented ali and mortality in mice subjected to polymicrobial infection. targeted inhibition of syk in anp-susceptible neutrophils had no detrimental effect on neutrophil-dependent host defense because the subset of anplow neutrophils effectively controlled polymicrobial infection. the results show that neutrophil heterogeneity can be leveraged therapeutically to prevent ali without compromising host defense. subsets of neutrophils differ markedly in their response to both homeostatic and inflammatory signals ( ) . neutrophil heterogeneity is apparent in the lungs of naïve mice, where a large proportion is marginated in the microvasculature, where they may function as immune sentinels, while other neutrophils circulate unimpeded ( ) . neutrophils are an essential component of the innate immune response to polymicrobial infection due to their ability to eliminate the infectious agents ( ) . however, neutrophils can also become pathogenic in diseases by promoting excessive inflammation such as in the case of acute lung injury (ali), a main cause of morbidity and mortality worldwide ( ) . excessive activation of neutrophils by bloodstream bacteria and their products, such as the bacterial endotoxin lipopolysaccharide (lps), results in tissue damage and organ dysfunction ( ) . therapeutic efforts of curbing this excessive neutrophilic inflammation have been frustratingly ineffective ( ) . administration of nitric oxide, norepinephrine, low dose corticosteroids, prostaglandin e , or recombinant activated protein c, when critically evaluated, did not significantly improve patient mortality ( ) . moreover, neutralizing key inflammatory mediators such as the cytokines tnf-α, and il- β, or reactive oxygen species (ros), has also failed ( ) . in experimental models, the elimination of neutrophils markedly decreases the severity of ali ( ) . on the other hand, there is the risk of compromising host defense in the setting of global neutrophil impairment and deterioration of pulmonary function during recovery from neutropenia ( ) . targeting specific subsets of neutrophils could represent an optimal therapeutic approach if one could identify deleterious neutrophil subsets without compromising the subsets essential for host defense. the unique ability of neutrophils to rapidly change their phenotype and function according to changes in their microenvironment ( ) ( ) ( ) ( ) ( ) is a manifestation of neutrophil heterogeneity, but the distinct roles of neutrophil subsets in the setting of sepsis or endotoxemia and not well understood. we hypothesized that subsets of neutrophils are primarily responsible for the maladaptive hyperinflammatory response that causes ali, multiple organ failure, and death. in the present study, we identified the subset of neutrophils that incorporated specially formulated albumin nanoparticles (anp) as the subset that could be therapeutically targeted without impairing the elimination of bacteria in experimental septicemia. heterogeneous response of neutrophils to endotoxin and septicemia. after i.v. injection of albumin nanoparticles (anp) to naive mice, we observed anp-uptake in liver and spleen, whereas lungs, heart and kidney remained mostly free of anp (supplemental figure ) . in response to i.p. challenge with the endotoxin of gram-negative bacteria, lipopolysaccharide (lps), uptake of i.v.injected anp in heart, kidney, liver and spleen did not increase compared to naïve mice. in lungs, however, anp uptake increased significantly after lps challenge (supplemental figure ). ly g + polymorphonuclear neutrophils (pmn) have the capacity to internalize anp ( ) . we next determined whether uptake of anp in the lung was restricted to ly g + pmn and whether there was heterogeneity in the endocytosis of anp among ly g + pmn. in response to i.p. lps, only cd + leukocytes endocytosed i.v. injected anp whereas parenchymal cells (cd neg ) did not ( figure a ). anp-endocytosis was restricted to ly g + pmn and largely absent in cd + monocytes/macrophages, nk . + nk cells, or lymphocytes (data not shown). pulmonary pmn endocytosed anp in a bimodal manner, with one subset showing highly efficient uptake (anp high ), and the other subset demonstrating minimal to no uptake (anp low ) ( figure a ). bacterial endotoxins amplify the neutrophil activation in septicemic mice, leading to increased pmn sequestration in lungs where pmn release pro-inflammatory mediators and further enhance the recruitment of immune cells ( ) . using cecal ligation and puncture (clp), a reproducible and clinically relevant mouse model of polymicrobial infection that causes ali, we found that in naïve control mice after sequential i.v. injections of anp only ~ % of lung pmn endocytosed anp as evidenced by anp-specific fluorescence ( figure b) . at h after a sham operation, laparotomy plus cecal ligation without puncture of the cecum, and sequential i.v. injections of anp, anp high pmn increased to only ~ % ( figure b) . induction of severe polymicrobial sepsis by clp, however, increased the frequency of anp high lung cells -fold over baseline conditions to ~ % ( figure b ). we consistently found that cd b expression levels on pmn in peripheral blood, lung, and liver, were greater on anp high pmn than on anp low pmn ( figure e ), and lung anp high pmn showed the highest cd b expression ( figure e) , indicating a higher level of inflammatory activation of the anp high pmn subset. moreover, in septicemic mice, the percentages of anp high pmn was significantly greater than in sham controls in blood, lung, and liver ( figure f ), consistent with an increased pro-inflammatory state as well as increased adhesiveness of the anp high pmn subset. this heterogeneity in cd b activation on pmn suggested that susceptibility to anpendocytosis delineated distinct subsets of pulmonary pmn. to define the differences between the pmn subsets, we next analyzed whether anp high pmn had a transcriptomic profile different from anp low pmn. we performed an unbiased analysis of lung pmn transcriptomic profiles using rna-seq. we challenged mice with i.p. injections of lps or saline and administered anp i.v. h later. at h after anp injection, we euthanized mice and harvested pmn from single cell suspension of their lungs and sorted the ly g + pmn by flow cytometry according to their anp uptake into anp low and anp high pmn. immediately after sorting, we prepared pmn mrna for rna-seq analysis. we generated a heat map and dendrogram ( figure a ) to represent the normalized pmn gene expression data. we found that the biological replicates clustered into groups with distinct transcriptomic profiles; i.e., the mrna profiles defined pmn from lps-challenged or saline-injected mice, and were distinct in anp low and anp high pmn ( figure a ). using metacore pathway analysis to identify pathways that were different between anp high pmn and anp low pmn, we found that the pathways regulating immune response and immune cell migration were significantly overrepresented in the anp high pmn (supplemental table) . pathways containing chemokine receptors were significantly enriched in anp high pmn, consistent with the concept that pmn heterogeneity is a function of differential pmn trafficking into tissue presumably facilitated by different chemoreceptor expression. we also found that chemokine receptors were over-represented . to identify the chemokine receptors for each pmn subset, we generated separate heatmaps for chemokine receptors, plotting all genes with cpm > . ( reads at sequencing depth of m reads) regardless of differential expression levels. in naïve mice, anp high pmn showed relative over-expression of chemokine receptors cxcr , cxcr , and ccrl ( figure b ). in lpschallenged mice, anp high pmn showed relative over-expression of chemokine receptors ccr , ccr , ccr , ccr , ccrl , cxcr and cxcr ( figure c ). we next assessed the expression of chemokines in anp high pmn and anp low pmn. in saline injected control mice, anp high pmn were significantly enriched for the expression of the chemokines ccl , ccl , and cxcl ( figure d ). in lps-challenged mice, anp high pmn demonstrated relative over-expression of the chemokines ccl , ccl , ccl , ccl , ccl , cxcl , cxcl , cxcl , cxcl ( figure e ). of note, mice were only exposed to anp for the last hour of the h lps-challenge, and the differences in gene expression between anp high pmn and anp low pmn in lps challenged mice were far greater than those in the naïve mice, indicating that the anp uptake itself likely did not affect the gene expression profiles. these rna-seq data unequivocally demonstrated the existence of lung pmn subsets with a distinct response to the inflammatory stimulus lps. based on our rna-seq data and metacore pathway analysis, we selected a group of chemokine receptors and cytokines to determine the kinetics of their expression after lps-stimulation. we performed this independent validation by quantitative pcr (qpcr) and flow cytometry. we found that mrna expression of the chemokine receptor ccr was significantly greater in anp high pmn than in anp low pmn at h, h, and h after lps challenge ( figure a ). importantly, we found that ccr receptor cell surface expression, consistent with the mrna data, was significantly greater on lung anp high pmn than in anp low pmn before and h, h, and h after lps stimulation ( figure b ). cxcr expression was reduced at h after lps-stimulation compared to h stimulation, cxcr receptor cell surface expression increased at h after lps-stimulation and was greater in anp high than in anp low pmn, and decreased to expression levela of unstimulated pmn thereafter ( figure b ). these data demonstrate that the observed mrna expression heterogeneity translated into cell surface protein expression heterogeneity. the mrna levels of the chemokines ccl , ccl , cxcl , cxcl ( figure c -f) were significantly greater in anp high pmn than in anp low pmn h, h, and h after in vivo lps challenge. ccl ( figure c ) and ccl ( figure d ) expression, in particular, was vastly greater in anp high than in anp low pmn, suggesting that anp high pmn are specialized cells of inflammation, which recruit and activate additional pmn. in addition, heterodimers of ccl and ccl are known to attract monocytes/macrophages ( ) . the cytokine il- β is essential for antibacterial function and expression of il β was induced ~ -fold in anp low pmn h after lps challenge compared to anp low pmn from saline injected control mice ( figure g ); in anp high pmn, il β was induced ~ fold over anp high pmn from saline injected control mice ( figure h ). expression of the pleiotropic cytokine il- expression was significantly greater in the anp high than in the anp low pmn in lungs h, h, and h after lps challenge ( figure h ). these data demonstrate that pulmonary anp high pmn can markedly amplify the inflammatory response. we next determined whether adoptively transferring anp high pmn from donors into syngeneic recipient mice would induce inflammation in these mice. donor balb/c mice were challenged with a lethal dose of lps [ mg/kg] and injected with two doses of anp labeled with the stable fluorochrome af at h and h after lps challenge ( figure a ). at h after lps challenge, donor mice were euthanized and lung single cell suspensions were prepared for flow cytometric sorting into anp high and anp low neutrophils ( figure b ). syngeneic recipient mice were injected i.v. with x pulmonary anp high pmn or, as controls, with an equal number of pulmonary anp low pmn from the same donors (three donors per recipient mouse were required to achieve the necessary cell number). at h prior to transfer of donor cells, recipient mice were treated with a sublethal dose of lps [ mg/kg] to activate their endothelium, a prerequisite for initiating neutrophilic lung inflammation ( ) . at h after the adoptive transfer, we assessed lung inflammation in recipient mice ( figure a ). we found anp + ly g + pmn in lungs of recipient mice, confirming successful transfer of donor cells to recipient mice and their homing to the lung ( figure c ). transfer of donor anp high pmn significantly increased lung inflammation in the recipient mice when compared to mice that received anp low cells ( figure c ). moreover, after transfer of anp high pmn, lung ly g + pmn produced more ros when compared to controls receiving anp low pmn ( figure d ). because ros induce tissue inflammation ( , ) , and anp high cells are carriers of large amounts of mrna for inflammatory cytokines and chemokines ( figure ), we measured the inflammatory mediators il- β and cxcl in lung tissue extracts. il- β, released during activation of nlrp inflammasome, mediates tissue injury, whereas the chemokine cxcl amplifies the inflammatory cycle by attracting additional pro-inflammatory neutrophils ( ) ( ) ( ) . mice receiving anp high pmn had significantly greater concentrations of il- β ( figure e ) and cxcl ( figure f ) in their lungs than recipients of anp low pmn. these data demonstrated the intrinsic ability of anp high pmn to promote lung inflammation. firm pmn adhesion on microvascular endothelium, induced by endotoxin or bacteremia, upregulates mac- (a heterodimer of cd b and β -integrin cd ), contributing to maximal activation of pmn ( ). syk activity is required for β -integrin-mediated neutrophil activation ( ) . given our data above, we reasoned that inhibiting integrin signaling specifically in the subset of anp high pmn would reduce lung inflammation in the polymicrobial sepsis model. we thus used the drug piceatannol, a syk inhibitor ( , ) , that is readily incorporated into anp due to its poor water solubility ( , ) , to inhibit syk-mediated β -integrin-dependent neutrophil adhesion. we found that therapeutic administration of piceatannol loaded anp high pmn protected cd mice from lethal polymicrobial sepsis ( figure a ). treatment with two i.v. injections of piceatannol incorporated into anp (panp), h and h after clp, significantly reduced mortality of mice when compared to control groups treated with anp without any drug after challenge with clp ( figure a ). treatment using panp reduced clp lethality to the rate of sham-operated (laparotomy plus cecal ligation without puncture of the cecum) mice ( figure a ). injections of panp alone had no effect on the survival rate compared to saline injected controls ( figure a ). clp challenged mice treated with anp, without any drug, had the same mortality rate as saline-injected controls ( figure a ), demonstrating that targeting specifically the anp high subset of pmn is sufficient to prevent clp-induced mortality. similarly, in the absence of polymicrobial infection but after i.p. challenge with a lethal dose of the endotoxin lps (ld ), mice treated with sequential i.v. injections of panp h and h after lps challenge, showed significantly reduced mortality when compared to control mice injected with anp alone ( figure b ). reduced mortality after panp treatment was correlated with the presence of significantly fewer highly inflammatory cd b high cd high pmn ( figure c ), and reduced cd b expression on lung pmn when compared to anp-treated controls ( figure d ). furthermore, while cd b expression on pmn was reduced by panp treatment in the lungs, in peripheral blood pmn cd b surface-expression was higher when compared to pmn from anp-treated mice ( figure d ). to test whether augmented cd b expression on pmn in peripheral blood was a consequence of panp-induced pmn trafficking from the lung to the peripheral blood, we used two-photon microscopy to visualize pmn trafficking in the lung in vivo ( ) . we determined the number of ly g + pmn in microvasculature and velocity of their migration through the microvasculature in lungs (video, figure e ). in lps challenged cd mice, the number of pmn increased, as measured by pmn-specific fluorescence (video). treatment of pmn with panp, however, significantly increased the velocity of ly g + pmn in the lung microvasculature and reduced the number of ly g + pmn as compared to anp treated controls (movie, figure e ,f). cell targeted treatment of anp high pmn by inhibiting β -integrin signaling, and accelerated the transit of pmn through lungs, and thus reduced exposure time of lung tissue to noxious pmn-derived mediators. ros production is a potent mediator of tissue damage ( , ) . we found that anp high cells were characterized by high ros production ( figure d ). syk, whose enzymatic activity is the cellular target of piceatannol ( ), is required for integrin-mediated neutrophilic superoxide production ( ). we measured ros production by bone marrow ly g + pmn in vitro (supplemental figure ). bone marrow pmn responded to stimulation with the bacterial peptide fmlp with ros production (supplemental figure ) . pmn with higher uptake of panp showed greater reduction in ros production (supplemental figure ) . moreover, the delivery of piceatannol via panp increased drug efficacy by orders of magnitude when compared to free drug because of its incorporation in the toxic pmn subset (supplemental figure ) . we therefore examined whether panp treatment reduced superoxide production by lung pmn of endotoxemic mice. we challenged mice with a lethal dose of lps and analyzed the production of ros by the lung ly g + pmn ex vivo. we found that anp high pmn had significantly higher intracellular ros levels than anp low pmn ( figure a ). panp treatment, however, almost completely blocked ros production in these cells ( figure a ). these data demonstrated that anp high pmn are largely responsible for ros production by lung inflammatory cell in endotoxemia because targeted treatment via panp markedly reduced ros production. mice doubly deficient for nadph oxidase and inos (gp phox −/− nos −/− ) develop spontaneous infections ( ) . ros production and complementary no production by pmn are essential to control host microbial diversity and microbial infection ( ) , and functional lung pmn are essential for the task of clearing bloodstream bacteria because resident macrophages in liver and spleen alone are insufficient ( , ) . we therefore determined the effects of panp treatment on the bacterial burden of cd mice in the clp model of polymicrobial infection. we found no exacerbation or amelioration of the bacterial burden as a result of panp treatment when compared to anp treatment ( figure b ), suggesting that selective inhibition of integrin signaling in the anp high pmn subset did not compromise bacterial elimination. two consecutive i.v. injections of panp, given h and h after clp, did not increase bacteremia when compared to anp injected controls ( figure b ). the bacterial burden of lungs, livers, and spleens of bacteremic mice was similar between panptreated mice and anp-treated controls ( figure b ). pmn-dependent antimicrobial function was fully preserved after panp treatment, suggesting that antimicrobial functions, ingestion and elimination of bacteria are mainly performed by anp low pmn (supplemental figure ). because panp treatment did not weaken anti-microbial resistance, we analyzed parameters of tissue inflammation and damage we measured crucial inflammatory mediators, il- β, and cxcl . in lung tissue extracts of mice subjected to clp. we found a substantial reduction in the concentration of il- β and cxcl after panp treatment when compared to anp treated controls ( figure c ). we next measured nitrotyrosine formation in lungs and livers of septicemic mice. in the experimental mice as well as in septicemic patients, activated lung myeloid cells, inflammatory or resident, and epithelial type ii cells, release both no and superoxide which react to form peroxynitrite, a potent oxidant causing tissue damage ( ) . peroxynitrite (onoo − ), but not no or superoxide alone, nitrates tyrosine residues ( ) . we observed that nitrotyrosine-specific staining in inflammatory and parenchymal cells was significantly reduced in lungs and livers of mice treated with panp when compared to anp-treated controls ( figure d ,e). these data suggest that antimicrobial function and tissue damaging function are performed by distinct subsets of pmn. we next determined the effect of panp treatment on pulmonary edema which is a characteristic feature of inflammatory lung injury. a marked increase in lung wet-to-dry weight ratio is indicative of breakdown of the alveolar capillary barriers, the hallmark of ali. pneumonia is the most common cause of ali in patients ( ) and also the most common cause of sepsis ( ) . in the model of pneumonia induced by i.t. instillation of live p. aeruginosa bacteria, panp treatment significantly reduced pulmonary edema when compared to treatment with the control anp ( figure f ). photomicrographs of lung or liver sections from septicemic mice treated with anp or panp showing nitrotyrosine formation. paraffin embedded sections were stained with specific ab to nitrotyrosine (red staining), and with dapi to visualize nuclei (blue staining). polymicrobial sepsis was induced by clp, mice were treated with panp or anp h and h after challenge and were sacrificed for tissue processing and staining h after challenge. bar furthermore, treatment with panp significantly reduced pulmonary edema in endotoxemic or septicemic mice when compared to lungs from anp treated controls ( figure f) . a reduction of tissue damage, because of reduced lung inflammation, could be the proximate cause of reduced inflammatory lung injury after treatment. measuring a markers of overall cell damage, lactic dehydrogenase (ldh) ( ) , revealed that the polymicrobial sepsis-induced increased serum activity of ldh was significantly reduced by panp treatment when compared to anp treated controls ( figure g ). in addition, hepatocyte-specific sorbitol dehydrogenase (sdh) activity, a marker of hepatocyte damage ( ) , was also significantly reduced by panp treatment of septicemic mice ( figure g ). endocytosis of anp delineates two distinct subsets of pmn. anp high pmn cause tissue damage whereas anp low pmn control microbial infection ( figure h ). targeting neutrophilic inflammation that is pathogenic in ali remains an important unmet clinical need. our observation that neutrophils primed by bacterial infections or bacterial derived products exhibit heterogeneity in their capacity to endocytose anp lead to the discovery of two distinct neutrophilic subsets, one that causes inflammatory injury and one that controls microbial infection. functional and phenotypic pmn heterogeneity ( ) ( ) ( ) ( ) ( ) led us to test the hypothesis that immunopathology and severe tissue inflammation of endotoxemia and septicemia can be treated by targeting a distinct neutrophilic subset. increased expression of chemokines and chemokine receptors in anp high pmn was consistent with their role in promoting tissue inflammation. several of the chemokines such as ccl and ccl or cxcl and cxcl are members of the macrophage inflammatory protein family and are typically thought to be released by macrophages to increase the influx of pro-inflammatory cells such as neutrophils ( ) . our data suggest that a subset of neutrophils might be a substantial source of these inflammatory proteins in models of ali. all cells need to express genes that are required for their intrinsic functions, whereas production of secreted factors can be delegated to subsets ( ) . our results indicate that a specific subset of phenotypic and functionally distinct neutrophils is responsible for lethality in experimental polymicrobial sepsis. anp high pmn were characterized by higher expression of the chemokine receptors for the ligands released following lps activation such as ccr and ccr (the receptors for ccl ) which could point to a possible positive feedback loop in which inflammatory anp high pmn attract additional inflammatory pmn, and thus actively promote a vicious cycle of hyperinflammation and tissue injury ( ) . given their phenotypic and functional profile, anp high pmn might play a pathogenic role in covid- , the disease caused by coronavirus sars-cov- ( ) . the main cause of covid- -mortality is acute respiratory failure ( ) . in patients with severe covid- , activated neutrophils, recruited to the pulmonary microvasculature, produce histotoxic mediators including ros ( ) . activation of neutrophils might contribute to cytokine release syndrome ("cytokine storm") that characterizes severe covid- disease ( ) . therapy targeting anp high pmn might prevent a patient's hyperinflammatory response to sars-cov- without weakening the antiviral response. it has been shown that the incorporation of denatured albumin beads depends on mac- expression ( ) . anp-incorporation, by contrast, is mac- -independent ( ) suggesting a distinct molecular mechanism of anp-endocytosis. "aged neutrophils", were first described in vitro as functionally deficient ( ) and have been subsequently shown to promote disease in vivo in models of sickle-cell disease or endotoxin-induced septic shock ( ) . while "aged neutrophils" home to the bone marrow under steady-state conditions anp high pmn do not home to the bone marrow under steady-state conditions and markers of aged peripheral blood neutrophils, cxcr , cxcl , cd l, and tlr , do not distinguish anp high from anp low pmn under steady-state or inflammatory conditions in the lung (figure ). these findings thus show that the previously described subset of aged neutrophils is distinct from the anp high pmn subset we identified. administration of anp carrying piceatannol, a syk inhibitor, dramatically improved survival in polymicrobial sepsis but, critically, did not increase the host's bacterial burden. syk function is required for the essential antibacterial functions of neutrophils ( ) . we found that anp low pmn are more efficient in ingestion and elimination of e. coli bacteria in vitro than anp high pmn and inhibition of syk in anp high pmn does not impair control of polymicrobial infection in vivo. by therapeutically leveraging the preferential anp uptake of the inflammatory pmn subset, we succeeded in limiting immunopathology caused by polymicrobial infection. pathogens adapt to host resistance mechanisms, but not host tolerance mechanisms ( ) . it is conceivable that an anp-based approach of targeting toxic subset of neutrophils, by improving host tolerance, might assist the host in combating antibiotic-resistant microbial infections ( ) . anp could also be used to deliver compounds other than piceatannol or to specifically deliver sirnas or micrornas. furthermore, anp-based therapy might be useful in other forms of neutrophilic injury; e.g., ischemia reperfusion injury of the myocardium or the kidney ( ) . the ability to tolerate pathogens in experimental polymicrobial sepsis was greatly strengthened by targeting anp high pmn with a drug that accelerates the velocity of pulmonary pmn and abrogates their ros production. panp did not target β -integrins directly ( ) but mitigated downstream β -integrin signaling, and are thus more likely to be effective when β -integrin are already engaged; i.e., in the lung microvasculature of septic patients ( ) . further potential improvement of anp-based therapies over the current standard treatments of septic patients ( ) lies in the precision targeting of the relevant pathogenic subset of neutrophils. earlier efforts to neutralize reactive oxygen species , to use antibodies against key inflammatory cytokines such as tnf-α, il- β, or to inhibit the endotoxin lps ( , ( ) ( ) ( ) have failed to reduce mortality associated with ali/ards possibly because they did not discriminate between pmn subsets and may have compromised both host defense (resistance) and tissue repair (tolerance) ( ) . compared with the therapeutic administration of exogenous cells, generated from donors or from the patients themselves, for example, mesenchymal stromal/stem cells (mscs) ( ), anp-based therapy has the advantage of targeting the host's endogenous cells dependent on their pathogenic activation. one limitation of our approach is that we cannot distinguish whether the specification into anp high pmn occurs in the bone marrow prior to egress of pmn into circulation or whether it occurs in the tissue itself. it is also possible that the anp high state characterized by upregulation of chemokine receptors and chemokine ligands is reversible and that pmn can transition between these states, in response to environmental cues even during the short life-span of neutrophils ( ) . the present data support the concept that the generation of heterogeneous pmn subpopulations evolved as a host defense mechanism to avoid an indiscriminate response by all pmn to septicemia ( ) . ( , ) . we demonstrate here that neutrophil heterogeneity can be effectively leveraged for a nanoparticle-based therapy. mice. we used outbred male cd mice, at a body weight ranging from g to g and, for adoptive transfer experiments, male inbred balb/c mice between and wk of age. mice were treated in accordance with the nih guide for the care and use of laboratory animals and uic animal care committee's regulations. all procedures were approved by the uic iacuc. synthesis of uniform-sized spheric albumin nanoparticles. anp and panp, synthesized as described ( ) , were of consistent hydrodynamic size ( nm ± nm diameter and zeta potential (- ± . mv) distribution. we injected i.v. . mg/kg body weight of anp, or of anp loaded with . µm piceatannol (panp) h and h after challenge with lps or h and h after clp. flow cytometry and cell sorting. single cell suspensions were prepared as described ( ) tissue damage markers. the activity, ldh and sdh was determined using commercial kits according to manufacturers' instructions. histopathology was evaluated in sections from paraffin embedded or frozen tissues using specific antibodies to nitrotyrosine as described ( ) . quantification of hydrogen peroxide production. we measured hydrogen peroxide production using the amplex red hydrogen peroxide kit (invitrogen) following the manufacturer's instructions. heterogeneity of neutrophils neutrophil kinetics in health and disease the role of neutrophils in immune dysfunction during severe inflammation neutrophils and acute lung injury sepsis: current dogma and new perspectives surviving sepsis campaign: international guidelines for management of severe sepsis and septic shock: drotrecogin alfa (activated) in adults with septic shock sepsis: pathophysiology and clinical management respiratory status deterioration during g-csf-induced neutropenia recovery phenotypic and functional change of cytokine-activated neutrophils: inflammatory neutrophils are heterogeneous and enhance adaptive immune responses getting to the site of inflammation: the leukocyte adhesion cascade updated neutrophil heterogeneity: implications for homeostasis and pathogenesis neutrophils in homeostasis prevention of vascular inflammation by nanoparticle targeting of adherent neutrophils identification of human macrophage inflammatory proteins α and β as a native secreted heterodimer endothelium-derived toll-like receptor- is the key molecule in lps-induced neutrophil sequestration into lungs reactive oxygen species in inflammation and tissue injury critical role for cxcr and cxcr ligands during the pathogenesis of ventilator-induced lung injury critical role of endothelial cxcr in lps-induced neutrophil migration into the lung caspase- -mediated endothelial pyroptosis underlies endotoxemia-induced lung injury annual review of immunology syk is required for integrin signaling in neutrophils piceatannol, a syk-selective tyrosine kinase inhibitor, attenuated antigen challenge of guinea pig airways in vitro piceatannol ( , , ', '-tetrahydroxy-trans-stilbene) is a naturally-occurring protein-tyrosine kinase inhibitor stabilized imaging of immune surveillance in the mouse lung phenotype of mice and macrophages deficient in both phagocyte oxidase and inducible nitric oxide synthase the lung is a host defense niche for immediate neutrophil-mediated vascular protection outcomes of bacteremia in patients with cancer and neutropenia: observations from two decades of epidemiological and clinical trials the acute respiratory distress syndrome prognostic significance of elevated serum lactate dehydrogenase (ldh) in patients with severe sepsis pneumotoxicity and hepatotoxicity of styrene and styrene oxide the mercurial nature of neutrophils: still an enigma in ards? neutrophils: between host defence, immune modulation, and tissue injury social networking of human neutrophils within the immune system neutrophil heterogeneity in health and disease: a revitalized avenue in inflammation and immunity neutrophil recruitment and function in health and inflammation role of inflammatory mediators in the pathophysiology of acute respiratory distress syndrome desynchronization of the molecular clock contributes to the heterogeneity of the inflammatory response monocyte recruitment during infection and inflammation the sars-cov- outbreak: what we know clinical characteristics of hospitalized patients with novel coronavirus-infected pneumonia in wuhan, china cytokine release syndrome in severe covid- the cytokine storm in covid- : an overview of the involvement of the chemokine/chemokine-receptor system heterotypic interactions enabled by polarized neutrophil microdomains mediate thromboinflammatory injury impairment of function in aging neutrophils is associated with apoptosis neutrophil ageing is regulated by the microbiome neutrophil-specific deletion of syk kinase results in reduced host defense to bacterial infection disease tolerance as a defense strategy two ways to survive infection: what resistance and tolerance can teach us about treating infectious diseases involvement of neutrophils in the pathogenesis of lethal myocardial reperfusion injury selected treatment strategies for septic shock based on proposed mechanisms of pathogenesis surviving sepsis campaign: international guidelines for management of sepsis and septic shock effect of eritoran, an antagonist of md -tlr , on mortality in patients with severe sepsis: the access randomized trial a randomized, double-blind, placebo-controlled trial of tak- for the treatment of severe sepsis anti-tumor necrosis factor therapy in sepsis: update on clinical trials and lessons learned mesenchymal stem (stromal) cells for treatment of ards: a phase clinical trial selective roles for tolllike receptor (tlr) and tlr in the regulation of neutrophil activation and life span neutrophil diversity in health and disease e ubiquitin ligase cblb regulates the acute inflammatory response underlying lung injury inos expression and nitrotyrosine formation in the myocardium in response to inflammation is controlled by the interferon regulatory transcription factor measurement of oxidative burst in neutrophils flow cytometric analysis of the granulocyte respiratory burst -a comparison study of fluorescent-probes star: ultrafast universal rna-seq aligner featurecounts: an efficient general purpose program for assigning sequence reads to genomic features differential expression analysis of multifactor rna-seq experiments with respect to biological variation edger: a bioconductor package for differential expression analysis of digital gene expression data controlling the false discovery rate: a practical and powerful approach to multiple testing analyzing real-time pcr data by the comparative ct method cd (tnfrsf ), c-f pyk (fak ), i-kb, mhc class ii tgf-beta , l-selectin ubiquitin, tap (psf ), beta-catenin, ap- , irf , rsad , lck, pkc-theta il- beta, fc gamma ri, tnf-r , hla-drb , tgf-beta, mhc class ii . e- fc gamma ri, l-selectin, pecam , mhc class ii, ccr , cd . e- ccl , il- , il- r beta chain, il- beta, ap- , il ra, ccl , ccr , fkhr . e- e- . e- mhc class ii alpha chain, il- beta, i-kb ccl , il- beta, tgf-beta , il- alpha, caspase- , pd-l , il rn, il r , . e- il- beta, i-kb, tgf-beta , mhc class ii psmb , tap (psf ) il- beta, mhc class ii, cd (tnfrsf ), ccr , il- , p mapk, c . e- . e- il- beta, tnf-r , mhc class ii, il- il- beta, i-kb, tpl (map k ), mhc class ii c-flip(long) mhc class ii, pd-l , slam, cd (tnfrsf ), cd (tnfrsf ) cox- (ptgs ), jak , mhc class ii, lck, cd (tnfrsf ) mhc class ii, cd (tnfrsf ) e- . e- tgf-beta , c-iap , c-flip(short) gro- , il- beta, i-kb il- r beta chain, i-kb, sil- ra, il- ra, lck, nik(map k ) cyclin d , bfl , nik(map k ), cd (tnfrsf ), p mapk il- beta, i-kb s p receptor, tgf-beta , cd , tie , pd-l ikk-gamma, il- beta il- r beta chain, jak , c-myc, il- r alpha chain, granzyme . e- il- , caspas . e- il- r beta chain, il- beta, sil- ra, mhc class ii cox- (ptgs ), pge r , ikk-gamma, il- beta, i-kb, tgf-beta , mhc cl ptpn , cd (tnfrsf ), c-flip nf-at (nfatc ) lck, cd (tnfrsf ), pkc-theta, nik(map k ) rig--drb , tgf-beta, mhc class ii, ptpn , cd (tnfrsf ), csf , il- , mhc class ii beta chain, il- r alpha chain lass ii, klf , slam, cd (tnfrsf ), pkc-theta, nik(map k ), fkhr, bcl- , irf , malt , cd , ifn-gamma, cd , blimp (pr - , il ra, ccl , ccr , fkhr, psgl- , ccr , nf-at (nfatc ), ifn-gamma , cd , lat, itk, p mapk, nf-at (nfatc ) cd (tnfrsf ), ccr , mhc class ii beta chain, p mapk, ask (map k ), cd , c-jun spase- , pd-l , il rn, il r , csf , il- , endothelin- , caspase- , msr , p mapk, pd-l , aml (runx ) il- r alpha chain, cd zeta, nf-at (nfatc ) mhc class ii beta chain, zap , cd , lat, ets , aiolos, cd zeta, nf-at (nfatc ) rar-alpha/rxr-alpha nik(map k ), fkhr, bcl- , cd , malt , il- r alpha chain, nf-at (nfatc ), cxcr , ifn-gamma il- , p mapk, cd , cxcr , ifn-gamma ase- , granzyme b, cd , ifn-gamma, fasr(cd ) mhc class ii, irak / , ap- , cd (tnfrsf ), hsp , p mapk, cd , c-jun cd , cd (tnfrsf ), cd , pd-l (tnfrsf ), il ra , zap , cd , lat, itk, nf-at (nfatc ), nf-at, fasr(cd ) , caspase- , dr (tnfrsf a), c-iap , tbid, bcl- , bid -beta , mhc class ii, ccr , cd (tnfrsf ), ccr , csf , cmklr , m-csf receptor d (tnfrsf ), smad , tieg , il- r alpha chain, nf-at (nfatc ) hgf receptor (met), c-iap , tbid nfkbia, ifn-gamma, bcl- , c-jun cd , itk, nf-at (nfatc ), vav- , ifn-gamma tcf(lef), dsh, frizzled c, il- r alpha chain, granzyme b, bcl- usp reg class ib (p ), hip , itk, p mapk, cxcr , c-jun, vil (ezrin) vav- , cd sl , c-jun, fasr(cd ) vav- , ifn-gamma, bcl- p mapk t (nfatc ), cxcr , c-jun, pd-l , p mapk, m-csf receptor, cd , socs , ifn-gamma supplemental figure . anp biodistribution measured by ivis in naïve mice, i.v.-injected anps were mostly found to liver and spleen. after i.p. administration of the endotoxin lps [ mg/kg], anp appeared prominently in lungs, and remained visible in liver and in spleen. albumin was labeled with vivotag (perkinelmer) and then formed into anps. organs were harvested h after µl tail vein injection of mg/ml vivotag labeled anps. epi-fluorescence radiance scale corresponding to images. fluorescence was measured by a xenogen ivis spectrum (caliper life sciences) and images were processed with living image software (ver. . . ). an excitation filter of nm and emission filter of nm with sec exposure times were used for all experiments. representative data obtained from at least mice per treatment group. flow cytometry histograms of bone marrow pmn stimulated with lps [ mg/ml] and incubated with panp, at the indicated doses, for min at °c, and then processed for flow cytometry. representative data obtained from at least mice per treatment group panp treatment reduces superoxide production by pmn in response to fmlp stimulation. pamp reduced fmlp stimulated ros production in a dose dependent manner. bone marrow pmn were incubated with the indicated dises of panp and stimulated with fmlp for the indicated time. ros production was measured using an amplex red hydrogen peroxide/peroxidase assay kit. representative data obtained from at least mice per treatment group. key: cord- -w x i im authors: volk, t.; kox, w.j. title: endothelium function in sepsis date: journal: inflamm res doi: . /s sha: doc_id: cord_uid: w x i im endothelial cells can be the prime target for an infection and infected endothelial cells may serve as an initiating system for a systemic response as these cells are able to secrete many mediators known to be of paramount importance. endothelial cell functions in turn are regulated by these circulating mediators. cellular interactions with leukocytes revealed protective and destructive functions. single cell and animal studies indicate that endothelial permeability is increased and apart from clinical obvious edema formation in septic patients, the endothelial component remains unknown. endothelial coagulation activation has been shown in vitro, however human data supporting an endothelial procoagulatory state are lacking. defects in endothelium dependent vasoregulation in animal models are well known and again human studies are largely missing.¶an imbalanced production of reactive oxygen species including nitric oxide has been found to be involved in all endothelial functions and may provide a common link which at present can be supported only in animal studies. sepsis is considered the leading cause of death in noncoronary intensive care units. it has been defined as a systemic inflammatory reaction to an infection. among many cellular disturbances endothelial cells play a major role in the pathogenesis of this disease as these cells are critically involved in maintaining a delicate balance between vasoconstriction and vasodilation, blood cell adherence and nonadherence, anticoagulation and procoagulation, permeability and thightness. all these functions are believed to be imbalanced and impairment is believed to precede clinically recognizable alterations (e.g. bleeding, edema, organ dysfunction and shock) but it is by no means clear whether these changes are the cause or consequence of endothelial dysfunction in sepsis. endothelial functions have largely been studied in vitro from the first successful attempts of culturing isolated human endothelial cells dating back to the early ties. endothelial cells from and within different organs or different species behave differently and tend to change properties the longer they are kept in culture. to overcome isolation variabilities several endothelial cell lines are currently available which have retained at least some features. however, experiments from single cell cultures are flawed in many ways and results from these may never be of any relevance to medical practice. it is inherent to any cell culturing that data obtained from these experiments often are restricted to cell type, time of culture and general working conditions. in vitro stressed endothelial cells almost uniquely tend to activate a functional program leading to a proinflammatory, procoagulatory and hyperpermeable phenotype. in this review we want to summarize investigations of disturbed endothelial functions including infection, mediator and cellular interactions, permeability, coagulation and vasoactivc properties from molecular findings to patient care. staphylococcus aureus is the most prevalent bacterial pathogen isolated from patients with blood stream infection in north america [ ] . s. aureus has been reported to directly infect human umbilical vein endothelial cells (huvec) thereby inducing secretion of cytokines and functional upregulation of adhesion molecules [ ] . internalized s. aureus may lead to apoptosis in huvec [ ] or persistence as small colony variants [ ] . group a streptococci can enter huvec [ ] , a process which may render these cells particularly sen-sitive to otherwise subtoxic concentrations of hydrogen peroxide [ ] . group b streptococci (gbs) are the most common cause of neonatal sepsis and pneumonia. gbs-induced endothelial cell injury can be confirmed by histological findings at autopsy, in animal studies and in vitro [ ] . gbs invasion and subsequent damage of endothelial cells may be inhibited by cytochalasin d in huvec implicating that cytoskeletal interactions are important for toxicity. however, invasion of brain microvascular endothelial cells by gbs may be dose dependently cytotoxic due to beta-hemolysin production [ ] . streptococcus pneumoniae may enter activated endothelial cells using paf-receptors [ ] . this receptor engagement may also serve as a sorting signal for endothelial transcytosis [ ] . infection and activation of endothelial cells by listeria monocytogenes is believed to be a critical component of the pathogenesis of this disease and includes ceramide generation, transcription factor activation and increases in adhesion molecule expression on huvec [ ] . listeria have been described to enter huvec either directly via internalin b or by a cell-to-cell spread from infected monocytes [ ] . gram negative bacteria have lipopolysaccarides (lps) within their cell wall. cellular binding of lps usually is accomplished by cd . endothelial cells lack cd receptors and lps effects on endothelial cells generally require the presence of cd in the serum. lps effects from gram negative live bacteria (b. fragilis, e. cloacae, h. influenzae, k pneumoniae) on endothelial cells have been demonstrated by transcription factor activation and subsequent surface expression of e-selectin and tissue factor which was not seen from viable or heat-killed gram-positive bacteria (s. aureus, e. faecalis, s. pneumoniae) [ ] . neisseria meningitidis adherence on endothelial cells has been reported to be influenced by pilus protein c expression and cd on endothelial surfaces [ , ] and may cause tissue factor expression due to the presence of lps within the cell wall. haemophilus influenzae generally is not toxic to endothelial cells except three clones of biogroup aegyptius causing brazilian purpuric fever [ ] . toxicity has been reported to be independent of endotoxin, phagocytosis and replication since irradiation, cycloheximide, cytochalasin d and methylamine have no effect on the ability of the bacterium to invade and cause a cytotoxic response. piliated pseudomonas aeruginosa adheres to and enters human endothelial cells leading to progressive damage [ ] or may persist by lysis of endosomal membranes [ ] . escherichia coli may invade human brain microvascular endothelial cells involving specialized proteins [ , ] . endothelial infection by chlamydia pneumoniae also activates endothelial cells to produce cytokines and adhesion molecules and become procoagulant [ ] [ ] [ ] . bartonella quintana, the cause of trench fever transmitted by the body louse, has recently been implicated in culture-negative endocarditis and bacteraemia amongst homeless people [ ] . infection and damage of endothelial cells by b. quintana has been demonstrated in vitro and in vivo [ ] . interaction of bartonella henselae with endothelial cells may result in bacterial aggregation on the cell surface and the subsequent internalisation of the bacterial aggregate by a unique struc-ture, the invasome [ ] . rickettsia rickettsii, an obligate intracellular gram-negative bacterium which causes rocky mountain spotted fever, inhibits endothelial apoptosis allowing to remain inside the host endothelium [ ] but is also known to cause a proinflammatory endothelial phenotype. rickettsia conorii causes mediterranean spotted fever which causes endothelial infection with secretion of cytokines, increases in adhesion molecules and induction of surface tissue factor [ , ] . attachment of borrelia burgdorferi, the agent inducing lyme disease, to endothelial cells may be accomplished by cd , alpha(v)beta and alpha beta integrins or different classes of proteoglycans [ ] [ ] [ ] . b. burgdorferi activates the transcription of chemokine and adhesion in molecule gene expression in endothelial cells [ ] . plosmodium infected erythrocytes may bind to endothelial cells via p-selectin, cd , intercellular adhesion molecule (icam- ) or platelet endothelial cell adhesion molecule (pecam- ) on the endothelial surface [ ] [ ] [ ] . phagocytosed live candida albicans stimulates cytokine secretion and inducible cyclooxygenase expression in endothelial cells [ , ] . some viral diseases are known to primarily infect endothelial cells and alter their function. dengue virus infection of huvec leads to production of chemoattractant proteins rantes and il- [ ] , herpes and measles virus infection of brain microvascular endothelial cells increases lymphocyte adhesion by increasing icam- [ ] and measles virus and cytomegalovirus increases tissue factor expression on huvec [ , ] . hemorrhagic fever caused by hantaviruses may be accomplished by integrin mediated endothelial infection [ ] . on the other hand, ebola virus infects endothelial cells with a transmembrane glycoprotein and inhibits inflammatory responses [ ] . exotoxins are known to directly activate endothelial cells. platelet activating factor (paf), no˙and pgi secretion from huvec has been demonstrated by e. coli hemolysin via inositolphosphate/diacylglycerol formation and by s. aureus alpha-toxin via transmembrane ca + entry [ ] . there is increasing evidence that hemolytic uremic syndrome results from the systemic action of verocytotoxin producing e. coli on vascular endothelial cells [ ] . alpha toxin from clostridium perfringens is a phospholipase c and has been reported to induce adhesion molecule expression and secretion of chemokines from endothelial cells [ ] . brain capillary endothelial cells have been reported to express mbec , a protein that may serve as the c. perfringens enterotoxin receptor [ , ] . the activity of small gtpase rho has been shown to be altered by c. difficile toxin b [ ] and pasteurella mulrocida toxin [ ] , which leads to alterations of endothelial permeability. p. aeruginosa exotoxin a may directly injure endothelial cells by a motif shared by many toxins [ ] . listeriolysin and phosphatidylinositol-specific phospholipase c secreted from l. monocytogenes has been shown to induce phosphatidyinositol metabolism and diacylglycerol formation in the absence of bacterial uptake by the endothelial cells [ ] . some bacterial exotoxins have been used for years as pharmacological tools like pertussis toxin for studying g-protein dependent endothelial cell functions. lipoteichoic acid and peptidoglycan from cell wall components of gram positive bacteria have been shown to induce sepsis in animal models. while lipoteichoic acid has been reported to directly activate endothelial cells [ ] , peptidoglycan seems monocyte dependent [ ] . most in vitro experiments, however, were performed with different sources of lps as a surrogate activator modelling gram negative bacterial infection. endothelial stimulation using lps in relevant concentrations are usually performed in the presence of serum containing soluble cd -receptor because endothelial cells generally lack cd . alternatively, proinflammatory cytokines including tumor necrosis factor alpha (tnf-a), interleukins (il- , il- ) and interferon gamma (ifng) acting on endothelial cells have extensively been investigated and shown to alter endothelial in vitro functions [ , ] . the inflammatory response in endothelial cells has been linked to an alteration in reactive oxygen production including superoxide (o -˙) , hydrogen peroxide (h o ), nitric oxide (no˙), hydroxyl radicals (oh˙) and secondary reaction products thereof. o -˙i s believed to be present in unstressed conditions in less than nanomolar quantities within cells. mitochondrial and cytoplasmatic superoxide dismutase readily reacts with o -˙f orming h o which has been measured in micromolar concentrations in human blood. sources of endothelial o -˙p roduction apart from mitochondrial leakage may include metabolism of cytp or other metabolic byproducts and production by a nadh oxidase system or by nitric oxide synthase in the absence of l-arginine. some bacterial pathogens are known to be able to produce h o by themselves, however interaction with the endothelium may greatly enhance cellular alterations. streptococcal hemolysin, streptolysin s, is capable of interacting with h o to injure vascular endothelial cells [ ] . iron bound to the p. aeruginosa siderophore, pyochelin, augments oxidantmediated endothelial cell injury by modification of transferrin to form iron complexes capable of catalyzing the formation of oh˙from o -˙a nd h o [ ] . r. rickettsii infection of the endothelial cell line ea.hy and huvec has been demonstrated to cause glutathione depletion, a major intracellular antioxidant, and reduced glutathione peroxidase activity leading to increased amounts of intracellular peroxide [ ] . an increase in reactive oxygen species production has been shown in endothelial cells after incubation with lps, il- , tnf-a and ifn-g [ ] [ ] [ ] [ ] [ ] [ ] . tnf-a has many times been reported to increase, e.g., adhesion molecule expression inhibitable by various antioxidants [ , [ ] [ ] [ ] . nitric oxide, like o -˙, is chemically not very reactive at all. endothelial production has been established by either constitutive no-synthase (nos) iii in a ca + and phosphorylation dependent manner or by inducible nos ii. direct biochemical actions of no˙include metalcomplex containing proteins, other radical species, oxygen and oxygen derivatives leading to oxidation, nitrosation and nitration. relevant concentrations in vivo have been reported to range from nm to mm. no˙directly reacts with oxyhemoglobin (fe -o ) leading to methemoglobin (fe + ) and nitrate (no -). no˙reacts with o forming nitrite (no -) via intermediate no and n o . mainly n o is participating in n-or s-nitrosylations leading to nitrosamines or nitrosothioles, the latter may serve as a circulating source or as a transporter across cell membranes. reduced glutathione has a high affinity to n o and may also be important in toxicity related to no˙autoxidation. toxicity related cellular targets of no˙may include inhibition of cytochrome c oxidase, inhibition of catalase or dna damage. on the other hand, no˙has been reported to inhibit iron catalysed fenton reaction and to inhibit lipid peroxidation. iron nitrosyl formation in heme containing proteins are the best characterized reactions for no˙in biology. this type of interaction includes very sensitive stimulation of guanylate cyclase and inhibition of cytochrome c oxidase. e. coli hemolysin and s. aureus alpha toxin induce no˙formation in cultured porcine pulmonary endothelial cells [ ] . transformed mouse endothelial cells stimulated by the combination of ifn-g and tnf-a killed intracellular r.conorii by a mechanism that required the synthesis of no˙ [ ] . ifn-g, tnf-a and il- stimulated murine endothelial cells have been shown to kill schistosoma mansoni through the production of nitric oxide [ ] . both no˙and o -˙p roduction may have a limited influence on endothelial viability under resting conditions. cultured bovine and porcine aortic endothelial cells showed decreased no˙production after h of lps incubation which was related to decreases in capacitative ca + signals [ ] . posttranscriptional destabilization of nos iii mrna may have accounted for this early decrease in no˙production [ ] . nos ii, which is believed to produce larger amounts of no˙is also known to be regulated by many proinflammatory stimuli with significant cell type variablilities. at sites of endothelial involvement in an inflammatory process both vascular non-endothelial and non-resident cells are known to produce no˙. as the amount and physiological consequences of no˙produced from noss at the microcirculatory level is not known, it may well serve also to reduce inflammatory processes as recently implicated from a coculture experiment [ ] . that an increase in reactive oxygen species is present in human sepsis has been documented by almost any study trying to quantify secondary reaction products by several methods, however the cellular sources remain speculative ( table ). in beckman et al. showed that the presence of both no˙and o -˙p roduced peroxynitrite (onoo -) which may decompose to produce ho˙like molecules and thereby kill endothelial cells [ ] . at ph its lifetime is in the order of a second. half of the onoo formed is rapidly equilibrated to peroxynitrous acid (onooh) and breaks down to no - sepsis immunohistochemical endothelial nitrotyrosine≠ [ ] septic shock no -/no -, plasmatic nitrotyrosine≠ [ ] septic lung injury immunohistochemical endothelial nitrotyrosine≠ antioxidant capacity was defined as the ability of plasma to inhibit * ferryl myoglobin production by hydrogen peroxide addition to metmyoglobin or ** oxo-iron induced damage to deoxyribose, phospholipids and dna. tbars, thiobarbituric acid reactive substances; xod, xanthine oxidase. models and therefore question the view that this molecule solely is detrimental. toxicity induced by an interaction between hydrogen peroxide and nitric oxide has led to conflicting results. rat lung microvascular endothelial cells and porcine pulmonary artery endothelial cells exposed to h o have been reported to be protected by no˙donors [ , ] , whereas toxicity in bovine aortic endothelial cells [ ] and in rat liver microvascular endothelial cells [ ] was increased in the presence of no˙donors. no˙in the presence of h o may also produce oh˙like molecules independent of the presence of iron [ ] . neutrophils may add to the complexity of toxic reactions. myeloperoxidase from activated neutrophils, which produces hocl and oh˙molecules in the presence of o -˙, has recently been demonstrated to convert no into no , thereby damaging endothelial cells [ ] . high doses of reactive oxygen species (including no˙) have been shown to cause apoptosis of endothelial cells, whereas low doses were protective [ ] . in mice disseminated endothelial apoptosis has been suggested to be responsible for organ failure and shock induced by endotoxin or tnf-a [ ] . both lps or tnf-a usually do not cause endothelial cell death unless protein synthesis is blocked probably due to simultaneous increases in antiapoptotic protein synthesis [ ] . however, postmortal investigation in humans deceased from or with sepsis did not confirm these results [ ] . most of the genes activated in vitro during the endothelial stress response are controlled by at least two transcription factor families: activator protein (ap- ) and nuclear factor kappa b (nfkb). nfkb has gained wide interest as a target in inflammatory diseases as it seems to be invariably upregulated [ ] . a variety of agents including cytokines and reactive oxygen stress cause ikb to dissociate from the complex after phosphorylation by ikb-kinase complex. h o has been reported to activate transcription factor nfkb in porcine aortic endothelial cells [ ] whereas huvec were unresponsive [ ] . no˙has in most cases been shown to inhibit transcription factor nfkb and its influence on transcription factor ap- is unclear. inhibition of nfk-b as a therapeutic means has been suggested. however, as this transcription factor is also involved in protective gene regulation, its inhibition can make cells sensitive to e.g. tnf-a [ ] . data on activated intracellular signalling pathways in sepsis patients are scarce but include nfkb within mononuclear cells [ ] . usually any blood cell is kept off endothelial surfaces. this is believed to be accomplished by net electrical charge, biomechanical characteristics of flowing blood and the secretion of no˙. if blood cells touch the endothelium a ca + -signal is induced [ ] , but it is unclear at present whether this has any functional consequence. it is tempting to speculate that ca +signals may then in a context sensitive manner augment proadhesive processes or antiadhesive endothelial properties. activated endothelial cells are potent producers of cytokines like il- , a major proinflammatory cytokine, and chemotactic peptides including il- for neutrophils, macrophage inflammatory protein- alpha (mip- a), monocyte chemoattractant proteins - and rantes (regulated on activa-tion, normal t cell expressed and secreted) for monocytes, t-lymphocytes and dendritic cells, growth related protein (gro) and gamma-interferon-inducible protein (ip- ) for activated t-lymphocytes, epithelial neutrophil activating peptide (ena- ), vascular monocyte adhesion-associated protein (vmap- ) and endothelial monocyteactivating polypeptide ii (emap-ii) for monocytes [ ] . many adhesion molecules are expressed on the surface of endothelial cells in a highly complex yet regulated manner. p-selectin, e-selectin, icam- , vascular cell adhesion molecule (vcam- ), pecam- are well known for their stimulus-, cell-, time-and organ specific dependence of expression and their importance in regulation of leukocyteendothelial interactions. moreover, apart from being passive adhesion molecules, all of the above mentioned molecules have been shown to signal inside endothelial cells upon receptor engagement. shed receptors from endothelial surfaces may also serve as endogeneous antiadhesive molecules demonstrating even more the dynamic and complex nature of these processes. soluble forms of adhesion molecules have been shown to be present in high amounts in human sepsis (table ) , however whether this is beneficial or detrimental is not known and the assumption that these parameters may serve as practical indexes remains to be established. particularly ifn-g has been repeatedly shown to increase endothelial hla-dr expression rendering them capable of mhc class ii restricted interactions with cd + t-cells. costimulatory cd (b - ) and cd (b - ) are usually not present on endothelial surfaces leading to the conventional view that endothelial cells are semiprofessional antigen presenting cells. however, under certain conditions both costimulatory molecules and cd can be upregulated on endothelial surfaces indicating excessive antigen presentation [ ] . migration of lymphocytes into inflamed mouse tis-vol. , endothelial function in sepsis sues has been reported to depend on psgl- and esl- binding endothelial p-selectin and e-selectin, respectively [ ] . this phenotype was found to be restricted to th lymphocytes, but human sepsis seems to be predominated by a th type [ ] . both tnf-a and ifn-g are able to promote transmigration of leukocytes, whereas coapplication of both cytokines inhibit this process [ ] . t-cells migrating through the endothelial barrier in a pecam- -dependent manner are subject to inhibitory signals which may limit their activation in tissues [ ] . tnf bound to monocytes has been shown to inhibit endothelial apoptosis, whereas this process is promoted in the presence of lymphocytes [ ] . unperturbed endothelial cells express fas-ligand which has been implicated as a means of signalling apoptosis to constitutively fas receptor (cd ) bearing cells, whereas tnf-a treated endothelial cells decrease fas ligand expression thereby allowing leukocyte survival during extravasation [ ] . how the endothelium might interact specifically with lymphocytes or monocytes in septic patients can only be speculated on. neutrophils interacting with endothelial cells have repeatedly been shown to cause toxicity due to the release of enzymes and reactive oxygen species. if uncontrolled, these cells are believed to mediate significant tissue damage. however whether uncontrolled activation or rather deactivation is present in human sepsis is a matter of debate. endothelial cells have been reported to inhibit some neutrophil functions [ ] . transmigrated neutrophils may actively participate in the endothelial resealing process by the secretion of adenosin precursors [ ] . however, massive leukocyte extravasation as one would expect from most animal studies has never been shown in septic patients [ ] . endothelium is known to regulate transvascular fluid flux, flux of nutrients, mediators and cells by either paracellular or transcellular vacuolar channel related pathways. lateral junction proteins including the vascular endothelial cadherin-complex, platelet-endothelial cell adhesion molecule- , occludin, zona occludens- , recently described junctional adhesion protein, cd /platelet endothelial tetraspan antigen and cd /target of antiproliferative antigen are known to participate in this process. paracellular permeability is achieved by either an active contraction or the controlled release of an intrinsic tone mediated in most cases by the action of myosin light chain kinase (mlck) acting on non muscle myosin. generally an increase in the concentration of camp keeps cultured endothelial monolayers tight and cgmp has been reported to assist this function in human aortic and foreskin vessels [ ] . endothelial retraction may be initiated by increases in intracellular ca + concentration, but elevation of ca + in the presence of maintained camp-kinase dependent phosphorylation is not edemagenic. these antagonistic effects of ca + and camp in endothelial permeability regulation have recently been reviewed by moore et al. [ ] . many reports documented increases in endothelial permeability involving exotoxins like s. aureus alpha-toxin and p. aeruginosa cytotoxin [ , ] or endotoxins [ ] . for example, p. multocida toxin has been shown to activate rho/rho kinase, which inactivates mlc phosphatase. the resulting increase in mlc phosphorylation caused endothelial cell retraction and a rise in endothelial permeability [ ] . lps induced increases in paracellular permeability by caspase activated cleavage of adherens junction proteins [ ] . counteracting lipid peroxidation during lps activation may inhibit increases in permeability [ ] . tnf-a stimulated endothelial cadherin complex is disrupted in a proteasome dependent manner [ ] . the resulting increase in permeability has been reported to lower camp and activate phosphediesterase ii and iv [ ] . h o induced hyperpermeability of porcine pulmonary endothelial cells has been reported to be effectively reduced by cgmp elevating drugs including phosphodiesterase ii inhibition or no˙donators [ ] . the electroneutral na-k-cl cotransport system is thought to function in the maintenance of a selective permeability. il- , tnf-a and lps upregulate the expression of a bumetanide-sensitive na-k-cl cotransporter subtype in huvec and in murine lung and kidney endothelial cells [ ] . septic rats show different increases in albumin flux accross several endothelial beds [ ] . increases in venular permeability have been shown to be preventable by the antioxidants n-acetyl-cystein or tirilazad mesylate in e. coli infused rats [ , ] . il- , an antiinflammatory cytokine not produced by endothelial cells, was shown to participate as an inhibitor of endothelial permeability induced by lps in mice [ ] . clinically, increases in endothelial permeability may be obvious in many septic patients, but only recently venous congestion plethysmography showed a selectively elevated filtration capacity as a measure of endothelial dysfunction in septic patients [ ] . which of the many pathways of increased permeability might be turned on and whether it persists remains unknown. in principle endothelial cells are believed to be anticoagulatory by virtue of their surface expression of glycosaminoglycan-antithrombin iii complex, thrombomodulin, heparin releasable tissue factor pathway inhibitor and production of adenosine by ecto-adpases, their secretion of protein s, prostacyclin and no˙. no˙production was shown to participate in heparan sulfate preservation in porcine aortic endothelial cells [ ] and may be responsible for prostacyclin secretion by activating cyclooxygenase- under resting conditions [ ] . endothelial release of plasminogen activator (t-pa) and plasminogen activator inhibitor (pai- ) may determine the fibrinolytic potential of plasma. endothelial cells specifically bind coagulation factors xii, iia, ix, viia and xa. xa was shown to bind to endothelial effector cell protease receptor- and thereby cause release of no˙, il- , il- , mcp- and functional upregulation of icam- , e-selectin and vcam- [ ] . when endothelium is perturbed by physical or chemical factors transformation to a prothrombotic surface is invariably seen in in vitro models. thrombomodulin surface expression on huvec can easily be downregulated by lps, il- and tnf-a and upregulated by increasing camp [ ] . a tnf-a induced decrease in surface thrombomodulin has been suggested via activation of phosphodiesterase ii and iv thereby decreasing camp in baec [ ] . vascular endothelial growth factor may counteract il- , tgf-b and lps induced suppression of both thrombomodulin surface antigen and mrna [ ] . adenosin nucleotides are released from damaged as well as lps stimulated and shear stressed huvec [ ] . endothelial cells have atp diphosphohydrolase (cd ) on their surface to degrade atp via adp and amp to adenosine. adenosine is known to have antiaggregatory properties due to stimulation of prostacyelin and no˙production. however, activating endothelial cells with tnf-a has been reported to cause loss of atp diphosphohydrolase activity, which was preventable in the presence of antioxidants [ ] . tissue factor (tf) expression on endothelial cells by bacteria, lps, il- and tnf-a is well known. tissue factor pathway inhibitor (tfpi), a serine protease inhibitor of xa and xa/viia/tf complex on endothelial surfaces, which immediately blocks tissue factor activation, has been shown to be decreased under proinflammatory conditions. another counterbalancing mechanism includes shear stress in tnf-a stimulated huvec [ ] . however, an anticipated increase in endothelial tissue factor expression has not convincingly been demonstrated in animal or human sepsis [ , ] . fibrinolytic systems on endothelial surfaces are also believed to be altered in sepsis. increases in pai- has been reported after stimulation with il- or lps [ , ] . however soluble pai- in septic patients was not found to be different from nonseptic patients [ ] . once thrombin formation has occured, its cleavage of endothelial proteinase activated receptor (par) in turn may lead to secretion of il- and il- [ ] , upregulation of icam- and vcam- [ ] , relaxation via no˙production or to vasoconstriction by an as yet unidentified factor [ ] . these data may demonstrate an interconnection between coagulation activation, inflammation and vasoregulation mediated by the endothelium. coagulation activation is clearly present in septic patients, however endothelial participation in this process is unclear. potent procoagulatorv sources may well include bacterial surfaces per se [ ] or monocytes [ ] . up to the late ies the endothelium was viewed as a passive organ, which at best was able to remove vasoactive hormones in the lung. in - sir john vane's group (noble laureate ) reported that endothelial cells can synthesize i series prostaglandins (like prostacyclin) and thereby relax arteries and inhibit platelet aggregation. however, whether pgi regulates basal vascular tone is unclear. after a new technician used an unintended vessel preparation robert furchgott realized after a series of contradicting results that acetylcholine (ach) was no longer able to relax precontracted arteries when endothelium was removed and termed the nonprostanoid mediator an endothelium derived relaxing factor (edrf). in superoxide was shown to participate in vasoregulation. the presence of superoxide dismutase prolonged the action of edrf whereas addition of o -˙i nactivated edrf. even direct effects of several reactive oxygen species have been suggested to be relevant in cerebral or coronary circulation. collectively robert furchgott, louis ignarro and ferrid murad received the noble laureate in for demonstrating that no˙is a major edrf. endothelial cells produce more relaxing factors which pharmacologically can be separated from nitric oxide action and these were termed endothelium derived hyperpolarizing factors (edhfs). these factors may particularly be important in coronary and gastrointestinal vessels. epoxyeicosatrienoic acids, anandamide, the endogenous ligand of cannabinoid receptors or simply the release of k + may constitute edhfs. conceptually shear may in larger vessels primarily determine production of no˙, whereas cyclic strain determines physiological edhf release. soon after the discovery of edrf endothelin- (et- ), a vasoconstricting peptide produced from endothelial cells was isolated. low doses of et- can induce no˙release and subsequent relaxation via et breceptors on endothelial cells. endothelin secretion is thought to occur abluminally leading to et a -receptor activation on smooth muscle cells and subsequent vasoconstriction. many other factors clearly contribute to endothelial control of vasoregulation by e.g. transcellular production of vasoconstricting prostanoids like thromboxane a or prostaglandin h or the enzymatic conversion of angiotensin i to angiotensin ii by angiotensin converting enzyme. a hallmark of sepsis is the heterogeneous pattern of vasoconstriction and vasodilatation in different organs, culminating in a fall in total peripheral vascular resistance concomitant with regional maldistribution of blood flow. vasoactive substances produced by the endothelium under experimental septic conditions are known to be altered by factors such as no˙, pgi , angiotensin converting enzyme (ace) activity, endothelin and adrenomedullin. endothelium dependent vasoregulation has largely been studied in animal models (table ). in endothelium dependent vasoregulatory failure was seen as a defect in reactive hyperemia related vasodilator release [ ] and decreased dilatation of arterioles induced by ach [ ] . in a rat model of cecal ligation and puncture decreased vasoconstriction was found after administration of norepinephrine in septic animals which was largely reversible by removal of the endothelium [ ] . parker et al. [ ] showed in explanted coronary arteries and aortas of guinea pigs treated with lps intraperitoneally for h that endothelium dependent relaxation induced by acetylcholine and adp was depressed, whereas relaxation induced by substance p or receptor independent relaxations by ca + -ionophore a was unaffected. edrf release and bioactivity from explanted aortas of these animals was decreased after adp or ach stimulation, whereas a induced edrf release was unaltered [ ] . this group also demonstrated reduced adp and ach responses after h of lps endotoxemia in guinea pigs and that adp may produce constricting thromboxane in septic animals [ ] . in contrast, coronary arteries of rabbits treated for weeks with low doses of lps stimulation with ach but not adp showed increased relaxation of explanted vessels [ ] . wang et al. [ ] isolated subepicardial arterioles from rats treated h intraperitoneally with feces containing life e. coli and showed in a pressurized no-flow chamber that relaxation by alpha agonist clonidine and adp was reduced in an endothelium dependent manner, which could be inhibited by the nos inhibitor lnma. also, in this model mesenteric arter-vol. , endothelial function in sepsis iolar relaxation after adp and clonidine was decreased, whereas in skeletal muscle these agonists caused vasoconstriction [ ] . pulmonary arteries of rats treated with lps showed depressed endothelin- induced contractions which were even augmented in endothelium denuded vessels. the authors [ ] concluded that a vasoconstrictor eicosanoid is produced in lps treated animals by pulmonary endothelium upon et- stimulation. swine infused with live p. aeruginosa showed no alteration in endothelium dependent bradykinin and endothelium independent nitroprusside relaxation, whereas ach induced relaxation was found to be reduced in explanted peripheral arteries [ ] . chaudry's group investigated endothelium dependent relaxation in rats treated with cecal ligation and puncture. a time dependent alteration was demonstrated with increased ach induced vasorelaxation early after challenge whereas depressed vasodilatation after - h was found in explanted aortas with no alteration in nitroglycerine induced relaxation [ ] . the decreased endothelium dependent response to ach was also found in superior mesenteric arteries and small intestinal arteries [ ] . in the same model this group demonstrated a reduction of immunodetectable nos iii in explanted aortas [ ] . porcine coronary arterioles incubated for h with e. coli lps ( mg/ml) decreased bradykinin induced edhf secretion [ ] . carotid and coronary arteries from rabbits also showed decreases in edhf-release in an ex-vivo assay after treatment with lps, tnf-a and il- [ ] . collectively these data indicate that the majority of sepsis models consistently show a disruption of receptor coupled relaxation mechanism leading to an intraendothelial signalling deficit. to quantify endothelial function in humans several methods are available. endothelium dependent relaxation after pharmacological stimulation or flow dependent relaxation after vessel obstruction are frequently quantified by high resolution ultrasound techniques [ ] , alternatively flow and size can be determined angiographically. however, definite functional measurements in human sepsis are scarce. endothelium dependent relaxation has been investigated in isolated superficial hand veins of healthy volunteers after lps exposure. reduced vasorelaxation by bradykinin and arachidonic acid in noradrenalin precontracted vessels were noted which persisted for more than days [ ] . reactive hyperemia is believed to mainly test endothelial no˙production upon shear stress if vessel diameters and flow is monitored. implications from indirect measurements support an endothelial dysfunction in septic patients [ ] [ ] [ ] . however, data on pharmacological stimulation of endothelium dependent relaxation in humans are currently not available. t. volk +/-ec: presence or absence of endothelium; ne: norepinephrine; ach: acetylcholine; adp, adenosine diphosphate; sp, substance p; a , ca + -ionophore; cox, cycloogygenase; snp, sodium nitroprusside; ntg, nitroglycerine; et, endothelin; bk, bradykinin; edhf, endothelium derived hyperpolarizing factor; pres., preserved. table . endothelium dependent relaxation is impaired in animal sepsis models. a normal response to infection or other insults is a self limiting process that through temporal expression of regulators and effector molecules causes resolution. the failure to resolve the causative infection may lead to sepsis. cellular and animal models of sepsis using bacteria, endotoxins, exotoxins, cytokines or some peptides all consistently produce endothelial impairment which is usually regarded as dysfunctional. blocking the majority of pathways used by these inducing agents has often lead to the inhibition of such endothelial alterations. many aspects of these induced alterations can be expected to reveal exciting new pathways and complex interactions at various molecular and cellular level. the past has taught us that inhibitors of presumably activated pathways consistently failed to improve survival in septic patients. this has stimulated many researchers to reconcile the results of experimental and clinical models in sepsis. compared to activating pathways, considerably less is known about how an inflammatory response is endogenously counterregulated. cytokines induce a whole host of signal inhibiting proteins and endogenous counterregulating systems are just beginning to be elucidated. activation of endogenous counterregulatory systems may become the predominant feature of the so-called compensatory antiinflammatory response syndrome. endothelial responses to endogenously present antiinflammatory mediators have hardly been investigated in sepsis models. almost all endothelial cell studies in sepsis indicated that an imbalance in reactive oxygen species production is associated with the above described dysfunctions. animal studies in which endothelial function could be improved pharmacologically also consistently indicate that reversal of imbalanced reactive oxygen production may be a common link (table ). it seems that at times an adequate production of nitric oxide is lacking whereas superoxide and/or derivatives are overproduced. however, as endothelial functional measurements in septic humans become available, we will hopefully get a clearer picture of what might happen in our patients. [ ] clp, cecal ligation and puncture; lpo, lipid peroxidation; ros, reactive oxygen species. table . treatment of endothelial vasoregulatory dysfunction in animal sepsis models. bacterial pathogens isolated from patients wit bloodstream infection: frequencies of occurrence and antimicrobial susceptibility patterns from the sentry antimicrobial surveillance program (united states and canada, ) staphylococcus aureus infections internalization of staphylococcus aureus by endothelial cells induces apoptosis staphylococcus aureus small colony variants are induced by the endothelial cell intracellular milieu genetic inactivation of the extracellular cysteine protease enhances in vitro internalization of group a streptococci by human epithelial and endothelial cells interaction of viable group a streptococci and hydrogen peroxide in killing of vascular endothelial cells group b streptococci invade endothelial cells: type iii capsular polysaccharide attenuates invasion invasion of brain microvascular endothelial cells by group b streptococci streptococcus pneumoniae anchor to activated human cells by the receptor for platelet-activating factor pneumococcal trafficking across the blood-brain barrier. molecular analysis of a novel bidirectional pathway two distinct phospholipases c of listeria monocytogenes induce ceramide generation, nuclear factor-kappa acivation, and e-selectin expression in human endothelial cells internalin b is essential for adhesion and mediates the invasion of listeria monocytogenes into human endothelial cells activation of human endothelial cells by viable or heat-killed gram-negative bacteria requires soluble cd interaction of neisseria maningitidis with the components of the blood-brain barrier correlates with an increased expression of pilc the ndomain of the human cd a adhesion molecule is a target for opa proteins of neisseria meningitidis and neisseria gonorrhoeae human microvascular endothelial tissue culture cell model for studying pathogenesis of brazilian purpuric fever pseudomonas aeruginosa selective adherence to and entry into human endothelial cells endothelial function in sepsis cincomitant endosome-phagosome fusion and lysis of endosomal membranes account for pseudomonas aeruginosa survival in human endothelial cells endothelial cell glcnac beta - glcnac epitopes for outer membrane protein a enhance traversal of escherichia coli across the blood-brain barrier escherichia coli invasion of brain microvascular endothelial cells in vitro and in vivo: molecular cloning and characterization of invasion gene ibe characterization of a strain of chlamydia pneumoniae isolated from a coronary atheroma by analysis of the omp gene and biological activity in human endothelial cells chlamydia species infect human vascular endothelial cells and induce procoagulant activity signal transduction pathways activated in endothelial cells following infection with chlamydia pneumoniae bartonella (rochalimaea) quintana endocarditis in three homeless men bartonella quintana invades and multiplies within endothelial cells in vitro and in vivo and forms intracellular blebs interaction of bartonella henselae with endothelial cells results in bacterial aggregation on the cell surface and the subsequent engulfment and internalisation of the bacterial aggregate by a unique structure, the invasome nf-kappa b-dependent inhibition of apoptosis is essential for host cellsurvival during rickettsia rickettsii infection rickettsia conorii infection enhances vascular cell adhesion molecule- -and intercellular adhesion molecule- -dependent mononuclear cell adherence to endothelial cells il- and il- production from cultured human endothelial cells stimulated by infect on with rickettsia conorii via a cell-associated il- alpha-dependent pathway the role of cd in signaling mediated by outer membrane lipoproteins of borrelia burgdorferi integrins alpha(v)beta and alpha beta mediate attachment of lyme disease spirochetes to human cells different classes of proteoglycans contribute to the attachment of borrelia burgdorferi to cultured endothelial and brain cells borrelia burgdorferi upregulates the adhesion molecules e-selectin, p-selectin, icam- and vcam- on mouse endothelioma cells in vitro characterization of plasmodium falciparum-infected erythrocyte and p-selectin interaction under flow conditions intercellular adhesion molecule- and cd synergize to mediate adherence of plasmodium falciparum-infected erythrocytes to cultured human microvascular endothelial cells pecam- /cd , an endothelial receptor for binding plasmodium falciparum-infected erythrocytes candida albicans stimulates cytokine production and leukocyte adhesion molecule expression by endothelial cells secreted aspartyl proteinases and interactions of candida albicans with human endothelial cells dengue virus infection of human endothelial cells leads to chemokine production, complement activation, and apoptosis adhesion molecule expression and lymphocyte adhesion to cerebral endothelium: effects of measles virus and herpes simplex virus measles virus induction of human endothelial cell tissue factor procoagulant activity in vitro effects of viral activation of the vessel wall on inflammation and thrombosis cellular entry of hantaviruses which cause hemorrhagic fever with renal syndrome is mediated by beta integrins ebola virus inhibits induction of genes by double-stranded rna in endothelial cells human endothelial cell activation and mediator release in response to the bacterial exotoxins escherichia coli hemolysin and staphylococcal alpha-toxin infection by verocytotoxin-producing escherichia coli alpha toxin from clostridium perfringens induces proinflammatory changes in endothelial cells brain capillary endothelial cells express mbec , a protein that is related to the clostridium perfringens enterotoxin receptors phospholipase c and perfringolysin o from clostridium perfringens upregulate endothelial cellleukocyte adherence molecule and intercellular leukocyte adherence molecule expression and induce interleukin- synthesis in cultured human umbilical vein endothelial cells glucosylation of small gtp-binding rho proteins disrupts endothelial barrier function pasteurella multocida toxin increases endothelial permeability via rho kinase and myosin light chain phosphatase evidence for a structural motif in toxins and interleukin- that may be responsible for binding to endothelial cells and initiating vascular leak syndrome the listerial exotoxins listeriolysin and phosphatidylinositol-specific phospholipase c synergize to elicit endothelial cell phosphoinositide metabolism lipoteichoic acid-induced neutrophil adhesion via e-selectin to human umbilical vein endothelial cells (huvecs) endothelial and epithelial cells do not respond to complexes of peptidoglycan with soluble cd but are activated indirectly by peptidoglycan-induced tumor necrosis factor-alpha and interleukin- from monocytes cytokines and endothelial cell biology cytokine regulation of endothelial cell function: from molecular level to he bedside pseudomonas siderophore pyochelin enhances neutrophil-mediated endothelial cell injury superoxide dismutase-dependent, catalase-sensitive peroxides in human endothelial cells infected by rickettsia rickettsii superoxide release from interleukin- b-stimulated human vascular cells: in situ electrochemical measuremeut lipopolysaccharide enhances oxidative modification of low density lipoprotein by copper ions, endothelial and smooth muscle cells e-selectin expression in human endothelial cells by tnf-alpha-induced oxidant generation and nf-kappab activation superoxide responses of endothelial cells to c a and tnf-alpha: divergent signal transduction pathways lactosylceramide mediates tumor necrosis factor-alpha induced intercellular adhesion molecule- (icam- ) expression and the adhesion of neutrophil in human umbilical vein endothelial cells effect of antioxidants on lipopolysaccharide-stimulated induction of mangano superoxide dismutase mrna in bovine pulmonary artery endothelial cells ambient but not incremental oxidant generation effects intercellular adhesion molecule induction by tumour necrosis factor alpha in endothelium icam- and vcam- expression induced by tnf-alpha are inhibited by a glutathione peroxidase mimic glutathione peroxidase mimics prevent tnfalpha-and neutrophilinduced endothelial alterations pore-forming bacterial toxins potently induce release of nitric oxide in porcine endothelial cells cytokine-induced, nitric oxide-dependent, intracellular antirickettsial activity of mouse endothelial cells endothelial cells are activated by cytokine treatment to kill an intravascular parasite, schistosoma mansoni, through the production of nitric oxide escherichia coli endotoxin inhibits agonistmediated cytosolic ca + mobilization and nitric oxide biosynthesis in cultured endothelial cells expressional control of the "constitutive" isoforms of nitric oxide synthase (nos i and nos iii) inducible nitric oxide: an autoregulatory feedback inhibitor of vascular inflammation apparent hydroxyl radical production by peroxynitrite: implications for endothelial injury from nitric oxide and superoxide nitric oxide donor prevents hydrogen peroxide-mediated endothelial cell injury nitric oxide attenuates hydrogen peroxide-mediated injury to porcine pulmonary artery endothelial cells protective effects of tetrahydrobiopterin against nitric oxide-induced endothelial cell death endothelial damage induced by nitric oxide: synergism with reactive oxygen species hydroxyl radical formation resulting from the interaction of nitric oxide and hydrogen peroxide halliwell b van d v. formation of nitric oxide-derived inflammatory oxidants by myeloperoxidase in neutrophils nitric oxide inhibits lipopolysaccharide-induced apoptosis in pulmonary artery endothelial cells lipopolysaccharide induces disseminated endothelial apoptosis requiring ceramide generation lipopolysaccharide induces the antiapoptotic molecules, a and a , in microvascular endothelial cells apoptotic cell death in patients with sepsis, shock, and multiple organ dysfunction nuclear factor-kappab: a pivotal transcription factor in chronic inflammatory diseases oxidant-sensitive and phosphorylation-dependent activation of nf-kappa b and ap- in endothelial cells endothelial activation by hydrogen peroxide. selective increases of intercellular adhesion molecule- and major histocompatibility complex class i adenovirus-mediated expression of a dominant negative mutant of p /rela inhibits proinflammatory gene expression in endothelial cells without sensitzing to apoptosis role of nfkappab in the mortality of sepsis endothelial function in sepsis initial contact and subsequent adhesion of human neutrophils or monocytes to human aortic endothelial cells releases an endothelial intracellular calcium store chemokines and leukocyte traffic cd ligation induced phenotypic and functional expression of cd by human cardiac microvascular endothelial cells cd + t cells migrate into inflamed skin only if they express ligands for e-and p-selectin t helper cell subset ratios in patients with severe sepsis cutting edge: combined treatment of tnf-alpha and ifngamma causes redistribution of junctional adhesion molecule in human endothelial cells a new role for platelet-endothelial cell adhesion molecule- (cd ): inhibition of tcrmediated signal transduction monocytes stimulate expression of the bcl- family member, a , in endothelial cells and confer protection against apoptosis negative regulation of inflammation by fas ligand expression on the vascular endothelium regulatory effects of endogenous protease inhibitors in acute lung inflammatory injury neutrophil-derived ¢-adenosine monophosphat promotes endothelial barrier function via cd -mediated conversion to adenosine and endothelial a b receptor activation neutrophil migration during endotoxemia expression of cgmp-dependent protein kinase i and phosphorylation of its substrate, vasodilator-stimulated phosphoprotein, in human endothelial cells of different origin signal transduction and regulation of lung endothelial cell permeability interaction between calcium and camp bacterial exotoxins and endothelial permeability for water and albumin in vitro effects of escherichia coli hemolysin on endothelial cell function endotoxin-neutralizing protein protects against endotoxin-induced endothelial barrier dysfunction bacterial lipopolysaccharide disrupts endothelial monolayer integrity and survival signaling events through caspase cleavage of adherens junction proteins endotoxin-induced changes of endothelial cell viability and permeability: protective effect of a -aminosteroid endothelial-dependent mechanisms regulate leukocyte transmigration: a process involving the proteasome and disruption of the vascular endothelial-cadherin complex at endothelial cell-tocell junctions tnf modulates endothelial properties by decreasing camp role of nitric oxide and phosphodiesterase isoenzyme ii for reduction of endothelial hyperpermeability expression of the bumetanide-sensitive na-k-cl cotransporter bsc is differentially regulated by fluid mechanical and inflammatory cytokine stimuli in vascular endothelium endothelial barrier resistance in multiple organs after septic and nonseptic challenges in the rat n-acetylcysteine attenuates endotoxin-induced leukocyte-endothelial cell adhesion and macromolecular leakage in vivo effect of the -aminosteroid tirilazad mesylate on leukocyte adhesion and macromolecular leakage during endotoxemia endogenous interleukin- regulates hemodynamic parameters, leukocyte-endothelial cell interactions, and microvascular permeability during endotoxemia increased microvascular water permeability in patients with septic shock, assessed with venous congestion plethysmography (vcp) endothelial-derived nitric oxide preserves anticoagulant heparan sulfate expression in cultured porcine aortic endothelial cells does elevated nitric oxide production enhance the release of prostacyclin from shear stressed aortic endothelial cells? hypotension and inflammatory cytokine gene expression triggered by factor xa-nitric oxide signaling up-regulation of thrombomodulin in human umbilical vein endothelial cells in vitro thrombomodulin-dependent anticoagulant activity is regulated by vascular endothelial growth factor increased release of atp from endothelial cells during acute inflammation loss of atp diphosphohydrolase activity with endothelial cell activation fluid shear stress attenuates tumor necrosis factoralpha-induced tissue factor expression in cultured human endothelial cells cell biology of tissue factor, the principal initiator of blood coagulation on behalf of the subcommittee on tissue factor pathway inhibitor (tfpi) of the scientific and standardization committee of the isth effects of lipopolysaccharide on the expression of fibrinolytic factors in an established cell line from human endothelial cells induction of plasminogen activator inhibitor type and type collagen expression in rat cardiac microvascular endothelial cells by interleukin- and its dependence on oxygencentered free radicals plasminogen: an important hemostatic parameter in septic patients potential mechanisms for a proinflammatory vascular cytokine response to coagulation activation thrombin-activated human endothelial cells support monocyte adhesion in vitro following expression intercellular adhesion molecule- cd ) and vascular cell adhesion molecule- (vcam- ; cd ) dual endothelium-dependent vascular activities of proteinase-activated receptor- -activating peptides: evidence for receptor heterogeneity activation of the contact-phase system on bacterial surfaces -a clue to serious complications in infectious diseases reactive hyperemic responses of single arterioles are attenuated markedly after intestinal ischemia, endotoxemia and traumatic shock: possible role of endothelial cells failure of microscopic metarterioles to elicit vasodilator responses to acetylcholine, bradykinin, histamine and substance p after ischemic shock, endotoxemia and trauma: possible role of endothelial cells vascular endothelium contributes to decreased aortic contractility in experimental sepsis selective inhibition of endotheliumdependent vasodilator capacity by escherichia coli endotoxemia release of edrf and no in ex vivo perfused aorta: inhibition by in vivo e. coli endotoxemia inhibition of endothelium-dependent vasodilation by escherichia coli endotoxemia chronic endotoxemia and endothelium-dependent vasodilation in coronary arteries chronic septicemia alters alpha-adrenergic mechanisms in the coronary circulation mesenteric and skeletal muscle microvascular responsiveness in subacute sepsis contraction to endothelin- in pulmonary arteries from endotoxin-treated rats is modulated by endothelium pulmonary artery 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injury elevated von willebrand factor antigen is an early plasma predictor of acute lung injury in nonpulmonary sepsis syndrome increased plasma levels of soluble thrombomodulin in patients with sepsis and organ failure endothelial cell activity varies in patients at risk for the adult respiratory distress syndrome soluble e-selectin levels in sepsis and critical illness. correlation with infection and hemodynamic dysfunction die zirkulierenden adhäsionsmoleküle sicam- und sb-selectin bei patienten mit sepsis elevated circulating e-selectin, intercellular adhesion moleculc , and von willebrand factor in patients with severe infection increased circulating thrombomodulin in children with septic shock systemic endothelial activation occurs in both mild and severe malaria. correlating dermal microvascular endothelial cell phenotype and soluble cell adhesion molecules with disease severity increased plasma von willebrand factor in the systemic inflammatory response syndrome is derived from generalized endothelial cell activation blood levels of endothelin- and thrombomodulin in patients with disseminated intravascular coagulation and sepsis plasma levels of endothelial cell protein c receptor are elevated in patients with sepsis and systemic lupus or erythematosus: lack of correlation with thrombomodulin suggests involvement of different pathological processes demonstration of rickettsia conorii-induced endothelial injury in vivo by measuring circulating endothelial cells, thrombomodulin, and von willebrand factor in patients with mediterranean spotted fever influence of angiotensin-converting enzyme inhibitor enalaprilat on endothelial-derived substances in the critically ill mesenteric and skeletal muscle microvascular responsiveness in subacute sepsis influence of group b streptococci on piglet pulmonary artery response to bradykinin effects of antisense oligonucleotide to inos on hemodynamic and vascular changes induced by lps pentoxifylline maintains vascular endothelial cell function during hyperdynamic and hypodynamic sepsis a novel nonanticoagulant heparin prevents vascular endothelial cell dysfunction during hyperdynamic sepsis effect of endotoxin-enhanced hepatic reperfusion injury on endonthelium-dependent relaxation in rat aorta splanchnic vascular endothelial dysfunction in rat endotoxemia: role of superoxide radicals endothelial dysfunction in a rat model of endotoxic shock. importance of the activation of poly (adp-ribose) synthetase by peroxynitrite endothelial cell dysfunction in septic shock key: cord- -s s wb authors: noga, marek j.; büke, ferhat; van den broek, niels j. f.; imholz, nicole c. e.; scherer, nicole; yang, flora; bokinsky, gregory title: posttranslational control of plsb is sufficient to coordinate membrane synthesis with growth in escherichia coli date: - - journal: mbio doi: . /mbio. - sha: doc_id: cord_uid: s s wb every cell must produce enough membrane to contain itself. however, the mechanisms by which the rate of membrane synthesis is coupled with the rate of cell growth remain unresolved. by comparing substrate and enzyme concentrations of the fatty acid and phospholipid synthesis pathways of escherichia coli across a -fold range of carbon-limited growth rates, we show that the rate of membrane phospholipid synthesis during steady-state growth is determined principally through allosteric control of a single enzyme, plsb. due to feedback regulation of the fatty acid pathway, plsb activity also indirectly controls synthesis of lipopolysaccharide, a major component of the outer membrane synthesized from a fatty acid synthesis intermediate. surprisingly, concentrations of the enzyme that catalyzes the committed step of lipopolysaccharide synthesis (lpxc) do not differ across steady-state growth conditions, suggesting that steady-state lipopolysaccharide synthesis is modulated primarily via indirect control by plsb. in contrast to steady-state regulation, we found that responses to environmental perturbations are triggered directly via changes in acetyl coenzyme a (acetyl-coa) concentrations, which enable rapid adaptation. adaptations are further modulated by ppgpp, which regulates plsb activity during slow growth and growth arrest. the strong reliance of the membrane synthesis pathway upon posttranslational regulation ensures both the reliability and the responsiveness of membrane synthesis. activities have been proposed or identified. however, how each of these individual elements is used to regulate membrane synthesis during steady-state growth remains unclear ( ) . the issue of how membrane construction is coordinated with growth can be approached by considering how the metabolic pathways that synthesize membrane building blocks (pl and lps) are regulated. biosynthetic fluxes are regulated either by control of enzyme concentrations or by direct control of enzyme activity. examples of both forms of regulation can be readily found: in escherichia coli, the steady-state protein synthesis rate is controlled by ribosome concentration, which is transcriptionally regulated to balance amino acid supply with translation demand ( ) . in contrast, fluxes through central carbon metabolism are controlled by concentrations of substrates and inhibitors (i.e., posttranslational control) rather than by changes in enzyme concentration ( , ) . posttranslational control is also known to contribute to membrane synthesis regulation ( ) ( ) ( ) ( ) ( ) ( ) ; however, how cells use transcriptional and posttranslational control to coordinate membrane synthesis with growth has never been defined well. in order to understand how the model gram-negative species escherichia coli coordinates membrane synthesis with growth, we quantified substrates and enzymes of the fatty acid and pl synthesis pathways under both steady-state and dynamic conditions. we found that posttranslational control of the first enzyme in the pl synthesis pathway, plsb, is sufficient to ensure steady-state regulation of pl synthesis. in contrast, transcriptional regulation maintains stable enzyme concentrations regardless of growth rate. furthermore, due to feedback regulation of the fatty acid pathway, plsb exerts strong control over lps synthesis via its effects on concentrations of an lps fatty acid precursor. ( fig. d) . we also found no significant positive correlation between pl flux and plsb substrate c : -acp or c : -acp (pearson correlation coefficient r ϭ Ͻ . , two-tailed test of significance p ϭ Ͼ . ), while c : -acp concentrations correlated negatively with pl flux (r ϭ Ϫ . , p Ͻ . ) ( fig. e ; see also table ). although g p concentrations were found to correlate with pl flux (r ϭ Ͼ . , p ϭ Ͻ . ), growth in glycerol medium increased g p by -fold without increasing pl abundance or flux. the absence of any significant positive correlation between acp substrate concentrations and pl flux indicates that steady-state pl synthesis is not controlled by substrate concentrations. in contrast to pl precursors, concentrations of the fatty acid initiation and elongation substrate malonyl-acp correlated with pl flux (fig. e ; r ϭ . , p ϭ Ͻ . ) (table ), while the concentrations of most saturated acp species remained relatively constant (see fig. s in the supplemental material), suggesting that the fatty acid initiation and elongation reactions are controlled primarily by malonyl-acp concentrations and thus by the rate of malonyl-acp synthesis by acc and fabd. concentrations of c : -oh-acp, the lipid precursor of lps, also correlated with pl flux (r ϭ . , p ϭ Ͻ Ϫ ), suggesting that pl synthesis is naturally coupled with lps synthesis via concentrations of c : -oh-acp (fig. e ). concentrations of holo-acp, the most abundant acp species, declined with increasing pl flux (r ϭ Ϫ . , p ϭ Ͻ . ) (table ) , as levels of fatty acid synthesis intermediates increased due to increased fatty acid flux. as with substrates, positive correlations between enzyme concentrations and reaction flux may indicate that a reaction rate is controlled by enzyme concentrations, i.e., via transcriptional control. we measured concentrations of fatty acid synthesis pathway enzymes using lc/ms to determine whether fatty acid flux is coupled with by transcription-based regulation. while concentrations of one of the four acc subunits (acca) and several of the fatty acid synthesis pathway enzymes correlated with pl flux (table ; see also fig. s ), the increases were small ( %) relative to the pl flux increase ( -fold). we infer that the rate of malonyl-acp synthesis and thus the rate of fatty acid synthesis are determined primarily by posttranslational control of acc. the negative correlation between c : -acp and pl flux is consistent with long-chain acp inhibiting malonyl-coa synthesis by acc via negative-feedback inhibition ( ) . unlike long-chain acp, the concentrations of all pl synthesis intermediates downstream of plsb were found to correlate positively with pl flux (fig. f ; r ϭ . to . ) ( table ; see also fig. s ). the close match between the increase in the concentration and the level of pl flux is consistent with the rates of these reactions being under substrate control. the contrast between the trends in concentrations of the substrates and the products of plsb implies that plsb activity alone controls pl synthesis. plsb activity might be regulated either via transcriptional control of plsb concentration or via posttranslational control of plsb activity. transcription of plsb is controlled by the membrane stress-activated sigma factor rpoe ( ) and by the -sensitive regulator guanosine tetraphosphate (ppgpp) ( ) . concentrations of ppgpp are inversely correlated with (fig. d) , implying that pl flux may be coupled to by transcriptional control of the plsb gene by ppgpp. however, we found no correlation between pl flux and plsb concentration (table , p ϭ Ͼ . ). instead, plsb concentrations were nearly constant across all conditions studied (fig. g ). the invariance of plsb concentrations over a -fold range of pl flux indicates that plsb activity is regulated mainly via posttranslational control. unexpectedly, concentrations of lpxc, which catalyzes the committed step in the lps pathway ( ), also did not correlate with pl flux (table , p ϭ Ͼ . ) and also remained stable (fig. g) , indicating that changes in lps flux between steady-state growth conditions are not driven by changes in lpxc concentrations. can be modulated independently of nutrient conditions by artificially altering levels of ppgpp. titrating ppgpp above basal concentrations, but below the concentrations that occur during starvation, reduces steady-state by reducing rrna synthesis ( ) . furthermore, plsb activity is inhibited at the high ppgpp concentrations observed during amino acid starvation ( ) . we titrated by expressing the catalytic domain of the ppgpp synthesis enzyme rela (rela*) from an inducible promoter (p tet ) in glucose medium. as previously observed ( ) , rela*-titrated cultures exhibited ppgpp concentrations that were elevated -fold above those seen with wild-type cultures growing at similar rates (fig. d ). trends in pl abundance, pl flux, acp species, pl intermediates, and pl synthesis enzyme concentrations in ppgpp-limited cultures closely followed the trends observed when was adjusted by carbon source (fig. b , c, and e to g) (see also fig. s , s , and s ). notably, despite ppgpp regulation of plsb transcription, concentrations of plsb also did not substantially vary with increasing concentrations of steady-state ppgpp. interestingly, lpxc concentrations decreased with increasing pl flux under these conditions (fig. g) . the trends in substrate, enzyme, and intermediate concentrations observed in ppgpp-limited cultures are consistent with growth regulating pl flux via posttranslational control-not transcriptional control-of plsb. mathematical modeling supports plsb control of steady-state pl and lps synthesis. to test whether regulation of plsb activity is sufficient to control steady-state pl synthesis, we constructed a simplified differential equation model that describes fatty acid, lps initiation, and pl biosynthesis ( fig. a ; see also text s and table s ). the model includes competitive inhibition of acc by both c : -acp and c : -acp as the sole regulatory interactions. the model also includes a branch point at c : -oh-acp into lps synthesis. lps initiation is represented in the model by a single step that combines reactions catalyzed by lpxa and lpxc, as lpxc catalyzes the first irreversible reaction in the lps pathway. concentrations of g p and c : -acp are fixed in the model to reflect experimental observations of plsb saturation by g p and invariance of c : -acp concentrations, respectively (see text s in the supplemental material for model details, parameter sets, and sensitivity analyses). we used the model to identify enzymes and substrates that exert control over pl and lps synthesis. increasing either acc or plsb v max by -fold increased simulated pl and lps fluxes in parallel by -fold (fig. b) , while changing v max of all other enzymes in the simulated pathway had little or no effect on simulated pl flux. the insensitivity of pl flux to v max of all fatty acid and pl synthesis enzymes aside from acc and plsb suggests that the experimentally observed positive correlations between enzyme concentrations and pl flux did not actually result in increased pl flux. the unexpectedly strong control of lps flux by plsb is due to the natural coupling between pl flux and concentrations of the lps synthesis substrate c : -oh-acp. changes in c : -oh-acp dehydration/reduction v max (catalyzed in e. coli by fabz and fabi, respectively) and lps initiation v max (catalyzed by lpxa and lpxc) exert strong and opposing forms of control over lps flux. however, lpxc and fabz do not exert significant control over pl synthesis, indicating that substantial flux can be diverted into the lps pathway without depleting that required for pl synthesis. of the two substrates considered in the model (acetyl-coa and c : -acp), only acetyl-coa affects pl and lps flux (fig. b) . fig. ). line plots are offset to prevent overlap. differential equations and parameters of the simulation are provided in text s and table s . to determine which of the two enzymes with nonnegligible levels of pl flux control-acc or plsb-actually controls pl flux during steady-state growth, we compared the predicted trends in substrate concentrations caused by simulated v max variations against experimentally observed trends. the predicted trends driven by acc and plsb v max variation closely reproduce most experimentally observed trends in both fatty acid and pl intermediate concentrations (fig. c ). however, variations in acc and plsb v max cause opposing trends in predicted concentrations of plsb substrates c : -acp and c : -acp, allowing acc regulation to be distinguished from plsb-based regulation. increasing acc v max causes c : -acp and c : -acp concentrations to increase with pl flux, which contradicts our experimental observations. however, increasing plsb v max is predicted to decrease c : -acp and c : -acp concentrations, a trend that better follows the experimentally observed trends (fig. c) . we therefore conclude that pl synthesis is primarily regulated by plsb during steady-state growth. our model also suggests that plsb control over steady-state lps flux (achieved by its tight control over c : -oh-acp concentrations) may be sufficient to couple lps synthesis with growth as well. translation inhibition causes carbon overflow into fatty acid synthesis. we set out to evaluate whether a known regulator of pl synthesis, ppgpp, is able to directly regulate plsb activity during steady-state growth. high concentrations of ppgpp lead to plsb inhibition in vivo ( ) , although it remains unclear whether this inhibition acts via a ppgpp-plsb interaction or via an indirect route. low concentrations of ppgpp correlated inversely with (fig. d ). the notion that ppgpp might directly control pl synthesis even at the low basal concentrations present during steady-state growth was proposed previously but never tested ( ) . levels of acyl-acp substrates of plsb increased as ppgpp was titrated upwards with rela* whereas the level of the product of plsb (lpa) decreased, consistent with ppgpp inhibiting flux into the pl pathway ( fig. e and f) and closely following simulated variations in plsb v max (fig. c) . therefore, changes in driven by substringent concentrations of ppgpp must somehow influence steady-state plsb activity. however, the mode of control by ppgpp (transcriptional control of plsb, posttranslational inhibition of plsb, or indirect control via an unknown regulator of plsb) cannot be determined from steady-state data alone. we first wished to observe the effects of high concentrations of ppgpp on the fatty acid and pl synthesis pathways. synthesis of high concentrations of ppgpp by rela (the stringent response) is triggered by any stress or starvation conditions that lead to the specific biochemical cue of uncharged trna bound to the ribosome. during the stringent response, ppgpp accumulates by more than -fold over basal concentrations and pl synthesis rates are reduced by approximately half ( , ) , likely due to plsb inhibition ( ) . we triggered ppgpp synthesis by adding the trna aminoacylation inhibitor mupirocin to glucose cultures of wild-type e. coli. to distinguish effects of ppgpp from effects of translation inhibition, mupirocin was also added to a culture of Δrela e. coli. mupirocin caused a -fold accumulation of ppgpp in the wild-type strain within min to over pmol/od, reaching Ͼ pmol/od after min (fig. s ) . unexpectedly, mupirocin treatment also transiently increased concentrations of malonyl-acp and all hydroxyl-acp species at the expense of holo-acp in both strains ( c : -acp accumulation was followed in turn by an increase in holo-acp and a decrease in malonyl-acp levels, consistent with acc inhibition (fig. a) . pl intermediates phosphatidic acid (pa), ps, and pgp were rapidly depleted in the wild-type strain after briefly increasing in abundance (fig. b ). the transient carbon influx briefly shifts the population of the acyl chains incorporated into pl toward longer-chain fatty acids, likely due to increased malonyl-acp concentrations favoring fatty acid elongation over membrane incorporation by plsb and plsc (fig. s ). while suppression of fatty acid synthesis in the wild-type strain can be attributed to ppgpp, it is unclear what had attenuated the carbon influx in the Δrela strain; however, c : -acp accumulation after min likely contributed to acc inhibition. inhibition of fatty acid elongation also depleted the lps precursor c : -oh-acp, which is expected to decrease lps synthesis in parallel with pl synthesis (fig. a) . as malonyl-acp levels increased transiently after mupirocin addition in both the wild-type and Δrela strains, we hypothesized that translation inhibition somehow diverts carbon into lipid synthesis. we added the ribosome inhibitor chloramphenicol and the transcription initiation inhibitor rifampin to glycerol cultures of wild-type e. coli. both compounds inhibit translation via mechanisms that suppress ppgpp synthesis. as with mupirocin treatment of the Δrela strain, chloramphenicol triggered a rapid decrease in holo-acp and an increase in long-chain unsaturated acp species c : -acp and c : -acp levels (fig. c ). both antibiotics triggered an increase in the levels of pl synthesis intermediates pa and ps that resembled the response of the Δrela strain to mupirocin (fig. d) . what might have caused the transient increase in fatty acid synthesis observed after translation inhibition? interestingly, addition of both rifampin and chloramphenicol increased acetyl-coa concentrations in glycerol cultures by -fold (fig. e) , suggesting a possible cause. acetyl-coa concentrations also increased in both wild-type and Δrela strains after mupirocin treatment in glucose medium, before decreasing ϳ % in the wild-type strain (fig. s ) . while it is unclear why translation inhibition would increase acetyl-coa concentrations, the observed response of the fatty acid pathway is consistent with our mathematical model, which predicts fatty acid flux to be highly sensitive to changes in acetyl-coa concentrations (fig. b) . we confirmed the sensitivity of the fatty acid and pl synthesis pathways to environmental changes using a fast nutritional upshift. addition of glucose and amino acids to a glycerol culture also caused rapid accumulation of pa and ps species that resembled the increases observed following translation inhibition (fig. s ) . moderate to high concentrations of ppgpp regulate pl synthesis via posttranslational control. in order to clearly discern the effects of ppgpp on the fatty acid and pl synthesis pathways without complications introduced by translation inhibition, we monitored the fatty acid and pl synthesis pathways immediately after inducing rela*. the responses of the fatty acid and pl synthesis pathways were again consistent with plsb inhibition causing long-chain acp to accumulate, which depletes malonyl-acp by inhibiting acc (fig. a) . pl intermediates also responded in a manner consistent with plsb inhibition: levels of lpa species steadily decreased, followed by pa, ps, and pgp (fig. b) . addition of chloramphenicol min following rela* induction caused an increase in unsaturated long-chain acp levels, though ppgpp appears to attenuate the response of the pl pathway to translation inhibition. we next sought to evaluate whether ppgpp concentrations far below those occurring during the stringent response would also be capable of regulating plsb via posttranslational control. as the effects of posttranslational control can be distinguished from those of transcriptional regulation by response time, we closely followed the dynamics of the pl pathway after mildly inducing ppgpp synthesis with a low doxycycline concentration. ppgpp concentrations increased within min of rela* induction and reached the elevated steady-state concentration (ϳ pmol/od) by min. concentrations of ps begin to decrease within min (fig. c) , consistent with posttranslational inhibition of pl synthesis. to verify that the immediate ps depletion had occurred too quickly to be explained by transcriptional regulation, we compared the ps response kinetics with the kinetics of a ppgpp-driven response known to be mediated by transcriptional control. cyclopropyl pl is produced from unsaturated fatty acids of membrane pl by the activity of the enzyme cfa, expression of which is induced via transcriptional control by ppgpp ( ) . cyclopropyl pl began to accumulate min economy of membrane synthesis in e. coli ® after rela* induction, well after the ps decrease was established. we conclude that ppgpp concentrations well below stringent response concentrations inhibit plsb via a posttranslational mechanism. over several decades, biochemical and genetic research has extensively characterized the pathways and the enzymes that produce the building blocks of bacterial membranes ( , , ) . many control mechanisms acting on both the transcriptional and posttranslational levels have been proposed to maintain membrane homeostasis. for instance, each of the enzymes acc ( ), fabh ( ), fabz ( ), fabi ( ), lpxc ( ) , and plsb ( ) have been suggested to contribute to membrane synthesis regulation. however, the existence of a control mechanism does not necessarily indicate that it is used to regulate total membrane synthesis flux during steady-state growth. furthermore, models derived from biochemical and genetic studies can overlook selfregulating mechanisms that occur automatically in a metabolic pathway, such as the coupling of c : -oh-acp concentrations to pl flux. the primary value of our work is that it reveals the regulatory mechanisms that are actually used by growing e. coli cells to regulate membrane biosynthesis. both our data and our modeling indicate that only two of the many control mechanisms previously identified are sufficient to regulate total pl biosynthesis during steady-state growth. specifically, we found that cellular demand for membrane synthesis is communicated to plsb via an undiscovered posttranslational mechanism. in turn, plsb activity regulates fatty acid synthesis by consuming long-chain acp species, relieving feedback inhibition of acc ( ) and increasing fatty acid flux. this demand-level flux regulation is consistent with metabolic control theory ( ) . our measurements of enzyme concentrations indicate that pl flux is regulated primarily via posttranslational control of enzyme activity, rather than via transcriptional regulation. the concentrations of the pathway enzymes with the greatest control over pl flux (acc and plsb) are maintained at nearly invariant levels across a -fold range of . although it has been long accepted that plsb activity is adjusted via posttranslational control and not via transcriptional control ( ), the strong flux control of acc ( ) and the growth-regulated transcription of the accbc, acca, and accd genes ( ) inspired the suggestion that pl flux might be coupled with via transcriptional regulation. instead, we found that transcriptional regulation of fatty acid and pl synthesis genes stabilizes the concentrations of fatty acid and pl synthesis enzymes across . the correlations between concentrations of several enzymes (e.g., fabf) and pl flux that we observed are unlikely to increase total pl flux but rather likely regulate aspects of membrane metabolism aside from pl flux, such as membrane fluidity. maintaining a stable membrane synthesis capacity enables the cell to quickly respond to any change in membrane demand at the cost of expressing enzymes that remain less active at low or moderate ( ) . our data are also essential for evaluating proposed models of lps synthesis regulation. most importantly, both our model and our experiments indicate that pl fluxand thus, plsb activity-controls concentrations of lps precursor c : -oh-acp. this tightly links fluxes into the pl and lps synthesis pathways, as varying pl synthesis by adjusting either plsb or acc activity would naturally vary lps synthesis in parallel. surprisingly, we found that flux into the lps synthesis pathway is not adjusted by variations in lpxc concentrations, as lpxc concentrations remain constant despite a -fold change in . lps flux may be varied instead by fabz, concentrations, which increased by % over the -fold range of sampled (see fig. s in the supplemental material). our model predicts that this would attenuate the effects of increasing c : -oh-acp concentrations caused by elevated fatty acid synthesis and divert flux away from lps synthesis without affecting pl flux. lpxc concentrations may be stabilized across by degradation by the ftsh protease ( ) . degradation of lpxc by ftsh might be used as an emergency brake to rapidly halt lps synthesis during growth arrest or growth transitions to prevent accumulation of toxic lps intermediates. com-prehensive characterization and modeling ( ) of lps substrates and enzymes across several steady-state conditions, as we have done for pl synthesis, will be necessary to determine how lps synthesis is varied. although plsb determines pl flux during steady-state growth, this control is not absolute, as fatty acid and pl flux (and likely lps flux) are also sensitive to acetyl-coa concentrations and acc activity. the sensitivity of membrane synthesis to protein synthesis arrest demonstrates the responsiveness of membrane synthesis to carbon metabolism. while this sensitivity facilitates rapid adaptation of pl flux to environmental changes, the tight connection between protein synthesis, central carbon metabolism, and membrane biogenesis demands additional regulation (including inhibition by ppgpp) to rebalance the pathway with and prevent pl overflow. if protein synthesis were inhibited in a manner that does not decrease carbon flow into the fatty acid pathway (e.g., by nitrogen starvation), continued pl and lps synthesis would outpace synthesis of the lipoproteins that tether the outer membrane to the peptidoglycan layer. inhibition of pl and lps pathways by ppgpp would thus prevent production of excess membrane, enforcing the coupling of pl and lipoprotein synthesis. consistent with this notion, Δrela strains generate higher quantities of extracellular pl and lps, likely as outer membrane vesicles ( ) . identifying the specific signal that controls plsb will reveal how membrane synthesis is synchronized with growth. but what controls plsb? posttranslational control of plsb by moderately high concentrations of ppgpp (Ն pmol/od) at least partly contributes to steady-state membrane synthesis regulation, but likely only during nutritional downshifts, stress, or very slow steady-state growth ( Ͻ . h Ϫ ) when such concentrations are achieved. whether basal ppgpp concentrations observed during fast growth ( Ͼ . h Ϫ , ppgpp Ͻ pmol/od) also regulates pl synthesis via plsb control remains unclear. in vivo experiments such as ours cannot determine the mechanism by which ppgpp inhibits plsb. although it was demonstrated previously that high concentrations of ppgpp directly inhibit plsb ( ) , multiple groups were unable to observe ppgpp inhibition of plsb in vitro when acyl-acp was used as a substrate ( , ) . ppgpp may therefore inhibit plsb indirectly via a regulator or a cellular process that interacts with plsb. immunoprecipitation experiments revealed several proteins that interact with plsb, including acp and pssa, as well as some (plsx and ybgc) whose roles are unclear ( ) , suggesting that plsb forms part of a pl synthesis complex that may couple plsb activity with . as pl synthesis flux has been observed to oscillate with the cell division cycle in e. coli and other bacteria ( ) , plsb may also be regulated by the divisome or by septum formation. degradation of pl by the activity of phospholipases may also play an important role in membrane homeostasis during steady-state growth ( ) , as is known to occur during growth and division in eukaryotes ( ) . in addition to identifying the allosteric regulators of plsb, studies that integrate connections between pl catabolism and transport with pl synthesis are needed for a comprehensive understanding of membrane homeostasis. culture conditions. cultures were grown in -ml to , -ml erlenmeyer flasks filled to % of nominal volume with mops (morpholinepropanesulfonic acid) minimal medium ( ) with . mm nh cl or nh cl and . % (wt/vol) carbon source (acetate, succinate, malate, glycerol, glucose, u- c-glucose [cambridge isotope laboratories], or glucose supplemented with . % cas-amino acids). culture flasks were placed in a grant instruments sub aqua pro dual water bath at °c and agitated by stirring with a -mm-long magnetic stir bar (vwr), coupled to a magnetic stir plate ( mag mixdrive eco and mixcontrol ) set at , rpm. growth was monitored by optical density measurement at nm using an ultrospec cell density meter (ge healthcare). samples for acp, lipid analysis, and proteomics were collected using cultures without isotopic labeling. samples for nucleotide phosphate measurements were collected from u- n-labeled cultures, and samples for g p measurements were collected from u- c-labeled cultures. strains and plasmid prela*. all experiments were performed using escherichia coli k- strain ncm (cgsc catalog no. ) and its derivatives. ncm rela::kan was constructed by p phage transduction using strain cf [mg Δlac (rph ϩ ) rela ::kan] as a donor. plasmid prela* was created by cloning dna encoding residues to from the e. coli rela protein into bglbrick plasmid economy of membrane synthesis in e. coli ® pbbs k ( ) (sc * origin of replication, p tet promoter, kanamycin resistance). the fluorescent protein mvenus was fused by restriction-digestion to the c terminus of rela via a glycine-serine linker. metabolite sampling. samples for acp, proteomics, and lipid analysis were acquired by fast quenching of ml of culture sample into l of ice-cold % trichloroacetic acid. after min incubation at °c, cells were pelleted by centrifugation and stored at Ϫ °c until analysis. for analysis of nucleotide phosphates and polar metabolites, samples were acquired by the use of a modified fast vacuum filtration method ( ) . a -ml volume of culture was collected by vacuum on a prewetted . -cm-diameter . -m-pore-size hv durapore membrane filter. after rapid collection, the filter was immediately placed upside down in quenching solution. for measurement of the nucleotide phosphates, ml of ice-cold m formic acid was used with l internal standards (is) mix as a quenching solution, which was subsequently neutralized by the use of l of % ammonium hydroxide. for g p measurements, ml of a : : (vol/vol/vol) mixture of methanol, acetonitrile, and water with . % formic acid with l internal standard solution (cooled on dry ice) was used as a quenching solution. after min of incubation, cells were washed from the filter, transferred to a tube, and stored at Ϫ °c until analysis. preparation of internal standards. isotopically labeled internal standards (is) were used to control for sampling and measurement variation. for acp and proteomics assays, u- n e. coli whole-cell extracts were prepared using a mops minimal medium culture with nh cl as the sole nitrogen source. at od of ϳ . , % trichloroacetic acid (tca) was added at a : ratio to the culture to facilitate quenching of metabolism. after min of incubation on ice, -ml single-use is aliquots were collected by centrifugation and stored at Ϫ °c until the sample preparation step. for the phospholipid measurement, u- c lipid extract was prepared using a culture grown in minimal mops medium with . % u- c glucose as the sole carbon source. at od of ϳ . , % tca was added at a : ratio to the culture and insoluble cell material was collected by centrifugation after min of incubation on ice. pellets were resuspended in a mixture consisting of l meoh, l mm citric acid- mm dipotassium phosphate buffer, and l of methyl-t-butyl ether per ml of initial culture volume. after vortex mixing and min of sonication, phase separation was induced by addition of l/ml of mm citric acid/ mm dipotassium phosphate buffer. after further vortex mixing, sonication and min of incubation at room temperature, the phases were separated by min of centrifugation at , rpm at room temperature. the upper phase was collected, placed in a glass vial, and stored at Ϫ °c until the sample preparation step. instrumentation. all lc/ms runs were performed using a agilent lc/ms system consisting of a binary pump (g b), an autosampler (g a), a temperature-controlled column compartment (g a), and a triple-quadrupole (qqq) mass spectrometer (g c) equipped with a standard electrospray ionization (esi) source, all operated using masshunter data acquisition software (version . ). a mass spectrometer was operated in dynamic multiple-reaction monitoring (mrm) mode using transitions generated in silico by the use of a script written in python, an rdkit library, and chemical structures of the target compound as the input. transitions for targeted proteomics assays were developed using skyline ( ) based on protein sequences from the uniprot database. lc/ms quantification of acp intermediates. acyl-acp levels were measured using a published method ( ) with minor modifications. lysis buffer was prepared by suspending an appropriate number of frozen u- n-labeled e. coli pellets in ml of a mixture consisting of mm potassium phosphate buffer (ph . ), m urea, mm n-ethyl-maleimide, mm edta, and mm ascorbic acid. a -ml volume of lysis buffer was added to each of the preparations of tca-quenched and pelleted cells, and proteins were isolated by chloroform/methanol precipitation as described previously. protein pellets were resuspended in l of digestion buffer ( % -octyl-glucoside- mm potassium phosphate buffer, ph . ) and, after addition of l of . mg/ml gluc protease (promega), incubated overnight at °c. after quenching was performed by addition of l meoh, samples were centrifuged and l was injected in an lc/ms system. separation was performed on a csh c column (waters) ( . mm by mm, . -m pore size) held at °c using the following binary gradient: % b, -min ramp to %, -min increase to %, and -min hold at % b followed by min of reequilibration under the starting conditions (a, mm formic acid; b, mm formic acid) at a flow rate of . ml/min. lc/ms quantification of phospholipids. the sample preparation procedure used for the phospholipids consisted of a combination of an methyl tert-butyl ether (mtbe) extraction method ( ) and an established lc/ms method ( ) . pelleted e. coli cells were resuspended in a mixture containing l of meoh, l of u- c e. coli extract prepared as described above, and l of mtbe. after vigorous vortex mixing and sonication, l of mm citric acid- mm dipotassium phosphate buffer was added to homogenized pellets. following further vortex mixing and min of incubation at room temperature, liquid phases were separated by centrifugation for min at , ϫ g. a -l volume of the upper phase was moved to a new tube and dried in a vacuum centrifuge (labconco). dried lipid films were resuspended in l of : : (vol/vol/vol) isopropanol/acetonitrile/h o, supplemented with mm acetylacetone. after resuspension, l h o was added to reduce the organic content of the buffer and l of the resulting mixture was injected into the lc/ms system. separation was performed on a csh c column (waters) ( . mm by mm, . -m pore size) at °c with a flow rate of . ml/min using the following binary gradient: % b, ramp to % b in min followed by a linear increase to % b in min, -min hold at % b, and min reequilibration (a, . % nh oh-water; b, . % nh oh- % isopropanol- % acetonitrile [acn]). lc/ms quantification of nucleotides. frozen cell extracts were defrosted by to min of incubation in a °c water bath and sonicated for min in a water-ice slurry. after min of centrifugation at , ϫ g, samples were loaded on a ml/ mg oasis wax cartridge (waters) preconditioned with ml of meoh and ml mm ammonium acetate buffer (ph . ). after washing with ml ammonium acetate buffer was performed, analytes were eluted with l of . % ammonium hydroxide-meoh/acn/h o : : (vol/vol/vol). after addition of l of % trehalose and brief vortex mixing, the samples were dried in a vacuum centrifuge (labconco). dried trehalose-stabilized extracts were redissolved in l of meoh/acn/h o ( : : [vol/vol/vol]) and moved to an autosampler vial for analysis. separation was performed on an ihilic column (hilicon) ( . mm by mm, . -m pore size) or a sequant zic-chilic column (merck) ( . mm by mm, -m pore size) at . ml/min using the following binary gradient: % b, ramp to % b in . min followed by min of isocratic hold at % b and a linear decrease to % b in min with a -min hold at % b and min reequilibration under the initial conditions (a, . mm ammonium acetate- . mm acetic acid- mm acetylacetone-milli-q water, b, . mm ammonium acetate- . mm acetic acid- mm acetylacetone- % acn). the injection volume was l. lc/ms quantification of g p. stored metabolite extracts were dried down in a vacuum centrifuge (labconco), redissolved in l of meoh:acn:h o ( : : [vol/vol/vol]), and moved to an autosampler vial for analysis. separation was performed on an ihilic column (hilicon) ( . mm by mm, . -m pore size) at . ml/min using the following binary gradient: % b, ramp to % b in min followed by linear decrease to % b in min, -min hold at % b, and min reequilibration. the injection volume was l. lc/ms targeted protein quantification. relative concentrations of enzymes were measured by targeted proteomics using a modified version of the acp assay. lysis buffer was prepared by suspending an appropriate number of frozen u- n-labeled e. coli pellets in ml of mm potassium phosphate buffer (ph . )- m urea. to improve the detection of peptides from plsb and lpxc, u- n-labeled e. coli ncm strains overexpressing plsb and lpxc were used as internal standards. lpxc was overexpressed using the corresponding plasmid from the aska library ( ) (national bioresource project [nig, japan]). a -ml volume of lysis buffer was added to each of tca-quenched and pelleted cells, and proteins were isolated by a chloroform/methanol precipitation as described previously. protein pellets were resuspended in l of digestion buffer [ % -octyl-glucoside- mm tris buffer (ph . ) supplemented with mm cacl and mm tris( -carboxyethyl)phosphine hydrochloride (tcep)]. alkylation of cysteine residues was performed by adding l of mm iodoacetamide followed by min of incubation in darkness. digestion was performed by adding l of . mg/ml trypsin gold (promega) followed by overnight incubation at °c. samples were centrifuged, and l of the reaction volume was injected in an lc/ms system. separation was performed on a csh c column (waters) ( . mm by mm, . -m pore size) held at °c using a binary gradient: % b, min ramp to % b, min increase to % b, . ramp to %, and -min hold at % b before min of reequilibration under the starting conditions (a, mm formic acid; b, mm formic acid) at a flow rate of . ml/min. data analysis. all lc-ms data files were processed in versions of skyline ( .x) using a target list chosen on the basis of an in silico-generated transition list. each target compound had a matching isotopically labeled internal standard (is). processed data were exported as target compounds and is peak areas and processed further using a set of python scripts. growth rates were obtained from linear fits to log-transformed growth curves. od-corrected data were obtained by dividing the signal by the od value interpolated from the growth curve at the time of sampling. pe-corrected results were produced by dividing the signal by the sum of all of the signals for all phosphatidyl-ethanolamine species from the same measurement (in the case of the phospholipids) or matching sample (in the case of the other assays). in the nucleotide phosphate and g p assays, absolute concentrations were estimated based on amounts of internal standards in is-spike solution by assuming that rr ϭ implies equimolar amounts of target compound and is at the moment of quenching. correlations in table were calculated from log( )-normalized concentrations and pl fluxes using the descriptive statistics function (originpro v. ). mathematical modeling. the computational model was constructed and tested using copasi version . ( ) . full details of the model are described in text s in the supplemental material. results from sensitivity analysis are included in fig s . data availability. mass spectrometry data have been deposited in the embl-ebi metabolights database (https://academic.oup.com/nar/article/ /d /d / ; pmid ) with the identifier mtbls . the complete data set can be accessed here: https://www.ebi.ac.uk/metabolights/ mtbls . (the authors could not make this metabolights record available at the time of this paper's publication due to circumstances related to the covid- pandemic, but it will be made accessible as soon as possible after publication.) supplemental material is available online only. text s , docx file, . mb. outer membrane of salmonella typhimurium: accessibility of phospholipid head groups to phospholipase c and cyanogen bromide activated dextran in the external medium asymmetric localization of lipopolysaccharides on the outer membrane of salmonella typhimurium murein-lipoprotein of escherichia coli: a protein involved in the stabilization of bacterial cell envelope a retrospective: use of escherichia coli as a vehicle to study phospholipid synthesis and function interdependence of cell growth and gene expression: origins and consequences transcriptional regulation is insufficient to explain substrate-induced flux changes in bacillus subtilis systems-level analysis of mechanisms regulating yeast metabolic flux gene cloning for the isolation of enzymes of membrane lipid synthesis: phosphatidylserine synthase overproduction in escherichia coli cloning of genes involved in membrane lipid synthesis. effects of amplification of phosphatidylserine synthase in escherichia coli massive accumulation of phosphatidic acid in conditionally lethal cdp-diglyceride synthetase mutants and cytidine auxotrophs of escherichia coli construction of a lethal mutation in the synthesis of the major acidic phospholipids of escherichia coli increased synthesis of phosphatidylserine decarboxylase in a strain of escherichia coli bearing a hybrid plasmid regulation of the balanced synthesis of membrane phospholipids modulation of chemical composition and other parameters of the cell at different exponential growth rates dependence of the content of cell envelopes on the growth rate of bacillus megaterium dependence of the rate of synthesis of phosphatidylethanolamine and phosphatidylglycerol on the rate of growth of escherichia coli relation of turnover of membrane phospholipids to synthesis of membrane-derived oligosaccharides of escherichia coli relative rates of surface and volume synthesis set bacterial cell size mass-spectrometry-based quantification of protein-bound fatty acid synthesis intermediates from escherichia coli inhibition of escherichia coli acetyl coenzyme a carboxylase by acyl-acyl carrier protein conserved and variable functions of the sigma e stress response in related genomes antagonistic regulation of dgka and plsb genes of phospholipid synthesis by multiple stress responses in escherichia coli regulation of udp- -o-[r- -hydroxymyristoyl]-n-acetylglucosamine deacetylase in escherichia coli: the second enzymatic step of lipid a biosynthesis overexpression of the rela gene in escherichia coli guanosine tetraphosphate inhibition of fatty acid and phospholipid synthesis in escherichia coli is relieved by overexpression of glycerol- -phosphate acyltransferase (plsb) regulation of phospholipid synthesis in escherichia coli by guanosine tetraphosphate regulation of membrane phospholipid synthesis by the rela gene: dependence on ppgpp levels effect of ppgpp on escherichia coli cyclopropane fatty acid synthesis is mediated through the rpos sigma factor (sigmas) enzymology, genetics, and regulation of membrane phospholipid synthesis in escherichia coli bacterial membrane lipids: where do we stand? overproduction of acetyl-coa carboxylase activity increases the rate of fatty acid biosynthesis in escherichia coli inhibition of ␤-ketoacyl-acyl carrier protein synthase iii (fabh) by acyl-acyl carrier protein in escherichia coli mutants resistant to lpxc inhibitors by rebalancing cellular homeostasis enoyl-acyl carrier protein reductase (fabi) plays a determinant role in completing cycles of fatty acid elongation in escherichia coli ftsh-mediated coordination of lipopolysaccharide biosynthesis in escherichia coli correlates with the growth rate and the alarmone (p) molecular biology of bacterial membrane lipids regulating the cellular economy of supply and demand growth rate regulation of escherichia coli acetyl coenzyme a carboxylase, which catalyzes the first committed step of lipid biosynthesis lessons on enzyme kinetics from quantitative proteomics balanced biosynthesis of major membrane components through regulated degradation of the committed enzyme of lipid a biosynthesis by the aaa protease ftsh (hflb) in escherichia coli crosstalk between the lipopolysaccharide and phospholipid pathways during outer membrane biogenesis in escherichia coli relation between protein synthesis and phospholipid synthesis and turnover in escherichia coli the involvement of guanosine -diphosphate- -diphosphate in the regulation of phospholipid biosynthesis in escherichia coli. lack of ppgpp inhibition of acyltransfer from acyl-acp to sn-glycerol -phosphate regulation of fatty acid synthesis during the cessation of phospholipid biosynthesis in escherichia coli a protein network for phospholipid synthesis uncovered by a variant of the tandem affinity purification method in escherichia coli lipid synthesis during the escherichia coli cell cycle the escherichia coli phospholipase plda regulates outer membrane homeostasis via lipid signaling mechanisms of glycerophospholipid homeostasis in mammalian cells culture medium for enterobacteria bglbrick vectors and datasheets: a synthetic biology platform for gene expression systematic identification of allosteric protein-metabolite interactions that control enzyme activity in vivo skyline: an open source document editor for creating and analyzing targeted proteomics experiments lipid extraction by methyl-tert-butyl ether for high-throughput lipidomics lpp mediates self-generation of chemotactic lpa gradients by melanoma cells complete set of orf clones of escherichia coli aska library (a complete set of e. coli k- orf archive): unique resources for biological research copasi -a complex pathway simulator we thank michael cashel for providing e. coli strain cf . we thank christophe danelon, bertus beaumont, and frank bruggeman for invaluable suggestions for improving the manuscript. we thank the national bioresource project (nig, japan) for sharing the lpxc overexpression plasmid. this research was funded by a grant to g.b. from the netherlands organisation for scientific research (alw open . . ) and an award from the frontiers of nanoscience program.we key: cord- -ib z elq authors: ryter, stefan w.; otterbein, leo e.; morse, danielle; choi, augustine m.k. title: heme oxygenase/carbon monoxide signaling pathways: regulation and functional significance date: journal: mol cell biochem doi: . /a: sha: doc_id: cord_uid: ib z elq carbon monoxide (co), a gaseous second messenger, arises in biological systems during the oxidative catabolism of heme by the heme oxygenase (ho) enzymes. ho exists as constitutive (ho- , ho- ) and inducible isoforms (ho- ), the latter which responds to regulation by multiple stress-stimuli. ho- confers protection in vitro and in vivo against oxidative cellular stress. although the redox active compounds that are generated from ho activity (i.e. iron, biliverdin-ixα, and bilirubin-ixα) potentially modulate oxidative stress resistance, increasing evidence points to cytoprotective roles for co. though not reactive, co regulates vascular processes such as vessel tone, smooth muscle proliferation, and platelet aggregation, and possibly functions as a neurotransmitter. the latter effects of co depend on the activation of guanylate cyclase activity by direct binding to the heme moiety of the enzyme, stimulating the production of cyclic ′: ′-guanosine monophosphate. co potentially interacts with other intracellular hemoprotein targets, though little is known about the functional significance of such interactions. recent progress indicates that co exerts novel anti-inflammatory and anti-apoptotic effects dependent on the modulation of the p mitogen activated protein kinase (mapk)-signaling pathway. by virtue of these effects, co confers protection in oxidative lung injury models, and likely plays a role in ho- mediated tissue protection. carbon monoxide (co) arises in biological systems principally during heme degradation as the oxidation product of the αmethene bridge of heme, a process catalyzed by the heme oxygenase (ho) enzymes [ec : . . , heme, hydrogen donor: oxygen oxidoreductase, (α-methene hydroxylating, decyclizing)] [ , ] . the inducible form of ho, heme oxygenase- (ho- ), confers protection against oxidative stress conditions in vitro and in vivo, through anti-oxidative, antiapoptotic and anti-inflammatory actions [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . although the underlying mechanisms in ho-dependent cytoprotection remain incompletely understood, recent evidence has strongly implicated contributory role(s) for endogenous co generated from ho activity [ , ] . previously regarded as metabolic waste, co affects vascular function by influencing the regulation of vessel tone, platelet aggregation, and smooth muscle proliferation [ ] [ ] [ ] [ ] [ ] . studies of ho- localization in the brain have implicated ho-derived co as a neurotransmitter [ ] . these potential effects of co involve its complexation to the heme moiety of soluble guanylate cyclase (sgc), stimulating the production of guanosine ′, ′-cyclic monophosphate (cgmp), a second messenger molecule [ ] [ ] [ ] [ ] [ ] . as a consequence of heme binding, intracellular co potentially influences the activity of other cellular hemoproteins such as cytochrome p- , nitric oxide synthase (nos), nadph oxidase, and cytochrome-c oxidase, which are involved in vital processes including drug detoxification, inflammation, respiration, and possibly oxygen sensing [ ] [ ] [ ] [ ] . recent studies have discovered a potent anti-inflammatory effect of co: the inhibition of pro-inflammatory cytokine production following inducing stimuli, dependent on the modulation of mitogen activated protein kinase (mapk)signaling cascades [ , ] . the effects of co on mapk apparently occur independently of sgc activation and cgmp production; however the direct physical target of the co, in this case, remains unknown. in addition to co, redox-active heme metabolites may also participate in cellular defense mechanisms (fig. ) [ ] [ ] . ho exerts anti-oxidative functions by converting heme, whose intercellular accumulation may elevate intracellular pro-oxidant status [ ] , into the bile pigments, biliverdin-ixα, and bilirubin-ixα, which have potent antioxidant properties [ ] . the reactive iron released from heme by ho activity may follow detoxification pathways involving either sequestration or extracellular efflux [ ] [ ] [ ] . by inactivating iron regulatory protein (irp) activity, iron stimulates the synthesis of the iron sequestration protein ferritin [ ] [ ] , promoting a secondary cellular desensitization to oxidative stress [ , ] . this review will (i) describe the regulation of ho- as an inducible source of endogenous co, (ii) describe evidence that ho- acts as a mediator of cellular and tissue protection against oxidative stress, and (iii) emphasize recent studies that introduce novel anti-apoptotic and anti-inflammatory properties of ho-derived co in oxidative lung injury models. heme oxygenase activity generates equimolar co, ferrous iron (fe + ), and biliverdin-ixα per mole of heme-b oxidized, in a reaction requiring nadph: cytochrome p- reductase [ec . . . ] as electron donor [ ] [ ] [ ] [ ] . the reduction of biliverdin-ixα to bilirubin-ixα by nad(p)h: biliverdin reductase [ec : : : ] completes heme degradation [ ] [ ] ] . in addition to ho- , the inducible form, the ho system consists of two constitutively expressed isozymes (ho- , and ho- ) which represent the products of distinct genes [ ] [ ] [ ] [ ] . while the ho- gene responds to induction by a broad spectrum of chemical and physical agents, the ho- and ho- genes do not respond to xenobiotic induction [ ] . thus, ho- protein occurs at undetectable levels in most tissues and cell types until a stress condition arises, whereas ho- may exist at detectable levels in most tissues in the absence of stress. ho- occurs abundantly in the central nervous system and vasculature [ , ] , and responds to regulation by adrenal glucocorticoids in the brain [ ] [ ] . ho- and ho- differ in primary structure and molecular weight ( and kd respectively, for the rat isozymes), and in their k m values ( . , . µm, respectively) and reaction rates toward heme [ ] [ ] . ho- contains two high affinity heme-binding sites termed heme regulatory domains (hrd) that are distinct from the catalytic heme-binding site. accessory heme molecules bound to ho- hrd possibly act as a reservoir for small gas molecules, including no and co [ ] [ ] . the significance of ho- , a homolog of ho- , remains unclear as it demonstrates poor heme catalytic activity [ ] . ho enzymes perform a vital physiological function in the turnover of hemoglobinheme during the metabolism of senescent erythrocytes in reticuloendothelial tissues, especially the spleen, liver and kidney [ ] . ho regulates the intracellular concentration of heme, from the turnover of intracellular hemoproteins and cytochromes, and thus governs the redistribution of heme iron in tissues [ - ]. in keyse and tyrrell, using hybrid-selection cloning techniques, identified the major -kda mammalian stressprotein inducible by hydrogen peroxide, ultraviolet-a (uva, - nm) radiation, and sodium m-arsenite (naaso ), as identical to the rate limiting enzyme in heme degradation, ] . in addition to oxidants, the induction of the ho- gene also follows cellular exposure to agents such as heme [ the induction of ho- mrna by ros generating systems may be enhanced by the depletion of intracellular reduced glutathione gsh, using the drug d,l-buthionine-(s,r)-sulfoximine (bso) which inhibits γ-glutamyl-cysteinyl-synthetase (γ-gcs), the rate limiting step in gsh biosynthesis [ - ]. . functional consequences of ho activity. heme oxygenase degrades heme to biliverdin-ixα, carbon monoxide, and iron. biliverdin-ixα is converted to bilirubin-ixα by nad(p)h biliverdin reductase. both bile pigments have potent in vitro antioxidant activity. redox-active iron released from ho activity may promote oxidative damage. however, by inactivating iron regulatory protein (irp) activity, iron stimulates the synthesis of ferritin, an iron-sequestration protein and possible cytoprotectant. co derived from the ho reaction has possible significance in the regulation of vascular and neural functions. the stimulation of cgmp-dependent signal transduction pathways may account for the vasodilatory and anti-proliferative effects. co has potent anti-inflammatory effects, which depend on downregulation of pro-inflammatory cytokine production mediated by modulation of p mapk. the abbreviations used in this figure include: cgmp = guanosine ′, ′-cyclic monophosphate; co = carbon monoxide; fe(ii) = ferrous iron; fe (iii) = ferric iron; gtp = guanosine triphosphate; irp = iron regulatory protein; nos = nitric oxide synthase; p mapk = p mitogen activated protein kinase. other thiol reactive substances that induce ho- include chemicals which conjugate gsh in glutathione s-transferase (gst) catalyzed reactions (i.e. diethylmaleate, dem) to form mixed disulfides (gssr), many which undergo prior biotransformation to electrophilic intermediates by cytochrome p / p enzymes (i.e. halogenated hydrocarbons) [ ] . the complexation and subsequent depletion of gsh by dem to a degree exceeding % induced ho- in various cell types [ - , [ ] [ ] . sulphydril oxidants such as diamide, which promote the formation of gssg are typically ineffective at inducing ho- in cell culture [ ] [ ] . while gssg may be regenerated to gsh by nadph:glutathione reduct-ase, gssr species may not undergo enzymatic reduction, but are detoxified as n-acetyl-cysteine (mercapturic acid) derivatives. the -sh reactive substance n-ethylmaleimide has little effect on ho- induction, due to its preferential reactivity for protein -sh groups rather than gsh [ ] . metal salts (i.e. cdcl , cocl , nicl , sicl , hgcl etc.) potently activate ho- in vivo [ , , ] , as well as in many cell types [ , , , - , , - , ] . heavy metals form complexes with thiol groups including cysteine and gsh. when injected into rats, heavy metals depress hepatic gsh levels, which in turn rebound to elevated levels in a compensatory response. metal-dependent induction of hepatic ho activity may be inhibited by the prior complexation of the metals with thiol compounds, and potentiated by gsh depletion [ ] . transgenic mice lacking the metallothionein -l and -ll genes, which code for low molecular weight thiolrich proteins involved in metal detoxification, display more pronounced hepatic and renal ho- mrna and protein expression following cdcl injection, than wild-type mice [ ] . the induction of ho- expression by metals is regulated at the transcriptional level, demonstrated in vitro and in vivo using nuclear run-on analysis [ , , [ ] [ ] . certain metals (i.e. fe + , co + , cu + ) undergo ferrochelatasedependent incorporation into protoporphyrin ix (ppix) to form metalloporphyrins [ , ] . non-heme synthetic metalloporphyrins (i.e. snppix, znppix) paradoxically inhibit ho enzyme activity but stimulate ho- transcription [ , , [ ] [ ] . the free radical gas nitric oxide (no) mediates a number of physiological functions, including vasoregulation, neurotransmission, and inflammation. no serves as a cytotoxic effector species of the macrophage respiratory burst. at high concentrations, no may exert a 'nitrosative' cellular stress, reacting with thiols (including gsh) to form s-nitrosothiols, and with o -, to form the pro-oxidant peroxynitrite (onoo -) [ ] . exogenous no gas administered to human embryonic lung fibroblasts potently induced ho- protein and mrna levels in a concentration and time-dependent manner [ ] . no donor compounds such as sodium nitroprusside (snp), s-nitroso-nacetylpenicillamine (snap), -morpholinosydnonimine (sin- ), and spermine nonoate (snn) dose and time dependently increased ho expression in various cell culture systems [ - , [ ] [ ] . the activation of ho- by no donors or no gas is independent of cgmp production, since cgmp analogues had no effect and involves transcriptional regulation of the ho- gene [ - , - ]. in human fibroblasts, however, no donors or no gas stabilized ho- mrna in a no concentration-dependent fashion [ , ] . furthermore, no donation by snap increased detectible non-heme iron levels in paec and stimulated the synthesis of ferritin in a ho-activity dependent manner [ ] . the no metabolite peroxynitrite (onoo -) induced ho- in endothelial cells, which could be inhibited by the antioxidants nac or uric acid [ ] . ho- elevation may occur as a consequence of inflammation, infection, sepsis and other pathophysiological conditions associated with increased ros production and may play a protective role in these contexts [ , , [ ] [ ] . ho- elevation appears as a component of the hepatic acute phase response in humans, and rodents. the lipopolysaccharide (lps) component of bacterial endotoxin induces ho activity in rat peritoneal macrophages, and in hepatic parenchyma and sinusoidal cells following intraperitoneal injection [ ] . in mice, injection of lps, or the pro-inflammatory cytokines interleukin- (il- ), tumor necrosis factor-α, (tnfα), and interleukin- (il- ) induced hepatic ho- mrna, with the response to il- verified as a transcriptional regulation [ ] . the induction of hepatic ho- mrna levels by lps could be enhanced by gsh depletion and diminished by nac, suggesting an influence of cellular redox status in the induction mechanism [ ]. likewise, ho- expression responded in vitro to cellular stimulation with lps [ ], or pro-inflammatory cytokines (il- , il- , tnfα) [ - , ]. in huvec, the tnfα mediated induction of ho- required protein kinase-c and phospholipase a , and responded to inhibition by nac, and intracellular calcium chelation [ ] . interestingly, ho- induction (in huvec) also responded to treatment with the thrombopoietic cytokine interleukin- [ ] . growth factors that mimic cytokine responses with respect to ho- induction include transforming growth factor-β (tgf-β), which induced ho- protein in human retinal pigment epithelial cells [ ], and platelet derived growth factor (pdgf), which stimulated ho- mrna in vsmc [ ] . like the cytokine-mediated responses, the growth factor responses occurred in association with increased intracellular ros production, and responded to inhibition by nac treatment. ho- expression responds to fluctuations in the 'normal' or acclimated oxygen (o ) tension of the system [ ] [ ] [ ] ] . hypoxia, or lowered po , may occur in the cardiovascular system as a consequence of restricted oxygen intake, ischemia, or disease states such as atherosclerosis. acute hypoxia dilates the systemic vasculature, whereas chronic hypoxia may constrict the pulmonary vasculature, leading to pulmonary hypertension [ ] [ ] . the exposure of mammalian cells to hypoxia in vitro trig-gers cell type-specific alterations in protein expression patterns [ ] [ ] [ ] [ ] . following the original observation by murphy et al. that described ho- as the major hypoxia-inducible protein in cho cells [ ] , the response has also been demonstrated in vascular systems. for example, acute hypoxia induced ho- mrna accumulation in rat organs, including lung, liver, heart, and aorta [ ] . chronic hypoxia induced ho- mrna in both ventricles of the rat heart [ ] . in bovine aortic endothelial cells (baec), hypoxia treatment induces ho- protein levels and ho enzymatic activity, which persisted during subsequent reoxygenation [ ] . this response could be abolished by inclusion of iron chelators or nac in the hypoxic phase, and conversely increased by prior iron loading [ ]. inhibitors of inos or no scavengers, inhibited the induction of ho activity by hypoxia, while treatment with s-nitrosoglutathione augmented the response [ ] . these reports, taken together, suggest a critical role for iron and intracellular redox equilibrium in the hypoxic activation of ho- gene expression. hypoxia induced ho- transcription and ho- mrna accumulation in rat aortic vascular smooth muscle cells (vsmc) [ , ] , and pulmonary artery endothelial cells (paec) [ ] . in paec the response occurred in association with increased ap- dna binding activity, whereas in vsmc, involved activation of hif- dna binding activity [ , ] . in contrast to wild-type cells, mutant hepa cell lines deficient in hif- β did not exhibit ho- mrna accumulation in response to hypoxia [ ] . interestingly, hypoxic activation of the ho- gene in cho cells occurred independently of hif- as demonstrated in mutant cho cells deficient in hif- α [ ] . taken together, these results suggest that while hif- mediates the hypoxic induction of ho- in some cell types (i.e. vsmc), it may not be the sole factor involved. hyperoxia, or high o tension, used clinically for critical care applications, also activates a stress response in vitro and in vivo. hyperoxia causes oxidative injury to the lung, associated with increased production of mitochondrial ros [ ] . hyperoxia (> % o ) increased ho- mrna, protein, and enzymatic activity in the adult rat lung [ ] , and increased ho activity in neonatal rat lung [ ] . hyperoxia activated ho- transcription in vitro in cultured cells of lung origin (epithelial cells, fibroblasts, macrophages, and smooth muscle cells) [ ] . in human cell lines the activation of ho- by hyperoxia could be augmented by iron loading and diminished in the presence of iron-chelators [ , ] . thus, iron appears to represent a general requirement for the activation of ho- gene expression under either high or low o tension. the rat ho- protein classifies as a heat shock protein (hsp- ) since it responds to transcriptional regulation by heat ( °c); and the ′ regulatory region of its gene contains heat shock elements (hses) resembling those described in the promoter regions of heat shock genes (i.e. hsp ) [ ] [ ] [ ] ho- mrna and protein accumulate to a high degree after whole-body hyperthermia ( °c) in rat organs, including the liver, heart, and kidney, and brain [ ] [ ] [ ] [ ] . heat shock ( °c) increases the transcriptional rate of ho- mrna in cultured rat glioma cells [ ] . the rat ho- gene contains two hses, hse (- /- ) and hse (- /- ) which contain inverted repeats of the core element ′ngaan ′ [ ] . the rat, mouse, and human ho- genes differ in the number, position, and configuration of hses in their ′ regulatory regions [ ] [ ] [ ] . both the human and rat hses formed complexes with heat inducible nuclear proteins, and conferred heat responsiveness to reporter gene constructs in respective transient transfection assays [ , ] . human cell lines, however, generally failed to induce ho- in response to heat [ , [ ] [ ] . the signal transduction pathways that operate ho- gene activation under the multiplicity of inducing conditions remain only partially understood. existing studies often report contradictory data, or cell type-specific and inducer-dependent variations, which are based on known specificities of chemical inhibitors. mitogen activated protein kinase (mapk) pathways, including extracellular regulated kinases (erk) [ , ] and/or p mapk [ , [ ] [ ] , participate in the activation of ho- by inducing xenobiotics. for example, the cdcl induction of ho- transcription in murine mcf- cells, could be abolished by the p mapk inhibitor (sb ) and by dominant negative mutants of p α, but not by an erk kinase (mek ) inhibitor (pd ) [ ] . similar mapk inhibitor studies have demonstrated the requirement for both erk and p mapk pathways in the naaso -dependent transcriptional activation of the chicken ho- promoter [ ] . in this system, the overexpression of dominant negative forms of ras, mek , and p mapk inhibited transcriptional activation of ho- in response to naaso treatment [ ] . both p mapk and erk pathways participated in ho- activation in hela cells following exposure to no donors [ ] . in contrast, ho- activation by naaso , heme, or cdcl in hela cells required tyrosine kinase activity but not erk or p mapk pathways [ ] . the regulation of ho- under hypoxia required p mapk, but not erk or tyrosine kinase dependent pathways in cardiomyocytes [ ] . to the contrary, the p mapk inhibitor sb activated ho- mrna expression under hypoxia in rat paec, whereas a mek / inhibitor (uo ) strongly activated ho- under normoxic conditions in the absense of stimuli; indicating that mapk inhibitors alone may acti-vate ho- transcription under certain conditions [ ] . the over-expression of mapk kinase kinases (mekk , tak , and ask ) induced ho- in hepg cells [ ] . the murine ho- gene ′ flanking sequence contains two transcriptional enhancer sequences located at - kb (e ; formerly sx ) and - kb (e ; formerly ab ) of the transcriptional start site [ , [ ] [ ] [ ] . these elements maintain basal promoter activity and mediate the induction of ho- by many xenobiotics, including cdcl , -o-tetradecanoylphorbol- -acetate, endotoxin, heme, and h o [ , [ ] [ ] [ ] [ ] . both e and e consist of repeated essential cis-acting dna motifs designated as stress responsive elements (stre) with the consensus sequence (t/cgctgagtca). intrinsic to the stre appears several overlapping consensus sequences for transcription factor binding sites: ap- , v-maf oncoprotein, and the cap'n'collar/basic-leucine zipper family of proteins (cnc-bzip). the latter sequence resembles the antioxidant responsive element (gcnnngtca) [ ] . the stre elements of e are critical for the ho- transcriptional response to cdcl [ ] . transfection studies in l cells with candidate transcription factors demonstrated that only members of the cnc/bzip family of proteins effectively activate an e reporter construct, with nuclear regulatory factor- (nrf ) displaying the strongest activity. the over-expression of the dominant negative mutant form of nrf inhibited e enhancer activity (and endogenous ho- induction) in response to cdcl and other inducing agents in l and mcf- cells [ , ] . transcription factor atf has recently been identified as the possible binding partner of nrf in regulating ho- transcription, by yeast two-hybrid analysis [ ] . the hyperoxia-mediated induction of ho- in raw . cells requires e and the participation of e enhancer regions. the response is mediated by the intrinsic ap- elements acting in cooperation with stat (signal transducer and activator of transcription) elements located within the proximal promoter region [ ] . in contrast, the hypoxic activation of ho- in vsmc requires a sequence at - kb (hypoxia responsive element) distinct from e , that contains two functional binding sites for hif- [ ] . an increasing body of evidence supports the general hypothesis that ho- acts as an inducible mediator of cellular and systemic defenses against oxidative stress, in models of inflammation, ischemia-reperfusion, hypoxia, and hyperoxiamediated injury. for example, induction of endogenous ho- protein with hemoglobin infusion increased survival in a rat model of lps-induced inflammatory lung injury [ ] . pre-induction of ho- with either lps or hemoglobin infusion conferred protection in a rat model of renal injury (glycerolinduced rhabdomyolysis) [ ] [ ] [ ] . homozygous ho- null mice (ho- -/-) displayed increased mortality in a model of lung ischemia-reperfusion (i/r). inhalation co ( . %) compensated entirely for the ho- deficiency in ho- -/mice, and restored survival following i/r to that of the wild-type mice [ ] . the proposed mechanism involved the co/ cgmp-dependent inhibition of plasminogen activator inhibitor- (pai- ) leading to enhanced fibrinolysis [ ] . adenoviral mediated overexpression of ho- (adho- ) in pigs inhibited vascular cell proliferation and lesion formation in a model of arterial injury. conversely, ho- -/mice subjected to arterial injury displayed increased vascular cell proliferation, and developed hyperplastic lesions in comparison to ho- +/+ controls [ ] . chronic hypoxia treatment ( % o ) increased right ventricular dilation and caused right myocardial infarction in ho- -/mice relative to wild-type mice that withstood the treatment [ ] . in this model wild-type or ho- -/mice did not differ in their development of pulmonary hypertension following chronic hypoxia [ ] . the induction of ho- protein by chemical inducers (i.e. nicl or hemin) however, prevented the development of pulmonary hypertension in the rat lung as a consequence of chronic hypoxia treatment [ ] . transgenic mice with a lung-specific ho- overexpression phenotype, resisted the inflammatory and hypertensive effects of hypoxia [ ] . both ho- and ho- potentially contribute to pulmonary defenses against high o levels. the adenoviral mediated gene transfer of ho- into rat lungs protected against the development of lung apoptosis and inflammation during hyperoxia [ ] . heme oxygenase- null mice (ho- -/-), displayed increased sensitivity to the lethal effects of hyperoxia relative to wildtype mice, despite compensatory increases in ho- , and accumulated iron in their lungs [ ] . on the other hand ho- -/mice had low serum iron anemia, yet accumulated non-heme iron in the kidney and liver, suggesting that iron recycling by ho- is critical in maintaining blood iron levels [ ] . the mechanism by which ho- deficiency resulted in accumulation of tissue iron is unclear. these studies have indicated that animals deficient in either ho- and ho- display enhanced sensitivity to oxidative stress conditions, and aberrations in the distribution of intra-and extracellular iron [ , , ] . ho- also confers protection in animal models of arteriosclerosis, where it may be found in atherosclerotic lesions [ ] . the adenoviral-mediated transduction of ho- into apoe deficient mice inhibited the formation of arteriosclerotic plaques relative to control virus transduced mice [ ] . induction of endogenous ho- by chemical treatment (hemin) reduced the formation of atherosclerotic lesions in ldl-receptor knockout mice fed high fat diets, relative to untreated or snppix treated controls [ ] . evidence from in vitro studies also supports protective roles of ho- . for example, the overexpression of ho- in endothelial cells conferred protection against heme and hemoglobin-mediated toxicity [ ] . cultured cerebral granular neurons overexpressing ho- displayed resistance to glutamate toxicity relative to wild-type cells [ ] . embryo fibroblasts with the ho- -/genotype displayed hypersensitivity to heme and h o treatment and generated increased intracellular ros production in response to these agents [ ] . overexpression of ho- in lung epithelial cells or rat fetal lung cells conferred resistance against the cytotoxic effects of hyperoxia, associated with growth arrest [ , ] . the conditional overexpression of ho- in cultured l fibroblasts inhibited tnfα−induced apoptosis, a phenomenon that could be blocked by inhibitors of ho activity (snppix), and mimicked by exogenous co ( ppm) [ ] . finally, the administration of ho- antisense oligonucleotides inhibited the cytoprotective effect of uva-preconditioning against subsequent lethal uva exposures in human skin fibroblasts [ ] . on the other hand, not all model systems support a protective role for ho- . pro-oxidant effects of ho activity have been reported in over-expression systems, related to transient iron overload [ , [ ] [ ] . for example, the susceptibility of hela cells to uva radiation was increased in ho- overexpressing strains, when the uva was applied in combination with a substrate load (heme), in a fashion dependent on heme iron release [ ] . carbon monoxide carbon monoxide is a low molecular weight diatomic gas that occurs ubiquitously in nature as an air pollutant. environmental co arises from the oxidation or combustion of organic matter (i.e. wood, coal, gasoline, natural gas, tobacco). ambient co concentrations in the lower atmosphere occur in the range of . - . µl/l or < ppm; which may reach - ppm in urban areas, and still higher in heavily polluted areas [ ] [ ] . co is a major component of cigarette smoke, reaching yields of up to mg per cigarette [ ] . in man, endogenous co arises principally from heme degradation (> %). the remainder arises from other sources that may include lipid peroxidation, and xenobiotic metabolism [ ] . the field of small gas signal transduction was born with the realization that an endothelial derived relaxing factor responsible for the paracrine regulation of vascular smooth muscle tone, was identical to the diatomic free radical gas no. the nitric oxide synthase (nos) enzymes generate no during the conversion of l-arginine to l-citrulline. the effects of no on vasodilation involve the activation of soluble guanylate cyclase (sgc), increasing the production of guanosine ′, ′cyclic monophosphate (cgmp) [ ] . this paradigm led to the proposal that co, a small gas of similar structure, released directly from heme during ho activity, may function as a soluble messenger molecule in a similar fashion [ , - , , ] . unlike no however, co is not a radical, and therefore is relatively inert by comparison. both co and no stimulate sgc activity in vitro by binding to the ferrous heme moiety of the enzyme [ , [ ] [ ] . while no forms a pentacoordinate complex with the heme of sgc, co may initially form a hexacoordinate complex [ , ] . co has a relatively lower affinity for the heme-iron of sgc than no, and is one-thousandfold less potent than no with respect to vasodilation and the in vitro activation of sgc [ , ] . co signaling may become relevant under oxidative stress or pathophysiological conditions where ho- is dramatically induced, and/or where the bioavailability of no is reduced. little is known about how co is mobilized for signaling, apart from two intuitive mechanisms (i) the availability of substrate heme for enzymatic degradation, and (ii) the availability of active ho enzymes, a process which in turn may be regulated by the transcriptional activation of the ho- gene by stress, and the possible modulation of ho- by glucocorticoids [ ] . transient fluxes in the free heme pool have been reported following oxidative stress conditions such as uva ( - nm) radiation treatment [ ] . paradoxically, co may inhibit ho activity in reconstituted microsomal systems, implying that the production of co may be limited by negative feedback regulation [ ] . physiological roles for co, which directly involve modulation of cgmp levels, include neurotransmission, vasodilation, the inhibition of platelet aggregation, and anti-proliferative effects on smooth muscle [ , - , , ] . in brain slices, in situ hybridization studies demonstrated that the distribution of ho- matches that of nadph cytochrome p- reductase and guanylate cyclase [ ] . the induction of guanylate cyclase in cultured olfactory neurons by olfactory stimulants can be inhibited by metalloporphyrin inhibitors of ho such as znppix, but not inhibitors of nos [ ] . recent studies point to the involvement of co in cardiovascular signaling. in the rat, both whole body hyperthermia ( °c), or renal i/r triggered the elevation of cgmp levels in the heart in parallel with the transcriptional induction of ho- [ , ] . in vsmc, an elevation of cgmp occurred following exogenous co treatment [ ] . cgmp increased also following hypoxia in association with ho- elevation, an effect that could be inhibited by snppix, and the co scavenger hemoglobin, but not inhibitors of nos [ ] . vsmc derived co had paracrine effects on endothelial cells in co-culture, stimulating the production of endothelial cgmp, and suppressing the expression of endothelial-derived mitogens (pdgf, endothelin ) [ ] . both exogenously applied co, or hypoxia induced co had antiproliferative effects on vsmc, associated with elevation of cgmp, and inhibition of transcription factor e f, a regulator of cell cycle control [ ] . adho- infection in vsmc stimulated cgmp production, and inhibited cell proliferation in vitro by g /g o arrest, which required the g cyclin dependent protein kinase inhibitor p cip [ ] . the involvement of endothelial derived co in no-independent vasodilation has been suggested in inhibitor studies. in the presence of the nos inhibitor nω-nitro-l-arginine-methylester, (l-name), the ho inhibitor snppix further inhibits vasorelaxation elicited by acetylcholine in porcine aortic rings [ ] . conversely, the endothelium-dependent contractile response to phenylephrine in thoracic aortic rings was more pronounced in the presence of both znppix and nω-nitro-larginine (nna); than in the presence of nna alone [ ] . in this system, exogenously applied co relaxed the aortic rings in a cgmp-dependent fashion. overexpression of ho- by adho- infection in pigs inhibited phenylephrine-dependent vasoconstriction in isolated aortic rings. furthermore, adho- infection induced cgmp production in vsmc. the effects of ho- expression on vasoconstriction and cgmp production were subject to inhibition by znppix; but occurred in the presence of nos inhibitors (i.e. l-nna, l-name) [ ] . thus, these effects are dependent on heme degradation and independent of nos activity or no generation. exogenous co or heme treatment dilated pig cerebral artioles, the latter effect which could be blocked by chromium mesoporphyrin [ ] . znppix, but not nos inhibitors, inhibited smooth muscle relaxation in the opposum internal anal sphincter produced by nonadrenergic noncholinergic (nanc) nerve stimulation [ ] . in isolated perfused rat liver, znppix diminished co levels detectable in the effluent, and increased the perfusion pressure under the constant flow conditions. these effects were reversed by the addition of co or cgmp analogues in the perfusate [ ] . these studies support the existence of co/cgmp signal transduction cascades and their possible regulation by heme oxygenases, as potential pathways governing physiological processes. it remains possible, however, that a fraction of endogenous co originating from non-heme sources may contribute to a corresponding fraction of cgmp production. more discussion on the significance of co in the cardiovascular system under normal and pathophysiological states appears in other recent reviews [ , ] . ho- exerts a novel anti-inflammatory effect mediated by carbon monoxide (co) generated in the ho reaction [ ] . the effectiveness of bacterial lipopolysacharide (lps) (heretofore µg/ml), to stimulate the production of the pro-inflammatory cytokine tnfα, was inhibited in transfected raw . macrophage cells overexpressing ho- , compared to that in control transfectants. exogenously administered co (heretofore ppm) inhibited the production of tnfα in wild-type raw . cells after lps treatment, indicating that co can substitute for ho activity in mediating these effects. the treatment of raw . cells with exogenous co prior to lps treatment inhibited the expression of additional pro-inflammatory cytokines (i.e. il- β, and the macrophage inflammatory protein-β, mip- β), whereas increased the production of the anti-inflammatory cytokine interleukin- (il- ). the lps mediated stimulation of pro-inflammatory cytokines in macrophages involves the activation of mapk signaling pathways [ ] [ ] [ ] [ ] . lps treatment activated the p , erk /erk and c-jun n-terminal kinase, (jnk) pathways in raw . macrophages. in the presence of lps, co increased p mapk activation, but did not modulate erk /erk and jnk. of the map kinase kinases (mkk): (mkk , mkk , and mkk ) that activate p mapk [ ] [ ] , co enhanced the lps-mediated stimulation of mkk and mkk in raw . cells. co treatment did not significantly modulate cgmp production in raw . macrophages, but dramatically increased cgmp levels in control smooth muscle cells. pretreatment of the raw . macrophages with a non-hydrolysable cgmp analog or l-name did not compromise the ability of co to inhibit lpsinducible tnfα production. these anti-inflammatory effects of co were substantiated in vivo, in experiments where mice received injections of lps (heretofore mg/kg) with or without co pretreatment (heretofore ppm). co dose-dependently inhibited lps-inducible serum tnfα levels and increased lps-inducible il- production. the responsiveness of tnfα to lps treatment appeared downregulated in mkk -/mice compared to wildtype mice. co failed to further downregulate tnfα levels or upregulate il- levels in lps treated mkk -/mice. in il- -/mice, co inhibited tnfα levels within the first hour of lps treatment to a similar extent than in wild-type mice, excluding a role for il- in the early anti-inflammatory effects of co [ ] . these results, taken together, demonstrate that co exerts anti-inflammatory effects by inhibiting the synthesis of the pro-inflammatory cytokines under inducing conditions, by a mechanism that involves stimulation of the mkk /p mapk pathway, but excludes sgc/cgmp, inos, or no-dependent signaling. the direct physical target of co in initiating this pathway remains obscure. various intracellular hemoproteins (i.e. cytochrome p- , cytochrome c oxidase, nad(p)h: oxidase, peroxidases, and others) may serve as targets for co binding [ ] [ ] [ ] [ ] [ ] [ ] . future research may focus on elucidating the functional significance (with respect to cell signaling) of co-hemoprotein interactions in vivo. co, through anti-inflammatory action, protects the lung in a model of hyperoxia-induced lung injury [ ] , which evokes symptoms in mice similar to human acute respiratory distress syndrome (ards) [ ] . mice subjected to continuous hyperoxia treatment (heretofore > % o ), displayed signs of lung injury by - h, and all died within - h of exposure. the presence of co (heretofore, ppm) initiated prior to the hyperoxia, prolonged the survival of mice in the hyperoxic environment, increasing the ld to h exposure. co inhibited the appearance of markers of lung injury associated with hyperoxia (i.e. hemorrhage, fibrin deposition, edema, and protein accumulation in the airway), as well as markers of oxidative damage (i.e. lung lipid peroxidation) [ ] . co also inhibited the influx of neutrophils into the airways associated with hyperoxia treatment, as measured in bronchoalveolar lavage fluid. hyperoxia induced the expression of proinflammatory cytokines including tnfα, il- β, and il- , by h of exposure and activated stress kinases in lung tissue including erk / , jnk, p /mapk and mkk /mkk . the protection afforded by co treatment against the lethal effects of hyperoxia correlated with the inhibited expression of the pro-inflammatory cytokines, tnfα, il- β and il- . mkk -/mice, or wild-type mice injected with the selective inhibitor of p α/β mapk (sb ), displayed the accelerated manifestation of tissue damage markers (with the exception of neutrophil influx) and increased sensitivity to the lethal effects of hyperoxia, relative to untreated wild-type mice. cytokine mrna (tnfα, il- β and il- ) expression in response to hyperoxia appeared earlier in the mkk -/mice relative the wild-type mice exposed to continuous hyperoxia. co failed to inhibit the expression of the pro-inflammatory cytokines in the mkk -/mice, and furthermore failed to confer protection or extend survival against hyperoxia in mkk -/mice or in wild-type mice injected with sb . on the other hand, jnk -/mice behaved as wild-type mice with respect to the anti-inflammatory effects of co [ ] the co treatment of a lung epithelial cells in vitro increased mkk activation, and specifically the β-isoform of p . the presence of co increased the survival of a cells grown in continuous hyperoxia, relative to cells exposed to hyperoxia alone. treatment with the inhibitor of p α/β mapk or transient transfection with dominant negative mutants of p β or mkk abolished the cytoprotective effect of co against hyperoxia. currently no studies support the selective activation of antioxidant enzymes or stress proteins as an underlying mechanism for the anti-inflammatory properties of co in vivo. however, the treatment of endothelial cells in vitro with exogenous co ( ppm) stimulated the expression of manganese superoxide dismutase (mnsod) and ho activity [ ] . in summary, these experiments demonstrate that co protects against the lethal and inflammatory effects of hyperoxia in vivo and in vitro, by downregulating the expression of pro-inflammatory cytokines, through a mechanism dependent on activation of the p β/ mkk pathway [ ] . the functional significance of heme oxygenase- , which provides the rate-limiting step in heme degradation, and whose induction represents a general response to cellular stress, has remained a subject of debate for decades [ - , , ] . the overwhelming evidence described above supports the conclusion that ho- expression confers protection in animal models of oxidative stress. these studies taken together, suggest that ho- expression may have therapeutic value in gene therapy approaches. attempts to explain the cytoprotective action of ho- have implicated possible roles for all the products of ho-activity including redox active iron and bile pigments [ ] [ ] . co, formerly regarded as a toxic elimination product of the ho reaction has taken on a new significance as a possible autocrine and paracrine signaling molecule. co regulates vascular and neural processes by modulation of cgmp production [ ] . recent work has identified anti-inflammatory and antiapoptotic properties of ho-derived co [ , ] . in animal models of lung oxidative stress, including hyperoxia and ischemia/ 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and implication of a novel stress-inducible protein in oxidant-induced lung injury key: cord- -jd cyg authors: dos santos, gimena; rogel, micah r.; baker, margaret a.; troken, james r.; urich, daniela; morales-nebreda, luisa; sennello, joseph a.; kutuzov, mikhail a.; sitikov, albert; davis, jennifer m.; lam, anna p.; cheresh, paul; kamp, david; shumaker, dale k.; budinger, g. r. scott; ridge, karen m. title: vimentin regulates activation of the nlrp inflammasome date: - - journal: nat commun doi: . /ncomms sha: doc_id: cord_uid: jd cyg activation of the nlrp inflammasome and subsequent maturation of il- β have been implicated in acute lung injury (ali), resulting in inflammation and fibrosis. we investigated the role of vimentin, a type iii intermediate filament, in this process using three well-characterized murine models of ali known to require nlrp inflammasome activation. we demonstrate that central pathophysiologic events in ali (inflammation, il- β levels, endothelial and alveolar epithelial barrier permeability, remodelling and fibrosis) are attenuated in the lungs of vim(−/−) mice challenged with lps, bleomycin and asbestos. bone marrow chimeric mice lacking vimentin have reduced il- β levels and attenuated lung injury and fibrosis following bleomycin exposure. furthermore, decreased active caspase- and il- β levels are observed in vitro in vim(−/−) and vimentin-knockdown macrophages. importantly, we show direct protein–protein interaction between nlrp and vimentin. this study provides insights into lung inflammation and fibrosis and suggests that vimentin may be a key regulator of the nlrp inflammasome. supplementary information: the online version of this article (doi: . /ncomms ) contains supplementary material, which is available to authorized users. t he cytoskeleton comprises microfilaments, microtubules and intermediate filaments (ifs) . vimentin is the most abundant if protein and has a critical role in stabilizing intracellular architecture . until recently, the importance of vimentin remained elusive because vimentin knockout (vim À / À ) mice displayed a relatively normal phenotype . however, a thorough characterization of vim À / À mice under stress conditions revealed prominent abnormalities [ ] [ ] [ ] , complementing the metabolic, signalling and regulatory abnormalities displayed by patients with if-related diseases and mice expressing functional mutations in ifs , [ ] [ ] [ ] [ ] . recently, evidence to support a role for ifs as scaffolds for macromolecular complexes involved in critical cell signalling functions was reported , . in this study, we demonstrate that vimentin is required for the activation of the nlrp (nacht, lrr and pyd domains-containing protein ) inflammasome, a macromolecular complex that orchestrates early inflammatory responses of the innate immune system. the nlrp inflammasome is activated in response to a broad spectrum of infectious agents known to cause acute lung injury (ali), including the bacterial pathogens staphylococcus aureus, pseudomonas aeruginosa and mycobacterim tuberculosis [ ] [ ] [ ] and viral pathogens such as influenza a virus . the wide range of pathogens known to activate the nlrp inflammasome suggests that nlrp indirectly senses microbes by probing for hostderived danger-associated molecular patterns that are produced or released after cellular or tissue injury , . for example, uric acid released from cells exposed to bleomycin triggers the nlrp inflammasome, leading to maturation of interleukin b (il- b) and inflammation . in both human patients and animals treated with bleomycin, il- b released from injured cells results in production of the pro-fibrotic cytokine transforming growth factor b (tgf-b), which contributes to the development of pulmonary fibrosis . in addition, exposure to crystalline particles such as asbestos and silica also cause pulmonary fibrosis that is regulated by the nlrp inflammasome , . the nlrp inflammasome must be tightly regulated to avoid deleterious effects from the wide array of pathogens, pathogenassociated molecular patterns, danger-associated molecular patterns and environmental irritants, which are known to trigger its activation. unlike other inflammasome-associated nod-like receptor (nlr) proteins, nlrp is expressed at very low levels in naive macrophages. activation of the nlrp inflammasome requires two distinct pro-inflammatory stimuli. in the first step, toll-like receptor (tlr) signalling activates nf-kb and upregulates nlrp and pro-il- b . the second step involves the assembly of the multiprotein complex, which induces selfcleavage of caspase- , which in turn mediates the cleavage of pro-il- b into its biologically active form . the mechanism by which the inflammasome is ultimately activated is still a subject of intensive research. we postulate that vimentin is required for nlrp inflammasome assembly and activation. the experiments described below use three well-established rodent models of nlrp inflammasome activation and result in increased levels of biologically active il- b that contribute to lung injury. our data suggest that the if protein vimentin may act as an additional checkpoint to control the nlrp inflammasome. vimentin deficiency suppresses inflammation and injury. lipopolysaccharide (lps) is a potent inducer of inflammation and a known nlrp inflammasome activator [ ] [ ] [ ] . to determine whether vimentin is important in the inflammatory response, wild-type (wt) and vimentin knockout (vim À / À ) mice were subjected to a lethal dose of lps, which induces both proinflammatory cytokine expression and a systemic inflammatory response that is associated with significant mortality. wild-type mice displayed about a threefold increase in mortality compared with vim À / À mice. vim À / À mice had a median survival of h, whereas wt animals had a median survival of b h (fig. a) . surviving vim À / À mice exhibited mild lethargy, coat ruffling, febrile shaking and eye watering but recovered by day . to study whether vimentin affects lps-induced inflammation and ali, we used a sublethal dose of lps, as previously described , . histopathological examination showed that lungs of lps-treated wt mice had more severe alveolar damage characterized by interstitial oedema and more fluid and debris in the airspace than in lps-treated vim À / À mice (fig. b) . wild-type mice exposed to lps exhibited an increase in wet-todry lung weight ratio and total protein concentration in the bronchoalveolar lavage fluid (balf), whereas vim À / À mice failed to exhibit such an increase (fig. c,d) . exposure to lps is followed by large increases in immune cells in the airspace , . therefore, to further characterize the nature of immune cell recruitment into the lungs, we performed a systematic flow cytometric analysis of whole-lung lysates . no differences were observed in the myeloid cell population between wt and vim À / À mice treated with saline ( supplementary fig. a ). as expected, both wt and vim À / À mice challenged with lps had significant increases in neutrophils and moderate increases in macrophages compared with saline-treated animals ( supplementary fig. a ); no differences were observed between wt and vim À / À mice challenged with lps ( supplementary fig. a) . these data suggest that the protection from the lpsinduced increase in alveolar capillary barrier permeability observed in vim À / À mice was not associated with impaired recruitment of inflammatory cells to the lung. lps induces rapid production and release of pro-inflammatory cytokines and chemokines, which are sufficient to induce lung inflammation and ali in mice and humans . when we exposed wt and vim À / À mice to lps for h, significantly more caspase- and mature il- b were found in balf from wt mice than in vim À / À mice (fig. e,f) . furthermore, whole-lung homogenates prepared from wt and vim À / À mice exposed to lps had increased levels of il- b messenger rna (mrna, supplementary fig. b) . these data suggest that vimentin is not involved in the tlr-mediated nf-kb activation and upregulation of pro-il- b and nlrp (refs - ) . rather, vimentin must be required for the second step in lps-induced caspase- activation and il- b maturation via the nlrp inflammasome. vimentin required for asbestos-induced injury and fibrosis. the nlrp inflammasome has been implicated in the pathological increase of il- b production in a variety of pulmonary inflammatory diseases that lead to fibrosis, including asbestosis [ ] [ ] [ ] . in a model of asbestosis, we sought to determine whether vim À / À mice showed diminished cytokine production and whether these animals were protected from lung injury and pulmonary fibrosis. wild-type and vim À / À mice were exposed to crocidolite asbestos or an inert particle, titanium dioxide (tio ); markers of injury, inflammation, cytokine production and fibrosis were assessed. confirming previous reports , , asbestosexposed mice had increased total cell numbers in balf compared with tio -exposed animals. however, significantly fewer cells were recruited to the lungs of vim À / À mice after asbestos exposure (fig. a) . additional analysis of immune cell recruitment into the lungs of wt and vim À / À mice was performed by fluorescence-activated cell sorting analysis of whole-lung lysates ( supplementary fig. a) . wild-type mice had profound inflammatory cell infiltrates following exposure to asbestos as observed by haematoxylin and eosin (h&e) staining; in contrast vim À / À mice showed reduced inflammation ( supplementary fig. b ). wild-type mice exhibited an increase in total protein concentration in balf, whereas vim À / À mice failed to exhibit such an increase (fig. b) . the reduction in inflammation in vim À / À mice exposed to asbestos was associated with reduced levels of caspase- and il- b in balf compared with wt mice (fig. c,d) . these data suggest that vimentin is required for asbestos-mediated activation of the nlrp inflammasome (step ), but not for nf-kb activation and upregulation of pro-il- b and nlrp (step ). furthermore, we observed that wt mice exposed to asbestos develop pulmonary fibrosis, as assessed by masson trichrome and picrosirius red stain (a dye that binds specifically to collagen fibrils, fig. e ) and sircol assay (fig. f) . vimentin deficiency prevented pulmonary fibrosis, as no significant increase in collagen deposition was observed in vim À / À mice (fig. e,f) . collectively, these results demonstrate that loss of vimentin prevents asbestos-induced collagen deposition and fibrosis. vimentin required for bleomycin-induced injury and fibrosis. bleomycin is one of the best characterized rodent models of pulmonary fibrosis ; intratracheal delivery of the drug causes severe ali, which is followed by clearance of alveolar inflammatory cells, fibroblast proliferation and increased collagen deposition . moreover, bleomycin-induced fibrosis has been shown to be dependent on the nlrp inflammasome . wt and vim À / À mice were exposed to bleomycin for days. wt mice displayed intense lung inflammation as assessed by h&e staining (fig. a) , increased wet-to-dry lung weight ratio (fig. b) , and increased protein concentration in balf (fig. c) . all of these end points were significantly attenuated in vim À / À mice (fig. a-c) . exposure to bleomycin resulted in a large increase in immune cells in the airspace of both wt and vim À / À mice as assessed by flow cytometric analysis of whole-lung lysates ( supplementary fig. a ). vim À / À mice failed to exhibit an increase in caspase- (fig. d ) and il- b (fig. e) in balf following the exposure to bleomycin; in contrast, wt mice showed a robust activation of caspase- and production of mature il- b (fig. d,e) . as a proinflammatory cytokine, macrophage-produced il- b is known to induce production of interleukin (il- ). we observed that wt mice, but not vim À / À mice, had increased levels of il- following the exposure to bleomycin (fig. f ). tgf-b is a critical mediator of remodelling and fibrotic responses in the lung. total tgf-b was detected after activation in balf from wt mice days following the exposure to bleomycin, but remained at the baseline level in balf from vim À / À mice (fig. g ). no significant difference was observed between wt and vim À / À mice in nlrp inflammasome-independent cytokines tnf-a and mcp- (fig. h,i) . interestingly, il- b mrna levels were increased in the lungs of both wt and vim À / À mice following the exposure to bleomycin ( supplementary fig. b ). bleomycin exposure results in chronic inflammation and progressive pulmonary fibrosis , . the role of vimentin in pulmonary fibrosis was examined days following the administration of bleomycin. the lung architecture was similar for saline-treated wt and vim À / À mice, but in wt mice bleomycin induced extensive fibrotic areas with abundant collagen production, as assessed by masson trichrome stain ( fig. a and supplementary fig. a ). cellular infiltrates, alveolar wall destruction and collagen deposition were significantly reduced in vim À / À mice. similarly, we observed increases in collagen deposition in bleomycin-treated wt mice by staining with picrosirius red (fig. b) . in contrast, there was virtually no collagen deposition in the lungs of vim À / À mice (fig. b) . lung collagen concentrations, as assessed by sircol assay, were (a) survival of wt and vim À / À mice subjected to a lethal dose of lps ( mg kg À , intraperitoneally) was measured and compared. the survival curves were analysed using the log rank test, which calculates the chi-square (w ) for each event time for each group and sums the results. the summed results for each group were added to derive the ultimate chi-square to compare the full curves of each group. the log rank test for the entire data set was p ¼ . . (b-f) wt and vim À / À mice were challenged with a sublethal dose of lps and markers of ali were measured after h, as assessed by h&e staining (scale bar, mm) (b); by wet-to-dry lung weight ratio (c); and by protein content in the balf (d). inflammasome activation was measured by elisa for caspase- (e) or il- b (f) in balf from vim À / À and wt mice. data in b-e are from three independent experiments of n ¼ - animals per group and presented as mean±s.d. **po . , ***po . relative to wt versus vim À / À , by one-way analysis of variance with a correction provided by the bonferroni multiple comparisons test. nature communications | doi: . /ncomms article . ± . and . ± . mg per lung in bleomycin-treated wt and vim À / À mice, respectively (fig. d ). in patients with pulmonary fibrosis, pulmonary function tests typically reveal a parenchymal-restrictive ventilatory defect due to decreased lung compliance . lung compliance is influenced by the underlying connective tissue, which is rich in extracellular matrix components such as collagen . because vim À / À mice exhibited less collagen deposition following treatment with either bleomycin or asbestos, we reasoned that lungs of these mice would maintain normal compliance. to test this hypothesis, we measured the lung mechanics of wt and vim À / À mice using a scireq ventilator days following the bleomycin administration. in comparison with saline treatments, lungs of wt mice exhibited a . -fold decrease in quasi-static compliance following the bleomycin administration, whereas the compliance of lungs from vim À / À mice decreased only . -fold (fig. e) . bleomycin administration creates heterogeneous regions of lung fibrosis in mice (fig. a) , which is a pathologic feature of the human disease as well . to assess focal changes in lung tissue stiffness, we performed regional nano-indentations on lung sections ( - mm thick) using atomic force microscopy (afm). the micro-indentations provide a measure of the stiffness or elastic modulus (e) of lung tissue, which is inversely related to lung compliance. elasticity maps from afm micro-indentation from bleomycin-treated wt lungs clearly displayed the wide ranges of tissue stiffness (fig. c) , which corresponded to the heterogeneity of collagen deposition (fig. a) . the median elastic modulus of lung parenchymal tissue from wt mice increased . -fold in response to bleomycin compared with control lung tissue ( to . kpa, respectively), whereas the elastic modulus of lung tissue from bleomycin-treated vim À / À mice was significantly decreased (po . ) compared with wt bleomycin-treated lung tissue (fig. f) . the wide range of e-values is reflected in the frequency of occurrence and in the median and interquartile ranges for each condition ( supplementary fig. b ). in addition, there was a strong correlation (pearson r ¼ À . ) between the mean compliance values for each condition and the mean e-values for each condition (for example, wt-bleomycin-afm versus wtbleomycin-scireq). thus, the mechanical properties of the lung were preserved in vim À / À mice following the bleomycin treatment, which is evident at both the global tissue scale and in focal regions of lung. injury and fibrosis reduced in vim À / À bone marrow chimeras. because multiple cell types contribute to the inflammatory and fibrotic responses, we next addressed the respective roles of bone marrow-derived cells in bleomycin-induced lung injury and fibrosis by generating bone marrow chimeras (fig. a) . reconstitution of bone marrow from wt donor mice into irradiated recipient mice (wt) resulted in protein accumulation in balf in response to bleomycin, whereas in recipient mice reconstituted with bone marrow from vim À / À donor mice (vim À / À ), the balf protein was unchanged from saline controls (fig. b) . secretion of il- b (fig. c ), il- ( fig. d ) and tgf-b (fig. e ) in the balf of wt mice significantly increased in response to bleomycin; the response in vim À / À mice was significantly attenuated. finally, wt mice displayed a significant degree of collagen deposition and fibrosis, as shown by masson trichrome wt and vim À / À mice were treated with pbs containing either mg of asbestos crocidolite or the control particle titanium dioxide (tio ), intratracheally. markers of inflammation and fibrosis were assessed and weeks after instillation, respectively. (a) total cell count (macrophages, neutrophils, eosinophils, erythrocytes and lymphocytes ) and (b) protein levels were assessed in balf collected from vim À / À mice weeks after asbestos administration. inflammasome activation in wt and vim À / À mice was measured by elisa for caspase- (c) and il- b (d) levels in balf at the same time point. collagen deposition was evaluated in asbestos-treated vim À / À and wt mice by masson's trichrome and picrosirius red staining of lung slices (e; scale bar, mm) and by measuring total collagen content by sircol assay (f). data are from two independent experiments n ¼ - animals per group, and presented as mean ± s.d. *po . , **po . , relative to wt versus vim À / À , by one-way analysis of variance with a correction provided by the bonferroni multiple comparisons test. sircol assay ( fig. f ), whereas this response was severely attenuated in vim À / À mice ( fig. f ,g and supplementary fig. c ). all together, these results suggest that vimentin-expressing bone marrow-derived cells are important for bleomycin-induced activation of the nlrp inflammasome and pulmonary fibrosis. loss of vimentin prevents nlrp activation. as stated above, nlrp inflammasome activation requires two distinct stimuli. an inflammatory stimulus, such as lps, primes cells to transcribe and synthesize pro-il- b (signal ) and then another stimulus such as extracellular adenosine triphosphate (atp), monosodium urate (msu) or asbestos (signal ) is necessary for inflammasome complex formation, caspase- activation and il- b maturation , , . to further investigate the possibility that nlrp inflammasome activation requires vimentin, we isolated primary alveolar macrophages from wt and vim À / À mice. macrophages were primed with lps and stimulated with atp as previously described . levels of mature caspase- and il- b were significantly increased in the supernatants collected from wt macrophages following lsp and atp treatment compared with saline treatment, but in the supernatant collected from vim À / À macrophages there was only a slight increase (fig. a,b) . there was no difference in pro-il- b levels in total cell lysates between activated wt and vim À / À alveolar macrophages ( fig. c ; full blots are presented in supplementary fig. ) . as an independent approach, we expressed a small hairpin rna (shrna) targeted to vimentin (vim kd ) or a scrambled shrna (ctrl) in j a. macrophages and treated the cells with atp. caspase- is required to cleave pro-il- b into its biologically active form and like other caspases is proteolytically activated from a pro-enzyme to produce a tetramer of its two active subunits, p and p (ref. ). the levels of mature caspase- p , but not pro-caspase- , were considerably reduced in vim kd cells (fig. d ,e; full blots are presented in supplementary fig. ) compared with ctrl cells treated with atp, further supporting the hypothesis that vimentin deficiency prevents nlrp inflammasome activation. uric acid released from cells exposed to bleomycin triggers nlrp inflammasome assembly leading to il- b maturation and inflammation in the lungs , . in an in vitro model, we examined whether silencing vimentin would impair nlrp inflammasome activation in response to msu. a human macrophage-like cell line was treated with silencing rna (sirna) targeted to vimentin (vim kd ) or a scrambled sirna (ct; fig. a ; full blots are presented in supplementary fig. ) and subsequently activated with msu. no differences were observed in the levels of pro-il- b between ct and vim kd cells (fig. a) , suggesting that vimentin deficiency does not impair signal . nlrp inflammasome activation was repressed in vim kd cells as assessed by il- b and caspase- levels in cell supernatants (fig. b,c) . similar results were obtained when ct and vim kd cells were activated with asbestos (fig. b,c) . in addition, bone marrow-derived macrophages (bmdms) were isolated from wt and vim À / À mice, primed with lps and activated with msu. caspase- activation was assessed using a specific fluorescent probe, fam-yvad-fmk . wt bmdms showed robust caspase- activation following treatment with msu, with caspase- forming aggregates throughout the cytoplasm. however, caspase- activation was severely reduced in bmdms isolated from vim À / À mice (fig. d) . no caspase- activation was observed in untreated bmdms isolated from either wt or vim À / À mice. vimentin interacts with inflammasome components. inflammasome activation involves the physical interaction of nlrp , asc (apoptosis-associated speck-like protein containing a card) and caspase- (ref. ), but the mechanism by which the inflammasome assembles is not clear. if the vimentin network serves as a protein scaffold upon which the nlrp inflammasome components are assembled, then vimentin should interact with inflammasome proteins. immunoprecipitation of caspase- and nlrp from human macrophage lysates resulted in a robust co-precipitation of vimentin, which was enhanced following activation of cells with atp ( fig. a ; full blots are presented in supplementary fig. ). these interactions were specific for vimentin, as no actin was detectable in immunoprecipitates. we also used solution-binding assays to demonstrate a direct protein-protein interaction between vimentin and nlrp . bacterially expressed and purified s-tag his-vimentin was incubated with extracts prepared from either control or msutreated human macrophage cells. the s-tag vimentin was precipitated using s-protein agarose. the precipitated proteins (c) representative elastographs from afm micro-indentation of lung tissue from wt and vim À / À animals. the colour bar indicates elastic modulus, e. the darkest red corresponds to elastic modulus values of kpa and above. the data represent indentations per region, with at least two regions per tissue section from at least three mice per group. (d) collagen content in lungs from wt and vim À / À mice assessed by sircol assay. (e) mice were ventilated days after intratracheal instillation of bleomycin or pbs. shown are quasistatic compliance measurements of wt and vim À / À lungs. (f) elastic modulus frequency plot obtained from live, unfixed lung tissue of saline and bleomycin-treated wt and vim À / À animals. microindentation data were fit using the hertz model to acquire the elastic modulus of regions of lung tissue. images in a-b represent data obtained from at least three animals per group; data shown in d-e are mean ± s.d. of at least five animals. **po . , ***po . relative to wt versus vim À / À , by one-way analysis of variance with a correction provided by the bonferroni multiple comparisons test. were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page), transferred to nitrocellulose and then immunoprobed with antibodies directed against nlrp . we observed a . ± . -fold increase in binding between vimentin and nlrp in extracts prepared from msu-treated cells, as compared with untreated control cells (supplementary fig. ). article a complementary method, bio-layer interferometry (bli), was also used to investigate the interaction between nlrp and vimentin. bli is an optical technique that analyses the interference pattern of white light reflected from two surfaces: a layer of immobilized protein on the biosensor tip and an internal reference layer . any change in the number of molecules bound to the biosensor tip causes a shift in the interference pattern that can be measured in real time, providing detailed information on the kinetics of association and dissociation between the two molecules (k a , k d and k d ). commercially available nlrp was bound to the biosensor tip, then various concentrations ( mm, mm, mm, . mm, nm, nm and . nm) of full-length vimentin were added to determine the association kinetics with nlrp . in agreement with other methods (fig. a ,b,d and supplementary fig. ), the assay demonstrated that nlrp binds to vimentin with a measureable affinity; a global dissociation constant (k d ) of nm was determined using blitz pro. consistent with the co-immunoprecipitation and solutionbinding results, we observed co-localization of nlrp and vimentin in stimulated alveolar macrophages by confocal immunofluorescence microscopy (fig. d) . following activation, the wt cells, but not the vim kd cells, were spread and displayed lamellipodia. furthermore, the vimentin network spanned the cytoplasm in the wt activated cells, whereas in unstimulated cells, the network appeared to be collapsed around the nucleus and lamellipodia were not observed (fig. d) . vimentin filaments (red) strongly co-localized with nlrp (green) in both perinuclear and distal regions of the cells. co-localization studies were performed using fiji . r ( -bit, win) and the coloc test . co-localization analyses were calculated on rois drawn around the lamellipodia, determined by the vimentin image. the thresholded manders co-localization scores were . ± . for vimentin and . ± . for nlrp . nlrp and asc also colocalized in stimulated macrophages ( supplementary fig. ); the score for nlrp co-localization with asc in untreated cells was . ± . , indicating weak co-localization. in contrast, the score for nlrp co-localization with asc in cells treated with atp was . ± . , indicating strong co-localization. however, knockdown of vimentin resulted in a lack of co-localization between nlrp and asc, even in activated macrophages ( supplementary fig. ). taken together, these results demonstrate that vimentin associates with components of the inflammasome and is required for inflammasome activation. inflammation, the process aimed at restoring homeostasis after an insult, can be more damaging than the insult itself if uncontrolled, excessive or prolonged. growing evidence indicates that il- b is a critical mediator of acute inflammation, remodelling and fibrosis in the lung. using three well-characterized murine models of ali and pulmonary fibrosis, which were previously shown to be dependent on the nlrp inflammasome , , , , we show that nlrp inflammasome activation, which leads to il- b maturation, is dependent on the type iii if protein vimentin. our data obtained in vimentin knockout mice and vimentin-null cells suggest that vimentin is required for nlrp inflammasome activation. dysregulated inflammation, excessive leukocyte accumulation and increased permeability of endothelial and alveolar epithelial barriers are the central pathophysiologic events in ali and its most severe form, the acute respiratory distress syndrome (ards) . all these end points were observed in wt mice challenged with lps, bleomycin and asbestos. in contrast, vim À / À mice exposed to the same treatments experienced mild increases in protein levels in balf and limited alterations in lung histopathology, suggesting that they were protected from lps-, bleomycin-and asbestos-induced ali. inflammasome-regulated cytokines have a key role in the initiation and propagation of the aforementioned features of ali . in fact, alveolar macrophages from patients with ards produce excessive amounts of il- b , and il- was elevated in the plasma of patients with ards, which correlated with increased morbidity and mortality . since vimentin-deficient mice were protected from ali, we speculated that failure to produce inflammasome-mediated cytokines (for example, il- b) caused this protection. in fact, vimentin-deficient mice failed to increase mature il- b levels following exposure to lps, asbestos or bleomycin. moreover, we demonstrated that caspase- activation and mature il- b production in vitro requires vimentin, as no increase was observed in activated macrophages either isolated from vim À / À mice or in which vimentin was silenced using rna interference. resolution of ali/ards depends on a precise balance of inflammatory and molecular signalling ; an imbalance in that process caused by il- b overproduction might prevent cellular restoration and lead to pulmonary fibrosis. in fact, il- b produced by the inflammasome has been recently identified as an initiator of fibrosis in response to bleomycin , and administration of il- b alone recapitulates much of the lung pathology caused by bleomycin . because vimentin-deficient mice did not produce the same amount of il- b as wt mice, we reasoned that vim À / À mice would be protected from bleomycin-and asbestos-induced pulmonary fibrosis. indeed, lung pathology was significantly reduced in vimentin-deficient mice, pointing to the essential role of vimentin in pulmonary fibrosis. we found substantially less collagen deposition in vim À / À mice compared with wt mice. the excessive collagen deposition in lungs of wt mice led to loss of mechanical compliance, whereas the lungs of vim À / À mice maintained compliance similar to that of saline-treated mice. these results are consistent with previous reports demonstrating decreases in the compliance of fibrotic lungs . the elastic modulus measurements strongly suggest that increased tissue stiffness at the microscale corresponds to areas of increased collagen deposition. importantly, macroscale measurements of lung compliance were highly correlated with the microscale elastic modulus measurements obtained by afm. a number of studies have demonstrated that il- b can induce ali and contribute to the progression of pulmonary fibrosis , , , . il- b-induced fibrosis is associated with an increase in tgf- b, demonstrating that ali initiated by proinflammatory cytokines can result in progressive fibrosis , . vim À / À mice were treated with recombinant mouse il- b (rmil- b . mg kg À body weight, intranasal (i.n.)), but we were unable to detect latent tgf-b in balf from vim À / À mice after i.n. rmil- b at days , and (data not shown), whereas latent tgf-b was detected days after bleomycin administration in wt mice (fig. ) . gasse et al. reported that collagen deposition was increased by rmil- b in wild-type mice; vim À / À mice treated with rmil- b had no increase in collagen deposition. we reason that the incomplete suppression of mil- b in vim À / À mice is a reflection of incomplete chimera, with only b % engraftment. studies with il- r À / À , myd À / À , asc À / À , caspase- À / À and nlrp À / À mice have suggested that uric acid (induced by bleomycin), asbestos and silica are detected by the nlrp inflammasome in macrophages, likely leading to il- r /myd signalling in pulmonary epithelial cells, then to inflammation, neutrophil and lymphocyte recruitment and fibroblast activation . lung fibroblast activation results in collagen deposition and pulmonary fibrosis. importantly, it was reported that vimentin is required for the stabilization of collagen mrnas . we reason that the vim À / À mice treated with either rmil- b or wt bone marrow failed to increase collagen deposition because vimentin knockout fibroblasts fail to produce type i collagen due to decreased stability of collagen a (i) and a (i) mrnas . protection from injury, inflammation and fibrosis has been observed in mice lacking any components of the nlrp inflammasome, similar to our observations of vim À / À mice following injury , , , , . these phenotypes led us to investigate whether vimentin might serve as a scaffold for the inflammasome by binding to one or more proteins in the inflammasome complex. several studies have suggested that ifs, including vimentin, act as scaffolds for signalling molecules , , . in fact, vimentin interacts with and is involved in the translocation of perk / to the nucleus , limits the ability of - - to interact with raf and cdk , and regulates the release and translocation of roka following rhoa activation. these findings exemplify how vimentin ifs can act as scaffolds for signalling molecules. recently, vimentin was reported to bind to nod (nucleotide-binding oligomerization domain-containing protein ), a member of the nlr family, via the leucin-richrepeat (lrr) . the lrr is a protein-binding domain shared by other nlr family members, including nlrp . therefore, we sought to determine whether vimentin interacts with nlrp . in our experiments, immunoprecipitation of both nlrp and caspase- in lysates from wt alveolar macrophages resulted in a robust co-precipitation of vimentin, which was enhanced following activation of the nlrp inflammasome. these data were confirmed by immunofluorescent confocal microscopy of human and murine macrophages. the co-localization score for vimentin with nlrp was . ± . , indicating strong colocalization . moreover, we were able to demonstrate a direct protein-protein interaction between vimentin and nlrp by bli assays, further establishing a role for vimentin in assembly of the nlrp inflammasome. intermediate filaments, including vimentin, have been shown to provide a scaffold whereby active subunits of caspase- can activate caspase- , which, in turn, can activate more caspase- resulting in an amplification loop , . caspase- , caspase- and caspase- have also been shown to interact with vimentin ; to our knowledge, we are the first to report an interaction between caspase- and vimentin. we can only speculate about where and how vimentin associates with nlrp and caspase- during inflammasome assembly, since the crystal structures of the vimentin dimer and all components of the inflammasome except for pyd are unknown [ ] [ ] [ ] . nlrp proteins have been shown to form inactive preassembled complexes in unstimulated cells [ ] [ ] [ ] . under stimulation, these proteins undergo conformational changes whereby the inflammasome is activated. the vimentin if network could potentially associate with the nlrp inflammasome through one or more domains of nlrp and mediate the conformation change or stabilization of nlrp that leads to activation. we reason that vimentin may interact with nlrp in two distinct regions: the nacht domain and the lrr; vimentin has been previously shown to interact with nod in the lrr domain . because asc is localized to the nucleus in unactivated cells, no interaction with vimentin is anticipated in nonactivated cells. furthermore, on the basis of the structure of asc, a pyd and card domain with a short linker, we do not anticipate a direct interaction with vimentin as vimentin lacks either of the asc domains, therefore, any asc signal following cell activation is anticipated to be due to vimentin interaction with other proteins in the inflammasome protein complex. an unresolved issue is how vimentin mediates inflammasome activation. mitochondrial-derived reactive oxygen species (ros) have been linked to inflammasome activation . vimentin if are known to modulate mitochondrial motility , and lack of vimentin results in increased ros production . this suggests that vimentin-deficient cells should have more robust inflammasome signalling, the exact opposite of what we have demonstrated. in a very provocative paper, however, bauernfeind et al. demonstrated that mitochondrial-derived ros is required for priming, not activation, of the inflammasome, as ros inhibitors blocked the priming step required for nlrp expression, but activation was not affected. this correlates with our findings that signal is unaffected by the lack of vimentin. further studies on the relationship between vimentin, mitochondria and ros will likely provide insights into the regulation behind both signals and during inflammation, but currently they are beyond the scope of this paper. in summary, this study demonstrates a novel function for the type iii if, vimentin, in innate immunity and inflammation leading to ali and pulmonary fibrosis. in our experiments, vimentin deficiency resulted in a blunted inflammatory response in the early stages of injury, which was consistent among the different animal models of lung injury used: lps, bleomycin and asbestos. we further demonstrate that vimentin is required for il- b maturation through its interaction with the nlrp inflammasome. vimentin may act as a scaffold for assembly of the inflammasome protein complex and thus regulate its function. overall, this study provides new insights into lung inflammation and fibrosis, and importantly, suggests that vimentin may be a key regulator of the nlrp inflammasome. mice and induction of acute lung injury. age-and sex-matched -to -weekold mice were used in all the experiments. the /sv vimentin-deficient (vim À / À ) mice were a gift from albee messing (madison, wi). bleomycin ( . units in ml of pbs; hospira) and asbestos ( mg in pbs; us epa) were administered intratracheally via two -ml aliquots. lps ( mg kg À , lethal dose; mg kg À , sublethal dose; sigma-aldrich) was administered via intraperitoneal injection. wt and vim À / À mice were treated with a single intratracheal injection of ml of phosphate-buffered saline (pbs) or bleomycin ( . units in ml of pbs; hospira). all mice were bred in our animal facility and the experiments were approved by the northwestern university animal care and use committee. histology. a -gauge angiocath was sutured into the trachea, heart and lungs were removed en bloc and inflated with . ml of % paraformaldehyde. the heart and lungs were fixed and embedded in paraffin; -mm sections were stained with h&e, picrosirius red or masson trichrome by the mouse histology phenotyping laboratory (northwestern university, chicago, il). sections were visualized using the tissuegnostics tissue/cell high throughput imaging analysis system (vienna, austria) and captured using tissuefaxs software (tissuegnostics, los angeles, ca) at the northwestern university cell imaging facility. specific regions of interest were visualized using a zeiss axioskop microscope and captured using the cri nuance spectral camera software suite. wet-to-dry weight ratios. mice were anaesthetised and lungs were surgically removed en bloc. the left lung was ligated, excised and weighed in a tared container. the left lung was then dried at °c in a speed-vac sc evaporator (thermo scientific, waltham, ma) until a constant weight was obtained, and the wet-to-dry weight ratio was calculated. bronchoalveolar lavage. to perform bronchoalveolar lavage (bal), a -gauge angiocath ligated into the trachea and ml of sterile pbs was instilled, then removed through the angiocath; the process was repeated three times. the samples were pooled and a ml aliquot of the bal fluid was placed in a cytospin and centrifuged at g for min. the pellet was re-suspended in ml of pbs, plated on glass slides, stained with crystal violet and subjected to a blinded manual cell count and differential. cytokine and chemokine levels in the supernatant were measured using the bd cytometric bead array (bd biosciences). samples were analysed in triplicate using the mouse inflammation kit (bd biosciences) according to the instructions provided. this kit detects interleukin (il)- , mcp- and tumour necrosis factor (tnf)-a. total transforming growth factor (tgf)-b was measured using the tgf-b rii duoset enzyme-linked immunosorbent assay (elisa, r&d systems, minneapolis, mn) according to the manufacturer's instructions. total interleukin (il)- b was measured using the il-b ready-set-go elisa (ebioscience, san diego, ca) according to the manufacturer's instructions. caspase- activity was measured using the caspase- fluorometric assay (r&d systems), according to the manufacturer's instructions. sircol assay. total collagen content was determined by harvesting lungs from mice d after treatment with bleomycin or pbs, or weeks after treatment with asbestos or tio . exsanguination was accomplished by a left nephrectomy, followed by perfusion of the right ventricle with ml of cold pbs. both lungs were removed and homogenized ( min with a tissue homogenizer followed by strokes in a dounce homogenizer) in ml of . n acetic acid supplemented with roche edta protease inhibitor. aliquots of lung homogenate were then assayed for total lung collagen levels and compared with a standard curve prepared from rat tail collagen by using the sircol collagen dye binding assay (biocolor ltd, newtownabbey, uk), according to the manufacturer's directions. compliance measurements. wt and vim À / À mice were anaesthetised with ketamine and xylazine ( mg per g of body weight) and a tracheotomy was performed. pancuronium was administered intraperitoneally ( . mg kg À of body weight). the mouse was mechanically ventilated on a computer-controlled piston ventilator (flexivent; scireq, montréal, québec, canada) with a tidal volume of ml kg À at a frequency of breaths per minute against - cm h o positive end-expiratory pressure. following two total lung capacity manoeuvres to standardize volume history, pressure and flow data (reflective of airway and tissue dynamics) were collected during a series of standardized volume perturbation manoeuvres. these data were used to calculate both total lung resistance (r) and elastance (e) using the single-compartment model and used to derive further parameters of respiratory function. the flexivent calibration procedure removes cannula impedance from the reported data. residual impedence (i) is therefore negligible and is not reported herein. atomic force microscopy measurements. lungs were used at - days following bleomycin challenge. following instillation of . % low-gel point agar ( ml kg À ), lungs were sectioned using a vibratome (leica vt s) into mm or -mm thick slices. these slices were used within h of harvest; microindentations were performed using a bruker bioscope catalyst located at the northwestern university nuance facility. modified probes with -mm spheres (novascan, ames, ia) were used to indent mm  mm areas on the lung slices. force and indentation depth were converted to elastic modulus using the hertz spherical indentation model , and a custom matlab program (generated by dr esra roan, university of memphis). a poisson ratio of m ¼ . was assumed for the tissue - . cell culture. to isolate bone marrow-derived macrophages (bmdm), bone marrow was extracted from mice femurs and tibia then purified through a ficoll-paque gradient. upon purification, cells were differentiated on charge-free plates in dmem containing % endotoxin-reduced fetal bovine serum and % l cell supernatant for days . peritoneal macrophages were obtained from untreated mice. briefly, the abdomen of anaesthetised mice was exposed and ml of sterile pbs was instilled intraperitoneally using a -gauge syringe. following agitation, the fluid was removed and centrifuged. the cells were plated in dulbecco modified eagle medium containing % fetal bovine serum and used for western blotting. alveolar macrophages were obtained from balf of untreated mice. briefly, ml of sterile pbs was instilled through a -gauge angiocath ligated into the trachea and the fluid then removed; this process was repeated three times. the fluid was centrifuged and cells were used for elisa. the second-generation lentiviral pgipz expression vectors encoding shrnas against mouse vimentin (clones v lmm_ , v lhs_ and v lhs_ ) and scrambled shrna control (catalogue no. rhs ) originated from the thermo fisher scientific shrna library (waltham, ma) and were provided by rnai/throughput robotics core of northwestern university (evanston, il). lentiviral stocks (with titres of - tu ml À ) encoding shrnas against mouse vimentin were produced by the dna/rna delivery core (skin disease research center, northwestern university, chicago, il). western blotting. primary murine macrophages were cultured overnight in % serum and treated or not with lps ( ng ml À ) for h. atp ( mm) was added to lps-stimulated cells during the last min. cells were lysed in sample buffer and equal volumes loaded in % sds-page gels transferred to nitrocellulose membranes. the presence of indicated proteins in cell lysates and medium was assessed by western blotting using the following antibodies: mouse monoclonal anti-vimentin (clone v ; : ; sigma-aldrich no. treated according to manufacturer's instructions. bound proteins were prepared for western blotting. protein-protein interaction. the interaction between nlrp and vimentin was analysed using a bli, blitz (fortebio, menlo park, ca). antibodies directed against nlrp (adipogen) were diluted : then attached to a protein a biosensor. next, commercially available, purified nlrp (abnova) was attached to the biosensor for min. finally, each labelled biosensor was placed into a specific concentration of purified vimentin and binding was measured for s followed by dissociation in pbs for s. at the start of each binding experiment, vimentin was diluted into pbs to assemble filaments real-time reverse transcriptase polymerase chain reaction. total rna extraction and quantitative pcr were performed as previously described (). briefly, total rna was isolated using the aurum total rna mini kit (qiagen). cdna was synthesized using the qscript cdna supermix (quanta biosciences) and amplified using -cycle two-step pcr with sequence-specific primer pairs (idt). sybr green fluorescence was quantified with a cfx real-time system thermal cycler (bio-rad) using ddct analysis. relative il- b mrna expression was determined via normalizing to b-actin mrna expression. statistical analysis. data are expressed as means ± s.d. or means ± s.e.m. differences between two groups were assessed by using a student's t-test. differences between three or more groups were assessed using one-way analysis of variance with a bonferroni multiple comparisons test. values of po . were considered to be significant. the log rank test, which calculates the chi-square (w ) for each event time for each group and sums the results, was used in the analysis of the kaplan-meier curve was used. all analyses were performed using graphpad prism software version . for windows (graphpad software, san diego, ca). wild-type j . cells and j . cells expressing shrna against vimentin were fixed in methanol ( À °c) for min and processed for indirect immunofluorescence as described previously , using the following antibodies: mouse monoclonal anti-nlrp (adipogen international no. ag- b- images of fixed, stained preparations were taken with either a zeiss lsm microscope or a nikon a r microscope immunoprecipitated caspase- in the right panel could not be detected due to masking by the signal from the heavy chain of the antibody. protein a/g agarose was incubated with cell lysates in the absence of an antibody to control for non-specific binding. to enable caspase- detection in immunoprecipitates, caspase- antibodies were crosslinked to protein a/g agarose using crosslink ip kit (thermo) cell mechanics and the cytoskeleton introducing intermediate filaments: from discovery to disease mice lacking vimentin develop and reproduce without an obvious phenotype impaired wound healing in embryonic and adult mice lacking vimentin a role for vimentin in crohn disease functions of the intermediate filament cytoskeleton in the eye lens if-pathies": a broad spectrum of intermediate filamentassociated diseases intermediate filaments: primary determinants of cell architecture and plasticity dysfunctions of neuronal and glial intermediate filaments in disease novel functions of vimentin in cell adhesion, migration, and signaling intermediate filaments as signaling platforms inflammasome-mediated production of il- beta is required for neutrophil recruitment against staphylococcus aureus in vivo activation of inflammasome signaling mediates pathology of acute p. aeruginosa pneumonia mycobacterium tuberculosis protein esat- is a potent activator of the nlrp /asc inflammasome the nlrp inflammasome mediates in vivo innate immunity to influenza a virus through recognition of viral rna the inflammasomes inflammasomes: guardians of cytosolic sanctity uric acid is a danger signal activating nalp inflammasome in lung injury inflammation and fibrosis integrating mechanisms of pulmonary fibrosis molecular basis of asbestos-induced lung disease innate immune activation through nalp inflammasome sensing of asbestos and silica the nalp inflammasome is essential for the development of silicosis cutting edge: reactive oxygen species inhibitors block priming, but not activation, of the nlrp inflammasome the inflammasomes regulation of inflammasome signaling inflammasomes in health and disease the inflammasome in lung diseases suppression of inflammation and acute lung injury by miz via repression of c/ebp-d the intrinsic apoptotic pathway is required for lipopolysaccharide-induced lung endothelial cell death flow cytometric analysis of macrophages and dendritic cell subsets in the mouse lung cytokine storm in a phase trial of the anti-cd monoclonal antibody murine models of pulmonary fibrosis an official american thoracic society workshop report: features and measurements of experimental acute lung injury in animals il- r /myd signaling and the inflammasome are essential in pulmonary inflammation and fibrosis in mice idiopathic pulmonary fibrosis lung parenchymal mechanics in health and disease mechanisms of pulmonary fibrosis cutting edge: nitric oxide inhibits the nlrp inflammasome gout-associated uric acid crystals activate the nalp inflammasome cell volume regulation modulates nlrp inflammasome activation bio-layer interferometry for measuring kinetics of protein-protein interactions and allosteric ligand effects briding the gap between qualitative and quantitative colocalization results in fluorescence microscopy studies the acute respiratory distress syndrome inflammasome-regulated cytokines are critical mediators of acute lung injury elevated interleukin- release by human alveolar macrophages during the adult respiratory distress syndrome bleomycin and il- beta-mediated pulmonary fibrosis is il- a dependent transient expression of il- b induces acute lung injury and chronic repair leading to pulmonary fibrosis a novel role of vimentin filaments: binding and stabilization of collagen mrnas the highly conserved nuclear lamin ig-fold binds to pcna: its role in dna replication vimentin binding to phosphorylated erk sterically hinders enzymatic dephosphorylation of the kinase the intermediate filament protein, vimentin, is a regulator of nod activity intermediate filaments control the intracellular distribution of capases during apopptosis caspase cleavage of vimentin disrupts intermediate filaments and promotes apoptosis a role for mitochondria in nlrp inflammasome activation vimentin intermediate filaments modulate the motility of mitochondria vimentin is secreted by activated macrophages proapoptotic bid is required for pulmonary fibrosis norepinephrine increases alveolar fluid reabsorption and na,k-atpase activity poissons' ratio of lung parenchyma and parenchymal interaction with bronchi feedback amplification of fibrosis through matrix stiffening and cox- suppression oxidative stress, plasminogen activator inhibitor , and lung fibrosis a nuclear receptor atlas: macrophage activation shear stress induced reorganization of the keratin intermediate filament network requires phosphorylation by protein kinase c zeta vimentin is sufficient and required for wound repair and remodeling in alveolar epithelial cells structure and assembly properties of the intermediate filament protein vimentin: the role of its head, rod and tail domains vimentin intermediate filament formation: in vitro measurement and mathematical modeling of the filament length distribution during assembly dr esra roan (university of memphis, tn) generously assisted with the afm microindentation matlab code and analysis of results. we thank mr luke skertich for assistance with immunofluorescence confocal microscopy. histological staining was performed by the mouse histology phenotyping laboratory. imaging work was performed at the northwestern university cell imaging facility generously supported by nci ccsg p ca awarded to the robert h. lurie comprehensive cancer center. afm measurements were produced in the nifti facility (nuance center, northwestern university), which has received support from the nsf-nsec, nsf-mrsec, keck foundation, the state of illinois and northwestern university. key: cord- -r cgpvs authors: wu, fengjiao; zhao, yawei; jiao, tian; shi, dongyan; zhu, xingxing; zhang, mingshun; shi, meiqing; zhou, hong title: cxcr is essential for cerebral endothelial activation and leukocyte recruitment during neuroinflammation date: - - journal: j neuroinflammation doi: . /s - - - sha: doc_id: cord_uid: r cgpvs background: chemokines and chemokine receptors cooperate to promote immune cell recruitment to the central nervous system (cns). in this study, we investigated the roles of cxcr and cxcl in leukocyte recruitment to the cns using a murine model of neuroinflammation. methods: wild-type (wt), cxcl (−/−), and cxcr (−/−) mice each received an intracerebroventricular (i.c.v.) injection of lipopolysaccharide (lps). esterase staining and intravital microscopy were performed to examine neutrophil recruitment to the brain. to assess endothelial activation in these mice, the expression of adhesion molecules was measured via quantitative real-time polymerase chain reaction (pcr) and western blotting. to identify the cellular source of functional cxcr , chimeric mice were generated by transferring bone marrow cells between the wt and cxcr (−/−) mice. results: expression levels of the chemokines cxcl , cxcl , and cxcl were significantly increased in the brain following the i.c.v. injection of lps. cxcr or cxcl deficiency blocked neutrophil infiltration and leukocyte recruitment in the cerebral microvessels. in the cxcr (−/−) and cxcl (−/−) mice, the cerebral endothelial expression of adhesion molecules such as p-selectin and vcam- was dramatically reduced. furthermore, the bone marrow transfer experiments demonstrated that cxcr expression on cns-residing cells is essential for cerebral endothelial activation and leukocyte recruitment. compared with microglia, cultured astrocytes secreted a much higher level of cxcl in vitro. astrocyte culture conditioned medium significantly increased the expression of vcam- and icam- in cerebral endothelial cells in a cxcr -dependent manner. additionally, cxcr messenger rna (mrna) expression in cerebral endothelial cells but not in microglia or astrocytes was increased following tumor necrosis factor-α (tnf-α) stimulation. the intravenous injection of the cxcr antagonist sb significantly inhibited endothelial activation and leukocyte recruitment to cerebral microvessels. conclusions: cxcl secreted by astrocytes and endothelial cxcr play essential roles in cerebral endothelial activation and subsequent leukocyte recruitment during neuroinflammation. immune cell recruitment is a key event in the development of many types of central nervous system (cns) inflammatory diseases, such as bacterial meningitis [ ] , stroke [ ] , and multiple sclerosis [ ] . following the detection of pathogen-derived components or danger signals, leukocyte recruitment to the brain via chemotaxis [ , ] occurs via a cascade-like process that involves the expression of endothelial cell-and leukocyte-expressed adhesion molecules such as selectins and integrins [ ] [ ] [ ] . during the early stage of cns inflammation, the interactions between chemokines and their receptors also exert a profound effect by attracting immune cells to migrate across the blood-brain barrier (bbb) [ , ] . cxcr is a g protein-coupled receptor that is activated by cxc chemokines, including murine cxcl , cxcl , and cxcl [ , ] . interactions between cxcr and its ligands play an essential role in mediating neutrophil migration to sites of inflammation. although extensive studies have focused on the role of cxcr in inflammatory responses in different organs, the involvement of individual chemokines in different types of inflammatory responses remains contentious. for example, cxcl is essential for the host pulmonary defense to klebsiella infection [ ] and mediates neutrophil recruitment during the progression of experimental lyme arthritis [ ] . however, even in the presence of high levels of cxcl expression, the interaction between cxcl and cxcr is still essential for neutrophil migration in response to specific antigen challenge [ ] . additionally, cxcl plays a more important role than cxcl in a viral antigen-induced delayed-type hypersensitivity response [ ] . furthermore, cxcr is widely expressed on neutrophils [ ] , lymphocytes [ ] , and other types of non-hematopoietic cells, including epithelial [ ] and endothelial cells [ , ] . most studies have focused on the functions of cxcr expressed on hematopoietic cells, such as monocytes [ , ] and neutrophils [ ] [ ] [ ] [ ] . however, recent studies have revealed a critical role of cxcr expressed on nonhematopoietic cells during inflammatory responses. in a murine model of acute kidney infection, cxcr on non-bone marrow-derived cells influenced the neutrophil response [ ] . additionally, cxcr expression on resident cells is essential for the migration of mast cell progenitors in the lung of antigen-challenged mice [ ] . however, the roles of cxcr and its ligands in cns inflammation remain to be addressed. in this study, we performed intravital microscopy to examine the role of cxcr in leukocyte recruitment during neuroinflammation. we observed reduced neutrophil infiltration and attenuated leukocyte-endothelial cell interactions in cxcr −/− and cxcl −/− mice following the intracerebroventricular (i.c.v.) injection of lipopolysaccharide (lps). moreover, cxcr or cxcl deficiency impaired endothelial activation by attenuating the expression of adhesion molecules. using chimeric mice expressing cxcr on either hematopoietic cells or radiation-resistant non-hematopoietic cells, we showed that cxcr expression on radiation-resistant cells in the cns is essential for endothelial activation and subsequent leukocyte recruitment during neuroinflammatory responses. furthermore, a high level of cxcl was detected in primary astrocyte culture and culture conditioned medium significantly increased the expression of vcam- and icam- on cerebral endothelial cells. taken together, our findings revealed a previously unrecognized role of cxcr expressed on cerebral endothelial cells in the regulation of endothelial activation and immune cell recruitment across the bbb during cns inflammation. animals c bl/ j mice ( to weeks old, to g) used as wild-type controls were purchased from the model animal research center of nanjing university. cxcr −/− mice (on the c bl/ j background) were purchased from the jackson laboratory (bar harbor, me, usa). all mice were maintained under environmentally controlled conditions (ambient temperature, ± °c; humidity %) in a pathogen-free facility with a -h light/dark cycle and had access to water and food ad libitum. all experimental procedures were performed in strict accordance with the institutional animal care and use committee of nanjing medical university. to target the cxcl gene in the mouse genome, we designed and synthesized highly active talens specific to the cxcl gene. the talen target sequence for cxcl was gatcccagccacccgc. talen messenger rnas (mrnas) were diluted in rnase-free phosphate-buffered saline (pbs) and then injected into the cytoplasm of mouse pronuclear stage embryos to produce mutant founders (f ). heterozygous f offspring were interbred to produce homozygous f animals. to functionally validate the talen-induced mutations, we intracerebroventricularly injected lps into these mice and measured the level of cxcl in the brain. no expression of cxcl was detected in the cxcl mutant founder. the cxcl −/− mice were viable and fertile and did not exhibit any gross abnormalities. intracerebroventricular injections into the mice were performed as previously described [ ] . briefly, the mice were anesthetized via intraperitoneal (i.p.) injection with a mixture of mg/kg ketamine and mg/kg xylazine. then, μg of lps (dissolved in sterile saline at a concentration of μg/μl; escherichia coli serotype :b strain; invivogen, carlsbad, ca, usa) was injected into the left ventricles using a microsyringe over a -min period. sham mice received isovolumetric sterile saline injection. after lps injection, the mice were maintained under anesthesia at ± °c on a thermostatic heating system (harvard apparatus, ma, usa) for h before intravital microscopy was performed. the mice were anesthetized after lps injection and subsequently perfused through the heart with ml of cold pbs over a period of min to remove protein from the blood circulation. mouse brains were homogenized in ml of cold pbs and centrifuged at , rpm for min at °c. the cxcl , cxcl , and cxcl concentrations in the supernatant were measured using commercial enzyme-linked immunosorbent assay (elisa) kits (r&d systems, minneapolis, mn, usa) according to the manufacturer's instructions. the detection limit was . pg/ml for all assays. flow cytometric analysis of single-cell suspensions prepared from peripheral blood or spleens of wild-type or cxcl −/− mice was performed on a beckman cytoflex (beckman coulter, suzhou, china). antibody clones used for staining were specific for gr- (rb - c , ebioscience, san diego, usa), cd ( -f , ebioscience), and cxcr (tg , biolegend, san diego, ca, usa). after anesthetization, the mice were transcardially perfused with ice-cold % formalin. then, the brains were removed and fixed in % formalin for h. the formalin-fixed tissues were embedded in paraffin and then sliced into μm sections. infiltrating neutrophils were stained using a naphthol as-d chloroacetate specific esterase kit (sigma, st. louis, mo, usa). we selected more than four fields of view at a primary magnification of × in the cortex or hippocampus of every brain section. cells were counted under a nikon e microscope, and the data are presented as the means ± sem. intravital microscopy of the mouse brain intravital microscopy was performed as previously described [ ] . briefly, after anesthetization, the right parietal bone was subjected to craniotomy using a highspeed drill. subsequently, the dura were removed from this site to expose the pial brain vessels. rhodamine g (sigma) was injected intravenously ( . mg/kg) into the mouse to label the leukocytes. then, a microscope equipped with a fluorescent light source was used to detect the leukocytes. the data were collected through a scmos camera (orca-flash . , hamamatsu) mounted on the microscope and stored for subsequent analysis. rolling leukocytes were defined as those cells moving at a slower velocity than the erythrocytes; adherent cells were defined as those that remained stationary for at least s. after perfusion through the heart, the brains were homogenized in ml of trizol (takara bio, inc., shiga, japan) on ice, and rna was extracted using trizol reagent according to the protocol supplied by the manufacturer. a total of μg of total rna was reversetranscribed into cdna. then, sybr® green-based quantitative real-time polymerase chain reaction (pcr) was performed with a bio-rad cfx touch (bio-rad laboratories, hercules, ca, usa) according to the manufacturer's instructions. β -macroglobulin (β -mg) was used as a housekeeping gene because its expression was not influenced by the treatments. the amplification conditions were as follows: °c ( min) followed by cycles of °c ( s), . °c ( s), and °c ( s). quantitative pcr assays were conducted in triplicate for each sample and were performed using the −ΔΔct method. the amplified products were verified on a . % agarose gel by electrophoresis. the data are expressed as the n-fold differences relative to the standard. after i.c.v. lps injection, the mice were anesthetized and perfused with ice-cold pbs to clear blood-borne proteins. next, the brain was homogenized in ml of cold pbs on ice, and the homogenate was centrifuged ( , rpm, min). cells were digested with radioimmunoprecipitation assay (ripa) lysis buffer ( mmol/l tris-hcl, mmol/l nacl, % nonidet- , . % sodium deoxycholate, mmol/l edta, mmol/l pmsf) for min on ice and centrifuged at , rpm for min at °c. the brain homogenates or cell lysate were diluted in pbs and loading buffer, boiled ( °c, min), loaded on a % acrylamide-sds gel, and transferred to a protran nitrocellulose membrane (millipore, billerica, ma, usa). the membranes were blocked with % dry milk in pbs for h at room temperature, incubated in primary antibodies against p-selectin (ab , abcam, cambridge, usa), e-selectin (ab , abcam),vcam- (ab , abcam), icam- (ab , abcam), cxcr (ab , abcam), albumin (ab , abcam), and β-actin (cell signaling, beverly, ca, usa) overnight at °c, washed, incubated in species-appropriate hrp-conjugated secondary antibodies for - h at room temperature in the dark, and washed three times. then, the membranes were subjected to immunodetection using enhanced chemiluminescence reagents (perkinelmer, waltham, ma, usa). the mice were anesthetized and perfused with ml of cold pbs over a period of min to remove proteins from the blood circulation. then, the concentration of albumin, a serum protein that is normally excluded from the brain by the intact blood-brain barrier, was measured in brain homogenates by western blotting as previously described [ ] . after the neonatal cerebra were harvested, cerebral cortices devoid of cerebella, white matter, and leptomeninges were trypsinized for min and then filtered through a -μm pore size filter. the cells from seven cerebra were seeded on an uncoated -cm culture flask and incubated in ml of dulbecco's modified essential media (dmem)/f containing % fbs. the medium was replenished every - days after cell seeding. on days - , microglia were isolated by shaking the flask at rpm for h as described [ ] . then, the cells were centrifuged and seeded at the appropriate density in six-well plates for further stimulation. after the mixed glial cells were passaged two to three times and shaken at rpm for h, the supernatants were discarded; the remaining adherent cells that remained consisted predominantly of astrocytes [ ] . isolation and culture of primary mouse brain microvascular endothelial cells primary cerebral endothelial cells were prepared as previously described [ ] . in brief, cortices from -to week-old c bl/ j mice were isolated by removing the cerebellum, striatum, optic nerves, and white matter. the outer vessels and the meninges were removed using dry cotton swabs. then, the tissue sample was fragmented into -mm -thick pieces and digested in ml of . % collagen b (roche, indianapolis, in, usa) supplemented with u/ml dnase i (sigma, st. louis, mo, usa) for . - h at °c with occasional agitation. the suspension was centrifuged at rpm for min. the resulting homogenate was mixed with % bsa in dmem and centrifuged at rpm for min at °c. the neural component and the bsa layer were discarded, and the pellet containing the vascular component was further digested in . % collagenase/dispase (roche, indianapolis, in, usa) supplemented with u/ml dnase i for . - h at °c. the final microvessel pellets were resuspended in dmem supplemented with % fbs (life technologies, carlsbad, ca, usa), ng/ ml bfgf (peprotech, rocky hill, nj, usa), u/ml heparin, u/ml penicillin, and mg/ml streptomycin. the medium was refreshed every days. the endothelial cells grew to confluency after days. the purity of the endothelial cells was > %. prior to irradiation, the mice were treated with antibiotics with the intention of eliminating pseudomonas aeruginosa from the gastrointestinal tract. neomycin was added to the drinking water weeks post-irradiation. the recipient mice were lethally irradiated with two doses of rad (separated by - h) as previously described [ ] . bone marrow cells were harvested from both the femora and tibiae of the donor mice, and approximately - million cells were intravenously injected into the recipient mice. bone marrow transfers were performed as follows: ( ) bone marrow cells from the cxcr −/− mice were transferred into the wild-type (wt) mice (chimeric, expressing cxcr on only the non-hematopoietic cells) and ( ) bone marrow cells from the wt mice were transferred into the cxcr −/− mice (chimeric, expressing cxcr on only the hematopoietic cells). all chimeric mice were used for intravital microscopy experiments - weeks after bone marrow transfer. to block endothelial cxcr , wt mice were intravenously injected with cxcr antagonist sb (cayman, ann arbor, mi, usa) at a dose of mg/kg . h prior to i.c.v. lps injection [ ] . the mice in the control group were intravenously injected with % dmso . h prior to i.c.v. lps injection. sb was dissolved in dmso and diluted with . % saline, achieving a final concentration of % dmso. the data were analyzed using spss software ( . for windows, ibm inc., chicago, il, usa). data shown represent the means ± standard error of the mean (sem). statistical significance was determined using student's t tests for comparisons between two groups or by oneway anova with bonferroni correction for multiple groups of treatments. the differences were considered to be significant when p < . . to target the cxcl gene in the mouse genome, talen constructs that targeted the dna sequence of the murine cxcl gene were created as illustrated in fig. a . the founders (# , # , and # ) from the newborns were verified by t endonuclease i (fig. b) talen-induced mutations were deletions of variable lengths that induced frameshifts in the cxcl gene. bi-allelic mutations were observed in three mutant mice (fig. c ). cxcl −/− mice were healthy, fertile, displayed no overt phenotype, and had normal leukocyte and neutrophil counts in the peripheral blood and spleen (fig. d ). flow cytometry also revealed that neutrophils from cxcl −/− mice expressed cxcr at a similar level to wild-type mice (fig. e ). in addition, cxcl −/− mice showed normal tlr and cxcr mrna expression levels in the brain (fig. f) . in response to systemic or i.c.v. lps treatment, il- and tumor necrosis factor-α (tnf-α) in the brain and plasma were increased to a similar extent in both cxcl −/− and wild-type mice (fig. g ). intracerebroventricular injection of lps induces neutrophil recruitment and cxcl chemokine (cxcl , cxcl , and cxcl ) expression intracerebroventricular injection of lps strongly induced the expression of inflammatory cytokines and chemokines in the cns. as shown in fig. a -c, lps injection significantly induced the expression of cxcl chemokines, including cxcl , cxcl , and cxcl . the levels of both cxcl ( fig. a) and cxcl (fig. b ) gradually increased and peaked h after lps injection, then gradually decreased thereafter. the level of cxcl (fig. c ) peaked at h. among all of these cxcl chemokines, cxcl exhibited the highest expression level following lps injection. the peak concentration of cxcl was -fold less than that of cxcl . at h after i.c.v. lps injection, neutrophils began to migrate into the brain. infiltrating neutrophils, as detected by esterase-specific staining, were observed h posttreatment in the cortex (fig. d) ; this infiltration peaked at h in the cortex (fig. d ) and the hippocampus (fig. e) and decreased thereafter. deficiency in cxcr or cxcl affects neutrophil infiltration induced by the i.c.v. injection of lps to investigate the role of cxcr in neutrophil recruitment to the brain, we compared neutrophil infiltration in the wt and cxcr −/− mice after i.c.v. lps injection (fig. f ). cxcr −/− mice displayed significantly reduced neutrophil infiltration into the cerebral cortical (fig. g) , hippocampal regions (fig. h ) and choroid plexus (fig. i) . as cxcl was most highly expressed during lps-induced cns inflammation, we generated cxcl deficient mice using the talen knockout technique and compared neutrophil infiltration in these mice with that in the wt mice. interestingly, the cxcl −/− mice also exhibited a complete lack of neutrophil recruitment to the cortex, the hippocampus, and choroid plexus ( fig. g-i) . therefore, both cxcl and cxcr play essential roles in lps-induced neutrophil recruitment into the brain. deficiency in cxcr or cxcl affects leukocyte recruitment in brain vessels chemokines regulate immune cell trafficking by assisting the activation, adhesion, crawling, and transmigration of leukocytes across the cerebral endothelial barrier. to verify the role of cxcr in the neutrophil recruitment cascade in brain microvessels, we performed intravital microscopy to examine leukocyte recruitment in brain vessels during lps-induced cns inflammation. as expected, i.c.v. lps injection caused significant rolling and adhesion of leukocytes in post-capillary venules in brains of wt mice (fig. a) . interestingly, leukocyteendothelial cell interactions appeared to be reduced in the cxcr −/− (fig. b ) and cxcl −/− mice (fig. c) . rolling (fig. d ) and adherent cells (fig. e) were almost completely absent in the cxcr −/− and cxcl −/− mice, indicating that both cxcl and cxcr are essential for the leukocyte recruitment cascade in cerebral microvessels in the lps-induced neuroinflammation. to investigate the molecular mechanisms underlying the effects of cxcr and cxcl deficiency on leukocyteendothelial cell interactions, the expression of adhesion molecules in cxcr −/− and cxcl −/− mice was assessed via real-time pcr h after i.c.v. lps injection. interestingly, the mrna expression levels of p-selectin (fig. a ), e-selectin (fig. b) , vcam- (fig. c) , and icam- (see figure on previous page.) fig. generation of talen-mediated cxcl knockout mice. a dna-binding sequences are presented in red or green, and the spacer region for cxcl -talen where a double-strand break will occur is underlined. b t endonuclease i (t ei) assays were conducted using genomic dna from the founder mice. the arrow shows the size ( bp) of t ei-digested dna fragments. # , # , and # are the mutant founder (f ) mice generated by injection with cxcl -talen mrna. c dna sequences of the cxcl locus from live founder mice identified by t e assays in b. "-" shows the deleted nucleotides. d peripheral blood mononuclear cells and splenocytes were collected from wild-type or cxcl −/− mice. numbers of cd + and gr. + cells were quantified via flow cytometry. e peripheral blood mononuclear cells were collected from wild-type or cxcl −/− mice, cxcr expression in neutrophils from wt and cxcr −/− mice were analyzed via flow cytometry. f mrna expression levels of tlr and cxcr in the brain of wt or cxcl −/− mice were analyzed by rt-pcr. g wild-type and cxcl −/− mice were treated with i.p. or i.c.v. lps injection. four hours later, levels of tnf-α and il- in the brain tissue or plasma were determined by elisa. data are expressed as the mean ± sem, n = mice in all of the groups d) were significantly reduced in cxcr −/− and cxcl −/− mice. additionally, the protein expression levels of these adhesion molecules were assessed by western blotting (fig. a) . consistent with the realtime pcr results, both cxcr deficiency and cxcl deficiency dramatically reduced the protein expression of p-selectin (fig. b ) and vcam- (fig. c ) in the brain. a significant reduction in the levels of icam- ( fig. d ) was also observed in the cxcr −/− mice, albeit to a lesser extent. taken together, these results suggest that both cxcr and cxcl are critical effectors that mediate the expression of adhesion molecules on cerebral endothelial cells during cns inflammation. it is well established that cns inflammation induces permeability changes to the blood-brain barrier. the i.c.v. injection of lps induced a significant change in brain albumin concentration and h post-treatment in both wt and cxcr −/− mice. however, no significant difference in albumin concentrations between the wt and cxcr −/− mice was observed , , and h after i.c.v. lps injection (fig. e) . therefore, it is likely that rather than affecting the integrity of blood-brain barrier, cxcr deficiency affected neutrophil recruitment by attenuating endothelial activation. to identify the source of functional cxcr that mediates leukocyte recruitment, we generated chimeric mice by transferring bone marrow cells between wt and (see figure on previous page.) fig. chemokine levels and the effect of cxcr or cxcl deficiency on neutrophil recruitment to the brain parenchyma after i.c.v. lps injection. wild-type mice were i.c.v. injected with lps. cxcl (a), cxcl (b), and cxcl (c) concentrations in the brains of wt (c bl/ j) mice were determined via elisa at various time points after i.c.v. lps injection. wt mice i.c.v. injected with saline for h served as negative controls. infiltrating neutrophils in the cortex (d) and hippocampus (e) were quantified via esterase staining of the brain sections , , , or h after i.c.v. lps or saline injection. (f) the wt, cxcr −/− , and cxcl mice were i.c.v. injected with lps h before the quantification of infiltrating neutrophils. representative photomicrographs of brain sections stained for esterase positive neutrophils (arrows) from wild-type, (g-i) cxcr −/− and cxcl mice. (fig. a) . intravital microscopy was performed on all chimeric mice h after i.c.v. lps injection. cxcr −/− mice that were reconstituted using wt bone marrow cells exhibited reduced leukocyte rolling and adhesion. by contrast, the chimeric mice generated by reconstituting the wt mice using cxcr −/− bone marrow cells exhibited normal leukocyte recruitment to the cerebral microvessels (fig. b) . consistent with our observations from intravital microscopy, the cxcr −/− mice reconstituted with wt bone marrow cells displayed significantly reduced levels of p-selectin and vcam- expression, whereas the levels of p-selectin and vcam- expression in the wt mice reconstituted using cxcr −/− bone marrow cells were almost similar to that of wt mice (fig. c) . the reductions in endothelial activation and subsequent leukocyte-endothelial cell interactions in cns microvessels resulted from a lack of cxcr expression on cns-residing cells, but not on circulating neutrophils. therefore, the functional cxcr that mediates endothelial activation is likely localized to radiation-resistant non-hematopoietic cells, such as endothelial or glial cells. lps i.c.v. injection induced significant levels of cxcr mrna (fig. a) and protein (fig. b) . upon tnf-α stimulation, primary endothelial cells, compared with glial cells, exhibited much higher expression of cxcr mrna. no significant change in cxcr transcription was noted in primary microglia or astrocytes (fig. c ). following stimulation with tnf-α or lps, the cerebral endothelial cells, compared with astrocytes and microglia, also expressed much higher level of cxcr protein (fig. d ). astrocytes secreted much higher levels of cxcl than microglia in response to tnf-α and lps (fig. e) . astrocyte culture conditioned medium stimulated strong expression of vcam- and icam- in wt cerebral endothelial cells. reduced expression of these molecules was observed in cxcr −/− endothelial cells (fig. f) . these results suggest that astrocyte-derived cxcl and to validate the critical role of endothelial cxcr in cerebral endothelial activation, we intravenously infused the cxcr antagonist sb at a dose of mg/kg . h prior to i.c.v. lps injection to block cxcr signaling [ ] from the luminal surface of the cerebral microvessels. as detected by western blotting, sb treatment decreased the levels of vcam- and e-selectin expression but not that of p-selectin (fig. a ). in addition, intravital microscopy also revealed that the injection of sb significantly decreased leukocyte rolling and adhesion in brain microvessels (fig. b) . these results further indicate that endothelial cxcr plays a critical role in endothelial activation and subsequent leukocyte recruitment. leukocyte recruitment is a hallmark of various cns inflammatory diseases. the chemokine receptor cxcr and its ligands cxcl , cxcl , and cxcl play crucial roles in the trafficking of neutrophils. in the current study, we showed that lps injection into the brain significantly induced the production of cxcl , cxcl , and cxcl . cxcl , the most potent neutrophilchemoattracting cxcr ligand, was upregulated in the cns at the earliest time point and was correspondingly expressed at the highest level. the i.c.v. injection of lps has been widely applied as an animal model for the study of brain inflammation. the dosage of μg of lps fig. effects of cxcr or cxcl deficiency on the expression of p-selectin, e-selectin, and adhesion molecules in vivo and on bbb permeability. a the protein expression of p-selectin, e-selectin, vcam- , and icam- ( h after i.c.v. saline injection) in the saline-treated control group of wt mice and lps-treated ( h after i.c.v. lps injection) wt, cxcr −/− , and cxcl −/− mice was determined via western blot analysis. effects of cxcr or cxcl deficiency on p-selectin (b), vcam- (c), and icam- (d) expression in the brain. e western blotting analysis of the albumin levels in the brains of wt and cxcr −/− mice , , and h after the intraventricular injection of lps was performed. optical densities were determined using a computer imaging analysis system. n = mice per group. **p < . ; ***p < . is considerably above what is observed in most infections and results in robust neutrophil recruitment. however, a significant reduction in the number of infiltrating neutrophils was observed in the brain parenchyma of cxcr −/− and cxcl −/− mice after i.c.v. lps injection. therefore, cxcl acts as the principal mediator of neutrophil recruitment during lps-induced cns inflammation. the leukocyte recruitment cascade in brain vessels is directed by the complex interactions between adhesion molecules and their receptors [ , ] . intravital microscopy revealed that a deficiency in either cxcr or cxcl significantly reduced leukocyte-endothelial cell interactions in brain vessels. additionally, reduced expression of p-selectin, vcam- , and icam- was observed in the brain of cxcr −/− mice. therefore, it is likely that the functional cxcr that mediates leukocyte recruitment is located on the cns endothelium. our previous study reported that tnf-α in the lps-treated brain activated the endothelium to cause an increase in adhesion molecule expression and leukocyte recruitment [ ] . in response to i.c.v. lps injection, cxcr deficiency did not reduce tnf-α levels in the brain. clearly, a deficiency in cxcr or cxcl directly affected cerebral endothelial activation, but not microglial activation. in addition to its chemotactic properties, cxcl also exerts direct effects on bbb permeability. the exposure of brain microvascular endothelial cells to cxcl in vitro altered endothelial permeability and facilitated transendothelial monocyte migration [ ] . however, in our study, cxcr deficiency did not affect albumin leakage across the bbb. therefore, the reduction in neutrophil infiltration was not due to a change in the fig. leukocyte recruitment and the expression of p-selectin and vcam- in the wt, cxcr −/− , and chimeric mice. a chimeric mice were generated by transferring bone marrow cells between wt and cxcr −/− mice. b intravital microscopy was performed on wt, cxcr −/− , and chimeric mice, h after i.c.v. lps injection. the number of rolling and adherent leukocytes is presented as the mean ± sem. c the expression of p-selectin and vcam- in the wt, cxcr −/− , and chimeric mice was compared by western blotting, n = mice per group. data are presented as the means ± sem, *p < . ; **p < . integrity of bbb but to a lack of cerebral endothelial activation resulting from cxcl or cxcr deficiency. among the chimeric mice generated by transferring bone marrow precursors between cxcr −/− and wt mice, wt mice reconstituted using cxcr −/− bone marrow cells exhibited normal cell recruitment to the brain vessels. interestingly, functional cxcr is not expressed on circulating leukocytes from the bone marrow. therefore, the activation of cxcr on leukocytes is not required for their recruitment in cerebral blood vessels. earlier studies have identified the expression of cxcr on many types of cns-residing cells [ ] [ ] [ ] . fig. astrocyte-derived cxcl and endothelial cxcr are essential for cerebral endothelial activation. a i.c.v. lps injection ( h) induced significant cxcr mrna expression in wt mice. b levels of cxcr protein after i.c.v. lps injection from to h gradually increased compared with the control group ( h after i.c.v. saline injection). c the expression of cxcr mrna in primary brain microvascular endothelial cells, microglia, and astrocytes stimulated with either vehicle or tnf-α ( ng/ml) was measured via real-time pcr. the results are represented as the means ± sem of three independent experiments; *p < . . d primary endothelial cells, astrocytes, and microglia were seeded at × cells/well in six-well plates and were incubated overnight. the following day, the cells were stimulated with ng/ml lps or ng/ml tnf-α for h. cell lysates were collected and analyzed for cxcr expression via western blotting. e primary astrocytes and microglia from wild-type mice were seeded at × cells/well in six-well plates and were incubated overnight. the following day, the cells were stimulated with ng/ml lps or ng/ml tnf-α for h. then, the conditioned supernatants and cell lysates were collected and analyzed for cxcl expression via elisa. the results are represented as the means ± sem of three independent experiments; **p < . . f astrocyte culture conditioned medium was added into primary cerebral endothelial cells from wild-type or cxcr −/− mice, and the levels of vcam- and icam- were measured via western blotting this finding demonstrated that the expression of cxcr on cns-residing cells, including endothelial cells, astrocytes, and microglia, is more important than its expression on circulating cells during cns inflammation. moreover, tnf-α robustly induced a high expression of cxcr mrna in primary murine endothelial cells, but not in primary microglia or astrocytes. high levels of expression of the cxcr mrna and protein were detected in wild-type cerebral endothelial cells, which strongly indicates that endothelial cxcr is a key player mediating cerebral endothelial activation. to further validate the role of endothelial cxcr , we intravenously injected sb to block the function of cxcr in brain endothelial cells, as sb in the blood circulation can easily access the brain endothelium. compared with mice treated with lps alone, both cerebral endothelial activation and leukocyte recruitment in the cerebral vessels were reduced in the mice treated with both sb and lps. taken together, these data indicate that sb potently inhibited cxcr function on brain endothelial cells, thereby blocked leukocyte recruitment. astrocytes, which are more abundant than microglia in the brain [ ] , released much higher levels of cxcl than microglia in response to stimulation with lps or tnf-α. our study confirmed that astrocytes released significantly higher levels of cxcl than microglia in response to stimulation with lps or tnf-α, suggesting that the main source of cxcl may be astrocytes. cxcl deficiency reduced leukocyte recruitment and endothelial activation by over % in vivo. astrocytes are essential structural components of the bbb [ , ] ; among all cell types in the brain, they have the easiest access to the brain endothelium and can release cxcl , which possibly accumulate in the perivascular space at an extremely high concentration to activate cerebral endothelial cells. therefore, the cxcl secreted from astrocytes and cxcr expressed on the endothelium may cooperate in contributing to cerebral endothelial activation and the subsequent leukocyte recruitment cascade during cns inflammation. endothelial activation is a critical step in the process of leukocyte recruitment during cns inflammation. in the current study, we found that either cxcr or cxcl deficiency resulted in reduced neutrophil infiltration and leukocyte-endothelial cell interactions in the brain. a dramatic reduction in the endothelial expression of adhesion molecules was also noted in these mice. our results demonstrate that cxcl , an important factor secreted by astrocytes, also plays a critical role in leukocyte recruitment to the cns by cooperating with cxcr expressed on cerebral endothelial cells. the cxcl -cxcr axis may represent another potential therapeutic target for the treatment of cns inflammatory diseases. abbreviations bbb: blood-brain barrier; cns: central nervous system; cxcl: chemokine (cxc motif) ligand; cxcr : cxc chemokine receptor ; elisa: enzyme-linked immunosorbent assay; i.c.v.: intracerebroventricular; il: interleukin; lps: lipopolysaccharide; pcr: polymerase chain reaction; tnf-α: tumor necrosis factor-α; wt: wild-type; β -mg: β -macroglobulin. the authors declare that they have no competing interests. authors' contributions hz designed the experiments, supervised the project, and drafted the manuscript. fw performed most of the experiments and participated in the study design. yz and xz performed the flow cytometry, elisa, and real-time pcr. tj performed real-time pcr. ds and mz contributed to the experimental design and data analysis. ms was involved in the study design and helped to draft the manuscript. all authors read and approved the final manuscript. fig. the effect of cxcr antagonist infusion on leukocyte rolling and adhesion in cns vessels. wt mice received an intravenous injection of the cxcr antagonist sb ( mg/kg) . h prior to i.c.v. lps injection. four hours after i.c.v. lps injection, the protein expression of pselectin, vcam- , and e-selectin in the brain was determined by western blot analysis (a). intravital microscopy was performed on the mice. the results of leukocyte recruitment (b) are presented as the mean ± sem. n = mice for all groups. *p < . ; **p < . leukocyte and bacterial interrelationships in experimental meningitis inflammatory mechanisms in ischemic stroke: role of inflammatory cells how do immune cells overcome the blood-brain barrier in multiple sclerosis? neutralization of macrophage inflammatory protein (mip- ) and mip- alpha attenuates neutrophil recruitment in the central nervous system during experimental bacterial meningitis chemokines: sirens of neutrophil recruitment but is it just one song? recruitment of neutrophils across the blood-brain barrier: the role of e-and p-selectins getting to the site of inflammation: the leukocyte adhesion cascade updated the physiology of leukocyte recruitment: an in vivo perspective lack of ccr results in increased mortality and impaired leukocyte activation and trafficking following infection of the central nervous system with a neurotropic coronavirus c-c chemokine receptor -regulated entry of th- cells into the cns through the choroid plexus is required for the initiation of eae the cxc chemokine receptor , cxcr , is the putative receptor for elr + cxc chemokine-induced angiogenic activity the chemokine receptors cxcr and cxcr couple to distinct g proteincoupled receptor kinases to mediate and regulate leukocyte functions cxcl regulates pulmonary host defense to klebsiella infection via cxcl , cxcl , nf-kappab, and mapks the chemokine receptor cxcr ligand kc (cxcl ) mediates neutrophil recruitment and is critical for development of experimental lyme arthritis and carditis neutrophil recruitment in immunized mice depends on mip- inducing the sequential release of mip- alpha, tnf-alpha and ltb( ) role for macrophage inflammatory protein (mip- ), mip- alpha, and interleukin- alpha in the delayed-type hypersensitivity response to viral antigen on the mechanism and significance of ligand-induced internalization of human neutrophil chemokine receptors cxcr and cxcr human t lymphocytes and mast cells differentially express and regulate extra-and intracellular cxcr and cxcr effects of epithelial and neutrophil cxcr on innate immunity and resistance to kidney infection angiogenic effects of interleukin (cxcl ) in human intestinal microvascular endothelial cells are mediated by cxcr critical role of endothelial cxcr in lps-induced neutrophil migration into the lung oxldl upregulates cxcr expression in monocytes via scavenger receptors and activation of p mitogen-activated protein kinase induction of functional il- receptors by il- and il- in human monocytes cxcr and cxcr antagonistically regulate neutrophil trafficking from murine bone marrow defective regulation of cxcr facilitates neutrophil release from bone marrow causing spontaneous inflammation in severely nf-kappa b-deficient mice decreased cxcr and cxcr expression on neutrophils in anti-neutrophil cytoplasmic autoantibody-associated vasculitides potentially increases neutrophil adhesion and impairs migration cxcr and cxcl regulate the il- /g-csf axis and neutrophil homeostasis in mice pulmonary cxcr regulates vcam- and antigen-induced recruitment of mast cell progenitors a requirement for microglial tlr in leukocyte recruitment into brain in response to lipopolysaccharide myd is required for mounting a robust host immune response to streptococcus pneumoniae in the cns microglia repetitively isolated from in vitro mixed glial cultures retain their initial phenotype isolation and culture of mouse cortical astrocytes a simple method for isolation and characterization of mouse brain microvascular endothelial cells macrophage and t cell dynamics during the development and disintegration of mycobacterial granulomas cxcl and its receptor, cxcr , mediate murine sickle cell vaso-occlusion during hemolytic transfusion reactions three or more routes for leukocyte migration into the central nervous system vascular inflammation in central nervous system diseases: adhesion receptors controlling leukocyte-endothelial interactions cxcl contributes to β-amyloid-induced transendothelial migration of monocytes in alzheimer's disease glioblastoma cell-secreted interleukin- induces brain endothelial cell permeability via cxcr expression of cxcl , cxcr , and cxcr in neurons and glial cells of the human and rabbit retina role for cxcr and cxcl on glia in multiple sclerosis microglia and astrocytes in the adult rat brain: comparative immunocytochemical analysis demonstrates the efficacy of lipocortin immunoreactivity astrocyte-endothelial interactions at the blood-brain barrier submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution this work was supported by the national natural science foundation of china (grant nos. and ) and the natural science foundation of jiangsu province (grant no. bk ). key: cord- -ygb yxc authors: williams, alexander t.; muller, cynthia r.; govender, krianthan; navati, mahantesh s.; friedman, adam j.; friedman, joel m.; cabrales, pedro title: control of systemic inflammation throughearly nitric oxide supplementation with nitric oxide releasing nanoparticles date: - - journal: free radic biol med doi: . /j.freeradbiomed. . . sha: doc_id: cord_uid: ygb yxc amelioration of immune overactivity during sepsis is key to restoring hemodynamics, microvascular blood flow, and tissue oxygenation, and in preventing multi-organ dysfunction syndrome. the systemic inflammatory response syndrome that results from sepsis ultimately leads to degradation of the endothelial glycocalyx and subsequently increased vascular leakage. current fluid resuscitation techniques only transiently improve outcomes in sepsis, and can cause edema. nitric oxide (no) treatment for sepsis has shown promise in the past, but implementation is difficult due to the challenges associated with delivery and the transient nature of no. to address this, we tested the anti-inflammatory efficacy of sustained delivery of exogenous no using iv infused no releasing nanoparticles (no-np). the impact of no-np on microhemodynamics and immune response in a lipopolysaccharide (lps) induced endotoxemia mouse model was evaluated. no-np treatment significantly attenuated the pro-inflammatory response by promoting m macrophage repolarization, which reduced the presence of pro-inflammatory cytokines in the serum and slowed vascular extravasation. combined, this resulted in significantly improved microvascular blood flow and -hour survival of animals treated with no-np. the results from this study suggest that sustained supplementation of endogenous no ameliorates and may prevent the morbidities of acute systemic inflammatory conditions. given that endothelial dysfunction is a common denominator in many acute inflammatory conditions, it is likely that no enhancement strategies may be useful for the treatment of sepsis and other acute inflammatory insults that trigger severe systemic pro-inflammatory responses and often result in a cytokine storm, as seen in covid- . sepsis represents a dynamic progression of host-pathogen interactions that progressively causes a systemic inflammatory response syndrome (sirs) and ultimately leads to multi- organ dysfunction syndrome (mods) even after the initial insult has been controlled. the complications associated with sepsis are the most common cause of death in non-coronary intensive care units (icus) worldwide. medical care costs related to sepsis treatment add up to approximately $ billion in the united states. , however, the development of a comprehensive study to target key aspects of sepsis development and progression has been challenging and most of the results obtained from bench top experiments are hard to translate to the bedside. the difficulty to generate translatable data in experimental studies comes mainly from two reasons. first, the large number of variables that play a role in the deterioration of cell and tissue function during sirs and sepsis makes it almost impossible to pinpoint a single therapeutic target. , second, the complexity and diverse sources of human sepsis makes the development of an animal model that is reproducibly translatable, in which different theories could be tested, almost impossible. thus, a middle ground should be set in which individual components of sepsis can be used to understand the response of the insulted organism. a well-described hallmark of sepsis is endothelial dysfunction in response to a cytokine 'storm', which is associated with an increase in a series of negative consequences arising from overproduction of reactive oxygen species (ros), disruption of the glycocalyx, and endothelial nitric oxide synthase (enos) uncoupling, all contributing to increased adhesion of red blood cells (rbcs), white blood cells (wbcs), and platelets to the endothelium lining, enhanced platelet activation, blood stagnation, decreased tissue perfusion and increased vascular permeability. , , experimental therapies designed to target this aspect have been promising and represent pathways that could be targeted to increase survival. , from a clinical standpoint, several studies have highlighted that microvascular function is drastically impaired in patients in different stages of sepsis. the evidence indicates that the gold standard, fluid replacement therapies, fail to recover microvascular blood flow causing poor perfusion, which has been associated with increased mortality. , routinely monitored clinical variables poorly reflect the true state of the microcirculation in central and peripheral tissues. recently it has been proposed that in order to properly restore cardiovascular homeostasis, the microcirculation should be targeted as a functional unit. intravital microscopy and microvascular measurements. the window chamber was studied using transillumination on a custom intravital microscope. briefly, the animals were restrained on a plexiglass tube with a longitudinal opening from which the window protruded and then they were fixed to the stage of an upright microscope (bx wi, olympus, new hyde park, ny) as described and depicted elsewhere. measurements were carried out using a ˣ (lumpfl-wir, numerical aperture . , olympus) water immersion objective. the microscope was equipped with a high-speed video camera (fastcam pci, photron usa), which was used to record videos of the microvascular blood flow at , frames per second (fps). briefly survival. in addition to measuring functional parameters, we also assessed survival of mice dosed with lps over hours (figure ) cd , indicating an m -like macrophage phenotype, which is considered to be inflammatory. in fact, over % of macrophages tested via facs presented an m -like phenotype for animals treated with control-np, but only % of macrophages represented an m -like phenotype for animals treated with no-np. however, animals treated with no-np showed a larger population of m -like macrophages (representing % of macrophages harvested), which are typically considered to be anti-inflammatory, and are associated with tissue repair. to supplement these macrophage phenotypes, we also measured the cytokine profile of treated mice after lps injection. cytokine profile. cytokines were measured and hours after lps injection (figure ) . after hours, animals treated with no-np showed significantly higher levels of cytokines traditionally considered to be anti-inflammatory (interleukin [il]- , tgf-beta), and significantly lower levels of cytokines associated with a proinflammatory response (il- , , , mcp, and tnf alpha) than animals treated with control-np. however, after hours, all cytokines measured were elevated in animals dosed with control-np compared to no-np, but not all of these comparisons were statistically significant. the principle finding of this study is that sustained delivery of exogenous no using no releasing nanoparticles improved microvascular flow and capillary transit compared to animals treated with control nanoparticles during lps-induced endotoxemia. additionally, this study demonstrates that the adverse microcirculatory changes from lps-induced endotoxemia there are a number of mechanisms that may be responsible for the improved survival and maintenance of microvascular perfusion seen with no-np treatment in this study, but the exact mechanism or set of mechanisms is unclear. as we saw, and as others have demonstrated, no treatment decreased m , and increased m polarization of macrophages. this can have a multitude of downstream effects as m macrophages produce proinflammatory cytokines, upregulate inducible nitric oxide synthase (inos), and enhance production of ros and reactive nitrogen species, which disrupt the glycocalyx, expose the endothelial layer, decrease no production from enos, and destroy endothelial cells. , disruption of the glycocalyx undermines vascular integrity and eliminates the shear stress- based trigger for production of no from enos. mice treated with control-np in our study experienced significantly increased vascular permeability, as shown in figure , suggesting endothelial cell and glycocalyx disruption in these animals. the glycocalyx has a number of vital physiological roles, including acting as a barrier to preserve intravascular oncotic pressure, promoting rbc marginalization and the presence of a red cell-free layer, , reducing leukocyte adhesion and infiltration, and preventing thrombus formation and attachment, so its disruption is likely a primary determinant for many of the clinical presentations of sepsis, such as edema. as such, it is logical that the glycocalyx has been a common drug target in improving outcomes from sepsis. others have explored individual mechanisms as a method to prevent glycocalyx degradation during sepsis, but these j o u r n a l p r e -p r o o f strategies have not shown much clinical success. however, no-np treatment seems particularly promising as it attempts to treat a more upstream target than previous therapies by preventing the initial insult to the glycocalyx, and thus preserving endogenous no signaling. endogenous no may function to upregulate and activate nuclear factor erythroid -related factor (nfr ) which represents a potent signaling pathway to counter an lps-induced inflammatory cascade, and to down regulate pro-inflammatory pathways and thus decrease ros production. limitations: lps induced endotoxemia does not fully replicate the cascade of events that occurs during septic shock, but no study can fully replicate the morbidity of septic shock due to its distinct etiologies. however, this study did demonstrate that this mouse model replicates many of the early changes that occur in septic shock, including increased vascular permeability, and impaired systemic oxygen transport, and allowed us to observe these changes in an awake, unanesthetized model. since anesthetics have a poorly characterized influence on inflammation, the awake unanesthetized state is most representative of changes that may occur during sepsis. unfortunately, this study did not measure any parameters examining the status of the vascular endothelial glycocalyx, since sepsis damages the glycocalyx, and exogenous no could help protect the glycocalyx. future studies should examine changes in the glycocalyx by measuring its breakdown molecules, such as heparan sulfate and sydecan- , in the plasma, or measure glycocalyx integrity and thickness via fluorescent lectin binding, in order to directly study glycocalyx disruption during lps-induced endotoxemia, and its protection by no-nps. conclusion pathogenetic mechanisms of septic shock epidemiology of severe sepsis in the united states: analysis of incidence, outcome, and associated costs of care epidemiology of severe sepsis severe sepsis and septic shock sepsis and endothelial permeability novel therapies for microvascular permeability in sepsis the microcirculation is the motor of sepsis severe abnormalities in microvascular perfused vessel density are associated to organ dysfunctions and mortality and can be predicted by hyperlactatemia and norepinephrine requirements in septic shock patients microvascular resuscitation as a therapeutic goal in severe sepsis early difference in tissue ph and microvascular hemodynamics in hemorrhagic shock resuscitation using polyethylene glycol-albumin-and hydroxyethyl starch-based plasma expanders b. & intaglietta, m. microcirculation: its significance in clinical and molecular microcirculation: physiology, pathophysiology, and clinical application clinical review: clinical imaging of the sublingual microcirculation in the critically ill -where do we stand? lps/tlr signal transduction pathway animal models of sepsis: setting the stage sustained release nitric oxide releasing nanoparticles: characterization of a novel delivery platform based on nitrite containing hydrogel/glass composites a nanoparticle delivery vehicle for s-nitroso-n-acetyl cysteine: sustained vascular response sustained release nitric oxide from long-lived circulating nanoparticles. free radical biology & medicine technical report-a new chamber technique for microvascular studies in unanesthetized hamsters a. & leunig, m. dorsal skinfold chamber preparation in mice: studying microhemodynamic parameters quantification from intravital microscopy videos macrophage polarization mediates anti-inflammatory effects of endothelial nitric oxide signaling glycocalyx and sepsis-induced alterations in vascular permeability the m and m paradigm of macrophage activation: time for reassessment implications enzymatic degradation of the endothelial glycocalyx on the microvascular hemodynamics and the arteriolar red cell free layer of the rat cremaster muscle modulates immobilization of leukocytes at the endothelial surface the glycocalyx: a novel diagnostic and therapeutic target in sepsis activation of endothelial nitric oxide synthase by dietary isoflavones: role of no in nrf -mediated antioxidant gene expression the coagulopathy of acute sepsis the role of microvascular thrombosis in sepsis endogenous and exogenous nitric oxide protect against intracoronary thrombosis and reocclusion after thrombolysis nitric oxide regulates exocytosis by s-nitrosylation of n- ethylmaleimide-sensitive factor nitric oxide and its relationship to thrombotic disorders exposure of fibrinogen and thrombin to nitric oxide donor prolinonoate affects fibrin clot properties the role of the microcirculation in multiple organ dysfunction syndrome (mods): a review and perspective nitroglycerin in septic shock after intravascular volume resuscitation nitrite protects against morbidity and mortality associated with tnf-or lps-induced shock in a soluble guanylate cyclase-dependent manner reducing ischemia/reperfusion injury by the targeted delivery of nitric oxide from magnetic-field-induced localization of s-nitrosothiol-coated paramagnetic nanoparticles covid- : consider cytokine storm syndromes and immunosuppression interactions of coronaviruses with ace , angiotensin ii, and ras inhibitors-lessons from available evidence and insights into covid- cov- cell entry depends on ace and tmprss and is blocked by a clinically proven protease inhibitor antigenicity of the sars-cov- spike glycoprotein a crucial role of angiotensin converting enzyme (ace ) in sars coronavirus-induced lung injury ace -angiotensin-( - )-mas axis and oxidative stress in cardiovascular disease harnessing nitric oxide for preventing, limiting and treating the severe pulmonary consequences of covid- inflammatory response was reduced with nitric oxide releasing nanoparticles (no-nps) endotoxemia-induced microvascular deficits were alleviated by no-nps no-nps promoted m macrophage repolarization, and reduced m macrophage population no-nps could improve outcomes of insults that cause a cytokine storm key: cord- -dyk mr q authors: zheng, yong; deng, yan; gao, jian-mei; lv, chun; lang, ling-hu; shi, jing-shan; yu, chang-yin; gong, qi-hai title: icariside ii inhibits lipopolysaccharide-induced inflammation and amyloid production in rat astrocytes by regulating ikk/iκb/nf-κb/bace signaling pathway date: - - journal: acta pharmacol sin doi: . /s - - - sha: doc_id: cord_uid: dyk mr q β-amyloid (aβ) is one of the inducing factors of astrocytes activation and neuroinflammation, and it is also a crucial factor for the development of alzheimer’s disease (ad). icariside ii (ics ii) is an active component isolated from a traditional chinese herb epimedium, which has shown to attnuate lipopolysaccharide (lps)-induced neuroinflammation through regulation of nf-κb signaling pathway. in this study we investigated the effects of ics ii on lps-induced astrocytes activation and aβ accumulation. primary rat astrocytes were pretreated with ics ii ( , , and μm) or dexamethasone (dxms, μm) for h, thereafter, treated with lps for another h. we found that ics ii pretreatment dose dependently mitigated the levels of tumor necrosis factor-alpha (tnf-α), interleukin- beta (il- β), inducible nitric oxide synthase (inos), cyclooxygenase- (cox- ) in the astrocytes. moreover, ics ii not only exerted the inhibitory effect on lps-induced iκb-α degradation and nf-κb activation, but also decreased the levels of aβ( – ), aβ( – ), amyloid precursor protein (app) and beta secretase (bace ) in the astrocytes. interestingly, molecular docking revealed that ics ii might directly bind to bace . it is concluded that ics ii has potential value as a new therapeutic agent to treat neuroinflammation-related diseases, such as ad. alzheimer's disease (ad) is a progressive neurodegenerative disease characterized by beta-amyloid (aβ) peptide fibrils, which are extracellular depositions of a particular protein and are accompanied by extensive neuroinflammation [ ] [ ] [ ] . a number of studies have reported that inflammation, which may precede amyloid deposition, exerts vital effects in the pathogenesis of ad [ , ] . moreover, inflammatory mediators increase the expression of amyloid precursor protein (app) and the formation of aβ and upregulate beta-secretase activity [ ] . therefore, antiinflammatory drugs may prevent or treat ad by inhibiting neuroinflammation, thereby reducing the production or deposition of aβ [ ] [ ] [ ] [ ] . however, there are still no ideal antiinflammatory drugs to prevent or treat ad. astrocytes and microglia are important components of homeostasis in the brain [ ] . when the brain is exposed to undesirable environmental conditions, both astrocytes and microglia, which are crucial perpetrators of inflammation and potential neuronal dysfunction [ , ] , acquire special "response" or "activation" phenotypes [ , ] . in ad, the interaction between microglia and astrocytes may be of great significance for the development of neurodegenerative disease. in particular, astrocytes represent the most plentiful cell type in the brain and play an imperative role in maintaining the homeostasis of the central nervous system [ ] [ ] [ ] . under physiological conditions, astrocytes play a role in supporting and separating nerve cells [ ] [ ] [ ] [ ] . nevertheless, under neuroinflammatory conditions, the excessive activation of astrocytes is involved in the inflammatory response through its ability to release multiple molecules and further lead to neurodegenerative diseases [ ] [ ] [ ] . epimedium is a traditional chinese herb used for the treatment of cardiovascular diseases, osteoporosis, and sexual and neurological disorders [ ] . icariside ii (ics ii) is known as one of the major active pharmaceutical ingredients of epimedium, and it has been indicated to have an extensive range of pharmacological effects, including antiinflammatory [ , ] , anticancer [ , ] , antioxidative [ , ] , and antiaging activities [ ] . our previous study found that ics ii attenuates streptozotocin-or aβ - -induced cognitive deficits in rats by increasing the number of surviving neurons in the hippocampus [ ] or inhibiting neuronal apoptosis and reducing pde protein expression [ ] . in our previous in vivo studies, we proved that ics ii exerts beneficial effects on lipopolysaccharide (lps)-induced neuroinflammation by regulating the tlr /myd /nf-κb signaling pathway in rats and inhibiting lps-induced astrocyte overactivation [ ] . however, whether ics ii can suppress neuroinflammatory responses in vitro remains unclear. thus, in the current study, we investigated the effects of ics ii on lipopolysaccharide (lps)-induced neuroinflammation in primary astrocytes and the underlying molecular mechanism. ics ii (purity ≥ %) was obtained from nanjing zelang medical technology corporation ltd (nanjing, china). dmem/f was obtained from hyclone (logan, ut, usa), and fetal bovine serum (fbs) was purchased from gibco (thermo fisher scientific, ma, usa). dexamethasone (dxms) and lps (l ) (escherichia coli o :b ) were purchased from sigma-aldrich (st louis, mo, usa). dxms and lps were dissolved in normal saline at concentrations of mm and mg/ml, respectively, and ics ii was dissolved in dimethyl sulfoxide (dmso) at a concentration of mm. anticyclooxygenase- (cox- ) (#ab ), anti-nitric oxide synthase (inos) (#ab ), antinuclear factor-κb (nf-κb) (p ) (#ab ), anti-p-nf-κb (p ) (#ab ), anti-iκb-α (#ab ), anti-ikk-α (#ab ), anti-p-ikk-α (#ab ), anti-ikk-β (#ab ), anti-p-ikk-β (#ab ), and anti-β-site app cleaving enzyme (bace ) (#ab ) antibodies were purchased from abcam (cambridge, uk). anti-app (#ab b) was purchased from bbi life sciences corporation (shanghai, china). tumor necrosis factor-alpha (tnf-α) enzyme-linked immunosorbent assay (elisa) kits (an interleukin- beta (il- β) elisa kit, an aβ - elisa assay kit (jl ), and an aβ - elisa kit (jl )) were purchased from shanghai jianglai biotechnology (shanghai, china). a nitric oxide (no) detection kit (s ) was purchased from beyotime biotechnology (shanghai, china). astrocyte culture and drug treatment sprague-dawley rats ( ± g) were housed under a -h-light/ dark cycle at a humidity of % ± % and a temperature of °c. all animals were given free access to water and food. animal experiments were performed in compliance with the state committee of science and technology of the people's republic of china order no. of november , , and the protocol was approved by the experimental animal ethics committee of the zunyi medical university. primary astrocytes were extracted from the safe concentration range of ics ii within h (n = ). c the safe concentration range of ics ii within h (n = ). d the safe concentration range of ics ii within h (n = ). * p < . , ** p < . versus control group neonatal rat brains as previously described [ ] . primary astrocytes were isolated from the cerebral cortex of -h-old neonatal rats. briefly, suckling rats were repeatedly disinfected with % alcohol three times, and then the brains were removed, and the brain tissues were separated. then, the cerebral cortex was collected under low temperatures, and the meninges and blood vessels were slightly removed. the cerebral cortex was fully dissociated by the addition of ml of trypsin, the flask was treated with polylysine, and the suspension was spread in a culture flask at a cell density of × cells/ml. the differential adherence method was used to remove other types of cells in the tissue, and then astrocytes were cultured in dmem/f containing % fbs. the dmem/f was changed every days until the th day. the primary cells were shaken at °c at a constant temperature to remove other glial cells, such as microglia and oligodendrocytes. thereafter, the astrocytes were fixed with % paraformaldehyde, incubated with an anti-gfap antibody ( : , abcam), and visualized using an alexa fluor-conjugated secondary antibody ( : , abcam). more than % of the cultured astrocytes were identified by gfap staining, and the astrocytes were pretreated with different concentrations of ics ii or dxms ( μm) for h. thereafter, they were treated with lps for another h. since dxms is an effective and classical antiinflammatory drug, it was selected as a positive control drug to evaluate the antiinflammatory effect of ics ii, and μm was chosen as the desired concentration of dxms according to a previous study [ ] . cell viability was assessed using the -[ , -dimethylthiazol- -yl]- , -diphenyl tetrazolium bromide (mtt) assay as described in our previous study [ ] . in brief, astrocytes were seeded in a -well plate at × cells/well and treated as described above. then, μl of mtt ( mg/ml) was added to fbs-free medium and cultured for another h. the mtt was removed, and the cells were lysed with μl of dmso in each well. the optical density was measured at nm using a microplate reader. the results of the treatments were expressed as a percentage of the control. in brief, astrocytes were inoculated into -well plates at a density of × cells/well and pretreated with ics ii ( , , μm) for h. lps ( mg/ml) and ics ii were added to the plates for h. thereafter, the culture medium was collected and centrifuged for min at × g. the supernatants were then collected and used to measure tnf-α, il- β, aβ - , and aβ - levels using elisa kits. all data were obtained from at least three independent experiments. measurement of nitric oxide (no) astrocytes were inoculated in -well plates at a density of × cells/well and pretreated with ics ii ( , , μm) for h. lps ( mg/ml) and ics ii were added to the plates for h. the accumulation of nitrite in the supernatant was evaluated using the griess reaction. each μl of supernatant was reacted with an equal volume of griess reagent and cultured for min at room temperature. the absorbance was detected in a microplate absorbance reader at a wavelength of nm, and a series of known concentrations of sodium nitrite was utilized as a standard. western blot analysis western blot analysis was performed as described in our previous study [ ] . briefly, astrocytes were treated as mentioned above, homogenized with protein extraction solution, and lysed for min on ice. the lysate was centrifuged for min at × g. ) . then, the blots were subjected to the corresponding horseradish peroxidase-conjugated anti-rabbit or mouse immunoglobulin g. thereafter, immunoreactive proteins were measured using an enhanced chemiluminescence western blotting detection system. statistical analysis all data were analyzed by spss . statistics software and are expressed as the mean ± sd. the data were analyzed using oneway anova followed by the post hoc least significant difference. all data were obtained from at least three independent experiments. p < . was considered significant. we extracted the cultured cells and identified them by staining for gfap, which is a specific marker of astrocytes [ ] . the results showed that astrocytes made up % of the cultured cells (fig. a) . furthermore, the results showed that ics ii ( . , . , . , . , , or μm) for , , or h had no effect on the astrocytes; however, ics ii ( , , or μm) for , , or h was cytotoxic to the astrocytes. since the concentration of dmso used to dissolve ics ii in the experiment did not exceed . %, it was assumed that the concentration of dmso itself was not toxic to the cells and that the toxicity to the cells therefore resulted from the high concentration of ics ii. thus, a concentration of ics below μm was considered to be the safe concentration range (fig. b-d) . thereafter, , , and μm ics ii were used in subsequent experiments, and dxms ( μm) was used as a positive control agent. the effect of ics ii on the lps-induced production of tnf-α and il- β in astrocytes was determined using an elisa assay. the results showed that ics ii ( , , or μm) slightly mitigated the production of tnf-α (fig. a) and il- β (fig. b) . following lps stimulation, the production of tnf-α and il- β was substantially elevated compared with that in the control group, while ics ii markedly alleviated lps-induced tnf-α and il- β overproduction in a concentration-dependent manner. the effect of ics ii on lps-induced no production and inos and cox- expression in astrocytes was determined using the griess reaction and western blot analysis, respectively. the results showed that ics ii decreased lps-induced no production in astrocytes (fig. c) . moreover, inos and cox- expression levels were dramatically increased after lps insult. however, ics ii ( , , and μm) induced a concentration-dependent decrease in the expression of inos and cox- in astrocytes compared with that in the lps group ( fig. d-f ). the results suggested that ics ii not only suppressed the lpsinduced phosphorylation of p (fig. a, b) but also prevented the nuclear translocation of p (fig. c-f ). furthermore, ics ii also suppressed the lps-induced degradation of iκb-α (fig. a, b) , ikk-α, and ikk-β (fig. c, d) . these results indicate that ics ii mitigates the lps-induced activation of nf-κb via inhibiting iκb-α phosphorylation and the translocation of p to the nucleus. the effect of inflammation on amyloid formation in vitro was also investigated because neuroinflammation can cause amyloid production, whereas the aberrant activation of astrocytes is a major source of neuroinflammation. astrocytes provide both mechanical and metabolic support to neurons, regulating the environment in which they function. to determine the relationship between neuroinflammation and amyloidogenesis, we investigated whether the antiinflammatory effect of ics ii can result in antiamyloidogenesis. as shown in fig. a , b, when the cells were unstimulated, they expressed low protein levels of app and bace , whereas the protein expression of app and bace increased in response to lps after h. in addition, ics ii also decreased lps-induced aβ - and aβ - secretion into the culture media of astrocytes (fig. c, d) . in astrocytes, we also found that ics ii inhibited the lps-induced expression of app and bace , as well as aβ - and aβ - levels in a concentration-dependent manner. these results further indicate that the amyloidogenic pathway can be promoted by neuroinflammatory stimulation and that the antiinflammatory effect of ics ii can result in antiamyloidogenesis. interestingly, mock molecular docking was used to verify whether ics ii binds to bace protein, and the results showed that the binding energy of ics ii and bace was − . kcal/mol, which confirmed that ics ii can bind to bace , as the standard threshold for a molecule to bind to a protein is thought to be greater than or equal to − kcal/mol (fig. a, b) . we further investigated the supposed binding modes and interactions within the amino acid pocket, including gly , gly , gly , thr , tyr , gly , lys , asp , and phe (fig. c, d) . taken together, these findings show that ics ii may directly affect bace and thus exert neuroprotective effects. the present study revealed that ( ) ics ii protects against lpsinduced inflammation in primary-cultured astrocytes; ( ) the inhibitory effect of ics ii is due to regulation of the ikk/iκb/nf-κb signaling pathway; and ( ) ics ii decreases aβ - and aβ - levels by downregulating app and bace expression (fig. ) . lps is an effective component of gram-negative bacilli endotoxin, which can cause a series of inflammatory reactions in the body and is widely applied to establish animal or cellular inflammatory models. moreover, astrocytes represent the most abundant cell type in the central nervous system, and they exert a variety of physiological functions through close association with neurons and other brain structures. similar to microglia, the immune and inflammatory properties of astrocytes, which can promote the secretion of various neuroinflammatory factors, such as tnf-α and il- β, are activated by lps. accumulating evidence has demonstrated that the production of multiple neuroinflammatory factors, including tnf-α, il- β, and inos, can activate the nf-κb pathway, resulting in neuroinflammation [ ] . moreover, the excessive production of neuroinflammatory factors leads to neuronal damage through the activation of glial cells. notably, astrocytes play a key role in central nervous system inflammation by producing cytokines, such as tnf-α, il- β, and no, leading to neuronal injury [ ] . therefore, the suppression of astrocyte activation may be an effective treatment for neuroinflammation-related diseases [ ] . our findings showed that lps induces an increase in proinflammatory factors, which is consistent with the results of previous studies [ , ] . ics ii significantly inhibited the lps-induced accumulation of tnf-α, il- β, no, inos, and cox- in primary culture astrocytes. in addition, these effects were not due to the cytotoxic effect of ics ii, as evidenced by the observation that there was no effect on astrocytes at concentrations up to μm. moreover, ics ii also inhibited inos and cox- protein notably, nf-κb not only regulates a variety of inflammatory factors, such as il- β and tnf-α but also plays a key role in mediating cox- and inos expression [ ] . nf-κb (p /p ) heterodimers exist in the cytoplasm of resting cells. however, under stimulation by lps, the phosphorylation of ikk was induced, which subsequently phosphorylates the iκb protein, resulting in the release of nf-κb (p /p ) heterodimers. then, nf-κb-p translocates from the cytoplasm to the nucleus, thereby promoting the release of proinflammatory factors, including tnf-α and il- [ , ] . consistent with these studies, the present study showed that lps induces the phosphorylation of nf-κb-p , iκb, and ikk. however, ics ii abolished these changes, suggesting that ics ii exerts potent antiinflammatory effects, at least partly through modulating the ikk/iκb/nf-κb pathway. app is a membrane-intrinsic protein expressed in a variety of tissues and is closely related to ad. app can be cleaved by α, β, and γ proteases, and the continuous action of β-protease and γprotease can cause app to be cleaved to produce aβ [ ] . however, aβ can cause the formation of senile plaques in the brain and the apoptosis of neuronal cells, thereby causing ad. most importantly, mounting evidence has shown that neuroinflammation is related to the accumulation of aβ in the brains of ad patients [ , ] . in addition, app is first cleaved by β-secretase at its β-cleavage site and is then proteolyzed by the second enzyme, γ-secretase, to produce aβ - and aβ - . in particular, the aβ - and aβ - peptides are involved in the amyloidogenic pathway; aβ - is the most plentiful species, and aβ - shows the strongest neurotoxicity [ , ] . of note, bace is an important rate-limiting enzyme in the app-aβ pathway and plays an important role in the production of aβ. bace was overexpressed in the brains of animal models of ad [ ] . in contrast, in the brains of bace knockout mice, the levels of aβ and sappβ are decreased [ ] . in addition, activated astrocytes that release mediators can induce app and bace expression, 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in appswe/ps de mice by targeting multiple pathogenic mechanisms in vivo beta-secretase inhibition leads to brain abeta lowering and increased alpha-secretase processing of amyloid precursor protein without effect on neuregulin- inflammation in alzheimer disease: driving force, bystander or beneficial response large-scale production of soluble recombinant amyloid-beta peptide - using cold-inducible expression system regional and sub-regional differences in hippocampal gabaergic neuronal vulnerability in the tgcrnd mouse model of alzheimer's disease chitosan oligosaccharides inhibit/ disaggregate fibrils and attenuate amyloid beta-mediated neurotoxicity gene structure and organization of the human beta-secretase (bace) promoter increased nf-kappab signalling up-regulates bace expression and its therapeutic potential in alzheimer's disease neuro-inflammation induced by lipopolysaccharide causes cognitive impairment through enhancement of beta-amyloid generation qhg and jss designed the experimental protocols. yz carried out all the studies except the western blot analysis, which was performed by cl, lhl, and yd. yz wrote the manuscript with help from qhg, cyy, and jmg. competing interests: the authors declare no competing interests. key: cord- -sic gsg authors: turzo, maurizio; metzger, karin; lasitschka, felix; weigand, markus a.; busch, cornelius j. title: inhibition of overexpressed kv . augments hpv in endotoxemic mice date: - - journal: bmc pulm med doi: . /s - - - sha: doc_id: cord_uid: sic gsg background: hypoxic pulmonary vasoconstriction (hpv) is a reaction of the pulmonary vasculature upon hypoxia, diverting blood flow into ventilated areas to preserve oxygenation. it is impaired in endotoxemia or ards. voltage gated potassium channels have been shown to play a key role in the regulation of hpv. the aim of the study was to identify a voltage gated potassium channel involved in dysregulated hpv during endotoxemia. methods: lungs of male c bl/ mice with and without endotoxemia (n = ea. group) were analyzed for kv . gene and protein expression. hpv was examined in isolated perfused lungs of mice with and without endotoxemia and with and without selective kv . blocker bds-i (n = ea. group). pulmonary artery pressure (pap) and pressure-flow curves were measured during normoxic (fio( ) . ) and hypoxic (fio( ) . ) ventilation. hpv was quantified as the increase in perfusion pressure in response to hypoxia in percent of baseline perfusion pressure (Δpap) in the presence and absence of bds-i. results: kv . gene ( . ± . -fold, p < . ) and protein ( . ± . -fold p < . ) expression levels were increased in endotoxemic mouse lungs. endotoxemia reduced hpv (∆pap control: . ± . % vs. lps . ± . %, means ± sem) while inhibition of kv . with nm bds-i augmented hpv in endotoxemic but not in control lungs (∆pap control bds-i: . ± . % vs. lps bds-i . ± . %, means ± sem). conclusions: kv . gene and protein expressions are increased in endotoxemic mouse lungs. selective inhibition of kv . augments hpv in lungs of endotoxemic mice, but not in lungs of control mice. in heterologous expression systems, produce sustained currents, while kv . and kv . produce currents respectively with a slow and fast rate of inactivation [ ] . activation of kv -channels increases efflux of k + , resulting in increased closure of ca + channels, decreased ca + −influx and reduced intracellular ca + , which in turn leads to smooth muscle relaxation. in humans, expression of kv . has been shown in pulmonary [ ] and uterine smooth muscle cells, as well as in purkinje cells [ , ] . besides vascular tone of pulmonary and splanchnic arteries, kv . is involved in the control of the cell cycle and proliferation of cells [ ] . the aim of the study was to evaluate the role of kv . , kv . and kv . in endotoxemic mouse lungs. we report that kv . gene as well as protein expressions were induced in endotoxemic mouse lungs. gene expressions of kv . and kv . were unchanged. immunohistochemistry showed a kv . immunoreactive protein in smooth muscle cells of mouse pulmonary arteries. hpv was reduced in lungs of endotoxemic mice. inhibition of kv . with the specific blocker bds-i increased hpv in isolated perfused endotoxemic mouse lungs. all animal experiments were conducted under protocols reviewed and approved by the subcommittee on research animal care of the university of heidelberg (regierungspräsidium karlsruhe, germany, az . /g- / ) and handling of the animals was in accordance with the european community guidelines. the study was performed with male c bl/ mice (body weight . ± . g), animals were obtained from charles river gmbh, sulzfeld, germany. mice for lung perfusion received endotoxin (lps i.p. injection . ml; e.coli :b , mg/kg bw; sigma-aldrich, st. louis, mo, usa) h before isolated lung perfusion experiments (lps groups). during lung perfusion, bds-i (sigma-aldrich) dissolved in hanks' balanced salt solution (life technologies, paisley, uk) was added to give a final concentration in the perfusate of nm (n = animals in each group). animals injected with . ml . % nacl solution served as controls (n = animals in each group). for analysis of gene-and protein expression, as well as immunoenzyme staining, extra animals received an i.p. injection of endotoxin (n = ) or . % nacl solution (n = ). receiving pentobarbital sodium ( mg/kg body weight i.p., merial, hallbergmoos, germany), mice were sacrificed and lungs were explanted and buffer perfused as previously described ( ) . an in-line flow probe was used to adjust perfusate flow (transonic systems, ithaca, ny, usa). hanks' balanced salt solution (life technologies) supplemented with bovine serum albumin ( %; serva, heidelberg, germany) and dextran ( %; sigma-aldrich) to prevent pulmonary edema was used as perfusate as previously described [ ] . in order to inhibit endogenous nitric oxide and prostaglandin synthesis, the nonselective nitric oxide synthase inhibitor l-name ( mm l-arginine methyl ester; sigma-aldrich) and indomethacin ( mm; sigma-aldrich) were added to the perfusate. the perfusate ph was adjusted to . - . with sodium bicarbonate. lungs were included in the study if they had a white homogenous appearance without signs of hemostasis or atelectasis, showing a stable perfusion pressure less than mmhg during the second min of the initial min baseline perfusion period. using these two criteria, three animals were excluded. pulmonary artery pressure (pap) and left atrial (la) pressure were measured via . % nacl solution-filled membrane pressure transducers connected to a side port of the inflow and outflow cannulae. pressure transducers were connected to a biomedical amplifier ( channel bridge amplifier tbm m, world precision instruments, berlin, germany), and data were recorded at hz on a personal computer using an analog-to-digital interface with a data acquisition system (di- ; dataq instruments, akron, oh, usa). before each experiment, the system was calibrated. hpv responsiveness (Δpap) was quantified as the difference between basal pulmonary arterial pressure (pap) and pap at the end of min hypoxic ventilation (fio . ). both lungs of the studied animals (excluding their hilar structures) were excised and immediately weighed at the end of the experiment, dried in an oven at °c overnight, and then re-weighted. lung wet/dry weight ratios were calculated by dividing the wet weight by the dry weight as described previously [ ] . mice were sacrificed after h of lps exposure (lps; e.coli :b , mg/kg bw i.p.; sigma-aldrich) with a lethal injection of pentobarbital sodium (merial, mg/ kg bw i.p., n = ). with . % nacl solution injected mice served as controls (n = ). lungs were exposed via median sternotomy and heparin ( u) was injected into the right ventricle. lungs were perfused with iced . % nacl solution for min at ml*kg- *min- flow, dissected (excluding hilar structures), quick-frozen, and stored at − °c. rna was isolated using the trizol reagent (invitrogen, thermo fisher scientific, waltham, ma, usa), and cdna was generated with mmlv reverse transcriptase (promega, madison, wi, usa) and random primers (promega). quantitative rt-pcr was performed with the abi prism sequence detection system (applied biosystems, foster city, ca, usa), using specific primers (see table ) and sybr®green pcr master mix (applied biosystems). to verify the presence of a single amplification product in the absence of dna contamination, postamplification dissociation curves were performed. changes of expression of the gene of interest were determined using the ΔΔct method with normalization to s ribosomal rna. immunoblots were performed with tissue of mouse lungs to assess protein levels of kv . as well as the housekeeping protein gapdh (glycerinaldehyd- -phosphat-dehydrogenase). samples of mouse lungs with and without endotoxemia (n = ) were homogenized at °c in ripa buffer ( mmol/l tris-hcl ph . , mmol/l nacl, % triton x- , . % sds and protease inhibitor mix (roche diagnostics gmbh, mannheim, germany)) and centrifuged at , g, °c for min. supernatant protein was subjected to electrophoresis, transferred to a pvdf membrane, and probed with rabbit anti-kv . ( : , sigma-aldrich) and mouse anti-gapdh antibodies ( : , merck, darmstadt, germany). as secondary antibodies served irdye® cw goat anti-rabbit igg ( : ) and irdye® rd goat anti-mouse ( : , irdye®, li-cor biosciences, lincoln, ne, usa). proteins were visualized with a licor infrared imager (odyssey, li-cor biosciences), quantitative densitometric analysis was performed by applying odyssey version . infrared imaging software and signals were normalized to gapdh. lungs of mice with and without endotoxemia were fixed in paraformaldehyde (n = each group). after embedding in paraffin, μm sections were cut using an automatic rotary microtome (microm hm , thermo fisher scientific). immunoenzyme stainings were performed using standard avidin-biotin anti-alkaline phosphatase technique (vector laboratories, burlingame, ca, usa) according to the manufacturer's instructions. tris-buffered saline supplemented with . % bovine serum albumin (biotrend, cologne, germany) was used as buffer. primary antibody dilutions of rabbit anti-kv . , / (anti-kcnc , sab , sigma-aldrich) and an isotype-and concentration-matched rabbit control ig (dianova, hamburg, germany) were prepared in this buffer and incubated for h at room temperature. a biotinylated donkey anti-rabbit igg ab, / (amersham™, ge healthcare uk), was used as a secondary reagent ( min at room temperature). naphthol as-biphosphate (sigma-aldrich) with new-fuchsin (merck, darmstadt, germany) was used as the substrate for alkaline phosphatase. slides were counterstained with hematoxylin (sigma-aldrich). magenta positive staining (corresponding to kv . positive staining) was defined as the area of interest and was quantified (bx upright microscope; cellsens dimension . software, olympus, tokyo, japan). data are reported as mean ± sem. after approving the assumption of normality and equal variance across groups, differences were assessed using anova followed by an appropriate post hoc comparison test. groups were compared by a two-way anova statistical test. when significant differences were detected by anova, a post hoc least difference test for planned comparisons was used (spss , ibm, armonk, usa). statistical significance was assumed at a p value of less than . . extracts of total lung tissue from mice with lps i.p. showed an increase of kv . gene expression ( . ± . fold, n = , mean ± sem, p < . ) ( fig. ) in comparison to . % nacl solution treated controls ( . ± . -fold, normalized on , n = , means ± sem). gene expression levels of kv . and . were unaltered in lps treated animals (kv . : . ± . ; kv . : . ± . , n = , means ± sem). these results demonstrate that lps increases mrna expression of the voltage gated potassium channel kv . . in order to measure if increased kv . gene expression is followed by increased protein expression, tissue of mouse lungs was extracted after h of endotoxemia, . % nacl solution injected animals served as controls. kv . immunoreactive protein was increased in whole lung extracts in the lps group (control . ± . vs. lps . ± . -fold; n = , p < . , means ± sem, fig. ) . these results show that lps induces an increase of kv . protein expression in whole mouse lung extracts. pulmonary vascular response to hypoxic ventilation in control mice as well as endotoxemic mice hypoxic ventilation of lungs of control mice caused an increase of pap from . ± . to . ± . mmhg (Δpap: . mmhg, fig. ). these results demonstrate taken together, this data shows that hypoxia induces hpv in lungs of control mice and endotoxemia attenuates hpv. in order to test the role of kv . in the pulmonary vasculature, lungs of mice with and without endotoxemia were perfused with and without nm of the kv . specific inhibitor bds-i. perfusion of untreated control mice with nm bds-i did not affect baseline pap under normoxia (control . ± . mmhg vs. bds-i . ± . mmhg). again, rise of pap started within min of hypoxia and reached its maximum within min. perfusion with the inhibitor bds-i did not augment hpv in control mice ( fig. ; control without bds-i Δpap . ± . mmhg vs. control with bds-i Δpap . ± . mmhg). blockade of kv . with bds-i in lungs of endotoxemic mice did not alter baseline pressures (lps . ± . mmhg vs. lps/bds-i . ± . mmhg). in contrast, specific inhibition of kv . in lungs of endotoxemic mice augmented hpv (Δpap . ± . mmhg) compared to lungs perfused without inhibitor (Δpap . ± . mmhg) (fig. ) . this shows that bds-i does not alter hpv in control but augments hpv in endotoxemic mice. this study investigated the role of the voltage-gated potassium channel kv . on hpv in endotoxemic mice. while lungs of control mice showed solid hpv, it was impaired in endotoxemia. exposure of mice to lps increased gene as well as protein expressions of kv . in lung tissue. this induced kv . expression was attributable to pulmonary artery smooth muscle cells. pharmacological inhibition of kv . in lps treated mice restored in part hpv, while it did not affect hpv in control animals. in line with other studies, a strong hpv was found in buffer-perfused isolated mouse lungs of healthy animals [ , ] . consistent with previous experiments, a distinctive reduction of hpv was observed in lungs of endotoxemic mice [ , ] . it has been shown that hpv is modulated by several vasoactive mediators including nitric oxide and arachidonic acid metabolites, but the precise mechanisms mediating hpv are still incompletely understood [ ] [ ] [ ] [ ] [ ] . increased activity of nos [ ] results in elevated sgc activity with subsequent induction of the effector proteins of sgc in smooth muscle cells. this alters pkg and ca + signaling creb dependent in pasmc, which in turn leads to vasodilatation and reduced hpv. moreover, expression of voltage gated potassium channels is regulated by creb [ ] . further mechanisms altering hpv in endotoxemia include leukotrienes, which are induced and have been shown to reduce hpv in mice. inhibition of lipoxygenase ( -lo) \with mk in endotoxemic mice augmented hpv and endotoxemic -lo knock-out mice showed persistent hpv [ ] . another regulatory protein of hpv is pkc [ ] . inhibition of pkc reduced hpv in isolated blood perfused dog lungs while activation of pkc increased hpv [ ] . ahn et al. found a similar proinflammatory cytokine response in pkc-δ knock-out mice after intratracheal lps administration when compared to wild type mice, but increased pulmonary neutrophil infiltration and perivascular edema [ ] . this suggests that pkc-δ is not involved in the regulation of proinflammatory mediators, but plays a role in the control of vascular permeability and transmigration of neutrophils through the endothelium [ ] . potassium channels have been shown to play a key role in hpv [ ] . endotoxemia induced overexpression of one or more kv channels in the pulmonary vascular bed contribute to loss of hpv [ ] . numerous voltage gated potassium channels, like kv . , kv . , kv . , kv . , kv . , kv . as well as kv . have been reported to be oxygen sensitive and expressed in the pulmonary vasculature [ , , , ] . of these, gene expressions of kv . , kv . , kv . and kv . were found to be unchanged or decreased in extracts of endotoxemic whole mouse lungs [ ] . although it seems less likely that these channels are responsible for loss of hpv in endotoxemia, it cannot be ruled out that heterogenic expression of a channel within the course of a vessel is missed by qpcr of whole lung tissue [ ] . another possibility is an alternated function of oxygen sensitive potassium channels, like it has been reported for kv . /kv . [ ] . in xenopus oocytes and transfected cos cells, coexpression of kv . /kv . consistently increased channel current amplitude and shifted steady-state activation towards negative values. since kv . gene expression was unaltered by lps, this mechanism is unlikely responsible for loss of hpv in endotoxemia. presence of kv . was first described in the tissue of rat brains [ ] , but is also present in pasmc [ ] and functions as a modulatory subunit for kv . [ ] . heterodimeric kv . /kv . channels showed a reduced rate of deactivation compared to kv . channels alone [ ] . since kv . gene expression was unchanged in lps lungs, alteration of hpv through kv . in endotoxemia is unlikely. further oxygen sensitive channels expressed in the pulmonary vasculature include kv . and kv [ , ] , which have not been investigated in these experiments. kv . was not only present in pulmonary arteries of mice, but also detected in rat pasmc [ ] . kv . is reported to be expressed in cultured human pasmc as well as in intact human main and small pulmonary arteries [ , ] . reduced kv . expression has been reported in cultured rat pasmc after h of hypoxia, this effect was dependent on -lo [ ] . we did not measure kv . expression after the brief hypoxia in our experimental setting, but it is unlikely that expression changed within or shortly after min of hypoxia. kv . channel activity was altered in vitro by phosphorylation of the n-terminus through pkc, converting kv . to a non-inactivating delayed rectifier type channel [ ] . since lps induced pkc in vitro within h in mouse microglia [ ] and after h in mouse lungs [ ] , it is possible that not only the number of kv . channels is increased by lps, but also the amount of open channels. the sea anemone venom bds-i is a amino acid peptide and described as selective kv . inhibitor, mostly inactive to other kv subchannels [ ] . in xenopus oocyte expressed kv channels, highest selectivity was described for kv . with an ic of nm, some inhibitory effects were found for kv . , kv . and kv . at a dose of μm [ ] . the k . value for bds-i in rat brain receptors was nm [ ] . besides kv . , bds-i showed inhibiting properties for kv . a and kv . b at a dose of nm ( and %) [ ] . since a ten times lower concentration was used in the isolated perfused mouse lung, inhibition with nm bds-i most likely blocked predominantly kv . . besides kv . channels, bds-i also shows inhibitory properties to voltage-sensitive na + -channels, especially nav . at a concentration of nm. this voltage gated sodium channel can be found in the heart, skeletal muscle, placenta, pancreatic β-cells as well as the brain and sensory and sympathetic neurons of the peripheral nervous system [ ] . only the neuronal nav-channels show relevant sensitivity to bds-i [ ] . since nav . expression is not described in pulmonary arteries, it reduces the possibility that the effect by nm bds-i in the isolated perfused mouse lung are provoked via inhibition of nav . . in inactive smooth muscle cells, membrane potential is stabilized by open k-channels maintaining an outward flux of k + . this prevents voltage sensitive ca + -channels from opening, resulting in vasodilatation. however, if outward k + flux is decreased through inhibition of kv channels, membrane potential is shifted to more positive potential, which in turn opens more ca + -channels causing vasoconstriction induced by ca + -influx. a hypothetical mechanism by which hpv is increased in endotoxemia is the inhibition of overexpressed kv . that would augment hpv through depolarizing the membrane and increasing the open-state probability of ca + -channels. an isolated, perfused mouse lung model was used to study pulmonary vasoreactivity in response to acute hypoxia in lps exposed mice. several limitations of this experimental setup have to be considered when extrapolating our data to mice in vivo or even to septic humans. the initial normoxic pap showed no difference between untreated and lps-treated mice. in contrast, patients with ards or animals with endotoxemia suffer from elevated pap and vascular hyperreactivity [ ] . this is most likely due to the model of the isolated perfused mouse lung that was used. hanks' balanced salt solution as perfusate without recirculation excludes thrombosis and perfusion with vasoconstrictive mediators. furthermore a stable outflow pressure of mmhg (equaling left atrial pressure) and avoiding atelectasis by explanting and expanding the lung also contribute to the missing increase of pap in endotoxemia. the cyclooxygenase inhibitor indomethacin as well as the inhibitor of soluble guanylate cyclase, l-name, were added to the perfusate to generate a robust hpv [ ] . simultaneous inhibition of no and prostaglandin production do not alter baseline pulmonary pressures in healthy isolated mouse lungs, but upon hypoxia [ ] . an i.p. lps model was used to induce inflammation. although it does not reproduce the full clinical scale of sepsis in humans, it has advantages. it is better reproducible compared to cecal ligation and puncture or colon ascendens stent peritonitis models and has no need for a surgical intervention before starting an experiment [ , ] . one of the disadvantages of the lps model is the difficulty of transferability into human pathophysiology. almost % of the patients admitted to intensive care units due to severe sepsis develop ards [ ] . in these patients, at least part of their hypoxemia can be attributed to loss of hpv in sepsis. therapeutic pulmonary vasoconstriction for the treatment of hypoxemia due to ards has been tested before using vasoconstrictors such as norepinephrine, phenylephrine or almitrine in combination with inhaled nitric oxide, without clinical benefit [ ] [ ] [ ] . so far there is still no data on the expression of potential target channels in septic patients with acute lung injury, but selective inhibition of upregulated voltage gated potassium channels in these patients might be a therapeutic tool to restore hpv and to prevent hypoxia. in summary, endotoxemia induces in mouse lungs expression of kv . . this increased expression is located in pulmonary arteries. the kv . inhibitor bds-i acutely increased hpv in endotoxemic mice but not in controls. selective inhibition of in endotoxemia overexpressed voltage gated potassium channels are potential targets to increase hpv in gram negative induced respiratory failure. 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almitrine combined with inhaled nitric oxide for acute respiratory distress syndrome. the no almitrine study group publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the authors thank jutta scheuerer for expert technical assistance. we acknowledge financial support by the baden-württemberg ministry of science, research and the arts and by ruprecht-karls-universität heidelberg.author details department of anesthesiology, heidelberg university hospital, im neuenheimer feld , heidelberg, germany. institute of pathology, heidelberg university hospital, heidelberg, germany. authors' contributions mt and cjb: study conception and design, acquisition of data, analysis and interpretation of data, drafting of manuscript; km: acquisition of data, analysis and interpretation of data; fl: immunohistochemistry, interpretation of data, critical revision of manuscript. maw: interpretation of data, critical revision of manuscript. all authors read and approved the final manuscript. histology and immunohistology services were provided by the tissue bank for inflammatory diseases heidelberg (gezeh), which was funded by the dfg (sfb tp z to f.l.). open access funding provided by projekt deal. the datasets used and analysed during the current study are available from the corresponding author on request.ethics approval and consent to participate all animal experiments were conducted under protocols reviewed and approved by the subcommittee on research animal care of the university of heidelberg (regierungspräsidium karlsruhe, germany, az - . /g- / ) and handling of the animals was in accordance with the european community guidelines.consent for publication not applicable. we have no competing interests to report. key: cord- -e s racp authors: wu, xiaojing; kong, qian; zhan, liying; qiu, zhen; huang, qin; song, xuemin title: tipe ameliorates lipopolysaccharide-induced apoptosis and inflammation in acute lung injury date: - - journal: inflamm res doi: . /s - - - sha: doc_id: cord_uid: e s racp objective: tumour necrosis factor-α-induced protein -like (tipe ) has strong anti-inflammatory properties. however, it is unknown whether increased tipe is protective against lipopolysaccharide (lps)-induced ali. in the current study, we aimed to investigate whether increased tipe can exert protective effects in a mouse model of ali induced by lps. methods: we administered tipe adeno-associated virus (aav-tipe ) intratracheally into the lungs of mice. three weeks later, ali was induced by intratracheal injection of lps into balb/c mice. twenty-four hours later, lung bronchoalveolar lavage fluid (balf) was acquired to analyse cells and protein, arterial blood was collected for arterial blood gas analysis and the determination of pro-inflammatory factor levels, and lung issues were collected for histologic examination, transmission electron microscopy (tem), tunel staining, wet/dry (w/d) weight ratio analysis, myeloperoxidase (mpo) activity analysis and blot analysis of protein expression. results: we found that tipe overexpression markedly mitigated lps-induced lung injury, which was evaluated by the deterioration of histopathology, histologic scores, the w/d weight ratio, and total protein expression in the balf. moreover, tipe overexpression markedly attenuated lung inflammation, as evidenced by the downregulation of polymorphonuclear neutrophils (pmns) in the balf, lung mpo activity, and pro-inflammatory cytokine levels in the serum. moreover, tipe overexpression not only dramatically prevented lps-induced pulmonary cell apoptosis in mice but also blocked lps-activated jnk phosphorylation and nf-κb p nuclear translocation. conclusions: our study shows that the increased expression of aav-mediated tipe in the lungs of mice inhibits acute inflammation and apoptosis and suppresses the activation of nf-κb and jnk in a murine model of ali. acute lung injury (ali) and acute respiratory distress syndrome (ards) are life-threatening medical conditions with high morbidity and mortality rates, and they are triggered by common pathologies such as sepsis, trauma and pneumonia [ ] . ali pathophysiology is characterised by the increased permeability of the alveolar-capillary barrier, interstitial edema, neutrophil recruitment, and inflammatory stress-induced cell apoptosis [ , ] . the pathophysiology of sepsis-induced ali is characterised by complex mechanisms that involve cell inflammation, cytokine production, and abnormal apoptosis [ ] . to date, there is no effective pharmacological approach to treat ali. apoptosis, known as programmed cell death, is essential for the selective elimination of cells. however, the dysregulation of apoptosis pathways has been demonstrated to contribute to epithelial and endothelial injury, which are characteristic of ali [ ] . the inhibition of apoptosis increases the animal survival rate in an lps-induced ali model [ ] . lipopolysaccharide (lps), a main component of the outer membrane of gram-negative bacteria, is widely used to induce animal models of ali through intratracheal instillation [ ] . after binding with toll-like receptor (tlr ), lps induces the activation of downstream signalling pathways responsible for the infiltration of inflammatory cells (i.e., neutrophils) into the lungs and the production of proinflammatory cytokines [ ] . the binding of lps to tlr also induces iκb-α phosphorylation and degradation, promotes the nuclear translocation and activation of nf-κb, and subsequently leads to the excessive release of pro-inflammatory cytokines [e.g., tumour necrosis factor-α (tnf-α), interleukin (il)- β, il- ] [ ] . moreover, lps can activate jnk, a member of the mapk family. activated jnk can phosphorylate numerous mitochondrial proteins, including bcl- and bcl-xl [ , ] . subsequently, cytochrome c is released from the mitochondria, which leads to the activation of death signals [ ] . jnk activation is essential for lps-induced macrophage apoptosis during sepsis [ ] . tumour necrosis factor-α-induced protein- (tnfaip )like (tipe ), which is an essential negative regulator of tlr and tcr function, has been confirmed to inhibit caspase-mediated apoptosis [ ] . tipe has been reported to be a negative regulator of the activating protein (ap)- , nf-κb, jnk, and p pathways [ ] . whether tipe has a therapeutic effect on lps-induced ali has not been reported. the current study was designed to test the hypothesis that tipe attenuates lps-induced ali through the inhibition of lung inflammation and apoptosis, which may be associated with suppressing nf-κb and jnk activation. reagents lps (e. coli :b ) was obtained from sigma-aldrich (st. louis, mo). rabbit polyclonal bax, caspase , tipe and lamin a antibody were sourced from abcam limited. rabbit polyclonal jnk, rabbit monoclonal p-jnk and β-actin antibody were from cell signaling technology (boston, ma). rabbit polyclonal bcl- , nf-κb p , and caspase antibodies were from proteintechgroup, inc (wuhan, china). hrpconjugated secondary antibody was obtained from boster biological technology co. ltd (wuhan, china). enzymelinked immunosorbent assay (elisa) kits were purchased from abclonal technology (usa). mpo kit was obtained from nanjing biohelper co., ltd. (nanjing, china). adult male babl/c mice ( - weeks) weighing - g were purchased from the wuhan institute of biological products co., ltd. (wuhan, china). the mice were housed under specific pathogen-free (spf) conditions that provide relative humidity ranging between % and %, temperature of ± °c, a : h light-dark cycle, with free access to food and water. the animals were adapted to this environment for week before the experiment. this study was approved by medical ethics committee of renmin hospital of wuhan university and was performed in accordance with the national institutes of health guidelines for the care and use of laboratory animals. a recombinant adeno-associated virus containing the mouse tipe gene was purchased from hanheng company (hanheng biotechnology co., ltd., shanghai, china). an adeno-associated virus expressing no transgene was used as a negative control (paav-ires-zsgreen). twenty-one days before lps instillation, balb/c mice were anaesthetized using sodium pentobarbital and given × vector genomes (vg) of raav -flag-mtipe ( × vg/ml) in μl of pbs via intratracheal (i.t.) administration to induce the overexpression of pulmonary tipe . control mice were treated with the control adeno-associated virus. the efficacy of the fusion protein was evaluated by western blotting. forty mice were randomly divided into four groups (n = per group): the control group (pbs); the lps group (lps); the tipe + pbs group (aav-tipe + pbs); and the tipe + lps group (aav-tipe + lps). as previously described by matute-bello et al. [ ] , to establish the ali model, mice were anaesthetized with an intraperitoneal (i.p.) injection of pentobarbital sodium ( mg/kg), orally intubated with a sterile plastic catheter and subsequently intratracheally injected with lps (escherichia coli : b ; sigma, st. louis, mo, usa) at a dose of mg/kg body weight. control mice were intratracheally administered μl of sterile phosphatebuffered saline (pbs). twenty-four hours after lps treatment, the mice were sacrificed by an i.p. injection of pentobarbital ( mg/kg; sigma). arterial blood was collected, and then a median sternotomy was performed to expose the lungs. in each mouse, after the hilum of the right lung was ligated, the left lung was lavaged to obtain the balf. the right upper lobe of the lung was excised to calculate the lung wet/dry weight ratio. lung tissues from part of the right middle lobe of the lung were taken for he staining, immunohistochemical staining, transmission electron microscopy, and tunel staining. lung tissues from part of the right lower lobe of lung were taken for mpo activity detection and western blotting. lung tissues were snap-frozen in liquid nitrogen and stored at − °c for later analysis. lung tissues were harvested for observing morphologic alterations at h after lps or pbs administration. the right middle lobe of lung were excised, washed and fixed with % (v/v) paraformaldehyde for h at °c. lung tissues were embedded in paraffin, sectioned at μm thickness, dewaxed and rehydrated, and stained with hematoxylin and eosin (h&e) solution (hematoxylin, mhs ; eosin, ht ; sigma-aldrich, usa) to estimate inflammation in alveolar and peribronchial lesions. the stained slides were then observed with the light microscope and the digital micrographs were taken for analyzing. histologic changes were evaluated by a pathologist blinded to the experiment. the degree of lung injury was graded using a histologic ali scoring system based on histologic features, including neutrophils infiltration, hyaling membranes, proteinaceous debris, and alveolar septal thickening [ ] . sections of paraffin-embedded tissue were subjected to immunohistochemical staining. the rabbit polyclonal tipe antibody ( - -ap, proteintech group, inc. usa) was used at : . sections were incubated with primary antibody ( °c, h), followed with a poly-horseradish peroxidase anti-rabbit secondary antibody (dilution ratio : , ; a , thermo fisher scientific inc. usa) incubated ( °c, min), and diaminobenzidine (dab) was used to visualize the complex. subsequently, the sections were counterstained with hematoxylin, dehydrated, and mounted. the expression of tipe was evaluated using a light microscope (bx ; olympus corporation, tokyo, japan). the lungs were isolated and cut into - -mm cubes. lung tissue samples were fixed by immersion in . % glutaraldehyde buffer for h at °c, washed with pbs solution three times, post-fixed for h in % osmium tetroxide, dehydrated in graded solutions of ethyl alcohol ( %, %, %, % and %), and embedded in epoxy resin. ultrathin sections ( nm) that were double-stained with uranyl acetate and lead citrate were examined under a transmission electron microscope (hitachi h- , hitachi, tokyo, japan). apoptosis was detected and quantified by the tunel assay using the in situ cell death detection kit (roche diagnostics gmbh, mannheim, germany.) according to the manufacturer's protocol. apoptotic cells exhibited brownish staining in the cell nuclei. ten random sections of the lung from each mouse were analysed without knowledge of the group of mice from which the lung tissue was taken, and the apoptosis index was expressed as a percentage of tunel-positive cells. the examination was performed by two pathologists blinded to the experimental design. to obtain the balf, the lungs were lavaged three times with ice-cold pbs ( . ml) and withdrawn each time using a tracheal cannula (a total volume of . ml). the collected balf was centrifuged at ×g for min at °c, and the supernatants were collected and frozen at − °c for subsequent assays. the cell pellet was resuspended in pbs, and after excluding the dead cells by trypan blue staining, the total number of inflammatory cells in the balf was determined by counting the cells with a haemocytometer (beckman coulter, inc). to analyse the cell numbers, μl of balf was centrifuged onto slides by a cytospin (thermo fisher scientific, waltham, usa). after the slides were dried, the cells were fixed and stained using wright stain solution ( , sigma, usa) according to the manufacturer's instructions. the number of polymorphonuclear neutrophils (pmns) was classified by a laboratory technologist blinded to the experimental design to determine the percentage of neutrophils. the frozen balf supernatant was thawed and thoroughly mixed, and the total protein concentration was determined by the bca (bicinchoninic acid) method. after mice were anesthetized, the arterial blood sample was collected with a heparinized syringe from the carotid artery. the arterial blood samples were immediately injected into an abl radiometer (radiometer america, usa) to measure ph value, partial gas pressures of oxygen (pao ), pao /fraction of inspired oxygen (fio ), and carbon dioxide (paco ). the magnitude of pulmonary edema was determined by calculating the lung wet/dry weight ratio. the right upper lobe of the lung was excised, washed with phosphate-buffered saline (pbs), blotted and then weighed to obtain the "wet" weight. the lung was then placed in an oven for h at °c and weighed to obtain the "dry" weight. the wet/dry ratio was calculated to quantify the degree of pulmonary edema. ripa lysis buffer was used for lysing the lung tissues, and mg of tissue was used for each test sample. after washing with cold pbs, the tissues were resuspended in four volumes of mpo assay buffer and then centrifuged ( , ×g for min, °c). the supernatant was collected and transferred to clean tubes, which were placed on ice. the mpo activity was assayed using a myeloperoxidase activity assay kit (abcam, ab ) by measuring the absorbance of the sample at nm using a microplate reader (bio-rad laboratories, hercules, ca, usa). the specific mpo activity in the lungs is expressed as unit/mg protein. blood was collected from the carotid artery, and serum was obtained following centrifugation ( ×g for min). the levels of tnf-α, il- and il- β in serum were determined using enzyme-linked immunosorbent assay (elisa) kits according to the manufacturer's instructions (r&d systems, minneapolis, mn, usa). the absorbance was measured at nm using an elisa reader (biotek instruments, inc., usa). h after the injection of lps, the lung tissues were harvested and snap-frozen in liquid nitrogen until homogenization. the lung tissues were homogenized using a homogenizer with tissue nuclear and cytoplasmic extraction reagents (sigma-aldrich, usa), according to the manufacturer's instructions. protein concentrations were determined using the bca protein assay kit (invitrogen; thermo scientific). equal amounts of protein ( μg) were loaded per well on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (sds-page) and transferred onto polyvinylidene difluoride membranes. the resulting membranes were blocked by incubation with % skim milk in tbst at room temperature for h on a rotary shaker, followed by washing with tbst. subsequently, the membranes were incubated with specific primary antibody overnight at °c. the membranes were washed with tbst followed by incubation with horseradish peroxidase (hrp)-conjugated secondary antibody at room temperature for h. the blots were washed tbst and detected using an enhanced chemiluminescence (ecl) western blotting detection kit. the proteins bands were observed using an ecl western blotting analysis system (bio-rad laboratories, inc., usa) and quantified by densitometry (image lab software version . . ). the data are expressed as the mean ± sem. the statistical analysis was performed using graphpad prism (version . ; graphpad software, inc., la jolla, ca, usa) by one-way analysis of variance (anova) followed by dunnett's least significant difference post hoc test. p < . was considered statistically significant. in the present study, we first investigated the effects of tipe overexpression on lung histopathology and function in mice challenged with lps (fig. ) . the lung tissues were harvested h after lps stimulation and subjected to h&e staining. there were no obvious histological changes in the lung tissues of the mice group a and group c. significant pathological changes, including pulmonary capillary congestion, pulmonary interstitial edema, mass inflammatory cell infiltration into the alveolar space and lung interstitium, and alveolar wall thickening, were observed in the lung tissues of lps-challenged mice. a subsequent western blot assay further demonstrated that tipe protein levels were significantly increased in the lung tissues of aav-tipe infected mice. treatment with aav-tipe significantly attenuated the histopathological changes induced by lps (fig. a) . in addition, a scoring system was used to assess the degree of lung injury. as shown in fig. b , the quantitative scoring of histological lung injury in the ali mice was markedly increased compared with that in the control group h after lps challenge. however, recombinant adenoassociated virus-mediated tipe overexpression markedly decreased the pathological scores compared with those of the lps group. transmission electron microscopy was used to examine the ultrastructural changes of lung tissues (fig. ) . in the control group, there were abundant mitochondria with regularly arranged mitochondrial cristae and homogeneous matrix. lungs from lps-treated mice exhibited a disordered arrangement of mitochondrial cristae, and the number of lamellar bodies was decreased. in the tipe + lps group, the pathologic damage was significantly alleviated compared with that in the lps group. we investigated the effects of tipe overexpression on lung cell apoptosis in lps-challenged mice by tunel staining. tunel staining revealed few apoptotic cells in the lungs of the control group. after the administration of intratracheal lps, unlike in control mice, numerous lung cells were strongly positive for tunel staining. however, in the lung tissues of aav-tipe -treated mice, a few of the lung cells were tunel-positive (fig. a) . for quantitative measurement, the percentage of tunel-positive lung cells was analysed for each specimen (fig. b) . the results showed that lps-challenged mice showed a significant increase in the number of apoptotic cells, which was reduced by aav-tipe treatment. these data indicated that tipe overexpression inhibited apoptosis in the lung after lps challenge. the lung w/d ratio and balf protein concentration are two commonly used indicators of pulmonary vascular permeability, which is an important characteristic of ali/ards. lps-challenged mice showed a significant increase in the lung w/d ratio (fig. a) and balf protein concentration (fig. b) when compared with those of the control group, and these levels were decreased by aav-tipe treatment. we also detected the ratio of the number of pmns relative to the number of total cells in the balf and the activity of mpo, an indicator of neutrophil infiltration, in the lung h after lps administration. compared with those in the control group, the pmn/total cell ratio in the balf (fig. c) and lung mpo activity (fig. d) in lps-challenged mice were dramatically increased, and these levels were inhibited by aav-tipe treatment. these results suggest that tipe overexpression attenuates lung edema and inflammation in lps-challenged mice. as shown in fig. , the arterial blood gas analysis of mice that received lps treatment showed significant changes compared with that of the control group, with the ph (fig. a) , partial pressure of arterial oxygen (pao ) (fig. b) and pao /fio (fig. c) decreasing and the partial pressure of arterial carbon dioxide (paco ) (fig. d) increasing. the pao /fio of the mice in the lps group however, tipe overexpression effectively mitigated the change in arterial oxygenation. compared with those in the lps group, the ph and pao were increased, and paco was decreased in the tipe + lps group. the pao /fio in the tipe + lps group recovered to normal levels and was higher than that in the lps group. as depicted in fig. , we found that the levels of the proinflammatory cytokines tnf-α (fig. a) , il- ( fig. b) and il- β (fig. c) in the serum were significantly increased h after lps challenge in mice. aav-tipe treatment significantly downregulated the levels of pro-inflammatory cytokines in the serum of lps-challenged mice. previous studies have shown that tipe is a negative regulator of the nf-κb, jnk, and p mapk pathways in macrophages [ ] . the effect of tipe on nf-κb and jnk activation was assessed in mice after lps challenge. to evaluate the effect of tipe overexpression on lung cell apoptosis in lps-challenged mice, the protein expression levels of anti-apoptotic proteins (bcl- ) and proapoptotic proteins (bax, cleaved caspase- , cleaved caspase- ) in the lung tissues were analysed by western blotting. as shown in fig. , compared with control mice, mice with lps-induced ali exhibited decreased bcl- expression and increased bax, bax/bcl- , cleaved caspase- , and cleaved caspase- protein expression in lung tissue samples, whereas the changes in the expression of the proteins were reversed by aav-tipe treatment. in addition, lps stimulation markedly increased nuclear nf-κb p and phosphorylated jnk (p-jnk) expression and decreased tipe expression in the lungs compared with that in the control group. however, adeno-associated virus-mediated tipe overexpression suppressed the increase in the expression of nuclear nf-κb p and p-jnk induced by lps. the expression of the tipe protein was significantly increased in the alveolar epithelium after aav-tipe administration (fig. ) . the expression of tipe was decreased h after lps challenge. the tipe + lps group exhibited higher expression of tipe compared with that in the lps group. ali and ards are the two main causes of acute lung failure, which is characterised by high morbidity and mortality and for which effective therapeutic strategies are lacking [ ] . thus, identifying novel therapeutic treatments for ali is urgently needed. in the current study, we found that tipe overexpression attenuated lps-induced ali in mice via its anti-inflammatory and anti-apoptotic effects. first, tipe overexpression significantly improved lps-induced lung injury, as evidenced by changes in histopathology, the lung w/d weight ratio, balf protein concentration, and arterial blood gas. second, tipe overexpression decreased inflammatory cell infiltration into the lungs and pulmonary cell apoptosis. third, the levels of pro-inflammatory cytokines in the serum of lps-challenged mice were reduced by aav-tipe treatment. fourth, tipe overexpression inhibited lps-induced nf-κb p nuclear translocation and jnk phosphorylation in the lungs. finally, tipe overexpression prevented lung cell apoptosis by downregulating the expression of pro-apoptotic proteins (bax, cleaved caspase- , and cleaved caspase- ) and upregulating the expression of an anti-apoptotic protein (bcl- ) in the lungs of lps-challenged mice. in conclusion, these results demonstrate that tipe overexpression ameliorates lps-induced ali via reducing fig. tipe overexpression reduced lps-induced proinflammatory cytokine levels in mice. the mice were treated as described in fig. . blood sample was collected for measuring the pro-inflammatory cytokines levels at h after lps or pbs administration. a tnf-α; b il- ; c il- β. the data are presented as mean ± sem. n = /group, * p < . versus pbs group; # p < . versus lps group lung inflammation and apoptosis, which may be associated with decreased nf-κb and jnk activity. the characteristics of ali/ards can be reproduced by the intracerebral administration of lps [ ] , which acts via tlr to induce the production of inflammatory cytokines, resulting in damage to microvascular and epithelial integrity and increased alveolar and interstitial edema [ ] . in this study, we successfully produced a mouse model of ali by the intratracheal administration of lps. we found that acute lung injury, which was characterised by pathological changes in lung tissue (by h&e staining and tem), increased lung water content, the infiltration of inflammatory cells, and pulmonary dysfunction was present h after lps administration. however, aav-tipe treatment markedly reduced lps-induced lung injury. tipe is a critical regulator of immune homeostasis that negatively regulates t cell receptor (tcr) and toll-like receptor (tlr) signalling [ , ] . tipe deficiency in mice induces foetal inflammatory diseases, and tipe downregulation in humans causes systemic autoimmunity [ , ] . moreover, tipe has also been identified as an apoptosis regulator that contains a death effector domain (ded) and is able to inhibit the activities of the apoptotic enzymes caspase- and caspase- [ ] . sun et al. demonstrated that the deletion of tipe amplifies jnk and p mapk phosphorylation and nf-κb activation, suggesting that tipe is a negative regulator of jnk, p mapk and nf-κb [ ] . in lps-induced ali, lps is recognized by tlr and subsequently promotes the activation of nf-κb [ ] , which is a pivotal transcription factor in the pathogenesis of ali. when activated, nf-κb p translocates to the nucleus, where it triggers the transcription of inflammatory cytokines, such as tnf-α, il- , and il- β [ ] . the inactivation of nf-κb p inhibits inflammation-induced inflammatory cell infiltration, edema, and pro-inflammatory cytokine production in the lungs [ ] . in the current study, tipe fig. tipe overexpression suppressed the activation of jnk and nf-κb as well as the protein expression of genes involved in apoptosis and lung injury induced by lps. the mice were treated as described in fig. . lung tissues were collected for western blotting analysis at h after lps or pbs administration. a western blotting analysis of protein expression of bax, cleaved caspase- , cleaved cas-pase- , bcl- , jnk, p-jnk, nuclear nf-κb p , and tipe ; b-h the relative ratio of bax, cleaved caspase- , cleaved caspase- , bcl- , p-jnk, nuclear nf-κb p , and tipe protein expression. the data are presented as mean ± sem. n = /group, * p < . versus pbs group; # p < . versus lps group overexpression significantly inhibited the lps-induced levels of tnf-α, il- , and il- β in the serum-induced and concomitantly decreased nf-κb activation. therefore, the inhibitory effect of tipe overexpression on lps-induced increases in the levels of pro-inflammatory cytokines may be ascribed to its suppression of nf-κb activation. liu mw et al. found that, after lps challenge, the increased expression of tipe is associated with the inhibition of nf-κb expression, the production of tnf-α and il- and decreased ros activity in raw . cells [ ] . in another study by liu et al., melilotus extract was shown to have protective effects against clp-induced lung injury, possibly by upregulating the expression of tipe [ ] . thus, tipe may have a protective effect against sepsis-induced lung injury and lps challenge in cells. in our present study, aav-tipe treatment markedly reduced lps-induced lung injury. pulmonary cell apoptosis also plays a critical role in the pathogenesis of ali [ ] . lps-induced cell apoptosis may be partly dependent on the mitochondria pathway [ ] . jnk, a member of the mapk family, is critical for lps-induced apoptosis in macrophages [ ] . lps triggers jnk phosphorylation, which subsequently mediates the phosphorylation of anti-apoptotic proteins (bcl- /bcl-xl) and upregulates proapoptotic bax. when the bax/bcl- ratio is elevated, the mitochondrial membrane potential changes, causing the release of cytochrome c into the cytosol from the mitochondria [ ] and resulting in the activation of caspase- and then caspase- to induce apoptosis [ ] . several previous studies have shown that tipe contains a death effector domain (ded) and that the overexpression of tipe is associated with enhanced survival and the inhibition of caspasemediated apoptosis through the inhibition of the activities of the apoptotic enzymes caspase- and caspase- [ ] [ ] [ ] . in addition, the depletion of tipe enhances cell death [ , ] . in the current study, we found that aav-tipe administration remarkably inhibited the expression of pro-apoptotic proteins (bax, cleaved caspase- , and cleaved caspase- ) and jnk phosphorylation and restored the expression of the antiapoptotic protein bcl- . these findings suggest that epithelial tipe drives the protective effects against lps-induced injury and that the inhibitory effect of tipe overexpression on lps-induced cell apoptosis may be attributed to its suppression of jnk activation. together, tipe exerts a protective effect against lpsinduced ali possibly through its anti-inflammatory and antiapoptotic activities, and it may be a potential therapeutic target for ali. in conclusion, our study demonstrated that tipe expression was significantly decreased in lung tissues in ali mice after lps challenge. moreover, adeno-associated virus-mediated tipe overexpression remarkably inhibited inflammation and cell apoptosis induced by lps. we also provided evidence that the anti-inflammatory and anti-apoptotic effects of tipe might at least partly involve the inhibition of nf-κb and jnk activation, respectively. therefore, our results indicate that tipe might be useful as a potential therapeutic target for sepsis-induced ali. fig. immunohistochemical staining of tipe protein expression in the lung. the mice were treated as described in fig. . lung tissues were collected for immunohistochemical staining at h 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novel tumor necrosis factor alpha-regulated genes in rheumatoid arthritis role of scc-s in experimental metastasis and modulation of vegfr- , mmp- , and mmp- expression publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations acknowledgements this work was supported by research grants from the national natural science foundation of china (no. ). conflict of interest the authors declare no conflict of interest. key: cord- -mi uf vk authors: zhang, wei; zhuang, ningtong; liu, xiaoyi; he, long; he, yan; mahinthichaichan, paween; zhang, hang; kang, yanhua; lu, yin; wu, qinan; xu, dakang; shi, liyun title: the metabolic regulator lamtor suppresses inflammatory signaling via regulating mtor-mediated tlr degradation date: - - journal: cell mol immunol doi: . /s - - - sha: doc_id: cord_uid: mi uf vk comprehensive immune responses are essential for eliminating pathogens but must be tightly controlled to avoid sustained immune activation and potential tissue damage. the engagement of tlr , a canonical pattern recognition receptor, has been proposed to trigger inflammatory responses with different magnitudes and durations depending on tlr cellular compartmentalization. in the present study, we identify an unexpected role of lamtor , a newly identified component of the amino acid-sensing machinery, in modulating tlr signaling and controlling inflammation. specifically, lamtor associated with tlr via their lz/tir domains and facilitated their colocalization at autolysosomes, preventing lysosomal tethering and the activation of mtorc upon lps stimulation and thereby derepressing tfeb to promote autophagic degradation of tlr . the loss of lamtor was unable to trigger the tfeb-driven autolysosomal pathway and delay degradation of tlr , leading to sustained inflammation and hence increased mortality among lamtor haploinsufficient mice during endotoxic shock. intriguingly, nutrient deprivation, particularly leucine deprivation, blunted inflammatory signaling and conferred protection to endotoxic mice. this effect, however, was largely abrogated upon lamtor deletion. we thus propose a homeostatic function of lamtor that couples pathogenic insults and nutrient availability to optimize the inflammatory response; this function may have implications for tlr -associated inflammatory and metabolic disorders. mammalian toll-like receptors (tlrs) are evolutionally conserved sensors of pathogen-associated molecular patterns that play a central role in host defense against infection and injury. , appropriately controlled immune responses are essential for eliminating injurious agents and maintaining homeostasis, whereas excessive immune and inflammatory responses cause tissue damage. dysregulated tlrs and downstream signaling molecules are therefore major players in various disorders such as infections, inflammatory, and metabolic diseases and cancers. among known tlrs, tlr is distinguished by its ability to recognize a variety of exogenous and endogenous agents and activate different signaling pathways depending on its cellular localization. upon ligation, tlr dimerizes and associates with the tir domain-containing adaptors tirap and myeloid differentiation primary response (myd ), initiating signaling at the plasma membrane to drive nf-κb-mediated production of proinflammatory cytokines. concurrently, engaged tlr is transferred to endosomes, where it activates toll/il- r domain-containing adaptor and inducing ifn (trif) and the interferon factor (irf) -dependent production of interferon (ifn)-β. [ ] [ ] [ ] alternatively, tlr is transported to the degradative lysosome, where it contributes to signaling termination, or sorted to the recycling endosome for return to the cellular surface. [ ] [ ] [ ] [ ] thus, the intracellular trafficking and subcellular compartmentalization of tlr constitute a key mechanism that dictates the direction and amplitude of inflammatory signaling. , in particular, cellular degradation systems, such as lysosome-and proteasomemediated catabolic processes, play a pivotal role in controlling tlr destiny and immune homeostasis. autophagy is a conserved cellular catabolic process that can involve the digestion of intracellular protein aggregates, damaged organelles, and invasive microorganisms. autophagy is initiated by the engulfment of large cytoplasmic portions and the formation of double membrane autophagosomes that subsequently fuse with endosomes and lysosomes, leading to the degradation and clearance of the sequestered contents. the disruption of autophagy, which has been described as a cytoprotective response to stressful conditions, is associated with a myriad of diseases including inflammatory disorders. , loss of the autophagic components autophagy-related like , beclin , and microtubule-associated protein a/ b light chain (lc ) enhanced the macrophage response to lps, a prototypical agonist of tlr , and caused sustained inflammatory signaling. , tlr engagement has been demonstrated to trigger the formation of lc + structures and activate the autophagosomal pathway, which in turn causes the eradication of inflammatory initiators such as invading pathogens and mitochondrial ros and dna release , or the degradation of key signaling molecules such as trif, nf-κb, and pellino e ubiquitin protein ligase family member (peli ). [ ] [ ] [ ] studies have shown the critical involvement of autophagy in tlr signaling, but the direct relevance of autophagy to tlr turnover has not been explored. late endosomal/lysosomal adaptor, mitogen-activated protein kinase (mapk) and mtor activator (lamtor) is a highly conserved, ∼ kda protein originally identified as a hepatitis type b x proteininteracting protein. lamtor associates with the oncogenic proteins c-fos and c-myc as well as survivin and p , promotes proliferation and exerts antiapoptotic effects in cancer cells. [ ] [ ] [ ] a recent study identified lamtor as an indispensable component of the ragulator complex that interacts with small gtpase ras-related gtp binding protein (rag), contributing to lysosomal recruitment and the activation of mechanistic target of rapamycin complex (mtorc ) in response to amino acids. , mtorc is a master growth regulator whose translocation to the lysosomal surface, the site of mtorc activation, is stimulated by amino acids and gtpase. as a result, the mtor pathway couples amino acid availability to cell growth and autophagy. , in this process, lamtor , in combination with other lamtor proteins, serves as a guanine exchange factor (gef) for rag gtpase to transduce nutrient-sensing signaling. gefs, molecular switches that control the gtp-bound 'on' and gdp-bound 'off' statuses of gtpase, play a key role in integrating extracellular signals initiated from surface receptors to intracellular pathways. gefs have been implicated in diverse cellular processes such as receptor recycling, synaptic vesicle endocytosis, and protein transport. recent studies have indicated that the gefs rab guanine nucleotide exchange factor , ras guanine nucleotide releasing protein , and denn domaincontaining b are critically involved in inflammatory and allergic processes, [ ] [ ] [ ] implying the expanding roles of gefs in immunological contexts. in the present study, we reveal the unexpected key role of lamtor in regulating tlr intracellular fate and immune homeostasis. specifically, lamtor directly interacts with internalized tlr and facilitates their colocalization at autolysosomes, preventing mtorc recruitment to lysosomes and facilitating nuclear translocation of transcription factor eb (tfeb) , hence promoting the autophagic degradation of tlr . accordingly, the depletion of lamtor delays tlr degradation and sustains inflammatory signaling, leading to increased susceptibility to endotoxic sepsis. in addition, lamtor was shown to largely mediate the antiinflammatory action of nutrient deprivation, particularly leucine depletion. we have thus identified a critical role for lamtor in tlr biology and inflammatory regulation that provides a potential molecular link between immune and metabolic processes. reagents and antibodies lps from escherichia coli o :b , chloroquine (cq) (c ), lleucine (l ), and a myeloperoxidase (mpo) fluorometric activity assay kit (mak ) were purchased from sigma (st. louis, mo). bafilomycin a (baf-a ) (s ), torin (s ), u (s ), sb (s ), ly (s ), bay - (s ), sp (s ), and vinblastine (s ) were obtained from selleck chemicals (shanghai, china). dynabeads™ protein a for immunoprecipitation ( d) and dq-bsa (d- ) were purchased from thermo fisher scientific (waltham, ma). enzymelinked immunoassay kits for il- , il- β, and tnf-α were obtained from r&d systems (minneapolis, mn). antibodies (abs) specific for total and phosphorylated forms of p (thr /tyr ), erk / (thr /tyr ), jnk (thr /tyr ), p (ser ), ikkα/β (ser ), tak (thr ), akt (ser ), mtor (ser ), p s kinase (thr /ser ), e-binding protein ( e-bp ) (thr / ), ulk (ser ), irf (ser ), and iκbα (ser ) were obtained from cell signaling technology (beverly, ma). abs against lamtor (ab ), tlr (ab and ab ), tfeb (ab ), rab (ab ), p (ab ), and eea (ab ) were obtained from abcam. abs against flag (# and # ), gfp (# and # ), lc a/b (# ), lc b (# ), lysosomalassociated membrane protein (lamp ) (# ), and β-actin (# ) were obtained from cell signaling technology (beverly, ma). horseradish peroxidase-conjugated secondary antibody was purchased from santa cruz biotechnology (santa cruz, ca). alexa fluor-conjugated antibodies were purchased from life technologies (carlsbad, ca). lamtor -, tfeb-, atg -, and rab -targeted sirnas and nonspecific sirna were synthesized from genepharma (shanghai, china). the pegfp-lc (# ) and ptflc (# ) plasmids were purchased from addgene. the prl-tk-renilla-luciferase plasmid was obtained from promega (madison, wi). the fulllength fragments of lamtor (nm_ . ) and tlr (nm_ . ) were cloned and inserted into the pcmv-tag b or pegfp vectors, respectively, using standard molecular biology protocols. expression plasmids encoding lamtor , lamtor mutants (Δlz), and truncated tlr proteins were constructed by genechem (shanghai, china). generation of lamtor knockout mice the crispr/cas technique was used to edit lamtor in mice by casgene biotech (beijing, china). small guide rnas (sgrnas) targeting exons - of lamtor were designed and constructed. sgrna-cas coexpression plasmids were injected into mouse zygotes, which were then transferred into pseudopregnant mice. neonatal mutant mice were identified by genotyping and sequencing. cell culture and transfection raw . cells (atcc ® tib- ™) and t cells (atcc ® crl- ™) were cultured in dmem-containing % fetal bovine serum (fbs). thp- cells (atcc ® tib- ™) were cultured in rpmi- medium supplemented with % fbs in a humidified incubator at °c with % co . to generate peritoneal macrophages, -week-old mice were injected i.p. with % thioglycolate broth. after h, peritoneal cells were harvested, and macrophages were enriched by quick adherence. for cell transfection, raw . or t cells were transiently transfected with plasmids using x-tremegene transfection reagent (roche) or sirnas using siport tm transfection agent (thermo fisher scientific) according to the manufacturer's instructions. to establish stable lamtor -expressing or lamtor silenced cell lines, the transfected cells were subjected to selection with g ( μg/ml) for - weeks. rna isolation, reverse transcription pcr, and quantitative pcr total cellular rna was isolated using trizol reagent (invitrogen). sybr green pcr master mix (toyobo) was used to detect mrna levels. mrna levels of the tested genes were normalized to those of β-actin and determined by applying the ΔΔct method. the following primer sequences were used (forward and reverse): il- β: ′-ctcgtgctgtcggacccat and ′-caggcttgtgctct gcttgtga; il- : ′-ccacttcacaagtcggaggc and ′-tgcaa gtgcatcatcgttgttc; tnf-α: ′-atccgcgacgtggaactggc immunoblot analysis and coimmunoprecipitation assay cell lysates were prepared in a lysis buffer containing mm nacl, mm tris-hcl (ph . ), % np- and protease inhibitor cocktail (roche diagnostics). proteins were separated by sds-page and transferred to pvdf membranes followed by incubation with the appropriate primary antibodies. the membranes were then incubated with hrp-conjugated secondary antibody (santa cruz, ca). signals were visualized with an ecl kit (amersham biosciences). images were captured using a chemidoc™ xrs+ system (bio-rad, usa). band detection was within the linear range. for immunoprecipitation studies, cell lysates were incubated at °c for h with a capture antibody or control igg, followed by overnight incubation with dynabeads™ protein a. the immunocomplexes were collected by centrifugation, washed with ice-cold pbst (pbs- . % tween- ), and separated by sds-page. proteins in the samples were detected by standard immunoblotting methods. luciferase reporter assay to assess nf-κb-driven promoter activity, the pgl -nf-κb, and pgl -basic (promega) plasmids were transfected into raw . cells using x-tremegene dna transfection reagent (roche). the prl-tk plasmid was used as an internal control. twenty-four hours later, the cells were stimulated with or without lps ( ng/ml) for h. luciferase activities were determined with dual-glo reagent (e , promega). determination of cytokine and mpo levels the levels of tnf-α, il- , and il- β in the culture supernatants or bronchoalveolar lavage fluid (balf) were measured by elisa (r&d systems). lung mpo levels were determined using mouse mpo elisa (hycult biotech) following the manufacturers' instructions. animal experiments all of the animal experiments were performed in accordance with the national institutes of health guide for the care and use of laboratory animals with the approval of the animal care and use committee of nanjing university of chinese medicine. the crispr/cas technique was used to edit lamtor in mice. mice were housed in a temperature-controlled room with a -h-light/ dark cycle and allowed food and water ad libitum. six-to eightweek-old lamtor +/− or wild-type (wt) mice were instilled with pbs or lps ( mg/kg, i.t.) and killed or h after exposure. balf and lung tissues were then collected for functional analysis. to induce endotoxic sepsis, mice were intraperitoneally injected with lps ( mg/kg), and their survival was monitored every h. alternatively, prior to endotoxin challenge, mice were fed a rodent diet (research diet, a b) or leucine-free diet (research diet, a ) for a week. bronchoalveolar lavage and cell differentiation briefly, the trachea was exposed through a midline incision and cannulated with a sterile -gauge needle. balf was obtained by flushing three times with ml of . mm edta/pbs. the supernatants were stored at − °c until use. total cell numbers in balf were counted with a hemocytometer. neutrophils and macrophages were assessed through immunostaining and flow cytometry. immunohistochemistry (ihc) staining five-micron-thick sections of murine lung tissue were deparaffinized, hydrated, and blocked in dpbs with % normal goat serum. the slides were then stained with anti-lamtor ( : ) and biotinconjugated secondary antibodies, followed by incubation with streptavidin-conjugated hrp. sections were finally incubated with dab reagent and counterstained with hematoxylin. flow cytometry and lysosome labeling after blocking unspecific binding with mouse igg, cells were incubated with . μg of pe-conjugated anti-mouse tlr antibody (ebioscience) for h at room temperature. appropriate isotype controls were used. after washing twice in flow cytometry buffer, cells were analyzed on a facscalibur flow cytometer (bd biosciences). to evaluate functional lysosomes, the lysosensor green probe, which fluoresces in an acidic environment (ph ≤ . ), was used. raw . cells were loaded with μm lysosensor in prewarmed medium for h at °c, washed twice with pbs and immediately analyzed by flow cytometry. data were analyzed with flowjo (tree star). immunofluorescence staining and dq-bsa quench assay cells were grown on cover glasses, fixed with % paraformaldehyde, and permeabilized with . % triton x- in pbs. after blocking with % bsa, cells were stained by incubation with primary antibodies (against tlr , lamp , lc , eea , mtor, lamtor , or tfeb) for h at °c. after washing, samples were incubated with alexa fluor -conjugated anti-rabbit antibody and/or alexa fluor -conjugated anti-mouse antibody. cell nuclei were visualized with dapi (sigma). slides were mounted with slowfade gold anti-fade reagent (invitrogen) and detected under a lsm laser scanning confocal microscope (carl zeiss). for the dq-bsa quench assay, cells were treated with . mg/ml dq green bsa (molecular probe) at °c for h. green fluorescence was detected by a zeiss lsm live. electron microscopy cells were first fixed with . % glutaraldehyde in pbs ( . m, ph . ) for h, washed, and postfixed with % oso for h. the specimen was dehydrated with a graded ethanol series for min at each step, transferred to absolute acetone for min, placed in a mixture of absolute acetone and resin overnight, and embedded in resin at °c for more than h. the specimen was then sectioned with a leica em uc ultratome and stained with uranyl acetate and alkaline lead citrate for - min. electron micrographs were taken on a jeol model jem- transmission electron microscopy (tem). the structural modeling and molecular dynamics (md) simulation the lamtor dimer was modeled using the . -Å resolution crystal structure deposited as pdb entry msh. a disulfide bond was modeled between the two cys residues located at the dimer interface and adjacent to each other. because the structure of the tir domain of tlr (residues - ) was not available in the pdb, a refined homology model was obtained using the i-tasser webserver. using the first of five predicted models, the tir-tlr dimer was modeled using the matrix operator provided for the tir domain of tlr (pdb entry fyw), the sequence of which is most similar to that of tir-tlr . a disulfide bond was modeled between the two cys residues of tir-tlr , which are located at the dimer interface. the cluspro webserver, which is widely accepted as a reliable docking program by capri (critical assessment of predicted interactions), was used for protein-protein docking. lamtor dimer, tir-tlr dimer, and lamtor -tlr complex simulations were performed using namd with a time step of fs and the charmm force field. these simulations were all performed under the generalized born/solvent-accessible surface area (gb/sa) implicit solvent model implemented in namd with both hydrophilic and hydrophobic effects taken into account. the solvent (ions and water molecules) was represented as a continuous medium with a solvent dielectric constant of . the metabolic regulator lamtor suppresses inflammatory signaling via. . . w zhang et al. instead of individual explicit molecules. the born radius cutoff was set to Å. all bonds involving hydrogen atoms were kept rigid using the shake algorithm. the cutoff for van der waals interactions was set to Å with the switching distance set to Å. the temperature was maintained at k by langevin dynamics with a damping coefficient of ps − . all experiments were performed at least three times. the results are expressed as the mean ± sd. differences between two groups were established by student's t-test. multiple group comparisons were performed by one-way anova followed by bonferroni post hoc t test. p < . indicated statistical significance. to explore the potential role of lamtor in innate immune responses, we first examined its expression in macrophages in response to an array of tlr ligands. strikingly, lamtor expression was induced by lps, pam csk , poly i:c, cpg (supplementary fig. s a -d) and the pathogenic agents staphylococcal aureus (s. aureus) and vesicular stomatitis virus ( supplementary fig. s e , f). these findings indicated the involvement of lamtor in prrmediated immune responses, and our attention was focused on tlr signaling given its central importance in immune and inflammatory responses. we further confirmed that lps stimulation induced lamtor in murine raw . macrophages and primary macrophages in a time-and dose-dependent manner ( fig. a-d) . a similar increase in lamtor levels was observed in human monocytic thp- cells and peripheral blood mononuclear cells ( fig. e-g) . moreover, the in vivo induction of lamtor was demonstrated in murine lungs intratracheally challenged with endotoxin (fig. h) . to understand the mechanism underlying tlr -driven lamtor expression, we applied specific inhibitors of phosphoinositide kinase, mapk, nf-κb, and mtor signaling pathways, the key pathways downstream of tlr . lamtor expression was impaired by treatment with the nf-κb inhibitor bms and the erk inhibitor u , although the effect of u was less pronounced (fig. i) . since lamtor is a component of the amino acid-sensing machinery, we then assessed the impact of nutrient status on lamtor expression. unexpectedly, the deprivation of serum or leucine, an essential amino acid (eaa), profoundly increased lamtor levels, whereas the addition of leucine reduced lamtor expression (fig. j, k) . taken together, our data indicate that lamtor is induced in macrophages in response to cellular stresses such as pathogenic insult or nutrient insufficiency. to investigate the function of lamtor in tlr -induced immune and inflammatory responses, we generated lamtor -expressing and lamtor -silenced macrophage cell lines ( supplementary fig. s a, b) . remarkably, the enforced expression of lamtor reduced the expression of the proinflammatory cytokines interleukin (il)- , il- β, and tumor necrosis factor (tnf)-α in response to lps stimulation, whereas knockdown of lamtor increased their expression (fig. a-d) . a similar increase in proinflammatory cytokine expression was induced in macrophages isolated from mice following the monoallelic depletion of lamtor , as homozygous gene knockout was embryonically lethal ( supplementary fig. s ). this suppressive effect of lamtor was also confirmed in human thp- cells following lps challenge, implying its conserved role in inflammatory responses (fig. e) . to further understand the in vivo relevance of this finding, we exploited a murine model of acute lung inflammation and injury induced by endotoxins. compared with their control littermates, lamtor haploinsufficient mice displayed more severe airway inflammation and tissue damage, as evidenced by increased inflammatory cell infiltration, proinflammatory cytokine production, protein leakage, and lung mpo activity (fig. f-j) . furthermore, lamtor +/− mice exhibited elevated mortality compared with wt controls when challenged with lethal doses of endotoxin (fig. k) . taken together, our data indicated that lamtor plays a key role in restraining tlr -mediated inflammatory signaling and maintaining immune homeostasis. lamtor restrains tlr -induced inflammatory signaling to gain mechanistic understanding of the lamtor -mediated regulation of inflammatory responses, we next analyzed the key signaling pathways downstream of tlr . as revealed in fig. a , b, enforced expression of lamtor increased lps-stimulated activation of iκb kinase (ikk), nf-κb inhibitor alpha (iκbα), and p , the key signaling molecules required for nf-κb activation. in contrast, knockdown of lamtor enhanced the phosphorylation of these molecules upon lps stimulation. in addition, our data showed that nf-κb-driven promoter activity and the nuclear translocation of p , the essential step for the activation of proinflammatory genes, were consistently repressed upon lamtor overexpression but increased upon lamtor silencing (fig. c, d) . the suppression of nf-κb activity by lamtor was further confirmed in lamtor +/− macrophages (fig. e) . activation of the mapks p , erk / , and jnk, however, was slightly affected by lamtor (fig. f, g) . in addition, lamtor overexpression decreased, while lamtor knockdown increased, the phosphorylation of tank binding kinase and irf , which was consistent with the modulated ifn-β expression upon lps stimulation (fig. h-k) . thus, our data indicated that lamtor exerts an inhibitory effect on both nf-κband irf -dependent signaling upon the engagement of tlr . lamtor promotes tlr degradation through the autophagic pathway given the comprehensive inhibition of tlr signaling by lamtor observed as described above, we hypothesized that lamtor affects key upstream signaling events and, in particular, the dynamic expression of tlr . indeed, our data demonstrated that enforced expression of lamtor reduced, while the knockdown of lamtor increased, tlr levels upon tlr engagement (fig. a) . lamtor +/− macrophages consistently displayed increased tlr levels (fig. b) . notably, despite the prominent suppression of the tlr protein induced by lamtor , the tlr mrna level was only mildly affected ( supplementary fig. s a, b) . furthermore, by fluorescent cytometry, we observed that surface tlr levels were reduced upon lamtor overexpression but increased upon lamtor deletion (fig. c) . taken together, these data implied that lamtor exerts a regulatory effect on tlr turnover upon tlr ligation. since tlr is internalized upon engagement and subjected to different intracellular fates, we then evaluated the impact of lamtor on tlr endocytosis by staining with eea , an endocytic marker. tlr localization at the endosome was not affected by lamtor (supplementary fig. s c ). however, using the self-quenched fluorophore dq-bsa , we showed that bsa hydrolysis was enhanced in lamtor -expressing cells but decreased in lamtor -silenced cells (fig. d) , indicating that lamtor affects cellular degradation. given that autophagy is a major cellular catabolic process essential for the degradation of cellular components and proteins, we then assessed the potential role of the autophagic pathway in lamtor -mediated tlr regulation. critically, lamtor expression promoted, while lamtor silencing impeded, the conversion of lc -i to lc -ii and the stability of p , which are key events representative of the autophagic pathway (fig. e ). in addition, the amplitude of the fluorescence at lc puncta was increased upon lamtor overexpression but decreased by lamtor deletion in lps-stimulated macrophages the metabolic regulator lamtor suppresses inflammatory signaling via. . . w zhang et al. (fig. f) . we also confirmed that autophagic flux was induced in lamtor -expressing macrophages, as detected upon treatment with baf-a , a putative autophagy inhibitor (fig. g) . to corroborate the potential role of lamtor in autophagy, we then analyzed its effect on the activity of mtor, a critical regulator of autophagy induction. the activation of mtorc , along with its downstream signaling molecules ribosomal protein s kinases, eukaryotic translation initiation factor e-bp , and unc- like kinase (ulk) , was repressed upon lamtor overexpression but increased upon lamtor deletion (fig. h, i) . therefore, our data demonstrated that lamtor contributes to the mtor-dependent autophagic pathway in response to lps stimulation. next, we determined whether inhibition of the lamtor mediated autophagic pathway affects tlr -driven signaling. to this end, baf-a was used to block the autophagic process, which restored the tlr level and nf-κb activity (fig. j) and led to the subsequent elevation of lps-induced il- and tnf-α in lamtor -expressing macrophages (supplementary fig. s ). in contrast, the application of torin , a putative mtor inhibitor, downregulated tlr levels and p activity in lamtor deficient macrophages following lps stimulation (fig. k) . consistent with this finding, the knockdown of autophagy protein (atg) , a key factor required for autophagosome elongation, largely abolished the lamtor -mediated suppression of tlr (fig. l) . collectively, our data indicated that lamtor plays a pivotal role in promoting the mtor-dependent autophagic pathway and hence reduces tlr levels and subsequent signaling. lamtor directly binds and recruits tlr to autophagosomes through the tir/lz interaction during the induction of autophagy, substrate proteins or targeting organelles are sequestered in double membrane autophagosomes that then fuse with lysosomes for digestion. to further understand the mode of action of lamtor in the autophagic process, we next examined its localization at autolysosomal compartments. importantly, lamtor colocalized with the autophagosome marker lc b and lamp (fig. a, b) , suggesting lamtor tethering at autophagic/lysosomal vacuoles upon lps stimulation in particular. furthermore, upon its ligation, tlr was internalized and transported to the autolysosomal compartments, as inferred from its colocalization with lc b and lamp , particularly that upon lamtor overexpression (fig. c, d) . using immunofluorescence confocal microscopy and coimmunoprecipitation (co-ip), we further revealed that lamtor is prominently associated with tlr during tlr engagement (fig. e, f) . thus, we have demonstrated for the first time that internalized tlr is trafficked to and resides at the autolysosome through the recruitment of lamtor . to further understand the interaction between lamtor and tlr , we generated a series of expression constructs containing truncated tlr fragments or lamtor fragments (fig. g) . loss of the leucine zipper (lz) domain significantly impaired the association of lamtor with tlr ( fig. h-j) . more specifically, when we substituted leu with leu (l → a), the key residue essential for the lz structure, the interaction between lamtor and tlr was inhibited (fig. k) . we further confirmed that the toll/il- receptor homology (tir) domain, rather than leucine-rich repeat (lrr) domain, within tlr is indispensable for this association (fig. l, m) . accordingly, mutation of the lamtor lz domain or deletion of the extracellular domain of tlr caused a profound reduction in the tir/lz interaction (fig. n, o) . in addition, md simulation , provided a structural basis to support the ligation of lamtor and tlr (fig. p) . therefore, our data demonstrated that lamtor directly associates with tlr and contributes to their colocalization at the lc + lamp + autophagosome for subsequent tlr hydrolysis. in support of this conclusion, lz-mutant lamtor lost its ability to interact with tlr and hence failed to suppress tlr levels (fig. q) . lamtor enhances lysosomal biogenesis through modulating the mtor/tfeb axis since lamtor was shown to recruit and colocalize with tlr at the autolysosome, a site generally thought to be occupied by mtorc upon amino acid induction, - we thus wondered whether mtorc participates in the lamtor /tlr interaction. indeed, our data demonstrated the docking of mtorc at the lamp + lysosome following lps stimulation; however, this fig. lamtor inhibits the lps-induced inflammatory response in vitro and in vivo. expression of il- , il- β, and tnf-α in control, lamtor expressing, and lamtor -silenced raw . cells following lps ( ng/ml) stimulation at either the mrna (a, b) or protein level (c, d). e levels of il- , il- β, and tnf-α in control or lamtor -expressing thp- cells stimulated with lps ( ng/ml) for the indicated time periods. f-j wt and lamtor +/− mice (n = ) were intratracheally instilled with lps ( mg/kg) and killed h later. representative h&e staining of lung sections (f); balf cytokine levels (g), cell counts (h) and protein leakage (i); and lung mpo activity (j). k wt and lamtor +/− mice (n = - ) were challenged with lps ( mg/kg, i.p.), and their survival was analyzed by the kaplan-meier method. all the data are from three independent experiments (a-e) and expressed as the means ± sds. *p < . , **p < . by student's t-test the metabolic regulator lamtor suppresses inflammatory signaling via. . . w zhang et al. docking was prevented when wt lamtor , but not mutant lamtor (Δlz), was expressed (fig. a) . this finding prompted us to further examine whether the lamtor /tlr association affects mtorc localization at the lysosome. intact tlr , but not the truncated fragment (lrr) of tlr , prevented the localization of mtorc at the lysosome (fig. b) . accordingly, associationincompetent lamtor or tlr , unlike the intact lamtor or tlr fragments (fig. h) , had no apparent effect on mtorc activation upon lps stimulation (fig. c) . activated mtorc has been demonstrated to bind and phosphorylate tfeb, a master transcription factor responsible for the expression of autophagy-lysosome genes, which then causes the cytoplasmic retention of tfeb and hinders its nuclear translocation to trigger gene transcription. , given that fig. lamtor negatively regulates tlr -driven signaling. a, b immunoblotting of total and phosphorylated ikk, iκbα, and p in control, lamtor -expressing and lamtor -silenced raw . cells stimulated with lps ( ng/ml) for the indicated time periods. c relative nf-κb promoter activity induced by lps in control, lamtor -expressing and lamtor -silenced raw . cells. d confocal microscopy analysis of p nuclear translocation upon stimulation with p . p immunoreactivity is shown in red, and nuclei are stained with dapi (blue). e immunoblotting of total and phosphorylated ikk, iκbα, and p in wt and lamtor +/− cells stimulated with lps ( ng/ml) for the indicated time periods. f, g immunoblotting of total and phosphorylated jnk, p , and erk in control, lamtor -expressing or lamtor -silenced raw . cells stimulated with lps ( ng/ml) for the indicated time periods. h, i immunoblotting of total and phosphorylated tbk and irf and j, k quantitative pcr to assess ifnβ production in control, lamtor -expressing, or lamtor -silenced raw . cells stimulated with lps ( ng/ml) for the indicated time periods. representative images are shown, and the data from three independent experiments are expressed as the means ± sds. *p < . , **p < . by student's t-test the metabolic regulator lamtor suppresses inflammatory signaling via. . . w zhang et al. quantification of lc puncta per cell is also shown (n = cells). g immunoblotting for lc in control and lamtor -expressing raw . cells pretreated with dmso or baf-a and subjected to lps stimulation for the indicated time periods. h, i immunoblotting for the indicated proteins in control, lamtor -expressing, and lamtor -silenced raw . cells stimulated with lps for the indicated time periods. j immunoblotting for tlr and total and phosphorylated p in control and lamtor -expressing raw . cells pretreated with dmso or baf and then stimulated with lps for the indicated time periods. k immunoblotting for tlr and total and phosphorylated p in control and lamtor -silenced raw . cells pretreated with dmso or torin and then stimulated with lps for the indicated time periods. l immunoblotting for tlr in control, lamtor -expressing, and lamtor -silenced raw . cells transfected with nonspecific control (nc) or atg -specific sirna, followed by lps stimulation for h. representative images are shown, and data from three independent experiments are expressed as the means ± sds. *p < . , **p < . by student's t-test lamtor interacts with tlr and promotes its tethering at autolysosomes upon lps stimulation. a, b colocalization of lamtor with lc or lamp and c, d colocalization of tlr with lc or lamp in control and lamtor -expressing raw . cells stimulated with lps for h. e colocalization of tlr with lamtor in raw . cells stimulated by lps for h. f coimmunoprecipitation of lamtor and tlr in t cells transfected with control, gfp-tlr -and/or flag-lamtor -expressing plasmids. igg was used as a control. g schematic illustration of the tlr structure and truncated tlr fragments (upper), the lamtor structure and truncated lamtor fragments (middle), and the lzmutant fragment (lower). h-k coimmunoprecipitation of lamtor and tlr in t cells transfected with plasmids harboring intact lamtor or truncated/mutant lamtor -expressing plasmids with or without tlr -expressing plasmids; l, m coimmunoprecipitation of lamtor and tlr in t cells transfected with plasmids encoding truncated tlr constructs and/or lamtor . n, o colocalization of tlr with lamtor in t cells transfected with intact lamtor -or mutant lamtor -expressing plasmid with or without tlr -expressing plasmid. p stereoview of the interaction between tlr (tir) and lamtor fragments using the i-tasser server. both tlr (tir) and the lamtor fragments are modeled as dimers, and the leucine zipper (lz) structure at the interactive interface is shown. q immunoblotting for tlr in raw . or thp- cells transfected with lamtor -or lamtor (Δlz)-expressing plasmids. quantified relative band intensities are also shown. representative images are shown, and the data from three independent experiments are expressed as the means ± sds. *p < . , **p < . by student's t-test mtorc activation was suppressed upon lamtor expression, the activity of its substrate, tfeb, is expected to be affected upon lamtor expression. indeed, the phosphorylation of tfeb was decreased upon lamtor overexpression but enhanced by lamtor knockdown following lps stimulation (fig. d) . the nuclear translocation of tfeb was thus boosted upon lamtor overexpression (fig. e) . consequently, the expression of autophagy-lysosome genes was upregulated in lamtor expressing macrophages but reduced in lamtor -silenced cells in response to lps stimulation (fig. f) . tem consistently demonstrated increased amounts of lysosome upon lamtor expression (fig. g) . moreover, our data indicated that lamtor overexpression increased, while lamtor knockdown hindered, lysosomal maturation, as detected with lysosensor green, a phsensing dye (fig. h) . to establish functional relevance, we showed that knockdown of tfeb largely abolished the lamtor mediated reduction in tlr levels ( fig. i and supplementary fig. s a ). this result appeared to be related to abrogation of the lamtor -induced increase in lc fluorescence (fig. j) . thus, the tfeb-driven autolysosomal pathway appears to be essential for lamtor -mediated regulation of tlr stability. we further tested this assumption by interfering with the autophagic pathway. treatment with rab sirna or the autophagic inhibitor vinblastin remarkably abrogated the suppressive effect of lamtor on tlr levels ( supplementary fig. s b, c) . in addition, the application of cq, a putative neutralizer of lysosomal ph, or leupeptin, a lysosomal protease inhibitor, restored tlr levels in lamtor -expressing macrophages ( supplementary fig. s d, e) . collectively, our data indicated that lamtor , through recruiting and binding tlr at the autolysosome, dislodges, and inactivates mtor and hence facilitates tfeb-driven autolysosomal processes. amino acid starvation reduces endotoxic shock via a lamtor dependent mechanism as lamtor plays an essential role in the amino acid-sensing pathway and nutrient status was shown to regulate the level of lamtor in macrophages (fig. j, k) , we wondered whether lamtor plays a role in coordinating immune and metabolic signaling. intriguingly, deprivation of the eaa leucine significantly repressed the production of il- , tnf-α, and il- β in lps-stimulated macrophages, which was at least partially abrogated upon the knockdown of lamtor (fig. a ). in addition, leucine insufficiency had an anti-inflammatory effect on macrophages from wt control but not lamtor +/− mice (fig. b ). in addition, tlr levels were reduced upon leucine deprivation in control but not lamtor silenced macrophages (fig. c) , implying that tlr is regulated by leucine deprivation in a lamtor -dependent manner. to further test this hypothesis in an in vivo system, we exploited a murine model of lung inflammation and injury induced by endotoxin challenge. lung inflammation and injury, as exhibited by proinflammatory cell infiltration, proinflammatory cytokine production, protein leakage and lung mpo activity, were alleviated in mice fed a leucine-free diet (leu-), but this effect was largely abrogated in lamtor haploinsufficient mice (fig. d-h) . moreover, mice fed a leucine-free diet exhibited a survival advantage over littermates fed a normal diet when challenged with a lethal dose of endotoxin. however, this elevated survival rate upon leucine deprivation appeared to be abrogated in lamtor +/− mice (fig. i) , implying that the antiinflammatory effect of leucine deletion is at least partially mediated through lamtor functioning. in the present study, we elucidate an unexpected key role for lamtor , a newly identified amino acid-sensing protein, in modulating tlr signaling and inflammatory responses. importantly, lamtor impacted tlr intracellular trafficking, promoted its autophagic degradation, and controlled inflammatory signaling. we demonstrate that the physical association of lamtor with tlr facilitated their colocalization at lc + lamp + autolysosomes and thereby hindered mtorc lysosomal tethering and activation upon lps stimulation, which in turn derepressed tfeb to activate the autolysosomal program and promote tlr degradation. accordingly, lamtor deficiency caused uncontrolled tlr signaling and increased susceptibility to endotoxic shock and lung injury; we have thus elucidated a hitherto unknown role of lamtor in tlr biology and identified a novel catabolic mechanism essential for immune homeostasis (fig. j) . interestingly, lamtor was induced by not only inflammatory insult but also by nutrient stress, specifically deprivation of the eaa leucine. lamtor , mostly likely through promoting tlr degradation, mediated the anti-inflammatory effect of leucine deprivation and contributed to the alleviation of inflammatory pathology in mice fed a leucine-free diet. we thus propose a lamtor -centered regulatory paradigm that integrates inflammatory and metabolic cues to shape immune and inflammatory responses; this model may have implications for tlr -associated metabolic inflammation. , an increasing amount of data indicate that cellular degradative system is a dynamic and complex vacuolar network that also involves the autophagosome, a canonical catabolic structure responsible for the clearance of intracellular molecules or organelles. tlr engagement on macrophages has been demonstrated to trigger the generation of lc + autophagosomes or promote lc recruitment to endosomes or phagosomes, resulting in activation of the autolysosomal pathway in an atg / dependent manner. , the autolysosomal machinery has been implicated in tlr-mediated responses, but its exact role and the involved mechanism remain largely unknown. our present study demonstrates that the cellular catalytic process controlled by lamtor is essential for the autophagic degradation of tlr and homeostatic control of inflammation. lamtor regulates tlr on at least two levels: the association and recruitment of tlr to degradation vacuoles and regulation of the autolysosomal pathway. lamtor specifically associates with tlr through the tir/lz interaction, as disruption of the lamtor lz domain or deletion of the tlr tir fragment almost completely abrogated their association and subsequent tlr degradation. computer-aided structural predictive analysis further substantiated the feasibility and reliability of the tir/lz interaction between these two molecules. we have thus identified lamtor as an unconventional adaptor without a tir domain capable of interacting with tlr in a site-specific manner that is essential for tlr recruitment to autolysosomes. indeed, lamtor has been reported to act as a scaffold in its interactions with rag gtpases, c-fos, c-myc, survivin, and p . [ ] [ ] [ ] intriguingly, the lz domain is required for the association of lamtor c-fos and c-myc but not survivin, implying that lamtor recognizes and interacts with different substrates with different site preferences. in addition, environmental conditions, particularly ph, were shown to affect the conformation of lamtor and hence its affinity to binding partners. a low ph ( . - . ) was favorable for a folded and hence more stable and accessible lamtor structure, whereas lamtor was more unstructured and unstable and bound its partners with a lower affinity in basic conditions (ph . ). this finding seems compatible with our currently proposed model that lamtor promotes the biogenesis and maturation of lysosomes upon lps stimulation, generating acidic, and amenable conditions for its association with tlr , whereas low levels of lamtor in resting macrophages yield immature and nonacidic lysosomes, preventing the binding of tlr to lamtor . thus, lamtor functioning serves as a ph-sensitive and lysosome-dependent feedback mechanism to control ongoing inflammation. aside from its role as an anchor for internalized tlr , lamtor also plays a critical role in promoting the autolysosomal pathway through regulating the mtor/tfeb axis. tlr signaling has been , our current study reveals that mtorc , which is controlled by lamtor , plays a unique role in promoting tlr signaling through counteracting autophagy. although the exact mechanism by which lamtor modulates mtorc is not understood at this stage, our initial data indicate that the association and colocalization of lamtor and tlr significantly hinders the lysosomal docking of mtorc and thus prevents its activation. mtorc may be inaccessible due to altered lysosomal architecture upon lamtor /tlr ligation or the formation of a compact tlr /lamtor structure that blocks mtorc tethering. this hypothesis is likely supported by the recent finding that the lamtor -containing ragulator complex forms a tightly bound supercomplex with v-atpase and rag gtpases that inhibits lysosome docking and activation of mtorc upon amino acid insufficiency. alternatively, the suppression of mtorc by lamtor might be attributed to its promotion of the activation of amp-activated protein kinase (ampk), a putative mtorc inhibitor, in response to lps stimulation (supplementary fig. s ) . intriguingly, the lamtor -mediated inhibition of mtorc is seemingly at odds with the requirement of lamtor for mtorc activation upon amino acid stimulation. this discrepancy might be attributed to the context-dependent function of lamtor . in fact, lamtor is not the sole ragulator member that exerts an inhibitory effect on mtorc activity. lamtor , another ragulator molecule, fig. lamtor partially mediates the anti-inflammatory effect of leucine deprivation. a production of proinflammatory cytokines by control and lamtor -silenced raw . cells or b macrophages from wt and lamtor +/− mice. cells were cultured in complete (leu + ) and leucinedepleted (leu − ) media and stimulated with lps for h. levels of the indicated cytokines in cellular supernatants were analyzed by elisa. c tlr levels in control and lamtor -silenced raw . cells cultured in complete (leu + ) and leucine-depleted (leu − ) media stimulated with lps for the indicated time periods. relative intensities were quantified and are shown below each corresponding band. d-h wt and lamtor +/− mice (n = ) were fed a normal (leu + ) or leucine-deficient (leu − ) diet for week and then received lps treatment ( mg/kg, i.t.) for h. cell counts (d), h&e staining of lung sections (e), balf cytokine levels (f) and protein leakage (g), lung mpo activity (h). all the data are from three independent experiments and expressed as the means ± sds. *p < . , **p < . by student's t-test. i wt and lamtor +/− mice (n = - ) were fed a normal (leu + ) or leucine-depleted diet (leu − ) and challenged with lps ( mg/kg, i.p.), following which the survival rate was analyzed by the kaplan-meier method. j a proposed working model indicating that lamtor plays dual roles in both associating with/ recruiting tlr and regulating the tfeb-driven autolysosomal pathway, thereby contributing to the autophagic degradation of tlr and inflammation termination. aa amino acid, ap autophagosome, al autolysosome, ly lysosome, lam lamtor , leu leucine, nuc nucleus the metabolic regulator lamtor suppresses inflammatory signaling via. . . w zhang et al. was shown to repress mtorc activity in dendritic cells (dcs), leading to rapid degradation of the fms-like tyrosine kinase receptor and limiting dc activity. lamtor can also promote the autolysosomal pathway, a process presumably repressed by mtorc , and plays a central role in epidermal development. these results reveal the intimate relationship between mtor and lamtor family members, which appears to be essential for immune homeostasis. despite an increasing appreciation for the importance of autolysosomes in immune and inflammatory responses, how this degradative machinery reacts to external signals and contributes to optimized immune and inflammatory responses remains elusive. we herein have identified tfeb, a master transcriptional factor for autolysosome genes, as an effector molecule of lamtor that translates inflammatory information into the regulatory system for tlr signaling. in response to nutrients and growth factors, tfeb is phosphorylated by mtorc and subsequently bound by - - proteins for cytosolic sequestration. , nutrient starvation, however, leads to mtorc inaction and the consequent depression of tfeb, allowing its entry into the nucleus to activate autolysosomal genes. our current study expands upon the functions of tfeb by demonstrating its importance in mediating the cellular response to pathogenic insults such as bacterial endotoxin. upon lps stimulation, tfeb is released from mtorc -mediated inhibition, which is suppressed by lamtor , promoting autolysosome biogenesis and expediting tlr processing. the mtor/tfeb axis appears to function as a balanced system that guarantees the tlr -driven response to be comprehensive but not excessive. another interesting finding of our present study is that lamtor plays a role in coordinating inflammatory and metabolic cues to shape the optimized host response. lamtor , most likely through accelerating the autophagic degradation of tlr , mediated the anti-inflammatory effect of leucine deprivation and protected against endotoxic sepsis in mice fed a leucine-free diet; these results may confer mechanistic insight into so-called "starving inflammation". recently, zhao et al. identified transmembrane bax inhibitor motif-containing (tmbim ) as a negative regulator of tlr signaling that also plays a role in suppressing hepatic steatosis and inflammation. similar to our findings, the multivesicular body-lysosomal pathway was identified as a key mechanism responsible for tmbim -mediated tlr degradation and immune and metabolic balance. given that the tlr pathway is frequently aberrantly activated during metabolic diseases, , further studies on the potential role of lamtor in tlr -associated metabolic inflammation may be warranted. in summary, we have uncovered hitherto unknown roles of lamtor in regulating tlr fate and maintaining immune homeostasis. we also show that lamtor is essential in coordinating nutrient and inflammatory cues to shape the optimized immune response, which may shed new light on immunometabolic disorders. the role of pattern-recognition receptors in innate immunity: update on toll-like receptors cellular and molecular regulation of innate inflammatory responses origin and physiological roles of inflammation innate immune pattern recognition: a cell biological perspective assembly and localization of toll-like receptor signalling complexes tram couples endocytosis of toll-like receptor to the induction of interferon-beta the history of toll-like receptorsredefining innate immunity lysosome-associated small rab gtpase rab b negatively regulates tlr signaling in macrophages by promoting lysosomal degradation of tlr recycling endosomes and tlr signaling-the rab gtpase leads the way ras-related protein rab facilitates tlr signaling by promoting replenishment of tlr onto the plasma membrane the cop ii adaptor protein tmed is required to initiate and mediate the delivery of tlr to the plasma membrane a promiscuous lipid-binding protein diversifies the subcellular sites of toll-like receptor signal transduction cytoplasmic linker protein clip negatively regulates tlr signaling by targeting the tlr adaptor protein tirap autophagy at the crossroads of catabolism and anabolism autophagy: renovation of cells and tissues autophagy and human diseases crosstalk between autophagy and inflammatory signalling pathways: balancing defence and homeostasis loss of the autophagy protein atg l enhances endotoxininduced il- beta production alphav integrins combine with lc and atg to regulate toll-like receptor signalling in b cells toll-like receptor is a sensor for autophagy associated with innate immunity toll-like receptor signalling in macrophages links the autophagy pathway to phagocytosis trim -tax bp -dependent selective autophagic degradation of trif negatively regulates tlr / -mediated innate immune responses nrf -mediated induction of p controls toll-like receptor- -driven aggresome-like induced structure formation and autophagic degradation autophagy-dependent peli degradation inhibits proinflammatory il b expression cloning and characterization of a novel hepatitis b virus x binding protein that inhibits viral replication hbxip and lsd scaffolded by lncrna hotair mediate transcriptional activation by c-myc hbxip functions as a cofactor of survivin in apoptosis suppression the oncoprotein hbxip modulates the feedback loop of mdm /p to enhance the growth of breast cancer the metabolic regulator lamtor suppresses inflammatory signaling via ragulator is a gef for the rag gtpases that signal amino acid levels to mtorc ragulator-rag complex targets mtorc to the lysosomal surface and is necessary for its activation by amino acids mtorc senses lysosomal amino acids through an inside-out mechanism that requires the vacuolar h(+)-atpase slc a is a component of the lysosomal amino acid sensing machinery that controls mtorc guanine nucleotide exchange factor rabgef regulates keratinocyte-intrinsic signaling to maintain skin homeostasis rasgrp limits toll-like receptor-triggered inflammatory response in macrophages by activating rap small gtpase regulation of t cell receptor signaling by dennd b in th cells and allergic disease tfeb links autophagy to lysosomal biogenesis multiplex genome engineering using crispr/cas systems mapk kinase potentiates chlamydia hsp -induced inflammatory response through distinct activation of nf-kappab the amino acid sensor gcn controls gut inflammation by inhibiting inflammasome activation snapin is critical for lysosomal acidification and autophagosome maturation in macrophages beclin -binding uvrag targets the class c vps complex to coordinate autophagosome maturation and endocytic trafficking i-tasser server: new development for protein structure and function predictions structural basis for signal transduction by the toll/interleukin- receptor domains scalable molecular dynamics with namd parallel generalized born implicit solvent calculations with namd structural characterization of hbxip: the protein that interacts with the anti-apoptotic protein survivin and the oncogenic viral protein hbx the structural basis of lipopolysaccharide recognition by the tlr -md- complex a lysosome-to-nucleus signalling mechanism senses and regulates the lysosome via mtor and tfeb a novel assay to study autophagy: regulation of autophagosome vacuole size by amino acid deprivation toll-like receptors: linking inflammation to metabolism immunometabolism governs dendritic cell and macrophage function toll-like receptors control autophagy modulation of inflammation by autophagy: consequences for human disease scimp is a transmembrane non-tir tlr adaptor that promotes proinflammatory cytokine production from macrophages the tsc-mtor signaling pathway regulates the innate inflammatory response rab a interacts directly with pi kgamma to modulate tlr -driven pi k and mtor signalling disruption of the ragragulator complex by c orf inhibits mtorc ampk phosphorylation of raptor mediates a metabolic checkpoint lamtor regulates dendritic cell homeostasis through flt -dependent mtor signalling the lysosomal signaling anchor p /lamtor controls epidermal development by regulating lysosome-mediated catabolic processes mtorc functions as a transcriptional regulator of autophagy by preventing nuclear transport of tfeb metabolic instruction of immunity tmbim is a multivesicular body regulator that protects against non-alcoholic fatty liver disease in mice and monkeys by targeting the lysosomal degradation of tlr the serine protease prostasin regulates hepatic insulin sensitivity by modulating tlr signalling fetuin-a acts as an endogenous ligand of tlr to promote lipidinduced insulin resistance the online version of this article (https://doi.org/ . /s - - - ) contains supplementary material.competing interests: the authors declare no competing interests. key: cord- -btf ju authors: sun, zhiheng; pan, yuchen; qu, junxing; xu, yujun; dou, huan; hou, yayi title: β-estradiol promotes trained immunity in females against sepsis via regulating nucleus translocation of relb date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: btf ju sepsis is more common among males than females, and the unequal estrogen levels have been suspected to play a vital role in gender differences. recently, trained immunity is reported to be a novel strategy for the innate immune system to fight infection. however, it has not been clarified whether β-glucan-induced trained immunity causes different responses to early sepsis between male and female mice. in this study, sepsis was induced in mice by intraperitoneal injection of escherichia coli (e. coli). the changes of inflammatory cytokines expression, and macrophage polarization in male, female, and ovariectomized c bl/ mice in sepsis model were investigated. for in vitro studies, different macrophages were treated with lps. the function of estradiol (e ) on macrophage cell lines was verified and the mechanism of e affecting trained immunity was explored. we demonstrated that β-glucan-induced trained immunity was more resistant to sepsis in female than male mice. macrophage polarization toward the m phenotype, which exhibited enhanced trained immunity, was related to the difference in sepsis resistance between female and male mice. moreover, ovariectomized (ovx) mice manifested serious sepsis consequences with a weaker trained immunity effect than female mice. female bone marrow-derived macrophages (bmdms) were also apt to be polarized to the m phenotype in response to trained immunity in vitro. furthermore, e promoted trained immunity in macrophage cell lines j and raw . . e was also verified to facilitate trained immunity in primary bmdms from female and male mice. mechanistically, we found that e inhibited the nuclear translocation of relb, which is a member of non-canonical pathway of nfκb and contributes to macrophage polarization to change the intensity of trained immunity. this study is the first to indicate the role of e in the trained immunity induced by β-glucan to protect against e. coli-induced sepsis via the non-canonical nfκb pathway. these results improve our understanding of the molecular mechanisms governing trained immunity in gender differences. sepsis is a systemic reaction, which can even be caused by an ordinary infection ( ) . the infection is most commonly bacterial but also can be fungal, viral, or parasitic. since many organs are affected, the mortality rate of sepsis is - % ( ) . with the development of modern medicine, the mortality rates have declined to about % ( ), but this is still far from acceptable. the biomarker of sepsis is widely discussed by a number of reviews ( ) ( ) ( ) ( ) . several important signs of sepsis are damage to the kidney, liver, and lung, as well as an increased bacterial load in the kidney and elevated levels of transaminase and lactate in serum ( ) . moreover, the common model of sepsis is an immune response to escherichia coli or endotoxin, lipopolysaccharide (lps), found in the cell wall of gram-negative bacteria. endotoxin is an excellent example of a pathogen-associated molecular pattern (pamp). of note, a number of studies indicate gender dimorphism in terms of response to sepsis ( ) . the intuitive result is that the incidence of female sepsis is much lower than that of males ( , ) . furthermore, some studies attributed these differences to the prevailing hormonal milieu of the victim ( ) ( ) ( ) . thus, it is necessary to explore the mechanism of estrogen in modulating immunity, which allows females to resist sepsis. the nfκb (nuclear factor kappa-b) family of transcription factors constitutes five members (rela or p , relb, crel, nfκb or p , and nfκb or p ), all of which play important roles in cell homeostasis, especially in the immune process ( ) . apart from the well-known canonical signaling pathway, the noncanonical pathway is also involved in vital immune processes, which could be triggered by signaling from a subset of tnfr members. this pathway mediates the persistent activation of relb/p complex with the ability to modulate a series of gene expression, including macrophage polarization-associated cytokines. to date, a growing number of studies indicated that estrogen is involved in some immune processes. estrogen can bind to and activate estrogen receptors (ers), which regulate the expression of downstream genes ( ) . the interaction between ers and nfκb is mainly discussed in breast cancer articles ( ) . a highly significant negative correlation between the expression of nfκb target genes and er activation was found. some studies abbreviations: e , -β-estradiol; ti, trained immunity; lps, lipopolysaccharide; pbs, phosphate buffer saline; ast, aspartate aminotransferase; alt, alanine aminotransferase; il- , interleukin- ; tnfα, tumor necrosis factor α; il- , interleukin- ; il- , interleukin- ; m-csf, macrophage colony-stimulating factor. demonstrated the interaction of er with nfκb; ers can compete with nfκb for binding to transcriptional coactivators (ie, creb) or ers to recruit co-suppressors into nfκb complexes ( ) . ers can inhibit de novo relb synthesis in breast cancer tissues and cell lines. in addition, e inhibits the nucleus translocation of p , c-rel, and relb without affecting p in mouse splenocytes ( ) . these data suggest that estrogen-er signaling regulates the nfκb pathway at the transcriptional level of its constituents. the host's immune defense mechanisms can be divided into innate immunity and adaptive immunity. adaptive immunity, although slower than the innate immune response, has good specificity and produces immunological memory ( ) . recently, researchers discovered an ability in innate immunity, similar to immune memory in adaptive immunity, called trained immunity ( ) . trained immunity is found in mammals and its basic features have been identified by several researchers. trained immunity mainly involves a set of cells (myeloid cells, natural killer cells, and innate lymphoid cells) ( ) . antituberculosis vaccine bacillus calmette-guérin (bcg) is a wellknown immune modulator that induces trained immunity. it was reported that estrogen did not influence the induction of trained immunity by bcg, and did not induce training or tolerance in monocytes themselves, indicating that the estrogen is unlikely to explain the sex-differential effects after bcg vaccination ( ) . in fact, β-glucan is a major cell wall component of c. albicans and can induce trained immunity in monocytes. the initiation of trained immunity is associated with enhanced signaling of the akt (protein kinase b)-mtor (mammalian target of rapamycin)-hif- α (hypoxia-inducible factor- α) pathway ( ) , modifications in metabolic pathways (conversion to glycolysis), and epigenetic rewriting ( , ) . however, the effect of estrogen on β-glucan-induced trained immunity to resist sepsis has not been clarified. β-glucan (g- ) and β-estradiol ( - - ) were purchased from xiensi biotechnology company (tianjin, china). m-csf (#cb ), ifn-γ (#c ), and tnfα (#cf ) were purchased from novoprotein (shanghai, china). e and lps was purchased from sigma (st. louis, mo, usa). antibody against p-akt (ser , # ), akt (# ), p- ebp (# ), ebp (thr / , # t), p-s (ser / , # ), s (# ), pcna (# ), p (# ), estrogen receptor α ( s), gapdh (# ), and β-actin (# ) were purchased from cell signaling (boston, mo, usa). relb (sc- ) was purchased from santa cruz biotechnology (dallas, tx, usa). f / -apc, f / -fitc, cd -pe, cd -apc, cd -fitc, inos-pe were all from biolegend (san diego, ca, usa). raw . , j , and hek t cells were obtained from the type culture collection of the chinese academy of sciences, shanghai, china. cells were cultured in phenol red-free dmem with % fbs, % ( u/ml penicillin and ug/ml streptomycin) at • c in an atmosphere of % air and % co . cells were seeded onto different types of plates for further experiments when the cell density reached ∼ %. the cells were used within a maximum of five passages. the in vitro trained immunity model was established with raw . and j . cells were challenged with µg/ml βglucan for h. the cells were then washed and rested in culture medium for days. next, cells were treated with ng/ml lps for h, and then the cytokines were measured in mrna level. × bmdms were seeded in -well plates ( µl final volume; corning) and stimulated with µg/ml β-glucan for h. then cells were washed and rested for days in culture medium. on day , bmdms were washed again and treated with ng/ml ifn-γ for h. on day , a final wash was performed, and cells were primed with µg/ml lps. the supernatants were harvested for elisa assay after h of lps stimulation. other experiments were all based on the same model but with different numbers of bmdms. to assess mrna expression and cell viability, × bmdms were plated in non-treated well plates ( , ml final volume; corning) and followed the training scheme described above. for western blotting (wb) assays × bmdms were plated in six-well plates ( ml final volume; corning). male and female c bl/b mice were purchased from the model animal research center of nanjing university. mice used in the model of trained immunity and sepsis were weeks old. all animal procedures were performed in accordance with guidelines of the us nih with specific pathogen free conditions. soyfree standard rodent chow and water were provided ad libitum. female mice were anesthetized with % chloral hydrate and then underwent ovariectomy (ovx) at weeks of age. when they were weeks old, the same model of trained immunity and sepsis was established. peripheral blood mononuclear cells (pbmcs) were separated from mouse plasma by ficoll centrifugation using lymphocyte separation medium from mp biomedicals (solon, usa) according to the standard procedures. mice were sacrificed via cervical dislocation and sterilized by soaking in % ethanol. dissected the legs and bone marrow was extracted from tibia and femur bones by using a -gauge needle and a ml syringe filled with pbs following removal of surrounding muscle. blow the bone marrow gently and spread it through a µm cell strainer. the cell suspension was centrifuged at g for min at room temperature. cells were cultured in phenol red-free dmem with % ultra-low endotoxin fbs and m-csf ( ng/ml). after changing the fresh medium on the third and fifth day, bmdm was induced. the e. coli strains were purchased from atcc, collected, and identified by the medical laboratory center of zhongda hospital in nanjing jiangsu, china, and stored at − • c. bacterial strains were prepared in lb medium. a nuclear protein extraction kit was purchased from biyuntian (wuhan, china) and used according to the manufacturer's instructions. sirna transfection sirna was transfected according to the product instructions (ruibo company, china). the concentration of sirna used in the study was nm. the erα sirna target sequence is tgcacattgaagatgctga. the target sequence of non-coding (nc) sirna was a random sequence with no biological effects. to assess the effect of e on cell viability, a cck- assay was used according to the manufacturer's instructions (bioss company, china). raw . /j were seeded onto -well plates at a concentration of ∼ × cells/well. different concentrations of e were used to treat cells for different length of time as described in the article. total rna was extracted from cells using trizol reagent, and reverse transcriptions were performed in a µl mixture with µg of total rna according to the manufacturer's instructions (vazyme company, china). the oligonucleotide primers used for pcr amplification are listed in table . pcr amplification consisted of cycles of denaturation at • c for min, annealing at • c for s, and extension at • c for min. all reactions were run in triplicate. the gene expression levels were normalized to ß-actin. the protein samples were obtained from lysis buffer treated cells. cell lysates were put on ice for min and then centrifuged at , × g for min. subsequently, µg of protein per lane was separated on % polyacrylamide gels and transferred onto polyvinylidene difluoride membranes (millipore, billerica, ma, usa). membranes were blocked with % bovine serum albumin (bsa) in tris-buffered saline containing . % tween , and then the membranes were incubated with specific antibodies. the values were normalized to the β-actin/gapdh intensity levels. mice were trained with two intraperitoneal (i.p.) injections of mg β-glucan particles on days − and − . sterile pbs was used as a control. on day , mice were challenged with . × e. coli. the lung, kidney, and serum were harvested h after e. coli treatment. the fresh lung tissues were fixed in % paraformaldehyde (pfa). then, the samples were gradually dehydrated and embedded in paraffin. after that, the samples were cut into µm sections and stained with hematoxylin and eosin for further light microscopy observation. scores were evaluated by a pathologist based on the lung tissue integrity, alveolar integrity, and mononuclear infiltration ( = none; = mild; = moderate; = severe). cultured cells were seeded on glass coverslips in six-well plates. after three pbs washes, the samples were fixed for min at room temperature with % paraformaldehyde. fixed cells were rinsed with pbs and then incubated for min at • c with . % triton x- and . % bsa in pbs. following permeabilization, non-specific binding in the cells was blocked by % bsa in pbs for h at room temperature. cell samples were incubated with anti-erα, anti-relb, and anti-p primary antibodies at a : dilution for h at room temperature. samples were further incubated with alexa fluor -conjugated and alexa conjugated secondary antibody at a : dilution for . h in the dark. after washed with pbs, the nuclei were stained by dapi. slides were visualized using a nikon eclipse ti-u fluorescence microscope equipped with a digital camera (ds-ri , nikon). the protein concentration of il- , lactate, tnfα, and estrogen in cell supernatant or mouse serum were detected using the corresponding mouse enzyme-linked immunosorbent assay (elisa) kit according to the manufacturer's instructions (biolegend, china). the kidney e. coli burden at indicated time points was measured by plating organ homogenates obtained mechanically over µm cell strainers (bd biosciences) following slicing the tissue, in serial dilutions on lb agar plates; colony-forming units (cfus) were counted after growth at • c for h, and data are shown as cfus in total kidney. pbmcs or bmdms were filtered through a µm cell strainer and then washed with complete rpmi medium to generate single-cell suspensions. an fc-receptor blocker (cd / , ebioscience) was used to reduce non-specific antibody binding. antibodies used in these experiments included f / -apc, f / -fitc, inos-pe, cd -apc, and cd b-pe. stained samples were detected by a facs calibur flow cytometer (bd bioscience) and data were analyzed using flowjo software (treestar, ashland, or). the statistical analysis was performed using prism (prism for windows, graphpad software inc., usa). unless specified, statistical significance for comparison between two sample groups with a normal distribution (shapiro-wilk test for normality) was determined using two-tailed paired or unpaired student's t-test. differences were considered significant at p < . as indicated. except when specified, only significant differences are shown. as indicated in figure legends, either a representative experiment or a pool is shown, and the number of repetitions of each experiment and number of experimental units (either cultures or mice) is indicated. the results are presented as the means ± standard error (sem). several studies have shown that women have better survival and tolerance to sepsis than men ( , ) . thus, we further verified this phenomenon by establishing a sepsis model in mice ( figure a) . the difference in lung injury between male and female mice in sepsis was determined by h&e analysis of lung sections (figure ba) . the histochemical scores (figure bb ) indicated that male mice had more severe lung damage than female mice; trained immunity prevented lung injury in mice and the protective effect of trained immunity was better for females than males. notably, we observed a decreased renal e. coli burden in females than males. in addition, in trained immunity mice, females had less renal e. coli burden than males (figures c,d) . in addition, liver damage markers, aspartate aminotransferase (ast), and alanine aminotransferase (alt) were increased in males than females; and trained immunity markedly decreased ast and alt concentration, with a greater decrease in females than in males (figures e,f) . we found that in the sepsis model, the serum lactate concentration of the male mice was higher than that of the female mice. in the trained immunity group, serum lactate was decreased, with the level in the females lower than males ( figure g ). as expected, male serum e content is much lower than female mice ( figure h) . our results showed that females expressed higher il- and tnfα than males in sepsis, and trained immunity exacerbated this trend (figures i,j) . macrophages are the most significant cells in the body in regard to trained immunity ( ) . the promoted trained immunity is related to the increased m phenotype macrophage. in order to understand the different response to sepsis between the different genders, we further investigated the polarization of macrophages during the process. thus, we focused on macrophage content and polarization. by detecting the percentage of macrophages in the spleen, we found that macrophages in female mice are more abundant than male mice, and trained immunity significantly increased the content of female macrophages (figure a) . sepsis promoted the polarization of macrophages to m and trained immunity aggravated this trend. notably, females showed more changes than males (figure b) . on the other hand, females inhibited the polarization of m macrophages ( figure c) . trained immunity simulated m type polarization of macrophages, and this effect was more pronounced for females. by analyzing the experiments above, we found that the difference between male and female responses to sepsis was due to the difference in macrophage polarization in vivo, and the difference in estrogen content in the different genders was very significant. to verify that differences in the estrogen level play a vital role, ovx mice were made, since the ovary is the main organ to produce e . the same sepsis model was established with ovx mice (figure a) . firstly, the serum e concentration was significantly decreased (figure a) . the degree of lung injury was much higher than that of female mice as well as ti + e. coli group (figure b) . the kidney e. coli burden was also evaluated. the results showed that ovx mice also had more severe kidney injury ( figure c ) as well as lung injury, as measured by serum alt (figure d ) and ast ( figure e ) concentrations, in comparison to female mice. with a lower serum il- and tnfα concentration in ovx mice in the ti + e. coli group, the data suggested that ovx mice have a lower trained immunity response than female mice (figures f,g) . finally, similar to male mice, ovx mouse pbmcs began polarizing to m -type at h after i.p. e. coli ( figure h ). taken together, these results demonstrated that e does facilitate trained immunity in the body to resist sepsis, and simultaneously inhibits macrophage polarization to m . since the macrophages are mostly derived from bone marrow cells, and also the cytokines up-regulated in trained immunity are pro-inflammatory cytokines. although the upregulation of inflammatory cytokines after trained immunity of macrophages is determined, no research has studied the polarization of macrophages during this process. therefore, exploring the polarization of bmdm as an important primary macrophage in trained immunity is very significant and necessary to understand the nature of trained immunity. to determine whether bmdms are similar to macrophage polarization in pbmcs, we tested the polarization of microphages from male or female mice when challenged with lps or trained immunity plus lps. we considered f / + inos + bmdms as m -type bmdms and f / + cd + bmdms as m -type bmdms. the results demonstrated that female bmdms are more polarized to m in both the lps group ( . - . %) and ti + lps group ( . - . %) ( figure a) . also, male bmdms began to transform to m in h, which did not occur in female bmdms. trained immunity also prevented both male and female bmdms from m polarization as well as pbmcs (figure b ). macrophage cell lines j and raw . in order to examine the effect of estradiol on trained immunity, we decided to pre-treat the macrophage cell lines with estradiol before trained immunity model. since the concentration of estradiol that can stimulate the macrophage cell lines raw . and j is unknown, we made a concentration gradient of estradiol on the activity of the macrophage cell lines. the results showed that the response of the macrophage cell lines to the stimulation of estradiol is not particularly sensitive, and the viability of j begins to increase significantly under the stimulation of nm estradiol. thus, the concentration of e used in cell experiments was verified as nm (figures a,b) . the in vitro trained immunity model was established with raw . and j ( figure c ) cell lines derived from male and female mice, respectively. using tnfα and il- β as the marker of inflammation, our data suggested that e can induce stronger trained immunity both in raw . and j (figures d,e) . we also made a trained immunity model of bmdms (figure a ). m-csf was used to induce bmdm with a purity of over % from both male and female mice ( figure b) . then, the mrna levels ( figure c ) and protein levels ( figure d ) of tnfα and il- were measured. the results suggested that female bmdms can have a more intensive response to lps as well as a trained immunity response than male bmdms. phosphorylation of akt and mtor targets (s and ebp ) are considered to be key hallmarks of trained immunity ( ) . to clarify the effect of e on trained immunity, these hallmarks are checked by western blot, which indicated that e as well as β-glucan induced trained immunity in bmdms ( figure e ). in addition, the data showed that e further boosted the polarization of m -type macrophage in trained immunity. consistently, this effect of e is more apparent in female bmdms, and e inhibited m polarization in trained immunity (figure f ). nfκb signaling pathway is involved in the regulation of many cellular physiological processes. previous articles have reported that relb −/− mice, or inhibiting the nucleus translocation of relb, led to a higher basal inflammatory level ( , ) . other reviews declared that e could regulate nuclear translocation of nfκb through ers. since it has been widely studied that tnfα can combine with tnfr on the cell membrane surface to promote nfκb translocate into the nucleus. also, because tnfα as a representative inflammatory cytokine is secreted by macrophages and will stimulate back to macrophage. we use tnfα as a positive control to study nfκb entry into the nucleus. we use immunofluorescence to explore the intracellular localization of nfκb protein. in immunofluorescence, the results indicated that when tnfα is used to stimulate cells, it does not affect the colocalization of erα and the nucleus but increases the colocalization area of p and relb with the nucleus. on the contrary, e treatment appeared to induce nuclear positioning of erα and kept relb distribution in the cytoplasm in both hek t and bmdms (figures a,b) . however, stimulation of e did not affect the distribution of p in cells. next, we used the rna interference system to further verify the impact of e on the nuclear translocation of relb. the silencing performance of interfering small rna sequence was evaluated both at the mrna level ( figure c ) and protein level ( figure d) . by extracting nuclear protein, we found that estrogen could reduce the content of relb protein in the nucleus; by silencing the expression of erα, the content of relb protein in the nucleus was also restored ( figure e ). as markers of m macrophage polarization, il- and il- are also the downstream genes regulated by relb ( , ) . we found that e inhibited the mrna expression level of il- and il- by regulating nuclear translocation of relb ( figure f ). in conclusion, these results indicated that e could block relb translocation to the nucleus; to further inhibit m -associated gene expression, e suppressed bmdms m polarization. during the development of sepsis, many systemic organ abnormalities occur including damage to the lung, liver and kidney; the normal operation of the body's neurosensory system may even be affected ( ) . since lps is a component in the cell wall of gram-negative bacteria, it is wildly used as an ideal stimulus to establish sepsis models. when tlr on figure | female bmdms are apt to be polarized to m phenotype in responses to trained immunity in vitro. (a) female bmdms m polarization was stronger than male bmdms treated with lps or ti + lps groups. (b) very few female bmdms to polarized to m in lps group, while male bmdms showed the polarization of m phenotype. ti suppressed bmdms to m polarization from both male and female mice (n ≥ /group). ***p < . , paired student's t-test comparing lps group and ti + lps group in the same gender. # p < . , ### p < . , paired student's t-test comparing same group between different genders. the macrophage cell membrane recognizes lps, a series of signaling pathways will be initiated to stimulate macrophages to produce pro-inflammatory cytokines, which can induce potent systemic inflammatory responses in vivo. therefore, tnfα, il- β, and il- are currently considered to be markers of the early phase of sepsis ( ) . the development of sepsis begins with systemic inflammatory response syndrome (sirs) ( ) , which is considered as the early phase of sepsis. elevated expression of pro-inflammatory cytokines could help the body return to a normal state in early sepsis ( ) . after sirs, the body enters the cars (compensatory anti-inflammatory response syndrome) stage. in the cars stage, the immune system will undergo the decline-regulation ( ) . the hyper-inflammatory response of sirs will normally be restored by the subsequent cars stage. the patients can return to normal state after the cars stage ( ) . however, if the cars phase fails, it can cause death from sepsis. as one of the important ways to protect the body from foreign microbes and antigens, the role of trained immunity is very significant. this study focused on the effects of e on early sepsis by promoting trained immunity. in the development of early sepsis, estrogen induced a stronger trained immunity response of macrophages to remove antigens and microorganisms from the body. simultaneously, estrogen promoted the polarization of macrophages to m in vivo and inhibited the polarization of m , which is considered anti-inflammatory in the early phase of sepsis. in summary, estrogen can maintain the higher proinflammatory state of macrophages. this also illustrates the reason why females are more tolerant to sepsis than males in one aspect. however, further research is needed to understand the role of estrogen in the later stages of sepsis. the mrna levels of tnfα and il- β in raw . were detected by qpcr to determine the trained immunity effect with or without e (n ≥ /group). # p < . , ### p < . , paired student's t-test comparing β-glucan + lps group and lps group. *p < . , ***p < . , paired student's t-test comparing with control group. ∧p < . , ∧∧p < . , and ∧ ∧ ∧p < . , paired student's t-test comparing between β-glucan + lps groups with or without e . here, we demonstrated that β-glucan-induced trained immunity was more resistant to sepsis in female than male mice. ovx mice manifested serious sepsis consequences with a weaker trained immunity effect than female mice. macrophage polarization toward m phenotype is thought to exhibit the enhanced trained immunity. e promoted trained immunity in vitro and in vivo. inhibition of e on the nucleus translocation of relb contributed to macrophage polarization to change the intensity of trained immunity. our results indicated that e is involved in the trained immunity in gender differences. since trained immunity was discovered in , although there are many articles about trained immunity, there is no article to find the fundamental way of trained immunity activation but only about the changes of immune cell characterization after trained immunity activation. such as epigenetic changes, upregulation of glycolysis, autophagy level, akt phosphorylation, and so on. in this study, we found that e promotes trained immunity by suppressing relb entry into nucleus, while inhibiting macrophage polarization to m in inflammation (a way reverse the inflammatory response). it is possible that e can change the epigenetics of macrophages through estrogen receptors to affect the trained immunity. this hypothesis needs further researches. trained immunity is a way of immunization that occurs in the innate immune cells through the first stimulation to fight the later secondary infections. by obtaining trained immunity, the body can fight various infections from bacteria, fungi and even viruses timely. it is worth mentioning that in countries and regions with underdeveloped scientific research capabilities, it may be an economical and quick means of protection against covid- to enhance people's trained immunity ability. sepsis is a clinically common infectious disease with obvious gender differences. in this article, sepsis model is used as an infection model to evaluate the role of estradiol in enhancing trained immunity. the mechanism and effects of estradiol in promoting trained immunity also need subsequent study in other types of infectious diseases. the purpose of this paper was to explore the reasons why different genders have different tolerance effects on sepsis. an article reported that estradiol and dht (dihydrotestosterone) did not influence bcg-induced trained immunity, and the article only focused on the inflammatory cytokines of monocytes induced by bcg in vitro ( ) . apart from this, almost no articles study the effects of e on trained immunity and its biological significance. our research attempted to explain its effects on trained immunity and understand how it causes women to be more tolerant of sepsis than men. however, in terms of gender differences, many factors, such as androgen concentration, genetic differences, metabolic differences, and physiological differences may all be the result of different tolerances for sepsis between genders ( , , ) . gender differences are a very complex and exquisite scientific issue ( ) , and all of the factors that contribute to gender differences are worth exploring for subsequent experiments. in summary, we demonstrated that as one of the key factors for gender differences, e plays a vital role in the stronger tolerance of females to sepsis than males. one the one hand, e can better facilitate trained immunity by increasing expression of inflammatory cytokines and inducing macrophage m -type polarization; on the other hand, e can inhibit or delay the macrophage m -type polarization by regulating nucleus translocation of relb and its down-stream m -associated genes. thus, this may provide a new idea for the clinical treatment of sepsis. drugs with a structure similar to estrogen may be developed to treat sepsis. more importantly, the treatment of some diseases may able to find better solutions by addressing the different characteristics of different genders. the datasets generated for this study are available on request to the corresponding author. the animal study was reviewed and approved by model animal research center of nanjing university. all authors reviewed the manuscript, contributed to data analysis, drafting and revising the article, gave final approval of the version to be published. zs conceived and designed the project. zs, yp, jq, and yx participated in the animal experiments. zs and jq wrote the manuscript. yh and yx revised it. biomarkers of sepsis two decades of mortality trends among patients with severe sepsis: a comparative meta-analysis incidence and trends of sepsis in us hospitals using clinical vs claims data biomarker cruises in sepsis: who is the captain? discussion on "circulating biomarkers may be unable to detect infection at the early phase of sepsis in icu patients: the captain prospective multicenter cohort study sepsis pathophysiology, chronic critical illness, and persistent inflammationimmunosuppression and catabolism syndrome penkid: a novel biomarker of reduced gfr in sepsis adaptation of a biomarker-based sepsis mortality risk stratification tool for pediatric acute respiratory distress syndrome reconsidering lactate as a sepsis risk biomarker gender differences in sepsis: genetically determined? shock effects of gender on the severity of sepsis sexual dimorphism in bacterial 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bcg-induced trained immunity mtor-and hif- alpha-mediated aerobic glycolysis as metabolic basis for trained immunity epigenetic programming of monocyte-to-macrophage differentiation and trained innate immunity increased acetylation of h k in the genomic regions that encode trained immunity enzymes in lysophosphatidylcholine-activated human aortic endothelial cells-novel qualification markers for chronic disease risk factors and conditional damps severe sepsis and septic shock epidemiology of severe sepsis in the united states: analysis of incidence, outcome, and associated costs of care trained immunity: a smart way to enhance innate immune defence differential involvement of relb in morphine-induced modulation of chemotaxis, no, and cytokine production in murine macrophages and lymphocytes relb regulation of chemokine expression modulates local inflammation relb cellular regulation and transcriptional activity are regulated by p year in review in intensive care medicine, . i. brain injury and neurology, renal failure and endocrinology, metabolism and nutrition, sepsis, infections and pneumonia sepsis and septic shock: a review sepsis: a review of advances in management review: immunology of sinusitis, trauma, asthma, and sepsis review on sepsis in children did not mention important trial clinical outcome and risk factors of neonatal sepsis among neonates in felege hiwot referral hospital systematic review of gender differences in sepsis management and outcomes the influence of gender on the epidemiology of and outcome from severe sepsis age and sex influence the hippocampal response and recovery following sepsis the authors appreciate the warm-hearted help from all the co-authors and the professor. key: cord- -ujzrqzv authors: lu, xiaying; wang, juan; chen, xiaohuan; jiang, yong; pan, zhixing k. title: rolipram protects mice from gram-negative bacterium escherichia coli-induced inflammation and septic shock date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: ujzrqzv sepsis is typically triggered by an overwhelming systemic inflammatory response to pathogens, and may lead to severe organ dysfunction and/or death. sepsis consequently has a high mortality rate and a high rate of complications for survivors, despite modern medical advances. therefore, drug identification and validation for the treatment of sepsis is of the utmost importance. as a selective phosphodiesterase- inhibitor, rolipram also exhibits the abilities of inhibiting multiple pro-inflammatory cytokines production in macrophages and toxin-induced inflammation in mice. however, this drug has never been studied as a sepsis treatment method. we found that rolipram significantly improves survival in mice challenged with gram-negative bacterium e. coli, clp, or e. coli derived lipopolysaccharide. we have also found that rolipram inhibits organ damage, pro-inflammatory cytokine production, and intracellular migration of early-stage inflammatory elements. our results also show that rolipram increases anti-inflammatory cytokine production. the protective effects of rolipram on septic mice may result from inhibition of the map kinase and nf-κb signaling pathways. rolipram may therefore be a potential novel sepsis treatment, one that would bypass the time-consuming and costly drug-discovery process. rolipram significantly reduces the mortality rates in multiple mice septic models. to assess the protective effect of rolipram on sepsis induced by e. coli, clp, or e. coli derived lps, we investigated the effect of the drug on survival rate. first, mice were injected intraperitoneally (i.p.) with rolipram or vehicle hr before e. coli injection. in the absence of rolipram, % of infected mice died within hr of e. coli injection (fig. a) . in contrast, injection with rolipram resulted in % mortality over days, suggesting that rolipram pretreatment can prevent e. coli -induced septic shock in mice. pretreat the clp model mice can also significant improve the mice survival rate, from % to % (fig. b) . to confirm the protective effect of rolipram both in e. coli-induced and clp-induced septic mice, we assessed the role of rolipram in mouse sepsis induced by lipopolysaccharide (lps) derived from e. coli. mice were i.p. injected with rolipram hr before lps injection. in the absence of rolipram, % of endotoxic mice died within hr of lps injection, but % mice survival in the rolipram pretreated group (fig. c) . these results suggest that rolipram may have a protective effect in sepsis. the survival dose-response curve for rolipram indicates that the mice receiving the highest dose, mg/kg rolipram, experienced the most benefit (fig. d ). taking into account the differential survival rates and occurrence of first mortality, mg/kg rolipram showed the highest efficacy of all tested concentrations. rolipram at mg/kg significantly improved the survival rate to % as opposed to % (p < . ). mg/kg rolipram did not significantly improve the survival rate ( %) compared to the lps-only group. rolipram significantly reduces e. coli derived lipopolysaccharide-induced release of serum pro-inflammatory cytokines in mice. the overwhelming release of pro-inflammatory cytokines plays an important role in the pathology of sepsis. therefore, the serum levels of multiple pro-inflammatory cytokines male c bl/ mice were injected with different doses of rolipram ( mg/kg, mg/kg, and mg/kg, i.p.) hr before lps injection. survival of mice was monitored for days. kaplan-meier analysis, followed by a log-rank test, was used for survival time analysis. *represents p < . in treatments vs. lps groups. were examined. results show that the concentrations of il- β, il- , il- , il- (p ), tnf-α, mcp- , mip- , eotaxin, kc, mig, lif, and vegf, as well as the anti-inflammatory cytokine il- , were all significantly elevated in serum after hr and hr of lps challenge (fig. ) . in contrast, administration of rolipram effectively reduced the production of pro-inflammatory cytokines and chemokines, and further increased the levels of il- . rolipram prevents e. coli derived lipopolysaccharide-induced lung injury in mice. lps-induced endotoxic shock is known to cause a number of effects in the murine host, including severe lung injury. histopathological analysis, consisting of hematoxylin and eosin (h&e) staining of lung sections from lps-only mice, revealed signs of extreme inflammation. edema, neutrophil recruitment, and hemorrhage in the lung samples were also seen ( fig. a ). lung injury in the lps group was visibly increased in comparison with the control group. rolipram treatment, however, markedly attenuated lps-induced lung injury. in bronchiolar lavage fluid, total protein ( rolipram alleviates e. coli derived lipopolysaccharide-induced liver and kidney damage. h&e staining of liver tissue from control mice showed that the liver plate was radially arranged around the central vein in normal hepatic lobules. the livers of lps-only mice sacrificed at the -hr time point showed structural disorder of the hepatic lobule, narrowing or even disappearance of the hepatic sinusoidal space, vacuolar degeneration of hepatocytes, and nuclear pyknosis. however, rolipram pre-treatment for hr significantly reduced these changes (fig. a ). renal tissue h&e staining (fig. b ) clearly showed normal structure and morphology in glomeruli, renal tubules, and renal tubular epithelial cells in control mice. the renal lumen was regular, and no inflammatory cells infiltrated the renal interstitial cells. the kidneys of lps-only mice at the -hr time point showed inflammatory infiltrate in the renal interstitial cells, swelling of renal tubular epithelial cells, and an unclear cell gap. rolipram pretreatment for hr significantly reduced these changes. we also examined biochemical markers for liver and kidney injury in serum from each group of mice treated with lps for hr. rolipram inhibited levels of aspartate and alanine aminotransferases (ast and alt), creatinine (cr), and brain natriuretic peptide (bun) in serum. this indicates that rolipram can ameliorate liver and kidney injury induced by lps ( fig. c-f ). mapk signaling pathways in mouse lungs. immunofluorescence microscopy using anti-nf-κb/p antibody, revealed that lps induces nf-κb/p nuclear translocation in the mouse lung hr after injection. however, rolipram markedly inhibited this translocation (fig. a ). three hours after lps treatment, phosphorylation of nf-κb/p significantly increased in the mouse lung as compared to controls. rolipram markedly suppressed phosphorylation of this molecule (fig. b ). western blot results showed that lps treatment induced we demonstrate that rolipram may protect against lps-induced inflammation and shock in mice, likely through the inhibition of the nf-κb and mapk signaling pathways. we found that rolipram significantly improved animal survival, decreased inflammatory cytokines essential to the process of shock, alleviated organ injury, and dephosphorylated critical inflammatory pathways. these results indicate that rolipram suppresses inflammatory responses that are essential to the process of sepsis, and thus may be protective against sepsis. therefore, rolipram may serve as a novel drug treatment for inflammatory disease, such as septic shock. sepsis is induced by a dysregulated innate immune reaction, leading to a harmful host response to pathogens. this is includes excessive amounts of pro-inflammatory cytokines, such as tnf-α and il- β , , as well as other inflammatory mediators. these molecules can in turn trigger secondary inflammatory processes, leading to inflammatory pathology and life-threatening organ damage. proteins that induce the migration of inflammatory cells into tissue are also upregulated. we have found that rolipram inhibits pro-inflammatory cytokines and chemokines released by the administration of lps, leading to the suppression of excessive inflammatory responses, cell adhesion and migration, and the further sequelae of shock and multiple organ failure in the mouse host. sepsis-induced multi-organ dysfunction and injury are the main mechanisms of patient shock and death. therefore, sepsis treatment guidelines consider multi-organ dysfunction a key point of attack in sepsis treatment . it has previously been reported that rolipram significantly decreased hyperoxia-induced neutrophil numbers in balf and inhibits il- and mcp- transcription in rat lungs . rolipram also alleviates pulmonary edema and reduces neutrophil numbers in balf during chlorine-induced mouse shock . our results go further in showing that rolipram inhibits pulmonary edema, neutrophil infiltration in alveoli, and protein concentrations and neutrophil numbers in balf. liver and kidney function is also improved, indicating that rolipram can prevent lps-induced multi-organ failure. the exact mechanism by which rolipram controls the inflammatory cascade, thereby preventing shock and multiple organ failure in the host, is unknown. however, it is known that the nf-κb pathway is crucial in the regulation of inflammatory gene expression. nuclear translocation activates the transcription and expression of a variety of cytokines and adhesion molecules, all of which are closely associated www.nature.com/scientificreports www.nature.com/scientificreports/ with inflammation and the immune response . the nf-κb family, including p (rela), p /p (nf-κb ), p /p (nf-κb ), relb, and c-rel, exist in the cytoplasm in homo-or heterodimers that can bind with iκb. during conditions of stimulation by lps or other pro-inflammatory factors, iκb kinase (ikk) phosphorylates iκb, leading to nf-κb nuclear translocation and further activation of inflammatory genes . it has been confirmed that toll-like receptor -nfκb signaling plays a key role in acute lung injury . our studies show that lps activates nf-κb and its signaling pathways, while rolipram restrains activation. this indicates that the nf-κb may be part of the mechanism by which rolipram exerts its effects. the mapk signaling pathway also plays a critical role in regulating inflammation and the immune response. as a conserved signaling cascade, the mapk pathway exists in almost all eukaryotic cells. mapks can regulate target protein function through phosphorylation, thus participating in cell proliferation, growth, differentiation, and function. mapk signaling is activated through three levels, including map k, mapkk, and mapk. at present, four mapk signaling pathways have been identified: erk / , erk , p mapk, and jnk . it has been found that rolipram promotes the maturation of glial progenitor cells and regeneration of myelin through enhancement of erk phosphorylation . studies have also found that rolipram restrains bone cancer pain through inhibition of the jnk signaling pathway in the bone marrow, suppressing neuron-stellate cell activation . in addition, rolipram can inhibit lps-induced p mapk phosphorylation in j cells . however, the role of rolipram on phosphorylation of the mapk downstream signaling pathways has not yet been reported. our results indicated that rolipram suppresses lps-induced erk, jnk, and p mapk phosphorylation in lung tissue, suggesting that the mapk signaling pathway -as well as nf-κb -may mediate the anti-inflammatory actions of rolipram. in conclusion, we have found that rolipram protects mice from massive inflammatory responses and endotoxic shock through inhibition of nf-κb and mapk signaling pathway activation. rolipram may thus serve as a novel drug treatment for inflammatory disease, namely sepsis and septic shock. as rolipram is an approved drug in the united states for chronic obstructive pulmonary disorder, this drug repositioning strategy may circumvent the lengthy and expensive drug discovery process, and allow faster and better treatment for sepsis patients. animals. male c bl/ mice, weighing - g, all - weeks old, were used for experiments. the mice were purchased from the department of laboratory animal science of southern medical university (guangzhou, china). all animals were housed with free access to food and water under conditions of optimal light, temperature, and humidity ( : -hr light-dark cycle, approximately degrees celsius, - % humidity). all experimental procedures were approved by the ethics committee of animal research at the college of medicine, southern medical university, and were conducted in accordance with the international guidelines for care and use of laboratory animals. animal model. lipopolysaccharide was dissolved in phosphate-buffered saline (pbs) and stored at − degrees celsius. rolipram was dissolved in dimethyl sulfoxide (dmso) and further diluted in pbs (final dmso concentration: < . %). first, male c bl/ mice were intraperitoneally (i.p.) injected with µl rolipram ( mg/kg) hr before, hr after, or at the same time as lps ( mg/kg in µl). survival rate was monitored for days. the experiments were repeated using variable doses of rolipram ( , , and mg/kg). at , , , , and hr after lps injection, mice were anesthetized and sacrificed. blood was collected for analysis of serum cytokines. for histological analysis, normal saline ( ml) was perfused through the right heart ventricle. lung, liver, and kidney samples were collected and processed as described later. mouse blood samples were left at room temperature for hr, then spun at × g for minutes at degrees c. serum was obtained and stored at − degrees c until required. the chemiluminescent signal was quantified; phosphorylated erk, jnk, and p mapk were normalized against total erk, jnk, and p mapk. phosphorylated protein levels are expressed in arbitrary units. lpsstimulated cells were set at %, and other values relate to that setting. data represent the mean and sd of at least three independent experiments, performed in triplicate. significant differences between more than two groups were performed using anova. comparisons between two groups were performed using two-tailed unpaired student's t-tests. *represents p < . vs. control, # represents p < . vs. lps alone. ( ) : | https://doi.org/ . /s - - - www.nature.com/scientificreports www.nature.com/scientificreports/ cytokine assays. cytokines from mouse serum were measured using the milliplex ® map mouse cytokine/chemokine magnetic bead panel kit for -well plate assay, run on a luminex platform. cytokines include the three primary cytokines of interest: tumor necrosis factor-α (tnf-α), interleukin- -β (il- β), and il- . multiple cytokines were also explored: il- α, il- , il- , il- , il- , il- , il- (p ), il- (p ), il- , il- , il- , ifn-γ inducible protein (ip- ), eotaxin, interferon-γ (ifn-γ), monocyte chemoattractant protein- (mcp- ), macrophage inflammatory protein- α (mip- α), mip- β, mip- , migration-inducing protein (mig), keratinocyte chemoattractant (kc), leukemia inhibitory factor (lif), lipopolysaccharide-induced cxc chemokine (lix), lipopolysaccharide-induced cxc chemokine (rantges), granulocyte-macrophage colony-stimulating factor (gm-csf), g-csf, m-csf, and vascular endothelial growth factor (vegf). for quality assurance, each sample was run twice. liver and renal function tests. serum activity of aspartate aminotransferase (ast, a nonspecific marker for hepatic injury), alanine aminotransferase (alt, a specific marker for hepatic parenchymal injury), blood urea nitrogen (bun, a marker for glomerular renal function), and creatinine (cr, a marker for glomerular filtration rate and renal failure) were measured by fengrui biotechnology company kits (hunan, china). hr of lps challenge. lungs were lavaged with pbs ( . ml × ). the recovery rate of bal fluid (balf) was approximately %. cells were isolated from the balf by centrifugation of x g for minutes, and the cell pellets were resuspended. the total cells in the balf were counted using a hemocytometer. subsequently, the cell suspension was cytospin-centrifuged onto microscope slides and stained using liu stain (baso, china). the percentage of neutrophils was determined by counting cells. the balf supernatant was analysed for total protein concentration by using bicinchonic acid (bca) assay, according to the manufacturer's instructions. histology. lungs, livers, and kidneys were fixed in % paraformaldehyde (ph . ). the organs were then dehydrated and embedded in paraffin. sections µm thick were cut. the slides were then stained with hematoxylin and eosin (h&e), and examined by a light microscope. extraction reagent (pierce, il, usa). protein concentrations were determined using a bca assay kit (keygen, china). equal amounts of lung tissue protein ( µg per animal) were run on a % sds-page gel and transferred onto polyvinylidene difluoride (pvdf) membranes. the membranes were blocked with % bovine serum albumin (bsa) in tbst at room temperature for hr, and then incubated with primary antibody against mouse erk / , p-erk / , jnk, p-jnk, p , p-p , or p (all at a : antibody:bsa ratio) at degrees c overnight. after washes with tbst, the membranes were incubated in secondary hrp-conjugated anti-rabbit igg at room temperature for hr. the membranes were then washed with tbst, processed with an ecl detection kit (pierce, il, usa), and measured on film in a darkroom. immunofluorescence. all tissue stainings were performed on frozen µm-thick oct-embedded mouse lung tissue sections. sections were blocked to eliminate non-specific binding with . % bsa in pbs for hr, then incubated with primary antibody against mouse phosphorylated p ( : ) at degrees overnight. after subsequent washes with pbs, the sections were incubated with alexa fluor ® goat anti-rabbit igg (h + l) for hr at degrees c, then washed with pbs. the slides were stained with dapi (santa cruz biotechnology, ca, usa) for min. slides were then washed in pbs. coverslips were mounted onto the slides using anti-fade reagent (beyotime biotechnology, china). the images were acquired using a zeiss lsm confocal microscope (zeiss, germany). data are expressed as the mean plus or minus standard deviations. statistical evaluation was performed with spss software (version . ). significant differences between more than two groups were performed using anova. comparisons between two groups were performed using two-tailed unpaired student's t-tests. kaplan-meier analysis, followed by a log-rank test, was used for survival time analysis. p < . was taken as the maximum significant difference. epidemiology of severe sepsis in the united states: analysis of incidence, outcome, and associated costs of care epidemiology of severe sepsis long-term cognitive impairment and functional disability among survivors of severe sepsis coordinated molecular cross-talk between staphylococcus aureus, endothelial cells and platelets in bloodstream infection how bacterial pathogens colonize their hosts and invade deeper tissues the response of cd d-restricted invariant nkt cells to microbial pathogens and their products anti-endotoxin vaccines: back to the future nf-kappab at the crossroads of life and death tnf-alpha induction by lps is 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inhibition of chlorine-induced lung injury by the type phosphodiesterase inhibitor rolipram shaping the nuclear action of nf-kappab signaling to nf-kappab identification of oxidative stress and toll-like receptor signaling as a key pathway of acute lung injury mitogen-activated protein kinase phosphatase as an inflammatory factor and drug target rolipram promotes remyelination possibly via mek-erk signal pathway in cuprizoneinduced demyelination mouse the analgesic effect of rolipram is associated with the inhibition of the activation of the spinal astrocytic jnk/ccl pathway in bone cancer pain we would like to acknowledge ms. hallie hanna dolin at the university of toledo for helping us modify the manuscript. this work was supported by national natural science foundation of china, grant number . z.k.p. and y.j. designed the experiments; x.y.l. and j.w. performed the experiments and collected the samples; x.y.l., j.w., and x.h.c. analyzed the results; x.y.l. and z.k.p. prepared and wrote the manuscript, z.k.p., y.j. and x.h.c. modified the manuscript. all authors read and approved the final manuscript for publication. the authors declare no competing interests. correspondence and requests for materials should be addressed to y.j. or z.k.p.reprints and permissions information is available at www.nature.com/reprints.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- - zrnfaad authors: giese, matthias title: types of recombinant vaccines date: - - journal: introduction to molecular vaccinology doi: . / - - - - _ sha: doc_id: cord_uid: zrnfaad the original scientific strategy behind vaccinology has historically been to “isolate, inactivate, and inject,” first invoked by louis pasteur. new vaccines and vaccination strategies are being developed including the use of attenuated live mycobacteria, recombinant microorganisms, and subunits, prime-boost strategies based on the successive administration of a certain mycobacterial antigen under two different vaccine vectors, and dna vaccines [ ] . the various types of vaccines differ in eliciting an immune response. live attenuated vaccines (lavs) mimic a natural infection without being virulent and trigger the activation of the innate immune system through pamps. following injections, lavs rapidly disseminate throughout the vascular network to the draining lymph nodes. therefore, the route of application of lavs does not specifi cally infl uence the immune response. lavs also don't need an adjuvant; they possess a natural intrinsic adjuvancy. safety concerns exist because of the replication competence and the possibility of recombination with a wild type. non-live vaccines, inactivated and most recombinant vaccines, whether containing proteins or carbohydrates (−conjugates), are less effective. in the absence of replication, vaccine-induced immune reactions remain more limited, and therefore the route of vaccination infl uences the effi cacy and the duration of the immune reaction. nonlive vaccines induce a lower antibody response and generally no cytotoxic t lymphocyte activation. compared to lav, all non-live vaccines are regarded as biologically safe ( fig. . ). dna vaccines entail the direct, in situ inoculation of dnabased eukaryotic expression vectors that encode the sequence of a pathogenic protein antigen. the constructed plasmids are then subsequently grown in bacteria like e. coli and highly purifi ed via chromatographic methods. lps contamination of plasmids has to be prevented because of the immunotoxic properties of natural lps. after purifi cation the circular double-stranded dna plasmids are ready for vaccination. the de novo production of the encoded antigens in the host results in the elicitation of both the antibody and the cellular response by activating cytotoxic t lymphocytes (ctls). vaccine proteins made by the host are natural proteins and contain important posttranslational modifi cations such as the correct glycosylation. but like subunit vaccines, dna vaccines must be adjuvanted. naked dna does not work. the unique advantage of dna vaccines is their ability to mimic the effects of live attenuated vaccines without the risk associated with the administration of infectious albeit attenuated material. dna vaccines are able to stimulate a complete, humoral and cellular immune response. peptide fragments are processed via the endogenous pathway, resulting in the presentation of antigen on the cell surface by mhc class i molecules. plasmid dna is very stable also beyond a cold chain. therefore, the storage, transportation, and distribution of dna vaccines are more practical and also cheaper [ ] . mostly all plasmid dna constructs ( fig. . ) used for vaccination share fi ve main characteristics: • strong promoter/enhancer sequence for driving the incorporated foreign gene • convenient cloning site for insertion of foreign genes • origin of replication for initiation of plasmid replication • polyadenylation/termination sequence for production of mature mrna • resistance/antibiotic marker for selection • immunomodulators, e.g., cpgs, interleukins, ubiquitin, etc. • (on the same plasmid or on extra plasmids) uptake of plasmid dna. some biological barriers have to be overcome by dna vaccines on the way to the cell nucleus where the plasmid dna is translated into cellular mrna. after delivery of plasmid dna to the target tissue, e.g., skeletal muscle or skin, lots of tissue nucleases attack . dna vaccines and degrade a large amount of the applicated dna. also the extracellular matrix with collagen and hyaluronic acid infl uences the passage from the application site to the cell membrane. only a small portion ( % estimated) of the still intact plasmid dna will cross the cell membrane by phagocytosis or pinocytosis. inside the cell the route toward the nucleus is also spiked with exo-and endonucleases so that probably only . % (estimated) is successfully and actively transported through the nucleus pore membrane (npc). small particles (<~ kda) are able to pass through the nuclear pore complex (npc) by passive diffusion; larger particles need the support of carrier proteins for effi cient passage through the complex. because of this enormous loss of plasmid dna (up to . %), various tools were developed to protect the plasmid dna and thus increase the effi cacy such as encapsulation into liposomes or binding of dna to dendrimers. figure . illustrates the passage of plasmid dna from the extracellular matrix (ecm) to the nucleus. whereas in human medicine clinical trials with dna vaccines are still ongoing without any registered product on the market, the fi rst approved dna vaccines for the veterinarian medicine are available since and are discussed now. the fi rst veterinarian dna vaccines were developed for horses (davis b.s., for wnv [ ] ; giese m., for eav [ ] ). today the number of current clinical trials worldwide with veterinary dna vaccines is unmanageable and probably all species are hit. canine malignant melanoma (cmm) typically begins in the mouth or around the toes and can spread within the body to the heart, lungs, intestines, and other organs. canine malignant melanoma is known for being one of the most aggressive cancers in dogs and deadly. cmm is most commonly seen in golden retrievers, scottish terriers, dachshunds, labradors, and poodles ( fig. . ). metastases of the tumors will be found very often in distant parts of the body. the overall biology of cmm is similar to the biology of human melanoma. however the melanomas in dogs have diverse biologic behaviors due to the race and a variety of factors. standardized treatments such as surgery, radiation, and chemotherapy are the common tools to fi ght canine malignant melanoma. these traditional tools have afforded minimal to modest stage-dependent clinical benefi ts. xenogeneic dna vaccine. the plasmid dna contains a cdna for the human tyrosinase, hutyr, a tumor antigen (ta). this is a non-mutated differentiation antigen and specifi c to melanoma. tyrosinase is a glycoprotein and essential in the process of melanin synthesis ( fig. . ). like other tas tyrosinase is overexpressed in tumor cells and therefore an ideal target in cancer therapy. normally there is no strong immune reaction against the body's own protein. but immunization of dogs with xenogeneic hutyr cdna can break the immune tolerance against this self tumor differentiation antigen and induce antibody and cytotoxic t cell response against melanoma cells [ ] . tyrosinase is highly conserved from dog to mouse to man. radiotherapy in cases with positive surgical margins or positive regional lymph nodes. one dose contains μg dna given in a volume of . ml by the transdermal route via a needle-free vaccination device. booster immunizations were given at -month intervals. in march the drug manufacturer received a conditional license for oncept from the usda and a full license in . the results of the xenogeneic immunization of dogs with hutyr cdna as an adjunct therapy for cmm demonstrate a signifi cant increase of survival time compared to the control group. none of the dogs developed systemic adverse reactions; no toxicity was seen. the overall safety of this dna vaccine is confi rmed. this vaccine development represents a tremendous milestone in dna science and technology. virus. west nile virus (wnv) is a mosquito-borne member of the family flaviviridae , genus flavivirus , and was fi rst identifi ed in in uganda, africa. it is a positive-sense, single-strand rna virus, (+)ssrna, of about kb that encodes a single polyprotein with seven nonstructural proteins and three structural proteins. the rna strand is held within a nucleocapsid. wnv replicates in the cytoplasm of infected cells. wnv is a zoonotic virus. the primary reservoir is birds with a signifi cant impact to spread the infection across countries and continents. more than different species are described as carrier of this virus. wnv is spread from bird to bird by mosquitoes when they bite, or take a blood meal, from birds that are infected with the virus. birds from some species get ill and die; others have no clinical signs and survive. mosquitoes are also capable of spreading the virus to horses, dogs, cats, mice, alligators, and lots of other mammals but also to humans. one-third of all horses bitten by carrier mosquitoes develop the disease and die or are so affected that euthanasia is required. the incubation period ranges from to days. horses that do become ill vary in symptoms: muscle trembling, skin twitching, ataxia, sleepiness, dullness, and listlessness. wnv may cross the placenta from mother to gestating foal. horses cannot spread the disease to humans. wnv produces different outcomes in humans like in horses: fever, headache, chills, diaphoresis, weakness, swollen lymph nodes, drowsiness, and pain in the joints comparable to symptoms of infl uenza. more severe neuroinvasive infection includes meningitis and encephalitis. wnv-dna vaccine. the surface envelope protein e is the main target for the antibody response. there are more than copies of the e protein in a mature wnv virion. the e function is the interaction between the cell surface and the fusion between virus and cellular membrane. the premembrane protein prm is cleaved during viral maturation into a smaller membrane m peptide. the expression of prm and e protein in cells results in the formation of virus-like particles, vlp. these vlp share many of the antigenic and structural properties of fully mature viruses and are of special interest for a vaccine development ( fig. . ) . the fi nal expression plasmid for immunization of horses contains the human cytomegalovirus early gene promoter, signal sequences from japanese virus, and a fusion gene of part of the fi shing industry is aquaculture, also known as aqua farming, but it can be contrasted with commercial fi shing, which is the harvesting of wild fi sh. aquaculture involves cultivating freshwater and saltwater fi sh and other populations (shrimp, oyster) under controlled conditions. salmon is one of the main food-producing fi sh in the world. a dna vaccine for fi sh must be not only safe for the animal but especially safe for the fi sh consumer. salmon is the major economic contributor to the world production of farmed fi sh, representing over u$ billion annually in the united states. salmon farming is also very big in norway, scotland, canada, and chile and is the source for most salmon consumed in the united states and europe. like all other animals also fi sh is threatened by viruses, bacteria, and parasites. one major problem for salmons is the infectious hematopoietic necrosis (ihn) virus [ ] . virus. infectious hematopoietic necrosis (ihn) virus is a common viral pathogen of both wild and farmed salmonids, in particular pacifi c salmonids, rainbow trout, and atlantic salmon. ihn virus is enzootic to the pacifi c northwest; however it has varying effects on different pacifi c salmonids. it is a negative-sense single-stranded, (−)ssrna virus that is a member of the rhabdoviridae family, genus novirhabdovirus . the rna genome is , nucleotides long and contains a leader (l) and trailer (t) sequences at its ′-end and ′-end, respectively. the coding regions are n, p, m, g, nv, and l genes. g encodes the surface glycoprotein, so-called spikes, main target for the immune response. transmission. ihnv is transmitted following shedding of the virus in the feces, urine, sexual fl uids, and external mucus and by direct contact or close contact with surrounding contaminated water. the virus gains entry into fi sh at the base of the fi ns. salmons are carnivorous and are currently fed a meal produced from catching other wild fi sh and other marine organisms -a permanent origin of possible infections with ihnv. clinical signs of infection with ihnv include anemia, skin darkening, bulging of the eyes, fading of the gills, and abdominal distension. infected fi sh commonly hemorrhage in several areas, like the mouth, the pectoral fi ns, muscles near the anus, and the yolk sac of fry. diseased fi sh weaken, eventually fl oating on the surface of the water. necrosis is common in the kidney and spleen and sometimes in the liver. mortality rates in older fi sh ( - kg) tend to range from to %; in smolts the mortality rate often exceeds %. the average cumulative mortality following an outbreak is estimated at %. ihnv-dna vaccine. the antigen is the viral surface glycoprotein (g) capable of eliciting neutralizing antibody and the production of a protective immune response. the g gene was cloned into a eukaryotic expression vector by insertion of an intermediate-early promoter and a polyadenylation signal. but the speciality of this vaccine is to be prepared as a two-component vaccine in a single vaccine, one plasmid or more. the second component is a portion of the nucleic acid sequence encoding a second peptide, derived from a fi sh pathogen other than the said rhabdovirus resulting in a fusion. this second pathogen can be any fi sh pathogen, e.g., isav, ipnv, iridovirus, nnv, spdv, svcv, vhsv, koi herpes virus, and more. the rationale behind this is that the presence of the ihnv g protein boosts the immune response to the second protein, resulting in a protective effect against infection by this fi sh pathogen. the vaccine is given intramuscularly with a dosage of only μg in μl on the left dorsal fl ank, in the area just below the dorsal fi n [ , ] . this fi rst dna fi sh vaccine was licensed in in canada by the veterinary biologics section (vbs), animal health and production division, canadian food inspection agency (cfia) and is also used now in studies in norway. there are many environmental stressors and diseases which infl uence and seriously threat the life of european honey bees, apis mellifera. the european honey bee is professionally managed worldwide for honey production and pollination. the bee was imported to the united states years ago with the fi rst european settlers and called "white man's fl y" by the native americans, the indians. first reported in the united states, a mysterious socalled colony collapse disorder (ccd) decimated the bee colonies there between and %, fi rst observed during the winters of - and then - and without interruptions up to now. a similar situation is also given in europe. about , - , bees live in a colony. the fi rst description originated from the s. in the early nineteenth century, the colony losses were known in england as "isle of wight disease," and the americans called this phenomenon "disappearing disease" in the s, whereas these colony losses in france in the late s were called "mysterious bee losses." where have all the bees gone? economic value. the huge loss of honey bees as pollinators has a dramatic impact on agricultural pollination. about crops, nuts, fruits, and vegetables are pollinated by a. mellifera , with an overall value of more than $ billion in the united states and more than € billion for the eu in . a bee colony produces some kg/ lb honey per day. in return, these bees have to pollinate - million fl owers. one should keep in mind that besides european honey bees, wild insects, among them , species of wild bees, have also a very great impact on pollination and seem to be more effi cient in pollination as managed honey bees [ ] . the industrial farming threatens also the natural biotope of wild insect pollinators. ecologic value. the total global economic value of honey bee pollination was calculated in to more than € billion or $ billion. the food and agriculture organization (fao) of the united nations estimates that there are million managed honey bee colonies worldwide. beside this professional agriculture, honey bees are irreplaceable for the biodiversity. this organism appeared during evolution with the fi rst fl ower plants and exists since million years as described in chap. , fig. . . after swine and cattle, bees are in europe and north america the third important farm animal and since formally listed as farm animal in switzerland. therefore, ccd is not only an economical but also an essential global ecological problem which urgently must be solved in the future. "the bee is more than honey." what is causing ccd? the colony collapse disorder of the last years seems to differ from past outbreaks: the worker bees disappear instead of dying in place, leaving behind the queen and young bees. high levels of bacteria, viruses, and fungi are measured in the gut of the remaining bees. collapses can occur within days. a complex problem. different theories are discussed about what is causing ccd. pesticide contamination, hotly debated to interfere with the nerve system affecting foraging behavior of bees, lead them to abandon their hives. fungal diseases such as nosema spp. is known for big bee losses in spain. monocultures or gene-manipulated crops. electro smoke (radio waves) caused by cell phones destroys the bee's compass. the rigors of travelling in trucks from crop to crop in the usa. down from february professional us beekeepers travel with their colonies through the country until december. thereby the bees must relocate up to times. in europe the bee colonies begin the winter sleep around september. also the climate change, the temperature sensitivity is discussed to have an impact on crop pollination. ccd is likely caused by a combination of factors [ , ] . varroa destructor . but in all ccd cases, an overload of bloodsucking varroa mites is detectable and varroa is currently considered the major threat for apiculture. the infection and disease is called varroosis. varroa destructor is an ectoparasite, has a reddish-brown fl at shape, and is - . mm long and . - mm wide, with eight legs. v. destructor infest worker bees and drones and its brood. the mite develops inside the brood cells. varroa is a real colossus compared to the size of bees as can be seen in fig. . . varroa mites belong to the scientifi c class of arachnida, subclass acari. there are , species described alone from mites. some mites prefer carbohydrates as food such as meal or crops. the house dust mites feed fl akes of shed human skin. varroa mites prefer fresh "blood" and the hemolymph of bees and can feed . mg/ . lb within h. varroa is transported into the hives via piggyback by worker bees. the female mite enters broad cells, preferentially drone cells. once the cell is capped, varroa lays eggs on the larvae. the development from egg to insect takes days. bee larvae and mites hatch in about the same time and the newborn varroa mites spread to other bees [ , ] . the lifetime of summer mites are - weeks, whereas fall mites can live for several months. varroa can only reproduce in honey bees and thus are considered harmless to other insects. varroa is more than a disease. it is a global pest having devastating effects on bees ( fig. . ). varroa as vector. varroa may be not considered as isolated agent for the disease. the mortality of adult bees and its brood must be considered in the context with secondary viral infections. at least various viruses are able to infect honey bees, mostly ssrna viruses. eight viruses are known to be associated with varroa mites: acute bee paralysis virus (abpv), black queen cell virus (bqcv), chronic bee paralysis virus (cbpv), deformed wing virus (dwv), kashmir bee virus (kbv), sacbrood bee virus (sbv), cloudy wing virus (cwv), and slow bee paralysis virus (sbpv) [ - ] . varroa control. a number of natural and synthetic chemicals are commercially available for the control of varroa infestations. the fi rst compounds were bromopropylate, fl uvalinate, or other pyrethroid insecticides. and to make a long story short, varroa mites became resistant not only against one product of a given chemical class; the resistance was against the entire class with several related synthetic products. also the use of natural products, such as formic acid, mineral oil, or thymol, is only partially and temporally effective and show adverse effects [ ] . there is no successful chemical treatment. mites will quickly develop resistance to all chemicals. the immune system of insects. the basic difference between insect and vertebrate immunity is the missing highly specifi c antigen response of the acquired immune system in insects. nevertheless, in the million years of evolution, insects developed a powerful defense strategy against bacteria, fungi, viruses, and parasites. only protected by this "primitive" immune system insects were so successful that they colonized all terrestrial ecosystems. the insect innate immunity shows many similarities to the vertebrate and to the human innate immunity, is multifaceted, and involves both humoral and cellular components [ ] . most insights on insect immunity are provided by drosophila melanogaster research. the key mechanism is also observed in honey bees. the humoral and systemic response to bacterial and fungal infections is controlled by antimicrobial peptides (amps). there are circulating receptors sensing a danger signal and activating the toll pathway, whereas membranebound receptors activate the imd pathway. both pathways lead to the translocation of nf-kb-like transcription factors and the production of amps. nf-kb response elements can be detected in the promoter region of the diptericin gene. the cellular immune response is mediated by specialized blood cells, the hemocytes, plasmatocytes, crystal cells, and lamellocytes [ ] . plasmatocytes represent % of the majority of hemocytes. they express phagocytic receptors and patrol through the body, clear microorganism and cell a b c debris, and signal infections to the fat bodies. the bee genome was completely sequenced in [ ] . the bee dna vaccine. an expression plasmid was constructed with a cmv promoter. surprisingly, no bee or other insect specifi c promoter was essential to drive the expression of the protein. the enhanced green fl uorescent protein (egfp) was chosen as reporter gene and inserted into the multiple cloning site, together with an sv enhancer element. the plasmid construct was produced in e. coli and highly purifi ed by standard techniques. european honey bees ( apis mellifera ) were obtained from local beekeepers and cultivated under lab conditions. varroa mites were collected from infested bees. the oral vaccination of the egfp plasmid was operated by feeding the bees with a mixed solution of sugar and plasmid dna (vaccine sugar). standard sugar solutions made by the beekeeper are the normal food for winter bees. results. over days after onset of feeding, we measured the expression of egfp by immunofl uorescence and western blot analysis with egfp antibodies. between day and , a clear egfp signal was detected in the thorax and especially in the malpighian tubules. control bees fed with dna lacking the reporter did not show any signal. in parallel, control experiments with transformed e. coli were done to study the possibility of egfp expression in gut bacteria instead of bee cells. no egfp signal was detected in transformed bacteria. most surprisingly, we found the egfp signal after days in varroa mites sucking hemolymph of bees which were fed by the vaccine sugar solution and no signals in control mites of infested control bees. feeding of plasmid dna results in expression of a reporter gene in different bee tissues over a period of several days, and fi nally varroa absorbs this protein via bloodsucking. the bee blood is not carried by arteries and veins but fl ows loosely around the body. no egfp signals were detected either in the honey stomach or in the feces. figure . illustrates the egfp passage through the bee body and toward the varroa mite. we started with the simple idea that the biochemistry in eukaryotic cells remains the same, irrespective of the organism. a difference is given in the confi guration of the immune system. that means, an insect can successfully fi ght against parasites and infections but with different weapons. no t cells, no b cells, and consequently no antibodies and no memory. we are able to stimulate targeted immune genes of bees and measure an insect typical immune response. a standard plasmid dna vaccine, fi rst developed for horses, bridges the evolution from fi sh to insects to mammals. no other vaccine type is able to do this job. how fascinating biology is! a protein subunit is based on a single protein molecule and able to stimulate a humoral immune response, but usually not a cellular response. after phagocytosis proteins are degraded by acid-dependent proteases in endosomes (endosomal or exogenous pathway), resulting in an mhc ii presentation of the antigenic peptides. a peptide is one form of a subunit. carbohydrates are also used as subunits with a poor and age-dependent immunogenicity. carbohydrate antigens induce a t cell-independent b cell response as discussed in chap. . therefore carbohydrates are mainly linked to a protein (conjugation) to enhance toe immune reaction as discussed here with the hib conjugate vaccine. conjugate vaccines. the polyribosylribitol phosphate (prp) capsule of haemophilus infl uenzae type b (hib) is a major virulence factor for the organism. prp is a t cellindependent antigen characterized by, e.g., induction of a poor antibody response in less than -month-old infants and children and the inability to induce a booster response. polysaccharide vaccines based on prp alone were developed in the s. by covalent linkage of prp with t cell dependent protein antigens, a conjugated vaccine was created to overcome the t cell independent characteristics of prp. at present three different licensed protein carriers are linked to prp: • hboc: diphtheria crm protein , mutant corynebacterium -linkage: no spacer • hbomp: outer membrane protein, omp, neisseria meningitidis -linkage: spacer • prp-d: diphtheria toxoid, d -linkage: spacer these hib conjugate vaccines differ by protein carrier, polysaccharide size, and method of chemical conjugation, including use of a spacer between the prp and protein carrier. a standard chemical conjugation between a polysaccharide and a protein is illustrated in fig. . . subunit vaccines, while offering greater safety, are intrinsically poorly immunogenic and strong adjuvants are essential to boost the activation of immune responses. serotype variability is dictated by modifi cations of the o-antigen portion of lps. o antigens vary in the number of oligosaccharide unit repeats, the types and distribution of carbohydrates, and the intra-and intermolecular linkages [ ] . in s. fl exneri , these genes are encoded in the bacterial chromosome. in contrast, s. sonnei , which shows no serotype variability, expresses plasmid-encoded o-antigen modifi cation enzymes. the o antigen is one of the major immunogenic components of shigella and is a virulence factor, in part, due to masking the exposure of type three secretion apparatus [ ] . the inclusion of conserved proteins in vaccine compounds potentially solves the issue of serotype specifi city, thus allowing the generation of a highly desirable pan-shigella vaccine. in addition, recombinant proteins usually have increased safety profi les. another important impact in shigella epidemiology that prompts vaccine development is the increasing frequency of antibiotic-resistant strains. antibiotic resistance is continually rising for this pathogen [ ] . shigella spp. as causative agent of shigellosis. first defi ned as a causative agent of bacillary dysentery by shiga in japan, shigella is a gram-negative bacillus that is noncapsulated and nonmotile. diagnosis is generally based on symptoms [ ] since bloody, mucoid stools are indicative of shigella infections. however, because several diarrheal infections caused by other microorganisms share these symptoms (enteroinvasive e. coli and campylobacter , among others), the sole analysis of symptoms is insuffi cient for an accurate diagnosis. therefore, clinical diagnosis must be complemented with microbiological isolation from culture. shigella invasion and pathogenesis. shigella is transmitted through the fecal-oral route by consumption of contaminated food and water. following ingestion, the acid-tolerant shigella passes through the stomach and small intestine into the large intestine [ ] (fig. . ). here, they are taken up by m cells, transcytosed to the basolateral face of the colonic epithelium, and presented to resident macrophages wherein ipab of the type three secretion system (t ss) induces apoptosis by caspase activation, thereby escaping killing by the macrophage [ ] . shigella then invades epithelial cells using its t ss to create a translocation pore in the host cell membrane to initiate an orchestrated fl ow of effectors into the host cell cytoplasm to induce actin rearrangements that ultimately result in uptake of bacteria. once inside, shigella quickly escapes its vacuole, replicates, and moves about the cytoplasm via actin-based motility. in a t ssdependent manner, the shigella then forms a protrusion into a neighboring uninfected cell with the resulting vacuole being quickly lysed to complete the process of intercellular spread. the genes associated with the t ss are encoded on a -kb plasmid which is highly conserved among the shigella species. at the heart of the t ss is the type three secretion apparatus (t sa) which is composed of a basal body similar to that of fl agellar systems and an extracellular needle [ ] . invasion plasmid antigen d (ipad) is a kda protein that forms a pentameric ring at the tip of the needle. it controls secretion of effector proteins and is the environmental sensor for mobilization of ipab to the t sa tip complex. ipab is a kda translocator that forms a ring atop the ipad ring and is responsible for host cell contact. this contact is required for mobilization of ipac to the needle tip and formation of a complete unidirectional conduit from the bacterial cytoplasm to the host cell cytoplasm. the initiation of infl ammation and invasion processes occurs exclusively at the basolateral side of host cells, highlighting the importance of the previous steps of macrophage subversion in shigella colonization of the gut. animal models. shigellosis is strictly a human disease. while the basis of this restriction is unknown, it complicates the ability to investigate the pathogenesis of shigella . however, several animal models have been developed to study the pathogenesis of shigella , the resulting immune response against shigella antigens, and the protection efficacy of candidate vaccines against shigellosis: [ ] . nonhuman primate (nhp) models : nhp models have been used to defi ne the ability of vaccines to elicit immune responses and protection (rhesus and cynomolgus monkeys) [ ] . the main advantage of this model is that shigella is able to colonize the large intestine and generate symptoms that these bacteria generate in human infection. o antigen/proteosome : o antigen represents the variable portion of shigella lps ( fig. . ). administration of lps or o antigen alone in animal models is not enough to elicit immune responses, making them ineffective immunogens. to solve this limitation, these molecules have been used in conjunction with different proteins as carriers. several variants of lps/oantigen mixtures have been developed and characterized. one of these protein combination approaches uses s. fl exneri and s. sonnei lps complexed with neisseria meningitidis outer membrane protein proteosomes [ , ] . lps is extracted from s. fl exneri or s. sonnei by hot phenol extraction and mixed with detergent-extracted outer membrane proteins from n. meningitidis . the complex was then separated from free lps present in the mixture by gel fi ltration chromatography. the concept behind this vaccine is that the proteins present in the n. meningitidis proteosome are able to act as carriers for t cell stimulation, thus allowing the recognition of lps. shigella outer membrane vesicles. outer membrane vesicles (omvs) are particles composed of lps, proteins, and nucleic acids. in a proposed vaccine formulation, these particles were purifi ed from liquid cultures of s. boydii by centrifugation with subsequent fi ltering ( fig. . ). the precise identity and amount of the proteins included in this preparation is not currently known, although the presence of proteins having the same mass as ipab, ipac, and ipad suggests its composition includes these proteins. when these omvs are administered orally to mice, antibodies are generated against omv lysates. this vaccine has the advantage of heterologous protection (as shown by challenge against strains from each shigella serogroup) and the absence of adjuvant dependency. in addi-tion, immunity can be passively transferred to offspring, suggesting that the protective mechanism involves antibodies and raising the possibility that this vaccine can be used in infants, which is the main target population for a shigella vaccine. the use of live, fully virulent shigella during its formulation process, the presence of lps, and lot-to-lot consistency are possible downsides of this preparation. invaplex. another vaccine candidate that uses t ss proteins and lps as part of the formulation is the invaplex [ ] . these complexes are obtained by aqueous extraction followed by ion exchange chromatography (fig. . ). the precise composition of these extracts has not been completely characterized but includes lps, ipab, and ipac [ ] . these complexes are able a b d c fig. . depiction of lps/o-antigen-based vaccines. lps ( a ) extracted from shigella fl exneri or sonnei is admixed with protein preparations from n. meningitides and used as a carrier. when this complex is administered orally and intranasally to mice and guinea pigs [ ] , serum igg and mucosal iga in intestines and lungs are vaccine com-pound ( b ). o antigen purifi ed from lps is delivered in combination with exoprotein a from p. aeruginosa ( c ). finally, o antigen from different shigella serogroups is combined with ribosomes from shigella and is depicted in ( d ) to elicit igg and iga responses against ipa proteins as well as lps in both mice and guinea pigs. in addition, they are protective against the shigella species/serotype used for extract generation [ ] in the mouse and guinea pig challenge models. two phase one studies have been performed using the invaplex vaccine on adult volunteers [ ] and showed no major side effects to delivery of intranasal doses of up to μg. the highest dose employed in these studies generated an asc response to lps in % of the volunteers. an advantage of this approach is that, other than the invaplex itself, no additional adjuvants need to be administered. a drawback of this vaccine consists in a challenging production process that includes cultures of virulent shigella as well as the presence of bacterial lps products in the intermediate steps and fi nal formulation. another possible caveat is the uniformity of protein composition in these complexes through manufacturing lots. finally, this vaccine was not designed to protect against multiple serotypes. a solution for this possible drawback, however, is the generation of formulations that include invaplex complexes generated from more than one particular serotype, which increases an already diffi cult manufacturing process. this would allow the generation of vaccine formulations specifi c for the serotypes prevalent in a particular region. a vaccine candidate that targets conserved shigella virulence proteins includes some of the t ss ipa proteins (fig. . ) . recombinant ipab and ipad can be expressed in e. coli at high levels. ipad is then easily purifi ed from the e. coli cytosol while ipab must be purifi ed as a complex with its cognate chaperone ipgc. the chaperone is needed to maintain the hydrophobic ipab in a soluble state and to provide stability for ipab from proteolytic degradation. ipab can then be further purifi ed after separation from ipgc in low concentrations of detergent. analyses have indicated that ipab is greater than % pure following this scheme. in its fi nal formulation, this ipa-based vaccine also contains a double mutant of heat-labile enterotoxin from e. coli (dmlt) [ ] as an adjuvant. the mechanism of protection for this vaccine has not yet been worked out. nevertheless, it was tested in the mouse lethal pulmonary model [ ] where it exhibited over % homologous protection (against s. fl exneri ) and greater than % heterologous protection (using s. sonnei during the challenge experiments). igg and mucosal iga were generated after intranasal administration along with antigen-specifi c ifn-γ-secreting cells. ompa. a -kda outer membrane protein (omp) was purifi ed from s. fl exneri a using ion exchange chromatography. incubation of macrophages with this -kda protein induced the production of nitric oxide and increased production of il- and tnf-α. this protein was delivered parenterally fi ve times in rabbits, giving protection against challenge by s. fl exneri in the rabbit cecal ligation model [ ] . subsequent work using a recombinant protein purifi ed by affi nity chromatography identifi ed this -kda omp as ompa, part of a family of immunomodulating proteins present in numerous gram-negative bacteria. this protein showed high protective effi cacy in the mouse lethal pulmonary model [ ] where it elicited serum igg and mucosal iga. ticks are widely distributed throughout the world, affecting % of the world's cattle population [ ] . the economic importance of ticks and tick-borne diseases (tbds) has been estimated by a number of studies; however they most likely represent an underestimation of the real impact of these arthropod vectors and their transmitted diseases. tick feeding has devastating effects including disease transmission, paralysis, toxicosis, and secondary infections of the tick-feeding site [ ] . the effect of ticks and tickborne diseases is particularly pronounced in the livestock sector where it is repeatedly rated highly for its impact on the livelihood of farmers, particularly in countries of the south which are heavily dependent on agricultural production. there are six genera of ixodid ticks of importance, namely, amblyomma , dermacentor , haemaphysalis , hyalomma , rhipicephalus , and ixodes . historically, tick and tick-borne disease control has focused on the control of ticks at tolerable levels through acaricide use and treatment of disease with appropriate drugs. in some cases acaricide-based tick control is often the only method of reducing tick populations without sacrifi cing productivity [ ] . acaricides are commercially available in a number of formulations that are applied either directly onto livestock or in dipping vats where multiple animals can be passed through at regular time intervals. acaricide application relies heavily on correct formulation and administration to be effective. a large number of chemical compounds have been found to be effective against ticks including arsenic (introduced ~ ), ddt (~ ), cyclodienes and toxaphene (~ ), organophosphates-carbamate group (~ ), formamides (~ ), and macrocyclic lactones (~ ). the potency and usefulness of many of the abovementioned compounds is gradually eroding with resistance developing in many tick species of rhipicephalus , amblyomma , and hyalomma . multiple acaricide-resistant tick stocks have been identifi ed, limiting or entirely excluding the use of many acaricides [ ] . in addition to resistance, chemical control through the guiding principle for anti-tick vaccination stems from early studies conducted on acquired host resistance to tick infestations. repeated exposure of hosts to ticks or tick organ homogenates induced resistance to tick re-infestation. while the degree of resistance may vary between different tick and host species, evidence strongly suggests that natural resistance against tick infestation develops based on adaptive immune response mechanisms [ ] . ticks feeding from hosts vaccinated with tick components take up effector molecules during feeding that mediate deleterious effects on the ticks. this effect manifests as reduction of feeding time, tick mortality (during or after feeding), reduced engorgement weights, and reduced reproductive capacity of adult females. eggs laid from ticks fed on vaccinated hosts may also show reduced hatching rates. the overall result culminates in reduction of tick populations and tick-borne diseases. many of the anti-tick vaccine targets have been identifi ed using conventional immune-screening techniques. immunization of vertebrate hosts with tick homogenates or purifi ed tick extracts generates immune sera. these sera are used to screen for tick antigens detected by the host. the identifi cation of tick proteins essential for tick survival is a useful method for more targeted antigen discovery, which is made increasingly possible as information is gathered on tick biology. with the availability of genome sequences for a number of tick species, the number of candidates for discovery is expanding through reverse vaccinology. the use of other techniques such as rna interference (rnai) has been useful in confi rming the importance of antitick vaccine candidates and is likely to play a role in future anti-tick vaccine antigen discovery [ ] . exposed or concealed antigens. anti-tick vaccine candidates have been classifi ed into two categories: exposed or concealed antigens. exposed antigens are secreted in tick saliva during attachment and feeding on a host while concealed antigens are normally hidden from the host immune response. molecular mimicry by ticks of host components has been observed, and vaccination may induce host sensitivity and autoimmune reactions when exposed antigens are used [ ] . one advantage of using exposed antigens is that natural boosting occurs through tick feeding. mechanistically, vaccination with exposed antigens is thought to induce a focal hostile environment unsupportive for tick attachment and feeding. concealed antigens do not come into contact with the host immune response during natural tick feeding. although often contained within the thoracic cavity of the tick, some salivary gland proteins can be characterized as concealed if they are not secreted into the tick-feeding site. one diffi culty in the development of concealed anti-tick vaccines is that the antigen must be accessible to the induced humoral vaccine response. this often limits the number of candidates to those coming into prolonged and direct contact with the blood meal or where the humoral response can be transported over the gut barrier into the hemolymph [ - ] . the second limitation of concealed antigens relates to natural boosting of the immune response. as the antigens do not come into contact with the immune response within the host, suffi ciently high antibody levels must be induced through repeated vaccination. as the blood meal acts as the carrier for the effector immune responses, the anti-tick effect can take place over a longer period of time compared to exposed antigens. this effect may even extend beyond the mere feeding period into the inactive stages where digestion and molting/egg laying takes place. bm anti-tick vaccine. the bm -based anti-tick vaccine remains the only anti-tick vaccine commercially produced and has become the benchmark for future anti-tick vaccine development and evaluation. the gut-associated bm glycoprotein was fi rst identifi ed in r. microplus although homologues in other tick species have since been identifi ed [ - ] . the biological function of bm remains unknown although it is thought to play a role in the digestion of the blood meal [ ] . in r. microplus , expression of bm is increased during embryogenesis, reaching the highest level in unfed larvae. expression decreases during feeding and molting with lowest levels of expression detected during the resting stages of the tick. bm has a translated coding sequence of amino acids and a size of . kda. the protein contains four potential n-linked glycosylation sites and a leader peptide suggesting transport to the cell surface. localization studies have shown the molecule is located predominantly on the microvilli of gut epithelial cells. a single c-terminal transmembrane sequence is present in the unprocessed protein which is replaced by a glycosylphosphatidylinositol anchor in the mature protein. the protein also contains multiple predicted egf repeats rich in cysteine residues. vaccination has been performed mostly with the whole molecule, and protective epitopes for bm have not been well determined. the site of a protective b cell epitope was defi ned and additional epitopes are likely to exist. overlapping cross-reactive immune-reactive epitopes have been found between bm and the r. decoloratus homologue, bd . vaccine effi cacy is directly related to anti-bm antibody titer and the ability to control tick populations is directly related to achieving a strong antibody response. substantial animal-to-animal variation has been observed in the ability to generate anti-bm antibody titers which is likely related to the mhc class ii haplotypes expressed. antibodies to bm and cattle complement system are taken up during the blood meal. antibody binding results in lysis of the gut epithelial cells culminating in impaired blood meal digestion. strong antibody responses may induce tick mortality due to blood leakage from the gut into the hemolymph and ticks may turn reddish instead of gray. the development of the antibody response in cattle [ ] after immunization with rbm is demonstrated in fig. . . recombinant expression of bm has been attempted in several expression systems including escherichia coli , aspergillus nidulans , aspergillus niger , and pichia pastoris . vaccine trials showed that bm vaccination targeted mainly the adult stage of r. microplus , particularly the number of adult females fully engorging and post-engorgement mortality. reproductive capacity of adult r. microplus females was affected in terms of egg-laying capacity and hatching of eggs [ ] . under fi eld situations, vaccination of cattle reduced tick numbers by % within a single generation and reduced the reproductive capacity by %. reversal of negative effects of tick feeding on live weight of vaccinated animals by an average increase in live weight of . kg over a -month period was observed. extensive fi eld trials in cuba, brazil, argentina, and mexico showed between and % control of r. microplus ticks within a -week period [ ] . importantly, complete control of acaricide-resistant ticks could be accomplished by integrating bm vaccination with acaricide use [ ] , showing that integrated control systems are effective in controlling tick populations. vaccination also decreased the amount of acaricides required to control tick populations and prolonged the time interval between cattle dipping. bm vaccination has been extensively evaluated for its ability to control other tick species. almost complete cross-protection against rhipicephalus annulatus has been reported [ ] . signifi cant protection against hyalomma anatolicum , h. dromedarii , and r. decoloratus has been observed; however no cross-protection was seen against r. appendiculatus or amblyomma variegatum . genetically attenuated microorganisms, viruses and bacteria, can be engineered to deliver recombinant heterologous antigens to stimulate the host immune system. some experimental vector systems are summarized in work with vectors classifi ed as bsl- does not require biosafety program approval. work with vectors classifi ed as bsl- or higher requires approval by the local biosafety committee. safety concerns. as demonstrated for adenovirus (ad ), following i.m. injection, the vector persisted mainly near the injection site and in draining lymph nodes for up to months. low levels of integration into chromosomal dna were observed, with a calculated mutation rate of × − mutations per cell. the spontaneous mutation rate of a cell is × − and therefore tenfold higher. ad is classifi ed as biosafety level (bsl- ). live vectors are able to stimulate the mucosal as well as a systemic humoral and cellular immunity. a severe drawback of the vector technology is that, once used, the vector cannot be effectively used in the patient again because it will be recognized by antibodies. repeated booster immunization will fail. also preexisting immunity in the patient against the vector could render the vaccination ineffective. a heterologous prime-boost and vector priming as described in chap. could circumvent this barrier. disease lactic acid bacteria have generally recognized as safe (gras) status and have been developed in the past decade as potent adjuvants for mucosal delivery of vaccine. a platform technology based on lactobacillus plantarum ( fig. . ) was developed to deliver antigens against plague disease caused by yersinia pestis , an aerobic, nonmotile, gram-negative bacillus belonging to the family enterobacteriaceae , which is transmitted to humans via fl eabite or via aerosol droplet, causing bubonic or pneumonic plague, respectively [ ] . most human plague cases present as one of three primary forms -bubonic, septicemic, or pneumonic. secondary plague septicemia, pneumonia, and meningitis are the most common complications. the pathogenicity of y. pestis results from its impressive ability to overcome the defenses of the mammalian host and to overwhelm it with massive growth. plague is enzootic in rodents in africa, asia, south america, and north america. y. pestis is transmitted from host to host by fl eas via blood feeding, through consumption or handling of infectious host tissues, or through inhalation of infectious materials. y. pestis infects an astonishingly broad range of mammals and uses rats, squirrels, mice, prairie dogs, marmots, or gerbils as reservoirs and several arthropod vectors for transmission [ ] (fig. . ) . humans acquire this zoonotic infection via an atypical bite from animal fl eas, sometimes prompted by an animal's death from plague, after which the fl ea seeks a new source of blood. most infected fl eas come from the domestic black rat rattus rattus or the brown sewer rat rattus norvegicus. y. pestis cells spread from the site of the infected fl eabite to the regional lymph nodes, grow to high numbers causing the formation of a bubo, and spill into the bloodstream where bacteria are removed in the liver and spleen. growth continues in these organs, spreads to others, and causes septicemia. fleas feeding on septicemic animals complete the infection cycle. in humans, bubonic plague can develop into an infection of the lung (secondary pneumonic plague) that can lead to aerosol transmission (primary pneumonic plague) [ ] . multiple antibiotic-resistant strains of y. pestis occur naturally and they can be easily bioengineered. thus, plague is a category a bioterrorism agent in need for novel strategies for its prevention. bubonic plague is the classic form of the disease. patients usually develop symptoms of fever, headache, chills, and swollen, extremely tender lymph nodes (buboes) within - days of contact with the organism either by fl eabite or by exposure of open wounds to infected materials. primary septicemic plague is generally defi ned as occurring in a patient with positive blood cultures but no palpable lymphadenopathy. patients are febrile, and most have chills, headache, malaise, and gastrointestinal disturbances. primary pneumonic plague is a rare but deadly form of the disease that is spread via respiratory droplets through close contact ( - ft) with an infected individual. it progresses rapidly from a febrile fl u-like illness to an overwhelming pneumonia with coughing and the production of bloody sputum. the incubation period for primary pneumonic plague is between and days. in general, patients who develop secondary plague pneumonia have a high fatality rate. fig. . schematic of the plague cycle with small mammals as hosts and fl eas as vectors. arrows represent connections affected by climate with a color coding depending on the most infl uential climate variable on this link (i.e., precipitation, temperatures, and other variables indirectly depending on them such as soil characteristics and soil moisture). grey rectangles somewhat arbitrarily delimit epizootic, enzootic, and zoonotic cycles. note that despite their location at the far end of the cycle, humans often provide the only available information on plague dynamics the laboratory diagnosis of plague is based on bacteriological and/or serological evidence [ ] . samples for analysis can include blood, bubo aspirates, sputum, cerebrospinal fl uid in patients with plague meningitis, and scrapings from skin lesions, if present. staining techniques such as the gram, giemsa, wright, or wayson stain can provide supportive but not presumptive or confi rmatory evidence of a plague infection. lactic acid bacteria (lab) are a group of gram-positive, nonpathogenic, non-sporulating bacteria that include species of lactobacillus (fig. . ) , lactococcus , leuconostoc , pediococcus , and streptococcus . they have limited biosynthetic abilities and require preformed amino acids, b vitamins, purines, pyrimidines, and a sugar as a carbon and energy source. these nutritional requirements restrict their habitats to those in which the required compounds are abundant. thus, these highly specialized bacteria occupy a range of niches including milk, plant surfaces, the oral cavity, the gastrointestinal tract, and the vagina of vertebrates [ ] . lab have been consumed for centuries by humans in fermented foods and have an extraordinary safety profi le. these intrinsic advantages turn lab into excellent delivery vectors of novel preventive and therapeutic molecules for humans. a number of studies of oral vaccines generated from genetically engineered pathogenic or commensal bacteria have been reported [ , ] . live attenuated pathogenic bacteria, such as derivatives of mycobacterium , salmonella , and bordetella spp., are the most popular live delivery vectors used currently. they are particularly well adapted to interact with mucosal surfaces as they have specialized machinery to initiate the infection process. the major disadvantages of live vaccines include inadequate attenuation and the potential to revert to virulence. lactic acid bacteria-based vaccines act as live attenuated vaccines but without the safety concern. lab have a generally recognized as safe (gras) status and thus are not likely to cause harm. the production of a desired antigen by lab can occur in three different cellular locations: • intracellular , which allows the protein to escape harsh external environmental conditions (such as gastric juices in the stomach) but requires cellular lysis for protein release and delivery • extracellular , which allows the release of the protein into the external medium, resulting in direct interaction with the environment (food product or the digestive tract) • cell wall anchored , which combines the advantages of the other two locations (i.e., interaction between the cell wall-anchored protein and the environment, in addition to protection from proteolytic degradation) in this context, several studies have compared the production of different antigens in lab, using all three locations, and evaluated the subsequent immunological impact. these studies demonstrated that the highest immune response was obtained with cell wall-anchored antigens exposed on the surface of lab. therefore, most of the recent lab vaccination studies have selected surface exposure of the antigen of interest, rather than intra-or extracellular production [ ] . dendritic cells (dcs) play a central role in bridging the innate immune system with the adaptive immune system. dcs are found throughout the body and are especially common at mucosal surfaces. with only a single layer of epithelial cells separating the external from the internal world amid the constant need for particle exchange, intestinal dendritic cells (dcs) play a key role in maintaining intestinal homeostasis as well as governing protective immune responses against invading pathogens. to avoid activation of selfreactive t cells and to limit unnecessary responses, such as those against commensal fl ora, dcs can imprint tolerance onto t cells (fig. . ). immature-type dcs are enriched underneath the epithelium of mucosal inductive sites and are poised to capture antigens. they extend protrusions between epithelial cells, enabling direct sampling of luminal antigens [ ] . through upregulation of mhc and co-stimulatory molecules, matured dcs convert into highly effi cient antigen-presenting cells. successful antigen presentation to cd + t cells requires recognition of cognate peptide in the context of mhc class ii molecules, whereas epitopes presented on mhc class i molecules stimulate ag-specifi c cd + t cells. when antigen uptake occurs, these dcs change their phenotype by expressing higher levels of co-stimulatory molecules and move to t cell areas of inductive sites for antigen presentation. thus, dcs and their derived cytokines play key roles in the induction of antigen-specifi c effector th cell responses. in this regard, targeting mucosal dcs is an effective strategy to induce mucosal and systemic immune responses. lab persistence. the ability of some lab to persist in the gastrointestinal tract may be critically important in the effectiveness of lab-based vaccines. a comparison of a persisting lab strain, l. plantarum , with a nonpersisting lab strain, l. lactis , identifi ed l. plantarum to be more effective at eliciting antigen-specifi c immunity, suggesting that persistence promoted immunogenicity [ ] . furthermore, it has been shown that particular lactobacillus species induced critical infl ammatory cytokines and induced activation and maturation of dendritic cells [ ] . it has also been shown that immature dcs effi ciently capture lactobacillus species, and these bacteria activated human dcs, resulting in the production of pro-infl ammatory cytokines like il- , increased proliferation of cd + and cd + cells, and skewed t cell response toward a th pathway believed to be involved in effective clearance of microbial pathogens [ , ] ( fig. . ). evidence suggests that the peptidoglycan layer of some lab promote natural immuno-adjuvanticity [ ] , and antigen localization on the cell wall makes it more accessible to the immune system as compared to intracellular or secreted proteins. leader peptides mark proteins for translocation across the cytoplasmic membrane, and lipid modifi cation is of major importance both for anchoring exported proteins to the membrane and for protein function [ ] . it has been shown that lipidation at the fi rst amino acid of the mature borrelia burgdorferi ospa protein is essential to induce an immune response via tlr [ ] . the leader peptide of ospa targets the protein to the cell envelope of lactobacillus and that the cys [ ] is recognized by the l. plantarum cell wall-sorting machinery that lipidates and anchors the protein to the cell envelope. the end result is a delivery system that exerts a potent adjuvant effect [ ] . virus-like particles (vlps) that mimic the antigenic architecture of authentic virions, however, can be produced in insect, mammalian, and plant cells by the expression of the capsid protein. the particulate nature and high-density presentation of viral structure proteins on their surface render vlps as a premier vaccine platform with superior safety, immunogenicity, and manufacturability. therefore, this chapter focuses on the development of effective nov vaccines based on vlps of capsid proteins. the expression and structure of nov vlps, especially vlps of norwalk virus, the prototype nov, are extensively discussed. the ability of nov vlps in stimulating a potent systemic and mucosal anti-nov immunity through oral and intranasal delivery in mice is presented. gastroenteritis (ge) is a worldwide health problem that affects people of all ages. as its name implies, ge is characterized by infl ammation of the gastrointestinal tract and often associated with symptoms of diarrhea, nausea, vomiting, and abdominal cramping and pain. ge and its associated diarrheal diseases remain as one of the top causes of death in the world especially in developing countries and in young children with an estimated death toll of four to six million per year [ ] . ge can be caused by a variety of pathogens including viruses, bacteria, and parasites and by ingestion of noninfectious toxins or medications, with viruses as the most common offending agents. norovirus (nov) and rotavirus are the most common viruses that cause viral ge, while adenovirus, astrovirus, coronavirus, and parechovirus are also known to cause ge in humans. novs are a group of genetically diverse rna viruses that belong to the genus of norovirus in the caliciviridae family [ ] . they were fi rst discovered and characterized in their prototype virus, the norwalk virus (nv), in [ ] . studies of nv revealed that novs are non-enveloped viruses with a rna genome surrounded by a round capsid protein shell approximately nm in diameter. novs are divided into genogroups and clusters with clusters in genogroup i (gi), in gii, in giii, and each in giv and gv. within the fi ve genotypes, gi and giv strains are found to infect humans exclusively and gii are found in both humans and pigs, while giii and gv strains are animal viruses that infect cattle and murine species, respectively [ ] . currently, strains in cluster of gii (gii ) are the most prevalent novs in human population [ ] . the genome of novs, which was fi rst characterized in nv, contains a single-stranded positive-sense rna of . - . kb with three open reading frames (orfs) and a poly a tail at its ′ end [ ] (fig. . ). orf encodes a polyprotein that is processed by viral protease clpro into the rnadependent rna polymerase and approximately fi ve other nonstructural proteins including p , the nucleoside triphosphatase, p , vpg, and clpro. the two structural proteins, the major (vp ) and minor (vp ) capsid proteins, are encoded by orf and orf , respectively [ ] . structural analysis of nov has revealed that each viral capsid is composed of dimers of vp in a t = icosahedral symmetry. vp folds into two domains: a shell (s) domain that is responsible for initiating capsid assembly and icosahedral contacts and a protruding domain (p), containing two subdomains of p and p , that enhance the stability of the capsid by providing intermolecular contacts between vp dimers. studies of nv also indicate that the vp protein enhances the expression level of vp and stabilizes the vp in the viral capsid. novs are highly contagious and spread rapidly, and their outbreaks commonly occur in various social places and settings where people share common food and water sources or are in close physical proximity, such as cruise ships, schools, military units, nursing homes, daycare centers, hospitals, restaurants, and catered events . the life cycle of nov has not been fully understood due to the lack of an in vitro cell culture system and a small animal model of infection. the failure of nov replication in mammalian cell cultures is not due to the lack of host factors to support intracellular expression of nov rna. instead, the problem may lie in the steps of viral binding to cellular receptors, virus entry into cells. nov [ ] . while serum antibodies to nov can be readily detected, this method has little clinical relevance due to the cross-reactivity of antibodies. since there is no culture system available for nov, the detection of virus in stool samples has become the preferred method of diagnosis. traditionally, nov infection was diagnosed by detecting the virus by immune transmission electron microscopy (tem). tem offers the advantage of direct visualization of any potentially responsible virus particles in stool samples. however, it does have the disadvantage of requiring sophisticated and expensive equipment and highly specialized technicians for its operation. several enzyme-linked immunosorbent assays (elisas) that detect nov antigens were later developed for nov diagnosis. studies have shown that nov antigen-detecting elisas have high specifi city ( - %) but poor sensitivity ( - %), most likely due to the antigenic diversity of nov strains [ ] . similar to elisa-based assays, rt-pcr is rapid and robust, because it can process large numbers of samples simultaneously and results can be obtained within a working day. however, it requires rna extraction from fecal samples and needs expensive equipment and skilled workers to operate. therefore, rt-pcr is more labor intensive and less economical than elisas. overall, tem, elisa, and rt-pcr-based methods all have their advantages and challenges. the three methods detect different components of the virus and therefore are complementary to each other. immunology of nov infection. the immunological knowledge of nov is mostly obtained from human challenge studies and natural outbreaks due to the lack of small animal models. observations of repeat infections in adults suggest the scarcity of long-term immunity against these viruses. however, other studies showed that close to % of the genetically susceptible subjects were not infected by nov challenge, which support the possibility of long-term immunity [ ] . nov. the lack of a tissue culture system also impedes the development of vaccines against nov. fortunately, the discovery of the spontaneous assembly of expressed vp into virus-like particles (vlps) that are morphologically and antigenically similar to the native viruses has facilitated vaccine development. vlps combine the best traits of wholevirus and subunit antigens for vaccine development. vlps are noninfectious, therefore, safer than inactivated or attenuated virus due to the lack of viral nucleic acid genome. importantly, vlps can induce potent cellular and humoral immune responses without adjuvants and are more effective vaccines than other subunit antigens because their architectures mimic infectious viruses. vlps can be produced by recombinant technology in heterologous expression systems without requiring the ability to support viral replication. this is particularly important for nov because no such culture system has been developed to support the growth of these viruses. studies have demonstrated that viruses and corresponding vlps have a particle size ideal for dc and macrophage uptake to initiate antigen processing [ ] . thus, the particulate nature of vlps favors their targeting to relevant apcs for optimal induction of t cell-mediated immune responses. vlps can also be presented effi ciently to b cells and induce strong antibody responses. like live viruses, the quasicrystalline surface of vlps, with its arrays of repetitive epitopes, presents a prime target that vertebrate b cells have evolved to specifi cally recognize. this recognition triggers the cross-linking of surface membrane-associated immunoglobulins (ig) on b cells and leads to their proliferation and migration, t helper-cell activation, antibody production and secretion, and the generation of memory b cells. thus, vlps can directly activate b cells at much lower concentrations than other subunit antigens and induce high titer and durable b cell responses in the absence of adjuvants. these inherent advantages of vlps have made them one of the most successful recombinant vaccine platforms. for example, fi ve vlp-based vaccines for hepatitis b virus characterization of nov vlps. vlps of novs were fi rst produced in insect cells using baculovirus vectors [ ] and then in plants using tobamovirus and geminivirus vectors and in mammalian cells using the venezuelan equine encephalitis (vee) replicon system [ ] . these studies demonstrated that expression of the major capsid protein vp alone can drive the self-assembly of vlps that morphologically and antigenically resemble native virus particles (fig. . ) . vlps generated by all three expression systems are similar to each other. the structure of nov vlps is exemplifi ed by the vlp of nv capsid protein (nvcp). studies of insect cellbaculovirus-derived nvcp vlps by cryo-electron microscopy and x-ray crystallography reveal that the nv capsid is a nm icosahedral arrangement of copies of the kda capsid protein vp organized into dimers in a t = symmetry. while all dimers are formed from two identical nvcp monomers, two different dimer confi gurations are required to correctly form the complete assembled capsid [ ] . as in native nv particles, the nvcp also folds into two distinctive domains in vlps, with s domain forming the inner core of the shell and p domain protruding out from the capsid [ ] . similarly, the p subdomain is also the most surface-exposed region in nv vlps and may contain hbga and neutralizing antibody-binding sites and determinants of strains specifi city [ , ] . the similarity between vlps of nv and other novs including gii. viruses has been demonstrated [ ] . insect cell-baculovirus vector-produced nvcp vlps. it was shown that four oral doses of as little as μg nvcp vlps without any adjuvant triggered serum nv-specifi c anti-igg response in the majority ( / ) of vlpfed icd outbred mice [ ] . systemic igg response was observed after two oral dosages and the highest titer was induced by four doses of μg vlps. moreover, mice in the μg dosage group developed nv-specifi c intestinal iga in a level up to . % of total iga. inclusion of the mucosal adjuvant cholera toxin (ct) did not signifi cantly change the number of positive responders of serum igg or intestinal iga but signifi cantly enhanced the amplitude of serum igg response, especially for higher doses of vlps. thus, nvcp vlp is clearly a potent oral immunogen and can induce both systemic and gut mucosal antibody responses. nanovaccines: gas infections group a streptococcus ( streptococcus pyogenes) (gas) is an important human mucosal pathogen that is responsible for a wide spectrum of diseases with varying clinical manifestations and severity [ , ] : pharyngitis (strep throat) is a common minor complication of gas infection but when left untreated can lead to life-threatening diseases including the autoimmune sequelae rheumatic fever (rf) and rheumatic heart disease (rhd). rhd results in permanent damage to the heart tissues and valves. gas infections cause > , deaths each year mostly in developing countries and indigenous populations within developed nations where poor socioeconomic conditions and overcrowding contribute to the high rates of gas diseases. in developing countries, rf is the leading cause of heart disease among children [ ] . there is currently no available vaccine to prevent infection with gas and consequently prevent gas diseases. a successful mucosal gas vaccine would need to stimulate the appropriate humoral and cellular immunity for protection against gas infection ( fig. . ). this is especially diffi cult due to a lack of human-compatible mucosal vaccine adjuvants that are essential to boost immune responses. researchers have therefore focused mainly on parenteral gas vaccine delivery approaches, for which suitable adjuvants are available, designed to provide protection against systemic infection via the induction of opsonic igg antibodies. antigen. an effective gas vaccine needs to have broad antigenic reach because of the many different gas strains (> different m types) circulating in a population fig. . gas vaccination approaches. gas that breaches the physical barrier of the mucosal epithelium of the nasal-associated lymphoid tissue, functionally analogous to human tonsils, is purported to be transported to the underlying lymphoid tissue via association with membranous (m) cells. cells of the innate immune system sense gas and produce cytokines and chemokines to contain the infection to the mucosa. gas antigens are delivered to antigen-presenting cells such as dcs and b cells. iga-committed b cells are activated and initiate antigen-specifi c iga responses. dcs play a fundamental role in the development of immunity to gas and present antigen to t cells to induce a th response that is integral along with iga for mucosal defense against pharyngeal gas colonization. mucosal vaccination is designed to mimic these responses and effectively clear gas from the mucosal surface upon infection to prevent gas colonization and carriage. gas that escapes the host's defense mechanisms can disseminate into the lymphatics and blood, leading to systemic infection. mucosal vaccination is also able to induce a systemic immune response characterized by the induction of opsonic igg antibodies, which destroy the pathogen by opsonophagocytosis. parenteral gas vaccination induces serum igg but is not able to induce mucosal immunity and should not induce immune responses that are potentially cross-reactive with self tissue proteins. the gas m protein is the major protective antigen and an ideal target for vaccine development; however it contains heart tissue cross-reactive epitopes particularly in the conserved region [ ] . evidence suggests that cross-reactive t cells especially play a pivotal role in the pathogenesis of rhd. the m protein is an α-helical coiled-coil surface protein consisting of a hypervariable amino-terminal region and a highly conserved (> % sequence identity) carboxyterminal c-repeat region [ ] (fig. . ) . functionally, the m protein is important in preventing bacterial clearance by complement-mediated phagocytosis, which limits host defense mechanisms. previous studies indicate that protective immunity to gas can be evoked by opsonic antibodies to serotypic epitopes at the amino-terminal region that are m-type specifi c [ ] . nanovaccine. combined vaccine/adjuvant delivery systems offer the potential of mucosal vaccine delivery. for example, the lipid-core-peptide (lcp) system is a novel, synthetic, selfadjuvanted vaccine delivery system that incorporates the adjuvant (prr agonist), carrier, and antigenic peptides of a vaccine into a single molecular entity ( fig. . ). this system has been previously shown to effi ciently deliver gas vaccines and induce immunity [ ] . evidence suggests the adjuvant activity of lcp involves the induction of dc activation. preclinical developments. three approaches are currently being investigated in the development of a subunit gas vaccine based on the m protein: . multivalent gas vaccine . a multivalent approach employs a combination of amino-terminal protein fragments representing different m types and is designed to target prevalent gas strains in a population. using this approach, a recombinant multivalent gas vaccine containing m protein peptides from different gas serotypes prevalent in north america was demonstrated to evoke opsonic antibodies in animals [ ] . from epidemiological data, the -valent vaccine would cover the majority of pharyngitis and invasive gas diseases, including rf, invasive fasciitis, and toxic shock syndrome. recently, a new -valent gas vaccine was shown to be immunogenic in rabbits and evoked opsonic antibodies against "non-vaccine" serotypes [ ] potentially creating a vaccine with much broader coverage. this type of vaccine is population specifi c and therefore may not be effective universally. it may also need to be re-designed periodically to refl ect changes in the epidemiology of gas infections. (ch ) (ch ) ch ch ch fig. . the lipid-core-peptide (lcp) system. the lcp system contains an adjuvant component (lipid-core made of lipoamino acids) and a polylysine carrier onto which peptide epitopes are attached. the example shows a gas vaccine candidate containing j and an amino-terminal serotypic epitope called . the adjuvant has three -amino-dodecanoic ( n = ) lipoamino acids separated by glycine amino acid spacers . j vaccine . a gas vaccine that employs peptide epitopes from the conserved c-repeat region of the m protein is the second approach and has the potential in theory for greater coverage of m types. immunization of mice with a c-region peptide gas vaccine candidate called j conjugated to the carrier protein diphtheria toxoid (dt) and co-delivery with an appropriate adjuvant led to protection against systemic and mucosal gas infection [ ] (fig. . ). j also elicited protective immunity against gas when linked to lipopeptides [ , ] . other studies have shown that intranasal immunization of mice with c-region peptides conjugated to the experimental mucosal adjuvant cholera toxin b subunit (ctb) evoked protective immunity against gas at the mucosal level [ ] . ctb could possibly enter olfactory regions of the central nervous system and cause neuronal damage following intranasal delivery [ ] and therefore is not suitable for human use. vector delivery approaches have included expressing the c-repeat region on vaccinia virus [ ] , the commensal bacterium lactococcus lactis [ ] , or streptococcus gordonii [ ] . . j . the third combination vaccine approach uses both serotypic and conserved m protein peptide epitopes. initially, a heteropolymer gas vaccine construct was synthesized by free radical-induced polymerization of acryloyl peptides to combine seven serotypic epitopes and a highly conserved c-region peptide epitope called j [ ] . the m types that were targeted in the heteropolymer represented gas infections prevalent in the northern territory of australia -a region highly endemic for gas. immunization of mice with the heteropolymer demonstrated excellent immunogenicity and protection fig. . preclinical evaluation of gas vaccine candidates. intranasal immunization of mice with the j -dt vaccine candidate led to significantly greater survival after intranasal challenge with gas versus control groups but the mucosal adjuvant ctb was essential for protection ( a ). a multiepitope lcp-based gas vaccine candidate elicited protective immunity against mucosal gas infection even in the absence of ctb ( b ) against homologous and heterologous gas strains, indicating its potential to provide broad coverage. however, batch-to-batch variation led to altered immune responses, which limited its applicability for human use. the vaccine also required the addition of an adjuvant to be effective, further limiting its use as a mucosal vaccine due to a lack of safe and effective mucosal adjuvants. later, multiepitope gas vaccine candidates were synthesized based on the lcp system that induced highly opsonic antibodies following parenteral delivery to mice [ ] , as well as protection against mucosal gas infection following intranasal immunization [ ] (fig. . ). safety and effi cacy. the main concern when using large regions of the m protein in a gas vaccine is the potential for inducing an autoimmune response due to immunological cross-reactivity with host proteins. it is therefore important to identify protective antigenic determinants and to separate the biological relevant epitopes from those that are host tissue cross-reactive and potentially harmful. epitope mapping studies were used to identify the conserved gas vaccine candidate j , which contains a conformational protective b cell epitope and was designed to lack a human heart cross-reactive t cell epitope [ , ] . tb vaccines -state of the art and progresses dna-antiviral vaccines: new developments and approaches -a review west nile virus recombinant dna vaccine protects mouse and horse from virus challenge and expresses in vitro a noninfectious recombinant antigen that can be used in enzymelinked immunosorbent assays stable and long-lasting immune response in horses after dna vaccination against equine arteritis virus vaccination with human tyrosinase dna induces antibody responses in dogs with advanced melanoma infectious haematopoietic necrosis epidemic ( to ) in farmed atlantic salmon salmo salar in british columbia naked dna vaccination of atlantic salmon salmo salar against ihnv effi cacy of an infectious hematopoietic necrosis (ihn) virus dna vaccine in chinook oncorhynchus tshawytscha and sockeye o. nerka salmon wild pollinators enhance fruit set of crops regardless of honey bee abundance a metagenomic survey of microbes in honey bee colony collapse disorder colony collapse disorder: a descriptive study the reproductive program of female varroa destructor mites is triggered by its host, apis mellifera brood cell size of apis mellifera modifi es the reproductive behavior of varroa destructor the transmission of deformed wing virus between honeybees (apis mellifera l.) by the ectoparasitic mite varroa jacobsoni oud detection of acute bee paralysis virus and black queen cell virus from honeybees by reverse transcriptase pcr varroa destructor is an effective vector of israeli acute paralysis virus in the honeybee, apis mellifera prevalence and transmission of honeybee viruses age-related changes in the behavioural response of honeybees to apiguard(r), a thymolbased treatment used to control the mite varroa destructor the immune response of drosophila drosophila hemopoiesis and cellular immunity c: insights into social insects from the genome of the honeybee apis mellifera structure and genetics of shigella o antigens optimization of virulence functions through glucosylation of shigella lps growing antimicrobial resistance of shigella isolates role of m cells in initial antigen uptake and in ulcer formation in the rabbit intestinal loop model of shigellosis ipab mediates macrophage apoptosis induced by shigella fl exneri molecular pathogenesis of shigella spp.: controlling host cell signaling, invasion, and death by type iii secretion mucosal lymphoid infi ltrate dominates colonic pathological changes in murine experimental shigellosis not available development of an improved animal model of shigellosis in the adult rabbit by colonic infection with shigella fl exneri a a challenge model for shigella dysenteriae in cynomolgus monkeys (macaca fascicularis) immunogenicity and effi cacy of oral or intranasal shigella fl exneri a and shigella sonnei proteosome-lipopolysaccharide vaccines in animal models enhancement of anti-shigella lipopolysaccharide (lps) response by addition of the cholera toxin b subunit to oral and intranasal proteosome-shigella fl exneri a lps vaccines isolation and characterization of a shigella fl exneri invasin complex subunit vaccine immunogenicity and effi cacy of highly purifi ed invasin complex vaccine from shigella fl exneri a development and evaluation of a shigella fl exneri a and s. sonnei bivalent invasin complex (invaplex) vaccine safety and immunogenicity of an intranasal shigella fl exneri a invaplex vaccine characterization of a mutant escherichia coli heat-labile toxin, lt(r g/l a), as a safe and effective oral adjuvant broadly protective shigella vaccine based on type iii secretion apparatus proteins purifi cation and characterization of an immunogenic outer membrane protein of shigella fl exneri a outer membrane protein a (ompa) of shigella fl exneri a, induces protective immune response in a mouse model global aspects of the management and control of ticks of veterinary importance the global importance of ticks chemical control of ticks on cattle and the resistance of these parasites to acaricides factors that infl uence the prevalence of acaricide resistance and tick-borne diseases the molecular revolution in the development of vaccines against ectoparasites rna interference for the study and genetic manipulation of ticks proteins in the saliva of the ixodida (ticks): pharmacological features and biological signifi cance comparison of the proteins in salivary glands, saliva and haemolymph of rhipicephalus appendiculatus female ticks during feeding immunoglobulin-binding proteins in ticks: new target for vaccine development against a blood-feeding parasite amblyomma americanum : specifi c uptake of immunoglobulins into tick hemolymph during feeding synthetic vaccine (sbm ) against the cattle tick rhipicephalus (boophilus) microplus : preservation of immunogenic determinants in different strains from south america cloning, expression and immunoprotective effi cacy of rhaa , the homologue of the bm tick vaccine antigen, from hyalomma anatolicum anatolicum differential transcription of two highly divergent gut-expressed bm antigen gene homologues in the tick rhipicephalus appendiculatus (acari: ixodida) cloning and expression of a protective antigen from the cattle tick boophilus microplus bovine immunoprotection against rhipicephalus (boophilus) microplus with recombinant bm -campo grande antigen tick vaccines and the transmission of tick-borne pathogens vaccination against ticks (boophilus spp.): the experience with the bm -based vaccine gavac immunity against boophilus annulatus induced by the bm (tick-gard) vaccine developing live vaccines against plague yersinia pestis--etiologic agent of plague yersinia: strategies that thwart immune defenses plague . in: crc handbook series in zoonoses . section a. bacterial, rickettsial, chlamydial and mycotic diseases mucosal delivery of therapeutic and prophylactic molecules using lactic acid bacteria the induction of hiv gag-specifi c cd + t cells in the spleen and gutassociated lymphoid tissue by parenteral or mucosal immunization with recombinant listeria monocytogenes hiv gag mucosal immunization with surface-displayed severe acute respiratory syndrome coronavirus spike protein on lactobacillus casei induces neutralizing antibodies in mice lactococci and lactobacilli as mucosal delivery vectors for therapeutic proteins and dna vaccines dendritic cells express tight junction proteins and penetrate gut epithelial monolayers to sample bacteria protection against tetanus toxin after intragastric administration of two recombinant lactic acid bacteria: impact of strain viability and in vivo persistence lactobacilli differentially modulate expression of cytokines and maturation surface markers in murine dendritic cells lactobacilli as natural enhancer of cellular immune response lactobacilli activate human dendritic cells that skew t cells toward t helper polarization instruments for oral disease-intervention strategies: recombinant lactobacillus casei expressing tetanus toxin fragment c for vaccination or myelin proteins for oral tolerance induction in multiple sclerosis surface proteins of gram-positive bacteria and mechanisms of their targeting to the cell wall envelope. microbiol treponema pallidum and borrelia burgdorferi lipoproteins and synthetic lipopeptides activate monocytic cells via a cd -dependent pathway distinct from that used by lipopolysaccharide immune response to lactobacillus plantarum expressing borrelia burgdorferi ospa is modulated by the lipid modifi cation of the antigen a review of viral gastroenteritis noroviruses: a comprehensive review biological properties of norwalk agent of acute infectious nonbacterial gastroenteritis norovirus classifi cation and proposed strain nomenclature mechanisms of gii. norovirus persistence in human populations sequence and genomic organization of norwalk virus norwalk virus genome cloning and characterization methods for the detection and characterisation of noroviruses associated with outbreaks of gastroenteritis: outbreaks occurring in the north-west of england during two norovirus seasons european multicenter evaluation of commercial enzyme immunoassays human susceptibility and resistance to norwalk virus infection t cellindependent type i antibody response against b cell epitopes expressed repetitively on recombinant virus particles threedimensional structure of baculovirus-expressed norwalk virus capsids expression and self-assembly of norwalk virus capsid protein from venezuelan equine encephalitis virus replicons conformational stability and disassembly of norwalk virus like particles: effect of ph and temperature structural studies of recombinant norwalk capsids e. coliexpressed recombinant norovirus capsid proteins maintain authentic antigenicity and receptor binding capability c-terminal arginine cluster is essential for receptor binding of norovirus capsid protein structural basis for the recognition of blood group trisaccharides by norovirus pathogenesis of group a streptococcal infections group: a streptococcal infections and acute rheumatic fever multiple cross reactive epitopes of streptococcal m proteins streptococcal m protein: molecular design and biological behavior localization of protective epitopes of the amino terminus of type streptococcal m protein towards the development of a broadly protective group a streptococcal vaccine based on the lipid-core peptide system immunogenicity of a -valent group a streptococcal vaccine new -valent m protein-based vaccine evokes cross-opsonic antibodies against non-vaccine serotypes of group a streptococci protection against group a streptococcus by immunization with j -diptheria toxoid: contribution of j -and diphtheria toxoid-specifi c antibodies to protection intranasal vaccination with a lipopeptide containing a minimal, conformationally constrained conserved peptide, a universal t-cell epitope and a self-adjuvanting lipid protects mice from streptococcus pyogenes and reduces throat carriage immunisation of mice with a lipid core peptide construct containing a conserved region determinant of group a streptococcal m protein elicits heterologous opsonic antibodies in the absence of adjuvant epitopes of group a streptococcal m protein that evoke cross-protective local immune responses cutting edge: the mucosal adjuvant cholera toxin redirects vaccine proteins into olfactory tissues protection against streptococcal pharyngeal colonization with a vaccinia: m protein recombinant mucosal vaccine made from live, recombinant lactococcus lactis protects mice against pharyngeal infection with streptococcus pyogenes clinical and microbiological responses of volunteers to combined intranasal and oral inoculation with a streptococcus gordonii carrier strain intended for future use as a group a streptococcus vaccine new multi-determinant strategy for a group a streptococcal vaccine designed for the australian aboriginal population immunization with a tetraepitopic lipid core peptide vaccine construct induces broadly protective immune responses against group a streptococcus intranasal administration is an effective mucosal vaccine delivery route for self-adjuvanting lipid core peptides targeting the group a streptococcal m protein mapping a conserved conformational epitope from the m protein of group a streptococci mapping the minimal murine t cell and b cell epitopes within a peptide vaccine candidate from the conserved region of the m protein of group a streptococcus key: cord- -gq fss l authors: hsueh, wei; caplan, michael s.; qu, xiao-wu; tan, xiao-di; de plaen, isabelle g.; gonzalez-crussi, f. title: neonatal necrotizing enterocolitis: clinical considerations and pathogenetic concepts date: - - journal: pediatr dev pathol doi: . /s - - -z sha: doc_id: cord_uid: gq fss l necrotizing enterocolitis (nec), a disease affecting predominantly premature infants, is a leading cause of morbidity and mortality in neonatal intensive care units. although several predisposing factors have been identified, such as prematurity, enteral feeding, and infection, its pathogenesis remains elusive. in the past years, we have established several animal models of nec in rats and found several endogenous mediators, especially platelet-activating factor (paf), which may play a pivotal role in nec. injection of paf induces intestinal necrosis, and paf antagonists prevent the bowel injury induced by bacterial endotoxin, hypoxia, or challenge with tumor necrosis factor-a (tnf) plus endotoxin in adult rats. the same is true for lesions induced by hypoxia and enteral feeding in neonatal animals. human patients with nec show high levels of paf and decreased plasma paf-acetylhydrolase, the enzyme degrading paf. the initial event in our experimental models of nec is probably polymorphonuclear leukocyte (pmn) activation and adhesion to venules in the intestine, which initiates a local inflammatory reaction involving proinflammatory mediators including tnf, complement, prostaglandins, and leukotriene c . subsequent norepinephrine release and mesenteric vasoconstriction result in splanchnic ischemia and reperfusion. bacterial products (e.g., endotoxin) enter the intestinal tissue during local mucosal barrier breakdown, and endotoxin synergizes with paf to amplify the inflammation. reactive oxygen species produced by the activated leukocytes and by intestinal epithelial xanthine oxidase may be the final pathway for tissue injury. protective mechanisms include nitric oxide produced by the constitutive (mainly neuronal) nitric oxide synthase, and indigenous probiotics such as bifidobacteria infantis. the former maintains intestinal perfusion and the integrity of the mucosal barrier, and the latter keep virulent bacteria in check. the development of tissue injury depends on the balance between injurious and protective mechanisms. cumstances are not uniform, nec may represent a syndrome, with common findings and a variety of etiologies. intestinal necrosis, representing a latestage response, is consistent with a common pathogenesis, but disparate etiologies are possible. necrosis of the intestine can occur at any age following the sudden, complete occlusion of the blood supply to the bowel. in the newborn, thromboemboli secondary to intravascular catheters may cause bowel infarction. however, since neonatal nec cannot be traced to thromboemboli, it is considered nosologically distinct from this kind of bowel infarction. therefore, in the following discussion, nec is understood to exclude cases of bowel infarction associated with thromboembolic lesions. nec remains a leading cause of morbidity and mortality in neonatal intensive care units, with a reported incidence of approximately % among very low birth weight infants (Ͻ g) [ ] , and a mortality of % [ ] . a disease of serious prognosis, advanced cases of nec may cause multisystem organ failure [ ] . of the cases occurring annually in the united states [ , ] , - % require surgical treatment [ ] . at least % of patients are preterm, or have low, or very low birth weight, and the incidence of the disease is inversely proportional to the gestational age [ , , ] . advances in the supportive care of premature babies, such as the use of surfactant, improved technologies for mechanical ventilation, and wider availability of skilled personnel, enable the very premature to survive, and in so doing increase the population of patients susceptible to nec. thus, it may be that despite medical advances that would potentially reduce the incidence of the disease, the incidence of nec remains unchanged over the last years [ , ] . infants of extremely low birth weight (Ͻ g) and those wk or less of gestational age are at greater risk of nec than infants born closer to term. the severity of the disease, risks of complications, and mortality are greater in infants of extremely low birth weight [ ] . nec is uncommon in term infants, in whom it usually appears within to days after birth, whereas in the preterm it begins at to days after birth [ ] . presumably, a postnatal insult is followed by the pathogenetic events that lead to the tissue devastation characteristic of nec. the initi-ating and pathogenetic factors may differ in patients of different age groups. in any case, the clinical consequences do not differ substantially in the various patient populations, including the infants of extremely low birth weight or extreme prematurity [ ] . the symptoms have been staged according to widely used criteria [ , ] . the infant manifests abdominal distension (among the most common signs of nec), vomiting, increased gastric residual, lethargy, apnea, bradycardia, or guaiac-positive stools. in stage i, there are no clear radiological signs, and these nonspecific manifestations suggest the disease but give no indication of the status of the bowel or the prognosis. in stage ii, the diagnosis is clearly established, with the appearance of pneumatosis intestinalis or free air in the portal vein. stage iii indicates more advanced disease, as manifested by shock, disseminated intravascular coagulation, acidosis, thrombocytopenia, and sometimes intestinal perforation. the predominant anatomic lesion of nec is coagulative or ischemic necrosis [ - ] (fig. a-c) . the usual site is the ileocolic region. this may be because of remoteness of the ileocolic arterial branches from the main blood supply of the superior mesenteric artery, which also supplies the proximal intestine. in about half the cases, the necrosis involves both the small and large intestine; continuous or discontinuous involvement occurs in approximately equal proportions [ , ] . the affected bowel is grossly distended, lusterless, and gray or greenish-gray, but it may be dark purple or black in the areas containing extensive hemorrhage. the soft, fragile wall may perforate when the involvement is severe and transmural. perforation tends to occur at the junction between normal and necrotic bowel, but it may appear in the midst of a devitalized region, and sometimes at more than one site. gas bubbles, which may be grossly visible in the intestinal wall, involve the entire colon more commonly in the term infant than in the premature infant [ ] . ischemia in nec accounts for the necrosis, but the mechanism remains unresolved. nowicki [ ] distinguished extrinsic and intrinsic mecha-neonatal necrotizing enterocolitis nisms of vascular regulation. extrinsic vascular regulation integrates the circulation of the intestine with systemic cardiovascular reflexes. an atavistic "diving reflex" (so named after the physiological changes noted in seals upon diving) [ ] has been hypothesized in neonates who experience severe anoxic episodes, during which blood is diverted preferentially to the heart and the brain, to the detriment of the abdominal organs. although the diving reflex hypothesis is supported by much experimental evidence in animals [ ] , it cannot satisfactorily explain all the clinical observations in nec. presumably, the reflex takes place as a result of a postulated ischemic insult during parturition [ ] , whereas the manifestations of nec usually start during the nd wk of postnatal life. vascular reactivity in early postnatal life has been assumed to differ from that of older subjects. however, the intestinal vasculature of - -day-old piglets manifests autoregulatory "escape" from sustained sympathetic stimulation, in the same manner as the intestine of older swine. experimentally, sympathoadrenergic stimulation causes transient intestinal vasoconstriction, and normal oxygen uptake is restored after to min [ , ] . moreover, prospective clinical studies do not always establish an association between neonatal hypoxia or asphyxia and the development of nec: most patients with nec have no clinically apparent hypoxemia at birth [ , , ] . these discrepancies by no means exclude an important participa-tion of autonomic neural influences in the development of nec. other extrinsic regulatory mechanisms, such as the participation of the renin-angiotensin axis in bowel ischemia deserve serious investigation. angiotensin receptors are densely distributed in the bowel [ ] . this may explain why ischemic colitis that develops from mesenteric vasoconstriction during experimental cardiogenic shock cannot be prevented by total adrenergic blockade but is completely abolished by drugs such as captopril, which ablate the reninangiotensin axis [ ] . the intrinsic vasoregulation of the intestine, defined as that "mediated by effector mechanisms produced and released within the intestine and its attendant circulation," has been studied in denervated intestinal segments and other in vivo and in vitro models [ ] . a "metabolic theory" stresses homeostatic control by local tissue need for oxygen, and a "myogenic reflex theory" proposes vasoconstriction in the intestinal circulation in response to changes in venous pressure. presumably, labile, active myogenic vascular responses in the very young increase their susceptibility to intestinal ischemia [ ] . other "intrinsic" vasoregulatory influences leading to intestinal ischemia include the potent agents that are considered central to a theoretical pathogenesis of nec (vide infra). the clinical situation is more complex than any hypothetical model centered upon experimental observations. as kosloske [ ] observed, the chronology of clinical events is not always clear. in patients with congenital heart disease or cardiogenic shock, hemodynamic disturbances acquire a significant role in the causation of nec, but this does not gainsay the utility of clarifying the basic steps by which the disease is initiated and maintained. necrosis of the bowel can develop secondary to mesenteric thromboembolism. in neonates, thrombosis is usually an untoward effect of the placement of an umbilical artery catheter. however, in most patients with nec, no occlusion of large arteries can be identified. nec and infarction are probably different clinicopathological entities, even though both manifest coagulative necrosis. an infarct is usually single and should follow the distribution of the arterial blood supply. in contrast, nec is basically an inflammatory process, and a venule may be the initiating site of the pathophysiology. the affected areas are often multiple, are random, and are not necessarily related to the arterial supply. the early histological change of nec is coagulative necrosis, but inflammatory cells infiltrate when the disease progresses [ ] . bacteria are important in nec, since the disease does not occur before the colonization of the intestine by bacteria. in a fetus whose intestinal contents are sterile, compromise of the blood supply may result in intestinal injury. in the healing process, atresia or stenosis may develop. anaerobic bacteria in the lumen of the bowel might be expected to proliferate in a segment of devitalized bowel. bacterial overgrowth in nec seems to exceed that in other diseases with ischemic bowel [ ] . intestinal pneumatosis, the peculiar and characteristic finding seen in many cases of nec, is not observed in infarcts. the formation of gas bubbles within the wall of the intestine (fig. d) , develops largely as a result of the fermentation of intraluminal contents by bacteria, and is associated more with nec than with any other necrotizing conditions affecting the intestine. bacterial production of p-galactosidase, which reduces ph by fermentation of lactose, has been suggested to contribute to the development of nec [ ] . the ability of colonizing bacteria to ferment lactose is not correlated with the production of nec [ ] ; moreover, the endemic cases of nec are not consistently associated with a single infectious agent or with a particularly virulent organism that produces highly damaging toxins or that displays great entero-invasiveness or entero-aggregative ability. disparate microorganisms have been isolated from the stools of nec patients, and in some cases from both blood and stools: escherichia coli, klebsiella, enterobacter, pseudomonas, salmonella, clostridium perfringens, clostridium difficile, clostridium butyricum [ ] , coagulase-negative staphylococci [ ] , coronavirus, rotavirus, and enteroviruses [ ] . intestinal inflammation affects about % of the patients with nec and is considered an appropriate host response to necrosis and proliferating bacteria [ ] . inflammation tends to be less severe following sudden occlusion of the arterial circulation, as with thromboembolism, and much more conspicuous when devitalization of the bowel is gradual. according to ballance et al. [ ] , the character of the inflammation in colitis of infectious origin differs from that in nec. microabscesses and crypt abscesses are common in infectious colitis, but they affect only % of patients with nec. moreover, extensive necrosis beyond the inflammation is a feature of nec that is generally absent in cases of infectious enterocolitis. regenerative changes in nec are usually marked by replacement of the mucosa by a cuboidal or tall epithelium displaying hyperchromatic nuclei, with mitotic activity and without mucin production. this layer covers granulation tissue or a partly reconstituted lamina propria with distorted, morphologically aberrant glands [ , ] . regenerative changes may appear even in cases without a protracted history. ballance et al. [ ] found reparative activity of recent onset in % of the patients, all undergoing surgery for the first time. these findings suggest that the disease may have started earlier than could be inferred from the degree of severity and/or duration of clinical symptoms. we developed a model of bowel necrosis in adult rats and mice by injecting endotoxin (lipopolysaccharide, lps) [ ] , paf (platelet-activating factor, paf-acether) [ , ] , tumor necrosis factor-␣ (tnf) [ ] , or a combination of these agents. the rationale for using these agents is as follows: lps: nec is clearly associated with intestinal bacterial growth, since nec usually develops following oral feeding, and oral feeding markedly increases the growth of e. coli in the intestinal tract [ ] . no single infectious agent has been isolated consistently from patients with nec. we hypothesized that resident intestinal flora such as e. coli and its toxic product, lps, would be causative agents of nec. paf: injection of lps induces endogenous production of paf [ , ] , systemic administration of paf [ - ] to animals mimics signs of shock, and paf antagonists prevent lps-induced shock [ , ] . tnf: lps induces endogenous tnf production [ , , ] and administration of tnf causes shock [ , ] , whereas pretreatment of the animal with anti-tnf [ ] ameliorates endotoxin shock and increases survival. paf is an endogenous phospholipid mediator produced by inflammatory cells, endothelial cells, platelets [ , , ] , and bacteria of the intestinal flora, such as e. coli [ ] . systemic administration of paf induces an immediate and sometimes transient hypotensive response. with large doses, the shock becomes profound, irreversible, and intestinal necrosis develops rapidly. early injury is usually detectable within min. paf is probably the most potent systemically administered agent for inducing intestinal injury. in our experiments, as little as . g/kg often caused small intestinal necrosis of varying degree in the rat. since rat platelets are refractory to paf [ , ] , the pathogenesis of necrosis cannot be due to the thromboembolic effect of paf. the necrosis, usually focal, involved the jejunum, ileum, especially the distal ileum, and/or cecum. with high doses, the entire small bowel could be affected. the necrosis began at the villus tip ( fig. a ) [ ] , often involved more than half of the villus (fig. b) , and sometimes extended to the submucosa or even the serosa (fig. c ). although lps alone can cause hypotension and intestinal necrosis, the required dosage is often high (Ͼ mg/kg). lps is a potent "priming" agent for paf: a small dose of lps ( . mg/kg) acts synergistically with a low dose of paf (table ) [ , , ] . lps-induced intestinal injury is blocked by pretreatment with paf antagonists [ ] , suggesting that this effect is mediated by endogenous paf. one probable reason why the small intestine, in particular the ileum, is especially sensitive to paf action, is its high content of paf receptors (paf-r). using quantitative polymerase chain reaction (pcr), we found that the ileum has the highest number of paf-r transcripts: ( . Ϯ . ) ϫ molecules/g rna [ ] . the paf-r content of jejunum was only % of that of the ileum, and the spleen was only %. other organs, e.g., lung, kidney, heart, stomach, and liver, had less than % of that of ileum [ ] . paf, even at doses below those causing bowel necrosis, almost doubled paf-r mrna in the intestine [ ] . the increase was biphasic; the second peak (at h) seemed dependent on endogenous paf and tnf [ ] . in the small intestine, paf receptor was localized mainly in epithelial cells and eosinophils of the lamina propria [ ] . paf has a short half-life in the blood, being rapidly degraded by serum acetylhydrolase into the biologically inactive lyso-paf [ ] [ ] [ ] . paradoxically, the in vivo action of paf is prolonged. one mechanism that may account for this prolonged action is that paf induces its own production in tissues [ ] . when paf antagonists were given before paf challenge, the production of paf (and paf-like phospholipids) was markedly reduced (table ) [ , ] . (since in these studies we assessed the biological activity, rather than chemical analysis of paf, we could not differentiate paf from paf-like phospholipids; the latter bind to paf receptor and have effects that are much like those of paf [ ] ). the initial event following paf challenge is probably polymorphonuclear leukocyte (pmn) activation and pmn-endothelial adhesion. pmn-depletion markedly reduced paf-induced bowel injury [ - ] . the major adhesion molecule involved in the paf effect is leukocyte ␤ -integrin, especially cd b/cd , since pretreatment with anti-cd b or anti-cd antibody largely prevents pmn influx as well as paf-induced bowel injury [ ] . anti-cd also prevents the paf-induced increase in endothelial [ ] and mucosal [ ] permeability. p-selectin-deficient mice and fucoidin-treated intercellular adhesion molecule- (icam- ) deficient mice are also protected from the adverse effects of paf [ ] , suggesting a possible role of p-selectin. paradoxically, fucoidin, a potent inhibitor of selectins, shows no protective effect [ , ] . the marked increase in pmn influx (adhered to vessels) is reflected by the increased myeloperoxidase (mpo) content in the intestine [ ] . yet extravascular pmn infiltration is not found by histological examination, indicating that pmn transmigration into tissues is not required for necrosis. paf, platelet-activating factor; lps, lipopolysaccharide (bacterial endotoxin); tnf, tumor necrosis factor-␣. a all values were obtained min after the injection of paf [ ] . (in later studies, the dose of paf used to induce the same degree of bowel necrosis was reduced to . - . mg/kg, when pure c -paf was used.) b all values were obtained h after the injection of tnf [ ] . c mild necrosis: involving top third of villi. d moderate necrosis: involving more than top one-third of villi, but confined to the mucosa. the final effector of paf causing cell injury is most likely reactive oxygen species (ros). one of the major endogenous sources of ros in the intestine is the xanthine dehydrogenase/xanthine oxidase system (xd/xo) [ ] . xd, the precursor of xo, is constitutively and abundantly expressed in the intestinal villus epithelium [ , ] , which catalyzes the conversion of hypoxanthine to xanthine, coupled with the reduction of nad ϩ to nadph. because xo uses molecular oxygen rather than nad ϩ as an electron receptor and thereby generates superoxide, xd to xo conversion (during ischemia) has been suggested to play the central role in intestinal reperfusion injury [ ] . in normal rat intestine, the total xdϩxo content (xd/xo ratio approximately : ) is higher in the jejunum than in the ileum (the colon has low xdϩxo) [ ] . interestingly, following paf challenge, it is the ileum that shows the most dramatic xd to xo conversion (more than twofold increase in xo) [ ] . this change is rapid, detected at min, and by min, more than % of the total xdϩxo activity has converted to xo [ ] . the conversion takes place mainly in the villus epithelial cells, but not in the crypt epithelium, and the major pathway is probably via activated protease [ ] . how this activation is related to pmn activation and adhesion to endothelial cells, remains enigmatic. the central role of xo and ros in causing the injury is supported by pretreatment with allopurinol [ , ] , a xanthine oxidase inhibitor, which largely prevents paf-induced bowel necrosis ( table ). infusion of superoxide dismutase plus catalase also alleviates the injury [ ] (table ) . tnf has many proinflammatory actions [ ] [ ] [ ] , such as inducing leukocyte and endothelial adhesion molecules, activating pmns and endothelial cells, and causing production of other cytokines [ ] [ ] [ ] , including tnf itself [ ] [ ] [ ] , eicosanoids [ , ] , and paf [ , ] . intravenous injection of tnf ( mg/kg) also induces hypotension and mild intestinal injury in rats [ ] . the effects of tnf and lps are synergistic: tnf ( . mg/kg), when combined with lps ( mg/kg), causes profound shock and severe intestinal necrosis in rats [ ] and mice [ ] . paf is probably the endogenous the splanchnic bed is considered a major source of tnf production in vivo [ ] . we have shown that lps ( mg/kg) and paf ( g/kg), at doses below those causing shock and intestinal injury, stimulate tnf gene expression and protein production in the rat's liver and small intestine, predominantly in the ileum [ ] (fig. ) . web- , a paf antagonist, only partially blocked lps-induced tnf mrna formation (fig. ) , suggesting that lps induces tnf formation via both paf-dependent and paf-independent pathways. in normal intestine, tnf is constitutively expressed at very low levels within paneth cells [ ] . during the acute stage of nec, tnf gene transcripts markedly increase not only within paneth cells, but also in lamina propria eosinophils, and infiltrating (but not resident) macrophages [ ] . paneth cells are also rich in group iia phospholipase a (pla -iia) [ ] , an acute phase protein, which is also upregulated by paf [ ] . production of many proinflammatory cytokines, including tnf, is upregulated by transcription factors, such as nuclear factor b (nf-b) [ ] . tnf activates nf-b in vitro [ , ] , a pathway that may be involved in tnf self-activation. low doses of tnf ( mg/kg) or paf ( g/kg), which are below those causing shock and intestinal injury, increase the mrna of nf-b precursor, p /p , in the small intestine [ ] . the action of paf is as potent as, but more rapid than, that of lps [ ] . paf also rapidly induces nf-b nuclear translocation and activation in the intestine, mainly as p homodimers [ ] . lps also activates nf-b, but as p -p dimer, and its effect is partly mediated via endogenous paf and tnf [ ] . the role of this transcription factor is unclear, but preliminary experiments show that blocking nf-b with decoy [ ] or with nf-b essential modulator (nemo) (ikk␥) binding peptide [ ] attenuates paf-induced injury. a paf challenge increases gut mucosal permeability [ ] . this event precedes cell necrosis, and occurs at doses below that causing necrosis [ ] . paf alters the cytoskeletal structure of the intestinal epithelium and induces tyrosine phosphorylation of e-cadherin, an epithelial membrane component of the zona adherens [ ] . this may be physiologic, since glucose-induced increased mucosal permeability is blocked by paf antagonists [ ] . in nec, this action of paf may facilitate the entry of bacterial products, e.g., lps from the gut lumen into the tissues, triggering the inflammatory cascade. indeed, our data suggest that endogenous bacterial toxins from the intestinal lumen play a central role in paf-induced shock and bowel injury: ( ) endotoxin-resistant mice are protected from paf-induced intestinal injury [ ] ; ( ) germ-free rats are protected from paf-induced prolonged shock and bowel injury, and the protection is lost when these animals are primed with exogenous lps [ ] ; and ( ) conventional rats, treated with combined anti- paf induces tumor necrosis factor (tnf) mrna production in the rat liver and ileum. mrna from the liver and the small intestine was extracted min after injection of a low dose ( g/kg, iv) of paf, lps ( mg/kg, iv), or web- ( mg/kg) plus lps. results calculated from northern blot analysis [ ] . ␤-actin, a housekeeping gene, is used as the common denominator for calculation, and quantity of tnf mrna is expressed as ratio of tnf mrna/␤-actin mrna. sham-op., sham operation; lps, lipopolysaccharide (bacterial endotoxin); web, web- . (from hsueh et al. [ ] , with permission.) biotics which markedly decrease intestinal bacteria, are protected to a large extent from the injurious effects of paf [ ] (table ) . paf probably causes intestinal injury and deleterious systemic changes via a synergistic action with endogenous bacterial toxins from intestinal bacteria [ , ] . lps may not be the only bacterial product that synergizes with paf to produce tissue damage, since polymyxin b (which inhibits lps) alone was without protective effects [ ] . paf has a prolonged in vivo action despite its short half-life in the circulation. furthermore, paf is a vasodilator in vitro [ ] , but high doses cause sustained vasoconstriction of the splanchnic bed in vivo [ , ] . these apparently paradoxical effects could be reconciled by the observations that leukotriene c (ltc ) [ ] and norepinephrine [ ] , which cause splanchnic vasoconstriction, are released after paf injection. moreover, in vivo administration of antagonists to peptide leukotrienes [ , ] , or alpha blockers [ ] , do not reverse shock, but prevent paf-induced intestinal injury ( table ). the complement system, especially c , may also participate in producing nec, since the injection of paf activates the complement system in vivo [ ] , and c deficient mice are protected from tnf/lps-or paf-induced injury [ , ] . nitric oxide (no) is produced endogenously by three nitric oxide synthase (nos) isoforms: the constitutive neuronal (type i) nnos, the inducible (type ii) inos, and the endothelial (type iii) enos [ ] . more than % of the total nos in the small intestine is nnos [ ] (fig. a,b) . although inos is constitutively present (mainly in the epithelial cells), it accounts for less than % of the total nos activity [ , ] , and enos is barely detectable in the intestine. paf rapidly decreases intestinal nnos protein, mrna, and enzyme activity [ ] (fig. c) , but has little effect on inos [ ] . interestingly, the degree of injury is inversely re-lated to the nnos activity, suggesting a protective role of nnos. the protective role of no is supported by the following observation: ( ) nos inhibitor l-name aggravates paf-induced necrosis [ ] ; ( ) inos inhibitors are protective only when there is "sufficient" nnos activity [ ] ; and ( ) no donors significantly reduce paf-induced bowel injury [ ] . no may help to maintain the integrity of the mucosal barrier and the microvasculature, to increase blood flow, and to inhibit leukocyte adhesion [ ] . several conditions that involve decreased oxygen delivery to the mesenteric circulation are associated with an increased risk of nec in human infants. these include asphyxia [ ] , cyanotic congenital heart disease [ ] , decreased mesenteric blood flow as reported in intrauterine growth re- tardation [ ] , and maternal cocaine use [ ] . in animal models of nec, hypoxia is associated with the development of ischemic bowel necrosis [ ] but does not define the mechanism of bowel injury. we first explored the role of hypoxia in ischemic bowel necrosis using young ( - -day-old) adult male sprague-dawley rats [ ] . the animals were exposed to acute severe hypoxia by placing them in % nitrogen for min, or to subacute moderate hypoxia by placing them in a % oxygen atmosphere for or min. plasma levels of paf were markedly elevated in the animals treated with min of moderate hypoxia when compared with controls, and were also elevated in animals treated with only min of severe hypoxia [ ] . thirty minutes of moderate hypoxia produced mild to moderate ischemic bowel necrosis, with no evidence of necrosis in any other organs. (two minutes of severe hypoxia were not sufficient to induce bowel injury.) the bowel injury was prevented by two structurally unrelated paf antagonists, web and sri - . we concluded that hypoxia results in a rapid increase in endogenous paf levels and that paf is a mediator of hypoxic intestinal injury. in addition to decreased mesenteric oxygen delivery, bacterial colonization of the gastrointestinal tract is generally held to be an important prerequisite for the development of nec [ ] . the importance of bacteria can be inferred from the observations that full-blown ischemic bowel necrosis cannot be reproduced in a sterile animal model [ ] , and, although bowel infarction occurs in the fetuses, typical nec has never been reported as present at birth or in a stillborn infant [ ] . because hypoxia alone produced relatively mild bowel injury in our model, we hypothesized that hypoxia and bacterial endotoxin (lps) might act synergistically to produce more severe bowel injury. we treated young adult male sprague-dawley rats with either hypoxia alone ( % oxygen for min), lps alone ( mg/kg salmonella typhosa endotoxin, i.v.), or lps ϩ hypoxia (lps given at min followed min later by hypoxia for min) [ ] . both lps alone and hypoxia alone caused little or no intestinal injury, whereas combined treatment with lps and hypoxia resulted in significantly worse gross and microscopic intestinal in-jury. this injury was ameliorated by treatment with the paf antagonists, either web or sri - . animals treated with lps ϩ hypoxia tended to have higher plasma paf levels than animals in the other groups, but the difference did not reach statistical significance in this study. both lps and lps ϩ hypoxia caused a significant increase in plasma tnf levels. we concluded that lps and hypoxia act synergistically to produce bowel necrosis and that paf is an important mediator in this process [ ] . we explored the role of endogenous no in the pathogenesis of hypoxia-induced intestinal injury [ , ] . inhibition of endogenous no production with l-arginine analogs significantly worsened the bowel injury produced by min of % oxygen exposure, suggesting that endogenous no production constitutes an important defense mechanism against hypoxia-induced intestinal injury. paf levels were significantly elevated in the intestines of animals treated with hypoxia and a no synthase inhibitor, and the intestinal injury seen in these animals was prevented with the paf antagonist web . in the vascular endothelium, no synthesized from l-arginine by the constitutive form of nitric oxide synthase (cnos) limits neutrophil adhesion, promotes microvascular integrity, and maintains basal vasodilator tone [ ] . in a related study, inhibition of endogenous no production markedly worsened the bowel injury and intestinal neutrophil accumulation caused by paf [ ] . a major challenge in understanding nec in human infants is the absence of a perfect experimental animal model. although several animal models have been used, most lack some or all of the cardinal features of the human condition. the adult rat model characterizes paf and other mediators in acute ischemic bowel necrosis, but it lacks the critical predisposing feature of prematurity. the role of paf in human nec remains speculative. in published experiments on neonatal animals, the model of barlow et al. [ ] , first described in , most closely resembles human nec [ ] [ ] [ ] . in this model, newborn rat pups were removed from their mothers, exposed to maternal milk, stressed briefly with asphyxia, colonized with gram-negative enteric bacteria, and fed with artificial formula. by the rd day of life, most animals developed abdominal distention and discoloration, bloody stools, respiratory distress, cyanosis, hemorrhagic intestinal necrosis, and microscopic evidence of severe necrosis identical to the pathology observed in neonatal nec. maternal milk, milk leukocytes, immunoglobulin, and oral antibiotics were identified as important for the prevention of nec [ , ] . we set out to reproduce the findings of barlow et al. and to better characterize the pathologic findings [ ] . neonatal rats delivered via abdominal incision were maintained in a neonatal incubator and received the following stresses: ( ) artificial formula feedings ( . ml every h via orogastric tube, cal/kg/d, advanced as tolerated); ( ) asphyxia ( % n for s twice daily); and ( ) e. coli inoculation ( ϫ organisms/day via orogastric tube). our data (table ) confirm that asphyxia and formula feeding together are necessary to produce nec in this model. enteral bacterial inoculation was not a critical factor in our model, since more than half of the animals treated with asphyxia and formula alone developed disease compared to those treated with asphyxia, formula, and bacteria (p ϭ ns, not significant). pathologic findings were similar to human nec. grossly, the intestine was hemorrhagic, with friable, occasionally segmental lesions, but often involving most of the intestinal length. in most ani-mals, necrosis extended from villus to the submucosa (fig. a,b) and often transmurally (fig. c,d) . to evaluate the role of paf, animals stressed with asphyxia, formula feeding, and bacterial inoculation were compared with those pretreated with the paf receptor antagonists web and web [ ] . web in appropriate enteral dosing ( mg/kg q am/ mg/kg q pm) significantly reduced the incidence of nec and death compared with controls (table ) . a four-fold higher web dosing regimen did not alter the incidence of nec, presumably because of an agonist effect on the paf receptor at very high doses [ ] . in contrast, web did not reduce the incidence of nec in stressed animals, presumably because web has a much shorter half-life than web . intestinal paf concentrations were elevated ( Ϯ pg/g) in animals stressed with asphyxia, formula feeding, and bacterial inoculation compared with age-matched, healthy, maternally fed controls ( Ϯ pg/g, p Ͻ . ). to further clarify the role of endogenous paf in nec, neonatal rats were treated with the paf degrading enzyme, paf-acetylhydrolase (paf-ah), as enteral supplementation in doses approximately -fold higher than found in human breast milk. this intervention markedly reduced the incidence of nec from / in controls to / (p Ͻ . ) [ ] . in addition, paf-ah (human, recombinant protein) was identified by immunohistochemistry throughout the intestinal tract and remained functionally active for greater than h after dosing [ ] . interestingly, there was no measurable human paf-ah in the circulation of animals using a sensitive monoclonal antibody/enzyme linked immunosorbent assay (elisa) technique [ ] . taken together, the data support the hypothesis that endogenous paf acts as a critical mediator in this neonatal rat model of nec. experimental studies on phospholipase further support the role of paf in the neonatal rat model. phospholipase a (pla ) consists of a diverse family of enzymes with potent biological activity [ ] . group iia pla , a secretory form of pla , appears to be important in the inflammatory cascade and may regulate paf production [ ] . the regulation of group iia pla mrna in intestine from animals stressed with asphyxia, formula feeding, and bacterial inoculation was compared with control, maternally fed animals. northern blot analysis using a cdna probe for group ii pla showed an almost . -fold increase in mrna in the stressed animals compared with controls (n ϭ in each group; caplan et al., unpublished observations), further supporting the role of paf activation in the development of nec. additional studies were performed to understand the role of bacterial flora and long chain polyunsaturated fatty acids (pufa) on the pathophysiol-ogy of nec. since healthy breast-milk fed neonates are colonized with multiple flora including a predominance of bifidobacteria and lactobacilli, neonatal animals were treated with bifidobacteria infantis organisms/day and evaluated for the development of nec, endotoxin translocation, mucosal permeability, and pla -ii mrna expression. bifidobacteria infantis supplementation reduced the incidence of nec ( / vs. / control, p Ͻ . ) but did not alter the colonization pattern of gram negative organisms [ ] . bifidobacteria infantis were identified in the stool and intestinal lumen of treated animals but absent in controls. in addition, bifidobacteria infantis treatment markedly reduced pla -ii gene expression in intestinal tissue ( Ϯ mol/g tissue vs. Ϯ control, p Ͻ . ) but had no effect on mucosal permeability [ ] . the results suggest that bifidobacteria infantis reduces the incidence of nec by altering paf metabolism and bacterial translocation. the role of polyunsaturated fatty acids on the inflammatory cascade, especially the omega- fish oil preparations, have been well recognized. since these compounds seem to reduce the incidence of nec in a human trial, we evaluated them in the neonatal rat model. animals were treated with arachidonic acid ( mg/ ml) and docosohexanoic acid ( mg/ ml) and studied for the development of nec, pla gene expression, apoptosis, and endotoxin translocation. pufa supplementation did not alter the semiquantitative assessment of intestinal epithelial apoptosis, but it reduced the incidence of nec and death compared to controls, and decreased endotoxinemia at and h. furthermore, pufa decreased pla mrna synthesis but had no effect on inos gene expression in intestinal homogenate [ ] . experimental evidence strongly supports the role of paf, lps, and tnf in acute ischemic bowel necrosis and in the neonatal rat model of nec. some data from human studies suggest a similar pathophysiology in neonatal nec. local and systemic paf concentrations are elevated in neonates with nec, and feeding alone promotes paf production. we and other investigators found higher circulating plasma levels of paf and/or paf-like phospholipid in nec patients compared with agematched, illness-matched controls [ , ] . these nec patients also had higher circulating tnf-␣ levels [ ] and lower plasma acetylhydrolase activity (paf-degrading enzyme) than control babies. enteral feeding itself caused elevations of circulating paf levels in a significant percentage of preterm infants [ ] , although the circulating acetylhydrolase activity was not affected by the feeding regimen. circulating paf may not adequately reflect the activity in the local environment (intestinal lumen/mucosa), but stool paf concentrations also increased with feedings [ ] . fourteen days after feedings were begun, the paf levels were approximately threefold higher than prefeed-ing values ( Ϯ pg/g vs. Ϯ pg/g, p Ͻ . ) [ ] . stool samples from seven patients with nec (stage ii or iii) had the highest levels, with a mean paf concentration eightfold higher than controls ( Ϯ pg/g) [ ] . the apparent increased paf production in experimental and human nec fails to explain why nec exclusively afflicts newborn infants. several factors may predispose newborns and especially premature infants to nec, e.g., immature gastrointestinal host defense, dysfunctional mesenteric blood flow autoregulation, and low paf-degrading enzyme acetylhydrolase (paf-ah) [ , ] . although plasma paf-ah activity is lower in nec patients than in controls [ ] , paf-ah activity is low in newborns as a group, reaching normal adult values at wk of life [ ] . infants fed with breast milk (containing significant paf-ah activity) have a much lower risk of nec than infants fed with formula (without measurable paf-ah activity) [ ] . in animal experiments, upregulation of paf-ah can prevent ischemic bowel necrosis following exogenous paf infusion [ ] . these data strongly support the role of paf in neonatal nec and suggest that low neonatal paf-ah activity may in part explain the neonates' predilection of nec. we hypothesize that the initial insult in the chain of events leading to nec could be perinatal hypoxia or a mild postnatal infection, either of which results in mild mucosal damage (fig. ). following formula feeding and the proliferation of the intestinal flora, bacteria may attach to the damaged intestinal epithelium because of immaturity of the "mucosal barrier," thus eliciting endogenous production of paf (and paf-like phospholipids) and tnf. the major source of paf may be epithelial cells, lamina propria cells, or endothelial cells. although gut bacteria may themselves form paf, the normal mucosal barrier probably prevents any deleterious action on the epithelium. however, in immature or mildly damaged mucosa, the close proximity of bacteria and intestinal epithelial cells may facilitate transcellular permeation of paf into the mucosa. if the acetylhydrolase is low (as in the case of premature infants), paf, which increases the intestinal epithelial permeability in vivo, may ac-cumulate locally, leading to focal mucosal "leak" and local entry of bacteria or bacterial products. paf may then synergize with lps and/or tnf, reaching the threshold necessary to trigger a cascade of inflammatory events: pmn activation and adhesion to venular endothelium, increase in vascular permeability, complement activation, nf-b translocation, induction of proinflammatory cytokines and adhesion molecules, and release of ros and inflammatory mediators (including ltc , prostaglandins, and paf). eventually, vasoconstriction occurs leading to ischemia and subsequent reperfusion. activation of xanthine oxidase with massive reactive oxygen species production occurs as a consequence of ischemia and/or protease activation. the final result depends on the balance between the injurious mechanisms (inflammatory mediators, cytokines, ischemia) and the protective mechanisms (mainly nnos). an imbalance favoring the former will result in serious breakdown of the mucosal barrier and bacterial entry, thereby launching a self-perpetuating vicious cycle, leading to shock, sepsis and, sometimes, death. this work was supported by nih grants dk , hd , and hd . we thank dr. h. heuer, boehringer ingelheim, mainz, germany, for his generous gift of web and web . paf-ah, paf acetylhydrolase; no, nitric oxide; c', complement; nf-b, nuclear factor-b; ltc , leukotriene c ; ros, reactive oxygen species; nec, necrotizing enterocolitis; pmn, polymorphonuclear leukocyte. 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and protein–protein interactions in the pulmonary surfactant system and their role in lung homeostasis date: - - journal: int j mol sci doi: . /ijms sha: doc_id: cord_uid: em tiz s pulmonary surfactant is a lipid/protein complex synthesized by the alveolar epithelium and secreted into the airspaces, where it coats and protects the large respiratory air–liquid interface. surfactant, assembled as a complex network of membranous structures, integrates elements in charge of reducing surface tension to a minimum along the breathing cycle, thus maintaining a large surface open to gas exchange and also protecting the lung and the body from the entrance of a myriad of potentially pathogenic entities. different molecules in the surfactant establish a multivalent crosstalk with the epithelium, the immune system and the lung microbiota, constituting a crucial platform to sustain homeostasis, under health and disease. this review summarizes some of the most important molecules and interactions within lung surfactant and how multiple lipid–protein and protein–protein interactions contribute to the proper maintenance of an operative respiratory surface. in the lung, the essential function of respiration takes place through gas exchange between air and blood. to allow this, a perfect architecture has been developed in which alveoli constitute the functional units of the pulmonary system. there, a thin exchange surface structure is needed to ensure an effective diffusion of oxygen to the capillaries, which is achieved by the existence of flattened alveolar epithelial type i cells (ae c) that constitute % of the total alveolar area [ ] . on the other hand, the presence of an air-water interface physically requires a reduction of the surface tension generated by the cohesive forces between liquid molecules that, otherwise, would lead to shrinkage of alveoli [ ] . to fulfill this crucial role, alveolar epithelial type ii cells (ae c) secrete pulmonary surfactant, a lipid-protein complex that forms a surface active film at the respiratory interface, allowing its stability and avoiding alveolar collapse during expiration [ , ] . moreover, beyond this main function, the huge respiratory area exposed to external potentially harmful particles and microorganisms, entails an important role of the lung in protection against pathogens, for which alveolar macrophages (am) are critical, together with the first line of defense constituted by surfactant [ ] . pulmonary surfactant is mainly composed of lipids (approximately % by mass), with phospholipids constituting the principal class, with phosphatidylcholine (pc) as the predominant species ( - %) . among these, the saturated dipalmitoylphosphatidylcholine (dppc) accounts for % in average (although it can be as much as - % of surfactant mass in humans, pigs, rats or mice), whereas unsaturated [ , ] . upon secretion to the alveolar fluid, surfactant is efficiently adsorbed into the air-liquid interface, in a process highly dependent on the presence of proteins sp-b and sp-c [ , ] . multilayered membranous surfactant structures associate to the interfacial film forming a reservoir, which provides mechanical stability and a source of material upon respiratory dynamics [ ] . extracellular surfactant structures include a membrane network promoted by sp-a and sp-b (tubular myelin) [ ] and membrane arrangements of different complexity [ ] . after exposure to air, surfactant "used" membranes are converted into small vesicles [ ] in a sp-d-mediated process [ ] that can be at least partly re-uptaken by ae c, a process promoted by sp-a [ ] . sp-a also acts as a secretion inhibitory signal [ ] , in an opposing role to that of sp-b as secretion inducer [ ] . components captured by ae c are routed to the recycling pathway or degraded [ ] . besides, alveolar macrophages account for a % of surfactant clearance [ ] , for which the presence of granulocyte-macrophage colony stimulating factor (gm-csf) is required [ ] . alveolar homeostasis highly depends on the effective cross-talk between alveolar macrophages (am) and ae c and the maintenance of lipid homeostasis in both cells, involving a proper reverse cholesterol transport (rct) mediated by abca and abcg lipid transporters [ ] [ ] [ ] . sp-a is represented as its most abundant octadecameric form; sp-b as double rings, each formed by six dimers; sp-c as monomers; sp-d as dodecamers. (lb) . proper formation of these highly packed lipid organelles requires the presence of sp-b and abca [ , ] . upon secretion to the alveolar fluid, surfactant is efficiently adsorbed into the air-liquid interface, in a process highly dependent on the presence of proteins sp-b and sp-c [ , ] . multilayered membranous surfactant structures associate to the interfacial film forming a reservoir, which provides mechanical stability and a source of material upon respiratory dynamics [ ] . extracellular surfactant structures include a membrane network promoted by sp-a and sp-b (tubular myelin) [ ] and membrane arrangements of different complexity [ ] . after exposure to air, surfactant "used" membranes are converted into small vesicles [ ] in a sp-d-mediated process [ ] that can be at least partly re-uptaken by ae c, a process promoted by sp-a [ ] . sp-a also acts as a secretion inhibitory signal [ ] , in an opposing role to that of sp-b as secretion inducer [ ] . components captured by ae c are routed to the recycling pathway or degraded [ ] . besides, alveolar macrophages account for a % of surfactant clearance [ ] , for which the presence of granulocyte-macrophage colony stimulating factor (gm-csf) is required [ ] . alveolar homeostasis highly depends on the effective cross-talk between alveolar macrophages (am) and ae c and the maintenance of lipid homeostasis in both cells, involving a proper reverse cholesterol transport (rct) mediated by abca and abcg lipid transporters [ ] [ ] [ ] . sp-a is represented as its most abundant octadecameric form; sp-b as double rings, each formed by six dimers; sp-c as monomers; sp-d as dodecamers. hydrophobic proteins sp-b and sp-c are required for efficient surfactant unpacking upon secretion, and for the formation of the surface film [ ] . sp-b in particular plays a vital function in surfactant biophysics. this protein, which belongs to the saposin family, shows lipid membrane fusion and lysis activities that have been ascribed to the different segments of the sequence [ ] [ ] [ ] . the lipid-protein and protein-protein interactions behind these sp-b properties are responsible for its role in promoting efficient surfactant adsorption and re-extension as well as in maintaining film stability at low surface tensions [ , ] . sp-b is known to be able to generate contacts among membranes [ ] , and we have shown that sp-b is able to oligomerize into ring structures formed by association of dimers, with a hydrophobic cavity inside [ ] . docking of sp-b oligomers would allow the connection of contiguous surfactant structures and the generation of a hydrophobic tunnel through which lipids could rapidly be transferred across the alveolar fluid phase, adsorbing towards the interfacial film [ , ] . the cohesivity among surfactant membranes provided by sp-b protein-protein interactions would also be important for supporting film stability [ ] . besides offering a hydrophobic pocket, the orientation of the amphipatic α-helices of sp-b at the surface of surfactant layers allows electrostatic interaction of positively-charged residues at the protein with the polar heads of surfactant anionic phospholipids, mainly pg. within the literature, there is some evidence suggesting a preferential interaction of sp-b and sp-c with anionic phospholipids [ , [ ] [ ] [ ] . this pg/sp-b interaction could be important for the orientation of the protein in the membrane. as a matter of fact, the effect of pg to facilitate surfactant adsorption and in maintaining the stability of the film, was early related to its interaction with cationic hydrophobic proteins, and in particular, with sp-b [ ] . the possibility that sp-b oligomer could form a proteolipidic pore implies also the involvement of phospholipids, especially pg, as essential determinants of lipid transfer/adsorption properties of surfactant [ , , ] . apart from pg, unsaturated phospholipids play also a role in favoring surfactant adsorption, facilitating a dynamic insertion of the material into the interfacial film [ ] . on the other hand, molecular dynamics simulations have very recently proposed potential selective interactions of sp-b with cholesterol, an interesting possibility whose functional implications have still to be clarified [ ] . protein/protein and lipid/protein interactions involved in the formation, stability and dynamics of the surfactant film are summarized in table . regarding sp-c, beyond its implication in promoting surfactant adsorption [ , ] , it has been ascribed a role in maintaining attachment of the surfactant reservoir to the interfacial monolayer, thus providing stability [ ] [ ] [ ] [ ] . a potential cooperation between sp-b and sp-c in biophysical functions in surfactant could be inferred from several studies, including their synergic action in permeability and lipid transfer properties of surfactant [ , , , ] . as a matter of fact, a surfactant containing both proteins, sp-b and sp-c, exhibits superior functional properties to facilitate breathing, as tested in vivo, than surfactant preparations lacking one of the hydrophobic proteins [ , ] . thus, the existence of direct interactions between both proteins has been considered as an interesting possibility, which finally was recently demonstrated by advanced fluorescence spectroscopy techniques [ ] . a model has been proposed in which sp-c could also regulate the interaction between sp-b oligomers. the specific and opposing roles of both hydrophobic proteins is revealed by the destabilizing effect of sp-c upon lipid vesicles versus the fusogenic activity of sp-b, and might have functional implications along the surfactant cycle [ , ] . concerning the preferential interactions of sp-c with lipids, besides pg the protein has been proposed to specifically interact with cholesterol [ ] . this neutral lipid is important for maintaining a proper fluidity and viscosity in surfactant, thus facilitating the dynamical properties of the membranes [ ] [ ] [ ] . however, cholesterol proportion in surfactant must be tightly controlled, so that its increase has been related to functional inhibition [ ] [ ] [ ] . the presence of sp-c seems to be critical to ensure the proper function of surfactant in the presence of cholesterol [ , , ] , and the combined action of sp-b and sp-c could be important for the removal of cholesterol during film refining at expiration [ ] . despite hydrophobic proteins are the principal players in the biophysical activities of surfactant, the hydrophilic sp-a is also important for an optimal function. this protein, which forms oligomers consisting of six trimers, is able to bind dppc and cholesterol through its carbohydrate recognition domain (crd) [ ] . sp-a induces calcium-dependent vesicle aggregation and enhanced surface adsorption [ , ] , requiring for it a supratrimeric assembly [ ] . the interaction of the protein with cholesterol could be involved in the protecting role of sp-a against the surfactant dysfunction produced as a consequence of supraphysiological concentrations of this neutral lipid [ ] . interestingly, slightly different biophysical activity has been found in the two proteins encoded by the two sp-a-related human genes, sp-a and sp-a , which differ in several features of its structure [ , ] . in particular, sp-a has been shown to be important for the efficient adsorption of surfactant phospholipids and for the reorganization of the surface film during breathing-like compression-expansion cycles. this role could be related to the high level of oligomerization of sp-a , which could assist in the formation of the highly cohesive multilayer film, for which sp-b is essential [ ] . the existence of recently found protein-protein interactions between sp-a and sp-b [ ] would also support the concerted action of both proteins in surfactant processes, and some evidences even suggest the possibility of different modes of interaction of sp-a and sp-a with sp-b [ ] , although precise information is still lacking. thus, protein-protein cooperation could be crucial for developing sp-a physiological roles in surfactant at different levels, including enhancing surfactant adsorption, optimizing film stability and allowing the formation of specific surfactant structures at the alveolar spaces, as tubular myelin [ ] . finally, sp-d, which is not involved in the biophysical activity of surfactant, is not lipid-associated although preferential binding to pi has been proposed, with potential implications for the regulation of surfactant homeostasis, as will be discussed later [ ] . interfacial adsorption [ ] cholesterol cholesterol removal from interfacial film (refining) cholesterol removal from alveolar spaces * crosstalk ae c-am [ , , ] [ ] [ ] sp-d pi regulation of re-uptake to ae c [ , , ] gpr * regulation of secretion and re-uptake * [ ] * suggested potential interactions or roles. as previously described, the surfactant film is essential for reducing surface tension avoiding collapse of alveoli. the presence of surfactant itself is therefore the first indispensable requirement for maintaining lung homeostasis, stabilizing alveoli and preventing edema formation. besides this, surfactant precludes interfacial stress forces that could deform and cause dysfunction of ae c as a consequence of their contact with the air-liquid interface [ , ] . this role of surfactant in alveolar micromechanics entails that its deficiency or dysfunction, either by primary (e.g., absence of sp-b, ae c injury) or secondary (as lung inflammation) causes, triggers lung injury [ , ] . surfactant deficiency or alteration has been associated with the leading causes of acute respiratory distress syndrome (ards), which refers to inflammatory processes frequently associated with lung injury. inflammation itself mediates ae c injury, but also liberation of pro-inflammatory mediators and cell and tissue debris, all contributing to an altered composition of surfactant. a complex group of alterations are found in alveolar spaces at ards: reduced amount of surfactant lipids and proteins, altered profiles of surfactant protein intermediates and byproducts, increased levels of plasma proteins (because of the increased capillary permeability) that produce surfactant inhibition, increased surfactant hydrolysis of surfactant by secretory phospholipase a (pla ) and release of reactive oxygen species [ ] [ ] [ ] [ ] [ ] . absence of sp-c and decreased levels of sp-a and sp-b are associated with familial interstitial lung disease [ ] . decreased levels of sp-a and/or sp-d are also observed in neonates with respiratory distress syndrome (rds) and patients with idiopathic pulmonary fibrosis, bacterial pneumonia, chronic obstructive pulmonary disease, and asthma (reviewed in [ ] [ ] [ ] ). reduced amounts of lung collectins in the lung increase the susceptibility to respiratory infections, promote inflammation and hamper the elimination of apoptotic cells leading to abnormal lung tissue regeneration as described in the following sections. the finding that elderly people show reduced levels of sp-a in the lungs [ , ] suggests that ageing may aggravate the clinical consequences of surfactant deficiency, particularly in chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis [ ] . on the other hand, since lung collectins levels are usually reduced in the lung because of protein leakage towards the vascular compartment, serum levels of these proteins are considered biomarkers of disease severity and predictive factors of unfavorable disease outcome [ ] [ ] [ ] [ ] . genetic deficiency of sp-b is rare, and occurs as a consequence of mutations leading to the absence of sp-b or prosp-b proteins. the disease is characterized by the lack of properly packed lamellar bodies, absence of mature sp-c and appearance of unprocessed prosp-c forms [ , [ ] [ ] [ ] . unless partial deficiency in sp-b production still allows a certain production of sp-b [ , ] , this genetic deficiency gives rise to a lethal rds upon birth, requiring early lung transplantation to survive [ ] . gene therapy would be thus an essential tool to reverse the usually lethal sp-b deficiency, and promising progress has been recently made in the production of human lung organoids [ ] . in the case of adults carrying mutations in heterocygosis, susceptibility to lung diseases in the adulthood has been described, particularly among smokers [ ] . unlike sp-b, genetic sp-c deficiency is not usually a cause of neonatal rds, but interstitial lung disease, including pulmonary fibrosis, can appear at long-term [ ] [ ] [ ] . pathophysiology of the disease caused by sp-c mutations is mainly related to ae c cell damage, produced by endoplasmic reticulum (er) stress due to prosp-c misfolding, or to its altered traffic to lamellar bodies [ ] . on the other hand, different studies have shown that mutations in the genes encoding sp-a and sp-a are also involved in lung diseases. for example, mutations that affect the crd domain of sp-a (w r) or sp-a (g v and f s) are associated with idiopathic interstitial pneumonia [ ] and pulmonary fibrosis [ , ] , respectively. these mutations impair protein secretion and promote protein aggregation within the er, increasing er stress at resident ae c [ ] . in addition, genetic polymorphisms of lung collectins are known to be associated with susceptibility to acute lung injury [ ] , rds [ ] and infections [ , ] . in the case of acute lung injury, a polymorphism that causes the insertion or deletion of three amino acids within the collagen-like domain of sp-a has been related to the reduced water levels in the lung characteristic of this disease, probably due to alterations in the structure and binding properties of the protein [ ] . complex interactions of single nucleotide polymorphisms of the genes that encode all surfactant proteins seem to contribute to the pulmonary disease in cystic fibrosis patients [ ] . similarly, interactions between sp-a and sp-b polymorphisms are involved in genetic susceptibility to rds [ ] . to ensure the presence of a functional surfactant film at the respiratory interface, synthesis, secretion, reuptake and degradation of surfactant components must be perfectly regulated and balanced. this includes achieving both a precise composition of lipids and proteins and their proper organization in defined surfactant structures at the alveolar spaces, together with an efficient cross-talk established between epithelial cells and alveolar macrophages. protein and lipid interactions involved in surfactant homeostasis are also summarized in table . de novo synthesis of surfactant phospholipids takes place at the endoplasmic reticulum in ae c. pc is synthesized from choline by the kennedy pathway, which includes several enzymatic steps, with the cytidine triphosphate-phosphocoline cytidyltransferase (cct) controlling the synthesis rate. a parallel formation of diacylglycerols occurs coupled to this pathway (for a recent review on surfactant metabolism see [ ] ). direct interaction of acyl-coa:lysophosphatidylcholine acyltransferase (lpcat ) with the star (steroidogenic acute regulatory-related lipid transfer protein ) protein has been found to be important for trafficking of newly synthesized dppc from endoplasmic reticulum to lb [ ] , which seems to occur by direct non vesicular transport between both organelles, apparently skipping golgi and multivesicular bodies [ ] . in fetal mouse lung, glycogen storages present in ae c, in which cct is also found, seem to provide substrate for pc biosynthesis [ , ] . besides this, triglycerides have been also shown to be directed from alveolar lipofibroblasts to ae c and contribute to the fetal surfactant phospholipid pool, pointing to a cross-talk between these two cell types to ensure surfactant homeostasis [ , ] . apart from this de novo synthesis, in the lands cycle dppc is also obtained by remodeling from unsaturated pc by pla and lpcat , being the latter also involved in the synthesis of pg. these two enzymes, together with cct, have been shown to be key players for the proper regulation of surfactant synthesis, so that their alteration is cause of respiratory distress [ ] [ ] [ ] . in the case of lpcat , it has been ascribed a role in coordinating the two pathways of dppc synthesis according to the physiological demands of surfactant at the alveolar spaces [ ] . besides this, the finding that peroxiredoxin , a multifunctional enzyme with pla and lpcat activities, is located in lamellar bodies, pointed to these organelles as a sufficient location for dppc remodeling [ ] . regarding to pla , not only this enzyme has a key role in regulating the turnover of surfactant phospholipids because of its implication in their synthesis and degradation, but it is also involved in antioxidant and cell signaling roles [ ] . finally, the interaction of sp-a with peroxiredoxin leads to inhibition of its pla activity, pointing to a potential role of sp-a in regulating surfactant phospholipids turnover [ , ] . with regard to cholesterol, apart from the principal plasmatic source, it can be also synthesized in peroxisomes in ae c, whereas cholesterol-binding proteins have been found in lamellar bodies, suggesting a potential implication of the niemann-pick c pathway in the regulation of surfactant cholesterol and homeostasis [ , ] . finally, alveolar lipofibroblasts could also participate in cholesterol supply to surfactant under certain conditions [ ] . the synthesis and complex processing of proteins sp-b and sp-c initiate in the endoplasmic reticulum of ae c, and occur coupled to surfactant lipid synthesis and intracellular trafficking. both proteins are produced as large soluble precursors where the mature hydrophobic modules are protected by flanking n-and c-terminal domains [ ] . processing of precursors takes place in transit to lbs via multivesicular bodies, and consists of several proteolytic cleavages and post-translational modifications, in a route coupled to sequential acidification. thus, maturation of both proteins is conducted in a concerted way, being sp-b required for proper sp-c processing [ ] by mechanisms that are still unknown, although they could well involve the previously described sp-b/sp-c interactions [ ] , or even potential interactions between its precursors. furthermore, the fusogenic and lytic properties behind membrane remodeling abilities of sp-b, together with its role in generating membrane contacts, turn sp-b into an essential requirement for the formation of correctly assembled lbs [ , ] . these organelles are formed by packed lipid membranes in a well-defined structure that, in the case of humans, is arranged as concentric lamellae [ ] . surfactant lipids, at least pc, pg and cholesterol, are imported and accumulated into lbs by their limiting membrane transporter atp binding cassette subfamily a member (abca ), in an atp-depending process [ , , ] . the coupling between lipid and protein synthesis, processing and assembly in lbs places all these actors as essential providers of surfactant homeostasis, so that their disruption leads to alteration affecting the rest of components of the system and inadequate surfactant production. thus, absence of cct is cause of changes in surfactant protein levels and aberrant lbs [ ] , mutations in abca , or in its interacting protein emc (endoplasmic reticulum membrane protein complex subunit ), which is required for abca stabilization, produce absence of lamellar bodies and misprocessed surfactant proteins [ , ] , and decrease of sp-b levels affects sp-c processing and lb formation, as previously stated. on the other hand, mutations in the sp-c gene, as previously described, are related to long-term lung diseases, including fibrosis, and injury to ae c by endoplasmic reticulum stress due to protein misfolding (mutations affecting sp-c precursor) [ ] . finally, absence of abca has been also related to ae c apoptosis by endoplasmic reticulum stress due to the accumulation of phospholipids and cholesterol, which are then rerouted to the endoplasmic reticulum from lb [ ] . hydrophilic proteins sp-a and sp-d are synthesized in the endoplasmic reticulum, where post-translational modifications and oligomerization also occurs. the main route of secretion of these proteins is via constitutive non-regulated vesicular transport, bypassing lbs, although a certain proportion of synthesized sp-a seems to be directed to these organelles, together with the recycled protein recovered from alveolar spaces [ ] . due to the role of hydrophilic proteins in lung immunomodulation, their absence produces increased inflammation [ ] , although alterations in surfactant homeostasis are also found, related to their involvement in surfactant catabolism [ , ] , as it will be discussed later. lbs are secreted to the alveolar fluid in response mainly to alveolar stretching produced during inspiration, through stimulation by purinergic agonists such as atp. the stimulation of ae c by atp leads to an increase in cytoplasmic calcium, both by entry from the alveolar medium and by release from intracellular stores, mainly endoplasmic reticulum. calcium triggers lb secretion by regulating the direct fusion of lbs with the plasma membrane and by cytoskeleton activation. generation of the fusion pore, involving soluble snare (n-ethylmaleimide-sensitive factor attachment receptor) proteins at both the lb and the cell membrane, triggers a local entry of extracellular calcium (fusion-activated calcium entry) that expands the pore and allows surfactant release, which requires compression of the vesicle by an actin coat [ ] . regulation of surfactant secretion is essential to maintain alveolar homeostasis. extracellular sp-a was described as an inhibitor of secretion by interaction with its receptor tumor protein (p ) at the ae c membrane [ , ] , although little is known about the mechanisms involved in sensing the availability of surfactant at the alveolar spaces and the possible need for secretion of material. stretching sensing has been traditionally ascribed to ae c, which would as a result induce lb secretion through the increase of extracellular atp [ ] . however, an implication of the air-liquid interface itself has been reported, so that ae c could be able to sense the mechanical forces produced by the thinning of the aqueous phase and the increase in surface tension, responding with intracellular calcium increase, thus promoting the secretion of a surplus of new surfactant material. besides this, ae c could be also capable of activating cellular responses involved in cell repair, to prevent lung injury [ , ] . on the other hand, extracellular sp-b has been recently found to stimulate surfactant secretion by the purinergic pathway, so that the protein could ensure the availability of enough density of surfactant and a proper membrane network at the alveolar spaces, and thus, surfactant homeostasis [ ] . the molecular mechanism of this regulation needs still to be investigated, as well as the potential existence of a "sp-b receptor" at the ae c plasma membrane. the possibility that sp-b/sp-a interactions [ ] , as a secretion activator/inhibitor couple, might be involved in a concerted regulation of surfactant secretion will require further investigation. finally, a potential role as sensor of the surfactant pool size or its composition has been postulated for g-protein coupled receptor (gpr ), a transmembrane receptor of ae c that has been identified as a negative regulator of surfactant secretion. the sensing mechanism of this protein could rely on its interaction with some surfactant component(s) which, up to now, has not been yet identified [ , ] . however, suggested possibilities for gpr sensing include interaction with sp-d or its direct modulation by mechanical stretching upon inspiration [ ] . once surfactant is secreted into the alveolar fluid, fast adsorption of material takes place and new components are transferred into the air-liquid film. the specific mechanisms converting the secreted highly packed membrane structures into part of the interfacial film are not still completely understood. sp-b/sp-c common machineries have been shown to be essential for unpacking of secreted surfactant lb-like particles and formation of the surface film, pointing to the possibility that, upon contact with the air-water interface, sp-b nanorings, regulated by sp-c, would trigger the fast transfer of lipids into the film [ ] . apart from these freshly secreted structures, another surfactant assembly that could act as an intermediate between lb and the interfacial film, is tubular myelin (figure ). tubular myelin is a complex three-dimensional membranous tubular structure, whose formation requires the presence of sp-a (in humans, both sp-a and sp-a ), sp-b, and anionic lipids, highlighting once again the essential role of the interactions among these surfactant components [ , , ] . although the particular function played by tubular myelin is still unknown, as its presence is not essential for respiration [ ] , potential roles could be related with the involvement of sp-a in antimicrobial defense. the study of the extracellular surfactant recovered from alveolar spaces led to the finding that two different pools of surfactant are present, in terms of size, composition and structural complexity. the more dense surfactant material known as large aggregates (la), which contains secreted lbs, tubular myelin and sp-a, sp-b and sp-c, is thought to be somehow metabolically converted into another form rich in small vesicles (small aggregates or sa) with decreased content in proteins and a lower adsorption activity [ , ] . of note, an increased sa/la ratio is found in patients suffering ards [ ] . the conversion of la to sa can be reproduced in vitro by compression-expansion cycling of the interfacial film, so that it is generally assumed that the smaller structures are generated by the "used" surfactant components once surfactant has been exposed to a dynamic air-water interface ( figure ). the physiological role of these structural changes of surfactant seems to be encompassed in an important mechanism of surfactant homeostasis regulation, as it will be discussed below. the proper regulation of surfactant pool size includes the clearance of the used surfactant from the alveolar spaces, thus avoiding the excessive accumulation of a spent, substantially altered (including extensive oxidation) material that can lead to lung injury and inflammation. surfactant is removed from the alveolar fluid by re-uptake to ae c for recycling or degradation, and to alveolar macrophages for its catabolism. surfactant pool size itself has been shown to induce the increase of catabolic rate, thus maintaining alveolar homeostasis, although the molecular pathways involved in such response are not clear [ ] . apart from inhibiting surfactant secretion, sp-a contributes to alveolar homeostasis by stimulating the phospholipid uptake by ae c via clathrin-mediated endocytosis upon interaction with its receptor p [ ] . a similar case is gpr , which acts both as secretion inhibitor and as stimulator of pneumocyte re-uptake, for which potential interactions with sp-d may be involved [ , ] . as a matter of fact, sp-d seems to play an essential role in the regulation of surfactant pool size. this protein is involved in the structural remodeling of surfactant structure into sa, which is the form taken up by ae c to proceed with surfactant recycling or degradation ( figure ). to achieve this role, for which sp-d oligomeric structure is required [ ] , the protein seems to induce fragmentation of surfactant membranes, for which its interaction with pi has been suggested to be a determinant factor [ , , ] . on the other hand, the structure of surfactant aggregates does not influence its uptake by ams [ ] , and although sp-d is able to bind to the surface of these cells to modulate inflammation, the homeostasis/immune roles of the protein seem to be independently regulated [ ] . the regulation of surfactant homeostasis carried out by sp-d is particularly important upon birth. newborn mice show a much larger pool size and an ultrastructure of sa and la pools different to that observed in adults. the postnatal change leading to a normalization of the pools has been suggested to be initiated by the increased amount of pi of newly secreted surfactant (both in newborns and adults) that promotes the association of sp-d to surfactant lipids, triggering the structural conversion to small vesicles [ , ] . all these findings highlight how the particular composition and structure of surfactant at the different levels of its functional cycle regulate alveolar homeostasis in detail, and how the interactions between surfactant proteins and lipids are particularly essential for this task. maintaining lipid homeostasis inside the cell is crucial both for ae c and am, mainly through the atp-binding cassette transporters abca and abcg . these proteins are involved in the reverse cholesterol transport by mediating efflux of excess cholesterol and phospholipids towards the plasma (figure ). in ae c, alteration of these proteins produces lipid accumulation and affects lb morphology, secretion and recycling, suggesting an important role in surfactant homeostasis [ , ] . alveolar macrophages are essential for surfactant lipids catabolism, accounting for around % of its clearance [ ] . alterations affecting lipid degradation by am entails accumulation of lipids in these cells (the so-called foamy macrophages) that causes inflammation, and reduced clearance of surfactant, leading to accumulation of impaired surfactant at the alveolar spaces. factors involved in these pathways, such as cluster of differentiation cd , are thus regulators of lung homeostasis and inflammation [ ] . the main regulator of lipid catabolism in am has been shown to be granulocyte-macrophage colony stimulating factor (gm-csf) (figure ). this factor does not affect surfactant uptake by am directly but promotes the maturation of these cells and activates proteins involved in phospholipid and cholesterol clearance, as abca and abcg [ , , ] . on the other hand, the administration of gm-csf to mice with impaired am development has been recently shown to produce a decrease in the amount of sp-d, pointing to a cross-regulation of ae c re-uptake and am catabolic pathways [ ] . surfactant would then mediate somehow the communication between both cells, as it seems to occur also in relation to cholesterol metabolism, so that the increase of cholesterol in surfactant has been described to trigger alteration of cholesterol metabolism in am [ ] . on the other hand, an involvement of sp-b has been suggested in facilitating cholesterol efflux from am to surfactant, which would act as acceptor with the aim of reducing inflammation-induced cholesterol accumulation in these cells [ ] . a potential role of sp-b/cholesterol interactions in this activity has still to be demonstrated. regarding sp-c, its reported membrane fragmentation ability, together with its tendency to partition into cholesterol-rich environments have been also suggested to be potentially involved in regulatory mechanisms of surfactant homeostasis [ ] . intrinsic absence of sp-c in am with cholesterol accumulation in sp-c-ko mice leads to an impaired metabolism and the generation of cholesterol crystals in am cytoplasm, a phenotype that has been suggested to be the consequence of the impairment of ae c/am cross-talk, and that could be mediated by the alveolar instability generated by the absence of sp-c [ ] . an altered lung surfactant homeostasis as a consequence of unbalanced anabolism/catabolism leads to the generation of pulmonary alveolar proteinosis (pap). pap is a syndrome characterized by the accumulation of alveolar surfactant and dysfunction of am, thus producing respiratory deficiency and alteration of the lung immune defense. the existence of such numerous proteins involved in the regulation of lung lipid homeostasis entails a broad etiology for pap, including alterations of re-uptake/catabolism by ae c or am (as autoantibodies to gm-csf, absence or alteration in gm-csf receptors colony stimulating factor receptors alpha and beta, abca , abcg , sp-d and gpr ) or of surfactant secretion (gpr ). besides this, mutations in genes required for the proper production of surfactant (abca , sp-b or sp-c) also leads to congenital pap [ ] . regarding to the nature of the accumulated lipids, the analysis of the lipidome of pap samples from different etiologies revealed similar results, including drastic increase of sphingolipids (mainly ceramide), free and sterified cholesterol, pc and lysophosphatidylcholine [ ] . alteration of surfactant homeostasis can also occur upon exposure to external toxicity. electronic cigarettes have been found to alter lipid homeostasis in ae c and am, with effects in abca , abca , lb structure and immune functions [ ] . finally, chronic alcohol consumption also disrupts lipid homeostasis and immune responses in am, increasing susceptibility to pulmonary infections [ ] . in conclusion, the importance of a proper lipid homeostasis in the lung goes beyond the necessity of maintaining sufficient but proportionate lipid levels to ensure surfactant function and alveolar stability. lipids play essential roles in lung physiology, and constitute a link between metabolism and host defense, sensing and translating extracellular information to immune cells. as will be detailed later, anionic phospholipids influence am response to inflammatory stimuli and oxidized lipids and cholesterol mediate and expand inflammatory responses [ ] . of note is the role of pla , involved in surfactant lipid remodeling and degradation, in connecting surfactant turnover and oxidative stress responses in am [ ] . the constant exposure of the lung to microorganisms from inhaled air at the upper respiratory tract activates the immune response, which leads to transcriptional expression of inflammatory mediators that coordinate the elimination of infected cells and pathogens. resolution of inflammation and return to homeostasis is induced by activation and secretion of endogenous anti-inflammatory factors [ ] . however, aberrant activation of the inflammatory response leads to immunodeficiency, septic shock, or induction of autoimmunity [ ] . therefore, the activation and resolution of inflammation must be tightly regulated by immune mechanisms that decrease microbial burden by facilitating clearance of inhaled pathogens, and control tissue damage caused by pathogens or by the host immune response by modulating inflammatory responses [ ] . surfactant proteins and lipids have been shown to provide immune protection against respiratory pathogens, both directly by limiting inflammation and promoting pathogen clearance, and indirectly by activating molecular and cellular mechanisms that contribute to restore lung homeostasis [ , [ ] [ ] [ ] . this part of the review summarizes the role of surfactant proteins and lipids on the immune response against bacterial lipopolysaccharide (lps), the most potent pathogen-associated molecular pattern among bacterial cell wall components. bacterial lipopolysaccharide, the major component of the outer membrane of gram-negative bacteria, strongly stimulates the innate or natural immunity in eukaryotic species [ ] . lps is composed of a poly-or oligo-saccharide part linked to a membrane-anchoring lipophilic domain known as lipid a or endotoxin (figure ). the polysaccharide part is commonly subdivided into the terminal o-specific chain, whose composition varies within bacterial species, and the rather invariable core region, most proximal to lipid a. bacteria express either smooth lps, which contains the o-antigen and complete core oligosaccharides, or rough lps, which lack o-antigen and possess progressively shorter core oligosaccharides. rough lps is designated as ra, rb, rc, rd, and re in order of decreasing core length [ ] . strains with rough type lps are more common among pathogens colonizing pulmonary compartments [ , , ] . lipid a, which represents the conserved molecular pattern of lps, consists of a β( → )-linked diglucosamine backbone carrying two phosphate groups at positions and ' of the glucosamine residues and six or seven ester-and amide-linked saturated acyl chains of and mostly carbon atoms in length [ ] (figure ). both phosphate groups and the myristoyl and lauroyl acyl chains have been shown to be major determinants for specific recognition and activation of innate immunity. lps recognition by cell receptors requires a specific three-dimensional non-lamellar supramolecular structure of lps aggregates [ , [ ] [ ] [ ] (figure ) . host responses to lps are mainly mediated by the pattern recognition receptor toll-like receptor (tlr ) [ ] . the activation of tlr by lps requires the interaction of lps with several receptor-proximal proteins (figure ). the lipopolysaccharide binding protein (lbp) binds to the aggregated structures of lps and transfers lps monomers to the soluble or membrane-bound forms of cluster of differentiation (cd ) via specific recognition of the lipid a domain. cd subsequently transfers lps to the myeloid differentiation factor (md ), which forms a complex with tlr . lps binding to md induces a conformational change in tlr that promotes tlr /md dimerization and the subsequent cd -controlled internalization of tlr into endosomes [ ] , initiating the signalling cascade [ ] . using different lps analogs, triantafilou and co-workers [ ] showed that the recruitment of these receptors is triggered by the -phosphoryl lipid a moiety of lps. and an inner core region proximal to lipid a that contains -deoxy-d-manno-octulosonic acid [ ] . the o-antigen, also termed o-specific chain, contains up to highly variable oligosaccharide units. both the acyl chains and the negative charges of the phosphate groups bound to the diglucosamine backbone of lipid a are required for the recognition of lps by cell receptors and the subsequent activation of the host immune system. lps molecules associate forming non-lamellar supramolecular structures that are recognized by lbp, which transfers lps monomers to cd . lps is subsequently transferred to the tlr /md complex, triggering complex dimerization and initiating the signaling cascade. interaction of surfactant protein sp-a (functional form, octadecamer) with lps modifies the aggregated lps structure, inducing formation of lamellar phases, and therefore preventing the recognition of lps by lbp. [ ] [ ] [ ] , contributing to the clearance and inactivation of lps released by gram-negative bacteria. these proteins interact with different lpss [ ] [ ] [ ] [ ] [ ] [ ] . while sp-d binds to the acyl chains of lipid a [ ] as well as to the heptoses and mannoses of the core [ , ] and o-antigen [ ] , respectively, sp-a only binds to lipid a [ , ] . as a result, sp-a only binds to rough lps since the bulky headgroup of smooth lps hinders the access of sp-a to the lipid a moiety. although the interaction of sp-a and sp-d with lps was firstly described as being mediated by calcium [ ] [ ] [ ] , it has been shown that both collectins bind to re-lps in the absence of this cation [ , , ] . on the other hand, sp-c binds to the lipid a moiety of lps by its n-terminal segment, probably through electrostatic interactions between the polar and basic residues of the protein and the negatively charged terminal -phosphate group at the reducing end of the lipid a disaccharide [ ] . similarly, sp-a binding to the -phosphate of lipid a [ ] may involve electrostatic interactions with a cluster of basic residues located in the crd of the protein [ , ] . since the negative charges of the phosphate groups are required for lps binding to lbp [ ] and the activation of the tlr /md complex [ ] , the interaction of sp-c and sp-a with -phosphate may shield this functional group, blocking some of the physiological responses to lps in the lungs. this hypothesis is supported by the finding that sp-a prevents the interaction of re-lps with lbp [ ] . sp-a also agglomerates rough lps aggregates in the presence of calcium [ ] and rearranges the aggregated structure of lps, figure . host response to lipopolysaccharide (lps). lps is composed of lipid a, the hydrophobic membrane-anchoring region, and a hydrophilic part formed by the core oligosaccharide region and the o-antigen. the core oligosaccharide region includes an outer core region, enriched in hexoses, and an inner core region proximal to lipid a that contains -deoxy-d-manno-octulosonic acid [ ] . the o-antigen, also termed o-specific chain, contains up to highly variable oligosaccharide units. both the acyl chains and the negative charges of the phosphate groups bound to the diglucosamine backbone of lipid a are required for the recognition of lps by cell receptors and the subsequent activation of the host immune system. lps molecules associate forming non-lamellar supramolecular structures that are recognized by lbp, which transfers lps monomers to cd . lps is subsequently transferred to the tlr /md complex, triggering complex dimerization and initiating the signaling cascade. interaction of surfactant protein sp-a (functional form, octadecamer) with lps modifies the aggregated lps structure, inducing formation of lamellar phases, and therefore preventing the recognition of lps by lbp. [ ] [ ] [ ] , contributing to the clearance and inactivation of lps released by gram-negative bacteria. these proteins interact with different lpss [ ] [ ] [ ] [ ] [ ] [ ] . while sp-d binds to the acyl chains of lipid a [ ] as well as to the heptoses and mannoses of the core [ , ] and o-antigen [ ] , respectively, sp-a only binds to lipid a [ , ] . as a result, sp-a only binds to rough lps since the bulky headgroup of smooth lps hinders the access of sp-a to the lipid a moiety. although the interaction of sp-a and sp-d with lps was firstly described as being mediated by calcium [ ] [ ] [ ] , it has been shown that both collectins bind to re-lps in the absence of this cation [ , , ] . on the other hand, sp-c binds to the lipid a moiety of lps by its n-terminal segment, probably through electrostatic interactions between the polar and basic residues of the protein and the negatively charged terminal -phosphate group at the reducing end of the lipid a disaccharide [ ] . similarly, sp-a binding to the -phosphate of lipid a [ ] may involve electrostatic interactions with a cluster of basic residues located in the crd of the protein [ , ] . since the negative charges of the phosphate groups are required for lps binding to lbp [ ] and the activation of the tlr /md complex [ ] , the interaction of sp-c and sp-a with -phosphate may shield this functional group, blocking some of the physiological responses to lps in the lungs. this hypothesis is supported by the finding that sp-a prevents the interaction of re-lps with lbp [ ] . sp-a also agglomerates rough lps aggregates in the presence of calcium [ ] and rearranges the aggregated structure of lps, which changes from inverted to lamellar phases [ ] (figure ). both the clustering of lps aggregates and their lamellar structure would reduce lps toxicity by preventing the recognition of lps by lbp, and the subsequent initiation of the lbp/cd pathway towards inflammatory reactions. surfactant lipids can facilitate the scavenger action of surfactant proteins. in this regard, it has been shown that lps incorporates in vesicles of a modified porcine pulmonary surfactant [ ] and films of surfactant lipids [ , ] . repulsive interactions between smooth lps and surfactant lipids facilitate the interaction of sp-a with the lipid a moiety of smooth lps, which results in the extraction of lps molecules [ ] . additionally, incorporation of lps into surfactant membranes would decrease the endotoxicity of lps by abrogating the interaction of lipid a with its cellular receptors. this hypothesis is supported by earlier studies reporting that the biological activity of lps is reduced when lps is incorporated into liposomes [ ] . alternatively, surfactant lipids may incorporate into lps aggregates, changing the aggregated structure of lps, and hence, reducing its ability to stimulate immune cells [ ] . in addition to the direct interaction with lps, surfactant proteins also modulate the cellular inflammatory response via interaction with pattern-recognition receptors and associated molecules. for instance, sp-a modulates the cellular response to smooth lps, although this lps is not a ligand for sp-a, by binding to cd [ ] and the tlr /md complex [ ] . thus, the interaction between sp-a and lps receptors significantly decreases the binding of smooth lps and consequently prevents the smooth lps-elicited cellular response [ , ] . in contrast, sp-a significantly enhances the binding of rough lps to cd [ ] and does not attenuate rough lps binding to tlr /md [ ] . this effect has been related to the formation of a sp-a/cd /rough lps complex in which the amphipathic neck domain of sp-a would bind to the leucine-rich region of cd , whereas the crd would bind to the lipid a moiety of rough lps, allowing the transfer of lps molecules to the tlr /md complex and initiating physiological responses against pathogens [ ] . formation of a ternary complex with cd and lps has also been proposed for sp-c, which, like sp-a, binds to cd increasing the binding of rough lps [ ] . sp-c-containing phospholipid vesicles, but not lipid vesicles alone, block lps-induced cytokine production by a tlr -dependent mechanism in human embryonic kidney hek t cells [ ] . whether this effect is due to the direct interaction of sp-c with tlr and/or md needs to be clarified. on the other hand, sp-d binds to cd and tlr /md through its crd [ , , ] , decreasing the binding of both smooth and rough lps to cd [ ] and tlr /md [ ] . this suggests that sp-d may act as a negative regulator to protect against a chronic inflammatory state induced by the release of numerous inflammatory mediators as a consequence of the interaction of sp-a and sp-c with rough lps and its receptors. on the other hand, anionic surfactant lipids pg and pi inhibit lps-induced cell activation by competitive binding to lps receptors [ , ] . while pi interferes with the interactions between lps and cd [ ] [ ] [ ] , pg inhibits lps binding to lbp, cd and md [ , , , ] . alternatively, surfactant lipids can prevent activation of tlr by lps by incorporating it into the plasma membrane. thus, dppc has been shown to inhibit the translocation of tlr to raft domains [ ] , precluding the co-localization of cd and tlr in lipid rafts that is required for lps-induced activation of tlr [ ] . voelker and co-workers [ ] have suggested that inhibition of toll-like receptor function by surfactant lipids would set a threshold for the engagement of inflammatory cascades in the lung, preventing inflammation by casual environmental stimuli like environmental levels of particulated lps while allowing a robust inflammatory response during established bacterial infections. surfactant proteins and lipids also contribute to lung homeostasis by exerting antimicrobial effects (figure ). animal models of sp-c and collectin deficiencies show a significant defect in host defence since: (i) sp-c, sp-a, and sp-d knock-out mice are more susceptible to bacterial, fungal, and viral infections [ , [ ] [ ] [ ] [ ] , (ii) sp-a −/− and sp-d −/− mice show reduced uptake of these microbes by alveolar macrophages (reviewed in [ ] ), and (iii) bacterial clearance is restored by intratracheal administration of sp-a [ ] . lung collectins sp-a and sp-d prevent microbial dissemination by direct binding to a broad range of microorganisms, including viruses, fungi and gram-positive and gram-negative bacteria (reviewed in [ , , ] ). these proteins recognize different pathogen-associated molecular patterns on the surface of microorganisms: lps in gram-negative bacteria, lipoteichoic acid and peptidoglycan in gram-positive bacteria, lipoarabinomanan in mycobacteria, phospholipids in mycoplasma, glucan and mannose in fungi, and glycoproteins in fungi and viruses [ , [ ] [ ] [ ] [ ] [ ] . the association of sp-a and -d with pathogens may result in microbial aggregation, which facilitates mucociliary clearance by the respiratory tract, prevents attachment of pathogens to cell surfaces, inhibits microbial colonization and invasion, and may favor microbial phagocytosis [ ] . sp-a and sp-d also act as opsonins, increasing the association, uptake and killing of the microorganisms [ ] . this requires specific interactions with different receptors on phagocytic cells, such as surfactant protein receptor (sp-r ) [ ] , a specific receptor for sp-a [ ] , the putative opsonin receptor glycoprotein (gp ), a macrophage scavenger receptor family member, to which both sp-a and sp-d bind [ , ] , or the immunoglobulin g [ ] . interaction of the globular heads of lung collectins with pathogen-associated molecular patterns promotes the presentation of the collagenous tails to calreticulin/cluster of differentiation (cd ) on the surface of phagocytic cells, stimulating phagocytosis and pro-inflammatory responses [ ] . lung collectins can also stimulate phagocytosis through upregulation of the expression of cell-surface receptors involved in microbial recognition in phagocytic cells [ ] , such as the mannose receptor [ , ] or the class a scavenger receptor [ ] . the precise receptor(s) involved in the activation of the signal transduction pathways that lead to an increased trafficking of pathogen recognition receptors to the plasma membrane remains elusive. alternatively, lung collectins may promote microbial clearance by regulating the complement system. thus, sp-d enhances the activity of complement component q (c q) by forming additional binding sites for c b and c b [ ] , two complement binding protein complexes involved in opsonization [ ] . on the contrary, association of sp-a with c q may down-regulate complement activity, preventing c q-mediated complement activation and inflammation [ ] . lung collectins are also involved in pathogen clearance by neutrophil extracellular traps (nets), dna-based extracellular traps that capture and kill microbes. simultaneous binding of sp-d to bacteria and nets promotes bacterial trapping by the nets [ ] . on the other hand, since lps efficiently induces the formation of nets [ ] , binding of sp-d to lps has been recently shown to attenuate nets formation [ ] . this suggests that sp-d may regulate the balance between the antimicrobial activity of nets and the exacerbated immune response and tissue injury as well as surfactant inhibition induced by an excessive presence of nets in the lung. sp-a and sp-d directly affect the growth and viability of microorganisms [ ] . lung collectins have been found to inhibit the growth of different bacteria like escherichia coli, klebsiella pneumoniae, enterobacter aerogenes, legionella pneumophila, mycobacterium avium, and mycoplasma pneumoniae [ , ] . this bacteriostatic effect has been related to the ability of sp-a and sp-d to increase the permeability of the microbial cell membrane [ ] . gram-negative bacterial strains decorated with rough lps are permeabilized more effectively by sp-a and sp-d than strains containing smooth lps [ ] . although the mechanism of bacterial permeabilization by lung collectins is unclear, it seems to require a direct interaction between sp-a and lps. in this regard, kuzmenko and co-workers [ ] suggested that sp-a distorts or perturbs membrane structure, creating defects that allow small hydrophilic molecules to enter and translocate through the bilayer. such defects seem to be related to the formation by sp-a of extensive lattice-like structures on the surface of lps-containing layers [ , ] . pulmonary collectins also decrease the viability of histoplasma capsulatum by increasing the permeability of the fungal cell wall in a calcium-dependent manner [ ] . although the precise mechanism involved in membrane permeabilization of this fungi is unknown, mccormack and co-workers [ ] have suggested that membrane disruption could be due to the formation of calcium bridges between the collectins and fungal phospholipids. growth of microorganism is also affected by the agglutinating activity of lung collectins, i.e., sp-d agglutinates candida albicans [ , ] decreasing the availability of substrate to agglutinated yeasts [ ] . sp-a and sp-d directly affect the growth and viability of microorganisms [ ] . lung collectins have been found to inhibit the growth of different bacteria like escherichia coli, klebsiella pneumoniae, enterobacter aerogenes, legionella pneumophila, mycobacterium avium, and mycoplasma pneumoniae [ , ] . this bacteriostatic effect has been related to the ability of sp-a and sp-d to increase the permeability of the microbial cell membrane [ ] . gram-negative bacterial strains decorated with rough lps are permeabilized more effectively by sp-a and sp-d than strains containing smooth lps on the other hand, a surfactant lipid mixture composed of dppc, palmitoyloleoylphosphatidylcholine and palmitic acid has been shown to interfere with the interaction of non typable haemophilus influenzae with pneumocytes by binding to the bacteria and preventing bacterial adhesion and internalization in alveolar epithelial cells [ ] . in addition, these lipids can be endocytosed by pneumocytes by binding to the scavenger receptor class b type i, blocking bacterial uptake [ ] . in vivo studies show that administration of the hydrophobic fraction of native surfactant, containing surfactant lipids and proteins sp-b and sp-c, significantly diminishes bacterial load in bronchoalveolar lavage and lung tissue of mice infected with this pathogen [ ] . although this antimicrobial activity has been ascribed to surfactant lipids, it is plausible that surfactant proteins sp-c and sp-b may play a bactericidal role against other pathogens. in this regard, bovine lung surfactant extract, a mixture of dppc, palmitoyloleoylphosphatidylglycerol (popg) and sp-b/-c, and a synthetic model lung surfactant, but not the lipids alone, all show antimicrobial activity against e. coli and staphylococcus aureus [ ] . sp-b and sp-c have a strong affinity for iron-regulated surface determinant proteins a and c [ ] , cell-wall receptors of s. aureus involved in the process of heme-iron acquisition. binding to these receptors would disturb the bacterial surface, enhancing bacterial killing [ ] . sp-b and sp-c also bind to β-barrel assembly machinery protein a and lipopolysaccharide transport protein d, two β-barrell proteins at the outer membrane of e. coli, lethally disrupting the bacterial outer membrane [ ] . additionally, incubation of e. coli with sp-c incorporated into liposomes of dppc and popg, but not the lipids alone, decreases bacterial growth by altering the membrane [ ] . although the mechanism by which sp-c disturbs the outer membrane is not clear, sp-c binding to lps could be a key feature to disrupt the outer membrane and induce the release of lps, rendering the cells permeable. this would facilitate the access of sp-c to the plasma membrane, where this protein would exert an antimicrobial activity [ ] . the antimicrobial action of sp-b, which has been shown to aggregate and kill clinical isolates of k. pneumoniae, p aeruginosa, s. aureus and group b streptococcus by increasing membrane permeability [ ] , is inhibited by surfactant lipids, mainly popg [ ] . this suggests that endogenous sp-b may not play by itself a significant role in alveolar host defense. however, prosp-b, the sp-b precursor may be proteolitically cleaved into smaller peptide fragments that could retain its antimicrobial activity in the presence of surfactant lipids. in this regard, sp-b n , the n-terminal propeptide of sp-b (residues , which encodes a saposin-like domain, promotes the uptake of bacteria by macrophages and exerts a bactericidal effect at acidic ph, consistent with a lysosomal, antimicrobial function [ ] . binding of surfactant lipids and proteins to viruses results in enhanced phagocytosis and viral neutralization at the initial stages of infection [ , ] . regarding lung collectins, the viral neutralizing activity of sp-d is enhanced compared to sp-a, playing an important role in the innate immune response to different viruses such as vaccinia or influenza a virus. the antiviral activity of lung collectins is related to their ability to bind to different proteins on the viral surface [ ] . in general, interactions of lung collectins with viral proteins are calcium-dependent, involve the crd and are facilitated by the multimerization of the full-length protein. the collagen domain of sp-a may also play a role in the antiviral action of the protein, since a recombinant variant of human sp-a composed of the neck and carbohydrate recognition domains can promote the replication of influenza a virus [ ] . while sp-a prevents the recognition of influenza a virus by host cell receptors through binding of the sialylated asparagine residue of sp-a to hemagglutinin [ , ] , binding of sp-d to mannose-rich oligosaccharides close to the sialic acid-binding sites of hemagglutinin reduces viral uptake into epithelial cells [ , ] , and sterically blocks the access of neuraminidase to surface-bound substrates [ , ] thereby preventing viral dissemination. this inhibitory effect has been related to the bulky collagen domain in the extended structure of dodecameric sp-d [ ] . sp-a and sp-d also bind to the human immunodeficiency virus envelope glycoprotein [ ] [ ] [ ] and to the fusion and attachment glycoproteins of respiratory syncytial virus, f and g, respectively [ ] [ ] [ ] [ ] , neutralizing viral infectivity and inhibiting viral replication [ ] . sp-a can mediate phagocytosis of herpes simplex virus type by alveolar macrophages via its n-linked oligosaccharides [ ] . to which viral surface protein(s) sp-a binds is not known. however, since sp-a binding to herpes simplex virus is inhibited by heparin, and heparin is known to bind to viral glycoproteins b and c, these proteins are likely candidates [ ] . other viral surface proteins recognized by lung collectins are the spike glycoprotein of the severe acute respiratory syndrome (sars) coronavirus [ ] , the outer capsid glycoprotein of non-enveloped rotavirus [ ] , and the a protein of vaccinia virus [ ] . the potential interaction of surfactant proteins with proteins at the surface of the sars-cov- virus responsible of the emergent covid pandemic is currently under intensive scrutiny. interactions of lung collectins with viral proteins can also result in deleterious effects during viral infections. in this regard, binding of sp-d to the glycoprotein of ebola virus enhances infection in mammalian cells, facilitating the attachment of sp-d-bound virus to host cell co-receptors and probably affecting the inflammatory response [ ] . on the other hand, the finding that constitutive expression of misfolded sp-c increases susceptibility to viral-induced cell death [ ] suggests an antiviral activity for sp-c. this hypothesis is supported by the increased susceptibility of sp-c deficient mice to respiratory virus infection [ ] , which is reduced upon transgenic restoration of sp-c [ ] . surfactant lipids also play antiviral roles. different molecular species of pg and pi have been shown to bind to respiratory syncytial virus and influenza a virus, inhibiting their interaction with the epithelial cell surface [ , [ ] [ ] [ ] [ ] . neither the structural basis for lipid antagonism nor the inhibition mechanisms are known. it has been proposed that popg and pi could act as decoy ligands for toll-like receptors, given the structural similarities between the lipid a moiety of lps and the anionic surfactant lipids [ ] . likewise, the structural similarities between anionic surfactant lipids and sialic acid containing lipid receptors for the hemagglutinin of h n influenza a virus suggest that these lipids could also act as decoy receptors for viral attachment proteins [ ] . the antimicrobial function of surfactant components may be modulated by interaction with other antimicrobial components of the alveolar fluid. cooperative interactions of sp-a and sp-d with antimicrobial peptides have been suggested. for example, binding of sp-a to sp-b n results in synergistic activity against sp-a-and sp-b n -resistant capsulated k. pneumoniae both in vitro and in vivo [ ] . on the other hand, binding of sp-d to human neutrophil defensins results in antagonistic or cooperative effects on the hemagglutinin inhibitory and neutralizing activity of sp-d [ ] . these effects depend on the viral strain and the multimeric forms of both proteins, with higher weight multimers precipitating sp-d in bronchoalveolar lavage fluid and thereby, reducing its hemagglutinin inhibitory activity [ ] . on the contrary, combinations of sp-d with human β-defensins and show additive activity against influenza a virus [ ] . sp-a, sp-d and the scavenger receptor-rich gp act cooperatively against influenza a virus, inducing viral aggregation and inhibiting hemagglutination [ ] . although both collectins bind to gp [ , ] , this cooperative effect does not appear to require a direct interaction with gp , at least for sp-d [ ] . whether this is also true for sp-a, needs to be clarified. additionally, lung collectins can modulate the activity and toxicity of some antimicrobial peptides. by binding to β-defensin , sp-a negatively regulates the cytotoxicity of this peptide on epithelial cells without affecting its antimicrobial activity, protecting the lung epithelium from injury caused by an excess amount of β-defensin liberated during inflammation [ ] . surfactant proteins also contribute to the homeostasis of the alveolar epithelium by modulating apoptosis, promoting clearance of apoptotic cells, and enhancing tissue repair [ ] . alveolar epithelial apoptosis is an important contributor to the pathophysiology of lung diseases since severe alveolar epithelial apoptosis increases alveolar capillary permeability, and causes leakage of serum into alveoli and loss of compartmentalization with unrestricted mechanical transduction and signaling from the airways [ ] . excessive apoptosis may therefore affect the outcome of lung diseases, including adult respiratory distress syndrome [ ] , chronic obstructive pulmonary disease [ ] , and pulmonary fibrosis [ ] . therefore, a well-balanced interplay of cell apoptosis and proliferation is required to maintain the integrity and function of the lung. lung collectins have been shown to play a key role regulating apoptosis of alveolar epithelial cells apoptosis. in this regard, sp-d has been shown to decrease apoptosis both in vitro and in vivo. a recent study shows that sp-d-deficient mice have higher level of apoptotic cells in their alveolar spaces than wild type mice and intratracheal administration of recombinant sp-d reduces levels of apoptotic cells [ ] . this reduction in apoptosis is due to a delayed activation of caspase and and externalization of phosphatidylserine [ ] , the aminophospholipid that in normal conditions is located in the inner leaflet of the plasma membrane, but flips to the outer leaflet in dying cells. janssen et al. [ ] have proposed that sp-a and sp-d suppress the phagocytic function of alveolar macrophages through tonic interaction with the transmembrane receptor signal inhibitory regulatory protein alfa (sirpα) in the resting, noninflamed lung. binding of sp-a or sp-d to sirpα may facilitate the interaction of this receptor with cluster of differentiation cd , and the subsequent negative control of phagocytosis by innate immune cells [ ] . in type ii cells, binding of sp-a to its receptor initiates a signaling pathway that regulates apoptosis through tyrosine phosphorylation and activation of phosphatidylinositol -kinase [ ] . on the other hand, it has been recently demonstrated that the interaction of interleukin , a chemokine produced by macrophages and epithelial cells, with sp-a exacerbates cell damage in acute lung injury through inhibition of sp-a, promoting cell viability and suppressing apoptosis [ ] . interleukin also binds to sp-b [ ] , inhibiting the protective effect of this protein on calcium ion transport. this would result in an increase of intracellular calcium ions that may induce passive depolymerization of cytoskeleton and destroy the endothelial barrier [ ] . regarding surfactant lipids, little is known about their role in apoptosis. supplementation of poractant alfa (curosurf), an exogenous porcine-derived surfactant composed of surfactant lipids, sp-b, and sp-c, with popg or dioleoylphosphatidylglycerol reduces alveolar epithelial cell apoptosis [ , ] . how these phospholipids regulate apoptosis is unknown, but they are supposed to act directly on alveolar epithelial cells [ ] . surfactant proteins sp-b and sp-c are not involved in principle in regulation of apoptosis. however, it has been shown that mutations in the sp-c gene produce aberrant forms of sp-c that can generate accumulation of aggregated forms of prosp-c, causing misfolded protein stress responses, inhibiting proteasome function, and activating caspase- -mediated apoptosis [ ] . non-inflammatory removal of dying cells is important for maintenance of lung tissue homeostasis since apoptotic cells can undergo necrosis, releasing potentially damaging pro-inflammatory molecules and antigens that cause autoimmunity [ ] and the eventual destruction of alveolar walls [ ] . lung collectins enhance the ingestion of apoptotic cells by alveolar macrophages [ , ] , being sp-d a more potent promoter of apoptotic cell clearance than sp-a [ ] . removal of apoptotic cells by sp-a and sp-d occurs through simultaneous interaction with apoptotic cells and surface receptors on alveolar macrophages. the interaction of sp-a and sp-d with dying cells involves recognition of intracellular molecules that become exposed on the outside of the cell upon apoptosis. in this regard, both collectins have been shown to bind to dna [ , ] and myeloperoxidase [ ] , a specific intracellular defence molecule of neutrophils that has been proposed to act as a bridging molecule between phosphatidylserine epitopes on apoptotic cells and sp-a, facilitating the internalization of dying cells by macrophages. on the other hand, the macrophage receptors calreticulin/cd and myosin a are involved in collectin-mediated clearance of dead cells [ , ] . interestingly, the interactions of sp-a and sp-d with these receptors, as well as the binding of sp-d to dna, occur through their collagenous domains [ , , , ] . therefore, the potency of sp-d as promoter of apoptotic cell clearance may be linked to its large and extended collagen steams, which would facilitate the interaction between the macrophage receptors and apoptotic cells. this would allow the binding of calreticulin to dying cells and their subsequent targeting for removal by phagocytosis [ ] . in vivo administration of poractant alfa increases the clearance of apoptotic neutrophils [ ] . whether this effect is due to surfactant lipids or to surfactant proteins sp-b and/or -c is not clear. although the mechanisms involved in this effect remain elusive, willems and co-workers [ ] have proposed that surfactant lipids could compete with sirpα for binding sp-a and sp-d, thereby preventing their suppressive effects on phagocyte function. pathogens, damaged cells or toxic compounds may trigger an exacerbated or chronic inflammatory response that damages lung tissue, playing a critical role in the pathology and progression of different lung diseases such as emphysema [ ] and lung fibrosis [ ] . resolution of inflammation and the subsequent regeneration of lung tissue requires replacement of injured cells by cells of the same type and the subsequent fibroplasia or fibrotic scarring in which connective tissue replaces normal parenchymal tissue, associated with an alternative, anti-inflammatory m polarization of macrophages [ ] [ ] [ ] . willems and co-workers demonstrated that pulmonary surfactant plays a role in the regeneration and remodeling of lung parenchyma [ ] . sp-a has been shown to participate in the processes that control lung epithelial cell proliferation and apoptosis by binding to transforming growth factor β (tgf-β) [ , ] , a potent inhibitor of epithelial cell proliferation. formation of the sp-a/tgf-β complex protects tgf-β from inactivation by the latency-associated peptide [ ] and facilitates tgf-β binding to its receptors, inducing signaling cascades involved in cell cycle progression and proliferation [ ] . sp-a and sp-d also contribute to tissue repair by activating the polarization of alveolar macrophages to a m phenotype [ , ] . this effect has been related to the binding of the collagen domain of sp-a to myosin [ ] . this receptor, also known as sp-r , forms a pro-inflammatory complex with cd and class a scavenger receptor (sr-a) that promotes internalization of lps and subsequent signaling [ ] . therefore, sp-a may inhibit the sp-r /cd /sr-a complex, contributing to regulation and resolution of the inflammatory response [ ] . in addition, through binding to interferon gamma (ifn-γ), sp-a suppresses ifn-γ interaction with its receptor, inhibiting the polarization of alveolar macrophages to a pro-inflammatory m phenotype [ ] that has been shown to hinder fibroplasias [ ] . on the other hand, sp-c [ ] also plays a key role in maintaining alveolar integrity and repair since sp-c-null patients and animal models of sp-c deficiency develop fibrosis [ , ] . moreover, mutations or decreased expression of the gene encoding sp-c, causes alveolar type ii cell injury and aberrant repair of lung tissue to develop pulmonary fibrosis [ ] [ ] [ ] . in this regard, glasser and co-workers [ ] showed that sp-c-deficient mice express markers of macrophage alternative activation associated with advancing pulmonary fibrosis. this suggests that sp-c negatively regulates the alternative activation of macrophages, preventing the development of fibrosis. although the role of sp-c deficiency in fibrosis development has been documented by numerous lines of evidence, little is known about the interactions of sp-c with macrophage receptors involved in regulation of macrophage polarization. the extensive network of lipid-protein and protein-protein interactions described by this review to occur between lung surfactant molecules and host and pathogen cells at the alveolar spaces put surfactant structures at the center of lung homeostasis. through these interactions, the surfactant network likely integrates signals from epithelial cells and the cells responsible for the innate and induced immune response, leading to a coordinated modulation of the respiratory scenario. this includes a proper control over an environment that needs to be at the same time protective against mechanical and biophysical demands and against infection, but relatively tolerant with the occasional entrance of exogenous particles and microorganisms inhaled within the thousands of liters of air breathed every day. on the other hand, lung surfactant-associated interactions and processes have to coordinate appropriate responses to challenges triggered by different physiopathological contexts, whose resolution defines the edge between health and illness. a full understanding of the different levels of complexity of the surfactant network will open unprecedented diagnostic and therapeutic opportunities. funding: research in the laboratory of the authors is currently funded by grants from the spanish ministry of science, universities and innovation (rti - -b-i ) and the regional government of madrid (p /nmt- ). the authors declare no conflict of interest. the funders had no role in the writing of the manuscript, or in the decision to publish this review. steroidogenic acute regulatory-related lipid transfer protein tgf-β transforming growth factor β tlr toll-like receptor cell number and cell characteristics of the normal human lung pulmonary surfactant: functions and molecular composition surface properties in relation to atelectasis and hyaline membrane 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deficiency inhibits lamellar body formation the closer we look the more we see? quantitative microscopic analysis of the pulmonary surfactant system identification of lbm , a lamellar body limiting membrane protein of alveolar type ii cells, as the abc transporter protein abca abca protects alveolar epithelial cells against free cholesterol induced cell death abca is critical for lamellar body biogenesis in vivo surfactant protein and lipid homeostasis required for respiration distinct effects of surfactant protein a or d deficiency during bacterial infection on the lung surfactant metabolism in sp-d gene-targeted mice ca( +)](i) oscillations regulate type ii cell exocytosis in the pulmonary alveolus orphan g protein-coupled receptor gpr regulates pulmonary surfactant pool size essential regulation of lung surfactant homeostasis by the orphan g protein-coupled receptor gpr epithelial gpr regulates pulmonary alveolar homeostasis via gq/ signaling a stereologic electron microscope study of "tubular myelin figures" in alveolar fluids of rat lungs humanized sftpa and sftpa transgenic mice reveal functional divergence of sp-a and sp-a : formation of tubular myelin in vivo requires both gene products characteristics of surfactant from sp-a-deficient mice surfactant phospholipid catabolic rate is pool size dependent in mice activity of pulmonary surfactant protein-d (sp-d) in vivo is dependent on oligomeric structure preferential uptake of small-aggregate fraction of pulmonary surfactant in vitro degradation of pulmonary surfactant disaturated phosphatidylcholines by alveolar macrophages abcg regulates pulmonary surfactant metabolism in mice and men cd loss disrupts lung lipid surfactant homeostasis and exacerbates oxidized lipid-induced lung inflammation. front the molecular basis of pulmonary alveolar proteinosis aibp augments cholesterol efflux from alveolar macrophages to surfactant and reduces acute lung inflammation quantitative lipidomics in pulmonary alveolar proteinosis electronic cigarettes disrupt lung lipid homeostasis and innate immunity independent of nicotine alcohol-induced lipid dysregulation impairs glycolytic responses to lps in alveolar macrophages surfactant lipids at the host-environment interface. metabolic sensors, suppressors, and effectors of inflammatory lung disease conceptual approaches to lung injury and repair pattern recognition receptors and inflammation evolutionary convergence and divergence in nlr function and structure the role of collectins and galectins in lung innate immune defense surfactant protein c dampens inflammation by decreasing jak/stat activation during lung repair phospholipid regulation of innate immunity and respiratory viral infection bacterial lipopolysaccharides and innate immunity intrinsic conformation of lipid a is responsible for agonistic and antagonistic activity pseudomonas aeruginosa isolates from patients with cystic fibrosis: a class of serum-sensitive, nontypable strains deficient in lipopolysaccharide o side chains comparative immunochemistry of lipopolysaccharides from typable and polyagglutinable pseudomonas aeruginosa strains isolated from patients with cystic fibrosis a conserved structural motif for lipopolysaccharide recognition by procaryotic and eucaryotic proteins biological activities of lipopolysaccharides are determined by the shape of their lipid a portion endotoxin: physical requirements for cell activation early innate immune responses to bacterial lps cd controls the lps-induced endocytosis of toll-like receptor regulation of innate immune signalling pathways by the tripartite motif (trim) family proteins combinational clustering of receptors following stimulation by bacterial products determines lipopolysaccharide responses aerosolized endotoxin is immediately bound by pulmonary surfactant protein d in vivo bacterial lipopolysaccharide promotes destabilization of lung surfactant-like films persistence of lps-induced lung inflammation in surfactant protein-c-deficient mice interaction of bacterial lipopolysaccharide with mouse surfactant protein c inserted into lipid vesicles structural basis for interactions between lung surfactant protein c and bacterial lipopolysaccharide crystal structure of a complex of surfactant protein d (sp-d) and haemophilus influenzae lipopolysaccharide reveals shielding of core structures in sp-d-resistant strains interaction of surfactant protein a with bacterial lipopolysaccharide may affect some biological functions interactions of surfactant protein d with bacterial lipopolysaccharides. surfactant protein d is an escherichia coli-binding protein in bronchoalveolar lavage interaction of sp-a (surfactant protein a) with bacterial rough lipopolysaccharide (re-lps), and effects of sp-a on the binding of re-lps to cd and lps-binding protein interactions of surfactant protein d with fatty acids structure binding relationship of human surfactant protein d and various lipopolysaccharide inner core structures structural definition of hsp-d recognition of salmonella enterica lps inner core oligosaccharides reveals alternative binding modes for the same lps surfactant protein d binds selectively to klebsiella pneumoniae lipopolysaccharides containing mannose-rich o-antigens surfactant proteins a and d bind cd by different mechanisms binding of surfactant protein a to the lipid a moiety of bacterial lipopolysaccharides pulmonary surfactant protein a modulates the cellular response to smooth and rough lipopolysaccharides by interaction with cd surfactant protein a forms extensive lattice-like structures on , -dipalmitoylphosphatidylcholine/rough-lipopolysaccharide-mixed monolayers pulmonary surfactant protein a-induced changes in the molecular conformation of bacterial deep-rough lps lead to reduced activity on human macrophages elucidation of lipid binding sites on lung surfactant protein a using x-ray crystallography, mutagenesis, and molecular dynamics simulations reconstruction of lps transfer cascade reveals structural determinants within lbp, cd , and tlr -md for efficient lps recognition and transfer the structural basis of lipopolysaccharide recognition by the tlr -md- complex surfactant protein a inhibits lipopolysaccharide-induced immune cell activation by preventing the interaction of lipopolysaccharide with lipopolysaccharide-binding protein the perturbation of pulmonary surfactant by bacterial lipopolysaccharide and its reversal by polymyxin b: function and structure altered in vivo activity of liposome-incorporated lipopolysaccharide and lipid a surfactant protein a directly interacts with tlr and md- and regulates inflammatory cellular response. importance of supratrimeric oligomerization interaction of pulmonary surfactant protein c with cd and lipopolysaccharide human pulmonary surfactant protein d binds the extracellular domains of toll-like receptors and through the carbohydrate recognition domain by a mechanism different from its binding to phosphatidylinositol and lipopolysaccharide pulmonary surfactant protein d binds md- through the carbohydrate recognition domain pulmonary surfactant protein d inhibits lipopolysaccharide (lps)-induced inflammatory cell responses by altering lps binding to its receptors treponemal phospholipids inhibit innate immune responses induced by pathogen-associated molecular patterns anionic pulmonary surfactant phospholipids inhibit inflammatory responses from alveolar macrophages and u cells by binding the lipopolysaccharide-interacting proteins cd and md- inhibitory effect of pulmonary surfactant proteins a and d on allergen-induced lymphocyte proliferation and histamine release in children with asthma regulatory roles for cd and phosphatidylinositol in the signaling via toll-like receptor -md- structural analogs of pulmonary surfactant phosphatidylglycerol inhibit toll-like receptor and signaling surfactant lipids regulate lps-induced interleukin- production in a lung epithelial cells by inhibiting translocation of tlr into lipid raft domains lps-induced clustering of cd triggers generation of pi( , )p anionic pulmonary surfactant lipid regulation of innate immunity host defense against fungal infections and allergies surfactant protein (sp)-a and sp-d as antimicrobial and immunotherapeutic agents. recent patents anti-infective drug discov macrophage dysfunction and susceptibility to pulmonary pseudomonas aeruginosa infection in surfactant protein c-deficient mice surfactant protein c-deficient mice are susceptible to respiratory syncytial virus infection diverse functions of pulmonary collectins in host defense of the lung pulmonary collectins and innate host defense of the lung role of soluble innate effector molecules in pulmonary defense against fungal pathogens an insight into the diverse roles of surfactant proteins, sp-a and sp-d in innate and adaptive immunity. front surfactant protein a binds mycoplasma pneumoniae with high affinity and attenuates its growth by recognition of disaturated phosphatidylglycerols characteristics of surfactant protein a and d binding to lipoteichoic acid and peptidoglycan, major cell wall components of gram-positive bacteria respiratory fungal infections: their role in the inflammatory response surfactant proteins a and d: trimerized innate immunity proteins with an affinity for viral fusion proteins collectins and fungal pathogens: roles of surfactant proteins and mannose binding lectin in host resistance surfactant protein d binding to terminal alpha - -linked fucose residues and to schistosoma mansoni opsonic activities of surfactant proteins a and d in phagocytosis of gram-negative bacteria by alveolar macrophages surfactant protein a (sp-a)-mediated clearance of staphylococcus aureus involves binding of sp-a to the staphylococcal adhesin eap and the macrophage receptors sp-a receptor and scavenger receptor class a purification of a cell-surface receptor for surfactant protein a lung surfactant proteins (sp-a and sp-d) in non-adaptive host responses to infection glycoprotein- binds surfactant protein-a (sp-a) and stimulates alveolar macrophage migration in an sp-a-independent manner collectin surfactant protein d binds antibodies and interlinks innate and adaptive immune systems by binding sirpalpha or calreticulin/cd , lung collectins act as dual function surveillance molecules to suppress or enhance inflammation natural anti-infective pulmonary proteins: in vivo cooperative action of surfactant protein sp-a and the lung antimicrobial peptide sp-bn pulmonary surfactant protein a up-regulates activity of the mannose receptor, a pattern recognition receptor expressed on human macrophages pulmonary collectins enhance phagocytosis of mycobacterium avium through increased activity of mannose receptor pulmonary surfactant protein a augments the phagocytosis of streptococcus pneumoniae by alveolar macrophages through a casein kinase -dependent increase of cell surface localization of scavenger receptor a ten haken, b.; et al. pulmonary surfactant protein sp-d opsonises carbon nanotubes and augments their phagocytosis and subsequent pro-inflammatory immune response overview of complement activation and regulation surfactant protein a regulates complement activation innate immune collectin surfactant protein d simultaneously binds both neutrophil extracellular traps and carbohydrate ligands and promotes bacterial trapping hemoglobin induction in mouse macrophages sp-d attenuates lps-induced formation of human neutrophil extracellular traps (nets), protecting pulmonary surfactant inactivation by nets surfactant protein d attenuates acute lung and kidney injuries in pneumonia-induced sepsis through modulating apoptosis, inflammation and nf-kappab signaling surfactant proteins a and d inhibit the growth of gram-negative bacteria by increasing membrane permeability pulmonary collectins selectively permeabilize model bacterial membranes containing rough lipopolysaccharide sp-a permeabilizes lipopolysaccharide membranes by forming protein aggregates that extract lipids from the membrane macrophage-independent fungicidal action of the pulmonary collectins paradoxical bactericidal effects of hydrophobic lung surfactant proteins and their peptide mimics using liposome molecular trojan antimicrobial activity of native and synthetic surfactant protein b peptides surfactant protein b propeptide contains a saposin-like protein domain with antimicrobial activity at low ph the role and molecular mechanism of action of surfactant protein d in innate host defense against influenza a virus full-length human surfactant protein a inhibits influenza a virus infection of a lung epithelial cells: a recombinant form containing neck and lectin domains promotes infectivity interactions of surfactant protein a with influenza a viruses: binding and neutralization impact of ozone exposure on the phagocytic activity of human surfactant protein a (sp-a) and sp-a variants evidence for a protective role of pulmonary surfactant protein d (sp-d) against influenza a viruses mechanisms of anti-influenza activity of surfactant proteins a and d: comparison with serum collectins collectin-mediated antiviral host defense of the lung: evidence from influenza virus infection of mice inhibition of influenza viral neuraminidase activity by collectins surfactant protein a binds to hiv and inhibits direct infection of cd + cells, but enhances dendritic cell-mediated viral transfer surfactant protein d binds to human immunodeficiency virus (hiv) envelope protein gp and inhibits hiv replication surfactant protein d modulates hiv infection of both t-cells and dendritic cells surfactant protein a binds to the fusion glycoprotein of respiratory syncytial virus and neutralizes virion infectivity surfactant protein-d enhances phagocytosis and pulmonary clearance of respiratory syncytial virus lactoferrin and surfactant protein a exhibit distinct binding specificity to f protein and differently modulate respiratory syncytial virus infection lung surfactant protein a provides a route of entry for respiratory syncytial virus into host cells binding of surfactant protein a (sp-a) to herpes simplex virus type -infected cells is mediated by the carbohydrate moiety of sp-a the sars coronavirus spike glycoprotein is selectively recognized by lung surfactant protein d and activates macrophages antiviral activity of bovine collectins against rotaviruses protective effect of surfactant protein d in pulmonary vaccinia virus infection: implication of a viral protein involvement of surfactant protein d in ebola virus infection enhancement via glycoprotein interaction adaptation and increased susceptibility to infection associated with constitutive expression of misfolded sp-c pulmonary surfactant phosphatidylglycerol inhibits respiratory syncytial virus-induced inflammation and infection phosphatidylinositol inhibits respiratory syncytial virus infection pulmonary surfactant lipids inhibit infections with the pandemic h n influenza virus in several animal models phosphatidylglycerol provides short-term prophylaxis against respiratory syncytial virus infection innate defense against influenza a virus: activity of human neutrophil defensins and interactions of defensins with surfactant protein d interactions of alpha-, beta-, and theta-defensins with influenza a virus and surfactant protein d respiratory innate immune proteins differentially modulate the neutrophil respiratory burst response to influenza a virus pulmonary surfactant protein a protects lung epithelium from cytotoxicity of human beta-defensin role of apoptosis in amplifying inflammatory responses in lung diseases the role of apoptosis in the pathophysiology of acute respiratory distress syndrome (ards): an up-to-date cell-specific review role of apoptosis in the pathogenesis of copd and pulmonary emphysema apoptosis in lung injury and fibrosis surfactant protein d regulates caspase- -mediated cascade of the intrinsic pathway of apoptosis while promoting bleb formation surfactant proteins a and d suppress alveolar macrophage phagocytosis via interaction with sirp alpha functional cd /signal regulatory protein alpha (sirp(alpha)) interaction is required for optimal human t-and natural killer-(nk) cell homeostasis in vivo elevated expression of surfactant proteins in newborn rats during adaptation to hyperoxia mechanism of il- -induced acute lung injury through pulmonary surfactant proteins a and b endothelial mitochondria determine rapid barrier failure in chemical lung injury novel therapeutic roles for surfactant-inositols and -phosphatidylglycerols in a neonatal piglet ards model: a translational study : / : -dioleoyl-phosphatidylglycerol prevents alveolar epithelial apoptosis and profibrotic stimulus in a neonatal piglet model of acute respiratory distress syndrome misfolded brichos sp-c mutant proteins induce apoptosis via caspase- -and cytochrome c-related mechanisms efferocytosis and lung disease functional significance of apoptosis in chronic obstructive pulmonary disease role of surfactant proteins a, d, and c q in the clearance of apoptotic cells in vivo and in vitro: calreticulin and cd as a common collectin receptor complex surfactant protein a enhances alveolar macrophage phagocytosis of apoptotic neutrophils surfactant protein d binds genomic dna and apoptotic cells, and enhances their clearance, in vivo nucleic acid is a novel ligand for innate, immune pattern recognition collectins surfactant proteins a and d and mannose-binding lectin surfactant protein a (sp-a) binds to phosphatidylserine and competes with annexin v binding on late apoptotic cells cell-surface calreticulin initiates clearance of viable or apoptotic cells through trans-activation of lrp on the phagocyte surfactant protein a inhibits t cell proliferation via its collagen-like tail and a -kda receptor local amplifiers of il- ralpha-mediated macrophage activation promote repair in lung and liver programmed cell removal by calreticulin in tissue homeostasis and cancer poractant alfa (curosurf(r)) increases phagocytosis of apoptotic neutrophils by alveolar macrophages in vivo immune-mediated inflammation in the pathogenesis of emphysema: insights from mouse models inflammation in cystic fibrosis: an update p-selectin suppresses hepatic inflammation and fibrosis in mice by regulating interferon gamma and the il- decoy receptor macrophage plasticity and polarization in tissue repair and remodelling dual polarization of human alveolar macrophages progressively increases with smoking and copd severity surfactant protein a binds tgf-beta with high affinity and stimulates the tgf-beta pathway surfactant protein-d is essential for immunity to helminth infection sp-r (myo a) isoforms as intrinsic modulators of macrophage priming and activation surfactant protein a prevents ifn-gamma/ifn-gamma receptor interaction and attenuates classical activation of human alveolar macrophages histone deacetylase inhibitor restores surfactant protein-c expression in alveolar-epithelial type ii cells and attenuates bleomycin-induced pulmonary fibrosis in vivo expression of mutant sftpc in murine alveolar epithelia drives spontaneous lung fibrosis genetics of fibrosing lung diseases key: cord- -ezrkg dc authors: myerson, jacob w.; patel, priyal n.; habibi, nahal; walsh, landis r.; lee, yi-wei; luther, david c.; ferguson, laura t.; zaleski, michael h.; zamora, marco e.; marcos-contreras, oscar a.; glassman, patrick m.; johnston, ian; hood, elizabeth d.; shuvaeva, tea; gregory, jason v.; kiseleva, raisa y.; nong, jia; rubey, kathryn m.; greineder, colin f.; mitragotri, samir; worthen, george s.; rotello, vincent m.; lahann, joerg; muzykantov, vladimir r.; brenner, jacob s. title: supramolecular organization predicts protein nanoparticle delivery to neutrophils for acute lung inflammation diagnosis and treatment date: - - journal: biorxiv doi: . / . . . sha: doc_id: cord_uid: ezrkg dc acute lung inflammation has severe morbidity, as seen in covid- patients. lung inflammation is accompanied or led by massive accumulation of neutrophils in pulmonary capillaries (“margination”). we sought to identify nanostructural properties that predispose nanoparticles to accumulate in pulmonary marginated neutrophils, and therefore to target severely inflamed lungs. we designed a library of nanoparticles and conducted an in vivo screen of biodistributions in naive mice and mice treated with lipopolysaccharides. we found that supramolecular organization of protein in nanoparticles predicts uptake in inflamed lungs. specifically, nanoparticles with agglutinated protein (naps) efficiently home to pulmonary neutrophils, while protein nanoparticles with symmetric structure (e.g. viral capsids) are ignored by pulmonary neutrophils. we validated this finding by engineering protein-conjugated liposomes that recapitulate nap targeting to neutrophils in inflamed lungs. we show that naps can diagnose acute lung injury in spect imaging and that nap-like liposomes can mitigate neutrophil extravasation and pulmonary edema arising in lung inflammation. finally, we demonstrate that ischemic ex vivo human lungs selectively take up naps, illustrating translational potential. this work demonstrates that structure-dependent interactions with neutrophils can dramatically alter the biodistribution of nanoparticles, and naps have significant potential in detecting and treating respiratory conditions arising from injury or infections. the covid- pandemic tragically illustrates the dangers of acute inflammation and infection of the lungs, for both individuals and societies. acute alveolar inflammation causes the clinical syndrome known as acute respiratory distress syndrome (ards), in which inflammation prevents the lungs from oxygenating the blood. severe ards is the cause of death in most covid- mortality and was a major cause of death in the influenza epidemic, but ards is common even outside of epidemics, affecting ~ , american patients per year with a ~ - % mortality rate. - ards is caused not just by viral infections, but also by sepsis, pneumonia (viral and bacterial), aspiration, and trauma. , largely because ards patients have poor tolerance of drug side effects, no pharmacological strategy has succeeded as an ards treatment. , [ ] [ ] [ ] therefore, there is an urgent need to develop drug delivery strategies that specifically target inflamed alveoli in ards and minimize systemic side effects. neutrophils are "first responder" cells in acute inflammation, rapidly adhering and activating in large numbers in inflamed vessels and forming populations of "marginated" neutrophils along the vascular lumen. [ ] [ ] [ ] [ ] [ ] [ ] [ ] neutrophils can be activated by a variety of initiating factors, including pathogen-and damage-associated molecular patterns such as bacterial lipopolysaccharides (lps). , after acute inflammatory insults, neutrophils marginate in most organs, but by far most avidly in the lung capillaries. , , , , neutrophils are therefore key cell types in most forms of ards. in ards, marginated neutrophils can secrete tissue-damaging substances (proteases, reactive oxygen species) and extravasate into the alveoli, leading to disruption of the endothelial barrier and accumulation of neutrophils and edematous fluid in the air space of the lungs ( figure a ). , , , [ ] [ ] [ ] targeted nanoparticle delivery to marginated neutrophils could provide an ards treatment with minimal side effects, but specific delivery to marginated neutrophils remains an open challenge. antibodies against markers such as ly g have achieved targeting to neutrophils in mice, but also deplete populations of circulating neutrophils. [ ] [ ] [ ] [ ] additionally, while ly g readily marks neutrophils in mice, there is no analogous specific and ubiquitous marker on human neutrophils. therefore, antibody targeting strategies have not been widely adopted for targeted drug delivery to these cells. as another route to neutrophil targeting, two previous studies noted that activated neutrophils take up denatured and agglutinated bovine albumin, concluding that denatured protein was critical in neutrophil-particle interactions. , nanoparticle structural properties such as shape, size, and deformability can define unique targeting behaviors. [ ] [ ] [ ] [ ] [ ] here, we screened a diverse panel of nanoparticles to determine the nanostructural properties that predict uptake in pulmonary marginated neutrophils during acute inflammation. as a high-throughput animal model for ards, we administered lps to mice, causing a massive increase in pulmonary marginated neutrophils. we show that two initial leads in our screen, lysozyme-dextran nanogels (ldngs) and crosslinked albumin nanoparticles (anps), selectively home to marginated neutrophils in inflamed lungs, but not naïve lungs. in our subsequent screen of over diverse nanoparticles, we find that protein nanoparticles, all defined by agglutination of protein in amorphous nanostructures (nanoparticles with agglutinated proteins, naps), but not by denatured protein, have specificity for lps-inflamed lungs. in contrast to naps, we demonstrate that three symmetric protein nanostructures (viruses/nanocages) have biodistributions unaffected by lps injury. we show that polystyrene nanoparticles and five liposome formulations do not accumulate in injured lungs, indicating that nanostructures that are not based on protein are not intrinsically drawn to marginated neutrophils in acute inflammation. we then engineered liposomes (the most clinically relevant nanoparticle drug carriers) as naps, through conjugation to protein modified with hydrophobic cyclooctynes, encouraging protein agglutination on the liposome surface by hydrophobic interactions. we thus show that supramolecular organization of proteins, rather than chemical composition, best predicts uptake in marginated neutrophils in acutely inflamed lungs. we then demonstrate proof of concept for naps as diagnostic and therapeutic tools for ards. we show; a) in-labeled naps provide diagnostic imaging contrast that distinguishes inflammatory lung injury from cardiogenic pulmonary edema; b) napliposomes can significantly ameliorate edema in a mouse model of severe ards; c) naps, but not crystalline protein nanostructures, accumulate in ex vivo human lungs rejected for transplant due to ards-like conditions. collectively, our results will demonstrate that supramolecular organization of protein, namely protein agglutination, predicts strong, intrinsic nanoparticle tropism for marginated neutrophils. this finding indicates that naps, encompassing a wide range of nanoparticles based on or incorporating protein, have biodistributions that are responsive to inflammation. naps could be useful beyond ards, since marginated neutrophils play a pathogenic role in a diverse array of inflammatory diseases, including infections, heart attack, and stroke. [ ] [ ] [ ] [ ] [ ] but our findings provide a clear path forward for using naps to improve diagnosis and treatment of ards. to quantify the increase in pulmonary marginated neutrophils after inflammatory lung injury, radiolabeled clone a anti-ly g antibody (specific for mouse neutrophils) was administered intravenously (iv) to determine the location and concentration of neutrophils in mice. iv injection of lps subjected mice to a model of mild ards. accumulation of anti-ly g antibody in the lungs was dramatically affected by iv lps, with . % of injected antibody adhering in lps-injured lungs, compared to . % of injected antibody in naïve control lungs ( figure b) . agreeing with previous studies addressing the role of neutrophils in systemic inflammation, biodistributions of anti-ly g antibody indicated that systemic lps injury profoundly increased the concentration of neutrophils in the lungs. , , , single cell suspensions prepared from mouse lungs were probed by flow cytometry to further characterize pulmonary neutrophils in naïve mice and in mice following lps-induced inflammation. to identify intravascular populations of leukocytes, mice received iv fluorescent cd antibody five minutes prior to sacrifice. single cell suspensions prepared from iv cd -stained lungs were then stained with anti-ly g antibody to identify neutrophils. a second stain of single cell suspensions with cd antibody indicated the total population of leukocytes in the lungs, distinct from the intravascular population indicated by iv cd . flow cytometry showed greater concentrations of neutrophils in lps-injured lungs, compared to naïve lungs ( figure c , counts above horizontal threshold indicate positive staining for neutrophils, figure d , rightmost peak indicates positive staining for neutrophils). comparison of ly g stain to total cd -positive cells indicated . % of leukocytes in the lungs were ly g-positive after lps injury, compared to . % in the naïve control ( figure d , center panel). comparison of ly g stain to iv cd stain indicated that the majority of neutrophils were intravascular, in both naïve and lpsinjured mice. in naïve mice, . % of neutrophils were intravascular and in lps-injured mice, . % of neutrophils were intravascular ( figure d , right panel). the presence of large populations of intravascular neutrophils following inflammatory injury is consistent with previously published observations. , , , , histological analysis confirmed results obtained with flow cytometry and radiolabeled anti-ly g biodistributions. staining of lung sections indicated increased concentration of neutrophils in the lungs following iv lps injury ( figure e , left panels). co-registration of neutrophil staining with tissue autofluorescence (indicating tissue architecture) broadly supported the finding that pulmonary neutrophils reside in the vasculature ( figure e , right panels). previous work has traced the neutrophil response to bacteria in the lungs, determining that pulmonary neutrophils pursue and engulf active bacteria following either intravenous infection or infection of the airspace in the lungs. , , we injected heat-inactivated, oxidized, and fixed e. coli in naïve and iv-lps-injured mice. with the bacteria stripped of their functional behavior, e. coli did not accumulate in the lungs of naïve control mice ( . % of initial dose in the lungs, blue bars in figure f ). however, pre-treatment with lps to recapitulate the inflammatory response to infection led to enhanced accumulation of the deactivated e. coli in the lungs ( . % of initial dose in the lungs, red bars in figure f ). with e. coli structure maintained but e. coli function removed, the inactivated bacteria were taken up more avidly in lungs primed by an inflammatory injury. in order to identify nanostructural parameters that correlate with nanoparticle uptake in inflamed lungs, we conducted an in vivo screen of a diverse array of nanoparticle drug carriers. the screen was based on the method used above for tracing inactivated bacteria: inject radiolabeled nanoparticles into mice and measure biodistributions, comparing naïve with iv-lps mice. to validate that the radiotracing screen would measure uptake in pulmonary marginated neutrophils, we more fully characterized the in vivo behavior of two early hits in the screen. lysozyme-dextran nanogels (ldngs, ngs) and poly(ethylene)glycol (peg)crosslinked albumin nps have been characterized as targeted drug delivery agents in previous work. [ ] [ ] [ ] here, ldngs ( . ± . nm diameter, . ± . pdi, supplementary figure a ) and peg-crosslinked human albumin nps ( . ± . nm diameter, . ± . pdi, supplementary figure b) were administered in naïve and iv-lps-injured mice. neither np was functionalized with antibodies or other affinity tags. the protein component of each particle was labeled with i for tracing in biodistributions, and assessed minutes after iv administration of nps. both absolute ldng lung uptake and ratio of lung uptake to liver uptake registered a ~ -fold increase between naïve control and lps-injured animals (figure a , supplementary table ) . specificity for lps-injured lungs was recapitulated with peg-crosslinked human albumin nps. albumin nps accumulated in naïve lungs at . % injected dose per gram organ weight (%id/g), and in lps-injured lungs at . %id/g, accounting for a -fold increase in lung uptake after intravenous lps insult ( figure b , supplementary table ) . single cell suspensions were prepared from lungs after administration of fluorescent ldngs or peg-crosslinked albumin nps. flow cytometric analysis of cells prepared from lungs after np administration enabled identification of cell types with which nps associated. firstly, the total number of cells containing ldngs or albumin nps increased between naïve and lps-injured lungs. in naïve control lungs, . ly g stain for neutrophils indicated that the bulk of ldng and albumin np accumulation in lps-injured lungs could be accounted for by uptake in neutrophils. in figure c and d, counts above the horizontal threshold indicate neutrophils and counts to the right of the vertical threshold indicate cells containing ldngs ( figure c ) or albumin nps ( figure d ). in iv-lps-injured lungs, ldng and albumin np uptake was dominated by neutrophils ( figure c , figure d , upper right quadrants indicate nppositive neutrophils). in lps-injured lungs, the majority of neutrophils, > % of cells, contained significant quantities of nanoparticles, compared to < % in naïve lungs. likewise, the majority of nanoparticle uptake in the lungs (> %) was accounted for by nanoparticle uptake in neutrophils ( figure e , f, g, h, supplementary table ) . for np uptake not accounted for by neutrophils, cd staining indicated that the remaining np uptake was attributable to other leukocytes. co-localization of albumin np fluorescence with cd stain showed that . % of albumin np uptake was localized to leukocytes in naïve lungs and . % of albumin np uptake was localized to leukocytes in injured lungs (supplementary figure c, supplementary figure d ). for ldngs, localization to neutrophils in injured lungs was confirmed via histology. ly g staining of lps-injured lung sections confirmed colocalization of fluorescent nanogels with neutrophils in the lung vasculature ( figure i ). slices in confocal images of lung sections indicated that ldngs were inside neutrophils ( figure j ). intravital imaging of injured lungs allowed real-time visualization of ldng uptake in leukocytes in injured lungs. ldng fluorescent signal accumulated over minutes and reliably colocalized with cd staining for leukocytes ( figure k , supplementary movie ). ldng pharmacokinetics were evaluated in naïve and iv-lps-injured mice (supplementary figure ) . in both naïve and injured mice, bare ldngs were rapidly cleared from the blood with a distribution half-life of ~ minutes. in naïve mice, transient retention of ldngs in the lungs ( . %id/g at five minutes after injection) leveled off over one hour. in iv-lps-treated mice, ldng concentration in the lungs reached a peak value at minutes after injection, as measured either by absolute levels of lung uptake or by lungs:blood localization ratio. ldng biodistributions were also assessed in mice undergoing alternative forms of lps-induced inflammation. intratracheal (it) instillation of lps led to concentration of ldngs in the lungs at . %id/g. liver and spleen ldng uptake was also reduced following it lps injury, leading to a -fold increase in the lungs:liver ldng localization ratio induced by it lps injury (supplementary figure ) . as with iv lps injury, it lps administration leads to neutrophil-mediated vascular injury focused in the lungs. mice were administered lps via footpad injection to provide a model of systemic inflammation originating in lymphatic drainage. ldng uptake in the lungs and in the legs was enhanced by footpad lps administration. at hours after footpad lps administration, ldngs concentrated in the lungs at . %id/g, an -fold increase over naïve. at hours, ldngs concentrated in the lungs at . %id/g (supplementary figure a) . total ldng accumulation in the legs accounted for . % of initial dose (%id) in naïve mice, . %id in mice hours after footpad lps injection, and . %id at hours after footpad injection (supplementary figure b) , indicating ldngs can concentrate in inflamed vasculature outside the lungs. previous work has indicated that nps based on denatured albumin accumulate in neutrophils in inflamed lungs and at sites of acute vascular injury, whereas nps coated with native albumin do not. , we have characterized lysozyme-dextran nanogels and crosslinked human albumin nps with circular dichroism (cd) spectroscopy to compare secondary structure of proteins in the nps to secondary structure of the native component proteins (supplementary figure a-b) . identical cd spectra were recorded for ldngs vs. lysozyme and for albumin nps vs. human albumin. deconvolution of the cd spectra via neural network algorithm trained against a library of cd spectra for known structures verified that secondary structure composition of lysozyme and albumin was unchanged by incorporation of the proteins in the nps. free protein and protein nps were also probed with -anilino- naphthalenesulfonic acid (ansa), previously established as a tool for determining the extent to which hydrophobic domains are exposed on proteins. consistent with known structures of the two proteins, ansa staining indicated few available hydrophobic domains on lysozyme and substantial hydrophobic exposure on albumin (supplementary figure c -d, blue curves). ldngs had increased hydrophobic accessibility vs. native lysozyme whereas albumin nps had reduced hydrophobic accessibility compared with native albumin. therefore, our data indicate that lysozyme and albumin are not denatured in ldngs and albumin nps, but the nps composed of the two proteins present a balance of hydrophobic and hydrophilic surfaces differing from the native proteins. the previous section demonstrates that two different nanoparticles based on protein, shown not to be denatured in cd spectroscopy studies, have uptake in lpsinflamed lungs driven by uptake in marginated neutrophils. we next undertook a broader study considering how aspects of np structure including size, composition, surface chemistry, and structural organization impact np uptake in lps-injured lungs. as examples of different types of protein nps, variants of ldngs (representing nps based on hydrophobic interactions between proteins), crosslinked protein nps, and nps based on electrostatic interactions between proteins were traced in naïve control and iv-lps-injured mice. as examples of nps based on site-specific protein interactions (rather than site-indiscriminate interactions leading to crosslinking, gelation, or chargebased protein nps), we also traced viruses and ferritin nanocages in naïve and lpstreated mice. liposomes and polystyrene nps were studied as examples of lipid and polymeric nanostructures. nanoparticles based on hydrophobic protein interactions ldng size was varied by modifying lysozyme-dextran composition of the nps and ph at which particles were formed. figure , all sizes of ldngs accumulated in lps-injured lungs at higher concentrations than in naïve lungs, with accumulation in injured lungs reaching ~ % of initial dose for all types of ldngs (supplementary table ). variations in size and composition of ldngs therefore did not affect ldng specificity for lps-injured lungs. expanding on data with peg-nhs ester-crosslinked human serum albumin particles, we varied the geometry and protein composition of nps based on peg-nhs protein crosslinking. human serum albumin nanorods (aspect ratio : ), bovine serum albumin nps ( . table ). lysozyme nps accumulated in naïve lungs at a uniquely high concentration of . %id/g, compared to . %id/g in inflamed lungs. degree of uptake in injured lungs, along with injured vs. naïve contrast, did vary with protein np composition. however, acute inflammatory injury resulted in a minimum three-fold increase in lung uptake for all examined crosslinked protein nps, excluding crosslinked lysozyme, which still accumulated in injured lungs at a high concentration ( . % of initial dose). we traced recently-developed poly(glutamate) tagged green fluorescent protein (e-gfp) nps, representing a third class of protein np based on electrostatic interactions between proteins and carrier polymer or metallic particles. negatively-charged e-gfp was paired to arginine-presenting gold nanoparticles ( . ± . nm diameter, pdi . ± . ) or to poly(oxanorborneneimide) (poni) functionalized with guanidino and tyrosyl side chains ( . ± . nm diameter, pdi . ± . ) (supplementary figure d) . for biodistribution experiments with poni/e-gfp hybrid nps, tyrosine-bearing poni was labeled with i and e-gfp was labeled with i, allowing simultaneous tracing of each component of the hybrid nps. the two e-gfp nps, with structure based on charge interactions, had specificity for iv lps-injured lungs. comparing uptake in lps-injured lungs to naïve lungs, we observe an lps:naïve ratio of . for poni/e-gfp nps as traced by the poni component, . for poni/e-gfp nps as traced by the e-gfp component, and . for au/e-gfp nps ( figure c , supplementary figure ). poni/e-gfp particles, specifically, accumulated in lpsinjured lungs at . % initial dose as measured by poni tracing and . % initial dose as measured by gfp tracing, indicating effective co-delivery in the inflamed organ. acute inflammatory injury therefore resulted in a two-to three-fold increase in pulmonary uptake of nps constructed via electrostatic protein interactions. nanoparticles based on symmetric protein organization adeno-associated virus (aav), adenovirus, and horse spleen ferritin nanocages were employed as examples of protein-based nps with highly symmetrical structure (see supplementary figure d for dls confirmation of structure). [ ] [ ] [ ] for each of these highly ordered protein nps, iv lps injury had no significant effect on biodistribution and levels of uptake in the injured lungs were minimal ( figure d table ). therefore, highly ordered protein nps traced in our studies did not have tropism for the lungs after acute inflammatory injury. liposomes and polystyrene nps were studied as example nps that are not structurally based on proteins. dota chelate-containing lipids were incorporated into bare liposomes, allowing labeling with in tracer for biodistribution studies. carboxylate polystyrene nps were coupled to trace amounts of i-labeled igg via edci-mediated carboxy-amine coupling. liposomes had a diameter of . ± . nm (pdi . ± . ) and igg-polystyrene nps had a diameter of . ± . nm (pdi . ± . ) (supplementary figure c -d). liposomes accumulated in inflamed lungs at a concentration of . %id/g, accounting for no significant change against naïve lungs. lps injury actually induced a reduction in the lungs:liver metric, from . for naïve mice to . for lps-injured mice. polystyrene nps accumulated in inflamed lungs at . %id/g ( . % initial dose), so iv lps injury did in fact induce increased levels of np uptake in the lungs, from a concentration of . %id/g in the naïve lungs ( figure e, supplementary figure ). however, neither bare liposomes nor polystyrene nps were drawn to lps-injured lungs in significant concentrations. significantly, isolated proteins did not home to lps-inflamed lungs themselves. we traced radiolabeled albumin, lysozyme, and transferrin in naïve control and iv lpsinjured mice (supplementary figure , supplementary table ). in injured mice, albumin, lysozyme, and transferrin localized to the lungs at low concentrations and no significant differences were recorded when comparing naïve to lps-injured lung uptake. the data presented in figure and supplementary figures - indicate that a variety of protein-based nanostructures have tropism for acute inflammatory injury in the lungs. nps based on agglutination of proteins in non-site-specific interactions (naps, figure a -c, supplementary figures - ) all exhibited either significant increases in lung uptake after lps injury or high levels of lung uptake in both naïve control and lpsinjured animals. nanostructures based on highly symmetrical protein organization had no specific tropism for inflamed lungs ( figure d ). representative nanostructures not based on proteins, bare liposomes and polystyrene beads, did not home to inflamed lungs ( figure e ). we next engineered naps from liposomes, a nanoparticle shown above to have no intrinsic neutrophil tropism. our methods for engineering nap-like liposomes serve to validate the finding that supramolecular organization of protein in nanoparticles predicts neutrophil tropism. liposomes were functionalized with rat igg conjugated via sata-maleimide chemistry (sata-igg liposomes) or via recently demonstrated copper-free click chemistry methods. briefly, click chemistry methods entailed nhs-ester conjugation of an excess of strained alkyne (dibenzocyclooctyne, dbco) to igg, followed by reaction of the dbco-functionalized igg with liposomes containing peg-azide-terminated lipids (dbco-igg liposomes, figure a ). dbco-igg liposomes had a diameter of . ± . nm and a pdi of . ± . and sata-igg liposomes had a diameter of . ± . nm and a pdi of . ± . (supplementary figure c) . in mice subjected to iv-lps, sata-igg liposomes accumulated in the lungs at a concentration of . %id/g ( figure b , yellow bars). dbco-igg liposomes, by contrast, concentrated in the lungs at . %id/g, corresponding to . % of initial dose and roughly matching the accumulation of nm ldngs in the inflamed lungs ( figure b , brown bars). for comparison, bare liposomes, as in figure e , concentrated in the inflamed lungs at . %id/g ( figure b , green bars). for dbco-igg liposomes, the inflamed vs. naïve lung uptake accounted for a twelve-fold change. dbco-igg liposomes specifically accumulated in injured lungs, whereas sata-igg liposomes and bare liposomes did not (supplementary figure , supplementary table ) . it lps instillation also led to elevated concentrations of dbco-igg liposomes in the lungs. biodistributions of the dbco-igg liposomes indicated a pulmonary concentration of . %id/g at hour after it lps, . %id/g at hours after it lps, and . %id/g at hours after it lps (supplementary figure ). even at early time points after direct pulmonary lps insult, dbco-igg liposomes accumulated in the inflamed lungs. results in figure b were obtained by introducing a -fold molar excess of nhs-ester-dbco to rat igg before dbco-igg conjugation to liposomes (dbco( x)-igg liposomes). optical density quantification of dbco indicated ~ dbco per igg following reaction of dbco and igg at : molar ratio (supplementary figure ) . to test the hypothesis that dbco functions as a tag that modifies dbco-igg liposomes for neutrophil affinity in settings of inflammation, we varied the concentration of dbco on igg prepared for conjugation to azide liposomes. dbco was added to igg at -fold, five-fold, and . -fold molar excesses. a -fold molar excess resulted in ~ dbco per igg, a -fold molar excess resulted in ~ dbco per igg, and a . -fold molar excess resulted in ~ dbco per igg (supplementary figure ) . igg with different dbco loading concentrations was conjugated to azide liposomes. dbco-igg liposomes had similar sizes across all dbco concentrations (supplementary figure c) , with diameters of ~ nm and pdis < . . the different types of dbco-igg liposomes were each traced in iv-lps injured mice. titrating the quantity of dbco on dbco-igg liposomes indicated that liposome accumulation in the lungs of injured mice was dependent on dbco concentration on the liposome surface. concentration of dbco-igg liposomes in inflamed lungs attenuated with decreasing dbco concentration on igg (supplementary table , figure c ). therefore, only igg with high concentrations of dbco served as a tag for modifying the surface of liposomes for specificity to pulmonary injury. flow cytometry verified the specificity of dbco-igg liposomes for neutrophils in injured lungs ( figure d -e). as with ldngs and albumin nps in figure c -h, single cell suspensions were prepared from lps-inflamed and naïve control lungs after circulation of fluorescent dbco-igg liposomes. confirming the results of biodistribution studies, . % of cells were liposome-positive in naïve lungs, compared to . % of all cells in lps-inflamed lungs (supplementary figure a-b) . dbco-igg liposomes predominantly accumulated in pulmonary neutrophils after iv lps. there were more neutrophils in the injured lungs and a greater fraction of neutrophils took up dbco-igg liposomes in the injured lungs, as compared to the naïve control ( figure d -e). approximately one half of neutrophils in iv lps-injured lungs contained liposomes. dbco-igg liposomes were also highly specific for neutrophils in inflamed lungs, with ~ % of liposome-positive cells in the injured lungs being neutrophils (supplementary table ). the remaining dbco-igg liposome uptake in the lungs was accounted for by other cd -positive cells (supplementary figure c -e). . % of liposome uptake colocalized with cd -positive cells in lps-injured lungs and . % of liposome uptake in the naïve lungs was associated with cd -positive cells. accordingly, less than % of liposome uptake was associated with endothelial cells (supplementary figure f -g). dbco( x)-igg itself did not have specificity for inflamed lungs (supplementary figure ). uptake of dbco( x)-igg in naïve and injured lungs was statistically identical and the biodistribution of the modified igg resembled published results with unmodified igg. these results verify that dbco-igg modifies the structure of immunoliposomes, but does not function as a standard affinity tag by acting as a surface motif with intrinsic affinity for neutrophils. indeed, cd spectroscopic and ansa structural characterization of dbcomodified igg and dbco-igg liposomes resembled results obtained for ldngs and crosslinked albumin nps. igg secondary structure, as assessed by cd spectroscopy, was unchanged by dbco modification (supplementary figure a) . deconvolution of cd spectra via neural network algorithm indicated identical structural compositions for dbco( x)-igg, dbco( x)-igg, dbco( x)-igg, dbco( . x)-igg, and unmodified igg, showing that igg was not denatured by conjugation to dbco. ansa was used to probe accessible hydrophobic domains on dbco( x)-igg and dbco( x)-igg liposomes (supplementary figure b) . ansa fluorescence indicated more hydrophobic domains available on dbco( x)-igg liposomes than on dbco( x)-igg itself, resembling results for lysozyme and ldngs. therefore, addition of a hydrophobic moiety to protein on the surface of liposomes led to uptake of the liposomes in pulmonary marginated neutrophils after inflammatory insult. this result indicates that hydrophobic interactions between proteins on the surface of functionalized liposomes, like the protein interactions in naps, predict liposome tropism for marginated neutrophils in inflamed lungs. including nps from our four classes of protein-based nps, two non-protein nps (bare liposomes and polystyrene nps), and five types of igg-coated liposomes, we traced nanoparticles in naïve and inflamed mice. direct assessment of naïve-toinflamed shifts in lung uptake led us to identify naps with specificity for inflamed lungs. to verify this assessment and derive additional patterns in the broader data set, we undertook linear discriminant and principal components analyses of the biodistribution data for our nanoparticles, along with three isolated proteins. grouping the nanoparticles and three proteins according to the classes defined in figure and supplementary figures - , we completed a linear discriminant analysis of the naïve-to-inflamed shift for particle retention in the lungs, blood, liver, and spleen (supplementary figure a) . data for particle uptake in each organ was normalized by subtracting and then dividing by the mean uptake over all particles. the first two eigenvectors, dominated by splenic uptake and a combination of liver and lung uptake, respectively, accounted for % of variation in the data. the resulting projection of the data along the first two linear discriminant analysis eigenvectors was analyzed by k-means clustering to confirm the classes of nanoparticle with specificity for the inflamed lungs (supplementary figure b) . indeed, division of the data into two clusters supported the delineation of the nanoparticles with specificity for inflamed lungs. naps, nanoparticles based on protein gelation, crosslinking, and charge association, all aligned in one cluster. as an exception, dbco( x)-igg liposomes were considered as a unique class of particle and the linear discriminant analysis indicated that the inflammation-specific liposomes had in vivo behavior resembling that of ldngs or poni-gfp nanoparticles. this analysis of the liposome biodistributions supports the classification of dbco( x)-igg liposomes as naps. igg-coated polystyrene nanoparticles and dbco( x)-igg liposomes were part of the k-means cluster without inflammation specificity, but data for these two particles resided close to the voronoi boundary distinguishing the two clusters. principal component analysis comparing normalized nanoparticle uptake in inflamed lungs to normalized retention in liver, spleen, and blood provided a reductive metric to compare the distinct in vivo behavior of nanoparticles in the classes identified by linear discriminant analysis. most variation in the biodistribution data was accounted for by an eigenvector closely aligned to variation in pulmonary uptake (supplementary figure a) . data was projected along that first eigenvector and magnitude of the projection was determined for each nanoparticle (supplementary figure b) . first eigenvector projection values were then grouped according to the classes examined above via linear discriminant analysis. only the classes in the inflammation-specific kmeans cluster had positive average first eigenvector projections. all other particle classes had average first eigenvector projections indistinguishable from isolated protein (supplementary figure b) . principal component and linear discriminant analyses of our compiled biodistributions confirmed; a) identification of naps as nanoparticles with distinct tropism for inflamed lungs and; b) alignment of dbco( x)-igg liposome in vivo behavior with that of other naps. computerized tomography (ct) imaging is a standard diagnostic tool for ards. ct images can identify the presence of edematous fluid in the lungs, but ct cannot distinguish between the two major types of pulmonary edema: non-inflammatory cardiogenic pulmonary edema (cpe) and ards-associated edema. we sought to use naps to distinguish inflammatory lung injury from cpe in diagnostic imaging experiments. we induced cpe in mice via prolonged iv propranolol infusion. edema was confirmed via ct imaging of inflated lungs ex vivo and in situ. three-dimensional reconstructions of chest ct images were partitioned to distinguish airspace and lowdensity tissue, as in normal lungs (white, yellow, and light orange signal in figure a ), from high-density tissue and edema (red and black/transparent signal in figure a ). quantification of ct attenuation and gaps in the reconstructed three-dimensional lung images indicated profuse edema in lungs afflicted with model cardiogenic pulmonary edema ( figure a nm ldngs were traced in mice with induced cardiogenic pulmonary edema. ldngs accumulated in the edematous lungs at . %id/g concentration, statistically indistinguishable from lung uptake in naïve mice and an order of magnitude lower than the level of lung uptake in mice treated with iv lps ( figure c ). naïve and iv lps-injured mice were dosed with ldngs labeled with in via chelate conjugation to lysozyme. in uptake in naïve and lps-injured lungs was visualized with ex vivo spect-ct imaging to indicate capacity of ldngs for imagingbased diagnosis of inflammatory lung injury ( figure d ). in signa was colocalized with anatomical ct images for reconstructions in figure d . in spect signal was detectable in lps-injured lungs, but in spect signal was at background level in naïve lungs (supplementary movies and ). reduced spect signal in the liver of lps-injured mice, in agreement with biodistribution data, was also evident in coregistration of spect imaging with full body skeletal ct imaging (supplementary movies and ). therefore, naps with tropism for marginated neutrophils have the ability to detect and assess ards-like inflammation via spect-ct imaging. since those same naps do not accumulate in lungs afflicted with cpe, naps have potential for differential diagnosis of acute lung inflammation against cpe. in recent work, we demonstrated that human donor lungs rejected for transplant due to ards-like phenotypes can be perfused with nanoparticle solutions. these perfusion experiments evaluate the tendency of nanoparticles to distribute to human lungs ex vivo. we used this perfusion method to evaluate nap retention in inflamed human lungs. first, fluorescent ldngs were added to single cell suspensions prepared from human lungs. µg, µg, or µg of ldngs were incubated with x cells in suspension for hour at room temperature. after three washes to remove unbound ldngs, cells were stained for cd and analyzed with flow cytometry ( figure a -b). the majority of ldng uptake in the single cell suspensions was attributable to cd positive cells. ldngs accumulated in the human leukocytes, extracted from inflamed lungs, in a dose-dependent manner, with . % of leukocytes containing ldngs at a loading dose of µg. therefore, our prototype nap was retained in leukocytes from human lungs. to test ldng tropism for inflamed intact human lungs, fluorescent or i-labeled ldngs were infused via arterial catheter into ex vivo human lungs excluded from transplant. immediately prior to ldng administration, tissue dye was infused via the same arterial catheter to stain regions of the lungs directly perfused by the catheterized branch of the pulmonary artery ( figure c ). after infusion of ldngs, phosphate buffered saline infusion was used to rinse away unbound particles. perfused regions of the lungs were dissected and divided into ~ g segments, then sorted into regions deemed to have high, medium, or low levels of tissue dye staining. for lungs receiving fluorescent ldngs, well-perfused and poorly-perfused regions were selected for sectioning and fluorescent imaging. fluorescent signal from ldngs was clearly detectable in sections of well-perfused tissue, but not poorly-perfused tissue ( figure d ). in experiments with i-labeled ldngs, i-labeled ferritin was concurrently infused (i.e. a mix of ferritin and ldngs was infused) as an internal control particle shown to have no tropism for injured mouse lungs. with ldngs and ferritin infused into the same lungs via the same branch of the pulmonary artery, ldngs retained in the lungs at . % initial dose and ferritin retained at . % initial dose ( figure e ). ldng accumulation in human lungs was focused in regions of the lungs with high levels of perfusion stain, with concentrations of . %id/g in the "high" perfusion regions, compared to . %id/g in the "medium" perfusion regions. ferritin accumulation was more diffuse, with . %id/g in the "high" perfusion regions, compared to . %id/g in the "medium" perfusion regions (supplementary figure ) . ldngs, a prototype nap shown to home to neutrophils in acutely inflamed mouse lungs, specifically accumulated in perfused regions of inflamed human lungs, but ferritin nanocages, a particle with no tropism for neutrophils, concentrated at much lower levels in injured human lungs. our data thus indicate that nap tropism for neutrophils in inflamed mouse lungs may be recapitulated in human lungs. previous studies indicate that nanoparticles can interfere with neutrophil adhesion in inflamed vasculature. we designed studies to evaluate whether or not naps mitigate the neutrophil-mediated effects of lung inflammation. namely, we administered ldngs, dbco( x)-igg liposomes, or bare liposomes in mice subjected to model ards and determined whether or not the nanoparticles prevented lung edema induced by inflammation. mice were treated with nebulized lps as a high-throughput model for severe ards. to evaluate physiological effects of the model injury, bronchoalveolar lavage (bal) fluid was harvested from mice at hours after exposure to lps. in three separate experiments, nebulized lps induced elevated concentrations of neutrophils, cd -positive cells, and protein in the bal fluid. in naïve mice, cd -positive cells concentrated at . x cells per ml bal and neutrophils concentrated at . x cells per ml bal. after lps injury, cd -positive cells and neutrophils concentrated at . x and . x cells per ml bal, respectively. in naïve mice, protein concentrated in the bal fluid at . mg/ml and in lps-injured mice, protein concentrated in the bal at . mg/ml ( figure , white and grey bars). vascular disruption after nebulized lps treatment thus led to accumulation of protein-rich edema in the alveolar space. dbco( x)-igg liposomes, ldngs, and bare liposomes were compared for effects on vascular permeability in model ards. nps were administered as an iv bolus ( mg per kg body weight) two hours after nebulized lps administration. as in untreated mice, bal fluid was harvested and analyzed at hours after exposure to nebulized lps. bare liposomes or ldngs did not have significant effects on vascular injury induced by nebulized lps, as measured by either leukocyte or protein concentration in bal fluid ( figure , red and green bars). dbco( x)-igg liposomes, however, had a significant salient effect on both protein leakage and cellular infiltration in the bal ( figure , brown bars). with dbco( x)-igg liposomes administered two hours after nebulized lps, cd -positive cells and neutrophils in bal were reduced to concentrations of . x and . x cells per ml, respectively. protein concentration in the bal was reduced to . mg/ml by dbco( x)-igg liposome treatment. as measured by protection against cellular or protein leakage, relative to untreated mice, dbco( x)-igg liposomes provided . % protection against leukocyte leakage, . % protection against neutrophil leakage, and . % protection against protein leakage. dbco( x)-igg liposomes, without any drug, altered the course of inflammatory lung injury to limit protein and leukocyte edema in the alveoli. our results with dbco( x)-igg liposomes indicate that some naps can interfere with neutrophil extravasation into the alveoli and thus limit edema following inflammatory injury. however, our results with ldngs show that tropism for marginated neutrophils is not alone sufficient to limit the neutrophil-mediated effects of inflammatory lung injury. neutrophils concentrate in the pulmonary vasculature during either systemic or pulmonary inflammation. , , , , these marginated neutrophils can recognize and engulf bacteria. , , therefore, neutrophils surveil the vasculature for potentially pathogenic foreign species, with the pulmonary vasculature serving as a "surveillance hub" in the case of systemic or pulmonary infection and inflammation. , , , our results with e. coli are noteworthy in this context: when e. coli are stripped of functional properties by heat treatment, oxidation, and fixation, but maintain their structure, uptake of the bacteria in the lungs only occurs after systemically prompting neutrophils with an inflammatory signal, lps. inflammation thus leads to pulmonary uptake of the e. colishaped particles. in large part, the overall outcome of this study is an accounting of nanoparticle structural properties that lead to recognition by "surveilling" neutrophils in the inflamed lungs, analogously to e. coli recognition by pulmonary neutrophils. including different liposomal formulations, nanoparticles were screened in our biodistribution studies comparing pulmonary nanoparticle uptake in naïve and lps-inflamed mice. thirteen different nanoparticles exhibited specificity for inflamed lungs over naïve lungs, with flow cytometry data indicating that at least three of those nanoparticle species specifically and avidly gather in neutrophils. the thirteen nanoparticles with specificity for the inflamed lungs have a range of properties. seven different proteins were used in the inflammation-specific particles. the particles have sizes ranging from ~ nm to ~ nm, include both spheres and rods, and have a range of zeta potentials. however, our analyses classify the inflammation-specific nanoparticles as; ) nanoparticles with structure based on hydrophobic interactions between proteins; ) nanoparticles with structure based on non-site-specific protein crosslinking; ) nanoparticles based on charge interactions between proteins. put broadly, these three classes can all be grouped as structures based on protein agglutination, without regard for site-specific interactions or symmetry in the resulting protein superstructure. we define the term nanoparticles with agglutinated proteins (naps) to indicate that particles with tropism for pulmonary marginated neutrophils during inflammation share commonalities in supramolecular organization. we identify naps as a broad class, rather than a single particle type. accordingly, we have presented diverse nap designs, implying a diversity of potential nap-based strategies for targeted treatment and diagnosis of ards and other inflammatory disorders in which marginated neutrophils play a role (e.g. local infections or thrombotic disorders). , , , , the diversity of naps will allow versatile options for engineering neutrophil-specific drug delivery strategies to accommodate different pathologies. in contrast to naps, three particles (adenovirus, aavs, and ferritin) characterized by highly symmetric arrangement of protein subunits into a protein superstructure [ ] [ ] [ ] did not accumulate in the inflamed neutrophil-rich lungs. these three particles have evolved structures that lead to prolonged circulation or evasion of innate immunity in mammals. [ ] [ ] [ ] [ ] it is conceivable that neutrophils more effectively recognize less patterned and more variable protein arrangements that may better parallel the wide variety of structures presented by the staggering diversity of microbes against which neutrophils defend. , to support our conclusions regarding supramolecular organization and neutrophil tropism, we re-engineered liposomes, particles with no intrinsic neutrophil tropism, to behave like naps. protein arrangement on the surface of dbco-igg liposomes was predicted to recapitulate protein agglutination seen in naps based on hydrophobic interactions. introduction of dbco to igg entails conjugation of a highly hydrophobic moiety to hydrophilic residues on the igg. replacing dbco with the less hydrophobic modifying group used in sata-maleimide conjugation abrogates the inflammation specificity observed with dbco-igg liposomes. likewise, titrating down the amount of dbco on the igg, thus limiting the hydrophobic groups on the protein, also ratchets down the targeting behavior of the dbco-igg liposomes. our data therefore points towards hydrophobic interactions between proteins on the liposome surface being a determinant in liposome uptake in neutrophils in the inflamed lungs. essentially, the dbco-igg liposomes may reproduce the hydrophobic interaction structural motif seen in naps produced by protein gelation (i.e. ldngs). nap-liposomes may be particularly attractive for future clinical translatability. liposomes are prominent among fda-approved nanoparticle drug carriers. further, even without cargo drugs, nap-liposomes conferred significant therapeutic effects in a mouse model of severe ards. ldngs, despite high levels of uptake in inflamed lungs, did not have the same therapeutic effect as the nap-liposomes. this result suggests that the composition of the liposomes may be important for their therapeutic effect. among possible mechanisms for the therapeutic effect, we note that lipid rafts are major signaling hubs in neutrophils. , the lipid content of the nap-liposomes (particularly the cholesterol content) may modulate neutrophil lipid rafts dependent on cholesterol. we have also observed that neutrophil content in the inflamed alveoli is markedly reduced by nap-liposomes. in this context, we note published work demonstrating that certain nanoparticles, in a still undetermined manner dependent on particle composition, can drive redistribution of neutrophils from the lungs to the liver. as a major corollary, our findings indicate many protein-based or proteinincorporating nanoparticles developed for therapeutic applications may accumulate in inflamed lungs, even when those nanoparticles were designed to accumulate elsewhere. the variety of protein nanostructures accumulating in inflamed lungs in our data includes particles that have been investigated as targeted drug delivery vehicles where marginated neutrophils are not the intended site of accumulation. , , , , the patterns in our data indicate that future studies may reveal additional nanoparticles that accumulate in the lungs following inflammatory insult. this study therefore serves as evidence that inflammatory challenges may prompt profound off-target changes in the biodistributions of nanomaterials, including dramatic shunting of nanoparticles and any associated drug payload to the lungs. the nanoparticle targeting profiles documented in naïve or, for instance, tumor model studies may be overturned by, for instance, bacterial infection in a patient receiving the nanoparticle. in conclusion, supramolecular organization in nanoparticle structure predicts nanoparticle uptake in pulmonary marginated neutrophils during acute inflammation. specifically, nanoparticles with agglutinated protein (naps) accumulate in marginated neutrophils, while nanoparticles with more symmetric protein organization do not. nap tropism for neutrophils allowed us to develop naps as diagnostics and therapeutics for ards, and even to demonstrate nap uptake in inflamed human lungs. future work may more deeply explore therapeutic effects of naps in ards and other diseases in which neutrophils play key roles. this study also obviates future testing of supramolecular organization as a variable in in vivo behavior of nanoparticles, including screens of tropism for other pathologies and cell types. these studies could in turn guide engineering of new particles with intrinsic cell tropisms, as with our engineering of nap-liposomes with neutrophil tropism. these "targeting" behaviors, requiring no affinity moieties, may apply to a wide variety of nanomaterials. but our current findings with neutrophil-tropic naps indicate that many protein-based and protein-coated nanoparticles could be untapped resources for treatment and diagnosis of devastating inflammatory disorders like ards. lysozyme-dextran nanogels (ldngs) were synthesized as previously described. , kda rhodamine-dextran or fitc-dextran (sigma) and lysozyme from hen egg white (sigma) were dissolved in deionized and filtered water at a : or : mol:mol ratio, and ph was adjusted to . before lyophilizing the solution. for maillard reaction between lysozyme and dextran, the lyophilized product was heated for hours at °c, with % humidity maintained via saturated kbr solution in the heating vessel. dextran-lysozyme conjugates were dissolved in deionized and filtered water to a concentration of mg/ml, and ph was adjusted to . or . . solutions were stirred at °c for minutes. diameter of ldngs was evaluated with dynamic light scattering (dls, malvern) after heat gelation. particle suspensions were stored at °c. crosslinked protein nanoparticles and nanorods were prepared using previously reported electrohydrodynamic jetting techniques. the protein nanoparticles were prepared using bovine serum albumin, human serum albumin, human lysozyme, human transferrin, or human hemoglobin (all proteins were purchased from sigma). protein nanorods were prepared using chemically modified human serum albumin. for electrohydrodynamic jetting, protein solutions were prepared by dissolving the protein of interest at a . w/v% (or . w/v% for protein nanorods) concentration in a solvent mixture of di water and ethylene glycol with : (v/v) ratio. the homobifunctional amine-reactive crosslinker, o,o′-bis[ -(n-succinimidylsuccinylamino)ethyl]polyethylene glycol with molecular weight of kda (nhs-peg-nhs, sigma) was mixed with the protein solution at w/w%. protein nanoparticles were kept at °c for days for completion of the crosslinking reaction. the as-prepared protein nanoparticles were collected in pbs buffer and their size distribution was analyzed using dynamic light scatting (dls, malvern). glutamic acid residues (e -tag) were inserted at the c-terminus of enhanced green fluorescent protein (egfp) through restriction cloning and site-directed mutagenesis as previously reported. proteins were expressed in an e. coli bl strain using standard protein expression protocol. briefly, protein expression was carried out in xyt media with an induction condition of mm iptg and °c for h. at this point, the cells were harvested, and the pellets were lysed using % triton-x- ( min, °c)/dnase-i treatment ( minutes). proteins were purified using hispur cobalt columns. after elution, proteins were preserved in pbs buffer. the purity of native proteins was determined using % sds−page gel. polymers (poni) were synthesized by ring-opening metathesis polymerization using third generation grubbs' catalyst as previously described. in brief, solutions in dichloromethane of guanidium functionalized monomer and grubbs' catalyst were placed under freeze thawing cycles for degassing. after warming the solutions to room temperature, the degassed monomer solutions were administrated to degassed catalyst solutions and allowed to stir for minutes. the polymerization reaction was terminated by the addition of excess ethyl vinyl ether. the reaction mixture was further stirred for another min. the resultant polymers were precipitated from excess hexane or diethyl ether anhydrous, filtered, washed and dried under vacuum to yield a light-yellow powder. polymers were characterized by h nmr and gel permeation chromatography (gpc) to assess chemical compositions and molecular weight distributions, respectively. subsequent to deprotection of boc functionalities, polymer was dissolved in the dcm with the addition of tfa at : ratio. the reaction was allowed to stir for hours and dried under vacuum. excess tfa was removed by azeotropic distillation with methanol. afterwards, the resultant polymers were re-dissolved in dcm and precipitated in anhydrous diethyl ether, filtered, washed and dried. polymers were then dissolved in water and transferred to biotech ce dialysis tubing membranes with a g/mol cutoff and dialyzed against ro water ( − days). the polymers were then lyophilized dried to yield a light white powder. poni polymer/e-tag protein nanocomposites (ppncs) were prepared in polypropylene microcentrifuge tubes (fisher) through a simple mixing procedure. . nmol of kda poni was incubated with . nmol of egfp at room temperature for minutes prior to dilution to µl in sterile pbs and subsequent injection. similarly, . nmol of arginine-tagged gold nanoparticles, prepared as described, were combined with . nmol of egfp to prepare egfp/gold nanoparticle complexes. azide-functionalized liposomes were prepared by thin film hydration techniques, as previously described. the lipid film was composed of mol% dppc ( , dipalmitoyl-sn-glycero- -phosphocholine), mol% cholesterol, and mol% azide-peg -dspe (all lipids from avanti). . mol% top fluor pc ( -palmitoyl- -(dipyrrometheneboron difluoride) undecanoyl-sn-glycero- -phosphocholine) was added to prepare fluorescent liposomes. . mol% dtpa-pe ( , -distearoyl-sn-glycero- phosphoethanolamine-n-diethylenetriaminepentaacetic acid) was added to prepare liposomes with capacity for radiolabeling with in. lipid solutions in chloroform, at a total lipid concentration of mm, were dried under nitrogen gas, then lyophilized for hours to remove residual solvent. dried lipid films were hydrated with dulbecco's phosphate buffered saline (pbs). lipid suspensions were passed through freezethaw cycles using liquid n / °c water bath then extruded through nm cutoff tracketched polycarbonate filters in cycles. dls assessed particle size after extrusion and after each subsequent particle modification. liposome concentration following extrusion was assessed with nanosight nanoparticle tracking analysis (malvern). for conjugation to liposomes, rat igg was modified with dibenzylcyclooctyne-peg -nhs ester (dbco, jena bioscience). igg solutions (pbs) were adjusted to ph . with m nahco buffer and reacted with dbco for hour at room temperature at molar ratios of . : , : , : , or : dbco:igg. unreacted dbco was removed after reaction via centrifugal filtration against kda cutoff filters (amicon [def] . dbcomodified igg was incubated with azide liposomes at igg per liposome overnight at room temperature. unreacted antibody was removed via size exclusion chromatography, and purified liposomes were concentrated to original volume against centrifugal filters (amicon). maleimide liposomes were also prepared via lipid film hydration. lipid films comprised % dppc, % cholesterol, and % mpb-pe ( , -dioleoyl-sn-glycero- phosphoethanolamine-n-[ -(p-maleimidophenyl) butyramide]), with lipids prepared, dried, resuspended, and extruded as described above for azide liposomes. igg was prepared for conjugation to maleimide liposomes by one-hour reaction of sata (n-succinimidyl s-acetylthioacetate) per igg at room temperature in . mm edta in pbs. unreacted sata was removed from igg by passage through kda cutoff gel filtration columns. sata-conjugated igg was deprotected by one-hour room temperature incubation in . m hydroxylamine in . mm edta in pbs. excess hydroxylamine was removed and buffer was exchanged for . mm edta in pbs via kda cutoff gel filtration column. sata-conjugated and deprotected igg was added to liposomes at igg per liposomes for overnight reaction at °c. excess igg was removed by size exclusion column purification, as above for azide liposomes. nm carboxylate nanoparticles (phosphorex) were exchanged into mm mes buffer at ph . via gel filtration column. n-hydroxysulfosuccinimide (sulfo-nhs) was added to the particles at . mg/ml, prior to incubation for minutes at room temperature. edci was then added to the particles at . mg/ml, prior to incubation for minutes at room temperature. igg was added to the particle mixture at igg per nanoparticle, prior to incubation for hours at room temperature while vortexing. for radiotracing, i-labeled igg was added to the reaction at % of total igg mass. the igg/particle mixture was diluted with -fold volume excess of ph . mes buffer and the diluted mixture was centrifuged at xg for minutes. supernatant was discarded and pbs with . % bsa was added at desired volume before resuspending the particles via sonication probe sonication (three pulses, % amplitude). particle size was assessed via dls after resuspension, and particles were used immediately after dls assessment. top e. coli were grown overnight in terrific broth with ampicillin. bacteria were heat-inactivated by -minute incubation at °c, then fixed by overnight incubation in % paraformaldehyde. after fixation, bacteria were pelleted by centrifugation at xg for minutes. pelleted bacteria were washed three times in pbs, prior to resuspension by pipetting. bacterial concentration was verified by optical density at nm, prior to radiolabeling as described for nanoparticles below. bacteria were administered in mice ( . x colony forming units in a µl suspension per mouse). protein, horse spleen ferritin nanocages (sigma), or adeno-associated virus (empty capsids, serotype ) were prepared in pbs at concentrations between and mg/ml in volumes between and µl. films of oxidizing agent were prepared in borosilicate tubes by drying µl of . mg/ml iodogen (perkin-elmer, chloroform solution) under nitrogen gas. alternatively, iodobeads (perkin-elmer) were added to borosilicate tubes (one per reaction). protein solutions were added to coated or beadcontaining tubes, before addition of na / i at µci per µg of protein. protein was incubated with radioiodine at room temperature for minutes under parafilm in a ventilated hood. iodide-protein reacottions were terminated by purifying protein solutions through a kda cutoff gel column (zeba). additional passages through gel filtration columns or against centrifugal filters (amicon, kda cutoff) were employed to remove free iodine, assuring that > % of radioactivity was associated with protein. lysozyme-dextran nanogels, crosslinked protein nanoparticles, e. coli, or adenovirus were similarly iodinated. at least µl of particle suspension was added to a borosilicate tube containing two iodobeads, prior to addition of µci of na i per µl of suspension. particles were incubated with radioiodine and iodobeads for minutes at room temperature, with gentle shaking every minutes. to remove free iodine, particle suspensions were moved to a centrifuge tube, diluted in ~ ml of buffer and centrifuged to pellet the particles ( xg/ minutes for nanogels, xg/ minutes for crosslinked protein particles, xg/ minutes for adenovirus, and xg/ minutes for e. coli). supernatant was removed and wash/centrifugation cycles were repeated to assure > % of radioactivity was associated with particles. particles were resuspended by probe sonication (three pulses, % amplitude) for nanogels or crosslinked protein nanoparticles or pipetting for adenovirus or e. coli. nanoparticle labeling with in in labeling of nanoparticles followed previously described methods, with adaptation for new particles. all radiolabeling chelation reactions were performed using metal free conditions to prevent contaminating metals from interfering with chelation of in by dtpa or dota. metals were removed from buffers using chelex metal affinity resin (biorad, laboratories, hercules ca). lysozyme-dextran nanogels were prepared for chelation to in by conjugation to s- -( -isothiocyanatobenzyl)- , , , -tetraazacyclododecane tetraacetic acid (p-scn-bn-dota, macrocyclics). nanogels were moved to metal free ph . m nahco buffer by three-fold centrifugation ( xg for minutes) and pellet washing with metal free buffer. p-scn-bn-dota was added to nanogels at : mass:mass ratio, prior to reaction for minutes at room temperature. free p-scn-bn-dota was removed by three-fold centrifugal filtration against kda cutoff centrifugal filters, with resuspension of nanogels in metal-free ph citrate buffer after each centrifugation. dota-conjugated nanogels or dtpa-containing liposomes in ph citrate buffer were combined with incl for one-hour chelation at °c. nanoparticle/ incl mixtures were treated with free dtpa ( mm final concentration) to remove in not incorporated in nanoparticles. efficiency of in incorporation in nanoparticles was assessed by thin film chromatography (aluminum/silica strips, sigma) with µm edta mobile phase. chromatography strips were divided between origin and mobile front and the two portions of the strip were analyzed in a gamma counter to assess nanoparticleassociated (origin) vs. free (mobile front) in. free in was separated from nanoparticles by centrifugal filtration and nanoparticles were resuspended in pbs (liposomes) or saline (nanogels). for spect/ct imaging experiments (see spect/ct imaging methods below) with nanogels, µci of in-labeled nanogels, used within one day in labeling as described above, were administered to each mouse. for tracing in-labeled liposomes in biodistribution studies, liposomes were labeled with µci in per µmol of lipid. nanoparticle or protein biodistributions were tested by injecting radiolabeled nanoparticles or protein (suspended to µl in pbs or . % saline at a dose of . mg/kg with tracer quantities of radiolabeled material) in c bl/ male mice from jackson laboratories. biodistributions in naïve mice were compared to biodistributions in several injury models. biodistribution data were collected at minutes after nanoparticle or protein injection, unless otherwise stated, as in pharmacokinetics studies. briefly, blood was collected by vena cava draw and mice were sacrificed via terminal exsanguination and cervical dislocation. organs were harvested and rinsed in saline, and blood and organs were examined for nanoparticle or protein retention in a gamma counter (perkin-elmer). nanoparticle or protein retention in harvested organs was compared to measured radioactivity in injected doses. for calculations of nanoparticle or protein concentration in organs, quantity of retained radioactivity was normalized to organ weights. mice subject to intravenous lps injury were anesthetized with % isoflurane before administration of lps from e. coli strain b at mg/kg in µl pbs via retroorbital injection. after five hours, mice were anesthetized with ketamine-xylazine ( mg/kg ketamine, mg/kg xylazine, intramuscular administration) and administered radiolabeled nanoparticles or protein via jugular vein injection to determine biodistributions as described above. for mice subject to intratracheal (it) lps injury, b lps was administered to mice (anesthetized with ketamine/xylazine) at mg/kg in µl of pbs via tracheal catheter, followed by µl of air. biodistributions of lysozyme-dextran nanogels in it-lps-injured mice were assessed as above hours after lps administration. biodistributions of liposomes in it-lps-injured mice were assessed at , , or hours after lps administration. mice subject to footpad lps administration were provided b lps at mg/kg in µl pbs via footpad injection. biodistributions of lysozyme-dextran nanogels were obtained at or hours after footpad lps administration. lysozyme-dextran nanogel biodistributions were also traced in a mouse model of cardiogenic pulmonary edema. to establish edema, mice were anesthetized with ketamine/xylazine and administered propranolol in saline ( µg/ml) via jugular vein catheter at µl/min over minutes. lysozyme-dextran nanogel biodistributions were subsequently assessed as above. single cell suspensions were prepared from lungs for flow cytometric analysis of cell type composition of the lungs and/or nanoparticle distribution among different cell types in the lungs. c bl/ male mice were anesthetized with ketamine/xylazine ( mg/kg ketamine, mg/kg xylazine, intramuscular administration) prior to installation of tracheal catheter secured by suture. after sacrifice by terminal exsanguination via the vena cava, lungs were perfused by right ventricle injection of ~ ml of cold pbs. the lungs were then infused via the tracheal catheter with ml of a digestive enzyme solution consisting of u/ml dispase, . mg/ml collagenase type i, and mg/ml of dnase i in cold pbs. immediately after infusion, the trachea was sutured shut while removing the tracheal catheter. the lungs with intact trachea were removed via thoracotomy and kept on ice prior to manual disaggregation. disaggregated lung tissue was aspirated in ml of digestive enzyme solution and incubated at °c for minutes, with vortexing every minutes. after addition of ml of fetal calf serum, tissue suspensions were strained through µm filters and centrifuged at xg for minutes. after removal of supernatant, the pelleted material was resuspended in ml of cold ack lysing buffer. the resulting suspensions were strained through µm filter and incubated for minutes on ice. the suspensions were centrifuged at xg for minutes and the resulting pellets were rinsed in ml of facs buffer ( % fetal calf serum and mm edta in pbs). after centrifugation at xg for minutes, the rinsed cell pellets were resuspended in % pfa in ml facs buffer for minutes incubation. the fixed cell suspensions were centrifuged at xg for minutes and resuspended in ml of facs buffer. for analysis of intravascular leukocyte populations in naïve and inflamed lungs, mice received an intravenous injection of fitc-conjugated anti-cd antibody five minutes prior to sacrifice and preparation of single cell suspensions as described above. populations of intravascular vs. extravascular leukocytes were assessed by subsequent stain of fixed cell suspensions with percp-conjugated anti-cd antibody and/or apcconjugated clone a anti-ly g antibody. to accomplish staining of fixed cells, µl aliquots of the cell suspensions described above were pelleted at xg for minutes, then resuspended in labeled antibody diluted in facs buffer ( : dilution for apcconjugated anti-ly g antibody and : dilution for percp-conjugated anti-cd antibody). samples were incubated with staining antibodies for minutes at room temperature in the dark, diluted with ml of facs buffer, and pelleted at xg for minutes. stained pellets were resuspended in µl of facs buffer prior to immediate flow cytometric analysis on a bd accuri flow cytometer. all flow cytometry data was gated to remove debris and exclude doublets. control samples with no stain, obtained from naïve and iv-lps-injured mice, established gates for negative/positive staining with fitc, percp, and apc. single stain controls allowed automatic generation of compensation matrices in fcs express software. comparison of percp anti-cd signal with fitc anti-cd signal indicated intravascular vs. extravascular leukocytes. comparison of apc anti-ly g signal with fitc anti-cd signal indicated intravascular vs. extravascular neutrophils, with percp and apc co-staining verifying identification of cells as neutrophils. similar staining and analysis protocols enabled identification of nanoparticle distribution among different cell types in the lungs. to enable fluorescent tracing, lysozyme-dextran nanogels contained fitc-dextran, dbco-igg liposomes contained green fluorescent top fluor pc lipid, and crosslinked albumin nanoparticles were labeled with nhs ester alexa fluor . alexa fluor labeling of albumin nanoparticles was accomplished by incubation of the nhs ester fluorophore with nanoparticles at : mass:mass fluorophore:nanoparticle ratio for two hours on ice. excess fluorophore was removed from nanoparticles by -fold centrifugation at xg for minutes followed by washing with pbs. nanoparticles were administered at . mg/kg via jugular vein injection and circulated for minutes, prior to preparation of single cell suspensions from lungs as above. fixed single cell suspensions were stained with apc-conjugated anti-ly g or percp-conjugated anti-cd as above. additional suspensions were stained with : dilution of apc-conjugated anti-cd , in lieu of anti-ly g, to identify endothelial cells. association of nanoparticles with cell types was identified by coincidence of green fluorescent signal with anti-cd , anti-ly g, or anti-cd signal. as described previously, thirty minutes after injection of µci of in-labeled nanogels, anesthetized mice were sacrificed by cervical dislocation. mice were placed into a milabs u-spect (utrecht, netherlands) scanner bed. a region covering the entire body was scanned for min using listmode acquisition. the animal was then moved, while maintaining position, to a milabs u-ct (utrecht, netherlands) for a fullbody ct scan using default acquisition parameters ( µa, kvp, ms exposure, . ° step with projections). for naïve mice and mice imaged after cardiogenic pulmonary edema, ct data was acquired as above without spect data. the spect data was reconstructed using reconstruction software provided by the manufacturer, with µm voxels. the ct data were reconstructed using reconstruction software provided by the manufacturer, with µm voxels. spect and ct data, in nifti format, were opened with imagej software (fiji package). background signal was removed from spect images by thresholding limits determined by applying renyi entropic filtering, as implemented in imagej, to a spect image slice containing ngassociated in in the liver. background-subtracted pseudo-color spect images were overlayed on ct images and axial slices depicting lungs were selected for display, with ct thresholding set to emphasize negative contrast in the airspace of the lungs. imagej's built-in d modeling plugin was used to co-register background-subtracted pseudo-color spect images with ct images in three-dimensional reconstructions. ct image thresholding was set in the d modeling tool to depict skeletal structure alongside spect signal. for three-dimensional reconstructions of lung ct images, thresholding was set, as above, for contrast emphasizing the airspace of the lungs, with thresholding values standardized between different ct images (i.e. identical values were used for naïve and edematous lungs). images were cropped in a cylinder to exclude the airspace outside of the animal, then contrast was inverted, allowing airspace to register bright ct signal and denser tissue to register as dark background. three-dimensional reconstructions of the lung ct data, and co-registrations of spect data with lung ct data, were generated as above with imagej's d plugin applied to ct data cropped and partitioned for lung contrast. quantification of ct attenuation employed imagej's measurement tool iteratively over axial slices, with measurement fields of view manually set to contain lungs and exclude surrounding tissue. mice were exposed to nebulized lps in a 'whole-body' exposure chamber, with separate compartments for each mouse (mpc- aero; braintree scientific). to maintain adequate hydration, mice were injected with ml of sterile saline warmed to °c, intraperitoneally, immediately before exposure to lps. lps (l - mg, sigma aldrich) was reconstituted in pbs to mg/ml and stored at - °c until use. immediately before nebulization, lps was thawed and diluted to mg/ml with pbs. lps was aerosolized via a jet nebulizer connected to the exposure chamber (neb-med h, braintree scientific, inc.). ml of mg/ml lps was used induce the injury. nebulization was performed until all liquid was nebulized (~ minutes). liposomes or saline sham were administered via retro-orbital injections of µl of suspension ( mg/kg liposome dose) at hours after lps exposure. mice were anesthetized with % isoflurane to facilitate injections. bronchoalveolar lavage (bal) fluid was collected hours after lps exposure, as previously described. briefly, mice were anesthetized with ketamine-xylazine ( mg/kg ketamine, mg/kg xylazine, intramuscular administration). the trachea was isolated and a tracheostomy was performed with a -gauge catheter. the mice were euthanized via exsanguination. . ml of cold bal buffer ( . mm edta in pbs) was injected into the lungs over ~ min via the tracheostomy and then aspirated from the lungs over ~ min. injections/aspirations were performed three times for a total of . ml of fluid added to the lungs. recovery bal fluid typically amounted to ~ . ml. bal samples were centrifuged at xg for minutes. the supernatant was collected and stored at - °c for further analysis. protein concentration was measured using bio-rad dc protein assay, per manufacturer's instructions. the cell pellet was fixed for flow cytometry as follows. µl of . % pfa in pbs was added to each sample. samples were incubated in the dark at room temperature for minutes, then ml of bal buffer was added. samples were centrifuged at xg for min, the supernatant was aspirated, and ml of facs buffer ( % fetal calf serum and mm edta in pbs) was added. at this point, samples were stored at °c for up to week prior to flow cytometry analysis. to stain for flow cytometry, samples were centrifuged at xg for min, the supernatant was aspirated, and µl of staining buffer was added. staining buffer used was a : dilution of stock antibody solution (apc anti-mouse cd ; alexa fluor anti-mouse ly g, biolegend) into facs buffer. samples were incubated with staining antibody for minutes at room temperature in the dark. to terminate staining, ml of facs buffer was added, samples were centrifuged at xg for minutes, and supernatant was aspirated. cells were resuspended in µl of facs buffer and immediately analyzed via flow cytometry. flow cytometric analysis was completed with a bd accuri flow cytometer as follows: sample volume was set to µl and flow rate was set to 'fast'. unstained and single-stained controls were used to set gates. forward scatter (pulse area) vs. side scatter (pulse area) plots were used to gate out non-cellular debris. forward scatter (pulse area) vs. forward scatter (pulse height) plots were used to gate out doublets. the appropriate fluorescent channels were used to determine stained vs. unstained cells. the gates were placed using unstained control samples. single-stain controls were tested and showed there was no overlap/bleed-through between the fluorophores. final analysis indicated the quantity of leukocytes (cd -positive cells) and neutrophils (ly g-positive cells) in bal samples. human lungs were obtained after organ harvest from transplant donors whose lungs were in advance deemed unsuitable for transplantation. the lungs were harvested by the organ procurement team and kept at °c until the experiment, which was done within hours of organ harvest. the lungs were inflated with low pressure oxygen and oxygen flow was maintained at . l/min to maintain gentle inflation. pulmonary artery subsegmental branches were endovascularly cannulated, then tested for retrograde flow by perfusing for minutes with steen solution containing a small amount of green tissue dye at cm h o pressure. the pulmonary veins through which efflux of perfusate emerged were noted, allowing collection of solutions after passage through the lungs. a ml mixture of i-labeled lysozyme-dextran nanogels and ilabeled ferritin nanocages were injected through the arterial catheter. ~ ml of % bsa in pbs was passed through the same catheter to rinse unbound nanoparticles. a solution of green tissue dye was subsequently injected through the same catheter. the cannulated lung lobe was dissected into ~ g segments, which were evaluated for density of tissue dye staining. segments were weighed, divided into 'high', 'medium', 'low', and 'null' levels of dye staining, and measured for i and i signal in a gamma counter. for experiments with cell suspensions derived from human lungs (chosen for research use according to the above standards), single cell suspensions were generously provided by the laboratory of edward e. morrisey at the university of pennsylvania. aliquots of , cells were pelleted at xg for minutes and resuspended in µl pbs containing different quantities of lysozyme-dextran nanogels synthesized with fitc-labeled dextran. cells and nanogels were incubated at room temperature for minutes before two-fold pelleting at xg with ml pbs washes. cells were re-suspended in µl facs buffer for staining with apcconjugated anti-human cd , applied by -minute incubation with a : dilution of the antibody stock. cells were pelleted at xg for minutes and resuspended in µl pbs for immediate analysis with flow cytometry (bd accuri). negative/positive nanogel or anti-cd signal was established by comparison to unstained cells. singlestained controls indicated no spectral overlap between fitc-nanogel fluorescence and anti-cd apc fluorescence. proteins were prepared in deionized and filtered water at concentrations of . mg/ml for human albumin, . mg/ml for hen lysozyme, and . mg/ml for igg. crosslinked albumin nanoparticles, lysozyme-dextran nanogels, and igg-coated liposome suspensions were prepared such that albumin, lysozyme, and igg concentrations in the suspensions matched the concentrations of the corresponding protein solutions. protein and nanoparticle solutions were analyzed in quartz cuvettes with mm path length in an aviv circular dichroism spectrometer. the instrument was equilibrated in nitrogen at °c for minutes prior to use and samples were analyzed with sweeps between and nm in nm increments. each data point was obtained after a . s settling time, with a s averaging time. cdnn software deconvolved cd data (expressed in millidegrees) via neural network algorithm assessing alignment of spectra with library-determined spectra for helices, antiparallel sheets, parallel sheets, beta turns, and random coil. -anilino- -naphthalenesulfonic acid (ansa) at . mg/ml was mixed with lysozyme, human albumin, or igg at . mg/ml in pbs. for nanoparticle analysis, nanoparticle solutions were prepared such that albumin, lysozyme, and igg concentrations in the suspensions matched the . mg/ml concentration of the protein solutions. protein or nanoparticles and ansa were reacted at room temperature for minutes. excess ansa was removed from solutions by centrifugations against kda cutoff centrifugal filters (amicon). after resuspension to original volume, ansa-stained protein/nanoparticle solutions/suspension were examined for fluorescence (excitation nm, emission - nm) and absorbance ( - nm) maxima corresponding to ansa. for imaging neutrophil content in naïve and iv-lps-injured lungs, mice were intravenously injected with rat anti-mouse anti-ly g antibody (clone a ) and sacrificed minutes later. lungs were embedded in m medium, flash frozen, and sectioned in µm slices. sections were stained with percp-conjugated anti-rat secondary antibody and neutrophil-associated fluorescence was observed with epifluorescence microscopy. similar procedures enabled histological imaging of lysozyme-dextran nanogels in iv-lps-injured lungs. nanogels synthesized with rhodamine-dextran were administered intravenously in injured mice minutes prior to sacrifice. lungs were sectioned as above and stained with clone a anti-ly g antibody, followed by briliant violetconjugated anti-rat secondary antibody. sections of human lungs were obtained after ex vivo administration (see nanoparticle administration in human lungs above) of lysozyme-dextran nanogels synthesized with rhodamine dextran. regions of tissue delineated as perfused and nonperfused, as determined by arterial administration of tissue dye as above, were harvested, embedded in m medium, flash frozen, and sectioned in µm slices. epifluorescence imaging indicated rhodamine fluorescence from nanogels, coregistered to autofluorescence indicating tissue architecture. a mouse was anesthetized with ketamine/xylazine five hours after intravenous administration of mg/kg b lps. a jugular vein catheter was fixed in place for injection of lysozyme-dextran nanogels, anti-cd antibody, and fluorescent dextran during imaging. in preparation for exposure of the lungs, a patch of skin on the back of the mouse, around the juncture between the ribcage and the diaphragm, was denuded. while the mouse was maintained on mechanical ventilation, an incision at the juncture between the ribs and the diaphragm, towards the posterior, exposed a portion of the lungs. a coverslip affixed to a rubber o-ring was sealed to the incision by vacuum. the exposed portion of the mouse lung was placed in focus under the objective by locating autofluorescence signal in the "fitc" channel. with ms exposure, fluorescent images from channels corresponding to violet, green, near red, and far red fluorescence were sequentially acquired. a mixture of rhodamine-dextran nanogels ( . mg/kg), brilliant violet-conjugated anti-cd antibody ( . mg/kg), and alexa fluor labeled kda dextran ( mg/kg) for vascular contrast was administered via jugular vein catheter and images were recorded for minutes. images were recorded in slidebook software and opened in imagej (fiji distribution) for composition in movies with coregistration of the four fluorescent channels. all animal studies were carried out in strict accordance with guide for the care and use of laboratory animals as adopted by national institute of health and approved by university of pennsylvania institutional animal care and use committee (iacuc). male c bl/ j mice, - weeks old, were purchased from jackson laboratories. mice were maintained at - °c and on a / hour dark/light cycle with food and water ad libitum. ex vivo human lungs were donated from an organ procurement agency, gift of life, after determination the lungs were not suitable for transplantation into a recipient, and therefore would have been discarded if they were not used for our study. gift of life obtained the relevant permissions for research use of the discarded lungs, and in conjunction with the university of pennsylvania's institutional review board ensured that all relevant ethical standards were met. error bars indicate standard error of the mean throughout. significance was determined through paired t-test for comparison of two samples and anova for group comparisons. linear discriminant analysis and principal components analysis were completed in gnu octave scripts (adapted from https://www.bytefish.de/blog/pca_lda_with_gnu_octave/, and made available in full in the supplementary materials). findings in this study contributed to united states provisional patent application number / . raw imaging, flow cytometry, gamma counter, and spectroscopy data supporting the findings of this study are available from the corresponding author upon reasonable request. all other data supporting the findings of this study are available within the paper and its supplementary information files. covid- in critically ill patients in the seattle region -case series the influenza pandemic: insights for the st century lung safe investigators; esicm trials group. epidemiology, patterns of care, and mortality for patients with acute respiratory distress syndrome in intensive care units in countries incidence and outcomes of acute lung injury. b. engl nanomedicine for the treatment of acute respiratory distress syndrome. the ats bear cage award-winning proposal the mercurial nature of neutrophils: still an enigma in ards? endothelial nanomedicine for the treatment of pulmonary disease balti- study investigators. effect of intravenous β- agonist treatment on clinical outcomes in acute respiratory distress syndrome (balti- ): a multicentre, randomised controlled trial national heart, lung, and blood institute acute respiratory distress syndrome (ards) clinical trials network. randomized, placebo-controlled clinical trial of an aerosolized β -agonist for treatment of acute lung injury neutrophil function in inflammation and inflammatory diseases paradoxical roles of the neutrophil in sepsis: protective and deleterious targeting neutrophils in ischemic stroke: translational insights from experimental studies neutrophil function in ischemic heart disease contribution of neutrophils to acute lung injury neutrophils kinetics in health and disease neutrophil-endothelial cell interactions in the lung the multifaceted functions of neutrophils neutrophils in the activation and regulation of innate and adaptive immunity what drives neutrophils to the alveoli in ards? pulmonary retention of primed neutrophils: a novel protective host response, which is impaired in the acute respiratory distress syndrome neutrophil margination, sequestration, and emigration in the lungs of l-selectin-deficient mice ly family proteins in neutrophil biology use of ly g-specific monoclonal antibody to deplete neutrophils in mice neutrophil targeted nano-drug delivery system for chronic obstructive lung diseases therapeutic targeting of neutrophil granulocytes in inflammatory liver disease prevention of vascular inflammation by nanoparticle targeting of adherent neutrophils neutrophil-mediated delivery of therapeutic nanoparticles across blood vessel barrier for treatment of inflammation and infection non-affinity factors modulating vascular targeting of nano-and microcarriers physical approaches to biomaterial design impact of particle elasticity on particle-based drug delivery systems factors controlling the pharmacokinetics, biodistribution and intratumoral penetration of nanoparticles cell-mediated delivery of nanoparticles: taking advantage of circulatory cells to target nanoparticles neutrophil sequestration and migration in localized pulmonary inflammation. capillary localization and migration across the interalveolar septum neutrophil recruitment to the lungs during bacterial pneumonia the lung is a host defense niche for immediate neutrophilmediated vascular protection icam- targeted nanogels loaded with dexamethasone alleviate pulmonary inflammation flexible nanoparticles reach sterically obscured endothelial targets inaccessible to rigid nanoparticles long-circulating janus nanoparticles made by electrohydrodynamic co-jetting for systemic drug delivery applications the transport and inactivation kinetics of bacterial lipopolysaccharide influence its immunological potency in vivo quantitative analysis of protein far uv circular dichroism spectra by neural networks selective staining of proteins with hydrophobic surface sites on a native electrophoretic gel lysozyme-dextran core-shell nanogels prepared via a green process in vivo editing of macrophages through systemic delivery of crispr-cas -ribonucleoprotein-nanoparticle nanoassemblies adeno-associated virus structural biology as a tool in vector development structure of human adenovirus cisplatin encapsulation within a ferritin nanocage: a high-resolution crystallographic study vascular targeting of radiolabeled liposomes with bio-orthogonally conjugated ligands: single chain fragments provide higher specificity than antibodies targeting superoxide dismutase to endothelial caveolae profoundly alleviates inflammation caused by endotoxin acute respiratory distress syndrome: diagnosis and management novel role for cftr in fluid absorption from the distal airspaces of the lung red blood cell-hitchhiking boosts delivery of nanocarriers to chosen organs by orders of magnitude neutrophil-particle interactions in blood circulation drive particle clearance and alter neutrophil responses in acute inflammation the tlr -myd pathway is critical for adaptive immune responses to adeno-associated virus gene therapy vectors in mice adeno-associated viral vectors at the frontier between tolerance and immunity serum ferritin: past, present and future facile double-functionalization of designed ankyrin repeat proteins using click and thiol chemistries a new reagent which may be used to introduce sulfhydryl groups into proteins, and its use in the preparation of conjugates for immunoassay doxil®--the first fda-approved nano-drug: lessons learned lipid rafts regulate lipopolysaccharide-induced activation of cdc and inflammatory functions of the human neutrophil alterations in membrane cholesterol cause mobilization of lipid rafts from specific granules and prime human neutrophils for enhanced adherence-dependent oxidant production generation of targeted adenoassociated virus (aav) vectors for human gene therapy biphasic janus particles with nanoscale anisotropy direct cytosolic delivery of crispr/cas -ribonucleoprotein for efficient gene editing direct cytosolic delivery of proteins through coengineering of proteins and polymeric delivery vehicles antioxidant protection by pecam-targeted delivery of a novel nadph-oxidase inhibitor to the endothelium in vitro and in vivo red: anti-ly g stain. green: tissue autofluorescence. (f) biodistributions of heat-inactivated, fixed, and ilabeled e. coli in naïve (n= ) and iv-lps-injured (n= ) mice tissue autofluorescence). (k) single frame from real-time intravital imaging of ldng (red) uptake in leukocytes (green) in iv-lps-inflamed pulmonary vasculature (blue, alexa fluor -dextran) biodistributions in iv-lps-injured mice for azide-functionalized liposomes conjugated to igg loaded with . , , , and dbco molecules per igg (bars further to the right correspond to more dbco per igg). (d) mouse lungs flow cytometry data indicating ly g anti-neutrophil staining density vs. levels of dbco( x)-igg liposome uptake. (e) flow cytometry data verifying increased dbco( x)-igg liposome uptake in and specificity for neutrophils following lps insult (inset: verification of increased concentration of neutrophils in the lungs following lps key: cord- -yp ehpeg authors: zhang, dong; qi, bo-yang; zhu, wei- wei; huang, xiao; wang, xiao-zhi title: crocin alleviates lipopolysaccharide-induced acute respiratory distress syndrome by protecting against glycocalyx damage and suppressing inflammatory signaling pathways date: - - journal: inflamm res doi: . /s - - -z sha: doc_id: cord_uid: yp ehpeg objective: to explore the mechanisms of crocin against glycocalyx damage and inflammatory injury in lipopolysaccharide (lps)-induced acute respiratory distress syndrome (ards) mice and lps-stimulated human umbilical vein endothelial cells (huvecs). methods: mice were randomly divided into control, lps, and crocin + lps ( , , and mg/kg) groups. huvecs were separated into eight groups: control, crocin, matrix metalloproteinase inhibitor (mmp- inhib), cathepsin l inhibitor (ctl inhib), lps, mmp- inhib + lps, ctl inhib + lps, and crocin + lps. the potential cytotoxic effect of crocin on huvecs was mainly evaluated through methylthiazolyldiphenyl-tetrazolium bromide assay. histological changes were assessed via hemotoxylin and eosin staining. lung capillary permeability was detected on the basis of wet–dry ratio and through fluorescein isothiocyanate-albumin assay. then, protein levels were detected through western blot analysis, immunohistochemical staining, and immunofluorescence. results: this study showed that crocin can improve the pulmonary vascular permeability in mice with lps-induced ards and inhibit the inflammatory signaling pathways of high mobility group box, nuclear factor κb, and mitogen-activated protein kinase in vivo and in vitro. crocin also protected against the degradation of endothelial glycocalyx heparan sulfate and syndecan- by inhibiting the expressions of ctl, heparanase, and mmp- in vivo and in vitro. overall, this study revealed the protective effects of crocin on lps-induced ards and elaborated their underlying mechanism. conclusion: crocin alleviated lps-induced ards by protecting against glycocalyx damage and suppressing inflammatory signaling pathways. acute respiratory distress syndrome (ards) is a wellknown disease with high morbidity and mortality. the main features of ards are lung endothelial cell injury, severe inflammatory responses, neutrophil adhesion or infiltration, and interstitial edema [ ] . at present, ards pathogenesis is complicated with lps-mediated sepsis being a common etiology [ ] . endothelial glycocalyx and inflammatory responses are crucial for the pathogenesis of ards. the endothelial glycocalyx, a layer of negatively charged gel-like substance, exists on surfaces of pulmonary vascular endothelial cells. heparan sulfate (hs) and syndecan- (sdc- ) are highly abundant in the glycocalyx [ , ] . the endothelial glycocalyx plays an important role in albumin exudation formation, leukocyte adhesion, and anticoagulation [ ] [ ] [ ] [ ] . however, the glycocalyx undergoes dynamic changes and is susceptible to various factors. enzymes are a key factor in glycocalyx degradation. glasner et al. [ ] reported that heparanase (hpa) and cathepsin l were connected with hs shedding in dengue virus non-structural protein -induced endothelial hyperpermeability. reine et al. [ ] reported that matrix metalloproteinase (mmp- ) was related to the degradation and shedding of sdc- in glomerular endothelial cells. excessive inflammation plays an important role in the development of ards. neutrophils are among the first inflammatory responders against acute bacterial inflammation. however, sepsis can induce changes in the deformation ability of neutrophils in pulmonary capillary and their retention on the surfaces of endothelial cells, thereby increasing the capillary permeability of alveoli and inducing and promoting the production of inflammatory cytokines [ ] . high mobility group box (hmgb- ), nuclear factor κb (nf-κb), and mitogen-activated protein kinase (mapk) signaling pathways are important and common inflammatory signaling pathways in lung injury [ ] [ ] [ ] . activation of the inflammatory signaling pathways promotes the production and release of high levels of pro-and anti-inflammatory factors. imbalance in the pro-and anti-inflammatory internal environment of the body leads to damage to vascular cells, cell dysfunction, adhesion or infiltration of inflammatory cells, and hemodynamic changes. crocin is an ester glycoside extracted from saffron. as one of the main active constituents of saffron, crocin has many pharmacological effects, such as anti-oxidation [ ] , anti-arteriosclerosis [ ] , anti-apoptosis [ ] , and anti-inflammatory [ ] . however, no studies have reported the effects of crocin on endothelial glycocalyx, hmgb- , nf-κb, and mapk signaling pathways in lps-induced ards mice and lps-stimulated huvecs. this study conducted an in-depth analysis of the protective effects and mechanisms of crocin on lps-induced ards. overall, the results of this study may provide a pharmacological basis for crocin in ards treatment. crocin (purity assay of ≥ %) was purchased from shanghai yuanye biological technology co., ltd. methylthiazolyldiphenyl-tetrazolium bromide (mtt), lps, , -diamidino- -phenylindole dihydrochloride (dapi), fluorescein isothiocyanate (fitc)-albumin, and dimethyl-sulfoxide (dmso) were obtained from sigma-aldrich. rabbit monoclonal antibody to hmgb (ab ), nf-κb p (ab ), iκbα (ab ), p-iκbα (ab ), jnk (ab ), erk (ab ), p (ab ), p-erk (ab ), p-jnk (ab ), p-p (ab ), laminb (ab ), mmp- (ab ), hpa (ab ), sdc- (ab ), ly g (ab ), β-actin (ab ), gapdh (ab ), mmp- inhibitor (mmp- inhib, ab ), and fitc (ab ) were acquired from abcam trading company ltd. mouse polyclonal antibody to cathepsin l (ctl) was purchased from santa cruz biotechnology. mouse polyclonal antibody to hs was purchased from ams biotechnology (switzerland, ma). thrombomodulin/bdca- was obtained from r&d systems (usa). hrp-conjugated goat anti-rabbit igg, goat anti-rabbit igg/alexa fluor , and rabbit anti-goat igg/alexa fluor were purchased from beijing biosynthesis biotechnology co., ltd. cathepsin l inhibitor (ctl inhib, cas - - ) was obtained from santa cruz biotechnology. huvecs were purchased from cell line bank (shanghai, china). huvecs were cultured in complete culture medium (allcells, shanghai, china) in an incubator with a humidified atmosphere of % air and % co at °c. cells at - % confluence were used for all assays. mtt assay, also known as mtt colorimetric assay, is a method used to detect cell survival and growth. the potential cytotoxic effect of crocin on huvecs was mainly evaluated by mtt assay. the concentration range of - µmol/l was randomly selected. as shown in fig. f , the concentration of - µmol/l crocin showed slight cytotoxicity to the huvecs. thus, µmol/l was selected as the pretreatment concentration of huvecs. for pretreatment, huvecs were separated into eight groups: control, crocin, mmp- inhibitor (mmp- inhib), ctl inhibitor (ctl inhib), lps, mmp- inhib + lps, ctl inhib + lps, and crocin + lps. in the control group, huvecs were only cultured in the complete culture medium without intervention. in the crocin group, huvecs were cultured in the complete culture medium with µm concentration of crocin. in the mmp- inhib group, huvecs were cultured in the complete culture medium with µm concentration of mmp- inhibitor. in the ctl inhib group, huvecs were cultured in the complete culture medium with µm concentration of ctl inhibitor. in the lps group, huvecs were cultured in the complete culture medium with µg/ml concentration of lps. in the mmp- inhib + lps group, huvecs were pretreated with µm concentration of mmp- inhibitor for h before the lps ( µg/ml) stimulation for h. in the ctl inhib + lps group, huvecs were pretreated with µm concentration of ctl inhibitor for h before the lps ( µg/ml) stimulation for h. in the crocin + lps group, huvecs were pretreated with µm concentration of crocin for h before the lps ( µg/ml) stimulation for h. male mice (c bl/ , - weeks old, weighing - g) were purchased from jinan animal feed center (shandong, china). all mice were housed in plastic cages with ± °c and supplied with clean food and purified water. the use of mice accorded with the national institute of health guide for the care and use of laboratory animals. the experimental mice were assorted into control, lps, and crocin + lps ( , , and mg/kg) groups. the doses and administration form of crocin were derived from the pre-experiments and previous reports [ , ] . the lps group mice were injected intraperitoneally with the lps ( mg/kg) for h to induce ards [ ] . for the crocin + lps groups, crocin at , , or mg/kg was administered intraperitoneally for days prior to intraperitoneal injection of the lps for h. at the same time, the control group mice were treated intraperitoneally with the same volume of normal saline. after anesthetizing with % chloral hydrate, lungs were collected and used for experiments. to assess the effect of crocin on the huvec viability, huvecs were cultured and treated with different concentrations ( - µmol/l, µl /well) of crocin for h and followed by lps stimulation ( µg/ml, µl /well). after h, the mtt ( µl, mg/ml) was added and incubated for h. supernatant was removed, and the formation of formazan was resolved with µl/well of dmso. optical density was measured at nm on a micro-plate reader. intraperitoneal injection of % chloral hydrate was conducted to anesthetize mice. mouse lungs were taken out and immersed in % paraformaldehyde for days. these portions were processed and embedded in paraffin. the lung sections ( µm thickness) were used for hematoxylin and eosin (he) staining. the lung injury score criteria were in accordance with the method described by aeffner et al. [ ] . the characteristics of lung injury were alveolar congestion, hemorrhage, infiltration, aggregation of neutrophils in the airspace or vessel wall, and thickness of the alveolar wall. the lung paraffin sections ( µm thickness) were treated via acetone-benzol dewaxing. then, the lung sections were placed in a microwave box with a certain ph . citrate buffer for the antigen retrieval. after the antigen retrieval, lung sections were washed with pbs and incubated with % h o at room temperature for min to block the activity of endogenous peroxidase. the lung sections were washed again with pbs and incubated with anti-ly g antibody at room temperature for h. then, the lung sections were incubated with secondary antibody and the dab solutions and hematoxylin. immunohistochemical images were observed and photographed with a microscope (olympus bx , japan). wet-dry (w-d) ratio was used as an indicator of lung tissue edema [ ] . intraperitoneal injection of % chloral hydrate was used to anesthetize mice; lungs were removed, washed, blotted dry, weighed to obtain the wet weight, and subsequently placed in an oven at °c for days to gain the dry weight. fitc-albumin osmosis analysis is a common method for measuring lung capillary permeability [ ] . mice were injected intraperitoneally with lps for h, followed by tail vein injection of fitc-albumin ( mg/ml). after h of the fitc-albumin blood circulation, intraperitoneal injection of % chloral hydrate was used to anesthetize mice. the lungs were removed and immersed in % paraformaldehyde for days. these portions were processed and embedded in paraffin and used for fitc-albumin osmosis analysis. after the lung sections had undergone retrieval antigen and purity antigen, the samples were incubated overnight with anti-bdca- and anti-fitc antibodies. the lung sections were washed with pbs ( min/each time) and incubated with fluorescent secondary antibodies for min. after the dapi staining, the lung sections were observed immediately under a fluorescent microscope (olympus, japan). finally, relative quantification of immunofluorescence was conducted by using imagej. to detect the contents of hs, sdc- , and ctl in huvecs, we seeded cells on coverslips in -well plates. after different pretreatments and treatments, the cells were fixed with % paraformaldehyde and permeabilized with . % triton x- . subsequently, the coverslips were incubated in serum blocking solution and then incubated with hs, sdc- , and ctl antibodies to overnight at °c. primary antibody binding was detected by using fitc-conjugated secondary antibodies. the cell nuclei were stained with dapi. finally, images were obtained by using a fluorescence microscope (olympus bx , japan). to detect the levels of hs, sdc- , and ctl in lungs, we deparaffinized, dehydrated, and treated for antigen retrieval in the -µm lung sections, and then, we permeabilized them with . % triton x- . the sections were washed with pbs and incubated with serum blocking solution and then treated with hs, sdc- , and ctl antibodies overnight at °c. thereafter, the sections were washed with pbs and fitc-conjugated secondary antibodies were incubated for min at room temperature. lastly, cell nuclei were stained with dapi. images were obtained by using a fluorescence microscope (olympus bx , japan). intraperitoneal injection of % chloral hydrate was used to anesthetize mice, whose lungs were removed and frozen in liquid nitrogen. the extraction of nuclear and cytoplasmic protein was performed with a protein extraction kit (beyotime, china), and its concentration was determined by the bca protein assay kit (beyotime, china). equal amounts of protein were loaded into each well and separated by % sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) and transferred to polyvinylidene fluoride (pvdf) membranes. the pvdf membranes were washed by tris-buffered saline with tween (tbst) and incubated with % nonfat milk for h. the pvdf membranes were incubated with primary antibodies (hmgb , nf-κb p , iκbα, p-iκbα, p , p-p , jnk, p-jnk, erk, p-erk, laminb , mmp- , hpa, β-actin, and gapdh) overnight. the pvdf membranes were washed again with tbst and incubated with hrp-conjugated goat anti-rabbit igg at °c for h. the protein band at the pvdf membranes was visualized by the chemical exposure solution. all data are presented as the mean ± standard deviation (sd). the differences between groups were analyzed using student's t test and one-way anova followed by the snk test. p < . was considered to indicate statistical significance. all statistical analyses were performed using spss . (ibm corp.). as shown in fig. b -e, the lung tissues of the control group had complete alveolar structure and almost no neutrophil infiltration. however, the lung tissues of the lps group showed pulmonary congestion and edema, obvious infiltration of neutrophil cells, and alveolar collapse (fig. b-e) . compared with the lps group, lung structural damage and neutrophil infiltration significantly decreased with increased pretreatment concentration of crocin in crocin + lps groups (fig. b-e) . these data indicated that pretreatment with crocin can significantly decrease lung injury in lps-induced ards mice. vascular permeability increase is an important pathological change in ards. to study the effect of crocin on vascular permeability in lps-induced ards mice, lung w-d ratio and fitc-albumins were detected. the lung w-d ratio in the lps group was significantly higher than that of the control group (fig. c) . however, pretreatment with crocin significantly reduced the w-d ratio (fig. c) . the detection of fitc-albumin further indicated that vascular permeability in the lps group was significantly higher than that in the control group, and pretreatment with crocin significantly reduced albumin permeability (fig. a, b) . therefore, pretreatment with crocin can improve pulmonary vascular permeability in lps-induced ards mice. the results in vivo showed that sdc- and hs were significantly abscised after lps stimulation compared with the control group (fig. a-d) . in pretreatment with crocin groups, sdc- and hs abscission were significantly decreased (fig. a-d) . the results in vitro showed that hs and sdc- had no significant difference among the control, crocin, mmp- fig. a , lung permeability was determined by fitc-albumin osmosis analysis (a, magnification × , scale bar µm) and lung w-d ratio (c). b fluorescence intensity analysis of a. all data are presented as means ± sd of three independent experiments. # p < . vs. control group, *p < . vs. lps group inhibitor, and ctl inhibitor groups (fig. e-h) . after lps treatment, hs and sdc- showed significant shedding in vitro (fig. e-h) . pretreatment with mmp- inhibitor significantly reduced the shedding of sdc- but had no effect on the shedding of hs (fig. e-h) . pretreatment with ctl inhibitor significantly reduced the shedding of hs but had no effect on the shedding of sdc- ( fig. e-h) . however, hs and sdc- shedding with pretreatment crocin were significantly reduced (fig. e-h) . the preceding results indicated that crocin protected hs by inhibiting ctl and hpa and protected sdc- by inhibiting mmp- . the in vivo results showed that ctl and hpa were significantly expressed after lps stimulation compared with the control group (fig. a-d) . however, pretreatment with crocin, the expressions of ctl and hpa by lps stimulation were significantly inhibited (fig. a-d) . the results of ctl and hpa in vitro showed no significant difference among the control, crocin, and mmp- inhibitor groups (fig. e, g-i) . however, the expression of ctl and hpa was inhibited in the ctl inhibitor group (fig. e, g-i) . the expressions of ctl and hpa induced by lps were significantly inhibited in pretreatment with the ctl inhibitor group (fig. e, g-i) . after lps stimulation, pretreatment with the mmp- inhibitor had no significant inhibition on the expressions of ctl and hpa. however, pretreatment with ctl inhibitor significantly inhibited the expressions of ctl and hpa by lps stimulation (fig. e, g-i) . the preceding results indicated that crocin might inhibit the expression of hpa by inhibiting the upstream protein of ctl in lps-induced ards mice and lps-stimulated huvecs. the results in vivo showed that the expression of mmp- by lps stimulation was significantly increased compared with that of the control group (fig. c, d) . however, pretreatment with crocin significantly inhibited lps stimulation mmp- expression compared with lps treatment (fig. c, d) . mmp- expression in vitro showed no significant difference among the control, crocin, and ctl inhibitor groups (fig. h, i) . however, mmp- expression was suppressed in the mmp- inhibitor group (fig. h, i) . compared with ctl inhibitor treatment, the pretreatment with mmp- inhibitor significantly inhibited lps-stimulated mmp- expression (fig. h, i) . the preceding results indicated that crocin might protect against sdc- shedding by inhibiting lps-stimulated mmp- expression. mapk signaling pathway plays an important role in regulating the inflammatory response of lung injury. in this study, animal results showed that lps stimulation significantly enhanced the phosphorylation of p , erk, and jnk in lungs compared with the control group (fig. a, c) . pretreatment with crocin inhibited the phosphorylation of p , erk, and jnk, and the effect was more obvious with increased drug concentration (fig. a, c) . similarly, cell results showed that only crocin had no effect on the mapk signaling pathway compared with the control group. however, the significant phosphorylation of p , erk, and jnk was observed after lps stimulation (fig. b, d) . pretreatment with crocin inhibited the phosphorylation of p , erk, and jnk (fig. b, d) . these results suggested that crocin can inhibit the activation of mapk pathway in lps-induced ards mice and lps-stimulated huvecs. the hmgb and nf-κb signaling pathway also regulate lung injury of the inflammatory process. results in lungs showed that lps stimulation significantly enhanced iκbα phosphorylation, nf-κb p nuclear transfer, and hmgb cytoplasmic transfer compared with that of the control group (fig. a, c) . pretreatment with crocin can inhibit iκbα phosphorylation, nf-κb p nuclear transfer, and hmgb cytoplasmic transfer compared with that of the lps group (fig. a, c) . the inhibitory effect was more obvious with increased drug concentration (fig. a, c) . fig. effects of crocin on sdc- and hs in lps-induced ards mice and lps-stimulated huvecs. following the process shown in fig. a , immunofluorescence images of sdc- in mice (a) and huvecs (e) (magnification × , scale bar µm). g, b fluorescence intensity analysis of e, a, respectively. immunofluorescence images of hs in mice (c) and huvecs (f) (magnification × , scale bar µm). h, d fluorescence intensity analysis of f, c, respectively. all data are presented as means ± sd of three independent experiments. # p < . vs. control group, *p < . vs. lps group ◂ likewise, the results of cell experiments showed no significant changes in the crocin and control groups. however, lps stimulation significantly increased iκbα phosphorylation, nf-κb p nuclear transfer, and hmgb cytoplasmic transfer (fig. b, d) . pretreatment with crocin significantly inhibited iκbα phosphorylation, nf-κb p nuclear transfer, and hmgb cytoplasmic transfer (fig. b, d) . these results suggested that crocin can inhibit the activation of the nf-κb and hmgb inflammatory pathway in lps-induced ards mice and lps-stimulated huvecs. increased vascular permeability is one of the important pathological features of ards. the pulmonary edema associated with albumin leakage is closely related to the endothelial glycocalyx degradation [ ] . the endothelial glycocalyx acts not only as a physical barrier to prevent albumin exudation but also as an information molecule to fig. effects of crocin on ctl and hpa, mmp- in lps-induced ards mice and lps-stimulated huvecs. following the process shown in fig. a , the expression levels of ctl (a) and hpa, mmp- (c) in mice were detected (a, magnification × , scale bar: µm). d, b protein intensity analysis of c, a, respectively. the effect of crocin on cytotoxicity in huvecs was determined by mtt assay (f). the expression levels of ctl (e) and hpa, mmp- (h) in huvecs were detected (a, magnification × , scale bar µm). i, g protein intensity analysis of h, e, respectively. all data are presented as means ± sd of three independent experiments. # p < . vs. control group, *p < . vs. lps group ◂ fig. effects of crocin on mapk pathway activation in lps-induced ards mice and lps-stimulated huvecs. following the process shown in fig. a , the levels of phosphorylation and non-phosphorylation of p , erk, and jnk in lung tissues (a) and huvecs (b) were detected by western blot. c, d protein quantification of a, b, respectively. gapdh was used as internal control. all data are presented as means ± sd of three independent experiments. # p < . vs. control group, *p < . vs. lps group participate in hemodynamics [ ] [ ] [ ] [ ] ] . as shown in previous reports, crocin had an important protective effects in several diseases by affecting on cell apoptosis, arteriosclerosis, oxidation, and inflammation [ ] [ ] [ ] [ ] [ ] . the present study demonstrated that pretreatment with crocin effectively improved the pulmonary edema associated with albumin leakage by maintaining the integrity of glycocalyx in lps-induced ards mice. to our knowledge, this study is the first to reveal its protective effects and mechanisms of the endothelial glycocalyx. enzymatic degradation had been found to be involved in the endothelial glycocalyx degradation. as shown in previous reports, mmp- can directly cause the shedding of sdc- in lung endothelial cells or sdc- in glomerular endothelial cells [ , ] . additionally, ramani et al. [ ] found that the enzymatic hydrolysis of hs chain can promote sdc- shedding in the cell lines cag, thereby indicating that non-mmp mechanism is also involved in the process of sdc- shedding. in the present study, cathepsin l inhibitor did not alleviate lps-mediated sdc- fig. effects of crocin on hmgb and nf-κb pathway activation in lps-induced ards mice and lps-stimulated huvecs. following the process shown in fig. a , the levels of hmgb and nf-κb p as well as the phosphorylation and non-phosphorylation of iκbα in lung tissues (a) and huvecs (b) were detected by western blot. c, d protein quantification of a, b, respectively. gapdh or lamin b was used as internal control. all data are presented as means ± sd of three independent experiments. # p < . vs. control group, *p < . vs. lps group shedding, thereby suggesting that non-mmp mechanisms may not be involved in sdc- shedding. more importantly, the inhibition of mmp- expression by pretreatment with crocin is closely related to the reduction in sdc- shedding, and the inhibition of cathepsin l and hpa expression by pretreatment with crocin is closely related to the reduction in hs shedding. nf-κb and mapk are representative and important inflammatory pathways in the onset and development of ards pathogenesis, and they promote the production and secretion of inflammatory cytokines after activation [ , ] . otherwise, hmgb- as an important late inflammatory factor can trigger the intracellular nf-κb signaling pathway and aggravate serious injury [ ] . as shown in previous reports, crocin had a strong anti-inflammatory effect in diabetic nephropathy model [ ] . the results of this study also showed that crocin had the same strong anti-inflammatory effect in lps-induced ards cell and animal models. however, inflammatory injury also induces the production and release of glycocalyx shedding factors (such as mmp- and hpa) [ , ] . an incomplete glycocalyx may aggravate the inflammatory reaction and further spread inflammatory factors, resulting in persistent lung injury [ , ] . the present study showed that pretreatment with crocin effectively reduced glycocalyx damage, and this protective effect may be related to inhibiting the activation of inflammatory pathways. during the ards process, activated neutrophils release intracellular toxic components so as to amplify the inflammatory response by recruiting more inflammatory cells into the site of damage [ ] . additionally, ma et al. [ ] reported that neutrophils were involved in regulating the microvascular endothelial permeability. suzuki et al. [ ] reported that neutrophil elastase damaged the pulmonary endothelial glycocalyx in lps-induced endotoxemia. in the present study, pretreatment with crocin significantly improved the neutrophil adhesion or infiltration induced by lps in ards mice. the ability of crocin to alleviate glycocalyx damage may be closely related to the reduction in neutrophil retention. oxidative damage is another important factor in the pathogenesis of ards. oxidation/antioxidant imbalance can aggravate oxidative damage to tissues and cells [ ] . previous study reported that reactive oxygen species can damage the endothelial glycocalyx of the kidneys and lungs and modulate hpa expression of glomerular adriamycin nephropathy [ , ] . additionally, crocin has been reported to improve methotrexin-induced liver injury by inhibiting oxidative stress in rats [ ] . however, further studies are needed to determine whether crocin's protective effect on glycocalyx degradation of lps-induced ards mice and lps-stimulated huvecs is also related to oxidative injury. interestingly, how to repair the damaged endothelial glycocalyx is another important topic in the study of endothelial glycocalyx [ , ] . the present study did not involve how the endothelial glycocalyx was repaired, and further studies are needed to determine whether crocin promotes the repair of the endothelial glycocalyx. the full story is still incomplete. given the limitations of laboratory conditions, the pharmacokinetic and hemodynamic changes in mice were not analyzed in the present study. the complete analysis of the pharmacokinetic and hemodynamic changes in mice will provide additional insights into crocin and endothelial glycocalyx. overall, the present study demonstrated that crocin can protect against hs and sdc- degradation by inhibiting enzyme expression. enzyme inhibition may be related to the decrease in inflammatory responses or oxidative damage. these findings add new pharmacological functions to crocin, providing potential targets for new therapies to inhibit enzymes and potential pathways that lead to endothelial dysfunction and vascular leakage in ards, which may contribute to the treatment of this disease. β -na(+), k(+)-atpase gene therapy upregulates tight junctions to rescue lipopolysaccharide-induced acute lung injury , , '-tri-o-acetylresveratrol attenuates lipopolysaccharide-induced acute respiratory distress syndrome via mapk/sirt pathway. mediators inflamm fibroblast growth factor signaling mediates pulmonary endothelial glycocalyx reconstitution shedding of syndecan- promotes immune cell recruitment and mitigates cardiac dysfunction after lipopolysaccharide challenge in mice glycocalyx degradation induces a proinflammatory phenotype and increased leukocyte adhesion in cultured endothelial cells under flow role of the endothelial surface layer in neutrophil recruitment protection of the endothelial glycocalyx by antithrombin in an endotoxin-induced rat model of sepsis the 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stress mediated neutrophil apoptosis: a biochemical insight role of neutrophil extracellular traps and vesicles in regulating vascular endothelial permeability neutrophil elastase damages the pulmonary endothelial glycocalyx in lipopolysaccharide-induced experimental endotoxemia lycium barbarum polysaccharide protects against lps-induced ards by inhibiting apoptosis, oxidative stress, and inflammation in pulmonary endothelial cells heparan sulfates mediate pressure-induced increase in lung endothelial hydraulic conductivity via nitric oxide/reactive oxygen species induction of glomerular heparanase expression in rats with adriamycin nephropathy is regulated by reactive oxygen species and the renin-angiotensin system crocin ameliorates methotrexate-induced liver injury via inhibition of oxidative stress and inflammation in rats therapeutic restoration of endothelial glycocalyx in sepsis acknowledgements this work was supported by funding from the national natural science foundation of china (no.: ). the authors declare no conflict of interest. key: cord- - e rlq authors: belančić, andrej title: gut microbiome dysbiosis and endotoxemia - additional pathophysiological explanation for increased covid- severity in obesity date: - - journal: obes med doi: . /j.obmed. . sha: doc_id: cord_uid: e rlq the overall intestinal lipopolysaccharide (lps) composition in the individuals with obesity could be shifted away from immunosilent/immunoinhibitory bacteroidetes lps subtypes, in favor of various proinflammatory lps subtypes due to gut microbiome dysbiosis. what is more, high-fat diet, as well as obesity per se, enhance intestinal permeability through various mechanisms. latter results in increased paracellular absorption and transcellular (via chylomicrons) transport of endogenous endotoxin in the circulatory system (endotoxemia). in addition, it is known that lipid a initiates a signaling cascade resulting in activation of various proinflammatory pathways and increases oxidative stress upon binding to tool-like receptor (tlr ). taking everything into consideration, it is very likely that gut microbiome dysbiosis and endotoxemia represent the additional pathophysiological explanation for increased covid- severity in obesity. abstract: the overall intestinal lipopolysaccharide (lps) composition in the individuals with obesity could be shifted away from immunosilent/immunoinhibitory bacteroidetes lps subtypes, in favor of various proinflammatory lps subtypes due to gut microbiome dysbiosis. what is more, highfat diet, as well as obesity per se, enhance intestinal permeability through various mechanisms. latter results in increased paracellular absorption and transcellular (via chylomicrons) transport of endogenous endotoxin in the circulatory system (endotoxemia). in addition, it is known that lipid a initiates a signaling cascade resulting in activation of various proinflammatory pathways and increases oxidative stress upon binding to tool-like receptor (tlr ). taking everything into consideration, it is very likely that gut microbiome dysbiosis and endotoxemia represent the additional pathophysiological explanation for increased covid- severity in obesity. emerging body of literature indicates that individuals with obesity are more at risk for covid- infection, as well as associated hospitalization, intensive care unit admission, and mortality ( ) . hence, an improved understanding of the pathophysiological interconnection between obesity and covid- would definitely guide preventive and therapeutic strategies for this vulnerable population. bearing this in mind, we have recently proposed obesity-related low-grade chronic inflammation, higher expression of ace- and pathway associated components, and decreased vitamin d bioavailability as potential pathophysiological mechanisms leading to increased susceptibility and severity in obesity ( ) . what is more, in the meantime, i have recognized gut microbiome dysbiosis and endotoxemia as additional plausible explanation for more severe forms of the covid- infection among individuals with obesity, which then encouraged me to provide the present brief overview of the novel findings. to clarify, lipopolysaccharide (lps), commonly known as endotoxin, is a glycolipid found on the outer membrane of gram-negative bacteria that is essential for bacterial cell integrity, viability, and defense against environmental stress. latter amphimpilic molecule is composed of three structural domains: lipophilic lipid a (immunostimulatory component), hydrophilic polysaccharides or oligosaccharide core, and o-antigen. it is worth mentioning that the structure of lipid a is diverse among bacterial species, and that the number of acyl chains determines its immunostimulatory potential ( ). the overall collection (> ) of microorganisms (bacteria, archaea, viruses, and eukaryotic microbes) colonizing the gastrointestinal tract is commonly known under term 'gut microbiome'. the human gut microbiota is mostly composed by two dominant (represent > % of the total community) bacterial phyla -bacteroidetes (gram-negative bacteria) and firmicutes (mostly gram-positive bacteria) ( ). what is more, eva d'hennezel et al. reported that bacteroidetes contribute % of the lps biosynthesis in healthy volunteers and . % in human microbiome project samples, and that a total lps produced in the healthy adult human gut is immunosilent/immunoinhibitory (has a very limited capacity to activate tlr -nf-κb pathway and elicit the production of inflammatory cytokines). furthermore, underacylated lipid a structures across the order bacteroidales are probably the main rationale for latter immunosilent/immunoinhibitory properties ( ). however, a relevant body of literature is generally consistent that individuals with obesity have a significantly higher level of firmicutes and lower level of bacteroidetes [increased firmicutes/bacteroidetes (f/b) ratio] compared to normal-weight/lean individuals ( ) ( ) ( ) . hence, the intestinal lps composition in the individuals with obesity could be shifted away from immunosilent/immunoinhibitory bacteroidetes lps subtypes, in favor of various proinflammatory lps subtypes (phyla producing more inflammatory lps) due to gut microbiome dysbiosis ( ) . in addition, it is known that high-fat diet enhances intestinal permeability through various mechanisms: (i) alters the distribution and expression of tight junctions, (ii) stimulates a shift to barrier-disrupting hydrophobic bile acids, (iii) induces intestinal epithelial cells oxidative stress and apoptosis, (iv) stimulates proinflammatory signaling cascades directly and indirectly by increasing barrier-disrupting cytokines and decreasing barrier-forming cytokines, (v) negatively modulates the intestinal mucus composition and enriches the gut microflora with barrier-disrupting species ( ) . it is also worth mentioning that nagpal et al. even reported a close link between obesity-associated gut microbiome dysbiosis to cause derangements in the intestinal cellular turnover homeostasis and functions to regulate gut permeability, independent of dietary ingredients such as high fat ( ) . thus, lpss (endotoxins) could move into the circulatory system through direct diffusion due to intestinal paracellular permeability or via absorption by enterocytes during chylomicron secretion ( ) . its presence in the bloodstream is then called endotoxemia. lipid a then initiates a signaling cascade resulting in activation of various proinflammatory pathways (predominantly nf-κb) and increases oxidative stress upon binding to tlr ( , ) . last j o u r n a l p r e -p r o o f but not least, petruk et al. in their preprint reported an interconnection between the s-glycoprotein of sars cov- and lps, and their link to induction of nf-κb and cytokine responses in monocytes and human blood, as well as significantly increased nf-κb responses in experimental animal models ( ) . taking everything previously mentioned into consideration, it is very likely that gut microbiome dysbiosis and endotoxemia represent the additional pathophysiological explanation for increased covid- severity in obesity (figure ). to deduce, reducing caloric intake (especially trans-fat and saturated fat consumption) and modulating gut microbiota (with prebiotics/probiotics/synbiotics and anti-inflammatory dietary pattern) may reduce endotoxemia and consequently the associated risk for more severe forms of covid- infection in obesity ( ) . however, novel large-scale randomized controlled trials and well-designed meta-analyses are highly needed to draw final conclusions. funding: this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. individuals with obesity and covid- : a global perspective on the epidemiology and biological relationships potential pathophysiological mechanisms leading to increased covid- susceptibility and severity in obesity recent advances in lipopolysaccharide recognition systems a human gut microbial gene catalog established by metagenomic sequencing total lipopolysaccharide from the human gut microbiome silences toll-like receptor signaling microbial ecology: human gut microbes associated with obesity association between body mass index and firmicutes/bacteroidetes ratio in an adult ukrainian population human intestinal microbiota composition is associated with local and systemic inflammation in obesity negative effects of a high-fat diet on intestinal permeability: a review obesity-linked gut microbiome dysbiosis associated with derangements in gut permeability and intestinal cellular homeostasis independent of diet influence of a high-fat diet on gut microbiota, intestinal permeability and metabolic endotoxemia involvement of reactive oxygen species in toll-like receptor -dependent activation of nf-kappa b metabolic endotoxemia with obesity: is it real and is it relevant? sars-cov- spike protein binds to bacterial lypopolysaccharide and boosts proinflammatory activity obesity-related low-grade chronic inflammation: implementation of the dietary inflammatory index in clinical practice is the milestone? j o u r n a l p r e -p r o o f highlights:• covid- susceptibility and severity are increased in obesity dear editor-in-chief, the author of the manuscript (commentary) entitled gut microbiome dysbiosis and endotoxemia -additional pathophysiological explanation for increased covid- severity in obesity declares no conflict of interest.sincerely yours, andrej belančić, md key: cord- -sbdtpsz authors: ramírez-pérez, sergio; hernández-palma, luis alexis; oregon-romero, edith; anaya-macías, brian uriel; garcía-arellano, samuel; gonzález-estevez, guillermo; muñoz-valle, josé francisco title: downregulation of inflammatory cytokine release from il- β and lps-stimulated pbmc orchestrated by st , a myd dimerisation inhibitor date: - - journal: molecules doi: . /molecules sha: doc_id: cord_uid: sbdtpsz the inflammatory process implicates homeostasis disruption and increased production of inflammatory mediators. myeloid differentiation primary response (myd ) is an essential protein recruited after lipopolysaccharide (lps) and interleukin (il)- β stimulation, a process that converges in nuclear factor kappa b (nf-κb) activation, as well as a transcription of several genes of both pro- and anti-inflammatory cytokines. the inhibition of myd has shown efficacy by decrease inflammatory response, and has demonstrated potential application as a therapeutic target in chronic diseases. in this study, we investigate the effect of myd dimerisation inhibitor st on cytokine production from rhil- β and lps-stimulated peripheral blood mononuclear cells (pbmc) from healthy blood donors (hbd). st significantly downregulates the production of ifn-γ, il- , il- , il- , il- , il- , vegf, il- ra, il- , il- , il- and il- (p < . ) in lps-stimulated pbmc. moreover, st had a relatively low impact on il- β signalling pathway inhibition, showing that only a few specific cytokines, such as ifn-γ and il- ra, are inhibited in rhil- β-stimulated pbmc (p < . ). in conclusion, myd dimerisation inhibitor st showed high efficacy by inhibiting pro- and anti-inflammatory cytokine production in lps-stimulated pbmc. moreover, although rhil- β induced a sustained cytokine production (p < . ), st did not show a significant effect in the secretion of neither pro- nor anti-inflammatory cytokines in rhil- β-stimulated pbmc. myeloid differentiation primary response (myd ) represents an important molecule associated with activation of several signalling pathways, which are implicated in the inflammatory immune response [ ] . interleukin (il)- β and toll-like receptors (tlr) signalling pathways are one of the most strongly studied mechanisms in which myd plays a primary role [ ] [ ] [ ] [ ] . the il- β activity implicates binding between this cytokine and the il- r type i (il- ri), this binding triggers the interaction of toll-il- -receptor (tir) domains followed by myd recruitment and downstream signalling cascades, which converge in the activation of several transcription factors and pro-inflammatory cytokine production [ , ] . regarding inflammation-associated tlr activation, molecules , , of tlr has been the best-studied molecule in both innate and adaptive immunity [ , ] . tlr signalling pathway can be activated myd -dependent or independent manner [ ] ; nevertheless, tlr -dependent lps-regulated signalling involves the recruitment and homodimerisation of myd and leads to pathways of intracellular signal transduction which converge in the production of inflammatory mediators implicated in the regulation of the inflammatory process [ , ] . overactivated myd -dependent lps and il- β inflammatory signalling pathways have displayed high expression of pro-inflammatory mediators not only in healthy blood donors (hbd), but also in patients with chronic, systemic and autoimmune diseases [ ] [ ] [ ] [ ] [ ] [ ] . due to this fact, the identification of new molecules that regulate these signalling pathways has taken high relevance [ , , [ ] [ ] [ ] . the chemical molecule st acts as an inhibitor of myd dimerisation and its activity has been demonstrated through the inhibition of tlr -dependent cpg-regulated signalling, and inhibition of il- , il- β, il- and tumor necrosis factor alpha (tnf-α) expression in lps-stimulated raw . cells [ ] [ ] [ ] [ ] . however, the effect of the st molecule on cytokine production mediated by il- β and lps-stimulated peripheral blood mononuclear cells (pbmc) has yet been clarified. in the present study, pbmc obtained from hbd were stimulated with both rhil- β and lps to identify pro-and anti-inflammatory cytokine profiles, as well as, the inhibitory activity of st molecule on the cytokine secretion. our results indicate that inhibition of myd dimerisation mediated by st molecule causes a decrease secretion of both pro-and anti-inflammatory cytokines in supernatants of lps-stimulated pbmc; however, st showed high impact neither pro-nor anti-inflammatory cytokine secretion in rhil- β-stimulated pbmc. to determine the specific concentration of st in which the secretion of cytokines was inhibited, curves of different concentrations of st were performed ( figure ). tnf-α quantification was taken as a positive control, and the concentration of this cytokine was determined in supernatants of lps-stimulated pbmc after h ( figure a ). the concentration of tnf-α in supernatants of pbmc without stimuli was . pg/ml; whereas, a high concentration of tnf-α in lps-stimulated pbmc was identified ( . pg/ml). regarding st stimuli three different concentrations were taken , and µm and tnf-α levels were determined, the medium values were . pg/ml (p = not significant [n.s.]), . pg/ml (p < . ), and . pg/ml (p < . ), respectively. similarly, the concentration of tnf-α was determined in supernatants of rhil- β-stimulated pbmc ( figure b ). tnf-α levels from rhil- β-stimulated pbmc were . pg/ml, for rhil- β plus µm of st were . pg/ml (p = n.s.), and after add and µm of st to rhil- β-stimulated pbmc, the tnf-α levels were pg/ml for both (p < . ) ( figure b ). lps has been implicated in the production of pro-inflammatory cytokines through tlr activation. our results indicate that lps is a potent inductor of several pro-inflammatory cytokines in pbmc. statistically significant differences were found between pbmc treated with rpmi alone and lps (p < . ). in addition, st molecule was used as a negative regulator of tlr -dependent lps-regulated signalling pathway. st in lps-stimulated pbmc decreased secretion of interferon gamma (ifn-γ) (p < . ), il- (p < . ), il- (p < . ), il- (p < . ), vascular endothelial growth factor (vegf) (p < . ), il- (p < . ) and il- (p < . ) ( figure ; table s ). since our study included males and females; in order to identify differential effects on cytokine production release a statistical analysis by gender was performed. however, our results showed non-statistically significant differences between males and females (data not shown). as a control for the effect of st alone, a statistical analysis was performed by comparing the production of inflammatory cytokines studied in the presence or absence of st . for ifnγ, tnfα, il- ra and il- , statistically significant differences were found (p < . ). the levels of these cytokines in the presence of st were significantly lower than in pbmc treated with rpmi alone; furthermore, a higher cytokine secretion as an effect of st than in the basal response of untreated pbmc was not observed (table s ) . molecules , , x of as a control for the effect of st alone, a statistical analysis was performed by comparing the production of inflammatory cytokines studied in the presence or absence of st . for ifnγ, tnfα, il- ra and il- , statistically significant differences were found (p < . ). the levels of these cytokines in the presence of st were significantly lower than in pbmc treated with rpmi alone; furthermore, a higher cytokine secretion as an effect of st than in the basal response of untreated pbmc was not observed (table s ) . the concentration of anti-inflammatory cytokines was determined in supernatants of lpsstimulated pbmc. interestingly and contrary to expectations, after h of stimulation with lps in pbmc, we observed anti-inflammatory cytokine production of il- ra, il- , il- , il- , il- and il- (p < . ). additionally, st inhibited the secretion of il- ra (p < . ), il- (p < . ), il- (p < . ), il- (p < . ) and il- (p < . ), but not il- ( . pg/ml, p = n.s.) ( figure ; table s ). moreover, it was observed that anti-inflammatory cytokine secretion from pbmc stimulated with st alone, have a similar response to unstimulated cells (table s ). based on these results, pbmc stimulated with lps can secrete pro-and anti-inflammatory cytokines and st can inhibit the observed response; this is a relevant finding that has not been reported till date. (b) the soluble levels of tnf-α in the supernatant of rhil- β-stimulated pbmc at ng/ml and rhil- β ( ng/ml) plus different concentrations of st ( , and µm) were determined. significant inhibition was identified at µm (p < . ) and µm (p < . ) of st for lps; while for rhil- β significant inhibition was identified at µm (p < . ) and µm (p < . ) of st . data provided in medians and interquartile ranges (n = ), Φ kruskal-wallis test was performed, and dunn's test obtained statistically significant differences. the concentration of anti-inflammatory cytokines was determined in supernatants of lps-stimulated pbmc. interestingly and contrary to expectations, after h of stimulation with lps in pbmc, we observed anti-inflammatory cytokine production of il- ra, il- , il- , il- , il- and il- (p < . ). additionally, st inhibited the secretion of il- ra (p < . ), il- (p < . ), il- (p < . ), il- (p < . ) and il- (p < . ), but not il- ( . pg/ml, p = n.s.) ( figure ; table s ). moreover, it was observed that anti-inflammatory cytokine secretion from pbmc stimulated with st alone, have a similar response to unstimulated cells (table s ). based on these results, pbmc stimulated with lps can secrete pro-and anti-inflammatory cytokines and st can inhibit the observed response; this is a relevant finding that has not been reported till date. the role of il- β has been widely described in several chronic conditions, as well as in several cell types. our results showed high secretion of pro-inflammatory cytokines in rhil- β-stimulated pbmc (table ). in an exciting way and as previously reported, il- β represents an important cytokine capable of inducing th -related cytokine profile. in this study, we observed high production of these cytokines: however, only il- a, granulocyte-monocyte colony-stimulating factor (gm-csf) and granulocyte colony-stimulating factor (g-csf) increased significantly after h of rhil- β stimulation (p < . ). furthermore, the concentration of cytokines, such as il- (p < . ), vegf (p < . ) and il- (p < . ), were found higher after rhil- β stimulation. on the other hand, only il- and il- significantly increased after rhil- β stimulation (p < . ). regarding the st effect on rhil- β-stimulated pbmc and contrary to our expectations; our results showed that this molecule had a relatively low impact on the il- β signalling pathway inhibition (table ) . st molecule only inhibited the secretion of ifn-γ (p < . ) and il- ra molecules , , of (p < . ). the present study shows that the specific inhibition of critical components in the il- signalling pathway is not enough to avoid the secretion of inflammatory mediators, the above suggests that various myd -independent mechanisms could regulate the production of cytokines in pbmc. the role of il- β has been widely described in several chronic conditions, as well as in several cell types. our results showed high secretion of pro-inflammatory cytokines in rhil- β-stimulated pbmc (table ). in an exciting way and as previously reported, il- β represents an important cytokine capable of inducing th -related cytokine profile. in this study, we observed high production of these cytokines: however, only il- a, granulocyte-monocyte colony-stimulating factor (gm-csf) and granulocyte colony-stimulating factor (g-csf) increased significantly after h of rhil- β stimulation (p < . ). furthermore, the concentration of cytokines, such as il- (p < . ), vegf (p < . ) and il- (p < . ), were found higher after rhil- β stimulation. on the other hand, only il- and il- significantly increased after rhil- β stimulation (p < . ). regarding the st effect on rhil- β-stimulated pbmc and contrary to our expectations; our results showed that this molecule had a relatively low impact on the il- β signalling pathway inhibition (table ) . st molecule only inhibited the secretion of ifn-γ (p < . ) and il- ra (p < . ). the present study shows that the specific inhibition of critical components in the il- signalling pathway is not enough to avoid the secretion of inflammatory mediators, the above suggests that various myd -independent mechanisms could regulate the production of cytokines in pbmc. the role of inflammatory key mediators, such as il- β and lps, in activating the innate immune response and subsequently leading to inflammation has been widely described in several studies [ , ] . il- β and lps-signalling pathways trigger cascades of intracellular activation mediated by myd recruitment, which converges in the activation of transcription factors, such as nf-κb and its translocation to the nucleus [ , , , ] . the expression of receptors associated with these pro-inflammatory mediators can be given in a variety of cells of the immune system, within the main ones are professional antigen-presenting cells, treg cells and effector t cells [ ] [ ] [ ] [ ] . our results show that after h, pro-inflammatory cytokine levels of il- β, tnf-α, ifn-γ, il- , il- , il- a, g-csf, gm-csf, il- , vegf, il- and il- significantly increased in lps-stimulated pbmc. recent studies performed in vitro have described that in both pbmc and thp- cells can be possible to carry out a differentiation towards m macrophages by stimulation with lps, ifn-γ or gm-csf; m macrophages have been characterised by the expression of pro-inflammatory cytokines, such as il- , il- , tnf-α and il- β [ , , ] . regarding cytokines, such as il- and tnf-α, previous studies performed in lps-stimulated pbmc from hbd reported increased levels of these cytokines in comparison with those levels found in unstimulated pbmc [ , ] . an important cytokine involved in the innate immune response mediated by nk and nkt cells is il- , which increased after lps stimulation; concerning this result, one study reported that lps-stimulated monocytes could produce il- [ ] . concerning the expression of gm-csf and vegf, previous studies conducted in lps-stimulated pbmc after h showed high levels of both cytokines, as we observed in this study [ ] . an earlier report indicated that gm-csf expression increased in lps-stimulated pbmc; nonetheless, its expression remains low compared with other pro-inflammatory cytokines, such as tnf-α or il- , which could suggest that this cytokine is produced only for a specific population of monocytes [ ] . the activation of several transcription factors after lps stimulation has been demonstrated in a large number of studies. rothfuchs et al. reported that ifn-γ mrna expression was strongly diminished in tlr -/macrophages compared with the wild type phenotype; moreover, tlr activation causes myd -dependent ifn-α production, which results in an autocrine effect that regulates ifn-γ mrna expression by stat activation [ ] . on the other hand, increased expression of ror-γt and the phosphorylated form of nf-κb were found after lps stimulation, and this effect was associated with the differentiation of th cells and high expression of il- a [ ] . peyssonnaux et al. reported that hif- α was highly expressed in lps-stimulated macrophages; interestingly, hif- α/rorγt/p complex can be linked to the promoter region of il a and can also promote its transcription [ , , ] . vegf is a cytokine produced in response to the hypoxia process and subsequently to hif- α activation which, as mentioned above, hif- α is a transcription factor induced in response to lps [ , ] . transcription factor activation followed by high production of pro-inflammatory cytokines in a tlr -lps-dependent pathway has been widely described. however, posttranscriptional mechanisms implicated in tlr activation have also recently been described. for example, arid a protein is a crucial factor for the production of il- ; this protein binds to the '-utr region of the il- mrna and leads to its stabilisation and subsequently, to its efficient expression in vivo [ ] . moreover, nyati et al. described an alternative lps signalling pathway that is independent on p activation, but dependent on the mitogen-activated protein kinase (mapk) phosphatase (mkp- ) [ ] . this phosphatase mkp- can induce the translocation from the nucleus to the cytoplasm of au-rich element rna-binding protein (auf- ), and auf- acts by stabilising the mrna of il- , tnf-α and il- [ ] . according to the present study, we observed that the production of anti-inflammatory cytokines significantly increases in lps-stimulated pbmc. several studies have reported that tlr -dependent lps-regulated signalling pathway causes a predominantly pro-inflammatory response in pbmc. however, some interesting studies have reported that the expression of irf could also be expressed after lps/ifn-γ or il- stimulation; irf is a key transcription factor involved in the differentiation of m macrophages [ , , ] . however, a significant limitation in our study is that the expression of cytokines was determined after h of stimulation. in this regard, several reports indicate that pro-inflammatory cytokines have an early maximum expression peak within the first eight hours of lps stimulation (il- , il- β, il- , tnf-α and il- ) and expression of cytokines, such as il- and il- ra, exhibit a maximum expression peak at and h, respectively [ ] . this behaviour on the cytokine profile could also be explained by a process known as "tlr tolerance", which is characterised by reducing expression of pro-inflammatory cytokines and high expression of m activation markers after sustained exposure to tlr ligands [ ] . additionally, a specific type of m polarisation in human mononuclear cells after induction of lps tolerance has been reported [ ] , and after lps tolerance recovering, macrophages can express both m and m polarisation states [ ] . stimulation with rhil- β triggered the production of several pro-inflammatory cytokines. interestingly, as previously described in other studies, this cytokine induces the release of th -related cytokine profile; il- a, gm-csf and g-csf were significantly expressed in our research [ ] [ ] [ ] . nevertheless, a new t helper cell subset characterised by high production of gm-csf has been currently described [ ] . this cell subset was identified as gm-csf producing cd + t cell (th-gm-csf), and its differentiation depends on il- β signalling pathway, and subsequent irak and nf-κb activation [ ] . furthermore, th-gm-csf cells are able to produce high concentrations of pro-inflammatory cytokines, such as il- , il- and tnf-α [ ] . our results also showed increased expression of il- and vegf; regarding il- production, langlet et al. reported that this cytokine could be expressed in an il- β-dependent activation [ ] . moreover, previous reports have shown that vegf expression requires activation of hif- α, and activation of hif- α occurs after il- β stimulation [ ] [ ] [ ] . concerning the results of the inhibition of myd dimerisation and according to expectations, st molecule significantly inhibits the pro-inflammatory and anti-inflammatory cytokine production mediated by the activation of lps-tlr pathway. about these results, long et al. reported a significant decrease in cytokine expression of il- , il- β, il- and tnf-α from raw . cells treated with lps plus st [ ] . several studies have reported that st causes decreased recruitment and activation of specific molecules and transcription factors involved in the activation of tlr and the lps-dependent immune response activation. the main inhibited molecules, due to the activity of st , are irak , irak , traf , p-ikk, p-ikbα, p-nf-κb and hif- α [ , ] . st has also been proposed as a novel drug targeting in diseases like lymphoma, leukaemia, human hepatocellular carcinoma, and traumatic brain injury [ ] [ ] [ ] . however, its role as a possible inhibitor in the production of cytokines produced after stimulation with lps remains undetermined. the decrease recruitment of molecules implicated in the myddosome formation may explain the inhibition of pro-and anti-inflammatory cytokines as a consequence of tlr -dependent lps-regulated signalling. at the same time, signalling pathway activation after il- -il- ri-il- racp complex formation has been widely described [ , , , , ] . this process implicates recruitment and homodimerisation of myd , as well as intracellular signalling cascades that converge in the transcription of genes of pro-inflammatory mediators [ , , , , ] . specific myd dimerisation inhibition has been tested in many studies where the role of this molecule in an il- β-dependent activation pathway was evident [ , , ] . another study performed in healthy human articular chondrocytes reported decreased map kinase (mapk) activation after myd dimerisation inhibition and il- β stimulation [ ] . an interesting study conducted by wang et al. ( ) previously reported that st decreases the expression of several molecules involved in the myddosome formation, such as phosphorylated btk and iκb, along with the decreased secretion of il- and ifn-β from b-cell lymphoma cell lines [ ] . in our study, il- release decreases on both il- β and lps-stimulated pbmc treated with st ; nonetheless, the effect was not statistically significant. in relation to the ifn response, a decreased secretion of ifn-γ on both il- β and lps-stimulated pbmc was observed our study as an effect of st . however, our results did not show a substantial inhibitory effect on cytokine production from pbmc treated with il- β plus st . in regard to this result, loiarro et al. ( ) previously described an inhibitory effect observed on nf-κb activity after stimulation with il- β stimulation ( ng/ml) plus st ( µm) on hek t cells [ ] . nevertheless, although nf-κb activity decreases, st did not deplete nf-κb activation, which could provide signalling activation enough to produce some of the inflammatory cytokines observed in our study [ ] . indeed, since this st chemical compound affects only the association of tir domains of myd and the disruption of this tir domain interaction inhibits the recruitment of irak . by extension, irak and the subsequent signalling cascade, dimerisation inhibition of myd might affect only one specific signalling pathway [ ] . these results might suggest that alternative il- β signalling pathways independent of myd homodimerisation and recruitment in pbmc could be active as well. this approach arises from previous studies in which new receptors associated with the recognition of il- β have been observed. il- racpb is a unique receptor expressed on neurons, and its expression implicates activation of certain signalling pathways, such as p mapk, but not nf-κb; it has even been observed that this alternative signalling pathway is independent on myd , irak and traf [ , ] . moreover, heinz et al. reported a new molecule known as unc cl; this protein contains death domains (dd) similarly to those found in myd [ ] . unc cl is considered as a pro-inflammatory signalling inducer and involves recruitment of irak , irak , traf and converges in nf-κb and jnk activation in a myd -independent manner [ ] . currently, there are not reports in which these new molecules have been reported in pbmc; however, the possibility to perform future studies that elucidate this observation remains open. moreover, in spite of the fact that st did not show a substantial inhibitory effect on downregulation of inflammatory cytokine from il- β-stimulated pbmc, it might be necessary to consider the use of other il- inhibitors, such as il- and il- [ , ] , which could be useful to compare a differential response orchestrated by st on il- β-stimulated pbmc and the inhibitory effect of both il- and il- . in this regard, conti et al. mentioned that il- suppresses the innate and acquired immune response and inhibits the inflammation through its binding with il- receptor-α chain (il- rα) [ ] . additionally, il- can activate the mtor signalling pathway and increase the adenosine monophosphate (amp) kinase [ ] ; therefore, il- and il- might represent cytokines of great interest in experimental models where the suppressive effect of several inflammatory mediators is required. furthermore, a weakness of this study was not to measure cell viability after stimulation with st . differences (that were not statistically significant) have been previously reported for st in other studies at , or µm on cell viability [ , , ] . our study used pbmc, reduction on specific pbmc subpopulations might contribute to the behaviour observed on these inflammatory cytokines. therefore, considering this limitation will be important in future studies to analyse the observed response of this molecule on cell viability and the percentage of apoptosis by the effect of st . the understanding of inflammatory mechanisms regarding activation and effector function on physiological processes in the first instance can provide us with an overview of the behaviour of specific factors involved in the regulation and maintenance of the inflammatory process and how it can lead to the development of chronic inflammation. this study provides information about the inhibitory activity of st on cytokine production (figure ) , possibly through the tlr signalling pathway regulation in lps-stimulated pbmc. moreover, a relatively low impact on il- β signalling pathway inhibition orchestrated by st in pbmc was observed ( figure ) ; possibly due to the various mechanisms of activation that remain unexplored on this signalling pathway, which could be cell subset-dependent. nevertheless, future studies focused on the identification of specific down-stream factors implicated in both il- β and lps signalling pathway activation to elucidate how the regulation of cytokine production takes place in distinct pbmc subpopulations will be required. therefore, our results not only provide valuable evidence about the potential use of the st chemical molecule to inhibit the inflammatory cytokine release in pbmc from hbd, but also leave open the possibility to study the effect of this molecule in chronic diseases, such as rheumatic and autoimmune diseases, in which the inflammatory process plays a critical role. regulation of cytokine production takes place in distinct pbmc subpopulations will be required. therefore, our results not only provide valuable evidence about the potential use of the st chemical molecule to inhibit the inflammatory cytokine release in pbmc from hbd, but also leave open the possibility to study the effect of this molecule in chronic diseases, such as rheumatic and autoimmune diseases, in which the inflammatory process plays a critical role. ten hdb over years of age were included in the study, five females and five males. a blood sample ( ml) was collected from each participant to obtain pbmc and perform cell culture experiments. the presence of infections and body mass index ≥ kg/m were taken as exclusion criteria. all subjects included in the present study signed the informed consent letter before their inclusion, and the present study was performed following the declaration of helsinki amendments. the pbmc were isolated from a blood sample following the density gradient separation method using histopaque ® - (sigma aldrich, st. louis, mo, usa; ρ . - . g/ml). the obtained pbmc were washed and resuspended in rpmi- medium supplemented with penicillin ( u/ml, sigma aldrich, st. louis, mo, usa) and streptomycin ( µg/ml, sigma aldrich, st. louis, mo, usa) at a concentration of % for both. the trypan blue test analysed the cell viability, and the total of separated cells per ml was quantified directly in a neubauer chamber. the pbmc were placed in -well plates after the density adjustment at × cells/ml (final volume of µl). cells were cultured in rpmi- medium without serum and supplemented with antibiotic and antifungal. untreated cells were taken as the control group for each experiment. the effect of lps ( ng/ml) in the secretion of tnf-α was studied in samples from four hbd to demonstrate the positive stimulation of the cells. the concentration of st necessary to inhibit the rhil- β and lps response in pbmc was determined at , , and µm. subsequently, pbmc culture was performed with lps ( ng/ml), lps ( ng/ml) plus st ( µm) or without stimulation (control group). similarly, pbmc were stimulated with rhil- β ( ng/ml), rhil- β ( ng/ml) plus st ( µm), st ( µm) alone or without stimulation (control group). st molecule was added to the corresponding well min before the stimuli with rhil- β or lps. each experiment was done with two biological replicates and was incubated for h at • c in a humidified % co atmosphere. once the incubation time had elapsed, supernatants were collected and stored at − • c for the subsequent quantification of pro-inflammatory and anti-inflammatory cytokine secretion. the concentration of il- β, tnf-α, ifn-γ, il- , il- , il- a, m-csf, gm-csf, il- , vegf, il- , il- , il- ra, il- , il- , il- , il- and il- and were quantified from culture supernatants by multiplex immunoassay method using the bio-plex pro tm human cytokine -plex assay (bio-rad laboratories, inc., hercules, ca, usa) according to the manufacturer's instructions. luminex magpix ® (luminex corporation, austin, tx, usa) instrument was used for xmap assays. all data were shown as medians and interquartile ranges. the differences between the non-parametric quantitative variables were analysed by u of mann-whitney test to compare two groups and both the kruskal-wallis test and the dunn's post hoc test for multiple comparisons. data analysis was performed using graphpad prism v . software, and a p-value < . was considered statistically significant. myd : a central player in innate immune signaling understanding early tlr signaling through the myddosome the il- family of cytokines and receptors in rheumatic diseases signaling in innate immunity and inflammation future potential therapeutic targets for ra the interleukin- receptor family toll-like receptors: critical proteins linking innate and acquired immunity toll-like receptors in immunity and inflammatory diseases: past, present, and future lps-induced cytokine production in human monocytes and macrophages lipopolysaccharide and il- β coordinate a synergy on cytokine production by upregulating myd expression in human gingival fibroblasts the il- β signalling pathway and its role in regulating pro-inflammatory and pro-labour mediators in human primary myometrial cells effect of pro-inflammatory/anti-inflammatory agents on cytokine secretion by peripheral blood mononuclear cells in rheumatoid arthritis and systemic lupus erythematosus elispot analysis of lps-stimulated leukocytes: human granulocytes selectively secrete il- , mip- beta and tnf-alpha increased production of il- and il- in lipopolysaccharide-stimulated peripheral mononuclears from patients with rheumatoid arthritis analysis of cytokines and chemokines produced by whole blood, peripheral mononuclear and polymorphonuclear cells discovery of small molecule inhibitors of myd -dependent signaling pathways using a computational screen the role of intermediary domain of myd in cell activation and therapeutic inhibition of tlrs toll-like receptors as therapeutic targets for autoimmune connective tissue diseases advance: inhibition of myd dimerisation and recruitment of irak and irak by a novel peptidomimetic compound pharmacological inhibition of tlr activation blocks autoantibody production in human b cells from sle patients targeting the 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induce innate il- production from gammadelta t cells, amplifying th responses and autoimmunity pivotal role of dermal il- -producing γδ t cells in skin inflammation critical regulation of early th cell differentiation by interleukin- signaling interleukin- β-induced irak ubiquitination is required for th-gm-csf cell differentiation in t cell-mediated inflammation pkc-alpha controls myd -dependent tlr/il- r signaling and cytokine production in mouse and human dendritic cells interleukin induces hypoxia-inducible factor in human gingival and synovial fibroblasts il- beta-mediated up-regulation of hif- alpha via an nfkappab/cox- pathway identifies hif- as a critical link between inflammation and oncogenesis regulation of hypoxia-inducible factor- α (hif- α) expression by interleukin- β (il- β), insulin-like growth factors i (igf-i) and ii (igf-ii) in human osteoarthritic chondrocytes dioscin reduces lipopolysaccharide-induced inflammatory liver injury via regulating tlr /myd signal pathway tlr promotes the expression of hif- α by triggering reactive oxygen species in cervical cancer cells in vitro-implications for therapeutic intervention effect of st on the proliferation and apoptosis of human hepatocellular carcinoma cells inhibition of myeloid differentiation factor (myd ) by st provides neuroprotection after experimental traumatic brain injury in mice myd inhibitor st suppresses the growth of lymphoma and leukaemia cells overview of the interleukin- family of ligands and receptors the interleukin (il)- cytokine family-balance between agonists and antagonists in inflammatory diseases interactive sites in the myd toll/interleukin (il) receptor domain responsible for coupling to the il beta signaling pathway peptide-mediated interference of tir domain dimerisation in myd inhibits interleukin- -dependent activation of nf-{kappa}b irak and traf knockdown in human chondrocytes inhibits interleukin- -induced matrix metalloproteinase- gene expression and promoter activity by impairing map kinase activation disrupting myddosome assembly in diffuse large b-cell lymphoma cells using the myd dimerisation inhibitor st neuron-specific effects of interleukin- β are mediated by a novel isoform of the il- receptor accessory protein a central nervous system-restricted isoform of the interleukin- receptor accessory protein modulates neuronal responses to interleukin- the death domain-containing protein unc cl is a novel myd -independent activator of the pro-inflammatory irak signaling cascade interleukin- family cytokines and mast cells: activation and inhibition car-t cell therapy causes inflammation by il- which activates inflammatory cytokine mast cells: anti-inflammatory role of il- induction of pro-inflammatory cytokines (il- and il- ) and lung inflammation by coronavirus- (covi- or sars-cov- ): anti-inflammatory strategies this article is an open access article distributed under the terms and conditions of the creative commons attribution the authors declare no conflict of interest.molecules , , key: cord- -iiqyzqsb authors: li, jin-ze; meng, shan-shan; xu, xiu-ping; huang, yong-bo; mao, pu; li, yi-min; yang, yi; qiu, hai-bo; pan, chun title: mechanically stretched mesenchymal stem cells can reduce the effects of lps-induced injury on the pulmonary microvascular endothelium barrier date: - - journal: stem cells int doi: . / / sha: doc_id: cord_uid: iiqyzqsb mesenchymal stem cells (mscs) may improve the treatment of acute respiratory distress syndrome (ards). however, few studies have investigated the effects of mechanically stretched -mscs (ms-mscs) in in vitro models of ards. the aim of this study was to evaluate the potential therapeutic effects of ms-mscs on pulmonary microvascular endothelium barrier injuries induced by lps. we introduced a cocultured model of pulmonary microvascular endothelial cell (ec) and msc medium obtained from mscs with or without mechanical stretch. we found that wright-giemsa staining revealed that msc morphology changed significantly and cell plasma shrank separately after mechanical stretch. cell proliferation of the ms-msc groups was much lower than the untreated msc group; expression of cell surface markers did not change significantly. compared to the medium from untreated mscs, inflammatory factors elevated statistically in the medium from ms-mscs. moreover, the paracellular permeability of endothelial cells treated with lps was restored with a medium from ms-mscs, while lps-induced ec apoptosis decreased. in addition, protective effects on the remodeling of intercellular junctions were observed when compared to lps-treated endothelial cells. these data demonstrated that the ms-msc groups had potential therapeutic effects on the lps-treated ecs; these results might be useful in the treatment of ards. to date, the emerging virus sars-cov- is causing a worldwide public health emergency; % critically ill patients developed acute respiratory distress syndrome (ards) [ ] . despite numerous efforts towards reducing mortality in established ards, in hospital mortality still remains near % [ ] . the main pathophysiology associated with ards in critically ill patients is the failure of pulmonary microvascular endothelium barrier integrity [ ] . therefore, maintaining the integrity of the endothelium barrier is critical for ards treatment. mesenchymal stem cell (msc) therapy is a potential method to treat ards [ ] , and our previous studies [ , ] have shown concrete benefits both in vitro and in vivo. however, clinical trials of allogeneic msc transplantation have provided conflicting evidences. in one trial, msc treatment in patients with ards produced a short-term improvement in oxygenation [ ] . yet, another trial demonstrated no significant difference in the -day mortality between patients treated with mscs and a control group ( % in the msc group versus % in the placebo group) [ ] . when injected intravenously, mscs preferentially homed to the lungs and engrafted at sites of injury in the pulmonary microvascular endothelium layer [ ] . the therapeutic function of mscs presents from the beginning of their engraftment on the endothelium layer to their merger with the layer [ ] . during this period, mscs are not only affected by biochemical factors but also by different kinds of mechanical stimulation coming from the microenviroment they have lived in [ ] . better integration of experimental and clinical data could provide further insight into the use of msc-based therapy in this setting. mechanical stimulation on the lung tissue exists constantly in physiological and pathological states, such as ards [ , ] . when utilized with mechancial ventilation to maintain essential oxygenation of ards patients, mechanical stimulation conducted through the lung tissue microenvironment varies from mild to severe levels and generates different degrees of lung compliance [ , ] . mechanical stretch may approximate the mechanical ventilation with low tidal volumes that are commonly used in the lungprotective mechanical ventilation required to treat ards previously [ ] and nowadays [ ] . when mscs are introduced into the lung microenvironment to treat ards, they have to encounter different degrees of mechanical stimulation. evidences have shown that mechanical stimulation can affect behavior of mscs, such as morphology [ ] , adhension [ ] , and differentiation [ , ] . therefore, mechanical stretch on mscs could play an important role on the treatment of lps-induced ec injuries. the aim of the this study was to present evidences of ms-msc therapeutic effects on ec injuries treated by lps. to test this hypothesis, we conducted a cocultured model of the ec and msc medium obtained from mscs with or without mechanical stretch. and, we evaluated the repair ability of the medium from mscs or ms-mscs on lps-induced ec injuries. first passage human bone marrow mesenchymal stem cells (mscs) were obtained from sciencell research laboratories (san diego, california, usa). the cells were characterised by the supplier. mscs were maintained in the mesenchymal stem cell medium (mscm; %fbs, % mesenchymal stem cell growth supplement, and % penicillin/streptomycin solution). the media were purchased from sciencell research laboratories (san diego, california, usa). cells were cultured at °c in an incubator with an atmosphere of % co air. every to days, cells were passaged when they reached - % confluency and passages from to of the cells were used for all experiments. mscs were preconditioned by mechanical stretch (ms) in vitro with a bioflex strain unit (bioflex, flexcell international corporation, hillsborough, nc, usa) as described previously [ ] . mscs were seeded onto a six-well plate containing flexible collagen type i-coated silicone rubber membranes at the bottom of each well and incubated at °c in % co atmosphere with % humidity (bioflex, flexcell international corporation, hillsborough, nc, usa). mscs were cultured for or days to reach - % confluency and subjected to mechanical stretch of % or % elongation for h or h using a computer-controlled vacuum stretch apparatus (fx- tension plus system, flexcell international corporation, hillsborough, nc, usa). the untreated msc group did not receive mechanical stretch and was incubated in the same incubator. mscs and supernatant from all groups were collected at scheduled time points and prepared for use in this study. supernatants from the stretched msc and control groups were collected and centrifugated to remove dead cells and cell debris. cell and dermal fibroblast culture. first passage human pulmonary microvascular endothelial cells (ecs) and human dermal fibroblasts (hdf) were obtained from sciencell research laboratories (san diego, california, usa), and the cells were cultured in an endothelial cell medium (ecm; %fbs, % endothelial cell growth supplement, and % penicillin/streptomycin solution) and fibroblast medium (fm; %fbs, % fibroblast growth supplement, and % penicillin/streptomycin solution), respectively. all mediums were purchased from sciencell research laboratories (san diego, california, usa). cells were cultured at °c in the incubator with an atmosphere of % co air. every to days, cells were passaged when they reached - % confluency. system. ecs at a density of , per well were seeded in the upper chambers ( . μm pore size polyester membrane from corning, inc.) and cultured for to days to produce a confluent monolayer, and mscs were seeded in the lower chambers,. then, cells were treated with lps ( ng/ml, sigma) for hours before permeability was tested, as previously described [ ] . after adding μl kda fluorescein isothiocyanate-(fitc-) dextran (sigma-aldrich) to each upper insert and incubating for minutes in an incubator, μl medium from the upper and lower chambers was withdrawn. then, the medium was transferred to a -well plate and read using excitation and emission wavelengths of nm and nm, respectively. to observe cell morphology, cells were stained with wright-giemsa stain (sigma aldrich) according to the manufacturer's protocols as previously described [ ] . after air drying the wells, mscs were inspected under a light microscope (olympus, tokyo, japan). assay. untreated and mechanically stretched mscs were seeded at cells per well onto well plates and cultured in an incubator with a humidified atmosphere of % co at °c. μl of cell counting kit- (cck- ) solution (beyotime, china) was added per well, and cells were cultured for hour at °c, before measuring absorbance at nm with a microplate reader. . . identification of mscs by flow cytometry. untreated and mechanically stretched mscs were identified by flow cytometry (bd bioscience, san diego, ca) as described previously [ ] . harvested mscs were washed with pbs and resuspended to × cells/ml, and μl of cell suspension was incubated with fluorescein-conjugated monoclonal antibodies against cd , cd , and cd (bd pharmingen, san diego, ca), respectively. samples were mixed in the dark stem cells international for minutes, then resuspended and centrifuged at rpm for minutes at room temperature. supernatants were removed, and cells were resuspended with pbs to μl for flow cytometry analysis. . . enzyme-linked immunosorbent assay. the supernatants from all msc groups were collected and centrifuged to remove cell fragments. levels of tumor necrosis factor (tnf-α), interleukin- (il- ), and interleukin- (il- ) in the culture medium were detected by elisa (excellbio, shanghai, china). all tests were performed according to the manufacturer's instructions. all samples were examined in duplicate. ecs were seeded in the upper chamber in -well culture plates ( . μm pore size polyester membrane from corning, inc.) and cultured for to days to produce a confluent monolayer. then, cells were treated with lps ( ng/ml, sigma) for hours before permeability was tested, as previously described [ ] . after adding μl kda fluorescein isothiocyanate-(fitc-) dextran (sigma-aldrich) to each upper insert and incubating for minutes in an incubator, μl medium from the upper and lower chambers was withdrawn. then, the medium was transferred to a -well plate and read using excitation and emission wavelengths of nm and nm, respectively. an annexin v-fitc assay kit (sigma-aldrich) was used to assess the percentage of ecs undergoing apoptosis, according to the manufacturer's instructions. ecs were harvested and washed with pbs and suspended in x binding buffer at a cell concentration of × cells/ml. then, μl propidiumiodide solution (pi) and μl annexin v-fitc conjugate (annexin v) were added to each sample and gently mixed. after minutes incubation in the dark at room temperature, samples were analyzed using a flow cytometer (bd biosciences, usa). western blotting was used to detect the expression of ve-cadherin and connexin- on ecs as previously described [ ] . total proteins from ecs after different treatments were extracted with ripa lysis buffer (beyotime institute of biotechnology, shanghai, china) supplemented with mmol/l phenylmethylsulfonyl fluoride (pmsf), and then separated with % sodium dodecyl sulphate-polyacrylamide gel electrophoresis and transfered onto polyvinylidene fluoride membranes (beyotime, china). afterwards, membranes were blocked in % bsa for hours at room temperature and incubated at °c overnight with primary antibodies against ve-cadherin (abcam) or connexin- (cell signaling technology). the next day, membranes were washed in tbs-t and incubated in hrpconjugated secondary antibody (boster biotechnology, wuhan, china) for hour at room temperature. then, ecl (beyotime, china) was applied to detect the bands with a chemiluminescence imaging system (chemiq mini; ouxiang, china). . . immunofluorescence staining. in a transwell system, ecs were seeded on the upper inserts and cultured to form a confluent monalayer for or days. after treating with lps for hours, cells were then washed with cold pbs and fixed with % paraformaldehyde for minutes. samples were permeabilized with . % triton x- for minutes, blocked with % bovine serum albumin (bsa), and incubated overnight with ve-cadherin primary antibody (ab) ( : rabbit polyclonal anti-ve-cadherin) (abcam, ab , ireland) at °c. after incubation for hours, samples were incubated with a secondary fitc-conjugated goat anti-rabbit igg ( : alexa fluor igg) (biosciences, ireland) and stained with (vwr, ireland) for h at room temperature. cell nucleis were stained with dapi (vwr, ireland) for min at room temperature, washed in pbs, and imaged using confocal microscopy (leica sp , ireland). . . statistical analyses. statistical analyses were performed using the spss . software package (spss inc., chicago, il, usa). results were presented as the mean ± standard deviation. group comparison was analyzed by one-way analysis of variance, followed by tukey's test. p < : was considered statistically significant. to determine if mscs protected ecs from lps-induced injury, we used a transwell coculture system (figure (a)) to assess paracellular permeability when mscs were added at varying seeding concentrations, from × cells per well to × cell per well. permeability significantly decreased when mscs were plated at × cell per well (figure (b); * p < : ) and decreased further as the density of mscs increased. this suggested that the therapeutic effect of mscs on endothelial cell permeability improved as the density of mscs increased. mscs were seeded on six-well mechanical stretch plates with collagen type i-coated flexible silicon rubber membranes placed at the bottom of each well and were preconditioned by mechanically stretching these plates during cell culture (figures (a) and (b) ). an example of a six-well mechanical stretch plate is presented in figure (c). mscs were plated on the silicon rubber membrane and stained with wright-giemsa stain. a schematic view of a well under mechanical stretch is presented in figure (d). the first column shows the side view of two wells containing either untreated or mechanically stretched mscs. the second column presents an illustration of untreated or mechanically stretched mscs, respectively. stem cells international following treatment (figure (a) ). cells in all groups remained firmly adhered to the seeding surface. compared to untreated mscs, the ms-mscs showed signs of atrophy, appearing thinner and flattened, and have increasingly shrunk in a time-and magnitude-dependent manner. moreover, cell proliferation significantly increased in the ms- % groups (figure (b); * p < : ) but decreased in the ms- % groups ( * * p < : ). proliferation in the ms- %- h group was significantly lesser than that in the ms- %- h group ( * p < : ). these data suggest that ms affected the morphology and proliferation of mscs significantly. markers on mscs. surface markers on mscs served as an index parameters for the identification of mscs [ ] . to determine if surface marker expression changed when mscs were preconditioned to mechanical stretch, we used flow cytometry to analyze major surface markers of mscs for identification, such as cd , cd , and cd ( figure ) . the results showed no statistical change in the expression with high levels of cd and cd expressions on nearly % of cells in all treatment groups and low levels of cd expression on fewer than % of cells for all treatment groups. the results suggest that ms did not alter the expression of surface markers. studies have shown that ms can induce biological function change [ ] . to evaluate the effects of ms on the inflammatory function of mscs, we examined the inflammatory mediators tnf-α, il- , and il- presented in the msc supernatants by enzyme-linked immunosorbent assay. the results showed that tnf-α and il- increased statistically as time and magnitude of mechanical stretch increased (figures (a) and (b); * p < : ), but the ms- %- h group did not produce significant differences when compared to the untreated msc group. however, il- did not significantly change in all groups ( figure (c) ). these results showed that ms could statistically increase the tnf-α and il- levels. stem cells international in assessing the integrity of the pulmonary microvascular endothelium barrier [ ] . we introduced a transwell coculture system to evaluate the effects of ms-mscs on the paracellular permeability of lps-treated ecs. treatment with lps significantly increased the paracellular permeabil-ity of the pulmonary microvascular endothelium barrier (figure (a); * * p < : ). and mscs significantly attenuated the increased paracellular permeability induced by lps (figure (a); * p < : ), while hdf showed no effect on the increased permeability. these results suggested that lps is a useful agent to induce injury and apoptosis on pulmonary microvascular endothelial cells [ ] . in this study, we applied the flow cytometry to evaluate the effect of mscs on apoptosis of ecs treated with lps (figure (a) ). lps could significantly induce the apoptosis of ecs both in early and late states (figures (b) and (c); * * p < : ), but mscs decreased the apoptosis of lps-treated ecs ( * p < : ). furthermore, the ms- %- h msc group could significantly attenuate both early and late apoptosis of ecs ( * p < : ), similar to the untreated msc group (figure (b) ). however, the ms- %- h group significantly decreased early apoptosis ( * p < : ) but not late apoptosis of ecs, although it showed a trend towards attenuating apoptosis (figure (c) ). intercellular junction proteins play an important role in maintaining the integrity of the pulmonary microvascular endothelium barrier. ve-cadherin [ ] and connexin- [ ] present critical effects on regulating the permeability of the barrier. to investigate the effects of ms-mscs on endothelium barrier integrity, we examined the expression of these two key proteins. compared with the lps-treated ecs, mscs increased the expression of ve-cadherin and connexin- (figures (b) and (c); * * p < : ). we also applied immunofluorescent staining to detect the protein expression of ecs and observed the cells under confocal microscopy. the results showed that ve-cadherin located on the surface of ecs were destroyed after lps treatment, thus leading to the loss of integrity of the pulmonary microvascular endothelium barrier (figure ). these data indicated that ms-mscs restored the intercellular junction of lpstreated ecs. ards is the leading cause of mortality in icu patients [ ] and featured with acute diffuse lung injury, which results in severely injured lung compliance and increased pulmonary vascular permeability [ ] . msc is a promising method to restore endothelial function [ ] , but when engrafted on the alveolocapillary barrier, the efficacy of mscs under mechanical stretch in the context of decreased lung compliance remains unproven. our study tried to reveal the effect of mechanically stretched mscs on restoring the injured alveolocapillary barrier. we applied a mechanical stretch system to simplify yet still mimic the mechanical microenviroments present within the lung in a simplified way. we demonstrated that mechanical stretch could impact msc morphology and biological function in a time-and magnitude-dependent manner and that ms-mscs could restored the increased permeability of endothelial cells induced by lps. the alveolocapillary barrier provides an essential function in regulating the diffusive exchange of molecules. loss of barrier integrity could lead to excessive leakage of fluid and proteins from the vasculature to the alveoli, producing the pulmonary edema common in ards [ ] . sepsis plays a major role in extrapulmonary edema, and the endothelial barrier stands as the first line of defense in keeping lps out of the vascular system [ ] . studies have demonstrated that endothelial injury is a more important consideration in extrapulmonary ards than pulmonary ards [ , ] . as a major factor driving sepsis and lethal septic shock, lps has been studied in in vivo, in vitro, and ex vivo settings [ , ] . hereby, we adopted lps and a transwell coculture system to investigate the effects of mscs on the permeability of the alveolocapillary barrier. we found that increased permeability by lps was significantly decreased by mscs as the cell density increased accordingly. manipulation of mesenchymal stem cell functions is important for tissue engineering and regenerative medicine. heterogeneous mechanical properties of the alveolocapillary barrier in ards caused a complicated microenviroment for the engraftment of mscs [ ] . so, we used an apparatus to mimic and simplify the mechanical properties within the lung tissue in clinical field, as % mechanical stretch for physical stimulation and % for severe pathological status. our previous research had applied this method and acquired positive therapeutic results of pulmonary fibrosis investigation [ ] . in this study, we tried to discover evidences of mechanically stretched mscs in restoring increased permeability of endothelial barrier induced by lps. while mscs injected via the bloodstream preferred to engraft on and merge into the injured sites of the pulmonary microvascular endothelial barrier [ ] . studies have proved that mscs can coexist with endothelial cells and other kind of cells in the barrier for about to hours. [ , ] therefore, we investigated mscs under these time durations of % and % mechanical stretch as used previously [ ] . we demonstrated that mechanical stretch affects cell morphology and cell proliferation, suggesting that mechanical stretch is important for the maintenance of msc functions. the most attractive charateristics of mscs are the stemness and self-renewal. these properties make them a promising therapeutic tool in many clinical field, such as the kidney [ ] , liver [ ] , and lung [ ] . the stemness of ms-mscs is analyzed by the expression of surface markers. when under the mechanical stretch modes in this study, whether mscs could maintain their stemness is crucial for the lps-treated ec therapy. the expression of cd , cd , and cd did not change with the intervention of different mechanical stretch patterns. all groups exhibit similar expression of the surface markers. the results indicate that, in h with the maximum ms- %, the mscs could maintain the stemness. increased permeability resulted from the disruption of the pulmonary endothelial barrier [ ] . ec apoptosis played stem cells international a vital role in ec barrier integrity [ ] we have been proved that mscs without mechanical stretch could repair the injured ec barrier in a cell density-dependent manner. nowadays, tissue engineering shows great impact on msc therapy and achieved great advances [ ] . mechanical stretch, a method to mimic the mechanical properties of the lung tissue, played an important role in the microenviroment where mscs engrafted. novel strategies to isolate the mechanical factor could shed some light on msc application. our results indicated that lps can induce both early and late apoptosis, and mechanically stretched mscs decreased ec apoptosis but decreased slightly as the time and magnitude of ms increased. thus, mechanical stretch may account for the restoring effect on the injured ec barrier through ec apoptosis. constant remodeling of intercellular junctions to regulate the transendothelial permeability is essential in maintaining endothelium barrier functions. treatment with lps can also alter the apoptotic status of endothelial barrier cells and badly damage the paracellular architecture of causing the endothelial barrier to function abnormally and producing pulmonary edema [ ] . of the intercellular juntion proteins, vecadherin and connexin- are vital for the barrier integrity and could act as index parameters to evaluate the disruption of barrier [ ] . the results presented that endothlial barrier integrity was severely damaged by lps, but ms-mscs increased ve-cadherin and connexin- expressions, which favored the integrity of the endothelial barrier. therefore, ms-mscs restored the increased permeability of the endothelial cell partially by remodeling of ve-cadherin and connexin- . the following limitations to this research should be noted. the mechanical stretch system that we used in this study applies a vacuum to generate mechanical stretch, but as the culture medium is flowing between the mscs, other categories of mechanical stimulations, including shear force and pressure force, are not absent and may influence cells in some level. these forces could also produce a biological response and may thus affect msc function. however, by their nature, these three forces are often mixed together and are difficult to study separately. this could be addressed in future studies. in conclusion, our experiments reveal that ms-mscs statistically improved the increased permeability of the ec barrier induced by lps, through decreasing the apoptosis of ecs and increasing the remodeling of intercellular junctions. these findings provide additional in vitro evidences for the therapeutic potential of mscs and may be useful for the clinical utilization of mscs. endothelial cells ards: acute respiratory distress syndrome mscs: mesenchymal stem cells ms: mechanical stretch lps: lipopolysaccharide elisa: enzyme-linked immunosorbent assay fitc: fluorescein isothiocyanate tnf-α: tumor necrosis factor-alpha il: interleukin ve-cadherin: vascular endothelial-cadherin. the data used to support the findings of this study are included within the article. the authors declare that they have no competing interests. epidemiological and clinical characteristics of cases of novel coronavirus pneumonia in wuhan, china: a descriptive study epidemiology, clinical course, and outcomes of critically ill adults with covid- in new york city: a prospective cohort study mind the gap: mechanisms regulating the endothelial barrier fifty years of research in ards. cell-based therapy for acute respiratory distress syndrome. biology and potential therapeutic value the hepatocyte growth factorexpressing character is required for mesenchymal stem cells to protect the lung injured by lipopolysaccharide in vivo mtor/stat- pathway mediates mesenchymal stem cell-secreted hepatocyte growth factor protective effects against 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acute respiratory distress syndrome: pathophysiology, monitoring, and therapeutic interventions surviving sepsis campaign: guidelines on the management of critically ill adults with coronavirus disease (covid- ) mechanical stimuli differentially control stem cell behavior: morphology, proliferation, and differentiation mechanically induced formation and maturation of d-matrix adhesions ( dmas) in human mesenchymal stem cells response of mesenchymal stem cells to the biomechanical environment of the endothelium on a flexible tubular silicone substrate mechanical stimulation induces morphological and phenotypic changes in bone marrow-derived progenitor cells within a three-dimensional fibrin matrix mechanical stress and the induction of lung fibrosis via the midkine signaling pathway co-regulation of transcellular and paracellular leak across microvascular endothelium by dynamin and rac fip is required for the cellautonomous maintenance of fetal hematopoietic stem cells minimal criteria for defining multipotent mesenchymal stromal cells. the international society for cellular therapy position statement interaction between mesenchymal stem cells and endothelial cells restores endothelial permeability via paracrine hepatocyte growth factor in vitro preconditioning of human mesenchymal stem cells to enhance their regulation of the immune response bone marrow derived mesenchymal stem cells inhibit inflammation and preserve vascular endothelial integrity in the lungs after hemorrhagic shock asef mediates hgf protective effects against lps-induced lung injury and endothelial barrier dysfunction endothelial connexin mediates acidinduced increases in pulmonary microvascular permeability epidemiology, patterns of care, and mortality for patients with acute respiratory distress syndrome in intensive care units in countries mesenchymal stem cells: mechanisms of potential therapeutic benefit in ards and sepsis respiratory distress syndrome, stat-pearls human lipopolysaccharide models provide mechanistic and therapeutic insights into systemic and pulmonary inflammation report from an nih-nhlbi workshop extravascular lung water in critical care: recent advances and clinical applications mechanisms differential role for p -catenin in regulation of tlr signaling in macrophages stem cells, cell therapies, and bioengineering in lung biology and diseases mesenchymal stem cell-based therapy for kidney disease: a review of clinical evidence strategies to improve the efficiency of mesenchymal stem cell transplantation for reversal of liver fibrosis treatment of acute lung injury: clinical and experimental studies acute respiratory distress syndrome: advances in diagnosis and treatment modulating the stem cell niche for tissue regeneration signaling mechanisms regulating endothelial permeability the authors would like to thank dr. ruoyu hu for his valuable support. key: cord- -xtsnlu f authors: drago-serrano, maria elisa; campos-rodríguez, rafael; carrero, julio césar; de la garza, mireya title: lactoferrin: balancing ups and downs of inflammation due to microbial infections date: - - journal: int j mol sci doi: . /ijms sha: doc_id: cord_uid: xtsnlu f lactoferrin (lf) is a glycoprotein of the primary innate immune-defense system of mammals present in milk and other mucosal secretions. this protein of the transferrin family has broad antimicrobial properties by depriving pathogens from iron, or disrupting their plasma membranes through its highly cationic charge. noteworthy, lf also exhibits immunomodulatory activities performing up- and down-regulation of innate and adaptive immune cells, contributing to the homeostasis in mucosal surfaces exposed to myriad of microbial agents, such as the gastrointestinal and respiratory tracts. although the inflammatory process is essential for the control of invasive infectious agents, the development of an exacerbated or chronic inflammation results in tissue damage with life-threatening consequences. in this review, we highlight recent findings in in vitro and in vivo models of the gut, lung, oral cavity, mammary gland, and liver infections that provide experimental evidence supporting the therapeutic role of human and bovine lf in promoting some parameters of inflammation and protecting against the deleterious effects of bacterial, viral, fungal and protozoan-associated inflammation. thus, this new knowledge of lf immunomodulation paves the way to more effective design of treatments that include native or synthetic lf derivatives, which may be useful to reduce immune-mediated tissue damage in infectious diseases. lactoferrin (lf) is a conserved iron-binding mammalian glycoprotein with antimicrobial activity, present in secretions that recover mucosal sites regarded as portals of entry and/or invasion of pathogenic agents [ ] . antimicrobial activity has been mostly characterized in lf of bovine and human origin isolated from milk [ , ] . mechanisms underlying the antimicrobial action of lf result from both direct (microbiostatic and/or microbicidal) and indirect (immunomodulatory) effects [ ] [ ] [ ] . at present, the therapeutic and prophylactic treatments for microbial infections that ameliorate both the antibiotic multiresistance and the inflammatory response have prompted the searching of agents that display both antimicrobial and modulatory properties such as lf. this review is focused on the modulatory impact of lf on the inflammatory response induced by infectious microorganisms, mainly in the lf was initially named lactotransferrin, due to being a milk glycoprotein that chelates iron. this protein belongs to the transferrin family, which includes the avian egg ovotransferrin (ovotf) and the mammalian serum and lymph transferrin (tf), but differs from other members of the family in its higher affinity for iron. lf is synthesized by the mammary gland and then it is abundant in colostrum and milk, through which it has been suggested to participate in the initial protection in newborns [ ] [ ] [ ] [ ] [ ] [ ] . regarding the content, human mature milk is highly enriched in lf ( . mg/ml) in comparison with bovine milk ( . mg/ml) [ ] [ ] [ ] . the amino acid sequences of both proteins exhibits approximately % identity [ , ] . lf is also present in many fluids and exocrine secretions, such as tears, saliva, and mucosal surfaces of the respiratory, urinary-reproductive and intestinal tracts; in these sites, lf contributes to the primary innate-immune defense system of mammals that exerts antimicrobial activity against an extensive variety of pathogens [ , , [ ] [ ] [ ] [ ] [ ] . lf is also synthesized during the natural cellular development of promyelocytes to myelocytes, and was early recognized as an important component of the secondary granules of polymorphonuclear (pmn) neutrophils [ , ] . these cells store lf ( - μg/ neutrophils) and release it at the sites of infection, which are acidic due to the activity of pathogens [ , , , ] . in plasma, lf derives from neutrophils and its concentration is very low ( . - μg/ml) [ ] ; nevertheless, in patients with sepsis the degranulation of activated neutrophils leads to secretion of significant levels of lf (~ . mg/ml) into the bloodstream [ ] . neutrophils also release lf in feces whose concentrations markedly increase during inflammatory processes such as inflammatory bowel disease (ibd), ulcerative colitis and crohn's disease, due to the response against pathogenic bacteria [ ] . lf was initially named lactotransferrin, due to being a milk glycoprotein that chelates iron. this protein belongs to the transferrin family, which includes the avian egg ovotransferrin (ovotf) and the mammalian serum and lymph transferrin (tf), but differs from other members of the family in its higher affinity for iron. lf is synthesized by the mammary gland and then it is abundant in colostrum and milk, through which it has been suggested to participate in the initial protection in newborns [ ] [ ] [ ] [ ] [ ] [ ] . regarding the content, human mature milk is highly enriched in lf ( . mg/ml) in comparison with bovine milk ( . mg/ml) [ ] [ ] [ ] . the amino acid sequences of both proteins exhibits approximately % identity [ , ] . lf is also present in many fluids and exocrine secretions, such as tears, saliva, and mucosal surfaces of the respiratory, urinary-reproductive and intestinal tracts; in these sites, lf contributes to the primary innate-immune defense system of mammals that exerts antimicrobial activity against an extensive variety of pathogens [ , , [ ] [ ] [ ] [ ] [ ] . lf is also synthesized during the natural cellular development of promyelocytes to myelocytes, and was early recognized as an important component of the secondary granules of polymorphonuclear (pmn) neutrophils [ , ] . these cells store lf ( - µg/ neutrophils) and release it at the sites of infection, which are acidic due to the activity of pathogens [ , , , ] . in plasma, lf derives from neutrophils and its concentration is very low ( . - µg/ml) [ ] ; nevertheless, in patients with sepsis the degranulation of activated neutrophils leads to secretion of significant levels of lf (~ . mg/ml) into the bloodstream [ ] . neutrophils also release lf in feces whose concentrations markedly increase during inflammatory processes such as inflammatory bowel disease (ibd), ulcerative colitis and crohn's disease, due to the response against pathogenic bacteria [ ] . physiologically, lf can be found as a fully iron-loaded (holo-lf) or iron-free protein (apo-lf) [ , [ ] [ ] [ ] . the holo-lf is conformationally more rigid and is more resistant to denaturation and proteolysis than the apo-lf, but instead, apo-lf is generally more effective against bacteria than holo-lf [ , [ ] [ ] [ ] [ ] . in this regard, it has been reported that holo-lf can be utilized as an iron source by several groups of microorganisms [ , [ ] [ ] [ ] . however, this is not always the case because studies on intestinal epithelial-barrier function and mucosal inflammation carried out in a caco- cells model and macrophages activated with lipopolysaccharide (lps) showed that both lf forms effectively inhibited the pro-inflammatory response. nevertheless, apo-lf was more effective in downregulating inflammation, probably due to its ability to bind and neutralize lps, as well as to neutralize microbial-derived antigens, thereby potentially reducing their pro-inflammatory effect [ ] . much evidence exists of the successful experimental use of lf from different origins (human, bovine, porcine, caprine, camelid, and buffalo) against the growth of diverse pathogens. as mentioned above, most results indicate that lf from different origins can exert bacteriostatic effects due to its iron-chelating activity, but it can also be bactericidal due to its interaction with lps and porins in gram-negative bacteria, or with teichoic acids in gram-positive bacteria. these interactions lead to membrane damage and bacterial death [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . moreover, the antimicrobial activity of lf is also highly dependent on its cationic properties, because the addition of positive charges to lf via amidation enhances its antibacterial and antiviral properties and, in contrast, the addition of negative charges by acylation abolishes them [ ] . as mentioned, lf displays antiviral properties against common virus infections. these antiviral properties are related to its ability to block the cellular attachment or replication of virus by inducing type i interferons (α/β) with antiviral action [ ] . thus, lf from diverse mammals shows a potent activity against replication of human immunodeficiency virus, cytomegalovirus, and hepatitis c virus [ , ] . less information exists about the microbicidal action of lf against fungi and protozoa [ ] [ ] [ ] [ ] . very important is the finding that lf synergizes with antibiotics and drugs, and even with other proteins of the innate immune system such as lysozyme and natural secretory iga (siga) antibodies, potentiating the antimicrobial effect [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the largest external source of lf is milk consumption. when consumed, lf can be enzymatically cleaved by pepsin in the stomach and by trypsin in the small intestine. in adults, whereas hlf is completely degraded, about % of blf resists proteolytic digestion mediated by pepsin [ ] . noteworthy to mention is that digestive tract of babies and infants has a relatively high ph and secretes low levels of pepsin, which allows for the innocuous transit of hlf and blf into epithelial cells, which can be extremely important at this stage of life [ , ] . nevertheless, native hlf derived from pancreatic juices and neutrophils is discharged into exocrine secretions of mucosal surfaces in adults, and thus it acts protecting those sites from invaders. in addition, diverse biological activities of lf including the facilitation of iron absorption, modulation of mucosal immunity and stimulation of mucosal differentiation result of its interaction with lf receptors (lfrs) expressed in the gastrointestinal cells [ , ] . one reason why lf can be used as a pharmaceutical is because its activity is maintained in some of its component peptides after being cleaved by proteolytic enzymes, e.g., the peptides derived from the n terminus of lf by pepsin, so-called lactoferricins (lfcins), lack the iron-chelating activity, and are characterized by their strong cationic charge. remarkably, lfcins often show a higher microbicidal activity than the parental lf as well as synergistically act with drugs and antibiotics against microbes [ , , ] . moreover, several lfcins have been synthesized and experimented against microbes [ ] . on the other hand, synthetic lfcin - , lactoferrampin (lfampin - ), and a fusion peptide of both called lfchimera, have been successfully assayed against multiresistant bacteria, and also against bacteria that typically form biofilms [ , ] . this synthetic lfchimera has also been shown to be effective against parasitic protozoa [ ] [ ] [ ] . another synthetic lfchimera prepared by the fusion between lfcin - and lfampin - , was effective against pseudomonas aeruginosa by down-regulating pyocyanin, elastase and biofilm formation [ ] . the presence of lf in secretions and its various mechanisms of action allow this glycoprotein to combat all types of microbes that colonize mucosae in the different bodily regions. however, depending on the site, microbes can be exposed to different concentrations of lf, to complexes of lf with other proteins, or to diverse levels of lf derivatives [ ] . at the same time, lf can help against the inflammatory process produced by strong immune reaction in infections. therefore, all findings on lf activities suggest that lf and lfcins can be of potential use as antimicrobial and anti-inflammatory compounds, either alone or as adjuncts to conventional antibiotics and drugs. in this sense, lf is one of the most studied proteins since the commercial point of view, being highly appreciated as a nutraceutical in some countries, promoted as a supplement in diarrheic diseases, cancer, increasing immunity, improvement of memory, and several other conditions. human lf has been cloned in different vectors and expressed as recombinant (r-hlf) overall in eukaryotic systems which can glycosylate it, such as yeasts and fungi [ , ] ; however, the best product is obtained from transgenic cows and plants [ ] [ ] [ ] . interestingly, r-hlf expressed in the cow mammary gland, enhanced systematic and intestinal immune responses in piglets used as a model of infants [ ] . in addition, when the meat from the progeny of hlf transgenic cows was analyzed, no abnormalities of its nutrient composition were found [ ] . thus, the wide use of lf in human health care is promissory. next, we will review the effects of lf as an anti-inflammatory protein in a number of infectious diseases in which it has been studied, mainly of gastrointestinal and respiratory tracts. inflammatory response is elicited by germ-line encoded pattern-recognition receptors (prrs) expressed in many cell types that interact with their ligands from exogenous or endogenous origin, namely pathogen-associated molecular patterns (pamps), or danger-associated molecular patterns (damps), respectively. some prrs comprise a large family of receptors such as toll-like receptors (tlrs) [ ] [ ] [ ] . upon ligand binding, tlrs lead to signaling pathways resulting in the activation and translocation of the nuclear factor (nf)-κb to the nucleus. nf-κb modulates the expression of pro-inflammatory cytokines such as interleukin (il)- , il- , type-i interferon (ifn-α, and ifn-β), tumor necrosis factor (tnf) α, as well as chemoattractant cytokines (chemokines). another class of prrs includes nod-like receptors (nlrs), some of which, such as nlrp , nlrp and nlrp , function as sensors or adaptors forming the "inflammasomes" [ ] . activation of inflammasomes by pamps and/or damps induces signal pathways resulting in the activation of caspase- that cleaves the inactive pro-forms of cytokines (il- , and il- ) to generate their active forms. besides to generate active pro-inflammatory cytokines, some inflammasomes regulate cell death in response to microbial and endogenous danger signals [ ] [ ] [ ] [ ] . although lf displays direct microbiostatic and/or microbicidal activities, indirect antimicrobial mechanisms have also been ascribed to its capability of modulating a wide array of humoral and cellular components of the innate and adaptive immunity [ , ] . immunomodulatory role of lf is due, in part, to its interactions with cell surface receptors that favor either elicitation of signal pathways, or lf translocation into nucleus and gene targeting [ ] [ ] [ ] . a summary of the modulatory effects of lf on inflammation due to microbial infections is shown in table . gastrointestinal infections blf treatment of cultured cells infected with e. coli lf and biopsies from patients with crohn's disease ↓il- , ↓il- and ↓tnfα mrna expression [ ] blf treatment of intestinal cell cultures infected with e. coli lf isolated from crohn's disease patients ↑ferroportin (fpn) in infected cells suggesting that blf action on inflammatory response in epithelial cells involves the iron homeostasis [ ] blf-nanoparticles (blf-nano) administration to balb/c mice infected with s. enterica serovar typhimurium ↑tnf α, ↑interferon (ifn) β and ↑ifniii levels (proinflammatory cytokines) [ ] blf administration to c h/hej mice infected with entamoeba histolytica (e. histolytica) ↑il- (th ), ↑il- , ↑iga ↓damage and ↓inflammation [ ] blf treatment to balb/c mice infected with helicobacter pylori (h. pylori) ↓gastric colonization and ↓inflammation (histopathology score) [ ] blf treatment of rotavirus infection children ↔ifnγ, ↔il- and ↔rotavirus incidence in children whether fed or unfed with blf [ ] gut-related systemic infections (sepsis) ↓lung infection, ↑ifnγ, ↑il- in spleen cell cultures, ↓tnfα and ↓il- β correlated with ↓lung pathology. ↑lymphocytic recall response towards bcg [ , ] recombinant human lf mixed with bcg vaccine in mice infected with m. tuberculosis early↑ and late↓ of pro-inflammatory cytokines that correlated with the ↓lung pathology [ ] blf effect in enhancing bcg vaccine by oral route in mice infected with m. tuberculosis ↓colony forming units (cfu) and ↓inflammation in the lungs, ↑ifnγ producing t cd and cd cells and ↑il- lymphocytes [ ] blf effects on cystic fibrosis and bronchial ib - cell cultures infected with burkholderia cenocepacia (b. cenocepacia) ↓il- β (pro-inflammatory cytokine), ↓il- (anti-inflammatory cytokine) [ ] blf administration to a murine model of lung injury by lps ↓bronchioalveolar leukocytes, ↓tnf-α, ↓myeloperoxidase (mpo) activity, ↑il- , ↓lung edema and inflammation [ ] blf administration to a murine model of respiratory syncytial virus infection ↔viral loads and ↔lung inflammation [ , ] blf administration to a murine model of influenza ↔viral load and ↔ifnγ, il- and il- in the lungs [ ] other mucosal and systemic sites blf effects on mammary gland in cows with staphylococcous aureus (s. aureus) mastitis ↓bacterial load, ↑c levels, ↓tnfα mrna expression via nuclear factor κb (nfκb) inhibition, ↑curation, ↑proinflammatory cytokines is correlated with ↑peptides derived from blf-elastase proteolysis [ ] [ ] [ ] [ ] blf effects on oral candidiasis in immunosuppressed mice infected with candida albicans (c. albicans) blf blocked the suppressive effects of candidiasis in polymorphonuclear (pmn) neutrophils; ↑ifnγ and tnfα production in cervical lymph nodes [ ] blf effects on hamsters with amoebic liver abscess by e. histolytica no damage or inflammation in the liver [ ] human lf (hlf) effects on balb/c mice infected with listeria monocytogenes (l. monocytogenes) ↓bacterial load and ↓necrotic foci in the liver, ↔necrotic foci in the spleen, ↓tnfα, il- β and ifnγ mrna [ ] hlf and peptide-hlf derivatives administration to c h/tif mice infected with e. coli o k uropathogenic strain ↓bacterial load in the bladder and kidneys, ↓leukocyte in urine, ↓urinary il- levels at h and systemic il- levels at h post-infection [ ] hlf expressing transgenic mice infected with s. aureus ↓bacterial growth, ↓septicemia, ↓mortality than congenic litter mates. ↑th polarization in the spleen, given that: ↑tnfα and ↑ifnγ, ↓il- and ↓il- upon stimulation ex vivo with exotoxin toxic shock syndrome toxin- compared with congenic controls [ ] ↓ decrease; ↑ increase; ↔ no changes. throughout the gastrointestinal tract, lf is present as an iron-binding multifunctional glycoprotein regarded as a natural compound able to inhibit the pathogens growth. lf is also able to up-and down-modulate both humoral and cellular components of immunity involved in the regulation of the inflammatory response having a key role in maintaining gut homeostasis [ ] . balance of homeostasis results from the tight regulation of several events, since too little inflammation disrupts the process of tissue repairing and remodeling, whereas too much inflammation entails collateral impact by causing tissue damage with life-threatening consequences [ ] . mucosal compartment of the small intestine is a scenario where takes place a physiologic inflammatory response orchestrated by innate and adaptive mechanisms mediated by intestinal epithelial cells and by a wide array of immunocompetent cells at lamina propria, such as dendritic cells, macrophages and tγδ lymphocytes, all with a key role in maintaining the gut homeostasis and combating infections [ ] . however, in some infectious clinical conditions of the large intestine, such as ibd, inflammation has a double-edged sword role by either enabling or inhibiting cancer development and progression [ , ] . as it was commented before, antimicrobial activity of blf against a wide array of pathogens has been profusely evidenced. in contrast, data about the regulatory role of blf on the parameters of gut inflammation caused by enteropathogenic microorganisms have been provided by a limited number of articles. findings from in vitro and in vivo models of infection show that blf displays up-and down-modulatory effects on pro-inflammatory th cytokine profile. the role of blf on the resolution of infections by modulating mediators of inflammation has been documented in models of infection caused by several strains of enteropathogenic bacteria [ ] [ ] [ ] [ ] [ ] , and parasites [ ] . additionally, it has been found that lf promotes the development of bifidobacterium, one of the major genera of bacteria of the colon flora used as probiotics, in a manner independent of the iron saturation level of lf [ , ] . this effect is believed to help maintaining the gut homeostasis. regarding to the gastric inflammation, the treatment with blf or hlf as single agents or in combination with antimicrobial drugs, was found to favor eradication of bacteria and to protect against gastritis caused by helicobacter pylori or helicobacter felis, as described in murine models [ , ] . however, other human and murine trials did not support this finding, and even more, bacterial growth and gastric inflammation seemed to be enhanced by blf or hlf administration [ , ] . these controversial data may reflect experimental or clinical settings of lf treatment. interestingly, lf as a single component failed to eradicate the h. pylori infection but in combination with triple esomeprazole, clarithromycin and amoxicillin therapy and with probiotics, favored the resolution of infection and ameliorated the inflammatory response more effectively than in combination with two-antibiotic treatment, as described in experimental mice treated with r-hlf from transgenic goats, and also in trails of patients treated with native blf. interestingly, r-hlf not only inhibited the growth of h. pylori, but also suppressed the expression of two of its major virulence factors [ , ] . on the other hand, several studies have demonstrated the effect of lf on modulating inflammation in the small intestine. for example, the anti-inflammatory activities of both r-hlf and native blf have been tested in models of bacterial infection by shigella flexneri in rabbits [ ] , and salmonella enterica serovar typhimurium in susceptible balb/c mice [ ] . unlike with lf-untreated animals, macroscopic and microscopic observations evidenced that both lf treatments favored the resolution of infection and protected mice from tissue damage caused by the intestinal inflammation [ , ] . mechanisms accounting for the anti-inflammatory role of blf may result, in part, from the elicitation of siga response with a key role in luminal clearance of pathogens and in down-modulation of intestinal inflammation [ , , , , ] . although lf may modulate inflammation by inhibiting the growth of pathogens through the iron chelating ability of apo-lf, the iron-free form of lf used in most studies, assays based on the murine typhoid model showed that pharmaceutical formulation of iron-saturated blf (holo-blf) enclosed in nanocapsules displayed both antimicrobial activity and modulatory properties on the inflammation. the latter was evidenced by up-and down-modulation of cytokines involved in innate and adaptive immune responses as well on hematopoietic cytokines, with a key role in the generation of both granulocyte (pmn neutrophils) or agranulocyte (monocytes/macrophages) phagocytes [ ] . thus, iron-loaded blf nanocapsules seem to evoke the convergence of innate and adaptive immune responses of pro-and anti-inflammatory cytokines resulting in the protection toward typhoid infection and concomitant intestinal inflammation. down-modulatory effects of blf on pro-inflammatory cytokines has also been documented in cultures of caco- monolayer cells infected with recombinant escherichia coli invasive strain harboring inv gene from yersinia pestis; this strain is able to accomplish invasion but not intracellular multiplication within epithelial cells [ ] . in this model, apo-blf as well as holo-blf decreased levels of il- elicited by non-invasive e. coli wild type strain, and il- , il- and tnfα by e. coli invasive. in addition, both apo-and holo-blf inhibited an il- increased response caused by e. coli invasive strain, but levels of this cytokine remained elevated. these findings suggested that the effect of blf toward inflammatory mediators was iron-independent and that constant high il- levels provided protection by inducing recruitment of phagocytes to combat the infection [ ] . in the large intestine, the regulatory impact of blf toward inflammatory response has been described in in vitro models of infection by adherent invasive e. coli (aiec) strains with a presumable role in the pathogenesis of crohn's disease. this disease, along with ulcerative colitis, are two clinical entities of ibd characterized by an abnormal response to commensal bacteria colonizing the intestinal lumen [ , ] . aiec lf strain is found in lesions of inflamed colon tissue in children suffering crohn's disease with a preponderant th pro-inflammatory response [ ] . findings in the model of infection by aiec lf indicated that blf inhibited the bacterial invasion and the pro-inflammatory cytokine response of tnfα, il- and il- in epithelial monolayers and in cultures from colonic biopsies from patients with crohn's disease, suggesting a potential therapeutic role for blf as antibacterial and anti-inflammatory agent [ ] . up-modulatory effects of blf on inflammatory cytokines in response to bacterial infections have been found in assays of infection with aiec lf in caco- monolayers stimulated with ifn-γ to mimic the preponderant response of th associated cytokines found in crohn's disease patients [ ] . data from these assays showed that in unstimulated infected cells, blf inhibited both bacterial invasion and survival, while in infected cells primed with ifn-γ, blf increased il- production whereas counteracted the inhibitory effect of aiec infection on ferroportin protein expression. ferroportin is an iron exporter protein regulated by the inflammation that determines the survival of intracellular pathogens by reducing the intracellular iron levels. apparent conflicting data described in the infection model with aiec lf strain regarding the anti-inflammatory [ ] versus pro-inflammatory [ ] role of blf seem to evidence two sides of the same coin, i.e., the ability of blf to up-and down-modulate the inflammatory response and iron availability resulting in the resolution of infection. in parasitic infections of the large intestine, we previously developed a model of intracecal infection by the protozoan entamoeba histolytica in susceptible c h/hej mice (a strain with a spontaneous mutation in the tlr gene) to simulate the intestinal infection caused by this parasite in humans [ ] . in this model, the oral therapeutic administration of blf resolved the intestinal infection with amoebae at high efficiency, effect that was shown to be associated to an increased local expression of il- and il- and the elicitation of siga in cecum [ ] , as happened with the salmonella infection. both il- and il- are th interleukins involved in the up-regulation of siga and il- , and also is a secondary inflammatory cytokine that exhibits pro-and anti-inflammatory properties [ , ] . thus, oral blf administration led to the anti-inflammatory response of mediators such as th cytokines and iga that collaborate in the protection against the parasite and maintenance of homeostasis with protective impact on tissue integrity. on the other hand, in experimental assays conducted in mice infected with enterovirus, blf treatment displayed protective action resulting from hampering the viral interaction with host cells [ ] . however, in vitro assays indicated that blf did not exhibit any suppressive activity against rotavirus [ ] . in addition, assessment of rotavirus infection incidence in children, either treated or not with blf, showed that blf neither prevented the viral infection nor had an effect on the levels of ifn-γ or il- as markers of pro-and anti-inflammatory interleukins, respectively [ ] . thus, therapeutic action of blf by itself on viral enteritis is unclear; however, assays in suckling rats showed that administration of whey protein concentrate containing blf as well as other bioactive components, favored the resolution of acute gastroenteritis caused by rotavirus infection [ ] . this result suggests a synergic effect from the bulk rather than each single bioactive component that provided protection to enterovirus. sepsis is a life-threatening syndrome that results by the harmful effects of inflammatory response associated to gut systemic infections [ , , ] . as in the case of infectious intestinal diseases, assessment of inflammatory parameters regulated by blf in response to systemic infections have been analyzed in a limited number of infection models, induced by e. coli strains administered by enteral or parenteral routes [ , , ] . in most cases, models mimic sepsis conditions found in human systemic infections by invasive enteropathogenic bacteria in immunocompetent hosts, or by microbiota members causing opportunistic nosocomial infections in neonatal or adult patients underwent solely parenteral nutrition, post-surgery antibiotic treatment, or immunosuppression, among other conditions [ , ] . therapeutic action of blf and the synthetic peptide lfchimera was tested in balb/c mice infected by intragastric route with enterohemorrhagic e. coli (ehec); the latter is a food borne pathogen causing mild diarrhea, hemorrhagic colitis and, in some patients, hemolytic uremic syndrome characterized by anemia, thrombocytopenia and kidney injury [ ] . in infected mice that underwent both lf and lfchimera treatments, fecal bacterial output and kidney colonization were found reduced, while lfchimera significantly decreased mortality rate. histological analysis in hematoxylin-eosin stained tissue slices to assess the inflammatory response reveled that both lf and lfchimera treatments decreased kidneys damage in comparison with mice without blf treatments that showed tubular necrosis, glomerular injury and intratubular hyaline cast. furthermore, in vivo and in vitro assays have documented the role of r-hlf against gut-related systemic infection by e. coli strain ec causing meningitis in orogastrically infected neonatal rats [ ] . this model mimics clinical observations in neonates which evidence that parenteral feeding as sole route of nutrition is a risk factor of sepsis, resulting from translocation of microbiota members, e.g., gram-negative enterobacteria, from intestinal tract to systemic organs via bloodstream, with potentially fatal consequences. treatment with r-hlf decreased the clinical sepsis illness and bacterial loads in the kidney and blood while in vitro assays in macrophage cultures showed that levels of nitric oxide, tnfα and nf-κb expression elicited by lps were even higher following the addition of r-hlf. these findings suggest that protective action of r-hlf resulted from an optimal activation of macrophages via pro-inflammatory cytokine elicitation to enhance their bacterial killing activity [ ] . effect of blf against sepsis caused by e. coli and staphylococcus aureus has been demonstrated in cyclophosphamide immunosuppressed mice, as a model to mimic immunosuppressive therapy for autoimmune and neoplastic diseases. antisepsis action of blf evidenced by the reduction of bacterial load in the spleen and liver was associated with a rise of circulating leukocytes induced by the elicitation of il- in the spleen as well as peritoneal macrophages in immunosuppressed mice treated with blf [ ] . studies in mice systemically infected with e. coli evidenced that blf enhanced the killing activity and recruitment of neutrophils [ , , ] . action of blf against e. coli sepsis resulted from the elicitation of il- involved in the production of acute phase proteins, as well as from the amelioration on tnfα levels increased in response to the infection [ ] . since high levels of circulating pro-inflammatory cytokines such as tnfα have lethal consequence, the results described above suggest that the ability of blf to control the tnfα increase may underlie its protective action to sepsis, as found in in vitro and in vivo assays [ , ] . clinical trials have confirmed the potential application of blf to prevent nosocomial sepsis and necrotizing enterocolitis of premature neonates, diseases that were associated with the up-modulation of t regulatory cells (foxp + cd +, and cd hi) involved in the control of intestinal immune response against pathogens, strengthening the essential role of blf in the control of the intestinal homeostasis [ ] . pro-and anti-inflammatory properties of lf in bacterial infections have been ascribed to its ability to act as a lps-binding protein (lbp). modulatory effects of either hlf or blf, including derived lfcins, on parameters associated with inflammation, has been evidenced in models of intestinal and systemic-related endotoxemia induced by lps (also known as endotoxin) from gram-negative enterobacteria [ , ] . these experimental models are intended to assess the impact of lf at intestinal and systemic levels on inflammatory markers elicited by lps that affect the gut barrier, bacterial translocation, diarrhea, tissue damage and endotoxic shock, among others [ , [ ] [ ] [ ] [ ] . in some cases, experimental assays with lps administration encompass the use of zymosan, d-galactosamine (d-galn) or carrageenan to increase the sensibility of animals toward the toxic effects of low lps doses [ , ] . porcine lf (plf) bioactive derivatives such as plf peptide (lfp ) have been proved to confer protection in mice against lps damage to colon, associated with its down-modulatory action on pro-inflammatory cytokines tnfα, il- and ifnγ. lfp elicited the expression of tight junction proteins involved in the regulation of intestinal permeability, i.e., zonula occludens- , occludin and claudin- , as well as decreased colonic apoptosis [ , [ ] [ ] [ ] [ ] [ ] [ ] . protective action of hlf on the gut barrier function against the lps-induced intestinal damage has also been described for hlf in in vitro cultures of caco- cells and jejune segments of mice underwent lps-endotoxemia [ , ] . iron status of blf had no impact on parameters of the gut barrier function, as documented in caco- cell cultures incubated with murine (j a. ) macrophages; however, apo-blf displayed stronger neutralizing effects than holo-blf against the pro-inflammatory cytokine generation in response to lps and thermally inactivated e. coli cm antigens, suggesting that iron content may determine the protective role of lf toward the inflammation caused by gut endotoxemia and/or sepsis [ ] . at intestinal level, neutralizing activity of blf or hlf has been tested in models of endotoxemia in mice induced by the intraperitoneal administration of lps [ , ] . other models of lps neutralization by blf include enterogenic endotoxemia in rats injected with carrageenan by intraperitoneal route, and also infection with e. coli in rats treated with nebacetin by intraduodenal via to enable the lps release [ ] . in the model of mice endotoxemia, intraperitoneal administration of hlf provided protection against deleterious effects of lps on the intestinal integrity [ ] . moreover, blf administered by intraperitoneal route in mice attenuated the lps-induced diarrhea by decreasing the production of pro-inflammatory mediators with powerful diarrheagenic activity, i.e., prostaglandin e (pge) by enterocytes in the small intestine and nitric oxide in plasma [ ] . in the enterogenic endotoxemia model in rats, blf decreased the endotoxic activity of lps, as measured in plasma on a dose-dependent manner, and also decreased the bacterial loads in the mesenteric lymph nodes [ ] . some experimental assays in neonatal mice indicated that feeding with blf and/or bifidobacteria decreased the intestinal levels of lps without changes in cell populations producing tnfα, ifnγ and il- in peyer's patches [ ] . at the systemic level, the neutralizing activity of hlf-derived lf peptide has been tested in models of endotoxemia in mice, induced by intravenous administration of lps and d-galn. in this assays, lf exhibited protective activity against lethal intravenous lps challenge by decreasing circulating levels of tnfα [ , ] . the dual action of lf on circulating tnfα seems to underlie its protective role, since down-modulation of tnfα provided protection against the deleterious impact of endotoxemia, whereas the modulatory role of lf in the control of tnfα elicitation is critical for eradication of gut-related systemic infections [ , , ] . in another example, blf displayed prophylactic action against lethal shock occurring upon intravenous injection of lps in germ-free piglets; this is a valuable model to study the primary toxicity of endotoxin portion of lps, i.e., lipid a, rather than the secondary toxicity of o and r polysaccharide portions [ ] . in cultures of monocytes from lps-treated piglets, blf inhibited the interaction of lps with cd , an antigen surface marker expressed by mononuclear phagocytes. priming of these phagocytic cells by lps via cd ligation, resulted in the elicitation of powerful pro-inflammatory cytokines including tnfα, il- and il- with lethal outcome for host in endotoxemic conditions [ ] . moreover, oral administration of blf prior to an intravenous lps challenge in piglets provided protection against the mortality caused by lps-induced hypothermia [ ] . like blf, protective activity against hypothermia has been described with hlf in mice underwent lps-endotoxemia [ ] . effect of blf on lps-neutralization and e. coli bacteremia has been explored in lps toxicity susceptible c h/hecr mice carrying a defective tlr- gene as well as in cba mice resistant to lps [ , ] . unlike its protective activity in cba mice, blf failed to confer protection against endotoxemia and e. coli bacteremia in c h/hecr mice due to its inability to ameliorate the elicitation of tnf-α and ifnγ, and to prevent il- decrease. thus, an unbalanced cytokine response may be responsible of the high susceptibility to lps endotoxemia in c h/hecr mice [ ] . differential impact of concurrent, prophylactic, and therapeutic effects of intraperitoneal administration of hlf on endotoxemia were analyzed in mice by intravenous administration of lps [ ] . in the concurrent scheme, hlf administered prior to the lps challenge decreased the serum levels of tnfα, nitric oxide, il- and il- . in the prophylactic and therapeutic protocols, hlf significantly down-modulated serum tnfα and nitric oxide, but no significant fluctuations were seen in the levels of il- and il- . in a mice model of hepatitis induced by intraperitoneal co-administration of lps and zymosan, orally administered blf decreased the serum aspartate aminotransferase activity (a marker of liver inflammation), and increased in the small intestine the production of il- , an anti-inflammatory cytokine with a role in the amelioration of inflammatory response [ , ] . the findings indicate that the up-modulation of il- levels by blf seems to provide therapeutic action in the small intestine induced by lps-zymosan in this model of hepatitis. treatment with r-hlf has also been analyzed in the same mice models of induced hepatitis [ ] . in the experimental scenario, r-hlf provided protection against hepatitis development as determined by the decrease in alanine transaminase activity as the marker of liver damage, which was associated with down-modulation of serum tnfα levels. moreover, the protective effect of r-hlf was not found in mice pre-treated with gadolinium chloride that destroys kupffer cells, suggesting that these cells are the source of tnfα and the targets of r-hlf [ ] . the mechanisms of lf action on the inflammatory response have been analyzed in regarding to the ability of lf to interact with lps and block its interaction with tlr in in vitro cultures of intestinal cell lines and murine peritoneal or cell line macrophages [ , , [ ] [ ] [ ] [ ] [ ] . these assays have evidenced that lf down-or up-modulates lps-mediated inflammatory response via dependent or independent tlr /nf-κb signal pathway. down-modulatory impact on lps-mediated inflammation was found in assays of intestinal porcine epithelial cell line (ipec- ) treated with the plf-derivative peptide lfp , effect that resulted from its ability to inhibit myd /nf-κb and myd /mapk signaling pathways [ , ] . similarly, in human monocyte cell lines (thp- and mono mac ), hlf also displayed down-modulatory activity on lps-associated cytokine response by blocking the binding of nf-κb to the tnfα promoter [ ] . assays in cultures of human peripheral blood mononuclear cells showed that blf displayed an anti-inflammatory response by driving the differentiation of lps-treated monocytes toward dendritic cells with low capacity of both differentiation and elicitation of th response by counteracting the tlr mediated activation signals [ ] . on the other hand, up-modulatory impact of lf on inflammatory effects of lps was documented in assays of raw . macrophage cell line and peritoneal macrophages, indicating that blf, in the form of complex with lps, enhanced cytokine response of tnfα, il- and il- via tlr -nf-κb signal pathway [ ] . however, another study suggests that the up-modulatory effect of lf on lps-mediated inflammation may involve pathways other than tlr signaling. thus, assays on raw . macrophage cell line treated with lps showed that elicitation of il- levels by blf was tlr -independent [ ] . therefore, lf displayed a dual role on the lps-mediated response of pro-inflammatory cytokines that encompasses alternative routes of tlr signalization. other presumable mechanism of lps-mediated inflammation includes the ability of lf to control the expression of iron-regulating proteins as found in thp- monocyte/macrophage cultures; in these cells, lf prevented the lps-induced decreased of ferroportin by reducing the il- levels [ ] . as we mentioned before, ferroportin is an iron binding protein involved in the control of iron levels during inflammatory response. in summary, the findings described above provide the experimental evidence that support the protective role of lf against the deleterious effects of lps-induced pro-inflammatory cytokine response on the gut-barrier function, diarrhea, bacterial translocation, and tissue damage. having in mind the antimicrobial and lps-binding protein activities of lf, its application either alone or in combination with probiotics, or as an adjunctive compound of antibiotics, may represent a very promising strategy for the treatment and prevention of sepsis and endotoxic shock. the human respiratory tract (rt) is responsible for the mobilization of millions of liters of gases throughout life. delivery of life-requiring oxygen to the systemic circulation and organs implies the potential incorporation of countless particles, toxicants and microbes, which are countered by local innate and adaptive immune responses that avoid their entry into the lung tissue and circulation and protect the lung structure and function [ ] . infections in the rt are very frequent in the population and represent a considerable cause of worldwide morbidity and mortality [ ] . infections of the upper rt such as common cold, laryngitis, pharyngitis, epiglottitis, otitis and sinusitis are typically caused by virus, bacteria, and, at less extension, by fungi. as an example, the common cold is a viral disease considered as the most frequent infection in humans, which can be caused by rhinoviruses, coronavirus, parainfluenza and adenovirus, and less frequently by respiratory syncytial virus and enterovirus. however, the influenza virus, a common cause of seasonal flu, can simultaneously affect other parts of the rt, including the lower tract. infections of the lower rt are mainly caused by bacteria, but also by virus, fungi and even parasites. they include bronchitis, pneumonia and pulmonary abscesses, among others. tuberculosis, caused by the bacterium mycobacterium tuberculosis, is among the most prevalent infections in the lower rt, causing mainly pneumonia, but also affecting other organs [ ] . all rt infections are accompanied with an inflammatory process whose intensity depends on many variables, including the type of pathogen and its virulence, the inoculum, the affected tissue, the host immunological status and whether the infection is acute or chronic. although the inflammatory process is essential for the control of invasive infectious agents, the development of an exacerbated or chronic inflammation results in alterations of the respiratory capacity due to the lung tissue damage, including edema, increased airway resistance and mucus production, such as in the infection with influenza virus [ ] . understanding how inflammation alters the respiratory system is indispensable for the development of better therapeutic interventions to support breathing and lung plasticity as a clinical treatment. in this regard, evidence of the modulation of rt inflammation by blf associated to certain microbes has been reported using several in vitro and in vivo models. moreover, lf is considered the second most important antimicrobial and anti-inflammatory peptide after lysozyme in the upper rt [ ] . tuberculosis is by far the most studied in vivo model of microbial pulmonary infection. this bacterial infection affecting nearly a third of the world's population and having a rate for new infections of approximately . % per year [ ] , is targeted by the bacillus calmette-guerin (bcg) vaccine, the most widely used vaccine in the world which has remained almost unchanged since [ ] . a study carried out in showed that a single subcutaneous immunization of mice with a mix of bcg and blf emulsified with freund's adjuvant, followed by a challenge with m. tuberculosis in aerosol, resulted in decreased mycobacterial loads in the lungs and spleen [ ] . splenocyte proliferative response to heat-killed bcg showed increased il- and ifnγ production. in a subsequent study by the same group, it was showed that blf admixed to the bcg vaccine, in incomplete freund's adjuvant or pbs, increased mice protection against a m. tuberculosis challenge when compared with mice that received bcg alone [ ] . in addition, there was a significant reduction of lung bacterial load associated to increased production of ifnγ and il- by splenocytes, as mentioned before; in this study, blf addition to the vaccine also resulted in a clear reduction in lung pathology, concomitant with down-regulation of pro-inflammatory mediators tnfα or il- β, suggesting that the main action of lf to enhance the bcg vaccine relied on its immunomodulatory properties reducing the immune-related tissue pathology, in part, by modulating macrophages and dendritic cells ability to present antigens and stimulate t-cells [ ] . noteworthy, the lymphocytic recall response towards bcg antigens two months after infection was higher in the mice that received lf as adjuvant, suggesting that lf improved the specific t-cell th response as determined by the increase in infγ production [ ] . the potential of r-hlf to reduce the m. tuberculosis tissue damage and pulmonary histopathology was also demonstrated in ulterior studies of the same group. they showed that r-hlf produced in the yeast pichia pastoris expression system with a glycosylation pattern similar to its natural human neutrophil counterpart, in contrast to the non-glycosylated r-hlf, was able to improve the efficacy of the bcg vaccine in protecting against the challenge with m. tuberculosis in aerosol, as manifested primarily in a significant reduction in the associated pulmonary pathology [ ] . in this case, the mycobacterial loads in the lung and spleen were not significantly reduced in the bcg-r-hlf group compared with the controls treated with bcg alone, but rather, the protection was associated with changes in the pathological manifestation of the lung disease; this was probably due to the notable immunomodulatory function of the granulocytic lf used in this study, when compared with the lf form from secretions, which is more microbicidal [ ] . recently, a r-hlf expressed in chinese hamster ovary (cho) cells was also used in the mixture with the bcg vaccine, showing a slight decrease over the time in the lung pathology after aerosol challenge with m. tuberculosis, which correlated with an initial increase in the secretion of inflammatory cytokines followed by their posterior decrease [ ] . the efficacy of blf in enhancing the bcg vaccine action was more recently analyzed using a more amenable route of administration. in this study, mice that received drinking water containing . % blf at day or post-infection had lower colony forming units (cfu) and lower inflammation in the lungs, with increased numbers of ifnγ producing t cd and cd cells and il- producing lymphocytes when compared with animals vaccinated with bcg alone [ ] . noteworthy, blf did not affect the in vitro replication of m. tuberculosis but instead enhanced the killing of bacteria by macrophages in a nitric oxide dependent way [ ] . these studies using the models of pulmonary infection with m. tuberculosis suggest that lf promotes certain up-regulation of pro-inflammatory response, while down-regulating overall tissue immunopathology. the role of lf in the inflammation in other pulmonary infections has been less addressed. homeostatic effect of blf on inflammation was reported in in vitro cultures of cystic fibrosis (cf) bronchial cells (ib - ) infected with burkholderia cenocepacia, a gram-negative opportunistic bacterium that recurrently infects patients with cf forming biofilms and is usually highly resistant to currently available broad-spectrum antibiotics. thus, even though the addition of blf did not reduce the rates of bacterial invasion, it decreased the release of pro-inflammatory il- β, while augmented the secretion of anti-inflammatory il- , suggesting a role for blf in protecting cf bronchial infected cells from the inflammation-associated damage [ ] . noteworthy, this study correlated with a previous one addressed on sputum samples from patients with cf which showed an inverse association between the levels of lf in the secretions and the inflammation burden [ ] . another study showed that the decrease of lf levels in patients with cf was due to its cleavage by the increased cathepsin activity in pseudomonas aeruginosa-positive sputum samples, another biofilm-forming opportunistic pathogen of these patients. a similar result with lf and tf undergoing proteolysis was previously reported in bronchioalveolar lavage of p. aeruginosa infected cf patients [ ] . these results suggest that the proteolytic cleavage of lf in patients with cf can contribute to b. cenocepacia and p. aeruginosa-associated lung damage, and that infection-associated lung damage can be improved by the exogenous therapeutic administration of lf, due to its potent immunomodulatory properties. similar protective effect of blf has been found in a murine model of lung injury induced by intraperitoneally administered lps [ ] . in this study, the intraperitoneal injection of blf ( mg/mouse) h before (prophylactic effect) or h after (therapeutic effect) lps challenge, were associated with significant reduction of the total number of leukocytes in bronchioalveolar lavage samples, increased il- , and decreased tnfα concentrations and myeloperoxidase activity [ ] . these changes paralleled attenuation of lung edema and inflammatory infiltration, suggesting a protective role of blf by avoiding the damage caused by the lps-induced acute inflammatory response. however, the protective role of lf based on the regulation of inflammation is not observed in all cases of rt infections. there are several viral infection models where the immunomodulatory effect of lf has not been documented. thus, although lf has in vitro antiviral activity against the respiratory syncytial virus (rsv) [ ] , as well as an immunomodulatory effect reducing the release of il- by hep- cells infected with rsv [ ] , the oral or intraperitoneal administration of different doses ( to mg/animal/day) of blf to mice from h before until h post-rsv infection did not have any effect on viral loads, pulmonary airflow resistance or obstruction, degree or type of pulmonary inflammation and serum t cellular responses, evaluated on day post-rsv infection [ ] . similar result was observed in a study carried out in mice treated by intranasal route with blf on days - , and evaluated on day post-rsv infection [ ] . another example is a mouse model of influenza infection, where the daily oral administration of . mg of blf from h before infection, did not have any effect on viral load and concentration of ifnγ, il- and il- cytokines evaluated at six days post-infection, when compared with untreated infected mice [ ] . the reason for the lack of blf protecting effect in these viral infection mice models is unknown, but possible explanations could be related to timing, dosing, and route of blf administration. in contrast, oral blf and curcumin supplementation to children with recurrent viral rt infections resulted in immune modulation by modifying the lymphocyte population and cytokine responses that reduce the rate of infections [ ] . a recent study aimed to determine the effect of three months supplementation with blf-fortified formula on respiratory tract infections and diarrhea in chinese weaned infants ( - months age), showed similar results, with a reduction in the incidence rate of respiratory-related illnesses when compared with a placebo group [ ] . moreover, a study in patients with chronic rhinosinusitis has established an association between the genetic deficiency of lf synthesis in the upper rt and the increased susceptibility of certain individuals to bacterial colonization, biofilm development, and recalcitrant sinus disease [ ] . this new knowledge of lf immunomodulation paves the way to more general design of t cell-dependent vaccines that incorporate naturally occurring granulocytic components, which may be useful in infectious diseases to reduce immune-mediated tissue damage. the role of blf on inflammatory response associated to infection has been described in other mucosal sites, such as the mammary gland of cows suffering staphylococcal mastitis [ ] . assessment of mammary gland secretions showed that, in sick cows, intramammary infusion of blf decreased the numbers of staphylococci and increased c levels, whereas in healthy animals blf infusion increased the numbers of pmn leukocytes expressing cd b, an integrin when complexed with cd (cr ) acts as receptor for the ic b complement fragment [ ] . according to these findings, up-modulatory effect of blf on pro-inflammatory components of innate immunity may underlie its therapeutic action toward mastitis; in fact, alternative approaches have also been tested to decrease the deleterious effect of inflammation on tissue integrity. combination of blf and antibiotics was found to be effective to control the staphylococcal mastitis and to attenuate the mrna expression of tnfα via the inhibition of nf-κb activation [ ] . thus, this approach may contribute to decrease the effects of inflammation on tissue damage and also to reduce the antibiotic dosage for the eradication of staphylococcal infection by multiresistant strains. inflammatory response in staphylococcal mastitis seems to be correlated with the elicitation of peptides derived from blf-elastase proteolysis that display low concanavalin a and low iron-binding affinities, as well as antibacterial properties, but induce the expression of pro-inflammatory cytokines il- and tnfα leading to neutrophil infiltration [ , ] . like staphylococcal mastitis, elicitation of low cona affinity lf-peptide derivatives in parotid saliva was correlated with the severity of symptoms of periodontitis patients [ ] . in oral cavity, the modulatory action of blf on infection-associated inflammatory response has been documented in mice infected with candida albicans [ ] . experimental settings of oral candidiasis in immunosuppressed mice showed that oral administration of blf displayed therapeutic effect by inhibiting the suppressive effects of infection on inflammatory parameters of innate response, such as circulating pmn neutrophils and cervical lymph-node cells; moreover, generation of ifnγ and tnfα was found increased in cervical lymph-nodes cultures primed with heat-killed c. albicans from blf-treated mice [ ] . according to in vitro assays testing full length blf and blf-derived peptides, therapeutic effect of parental blf relies on the n-terminal portion associated with its ability to up-modulate the killing action of pmn neutrophils by increasing the superoxide generation, protein kinase c, p mapk activity, and the expression of p phox [ ] . these findings suggest that, in the murine model of candidiasis, the up-modulatory effect of blf on parameters of inflammation provides protection to c. albicans infection. data from in vivo and in vitro assays support the role of blf and hlf in up-and down-modulation of inflammatory response to extra-intestinal infections, such as hepatic amoebiasis [ ] , listeriosis [ , ] , urinary tract infections [ ] , legionellosis [ ] , and staphylococcal septicemia [ ] . in experimental amoebic liver abscess in hamsters, blf treatment by gavage had protective action against the hepatic lesions by e. histolytica and favored the normalization of the liver function [ ] . having in mind its intrinsic anti-inflammatory and therapeutic action, as well as its low toxicity, the use of blf as adjunct of conventional drugs such as metronidazole, may contribute to decrease the drug dosage in the treatment of amoebiasis and even decrease the drug resistance of the parasite. in vitro experiments on listeria monocytogenes infection in ifnγ primed thp cell cultures indicated that blf displayed a protective action against cell death by necrosis, whereas bovine lfcin b diverted the death cell from necrosis to apoptosis [ ] . findings indicated a protective role of blf and lfcin b by reducing the inflammatory response associated to necrosis caused by the intracellular infection of the pathogenic bacteria in macrophages, and favored the anti-inflammatory conditions of cell death by apoptosis on these cells. data from studies of infection with l. monocytogenes in mice indicate that treatment with hlf displayed significant effects on one main organ target of this pathogen in regards to bacterial colonization, necrosis, and mrna expression of pro-inflammatory cytokines tnfα, il- β and ifnγ [ ] . although the majority of the experimental data on lf properties has been obtained with blf, some experimental studies indicate that hlf displays even more potent antibacterial and anti-inflammatory action. murine model of infection of urinary bladder with the uropathogenic e. coli o k strain showed that perorally administered hlf decreased the bacterial load in the kidneys and urinary bladder as well as the inflammatory response, as evidenced by reduced il- levels in urine at h post-infection, and in plasma, at h post-infection [ ] . thus, hlf administered by peroral route provides therapeutic action against infection and inflammation in remote sites such as the urinary tract. in vitro assays of legionella pneumophila infection in cultures of monocytes from healthy volunteers indicated that unlike holo-hlf that promoted the bacterial growing, apo-hlf inhibited the intracellular multiplication of the pathogen in both inactivated and ifnγ activated monocytes [ ] . these findings suggest a synergy between the antibacterial effect of iron-free human lactoferrin and the stimulating killer effects of the pro-inflammatory cytokine ifnγ on infected cells, leading to conditions for control of bacterial multiplication inside cells. a model of s. aureus infection in hlf-transgenic mice showed the pivotal role of hlf in the elicitation of a th profile of cytokines, which determined the resolution of systemic infection. polarization of the immune response to the th profile in hlf-transgenic mice was evidenced by up-modulation of tnfα and ifnγ levels and down-modulation of th cytokines il- and il- in culture supernatants of spleen cells [ ] . in summary, the studies mentioned above show that lf and its derivative peptides display bimodal effects that provide conditions for the up-and down-regulation of inflammation leading to the resolution of extra-intestinal infections. the biotechnological development of formulations of lf as nanoparticles for potential clinical use, in addition to lf-hydrolysate, and native lf alone or in combination with probiotics, may have application for the control of infections and inflammation [ , , , , , ] . evidence from the basic studies in animals of experimentation about the prophylactic and therapeutic activity of lf as antimicrobial and modulatory agent on inflammatory response, have promoted this glycoprotein from the innate immune system as a focus of interest for the biotechnological development of nanoparticle-based formulations for potential clinical use. in addition, lf-hydrolysate, and native lf alone or in combination with antibiotics and probiotics, may have potential application in the control of neonatal infections, and in inflammation. more studies are necessary to support the generalized practical application of lf, mainly in the control of inflammation associated to infections. the authors thank to the mexican council of science and technology (conacyt) for the supporting grants (mireya de la garza) and (julio césar carrero). julio césar carrero also thanks to programa de apoyo a proyectos de investigación e innovación tecnológica (papiit-unam) for grant no. in and to the institutional program "nuatei" for financial support. thanks also to carlos villasana and luisa samaniego-barrón for their technical assistance and pavel petrosyan for the english editing of the manuscript. the authors declare no conflict of interest. lactoferrin: a multifunctional glycoprotein lactoferrin from milk: nutraceutical and pharmacological properties overview of lactoferrin as a natural immune modulator lactoferrin-a multifunctional protein with antimicrobial properties immunomodulatory effects of lactoferrin on antigen presenting cells nanocapsules loaded with iron-saturated bovine lactoferrin have antimicrobial therapeutic potential and maintain calcium, zinc and iron metabolism the isolation of a red protein from milk preparation and properties of lactosiderophilin (lactotransferrin) of human milk a structural framework for understanding the multifunctional character of lactoferrin relative molecular mass, isoelectric point, iron-binding properties and uptake by the liver occurrence, structure, biochemical properties and technological characteristics of lactoferrin metal-combining properties of human lactoferrin (red milk protein). . the involvement of bicarbonate in the reaction proteins of iron metabolism the physiology of lactoferrin lactoferrin in milk from different species an iron-binding protein common to many external secretions lactoferrin and host defence: an overview of its immuno-modulating and anti-inflammatory properties biological role of lactoferrin lactoferrin concentration during involution of the bovine mammary gland concentrations of lactoferrin and iron in human milk at different stages of lactation concentration of lactoferrin and transferrin throughout lactation in cow's colostrum and milk molecular evolution of the transferrin family and associated receptors swiss-prot database the significance of iron in infection the involvement of lactoferrin in the hyposideremia of acute inflammation lactoferrin: a general review antimicrobial properties of lactoferrin the therapeutic potential of lactoferrin lactoferrin content of peripheral blood cells lactoferrin biosynthesis during granulocytopoiesis lactoferrin turnover in man lactoferrin, an iron-binding protein in neutrophilic leukocytes crohn's disease activity assessed by fecal calprotectin and lactoferrin: correlation with crohn's disease activity index and endoscopic findings structure of human lactoferrin at . -a resolution three-dimensional structure of diferric bovine lactoferrin at . a resolution heterogeneity of bovine lactotransferrin glycans the effects of differences in pspa alleles and capsular types on the resistance of streptococcus pneumoniae to killing by apolactoferrin bactericidal activity of human lactoferrin: differentiation from the stasis of iron deprivation in vivo and in vitro effects of lactoferrin on yersinia pseudotuberculosis antibacterial activity of lactoferrin and a pepsin-derived lactoferrin peptide fragment human hololactoferrin: endocytosis and use as an iron source by the parasite entamoeba histolytica iron-saturated lactoferrin and pathogenic protozoa: could this protein be an iron source for their parasitic style of life? ability of neisseria gonorrhoeae, neisseria meningitidis, and commensal neisseria species to obtain iron from lactoferrin the impact of lactoferrin with different levels of metal saturation on the intestinal epithelial barrier function and mucosal inflammation lactoferrin: mechanism of action, clinical significance and therapeutic relevance une protéine multifonctionnelle molecular structure and biological function elucidation of the antistaphylococcal action of lactoferrin and lysozyme in vivo antimicrobial and antiviral activity of components in bovine milk and colostrum involved in non-specific defence lactoferrin is a lipid a-binding protein lactoferrin inhibits the endotoxin interaction with cd by competition with the lipopolysaccharide-binding protein two outer membrane proteins are bovine lactoferrin-binding proteins in mannheimia haemolytica a in vitro studies of the digestion of caprine whey proteins by human gastric and duodenal juice and the effects on selected microorganisms antibacterial and antiviral activity of camel milk protective proteins comparison of the effects of acylation and amidation on the antimicrobial and antiviral properties of lactoferrin lactoferrin for prevention of common viral infections antiviral effects of plasma and milk proteins: lactoferrin shows potent activity against both human immunodeficiency virus and human cytomegalovirus replication in vitro effectiveness of human, camel, bovine and sheep lactoferrin on the hepatitis c virus cellular infectivity: comparison study lactoferrin: a protein of the innate immune system capable of killing parasitic protozoa twenty-five years of research on bovine lactoferrin applications microbicidal action of lactoferrin and lactoferricin and their synergistic effect with metronidazole in entamoeba histolytica effect of bovine lactoferrin in a therapeutic hamster model of hepatic amoebiasis bactericidal synergy of lysostaphin in combination with antimicrobial peptides synergy and antagonism between iron chelators and antifungal drugs in cryptococcus effector mechanisms of iga amoebicidal activity of milk, apo-lactoferrin, slga and lysozyme enhancement of the activity of novobiocin against escherichia coli by lactoferrin effect of bovine apo-lactoferrin on the growth and virulence of actinobacillus pleuropneumoniae antifungal activities of natural and synthetic iron chelators alone and in combination with azole and polyene antibiotics against aspergillus fumigatus gastric digestion of bovine lactoferrin in vivo in adults characterization of mammalian receptors for lactoferrin bioactive proteins/peptides and functional properties evaluation of synergistic activity of bovine lactoferricin with antibiotics in corneal infection effects of bovine lactoferrin hydrolysate on the in vitro antimicrobial susceptibility of escherichia coli strains isolated from baby pigs structural features governing the activity of lactoferricin-derived peptides that act in synergy with antibiotics against pseudomonas aeruginosa in vitro and in vivo bactericidal activity of lfchimera is stronger and less sensitive to ionic strength than its constituent lactoferricin and lactoferrampin peptides bactericidal effect of bovine lactoferrin, lfcin, lfampin and lfchimera on antibiotic-resistant staphylococcus aureus and escherichia coli microbicidal effect of the lactoferrin peptides lactoferricin - , lactoferrampin - , and lactoferrin chimera on the parasite entamoeba histolytica reduction of the infectivity of toxoplasma gondii and eimeria stiedai sporozoites by treatment with bovine lactoferricin parasiticidal effect of synthetic bovine lactoferrin peptides on the enteric parasite giardia intestinalis lactoferrin-derived peptides and lactoferricin chimera inhibit virulence factor production and biofilm formation in pseudomonas aeruginosa perspectives on interactions between lactoferrin and bacteria expression of cloned human lactoferrin in baby-hamster kidney cells research progress in physicochemical characteristics of lactoferrin and its recombinant expression systems expression, characterization, and biologic activity of recombinant human lactoferrin in rice recombinant human milk proteins plant-based biopharming of recombinant human lactoferrin supplementation transgenic cow's milk containing recombinant human lactoferrin enhances systematic and intestinal immune responses in piglets nutritional composition analysis of meat from human lactoferrin transgenic bulls mechanisms and pathways of innate immune activation and regulation in health and cancer innate immunity gone awry: linking microbial infections to chronic inflammation and cancer recognition of bacterial pathogens and mucosal immunity converging roles of caspases in inflammasome activation, cell death and innate immunity interactions of lactoferrin with cells involved in immune functionthis paper is one of a selection of papers published in this special issue bovine lactoferrin can be taken up by the human intestinal lactoferrin receptor and exert bioactivities the n domain of human lactoferrin is required for internalization by caco- cells and targeting to the nucleus mammalian lactoferrin receptors: structure and function lactoferrin protects rabbits from shigella flexneri-induced inflammatory enteritis effect of bovine lactoferrin in salmonella ser. typhimurium infection in mice lactoferrin downregulates pro-inflammatory cytokines upexpressed in intestinal epithelial cells infected with invasive or noninvasive escherichia coli strains lactoferrin prevents invasion and inflammatory response following e. coli strain lf infection in experimental model of crohn's disease. dig. liver dis lactoferrin differently modulates the inflammatory response in epithelial models mimicking human inflammatory and infectious diseases oral lactoferrin treatment resolves amoebic intracecal infection in c h/hej mice inhibition of helicobacter pylori infection by bovine milk glycoconjugates in a balb/ca mouse model effects of lactoferrin-containing formula in the prevention of enterovirus and rotavirus infection and impact on serum cytokine levels: a randomized trial protective effects of lactoferrin chimera and bovine lactoferrin in a mouse model of enterohaemorrhagic escherichia coli o :h infection protective effects of lactoferrin in escherichia coli-induced bacteremia in mice: relationship to reduced serum tnf α level and increased turnover of neutrophils enhanced clearance of escherichia coli and staphylococcus aureus in mice treated with cyclophosphamide and lactoferrin differential effects of prophylactic, concurrent and therapeutic lactoferrin treatment on lps-induced inflammatory responses in mice neutralization of endotoxin in vitro and in vivo by a human lactoferrin-derived peptide lactoferrin augments bcg vaccine efficacy to generate t helper response and subsequent protection against challenge with virulent mycobacterium tuberculosis lactoferrin enhanced efficacy of the bcg vaccine to generate host protective responses against challenge with virulent mycobacterium tuberculosis cho expressed recombinant human lactoferrin as an adjuvant for bcg influence of oral lactoferrin on mycobacterium tuberculosis induced immunopathology lactoferrin decreases inflammatory response by cystic fibrosis bronchial cells invaded with burkholderia cenocepacia iron-modulated biofilm lactoferrin protects against lipopolysaccharide-induced acute lung injury in mice lack of effect of bovine lactoferrin in respiratory syncytial virus replication and clinical disease severity in the mouse model lactoferrin reverses respiratory abnormalities in respiratory syncytial virus (rsv) infection of mice effects of orally administered bovine lactoferrin and lactoperoxidase on influenza virus infection in mice effects of bovine lactoferrin by the intramammary infusion in cows with staphylococcal mastitis during the early non-lactating period effect of combination therapy with lactoferrin and antibiotics against staphylococcal mastitis on drying cows small molecule lactoferrin with an inflammatory effect but no apparent antibacterial activity in mastitic mammary gland secretion inflammatory effect of cleaved bovine lactoferrin by elastase on staphylococcal mastitis effect of orally administered bovine lactoferrin on the immune response in the oral candidiasis murine model potential antimicrobial effects of human lactoferrin against oral infection with listeria monocytogenes in mice human lactoferrin and peptides derived from a surface-exposed helical region reduce experimental escherichia coli urinary tract infection in mice enhanced th response to staphylococcus aureus infection in human lactoferrin-transgenic mice intestinal inflammation and colorectal cancer: a double-edged sword? ability of lactoferrin to promote the growth of bifidobacterium spp. in vitro is independent of receptor binding capacity and iron saturation level effect of lactoferrin on helicobacter felis induced gastritis lactoferrin and desferrioxamine are ineffective in the treatment of helicobacter pylori infection and may enhance h. pylori growth and gastric inflammation in mice human recombinant lactoferrin is ineffective in the treatment of human helicobacter pylori infection recombinant human lactoferrin enhances the efficacy of triple therapy in mice infected with helicobacter pylori multi-faceted functions of secretory iga at mucosal surfaces. front. immunol. , , lactoferrin increases both resistance to salmonella typhimurium infection and the production of antibodies in mice characterization of adherent-invasive escherichia coli isolated from pediatric patients with inflammatory bowel disease protection against murine intestinal amoebiasis induced by oral immunization with the kda antigen of entamoeba histolytica and cholera toxin transcriptional regulation of the mucosal iga system cytokines in sepsis: potent immunoregulators and potential therapeutic targets-an updated view lactoferrin inhibits enterovirus infection by binding to vp protein and host cells inhibitory effects of human and bovine milk constituents on rotavirus infections supplementing suckling rats with whey protein concentrate modulates the immune response and ameliorates rat rotavirus-induced diarrhea sepsis: current dogma and new perspectives the blessings and curses of intestinal inflammation lactoferrin protects neonatal rats from gut-related systemic infection lactoferrin can protect mice against a lethal dose of escherichia coli in experimental infection in vivo antibacterial system generated by lactoferrin in mice in vivo is primarily a killing system oral lactoferrin to prevent nosocomial sepsis and necrotizing enterocolitis of premature neonates and effect on t-regulatory cells lactoferrin-lipopolysaccharide (lps) binding as key to antibacterial and antiendotoxic effects reciprocal interactions between lactoferrin and bacterial endotoxins and their role in the regulation of the immune response bovine lactoferrin protects lipopolysaccharide-induced diarrhea modulating nitric oxide and prostaglandin e in mice. can bovine lactoferrin ingestion protects against inflammation via il- induction in the small intestine of mice with hepatitis treatment of enterogenic endotoxinemia with lactoferrin in rats lactoferrin protects gut mucosal integrity during endotoxemia induced by lipopolysaccharide in mice porcine lactoferrin-derived peptide lfp- protects intestinal barrier by maintaining tight junction complex and modulating inflammatory response lfp- , a porcine lactoferrin peptide, ameliorates lps-induced inflammation via the myd /nf-κb and myd /mapk signaling pathways protective effects of lactoferrin against intestinal mucosal damage induced by lipopolysaccharide in human intestinal caco- cells in vivo effects of bifidobacteria and lactoferrin on gut endotoxin concentration and mucosal immunity in balb/c mice the protective effects of lactoferrin feeding against endotoxin lethal shock in germfree piglets gastrointestinal and hepatic mechanisms limiting entry and dissemination of lipopolysaccharide into the systemic circulation variations in susceptibility to proteoglycan-induced arthritis and spondylitis among c h substrains of mice: evidence of genetically acquired resistance to autoimmune disease lethality in lps-induced endotoxemia in c h/hecr mice is associated with prevalence of proinflammatory cytokines: lack of protective action of lactoferrin bovine lactoferrin induces interleukin- production in a hepatitis mouse model and human intestinal myofibroblasts lactoferrin protects against development of hepatitis caused by sensitization of kupffer cells by lipopolysaccharide lactoferrin prevents lps-induced decrease of the iron exporter ferroportin in human monocytes/macrophages bovine lactoferrin counteracts toll-like receptor mediated activation signals in antigen presenting cells lactoferrin works as a new lps-binding protein in inflammatory activation of macrophages lactoferrin activates macrophages via tlr -dependent and -independent signaling pathways mattsby-baltzer, i. lactoferrin down-regulates the lps-induced cytokine production in monocytic cells via nf-κb respiratory epithelial cells orchestrate pulmonary innate immunity infecciones respiratorias. in temas de bacteriología y virología médica; oficina del libro fefmur the inflammatory response triggered by influenza virus: a two edged sword the effects of corticosteroid on tissue lactoferrin in patients with nasal polyposis global epidemiology of tuberculosis bcg-different strains, different vaccines? influence of bovine lactoferrin on expression of presentation molecules on bcg-infected bone marrow derived macrophages a novel recombinant human lactoferrin augments the bcg vaccine and protects alveolar integrity upon infection with mycobacterium tuberculosis in mice loss of microbicidal activity and increased formation of biofilm due to decreased lactoferrin activity in patients with cystic fibrosis transferrin and lactoferrin undergo proteolytic cleavage in the pseudomonas aeruginosa-infected lungs of patients with cystic fibrosis effect of compounds with antibacterial activities in human milk on respiratory syncytial virus and cytomegalovirus in vitro lactoferrin and surfactant protein a exhibit distinct binding specificity to f protein and differently modulate respiratory syncytial virus infection immune modulation by lactoferrin and curcumin in children with recurrent respiratory infections effect of bovine lactoferrin from iron-fortified formulas on diarrhea and respiratory tract infections of weaned infants in a randomized controlled trial reduced levels of lactoferrin in biofilm-associated chronic rhinosinusitis cleaved inflammatory lactoferrin peptides in parotid saliva of periodontitis patients a novel bovine lactoferrin peptide, fkcrrwqwrm, suppresses candida cell growth and activates neutrophils apoptotic death of listeria monocytogenes-infected human macrophages induced by lactoferricin b, a bovine lactoferrin-derived peptide lactoferrin inhibits or promotes legionella pneumophila intracellular multiplication in nonactivated and interferon γ-activated human monocytes depending upon its degree of iron saturation. iron-lactoferrin and nonphysiologic iron chelates reverse mon biological activity of lactoferrin-functionalized biomimetic hydroxyapatite nanocrystals anti-inflammatory mechanisms of bioactive milk proteins in the intestine of newborns key: cord- -i n x uc authors: hwang, ji young; silva-sanchez, aaron; carragher, damian m.; garcia-hernandez, maria de la luz; rangel–moreno, javier; randall, troy d. title: inducible bronchus–associated lymphoid tissue (ibalt) attenuates pulmonary pathology in a mouse model of allergic airway disease date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: i n x uc inducible bronchus associated lymphoid tissue (ibalt) is an ectopic lymphoid tissue associated with severe forms of chronic lung diseases, including chronic obstructive pulmonary disease, rheumatoid lung disease, hypersensitivity pneumonitis and asthma, suggesting that ibalt may exacerbate these clinical conditions. however, despite the link between pulmonary pathology and ibalt formation, the role of ibalt in pathogenesis remains unknown. here we tested whether the presence of ibalt in the lung prior to sensitization and challenge with a pulmonary allergen altered the biological outcome of disease. we found that the presence of ibalt did not exacerbate th responses to pulmonary sensitization with ovalbumin. instead, we found that mice with ibalt exhibited delayed th accumulation in the lung, reduced eosinophil recruitment, reduced goblet cell hyperplasia and reduced mucus production. the presence of ibalt did not alter th priming, but instead delayed the accumulation of th cells in the lung following challenge and altered the spatial distribution of t cells in the lung. these results suggest that the formation of ibalt and sequestration of effector t cells in the context of chronic pulmonary inflammation may be a mechanism by which the immune system attenuates pulmonary inflammation and prevents excessive pathology. inducible bronchus associated lymphoid tissue (ibalt) is an ectopic lymphoid tissue that forms in the lung following inflammation or infection ( , ) . large b cell follicles that often contain germinal centers (gcs) and a dense network of follicular dendritic cells (fdcs) are the most prominent features of ibalt ( ) . unlike conventional lymph nodes, ibalt is not encapsulated, but is instead embedded in the lung tissue, most often along large bronchi or filling the peri-vascular space of pulmonary artieries ( , ) . despite being in a mucosal tissue, ibalt often lacks a welldefined m cell-containing dome epithelium ( ) and its high endothelial venules (hevs) express peripheral lymph node addressin (pnad) ( ) rather than mucosal addressin cell adhesion molecule (madcam). thus, although ibalt clearly participates in pulmonary mucosal immune responses, it lacks some of the features often associated with classic mucosal lymphoid organs. unlike conventional lymphoid organs, which develop independently of antigen during embryogenesis ( , ) , ibalt forms following pulmonary infection or inflammation ( , ) . although ibalt may develop at any time after birth given the proper pulmonary insult, it most easily forms in neonatal mice due to the relative paucity of tregs and the relative abundance of il- -producing γδt cells ( ) ( ) ( ) . similarly, infant humans also seem to develop (or maintain) ibalt more easily than adults, as the frequency of ibalt areas in the lungs of healthy subjects progressively declines with age ( , ) . interestingly, the neonatal period is the same developmental window when microbial exposure, or lack thereof, imprints the immune system to be more or less susceptible to developing atopic responses to foreign antigens ( , ) or developing autoreactive responses to self antgens-the so-called hygiene effect. given that it functions as a lymphoid tissue ( ) , it is not too surprising that ibalt plays an important role in immunity to pulmonary infections. for example, mice lacking conventional lymphoid organs develop ibalt following infection with influenza and are able to resist doses of virus that would normally be lethal to mice lacking ibalt ( ) . conversely, mice lacking the chemokines that promote ibalt formation succumb to much lower doses of influenza virus ( ) . those same chemokines are essential for the proper formation of pulmonary granulomas, the recruitment of antigen-specific t cells to the lung and the development of protective immunity to mycobacterium tuberculosis ( , ) . moreover, the pulmonary administration of inflammatory agents that trigger ibalt formation also leads to enhanced resistance to influenza, sars corona virus, pneumovirus, francicella tulerensis and coxiella burnetti ( , , ( ) ( ) ( ) . thus, the presence of ibalt is clearly beneficial in the context of pulmonary infections. in contrast, ibalt is a feature of pulmonary pathology that is associated with a variety of chronic lung diseases, including chronic obstructive pulmonary disease (copd) ( , ) , rheumatoid lung disease ( ), hypersensitivity pneumonitis ( ) , and asthma ( , ) , suggesting that ibalt may exacerbate these clinical conditions. for example, copd patients make autoantibodies that react with pulmonary antigens ( ) and patients with advanced disease have reactive ibalt areas with well-developed gcs that show evidence of antigendriven selection ( ) . similarly, patients with rheumatoid lung disease develop large gc-containing areas of ibalt that are surrounded by plasma cells that produce autoantibodies ( ) . thus, many investigators have concluded that the presence of ibalt contributes to pathology in the context of chronic inflammatory diseases. however, it is not clear whether ibalt is a cause or consequence of pulmonary inflammation or whether it contributes in any way to pulmonary pathology ( ) . given the link between ibalt and pulmonary pathology, we tested whether pre-existing ibalt would affect acute allergic responses to a pulmonary allergen. surprisingly, we found that the presence of ibalt did not exacerbate allergic responses to repeated pulmonary sensitization with ovalbumin (ova). instead, mice with ibalt had reduced th -associated mrna expression, less eosinophil recruitment to the lungs and airways, attenuated goblet cell hyperplasia and reduced mucus production following pulmonary sensitization and challenge with ova. interestingly, the presence of ibalt did not alter the initial priming of th cells, but instead delayed their recruitment to the lung and altered their spatial distribution following challenge-th cells were preferentially sequestered in ibalt, which reduced the numbers of th cells in the lung parenchyma. these results suggest that the formation of ibalt and sequestration of effector t cells in the context of pulmonary inflammation may be a mechanism by which the immune system attenuates pulmonary inflammation and prevents excessive pathology. to test the role of ibalt in a mouse model of allergic lung inflammation, we intranasally administered µg lps times over the course of days starting on day after birth and analyzed the lungs by histology on day ( figure a) . as expected ( ) , we observed ibalt structures with separated b and t cell zones and distinct fdc networks in the lpstreated lungs, but not in the control lungs ( figure b) . we also quantified the number of gc b cells by flow cytometry and found that gc b cells were present in lps-treated lungs, but not in control lungs (figures c,d) . given that gcs only form in organized lymphoid tissues, we conclude that pulmonary exposure of neonates to lps promotes the formation of functional ibalt areas. to test the effect of ibalt on the immune response to a pulmonary allergen, we administered lps (or pbs) to neonatal mice as described above, allowed the mice to rest until they were weeks old, then intranasally sensitized the ibalt and control groups with µg ova in combination with low dose ( . µg) lps on days , , and and challenged them on days , , , and with µg ova ( figure e ). we first examined the gc b cell response in the lungs of sensitized and challenged mice and found that gc b cells were abundant in the lungs of mice with ibalt, but were rare in control mice and almost undetectable in completely naïve mice (figures f,g) . we also examined the inflammatory response in the lung and airways by cytospin and found that the proportion (figures h,i) and number (figures j,k) of eosinophils were elevated in mice that had been sensitized and challenged with ova compared to those in naïve mice, however, the proportion (figures h,i) and number (figures j,k) of eosinophils in mice with ibalt were reduced compared to those in control mice. reductions in eosinophils were observed in both the lungs (figures h,j) and the airways (figures i,k) of mice with ibalt compared to those in control mice. the proportions of macrophage/monocytes, lymphocytes, and neutrophils are also shown in figure h and the numbers of those cells are shown in supplementary figure . we also found that total serum ige concentration was elevated in control mice, but less ige was detected in mice with ibalt ( figure l) . upon a histological examination of lung sections, we found that ovaexposed mice with pre-existing ibalt had densely-packed areas of lymphocytes to one side of the bronchi, but relatively clear airspaces, whereas ova-exposed control mice had extensive areas of diffuse inflammation that surrounded the bronchi and extended into the alveolar airspaces ( figure m) . thus, contrary to our expectations, the presence of ibalt did not exacerbate ova-induced pulmonary inflammation, but rather reduced the inflammatory response. the presence of ibalt could have altered t cell priming so that fewer th cells were initially generated after antigen exposure. to test this possibility, we next used il- reporter ( get) mice to enumerate th cells. the get mice have the enhanced green fluorescent protein (egfp) targeted into the il- locus with an internal ribosome entry site (ires) separating the il- coding sequence from the egfp coding sequence ( ) . thus, any cells that express il- mrna will also express egfp and can be easily identified using flow cytometry ( ) . therefore, we treated neonatal get mice with lps to initiate ibalt formation, waited until they were adults and then intranasally sensitized ibalt and control mice with µg ova and . µg lps on days , , and (figure a) . two days after the last sensitization, we enumerated il- reporter (egfp)-expressing cd + t cells. we found a significant expansion of egfp + cd + t cells in both ibalt and control mice relative to naïve mice, but did not observe any difference in the frequencies ( figure b ) or numbers ( figure c ) of egfp + cd + t cells between control and ibalt groups. these data suggested that ibalt did not dramatically affect th priming in the lung. although the previous assay evaluated th priming, we had no way of enumerating antigen-specific cells. therefore, to test this possibility in another way, we transferred x naïve ovaspecific cd + t cells (ot-ii cells) into control and ibalt mice on day after birth, sensitized the recipient mice with ova on days , , and , challenged them on days , , , and and enumerated the responding cells days later in the lung ( figure d ). we found that the frequency ( figure e ) and number ( figure f ) of ot-ii cells that accumulated in the lungs of sensitized and challenged mice were the same in control and ibalt groups. these data suggest that the presence of ibalt does not affect ova-specific t cell accumulation in the lung after sensitization and challenge. although th priming and expansion appeared normal in mice with ibalt, it was not clear whether the final effector cells could actually make th cytokines. therefore, to determine whether cd t cells primed by ova sensitization and challenge could actually produce th cytokines, we sensitized and challenged mice with and without pre-existing ibalt and measured the mrna expression of th cytokines in the lungs h after the last ova challenge ( figure a) . we found that mrnas encoding il- , il- , and il- were increased in ova-exposed and challenged lungs relative to their expression in naïve lungs, but we did not observe a significant difference between the control and ibalt groups ( figure b) . we next tested whether cd + t cells from ova sensitized and challenged control and ibalt mice were equally capable of making th cytokines. to test this possibility, we collected cells from the lung tissue, restimulated them with pma and calcimycin for h and assessed the production of il- , il- , and il- by intracellular staining. we found that the total number of activated cd hi cd + t cells was increased in control and ibalt mice relative to that in naïve mice (figures c,d) , but there was little difference between the control and ibalt groups. we also found that the numbers of cd hi cd + t cells that made il- , il- , or il- were increased in control and ibalt mice relative to those in naïve mice ( figure e ), but there was little difference between the control and ibalt groups. these data suggest that the presence of ibalt does not significantly alter the number of t cells capable of producing th cytokines following sensitization and challenge. th cytokines act on other cells in the lung, such as epithelial cells and macrophages, and promote their activation and differentiation, which changes gene expression. for example, genes like chitinase- -like- (chi l ) and mucin ac (muc ac) are expressed in in the lung following il- signaling ( ) . thus, we next examined the expression of mrnas encoding these genes in the lungs of ibalt and control mice after ova sensitization and challenge as in figure a . we found that mrnas encoding chi l and muc ac were increased in ova-exposed and challenged lungs relative to their expression in naïve lungs, but their expression in the ibalt group was significantly less than that in the control group ( figure f) . thus, the expression of genes that are responsive to th cytokines were reduced in the lungs of ibalt mice relative to those in control mice. these data are more consistent with the reduced histopathology that we see in the lungs of ibalt mice compared to those in the lungs of control mice, suggesting that the difference between the groups is not in th priming and expansion, but in the response to th cytokines. in the experiments above, we were unable to independently control the initial steps of t cell priming and expansion. to rigorously show that these steps were unaffected by the presence or absence of ibalt, we next generated ova-specific th effector t cells in vitro and showed that, upon restimulation, a high frequency of these cells made il- , whereas less than % made ifnγ and almost none of them made il- ( figure a ). we next adoptively transferred x of these th cells into -day-old control or ibalt mice and challenged them with ova on days , , , and ( figure b ). one day after the final challenge, we enumerated donor t cells in the lungs and found that a similar frequency ( figure c ) and number ( figure d ) of donor t cells had accumulated in the lungs of both groups. we also enumerated gc b cells in the lungs and found that a high frequency ( figure e ) and number ( figure f ) of gc b cells accumulated in the lungs of mice in the ibalt group, but not in the lungs of control or naïve mice. despite having similar numbers of donor th cells in the lungs of each group, the histology of the lungs was dramatically different, with the control group exhibiting extensive goblet cell hyperplasia and diffuse inflammatory infiltrate throughout the lungs, whereas the ibalt mice exhibited minimal goblet cell hyperplasia and dense lymphoid accumulations (ibalt) adjacent to, but not surrounding, the airways and in the perivascular space ( figure g) . we also quantified the amount of th cytokines in the bal fluid h after the last challenge and found that, although the amount of il- , il- , and il- was slightly less than in the bal fluid of ibalt mice than in control mice, the differences were not significant ( figure h ). together, these data demonstrate that, despite starting with the same number of th cells in the lung, the biological outcome of inflammation was dramatically different in control and ibalt mice. given that the biological outcome of disease was so different despite starting with similar numbers of fully differentiated th cells, we reasoned that ibalt must engage some sort of regulatory mechanism. one important cell type that has the ability to powerfully attenuate inflammatory responses is the treg population ( ) . tregs are immunosuppressive cd + t cells that have differentiated in a way that promotes the expression of the transcription factor, foxp ( ) . cd + foxp + tregs can reduce eosinophilia ( ) , impair humoral responses and secret an inhibitory cytokines, such as il- and tgfβ ( ) , which suppress the effector functions of activated t cells and reduce inflammation. thus, changes in treg number or activity can have dramatic consequences on immune responses regardless of the number of antigen-specific effector t cells. given the immunosuppressive capacity of tregs, we enumerated cd + foxp + t cells in the lungs of ibalt and control mice following ova sensitization and challenge. we found that although the frequency ( figure a ) and number (figure b ) of tregs increased in the lungs following ova sensitization and challenge, there was no difference in tregs between ibalt and control groups. thus, alterations in treg numbers do not explain why mice with ibalt exhibit attenuated allergic responses in their lungs. previous reports show that pulmonary administration of lps to the lung conditions epithelial cells in a way that promotes the over-expression of a ( ) , an ubiquitinmodifying enzyme that blunts nf-kb signaling thereby reducing expression of pro-th cytokines like il- ( ) . thus, we were concerned that the exposure of neonatal mice to lps may permanently suppress pulmonary inflammatory responses. to test this possibility, we treated neonatal mice with lps or pbs to trigger ibalt formation and, when the mice were -weeks old, transferred ova-specific th effector cells and challenged the recipients times with either ova or pbs ( figure c) . we then enzymatically digested the lung tissue, sorted epcam + cd − sca − cd − bronchial epithelial cells and performed qpcr for tnfaip , the gene encoding the a enzyme. we found that tnfaip expression was similar in all groups of bronchial epithelial cells (figure d) , regardless of whether they came from mice that received lps as neonates or whether they came from mice that were challenged with ova as adults. we also performed qpcr to quantify the expression of the inflammatory cytokines, il- and tslp. we found that although il- expression was strongly increased following ova challenge, it was not altered by previous exposure to lps (figure e ). we also found that tslp expression was not changed by either ova challenge or previous exposure to lps ( figure e) . thus, the pulmonary exposure of neonatal mice to lps did not permanently alter the ability of bronchial epithelial cells to express inflammatory cytokines. despite the overall reduction in th -driven pathology in the mice with ibalt, the number of th effector cells seemed to be similar at the endpoint of the experiment. however, we didn't know whether they accumulated in the lung at the same rate. to determine whether the kinetics of t cell recruitment was the same in mice with and without ibalt, we generated th effectors from naïve otii cells in vitro and then adoptively transferred x th effectors h prior to a series of ova challenges ( figure a) . we then enumerated the transferred th cells and the recruited eosinophils in the lungs and bal h after each ova challenge. not surprisingly, the numbers of donor t cells and eosinophils increased over time following each challenge (figures b-e) . unexpectedly however, we observed significant reductions in the numbers of th effectors (figures b,d) and eosinophils (figures c,e) in both the lungs (figures b,c) and airways (figures d,e) of ibalt mice relative to controls after the first challenges. however, by the fourth challenge, there was no difference in the numbers of t cells. these data suggest that the presence of ibalt slows the accumulation of th effectors as well as eosinophils following pulmonary allergen exposure. these data may also explain why we saw little difference in t cell numbers at the endpoints of earlier experiments, even though we observed significant differences in lung pathology. the most unique attribute of ibalt is its dense accumulations of spatially ordered lymphocytes in a tissue (the lung) that normally has relatively few lymphocytes and rarely has them so densely organized. therefore, we next asked whether the specialized environment of ibalt alters the spatial organization of t cells in the lung following ag exposure. to test this possibility, we generated ova-specific th effector cells in vitro using otii cells. these cells were subsequently transferred into ibalt and control mice and the recipients were challenged with ova times before we examined the lungs by histology (figure a) . we performed immunofluorescence looking for the donor t cell marker (cd . ), b cells (b ), and fdcs (cd ). we found that the donor effector th cells preferentially accumulated in the ibalt areas of ibalt mice and were relatively dilute in the rest of the lung (figure b ). in contrast, the donor th effector cells were distributed more evenly in the control mice. these data were quantified in figure c . importantly, we found that the total number of donor th effector cells in the lungs of mice with and without ibalt were indistinguishable at this time ( figure d) . we also examined cd +foxp + tregs in tissue sections from mice with and without ibalt. we found that tregs were densely clustered in areas of ibalt, but were relatively dilute in the rest of the lung (figures e,f) , despite the similar numbers of tregs in the lungs of mice with and without ibalt (figures a,b) . together, these data suggest that the spatial distribution of effector th cells and tregs is affected by the presence of ibalt (they cluster together), which may explain how ibalt and control mice can have similar numbers of th cells in their lungs, but have so profoundly different outcomes in terms of eosinophil accumulation and histopathology. the above experiments used mice that had been administered lps as neonates in order to form ibalt. however, one concern with these experiments is that neonatal exposure to lps might have altered systemic immunity in some way that ameliorated subsequent inflammatory responses independently of ibalt formation. to rule out this possibility, we generated in vitrodifferentiated otii th cells and transferred x th effector cells into control mice, mice that received pulmonary lps as neonates (ibalt mice) and mice that received peritoneal lps as neonates (i.p. lps mice). the following days, we administered µg ova intranasally to all groups and measured germinal center b cells and eosinophils in the lungs h later ( figure a) . when we enumerated th effectors ( figure b ) and eosinophils ( figure c ) in the lung tissue, the numbers were reduced in the presence of ibalt compared to control mice, but in mice treated with lps intraperitoneally, the number of effector t cells and eosinophils were the same as in the control mice (figures b,c) . thus, the exposure of neonates to pulmonary lps dramatically decreased the inflammatory response in adults, whereas the exposure of neonates to peritoneal lps did not. we also observed that mice that receive pulmonary lps as neonates (ibalt mice) had a significantly higher frequency ( figure d ) and number (figure e ) of gc b cells in the lungs, whereas mice that received lps intraperitoneally as neonates (i.p. lps mice) did not accumulate gc b cells and were comparable to control mice (figures d,e) . these data indicate that systemic exposure to lps does not lead to ibalt formation (as monitored by the germinal center response in the lungs). these data are consistent with the conclusion that lps-mediated ibalt formation alleviates th -dependent inflammatory responses in the lung, independently of any systemic effect of lps on immunity. our data show that the presence of ibalt in the lungs prior to pulmonary sensitization and challenge with ova does not exacerbate th responses, but rather attenuates th -driven pulmonary pathology. in fact, mice with ibalt exhibit delayed th accumulation, reduced eosinophil recruitment, reduced goblet cell hyperplasia and reduced mucus production compared to their control counterparts. although the initial priming of th cells is not affected by the presence of ibalt, the accumulation of th cells in the lung is delayed following pulmonary challenge. more importantly, the spatial distribution of effector t cells is altered by the presence of ibalt, such that effector cd t cells as well as tregs become concentrated in ibalt areas and relatively diluted in the rest of the lung. these results suggest that ibalt functionally sequesters effector t cells, thereby limiting the exposure of the lung parenchyma to t cellproduced inflammatory cytokines, which attenuates pulmonary inflammation and prevents excessive pathology. ibalt is associated with a wide variety of inflammatory lung diseases, including copd ( ), hypersensitivity pneumonitis ( ) and rheumatoid lung disease ( ), all of which are the result of chronic exposure to antigens or inflammatory agents. although asthma is also a chronic lung disease, the mouse model of repeated sensitization and challenge with ova is more like an acute allergic response. however, our data demonstrate that the mere presence of ibalt does not necessarily lead to an increased inflammatory response or pulmonary pathology in the context of pulmonary allergen exposure as one might expect if ibalt was facilitating antigen presentation and t cell activation. therefore, despite participating in local immune responses, ibalt may be beneficial in the context of inflammatory diseases by sequestering activated lymphocytes. the idea of reducing inflammation by sequestering t cells in lymphoid tissue is consistent with the activities of the immunosuppressant drug, fty , which prevents t cells from exiting lymphoid organs-effectively sequestering them and preventing their recirculation through peripheral tissues ( , ) . in fact, treatment with fty reduces airway remodeling and pulmonary inflammation in a rat model of ova-induced asthma ( ) . interestingly, ibalt areas are expanded in the lungs of fty -treated animals, suggesting that the fty -mediated sequestration of lymphocytes can occur in either conventional lymphoid organs or in areas of ibalt in the lung ( ) . another physical change that occurs in the lung concomitantly with the development of ibalt is the formation of additional lymphatic vessels surrounding the ibalt follicles ( ) . live imaging of ibalt suggests that these lymphatic vessels may facilitate the collection of dcs from the airways ( ) . static imaging also shows that ibalt areas collect inhaled antigens and particulates ( ) , suggesting that ibalt may sequester antigen as well as t cells. in this context, it is interesting to note that mice that spontaneously develop ibalt in the context of rheumatoid lung disease are highly resistant to developing fibrosis following pulmonary challenge with bleomycin ( ) . if ibalt areas efficiently collect and sequester intratracheally administered bleomycin or remove it from the lung via lymphatics, then one would expect to observe reduced fibrosis. similarly, if ibalt areas collect and sequester pulmonary antigens like ova and remove them from the lung parenchyma, one would expect reduced pulmonary inflammation. thus, ibalt may protect the lung by collecting antigens and inflammatory substances as well as t cells. our data are also consistent with observations that ibalt attenuates inflammatory responses and improves clinical outcomes following infection with a variety of pulmonary pathogens. for example, the presence of ibalt promotes the survival of mice that are challenged with normally lethal doses of influenza virus, pneumovirus and sars corona virus ( ) and attenuates the pulmonary pathology associated with these infections. much of the clinical pathology, such as weight loss, associated with pulmonary viral infections is linked to the production of inflammatory cytokines by t cells. thus, if ibalt sequesters virus-specific effector t cells, it may limit the exposure of the rest of the lung to inflammatory cytokines and chemokines and reduce the recruitment of additional inflammatory cells-thereby attenuating pathology. however, ibalt-mediated resistance to pneumovirus is associated with impressive increases, rather than decreases, in the mrna expression of ifnγ, cxcl , and ccl in the lung ( ) . these responses were measured by pcr using mrna extracted from whole lungs, so it is difficult to know whether the increases were predominantly confined to ibalt areas or not. in addition, it is not clear whether the reported increase is due to an overall increased magnitude of the response or altered kinetics, such that more t cells are responding earlier. this last possibility seems plausible, given that accelerated antibody responses are also observed in mice with ibalt ( ) . interestingly, we also observe altered kinetics of effector t cell recruitment to the lungs following pulmonary challenge with allergen. however, we find that fewer effector th cells accumulate in the lungs at early times after allergen exposure. the differences between our results and those reported for pneumovirus may be due to the types of antigens (replicating virus vs. inert allergen) or to the types of t cells (virus-specific cd and th cells vs. allergen-specific th cells). in any case, the presence of ibalt clearly has an impact on the kinetics of pulmonary immune responses. t cells are often imprinted with particular effector functions that differ depending on the secondary lymphoid organ in which they were primed ( , ) . thus, one could envision that ibalt skews cd t cell differentiation away from a th pathway and thereby attenuates the symptoms of allergic disease. however, we found equivalent numbers and frequencies of th cells primed in mice with or without ibalt, suggesting that cd t cell differentiation is not deviated toward an alternative effector function. moreover, we observed reduced pathology in mice with ibalt following the adoptive transfer of in vitro differentiated th effector cells, demonstrating that even when t cell priming occurs in vitro, the functional outcome of the effector response is altered beneficially in mice with ibalt. interestingly, naïve t cells primed in aortaassociated lymphoid tissues in apoe −−/−− mice preferentially differentiate into tregs that suppress atherosclerosis ( ) . although the preferential formation of tregs in ibalt areas would help to explain the reduced inflammation and pulmonary pathology in mice with ibalt following ova sensitization and challenge, we did not observe changes in either the number and frequency of tregs in the lung, regardless of whether ibalt was present. nevertheless, we did find that tregs were preferentially concentrated in ibalt areas and co-localized with effector t cells, suggesting that tregs in ibalt may more effectively suppress the inflammatory functions of effector t cells in this location. thus, in the context of pulmonary sensitization with allergens such as ova, ibalt seems to alter the placement of t cells rather than changing their differentiation pathways. considering that many of the adoptively transferred th cells are being sequestered in the ibalt areas and seem to be located in the b cell follicle, it is possible that they are converting to il- -expressing tfh cells ( ) . this possibility would be consistent with the large gcs and high frequencies of gc b cells that we observe in the lungs following adoptive transfer of th cells and sensitization with antigen. in addition, ibalt-induced resistence to influenza and sars corona virus is associated with accelerated antibody responses ( ) , suggesting that local tfh cells are promoting b cell differentiation. although th -tfh cells in b cell follicles are potent producers of il- ( ), they are directing cytokine secretion toward b cells rather than pulmonary epithelial cells, which would likely lead to accelerated antibody secretion, but reduced pulmonary pathology. thus, ibalt may be sequestering t cells and directing cytokine secretion toward alternative cell types, both of which should attenuate pulmonary pathology. in summary, our data show that the presence of ibalt does not exacerbate pulmonary pathology following sensitization and challenge with an allegen, but rather attenuates th induced inflammatory responses. this same mechanism may also explain the ability of ibalt to ameliorate pulmonary pathology in the context of infection and may be a general mechanism by which ectopic follicles in a variety of tissues attempt to cope with chronic inflammatory responses. thus, we may want to develop treatments that promote, rather than prevent, ectopic follicle formation in the context of chronic inflammatory conditions. to induce ibalt, pups were intranasally administered µg lps from escherichia coli ( :b ; sigma) in µl pbs every other day starting on day after birth for a total of times. in control experiments, pups were intraperitoneally injected with µg lps in µl pbs every other day starting on day after birth for a total of times. control mice received µl pbs or nothing according to the same schedule. to induce allergic airway inflammation, adult mice were sensitized intranasally with µg ova (sigma) plus . µg lps in µl pbs and subsequently challenged intranasally with µg ova in µl pbs according to the schedule indicated in each experiment. in the dc labeling experiments, we intratracheally administered µg ova labeled with alexa flour r (invitrogen). cd + t cells were purified from the spleens of naive cd . + b congenics using ls columns and anti-cd macs beads (miltenyi biotec) according to the manufacturer's instructions. single t cell preparations were > % pure as determined by flow cytometry. th effectors were generated by activating naïve t cells for - h with plate-bound anti-cd ( µg/ml; - c ; bioxcell) and anti-cd ( . µg/ml; . ; ebioscience) in the presence of il− ( u/ml; dnax) and anti-ifnγ ( µg/ml; xmg . ; ebioscience) followed by feeding with il− ( u/ml; peprotech) for additional h. naïve and effector cd + (cd . + ) t cells ( x /mouse) were injected intravenously into no ibalt and ibalt recipients that were subsequently immunized with ova. lungs were cut into small fragments and digested with . mg/ml collagenase a (sigma) and µg/ml dnase i (sigma) in rpmi medium (gibco) at • c for min to obtain single-cell suspensions. digested tissues were mechanically disrupted by passage through a metal strainer. cell suspensions from lungs and mediastinal lns were centrifuged and resuspended in mm nh cl, mm khco , and . mm ethylenediaminetetraacetic acid (edta; lonza) for min to lyse red cells. cell suspensions were then filtered through a µm nylon cell strainer (bd biosciences), washed, and resuspended in phosphate-buffered saline (pbs) with % donor calf serum and µg/ml fc block ( . g ; trudeau institute) on ice for min before staining with fluorochromeconjugated antibodies against the following antigens: cd (rm - ), cd b (m / ), cd (pc ), cd r (b ; ra - b ), cd (fas; jo ), cd ( - ), siglec-f (e - ), and i-a b (af - . ; all from bd biosciences); cd c (n ), cd (im ), cd . (a ), cd ( e ), and foxp ; (fjk− s); all from ebioscience; cd ( d ); from biolegend; cd (nimr− ); from southernbiotech; and goat anti-rabbit-alexa flour r (invitrogen). for intracellular staining, single-cell suspensions from the lungs were stimulated on plates coated with anti-cd ( µg/ml; - c ; bioxcell) in the presence of brefeldin a ( . µg/ml; sigma) for h. the restimulated cells were surface stained, then fixed in % paraformaldehyde, made permeable with . % triton tm x− (sigma), and stained with anti-il− ( b ), anti-il− (trfk ; all from bd biosciences) or anti-il− ( a; ebioscience). cells were analyzed with a lsr ii (bd biosciences) or facscanto ii (bd biosciences) located at the university of rochester and university of alabama at birmingham flow cytometry core facility. to isolate lung epithelial cells, we perfused the lungs with pbs containing . mm edta (lonza), instilled with ml dispase ( µg/ml; corning), and incubated them in a shaker at • c for min. using forceps, lungs were torn into small pieces, and put back in the shaker at • c for an additional min in dispase ( µg/ml; corning) and dnasei ( µg/ml; worthington biochemical). digested tissues were red cell lysed, filtered through a µm nylon cell strainer (bd biosciences), resuspended in pbs with % donor calf serum, µg/ml fc block ( . g ; trudeau institute) and anti-cd macs beads (miltenyi biotec) on ice for min. cell suspensions were depleted of cd -expressing cells using ls columns (miltenyi biotec) according to the manufacturer's instructions. the flow through cells were stained with anti-cd ( ; invitrogen); anti-cd . ( ) and anti-cd (epcam; g . ; all from biolegend) and anti-sca- (ly a/e; d ; invitrogen). lung epithelial cells were sorted with facsaria ii (bd biosciences) located at university of alabama at birmingham flow cytometry core facility. lungs were instilled with ml hank's balanced salt solution (hbss; corning) containing . mm edta (lonza) and cell suspensions (≈ , cells in µl) were centrifuged at rpm for min in a shandon cytospin cytocentrifuge (cell preparation system). the cytospin pellets were air dried on glass slides, stained with shandon kwik-diff tm stain kit (thermo scientific) and were mounted with richard-allan scientific tm cytoseal tm (thermo scientific). total ige levels were determined by coating plates with µg/ml purified rat anti-mouse ige (r - ; bd biosciences). standard curve was prepared with purified mouse ige κ (c - ; bd biosciences) and bound abs in serum samples were detected with biotin rat anti-mouse ige (r - ; bd biosciences) and streptavidin-alkaline phosphatase (invitrogen). alkaline phosphate substrate (moss, inc.) was added, and color development was detected with spectramax r m (molecular devices) at nm. paraffin-embedded sections ( µm in thickness) were stained with hematoxylin and eosion (h&e) for standard histology and periodic acid-schiff (pas) for airway mucus production. frozen sections ( µm in thickness) were prepared from lungs perfused with a mixture of optimum cutting temperature (oct) compound (sakura finetek) in pbs at a : ratio. sections were fixed in cold acetone for min and blocked with fc block ( µg/ml) and % (vol/vol) normal donkey serum in pbs at • c for min. sections were then stained with the following primary abs in pbs at rt for min: goat anti-cd ε (m− ; santa cruz biotechnology), anti-cd c (hl ), and anti-cd r (b ; ra - b ; all from bd biosciences); anti-cd /cd ( e ; biolegend); and peanut agglutinin (pna; sigma). primary abs were detected with donkey antigoat-alexa flour r , rabbit fluorescein-alexa fluor r , streptavidin-alexa fluor r , and streptavidin-alex flour r (all from invitrogen) at rt for min. slides were mounted with slowfade r gold antifade mountant with ′ , diamidino− -phenylindole (dapi; invitrogen). images were collected with a zeiss axiocam digital camera (zeiss) or nikon andor clara camera (nikon). the images were obtained with a x objective for a final magnification of x . t cells in immunofluorescent images were quantified by counting cd . + cells in ibalt areas (defined manually using the outline tool) and by counting cd . + cells in non-ibalt areas (excluding large empty airways). the mean number of t cells per µm was determined using a mosaic of images obtained from control and ibalt-containing lungs that were sectioned at a similar depth and orientation. data were obtained from multiple sections from each mouse and mice per group. total rna was extracted from lungs with trizol according to the manufacturer's specifications (invitrogen) and was repurified with an rneasy mini kit (qiagen). ribonucleic acid (rna; . µg) was reverse-transcribed with superscript ii and random primers (invitrogen) and complementary deoxyribonucleic acid (cdna; ng) was amplified with following primers and probes: glyceraldehyde− -phosphate dehydrogenase (gapdh; trudeau institute), il , il , il , chi l , muc ac. tnfaip , and tslp (all from applied biosystems), with taqman r gene expression master mix (applied biosystems) and all reactions were run on a lightcycler realtime pcr system (roche). the relative level of messenger rna (mrna) expression for each gene was first normalized to the expression of gapdh and then compared to the average level of mrna expression in lungs from naïve b mice. data is expressed as logarithmic fold changes in mrna expression. lungs were instilled with ml hbss (corning) containing . mm edta (lonza) and sigmafast tm protease inhibitor cocktail tablets (sigma). total protein levels of cytokines il− , il− , and il− in lavage were quantified by mousespecific milliplex r multi-analyte kits (emd millipore) using a magpix r instrument platform and related xponent r software (luminex corporation). the readouts were analyzed with the standard version of the milliplex analyst software (emd millipore). the difference in mean values between two groups was analyzed with a two-tailed student's t-test. if three or more groups were compared, tukey's multiple comparison test was used (graphpad prism version . ). p-values of less than . were considered statistically significant. the raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. the animal study was reviewed and approved by institutional animal care and use committee, university of alabama at birmingham. bronchus-associated lymphoid tissue bronchus-associated lymphoid tissue (balt) structure and function inducible bronchus-associated lymphoid tissue (ibalt) in patients with pulmonary complications of 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sudden infant death syndrome and other causes exposure to environmental microorganisms and childhood asthma farm living: effects on childhood asthma and allergy role of inducible bronchus associated lymphoid tissue (ibalt) in respiratory immunity in a murine tuberculosis model, the absence of homeostatic chemokines delays granuloma formation and protective immunity cxcr (+) t helper cells mediate protective immunity against tuberculosis bronchus-associated lymphoid tissue (balt) and survival in a vaccine mouse model of tularemia persistence and responsiveness of immunologic memory in the absence of secondary lymphoid organs inducible bronchus-associated lymphoid tissue elicited by a protein cage nanoparticle enhances protection in mice against diverse respiratory viruses increased number of b-cells in bronchial biopsies in copd the nature of small-airway obstruction in chronic obstructive pulmonary disease development of bronchus-associated lymphoid tissue in chronic hypersensitivity pneumonitis aggregations of lymphoid cells in the airways of nonsmokers, smokers, and subjects with asthma interleukin- expression in the lung epithelium of transgenic mice leads to pulmonary changes pathognomonic of asthma autoantibodies in patients with chronic obstructive pulmonary disease role of lymphotoxin-alpha in cigarette smoke-induced inflammation and lymphoid neogenesis lymphoid follicles in (very) severe copd: beneficial or harmful? analysis of type immunity in vivo with a bicistronic il- reporter a two-step process for cytokine production revealed by il- dual-reporter mice interleukin- : central mediator of allergic asthma regulatory t cells and immune tolerance foxp + regulatory t cells in the human immune system cd +cd + t cells regulate airway eosinophilic inflammation by modulating the th cell phenotype antigen-specific regulatory t cells develop via the icos-icos-ligand pathway and inhibit allergen-induced airway hyperreactivity farm dust and endotoxin protect against allergy through a induction in lung epithelial cells house dust mite allergen induces asthma via toll-like receptor triggering of airway structural cells fty : sphingosine -phosphate receptor- in the control of lymphocyte egress and endothelial barrier function fty blocks egress of t cells in part by abrogation of their adhesion on the lymph node sinus treatment with a sphingosine- -phosphate analog inhibits airway remodeling following repeated allergen exposure preferential lymphatic growth in bronchus-associated lymphoid tissue in sustained lung inflammation induced bronchus-associated lymphoid tissue serves as a general priming site for t cells and is maintained by dendritic cells inhalation of diesel exhaust for three months affects major cytokine expression and induces bronchus-associated lymphoid tissue formation in murine lungs autoreactive t and b cells induce the development of bronchus-associated lymphoid tissue in the lung a functionally specialized population of mucosal cd + dcs induces foxp + regulatory t cells via a tgf-beta and retinoic aciddependent mechanism selective imprinting of gut-homing t cells by peyer's patch dendritic cells artery tertiary lymphoid organs control aorta immunity and protect against atherosclerosis via vascular smooth muscle cell lymphotoxin beta receptors t follicular helper cells differentiate from th cells in response to helminth antigens il- -producing cd + t cells in reactive lymph nodes during helminth infection are t follicular helper cells the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/ . /fimmu. . /full#supplementary-material the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © hwang, silva-sanchez, carragher, garcia-hernandez, rangel-moreno and randall. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -wurzy k authors: van der merwe, rené; molfino, nestor a title: challenge models to assess new therapies in chronic obstructive pulmonary disease date: - - journal: int j chron obstruct pulmon dis doi: . /copd.s sha: doc_id: cord_uid: wurzy k chronic obstructive pulmonary disease (copd) is a major cause of morbidity and mortality. current therapies confer partial benefits either by incompletely improving airflow limitation or by reducing acute exacerbations, hence new therapies are desirable. in the absence of robust early predictors of clinical efficacy, the potential success of novel therapeutic agents in copd will not entirely be known until the drugs enter relatively large and costly clinical trials. new predictive models in humans, and new study designs are being sought to allow for confirmation of pharmacodynamic and potentially clinically meaningful effects in early development. this review focuses on human challenge models with lipopolysaccharide endotoxin, ozone, and rhinovirus, in the early clinical development phases of novel therapeutic agents for the treatment and reduction of exacerbations in copd. the global initiative for chronic obstructive lung disease defines chronic obstructive pulmonary disease (copd) as a … common preventable and treatable disease characterized by persistent airflow limitation that is usually progressive and associated with an enhanced chronic inflammatory response in the airways and the lung to noxious particles or gases. exacerbations and comorbidities contribute to the overall severity in individual patients. exacerbations are associated with increased airway and systemic inflammation, which lead to airway wall edema, sputum plugging, and bronchoconstriction. copd remains a major global health and economic burden that is expected to be the third leading cause of death, and the fifth leading cause of disability by . in , copd accounted for $ . billion in health care expenditures in the united states alone ($ . billion in direct health care expenditures, $ . billion in indirect morbidity costs, and $ . billion in indirect mortality costs). in europe, copd accounts for . billion euros in health care spending a year. pharmacological therapy is used to control symptoms, as well as to reduce exacerbations, and to improve exercise tolerance. ambulatory copd patients are currently treated with long-acting bronchodilators and inhaled corticosteroids, along with systemic corticosteroids during exacerbations. there is a pressing need to develop novel approaches for the treatment of copd and the prevention or reduction of acute exacerbations of copd. existing therapies give partial benefits either by incompletely improving airflow limitation or reducing acute exacerbations, hence the need for newer, more effective therapies. evidence that existing medications reduce lung function decline in the long term has been inconclusive. the torch study investigated the effects of combined salmeterol plus fluticasone, either component alone, and placebo, on the rate of postbronchodilator forced expiratory volume in second (fev ) decline. the investigators found that salmeterol plus fluticasone reduced the rate of fev decline by ml/year compared with placebo. despite this large study, a meta-analysis conducted by soriano et al concluded that inhaled corticosteroids showed a significant improvement in lung function decline compared with placebo at months, however, after months there was no significant difference between placebo and inhaled corticosteroid treatment. one of the main challenges in developing new therapeutic agents for the treatment or prevention of acute exacerbations of copd is that their potential success cannot be entirely known until the investigational therapies enter relatively large phase ii studies, assessing clinical outcome over a -to -month period or longer. , this article reviews the experimental challenges that can be performed relatively early in drug development for the treatment of copd in order to obtain preliminary signals of safety and efficacy in humans. these challenge models are representative of the local inflammatory response caused by an exacerbation of copd; however it is important to note that these models do not reflect the actual exacerbation milieu. the models chosen are those models that have successfully been used to date in copd drug development. depending on the dose given during inhalation, these challenge models may also cause local and systemic inflammation, thus making them ideal for assessing the inflammatory processes in the lungs during an exacerbation and the potential therapeutic benefit of novel agents, as they mimic the local inflammatory response in the lung during an exacerbation. lipopolysaccharide (lps) is a macromolecular cell wall surface antigen of gram-negative bacteria. it is made up of three components: the o antigen (or o polysaccharide) side chain, the core oligosaccharide, and the lipid a moiety. lps is an extremely biologically active substance and has been used for many years in preclinical and clinical research, due to its role in activating many transcription factors. in the serum, lps binds to a lipid-binding protein, which facilitates the association between lps and cd on the cell membranes. this in turn facilitates the transfer of lps to the trl /md complex ( figure ) . this triggers a signaling cascade in macrophage lineage and endothelial cells, resulting in the secretion of proinflammatory cytokines and nitric oxide, and the activation of complement and the coagulation systems that contribute to characteristic features of inflammation, and with excessive stimulation, "endotoxic shock." in monocytes and macrophages, lps triggers the production of powerful inflammatory mediators including cytokines (eg, interleukin [il]- , il- , il- , tumor necrosis factor [tnf]-α and platelet-activating factor), which stimulate production of prostaglandins and leukotrienes. in addition, lps activation results in enhanced macrophage phagocytic and cytotoxic activity. activation of alternative complement pathway factors c a and c a induce histamine release, and affect neutrophil chemotaxis and accumulation. kinin activation releases bradykinins and other vasoactive peptides, which cause hypotension. the release of these mediators and subsequent systemic response make lps a powerful research tool in evaluating inflammatory pathways. healthy subjects inhaling endotoxin show a systemic and pulmonary inflammatory response, recruiting neutrophils and macrophages to the lung tissue. inhalation of nebulized doses (up to µg) of lps via a dosimeter in healthy volunteers, leads to an increase in temperature, blood c-reactive protein (crp), blood and sputum neutrophils, blood monocytes and lymphocytes, and blood and sputum proinflammatory mediators, including: il- , tnfα, myeloperoxidase (mpo), matrix metalloproteinase- , il- , il- β, monocyte chemotactic protein- , and macrophage inflammatory protein- β. , bronchial segmental instillation induces an early phase response ( - hours), resulting in a statistically significant increase in neutrophils, tnf, il- β, il- r antagonist, il- , and granulocyte colony-stimulating factor. neutrophils, macrophages, and monocytes increase - hours post instillation. , intranasal lps challenge may be the least invasive and best tolerated model. clinical symptoms are minimal; however, very little or no data have been published to date using this model. further validation of the intranasal model needs to be performed to compare it to the lps inhalation challenge model, to better understand the relevance of the lps-triggered nasal inflammation to the phenomena that occur in the central and distal airways in copd. many of the effects of exogenous lps can be blocked by medications. in one study, pretreatment with oral prednisolone or cilomilast had no effect on the local lps-induced inflammatory response in the lung; pretreatment with prednisolone alone significantly inhibited the lps-induced crp response. cilomilast attenuated the increase in crp, but not significantly. similarly, in another study, subjects who received simvastatin - mg demonstrated a reduction in neutrophils, mpo, tnfα, matrix metalloproteinase- , - , and - in the bronchoalveolar lavage fluid (balf), as well as a reduction in plasma crp, versus placebo. hohlfeld et al used lps to show that roflumilast reduced the influx of total cells, neutrophils, and eosinophils into the airways of healthy subjects after segmental challenge with endotoxin, with statistical significance. it is important to remember that inhaled lps challenge is a model of acute neutrophilic inflammation and not a model of copd. the model can be used to understand the biological effects of compounds that inhibit the lps pathway (table ) . ozone (o ) is a major component of urban environmental air pollution. it is formed in the troposphere from primary precursor pollutants. in the presence of light, no is cleaved by sunlight to no figure ). in epidemiological studies, o levels have been associated with exacerbations of asthma, copd, and pneumonia. [ ] [ ] [ ] experimental o exposure in healthy human subjects is known to elicit a reversible impairment in lung function, as well as acute proximal airways neutrophilic inflammation, and an increase in the concentration of several cytokines and mediators of inflammation within the airways. in the first reported study of the inflammatory effects of low-level o exposure ( ppb o for . hours) in healthy volunteers, there were statistically significant increases in polymorphononuclear neutrophils, prostaglandin e , lactate dehydrogenase, il- , α -antitrypsin, and decreased phagocytosis via the complement receptor. this is similar to a more recent study with low-level exposure to o at ppb for . hours, in which there were increased airway neutrophils, monocytes, and dendritic cells, as well as modifications of the expression of cd , hla-dr, cd , and cd on monocytes. in another study examining whether circulating cd b plays a role in the inflammatory response following inhaled o exposure, volunteers underwent controlled exposure to o ( ppb for hours) and to clean air on two separate occasions. induced sputum collected from subjects exposed to o revealed marked neutrophilia, and increased expression of mcd on airway macrophages and monocytes. circulating cd b levels also predicted the magnitude of the airway neutrophil response following inhaled o exposure. a number of different classes of therapeutic agents have been studied in the o challenge model in healthy volunteers. therapeutic classes include corticosteroids (administered orally and by inhalation) and nonsteroidal anti-inflammatory drugs. more recently, studies investigating the effects of cxc chemokine receptor (cxcr) , antagonists have been reported. [ ] [ ] [ ] [ ] [ ] holz et al conducted a double-blind, double-dummy, placebo-controlled, three-period crossover study. eighteen healthy subjects, who had been shown at screening to produce more than a % increase in sputum neutrophils in response to exposure to ppb o , were randomly assigned to receive alternating single orally inhaled doses of fluticasone mg, mg of prednisolone orally, and placebo at least weeks apart. compared with placebo, pretreatment with inhaled or oral corticosteroids resulted in a significant reduction of sputum neutrophils, by % and %, respectively. this was associated with statistically significant reductions in sputum mpo, by % for inhaled corticosteroids and % for oral steroids. compared with placebo, there was a mean reduction in sputum il- levels, by % after inhaled corticosteroids and % after oral corticosteroids. similar results were obtained in a study conducted by alexis et al. in subjects receiving fluticasone . mg and mg, sputum neutrophilia was significantly reduced by % and %, respectively. the following inflammatory markers were also significantly reduced in a dose-dependent manner in subjects receiving fluticasone: cd b, mcd , cd , cd , hla-dr, and cd on sputum monocytes. serum clara cell protein levels (a marker of pulmonary damage) were significantly increased post-o challenge. schelegle et al pretreated healthy volunteers with indomethacin, which was shown to significantly reduce o -induced decrements in fev and forced vital capacity, when compared to no drug or placebo. this was associated with reductions in subjective symptoms of cough, shortness of breath, and throat tickle on indomethacin treatment, suggesting that cyclooxygenase products play a partial role in subjective symptoms associated with o exposure. hazucha et al demonstrated a similar reduction of o -induced decrements in fev and forced vital capacity following singledose treatment with either mg or mg of ibuprofen compared to placebo, which was associated with reduced post-o balf levels of prostaglandin e , thromboxane b , and il- . sch is a novel, selective, oral cxc chemokine receptor antagonist that inhibits neutrophil activation and modulates neutrophil trafficking in animal models. eighteen healthy o responders (. % increase in sputum neutrophils) underwent o challenge tests ( ppb, hours intermittent exercise) hour after the last treatment dose, and sputum was induced at hours postchallenge following sch treatment. the o challenge resulted in statistically significantly lower sputum neutrophil counts ( . × ml − ) compared with prednisolone ( . × ml − ; p = . ) or placebo ( . × ml − ; p = . ). comparable results were obtained for total cell count, percentage of sputum neutrophils, and for il- and myeloperoxidase in sputum supernatant. post challenge, sch inhibited neutrophilia in peripheral blood, but significantly less than in sputum. gaga studies in copd have been conducted using o concentrations in the range of - ppb with . - minutes of exercise every minutes, aiming to maintain a ventilatory rate of between - l/min. [ ] [ ] [ ] in a study of nine subjects with copd and ten age-matched controls, gong et al found an increase in specific airway resistance and a statistically significant decrease in fev in the copd subjects versus the age-matched controls. in summary, o challenge has been well tolerated in healthy volunteers and in older subjects, as well as subjects with asthma or copd. the o -challenge model potentially provides critical decision-making data in understanding whether new compounds have the desired biological effect in healthy volunteers and patients with copd; hence it can de-risk decisions to move forwards into large phase ii safety and efficacy trials. rhinovirus is responsible for the common cold and is spread through infected respiratory secretions from one person to another. human rhinovirus (hrv) replicates at °c- °c and thus has been linked to upper airway infections, where mucosal surfaces are cooler. evidence exists that hrv is not limited to the upper airways. gern et al hrv binds to intracellular adhesion molecule (icam)- , the major hrv receptor. the low-density-lipoprotein receptor binds to a minor group of hrv (hrv ). the gene for icam- maps to human chromosome , as do the genes for a number of other picornavirus receptors. several studies have shown induction of proinflammatory genes implicated in neutrophil activation following rhinovirus induction of bronchial epithelial cells (eg, il- regulated by nf-κβ signaling pathways and groα). [ ] [ ] [ ] rhinovirus infection of epithelial cells leads to the release of proinflammatory cytokines and chemokines including il- and il- . chemokines attract inflammatory cells (eg, neutrophils, eosinophils). these cells release toxic products, stimulating mucus production and leading to tissue damage, with possible long-term loss of lung function. some mediators, such as endothelin- , have a direct effect in causing bronchoconstriction and vasoconstriction, resulting in airflow obstruction and impaired gas exchange. healthy subjects, subjects with asthma, and subjects with allergic asthma have been intensively studied in clinical trials inoculating them with rhinovirus or other rhinovirus serotypes. [ ] [ ] [ ] these studies demonstrated that rhinovirus infection of the lower airways is common after experimental inoculation. several studies looking at causes of exacerbations in copd have shown that viruses account for up to % of exacerbations, and that hrv is numerically the most important virus type. , [ ] [ ] [ ] [ ] [ ] [ ] figure depicts the total viral and hrv exacerbation rate in seven exacerbation studies. other viruses associated with acute exacerbations of copd are coronavirus, influenza a and b, parainfluenza, adenovirus, and respiratory syncytial virus. , [ ] [ ] [ ] [ ] [ ] to develop a model of viral exacerbation in subjects with copd, mallia et al conducted a virus dose-escalating study infecting four copd subjects with rhinovirus. in this study, the median tissue culture infective dose (tcid ) of rhinovirus was administered by the inhaled route using a nebulizer, to elicit a copd exacerbation. although there was a decrease in fev ( %) and peak expiratory flow ( %) maximal on day , there was not a statistically significant increase in total sputum cell count or peripheral neutrophil count. symptoms of cold and lower respiratory tract symptoms, as well as lung function changes that are characteristic of viral-induced exacerbations of copd, were observed. there was an increase, although not statistically significant, in the proinflammatory cytokines il- and il- . in another study, there were significant increases in total respiratory scores in both copd subjects and submit your manuscript | www.dovepress.com dovepress dovepress healthy controls. peak expiratory flow fell by . ml in the controls (p = not significant) and by . ml (p , . ) in the copd patients. peripheral white cell counts and neutrophils increased in both groups. sputum neutrophil count also increased in the copd patients but not in the controls. more recently, both the upper and lower symptom scores were found to be significantly higher in the copd subjects. in this study, ten of the infected copd subjects met the criteria defining an exacerbation of copd. subjects in the copd group demonstrated significant decreased peak expiratory flow from baseline, while those in the control group did not. the blood and sputum showed a significant increase in peripheral neutrophils in the copd subjects but not the controls. however, crp was significantly increased in both groups on day . subjects in the copd group had significantly increased sputum neutrophil elastase levels over baseline on days and , as well as il- levels on day . sputum neutrophil elastase levels were significantly higher in subjects with copd, compared with control subjects on days - . to date, only one laboratory has published on this model and as such, the data should be interpreted with caution. the main advantage of this model is that it will give a clear understanding and insight into the molecular and cellular inflammatory processes that take place during a viral-associated exacerbation of copd. there are no published data to date on the effect of pharmacological interventions in this model. the rhinovirus challenge model has the potential for use as a preclinical and clinical tool to identify and investigate novel drug targets and establish whether new therapeutic agents have potential clinical utility. these include (but are not limited to) targets against soluble icam- and thus inhibit interaction of hrv with icam- , , inhibitors of rhinovirus rna-dependent rna polymerases d, activators of retinoic acid-inducible gene , inhibitors of rhinovirus capsid protein vp- and inhibitors of different rhinovirus proteases (eg, a, c). , currently, this is the only model that reflects the underlying mechanism of viral exacerbations of copd. the use of challenge models has the potential to significantly inform early decision making, before embarking on longterm phase ii and iii clinical trials designed to test interventions that may treat or avert exacerbations of copd. although challenge models are good predictive models of acute exacerbations of copd, there are ethical considerations associated with inducing exacerbations in subjects with copd. therefore, safety boards may be advised to only consider subjects with mild copd for inclusion in these studies. healthy subjects could be used as an alternative, when determining the effects of a developmental drug's mechanism of action on lung inflammation. the lps and o models have been used successfully in healthy subjects. [ ] [ ] [ ] , , [ ] [ ] [ ] as these models represent the local inflammation in the lung during an exacerbation, and test the mechanism of action of potential novel drugs, these data may be used for future decision making. the lps challenge model is the best validated model in subjects with copd. pharmaceutical companies have used lps models as a means to establish proof of principle early during the clinical development process, because they are relatively simple to perform and have few adverse events. this model is cost effective because it can be conducted in healthy subjects, who are easy to recruit. lps challenge data, whether positive or negative, can provide valuable information to aid investment decision making. one disadvantage of the lps model is that it is a model of lung inflammation, but not of the disease state, thus preclinical validation of the developmental drug's effects on lps pathways is essential. for anti-inflammatory targets that are involved in the tolllike receptor , nf-κβ pathway, the lps challenge model is the model of choice. despite the longstanding knowledge and understanding of the adverse effects of o on pulmonary biology, the use of o as a challenge model to assess the potential of new drugs for the prevention of acute exacerbations in copd, is relatively new. the model has been shown to be safe and to have few side effects in healthy volunteers, and in patients with asthma and copd. , , , additionally, it is reliable and reproducible. it has been used successfully to generate biological effect and systemic effect for fluticasone and the cxcr antagonists, sch and sb- . , a limitation of the o model is that it has yet to be determined whether inhibition of neutrophilia translates into clinical benefits for patients with copd. preliminary data indicate that inhibition of the neutrophil response following o challenge may be associated with beneficial changes in system scores obtained in subjects with copd. challenge with rhinovirus to elicit mild exacerbations in subjects with copd appears to be safe and well tolerated, but only a few copd subjects have been exposed to this model. the observation that fev does not always return to baseline after inducing an exacerbation in copd subjects may call to question the feasibility of using the challenge in a broader population of patients with copd, in addition to raising ethical considerations. this remains an exciting model with a great deal of potential, as rhinovirus models are good predictive models of viral-induced acute exacerbations of copd. in order for pharmaceutical companies to succeed in the copd arena, innovative approaches to clinical trial design and conduct are required that will generate critical, highquality proof of efficacy and biologic target engagement data to support early investment decision making, early drug termination, and facilitation of better-informed decisions regarding those drugs in which proof of effect has been clearly demonstrated. challenge models in copd, which expose fewer individuals for short periods of time to eliciting agents, may serve as surrogate of potential efficacy and thus may help early decision making and reduction in clinical development timelines. global initiative for chronic obstructive lung disease. global strategy for the diagnosis, management and prevention of copd chronic obstructive pulmonary disease (copd) fact sheet key facts effect of pharmacotherapy on rate of decline of lung function in chronic obstructive pulmonary disease: results from the torch study a pooled analysis of fev decline in copd patients randomized to inhaled corticosteroids or placebo salmeterol and fluticasone propionate and survival in chronic obstructive pulmonary disease for uplift study investigators. mortality in the -year trial of tiotropium (uplift) in patients with chronic obstructive pulmonary disease bacterial endotoxins: chemical structure, biological activity and role in septicaemia lps/tlr signal transduction pathway dose-response relationship to inhaled endotoxin in normal subjects inhaled endotoxin in healthy human subjects: a dose-related study on systemic effects and peripheral cd + and cd + t cells local inflammatory responses following bronchial endotoxin instillation in humans evaluation of oral corticosteroids and phosphodiesterase- inhibitor on the acute inflammation induced by inhaled lipopolysaccharide in human available at: http:// clinicaltrials.gov/ct /show/nct ?term=nct &ran k= roflumilast attenuates pulmonary inflammation upon segmental endotoxin challenge in healthy subjects: a randomized placebo-controlled trial blunting airway eosinophilic inflammation results in a decreased airway neutrophil response to inhaled lps in patients with atopic asthma: a role for cd health effects of air pollution air pollution in asthma: effect of pollutants on airway inflammation committee of the environmental and occupational health assembly of the ozone-induced airway inflammation in human subjects as determined by airway lavage and biopsy exposure of humans to ambient levels of ozone for . hours causes cellular and biochemical changes in the lung low-level ozone exposure induces airways inflammation and modifies cell surface phenotypes in healthy humans responses of subjects with chronic obstructive pulmonary disease after exposures to . ppm ozone validation of the human ozone challenge model as a tool for assessing anti-inflammatory drugs in early development fluticasone propionate protects against ozone-induced airway inflammation and modified immune cell activation markers in healthy volunteers indomethacin pretreatment reduces ozone-induced pulmonary function decrements in human subjects sch , a novel cxcr antagonist, inhibits ozone-induced neutrophilia in healthy subjects no effect of inhaled budesonide on the response to inhaled ozone in normal subjects effects of cyclo-oxygenase inhibition on ozone-induced respiratory inflammation and lung function changes sch , a novel treatment option for severe neutrophilic asthma sb- , a novel cxcr selective antagonist, inhibits ex-vivo neutrophil activation and ozone-induced airway inflammation in humans short-term respiratory effects of . ppm ozone exposure in volunteers with chronic obstructive pulmonary disease the acute effects of . ppm ozone in patients with chronic obstructive pulmonary disease response to ozone in volunteers with chronic obstructive pulmonary disease responses of older men with and without chronic obstructive pulmonary disease to prolonged ozone exposure detection of rhinovirus rna in lower airway cells during experimentally induced infection the major human rhinovirus receptor is icam- members of the low density lipoprotein receptor family mediate cell entry of a minor-group common cold virus role of p mitogen-activated protein kinase in rhinovirus-induced cytokine production by bronchial epithelial cells rhinovirus induction of the cxc chemokine epithelial-neutrophil activating peptide- in bronchial epithelium low grade rhinovirus infection induces a prolonged release of il- in pulmonary epithelium quantitative and qualitative analysis of rhinovirus infection in bronchial tissues rhinoviruses infect the lower airways lower airways inflammation during rhinovirus colds in normal and in asthmatic subjects respiratory viruses in exacerbations of chronic obstructive pulmonary disease requiring hospitalisation: a case-control study infections and airway inflammation in chronic obstructive pulmonary disease severe exacerbations a -year prospective study of the infectious etiology in patients hospitalized with acute exacerbations of copd effect of interactions between lower airway bacterial and rhinoviral infection in exacerbations of copd viral pathogens in acute exacerbations of chronic obstructive pulmonary disease a one-year prospective study of infectious etiology in patients hospitalized with acute exacerbations of copd and concomitant pneumonia rhinovirus infections in chronic bronchitis: isolation of eight possibly new rhinovirus serotypes role of infection in chronic bronchitis infectious agents associated with exacerbations of chronic obstructive bronchopneumopathies and asthma attacks respiratory viruses, symptoms, and inflammatory markers in acute exacerbations and stable chronic obstructive pulmonary disease respiratory viral infections in adults with and without chronic obstructive pulmonary disease an experimental model of rhinovirus induced chronic obstructive pulmonary disease exacerbations: a pilot study an experimental model of virus induced chronic obstructive pulmonary disease exacerbation experimental rhinovirus infection as a human model of chronic obstructive pulmonary disease exacerbation comparative antirhinoviral activities of soluble intercellular adhesion molecule- (sicam- ) and chimeric icam- /immunoglobulin a molecule efficacy of tremacamra, a soluble intercellular adhesion molecule , for experimental rhinovirus infection: a randomized clinical trial genetic clustering of all human rhinovirus prototype strains: serotype is close to human enterovirus regulation of innate antiviral defenses through a shared repressor domain in rig-i and lgp for pleconaril respiratory infection study group. efficacy and safety of oral pleconaril for treatment of colds due to picornaviruses in adults: results of double-blind, randomized, placebo-controlled trials implications of the picornavirus capsid structure for polyprotein processing purification and partial characterization of poliovirus protease a by means of a functional assay acute lps inhalation in healthy volunteers induces dendritic cell maturation in vivo this review is part of a dissertation for a master's degree (rvdm) at the university of surrey, uk. editorial assistance was provided by lourdes briz and carrie lancos, medimmune, llc. sponsorship: this manuscript was sponsored by medimmune. conflict of interest: rvdm is an employee of medimmune; nam is a former employee of medimmune. submit your manuscript here: http://www.dovepress.com/international-journal-of-copd-journalthe international journal of copd is an international, peer-reviewed journal of therapeutics and pharmacology focusing on concise rapid reporting of clinical studies and reviews in copd. special focus is given to the pathophysiological processes underlying the disease, intervention programs, patient focused education, and self management protocols.this journal is indexed on pubmed central, medline and cas. the manuscript management system is completely online and includes a very quick and fair peer-review system, which is all easy to use. visit http://www.dovepress.com/testimonials.php to read real quotes from published authors.international journal of copd : key: cord- - pc p az authors: hwang, eun-ha; kim, tae-hyoun; oh, sang-muk; lee, kyung-bok; yang, soo-jin; park, jong-hwan title: toll/il- domain-containing adaptor inducing ifn-β (trif) mediates innate immune responses in murine peritoneal mesothelial cells through tlr and tlr stimulation date: - - journal: cytokine doi: . /j.cyto. . . sha: doc_id: cord_uid: pc p az abstract mesothelial cells are composed of monolayer of the entire surface of serosal cavities including pleural, pericardial, and peritoneal cavity. although mesothelial cells are known to express multiple toll-like receptors (tlrs) which contribute to trigger innate immune responses against infections, the precise molecular mechanism remains still unclear. in the present study, we investigated the role of toll/il- domain-containing adaptor inducing ifn-β (trif), one of the two major tlrs–adaptor molecules, on innate immune response induced by tlr and tlr stimulation in murine peritoneal mesothelial cells (pmcs). trif was strongly expressed in pmcs and its deficiency led to impaired production of cytokines and chemokines by poly i:c and lps in the cells. activation of nf-κb or mapks through poly i:c and lps stimulation was reduced in trif-deficient pmcs as compared to the wt cells. trif was also necessary for optimal nitric oxide synthesis and gene expression of inducible nitric oxide synthase (inos) and ifn-β in pmcs in response to poly i:c and lps. furthermore, both escherichia coli and pseudomonas aeruginosa induced high level of il- , cxcl , and ccl production in pmcs, which was significantly impaired by trif deficiency. these results demonstrated that trif is required for optimal activation of innate immune responses in mesothelial cells against microbial infections. peritonitis is an inflammation of the peritoneum which lines the inner wall of the abdomen and covers and supports most of abdominal organs. the two major types of life-threatening peritonitis are: (i) primary spontaneous peritonitis, an infection that develops in the peritoneum [ ] ; and (ii) secondary peritonitis, which usually develops when an injury or infection in the abdominal cavity allows infectious organisms into the peritoneum [ , ] . mesothelial cells are monolayer of specialized cells which extends over the entire surface of the three serosal cavity (pleural, pericardial, and peritoneal) and the organs contained within these cavities [ ] . the predominant role of the mesothelium is to act as a protective barrier against physical damage and invading pathogens. recent studies have shown many different roles of mesothelial cells, which include antigen presentation, tumor cell adhesion and growth, initiation and resolution of inflammation, and tissue repair [ ] . under physiologic conditions, the mesothelial cells secrete numerous glycosaminoglycans, proteoglycans, and phospholipids that constitute a glycocalyx surrounding the cells and provide a protective barrier against abrasion and a slippery nonadhesive surface for intracoelomic movement [ ] . when faced with an infection, mesothelial cells express specific surface markers that enable them to promote the migration of neutrophils, to interact with extracellular matrix proteins, to present antigens to immune cells, and to produce biologically important molecules such as proinflammatory cytokines, chemokines, and nitric oxide (no) [ , ] toll-like receptors (tlrs) are type i transmembrane proteins and comprise an ectodomain, which contains leucine-rich repeats that mediate the recognition of pathogen-associated molecular patterns (pamps); a transmembrane region; and a cytosolic toll/ il- receptor (tir) domain that activates downstream signaling pathways [ ] . recognition of microbial components (e.g. lps, lipoprotein, flagellin, and nucleic acids) by tlrs initiates inflammatory signal cascades via two distinct pathways: (i) myeloid differentiation primary response (myd )-dependent; and (ii) toll/ il- receptor domain-containing adaptor inducing ifn-beta (trif)-dependent pathways. myd is an adapter molecule that triggers inflammatory signals commonly utilized by various tlrs with the exception of tlr . recruitment of myd leads to the activation of nuclear factor-kappa b (nf-jb) and mitogen activated protein kinases (mapks) to regulate the pro-inflammatory cytokines genes. on the other hand, trif is recruited to tlr and tlr and activates an alternative pathway that triggers the activation of nf-jb, mapks, and irf . these signaling cascades lead to the production of proinflammatory cytokines, type i interferons (ifns), chemokines, and antimicrobial peptides to remove the invading pathogens [ , ] . mesothelial cells have been known to express multiple tlrs [ , ] . tlr - are expressed in murine peritoneal mesothelial cells (pmcs) [ ] and each ligand induce the production of chemokines such as cxcl and ccl [ ] . in human pmcs, tlr is functionally expressed and its agonist poly i:c leads to the production of il- , ccl , and ccl [ , ] . the role of tlr is somewhat controversial. colmont et al. demonstrated that tlr was not expressed in primary cultured human pmcs and lps also did not induce any inflammatory molecules tested [ ] , whereas it was strongly expressed in a cell line (hmrsv cells) and lpsinduced autophagy was dependent on tlr in the cells [ , ] . bacterial and viral peritonitis can occur in patients with intestinal perforation or undergoing continuous ambulatory peritoneal dialysis (capd) [ , ] . corona virus and herpes simplex virus can cause peritonitis in ifn-c-deficient mice and diabetics patient [ , ] . in addition to its role in response to viral infection, tlr is involved in immune response to bacterial infection-induced tissue damage which is one of the major cause of peritoneal inflammation [ ] [ ] [ ] . therefore, tlr and tlr -mediated signal pathways are considered to play central roles in activation of host immune system in response to various pathogenic microbes in pmc. although trif is the only adaptor molecule mediating both tlr and tlr signaling pathways, investigations on the detailed function of trif in mesothelial cells have not been available. to address this limitation, in the current study, we characterized molecular mechanisms of trif-mediated innate immune response in murine pmcs. polyinosinic-polycytidylic acid (poly(i:c)) and ultrapure lps were purchased from invivogen (san diego, ca). escherichia coli o :b and pseudomonas aeruginosa were grown overnight in luria bertoni (lb) broth grown anaerobic conditions at °c. the culture was centrifuged at rpm for min, and the cell pellet was washed twice with cold pbs. the pellet was suspended in onetenth the original volume in pbs and the od nm was adjusted to give the approximate desired inocula. the inoculum concentrations were verified by serial -fold dilutions of the bacterial suspensions. the bacterial concentrations of the suspensions were adjusted to  cfu/ml. the bacteria were diluted to the desired concentration and used in subsequent experiments. trif-deficient mice on a c bl/ background were kindly provided by dr. shizuo akira (osaka university, japan). wild-type (wt) c bl/ mice were purchased from koatech (pyeongtaek, korea). bone marrow-derived macrophages (bmdms) were prepared as previously described [ ] . mesothelial cells were prepared from the peritoneum and external surfaces of the liver, spleen, and kidney of adult mice as previously described [ ] . briefly, samples of peritoneum and intact organs were obtained from sacrificed mice and digested with . %-trypsin-edta (invitrogen, grand island, ny, usa) solution for min at °c. intact tissues and tissue debris were discarded and the cell suspension was centrifuged at g for min. the pellet was resuspended in dulbecco's modified eagle's medium supplemented with % heat-inactivated fbs and % penicillin/streptomycin and cultured overnight. the next day, floating cells were removed by washing twice with pbs and adherent cells were cultured for five additional days. mesothelial cells were used between passages and . total rna was extracted from the cells using easyblue (intron biotechnology, daejeon, korea) according to the manufacturer's instruction. one microgram of total rna was reverse transcribed into cdna and pcr was performed using the power cdna synthesis kit (intron biotechnology) and one-step rt-pcr with accupower Ò hotstart pcr premix (bioneer, daejeon, korea). the following primer sets were used. mouse tlr forward, -gtggta cctgagaatgatgtggg- ; mouse tlr reverse, -gttaaggaagt caggaactgggtg- ; mouse tlr forward, -aggtacctgagttt gaagcgagc- ; mouse tlr reverse, -gagcatcagtcttt gaaggctgg- ; mouse tlr forward, -ctgggtgagaaat gagctgg- ; mouse tlr reverse, -gatacaattc cacctgctgcc- ; mouse trif forward, -atggataacc cagggcctt- ; mouse trif reverse, -ttctggtcactgcaggg gat- ; mouse inos forward, -cagcccaacaatacaagatgaccc- ; mouse inos reverse, -cagttccgagcgtcaaagacctgc- ; mouse ifn-b forward, -atgaactccagcagacag- ; mouse ifn-b reverse, -accaccatccaggcgtagc- ; mouse gapdh forward, -gtcggagrcaacggatt- ; mouse gapdh reverse, -aagcttcc cgttctcag- . the pcr reaction condition included predenaturing at °c for s, then - cycle of °c for s, °c for min. pcr products were then electrophoresed on a . % agarose gel and visualized using a gel documentation system. the concentration of il- and cxcl , ccl in the culture supernatants was determined using a commercial elisa kit (r&d systems, minneapolis, mn, usa). no synthase activity in the supernatant of cultured cells was assayed for nitrite accumulation by the griess reaction [ ] . pmcs (  /well) and bmdms (  /well) were plated in mm culture dishes. the cells were treated with polyi:c ( lg/ml) or lps ( ng/ml) were lysed in buffer containing % nonidet-p supplemented with a complete protease inhibitor cocktail (roche diagnostics ltd, mannheim, germany), and mm dithiothreitol. lysates were resolved by % sds-page, transferred onto a polyvinylidene fluoride (pvdf) membrane, and immunoblotted with primary antibodies against regular-and phospho-ijb-a, jnk (cell signaling technology inc., beverly, ma, usa), p , erk, b-actin, calretinin (santa cruz biotechnology, santa cruz, ca). after immunoblotting with secondary antibodies, proteins were detected with an enhanced chemiluminescence (ecl) reagent (intron biotechnology). pmcs's phenotypes were characterized by their cell surface markers using fluorescently labeled monoclonal antibodies (mabs) and analyzed by flow cytometry. the cells were stained with the following mabs (ebioscience, san diego, ca, usa): pe-cy congugated mab. isotype-matched controls were run in parallel. cell debris was eliminated by forward and side scatter gating. the samples were acquired on a facs calibur cell sorter (becton dickinson, mountain view, ca, usa) and the data was analyzed using flowjo software (tree star, inc., ashland, or, usa). the differences among the mean values of the different groups were tested and the values were expressed as the mean ± sd. all of the statistical calculations were performed by one-way anova followed by the bonferroni post hoc test for multigroup comparisons using graph pad prism version . (graph pad software, san diego, ca). values of p < . were considered significant. to determine phenotypic characteristics of pmcs, the expression of calretinin that is known as a selective marker for mesothelial cells [ , ] was evaluated by western blot. calretinin was strongly expressed in pmcs, but there was no expression in bmdms (fig. a) . flow cytometry analysis also showed that . % cells in bmdms preparation was positive for f / , which is a well-known marker of murine macrophages population [ ] , whereas only . % was positive in the pmcs preparation (fig. b) . the gene of tlr , tlr , and tlr was strongly expressed in pmcs as well as bmdms (fig. c) , which was consistent with a study kato et al. showing that tlr - were strongly expressed in pmcs derived from c h/hen mice [ ] . moreover, as shown in fig. d , the trif gene was highly expressed in the wt pmcs at levels comparable to the wt bmdms. as expected, the trif gene expression was not detected in both trif-deficient bmdms and pmcs (fig. d ). to assess levels of il- , cxcl , and ccl secretion in response to poly i:c or lps, elisa assays were performed on supernatants derived from the wt and trif-deficient mice. both poly i:c and lps increased the il- , cxcl , and ccl secretion in the wt pmcs in a dose-dependent manner (fig. a-f ). in contrast to the wt pmcs, poly i:c-induced production of il- and cxcl was completely abolished in trif-deficient pmcs ( fig. a and b) . furthermore, ccl production by poly i:c was also partially impaired in trif-deficient pmcs (fig. c) . although trif deficiency did not affect lps-induced production of cxcl and ccl in pmcs ( fig. e and f) , level of il- was decreased significantly with lps stimulation as compared to the wt pmcs. these findings suggest that trif may distinctly regulate cytokines and chemokines production in pmcs in response to tlr and tlr stimulators. nf-jb and mapks are essential for chemokines production in pmcs in response to bacterial infection [ ] . accordingly, we investigated whether trif is involved in the activation of nf-jb and mapks in pmcs in response to poly i:c or lps. poly i:c stimulation led to degradation of ijb-a and its phosphorylation in wt pmcs, but not in trif-deficient cells (fig. a) . phosphorylation of p , jnk, and erk mapks was also detected in both wt and trifdeficient pmcs in response to poly i:c at min after treatment (fig. a) . however, at min after treatment, such phosphorylation was much strongly detected in wt pmcs, whereas it was gradually diminished in trif-deficient cells (fig. a) . lps-induced phosphorylation of ijb-a was found in wt pmcs since min after treatment, whereas it was detected only at min after treatment in trif-deficient cells (fig. b ). ijb-a degradation was also found only in wt pmcs at min after treatment (fig. b) . in contrast, kinetics of phosphorylation of p , jnk, and erk mapks were similar in both wt and trif-deficient pmcs in response to lps, although densities of phosphorylated jnk and erk were slightly stronger in trif-deficient cells at min after stimulation (fig. b) . these findings suggest that trif may differently mediate the activation of nf-jb and mapks in pmcs depending on upstream tlrs. the inducible form of nitric oxide synthase (inos) is generally associated with no production related to immune responses [ ] . no synthesis is enhanced by certain types of cytokines and bacterial product in mesothelial cells [ , ] . we thus assessed whether stimulation of tlr and tlr promotes inos expression and no synthesis in a trif dependent manner in pmcs. as shown in fig. a , poly i:c-induced expression of inos was detected at h after stimulation and continuously increased by h in the wt pmcs. in contrast, the inos gene expression was not detected until h post-stimulation in trif-deficient cells (fig. a) . lps could induce strong inos expression in both wt and trif-deficient pmcs at h after stimulation (fig. b) . however, the high level of gene expression persisted only in the wt pmcs through h poststimulation, whereas it was gradually diminished in trifdeficient cells after h time point (fig. b) . we next examined no synthesis by measuring nitrite concentration. although poly i:c and lps induced inos gene expression ( fig. a and b) , either poly i:c or lps alone failed to induce no synthesis (data not shown). previously, it has been shown that ifn-c modulates no synthesis by mesothelial cells in response to bacterial products such as lps and kf b (a nod ligand) [ , ] . we then measured nitrite concentration in culture supernatant of pmcs stimulated by poly i:c or lps in the presence of ifn-c ( ng/ml). in our pilot experiments, nitrite was not detectable in pmcs in response to any dose of poly i:c ( - lg/ml) by h post-stimulation (data not shown). thus, culture supernatant was collected at h post-stimulation and nitrite was measured. as shown in fig. c -e, ifn-c alone could not induce no synthesis in pmcs at any time points, which is consistent with a study by park et al. [ ] . poly i:c induced no synthesis in the wt pmcs in a dose-dependent manner at h time point, while the trifdeficient cells exhibited no detectable level of no (fig. c) . similarly, lps also induced no synthesis in pmcs in a dose dependent manner. interestingly, no synthesis was significantly reduced in the trif-deficient pmcs in response to ng/ml of lps as compared to the wt cells (fig. d) . however, the impaired production of no at ng/ml of lps was restored at high concentration of lps ( ng/ml) in the trif-deficient pmcs (fig. d) . since inos expression was impaired in trif-deficient pmcs when treated with same concentration of lps ( ng/ml), we measured nitrite at earlier time points ( - h) . similarly, there was no significant difference in nitrite production between wt and trif-deficient pmcs in response to ng/ml lps (fig. e) . these finding suggest that trif may be critical for tlr -mediated no production in pmcs and its influence on no production induced by tlr activation may depend on concentration of stimuli. trif is a well-known myd -independent pathway responsible for expression of ifn-b in immune cells stimulated by poly i:c or lps [ ] . in addition, poly i:c can induce gene expression of type i ifns in human mesothelial cells [ ] . therefore, we investigated whether trif is required for ifn-b gene expression in pmcs in response to poly i:c and lps. rt-pcr analyses revealed that poly i:c strongly induced mrna expression of ifn-b in wt pmcs from h post-stimulation, which lasted until h time point (fig. a) . in contrast, the gene expression by poly i:c was significantly reduced in trif-deficient cells (fig. a) . lps-induced expression of ifn-b peaked at h post-stimulation in wt pmcs, but was not induced in trif-deficient cells (fig. b) . . . trif is involved in optimal production of il- , cxcl , and ccl by pmcs in response to live p. aeruginosa and e. coli live bacteria possess multiple pamps including lipoprotein, dsrna, and lps. we wondered the role of trif on innate immune response against live bacteria. p. aeruginosa and e. coli are the most frequent bacteria causing gram-negative peritonitis [ ] . accordingly, we finally sought to determine the effect of trif on production of il- , cxcl , and ccl by live p. aeruginosa and e. coli in pmcs. as shown in fig. , unlike the wt pmcs, il- production was completely impaired in trif-deficient pmcs infected with low number of p. aeruginosa (moi . ) or e. coli (moi ). although trif-deficient cells could produce il- under higher mois of the bacterial pathogens, the level was significantly lower than that of the wt cells ( fig. a and b) . these data were well correlated with the result of lps response shown in fig. d . interestingly, the production of cxcl and ccl by p. aeruginosa and e. coli was also impaired in trif-deficient pmcs, as compared to the wt pmc level (fig. c-f ). these findings indicate the existence of more complex mechanisms in trif-mediated signaling pathways in response to bacterial infections. peritonitis is a common cause of pathology and mortality in humans. mesothelial cells are a sentinel for inflammatory response of the mesothelium caused by bacterial products or infection, however, the precise mechanism by which mesothelial cells trigger innate immune responses against microbial infection still remains to be elucidated. in the present study, we provided evidences that trif is essential for optimal induction of innate immune responses in response to tlr and tlr stimulation in pmcs. production of il- and cxcl by pmcs in response to poly i:c was entirely trif-dependent. however, poly i:c could induce ccl in trifdeficient pmcs, although the amount was still lower than that of wt cells, indicating the existence of a trif-independent pathway responsible for poly i:c-induced ccl production in mesothelial cells. a previous study revealed expression of cytosolic receptors such as rig-i and mda in human mesothelial cells [ ] , which are responsible for cytosolic recognition of nucleic acids. inhibition of rig-i using sirna reduced gene expression and protein production of ccl induced by poly i:c in mesothelial cells [ ] . in addition, although its expression and functionality have not been studied in mesothelial cells, cd b/cd (mac- ) should be considered as a receptor responsible for poly i:c-induced immune responses in pmcs, because extracellular dsrna activates mac- to enhance tlr -dependent signaling and to trigger inflammatory oxidative signaling in macrophages via mac- -dependent pathway [ ] . bacterial lps can also lead to the production of various chemokines such as cxcl , cxcl , and ccl in mesothelial cells [ , ] . in the current study, we demonstrated that trif is not required for lps-induced production of cxcl and ccl in murine pmcs, whereas il- production by lps was partially impaired in fig. . trif is required for inos expression and no production by poly i:c or lps stimulation in pmcs. pmcs isolated from wt and trif-deficient mice were incubated with poly i:c and lps. mrna was extracted from the cells at indicated time points and inos expression was examined by rt-pcr (a, b). indicated levels were obtained by dividing densities of ifn-b by those of gapdh (a, b) . at the present of ifn-c ( ng/ml), the cells were incubated with various doses of poly i:c (c) and lps (d) for h and nitrite concentration in culture supernatants was measured. an additional experiment with stimulation of lps ( ng/ml) and ifn-c ( ng/ml) was performed at earlier time points (e). the data (c-e) were from one representative result of three independent experiments and are presented as the means ± sd. ⁄ p < . and ⁄⁄⁄ p < . . trif-deficient pmcs. pro-inflammatory genes expression induced by lps seems to be differently regulated via myd -or trifdependent signaling. in macrophages, expression of ccl and cxcl by lps stimulation was entirely trif-dependent [ ] . in contrast, genes regulated by myd alone included tnf, il b, cxcl , cxcl , and ccl [ ] . lps-induced il expression required both myd and trif [ ] . ccl mrna expression was also enhanced by lps in wt macrophages, which was absolutely abolished in myd deficient cells [ ] . these published data are in line with our results in pmcs. taken together, our poly i:c induction data suggest that the production of specific cytokines and chemokines seem to be regulated in differentially based on types of stimulators or associated receptor signaling pathways. trif is essential for poly-induced nf-jb activation in lung fibroblasts [ ] , which is consistent with our results showing that the activation of nf-jb and mapks induced by poly i:c was impaired in trif-deficient pmcs. lps-induced activation of macrophages was temporally regulated by myd and trif [ ] . myd was involved in early activation of nf-jb and mapks, whereas late phase activation of such molecules was controlled by trifdependent pathway [ ] [ ] [ ] . lps stimulation also resulted in almost normal activation of nf-jb and jnk in trif-deficient lung fibroblasts [ ] . moreover, nf-jb and mapks activation in response to lps was delayed in myd -deficient cells [ , ] and diminished in trif-deficient macrophages at late time of . trif is essential for optimal production of il- , cxcl , and ccl induced by live p. aeruginosa and e. coli in pmcs. wt and trif-deficient pmcs were stimulated with different mois of p. aeruginosa (a-c) and e. coli (d-f) for h. the level of il- , cxcl , and ccl in culture supernatants was measured by elisa. the data were from one representative result of three independent experiments and are presented as the means ± sd. ⁄⁄ p < . and ⁄⁄⁄ p < . . stimulation [ ] . accordingly, we predicted that lps-induced activation of nf-jb and mapks may not be different between the wt and trif-deficient pmcs at early time. however, in this study, nf-jb activation by lps was delayed in trif-deficient pmcs, although impact of trif on lps-induced mapks activation was very limited, suggesting that trif may be involved in rapid activation of nf-jb in pmcs. future studies will need to determine whether this inconsistency is due to difference in cell types. no production in macrophages is important for the control of bacterial clearance [ ] . it also has antiviral property by inhibiting a viral protease [ ] . moreover, no participates in intraperitoneal inflammatory reaction and is involved in the modulation of peritoneal permeability during peritonitis [ ] . endothelial cells of peritoneal vasculature or peritoneal macrophages are considered as major source of local no production during peritonitis [ , ] . however, there are evidences showing that mesothelial cells can produce no after stimulation by combination of cytokines and bacterial products [ , , ] . the current study for the first time demonstrates that dsrna can induce inos gene expression and no production in pmcs via a trif-dependent manner, which is consistent with a previous study showing that poly i:c induces intracellular reactive oxygen species (ros) in macrophages via tlr -and trif-dependent manner [ ] . in addition, contribution of trif to lps-induced no synthesis in pmcs was specific dosedependent response. no synthesis was impaired in trif-deficient pmcs in response to ng/ml lps, whereas high dose lps ( ng/ml) led to synthesis of comparable level of nitrite between the wt and trif-deficient pmcs. unreasonably, at this concentration, lps-induced expression of inos was diminished in trifdeficient pmcs at late time points ( and h), as compared to the wt cells. it is likely that early induction of inos is sufficient for optimal no synthesis by lps in pmcs. moreover, although myd is dispensable for endotoxin-induced nitrite synthesis in a human macrophage cell line [ ] , its role on pmcs should not be disregarded. the precise mechanism of lps-induced no synthesis in pmcs remains to be elucidated. future studies will be designed to adjudicate these limitations. type i ifns (ifn-a/b) play important roles in both innate and adaptive antiviral immune responses [ ] . gene expression of type i ifns is enhanced by poly i:c in mesothelial cells [ ] . in current study, we provided evidence that trif is critical for poly i:c-induced gene expression of ifn-b, which is in agreement with previous studies in other cell types such as epithelial cells and dendritic cells [ , ] . remarkably, ifn-b expression by poly i:c was detected in trif-deficient pmcs at h time point after infection, indicating that trif is required for earlier expression of type i ifns by poly i:c and trif-independent pathway may contribute to their expression in late response in pmcs. involvement of rig-i and mda in such a late response should be clarified, as both cytosolic receptors are functionally expressed in mesothelial cells [ , , ] . in addition, as expected, lps induced ifn-b expression in wt pmcs in a trif-dependent manner. in macrophages, trif is required for cytokines and chemokines production in response to p. aeruginosa and e. coli infections [ , ] . in the present study, il- , cxcl , and ccl production was impaired in trif-deficient pmcs, which was inconsistent with the lps-induced responses, as trif deficiency did not affect cxcl and ccl production in pmcs under lps stimulation. one possibility is that live bacteria may possess other molecules to stimulate tlr -independent and trif-mediated signaling. more recent studies also revealed that trif is involved in tlr -mediated immune responses [ , ] . in addition, trif mediates tlr -induced signalings such as nf-jb and mapks activation in intestinal epithelial cells [ ] . as tlr and tlr ligands are known to induce chemokines production in mesothelial cells [ , ] , it is likely that trif mediates multiple tlrs-mediated immune responses in mesothe-lial cells. extracellular rna released from necrotic cells can also induce various cellular signalings in host cells via tlr dependent manner. necrotic macrophages and cardiomyocytes induced robust production of cytokines and chemokines in rat cardiomyocytes, which was impaired trif-deficient cells [ ] . however, eigenbrod et al. demonstrated that trif is not required for cxcl production in mesothelial cells in response to necrotic cell extracts [ ] . in conclusion, our results showed that trif is essential for tlr and tlr -mediated innate immune responses in pmcs. in addition to the roles in inflammatory response, mesothelial cells are involved in the process of tissue repair, antigen presentation, and tumor growth and dissemination [ , ] . at present, we are attempting to define the role of pattern recognition receptors in the context of diverse physiological role of mesothelial cells. the authors declare no financial or commercial conflict of interest. diagnosis, treatment and prophylaxis of spontaneous bacterial peritonitis: a consensus document viridans group streptococci: an underestimated cause of spontaneous bacterial peritonitis in cirrhotic patients with ascites the role of virulence factors in the outcome of staphylococcal peritonitis in capd patients the mesothelial cell mesothelial cells structure and function of mesothelial cells mesothelial cells: their structure, function and role in serosal repair toll-like receptors and their crosstalk with other innate receptors in infection and immunity pathogen recognition in the innate immune response human peritoneal mesothelial cells respond to bacterial ligands through a specific subset of toll-like receptors endotoxin-induced chemokine expression in murine peritoneal mesothelial cells: the role of toll-like receptor nod /rick and tlr signaling regulate chemokine and antimicrobial innate immune responses in mesothelial cells novel role of toll-like receptor , rig-i and mda in poly (i:c) rna-induced mesothelial inflammation synthetic double-stranded rna stimulates the expression of interferoninducible protein in human mesothelial cells glucose-based peritoneal dialysis fluids downregulate toll-like receptors and trigger hyporesponsiveness to pathogen-associated molecular patterns in human peritoneal mesothelial cells lipopolysaccharide (lps)-induced autophagy is involved in the restriction of escherichia coli in peritoneal mesothelial cells complications of the peritoneal access and their management non-infectious complications of continuous ambulatory peritoneal dialysis and their impact on technique survival murine coronavirus-induced subacute fatal peritonitis in c bl/ mice deficient in gamma interferon herpes simplex virus type ii infection of ileum mesothelium: a case report and review of the literature tlr is an endogenous sensor of tissue necrosis during acute inflammatory events commensal bacteria regulate toll-like receptor -dependent inflammation after skin injury identification of a key pathway required for the sterile inflammatory response triggered by dying cells evidence for a gammainterferon receptor that regulates macrophage 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oxide production in peritoneal macrophages from peritoneal dialysis patients with bacterial peritonitis zymosan induces nitric oxide production by peritoneal mesothelial cells tlr -triggered reactive oxygen species contribute to inflammatory responses by activating signal transducer and activator of transcription- differential induction of the toll-like receptor -myd -dependent and -independent signaling pathways by endotoxins type i interferons in host defense interferon-beta induction through toll-like receptor depends on doublestranded rna structure toll-like receptor agonist poly(i:c)-induced antiviral response in human corneal epithelial cells interferon-gamma upregulates retinoic acid-inducible gene-i in human pericardial mesothelial cells role of viral receptors tlr , rig-i and mda in mesothelial tissue-type plasminogen activator and plasminogen activator inhibitor- synthesis a role of toll-il- receptor domain-containing adaptor-inducing ifn-beta in the host response to pseudomonas aeruginosa lung infection in mice a role for the adaptor proteins tram and trif in toll-like receptor signaling trif mediates toll-like receptor -dependent inflammatory responses to borrelia burgdorferi trif mediates toll-like receptor -induced signaling in intestinal epithelial cells role of extracellular rna and tlr -trif signaling in myocardial ischemia-reperfusion injury cutting edge: critical role for mesothelial cells in necrosis-induced inflammation through the recognition of il- alpha released from dying cells this study was financially supported by chonnam national university, south korea (grant no. - ). key: cord- -nblmshni authors: savva, athina; roger, thierry title: targeting toll-like receptors: promising therapeutic strategies for the management of sepsis-associated pathology and infectious diseases date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: nblmshni toll-like receptors (tlrs) are pattern recognition receptors playing a fundamental role in sensing microbial invasion and initiating innate and adaptive immune responses. tlrs are also triggered by danger signals released by injured or stressed cells during sepsis. here we focus on studies developing tlr agonists and antagonists for the treatment of infectious diseases and sepsis. positioned at the cell surface, tlr is essential for sensing lipopolysaccharide of gram-negative bacteria, tlr is involved in the recognition of a large panel of microbial ligands, while tlr recognizes flagellin. endosomal tlr , tlr , tlr , tlr are specialized in the sensing of nucleic acids produced notably during viral infections. tlr and tlr are favorite targets for developing anti-sepsis drugs, and antagonistic compounds have shown efficient protection from septic shock in pre-clinical models. results from clinical trials evaluating anti-tlr and anti-tlr approaches are presented, discussing the challenges of study design in sepsis and future exploitation of these agents in infectious diseases. we also report results from studies suggesting that the tlr agonist flagellin may protect from infections of the gastrointestinal tract and that agonists of endosomal tlrs are very promising for treating chronic viral infections. altogether, tlr-targeted therapies have a strong potential for prevention and intervention in infectious diseases, notably sepsis. sepsis is one of the leading causes of death worldwide. incidence of severe sepsis is increasing and mortality rates remain significantly high despite early care management ( ) . moreover, more than % of survivors develop long-term functional disabilities and cognitive impairments ( ) . the surviving sepsis campaign is a global initiative incepted in early s with the aim to improve sepsis diagnosis and treatment in order to enhance the awareness of sequelae and to decrease high mortality rates associated with sepsis . in collaboration with many countries in europe and the united states, the surviving sepsis campaign suggests evidencebased guidelines and bundles. the most recent guidelines recommend acute resuscitation of septic patients, administration of antibiotics and support of organ failure. yet, no treatment targeting the underlying mechanism of sepsis is actually available ( ) . recombinant human activated protein c (rhapc, xigris®, eli lilly), the only drug specifically registered for sepsis, has recently been withdrawn from the market following the negative results from the prowess-shock study that did not show reduction in mortality at or days in patients with septic shock ( ) . it is generally admitted that sepsis results from a dysregulated host response to an initial insult, characterized by inflammation mediating tissue damage and organ failure and an immune suppression state responsible for the development of secondary infections ( ) ( ) ( ) . the immune response to an infection is initiated by the sensing of microbial structures through families of receptors collectively called pattern recognition receptors (prrs). the most well-described families comprise toll-like receptors (tlrs), nucleotide binding oligomerization domains (nods)-like receptors (nlrs), c-type lectin receptors (clrs, such as dectin- , dectin- , dc-sign), rig-i-like receptors (rlrs, rig-i, and mda ), and intra-cytosolic dna sensors. prrs are expressed by innate immune cells like dendritic cells and macrophages. the binding of microbial ligands to prrs promotes the release of mediators, among which cytokines, that initiate and regulate the inflammatory response necessary to eliminate invasive pathogens and coordinate the development of the adaptive immune response ( , ) . innate immune cells are also triggered by damage (or danger)associated molecular patterns (damps), known as alarmins. damps are endogenous components commonly released by injured or stressed cells, such as nucleic acids, histones, uric acid crystals, atp, cytochrome c, s molecules, and hmgb . damps are primarily sensed through the nlrp inflammasome, which controls the secretion of il- β and il- ( ) . the concept of prrs sensing microbial-associated molecular patterns (mamps) and discriminating self from non-self molecular structures was proposed by janeway ( ) . two major cornerstone discoveries largely confirmed janeway's concept. the first one was the demonstration of the essential antifungal role of www.frontiersin.org the toll protein in drosophila ( ) . the second one arose from the positional cloning linking lps (commonly called endotoxin) unresponsive phenotype of c h/hej and c bl/ sccr strains of mice to missense and null mutations of the toll-like receptor (tlr ) gene ( ) . the importance of these discoveries and of the role of dendritic cells as central regulators of innate and adaptive immunity has been acknowledged by the nobel prize in physiology or medicine attributed to bruce a. beutler and jules a. hoffmann "for their discoveries concerning the activation of innate immunity" and to ralph m. steinman "for his discovery of the dendritic cell and its role in adaptive immunity" ( ) . toll-like receptors belong to the most studied family of prrs, due to their central role in host defenses and involvement in a number of pathological processes that include sepsis. tlrs are type i trans-membrane proteins composed of an extracellular leucine-rich repeat (lrr) domain involved in ligand recognition, a trans-membrane domain, and a toll-interleukin receptor (tir) domain involved in signaling ( , ) . about functional human tlrs (tlr - ) and functional mouse tlrs (tlr - , tlr - ) have been described, each one being involved in the sensing of distinct microbial products ( table ) . expressed at the cell surface, tlr detects lps from gram-negative bacteria. tlr shuttles to late endosome to induce alternative signaling following lps sensing. tlr as heterodimers in association with either tlr or tlr (and possibly tlr ) senses a variety of microbial products, such as lipopeptides, lipoproteins, peptidoglycan, porins, β-glucan, glycosylphosphatidylinositol (gpi) anchors, and glycoproteins from gram-positive bacteria, gram-negative bacteria, mycoplasma, mycobacteria, fungi, parasites, and viruses. tlr senses flagellin of bacterial flagella. tlr , tlr , tlr , and tlr are strategically expressed in endosomal compartments to recognize microbial nucleic acids: double-stranded rna (dsrna) by tlr , single-stranded rna (ssrna) by tlr and tlr , and unmethylated cpg motif containing dna by tlr ( table ) . it is therefore not surprising that endosomal tlrs have been primarily involved in host defenses against viruses, whereas tlr , tlr , and tlr - have been mainly involved in host response to bacterial and fungal infection. tlrs cooperate with other molecules to recognize microbial ligands. for example, tlr requires cd , cd , and dectin- for the recognition of peptidoglycan, lipopeptides, and β-glucan, respectively. of note, tlrs are also triggered by damps released by injured or stressed cells during infection ( ) ( table ) . tlr activation enables pathogen elimination by promoting bactericidal activity of leukocytes, and maturation and function of antigen presenting cells, thus orchestrating the development of adaptive immune responses ( ) . the signaling pathways resulting from tlr triggering engage adaptors that are recruited by tir/tir domain interactions ( table ) : myeloid differentiation primary response gene ( ) (myd ), tir domain-containing adaptor protein (tirap, also known as mal), tir domain-containing adaptor inducing interferon (ifn)β (trif), and trif related adaptor molecule (tram) ( ) . myd is essential for signaling through all tlrs except tlr and is involved in early nuclear factor-κb (nf-κb) and mitogen-activated protein kinases (mapks) activation and pro-inflammatory gene expression. tirap serves as a bridge to recruit myd to tlr and tlr . trif initiates myd independent ifn regulatory factor (irf ) and late nf-κb activation involved in the production of type i ifns and ifn-inducible genes ( , ) . trif is recruited to the cytoplasmic domain of tlr and, in late endosome, through tram that bridges trif to tlr . a fifth tir domain-containing adaptor, sterile α-, and armadillo-motif containing protein (sarm) acts as a negative regulator of tlr and tlr signaling. sarm interacts with trif and inhibits the induction trif-dependent genes. the signaling pathways activated downstream tlrs have some redundancy. yet, the engagement of multiple tlrs, especially myd and trif-dependent tlrs, have synergistic effects on host responses ( , ) . intracellular cross talk between signaling pathways may also occur when different families of prrs are involved. for example, dectin- synergizes with tlr and tlr and increases cytokine production through canonical and noncanonical nf-κb pathways ( , ) . moreover, a single mamp can be detected by different prrs. this differential sensing is primarily depending on the localization of the mamp. indeed, flagellin is sensed by tlr expressed at the cell surface and by the naip /nlrc (also known as ipaf) inflammasome when localized in the cytoplasm ( ) ( ) ( ) . similarly, peptidoglycans stimulate membrane tlr and intra-cytosolic nod /nod dependent cell activation ( ) . interestingly, some cpg and non-cpg oligodeoxynucleotides directly stimulate and polarize t-cells through tlr and myd -independent mechanisms ( ), possibly through intracellular dna sensors. recently, hagan et al. and kayagaki et al. demonstrated non-canonical tlr -independent recognition of intracellular lps through an uncharacterized receptor ( , ) . this unconventional mode of lps sensing activates caspase- -dependent il- β secretion and sensitizes mice to endotoxic shock. all these observations indicate that the host has evolved different strategies to sense invading microorganisms. ideally, all possible interactions should be characterized and/or anticipated, so that the effect of treatment application can be predicted and/or translated. this mandates carefully planned experiments that represent real-life conditions and a detailed knowledge of compound's mode of action. obviously, the redundancy of microbial sensing pathways should be taken into consideration when developing or applying targeted-treatment strategies to a single prr. experimental animal models and human clinical studies support a crucial role for tlrs in infectious diseases. the first evidence came from the observation that tlr defective c h/hej and c bl/ sccr mice are hyporesponsive to lps and susceptible to otherwise non-lethal infection with escherichia coli and salmonella typhimurium. subsequent studies with mice knockout in tlrs or tlr adaptor molecules have demonstrated the importance of the tlr pathway in host defenses. for example, tlr knockout mice are highly susceptible to infections by staphylococcus aureus and streptococcus pneumoniae ( ) . more recently, human association studies have linked polymorphisms affecting tlr expression or tlr structure with an augmented propensity to develop infections ( ) ( ) ( ) ( ) . the discovery of tlrs and their involvement in innate immune responses has attracted much interest into the development of drugs for controlling infections and improving sepsis management. this field of research has been very dynamic, and ( ) . these two particular aspects of the tlr-targeting field will not be addressed in this review. herein, we will review the most popular agonist (tlr , tlr , tlr , tlr , tlr ) and antagonist (tlr , tlr , tlr , tlr ) agents used in pre-clinical and clinical models of acute and chronic infections, including sepsis. relevant registered clinical trials are listed in table . frontiers in immunology | microbial immunology lps is the main pro-inflammatory molecule anchored in the outermembrane of gram-negative bacteria ( ) . neutralization of bacterial lps, inhibition of its recognition by host cells or inhibition of signaling downstream lps binding to its receptor has long been considered a promising approach for the treatment of severe sepsis and septic shock. interestingly, endotoxemia is prevalent in septic patients, not only in those with gram-negative infection. indeed, translocation of viable bacteria and lps from the gastrointestinal tract has been proposed to participate in the pathophysiology of sepsis. tlr was identified years ago as the signal-transducing molecule of the lps receptor complex ( ) , which also comprises md- and cd . thus, tlr is regarded as a primary target for treating sepsis ( ) . tlr expression is increased in human monocytes of healthy volunteers challenged with lps ( ), as well as in patients with sepsis ( ) . moreover, polymorphisms in the tlr gene have been associated with gramnegative sepsis ( , ) . in the following sections, we present the most advanced tlr antagonists developed for the treatment of sepsis. strategies to inhibit lps-mediated toxic effects have been initiated years before the discovery of tlr ( ) and the unraveling of the crystal structure of the tlr -md- -lps complex ( ) . lipid a, the toxic moiety of lps, is highly conserved among endotoxins and constitutes an ideal therapeutic target ( ) . e , developed by eisai research institute of boston (andover, ma, usa), was the first-generation lipid a antagonist derived from rhodobacter capsulatus endotoxin. e conferred protection in experimental models of endotoxemia and lethal infection with e. coli ( ) . the protective effect likely occurred through the binding of e to the tlr -md- complex and the inhibition of the interaction between lps and tlr -md- ( ) . e also blocked endotoxin response in human healthy volunteers challenged intravenously with lps ( ). e development went through phase clinical trial, but was stopped due to issues of bioavailability. a second-generation lps antagonist drug candidate developed by eisai is eritoran tetrasodium (known as eritoran or e ), a synthetic lipid a analog of rhodobacter sphaeroides ( ) . eritoran blocked lps-induced cytokines in vitro and in experimental animal models ( ) ( ) ( ) . in a phase clinical trial enrolling healthy volunteers challenged with lps, eritoran inhibited pro-inflammatory cytokine production and diminished clinical symptoms of sepsis, including fever, chills, tachycardia, and headache. additionally, creactive protein levels and white blood counts were significantly decreased ( ) ( ) ( ) ( ) . the only adverse event observed was a dosedependent phlebitis, due to the fact that high doses of eritoran were used to achieve stable activity of the drug over time. a phase randomized control trial recruiting critically ill septic patients as assessed by the acute physiology and chronic health evaluation ii (apache ii) score disclosed a trend toward decreased mortality in the eritoran treated group ( ) . phase access (a controlled comparison of eritoran and placebo in patients with severe sepsis) clinical trial for severe sepsis started in , and results were published in . about patients were treated with eritoran and patients with placebo within h after the onset of the first organ dysfunction. unfortunately, analyses did not reveal reduced all-cause mortality in primary and secondary end-points (i.e., days and year mortality) ( ) . eisai (tokyo, japan) waived to submit eritoran to marketing authorization for the treatment of severe sepsis in january , based on preliminary results of the access trial. several reasons may account for the lack of efficacy of eritoran ( ) ( ) ( ) . for instance, patients were not enrolled or monitored based on the circulating levels of lps, questioning about the appropriateness of inclusion criteria. it is also possible that eritoran would be more efficient if administrated rapidly, before septic shock is underway, pointing the early and aggressive sepsis management as a possible interfering factor. other factors to take into account include the heterogeneity of patients for genetic background, underlying diseases, inflammatory and immune status, sepsis severity, infectious agent, and site of infection. as mentioned earlier, intracellular lps sensed in a tlr -independent manner sensitizes mice to endotoxic shock ( , ) . this non-canonical lps detection may have limited the efficacy of the anti-tlr strategy. moreover, upon infection, innate immune cells will likely sense several mamps via several tlrs and non-tlr prrs. for example, gram-negative bacteria express mamps that may trigger redundant inflammatory pathways through tlr (lipopeptides), tlr (lps), tlr (flagellin), tlr (ssrna), and tlr (bacterial dna). all these observations suggest that blocking one single pathway may be insufficient to interfere with the deleterious cascade of events observed in sepsis. the positive side of the access trial failure was a rethink of the design of sepsis clinical trials ( ) ( ) ( ) . clearly, a drug like eritoran should be tested in selected patients and treatment efficacy examined and adjusted according to predefined appropriate biomarkers (such as lps blood levels and genetic polymorphisms affecting the tlr pathway). a rigorous approach combining the power of "omics" technologies would allow the selection of homogeneous cohorts and the follow-up of the response to treatment, both of which are mandatory for the successful development of anti-sepsis drugs. another anti-sepsis agent that exhibited promising therapeutic properties is tak- [ethyl-( r)-[n -( -chloro- -fluorophenyl) sulfamoyl] or resatorvid] from takeda pharmaceutical company (osaka, japan). tak- was originally characterized as a suppressor of nitric oxide (no) and cytokine production by lpsstimulated macrophages and during endotoxic shock in mice ( ) . tak- binds to cysteine in the intracellular domain of tlr , thereby inhibiting both myd -dependent and myd independent pathways activated by lps ( ) . when administered in conscious guinea pigs following lps challenge, tak- significantly improved septic shock symptoms, decreasing hmgb systemic levels, and increasing survival in a dose-dependent manner ( ) . tak- also increased survival rates from to % and improved organ dysfunction when co-administered with antibiotics in a mouse model of cecal ligation and puncture (clp). no effect on circulating bacterial counts was observed ( ) . a double-blind, randomized, placebo-controlled trial was initiated with tak- ( ) . inclusion criteria comprised symptoms of severe sepsis accompanied with either shock and/or respiratory frontiers in immunology | microbial immunology failure. the study was stopped prematurely due to failure to achieve significant decrease of systemic cytokine levels at stage of the analysis ( ) . a phase clinical study was designed but never launched based on business decision and not due to safety or efficacy concerns. antibodies directed against tlr or the tlr -md- complex have been generated and showed promising results in several pre-clinical studies. we engineered a soluble chimeric protein composed of the n-terminal and central domains of mouse tlr (amino acid - ) fused to the fc domain of human igg ( ) . the chimeric molecule was used to generate high titer anti-mouse tlr rabbit polyclonal antibodies. the anti-tlr antibodies powerfully inhibited nf-κb and mapk activation and cytokine production by lps-stimulated cells in vitro. the antibodies also hampered cytokine production and protected mice from lethal endotoxemia when administered both prophylactically and therapeutically h after lps. prophylactic administration of anti-tlr antibodies blunted tnf production and strikingly increased survival in e. coli sepsis, from % in the control antibody group to % in the anti-tlr group. even more impressive, anti-tlr therapy initiated as much as h after the onset of infection in a model of e. coli peritoneal infection improved survival from to % ( ) . our studies demonstrate that anti-tlr antibodies are efficient as adjunctive therapy for e. coli sepsis, with a window of clinical application comprising prophylactic and therapeutic intervention opportunities. several anti-tlr monoclonal antibodies have been produced. the group of miyake (university of tokyo, japan) reported in the generation of mts , the first rat monoclonal antibody specific of the mouse tlr -md- complex ( ) . mts was shown to inhibit lps-induced nf-κb activation and tnf production by macrophages. e is a rat monoclonal antibody produced by novimmune sa (geneva, switzerland) that reacts with the tlr -md- complex ( ). e inhibited lps-induced cell activation, and protected mice from lethal endotoxemia when injected up to h after lps challenge. moreover, administration of e at the time of surgery improved the outcome of mice with colon ascendens stent peritonitis, a model of polymicrobial abdominal sepsis. finally, the rat monoclonal antibody a , that recognizes both mouse and human tlr -md- complexes, conferred protection in a model of e. coli sepsis, but not salmonella enterica, sepsis ( ) . although some tlr inhibitors have entered clinical and pre-clinical trials, others remain in the developmental stage. lps-trap-fc antibodies (comprising the extracellular domain of mouse tlr fused with md- and linked to human igg fc) dose-dependently decreased il- release by macrophages, opsonized gram-negative bacteria, and enhanced phagocytosis and complement-mediated bacterial killing ( ) . cell-penetrating peptides comprising the translocating segment of drosophila antennapedia homeodomain fused with bb loop sequences of tlr (i.e., tlr -bb peptides) inhibited lps-induced nf-κb and mapk activation and cytokine production ( , ) . further studies will be required before advancing these products toward the clinical level. altogether, the experimental data reported above provided strong support for the concept of tlr -targeted therapy for gram-negative sepsis. in the gloomy context following the withdrawn of rhapc and eritoran from the sepsis field, it is hopeful that ni- has entered clinical development. ni- is an anti-tlr monoclonal antibody produced by novimmune able to block tlr dimerization and tlr -mediated signaling triggered by lps and endogenous and chemical ligands of tlr . data from pre-clinical studies in models of arthritis, respiratory inflammation, and organ injury have highlighted the potential favorable action of this agent . a phase clinical study is currently recruiting participants to evaluate drug safety and tolerance in healthy volunteers before and after ex vivo and in vivo lps challenge. pharmacokinetics and pharmacodynamics will also be assessed. results from these studies are eagerly awaited. albeit less well characterized, tlr has been implicated in the sensing of non-bacterial microorganisms such as viruses and fungi. tlr recognizes o-linked mannan from candida albicans, and human studies have linked asp gly tlr polymorphism with susceptibility to bloodstream candidiasis and pulmonary aspergillosis ( ) ( ) ( ) . in a model of disseminated infection with c. albicans, c h/hej tlr -deficient mice exhibited a -fold increased fungal load in the kidneys, which was associated with reduced production of the chemokines kc and mip- and an impaired recruitment of neutrophils ( ) . treatment with hta , an anti-human tlr mouse monoclonal antibody ( ), interfered with neutrophil-mediated protection against c. albicans invasion and cell injury in an in vitro epithelial model of oral candidiasis ( ) and inhibited tnf production by human pbmcs stimulated with aspergillus hyphae ( ) . it is still unclear whether targeting tlr may be beneficial in the context of fungal infections. a more clear yet unexpected picture has arisen from viral infection studies. reactive oxygen species (ros) produced by the nadph oxidase generates oxidized host phospholipids that stimulate tlr and the production of cytokines involved in acute lung injury ( ) . using a mouse model of lethal infection with influenza, the group of stephanie vogel (university of maryland, baltimore, ma, usa) reported that eritoran significantly increases survival in a dose-dependent manner even when administered days after viral challenge. lung pathology and clinical symptoms were improved while viral titers and influenza-induced cytokine gene expression in lung homogenates were decreased compared to the placebo-treated group. these data suggest that the therapeutic effect of eritoran in a more practical timing of severe sepsis treatment remains substantial ( ) . they also suggest that, despite the failure of eritoran in the access trial, new therapeutic potentials might still emerge for this agent. toll-like receptor has been implicated in the recognition of an amazingly broad spectrum of microbial ligands originating from bacteria, fungi, viruses, and parasites ( ) . this property is at least partly due to the fact that tlr forms heterodimers with tlr , tlr , and possibly tlr ( , , ) . the biological relevance of tlr homodimers is controversial. indeed, some ligands have been reported to trigger cells through tlr independently of tlr and tlr . yet, only tlr /tlr and tlr /tlr heterodimers have been successfully crystallized ( , ) . tlr represents an interesting target for numerous conditions, but clinical development of tlr -targeting drugs has been less extensive than that of tlr . t . is a tlr neutralizing mouse monoclonal antibody. t . blocked pam csk lipopeptide (a tlr /tlr -ligand)stimulated nf-κb nuclear translocation and mapk phosphorylation in vitro. in models of pam csk -induced toxic shock and microbial challenge with a high inoculum of heat-inactivated bacillus subtilis, t . prevented lethal shock-like syndrome and increased survival when administered h before or up to h after infection ( ) . furthermore, t . used in combination with the a anti-tlr /md- antibody and antimicrobial therapy protected mice from sepsis caused by s. enterica and e. coli ( ) . intracellular antibodies, i.e., intrabodies, have been designed to block the intracellular translocation of tlrs from the endoplasmatic reticulum to the cell surface. αt ib is a functional anti-tlr scfv intrabody comprising the variable domains of the heavy and light chains of t . linked together by a synthetic (gly ser) amino acid sequence. αt ib bound intracellularly to tlr and led to retention and accumulation of tlr inside the endoplasmatic reticulum. adenovirus-mediated expression of αt ib in raw . macrophages and mouse bone marrow derived macrophages inhibited tlr surface expression and tlr -ligand-driven tnf production ( ) . these data suggest for a therapeutic potential of t . or αt ib in microbial infections. many studies have attempted to elucidate the pathogenesis of acute kidney injury associated with sepsis, which involves mechanisms similar to those occurring during ischemia/reperfusion ( ) . damps released during infection are detected through tlr by immune cells recruited to the ischemic tissue and/or by cells of the ischemic tissue itself, amplifying the inflammatory response and inducing injury upon reperfusion ( , ) . blocking tlr under these conditions may be cytoprotective ( ) . opn- is a humanized anti-tlr igg monoclonal antibody [derived from opn- ( ) developed by opsona therapeutics (dublin, ireland)]. opn- reduced tlr -driven pro-inflammatory cytokine production through blocking of tlr / and tlr / mediated signaling. in a porcine model of myocardial ischemia/reperfusion injury, pretreatment with opn- or administration of opn- h after ischemia was associated with a % decrease in infarct size ( ) . results from a first in human phase trial evaluating safety, tolerability, pharmacokinetics, and pharmacodynamics of ascending doses of opn- given intravenously in healthy adult subjects have just been released ( ) . tlr occupancy and inhibition of il- secretion induced by heat-killed listeria monocytogenes were assessed in whole blood collected up to days after treatment with either the antibody or placebo. opn- was well tolerated, with no significant toxicity even at the highest dose tested. impressively, opn- at doses of . and mg/kg occupied % of tlr molecules expressed on monocytes collected and days after challenge, respectively. il- release was inhibited in a parallel manner. these results suggest that treatment with opn- could provide short-term protection against ischemia/reperfusion and be adjusted to confer long-lasting blockage in the case of tlr -mediated chronic diseases ( ) . a phase trial assessing safety, tolerability, and efficacy of opn- in kidney transplant patients has been initiated (nct ). new techniques are continuously implemented to facilitate the identification of therapeutic targets for adjunctive treatment in sepsis. immunoprecipitation with systematic evolution of ligands by exponential enrichment (selex) was developed to screen and identify high-affinity dna and rna molecules that bind to tlr and could be used to detect other molecules influencing tlrdriven activity. a most promising candidate, ap- , was shown to interact with tlr , thereby obstructing ligand binding to the receptor and inhibiting tlr -ligand-induced nf-κb activity and il- and il- production in thp- and hek cells ( ) . cellpenetrating tlr -bb peptides have been generated and shown to interfere with tlr -ligand-induced activation of nf-κb and mapk and cytokine production ( ) . whether these compounds will undergo clinical evaluation is unknown. toll-like receptor is an endosomal prr that senses dsrna typically produced during viral infection ( ) . experimental models comparing tlr wild-type and tlr knockout mice revealed either a protective role (west nile virus, encephalomyocarditis virus, poliovirus, coxsackievirus, murine cytomegalovirus, herpes simplex virus), a deleterious role (west nile virus, influenza a virus, phlebovirus), or no influence (lymphocytic choriomeningitis virus, vesicular stomatitis virus, murine cytomegalovirus, reovirus) of tlr on anti-viral responses ( ) . therefore, tlr agonists and antagonists might be efficient adjunctive therapies for viral infections depending on the context. in the following sections, we describe the development of synthetic dsrna tlr agonists (see tlr agonists) and of synthetic ssdna tlr antagonists and anti-tlr neutralizing antibodies (see tlr antagonists). the dsrna synthetic analog polyinosinic:polycytidylic acid [poly(i:c)] is a potent immunostimulant. for clinical development, poly(i:c) was stabilized with polylysine and carboxymethylcellulose (poly-iclc) (hiltonol, oncovir, washington, dc, usa) and used to generate poly(i:c u) (rintatolimod, tradename ampligen, hemispherx biopharma, philadelphia, pa, usa) by substituting an uridylic acid at a molar ratio of : in the synthesis of the polycytidylic acid strand ( ) . poly(i:c) and its derivatives have been tested in several clinical trials as adjuvants for vaccines (for both infectious diseases and cancer) and complement to haart (highly active anti-retroviral therapy) in human immunodeficiency virus (hiv) infected patients, topics that we do not discuss here. poly(i:c u) is highly specific for tlr and, unlike its parental molecule poly(i:c), does not require melanoma differentiationassociated protein (mda , a cytosolic prr for viruses), for the induction of the signaling cascade leading to type i ifn production ( ) . poly(i:c u) has some anti-viral activity against hiv, hepatitis b virus (hbv), coxsackie b virus, and several flaviviruses. results from animal models of lethal respiratory viral infection by severe acute respiratory syndrome coronavirus (sars-cov) and punta toro virus highlighted the favorable impact of intranasal treatment with poly(i:c) or poly(i:c u) on survival and viral loads in infected mice ( , ) . the mode of action of poly(i:c) in the respiratory tract was linked to the induction of caspase-mediated apoptosis and ros, which are involved in the cleavage and shedding of soluble tnf receptor blocking tnf bioactivity ( ) . interestingly, poly(i:c) protected against bacterial infections in the respiratory tract as well as in the central nervous system (cns). in a mouse model of pseudomonas aeruginosa pneumonia secondary to clp, intranasal administration of poly(i:c) improved immune activation and lowered bacterial load in the lungs compared to the untreated animals ( ) . corroborating results showing enhanced phagocytosis and killing of e. coli by microglial cells suggest that tlr activation is crucial for the immune response of cns against invading pathogens ( , ) . these data support the development of tlr agonists as adjuvant therapies to prevent or reduce the severity of respiratory tract infections caused by viruses and possibly bacteria. in connection with that particular field, a phase safety, tolerability, and pharmacokinetic trial of nasally applied poly-iclc in human volunteers is ongoing and will explore immune activation markers. a phase / clinical trial is assessing the immunogenicity and safety of flumist®(live attenuated influenza vaccine, med-immune, gaithersburg, md, usa) intranasal influenza vaccine administered with and without a poly(i:c u). more recently, tlr antagonists have been developed taking into consideration that tlr over-activation by viral dsrna may have detrimental consequences in some situations. indeed, it has been reported that administration of poly(i:c) in mice prior to intratracheal challenge with s. pneumoniae impaired bacterial clearance and increased mortality. excessive production of type i ifn was involved in this phenomenon ( ) . single-stranded dna oligonucleotides (ssdna odns) efficiently competed with dsrna for binding to tlr , thus inhibiting cytokine production and costimulatory molecule expression by epithelial cells, pbmcs, and dendritic cells ( , ) . the efficacy of ssdna odns was demonstrated in cynomolgus macaques, where intranasal injection of ssdnas odns inhibited poly(i:c)-induced cytokine production in nasal secretions ( ) . in addition to microbial ligands, tlr senses damps released from injured tissue during inflammation, for example rna from necrotic cells, promoting an excessive inflammatory response. interestingly, administration of a tlr neutralizing antibody to mice reduced cecal damage induced by gut ischemia and improved survival of animals with polymicrobial sepsis when the antibody was given and h after clp surgery ( ) . collectively, these data demonstrate that tlr works as an endogenous sensor of necrosis and a regulator of the immune response, pointing to receptor modulation as a possible adjuvant therapy for sepsis. work remains to be done to clearly delineate the precise role of tlr in viral and bacterial infections and to appraise the benefit afforded by tlr agonistic or antagonistic strategies for infectious diseases, especially septic shock. a single tlr agonist has had clinical development, namely cblb . flagellin is the only ligand of tlr described to date. detailed structural basis of flagellin recognition by tlr has been obtained through crystallographic analyses, unraveling a unique mode of interaction between the two molecules as depicted from stoichiometry, ligand arrangement, and binding interfaces ( ) . the role of structural constraints for induction of the nf-κb signaling cascade downstream tlr was supported by structure-guided mutagenesis and deletion analyses on cblb (entolimob), a therapeutic agent derivative of s. enterica flagellin implemented by cleveland biolabs (buffalo, ny, usa). cblb is currently tested in a phase trial in late stage cancer patients (nct ). several clinical trials have investigated the safety and adjuvant efficacy of recombinant flagellin in a number of vaccine settings (against influenza virus, helicobacter pylori, campylobacter, yersinia pestis, west nile virus, etc) ( ) . cblb plays a protective role against radiation-induced tissue injury, probably by suppressing apoptosis, attenuating ros generation, and promoting tissue regeneration ( ) . these properties could explain the beneficial effect of this agonist in a murine model of acute ischemic renal failure when administered min after reperfusion ( ) . highlighting the favorable role of flagellin against tissue damage, results from two mouse studies suggested that protection and repair of the intestinal mucosa that serves as a first line defense barrier is the key mode of action of flagellin. in the first study, flagellin was shown to induce the expression of regiiiγ, a c-type lectin with bactericidal activity, and to restrict small intestine colonization with vancomycin-resistant enterococcus (vre) in animals inoculated with vre via oral gavage ( ) . in the second study, treatment with flagellin reduced intestinal epithelium destruction induced by dextran sodium sulfate (a chemical used to induce severe acute colitis) and increased survival of mice inoculated with s. typhimurium by oral gavage ( ) . these data suggest that flagellin or tlr agonists may represent attractive tools for treating pathologies that injure the intestinal tract, including severe sepsis. no tlr antagonists have been reported. toll-like receptor agonists tested in clinical trials are synthetic cpg oligodeoxynucleotides (cpg odns) among which cpg , imo- , sd- , and cpg that mimic unmethylated cpg dinucleotide-rich sequences enriched in microbial dna. cpg odns are powerful immunostimulants, exploited for their adjuvant properties in vaccines against infectious diseases (flu, malaria, hiv infection, pneumococcal and meningococcal diseases) and cancer (melanoma, leukemia, glioblastoma, and colorectal, prostate and breast cancer) ( ) . the adjuvant properties of cpg odns have been used to enhance www.frontiersin.org the phagocytosis and the killing of bacteria (s. typhimurium and s. pneumoniae) by phagocytic cells ( , ) . interestingly, a recent study showed that cpg odn given h prior to clp surgery prevented clp-induced cardiac dysfunction in mice. the authors proposed that targeting tlr could be a useful approach for the management of cardiovascular dysfunction in severe sepsis patients ( ) . therapeutic strategies focusing on tlr -mediated immunomodulation are currently being implemented for chronic viral infections, such as chronic hepatitis c (hcv). plasmacytoid dendritic cells are the main cells producing type i ifn and are therefore considered to play an important role in viral infections. tlr agonists stimulate plasmacytoid dendritic cells to produce large amounts of type i ifn, especially ifnα, which is the backbone of therapy for hcv. indeed, ifnα powerfully inhibits viral replication and promotes innate and adaptive host immune responses. moreover, ifn production appears to be impaired in plasmacytoid dendritic cells of hcv patients ( , ) . cpg was originally developed under the trade name actilon by coley pharmaceuticals (wellesley, ma, usa), a company recently incorporated by pfizer (new york city, ny, usa). cpg has undergone two phase studies with promising results. a phase a study for drug safety and pharmacokinetics conducted in healthy volunteers revealed well tolerated immunostimulatory effects without serious adverse events even when using high doses of cpg ( ) . cpg was also tested in hcv patients, infected with genotype hcv. cpg was administered subcutaneously to four randomized groups at different doses twice per week for weeks alone or in combination with pegylated ifnα and ribavirin. the tlr agonistic effect of cpg was associated with the induction of ifnγ and ifnα and the decrease of viral loads. the only serious adverse events were urticaria and pruritis, without manifestation of respiratory complications ( ) . a phase study enrolling non-responders genotype hcv patients has been completed, but results have not been released yet. imo- , manufactured by idera pharmaceuticals (cambridge, ma, usa), has undergone two phase trials: one for dose estimation and one for safety, pharmacokinetics, and pharmacodynamics, enrolling and treatment-naïve genotype hcv patients, respectively. imo- administration dosedependently decreased viral loads, increased the production of anti-viral cytokines and chemokines especially ifnα, and activated nk and t-cell responses ( , ) . a phase trial was planned, but in april the company postponed its initiation. the decision was made based on histological data from a -week non-clinical toxicology study of imo- in rodents and non-human primates . preliminary analyses suggested evidence of atypical lymphocytic proliferation, although no adverse events were reported in humans. thorough analysis results are pending. a phase b study sponsored by dynavax (berkeley, ca, usa) investigated the safety and efficacy of sd- in chronic hcv. sd- was administered as monotherapy or in combination with ribavirin to chronically infected, treatment-naïve, genotype hcv patients. results released in indicated that sd- was well tolerated and safe without any serious adverse events. the drug had significant anti-viral activity based on dose-dependent anti-viral response, with % of patients at the highest dose showing more than one log reduction in viral load, and increased expression of type i ifn-dependent anti-viral genes (ip- , mcp- mx-b, isg- k). these data comfort results from in vitro studies showing that sd- stimulated human pbmcs to produce -fold higher levels of both ifnα and ifnλ in comparison with first-generation tlr agonists ( ) . bacterial dna released during infection is a mamp, and exuberant activation of tlr may participate to the sepsis pathophysiology. hence, drug inhibitors of tlr may have therapeutic potential in human sepsis. as a proof of concept, administration up to h after surgery of a single dose of an inhibitory cpg odn blocking tlr signaling protected mice from polymicrobial sepsis following clp ( ) . hmgb proteins are essential for triggering nucleic acid receptor-mediated innate immune responses. hmgb-binding non-immunogenic-odns have been designed to inhibit hmgb-mediated pathologies. a non-immunogenic odn termed ism odn was tested in a mouse model of endotoxemia. impressively, % of mice treated with ism odn survived up to h after lps challenge, while all mice from the control group died within h ( ). these data argue for a possible use of non-immunogenic-odns in therapeutic interventions. antimalarial drugs such as chloroquine are well known for their anti-inflammatory properties in autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus ( ) . chloroquine blunted cytokine production and protected mice from toxic shock induced by cpg odn and lps. chloroquine down-regulated the expression of both tlr and tlr , suggesting that it acts at multiple levels to inhibit inflammation ( , ) . additionally, when administered h after clp in elderly mice treated with fluids and antimicrobials, chloroquine significantly improved survival, strengthened renal function and protected from multiple organ dysfunction ( ) . these results support clinical evaluation of chloroquine in patients with severe sepsis, especially those presenting with acute renal failure. based on its anti-inflammatory activity and because it inhibits endosomal acidification which are important for cell infection by viruses, chloroquine is also tested in multiple trials for prevention and/or treatment of viral infections (hiv, influenza, dengue, chikungunya). yet, the mode of action of chloroquine is multi-factorial and not only through tlr . toll-like receptor and are closely related tlrs well known for their capacity to recognize ssrna from viruses such as hcv, hbv, hiv, influenza virus, herpes simplex virus, epstein-barr virus, vesicular stomatitis virus, papilloma virus, respiratory syncytial virus, and sendai virus. in agreement, tlr is primarily expressed in plasmacytoid dendritic cells. more recently, tlr and tlr were shown to sense bacterial rna released within phagosomal vacuoles ( ) . tlr and tlr triggering induces potent antiviral immune responses characterized by the production of type frontiers in immunology | microbial immunology i ifns and nf-κb-dependent cytokines. tlr / agonists are primarily developed for treating viral diseases, but also as adjuvants for cancer and infectious disease vaccines. imiquimod (aldara, originally developed by m pharmaceuticals, maplewood, mn, usa) is the only tlr agonist marketed for anti-viral treatment, i.e., external ano-genital warts caused by human papilloma virus. numerous tlr / agonists are in clinical development, like cl , isatoribine, ana , ana , pf- ( a), r- (resiquimod), and gs- . although therapeutic strategies for hcv have evolved in the recent years, quest of new immunomodulatory targets remains mandatory. treatment with new agents such as protease inhibitors appears to be efficient but presents with issues of resistance in the long run ( ) . moreover, current protocol therapy with ifnα provides replenishment with a specific subtype of this cytokine. however, use of tlr agonists is able to induce a variety of ifn subtypes, possibly providing a more radical and integrated anti-viral activity ( ) . finally, administration of tlr / agonists may overcome the adverse events caused by ifnα, like the suppression of granulocyte colony stimulating factor (g-csf) leading to neutropenia. indeed, cl reversed ifnα-mediated inhibition of g-csf production by pbmcs obtained from hcv patients and healthy volunteers ( ) . cl also restored defective cytokine production by myeloid dendritic cells from hiv patients ( ) . isatoribine (anadys pharmaceuticals, san diego, ca, usa) was one of the first guanosin analog selective tlr agonists to be implemented and tested on humans ( ) . intravenous administration of isatoribine to hcv patients during a day treatment plan resulted in reduced plasma concentration of hcv rna, regardless virus genotype. adverse events comprised dose-dependent joint pain, decreased white blood cells, and platelets counts, insomnia, and headache ( ) . the development of an oral prodrug that could lack the detrimental effects of isatoribine, especially in the gastrointestinal tract, pointed to a new candidate, ana . preliminary results from a study conducted with ana were promising. oral administration of ana presented with elevated plasma levels of isatoribine at a concentration able to reduce hcv rna in the plasma of infected patients ( ) . unfortunately, anadys pharmaceuticals and novartis (basel, switzerland) announced in discontinuation of drug development due to unacceptable toxicity in pre-clinical animal studies ( ) . subsequent elaboration of an oral prodrug of isatoribine by anadys pharmaceuticals led to the generation of ana . ana exhibits efficient induction of endogenous type i ifns. a double-blind, placebo-controlled study was conducted in patients with chronic hcv, either treatment-naïve or relapsed from ifn-based therapy. interestingly, ana was safe and well tolerated and presented only with grade and adverse events. moreover, ana dose-dependently decreased hcv rna levels ( ) . in another study, repeated treatment with ana was associated with transient decrease of myeloid and plasmacytoid dendritic cells and increased levels of ifnα and ip- in the blood of patients achieving a reduction in the viral load, suggesting an impairment in ifnα production in the case of non-responders ( ) . pf- , formerly known as a, is a tlr agonist generated by pfizer and implemented so that repeated low doses of the drug would be accompanied with the benefits of the agonistic activity without adverse events. a proof of concept study was conducted to evaluate safety and tolerability of the drug and to determine pharmacokinetics and pharmacodynamics. twentyfour healthy volunteers received orally increasing doses of pf- . pf- induced immune response in a dosedependent and frequency-related manner. however, two of the subjects exhibited severe lymphopenia along with flu-like symptoms and hypotension ( ) . in an attempt to decide on the future perspectives of this compound, a model was used to predict the safety and efficacy of pf- in hcv patients. this model exploited clinical results from the former study in healthy volunteers along with those reported from the use of cpg . further optimization will be required before entering the drug in a phase study ( ) . other tlr / agonists have reached phase trials, but demonstrated lack of efficacy and serious adverse effects. when monocytes from hiv patients were stimulated with resiquimod, il- secretion was augmented while tnf production was decreased compared to the control group. additionally, hiv replication in cultured monocytes was inhibited ( ) . these promising in vitro results were reproduced in a phase trial that enrolled patients with herpes simplex virus type . topical application of resiquimod protected from viral lesion spreading ( ) . however, a phase trial disclosed lack of efficacy of the drug and, although a phase study for the treatment of hcv demonstrated decreased viral loads, adverse side effects similar to those resulting from ifnα treatment were of serious concern ( ). two phase clinical trials for the safety and pharmacokinetics of a novel compound, gs- (gilead sciences, foster city, ca, usa), are currently enrolling treatment-naïve and viral suppressed hbv patients respectively, while another one enrolling hcv patients has been recently approved. gs- is a tlr agonist tested originally in hbv infected chimpanzees. drug administration was associated with reduced viral loads both in plasma and liver, probably through enhanced apoptosis of hepatocytes ( ) . testing of ascending dosages of the drug in healthy volunteers presented with flu-like adverse events at a dose of - mg, but cytokine induction was achieved even at administration of mg pointing to a promising adjunctive treatment for chronic viral infections ( ). all the above data illustrate the efforts devoted to the development of tlr / agonists for treating viral pathologies. taking into account that bacterial rna triggers tlr and tlr pathways, one may speculate that tlr / agonists would impact on bacterial sepsis. unfortunately, there are almost no data available on that subject. in one report, intravenous injection of r- prior to sepsis induction using the colon ascendens stent peritonitis model increased cytokine release and bacterial clearance, but the effect on survival was not reported ( ) . finally, considering that excessive tlr activation induced by viruses or bacteria may have www.frontiersin.org detrimental effects for the host, it might be of interest to generate tlr / antagonists to counteract overwhelming immune activation in conditions of severe infections. targeting tlrs is a promising field for sepsis management and infection control. tlr agonists are widely used to optimize vaccine efficacy, taking advantage of their powerful adjuvancity. whether tlr agonists and antagonists will have such success for treatment therapies, especially for sepsis, has to be established. agonists of tlr and tlr - yielded very promising results for treating viral infections. only few studies tested antagonistic drugs. mainly for historical reasons and because of their ligand specificity, tlr and to a lesser extent tlr are the favorite targets for developing antisepsis drugs. this domain of research has monopolized important resources, but unfortunately many drugs tested in animal models have not entered human studies. those that proceeded, like eritoran and tak- , did not achieve primary endpoint goal and/or were accompanied with manifestation of adverse events. animal models provide invaluable information about the role of tlrs and the mechanism underlying infection pathogenesis, but lack complexity in terms of comorbidities and host response compared to human disease ( ) . so, how to explore more efficiently new treatment strategies? improving the design of clinical studies is mandatory. we should discriminate those patients that could benefit from therapy based on their genotype (for example affecting tlr pathways) and the expression of the targeted molecule, and select homogeneous, well-described population bearing the same underlying conditions and disease severity ( ) ( ) ( ) ) . ideally, sepsis studies should use specific biomarkers to help patient enrollment and weigh treatment efficacy in realistic conditions ( ) . the failure of the recent trials in patients with severe sepsis has taught us valuable lessons regarding patient selection, time of intervention, and follow-up ( ) ( ) ( ) . blocking one single mediator may also not be sufficient to interfere with sepsis. developing sepsis-specific tlr-targeted therapies for patients is a path strewn with obstacles, but an exciting and promising area of research. these studies offer new possibilities for prevention and intervention in infectious diseases, but also for other conditions, such as cancer, allergy, asthma and autoimmune diseases either directly or through improvement of vaccines. benchmarking the incidence and mortality of severe sepsis in the united states long-term cognitive impairment and functional disability among survivors of severe sepsis surviving sepsis campaign: international guidelines for management of severe sepsis and septic shock drotrecogin alfa (activated) in adults with septic shock epigenetics in sepsis: targeting histone deacetylases sepsis: something old, something new, and a systems view harmful molecular mechanisms in sepsis regulation of adaptive immunity by the innate immune system toll-like receptors and their crosstalk with other innate receptors in infection and immunity inflammasomes and their roles in health and disease approaching the asymptote? evolution and revolution in immunology the 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dectin- and toll-like receptors dectin- directs t helper cell differentiation by controlling noncanonical nf-kappab activation through raf- and syk cytosolic flagellin requires ipaf for activation of caspase- and interleukin beta in salmonella-infected macrophages innate immune recognition of bacterial ligands by naips determines inflammasome specificity the nlrc inflammasome receptors for bacterial flagellin and type iii secretion apparatus signalling pathways and molecular interactions of nod and nod cpg and non-cpg oligodeoxynucleotides directly costimulate mouse and human cd + t cells through a tlr -and myd -independent mechanism cytoplasmic lps activates caspase- : implications in tlr -independent endotoxic shock noncanonical inflammasome activation by intracellular lps independent of tlr how important are toll-like receptors for antimicrobial responses? innate immunogenetics: a tool for exploring new frontiers of host defence human genetic susceptibility to infectious disease genetic variation in toll-like receptors and disease susceptibility targeting toll-like receptors: emerging therapeutics? phase trial of eritoran tetrasodium (e ), a toll-like receptor antagonist, in patients with severe sepsis effect of eritoran, an antagonist of md -tlr , on mortality in patients with severe sepsis: the access randomized trial a randomized, double-blind, placebo-controlled trial of tak- for the treatment of severe sepsis phase b dose-secalation study of sd- , a novel therapeutic tlr- agonist, in treatment-naive chronic hepatitis c patients a phase , multi-center, randomized, placebo-controlled, dose-escalation study of imo- , a tlr agonist, in hepatitis c-nonresponders imo- plus ribavirin gives substantial first-dose viral load reductions, cumulative anti-viral effect, is well tolerated in naive genotype hcv patients: a phase trial chloroquine for influenza prevention: a randomised, double-blind, placebo controlled trial chloroquine use improves dengue-related symptoms imiquimod . % cream applied daily to treat anogenital warts: combined results from women in two randomized, placebo-controlled studies imiquimod % cream for external genital or perianal warts in human immunodeficiency virus-positive patients treated with highly active antiretroviral therapy: an open-label, noncomparative study first-line therapy for human cutaneous leishmaniasis in peru using the tlr agonist imiquimod in combination with pentavalent antimony randomised clinical trial: anti-viral activity of ana , an oral inducer of endogenous interferons acting via tlr , in chronic hcv potent immune activation in chronic hepatitis c patients upon administration of an oral inducer of endogenous interferons that acts via toll-like receptor the innate immune response, clinical outcomes, and ex vivo hcv antiviral efficacy of a tlr agonist (pf- ) innate immune sensing and its roots: the story of endotoxin toll-like receptor modulation as a strategy to treat sepsis expression of tumour necrosis factor receptor and toll-like receptor and on peripheral blood leucocytes of human volunteers after endotoxin challenge: a comparison of flow cytometric light scatter and immunofluorescence gating increased expression of tolllike receptor- and - on leukocytes from patients with sepsis the structural basis of lipopolysaccharide recognition by the tlr -md- complex bacterial endotoxin: molecular relationships of structure to activity and function lipopolysaccharide interaction with cell surface toll-like receptor -md- : higher affinity than that with md- or cd a lipid a analog, e , blocks the endotoxin response in human volunteers with experimental endotoxemia eritoran tetrasodium (e ) treatment for sepsis: review of preclinical and clinical studies inhibition of endotoxin response by e , a novel toll-like receptor -directed endotoxin antagonist toll-like receptor antagonist (e ) prevents the chronic airway response to inhaled lipopolysaccharide physiologic, biochemical, and imaging characterization of acute lung injury in mice pharmacokinetics of e , a lipopolysaccharide antagonist, in patients with impaired hepatic function extended in vivo pharmacodynamic activity of e in normal volunteers with experimental endotoxemia safety, pharmacokinetics, pharmacodynamics, and plasma lipoprotein distribution of eritoran (e ) during continuous intravenous infusion into healthy volunteers continuous pharmacodynamic activity of eritoran tetrasodium, a tlr antagonist, during intermittent intravenous infusion into normal volunteers sepsis studies need new direction recombinant human activated protein c as a therapy for severe sepsis: lessons learned? trial watch: sepsis study failure highlights need for trial design rethink discovery of novel and potent small-molecule inhibitors of no and cytokine production as antisepsis agents: synthesis and biological activity of alkyl -(n-substituted sulfamoyl)cyclohex- -ene- -carboxylate analysis of binding site for the novel small-molecule tlr signal transduction inhibitor tak- and its therapeutic effect on mouse sepsis model the novel selective toll-like receptor signal transduction inhibitor tak- prevents endotoxaemia in conscious guinea-pigs combination of imipenem and tak- , a tolllike receptor signal transduction inhibitor, improves survival in a murine model of polymicrobial sepsis protection from lethal gram-negative bacterial sepsis by targeting toll-like receptor cutting edge: cell surface expression and lipopolysaccharide signaling via the toll-like receptor -md- complex on mouse peritoneal macrophages tlr /md- monoclonal antibody therapy affords protection in experimental models of septic shock tlr -induced ifn-gamma production increases tlr sensitivity and drives gramnegative sepsis in mice lipopolysaccharide-trap-fc, a multifunctional agent to battle gram-negative bacteria cutting edge: differential inhibition of tlr signaling pathways by cell-permeable peptides representing bb loops of tlrs targeting tlr signaling by tlr toll/il- receptor domain-derived decoy peptides: identification of the tlr toll/il- receptor domain dimerization interface immunity to fungal infections interplay between candida albicans and the mammalian innate host defense genetic susceptibility to candida infections the role of toll-like receptor (tlr) and tlr in the host defense against disseminated candidiasis md- , a molecule that confers lipopolysaccharide responsiveness on toll-like receptor human epithelial cells establish direct antifungal defense through tlr -mediated signaling involvement of cd and toll-like receptors in activation of human monocytes by aspergillus fumigatus hyphae identification of oxidative stress and toll-like receptor signaling as a key pathway of acute lung injury the tlr antagonist eritoran protects mice from lethal influenza infection the role of tlr in infection and immunity human tlr is a functional receptor, expressed by b cells and plasmacytoid dendritic cells, which activates gene transcription through myd crystal structure of the tlr -tlr heterodimer induced by binding of a tri-acylated lipopeptide recognition of lipopeptide patterns by toll-like receptor -toll-like receptor heterodimer antagonistic antibody prevents toll-like receptor -driven lethal shock-like syndromes generation of anti-tlr intrabody mediating inhibition of macrophage surface tlr expression and tlr -driven cell activation searching for mechanisms that matter in early septic acute kidney injury: an experimental study myocardial ischemia/reperfusion injury is mediated by leukocytic tolllike receptor- and reduced by systemic administration of a novel anti-toll-like receptor- antibody ischaemia-induced up-regulation of toll-like receptor in circulating monocytes in cardiogenic shock treatment with opn- , a humanized anti-toll-like receptor- antibody, reduces myocardial ischemia/reperfusion injury in pigs randomized, double-blind, placebo-controlled, dose-escalating phase i, healthy subjects study of intravenous opn- , a humanized anti-tlr- antibody identification and characterization of oligonucleotides that inhibit toll-like receptor -associated immune responses toll-like receptor, rig-i-like receptors and the nlrp inflammasome: key modulators of innate immune responses to double-stranded rna viruses antiviral responses induced by the tlr pathway structural requirements of the ri n-rc n complex for induction of human interferon essential role of mda- in type i ifn responses to polyriboinosinic:polyribocytidylic acid and encephalomyocarditis picornavirus tlr is essential for the induction of protective immunity against punta toro virus infection by the double-stranded rna (dsrna), poly(i:c u), but not poly(i:c): differential recognition of synthetic dsrna molecules intranasal treatment with poly(i*c) protects aged mice from lethal respiratory virus infections double-stranded rna induces shedding of the -kda soluble tnfr from human airway epithelial cells via tlr -trif-rip -dependent signaling: roles for dual oxidase -and caspasedependent pathways tlr agonist improves survival to secondary pneumonia in a double injury model the viral tlr agonist poly(i:c) stimulates phagocytosis and intracellular killing of escherichia coli by microglial cells microglia recognize doublestranded rna via tlr poly i:c enhances susceptibility to secondary pulmonary infections by gram-positive bacteria single-stranded oligonucleotides can inhibit cytokine production induced by human toll-like receptor singlestranded dna oligonucleotides inhibit tlr -mediated responses in human monocyte-derived dendritic cells and in vivo in cynomolgus macaques tlr is an endogenous sensor of tissue necrosis during acute inflammatory events structural basis of tlr -flagellin recognition and signaling flagellin as an adjuvant: cellular mechanisms and potential an agonist of toll-like receptor has radioprotective activity in mouse and primate models tlr agonist inhibits acute renal ischemic failure bacterial flagellin stimulates toll-like receptor -dependent defense against vancomycin-resistant enterococcus infection flagellin treatment protects against chemicals, bacteria, viruses, and radiation cpg still rocks! update on an accidental drug tlr activation in dendritic cells enhances salmonella killing and antigen presentation via involvement of the reactive oxygen species toll-like receptor stimulation enhances phagocytosis and intracellular killing of nonencapsulated and encapsulated streptococcus pneumoniae by murine microglia the toll-like receptor ligand, cpg oligodeoxynucleotide, attenuates cardiac dysfunction in polymicrobial sepsis, involving activation of both phosphoinositide kinase/akt and extracellular-signal-related kinase signaling rational design of new cpg oligonucleotides that combine b cell activation with high ifn-alpha induction in plasmacytoid dendritic cells a class c cpg toll-like receptor agonist successfully induces robust interferon-alpha production by plasmacytoid dendritic cells from patients chronically infected with hepatitis c safety, pharmacokinetics and immune effects in normal volunteers of cpg (actilon), an investigational synthetic toll-like receptor agonist phase b, randomized, double-blind, dose-escalation trial of cpg in patients with chronic hepatitis c virus toll-like receptor inhibition reduces mortality in polymicrobial sepsis suppression of immune responses by nonimmunogenic oligodeoxynucleotides with high affinity for high-mobility group box proteins (hmgbs) mechanism of endosomal tlr inhibition by antimalarial drugs and imidazoquinolines chloroquine inhibits proinflammatory cytokine release into human whole blood chloroquine protects mice from challenge with cpg odn and lps by decreasing proinflammatory cytokine release chloroquine and inhibition of toll-like receptor protect from sepsisinduced acute kidney injury detection of prokaryotic mrna signifies microbial viability and promotes immunity telaprevir and pegylated interferon-alpha- a inhibit wild-type and resistant genotype hepatitis c virus replication in patients stimulation of interferon and cytokine gene expression by imiquimod and stimulation by sendai virus utilize similar signal transduction pathways interferon-alpha suppressed granulocyte colony stimulating factor production is reversed by cl , a tlr / agonist tlr /tlr activation restores defective cytokine secretion by myeloid dendritic cells but not by plasmacytoid dendritic cells in hiv-infected pregnant women and newborns molecular basis for the immunostimulatory activity of guanine nucleoside analogs: activation of toll-like receptor isatoribine, an agonist of tlr , reduces plasma virus concentration in chronic hepatitis c infection discovery of ana : an oral prodrug of the tlr- agonist isatoribine masked oral prodrugs of toll-like receptor agonists: a new approach for the treatment of infectious disease use of modelling and simulation techniques to support decision making on the progression of pf- , a tlr agonist being developed for hepatitis c r- triggers the expression of tlr / and suppresses hiv replication in monocytes topical resiquimod . % gel decreases herpes simplex virus type genital shedding: a randomized, controlled trial oral resiquimod in chronic hcv infection: safety and efficacy in placebocontrolled, double-blind phase iia studies gs- , an oral agonist of toll-like receptor- , induces prolonged suppression of hepatitis b virus in chronically infected chimpanzees safety, pharmacokinetics and pharmacodynamics of gs- , an oral toll-like receptor agonist stimulation of tlr prior to polymicrobial sepsis improves the immune control of the inflammatory response in adult mice the search for effective therapy for sepsis: back to the drawing board? sepsis: time to reconsider the concept biomarkers in sepsis we would like to thank all our collaborators that participated in our studies. thierry roger is supported through grants from the swiss national science foundation ( _ , _ , _ , and _ ), an interdisciplinary grant from the faculty of biology and medicine of the university of lausanne (switzerland) and the european com the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. frontiers in immunology | microbial immunology key: cord- - c z gk authors: wu, chieh-shan; li, yi-rong; chen, jeremy j.w.; chen, ying-che; chu, chiang-liang; pan, i-hong; wu, yu-shan; lin, chi-chen title: antihelminthic niclosamide modulates dendritic cells activation and function date: - - journal: cellular immunology doi: . /j.cellimm. . . sha: doc_id: cord_uid: c z gk abstract dendritic cells (dcs) link the sensing of the environment by the innate immune system to the initiation of adaptive immune responses. accordingly, dcs are considered to be a major target in the development of immunomodulating compounds. in this study, the effect of niclosamide, a food and drug administration-approved antihelminthic drug, on the activation of lipopolysaccharide (lps)-stimulated murine bone marrow-derived dcs was examined. our experimental results show that niclosamide reduced the pro-inflammatory cytokine and chemokine expression of lps-activated dcs. in addition, niclosamide also affected the expression of mhc and costimulatory molecules and influenced the ability of the cells to take up antigens. therefore, in mixed cell cultures composed of syngeneic ova-specific t cells and dcs, niclosamide-treated dcs showed a decreased ability to stimulate t cell proliferation and ifn-γ production. furthermore, intravenous injection of niclosamide also attenuated contact hypersensitivity (chs) in mice during sensitization with , -dinitro- -fluorobenzene. blocking the lps-induced activation of mapk-erk, jnk and nf-κb may contribute to the inhibitory effect of niclosamide on dc activation. collectively, our findings suggest that niclosamide can manipulate the function of dcs. these results provide new insight into the immunopharmacological role of niclosamide and suggest that it may be useful for the treatment of chronic inflammatory disorders or dc-mediated autoimmune diseases. dendritic cells (dcs) are potent antigen presenting cells that are required for the initiation of t cell responses and function as a bridge between the innate and adaptive immune systems [ ] . they are localized to and circulated within distinct compartments of the lymphoid and peripheral nonlymphoid organs at different stages of maturation. dcs normally reside in an immature state within the peripheral nonlymphoid tissues, where they serve as sentinels for incoming antigens, such as those associated with microbial pathogens and tumors. therefore, upon antigen capture and process in the peripheral nonlymphoid tissues, dcs migrate through the afferent lymphatic vessels to the secondary lymphoid organs, where they stimulate naïve t cells and undergo the morphological, phenotypic, and functional changes characteristic of maturation [ ] . mature dcs possess immunostimulatory properties such as reduced phagocytic activity, increased surface expression of the major histocompatibility complexes (mhc) that present agpeptides, increased expression of costimulatory molecules and increased secretion of cytokines and chemokines [ , ] . in contrast to the enhanced t cell immunity associated with mature dcs, immature dcs fail to stimulate t cell responses and are involved in the induction of peripheral t cell tolerance by generating functional treg cells [ , ] . thus, the potential to harness the power of dcs makes these cells attractive pharmacological targets [ ] [ ] [ ] [ ] [ ] [ ] . http recently, the development of other indications or rediscovery of the inherent values of fda-approved drugs is a growing trend in the pharmaceutical industry. the repurposing or repositioning of an existing drug can accelerate the timeline and reduce the cost of bringing the drug to market because it eliminates the need for additional toxicological and pharmacokinetic assessments [ , ] . niclosamide is a food and drug administration-approved oral antihelminthic drug used to treat most tapeworms, including beef tapeworms and dwarf tapeworms. it has been used in humans for nearly years. it is also used as a molluscicide for water treatment in schistosomiasis control programs [ ] . the activity of niclosamide against these parasites is believed to be due to the inhibition of mitochondrial oxidative phosphorylation and anaerobic atp production [ ] . however, niclosamide is receiving renewed attention in light of new data showing that it has strong anti-neoplastic activity against a broad spectrum of cancer types. for example, several reports have indicated that niclosamide can suppress wnt/b-catenin signaling by targeting the wnt co-receptor lrp on the cell surface, an activity that is closely associated with anti-proliferation and pro-apoptotic activities in prostate and breast cancer cells [ , ] . in addition, jin et al. reported that niclosamide can inhibit the nfÀjb pathway and increase ros levels in acute myelogenous leukemia stem cells; niclosamide also potently inhibited the growth of aml cells in vitro and in nude mice [ ] . a study by ren et al. demonstrated that niclosamide is a selective inhibitor of stat . treatment with niclosamide inhibited the egf-mediated stat activity and inhibited the growth of several types of cancer cells with constitutive stat activation (e.g., du , hela, a ) [ ] . moreover, using an automated cell-based screening assay, balgi et al. observed that niclosamide can increase autophagy by inhibiting mammalian target of rapamycin complex (mtorc ) signaling [ ] . independently, fonseca et al. demonstrated that niclosamide suppresses mtorc signaling through the modulation of cytoplasmic ph in mcf- breast cancer cells [ ] . furthermore, wang et al. reported that niclosamide potently suppresses the notch-regulated gene c promoter-binding factor- (cbf- ) in k leukemia cells [ ] . taken together, these data suggest that the antitumor activity of niclosamide may be due to its ability to target multiple signaling pathways. however, until now, the cellular and molecular targets of this drug in the immune system have remained unknown, and the ability of niclosamide to modulate the functions of dcs, the most potent of the apcs, has not been defined. in this initial study, we designed experiments to examine the potential therapeutic effects of niclosamide on the functional properties of dcs and to elucidate the molecular mechanisms involved. the present study demonstrates that niclosamide inhibits the ability of lps-induced mouse bone marrow-derived dcs to secrete proinflammatory cytokines and affects the expression of costimulatory molecules, the ability of dcs to prime a t cell immune response in an syngeneic dc/t cell coculture and prevents , -dinitro- -fluorobenzene-induced contact hypersensitivity (chs) in vivo. consequently, blocking the lps-induced nf-jb, erk and jnk activation in dcs may explain the inhibitory effect of niclosamide on dc activation. these results show for the first time that niclosamide can manipulate the immunostimulatory properties of dcs and may be useful in suppressing chronic inflammatory disorders or autoimmune diseases. female c bl/ (h- b ) mice ( - weeks old) were purchased from the national laboratory animal center (taipei, taiwan). ot-i tcr transgenic mice were purchased from jackson laboratory (bar harbor, me, usa). ot-ii tcr transgenic mice were provided by dr. clifford lowell (ucsf, san francisco, ca). the mice were housed in a barrier facility at taichung veterans general hospital (taiwan) under the guidelines of the institutional animal care and use committee, and all experiments were conducted in accordance with the institution's guidelines for animal experimentation. mouse bone marrow-derived dcs were generated using a previously described method [ , ] . briefly, bone marrow cells were flushed from the femurs and tibias of c bl/ mice with rpmi using a syringe and a -gauge needle, and the erythrocytes were lysed with red blood cell lysis buffer. the bone marrow cells were suspended in rpmi- medium supplemented with % heat-inactivated fetal bovine serum, u/ml penicillin g, lg/ml streptomycin, ng/ml recombinant mouse granulocyte macrophage colonystimulating factor (peprotech) and ng/ml recombinant mouse il- and placed into a -well plate. the cells were incubated at °c in a % co atmosphere. fresh culture medium was added to the cells every days. on day , the non-adherent or loosely adherent cells were harvested and classified as immature dcs. more than % of the cells expressed cd c, as determined by flow cytometry (data not shown). cd c + dcs were further prepared by immunomagnetic selection from cd c + -enriched bm cells by positive selection using anti-cd -coated beads (miltenyi biotec, auburn, ca, usa) according to the manufacturer's instructions; these cells were used for experiments. the purity of the cd c + cells was confirmed to be > % by flow cytometry. dcs were cultured in the presence of . % dmso or . lm niclosamide (sigma, st. louis, mo, usa; stock solution of mm in dmso) for h followed by stimulation with ng/ml of lipopolysaccharide (lps) for h. the control was untreated group. after incubation, the dcs were harvested and stained with specific antibodies. after mab staining, the samples were analyzed for fluorescence on a facscalibur flow cytometer (bd biosciences, heidelberg, germany). we used fitc-or phycoerythrin (pe)-conjugated monoclonal antibodies (mabs) to stain for mouse cd c, cd , cd , cd , and mhc class ii and class i expression (all from bd biosciences, mountain view, ca). culture supernatants were collected from dcs propagated in the presence of . % dmso or indicated dose of niclosamide for h before or after stimulation with lps ( ng/ml) or other tlr ligands, including peptidoglycan ( lg/ml, tlr /tlr ), polyi:c ( lg/ml, tlr ), cpg odn ( nm, tlr- ) or imiquimod ( lg/ml, tlr- ) for h or tnf-alpha for h. all tlr ligands were purchased from invivogen, san diego, ca. after incubation, the cytokine and chemokine levels in the supernatants of the dc cultures were determined using sandwich elisa kits (r&d systems, minneapolis, mn). the viability of the cultured cells was assessed using the cck- colorimetric assay kit according to the manufacturer's instructions (sigma, st. louis, mo). cell viability of greater than % was observed in all of the experiments in this study. the protocol for this assay was modified from our previous report [ ] . the bmdcs generated from c bl/ were incubated with lg/ ml ova - (ovap ) or ova - (ovap ) (synthesized by echo chemical co., taiwan) in the presence of . % dmso or niclosamide ( . lm) at °c for h. subsequently, dcs were stimulated with lps ( ng/ml) for h. control was untreated group. after incubation, the cells were harvested and washed with pbs. ovap -specific cd + t cells or ovap -specific cd + t cells were enriched from the splenocytes of ot-i and ot-ii tcr transgenic mice (c bl/ ) by macs cell separation according to the manufacturer's protocol (miltenyi biotec). the enriched cd + and cd + t cells were cultured with stimulated dcs at various dc:t cell ratios for h, and the proliferation of the cells was estimated based on [ h] thymidine uptake. in addition, the supernatants from the dc/t cell cultures were collected after h, and the levels of ifn-gamma production were measured using an elisa kit. the proportion of the cd + ifngamma + or cd + ifn-gamma + double-positive t cells were measured by intracellular cytokine staining using a facscalibur flow cytometry (bd biosceince, ma, usa) as previous described [ ] . a total of  cfse ( lm, molecular probes)-stained ot-i or ot-ii spleen cells were injected intravenously (i.v.) into syngeneic c bl/ recipient mice on day . on day , bmdcs from c bl/ mice activated with with lg/ml ova - (ovap ) or ova - (ovap ) for h at °c, and  peptide-loaded bmdcs were i.v injected into the mice. at days , ot-i and ot-ii cell proliferation were analyzed by dilution of cfse labeling in the cd + or cd + population using flow cytometry. the endocytosis by dcs was assessed by dextran-fitc or ova-fitc uptake, as described previously [ ] . briefly, enriched cd c + mouse bone marrow-derived dcs were treated with niclosamide ( . lm), . % dmso plus lps ( ng/ml) or niclosamide plus lps for h. the control was untreated group. the cells were then harvested and incubated at °for h with mg/ml dextran-fitc (molecular weight , ; sigma) or lg/ml ova-fitc (invitrogen-molecular probes). the uptake of dextran-fitc or ova-fitc by the dcs was analyzed using a facscalibur flow cytometer. in addition, parallel experiments were performed at °c to demonstrate that the uptake of dextran by dcs is inhibited at low temperatures. dcs were cultured in the presence of . % dmso or niclosamide ( . lm) for h and subsequently stimulated with lps ( ng/ ml). the cells were harvested, and whole cell lysates were prepared at the min after lps stimulation. the control was untreated group. for protein detection, protein extracts ( lg/ml) were boiled, electrophoresed on % sds-polyacrylamide gels and transferred to nitrocellulose membranes. the membranes were blocked for h with % skim milk in tbs + . % tween . after blocking, the membranes were incubated with a primary antibody against phospho-p , p , phospho-p / , total p / , phospho jnk, b-actin (cell signaling, beverly, ma) or total jnk (sc- ; santa cruz biotechnology, santa cruz, ca) overnight. the membranes were then washed and incubated with horseradish peroxidase-labeled secondary abs (jackson immunoresearch, west grove, pa). the proteins were detected using a western lightning chemiluminescence reagent (perkin elmer life sciences, boston, ma) and analyzed with an las system (fujifilm, tokyo, japan). densitometric analysis was performed with imagej software (national institutes of health, bethesda, md, usa). to measure the nf-jb transcriptional activity, purified bmdcs ( .  cells/well) were stimulated with . % dmso or niclosamide ( . , . lm) for h and subsequently stimulated with lps ( ng/ml) for min in -well plates. the cells were then harvested, and nuclear extracts were prepared using the ne-per nuclear and cytoplasmic extraction system (pierce) according to the manufacturer's instructions. a bca protein assay kit (pierce, rockford, il, usa) was used to determine the protein concentration. for each assay, a total of lg nuclear extract was used in a transam nf-jb p elisa kit (active motif, carlsbad, ca) according to the manufacturer's instructions. the , -dinitro- -fluorobenzene (dnfb; sigma-aldrich)-induced contact hypersensitivity (chs) model was constructed using previously described methods, with some modifications [ ] . the shaved belly of mice was painted daily with ll vehicle (acetone/olive oil at ratio of : ) or . % (w/v) dnfb plus with i.v daily ll solvent solution ( % cremophor el; . % nacl) or ll niclosamide ( mg/kg). five days after sensitization, ll of dnfb . % (w/v) were painted on both sides of the right ears of all of the mice. the chs response was determined h after exposure to dnfb through histological analysis using h&e staining and by evaluating the increased thickness of the ear (thickness of the right (challenged) ear minus the thickness of the left (unchallenged) ear) using an engineer's spring-loaded micrometer (mitutoyo, tokyo, japan). all statistical analyses were performed using graphpad prism software package version . (graphpad software; san diego, ca, usa). cytokine production, t cell proliferation in vitro and in vivo, ifn-gamma production, immunoblotting assay, and increased thickness of the ear were evaluated by one-way anova followed by tukey's post hoc test. when p < . , the difference was considered to be statistically significant. a major attribute of mature dcs is the synthesis and secretion of cytokines and chemokines that modulate inflammatory responses and t cell differentiation and are responsible for the adaptive immune response [ ] [ ] [ ] . therefore, in the first series of experiments, we investigated the effect of niclosamide on the production of pro-inflammatory cytokines and chemokines by lps-stimulated mature mouse bone marrow-derived dcs. first, the supernatants were collected from lps-activated mouse dcs propagated in the presence or absence of niclosamide, and the cytokine and chemokine levels in the supernatants were quantified by eli-sa. bacterial lps is widely used as a strong inducer of dc activation. as shown in fig. , dcs exposed to niclosamide exhibited a significant dose-dependent decrease in the levels of the lps-induced pro-inflammatory cytokines (tnf-alpha, il- , il- ) and chemokines (rantes, mcp- , mip- alpha). in addition, the level of these cytokine or chemokine did not significant differ between the niclosamide-treated and dmso-treated dcs in the absence of lps. moreover, the decrease in cytokine and chemokine levels was not due to an increase in the number of dead cells, as determined by cck- , because there were no marked differences in cell viability between . lm niclosamide-treated cultures and dmso-treated control cultures (supplemental fig. ) . thus, these results suggest that niclosamide suppresses the inflammatory and immunostimulatory activity of dcs stimulated with lps. the activation of dcs is accompanied by the enhanced expression of surface molecules including the costimulatory molecules and major histocompatibility complex molecules (mhc) that mediate interactions with naïve t cells and contribute to t cell activation [ ] . thus, we determined whether treatment with niclosamide affected the expression of surface molecules on mouse dcs. as shown in fig. a , niclosamide treatment significantly altered the expression of the costimulatory molecules (cd , cd and cd ) and the major histocompatibility complex molecules (mhc class ii, mhc class i) (fig. b ) within h compared to lps-stimulated mature dcs that were not exposed to niclosamide. thus, we suggest that niclosamide also impairs the lps-induced phenotypic maturation of dcs. however, it is also important to note that immature dcs possess stronger endocytic activity than mature dcs [ , ] . therefore, we examined the ability of niclosamide-treated dcs to endocytose dextran-fitc or ova-fitc. as show in fig. a and b, the percentage of dextran-fitc-and ova-fitc positive cells did not significant differ between the niclosamide-treated dcs and dmso-treated dcs at °c. therefore, a lower percentage of lps-stimulated dcs than dmso-treated dcs were endocytic. moreover, additional pretreatment with niclosamide also increased the endocytic capacity of lps-stimulated dcs for dextran-fitc and ova-fitc. these findings pertaining to the expression of surface molecules and endocytotic activity strongly suggest that niclosamide prevents the maturation of dcs. one critical function of activated dcs is to induce t cell proliferation and differentiation. thus, we next studied the effects of niclosamide on the ability of dcs to induce ova-specific t cell responses. ova - (ovap ) or ova - (ovap ) peptideloaded immature mouse dcs were pretreated in the presence or absence of niclosamide, stimulated with lps, and tested for their capacity to stimulate allogeneic ova-specific cd + ot-i and cd + ot-ii t cells. t cell proliferation was measured by [ h] thymidine incorporation. as shown in fig. a , lps-activated dcs promoted t cell proliferation, and this effect was abrogated by niclosamide. this suggests that niclosamide decreases the ability of lps-treated dcs to stimulate cd + t cells. we also evaluated the effects of cd + t cell coculture with niclosamide-treated dcs on ifn-gamma (th cytokine) synthesis. as shown in fig. b and c, a cytokine analysis by elisa and intracellular cytokine staining revealed that cd + t and cd + t cells cocultured with niclosamide-treated dcs produced significantly lower amounts of ifn-c in the medium (fig. b ) and percentage of ifn-gamma producing cd + and cd + t cells (fig. c ) than the dmso-treated dcs did in the presence of lps. next, we extended the in vitro results obtained for the proliferation of t cells to in vivo model. for this, cfse-labeled spleen cells either from ot-i or ot-ii mice were i.v injected into syngeneic c bl/ mice. h later, ova - (ovap ) or ova - (ovap ) peptide-loaded immature mouse dcs were pretreated in the presence or absence of niclosamide, stimulated with lps and administrated by the same route. three days after dcs injection, proliferation of cd + and cd + t cells was evaluated in spleens of recipient mice by dilution of the cfse signal. as shown in fig. d , proliferation of cd + or cd + was observed in mice injected with lps-matured dcs, however this effect was attenuated by niclosamide. overall, these data suggest that niclosamide impedes the ability of dcs to prime a t cells-biased immune response. as shown previously, the activation of mapks and nf-jb plays a pivotal role in the production of cytokines by dcs in response to inflammatory stimuli, including lps, tnf-alpha, and interleukin- (il-l) [ , ] . to gain insight into the mechanism of this inhibitory action of niclosamide, we investigated whether the activation of mapks and nf-jb were altered by niclosamide in lps-stimulated mouse dcs. our results showed that pretreatment with niclosamide attenuated lps-induced erk and jnk-mapk phosphorylation but did not affect p phosphorylation (fig. a) . to further determine whether niclosamide decreases the lps-induced activation of the nf-jb pathway, nuclear extracts were prepared, and nf-jb binding activity was assessed using the transam nf-jb transcription factor assay kit. as shown in fig. b , lps induced a significant level of p nuclear translocation and upregulated nf-jb binding activity by min after stimulation. however, the nf-jb binding activity was dramatically reduced in niclosamide-treated bmdcs. therefore, these results may partially explain the inhibitory effect of niclosamide on the maturation of lps-induced dcs. contact hypersensitivity (chs) is a typical dc mediated t cell-dependent, ag-specific inflammatory response that is induced when the skin is exposed to haptens, such as dnfb [ ] . this model was used to investigate the in vivo effect of niclosamide on the dc-mediated immune response. our results showed that i.v injection of niclosamide ( mg/kg) elicited less ear swelling than injection with vehicle control group (fig. ) , suggests that niclosamide could potentially be used to prevent delayed-type hypersensitive diseases, such as allergic contact dermatitis. niclosamide is a food and drug administration-approved antihelminthic agent of choice for the treatment of most tapeworm infections. it was recently shown to have large spectrum of biological effects, including activity against the sars virus and anti-toxin and anti-tumor activities in various systems [ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] . our present data show that niclosamide attenuates the lps-induced pro-inflammatory cytokines and chemokines production, surface costimulatory and mhc molecules expression, the reduction in antigen uptake by dcs, and inhibits dc-triggered allogeneic t cell proliferation. to the best of our knowledge, this is the first study to report that niclosamide can modulate the phenotype and functional properties of dcs. both il- and tnf-alpha are important mediators of a wide range of biologic activities and play numerous beneficial roles in the modulation of pro-inflammatory immune responses [ ] . however, the dysregulation of the production of these two cytokines by dcs has been implicated in the pathogenesis of a variety of acute and chronic inflammatory diseases, as well as autoimmune diseases such as rheumatoid arthritis, ankylosing spondylitis, psoriasis and refractory asthma [ ] [ ] [ ] . therefore, small molecule inhibitors or monoclonal antibodies targeting these cytokines have been used in the clinical treatment of immunerelated diseases. for example, multiple monoclonal antibodies against tnf, including infliximab, adalimumab and certolizumab pegol, have been used therapeutically, and the circulating receptor fusion protein etanercept (enbrel) also targets tnf [ ] . other drugs, such as tocilizumab, a humanized anti-il- r monoclonal antibody, target il- [ ] . because our results indicate that niclosamide strongly inhibits the functions of the inflammatory cytokines tnf-alpha and il- without toxicity, niclosamide should be further evaluated as a clinical immunosuppressive therapy or adjuvant immunotherapy drug. il- has been identified as a cytokine that strongly influences the differentiation of naïve cd cells into t helper (th ) cells that produce ifn-gamma and aid in cell-mediated immunity [ ] . the present study demonstrated that niclosamide can significantly reduce lps-induced il- production in bmdcs and can also down regulate the capacity of lps-stimulated dcs to induce ifn-gamma expression by allogeneic cd + t and cd + t cells (fig. b) . these observations suggest that niclosamide may be effective in several chronic inflammatory diseases described as th -dominant diseases. however, we cannot exclude the possibility that niclosamide may modulate the responses of other types of t lymphocytes. specifically, the tlr- ligand lps was used in our study to stimulate dc maturation. lps has been shown to induce strong th -like responses instead of th immune responses [ ] . our data did not indicate that il- was expressed in dcs after lps stimulation (data not shown). therefore, the utilization of agents capable of stimulating a th response, such as dust mite allergens [ ] , to further determine the effects of niclosamide on dc-mediated th polarization should be examined in the future. to determine the mechanism by which niclosamide inhibits dc function, we examined tlr -related downstream signaling pathways. nf-jb has been demonstrated to regulate the expression of pro-inflammatory mediators, including costimulatory molecules, cytokines and adhesion molecules, in dcs, and it is upregulated as dcs mature [ ] . in addition, experiments in nf-jb subunit knockout mice further indicated that c-rel and p control the expression of costimulatory molecules (cd ) and cytokines (il- , il- ) during the t cell responses induced by lipopolysaccharide (lps)-stimulated dcs. [ ] . a previous study demonstrated that the tnf-alpha-induced nf-jb activity in acute myelogenous leukemia stem cells was significantly suppressed by niclosamide [ ] . our present study showed that niclosamide inhibited nf-jb activity in lps-treated dcs (fig. b) , suggesting that the inhibition of nf-jb activation might be an important mechanism of the inhibition of dc maturation by niclosamide. in addition, the mitogen-activated protein kinase (mapk) pathways: c-jun n-terminal (jnk), extracellular signal-regulated kinase (erk) and p , which direct the expression of various genes related to dc maturation and plays a major role in dcs function [ , ] . in this study, we also observed that niclosamide moderately decreased erk and jnk but did not change p activation in lps-treated bmdcs, suggesting that niclosamide inhibits tlr-related signaling, perhaps including the nf-jb and mapk-erk, jnk signaling pathways. however, niclosamide has been demonstrated to regulate multiple signaling pathways, and some of these pathways are involved in immune responses, such as akt [ ] and stat [ ] . thus, additional studies are needed to determine whether other pathways are involved in this effect and to fully elucidate the signaling pathway(s) responsible for the inhibition of dc maturation by niclosamide. it is worth noting that although our current study demonstrates for the first time that niclosamide can inhibit the lps-induced mapk-erk signaling pathway in bmdcs, it was recently reported that niclosamide dose not affect the induction of the mek/erk/ mnk pathway by serum starvation in mcf- cells [ ] . our preliminary inferences suggest at least two possible causes for the differences in the results. first, the difference in the effect of erk activation by niclosamide may be due to species-specific or cell-type differences. second, niclosamide may be not directly target erk but may instead regulate the pathway upstream of erk. thus, niclosamide has different effects on erk activation in serum-starved mcf- cells and lps-treated bmdcs. however, the identification of possible mechanisms responsible for these different effects may lead to future applications for niclosamide in the treatment of clinical diseases. in a separate study, we found that niclosamide was able to repress the expression of pro-inflammatory cytokines regardless of whether it was administered before or after lps stimulation the level of mapk phosphorylation was examined by western blot analysis with antibodies against erk, jnk, p and mapk (both phosphorylated and unphosphorylated). bands were analyzed by imagej and normalized to actin. (b) the nf-jb assay was performed as described in the materials and methods. the od was measured at nm. the data shown are the mean ± sd of samples from three wells. n.s p > . , ⁄ p < . , ⁄⁄⁄ p < . indicates a significant difference between the niclosamide-treated and . % dmso-treated lps-activated dcs. all data are representative of three independent experiments with similar results. (supplemental fig. ). this suggests that the anti-inflammatory and immunomodulatory effects of niclosamide could be clinically effective for the prevention or treatment of various diseases. in addition, we also tested whether niclosamide could modulate the activation of immature dcs by other tlr ligands, namely peptidoglycan (tlr /tlr ), polyi:c (tlr ), cpg odn (tlr- ) and imiquimod (tlr- ). each tlr ligand increased the release of pro-inflammatory il- and il- into the culture medium, and this release was reduced by treatment with . lm niclosamide (supplemental fig. ). although the mechanism through which niclosamide interferes with dc activation upon stimulation with different tlr ligands is largely unknown, we suggest that it may be related to the effect of niclosamide via the suppression of the mapk and nf-jb signaling pathway. in this pathway, nf-jb is believed to play an important role in various tlr ligand-stimulated selective cytokine and chemokine secretions from dendritic cells [ ] . previous studies showed that niclosamide has very low toxicity in mammals (oral ld in rats > mg/kg) [ , ] . in addition, a single oral administration of mg/kg niclosamide can generate a maximal plasma concentration of . lm in rat [ ] . another possibility to achieve higher plasma concentrations of the drug may be intravenous administration. for instance, a single intravenous administration of mg/kg niclosamide to rats gave rise to a peak plasma concentration of lm [ ] , which is sufficient to modulate dc function, as extrapolated from our data. naïve dendritic cells when activated using tlr ligands or proinflamtory cytokines, shift their metabolism from oxidative phosphorylation to aerobic glycolysis (i.e., the warburg effect). krawczyk et al. described this phenomenon in dcs and also demonstrated how tlr stimulation is essential for dendritic cell maturation [ ] . consequently, according to previous studies, uncoupling of mitochondrial oxidative phosphorylation was also believed to be its antihelminthic mechanism of action [ ] . thus, we hypothesized that immunomodulatory effects of niclosamides on dendritic cells could, in part, be due to its disruption of metabolism. niclosamide has been fda approved for human use against parasitic helminthic infestations for decades. in recent years, niclosamide was shown to have antiviral effects. for example, jurgeit et al. found that niclosamide is an entry inhibitor for a number of phdependent respiratory viruses, including influenza virus and human rhinoviruses [ ] . imperi et al. showed that at micromolar concentrations, niclosamide has high inhibitory activity against pseudomonas aeruginosa qs and virulence both in vitro and in vivo [ ] . moreover, wu et al. demonstrated that niclosamide was able to inhibit replication of sars-cov; viral antigen synthesis was totally abolished at a niclosamide concentration of . lm, suggesting that this drug is a possible candidate for the treatment of sars-cov infection [ ] . this is the first study demonstrating the ability of niclosamide in modulating the activation of immature dcs by several types of tlr ligands. since dcs are crucial for the elimination of pathogens and tumors [ , ] , further studies also should examine the risks associated with long-term niclosamide use. in summary, our results demonstrate for the first time that the fda-approved drug niclosamide inhibits lps-induced dc maturation and cytokine, costimulatory molecule, and mhc molecule expression. in addition, niclosamide-treated dcs inhibited antigen specific t cell responses, prompting us to speculate that this might be an important function of niclosamide. however, we also believe that additional issues warrant future investigation including the molecular mechanisms underlying the ability of niclosamide to suppress dc function. the increase in ear thickness was measured after h. the data are shown as mean ± sd from six mice and representative of two independent experiments yielding similar result, ⁄⁄ p < . indicates a significant difference between the dnfb + solvent (i.v) and dnfb + (niclosamide). 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interferon is selectively required by dendritic cells for immune rejection of tumors this study was supported by grants from the vghks - - , - and ministry of education, taiwan under the atu plan chieh-shan wu, yi-rong li and jeremy j. w. chen contributed equally to this work. the authors declare that they have no conflict of interests. supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/ . /j.cellimm. . . . key: cord- -ay cdf w authors: cho, chang-won; ahn, sungeun; lim, tae-gyu; hong, hee-do; rhee, young kyoung; yang, deok-chun; jang, mi title: cynanchum wilfordii polysaccharides suppress dextran sulfate sodium-induced acute colitis in mice and the production of inflammatory mediators from macrophages date: - - journal: mediators inflamm doi: . / / sha: doc_id: cord_uid: ay cdf w we recently reported the immune-enhancing effects of a high-molecular-weight fraction (hmf) of cw in macrophages and immunosuppressed mice, and this effect was attributed to a crude polysaccharide. as polysaccharides may also have anti-inflammatory functions, we investigated the anti-inflammatory effects and related molecular mechanisms of a crude polysaccharide (hmfo) obtained from hmf of cw in mice with dextran sulfate sodium- (dss-) induced colitis and in lipopolysaccharide-induced raw . macrophages. hmfo ameliorated the pathological characteristics of colitis and significantly reduced production of proinflammatory cytokines in the serum. histological analysis indicated that hmfo improved the signs of histological damage such as abnormal crypts, crypt loss, and inflammatory cell infiltration induced by dss. in addition, hmfo inhibited inos and cox- protein expression, as well as phosphorylated nf-κb p levels in the colon tissue of mice with dss-induced colitis. in macrophages, hmfo inhibited several cytokines and enzymes involved in inflammation such as prostaglandin e( ), nitric oxide, tumor necrosis factor-α, interleukin- , inducible nitric oxide synthase, and cyclooxygenase- by attenuating nuclear factor-κb (nf-κb) and mitogen-activated protein kinases. hmfo attenuated inflammation both in vitro and in vivo, primarily by inhibiting nf-κb activation. our findings indicate that hmfo is a promising remedy for treating inflammatory bowel diseases, such as colitis. excessive environmental and industrial development has created hazardous conditions, such as viral diseases (e.g., middle east respiratory syndrome (mers)) [ ] and particulate matter air pollution, which has been classified as carcinogenic to humans (iarc group ) [ ] . thus, the importance of building a strong immune system is becoming more important for good health, and the demand for health supplements that can help improve the immune system has been rapidly increasing. there is a close correlation between immune responses and inflammation. when immunity decreases, the body is easily exposed to infectious and noninfectious inflammatory diseases. ulcerative colitis (uc) is a type of inflammatory bowel disease (ibd). it is estimated that - million people in the united states suffer from ibd, approximately half of which have uc. the exact cause of uc was unknown, but it appears to be related to a combination of genetic and environmental factors [ ] . the goal of conventional ibd therapies is mainly to downregulate aberrant immune responses and inflammatory cascades. conventional drugs for ibd include corticosteroids and -aminosalicylic acid ( -asa; mesalazine), but these drugs have undesirable side effects, especially steroid therapy, which can cause infertility and developmental disability [ , ] . ibd patients present diverse symptoms throughout the course of the disease. some symptoms are directly related to disease activity, while others may be a consequence of therapy. the disease imposes a substantial burden on patients and significantly impacts their functioning and health-related quality of life [ ] . in spite of advances in modern medical science, traditional herbal medicine has continued to be widely used for health maintenance, disease prevention, and even disease treatment in china, japan, and korea for thousands of years. previous studies have shown that out of ibd patients turns to complementary and alternative medicine [ ] [ ] [ ] [ ] . cynanchum is a genus that includes approximately species that are distributed worldwide, including east africa, the mediterranean, the tropical zone of europe, and the subtropical and temperate zones of asia [ ] . many cynanchum species have been used as traditional medicines in korea toprevent and treat various diseases such as rheumatic arthritis and geriatric, vascular, and ischemia-induced diseases [ ] [ ] [ ] . the roots of cynanchum wilfordii hemsley are referred to as cynanchi wilfordii radix in the korean pharmacopoeia [ ] . c. wilfordii has received substantial attention as a traditional herbal medicine that can be used to treat various diseases. it has many potentially beneficial effects such as antitumor, antioxidant, and anti-inflammatory effects; guards against diabetes mellitus, gastric disorders, neuronal damage, and hypercholesterolemia; and causes vascular relaxation effects [ ] [ ] [ ] [ ] [ ] [ ] . this plant is well-known to contain biologically active compounds, including gagaminine and its glycosides, wilfosides, and cynauricuosides, as well as sarcotine, penupogenin, and cynandione a [ ] . polysaccharides of plant origin have proven to be an important class of natural bioactive products in recent years [ ] , and previous studies have demonstrated that they possess antitumor [ ] , immunity-enhancing [ ] , anticoagulant, antithrombotic, antioxidant, and anti-inflammatory activities [ ] . previously, we showed that a high-molecularweight fraction (hmf) of c. wilfordii exerted immuneenhancing effects on immunosuppression induced by cyclophosphamide and on macrophages, and this effect was attributed to the crude polysaccharide [ ] . however, the anti-inflammatory effects of polysaccharides of c. wilfordii in a dextran sulfate sodium-(dss-) induced mouse model of colitis and the related mechanism of action have not yet been reported. as polysaccharides may also have anti-inflammatory functions, we investigated the anti-inflammatory effects of polysaccharides from c. wilfordii on lipopolysaccharide-(lps-) induced raw . macrophages and on dss-induced chronic colitis in mice. hmfo was prepared as previously described [ ] . briefly, hmfo was obtained from a high-molecular-weight fraction of c. wilfordii that was processed through polyethersulfone ultrafiltration membranes with a molecular weight (m.w.) cutoff of kda in a crossflow filtration system. the highmolecular-weight fraction was precipitated by the addition of volumes of % ethanol. the mixture was allowed to stand at °c overnight and was then centrifuged at rpm for min to obtain the precipitate. the precipitate was lyophilized to produce hmfo, and the chemical and monosaccharide compositions of hmfo were analyzed in previous study [ ] . the molecular weight range of hmfo was estimated to be between . and . kda. model. experiments were performed using -week-old female balb/c mice after a -week acclimatization period. the mice were purchased from koatech animal inc. (pyeongtaek, korea) and maintained in a temperature-and light-controlled facility with free access to standard rodent chow and water. all experimental protocols were in compliance with the guiding principles for the care and use of laboratory animals of the ethics committee of the korea food research institute as table : criteria for scoring the dai ( ) . score weight loss (%) stool consistency ( ) bloodstain or gross bleeding ( ) disease activity index = (combined score of weight loss, stool constancy, and bleeding)/ . ( ) normal stool = well-formed pellets; loose stool = pasty stool that did not stick to the anus; and diarrhea = liquid stool that stuck to the anus. erosions and marked inflammatory cell infiltration mucosal damage was scored - based on the loss of crypts (mucosa) and infiltration of inflammatory cells (maximum score = ). . . disease activity index (dai). the dai was calculated by scoring weight loss, diarrhea, and rectal bleeding based on a previously described scoring system [ ] , as shown in table . weight loss was defined as the difference between the initial and final weights, and diarrhea was defined as the absence of fecal pellet formation and the presence of continuous fluid fecal material in the colon. rectal bleeding was assessed based on the presence of visible blood in diarrhea or gross rectal bleeding. dai values were calculated as (combined score of weight loss, stool constancy, and bleeding)/ . the mice were sacrificed at the end of the experiment, and the colons were separated from the proximal rectum, close to where they passed under the pelvis sternum. the colon length was measured between the ileocecal junction and the proximal rectum. activity. the accumulation of mpo in colon tissues was measured as a marker of neutrophil influx into the colon. colon tissues were thawed and homogenized in lysis buffer. subsequently, the homogenates were centrifuged at ×g for min, and the resulting supernatants were assayed for mpo assay using the colorimetric activity assay kit (sigma, st. louis, mo, usa) according to the manufacturer's recommended protocol. analysis. the entire colon was dissected and flushed with ice-cold phosphate-buffered saline (pbs). samples of the rectum were obtained and fixed in % neutral-buffered formalin (sigma-aldrich) for h at room temperature, embedded in paraffin, and sectioned for histological evaluation. the paraffin-embedded tissues were cut into μm sections and stained with hematoxylin and eosin (h&e). the severity of colitis was evaluated in h&estained sections by independent observers blinded to the experimental conditions, according to modified criteria [ ] , and the results are summarized in table . to perform immunohistochemical analysis for tnf-α and il- expression in the colon tissue, the μm-thick tissue sections were deparaffinized using xylene and dehydrated in a gradient of alcohol solutions. to exclude endogenous peroxidase activity, the sections were incubated in . % h o for min and then incubated with primary antibodies (diluted : ) against proteins of interest for h. the detection system visualized anti-mouse antibodies (k ; dako, glostrup, denmark) and was applied according to the manufacturers' instructions. slides were stained with liquid diaminobenzidine tetrahydrochloride (dab+), a highsensitivity substrate-chromogen system (k ; dako). the images on the slides were visualized under an olympus bx light microscope. . . cell culture and treatment. raw . cells were grown at °c and % co in dulbecco's modified eagle's medium (dmem) containing % fetal bovine serum, mg/ml streptomycin, and u/ml penicillin. the cells were seeded in -well plates at a density of × cells/well in normal dmem and pretreated with the indicated concentrations of hmfo for h. then, the cells were incubated with lps ( μg/ml) at °c for h. prostaglandin e (pge ) production. the inhibitory effects of hmfo on no and pge production were determined using the griess reagent [ ] and enzyme-linked immunosorbent assay (elisa) kits (r&d systems, minneapolis, mn, usa), respectively, according to manufacturer's instructions. and interleukin- (il- ) production. cell culture supernatants and mouse sera were collected, and tnf-α and il- levels were measured with elisa kits (bd biosciences, san jose, ca) targeting either protein, according to the manufacturer's protocols. . . western blot analysis. equal amounts of lysates were resolved by sodium dodecyl-polyacrylamide gel electrophoresis and then transferred to a nitrocellulose membrane. the blots were visualized using an enhanced chemiluminescence system (amersham biosciences inc., piscataway, nj, usa) followed by exposure to x-ray film (fuji photo film co. ltd., tokyo, japan), as previously described [ ] . primary antibodies against inos, total p , phosphorylated jnk, phosphorylated erk / , total jnk, total erk / , and β-actin were purchased from santa cruz biotechnology (dallas, tx, usa dss-induced colitis in mice is a well-established preclinical model that exhibits many of the phenotypic features of human ulcerative colitis [ ] . on day , mice with dssinduced colitis showed a % body weight loss compared to the weight of mice in the control group, while mice treated with mg/kg hmfo showed a % loss (figure (a) ). also on day , the dai scores of mice in the dss-treated group were . ± . points higher than those of mice in the control group. treatment with hmfo attenuated the dssmediated increase in dai scores on day by . ± . and . ± . points in mice treated with mg/kg or mg/ kg hmfo, respectively (figure (b) ). colon length shortening reflects the extent of colon damage that has occurred during acute dss-induced colitis [ ] . the average colon length was approximately mm in the normal control group, whereas it was decreased to mm in the dsstreated group. mice treated with high ( mg/kg) and low ( mg/kg) doses of hmfo showed significantly longer colon lengths than mice in the dss-treated group ( and mm in the groups treated with and mg/kg hmfo, resp., versus mm in the dss-treated group; figures (c) and (d) ). these results demonstrated that hmfo treatment reduced the severity of dss-induced acute colitis. in mice with dss-induced colitis. histological analysis was performed to assess the therapeutic effects of hmfo on colonic inflammation. mucosal thickness is regarded as an indicator of normal mucosal conditions. treatment with dss causes epithelial injury and infiltration of inflammatory cells, such as mast cells [ ] . colonic inflammation and mucosal injury were assessed by pathological examination of the colon after h&e staining, and representative results are shown in figure (a). representative colon tissue sections from mice in the control group showed intact surface epithelium, cryptal glands, stroma, and submucosa. however, colon sections from dss-treated mice showed branched crypts, cryptitis, decreased numbers of crypts and goblet cells, inflammatory cell infiltration, and extensive submucosal edema. in contrast, hmfo treatment improved the signs of histological damage, such as abnormal crypts, crypt loss, and inflammatory cell infiltration (figure (a) ). mice treated with dss alone had significantly elevated histological scores, compared with those of healthy control mice. however, the histological score decreased to approximately % of that in the dss-treated group when treated with the highest hmfo concentration ( mg/kg) (figure (b) ). these results suggested that hmfo treatment protected against acute dssinduced colitis. to evaluate the protective effects of hmfo against leukocyte infiltration in colonic tissue, we measured the levels of mpo, a marker of leukocyte infiltration in the colon tissue. mpo is an enzyme that is produced mainly by polymorphonuclear leukocytes and is specific to granulocyte lysosomes. therefore, mpo is directly correlated with the number of neutrophils [ ] . as shown in figure (c), dsstreated mice showed a significant increase in mpo activity compared to that in control mice, and this increase was drastically decreased in the hmfo-treated group. therefore, the increase in mpo activity in tissues induced by dss correlated with the development of colonic inflammation, and hmfo administration markedly suppressed mpo accumulation in colonic tissues. staining for proinflammatory cytokines. a key characteristic of dss-induced colitis is the increased secretion of proinflammatory cytokines into the serum [ , ] ; thus, we measured serum cytokine levels in this study. serum il- levels were significantly higher in the dss-treated group than in the control group. however, il- levels in mice treated with hmfo ( and mg/kg) were lower than those in the dss-treated group (figure (a) ). serum tnf-α levels were also increased in dsstreated mice compared with the normal control group, while hmfo-treated mice had lower serum tnf-α concentrations than dss-treated mice (figure (b) ). moreover, immunohistochemistry analysis indicated that hmfo at mg/kg markedly reversed the increase in il- -and tnf-α-positive cells (brown stained) in the colonic mucosa of dss-induced mice (figures (c) and (d) ). high levels of proinflammatory cytokine production are a major characteristic of dss-induced colitis [ , ] . in dss-induced acute colitis, enormous infiltrates appeared in inflammatory lesions, mainly consisting of t and b lymphocytes, macrophages, and neutrophils, which produce various proinflammatory cytokines, including tnf-α, ifn-γ, il- , il- , il- , and il- [ , , ] . in the colon of patients with ibd, markedly increased numbers of mast cells and macrophages were observed, and these cells might contribute to abnormal production of inflammatory cytokines and mediators, such as no and tnf-α [ ] . colon of dss-treated mice. the effects of hmfo on the expression of inflammatory proteins such as cox- and inos in cytosolic extracts from the colon of colitis mice were detected by western blot analysis. as shown in figures (a) and (b), inos and cox- expression levels significantly increased in the colon tissues from dss-treated mice compared to those in normal mice. however, hmfo administration remarkably reduced inos protein upregulation and significantly decreased dss-induced cox- expression, down to normal levels with different doses of hmfo (figures (a) and (b) ). the cox- and inos enzymes represent important molecular targets in the treatment and prevention of ibd, and their expression and activity are associated with disease severity, suggesting their potential as anti-inflammation drug targets. during active inflammation, bacteria can enter the lamina propria due to cell destruction and increased permeability. inflammatory cytokines and bacterial antigens induce and drive the transcription of both cox- and inos, and these proinflammatory enzymes are also upregulated in murine experimental colitis and active human ibd [ , ] . our data clearly demonstrated that colonic damage was associated with elevated expression of both cox- and inos proteins, and hmfo administration potently downregulated their expression levels. in a similar study, pomegranate polyphenols and resveratrol decreased cox- and inos overexpression in murine experimental colitis [ , ] . in mice with dss-induced colitis. nf-κb is a major transcription factor that, by regulating the expression of multiple inflammatory and immune genes, plays a key role in host defense and chronic inflammatory diseases [ ] . nf-κb was activated in the mucosal cells of ibd patients [ ] and in experimental colitis models [ ] and, thus, may be an ideal target for uc therapy. therefore, the effect of hmfo on nf-κb activation in mice with dss-induce colitis was investigated. dss administration significantly elevated the phosphorylated nf-κb p levels in the colon tissue compared to those in the control group. however, hmfo treatment drastically attenuated nf-κb phosphorylation, which was comparable in hmfo-treated and control mice (figure (c) ). these results suggested that the effects of hmfo on lps-induced no production in raw . macrophages were investigated by measuring no release into the culture supernatant, using the griess reagent. as shown in figure (a), hmfo significantly decreased lps-induced no production in a dosedependent manner, with > % inhibition at μg/ml hmfo, a nontoxic concentration. no is synthesized by inos and is a well-known proinflammatory mediator involved in various physiological and pathological processes. suppression of no production has been suggested as a new pharmacological strategy for treating inflammation-related diseases [ ] . as shown in figures (b) and (c), inos expression at both the mrna and protein levels significantly increased following lps stimulation. however, inos mrna and protein levels in lps-stimulated raw . macrophages were significantly reduced by treatment with hmfo. these results indicated that hmfo suppressed no production by suppressing inos gene expression in lps-induced raw . macrophages. the inos gene is the primary regulator of no production in macrophages, and inos inhibitors have been found to attenuate osteoarthritis [ ] , periodontitis [ ] , septic shock [ ] , and other chronic inflammatory diseases. suppressing cox- expression in raw . macrophages. we examined whether pge production was suppressed by hmfo in lps-stimulated raw . cells. lps ( μg/ml) significantly increased pge production compared to the basal production level in the absence of lps. treatment with hmfo significantly inhibited lps-induced pge production in a concentration-dependent manner ( figure (d) ). cox- mrna expression and protein levels increased markedly in response to lps ( μg/ml), and treatment with hmfo inhibited lps-activated cox- gene and protein expression in a dose-dependent manner (figures (e) and (f)). pge , which is generated by cox- , plays important roles in the inflammatory response, and lps treatment resulted in elevated pge levels at inflammation sites [ ] . cox- can also be activated by high concentrations of no, which intensifies the inflammatory responses in many chronic inflammatory disorders [ ] . production in raw . macrophages. to examine the ability of hmfo to inhibit the expression of major proinflammatory cytokines, we measured tnf-α and il- production in untreated and hmfo-pretreated, lps-stimulated raw . macrophages. treatment with hmfo inhibited lpsinduced tnf-α (figure (a) ) and il- production ( figure (c) ) in a dose-dependent manner. we next investigated the effects of hmfo on lps-induced tnf-α and il- gene expression in raw . macrophages. the mrna levels of tnf-α and il- were upregulated by lps stimulation, but treatment with hmfo suppressed tnf-α ( figure (b) ) and il- ( figure (d)) mrna expression, implying that hmfo could block transcriptional activation induced by inflammation-regulating transcription factors, such as nf-κb and ap- [ ] . . . inhibitory effects of hmfo on lps-induced nf-κb activation in raw . macrophages. to examine the inhibitory effects of hmfo on nf-κb activation, we measured nuclear nf-κb and cytoplasmic ikk α/β and iκb α/β levels by western blotting. as shown in figure (a), stimulation of raw . macrophages with lps increased nuclear translocation of nf-κb; however, this effect was inhibited by hmfo treatment. our findings indicate that hmfo decreased nf-κb activation by blocking the nuclear translocation of p via ikk-α/β-dependent iκb phosphorylation (figures (c) and (d) ). nf-κb is a ubiquitous transcription factor that is constitutively expressed in many cell types, including cells of the monocyte/macrophage lineage [ ] . nuclear translocation of nf-κb is involved in initiating the transcription of several genes, including tnf-α and inos [ ] . . . inhibitory effects of hmfo on lps-induced mapk activation in raw . macrophages. mapks play critical roles in regulating cell growth and differentiation, cellular responses to cytokines [ ] , and modulating nf-κb activity [ ] . to investigate whether hfmo-dependent inhibition of nf-κb activation and no production occurred through the mapk pathway, we examined the effects of hmfo on the lps-induced phosphorylation of jnk, p , and erk by western blot analysis. as shown in figures (a) and (c), pretreating cells with hmfo inhibited lps-induced phosphorylation of jnk, erk, and p in a concentrationdependent manner. these results suggest that hmfo may inhibit nf-κb activation and no production in raw . macrophages by suppressing mapk phosphorylation. in summary, our results suggest that hmfo inhibited the expression of several cytokines and enzymes involved in inflammation, such as pge , no, tnf-α, il- , inos, and cox- by attenuating nf-κb and mapks in raw . macrophages. furthermore, hmfo ameliorated the pathological characteristics of colitis, such as shortened colon length, and significantly reduced the serum levels of proinflammatory cytokines. histological analysis indicated that hmfo improved the signs of histological damage such as abnormal crypts, crypt loss, and inflammatory cell infiltration induced by dss. in addition, hmfo inhibited inos and cox- protein expression and the levels of phosphorylated nf-κb p in the colon tissue of mice with dssinduced colitis. these results indicated that hmfo attenuated inflammation both in vitro and in vivo primarily by inhibiting nf-κb activation. thus, our findings indicate that hmfo is an effective and promising remedy for ibds, such as colitis. advancing priority research on the middle east respiratory syndrome coronavirus the carcinogenicity of outdoor air pollution inflammatory bowel disease part : ulcerative colitis-pathophysiology and conventional and alternative treatment options intestinal permeability in patients with crohn's disease and ulcerative colitis and their first degree relatives serial biopsy in ulcerative colitis longitudinal study of quality of life and psychological functioning for active, fluctuating, and inactive disease patterns in inflammatory bowel disease use of complementary and alternative medicine in patients with inflammatory bowel disease: results of a crosssectional study in norway use of complementary and alternative medicine in swedish patients with inflammatory bowel disease: a controlled study the manitoba inflammatory bowel disease cohort study: a prospective longitudinal evaluation of the use of complementary and alternative medicine services and products characterisation of complementary and alternative medicine use and its impact on medication adherence in inflammatory bowel disease of flora reiplicae popularis sinicae the effect of herbal extract (estrog- ) on pre-, peri-and post-menopausal women: a randomized double-blind, placebo-controlled study anthraquinone production by cell suspension cultures of morinda citrifolia rapid identification of acetophenones in two cynanchum species using liquid chromatography-electrospray ionization tandem mass spectrometry improved endothelial dysfunction by cynanchum wilfordii in apolipoprotein e(−/−) mice fed a high fat/cholesterol diet pregnane glycoside multidrug-resistance modulators from cynanchum wilfordii effects of cynanchum wilfordii extract on serum lipid components and enzyme activities in hyperlipidemic and streptozotocin-induced diabetic rats observation of the protection effect of baishouwu to the liver of the high serum cholesterol mouse gastroprotective effect of a traditional chinese herbal drug "baishouwu" on experimental gastric lesions in rats antitumor activity of crude extract and fractions from root tuber of cynanchum auriculatum royle ex wight potent in vivo antifungal activity against powdery mildews of pregnane glycosides from the roots of cynanchum wilfordii bioactive polysaccharides from plants structural characterisation of polysaccharides from tricholoma matsutake and their antioxidant and antitumour activities immune-enhancing effects of a high molecular weight fraction of cynanchum wilfordii hemsley in macrophages and immunosuppressed mice a sulfated polysaccharide, fucans, isolated from brown algae sargassum vulgare with anticoagulant, antithrombotic, antioxidant and anti-inflammatory effects treatment of dextran sulfate sodium induced murine colitis by intracolonic cyclosporine inhibition of dextran sulphate sodium (dss)-induced colitis in mice by intracolonically administered antibodies against adhesion molecules (endothelial leucocyte adhesion molecule- (elam- ) or intercellular adhesion molecule- (icam- )) measurement of nitric oxide production in biological systems by using griess reaction assay a novel method in the induction of reliable experimental acute and chronic ulcerative colitis in mice clinical aspects and pathophysiology of inflammatory bowel disease ixeris dentate nakai reduces clinical score and hif- expression in experimental colitis in mice effects of antioxidant therapy on leukocyte myeloperoxidase and cu/zn-superoxide dismutase and plasma malondialdehyde levels in experimental colitis characterisation of acute murine dextran sodium sulphate colitis: cytokine profile and dose dependency cytokines in experimental colitis interferon-gamma is causatively involved in experimental inflammatory bowel disease in mice an experimental model of colitis induced by dextran sulfate sodium from acute progresses to chronicity in c bl/ : correlation between conditions of mice and the environment functional abnormalities in the intestine associated with mucosal mast cell activation increased mucosal nitric oxide production in ulcerative colitis is mediated in part by the enteroglial-derived s b protein acute and chronic responses associated with adrenomedullin administration in experimental colitis dietary supplementation of an ellagic acid-enriched pomegranate extract attenuates chronic colonic inflammation in rats dietary supplementation of resveratrol attenuates chronic colonic inflammation in mice nuclear factor-kappa b activation of nuclear factor kappa b inflammatory bowel disease selective inhibitors of inducible nitric oxide synthase: potential agents for the treatment of inflammatory diseases? reduced progression of experimental osteoarthritis in vivo by selective inhibition of inducible nitric oxide synthase protective effects of mercaptoethylguanidine, a selective inhibitor of inducible nitric oxide synthase, in ligature-induced periodontitis in the rat beneficial effects of l-canavanine, a selective inhibitor of inducible nitric oxide synthase, on lactate metabolism and muscle high energy phosphates during endotoxic shock in rats oenothera laciniata inhibits lipopolysaccharide induced production of nitric oxide, prostaglandin e , and proinflammatory cytokines in raw . macrophages in vitro and in vivo anti-inflammatory activities of columbin through the inhibition of cycloxygenase- and nitric oxide but not the suppression of nf-κb translocation molecular mechanisms of nf-kappab activation induced by bacterial lipopolysaccharide through toll-like receptors constitutive nuclear nf-kappa b in cells of the monocyte lineage nuclear factor kappa b, a mediator of lipopolysaccharide effects p and extracellular signal-regulated kinase mitogen-activated protein kinase pathways are required for nuclear factor-kappab p transactivation mediated by tumor necrosis factor molecular mechanisms underlying chemopreventive activities of anti-inflammatory phytochemicals: down-regulation of cox- and inos through suppression of nf-kappa b activation disease activity index dss:dextran sulfate sodium lps:lipopolysaccharide nf-κb: nuclear factor-κb ibd:inflammatory bowel disease uc:ulcerative colitis tnf-α: tumor necrosis factor-α il:interleukin inos: inducible nitric oxide synthase cox- : cyclooxygenase- no:nitric oxide mpo: myeloperoxidase pge : prostaglandin e . the authors declare no conflicts of interests. chang-won cho and sungeun ahn contributed equally to this work. key: cord- -zir q authors: chung, t. philip; laramie, jason m.; meyer, donald j.; downey, thomas; tam, laurence h.y.; ding, huashi; buchman, timothy g.; karl, irene; stormo, gary d.; hotchkiss, richard s.; cobb, j. perren title: molecular diagnostics in sepsis: from bedside to bench date: - - journal: j am coll surg doi: . /j.jamcollsurg. . . sha: doc_id: cord_uid: zir q background: based on recent in vitro data, we tested the hypothesis that microarray expression profiles can be used to diagnose sepsis, distinguishing in vivo between sterile and infectious causes of systemic inflammation. study design: exploratory studies were conducted using spleens from septic patients and from mice with abdominal sepsis. seven patients with sepsis after injury were identified retrospectively and compared with six injured patients. c bl/ male mice were subjected to cecal ligation and puncture, or to ip lipopolysaccharide. control mice had sham laparotomy or injection of ip saline, respectively. a sepsis classification model was created and tested on blood samples from septic mice. results: accuracy of sepsis prediction was obtained using cross-validation of gene expression data from human spleen samples and from mouse spleen samples. for blood studies, classifiers were constructed using data from a training data set of microarrays. the error rate of the classifiers was estimated on seven de-identified microarrays, and then on a subsequent cross-validation for all blood microarrays. estimates of classification accuracy of sepsis in human spleen were . %; in mouse spleen, %; and in mouse blood, . % (all estimates were based on nested cross-validation). lists of genes with substantial changes in expression between study and control groups were used to identify nine mouse common inflammatory response genes, six of which were mapped into a single pathway using contemporary pathway analysis tools. conclusions: sepsis induces changes in mouse leukocyte gene expression that can be used to diagnose sepsis apart from systemic inflammation. the incidence of sepsis and the number of sepsisrelated deaths is increasing at substantial cost ($ billion annually in the us), as reported recently. the cornerstone of successful therapy is early, accurate diagnosis, coupled with eradication of the source of infection and appropriate antibiotic therapy. unfortunately, efforts to identify specific and sensitive diagnostic markers for sepsis have met with limited success. the diagnosis of sepsis in icus is especially challenging because it is frequently difficult to distinguish between systemic inflammation and systemic infection. clinicians are unable to identify the pathogen responsible for sepsis in up to % of patients or to determine whether patients are responding to antibiotic therapy ; and the traditional means of identifying the organism responsible for bacterial infections are nonspecific (gram stain), slow (culture), or insensitive (both gram stain and culture). there is compelling evidence that efforts to identify clinically important biomarkers in sepsis will prove successful ultimately, - especially given the recent successes of high-throughput, genomic technologies in other fields that face diagnostic challenges (eg, leukemia, breast , and colon cancer ). this molecular classification strategy involves searching for expression patterns in a subset of genes from tissues of known phenotype (the "training" data set), constructing a prediction rule, and then using these "biomarker" genes to predict the phenotype of new samples (the "test" data set). studies that apply these genome-wide technologies to inflammation and sepsis in animal models and patients are now underway, [ ] [ ] [ ] [ ] as reviewed recently. , these reports suggest that genome-wide profiling of gene expression holds promise as a molecular diagnostic tool, capable of generating profiles from leukocytes that are sensitive, specific, and timely for pathogen detection. despite provocative in vitro findings, , there are few reports of using microarrays to study sepsis in vivo. these reports indicate that the transcriptional response during polymicrobial sepsis is organ-specific in mice and rats. there are no reports of using microarrays to make a diagnosis of sepsis, although very recent reports suggest that this will be feasible in patients. , we hypothesized that leukocyte gene expression profiles obtained using dna microarrays could be used to predict septic states; in particular, distinguishing between sterile and infectious sources of systemic inflammation, a common conundrum in caring for the critically ill or injured. tissue samples from both septic patients and clinically relevant mouse models of sepsis were tested. using an investigational protocol approved by the washington university human studies committee, informed consent was obtained to collect samples of splenic tissue intraoperatively or immediately postmor-tem as described previously. seven specimens for expression profiling were chosen retrospectively from patients with injury (trauma or operation) complicated by sepsis and organ dysfunction (sepsis group). these were compared with six age-and gender-matched control specimens from patients with injury (trauma) requiring splenectomy (injury group). a total of patient spleen samples were collected. care and use of laboratory animals were conducted in accordance with a protocol approved by the washington university animal studies committee. seven-to nineweek-old, male c bl/ mice were purchased (harlan, inc) and allowed to acclimatize for at least week. male mice were used to avoid the confounding effect of the female estrous cycle and the well-characterized sexual dimorphism of the adaptive response to cecal ligation and puncture (clp). the seven experimental groups were designed to make classification difficult, reflecting clinically important distinctions relevant to care of icu patients: lethal abdominal inflammation from a sterile source, lethal abdominal infection, and antibiotictreated abdominal infection. mice were assigned to the seven treatments listed in table , grouped into those with no deaths (controls) and those that were "sick" with substantial associated mortality (sterile or infectious causes of systemic inflammation). normal animals were untreated. previously reported protocols were used to perform clp and sham laparotomy. , the ceca of some animals were punctured twice using a -gauge needle (clp), and others were punctured using a gauge needle to produce a "milder" sepsis that caused much lower mortality (mild clp). animals that had laparotomy and cecal manipulation without ligation or puncture were included in the sham laparotomy group (sham). another group of animals had clp and treatment with a standard antibiotic regimen for mouse abdominal sepsis: ip ceftriaxone . mg/kg and metronidazole . mg/kg, delivered , , and hours after clp (clp ϩ antibiotics group). to induce severe systemic inflammation without infection, ip injection of escherichia coli lipopolysaccharide (lps) serotype :b mg/kg (sigma, inc) was performed (lps group). the dose of lps ( mg/kg) was used because it reliably produces death over several days in the animals; smaller doses in pilot experiments tended to produce morbidity without mortality. mice injected ip with abbreviations and acronyms clp ϭ cecal ligation and puncture fdr ϭ false discovery rate est ϭ expressed sequence tag ip ϭ intraperitoneal lps ϭ lipopolysaccharide pca ϭ principal components analysis normal saline vehicle (saline group) acted as the concurrent control group for lps treatment. the census of surviving mice was recorded at -hour intervals for days. in a separate cohort, mice were sacrificed at hours after injury by cervical dislocation under halothane general anesthesia. the -hours time point after injury was chosen because pilot experiments showed that the ability to distinguish between spleen expression profiles was greater at hours than at or hours after injury. whole spleen tissue from eight clp and eight sham mice was harvested and flash-frozen in liquid nitrogen and stored at Ϫ °c. in another cohort of animals, intracardiac blood from eight animals per group was pooled using the paxgene blood rna system to derive total rna from whole blood (preanalytix gmbh). while genechips from blood for each of the seven groups were prepared, there were a total of mouse blood genechips from animals: gene-chips were in the training data set and genechips were in the test (de-identified) data set. whole blood was collected for automated white blood cell counts from eight animals in each group over two replicates, each run performed with samples from concurrent control animals. a hemavet multispecies hematology analyzer (cdc technologies) provided counts and an automated differential. data were expressed as the mean Ϯ sem. total rna from human and mouse spleens was extracted per trizol protocol (life technologies, inc). total rna from mouse blood was extracted using the paxgene kit as recommended by the manufacturer. crna target for genechip hybridization was prepared from total rna using the protocol recommended (affymetrix, inc). the human spleen crna was hybridized with the human full-length genechip (approximately , probe sets). mouse spleen and blood crna were hybridized with the u av genechip (approximately , probe sets). fluorescent hybridization signal was detected using a genearray scanner (hewlett-packard, inc) at the washington university alvin j siteman cancer center and, for the prospective mouse cohort, the genechip scanner (affymetrix). the data discussed in this publication have been deposited in ncbi's gene expression omnibus (geo, http://www.ncbi.nlm.nih.gov/ geo/) and are accessible through geo series accession number gse . tests for differential expression, class prediction, and pathway analysis expression values were calculated from genechip.cel files using dna chip analyzer software (version . ) and default settings (only perfect match probes were used). principal components analysis (pca) was used to visually explore global effects for genome-wide trends, unexpected effects, and outliers in the expression data (partek software). differentially expressed genes were identified using a mixed-model anova and linear contrasts. we next determined whether it was possible to accurately distinguish tissue samples resulting from models of sterile systemic inflammation or models of systemic infection in human spleen, mouse spleen, and mouse blood. to produce unbiased estimates of prediction accuracy while also optimizing the set of predictor genes, classifier type, and classifier parameters, we used a two-level, nested cross-validation procedure that produces prediction estimates that are not biased by gene, classifier, and parameter selection. this procedure makes use of an "outer" cross-validation to produce the estimate of accuracy, and an "inner" cross-validation to perform classifier and gene selection. for class prediction, we compared k-nearest neighbor, nearest centroid, and linear and quadratic discriminant analysis classifiers. two complementary programs were used to query regulatory networks: pathwayassist (ariadne genomics) and pathway analysis (ingenuity systems). both path-wayassist and pathway analysis use the published literature or publicly available databases to identify relationships between genes, small molecules, or other objects. this information in turn is used to map de novo, putative, interaction networks from a given list of input gene survival data were calculated using the kaplan-meier method and survival curves were compared using logrank test (prism v . , graphpad software, inc). cbc and cell differential data were analyzed by one-way anova using the tukey-kramer post-test to correct for multiple comparisons (prism v . ). modified multiple organ dysfunction syndrome (mods) score are reported as the mean Ϯ sem and analyzed using the mann-whitney u test for nonparametric data (prism v . ). the significance of change in mouse blood relative rna abundance was measured across all seven groups using two-way anova for the effects of treatment and batch (time). from this analysis, a step-up false discovery rate (fdr) of . was used to identify a subset of discriminating genes for treatment effect, visualized using pca. for pair-wise group comparisons across mouse blood, a two-way anova for the effects of treatment and batch was used. because there was no batch effect for spleen, one-way anova was used. an fdr of . was applied to the raw p value for each pair-wise comparison, giving a list of informational genes for each comparison. the characteristics of the patients sampled are found in table . none of the injury (control) patients had positive cultures or signs of infection or sepsis at the time of sample collection, and all but one recovered uneventfully after splenectomy. in contrast, all sepsis patients had positive bacterial or fungal cultures or obvious signs of infection (pus or necrotizing fasciitis) complicated by sepsis-induced organ dysfunction at the time of operation (modified mods score, p ϭ . versus injury). dna chip analyzer hybridization signal analysis using default filters flagged of human genechips as a statistical outlier (Ͼ % probe pair expression values calculated as statistical outliers, suggesting unreliable hybridization signal). this sample (patient ) was omitted from additional analysis. pca was used to explore differences in gene expression for all genes across the remaining microarrays, demonstrating considerable differences between the sepsis and injury classes (data not shown). consistent with the large variance in expression observed across subjects, there were no genes identified as differentially expressed between these two classes using a fdr of . . to estimate sepsis class prediction accuracy among these samples, a ϫ leaveone-sample-out nested cross-validation was performed. the average predictive accuracy was . %. all control group animals (saline, sham, normal) survived the duration of the experiment. in contrast, "sick" animals with systemic infection (clp, clp ϩ antibiotics, mild clp, and lps) exhibited typical signs of piloerection, anorexia, and lethargy followed by -day mortality rates of %, %, %, and %, respectively ( fig. ) . absolute white blood cell counts varied among the seven experimental groups (p Ͻ . ), in particular between control and sick animals (fig. ) . there was no difference among animals with sterile or infectious causes of systemic inflammation (lps, clp, mild clp, or clp ϩ antibiotics). likewise, there was no difference in cell differentials among controls or among animals with sterile or infectious causes of systemic inflammation, except for lps versus clp ϩ antibiotics (neutrophils and lymphocytes, p Ͻ . ). mouse differential gene expression dna chip analyzer hybridization signal analysis flagged of the mouse blood genechips as statistical outliers: one saline and one sham genechip from the training data set. these mouse outliers genechips were omitted from additional analysis. thirty-three gene-chips remained in the mouse blood data set. pca was used to visualize treatment differences in expression for all genes in blood and spleen, demonstrating batch and any-clp treatment effects (p Ͻ . , fig. ). we applied a stringent multiple test correction (bonferroni, . ) to the p values from the two-way anova to identify a small set of genes in blood differentially expressed between the seven treatment groups ( probe sets corresponding to genes, as probe sets for lipocalin appeared twice [ table ]). pca analysis of these probe sets revealed that the seven experimental groups were clustered into three apparent phenotypes (fig. ) : control animals, lps-treated animals (sterile source of systemic inflammation), and those that had any clp treatment (sepsis). comparisons of gene expression across groups generated several informational gene lists (fdr ϭ . ); each indicated apparent increases and decreases in gene expression induced by clp or lps (fig. ). using mouse spleen samples for gene expression analysis, we were able to classify the samples as clp or sham with . % accuracy, estimated using cross-validation. for the blood data, the experimental design dictated that the first four replicates (batches) were used to train the classifiers. the best classifiers were trained on all training samples (batches to ) and used to predict the seven de-identified test samples (batch ). all seven test samples were predicted correctly as any-clp (septic) versus non-clp. we also performed fivefold leave-onebatch-out cross-validation, which produced an overall accuracy estimate of . % (fig. ) . the prediction accuracy differentiating lps from controls was . %. the ability to predict low versus high mortality after clp was substantially lower ( . %). to obtain the final mouse blood classifier, leave-one-batch-out crossvalidation was performed for the purpose of classifier selection. sixty-four classifiers tied for best prediction. the median number of genes for these classifiers was . of these genes, only genes showed changes greater than twofold, and the majority ( . %) were altered by twofold or less. there were nine genes that demonstrated increased rna abundance across spleen and blood and across clp and lps (fig. ) . we call this cluster of genes the "common inflammatory response cluster" (table ). pathway analysis tools also were used to put this list into a biologic (functional) context. all but one of these genes is annotated and has been associated previously with a gene product, small molecule, or cellular process linked to inflammation (fig. ) . a single expressed sequence tag (est) completed the list of nine genes (genechip identifier, _at). a blast search identified this sequence as retroviral. a search of the ncbi gene expression omnibus microarray database showed that rna for this affymetrix probe set is increased in a number of models of inflammation, both animal and human. the ability to diagnose sepsis more accurately would allow appropriate treatment to be instituted earlier, thereby improving the likelihood of survival. , we hypothesized that expression profiles could distinguish between septic and nonseptic states in vivo, and that expression profiles could define lists of common responder genes using a systematic, unbiased approach. , recent , saline, sham, and normal) is apparent, but less notable than the batch effect (appearing on pc and pc , explaining % ϩ % of variance, respectively). no pcs besides pc and pc showed a substantial difference between any-clp and non-clp groups (determined by t-test on each pc). note that the genechip scanner used for batches to was different than that used for batch (latest generation), which is one explanation why batch is most different from the other four batches. reports using microarray technology in vitro indicated that inflammatory and infectious insults produce distinct transcriptional signatures. , the current study is the first examining the ability of microarray gene expression profiles to distinguish sterile from infectious causes of systemic inflammation in vivo. we examined gene expression profiles of splenic tissue from patients with injury versus those with injury complicated by sepsis and organ dysfunction. differences in apparent gene expression between the sepsis and injury (control) phenotypes were used to construct a classifier, the accuracy estimate of which was . %. this small, exploratory clinical study provided "bedside" proof of feasibility using human transcriptional profiles to model the septic phenotype, but also demonstrated a large degree of variance in gene expression between subjects, because of both technical and biologic differences. to control more of this variance we moved from the bedside to the bench, performing a systematic examination of the diagnostic potential of spleen and blood gene expression profiling in inbred mice. consistent with the human data, spleen samples from mice after clp exhibited microarray patterns that could be modeled to predict the septic phenotype. the nested cross-validation accuracy estimate of . % for sepsis prediction using mouse spleen was considerably better than that found using human spleen, likely because of the mouse experimental design that exploited fresh tissues and identical age, gender, and genotype across subjects. because the clinical use of gene expression analysis using splenic tissue is severely limited, we explored next the use of circulating blood for class prediction in our mouse models. the combined accuracy of the predictions for any-clp versus non-clp and lps versus controls was high at . % and . %, respectively. the accuracy estimate for distinguishing between the high and low mortality clp groups (clp versus mild clp and clp plus antibiotics) at this -hour time point was much less at . %. these conclusions are consistent with the pca analysis in figure , in which we sought differences in apparent table . abx, antibiotics. gene expression across the seven experimental groups. interestingly, the only samples that were cluster outliers were in the clp plus antibiotics group, consistent with an effect of antibiotics to change the septic phenotype toward the control phenotypes. we conclude that circulating blood gene expression profiles can be used to predict clp and non-clp phenotypes in prospective cohorts, in particular, distinguishing controls from lethal endotoxicosis (lps) from lethal infection (clp). it is important to note that at this -hour time point, there were no substantial differences between lps or any of the clp groups in clinical presentation or complete blood counts. microarray analysis could make the diagnosis of sepsis (distinguishing between sterile and infectious sources of systemic inflammation) when clinical criteria and white blood cell counts could not, a frequent occurrence in icus. in addition, the differences between groups as measured by absolute fold-changes in individual gene expression were small (eg, in the final model for blood, . % of genes were altered less than twofold), yet the changes in the patterns of expression across hundreds of genes were robust predictors of phenotype. to discover changes in expression generic to the inflammatory and septic responses, we used the intersection of gene lists identified by the pair-wise group comparisons (fig. ) . nine genes were commonly increased, validated across two tissue types (spleen and blood) and two insults (clp and lps). in contrast, there were no genes that were commonly decreased. given that this list of nine genes was based on changes in relative rna abundance across a number of cell types, the network analysis performed served as an exploratory tool, validating in silico the role of six of nine genes in canonical pathways for inflammation, apoptosis, and signal transduction: inhibitor of dna binding , calgranulin a and b, interferon regulatory factor , lipocalin , and formyl peptide receptor-like ( table ) . several characteristics of these nine common inflammatory response genes are notable. inhibitor of dna binding is required for normal mouse immune development, especially of lymph nodes and peyer's patches. calgranulin a and b, which belong to a recently described group of proinflammatory molecules, form extracellular complexes that bind to and activate endothelial cells, promoting chemotaxis and phagocytic adhesion in a positive feedback manner. , interferon regulatory factor is a key regulatory of monocyte development, essential to differentiation of monocytes to macrophages. lipocalin is a secreted protein that undergoes transcriptional induction after cytokine withdrawal and induces leukocyte-specific apoptosis. lipocalin transcription, translation, and secretion are induced by ligation of toll-like receptors on leukocytes, with secreted lipocalin acting to sequester siderophores, thereby limiting bacterial growth. formyl peptide receptor-like is a member of the chemoattractant subfamily of g protein- coupled receptors that are involved in controlling leukocyte migration. the other two annotated genes not listed in the network, neutrophilic granule protein and serum amyloid a , have also been associated with inflammation and cellular defense, although less is known about their functions and protein interactions. neutrophilic granule protein is a cysteine protease inhibitor that has been associated with myeloid differentiation. serum amyloid a is a highdensity apolipoprotein, the only amyloid made by both hepatocytes and peripheral monocytes and macrophages. it is believed to function by retargeting transported lipids, including cholesterol, in the disposal of toxins. the function of the ninth gene, a retroviral species that is increased in a number of different models of infection and inflammation, is not known. we compared this common inflammatory response cluster with the list of proteins recently reported to diagnose intra-amniotic infection in patients, as another means of validating the importance of these nine genes. of the proteins and polypeptides detected in that study, were also identified in our study, specifically calgranulin a and b and lipocalin (neutrophil gelatinase-associated lipocalin). calgranulin a (s a ) was also the most differentially expressed gene in blood in a small microarray study comparing eight septic patients with four surgical controls with-out systemic inflammation. serum amyloid a protein was identified as a plasma proteome biomarker in patients with coronavirus (severe ards). together, these studies support our hypothesis that there are a group of common inflammatory response genes that can be used as novel biomarkers to diagnose inflammation across species, tissue, and different types of infecting organisms, at either the rna or protein level. because of the large degree of variation (noise) in human spleen gene expression, no individual genes surveyed passed the fdr filter. nevertheless, the data from these patients have proved invaluable for the study of immune dysfunction in human sepsis and provided proof-ofprinciple here that molecular profiles of human lymphoid tissues could be used to distinguish between septic and nonseptic phenotypes. because cellular populations of mammalian tissues are heterogeneous, use of microarray profiles to study the cellular response to a given stimulus must be understood in the context of changes in cell populations. this substantially limits conclusions about whole spleen data, given our reports and those of others that sepsis accelerates splenocyte apoptosis. , , in contrast, changes in blood cellular heterogeneity are measured routinely. although clp and lps stimuli changed both absolute wbcs and cell differentials compared with the controls, among the four groups of "sick" animals cell counts were indistinguishable (fig. ) . this mirrors the clinical situation where differentiating between sterile (lps) and infectious (any-clp) sources of systemic inflammation is not possible based on clinical grounds or cell counts. microarray profiles, as we have discussed, were very successful at making this distinction. many questions remain unanswered. what are the optimal computational methods to identify robust predictors from microarray or proteomic data? can gene or protein expression profiles be used to diagnose sepsis in other animal models? if so, are there predictive gene sets that are common to different types of infection (eg, gram-positive versus gram-negative infections)? once the diagnosis has been made, are there markers that indicate response to therapy or prognosis? there are sufficient preclinical and preliminary patient data to justify testing these hypotheses. because of the heterogeneity of expression profiles, large-scale collaborative studies will be required to enroll sufficient patients to identify robust sepsis markers, and in the process, untangle the biology of infection from inflammation. back to the bedside-the promise of molecular profiling for sepsis diagnosis in conclusion, our in vivo data corroborate in vitro findings indicating that microarray analysis holds promise as a means of identifying distinct expression profiles ("molecular fingerprints") that can diagnose the septic phenotype. our human spleen data join recent blood data from septic patients and serum and amniotic fluid data from patients with intra-amniotic fluid infection, indicating that transcriptome and proteome studies will deliver on the promise of novel inflammation diagnostics. , a single inflammation gene, calgranulin a (s a ), was detected in all three studies at either the rna or protein level. our data are unique in that they show that transcriptome molecular profiles can distinguish between sterile and infectious causes of systemic inflammation and can make a diagnosis of sepsis in pro- spective cohorts. importantly, we observed that the magnitude of change in gene expression that was needed to predict the septic phenotype was very small. it was the pattern of these small changes in expression that were predictive, not the magnitude of any single change. we and others reported recently validated clinical protocols for blood gene expression profiling used to characterize the human systemic inflammatory response. , the data presented here suggest that these protocols should be extended to clinical trials, testing the efficacy of microarray gene expression profiling to diagnose human sepsis. we expect that these studies will provide new insight into how specific pathogens uniquely perturb the physiology of cir-culating leukocytes and how the host successfully mounts pathogen-specific defenses. . mouse common inflammatory response cluster. nine probe sets (red genes) were commonly altered regardless of tissue or insult. a contemporary pathway analysis tool was used to automatically create a network of interactions among these nine genes, based on previously reported interactions in the literature. eight of the nine probe sets are known inflammation genes; the ninth probe set is an expressed sequence tag. this network validates in silico that six of these genes are involved in canonical pathways of inflammation, apoptosis, and regulation of signal transduction. the epidemiology of sepsis in the united states from through the pathophysiology and treatment of sepsis sccm/esicm/ accp/ats/sis international sepsis definitions conference the plasticity of dendritic cell responses to pathogens and their components human macrophage activation programs induced by bacterial pathogens measures, markers, and mediators: toward a staging system for clinical sepsis. a report of the fifth toronto sepsis roundtable molecular classification of cancer: class discovery and class prediction by gene expression monitoring molecular portraits of human breast tumours gene expression profiles in hereditary breast cancer broad patterns of gene expression revealed by clustering analysis of tumor and normal colon tissues probed by oligonucleotide arrays molecular signatures of sepsis: multiorgan gene expression profiles of systemic inflammation sepsis gene expression profiling: murine splenic compared to hepatic responses determined using cdna microarrays application of genome-wide expression analysis to human health and disease a network-based analysis of systemic inflammation in humans functional genomics of critical illness and injury injury research in the genomic era expression profiling: toward an application in sepsis diagnostics diagnosis of intraamniotic infection by proteomic profiling and identification of novel biomarkers apoptotic cell death in patients with sepsis, shock, and multiple organ dysfunction effect of gender and sex hormones on immune responses following shock evaluation of factors affecting mortality rate after sepsis in a murine cecal ligation and puncture model inducible nitric oxide synthase (inos) gene deficiency increases the mortality of sepsis in mice overexpression of bcl- in transgenic mice decreases apoptosis and improves survival in sepsis sequence makes a difference: paradoxical effects of stress in vivo physiological genomics model-based analysis of oligonucleotide arrays: expression index computation and outlier detection primary and secondary transcriptional effects in the developing human down syndrome brain and heart multiple organ dysfunction score: a reliable descriptor of a complex clinical outcome controlling the false discovery rate: a practical and powerful approach to multiple testing simultaneous statistical inference treating sepsis reducing mortality in sepsis: new directions determination of infection probability versus the diagnosis and treatment of antibiotic-responsive diseases development of peripheral lymphoid organs and natural killer cells depends on the helix-loop-helix inhibitor id s a : emerging functions and regulation phagocyte-specific s proteins: a novel group of proinflammatory molecules monocyte differentiation to macrophage requires interferon regulatory factor induction of apoptosis by a secreted lipocalin that is transcriptionally regulated by il- deprivation lipocalin mediates an innate immune response to bacterial infection by sequestrating iron agonist-induced trafficking of the low-affinity formyl peptide receptor fprl identification of a series of differentiation-associated gene sequences from gm-csf stimulated bone marrow murine serum amyloid a is a high density apolipoprotein and is secreted by macrophages plasma proteome of severe acute respiratory syndrome analyzed by two-dimensional gel electrophoresis and mass spectrometry apoptosis in lymphoid and parenchymal cell during sepsis: findings in normal and t & b cell deficient mice we thank ms alice tong, ms sandra k macmillan, and ms tracey h wagner for expert technical assistance. critical revision: chung, downey, buchman, stormo, hotchkiss, cobb tests for differential expression, class prediction, and pathway analysis: for the mouse blood study, a mixed-model anova was used to detect differential expression between treatment groups, with a linear contrast between the any-clp and non-clp groups. the anova model was chosen to partition treatment group and technical batch variability from variability due to biological and experimental noise. the following linear mixed model was used to detect differential expression on a gene-bygene basis in the mouse blood data:where y gij is the expression of the gth gene for ith treatment and jth batch. the mean expression for the gth gene is given by g . the symbols t and b represent effects due to treatment and batch respectively. the error for the gth gene for sample ij is designated as gij . for the mixed-model analysis of variance, treatment is a fixed effect and batch is a random effect. a batch constitutes samples (one from each treatment group) which were processed and hybridized at the same time. in the case of the last batch (batch ), the genechips were scanned on a different scanner. for the mouse and human spleen studies samples were processed in a single batch, so a simple one-way analysis of variance with a contrast between any-clp and non-clp was used to identify differentially expressed genes. the linear contrast between any-clp and non-clp is given by:where clp is the mean of the clp group, clpϩabx is the mean of the clpϩabx group, etc.where possible, the following competing classifiers were considered for all tasks, and the optimal classifier was selected: number of genes ( , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , and ), prior probabilities for nearest centroid (equal and proportional), functions for discriminant analysis (liner and quadratic), prior probabilities for discriminant analysis (equal and proportional), number of neighbors (k) for k-nn ( , , and ), and distance functions for k-nn (euclidean distance, pearson's linear correlation, and absolute value distance). thus as many as classification models were considered for each classification task.for the mouse blood data, we used a leave-one-batchout ( -fold, one for each of the batches) outer crossvalidation, while the inner cross-validation is leave-onesample-out. we refer to this as nested cross-validation with an outer "leave-one-batch-out" layer and an inner "leave-one-sample-out" layer. ( ) using this method, the determination of how many and which genes to use for classification were determined using only the training samples. in addition, all additional classifier parameters (e.g., number of neighbors and distance measure) were determined using only the training samples. for each held-out batch in the outer -fold cross-validation, the classifier and genes that performed best on inner cross-validation were selected and applied to the or held-out test samples (two batches only had samples due to removal of an outlier sample). for the mouse and human spleen data where all samples were processed in a single batch, both the outer and inner cross-validation used full leave-one-sample-out. .e key: cord- -xjq j gv authors: gamazo, carlos; martín-arbella, nekane; brotons, ana; camacho, ana i.; irache, j.m. title: mimicking microbial strategies for the design of mucus-permeating nanoparticles for oral immunization date: - - journal: eur j pharm biopharm doi: . /j.ejpb. . . sha: doc_id: cord_uid: xjq j gv dealing with mucosal delivery systems means dealing with mucus. the name mucosa comes from mucus, a dense fluid enriched in glycoproteins, such as mucin, which main function is to protect the delicate mucosal epithelium. mucus provides a barrier against physiological chemical and physical aggressors (i.e., host secreted digestive products such as bile acids and enzymes, food particles) but also against the potentially noxious microbiota and their products. intestinal mucosa covers m( ) in the human host, and, as a consequence, is the major portal of entry of the majority of known pathogens. but, in turn, some microorganisms have evolved many different approaches to circumvent this barrier, a direct consequence of natural co-evolution. the understanding of these mechanisms (known as virulence factors) used to interact and/or disrupt mucosal barriers should instruct us to a rational design of nanoparticulate delivery systems intended for oral vaccination and immunotherapy. this review deals with this mimetic approach to obtain nanocarriers capable to reach the epithelial cells after oral delivery and, in parallel, induce strong and long-lasting immune and protective responses. the oral administration of bioactive products (i.e., drugs, antigens or immunomodulators) is an attractive and desirable option under diverse points of view: economic, safety (needle-free), easiness and efficiency, particularly for vaccine delivery, taking into account that oral vaccination can induce a systemic, including mucosal, immune response [ ] . however, this practice has to face with a hard and very well organized frontier, the mucosa: a mucus secreting epithelium that lines the internal parts of the body. the intestinal mucosa is made up of epithelium, lamina propria, and muscularis mucosae. the epithelium is constituted by cells that are held together by tight junctions, which effectively form a seal against the external environment. in addition, there are two extra levels of protection against the outer milieu, the secreted mucus layer and the apical glycocalyx (fig. ) . globally considered these layers constitute the mucus that covers the tips of microvilli on the apical surfaces of intestinal enterocytes [ ] . mucus provides a barrier against physical and chemical aggressors, such as food res-idues, host secreted digestive products (e.g. bile acids and enzymes), but also against the potentially noxious microbiota and their products. not surprisingly, pathogens have evolved many ways of evading the mucosal barrier. in fact, mucosae cover m in the human host, and as a consequence is the major portal of entry of the majority of known pathogens [ , ] . this review will deal with the generation of nanocarriers, based on microorganism-mimicking approaches, for the oral delivery of either antigens or allergens for vaccination and immunotherapy purposes. mucus is a complex viscous secretion basically formed by water (approx. %), salts, lipids and various kinds of macromolecules including the so-called mucins [ , ] . mucins, secreted by goblet cells, are densely glycosylated proteins in which the protein backbone (apomucin) is linked to a number of carbohydrate chains ( - % by weight) [ , ] . in addition the carbohydrate structures themselves can be either linear or branched, and can be acidic (containing sialic acid or sulphate groups) or neutral in nature [ , ] . the degree and type of glycosylation differs depending on the type of mucin and its localization throughout the gut [ ] . these glycoproteins can be found as oligomers or non-oligomers, and are http://dx.doi.org/ . /j.ejpb. . . - /Ó elsevier b.v. all rights reserved. initially classified into three subfamilies: soluble ( - nm long), membrane-bound ( - nm), and, gel-forming mucins (up to several micrometres). gel-forming mucins are the major constituent of mucus and responsible for its viscoelastic properties [ ] . from a functional point of view, mucus appears as a dense fluid matrix that requires to be ineludibly porous, as a gel, since it needs to allow the diffusion of molecules to both orientations, into the cells (absorption of nutrients) and from the cells (secretion). however, at the same time, it needs to provide an effective physical barrier to foreign particulate matter, including microorganisms. to achieve successfully both functions, mucus is disposed in an arrangement that comprise two different layers: the external, which is named mucus layer, and the internal one or glycocalyx, that corresponds with the glycoproteins attached to the epithelial cell surface [ ] . the mucus layer constitutes then the first line of defence against epithelia damage by physical, chemical or biological aggression. it is thick ( - lm in the small intestine, lm in the large intestine) and constantly renewed by the host (approx. l/day) [ , ] . topographically comprise two layers: (i) the outer layer ( - lm diameter), which is loosely attached with large functional pores that allow the residence of normal microbiota, and (ii) the inner layer attached to the subjacent glycocalyx and, therefore, densely packed, with a very small functional pore that impede microbial and particle penetration. the predicted model for the physical mucin pore at this level is around nm, although native mucin fibres may aggregate under certain circumstances to create larger pores which allow larger particles to transit [ ] . the glycocalyx consists in long filaments of diverse glycoproteins and glycolipids well attached to the cell surface of enterocytes as a thin but very robust and compact layer ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) lm thick in the small intestine and around lm in the large intestine). in fact, this layer would be able to detain any macromolecule above nm [ ] . the glycocalyx is renewed every - h, being then release to the lumen, where is trapped and concentrated at the mucus layers. in addition, epithelial cells actively secrete mucins to block microorganisms in the lumen, before reaching the epithelial cells. fig. shows a schematic representation of the intestinal mucosa. summing up, the structure of a mucin fibre contains hydrophobic domains alternating with hydrophilic glycosidic regions that allow interactions with empathic areas on adjacent mucins or even on other molecules. consequently, mucin fibres are flexible and sticky. the energy invested by mucosal tissues in the production of mucins, and the finely tuned modulation in response to chemical physical or biological challenges, such as infections, reflects the importance of these glycoproteins. in fact, changes in mucin glycosylation are considered as mechanisms of the innate immune response to mucosal infections [ ] . in any case, mucus layers are not insurmountable for the microbial world. motility and degradative enzymes are main strategies used by many microbial pathogens to penetrate the mucus layers that we will considered in the following section. the harsh conditions of the gastrointestinal tract as well as the presence of intense peristaltic wave forces compromise the viability and survival of microorganisms within the gut. many of them have developed different tricks to interact and even penetrate through the mucus layer in order to adhere to and colonize the host mucosa, including the disruption of the balance between mucus erosion and mucus production/secretion or the degradation of mucins by specific proteinases and glycosidases [ ] . in view of that, microorganisms have evolved one or several of the following strategies ( fig. ) : (i) interaction with mucin, (ii) alteration of mucin synthesis or mucin assembly into a gel, (iii) degradation of the mucus layer and, (iv) evasion of mucin (alternatives pathways). within mucus, mucins form complex networks that act as a trap for foreign particulates and compounds. the main mechanisms by which mucins interact with foreign bodies are the following: size exclusion, unspecific polyvalent hydrophilic and hydrophobic interactions, and specific bonds. the mucus gel network is a heterogeneous structure with a wide range of pore sizes, which limit particulates above nm to travel through [ , , ] . some great examples of how to avoid size exclusion conferred by the mucus mesh are obtained from enteric virus. in fact, virions that use intestine mucosa to colonize the host have regular sizes below nm (i.e., rotavirus, coronavirus, influenza, and norwalk). this fact suggests that the mucus barrier has positively selected virus with sizes that better penetrate the mesh to reach efficiently the subjacent target cells. however, some particulates of up to nm are also able to diffuse through mucus [ ] . these circumstances are related with the physicochemical properties of mucin, such as the molecular charge, density of anionic and cationic groups, and the numerous hydrophobic domains distributed over the surface of the fibres [ , ] . as a result, adhesive interactions of particles with mucus can be achieved by electrostatic and/or hydrophobic interactions as follows. mucin filaments are covered with glycosylated residues that, in some extent, show an acidic character due to the presence of sialic and sulphate groups (fig. a) . thus, negatively charged mucin can bind with high avidity to positively charged particulates. to counteract this anionic character, a number of enteric virions display an external neutral net charge and, therefore, they are neither repelled nor wrapped into the mucus [ ] . larger organisms, like bacteria, need to use different strategies. for example, helicobacter pylori secretes a glycosulphatase that release sulphate groups from mucin to avoid an electrostatic adsorption and freely migrate through the mucus [ , ] . mucin layers also contain hydrophobic domains along the fibre structure [ ] . thus, and continuing with the virus examples, nature favours enteric naked virus, that means, non-enveloped particles. in contrast, blocking hydrophobic bonds will be established between mucin hydrophobic domains and enveloped viral particles [ ] (fig. b ). in fact, most microbial cells suffer from this limitation being immobilized by mucus via hydrophobic interactions. the complex chemical composition of mucin facilitates the specific linkages via conformational interactions (fig. c ). in fact, the selective pressure to handle pathogens may have modulated the ample variety of mucin glycosylation patterns [ ] . some examples are found in bacteria and protozoa for which the adherence to mucin could be a desirable strategic step to avoid natural peristaltic flowing. campylobacter jejuni is a motile bacterium that colonizes the intestine of vertebrates, being a main cause of human acute bacterial gastroenteritis. the evolutionary adaptation to mucosa is such that mucins are chemoattractants for campylobacter [ ] and the bacterium binds avidly to them through specific ligands, including lipopolysaccharide (lps) [ , ] . similarly, the intestinal pathogenic protozoa cryptosporidium parvum and giardia duodenalis express surface lectins to adhere to the intestinal mucins [ , ] . in a similar way, h. pylori attaches to mucin carbohydrates by the blood-group antigen-binding and the sialic acid binding adhesins at neutral ph. however, when the mucus gel is released into the acidic gastric milieu the interaction is weak, allowing the bacteria to detach and go further to the epithelium [ ] . a similar approach has been described for candida and salmonella species. once the specific contact between candida albicans and mucins has been established, the microorganism releases an aspartyl proteinase to degrade the surrounding glycoprotein and to move deeper into the mucus layer [ ] . on the other hand, salmonella typhimurium binds specifically to sialomucins. then, it expresses a sialidase to degrade the mucins, which liquefies the mucus, facilitating its penetration into the protective layer [ , ] . as stated above, a sign on the importance of mucin in the host defence is that its secretion is enhanced in response to intestinal microbes. in order to solve this drawback, some specialized intestinal pathogens are capable of expressing virulence factors to either alter mucin production (decreasing or increasing) or modify mucin assembly. outer exposed bacterial components, such as exotoxins, flagellin, lps or lipoteichoic acid, are known modulators of mucin production. for example, h. pylori is able to decrease mucin biosynthesis by lps and a cytosolic phospholipase a [ , ] , whereas clostridium difficile uses the so-called toxin a to obtain the same effect [ ] . some other pathogens cause mucin hypersecretion in order to produce mucus depletion. the well-known vibrio cholerae enterotoxin, which increases the intracellular levels of adenosine , cyclic monophosphate, activates mucus secretion mechanisms in intestinal goblet cells [ ] . listeria monocytogenes produces the exotoxin listeriolysin o to promote the synthesis and secretion of mucins with the same purpose [ ] . for the colonization of gastric and duodenum mucosae, h. pylori bacteria release urease that neutralizes the acid ph by generating ammonium from urea. this increase of the ph triggers the transition from mucin-gel to mucin-solution, allowing the bacteria to swim through the mucus [ ] . in addition, its outer membrane lps can also inhibit mucin glycosylation which may have deleterious effects on mucin assembly [ ] . an obvious direct mechanism to freely move through the mucus barrier is to degrade it. thus, some symbiotic intestinal bac-teria have mucolytic activity by glycosidases and proteases, with the purpose of getting monomers to be used as a source of energy [ ] . in turn, some intestinal pathogens use similar specific enzymes to open small breaches in the mucin network with the purpose of disassemble the oligomerized mucin. for example, some intestinal protozoa (e.g. tritrichomonas, giardia lamblia and entamoeba histolytica) may express several mucin-degrading enzymes [ ] . thus, e. histolytica secretes glycosidases [ ] and proteases that cleave mucin in the non-glycosylated oligomerization domains, breaking down the macromolecular structure and reducing mucus viscosity [ ] . bacteria also carry specific weapons against mucins. in this way, h. pylori releases a glycosulphatase to disrupt the oligomeric structure of mucin [ ] , whereas v. cholerae uses a taga protease for the same purpose [ ] . similarly, salmonella typhimurium possesses a sialidase [ , ] and pseudomonas aeruginosa and the enterotoxigenic escherichia coli strains (the most common causes of diarrhoea in children) secrete specific proteases [ , ] . finally, in the subcellular world, we can also find interesting examples such as the reovirus that also release mucolytic proteases to facilitate their penetration through the protective mucus barrier [ ] . some enteric pathogens are capable of reaching epithelial cells, travelling through the follicle-associated epithelium (fae). fae overlays the gut-associated lymphoid tissue (galt), in which peyer's patches (pp) and isolated lymphoid follicles are integrated, as a part of the mucosal-associated lymphoid tissue (malt) of the intestine. in order to mount an efficient immune response against luminal antigens, microfold (m) cells are strategically sited in the dome epithelium of pp. these m cells are specialized for ''antigen sampling'', presenting a reduced density of microvilli [ , ] . in addition, this dome epithelium lacks goblet cells, thus making a specialized sampling area where the mucus barrier is minimal. another related special feature of m cells is that they present a deep invagination at the basolateral side, forming an intraepithelial pocket containing immunocompetent cells. in spite of this, penetration of the gut mucosa by pathogens is believed to occur mainly through m cells [ ] . as a first step, previous to the invasion, pathogens interact with different pattern recognition receptors (prr) that recognize molecules that are broadly shared by pathogens but distinguishable from host molecules (pathogen-associated molecular patterns, pamps) [ , ] . these prr include toll-like receptors (tlr), nod-like receptors and c-type lectin receptors [ ] . the tlr family is particularly expressed by m cells and they detect, for example, lps from gram negative bacteria (tlr mediated), lipoteichoic acid from gram positive bacteria (tlr- ), or bacterial flagellin (tlr- ), among many others pamps [ , ] . enteric pathogens exploit those receptors for invading and colonizing the host. other receptors localized on m-cells that are used by microorganisms include specific glycoconjugates and the complement component a receptor (c ar). thus, reovirus specifically targets m cells through the interaction between r hemagglutinin with glycoconjugates terminated in sialic acid residues [ ] , whereas e. coli and s. typhimurium use the fimbriae adhesins fimh+ to specifically interact with the glycoprotein- also expressed on m cells [ ] . on the other hand, the outer membrane protein omph of yersinia enterocolitica recognizes the c ar [ ] and the shock protein hp- of brucella abortus interacts with a cellular prion protein also localized on m cells [ ] . in sum, the gut is covered by a mucosal absorptive epithelium that maintains homoeostasis by restricting the transit of macromolecules and foreign particles. however, most of infections occur along this area. in these circumstances, the obvious approach would be to use the whole natural or recombinant attenuated pathogens as antigen carriers for oral vaccination. however, there are many intrinsic factors that preclude its use: reversion to virulence, immunogenicity to the carrier that neutralizes booster immunizations, and the potential risks associated with the use of recombinant dna [ , ] . a possible safer solution for the oral delivery of antigens and allergens would be the use of microorganism-like nanocarriers. in the last years, efforts have been directed toward the enhancement of mucosal/oral vaccine delivery to the host using a variety of particulate delivery systems such as liposomes, immune-stimulating complexes (iscoms) or nanoparticles [ ] [ ] [ ] . from a general point of view, these nanocarriers offer some advantages that are of interest for the oral delivery of antigens and allergens for vaccination or immunotherapy purposes, respectively. thus, the encapsulation of these biomacromolecules in nanocarriers effectively protects them from the harsh conditions of the gastrointestinal tract, minimizing their degradation by hydrolysis or digestive enzymes [ , ] . from a biological point of view, some nanoparticles may also act as adjuvants because they may facilitate both the antigen uptake and internalization by the galt [ , ] and, further, the antigenic cross-presentation by antigen presenting cells (apcs) via both mhc class i and ii pathways [ ] . unfortunately, conventional nanocarriers interact with mucins and can remain immobilized in the mucus layer. under these circumstances, nanoparticles are cleared as fast as the mucus is removed, following advancing movements by peristaltic forces (fig. a) [ ] . thus, they display a low capability to target specific sites within the gastrointestinal tract (e.g. pp, mucosal dendritic cells [dcs]). as a consequence of this, the elicited immune response with these antigen carriers is usually not as high as necessary to offer the adequate degree of protection to the host, and consequently, high and multiple oral doses are required. in order to overcome these drawbacks and render nanoparticles more efficient as adjuvants for vaccination, one possible outcome can be their coating or ''decoration'' with compounds or molecules involved in the strategies developed by microorganisms to reach the fae (fig. ; section ) . for this purpose, ligands capable to target and interact with either epithelial glycoconjugates that are specifically activated by pathogens, such as the tlr family or the mannose receptors (mr) have been proposed. it is interesting to note that, in general, these pamps are also immunopotentiators and, thus, may improve the intensity and quality of the immune response induced by the antigen-loaded nanoparticles. in parallel, many of these ligands may confer mucus-permeating properties to the resulting nanocarriers by, at least, two different mechanisms. in the former, hydrophilic ligands yield particulates' surfaces less liable to the development of hydrophobic interactions with mucin fibres and other components of the mucus layer. in the later, some ligands (e.g. some types of bacterial lps or flagellin) may also inhibit the production of mucus glycoproteins (see section . ). in this biomimetic approach different ligands have been proposed including the use of flagellin [ , ] and lps or its derivatives [ ] . apart from their capabilities to modulate the production of mucins and their specificity for tlrs, these ligands have also an important effect as immunomodulators (adjuvants). in fact, these compounds alert the apcs to the presence of ''pathogenic'' material and, thus, facilitate the induction of the adequate immune response [ ] . the lipopolysaccharides are globally recognized as one of the main pamps [ , ] . these macromolecules are located on the outer membrane of gram negative bacteria and show the capability of activating apcs, through receptors on their membrane such as the tlr [ ] , potentiating th (cellular) responses [ ] . however, lps, which is also known as endotoxin, shows a potent biological activity with deleterious side effects. these effects are related to the presence of lipid a. nonetheless, natural lps from different bacteria may exhibit different biological properties, including their capability as tlr agonist or their effect as pyrogenic material [ , ] . on the other hand, nontoxic alternatives have been developed including the synthesis of modified products structurally related to lps but devoid of its toxicity. in this way, monophosphoryl lipid a (mpl, derivative of lps from salmonella enterica serovar. minnesota) was the first tlr ligand and biological adjuvant approved for human use for its safety and effectiveness [ , ] . moreover, it has been tested in numerous human trials against different infectious diseases (hepatitis b, malaria or herpes simplex virus) and allergen immunotherapy [ , ] . regarding its use to improve the immune response of antigens encapsulated in nanocarriers, mpl was incorporated into the external bilayers of liposomes containing the glucosyltransferase antigen from streptococcus mutans. when administered orally, the liposomes induced high levels of salivary, plasma, and vaginal iga, demonstrating the capability of the combination between nanocarriers and the lps derivative to induce strong mucosal immune responses [ ] . in a similar way, sarti and co-workers, using plga nanoparticles associated to mpl, demonstrated an important improvement of the immune response against ovalbumin (ova) only when the lps derivative was present [ ] . interestingly, these results were obtained with the administration of one single oral dose. in another study, plga-lipid nanocarriers functionalized in surface with mpl and a m-cell specific lectin stimulated effective mucosal and serum antibodies against the model antigen in mice [ ] . again, the presence of mpl appeared to be the key factor to elicit the immune response. if nanoparticles are capable of penetrating the mucus layer and reach the inner mucus layer (iml) the residence would be longer in time, since iml renewal is slower, thus facilitating greater doses of particle cargo to be released nearby the underlaying cells (b). some of these particles can even reach the glycocalyx, and the cellular membranes as some microorganisms do (c). (for the interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) nowadays, new generations of tlr agonists are being developed such as glucopyranosyl lipid adjuvant and aminoalkyl glucosaminide -phosphates [ , ] . however, to the best of our knowledge, researches about their use as an oral vaccine delivery system are limited. another approach to take advantage of the adjuvant potential of lps is the use of molecules with low toxicity. in this context, it has been proposed the use of the rough lps from brucella ovis, which shows a very low endotoxicity [ ] . this lps was used to decorate poly(anhydride) nanoparticles carrying ova as model allergen. orally administered to mice, lps-coated nanoparticles were capable to reach in a large extent the surface of the intestinal epithelium, including pp [ ] . more important, this capability to reach the epithelium was in line with the very high degree of protection offered by lps-nanoparticles (close to %) against an anaphylactic shock in ova-sensitized animals [ ] . in a similar approach, but using a lolium perenne protein extract, the coating of poly(anhydride) nanoparticles with lps from b. ovis shifted the immune response from a th (observed with naked nanoparticles) to a th profile in a sensitized murine model to this allergen [ ] . this cellular response induced with lps-coated nanoparticles was identified as the key aspect responsible for the efficacy of the nanoparticles. in fact, in the challenge experiment with sensitized mice, lps-nanocarriers decreased both the levels of mmcp- (mouse mast cell protease ) and the severity of the anaphylactic symptoms, increasing the survival rate of animals compared with the controls [ ] . flagellin is the monomeric protein that conforms the bacterial flagellum, which is a key virulence factor in some pathogens by providing motility and increasing adhesion [ ] . some examples of flagellated bacteria include h. pylori, vibrio, salmonella and pseudomonas species. flagellin has been extensively investigated as a pamp, since it binds tlr [ , ] . furthermore, flagellin induces the maturation of intestinal dcs, activates cd + t cells in vivo and promotes the development of mixed effector th cell responses [ , ] . as a mucosal adjuvant, flagellin is almost as potent as v. cholerae and e. coli heat-labile toxins but much safer than these two compounds [ ] . in order to evaluate the advantages offered by the combination between this pamp and nanocarriers as antigen oral delivery systems, flagellin from the flagella of salmonella enteritidis was used to functionalize poly(anhydride nanoparticles [ ] . when administered orally, these nanocarriers displayed an important capability to reach the surface of the epithelium, mainly in the ileum of laboratory animals. interestingly, the distribution profile of these nanoparticles within the gut correlated well with the described colonization profile for salmonella enteritidis [ , ] , including a broad concentration in pp. using ovalbumin as model antigen, these flagellin-coated nanoparticles elicited a strong and balanced secretion of both igg a (th ) and igg (th ) specific antibodies. furthermore, these nanoparticles were able to induce a much more strong mucosal iga response than naked nanoparticles [ ] . flagellin and other related compounds have been also used to decorate other type of nanocarriers including liposomes [ ] , virus like particles [ ] and polypropylene sulphide nanoparticles [ ] . more recently, flagellin-functionalized calcium phosphate nanoparticles induced a significantly higher immunostimulatory effect, mainly related with high levels of proinflammatory cytokines (il- , il- b and il- ) than controls [ ] . glycoconjugates enriched in mannose residues promote the interaction of a number of microorganisms (e.g. c. albicans, l. monocytogenes, leishmania donovani, hiv, enterobacteriaceae or bifido-bacterium) with different tissues and substrates, including lymphoid and non-lymphoid cells of different mucosal surfaces [ , ] . this binding is mediated by the high affinity between either mannose or glycoconjugates ending in mannose and the so-called mannose-binding lectins (or mr). in immune cells (i.e. dcs and macrophages), the mr mediate endocytosis, function as antigen capture receptors and are involved in antigen capture and presentation [ ] [ ] [ ] . in this context, mannosylated nanocarriers obtained by the decoration of particulates with mannose or its derivatives have been considered as promising non-live vectors for mucosal vaccination. thus, mannosylated niosomes loaded with tetanus toxoid (tt) were evaluated as oral vaccines against tetanus [ ] . the coating of these vesicles with a linear polymer of mannose (o-palmitoyl mannan) improved their stability in the presence of bile salts and digestive enzymes. furthermore, the functionalized nanocarriers were capable to target pp and to elicit important humoral and cellular responses as measured of the igg a/igg sera levels. similarly, the iga levels in mucosal secretions were also high against tt [ ] . in a similar work, the same mannosylated niosomes were evaluated as oral vaccine carrier of a plasmid designed for the expression of hepatitis b virus proteins. only animals immunized with these mannosylated niosomes offered adequate antibody levels to get seroprotection against hepatitis b virus infection [ ] . mannosylated liposomes have also been proposed for oral vaccination. thus, liposomes functionalized with a mannose derivative (mannose-peg-cholesterol conjugate) induced potent immune responses against a model antigen (bovine serum albumin, bsa) when orally administered. these immune responses were characterized by high levels of both sera igg and siga in different mucosal secretions [ ] . in a more recent study, mannosamine-coated polymeric nanoparticles were used to load a hot saline extract from b. ovis (hs). the vaccination of mice with a single oral dose of these nanocarriers offered an important protection against an experimental infection with the bacteria. in fact, the degree of protection (measured as reduction of b. ovis cfu in the spleen) obtained with mannosylated nanoparticles was about -times higher than for naked nanoparticles and -times higher than for the control [ ] . however, when the animals were conjunctivally vaccinated with mannosylated nanoparticles the degree of protection against the challenge was the highest, even than that observed for the commercial vaccine intramuscularly administered. this degree of protection was related with the fact that mannosylated nanoparticles, after their instillation in the eyes, were distributed (via the nasolacrimal duct) to both the nose and the gastrointestinal tract. in fact, h after instillation, nanoparticles were visualized in the cornea, nose and intestinal mucosa, including pp [ ] . it is important to highlight that in all of these areas, nanoparticles can encounter apcs and, thus, induce and potentiate the immune response. glucomannan (a water soluble polysaccharide comprised of glucose and mannose) has also been proposed to decorate different nanocarriers including bilosomes [ ] and chitosan nanoparticles [ ] . in both cases, using tt, it was demonstrated that these functionalized nanocarriers elicited significantly higher systemic and mucosal immune responses than controls. in addition, these ttloaded in glucomannan nanocarriers also induced a cell mediated immune response (il- and interpheron-gamma), which was not induced by the conventional vaccine based on alum intramuscularly injected. the intestinal epithelial cells possess a cell surface glycocalyx composed of membrane anchored glycoconjugates. it may, therefore, be possible to exploit these surface exposed carbohydrate res-idues as targets for lectin-mediated delivery to specific regions and cell-types within the gastrointestinal tract. several studies have revealed that, in many species and at many malt sites, the m cell surface glycocalyx differs in carbohydrate composition from that of enterocytes [ , ] . one of the first attempt to evaluate the capability of lectins to specifically target m-cells was the coating of liposomes [ ] and nanoparticles [ ] with ulex europaeus i agglutinin (uea ), a lectin specific for a-l-fucose residues [ ] . using model antigens (e.g. ova or bsa), uea coated particulates were capable to reach and target m cells in pp [ ] and induced systemic humoral responses significantly higher than those elicited with non-targeted antigen [ , ] . more recently, malik and coworkers have demonstrated that the coating of bsa-loaded chitosan nanoparticles with uea conjugated alginate produced nanocarriers capable to induce superior systemic responses in laboratory animals along with a mucosal immunity significantly higher than that induced by a conventional aluminium-based vaccine [ ] . all of these immunity effects would be consequence of a rapid endocytosis process of these nanocarriers after adhesion to m cells that would facilitate their capture by mucosal dcs and other immunocompetent cells in the subepithelial dome of the intestinal pp tissue [ , ] . other lectins that have demonstrated an important ability to both target and enhance pp uptake when associated to nanocarriers are the following: wheat germ agglutinin [ ] , peanut agglutinin [ ] , asparagus pea lectin [ ] and aleuria aurantia lectin [ ] . table summarizes some examples related with the functionalization of nanocarriers with lectins for oral vaccination purposes. the oral administration of antigens or allergens, for vaccination or immunotherapy, is very attractive for patient compliance (needle free systems), logistical (no cold-chain requirements) and it is supported by immunological foundations. in fact, gut mucosal surfaces are the major portal of entry for the majority of known pathogens and allergens, acting mucosae as the first line of the immune response (galt). nevertheless, the arrival of antigens or allergens to the galt has to face to a number of barriers. first, these compounds are highly sensitive to the harsh conditions of the gut and, in general, they are rapidly degraded by extreme ph conditions and/or digestive enzymes. second, the mucus layer constitutes a formidable hurdle that greatly hampers the encounter and interaction of these antigens and/or allergens with the antigen presenting cells. third, the antigens have to elicit a strong, longlasting and adequate (protective) immune response. in order to solve these barriers, different strategies have been proposed including the use of nanocarriers. nanocarriers (e.g. polymeric nanoparticles, liposomes, and iscoms) are a good option to protect the cargo against its early degradation within hostile environmental conditions (e.g. acidic ph, enzymes). however, when these nanocarriers are orally administered, they interact with the mucin fibres and, then, an important fraction of the given dose remains trapped in the protective mucus layer. as a consequence, these nanocarriers are rapidly eliminated by the physiological mucus turn-over and the gut peristaltism. one possible solution to minimize this problem would be the ''decoration'' of nanocarriers with ligands capable of mimicking the ability of some microorganisms to cross the protective mucus layer and reach the epithelium. basically, bacteria and virus use two types of mucus-permeating strategies. the first set includes particular physico-chemical properties to minimize the interaction with components of the mucus layer (e.g. size, surface charge and a hydrophilic character). the second set encompasses biological solutions such as the release of proteolytic/glycosidic enzymes, the use of propeller systems, and the presence of compounds capable of specifically interact with receptors of the host. however, it is interesting to note that, in general, microorganisms do not use a simple and unique strategy but a combination of them to cross the mucus layer. from our point of view, the association of nanocarriers with compounds with a particular specificity for certain receptors localized at the galt such as tlr, mr or particular glycoconjugates, can be a good option to induce the adequate immune response. for this purpose, the ligands (e.g. glycoconjugates, flagellin, lps, lectins) should be covalently bound to the surface of nanocarriers loaded with the biologically active molecules. important advantages can be obtained from this combination. first, the hydrophilic nature and neutral character of these ligands attached on the surface of nanocarriers would decrease both the electrostatic and hydrophobic interactions with mucins, increasing the possibilities of these nanocarriers of reaching the epithelial surface to deliver their cargo. in case of flagellin and lps, and due to their capability to decrease the synthesis of mucins, it can be hypothesized that they could also decrease the viscosity of the mucus layer, favouring the arrival of the nanocarriers to the epithelium. second, the capability of these ligands to specifically interact with prr on the cell surface (e.g. tlr, mr, glycoconjugates) would improve the possibilities of the resulting nanocarriers to reach the galt, including m cells in pp. thus, these targeting properties would be in line with the colonization pattern observed for microorganisms in their colonization process of the host gut mucosa, facilitating the antigen presentation and the activation of the immune system. last but not least, the immunoadjuvant properties of these ligands would boost the protective immune response. another interesting alternative to this biomimetic approach would be the incorporation of proteases or glycosidases (specific to mucins) to these pamps-coated devices. this combination should increase the fraction of such nanocarriers capable of reaching the surface of the epithelium and, thus, the efficacy of the antigen/allergen delivery system. however the binding of a second compound to the surface of nanocarriers may negatively affect to their targeting properties and, indeed, their efficacy. further table examples of oral immunizations using lectin-functionalized nanocarriers. both lectin-coupled microspheres displayed an affinity for m-cells and showed preferential binding to pp [ ] ulex europaeus agglutinin bsa liposomes uea -functionalized liposomes induced simultaneously both systemic and mucosal immune responses in mice [ ] aleuria aurantia lectin birch pollen proteins plga microspheres only allergic mice treated with lectin-functionalized microparticles induced important levels of igg a and il- and ifn-c [ , ] research is necessary in order to implement adequate methodologies to ''decorate'' nanocarriers with different compounds without loss of their efficacy as mucosal delivery systems as well as to select the most adequate ligand to 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potential oral vaccine carriers ulex europaeus lectin targets microspheres to mouse peyer's patch m-cells in vivo transgene vaccination using ulex europaeus agglutinin i (uea- ) for targeted mucosal immunization against hiv- envelope targeting polymerised liposome vaccine carriers to intestinal m cells toward targeted oral vaccine delivery systems: selection of lectin mimetics from combinatorial libraries application of ulex europaeus agglutinin i-modified liposomes for oral vaccine: ex vivo bioadhesion and in vivo immunity microfoldcell targeted surface engineered polymeric nanoparticles for oral immunization cholera toxin induces migration of dendritic cells from the subepithelial dome region to t-and b-cell areas of peyer's patches lectin anchored stabilized biodegradable nanoparticles for oral immunization - . development and in vitro evaluation m cell targeting with ateuria aurantia lectin as a novel approach for oral allergen immunotherapy lectin anchored plga nanoparticles for oral mucosal immunization against hepatitis b m-cell targeted biodegradable plga nanoparticles for oral immunization against hepatitis b in vitro and ex vivo characterization of lectin-labeled mycobacterium tuberculosis antigen-containing microspheres for enhanced oral delivery preparation and investigation of ulex europaeus agglutinin i-conjugated liposomes as potential oral vaccine carriers mucosal targeting of allergen-loaded microspheres by aleuria aurantia lectin the research leading to these results has received funding from the european community's seventh framework programme [fp / [fp / - for alexander under grant agreement n°nmp- - . - - , and project pi / from the ''plan nacional de investigación científica, desarrollo e innovación tecnológica - '' co-financed by ''isciii-subdirección general de evaluación y fomento de la investigación'' and the european regional development fund (erdf). key: cord- -kagggou authors: liu, chang; yin, zhigang; feng, tingting; zhang, min; zhou, zhi; zhou, ying title: an integrated network pharmacology and rna-seq approach for exploring the preventive effect of lonicerae japonicae flos on lps-induced acute lung injury date: - - journal: j ethnopharmacol doi: . /j.jep. . sha: doc_id: cord_uid: kagggou ethnopharmacological relevance: lonicerae japonicae flos (ljf, the dried flower bud or newly bloomed flower of lonicera japonica thunb.), a typical herbal medicine, targets the lung, heart and stomach meridian with the function of clearing heat and detoxication. it ameliorated inflammatory responses and protected against acute lung inflammation in animal models. acute lung injury (ali) is a kind of inflammatory disease in which alveolar cells are damaged. however, a network pharmacology study to thoroughly investigate the mechanisms preventing ali has not been performed. aim of the study: in this study, we examined the main active ingredients in ljf and the protective effects of ljf on lps-induced ali in rats. materials and methods: first, the main active ingredients of ljf were screened in the tcmsp database, and the ali-associated targets were collected from the genecards database. then, we used compound-target and target-pathway networks to uncover the preventive mechanisms of ljf. furthermore, we assessed the preventive effects of ljf in an lps-induced rat model with the rna-seq technique to validate the possible molecular mechanisms of the effects of ljf in the treatment of ali. results: the network pharmacology results identified main active compounds in ljf, and eight chemical components highly related to the potential targets, which were potential active compounds in ljf. in all, potential targets were recognized, including il , tnf, ptgs , app, f , and grm . the pathways revealed that the possible targets of ljf involved in the regulation of the il- signalling pathway. then, in vivo experiments indicated that ljf decreased the levels of proinflammatory cytokines (tnf-, il- , and il- ) in serum and bronchoalveolar lavage fluid, decreased the levels of oxidative stress factors (mda and mpo) and increased the activities of sod and gsh-px in lung tissue. the rna-seq results revealed that , and differentially expressed genes (degs) in ctrl (control group), ali-ljf (lonicerae japonicae flos group) and ali-dxsm (dexamethasone group), respectively. kegg pathway analysis showed that the degs associated with immune response and inflammation signalling pathways and the il- signalling pathway were significantly enriched in ljf. compared with those in ali, the expression of cxcl , cxcl , cxcl , nfkbia, ifng, il , il a, il f, il c, mmp and tnfaip , which are involved in the il- signalling pathway, were significantly decreased in the ljf group according to the qrt-pcr analyses. conclusions: in view of the network pharmacology and rna-seq results, the study identified the main active ingredient and potential targets of ljf involved in protecting against ali, which suggests directions for further research on ljf. f , and grm . the pathways revealed that the possible targets of ljf involved in the regulation of the il- signalling pathway. then, in vivo experiments indicated that ljf decreased the levels of proinflammatory cytokines (tnf-, il- , and il- ) in serum and bronchoalveolar lavage fluid, decreased the levels of oxidative stress factors (mda and mpo) and increased the activities obtain the aqueous extract, which was kept at - ℃ until use. prior to hplc analysis, the solution was filtered through a . μm membrane. the chromatographic fingerprinting of ljf showed main peaks, which were identified as (neochlorogenic acid), (chlorogenic acid), (chlorogenic acid), (secoxyloganin), (isochlorogenic acid b), (isochlorogenic acid a), and (isochlorogenic acid). the method could be used to identify the characteristics of ljf (fig. s ). for further analyses, the bronchoalveolar lavage fluid (balf), lung tissue and blood were collected and stored at − °c. after treatment, the levels of il- β, il- , and tnf-α in the serum and balf were determined with an enzyme-linked immunosorbent assay (elisa) kit, and an elisa kit was used to determine the levels of gsh-px, mpo, mda, and sod in the lung tissue. all data are presented as the mean ± sd. graphpad prism software was employed for one-way anova, and a p-value ≤ . was confirmed to be statistically significant. the rat lung tissues were fixed in % formaldehyde, embedded in paraffin and cut into μm sections. then, the sections underwent haematoxylin-eosin (h&e) staining. the changes in pulmonary histopathology were visualized under a microscope, and the pathological scores were obtained. the degree of pulmonary injury was evaluated according to a previous report (liu et al., ) . total rna was extracted with trizol reagent (tiangen, china) and detected on a % agarose gel. the purity, concentration, and integrity of the total rna samples were assessed prior to further analysis. after cluster generation, the library preparations were sequenced on the illumina hiseqtm platform by biomarker technologies (beijing, china), and the raw reads were generated. then, the raw reads were filtered by removing adapter and poly-n sequences and inferior quality reads from the raw reads. the clean reads were mapped to the rattus norvegicus (rnor_ . ) reference genome sequence by the hisat tools. the levels of quantitative gene expression were estimated by determining the fragments per kilobase of transcript per million fragments mapped. gene expression analysis of the different groups was performed by deseq . genes with a p-value < . were defined as differentially expressed genes (degs). then, we used the kobas . platform to perform the enrichment analysis of the degs (xie et al., ) . the volcano plot and heatmap were generated with the omicshare online tools the mrna expression of bcl a , cxcl , cxcl , cxcl , dnajc , fadd, kit, ifng, il r , il , il a, il f, il c, itga , mmp , nfkbia, nploc , ppp r a, thbs , tnfaip , tradd, and gapdh were detected by qrt-pcr in the lung tissues. the genes primer sequences (table s ) j o u r n a l p r e -p r o o f for qrt-pcr were designed the primer platform (http://frodo.wi.mit.edu/primer /). all qrt-pcr was repeated three times, the expression levels of candidate genes were determined using the -ΔΔct method. expression levels were normalized against the reference gene gapdh. data are represented as mean values ± sd, and graphpad prism software was employed for one-way anova, *p< . , ** p< . . in total, ingredients in ljf fulfilled the two criteria simultaneously (dl ≥ . and ob ≥ %). however, five ingredients (scolymoside, hederagenol, heriguard, hyperin, and luteolin- -o-glucoside) did not meet the criteria of ob ≥ %, but they were also presented as candidates. hence, main ingredients were obtained as potentially active constituents in ljf, including flavonoids and organic acids (table s ). these active components were used to identify the targets in swisstargetprediction and sea. after removing duplicates, we finally acquired targets (table s ). the ali-related genes were collected from the genecards database, and ali-related genes were identified (table s ). the shared targets of the active compounds and ali-related genes were identified by generating venn diagrams. ultimately, genes were identified as both the targets of active ingredients and ali-related genes. the identified genes were used to construct the compound-target network with cytoscape . . software. among the active compounds in ljf, eight active compounds demonstrated a higher number of connections and were connected with more than targets, including ethyl-linolenate, hederagenol, chrysoeriol, kaempferol, luteolin, mandenol, quercetin and zinc . the network analysis showed that one compound in the herbs can be linked with more than one target. in the compound-target network, alox (arachidonate -lipoxygenase) was simultaneously targeted by active ingredients, akr b (aldo-keto reductase family member b ) was targeted by active ingredients, and ptgs (prostaglandin g/h synthase ) was targeted by active ingredients (fig. .) . in the ppi network, eleven targets were linked ptgs might be identified as hub genes. the potential target proteins were subjected to enrichment analysis by kobas . . as a result, significant pathways related to ljf were identified by kobas . (table s ) . then, the target-pathway network was constructed (fig.s ), containing targets and corresponding pathways. it was obvious that the targets for ljf were mainly interlinked in three pathways, including metabolic pathways (degree = ), neuroactive ligand-receptor interaction pathways (degree = ), and pathways involved in cancer (degree = ). moreover, we identified the calcium signalling pathway (degree = ) and nod-like receptor signalling pathway (degree = ). fortunately, we also observed that these targets participated in pathways linked to immune and inflammatory signalling pathways, including human cytomegalovirus infection-associated pathways, the il- signalling pathway, the ppar signalling pathway, the pi k-akt signalling pathway, human t-cell leukaemia virus infection-associated pathways, pathways involved in inflammatory mediator regulation of trp channels, the jak-stat signalling pathway, natural killer cell-mediated cytotoxicity-associated pathways, t cell receptor signalling pathway, nf-kappa b signalling pathway, tnf signalling pathway and the toll-like receptor signalling pathway. to detect the anti-inflammatory effects of ljf, we examined the levels of il- , tnf-α, and il- β in serum and balf after treatment with lps, ljf and dxsm. the results showed that exposure of serum and balf to lps increased the production of these inflammatory cytokines (p< . ). however, production of these inflammatory cytokines (il- , tnf-α, and il- β) were significantly inhibited by ljf and dxsm (p< . ) ( fig. a and b) . then, the level of oxidative stress was measured, and lps-induced ali could increase the levels of mda and mpo and decrease the activities of sod and gsh-px in the lung tissue (p< . ) (fig. c) . interestingly, the levels of sod and gsh-px were significantly enhanced by ljf and dxsm (p< . ). h&e staining was used to detect the pathological changes in the lungs. after treatment with lps, the rat lung tissue showed increases in inflammatory cell infiltrates and alveolar histological structure damage compared with the lung tissue in the ctrl group. in our study, the ali group showed severe alveolar oedema fluid accumulation, alveolar capillary congestion and bronchial epithelial detachment (fig. b) . ljf was superior to dxsm in alleviating lps-induced ali ( (table s ) . to determine the differentially expressed genes (degs), a p-value < . was used as the cut-off value for gene expression in the ctrl, ali, ali-ljf, and ali-dxms groups, which was detected by pairwise comparisons between the ali group and the ctrl, ali-ljf, and ali-dxms groups. as a result, , degs in rat lung were identified after lps stimulation, whereas and , degs were identified in rat lung tissue treated with ljf and dxsm, respectively. overall, , upregulated and , downregulated degs were identified in the ali vs. ali-dxsm groups, and upregulated and downregulated degs were identified in the ali vs. ali-ljf groups ( fig. a-c) . in brief, after removing the duplicate genes, deg genes were associated with the ali group that were also affected in the ctrl, ali and ali-ljf groups; , genes were associated with the ctrl, ali and ali-dxsm groups; and only key deg genes were associated with the ctrl, ali, ali-ljf and ali-dxsm groups (fig. d) . to characterize these differentially expressed genes, trend analysis was applied to determine the expression patterns of the degs in the ctrl, ali and ali-ljf groups (table s ). in the j o u r n a l p r e -p r o o f ali vs. ctrl groups, the expression of genes displayed an initial increase, but there was a decrease in the ali vs ali-ljf groups; however, the expression of genes exhibited a reduction in the ali vs. ali-ljf groups, but there was a subsequent increase in the ali vs. ali-ljf groups (fig. ) . specifically, eight genes (dyrk a, ca , il , ptafr, arg , mgll, ltb r, and tymp) among the degs of the ali-ljf group were predicted as targets of active ingredients of ljf. to verify the reliability of the gene expression data obtained by rna-seq, eight genes (il r , dnajc , thbs , nploc , bcl a , ppp r a, kit, and itga ) were randomly selected for qrt-pcr detection. the qrt-pcr results showed that the tendency of gene expression was consistent with the rna-seq results. for each gene, the qrt-pcr expression results showed a similar tendency to the rna-seq results (fig.s ) . the results showed that the rna-seq results were credible in this study. to thoroughly investigate the potential pathways involved in the immune and inflammatory responses, the kobas . platform was employed for kegg pathway analysis of these degs. the kegg analysis showed that kegg pathways were significantly enriched for these degs (table s ; signalling pathway (fig. ) . within the four classic immune pathways (rno , rno , rno , and rno ), we identified candidate genes associated with ali: cxcl , cxcl , cxcl , il , lif, il rb , il , nfkbia, ifng, il a, il , birc , il a, il c, il f, cxcl , il r , tradd, mmp , ccnd , il , fadd, bcl a , and tnfaip . in this study, il- signalling pathway involved in thirteen degs, including cxcl , cxcl , cxcl , nfkbia, ifng, il , il a, il f, il c, mmp , tnfaip , fadd and tradd. ljf significantly inhibited cxcl , cxcl , cxcl , nfkbia, ifng, il , il a, il f, il c, mmp , and tnfaip mrna expression in lung tissue homogenates according to rna-seq, which indicates that the il- signalling pathway is critical for treatment of lps-induced ali with ljf (fig.s ) . interestingly, the involved il- family members, including il- a, il- c and il- f, played a significant role in the acute inflammatory responses (fig.s ) . consistent with the rna-seq data, the expression of cxcl , cxcl , cxcl , nfkbia, ifng, il , il a, il f, il c, mmp and tnfaip in lung tissue was significantly decreased compared with that in the ali and ljf groups according to the qrt-pcr analyses (p< . ) (fig. ) . the expression of tradd and fadd was increased compared with that in the ali and ljf groups by the qrt-pcr analyses (fig. ) , and associated with apoptosis in the il- signalling pathway (fig.s ) . we employed network pharmacology to determine the potential active ingredients and targets of ljf. then, we performed compound-target and target-pathway network analyses to explore the mechanisms of ljf. furthermore, an lps-induced rat model was constructed to evaluate the effect of ljf in the treatment of ali. according to the degree of the nodes in the compound-target network, we identified eight compounds as potential active ingredients that might participate in the regulatory processes of ljf in ali. obviously, the active ingredients chrysoeriol (wei et qrt-pcr analysis showed that the trends for eight genes (il r , dnajc , thbs , nploc , bcl a , ppp r a, kit, and itga ) were consistent with the rna-seq results. these findings indicated that the rna-seq results were credible. by comparative analysis, we found that there were more differentially expressed genes in the ali, ali-ljf and ali-dxsm groups. in all, degs were unique to the ali-ljf group, and degs were shared among the three groups. through kegg enrichment analysis, we found that the il- signalling pathway was significantly enriched in the ali-ljf group. thirteen genes were found, such as cxcl , cxcl , cxcl , nfkbia, ifng, il , il a, il f, il c, tradd, mmp , kaempferol regulates mapks and nf-κb signaling pathways to attenuate lps-induced acute lung injury in mice eupatorium lindleyanum dc. flavonoids fraction attenuates lipopolysaccharide-induced acute lung injury in mice mast cell and macrophage chemokines cxcl /cxcl control the early stage of neutrophil recruitment during tissue inflammation mmp- and mmp- in bronchoalveolar lavage fluid is associated with acute lung injury after cardiopulmonary bypass in a swine model shuang-huang-lian attenuates lipopolysaccharide-induced acute lung injury in mice involving anti-inflammatory and antioxidative activities. evid based complement alternat med local stimulation of alpha cholinergic receptors inhibits lps-induced tnf-alpha release in the mouse lung enforced expression of mir- b attenuates lps-induced acute lung injury network pharmacology: the next paradigm in drug discovery puerarin inhibits inos, cox- and crp expression via suppression of nf-kappa b activation in lps-induced raw . macrophage cells il- cytokines in immunity and inflammation acute lung injury: epidemiology, pathogenesis, and treatment protective and immunomodulatory effect of flos lonicerae japonicae by augmenting il- expression in a murine model of acute lung inflammation hisat: a fast spliced aligner with low memory requirements myeloperoxidase: friend and foe lonicera japonica resolution of acute inflammation in the lung exerts anti-viral and anti-inflammatory activity against novel coronavirus (sars-cov- ) sini decoction alleviates e. coli induced acute lung injury in mice via equilibrating ace-angii-at r and ace -ang-( - )-mas axis can chinese medicine be used for prevention of corona virus disease (covid- )? a review of historical classics, research evidence and current prevention programs acute lung injury and the acute respiratory distress syndrome -four decades of inquiry into pathogenesis and rational management stlr /smd- complex alleviates lps-induced acute lung injury by inhibiting pro-inflammatory cytokines and chemokine cxcl expression chinese traditional patent medicine in treating a family case of covid- in wuhan kaempferol reduces k -linked polyubiquitination to inhibit nuclear factor-κb and inflammatory responses in acute lung injury in mice glycyrrhizic acid ameliorates lps-induced acute lung injury by regulating autophagy through the pi k/akt/mtor pathway tcmsp: a database of systems pharmacology for drug discovery from herbal medicines incidence and outcomes of acute lung injury luteolin attenuates acute lung injury in experimental mouse model of sepsis cytoscape: a software environment for integrated models of biomolecular interaction networks lonicera japonica thunb.: ethnopharmacology, phytochemistry and pharmacology of an important traditional chinese medicine kobas . : a web server for annotation and identification of enriched pathways and diseases trifunctional inhibition of cox- by extracts of lonicera japonica: direct inhibition, transcriptional and post-transcriptional down regulation anti-inflammatory and protective properties of daphnetin in endotoxin-induced lung injury systems pharmacology for investigation of the mechanisms of action of traditional chinese medicine in drug the rna sequencing results revealed differentially expressed genes in lung tissue. (b) differentially expressed genes were confirmed by qrt-pcr. data are represented as mean values ± sd key: cord- - awvmpyh authors: kosukegawa, ima; okazaki, shunichiro; yamamoto, motohisa; nagoya, satoshi; suzuki, chisako; shimizu, junya; takahashi, hiroki; yamashita, toshihiko title: the proton pump inhibitor, lansoprazole, prevents the development of non-traumatic osteonecrosis of the femoral head: an experimental and prospective clinical trial date: - - journal: eur j orthop surg traumatol doi: . /s - - - sha: doc_id: cord_uid: awvmpyh background: an effective prevention strategy for osteonecrosis of the femoral head (onfh) has yet to be established. we previously reported that the innate immune system via the toll-like receptor (tlr) response induced by corticosteroids leads to the development of onfh and that repression of irf activity by an inhibitor could interfere with the development of onfh while maintaining the therapeutic effect of the corticosteroids. objective: in the present study, we hypothesize that lansoprazole has the potential to suppress irf activity and prevent corticosteroid-induced onfh in rats. furthermore, we conducted a preliminary clinical trial to prevent corticosteroid-induced onfh in autoimmune disease patients. methods: male wistar rats were randomly divided into four groups. on day , each rat was injected with tlr ligand (lps) or tlr ligand (imiquimod), followed by methylprednisolone with or without lansoprazole on day . they were killed at or days after the last injection.we prospectively recruited patients requiring primary high-dose corticosteroid treatment for immune diseases. all patients were administered lansoprazole, starting the night before corticosteroid treatment began. mri was performed before corticosteroid treatment, and at , and weeks afterward. results: in rats, co-treatment of lansoprazole with corticosteroids significantly repressed both irf activity and the development of onfh. moreover, in the human patients, the incidence of onfh was significantly decreased from . to . %. conclusions: although the present study is preliminary, the results show that co-treatment of lansoprazole with corticosteroids prevents onfh development. lansoprazole may be both safe and effective in preventing osteonecrosis of the femoral head in patients needing corticosteroid treatment. high-dose corticosteroid therapy for inflammatory diseases and alcohol-abuse was reported to be a risk factor for non-traumatic osteonecrosis of the femoral head (onfh) [ ] [ ] [ ] . the pathogenesis of onfh in patients with inflammatory diseases treated with corticosteroids remains unclear, although there has reported as factors caused ischemia due to fat emboli in peripheral blood vessels, reduction in arterial flow through the recruitment of increased adipose tissue and/ or an increase in intramedullary pressure [ , ] . on the other hand, onfh frequently results in osteoarthritis in young patients [ , ] , and no effective prevention strategy or effective nonsurgical treatment for onfh has been established to date. we previously reported corticosteroid-induced onfh rat models treated with a toll-like receptor (tlr) ligand and corticosteroid and that tlr signaling pathways contribute to the pathogenesis of corticosteroid-induced onfh in rats [ , ] . we also reported an alcohol-induced onfh rat model and that the tlr signaling pathway contributes to the pathogenesis of alcohol-induced onfh [ ] . furthermore, we reported that onfh results from the activation of nuclear factor kappa b (nf-κb) and interferon regulatory factor (irf ) via the tlr signaling pathways, followed by a subsequent repression in nf-κb activity by corticosteroid treatment, whereas irf activity is unaffected by corticosteroid treatment. further, the suppression of irf activity by the use of an inhibitor, bay - , could interfere with the development of onfh while maintaining the therapeutic effect of corticosteroids [ ] . however, as rats co-administered with bay - and corticosteroids showed high mortality, bay - may be unsafe for human use. as new drug development requires an enormous amount of time and money, drug repositioning has been attracting a good deal of attention in recent years [ , ] . the drug lansoprazole (lpz) is a proton pump inhibitor used to treat and prevent stomach ulcers by suppressing acid secretion through proton pump inhibition [ ] . it has, however, also been reported to have anti-inflammatory effects and suppress inflammatory responses via tlr signaling [ , ] . in the present study, we hypothesized that lpz has the potential to suppress irf and nf-κb, in the same manner as bay - , and so prevent corticosteroid-induced onfh in rats. moreover, we conducted a preliminary clinical trial for the prevention of onfh in patients with corticosteroid-treated immune diseases. all experiments observed the guidelines of the ministry of sports, culture, science, and technology of japan, and followed protocols approved by the animal ethics committee of sapporo medical university (# - ). male wistar st rats ( - g), obtained from sankyo labo service co., ltd. (sapporo, japan), were housed in temperature-and humidity-controlled rooms with unlimited normal food and water and a -h light/dark cycle. animals (n = ) were divided into four groups and treated, as in previous reports [ , ] , as follows: lipopolysaccharide (lps) + methylprednisolone (mpsl) rats (n = ) were given . mg/kg lps (from escherichia coli serotype : b ; sigma, st. louis, mo, usa), a ligand for tlr , intravenously on day and mg/kg mpsl (sigma, st. louis, usa) intramuscularly on day ; lps + lpz + mpsl rats (n = ) were given . mg/kg lps intravenously on day and mg/kg lpz (takepron ® intravenous mg, takeda pharmaceutical company limited, osaka, japan) intravenously with mg/kg mpsl intramuscularly on day ; imiquimod + mpsl rats (n = ) were given mg/kg imiquimod (tokyo chemical industry, tokyo, japan), a ligand for tlr , subcutaneously on day and mg/kg mpsl intramuscularly on day ; and imiquimod + lpz + mpsl rats (n = ) were given mg/kg imiquimod subcutaneously on day and mg/kg lpz intravenously with mg/kg mpsl intramuscularly on day . all injections were performed at : p.m. animals were killed at or days after the last injection. the femurs and livers were harvested and fixed with % formalin- . m phosphate buffer (ph . ). a portion of the liver from each rat killed at day after the last injection was harvested and stored at − °c until analysis. bone samples were decalcified with kalkitox™ (wako pure chemical industries, ltd., osaka, japan), neutralized with a % sodium sulfate buffer, and then processed for routine hematoxylin and eosin staining to assess onfh. osteonecrosis was defined as the diffuse presence of empty lacunae or pyknotic nuclei in osteocytes within the bone trabeculae, accompanied by surrounding bone marrow cell necrosis [ , , ] . nf-κb and irf activity was assessed by emsa, as described previously [ ] . briefly, equal amounts of liver nuclear extract ( . mg of protein) were incubated for h at room temperature with p-labeled nf-κb or irf consensus oligonucleotide probes ( ′-agt tga ggg gac ttt ccc aggc - ′ or ′-act gat cgg aac cga acg atc tat g- ′, respectively) in binding buffer ( mm hepes [ph . ], mm kcl, . mm ethylenediaminetetraacetic acid, . mm dithiothreitol, % glycerol, and . % np- ). the dna protein complexes were separated on % non-denaturing polyacrylamide gels at a constant v at room temperature. the gels were then exposed to an image plate (fuji film, tokyo, japan) at room temperature, and the radioactivity of their dna-binding complexes was analyzed using an fla image analyzer (fuji film) and imagequant software (molecular dynamics, sunnyvale, ca, usa). data represent the mean ± sem. comparisons between two groups were performed using -tailed fisher's exact test or the -tailed nonparametric mann-whitney test, using graphpad prism . f software for mac os x (graphpad software, inc., la jolla, ca, usa). a p value < . was considered significant. the study was approved by the institutional review board of sapporo medical university hospital (approval number: # - ) and observed the standards of the declaration of helsinki. our study was a prospective, single-center, historically controlled trial. all patients required primary high-dose prednisolone treatment for immune diseases and were recruited in the departments of gastroenterology, rheumatology and clinical immunology of sapporo medical university hospital in sapporo, japan, between july and september . inclusion criteria required a prednisolone dose of mg/day or more and an age of - years. exclusion criteria were as follows: current onfh, hip joint disease requiring surgery, alcohol-abuse, dementia, past allergy to lpz, and treatment with atazanavir sulfate. all patients recruited provided written informed consent. all patients were administered lpz (takepron ® intravenous mg, takeda pharmaceutical company limited, osaka, japan) intravenously a total of times (once the night before corticosteroid treatment started, and thereafter twice a day). subsequently, all were administered lpz (takepron ® od mg, takeda pharmaceutical company limited) orally once a day for days. routine magnetic resonance imaging (mri) of the hips was performed before corticosteroid treatment, and at , and weeks thereafter using a ge signa hdx . t (ge healthcare, milwaukee, wi, usa). t -weighted images, t -weighted images, and fat suppression images on the axial and coronal plane were obtained. a low signal intensity band on t -weighted images was defined as onfh. two trained orthopedists and a trained radiologist assessed all radiographs. one patient was excluded because of a physical condition that precluded the mri at weeks. onfh was diagnosed using the classifications for the osteonecrosis of the femoral head of the japanese ministry of health, labor, and welfare [ ] , in which type a lesion occupies the medial one-third or less of the weight-bearing portion, type b lesion occupies the medial two-thirds or less of the weight-bearing portion, type c and type c lesions both occupy more than the medial twothirds of the weight-bearing portion, with type c lesions extending laterally to the acetabular edge, whereas type c lesions do not. the margins of the necrotic areas were determined as a low signal intensity band at the coronal slice of the center of the femoral head on the t -weighted images. table shows patient demographic data including age, gender, underlying diseases, maximal daily prednisolone dosage, total prednisolone dosage within months, days of corticosteroid treatment at mg/day, and occurrence of onfh. the patients consisted of men and women (mean age . years). the underlying diseases were igg related disease (n = ), dermatomyositis (n = ), systemic lupus erythematosus (sle, n = ), and others (n = ). the maximal mean prednisolone dosage was . ( - ) mg/ day, excluding days of treatment at mg/day in two patients. owing to the lack of an effective nonsurgical treatment for onfh, we did not conduct a randomized control study. we used a historical control group ( men and women, mean age . years) of patients from the same institute, who were the subject of a previous report [ ] . their underlying diseases were sle (n = ), igg -related disease (n = . mikulicz's disease, as described in previous reports), adult-onset still's disease (n = ), dermatomyositis (n = ), microscopic polyangitis (n = ), and others (n = ). the maximal mean prednisolone dosage was . mg/day, excluding days of treatment at mg/day in ten patients. these parameters, excluding gender consistency (p < . , fisher's exact test), were not significantly different compared to the lpz treatment group (age: p = . , mann-whitney test, underlying diseases: p = . , chi-square test, maximal steroid dosage: p = . , mann-whitney test). .comparisons between the two groups was performed with a -tailed fisher's exact test, chi-square test or a -tailed nonparametric mann-whitney test using graphpad prism . f software for mac os x (graphpad software, inc., la jolla, ca, usa). a p value < . was considered significant. to evaluate the activity of transcription factors nf-κb and irf in the liver, the lps + mpsl (n = ) and imiquimod + mpsl (n = ) (control groups) and lps + lpz + mpsl (n = ) and imiquimod + lpz + mpsl (n = ) (experimental groups) rats were killed at day after the last injection. figure shows the activity of transcription factors nf-κb and irf in the liver. lpz treatment significantly suppressed the activity of nf-κb in the lps + lpz + mpsl group compared to that in the lps + mpsl group at day after the last injection ( fig. : lps + mpsl vs. lps + lpz + mpsl, ± . vs. . ± . ; p = . ). however, there was no significant difference in nf-κb activity between the imiquimod + lpz + mpsl and imiquimod + mpsl groups at that time ( fig. : imiquimod + mpsl vs. imiquimod + lpz + mpsl, ± . vs. . ± . ; p = . ). on the other hand, lpz treatment significantly suppressed the activity of irf in the lps + lpz + mpsl group compared to that in the lps + mpsl and imiquimod + lpz + mpsl groups as well as the imiquimod + mpsl group at day after the last injection ( fig. : lps + mpsl vs. lps + lpz + mpsl, ± . vs. . ± . ; p = . . imiquimod + mpsl vs. imiquimod + lpz + mpsl, ± . vs. . ± . ; p = . ). to evaluate the incidence of onfh, the lps + mpsl (n = ) and imiquimod + mpsl (n = ) (control groups) and lps + lpz + mpsl (n = ) and imiquimod + lpz + mpsl (n = ) (experimental groups) rats were killed days after the last injection. onfh was observed in of (one had died prematurely) rats in the lps + mpsl group and of rats in the imiquimod + mpsl group. by contrast, no onfh was observed in the lps + lpz + mpsl ( of ) or imiquimod + lpz + mpsl groups ( of ). the incidence in the lpz-treated groups ( of ) was significantly lower than in the control groups ( / ) (p < . ; fisher's exact test). figure shows the histopathological appearance of the femoral head after hematoxylin and eosin staining in the lps + mpsl (a), lps + lpz + mpsl (b), imiquimod + mpsl (c) and imiquimod + lpz + mpsl (d) rats. in the lps + mpsl and imiquimod + mpsl groups, empty lacunae and pyknotic nuclei were observed within the necrotic bone trabeculae, and bone marrow cell necrosis was present in the medullary space across most areas of the femoral head (fig. a, c) . normal trabeculae as well as hematopoietic and fat cells were observed in the lps + lpz + mpsl and imiquimod + lpz + mpsl rats (fig. b, d) . as described above, we found that lpz has the potential to prevent onfh development in rats. thus, we conducted a preliminary clinical trial. in this clinical study, no adverse events associated with lpz administration were observed. onfh was found in of ( . %) patients treated with high-dose corticosteroids, which was a significantly lower incidence (p = . ; fisher's exact test) than that in the control group; i.e., of ( . %). no low signal intensity band was observed on t -weighted images pre-corticosteroid treatment or at weeks after treatment. however, a band was observed in patients at weeks after treatment and in another patient at weeks after treatment. the patients with onfh consisted of males and one female, with mean age of . ( - ) years, and observation was continued in the department of orthopedic surgery, sapporo medical university hospital in sapporo, japan. the underlying diseases in the patients with onfh were igg -related disease (n = , three males) and sle (n = , female) ( table ). the mean dosage of prednisolone over the -month period did not differ significantly in the patients with onfh (table ; onfh vs. non-onfh, ± . vs. ± . ; p = . ; mann-whitney test). the patients with onfh did not receive mg/day corticosteroid treatment. two developed bilateral onfh, and two developed unilateral onfh. according to the classifications of the osteonecrosis of the femoral head of the japanese ministry of health, labor, and welfare, patients developed type b, patients type c- , and patient type c- , with no collapse of the femoral head or other symptoms. in all patients, onfh lesions with a low signal intensity band on t weighted images decreased in size during the fig. transcription factor activity. emsa for nf-κb and irf in lps + mpsl, lps + lpz + mpsl, imiquimod + mpsl and imiquimod + lpz + mpsl rats on day . lane contains no extract. lane contains extract from untreated rats. lane contains extract from rats treated with lps/imiquimod + mpsl. lane contains extract from rats treated with lps/imiquimod + lpz + mpsl. a significant suppression of nf-κb activity was observed at day after the last injection in the lps + lpz + mpsl group compared with that in the lps + mpsl group. a significant suppression in irf activity was also observed at day after the co-administration of lpz with corticosteroids. data represent the mean ± sem. *p < . , **p < . compared with lps/imiquimod + mpsl rats observation period. a -year-old man with igg -related disease developed bilateral onfh at weeks after corticosteroid treatment. however, the onfh lesions had become smaller in the mr images at weeks. the left-side lesion disappeared at months after corticosteroid treatment, and the right-side lesion at months (fig. ) . a -year-old man with igg-related disease developed right-side onfh at weeks after corticosteroid treatment. however, the onfh lesion had become smaller at months, and observation was continued. a -year-old man with igg-related disease developed right-side onfh at weeks after corticosteroid treatment. however, the onfh lesion had become smaller at months, and once again observation was continued. a -year-old woman with sle developed right-side onfh at weeks and left-side onfh at weeks. however, the onfh lesions had decreased in size at months, and observation was continued. none of these patients received any special treatment such as avoidance of weight bearing. onfh leads to collapse of the femoral head in % of cases, results in osteoarthritis in young patients and affects the patient's quality of life [ , ] . the present study showed that the co-treatment of lpz with corticosteroids prevents the development of onfh in experimental animals, as well as in patients with immune disease treated with corticosteroids. we believe that this is the first report to describe the prevention of corticosteroidinduced onfh development in a clinical trial based on experimental outcomes. it has been reported that innate immune signaling via tlr pathways contributes to the pathogenesis of underlying diseases in patients with corticosteroid-induced onfh [ , ] . consequently, we previously reported that corticosteroid treatment after tlr , tlr or tlr ligand injection induces onfh in wistar rats and that onfh results from the activation of nf-κb and irf via the myd -dependent pathway, followed by a subsequent repression in nf-κb activity by corticosteroid treatment, whereas irf activity is unaffected by corticosteroid treatment. furthermore, suppression of irf activity using bay - , ikk-α inhibitor and ikk-β inhibitor with the potential to inhibit nf-κb and irf activity [ ] interferes with the development of onfh [ ] . thus, we believe that a proinflammatory response induced by corticosteroids leads to the development of onfh. however, as the safety of bay - has not been approved for humans, its clinical application is difficult. therefore, we explored the use of an approved, commercially available drug, which has the potential to suppress irf activity. it has been reported that lpz exerts anti-inflammatory effects by suppressing the induction of inflammatory cytokines via the suppression of nf-κb activity [ ] . consequently, we hypothesized that lpz also inhibits irf activity and prevents onfh development in rats, and this study showed that lpz significantly suppressed irf activity in the lps + lpz + mpsl and imiquimod + lpz + mpsl groups compared with that in the lps + mpsl and imiquimod + mpsl groups. this is consistent with previous reports on the effect of bay - [ ] and confirms that lpz inhibits irf activity. further, nf-κb activity was significantly suppressed in the lps + lpz + mpsl group only. we previously reported that corticosteroid treatment suppressed nf-κb activity in imiquimod + mpsl rats compared with that in imiquimod + saline rats [ ] . this result indicates that lps activates nf-κb activity more than does imiquimod. therefore, nf-κb activity was significantly suppressed by lpz treatment, and the cotreatment of lpz with corticosteroids prevents the development of onfh in rats. the results of the experimental study indicate that lpz may also prevent the development of onfh in patients with immune diseases needing corticosteroid treatment. it has been reported that antihyperlipidemic, antiplatelet and antioxidant agents prevent the development of osteonecrosis in experimental animals [ ] [ ] [ ] . although clinical studies about prevention for onfh by statin therapy and anti-coagulant were reported [ , ] , there are no standard preventive methods today. unfortunately, no effective prevention strategy for onfh has been clinically established. in the present study, lpz also prevented the development of corticosteroid-induced onfh in a clinical study. however, onfh did develop in four patients, although not in any rats. patients were administered with lpz at the approved dosage, suggesting the necessity to reconsider the dosage and usage period of lpz. additionally, the onfh lesions in the four patients were found to decrease in size, as evaluated by mri, during onfh was detected at months after corticosteroid treatment (type c lesion in the right femoral head and type b lesion in the left side). six months after treatment, the onfh lesion had decreased in size. the left femoral head onfh lesion disappeared at months after corticosteroid treatment and the right femoral head onfh lesion disappeared at months after corticosteroid treatment the observation period. reports exist of onfh lesions, observed by mri, regressing spontaneously over several years [ ] [ ] [ ] [ ] . these reports studied specific diseases, such as renal recipients, severe acute respiratory syndrome and sle, so there is a possibility that specific treatments were provided for each group of patients. the present study could not determine whether the disappearance of onfh was due to its natural course or the effects of lpz, and onfh patients need to be observed in a future clinical study. several limitations of our clinical study should be noted. first, it was unclear whether the pharmacological effect of lpz as prevention agent for osteonecrosis in rat models can be transferred to humans and there have been no reports to date. second, the sample size was small, and it was a single-center and historically controlled study. further, there were differences in underlying diseases in this study group from those in past reports, particularly, as a large number of patients with igg -related disease were included in this study. this is due to the high rate of patients treated for igg -related disease in our hospital. furthermore, there was some bias in terms of underlying diseases between the study and control groups. last, we could not evaluate transcriptional factor activity in the patients. nevertheless, our clinical trial did show a decrease in the incidence of onfh in our institute. the present study shows that the co-treatment of lpz with corticosteroids prevents the development of onfh through the suppression of irf activity in rats. moreover, lpz also prevents onfh development in immune disease patients treated with corticosteroids, although the study was only preliminary. consequently, it is necessary to conduct a multicenter clinical trial with large sample size. nevertheless, we believe that lpz is safe to use and could be effective in preventing osteonecrosis of the femoral head in patients needing corticosteroid treatment. we anticipate that further clinical trials will show that this protocol is both consistent and reproducible. influence of alcohol intake, cigarette smoking, and occupational status on idiopathic osteonecrosis of the femoral head temporal trends in 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autoimmune disease patients showing no immediate increase in hepatic enzyme under steroid therapy quality of life of patients with osteonecrosis of the femoral head: a multicentre study inhibitor of ikappab kinase activity, bay - , interferes with interferon regulatory factor nuclear translocation and type i interferon production by plasmacytoid dendritic cells pitavastatin may reduce risk of steroid-induced osteonecrosis in rabbits: a preliminary histological study prevention of steroid-induced osteonecrosis by intravenous administration of vitamin e in a rabbit model effects of an anti-platelet drug on the prevention of steroid-induced osteonecrosis in rabbits prevention of steroid-induced osteonecrosis of femoral head in systemic lupus erythematosus by anticoagulant stain therapy decreases the risk of osteonecrosis in patients receiving steroids multimodality approach to osteonecrosis of the femoral head apparent avascular necrosis of the hip: appearance and spontaneous resolution of mr findings in renal allograft recipients spontaneous repair of asymptomatic osteonecrosis associated with corticosteroid therapy in systemic lupus erythematosus: -year minimum follow-up with mri lesion size changes in osteonecrosis of the femoral head: a long-term prospective study using mri funding the author s nagoya has received research grants from smith and nephew, zimmer biomet, and daiichi sankyo. conflict of interest the authors i kosukegawa, s okazaki, m yamamoto, c suzuki, h takahashi and t yamashita declare that they have no conflicts of interest.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/ . /. key: cord- -vmbhok u authors: sichelstiel, anke; yadava, koshika; trompette, aurélien; salami, olawale; iwakura, yoichiro; nicod, laurent p.; marsland, benjamin j. title: targeting il- β and il- a driven inflammation during influenza-induced exacerbations of chronic lung inflammation date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: vmbhok u for patients with chronic lung diseases, such as chronic obstructive pulmonary disease (copd), exacerbations are life-threatening events causing acute respiratory distress that can even lead to hospitalization and death. although a great deal of effort has been put into research of exacerbations and potential treatment options, the exact underlying mechanisms are yet to be deciphered and no therapy that effectively targets the excessive inflammation is available. in this study, we report that interleukin- β (il- β) and interleukin- a (il- a) are key mediators of neutrophilic inflammation in influenza-induced exacerbations of chronic lung inflammation. using a mouse model of disease, our data shows a role for il- β in mediating lung dysfunction, and in driving neutrophilic inflammation during the whole phase of viral infection. we further report a role for il- a as a mediator of il- β induced neutrophilia at early time points during influenza-induced exacerbations. blocking of il- a or il- resulted in a significant abrogation of neutrophil recruitment to the airways in the initial phase of infection or at the peak of viral replication, respectively. therefore, il- a and il- β are potential targets for therapeutic treatment of viral exacerbations of chronic lung inflammation chronic obstructive pulmonary disease (copd) is currently ranked the th leading cause of death worldwide by the world health organization (who), and its incidence is increasing. the main risk factor of copd is exposure to tobacco smoke which triggers a cascade of inflammatory pathways leading to disease induction in susceptible people. major hallmarks of the disease pathology are the development of emphysema and chronic bronchitis that lead to a progressive and irreversible airflow limitation resulting in a continuous decline of lung function [ ] . copd severity has been associated with acute periods of disease worsening [ , ] , so-called exacerbations, a key factor in copd morbidity and mortality [ , ] . by causing acute respiratory distress, they impact on the quality of patient's health [ ] and are responsible for most hospital stays related to the disease [ ] . exacerbations are primarily caused by respiratory viral or bacterial infections. amongst those, viral-induced exacerbations account for approximately half of the cases [ , ] and are associated with more severe acute episodes and prolonged recovery time [ ] [ ] [ ] . the most common viral pathogen in exacerbated patients is rhinovirus, followed by influenza virus, rsv and coronavirus [ , , , ] . due to targeted vaccination of high risk groups, influenza infections occur less frequently in copd patients of westernized countries [ ] . however, they continue to be the predominant cause of viral exacerbations in hong kong [ ] and singapore [ ] . copd exacerbations have been linked to excessive inflammatory responses, including enhanced recruitment of inflammatory cells [ ] and upregulation of a variety of proinflammatory mediators [ , ] . nevertheless, the underlying mechanisms and the most effective therapeutic strategies are still poorly understood and first-line therapy still predominantly relies on corticosteroids and bronchodilators [ ] , which are limited in their efficacy [ , ] . thus, the study of cellular and molecular mechanisms leading to exacerbations is key for the identification of urgently required therapeutic targets. one of the proinflammatory cytokines that has been associated with copd is il- b, a major player in initiation and persistence of inflammation. in animal models mimicking features of copd, il- has been shown to be key to the induction of emphysema and inflammation [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . furthermore, its expression is significantly enhanced in copd patients during acute episodes of exacerbations [ , , , ] . unraveling the role of il- b in viral exacerbations might therefore not only result in an overall better understanding of mechanisms of exacerbations, but also indicate whether it qualifies as a valid therapeutic target. a promising candidate for therapeutic inhibition of il- b signaling is one of its inhibitors, the interleukin- receptor antagonist (il- ra) anakinra (kineret, amgen), which has been used effectively in treatment of rheumatoid arthritis. in order to investigate the role of il- b during copd exacerbations we utilized a model of lps and elastase induced chronic lung inflammation, followed by infection with influenza in wild type or il- b deficient mice. we found that il- b was a key driver of pulmonary inflammation, primarily concerning recruitment of neutrophils and lung dysfunction. il- b driven neutrophilia was mediated by il- a in the initial phase of viral infection, but became independent of il- a during the peak phase of viral replication. treatment with the il- ra, anakinra, proved efficient in reducing neutrophilic inflammation at the peak of viral replication while blocking of il- a abrogated neutrophilia in the early phase of viral infection. taken together our data indicate that blockade of il- b and il- a could be valid therapeutic approaches for treatment of virus-induced copd exacerbations. all animal experiments were performed according to institutional guidelines and swiss federal and cantonal laws on animal protection. animal experiments were approved by the following ethical committee: service de la consommation et des affaires vétérinaires, affaires vétérinaires, canton de vaud, switzerland (permit numbers and ). c bl/ or balb/c mice were between - weeks of age and were purchased from charles river laboratories. il- b deficient mice on c bl/ background were received from prof. iwakura [ ] , tokyo university of science, japan, and bred in house. mice were exposed intranasally to a mixture of mg lps from e. coli o :b (sigma-aldrich) and . u porcine pancreatic elastase (epc) in a total volume of ml, and treated once per week for four consecutive weeks ( figure a ). control mice were treated with pbs. two weeks after the last lps/elastase challenge, mice were infected with pfu influenza a virus, strain pr (a/puerto rico / , h n , viropur). the virus was administered intranasally in a total volume of ml pbs; control mice received only pbs. for all intranasal administrations c bl/ mice were anesthetized by intraperitoneal injection of . mg/kg ketamin (ketasol- , graeub) and . mg/kg xylazin (xylasol, graeub) and balb/c mice with mg/kg ketamin and . mg/kg xylazin. lungs were inflated with ml % formalin, embedded into paraffin and stained with hematoxylin and eosin. stained slides were analyzed by light microscopy. pulmonary emphysema was quantified using image j software by measuring the mean linear intercept for airspace enlargement and destruction index for alveolar wall destruction. fields of view at x magnification per section of lung were used for quantification as described previously [ , ] . airway cells were recovered by bronchoalveolar lavage and either analyzed by flow cytometry as described below or spun onto slides for differential cell counts. slides were stained with diff-quik (dade) and counts were performed according to standard criteria. total lung resistance was measured using the whole body restrained plethysmograph system flexivent from scireq. mice were anesthetized by intramuscular injection of mg/kg ketamine (ketasol- , graeub) and intraperitoneal injection of mg/kg pentobarbital (esconarkon, streuli pharma). subsequently, mice were tracheotomized and mechanically ventilated at a rate of breaths/min and a tidal volume of ml/kg bodyweight. single cell suspensions from the whole lung including airways and trachea were obtained by digestion with mg/ml collagenase iv (invitrogen) and u/ml dnasei (roche). neutrophils and monocytes in lung and bronchoalveolar lavage fluid were distinguished by staining with cd c apc-cy , cd b percp-cy . , ly- g biotin, ly- c pacific blue, streptavidin pe-cy . neutrophils were defined as cd c cd b + ly- c + ly- g + and inflammatory monocytes as cd c cd b + ly- c + ly- g low intermediate as precised in detail in figure s a . to analyze il- a production, cells from lung digests were stimulated with m pma, mg/ml ionomycin and m monensin for h at uc (indicated chemicals were purchased from sigma-aldrich). subsequently, cell subsets were distinguished by surface staining for cd percp-cy . , cd b fitc, cd tcr biotin, cd pacific blue, streptavidin pe-cy ( figure s b ) and il- a production was characterized by intracellularly staining with il- a alexa after fixation with bd lysis buffer (bd biosciences). all antibodies were purchased from biolegend. stained cells were acquired on a bd facs canto or bd facs lsrii and analyzed by using flowjo software (tree star). for neutralization of il- a, mice were treated with mg of anti-il- a (clone f ) or the corresponding isotype control antibody (clone mpoc- ) from bioxcell. the clone f uniquely reacts with the il- a and no other il- isoform [ ] . antibodies were administered intraperitoneally one day before viral infection and two days post infection. to block il- b signaling mice received mg of the interleukin- receptor antagonist (il- ra) anakinra (kineret, amgen) twice daily while control mice received only pbs. anakinra was kindly provided by prof. alexander so (centre hospitalier universitaire vaudois, lausanne, switzerland) and mme ghislaine aubel (centre hospitalier universitaire vaudois, lausanne, switzerland). total rna was isolated from lung and trachea with tri reagent (molecular research). all rna samples were checked for purity using a nanodrop spectrophotometer (thermo scientific) and met standard quality criteria. rna was subsequently transcribed into cdna by the iscript cdna synthesis kit (bio-rad) and quantitative real-time pcr was performed according to the manufacturer's instructions utilizing the ssoadvanced sybr green supermix from bio-rad. expression was determined by comparative delta-threshold cycle method using gapdh as a comparator. the following primers were used: to determine cytokine levels whole lung and trachea were collected and stored in protease inhibitor solution (roche) at uc until use. tissue homogenate was prepared using a tissuelyser (qiagen). il- b protein was determined using the mouse il- b elisa kit ready-set-go! from ebioscience by following the manufacturer's instructions. il- and tnfa were measured in a sandwich elisa using . mg/ml anti-mouse il- or tnfa (biolegend) for coating. bound protein was detected by mg/ml biotin labeled anti-il- or anti-tnfa antibody (biolegend) and subsequent incubation with alkaline phosphatase conjugated streptavidin (biolegend). plates were developed using the substrate p-nitrophenyl phosphate (sigma-aldrich) and od was measured using an elisa reader (biotek). statistical significant differences were assessed using the student's t test (two tailed, unpaired). p-values below . were considered significant and were depicted with p# . (*), p# . (**), p# . (***). standard error of the mean was applied. copd is a heterogeneous disease in humans but core features of its pathology can be reproduced in mice by repetitive exposure to lipopolysaccharide (lps) and elastase [ ] . lps is a bacterial endotoxin present in tobacco smoke [ , ] , the predominant risk factor of copd. it has been shown to cause inflammation, and particularly when co-administered with elastase, chronic emphysema-like changes develop in mouse lungs [ ] . as such, we exposed mice once a week for consecutive weeks to a mixture of mg lps and . u porcine pancreatic elastase via the intranasal route, as depicted in figure a . by this we induced strong emphysema ( figure b ,c) and sustained pulmonary inflammation ( figure b ,d) in balb/c and c bl/ mice. the development of emphysema was scored by increases in the mean linear intercept and the destructive index ( figure c ) from lung histology. in addition, we observed a strong airway inflammation, mainly driven by neutrophils and lymphocytes ( figure d ). both, emphysema and inflammation, remained above baseline levels for at least months (data not shown). thus, the response induced by repeated challenges with lps/elastase resembled the pathology of copd. as the disease severity was more pronounced in the balb/c strain ( figure b-d) , all experiments performed in wild type mice were conducted in balb/c mice. to study viral-induced exacerbations, mice were infected with influenza virus weeks after the last lps/elastase challenge, when the acute inflammation caused by lps/elastase exposure had subsided ( figure a ). the peak of viral replication was reached days after the infection ( figure b) and was followed by a rapid decline in viral titers ( figure b ) until complete viral clearance at day post infection (data not shown). the efficiency of the influenza infection was strikingly reduced in mice pretreated with lps/elastase compared to naïve mice, as revealed by significantly lower viral titers at the peak of viral infection, day and day post infection ( figure s ) which was also reflected by reduced amounts of viable virus in the lung on day (data not shown). this is likely to be due to the development of a polyclonal antibody response observed upon lps/elastase treatment ( [ , ] and data not shown). thus, the response to the influenza infection was not comparable between lps/elastase pretreated and naive mice, and was therefore not included in the remainder of the study. invasive measurements of pulmonary resistance in lps/elastase pretreated mice revealed a viral-induced acute exacerbation of airway dysfunction ( figure c ). pulmonary resistance can be influenced by a variety of factors, of which the severity of inflammation is likely to play one of the key roles during exacerbations. in line with this we detected a strong inflammatory response upon infection with influenza virus associated with an augmented absolute number of cells infiltrating into the airways and the lung ( figure d ). the cells were primarily neutrophils ( figure e , figure s a ) and inflammatory monocytes ( figure f , figure s a ). the peak of neutrophilic inflammation was reached at day post infection and neutrophil numbers declined afterwards ( figure e , figure s a ), directly correlating with the kinetics of viral replication ( figure b ). in line with this we observed increased expression of the proinflammatory cytokines il- and tnfa peaking at day or post infection, respectively, and subsiding at day after the infection ( figure g ). emphysematous damage upon lps/elastase treatment also resulted in an increase in the lung compliance. however the acute inflammatory changes induced by influenza infection had no impact on the increase in lung compliance (data not shown). acute pulmonary dysfunction, neutrophilic inflammation and enhanced levels of proinflammatory cytokines such as il- and tnfa have all been observed during exacerbations of copd patients, indicating that the viral-induced inflammation in our mouse model is in line with that seen in humans. il- has previously been shown to be a driving factor in the development of emphysema and inflammation in animal models of copd [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . as we observed an increase of il- b protein in the lungs of lps/elastase treated mice upon influenza infection ( figure a ), we hypothesized that it also promotes innate immune responses and influences pulmonary function during exacerbations. consequently, we exposed il- b deficient and c bl/ wild type mice to lps/elastase and infected them with influenza virus as described above. the c bl/ strain showed similar kinetics of neutrophil and inflammatory monocyte recruitment upon viral exacerbation as observed for the balb/c mice ( figure d , figure s b , s b). of note, viral replication was not altered in il- b deficient mice ( figure b ), demonstrating that any effects seen in the absence of il- b were not due to differences in the infection rate. c bl/ wild type mice exhibited a smaller change in pulmonary resistance in response to viral infection ( figure c ) in comparison to balb/c mice ( figure c) , with a slight increase at day post infection ( figure c ). nevertheless, pulmonary resistance in mice lacking il- b was significantly reduced already after lps/elastase exposure alone ( figure c ), thus supporting the described role for il- b in the development of chronic lung disease [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . furthermore, pulmonary resistance in il- b-deficient mice was also completely unaffected by the viral challenge ( figure c ). we did not detect any impact of il- b on inflammatory monocytes ( figure s b ); however, we found a decreased frequency and number of neutrophils in non-infected il- b deficient mice upon exposure to lps/elastase ( figure d , figure s c) . similarly, the recruitment of neutrophils to the airways and lung following viral infection was also significantly abrogated in the absence of il- b ( figure d , figure s c ). we observed significantly lower frequencies and absolute numbers of neutrophils including during the peak of neutrophil infiltration and viral replication at day post infection ( figure d , figure s c ). nevertheless, the control of the virus was unaffected as displayed by unaltered viral titers ( figure b ). considering its strong impact particularly upon neutrophils, we sought to address the mechanisms through which il- b mediated this neutrophilic inflammation. expression of the main neutrophil chemoattractants cxcl , cxcl , and cxcl were induced upon influenza infection, but were unaffected by il- b ( figure s ). in addition we did not detect any influence of il- b or the viral infection on the expression of g-csf ( figure s ). given there is substantial redundancy in neutrophil chemoattractants we thus next looked upstream at the proinflammatory cytokines il- a, il- , and tnfa, which can all stimulate neutrophilic inflamma-tion by inducing chemotactic, growth or survival factors [ , , ] . as expression of il- and tnfa were induced in our mouse model upon influenza infection ( figure g ), we hypothesized that il- b drove the observed inflammation by altering their production. the peak expression of il- and tnfa upon influenza infection was reached faster in c bl/ wild type mice at day post infection ( figure g ) as compared to day in balb/c mice ( figure e) , and thus coincided with the earlier response in pulmonary resistance observed in the c bl/ strain ( figure c ). il- protein levels followed similar kinetics in both mouse strains ( figure s a,b) , while tnfa protein production was below the sensitivity limit of our assay. however, lack of il- b did not impair the expression and production of il- ( figure e , figure s b ) and in fact increased the expression of tnfa ( figure e ). we therefore focused on il- a, a proinflammatory cytokine that has been shown to be elevated in copd patients [ , ] and whose induction partially depends on il- b [ ] [ ] [ ] . we found significantly reduced expression levels of il- a in lung homogenate in the absence of il- b, in both non-infected as well as influenza-infected lps/elastase exposed mice ( figure f ). il- a production was significantly reduced in the predominant cellular sources of il- a, such as the cd + t cells and the cd t cells in il- b deficient mice ( figure g ). further sources of il- a such as cd + cd cd cdtcr cells that might comprise nkt cells, and cd cells also showed reduced levels of il- a in the absence of il- b ( figure s a ). however, even in the wild type animals these cells represented a very minor population of cells relative to the il- a-producing t cells ( figure s a ). taken together our data showed that in addition to contributing to lung dysfunction, il- b played a key role in driving neutrophilic inflammation during influenza-induced exacerbations, an effect that was tightly linked to il- a expression. to assess whether il- a was indeed a mediator of il- b driven neutrophilia we neutralized il- a during influenza infection of lps/elastase treated mice. neutralization assays were performed in balb/c mice, which exhibited a similar induction of il- a as c bl/ mice ( figure s b ). mice received either an il- a neutralizing antibody or an isotype control antibody one day before and two days after the viral infection ( figure a ). il- a neutralization did not impact on the control of viral replication, as viral burden was comparable to the isotype control treated animals ( figure b ). we found that influenza-induced neutrophil recruitment to the airways and lung was indeed entirely attenuated h after the infection in absence of il- a ( figure c , figure s d ). however, neutrophils infiltrated into the lung and airways during the later stages of infection (day and respectively) to finally reach the same frequencies as in mice treated with the isotype control ( figure c, figure s d ); thus indicating that il- a was only required for the initial but not for the later recruitment of neutrophils. hence, il- b driven neutrophilia during influenza infection of lps/elastase exposed mice was mediated by il- a in the early phase of infection, but became independent of il- a. our data showed that a constitutive lack of il- b substantially impaired neutrophil infiltration into the airways and lung during influenza-induced exacerbations of chronic lung inflammation. thus, we sought to assess whether it is sufficient to block il- b signaling only during the course of infection, an important determinant regarding a potential therapeutic intervention. accordingly, recombinant il- ra (anakinra) or pbs was administered twice daily, starting two days prior to the viral infection ( figure a ). mice receiving anakinra displayed an impaired early control of viral infection leading to a higher viral burden at day post infection, although viral titers rapidly declined afterwards to levels similar to non-treated mice at day post infection ( figure b) , and virus was completely cleared at day post infection (data not shown). treatment with anakinra was efficient in reducing neutrophil frequencies and numbers in the airways at day post infection, the peak of neutrophilic infiltration and viral replication ( figure c , figure s e ). we did not observe an effect of anakinra on the recruitment of inflammatory monocytes ( figure s c ). similarly, we did not detect significant changes in lung function upon the treatment with anakinra (data not shown). in conclusion our data demonstrated that il- b influenced neutrophilic inflammation during influenza-induced exacerbation of chronic lung inflammation in mice throughout the entire phase of viral replication. neutrophil recruitment was mediated by il- a in the first h following viral challenge and could efficiently be blocked in the early phase of infection by antibodies neutralizing il- a. during the peak of inflammation and viral replication, il- b driven neutrophilia was independent of il- a, but could be significantly reduced by treatment with the il- ra anakinra ( figure ). in addition to a constant disease burden, copd patients suffer from episodes of acute symptom worsening causing a rapid decline in respiratory function that can necessitate hospitalization and even lead to death [ ] . indeed, a meta-analysis study estimated a case-fatality rate of . % following hospitalization due to an exacerbation [ ] . as there is a clear need to understand the mechanisms driving exacerbations and responsiveness to therapy, we examined viral-induced exacerbations in mice. in our model (figure a ), mice developed a strong inflammatory response characterized by a neutrophilic infiltrate into the airways and lung ( figure e ), enhanced expression of proinflammatory cytokines such as tnfa and il- ( figure g ), and impairment in lung function ( figure c ). decline in lung function and neutrophil accumulation are characteristic of exacerbations in humans [ ] [ ] [ ] , and elevated levels of tnfa and il- have similarly been measured in the sputum of patients undergoing an exacerbation [ , ] . thus, our mouse model reflects key pathological characteristics of copd exacerbations in humans. given the significant differences in susceptibility between the pbs treated and lps/elastase treated mice ( figure s and data not shown) which might be due to the development of a polyclonal b-cell response upon lps/elastase administration, we believe that conclusions can only be drawn from the comparison between stable disease versus episodes of exacerbation. notably, this is also supported by findings in cigarette smoke (cs) models of pulmonary inflammation where cs exposed mice showed a considerably altered response to influenza infection [ ] as well as the accelerated clearance of haemophilus influenza [ ] in comparison to control mice. in this study we specifically focused on the role of the proinflammatory cytokine il- b. il- signaling has been shown to be essential for the recruitment of neutrophils in other mouse models mimicking the pathology of stable copd, such as exposure to cigarette smoke [ , , , ] or to elastase alone [ ] . building upon these studies we found that during influenzainduced exacerbations, pulmonary accumulation of neutrophils was also driven by il- b ( figure d , figure s c ). our data is in line with results from a study of botelho et al. [ ] who investigated il- in a mouse model of acute cigarette smoke exposure. they found that interleukin- receptor (il- r) deficiency led to a reduction of neutrophils following infection with influenza and that this effect was independent of il- a. one could therefore speculate that il- b may play a critical role in neutrophil recruitment in their model as well. il- a, a cytokine that plays a central role in amplifying inflammatory cascades by inducing a variety of chemokines and cytokines, has also been reported to contribute to the development of emphysema [ , ] and to the immunopathology following influenza infections [ ] . in line with this, we found that il- a mediated early inflammation in our model of influenza-induced exacerbations of chronic lung disease ( figure c ). our data showed that il- b driven neutrophil recruitment during the first h of infection was mediated by ll- a, while it became independent of il- a at later time points during the exacerbation. we found that in the absence of il- b the expression of il- a was completely abrogated upon lps/elastase exposure as well as during exacerbations ( figure f ), thus demonstrating that il- b is required for the induction of il- a. il- a expression was induced by lps/elastase exposure alone, and levels were maintained throughout the viral-induced exacerbation ( figure f,g) ; however, while il- b levels increased during the later phase of infection ( figure a) , il- a expression surprisingly remained unaltered and even decreased at the peak of viral replication ( figure f ). nevertheless, we could block the recruitment of neutrophils at early time points following the influenza infection by neutralizing il- a ( figure c ). during the peak of viral replication, and thus a more severe state of inflammation, il- a neutralization could not prevent neutrophil influx. this might be due to an induction of cytokines during the progression of the infection, which could overcome the effect of il- a. therefore, treatment with anti-il- a seems to be favorable in the early phases of exacerbations while blocking il- b might be more advantageous during the ongoing infection. whether these findings translate into a clinical setting remains to be investigated. keeping in mind that neutrophil recruitment is elevated in the vast majority of cases of copd exacerbations regardless of their etiology [ ] , and furthermore that the increase of neutrophils in the sputum correlates with exacerbation severity [ ] , attenuating neutrophilia could be beneficial in patients undergoing a viralinduced exacerbation. current treatment relies mainly on corticosteroids and bronchodilators that have been shown to reduce the frequency of exacerbations [ ] [ ] [ ] , but have no positive effect on an ongoing episode of exacerbation. indeed, no reduction in the inflammatory response including neutrophil influx in mice and cytokine expression in humans could be achieved by treatment with steroids during copd exacerbations [ , ] . in contrast, corticosteroids have been shown to actually support neutrophil survival [ , ] . moreover, treatment with corticosteroids, efficient in reducing il- b levels in stable copd, did not affect the amount of il- b protein in exhaled breath condensate during exacerbations [ ] . as blocking of either il- a ( figure c ) or il- b ( figure d , figure s c ) efficiently reduced neutrophilic inflammation during viral exacerbation, these two molecules could be considered potential targets for therapeutic intervention. given the redundancies in these two inflammatory pathways, a combination therapy of anti-il- a and anti-il- b may prove more beneficial in improving lung function. within the context of targeting il- a or il- b in the clinic, one has to keep in mind that altering proinflammatory immune responses harbors the risk of interfering with the control of acute infection and of susceptibility to opportunistic infections. to shorten the duration of intervention and thereby reducing the risk of prolonged or secondary infections, we treated mice directly before and during the viral infection with anti-il- a ( figure a ) or il- ra ( figure a ). this was sufficient to abrogate neutrophil recruitment at the indicated time points. furthermore, neutralizing il- a did not promote elevated viral replication ( figure b ). treatment with il- ra, and thereby blocking both il- a and il- b, led to increased viral titers in the initial phase of infection ( figure b ), whereas the absence of il- b alone did not affect viral replication ( figure b ). it is therefore likely that in our model the initial control of the virus might be mediated rather by il- a than by il- b. this is in line with data from botelho et al. [ ] , who found similarly elevated viral titers upon neutralization of il -a as in the complete absence of il- r. our data thus suggest that targeting specifically il- b, and not its receptor, would be favorable in a therapeutic application. however, as treatment with anakinra did not interfere with final viral clearance it still could be considered as a therapeutic approach with the additional advantage of already being used in the clinic for other indications. taken together our data demonstrated that blocking of il- a or il- b signaling during influenza-induced exacerbations diminished neutrophilic infiltration at distinct phases of infection. whether those mechanisms apply also to other respiratory viral infections remains to be elucidated, however is plausible given the common early inflammatory pathways induced by respiratory viral infections. overall, blockade of il- a and il- b could be valuable therapeutic options for future treatment of viral induced exacerbations of chronic 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and severity of chronic obstructive pulmonary disease il- ra is required for ccl expression, macrophage recruitment, and emphysema in response to cigarette smoke cigarette smoke induction of osteopontin (spp ) mediates t(h) inflammation in human and experimental emphysema critical role of il- ra in immunopathology of influenza infection maintenance therapy with budesonide and formoterol in chronic obstructive pulmonary disease efficacy and tolerability of budesonide/formoterol in one hydrofluoroalkane pressurized metered-dose inhaler in patients with chronic obstructive pulmonary disease: results from a -year randomized controlled clinical trial efficacy and safety of budesonide/formoterol in the management of chronic obstructive pulmonary disease medicine. neutrophils find smoke attractive beta -agonists potentiate corticosteroid-induced neutrophil survival key: cord- -ncvvmkca authors: labarque, g; van reeth, k; van gucht, s; nauwynck, h; pensaert, m title: porcine reproductive–respiratory syndrome virus infection predisposes pigs for respiratory signs upon exposure to bacterial lipopolysaccharide date: - - journal: vet microbiol doi: . /s - ( ) - sha: doc_id: cord_uid: ncvvmkca this study examined whether an infection with porcine reproductive and respiratory syndrome virus (prrsv) potentiates respiratory signs upon exposure to bacterial lipopolysaccharides (lps). five-week-old conventional pigs were inoculated intratracheally with the lelystad strain of prrsv and received days later one or two intratracheal lps administrations. the necessary controls were included. after lps administration, pigs were intensively monitored for clinical signs. additionally, some pigs were euthanatized after a second lps administration for broncho-alveolar cell analysis and virological examinations of the lungs. broncho-alveolar lavage (bal) cells were counted and differentiated. lung suspensions and bal fluids were titrated for prrsv. exposure of pigs to prrsv only resulted in a fever for time periods ranging from to days and slight respiratory signs. exposure of pigs to lps only resulted in general signs, characterized by fever and depression, but respiratory signs were slight or absent. prrsv–lps exposed pigs, on the other hand, developed severe respiratory signs upon lps exposure, characterized by tachypnoea, abdominal breathing and dyspnoea. besides respiratory signs, these pigs also showed enhanced general signs, such as fever and depression. lung neutrophil infiltration was similar in non-infected and prrsv-infected pigs upon lps exposure. prrsv quantities were similar in lungs and bal fluids of pigs infected with prrsv only and prrsv–lps exposed pigs. these data show a clear synergism between prrsv and lps in the induction of respiratory signs in conventional pigs. the synergism was observed in % of the pigs. so, it can be considered as reproducible and may be used to test the efficacy of preventive and therapeutic measures. porcine reproductive and respiratory syndrome virus (prrsv), an arterivirus, causes infections in pigs worldwide. the virus replicates highly in the respiratory tract and shows a distinct tropism for broncho-alveolar macrophages (duan et al., ) . however, a single prrsv infection, particularly under experimental circumstances and with european isolates, fails to induce overt respiratory disease (done and paton, ) . also under field circumstances, most pigs become infected with prrsv at growing age without respiratory disease. still, the frequency and severity of respiratory disease have increased since the enzootic occurrence of prrsv (done and paton, ) . this has stimulated research into the combined effects of prrsv and other infectious agents. consequently, experimental dual infections have been performed with prrsv followed by various bacteria such as haemophilus parasuis, pasteurella multocida, streptococcus suis, bordetella bronchiseptica, and salmonella choleraesuis (cooper et al., ; galina et al., ; brockmeier et al., ; wills et al., ) . we ourselves have performed dual infections with prrsv followed by enzootic viruses, notably porcine respiratory coronavirus (prcv) or swine influenza virus (siv) (van reeth et al., ) . the clinical effects of these combinations were extremely severe in some cases, but almost completely subclinical in other. most important, none of the dual infections mentioned provides a reliable model to study pathogenetic features or to test control measures. we hypothesized therefore that the clinical outcomes of dual inoculations with two infectious agents are influenced by factors that are too difficult to control, such as the stage of replication and the viral or bacterial load. bacterial lipopolysaccharides (lps) or endotoxins, a major constituent of the cell wall of gram-negative bacteria, are released in high concentrations in the lungs upon infection with gram-negative bacteria (lamp et al., ; kadurugamuwa and beveridge, ) and these endotoxins are present in varying concentrations in dust in swine buildings (rylander, ; zejda et al., ) . the release of lps by gram-negative bacteria, such as h. parasuis, p. multocida, b. bronchiseptica, and s. choleraesuis may explain the more severe disease in the experimental dual infections with prrsv and these bacteria (cooper et al., ; brockmeier et al., ; wills et al., ) . van reeth et al. ( ) recently demonstrated that dual inoculations with prcv followed by bacterial lps seriously aggravate respiratory signs in gnotobiotic pigs, while the respective single inoculations were subclinical. therefore, we wanted to examine if exposure of prrsvinfected pigs to lps similarly enhances respiratory signs. prrsv may lend itself excellently as a predisposing agent for synergism with lps, because all pigs become infected at ages varying from weeks to fattening age (albina et al., ; houben et al., ) . also, prrsv persists in the lungs for (labarque et al., ) to (mengeling et al., ) days. we have examined the clinical course of inoculations with prrsv followed by lps, and the effect of the timing and frequency of lps administrations. additionally, some preliminary investigations of cellular and virological aspects in the lungs were performed. a fifth passage on pulmonary alveolar macrophages (pams) of the lelystad strain of prrsv (wensvoort et al., ) was used in this study. the inoculation dose was . tcid /pig. escherichia coli lps (o :b ) was obtained from difco laboratories and used at a dose of mg/kg body weight. this dose was based on data from previous experiments in gnotobiotic pigs, and selected to cause no respiratory signs . forty-six conventional pigs, originating from prrsv-negative sows, were used. pigs were weaned at weeks of age and placed in isolation. they were allowed to acclimatize during days before initiation of the experiments. prrsv inoculations and lps administrations occurred intratracheally as described by van reeth et al. ( ) . briefly, the pigs were held in vertical position with their neck extended. a needle was inserted through the skin cranial to the sternum and the inoculum was injected. the intratracheal administration was chosen to ensure that all the pigs received exactly the same dose in the lungs. three experiments were performed. in the first experiment, pigs were inoculated with prrsv and received one lps administration days later. seven pigs were inoculated with prrsv only. eight pigs, not previously inoculated with prrsv, received one lps administration. clinical monitoring was performed daily during consecutive days after prrsv inoculation and every h during the first h after lps administration. in the second experiment, eight pigs were inoculated with prrsv and, days later they received two lps administrations with a h interval. four pigs, not previously inoculated with prrsv, received two lps administrations with a h interval. clinical monitoring was performed daily during consecutive days after prrsv inoculation and at , , , and h after the second lps administration. in the third experiment, out of the prrsv-lps exposed pigs, described in the first experiment, received a second lps administration, h after the first one. these pigs were divided in two subgroups. one subgroup of six pigs was again monitored for clinical signs every h until h after the second lps administration. one subgroup of five pigs was euthanatized between and h after the second lps administration for study of the broncho-alveolar lavage (bal) cell population and for virological and bacteriological examinations of the lungs. from the seven prrsv control pigs, described in the first experiment, four pigs were again monitored for clinical signs every h for h at day after prrsv inoculation. the remaining three pigs were euthanatized at time points corresponding to those of the prrsv-lps exposed pigs and served as controls for the broncho-alveolar cell and virological examinations. all eight lps exposed pigs, described in the first experiment, received a second lps administration, h after the first one. four pigs were again monitored for clinical signs every h until h after the second lps administration and four pigs were euthanatized between and h after the second lps administration for broncho-alveolar cell and virological examinations. four non-inoculated pigs were euthanatized for the same purpose. pigs were monitored for general signs, notably fever and depression, and for respiratory signs, notably tachypnoea, abdominal breathing and dyspnoea. scores were given for these five clinical parameters. body temperatures . c were scored as , temperatures between ! . and . c were scored as and temperatures ! . c were scored as . respiration rates were scored as , rates between ! and were scored as and rates ! were scored as . depression, abdominal breathing and dyspnoea were scored as (absent) or (present). scores were added up and a mean of the cumulative general and respiratory scores per group was calculated. at necropsy, the lungs were removed. the right lung was used for broncho-alveolar cell examination after bal using the method described by van reeth et al. ( ) . the bal fluid was centrifuged (  g, min, c) to separate the cells and the cell-free lavage fluid. aliquots of the cell-free lavage fluid were stored at À c until virus titration on pams. bal cells were counted in a türk chamber and cytocentrifuge preparations were stained with diffquik (baxter, düdingen, switzerland) to determine the percentage of mononuclear cells and neutrophils. the left lung was used for virological and bacteriological examinations. twenty percent suspensions of lung lobes were made in a phosphate-buffered saline, clarified by centrifugation and the supernatant was used for prrsv titration. virus titration of lung suspensions or bal fluids was performed on pams, as described by labarque et al. ( ) . for bacteriology, samples of lung tissue were plated on bovine blood agar and cultured aerobically. a nurse colony of coagulase-positive staphylococcus species was streaked diagonally on each plate. plates were inspected for bacterial growth after and h. colonies were then identified by standard techniques. non-parametric tests were used, because of lack of normality in the data. standard twosample mann-whitney tests were used to compare general and respiratory clinical scores between groups. p < : was taken as the level of statistical significance. statistical analyses were performed using spss . . twenty-six of the total of prrsv-infected pigs developed fever for time periods ranging from to days. respiratory signs were absent, except for two pigs, which showed tachypnoea and abdominal breathing. in experiment , six of the seven prrsv control pigs showed fever until the end of the monitoring period. respiratory signs were slight in one pig and absent in the other pigs. the mean respiratory score was . (table ) . in experiment , all four prrsv control pigs showed fever until the end of the monitoring period. respiratory signs, characterized by increased respiration rates, were observed in one of the four pigs. the mean respiratory score was . (table ) . in non-infected pigs, a single lps administration induced transient general signs (fig. ) . respiratory signs were slight or absent and the mean respiratory score was only . ( table ). in prrsv-infected pigs, however, lps induced severe clinical signs with fever table mean general and respiratory scores after the last lps administration in prrsv-lps exposed pigs and their controls in all the pigs and respiratory signs in % of the pigs (fig. ) . respiratory signs were characterized by tachypnoea (peak breaths/min), abdominal breathing and dyspnoea and lasted until the end of the monitoring period. two out of the pigs did not show respiratory signs after lps administration. mean general and respiratory scores were significantly higher in prrsv-lps exposed pigs than in singly lps exposed pigs (table ) . in non-infected pigs, which received two lps administrations with a h interval, both general and respiratory signs were observed (fig. ) . clinical signs were significantly higher in prrsv-infected pigs not only with regard to the number of affected pigs but also with regard to the clinical scores. all pigs reacted severely. the mean clinical scores after the second lps administration are presented in table . non-infected pigs had recovered at the time of the second lps administration, h after the first one. this second lps administration again induced general signs within h, but no respiratory signs (fig. ) . prrsv-infected pigs had not yet recovered h after the first lps administration (fig. ) . the second lps administration however increased the number of pigs with general and respiratory signs and mean clinical scores (fig. ) . here again, mean general and respiratory scores were significantly higher in prrsv-lps exposed pigs than in singly lps exposed pigs. total bal cell numbers and differentials are shown in table . bal cell numbers and differentials in prrsv-lps exposed pigs were essentially similar to those of pigs, exposed to prrsv or lps only. however, there was great individual variation within all three groups. mean prrsv titres in lungs and bal fluids are shown in table . virus titres were similar in lungs and bal fluids of singly prrsv-inoculated pigs and prrsv-lps exposed pigs. the lungs and bal fluids of lps controls and non-inoculated controls were negative for prrsv. bacterial culture of lung tissue yielded negative results for all pigs. it has become generally accepted that prrsv plays an important role in respiratory disease problems in the field, particularly in multi-factorial respiratory disease. however, it has been most difficult to reproduce respiratory signs in experimental infection studies with prrsv and a second infectious agent. the present prrsv-lps combination induces clear respiratory signs in % of the pigs. unlike in our previous studies with prrsv-siv table bal cell study of prrsv-lps exposed pigs and their controls at - h after a second lps administration table virological study of lungs and bal fluids of prrsv-lps exposed pigs and their controls at days after prrsv inoculation exposure number of pigs mean prrsv titres (range) lungs (log tcid /g) bal fluids (log tcid /ml) prrsv-lps . ( . - . ) . ( . - . ) prrsv only . ( . - . ) . ( . - . ) lps only negative negative none negative negative and prrsv-prcv combinations (van reeth et al., ) , mean clinical scores were higher than those of control pigs in every experiment. only two out of the prrsvinfected pigs did not develop respiratory signs upon lps exposure. it is important to mention, however, that individual variation in disease severity is unavoidable with respiratory pathogens. such an individual variation has even been reported in experimental infection studies with primary respiratory pathogens such as actinobacillus pleuropneumoniae (baarsch et al., ) or siv (van reeth et al., ) . we used two lps administrations with the purpose to extend the duration of clinical signs. the clinical effect of a second lps administration was dependent on the time interval between the two lps administrations. in non-infected pigs, a second lps administration at a h interval caused milder clinical signs than the first one. on the other hand, a second lps administration within a h interval seriously aggravated and prolonged general and respiratory signs. these observations suggest that two lps administrations within a short time interval lead to an accumulation of lps in the lungs. indeed, it has been demonstrated that the clinicopathological manifestations of lps are strictly dose-dependent. for example, if sufficient amounts of lps are given to animals and man, cytokine induction, lung inflammation and decreased lung function are observed. slightly smaller lps doses, on the other hand, will cause only a mild lung inflammation (michel et al., ) . in prrsv-infected pigs, the clinical effect of a second lps administration was difficult to assess since pigs had not yet recovered at the moment of the second lps administration, or h after the first one. there is little information on the effect of repeated lps administrations to farm animals in the literature. it appears logical, however, that the mediators or mechanisms responsible for the clinical effects of lps may become exhausted if high lps doses are administered frequently. respiratory signs following prrsv-lps exposure could not be explained by the extent of virus replication or inflammatory changes in the lungs. indeed, virus titres were similar in prrsv-lps or singly prrsv-inoculated pigs. total bal cell numbers and neutrophil infiltration were similar in prrsv-lps or singly lps exposed pigs. also, the two prrsv-lps exposed pigs, which remained healthy, had similar bal cell profiles as their clinically affected group mates. this suggests that inflammatory changes in the lungs have little or no effect on the synergism between prrsvand lps. we hypothesize therefore that functional lung changes, such as bronchial hyper-responsiveness, are more important in the pathogenesis of prrsv-lps induced disease than structural changes. similar findings were made in a previous experimental infection study with prcv followed by lps . in this study, disease development was tightly correlated with lung production of proinflammatory cytokines, among which tumor necrosis factor-alpha (tnfa). interestingly, tnf-a has been shown to cause bronchial hyper-responsiveness in laboratory animal models (kips et al., ; thomas et al., ) . under field circumstances, most pigs become infected with prrsv at growing age and they are continuously exposed to airborne endotoxins. also, during gram-negative infections of the lungs, excessive amounts of endotoxins are released locally. the present prrsv-lps infection model therefore is relevant for the study of prrsv-induced respiratory problems in the field. the synergism was observed in % of the pigs. so, it can be considered as reproducible and may be used to test the efficacy of preventive and therapeutic measures. immune response and persistence of the porcine reproductive and respiratory syndrome virus in infected pigs and farm units pathophysiologic correlates of acute porcine pleuropneumonia effects of intranasal inoculation 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reproductive and respiratory syndrome virus in the lungs of pigs influence of antibiotic and e monoclonal immunoglobulin m interactions on endotoxin release from escherichia coli and pseudomonas aeruginosa diagnosis of porcine reproductive and respiratory syndrome dose-response relationship to inhaled endotoxin in normal subjects endotoxins tumor necrosis factor-a increases airway responsiveness and sputum neutrophilia in normal human subjects dual infections of feeder pigs with porcine reproductive and respiratory syndrome virus followed by porcine respiratory coronavirus or swine influenza virus: a clinical and virological study broncho-alveolar interferon-a, tumor necrosis factor-a, interleukin- and inflammation during acute influenza in pigs: a possible model for humans? a potential role for tumour necrosis factor-a in synergy between porcine respiratory coronavirus and bacterial lipopolysaccharide in the induction of respiratory disease in pigs mystery swine disease in the netherlands: the isolation of lelystad virus synergism between porcine reproductive and respiratory syndrome virus (prrsv) and salmonella choleraesuis in swine respiratory health status in swine producers relates to endotoxin exposure in the presence of low dust levels this work was supported by grant a from the belgian ministry of agriculture. the authors would like to thank fernand de backer, krista de winne, lieve sys, chantal vanmaercke and carla de winter for excellent technical assistance. geoffrey labarque was supported by grant o d from the research council of the ghent university. key: cord- -ew uid e authors: he, xinran; li, wangen; xie, yunliang; zhao, yunjuan title: long-term inhibition of dipeptidyl-peptidase reduces islet infiltration and downregulates il- β and il- in nod mice date: - - journal: int immunopharmacol doi: . /j.intimp. . sha: doc_id: cord_uid: ew uid e dipeptidyl-peptidase (dpp- ) inhibitor (sitagliptin) is a novel anti-hyperglycemia drug in the treatment of type diabetes. however, its potential in type diabetes is still unclear. recent studies show that increased infection, especially respiratory tract infection, is significantly associated with dpp- inhibitors. in this study, we aimed to explore the effects of long-term inhibition of dpp- on innate immunity in type diabetes. forty mice were randomly divided into groups (n = in each group): control group, lipopolysaccharide (lps) group, sitagliptin group and sitagliptin + lps group. the concentrations of il- β, il- , il- , il- , il- , il- , il- , tnf-α and ifn-γ were measured with mesco scale discovery multiplexed-assay kit. immunohistochemistry staining of pancreases was performed and insulitis scores for each islet were determined. the results showed that dpp- inhibition has no effect on incident rate of diabetes and metabolic parameters in nod mice. long-term inhibition of dpp- reduced cd +t cells to infiltrate into islets and ameliorated insulitis in nod mice. dpp- inhibition downregulated serum interleukin il- β and il- in nod mice. however, it had no significant effect on lps-induced il- β, il- , il- , il- , tumor necrosis factor (tnf)-α and interferon (ifn)-γ in nod mice. in conclusion, long-term inhibition of dpp- exists anti-inflammatory effect in type diabetes probably by reducing cd +t cells to infiltrate into islets and downregulating l- β and il- in serum. type diabetes is an organ-specific autoimmune disease characterized by the selective destruction of islet β-cell destruction and dysfunction of insulin secretion [ ] . over the past two decades, studies about the pathogenesis of type diabetes focus on adaptive immunity. wen l et found that myd deficiency prevents the onset of type diabetes in nonobese diabetic (nod) mice (an automatic autoimmune diabetes mouse model) [ ] . their further study shows that deficiency of the nucleotidebinding oligomerization domain, leucine-rich containing family and pyrin domaincontaing protein (nlrp ) prevents type diabetes by inhibiting pathogenic t cell migration to the islets in nod mice [ ] . these studies suggest a critical role of innate immunity in the pathogenesis of type diabetes. toll-like receptors (tlrs) are pattern recognition receptors which plays a key role in innate immune responses [ ] . tlr is considered the core molecular directly modulating innate immunity with subsequent activation of adaptive immunity [ ] . tlr is localized on the cell surface and functions as a specific receptor for lipopolysaccharide (lps) [ ] . lps binds to tlr , which causes activation of myd and trif adaptor molecules, leading to secretion of inflammatory cytokines il- β, tnf-α and il- [ ] . tlrs primarily regulates both pro-and antidiabetic signals which are triggered by microbiota in type diabetes [ ] . tlr selectively damages β cells and involves initiation of type diabetes in nod mice [ ] . tlr- knockout downregulates myd levels, nf-κb activity and reduced macrophage to secrete il- , il- β, tnf-α, and ifn-β in streptozotocin-induced diabetic mouse [ ] . tlr -ab reserves type diabetes, alleviates insulitis and preserves pancreatic islets in nod mice [ ] . dipeptidyl peptidase- (dpp- ) inhibitor is a kind of anti-diabetic drug for the treatment of type diabetes. in recent years, many studies have tried to use dpp- inhibitors in type diabetes. dpp- inhibitor and quercetin co-administration alleviates insulitis and improves glucose metabolism in type diabetic rats [ ] . our previous study shows that sitagliptin preserves β cell function in patients with latent-autoimmune diabetes in adults [ ] . combination therapy with anti-cd and mk increases cd + cd + foxp + t cells in pancreatic draining lymph nodes, alleviates insulitis and preserves islet β -cell function in recent-onset diabetic nod mice [ ] . to investigate the immunoregulatory effect of dpp- inhibitor, pinheiro mm et al cultured peripheral blood mononuclear cells of healthy volunteers with sitagliptin, and they found that sitagliptin suppresses lymphocytes proliferation and inhibits the differentiation of th , th and th cells [ ] . these studies suggest dpp- inhibitor is a promising drug for type diabetes, probably involving its immunoregulatory effect. however, there are very few studies about the effect of dpp- inhibitor on innate immunity in type diabetes [ ] . a nested case-control study based on adverse drug reaction database of world health organization (who) shows that increased infection (especially respiratory tract infection) is significantly associated with dpp- inhibitors, which suggests dpp- inhibitors may play a potential role in innate immunity [ ] . in this study, we aimed to explore the effect of long-term dipeptidyl-peptidase inhibition on innate immunity in type diabetes. all experiments complied with institutional guidelines and was approved by institutional animal ethics committees of guangzhou medical university. female nod mice ( - -wk-old) (nanjing biomedical research institute of nanjing university) were used in the experiment. all mice were housed in an spf environment and had free access to water and food. mice were maintained in each cage and received light for h every day. all animal procedures were approved by the animal ethics committee of guangzhou medical university. mice (n = ) were randomly divided into groups (n = per group): control group, lps group, sitagliptin group and sitagliptin + lps group. among the groups, mice in sitagliptin and sitagliptin + lps group received intragastric administration with sitagliptin ( mg/kg) (januvia ®, merck pharmaceuticals) every day. mice in control group and lps group received intragastric administration with an equal volume of . % saline. the mice were treated for weeks. the information random blood glucose, weight, food intake and water intake were collected each week. the mice in lps group and sitagliptin + lps group received intraperitoneal injection of lps ( mg/kg) (lipopolysaccharide, sigma, usa) for h before sacrificing. bodyweight, food intake and water intake were measured once a week. blood samples were collected from the tail vein of the mice for the measurement of blood glucose with a glucometer (accu-chek performa). random capillary blood glucose was determined once a week. if two consecutive glucose levels ≥ . mmol/l, the mouse was diagnosed with diabetes. the intraperitoneal glucose tolerance test (ipgtt) was performed on nod mice at week ( days after treatment with sitagliptin). for the ipgtt, mice were fasted for h and blood glucose were measured at , , and min after intraperitoneal injections of glucose ( g/ kg, % glucose solution). blood samples were collected before sacrificing the mice. serum was separated by centrifugation and stored at − ℃ until assayed. serum levels of pro-inflammatory and inflammatory cytokines were determined with mesco scale discovery multiplexed-assay kit (meso scale discovery, gaithersburg, md, usa). serum levels of il- β, il- , il- , il- , il- , il- , il- , tnf-α and ifn-γ were measured according to the references [ ]. . . histology, immunohistochemistry and insulitis score . . . h&e staining pancreases were harvested at weeks after treatment with sitagliptin. after isolation, pancreas tissue was fixed with % paraformaldehyde and embedded in paraffin. h&e staining was performed. histology slides were observed using a microscope (bx - p -sd , olympus) and photomicrographs were captured using canon utilities software. insulitis scores for each islet were determined as follows: , no mononuclear cells infiltrate in islet; , mononuclear cells infiltrate around the islet but not in islet; , mononuclear cells infiltrate in and around the islet, but the infiltrated area is less than one-third of the islet; , intra-islet infiltration of mononuclear cells is / to / of the islet area; , intra-islet infiltration of mononuclear cells is more than / of the islet area [ ] . immunohistochemistry was done according to the reference [ ] . primary specific antibodies for cd (gb - , : dilution, servicebio, wuhan, china), cd (gb , : dilution, servicebio, wuhan, china), cd b (gb , : dilution, servicebio, wuhan, china), cd c(gb , : dilution, servicebio, wuhan, china), second antibody: hrp-labeled goat anti-rabbit igg(gb , : dilution, servicebio, wuhan, china), il- (gb , : dilution, servicebio, wuhan, china), il- (pa ml, : dilution, servicebio, wuhan, china). the positive express was shown by diaminobenzidine (dab, g , brown color, servicebio, wuhan, china) after second antibody incubation. an immunohistochemical evaluation was conducted by two pathologists blindly. we randomly take three areas at least for each sample to take photograph at a magnification of and (figs. and ). the mean of values obtained in these areas was used for data analysis. all these data were presented as mean ± standard error of the mean. data were analyzed by using the spss . software (ibm spss statistics). diabetes incidence was compared between different groups using chi-square test. anova or non-parametric tests were used to compare differences between group, depending on the distribution and characteristics of data. p value < . was considered statistically significant. nod mice were diagnosed with diabetes after two sequential glucose levels ≥ . mmol/l. in the end of the experiment, there are mice alive in control group, mice alive in lps group, mice alive in sitagliptin group and mice alive in sitagliptin + lps group. by weeks, the incident rate of diabetes is % in control group, % in lps group, % in sitagliptin group and % in sitagliptin + lps fig. . a, sitagliptin has no effect on incident rate of diabetes in nod mice. fig. b -e, sitagliptin has no effect on metabolic parameters in nod mice. we measured body weight, food intake, water intake, and random blood glucose in nod mice. sitagliptin has no significant difference on body weight, food intake, water intake, and random blood glucose. fig. f , sitagliptin/sitagliptin + lps has no effect on glucose tolerance in nod mice. ipgtts were performed with glucose ( g/kg) in week-old non-diabetic or diabetic nod mice in control group (n = ), sitagliptin group (n = ), lps group (n = ) and sitagliptin + lps group (n = ). the glucose tolerance has no significant difference between the groups (p > . ). group, which has no significant difference between the groups (fig. a) . we measured body weight, food intake, water intake, and random blood glucose in nod mice. dpp- inhibitor has no effect on body weight, food intake, water intake, and random blood glucose (fig. b, c, d and e). ipgtt was performed at weeks after sitagliptin treatment. there was no significant difference in blood glucose level during an ipgtt between the groups ( fig. f) . pancreases of nod mice were separated and stained with h&e. islets of the mice in control group, lps and sitagliptin + lps group long-term inhibition of sitagliptin ameliorates insulitis in nod mice. a insulitis scores were calculated as follows: , no mononuclear cells infiltrate in islet; , mononuclear cells infiltrate around the islet but not in islet; , mononuclear cells infiltrate in and around the islet, but the infiltrated area is less than one-third of the islet; , intra-islet infiltration of mononuclear cells is / to / of the islet area; , intra-islet infiltration of mononuclear cells is more than / of the islet area. b. representative images of pancreases were stained with h&e. the arrow points to the islets infiltrated with mononuclear cells. magnification of images: × . showed widespread infiltration of mononuclear cells. mice receiving sitagliptin showed significantly less infiltration of mononuclear cells, compared with control group ( fig. a and b) . these findings were furthered confirmed by insulitis scoring, which suggested that longterm dpp- inhibition ameliorates insulitis in nod mice. to explore the effect of sitagliptin on the pro-inflammatory and inflammatory cytokines in nod mice, we determined the secretion of il- β, il- , il- , il- , il- , il- , il- , tnf-α and ifn-γ in the serum of nod mice. sitagliptin significantly downregulates serum il- β and il- in nod mice in sitagliptin group compared with control group, lps group and sitagliptin + lps group (p < . ) (fig. a and b) . in order to explore the effect of long-term inhibition of dpp- on the expression of il- and il- in pancreases, we determined il- and il- levels by immunohistochemistry (fig. c) . unfortunately, there was no significantly difference in sitagliptin, compared with control group, lps group and sitagliptin + lps group (p> . ) (fig. a and b) . to identify the effect of pretreatment with sitagliptin on lps-induced inflammation, we analyzed the levels of il- β, il- , il- , il- , il- , il- , il- , tnf-α and ifn-γ in the serum of nod mice between sitagliptin and sitagliptin + lps group. there was no difference of the levels of il- β, il- , il- , il- , il- , il- , il- , tnf-α and ifn-γ between sitagliptin and sitagliptin + lps group (fig. c, d, e , f, g, h and i). it suggests that pretreatment with sitagliptin has no effect on the levels of il- β, il- , il- , il- , il- , il- , il- , tnf-α and ifn-γ in the serum of nod mice. the immune cells infiltrated into islets and led insulitis in the characterization of type diabetes. in the present study, long-term inhibition of dpp- reduced cd +t cells to infiltrate into islets in nod mice (p< . ) (fig. a and e) . lps injection induces cd b + cells to fig. c -i, pretreatment with sitagliptin has no effect on lpsinduced il- β, il- , il- , il- , tnf-α and ifn-γ in nod mice (p > . ). international immunopharmacology ( ) infiltrate into islets in nod mice ( fig. c and e) . long-term inhibition of dpp- had no effect on cd + t and cd c + cells infiltration in islets in nod mice ( (fig. b , d and e) (p> . ). in the present study, long-term inhibition of dpp- significantly reduces cd +t cells to infiltrate into islets, ameliorates insulitis and downregulates serum levels of il- β and il- in nod mice. long-term treatment of sitagliptin, is more effective than insulin in β cell preservation for at least years in type diabetes, probably due to the immunoregulatory effect of sitagliptin [ ] . however, there are few studies about the immunoregulatory effect of dpp- inhibitors on innate immunity in type diabetes. recently, davanso mr et al. found that treatment of dpp- inhibitor for days had no effect on cd + cd +t cells and cd + cd + t cells in the spleen in streptozotocin-induced type diabetic mouse [ ] . however, in the present study, we found that long-term inhibition of dpp- (more than fig. . long-term inhibition of dpp- has no effect on il- and il- expression in islets in nod mice. a. long-term inhibition of dpp- has no effect on il- expression in islets in nod mice (p> . ). b. long-term inhibition of dpp- has no effect on il- expression in islets in nod mice (p> . ). c. protein expression was assessed by immunohistochemistry staining. brown color was considered positive. (n = in control group, n = in lps group, n = in sitagliptin group, n = in sitagliptin + lps group). months) significantly reduces cd +t cells to infiltrate into islets and ameliorates insulitis. many immunoregulatory drugs were effective in early stage of type diabetes, however, very few drugs were still effective in the late stage of disease. in the present study, we used sitagliptin to treat nod mice for weeks (more than months), which showed to reduce cd +t cells infiltration. it is a highlight of our study. long-term inhibition of dpp- had no effect on cd + t and cd c + cells infiltration in islets in nod mice. lps injection induces cd b + cells to infiltrate into islets in nod mice, which suggested lps induced inflammation in type diabetes. il- gene may involve in the pathogenesis of type diabetes [ , ] . administration of high dose il- β significantly increases the onset of type diabetes in the biobreeding rat [ ] . il- signaling plays an important role in β cell dysfunction probably via the mitogen activated protein kinase (mapk) and nf-κb pathway [ ] . il- block in the early stage of type diabetes prevents virus-triggered insulitis in kilham rat virus (krv)-induced diabetes [ ] . in the present study, we found the od value of serum il- β by msd in sitagliptin group is significantly decreased compared with control group, which suggests sitagliptin downregulates serum il- βlevel. meanwhile, we found sitagliptin protects βcell function in nod mice. the decrease of il- βpossibly contributes to the protection of βcell function in nod mice. il- is mainly produced by phagocytes (macrophages, monocytes and neutrophils) and dendritic cells [ ] . it is a key proinflammatory cytokine which promotes naïve cd + t cells to differentiate into t helper (th ) cells [ ] . it is considered as a link between innate and adaptive immunity [ ] . in the present study, we found sitagliptin downregulates serum il- level, which might play some role in innate and adaptive immunity in nod mice. il- is positively associated with risk of type diabetes [ , , ] . il- mrna expression of mononuclear leukocytes isolated from islets is correlated with β cell destruction in nod mice [ ] . administration of il- induces severe lymphocyte infiltration in the pancreatic islets and accelerates type diabetes in nod mice [ ] . in the present study, we found sitagliptin downregulates il- β and il- in the serum, but not in the pancreases, which might own to the different distribution of these cytokines in the body. down regulation of il- β and il- was probably related with amelioration of insulitis by an indirect effect in type diabetes. in the present study, we determined serum levels of il- , il- , il- , il- , il- , tnf-α and ifn-γ, which shows no significant difference between sitagliptin and control group. there were few studies about the effect of dpp- inhibitor on cytokine profile in autoimmune diseases. dpp- inhibitor (pkf - ) does not affect il- in kidney and serum il- in sprague-dawley (sd) rats of type diabetes [ ] . sitagliptin treatment for days decreased tnfand il- in pancreatic homogenate in streptozotocin-induced type diabetic mice [ ] , which was different with our result. we considered it was owing to: ( ) the concentrations of tnfand il- were higher in pancreas than in serum in type diabetes; ( ) there were different effects of cytokines in different stages of disease. marjolein j et al. found that increased infection (especially respiratory infection) is significantly associated with dpp- inhibitors [ ] . corona virus infectious disease (covid- ) is a new epidemic disease with high mortality. diabetes is one of the most common comorbidities among the patients with covid- [ ] . the patients suffered from severe covid- with diabetes had higher mortality than patients without diabetes [ ] . icu patients infected with covid- had higher inflammatory cytokines such as il- , il- , il- and tnf-, compared with non-icu patients [ ] . dpp- has been identified as a functional receptor for human coronavirus-erasmus medical center [ ] , which is essential for coronavirus infection [ ] . it means there is intense contact between dpp- and coronavirus infection. mice expressing human dpp- were susceptible to middle east respiratory syndrome coronavirus (mers-cov) [ ] . human dpp- transgenic diabetic mice exhibit prolonged severe disease and immune dysregulation following mers-cov infection [ ] . however, rinkoo dalan presents that dpp- inhibitors might not be beneficial to covid- infection for the following reasons: ( ) dpp- i may not effectively reduce infection transmission; ( ) dpp- i may be a disadvantage for suppressing t cell immunity; ( ) dpp- i may increase fibrotic lesion in the lung; ( ) dpp- i recruits regenerative stem cells; ( ) dpp- i is related to a prothrombotic state [ ] . to date there is no direct consequences between covid- infection and dpp- inhibitors. a case control study shows that treatment with dpp- inhibitors in t d patients with covid- has no influence to disease outcome, compared with those without dpp- inhibitors treatment [ ] . because the intense contact of dpp- and coronavirus infection, further research between dpp- i and coronavirus in diabetes is warranted. stervbo et al found that gravitation stress decreased serum levels of inflammatory mediators and they provides promising design to assess the effect of cytokines with or without lps stimulation ex vivo [ ] . in this study, we injected intraperitoneally the mice in lps group and sitagliptin + lps group with lps ( mg/kg) h before sacrificing these mice to initiate innate immune response. after lps injection, the serum concentration of il- β, il- , il- , il- , tnf-α and ifn-γ is significantly increased. however, pretreatment with sitagliptin for weeks does not decrease the production of pro-inflammatory cytokines, probably owing to the following reasons: first, lps is strong activator of serious inflammatory reaction, which cann , t be reversed by pretreatment of sitagliptin; second, treatment with sitagliptin is before lps injection, which acts like effects of pretreatment. long-term inhibition of dpp- exists anti-inflammatory effect in type diabetes probably by reducing cd +t cells to infiltrate into islets and downregulating l- β and il- in serum. the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. . long-term inhibition of dpp- significantly reduced cd +t cells to infiltrate into islets in nod mice. a. compared with control group, sitagliptin treatment significantly reduced cd +t cells to infiltrate into islets in nod mice (p< . ). administration of lps following with sitagliptin treatment also reduced cd +t cells to infiltrate into islets in nod mice (p< . ). b. sitagliptin treatment has no effect on cd + t cells infiltration in islets (p> . ). c. administration of lps following with sitagliptin treatment increased cd b + cells to infiltrate into islets in nod mice, compared with control group and sitagliptin treatment alone (p< . ). administration of lps increased cd b + cells to infiltrate into islets in nod mice, compared with control group (p< . ). d. sitagliptin treatment has no effect on cd c + cells infiltration in islets (p> . ). e, protein expression assessed by immunohistochemistry staining of the immune cells infiltrating into pancreatic islets, for cd +t cells, cd + t cells, cd b + cells as well as cd c + cells. brown color was considered positive. cd +t cells, cd + t cells and cd c + cells were the dominant immune cell types infiltrating into the islet. normal islet structure was destroyed when immune cells infiltrated into the islets. 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covid- with diabetes clinical features of patients infected with novel coronavirus in dipeptidyl peptidase is a functional receptor for the emerging human coronavirus-emc acute respiratory infection in human dipeptidyl peptidase -transgenic mice infected with middle east respiratory syndrome coronavirus comorbid diabetes results in immune dysregulation and enhanced disease severity following mers-cov infection is dpp inhibition a comrade or adversary in covid- infection exposure to dpp- inhibitors and covid- among people with type diabetes. a case-control study repeated changes to the gravitational field negatively affect the serum concentration of select growth factors and cytokines we thank for the technicians of animal house of guangzhou medical university for taking care of our mice. the study was supported by national natural science foundation of all experiments were complied with institutional guidelines and approved by institutional animal ethics committees of guangzhou medical university. supplementary data to this article can be found online at https:// doi.org/ . /j.intimp. . . key: cord- -s c h lm authors: hong, joung-woo; yang, ga-eun; kim, yoon bum; eom, seok hyun; lew, jae-hwan; kang, hee title: anti-inflammatory activity of cinnamon water extract in vivo and in vitro lps-induced models date: - - journal: bmc complement altern med doi: . / - - - sha: doc_id: cord_uid: s c h lm background: cinnamon bark is one of the most popular herbal ingredients in traditional oriental medicine and possesses diverse pharmacological activities including anti-bacterial, anti-viral, and anti-cancer properties. the goal of this study is to investigate the in vivo and in vitro inhibitory effect of cinnamon water extract (cwe) on lipopolysaccharide (lps)-induced tumor necrosis factor (tnf)-α and its underlying intracellular mechanisms. methods: cwe was orally administrated to mice for days prior to intraperitoneal injection of lps. serum levels of tnf-α and interleukin (il)- were determined hour after lps stimulation. peritoneal macrophages from thioglycollate-injected mice were isolated and assayed for viability, cytokine expression and signaling molecules upon lps stimulation. cwe was further fractioned according to molecular size, and the levels of total polyphenols and biological activities of each fraction were measured. results: the oral administration of cwe to mice significantly decreased the serum levels of tnf-α and il- . cwe treatment in vitro decreased the mrna expression of tnf-α. cwe blocked the lps-induced degradation of iκbα as well as the activation of jnk, p and erk / . furthermore, size-based fractionation of cwe showed that the observed inhibitory effect of cwe in vitro occurred in the fraction containing the highest level of total polyphenols. conclusions: treatment with cwe decreased lps-induced tnf-α in serum. in vitro inhibition of tnf-α gene by cwe may occur via the modulation of iκbα degradation and jnk, p , and erk / activation. our results also indicate that the observed anti-inflammatory action of cwe may originate from the presence of polyphenols. the bark of cinnamon has been used not only as a spice and tea, but also as one of the key components of herbal remedies for the common cold, cardiovascular disease, and chronic gastrointestinal and gynecological disorders in oriental herbal medicine. accordingly, extensive studies on the pharmacological activities of the cinnamon bark have been conducted, indicating that cinnamon bark is involved in a vast range of pathological and physiological events. for instance, essential oil and water-based extracts from cinnamon have been shown to be effective against pathogenic microbes, viruses, and various types of tumor cell lines [ ] [ ] [ ] [ ] . furthermore, it has been also reported that cinnamon bark reduces the level of serum glucose through the enhancement of insulin-regulated glucose utilization in vivo [ , ] . inflammation is a protective response for the purpose of removal of exogenous and endogenous harmful substances produced by injurious stimuli and is a part of the healing process in wounded tissues [ ] . since proinflammatory cytokines such as tumor necrosis factoralpha(tnf-α), interleukin(il)- and il- , lipid mediators, proteases, and oxidants produced during the typical response can cause damage to normal tissues regardless of how and where the inflammatory response is triggered, the substances involved in the inflammatory response need to be tightly regulated. if the scavenging reaction is delayed, the inflammatory response may evolve into a variety of chronic inflammatory diseases, such as atherosclerosis, rheumatoid arthritis, asthma, and neurodegenerative diseases. a vast number of molecular studies have identified several target molecules involved in inflammatory changes, and most anti-inflammatory drugs currently used suppress the biosynthesis of the inflammatory mediators mentioned earlier [ ] . previous studies have indicated that the major pharmacological activities of cinnamon bark, such as its anti-bacterial, anti-inflammatory, anti-viral, and anticancer effects are derived from essential oils such as cinnamaldehyde [ , [ ] [ ] [ ] . however, since cinnamon bark has been typically used as in the form of a water extract, where the volatile ingredients are seldom found, it is likely that the established pharmacological activities of cinnamon bark depend on a mixture comprised of a variety of water-soluble components, thereby ensuring its safety as a traditional remedy. recently, it has been found that cinnamon bark water extract (cwe) elevates glucose uptake through the promotion of insulin sensitivity and inhibits angiogenesis through blocking vascular endothelial growth factor signaling [ , ] . these results indicate that the observed pharmacological activities may have originated from polyphenolic compounds in cwe. in this study, we investigated the in vivo and in vitro effects of cwe on lipopolysaccharide (lps)-induced tnf-α and its underlying intracellular mechanisms. we also fractioned cwe according to molecular size to determine whether there exists a positive correlation between the anti-inflammatory activity of cwe and the amount of polyphenolic compounds. cinnamon bark (cinnamomi cassia p resl ) of vietnamese origin was purchased from omni herb (daegu, south korea). the plant was identified by professor choi of the department of herbology at kyung hee university. a voucher specimen sample (cc- ) was deposited at the laboratory of herbology at kyung hee university. the plant was pulverized and soaked in one volume of water for hours at room temperature, and further dissolved by sonication for hour. the extract was filtered and evaporated using a freeze dryer (eyela, japan) at − °c. the yield of cwe was about . %. for size fractionation, . g of cwe was dissolved in ml of distilled water and fractions were collected using kda and kda amicon ultra centrifugal filter device (millipore, ireland). the yields of a low molecular weight (mw) fraction (below kda), a middle mw fraction (between kda and kda), and a high mw fraction (over kda) were %, % and % of cwe, respectively. all the final samples were dissolved in pbs and sterilized by passing through a . -μm syringe filter. eight-week-old male balb/c mice were purchased from the korean branch of taconic, samtaco (osan, korea) and fed rodent chow and water ad libitum in a temperature-and humidity-controlled pathogen-free animal facility at the medical center of kyung hee university hospital. mice were maintained in accordance with the guide for the care and use of laboratory animals issued by the us national research council ( ) , and the protocol khmc-iacuc - was approved by the kyung hee university medical center institutional animal care and use committee. in vivo lps injection cwe ( , or mg/kg of body weight) was given to mice via oral gavage for days. control mice received an equal volume of normal saline during the experimental period. each group consisted of mice. on day , lps (serotype :b ; sigma, st. louis, mo, usa) ( . mg/kg) was injected intraperitoneally hour before blood sampling. blood was obtained by cardiac puncture. as a reference drug, dexamethasone (sigma) ( mg/kg) was injected intraperitoneally hours before the lps injection. blood samples were centrifuged at g for min. the serum samples obtained were stored at − °c until used. for the use of in vitro culture of macrophages, normal mice were injected intraperitoneally with ml of sterile thioglycollate medium (bd, france), and macrophages were collected three days later by peritoneal gavage with cold dulbecco's modified eagle's medium (dmem). the recovered peritoneal fluid was washed by centrifugation. the cells were resuspended in dmem with % fetal bovine serum and incubated for hours at °c with % co . non-adherent cells were removed. cell viability was determined using the mtt method. macrophages were seeded in -well plates and treated with , , , , and μg/ml cwe in the presence or absence of lps for hours. ten microliters of mtt solution ( mg/ml) (sigma) was added to each well and, after hours of incubation, media was aspirated and μl of dimethyl sulfoxide (dmso) (sigma) was added. the optical density was read at nm using a microplate reader (molecular devices, sunnyvale, ca, usa). peritoneal macrophages were seeded in -well plates and pre-treated with cwe for hour, then stimulated with ng/ml lps for hours. total rna was isolated using an rneasy mini kit (qiagen, germany) and cdna was reverse-transcribed using superscript iii reverse transcriptase (invitrogen, carlsbad, ca, usa). diluted cdna was mixed with power sybr green pcr master mix (applied biosystems, foster city, ca, usa) and pmol of primers for tnf-α or gapdh and. the following forward and reverse primer sequences were used: tnf-α, forward : -atg atc gcg gac gtg gaa- and reverse: -agg gcc tgg agt tct gga a- ; gapdh, forward: -ggc atg gac tgt ggt cat ga- and reverse: -ttc acc acc atg gag aag gc- . amplification of cdna was performed in triplicate using a stepone realtime pcr system (applied biosystems). after an initial heat denaturation at °c for min, the pcr conditions were set at °c for s and °c for min for cycles. for each pcr, a corresponding mrna sample without rt was included as a negative control. quantification of each cdna copy number was determined according to the manufacturer's protocol. the gapdh gene was used as an endogenous control. the levels of cytokines from serum or cell supernatants were measured by enzyme-linked immunosorbent assay (elisa), according to the manufacturer's protocol (bd pharmingen, usa). peritoneal macrophages were seeded and pretreated with cwe for hour and then stimulated with lps for min. cells were rinsed in cold pbs and then lysed on ice in . ml of ripa buffer ( mm tris-hcl, ph . ; mm nacl; mm edta; mm naf; . % np- ; and % triton x- ) containing phosphatase inhibitor cocktail (sigma) and protease inhibitor cocktail (roche diagnostics, mannheim, germany). after centrifugation at , g for min, supernatants were collected. protein concentrations were determined using the bradford protein assay reagent (bio-rad, usa) and the samples were diluted with x sodium dodecyl sulfate(sds) buffer and boiled for min. the samples were separated on a % sds-polyacrylamide gel and were transferred to polyvinylidene fluoride membranes. the membranes were blocked with % skim milk in tris-buffered saline with . % tween (tbst) for hour. the membranes were incubated with iκbα, ikk, tubulin (santa cruz biotechnology, ca, usa), phospho-ikk, phospho-iκbα, phospho-jnk, jnk, phospho-erk / , erk / , phospho-p , and p diluted in % skim milk in tbst overnight at °c. the blots were washed with tbst and incubated for hour with anti-rabbit horseradish peroxidaseconjugated antibodies. immunoreactive bands were visualized by chemiluminescence using ecl (ge healthcare, little chalfont, buckinghamshire, uk), according to the manufacturer's instructions. total polyphenols from cwe and the size-based fractions were determined by folin-ciocalteau (fc) colorimetry as described previously [ ] . gallic acid solutions were used for a calibration standard curve. twenty microliters of each fraction or total cwe in . ml of water was incubated with μl of fc reagent (sigma) for min at room temperature. three hundred microliters of . m sodium carbonate solution was used to quench the fc reagent-mediated reaction to form chromogens. after reading absorbance at nm, the concentration of polyphenols was calculated as gallic acid equivalent per gram of extract. in vivo data are presented as mean ± sem. statistical differences among the means of multiple groups were determined by using one-way anova followed by the scheffe test. in vitro data are presented as mean ± sd. the difference between the two means was assessed using a non-paired student's t-test. calculations were carried out using spss version . p values of less than . were considered significant. lps is an endotoxin originating from the cell walls of gram-negative bacteria, which stimulates the expression of tnf-α and il- in monocytes and macrophages. these cytokines provide protective effects for the body by inducing blood clotting, leukocyte recruitment and activation of adaptive immunity, but a large quantity of such cytokines in serum results in provoking disseminated intravascular coagulation, multiple organ failure or septic shock [ ] . first, we tested whether cwe treatment affects systemic inflammatory response to lps stimulation. to this end, cwe was orally administrated to mice for days before intraperitoneal injection of lps and the release of serum tnf-α and il- was measured by elisa. based on our previous study, doses of , , and mg/kg were chosen for oral administration [ ] . dexamethasone was used as a reference drug to compare the suppressive activity of cwe in the presence of lps. serum levels of tnf-α were significantly reduced with the and mg/kg doses, while a higher dose ( mg/kg) showed a lesser reduction than the lower dose points and was not statistical significant ( figure ). this pattern was observed in our previous work in which a dose-dependent reduction in serum ifn-γ occurred only between - mg/kg of cwe [ ] . in the case of il- , a dose-dependent decrease occurred in the and mg/kg dosage groups although the latter group reached statistical significance. the mg/kg group showed a higher level of il- than the control group. it seems that unidentified compounds beyond a critical level may interfere with the antiinflammatory components of cwe. next, we wanted to examine the in vitro effect of cwe in macrophages, which are the major cell source of tnf-α. we used peritoneal macrophages isolated from thioglycollate-injected mice to determine the range of cwe concentration representing no cytotoxic activity using the mtt method. concentrations of cwe up to μg/ml was not cytotoxic to peritoneal macrophages treated with or without lps (figure ). many herbal water extracts contain polysaccharides, which induce the secretion of tnf-α in non-stimulated macrophages in vitro [ ] . we found that treatment with cwe alone ranging from to μg/ml stimulated the release of tnf-α into media in a concentration-dependent manner ( figure a ). such phenomenon must be due to the presence of water soluble polysaccharides in cwe. subsequently, we examined the effects of cwe on figure in vitro effect of cwe on lps-induced iκbα degradation and map kinase activation. peritoneal macrophages were pretreated with the indicated concentration of cwe for h and then stimulated with lps for min. phospho-ikk, iκbα, phospho-iκbα,phospho-jnk, jnk, phospho-p , p , phospho-erk / , and erk / in whole protein extracts were examined by western blot analysis. tubulin was used as an internal control. one of the five experiments is shown. lps-stimulated tnf-α secretion. there was no apparent decrease in tnf-α secretion by cwe ( figure a ). however, tnf-α mrna expression at h after lps challenge was decreased in cells treated with and μg/ml of cwe ( figure b ). despite its inhibition of lps-induced tnf-α transcription, the unabated tnf-α levels in supernatant must have been caused by prior exposure of cells to cwe. in vitro effect of cwe on activation of iκbα, jnk, p and erk / upon lps stimulation in the absence of stimuli, iκbα acts as an inhibitor to block the nuclear translocation of nfκb by masking its nuclear localization signal. upon inflammatory stimulation, iκbα is subjected to phosphorylation mediated by an upstream kinase, iκbα kinase (ikk), and is subsequently released from nf-κb, followed by phosphorylation-induced proteosomal degradation [ ] . we tested the effect of cwe on iκbα degradation min after lps stimulation. a time course study of iκbα activity in lps-stimulated macrophages shows that phosphorylation of iκbα reaches its peak at min after lps stimulation and then disappears at min while a complete degradation of iκbα occurs within min [ ] . as expected, an almost complete loss of phospho-iκbα as well as iκbα was identified in macrophages treated with lps alone (figure ). cwe at doses of and μg/ml inhibited iκbα degradation, although phospho-iκbα was still detected. phosphorylation of ikk was not affected. these results strongly indicate that the inhibitory effect of cwe on nf-κb signaling may occur downstream of the phosphorylation of iκbα. mitogen-activated protein (map) kinases resident in the cytoplasm of mammalian cells relay extracellular signals to the nucleus through various signal transduction pathways [ ] . jnk, p , and erk / , representing the family of map kinases, play a critical role in lpsinduced cytokine gene expression. we tested whether cwe can inhibit the activation of jnk, p , and erk / min after lps stimulation. jnk phosphorylation was reduced at all concentrations tested and phosphorylation of p and erk / was inhibited at μg/ml of cwe, implying that different constituents of cwe may act on these pathways (figure ). taken together, these results show that such inhibition occurred upstream of the map kinases. inhibitory effect of the cwe fraction containing highmolecular-weight compounds on signaling molecules in lps-stimulated macrophages since the effective oral dose of cwe is relatively low, it is reasonable to speculate that the nature of the active components might be small-molecule compounds. thus we separated cwe into a low mw fraction (below kda), a middle mw fraction (between kda and kda), and a high mw fraction (over kda). since the insulin-like and antioxidant activities of cwe originate from polyphenolic compounds, we wanted to determine the polyphenol content in these fractions [ ] . as shown in table , the highest concentration of total polyphenols was observed in the high mw fraction, being more than ten-fold than that of the low or middle mw fraction. next, based on the fraction yield ratio relative to the total cwe, μg/ml, μg/ml and μg/ml of the low, middle and high mw fractions were added to macrophages and their effects on signaling molecules were examined. suppression of the degradation of iκbα and phosphorylation of jnk, p and erk / was detected only in cells treated with the high mw fraction ( figure ). these findings indicate that the inhibitory activity of cwe in vitro is derived from the polyphenol-rich fraction. in this study, we present in vivo and in vitro evidence that cwe inhibits expression of tnf-α. in addition, lps-induced iκbα degradation and map kinase phosphorylation in macrophages was strongly inhibited by the polyphenol-rich cwe fraction. macrophages are phagocytic cells that play a critical role in clearing foreign materials, invading bacteria and cellular debris produced by tissue injuries [ ] . phagocytes such as macrophages contain a variety of pattern-recognition receptors (prrs), which specifically recognize foreign organisms and modified self ligand. toll-like receptors (tlrs), complement receptor and scavenger receptors are affiliated members of the prr family. among them, lps uses tlr-mediated signaling pathways such as nf-κb and map kinases to stimulate tnf-α and il- in macrophages. oral administration of cwe decreased serum levels of lps-induced tnf-α and il- , but such anti-inflammatory activity was attenuated in the high dose group. in the clinical setting, cwe is used in combination with other herbal agents and thus different results could be produced. however, our experimental data imply that when used singularly the anti-inflammatory activity of cwe is subjected to dose ranges. chronic inflammatory responses found in most autoimmune diseases and metabolic diseases exhibit common characteristic processes where macrophages are initially activated and interferon (ifn)-γ-producing type- t helper cells subsequently stimulate macrophages to release more inflammatory cytokines. together with our previous findings that cwe prevented anti-cd stimulated t cells from secreting ifn-γ, our current study clearly shows that cwe is able to interfere with the chronic activation of macrophages [ ] . the inhibitory effect of cwe on the signaling pathways mediated by nf-κb and map kinases occurred in its polyphenol-rich high mw fraction. there is increasing evidence that polyphenols exert anti-inflammatory effects. since cwe is rich in polyphenols such as flavonoids and tannins, the anti-inflammatory effect of cwe may originate partly from polyphenolic compounds. although the high mw fraction accounts for only % of the yield of cwe, its polyphenol content is four times more than that of cwe. the high mw fraction may contain polyphenols conjugated with polysaccharides or tannins. procyanidins, known as condensed tannins, consist of oligomer or polymers of (epi)catechin. a higher degree of polymerized procyanidins exhibited stronger inhibition of macrophage activity [ ] . therefore, it is conceivable that the polyphenol-rich high mw fraction of cwe may contain the anti-inflammatory compounds that play a major role in suppressing lpsinduced nfκb and map kinase signaling pathways. further study is required to examine whether the macromolecular polyphenols of cwe exert these antiinflammatory effects in animal models. in summary, oral treatment of cwe decreased lpsinduced tnf-α and il- release in serum. cwe inhibited iκbα degradation and map kinase activation in lps-stimulated macrophages in vitro. in particular, the inhibitory activity of cwe in vitro occurred in the polyphenol-rich high molecular weight fraction. mechanism of action of spanish oregano, chinese cinnamon, and savory essential oils against cell membranes and walls of escherichia coli o :h and listeria monocytogenes procyanidins and butanol extract of cinnamomi cortex inhibit sars-cov infection cinnamon extract induces tumor cell death through inhibition of nfkappab and ap water-soluble polymeric polyphenols from cinnamon inhibit proliferation and alter cell cycle distribution patterns of hematologic tumor cell lines anti-diabetic effect of cinnamon extract on blood glucose in db/db mice cinnamon extract (traditional herb) potentiates in vivo insulin-regulated glucose utilization via enhancing insulin signaling in rats points of control in inflammation targeting innate immunity protein kinase signalling in inflammation suppression effect of cinnamomum cassia bark-derived component on nitric oxide synthase inhibitory effect of cinnamaldehyde, derived from cinnamomi cortex, on the growth of influenza a/pr/ virus in vitro and in vivo the cinnamon-derived michael acceptor cinnamic aldehyde impairs melanoma cell proliferation, invasiveness, and tumor growth isolation and characterization of polyphenol type-a polymers from cinnamon with insulin-like biological activity novel angiogenesis inhibitory activity in cinnamon extract blocks vegfr kinase and downstream signaling screening of dried plant seed extracts for adiponectin production activity and tumor necrosis factor-alpha inhibitory activity on t -l adipocytes bench to bedside: tumor necrosis factor-alpha: from inflammation to resuscitation immunomodulatory effect of water extract of cinnamon on anti-cd -induced cytokine responses and p , jnk, erk / , and stat activation botanical polysaccharides: macrophage immunomodulation and therapeutic potential shared principles in nf-kappab signaling distinct role of spleen tyrosine kinase in the early phosphorylation of inhibitor of kappab alpha via activation of the phosphoinositide- -kinase and akt pathways mammalian mitogen-activated protein kinase signal transduction pathways activated by stress and inflammation the macrophage: past, present and future grape-seed procyanidins act as antiinflammatory agents in endotoxin-stimulated raw . macrophages by inhibiting nfkb signaling pathway anti-inflammatory activity of cinnamon water extract in vivo and in vitro lps-induced models the authors have no conflict of interests. key: cord- -o a q authors: xu, suming; wang, xu; wang, yaoqin; lutgendorf, susan; bradley, catherine; schrepf, andrew; kreder, karl; o'donnell, michael; luo, yi title: transgenic mice expressing mcp- by the urothelium demonstrate bladder hypersensitivity, pelvic pain and voiding dysfunction: a multidisciplinary approach to the study of chronic pelvic pain research network animal model study date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: o a q monocyte chemoattractant protein- (mcp- ) is one of the key chemokines that play important roles in diverse inflammatory and chronic pain conditions. interstitial cystitis/bladder pain syndrome (ic/bps) is a chronic and debilitating inflammatory condition of the urinary bladder characterized by the hallmark symptoms of pelvic pain and voiding dysfunction. to facilitate ic/bps research, we used transgenic technology to develop a novel urothelial mcp- secretion mouse model (uro-mcp- ). a transgene consisting of the uroplakin ii gene promoter and the mouse mcp- coding sequence with a secretory element was constructed and microinjected. uro-mcp- mice were found to express mcp- mrna in the bladder epithelium and mcp- protein in the urine, and developed bladder inflammation hours after intravesical administration of a single sub-noxious dose of lipopolysaccharide (lps). the inflamed bladders of uro-mcp- mice exhibited elevated mrnas for interleukin (il)- ß, il- , substance p precursor, and nerve growth factor as well as increased macrophage infiltration. in parallel with these phenotypic changes, uro-mcp- mice manifested significant functional changes at days and after cystitis induction. these functional changes included pelvic pain as measured by von frey filament stimulation and voiding dysfunction (increased urinary frequency, reduced average volume voided per micturition, and reduced maximum volume voided per micturition) as measured by micturition cages. micturition changes remained evident at day after cystitis induction, although these changes were not statistically significant. control wild-type c bl/ mice manifested no clear changes in histological, biochemical and behavioral features after similar cystitis induction with lps. taken together, our results indicate that uro-mcp- mice are hypersensitive to bladder irritants such as lps and develop pelvic pain and voiding dysfunction upon cystitis induction, providing a novel model for ic/bps research. aberrant overexpression of monocyte chemoattractant protein- (mcp- ; also named ccl ) has been observed in diverse inflammatory and chronic pain conditions [ ] . mcp- is produced by multiple cell types including bladder epithelial cells [ , ] and plays an important role in recruiting monocytes/macrophages as well as other leukocytes to sites during an inflammatory process. the fundamental importance of mcp- and its cognate receptor ccr are underscored by studies using genetic knockout mice lacking either of these two proteins. these genetically deficient mice display decreased macrophage recruitment and activation, increased susceptibility to mucosal infection, and reduced t cell responses [ ] [ ] [ ] . moreover, transgenic models of tissue specific mcp- expression have recapitulated many inflammatory disorders such as insulitis, pneumonitis and encephalitis but typically only after an additional inflammatory stimulus is introduced [ ] [ ] [ ] [ ] . in humans, elevated mcp- has been observed in various inflammation and autoimmune associated diseases such as inflammatory bowel disease, multiple sclerosis, diabetes, rheumatoid arthritis, and allergic asthma [ ] . elevated mcp- has also been reported for overactive bladder (oab) [ , ] and chronic prostatitis/chronic pelvic pain syndrome (cp/cpps) [ ] . similarly, animal studies have demonstrated elevated mcp- in bladder outlet and ureteral obstruction, which correlate with bladder and renal pathology, respectively [ , ] . in addition, elevated mcp- in the bladder has been observed to associate with bladder inflammation, reduced bladder capacity, and pelvic pain in a cyclophosphamide (cyp)-induced cystitis model [ ] . moreover, one study demonstrated that elevated mcp- in the bladder promoted histamine release from mast cells in a protamine sulfate and lipopolysaccharide (lps)-induced cystitis model [ ] . another study demonstrated that mast cell associated mcp- mediated bladder inflammation and chronic pelvic pain in a uroplakin peptide-induced autoimmune cystitis model [ ] . all these findings suggest that aberrant overexpression of mcp- may contribute to pelvic pain and voiding dysfunction in interstitial cystitis/bladder pain syndrome (ic/bps), a chronic and debilitating inflammatory condition of the urinary bladder characterized by the hallmark symptoms of pelvic pain and voiding dysfunction [ ] . here we report the development of a transgenic mouse model (uro-mcp- ) that secretes mcp- by the bladder epithelium and develops bladder inflammation, pelvic pain and voiding dysfunction upon intravesical administration of a single sub-noxious dose of lps. the uro-mcp- model demonstrates the hallmark symptoms of ic/bps and provides a novel model for ic/ bps research. all animal experiments were approved by university of iowa animal care and use committee (permit number: ) and performed according to the guide for the care and use of laboratory animals of the national institutes of health. the schematic structure of the transgenic mcp- construct is shown in fig a. a plasmid containing the uroplakin ii (upii) gene promoter was kindly provided by dr. t. sun at the new york university school of medicine [ ] . the . kb fragment containing the upii gene promoter was excised and placed upstream to the mouse mcp- coding sequence ( . kb). an intron sequence was inserted between the upii gene promoter and the mcp- transgene to facilitate discriminating mcp- mrna from its genomic dna in rt-pcr analysis [ ] . the . kb kpnl-draiii dna fragment consisting of the above-mentioned sequences and a poly a additional site was microinjected into fertilizing eggs (b /sjl background; the jackson laboratory, bar harbor, maine). transgenic founders were backcrossed with c bl/ mice (charles river laboratories, wilmington, ma) for generations to generate c bl/ congenic uro-mcp- mice. each generation was confirmed for the presence of the mcp- transgene by pcr genotyping using a sequence-specific primer pair ( '-cgaggtcgactgcagaag- ' and '-tgaggtggttgtggaaaagg- '; bp). pcr was performed for cycles at °c for seconds, °c for seconds, and °c for one minute. the pcr products were analyzed by % agarose gel electrophoresis. rt-pcr was performed on various tissues from an uro-mcp- mouse. mcp- mrna product is indicated by a red arrow. gapdh was used as an internal control. pupii-mcp- , a plasmid containing the transgenic mcp- dna sequence (a control for mcp- genomic dna). c bl/ bladder, a c bl/ mouse bladder (a negative control). (c) uro-mcp- mice express mcp- mrna in the bladder epithelium. the bladder epithelium from an uro-mcp- mouse was processed for rt-pcr. mcp- mrna product is indicated by a red arrow. gapdh was used as an internal control. pupii-mcp- served as a control for mcp- genomic dna. c bl/ bladder epithelium served as a negative control. (d) uro-mcp- mice express mcp- protein in the urine. urine was collected from both c bl/ (n = ) and uro-mcp- mice (n = ) and analyzed for mcp- by elisa. data are shown as mean ± s.d. *p< . as compared to the urine of c bl/ mice. female mice ( - weeks old) were used due to a higher incidence of ic/bps in females than males in humans and the easier feasibility of intravesical procedures in females. mice were anesthetized by intraperitoneal (i.p.) injection with μl of a mixture solution of ketamine ( . mg/kg) and xylazine ( . mg/kg). the bladder was then catheterized urethrally with a gauge / " long plastic intravenous catheter (smiths medical, southington, ct), instilled with μg of lps (e. coli :b , sigma-aldrich, st. louis, mo) in μl phosphate-buffered saline (pbs), and retained for hour. mice instilled with μl pbs in the bladders served as controls. bladders were collected and processed for formalin fixation, paraffin embedment, section preparation, hematoxylin and eosin (h&e) staining, and photography as described previously [ ] . bladder inflammation was scored in a blinded manner based on infiltration of inflammatory cells in the lamina propria and the presence of interstitial edema as described previously: + (mild infiltration with no or mild edema), + (moderate infiltration with moderate edema), and + (moderate to severe infiltration with severe edema) [ ] . bladder immunohistochemistry was performed as described previously [ ] . briefly, the bladders were fixed in % neutral formalin, embedded in paraffin, and cut into μm sections. after antigen retrieval and blocking, slides were incubated with biotinylated rat anti-mouse f / antibody (biolegend, san diego, ca; clone: ci:a - ; rat igg b) or control biotinylated rat igg b (biolegend, san diego, ca; clone: rtk ;) overnight. slides were developed conventionally using streptavidin-horseradish peroxidase complex (sav-hrp) and diaminobenzidine (dab) substrate solution (bd pharmingen). after rinsing, slides were counterstained with hematoxylin solution and photographed using an olympus bx- microscope. total rnas were extracted from the bladder and bladder epithelium using qiagen rneasy mini kit (qiagen, valencia, ca) as described previously [ ] . cdna was synthesized using invitrogen superscript iii reverse transcriptase (invitrogen, carlsbad, ca) and oligo dt. pcr amplification was performed on cdna products using taq dna polymerase (new england biolabs, ipswich, ma) and sequence-specific primer pairs for mcp- ( '-cgaggtcgactgc agaag- ' and '-tgaggtggttgtggaaaagg- '; bp), il- ß ( '-gcccatcc tctgtgactcat- ' and '-aggccacaggtattttgtcg- '; bp), il- ( '-gttctctgggaaatcgtgga- 'and '-ggaaattggggtaggaagga- '; bp), tachykinin- (substance p precursor) ( '-gccaatgcagaactacgaaa- ' and '-gcttggacagctccttcatc- '; bp), nerve growth factor (ngf) ( '-ctgtggaccccagactgttt- ' and '-cactgagaactcccccatgt- '; bp), and glyceraldehyde- -phosphate dehydrogenase (gapdh) ( '-gttc cagtatgactccact- ' and '-gtgcaggatgcattgctg- '; bp). gapdh was amplified for cycles and other molecules were amplified for cycles. the pcr products were run on a % agarose gel, stained with ethidium bromide, and imaged by gel doc ez imager (bio-rad laboratories, hercules, ca). urine was collected using micturition cages (see below) and levels of urinary mcp- were measured using elisa with paired capture and detecting antibodies (r&d systems, minneapolis, mn). mice were placed in individual micturition cages (columbus instruments, columbus, oh) for -hour real time recording of voiding habits with -hour light and -hour dark cycles as described previously [ ] . the cages consist of a fluid receptacle that funnels the captured urine droplets into a specimen freezer to prevent urine evaporation and chemical changes. a balance beneath the specimen freezer measures the weight of the collected urine. mice had free access to drinking water but were restrained from solid food to prevent feces from interfering with measurement of urine output. the entire system was computer interfaced for automated data acquisition in -minute intervals using oxymax software (columbus instruments). urinary frequency, voided volume per micturition, and total urine volume were recorded. pelvic pain was assessed by quantifying referred tactile allodynia of the lower abdominal region in response to applied force with a series of calibrated von frey filaments as described previously [ ] . mice were kept in individual plexiglas chambers ( x x cm) with a stainless steel wire grid floor and allowed to acclimate for minutes before testing. five individual filaments (stoelting co., wood dale, il) with forces of . , . , . , and grams were used in ascending order of force. the filament was applied perpendicularly to the skin for - seconds with intervals of seconds between each stimulus for a total of applications. stimulation was confined to the lower abdominal area in the general vicinity of the bladder. a positive response to filament stimulation was considered when mice showed sharp abdominal retraction, instant licking or scratching of the stimulated area, or jumping. response frequency was calculated as the percentage of positive response to each filament. tactile sensitivity of the plantar region of the hind paw was assessed using the same calibrated von frey filaments. the % withdrawal threshold was calculated and presented. a positive response to hind paw stimulation was defined as either a sharp withdrawal or licking of the tested paw. results were analyzed using statistics package for social sciences (spss . , chicago, il), and presented as mean ± s.d. for urinary mcp- levels and mean ± sem for both voiding habit and pelvic pain changes. data was compared using student's t-test (two groups) or anova followed by lsd post hoc tests (multiple groups). a value of p< . was considered statistically significant. uro-mcp- mice were generated through microinjection of a . kb dna construct consisting of the upii gene promoter fused to the mouse mcp- coding sequence with a secretory element (fig a) . the upii gene promoter is an urothelium-specific promoter and facilitates the expression of mcp- by the bladder epithelium [ ] . uro-mcp- mice were found to express mcp- mrna in the bladder but not in other organs tested (fig b) . further analysis indicated the expression of mcp- mrna by the bladder epithelium of uro-mcp- mice but not that of wild-type c bl/ mice (fig c) . urine from uro-mcp- mice contained a significantly higher level of mcp- protein ( . ± . ng/ml) compared to urine from wild-type c bl/ mice ( . ± . ng/ml) (fig d) . uro-mcp- mice appear healthy and do not spontaneously develop bladder inflammation and symptoms during their lifespan. uro-mcp- mice exhibit bladder hypersensitivity and develop bladder inflammation upon intravesical administration of a single sub-noxious dose of lps the bladders of uro-mcp- mice show a hypersensitive response to intravesical lps. compared to wild-type c bl/ mice (n = ; score: -+), uro-mcp- mice developed clear histological bladder inflammation (n = ; score: ++-+++) hours after intravesical instillation of a single sub-noxious dose of lps ( μg of lps in μl pbs) (fig a, table ). intravesical instillation of pbs ( μl) did not induce bladder histological changes in either strain of mice. the lps-treated bladders of uro-mcp- mice exhibited severe interstitial edema, mucosal hyperemia, and cellular infiltration in the lamina propria. in parallel with bladder histopathology, the inflamed bladders expressed elevated levels of mrnas for il- ß, il- , substance p precursor (pre-sp), and ngf as detected by rt-pcr (fig b) . immunohistochemistry revealed increased f / positive cell (macrophages) infiltration in the inflamed bladders bladder inflammation is associated with voiding dysfunction in uro-mcp- mice uro-mcp- mice were evaluated for voiding habits using micturition cages before (baseline) and , and days after intravesical pbs or lps treatment (table , s table) . for comparison, wild-type c bl/ mice were evaluated in parallel for voiding habits (s and s tables). there were no significant changes in voiding habits after intravesical pbs treatment compared to baseline voiding habits for both uro-mcp- (s table) and c bl/ mice (s table) . our prior analysis also showed no significant differences in baseline voiding habits between c bl/ and uro-mcp- mice (s table) . while a single sub-noxious dose of lps failed to induce clear changes in voiding habits in c bl/ mice (s table) , the lps treatment induced significant changes in voiding habits in uro-mcp- mice at days and ( table ) both wild-type c bl/ and uro-mcp- mice were evaluated for pelvic pain using von frey filament stimulation before (baseline) and , and days after intravesical pbs or lps treatment (fig a) . there were no significant differences in baseline pelvic responses between c bl/ and uro-mcp- mice or in pelvic response changes after pbs treatment in both mouse strains. intravesical lps at a single sub-noxious dose induced no clear changes in pelvic response in c bl/ mice. however, the same lps treatment induced significant changes in pelvic response in uro-mcp- mice at days ( . and uro-mcp- mice (right panels) were treated intravesically with μl pbs or μg of lps in μl pbs and evaluated for voiding habits using micturition cages at , and days after intravesical treatment (see table and s table) . the baseline voiding habits were included for comparison (see s and s tables). the results are representative of - mice for each of baseline, pbs-treated (day ), and lps-treated (day ) groups in both mouse strains. ( % threshold) of the plantar region of the hind paw before (baseline) and , and days after intravesical pbs or lps treatment in both wild-type c bl/ and uro-mcp-mice ( fig b) , suggesting that the pain developed in intravesical lps-treated uro-mcp- mice was restricted to the pelvis. ic/bps is one of the most refractory diseases in urology today and the effort to develop animal models that can reproduce the clinical correlates of the human disease is greatly needed for ic/bps research. since the etiology of ic/bps remains elusive and many factors appear to be causative for the disease, animal models with diverse pathological pathways have been developed [ ] . it is now generally agreed that a valid ic/bps animal model must present, at minimum, pelvic or bladder nociception and/or voiding dysfunction such as urinary frequency [ ] . in this study we created a novel transgenic cystitis model (uro-mcp- ) that secretes mcp- by the bladder epithelium and develops bladder inflammation upon intravesical instillation of a single sub-noxious dose of lps. besides bladder histopathology, the uro-mcp- model demonstrates both qualitative and quantitative changes in bladder functions such as increased pelvic pain sensitivity, increased urinary frequency, reduced average volume voided per micturition (urgency), and reduced maximum volume voided per micturition (bladder capacity). because of the genetic stability of the incorporated transgene, the uro-mcp- model is stable and reproducible and provides a unique translational model for ic/bps research. uro-mcp- mice do not spontaneously develop bladder inflammation, pelvic pain and voiding dysfunction in the unmanipulated state, suggesting that the urothelial expression of mcp- alone is not sufficient to cause bladder inflammation and functional changes in these mice. however, uro-mcp- mice readily develop phenotypical and functional changes upon intravesical administration of a single sub-noxious dose of lps. our observation is similar to those observed in other mcp- transgenic models. gunn and associates reported that transgenic mice expressing mcp- by type ii alveolar epithelial cells showed no morphologic evidence of inflammation in the lung but exhibited enhanced inflammatory response upon treatment with either intraperitoneal lps or intravenous yeast wall glucan [ ] . huang and associates reported that transgenic mice expressing mcp- by astrocytes only manifested neurological impairment and encephalopathy after treatment with intravenous pertussis toxin plus subcutaneous complete freund's adjuvant [ ] . similarly, trujillo and associates reported that transgenic mice expressing mcp- by oligodendrocytes predisposed mice to a defective immune response to a minimally lethal neurotropic coronavirus and developed encephalitis following intracranial infection by the virus [ ] . these observations indicated that development of inflammatory disorders is dependent on both genetic and environmental factors in the mcp- transgenic models. constitutive expression of mcp- by the bladder epithelium renders the bladders of uro-mcp- mice hypersensitive to otherwise sub-noxious irritative stimuli such as lps. we have observed that uro-mcp- mice exhibit a much lower threshold trigger for producing exaggerated responses to a single sub-noxious dose of lps as compared to wild-type c bl/ mice. intravesical administration of lps at μg in μl pbs efficiently induced profound bladder inflammation in uro-mcp- mice but not in wild-type c bl/ mice. this dose of lps used for cystitis induction in uro-mcp- mice was only one-tenth of the dose commonly used for cystitis induction in wild-type c bl/ mice [ , ] . this feature of the uro-mcp- model is clinically relevant, as subclinical infection can be a causative factor for ic/bps and patients with ic/bps are often found to be hypersensitive to minor bladder irritants [ ] . inflammation plays a central role in the pathogenesis of ic/bps and may directly affect bladder function in ic/bps patients [ ] . our recent studies supported the fundamental role of inflammation in ic/bps, as the production of toll-like receptor (tlr ) mediated proinflammatory cytokines il- ß and il- was significantly associated with multiple ic/bps pain indicators [ ] [ ] [ ] . as a clinically relevant model, the uro-mcp- model develops bladder inflammation and functional changes such as pelvic pain, urinary frequency and urgency. in addition to bladder histopathology, the inflamed bladders expressed elevated mrnas for proinflammatory cytokines il- ß and il- . moreover, the inflamed bladders also expressed elevated mrnas for ngf (a neurotrophic factor) and substance p precursor (a neurotransmitter), suggesting the presence of a strong neuro-immune interaction in the animal model. multiple cell types including urothelial cells, macrophages, mast cells, and neurons are known to express these inflammatory factors, reflecting an ongoing local inflammatory response in the bladders of uro-mcp- mice. all these inflammatory factors have been detected in the urine and/or bladder tissues of ic/bps patients [ ] . the uro-mcp- model described in this study represents an acute cystitis model. future studies will focus on extending this model to a chronic cystitis model to more closely mimic human ic/bps. the other limitation of the present study is the lack of direct evaluation of bladder nociception. although our data indicate the presence of increased pelvic pain sensitivity, we will continue demonstrating bladder pain by using more specific methods such as the bladder distention-evoked visceromotor response (vmr) method [ ] . future studies are also warranted to investigate the role of tlr in the uro-mcp- model and use this model for therapeutic development. the uro-mcp- model demonstrates bladder hypersensitivity, pelvic pain, and voiding dysfunction, providing a novel model for ic/bps research. supporting information s monocyte chemoattractant protein- (mcp- ): an overview mycobacterium bovis bacillus calmette-gué rin (bcg) induces human cc-and cxc-chemokines in vitro and in vivo wound healing in mip- alpha (-/-) and mcp- (-/-) mice mice lacking mcp- have enhanced susceptibility to an interstitial polymicrobial infection due to impaired monocyte recruitment impaired monocyte migration and reduced type (th ) c-c chemokine receptor knockout mice increased expression of ccl in insulin-producing cells of transgenic mice promotes mobilization of myeloid cells from the bone marrow, marked insulitis, and diabetes monocyte chemoattractant protein- is sufficient for the chemotaxis of monocytes and lymphocytes in transgenic mice but requires an additional stimulus for inflammatory activation pertussis toxin-induced reversible encephalopathy dependent on monocyte chemoattractant protein- overexpression in mice transgenic ccl expression in the central nervous system results in a dysregulated immune response and enhanced lethality after coronavirus infection urine cytokines suggest an inflammatory response in the overactive bladder: a pilot study differential profile analysis of urinary cytokines in patients with overactive bladder monocyte chemoattractant protein- and macrophage inflammatory protein- α as possible biomarkers for the chronic pelvic pain syndrome recruitment of bone marrow derived cells to the bladder after bladder outlet obstruction urinary concentration and tissue messenger rna expression of monocyte chemoattractant protein- as an indicator of the degree of hydronephrotic atrophy in partial ureteral obstruction expression and function of ccl /ccr in rat micturition reflexes and somatic sensitivity with urinary bladder inflammation mcp- -induced histamine release from mast cells is associated with development of interstitial cystitis/bladder pain syndrome in rat models chronic pelvic allodynia is mediated by ccl through mast cells in an experimental autoimmune cystitis model diagnosis and treatment of interstitial cystitis/bladder pain syndrome: aua guideline amendment a tissue-specific promoter that can drive a foreign gene to express in the suprabasal urothelial cells of transgenic mice vectors for high-level expression of cdnas controlled by tissuespecific promoters in transgenic mice urinary bladder epithelium antigen induces cd + t cell tolerance, activation, and autoimmune response animal models of urologic chronic pelvic pain syndromes: findings from the multidisciplinary approach to the study of chronic pelvic pain research network gene expression profiling of mouse bladder inflammatory responses to lps, substance p, and antigen-stimulation modulating bladder neuro-inflammation: rdp , a novel anti-inflammatory peptide, decreases inflammation and nerve growth factor production in experimental cystitis role of inflammation in bladder function and interstitial cystitis inflammation and inflammatory control in interstitial cystitis/bladder pain syndrome: associations with painful symptoms toll-like receptor and comorbid pain in interstitial cystitis/bladder pain syndrome: a multidisciplinary approach to the study of chronic pelvic pain research network study inflammation and symptom change in interstitial cystitis or bladder pain syndrome: a multidisciplinary approach to the study of chronic pelvic pain research network study potential urine and serum biomarkers for patients with bladder pain syndrome/interstitial cystitis we thank dr. tung-tien sun for providing the upii gene promoter-containing plasmid, dr. timothy l. ratliff for providing the micturition cages, and ms. kris greiner for editorial review of the manuscript. this work was supported in part by grants uo dk and ro dk from the national institute of diabetes digestive and kidney diseases.transgenic mice were generated at the university of iowa genome editing core facility directed by william paradee, phd and supported in part by grants from the nih and from the roy j. and lucille a. carver college of medicine. we wish to thank norma sinclair, patricia yarolem and joanne schwarting for their technical expertise in generating transgenic mice. key: cord- -tu gffr authors: wang, zhiyu; wang, yanfei; vilekar, prachi; yang, seung-pil; gupta, mayuri; oh, myong in; meek, autumn; doyle, lisa; villar, laura; brennecke, anja; liyanage, imindu; reed, mark; barden, christopher; weaver, donald f. title: small molecule therapeutics for covid- : repurposing of inhaled furosemide date: - - journal: peerj doi: . /peerj. sha: doc_id: cord_uid: tu gffr the novel coronavirus sars-cov- has become a global health concern. the morbidity and mortality of the potentially lethal infection caused by this virus arise from the initial viral infection and the subsequent host inflammatory response. the latter may lead to excessive release of pro-inflammatory cytokines, il- and il- , as well as tnf-α ultimately culminating in hypercytokinemia (“cytokine storm”). to address this immuno-inflammatory pathogenesis, multiple clinical trials have been proposed to evaluate anti-inflammatory biologic therapies targeting specific cytokines. however, despite the obvious clinical utility of such biologics, their specific applicability to covid- has multiple drawbacks, including they target only one of the multiple cytokines involved in covid- ’s immunopathy. therefore, we set out to identify a small molecule with broad-spectrum anti-inflammatory mechanism of action targeting multiple cytokines of innate immunity. in this study, a library of small molecules endogenous to the human body was assembled, subjected to in silico molecular docking simulations and a focused in vitro screen to identify anti-pro-inflammatory activity via interleukin inhibition. this has enabled us to identify the loop diuretic furosemide as a candidate molecule. to pre-clinically evaluate furosemide as a putative covid- therapeutic, we studied its anti-inflammatory activity on raw . , thp- and sim-a cell lines stimulated by lipopolysaccharide (lps). upon treatment with furosemide, lps-induced production of pro-inflammatory cytokines was reduced, indicating that furosemide suppresses the m polarization, including il- and tnf-α release. in addition, we found that furosemide promotes the production of anti-inflammatory cytokine products (il- ra, arginase), indicating m polarization. accordingly, we conclude that furosemide is a reasonably potent inhibitor of il- and tnf-α that is also safe, inexpensive and well-studied. our pre-clinical data suggest that it may be a candidate for repurposing as an inhaled therapy against covid- . covid- , a potentially lethal infection caused by the sars-cov- virus, has emerged as a public health crisis of global concern. currently, there are no effective curative treatments for covid- , affording little patient recourse beyond supportive care (cortegiani et al., ) . the morbidity and mortality of covid- arise from two competing pathological processes, the initial viral infection and the subsequent host inflammatory response, the latter of which may lead to excessive release of pro-inflammatory cytokines (interleukins (e.g., il- , il- ) or non-interleukins (e.g., tnf-a)) and may culminate in hypercytokinemia ("cytokine storm"), a self-targeting inflammatory response syndrome (conti et al., ; mehta et al., ; qin et al., ; huang et al., ) . reflecting these dichotomous disease processes, current therapeutic development strategies may be categorized into two broad groups: anti-viral and anti-inflammatory. arguably, truly effective treatments may require both agents, since an antiviral will fail to suppress uncontrolled pro-inflammatory cytokine release once the process has been triggered. to address the covid- immuno-inflammatory pathogenesis, multiple clinical trials have been proposed to evaluate anti-inflammatory biologic therapies targeting specific cytokines. agents under consideration include tocilizumab (peking university first hospital, ; tongji hospital et al., ; national cancer institute naples, ) and sarilumab (regeneron pharmaceuticals & sanofi, ) , both monoclonal antibodies that target the il- pathway (sebba, ; boyce et al., ) , as well as adalimumab, which binds with specificity to tnf-a (furst et al., ) . data from a chinese study in which tocilizumab was given to patients with severe covid- reported that the patients "improved remarkably" with patients being discharged within days or less following treatment, six patients within - days and only two patients stayed in the hospital for more than days . however, despite the obvious clinical utility of such biologics for many disorders, their specific applicability to covid- is hampered by various issues: they target only one of the multiple cytokines implicated in covid- 's immunopathy; if administered systemically, they can predispose patients to secondary infections or other toxicities, such as hepatoxicity; and they may be expensive to mass produce and distribute. therefore, they are of limited utility in the context of a global pandemic. accordingly, we set out to identify a small molecule with the following properties: broad-spectrum anti-inflammatory mechanism of action targeting cytokines of innate immunity; low toxicity and excellent safety profile; chemically stable; easily stored and administered; able to be rapidly adopted in clinical settings worldwide; and, widespread availability with inexpensive and efficient means of production. as described herein, a systemic series of in silico and in vitro studies have enabled us to identify furosemide as a candidate molecule. in silico studies molecular docking simulations on the endogenous molecule ( , , created in-house through literature search) and known drug datasets ( , , taken from drugbank.ca) against il- and tnf-a were carried out in two steps. the first step involved placement of the endogenous or ligand molecule in an identified binding pocket (docking) of il- and tnf-a. in the second step, the calculation of the binding energies of the docked molecules (scoring) was completed. preparation of tnf-a and il- d protein structures the tnf-a structure (pdb code: az ) and il- structure (pdb code: alu) were selected for molecular docking simulations in this work. we have employed these x-ray crystallographic structures because of three underlying motivations: ( ) these x-ray crystallographic structures are solved for human il- and tnf-a proteins. ( ) both of these x-ray structures have been solved at high resolution that is, alu for il- at . Å and az for tnf-a at . Å, which minimizes the error while solving the structure from electron density maps. ( ) the ligand molecules did not involve any metal atoms, which can otherwise bias the active site binding residues. the downloaded pdb structures were loaded into molecular operating environment (moe . ) software (molecular operating environment (moe), ). the loaded crystal structure was prepared employing the "quickprep panel" in moe, which contains the "structure preparation" feature. the "protonate d" function was used to optimize ionization states of the added hydrogen atoms. water molecules, which were present . Å away from ligand or receptor, were deleted. the mff x force field, with rms gradient of . kcal/Å, was used for energy minimization (panigrahi & desiraju, ) . a ramachandran plot was used to confirm the geometric and stereochemical qualities of the tnf-a and il- protein structures. the datasets of endogenous molecules ( , ) and drugs ( , ) were prepared using moe software. two functions 'wash at dominant ph of . ′ and "energy minimize" were used to obtain an energy minimized molecular form of molecules present at physiological ph of . . the active site of il- and tnf-a was identified using the "moe-site finder" module. the "triangle matcher" placement method and "london dg" scoring functions were used for docking simulations. the poses from ligand conformations were generated in the placement phase. after placement of poses, the refinement was done with the "gbvi/wsa dg" scoring method using "induced fit" option. induced fit refinement allows both the ligand molecule and protein active site to move freely to facilitate residue alignment during docking simulations. thirty docked poses of the placement retention simulation and poses of the refinement step were retained. the docking pose corresponding to the highest score for an endogenous or a drug molecule was considered for comparison of binding affinity within datasets. further details of descriptor calculation and docking simulations are provided in our recent publications (gupta et al., (gupta et al., , and in supporting information. validation of the docking protocol was carried out by re-docking the co-crystallized ligand in the active sites of the il- and tnf-a protein receptors. the following parameters were confirmed for validating the accurate docking protocol: . molecular superposition: the lowest energy docked conformation of a native ligand for il- and tnf-a is overlaid relative to the co-crystallized ligand present in the x-ray structure of the protein; the root-mean-square deviation (rmsd) has been calculated and is taken as an evaluation criterion for the validation of docking simulations. an rmsd value of < . Å between docked and crystallographic ligand conformation confirms successful docking (plewczynski et al., ; ramírez & caballero, ) . the docking protocol is validated if the docked ligand interacts with the active site of the protein similar to the corresponding co-crystallized ligand conformer and exhibits the same interactions with active site amino acid residues (plewczynski et al., ; ramírez & caballero, ) . inflammation activation on raw . macrophage raw . cells were purchased from atcc and maintained in dulbecco's modified eagle's medium (dmem) containing foetal bovine serum (fbs) at a final concentration of %. raw . cells were seeded in -well plates at . × cells/well seeding density, one day before experiments. to activate the cells, cell culture medium was changed to a lipopolysaccharide (lps) and interferon γ (ifnγ) containing medium with dimethyl sulfoxide (dmso) or furosemide at required concentrations, followed by h incubation at c; conditioned media and lysates were harvested for analysis. inflammation activation on thp- monocytic cells thp- cells (atcc) were maintained in rpmi , medium supplemented with -mercaptoethanol at a final concentration of . mm and fbs at a final concentration of %. thp- cells were seeded in each well of a -well tissue culture plate at a density . × cells/ml, one day before experiments. thp- monocytes were differentiated by nm pma (phorbol -myristate -acetate) for h. cells were then treated with lps+ifnγ with dmso or furosemide at the required concentration, followed by h incubation; conditioned media and lysates were collected for analysis. nitric oxide production from the conditioned media of raw . and sim-a cell cultures was examined by a griess assay. conditioned medium and sulfanilamide were mixed in a microwell plate to form a transient diazonium salt. then, n-naphtylethylenediamine was added to all wells to form a stable azo compound by incubating for - min in the dark at room temperature. the absorbance was measured between nm and nm. the concentration of no production was quantified by being plotted against a standard curve. cytokines were quantified using elisa kits following the manufacturer's instructions. briefly, the high-binding plates were coated at ml/well with diluted capture antibodies ( : ) at c overnight. the coated plates were then blocked with the diluent for h before assay. each sample was diluted accordingly and added to the plates for a h incubation period at room temperature. plates were then washed with ml/well pbs with . % tween- and incubated with detection antibodies ( : in assay diluent) for h at room temperature. after another washing step, : diluted avidin-hrp (horseradish peroxidase) was added and incubated for min. next, ml tmb-substrate ( , ′, , ′-tetramethylbenzidine) was added and the plate was incubated in the dark until the signal was sufficiently developed. the final concentration of tmb substrate solution was µg/ml. the reaction was stopped with ml of n sulfuric acid. absorbance was measured at nm with a correction of nm using a plate reader. cells were washed twice with ice-cold pbs and harvested in ripa buffer supplemented with a protease inhibitor cocktail. the whole-cell extracts were then centrifuged at , ×g for min at c to remove cell debris. protein concentrations were quantified using a micro bca protein assay kit. the absorbance was measured at nm using a microplate reader. equal amounts of cellular protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) and transferred onto polyvinylidene difluoride (pvdf) membranes at v for min. the sds-page was performed with % polyacrylamide gel for inos and . % for pro-il- β. the membranes were blocked for h in tris-buffered saline (tbs), ph . , with . % tween- (tbs-t) containing % skim milk. the membrane blot was then incubated overnight at c with primary antibodies against inos ( : , ), il- β ( : , ), actin ( : , ) and gapdh ( : , ) in tbs-t containing % skim milk. the membrane was washed with tbs-t × mins and incubated with goat anti-rabbit igg-horseradish peroxidase ( : , ) for hour. after the washing step, the immunoblotting was visualized by chemiluminescence hrp-substrate. cells were harvested and re-suspended with staining buffer. cells were stained with antibody ( : ) by incubating at c for min in the dark. stained cells were centrifuged, and the supernatant was discarded. the cell pellets were then re-suspended in cell flow buffer, transferred to facs tubes and analyzed by flow cytometry within h. to all cells in the experiment, fc blocker was added. sim-a (atcc) cells were maintained in dulbecco's modified eagle medium: nutrient mixture f- (dmem-f ) with % foetal bovine serum, % horse-serum and antibiotic-antimycotic. sim-a cells were seeded h before the experiment. culturing medium was replaced with dmem-f medium containing % fbs + . % horse serum with required lps concentration (final volume was one ml/well). the conditioned media and lysates were harvested for cytokine and cell marker examination. data are presented as mean ± sd or ±sem. statistical analysis was performed with graphpad prism software version . c, applying a two-tailed unpaired t-test. a p-value of > . was considered significant. a screening strategy was devised for identifying an initial compound with potential broad-spectrum anti-inflammatory activity targeting relevant cytokines of the pulmonary innate immune system. since immunologically-mediated inflammatory responses in the human body are subject to tight homeostatic regulation, there exist multiple endogenous compounds capable of either up-or down-regulation of innate immune processes. accordingly, we sought to identify an endogenous compound as an initial molecular platform around which to devise a therapeutic. a library of , small molecules endogenous to the human body was assembled, subjected to in silico molecular docking simulations and a focused in vitro screen to identify anti-pro-inflammatory activity via interleukin inhibition. multiple compounds within the tryptophan metabolic pathway were identified, both indoleamine and anthranilate metabolites. in particular, -hydroxyanthranilic acid ( -haa) was found to demonstrate significant anti-inflammatory potential (in accord with previous studies by others, such as lee et al. ( ) , who have shown significant activity of -haa in inhibiting il- and tnf-a). regrettably, -haa is a small polar molecule with poor drug-like properties and is not an approved therapeutic for human use. the task of converting -haa to a drug or drug-like compound can be addressed via two approaches: ( ) synthesis of new chemical entities with drug-like properties based on the -haa scaffold, or ( ) identification of known drugs with structural properties similar to the -haa scaffold with the goal of repurposing. because of the urgency imposed by the unfolding pandemic, approach was selected to provide a short-term solution. accordingly, , known drugs were computationally evaluated for anthranilate structural components. this screen identified mefenamic acid (n-( , -xylyl)-anthranilate) and furosemide ( -chloro- -sulfamoyl-n-furfuryl-anthranilate) as the two strongest leads. mefenamic acid is known to provide significant protection against elevated levels of tnf-a and il- β in radiation-induced genotoxicity of human lymphocytes (armagan et al., ; hosseinimehr et al., ) ; furosemide is known to significantly reduce production of il- , il- and tnf-a in bronchial inflammation of asthma (prandota, ; yuengsrigul, chin & nussbaum, ) . this screen also identified other loop diuretics structurally related to -haa and furosemide, including bumetanide, piretanide and azosemide (fig. ) . arising from the observation that furosemide can reduce bronchial inflammation, can be administered by inhalation (vide infra, discussion) (prandota, ; inokuchi et al., ; waskiw-ford et al., ) and is a widely available drug, furosemide was selected to be explored for repurposing in the treatment of covid- . furosemide has been used worldwide for decades as a loop diuretic working via the renal na + /k + -atp shuttle pathway. to pre-clinically evaluate furosemide as a putative covid- therapeutic, a series of in vitro efficacy assessments was performed to investigate its anti-inflammatory properties. first, we investigated if furosemide could reduce the release of pro-inflammatory cytokines induced by lps in macrophage cell line. raw . macrophages were stimulated with lps in the presence or absence of furosemide and the levels of tnf-a and no were measured from the conditioned media using elisa and griess assay, respectively. the results in figs. a and b show that lps induces the production of no and tnf-a, indicating macrophage polarization to an m pro-inflammatory phenotype. when cells were treated with lps in the presence of mm of furosemide, the production of no and tnf-a significantly decreased. to further investigate the reduction of no production by furosemide, we determined the expression level of inducible nitric oxide synthase (inos) which produces no in response to inflammatory stimulations. therefore, we stimulated raw . macrophages with lps and ifnγ. the expression of inos was significantly induced by stimulation with lps and ifnγ, as shown by the western blot results in fig. c . we found that furosemide was able to suppress the figure screening approach with chemical structures of important compounds. with the goal to find a small therapeutic with anti-inflammatory properties and endogenous to the human body, a library of , small molecules was screened by docking simulations and further investigated by in vitro experiments. this intensive screen yielded -hydroxyanthranilic acid ( -haa) as initial hit. since -haa is not approved for medical applications, , known drugs were screened computationally for a -haa substructure. besides mefenamic acid, which is not displayed in this figure, the diuretic furosemide was the strongest lead directly followed by bumetanide, azosemide and piretanide. full-size  doi: . /peerj. / fig- expression of inos during lps and ifnγ induced stimulation. densitometry analysis showed that normalized inos/gapdh ratio was reduced from to . by furosemide. lps is recognized by the cell surface pattern-recognition receptors such as the toll-like receptor protein (tlr ) and triggers downstream signaling pathways. lps induces expression of the pro-inflammatory cytokine ifnγ. ifnγ is known to increase tlr expression which may then promote the response to lps stimulation. we investigated the effect of furosemide on lps-induced tlr expression in raw . macrophage cells using flow cytometry. as shown in fig. , lps increased the tlr + cell population significantly, whereas il- , an anti-inflammatory cytokine, barely induced tlr expression from the cells. interestingly, furosemide completely blocked lps-induced tlr expression, suggesting the possible involvement of furosemide in the lps-or ifnγinduced inflammation. as the next stage of macrophage activation, we studied the activity of furosemide on the expression of il- β which plays a key role in modulating inflammatory response, as a downstream pro-inflammatory marker of the tlr signaling pathway by using differentiated thp- monocytes. we analyzed the expression of il- β precursor protein (pro-il- β) from thp- macrophage cells by using western blot analysis. furosemide, as shown in fig. , significantly decreases the expression of pro-il- β in differentiated thp- cells, demonstrating yet another inflammatory cytokine targeted by furosemide. to further explore the effect of furosemide on different macrophages, we next tested it on sim-a cells. we stimulated sim-a macrophages with lps and analyzed the release of pro-inflammatory markers such as no, il- and tnf-a. the results in fig. show that lps induced the production of no, il- and tnf-a from sim-a cells. similar to raw . cell results, furosemide significantly reduced the production of all these pro-inflammatory markers from sim-a cells as well. we have tested three different macrophage cell lines for the effects of furosemide on pro-inflammatory markers. furosemide consistently reduced pro-inflammatory markers such as no production, secretion of il- , tnf-a and expression of pro-il- β from different macrophage cell lines, implying that furosemide has broad inhibitory activity against pro-inflammatory cytokines. then, we evaluated if furosemide exhibits any effects on anti-inflammatory cytokines. we measured the levels of anti-inflammatory cytokines from the conditioned medium of differentiated thp- cells after h of stimulation with lps and ifnγ by multiplex assay using flow cytometry. furosemide induces the expression of il- , interleukin- receptor antagonist (il- ra) and arginase, which are anti-inflammatory markers, suggesting the polarization of thp- macrophage to an m phenotype (fig. ) . together with the experiments discussed above, these results show that furosemide inhibits the expression of m pro-inflammatory markers and promotes the expression of m anti-inflammatory markers. thus, furosemide is a broad-spectrum anti-inflammatory drug candidate targeting multiple cytokines. the molecular mechanism of action whereby -haa and furosemide inhibit il- and tnf-a activity is unknown at this time and lies outside the scope of the present study. interestingly, in an in silico screen of possible binding sites for -haa and related drug anthranilates, we noted favorable interactions with both the il- and tnf-a proteins. whilst it is improbable that such interactions are completely responsible for the observed anti-inflammatory effects, this notable observation is worthy of future experimental evaluation. the docking protocol needs to be validated before any conclusive in silico results can be obtained. to validate the docking methodology, co-crystalized ligands for tnf-a and il- have been re-docked into the active sites of the corresponding proteins. table shows the re-docking parameters that have been evaluated for confirmation of accuracy of the employed docking protocol. il- (pdb code: alu) has been re-docked with tartaric acid. after re-docking simulations, the docked conformer of tartaric acid is overlaid with the co-crystallized ligand in the pdb structure. figure a represents the overlay of the docked pose and the co-crystallized ligand conformer. the rmsd between the docked conformer and co-crystallized ligand is . Å, which is < . Å as recommended for successful docking method confirmation. furthermore, all the amino acid residues which are interacting with the native il- binding pocket (arg , arg and gln ) are shown to be interacting with the docked conformer of the tartaric acid in the re-docking simulation (as shown in fig. b ). this further confirms that the re-docking method is able to accurately predict the binding mode and the active site of il- . the tnf-a dimer inhibitor has also been re-docked with tnf-a for validation of the docking method. the overlay of the docked ligand with the co-crystallized ligand conformation is presented in fig. a. from fig. a it can be seen that the tnf-a dimer inhibitor is superposed with the co-crystallized ligand conformer with an rmsd value of . Å, which validates the docking simulation methodology for tnf-a. the ligand interaction diagram of the docked conformer of tnf-a, is shown in fig. b . the amino acid residues present within Å of the active site are labeled i.e. leu , tyr , ser , leu , gly , gly , tyr . these residues are present in the active site of the tnf-a dimer in the vicinity of co-crystallized ligand. the re-docking simulations of co-crystallized ligands for il- and tnf-a confirm the accuracy of the docking methodology employed in this study. the computational simulations of -haa and furosemide with tnf-a and il- are briefly outlined below. the binding of -haa in il- and tnf-a active sites is presented in fig. . the docking score (s) of minimum energy pose of -haa in il- is − . and in tnf-a is − . . in figs. a and c, the ligand -haa is shown in yellow and amino acid residues of binding pocket have been labeled. figures b and d represent the ligand interaction diagrams of -haa with active site residues of tnf-a and il- . -haa interacts with the arg and arg in il- and docks in the tnf-a dimer site with residues tyr , tyr , ser , leu , gly and tyr . this confirms that -haa interacts with il- and tnf-a in the same binding site as observed in re-docking simulations of co-crystallized ligands for the corresponding proteins. figure presents docking simulations of furosemide with tnf-a and il- , respectively. in figs. a and c, furosemide is shown in yellow and the amino acid residues of the binding site are labeled. the tnf-a protein complex (pdb: az ) contains four identical chains having amino acids each and two bound ligands. the a and b chains were retained (coloured orange and purple) and the co-crystalized ligand was removed while preparing the protein structure for docking. figure a shows the minimum energy docking pose of furosemide in the tnf-a active site. the amino acid residues leu b , tyr b , gly b , tyr a , tyr a and leu a are involved in forming hydrogen bonding and π-π interactions (arene-arene, arene-h, arene-cation) with furosemide in minimum energy docked poses. the docking score of the minimum energy pose is − . . the similar mode of binding in the active site of tnf-a as of co-crystallized ligand (as shown in fig. b ) and favorable binding energy indicates that furosemide fits well into the active site of tnf-a and inhibits its activity. figures c and d show the minimum energy docking pose of furosemide in the protein structure of human il- (pdb: alu), having amino acid residues and a co-crystallized ligand (tartaric acid). the co-crystallized ligand had been removed during the structure preparation step of the il- protein. furosemide interacts with arg , gln , leu and arg in most of the minimum energy poses, which agrees well with the il- co-crystallized ligand tartaric acid binding mode. the docking score (s) of the minimum energy pose of furosemide with il- is − . , which computationally supports the ability of furosemide to inhibit il- activity. the pathogenic mechanisms of covid- morbidity and mortality are diverse, though immuno-inflammatory contributions are likely a central player. it is now appreciated that covid- afflicted individuals with major respiratory symptoms have pathologically elevated levels of pro-inflammatory cytokines including il- , il- and tnf-a (conti et al., ; mehta et al., ; qin et al., ; huang et al., ) . a logical therapeutic approach to the management of covid- thus includes a need to modulate immunotoxicity. tryptophan and its metabolites, particularly via the indoleamine- , -dioxygenase initiated pathway, have a previously described role as endogenous modulators of innate immunity. of these various metabolites, -haa has been identified to exhibit a significant anti-inflammatory ability to suppress inflammation mediated via multiple pro-inflammatory interleukin cytokines, including il- β, il- , il- and tnf-a (lee et al., ) . this motivated our search for an anthranilate-based -haa-like agent from a library of known drugs; furosemide emerged as a possible candidate from this search. in this study, as part of its pre-clinical evaluation, we studied furosemide's anti-inflammatory activity on multiple macrophage cell lines involved in innate immunity. as pattern recognition receptors, tlrs contribute to the recognition of the molecules that are commonly shared by pathogens, such as lps and viral nucleotides (alexopoulou et al., ) . the activation of tlrs triggers downstream signaling through the myd -dependent pathway and eventually induces the activation of nuclear factor-кb (nf-кb) (oeckinghaus, hayden & ghosh, ) . nf-кb has been shown to play an important role in coronavirus infections. for instance, nf-кb activation was identified in the lungs of sars-cov infected mice and triggered the production of pro-inflammatory cytokines, such as tnf-a (dediego et al., ) . however, the mechanism of sars-cov pathogenesis is debated. an in vitro study suggested the nucleocapsid protein of sars-cov was crucial in the pathogenesis, although this correlation is cell-specific (liao et al., ) . on the other hand, sars-cov lacking the envelope protein (e protein) attenuated nf-кb activation and associated pro-inflammatory responses (dediego et al., ) . the pandemic outbreak of covid- is caused by the infection of sars-cov- that shares . % identity to sars-cov . to investigate the potential use of furosemide as a therapy for covid- , we developed in vitro assays using lps as exogenous stimulation to induce inflammatory responses. lps-induced tlrs activation is well studied: it interacts with tlrs and triggers nf-кb activation followed by pro-inflammatory responses (lu, yeh & ohashi, ) . in our in vitro assays, we aimed to ascertain if lps induces a similar "cytokine storm" as sars-cov- infection and if furosemide inhibits the production of pro-inflammatory cytokines or promotes the secretion of anti-inflammatory cytokine products. broadly conceptualized stimulated macrophages can be polarized to either an m pro-inflammatory phenotype or an m anti-inflammatory phenotype. we investigated if furosemide inhibits the production of pro-inflammatory cytokines (m ) or promotes the secretion of anti-inflammatory cytokines (m ) using various macrophage cell lines including raw . , thp- and sim-a . upon stimulation, these cell lines initiated an immune response by producing cellular stress signals and secreting pro-inflammatory cytokines. these pro-inflammatory markers were reduced upon treatment with furosemide, indicating that furosemide suppresses the m polarization, including no, il- and tnf-a. more importantly, our study results demonstrated furosemide promotes the production of anti-inflammatory cytokine products (il- ra, arginase), indicating m polarization. all these results strongly suggest the potential application of furosemide as an immunomodulating agent for such disease conditions in which the inflammatory burden of patients increases suddenly. it also suggests that furosemide can be used for treating in which the sudden increase of pro-inflammatory cytokines is part of the disease pathogenesis. furosemide is a small molecule with a molecular weight of . g/mol and relatively low lipophilicity (logp = . ) (hardman, goodman & gilman, ) . although the drug has low water solubility at ph , furosemide can be formulated in weakly basic buffer solution (ph ) to achieve mg/ml solutions suitable for intravenous administration. due to the presence of a primary sulfonamide and carboxylic acid group, furosemide is highly bound to albumin with a human plasma protein binding value of . ± . %. the drug has a very low volume of distribution (v d = . ± . l/kg) and a relatively short half-life of . ± . h (hardman, goodman & gilman, ) . these pharmacokinetic parameters along with high plasma protein binding equates to low tissue distribution with furosemide being retained with the blood. alveolar macrophages are front line innate immune cells, playing a crucial role in the maintenance of lung homeostasis and lung tissue defense through various immune responses. tissue-resident alveolar macrophages are derived from the foetal liver and populate the alveoli shortly after birth. these macrophages are self-renewing and persist over the life span. however, exposure to environmental challenges and injury induces recruitment of monocyte-derived alveolar macrophages from circulating monocytes. the intrinsic molecular properties of furosemide are ideal for targeting macrophages recruited from the blood stream prior to their tissue distribution. furosemide exhibits a large therapeutic window and is listed on the who's list of "essential medicines"; it is readily available worldwide, is easily manufactured, and has a long record of safety and efficacy when given orally or intravenously. more importantly, furosemide may also be administered safely by inhalation. more than years ago, the concept of inhaled furosemide was explored as an approach to reduce dyspnea, primarily based on the rationale that edematous airway mast cells would be reduced in size following diuresis (prandota, ) . however, further investigations established that the mechanism of action was not related to local diuretic effects or engagement of the na + /k + -atp shuttle. more recent studies have reported reduction in pulmonary il- , il- and tnf-a levels upon administering inhaled furosemide to patients with conditions including tachypnea (armed forces hospital pakistan, ; university of cologne, ), bronchopulmonary dysplasia (university of north carolina et al., ) and chronic lung disease (beth israel deaconess medical center, ; mcgill university, ; oxford brookes university, ) . a double-blind, placebo-controlled trial by grogono et al. ( ) evaluated inhaled nebulized furosemide ( mg furosemide in ml . % saline) in healthy adults, demonstrating effective relief of experimentally induced air hunger during dyspnea after multiple dosing per day with no untoward effects. therefore, accumulated data indicate that furosemide is a cytokine-targeting anti-inflammatory, which may be safely administered by inhalation multiple times per day. our in silico screening identified other loop diuretics with structural similarities to furosemide. bumetanide exhibits anti-inflammatory properties in lps stimulated raw . cells, reduces lps-induced production of cytokines following direct pulmonary administration, and lowers levels of tnf-a production in lung-injured mice (hung et al., ) . bumetanide however failed to inhibit sodium metabisulfite induced bronchoconstriction in asthmatic subjects (o'connor et al., ) . piretanide and azosemide have also been variously studied in models of cytokine-mediated inflammation and bronchoconstriction (yeo et al., ) . since none of these agents are in widespread clinical use, we have elected to pursue the development of furosemide in covid- because of its worldwide availability in the time of a global pandemic. the potential use of furosemide in the anti-inflammatory treatment of covid- has strengths and weaknesses. furosemide is inexpensive and available in every country in the world; it is safe and has profound anti-inflammatory cytokine activity, particularly against il- and tnf-a. if administered orally, furosemide can produce a profound diuresis which would be a clinical detriment in a febrile and potentially dehydrated person. when administered orally, the pharmacokinetics of furosemide indicate that it would have primarily intra-vascular distribution, suggesting greater utility early in the course of the disease, but less so in later-stage ventilator-supported individuals in whom macrophage migration from bloodstream to pulmonary tissue has occurred. as the disease progresses, direct administration of furosemide to the lungs by nebulized delivery adequately addresses the need to have furosemide reach intra-alveolar macrophages. arguably, truly effective treatments may require both anti-viral and anti-inflammatory agents since the early stages of the infection are dominated by high viral replication. however, application of an anti-inflammatory agent only, especially in the current situation where an effective anti-viral is still missing, may have the potential to reduce the number of patients requiring mechanical ventilation and reduce cough as a symptom which could help limiting the spread of the virus. care must be taken to prevent viral contamination and bystander exposure during the aerosolized administration of the drug, but this can be achieved with appropriate cautions in place. we are currently pursuing a clinical study of inhaled furosemide in people with covid- . covid- is a pandemic threatening global health. the need to identify innovative therapeutics which may be deployed rapidly and efficiently is a pharmacological priority. furosemide is a safe, inexpensive, well-studied small molecule which is a reasonably potent inhibitor of il- and tnf-a and may be administered locally to the lungs; pre-clinical data from in silico and in vitro experiments suggest that it may be a candidate for repurposing as an inhaled therapy against the immunopathologies of covid- . a list of physiochemical descriptors used as initial screening test of the dataset; a list of promising drug candidates along with corresponding physiochemical descriptors; docking score 's' of promising drug candidates with il- and tnf-a; figures and ligand interaction diagrams of mefenamic acid, bumetanide, piretanide and azosemide docking into binding site of tnf-a and il- . this information is available free of charge on peerj. this work was supported by the canada research chair program (donald f. weaver, canadian research chair tier ). the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. recognition of double-stranded rna and activation of nf-κb by toll-like receptor neuroprotection by mefenamic acid against d-serine: involvement of oxidative stress, inflammation and apoptosis role of salbutamol and furosemide in ttn aerosol inhalation treatment for dyspnea-patients sarilumab: review of a second il- receptor antagonist indicated for the treatment of rheumatoid arthritis induction of pro-inflammatory cytokines (il- and il- ) and lung inflammation by coronavirus- (covi- or sars-cov- ): anti-inflammatory strategies a systematic review on the efficacy and safety of chloroquine for the treatment of covid- inhibition of nf-κb-mediated inflammation in severe acute respiratory syndrome coronavirus-infected mice increases survival adalimumab, a fully human anti tumor necrosis factor-alpha monoclonal antibody, and concomitant standard antirheumatic therapy 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coronavirus lps/tlr signal transduction pathway inhaled nebulized furosemide & physical activity-related breathlessness covid- : consider cytokine storm syndromes and immunosuppression integrated computer-aided molecular design platform. montreal: chemical computing group ulc. national cancer institute naples. . tocilizumab in covid- pneumonia (tocivid- ) crosstalk in nf-κb signaling pathways specificity of dyspnoea relief with inhaled furosemide effect of inhaled furosemide and bumetanide on adenosine '-monophosphate-and sodium metabisulfite-induced bronchoconstriction in asthmatic subjects strong and weak hydrogen bonds in the protein-ligand interface favipiravir combined with tocilizumab in the treatment of corona virus disease can we trust docking results? evaluation of seven commonly used programs on pdbbind database furosemide: progress in understanding its diuretic, anti-inflammatory, and bronchodilating mechanism of action, and use in the treatment of respiratory tract diseases dysregulation of immune response in patients with covid- in wuhan, china is it reliable to take the molecular docking top scoring position as the best solution without considering available structural data? evaluation of the efficacy and safety of sarilumab in hospitalized patients with covid- tocilizumab: the first interleukin- -receptor inhibitor hubei xinhua hospital, wuhan no. hospital, wuhan central hospital. . tocilizumab vs crrt in management of cytokine release syndrome (crs) in covid- the emmes company llc. . safety of furosemide in premature infants at risk of bronchopulmonary dysplasia (bpd) effect of inhaled nebulized furosemide ( and mg) on breathlessness during exercise in the presence of external thoracic restriction in healthy men effective treatment of severe covid- patients with tocilizumab protective effect of loop diuretics, piretanide and frusemide, against sodium metabisulphite-induced bronchoconstriction in asthma immunosuppressive and cytotoxic effects of furosemide on human peripheral blood mononuclear cells the authors declare that they have no competing interests. the following information was supplied regarding data availability:the raw measurements are available in the supplemental files. supplemental information for this article can be found online at http://dx.doi.org/ . / peerj. #supplemental-information. key: cord- -gmcugd h authors: song, jian-xin; zhu, lin; zhu, chuan-long; hu, jin-hua; sun, zi-jian; xu, xiang; xin, min-you; zhang, qiong-fang; zhang, da-zhi; shang, jia; huang, jia-quan; xu, dong title: main complications of aechb and severe hepatitis b (liver failure) date: - - journal: acute exacerbation of chronic hepatitis b doi: . / - - - - _ sha: doc_id: cord_uid: gmcugd h this chapter describes the clinical features, and diagnosis of complications in aechb including secondary bacterial infections, coagulation disorder, water electrolyte disorder, hepatorenal syndrome, hepatic encephalopathy, hepatopulmonary syndrome and endotoxemia: . patients with severe hepatitis have impaired immunity and are therefore vulnerable to all kinds of infections. after infection, these patients may experience shock, dic and multiple organ failure, all of which seriously affect their prognosis and are major causes of death. concurrent infections consist primarily of infections of the lungs, intestines, biliary tract, and urinary tract, as well as spontaneous bacterial peritonitis and sepsis. . severe hepatitis may reduce the synthesis of coagulation factors and enhance their dysfunction and increase anticoagulants and platelet abnormalities, leading to coagulopathy. infection, hepatorenal syndrome and complications can further aggravate coagulopathy, resulting in dic and seriously affecting patient prognosis. . hepatorenal syndrome, which is characterized by renal failure, hemodynamic changes in arterial circulation and abnormalities in the endogenous vascular system, is a common clinical complication of end-stage liver disease, and one of the important indicators for the prognosis of patients with severe hepatitis. . water electrolyte disorder (water retention, hyponatremia, hypokalemia, hyperkalaemia) and acid-base imbalance are common in patients with severe hepatitis. these internal environment disorders can lead to exacerbation and complication of the illness. . hepatic encephalopathy is a neurological and psychiatric anomaly syndrome based on metabolic disorder, and an important prognostic indicator for patients with severe hepatitis. . the hepatopulmonary syndrome is an important vascular complication in lungs due to systemic hypoxemia in patients with cirrhosis and portal hypertension. the majority of patients with hps are asymptomatic. long-term oxygen therapy remains the most frequently recommended therapy for symptoms in patients with severe hypoxemia. . endotoxemia, an important complication of severe hepatitis, is not only a second hit to the liver, but also leads to other complications including sirs and mods. (nacseld) for survival analysis of prospectively enrolled cirrhosis patients hospitalized with infections, bajaj found that there is . % of nosocomial infection in all patients [ ] . domestic data showed significant difference of infection incidence in different times. it is related to raising diagnosis and treatment level. according to the data from shanghai public health clinical center, in patients who were treated as severe hepatitis from january to december , there were patients obtained nosocomial infection with a rate of . % [ ] . the incidences of infection complicated with acute liver failure, acute on chronic liver failure or chronic liver failure are similar. patients with old age or long hospitalization are more easily to get infection. there was rare report about infection in children patients with severe hepatitis. in , godbole et al. from uk reported that % in children patients with severe hepatitis were complicated with infection, and mostly are sepsis, respiratory tract infection and urinary infection. most of the infections occurred within weeks of admission, while patients with infection had longer hospitalization [ ] . reduced innate immunity the innate immune system is the first line of defense against premier environmental challenges and injury. in liver, it is a complex system that includes nk cells, nkt cells, kcs, neutrophils, eosinophils and complement components. the innate immune response acts much more rapidly compared with adaptive immunity. monocytes and macrophages: the liver plays an important role in defense and immune function. the main cellular components of the innate immune system within the liver are the kupffer cells. kupffer cells represent - % of the tissue macrophages in the human body [ ] . in normal condition, kupffer cells in liver help to clear the macromolecular substance such as pathogen, endotoxin, heteroantigen and immune complex to defense infection. in severe hepatitis, due to massive hepatocytes necrosis, the number and function of monocytes/macrophages are impaired, thus the activity of fibronectin, which is opsonin of macrophages, is decreased. therefore, bacterium, endotoxin and other poison from gut directly access into circulation. subrat kumar acharya from india reported that the plasma fibronectin (fn) level in severe hepatitis patients was significantly lower than that in healthy controls ( . μg/ml ± . μg/ml, vs. . μg/ ml ± . μg/ml). the fn level was remarkably correlated with incidence of infection and mortality [ ] . complement: liver is the organ where complements are mainly produced, such as c , c , c , c . the complements help to expand phagocytosis of phagocytes by chemotaxis, opsonization or adhesion, as well as help antibody to kill or solute some gram negative bacilli [ ] . report from wyke showed that the defect of complement closely correlated with impaired opsonization [ ] . defect of c or c can result to weakened movement of polymorphonuclear leucocyte. in severe hepatitis, the ability of liver to produce complement has been weakened due to massive injury of parenchymal hepatic cells, which leads to decreased activity of complement to % of normal condition. meanwhile, serum opsonization to e. coli or saccharomycetes are also diminished. besides, high ammonia level in severe hepatitis also restrains complement activity to impact germicidal effect. the most direct and also the most important result for decreased complement production and reduced opsonization is the susceptibility to infection. it was also reported that complement and the alternative pathway play an crucial role in lps/d-galn-induced fulminant hepatic failure [ ] . neutrophils: a majority of patients with severe liver disease have altered function of neutrophil granulocytes [ ] . the most common manifestation include abnormal ultrastructure or function of neutrophil granulocytes, as cytoplasm degeneration, organelle reduction, mitochondrial swelling, pyknotic nuclei, etc. decrease filtration and phagocytosis of reticuloendothelial system, as well as impaired chemotaxis of blood cells, making immunity weakened, lead to invasion of bacterium. therefore, patients with severe hepatitis are vulnerable to be infected with bacterium or fungi due to decreased phagocytosis and germicidal effect of neutrophil granulocytes, and impaired adhesion of macrophages and white cells [ ] . data from liu h demonstrated pretreatment neutrophil-lymphocyte ratio was associated with the prognosis of patients with hbv-aclf, and elevated nlr predicted poor outcome within weeks [ ] . natural killer and natural killer t cells: natural killer (nk) and natural killer t cells (nkt) are important components of the innate immune response. natural killer cells have potent cytolytic activities that are exerted through the death receptor and perforin/granzyme pathways. activated nkt cells have both perforindependent and fas-ligand dependent cytotoxic function that are triggered upon tcr recognition of an antigen [ ] . nk cells and nkt cells play an important role in many experimental models of liver injury, such as viral hepatitis, alcoholic liver disease, and autoimmune liver disease [ ] . however, their role in aclf has not yet been clearly elucidated, it was reported the median percentage of nk cells in the lymphocytes of patients with acute and fulminant liver failure were significantly lower compared to healthy controls. meanwhile, patients with acute and fulminant liver failure had significantly high and comparable nkt cells compared to control group [ ] . the important pathophysiological role of innate immune dysfunction in patients with acute-on-chronic liver failure (aclf) has been investigated in recent years. however, dysregulation of adaptive immunity remains poorly elucidated [ ] . patients with severe hepatitis has varying degrees of impaired cellular immunity, manifested as decreased cd + cell number and declined cd +/cd + ratio, which is pathogenesis of opportunistic infection. it was reported that there exists a reduction in cd (+) t lymphocytes in hbv-aclf patients. these cd (+) t cells predominantly are cd (+) tconv, and the development of suppressive cd (+) tregs greatly surpass tconv, which constitutes important characteristics of adaptive immune dysfunction of hbv-aclf [ ] . a report from china showed total amount of lymphocytes, cd (+) t cells, cd (+) t cells and nk cells in circulation were lower in the hbv-aclf patients compared to the chb patients [ ] . hbv-specific cd (+)t-cell responses are considered to be of great importance in viral control and immune-mediated liver damage [ ] . however, cd (+) t cell has seldom been studied in aclf. ye [ ] reported decreased activated cd (+) t cells may be related to poor outcomes in patients with sh. the frequency of circulating th cells increased with disease progression from chb to aclf patients compared to healthy control. th cells were also found largely accumulated in the livers of chb patients. the increases in circulating and intrahepatic th cells were positively correlated with plasma viral load, serum alanine aminotransferase levels and histological activity index. in addition, the serum concentration of th -associated cytokines was also augmented in both chb and aclf patients [ ] . in process of diagnosis and treatment for severe hepatitis, repeatedly abdominal paracentesis, retention catheterization, venous cannula, hemofiltration, or trachea cannula are usually necessary. unthoroughly sterilization or nonstandard sterile operation will lead to pathogen invading to develop an infection. in addition, artificial liver treatment is also an important cause of infection. it was reported that the incidence of fungal infection in severe hepatitis correlated with the number of artificial liver treatment. application of broad spectrum antibiotics is also a major cause of infection in severe hepatitis. antibiotics inhibit or kill sensitive normal bacterium as well as pathogens, especially normal bacterium colonized in natural orifice, leading to flora disproportionality. this time, nonpathogen could cause infection, or mass produced pathogen become dominant colony to develop infection. it was proved that dosage, exposure time, or varieties of antibiotics used in patients was closely correlated with severity of dysbacteriosis and incidence of sbp [ ] . nosocomial origin of infection, longterm of norfloxacin prophylaxis, history of recent infection by multiresistant bacteria and recent use of β-lactams were independent inducements associated with the development of multiresistant infections. bacterial translocation is defined as the migration of viable microorganisms or bacterial products (i.e., bacterial lps, peptidoglycan, and lipopeptides) from theintestinal lumen to the mesenteric lymph nodes and other extraintestinal sites [ ] . there are multiple mechanisms which are involved in defective gut functions and altered microbiota in patients with cirrhosis or liver failure. these include small intestine bacterial overgrowth (sibo), increased intestinal permeability, and impaired antimicrobial defense. additionally, decreased bile acids, due to decreased synthesis and defective enterohepatic circulation, contribute to altered gut microbiota [ ] . small intestinal bacterial overgrowth (sibo) was defined as ≥ total colonyforming units per milliliter of proximal jejunal aspirations, which presents in % of cirrhotic patients and is associated with systemic endotoxemia [ ] . in the condition of hepatic failure, due to impaired immunity, bacterium overgrowth and translocation happened, which increased incidence of infection [ ] . in vivo study from wang showed phagocytosis of monocytes were prominently and immediately diminished after resection of liver tissue up to %, accompanied by decreased oxygen ingestion [ ] . mass propagation of e. coli in the lower part of the small intestine and bacterium translocation were all happened h after resection. in rats with ascites in severe liver disease induced by carbon tetrachloride, the bacteria easily transferred from the intestine to extra-intestine, including local lymph nodes and blood circulation. these evidences proved that liver failure prompted the proliferation of intestinal bacterial overgrowth and translocation, which may be the main reason for the endogenous infections in patients with liver failure. another reason for bacterial translocation is that patients with cirrhosis or liver failure display increased intestinal permeability. in patients with severe hepatitis, gastrointestinal mucosa membrane has impaired ability for regeneration and repair, leading to dysfunction of intestinal barrier and declined anti-infection ability. patients with severe hepatitis have weakened regeneration and repair capacity of gastrointestinal mucosa, as well as decreased gastrointestinal barrier function and resistance to infection. the intestinal mucosa often manifested as congestion, edema or erosion. especially when intestine get infection, bacteria can invade to abdominal cavity directly or through slight mucosal defect [ ] . besides, ascites often present in severe hepatitis patients, which provide a good medium for bacteria to multiply. patients with severe hepatitis often have ascites combined with portal hypertension. due to lobular structural damage, abnormal construct of liver sinusoids or blood vessels, bacteria directly access into the systemic circulation without filtration and phagocytosis by liver, leading to formation of bacteremia. meanwhile, bacteria in blood circulation may access into the abdominal cavity and cause abdominal infections. hence, patients with severe hepatitis are not only prone to get endogenous infection but also prone to have intestinal endotoxemia. patients with severe hepatitis have decreased bile secretion, changes of bile composition, so that infection are prone to occur in epithelium of bile duct and gallbladder. in patients with severe hepatitis, intestinal edema and cellulitis are obvious, some may develop acute serositis. for patients with portal hypertensive gastrointestinal disease, regeneration and repair capacity of gastrointestinal mucosa are decreased, accordingly, the natural immune barrier function is reduced. once esophageal and gastric venous bleeding occur, gastrointestinal ischemia aggravate, which result in decreased resistance to infection. invasive procedures, complications, prophylactic use of broad-spectrum antibiotics, underlying diseases, long hospital stay, and old age are risk factors for infection in patients with severe hepatitis. elderly patients with other underlying diseases, decreased immune function, more severe primary disease, have a high risk of infection and may predispose to severe infections. in addition, albumin level is closely related to ascites production and sbp occurrence. irrational uses of immunosuppressive agents such as corticosteroids suppress immune function, lead to flora, and promote the formation of drug-resistant strains and pathogenic bacteria. mortality of severe hepatitis is closely related to infection, which directly affects the prognosis of severe hepatitis. to reduce the incidence of infection in patients with severe hepatitis, we should aim at a variety of risk factors, improve patients' immunity, rectify hypoproteinemia, treat the primary disease, prevent complications, strengthen disinfection and isolation, operate with strict aseptic technique and strict indications for invasive procedures, and use antibiotics rationally. once patients with severe hepatitis are infected, the consequences are often serious, as infection easily diffuse, which is difficult to control. after infection, the bacteria produce more toxins, which aggravate liver disease and cause a series of adverse effects, finally lead to severe complications (hepatic encephalopathy, hepatorenal syndrome, and etc.), which is the major cause for death in patients with severe hepatitis. bacterial endotoxin plays a major role in development and prognosis of severe hepatitis when infection happened [ ] . endotoxemia may directly or indirectly worsen liver injury. intrahepatic cholestasis positively correlated with endotoxin levels. endotoxin can trigger hepatorenal syndrome. for kidney, endotoxin is a potent renal vasoconstrictor substance, which can make a strong contraction of the renal artery and renal blood flow reduction. in addition, endotoxin can cause kidney capillary thrombosis and acute renal tubular necrosis, or even renal cortex necrosis. endotoxin can activate coagulation factor vii and factor vi, making the intrinsic coagulation system startup, as well as directly damage hepatocytes which release tissue thromboplastin to start the extrinsic coagulation system. meanwhile, endotoxin can also damage the vascular endothelial cells to cause bleeding, especially upper gastrointestinal bleeding. in the event of gastrointestinal bleeding, infections are more prone to happen or primary infection aggravates. in recent years, due to extensive use of broad-spectrum antibiotics, the mortality of severe hepatitis has been significantly reduced. however, infection is still an important cause of death. nosocomial infection control directly affects the prognosis of patients with severe hepatitis, which is an important part for the treatment of severe hepatitis. once the etiology and other evidence of infection appear, patients should be treated with antibiotics. effective prevention and treatment for nosocomial infections are positive to promote recovery and reduce mortality. bacterial translocation (bt) is the key mechanism in the pathogenesis of sbp, which is because of the concurrent failure of defensive mechanisms in liver failure [ ] . investigators have demonstrated pronounced impairment of gastrointestinal tract motility in cirrhosis [ ] . the disturbance of gut flora microecology was associated with changes in the (ultra)structure of the gastrointestinal tract [ ] . meanwhile, reduced cellular and humoral immunity make it easier for the free flow of microorganisms and endotoxins to the mesenteric lymph nodes [ ] . besides, it was reported that hypoalbuminemia was related to sbp. a prospective study from runyon reported that the incidence of sbp in patients with ascites protein less than . g/l was %, while the number in patients with ascites protein more than . g/l was only %. after years of follow-up, sbp incidence in patients with ascites protein higher than . g/l were negligible [ ] . the level of ascites protein correlates with opsonin activity, which means ascites protein < . g/l was an independent risk factor for sbp. the clinical manifestations of sbp are diverse. most patients have subtle and insidious onset, which usually manifested as abdominal pain and fever. acute onset may burst chills, fever and abdominal pain. generally the body temperature of patient is around °c, sometimes as high as °c. fever type usually presents as remittent fever. abdominal pain is always around the navel or lower abdomen, paroxysmal or persistent. nausea and vomiting are obvious, sometimes with diarrhea. ascites can occur in patients without ascites, or increased significantly. patients may also have significant bloating, abdominal muscle tension, tenderness, rebound tenderness, diminished or disappeared bowel sounds, and so on, or even shock in severe cases. according to clinical manifestations, sbp can be divided into five types: . common type: acute onset, protruding abdominal pain, followed by fever. or irregular fever take place followed by abdominal pain, abdominal tenderness and rebound tenderness, mild to moderate abdominal tension and growing ascites. increased wbc count and nuclear left shift. routine examination of ascites showed acute inflammatory changes. . shock: septic shock break out in a few hours to one day after abdominal pain or fever. the clinical manifestations include low temperature, lip cyanosis, abdominal tenderness, and hardly relieved shock. wbc count increases, blood culture is positive. . encephalopathy: fever and abdominal pain are often obscure, the early emergence of trance and other neuropsychiatric symptoms rapidly develop into a coma. careful examination of the abdomen in patients with light coma may still find in patients with pain expression. jaundice is deep, liver damage is serious. . refractory ascites: ascites progressively increase, which is difficult to subside with invalid diuretic therapy. abdominal distension is obvious, often without pain. carefully check the abdomen may still find slight peritoneal irritation. . asymptomatic: symptoms are mild, exhibited as slight bloating, occasional fever. mild tenderness can be found by deep palpation. in addition, a considerable part of the patients showed non-specific symptoms and signs, such as deepened coma, deepening jaundice, oliguria, azotemia or dramatically increased ascites. diagnosis of sbp should be considered if following conditions exist: ( ) fever, which can't be explained by other reasons or other parts of infection; ( ) abdominal pain, abdominal tenderness or rebound tenderness, but not serious; ( ) sudden increased ascites or poor diuretic effect manifested as refractory ascites; ( ) sudden onset of septic shock; ( ) rapid deterioration of general condition, or deteriorated liver and kidney function in a short term, deepening jaundice, or hepatic encephalopathy. the diagnoses of sbp mainly rely on ascites puncture. easl clinical practice guidelines recommend that a diagnostic paracentesis should be performed in all patients with new onset grade or ascites, and in all patients hospitalized for worsening of ascites or any complication of cirrhosis (level a ) [ ] . for patients with severe hepatitis, diagnostic paracentesis indications are: . in liver cirrhosis patients with ascites that paracentesis should be performed after admission to determine whether sbp exist. . if the following conditions happened during hospitalization, diagnostic paracentesis should be performed: ( ) abdominal sighs suggest abdominal infections, such as abdominal pain, rebound tenderness and gastrointestinal symptoms (such as vomiting, diarrhea, intestinal paralysis); ( ) systemic infection signs such as fever, leukocytosis, or septic shock; ( ) no clear incentive for hepatic encephalopathy, or rapidly emerged renal dysfunction. . in patients with ascites and gastrointestinal bleeding, paracentesis should be performed before prophylactic antibiotics. once ascites was acquired, polymorphonuclear cells (polymorphonuclears, pmn) count and ascites culture should be performed. diagnosis of sbp is made according to international guidelines [ , ] in patients with liver cirrhosis if the ascites polymorphonuclear (pmn) cell count exceeds cells/μl and other forms of peritonitis have been excluded. an ascites fluid polymorphonuclear (pmn) leukocyte count ≥ /mm should be considered as sbp, if pmn > /mm can be confirmed as sbp. if there is bloody ascites (erythrocytes more than , /mm ), pmn count are as / of red blood cells. the leukocyte esterase reagent strips (lers) test is based on the esterase activity of the leucocytes. since , studies have examined the validity of using leukocyte esterase reagent strips (lers) in sbp diagnosis. lers appeared to have low sensitivity for sbp. on the other hand, a high negative predictive value (> % in the majority of the studies)supported the use of lers as a preliminary screening tool for sbp diagnosis [ ] . there is bacteria colonization in ascites but no inflammation. the diagnosis is based on positive ascites culture, ascites pmn countless than /mm , without evidence of systemic or local infection. bacterascites have two outcomes: a shortterm or transient bacterial ascites (mostly asymptomatic), or the development of sbp (mostly with symptoms). once the diagnosis was established, paracentesis examination should be conducted again after - days. take appropriate action under the circumstances. if the second sample has a pmnl count > /mm , treat as for sbp. if the pmnl count is< /mm and a second set of cultures is positive, treat as for sbp. if the pmnl count is < /mm and the second set of cultures is negative, no further action is recommended [ ] . if ascites culture is positive but ascites pmn < /mm and there were signs of abdominal infection, it usually progress to sbp within a few days. these patients should be given appropriate antibiotic therapy. brolin first proposed the concept in . the mechanism: in early stage of severe hepatitis, ascites has not been exist yet, however because of necrosis of hepatocytes, dysfunction of kupffer cell, impairment of liver immune barrier, intestinal bacteria easily invasive into the systemic circulation through liver, causing spontaneous bacteremia, and then sbp. diagnostic criteria: ( ) primary disease was severe hepatitis, no ascites was detected by strict examination and ultrasound. ( ) clinical manifestation include fever, varying degrees of abdominal pain, diarrhea, diffuse abdominal tenderness and rebound tenderness, increased peripheral blood leukocyte, positive rivalta's test, ascites fluid leukocyte count> /mm , or pmn > /mm . ( ) exclude abdominal organ perforation or primary foci. it was used to describe the clinical situation when the ascitic pmnl count is > /mm but cultures fail to grow any bacteria. however, the term is now considered obsolete. in severe hepatitis with secondary infection, pneumonia is most common. patients with hepatic encephalopathy are susceptible to pulmonary infections as pneumonia since bed-ridden, impaired cough reflex and inadequate ventilation, especially comatose patients with intubation and tracheotomy. inpatients underwent thoracentesis, paracentesis and other invasive procedures, non-specific damage to immune barrier provide conditions for the bacterial invasion. reported in patients with invasive procedures, lung infection risk increased significantly, which demonstrated blood infection is an important way for pulmonary infection. furthermore, there was a significant increase of pulmonary infection in patients with intestinal infections or abdominal infection. pathogen: in recent years, pneumonia was classified as "community acquired pneumonia" (community acquired pneumonia, cap) "and" nosocomial pneumonia (hospital acquired pneumonia, hap). cap is the pneumonia acquired outside hospital [ ] . although cap can be caused by a wide variety of micro-organisms, the pneumococcus, mycoplasma pneumoniae and chlamydophila pneumoniae, staphylococcus aureus and certain gram-negative rods are more usual pathogens encountered [ ] . hap is the pneumonia that develops h or more after hospital admission and that was not incubating at hospital admission. hap infected with gram-negative bacilli accounts for more than %, of which pseudomonas aeruginosa, klebsiella pneumoniae, escherichia coli, acinetobacter baumannii, other aeruginosa are common bacteria. the primacy is pseudomonas aeruginosa, while gram-positive cocci accounted for about %, mainly are staphylococcus aureus, coagulase-negative staphylococci, viridans and streptococcus pneumoniae. anaerobic is rare. clinical manifestations and diagnosis: clinical manifestations are characterized as fever, cough, expectoration, dyspnea, and cyanosis. diagnostic criteria: . chest percussion of dullness, auscultation of rales, along with one of the following conditions: ( ) purulent sputum or change of phlegm; ( ) positive pathogens in sputum culture. . the chest x-ray and/or ct examination revealed new or progressive exudative lesions, and the emergence of one conditions above. urinary tract infection is divided into upper urinary tract infection and lower urinary tract infection. upper urinary tract infections are mainly pyelonephritis, which can be manifested as acute pyelonephritis and chronic pyelonephritis. lower urinary tract infections include urethritis and cystitis. the most common pathogen of urinary tract infection is escherichia coli, followed by enterococcus faecalis, klebsiella pneumoniae and streptococcus agalactiae. urinary tract infections often occur in patients with indwelling catheter, therefore, aseptic urethral catheterization and management and timely replacement of catheterization are effective to prevent such infections [ ] . . acute pyelonephritis: ( ) acute onset; ( ) chills, chills; ( ) fever; ( ) malaise, headache, fatigue; ( ) loss of appetite, nausea, vomiting; ( ) urinary frequency, urgency, dysuria; ( ) low back pain, kidney area discomfort; the ( ) tenderness of upper ureter point; ( ) tenderness of rib waist point; ( ) percussion pain in kidney area or bladder. . chronic pyelonephritis: ( ) acute onset of acute pyelonephritis with the same, but usually much lighter, even without fever, malaise, headache and other systemic manifestations, urinary frequency, urgency, dysuria and other symptoms are not obvious; ( ) edema; ( ) hypertension. . urocystitis and urethritis: frequent urination, urgency, dysuria, urinary bladder pain. urethral secretions. laboratory tests and diagnosis: diagnosis elements include: ( ) tenderness in rib waist point, percussion pain in kidney area. ( ) urine leukocytosis, pyuria. ( ) urinary sediment smear find bacteria. ( ) positive in urine culture. ( ) urinary colony counts> /ml; in patients with urinary frequency and other symptoms, colony counts> /ml are meaningful; counts - /ml also helpful in diagnosis; ( ) one hour urine wbc count> , . ( ) blood test showed leukocytosis, neutrophil nucleus left. ( ) increased esr. biliary tract infection is a common complication in severe hepatitis, which is often in company with cholelithiasis to reinforce each other. hepatitis b virus can directly violate bile duct cells and cause cholecystitis. on this basis, cholelithiasis and secondary bacterial infections are easy to happen and become a major focus, which can cause other parts of infection in severe hepatitis patients. furthermore, severe hepatitis patients often have reduced gastric acid secretion, so that e. coli in duodenum easily reproduce and cause ascending infection. biliary tract infection is usually a mixed infection of aerobic and anaerobic infections. enteric gram-negative bacteria include escherichia coli, klebsiella, enterobacter, proteus and enterococcus. anaerobic bacteroides include clostridium and fusobacterium, in which bacteroides is common, about - %, particularly bacteroides fragilis. common symptoms of biliary tract infection include chills, fever, nausea, vomiting, right hypochondrium pain and tenderness in gallbladder area. clinical performance of concurrent biliary tract infection in severe hepatitis is not always very clear, often unconfirmed by pathogen test. symptoms were epigastric or right upper quadrant pain, radiating to the right shoulder, hours after a heavy meal or a high fat meal. the patient may have severe colic, often accompanied by nausea and vomiting. patients with chronic biliary tract infection have indigestion symptoms as heartburn, belching, acid reflux and bloating, or sometimes fever and right upper quadrant pain. severe hepatitis patients have weakened intestinal resistance, especially for reduced immunoglobulin a secretions, which creates good invasion opportunities for bacterium. in addition, as mentioned above, intestinal flora are prone to happen in severe hepatitis patients, which makes intestinal infection very common [ ] . few patients exhibit cellulitis like colitis, which can further result in peritonitis and septicemia, and finally lead to death. pathogens in intestinal infections could be shigella spp., salmonella, campylobacter jejuni, clostridium difficile, and salmonella typhi etc. clinical manifestations are nausea, vomiting, abdominal pain, diarrhea, watery stool or bloody mucopurulent stool. some patients may have fever and tenesmus. depending on the pathogenesis and clinical manifestations, intestinal infections are classified as enterotoxigenic bacterial enteritis and invasive bacterial enteritis. pathogenesis of enterotoxigenic bacterial enteritis is that bacteria adhere but not invade intestinal mucosa. intestinal toxins are secreted in the process of bacteria growth and reproduction, which stimulate small intestinal epithelial cells secreting large amount of water and electrolytes. overuptake of water and electrolyte and retention in intestine makes watery stool, which is called "secretory diarrhea." secretory diarrhea is exhibited as frequent, large amount stool with no pus, usually without pain or tenesmus. it is often accompanied by vomiting, which is prone to bring out dehydration, electrolyte imbalance and acidosis, but less severe systemic toxic symptoms. stool examinations show less erythrocytes or leukocytes. invasive bacterial enteritis refers to pathogenic bacteria adhere and invade the intestinal mucosa and submucosa, causing significant inflammation. different pathogens violate different parts of intestine, small intestine, or colon, and sometimes cause inflammation both of small intestine and colon. the basic clinical manifestations include obvious systemic sepsis, high fever, and even septic shock in severe patients. stool can be mucus bloody or purulent, less amount and more frequency. abdominal pain are often severe, paroxysmal colic. if the lesion invades the rectum and distal colon in particular, there may be tenesmus. sigmoidoscopy examination showed diffuse inflammation and ulceration. if only the small intestine or upper part of colon are invaded, the stool is more moisture, and without tenesmus. stool examination show large amount of leukocytes. although diagnosis of intestinal infection is not difficult, it should be careful to distinguish the site of infection, make sure of pathogens, and pay particular attention to water, electrolyte imbalance and acid-base imbalance. therefore, in addition to routine examinations and stool culture, keeping abreast of the general condition is also important to avoid disturbance of the internal environment which aggravate liver damages. in severe hepatitis, the more severe hepatic damage and immune dysfunction, the higher of incidence of sepsis. bacterium most commonly enters through intestine into the portal vein and then into the systemic circulation, followed by skin, respiratory tract, urinary tract or other intrusion. pathogens are often opportunistic bacteria, in which gram-negative bacteria are more than gram-positive bacteria, especially escherichia coli. clinical manifestations of nosocomial infection concurrent with severe sepsis are not specific, easily masked by the primary disease and complications. in some cases primary focus is not obvious, therefore the diagnosis mainly rely on blood culture. clinical manifestations include: ( ) unexplained sudden chills, fever, shock, increased peripheral blood leukocytes or neutrophils; ( ) deepening jaundice, an increase in ascites, or appearance of hepatic coma, hepatorenal syndrome in a short term. when complications appear in severe hepatitis patients, sepsis should be alert. the mortality of sepsis in severe hepatitis is high. nosocomial infection not only aggravates liver damages, but also induces hepatic coma, hepatorenal syndrome, upper gastrointestinal bleeding and other serious complications, leading to multiple organ failure. nancy rolando reported mortality of septicemia in patients with liver failure was as high as %, in which % with septic shock [ ] . fungal infections can be classified into endogenous and exogenous infection. according to the source of the fungus, the former belongs to opportunistic pathogens, while the latter is in the environment, being infected through various routes of exposure. fungal infections in severe hepatitis are invasive fungal infection in most occasions, and the majority are nosocomial infection and endogenous pathogenic fungi infection [ ] . candida infection is most common, followed by aspergillus, again neoformans and histoplasma monocytogenes. candida albicans is widely present in normal human digestive tract. other fungi such as cryptococcus neoformans, aspergillus are widely found in nature, which can colonize in body surface, or non-enclosed cavity. they also exist in hospital work environments, increasing the chance of nosocomial infection in hospitalized patients. clinically, severe hepatitis with fungal infections are mostly superinfection. the rate of fungal infection in severe hepatitis is increasing in recent years. nancy rolando reported fungal infection was present in of acute severe hepatitis patients ( candida, aspergillus) and in seven cases was considered the major cause of death [ ] . domestic data reported that in a group of patients with liver failure, fungous infection was found in cases in which the rate of nosocomial infections was . %. in separated fungous strains, strains ( . %) were candida albicans, strains ( . %) were aspergillus fumigatus and ( . %) strains were non-candida albicans. the main sites of fungus infection were lungs ( cases) and oral cavity ( cases) [ ] . for severe hepatitis complicated by fungal infection, the mechanism is complex. there are many influencing factors such as immune dysfunction and reduced defensing ability, besides, application and abuse of broad-spectrum antimicrobial drugs are also related [ ] . because of broad-spectrum antibiotics destroy the imbalance between bacteria and fungi in digestive system, which inhibit the growth some gram-negative bacteria that had an anti-fungal effect and some bacteria that are able to synthesize vitamin b family. lack of vitamin b can lead to inhibition of oxidation coenzyme, enabling weakened immunity, which is conducive to fungal growth. research and experience have demonstrated that repeatedly use of glucocorticoid is also an important factor to induce fungal infection in patients with severe hepatitis [ ] . and therefore, the use of corticosteroids also need special care of. currently the use of glucocorticoids in patients with severe hepatitis is still controversial. the majority do not advocate glucocorticoids, or consider a short-term use in the early stages of the disease and be removed as soon as possible. otherwise it will cause a large increase of possibility of fungal infection. fungal infections are among the leading causes of death in patients with severe hepatitis. according to an analysis from rolando, among cases of severe hepatitis, seven cases of deaths directly related to fungal infections [ ] . a recent domestic research analyzed outcomes of patients with severe hepatitis, it was found that the mortality of patients with fungal infection was significantly higher than those without fungal infections ( . % compared to . %) [ ] . according to statistics, fungal infections often occur eight days after admission ( - days) or . days after broad-spectrum antibiotics usage ( - days). symptoms of fungal infection are often covered by severe liver injury related symptoms, while clinical systemic manifestation such as fever is also difficult to identify with the bacterial infection. the site of infection often occurs in mouth, respiratory, digestive or urinary tract. severe immunocompromised persons may appear disseminated infection. oropharyngeal is the site that fungal infections mostly occur, and candida albicans is the most common pathogen, followed by non-candida albicans and aspergillus infection. bedridden patients with severe hepatitis can not properly maintain oral hygiene, which results in change of oral local environmental ph and blood flow. candida albicans easily retains in the mouth, and multiplies, causing oral flora and opportunistic infections. usually general symptoms are mild. patients often have abnormal taste or loss of taste, followed by xerostomia, mucosal burning and other symptoms. candida stomatitis may have pseudomembrane formation which is not easy to peel, accompanied by angular cheilitis, or sometimes manifests as mucosal congestion, erosion or clumps shrink of tongue papillae, thickening of coating on the tongue. there are clear lines between oral pseudomembranous damages. if remove the pseudomembrane it will leave a bright red base, sometimes thick film is like a layer of cheese. take the film directly under microscopic examination shows hyphae and spores. oral fungal infection is often a prelude of deep fungal infection, which should be on the alert. simple oral candida albicans infection is not always accompanied with fever. oral fungal infection is easy to be found, therefore, if oral fungal infection exists with fever and increased leukocytes, it should be paid attention to merger of deep fungal infections such as the lungs or other organs [ ] . aspergillus is ubiquitous in nature which can be spread through air flow. the most common pathogen that causes invasive aspergillosis is aspergillus fumigatus, while aspergillus flavus, aspergillus niger and aspergillus soil are less common. inhalated aspergillus spores can reproduce in healthy or immunity weakened people. respiratory tract is an infective route of invasive aspergillosis, accounted for %. once tissue infection exists, blood vessels violation and bloodstream invasion are common. invasive aspergillosis infection has three characteristics: tissue necrosis, hemorrhage, dissemination [ ] . the mortality of invasive pulmonary aspergillosis accompanied by severe hepatitis is up to % [ ] . it is lack of specific clinical manifestations. in most patients fever is the first arisen symptom, mostly with middle or low heat, sometimes with sudden high fever. hemoptysis occurs with fever simultaneously or - days later, often accompanied with purulent tan sputum or bloody sputum, sometimes with a pinhead size of gray-green particles in it. shortness of breath and chest tightness often exist in late stage of infection, as well as dyspnea, cyanosis, hypoxemia, and expectoration of a lot of bright red bloody sputum or blood clots. pulmonary signs appear later, manifests as pulmonary wet or dry rales, occasional pleural friction sound. in some cases there are no pulmonary signs. x-ray examination of invasive pulmonary aspergillosis showed patchy infiltrates and (or) nodular lesions. typical nodules were like cotton, which can occur in unilateral or bilateral lobes. the lesion progresses rapidly to cause expanded infiltrates, and segmental or lobar consolidation. ct examination showed mass shadow, nodules, or exudative lesions. there are two typical imaging performance: ( ) a characteristic finding on chest ct is the halo sign: a solid nodule surrounded by a halo of ground-glass attenuation [ ] . ( ) the formation of voids, which appear in late infection. signs are hollow cavity lesions, the balloon "crescent" sign was seen around necrosis tissue [ , ] . in order to prevent hepatic encephalopathy, patients can be given high doses of laxatives such as lactulose, which can reduce the intestinal ph value, creating an environment for growth of fungi, thereby increasing the chances of fungal infection of the intestine. in particular, some antibiotics combination increases the incidence of intestinal candidiasis. patients usually have watery or jerry-like diarrhea, with foam or blood. diarrhea is accompanied by bloating, sometimes with vomiting and fever, however, abdominal pain is not obvious. in patients with severe hepatitis, due to decreased intestinal mucosal defense capability, candida may invade the muscle and cause intestinal bleeding, or even perforation. in part of patients, it may even progress to fungal peritonitis, which is similar to clinical manifestations of bacterial peritonitis. in patients with oral candidiasis, if there is dysphagia or pain, especially retrosternal burning, it should be considered that esophagus is invaded. incoordination motor of the upper and lower esophageal can be found by esophageal barium enema. gastroscopy helps to confirm the diagnosis. urinary fungal infection usually involves bladder and kidney, among which candida infection is the most common. however, all pathogenic fungi (such as cryptococcus neoformans, aspergillus species, mucor species, histoplasma, blastomycete, coccidioides) can spread to the urinary system as a systemic or part of disseminated fungal infection, which is more related to usage of broad-spectrum antibiotics and indwelling catheters. clinical manifestations include fever and urinary tract irritation, while some patients were asymptomatic with candidiasis urine. candida and bacterial infections often occur simultaneously. candida infections of the kidney, mostly secondary, are caused by the spread of blood candida. renal cortex and medulla abscesses can occur, which affect renal function in severe cases. patients may have low back pain, abdominal pain, fever and chills, accompanied by urgency, urinary frequency, proteinuria and hematuria. urine tests may find hyphae and fungal spores, culture for candida is positive. candida albicans is common, but now there is a growing trend of candida glabrata [ ] . the main pathogen of fungal sepsis is saccharomyces. most patients have high fever, often above °c. the thermal type varies, of which intermittent fever or remittent fever is more common. wbc count and neutrophil are usually increased. disease in patients with fungal sepsis followed by severe hepatitis is deteriorating rapidly, even progressing to shock. fungal septicemia may invade all the tissues and organs. involved organs have corresponding performance, such as fungal pneumonia, oral fungal infections, intestinal and urinary tract infections. disseminated fungal infection often occurs in severe immunocompromised patients who have long-term use of antimicrobial agents. candida, cryptococcus, aspergillus can disseminate along with blood to all of the organs, such as kidneys, lungs, heart, and liver. the condition is dangerous, which often lead to death in a short term. in addition to common bacterial and fungal infections, severe hepatitis can also be complicated by other pathogens, such as viruses, tuberculosis, protozoa and others. cmv (cytomegalovirus, cmv), herpes simplex virus (herpes simplex virus, hsv), or varicella -zoster virus (varicella-herpes and zoster virus, vzv) infections are three of common herpes viruses infections in severe hepatitis. their common characteristics rely on that once the host is infected, the virus can persist for long periods in the host. when the host immunity is weakened, the virus can re-proliferative, which leads to disease resurgence [ ] . hsv infections manifest as perioral or external genital herpes, oral and esophageal mucosa inflammation and ulcers, also viremia which leads to pneumonia and encephalitis. common clinical symptoms of herpes simplex are mild, only a few people show fatigue, fever and other symptoms. local manifestations are single or multiple blisters on skin or mucous membrane, with tingling. due to reduced immunity, skin rashes in patients with severe hepatitis perform as varicella-like rash, vaccination herpes, herpes keratoconjunctivitis and disseminated herpes simplex. severe herpes usually manifests as herpes simplex virus encephalitis with high mortality. there are fever, headache, mental disorders, coma and other clinical symptoms, often without skin herpes lesions. the sites of infection are commonly in the frontal and temporal lobes. elevated serum antibodies help confirm the diagnosis. cytomegalovirus infections are common in cirrhosis of the liver [ ] . interstitial pneumonia is the most common clinical manifestation in severe hepatitis concurrent with cmv infection. chest ct examinations mainly perform as diffuse interstitial or alveolar infiltrations, very few case show as nodular shadows, occasionally as pleural effusion [ ] . pulmonary consolidation reminds complicated bacterial or fungal infections. pathology manifestations show alveolar interstitial edema, with varying degrees of fibrosis, lymphocyte infiltration and epithelial cell proliferation. blood examination shows leukopenia. because viral pneumonia shares a certain similarity in clinical manifestations, diagnosis mainly depends on pathologic examination. pathogenic examinations for cmv often use methods below: ( ) detect of cmv inclusion body cells and viral particles: eosinophilic nuclear inclusions giant cells are found in respiratory secretions and bronchoscopy lung biopsy specimens. respiratory secretions, saliva, urine, cervical secretions, liver or lung biopsy specimens were inoculated to human embryonic fibroblast cell culture medium, where cytomegalovirus was separated. ( ) immunological methods: cmv antigen from secretions was detected by fluorescent antibody assay, or elisa, which is conducive to the early diagnosis. serum antibodies can also be detected by complement combined experiment, in which acute and convalescent serum antibody titer more than times are positive. ( ) molecular biology methods: pcr technology and nucleic acid hybridization, which helps to make distinction between a variety of different subtypes of the virus. most of vzv infections in patients with severe hepatitis are due to latent virus reactivated. in patients who have had chickenpox, there is a small amount of virus lurking in the spinal cord dorsal root ganglia or cranial nerve sensory ganglia. when severe hepatitis happened, latent virus is reactivated in ganglia due to decreased immunity. the activated viruses spread along with sensory nerve axons to downstream disposal areas, proliferate and cause shingles. in early time, there is paresthesia, itching, and pain in local skin. and then rashes and herpes break out, chaining into a strip, which distribute in denomination or trunk, unilateral, with duration of about three weeks or several months. part of patients with severe hepatitis show severe disseminated herpes zoster. varicella-like rashes appear in a few days, often accompanied by fever, which may be complicated by lung, brain damage with a high mortality rate. latent tuberculosis can burst to tuberculosis and extrapulmonary tuberculosis when cellular immune function gets weakened. under normal host immune function, lymphocytes, macrophages and common langerhans cell may promote granuloma formation and make infection localized. when host immunity is dysfunctional, tissue reaction is very small or even disappeared, leading to mycobacterium growing rather than formation of granulomas, nor any effective defense against infection. severe hepatitis patients with m. tuberculosis infection may develop acute miliary tuberculosis that manifest as fever, cough, expectoration, bloody sputum, chest pain, and shortness of breath in addition to deteriorated liver function. moreover, tuberculosis easily spread in patients with severe hepatitis, with poor anti-tuberculosis efficacy. common extrapulmonary tuberculosis can be lymphatic tuberculosis, intestinal tuberculosis, bone tuberculosis, renal tuberculosis, epididymal tuberculosis, genitourinary tuberculosis, nervous system tuberculosis, tuberculous meningitis [ ] . in patients with severe hepatitis concurrent with tuberculous mycobacterial infections, tuberculin reaction is negative in about half of patients, especially in those applied with glucocorticoid. diagnosis relies on sputum acid-fast staining or pcr, but the diagnostic yield is not high. interferon gamma release assays (igras), including quantiferon ® -tb gold in-tube, and the t-spot tb, have been extensively used for the auxiliary diagnosis of tuberculosis infection in adults. igras detect circulating t-cells responsive to specific mycobacterium tuberculosis antigens, which are absent in bcg and other non-tuberculosis mycobacteria, and exhibited similar sensitivity and higher specificity than tst in adults [ , ] . however, these igra tests are also affected by host immune status [ ] . in addition, the decision regarding whether to treat ltbi should be dependent not only on igras results but also on clinical histories. ntm generally causes local wound infections. however, in severe hepatitis patients with impaired immune function, non-tuberculous mycobacteria can invade the lungs, causing tuberculosis-like diseases, but rarely causes hematogenous dissemination. histological examination of the lesion is mainly characterized by epithelioid cell granulomas and foam cell-like balls of tissue proliferation, detection of non-tuberculous mycobacteria [ ] . protozoa and worms, such as toxoplasma gondii, giardia lamblia, cryptosporidium and stercoralis, can also infect patients with severe hepatitis, especially those with immunosuppression drugs or combined with cancer. main lesions of toxoplasmosis manifest as lymphadenopathy, hepatosplenomegaly, encephalitis and pneumonia [ ] . clinical manifestations of giardia lamblia infection are chronic diarrhea and malabsorption, also fever and cholecystitis [ ] . pathological changes are deformation of small intestine villi and lymphoid hyperplasia. parasites present in small intestinal surface and gallbladder, and the detection of parasites from the stool and duodenal drainage fluid that is eligible confirmed. strongyloides stercoralis is a weak virulent worm, there is few clinical stercoralis infection [ ] . but this worm infection in patients with severe hepatitis is a serious threat, even causing death. clinical manifestations include long-term nausea, vomiting, diarrhea, bloating, intestinal paralysis, dehydration, electrolyte imbalance, edema, weight loss and difficulty breathing in cases with extensive lung lesions. all patients have hypoproteinemia and anemia. patients with increased eosinophils have good prognosis, whereas eosinophils reduction often is a dangerous signal. varieties of complications, such as system or local infections, coagulopathy, hepatic encephalopathy, hepatorenal syndrome, hepatopulmonary syndrome, acid-base imbalance and water-electrolyte imbalance, are main causes of hbv-associated mortality during deterioration of liver function of aechb. reducing and effective treatments of these complications are still nodules in therapy of severe phase of aechb. infection is one of usual complications of aechb, which shows - % morbidity in civil china. the most common localities are respiratory system, especially lungs, and abdomen, which shows the incidence of spontaneous bacterial peritonitis (sbp) as much as %. others include urinary tract and bloodstream. gram-negative bacteria are the predominant causes including aerobic gram-negative bacilli, escherichia coli, klebsiella, enterococcus and anaerobic bacteroides fragilis, etc. however in recent years, gram-positive bacterial infections are found increasing in patients with aechb, including pneumococcus and other streptococcus, but staphylococcus aureus infection is relatively infrequent. fungal infections are usually secondary to bacterial infection. candida, aspergillus, sporotrichosis, histoplasmosis and coccidioidomycosis infection, especially candida albicans, are most frequent in occurrence. but, infections of cryptococcus and mucormycosis are relatively rare. beyond of bacterial and fungal infection, other infectious pathogens in aechb are virus, mycobacterium tuberculosis, mycoplasma, chlamydia, parasites and protozoa. bacterial infection is the most frequent complication in aechb. infections are more likely occurred in abdomen, including abdominal cavity, biliary tract, gastrointestinal tract, or other parts like respiratory tract, and urinary tract. keeping oral and perineal asepsis, unobstructed respiratory tract and urine tract, anti-bedsore care for patients with limited mobility, rational use of antibiotics, avoiding long-term use of broad-spectrum antibiotics, strict controlling usage of glucocorticoid, aseptic practices in invasive operation, parenteral nutrition and protecting the intestinal mucosa are all necessities to prevention of bacterial infection in aechb. empirical use of antimicrobial agents could be determined by the localities of infection without antibiotic susceptibility testing results. gram-negative infections are more frequent in peritoneal or biliary tract, in which cephalosporin or quinolone could be the first choice. penicillin and vancomycin could be considered in pulmonary infection. azithromycin and quinolone could be adopted in urinary tract infection. metronidazole or tinidazole could be used in anaerobic infection. broad-spectrum antibiotics can be used in serious infection, such as ceftriaxone, cefoperazone, cefepime, and carbapenems, but the secondary infections need to be highly concerned. initial antibiotics should be adjusted according to antibiotic susceptibility testing results as soon as possible. non-specific immune enhancer agents, such as thymosin, are well used in treatment of infection during aechb, but it currently still lack of evidence-based support. the incidence of fungal infection ranks secondly in aechb associated infection. respiratory tract, gastrointestinal tracts and genitourinary system are the major sites. keeping wards dry and ventilated can help to reduce environmental fungal growth. oral and perineal cleaning and regularly dental check are necessary. if oral fungal spots are found, alkaline mouthwash and nystatin with glycerol can be used in treatment. for patients with limited mobility, anti-bedsore care is terribly necessary. rational use of antibiotics, especially avoiding long-term usage of broadspectrum antibiotics, can prevent secondary fungal infection. glucocorticoid should be used strictly according to indication. during invasive procedures, aseptic operations are obligated. regular check of sputum, lungs, urine and feces will facilitate early diagnosis. empiric antifungal treatment is generally not recommended, but to patients with aechb with aids, especially with count of peripheral blood cd + t cell-less than /μl or oropharyngeal candidiasis found, the sulfamethoxazole (smz-tmp) should be chosen to prevent pneumocystis pneumonia. fluconazole should be considered when moderate amount of candida albicans has been indicated by sputum culture. empirical treatment. if candida albicans infection is highly susceptible, the initial treatment choice is fluconazole ( - mg/days). but if aspergillus infection is preferred, amphotericin b liposome, itraconazole, caspofungin or voriconazole could be considered as the initial treatment, in which process liver and kidney function should be intensively monitored. to patients with cryptococcal encephalopathy combining severe liver damage, fluconazole or flucytosine combined with intrathecal injection of amphotericin b treatment could be implemented. evidence based treatment. initial antifungal strategy should be adjusted according to antibiotic susceptibility testing results. for certain invasive pulmonary aspergillosis, combination treatment of several different types of antifungal agents should be considered under monitoring of liver and kidney function. however, for pulmonary mucormycosis infection, the combination of amphotericin b and flucytosine is the only effective strategy. amphotericin b is a type of polyene antifungals, mainly for aspergillus, candida, cryptococcus, histoplasma infection, but is invalid in aspergillus terreus or ringworm fungus infection. intravenous or intrathecal injection of amphotericin b should be administrated because of non-digestive absorbance. the recommended initial dose of intravenous administration is - mg/days, and gradually increases to . - mg/kg.days. the infusion track needs to be dark and process needs not less than h. the initial dose of intrathecal injection is . mg/days and gradually increases to . - mg/days. amphotericin b has an extra toxicity to liver and kidney, and also causing hypokalemia. intensive monitoring of liver and kidney function and serum potassium levels is necessary during treatment, and be sure largely avoiding combination with other agents with liver and kidney toxicity. itraconazole is one of triazole antifungal agents, mainly for aspergillus, candida, cryptococcus and histoplasma infection, but it is invalid in fungi fusarium or zygomycetes infection. the intravenous dose for the initial days is mg/days, administrated by twice, and then followed by mg/days for weeks. the oral doses of mg bid could be subsequent. the total curative course could be determined by the improvement of symptoms and largely absorption of radiographic lesions of infection. the monitoring of liver function is necessary and recommended, especially in long duration treatment. furthermore, combination treatment with other hepatotoxic drugs should be avoided. fluorocytosine is one of bacteriostatics, mainly for cryptococcosis and candida infection, frequently based on the combination with amphotericin b. the daily dose for adults is . g with intravenous dripping speed of - ml/min. a half dose should be conducted in renal insufficiency. the inhibited administration includes severe liver or kidney dysfunction and allergy to fluorocytosine, and also cautious treatment for pregnant or breastfeeding women. no combination treatment of fluorocytosine with cytarabine or bone marrow suppression agents is recommended. fluconazole is a triazole antifungal, mainly for candida albicans or cryptococcus infection, but not as good in candida glabrata infection, totally invalid in aspergillus or candida krusei infection. the recommended daily dose for adult is - mg, and the initial dose should be doubled. voriconazole belongs to triazole antifungals, mainly for candida, cryptococcus, aspergillus, fusarium and histoplasma capsulatum infection, especially used for invasive aspergillosis and invasive fluconazole-resistant candida infection, but is invalid for mucor or rhizopus infection. the recommended intravenous initial dose in adult is mg/kg, q h, with infusion rate of mg/kg within - h. the maintenance dose from the second day is mg/kg, q h. to patients without tolerance to normal dose, it could be reduced to mg/kg, q h. the reduction of daily dose could not be necessary in mild to moderate liver dysfunction. but intravenous administration should be avoided in patients with severe renal dysfunction. the side effects of voriconazole include transient visual disturbances, mental disorders, thrombocytopenia and so on. caspofungin belongs to echinocandin antifungals with antibiotic spectrum of pathogenic aspergillus, candida and pneumocystis, without in cryptococcus neoformans, fusarium and mucor. it is mainly used for invasive aspergillosis. the initial dose for adults is mgqd, and with subsequent mg qd. the infusion time is no less than h. no fixation curative course is suggested. caspofungin should be avoided for patients with severe liver function, because of highly hepatic distribution and metabolic pathways during treatment. liver is the largest solid organ in the body, and it plays a central role in the clotting process, except for general function such as metabolism, detoxification and choleresis [ ] . a majority of the coagulation factors are synthesized almost exclusively in the liver. in the pathogenesis of severe hepatitis, massive hepatic necrosis leading to reduced production and dysfunction of coagulation factor. in addition, the increased levels of anticoagulation and platelet abnormalities also contribute to occurrence of coagulopathy, which were further exacerbated by severe complications such as spontaneous bacterial peritonitis (sbp) and hepatorenal syndrome (hrs). coagulation abnormalities, even disseminated intravascular coagulation (dic) often occur in those patients with severe hepatitis. therefore, the changes in coagulation factors were usually used to evaluate prognosis of severe hepatitis [ ] . the normal procedure of coagulation includes two independent coagulation process, namely intrinsic and extrinsic pathway, which initiated by coagulation factor xii, and factor viia/tissue factor, respectively. the two pathways reach a confluence at the points of factor xa and va. wherein factor xa activates prothrombin to thrombin in the presence of ca + and factor va bound to membrane surfaces, and then thrombin converts fibrinogen to fibrin. thus, blood becomes clotted and the "y" shaped pathway was established [ ] . simultaneously, there are some anticoagulation systems existing in our body, which prevents the formation of thrombus, then keeps normal circulating of blood flow. it also participates in maintaining of normal permeability of the blood vessel [ ] . among anticoagulation systems, the most important one is the fibrinolytic system, whose basic process can divided into two stages, i.e. plasminogen activation and fibrin degradation [ ] . plasminogen activators include tissue-type plasminogen activator (tpa) and urokinase-type plasminogen activator (upa), and the principal function of these two plasminogen activators is to convert plasminogen to plasmin, then plasmin cleaves fibrin into soluble small peptides, namely fibrin degradation products [ ] . moreover, the process of fibrinolysis was affected by fibrinolysis inhibitors, such as plasminogen activator inhibitor (pai) and alpha antiplasmin (α -ap). those fibrinolysis inhibitors can either inhibit plasminogen activation, or reduce the function of plasmin [ ] . thus, there is a dynamic balance between coagulation and anti-coagulation systems under physiological conditions ( fig. . ) [ ] . many factors involved in coagulation process, i.e. clotting factors, thrombin inhibitor, fibrinolytic system and so on, are synthesized almost exclusively in the liver. thus, liver plays a pivotal role in remaining the balance between hemorrhage and coagulation under physiological conditions (table. . ) [ ] . it is the important basis for pathogenesis of coagulopathy that massive hepatocyte necrosis occurs in severe hepatitis patients, resulting in reduced production and dysfunction of those blood clotting factors [ ] . coagulation abnormalities, such as decreased production and dysfunction of coagulation factors, are commonly found in severe hepatitis, there are at least types of factors participated in the coagulation process, including classical coagulation factors, namely factor i/fibrinogen, factor ii/prothrombin, factor iii/tissue factor, factor iv/calcium ions, as well as factor v, vii, viii, ix, x, xi, xii, and factor xiii. some bradykinin factors, such as rk (prekallikrein) and hmwk (high molecular weight kininogen) are also associated with coagulation. among above factors, the others belong to proteins excepting factor iv. plasma coagulation factors are synthesized exclusively in the liver, excepting factor iii/tissue factor, factor iv, factor vi/activated factor v, factor viii and factor viiia [ ] . massive hepatic necrosis leads to the decreased production of clotting factor in patients with severe chronic hepatitis. moreover, the coagulation factors are sensitive indicators for clinical evaluation of severe hepatitis. it is common that the serum levels of factor v, vii, ix, x, xi and prothrombin decreased in the patients with severe hepatitis. on the contrary, clotting factor viii is synthesized, and then excreted increasingly by the mononuclear phagocytic cells with stimulation of various inflammatory cytokines in the patients with severe hepatitis [ ] . anorexia and antibiotics overuse lead to obstacles in assimilation, absorption, and application of vitamin k in these patients. vitamin k dependent coagulation factors includes factor ii, vii, ix, and x. the function of γ-hydroxylation was weaken by the deficiency of vitamin k and damage of hydroxylase, then the abnormal clotting factors without γ-hydroxy glutamate were synthesized. finally, these abnormal clotting factors lead to dysfunction of the vitamin k dependent coagulation factors [ ] . coagulation factor vii eliminated significantly in the initial stage of liver injury was usually used as an early diagnosis index [ ] . in addition, coagulation factors ii and x, then factor ix decreased markedly, along with exacerbation of liver injury. the deficiency of vitamin k induced by obstructive jaundice, malabsorption syndrome, can be rescued after intravenous injection of vitamin k, which is different from coagulopathy caused by liver injury. the plasma level of fibrinogen is within normal range in patients with compensatory cirrhosis. however, patients with severe hepatitis or liver failure have a significant reduction in the level of fibrinogen, and then develop dysfibrinogenemia [ ] . fibrinogen, as the main hydrolysis substrate of thrombin, is the crucial factor in the coagulation process. thus, decreased levels or abnormal structure of fibrinogen leads to coagulation abnormalities in patients with severe hepatitis [ ] . there are two clotting factors associated with thrombin except for fibrinogen, i.e. factor v and factor xiii. factor xiii induce a crosslink of sfm (soluble fibrin monomer), resulting in the formation of insoluble fibrin [ ] . it reports that the levels of circulating coagulation factor xiii were decreased in % of patients with liver disease, resulting in fibrin decline or abnormality. these patients with liver disease will receive a bad prognosis if the plasma level of fibrin was at level below % of normal concentration [ ] . it is uncommon that the plasma level of plasma factor v decreased markedly in patients with severe hepatitis, except for those complicated with dic or hyperfibrinolysis. the low level of factor v cannot induce the formation of the enzyme-substrate complex, which delay the activation of prothrombin [ ] . contact factors include classical coagulation factor xii and xi, as well as some bradykinin factors, i.e. pk and hmwk. these factors contact with vascular intima damage or abnormal surface in blood vessel walls, then active the intrinsic coagulation pathway [ ] . moreover, the contact factors can connect with bradykinin, fibrinolysis and complement system. in addition, coagulation factor xiii also activates fibrinolysis system as the initiating factor. factor xii deficiency does not cause excess bleeding, but induce thrombus due to its decreasing activation of fibrinolytic system [ ] . there is a dynamic balance between pro-and anti-coagulation systems in physiological conditions, which keep normal blood circulation in the body. the anticoagulation system includes physiological anti-coagulation factors (e.g. antiprothrombin-iii, protein c, and so on) and fibrinolysis factors (e.g. plasminogen, α- plasmin inhibitor, and so on) [ ] . at-iii, with a half-life period of . days, synthesized mainly in hepatocytes and partly in endotheliocyte, is responsible for about % of anticoagulation in plasma. it is the main reason for low plasma level of at-iii that decreased production and increased consumption of at-iii caused by hepatocytes necrosis in patients with severe hepatitis. the activity of at-iii obviously decreased in severe hepatitis [ ] . therefore, heparin, thrombin, activated coagulation factors (e.g. factor x, ix, xi, xii) cannot be inhibited, due to rare at-iii combining with them [ ] . there is a negative correlation between at-iii: a (the activity of at-iii) and pt (prothrombin time), which indicate that the level of at-iii: a decreased obviously along with exacerbation of liver injury [ ] . the plasma levels of pro-and anti-coagulant factors are low in patients with liver injury. when the necrotic tissue and cytolysis are released in the blood, the balance of pro-and anti-coagulant is destroyed. finally the depletion of at-iii by massive activated coagulation factors will lead to dic [ ] . massive hepatocytes necrosis, vitamin k deficiency, and protein c without γ-hydroxyglutamic acid result in blocking activation of protein c. thus, va, viiia and pai cannot be degraded, and the plasma level of them will rise up, due to reduction of activated protein c (apc) in the patients with severe hepatitis [ ] . plasminogen synthesis will decreased by about % in the liver when sever hepatitis occurs. as the activators of plasminogen, tpa and upa are synthesized by vascular endothelial cells, and their production will increase after vascular endothelial cells initiated with virus, immunocomplex or endotoxin in the patients with severe hepatitis b [ ] . with exacerbation of liver injury, pai synthesis decreased significantly in the liver. the main physiological activity of pai is to inhibit tpa induced plasminogen activation. therefore, the activity of tpa will increase with reduction of pai synthesis, resulting in promoting the conversion of plasminogen into plasmin [ ] . plasmin, as a kind of powerful proteolytic enzyme, can hydrolyse fibrinogen into fdps, degrade coagulation factors, and inhibit platelet aggregation, resulting in the aggravation of bleeding [ ] . hyperfibrinolysis can be caused by congenital or acquired reason, and it commonly leads to a rapid depletion of coagulation and anticoagulation factors, especially in those patients with dic [ ] . synthesis and secretion of tpa and upa markedly increase, while pai, namely tpa or upa inhibitor, has been a decrease in the plasma of severe hepatitis patients, resulting in hyperfibrinolysis [ ] . at the same time, mononuclear phagocyte system cannot degrade plasminogen activator, also leading to hyperfibrinolysis. it is not necessarily accompanied by hemorrhagic tendency, although there are many risk factors that can cause hyperfibrinolysis, even hyperfibrinolysis occurred in severe hepatitis [ ] . in addition, fdps inhibit fibrin monomer polymerize function, and also block platelet aggregation, then further worse the deficiency of hemostasis and coagulation, finally leading to the aggravation of bleeding tendency [ ] . studies have shown that low-level endogenous small molecule heparin in patients with chronic hepatitis may be associated with many factors, such as increased mastocyte, decreased production of heparinase in the liver and reduced activity of ph (platelet factor ) [ ] . when the disease progress to cirrhosis or chronic severe hepatitis, pt was significantly prolonged, while endogenous heparin wasn't increased markedly, indicating that low-level endogenous heparin has little effect on pt elongation and hemorrhagic tendency [ ] . however, if the patient is undergoing the following condition together, the endogenous small molecule heparin will increase significantly. ( ) esophageal variceal bleeding with serious infections (such as abdominal infection, biliary tract infection and pulmonary infection) or portal hypertension. ( ) combining with the significant increased white blood cell count. ( ) percentage of neutrophils > %. the level of endogenous small molecule heparin in plasmin will decrease after those infections were cured. taken together, these data indicate that increased level of endogenous small molecule heparin in blood circulation was closely related to severe hemorrhagic tendency when combining with infection [ , ] . platelet significantly promotes blood coagulation through connecting with many coagulation factors (such as fibrinogen, factor v, factor xi, factor xiii and so on). α-granule includes fibrinogen, factor xiii, and some platelet factor such as platelet factor (pf ) and platelet factor (pf ), which promote coagulation activation process of factor xii and factor xi can be accelerated by activated platelets [ ] . it is estimated that pf provided by platelets could accelerate activating of thrombin by twenty thousand times. xa and factor v could not be inhibited by at-iii and heparin if they were linked with pf . when platelets aggregated and formed platelet plugs, the process of coagulation was initiated at this site, and then platelets reveal a large number of phospholipid surfaces, which were helpful for activating of factor x and fibrinogen [ ] . various platelet factors were released from α-granule after platelet aggregating, and then hemostyptic fibre was produced increasingly. those hemostyptic fibre finally produced blood clot formation after capturing the other hemocytes. thus, platelet plugs would progress independently of platelet disintegrating gradually [ ] . two aspects of platelet abnormalities consist of depletion and dysfunction. in patients with chronic hepatitis and cirrhosis, the occurrence of decreased platelet level is usual through hypersplenism accompanied with other hemocyte decreasing [ ] . the ratio of which reaches - %. its incidence rate in severe hepatitis and explosive hepatic failure also reaches approximately by %. when severe hepatitis occur, myelosuppression, decreased megacaryocyte replication leading short-life platelet, lacking of hemopoietic material such as vitamin b, folate and so on, all of these can initiate thrombocytopenia [ ] . other reasons leading to thrombocytopenia contain low expression and metabolic disturbance of thrombopoietin (tpo), as well as platelet antibody production. researches show that the serum level of tpo related positively to platelet expression [ ] . we next studied platelet associated immunoglobulin (paig) and its sorts such as paigg, paiga, paigm etc., with chronic hepatitis. in line with previous reports, we found that serum levels of paig and paigg negatively correlated with blood platelet count, corroborating the crucial role of the paig-mediated autoimmunization in controlling thrombocytopenia in viral hepatitis [ ] . various factors impact platelet function forwardly or passively. increased expression of oxide and prostacyclin, two kinds of platelet inhibitor derived by endothelium, may inhibit platelet activation in vivo [ ] . on the other hand, increased serum level of von willebrand factor (vwf) can promote platelet adhering and aggregation in patients with cirrhosis. when severe hepatitis occur, . % and . % of patients have decreased levels of platelet adhesion rate and platelet aggregation rate (par), respectively. in addition, reduced effectiveness of pf and abnormal clot contraction occur in patients with severe hepatitis by an incidence of . % and . %, severally [ ] . patients with terminal liver disease significantly exert hemorrhagic tendency, especially in the digestive tract [ ] . the latest report indicated that basic laboratory examinations for coagulation function testing in common use at present, such as pt, aptt, international normalized ratio (inr) etc., have little correlation with occurrence of gastrointestinal bleeding in these patients, thereby revealing the importance to search and pay close attention to those complicating disease upregulating bleeding risk, such as bacterial infection, renal failure, hemodynamic change after portal hypertension, dysfunction of endotheliocyte as well as macrophagocyte and so on [ ] . it is common to see renal failure occurrence in advanced hepatopathy. when it happened, acquired platelet dysfunction, abnormal activation of platelet and vascular wall, anemia and so on, all of them significantly promote hemorrhage [ ] . as another severe complication, bacterial infection is also very important and common [ ] . when tumor necrosis factor (tnf) were injected into healthy individuals, we found that endotoxin have an important role to exert its function directly in clotting cascade reaction. early researches indicated that the body can express endogenous heparin-like substance through stimulation by endotoxin in patients with cirrhosis [ ] . furthermore, some studies revealed the relevance of endotoxin with prothrombin fragment, indicating that infection promoted occurrence of dic-like status in laboratory examination in cirrhosis [ ] . coagulation system became weaker in patients with chronic hepatitis. it can hardly mediate factors due to the relative deficiency of pro-and anticoagulation factors. when the balance was destroyed, it may tend to hemorrhage or thrombosis depending on related risk factors which were in the ascendant. to assess abnormality of blood coagulation in patients with liver disease, we should be in consideration of hypercoagulability, one state may easily be overlooked. otherwise, it will be unfair. current literatures indicated that, unlike previous concept that the body of patients initiated anticoagulation state automatically when infected by the hepatitis virus. various clinical evidences revealed that hepatitis virus infecting cannot inhibit thrombus forming. furthermore, it may increase dangerous to thrombus forming especially in the portal system, for existence of individuals having hereditary mutation to promote thrombus forming [ ] . slower bloodstream, abnormal fibrinolysis initiated by blood stasis, and decreased activity of anticoagulant accelerate formation of venous thrombosis. moreover, the changes of platelet phospholipids membrane activity were also helpful to the formation of thrombosis in patients with chronic liver disease [ ] . it is reported that the incidence of periphery deep venous thrombosis and pulmonary embolism was . and . % in patients with cirrhosis, respectively [ ] . the rate of portal vein thrombosis is about % in patients with compensated cirrhosis, but it reaches - % in the candidates waiting for liver transplantation [ ] . for mutations (such as leiden mutation of factor v, g a mutation of prothrombin, c mutation of methylenetetrahydrofolate reductase) or the existence of antiphospholipid antibody syndrome, the hypercoagulable state will be represented in patients with liver disease [ ] . the hypercoagulable state represents diverse modes in the body of patients with chronic hepatitis. among them, thrombosis is more traditional and common. the mechanism of dic complicated with severe hepatitis may contain several aspects as follows. . with attacks from endotoxin, virus and immune complex etc., endotheliocyte was injured, and then it activated plasmakinin system and complement system, leading to the aggravation and participation of dic progress. damaged endotheliocyte can simultaneously activate factor vii, intrinsic coagulation pathway and platelets, and participate in micro-thrombosis with platelets adhesion and aggregation beneath endothelium [ , ] . . in patients with severe hepatitis, massive necrotic hepatocytes activated extrinsic coagulation system with the releasing of various tissue thromboplastin-like substances [ ] . . hepatocellular necrosis or dysfunction decreased expression of anti-coagulation factors such as at-iii, pc, protein s and so on, and then it enhanced activation of thrombin and plasmin [ , ] . . impaired function of the mononuclear phagocyte system. mononuclear phagocytes can express activated tissue factor with the releasing of tnf, il- and platelet activating factor (paf) on its surface after stimulation by endotoxin, inflammatory cytokines and complement activation. tnf and il- can decrease the activation of protein c through its function to increase the expression of plasminogen activator and plasminogen activator inhibitor- and to inhibit the production of thrombomodulin (tm) [ ] . activated clotting factor and other factors with promoting coagulation can lead to the occurrence and aggravation of dic, since it cannot be promptly removed [ ] . . the onset and enhancement of fibrinolysis. massive clotting factors and platelets were depleted in the extensive formation in vivo microthrombus. thrombin promoted the conversion of fibrinogen into fibrous protein [ ] . simultaneously, it activated fragments which dropped in the formation of activated factor xiii, factor xa and factor xiia. all of them can activate plasmin, and it enhanced fibrinolysis with tpa, one factor released by damaged blood vessel endothelium [ ] . fibrinogen and fibrous protein were degraded after plasminogen activation to generate the corresponding fdps, one factor inhibit blood coagulation and also block platelet aggregation [ ] . taken together, the above process exacerbates bleeding initiated by coagulation factors depletion and platelet deficiency. coagulopathy, characterized by prolongation of blood clotting time, tendency of hemorrhage, or even dic, were commonly found in severe hepatitis patients. it may cause uncontrolled external or internal hemorrhage. as common clinical symptoms, the external hemorrhages include gingivale or nasal mucosal bleeding, skin petechiae, the punctures or injection-site ecchymosis, and so on. the internal hemorrhages include esophagogastric varices, intracranial, subcutaneous, and muscle interval bleeding. except for esophagogastric varices hemorrhage, the other internal hemorrhages rarely occur in those patients. in addition, massive hemorrhage from esophagogastric varices is a serious medical emergency, can potentially cause death and cardiac arrest if proper medical treatment is not received quickly [ ] . when dic occur, a wide range of thrombogenesis in microvasculature can cause circulatory collapse, characterized by low blood pressure and shock. microcirculatory dysfunction occur after microthrombosis producing, resulting in hypofunction of multiple organs (e.g. kidney, liver, lung and pancreas), which perform from dysfunction to failure, with illness progressing [ ] . crushed by the fibrous protein in the vessels, red blood cell destruction can lead to intravascular hemolysis. at the early stage of disease, it shows hypercoagulability. the blood in the needle is easy to coagulate, when getting blood sampling from the vein. after that, it comes with the stage of consumed hypocoagulation. the consuming of a great number of blood coagulation factors leads to significant tendency of hemorrhage [ ] . it is difficult to stop bleeding in many parts of the body, including visceral organs, operative sites, injection sites, puncture sites, and mini-invasiva sites [ ] . when the third stage, namely secondary fibrinolytic stage comes, a great number of the blood coagulation factors have already been used up, resulting in severe bleeding under the condition of low coagulation. shock, acidosis and mods make the patient's condition continue deteriorating, and are also the main reasons for death [ ] . the latest study indicates that thrombosis is also a noticeable state in cirrhosis and end-stage liver disease. portal thrombosis and peripheral vein thrombosis (e.g. deep vein thrombosis and lung embolism) are commonly seen. the deep vein thrombosis is more danger than lung embolism. anyway, the incidence rate of thrombosis in cirrhosis and end-stage liver disease is still very low. the clinical symptoms depend on the embolizing position after the thrombosis occurs [ ] . presently blood clotting factors test is the most maturely and frequently used test in the term of blood coagulation function in the world. the indicators including prothrombin time (pt), international normalized ratio (inr) and prothrombin time activity (pta) have always been chosen in the patients with severe hepatitis. prothrombin time (pt) reflects whether there are anticoagulant substances in the extrinsic blood coagulation system and blood circulation or not. the elongation of prothrombin time (pt) presents the declined activity of several blood clotting factors including factorii (fii), factorv (fv), factor vii (fvii), factorx (fx) or the existence of anticoagulant substances. in severe hepatitis patients, the incidence rate of prothrombin time (pt) elongated can reaches to %, thus it is regarded as a sensitive and frequently used indicator in the term of liver function [ ] . another index international normalized ratio (inr), a reckoned ratio calculated from prothrombin time (pt) and international sensitivity index (isi), making prothrombin time (pt) between different laboratories and different reagents comparable, is an international general indicator. the guides about acute-on-chronic liver failure and acute liver failure take the index, inr ≥ . , as one of the most significant diagnostic criteria in the american association for the study of liver diseases (aasld) and the asian pacific association for the study of liver (apasl). international normalized ratio (inr) can also be used as a monitor on blood coagulation function [ ] . hepaplastin test (hpt), reflecting not only blood coagulation mechanism in hepatitis patients but also the function of hepatocytes to synthetize vitamin k dependent clotting factors including factorii (fii), factor vii (fvii), factorix (fix), factorx (fx) synthetically, is a test about liver reserve function; but this indicator can't reflect the change of factorv (fv). when severe hepatitis occur, the function of liver to synthetize above-mentioned clotting factors declines and the time of hepaplastin test (hpt) elongates. with illness progressing, hpt continues elongating. survivors undergoing effective therapies can have gradual recovery in the time of hepaplastin test (hpt). so this test is regarded as the specific test of liver diseases or the optimal indicator reflecting liver reserve function [ ] . the severity of liver damage is positively correlated with the decline degree of prothrombin time activity (pta): the more severe damage occurs in hepatocytes, the more significantly prothrombin time activity (pta) will decline. therefore prothrombin time activity (pta) < % and total bilirubin (tbil)> μmol/l have always been used as the main laboratory indicators to diagnose severe hepatitis domestically. pta < % is also regarded as diagnostic criterion of blood coagulation dysfunction in the guideline of acute-on-chronic liver failure in the asian pacific association for the study of liver (apasl) [ ] . in severe hepatitis, total bilirubin (tbil), total cholesterol, prothrombin time activity (pta) and complications (e.g. rectory hyponatremia, hepatic encephalopathy, hepatorenal syndromes and so on) are all independent risk factors to evaluate prognosis. the lack of any factors including factori (fi), factorii (fii), factorv (fv), factorvii (fvii), factor x (fx), can lead to the decline of prothrombin time activity (pta). moreover the half-life time of those factors are extremely short, factorii (fii) - h, factorv (fv) - h, factorv (fv) - h, factorv (fv) - h, respectively. it means that when hepatocytes suffer from severe serious damage and necrosis in severe hepatitis, prothrombin time activity (pta) will have dramatic decline just in a few days. as a result, prothrombin time activity (pta), characterized by significant advantages in evaluating patients condition and judging prognosis over other laboratory indicators, has been widely used. elongation of aptt prompts the lack of any clotting factor belonging to intrinsic coagulation system or the existence of anticoagulant substances. the incidence of the elongation of aptt reaches to - % in severe hepatitis patients [ ] . fxii:c reflects liver synthetic function. fvii, characterized by the shortest halftime - h, is the first one to be affected when facing liver synthetic dysfunction. on the contrary, fv:c, characterized by a relatively long half-time, is one of the latest factors to be affected and is correlated with the degree of liver damage. it prompts severe liver failure, bad prognosis and even easy death when plasma levels of fv:c under %. some literatures report that fv:c and pta can be used as significant prognostic factors in liver failure and significant screening indicators in liver transplantation. however, the indicators-fxii:c and fv:c-are not listed as routine examination items. they are just selected on the condition of illness demand [ , ] . the main test about anti-clotting factors is the determination of antithrombin iii activity (at-iii:a). in the state of pathosis, the decline of antithrombin iii activity (at-iii:a) is not parallel with the decline of antithrombin iii (at-iii) content, namely the depletion of antithrombin iii activity (at-iii:a) more apparent. owe to this, it has more clinical value to determine antithrombin iii activity (at-iii:a) rather than antithrombin iii (at-iii) content. moreover, anti-clotting factors tests include the determination of protein c activity (pc:a) as well [ ] . serum levels of fdps are very low in normal people. significantly elevating of fdps indicates the existence of hyperfibrinolysis and reflects the occurring of dic indirectly. there are many assay methods including immunization fi test (namely latex particle agglutination test, normal titer< : ), fdps flocculation test, radial immunodiffusion staphylococcal clumping test, indirect hemagglutination inhibition test, enzyme-linked immunosorbent assay (elisa) and so on. if the serum levels of fdps elevate, it indicates acute dic may occur [ ] . plasma protamine paracoagulation test ( p test) and ethanol gel test (egt) reflect the soluble fibrin complexes in plasma. soluble fibrin complexes, combination of fdps and fibrin monomer, can't be solidified by thrombase. but protamine is able to make the complexes isolate and then fibrin monomers separate out again. the paracoagulation test means self-polymerization between fibrin monomers and fdps, then forming macroscopic flocks. ethanol gel test (egt) has the same principle with plasma protamine paracoagulation test ( p test), whereas the former has a lower positive rate. the two methods may have false negative results and false positive results. in contrast, egt has a relatively lower sensitivity, while p test has a relatively lower specificity. for example, relative small molecular mass of the shreds of fdps may lead to negative result using p test. so it is more valuable to compare the two indicators simultaneously [ , ] . euglobulin, a protein (including fibrinogen, plasminogen and other activins except for fibrinolysis inhibitor) separating out from plasma in acid circumstances, can be used to determine whether levels of plasminogen activators increase or not [ ] . when hyperfibrinolysis occurs, plasma levels of plasminogen decline, plasma levels of plasmin increase, and euglobulin suffer from accelerated dissolution by a great number of plasmin. the normal value of euglobulin lysis time (elt) is above h. that is to say dissolution within h means the occurrence of hyperfibrinolysis. domestic population data report the positive rate of elt test reaches - . %, when dic occur [ ] . furthermore there are other tests about fibrinolysis including tissue-type plasminogen activator test (t-pa), plasminogen activator inhibitor test (pai), plasminogen antigen test (plg:ag), plasmin activity test (pl:a), α -plasmin inhibitor test (α -pi) and so on [ , ] . blood platelet count reflects the absolute number of platelet in peripheral blood circulation. according to the reports at home and abroad, platelet count have a significant decline in patients with chronic severe hepatitis. moreover, studies on domestic population find that platelet count ranges from × /l to × /l in peripheral blood of severe hepatitis patients. some studies have already compared the platelet count among early stage, typical stage and late stage in severe hepatitis, and they have turned out to be × /l, × /l and × /l respectively. as a result, it indicates that platelet count is positively correlated with the severity of hepatitis [ ] . except for platelet count, other routine indicators including mean platelet volume (mpv), plateletcrit (pct) and platelet distribution width (pdw) also have significant reference value. when severe hepatitis occurs, the above-mentioned three indicators will be dramatically lower, and they have the tendency to continue declining with illness progressing. what's more, platelet quality tests include platelet aggregation rate, platelet factor validity tests and blood clot retraction test (table . ) [ ] . diagnosis of blood hypercoagulability in the early stage of dic relies on several molecular marks including plasma thrombinogen segment + (f + ), thrombinantithrombin complex (tat) and d-dimer, due to no significant changes in general laboratory tests. this stage is characterized by the elevated levels of those three molecular marks, and levels will increase more significantly with the occurrence of typical dic symptoms. dynamic monitoring of above-mentioned indicators is helpful for early diagnosis of dic [ ] . the stages are mainly characterized by the decline of blood clotting factors (including factorv (fv), factorvii (fvii), factorxii (fxii), factorix (fix), fac-torx (fx)), platelet count and plasminogen, and increasing of fibrinolytic the elevating levels of fibrin(−ogen) degradation products (fdps) and d-dimer; the shortening of euglobulin lysis time (elt) and the positive reaction of p test [ , ] . with the occurrence of dic, there will be a wide range of blood coagulation and highly-activated fibrinolysis in the patient's body [ ] . what's more, abnormal increased soluble fibrin monomer and fdp fragments will exist in plasma [ ] . the level of soluble fibrin monomer complex (sfmc), a complex combined fibrin monomer with fdp, is determined by p test. the p test shows positive with the occurrence of secondary fibrinolysis, whereas it shows negative with the occurrence of primary fibrinolysis. this means p test is negative when there is no blood coagulation. domestic population data report that the positive rate of p tests reach - %. however, the test can't be used as the ideal diagnostic indicator for dic, owing to many affected factors. false positive reactions are mainly found in the following conditions: gastrointestinal bleeding, massive hemoptysis, malignant carcinoma or blood sampling reserved improperly, whereas false negative reactions are usually found at the late period of the stage of secondary fibrinolysis [ , ] . as mentioned above, plasma thrombinogen segment + test (f + ), thrombinantithrombin complex test (tat) directly reflects production of intracorporeal thrombase, which increased in the early stage of dic. plasmin degrades the crosslinked fibrin to release fibrin degradation products and expose the d-dimer antigen, which reflects production of intracorporeal plasmin. d-dimer will have an apparent increase with the occurrence of secondary fibrinolysis, but d-dimer test shows negative when primary hyperfibrinolysis occurs. monitoring the above molecular markers dynamically is helpful to estimate therapeutic efficacy and guide treatment [ ] . as mentioned above, clinical manifestations of blood coagulopathy in patients with severe hepatitis are lack of specificity. the most common manifestation is bleeding, not only little hemorrhage from superficial sites, but also massive hemorrhage from internal sites, such as esophagogastric varices [ ] . the diagnosis of severe hepatitis complicated with blood coagulopathy is mainly based on the results of laboratory tests and clinical manifestations [ ] . according to current guidelines, basic diagnose conditions of dic contain the following points [ ] . . severe or multiple bleeding tendency. . microcirculation collapse or shock which is difficult to explain using protopathy. . a wide range of embolism in skin and mucosa, focal ischemic necrosis and ulcer, or unexplained dysfunction of kidney, lung, brain. . anticoagulant therapy is effective. if severe hepatitis b patients have one of the above-mentioned points except for ( ) and exhibit blood coagulating easily or prothrombin time (pt) shortening over s simultaneously, it can be considered as the early stage of dic in which the tendency of bleeding is not obvious. in addition, if severe hepatitis b patients have two of the above-mentioned four points, dic can be considered as the preliminary clinical diagnosis. furthermore, it can be definitely diagnosed when combined with the aforementioned items of laboratory tests ( table . ). firstly, we should use anti-viral therapy as a mainly method to treat primary disease of severe hepatitis b [ ] . then, we must eliminate the incentive, maintain the balance of water and electrolyte, and correct hypoxia and acidosis. it is very important to focus on massive upper gastrointestinal hemorrhage and disseminated intravascular coagulation when treating coagulation disorders [ ] . gastrointestinal bleeding, including esophageal varices bleeding and non-bleeding esophageal varices, were correlated with coagulation dysfunction and portal hypertension. prevention is still focused on improving the coagulation function, including adequate vitamin k supplements, coagulation factors, fibrinogen, fresh plasma or platelets supplements. it is particularly critical to control diet in patients with severe hepatitis. in which, light and easily digestible diet was recommended, but rough, hard and too greasy food was prohibited. for these patients, appropriate antacids can be used to protect the gastric mucosa [ ] . . general treatment: bed rest is necessary, meanwhile vital signs were closely monitored. the patients can eat liquid diet when bleeding mildly or having no active bleeding. however, abrosia is required when the patients have a heavy bleeding. . fluid resuscitation: firstly, intravenous access should be established rapidly in patients with gastrointestinal bleeding, then, the patients were given intravenous infusion of normal saline, lactated ringer's solution, plasma, whole blood or plasma substitute [ ] . . vaso-active agents: vaso-active agents such as dopamine and alamin may be given to maintain normal blood pressure, if blood circulation is still not stable after fluid resuscitation [ ] . . hemostatic: if mucosal bleeding was caused by portal hypertension, oral administration of norepinephrine and ice normal saline can promote mucosal vascular contraction and hemostasis. in addition, oral administration of yunnan baiyao may be effective [ ] . . acid suppression therapy: the proton pump inhibitors, such as omeprazole, pantoprazole and esomeprazole, are commonly used to inhibit gastric acid secretion [ ] . . reduction of portal pressure: patients with severe hepatitis complicated by gastrointestinal bleeding often accompanied with portal hypertension, so it may be considered to give them drugs to decrease portal pressure. especially in patients with portal hypertension gastropathy, reduction of portal pressure is more important than acid suppression therapy [ ] . . compression hemostasis via using three-chamber double-balloon catheter: after the above treatment, if there is still active bleeding in patients with bleeding esophageal varices, it can be considered to use three-chamber double-balloon catheter [ ] . . endoscopic hemostasis: if the above treatments have no effect in upper gastrointestinal bleeding, endoscopic hemostasis, including endoscopic hemostatic agents spray, endoscopic ligation or endoscopic injection sclerotherapy may be used [ ] . . others: surgery or interventional therapy may be considered, if internal medicine therapy is ineffective [ ] . firstly, we should treat original disease, then improve coagulative function in those patients. in addition, prevention and control of infection, correction of electrolyte disturbance, avoiding the hemorrhage and reduction of allergies and transfusion reactions should be considered [ ] . the key is early diagnosis and early treatment. . anticoagulant drugs: low molecular weight heparin is the most commonly used drug in earlier stage of dic. it is recommended to periodic test blood routine and coagulation, and dynamically observe coagulation status during medicine therapy. others such as dextran, anti-platelet aggregation ticlopidine, salvia injection, urokinase may be effective, but it should be support by more evidence-based medicine [ ] . . plasma and blood products: during the process of dic formation, the patients should be transfused with fresh plasma, each - ml/kg. when they developed the stage of secondary fibrinolysis, prothrombin complex containing coagulation factors, cryoprecipitate and platelets can be supplemented due to a large consumption of coagulation factors [ ] . . others: such as hemodialysis, anti-fibrinolytic -aminocaproic acid and tranexamic acid should be supported by more evidence-based medicine. hepatorenal syndrome (hrs) is a common and serious complication occurring in patients with decompensated cirrhosis and liver failure, who have overt circulatory dysfunction. the -year incidence of hrs in patients with ascites is about % [ ] . hrs may predict a poor prognosis in spite of it being functional reversible [ , ] . the clinical characteristics of hrs were first described in [ ] . in , hrs was defined by a group of international investigators as a progressive renal dysfunction that occurred in severe liver diseases, with features of prerenal failure (low urine sodium concentration and hyperosmolar urine) but without any improvement following volume expansion [ ] . the international ascites club (iac) developed hrs definition in , as a syndrome that occurs in patients with cirrhosis, portal hypertension and advanced liver failure, characterized by impaired renal function with marked abnormalities in the arterial circulation and activity of endogenous vasoactive systems [ ] . hrs is a potentially reversible syndrome that occurs mainly in patients with liver cirrhosis, ascites and all kinds of liver failure [ ] . it's clinical features include impaired renal function, marked changes in cardiovascular function and over activity of the renin-angiotensin systems and the sympathetic nervous. progressive hrs with severe renal vasoconstriction is able to cause a decrease of gfr [ ] . clinically, hrs can be divided into two types ( and ). type- , so-called acute hrs is a rapid progressive form of renal failure defined by doubling of the initial serum creatinine concentrations to a level higher than mmol/l ( . mg/dl) or a % reduction of the first h creatinine clearance to < ml/min within weeks [ ] . it appears mainly in patients with acute liver failure, but often develops after a precipitating event, such as bacterial infections (especially spontaneous bacterial peritonitis, sbp). the prognosis of type is poor with the median survival about month [ ] [ ] [ ] . type- , so-called chronic hrs is a steady or slowly progressive form of renal failure defined by serum creatinine from to mmol/l or from . to . mg/ dl [ ] . type- hrs is mostly related to refractory ascites. survival of patients with type- hrs is generally around - months, which is better than that of patients with type- hrs but shorter than that of non-azotaemic cirrhotic patients with ascites. patients with type- hrs tend to develop type- hrs while infections or other trigger events occurred [ , [ ] [ ] [ ] ]. portal hypertension is the essential factor of haemodynamic changes, which resulted from the development of cirrhosis associated with distortion, compression and even obliteration of the hepatic sinus and vessels [ ] . in patients with portal hypertension, bacterial translocation is increased and intrahepatic hypercontractile stellate cells activated [ , ] . this overall increased resistance to portal hypertensional causes increased local production of various vasodilators such as nitric oxide, leading to splanchnic vasodilation [ , ] . there are several other factors contributing to the splanchnic vasodilation, including hyporesponsiveness of the splanchnic vessels and mesenteric vascular hyperplasia [ ] . in addition, portal hypertension per se can cause renal vasoconstriction by activating sympathetic nervous. for example, when tips is used to reduce portal hypertension and improve renal blood flow, a corresponding reduction in sympathetic nervous activity has been observed [ , ] . as a result of splanchnic vasodilation, blood is accumulated in the splanchnic vascular bed just like a splanchnic steal syndrome [ ] . the combined effect leads to reduction in the effective arterial blood volume (relative hypovolemia) causing a relative inadequacy in the systemic circulation, which triggers a hyperdynamic circulation in these patients [ , ] . vasodilatation induces activation of neurohumoral systems including the reninangiotensin-aldosterone system (rass); sympathetic nervous system (sns); and non-osmotic release of antidiuretic hormone (adh) [ ] . relative hypovolemia initially causes sodium and water retention, increases intravascular volume, and simultaneously increases cardiac output. as cirrhosis progresses, vasodilatation aggravates, which activated vasoconstrictive systems, causing renal vasoconstriction and decreased renal blood flow [ , ] . local release of potent vasodilators such as nitric oxide (no) leads to splanchnic visceral vasodilation, as well as enables the splanchnic circulation against a variety of vasopressor agents, including norepinephrine, vasopressin, angiotensin ii and endothelin [ ] . the resistance of the splanchnic circulation to these vasopressor agents makes the control of arterial pressure during cirrhosis dependent on the extra-splanchnic effects produced by the endogenous vasoconstrictor systems. the role of vasoconstrictors in maintaining haemodynamic stability becomes pivotal as arterial vasodilatation increases during cirrhosis, which makes clear why cirrhotic patients with hrs are prone to develop renal, hepatic and cerebral vasoconstriction [ ] . the reduction of effective arterial blood volume leads to the compensatory activation of various vasoconstrictor systems. normally, the kidneys increase the production of renal vasodilators including prostaglandins and kallikrein to maintain blood flow. however, renal vasodilator production is generally reduced in patients with cirrhosis, thus contributing to renal vasoconstriction. this type of renal hypoperfusion further increases the production of various intrarenal vasoconstrictors such as angiotensin ii and endothelin, causing further decline of renal haemodynamics and renal function, occasionally accompanied by glomerular ischaemia and mesangial constriction [ ] . when blood pressure fluctuates, renal auto-regulation regulatory mechanisms initiate to make sure that the kidneys receive a relatively constant blood supply. when the critical threshold is below mmhg, renal blood flow is proportional to renal perfusion pressure which, in turn, is dependent on mean arterial pressure. in cirrhosis, with the development of liver disease in patients of cirrhosis, the renal auto-regulation curve gradually shifts to the right -which means as liver disease advances, the renal blood flow gradually decreases for each given renal perfusion pressure [ ] . furthermore, lumbar sympathetic blockade increases renal blood flow in patients with hrs, suggesting that the renal sympathetic activity is involved in this outgoing hepatorenal arm. insufficient cardiac output is considered one of the leading causes for renal hypoperfusion in patients with hrs in recent years. despite control of infection, the cardiac output of cirrhotic patients with sbp who developed progressive renal failure was lower than that in similar sbp patients without renal failure. similarly, when patients with non-azotaemic cirrhotic patients who developed hrs are compared with similar patients who did not, it is observed that low cardiac output and high plasma renin activity (pra) were independent predictors of hrs. in addition, in patients developing hrs, the evolvement of circulatory dysfunction leading to arterial hypotension and renal failure occurs in the setting of a continued decline in cardiac output and increase in pra. therefore, effective hypovolaemia occurs when cardiac output decreases, resulting in renal hypoperfusion and hrs [ ] . to summarize, the principal mechanisms leading to renal vasoconstriction include systemic circulation changes, accompanying portal hypertension which are characterized by decreased peripheral vascular resistance with subsequent vasodilatation, hyperkinetic circulation and the activation of compensatory mechanisms, i.e., the sns, raas, and adh. with the progression of cirrhosis, the combined effective of all the above factors will result in the gradual deterioration in renal function. any event that leads to a sudden deterioration in hemodynamics can cause a rapid renal dysfunction, precipitating type hrs [ ] . the diagnostic criteria for hrs have been first defined by iac in [ ] . the main findings include reduced glomerular filtration (creatinine clearance) less than ml/min or serum creatinine increased more than μmol/l after excluding the other causes of renal dysfunction. however, estimation of renal function by using creatinine clearance is not reliable, because these patients have lower levels of serum creatinine and higher renal tubular creatinine secretion compared with filtered creatinine. furthermore, it is often incomplete for the collection of a h urine. iac developed the new definition and diagnostic criteria for hrs in , which ( ) excludes creatinine clearance because of its inaccuracy of renal function estimation and the complicity to perform; ( ) includes renal failure at the time of combined bacterial infection (but absence of septic shock), indicating that hrs can be diagnosed before antibiotic treatment; ( ) determines by using albumin for plasma volume expansion better than saline. ( ) excludes minor diagnostic criteria (urinary indices) because of its poor sensitivity and specificity for the diagnosis [ , , ] . the diagnostic criteria of hrs for patients with liver cirrhosis are as follows: . cirrhosis with ascites; . serum creatinine > mmol/l ( . mg/dl); . no improvement in serum creatinine (decrease to a level of ≤ mmol/l or . mg/dl) after at least days of diuretic withdrawal and volume expansion with albumin. the recommended dose of albumin is g/kg body weight/day up to a maximum of g/day; . absence of shock; . no current or recent treatment with nephrotoxic drugs; . absence of parenchymal kidney disease as indicated by proteinuria > mg/ day, microhematuria (> red blood cells/high power field) and/or abnormal renal ultrasonography [ , ] . there are some other causes of renal failure in patients with cirrhosis that were not included, such as membranoproliferative glomerulonephritis and/or iga nephropathy in patients with chronic liver diseases. these chronic forms of kidney disease can cause acute rises in serum creatinine. determining whether it is a potential kidney disease or hrs that causes a sudden increase in serum creatinine in patients with cirrhosis and chronic kidney disease could be difficult [ ] . naturally, liver transplantation is the only rational solution in cases of advanced liver disease while it is also the treatment of choice for both type- and type- hrs. as calcineurin inhibitors (ciclosporin and tacrolimus) may induced gfr impairment, it is recommended to delay their administration until a partial recovery of renal function is recorded, usually - h after transplantation [ ] . clinically, the haemodynamic associated with hrs as well as neurohormonal abnormalities fade away within one month of transplantation, and the patients recover their ability to excrete sodium and free water. compared with patients without hrs, patients with hrs tend to have more complications, take more days in intensive care units and have higher in-hospital mortality rates after liver transplantation. nevertheless, their -year survival rate is acceptable ( % vs. - % in liver transplant patients without hrs [ ] . the primary limitation of liver transplantation is that most patients with type- hrs die before transplantation due to the shortage of donor liver and their extremely short survival time. reference to the model of end-stage liver disease (meld, including scr, tbil, inr) for organ prioritisation has partially addressed this problem, since patients with hrs have a high priority on the waiting list. in addition, the use of vasoconstrictors and albumin in the treatment of type- hrs can improve survival rate of these patients, and increase the likelihood of their transplantation [ ] . in patients with advanced liver disease and the renal insufficiency, simultaneous liver and kidney transplant (slkt) is taking into consideration. however the reversibility of renal function in some patients when they receive slkt should be taken into account. therefore, to ensure allocation of transplants only to those truly in need, the transplant community proposed an evaluation algorithm in , whose purpose is to determine the presence of kidney disease with structural damage (preferably on biopsy) before giving slkt. in the case of chronic kidney disease, a decreased creatinine clearance at ml/minute or less is considered an indication of slkt. slkt should not be performed for the patients with simple hrs, but for the patients with hrs who become dialysis dependent and without any recovery after to weeks of dialysis [ , ] . vasoconstrictors combined with albumin are the first line of therapy for type- hrs patients. it was recognized long time ago that the effective plasma volume was reduced when patients of advanced liver diseases complicated with hrs, and this led to many attempts to improve the patients' renal function by expanding their plasma volume, including a large dose of albumin or saline perfusion. with the advent of safer compounds including terlipressin, a vasopressin analogue with longer activity, and the α -agonist midodrine combined with octreotide the analogue, vasoconstrictors is widely used in the patients with hrs. these vasoconstrictors are able to ameliorate vasodilatation while increasing effective arterial blood volume, improving renal vasoconstriction and improving renal flow. in order to further increase effective blood volume, vasoconstrictors have been used in conjunction with intravenous albumin. the clinical results from uncontrolled studies including patients with hrs (type- , ) showed that a total of % were observed complete response (mostly defined as a decrease in scr to . mg/dl). interestingly, once the treatment is stopped, hrs relapses only in a few "responders" [ , ] . there were several randomized controlled trials (rcts) published, suggesting that terlipressin was associated with an increase in gfr compared with albumin alone or with a placebo. the rate of hrs reversal in the terlipressin group was higher than that in the control group ( % vs. %). as survival rate was not improved in the two largest rcts, liver transplantation is still the preferred treatment for hrs, but terlipressin seems to serve as a "bridging" treatment. two recent small, open-label rcts suggested that the incidence of hrs reversal and the rate of side effects showed no significant difference between the two groups of norepinephrine and terlipressin [ , , ] . the initial dose of terlipressin recommended in many studies ranged from . to mg per - h [ , ] . if the creatinine level did not decrease by % on the third day, the dose could be increased to mg every h or mg/days by continuous intravenous infusion, respectively. in some studies, the daily dose of albumin was generally - g by a load of g/kg body weight. some mentioned central venous pressure to establish albumin doses and to prevent body fluid from overloading. this treatment was maintained until hrs is reversed, but did not exceed weeks [ ] . about % of patients relapsed after the treatment withdrawal. however, these patients should be given repeated treatment with terlipressin, which is often effective [ ] . several studies have evaluated the role of transjugular intrahepatic portosystemic stent-shunt (tips) in hrs. these studies show that tips help decreasing in scr in most patients, even in a minority of organic renal failure, but it is slower compared to those obtained using terlipressin combined with albumin [ ] . recrudescence of hrs is rare provided the shunt remains patent, while hepatic encephalopathy often comes. it is worth noting that the vast majority of patients included in these studies suffered from alcoholic cirrhosis, many of whom have active alcoholism, and therefore the improvement observed may be caused by the improvement in an acute-on-chronic process. in addition, since all these studies excluded patients with a child-pugh score ≥ , resulting in a lack of data, the efficacy of tips should be further explored in rcts [ ] . extracorporeal albumin dialysis molecular adsorbent recirculation system (mars) is designed for making clearance of water-soluble cytokines (i.e., il- ) and albumin-bound toxins (i.e., bile acids) which is implicated in the pathogenesis of hrs. two studies showed that mars was ineffective in improving survival rate and emic haemodynamics in type- hrs [ , ] . another clinical observation including patients with type- hrs reported a rate of complete renal response of % [ ] . extracorporeal albumin dialysis (ecad) reduces serum creatinine levels, but it is not clear whether this effect is due to a real improvement of renal function or merely a filtration process. several studies demonstrated that patients' systemic haemodynamics improved after ecad, manifested as an increase in systemic vascular resistances and arterial pressure, as well as a decrease in cardiac output, pra and levels of norepinephrine. however, there were too few studies on the effect of ecad on survival in type- hrs patients to draw any definitive conclusions [ , ] . in addition, ecad is very expensive and therefore not suitable for wide and rapid clinical application [ ] . the treatment of type- hrs should take into account the survival rate as well as controlling the ascites. both hrs- and hrs- are indications for the tips treatment. the therapeutic effect of tips is excellent for its better controlled of complications of portal hypertension compared with other treatments. tips have been reported not only to improve renal function in patients with type- hrs but also to treat refractory ascites in patients with type- hrs [ ] [ ] [ ] . the contraindications to the creation of tips are shown in the followings [ ] . • contraindications to placement of a tips: - there were only a few studies evaluated the role of tips in type- hrs and the number of cases was quite low. in most patients, tips could decrease scr, even in some with organic renal failure [ ] . hrs recurrence is rare as long as the shunt remains patent, but hepatic encephalopathy often occurs [ ] . nine patients were followed-up for month after the treatment of tips in a study, eight cases were found with decreased scr decreased and notably controlled ascites. four patients died, two of them died within month, the other two died at months and months respectively. the remaining five patients survived for a long time. although tips can be used in improving refractory ascites which often contributes to type- hrs, data on the effect of tips on survival are still insufficient. therefore, the efficacy of tips should be further explored in randomized controlled trials (rcts) [ ] . the information about combining albumin and vasoconstrictive agents treated in type- hrs is limited. only a few patients with type- hrs have been specifically treated with terlipressin and albumin. in one clinical study, patients with hrs- were assigned to receive this treatment and of them achieved improvement of renal function. however after the treatment withdrawal, hrs patients showed relapsed during the follow-up. the most common side effects during terlipressin therapy are cardiovascular and ischemic and reported as an incidence of nearly %. the high recurrence rate of hrs after terlipressin and albumin treatment discontinuation suggests that they are less effective in treating type- hrs compared to type- hrs [ ] . prevention of hrs is important because it develops at a constant frequency in cases of spontaneous peritonitis (sbp) and advanced liver disease [ , ] . it becomes possible to prevent hrs if sbp is diagnosed and treated promptly [ ] . according to current data, using albumin in combination with antibiotics for the treatment of patients with sbp seems to be warranted but only for those with jaundice or renal dysfunction. the prophylactic use of antibiotics in cirrhosis with gastrointestinal bleeding also seems to be necessary, because the use of antibiotics contributes to reducing incidence of infection and rebleeding whereas improving survival rate. furthermore, the incidence of hrs in sbp patients decreases by albumin administration, and prevention of hrs can also be related to increased survival. the recommended dose of albumin is . g/kg body weight on the first day then g/kg body weight on the third day, a maximum of g and g, respectively. albumin administration is strongly recommended in sbp patients with serum bilirubin levels higher than . mmol/l ( mg/dl) or serum creatinine more than . mmol/l ( mg/dl). a placebo-controlled rct that enrolled the patients with low (< . g/l) ascites protein who also had advanced liver diseases or "renal dysfunction"(defined as scr ≥ . mg/dl or blood urea nitrogen≥ mg/dl, or serum sodium level ≤ meq/l) suggested that oral norfloxacin contributed to a reduced hrs incidence within year ( % vs. %) and an improvement in survival at the end of months [ ] . norfloxacin may ameliorate or prevent vasodilatation by reducing bacterial translocation and overt infections, as well as suppressing plasma renin activity, thereby prevent these patients from developing hrs. the concept that the severity of the clinical course of patients with cirrhosis complicated with serious bacterial infection is related to the degree of an impairment of circulatory function, which has led to new and effective approaches in the prevention and treatment of these complications. in patients with severe hepatitis, multiple causes may lead to disorders of internal environment, mostly manifesting fluid and electrolyte imbalance as well as acidbase imbalance, usually resulting in deterioration, greater complexity and even death. accurately recognizing the occurrence of severe hepatitis with complications such as fluid and electrolyte imbalance and/or acid-base imbalance, and therefore giving appropriate treatment to maintain balance of internal environment, is of great importance for improving prognosis of the patients [ ] . water is the major component of human body. electrolytes are substances that dissociate in solution to form charged particles, orions. body fluid comprise mainly of water and electrolytes and electrolytes comprise mainly of na + , k + , ca + , mg + , cl − , hco − , hpo − and so − . the primary function of electrolyte include: ( ) to maintain osmotic pressure and acid-base balance of body fluids; ( ) maintain nerve, muscle, cardiac cells resting potential, involved in the formation of action potentials; ( ) involved in metabolism and physiological activities [ ] . body fluid include intracellular fluid and extracellular fluid, the latter can be divided into plasma and interstitial fluid. intracellular and extracellular fluid differ in ion components. na + is major cation in extracellular fluid and its main anions are cl − and hco − . k + is major cation in intracellular fluid and its major anion is hpo − . the total number of ions in body fluids is called osmolality, its unit is mosm/l. if the osmolality on both sides of a semipermeable membrane is not equal, water moves toward the side with the higher osmolality. this phenomenon is called osmosis [ ] . osmosis of water can be opposed by applying a pressure across the semipermeable membrane in the direction opposite to that of the osmosis. the amount of pressure required to oppose the osmosis is defined to be osmotic pressure. in spite of various solute concentrations are different in extracellular and intracellular fluid, the osmotic pressure remain equal. normal plasma osmotic pressure is - mosm/l. steady osmotic pressure is the basic guarantee to maintain the fluid balance across the cell membrane. multiple mechanisms in nervous and hormonal system are involved in the regulation of body fluid and electrolyte balance [ ] . ( ) there exists sensation of thirst in central nervous system, which plays an important role in regulating body water. ( ) there is a powerful feedback system for regulating plasma osmolarity and sodium concentration that operates by altering renal excretion of water independently of the rate of solute excretion. a primary effector of this feedback is called antidiuretic hormone (adh) which plays an important role in regulation of renal concentration and dilution to maintain the body fluid homeostasis. ( ) reninangiotensin-aldosterone system (raas) is an important regulator of sodium reabsorption and potassium secretion by the renal tubules. human body fluid environment must be suitable ph value for maintaining normal metabolism and physiological function, under normal conditions, human body take in acidic and basic food and drinking water, produce acids and bases during metabolism and eliminate acidic or basic substances by the kidneys and lungs. in the plasma, the normal ph value ranges from . to . with an average value at . . the regulation of the acid-base balance is accomplished by the buffer system of the body fluid, the respiration of the lungs and the excretion of the kidney [ ] . ( ) blood buffer system is composed by a weak acid and its corresponding buffer base, includes bicarbonate buffer system, phosphate buffer system, plasma protein buffer system, hemoglobin and oxygen synthetic hemoglobin buffer system. ( ) the role of the lung in acid-base balance is to adjust the concentration of plasma carbonic acid by changing the amount of co , so that the ratio of hco − and h co in the plasma is close to normal, so that the ph is relatively constant. ( ) the major role of the kidneys in maintaining acid-base balance is to conserve circulating stores of bicarbonate and to excrete h + . the kidneys maintain ph by increasing urinary excretion of h′ and conserving plasma hco − when the blood is too acidic, or increasing urinary excretion of hco − , and decreasing urinary excretion of h + when the blood is too alkaline. patients with severe hepatitis are prone to develop water retention, with the main manifestation of seroperitoneum (ascites) as well as body weight gain. with the acatharsia of water becoming more serious, oliguria and edema of lower extremities occur. sbp (spontaneous bacterial peritonitis) can also occur, which is manifested with symptoms such as fever and abdominalgia. several factors contribute to ascites include an increase in capillary pressure due to portal hypertension, obstruction of venous and lymph flow through the liver, decrease in colloidal osmotic pressure due to impaired synthesis of albumin by the liver, salt and water retention by the kidney [ ] . some theories have been used to explain the increased salt and water retention by the kidney. because of vasodilation or an actual loss of fluid into the peritoneal cavity, the effective blood volume maybe reduced, which may in turn decrease the renal blood flow leading to a lower glomerular filtration rate (gfr) and an activated rennin-angiotensin-aldosterone system (raas). the diagnosis of water retention depends on typical clinical symptoms such as ascites, pleural effusion, and edema of lower extremities, when the body begins to excess water, blood pressure is increased, which leads to many complications such as congestive heart failure and pulmonary edema. hyponatremia is a common complication in severe hepatitis patients, and always incorporate with the retention of water and sodium, but the total sodium can be decreased, normal or even increased, that is dilutional hyponatremia. in laboratory test, serum sodium is below . mmol/l. the mechanism of hyponatremia probably depends on the following factors: ( ) the decreased function of adh inactivation in liver brings adh increase, enhancing the reabsorption of water in renal tubule, which causes the formation of water retention. this is the main cause of dilutional hyponatremia. ( ) water retention brings about volume extension, causing the aldosterone secretion decrease, which leads to the sodium egestion increase in urine. ( ) some severe hepatitis patients frequently vomit, and can not eat, bringing about a major loss of body fluid and electrolyte. ( ) severe hepatitis patients, serum albumin reduces, combining with the factors such as poor appetite, anorexia, fasting or limit sodium, bring about a state of low permeability in the cell, which causes the extracellular na + moving into the cell. ( ) iatrogenic factors, exhaust potassium diuretic such as hydrochlorothiazide and furosemide and spironolactone have a strong role in the excretion of sodium, so a large number diuretic is liable to hyponatremia in ascites patients, and in the treatment of cerebral edema, a large infusion of mannitol may cause hyponatremia either [ ] . hyponatremia due to the osmotic pressure of extracellular fluid decreased, water moves to the cells, causing cell edema, especially brain edema. so the symptoms of nerve system are the main manifestation in hyponatremia patients [ ] . generally dilutional hyponatremia in severe hepatitis patients develops slowly and progressively, and the symptoms are often covered by primary disease symptoms, like weak, feeble, nausea, vomiting, lethargy, significant body weight increase, pale and moist skin and sometimes saliva, tears increase. improper treatment will bring about a sharp serum sodium decrease in the short term, such as serum sodium rapidly decreasing to below mmol/l, acute hyponatremia syndrome comes, the manifestation include convulsions, coma, hypotension, pulse narrowing, tachycardia, oliguria even respiratory arrest and death. if cerebral hernia happens, corresponding nerve location signs will follow. hypokalemia refers to the condition in which the concentration of potassium (k + ) in serum is less than . mmol/l. it could occur during the whole period in severe hepatitis patients and is more common in early metaphase of disease. hypokalemia can be the result from one or more following medical conditions: ( ) insufficient intake of potassium due to the poor appetite or anorexia in the patients with severe hepatitis. ( ) frequent vomiting leads to excessive loss of stomach acid, which causes alkalosis and extracellular potassium is transferred into cells. ( ) the reduce of effective circulating blood volume can cause to high aldosterone levels and excessive urinary losses of potassium. ( ) the decreased function of aldosterone inactivation in liver brings aldosterone increase, enhancing urinary losses of potassium. ( ) some medications such as diuretics can also cause urinary losses of potassium. the clinical syndromes of hypokalemia are related to the degree of the shortage of intra/extracellular potassium and disorders of other electrolytes and acid-base, but more depends on how soon it occurs. in the early time it shows muscle weakness, first in limbs, then develops to the torso and respiratory muscle. deficiency of potassium also can lead to weak peristalsis, poor appetite, sick and constipation in mild hypokalemia but abdominal distention and paralytic ileus in severe situation. in cardiac syndromes, it mainly presents atrioventricular block and arrhythmia which including premature ventricular contraction or atrial premature beats, sinus bradycardia, paroxysmal auricular tachycardia or junctional tachycardia, even ventricular fibrillation. in hypokalemia state an increasing shift of potassium from extracellular fluids into cells and an obligate loss of potassium from kidney can cause metabolic alkalosis and abnormal acidic urine. long-term hypokalemia also can lead to hypokalemic nephropathy with proteinuria and cylindruria syndrome. the changes of ecg [ ] : in the early stage, flattened t wave and an obvious u wave, st-segment depression can be found, qu interval is widen. in severe situation, a wide pr interval, low voltage, wide qrs interval and ventricular arrhythmia can occur. hyperkalemia refers to the condition in which the concentration of potassium (k + ) in serum is higher than . mmol/l. it is more common in the middle and late period of severe hepatitis. the mechanism of hyperkalemia: ( ) the most usual way lead to hyperkalemia is oliguria or uroschesis which are caused by renal dysfunction among the patients have severe hepatitis with hepatorenal syndrome. ( ) metabolic acidosis and na + -k + -atp enzyme inactive lead to a shift of potassium out of cells also contribute to develop hyperkalemia. ( ) long-term and high dose potassium-sparing diuretics applied during the treatment lead to hyperkalemia is not rare, and easy to get sudden death in patients. hyperkalemia mainly influences myocardium and skeleton muscle. the most dangerous situation is fatal arrhythmia. when the concentration of k + is higher than . - . mmol/l, there are peaked t waves. when it is over - mmol/l, pr interval is widen and p wave is flattened even vanish. when it is up to - mmol/l, t waves and qrs complex can evolve to sinusoidal shape and cardiac arrest. hyperkalemia is a common critical and severe symptom in clinic. when it happens, all of the potassium-sparing diuretics and potassium uptake should be stopped. at meantime, the treatment against the toxicities to myocardium and skeleton muscle should be taken to accelerate a shift into cells and potassium excreting. there also can happen hypocalcemia, which shows neuromuscular excitability, cardiac electrical instability and instable emotion. the concentration of calcium in serum under mmol/l is significant in diagnosis. hypomagnesemia can be found too. it mainly presents similar symptoms as hypocalcemia such as weakness, muscle cramps, increased irritability, tetany and chvostek positive. hypomagnesemia can cause cardiac arrhythmia, when it occurs, the concentration of magnesium is less than . mmol/l, and it should be urgently treated. severe hepatitis patients prone to acid-base imbalance [ ] , mainly to alkalemia. the main types of acid-base imbalance include respiratory alkalosis, metabolic alkalosis, respiratory alkalosis plus metabolic alkalosis, secondly include respiratory alkalosis plus metabolic acidosis, triple acid-base disorders (tabd), metabolic alkalosis plus metabolic acidosis [ ] . history and clinical manifestations provides important clues for the judgment of acid-base imbalance. the result of blood gas monitoring is the decisive basis for judging the type of acid-base imbalance. serum electrolyte examination is an important reference. anion gap (ag) has important diagnostic value in determining the type of acidbase imbalance [ ] . ag = na + -(cl − + hco − ) is a simple formula for the value between the number of cations and anions in serum. its normal value was ± mmol/l. ag can not only help diagnose "potential" metabolic acidosis and to distinguish different types of metabolic acidosis, can also help determine special types of mixed acidosis, and has its unique role in the judgment of tabd. sometimes the indicators of blood gas analysis are normal, the calculation of ag value become the only evidence of diagnosis of metabolic acidosis. in addition, ag value can be used as reference for correction of acid-base imbalance. in severe hepatitis, the change of ag values can be used as an indicator to estimate the complications and prognosis. clinical observations indicate: ag value significantly increased often suggestive of severe infection, kidney dysfunction or severe bleeding, and the prognosis is poor. respiratory alkalosis refers to arterial paco decrease and ph > . as well as compensatory decrease of blood hco − . respiratory alkalosis occurred in early stage of severe hepatitis. in severe hepatitis, respiratory alkalosis related to hyperventilation: accumulated ammonia and other vasoactive peptides excited respiratory center, ascites and pleural fluid increase respiratory rate, hypoxemia excited respiratory center. compensatory mechanisms: co reduction, breathing shallow and slow, so that co retention, h co compensatory rise; when last longer, reduce renal row h + , hco − excretion increased, hco − /h co equilibrium at a low level. most patients have the performance of shortness of breath and heart rate increase. can have vertigo, hand, foot and mouth numbness, muscle tremor, hand and foot convulsions. convulsions associated with low calcium. dysfunction of nervous system is related to the damage of brain function and cerebral blood flow decrease. respiratory alkalosis diagnosis relies on the following: ( ) ph is normal when fully compensated, increased underdecompensation. ( ) paco lower (typically < mmhg or . kpa). ( ) hco − compensatory decline. ( ) ag value may have a slight increase. ( ) blood cl − may increase. metabolic alkalosis refers to the type of acid-base imbalance characterized by an increase hco − in extracellular liquid. the inappropriate application of basic drugs, potassium-sparing diuretics, dehydrating agents, hormones can often induce or aggravate metabolic alkalosis. severe gastrointestinal symptoms, anorexia, vomiting or diarrhea are also the reasons for the occurrence of metabolic alkalosis. compensatory mechanisms: when alkaline substances increased in body, buffer system instantly transfer strong base into weak base, increase hco − consumption, h co increased. inhibit respiratory center, decrease pulmonary ventilation, co retention, hco − compensatory increase. renal carbonic anhydrase activity decreased and h + formation and excretion decreased, nahco reabsorption is also reduced, so hco − /h co compensatory restore to : , ph value is normal. patients with mild metabolic alkalosis usually have no obvious symptoms. many disorders can occur in severe metabolic alkalosis. ( ) functional changes in the central nervous system, the patient may have irritability, confusion, delirium, consciousness disorders. ( ) slow and shallow breathing, hypoxemia. brain tissue is particularly sensitive to hypoxia, thus neurological symptoms first appeared. ( ) hypocalcemia and neuromuscular stress increased, the performance of tendon hyperreflexia, face and muscle twitching, and limbs twitching. ( ) hypokalemia can cause neuromuscular symptoms and arrhythmias. according to ph value, paco , hco − , level of k + and cl − , effective circulating blood volume and performance of primary disease, diagnosis of metabolic alkalosis is no difficult to make. metabolic alkalosis should be divided into two categories based on the urinary level of cl − . ( ) chloride positive metabolic alkalosis: supplement sodium chloride can correct the alkalosis. it indicates that the body has cl − deficiency, urinary cl − < mmol/l. ( ) chlorine negative metabolic alkalosis: alkalosis can not be corrected by supplement sodium chloride, urinary cl − > mmol/l. respiratory alkalosis plus metabolic alkalosis tend to occur on the early stage of severe hepatitis. in most cases, there is no obvious complication, more often metabolic alkalosis happens on the basis of respiratory alkalosis, or the other way around. due to respiratory and metabolic factors are inclined to alkaline change, name as reduce paco and elevated plasma hco − , there is no mutual compensation between them, so it is easy to present as severe decompensation and poor prognosis. main point of diagnosis: ( ) ph value of blood increase significantly. ( ) paco decrease. ( ) hco − increases, the value should be greater than . ×( -paco ) + . . ( ) hypokalemia and hypochloremia are common phenomenon. respiratory alkalosis plus metabolic acidosis is relatively rare. metabolic acidosis can be divided into types: ( ) value of ag is normal (high chlorine acidosis), commonly seen at long-term diarrhea, combined with renal tubular acidosis and a large amount of physiological saline input in patients or water intoxication. ( ) high ag value type (normal blood chlorine acidosis), regularly present in combination of hepatorenal syndrome, lactic acidosis and patients with ketoacidosis. ( ) a hybrid type (high ag merged high blood chlorine), mainly in patients with severe diarrhea following lactic acid or ketoacidosis. when respiratory alkalosis plus metabolic acidosis happens, paco and plasma concentration of hco − are higher than scope of compensation to each other. its characteristics as follows: ( ) the range of blood ph change is not large, normal, slightly higher or slightly lower. ( ) paco reduce to less than . × hco − + or hco − < -( -paco ) × . - . . ( ) ag values can be normal or elevated, the latter is more common. if the elevated blood cl − value is equal to the hco − decrease, ag value normal metabolic acidosis type can be diagnosed; the cases that the rising value of ag is equal to the decline of hco − values can be diagnosis as ag increased metabolic acidosis type. on the third occasion, the increase value of ag is equal to the sum of hco − and cl − drop, the diagnosis is mixed metabolic acidosis. for this type of offset mixed acid-base balance disorders, treatment should be moderate, the measure of the metabolic factors correcting should be precede to respiratory factors, avoid paco quickly returning to normal in the process of treatment, which would lead to blood ph drop rapidly and acidosis more worse. to patients with severe hepatitis, the harm of alkalosis is greater than acidosis, thus the blood ph should be kept slight acidic in a normal state. generally, the target of alkali supplement can be arterial blood ph value recovered to . . respiratory alkalosis tabd refers to respiratory alkalosis, metabolic acidosis and metabolic alkalosis three primary imbalances coexist in the same patient, which is one sort of serious acid-base imbalance, mostly develops in the late stages of severe hepatitis, fatality rate is high. respiratory alkalosis tabd characteristics as follows: ( ) blood ph value depends on the relative severity of these three primary imbalances, which can be normal, or slightly high generally. ( ) reduce paco , its value is less than . xhco − + . ( ) hco − can raise, normal or lower. ( ) value of ag rise significant, and extent of ag raise is greater than the hco − lower. ( ) cl − often lower than normal. the occurrence of metabolic alkalosis plus metabolic acidosis in patients with severe hepatitis is not uncommon. usually it is accompanied with existing lactic acidosis or ketoacidosis, and the patient may manifest frequent vomiting. since the causes for raising and lowering hco − coexist, they tend to cancel one another. the ph and hco − concentration can be normal, increased or decreased, depending on the relative severity of the two kinds of imbalances. severe hepatitis can also be accompanied by metabolic acidosis, metabolic acidosis plus respiratory acidosis, tabd of respiratory acidosis, and etc. pure metabolic acidosis refers to arterial blood ph < . and compensatory decline of paco due to primary decrease of hco − . typical manifestation is known as kussmaul breathing, characterized by deeper and faster breathing, as well as obvious contraction of respiratory muscle, and also ketone-smelled exhaled breath. the patients often flush, companied by increased heart rate and decreased blood pressure. there may be reduced or disappeared tendon reflexes, confusion or stupor. due to respiratory and metabolic factors both towards to acidic changes, there is no respiratory compensation for decrease in hco − , nor renal compensation for increase in paco , hence presenting severe decompensated status. the resulting distinct decrease in ph and vicious circle are the characteristics for metabolic acidosis plus respiratory acidosis. the characteristics for tabd of respiratory acidosis include significantly increased paco , elevated hco − , ag > mmol/l, and significantly decreased cl − . the incidence of the above types of acid-base imbalance is very low in patients with severe hepatitis. once it happens, it should be actively treated with corresponding methods, so that the blood ph quickly restores to the safety range. during therapeutic process against various pure acid-base imbalance, interactions among various treatments need to be taken into consideration, in order to avoiding the possibility that the treatment for one type of acid-base imbalance causes or aggravates another type. in summary, water-electrolyte imbalance and acid-base imbalance have a relatively high morbidity in patients at various stages of severe hepatitis. this often results in deterioration and complication of the disease, evermore, the death of the patient. therefore, the functions of heart, lung, kidney, blood circulation as well as changes of body weight in the patient need to be intently monitored. regular detection of k + , na + , cl − , carbon dioxide combining power (co cp), blood urea nitrogen, creatinine, ph, data about arterial blood gas analysis, and also detailed records of patient's input and output are demanded. during the process of diagnosis and treatment, careful analysis of the history, clinical manifestations and laboratory examination are necessary to achieve correct diagnosis, early prevention and prompt treatment. in normal condition, proper amount fluids can make a lubrication action on organs in peritoneal cavity. but in those patients who have severe hepatitis, especially with cirrhotic portal hypertension, too many fluids over ml can lead to ascites. the ascites can be categorized into uncomplicated ascites and refractory ascites. there is no infection in uncomplicated ascites and won't lead to hepatorenal syndrome, but refractory ascites is in the contrast. the refractory ascites includes diureticresistant and diuretic-intractable ascites. the diuretic-resistant ascites shows no response to diuretic and diuretic-intractable ascites limits the application of diuretic due to the complications induced by diuretic. mg antisterone per day as an initial dose can be given to those patients with moderate ascites, if it goes to no satisfied effect, mg can be added after every days till the maximum dose to mg/d. if the patients show a hyperkalemia or aldosterone antagonist-resistant, nicorol can be combined and with an increasing dose from to mg/d gradually. the patients without edema losing weight should be less than . kg and those with edema should be less than kg per day to avoid electrolyte disturbance or hepatorenal syndrome during the whole treatment period. diuretics should be withdrawn on the patients with severe hepatic encephalopathy, severe hyponatremia, progressive renal failure or severe muscle spasm. the patients should only take the minimum dose of diuretics to maintain the state after the syndrome controlled, or withdraw when it is necessary. due to the poor outcome and living quality, the median survival time of refractory patient is half year. so liver transplantation can be considered in the patients with refractory ascites induced by cirrhosis, which required more cautious before make a diagnosis. generally, if the patients meet the following conditions when they are receiving mg/days antisterone and mg/days nicorol treatment over week and sodium uptake limited in mmol/days, refractory ascites can be diagnosed. ( ) losing weight less than . kg/days over days ( ) sodium uptake is more than elimination ( ) grade - ascites is arisen again after -week long treatment ( ) hepatic encephalopathy, hepatorenal syndrome and severe electrolyte disorder induced by diuretics are shown up. refractory ascites patients without complications can be treated with abdominocentesis, but it will possibly induce circulation failure, and increases the risk of hepatic coma and hemorrhage. so low rate infusion of albumin with abdominocentesis is combined to avoid circulation failure, meantime diuretics also need to give after abdominocentesis. aldosterone antagonist combined with nicorol is a suitable strategy: the dosage of antisterone is increased from to mg/days and nicorol from to mg/days gradually. the goal of this strategy is to maintain the situation without ascites under the minimum dosage. but when severe hepatic encephalopathy and electrolyte disorder show up, which means serum sodium concentration is less than mmol/l, serum potassium concentration is less than . mmol/l, nicorol should be withdrawn, if serum potassium concentration is more than . mmol/l, antisterone should be withdrawn. to those patients who need abdominocentesis repeatedly, transjugular intrahepatic portosystemic shunt (tips) can be considered, but the risk of hepatic encephalopathy will be higher and the outcome is poor. so the patients with severe liver and renal failure, cardiorespiratory function failure or active infection should be in cautious. the mean survival time of refractory ascites patients complicated with hepatorenal syndrome is months, preventive antibiotics combined with albumin is an option for these patients. to those patients who have already showed hepatorenal syndrome, terlipressin combined with albumin could be useful. meantime, abdominocentesis, tips or artificial liver supporting treatment can improve patients' living quality in short term, but long-term outcome won't be good, so liver transplantation should be execute as soon as possible. in general, medicine can improve the symptoms in short term but with poor outcome, liver transplantation is more meaningful. patients with hypovolemic hyponatremia can have a supplement with sodium and decrease the dosage of diuretics, patients with hypervolemic hyponatremia can restrict fluids uptake (less than ml/d) and combined with vasopressin v receptor blocker or antidiuretic hormone receptor blocker. currently, vaptans, tolvaptans, conivaptan and satavaptan have already applied in clinical practice. there was research showed that vaptans could improve - % patients' symptoms significantly after patients took it for week to month, and main side reaction was thirsty. the patients complicated with hepatic encephalopathy should be used vaptans with cautious due to its high risk in dehydration and hypernatremia. meantime, vaptans is metabolized by cyp a, so rifampin, barbital and phenytoin can decrease its effect, and ketoconazole, clarithromycin can increase its plasma concentration. tolvaptans can give some relief but increase the risk of hemorrhage. satavaptan will decrease patients' survival rate. so the proper treat period and side reactions of these drugs in long-term using need to make clear. potassium uptake should be withdrawn immediately after hyperkalemia occurs, emergency treatment for detoxicating potassium should be taken to protect cardiac. the treatment depends on the plasma concentration of potassium. the treatment for patients with fulminant hepatitis b with cirrhosis complicated with hyperkalemia is to restrict the uptake of potassium, improve the microcirculation, correct renal filtration decrease induced by hepatorenal syndrome and increase the elimination of potassium. there are also some patients have abnormal distribution of potassium due to hypoxia, acidosis, catabolism, and deficiency of energy supplies, which leads to intracellular potassium is transferred into extracellular. the treatment for these patients is to correct hypoxia and acidosis, high glucose, insulin and atp are administered to boost glycogen synthesis to transfer potassium from extracellular to intracellular. peritoneal dialysis and plasmapheresis can be given to the patients with intractable hyperkalemia. hypokalemia can present during the whole period of fulminant hepatitis b, it will occur more often on the early and middle stage. long-term inappetency and abdominal distension lead to insufficient potassium uptake. nausea, vomiting and diarrhea lead to increase of potassium losing. renal filtration rate decreasing and aldosterone increasing lead to potassium elimination increase. complicated with alkalosis and anabolism increase also can cause hypokalemia. the treatment for hypokalemia should focus on comprehensive therapy, correcting alkalosis, increasing potassium supply, improving microcirculation. disturbance of acid-base balance occurs quit often in hepatitis b patients, especially with alkalosis. during the early stage, it can present in pure respiratory alkalosis, also can complicated with metabolic alkalosis. during the middle and late stage, both of above symptoms and metabolic acidosis occurs concurrently. treating idiopathy and correcting hyperventilation is a treatment for respiratory alkalosis. arginine hydrochloride injection is used to treat metabolic alkalosis to avoid secondary metabolic alkalosis. the general regulation is prefer acid to base, till ph value of arterial blood back to . . in prevention of disturbance of acid-base balance, the effective strategy is to correct hypokalemia and hypochloremia, control vomiting. meantime, controlling infection, endotoxemia and upper gastrointestinal hemorrhage are necessary. hepatic encephalopathy (he) due to metabolic disturbance is a complex neuropsychiatric syndrome caused by severe liver dysfunction or disorder and is one of the common complications and causes of death in severe liver diseases. patients with he mainly present with neuronal or mental abnormalities and disturbance of consciousness, even coma and death. the clinical manifestations and the severity of the disease vary because of its complex pathogenesis. hepatic encephalopathy is the result of acute and chronic hepatic failure caused by cirrhosis or various kinds of portosystemic shunt (pss) created. a diagnosis of he can be made after excluding encephalon diseases. the syndrome is caused by metabolic disorders and is potentially reversible. he clinical features differ due to the wide degree and range of neuropsychiatric symptoms that vary from subtle abnormalities detected only by intelligence tests or electrophysiological methods geared for detecting personality changes to abnormal behavior, intellectual impairment, and even different degrees of consciousness disorders. he was previously known as hepatic coma, but that is only one of the worst severe signs of he and does not represent all types of he. in , the world congress of gastroenterology (wcog) suggested that based on the cause he can be divided into three types (a, b, and c) [ , ] . type a: type a is acute liver failure-related he and the symptoms occur within weeks. in subacute liver failure-related he, the symptoms of he occur within - weeks with or without predisposing factors. type b: patients with type b he have obvious pss and normal liver histology without associated intrinsic liver disease. these clinical manifestations are similar to those in patients with he and cirrhosis. the pss may be spontaneous or caused by surgical or interventional procedures [ ] . common causes of pss include congenital vascular malformation, intrahepatic or extrahepatic portal vein obstruction (including trauma, carcinoid, and bone marrow hyperplastic disease caused by a high coagulation state due to portal vein branch embolization and thrombosis) and generation of portal hypertension by oppression of lymphoma, metastatic tumors, and bile duct carcinoma. type c: type c he is related to chronic liver diseases, with cirrhosis being the most common type, and is generally accompanied by portal hypertension and pss. type c he is mainly caused by liver function failure, rather than by pss. according to the clinical manifestations, duration and characteristics, type c can be divided into three types: episodic he, persistent he, and minimal he [ ] . episodic he, related to chronic hepatic disease, is defined as a disturbance of consciousness and cognitive change in a short time and can be alleviated by spontaneous remission or drug treatment in the short term, which cannot be explained by a relevant preexisting mental disorder. episodic he can be divided into three types according to the presence of known risk factors: ( ) incentive type: there is a clear history of predisposing factors; ( ) spontaneous type: there is no history of predisposing factors. ( ) recurrent type: he attacks more than two times within a year. persistent he related to chronic hepatic disease is defined as an occurrence of continuous neural mental abnormality, including cognitive decline, disturbance of consciousness, coma and even death. persistent he can be further divided into three types according to the severity of the disturbance in the patient's self-control and self-discipline: , mildest type, namely west haven level ; , severe type: namely west haven level - ; and , therapeutic resistance type: medication can alleviate he quickly, but withdrawal can aggravate he rapidly. patients with minimal he, with normal clinical manifestations and routine biochemical tests, have mild cognitive and psychomotor deficits detected by neuropsychology and neural physiology tests, and these patients usually have a history of chronic hepatic disease [ ] . the prevalence of minimal he in patients with cirrhosis is - %. patients with minimal he with reduced physical and mental ability have gained more and more attention recently because they have a high risk of accidents when engaged in occupations involving mechanical, or driving work. the pathogenesis of he has not been fully elucidated so far, and many theories have been put forward. it is generally believed that he is caused by acute and chronic liver failure and/or pss. when toxic substances absorbed by the intestines cannot be detoxified and cleared by (or through) the liver, they directly enter into the systemic circulation and pass through the blood-brain barrier to reach the brain tissue and cause central nervous system dysfunction. a variety of the risk factors mentioned above can result in he. hyperammonemia is still recognized as one of the most important factors, especially in he related to chronic liver disease, liver cirrhosis and/or pss. according to the ammonia intoxication theory several factors including false neurotransmitters, such as γ-aminobutyric acid/benzodiazepine (gaba/bz) receptor complex, an imbalance in the ratio of branched chain amino acids to aromatic amino acids, brain cell edema, astrocyte dysfunction, mercaptan, short chain fatty acid toxicity and manganese deposition are all involved in the occurrence of he [ ] . ammonia intoxication caused by an ammonia metabolism disorder is the most important factor in the pathogenesis of he [ ] . ammonia comes mainly from the gut and the generation and absorption of ammonia increase in a serious liver disease when excess ammonia cannot be cleared sufficiently by ornithine cycle due to serious damage to liver parenchyma. when pss occurs, intestinal ammonia directly enters the systemic circulation without liver detoxification, resulting in increased blood ammonia. high levels of blood ammonia can enter the brain through the blood-brain barrier and generate central nervous system toxicity by interfering with cerebral energy metabolism, neurotransmitter and nerve cell membrane ion transport; increasing cerebral edema; and changing gene expression (such as stellate cell glutamate carrier, stellate cell structural protein, glial fibrillary acidic protein, peripheral benzodiazepine receptor and aquaporin- ) and inducing the mitochondrial permeability transition (mpt). the main way of removing ammonia from the brain is through urea cycle. during glutamine synthesis, glutamic acid is formed from ammonia and α-ketoglutaric acid and the glutamic acid combines with ammonia to generate glutamine. this process requires atp and consumes a large amount of α-ketoglutaric acid, which interferes with the brain energy metabolism and causes an energy supply shortage in brain cells. glutamate is an important excitatory neurotransmitter in the brain, and lack of glutamate increases inhibition in the brain. glutamine synthetase is present in astrocytes, where glutamic acid is detoxified to glutamine. glutamine is a strong intracellular osmotic agent, and increases in glutamine can lead to brain cell swelling. reports have identified a strong correlation between the content of glutamine in cerebrospinal fluid (csf) and the degree of he [ ] . during he, excess ammonia under the effect of glutamine synthetase, not only reduces the formation of active glutamate but also consumes a lot of energy, leading to the accumulation of glutamine, which increases intracellular osmotic pressure and causes brain cell swelling. swollen astrocytes with impaired function further affect ammonia metabolism, reduce the ability of neurons to efficiently uptake or release extracellular ions and neurotransmitters, and stimulate glial cell synthesis of neurosteroids by upregulating their expression of the peripheral-type bz receptor (translocator protein, kda). neurosteroid is an endogenous bz that can enhance gaba nerve tension and cause symptoms in patients with he [ ] (fig. . shown that the metabolic rate of cerebral ammonia in he patients is increased. increased levels of blood ammonia enter the brain through the blood-brain barrier. brain dysfunction also occurs even if blood ammonia levels appear normal; this partially explains the occurrence of he in the case of normal blood ammonia and invalidates he treatment by simply reducing blood ammonia. in addition, increasing evidence suggests a synergistic effect between blood ammonia and its metabolic disorders with systemic inflammation, nerve steroids, oxidative stress, nitrification stress, manganese poisoning, and gaba/bz [ ] . the main inhibitory neurotransmitter in the mammalian brain is gaba. plasma gaba is derived from the conversion of glutamic acid by glutamate decarboxylase in intestinal bacterial. notably, gaba has dual role. on one hand, during liver function failure and pss, the removal of gaba in liver is significantly decreased; on the other hand, gaba can directly enter the systemic circulation bypassing the liver, resulting in increased concentration of gaba in blood. the concentration of gaba in csf and brain tissue increases as more gaba crosses the abnormal blood-brain barrier. in addition, endogenous bz was found in the blood and csf, and the gaba receptor on the membrane surface of the brain's postsynaptic neurons increased significantly in some patients with he and in animal models. this receptor not only combines with gaba but also binds to barbiturates (barb) and bz on different parts of the receptor surface; thus, it has been named the gaba/bz complex receptor or the super receptor complex. when liver function is severely impaired, the binding affinity of this complex receptor to its three ligands is also increased. binding of gaba, barb, or bz with the complex receptor can promote entry of chloride ions from neuronal membrane ion channels into the cytoplasm of postsynaptic neurons, causing membrane hyperpolarization and nerve conduction inhibition. he symptoms were relieved in about % of patients treated with a gaba receptor antagonist or bz receptor antagonist, and gaba/bz and ammonia were reported to act synergistically in he. recently, some studies focused on peripheral type bz receptors, which are different from central gaba [ , ] . some questions, including the source of endogenous bz, and the correlation between the increased degree of gaba or bz and the disease, remain to be answered. therefore, therapy targeted at reducing the blood ammonia concentration in patients with he and significantly reducing the increased gaba nerve tension seems reasonable [ ] , but may not be completely effective. treatment effects of reducing ammonia vary, because of the different levels of ammonia in he patients that can be produced by the interaction between various known or unknown factors and the different effects of bz receptor antagonists. this theory is related to the metabolism of aromatic amino acids (aaa), the precursors of true neurotransmitters, including norepinephrine and dopamine. due to the reduction in the liver's detoxification function or formation of pss, the amines (phenylethylamine and tyramine) produced in the intestine cannot be cleared completely, resulting in elevated concentrations of these amines in the systemic circulation and increased levels in the brain through the blood-brain barrier. under the effect of β-hydroxylase, phenethanolamine and β-hydroxytyramine (β-dopamine) are generated from phenylethylamine and tyramine, respectively and are similar to norepinephrine and dopamine in chemical structure. these amines can be taken up, stored and released by adrenergic neurons in the brainstem reticular structure. phenethanolamine and β-hydroxytyramine are called false neurotransmitters because of their low physiological effects on the postsynaptic membrane, which is about / of norepinephrine. when these false neurotransmitters accumulate in the nerve synapse, they can outcompete or replace normal neurotransmitters, resulting in a disorder of nerve conduction. it was reported that plasma aaa (such as phenylalanine, tyrosine, and tryptophan) increased and branched-chain amino acids (bcaa, such as valine, leucine, isoleucine) decreased in patients with decompensated liver cirrhosis, leading to an imbalance of amino acid metabolism. aaa are decomposed and metabolized in the liver, and liver failure decreases aaa decomposition resulting in an elevated concentration of aaa in the plasma. insulin can promote bcaas entering muscle, which is then broken down and metabolized in the skeletal muscle instead of the liver. insulin inactivation is decreased in patients with liver failure, promoting a large number of bcaas entering the muscle tissue and decreasing the concentration of bcaas in plasma. finally, the bcaa/aaa ratio is reduced from a normal - . : to : or lower. the above process reduces the bcaa concentration, but increases the aaa concentration, leading to an increase in synthesis of false neurotransmitters and reduction of the normal neurotransmitter [ ] [ ] [ ] . the epidemiological data suggests that manganese poisoning and he extrapyramidal have common clinical symptoms. the liver is an important organ for manganese excretion. the concentration of blood manganese can be increased when liver function is affected, during pss, or when excretion of bile is reduced. manganese content in plasma was sharply increased in more than % of patients with acute hepatitis and liver cirrhosis and the density of globus pallidus increased in the brain basal ganglia of he patients (partially - times higher by mri). based on histological results, the above changes were caused by manganese deposition, which disappears after liver transplantation. it has been suggested that manganese deposition may cause dopamine dysfunction. deposition of manganese not only cause direct brain injury, it can influence the function of -hydroxytryptamine ( -ht), norepinephrine and gaba neurotransmitters; impair astrocyte function; and have a synergistic effect with ammonia. however, there is no reliable correlation between the concentration of serum manganese and he severity, which may be due to the chronic deposition of manganese [ ] . the characteristic change in mri imaging as the deposition of manganese remains to be verified. the effectiveness of manganese removal to improve the symptoms and neurological signs of patients with he needs further validation. the synergistic toxic effects between toxins (ammonia and mercaptan) and short chain fatty acids [ ] , the -ht hypothesis, the effect of helicobacter pylori urease, opioids, endotoxin, tumor necrosis factor, melatonin, and hepatitis b virus termed additional theories of he syndrome. this theory also suggests the same hypothesis mentioned in the above theories. due to the extensive amount of liver cell damage caused by acute liver failure in type a he, the residual liver cells cannot effectively remove toxins leading to central nervous system dysfunction. type a he, known as non-ammonia encephalopathy, is endogenous he without clear causative agents. simple type b he is rare in mainland china; the liver can clear limited metabolic toxins in patients with chronic liver failure or pss, but once these toxins exceed the compensatory capacity of the liver, type c he occurs. the occurrence of type c he is largely related to the following risk factors, which are the most important factors in the prevention and treatment of he. patients with chronic liver failure or pss are less tolerant to the protein found in food, especially animal protein. a large amount of ammonia and aaa are produced by the decomposition of intestinal bacteria, which can induce he. oral ammonium salts, urea, and methionine can induce he by increasing the absorption of nitrogenous substances and elevating blood ammonia. intestinal production of ammonia can be increased by hemorrhage in the intestine ( ml of blood contains - g protein). at the same time, because of the lack of isoleucine in the blood, after digestion and absorption of a hemorrhage, extra blood leucine and valine increase bcaa decomposition by enhancing the activity of bcaa dehydrogenase, thereby exacerbating the imbalance in the bcaa/aaa ratio. loss of blood volume, cerebral ischemia and hypoxia also increase the sensitivity of the central nervous system to ammonia and other toxic substances [ ] . infections such as spontaneous peritonitis, pneumonia, and urinary tract infection can increase tissue decomposition and production of ammonia. secondary sepsis or sirs induce he through tnf-α, il- , il- and other inflammatory factors, exacerbates oxidative stress, and increases the blood-brain barrier permeability of ammonia and other toxic molecules to liver and brain [ ] . studies have shown that sirs is directly related to the deterioration of he in patients with liver cirrhosis, and its extent and mortality increase with the deterioration of sirs [ ] . similarly, sirs is a common factor in triggering chronic liver failure characterized by he and renal failure. in a study of patients with liver cirrhosis, artificially-induced hyperammonemia by oral administration of glutamine may have worsened the results of psycho-mental testing in cases of sepsis patients; while brain toxicity was not obvious after the inflammation was relieved, the observation of decreased cytokine levels indicated that infection and induced inflammatory mediators enhanced brain toxicity of hyperammonemia. accordingly, some researchers suggested that sirs could be an independent pathogenesis of he rather than a risk factor [ ] . hyponatremia can affect the intracellular osmotic pressure and lead to brain edema, which induces he. hypokalemia is often associated with metabolic alkalosis [ ] . mass use of diuretics or extraction of ascites can also cause alkalosis. ammonia is easily absorbed by the intestinal tract or through the blood-brain barrier inducing he [ ] . a variety of reasons can cause pre-renal azotemia such as hypovolemia, anorexia, diarrhea, limiting the amount of liquid, mass use of diuretics, or extraction of ascites. hepatorenal syndrome or other causes can result in renal azotemia. pre-renal azotemia and renal azotemia caused by hepatorenal syndrome or other causes can increase the concentration of ammonia in the blood. several other predisposing factors can contribute to he such as constipation, hypoglycemia, the use of sedatives and proton pump inhibitors, and epilepsy. after the occurrence of constipation and intestinal obstruction, the patient's intestinal mucosa is exposed to ammonia longer thus increasing the absorption of ammonia. hypoglycemia can reduce brain deamination. the binding of sedatives, hypnotics and the brain gaba/bz receptor produce an inhibitory effect on the brain. it was reported that proton pump inhibitors increase the risk of he in patients with cirrhosis in a population study [ ] . another study also suggested that epilepsy was associated with an increased risk of he in patients with cirrhosis [ ] . patients with type a he often have no obvious anatomical abnormalities in their brains, but - % of patients have brain edema, which may be a secondary change of the disease. hypertrophy and hyperplasia of the original plasma astrocytes in gray matter and subcortical tissue can be found in patients with type c he. patients with longer course of the disease will exhibit brain atrophy (especially in patients with alcoholic cirrhosis) of different degrees, thinning of the cerebral cortex, loss of neurons and nerve fibers, and deep cortical sheet necrosis, even the cerebellum and the base may also be involved. the majority of patients with cirrhosis may have different degrees of he at some stage in the course of the disease. the incidence of he in patients with liver cirrhosis is at least - % in mainland china while the incidence of post-tips (transjugular intrahepatic portosystemic shunt) he is - %. if patients with chronic liver disease have he, the outcome is poor; the one year survival rate is lower than % and the year survival rate is less than % [ , ] . the incidence of mild he is . % in mainland china in patients with liver cirrhosis, . % in patients with child-pugh a, . % in patients with child-pugh b, and . % in patients with child-pugh c. the incidence of mild he is not significantly associated with cirrhosis; however, with the increased degree of decompensated liver cirrhosis, the incidence of mild he increase. several studies have found that the incidence of depression and anxiety in patients also increased, with the increase of liver function damage, the incidence also increased, and the outcome is poor [ , ] . the clinical manifestations of he vary, because of the difference in the nature of underlying disease, the degree of liver cell damage, the speed of injury and incentives. they are not specific to he compared with other metabolic encephalopathies. early pathological changes of he are mild he. the neuropsychological and intelligence tests detect mild form of he, which exhibit no clear clinical symptoms and often develop symptomatic he. the main clinical manifestations seen in acute liver failure induced by type a he are rapid-onset jaundice, bleeding, decrease in prothrombin, and eventually, change in mental status that can start as mild confusion but progress to coma and even death. type c he is characterized by chronic recurrent episodes of changes in personality and behavior [ ] , stupor and coma, which is often accompanied by increased muscle tone, hyperreflexia, hepatic flap, ankle clonus or positive babinski sign and nervous system abnormalities. most patients in the early stages relapse, but then their symptoms become persistent. he often has a variety of risk factors such as consuming a high-protein diet or discontinuing treatment of he. patients with type c he not only have the clinical manifestations of encephalopathy, they also have chronic liver injury, cirrhosis and other clinical manifestations [ ] . observation of encephalopathy dynamic changes is beneficial for early diagnosis, treatment and analysis of treatment efficacy. he can be graded and quantified according to the degree of disturbance of consciousness, nervous system performance and eeg changes. according to the edition of the "consensus on the diagnosis and treatment of hepatic encephalopathy" in china, he is divided into - periods, but each period can be overlapping or distinct but each period can be overlapping (table . ). at present, scholars have stressed that the occurrence of he is a continuous progression of the disease and should be viewed as a continuum of a wide range of neuropsychiatric abnormalities, rather than isolated clinical stages. according to the traditional west haven criteria diagnosing grade he is based on clinical signs and physician assessments, resulting in diagnostic criteria confusion [ ] . (fig. . ) . covert he is diagnosed by a variety of neuropsychological and intelligence tests; the evaluation of overt he widely uses the modified west haven semi-quantitative grading table for the analysis of patients with neuropsychiatric state (table . ), the glasgow coma scale for the analysis of the degree of consciousness of patients, and the simple he severity rating scale for the disease in addition to abnormal liver function (such as increased bilirubin, enzyme bile separation, and decreased prothrombin activity) commonly used auxiliary examinations for he diagnosis include: determination of ammonia, amino acid analysis of plasma and csf, psychological intelligence test, neurophysiological test, electroencephalogram and neuroimaging. the normal level of fasting venous ammonia is - μg/l (serum) or - μg/l (whole blood) and arterial ammonia concentration may be . - times that of venous ammonia. generally, the determination of arterial ammonia is common in clinical practice than intravenous determination; however, if venous blood has been transported on ice box and detected in a timely manner after proper collection, the result is expected to be as effective as arterial detection. ammonia levels are increased in type b and c he, but are normal in type a he. thus, he cannot be ruled out based on having a normal ammonia level. the increased level of ammonia was reported to be associated with the degree of type a he, but significant overlaps in different clinical stages of patients were also found [ , ] . therefore, ammonia detection is not routinely recommended in the diagnosis of he. notably, we need to rule out falsely elevated levels of ammonia caused by lab error, renal failure, complete parenteral nutrition, gastrointestinal bleeding, the use of steroid hormones and other extrahepatic factors. the fischer ratio (bcaa/aaa) is used as a marker of he, the plasma bcaa levels decrease while aaa levels increase; resulting bcaa/aaa: < (normal > ). it was reported that the concentration of glutamate in csf in he patients is increased compared to healthy controls. the concentrations of phenylalanine and tyramine in csf were also significantly increased, and the level of phenylalanine was closely related to the degree of he [ ] . recently, it was reported that h-nuclear magnetic resonance spectroscopy could select biomarkers for these diseases, such as in patients with he [ ] , but this is not commonly used clinically because the the characteristic manifestations of cognitive dysfunction in patients with covert he are lack of attention, working memory problems, and deficits in executive function. therefore, various intelligence tests are used to assess the subtle changes in a patient's cognitive or precise movement, which is important for the diagnosis of covert he, but not for overt he. [ ] . at present, computer-aided psychological tests such as information and communication technology (ict), cognitive drug research test (cdr), and critical flicker fusion test (cff) are not influenced by the factors mentioned above and easily operated, which can be used as an alternative choice for pen and paper tests. ict with sensitivity % and specificity % was one of the most commonly used tests to diagnose minimal he. cff was originally used to detect the critical flicker frequency of alert patients, reflecting brain conduction dysfunction. based on a spanish study of cases, including patients with cirrhosis and healthy controls, cff was a sensitive method to diagnose covert he with sensitive, simple and reliable advantages [ , ] . because the diagnosis of minimal he has just started, the related diagnostic value still needs to be further evaluated. an abnormal eeg is often observed before biochemical abnormalities or mental abnormalities [ ] . the main abnormalities by eeg are slowed rhythm, sporadic or universal θ wave ( - times/s) and the occasional α wave ( - times/s). with the deepening of consciousness, symmetrical δ-wave and three-phase waves appear on both sides simultaneously. this change usually occurs on both sides of the forehead and the top, gradually moving backwards. although these eeg changes are not specific to he and can appear in uremic encephalopathy and other metabolic encephalopathy, the severity of changes have a good correlation with clinical stages of he. computer analysis of eeg frequency distribution, such as artificial neural network-expert system (aness) and short epoch dominant activity cluster analysis (sedaca), is more objective and valuable in diagnosing minimal he than conventional eeg [ ] . there are many kinds of evoked potential tests, including visual evoked potential (vep), brainstem auditory evoked potential (baep), somatosensory evoked potential (ssep) and endogenous event evoked potential (event-related potentials, erps) p , of which the p is the most sensitive test for the diagnosis of he. compared with intelligence tests, neurophysiologic tests, independent of age and education background, are more objective. however, they are only used in clinical studies and are limited by equipment, and professional operation. based on cerebral ct and mri, brain edema can be found in patients with type a he while brain atrophy in the frontal cortex, and the t -weighted signal enhancement in the globus pallidus can also be found, which may be associated with manganese deposition. detected by h-magnetic resonance spectroscopy (h-mrs), the metabolic changes of he patients in the brain include increased levels of glutamate and glutamine, and decreased levels of inositol, taurine and choline [ ] . using fluid attenuation inversion recovery (flair) and diffusion weighted imaging (dwi) techniques, diffuse t -weighted signal enhancement is found in the hemisphere white matter and corticospinal tracts, which may be associated with cerebral ischemia. however, the sensitivity and specificity of the above-mentioned imaging abnormalities remain unknown, and the correlation with he staging is not clear. therefore, the main significance of cranial nerve imaging is to exclude cerebrovascular accident, intracranial tumors and other diseases, rather than diagnose he. there is no gold standard diagnostic criteria of he, and diagnosis is mainly based on the exclusion of other diseases. but one should consider the following five factors [ ]: several forms of hepatic diseases may lead to different kinds of he. type a he is caused by acute hepatic failure, but without chronic hepatic disease. type b is caused by pss, but without any history of hepatic diseases. type c is caused by serious hepatic diseases and/or widespread pss, such as cirrhosis, liver cancer, post-tips and so on. psychiatric symptoms can be found such as change of mood and personality, dementia, behavior disorder and disorientation. drowsiness alternating with excitability, hypermyotonia, asterixis, ankle clonus, insanity and coma are physical signs could be present in progressed patients. some patients may lack related physical signs and psychiatric symptoms, but have deficits in ability of learning, understanding, concentration, and quick verbal response. upper gastrointestinal hemorrhage, ascites tapping, excessive diuresis, high protein diet, medicine (such as sedatives) and infection could lead to he. previous he symptoms could be helpful for the diagnosis. type a usually does not have any risk factors. metabolic encephalopathy includes ketoacidosis, hypoglycemia, uremia, pulmonary encephalopathy, serious electrolyte disturbances and toxic encephalopathy. nervous system diseases include intracranial hemorrhage, infection or tumors, mental diseases and excessive use of sedatives [ ] . but one should also watch out for the coexistence of he in these situations. an overt he should be considered if ( ), ( ), ( ), and ( ) coexist; and covert he is based on ( ), ( ), ( ), and ( ) [ ] . then, based on the degree of neuropsychiatric symptoms, determine the stage of he, or for he classification refer to the west have semi-quantitative classification table or ishen scores. the flow chart of diagnosis is shown in fig. . . he is a complex metabolic disorder caused by many factors and comprehensive measures should be taken to cure it from different aspects. according to the clinical type, inducements and the severity of the disease, different plans of treatment should be designed for he. at present, the treatment of overt he generally includes the following aspects: ( ) supportive treatment; ( ) identification of possible concurrent encephalopathy and removal of other precipitants; ( ) cause of treatment; and ( ) empirical treatment (fig. . ). the point of nutritional therapy is to promote anabolism, inhibit catabolism, and maintain a positive nitrogen balance, rather than simply limiting protein intake. to reduce the source of ammonia, it has been suggested that patients with he should limit protein intake. in critically ill patients, it has been suggested that they should stop all protein intake and, after the disease improves, gradually increase protein intake to the maximum clinical tolerance. these recommendations are now being questioned because most cirrhotic patients are malnourished and all long-term protein-restricted diets increase the severity of malnutrition. in addition, a negative nitrogen balance increases mobilization of skeletal muscle, resulting in a reduction in ammonia metabolism that may increase blood ammonia levels. recent studies have shown that normal ingestion of protein . g/(kg • d) can also improve the health-related quality of life (hrqol), especially in mhe [ ] ; and have no adverse effects on the recovery of blood ammonia and he fig. . the flow chart of diagnosis of he compared with the restricted protein intake. according to the guidelines of the european society of enteral nutrition in , the intake of protein should be guided by the following principles: patients with acute phase he on the first day should be put on a prohibited protein diet and given glucose to ensure energy supply and those who cannot eat food may be fed through a nasogastric tube without short-term fasting; patients with chronic he do not need to fast and their intake of protein should be - . g/(kg • d); oral or intravenous use of bcaa and essential amino acid preparations can be administered to adjust the balance of aaa/bcaa, promote the balance of nitrogen, and also reduce the risk of he recurrence [ ] ; probiotics and prebiotics can enhance the body's tolerance to protein; plant protein is superior to animal protein because it contains methionine, has less aaa, and more bcaa, but it also contains cellulose, which is conducive to maintain the normal flora in the colon and acidize the intestinal tract, shortening the transit time of the colon and reducing absorption of ammonia. the above points need further verification. additional supportive treatments include: maintaining adequate hydration, electrolytes and acid-base balance; ensuring an energy supply of - kal/(kg • d), which should be composed of - % sugar, - % protein, and - % fat; administering appropriate vitamins and trace elements; treating for hypokalemia, hyperkalemia, hyponatremia, hypocalcemia, hypomagnesemia and metabolic alkalosis as needed; strengthening the basis of treatment with the appropriate infusion of fresh plasma or albumin, increased plasma colloid osmotic pressure; treating for hypoxemia and cerebral edema; and preventing and treating any bleeding and bacterial infection. type c he has a variety of precipitents. actively finding and eliminating triggers can effectively prevent the development of he, such as esophageal variceal bleeding that can develop into he. active hemostasis, anemia correction, and removal of intestinal blood are also conducive to controlling he. in addition, active control of infection, correction of water and electrolyte imbalance, elimination of constipation and improving renal function are essential to control he. anesthetics, painkillers, sedatives, sleeping pills, and other drugs should be strictly controlled. patients with mania or convulsions can reduce the use of diazepam and scopolamine, and the frequency of administration can also be reduced for promethazine, chlorpheniramine, and other antihistamines. toxic substances causing he mainly come from the intestine. thus, in order to prevent and control he, it is very important to clean the intestinal tract to reduce the generation and absorption of ammonia and other toxic substances. saline or weak acidic solution enemas (such as a dilute acetic acid solution), or oral or nasal feeding of % magnesium sulfate ( - ml) can be used to clear intestinal hemorrhage, intestinal impaction, and other toxic substances. an enema composed of non-absorbable lactulose ( - ml) plus water ( ml) is also useful, especially when applied for type b he. a recent clinical trial suggested that polyethylene glycol was more effective than the current standard first-line therapy in treating these patients [ ] . another two studies showed that polyethylene glycol was more effective than the standard lactulose therapy in treating patients with acute he by cirrhosis [ , ] . other available drugs include pear liquors, mannitol, rhubarb, and so on, but excessive use of these substances may lead to dehydration and aggravate he. non-absorbable disaccharides include lactulose and lactitol. lactulose, a kind of synthetic ketone disaccharide, cannot be broken down in the stomach and small intestine due to a lack of enzymes that can break down galactose in the digestive tract. after entering the colon, lactulose can be broken down into acetic acid and lactic acid with the help of gut bacteria, leading to a reduction in the colonic ph and inhibition of the absorption of ammonia in the intestine. these non-absorbent disaccharides are decomposed into organic particles in the intestinal tract, which can increase the osmotic pressure of the intestine, and their acidic products stimulate the intestinal wall and can slightly promote intestinal excretion. these non-absorbent disaccharides, acting as prebiotics in the intestine, can inhibit the growth of bacteria, which can produce ammonia and urea, finally reducing the production of ammonia and reversing low-grade cerebral edema when combined with rifaximin [ ] . however, probiotics can benefit patients in the long-term [ ] . oral or nasal feeding ( - ml, or times daily) was recommended to adjust the daily defecation appropriately, about - times daily. main adverse reactions include abdominal discomfort, abdominal distension, abdominal pain, loss of appetite, nausea, vomiting, and diarrhea. lactulose can even be used in patients with diabetes or lactose intolerance when the purity of non-absorbable disaccharide was high (≥ %), but is not used in patients with intestinal obstruction. numerous randomized controlled studies showed that lactulose or lactitol can significantly alleviate overt he and improve the patient's cognitive function and quality of life [ , ] . lactulose is still the first-line therapy of anti-he, although its effect on improving the survival rate of patients is uncertain. antimicrobial agents can be used as a substitute for non-absorbable disaccharides in treating acute and chronic he. in the past, oral aminoglycoside antibiotics, such as neomycin, which are rarely orally ingested, were used to inhibit the overgrowth of bacteria in the intestine. however, recent randomized placebo-controlled studies have shown that neomycin may not benefit patients with he compared with placebo-treated patients and that long-term use of neomycin may lead to increased ear and renal toxicity risk and impair the function of small intestinal mucosa [ ] . metronidazole can inhibit anaerobic bacteria in the intestine and alleviate he, but long-term use may lead to disruption in the intestinal flora, gastrointestinal discomfort, or neurotoxicity. rifaximin, a derivative of rifamycin with a broad-spectrum, has a potent inhibitory effect on intestinal bacterial growth, is a minimally-absorbed oral antibiotic and only plays a role in the gastrointestinal part. administration of rifaximin ( mg, twice a day) can significantly prevent the occurrence of he compared with placebo-treated patients [ ] [ ] [ ] ; rifaximin was equivalent to or better than lactulose and neomycin in treating patients with chronic he [ ] . a study indicated that rifaximin-α in combination with lactulose was a cost-effective therapy for patients who had experienced at least two prior overt he episodes [ ] , and this therapy could also improve the driving ability of patients with covert he without toxicity to the auditory nerve and renal function [ , ] . thus, rifaximin has been recommended by the us food and drug administration (fda) for the prevention of recurrent he. the efficacy of rifaximin and relation between longterm use of rifaximin and intestinal flora in the treatment of he needs to be further investigated. however, a recent study in mice showed that rifaximin beneficially alters intestinal ammonia generation by regulating intestinal glutaminase expression in mhe [ ] . the study may provide a new opportunity to study intestinal flora in the treatment of he. microecologics with bifidobacterium and lactobacillus can regulate intestinal flora structure to inhibit the growth of bacteria that produce ammonia and urease. in combination with prebiotics, microecologics can reduce the production and absorption of intestinal ammonia and other toxic substances [ , ] . in a recent openlabel study, patients with cirrhosis were randomized to three groups and treated with lactulose ( - ml daily), probiotic capsules, or with both drugs. after a month of treatment, patients with he showed better results in the neuropsychological test, p auditory evoked potentials, and blood ammonia. however, there was no difference in the therapeutic effect among the three groups [ , ] . clinicians commonly use sodium glutamate, potassium glutamate, arginine hydrochloride and potassium magnesium aspartate, but the exact efficacy is highly controversial at present and effective drug reduced ammonia is described below. (a) l-ornithine-l-aspartate lola, a dipeptide, can lower blood ammonia by promoting ammonia consumption and the synthesis of urea, glutamic acid and glutamine in brain, liver and kidney [ , ] . ornithine, a substrate of the ornithine urea cycle, can increase activity of carbamyl phosphate synthetase and ornithine carbamyl transferase, and promote urea synthesis. n-methyl-d-aspartate (nmda) is a substrate of glutamine synthesis, and the conversion of glutamic acid to glutamine in the body can remove blood ammonia [ ] . nmda is also involved in nucleic acid synthesis in liver cells and indirectly improves the metabolism of the krebs cycle process in liver cells to facilitate the repair of liver cells. clinical studies show that, compared with a placebo group, g/days lola intravenously could noticeably reduce fasting blood ammonia (fnh ), postprandial blood ammonia, and improve the mental status of patients with he [ ] . patients with oral lola also had improved he examination results for the digital connection test, the flapping tremor, and eeg results [ ] . in addition, glycerol phenylbutyrate (gpb) can safely reduce the incidence of he as well as ammonia in patients with cirrhosis and he. the results showed that gpb had therapeutic potential in this population [ ] . zinc is an important cofactor in urea cycle enzyme catalysis. a study in he patients showed that serum zinc concentration is reduced, and showed a negative correlation with the blood ammonia concentration; the serum ammonia level is much lower after zinc supplementation in patients, and he can be improved in some patients. a new study suggests that antioxidant and zinc supplementation can improve mhe in patients with liver cirrhosis [ ] . oral zinc preparation can also reduce absorption of divalent cations such as manganese in the intestine; however, it has not been determined if zinc has a positive therapeutic effect on he. (c) sodium benzoate sodium benzoate can lower the blood ammonia concentration by activating the urea cycle for ammonia detoxification and promoting urinary ammonia. randomized controlled studies showed that sodium benzoate had the same efficacy as lactulose in treating patients with he. the recommended sodium benzoate dose is g twice a day; nevertheless, few patients can tolerate this dose because of its high gastrointestinal side effects [ ] . a recent study showed that tranilast could protect patients from thioacetamide-induced acute liver injury and alleviate he [ ] . endogenous bz analogues combine with the inhibitory neurotransmitter gaba receptor to depress the action on the cns, and is one of the occurring hallmarks of he pathogenesis. a large-scale clinical study on he cases showed that the improvement rate in brain function in treatment and control groups were % and %, respectively [ ] . the study showed that treatment of he with receptor antagonists such as fluorine marcie is feasible. a meta-analysis which included casecontrol studies of patients show that fluorine marcie can noticeably improve he, but didn't show any long-term benefits or improve patient survival rate. so fluorine marcie should only be considered for he patients who had used bz. although the reduction of dopamine neurotransmitter activity is also one of the pathogenesis, the application of bromocriptine, levodopa, has been unable to bring more benefits besides partly improving symptoms of patients. oral or intravenous infusion with a bcaa-based amino acid mixture can theoretically correct an imbalance in amino acid metabolism and control false neurotransmitter formation in the brain [ ] . a meta-analysis which included five studies showed that intravenous bcaa did not reduce the mortality rate of he. three studies with bcaa did not reduce the mortality rate of he; however, two larger studies (randomized controlled study about patients with liver cirrhosis in cases and cases, respectively) show that the application of bcaa not only reduced the occurrence of he and liver failure, but also improved the nutritional status, liver function and survival rate in patients. another study showed that bcaa could stimulate liver cell regeneration thus reducing the occurrence of liver failure. supplementation with a bcaa-rich amino acid mixture showed improved restoration of the patients' positive nitrogen balance, and increased the patient's susceptibility to protein food, improving cerebral perfusion [ ] . considerable progress has been made to understand treatment of he, studies of basic and clinical research are underway using newly discovered treatment strategies, such as toll-like receptor antagonists (with the ability to reduce systemic inflammation and oxidative stress) as well as non-steroidal anti-inflammatory drugs (ibuprofen) [ , ] , nmda antagonists, anticholinesterase. however, research using gene therapy should not be ignored [ ] . after tips, lola can significantly reduce the increase of venous ammonia concentration in patients with he [ ] . and the positive dietary intervention can significantly reduce the incidence of he [ ] . for patients with refractory he, embolization of pss is a safe and effective treatment strategy [ ] . improving liver function antiviral treatment with nucleos(t)ide analogues can reduce or eliminate liver inflammation and necrosis, promote the regeneration of liver cells, and help restore the functions of hepatic metabolism and detoxification in chronic liver failure caused by hepatitis b virus. artificial liver support systems can be divided into three types including nonbiological type, biological type and mixed type. the non-biological liver support system is the most widely used type, and consists of hemodialysis, hemofiltration, plasma exchange, blood perfusion, plasma adsorption, and the molecular adsorption recirculation system (mars) [ ] . the artificial liver support system can replace the partial function of the liver, remove the poison accumulated in the body, create conditions that allow for the regeneration of liver cells and provide enough time to wait for liver transplantation for patients with he. an artificial liver support system can be used to treat acute and chronic he, but patients with overt stage he should be especially careful with plasma exchange. liver transplantation remains the only promising therapy for patients with an acute liver failure or endstage liver disease. liver transplantation is an effective means for all kinds of persistent and severe he; however, in patients with he there is a significant increase in mortality among patients awaiting liver transplantation [ ] . recently, it has been reported that cognitive function was not fully recovered after liver transplantation in some patients with severe he [ ] . the key point in improving the prognosis of he is early recognition and timely treatment. active treatment should be given when diagnosing covert he. theoretically, for patients with serious pss, interventional therapy, surgery or permanently/temporarily and partially/totally blocking the pss can improve the patient's symptoms. the use of this therapy should be carefully weighed because it can increase the risk of gastrointestinal bleeding in case of portal hypertension. covert he has gained an increasing amount of attention in recent years. patients with covert he do not exhibit obvious signs and symptoms; however, their quality of life is reduced because of reduced operational ability or sleep disorders. without treatment, covert he will progress to overt he over time. the population with high risk should be examined and treated early, especially those engaged in potentially dangerous occupations. the following solutions can be referred to: (a) adjusting dietary structure (vegetable protein is the main intake); (b) oral administration of lactulose ( - ml, - times daily); (c) oral administration of rifaximin ( mg, twice a day); (d) oral administration of lola ( g, times a day); (e) oral administration of baaa; and (f) oral administration of probiotic preparations [ , , ]. although medical technology has made great progress and the research into he is also increasing in recent years, the pathogenesis of he is still unclear. due to a lack of specific methods, combination treatment is still the main therapy for he. it is generally believed that the onset of he may be a result of the synergistic effects of many factors. therefore, it is difficult to implement and draw convincing conclusions from randomized controlled trials with a single intervention targeting a specific pathogenesis and risk factor. ongoing issues remain, such as standardizing the research design of he treatment and evaluating the efficacy of he treatment more scientifically and objectively. some clinical studies may bring new hope for he treatment by new ongoing strategies of targeted systemic inflammation, oxidative stress, and neurosteroids. in addition, the key point in improving the prognosis of he is early recognition and timely treatment. active treatment should be given when diagnosing covert he. it is difficult to popularize the cognitive dysfunction detection methods of latent he. so the key and difficult point is to develop new method of assessments for clinicians in the future. jia shang hepatopulmonary syndrome (hps) is a syndrome of shortness of breath and hypoxemia induced by vasodilation in the lungs of patients with a variety of acute and chronic liver disease. essentially primary liver disease, pulmonary vasodilation and arterial oxygen lack of co-triad constituted. due to abnormal increase of vasodilators、ventilation/ blood flow disproportion and pulmonary hypertension caused by liver disease, the hypoxemia (pao < . kpa) ( mmhg) is included in hps. when fluckiger reported a -year-old syphilis female patient as early as in , he described cirrhosis, cyanosis and clubbing at the same time, while he was not aware of the intrinsic relationship between these clinical manifestations [ ] . in , snell reported decreased arterial oxygen saturation (sao , less than %) with abnormal hemoglobin in patients with liver parenchymal lesions and biliary obstruction, and years later, he proposed that such a phenomenon was associated with decreased affinity of o with hemoglobin. in , rydell and hoffbauer reported the detailed clinical diagnostic and treatment process of a -year-old male with "juvenile cirrhosis", and found multiple arterial-venous anastomoses in the lungs during autopsy, which he thought contributed to clinical cyanosis mainly. this provided a histological basis for the patient, and people conducted a large amount of studies thereafter. in , berthelot et al. injected opaque glue into the pulmonary vascular beds at the time of biopsy after the patient's death for the first time, and he found abnormal small arterial dilation in the lungs of the patient with cirrhosis, which he termed lung spider nevus. the term hepatopulmonary syndrome (hps) was first proposed by kennedy and knudson in [ ] . after nearly years of studies in a large number, people gradually developed a clear understanding of the mechanisms underlying its pathogenesis. in , eriksson used the term functional hepatopulmonary syndrome for the first time. in , the famous liver disease expert sherlock formally used this diagnostic term in his monograph hepatobiliary diseases, which has been recognized by many scholars [ ] . hps can occur in patients of any age groups, and various literature reports show conflicting incidences of hps in patients with cirrhotic portal hypertension, with the average incidence of various chronic liver diseases being about - %. the incidence of cirrhosis in patients is high, and - % of patients can additionally develop mild arterial hypoxia and - % develop arterial hypoxia. in the study by binay on indian cirrhotic populations arising from hepatitis b mainly, the incidence of this disease is relatively low ( . %) [ ] . the differences in incidence were mainly attributable to the different diagnostic criteria adopted. schenk et al. studied the incidence of hps by performing transthoracic contrast echocardiography (ttce), pulmonary function tests and blood gas analysis on patients with cirrhosis patients. the results showed that the incidence of hps patients in whom alveolar-arterial partial pressure of oxygen (aapo ) was used as an indicator of hypoxemia was significantly higher than those in whom arterial partial pressure of oxygen (pao ) was used [ ] . when arterial partial pressure of oxygen (pao ) was reduced to reflect hypoxemia, hps incidence was % when < mmhg and % when < mmhg, respectively. while when increase in alveolar-arterial partial pressure of oxygen (aapo ) was used to reflect hypoxemia, the incidence of hps was high, with % when > mmhg and % when > mmhg, respectively. hps is most common in cirrhosis due to various causes. pulmonary vascular abnormalities and arterial hypoxemia can occur in a variety of acute and chronic liver diseases, and this is true mainly when it comes to cirrhotic patients due to chronic liver diseases, especially cryptogenic liver cirrhosis, alcoholic cirrhosis, hepatitisinduced cirrhosis and primary biliary cirrhosis. besides, hps can also occur in chronic hepatitis, acute severe hepatitis, cholestasis, ɑ-anti-trypsin deficiency [ ] , tyrosinemia, wilson disease, and non-cirrhotic portal hypertension (such as idiopathic portal hypertension and schistosomiasis cirrhosis, etc.). arterial hypoxemia can also occur in extrahepatic portal vein occlusion. the observation of these patients suggests that portal hypertension may be the main factor for the pathogenesis of hps. hps can also occur in non-cirrhotic portal hypertension, and even cirrhosis-and portal hypertension-free chronic viral hepatitis. in , binay et al. found that patients with progressive liver failure with hyperdynamic circulation are most likely to suffer from hps, while they did not find the correlation with the severity of liver cirrhosis. hps is, in essence, hypoxemia due to anomaly in pulmonary vascular dilatation and arterial oxygenation when liver disease occurs. arterial hypoxemia occurs as the result of insufficient oxygenation by blood cells in the blood when blood flows through the lungs, or a proportion of blood fail to flow through the alveoli [ ] . since primary heart and lung diseases have been excluded when hps occurs, the abnormal pathways that red cells may pass through include: ( ) passing through the pleural and hilar bronchial vessels while not reaching the alveoli; ( ) blood flows directly into the pulmonary veins due to the high pressure portal system in the mediastinum, thereby bypassing the pulmonary circulation; ( ) flowing directly into the pulmonary veins through the expanded alveolar capillaries or the pulmonary-venous fistula. alveolar telangiectasia may be more important to the formation of hypoxemia, and existing study data show that the development of hps is at least associated with the systemic hyperdynamic state, portal hypertension, hepatic encephalopathy, hepatorenal syndrome and pulmonary hypertension [ ] . therefore, it is believed that the main causes of hps are systemic metabolism and hemodynamic disorders, and that it is involved in the formation of systemic metabolism and hemodynamic disorders, which is of important pathophysiological significance. . the basic pathological change of hps is pulmonary vascular dilatation, which is manifested as: (a) dilation of anterior capillaries in a large number. (b) formation and opening of the pulmonary basilar arterial -venous communicating branches. (c) formation of pleural "spider mole", which is mainly manifested as dilation of anterior capillaries. in autopsies, it was found that the basic pathological changes in patients with liver cirrhosis and other chronic liver diseases were extensive pulmonary vascular dilatation and arteriovenous communicating branches. some people found the pathological changes through vascular shaping, with pleural vasodilation at the basal aspect of the lungs or the formation of subpleural spider nevus. domestic professor gu changhai summarized these pathologic changes in as arterial dilation within the pulmonary acinus in a pattern of inhomogeneous distribution, thin-walled blood vessels, - μm in diameter, in the lower lobes of the whole lungs, extensive dilation of pulmonary vascular beds adjacent to the alveolar gas at the anterior capillary level, and significantly expanded pulmonary artery branches and pulmonary capillaries up to μm in diameter. electron microscopy showed thickened pulmonary capillaries, pulmonary arterial walls and the basal layers of small veins. . factors that affect the dilation of blood vessels: the mechanisms underlying pulmonary vascular dilatation have not yet fully elucidated, and the possible influencing factors include: (a) increased activity of vascular dilators various acute and chronic liver diseases, liver cell failure and metabolic disorders, particularly reduced inactivation of vasoactive substances which can enter directly into the systemic circulation through abnormal anastomotic collateral vessels, result in disorder of the systemic hemodynamics and increased contents of vasodilators in the blood circulation. just as visceral congestion in patients with portal hypertension, they can act on the intrapulmonary vessels, causing pulmonary vascular dilatation and pulmonary congestion. substances that cause vasodilation include glucagon, prostaglandin, vasoactive intestinal peptide, nitric oxide, angiotensin, bradykinin and endotoxin, etc. (b) reduced vasoconstrictors or decreased sensitivity of intrapulmonary vascular beds to the endogenous vasoconstrictors, such as norepinephrine, endothelin, atrial natriuretic peptide, vasopressin, serotonin and tyrosine, etc. the contents of the substances are not absolutely reduced because maybe their sensitivity is reduced. when chronic liver disease occurs, the anterior communicating branches of the originally closed non-functional capillaries may be opened, and a disorder occurs in the hypoxic pulmonary vascular systolic dysfunction which should have been normal, and it is only % of the normal state [ ] . (c) neurological factors cirrhotic patients show sympathetic nerve hyperactivity, but after the formation of portal hypertension, their sympathetic nerve function may be damaged, which play an important role. animals with portal hypertension often show abnormal pressure responses and reduced sensitivity of blood vessels to norepinephrine, resulting in increased cardiac output, and dilated pulmonary vascular volumes. besides, hemodynamics within the lungs is also a manifestation of the body's hyperdynamics. (d) decreased reactivity of intrapulmonary blood vessels to hypoxia recent inert gas dispersion tests show that cirrhotic patients with over two spider nevus are manifested as not only liver damage, but also decreased systemic intrapulmonary vascular resistance, decreased reactivity of blood vessels to hypoxia and dilated pulmonary vessels. however, it was also found using pulmonary angiography that in spite of the dilated vessels at the ending of arteries, the responses of vessels to oxygen were almost normal, which did not support this view. (e) intrahepatic angiogenesis or dysplasia may also be one of the factors for the formation of hps. to date, the mechanisms underlying pulmonary vascular dilatation caused by hps is still unclear. however, long-term administration of intrapulmonary vasoactive substances can cause significantly increased intracellular cyclic adenosine monophosphate (camp) and/or cyclic guanosine monophosphate (cgmp), resulting in hypoxic pulmonary vasomotor dysfunction and pulmonary artery dilatation, which may be an important cause of this disease and also pulmonary manifestations of systemic hyperdynamic circulation. due to the significant dilation of the pulmonary capillaries and the anterior capillaries, some of the blood around the capillaries in contact with the alveoli can still undergo exchanges with gases, while the central blood, due to the increased diffusion distance from the alveoli, leads to insufficient gas exchange, resulting in insufficient arterial oxygenation and thus a series of hypoxemic manifestations. to date, the pathogenesis underlying the pathogenesis of hps has not yet been elucidated. in view of the above pathophysiological changes and current studies, it is believed that the disease may be caused by insufficient ventilation, diffusion disorder, ventilation/blood flow imbalance and decreased oxygenated hemoglobin affinity, or the above factors in combination. under normal circumstances, insufficient ventilation due to a variety of reasons causes insufficient oxygen inhaled into the alveoli and reduced blood oxygen exchanges, which can result in hypoxemia [ ] , such as chronic bronchitis, foreign bodies in trachea, atelectasis and respiratory muscular paralysis, etc. and the presence of insufficient ventilation in patients with chronic liver disease and cirrhosis or not is still controversial. in , fujiwara studied the lung function in patients with decompensated liver cirrhosis and reported that vital capacity (vc), functional residual capacity (frc) and respiratory reserve volume (evr) in the patients were significantly reduced, that r/t was mildly increased, and that there was no changes in s forced expiratory volume (fev ). therefore, it was believed that mechanical compression and insufficient ventilation due to pulmonary interstitial edema in patients with liver cirrhosis was the main reason for impaired lung function. subsequently, edison et al. studied the pulmonary function of patients with decompensated liver cirrhosis, and found that their vc, maximum ventilation volume (mvv), frc, total lung volume, and r/t were significantly reduced, and they believed that patients with cirrhosis had obvious obstructive and limited insufficient ventilation, which were mainly caused by compression of lung tissue due to increased abdominal pressure, elevated diaphragm and increased chest volume and pressure when patients had additional ascites, and atelectasis [ ] . however, decreased fev resulted from compression of small trachea due to pulmonary interstitial edema and vasodilation, and early closure of expiration. theoretically, all of the above factors can lead to insufficient ventilation, one of the factors resulting in this disease. this was also substantiated by significantly increased arterial partial pressure of oxygen and decreased co partial pressure in cirrhotic patients with pleural effusion after pleural effusion extraction and recovery from atelectasis. however, there are also some people who do not think that hypoxemia results from insufficient ventilation, but because cirrhotic patients are not complicated by high concentrations of co when their arterial partial pressure of oxygen is decreased [ ] . this is likely because when patients have hypoxemia, compensation of hyperventilation causes arterial blood co partial pressure not to increase, and results in decreased paco or even respiratory alkalosis. besides, in some patients without decompensated liver cirrhosis, arterial hypoxemia can also occur. even it has been found that the lung function tests in patients with decompensated liver cirrhosis are normal. therefore, the majority of scholars currently believe that insufficient ventilation is not the main cause of hypoxemia in cirrhotic patients. for patients with hps, the inert gas exclusion technique should be performed to prove that there is a disorder in the diffusion of oxygen, which is determined by the basic pathological changes of hps -pulmonary vasodilatation. pulmonary angiography can show small spider-like to obviously cavernous diffuse vasodilation within the lungs. due to the significant dilation of the pulmonary capillaries and the anterior capillaries, the diffusion distance of the blood flow in central blood vessels and the alveoli is increased, preventing the gases in the alveoli from entering the pulmonary capillaries, thereby affecting the gas exchanges. studies have shown that hypoxemia often occurs in patients with cirrhosis or aggravates during exercises, and it is believed that diffusion disorder or limitation of oxygen occurs in patients. in fact, factors that affect o diffusion do occur in patients with cirrhosis, but they are still not sufficient to explain the apparent hypoxemia. although vascular dilatation occurs at arterial endings in patients with hps, their arterial partial pressure of oxygen can be reduced while inhaling air and increased when they are given oxygen inhalation, which further proves that although diffusion disorder does exist and it plays a role in the formation of this disease, the role is not important. to engage in gas exchanges is the most important biological function of lung tissues, and this gas exchange must be completed when there is an appropriate ventilation/blood flow ratio. under the normal circumstance (normal adult resting state), the most appropriate ventilation/blood flow ratio physiologically is . . changes in the ratio due to any cause can affect the gas exchange, and the imbalanced ventilation/blood flow ratio in hps patients with hypoxemia is mainly because of pulmonary vascular dilatation and arteriovenous shunt [ ] . . intravascular vascular dilatation: pulmonary vascular dilatation has been confirmed pathologically and by angiography. dilated blood vessels in the lungs leads to gas diffusion disorder. besides, since the oxygen molecules in the air can not be diffused to the central dilated blood for the gas exchange, causing decreased ventilation/blood flow ratio and pulmonary arterial partial pressure of oxygen. this decreased ventilation/blood flow, together with increased amount of reactive cardiac output, shortens the duration of blood that flows through the capillary network and insufficient oxygenation [ ] . excessive ventilation can in part enhance patients' pao . if the alveolar oxygen partial pressure is increased at this time, some oxygen molecules can reach the central areas of dilated blood vessels, increasing the arterial partial pressure of oxygen. therefore, it is called the diffusion -perfusion disorder or pulmonary arteriovenous functional shunt rather than the true lung shunt. . arterial -venous shunt: intrapulmonary vascular fistula and pleural spider nevus can occur in cirrhosis of the liver and allow the pulmonary arterial blood to circumvent gas exchange and directly flow into the pulmonary vein so that patients may develop hypoxemia. this hypoxemia can not be corrected by oxygen inhalation and represents the true pulmonary shunt, which has been confirmed by pulmonary histopathology, angiography, transthoracic echocardiography and other examinations. it is now believed that pulmonary vascular casting is still the most direct evidence for determining the arterialvenous shunt. this intrapulmonary arterial -venous shunt is the main cause of abnormal ventilation/blood flow ratio and insufficient gas exchange. although pleural spider nevus can also cause arterial -venous shunt, it generally does not suffice to cause significant hypoxemia due to the small amount of shunt. in addition, studies in recent years also show that portal-pulmonary vein shunt in a small amount occurs in some patients with cirrhosis, in whom blood flow circumvents the alveolar gas exchange and enters directly the systemic circulation. this can also cause ventilation/blood flow abnormalities, causing insufficient gas exchange . airway closure: in , ruff et al. proved that cirrhotic patients had significantly increased closed volume (cv) and total amount of closed gas (cc) and increased gases trapped in the lower fields of the lungs, resulting in an extremely low ratio of ventilation/blood flow, and they believed these were due to reduced airway ventilation [ , ] . in , furukawa et al. measured the lung function of patients with liver cirrhosis and did not find abnormalities; however, most patients had flow -volume abnormalities and significantly increased cv, suggesting the closed the airway in advance and decreased ratio of ventilation/blood flow, which might important causes of hypoxemia. . decreased affinity of oxygen with hemoglobin: some reports showed that patients with cirrhosis (mostly alcoholic cirrhosis) patients had mild systemic vascular or pulmonary vascular dilatation, normal pao , mild hypocapnia, mild right shift of the oxygenated hemoglobin dissociation curve, normal amount of carbon monoxide diffusion [ ] , and mild imbalance of the ventilation/pao blood flow, indicating that the right shift of the oxygen dissociation curve due to decreased affinity of oxygen with hemoglobin in the patients. this was possibly caused by the increased concentration of , -diphosphate glyceride in red blood cells, which, however, is not an important factor in the occurrence of hypoxemia [ ] . in summary, hypoxemia can result from many factors, while none of the factors can completely explain the pathogenesis underlying the disease. since the basic pathological changes in patients with hps are intrapulmonary vascular dilatation and opening of arterial -venous communicating branches, together with recent findings, it is suggested that the diffusion disorder of the alveoli and pulmonary capillaries and ventilation/blood flow imbalance may coexist, and are the main cause of hypoxemia in this disease. other factors may aggravate hypoxia and are secondary factors. therefore, it is believed that the disease occurs as result of the above factors. the pathological features of hps are dilation of capillaries in the anterior aspect of the lungs and telangiectasia. autopsies show arterial-venous short circuit within the lungs, vasodilation and thickened pulmonary muscles [ ] . at the same time, arterial hypoxemia is common in liver diseases, often attributable to a variety of factors (such as ascites, hepatic pleural effusion, and copd in patients with alcoholism); it shows unique pathophysiological characteristics under specific circumstances of hps. its prominent features are dilation of micro-arteries in the anterior aspect of pulmonary capillaries and true capillaries (the normal diameter of these vessels is to μm, which can reach - μm when patients rest), with the number of dilated vessels increased macroscopically. some patients show arterial-venous communicating between the pleura and lungs, vascular anastomosis in the liver and the lungs, and thickened walls of small veins and capillaries. pulmonary vascular dilatation is promoted, and mixed venous blood quickly or directly enter the pulmonary veins through the anastomosis in the lungs, leading to oxygenation defects. increased nitric oxide (no) is a key cause for pulmonary vasodilation, and whether other mediators, such as heme oxygenase-derived carbon monoxide, are causes of pulmonary vasodilatation are not yet confirmed. abnormal arterial oxygenation seriously affects the survival of patients, and is an important indicator that determines the timing and risk of liver transplantation as well as an important basis for the grading of severity of hps. causes of deaths associated with hps are often multifactorial and are associated with basic liver diseases, and there are few cases of respiratory failure due to severe hypoxemia. hps is a triad composed by intrapulmonary vascular dilatation and insufficient arterial oxygenation due to primary liver disease, and it is mainly clinically manifested as primary liver disease and pulmonary lesions. hps can occur in various liver diseases, mostly in chronic liver disease, especially cirrhosis caused by various causes, such as cryptogenic cirrhosis, alcoholic cirrhosis, liver cirrhosis, viral cirrhosis, postnecrotic cirrhosis and biliary liver cirrhosis, etc. the most common clinical manifestations include liver palms [ ] , spider nevus, jaundice, ascites, hepatosplenomegaly, gastrointestinal bleeding and abnormal liver function, etc., while they are not significantly correlated with hps. some patients with clinically stable liver disease may also develop the clinical manifestation of progressive pulmonary insufficiency. since hps patients have no primary cardiopulmonary diseases, most ( - %) patients gradually develop respiratory manifestations on the basis of various liver diseases, such as cyanosis, dyspnea, clubbing, orthodeoxidation and platypnea, etc. among them, progressive dyspnea is the most common lung symptoms of hps. binay et al. believed that cyanosis was the only reliable clinical sign, and that platypnea and orthostatic hypoxia are the most characteristic manifestations. pulmonary examinations generally show no obvious positive signs [ ] . a small number of patients (about - %) can present complaining dyspnea on exertion in the absence of clinical manifestations of a variety of liver diseases, to which attention should be paid clinically so as to prevent misdiagnosis. the domestic researchers gao zhi et al. reported that patients presented to hospitals with cyanosis, palpitation after exercises and shortness of breath; meanwhile, they found that the patients had clinical manifestations of liver cirrhosis [ ] (such as liver palms, spider nevus, hepatosplenomegaly and ascites), which were conducive to the diagnosis of this disease. if liver disease patients have other lung diseases (such as chronic bronchitis, emphysema and pneumonia, and pleural effusion, etc.), then significant respiratory symptoms may occur. therefore, differential diagnosis should be made. data show that the duration of initial dyspnea to the conformed diagnosis in patients with hps average - years; that is to say, about % of patients already have dyspnea at the time confirmed diagnosis. . orthodeoxidation: pao is decreased by > % when patients switch from the supine position to the standing position. . platypnea: when patients switch from the supine position to the standing position, they have palpation, chest tightness and shortness of breath, and when patients resume the supine position, the above symptoms are improved [ ] . krowka reported that about - % of patients with hps had the above two manifestations because vascular dilatation in the patients was mainly distributed in the middle and low lung fields. when patients switch from the supine position to the standing position, the blood flow in the middle and lower lobes of the lungs is increased under the action of gravity, aggravating hypoxemia [ ] . although the two manifestations are not unique to hps, they suggest the significant abnormality in the patients' pulmonary vascular system. if patients with a variety of liver diseases present with the above two manifestations, further examinations are needed for confirmation. patients may present with liver palms, hepatosplenomegaly, spider nevus and ascites; patients show palpitation, chest tightness, shortness of breath when switching from the supine position to the standing position due to hypoxemia. schenk et al. defined the values of pao for the diagnosis of hps, thinking that pao < mmhg suggested a high possibility of hps, and that for pao < mmhg, the diagnosis of hps could be made [ ] . pulmonary function tests mainly showed significantly decreased vc, frc, mvv and fev , but sometimes the total lung volume and fev were normal. chest x-ray, ct scan and transthoracic contrast echocardiography (ttce) hps patients are mostly normal on chest radiography or show diffuse small millet shadows predominantly in both lower lobes of the lungs, nodular shadows in both lower lung interstitium, dilated pulmonary arterial trunks, and thickened pulmonary markings, whereas these manifestations have no specific values to the diagnosis of this disease. ct scan shows certain diagnostic values in that it demonstrates distal vasodilation and even pleural blood vessels, and can suggest the presence of hps. arteriovenous communicating occurs in hps patients due to pulmonary vascular dilation, indicating that subclinical pulmonary vascular dilatation and abnormal gas exchanges occur in cirrhotic patients with normal pao [ ] . contrast echocardiography: when hps is suspected, transthoracic echocardiography can be used as a preliminary screening to determine whether the intrapulmonary vascular dilation occurs or not. the microbubble contrast material in the right atrium, after intravenous injection of dioxane isotonic saline, will develop images in the left atrium through the dilated vascular beds after - cardiac cycles, while the microbubbles cannot pass through the normal capillaries (normal capillaries are < - μm in diameter). approximately % of patients with cirrhosis had positive changes on contrast echocardiography, while only a small proportion of patients are in line with the diagnosis of hps due to the influence of dilated blood vessels. if contrast echocardiography is positive for liver cirrhosis or portal hypertension patients with hypoxemia and cardiopulmonary diseases in them can be ruled out, then the diagnosis of hps is established. this means is used to confirm the diagnosis of intrapulmonary vasodilatation. the pulmonary vascular abnormalities in hps patients are as follows: ( ) diffuse spider nevus images. patients of this type have severe hypoxemia and erectile hypoxia and respond well to inhalation of % oxygen; ( ) cavernous or spotted arterial dilatation mainly seen in the basal aspect of the lungs. patients during this period respond poorly to % oxygen; and ( ) intermittent local arterial malformation or communicating branches, isolated earthworm-like or bulk images. in addition to severe hypoxemia and erect hypoxia, patients of this this type respond extremely poorly to the inhalation of % oxygen. when hypoxemia is caused by hps and cardiopulmonary diseases concurrently, cm tc-maa scan can determine hypoxemia resulting from hps more likely. radiolabeled albumin cm tc, which is administered through intravenous injection, is about pm in diameter. when pulmonary vascular shunt occurs, a proportion of the polymerized albumin passes through the lungs and enters the systemic circulation, the intake of albumin by other organs can be simultaneously determined by scintigraphy [ ] . therefore, the amount of shunt can be calculated. a study showed that cm tc-maa scan was positive for hps patients with pa < mm hg, while the scan was negative for chronic obstructive pulmonary disease (copd) patients with the same degree of hypoxemia, a result indicative of the good specificity of this means. compared with contrast echocardiography, cm tc-maa scan, in spite of its low sensitivity, can be used for the diagnosis of hps in patients with copd. pathological examinations are the most reliable means for the diagnosis of hps, whose basic pathological change is pulmonary vasodilation manifested as diffuse anterior capillary dilation or discontinuous formation of arteriovenous branches [ ] . in addition, pulmonary perfusion scan and right cardiac catheterization are also valuable for the diagnosis of hps to a certain extent. there is no unified standard for hps diagnosis to date. diagnosis should be based on clinical manifestations plus imaging evidence of pulmonary angiography. (c) the domestic scholars gao zhi et al. thought in that the diagnosis of this disease should be based on the following manifestations in patients, hepatosplenomegaly, ascites, liver palms, spider nevus, dyspnea on exertion, hypoxia while breathing in the supine and orthostatic positions, increased mesenchyma in the basal aspects of the lungs and vascular markings on chest radiography, patchy or nodular shadows, dilated basilar pulmonary vessels and increased pulmonary vascular branches on ct, severe hypoxemia or not on blood gas analysis, increase in alveolar -arterial oxygen gradient by ≥ kpa ( mmhg), and % diffusion disorder on pulmonary function tests [ ] . in addition, shunt-related examinations should be performed, such as cm tc-maa scanning, contrast-enhanced two-dimensional echocardiography and pulmonary angiography, etc., while the last one does not show the same sensitivity as that of the former two because the small blood vessels in the lungs may not develop on angiography. most hps patients have a slow onset of the disease, are difficult to treat, and have a poor long-term prognosis, with a mortality of more than % after years. therefore, early diagnosis of the disease and its differential diagnosis are vital to improving the prognosis of patients. first of all, the previous liver and lung diseases in the patients should be ruled out, such as chronic obstructive pulmonary emphysema, pulmonary infection, interstitial pneumonia and silicosis, etc. at the same time, cirrhosis with pulmonary hypertension, infections secondary to pleural effusion, interstitial pulmonary edema, atelectasis and hyperventilation syndrome, etc. need to be ruled out. hps should be differentiated mainly from the following diseases: . liver cirrhosis following pulmonary heart disease: this is mainly because pulmonary diseases result in cardiac insufficiency and thus increased pulmonary venous pressure. repeated or long-term existence of liver congestion can lead to central venous hypertrophy and lobular central connective tissue hyperplasia, and further progression of the lesion will lead to the formation of liver cirrhosis following pulmonary heart disease. patients with pulmonary cirrhosis often have a long history of chronic lung disease and signs of cardiac insufficiency, such as edema of lower extremities, palpitation, shortness of breath and other symptoms. this patient has no history of chronic lung disease or edema of lower extremities, making him inconsistent with liver cirrhosis following pulmonary heart disease. . left heart insufficiency: both hps and left heart insufficiency can cause severe dyspnea and hypoxemia. a history of liver disease or evidence of chronic liver damage and decreased po can be found in patients with hps, especially such characteristics as orthostatic hypoxemia and intrapulmonary vascular dilatation. patients with left heart insufficiency have a history of heart disease, orthopnoea, pink frothy sputum and moist rales in the lungs, etc. this patient is inconsistent with such manifestations. . primary pulmonary hypertension: after inhalation of pure oxygen, hypoxemia in most of patients with hps will be significantly alleviated. the effects of oxygen are poor in patients with hypoxemia [ ] , and hps is manifested as orthostatic hypoxemia. the characteristics of hemodynamics of patients with hps are hyperdynamics and normal or decreased pulmonary artery pressure and pulmonary vascular resistance, while those in hypoxemic patients are increased. . others: ductus arteriosus, eisenmenger syndrome and pulmonary embolism, etc., need to be differentiated from this disease, and comprehensive judgments should be provided based on other clinical data of the medical history. hps patients can also have the above-mentioned diseases, and careful and meticulous examinations are needed for differentiation. because hps is developed on the basis of original liver disease, the frequency of its occurrence and its severity are mostly associated with the liver cell function of patients, while there are also hps patients in whom chronic liver disease is relatively stable and liver functions are normal. besides, pleural effusion, ascites and infections secondary to pulmonary edema after liver function decompensation can aggravate patients' respiratory function injury. therefore, under the current circumstances in which there are no effective measures for hps, active and effective treatment of primary liver diseases is the basis for the treatment of hps. therapy of primary diseases, including correction of hypoproteinemia, elimination of pleural effusion, improvement of liver function and treatment of complications, etc., can improve tissue oxygenation and improve arterial oxygen saturation. on this basis, the following treatment can be given. oxygen therapy also helps the differential diagnosis of pulmonary shunt: if pao is resumed after oxygen inhalation, then the diagnosis of intrapulmonary vascular dilatation (ipvd) can be made; for patients with partial improvement, pulmonary anatomical shunt and functional shunt may coexist; for patients in whom the oxygen therapy proves inefficacious, pulmonary arteriovenous fistula is a possible diagnosis. it is now believed that once the diagnosis is established, treatment should be given as soon as possible. in the early stage of correcting hypoxemia in patients with mild conditions, even in patients in whom the critical value of hypoxemia (pao , - kpa ( - . mmhg) is reached and who have ascites, the hemoglobin saturation may still be less than % when patients are in activities or even sleep. that is to say, nasal catheter oxygen inhalation at - l/min is needed so as to improve hypoxemia [ ] . with the development of the disease, oxygen flow needs to be gradually increased, and intratrachea oxygen supply can be offered when necessary. during the late stage, patients can receive pressurized oxygen through a ventilator or a hyperbaric oxygen chamber. for patients whose conditions are severe, the efficacy of oxygen therapy alone is not obvious. . vasoactive drugs vasoactive drugs for the treatment of patients with hps are most studied; however, since its pathogenesis has not been clarified to date and primary liver disease is difficult to reverse, it is hard to define the clinical efficacy of these drugs. the commonly used drugs include: used aerosolized ephedrine hydrochloride for the treatment of patients with hps, and the preliminary efficacy was significant. the mechanisms were that ephedrine could excite the pulmonary vascular α receptor, resulting in contracted bronchial mucosa and pulmonary capillaries and alleviated bronchial edema, so that the dilated blood vessels within the lungs were contracted and intrapulmonary shunt was reduced. meanwhile, the bronchial β receptors were excited and the bronchi were dilated so as to improve the ventilation/blood flow ratio and relieve hypoxia. further studies are merited. (f) others: there have been reports on sympathomimetic drugs (isoproterenol) and β-blockers (propranolol), etc. that improve the symptoms of hps. theoretically, vascular endothelin, estrogen suppressor (tamoxifen) and so on can relieve the spider nevus and pulmonary vascular dilatation in patients with liver cirrhosis and improve their respiratory symptoms, while further studies are needed. no is most studied currently, and there are reports indicating that no synthesis inhibitors can increase pulmonary vascular resistance. alexander et al. used no for the treatment of severe hypoxemia in patients after liver transplantation, and obtained good results. durand et al. also reported that an hps patient was cured by inhaling no, while its mechanisms and clinical efficacy needed to be further confirmed. . pulmonary embolism it is generally considered that pulmonary vascular dilatation can vanish after liver transplantation in hps patients who are normal on pulmonary angiography or who have cavernous vessels on imaging [ ] ; for patients who show diffuse pulmonary vascular dilation features on pulmonary angiography, embolization is usually not adopted since patients' lesions are extensive and the efficacy is poor; for patients with isolated and severe pulmonary vascular dilation or arterial-venous communicating branches, local pulmonary embolism therapy can yield a satisfactory effect. . liver transplantation it is currently considered that liver transplantation is still a possible fundamental measure for the treatment for hps. in the past, it was believed that serious hypoxemia was an absolute contraindication against liver transplantation, while recent studies show that liver transplantation is preferred for patients who have good alveolar gas diffusion function, who can respond well to pure oxygen inhalation and who can undergone oxygenation safely during anesthesia. recent reports further prove that hypoxemia can be cured after liver transplantation [ ] . through literature review and case reports, krowka et al. believed that progressive hypoxemia in hps could be used as an indication of liver transplantation. temporary hypoxemia following liver transplantation can be adjusted by using no and taking the head-down supine position and the alternate lateral decubitus position. and for hps patients who fail to respond to the inhalation of pure oxygen, who have direct pulmonary arterial communicating branches on pulmonary angiography and who have severe clinical hypoxia, liver transplantation cannot improve their hypoxic status, has limited efficacy, or even increases intraoperative and postoperative risks. therefore, liver transplantation should not be performed on them. tips was an effective method for the treatment of hps, and its effects of improving symptoms, enhancing oxygenation and reducing intrapulmonary shunt could last up to months. riegler et al. performed tips on an hps patient with diffuse intravascular dilatation who was not suitable for vascular embolization, and the results showed significantly increased pao and significantly improved hypoxemia. however, coley et al. also reported that a patient failed to respond to tips, and therefore, its exact effects remain to be studied. . other treatment options one patient with hps was once treated with garlic, and months later, his oxygenation was significantly improved and his symptoms were relieved. there are also patients who receive plasma replacement therapy, which has limited effects on the oxygenation of patients with hps [ ] . to sum up, no effective treatment options are currently available for hps. since the basic cause of hps is liver cell failure, the usual cause of patients' deaths is not lung failure, mostly complications such as gastrointestinal bleeding, renal failure, hepatic encephalopathy and sepsis. therefore, we consider that the therapy of primary liver disease is particularly important. oxygen inhalation alone can be given in the early stage of hypoxemia, or conservative treatment can be provided if additional drugs are effective. liver transplantation is the best solution whenever possible. it is generally accepted that liver transplantation is the most promising regimen with confirmed efficacy. if oxygen inhalation is less satisfactory and patients are diagnosed with local intrapulmonary vascular dilatation or arterial -venous fistula by such means as pulmonary angiography, pulmonary embolism should be carried out as soon as possible. for patients with additional obvious portal hypertension, tips treatment can also be given. the interval from chronic liver disease and cirrhosis in patients to the confirmed hps due to such respiratory symptoms as anoxic dyspnea is usually several years or even more than years [average interval, ( . ± . ) years], and a small number of patients can develop such a disease acutely in the short term. besides, signs of chronic liver disease can be traced in patients complaining breathing difficulties. once hps is established, obvious hypoxemia has occurred already. it confers a poor prognosis, and most patients die within - years often due to other complications of liver disease [ , ] . if patients' oxygenation is satisfactory and they have undergone liver transplantation, or with the improvement in liver function, their hypoxemia can be resolved or improved of its own volition with good prognosis. if patients' oxygenation deteriorates severely and they have a very poor prognosis, most of them will die in the short term. hps often progresses slowly. although it is not a direct cause of death in patients with cirrhosis, it can significantly aggravate the disease. therefore, cirrhotic patients, especially those with positive liver palms and spider nevus as well as patients with portal hypertension, should be careful of the possibility of hps. timely detection and symptomatic treatment (such as low flow oxygen inhalation) can improve the prognosis of patients. active and effective treatment of primary liver disease forms the basis for the prevention of this disease. education of common sense should be given to patients with liver diseases so as to avoid factors inducing hps in their life. for patients with liver disease, mild hps should be found as soon as possible and appropriate treatment should be given. jia-quan huang and dong xu lipopolysaccharide (lps) is a constituent of bacteria cell wall which plays an essential role in the pathogenesis of septic shock by generating endogenous mediators such as nitrous oxide, cytokines, superoxide anions, and lipid mediators. despite the recent advances in antibiotic treatment and hemodynamic monitoring, septic shock still remains a serious disorder that is associated with a high mortality rate, to more comprehensive definition of the mechanisms that underlie innate immunity against bacterial pathogens, lps has been extensively studied [ ] . the pathophysiological consequences of bacterial sepsis are contributed by the dysregulation of these same mechanisms. before we can hope to design effective anti-sepsis therapies, greater insight into the nature of host interactions with lps is extremely essential. the gram-negative bacterial envelope is composed of two bacterial membranes, outer and inner membrane. the outer membrane consists of the following substances, like lipopolysaccharide (lps), several kinds of outer membrane proteins, lipid a and metal ions. for most gram-negative bacteria, lps is a major component in the outer monolayer of the outer membrane which works like a tight shield. the shield is composed by unique molecules, such as polysaccharide, or long chain of sugar, and lipid a. during the process to evoke the signaling events of lps, lipid a plays a pivotal role. the entire lipid component of lps molecule, however, is required for optimal activity [ ] . the basic principles of lps bioactivity are nowadays well understood. endotoxins do not elicit their toxic effects -as we might suspect and as it is known for many proteinous exotoxins which can kill host cells or inhibit cellular functions. rather, lps requires the active response of host cells. according to present knowledge we get, lps interacts with various host cell types through lipid a, those cells include mononuclear cells, thrombocytes, endothelial and smooth muscle cells, and polymorphonuclear granulocytes, among which macrophages/monocytes are of particular importance. through the lps-induced activation, macrophages produce many substances, like bioactive lipids, reactive oxygen species, and in particular, cytokines such as tumor necrosis factor a (tnf), interleukin- , il- , il- , and il- . it appears that when low levels of mediators are produced, beneficial effects (e.g., induction of resistance to infection, adjuvant activity) are elicited and when high levels of mediators reach the circulation that detrimental effects (e.g., high fever, hypotension, irreversible shock) are induced. however, when the host organism is in a hyperreactive state lps, low mediator concentrations may also become harmful. the hyperreactivity to endotoxin may be caused by exo-toxins, chronic infection, and by growing tumors, and interferon-γ. to function properly, organism requires an immune system that must detect pathogens, from viruses to parasitic worms, and distinguish them from the organism's own healthy tissue. the immune system can be classified into humoral immunity versus cell-mediated immunity or the innate immune system versus the adaptive immune system. when microbes invade organism, the innate response is usually triggered by pattern recognition receptors, which recognize components that are conserved among broad groups of microorganisms, or when injured, damaged, or stressed cells send out alarm signals, many of which (but not all) are recognized by the same receptors as those that recognize pathogens. the innate immune system defenses are nonspecific, meaning that the system responds to pathogens in a generic way. this system does not confer long-lasting immunity against a pathogen. in most organisms, the innate immune system is the dominant system of host defense [ ] . they activate innate immune responses by identifying some conserved non-self molecules, so as to protect the host from infection,. bacterial lipopolysaccharide (lps), an endotoxin which is found on the bacterial cell membrane, is considered to be the prototypical pamp. lps is specifically recognized by toll-like receptor (tlr) , a recognition receptor of the innate immune system. the interaction of the lipid a moiety of lps with macrophages appears to be especially important because subsequent cellular activation results in the release of systemically active pro-inflammatory molecules, which in turn mediate systemic toxicity. lps has extreme potential in macrophages activated at concentrations of lps as low as pg/ml [ ] . host-defense peptides (hdps) could be a possible alternative solution since they possess the antimicrobial, antiseptic, and immunomodulatory properties [ ] . endotoxins lipopolysaccharide is released not only from dead gram-negative bacterial, but also from the growing ones. endotoxins are very stable molecules, which are resisted to extreme temperatures and ph values in comparison to proteins. endotoxins are shed largely during cell death as well as growth and division. they are highly heat-stable and are not destroyed under regular sterilizing conditions. endotoxin can be inactivated through exposed at temperature of ° c for more than min or ° c for more than h. acids or alkalis of at least . m strength can also be used to destroy endotoxin in laboratory scale [ ] . gut microbiota is composed of strict anaerobes, facultative anaerobes and aerobes. recent reports suggest the existence of over , bacterial species in the human gut microbiota. an important characteristic of gut microbes is their heterogeneity [ ] . the composition and the frequency of the microbiome changes with the different segments of the elementary tract. the composition is influenced by the environment, consumed diet and host factors. endotoxin to surrounding tissues and organs or blood shift that shift pathway include: ( ) via the portal vein, liver into the systemic circulation; ( ) through the intestinal tract into the lymphatic system lymphatic; ( ) through the intestinal wall into the peritoneal cavity and then absorbed into the bloodstream. under physiological conditions, although a small amount of toxins continued to parenteral shifted via the portal vein into the liver, but it does not cause endotoxemia; mild in gram-negative bacilli infections, although bacteria continue to release to the tissue or blood endotoxin, but it does not give rise to a strong inflammatory response, the above are dependent on the presence of an effective mechanism within the body to remove toxins and detoxification. the liver is the main organ of clearance of endotoxin, and the spleen, also removes toxins. molecules in lps removed include cationic antimicrobial peptides (cationic antimicrobial peptides, cap), acyloxy acyl hydrolase (acyloxyacyl hydrolase, aoah) lipoprotein binding protein and anti-lps antibodies are important endotoxin clearance methods [ , ] . liver blood endotoxin clearance, primarily through kupffer cells, hepatocytes internal toxin endocytosis achieved, but the specific metabolic process is not entirely clear. mediated by kupffer cells engulf toxins may be scavenger receptor, it may be a molecular weight of , and , protein; mediate phagocytosis liver toxin structure may be the lectin-like receptor (lectin-like receptor). endotoxin receptor hepatocyte sinusoidal plasma membrane on the surface: after one to one binding, is taken up within the liver cells endocytosis way to microtubule-dependent vesicular transport through the liver cells, transported to the liver cells bile duct surface to exocytosis into the bile duct, and then discharged into the biliary system through the intestine [ ] . splenic macrophages containing approximately % of the body to settle within the organization, and macrophages are important endotoxin removal cells. when endotoxin intravenously into the body, except gathering in the liver, a lot of endotoxin can be quickly gathered and taken up into macrophages in the spleen, the spleen and therefore equally important endotoxin removal organs. in addition to its clear role in the performance of its direct effect, but more importantly, spleen macrophages is the precursor cells of kupffer cells in the liver, having a very big impact on removing toxins within the liver [ ] . mechanisms for removing toxins in the intestine are related to the intestinal villus tip epithelial cells. under normal circumstances, injected into the intestinal, endotoxin in intestine does not enter intestinal epithelial cells, but after intravenous injection of endotoxin, endotoxin may enter intestinal epithelial cells inside. therefore, endotoxin receptor may identify ways by endotoxin and (or) simple diffusion way into the intestinal epithelial cells. endotoxin way into the intestinal epithelial cells intravenously may have two: ( ) displaced from the lamina propria macrophages to intestinal epithelial cells basolateral; and enter intestinal epithelial cells from the side; ( ) including lower toxin, intestinal lamina propria macrophages, intestinal epithelial cells release large amounts of no and oxygen free, resulting in intestinal lamina propria microvascular injury, increased permeability, extravasation of endotoxin and ultimately displaced into the outer intestinal epithelial cells. villus tip epithelial cells within a stronger uptake of toxins, thus endotoxin can be started from the crypt, moving along the intestinal villi, and finally to the top of the inner hair cells of the intestinal epithelium. uptake of endotoxin villus tip epithelial cell loss, while the endotoxin into the intestine, which constitutes one of the effective clearance mechanisms of endotoxin [ , ] . plasma lipoproteins in endotoxin detoxification mechanisms play an important role, in which the lipopolysaccharide binding protein (lipopolysaccharide binding protein, lbp) and high-density lipoprotein (high density lipoprotein, hdl) play a major role. endotoxin into the bloodstream within minutes there were half white blood circulation due to binding to the edge of the pool or the pool is cleared, the remaining residual endotoxin and rapidly bound to plasma lipoprotein is inactivated. in plasma, lipoproteins and endotoxin when hdl plays a major role in its binding of endotoxin to reach more than % of the total, hdl, and thus research endotoxin important [ ] . cationic antimicrobial peptides are an ancient ingredients in the natural evolution of the immune system, including bactericidal/permeability-increasing protein (bactericidal/permeability-increasing protein, bpi), cathelicidin, lactoferrin, defensins and other substances, with not only the activity against gram-negative bacteria, but also the ability to combine internal toxins. cationic antimicrobial peptides are mainly in regular contact with the pathogen site mammalian skin, digestive tract, respiratory tract, and inherently express or express induced by pathogens and their products in the blood, secretions and neutrophil granules. cationic antimicrobial peptides have two types of three-dimensional structure; one is a α-helix, having such a molecular structure include cathelicidin and lactoferrin; the other is β-fold, including mammals α and β-defensins, etc. [ ] aoah aoah is a glycoprotein produced by white blood cells with weight of . - , , the large subunit of , and small subunits of . million to , , the large and small subunits connected by covalent disulfide bond. aoah as a lipase with hydrolysis for toxin, can selectively hydrolyze the secondary acyl chain on lipid a acyl groups acyloxy. when hydrolyzing endotoxins, both the large and small subunits of aoah play an important role, and both are indispensable. in addition to directly destroying toxin, the deacylated lps after the hydrolysis of endotoxin by aoah is also involved in aoah's detoxification mechanism on endotoxin; the material can accumulate and inhibit endotoxin-induced inflammatory response in the cell. however, due to a limited number of secretion by local infiltration of leukocytes, the internal detoxification of toxins is also limited [ ] . when the body respond with endotoxin, one trigger inflammation, on the other hand can be cleared to produce or activate, specific polysaccharide inactivate toxins, including antimicrobial-specific polysaccharide antibody and anti-core polysaccharide antibody. after two antibodies binding with endotoxin, and then with the fc receptors on the cell membrane, inner source of the toxin-mediated, so that the endotoxin inactivated intracellularly. anti-endotoxin antibodies can interfere with toxin within lbp binding, thus preventing lbp endotoxin transport [ ] . in the body's defense system, the shift from the inhibition of intestinal endotoxin ingredients include intestinal mucosal mechanical barrier, intestinal mucosal immune barrier, the normal intestinal flora and liver hepatocytes and kupffer cells, in which intestinal mucosal mechanical barrier, intestinal mucosal immune barrier, hepatocytes and kupffer cells play a direct inhibitory effect, while the normal flora plays an indirect inhibition. the intestine is huge "endotoxin library", a special anatomical location determines the intestinal mucosa must be an effective defense barrier. immunological barrier intestinal barrier formed by the epithelial cells of mechanical barrier and secretory iga (siga) and the like components [ ] . intestinal epithelial cells and tight junction formation mucosal mechanical barrier, is a significant barrier in the intestine endotoxin translocation defense to maintain its integrity is it to play an important role in defense guarantee. in severe trauma, burns, infection, considerable loss of body fluids, hypovolemia, cause the body ischemia and hypoxia. in order to maintain blood pressure, to ensure that the blood supply to the heart brain and other vital organs, compensatory splanchnic vascular contraction, including gastrointestinal ischemia and hypoxia longer time than other organs, even after shock patients after resuscitation to restore normal hemodynamics, stomach intestinal still in a state of shock occult. therefore, when the intestinal microvascular perfusion recovery, intestinal ischemic/reperfusion injury, epithelial cells produce large amounts of reactive oxygen species and other media, resulting in intestinal epithelial cell apoptosis, destruction of tight junctions between cells, thus rapidly increasing intestinal permeability mechanical barrier function weakens, thus contributing to the intestine of the endotoxin absorbed through the intestinal wall to parenteral tissue displacement [ ] . intestinal mucosal intestinal immunology barrier is a defense of the invasion of pathogens and endotoxin important line of defense, siga plays an important role in intestinal mucosal immunity. siga is an important component of the protection of the intestinal mucosa, both to prevent bacteria in the intestine mucosal surface colonization, but also in endotoxin. studies have found that e. coli o infection suffered intestinal mucosa, the anti-endotoxin core polysaccharide-specific siga secretion, in convalescent patients has been particularly evident, suggesting siga endotoxin to prevent the transfer of the body has a protective effect. in addition, studies suggest that nitric oxide (nitrogen monoxide, no) preventing endotoxin translocation in intestinal mucosal barrier oxide formed locally. under physiological conditions, nitric oxide synthase (inducible nitric synthase, inos) expression only in the respiratory epithelium, the pregnant uterus and ileal mucosa and a few other parts. induced by endotoxin including but under normal colonic epithelial cells also express inos and catalytic synthesis of no, are formed in the local oxidation barrier to prevent bacterial translocation colon, thus effectively preventing bacterial translocation, also indirectly prevents endotoxin shift [ ] . under physiological conditions, intestinal flora forms a relatively balanced microecosystem. flora distribution in the intestine has certain rules: deep close to the intestinal mucosal surface, parasitic anaerobic bifidobacteria and lactobacilli, these anaerobic bacteria are sugar coated, relatively stable, known as membrane flora; middle class bacteria, streptococcus digest, veillonella and excellent bacilli; the surface of e. coli and enterococci, can swim in the intestine, known as cavity flora. the antagonism between the layers flora, mutual cooperation, to maintain a dynamic equilibrium, in which the film anaerobic flora is a very important body's natural defense barrier that can prevent opportunistic pathogens such as e. coli colonization in the mucosa, but also can inhibit the overgrowth of opportunistic pathogens. intestinal flora micro-ecosystem is a very sensitive system, in severe stress or long-term systemic administration of large doses of broad-spectrum antibiotics, etc., the film significantly reduced the number of anaerobic bacteria, e. coli and other bacteria thrive conditions and continuous release of endotoxin to the intestine, since the film flora defense decreased, these opportunistic pathogens to colonize the intestinal mucosa, resulting in intestinal mucosal barrier damage, followed by the occurrence of intestinal bacteria, endotoxin translocation [ ] . under normal circumstances, the liver is one of the major barriers preventing endotoxin translocation, via the portal vein into the liver hepatocytes and a small amount of the toxin can be kupffer cell depletion. in conditions such as stress, not only liver cell dysfunction, so the ability to reduce endotoxin detoxification and collaterals between the portal vein and the vena cava, causing an overflow of endotoxin from the liver into the systemic circulation. endotoxin absorbed into the bloodstream, which in turn may increase the intestinal epithelial cells of the intestinal microvascular endothelial cell damage and, in a vicious cycle [ ] . when the body's defense system to produce responses in endotoxin, the innate immune system plays a major role. pathogens can be identified conserved receptors called pattern recognition molecules (pathogen-associated molecular patterns, pamps), including endotoxins of gram-negative bacilli, peptidoglycan grampositive cocci, lta and other cell wall composition and gram-negative bacteria, gram-positive bacteria such as dna. although a variety of pattern recognition molecules of different chemical structures, but they have similar characteristics: ( ) characteristic structure in which different types of pathogens in a relatively constant conserved; produced by a pathogen, the host body without these molecules; survival or disease-causing pathogen is generally the essential, such as mutations, death or loss of a pathogen will pathogenicity. natural immune system to recognize the receptor molecule called pattern recognition receptors (pattern-recognition receptors, prrs), including cd , toll-like receptor family (toll-like receptor, tlrs) and scavenger receptors. but in recognition of toxins, some differences exist between the different kinds of cells [ ] . macrophages in addition to expressing cd , tlrs and scavenger receptors and other associated endotoxin receptors on the cell surface, but in the cytoplasm also express the protein molecules nod recognizing toxins. cd , tlrs are key receptors that mediate endotoxin within macrophage activation; and scavenger receptor has relationship with macrophage clearing and inactivating toxins [ ] . kupffer cell is the main cell that clears the endotoxin in the liver. under physiological conditions, although there is still a small amount of bacteria and endotoxin via the portal vein into the liver, but kupffer cells will clear. kupffer cells are the most resident macrophages in the liver and are the largest number of resident cells in tissues. there is a theoretical speculation that if kupffer cells and on is very sensitive to endotoxin as other macrophages, the cell will be in constant activation, but in fact when kupffer cell engulfs, removes endotoxin, its itself is not activated by endotoxin, which suggests that in the treatment of endotoxin, kupffer cells have different mechanisms with other macrophages: kupffer cells treats endotoxin mainly depending on its phagocytosis. in the absence of serum, the phagocytic effects of kupffer cell on endotoxin can play a normal; and with the appropriate increase in endotoxin concentration, the phagocytic activity of kupffer cell was enhanced. the effect has something to do with phosphorylation events of two protein tyrosine residue individually weighting . million and . million. cd is the main receptor that mediates endotoxin activating kupffer cell, scavenger receptor is kupffer cells' important defense of receptor mediated kupffer cell to remove and inactivate endotoxin. there are four stages of the activation of kupffer cells, of which cd is the characteristic marker of cellular activation and function change. ( ) the stationary phase; the performance of less number of kupffer cells, small shape, a number in the hepatic sinusoids, cd staining negative; ( ) reaction period: for the local kupffer cells stimulate hyperplasia and systemic mononuclear macrophage intrahepatic accumulation; ( ) pre excitation period: kupffer cell phenotype occurred transformation period, expressed cd cell membrane receptor, kupffer cell functional changes; ( ) the activation period: the performance of nuclear transcription factor nf kb activation, cellular secretion cytokines [ ] . the cd (cd membrane-bound, mcd ) and tlr endotoxin were activated by the neutrophil surface to activate the neutrophils by binding with the receptor. in addition to the expression of the high affinity endotoxin receptor cd , the surface of the neutrophils also expressed the low affinity endotoxin receptor l-and the activated cells were activated by the receptor. in addition, the integrin is considered to be a low affinity endotoxin receptor for the surface of the neutrophils [ ] . it is generally believed that the expression of mcd was not on the surface of endothelial cells and serum soluble cd (soluble cd (scd ) is mediated endothelial cell recognition of lps molecules, and tlr is involved in lps induced endothelial cell activation. lbp was transported to scd by endotoxin, and tlr was activated by lps/scd and activated endothelial cells in the endothelial cell membrane. scd in addition to mediated endothelial cell activation and also mediated by endothelial removal of endotoxin and lps/scd /lbp form trimers and binding to endothelial cells, following the lps/scd endogenise, thus the removal of endotoxin [ ] . the epithelial cells of the intestinal mucosa were consistently associated with the bacterial and its products, and the bacteria and its products could stimulate other types of cells and induce inflammatory response, but did not induce intestinal epithelial cell defense, this feature for colonic epithelial cells is particularly important, because if can react to the normal intestinal flora in intestinal epithelial cells, it will cause adverse effects on the body. but this does not mean that intestinal epithelial cells are immune cells, when suffered pathogens and their products invasion, intestinal epithelial cells produce normal response. description: intestinal epithelial cells with normal differentiation of natural bacteria and pathogenic ability, and the recognition system of subcellular localization. there are different endotoxin recognition mechanisms in the intestinal epithelium and the myeloid cells [ ] . uncontrolled inflammatory responses (uncontrolled inflammatory response) has a relationship with infection, bacteremia, septicemia, sepsis, systemic inflammatory response comprehensive sign (systemic inflammatory response syndrome (sirs), compensatory anti-inflammatory response syndrome (compensatory antiinflammatory response syndrome, cars) and other related terms used for a long time, but also has an essential difference. out of control including inflammatory reaction syndrome (msas anti-inflammatory response syndrome, mars), a dynamic process of sirs and cars and the mixed antagonistic response syndrome, at present clinical many diseases occurrence and development are closely related. uncontrolled inflammation is a common pathological phenomenon in clinic, which is the important mechanism of the development of the complication after trauma. lps is one of the main factors that induce the uncontrolled inflammatory reaction in the most common. lps receptor on the monocyte/macrophage surface is the he initial factor for the body to recognize and start inflammatory reaction, also is one of the key links for the induction of uncontrolled inflammatory response. the concept of uncontrolled inflammatory response refers to inflammatory disorders then resulting in multiple organ dysfunction syndrome (multiple organ dysfunction syndrome. mods), emphasizes the importance of the of balance inflammatory/anti-inflammatory mechanism in the body, changes the limitations that in the past we only attached importance of the pathogenic effects of inflammatory factors. it is believed that the response of the body to the inflammatory factor is the dominant factor in the development of the whole body inflammatory reaction and mods. this concept is more focused than the previous focus on the dynamic changes of the whole process of inflammation. this can be divided into two types of mods: one is the early stage after the injury, that is, the speed hair style. the main blame is a strong inflammatory reaction induced by proinflammatory factors, and the other is a late phase of the disease, which is "late style", mainly due to the immune paralysis or worse immune disorders caused by cars or mars. the inflammatory is actually a kind of medium disease mainly caused by the chain reaction of cytokine. endotoxin is thought to be one of the most important predisposing factors in the chain reaction and can be referred for chain reaction "trigger" (trigger). endotoxin induced inflammation mechanism is mainly mediated by pamps that can induce cytokines such as il- , tnf alpha and other active molecules synthesis, the formation of the cytokine network, has a very important role in the occurrence and development of infection. the excessive activation of cytokines can cause septic shock, and is the leading cause of death in patients with bacterial infections. accordingly, prrs plays an important role in innate immunity and inflammation, and it can distinguish the pathogens from self organization through prrs organism, which is characteristic of immune response [ ] . inflammatory response syndrome systemic (sirs) is a systemic inflammatory response caused by any pathogenic factor to the body. the concept is first proposed by coris in . august american college of chest physicians and critical care medicine to present the diagnostic standard of sirs, think to have the following each of the two or more than two, sirs can be established: ( ) temperature > deg c or < deg c; ii heart rate beats/min; ( ), the breathing frequency > times per minute or arterial blood carbon dioxide into pressure (paco ) < . kpa mmhg; ( ) peripheral white blood cell count > × /l or × /l < or immature myeloid cells > %. what should be paid attention to is that sirs is a common athophysiological state of body with severe inflammatory reactions, and should be differentiated from some abnormal factors such as leukemia or cause increase or reduction of white cells after chemotherapy. although the naming of sirs has been generally concerned, but some scholars have raised objection to the concept, for example sirs has following problems: the sensitivity and specificity of the diagnostic criteria is poor, has the same meaning with the "critical"; can not understand the pathophysiology of the original disease; is difficult to guide clinical trials and practice. the production of sirs can be divided into two cases, the sirs caused by the infection and the non infectious sirs. from the point of view of the clinical development process, sirs can be followed by injury immediately aroused, then known as the "single phase velocity hairstyle; also to start local, and later developed into a systemic sirs, namely after the initial shock is brief period of stability, later gradually intensified when sirs is called" dual phase delayed onset. either of the factors or the clinical development process, the systemic inflammation of the control of the uncontrolled, and ultimately can lead to mods [ ] . the intestine is the biggest bacterial and endotoxin warehouse in the body. in severe trauma, systemic infection, intestinal ischemia and liver disease, there may be the occurrence of endotoxin. the main source is due to intestinal gram-negative bacteria in the excessive growth and reproduction, or due to increased intestinal permeability lps entry into the portal vein increased. if hepatic kupffer cell phagocytic function is low, the amount of endotoxin over the liver ability to remove endotoxin can "flood" (spill over) into the body of the loop and the endotoxemia formated. because of the endotoxin from the gut, so it is called intestinal endotoxemia (intestinal endotoxemia, ietm) [ ] . hepatitis b patients are often accompanied with ietm. its formation mechanism is: the production and absorption of intestinal endotoxin increased. there is a large number of gram negative bacteria in the body's normal intestinal, so endotoxin in intestinal contents is very high, but the intestinal mucosal epithelial cells have stronger resistance to toxins so that endotoxin is not easy to run through the intestinal mucosa into the blood, even a small amount of endotoxin breaking through the intestinal mucosal barrier into the portal vein, will be swallowed up by the kupffer cells in the liver. severe hepatitis b when the intestinal flora disturbance, endotoxin increased, increased intestinal hyperemia, edema and the permeability of the intestinal mucosa; endotoxin itself can damage the mitochondria and lysosome of intestinal epithelial cells, leading to epithelial cell autolysis; endotoxin can cause intestinal microvascular contraction of the intestinal mucosa, reduce blood, intestinal ischemia, hypoxia, cause the intestinal mucosal barrier function decreased, increased the absorption of endotoxin; severe hepatitis, due to intrahepatic bile acid and bilirubin deposition in kupffer cell phagocytosis was inhibited, resulting in the removal of endotoxin in the endotoxin decreased; through the door body circulation circuit into the systemic circulation, resulting in blood within liver cells to escape kupffer toxin the phagocytosis and clearance, which aggravate endotoxemia; the endotoxin can also pass through the celiac lymphatic system into systemic circulation by thoracic duct. in addition, severe hepatitis patients with sepsis, spontaneous bacterial peritonitis, etc., in the release of endotoxin, so the formation of the endotoxin is exogenous [ ] . in viral hepatitis and other basic diseases complicated with ietm and liver function failure are closely related and endotoxin can directly cause arterial vasoconstriction, the organ ischemia; endotoxin can activating endogenous clotting system, coupled with kupffer cell dysfunction, decrease delimination of blood coagulation or fiber soluble substances, easily lead to dic, so as to damage to multiple organs. endotoxin activated phospholipase a mediated membrane phospholipid degradation and lipid peroxidation, which is an important part of liver cell damage. nolan has pointed out that the effect of kupffer cell dysfunction induced by intestinal endotoxemia on liver and body, far more than the direct action the endotoxin, and production and release of inflammatory mediators and factors from kupffer cells activated by endotoxin are closely related. the occurrence of ietm affect hepatic energy metabolism, resulting in liver cell damage and necrosis, also caused hepatic microcirculatory disturbance, performing liver hemorrhagic necrosis. on the basis of severe hepatitis, it can accelerate liver function failure [ ] . hepatic cellular jaundice the acute and chronic liver function is often accompanied by intrahepatic cholestasis jaundice, and ietm plays an important role in the occurrence of intrahepatic cholestasis.. endotoxin involves in liver cell damage mainly through activation of phospholipase a , and inhibits the activity of na + − k + − atpase on liver canalicular membrane to make the bile excretion disorder, and then to cause intrahepatic cholestasis in the liver cells. endotoxin can start the peroxidation of liver parenchymal cells mitochondrial membrane lipid so that an increase in the content of oxygen free radical in blood, resulting in the disorder of energy generation, atp was reduced, so that the active uptake, metabolism and secretion of bile acid by liver parenchymal are short of energy, resulting in cholestasis [ ] . the liver disease with ietm often causes the coagulation dysfunction, the serious person appears the different degree bleeding, in particular the severe liver disease patient may concurrent dic, endangers the life. violi found that, when liver dysfunction in patients with ietm, the expression of tissue factors on the surface of macrophages and endothelial cell factor induced by endotoxin increased, promoting the synthesis of tumor necrosis factor (tumor necrosis, factor, tnf) to increase, and thrombin generation increasing, activation of coagulation system in about % of patients, followed by hyperfibrinolysis, suggesting that ietm in liver dysfunction can be used as the warning signal as the activation of coagulation and fibrinolysis system and activate, and with the increased hepatic lesions, plasminogen activation decreases with endotoxin levels increased, thus plasminogen may decline with endotoxin induced by chronic consumption of dic on the microstructure of ii related factors, new blood can not correct. in fact, before variceal rupture bleeding, patients with liver function severely damaged already have the gastrointestinal mucosa extensive ischemia and erosion, which is the potential causes of bleeding, ietm in the process. and gastric h + at this time can occur abnormal reverse diffusion and stimulate mast cells to release histamine, may lead to mucosal blood vessel dilation and permeability enhanced. as a result, hemorrhage, edema; histamine and directly stimulate the secretion of gastric acid, so that an increase in the number of h + and reverse diffusion, lesions persisted, form a vicious circle [ ] . it is important for the formation of ascites in ascites due to the obstruction of the hepatic vascular outflow tract obstruction. the initial vascular response to endotoxin was the rapid obstruction of the hepatic venous outflow tract and increased the portal pressure which may be related to endotoxin induced swelling of kupffer cells, liver cells with microvilli swelling, platelet aggregation and fibrin deposition effect, while others think that is endotoxin of blood vessels of the liver has a direct effect. ietm continuous damage to the liver cells, resulting in albumin synthesis and of hormones such as aldosterone de live function obstacles, thus affecting the renal function, and led to the emergence of the refractory ascites plays an important role in the process [ ] . patients with severe liver disease always are complicated with the functional renal failure, hepatorenal syndrome (hrs). the patients with severe liver disease can be associated with the pre-renal azotemia and acute renal tubular necrosis, and there is a certain relationship with ietm. the clinical studies showed that the levels of no --no and endotoxin in serum of patients with liver cirrhosis were significantly higher. at the same time, plasma renin activity, aldosterone and vasopressin levels increased and urinary sodium excretion decreased. the mechanism about ietm induced by hrs is not clear, may be related to the following factors: leukotrienes (leukotrienes, lts) lts can lead to renal vasoconstriction, increased renal vascular resistance, reduced renal blood flow and renal blood redistribution, decreased glomerular filtration rate induced by hrs, in ietm lts generation and release increased obviously. in addition, liver dysfunction, liver's uptake, inactivation and excretory function of lts decline, causing blood concentration increased; the thromboxane a (thromboxane a txa )/i (prostaglandin i , prostaglandin pgi ) can contract renal arterioles, decrease glomerular filtration rate, while pgi and pge (prostaglandin e , pg e ) is caused by on the role of txa . in patients with severe hepatitis with ietm, elevated systemic levels of pgi , reducing the renal vascular resistance, leading to renal vascular resistance txa , reduce the renal blood flow and glomerular filtration rate, promote the formation of hrs; third, nitric oxide, nitrogen monoxide) nono can through vasodilatation of the systemic, resulting in effective circulating blood volume reduction and evoked hrs; endothelial endothelin (et) et caused renal cortical blood priming hrs; and platelet activating factor (growth factor (paf) endotoxin and platelet activating factor (paf) can lead to a decrease in cirrhotic rats cardiac ejection fraction, reducing blood flow to the kidney, and paf antagonist can improve hemodynamics changes [ ] . the mechanism of endotoxin induced hepatic encephalopathy is not clear. we have known that lps can increase the permeability of blood brain barrier, promote intestinal toxic substances through the blood brain barrier (bbb), damage mitochondrial oxidative metabolism in brain cells, reduce oxygen utilization in patients with liver cirrhosis and disrupt energy metabolism of brain cells, induce brain edema. the clinical symptoms of chronic severe hepatitis with endotoxemia included in addition to fatigue, anorexia, tiresome of the oil, nausea, vomiting, yellow skin and sclera and, performance of endotoxemia. endotoxin can cause the release of histamine, -hydroxytryptamine ( -ht), prostaglandin, bradykinin, resulting in micro circulation expansion, venous blood volume reduction, decreased blood pressure, inadequate tissue perfusion, hypoxia and acidosis, and main symptoms and signs are: fever, elevated white blood cell count, bleeding tendency, heart failure, renal dysfunction, hepatic injury, nervous system symptoms and shock [ ] . improve liver function this is the basic treatment of ietm. liver function improved can strengthen mononuclear phagocyte system function to help the endotoxin removal. it can also decrease portal vein pressure to relieve intestinal congestion, edema, hypoxia, improve the intestinal microenvironment, reduce production and lymph reflux, and lower door shunt. these all contribute to the prevention and treatment of ietm. clean the gut saline is available as enema if severe liver disease, which helps reduce intestinal endotoxin generation and absorption. decompensated cirrhosis is often accompanied by small intestinal bacterial overgrowth and intestinal flora disturbance. thus, the promotion of intestinal flora back to normal state help prevent and treat intestinal endotoxemia. a variety of bifidobacterium, lactobacillus can be selected. a synthetic disaccharide, it is not digested and absorbed in the small intestine, but can be broken down into lactic acid, acetic acid and other small molecules by the bacteria into the colon. such acidification of the intestine reduces the generation and absorption of endotoxin, and promotes the growth of intestinal bacteria, stimulates bowel movements so as to increases tool frequency and so on. in addition, the lactulose may have internal direct inactivation of toxins, prevents activation of macrophages to release cytokines. oral absorption of antibiotics can effectively suppress the generation of intestinal endotoxemia. patients with liver cirrhosis taking oral polymyxin e or neomycin, the level of plasma lps and no -/no -horizontal declines in synchronization. polymyxin b has an internal direct antitoxin effect [ ] . it play a role by reducing intestinal absorption of toxins, inactivating toxins and inhibiting those lps-induced media by monocyte macrophages. the new anti-endotoxin therapy including interrupt endotoxin synthesis, binding or neutralizing its activity, preventing its interaction with the host effector cells, or interfering with toxin-mediated signal transduction pathways. therapeutic formulations include endotoxin analogs, antibodies, subunit vaccines, polymyxin combination column, recombinant human protein, small molecule inhibitors of endotoxin synthesis and intracellular signal transduction. bacteriophage producing a piece of short nucleotide sequence which plays the role of an antisense rna, blocking the synthesis of bacterial lps synthase. current clinical studies carried out an experiments in cloning of human anti-endotoxin lipid a light and heavy chain variable region. it laid the foundation for the next screening and expression that recombination between dna of antibodies and phage's succeeded. the clone is one kind of anti-polymyxin b (polymyxin b) monoclonal antibody of the igm class. it can play the role of anti-endotoxin shock by imitating the surface antigen structure of lipid a so as to substitute receptor antagonist of lipid a and lps blocking the causative link that the endotoxin induces inflammatory mediators [ ] . isolating antibodies having a high affinity of various g-bacteria to prepare a chimeric monoclonal igg antibody sdz - which have a therapeutic effect on the human endotoxemia. anti-endotoxin core glycolipid monoclonal antibody (antimonoclonal antibody r ) can prevent and treat the metabolic disorders in peritoneal infection with mods; it plays a significant role in conditioning in high catabolism, and can significantly improve metabolic disorders under the condition of abdominal infection associated with mods. bactericidal/permeability-increasing protein (bpi) bpi is a human endogenous protein, found primarily in neutrophils primary particles. its molecular amino-terminal and carboxy-terminal appear v-shaped structure planar symmetry. many amino acids to form a hydrophobic capsule hold lps's lipid a. it was reported that bpi has an obvious protective effect on intra-abdominal infection induced sepsis, which might be related to its antagonism against endotoxin [ ] . reconstructing hdl (high density lipoprotein, hdl) hdl can be used as an endogenous lps scavenging system, binding of bacterial endotoxin with high affinity to form a stable hdl-lps. lps-hdl complexation may contribute to a reduction in endotoxic activities in vivo by preventing lps (lipid a) from generating important transmembrane signals after binding to cells [ ] . e is the first generation lipid a analogue, which is derived from the lipid a structure from the endotoxin of rhodobacter capsulatus. it can block lps in cell culture without any endotoxin-like activity. e can protect mice from lethal doses of lps, and viable e. coli infections in combination with antibiotics. in human healthy volunteers who are exposed to intravenous lps. e also blocks the endotoxin response [ ] . an anti-cd antibody cd has a very important role in monocyte-macrophage cell signaling. since epitopes of lps on the cell membrane at the binding site is the same material with soluble cd , so we can develop a monoclonal antibody interfering with lps binding to cd and blocking to pass activation signals from immune effector cell [ ] . tyrosine kinase and mitogen-activated protein kinase are involved in lps cellular signal transduction. build anti-endotoxin (lps) single-chain antibody gene and attempt to make it express in e, coli. the scfv gene was successfully constructed and gst scfv fusion protein highly expressed in e. coli was obtained. glucocorticoids, including the synthetic glucocorticoid dexamethasone, are recognized for their anti-inflammatory properties and have the ability to inhibit the production of proinflammatory cytokines such as tnf-α. in intestinal ischemia-reperfusion methylprednisolone pretreatment can prevent endotoxemia. combined lps and dexamethasone treatment at h significantly changed tnf-α [ ] . this points that glucocorticosteroids added before or during stimulation of macrophages can prevent tnf release, after which the administration would be invalid. in fact, it is very difficult to use corticosteroids before tnf release. current clinical anti -tnf antibodies and tnf antagonist use exists, and before many scholars have obtained a more satisfactory results of blocking or neutralizing excessive tnf with anti-tnf. although the clinical symptoms improved, yet the survival rate is not higher than expected. the effect of anti--tnf clinical application needs further evaluation. the first proposed concept is sequential organ failure, based on which multiple organ failure (multiple system organ failure, msof or mof) is put forward, and in diagnostic criteria is developed, but it reflected the end-stage, denied reversibility, ignore the dynamic development from organ dysfunction to failure. therefore, the us accp/sccm proposed to replace the concept with mods. mods emphasized an early phase of organ dysfunction before overt failure occurred. it is defined as "the presence of altered organ function in an acutely ill patient such that homeostasis cannot be maintained without intervention." [ ] organ dysfunction may be relative, or can be absolute; with extension of time, mods can increase or reverse. thus, the term. mods was coined to indicate the wide range of severity and the dynamic nature of this disorder, which contributes to early diagnosis and treatment of patients and is more in line with clinical practice. there are two relatively distinct (although not mutually exclusive) pathways by which mods can develop: in primary mods, there is a direct insult to the organ that becomes dysfunctional. examples of such direct insults include gastric aspiration in the lungs or rhabdomyolysis in the kidney. this direct insult causes: an inflammatory response that is localized, at least in the beginning, to the affected organ. secondary mods is a consequence of trauma or infection in one part of the system that results in the systemic inflammatory response and dysfunction of organs elsewhere [ ] . secondary mods means not directly caused by damage, but to experience the "second strike", the first blow can make the immune system in a preactivated state under which inflammation lost control and significant sirs appears, then the following second strike can quickly cause multiple distant organ dysfunction and easily form sepsis with the basis of sirs/cars. this type of mods often develops as the following model: the original cause → stress → immune wpw → sirs/cars → infection → sepsis → mods → mof. the process of severe hepatitis, can cause intestinal damage and massive release of endotoxins into the blood, leading to intestinal endotoxemia (ietm). endotoxin is a powerful trigger for complement activation, and then these complement can activate "cascade effect" (cascade) to release oxygen free radicals, prostaglandins, endorphins, paf, cytokines and other inflammatory mediators to cause cytotoxicity, the error of microcirculation and tissue metabolism, eventually leading to the occurrence of mods. in this case, we emphasize mods originated in continuous, uncontrolled inflammation and factors causing systemic inflammatory response can be induced mods, including bacteria, fungi, parasites, viruses, toxins and other infectious agents. it is a debate of long-century problems that whether endotoxin has effects on hearts. the late s a large number of experiments prove endotoxins has a role of heart. it enables reduction of coronary blood flow, decrease of total coronary vascular resistance, and have meanings in heart failure. studies have shown that endotoxin can damage myocardial mitochondria, muscle paddle net, muscle membrane, contractile proteins etc., leading to cell membrane system damage, energy metabolism. past research in cardiac dysfunction under endotoxin shock did not attract enough attention, for they think that the heart is the final failure among all organs so that clinical treatment value is little. now people's awareness has completely changed that heart dysfunction can occur early in sepsis or septic shock. therefore, early identification and prevention of the occurrence and development of cardiac dysfunction may have some clinical value for treatment of septic shock and other serious complications [ ] . clinical gram-negative bacteria sepsis often complicated by adult respiratory distress syndrome, or server development of mods. in this pathological process, the high biological activity of endotoxin plays an important role. endotoxins often involving the lungs firstly, and the pathogenesis of non-injury may be associated with the direct damage on endothelial cell by endotoxin through complement pathway and induction of cytokines [ ] . the liver is the major site of endotoxin on clearing and detoxifying and the place where clinical gram-negative bacteria sepsis often can be complicated, and it is also the primary organ suffering attacks by toxins. it is generally believed that toxins in the liver circulating mainly from the gut and liver dysfunction is closely related to the formation of endotoxemia. histological examination revealed the inner toxins damage the liver cells, showing sinusoidal congestion, dieldrin expansion chamber, kupffer cell swelling, endoplasmic reticulum、mitochondria swelling, crest destroy and lysosomal activation etc. [ ] . the liver is the body's largest metabolic organ, and many cases of acute liver failure often involve other organ complications, which has become an important factor in determining the prognosis. endotoxins may cause the reduction of liver nutritional blood flow, mitochondrial oxygen metabolism, interfering with sugar metabolic pathways in liver leading to metabolic disorders. various damaging factors (such as gastrointestinal disorders, ischemia, immunocompromised, dysbiosis) promote absorb of intestinal bacteria and toxins and displacement via the portal vein, the lymphatic system into the systemic circulation. on the one hand these infectious agents can directly damage liver cells, or mediate hepatic injury whether by kupffer cell; on the other hand it can induce systemic inflammatory response by the monocyte-macrophage cells to release the media, both leading to organ perfusion disorder, affecting protein synthesis and energy metabolism, eventually resulting in severe sepsis, mods and even death. the effect of endotoxin on kidneys is not clear yet. in the early stage, it can affect the kidneys, decreased its blood flow renal via vasoconstriction. when endotoxemia is complicated by renal failure, the mechanism of glomerular filtration rate decreasing is unclear. the pathophysiology of aki in sepsis is complex and multi-factorial and includes intrarenal hemodynamic changes, endothelial dysfunction, infiltration of inflammatory cells in the renal parenchyma, intraglomerular thrombosis, and obstruction of tubules with necrotic cells and debris [ ] . the relationship between endotoxin and dic is quite complicated. dic is considered to be an important incentive of mods, especially patients with severe sepsis with dic having a highly possibility of developing mods, what's more, the prognosis is very poor, and the mechanism is multifaceted. endotoxin can start the endogenous coagulation system directly or via activating factor xii (hageman factor) by damaged endothelial cells, also can act on the monocyte-macrophage cells, stimulate the release of tissue factor to trigger the extrinsic coagulation pathway [ ] . in clinical practice it has been noted that serious infections is very possible to be complicated by gastrointestinal failure. the intestine is the biggest reservoir of bacteria in the body and leakage of bacteria or microbial products, notably lps, from the lumen of the gut into the systemic compartment, leads to initiation or amplification of a deleterious inflammatory response and mods [ ] . after endotoxins challenging the gastrointestinal mucosa, it initially shows mucosal telangiectasia, interstitial edema and hemorrhage. microcirculation leading to damage of lysosomal and release of proteases in the cell, cell degeneration and necrosis. in addition, mucosal cellular energy metabolism decrease, h + reverse diffusion, prostaglandins and bradykinin further aggravate mucosal damage. at the same time destroy of the gastric mucosal barrier also make a lot of bacteria and endotoxins pass through the gastrointestinal mucosa, migrate to the blood circulation, the lymphatic system and the abdominal cavity etc., leading to systemic multi-system organ damage. clinical characteristics of mods . organ failure usually do not result directly from the primary injury. there is a certain time interval from the primary injury to organ failure. . not all of infection have bacteriological evidence, and more than % of patients and autopsy found no infected lesions. thus, to identify and treat the infection may not be able to improve the patient's survival. . mods may have perfectly healthy organ involved, and it is ferocious and rapidly progresses. once happened, it is difficult to depress in the event almost, so often with a high mortality rate. . in pathology, mods lacks features, the affected organ only showing acute inflammation, such as inflammatory cell infiltration and so on, and these changes are very inconsistent with severe clinical manifestations, and once restored, patients do not have any clinical sequelae. . mods is closely with shock and infection. shock, infection, injury (including trauma and surgery, etc.) are the three main causes of mods. . generally the later period of shock will typesetting idc and mods, and the order of occurrence of mods usually is the lungs, liver, kidney, gastrointestinal tract, finally the heart. the characteristic clinical manifestations . instability of circulation due to a variety of inflammatory mediators have effects on the cardiovascular system, the circulation is most likely involved. almost all cases, at least in the course of the early and middle will be in highpower type of cycle of "high ranked low resistance". cardiac output up to l/ min or more and low peripheral resistance cause shock and need vasopressors to maintain blood pressure. . high metabolic systemic infection and mods are usually accompanied by severe malnutrition. its metabolic mode has three salient features: ( ) persistent high metabolism, metabolic rate up to . times more than normal; ( ) abnormalities of energy pathway. in starvation, the body obtain energy mainly through the decomposition of. however, with systemic infection, the body will get energy by breaking down proteins while the use of sugar is limited and fat utilization may increase early, fall later; ( ) poor response to exogenous nutrient, supplement of exogenous nutrition can not effectively prevent itself consumption, which suggests that a high metabolism itself has a "mandatory" also known as "autophagy metabolism." high metabolic may have serious consequences. first, protein malnutrition result from it will cause serious damage to the structure and function of the enzyme system of organs; secondly, imbalance of branched-chain amino acids and aromatic amino acid which makes the latter formate into a pseudo-neurotransmitter, then further lead to dysfunction of nerve. . hypoxia in tissue cells at present many scholars believe that the high metabolic and circulatory disorders often cause oxygen supply and oxygen demand does not match, so that the tissues of bodies are in a hypoxic state, mainly clinically manifesting "oxygen supply dependency" and "lactic acidosis. ". currently mods still lacks an unified diagnostic criteria, and any one of the diagnostic criteria of mods is difficult to reflect the entire contents of organ dysfunction, so in clinical practice we can select one according to our own specific situation. . the main contents from national critical care medicine conference standard in are: ( ) respiratory failure: r > /min; pao < . kpa; pco > . kpa; pao /fio ≤ . ( mmhg); p (aa) do (fio . ) > . kpa ( mmhg); x-ray of chest shows alveolar consolidation ≥ / lung (which have more than three or three); ( ) renal failure: except prerenal factors, little or no urine, serum creatinine, increased blood urea and nitrogen levels, exceeding more than twice the normal value; ( ) heart failure: systolic blood pressure < mmhg ( . kpa), sustained more than h; ci < . l/(min · m ); ventricular tachycardia; ventricular fibrillation; degree atrioventricular block; resuscitation after cardiac arrest (with which three or more); ( ) liver failure: total bilirubin> μmol/l; liver enzymes increased more than times compared with the normal; prothrombin time > s; with or without hepatic encephalopathy; ( ) dic: platelets × /l; prothrombin time and partial thromboplastin time prolong . times, and fibrin degradation products increase; systemic hemorrhage; ( ) brain failure: glasgow score below means coma, and less than points means brain death. . the sooner the primary diseases or the primary risk factors are eliminated or controlled, the greater the possibility of organ recovery is. . to effectively rescue and debride as soon as possible, prevent infection, prevent ischemia-reperfusion injury, use a variety of supportive care; . to reduce stress response, mitigate and shorten high metabolism and the magnitude and duration of glucocorticoid receptor; . to pay attention to the patient's breathing and circulation, as soon as possible to correct hypovolemia and hypoxia; . to prevent infection is an important measure of preventing mods; . if possible, improve the nutritional status of patients. . early treatment of any starting organ failure. mods is a problem in the medical field with an acute onset, rapid progression, and high mortality rate. so far for mods, there is no specific treatment, but through clinical monitoring, early detection of possible organ dysfunction, early intervention, and taking effective measures can slow down or block the course, 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phagocytosis of apoptotic neutrophils is critically regulated by the opposing actions of pro-inflammatory and antiinflammatory agents: key role for tnf-α the accp-sccm consensus conference on sepsis and organ failure sepsis and the heart endotoxin and lung injury the role of intestinal endotoxin in liver injury: a long and evolving history pathophysiology of septic acute kidney injury: what do we really know? sepsis, thrombosis and organ dysfunction bile high-mobility group box contributes to gut barrier dysfunction in experimental endotoxemia key: cord- -trd rkxw authors: chen, na; wu, qianchao; chi, gefu; soromou, lanan wassy; hou, jinli; deng, yanhong; feng, haihua title: prime-o-glucosylcimifugin attenuates lipopolysaccharide-induced acute lung injury in mice date: - - journal: int immunopharmacol doi: . /j.intimp. . . sha: doc_id: cord_uid: trd rkxw prime-o-glucosylcimifugin is an active chromone isolated from saposhnikovia root which has been reported to have various activities, such as anti-convulsant, anticancer, anti-inflammatory properties. the purpose of this study was to evaluate the effect of prime-o-glucosylcimifugin on acute lung injury (ali) induced by lipopolysaccharide in mice. balb/c mice received intraperitoneal injection of prime-o-glucosylcimifugin h before intranasal instillation (i.n.) of lipopolysaccharide (lps). concentrations of tumor necrosis factor (tnf)-α, interleukin (il)- β and interleukin (il)- in bronchoalveolar lavage fluid (balf) were measured by enzyme-linked immunosorbent assay (elisa). pulmonary histological changes were evaluated by hematoxylin–eosin, myeloperoxidase (mpo) activity in the lung tissue and lung wet/dry weight ratios were observed. furthermore, the mitogen-activated protein kinases (mapk) signaling pathway activation and the phosphorylation of iκbα protein were determined by western blot analysis. prime-o-glucosylcimifugin showed promising anti-inflammatory effect by inhibiting the activation of mapk and nf-κb signaling pathway. the acute respiratory distress syndrome (ards), a clinically important complication of severe acute lung injury (ali) in humans, highly associated with sepsis pneumonias and severe acute respiratory syndrome (sars) is a significant cause of morbidity and mortality in critically ill patients [ ] [ ] [ ] . inflammatory stimuli from microbial pathogens, such as endotoxin (lipopolysaccharide [lps]), are well recognized for their ability to induce pulmonary inflammation, and experimental administration of lps, has been used to induce pulmonary inflammation in animal models of ali [ ] [ ] [ ] . the development of an ali model by way of i.n. lps instillation is well suited for preliminary pharmacological studies of new drugs or other therapeutic agents because i.n. instillation of lps into mice can produce a controlled ali without causing systemic inflammation and multi-organ failure [ , ] . lps-induced ali is considered a neutrophil-dependent ali that contributes to local recruitment and activation of neutrophils [ ] ; the release of pro-inflammatory cytokines such as tumor necrosis factor (tnf)-α, interleukin (il)- β, and il- ; and the formation of reactive oxygen and nitrogen species [ ] [ ] [ ] . neutrophil recruitment in the lungs is regarded as a histological hallmark in the progression of ali [ ] . several candidate therapy strategies such as fluid management, surfactants, glucocorticoids, and stem cells have been applied to treat acute lung injury and acute respiratory distress syndrome in the last decade [ , ] . however, the mortality resulting from these conditions remains high [ ] . fangfeng, the root of saposhnikovia divaricata (turcz) schischk, is widely applied for headache, febrility, vertigo and arthralgia as an important member of traditional chinese medicines (tcms). it has been proved that there are numerous pharmacologic effects of the extract from fangfeng, such as suppression of adjuvant arthritis, inhibitory effects on the peptic ulcers and analgesic, anti-convulsant, anticancer, anti-inflammatory and anticoagulant activities, etc in modern pharmacological experiments [ ] . abundant compounds were isolated from it, such as chromones, conmarins, and polyacetylenes [ , ] . prime-o-glucosylcimifugin is a major active chromone isolated from fangfeng. in recent years, it has been identified that chromones are the main active components which contribute most to its pharmacological efficacy [ ] , but so far, the anti-inflammatory effects of prime-o-glucosylcimifugin on ali has not yet been studied. therefore, with a mouse model of acute lung inflammation, the present study was undertaken to examine the effect of prime-o-glucosylcimifugin on acute lung injury induced by intranasal instillation of lps in balb/c mice and investigate its possible mechanisms. prime-o-glucosylcimifugin was purchased from the national institute for the control of pharmaceutical and biological products (jilin, china). fetal bovine serum (fbs), dulbecco's modified eagle's medium (dmem), penicillin and streptomycin for cell culture were purchased from invitrogen-gibco (grand island, ny, usa). -( , dimethylthiazol- -y )- , -diphenyltetrazolium bromide (mtt), dimethyl sulfoxide (dmso) and lipopolysaccharide (lps) (escherichia coli :b ) were purchased from sigma chemical co. (san diego, ca, usa). rabbit polyclonal anti-p erk, mouse monoclonal phosphospecific p -p erk antibodies, rabbit polyclonal anti-p jnk, mouse monoclonal phospho-specific p -p jnk antibodies, rabbit polyclonal anti-p , mouse monoclonal phospho-specific p antibodies, rabbit mab iκbα and mouse mab phospho-iκbα were purchased from cell signaling technology inc. (beverly, ma). hrp-conjugated goat antirabbit and goat-mouse antibodies were provided by ge healthcare (buckinghamshire, uk). mouse tnf-α, il- and il- β enzyme-linked immunosorbent assay (elisa) kits were purchased from biolegend (ca, usa). the myeloperoxidase (mpo) determination kit was purchased from jiancheng bioengineering institute of nanjing (nanjing, jiangsu province, china). all other chemicals were of reagent grade. balb/c male mice, weeks old and weighing approximately to g, were purchased from the center of experimental animals of baiqiuen medical college of jilin university (jilin, china), and maintained at an animal facility under pathogen free conditions. the mice were fed a standard diet and water ad libitum and housed in microisolator cages under standard conditions (temperature: ± °c, relative humidity: %- %). the mice were allowed to adapt themselves to their environment for - days before experimentation. all animal experiments were performed in accordance with the national institutes of health (nih) guide for the care and use of laboratory animals and approved by the jilin university animal administration committee. the raw . mouse macrophage cell line was obtained from the china cell line bank (beijing, china). cells were cultured in complete medium (dmem supplemented with % heat-inactivated fbs, mm glutamine and antibiotics ( u/ml penicillin and u/ml streptomycin)) at °c in a humidified incubator containing % co and % air. cells were treated with various concentrations of prime-o-glucosylcimifugin for h followed by stimulation with lps ( mg/l). cytotoxicity studies induced by prime-o-glucosylcimifugin were evaluated by the mtt assay. raw . macrophages were seeded in -well plates at a density of × cells/ml in complete medium and incubated for h ( μl/well). then the cells were treated with different concentrations of prime-o-glucosylcimifugin ( - μg/ml, μl/well) for h, followed by stimulation with lps ( mg/l, μl/well) for h. after h, μl mtt ( g/l) was added to each well and the cells were further incubated for h. the supernatant was removed and the cells were lysed with μl/well dmso. the optical density was measured at nm on a microplate reader. raw . cells were seeded in -well plates ( × cells/ml) and treated with . , or μg/ml of prime-o-glucosylcimifugin for h prior to stimulation of mg/l lps for h in a °c, % co incubator. cell-free supernatants were collected and assayed. the concentrations of tnf-α, il- β and il- in the supernatants of raw . cell cultures were measured by using an elisa kit, according to the manufacturer's instructions (biolegend, inc., camino santa fe, suite e, san diego, ca, usa). raw . cells ( × cells/ml) plated onto -well plates were incubated for h and treated with . , or μg/ml of prime-o-glucosylcimifugin for h and then stimulated with mg/l of lps for min. the cells were collected and washed three times with ice-cold pbs. the cells were treated with a cell lysis buffer [ mm tris (ph . ), mm nacl, mm edta (ph . ), . % np- , mm na vo , mm β-glycerophosphate, mm phenylmethylsulfonyl fluoride, mm p-nitrophenyl phosphate, and : complete mini protease inhibitor cocktail (boehringer, mannheim, germany)] and kept on ice for min. the cell lysates were centrifuged ( , g at °c) for min to obtain a cytosolic fraction. the protein concentration was determined by bca protein assay kit (beyotime, haimen, china). aliquots of the lysates were separated on % sodium dodecyl sulfate (sds)polyacrylamide gel electrophoresis (page) and then electroblotted onto a polyvinylidene difluoride (pvdf) membrane. the blots were blocked with % (w/v) non-fat dry milk for h at °c, followed by incubation with specific primary antibody at °c overnight. blots were washed with tween /tris-buffered saline [ttbs, mm tris-hcl buffer, ph . , containing mm nacl and . % (vol/vol) tween ] and incubated with a peroxidase-conjugated secondary antibody for h. blots were again washed with ttbs and the immunoactive proteins were detected using ecl plus (thermo, usa). the mice were randomly divided into five groups: control group; lps group; lps + prime-o-glucosylcimifugin ( . , or mg/kg bodyweight). prime-o-glucosylcimifugin was given intraperitoneally. one hour later, lps group and lps + prime-o-glucosylcimifugin group mice were given μl lps intranasally (i.n) ( mg/l) to induce acute lung injury. control mice were given μl pbs intranasally (i.n) without lps. all the mice were alive after h lps stimulation. collection of balf was performed three times through a tracheal cannula with . ml of autoclaved pbs, instilled up to a total volume of . ml. balf was centrifuged ( °c, rpm, min) to pellet the cells. the concentrations of tnf-α, il- β and il- in the balf were measured using sandwich enzyme-linked immunosorbent assay (elisa) kits according to the protocol recommended by the manufacturer. the cell pellets were resuspended in pbs, and the total cell number was counted using a standard hemocytometer. differences in cell numbers were examined by counting on a smear prepared by wright-giemsa staining. seven hours after intranasal instillation of lps, the mouse lungs were excised, and immediately weighed to obtain the wet weight. the dry weight was determined after heating the lungs at °c for h. the w/d ratio was calculated by dividing the wet weight by the dry weight. mpo activity, which reflects the parenchymal infiltration of neutrophils and macrophages, was measured as described previously [ , ] . mice were killed h after lps administration under diethyl ether anesthesia. lung tissues were homogenized in mm hydroxyethyl piperazine ethanesulfonic acid (hepes) (ph . ) containing . % cetyltrimethyl ammonium bromide (ctab) and subjected to three freeze-thaw cycles. the homogenate was centrifuged at , ×g for min at °c, and the cell-free extracts were stored at − °c until further use. the mpo activity was assayed using a mouse mpo elisa kit. samples were diluted in phosphate citrate buffer (ph . ). histopathologic examination was performed on mice that were not subjected to balf collection. the lungs were excised and fixed in % buffered formalin. then the tissues were dehydrated with graded alcohol, embedded in paraffin and sliced. the sections were stained with hematoxylin and eosin (h&e) and pathological changes of lung tissues were observed under a light microscope. all values are presented as mean ± sd. data were entered into a database and analyzed using spss software (spss for windows version . , chicago, usa) and comparison between groups was made with one-way anova (dunnett's t-test) and student's t-test. p-values of . or less were considered statistically significant. to assess the suitable concentration of prime-o-glucosylcimifugin for the study, raw . cells were incubated with prime-oglucosylcimifugin at concentrations ranging from . to mg/l in the absence or presence of lps and cell viability was measured by mtt test h later. the results showed that prime-o-glucosylcimifugin at concentrations from . to mg/l had no cytotoxic effect on raw . cells (fig. ) . tnf-α, il- β, il- and il- concentrations in the culture supernatant of raw . macrophages were measured by elisa kits (fig. ) . raw . macrophages treated with lps alone produced significant amounts of tnf-α, il- β, il- and il- compared to the control group. however, the production of tnf-α was slightly decreased while the levels of il- β and il- were significantly inhibited in a dose-dependent manner when the cells were treated with . , or mg/l of prime-o-glucosylcimifugin ( ⁎ p b . , ⁎⁎ p b . ). in contrast, the concentrations of il- was significantly increased when the cells were treated with mg/l of prime-o-glucosylcimifugin (p b . ⁎ ). in order to investigate the mechanism by which prime-oglucosylcimifugin inhibits lps-induced cytokine production, we examined the levels of lps-induced phosphorylation of erk / , jnk and p mapk in the cytoplasm by western blotting analysis using three different phosphor-special antibodies. in our study, fig. a showed that lps stimulation significantly increased the phosphorylation of erk / , jnk and p . however, prime-o-glucosylcimifugin inhibited the phosphorylation of erk / , jnk and p in lps-induced cells. there were no changes in the expression of non-phosphorylated mapks among groups. furthermore, we examined the effect of prime-o-glucosylcimifugin on iκbα phosphorylation and degradation. the results showed that lps-induced iκbα degradation was inhibited after pretreatment with prime-o-glucosylcimifugin in a dose-dependent manner (fig. b ). balf was collected at h after lps administration and the cytokine levels in balf were measured by elisa according to the manufacturer's instructions and as described in the materials and methods section. the levels of tnf-α, il- β and il- in balf were increased dramatically compared with control group (fig. ) . however, pretreatment with prime-o-glucosylcimifugin ( . , or mg/kg) significantly down-regulated the levels of tnf-α, il- β and il- in a dose-dependent manner ( ⁎ p b . , ⁎⁎ p b . ). seven hours after lps administration, the balf was collected to evaluate the total cell counts and the number of inflammatory cells in balf, such as macrophages and neutrophils. as shown in fig. , lps challenge markedly increased the number of total cells, neutrophils, and macrophages compared to the control group (p b . ). in addition, pretreatment with prime-o-glucosylcimifugin was found to significantly decrease the number of total cells, neutrophils and macrophages ( ⁎ p b . , ⁎⁎ p b . ). to evaluate lps-induced changes in pulmonary vascular permeability to water, we evaluated the wet weight to dry weight ratio of the lungs. the lung w/d ratio was evidently higher at h after lps administration compared with the control mice as illustrated ( ⁎⁎ p b . ). pretreatment of mice with prime-o-glucosylcimifugin significantly reduced the water gain (fig. ) ( ⁎ p b . , ⁎⁎ p b . ) . balf was collected at h following lps challenge to analyze the inflammatory cytokines tnf-α (fig. a) , il- β (fig. b ) and il- (fig. c) . the values presented are the mean ± sem (n = in each group). ## p b . vs. control group, ⁎ p b . , ⁎⁎ p b . vs. lps group. the mpo activity (fig. ) was determined to assess the effects of prime-o-glucosylcimifugin on neutrophil accumulation within pulmonary tissues. lps challenge resulted in significantly increased lung mpo activity compared with the control group ( ⁎⁎ p b . ). however, this increase was reduced by prime-o-glucosylcimifugin ( ⁎ p b . , ⁎⁎ p b . ). to evaluate the effect of prime-o-glucosylcimifugin on ali, we observed histological changes after prime-o-glucosylcimifugin treatment in lps-treated mice. in the lps group, the lungs were significantly damaged with inflammatory cell infiltration, alveolar wall thickening and interstitial edema. in contrast, prime-o-glucosylcimifugin ( . , or mg/kg) was found to decrease these histopathological changes (fig. ) . the results of this study indicate that prime-o-glucosylcimifugin had a promising anti-inflammatory activity. treatment with prime-o-glucosylcimifugin before lps challenge can attenuate lps-induced inflammatory responses in raw . cells and significantly protect mice against lps-induced ali. the study primarily focused on anti-inflammatory effects of prime-o-glucosylcimifugin on lps-stimulated raw . cells. lps, a glycolipid that constitutes the major portion of the outermost membrane of gram-negative bacteria, can bind to the cell membrane receptor of the monocytes/macrophages and endothelial cells, then activate the signal-transduction system, thus resulting in the synthesis and release of cytokines and inflammatory mediators [ ] . excessive production of pro-inflammatory cytokines not only enhances immune responses by fighting invading pathogens, but also has deleterious effects like perturbing regular hemodynamic and metabolic balances [ ] . thus, it is an important target in the treatment of inflammatory diseases to inhibit the pro-inflammatory mediator. as shown in fig. , the levels of the pro-inflammatory cytokines tnf-α, il- β and il- were down-regulated while the level of il- was up-regulated in the prime-o-glucosylcimifugin group. il- , an anti-inflammatory, is well known to down-regulate the production of tnf-α, il- β, il- , il- and no [ ] [ ] [ ] . a number of cytokines in combination with endotoxin can cause expression of inducible nitric oxide synthase (inos) in macrophages [ ] . inducible nos is an important pharmacological target in inflammatory and mutagenesis research. it has been proved that prime-o-glucosylcimifugin had inhibitory effect on the synthesis of no induced by lps in raw . cells [ , ] . combined with the finding of our study, it seems like prime-o-glucosylcimifugin inhibits the synthesis of no by producing il- in raw . cells. the anti-inflammatory actions of il- can interfere with the production of pro-inflammatory cytokines through the suppression of nf-κb activation by preserving the expression of iκb protein. nf-kb is a key transcriptional factor involved in regulating the expression of proinflammatory mediators, including cytokines, chemokines, and adhesion molecules, thereby playing a critical role in mediating inflammatory responses [ , ] . under resting conditions, nf-κb is held inactive by iκb. however, nf-κb can be activated by some stimulation of various receptors including tnf receptor, toll-like receptors (tlrs) and t-cell receptor (tcr). persistent activation of nf-κb is central to the pathogenesis of many inflammatory lung disorders including chronic obstructive pulmonary, asthma, pneumonia, and acute lung injury [ ] . the activation of nf-κb is implicated in the mapk signaling pathway. inhibition of mapk family pathway, such as erk, p , and jnk, alleviates the production of pro-inflammatory cytokines [ ] . therefore, we investigate the possibility that prime-o-glucosylcimifugin inhibits the production of tnf-α, il- β, and il- via interfering with the activation of mapk and nf-κb. the results showed that prime-oglucosylcimifugin obviously not only inhibited nf-κb activation, but also inhibited lps-induced phosphorylation of erk / , jnk and p in raw . cells (fig. ) . based on the above observations, our results suggest that prime-o-glucosylcimifugin had antiinflammatory ability by suppressing the expression of pro-inflammatory cytokines through blocking the activation of mapk and nf-κb pathways in vitro. acute lung injury is characterized by systemic airway inflammatory response including cytokines (e.g., tnf-α, il- , il- ), chemokines, pro-inflammatory mediators and a variety of cells, which regulate the migration and pulmonary infiltration of neutrophils into the interstitial tissue [ ] . neutrophils are an important component of the inflammatory response that characterizes acute lung injury (ali) [ ] . once an inflammatory response is initiated, neutrophils are the first cells to be recruited to sites of infection or injury. although neutrophils have beneficial actions in eradicating microbial infections, excessive neutrophil activation, with resultant release of cytokines and other pro-inflammatory mediators, results in tissue injury and contributes to the development of organ dysfunction, such as ali [ ] . high levels of pro-inflammatory cytokines, such as tnf-α, il- β and il- , perform a central role in the initiation and propagation of the inflammatory cascade in lps-induced ali [ ] . cytokines, such as tnf-α, il- β, il- , il- , and il- , that are secreted by alveolar macrophages stimulate more chemotaxis and attract more neutrophils to injured lungs [ ] [ ] [ ] . in our study, the levels of tnf-α, il- β and il- in balf were lower in the prime-o-glucosylcimifugin group than in the lps group. these reductions may have contributed to the decreased neutrophil count in balf in the lps-induced ali model treated with prime-o-glucosylcimifugin. edema is a typical symptom of inflammation both in systemic inflammation and in local inflammation [ ] . permeability edema which accompanies acute lung injury, severe pneumonia and acute respiratory distress syndrome (ards) is associated with alveolar fluid clearance capacity reduction, increase of capillary endothelial permeability, and alveolar epithelial barrier disruption [ ] . to quantify the magnitude of pulmonary edema, we determined the w/d ratio of the lung tissue and prime-o-glucosylcimifugin was shown to inhibit this ratio. on the other hand, mpo is an enzyme located mainly in the primary granules of neutrophils, thus mpo activity in the parenchyma reflects the adhesion and margination of neutrophils in the lung [ ] . in the lps-induced ali model, a large amount of pmn is recruited from peripheral blood into the lung, producing a substantial amount of mpo and reactive oxygen derivatives, and finally resulting in a cascade-like response and tissue damage [ , ] . by contrast, administration of prime-o-glucosylcimifugin markedly suppressed lpsinduced balf neutrophilia (fig. ) and mpo activity (fig. ) , as well as ameliorated the histopathologic changes in lung tissue produced by lps challenge (fig. ) . recent studies that have been conducted on pravastatin [ ] , ceftiofur [ ] , and pinocembrin [ ] showed the same regulatory effects as prime-o-glucosylcimifugin, suggesting that this agent may be an important regulator of inflammatory responses. taken together, these results indicate that the protective effects of prime-oglucosylcimifugin on acute lung injury in a mouse model induced by lps may be due to its inhibition of inflammatory mediators and limitation of inflammatory response in the lung. in conclusion, the findings of this study showed that prime-oglucosylcimifugin has a promising anti-inflammatory effect and a protective effect against lps-induced ali. pretreatment with prime-o-glucosylcimifugin inhibited the release of in vitro and in vivo inflammatory responses by counteracting mapk and nf-κb activation. these findings strongly suggest that prime-o-glucosylcimifugin has a potent anti-inflammatory activity and may represent a novel strategy for the modulation of inflammatory responses. further studies are warranted to investigate the clinical usefulness of prime-oglucosylcimifugin. pathology and pathogenesis of severe acute respiratory syndrome new insights into the pathology of acute respiratory failure prevention of lps-induced acute lung injury in mice by mesenchymal stem cells overexpressing angiopoietin fas/fasl-dependent apoptosis of alveolar cells after lipopolysaccharide-induced lung injury in mice endotoxin-induced lung injury in mice: structural, functional, and biochemical responses computational identification of key biological modules and transcription factors in acute lung injury effect of surfactant on pulmonary expression of type iia pla( ) in an animal model of acute lung 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acute lung injury myeloperoxidase: friend and foe pulmonary neutrophil infiltration in murine sepsis: role of inducible nitric oxide synthase protective effect of pravastatin on lipopolysaccharide-induced acute lung injury during neutropenia recovery in mice ceftiofur attenuates lipopolysaccharide-induced acute lung injury in vitro and in vivo protection provided by pinocembrin against lipopolysaccharide-induced inflammatory responses this work was supported by grants from the national nature science foundation of china (no. ) and the china postdoctoral science foundation (no. ) . key: cord- - wxih v authors: you, qinghai; wang, jinmei; jia, dan; jiang, lijuan; chang, yuanmin; li, wenmei title: mir- alleviates lipopolysaccharide-induced acute lung injury by targeting peli date: - - journal: inflamm res doi: . /s - - -z sha: doc_id: cord_uid: wxih v introduction: acute respiratory distress syndrome (ards) is a life-threatening medical condition. it is characterized by serious lung inflammation or injury. characterizing novel mirnas implicated in ards pathogenesis may provide new therapeutic strategy for managing ards. methods: we employed lps-induced lung injury model to profile mirnas associated with ards. we isolated one mirna candidate and characterized its role in lipopolysaccharide (lps)-induced proinflammatory cytokine production in lung macrophages. we further evaluated its functional role in ards model by assessing histological change, neutrophil activation, tissue permeability and tumor necrosis factor alpha (tnfα) production. we also characterized its downstream target using luciferase assay, western blotting, enzyme-linked immunosorbent assay and cell inflammation assay. results: microarray profiling revealed mir- was significantly downregulated in ards mouse model. lps-induced mir- downregulation was confirmed in lung macrophages. overexpression of mir- significantly suppressed lps-induced inflammatory cytokine production in vitro and alleviates lps-induced acute lung injury in vivo. peli was identified as a downstream target of mir- and found upregulated in ards model. overexpressing peli abolished the antagonizing effect of mir- on lps-mediated inflammatory response. conclusion: mir- carried a protective role against lps-induced acute lung injury by downregulating peli . mir- /peli axis may act as intervening targets to manage ards. acute respiratory distress syndrome (ards) is a severe lung inflammatory disorder commonly characterized by infection or injury inducing the development of diffuse alveolar damage that results in severe hypoxemia. it remains a lethal or disabling medical condition. in intensive care unit (icu), the incident rate of patients attributed to ards is high. the mortality rate of patients with severe ards is nearly % as estimated [ ] . even though patients who recover from this disorder are still at high risk for depression, cognitive decline, and persistent skeletal muscle weakness and post-traumatic stress disorder. risk factors for ards include factors that lead to direct or indirect lung injury. for instance, pulmonary contusion, inhalation injury, pneumonia, aspiration of gastric contents and drowning are the common direct lung-injury factors, while pancreatitis, sepsis, major burn injury, drug overdose, transfusion of blood products and cardiopulmonary bypass can indirectly cause lung injury that leads to ards. based on the recent clinical findings, more than % of ards cases are attributed to sepsis, pneumonia and aspiration of gastric contents [ ] . lung injury-induced inflammatory tissue damage is the core pathogenesis event of ards. initial response to injury is characterized by innate cell-mediated inflammation. the prominent innate cell type activated at this early stage is resident alveolar macrophages that subsequently secrete proinflammatory cytokines or chemokines to promote the activation of alveolar epithelial and endothelial cells and recruitments of monocytes and neutrophils. the subsequent activation of alveolar epithelial and endothelial cells contributes to the damage of barrier function and the accumulation of edema fluid flooding in injured lung tissue. the local accumulation of monocytes and neutrophils produces more inflammatory mediators and results in prolonged tissue damages. uncontrolled ards leads to increased mortality [ ] . though clinical validated biomarkers for ards are yet to be defined, common pathological features associated with ards include tissue inflammation, alveolar edema and increased lung permeability. therefore, diffuse alveolar damage is widely recognized as a histological hallmark associated with ards [ ] . animal models of ards, such as sepsis-or ventilator-induced acute lung injury, have been demonstrated to recapitulate these pathological features in human patients. they are very useful to study the ards pathological factors and mechanisms or evaluate the therapeutic factors or agents for managing ards [ , ] . the dysregulated mirna expression can contribute to the pathogenesis of many diseases including pulmonary disorders, chronic inflammation and cancers [ ] . the potential roles of mirnas in the pathogenesis and progression of ards have been reported recently, suggesting a complexity underlying the pathological mechanism of ards [ , ] . elucidating novel mirna players in ards may provide new biomarkers and therapeutic candidates for managing the disorder. in this study, our aim was to characterize a novel mirna associated with ards and provide scientific insight into therapeutic development to treat ards. we used sepsis-induced lung injury as ards animal model to profile mirna expression pattern. the mirna candidate was further functionally characterized in both in vitro and in vivo models. we also identified and validated a downstream target of the mirna in ards. the established signaling axis revealed a novel molecular mechanism underlying ards and provided a new avenue to develop therapeutics for ards. primary alveolar macrophages were isolated from lungs by bronchoalveolar lavage and cultured in dulbecco's modified essential medium (dmem)/ham's f- medium, supplemented with % fetal bovine serum (fbs), u/ ml streptomycin and u/ml penicillin. raw . and a were purchased from atcc and maintained in dmem or roswell park memorial institute (rpmi)- medium supplemented with % fbs, % glutamine and antibiotics at °c and % co . cells were passaged when they reached % confluence. cells were treated with µg/ml lipopolysaccharides (lps, sigma, st. louis, mo) or dmso following the time points as indicated in the study. bms , sb and sp were purchased from santa cruz. total rna from the sample tissue after the treatment was harvested using trizol method (invitrogen, waltham, ma usa). first-strand cdna was labelled with cy or cy and used for hybridization reaction on mouse genechip mirna . array from thermo fisher. fluorescence images were acquired using affymetrix gcs scanner. bioinformatics analysis was performed using the partek genomics suite software. only fold changes more than two between two groups were considered significant. we included three mice for the control and ards group, respectively. the ards mouse model was established by sepsis induction as previously described [ ] . briefly, male balb/c mice were divided randomly into four groups with subjects in each group: ( ) sham control group (receiving saline + scramble control mirna mg/kg); ( ) lps group (receiving lps mg/kg + scramble control mirna mg/ kg); ( ) sham/mirna group (receiving saline + mirna mg/kg); ( ) lps/mirna group (receiving lps mg/ kg + mirna mg/kg). the mirnas were delivered using invivofectamine . as described previously [ ] . h later, the drugs were delivered by intratracheal instillation. mice were killed at day upon lps challenge. the lung tissues were collected for further analysis. the animal study was carried out according to the ethical guidelines approved by animal care and use committee in the first affiliated hospital of anhui medical university. mice lung tissues were harvested at the end of the study. the neutrophil activation levels in the tissues were quantified by mpo activity assay kit (ab ) from abcam following the standard manual. we included eight mice for each group. to measure lung permeability, evan's blue dye ( mg/kg, sigma) was intravenously injected into the mice on the last day of the study. one hour after the injection, the mice were killed and the lungs were harvested and homogenized as previously described [ ] . the supernatants containing the dye were collected from the homogenates by centrifugation and quantified in a plate reader at nm and nm. the corrected value was calculated by abs (corrected) = abs − ( . × abs + . ). we included eight mice for each group. apoptotic cells in the harvested lung tissues were detected by dutp nick end-labelling (tunel) staining kit (roche diagnostics) following the manufacturer's manual. the apoptosis rate was calculated as the ratio of tunel-positive cells over total cells as stained by dapi. mir- mimic and scramble control were synthesized by qiagen. the cells were transfected with the mirnas using hiperfect transfection reagent (qiagen, valencia, ca, usa) following the manufacturer's manual. other gene transfections were done with lipofectamine . to measure tumor necrosis factor alpha (tnfα) secretion in lung tissue, balf from the study groups were collected and processed using mouse tnfα elisa kit from abcam according to the standard protocol. peli protein expression was determined in homogenized lung tissues using a customized elisa kit from cusabio biotech. we included eight mice for each group. the 'utr of peli containing the wild-type or mutated mir target site was cloned into the luciferase reporter plasmid (pmir-report). renilla luciferase vector (prl-sv ) was used as normalization control. both mir- mimic and luciferase reporter plasmids were co-transfected into balf macrophage. the luciferase activity was determined h post-transfection with the dual luciferase assay kit (promega, madison, wi, usa) using a plate reader following the manufacture's instruction. cells were harvested in radioimmunoprecipitation assay (ripa) buffer supplemented with protease inhibitor cocktail (roche, penzberg, upper bavaria, germany). µg protein lysate was first resolved in % sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) and then transferred onto a nitrocellulose membrane. the membrane was blocked with % non-fat milk for h at room temperature and then incubated with primary antibody ( : ) for overnight at °c. on the nd day, the membrane was washed with pbst before incubating with a nd antibody ( : ) for h at room temperature. the target protein was detected using supersignal west pico plus chemiluminescent kit. peli and nlrp antibodies were from abcam. ubiquitin and gapdh antibodies were purchased from santa cruz. total rna including mirna were harvested using mirvana mirna isolation kit (ambion). one microgram of mrna was converted to cdna using high-capacity cdna reverse transcription kit. and mirna first-stand cdna was synthesised using miscript ii rt kit from qiagen. the gene expression was measured by sybr green kit from sigma-aldrich on quantstudio (applied biosystems, waltham, ma, usa) following the standard protocol. the primer sequences are listed as follows: the statistical analysis was done using spss software. the data were presented as mean ± standard deviation (sd). the unpaired student's t test was employed to compare the differences between the groups. one-way anova analysis followed by a tukey's post hoc test was applied to the comparison of more than two groups. only p value less than . was considered significant. to elucidate the mirna candidates implicated in the pathogenesis of ards, we profiled the expression changes of mirnas in a lps-induced ards mouse model. lps was administrated intratracheally to induce acute lung injury. h later, the lung tissues were harvested and processed for microarray analysis of mirna gene expression. a list of mirnas showing most significant changes is shown in fig. a . among those, mir- was one of the most significantly downregulated targets in ards lung tissue. the role of this candidate in ards or other lung injury has not been reported, which made it a novel mirna in ards field. to explore its function in ards, we subsequently isolated alveolar macrophages from lungs by bronchoalveolar lavage. the primary macrophages were cultured and challenged with lps. we found lps stimulation reduced the expression of mir- to almost fourfold (fig. b) . this was consistent with the in vivo result. we also confirmed the lps-mediated suppression of mir- in raw . , an immortalized monocyte/macrophage line (fig. c) . interestingly, when we challenged a , an alveolar basal epithelial cell line, no mir- reduction was detected (fig. d) . therefore, mir- in lung tissue was suppressed by lps and the reduction was largely attributed to the response in macrophages. next, we proceeded to elucidate the role of mir- in lps-induced lung injury. macrophages are a key cell type in lung in response to lps challenge and proinflammatory cytokine production is a critical step that mediates lpsinduced tissue damage. since mir- was found reduced by lps in alveolar macrophages, we restored the mirna expression in the cells and examines its impact on proinflammatory response induced by lps. as shown (fig. a) , macrophages challenged with lps produced high level of tnfα, while overexpressing mir- before lps stimulation greatly suppressed the tnfα expression. we observed similar suppressing effects of mir- in lps-mediated interleukin- beta (il- β) and il- induction (fig. b, c) . therefore, we concluded mir- expression may antagonize proinflammatory cytokine production during lpsmediated lung inflammatory response and tissue damage. independently prolonged lps challenge could induce cell apoptosis. as shown (fig. d) , lps increased cell death rate in macrophages as measured by tunel assay. however, mir- expression did not change the lps effect on cell apoptosis. therefore, the regulatory function of mir- may be specific to inflammatory pathway. to evaluate the effect of mir- on lps-induced lung acute injury in vivo, the mice were administrated with mir- or scramble control intragastrically before being subjected to sepsis challenge. first, we analyzed the morphological change of lung tissues with h&e staining. as shown (fig. a) , sepsis group showed increasing neutrophil infiltration, alveolar hemorrhage, lung edema, and compromised epithelial/endothelial cell structure compared with the sham group. interestingly pre-treatment of mir- significantly reduced tissue damage and led to a significant improvement on lung morphology. second, we determined the neutrophils activation by measuring myeloperoxidase (mpo) activity in lung tissues. as shown (fig. b) , mpo activity increased greatly in lps-challenged lung tissues and the delivery of mir- reduced the increased mpo activity in damaged lung. lps challenge also increased the permeability of the lung tissues as measured by evan's blue staining (fig. c) . in mir- -treated group, the lps-mediated protein leakage in lung tissues was significantly improved (fig. c) . last but not least, since the suppression effect of mir- in inflammatory cytokine production was found in vitro, we proceeded the measure level in the lung tissues. as expected, lps administration caused a significant induction of tnfα in the damaged tissues and overexpressing mir- markedly antagonized the accumulation of the proinflammatory factor (fig. d) . taken together, the in vivo study suggested a protective role of mir- in lps-induced ards model. next, we sought to identify the downstream target of mir- in lung macrophages. we analyzed the putative targets of mir- in mirbase [ ] . among them peli carried a mir- target site in its 'utr, which was conserved in both mouse and human (fig. a) . peli is an e ligase that mediates the inflammatory response in response to lps [ ] . we hypothesized that mir- may target peli to suppress lps challenge. therefore, we constructed luciferase reporter gene (denoted as wt), in which peli 'utr containing the mirna target site was cloned to the downstream of the coding sequence. as comparison, we constructed the mut vector in which the mirna target site in peli 'utr was mutated (fig. a) . to access the binding, we co-transfected the wt vector with either mir- or scramble control. as shown (fig. b) , mir- but not scramble mirna markedly reduced the luciferase activity. in contrast, co-transfection of mir- with the mut vector did not impact on the luciferase activity (fig. b) . to confirm the direct regulation of mir- on peli , we expressed either the mirna or the scramble control in alveolar macrophages and then measured peli protein level. as shown (fig. c) , overexpression of mir- markedly decreased the protein expression of peli . functionally peli controls the activation nlrp inflammasome by promoting its ubiquitination in response to lps [ ] . thus, we proceeded to measure the effect of mir- in nlrp activation by lps. as shown (fig. d) , lps stimulation promoted the accumulation of k -linked ubiquitinated nlrp , which was an indicator of nlrp next, we sought to confirm the functional relevance of peli in mir- -mediated inflammation suppression in ards model. in ards mouse model, mir- was found downregulated. as the hypothetical target of the mirna, peli level would increase in response to lps challenge in lung tissues. we then compared peli protein expression in lung samples between lps or sham groups. as shown (fig. a) , as expected, peli protein concentration was significantly higher in lps group. in the rescue study, we examined the effect of peli in mir- -mediated inflammation inhibition in primary macrophages with lps challenge. as shown (fig. b) , co-transfection of peli with mir- abolished the antagonizing effect of the mirna on lps-induced tnfα expression. similarly, overexpression of peli was also found to abrogate the inhibition of mir- on il- β and il- production primed by lps (fig. c, d) . overall, these results indicated mir- targeted peli to suppress lps-induced lung inflammation reaction. last but not least, we explored the mechanistic regulation of lps on mir suppression. lps binds to toll-like receptor (tlr ) that leads to tak activation. tak is a pivotal regulator that subsequently activates nuclear factor kappa-light-chain enhancer of activated b cells (nfκb), p and c-jun n-terminal kinase (jnk) sub-pathways [ ] . we treated primary macrophages with specific inhibitors targeting these downstream factors, including bms , a selective inhibitor of iκb kinase to block nfκb activation, sb and sp that directly target p and jnk, respectively. after h, we challenged the cells with lps and measured the effect on mir suppression. as shown in fig. a , only bms but not the other two inhibitors could disrupt the suppression effect on the mirna. to confirm this, we also repeated the same treatment on raw . . consistently only nfƙb could specifically abolish lps-mediated mir reduction (fig. b) . taken together lps-induced mir suppression was nfƙb pathway dependent (fig. c ). with mirna profiling, we identified a list of candidates that may play parts in ards model. at the top of the list, mir- , mir- , mir- , mir- , mir- , and mir- are found significantly downregulated in the disease model. the upregulated candidates are mir- a and mir- b. in this study, we have elucidated the role of mir- in ards. specifically, the mirna downregulation has been confirmed in lps-challenged lung macrophages but not epithelial cells. restoring the mirna expressing level in lps-challenged macrophages can reduce the overproduction of proinflammatory cytokines such as tnfα, il- β and il- . however, the overexpression of mir- cannot inhibit the cell death challenged by lps, suggesting the regulation of mir- in macrophages may be specific to proinflammatory pathway. the benefits of overexpressing mir- in ards can be extended to the in vivo model. we have observed the alleviation in neutrophil activation, tissue permeability and luciferase reporter assay study of the interaction between mir- and 'utr of peli . lung macrophages were first transfected with luciferase reporter genes conjugated with either wild-type (wt) or mutant (mut) 'utr of peli . after h, the cells were transfected with either mir- mimic or scramble control. transfected cells were cultured for another h before the measurement of luciferase activities in a plate reader. c western blotting analysis of peli protein expression in cells trans-fected with mir- or scramble control. gapdh was used as loading control. the normalized peli expression was quantified by densitometry. d western blotting analysis of nlrp ubiquitination in cells transfected with mir- or scramble control. nlrp protein was pulled down with a monoclonal antibody. the immunoprecipitated complex was resolve in sds-page and immunoblotted with ubiquitin antibody. the ubiquitination level on peli was quantified by densitometry. data were expressed as mean ± sd. independent experiments were repeated in triplicate. ***p < . significantly different from control group proinflammatory cytokine secretion in addition to the clinical improvements by histological analysis. in the same study, we have identified peli , a positive regulator in lps/tlr pathway, as downstream target of mir- in macrophages. overexpressing mir- can repress peli expression and nlrp ubiquitination. interestingly, peli expression has been found upregulated in ards model that inversely correlates with the mir- expression. more importantly, coexpression of peli in mir- -transfected macrophages can abolish the antagonizing effect of the mirna in proinflammatory pathway. last but not least, inhibiting nfκb activation but not p or jnk phosphorylation specifically abolishes lps-mediated repression of mir expression. therefore, our study has shown strong evidence to support the involvement of mir- /peli signalling axis in ards (fig. c) . acute respiratory distress syndrome as a severe form of lung injury is commonly found in critically ill patients. lack of effective therapeutic targets for ards leads to the high mortality rate of ards patients. mirnas are emerging as promising drug targets for many diseases. they can regulate the singling proteins post-transcriptionally to control the disease progression. in ards, modified expression of mirnas has been studied to develop the diagnosis and treatment for a the protein expressions of peli in lung tissues between lps-induced ards model and sham group were compared by elisa analysis (n = , for each group). the primary lung macrophages were co-transfected with mirna or peli before lps challenge. h later, the gene expression levels of b tnfα, c il- β and d il- with or without lps stimulation were quantified by real-time pcr. independent experiments were repeated in triplicate. **p < . , ***p < . , compared with dmso control; ## p < . , ### p < . compared with no peli control in the same treatment condition the diseases [ , ] . microarray-based mirna profiling has been employed to study the modified mirna expression in rat ards model [ ] . many mirnas involved in the pathogenesis of ards regulate either tissue repair or inflammatory response in lung tissue. in lps-induced ards model, mir- has been found downregulated with a similar trend as mir- in our study. overexpressing mir- can attenuate lps-mediated inflammation in the disease model. interestingly, mir- has been also found to inhibit nlrp inflammasome by targeting rho-related gtpbinding protein rhob (rhob) [ ] . in another study, mir- has been reported being elevated in bronchoalveolar lavage fluid (balf) samples of ards patients. in this case, the elevation of the mirna is likely an adaptive mechanism to the stress. in the animal experiment, the overexpression of mir- alleviates septic lung injury by targeting transforming growth factor-β-activated binding protein (tab ), a regulatory molecule under tlr signalling [ ] . in another similar study, mir- a has been characterized as a suppressor in lps-induced acute lung injury [ ] . the upregulation of mir- a inhibits the inflammatory responses by suppressing the expression of traf- and irak- . interestingly, irak is acting upstream of peli under tlr activation. the activation of peli by irak enhances its e ligase activity. the activated peli mediates the ubiquitination and activation of nlrp inflammasome [ , ] . there are increasing evidences from ards animal models supporting this signalling axis can be regulated by multiple mirnas including mir- from our study. these studies indicate tlr signalling be critical for the initiation and development of acute lung injury and the signalling components in the pathway carrying the therapeutic potential for managing ards. mir- has been studied intensively in cancer biology. generally, it has been proposed as a tumor suppressor. for instance, in cervical cancer, mir- was downregulated and the restoration of the mirna inhibits serine-/argininerich splicing factor (srsf ) to induce cancer cell apoptosis [ ] . the enforced expression of mir- can antagonize gastric cancer oncogenesis by inhibiting rab expression [ ] . similarly, mir- can also act as a tumor suppressor against tongue squamous cell carcinoma growth and metastasis [ ] . in tongue squamous cells, mir- directly regulates the expression of map k . in addition, mir- has been demonstrated in human prostate cancer to regulate epithelial-mesenchymal transition process by targeting flotillin- [ ] . therefore, the role of mir- is cell type specific and depends on the molecular targets acting downstream. our study reveals that the mirna has a novel role in controlling inflammatory response. in lung tissue particularly lung macrophages, mir- targets peli in tlr pathway. the enforced expression of mir- seems unlikely to induce any unwanted risks such as oncogenesis. therefore, targeting mir- /peli axis holds a promise to develop therapeutics for ards. currently, the first-line treatment for severe ards is extracorporeal membrane oxygenation (ecmo) or extracorporeal co elimination [ ] . early recognition and targeting intervention are crucial to improve the clinical outcomes of ards treatment in the future. newly identified mir- / peli may shed light on precise intervention therapy. to test the validity of mir- /peli in treating ards, it will be important to confirm the mirna expression change in human patients for the next step. furthermore, using sirna or small molecule inhibitor to target peli in animal ards model will also be planned to evaluate whether the fig. lps-mediated mir suppression was through nfκb pathway. a primary macrophages or b raw . cells were incubated with µm each of the following inhibitors for overnight. on the next day, the cells were challenged with µg/ml lps for overnight and harvested to measure mir expression. the representative results from at least three biological repeats were shown. data were expressed as mean ± sd. independent experiments were repeated in triplicate. **p < . , ***p < . , compared with dmso control. c schematic diagram showing the biological role of mir in lpsinduced ards model as supported in our study axis is critical for the pathogenesis of ards. however, our study only focuses on sterile inflammatory ards model induced by lps. it will be important to evaluate the role of mir- in other mouse model of ards, such as an infectious model driven by streptococcus pneumoniae or ventilator-induced acute lung injury [ , ] . in addition, to be more clinically relevant, it is also critical in the future to validate the expression pattern of mir- /peli in human ards patients before developing the intervention targeting strategy on this promising pathway. in conclusion, mir- restoration or peli inhibition may provide a new avenue to treat ards. funding the study was supported by the national natural science foundation of china ( ). ethical approval the animal study was carried out according to the ethical guidelines approved by animal care and use committee in the first affiliated hospital of anhui medical university. acute respiratory distress syndrome: the berlin definition the acute respiratory distress syndrome acute lung injury: a clinical and molecular review the acute respiratory distress syndrome mouse models of acute respiratory distress syndrome: a review of analytical approaches, pathologic features, and common measurements development of animal models for the acute respiratory distress syndrome microrna functions identification of micrornas in acute respiratory distress syndrome based on microrna expression profile in rats microrna and mrna expression profiling in rat acute respiratory distress syndrome downregulation of mir- a protects mice from lps-induced acute lung injury by targeting bcl- evaluation of microrna delivery in vivo acute downregulation of mir- a attenuates sepsis-induced acute lung injury by targeting sirt mirbase: microrna sequences, targets and gene nomenclature kinase-active interleukin- receptor-associated kinases promote polyubiquitination and degradation of the pellino family: direct evidence for pellino proteins being ubiquitin-protein isopeptide ligases the e ubiquitin ligase pellino mediates priming of the nlrp inflammasome tak , more than just innate immunity micrornas as biomarkers of acute lung injury microrna regulation of acute lung injury and acute respiratory distress syndrome microrna attenuates lpsinduced inflammation in an acute lung injury model via the nlrp inflammasome and tlr /nfkappab signaling pathway via rhob mir- alleviates septic lung injury by inducing autophagy via inhibition of transforming growth factor-beta-activated binding protein upregulation of mir- a contributes to the suppression of inflammatory responses in lps-induced acute lung injury pellino proteins contain a cryptic fha domain that mediates interaction with phosphorylated irak microrna- inhibits cell proliferation and induces apoptosis in human cervical cancer by targeting serine/arginine-rich splicing factor upregulation of mir- suppresses gastric cancer oncogenicity via targeting rab expression microrna- plays a tumour suppressive role in tongue squamous cell carcinoma through directly targeting map k microrna- inhibits epithelialmesenchymal transition through targeting flotillin- in human prostate cancer acute respiratory distress syndrome: challenge for diagnosis and therapy key: cord- -hkopopa authors: dormont, flavio; brusini, romain; cailleau, catherine; reynaud, franceline; peramo, arnaud; gendron, amandine; mougin, julie; gaudin, françoise; varna, mariana; couvreur, patrick title: squalene-based multidrug nanoparticles for improved mitigation of uncontrolled inflammation in rodents date: - - journal: sci adv doi: . /sciadv.aaz sha: doc_id: cord_uid: hkopopa uncontrolled inflammatory processes are at the root of numerous pathologies. most recently, studies on confirmed covid- cases have suggested that mortality might be due to virally induced hyperinflammation. uncontrolled pro-inflammatory states are often driven by continuous positive feedback loops between pro-inflammatory signaling and oxidative stress, which cannot be resolved in a targeted manner. here, we report on the development of multidrug nanoparticles for the mitigation of uncontrolled inflammation. the nanoparticles are made by conjugating squalene, a natural lipid, to adenosine, an endogenous immunomodulator, and then encapsulating α-tocopherol, as antioxidant. this resulted in high drug loading, biocompatible, multidrug nanoparticles. by exploiting the endothelial dysfunction at sites of acute inflammation, these multidrug nanoparticles delivered the therapeutic agents in a targeted manner, conferring survival advantage to treated animals in models of endotoxemia. selectively delivering adenosine and antioxidants together could serve as a novel therapeutic approach for safe treatment of acute paradoxal inflammation. uncontrolled inflammation is a key health challenge and is associated with numerous diseases ( ) ( ) ( ) . recently, coronavirus disease (covid- ) infections have been recognized as leading to a hyperinflammatory state characterized by a fulminant cytokine storm (hypercytokinemia) before acute respiratory distress syndrome and death ( ) . a growing understanding of the pathophysiology accompanying acute inflammation can help devise novel therapeutics for inflammatory diseases ( ) . of particular relevance is that severe inflammation is associated with significant alterations to redox balance ( ) , inducing oxidative stress to tissues and cells. evidence accumulated over the past two decades has pointed to significant connections between inflammation and oxidative stress, both processes contributing to fuel one another, thereby establishing a vicious cycle able to perpetuate and propagate the inflammatory response ( , ) . inhibiting pathological inflammatory responses and the cross-talk between oxidative stress and inflammation presents various challenges ( ) . for instance, while potent anti-inflammatory agents-such as corticosteroids-already exist, these have fallen short in acute inflammatory conditions such as sepsis, because of their negative effects on tissue repair and the reported adrenocortical insufficiency common in patients with sepsis ( ) . adenosine (ad), an endogenous purine, and ad receptor agonists have shown promise by promoting the resolution of inflammation ( , ) , but their systemic administration is associated with rapid clearance ( ) and unacceptable medical side effects related to untargeted activation of their cognate receptors ( , ) . another challenge consists in the fact that, during the evolution of systemic inflammatory insults, the initial response performed by the innate immune system is transferred from the plasma to tissues and cells where it results in disturbed signaling, cell dysfunction, and eventually organ failure. therefore, efficient therapies against such processes need to be targeted to the inflammation sites. similarly, antioxidant supplementation has been attempted ( ) ( ) ( ) to scavenge reactive species during acute inflammation but remains limited by poor pharmacodynamics and tissue penetration ( ) . recently, multidrug treatments of low-dose hydrocortisone with antioxidants have emerged as a promising approach for the mitigation of uncontrolled inflammation ( ) , simultaneously inhibiting pro-inflammatory cascades and scavenging reactive oxygen species (ros). however, so far, most of the antioxidants used in this context perform their action predominantly in the plasma. while this is useful during the initial hyperinflammatory stages of the body response, it is ineffective at inhibiting the pathological redox cycles happening inside cells and tissues ( ) -as plasma antioxidant levels poorly correlate with intracellular antioxidant levels ( ) . to improve on these issues, and taking into account the potential of ad and multidrug therapies for the resolution of inflammation, we propose here a novel prodrug-based nanoparticle (np) formulation, enabling the targeted delivery of ad and tocopherol (vite) to the sites of acute inflammation. recently, conjugation of therapeutic molecules to squalene (sq), an endogenous lipid, has been shown to enhance blood circulation time ( ) , provide interesting targeting properties ( ) and lower toxicity ( ) . here, we show that the bioconjugation of ad to sq and further nanoformulation with vite led to the formation of stable multidrug nps allowing (i) efficient encapsulation of both drugs, (ii) reduced side effects, and (iii) promising anti-inflammatory and protective effects in models of endotoxemia and lethal systemic shock. furthermore, we also report evidence that these sq-based nps could target inflamed tissues in multiple murine models of inflammation for selective ad receptor activation and antioxidant action. these functionalities together enabled a therapeutic intervention with significant potential for the antioxidant management of acute inflammatory diseases and improved the use of ad as a proresolving pharmaceutical agent. tribution with a mean hydrodynamic diameter of . ± nm and a polydispersity index inferior to . (fig. c) . surface zeta potential was measured to be − . ± . (sem) mv, similar to sqad np only (− . ± . mv), ensuring proper colloidal stability (fig. d) . the obtained multidrug nps were also observed by cryogenic transmission electron microscopy (cryo-tem) imaging, revealing uniform nps (fig. e) . following their formulation, sqad/vite nps were suspended in % serum and showed satisfactory colloidal stability over days (fig. f ). to evaluate drug release, we measured the amount of free ad and vite released from the formulation over time by high-performance liquid chromatography (hplc). the incubation of sqad/vite nps in serum resulted in a slow, progressive decrease in detected sqad bioconjugate, which correlated with a release of free ad (fig g) . after hours of incubation, about % of the initial ad was found to have been released from sqad/vite nps, while no vite release could be detected (see supplementary discussion). sq is an endogenous precursor of cholesterol, which forms stable colloidal phases in water ( ) . vite is insoluble in water and usually colocalizes with cholesterol in low-density lipoproteins (ldls) in vivo ( ) . accordingly, our results showed that vite was efficiently encapsulated in sqad nps to form stable multidrug nps. overall, the preparation of sqad/vite nps is very straightforward because it simply requires an ethanolic solution of sqad and vite to be added in water medium before ethanol evaporation. the total synthesis also did not require any excipients, which makes the scaling-up affordable as shown previously with the ability of sqad bioconjugates to be produced as an industrial sample ( ) . noteworthy, this may represent an asset over the current trend of developing ever more sophisticated nanomedicines, whose physicochemical complexity represents an important factor to slow the speed and even the feasibility of nanomedicine translation into the clinic. to investigate whether sq nps could improve the bioavailability of the encapsulated therapeutic agents and direct them to the inflammation foci, we evaluated the in vivo biodistribution of sqad/vite nps in two different models, one of local acute inflammation and one of systemic inflammation. first, the in vivo circulation of sqad/vite nps was followed after intravenous injection of fluorescent did ( , ′-dioctadecyl- , , ′, ′-tetramethylindodicarbocyanine, -chlorobenzenesulfonate salt)-labeled sqad/vite nps in a murine lipopolysaccharide (lps)-induced paw inflammation model. animals received ng of lps in their right paw and a control saline injection in the left paw. the fluorescence in tissues was monitored noninvasively up to hours, from the abdomen side using an in vivo imaging system (ivis) lumina. the real-time in vivo imaging showed that, in comparison with the control healthy left paw, a strong increase in the radiant efficiency of the inflamed right paw could be detected after intravenous injection of fluorescent sqad/ vite nps (fig. , a to e). in a control experiment, when the mice received a free did solution, no significant accumulation of fluorescence was observed in the inflamed paw ( fig. b ) (see supplementary discussion). the relative fluorescence signals detected in the right (inflamed) paw versus left (noninflamed) paw of the studied animals can be found in fig. s . in another control experiment, comparing the observed accumulation of sq nps with that of plga [poly(lacticco-glycolic) acid] nps, it was observed that the accumulation of sqad/vite nps occurred faster, providing higher in a second study, we evaluated the ability of sqad/vite nps to accumulate in organs in a model of lps-induced sepsis. loss of endothelial integrity is one of the hallmarks of sepsis, and lps injection has been shown to induce capillary leakage in a  -integrindependent mechanism ( ) . here, rhodamine b covalently linked to sq (sqrho; figs. s and s ) was used as a fluorescent marker for sqad/vite nps. animals received lps intraperitoneally and, after hours, an intravenous injection of fluorescent sqad/vite nps. after hours, the mice were deeply anesthetized and intracardially perfused with ml of phosphate-buffered saline (pbs) to remove blood. the fluorescence signal in the different organs was measured using an ivis lumina. mice that did not receive an lps challenge were used as noninflamed controls. the fluorescent imaging showed that, in comparison with healthy mice, lps-inflamed animals had a significant increase in the levels of total radiant efficiency in the lungs, liver, and kidneys (fig. ) . in a control experiment, when lps-treated mice were intravenously injected with a free rhodamine probe solution, no significant accumulation occurred in the studied organs except the kidney, most probably because of the renal clearance of rhodamine. during inflammation, neutrophils interact with the vascular endothelium leading to barrier dysfunction and increased permeability. this constitutes a unique opportunity for nanomedicines to accumulate at the sites of organ injury and selectively deliver therapeutic agents through enhanced permeation and retention effects ( ) . in the present in vivo studies, sqad/vite nps were found to accumulate at the sites of acute inflammation and endothelial dysfunction in models of both local and systemic inflammation. when injected in vivo, free ad is readily catabolized into inosine and hypoxanthine, resulting in an extremely short blood half-life of s ( ). this requires ad to be administered continuously and in high doses to achieve a pharmacological response, resulting in side effects related to the unchecked activation of the ubiquitous ad receptors. the targeted accumulation achieved by sqad/vite nps could therefore potentially limit the side effects induced by ad treatment and enhance the bioavailability of both drugs at the sites of inflammation for improved therapeutic action. to investigate whether sqad/vite nps could effectively enact protection against oxidative stress, we first developed an in vitro model of oxidative insult. in inflamed tissue, immune cells produce ros such as hydrogen peroxide (h o ) that can cross the cellular membrane and produce intracellular oxidative stress ( ) to tissue cells. when h c murine cardiac cells were incubated with h o for min, a strong increase in intracellular ros was detected by flow cytometry using a ros-sensitive fluorescent probe. treatment with sqad/ vite nps was efficient at limiting intracellular ros production in a dose-dependent manner (fig. , a and b ). while sqad np and sq np controls did not provide this protection, free drug ad/vite in the medium did induce some protection against the oxidative insult, most probably because of the antioxidant action of vite. this effect was less pronounced than with sqad/vite nps. the reduction in intracellular ros correlated positively with improved cell survival to the oxidative insult as measured by propidium iodide straining. in sqad/vite np-treated samples, only % of cells were found to be necrotic, while nontreated cell populations contained % of necrotic cells (fig. , c and d). sqad/vite nps were found to be readily taken up by cells ( fig. s ), likely through ldl receptormediated mechanisms as shown previously with sqad-only nps ( ) . thus, after accumulating at the sites of inflammation, sqad/ vite nps are most likely able to enter cells, where they can deliver their therapeutic cargo intracellularly. as it has been suggested ( , ) , and as demonstrated here, the intracellular delivery of the antioxidant vite improved its capacity to diminish oxidative stress. this cellular uptake also likely allows sqad/vite nps to generate the active ad in a localized manner after hydrolysis of sqad ( ) . it was previously observed that after ldl receptor-dependent internalization, the sqad nps located in endo-lysosomal compartments where they acted as intracellular reservoirs for the encapsulated ad. once the hydrolysis of the amide bond released ad, equilibrative nucleoside transporters (ents) discharged the drug ad outside of the cell where it interacted with ad receptors ( ). data are mean ± sd. *p < . , **p < . , and ****p < . (two-way anova followed by tukey's multiple comparison test). we next evaluated the ability of sqad/vite nps to efficiently inhibit pro-inflammatory signaling. for this, raw . macrophages were used in an in vitro lps-induced inflammation model. raw macrophages respond to lps stimulation by releasing pro-inflammatory cytokines ( ) . accordingly, when raw macrophages were stimulated with lps ( g/ml), a significant increase in tumor necrosis factor- (tnf-) cytokine was measured in the supernatant. this pro-inflammatory response was inhibited by treatment with sqad/ vite nps at a concentration of g/ml for hours. here, sqad and sqad/vite nps had similar effects on the release of inflammatory cytokines, indicating that ad could be the main effector of the observed anti-inflammatory effect (fig. e) . when sqad/vite treatment was performed simultaneously to the lps challenge, no significant inhibition of pro-inflammatory signaling was observed, contrary to free drug ad/vite. this substantiated the idea that sqad is not active as an anti-inflammatory agent but needs to be activated in situ to perform anti-inflammatory activity ( ) . lps stimulation of macrophages also causes overproduction of nitric oxide by inducible nitric oxide synthase (inos) ( ) , which results in further pro-inflammatory signaling and oxidative stress by reactive nitrogen species (nox) ( ) . thus, raw . macrophages were stimulated with lps, and nitrite accumulation was monitored by griess reagent to evaluate the consequence of sqad/vite np treatment on nox formation. stimulation by lps ( g/ml) resulted in . m nitrite content in the supernatant, but nox production was significantly inhibited after incubation of sqad/vite nps at g/ml (fig. f) . free drugs ad/vite controls also showed efficient inhibition of nitrite accumulation, which likely resulted from free ad, acting on its cognate receptors a a and a b, inhibiting inos expression ( , ) . here, there was no difference in the sqad/vite data are the mean ± sd. n = independent experiments. *p < . , **p < . , ***p < . , and ****p < . (one-way anova followed by holm-sidak's multiple comparison test). groups between pretreated cells or cells simultaneously treated with nps at the time of lps challenge (see also supplementary discussion). noteworthy, nitrite production was significantly lower in sqad/vite np-treated cells compared to cells that only received sqad. this showed that increasing drug loading through the use of prodrug-based nanocarriers might be advantageous for inflammation therapy. overall, these results showed that multidrug sqad/vite nps could effectively scavenge ros in a concentration-dependent manner and established effective proresolving action through the combined effects of sqad and vite in vitro. by using a prodrug-based nanoformulation, sqad/vite nps did not trigger ad signaling until ad release, which could limit deleterious side effects associated with ad therapy. we then proceeded to evaluate the in vivo ability of sqad/vite nps to promote the resolution of inflammation in mice by examining their effect on the acute inflammatory response to endotoxin. in the blood, recognition of lps by circulating macrophages activates the redox-controlled nuclear factor b (nf-b) by toll-like receptor (tlr )-mediated mechanisms. this event potentiates downstream inflammation cascades, resulting in the pathological "cytokine storm." in our experiments, lps was injected in mice intraperitoneally, after which blood and organs were collected at various time points to measure the levels of pro-inflammatory and anti-inflammatory cytokines by enzyme-linked immunosorbent assay (elisa). in the blood, pro-inflammatory cytokine levels reached a maximum hour after the lps challenge, while anti-inflammatory cytokines followed with a peak hours after lps injection ( fig. s ). in the treatment group where sqad/vite nps were injected at a dose of mg/kg [corresponding to ad ( . mg/kg) and vite ( mg/kg)], a significant decrease in tnf- together with an increase in anti-inflammatory interleukin- (il- ) could be observed, comparatively to control groups that received either no treatment, free drugs ad/vite, sqad nps, or sqvite nps only at equivalent doses (fig. , a and b) . this effect took place in a dose-dependent manner ( fig. s ). free drug ad and vite have limited bioavailability due to extremely fast metabolization and poor cell localization, respectively. these initial in vivo results pointed to an improved pharmacological profile of these compounds, thanks to their formulation as nps and protection from early degradation. (c) pro-inflammatory cytokines including mcp- and il- from the lungs, liver, and kidneys, hours after lps challenge as measured by cytometric bead array. nontreated controls did not reach detection threshold and were not shown. n = to mice per group. data are mean ± sd. ns, not significant; *p < . , **p < . , and ***p < . significant difference to lps only control unless specified (one-way anova followed by holm-sidak's multiple comparison test). next, we evaluated in organ homogenates the levels of two key pro-inflammatory cytokines mcp- (monocyte chemoattractant protein- ) and il- , responsible, respectively, for recruiting immune cells to the sites of inflammation and mediating the acute-phase response ( ) . in the lungs and kidneys, hours after the initial lps challenge, treatment with sqad/vite nps at mg/kg significantly reduced the amount of mcp- and il- compared to nontreated or free drugtreated controls (fig. c ). in the liver, results failed to reach significance, but a tendency for mitigated acute inflammation could be observed. we next evaluated the antioxidant effects of sqad/vite nps in vivo by measuring the amount of lipid peroxidation products in the lungs following the lps challenge. during acute systemic inflammation, the immune cells recruited by the extensive pulmonary capillary bed induce high levels of oxidative stress in the lungs, resulting in substantial lipid peroxidation ( ) . lps challenge resulted in an increase in lipid peroxidation products as measured by reaction of malondialdehyde (mda) with thiobarbituric acid reactive substance (tbars test) ( . nmol of mda/mg protein versus . nmol of mda/mg protein). basal mda levels in mice typically range around nmol of mda/mg protein ( ) ( ) ( ) . although it could not be abrogated, the increase in mda was most strongly mitigated in the sqad/vite np treatment group, where mda levels reached only . nmol of mda/mg protein (fig. a) . together, these in vivo studies demonstrated an efficient and targeted resolution of inflammation by multidrug sqad/vite nps. while single-drug nps did display some efficacy at inhibiting plasma tnf-, they did not reach the therapeutic efficacy of sqad/vite nps. contrary to what was observed in in vitro studies, in vivo free drug ad/vite controls consistently failed to induce a significant therapeutic response. this could be explained by the quick metabolization of ad after a bolus injection ( ) and poor bioavailability of vite. our prodrugbased nps thus increased the efficacy of both drugs by simultaneously delivering them to the sites of inflammation. to further validate the in vivo capability of sqad/vite nps, we then investigated the hemodynamic effects of sqad/vite nps comparatively to free drugs ad/vite. the effects of a single injection of sqad/vite nps on blood pressure were measured noninvasively on healthy mice. while the np-encapsulated drugs had no significant effect on blood pressure compared to nontreated controls, free drugs ad/vite induced a measurable decrease in blood pressure due to ad, in accordance with published literature (fig. b) ( ) . these results confirmed that the sqad/vite np formulation helped to protect animals from the deleterious side effects induced by ad therapy. we therefore proceeded to evaluate the efficacy of sqad/vite nps in a model of lethal lps challenge ( ) . mice in the treatment groups received either sqad/vite nps at a dose of mg/kg or free drugs ad/vite at an equivalent dose. in the group treated with sqad/vite nps, all mice survived the lethal lps challenge, whereas free drug ad/vite failed to significantly improve the survival rate, compared to the untreated control animals (fig. d) . improvements in the clinical scores of the animals paralleled the improvements in survival rates for all groups (fig. c) . last, the consequence of the treatment with sqad/vite nps was histologically investigated regarding signs of inflammation. in the previous lps lethality model, organs from either sqad/vite np-treated mice or lps only-treated data are the mean ± sd. *p < . , **p < . , and ***p < . significant difference to lps only control unless specified (one-way anova followed by holm-sidak's multiple comparison test). for survival evaluation, a log-rank (mantel-cox) test was used, giving a p = . . controls were harvested at the -hour point and analyzed for histological signs of tissue stress following hematoxylin and eosin (h&e) staining. inflammatory changes including mononuclear cell infiltration, endothelial disruption, and hemorrhage were noticeably reduced in animals that received sqad/vite np treatment. in the liver, nontreated animals displayed severe injuries, which were not observed in sqad/vite np-treated animals (figs. s and s ); these included hemorrhagic sinusal occlusion, advanced hepatocellular stress, and disseminated steatosis. in the lungs, although both animal groups showed signs of inflammatory stress, the nontreated animals displayed more advanced loss of structure, alveolar thickening, and hemorrhage ( fig. s ) . as is the case with many animal models, lethal endotoxemia sepsis models, although widely used to study anti-inflammatory therapies ( ) ( ) ( ) , have inherent drawbacks. the main limitation of this model is the speed at which it induces a severe inflammatory insult. numerous studies have investigated therapeutic compounds in the context of sepsis by pretreating animals before lps injection ( , ) . other studies investigated treatment at the time of lps injection ( ) . in clinical practice, patients with sepsis usually receive treatment after the onset of the pro-inflammatory insult but before the insult reaches "peak severity." in this endotoxemia model, we found that pro-inflammatory cytokines such as tnf- reached a maximum about hour after lps injection (see fig. s ). as a result, we decided to inject our treatments at the -min time point, halfway between the onset and the peak of the inflammatory response, which is probably the best time point to fit with the clinical conditions. we presented here the first example of targeted delivery of ad, and of multidrug anti-inflammatory/antioxidant nps, for the mitigation of inflammation. bioconjugation of ad to sq allowed to obtain a prodrug-based nanocarrier, which, after nanoformulation with vite, yielded stable multidrug nps, improving the bioavailability of both drugs with significant pharmaceutical activity in models of acute inflammatory injury. with the ability to specifically target and deliver ad at tissue foci of acute inflammation and the capacity to react with intracellular reactive species at the target site, sqad/vite nps represent a promising therapeutic intervention overcoming limitations of both conventional ad and antioxidant therapy. our extensive in vivo data support this hypothesis that opens the way to explore the plethora of available specific ad receptor agonists and antioxidants. additional efforts will also allow to further characterize sq-based np biodistribution. overall, these sq-based multidrug nps represent a unique approach for inhibiting the pathological cross-talk between oxidative stress and inflammation, delivering therapeutic agents at the loci of inflammation, and thus afford a new tool in the fight against the complex and multifactorial phenomenon of uncontrolled inflammation. sqad was synthesized as previously described ( ) , and the resulting nps were prepared using the nanoprecipitation technique. briefly, sqad was dissolved in absolute ethanol ( mg/ml) and added dropwise under strong stirring to a % (w/v) dextrose solution. ethanol was then completely evaporated using a rotavapor ( rpm, °c, mbar) to obtain an aqueous suspension of pure sqad nps ( mg/ml). multidrug sqad/vite nps ( : wt %) were obtained by dissolving sqad ( mg/ml) and vite ( mg/ml) (-tocopherol, sigma-aldrich) in absolute ethanol and adding the solution dropwise under strong stirring to a % dextrose solution with subsequent ethanol evaporation to obtain an aqueous suspension of sqad/vite nps ( mg/ml). fluorescent nps were obtained by the same procedure, except % (w/w) of fluorescent probe (sqrho) or did perchlorate (thermo fisher scientific) was added to the ethanolic phase. all np sizes (hydrodynamic diameter) and surface charges (zeta potential) were measured using a malvern zetasizer nano zs . ( ° scattering angle, °c). for the size measurements by dls, a good attenuator value ( - ) was obtained when suspending l of nps in ml of distilled water. the mean diameter for each preparation resulted from the average of three measurements of s each. for zeta potential measurements, l of nps was dissolved in ml of mm kcl before filling the measurement cell. the mean zeta potential for each preparation resulted from the average of three independent measurements in automatic mode, followed by application of the smoluchowski equation. morphology was observed by cryo-tem. for this, drops of the np suspensions ( mg/ml) were deposited on electron microscopy grids covered with a holey carbon film (quantifoil r / ) previously treated with a plasma glow discharge. observations were conducted at low temperature (− °c) on a jeol field emission gun microscope operated at kv. images were recorded with a gatan camera. sqad/vite nps with different vite content were formulated in % dextrose solution and subsequently washed with water twice using an ultrafiltration device (molecular weight cutoff, , ). nps were dissolved in ethanol and measured by hplc to determine vite content. hplc waters alliance (waters, milford, ma), equipped with a waters dad photodiode array detector, hewlett-packard computer with a waters empower software, and a waters autosampler with a -l loop, was used, with simultaneous spectra detection wavelengths from to nm recorded for all peaks. a waters xselect lc- column ( . mm by mm, . m) was used with a nongradient mobile phase of acetonitrile and methanol ( : , v/v) at a constant flow rate of ml/min. the vite peak was measured at a wavelength of nm and quantitatively determined by comparing with a standard curve. fetal bovine serum ( ml) was prepared with l of ehna (erythro- -( -hydroxy- -nonyl)adenine) [ad deaminase inhibitor ( mg/ml), nacl . %; sigma-aldrich] and . l of dipyridamole [ad uptake inhibitor ( mg/ml), dimethyl sulfoxide; sigma-aldrich]. sqad/vite nps ( l; mg/ml) were incubated at different time intervals with l of the fetal bovine serum solution in various eppendorf vials, each for one time point. at the predetermined time intervals (i.e., min, min, hours, hours, hours, and hours), aliquots ( l) were collected and added into l of meoh to denature and precipitate the enzymes and proteins of the serum, which were removed after centrifugation ( , g for min). to quantify the remaining sqad bioconjugate and the released ad, the remaining supernatants ( l) were evaporated to dryness at °c under nitrogen flow and then solubilized in l of meoh. quantification was performed using a reversed-phase hplc on a halo c column ( . mm by mm, m; interchim), a binary lc pump (waters), a autosampler (waters), and a pda detector (waters). the hplc was carried out using a gradient elution with the mobile phase composed of % mm potassium phosphate in milli-q water (ph . ) % meoh (phase a) and meoh (phase b). elution was carried out at a flow rate of . ml/min. the system was held at % of a for min, followed by -min linear gradient from % a to % b, kept at % b for min, and brought back to initial condition by a -min linear gradient from % b to % a. the sample run was maintained for min with % a to equilibrate the column pressure. temperature was set at °c, and ultraviolet detection was monitored at nm for ad and nm for sqad. the detection limit of the hplc technique was g/ml for ad and g/ml for sqad. this method exhibited linearity (r = . ) over the assayed concentration ranges ( to g/ml). h c cells ( , cells per well) were seeded in a -well plate and cultured for hours at °c. the cells were treated for hours with sqad/vite nps or controls diluted in culture medium at a concentration of g/ml for standard tests or according to the described dose for dose-response experiments. cells were washed with pbs and incubated with medium containing hydrogen peroxide (h o ; final concentration, . mm; sigma-aldrich). after min, h o -containing medium was removed, and the cells were washed with pbs. intracellular ros production was detected using abcam cellular ros assay kit (deep red) per manufacturer's instructions. briefly, the ros detection probe was diluted in pbs, and cells were incubated with this staining solution for min. staining solution was removed, and cells were washed with pbs and treated with l of . % trypsin solution for min at °c. the trypsin solution was inhibited by adding . ml of medium, and cell fluorescence was recorded using an accuri flow cytometer c (accuri cytometers ltd.). necrotic cell death was assessed by propidium iodide staining at g/ml. nitrite (no − ) release was assessed with freshly prepared griess reagent. briefly, in -well plates ( , cells per well), raw . cells were treated for hours with sqad/vite nps or controls diluted in culture medium at a concentration of g/ml. lps (sigma-aldrich o :b ) was added at a final concentration of g/ml for hours for macrophage stimulation. for co-treatment experiments, sqad/vite np treatment was also performed concurrently to lps stimulation. after lps stimulation, the griess reagent was added in equal volume to culture supernatants. the absorbance at nm was measured on a perkinelmer absorbance reader after min of incubation in the dark at room temperature. the no − concentrations were determined using standard curves prepared from sodium nitrite (nano ) at various concentrations. in vitro evaluation of pro-inflammatory cytokine production lps stimulation and drug treatments were performed in the same way than with nitric oxide assays. after lps stimulation for hours, cell supernatants were retrieved and centrifuged for min at g to remove cell debris. inflammatory cytokine production was evaluated using a biolegend elisa mouse tnf- cytokine detection kit per manufacturer's instructions (biolegend, usa). supernatants were diluted × with assay diluent before detection experiment. male c bl/ j and female balb/c mice were purchased from janvier labs (france) for systemic inflammation and np tracking studies, respectively. animals were housed in a standard controlled environment ( ° ± °c, % relative humidity, -hour light/dark cycles) with food and water available ad libitum. experiments were approved by the animal care committee of the university paris-sud, in accordance with the principles of laboratory animal care and european legislation / /eu. all efforts were made to reduce animal numbers and minimize their suffering, as defined in the specific agreement (registration no. apafis# ). for the paw inflammation study, experiments were performed on -week-old female balb/c mice. paw inflammation was caused by intraplantar injection in the right paw of ng of lps (sigma-aldrich o :b ) dissolved at mg/ml in physiological saline (nacl, . %). animals received a control injection of l of saline in the left paw. in vivo imaging studies were performed after hours, following intravenous injection of fluorescent sqad/vite or plga nps ( l, mg/ml, containing % did) or control fluorescent did solution ( l, g/ml in % dextrose solution). the biodistribution of the nps was recorded at . , , , and hours with the ivis lumina lt series iii system (caliper life sciences) using -nm excitation and -nm emission filters. during imaging, mice were kept on the imaging stage under anesthesia with % isofluorane gas in oxygen flow ( liter/min) and were imaged in ventral position. images and measures of fluorescence signals were acquired and analyzed with living imaging software (caliper life sciences). for the systemic inflammation study, experiments were performed on -week-old male c bl/ j mice. systemic inflammation was caused by intraperitoneal injection of lps (sigma-aldrich o :b ) at a dose of . mg/kg. noninflamed control animals did not receive the lps injection. after hours, animals received intravenous injection of fluorescent sqad/vite nps ( l, mg/ml, containing % sqrho) or control fluorescent rhodamine b solution ( l, g/ml in % dextrose solution). after hours, animals were deeply anesthetized with an intraperitoneal sodium pentobarbital injection before euthanasia by intracardial perfusion of ml of saline ( ml/min), until the fluid exiting the right atrium was entirely clear. the liver, heart, lungs, kidneys, and spleen were excised and immediately imaged with the ivis imager using -nm excitation and -nm emission filters. images and measures of fluorescence signals were acquired and analyzed with living imaging software (caliper life sciences). the therapeutic efficacy of sqad/vite nps was evaluated in vivo in a mouse endotoxemia model with -to -week-old male c bl/ j mice. to evaluate the efficacy through cytokine production, endotoxemia was induced by intraperitoneal injection of a dose of lps ( . mg/kg; sigma o :b ) diluted at . mg/ml in buffered saline. mice injected with lps alone were used as controls. thirty minutes after lps injection, sqad/vite nps [sqad ( mg/kg), i.e., ad (equivalent . mg/kg) and vite ( mg/kg)], sqad nps [sqad ( mg/kg) and ad (equivalent . mg/kg)], or free ad/vite [ad ( . mg/kg) and vite ( mg/kg) with % pluronic f- vehicle for vite solubilization] were intravenously injected via the suborbital vein. following the injections, blood samples (~ l) were collected at predetermined time points via submandibular puncture before terminal cardiac puncture and organ collection. plasma was obtained by centrifuging blood samples at rcf (relative centrifugal force) for min and stored at − °c before further analysis. in the plasma, cytokines, including il- and tnf-, were quantified by biolegend elisa mouse kit per manufacturer's instructions. organ homogenates were obtained with a heidolph instruments (germany) rzr- organ homogenizer in pbs at a concentration of mg/ml. proinflammatory cytokines in organ homogenates were quantified using a cytometric bead array per manufacturer's instruction (bd biosciences). for mda content in the lungs, mg of lung tissues was homogenized on ice in l of mda lysis buffer containing l of bht (butylated hydroxytoluene) antioxidant solution as per manufacturer's instruction (biovision, lipid peroxidation mda fluorometric assay kit). to evaluate efficacy through survival in the lethal lps model, mice were sensitized to the lethal effects of lps with d-galactosamine hydrochloride (roth, germany) via intraperitoneal injection of an -mg dose concurrently to lps injection at g/kg. after min, nps or free drugs as controls were injected intravenously. signs of disease severity were evaluated at predetermined time points using a previously described disease scoring system ( ) . for histological evaluation, organs were fixed for hours in % paraformaldehyde and then embedded in paraffin. sections ( m) were deparaffinized and stained with h&e (vwr, france). slides were scanned with a digital slide scanner nanozoomer . -rs (hamamatsu, japan), which allowed an overall view of the samples. images were digitally captured from the scan slides using the ndp.view software (hamamatsu). for blood pressure measurements, a kent scientific (torrington, usa) coda tail-cuff vpr blood pressure measurement system was used. c bl/ j mice were acclimated to the procedure for days before measurements to avoid undue stress and experimental artifact. for all measurements, an experimental session of acclimation cycles and measurement cycles was used. only measurement cycles that passed coda software acceptance criteria were retained. for animals that received free drug ad/vite or sqad/vite ( mg/kg or equivalent), the blood pressure measurement was started immediately after intravenous injection. statistics were computed with graphpad prism . differences in group means were calculated by one-way analysis of variance (anova) followed by holm-sidak's multiple comparison test or kruskal-wallis test (nonparametric) followed by dunn's multiple comparison test when samples failed equality of variance or normality statistical tests (shapiro-wilk). a value of p < . was considered significant. for studies requiring grouped analyses, a two-way anova followed by tukey's multiple comparison test was performed. supplementary material for this article is available at http://advances.sciencemag.org/cgi/ content/full/ / /eaaz /dc view/request a protocol for this paper from bio-protocol. a unified theory of sepsis-induced acute kidney injury: inflammation, microcirculatory dysfunction, bioenergetics and the tubular cell adaptation to injury cytokine-mediated inflammation in acute lung injury covid- : consider cytokine storm syndromes and immunosuppression the immunopathology of sepsis and potential therapeutic targets oxidative stress in sepsis: pathophysiological implications justifying antioxidant co-therapy does the interdependence between oxidative stress and inflammation explain the antioxidant paradox? the role of oxidative stress during 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severity in an animal model inflammation and lymphatic function inos expression requires nadph oxidase-dependent redox signaling in microvascular endothelial cells the rise, the fall and the renaissance of vitamin e inhibition of nf-b transcriptional activity by -tocopheryl succinate inhibition of nf- b dna binding activity by alpha-tocopheryl succinate oxysterol-induced activation of macrophage nadph-oxidase enhances cell-mediated oxidation of ldl in the atherosclerotic apolipoprotein e deficient mouse: inhibitory role for vitamin e we wish to thank p. van tassel for helpful corrections to the manuscript and f. gobeaux of umr for help with cryo-tem imaging. funding: the authors gratefully acknowledge the financial support from the th euronanomed-ii call for proposals, project nanoheart no. anr- -enm - - . this work was further supported by la fondation pour la recherche médicale (frm) grant no. eco . author contributions: f.d., m.v., p.c., designed the research. f.d., r.b., c.c., f.r., a.p., a.g., f.g., m.v. conducted experiments. f.d., f.r., f.g., m.v., p.c. analyzed the data. f.d. wrote the paper, and all authors reviewed the manuscript. competing interests: the authors declare that they have no competing interests. data and materials availability: all data needed to evaluate the conclusions in the paper are present in the paper and/or the supplementary materials. additional data related to this paper may be requested from the authors. key: cord- - vbkttzx authors: li, xiao-jun; kim, kwan-woo; oh, hyuncheol; liu, xiang-qian; kim, youn-chul title: chemical constituents and an antineuroinflammatory lignan, savinin from the roots of acanthopanax henryi date: - - journal: evid based complement alternat med doi: . / / sha: doc_id: cord_uid: vbkttzx the phytochemical investigation on the roots of acanthopanax henryi (araliaceae) resulted in the discovery of twenty compounds whose chemical structures were elucidated by the analysis of d-, d-nmr, mass spectrometry data, other physicochemical properties, and a comparison of the spectral data with the literature. they were identified as (-)-sesamin ( ), helioxanthin ( ), savinin ( ), taiwanin c ( ), -methoxy- -hydroxycoumarin ( ), behenic acid ( ), -o-caffeoyl-quinic acid ( ), -o-caffeoyl-quinic acid ( ), , -di-o-caffeoyl-quinic acid ( ), , -di-o-caffeoyl-quinic acid ( ), , -di-o-caffeoyl-quinic acid ( ), (+)-threo-( r, r)-guaiacylglycerol-β-coniferyl aldehyde ether ( ), (+)-erythro-( s, r)-guaiacylglycerol-β-coniferyl aldehyde ether ( ), ferulic acid ( ), caffeic acid ( ), stigmasterol ( ), β-sitosterol ( ), adenosine ( ), syringin ( ), and trans-coniferin ( ). among these isolates, compound showed inhibitory activity against lipopolysaccharide- (lps-) induced nitric oxide (no) and prostaglandin e (pge( )) production with ic( ) values of . ± . and . ± . μm, respectively. the effects of compound were associated with the suppression of lps-induced expression of the inducible nitric oxide synthase (inos) and cyclooxygenase- (cox- ) protein. furthermore, compound negatively regulated the production of interleukin- (il-) β and tumor-necrosis factor- (tnf-) α at the transcriptional level in lps-stimulated bv microglial cells. these antineuroinflammatory effects of compound were mediated by p mitogen-activated protein kinase (mapk). neuroinflammatory responses are mainly mediated by microglial activation, and they are implicated in the development of neurodegenerative diseases such as alzheimer's diseases (ad), parkinson's disease (pd), multiple sclerosis (ms), and amyotrophic lateral sclerosis (als) [ ] . microglial cells are the primary resident immune cells in the central nervous system (cns) and are associated with defensive mechanisms to maintain homeostasis against brain injury [ ] . in steady state, microglia exert protective responses by regulating innate and adaptive immune responses. however, activated microglia produce excess immune reactions which are detrimental to brain tissue and can produce various proinflammatory mediators such as tumor necrosis factor-(tnf-) , interleukins (ils), nitric oxide (no), prostaglandin e (pge ), and reactive oxygen species (ros). released and accumulated, these proinflammatory mediators facilitate the development of neurodegenerative diseases [ , ] . therefore, it is important to suppress the secretion of proinflammatory mediators from activated microglial cells to prevent the neuroinflammation-related development of neurodegenerative diseases. acanthopanax spp. is one of the well-known medicinal resources in traditional oriental medicine in china, korea, japan, and far-east russia. its dried roots and stem barks are famous traditional folk medicine for treating rheumatism, arthritis, paralysis, sinew, and bone pain [ ] . acanthopanax henryi (oliv.) harms, a chinese endemic plant, has been used as a traditional remedy for the treatment of paralysis, arthritis, rheumatism, lameness, edema, injury from falls, hernia, and abdominal pain [ , ] . previous phytochemical studies evidence-based complementary and alternative medicine on a. henryi led to the isolation and identification of more than secondary metabolites, including five flavonoids, six caffeoylquinic acid derivatives [ ] , sixteen triterpenoid saponins [ , ] , one amide, one anthraquinone, one organic acid [ ] , three lignans, one diterpene, one phenylpropanoid, and two phytosterols [ ] . in addition, it has been reported that this plant exhibits diverse pharmacological activities due to the wide variety of chemical constituents. for example, metabolites of the leaves of a. henryi have strong antioxidant and antiacetyl cholinesterase activities [ ] , and the % methanol fraction of root bark and ciwujianoside c , which was isolated from leaves of this plant, have significant antiinflammatory effect in lipopolysaccharide-(lps-) induced raw . macrophage cells [ , ] . moreover, some glycosides from the leaves of a. henryi have antiadipogenic effect, decreasing lipid accumulation through the inhibition of proliferator-activated receptor gamma (ppar ) and ccaat/enhancer-binding protein alpha (c/ebp ) in t -l cells [ ] . however, it has not been investigated yet whether this plant has antineuroinflammatory effects. therefore, in this study, we purified twenty compounds from the roots of a. henryi and evaluated their antineuroinflammatory activity in vitro to continue our approach to contribute to the drug development for inflammationmediated neurodegenerative diseases. procedures. nmr spectra ( d and d) were recorded using a jeol jnm ecp- spectrometer (tokyo, japan) ( mhz for h and mhz for c). hmqc and hmbc experiments were optimized for ch = hz and n ch = hz, respectively. optical rotations were recorded using a jasco p- digital polarimeter (tokyo, japan). hplc (younglin-yl , younglin, anyang, korea) separation was performed on ymc-pack ods-a column ( × mm, m, and nm) with a flow rate of ml/min and the solvents used for hplc were analytical grade. esi-ms data were obtained using a q-tof micro-lc-ms/ms instrument (waters, milford, ma, usa) at korea university, seoul, korea. tlc was performed on kieselgel f ( . ; merck, darmstadt, germany) or rp- f s (merck) plates. spots were visualized by spraying with % aqueous h so solution followed by heating. column chromatography (cc) was performed on silica gel (kieselgel , - mesh and - mesh, merck) and ymc octadecyl-functionalized silica gel (c ). these procedures were based on our previous report [ ] . to give a residue ( g), which was suspended in distilled water and successively partitioned with petroleum ether (pe, b.p. - ∘ c), ethyl acetate (etoac), and n-butanol (buoh), respectively. the etoac extract ( g) was subjected to cc ( . × cm) on silica gel, eluted with a gradient of dichloromethane (ch cl )-methanol (meoh) ( : to : , v/v) to give ten fractions (fr. e -fr. e ). fr. e ( . g) was purified on silica gel cc ( . × cm), eluted with hexane-etoac ( : to : , v/v) to afford ( . mg), ( . mg), ( . mg), ( . the pe extract ( g) was subjected to cc ( . × cm) on a silica gel, eluted with a gradient of hexane-etoac ( : to : , v/v) to give thirteen fractions (fr. p -fr. p ). fr. p ( . g) was subjected to a silica gel cc ( . × cm) with a hexane-etoac gradient ( : to : , v/v) as the solvent to gain ( . mg). fr. p ( . g) was firstly performed on a silica gel cc ( . × cm) and then purified by recrystallization with hexane-etoac ( : ) to afford ( . mg) and ( . mg). the n-buoh extract ( g) was subjected to a c cc ( . × cm) elution with a gradient of mo) . primary antibodies such as anti-inducible nitric oxide synthase (inos) and anti-cyclooxygenase- (cox- ) antibodies were purchased from santa cruz biotechnology (dallas, tx, usa). anti-pextracellular signal-regulated kinase (erk), anti-erk, antip-c-jun n-terminal kinase (jnk), anti-jnk, anti-p-p , and anti-p were obtained from cell signaling technology (danvers, ma, usa). anti-mouse, anti-goat, and anti-rabbit secondary antibodies were supplied by merck millipore (darmstadt, germany). the enzyme-linked immunosorbent assay (elisa) kit for pge was purchased from r&d systems, inc. (minneapolis, mn, usa). culture. bv microglial cells were received from professor hyun park at wonkwang university (iksan, korea). bv cells were maintained at × cells in a mm dish with dmem supplemented with % (v/v) heat-inactivated fbs, penicillin g ( units/ml), streptomycin ( g/ml), and l-glutamine ( mm) and incubated at ∘ c in a humidified atmosphere containing % co . . bv cells were cultured in -well culture plates at a density of × cells/well. bv cells were pretreated with the isolated compounds from a. henryi and then stimulated with lps ( g/ml) for h. after incubation, l of each supernatant was collected and mixed with the same volume of the griess reagent. the absorbance at nm wavelength was measured using a microplate reader. the detailed procedures are described in our previous report [ ] . assay. the level of pge present in each sample was determined using a commercially available kit from r&d systems (minneapolis, mn, usa). three independent assays were performed according to the manufacturer's instructions. briefly, bv cells were seeded in -well culture plates at a density of × cells/well. prior to the stimulation with lps ( g/ml) for h, cells were treated with test compounds. after incubation, supernatant was collected and applied to the pge elisa kit for measuring the concentration of pge . the proteins inos, cox- , and mapk-associated proteins, including p-p , p , p-jnk, jnk, p-erk, and erk, were detected by a western blot analysis. the procedures of this experiment were based on our previous report [ ] . cells were lysed by ripa buffer (thermo fisher scientific, usa), and normalized for equal amounts of protein using the bradford protein assay (bio-rad laboratories, hercules, ca, usa). g of protein was loaded for each sample and separated on . or % sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) gels. then, proteins were transferred to nitrocellulose (nc) membranes (bio-rad laboratories). after that, membranes were incubated with tris-buffered saline containing . % tween- (tbs-t) with % skim milk (bd difco, usa) for h at ∘ c. then, membranes were probed with primary antibodies and incubated at ∘ c for min or overnight. after incubation, the membranes were washed with tbs-t, and then secondary antibodies were used for detecting primary antibodies. the protein bands on the nc membranes were covered by chemiluminescence with amersham ecl prime western blotting detection reagent (ge healthcare, little chalfont, uk). assays for il- , il- , and tnf-. the culture media were collected to determine the levels of il- , il- , and tnf-present in each sample using appropriate elisa kits (r&d systems, inc.), as per the manufacturer's instructions. briefly, bv cells were seeded in -well culture plates at a density of × cells/well. after incubation, the supernatant was collected and applied to the cytokine elisa kits for measuring the concentrations of il- , il- , and tnf-. the detailed procedures of qrt-pcr and the primer sequences, which were used in this investigation were reported in our previous investigations [ , ] . the analysis was conducted three times independently. analysis. data are presented as the mean ± standard deviation (sd) of at least three independent experiments. one-way analysis of variance (anova), followed by tukey's multiple comparison test, was used to compare three or more groups. statistical analysis was performed using graphpad prism software, version . (graphpad software inc., san diego, ca, usa). based on a comparison of these h-and c-nmr data with data previously reported [ ] , compound was identified as (-)sesamin. ( ) . evidence-based complementary and alternative medicine -och o-), . (c- , lactone, -ch -). the data of h-and c-nmr were consistent with those in previous report [ ] . compound was identified as helioxanthin. . based on a comparison of these nmr and esi-ms data with the data reported previously [ ] , compound was identified as savinin. all spectral data agreed with those previously reported [ ] . compound was identified as taiwanin c. . the data of h-and c-nmr were consistent with those in previous reports [ , ] . compound was identified as -methoxy- -hydroxycoumarin. - ) . the data of h-and c-nmr were consistent with those in previous report [ ] . compound was identified as behenic acid. ( ) reported [ , ] . compound was identified as , -di-ocaffeoylquinic acid. . (c- ). all spectral data agreed with those previously reported [ , ] . compound was identified as , -di-o-caffeoylquinic acid. . (c- -och ). all spectral data agreed with those previously reported [ ] . compound was identified as (+)-threo-( r, r)-guaiacylglycerol--coniferyl aldehyde ether. . (c- -och ). all spectral data agreed with those previously reported [ ] . compound was identified as (+)-erythro-( s, r)-guaiacylglycerol-coniferyl aldehyde ether. ( ) . . all spectral data agreed with those previously reported [ ] . compound was identified as ferulic acid. ( ) . all spectral data agreed with those previously reported [ ] . compound was identified as -sitosterol. . (c- ). all spectral data agreed with those previously reported [ ] . compound was identified as adenosine. ( ) . . all spectral data agreed with those previously reported [ , ] . compound was identified as syringin. . all spectral data agreed with those previously reported [ ] . compound was identified as trans-coniferin. the chemical structures of compoundsare shown in figure . in lps-induced bv microglial cells. among the isolated compounds from a. henryi, we selected compounds (compounds -, , , , , , , , and -) based on their number of publications, and these compounds were screened for anti-inflammatory effects, including the inhibition of no and pge production, in lps-stimulated bv microglial cells. bv cells were pretreated with the compounds for h and stimulated with lps ( g/ml) for h. the concentration required to inhibit the production of no by % (ic value) was calculated based on the concentrations of no and pge released into the culture figure : the effect of compound on lps-induced inos and cox- protein expression in bv microglial cells. cells were pretreated with/without the indicated concentrations of compound for h and then stimulated with lps ( g/ml) for h. the levels of inos and cox- were determined by western blot analysis. the experiment was repeated three times, and similar results were obtained. media as measured by the griess method and pge elisa kit, respectively. among the tested compounds, only compound showed inhibitory effects against lps-induced no production with an ic value of . ± . m and lpsinduced pge production with an ic value of . ± . m. the ic values of butein, a positive control, were . ± . m in no production and . ± . m in pge production, respectively. at tested concentrations, all test compounds did not show cytotoxic effects to bv cells (data not shown). therefore, compound was further investigated to elucidate the up-stream signaling pathways of its antineuroinflammatory effect. cells. inhibition of inos and cox- protein expression could be an important strategy to prevent neuroinflammation. in the present study, it was investigated whether compound inhibits lps-induced overexpression of inos and cox- protein in bv cells. bv cells were pretreated with compound for h and stimulated with lps ( g/ml) for h. as a result, lps augmented the expression of inos and cox- proteins; however, pretreatment with compound reversed these responses (figure ). to investigate whether compound inhibits the production of lps-induced proinflammatory cytokines including il- , il- , and tnf-, bv cells were pretreated with compound for h and stimulated with lps ( g/ml) for h. pretreatment with compound attenuated the lps-induced production of il- and tnf-, but it had little effect on il- production (figures (a)- (c) ). subsequently, to elucidate how compound inhibits proinflammatory cytokine production, the mrna levels of the cytokines were examined. bv cells were pretreated with compound for h and stimulated with lps ( g/ml) for h. consistent with the results obtained from the cytokine production data, pretreatment with compound reduced the lps-induced mrna levels of il- and tnf-, but it did not affect to mrna level of il- ( figures (d)- (f) ). these results suggested that compound exhibits the antineuroinflammatory effects by negatively regulating the production of il- and tnf-at the transcriptional level in lps-challenged bv microglial cells. we examined whether the antineuroinflammatory effects of compound contribute to the inactivation of mapk in the present study. bv cells were pretreated with compound for h and stimulated with lps ( g/ml) for min. lps markedly increased the phosphorylation of p , jnk, and erk. pretreatment with compound inhibited the phosphorylation of p (figure (a) ), but it had no effect on the phosphorylation of jnk and erk (figures (b) and (c)). the present study demonstrated that compound (savinin), one of the isolated compounds from the roots of a. henryi exerted antineuroinflammatory effects in lps-treated microglial cells. compound inhibited lps-induced the release of proinflammatory mediators, including no, pge , inos, cox- , il- , and tnf-. these inhibitory effects of compound were mediated by the inactivation of the p mapk pathway. no and pge , as well as their regulatory enzymes inos and cox- , are important mediators for neuroinflammation. they are involved in the pathogenesis of various neuroinflammatory pathophysiological conditions [ ] . in addition, proinflammatory cytokines, including il- , il- , and tnf-, are important factors in cns immune responses, and mediate neurodegeneration [ ] . microglial cells are activated by various stimuli. the activation of these cells leads to the release of these proinflammatory mediators and the exacerbation of neuroinflammation and neuroglia-induced neurotoxicity [ , ] . our investigation showed that compound , isolated from a. henryi, suppressed no and pge production in lps-stimulated bv microglial cells and also inhibited the lps-induced expression of the inos and cox- protein (figure ) . additionally, the present data indicated that compound significantly suppressed the release of il- and tnf-, but not il- , at the protein and mrna levels ( figure ). mapks are a family of serine/threonine protein kinases, and they consist of three major subunits: p , extracellular signal-regulated kinase (erk), and c-jun n-terminal kinase (jnk). mapks are involved in various cellular processes, including proliferation, differentiation, stress responses, and immune responses [ ] . in inflammatory responses, the activation of mapks leads to release of inflammatory-related factors, such as inos, cox- , ils, and tnf- [ ] . therefore, we further investigated whether the antineuroinflammatory effects of compound in lps-stimulated bv microglial cells were related to the inactivation of mapks. our results showed that compound significantly inhibited the phosphorylation of p mapk, but it did not affect the phosphorylation of erk and jnk mapks. nuclear factor kappa b (nf-b) is one of the major transcription factors regulating inflammatory responses. when microglial cells are stimulated with ils, tnf-, interferons, or lps, nf-b can be activated following increased translocation of p and p , which are subunits of the nf-b dimer, into the nucleus, as well as the phosphorylation and degradation of inhibitor of kappa b (i b)-in the cytosol [ ] . this response upregulates the expression of proinflammatory cytokines, chemokines, and adhesion molecules in microglial cells [ ] . therefore, nf-b can be an important target for the treatment of neuroinflammation-related neurodegenerative diseases. interestingly, pretreatment with compound did not decrease the nuclear translocation of p and p or the phosphorylation and degradation of i b-(data not shown). considering the effect of compound on the lps-induced activation of nf-b and mapk pathways, it is suggested that compound exerted antineuroinflammatory effects by specifically inactivating the p mapk pathway. various pharmacological activities of lignans, including compound (savinin), have been reported. these activities include cytotoxic effects against human cancer cell lines [ ] , cyp a inactivation [ ] , antisevere acute respiratory syndrome associated coronavirus (sars-cov) activity [ ] , inhibition of pge production in rat peritoneal macrophages [ ] , antiestrogenic [ ] , insecticidal [ ] , and inhibition of tnf-production in raw . macrophage cells [ ] ; however, this is the first finding on the antineuroinflammatory effects of compound (savinin) to the best of our knowledge. in summary, secondary metabolites were isolated from the etoac-, butanol-, and pe-soluble fractions of the methanol extract of acanthopanax henryi roots. among these compounds, savinin (compound ) was the only compound which inhibited the production of no and pge , and the expression of inos and cox- proteins in lps-stimulated bv microglial cells. in addition, this compound significantly suppressed the release of il- and tnf-at the protein and mrna levels. these inhibitory effects of compound were mediated by p mapk, but not erk and jnk mapks. taken together, our investigation showed that compound can be a promising candidate for the development of treatment options for neurodegenerative diseases. further studies elucidating the detailed mechanisms underlying the anti-inflammatory effects of compound are suggested. the data used to support the findings of this study are available from the corresponding author upon request. oxytocin inhibits lipopolysaccharide-induced inflammation in microglial cells and attenuates microglial activation in lipopolysaccharide-treated mice tetrahydroxystilbene glucoside attenuates neuroinflammation through the inhibition of microglia activation neuroinflammation induces neurodegeneration involvement of heme oxygenase- induction in the cytoprotective and immunomodulatory activities of , -dihydroxy- -methoxyflavanone in murine hippocampal and microglia cells cytotoxicity and anti-inflammatory effects of root bark extracts of acanthopanax henryi chemical constituents and their acetyl cholinesterase inhibitory and antioxidant activities from leaves of acanthopanax henryi: potential complementary source against alzheimer's disease editorial board of the state administration of traditional chinese medicine quantitative determination of bioactive triterpenoid saponins in different parts of acanthopanax henryi by hplc with charged aerosol detection and confirmation by lc-esi-tof-ms chemical constituents from leaves of acanthopanax henryi (ii) study on chemical constituents from root barks of acanthopanax henryi anti-inflammatory effects of ciwujianoside c , extracted from the leaves of acanthopanax henryi (oliv.) harms, on lps-stimulated raw . cells anti-adipogenic effect of glycoside st-e and glycoside st-c isolated from the leaves of acanthopanax henryi (oliv.) harms in t -l cells protein tyrosine phosphatase b inhibitors from the roots of cudrania tricuspidata inhibitory effects of alternaramide on inflammatory mediator expression through tlr -myd -mediated inhibition of nf-qb and mapk pathway signaling in lipopolysaccharide-stimulated raw . and bv cells anti-neuroinflammatory effects of cudraflavanone a isolated from the chloroform fraction of cudrania tricuspidata root bark phenylpropanoids from roots of acanthopanax sessiliflorus (rupr. et maxim.) seem intramolecular dehydrodiels-alder reaction affords selective entry to arylnaphthalene or aryldihydronaphthalene lignans constituents from the stems of ecdysanthera rosea chemical constituents from artemisia austro -yunnanensis rapid resolution liquid chromatography (rrlc) analysis and studies on the stability of shuang-huang-lian preparations chemical constituents from herbs of erigeron breviscapus a new caffeoyl quinic acid from aster scaber and its inhibitory activity against human immunodeficiency virus- (hiv- ) integrase cytotoxic activities of telectadium dongnaiense and its constituents by inhibition of the wnt/ -catenin signaling pathway the cytotoxicity of -o- neolignans from the seeds of crataegus pinnatifida chemical constituents from notopterygium incisum chemical constituents from leaves of perilla frutescens chemical constituents and in vitro antioxidant activity of crude extracts and compounds from leaves and stem bark of ficus burttdavyi chemical constituents from extract of isatidis radix two new phenylpropanoid glycosides from wikstroemia sikokiana lignan and phenylpropanoid glycosides from osmanthus asiaticus anti-neuroinflammatory effect of sophoraflavanone g from sophora alopecuroides in lps-activated bv microglia by mapk, jak/stat and nrf /ho- signaling pathways inducible isoforms of cyclooxygenase and nitric-oxide synthase in inflammation mapk phosphatasesregulating the immune response tiliroside, a dietary glycosidic flavonoid, inhibits traf- /nf-b/p -mediated neuroinflammation in activated bv microglia antiinflammatory activity of bee venom in bv microglial cells: mediation of myd -dependent nf-b signaling pathway a new lignan glycoside from juniperus rigida mechanism-based inactivation of cytochrome p a by methylenedioxyphenyl lignans from acanthopanax chiisanensis specific plant terpenoids and lignoids possess potent antiviral activities against severe acute respiratory syndrome coronavirus lignans from acanthopanax chiisanensis having an inhibitory activity on prostaglandin e production anti-estrogenic activity of lignans from acanthopanax chiisanensis root antimicrobial alkaloids from zanthoxylum tetraspermum and caudatum savinin, a lignan from pterocarpus santalinus inhibits tumor necrosis factor-production and t cell proliferation the authors declare that there are no conflicts of interest regarding the publication of this article. this research was supported by the national research foundation of korea (nrf) grants funded by the korea government (nrf- r a a ). xiao-jun li and kwan-woo kim contributed equally to this work. key: cord- -h o yqv authors: nan title: oral communications and posters date: - - journal: inflamm res doi: . /bf sha: doc_id: cord_uid: h o yqv nan the drosophila host defense is a multifaceted process which involves reprogramming of gene expression, activation of proteolytic cascades in the blood and uptake of microrganismsby professionalphagocytes. most of the recent studies have focused on challenge-induced expression of antimicrobial peptides and have addressed the following questions : ( ) which genes are upregulated during various types of bacterial, fungal or viral infections and what are their functions? ( ),what is the nature of the intracellular signalling cascades which lead togene transcription during these infections; ( ) how does drosophila recognize infections and does it discriminate between various types of aggressors (e.g. fungal versus bacterial or viral) to mount an appropriate response. over the last ten years we have gained significant insights into these various aspects and the presentation will review our current understanding of the drosophila immune response and put it into a phylogenetic perspective, namely by drawing some stringent parallels with the mammalian innate immune response. there is strong evidence that autoimmunity to myelin antigens plays a major role in the development of multiple sclerosis. several myelin-derived autoantigenic targets have been described and include myelin basic protein (mbp), proteolipid protein and myelin oligodendrocyte glycoprotein. there has been a particular focus on mbp for at least two reasons: mbp-specific cd + tcell receptors (tcrs) have been found in multiple sclerosis brains, and cells presenting an immunodominant mbp( - ) peptide in complex with hla-dr b have been shown to be present in multiple sclerosis lesions. also, humanized mice expressing the hla-dr b gene and a human t-cell receptor (tcr) that recognises the mbp - peptide in the context of hla-dr b either spontaneously or after immunization with mbp - develop experimental autoimmune encephalomyelitis, which has several features in common with multiple sclerosis. this talk will focus on, how humanized mice recently has been used to study the interplay between genetic and environmental risk factors in multiple sclerosis. to resolve the pathogenic mechanisms of rheumatoid arthritis (ra) we need to identify the causative genetic and environmental factors. this has however proven to be complex, with many factors and genes interacting. inbred animals are useful for studies of the identification of genes associated with complex traits and diseases such as ra. animal models offer a possibility to better define the traits, and to segregate the genes in a controlled way enabling linkage analysis. there are several arthritis models, which each may reflect various variants of the heterogeneity of ra in humans. examples are collagen induced arthritis (cia) and pristane induced arthritis (pia), which both fulfills the clinical diagnostic criteria for ra. both diseases are genetically complex and the susceptibility is, as ra, dependent on many polymorphic genes operating in concert. so far genes in this concert have been identified; the mhc class ii ab gene in the mouse ( ) and the ncf gene in the rat ( ) and in the mouse ( ) . the ncf protein is a part of the nadph oxidase complex mediating oxidative burst. the discovery of the ncf polymorphism led to a new proposed pathway in which oxygen radicals modify antigen presentation and the resulting activation of autoreactive t cells. mice with the deficient ncf allele are more susceptible to cia, and also developed a chronic form of arthritis. interestingly, the immune response to cii was enhanced by the ncf deficiency linking the ncf pathway to the adaptive immune response. oxidation of t cell membranes seem to be a key event in the pathogenic mechanism as reduction of t cell membranes induces arthritis in rats ( ). on the basis of these findings a new type of therapy myasthenia gravis is a prototypic autoimmune disease, caused in most cases by autoantibodies to the muscle acetylcholine receptor (achr) at the neuromuscular junction. the antibodies reduce the number of achr leading to failure of neuromuscular transmission and muscle weakness. the achr antibodies as measured in conventional immunoprecipitation assays are igg, high affinity, polyclonal and specific for human achr. they reduce the numbers of achr by antigenic modulation and by complement-mediated damage to the neuromuscular junction. myasthenia gravis has a very intriguing relationship with the thymus gland. in many younger patients, the thymus is hyperplastic with immune cell infiltrates and germinal centre formation. around the germinal centres, within the medulla, there are rare muscle-like cells called myoid cells that express achrs. there are many b cells and plasma cells and thymic lymphocyte preparations synthesise achr antibodies. the possibility that the thymic achr induces the germinal centre formation and achr antibody synthesis is supported by much evidence. some patients, however, have thymic tumours and in these the role of the thymus is less clear. moreover, older patients with typical myasthenia usually have thymic tissue which is normal for their age. there are up to % of myasthenia patients that do not have the typical achr antibodies. some of these have instead antibodies to muscle specific kinase, a receptor tyrosine kinase that is restricted to the neuromuscular junction. the pathogenic mechanisms by which the antibodies cause disease are not yet clearly identified and the evidence will be discussed. finally, among the patients who have neither achr nor musk antibodies by conventional testing, we have evidence for lower affinity antibodies to achr which can now be detected using molecular approaches which will be described. arry- (azd ) is an inhibitor of mek / currently in development for cancer. phase determined the msd ( mg) and the safety of the compound given continuously. in decreasing frequency, common treatment-related side effects were rash, diarrhea, nausea, peripheral edema, and vomiting. paired pre-and postdose tumor biopsies showed a reduction in perk (- %) and proliferative index (- %). the trough plasma concentration ( ng/ml) corresponded to~ % inhibition of perk. about % of pts had stable disease after months. these results demonstrate that arry- (azd ) is well-tolerated, hits its target, and produces a high incidence of long-lasting stable disease. there are several on-going phase studies, in melanoma, colorectal, lung and pancreatic cancer. arry- is another potent, selective mek / inhibitor, currently in development for inflammatory diseases. when human whole blood was stimulated with tpa, arry- inhibited tnfa, il- b and il- (ic s of , and nm, respectively). % inhibition of perk required nm. arry- was highly efficacious in cia and aia rat models, with ed s of and~ mg/ kg, respectively. in normal volunteers arry- was well-tolerated and there was a dose-proportional increase in drug exposure. in ex vivo blood samples, there was a dramatic time-and concentration-dependent inhibition of tpa-induced tnfa and il b. an on-going multiple ascending dose clinical study is further exploring the pharmacokinetics, pharmacodynamics and tolerability of arry- monotherapy. in addition, we have initiated a clinical trial designed to evaluate arry- in combination with methotrexate in patients with rheumatoid arthritis. rho kinases (rock) are involved in many physiological and pathological processes including smooth muscle contraction, cytoskeletal arrangement, cell adhesion, migration and proliferation.rocks prominent role in cytoskeletal architecture suggests that rock inhibitors should have therapeutic impact in oncology and fibrotic diseases which require cytoskeletal rearrangement to progress.we have synthesized small molecule inhibitors of rock which are specific for the rock- isoform.these rock- inhibitors, typified by slx- and slx- , are potent (ic < nm), selective for rock- (> fold selectivity for rock- over rock- ) and exhibit good oral bioavailability.this talk will focus on several areas in oncology and fibrotic disease where the ability to demonstrate an in vitro effect on the cytoskeleton translates into activity in the disease model in vivo.slx- inhibits cell proliferation and migration in several tumor cell lines including ht- , panc- and mda-mb- . moreover in xenograft studies using nude mice, slx- significantly inhibited tumor growth with these same cell lines. in liver fibrosis, slx- prevents the differentiation of rat primary hepatic stellate cells into myofibroblasts and inhibits the proliferation of myofibroblasts as well as inhibiting hepatic steatosis in an atherosclerosis model.slx- is an effective antifibrotic agent in the kidney unilateral urethral obstruction model and inhibits renal fibroblast differentiation and proliferation.these data suggest that rock- selective inhibition of cytoskeletal modification in key cell types (e.g. tumor cells, stellate cells and fibroblasts) by compounds such as slx- and slx- will provide effective treatment for oncology and fibrotic disease. cyclooxygenases (cox) catalyze the first step in the synthesis of prostaglandins (pg) from arachidonic acid.cox- is constitutively expressed.the cox- gene is an immediate early-response gene that is induced by variety of mitogenic and inflammatory stimuli.levels of cox- are increased in both inflamed and malignant tissues.in inflamed tissues, there is both pharmacological and genetic evidence that targeting cox- can either improve (e.g., osteoarthritis) or exacerbate symptoms (e.g., inflammatory bowel disease).multiple lines of evidence suggest that cox- plays a significant role in carcinogenesis.the most specific data that support a cause-and effect relationship between cox- and tumorigenesis come from genetic studies.overexpression of cox- has been observed to drive tumor formation whereas cox- deficiency protects against several tumor types.selective cox- inhibitors protect against the formation and growth of experimental tumors.moreover, selective cox- inhibitors are active in preventing colorectal adenomas in humans.increased amounts of cox- -derived pge are found in both inflamed and neoplastic tissues.the fact that pge can stimulate cell proliferation, inhibit apoptosis and induce angiogenesis fits with evidence that induction of cox- contributes to both wound healing and tumor growth.taken together, it seems likely that cox- induction contributes to wound healing in response to injury but reduces the threshold for carcinogenesis. ( ), k hagihara( ), t nishikawa( ), j song( ), a matsumura ( ) ( ) health care center, osaka university, japan ( ) osaka university graduate school of medicine, japan it is still less known about the actual pathogenic role of il- in the inflammatory status. to know the pathogenic role of il- and the efficacy of il- blockade in inflammation, a humanized anti-il- receptor antibody, tocilizumab, was used for the treatment in chronic inflammatory diseases, such as castlemans disease, rheumatoid arthritis and crohns disease. since il- blocking therapy improved the clinical symptoms and the laboratory findings, the il- function in inflammation could be analyzed for the induction of inflammatory molecules, such as serum amyloid a (saa). saamrna induction, saa promoter activity and assembling of transcriptional factors on saa promoter were analyzed by the real time rt-pcr, gel shift assay and dna affinity chromatography in hepatocyte stimulated with the proinflammatory cytokines, il- , il- and tnf-alpha. in result, il- was an essential cytokine in induction of saamrna through the activation of stat which constructed the complex with nf-kappab p and a cofactor p . although there was no stat consensus region on saa promoter, stat bound at the site of nf-kappab re. the above research proved that il- signal is essential on the synergistic induction of saa via newly discovered stat transcriptional mechanism, suggesting the presence of this stat mechanism in inflammation, and confirming the normalization of serum saa level by il- blocking therapy in inflammatory diseases. this research method develops a subsequent therapy for serious aa amyloidosis by inhibition of saa production, and elucidates the cytokine mechanism on immunopathogenesis of chronic inflammatory diseases. takashi wada( ), k matsushima( ), s kaneko ( ) ( ) kanazawa university, japan ( ) university of tokyo, japan accumulating evidence indicates that chemokine/chemokine receptor system plays a key role in the pathogenesis of various renal diseases via leukocyte migration. pathophysiological impacts of chemokines have shed light on molecular mechanisms of leukocyte trafficking and their activation in the inflammatory aspects of progressive renal injury.locally expressed chemokines are proven to be capable of inciting leukocyte migration to the kidney, resulting in initiating and promoting chronic kidney diseases.the possible positive amplification loop from cxc chemokines to cc chemokines may contribute to progressive renal injury, resulting in sclerosis/fibrosis.it is of note that monocyte chemoattractant protein (mcp)- / monocyte chemotactic and activating factor (mcaf)/ ccl , a prototype of cc chemokines, promotes and escalates chronic kidney diseases with any etiologies via the infiltration and activation of monocytes/macrophages, proteinuria and collagen synthesis.interactions between infiltrated inflammatory cells and resident renal cells eventually lead to the progression of fibrosis. new insights into renal fibrosis have been uncovered by the regulation of fibrocytes dependent on chemokine system. in addition, recent studies demonstrate that chemokines have been expanding their universe beyond leukocyte migration to the kidney, including homeostasis, development and repair of the kidney.the selective intervention of chemokines might have the therapeutic potential to alter inflammatory responses, thereby halting the progression of renal injury. we focus on recent progresses on the role of chemokines and their cognate receptors in renal injury in this symposium. ( ), p lacamera ( ), b shea ( ), g campanella ( ), b karimi-shah ( ) , n kim ( ), z zhao( ), v polosukhin ( ), y xu( ), t blackwell ( ) aberrant wound-healing responses to injury have been implicated in the development of pulmonary fibrosis, but the mediators directing these pathologic responses remain to be fully identified.here we demonstrate that lysophosphatidic acid (lpa) is induced by lung injury in the bleomycin model of pulmonary fibrosis, and that mice deficient for one of its receptors, lpa , are dramatically protected from pulmonary fibrosis and mortality following bleomycin challenge. the absence of lpa markedly reduced fibroblast responses to the chemotactic activity present in the airspaces following bleomycin, and attenuated the subsequent accumulation of fibroblasts in the lung.the increase in vascular permeability caused by lung injury was also markedly reduced in lpa -deficient mice, whereas bleomycin-induced leukocyte recruitment was preserved.these results demonstrate that lpa links pulmonary fibrosis to lung injury by mediating fibroblast recruitment and vascular leak, two of the wound-healing responses that are thought to be inappropriately excessive when injury leads to fibrosis rather than repair.lpa therefore represents a new target for lung diseases in which aberrant responses to injury contribute to the development of fibrosis, such as idiopathic pulmonary fibrosis and the acute respiratory distress syndrome. we have reported that inflammation is detrimental for survival of new hippocampal neurons early after they have been born. our data now show that microglia activation, as an indicator of inflammation, is not pro-or antineurogenic per se but the net outcome is probably dependent on the balance between secreted molecules with pro-and antiinflammatory action. we have found that a substantial fraction of the new hippocampal neurons formed after status epilepticus survive despite chronic inflammation. we have started to explore the role of tnf-alpha for adult neurogenesis. infusion of an antibody to tnf-alpha was shown to reduce survival of new striatal and hippocampal neurons generated after stroke, probably by interfering with action of the ligand on the tnf-r receptor. we have shown that tnf-r is a negative regulator of progenitor proliferation in basal and insult-induced hippocampal neurogenesis. we have also used patch-clamp technique to explore whether a pathological environment influences the synaptic properties of new granule cells. rats were exposed to either a physiological stimulus, i.e., running, or a brain insult, i.e., status epilepticus which is associated with inflammation. we found that new granule cells in runners and status epilepticus animals had similar intrinsic membrane properties. in contrast, the new neurons which had developed in the physiological and pathological tissue environments differed with respect to tonic drive and short-term plasticity of both excitatory and inhibitory afferent synapses. the role of inflammation for these differences is currently being explored. proteinase-activated receptor- (par- ) is cleaved within its aminoterminal extracellular domain by serine protei-nases such as trypsin, unmasking a new aminoterminus starting with sligkv that binds intramolecularly and activates the receptor. par- is implicated in innate defense responses associated with lung inflammation. we showed that par- is expressed by human alveolar (a ) and bronchial ( hbe) epithelial cell lines, and is activated by trypsin and by the activating synthetic peptide sligkv-nh . in cystic fibrosis patients, airspaces are invaded by polymorphonuclear neutrophils that release elastase and cathepsin g, two serine proteinases, and by pseudomonas aeruginosa that secretes an elastolytic metalloproteinase.we demonstrated that these three proteinases do not activate par- , but rather disarm this receptor, preventing its further activation by trypsin but not by sligkv-nh . preincubation of a par- transfected cell line with either proteinases leads to the disappearance of the cleavage/activation epitope. proteolysis by these three proteinases of synthetic peptides representing the aminoterminal extracellular domain encompassing the cleavage/activation sequence of par- , generate fragments that would not by themselves act as receptor-activating ligands and that would not yield receptor-activating peptides upon further proteolysis by trypsin. our data indicate that neutrophiland pathogen-derived proteinases can potentially silence the function of par- in the respiratory tract, thereby altering the host innate defense mechanisms. caspase- -dependent killing of host cells and to disrupt intestinal barrier function, which, at least in the case of giardiasis, ultimately causes lymphocyte-dependent intestinal malfunction, and the production of diarrheal symptoms. ongoing research is investigating whether par agonists and microbial pathogens may cause epithelial apoptosis, increased permeability, and overall epithelial malfunction in the gastrointestinal tract, via common or intersecting pathways. the intestinal epithelium is exposed to a variety of proteases in both health and disease, including digestive proteases such as trypsin.given that protease-activated receptor (par ) responds to trypsin and is expressed on intestinal epithelial cells, we investigated the effect of trypsin on intestinal epithelial barrier function.scbn, caco-ii and t epithelial cells were grown to confluence on filter supports and mounted in ussing chambers to study short circuit current (isc) and transepithelial resistance (rte).cell monolayers were incubated with inhibitors of transcellular ion transport in order to isolate the contribution of the paracellular pathway to rte.apical exposure to serine proteases including trypsin, elastase and chymotrypsin caused a rapid and sustained increase in rte and decreased the transepithelial flux of a mw dextran.interestingly, the effect of trypsin could not be replicated by activators of pars , and , suggesting that the effect on rte was not due to activation of pars.subsequent experiments showed that trypsin activated phosphatidylinositol-dependent phospholipase c.a trypsin-induced increase in intracellular calcium was not involved.inhibition of pkc-zeta, but not of typical pkcs, also blocked the response.our data point to a role for postprandial trypsin that extends beyond that of a digestive enzyme; it is also a participant in cellular pathways that control tight junction permeability. physiologically, the trypsin-induced increase in resistance could augment transcellular transport by reducing passive paracellular back-diffusion of ions. further studies will assess how these pathways might be disrupted in the barrier dysfunction characteristic of intestinal inflammation. clustering of inflammatory bowel disease in large families and the observation of an increased concordance between monozygotic twins suggests heritable components in these disorders. the high concordance in monozygotic twins (> %), which is not seen in dizygotic twins (< %) points to strong contribution of genetic susceptibility to the overall risk for disease. ibd represents a "complex disease" and may involve a large number of interacting disease genes. crohns disease has become an example for the successful molecular exploration of a polygenic etiology. crohns disease was not known before . incidence has increased since now leading to a lifetime prevalence of up to . % in western industrialized countries. the current hypotheses propose unknown trigger factors in the life style of western industrialized nations that interact with a polygenic susceptibility. it appears that increased expression and production of tnf and an enhanced state of activation of the nfkb system are main drivers of the mucosal inflammatory reaction. the exploration of inflammatory pathophysiology of crohns disease using full genome, cdna and oligonucleotide based arrays, respectively, has generated large sets of genes that are differentially expressed between inflamed mucosa and normal controls. while this may lead to new targets for a pathophysiology oriented therapy, it appears, however, that the dissection of the inflammatory pathophysiology does not allow to identify the multifactorial etiology of the disease. genome-wide linkage analysis has demonstrated eight confirmed susceptibility regions with the one on chromosome being most consistent between different populations. in three coding variations in the card gene were identified that are highly associated with development of the disease. all variants affect a part of the gene that codes for the leucin rich part of the protein, that appears to be involved in bacteria induced activation of nfkb in macrophages and epithelial cells. interestingly, the three disease associated snps are never found on the same haplotype. in compound heterozygotes or homozygotes they result in a rr of > to develop crohns disease as an adult. a particular subphenotype with localization of the disease in the ileocecal region is highly associated with the variants in the card gene. variations in the card gene do not fully explain the linkage finding in the pericentromeric region of chromosome . after stratification for card variants, the broad linkage peak is reduced to two more defined peaks on p and q, respectively. while the exploration of these regions has led to several association signals that are subject to further fine mapping a further disease gene progress has been greater in the other linkage regions (i.e. on chromosomes and , respectively). dlg- is the example of a low-risk susceptibility gene with a modest associated odds ratio ( . - . ) . interestingly, the associa-tion signal appears to be confined to young males. slc a / which encode the kation-transporters octn and have been suggested to represent the disease gene in the + kb haplotype block on chromosome q . mdr has also been implicated as a disease gene in ibd. although the human association studies have resulted in highly controversial findings a knockout mouse with a colitis phenotype makes mdr likely as a low risk susceptibility gene. with the advent of high-density, genome wide association studies enormous progress has been made to discover the remaining disease genes. recently a k illumina scan has been published identifying il- r as a further disease gene. we used a genome wide candidate gene approach (with appr. . csnps) to identify atg l as a further disease genes. both genes were confirmed and a further regulatory snp involving ptger was annotated by a belgian genome wide scan. by the time of presentation three further genome wide snp scans in crohn disease will most likely have entered the public domain. the further exploration of crohns disease (and other inflammatory conditions of barrier organs) will have to annotate the function and pathophysiologies based on genetic risk maps that are completed with amazing speed. the creation of a medical systems biology of disease will lead to new models and eventually new therapies. the chemokine receptor ccr plays a pivotal role in mediating the migration of t cells to the gastrointestinal mucosa. the ligand for ccr , teck, s highly expressed in the gi tract. the pathogenicity of intestinal ccr +/ cd + t cells has been demonstrated in animal models and this cell population is substantially increased in the peripheral circulation of crohns and celiac disease patients. ccx -b is a highly selective and potent, orally bioavailable, small molecule antagonist of ccr .the compound proved to be highly efficacious in the tnf-aare and mdr a-/-murine models of inflammatory bowel disease (ibd). in phase i trials, ccx -b was well-tolerated, and no drug-related saes were reported.a -day placebo-controlled phase ii study was recently completed in patients with moderate to severe crohns disease. ccx -b was shown to be both safe and to have encouraging clinical results: % of patients on ccx -b (cdai ! , crp > . mg/l) exhibited a -point drop in cdai compared to % on placebo. furthermore, a crp decrease of mg/l was seen in the ccx -b group compared to placebo. colonic biopsy samples were analyzed for expression of several pro-inflammatory cytokines. a mean decrease from baseline in the concentrations of tnf-alpha, il- p , ifn-gamma, and the chemokine rantes was shown in the ccx -b group while levels remained stable in the placebo group. ccx -b is the first chemokine-based inhibitor of leukocyte trafficking to be tested in ibd. the compound shows anti-inflammatory activity and encouraging evidence for clinical benefit in the treatment of crohns disease. the activating receptor nkg d seems to be implicated in the pathogenesis of several autoimmune diseases in humans and in animal models for type diabetes and multiple sclerosis. the aim of this study was to asses the role of nkg d in a model of inflammatory bowel disease, where cd +cd -t cells from balb/c mice are adoptively transferred to scid mice, and to evaluate the therapeutic effect of an anti-nkg d antibody therapy. the expression of nkg d was evaluated by flow cytometry, immunohistochemistry and by pcr. we found a marked up-regulation of nkg d on the cell surface as well as increased levels of nkg d mrna in cd + t cells from colitic scid mice as compared to normal balb/c mice. we next studied the effect of anti-nkg d antibody (cx ) treatment initiated either before onset of colitis, when the colitis was mild, or when severe colitis was established. cx treatment decreased the cellsurface expression of nkg d and prophylactic administration of cx attenuated the development of colitis significantly. a moderate reduction in the severity of disease was observed after cx administration to mildly colitic animals, whereas cx did not attenuate severe colitis. thus, nkg d may be involved in the pathogenesis of colitis in this model, particularly in the early phases, since the expression of nkg d in cd + t cells increased markedly during the development of disease and since administration of cx early but not late in the course attenuated the disease severity. proteins are used already for more than a century in the treatment of disease. the first generation were proteins derived from animals such as antisera used to treat infectious diseases as diphtheria and tetanus and later bovine and porcine insulin for the treatment ofdiabetes. the second generation were natural proteins from human source like the plasma derived clotting factors and human growth hormone. the development of the recombinant dna and cell fusion technology in the seventies of the th century opened up the possibilities to produce human proteins and monoclonal antibodies in unlimited amount in microbial and mammalian host cells. in human insulin was introduced as the first recombinant dna derived biopharmaceutical and since than more than have gained approval. the pipeline contains many more potential biopharmaceuticals and at present in new drug applications concerns a biotechnology derived product. a major problem of therapeutic proteins is the induction of antibodies. for foreign proteins such as the murine derived monoclonal antibodies thisimmunogenicity was to be expected. however the humanization of monoclonal antibodies has reduced but not solved the problem of immunogenicity. and also the proteins which are homologues of endogenousfactors such as gm-csf, interferons etc. induce antibodies, sometimes even in the majority of patients. by definition we are immune tolerant to products which are copies of endogenous proteins. the products not necessarily need to be exact copies of the natural proteins to share this immune tolerance. when human therapeutic proteins induce antibodies, they are breaking b cell tolerance, which starts with the activation of autoreactive b-cells. presenting the self-epitopes in an array form is very potent activator of these b-cells. this explains why aggregates of human proteins are the most important factor in induction of antibodies. these aggregates may not be immediately present in the product, but may appear during storage making stability and formulation an important issue in predicting the immunogenicity. there are only a few studies in experimental model systems on the properties of the aggregates which break b-cell tolerance, indicating that only multiple order aggregates (>trimers) are involved. we study the capacity of a protein product to break b-cell tolerance in mice made transgenic for the specific protein. these mice are immune tolerant and there is a good correlation of an immune response in these mice and in patients. although these models have helped to identify the factors important for breaking b-cell tolerance and also have been useful in improving the formulation of products, there is not yet enough experience to use them as absolute predictors of immunogenicity of human proteins. they also allow to study the involvement of tcells in breaking b cell tolerance. all data obtained untilnow indicate this process to be t-cell independent. contact information: dr huub schellekens, central laboratory animal institute, utrecht university, utrecht, the netherlands e-mail: h.schellekens@gdl.uu.nl biomonitor aps, and institute for inflammation research (iir), rigshospitalet, copenhagen, denmark using recombinant technology, one can now produce protein drugs which are almost identical with naturally occurring human proteins, including antibodies (abs). many have assumed that these drugs may be administered with little or no risk of triggering specific t-and/or b-lymphocyte reactivities, because patients according to immunological dogma are tolerant towards their own proteins. unfortunately, this is not the case, and even socalled % human biologicals are potentially immunogenic ( ) ( ) . i shall discuss two examples: ) recombinant human cytokines (ifn-beta- a and - b), and ) anticytokine ab constructs (anti-tumor necrosis factor (tnf)-alpha). ifn-beta has been used for treatment of patients with multiple sclerosis since the early nineties. though initially neglected as a clinical problem, ifn-beta like many other human proteins is indeed immunogenic, especially those produced by recombinant gene technologies. the reported frequencies and titers of anti-ifn-beta ab vary considerably depending upon ifn-beta preparations and administration, and the types of assays being used ( - ). it took more than years of clinical use before abmediated decrease in bioactivity of ifn-beta, a condition in which the clinical effect of continued injection of rec. ifn-beta is minimized or abrogated, was universally recognized ( , ). ) anti-tnf-alpha human ab constructs tnf-alpha is an inflammatory cytokine of central pathogenic importance in many immunoinflammatory conditions, and measures to diminish production and/or effects of tnf-alpha have long been a goal in the treatment of these conditions. currently, there are three approved and two other anti-tnf-alpha biopharmaceuticals in clinical use. unfortunately, response failure is frequently encountered. thus, - % of patients are primary non-or lowresponders to the anti-tnf constructs, and secondary response failure is commonplace, mostly due to induction of anti-abs. several different methods have been used to assess circulating levels of anti-tnf drugs as well as anti-abs. most of these have been based on elisa technology, with their inherent problems of false positivity, susceptibility to nonspecific interference, etc. interferon beta (ifnbeta) has been an important step forward in the treatment of multiple sclerosis(ms), an inflammatory disease of the human central nervous system. however, one of the problems of ifnbeta is its immunogenicity; a substantial percentage of ms patients treated this recombinant protein develop anti-ifnß antibodies, primarily of the igg class. the level of these antibodies tends to be low in the first month or two and peaks by six to eighteen months after initiation of therapy. most studies of these antibodies have measured their ability to neutralize ifnbetas effect in vitro, using assays in which sera from ms patients inhibit the protective effect of ifnbeta on viral killing of target cells. this antibody population is called neutralizing antibodies (nabs). tests measuring binding of antibodies to ifnß in vitro are called binding antibody (bab) assay. anti-ifnbeta antibodies detected by bab assays are present in a high percentage of ms patients, and can occur at low levels without any apparent adverse effect on ifnbeta bioactivity. the distinction between babs and nabs is artificial, and all binding antibodies are likely neutralizing, if the neutralizing assay system is adequately sensitive; i.e., the development of babs and nabs is a continuum with the assay systems simply measuring the strength of the antibody response. in many treated patients, the anti-ifnbeta antibody response is strong, despite the resemblance of the injected protein to the human homologue, and high levels of neutralizing antibodies develop. high levels of anti-ifnbeta antibodies with high affinity results in loss of ifnbeta bioactivity, a phenomenon which has been called antibody-mediated decreased bioactivity or adb. adb can be considered the in vivo correlate of the neutralizing effect of the anti-ifnß antibody population, while the nab assay measures the in vitro neutralization of this population of immunoglobulins in the serum. the three ifnbeta preparations have different incidences of nabs and different patterns of appearance and disappearance over time of nabs. because there is no direct correlation between nab levels and bioactivity at moderate levels of nab, in vivo bioactivity assays for ifnbeta have become increasingly utilized. in a large multicenter study in the us, called the insight study, bioactivity as measured by ifnbeta induced upregulation of the ifn-response genes mxa, viperin, and ifit , was shown to be highly correlated with nab levels, confirming a single center study (pachner, a.r., pak,e., narayan, k., multiplex analysis of expression of three ifnbeta-inducible genes in antibody-positive ms patients, neurology, : - , ) . multiple studies, including a large multicenter study in denmark and a recent study from our center using high resolution mri of the brain once a month, have demonstrated that nabs abolish the salutary effects of ifnbeta on clinical aspects of ms, especially inflammation. recent guidelines for european neurologists recommend stopping ifnbeta in nab-positive patients. in order to maintain bioactivity of this important medication for ms, some neurologists have attempted to use immunosuppressives either to prevent the development of nabs, or to treat them once they have developed. however, at this point in time, there is no clearly optimal way to treat nabs. major efforts have been underway to decrease the immunogenicity of ifnbeta and a new formulation of one of the higher immunogenicity products has been recently developed and tested. proteinase-activated receptors (pars). endogenous serine proteinases such as thrombin, mast cell tryptase, trypsins, kallikreins, cathepsin g, for example, as well as exogenous proteases released by mites or bacteria are involved in cutaneous inflammation, host defense or tumor cell regulation. thus, the expression of pars on keratinocytes, endothelial cells, nerves, and immune cells suggest important role of pars as a part of the communication system in the skin during inflammation and the immune response. for example, par activates nf-kb in keratinocytes and endothelial cells, stimulates the release of chemokines and cytokines, and is involved in proliferation and differentiation. on sensory nerves, this receptor controls neurogenic inflammation by modulating edema and extravasation via release of neuropeptides into the inflammatory site. par and par also modulate leukocyte-endothelial interactions in the skin, thereby regulating inflammatory responses such as leukocyte trafficking through the vessel wall. they also stimulate signal transduction pathways involved in cutaneous inflammation. in sum, this novel receptor family requires a paradigm shift in thinking about the role of proteases in cutaneous biology and disease. novel compounds regulating protease and par function may be beneficial for the treatment of several skin diseases such as atopic dermatitis, psoriasis or pruritus. serine proteinases are upregulated in arthritic joints where their enzymatic activity participates in the destruction of articular soft tissues. in addition to their degradative functions, serine proteinases can also act as signalling molecules by activating members of the gprotein coupled receptor family called the proteinase activated receptors (pars). these receptors are known to regulate tissue inflammation and pain, although their function in joints is unclear. our study examined the effect of par activation in joint inflammation and pain. male c bl/ mice received an intra-articular injection of either the par activating peptide aypgkf-nh or the inactive peptide yapgkf-nh ( mg) into the right knee. knee joint blood flow was then measured in these mice by laser doppler perfusion imaging while joint diameter measurements gave an indication of tissue oedema. mechanical allodynia was also assessed in these animals by application of von frey filaments onto the plantar surface of the ipsilateral hindpaw and a pain score was calculated. intra-articular injection of the par activating peptide caused knee joint blood flow to gradually increase by up to % over the succeeding hrs. knee joint swelling was also observed as well as the development of a mechanical allodynia. all responses could be blocked by pre-treatment with the selective par antagonist pepducin p pal ( mg i.p.). the control peptide yapgkf-nh had no discernible effect on joint inflammation or pain. these experiments show that peripheral activation of par receptors in mice knees causes joint inflammation and pain. vincent lagente ( ), e boichot ( ) ( ) air liquide, centre de recherche claude-delorme, jouy en josas, france ( ) inserm u , universitØ de rennes matrix metalloproteinases (mmps) are a major group of proteases known to regulate the turn-over of extracellular matrix and so they are suggested to be important in the process of lung disease associated with tissue remodelling. these led to the concept that modulation of airway remodeling including excessive proteolysis damage of the tissue may be of interest for future treatment. among metalloproteinases (mmps) family, macrophage elastase (mmp- ) is able to degrade extracellular matrix components such as elastin and is involved in tissue remodeling processes in chronic obstructive pulmonary disease (copd). pulmonary fibrosis has an aggressive course and is usually fatal for an average of three to six years after the onset of symptoms. pulmonary fibrosis is associated with deposition of extracellular matrix (ecm) components in the lung interstitium. the excessive airway remodeling as a result of an imbalance in the equilibrium of the normal processes of synthesis and degradation of extracellular matrix components could be in favor of anti-protease treatments. indeed, the correlation of the differences in hydroxyproline levels in the lungs of bleomycin-treated mice strongly suggests that a reduced molar pro-mmp- /timp- ratio in broncholaveolar lavage fluid is associated with collagen deposition, beginning as early as the inflammatory events at day after bleomycin administration. finally, these observations emphasize those effective therapies for these disorders must be given early in the natural history of the disease, prior to the development of extensive lung destruction and fibrosis. in addition to their degradative properties, proteases can act as signalling molecules that send specific messages to cells.recent work has demonstrated that proteases are able to signal to peripheral sensory neurons, thereby participating to neurogenic inflammation processes and to the transmission or inhibition of pain messages. serine proteases cleaving specifically at an arginin site are able to activate protease-activated receptors (pars), which then send specific messages to cells. we have demonstrated that members of the par family (par , par and par ) are present on peripheral sensory neurons, where they can be activated by different proteases.the activation of par and par in isolated sensory neurons provokes calcium mobilization and the release of substance p and cgrp, while the activation of par inhibited bradykinin-and capsaicin-induced calcium signal, and neuropeptide release.thrombin and pancreatic trypsin caused inflammation respectively through a par and par -dependent mechanism involving the release of neuropeptide.the extrapancreatic form of trypsin (mesotrypsin or trypsin iv) also caused neurogenic inflammation through a par and par dependent mechanism, and causes inflammatory hyperalgesia and allodynia, through a par -dependent mechanism.in contrast, activation of par on peripheral sensory neurons inhibited inflammatory hyperalgesia and allodynia. taken together, these results provide evidences that proteases can interfere with inflammatory and pain mechanisms through the activation of pars on peripheral sensory neurons.determining the role of each individual proteases and their receptors in sensory neuron signalling and above all inflammatory and pain mechanisms constitutes an important challenge to raise new anti-inflammatory and analgesic drugs. introduction: a scoring system for disseminated intravascular coagulation (dic) in humans has been proposed by the international society on thrombosis and haemostasis (isth). it was the objective of this study to develop and validate a similar scoring system for dic in dogs in order to establish the dog as a spontaneous animal model. methods: for the developmental study, consecutive dogs admitted to the intensive care unit (icu) were enrolled prospectively (group a). blood samples were collected daily and a broad panel of coagulation assays performed. diagnosis of dic was based on the expert opinion of one human physician and two veterinarians. a multiple logistic regression model was developed with the coagulation parameters as explaining variables for the diagnosis of dic. integrity and diagnostic accuracy was subsequently evaluated in a separate prospective study according to the stard criteria. the validation study prospectively enrolled consecutive dogs (group b). results: dogs were excluded from group a where / dogs ( %) had dic. final multiple logistic regression model was based on aptt, pt, d-dimer and fibrinogen and had a very high diagnostic sensitivity and specificity. diagnostic accuracy of the model was sustained by prospective evaluation in group b. conclusion: based on generally available assays, it was possible to design an objective diagnostic model for canine dic, which has both a high sensitivity and specificity. such a model will provide basis for treatment optimization and make it possible to conduct multicentered therapy studies with a minimum risk of systematic misclassification of patients. in , a coagulation independent change in light transmittance (biphasic waveform [bpw] ) was reported in automated activated partial thromboplastin time assays (aptt) in patients with disseminated intravascular coagulation (dic). a calcium-dependant precipitate of creactive protein and very-low-density-lipoprotein was causing the bpw. our group recently identified this phenomenon in dogs also. initially, bpw was introduced as a complementary tool to assist diagnosing dic. however, recent studies reported that bpw may have a stronger potential as a prognostic marker for survival. the aims of the study were to prospectively investigate (a) the diagnostic significance of bpw regarding dic and (b) the significance of bpw to outcome, in dogs with diseases known to predispose for dic. the study was performed as a prospective, observational study including consecutive dogs with a final diagnosis known to predispose for dic ( % were finally diagnosed with dic). outcome was -day survival. bpw was assessed by means of a hirudin-modified aptt assay (kjelgaard-hansen et al., jvim : ; - ) . relative risk according to bpw (rr [ % confidence interval]) for (a) a dic diagnosis and (b) -day mortality, were assessed. -day mortality in the study population was %. % were bpw positive. bpw was not a significant diagnostic factor for dic (rr= . [ . ; . ] ), but strongly so for outcome (rr= . [ . ; . ] ) with a % ( / ) mortality amongst bpw positive dogs. in conclusion, bpw was observed in dogs predisposed to dic, with a strong potential as a risk factor for outcome, a finding in line with recent findings in humans. ( ), b hideo ( ) ( ) department of molecular pathology, kumamoto university, japan ( ) department of gastroenterological surgery, kumamoto university, japan aeromonas species are facultative anaerobic gramnegative rods that are ubiquitous, waterborne bacilli, most commonly implicated as causative agents of gastroenteritis. aeromonas infections often develop sepsis and disseminated intravascular coagulation syndrome (dic) is a life-threatening complication of sepsis patients, causing multiple organ failure.however, a mechanism leading to coagulation induction in the bacterial infection has not been known. to study the dic induction by aeromonas species infection, we investigated coagulation activity of a serine protease (asp) from aeromonas sobria, predominantly isolated in patients blood. proteolytically active asp shortened both activated partial thromboplastin time and prothrombin time of human plasma in a dose-dependent manner starting at an enzyme concentration of nm. asp activated human prothrombin, releasing hydrolytic activity for thrombinspecific substrate boc-val-pro-arg-mca, but no enzymatic activity was produced from coagulation factors ix and x. analysis by sds-page revealed that asp released a prothrombin fragment with a molecular weight identical with that of f¿-thrombin in an incubation timedependent manner. western blotting using biotinylated phe-pro-arg-chloromethylketone, a thrombin inhibitor, showed that asp produced an enzymatically active fragment whose molecular weight was same as that of f¿-thrombin. prothrombin incubated with asp but not the protease itself caused platelet aggregation. these results indicate that asp activates prothrombin, producing f¿-thrombin that converts fibrinogen to fibrin clot, and suggest that asp coagulation-inducing activity contributes to dic development in sepsis caused by aeromonas sobria infection. the present study shows a link between inflammation and coagulation mediated by a bacterial protease. hemolytic episodes are often associated to high amounts of free heme in circulation (up to um) and the development of an inflammatory response that may develop to a chronic inflammation. our group has shown that free heme is a prototypical proinflammatory molecule, able to induce neutrophil migration, actin cytoskeleton reorganization and nadph oxidasederived reactive oxygen species (ros) generation, as well as pkc activation and interlukin- expression (graÅa-souza et al., ) . moreover, free heme inhibits human neutrophil spontaneous apoptosis, a feature that is closely related to the impairment of resolution of inflammation and consequent promotion of chronic inflammatory status. heme protective effect requires nadph oxidase-derived ros and involves the activation of mapk, pi k and nf-kb signaling cascades as well as heme oxygenase (ho) activity (arruda et al., ) . more recently, we have shown that heme antiapoptotic effect is closely related to the maintainance of mitochondrial stability, inhibition of bax insertion into mitochondria and a dramatic increase on bcl-xl/bad protein ratio in a ros-dependent manner, requiring the same signaling pathways that regulate heme anti-apoptotic effect. these findings attest to a prominent role of free heme in the onset of inflammation associated to hemolytic episodes as well as the statement of chronic inflammation related to these disorders. the recent advance on the study of free heme as a proinflammatory molecule brings up hope for the development of new strategies to ameliorate acute and chronic inflammation found during hemolytic episodes.financial support: faperj, cnpq, capes. ( ) ( ) university of melbourne, victoria, australia ( ) monash university, victoria, australia we have previously demonstrated that mice lacking the anti-oxidative enzyme, glutathione peroxidase (gpx ), show significantly larger infarcts after stroke. recent studies have demonstrated that adhesion molecule-mediated leukocyte recruitment is associated with increased tissue damage in stroke, while mice lacking key adhesion molecules conferred neuro-protection. nevertheless, the involvement of oxidative stress in leukocyte recruitment and subsequent regulated cell injury is yet to be elucidated. to explore this, gpx -/-mice were subjected to transient mid-cerebral artery occlusion (mcao) followed by cerebral intravital microscopy, for assessment of leukocyte-endothelium interactions in intact cerebral microvasculature. after hr mcao, leukocyte-endothelium interaction was significantly reduced in gpx -/-mice compared to wt counterparts during the second hour of reperfusion. laser doppler and direct measurement of blood flow in pial postcapillary venules revealed a reduction of reperfusion in gpx -/-mice following transient mcao. this suggests that the reduction in nutritive blood flow following stroke in gpx -/-mice may explain the enhanced injury in these mice as well as the reduced leukocyte-endothelium interaction. furthermore, matrix metalloproteinase- (mmp ) which has previously been shown to be implicated in endothelial dysfunction and the pathogenesis of stroke was found to be up-regulated in gpx -/-mice to a greater extent than in wt mice after mcao, suggesting a role for oxidative stress in cerebral microvascular injury. the data present here suggests oxidative stress may be one of the factors that contribute to reduced post-ischemic perfusion, via the disruption of the endothelial function as indicated by the increased level of mmp . chris bolton( ), c paul( ), s barker ( ), r mongru ( ) ( ) william harvey research institute, london, uk ( ) university west of england, bristol ( ) queen mary university of london adrenomedullin (am) acts as a vasodilator in many vascular beds including the cerebral circulation where the peptide is produced in larger amounts than in the periphery.in vitro work has shown that am beneficially regulates blood-brain barrier (bbb) characteristics including transendothelial electrical resistance, permeability and p-glycoprotein pump activation.our preliminary studies in acute experimental autoimmune encephalomyelitis (eae), a model of the human disease multiple sclerosis (ms), have demonstrated significant elevations in am peptide levels corresponding with am mrna changes during late, neurological disease where am production may be linked to the restoration of bbb function. however, am is not exclusively produced as result of am gene upregulation. furthermore, am peptide levels do not always match am mrna changes during other disease phases of eae.the current study has investigated, more closely, the relationship between am gene expression and subsequent levels of associated peptides. am mrna levels were determined, by rt-pcr, in the cerebellum, medulla-pons and spinal cord of normal and eae-inoculated lewis rats at the height of disease. am and proadrenomedullin peptide (pamp) levels were measured in the tissues by radioimmunoassay.all tissues examined showed an increase in am gene mrna compared to control levels.am and pamp changes were observed in the samples and differences between the peptide profiles were recorded.an understanding of alterations in the generation of am and related peptides during neuroinflammation may provide insight into mechanisms affecting bbb permeability and be of relevance to the changes in neurovascular function seen during ms. platelet-activating factor (paf) contributes to the robust inflammatory responses in acute phase and spread of secondary injury. although, paf is believed to be a potent edematous but non-painful mediator in peripheral tissues, we recently demonstrated that paf may be a mediator of noxious signaling in spinal cord in case of neuronal injury. paf-induced tactile allodynia may be mediated by atp, glutamate and the generation of nitric oxide (no). the present study elucidated down-stream signaling pathway for paf-induced tactile allodynia. paf-and glutamate-induced tactile allodynia was blocked by the pretreatment with no scavengers and inhibitors of no synthase, soluble guanylate cyclase or cgmp-dependent protein kinase (pkg). recent evidence attributes the generation of pain to specific disfunctions of inhibitory glycinergic neurotransmission. to explore the target molecule for induction of tactile allodynia, the effect of knockdown of glycine receptors containing the a subunit (glyr a ) by sirna spinally transfected with hvj-e vector was examined. in mice spinally transferred with sirna for glyr a , the reduction of glyr a was demonstrated in superficial layer of dorsal horn by immunohistochemical analysis. pcpt-cgmp, paf, glutamate failed to induce tactile allodynia in mice spinally transferred with sirna of glyr a , while these compounds produced tactile allodynia in mice transferred with mutant sirna of glyr a as a control. glycine tranporter inhibitors ameriolated paf-and pcpt-cgmp-induced allodynia. these results suggest that glutamate-no-cgmp-pkg pathway plays a key role for paf-induced tactile allodynia in spinal cord and glyr a may be a target molecule for pkg to induce allodynia. ( ), r leite( ), ys bakhle ( ) ( ) federal university of minas gerais, belo horizonte, brazil ( ) medical college of georgia, augusta, usa ( ) imperial college, london, uk selective cyclooxygenase inhibitors (coxibs) induce a characteristic increase in mechanical nociceptive threshold, referred to as "hypoalgesia", in inflammatory pain induced by carrageenan in rat paws.we have here assessed the role of the cytoskeleton in this hypoalgesia induced by celecoxib (cx). male holtzman rats ( - g; - animals/group) were injected in the right hind paw (ipl) with a range of cytoskeletal inhibitors (selective inhibitors of microtubules (taxol, nocodazole, colchicine), of actin microfilaments (latrunculin b, cytochalasin b) or of intermediary filaments (acrylamide) (pico to nanomoles per paw) and min later given cx ( mg/kg, s.c.). after a further min, rats were injected (ipl) with the inflammatory stimulus, carrageenan ( mg/paw). mechanical pain threshold was hourly measured over the next h, using the randall-sellitto method. the cxinduced hypoalgesia was reversed by low doses of latrunculin b or cytochalasin (latrunculin % reversal = . nanomoles), higher doses of microtubule inhibitors (taxol % reversal = . nanomoles) with no effect of acrylamide ( up to nanomoles).we conclude that ) local changes in (paw) cytoskeleton occurred during cxinduced hypoalgesia and ) actin microfilaments were the cytoskeletal components most critically involved in this hypoalgesia.financial support: cnpq, fapemig and capes there are reports regarding the up-regulation of cyclooxygenase isoenzyme particularly inducible isoform i.e. cox- in brain during neurodegenerative or neuropsychiatric disorders.in the present study, we examined the effect of nimesulide (a preferential cox- inhibitor) in subchronic immobilization stress. mice were subjected to immobilization stress for hrs daily for a period of seven days. nimesulide ( . mg/kg, i.p.) was administered daily for days before challenging them to immobilization stress. behavioral analysis revealed the hyperlocomotor activity and increased anxious response. subchronic stress decreased % retention of memory and also caused hyperalgesic response in mice. biochemical analysis revealed that chronic immobilization stress significantly increased lipid peroxidation and nitrite levels and decreased the reduced glutathione and adrenal ascorbic acid levels. chronic treatment with nimesulide significantly attenuated the immobilization stress-induced behavioral and biochemical alterations. these results suggested that the use of nimesulide could be a useful neuroprotective strategy in the treatment of stress. there is accumulated evidence for ngf role as a peripheral pain mediator. ngf is upregulated in diverse inflammatory conditions and evokes hyperalgesia when injected in humans and rats. ngf increase was also observed in temporomandibular join (tmj) after cfa injection, indicating its possible involvement in local hyperalgesic states. therefore, the objective here was to evaluate if ngf participate in the tmj nociception. to test this hypothesis, the ngf was injected into the tmj alone or after carrageenan (cg) and the spontaneous nociceptive behavior of head flinches was counted for up min. further evidence for the ngf nociceptive activity was obtained quantifying the local production of ngf after cg injection, by elisa, and the fos-like immunolabeling in the trigeminal sensory nucleus (including the caudalis, interpolaris and oralis) after ngf injection. injections were performed in . ul. ngf ( . , and ug) injected in the tmj challenged h prior by cg ( ug) induces a dose-dependent increase in the number of head flinches. this increase was reduced by k a ( and ug), indicating a trka receptor-mediated effect. we detected a significant increase in the ngf production and h after the tmj cg ( ug) injection. the tmj injection of ngf ( ug) alone did not induce detectable spontaneous nociceptive behavior. however, the ngf ( ug) injection induces a significant increase in the fos like immunolabeling (fli) in the sensory trigeminal nucleus compared to the saline injection. these results indicate that the ngf participates in the nociceptive activity in the tmj, specially in inflammatory conditions. mif was reported as a key cytokine in the pathogenesis of rheumatoid arthritis (ra) several years ago, but it now clear that mif is also involved in the pathogenesis of systemic lupus erythematosus (sle) and atherosclerosis. mif-deficient lupus-prone mrl/lpr mice exhibit prolonged survival and reduced renal and skin disease compared to mif-expressing mice. similarly, mif-deficient atheroma-pone ldlr-deficient or apoe-deficient mice are significantly protected from disease and antimif mab therapy is beneficial. ra and sle are each characterised both by an increased prevalence of atherosclerotic vascular disease and by overexpression of mif. given the effects of mif on atherosclerosis it can be hypothesised that mif overexpression participates in the risk of atherosclerotic vascular disease in ra and sle. recent data have provided insights into mechanisms of action for mif relevant to all these concepts. firstly, the newly described role of mif in the selective recruitment of monocyte-macrophage lineage cells is of particular relevance to ra, sle, and atherosclerosis, with evidence that mif mediates macrophage recruitment in sle and atherosclerosis. secondly, glucocorticoid (gc) therapy is possible risk factor for atherosclerosis in patients with ra and sle, and it is now clear that gc increase the expression and release of mif, potentially implicating mif in gc-related increases in atherosclerosis in ra and sle. specific therapeutic targeting of mif in ra and sle may address not only primary disease pathways but also the increased risk of atherosclerosis in these diseases. to enter inflamed tissues, leukocytes must undergo adhesion molecule-mediated interactions with the endothelial surface of vessels at the site of inflammation.cytokines such as tumour necrosis factor (tnf) are established as important mediators capable of promoting leukocyte-endothelial cell interactions.however, in inflammatory diseases such as atherosclerosis and rheumatoid arthritis, elevated expression of another cytokine, macrophage migration inhibitory factor (mif) occurs, yet the role of this cytokine in leukocyte recruitment is unknown.therefore we explored the ability of mif to regulate leukocyte recruitment.this was achieved using intravital microscopy to examine the intact microvasculature in mice following local mif treatment. these experiments showed that mif induced leukocyte adhesion and transmigration in vivo, resulting in accumulation of predominantly cd +/f / -ve/cd c-ve monocyte/ macrophage lineage cells.mif did not induce upregulation of adhesion molecules p-selectin and vcam- , although their constitutive expression contributed to recruitment.in contrast, mif-induced recruitment was blocked by antibodies to the monocyte-specific chemokine, ccl /mcp- , and its receptor ccr , and in response to anti-cxcr .this was supported by in vitro experiments showing that mif induced ccl /mcp- release from cultured murine endothelial cells.finally, mice lacking cd , the putative mif binding molecule, did not respond to mif.these data demonstrate a previously unrecognized function of this pleiotropic cytokine: induction of monocyte migration into tissues, and indicate the involvement of a pathway involving a complicated chemokine/chemokine receptor pathway with contribution from cd .this function may be critical to the ability of mif to promote diseases in which macrophages are key participants. gm-csf and m-csf (csf- ) can enhance macrophage lineage numbers as well as modulate their differentiation and function. of recent potential significance for the therapy of inflammatory/autoimmune diseases, their blockade in relevant animal models leads to a reduction in disease activity. what the critical actions are of these csfs on macrophages during inflammatory reactions are unknown. to address this issue, adherent macrophages (gm-bmm and bmm) were first derived from bone marrow precursors by gm-csf and m-csf, respectively, and stimulated in vitro with lps to measure secreted cytokine production, as well as nf-kb and ap- activities. gm-bmm preferentially produced tnfa, il- , il- and il- while, conversely, bmm generated more il- and ccl ; strikingly the latter population could not produce detectable il- and il- . following lps stimulation, gm-bmm displayed rapid ikba degradation, rela nuclear translocation and nf-kb dna binding relative to bmm, as well as a faster and enhanced ap- activation. each macrophage population was also pre-treated with the other csf prior to lps stimulation and found to adopt the phenotype of the other population to some extent as judged by cytokine production and nf-kb activity. thus gm-csf and m-csf demonstrate at the level of macrophage cytokine production different and even competing responses with implications for their respective roles in inflammation including a possible dampening role for m-csf. granulocyte macrophage-colony stimulating factor (gm-csf), initially discovered for its role in the differentiation of haematopoietic cells into granulocytes and macrophages, can also affect mature cell function and may be considered proinflammatory. gm-csf is able to prime macrophages for increased pro-inflammatory responses, including the increased release of tnfa and il- following stimulation with, for example, lps. in addition, gm-csf has been shown in vivo, using murine disease models, to play a key role in a number of inflammatory diseases. gm-csf-/-mice have been shown to be resistant to several diseases, including arthritis, and, most notably, blockade of gm-csf with a neutralizing monoclonal antibody was effective at ameliorating arthritis when given either prophylactically or therapeutically. t cells appear to be the major cell type responsible for gm-csf production required for arthritis, and gm-csf appears important in the effector phase of disease, subsequent to t cell activation. blockade of gm-csf results in fewer inflammatory cells, particularly macrophages, and cytokines such as tnfa, at the site of inflammation. these findings suggest that blockade of gm-csf may be an effective treatment in a range of inflammatory diseases. the autoimmune disease type diabetes mellitus (t dm) is thought to be mediated by autoreactive t cells recognizing islet autoantigens, including gad , ia- and proinsulin. this disease arises on a distinctive genetic background, mapping most notably to the mhc, and is also open to strong environmental influence. to investigate the pathogenesis of the disease, and in particular the prevailing paradigm that islet autoreactive t cells are important, we have developed an approach to epitope identification that is mhc allele and autoantigen specific, and operates for both cd and cd t cells. utilizing this, we have uncovered populations of islet antigen-specific t cells that have the immunological credentials to be both pathogenic (eg th , tc ) and protective (treg) in the disease. we have cloned some of these cell types, enabling us to analyse their function and provide an insight that will be important for an understanding of disease mechanisms, as well as guiding novel therapeutic interventions. tcr transgenic targeting b: - cause diabetes . knockouts of the insulin gene (expressed in thymus as well as islets) accelerates diabetes while knockout of insulin gene (islet expression) prevents % of diabetes . dual insulin knockout with transgenic insulin with altered peptide (b :a) prevents all diabetes . islets with native b: - sequence, but not altered sequence when transplanted into knockouts restore anti-insulin autoimmunity and diabetes transfer by t cells .anti-b - t cells have conserved valpha and jalpha chain usage but no conservation n region or beta chain . alpha chain as transgene sufficient to engender anti-insulin autoantibodies . kay and coworkers demonstrate insulin reactivity "upstream" of igrp and igrp reactivity nonessential.future studies in nod directed at deleting specific conserved alpha chains to test diabetes prevention and develop therapeutic.in man we can now identify at birth genetic risk as high as % of activating anti-islet autoimmunity with mhc analysis and restricted heterogeneity suggesting dominant target.insulin autoantibodies in prospective studies such as daisy usually appear initially and levels are related to progression to diabetes.analysis of cadaveric donors is underway to elucidate primary targets. (t d) is an autoimmune disease in which genes and environment contribute to cell-mediated immune destruction of insulin-producing beta cells in the islets of the pancreas. the holy grail of autoimmune disease prevention is negative vaccination against autoantigens to induce disease-specific immune tolerance. this has been achieved in rodents by administering autoantigen via a tolerogenic route (mucosal), cell type (stem cell or resting dendritic cell), mode (with blockade of t-cell co-stimulation molecules) or form (as an altered peptide ligand). compelling evidence demonstrates that proinsulin is the key autoantigen that drives beta-cell destruction in the non-obese diabetic (nod) mouse model of t d, and possibly in humans. proinsulin/ insulin dna, protein or t-cell epitope peptides administered in a tolerogenic manner to the nod mouse can delay or prevent the development of diabetes, via one or more mechanisms (deletion or anergy of effector t cells, induction of regulatory t cells). administration of autoantigen via the mucosal route, which induces anti-diabetic regulatory t cells in the rodent, is the most immediately translatable approach to humans. initial human trials of vaccination with oral autoantigens lacked evidence of bioeffect, probably due to inadequate dosage in end-stage disease. recently, however, the first evidence for a therapeutic effect of mucosal autoantigen has been seen in trials of oral and nasal insulin in islet autoantibodypositive individuals at risk for t d. combination autoantigen-specific vaccination also shows promise in combination with non-specific immunotherapy in established t d. leukocyte extravazation is an integral process both physiologically (immunosurveillance) and pathophysiologically (inflammation). the initial paradigm of a -step process comprising tethering/rolling, activation, firm adhesion, and diapedesis, each involving specific adhesion molecules, has repeatedly been modified in the light of more recent findings. additionally, organ-specific differences regarding the role of distinct molecules were established. finally, the skin became a good "model" to study due to its accessability and availability of powerful animal models. in-vitro adhesion assays, flow-chamber systems, intravital microscopy, animal models for delayed-type hypersensitivity, and transplantation approaches have successfully been employed to investigate leukocyte extravazation. numerous molecular interactions such as the cutaneous lymphocyte-associated antigen and sialyl-lewisx, or icam- and lfa- , have been proven sufficiently relevant to make them candidates for potential therapies. with the anti lfa- antibody efalizumab, approved for the treatment of psoriasis, the first therapeutic agent specifically targeting leukocyte extravazation is already on the market; other compounds are under development. moreover, novel data suggest that well-established anti-inflamamtory therapies such as fumarates also influence this process, thus contributing to their clinical efficacy. ongoing research aks for adopting a more "dynamic" view on leukocyte extravazation as several molecules obviously perform multiple tasks throughout this process rather than being limited to just one step of this multi-step cascade; this is particularly true for the so-called junctional adhesuíon molecules which obviously mediate more than just diapedesis. finally, similarities between leukocyte extravazation and hematogenic metastases are emerging. consequently, certain anti-inflammatory compounds may turn out to also exhibit striking anti-metastatic efficacy, and vice versa. department of dermatology, heinrich-heine-university, düsseldorf, germany atopic dermatitis, psoriasis vulagaris and cutaneous lupus erythematosus represent chronic inflammatory skin diseases showing distinct clinical phenotypes but sharing one aspect. the recruitment of pathogenic leukocyte subsets into the skin represents a prerequisite for their initiation and maintenance. during recent years, our knowledge of the immunopathogenesis of chronic inflammatory skin diseases increased significantly. with regard to the recruitment pathways of leukocytes, a superfamily of small cytokine-like proteins so called chemokines has attracted significant attention. here the complex interactions within the chemokine ligand-receptor network are introduced, the involvement of chemokines in memory t and dendritic cell trafficking is outlined and current concepts of their role in the immunopathogenesis of atopic dermatitis, psorasis vulgaris and cutaneous lupus erythematosus are summarized. the skin serves as a unique organ for studying general principles of inflammation because of its easy accessibility for clinical evaluation and tissue sampling. a network of pro-inflammatory cytokines including il- and tnf-a is known to play a key role in the pathogenesis of cutaneous inflammatory diseases through activation of specific signalling pathways. recently, progress in understanding the underlying mechanisms regulating inflammatory signalling pathways in the immunopathogenesis in skin carcinomas, psoriasis vulgaris and atopic dermatitis has been made. kinases have been identified to play a crucial role in regulating the expression and activation of inflammatory mediators in these inflammatory skin diseases. mitogen-activated protein kinases (mapks) are a family of serine/threonine protein kinases that mediate a wide variety of cellular behaviours in response to external stress signals. increased activity of mapks, in particular p mapk, and their involvement in the regulation of synthesis of inflammatory mediators at the transcriptional and translational level has recently been demonstrated. progress in our understanding of inflammatory signalling pathways has identified new targets for treating inflammatory diseases, but the challenge is to place a value on one target relative to another and to evolve strategies to target them. a careful examination of different signalling pathways in various inflammatory conditions is therefore needed. this presentation gathers recent advances in signal transduction in skin inflammation focusing interleukin- , tnf-µ, p mapk, msk / , mk , nf-kappab and ap- . histamine is an important inflammatory mediator in humans, and despite their relatively modest efficacy antihistamines are frequently used to treat allergic conditions, as well as other histamine-mediated reactions such as pruritus. in contrast, antihistamines are of very limited use for controlling other conditions where histamine production is abundant, including asthma. the discovery of the histamine h receptor (h r) prompted us to reinvestigate the role of histamine in pulmonary allergic responses, as well as in pruritus. h r deficient mice and mice treated with h r antagonists exhibited decreased allergic lung inflammation in several models, with decreases in infiltrating lung eosinophils and lymphocytes and decreases in th responses. ex vivo restimulation of primed t cells showed decreases in th cytokine production, and in vitro experiments suggest that decreased cytokine and chemokine production by dendritic cells after blockade of the h r was responsible for the the t cell effects. the influence of h r on allergic or histamine-induced pruritus was explored in mice using selective histamine receptor antagonists and h r deficient mice. the h r was found to mediate the majority of histamine-mediated and allergic itching, while the contibution by the h r was minor. surprisingly the h r effect was independent of mast cells or other hematopoetic cells. this work suggests that the h r can modulate both allergic responses via its influence on t cell activation, and pruritus through mechanisms that are independent of hematopoetic cells. the studies show that the h r mediates previously uncharacterized effects of histamine and highlight the therapeutic potential of h r antagonists. ( ), bsp reddy ( ) ( ) nizams institute of medical sciences, hyderabad; india ( ) genix pharma, india rupatidine, carries the majority of the histamine h receptor -blocking activity and has been introducedfor the treatment of allergic rhinitis and urticaria. objectives: the aim of this study was to compare the effect of two by measure of inhibition of histamine induced wheal and flare response. methodology: male volunteers were enrolled after written informed consent before to ethic committee approved protocol. in this randomised, double-blind, single oral dose, cross overstudy, they were randomized to receive either mg rupatidineformulation after overnight fast. washout was days. wheal and flare were induced on the forearm of the trial subjects, by histamine intradermally injection while the subject was lying comfortably with arm resting on the bed. ten minutes later, wheal and flare were visualized under a bright lamp. histamine induced wheal and flare skin test was performed before and regularly to hours after drug administration. results: administration of reference and test formulations of rupatidine, significantly inhibited the histamine induced cutaneous response in all the subjects. the least square mean ratio (%) t vs r for peak activity imax-% (maximum inhibition of histamine induced wheal and flare response); area under the activity time curve (auc - mmsq/hr and auc - %/hr) both for untransformed and log transformed data were found to be within - % of % ci limits both formulations well tolerated. conclusions: it can thus be concluded that be concluded that test formulation of rupatidine tablet is bioequivalent to reference rupatidine tablet ( ), h yoshimura( ), k ohara ( ), y mastui ( ), h hara ( ), h inoue ( ), h kitasato ( ), c yokoyama( ), s narumiya ( ), m majima ( ) ( ) kitasato university school of medicine, kanagawa, japan ( ) tokyo dental medical colledge, tokyo, japan ( ) kyoto university school of medicine, kyoto, japan thromboxane (tx) a is a potent stimulator of platelet activation and aggregation and vascular constriction. we have reported the magnitude of cytokine-mediated release of sdf- from platelets and the recruitment of nonendothelial cxcr + vegfr + hematopoietic progenitorsconstitute revascularization. we hypothesized that txa induces angiogenic response by stimulating sdf- and vegf which derived from platelet aggrega- inflamm. res., supplement ( ) tion.to evaluate this hypothesis, we dissected the role of the txa in angiogenesis response using mouse hind limb model. recovery from acute hind limb ischemia, as assessed by the ratio between the treated ischemic limb and the untreated control right limb was assessed in wild type mice (c bl/ wt) , prostaglandin i receptor (ip) knock out(ipko) and thromboxane (tx) a receptor (tp) knock out(tpko). blood recovery in tp-/-significantly delayed compared to wt and ipko. immunohistochemical studiesrevealed that tp-/-mice were less stained against pecam positive cells compared to wt and ipkoplasma sdf- and vegf concentration were significantly reduced in tp-/-mice. we observed during in vivo fluorescence microscopic study that compared to tpko, ipko and wt significantly increased platelet attachment to the microvessels around ligated area. tpko translpanted wt bone marrow cells increased blood recovery compared to tpko transplanted tpko bone marrow cells. in addition, mice injected with txa synthase c-dna expressing fibroblast increased blood flow recovery compared to control mice. these results suggested that tp signaling rescues ischemic condition by inducing angiogenesis by secreting sdf- and vegf from platelet aggregation. purpose: the s calcium-binding proteins, a , a and a are constitutively expressed in neutrophils and induced in activated macrophages. high levels are found in sera from patients with infection and several chronic inflammatory diseases. the calgranulin complex, a /a is anti-microbial; a has oxidant-scavenging functions. a is chemotactic for monocytes, and recruits leukocytes in vivo by activating mast cells (mc). effects of these mediators on mc and monocyte function were compared. methods: human pbmc or murine mc were activated in vitro with s and mediator release and cytokine induction (assessed by quantitative rt-pcr/elisa), determined. a cys to ala a mutant was used to determine whether effects on mc are mediated by redox. immunohistochemistry was used to demonstrate s s in asthmatic lung. the s s were expressed in asthmatic lung, particularly in eosinophils and alveolar macrophages. strong reactivity occurred with an antibody recognising predominantly the hypochlorite-oxidised from of a . a , a or the a /a complex had relatively low ability to induce il , tnf, il , and chemokines mrna from pbmc compared to a . only a induced significant levels of il ; none induced il or gm-csf mrna compared to lps. in contrast to a which is activating, a significantly inhibited mc degranulation provoked by ige cross-linking; suppression was dependent on cys . conclusions: the cytokine profile generated by a in mc and monocytes strongly supports a role the pathogenesis of asthma. in contrast, results strongly support a role from a in oxidant defence, particularly to hypobromite generated by activated eosinophils. ( ), d mankuta( ), g gleich ( ), f levi-schaffer ( ) ( ) hebrew university of jerusalem, israel ( ) hadassah university hospital, israel ( ) university of utah, usa the onset, amplitude and termination of allergic responses is regulated at the mast cell/eosinophil interface. eosinophil major basic protein (mbp), which activates mast cells in the late-chronic phase of allergic inflammation, is a central determinant in this interface. characterized more than two decades ago, the exact nature of this activation has not been clarified as yet. here we demonstrate that mbp exerts its activating effect on human mast cells and basophils through cd and hematopoietic cell kinase (hck). a genome-wide analysis showed that hck displays shifts in mrna levels specifically upon mbp-induced mast cell activation. hck also shows a unique priming pattern prior to this activation. cd is phosphorylated specifically upon activation with mbp and deploys a signaling complex that critically depends on hck. extracellular neutralization of cd interferes with mbp entry into the cell, and this as well as rna silencing of hck results in defective mbp-induced activation. finally, cd neutralization abrogates mbp-induced anaphylaxis in-vivo. these findings picture for the first time a chronic-phase specific pathway mediating eosinophil-induced mast cell activation with critical consequences for the therapy of chronic allergic inflammation. alexander robinson ( ), d kashanin ( ), f odowd( ), v williams( ), g walsh ( ) ( ) cellix ltd, institute of molecular medicine, dublin, ireland ( ) university of aberdeen, scotland, uk leukocyte adhesion to endothelial cell bound proteins, such as icam- and vcam- , is an initial step of the inflammatory response. we have developed an in vitro microfluidic system which mimics conditions found in blood vessels in vivo during an immune response. using this system, we can record leukocyte adhesion levels under physiologically relevant flow conditions (e.g. - dynes/cm ). the adhesion profiles of resting and pmastimulated peripheral blood lymphocytes (pbls) were recorded, with respect to vcam- , icam- , and bsa. images at each shear stress level were captured using a digital camera, and analysed using our in-house ducocell software package. distinct morphological changes in pma-stimulated pbls, compared to non-stimulated cells, can be observed. these include a less rounded appearance of the pma-stimulated pbls, and evidence of "uropod" formation, which anchor the t cell to the endothelium as part of the migration process. levels of adhesion to vcam- are high ( - %, compared to control), but there appears to be little difference between the adhesion profiles of non-stimulated and pma-stimulated pbls.however, there is a distinct difference between the adhesion levels of non-stimulated and pma-stimulated pbls to icam- , with pma-stimulated cells showing a higher affinity for icam- than nonstimulated cells (approx. % and %, respectively).pbl adhesion to bsa is negligible. we present a novel in vitro microfluidic pump system that can simulate leukocyte adhesion to the endothelium under flow conditions. this platform is a more efficient and economical system compared to those currently available, due to reduced material costs and style of construction. introduction: pulmonary aspiration of gastric contents is a common complication observed in icu patients and a potential trigger of ards. in this study we evaluated the course of lung inflammation induced by intranasal instillation of gastric juice (gj). methods: gj was obtained from donor rats (ph . ). male c bl/ were instilled with ml/kg of gj. after or h, the animals were sacrificed and lung and balf were collected. control group consisted of non-manipulated mice. . ae . , sg h: . ae . ; pg/ml). discussion: gj aspiration induced an initial adherence of pmn to lung tissue that is correlated with increased tnf-a/il- ratio in balf at the nd h. the reduction of mpo activity is correlated with the decrease in tnf-a/il- ratio. the late increase of pmn in balf might be a consequence of the early production of tnf-a. the results are suggestive that the treatment of patients exposed to acid aspiration should be focused in the initial period of the insult and in the blockage of tnf-a. objectives: intestinal i/r is implicated as a prime initiating event in the development of acute respiratory distress syndrome (ards) after trauma and hemorrhagic shock. we investigated the effects of lps challenge to mice previously submitted to i-i/r, a two-hit model of acute lung injury. methods: male c bl/ mice were subjected to min of intestinal ischemia and challenged with . mg/kg of intranasal lps at the th hour of reperfusion (two-hit). balf and culture of lung explants were performed h after lps challenge. mice subjected to i-i/r or lps alone were used as controls. results: two-hit mice showed marked increase in lung evans blue dye leakage compared to i-i/r ( . ae . vs . ae . , mg/mg). lung mpo was increased ( . ae . vs . ae . ; od nm) whereas the neutrophil recruitment to balf was inhibited in the two-hit group compared to lps group ( . ae . vs . ae . ; x e cells/mouse). the levels of nox-in the two-hit group were significantly increased when compared to i-i/ r controls in balf ( . ae . vs . ae . ; mm) and in lung explants ( . ae . vs . ae . ; mm/mg of tissue). conclusions: intestinal i/r predisposes the animal to an exacerbated response to a low dose lps insult. the exacerbated production of nitric oxide observed in the two-hit group may cause endothelial damage, thereby explaining the major increase in vascular permeability in the two-hit group. the results are suggestive that patients exposed to systemic inflammatory response might develop ards when in contact with secondary inflammatory stimuli. nitric oxide may play an important role in this process. ( ) ( ) novartis institutes for biomedical research, horsham, uk ( ) university of michigan, usa obligatory for using oxygen in energy transfer pathways was the simultaneous co-evolution of enzymes that detoxify the reactive species formed as by-products. thus, we hypothesized that individuals with low aerobic function will have reduced anti-oxidant capacity and, therefore, be more susceptible to smoking-related lung diseases like copd. to test this hypothesis, we exposed high capacity runner (hcr) and low capacity runner (lcr) rats to months of whole-body smoke exposures.the animals, bred over successive generations on the same background strain for high or low running capacity, differ by over % (p< x - ) for exercise capacity, measured by running on a treadmill.after months of exposures, inflammatory cells in bronchoalveolar lavage fluid were increased in both the hcr-and lcr-smokeexposed(se) animals compared to air-exposed controls (p< . ); however there was a - -fold increase in the number of neutrophils and lymphocytes in the lcr-over the hcr-se group (p< . ).histopathology revealed there was greater inflammation and lung damage present in the lcr-versus hcr-se group (p< . ). metabonomic (metabolite profiling) analysis revealed that while peroxidation of lung lipids occurred for both se groups, oxidative damage to the lung surfactant layer was significantly more extensive for the lcr-se. systemic oxidative damage was also more apparent in the lcr-se group, with metabolic profiling suggesting a reduced capacity to regenerate muscle glutathione. the metabolic data suggest that repair processes maybe more effective in the hcrs. in summary, these data support the concept that aerobic capacity may be central to ones susceptibility to developing smoking-related lung disease. ( ), ap ligeiro-oliveira( ), jm ferreira-jr( ), sr almeida( ), w tavares de lima( ), shp farsky ( ) ( ) university of s¼o paulo, brazil ( ) regional integrated university of alto uruguai and missðes, brazil methods: male wistar rats were exposed to vehicle or hq ( mg/kg; ip.;daily, days, two-day interval every five days). on day , animals were ip sensitized with ovalbumin (oa). assays were performed on day . results: hq-exposed rats presented reduced number of leukocytes in the bronchoalveolar fluid and by impaired in vitro oa-induced tracheal contraction. the latter effect suggests reduction on mast cell degranulation, and it was corroborated by in vivo decreased mesenteric mast cell degranulation after topical application of oa. the oa-specificity response was confirmed by normal ability of mast cells to degranulate in both groups of animals after topical application of compound / . in fact, lower levels of circulating oa-anaphylactic ige antibodies were found in hq-exposed rats. this latter effect was not dependent on number or proliferation of lymphocytes, nevertheless reduced expressions of costimulatory molecules cd and cd on oa-activated lymphocytes indicated the interference of hq exposure on signaling of the humoral response during an allergic inflammation. contact information: ms sandra manoela dias macedo, regional integrated university of alto uruguai and missðes / university of s¼o p, department of clinical and toxicological analyses, s¼o paulo, brazil e-mail: smdmacedo@yahoo.com.br ( ) ( ) radboud university nijmegen, medical centre, nijmegen, the netherlands ( ) university hospital, zürich, switzerland toll-like receptors (tlr) are essential in the recognition of invading microorganisms. however, increasing evidence shows involvement of tlr in autoimmunity, such as rheumatoid arthritis (ra), as well. here we investigated whether synovial expression of tlr and tlr was associated with the expression of ifna, tnfa, il- b, il- , il- , and il- and studied in what way these receptors and cytokines were associated in vitro. using immunohistochemistry, we found that tlr / tlr expression in synovial tissue was associated with the presence of ifna, il- b and il- , but not tnfa, il- and il- . to investigate whether ifna, il- b and il- could induce tlr / tlr upregulation in vitro, we incubated separate lymphocyte populations with these cytokines and subsequently determined tlr / mrna expression. ifna incubation resulted in significant tlr /tlr upregulation, whereas il- b and il- did not. pre-incubation with ifna and subsequent stimulation of tlr /tlr significantly enhanced il- , tnfa and ifna/b production, indicating that the ifn-induced tlr upregulation was functional. low amounts of biologically active il- b were produced upon stimulation with atp, but not upon tlr / tlr stimulation, although mrna levels were high. interestingly, ifna-priming significantly increased the atp-induced il- b production. here, we demonstrated a dual role for ifna in vitro, which could explain the association between tlr and il- b / il- in synovial tissue. first, involvement in tlr /tlr regulation and second, involvement in atp-induced production of biologically active il- b. these results suggest involvement of anti-viral immune responses in ra and ifna as a key player in chronic inflammation. the pathogenesis of chronic joint inflammation remains unclear although the involvement of pathogen recognition receptors (ppr) has been suggested recently. here, we described the role of two members of the nacht-lrr (nlr) family, nod (nucleotide/ binding oligomerization domain) and nod in model of acute joint inflammation induced by intraarticular injection of tlr (toll-like receptor) agonist streptococcus pyogenes cell wall fragments. we found that nod deficiency resulted in reduced joint inflammation and protection against early cartilage damage. in contrast, nod gene deficient mice developed enhanced joint inflammation with concomitant elevated levels of proinflammatory cytokines and cartilage damage. to explore whether the different function of nod and nod occurs also in humans, we exposed pbmcs carrying either nod frameshift or nod frameshift mutation with scw fragments in vitro. both tnfa and il- b production was clearly impaired in pbmcs carrying the nod fs compared to pbmcs isolated from healthy controls. in line with the nod gene deficient mice, human pbmcs bearing the nod mutation produced enhanced levels of proinflammatory cytokines after h stimulation with scw fragments. these data indicated that the nlr family members nod and nod have a different function in controlling tlr -mediated pathways. we hypothesize that intracellular nod -nod interactions determine the cellular response to tlr triggers. whether lack of controlling tlr -driven pathways by nod signalling is involved in the pathogenesis of autoinflammatory or autoimmune disease, such as rheumatoid arthritis (ra), remains to be elucidated. leukocyte immunoglobulin-like receptors (lilrs) are a family of receptors with potential immune-regulatory function. activating and inhibitory receptors play a role in maintaining immunological equilibrium and an imbalance may lead to the onset of autoimmune diseases such as rheumatoid arthritis (ra). ra is a chronic inflammatory disease of joints caused by mediators (i.e. tnf-a) produced by activated leukocytes. we recently demonstrated expression of activating lilra in synovial tissue macrophages from ra patients. the aim of this study was to determine expression and function of lilra in monocytes and macrophages. peripheral blood mononuclear cells (pbmc) were prepared by standard density gradient separation and in vitro-derived macrophages were generated by differentiating thp- cells with vitamin-d . lilra expression was measured by flow cytometry before and after modulation with cytokines. differentiation to macrophages significantly up-regulated lilra expression (p= . ). treatment of macrophages with lps, tnf-a, il- b and ifn-g but not il- caused significant down-regulation of lilra (p< . ). function of lilra was assessed by cross-linking with plate-immobilised lilra -specific mab. soluble tnf-a was measured by elisa. activation of cells elicited tnf-a production in a dose-dependent manner while time-course analysis shows maximal production at h. correlation between lilra expression and response to cross-linking indicates that level of expression may relate directly to the degree of activation. decrease expression in response to acute-phase cytokines suggests controlled regulation during inflammation. in ra, abnormal regulation of lilra could potentially exacerbate inflammation by inducing uncontrolled production of proinflammatory cytokines. pharmacological blocking of lilra could potentially provide therapeutic benefit. ( ) ( ) university of valencia, spain ( ) northwick park institute for medical research, uk co-releasing molecules (co-rms) mimic the biological actions of co derived from heme oxygenase activity. in the present work we studied the effects of a water-soluble co-releasing molecule (corm- ) on an animal model of human rheumatoid arthritis. dba- /j mice were treated with corm- ( , or mg/kg/day, i.p.) from day to after collagen-induced arthritis (cia) and sacrificed on day . administration of corm- resulted in a significant improvement of the clinical profile of this disease since it markedly reduced joint swelling and redness. histological analysis of the joints in control arthritic mice indicated the presence of granulocytes and mononuclear cells, cartilage erosion, chondrocyte death and proteoglycan depletion. all these parameters were significantly reduced by corm- treatment with the most pronounced protective effect observed at mg/kg. the levels of pro-inflammatory mediators (pge , il- beta, tnfalpha, il- and il- ) in the hind paw homogenates were significantly inhibited by corm- . in addition, comp levels in serum, a marker of cartilage degradation, was reduced by the co-releasing agent. our studies show that therapeutic administration of corm- alleviates the clinical features of murine cia at the late phase of this response. the beneficial action of co liberated from corm- appears to be associated with a decrease in inflammatory cytokines and reduction of cell infiltration into the synovial tissues ultimately leading to a protective effect on the cartilage. aim: to setup a bovine model for cytokine-induced articular cartilage collagen degradation, and characterize the model using a variety of compounds targeting different disease mechanisms relevant to arthritis. methods: full thickness bovine articular cartilage punches were cultured with or without ng/ml il- a, tnf-a and oncostatin m. after three weeks the cartilage and culture medium were analyzed for weight changes, water content, dna content, glycosaminoglycans (gag), hydroxyproline (hyp), damaged collagen molecules, mmp activity, ctx-ii and comp. diclofenac, dexamethasone, pioglitazone, remicade, risedronate, galardin and a - were tested for their effect on cartilage degradation. results: exposure of articular cartilage to cytokines resulted in a decreased cartilage weight, increased proteoglycans degradation, increased collagen degradation, increased percentage of denatured collagen, increased water content and increased levels of active mmps (all p < . ). comp release during the first week of culture showed a trend towards up regulation during the first week of culture for all three donors, this was however not significant due to the small number of donors. most of the described processes were modulated by one or more of the drugs tested, indicating that this model for articular cartilage destruction is sensitive to treatment. discussion: stimulation of bovine articular cartilage explants with a cocktail of il- a, tnf-a and osm results in clear and consistent changes in the cartilage, highly reminiscent of cartilage destruction during arthritis. further research needs to establish whether the model is also sensitive to anabolic factors that potentially could repair the damage. toll-like receptors (tlrs) may contribute to the progression of rheumatoid arthritis through recognition of hostderived damage-associated ligands that have repeatedly been found in arthritic joints. involvement of tlr and tlr activation in the expression of arthritis was studied using interleukin- receptor antagonist deficient (il- ra-/-) mice, which spontaneously develop an autoimmune t-cell mediated arthritis. spontaneous onset of arthritis was dependent on tlr activation by microbial flora, as germ-free mice did not develop arthritis. after crossing with tlr knockouts, il- ra-/-tlr -/-mice developed more severe arthritis compared to il-ra-/-tlr +/+ littermates; whereas, tlr -/-il- ra-/-mice were protected against arthritis. to clarify the mechanism by which tlr and tlr differentially regulated the disease expression, we studied the role of these tlrs in il- production and th development, both important in il- ra-/-arthritis. wild type bone-marrow-derived dendritic cells (bmdcs) produced similar levels of il- upon stimulation with tlr and tlr ligands; however, il- ra-/-bmdcs produced less il- than wild type dcs upon tlr stimulation and more il- than wild type dcs upon tlr stimulation. furthermore, il- ra-/-t cells produced lower amounts of il- when cultured with tlr -activated apcs and higher amounts of il- when cultured with tlr -stimulated apcs, both in combination with cd stimulation. facs analysis of th (cd +/il- +) cells from both spleen and draining lymph nodes revealed % reduction in il- ra-/-tlr -/-mice compared to il- ra-/-tlr +/+ littermates. specific cd /cd stimulation of non-adherent splenocytes confirmed lower il- production in il- ra-/-tlr -/-. these findings suggest important roles for tlr and tlr in regulation of th development and expression of arthritis. prostaglandine (pge ) stimulates the transactivational activity of p through p map kinase-dependent ser phosphorylation (jbc ) .p controls cell-cycle progression, in part, by differential regulation of ap- proto-oncogenes (jun/fos).currently we studied pge control of cyclin d promoter activity with particular attention to the role of ap- oncogenes.pge induced a . fold increase in junb mrna expression (northern blot), a . -fold increase in junb promoter activity (luciferase assay), and increased ser junb phosphorylation in human synovial fibroblasts (hsf) (western blot).c-jun was strongly inhibited while jund, c-fos, fra / , and fosb expression were upregulated by pge .in cell-cycle experiments, transformation with a constitutively active ha-ras construct (ras g v) resulted in a . fold increase in cyclin-d promoter activity, cyclin-d synthesis, thr /tyr phosphorylation of erk / ( . fold) and ap- (c-jun)-dependent transactivity ( . fold); cyclin d /cdk - inhibitor p ink a synthesis was suppressed. addition of excess rass n dominant negative mutant construct to the plasmid mix abrogated the aforementioned processes.ectopic expression of c-jun, c-fos and especially jund expression constructs stimulated cyclin d promoter activity/protein synthesis, blocked p ink a synthesis; the latter effects were reversed by the addition of excess junb.pge exerted temporal and bi-phasic dose-dependent control of the cyclin d promoter activity, largely through differential ap- activation and promoted cell cycle arrest and apoptosis in hsf at high physiological concentrations.the results provide further insight into the biology of the cpla / cox/pges biosynthetic axis and highlight the complexity of pge action in terms of cell-cycle progression. di-glucopyranosylamine (diga) is an antikeratitic (roberts et al., , acvo conference, scottsdale) immunomodulatory pyranosyl disaccharide with parenteral anti-rheumatic activity (bolton et al., , inflammation res. (s ) s ) and unknown mechanism of action.interestingly, anti-tnf therapy is anti-keratitic.-diga hydrolyses to monoglucosylamine (mga) and glucose, which is prevented by n-acetylation (nacdi-ga).lider ( , pnas. : - ) showed that sulphated disaccharides are orally active, inhibit tnf synthesis and the dth reaction.we have investigated the anti-tnf and anti-rheumatic activity of the sulphated and free digas.human whole blood (hwb) was stimulated with pha ( mg/ml) to synthesise tnf.antigen induced arthritis (aia) was induced in methylated bovine serum albumin (mbsa) sensitised c bl/ mice challenged i.a. into the stifle joint.collagen arthritis (cia) induced in dba mice by sensitisation to bovine collagen, were boosted i.p. with collagen at day . hwb tnf synthesis was inhibited by diga, mga and nacdiga(ic < . mm). diga ( ml, mm) i.a. prevented hour aia (- . +/- . mm).diga at mg/kg reduced aia when administered i.v. (- . +/- . mm, p< . ) and i.p. (- . +/- . mm, p< . ), but is hydrolysed p.o. ( . +/- . mm ns). polysulphated diga (diga s) was unstable, but stabilised by n-acetylation (nacdi-ga s).tnf synthesis was potently inhibited by both nacdiga and nacdiga s (ic < . mm).nacdiga s ( mg/kg p.o.) inhibited aia (- . +/- . mm), and nacdiga s with lower degrees of sulphation (mw and kda) inhibited the development of mouse collagen induced arthritis as assessed by clinical score. sulphated diglucosylamines represent a new class of heparinoid which are potent inhibitors of tnf synthesis and possess oral anti-rheumatic activity. excessive no appears to play a key role in the pathogenesis of chronic inflammatory diseases. in this study we aimed to evaluate no synthesis in rheumatoid arthritis (ra) before and after therapy. it was performed on persons, divided into groups: a negative control group of healthy volunteers, a positive control group with ra, a group with ra and physiotherapy (phys), a group with ra and low doses of cimetidine (cim) + doxycycline (dox), a group with ra and combined treatment phys + cim + dox, and a group treated with usual doses of ibuprofen (ibu). serum nitrite/nitrate (griess) was measured in order to evaluate no synthesis. results: compared to the positive control group, in all the treated groups no synthesis decreased significantly. there was no significant difference between phys and cim+dox effect alone. the combined treatment, phys + cim + dox had a much better inhibitory effect on no synthesis. between the phys, cim + dox and phys + cim + dox groups and that treated with ibuprofen, there was no significant difference in reducing no synthesis. conclusions: ) in ra phys + cim + dox treatment was as efficient as ibuprofen in reducing no synthesis. ) the low doses of cim and dox may allow a longer treatment due to the lower side effects risk enhanced socs expression following exposure of murine macrophages to lps implicated socs in the control of lps-mediated signaling. socs regulates nfkb signaling in murine macrophages, blocking at the level of mal or ikba phosphorylation. we investigated the role of socs in regulating the production tnf by lps and pam csk -activated primary human monocytes. blood monocytes were isolated by centrifugal elutriation and either infected with an adenoviral vector expressing socs (adv-socs ), control vector (adv-gfp) or left untreated. adv-socs monocytes were exposed to tlr and tlr ligands, lps ( ng/ml) or pam csk ( ng/ml). facs analysis demonstrated infection efficiencies of ae % and ae % (n= , mean ae sem) of monocytes expressing adv-gfp or adv-socs at moi . adv-socs blocked lps and pam csk induced tnf mrna and protein production in a dose-dependent manner. in contrast, il- and il- production by adv-socs -infected monocytes was not blocked. adv-socs also blocked lps and pam csk induced tnf production by macrophages isolated from synovial fluid. infection efficiencies of ae % or ae % were obtained. quantitative western blot analysis revealed that the classically defined nfkb pathway was not altered at the level of ikba or p activation. furthermore, the kinetics of lps and pam csk induced ikba phosphorylation and degradation in adv-socs monocytes remained unaffected (n= and donors, respectively). further, analysis of parallel mapk pathways demonstrated no block in p or erk mapk pathways. these data suggest that socs regulation of lps and pam csk -induced tnf production by human monocytes occurs downstream of tlrs, possibly at the level of transcription. recently, beta-nad+ has emerged as a novel extracellular player in the human urinary bladder. beta-nad+ is the natural substrate of cd which catalyzes the conversion of beta-nad+ to cadpr. under normal conditions in vivo, there is no or only very small quantities (submicromolar range) of extracellular beta-nad+ compared to intracellular levels ( - mm). during inflammation cell lysis may cause bursts of high local beta-nad+ levels. however, the effect of beta-nad+ on the human detrusor smooth muscle cells (hdsmc) was unknown. the effect of beta-nad+ on cultured (explant technique) hdsmc was determined by: ) measuring cytosolic free calcium ([ca +] i) in fura- loaded hdsmc using spectrofluorometry and ) force measurements in - mg detrusor strips. hdsmc responded to beta-nad+ ( - mm) with an immediate and transient increase in [ca +] i. the ca + transient was followed by one or two much slower and transient increases in [ca +] i, indicative of beta-nad+ enzymatic conversion into cadpr. the ca + responses persisted in the absence of extracellular calcium. the ca + responses to beta-nad+ were not affected by exposure of hdsmc to atp supporting the notion that the effects of beta-nad+ were not mediated via p x purinoceptors. furthermore, beta-nad+ caused a concentration-dependent detrusor muscle relaxation. this is the first study to report that extracellular beta-nad+ affect intracellular calcium homeostasis and force in hdsmc. these powerful actions of beta-nad+ suggest a role for beta-nad+/cadpr system as a novel extracellular player in the human detrusor during inflammation. aids remains a worldwide threat more than two decades after identification of hiv as the etiological agent. its wide dissemination can be partly attributed to its successful suppression of immunity resulting in disease progression and concomitant opportunistic infections including mycobacterial and cytomegalovirus infections. hiv trans-activator (tat) is one of the regulatory proteins that mediates hiv replication and dysregulates cellular functions such as apoptosis and cytokine expression. for example, tat induces tumor necrosis factor (tnf) and enhances gp -induced neurotoxicity. we recently showed that tat induces the overexpression of il- via cellular kinase pkr and activation of transcription factor ets- . in this study, we examined whether tat plays a role in perturbing interferon-& (ifn&) signal transduction. we showed that tat impaired ifn&-induced stat tyrosine phosphorylation, but had no effects on the serine residue of stat and jak kinases in primary human blood monocytes. furthermore, we found that the nuclear translocation of phospho-stat was abrogated by tat. the inhibition of phospho-stat led to the deformation of stat homodimers and subsequent stat-dna complex. to investigate the cellular consequences, we measured the expression of ifn&-stimulated genes including human leukocyte antigen (hla) and , oligoadenylate synthetase ( , oas), a key enzyme in the activation of latent ribonuclease l. the results showed that tat inhibited transcriptional activation of , oas and hla. taken together, we identified a new role for tat in which it impairs ifn& signal transduction and suppresses inflammation, thus crippling the immune system and contributing to hiv persistence, opportunistic infections and disease progression. caspase- belongs to the group of inflammatory caspases and is the activating enzyme for the pro-inflammatory cytokine interleukin- (il- ), a cytokine known to play an important role in the pathogenesis of psoriasis. the purpose of this study was to determine the expression of caspase- in psoriatic skin and the signaling mechanisms involved in stress induced activation of caspase- and il- . interestingly, increased caspase- activity in lesional compared with nonlesional psoriatic skin was seen as determined by western blotting. in vitro experiments in cultured human keratinocytes demonstrated anisomycin induced, p mapk dependent increased secretion of procaspase- and active caspase- . furthermore, anisomycin increased the mrna expression of il- through a p mapk dependent but caspase- independent mechanism, reaching a maximum level after hours of stimulation. finally, anisomycin caused a rapid ( hours) increase in the secretion of proil- and active il- . secretion of active il- was mediated through a p mapk/caspase- dependent mechanism, whereas secretion of proil- was mediated by a p mapk dependent but capsase- independent mechanism. these data demonstrate that the activity of caspase- is increased in psoriatic skin and that il- secretion is regulated by a p mapk/caspase- dependent mechanism, making caspase- a potential target in the treatment of psoriasis. prostaglandin e (pge ) regulates the stability of cyclooxygenase- (cox- ) mrna through adenylate/uridylate-rich elements (ares) in the untranslated region ( utr) by a positive autocrine/paracrine feed-forward loop. the principal objective of this study was to elucidate the molecular mechanisms involved in the pge dependent stabilization of cox- in human synovial fibroblasts (hsfs). transfection of well-known are binding proteins (aubps) demonstrated that tristetraprolin (ttp) potently destabilized a [luciferase-cox- utr] reporter fusion mrna ( ae . % decrease in luciferase activity vs. control). ttp protein levels in hsfs remained constant despite il- b-induced changes in ttp mrna levels, thus suggesting translational regulation of its expression. pge did not affect the transcription or translation of this gene in hsfs. western blot analysis of hsf ttp demonstrated the existence of a specific, covalent~ kda heterocomplex containing ttp (ttphcx). although ttphcxs exact composition and stoichiometry is yet to be defined, pge selectively regulated the amount of this heterocomplex in a time-dependent manner. furthermore, protein shuttling studies performed using real-time confocal microscopy revealed that pge can induce export of a small nuclear pool of ttp-gfp. finally, transfection of ttp into hsfs also influenced cox- gene transcription, thus enabling ttp to regulate cox- gene expression at both the transcriptional and post-transcriptional level. in conclusion, we have demonstrated that ttp is an rna binding protein capable of influencing cox- mrna stability and transcription and whose localization and interaction with other factors is regulated by pge . these data can provide important insight into deciphering the role of pge in fine-tuning physiological and pathophysiological gene regulation. ( ) ( ) chinese academy of sciences, shanghai, pr china ( ) ohio state university, usa mitogen-activated protein (map) kinases play a critical role in innate immune responses to microbial infection through eliciting the biosynthesis of proinflammatory cytokines. map phosphatases (mkp)- is an archetypical member of the dual-specificity phosphatase family that deactivates map kinases. induction of mkp- has been implicated in attenuating the lipopolysaccharide (lps) and peptidoglycan (pgn) responses, but how the expression of the mkp- is regulated is still not fully understood. here, we show that inhibition of p map kinase by specific inhibitor sb or rna interference (rnai) markedly reduced the expression of mkp- in lps or pgn-treated macrophages, which is correlated with prolonged activation of p and jnk. depletion of mapkap kinase (mk ), a downstream substrate of p , by rnai also inhibited the expression of mkp- . the mrna level of mkp- is not affected by inhibition of p , but the expression of mkp- is inhibited by treatment of cycloheximide. thus, p mapk plays a critical role in mediating expression of mkp- at a posttranscriptional level. furthermore, inhibition of p by sb prevented the expression of mkp- in lpstolerized macrophages, restored the activation of map kinases after lps restimulation. these results indicate a critical role of p -mk -dependent induction of mkp- in innate immune responses. the i-kb kinase (ikk) complex regulates the activation of nf-kb a key transcription factor in inflammation and immunity. whilst ikka activity is necessary for proinflammatory and anti-apoptotic gene expression, ikka has distinct roles in lymphorganogenesis and b cell maturation. here we describe a role for ikka in cell mediated immunity (cmi). paw inflammation in methylated bsa-induced cmi was significantly reduced in transgenic mice expressing a mutant ikka protein that cannot be activated (ikka aa/aa ) compared to wild-type (wt). antigen-induced il- and ifng production by ikka aa/aa splenocytes and ikka aa/aa t cell:dc cocultures were also significantly reduced ex vivo. this could be normalised by using wt t cell: ikka aa/aa dendritic cell (dc) but not ikka aa/aa t cell:wt dc combinations. this suggests that reduced cmi in ikka aa/ aa mice is due to a defect in ikka aa/aa t cells not dcs. this is not due to a requirement of ikka in tcrmediated activation of t cells, since anti-cd /cd mediated activation of ikka aa/aa t cells was unaltered. however, lps-induced production of the important th cytokine il- is impaired in ikka aa/aa dcs. we are currently addressing the hypothesis that ikka activity may be required for the generation and maintenance of antigen-specific t cells in vivo. recently we described a role for ikkµ in the negative regulation of innate immunity and acute inflammation, which is in contrast to its role shown here in promoting adaptive immunity and antigen-driven inflammation. ikka may represent an alternative target for the treatment of autoimmune disease which would not compromise host defence. as a latent transcription factor, nf-kb translocates from cytoplasm into nucleus upon stimulations and mediates expression of genes important in immunity, inflammation and development. although extensive studies have been done regarding how nf-kb is triggered into nucleus, little is known about how it is regulated inside nucleus. by twohybrid approach, we identify a prefolding-like protein snip that is expressed predominantly and interacts specifically with nf-kb inside nucleus. we show that rnai knockdown of snip leads to impaired nucleus activity of nf-kb and dramatically attenuates expression of nf-kb dependent genes. this interference also sensitizes cells to apoptosis by tnf-a. furthermore, snip forms a dynamic complex with nf-kb and is recruited to the nf-kb enhancesome upon stimulation. interestingly, snip protein level correlates with constitutive nf-kb activity in human prostate cancer cell lines. the presence of nf-kb within nucleus of stimulated or constitutive active cells is significantly diminished without endogenous snip. our results reveal that snip is an integral component of nf-kb enhancesome and essential for its stability in nucleus, which uncovers a new mechanism of nf-kb regulation. bone remodeling is a tightly regulated process that couples resorption of old bone by osteoclasts and the deposition of new bone by osteoblasts. an imbalance between bone formation and bone resorption can result in various metabolic bone diseases, such as rheumatoid arthritis and osteoporosis. osteoclasts are terminally differentiated cells that arise from a haematopoietic stem cell lineage, which also gives rise to monocytes and macrophages. osteoclast differentiation and regulation of this process to maintain bone homeostasis are central to the understanding of the pathogenesis and treatment of bone diseases, such as osteoporosis. in vitro, osteoclast formation from bone marrow macrophages is induced by rankl (receptor activator of nf-kappa b ligand) in the presence of m-csf (macrophage colony stimulating factor). osteoclastogenesis is markedly enhanced in bone marrow macrophages from ifnar -/-and ifnar -/-mice and results in increased number of multinucleated cells positive for osteoclast marker, trap (tartrate-resistant acid phosphatase). consequently, the mutant mice develop osteoporotic phenotype, characterised by reduced bone density. these findings suggest that the ifn alpha/beta system is critical for the negative feedback regulation of osteoclastogenesis and that rankl signaling is essential for the induction of osteoclast differentiation. atp acting on p x receptors in macrophage is one of the main physiological signals that lead to the processing and release of the pro-inflammatory cytokine, interleukin- beta (il- b), their activation also leads to rapid opening of a membrane pore permeable to dyes such as ethidium. here we identify pannexin- , a recently described mammalian protein that functions as an hemichannel when ectopically expressed, as this dye-uptake pathway and show that signalling through pannexin- is required for processing of caspase- and release of mature il- b induced by p x receptor activation. furthermore, maitotoxin and nigericin, two agents considered to evoke il- b release via the same mechanism were studied. maitotoxin evoked dye uptake whose kinetics were similar to a slow pannexin- -independent phase induced by p x receptor activation, and this was unaltered by pannexin- inhibition.nigericin did not induce dye uptake.inhibition of pannexin- blocked caspase- and il- b processing and release in response to this two stimuli.thus, while pannexin- is required for il- b release in response to maitotoxin, nigericin and atp, a mechanism distinct from pannexin- hemichannel activation must underlie the former two processes. introduction: saa is a classic acute-phase protein upregulated during inflammatory response. saa is active on leukocytes and modulates inflammation and immunity through the induction of cytokines, including the chemokine il- . here we verify the effect of saa on the mrna expression and release of mip- alpha, a chemokyne involved in the recruitment of dendritic cells. methods: peripheral blood mononuclear cells (pbmc) isolated from peripheral blood by density gradient were cultured in rpmi medium in the presence of saa. mip- alphaconcentration was determinated in the supernatant of cell cultures by elisa. mrnawas analyzed bythe ribonuclease protection assay (rpa). results: pbmc stimulated with saa ( ug/ml) induced the expression of mip- alpha mrna at , and hours. mip- alpha protein was found in the suppernatant of and hours cultures (p< , ) and the addition of sb (p inhibitor) and pd (erk / inhibitor) completely abolished the release of mip- alpha. conclusions: saa is an inducer of mip- alpha expression in pbmc and p and erk / are important pathway signaling to this effect. saa is one of the factors responsible by the recruitment of dendritic cells. the p pathway is activated in numerous inflammatory conditions, including ra, ibd asthma, acute coronary syndrome, and copd, and its activation helps drive the production of inflammatory mediators. inhibitors of p decrease mediator production and therefore can produce profound anti-inflammatory effects. arry- is a potent inhibitor of p enzyme (ic < nm) with a novel pharmacophore and physiochemical properties distinct from those of other p inhibitors, being very water soluble. it is extremely potent in human whole blood, blocking lps-stimulated tnf production with an ic < nm.in animal models of rheumatoid arthritis (cia and aia) the compound significantly normalized histologic endpoints, such as inflammation, bone resorption and cartilage damage (ed ~ mg/kg). a phase i single ascending dose clinical study was run in healthy volunteers. after an oral dose of , , , or mg), blood was drawn at various times, stimulated ex vivo with lps, and analyzed for cytokines and inflammatory mediatorsfj arry- was well-tolerated and drug exposure was proportional to dose. in ex vivo samples, there was both a time-and concentrationdependent inhibition of il , pge and tnffz with > % inhibition observed at the mg dose level. the plasma concentrations of drug peaked at~ ng/ml at the mg dose and cytokine inhibition was sustained for > hours, showing that low doses of arry- produced profound effects on clinical biomarkers. further evaluation of arry- in patients with inflammatory diseases is planned. introduction: we demonstrated that in vivo chronicle blockage of nos (l-name, mg/kg; oral route; days) impairs leukocyte-endothelial interactions and neutrophils migration into inflammatory focus. these effects may be depending, at least in part, on decreased expression of l-selectin on leukocytes and pecam- on endothelium. aimed to clarify the mechanisms involved on these inhibitory effects, we now investigated the role of l-name treatment on secretion of tnf and il- b; by circulating leukocytes and migrated peritoneal neutrophils. methods: male wistar rats were treated with l-name ( mg/kg; oral route; days) or sterile saline (control). circulating leukocytes were isolated from blood collected from abdominal aorta and migrated neutrophils were obtained hours after i.p. injection of oyster glycogen ( %; ml). no (griess reaction) and cytokines (elisa) were quantified in supernatants of x cultured cells before and hours after lps stimulation ( m;g/ml). results: levels of no, tnf and il- b; were reduced in circulating leukocytes from l-name-treated rats in both basal and lps stimulated conditions. on the other hand, only secretion of il- b; was impaired by migrated neutrophils. conclusions: results show that in vivo l-name treatment, which partially reduces no production, decreases the secretion of pro-inflammatory cytokines by circulating leukocytes. however, the same pattern of inhibition is not detected if neutrophils are in vivo primed. objectives: to investigate the ability and mechanism of ifn-g to suppress interleukin- (il- )-induced mmp- expression in articular chondrocytes. methods: human chondrocytes were treated with ifn-g or il- beta alone or in combination. mmp- mrna was analyzed by rt-pcr. mmp- protein, phospho-stat and p / mapk levels were measured by western blotting. mmp- promoter-luciferase, cmv-cbp/p plasmids and stat sirna were transfected by calcium phosphate method. ap- activity was monitored by elisa. stat -cbp/p interaction was studied by immunoprecipitation. results: ifn-gpotently suppressed il- -induced expression of mmp- and promoter activity. blockade with neutralizing ifn-gr antibody revealed that mmp- inhibition by ifn-¼ was mediated by the ifn-¼ receptor. ifn-beta-stimulated activation of stat was directly correlated with mmp- suppression. knockdown of stat gene by specific sirna or its inhibition with fludarabine partially restored the il- induction of mmp- expression and promoter activity. ifn-g did not alter activator protein (ap- ) binding ability but promoted physical interaction of stat and cbp/p co-activator. p overexpression reversed ifn-g inhibition of endogenous mmp- mrna expression and exogenous mmp- promoter activity. conclusions: ifn-g through its receptor activates stat , which binds with cbp/p co-activator, sequesters it from the cell system and thus inhibits transcriptional induction of mmp- gene in chondrocytes. ifn-g and its signaling pathways could be targeted therapeutically for ( ), p asmawidjaja ( ), r hendriks( ), erik lubberts ( ) ( ) erasmus medical center, department of rheumatology, rotterdam, the netherlands ( ) erasmus medical center, department of immunology, rotterdam, the netherlands the objective of this study was to identify the role of il- in th polarization in the prone autoimmune dba- mice with and without collagen-induced arthritis and to evaluate th specific cytokine and transcription factor expression. il- induced th cells in vitro from spleen cells of naïve and collagen-type ii (cii) immunized dba- mice. the percentage of th cells is markedly higher in cii-immunized versus naïve dba- mice. adding il- to tgf-beta/il- stimulated cd + t cells did not significantly increase the percentage of th cells. tgfbeta/il- in contrast to il- induced a relatively high percentage of il- +/ifn-gamma-cells and low il- -ifn-gamma+ cells. tgf-beta/il- did not increase il- receptor expression, which may explain why adding il- directly or two days after tgf-beta/il- did not result in an increase in the percentage of th cells. elevated expression of il- a and il- f as well as the th specific transcription factor rorgammat was found under il- as well as tgfbeta/il- conditions. interestingly, il- but not tgf-beta/il- is critical in the th cytokine il- expression in t cells from ciiimmunized dba- mice. these data show that il- was more pronounced in inducing il- +/ifn-gamma-(th ) cells under cii-immunized conditions. furthermore, il- did not markedly increase the percentage of th cells induced by tgf-beta/il- . however, il- is critical for the induction of il- expression, suggesting a unique role for il- in the induction of specific th cytokines ebi was initially discovered as a transcriptionally activated gene in epstein-barr virus-infected human b lymphocytes, and similar to p of il- . ebi protein has been shown to form heterodimers with p . p /ebi termed il- , can influence the function of multiple t cell subsets, including naive, effector, regulatory and memory t cells. however, previous studies showed that the overlapped expression of ebi and p is very limited. these data lead to the hypothesis that ebi may play a role independently from its association with p . thus, to define the function of ebi , we generated ebi transgenic (tg) mice expressing in multiple tissues. ebi tg mice exhibited no histologic abnormalities in various organs and normal numbers of naive and memory cd +, cd + t cells, b cells, nk cells and nkt cells. cd +t cells isolated from spleens of ebi tg mice, however, produced less ifn-g than cells from wt (wild type) control mice after in vitro stimulation with anti-cd and anti-cd antibodies. in vivo studies, delayed-type hypersensitivity (dth) and contact hypersensitivity (chs) responses were significantly reduced in ebi tg compared with wt mice. moreover, the chs responses in ebi tg mice were recovered with anti-ebi polyclonal antibody. notably, chs reaction in wt mice was increased by anti-ebi antibody. in contrast, anti-p antibody suppressed chs responses in wt mice. these data suggest that ebi acts in different from il- , and reduces th responses. ( ), o thaunat( ), x houard ( ), o meilhac ( ), g caligiuri( ), a nicoletti ( ) ( ) inserm u and university denis diderot-paris , chu xavier bichat, paris, france ( ) inserm umr s , universitØ pierre et marie curie-paris , centre de recherche des cordeliers, paris, france arteries are composed of three concentric tissue layers which exhibit different structures and properties. because arterial injury is generally initiated at the interface with circulating blood, most studies performed to unravel the mechanisms involved in injury-induced arterial responses have been focused on the innermost layer (intima). in contrast, the role of the outermost tunica, the adventitia, has attracted relatively little attention and remains elusive. in the present review, we focus on involvement of the adventitia in the response to various types of arterial injury leading to vascular remodeling. several lines of evidence show that the initial insult and the early intimal response lead to the genesis of (neo-) mediators that are centrifugally conveyed by mass transport towards the adventitia. these mediators trigger local adventitial responses including angiogenesis, immuno-inflammation, and fibrosis. we propose that these three processes sequentially interact and that their net balance participates in producing each specific pathological condition. hence, an adventitial adaptive immune response predominates in chronic rejection. inflammatory phagocytic cell recruitment and initiation of a shift from innate to adaptive immunity characterize the adventitial response to proteolysis products in abdominal aortic aneurysm. centripetal adventitial sprouting of neovessels, leading to intraplaque hemorrhages, predominates in atherothrombosis. adventitial fibrosis mediated by low inflammation characterizes the response to mechanical stress and is responsible for constrictive remodeling of arterial segments and initiating interstitial fibrosis in perivascular tissues. these adventitial events thus impact not only on the vessel wall biology but also on the surrounding tissue. atherosclerosis has many of the characteristics of an inflammatory disease, and thus would classically involve endothelial cox-derived prostaglandins such as pge and prostacyclin acting on ep and ip receptors, respectively.activation of vascular ip receptors is especially important in limiting the atherogenic properties of thromboxane a acting on tp receptors.more recently, expression of ghrelin receptors has been shown to be increased in atherosclerotic plaques, and ghrelin itself has anti-inflammatory properties in addition to its classical role as a hunger hormone.as well as the complex crosstalk between g-protein-coupled receptors (gpcrs), recent evidence indicates that many gpcrs exist constitutively as homodimeric complexes, and that the formation of heterodimers not only influences the classical cell signalling pathways used by these receptors, but also affects their subcellular distribution.we have found that ep -i, tp and ghrelin receptors readily form homodimers, but that co-transfection of hek cells with these receptors results in the formation of heterodimers with unpredictable effects on receptor distribution and cell signalling properties.since inflammatory conditions are thought to change the relative expression levels of gpcrs in the vasculature, and since varying the expression levels of gpcrs will affect their ability to form heterodimers, then one might predict that gpcr heterodimerization would indeed influence the reactivity of vascular tissue during inflammation. [this work was fully supported by grants from the research grants council of the hong kong special administrative region (cuhk / m and vascular inflammation leads to formation of leukotrienes through the -lipoxygenase pathway of arachidonic acid metabolism. leukotriene forming enzymes are expressed within atherosclerotic lesions and locally produced leukotrienes exert pro-inflammatory actions within the vascular wall by means of cell surface receptors of the blt and cyslt receptor subtypes. recent mechanistic studies have supported the notion of a major role of leukotriene signaling in atherosclerosis. leukotriene b (ltb ) is for example one of the most potent chemotactic mediators formed within the atherosclerotic lesion, inducing migration of a number different cell-types of both hematopoietic and non-hematopoietic origin. initially identified on neutrophils, blt receptor activation is involved in monocyte chemotaxis and adhesion as well as in vascular smooth muscle cell migration and proliferation, providing examples of potential mechanisms in ltb -induced atherogenesis. targeting ltb -induced activation of vascular smooth muscle cells has beneficial effects in models of intimal hyperplasia and restenosis after vascular injury. furthermore, blt receptor expression has been demonstrated on t-cells, suggesting ltb as a potential link between innate and adaptive immunological reactions. taken together, the local formation of leukotrienes within the atherosclerotic lesion and the potent pro-inflammatory effects of leukotriene receptor activation in target cells of atherosclerosis provide a rationale for a role leukotrienes in this disease. further experimental and clinical studies are however needed to develop therapeutic strategies of treatments targeting leukotriene signaling in atherosclerosis. in normal physiological conditions, the prostanoid (prostaglandin (pg) and thromboxane (tx)) synthesis is dependent on the constitutive isoform of cyclooxygenase (cox- ). this synthesis and release happen few minutes after cell or tissue stimulation. in vascular preparations submitted to pro-inflammatory conditions for some hours, the inducible isoform of cyclooxygenase (cox- ) and other prostanoid synthases can be observed. as an illustration of the previous experimental results, there is an increased presence of cox- and the inducible enzyme responsible for pge synthesis (mpges- ) detected by immunocytochemistry in the carotid atherosclerotic plaque with strong inflammation. in vascular cells in culture, pgi is the major biological active prostanoid produced in the normal physiological conditions. however, when cox- is induced, pgi and pge are equally produced. the role of cox- , cox- and mpges- activities is also dependent on the expression of the various prostanoid receptors in the considered vessel. there is increasing evidence for the presence and a role of the ep receptor subtypes (ep , ep , ep or ep ) preferentially stimulated by pge in the vascular wall. for these reasons, we have characterized the receptors activated by pge in human mammary arteries. in these vessels incubated with a pro-inflammatory cytokine (interleukin- â) and lipopolysaccharides a reduced contractility to norepinephrine has been observed. this effect is abolished by treatment of the vascular preparations with a selective cox- inhibitor, suggesting that prostanoid synthesis and/or prostanoid receptors could be involved. rheumatoid arthritis is a syndrome which probably consists of a number of diseases for which the risk factors differ. two major processes were identified: the generation of the anti-citrullinated antigens immune response (highly sepcific for ra).we show that the different hla class ii alleles contribute to the development of anti-ccp-positive and anti-ccp negative ra.the se alleles do not independently contribute to the progression to ra, but rather contributed to the development of anti-ccp antibodies. next we determined the effect of smoking and observed that smoking only conferred risk to contract ra in the ccp-positive group and not in the anti-ccp negative group. for the risk factor ptpn (a gene that regulates treshold of lumphocyte activation) the allele c t only contributed to ccp-positive ra. in contrast to hla two other risk factors were found to be associated with both ccp-positive and ccp-negative ra. the risk factor in the fcrl-gene as has been identified in the japanese population was also tested in dutch ra cases and unrelated dutch controls. carrier analysis of the snp (rs ) revealed association of cc genotype with higher risk of developing ra as compared to tt & tc carriers (p = . and or = . ). in a meta-analysis of all studies comparing individuals, the or for the cc genotype to develop ra was . and the p-value < . . in conclusion, different steps in pathogenesis of the syndrome ra can be delineated this talk will focus on recent advances in understanding primary genetic factors predisposing to inflammatory bowel disease (crohns disease and ulcerative colitis). proven genes containing genetic variants predisposing to crohns disease include ibd / q , card /nod and il r. data is suggestive but not yet as convincing for many other genes. a common theme is of genetic variants influencing early innate immune responses to intestinal bacterial components, and subsequent adaptive immune responses, leading to intestinal inflammation. only for card /nod is there (partial) understanding of how genetic variation influences biological function to cause chronic disease. some mouse models (gene targeted) of card appear to show opposite effects to other models and human systems. in humans, card mutations impair responses to bacterial components (muramyl dipeptide) mainly at low dose sensing. it is likely this receptor system normally maintains intestinal crypt sterility and protection from invasive infection. pathogen-recognition receptors (prrs) are key components of immune systems and are involved in innate effector mechanisms and activation of adaptive immunity. since their discovery in vertebrates, toll-like receptors (tlrs) have become the focus of extensive research that has revealed their significance in the regulation of many facets of our immune system. recently a new family of intracellular prrs, the nod-like receptors (nlrs), which include both nods and nalps have been described. mutations within the nalp /cryopyrin/ cias gene are responsible for three autoinflammatory disorders: muckle-wells syndrome, familial cold autoinflammatory syndrome, and cinca/nomid. the nalp protein associates with asc and caspase- (thereby forming a molecular machine termed inflammasome that displays high proil- beta-processing activity. macrophages from muckle-wells patients spontaneously secrete active il- beta. increased inflammasome activity is therefore likely to be the molecular basis of the symptoms associated with nalp -dependent autoinflammatory disorders. here we will emphasis on the ability of this protein complex to promote the development of autoinflammatory syndromes. allergic inflammation (ai) is a complex phenomenon initiated by allergen binding to ige sensitized mast cells and consequent mast cell activation. this causes the symptoms of the early phase of ai and the onset of the late phase characterized by the penetration in the inflamed tissue of inflammatory cells, notably the eosinophils. their subsequent activation is believed to cause tissue damage and to be the main responsible for the tissue remodeling, especially when the ai becomes a chronic process. we defined a novel functional unit that we termed the allergic synapse formed by mast celleosinophil couples. in the synapse these two old cellular players of ai have a cross talk via soluble mediators and receptor-ligand interactions. this results in mast celleosinophil functional synergism that consequently amplifies and prolongs the inflammatory response. in addition, mast cells and eosinophils are influenced and influence as in a sort of allergic niche the surrounding structural cells, i.e. fibroblasts and endothelial cells. we propose to view the allergic synapse/niche as a specialized effector unit worthy to be blocked for the treatment/prevention of allergic inflammation. ( ) ( ) erasmus mc, rotterdam, the netherlands ( ) department of immunology weizmann institute of science, rehovot, israel allergic asthma is one of the most common chronic diseases in western society, characterized by reversible airway obstruction, mucus hypersecretion and infiltration of the airway wall with th cells, eosinophils, and mast cells. if we are to devise new therapies for this disease, it is important to elucidate how th cells are activated and respond to intrinsically harmless allergens. dendritic cells (dcs) are the most important antigen presenting cells in the lung and are mainly recognized for their exceptional potential to generate a primary immune response and sensitization to aeroallergens. we have shown that intratracheal injection of ovalbumin (ova) pulsed dcs induces sensitization leading to eosinophilic airway inflammation upon ova aerosol challenge. we investigated the role of dcs in the secondary immune response in a murine asthma model. ova aerosol challenge in ova-dc sensitized mice, induced an almost fold increase in the number of airway dcs as well as an increase in eosinophils and t cells. to investigate the functional importance of dcs for the induction and maintenance of airway inflammation in response to allergen challenge, we conditionally knocked-out endogenous dcs in sensitised cd c-diphtheria toxin (dt) receptor (cd cdtr) transgenic mice by airway administration of dt h before ova aerosol ( x) challenge or during an ongoing inflammation (depletion after x ova aerosols continued with additional ova aerosols). numbers of balf eosinophils, th cytokine production by mediastinal lymph nodes and peribronchial and perivascular inflammatory infiltrates were dramatically decreased, illustrating an essential role for airway dcs during secondary challenge. karolinska institute, stockholm, sweden nk cells are innate lymphocytes with potent immunoregulatory functions. they are potent producers of several cytokines and chemokines, and also respond to similar molecules in the body and at inflammatory sites. even though traditionally best characterized for their role in anti-viral and anti-tumor immunity, they influence several other types of immune responses. for example, they are involved in, and affect, acute as well as chronic inflammatory responses. in the present talk, a general overview on our current knowledge of nk cell biology will be provided, with a special emphasis on the role of these cells in allergic inflammation. basophils are major effector cells in allergic reactions due to their ability to release substantial quantities of histamine and eicosanoids following activation of high affinity ige receptors (fc<epsilon>ri) with allergens. although these attributes are shared with their tissuefixed mast cell compatriots, basophils are unique in their ability to also rapidly elaborate th -type cytokines (e.g. il- and il- ), subsequently supporting ige synthesis and underlying atopy. importantly, these mediators are additionally secreted following primary exposure to certain parasites (e.g. s. mansoni) and immunoglobulin superantigens, suggesting a role for basophils in innate immunity and in assisting developing th -type adaptive immune responses. while we are beginning to understand the potential physiological functions of these cells regarding host defence, blocking their activity with respect to treating symptoms of allergic disease has remained an enigma. recent advances, however, have shed light upon the major intracellular signal transduction processes involved in fc<epsilon>ri activation and may lead to novel therapeutic strategies for inhibiting mediator secretions. an important discovery in this regard is the phosphatase ship, which downregulates pi -kinase signalling in both basophils and mast cells. recent data shows that ship expressions in basophils are reduced from donors with active allergic disease but that these levels may be increased, and the activity of basophils subsequently inhibited, by targeting receptors associated with ship recruitment (cd r, cd r). identifying the natural ligands for these inhibitory receptors may therefore pave the way for new therapies for the treatment of allergic inflammation. mitogenesis and proliferation of vsmc play an important role in atherogenesis. pro-inflammatory secretory phospholipases a (spla ) hydrolyse glycerophospholipids of hdl and ldl and release pro-inflammatory agents, lyso-lipids, oxidized and non-oxidized fatty acids and isoprostanes.spla s lipolysis products localize in vascular wall in vicinity of vsmc.we have tested the impact of spla , hdl and ldl and of their hydrolysis products on mitogenesis and pge and ltb release from vsmc.mitogenesis was significantly enhanced by native hdl, and ldl, and by group v spla . spla hydrolysis of hdl and ldl enhanced mitogenic activity in order v>x>iia.the release of pge from vsmc was enhanced by group x spla s but not iia or v.the greatest effect was seen for hdl hydrolysed by group v and x spla .native ldl and its spla hydrolysis products enhanced the release of pge in order x>v>iia.the release of ltb from vsmc was markedly increased by native ldl and hdl, and hydrolysis products of group v and x, but not iia spla .migration of vsmc was significantly enhanced by spla iia and inhibited by hdl.this study demonstrates a complex interaction of hdl and ldl with pro-inflammatory spla s, which affects mitogenesis, eicosanoid release and migration of vsmc.study of biocompatible spla blockers in the therapy of atherosclerosis is indicated. contact information: professor waldemar pruzanski, university of toronto, department of medicine, toronto, ontario, canada e-mail: drwpruzanski@bellnet.ca ( ) ( ) ipmc-cnrs umr , valbonne, france ( ) university of washington, seattle, usa ( ) inserm umrs , paris, france ( ) university of naples, italy the superfamily of phospholipase a comprises at least intracellular enzymes and up to secreted pla s (spla s). elucidating the biological roles of each pla member is currently the most challenging issue in the pla field. the different spla s are not isoforms and are likely to function either as enzymes producing key lipid mediators (eicosanoids and lysophospholipids) or as ligands that bind to specific soluble or membrane-bound proteins (like cytokines). increasing evidence suggests that spla s iia, iii, v, and x are involved in inflammatory diseases including atherosclerosis. among spla s, the human group x (hgx) enzyme has the highest enzymatic activity towards phosphatidylcholine, the major phospholipid of cellular membranes and low density lipoproteins (ldl). on human alveolar macrophages, hgx spla can trigger secretion of tnf alpha, il and il in a non-enzymatic manner. on colorectal cancer cells, hgx spla stimulates cell proliferation, produces potent eicosanoids including pge , and activates the transcription of key genes involved in inflammation and cancer. the enzyme can also hydrolyze pc and platelet-activating factor (paf) of ldl particles very efficiently. finally, hgx spla is present in human atherosclerotic lesions and converts ldl into a proinflammatory particle that induces macrophage foam cell formation, as well as map kinase activation, arachidonic acid release, and expression of adhesion molecules in huvec cells. some other key molecular features of spla s including hgx will be presented. we have reported preferential release of polyunsaturated fatty acids during hydrolysis of lipoprotein phosphatidylcholine (ptdcho) by spla s, but the mechanism of this selectivity is not known. since both sphingomyelin (sm) and lysoptdcho inhibit the activity while increasing fatty acid specificity of other pla s, we have examined fatty acid release by spla siia, v and x in relation to relative increases in proportion of endogenous sm and lysoptdcho during lipoprotein digestion. the analyses were performed by normal phase liquid chromatography with on-line electrospray mass spectrometry (lc/esi-ms) and lc/collision induced dissociation (cid)/esi-ms using conventional preparations ofldl and hdl. the highest preference for arachidonate release from ldl by group x spla was observed when the residual sm/ pdcho molar ratio had reached . compared to a starting ratio of . .group v spla showed preferential release of linoleate at residual sm/ptdcho molar ratio . , while at intermediate ratios, both arachidonate and linoleate were released at more comparable ratios. the relative increases in lysoptdcho and sm during the digestion with spla iia were much more limited, and a preferential hydrolysis of polyunsaturated fatty acids was not observed. these results suggest a lipid phase separation as a likely basis for a differential hydrolysis of molecular species of ptdcho. the residual sm/ptdcho ratios reached during group v and x spla digestion are similar to those observed for lesional ldl, which promote release of ceramides by smase leading to ldl aggregation. the above findings support a potential role of sphingomyelins in atherogenesis. although sphingomyelin (sm) is one of the most abundant phospholipids in lipoproteins and cell membranes, its physiological significance is unclear. because of its localization in the outer surface of the cells, and its structural similarity to phosphatidylcholine (pc),we proposed that it competitively inhibits phospholipolysis of cell membranes by external phospholipases (pla). we showed that sm inhibits several lipolytic enzymes including secretory pla iia, v, and x, and hepatic and endothelial lipases, all of which hydrolyze pc. treatment of sm in the substrate with smase c not only relieved the inhibition but also activated the pla reaction further, suggesting that ceramide, the product of smase c, independently stimulates pla , possibly by disrupting the bilayer structure. smase d treatment, which produces ceramide phosphate, did not stimulate the spla . the fatty acid specificity of pla is significantly affected by sm. thus spla x exhibited enhanced specificity for the release of arachidonic acid ( : ) in presence of sm, due to a preferential inhibition of hydrolysis of other pc species. in contrast, sm inhibited the release of : by spla v. ceramide selectively stimulated the release of : by both enzymes. only the long chain ceramides (> carbons) were effective, while ceramide phosphate did not stimulate spla activity. sm-deficient cells released more : in response to spla -treatment than normal cells, and pretreatment of normal cells with smase c increased their susceptibility to spla attack. these studies show that sm and ceramide regulate the activity and specificity of pla, and consequently the inflammatory response. secretory phospholipase a (spla ) types iia, v, or x, have been associated with inflammatory diseases and tissue injury including atherosclerosis in humans and mice.given the link between spla and atherogenesis, a mouse model of atherosclerosis (apoe-/-) was used to study the effects of a- , an inhibitor of spla enzymes, on atherosclerosis and cholesterol levels over weeks of treatment. mice were fed with a high-fat, high cholesterol diet alone during the study ( % fat; . % cholesterol, . % casein) and were treated with vehicle or a- bid at mg/kg or mg/kg by oral gavage. total cholesterol was significantly decreased after one month of treatment and remained lower throughout the study.treatment with a- significantly reduced aortic atherosclerotic plaque formation in apoe-/-mice fed a high fat diet when compared to the untreated control by approximately %. in a different model that used angiotensin ii in conjunction with a high fat diet in a background of apoe-/-deficient mice for weeks, oral dosing of a- ( mg/kg bid) significantly reduced aortic atherosclerosis and aneurysm rate when compared to vehicle. these data suggest that a- is a potential novel therapeutic agent for the treatment of atherosclerosis. ( ), s doty( ), c antonescu ( ), c staniloae ( ) ( ) saint vincents hospital manhattan, new york, usa ( ) hospital for special surgery, new york, usa ( ) sloan-kettering institute for cancer research, new york, usa tnf-stimulated gene (tsg- ) is induced by tnf-a during inflammation and its secreted product tsg- glycoprotein is involved in immune-mediated inflammatory diseases and fertility. it regulates cox- and prostaglandin synthesis, and participates in extracellular matrix remodeling. considering the chronic inflammatory nature of atherosclerosis we hypothesized that tsg- is expressed in atherosclerotic plaques and investigated tsg- protein expression and cellular distribution on superficial femoral artery endarterectomy specimens from diabetic and non-diabetic patients with peripheral vascular disease. six histologically normal radial artery specimens were analyzed as control. paraffin embedded samples were studied by immunohistochemistry using a goat polyclonal anti-human-tsg- antibody. tsg- expression was consistently present in all atherectomy specimens but not in control specimens. a distinct, strong cytoplasmic staining pattern was uniformly detected in the endothelial lining of the intima, as well as in the neo-vessel proliferation of the plaque. cytoplasmic staining was also identified in the smooth muscle cell proliferation of the neo-intima. patchy tsg- expression was noted in the extracellular matrix. within the inflammatory plaques from diabetic patients, tsg- stained the foamy macrophages. tsg- expression was also confirmed and quantified by qrt-pcr that showed a significant up-regulation of tsg- gene (more that fold induction compared to housekeeping genes). our study identifies for the first time the preferential expression of tsg- in atherosclerotic lesions and characterizes its distribution within the cellular and matrix components of the plaque. tsg- is a novel inflammatory mediator of atherosclerosis and a potentially new marker of endothelial / smooth muscle cell activation. ( ), r krohn ( ), h lue ( ), jl gregory( ), a zernecke ( ), rr koenen ( ), t kooistra ( ), p ghezzi( ), r kleemann ( ), r bucala( ), mj hickey ( ), c weber ( ) ( ) university hospital of the rwth aachen, germany ( ) centre for inflammatory diseases, monash university, melbourne, australia mediators, which cannot be classified into chemokine subfamilies but share functional patterns, e.g. signaling through chemokine receptors, constitute a group termed chemokine-like function (clf)-chemokines. the pleiotropic cytokine macrophage migration inhibitory factor (mif) plays a critical role in inflammatory diseases and atherogenesis. the underlying molecular mechanisms are poorly understood, but, interestingly, mif displays structural features resembling chemokines. we have identified the chemokine receptor cxcr as a functional receptor for mif. mif triggered galphai/integrin-dependent arrest and chemotaxis of monocytes specifically through cxcr , inducing rapid integrin activation. mif directly bound to cxcr with high affinity (kd of . nm). monocyte arrest mediated by mif in inflamed or atherosclerotic arteries involved cxcr as well as cd , a recently identified membrane receptor moiety for mif. accordingly, cxcr and cd were found to occur in a receptor complex. in vivo, mif deficiency impaired monocyte adhesion to the aortic/arterial wall in atherosclerosis-prone mice, as evidenced by intravital microscopy. thus, mif displays chemokine-like functions by acting as a non-cognate ligand of cxcr , serving as a regulator of inflammatory and atherogenic recruitment. these data harbor an intriguing novel therapeutic prospect by targeting mif in atherosclerosis and add a new dimension to mif and chemokine receptor biology. ( ), r toes ( ), h van bockel( ), paul quax ( , ) ( ) tno bioscienses, leiden, the netherlands ( ) department of vascular surgery, leiden university medical center, the netherlands ( ) department of rheumatoly, leiden university medical center, the netherlands the immune system is thought to play a crucial role in regulating collateral circulation (arteriogenesis), a vital compensatory mechanism in patients with arterial obstructive disease. here, we studied the role of lymphocytes in a murine model for artiogenenesis after acutehind limb ischemia. arteriogenesis was impaired in c bl/ mice depleted for natural killer (nk)-cells by anti-nk . antibodies and in nk-cell-deficient transgenic mice. arteriogenesis was, however, unaffected in jµ knockout mice that lack nk . + natural killer t (nkt)cells, indicating that nk-cells, rather than nkt-cells are involved in arteriogenesis. furthermore, arteriogenesis was impaired in c bl/ mice depleted for cd + tlymphocytes by anti-cd antibodies, and in major histocompatibility complex (mhc)-class-ii-deficient mice that lack mature peripheral cd + t-lymphocytes. this impairment was even more profound in anti-nk . treated mhc-class-ii-deficient mice that lack both nkand cd + t-lymphocytes. finally, collateral growth was severely reduced in balb/c as compared with c bl/ mice, two strains with different bias in immune responsiveness. correspondingly, fewer cd -positive lymphocytes accumulated around collaterals in balb/c mice. these data show that both nk-cells and cd + t-cells play an important role in arteriogenesis. moreover, our data hold promise for the development of novel clinical interventions as promoting lymphocyte activation might represent a powerful method to treat ischemic disease. post-interventional vascular remodeling in venous bypass grafts, seen as intimal hyperplasia (ih) and accelerated atherosclerosis, often causing graft failure. inflammation is an important trigger for these processe. complement is an important part of the immune system and participates in regulating inflammation. although involved in several other inflammatory diseases, the role of the complement cascade in vein graft remodeling is unknown. the involvement of the complement system in vein graft disease was studied here using a model in which caval veins are grafted in carotids arteries of hypercholesterolemic apoe leiden mice. in these veins ih and accelerated atherosclerotic lesions develop within days, consisting mainly of foamcells and smc. to study the functional role of complement in vein graft remodeling, cobra venom factor (cvf: u daily) was used to deplete complement starting one day prior to vein graft surgery. cvf-treatment reduced vein graft thickening by % (p= . ), when compared to saline treated controls (n= ).to confirm that the reduction by cvf was due to hampered complement function and not a direct effect of cvf, complement activation was blocked using crry-ig (inhibiting c convertases). crry-ig ( mg every other day) led to % decrease in vein graft thickening (p= . ) compared to controls receiving non-relevant control igg. these data prove that complement activation plays a major in intimal hyperplasia and accelerated atherosclerosis in vein grafts. ( ), m-c koutsing tine( ), p borgeat( ), h ong ( ), sylvie marleau ( ) ( ) universite de montreal, quebec, canada ( ) centre de recherche en rhumatologie et immunologie, canada we have previously shown that ep , a growth hormone-releasing peptide (ghrp) analogue binding selectively to the scavenger receptor cd , elicits a striking reduction in atherosclerosis development in apolipoprotein-deficient (apoe-/-), a condition associated with increased circulating numbers of primed/activated leukocytes. we investigated the effect of ghrp analogues on i/r-elicited remote lung injury in week-old apoe-/-mice fed a high fat high cholesterol (hfhc) diet from weeks of age. at weeks old, mice were treated daily with a s.c. injection of ep ( mg/kg) for days and were then subjected to unilateral hindlimb ischemia (by rubber band application) for minutes, followed by minutes reperfusion. our results show that ep significantly reduced leukocyte accumulation by % in the lungs, from . (ae . ) in vehicle-treated mice to . (ae . ) x leukocytes/g lung in ep -treated mice (n = - per group), as assessed by myeloperoxidase assay. this was associated with a % reduction of opsonized zymosan-elicited blood chemiluminescence. in contrast, neither blood chemiluminescence, nor leukocyte accumulation in the lungs were signicantly modulated in apoe-/-/cd -/-deficient mice, from . (ae . ) in vehicle-treated mice to . (ae . ) x leukocytes/g lung in ep -treated mice. we conclude that ep protects i/r-elicited circulating leukocyte priming/activation and remote lung injury, possibly through a cd -mediated pathway. glycogen synthase kinase beta (gsk- beta) is a serine/ threonine protein kinase that has recently emerged as a key regulatory switch in the modulation of the inflammatory response. dysregulation of gsk- beta has been implicated in the pathogenesis of several diseases including sepsis. here we investigate the effects of two chemically distinct inhibitors of gsk- beta, tdzd- and sb , on the circulatory failure and the organ injury and dysfunction associated with hemorrhagic shock. male wistar rats were subjected to hemorrhage (sufficient to lower mean arterial blood pressure to mmhg for min) and subsequently resuscitated with shed blood for h. hemorrhage and resuscitation resulted in an increase in serum levels of (a) creatinine and, hence, renal dysfunction, and (b) alanine aminotransferase and aspartate aminotransferase and, hence, hepatic injury. treatment of rats with either tdzd- ( mg/kg, i.v.) or sb ( . mg/kg, i.v.) min before resuscitation abolished the renal dysfunction and liver injury caused by hemorrhagic shock. the protection afforded by these compounds was confirmed by histological observations of lung, kidney and liver samples. in addition, tdzd- , but not sb , attenuated the increase caused by hemorrhage and resuscitation in plasma levels of the proinflammatory cytokine interleukin . neither of the gsk- beta inhibitors however affected the delayed fall in blood pressure caused by hemorrhagic shock. thus, we propose that inhibition of gsk- beta may represent a novel therapeutic approach in the therapy of hemorrhagic shock. ( ), y ito ( ), h yoshimura ( ), h inoue ( ), n kurouzu ( ), h hara ( ), y mastui ( ), h kitasato ( ), s narumiya( ), c yokoyama ( ), m majima ( ) ( ) kitasato university school of medicine, japan ( ) kyoto university school of medicine, japan ( ) tokyo medical and dental university, japan thromboxane (tx) a is a potent stimulator of platelet activation and aggregation and vascular constriction. we have reported cytokine-mediated release of sdf- from platelets and the recruitment of nonendothelial cxcr + vegfr + hematopoietic progenitors constitute the major determinant of revascularization. we hypothesized txa induces angiogenic response by stimulating sdf- and vegf which derived from platelet aggregation. to evaluate this hypothesis, we dissected the role of the txa in angiogenesis response using mouse hind limb ischemia. recovery from acute hind limb ischemia, as assessed in wild type mice (c bl/ wt) , prostaglandin i receptor (ip) knock out mice (ipko) and thromboxane (tx) a receptor (tp) knock out mice (tpko) by using lase doppler. blood recovery in tpko significantly delayed compared to wt and ipko. immunohistochemical studies revealed that the number of cd positive cells in the ischemic quadriceps were less stained in tpko compared to wt and ipko.plasma sdf- and vegf concentration were significantly reduced in tpko mice. we observed during in vivo fluorescence microscopic study that compared to tpko, ipko and wt significantly increased platelet attachment to the microvessels around ligated area. tpko translpanted wt bone marrow cells increased blood recovery compared to tpko transplanted tpko bone marrow cells. in addition, mice injected with txa synthase c-dna expressing fibroblast increased blood flow recovery compared to control mice. these results suggested that tp signaling rescues ischemic condition by inducing angiogenesis by secreting sdf- and vegf from platelet aggregation. administration of selective tp agonist may open new therapeutic strategy in regenerative cardiovascular medicine. during renal ischemia/reperfusion (i/r) injury, apoptosis has been reported as a very important contributor to the final kidney damage. the determinant role of cytoskeleton derangement in the development of apoptosis has been previously reported, but a clear description of the different mechanisms involved in this process has not been yet provided. the aim of the study is to know the role of peroxynitrite as inductor of cytoskeleton derangement and apoptosis during the inflammatory process associated to renal ischemia-reperfusion. based in a rat kidney i/r model, by experiments in which both the actin cytoskeleton and peroxynitrite generation were pharmacologically manipulated, results indicate that the peroxynitrite produced during the i/r derived oxidative stress state, is able to provoke cytoskeleton derangement and apoptosis development. thus, the control in the peroxynitrite generation during the i/r could be an effective tool for the improvement of cytoskeleton damage and reduction apoptosis incidence in the renal i/r injury. metabolomics, the global profiling of metabolites, may inform about the multiple interacting processes involved in inflammatory disease. using nmr spectroscopy we analysed metabolite fingerprints in serum from early arthritis, and at a site of inflammation, in the posterior segment of the eye. serum from patients with synovitis of "t months duration whose outcome was determined at clinical follow-up was used. vitreous samples were from patients undergoing vitrectomy for vitreoretinal disorders. one dimensional h nmr spectra were acquired. principal components analysis (pca) of the processed data was conducted along with a supervised classification. with the arthritis serum there was a clear relationship between each samples score in the pca analysis and the level of crp. supervised classification of the initial samples was able to predict outcome, whether rheumatoid arthritis, other chronic arthritis or self-limiting arthritis, with high specificity and sensitivity. a similar approach using the eye fluids was able to give a clear discrimination between two pathologically similar conditions lens-induced and chronic uveitis. in this case differences were not due to a straightforward relationship with inflammatory markers (il- , ccl ), which did not correlate with pca in these samples. similarly, certain molecules, such as lactate, were associated with ocular disease, but not rheumatoid arthritis. these results suggest that underlying inflammatory processes may differ in these conditions or may reflect predisposing metabolic patterns in individual patients. h-nmr-based metabolomics may provide a useful measure of outcome in inflammatory diseases and give novel insights into the pathological processes involved. ( ), am artoli( ), a sequeira( ), c saldanha ( ) ( ) instituto de medicina molecular,faculdade de medicina de lisboa, portugal ( ) cemat, instituto superior tØcnico, universidade de tØcnica de lisboa, portugal the recruitment of leukocytes from the blood stream and their subsequent adhesion to endothelial walls are essential stages to the immune response system during inflammation. the precise dynamic mechanisms by which molecular mediators facilitate leukocyte arrest are still unknown. in this study combined experimental results and computer simulations are used to investigate localized hydrodynamics of individual and collective behaviour of clusters of leukocytes. leukocyte-endothelial cell interactions in post-capillary venules of wistar rats cremaster muscle were monitorized by intravital microscopy. from these experiments the haemorheologic and haemodynamical measured parameters were used in time dependent three-dimensional computer simulations, using a mesoscopic lattice boltzmann solver for shear thinning fluids. the dynamics of leukocyte clusters under non-newtonian blood flow with shear thinning viscosity was computed and discussed. in this paper we present quantified distributions of velocity and shear stress on the surface of leukocytes and near vessel wall attachment points. we have also observed one region of maximum shear stress and two regions of minimum shear stress on the surface of leukocytes close to the endothelial wall. we verified that the collective hydrodynamic behaviour of the cluster of recruited leukocytes establishes a strong motive for additional leukocyte recruitment. it was found that the lattice boltzmann solver used here is fully adaptive to the measured experimental parameters. this study suggests that the influence of the leukocytes rolling on the increase of the endothelial wall shear stress may support the activation of more signalling mediators during inflammation. macrophages are essential for host defence, but when excessively and persistently activated, these cells contribute to the initiation and progression of inflammatory diseases such as rheumatoid arthritis. investigating the function of inflammatory genes in macrophages may identify novel therapeutic targets for inflammatory diseases. one family of transcripts that are highly expressed in activated macrophages are members of the schlafen (slfn) gene family; a recently identified family whose function is still unknown. this study examined the mrna expression of slfn in activated bone marrowderived macrophages in vitro, and in collagen-induced arthritis (cia) in vivo. real-time pcr expression analyses of bone marrow-derived macrophages stimulated with lipopolysaccharide (lps) over a time course, revealed differential expression of individual slfn family genes. in particular, slfn- , slfn- , and slfn- were maximally induced after hours. the maximal induction of slfn- and slfn- was observed after hours of lps treatment. individual members of the slfn family were also differentially expressed in cia, a model of rheumatoid arthritis. mrna levels of slfn- , slfn- , slfn- and slfn- were elevated in joints affected by cia. to investigate the role of slfn- , we have generated a transgenic mouse line, which over expresses slfn- specifically in cells of the mononuclear phagocyte system, by using a novel binary expression based on the c-fms promoter and gal . further characterisation of the slfn- over expressing mouse line will be used to assess the function of slfn- in macrophage biology and inflammation, and its potential as a therapeutic target. macrophages play an important role in resolving inflammation. it is known that the resolution of inflammation requires alternative activation of macrophages. but the precise events of phenotype switching in macrophages remain poorly understood. we show that lipocalin , lcn- , is able to provoke a switch in macrophage activation. in an in vitro co-culture model for renal epithelial cells and macrophages, we detected by sirna technique that the presence or absence of lcn- determines proliferation processes in damaged renal epithelial cells. the proliferative response was dependent on proinflammatory or anti-inflammatory environment. as lcn- is an acute phase protein synthesized during inflammation and unregulated in a number of pathological conditions, it may play an important role in survival and regeneration. we anticipate here that our results could be relevant for further research on the mechanisms of the phenotype switch induced by lcn- . ( ), y cao ( ), s adhikari ( ), m wallig ( ) ( ) national university of singapore, department of pharmacology, singapore ( ) university of illinois at urbana champaign, usa it has earlier been shown that the extent of apoptotic acinar cell death is inversely related to the severity of acute pancreatitis. our previous works have demonstrated that induction of pancreatic acinar cell apoptosis by crambene protects mice against acute pancreatitis. the current study aims to investigate the role of phagocytic receptors and the anti-inflammatory effect of phagocytosis in protecting mice against acute pancreatitis by crambene. acute pancreatitis was induced in the mouse by administering hourly injections of caerulein ( mg/kg) for , and hours respectively. neutralizing monoclonal anti-il- antibody ( . mg/kg) was administered either with or without crambene ( mg/kg) hours before the first caerulein injection. rt-pcr, western blotting and immunostaining were performed to detect cd expression. apoptosis in pancreatic sections was visualized by tunel. severity of acute pancreatitis was evaluated by estimation of serum amylase, pancreatic myeloperoxidase (mpo), water content, and morphological examination. pancreatic levels of inflammatory mediators were examined by elisa. the protective effect of crambene is mediated by reducing production of pro-inflammatory cytokines such as mcp- , tnf-a and il- â and up-regulating anti-inflammatory mediators like il- . phagocytotic clearance in mouse acute pancreatitis may be essentially through macrophage surface receptor cd .the anti-inflammatory mediator il- plays an important role in crambene-induced protective action against acute pancreatitis. the release of anti-inflammatory mediator il- is downstream of phagocytosis. these results show that induction of pancreatic acinar cell apoptosis by crambene treatment protects mice against acute pancreatitis via induction of anti-inflammatory pathways. ( , ) ( ) northern ontario school of medicine, thunder bay, ontario, canada ( ) lakehead university, canada integrin receptors and their ligands are involved in adhesion and internalization of several human pathogens, including pseudomonas aeruginosa. we have recently established that beta integrins in lung epithelial cells (lec) provide co-stimulatory signals regulating inflammatory responses (ulanova et al, am j physiol, , : l -l ). we hypothesized that lec integrins serve as receptors to recognize pathogen-associated molecules and mediate the innate immune response to p. aeruginosa. to determine molecular mechanisms of integrin involvement in innate immunity, we used an in vitro model of p. aeruginosa infection of a cells. to investigate interactions of bacteria with lec, p. aeruginosa strain pak was chromosomally labeled with a green fluorescent protein gene using a mini-tn delivery system.using several fluorescence-based detection systems, we established that the natural beta integrin ligand, fibronectin, mediates bacterial adhesion to lec.p. aeruginosa infection caused rapid transcriptional upregulation of alphav and beta integrin expression followed by the increased cell surface protein expression. the surface expression of integrin beta increased shortly following bacterial exposure without alterations of mrna expression, suggesting rapid protein redistribution within the cells. the data indicate that p. aeruginosa are capable to modulate integrin gene/protein expression in lec, potentially using fibronectin to alleviate bacterial binding to beta integrins. upon their engagement, integrin receptors can initiate intracellular signaling involved in innate immune and inflammatory responses to the pathogen. integrin receptors in lec may represent significant therapeutic targets in pulmonary infection caused by p. aeruginosa. the purine nucleoside adenosine has a major modulatory impact on the inflammatory and immune systems. neutrophils, which are generally the first cells to migrate toward lesions and initiate host defense functions, are particularly responsive to the action of adenosine. through activation of the a a receptor (a ar) present on neutrophils, adenosine inhibits phagocytosis, generation of cytotoxic oxygen species, and adhesion. also, recent work showed that adenosine can transform the profile of lipid mediators generated by neutrophils, inhibiting leukotriene b formation while potentiating that of prostaglandin e through the up-regulation of the cyclooxygenase (cox)- pathway. moreover, our laboratory determined that a ar engagement can dramatically modulate the generation and secretion of neutrophil-derived cytokines/chemokines, including tnf-and mips. in mice lacking the a ar, migrated neutrophils expressed less cox- than their wild type counterpart while displaying higher mrna levels of tnf-and mip- . mononuclear cells from a ar knock out mice, which eventually replace neutrophils into the air pouch, also displayed a more pro-inflammatory phenotype than those from wild-type animals. signal transduction experiments, aiming to delineate the intracellular events leading to the modulation of neutrophil functions following a ar engagement, implicate pivotal metabolic pathways such as intracellular cyclic amp, p and pi- k. together, these results indicate that adenosine may have a profound and multi-pronged influence on the phenotype of neutrophils and present this cell as being pivotal in mediating adenosines anti-inflammatory effects. the newest developments regarding adenosines effects on neutrophil functions will be presented.this work is supported by the canadian institutes of health research (cihr). human skin serves not only as a physical barrier against infection, but also as a "chemical barrier" by constitutively and inducibly producing antimicrobial proteins (amps). to identify human skin amps, we analysed extracts of healthy persons stratum corneum by reversed phase-hplc and purified a novel kda amp that showed sequence similarity to mouse hornerin. suggesting that it originates from the human ortholog, we cloned it. human hornerin encodes a amino acid protein that contains a s domain, an ef-hand calciumbinding domain, a spacer sequence and two types of tandem repeats, suggesting that it represents a novel member of the fused s protein family. strongest constitutive hornerin mrna expression was seen in differentiated keratinocyte cultures. to follow the hypothesis, that hornerin fragments represent amps, we recombinantly expressed three hornerin peptides, rhrnr (tandem repeat unit b), rhrnr (tandem repeat unit a) and rhrnr (c-terminus) and subsequently analysed their antimicrobial activity using the microdilution assay system. the rhrnr peptide, containing the sequence motif found in the purified natural hornerin fragment isolated from stratum corneum, exhibited antimicrobial activity at low micromolar concentrations against escherichia coli, pseudomonas aeruginosa and candida albicans. the other peptides were found to be not or nearly not antimicrobially active. our results suggest that hornerin may have a yet unknown protective function in healthy human skin as part of the "chemical barrier" with preformed amps, which are generated from parts of the tandem repeats of a hornerin precursor molecule by a yet unknown cleavage mechanism. ( ), n lu( ), r jonsson( ), d gullberg ( ) ( ) department of biomedicine, university of bergen, norway ( ) the gade institute, university of bergen, norway a ß is the latest addition to the integrin family of heterodimeric receptors for the extracellular matrix. previously, it has been shown that this collagen receptor takes part in processes such as cell migration and matrix contraction. in this study we investigated the factors that regulate mouse integrin a ß expression. specifically, we have analyzed the influence of cell passage, growth factors and the -d microenvironment. using sv immortalized as well as primary fibroblasts, we show that a ß integrin is up-regulated when these cells are cultured within stressed collagen type i lattices. however, a ß is downregulated when the collagen gels are made under relaxed conditions, allowing cells contract the lattice diameter. we also show here that a is upregulated by tgf-a on planar substrates. these findings suggest that mechanical tension and tgf-a are important factors in the regulation of a ß that need to be to taken into consideration when evaluating the role of a ß in wound healing and fibrotic disorders. ( ), n vergnolle ( ), p andrade-gordon ( ) ( ) inflammation research network, university of calgary, canada ( ) rw johnson pharmaceutical research institute, canada the objective of this study was to investigate the effects of par deficiency in various models of colonic inflammation in order to elucidate the role of endogenous par in the process of inflammation in the gut.colonic inflammation in c bl wildtype and par -/-mice was induced by treatment with . % dss (in drinking water) or tnbs ( mg or mg in ul of % ethanol, single intracolonic injection) or pre-sensitizing mice with % oxazolone (in olive oil) applied to the skin of the abdomen, and days later, a single intracolonic injection of % oxazolone (dissolved in % ethanol).intravital microscopy was performed, days (tnbs/dss) or days (oxazolone) after induction of colitis on the colonic venules to assess changes in leukocyte rolling, adhesion and vessel diameter.lastly, various parameters of inflammation were assessed following the intravital microscopy.par -/-mice showed significantly lower leukocyte adherence and vessel dilation compared to the wildtype mice in dss, and tnbs challenge. in all three challenges, mpo activity, macroscopic damage score and bowel thickness were significantly higher in wild-type mice, compared to par -/-.our evidences indicate that deficiency in par attenuates inflammatory responses in the experimental models of colitis associated with either th (tnbs/dss) or th (oxazolone) cytokine profile.therefore, par deficiency in the gut exerts antiinflammatory properties that are independent of th or th cytokine profile.the present study further highlights par as a potential target for inflammatory bowel diseases. ( ), n vergnolle ( ), p andrade-gordon ( ) ( ) inflammation research network, university of calgary, canada ( ) rw johnson pharmaceutical research institute, canada in a previous study, inflammatory responses induced by three different models of colitis (tnbs/dss/oxazolone) were significantly attenuated in mice deficient for par (par -/-). among the inflammatory parameters observed, infiltration of granulocytes to the colon was consistently reduced by par deficiency. aim of this study was to assess the effects of par deficiency (via par -/-mice) on the recruitment of leukocyte in colonic venules. in anaesthetized animals, leukocyte rolling/ adherence and vasodilation were induced, by topical administration of fmlp ( mm) or paf ( nm) or by intraperitoneal injection of tnf-a; ( . mg -given hours before the intravital microscopy). using intravital microscopy, we evaluated the ability of various leukocyte stimuli to induce leukocyte trafficking and vasodilation in colonic venules of par -/-versus par +/+ mice. fmlp and paf as well as tnf-a; induced significant vasodilation and an increase in rolling/adhesion of leukocytes in mouse colonic venules. par -/-mice showed significantly lower leukocyte rolling compared to the wildtype mice in response to fmlp topical administration. leukocyte adherence induced by fmlp and tnf-a; was significantly lower in par -/-mice compared to wild types as well. no difference was observed between par -/-and wildtype for leukocyte rolling/adherence-induced by paf. the lack of functional par attenuated leukocyte trafficking in response to fmlp and tnf-a; but not to paf. the involvement of par activation in mouse colon leukocyte trafficking highlights par as an important mediator of inflammatory cell recruitment and thereby a potential target for the treatment of inflammatory bowel diseases. ( ), kk hansen( ), k chapman( ), n vergnolle ( ), ep diamandis ( ), md hollenberg ( ) ( ) advanced center for detection of cancer, mount sinai hospital, university of toronto, toronto, on, canada ( ) proteinases and inflammation network, university of calgary, calgary, ab, canada kallikreins (klks) are secreted serine proteinases identified in many cancers and multiple sclerosis lesions. we have recently shown that klks can activate proteinaseactivated receptors (pars), a family of g-protein coupled receptors associated with inflammation. we hypothesized that like trypsin, kallikreins can trigger inflammation via the pars. we studied the ability of klks and to activate pars , and in vitro and to cause oedema in a mouse model of paw inflammation in vivo. we found that klk is able to activate both of pars and and to prevent thrombin from activating par . on the other hand, klk was a specific activator of par . kallikrein administration in vivo resulted in a paw oedema response comparable in magnitude and time to that generated by trypsin. the oedema was accompanied by a decreased threshold of mechanical and thermal nociception. our data demonstrate that by activating pars and and by inactivating par , kallikreins, like klks and , may play a role in regulating the inflammatory response and perception of pain. ( ), d park ( ), b short( ), n brouard( ), p simmons( ), s graves ( ), j hamilton ( ) ( ) melbourne university, melbourne, victoria, ( ) peter maccallum cancer institute. melbourne, australia mouse mesenchymal stem cell enriched populations can be isolated from bone tissue by employing lineage immuno-depletion followed by fluorescence-activated cell sorting based on the cell surface expression of the sca- antigen. such isolated cells can subsequently be cultured and differentiate towards the osteogenic, adipogenic or chondrogenic linage in vitro. using this model we investigated the influence of the proinflammatory cyto-kines, tnfa or il- b, on early osteogenesis in vitro. under osteogenic conditions, il- b was found to inhibit cell proliferation in a dose dependent manner, whereas tnfa exhibited no effect. histochemical examination revealed the presence of either tnfa or il- b to dramatically decreased mineralization in a dose dependent manner. q-pcr analysis indicated that in the presence of il- b, despite increased expression of bone-specific alkaline phosphatase (akp ) mrna, levels of other osteogenesis markers (runx , col a and sp ) were decreased. in the presence of tnfa, levels of akp , runx and sp were all decreased. our findings indicate that the influence of early mesenchymal progenitor cells on bone remodelling may be substantially altered in the presence of proinflammatory cytokines. using are-driven and nf-kb-targeted reporter genes, transfection of the nf-kb p subunit and nrf into hepg or other cells, as well as sirna technique to knockdown endogenous p in cells, we found that nf-kb p subunit repressed the anti-inflammatory and anticarcinogenetic nrf -are pathway at transcriptional level. in p -overexpressed cells, the are-dependent expression of heme oxygenase- was strongly repressed. in the cells where nf-kb and nrf were simultaneously activated, p unidirectionally antagonized nrf transcriptional activity. the p -mediated are inhibition was independent of the transcriptional and dna-binding activities of p . co-transfection and rna interference experiments revealed two mechanisms which coordinate the p -mediated repression of are: ( ) p selectively deprives creb binding protein (cbp) from nrf , but not mafk, by competitive interaction with the ch -kix domain of cbp, resulting in inactivation of nrf transactivation domain and concomitant abrogation of the nrf -stimulated coactivator activity of cbp; ( ) p promotes recruitment of histone deacetylases (hdac ) to are by enhancing the interaction of hdac with either cbp or mafk, leading to inactivation of cbp and deacetylation of mafk. this study may establish a novel pro-inflammatory and pro-carcinogenic model for the transrepression of the are-dependent gene expression by p subunit. since various inflammatory and tumor tissues constitutively overexpresses p in their nuclei, the finding in this study implies a strong repression of are-dependent gene expression must take place in those tissues. in this regard, the findings in this study may help to explain why oxidative stresses and toxic insults usually occur in those pathological loci. dendritic cells (dc) play a pivotal role in the induction of immune response and tolerance. it is less known that dc accumulate in atherosclerotic arteries, where they might activate t-cells and contribute to the progression of disease. the serine protease thrombin is the main effector protease of the coagulation cascade. thrombin is also generated at sites of vascular injury and during inflammation. hence, thrombin generation is observed within atherosclerotic and other inflammatory lesions including rheumatoid arthitis. thrombin activates various cells via protease-activated receptors (pars). immature dc do not express pars. upon maturation with lps, tnfalpha, or cd l, only lps-matured dc expressed par and par on their surface. stimulation of dc with thrombin, par -or par -activating peptides elicited actin polymerization and concentration-dependent chemotactic responses in lps-, but not in tnf-alphamatured dc. the thrombin-induced migration was a true chemotaxis as assessed by checkerboard analysis. stimulation of pars with thrombin or respective receptoractivating peptides led to activation of erk / and rho kinase i (rock-i) as well as subsequent phosphorylation of the regulatory myosin light chain (mlc ). the erk / -and rock-i-mediated phosphorylation of mlc was indispensable for the par-mediated chemotaxis as shown by use of pharmacological inhibitors of rock, erk and mlc kinases. in addition, thrombin significantly increased the ability of mature dc to activate proliferation of naive t-lymphocytes in mixed leukocyte reactions. in conclusion, our work demonstrates expression of functionally active thrombin receptors on lps-matured dc. we identified thrombin as a potent chemoattractant for mature dc, acting via rho/ erk-signaling pathways. data concerning the role of circulating modified low density lipoproteins (modldl) in atherogenesis and other pathologies are scarce. one reason for this is the lack of suitable radiolabeling methods for direct assessment of metabolic pathways of modldl in vivo. we report a novel approach for specific labeling of human native ldl (nldl) and modldl (iron-, hypochloriteand myeloperoxidase-oxidized, nitrated, glycated, and homocysteinylated ldl) with the positron emitter fluorine- ( f) by either nh -reactive n-succinimidyl- -[ f]fluorobenzoate or sh-reactive n-[ -( -[ f]fluorobenzylidene)-aminooxyhexyl]maleimide (radiochemical yields, - %; specific radioactivity, - gbq/ mmol). radiolabeling itself caused neither additional oxidative structural modifications of ldl lipids and proteins nor adverse alterations of their biological activity and functionality in vitro. the approach was evaluated with respect to binding and uptake of f-nldl and f-modldl in cells overexpressing various lipoproteinrecognizing receptors. the metabolic fate of f-nldl and f-modldl in vivo was delineated by dynamic small animal pet studies in rats and mice. the in vivo distribution and kinetics of nldl and modldl correlated well with the anatomical localization and functional expression of ldl receptors, scavenger receptors, and receptors for advanced glycation end products. the study shows that ldl modification, depending on type and extent of modification, in part or fully blocks binding to the ldl receptor, and reroutes the modldl to tissuespecific disease associated pathways. in this line, flabeling of modldl and the use of small animal pet provide a valuable tool for imaging and functional characterization of these pathways and specific sites of pathologic processes, including inflammatory processes, in animal models in vivo. the p mapk signaling pathway, which regulates the activity of different transcriptions factors including nuclear factor-Þb (nf-Þb), is activated in lesional psoriatic skin. the purpose of the present study was to investigate the effect of fumaric acid esters on the p mapk and the down stream kinases msk and in cultured human keratinocytes. cell cultures were incubated with dimethylfumarate (dmf), methylhydrogenfumarate (mhf) or fumaric acid (fa) and then stimulated with il- b before kinase activation was determined by western blotting. a significant inhibition of both msk and activations was seen after pre-incubation with dmf and stimulation with il- b whereas mhf and fa had no effect. also, dmf decreased phosphorylation of nf-kb / p (ser ), which is known to be transactivated by msk . furthermore, incubation with dmf before stimulation with il- b resulted in a significant decrease in nf-kb binding to the il- kb and the il- kb binding sites as well as a subsequent decrease in il- and il- mrna expression. our results suggest that dmf specifically inhibits msk and activations and subsequently inhibits nf-kb induced gene-transcriptions which are believed to be important in the pathogenesis of psoriasis. these effects of dmf explain the anti-psoriatic effect of fumaric acid esters. a humanized model of psoriasis was successfully established by transplanting non-lesional skin biopsies from psoriasis patients onto bg-nu-xid mice lacking b, t and nk cells. in this system, a psoriatic process is triggered by intradermal injection of activated autologous peripheral blood lymphocytes. inflammation is associated with the expression of activation markers and inflammatory medi-ators such as tnf-alpha, hla-dr and cd a and this results in increased proliferation and differentiation of keratinocytes, demonstrated by increased expression of ki- and ck- . epidermal hyperplasia is a typical readout in this model. in a series of studies, this model was found to be sensitive too a wide range of compounds, including inhibitors of tnf-alpha, antibodies directed against growth factors, mmp-inhibitors, calcipotriol, metothrexate, betamethasone and cyclosporine a.in addition, we showed that inhibition of fatty acid oxidation had an anti-psoriatic effect in this model (caspary et al. brit j dermatol ; , - ) . employing lesional skin it was demonstrated that inhibition can also be performed in a therapeutic setting.due to its humanized nature this model represents a powerful tool for the identification or validation of compounds with potential for the treatment of psoriasis. kristian otkjaer ( ), e hasselager( ), j clausen( ), l iversen ( ), k kragballe ( ) ( ) aarhus university hospital, denmark ( ) novo nordisk a/s, denmark interleukin- (il- ) is assumed to be a key cytokine in the pathogenesis of psoriasis. increased levels of il- are present in lesional psoriatic skin compared with nonlesional skin where it is barely detectable. whether il- is derived from antigen-presenting cells or keratinocytes remains unsolved. the aim of the present study was, therefore, to characterize il- expression in non-lesional psoriatic skin ex vivo. mm punch biopsies from nonlesional psoriatic skin were collected. biopsies were transferred to cacl enriched keratinocyte basal media and cultured with vehicle or il- beta ( ng/ml) for , , , , , and hours, respectively. the samples were analyzed by in situ hybridisation, qrt-pcr, immunofluorescent staining and elisa. incubation with il- beta rapidly induced il- mrna expression in the biopsies. the highest level of il- mrna was detected after hours and in situ hybridisation revealed that basal as well as suprabasal keratinocytes throughout the epidermis were the only cellular source of il- mrna. increased levels of il- protein were detected in the supernatant of the il- beta stimulated biopsies. immunofluorescent staining of the biopsies showed no il- protein in the keratinocytes, whereas the il- protein was present in epidermal cd a positive dendritic cells. our data emphasize the keratinocyte as the cellular source of il- expression in human skin. interestingly, immunofluorescent staining of our cultured biopsies showed il- protein in epidermal dendritic cells whereas no il- was detected in the keratinocytes. this indicates that epidermal dendritic cells are the target for keratinocytederived il- . one response of epidermal keratinocytes to inflammatory stress is the induction of matrix metalloproteinases (mmps) that participate in tissue remodeling. excessive proteolytic activity is associated with chronic wounds and tissue damage during persistent inflammation. calcitriol, the hormonally active form of vitamin d, is known to have beneficial effects during cutaneous inflammation. we hypothesized that one way in which calcitriol exerts its effect on inflamed skin is by attenuation of damages caused by excessive mmp proteolytic activity. our experimental model consists of hacat keratinocytes cultured with tnf to simulate an inflammatory state. pro-mmp- was quantified by gelatin zymography and mrna by real-time pcr. the levels and activation of signaling proteins were determined by immunoblotting. the increase in pro-mmp- activity and mrna levels induced by tnf was inhibited by~ % following h treatment with calcitriol. using specific inhibitors we established that the induction of mmp- was dependent upon the erk pathway, while p -mapk and pkc inhibited, and jun-kinase, pi- -kinase and src did not affect it. levels of c-fos, a component of ap- transcription complex known to mediate mmp- induction, were elevated by tnf and further increased by calcitriol. the induction of mmp- by tnf was abolished by inhibition of the egfr tyrosine kinase attesting to the requirement for egfr trans-activation. calcitriol also inhibited the induction of mmp- by egf. we conclude that calcitriol inhibits the induction of mmp- gene expression by tnf in keratinocytes by affecting an event downstream to the convergence of the egfr and the tnf signaling pathways. ( ), p verzaal ( ), t lagerweij ( ), c persoon-deen ( ), l havekes ( ), a oranje ( ) ( ) tno pharma, department of inflammatory and degenerative diseases, leiden, the netherlands ( ) erasmus medical center, department dermatology and venereology, rotterdam, the netherlands mice with transgenic overexpression of human apolipoprotein c in liver and skin display a strongly disturbed lipid metabolism. moreover, these mice show a loss of skin-barrier function evident from increased trans epidermal water loss. these mice develop symptoms of atopic dermatitis, i.e. scaling, lichenification, papules, excoriation and pruritus. both hyperplasia of epidermis and dermis are observed. histological analysis shows increased numbers of cd + t cells, eosinophils, mast cells and ige-positive cells in the dermis. serum levels of ige are increased as well. cytokine profiling of draining lymph nodes is in favor of a th -mediated disease. development of atopic dermatis in this model was found to be sensitive to topical treatment with triamcinoloneacetonide, fluticasone-proprionate and tacrolimus. moreover, oral treatment with dexamethasone successfully inhibits the development of disease in this model. impairment of the skin barrier is most likely the underlying cause of the development of atopic dermatitis in this model.this model is useful for identifying new therapeutic strategies and obtaining new insight into the pathogenesis of atopic dermatitis. topical immunosupppressants such as elidel and protopic are highly efficacious therapeutics for the treatment of atopic dermatitis and other dermatological conditions.-when delivered topically, these calcinuerin inhibitors offer several advantages over topical steroids; however, these marketed drugs have received a controversial "black box warning" because of a potential cancer risk. we speculated that systemic exposure of these drugs over long term use may contribute to the cancer risk.accordingly, we have designed and discovered a series of "soft" cyclosporin a (csa) derivatives as potentially safer alternatives.in general, soft drugs are engineered, via medicinal chemistry, to be effective upon local delivery but upon systemic exposure they are rapidly inactivated by metabolic pathways.in this way, exposure of active drug to distal organs is greatly minimized resulting in a significant enhancement in therapeutic index.the results or our drug discovery efforts around soft csa derivatives will be presented. ( ), y sawanobori ( ), u bang-olsen ( ), c vestergaard( ), c grønhøj-larsen ( ) background: a strain of japanese fancy-mice, nc/nga, serves as a model for atopic dermatitis. under specific pathogen-free conditions, the mice remain healthy, but when kept under non-sterile conditions, they exhibit pruritic lesions like atopic dermatitis. scratching behaviour of the mice precedes the development of dermatitis, and a correlation between registered scratching counts and expression of il- mrna has been shown. also, transgenic mice over-expressing il- exhibit increased scratching behaviour and develop severe dermatitis. consequently we decided to explore the therapeutic effect of an anti il- antibody on scratching behaviour and dermatitis in nc/nga mice. methods: prior to clinical manifestation of dermatitis, we commenced treatment of nc/nga mice with il- ratanti-mouse mg/kg intraperitoneally every fifth day for seven weeks. clinical dermatitis, scratching behaviour and weight gain, was assessed throughout the intervention period. serum analysis for ige and il- as well as histopathological and immunohistochemistry analysis on skin biopsies were also performed at end-point. results: taken over the entire intervention period, treatment with anti il- antibody in nc/nga mice from age seven weeks did not meet the primary end points, which were scratch, dermatitis and body weight. however, post hoc analysis revealed a significant reduc-tion of scratch by the anti il- antibody treatment in the time interval day - . our results suggest an anti pruritic role for il- antibody in an atopic dermatitis-like animal model. anti il- antibody is therefore a new therapeutic opportunity for the treatment of pruritus in atopic dermatitis and perhaps other pruritic diseases. ( ), p ferro ( ), hm asnagli ( ), v ardissone ( ), t ruckle ( ), f altruda ( ), ch ladel ( ) ( ) rbm merck serono/university of torino, italy ( ) merck serono pharmaceutical research institute, geneva, switzerland ( ) university of torino, dipartimento di genetica, biologia, biochimica, italy class-i phosphoinositide -kinases (pi ks) play a critical role in modulating innate and adaptive immune responses, as they are important transducers of external stimuli to cells, such as granulocytes and lymphocytes. since pi k-g plays a pivotal role in mediating leukocyte chemotaxis and activation, as well as mast cell degranulation, the pharmacological blockade of pi k-g might offer an innovative rationale-based therapeutic strategy for inflammatory skin disorders. in our study the inhibitory properties of a selective pi k-g inhibitor as on inflammation was applied to murine models modeling skin diseases like psoriasis and dermatitis. two mouse models were used: the first, irritant contact dermatitis (icd), is an innate inflammatory skin condition arising from the release of pro-inflammatory cytokines in response to haptens, usually chemicals. the second, contact hypersensitivity (chs) is a t-cell dependent model, modeling in part t-cell-mediated skin diseases such as psoriasis. we demonstrated the therapeutic effect of pi kg inhibition and subsequent inhibition of chemotaxis in models of skin diseases and showed that a selective pi k-g inhibitor can excert an important therapeutic efficacy (dose-dependent) in models of innate immunity (icd) -effective dose , mg/kg p.o. once -as well as in t-cell mediated skin pathology (chs)effective dose mg/kg p.o. bid. we conclude that the mechanism of action related to inhibition of pi k-g are demonstrable after oral administration of selective inhibitors like as in models of acute and chronic skin inflammation and are mediated by modulation of innate and acquired immunity. introduction: high mobility group box (hmgb ) has recently been identified as a late mediator of endotoxin lethality. we newly developed an extra corporeal hmgb absorber. the purpose of this study was to test the hypothesis that hmgb removal could prevent or reduce endotoxin induced lethality or tissue injury of rats. methods: all experiments were conducted in accordance with the institutional care and use committee.male wistar rats were randomly allocated into three groups; hmgb absorber group (group i),hmgb nonabsorber group (group ii), and vacant column group (group iii).we applied these columns for each groups at hours after lps injection. the rats were sacrificed hours after lps injection for pulmonary histology. we statistically analyzed survival rate with kaplan-meier and the levels of hmgb with anova. results: survival rate was % in the group i at hours after lps injection, as compared with % in the group ii and % in the group iii. the pulmonary histology in both group ii and group iii showed acute inflammatory injuries, whereas group i showed less inflammatory changes.the level of hmgb in the group i was significantly lower than those of group ii and iii. discussions:these results demonstratethat specific absorption of endogenous hmgb therapeutically reverses lethality of established sepsis indicating that hmgb inhibitors and absorber can be treated in a clinically relevant therapeutic window that is significantly wider than for other known cytokines. contact information: dr hideo iwasaka, oita university, anesthesiology and intensive care unit, yufu city, japan e-mail: hiwasaka@med.oita-u.ac.jp ( ) ( ) department of pharmacology, national university of singapore, singapore ( ) dso national laboratories, singapore hydrogen sulfide (h s) is increasingly recognized as a proinflammatory mediator in various inflammatory conditions. in this study, we have investigated the role of h s in regulating expression of some endothelial adhesion molecules and migration of leukocytes to inflamed sites in sepsis. male swiss mice were subjected to cecal ligation and puncture (clp) induced sepsis and treated with saline, dl-propargylglycine (pag, mg/kg i.p.), an inhibitor of h s formation or sodium hydrogen sulfide (nahs) ( mg/kg, i.p.), a h s donor. pag was administered either hour before or hour after induction of sepsis while nahs was given at the time of clp. using intravital microcopy, we found that in sepsis, prophylactic and therapeutic administration of pag significantly reduced the leukocyte rolling and adherence in mesenteric venules coupled with decreased mrna and protein levels of adhesion molecules (icam- , p-selectin and e-selectin) in lung and liver. in contrast, injection of nahs significantly upregulated leukocyte rolling and attachment as well as tissue levels of adhesion molecules in sepsis. on the other hand, normal mice were given nahs ( mg/kg i.p.) to induce lung inflammation with or without pretreatment of nf-x×b inhibitor, bay - . h s treatment enhanced the pulmonary level of adhesion molecules and neutrophil infiltration in lung. these alterations were reversed by pretreatment with bay - . moreover, expression of cxcr in neutrophils obtained from h s treated mice was significantly upregulated leading to an obvious elevation in mip- directed migration of neutrophils. therefore, h s act as an important endogenous regulator of leukocyte trafficking during inflammatory response. transient receptor potential vanilloid (trpv ) is primarily found on sensory nerves. we have demonstrated its pro-inflammatory potential in arthritis and now present evidence that it is protective in an endotoxininduced model of sepsis. selective trpv antagonists are not available for use in the mouse in vivo, thus established trpv knockout (-/-) mice were used. c bl wt and trpv -/-mice were matched for age and sex and injected intraperitoneally (i.p.) with lipopolysaccharide (lps). the response was monitored for h. blood pressure, measured before and at intervals after lps in conscious mice via a tail cuff was reduced in both wt and trpv -/-mice, with trpv -/-mice showing an enhanced drop at h. in a separate group temperature, a proposed pre-mortality marker was also reduced by h, again with a significantly increased drop in trpv ko. furthermore higher levels of two inflammatory markers tnfa and nitrite (as an indicator of no) were measured in peritoneal lavage and higher levels measured intrpv -/-as compared with wt samples. finally aspartate aminotransferase (ast) levels also enhanced in trpv -/-versus wt mice, although markers for kidney and pancreatic damage were similar in both genotypes. we conclude that trpv plays a protective role in sepsis. trpv is known to be present on nonneuronal (e.g. vascular components) and their relative involvement in sepsis is unknown. ( ), da souza-junior( ), l de paula ( ), mc jamur ( ), c oliver ( ), sg ramos ( ), cl silva ( ), lucia helena faccioli ( ) ( h rv) . infected balb/c mice developed an acute pulmonary inflammation and higher levels of tnf-a, il- , kc, mcp- and mip- were detected in the lungs by day . in vivo degranulation of mast cells by c / led to a reduction of the inflammatory reaction associated to a marked decline proinflammatory cytokine and chemokine levels in the lungs. the magnitude of cellular immune response was also partially impaired in infected mice and treated with c / . histologically, the exacerbated granulomatous inflammation shown in the lung parenchyma of infected mice was attenuated in infected mice and treated with c / . of interest, the number of mycobacterial bacilli recovered from the lungs was log higher after treatment of infected mice with c / . these findings suggest that mcs participate in host defense against m. tuberculosis infection through of the modulation of cytokines and chemokines, which are important for the recruitment and activation of inflammatory cells. ( ), ms chadfield ( ), db sørensen ( ), h offenberg ( ), m bisgaard ( ), he jensen ( ) ( ) department of veterinary pathobiology, faculty of life sciences, university of copenhagen, denmark ( ) novo nordisk a/s, cell biology, gentofte, denmark introduction: pasteurella multocida is an important cause of pneumonia in several animal species and may spread systemically. the aim of this study was to evaluate initial inflammatory reactions and the inocula effect due to strains of p. multocida of different origin in an aerogenous murine model. materials and methods: female balb/ c-j mice (app. g, taconic, denmark) were infected intranasally with two clinical isolates of p. multocida of avian (vp ) and porcine (p ) origin, at three different levels of inocula concentration. after euthanasia, specimens of lung and liver tissue were collected for bacteriological and histopathological evaluation. furthermore, lung tissue samples were taken for measurement of expression of metalloproteinase mmp- and metalloproteinase inhibitor timp- . results: all mice infected with the avian strain were euthanized after hours. viable counts recovered from lung and liver tissue were high, and histopathology revealed pronounced acute bronchopneumonia. in the liver, disseminated necrosis with formation of microabscess was also seen. on the contrary, a dose response was observed with the porcine strain with regard to recovery of viable counts and development of lesions was apparent after , and h. furthermore, differences were seen in the nature of the lesions caused by the two strains. there was a difference in expression of mmp- and timp- between infected and noninfected mice. the model proved suitable for the evaluation of pulmonary inflammatory reactions between the two different host-derived strains as demonstrated through viable counts, histopathology and expression of mmp- and timp- . ( ), r molinaro ( ), a franÅa ( ), m bozza ( ), f cunha( ), s kunkel ( ) ( ) universidade do rio de janeiro, brazil ( ) universidade de s¼o paulo, brazil ( ) university of michigan, usa introduction and objectives: studies reveal that regulatory t (treg) cells control immune responses; therefore these responses must be controlled to enable the effective protection against infections and cancer. ccr knockout mice (ccr -/-) are more resistant to lps shock. so, our aim is to study the mechanisms involved in the resistance of ccr -/-subjected to severe sepsis by cecal ligation and puncture (clp) and how tregs modulate this effect. results: c /bl mice were subjected to clp model, whereby the cecum was partially ligated and puncture nine times with a g needle. sham-operated mice were used as control. mice subjected to clp and sham surgery were treated with antibiotic since h after and until days. ccr -/-mice subjected to clp presented an increase in survival rate ( %) compared with wildtype mice ( %), and a marked improvement in the innate response concern to neutrophil migration to peritoneum and lung, bacteria load and cytokine levels compared to wild-type mice. besides, tregs from ccr -/-clp mice did not inhibit proliferation of t effector cells as observed for treg from wild clp mice, at a proportional ratio of teffector:treg. interesting, treg from ccr -/-clp mice did not inhibit neutrophil migration to bal when co-injected with fungal challenge as secondary infection, while the treg from wild clp mice did, as expected. conclusions: these results suggest that treg cells from ccr -/-mice did not present suppressive response and it could be an important factor in their survival. inflammation and oxidative stress are known to be one of the important causes that are responsible for many diseases. inflammation has been associated with diseases like cancer, diabetes and many other. proinflammatory cytokine (tnf -µ and il- â) and no are considered as pivotal mediators in inflammatory conditions like rheumatoid arthritis, sepsis and cancer etc. thus inhibition of pro-inflammatory cytokines and no production are important targets for treatment of inflammatory disorders. nowadays due to the emerging side effects of cox inhibitors, these targets have been paid more attention for the treatment of these conditions. some medicinal plants such as curcuma longa ( ), commiphora mukul ( ) in humoral memory, antibodies secreted into serum and other body fluids protect an individual against repeated challenges of previously encountered pathogens. antibody-secreting plasmacells are mostly considered to be shortlived, terminally differentiated b lymphocytes, eliminated after a few days or weeks by apoptosis. however, in secondary lymphoid organs and in the bone marrow, plasma cells can survive for months and years, without dna-synthesis and refractory to signals from antigen or antigen-antibody complexes. the lifetime of these longlived plasmacells depends on an intrinsic competence to survive in the distinct environment of those organs, which defines a specific survival niche. the niche provides survival signals like il- , cxcl and tnf. within a functional niche, the lifetime of a plasma cell is apparently not limited intrinsically. the number of niches in the body has to be limited, in order to maintain physiological concentrations of serum immunoglobulins. thus recruitment of new plasmacells to the pool of old memory plasma cells has to be competetive. this competition is probably controlled by a simple molecular mechanism, namely the dual functionality of chemokines like cxcl , which attract newly generated plasmablasts to a survival niche and at the same time are a survival signal for the plasma cell. plasmablasts and plasma cells express cxcr , the receptor for cxcl . while plasmablasts migrate in response to cxcl , plasma cells depend on it for survival in the niche, but are no longer migratory. thus once disloged from their niche, they will die. plasmablasts newly generated upon systemic secondary immunization, upon concommittant stimulation with interferon-gamma, can also express cxcr , the receptor for the interferon-gamma-induced chemokines cxcl , and , which may lure the plasmablasts into inflamed tissue. the switch in the potential to migrate provides also an efficient means to eliminate plasma cells of the peak of an immune response, which as plasmablasts had migrated to the tissue inflamed in that pathogenic challenge. inflamed tissue contains survival niches for plasma cells. in the inflamed tissue, plasma cells provide high local antibody concentrations while the tissue is inflamed. upon resolution of the inflammation the plasma cells will be dislodged and die. longlived plasma cells provide longlasting antibody titers (protective memory) and leave memory b cells a role in reactive memory, generating memoryplasmacells in secondary challenges, and if serum titers are not sufficient to protect. in chronic inflammation, this mechanism can contribute to pathogenesis. thus in the nzb/w model of lupus, longlived autoreactive plasma cells are generated early in pathogenesis, which survive in bone marrow and spleen. later, in established disease, autoreactive plasma cells are shortlived and continuously generated. they do not compete with the longlived plasma cells, and both populations coexist as prominent populations. interestingly, longlived plasmacells are resistant to therapeutic immunosuppression, while the generation of shortlived plasma cells is blocked. this may be the reason for the failure to cure antibodymediated immunopathology, e.g. in autoimmunity and allergy, by conventional immunosuppression, ( ), h lee ( ) c a is a potent inflammatory mediator produced during complement activation. unregulated c a signalling through its receptor (c ar) on neutrophils and other leukocytes is implicated in the pathogenesis of autoimmune diseases including rheumatoid arthritis and systemic lupus erythematosus. considerable effort has gone into development of c ar antagonists for human therapy. we took neutrophils from genetically modified human c ar knock-in mice in which the mouse c ar coding region was replaced with human c ar sequences and immunized wild-type mice to generate high affinity antagonist monoclonal antibodies (mabs) to human c ar. these mabs inhibit c a-induced neutrophil migration and calcium-flux, and bind to a region of the nd extracellular loop of c ar loop that seems to be critical for receptor activity. this study investigated the effectiveness of these mabs in the k/bxn serum-transfer model of inflammatory arthritis. human c ar knock-in mice were given - mg/kg mab intraperitoneally, before or after inflammatory arthritis developed. mice treated with anti-c ar mab one day before serum transfer did not develop swelling or clinical signs of arthritis in contrast to controls. histopathology of the joints in anti-c ar mab-treated mice revealed a complete block of the massive influx of leukocytes and cartilage erosion seen in controls. furthermore, and most significantly, a single mg/kg dose of anti-c ar mab given days after initiation of disease completely reversed inflammation. in the collagen-induced arthritis (cia) model, injection of anti-c ar mab after development of inflammation also reversed inflammation to baseline. these potent new antibodies to human c ar are in preclinical development. the cytokine macrophage migration inhibitory factor (mif) participates in fundamental events in innate and adaptive immunity. the profile of activities of mif in vivo and in vitro is strongly suggestive of a role for mif in the pathogenesis of many inflammatory diseases, including rheumatoid arthritis (ra), asthma, and sepsis. mif also has a unique relationship with glucocorticoids, in that despite antagonizing their effects, the expression of mif is in fact induced by glucocorticoids. thus, mif functions as a physiological counter-regulator of the anti-inflammatory effects of glucocorticoids. therapeutic mif antagonist may therefore provide a specific means of steroid sparing. since mif are highly conserved among different species, it is hard to develop high affinity antibodies due to immune tolerance.we developed a proprietary technique to break the immune tolerance and selected high affinity mouse monoclonal antibodies against mif.the antibody can neutralize mif activity in cell based assays, and is very effective in a lps induced mouse sepsis model. using this antibody as a tool, we are studying the function of mif in comparison with the function of lps.we found that lps induced inos expression and no secretion are dependent on the secretion of mif.we also found that although both lps and mif induce g arrest in macrophage cell line raw . , their functions are independent to each other. structure-based small molecule drug design. an effective agent would be the first orally active cytokine antagonist. methods: collagen-induced arthritis (cia) was induced in dba- mice by immunisation with bovine type ii collagen/adjuvant on day and . cor , synthesized on the basis of computer modeling of mif protein x-ray crystallographic data, was administered by daily oral gavage from day . etanercept ( mg/kg ip q d) was used as a positive control. the mek-erk pathway is activated in numerous inflammatory conditions, including ra, ibd and copd. arry- is a potent (ic = nm), selective, atp-uncompetitive mek / inhibitor.arry- is highly efficacious in cia and aia rat models, with ed s of and mg/kg, respectively, equal to or better than standard agents. addition of arry- to methotrexate, etanercept, ibuprofen or dexamethasone regimens in these models results in improved efficacy that is at least additive, if not synergistic. in tpa-stimulated human whole blood, this compound inhibited tnf, il- and il- production (ic s of , and nm, respectively). in contrast, inhibition of perk required nm to achieve a % reduction, demonstrating that inhibition of pro-inflammatory processes is very sensitive to perk inhibition.in clinical studies, healthy volunteers were administered a single oral dose of , , , or mg); blood was drawn at various times after dosing and stimulated ex vivo with tpa. arry- was well-tolerated and drug exposure was dose-proportional. in ex vivo blood samples, there was a dramatic time-and concentration-dependent inhibition of tpainduced il and tnffz with > % inhibition observed at plasma concentrations of and ng/ml, respectively. similar inhibition of perk required ng/ml. a multiple ascending dose clinical study has confirmed the pharmacokinetics and pharmacodynamics of arry- and helped define tolerability. clinical evaluation of arry- in combination with methotrexate in patients with rheumatoid arthritis is on-going. p (a mitogen-activated protein kinase) has been shown to play a key role in the release of cytokines such as tnfand il- a from monocytes in signaling cascades that are initiated due to extra cellular stress stimuli. inhibition of p activity is expected to regulate the levels of tnf-a and il- b thereby alleviating the effects of inflammation in ra. a new class of p inhibitors based on the naphthyridininone scaffold have been discovered. x-ray crystallography and site directed mutagenesis studies were critical tools that aided the evolution of the naphthyridinone lead class starting from a pyrido-pyrimidinone template. this presentation will discuss the derivation of key benchmark pre-clinical candidates in these novel scaffold classes (shown below) as influenced by structural biology studies, mutagenesis data and molecular modelling. efficacy studies in animal models for benchmark compounds will also be presented. ( ), h aaes ( ), w-h boehncke( ), j pfeffer( ), t skak-nielsen( ), i teige ( ), k abell ( ), ph kvist ( ), e ottosen ( ), tk petersen ( ), lars svensson ( ) ( ) discovery, leo pharma, industriparken, ballerup, denmark ( ) department of dermatology, johann wolfgang goethe-university, frankfurt am main, germany p map kinase plays an important role in mediating an inflammatory response in mammalian cells. as a consequence of activation, several inflammatory mediators are released including il- b] and tnfa. both cytokines have a central role in the pathogenesis of inflammatory conditions such as psoriasis. approximately % of psoriasis patients develop psoriatic arthritis. leo is a member of newly developed class of selective p map kinase inhibitors. the compound was tested orally in in vivo models relevant for psoriasis and psoriatic arthritis. the in vivo models selected include the cia arthritis model, the human psoriasis xenograft scid mouse model, the uvb-induced dermatitis model, the lps induced tnfa model and a local gvh model. treatment with leo led to an amelioration of the ongoing inflammation in all investigated in vivo models. in the cia model, a clear dose response effect was observed on the developing arthritis in both rats and mice ( % reduction in mice and % in rats at mg/kg p.o.). in the humanised psoriasis model, leo at a dose of mg/kg, had an effect on both the hyperplastic epidermis (epidermal thickness reduced by %) and on the infiltrating inflammatory cells. the anti-inflammatory effect of leo was even close to the effect of systemically delivered steroids in both models. we believe that the new highly selective class of p map kinase inhibitors has a strong potential as an orally delivered therapy for systemic inflammation diseases such as psoriasis and arthritis. slx- is a potent, selective, orally bioavailable inhibitor of the rho-kinase rock- . its ic for rock- and rock- inhibition is nm and > mm respectively. the ability of slx- to inhibit septic liver injury was investigated in c bl/ j mice challenged with lipopolysaccharide (lps) and d-galactosamine (d-gal). mice were given lps ( mg/mouse) and d-gal ( mg/ mouse) i.p and . hours later were sacrificed for analysis of liver injury. mice challenged with lps/d-gal had a > fold increase in serum alt and ast levels. this increase was reduced by > % in mice pretreated with slx- either orally ( mg/kg, and hrs prechallenge) or i.p. ( - mg/kg, min pre-challenge). slx- inhibited the increase in hepatic levels of tnffalpha produced by lps/d-gal by > %. to assess the kinetics of slx- s benefit, slx- ( mg/kg i.p.) was given min prior to, or or min after the lps/ d-gal. slx- was effective at inhibiting the rise in alt and ast levels at all time points suggesting that inhibiting rock- even after the initiation of the lps/d-gal driven cascade protects against septic liver injury. in a survival study, out of mice given lps/d-gal were dead by hrs whereas in mice given slx- ( mg/kg orally) animal died at hours and the remaining mice were alive hrs later. these results show that specific inhibitors of rock- may have therapeutic utility in the treatment of sepsis and subsequent liver injury. theta through three key hydrogen bond mediated interactions.they potently inhibit protein kinase c activity in vitro as demonstrated by inhibition of il- secretion in human purified t-cells stimulated with anti-cd and anti-cd or whole blood seb challenge.the pkc-theta inhibitors are orally bioavailable and demonstrate immunosuppressive activity in a mouse model of human delayed-type hypersensitivity responses. ( ), j zhang ( ), k henley ( ), m white ( ), d hilton ( ), b kile ( ) ( to investigate pathogenesis of rheumatoid arthritis, we used mice transgenic for the uniquely human fcgam-mariia in inducible and passively transferred models of arthritis. transgenic mice developed severe ra-like disease in both model systems, indicating that the transgene played a major role in arthritis pathogenesis. disease could be reduced by the administration of either specific monoclonal antibodies to fcgammariia or small chemical entities (sce) designed to bind to the fcgam-mariia dimer. to investigate the cause of this enhanced sensitivity to auto-immune stimuli, the phagocytic capacity of transgenic mice compared to c bl/ control mice was examined using phagocytosis of fluorescent beads coated with ova or ova/anti-ova immune complex or opsonised sheep red blood cells. in both assays macrophages from fcgammariia transgenic mice showed significantly increased phagocytosis comapred with cells from control mice at hours.this difference diminished over time andwas only seen where particles were opsonised. treatment of macrophages with specific fcgammariia blocking monoclonal antibody fragments . f(ab) or iv. f(ab) reduced phagocytosis to background levels . macrophages from transgenic mice also showed significantly greater production of inflammatory cytokines tnf-alpha and il- beta when stimulated in vitro with heat aggregated immunoglobulin (hagg). moreover, this response was also blocked by specific fcgammariia monoclonal antibody fragments or sce. thus, expression of the fcgammariia transgene in these mice leads to increased uptake of and reactivity to immune complexes, resulting in enhancement of inflammatory sequelae in the form of increased th cytokine secretion andamplification of the pro-inflammatory response leading to arthritic disease. harald burkhardt ( ), u hüffmeier ( ), i kçnig ( ), j lacorz ( ), a reis ( ), k reich ( ) ( ) johann wolfgang goethe university, frankfurt, germany ( ) friedrich-alexander-unisversity of erlangen-nuremberg, germany results: whereas the earlier described strong association of allele tnf*- a with psoriasis could be confirmed, our study revealed that this association was completely dependent on carrying the psors risk allele. for psa, but not psoriasis vulgaris without joint involvement strong association with the allele tnf*- t was detected (or= . , % ci . - . ; pcorr= . ) also in patients negative for the psors risk allele. our results indicate genetic differences between psoriasis vulgaris patients with and without joint manifestation. while the previously reported association between tnf*- a and psoriasis seems to primarily reflect ld with psors , tnf*- t may represent a risk factor for psa independent of psors . ( ), n modi ( ), m stanford ( ), e kondeatis ( ), r vaughan ( ), f fortune ( ), w madanat ( ), c kanawati( ), p murray ( ) methods: dna was obtained from patients with bd, from the uk and from the middle east (me), and controls individuals, from the uk and from me. dna was prepared by proteinase k digestion, and salt extraction and - and snp were detected by a pcr-ssp. results: there was no significant difference in expression of - c/t or a/g when all bd patients were compared to all control individuals, (p= . and . , respectively). as we have previously shown differences in snp expression in different patient groups we tested the uk and me patients separately. however, there was no significant difference in either snp in uk bd patients, (p= . , p= . ) or me bd patients (p= . , p= . ) when compared to the appropriate controls. ( ), da brown ( ), h johnen ( ), mr qiu ( ), t kuffner ( ), pgm curmi ( ), l brown ( ), m mazzanti ( ), sn breit ( ) ( introduction: cerebral palsy (cp) is a nonprogressive motor disorder caused by white matter damage in the developing brain. it is often accompanied with neurocognitive and sensory disabilities. the cause and pathogenesis of cp is multifactorial and continues to be poorly understood. chorioamnionitis, clinically silent or manifest, has been reported to be a risk factor for cp both in term and preterm infants. il- gene is single copy gene located on chromosome q . - in humans. interleukin- is synthesized by a variety of immune (t cells, eosinophils and dendritic cells) and non-immune (fibroblasts, epithelial and neuronal) cells. it is also detected in organ-specific secretions in a number of inflammatory processes. amniotic fluid interleukin- concentrations decreased with advancing gestational age. but, women with preterm labor and women with chorioamnionitis have higher interleukin amniotic fluid concentrations than those who delivered at term or those with sterile amniotic fluid. the aim of our study was to estimate allelic frequency for regulatory il - snp in the children with the cp. methods: dnas obtained from peripheral blood of cp patients and unrelated healthy volunteers were genotyped for the il - snp pcr-rflp method. results and conclusions: il - genotype fncc was more common in the population with cerebral palsy in the comparison to healthy volunteers. the significance of the association between il fngene polymorphisms and cerebral palsy has to be investigated in the future studies. ( ), d delbro ( ), e hansson ( ) ( ) kalmar university, sweden ( ) gçteborg university, sweden background: acetylcholine (ach) is a major signalling molecule, binding partly at nicotinic receptors (nachrs; a family of ions channels with nicotine as a selective ligand). one subtype, the alpha nachrs, has antiinflammatory effects by way of down-regulation of tnfalpha release from macrophages. the alpha nachrs have been demonstrated in neuronal as well nonneuronal tissues, e.g. astrocytes and microglia. aim. in rat astrocytes in primary culture, and in astrocytes cocultivated with primary microvessel cultures study: . the expression of alpha nachrs and alpha nachrs by immunofluorescence and western blot. . intracellular ca +-transients spread within the astroglial networks after stimulation with nicotine. . pro-inflammatory cytokines, il- b, il- , and tnfalpha, released from microglial cells after stimulation with nicotine. results: alpha nachrs expression was more evident in astrocytes co-cultivated with endothelial cells, suggesting that endothelial cells release factors, which increase the maturity of astrocytes. the ca + transient evoked by nicotine was also more pronounced in the co-cultured astrocytes. these ca + responses were blocked by the alpha nachr antagonist alpha-bungarotoxin. the release of pro-inflammatory cytokines was down-regulated after stimulation by nicotine. conclusion: alpha nachrs appear to be involved in some of the effects of nicotine administration to rat astrocytes. an anti-inflammatory action of cholinergic nerves on the astrocytes via nachrs seems probable, which may have therapeutic implications. university, department of natural sciences, kalmar, sweden e-mail: ann.pettersson@hik.se gedeon richter plc., budapest, hungary tramadol, an atypical opioid analgesic, is increasingly used for the treatment of osteoarthritis because it does not produce typical side effects of nsaids. review of clinical data show that it produces symptom relief and improves function, but these benefits are small and adverse events often cause participants to stop taking the medication (cohran database ). efficacy of tramadol in animal models of inflammatory pain is well established; however complex characterization of the drug regarding analgesic and side effects is missing in rats. our aim was to assess oral efficacy of tramadol at side effect free doses in rats. anti-hyperalgesic effect was determined in complete freunds adjuvant (cfa) induced thermal hyperalgesia test (th) and in model of cfa-induced knee joint arthritis. effect on inflammation was characterized in carrageenan induced paw edema test (et). for the characterization of side effect accelerating rotarod assay (arr) was used. significant impairment in arr was noted at mg/kg dose, and above. in the th test weak % effect was seen at side effect free dose ( mg/kg). in the arthritis model b.i.d. mg/kg dose of tramadol given on days - after cfa caused a maximum % effect on weight bearing incapacitance (day ). however, its effect seemed to diminish ( % on day ) upon repeated treatment. in et tramadol had significant anti-inflammatory effect at but not at mg/kg dose. these results show that efficacy of tramadol in rat inflammatory pain models is limited by its side effects, in accordance with clinical data. chronic relapsing experimental allergic encephalomyelitis (cr eae) is a disease that bears striking similarities to the human condition multiple sclerosis (ms). in particular, cr eae and ms have major inflammatory events in the central nervous system (cns) that culminate in demyelination and disruption of axonal function.one group of mediators involved in the progressive cns inflammation are the prostaglandins (pgs).pg generation is regulated in experimental non-immune conditions of the cns by n-methyl-d-aspartate (nmda) receptor activation.nmda receptor-mediated events are also evident in eae and may therefore have the potential to influence pg production.the study was designed to profile pge and pgd in cns tissues during the development of cr eae and to examine the role of the nmda receptor in pg production through the use of the specific antagonist mk- .biozzi mice were inoculated for cr eae and cns tissues were sampled during the course of disease.enzyme immunoassay of processed samples revealed pge and pgd levels within normal limits during the acute and subsequent remission phases of cr eae.in contrast, dramatic changes in pg concentrations were observed with a relapse of symptoms and a remission of disease.mk- was therapeutically administered to cr eae-diseased mice at the onset of relapse and changes in cns pg levels were recorded.the relapse phase of cr eae, but not the acute stage of disease, is characterised by an increase in cns pg production that may be influenced by nmda receptor activation. livia l camargo( ), lm yshii ( ) ( ), sk costa ( ) ( ) university of s¼o paulo, brazil ( ) king's college, london, uk ( ) butantan institute, s¼o paulo, brazil objectives: the neuropeptide substance p (sp) released by capsaicin-sensitive nerves (csn) plays a pivotal role in neurogenic inflammation. despite the prevalence of arthritis, the contribution of sp to the progression of arthritis has not been established. this study investigated the effect of csn ablation and sr , a sp antagonist, on knee joint inflammation and pain induced by intraarticular (i.a.) injection of kaolin ( %, h time course) in female wistar rats. the kaolin-injected knee (ipsilateral -ipsi) of vehicle-treated rats exhibited a significant, timedependent oedema as compared to the contralateral knee. in addition, increased pain score and high levels of myeloperoxidase (mpo, marker of neutrophil accumulation) and both pro-inflammatory (il- b and il- , but not tnfa) and anti-inflammatory (il- ) cytokines was detected in the ipsi synovial fluid of these animals. both destruction of knee joint csn fibres by neonatal capsaicin treatment and i.a. injection of rats with sr ( nmol/cavity) significantly attenuated the kaolin-induced pain score and knee oedema, suggesting that kaolin is acting, at least partially, via a neuronal mechanism. in contrast, the same treatment caused increased mpo activity and cytokine concentrations measured h post kaolin injection. conclusions: peripheral release of sp after kaolin injection acts to increase pain generation, oedema formation and inflammatory cell influx. however, chronic tachykininergic depletion by capsaicin treatment up-regulates the production of pro-inflammatory cytokines that are important in triggering cell influx in the synovial cavity. ( ) ( ) university of s¼o paulo, s¼o paulo, brazil ( ) butantan institute, s¼o paulo, brazil objectives: previously, intra-tracheal (i.tr.) injection of dep or , -naphthoquinone ( , -nq) evoked plasma extravasation and cell influx in rat airways. we now investigated whether simultaneous injection of these pollutants had a synergistic inflammatory action. we also determined the ability of dep and , -nq-induced airway inflammation to evoke changes in the rat isolated thoracic aorta (rta) and corpus cavernosum (rcc) reactivity using organ bath assays. results: capsaicin-or vehicle-treated male wistar rats received i.tr. injection of dep ( mg/kg) and , -nq ( nmol/kg). after min, dep and , -nq produced a potent (additive) plasma extravasation in the trachea and bronchi, but not lung, compared with each compound alone. in capsaicin-treated rats or treated with tachykinin antagonists, the response was inhibited, suggesting an important role for c-fibres, primarily tachykinins. increased mpo and cytokine levels were detected in bronchi of capsaicin-treated rats following h treatment with pollutants. this treatment contributes to augment the ach ( - - - m)-induced relaxation in the rta but not in the rcc. in capsaicin-treated rats, the rta response to the pollutants was not affected but it was capable of evoking a marked relaxation in rcc in animals challenged with pollutants. conclusions: , -nq exacerbates dep-induced plasma extravasation and mpo activity in the airways, indicating a neurogenic mechanism through tachykinins. the exposure to dep and , -nq affects the endotheliumdependent response in rta without interfering with rcc. neuropeptides are unlikely to affect the pollutantsinduced changes in the rta. acknowledgements: capes, cnpq, fapesp. we thank ma alves for technical assistance. trans-resveratrol (rv) is a naturally occurring polyphenolic compound present in certain foods that has anticancer and anti-inflammatory properties. the purpose of this study was to determine the effect of rv on the production of proinflammatory cytokines and reactive oxygen species stimulated by lipopolysaccharides (lps) in glial cells. rt-pcr showed that rv ( , , mm) dose-dependently inhibited ng/ml lps induced tnfa, il- b, il- , mcp- and inducible nitric oxide synthase mrna expression. rv also inhibited lps-induced production of these cytokines (elisa), nitric oxide and reactive oxygen species in a dose-dependent manner. western blot analyses showed that resveratrol could inhibit lps-stimulated phosphorylation of erk / and jnk but not p . nf-kb reporter assay showed rv could inhibit nf-kb activation by lps in microglia and astrocytes. these results suggest that rv may inhibit lps-induced microglial and astrocyte activation through erk / , jnk and nf-kb signaling pathways. therefore, rv is a natural product with therapeutical potential against disease conditions in cns that involve an overproduction of proinflammatory cytokines and reactive oxygen species. ( ), cs patil( ), sv padi ( ), vp singh ( ) ( ) university institute of pharmaceutical sciences, panjab university, chandigarh, india ( ) pharmacology r & d, panacea biotec ltd. lalru, punjab, india persistent stimulation of nociceptors and c-fibers by tissue injury causes hyperalgesia and allodynia by sensitization of nociceptors and facilitation of synaptic transmission in the spinal cord. the important participant in the inflammatory response of injured peripheral nerve may be nitric oxide (no). the aim of the present study was to test the sensitivity of pde inhibitor sildenafil in chronic constriction injury (cci) model, a rat model of neuropathic pain. sciatic nerve injury is associated with development of hyperalgesia days after the nerve ligation. sildenafil ( and fÝ g/rat, i.t.) produced a significant decrease in pain threshold, which in lower dose did not alter the nociceptive threshold. the hyperalgesic effect of sildenafil was blocked by l-name and methylene blue (mb), which on per se treatment showed antinociceptive effect in nerve ligated rats. the results from the present study indicated that the major activation of no cgmp pathway in the chronic constriction injury model of neuropathic pain. the aggravation of hyperalgesic response might be due to the increased cgmp levels resulting in pkg-i activation and its upregulation. glycine transporter (glyt) and glyt are expressed in glia and neurons, respectively. glyt make clearance of glycine released from glycinergic neuron in synapse and thus terminates the neurotransmission and also regulates over-stimulation by glycine spilled over to nmda receptors, while glyt supplies glycine into synaptic vesicles in glycinergic neurons. therefore, glyt inhibitors could modulate inhibitory glycinergic or excitatory glutamatergic neurotransmission. the present study examined the effects of glyt inhibitors on pain in animal models. inhibitors of glyt (sarcosine and org ) and glyt (alx and org ) by intrathecal (i.t.) injection reduced formalin-induced nociceptive behaviors. glyt inhibitors reduced allodynia score and reversed the reduction of paw withdrawal threshold in complete freund adjuvant (cfa)-induced inflammation mice but the antiallodynia effects appeared after latent period. on the other hand, glyt inhibitors produced antiallodynia effects immediately after the i.t. injection in cfa-treated mice. these inhibitors produced the similar antiallodynia effects in the partial sciatic nerve ligationinjury or streptozotocin-induced diabetic neuropathic pain models, either by i.t. injection or i.v. injection.pretreatment of specific antagonists of glycine site of the nmda receptors disappeared the latent periods of glyt inhibitors and potentiated the antiallodynia effect. glycine receptor antagonist, strychnine by i.t. injected reversed the antiallodynia effect of i.v. injected glyt inhibitors. these results suggest that both glyt and glyt inhibitors by enforcing glycinergic inhibitory neurotransmission in spinal cord produce potent antinociceptive effect and may be novel candidate for medicament of pain control. the tumour microenvironment in particular tumour associated macrophages (tams) play a role in determining tumour outcome. despite strong causative links between inflammation and human gastric cancer progression, little is known of the role of tams in this disease. we have utilized our mouse model of gastric tumourigenesis, the gp ff mouse to assess the effect of the gp ff mutation on macrophage function and ascertain the role of macrophages in tumor formation. this mouse has a knockin mutation in the il- family cytokine receptor gp preventing shp /ras/erk signaling and leading to constitutive stat transcriptional activation. tumour development is inflammation dependent, there is a requirement for il- , and development is inhibited by nsaid treatment or microbial eradication, however an adaptive immune response is s inflamm. res., supplement ( ) posters dispensable for tumorigenesis. in gastric antrum the gp ff mutation results, decreased erk / activation and constitutive phosphorylation of stat , abnormal signaling is replicated in macrophages. antral stat activation is unaffected by depletion of il- . however in macrophages an absence of il- results in higher stat activation demonstrating the anti-stimulatory role of il- on macrophages. the gp ff mutation results in macrophages with decreased il- and increased inos mrna expression reflecting a more basally activated phenotype. manipulations of the gp ff mouse that reduce tumor size (eg. antibiotic or nsaid treatment or stat hemizygosity) coincidently result in reduced macrophage infiltration of antral mucosa. macrophages of gp ff mice display aberrant gp signaling potentially resulting in exaggerated response to stimuli. the key difference between mutant gastric mucosa and macrophages are changes in transcription of target genes. ( ), y riffo-vasquez( ), s brain( ), s costa ( ) ( ) university of s¼o paulo, brazil ( ) king's college london, uk objectives: we have previously shown that simultaneous intra-tracheal injection of diesel exhaust particles (dep) and , -naphthoquinone ( , -nq) caused a potent inflammation in rat airways partially dependent on neurogenic-mediated mechanism. this study investigates the mechanism of action of thee pollutants using inflammatory assays and histopathological approach in the lung and trachea of wild type (wt) and trpv knockout (ko) mice exposed to dep and , -nq. dep ( mg/kg) and , -nq ( nmol/kg)-induced airway inflammation was assessed via i-labelled albumin h post pollutants injection. mpo assay and histopathology were performed h after treatment. staining of lung and trachea specimens with h&e provided a profile of cell infiltration in both wt and ko animals. results: injection of dep and , -nq evoked a potent plasma extravasation into the trachea and lung of wt, but not trpv ko, suggesting these pollutants act via a trpv -mediated mechanism. in contrast, mpo activity in the airways of trpv ko mice was exacerbated compared to wt mice data. likewise, the histopathology revealed high numbers of leukocytes and macrophages infiltrated into the lungs and trachea of trpv ko mice compared to wt. conclusions: inhibition of increased microvascular permeability in the airways of trpv ko mice treated with pollutants suggests that these receptors are the predominant mechanism involved in the inflammation. however, neutrophils/macrophages accumulate more in trpv ko, indicating that the lack of trpv receptors up-regulates production of inflammatory cells in response to pollutants, thus supporting a protective role for trpv receptors. acknowledgements: capes, cnpq, fapesp. ( ), n sato( , ), y endo ( ), s sugawara ( ) ( ) division of oral immunology, department of oral biology, tohoku university graduate school of dentistry, sendai, japan ( ) division of fixed prosthodontics, department of restorative dentistry, tohoku university graduate school of dentistry, sendai, japan biotin is a water-soluble vitamin of the b complex and functions as a cofactor of carboxylases.biotin deficiency causes alopecia and scaly erythematous dermatitis.moreover, serum biotin levels are significantly lower in atopic dermatitis patients than in healthy subjects, indicating that biotin deficiency is involved in inflammatory diseases.however, immunological effects of biotin on allergic inflammation remain unclear.in this study, we investigated the effects of biotin-deficiency on metal allergy using nickel (ni)-allergy model mouse and a murine macrophage cell line j . . female balb/c mice ( weeks old) received a basal diet or a biotin-deficient diet for weeks.ten days after sensitization with intraperitoneal injection of lps and nicl , the mice were challenged with intradermal injection of nicl into the pinnas.allergic inflammation was measured by ear swelling.the ethical board for nonhuman species of the tohoku university graduate school of medicine approved the experimental procedure followed in this study.j . cells were cultured in biotin-sufficient or deficient medium for weeks. ear swelling was significantly higher in biotin-deficient mice than biotin-sufficient mice.il- beta productions by splenocytes were significantly higher in biotin-deficient mice than in biotinsufficient mice.moreover, biotin-deficient j . cells produced il- beta significantly higher than biotinsufficient j . cells.to investigate the therapeutic effects of biotin-supplementation, biotin-deficient mice received biotin contained-water for weeks.ear swelling was significantly lower in biotin-supplement mice than biotin-deficient mice.these results indicated that biotindeficiency deteriorates allergic inflammation.the augmentation of il- beta production is probably involved in those deteriorations. one of the most important targets of cytokine action is the blood vessels, which undergo some structural and functional changes that result in activation of endothelium. applying the elisa technique, levels of il- , icam- and e-selectin were studied in patients with acute pancreatitis. mediators levels were studied in arterial, venous and pancreatic ascites samples. according the atlanta criterion the mild pancreatitis was established in patients and severe -in patients. the highest levels of il- were noted in ascites and lowest in arterial samples. the highest concentration of adhesion molecules was in venous samples and lowest in ascites. it was a clear correlation between levels of il- and adhesion molecules and severity of pancreatitis. during first week the levels of il- gradually increased in patients with severe pancreatitis, while in patients with the edematous pancreatitis its levels decreased starting from the third day. icam- levels gradually increased during first three day with the following decrease after this term. the highest levels of e-selectin were noted at the time of admission. the clear correlation between il- and adhesion molecules was noted in both groups of patients. besides that, the clear strong correlation was observed between il- and quantity of circulating granulocytes and between e-selectin and hematocrite in patients with necrotizing pancreatitis. our study confirms the importance of activation of endothelium as a part of the systemic inflammatory response in patients with acute pancreatitis. subsequently eos infiltrate the tissues, are activated, and release mediators inducing the late phase response. this is characterized by tissue damage and repair, and in chronic reactions, tissue remodeling and fibrosis. mc/eos cross-talk by physical and non-physical contact is an essential feature of late-phase and chronic allergic reactions. we have previously described an mc/eos interaction facilitated by soluble mediators and shown to enhance allergic inflammation. still, pathways that mediate mc/eos cross-talk in allergy are not fully characterized. methods: human cord-blood derived mc were cocultured with peripheral blood eos, and activated with anti-ige. mc/eos couples in co-culture and in human nasal polyp tissue sections were specifically stained and counted using microscopy. expression of surface molecules was analyzed by facs. mc activation was measured by chromogenic assays for â-hexosaminidase. relevant surface molecules were neutralized using antibodies, to assess interference with couple formation and activation. results: mc and eos physically interact, forming welldefined couples in-vitro and in-vivo. in the presence of eos, mc are more releasable under baseline or igeactivating conditions. this effect is partially mediated by cd and dnam- on mc, and b and nectin- on eos. we describe a novel physical interaction between mc and eos that we name "the allergic synapse". this synapse may upregulate allergic reactions, thus serving as a target for therapeutic intervention in allergic and inflammatory diseases. methods: mc were obtained from cord blood mononuclear cells ( - weeks with scf, interleukin- and prostaglandin e , cbmc). cbmc were activated, after their culture for days with myeloma ige ( mg/ml), by rabbit anti-human ige antibodies ( mg/ml), for , and h at c. activation was measured by b-hexosaminidase release, determined by enzymatic-colorimetric assay. the expression of flip, mcl- , bcl- , bcl-xl, bak and bax was assessed by immunoblot analysis. results: two anti-apoptotic proteins were found to be upregulated: flip, which is involved in the extrinsic apoptotic pathway and mcl- that is mainly implicated in the intrinsic apoptotic pathway. in contrast, the expression of two other anti-apoptotic proteins that we have examined (i.e. bcl- and bcl-xl) was not altered. likewise, the expression of pro-apoptotic proteins from the bcl- family (i.e. bak and bax) was either undetectable or unchanged. conclusions: our findings reveal that ige-dependent activation of human mc mainly induces the selective increase in the expression of two pro-survival molecules. this may be one of the mechanisms that underlay mc hyperplasia in the chronic allergic inflammation. ( ), c armishaw( ), z yang ( ), h cai ( ), p alewood( ), c geczy ( ) ( ) university of new south wales, faculty of medicne, sydney, australia ( ) university of queensland, institute for molecular biosciences, st lucia, australia purpose: human s a , s a and s a are closely related proteins associated with inflammation. s a is expressed in the human genome, but not in the mouse. s a is a potent monocyte chemoattractant and mast cell (mc) activator. mouse (m) s a and human (h) s a share % structural identity, but the hinge domains are more divergent. the ms a hinge (ms a - ) is a potent chemoattractant for leukocytes whereas this sequence in hs a (hs a - ) is inactive. methods: s a hinge domain and its alamine scan mutants were synthesized and their activities were tested by using thp cells or murine mc in vitro and mouse footpad injection. results: s a hinge domain (s a - ) was chemotactic for monocytes and mc and provoked mc degranulation in vitro and oedema, and leukocyte recruitment in vivo. in contrast to s a , the hinge domain only weakly induced cytokine production (il , il ) by macrophages. residues essential for oedema were hydrophobic in nature (leu , isoleu , isoleu and isoleu ). n and i were essential for responses provoked by - m s a - whereas mutation of k , l , n , i , d , k and i significantly reduced migration with - m s a - . conclusions: s a and ms a may be functional chemattractant equivalents; s a may have arisen by duplication of human s a . isoleucine residues in s a hinge domain are essential for its proinflammatory properties. ( ), g kiriakopoulou ( ), e tsimara( ), a voultsou( ), k zarkadis ( ) ( ) general hospital of zakynthos, greece ( ) medical centre of katastari, zakynthos, greece schistosome is a disease which pests in tropical countries. the endemic areas are south america, far and middle east, and africa. our aim is to present an incident which concerning a parasitic infection, not endemic in greece. patient: man years old who immigrated from pakistan (at the side of the aparkenar river) in greece, a year ago. he turned out with anlage, headache and weight loss. he is a swimmer. he mentioned also a fever before migrating to greece. clinically: largely-scaled paleness, stomach -mild and diffused sensitivity, with also lightly increased enteric sounds and a small degree of hepatomegaly, when palpated deeply. laboratory examination: leucocytes . eo . %, hb . g/dl, ht . %, mcv , fl, mch . pg, thrombocytes . , blood sedimentation mm, fe mg/ml, ferritin . ng/ml. normal biochemical examinations. u/s: livers size lightly increased . mm, hepatoportal vein with normal amplitude. parasitology of feces: in the immediate confection with lugol of the second sample, there were observed: big scattering oval ovules ( - mm) with a thin wall and quills on the side, as well as three imago worms (> mm): schistosoma mansoni. treatment: praziquantel was given. recheck in a months time: blood examinationleucosites . , eo . %, hb , g/dl, ht . %. parasitology of feces is negative. in the diagnosis of anemia combined with fever, to patients who are immigrants, schistosomiasis posters inflamm. res., supplement ( ) should be taken into account, especially when sideropenic anemia is accompanied with intense eosinophile, because the disease is mostly a reaction of retardate supersensitivity. bronchial asthma is a chronic airway inflammatory disease caused by immune cells such as t lymphocytes and eosinophils. recently, high-sensitivity crp (hs-crp) has become available for detecting small changes in crp levels within the normal range, allowing for assessment of subclinical inflammation. this study was undertaken to investigate the relationship between hs-crp and bronchial asthma. we collected blood samples from patients with bronchial asthma with or without attacks and measured serum eosinophil cationic protein (ecp) and pulmonary function as well as serum hs-crp. serum crp levels in patients without attacks (average . mg/ l; p < . ) and with attacks (average . mg/l; p < . ) were significantly higher than those of normal controls (average . mg/l). serum hs-crp levels were inversely correlated with fev . % in asthmatic patients (r = - . , p < . ). in conclusion, these results indicate that serum hs-crp as well as ecp may be related to the state of asthma exacerbation and allergic inflammation. objectives: the pshr assay can be used to test the biologic activity of allergens since it mimics the effector phase of a type i hypersensitivity reaction. in this study we tested methods for removing ige-antibodies (stripping) from donor basophils. moreover a method was developed for improving the antigen specificity profile in pshr. proof-of-concept was provided using absorption with complete allergen extracts. methods: buffy coats were screened to exclude reactivity against relevant allergens. subsequently pbmcs were purified and basophils were stripped with either lactate or a phosphate buffer. cells were passively sensitized with patient sera and challenged with allergen extracts in various concentrations. released histamine was measured spectrofluorometrically (hr-test, reflab). cutoff was % hr. for absorption experiments patient sera (from a peanut allergic and a codfish allergic patient) were incubated with streptavidin-coated sepharose beads coupled with biotinylated allergens (peanut, and bsa as control). after centrifugation the supernatant was used to passively sensitize stripped basophils. hr was measured as described above. results: stripping experiments using the two buffers only partially removed surface ige but passive sensitization of stripped basophils was equally effective enabling determinations of sub-nanogram quantities of peanut allergen. absorption experiments showed that it was possible to specifically remove peanut specific ige from patient serum. removal of specific ige reactivity to peanut extract was verified by western blotting. conclusions: using peanut extract as a model it was demonstrated that in pshr antigen specificity can be modified. ( ), sk bk( ), v sharma( ), rp bhandari ( ) ( ) nepal medical college teaching hospital, nepal ( ) all india institute of medical sciences, new delhi, background: nepal has one of the highest maternalmortality rates in the world. this study was to evaluate the incidence, disease pattern, and risk-factors for thromboembolism in pregnant nepalese women. methods: women with thromboembolic diseases were identified and their case records retrieved and reviewed s inflamm. res., supplement ( ) posters from january to december . demographiccharacteristics were compared between women with and without thromboembolism. the total number of deliveries over the study period was , , giving an incidence of . per deliveries. there were two cases of pulmonary-embolism and one resulted in a maternal-death. the others had deep-vein-thrombosis of which over % were limited to calf veins only. the ultrasound-examinations requested for suspected deep-venous-thrombosis before and after the event of maternal-death were . and . per deliveries (p <. );the corresponding cases of deepvenous-thrombosis diagnosed were . and . per deliveries, respectively (p <. ). the majority ( %) of cases were diagnosed in the postpartum period, mainly after cesarean-delivery. women with venous-thromboembolism were older, had higher bmi, and a higherincidence of preeclampsia. there were approximately twice as many postpartum as antepartum events. bloodgroup a, multiple-pregnancy, caesarean-section, cardiacdisease, delivery at gestational-age of < weeks, a bmi of , or more and maternal-age of or over were all found to increase incidence of venous-thromboembolism. the long-standing belief that thromboembolism is rare among nepalese is at least partly because of undiagnosis most of these events are deep-veinthromboses occurring in the postpartum period and it is very essential for primary prevention developing country like nepal. ( ), ch ladel ( ), t ruckle( ), c rommel( ), r cirillo ( ) ( ) rbm-merck serono, colleretto giacosa, italy ( ) serono pharmacological research institute, geneva, switzerland rheumatoid arthritis (ra) is a severe articular disease. massive leukocyte activation and infiltration into joints result in cartilage and bone destruction. blockade of pi k signalling pathway has been demonstrated to be curative in a murine model of ra, collagen-induced arthritis (cia). in this study we explored the molecular mechanisms by which pi k signalling inhibition resulted in clinical amelioration of disease symptoms. as , a novel isoform non-selective, yet specific class-i-pi k inhibitor, administered at mg/kg twice-a-day for days, to mice showing signs of arthritis, paw swelling and inflamed digits, induced a significant amelioration of disease course. at the end of treatment, post-arthritic paws were removed and phosphrylation levels of akt (p-akt), downstream target in pi k-mediated signal, were determined by semi-quantitative immunohistochemistry, also immunophenotyping of circulating cells by flowcytometry was conducted. akt phosporylation resulted to be significantly enhanced by disease induction and as was able to decrease its levels down to values comparable with naïve animals. controls and as treated mice were bled before treatment, after two days of treatment and at treatments end. no changes in cellular composition (morphology and hematology parameters) between experimental groups were observed. t cell number was not affected, however a significant decrease of natural-killer, memory and regulatory t cells was observed after as administration. finally, a non-significant, moderate reduction of bcell number was also observed. these data demonstrate that efficacy of as in arthritis models is mediated by direct modulation of the target resulting in a mixed anti-inflammatory (via pi kg) and immunosuppressive (via pi kd/a/b) activity. ( ) ( ) institute of biomedical science, university of sao paulo, brazil ( ) ibilce -sao paulo state university, brazil epidemiologic data suggest that female sex hormones are involved in the pathophysiology of allergic asthma. we investigated in rats the immunomodulatory potential of estradiol and progesterone on the expression of allergic asthma. seven days after being ovariectomized (ovx), groups of rats were sensitized with ovalbumin (oa). fourteen days after sensitization, the animals were oachallenged and used day thereafter. allergic,shamoperated animals were used as controls. some ovx animals were treated with estradiol ( ìg/kg) or progesterone ( ìg/kg) h before being challenged. mast cell degranulation was quantified in samples of isolated, oa-challenged bronchi. the airway reactivity of inner bronchi to methacholine and the functional activity of cells we analysed. ovx caused reduction of the allergic lung inflammation and bronchial hyperresponsiveness with regard to intact female rats. estradiol reverted the reduced cellular recruitment into lungs, whereas progesterone reduced the pulmonary inflammatory response and reverted the bronchial hyperresponsiveness. cultured bal and bone marrow cells from allergic rats increased posters inflamm. res., supplement ( ) s the release of il- and reduced that of il- and tnf. the release of il- by bone marrow cells was significantly reduced. these effects were reverted by estradiol, and progesterone reduced il- and increased il- production in bal and increased that of il- and tnf in bone marrow cells. bronchial mast cell degranulation upon direct contact with oa in ovx rats was less than in controls. it is suggested that female sex hormones can modulate the allergic lung inflammation in rats by acting on cellular migration/activity and airway responsiveness. objectives: to study the participation of fsh on modulation of e-and l-selectin, icam- and mac- expression in ovariectomized (ovx) rats made allergic. methods: female rats were sensitized (oa/alumen) after (ovx- ) or days (ovx- ) of ovx or sham-operated (sh). fourteen days thereafter animals were challenged (oa, %; aerosol) and sacrificed h after. bronchoalveolar lavage (bal) was collected and flow citometry analyses oficam- , mac- and l-selectin expression was studied. in parallel, lungs were frozen and sections were analysedfor e-selectin expression by immunohistochemistry. results: at day , e-selectin expression increased(ovx- = , ae , ) and at day , decreased (ovx- = , ae , ) as compared to respective controls (sh- = , ae , ; sh- = , ae , ). estrogen treatment reverted this profile in both groups (ovx- +e= , ae , ; ovx- +e= , ae , ). mean fluorescence intensity of bal cells showed increase of mac- expression (ovx- = ae , vs sh- = , ae , ), icam- (ovx- = , ae , vs sh- = , ae , ) and l-selectin (ovx- = , ae , vs sh- = , ae , ) at day i.e., ovx- group. on the other hand, we observed a decrease in icam- (ovx- = , ae , vs sh- = ae , ) and mac- expression (ovx- = , ae , vs sh- = , ae , ) was seen in ovx- group. conclusions: oscillation of hormone levels during immunization with oa increased (ovx- ) and decreased (ovx- ) expression of adhesion molecules. estradiol treatment reverted this effect. this results suggest that fsh modulates the ali in rats by acting on cell ( ), s lim ( ), y lin ( ), bp leung ( ), c thiemermann ( ), wsf wong ( ) ( ) national university of singapore ( ) the william harvey research institute, london, uk glycogen synthase kinase b (gsk- b) is known to regulate various cellular functions including inflammatory responses. we hypothesized that inhibition of gsk- b may have anti-inflammatory effects in a mouse asthma model. balb/c mice were sensitized with ovalbumin (ova) and challenged with aerosolized ova. tdzd- , a non-atp competitive gsk- b inhibitor, was administrated by i.v. injection one hour before ova challenge. tdzd- significantly reduced the ova-induced eosinophilia in a dose-dependent manner and inhibited the levels of il- , il- and eotaxin in bronchoalveolar lavage (bal) fluid. tdzd- also suppressed the mrna levels of icam- , vcam- and chitinase proteins in the lung. histological studies revealed that tdzd- substantially reduced the inflammatory cell infiltration and mucus secretion in the lung tissue. tdzd- also decreased ova-specific ige level in the serum. in addition, ova-induced increase in airway resistance and reduction in dynamic compliance were attenuated by tdzd- . our findings suggest that inhibition of gsk- b may have therapeutic potential for the treatment of allergic airway inflammation. ( ), a yildirim ( ), f ercan ( ), n gedik ( ), m yuksel( ), inci alican ( ) ( materials and methods: sprague-dawley rats ( - g) were exposed to oc (burn group) or oc water bath (control group) for s under ether anesthesia. adm ( ng/kg; s.c) was administered min before burn and all rats were decapitated h after the burn insult. trunk blood was collected for the measurement of tnf-alpha level and the lung, ileum and kidney samples were stored for microscopic scoring and for determination of lipid peroxidation (lp), myeloperoxidase (mpo) activity and formation of reactive oxygen metabolites (roms) using chemiluminescence assay. results: burn resulted in severe morphologic damage in tested tissues. lp increased in lung and kidney (p< . - . ) and mpo activity showed a marked increase in all tested tissues (p< . ) of the burn group. adm reversed these parameters effectively (p< . - . ). luminol chemiluminescence levels showed increases in both ileum and lung (p< . - . ) whereas lucigenin chemiluminescence levels increased in ileum and kidney (p< . ) of the burn group. adm treatment was also beneficial in reducing chemiluminescence levels near to controls (p< . ). adm reduced plasma tnf-alpha level (p< . ) which showed a significant increase in burned animals compared to controls (p< . ). conclusions: adm is beneficial in remote organ damage following burn insult via decreasing neutrophil infiltration, rom generation, lp, and the release of proinflammatory cytokine tnf-alpha. kalpana panday ( ), sd joshi ( ), kr reddy ( ) ( we determined the crystal structure of human hematopoietic prostaglandin (pg) d synthase (h-pgds) as the quaternary complex with glutathione (gsh), mg +, and an inhibitor, hql- , with anti-inflammatory activities in vivo, at . resolution. hql- was found to reside within the catalytic cleft between trp and gsh in the quaternary complex. hql- inhibited h-pgds competitively against the substrate pgh as well as noncompetitively with respect to gsh. surface plasmon resonance analysis revealed that hql- bound to h-pgds with an affinity that was -fold higher in the presence of gsh and mg + than in their absence. hql- inhibited selectively pgd production by human h-pgds-expressing megakaryocytes but only marginally affected the production of other prostanoids, suggesting the tight functional engagement between h-pgds and cyclooxygenase. orally administered hql- inhibited antigen-induced production of pgd , and airway inflammation in mice without affecting the production of pge and pgf f¿. knowledge about this quaternary structure should accelerate the structure-based development of novel anti-inflammatory drugs that inhibit pgd production specifically. introduction: it has been proved that high levels of mechanical ventilation produce lung injury as well as local inflammation. this study was designed to evaluate how the generation of inflammatory mediators by an over-stretched lung affects the non-hyperventilated lung. methods: male wistar rats ( - g) were anesthetized and paralyzed, and the two lungs were independently intubated. differential ventilation was applied for h ( breath/min). one lung was subjected to hyper-ventilation ( ml/kg/lung) and the other was ventilated with a normal volume ( ml/kg/lung). in a control group, both lungs were ventilated with a normal volume. after sacrifice, samples of lung, plasma and liver were collected. the expression of the pro-inflammatory chemokine mip- was evaluated by rt-pcr and the edema was assessed by the ratio between the wet and dry weight of the lung. systemic inflammation was estimated in liver by measuring the expression of tnfa by rt-pcr as well as its levels in plasma. the hiper-ventilated lung showed an increase in the ratio between the wet and dry weight and in mip- expression compared to the normal ventilated lung. no differences were found in edema, neither expression of mip- between the normally ventilated lung and control lungs. no significant changes were observed in liver expression and plasma levels of tnfa as a consequence of unilateral lung hyper-ventilation. the over-straining caused to the hyperventilated lung leaded to a local inflammatory response without systemic effects. the normal ventilated lung is not affected by the inflammatory process triggered on the over-strained lung. ( ), j-y gillon( ), v lagente ( ), e boichot ( ) ( ) university of rennes , rennes, france ( ) serono international s.a., geneva, switzerland macrophage elastase (mmp- ) is a metalloproteinase involved not only in emphysema but also in the inflammatory process associated with copd (chronic obstructive pulmonary disease). the mechanism of action of mmp- in the development of pulmonary inflammatory process is still unknown. in the present study, we investigated the effect of recombinant human mmp- (rhmmp ) on il- /cxcl release from alveolar epithelial cell line a and we explored the underlying mechanisms. a cells were stimulated with rhmmp- ( . - - . - u/ml) during hours and il- /cxcl level in supernatant was determined by elisa. involvement of map (mitogen activated protein)-kinases was studied by western-blotting and also using chemical inhibitors. nfkb activation was examined with the transam nfkb p kit. we observed that mmp- elicited il- /cxcl release in a dose-dependent manner. this production could be prevented by a pretreatment for hour with a selective mmp- inhibitor (as - mm) or with the nonselective inhibitor batimastat ( - mm) . the il- /cxcl production was also inhibited by actinomycin d ( mg/ml), erk / inhibitors (u mm and pd mm) and nfkb inhibitors including bay - ( mm) and nfkb activation inhibitor ( nm), whereas p kinase inhibitor (sb ) had no effect. stimulation with mmp- was rapidly followed by a phosphorylation of erk / ( min) and an nfkb nuclear translocation and activation ( h). the nfkb activation was not inhibited by a treatment with u . these data suggest that alveolar epithelium is a target of mmp- since it upregulates gene expres-s inflamm. res., supplement ( ) posters sion and release of il- /cxcl , via erk / and nfkb activation, but these two pathways appears to be distinct. agents which are associated with lung inflammation, such as cigarette smoke and lipopolysaccharide (lps), induce the production of pro-inflammatory chemokines in lung epithelial cells in vitro, and the induction of interleukin (il)- , in particular, is often used a measure of relative toxicity.in this study we have compared mrna expression and mediator release in nci-h human lung epithelial cells exposed to lung toxicants, namely: cigarette smoke total particulate matter (tpm), lps, bleomycin, diesel exhaust particles, residual oil fly ash (rofa), carbon black and vanadyl sulphate.polystyrene, poly(methyl-methacrylate) and the tpm vehicle, dimethyl-sulphoxide were used as negative controls.confluent monolayers of h cells were exposed to serial dilutions of test agents in serum-free medium for hours.the conditioned medium was then removed and assayed for a range of pro-inflammatory cytokines and other selected mediators by luminex technology.the levels of gene expression of il- , matrix-metalloprotease- (mmp- ), the gel-forming mucin muc ac, heparinbinding epidermal growth factor-like growth factor (hb-egf) and the cytochrome p s cyp a and cyp b were determined by quantitative-polymerase chain reaction.all of the toxicants induced similar responses whereas the negative controls were largely ineffective.-such a panel of biomarkers may enable an in vitro assessment of the potential to cause lung inflammation.-moreover the use of several biomarkers could give a more accurate picture of toxicity than the determination of il- alone, particularly in the case of agents such as tpm, where the conventional vehicle is found to have some biological activity. respiratory tract infections are a major public health issue. prevention in high risk populations relies mainly on vaccination. vaccination is highly recommended in decrease absenteeism. immunomodulating drugs are important tools in the treatment of infectious diseases. immunomodulatory agents are probably contributive in decreasing exacerbation rates. the authors present different classes of immunomodulators that are currently in use. the vaccine, created from a bacterial protein, reeducates the immune system to stop inflammation. by preventing infections, vaccines prevent the development of a strong t helper (th ) response. the challenge is now to inform about new possibilities of an optimal prevention respiratory tract infections. deoxypodophyllotoxin (dpt) is a medicinal herbal product that is isolated from anthriscus sylvestri. that inhibits cyclooxygenase- (cox- ) and cox- dependent phases of prostaglandin d (pgd ) generation in bone marrow-derived mast cells (bmmc) in a concentration-dependent manner with ic values of . mm and . mm, respectively. this compound inhibited cox- and -dependent conversion of the exogenous arachidonic acid to pgd in a dose-dependent manner with an ic values of . mm and . mm, respectively. however, dpt did not inhibit cox- protein expression up to a concentration of mm in the bmmc, indicating that dpt directly inhibits cox- activity. furthermore, this compound consistently inhibited the production of leukotriene c (ltc ) in a dose dependent manner, with an ic value of . mm. these posters inflamm. res., supplement ( ) s results clearly demonstrate that dpt has a dual cox- / -lox inhibitory activity in vitro. therefore, this compound might provide the basis for novel anti-inflammatory drugs. in order to determine anti-allergic and antiasthmatic activity of dpt in vivo, we used rat pca model which was activated by anti-dinirophenyl ige and ovalbumin/alum induced mouse asthmatic animal model, respectively. as a result, dpt strongly inhibited pca reaction as well as it reduced infiltrated eosinophil numbers in bronchoalveolar lavage fluid. furthermore, dpt decreased the mrna levels of the th cytokines in a murine asthmatic model. in addition, northern blot analysis showed that dpt also reduced both the eotaxin and arginase i mrna levels in a dose dependent manner. these results suggest that dpt may be beneficial in regulating various inflammatory diseases. an imbalance of proteases and anti-proteases in the lung has been implicated in the pathogenesis of chronic obstructive pulmonary disease (copd), a smokingrelated disorder associated with accelerated lung function decline.in particular, the activity of matrix metalloproteases (mmps) have been implicated in driving both the inflammation and parenchymal destruction observed in copd patients.here, we tested whether a broad spectrum mmp inhibitor, pkf- , could block the inflammation induced by an acute exposure to cigarette smoke in strains of mice, balb/c and c /bl .animals were administered the compound ( - mg/kg) either per os (p.o.) or intranasally (i.n.) hour before and hours after exposure to smoke on three consecutive days.bronchoalveolar lavage (bal) was performed and lungs were flash frozen for inflammatory marker analysis.pkf- dose-dependently reduced lung neutrophilia in balb/c mice when dosed either p.o. (~ % at mg/kg; p < . )or i.n. (~ % at mg/kg; p < . ).however, the compound had no clear effect on bal neutrophil infiltration in c /bl mice by either route of administration.interestingly, in both strains bal macrophages dose-dependently trended towards an increase when the compound was dosed p.o. and decreased when dosed locally (p < . ). examination of lung tissue cytokine levels revealed that while smoke-exposure increases il- beta, kc, and mip- , pkf- had little effect on these cytokines.these data suggests the ability of broad spectrum mmp inhibitors to inhibit smoke-induced acute neutrophil inflammation is strain-dependent, while its ability to limit macrophage infiltration may be route dependent. to investigate the role of seh in the regulation of the pulmonary inflammatory response, we have used seh deficient mice in a locally administered lps model. male seh deficient mice (ko) and their wildtype (wt) littermates were exposed to inhaled lps.four hours later they were sacrificed and bal was performed. differential counts and cytokine levels in bal were evaluated. results: lps induced a significant increase in total cell number and neutrophil number in bal in the seh deficient mice and in wt mice;no significant differences between the groups were seen (table ) cytokine analysis showed significant increases in tnfalpha, il- , kc, gm-csf, mcp- , il- beta and rantes in the lps-exposed wt mice. no significant differences were seen between lps exposed wt and ko mice except for a significant increase in tnfa in ko mice. our results show that seh has no pivotal role for the regulation of the acute inflammatory response to lung administered lps. fam.liliaceae. based on literature data which signaled the presence of steroidic saponins in the rhyzomes, isolation, identification and quantitative determination of these compounds were done. the antiinflammatory activity was tested in non-immune chronic inflammation model:the cotton-pellets granuloma test in rats, and in an immune chronic inflammation model:arthritis test induced by freunds adjuvant in rats. in the first test, the antiinflammatory effect of steroidic saponins mg/kg is weak, statistically insignificant. in the arthritis test, steroidic saponins mg/kg proved an antiinfalammatory activity, influencing especially the primary response, but also the secondary one, in the last part of the experiment (after days). in both tests, the suprarenal glands weight was modified. objectives: dendritic cells (dcs) are professional antigen presenting cells. many types of dc with subtle difference in phenotype have been reported in several organs. existence of dc was reported in synovial tissue of rheumatoid arthritis (ra), however, the details in ra dc still remains unclear. in this study, we generated a new lineage of dc with gmcsf (+tnfa) and investigated their functions. furthermore the ability of osteoclastgenesis was examined. methods: monocyte-derived dcs or macrophage were generated in ( ) tnfa + gm-csf ( ) gm-csf ( ) il- + gm-csf ( ) mcsf. the phenotypes of these cells were analyzed by morphological examination and flow cytometry (facs calibur). cell proliferation was examined by wst- assay. dc functions were assessed in antigen presenting ability (mlr assay), cytokine production (elisa), and endocytosis (fitc-dextran uptake). concerning osteoclastgenesis, monocyte-derived dcs were incubated with rankl and mcsf. trap stain was performed and the resorption ability was assessed on osteologic cell culture system and dentine slices. results: these cells were dendritic-like and their surface markers were cd a low cd b + cd c + cd + cd + cd low hla-dr + dc-sign low and different from conventional dc or macrophage. they had an antigen presenting ability to induce na*ve cd + t cell proliferation, il- production and endocytosis. in the presence of rankl and mcsf, they differentiated multinuclear trap-positive cells with bone resorption ability, which was strengthened by tnfa. we generated a new lineage of dc with gm-csf (+ tnfa). the dc seemed to play a pivotal role in inflammatory arthritis under tnf immunity. the clinical effectiveness of rituximab and other b cellattenuating rheumatoid arthritis (ra) therapies has increased interest in understanding the role of b cells in ra pathogenesis.the possible mechanisms underlying the effectiveness of rituximab were investigated by performing biosimulation research in the entelos ra physiolab platform, a mathematical model of the joint of an ra patient.the platform dynamically integrates the contributions of immune cells (t cells, b cells, and macrophages), resident cells (fibroblast-like synoviocytes and chondrocytes) and mediators to the joint inflammation and structural damage observed in ra.the b cell lifecycle is represented in the platform, as well as effector functions such as antigen presentation, mediator and autoantibody production, and immune complex formation.the dynamics of these b cell properties were calibrated to reproduce reported experimental behaviors and clinical outputs from ra patients (e.g., acr score and radiographic progression rates).an assessment of the contribution of individual b cell functions to clinical outcome suggests that plasma cell-derived immune complexes are key modulators of inflammation in ra patients. in contrast, proinflammatory cytokine production by b cells contributes minimally to synovial hyperplasia, but plays a role in the progression of structural damage.immune complex formation leads to monocyte activation, increased mediator production by macrophages and an increase in antigen availability to t cells.biosimulation research in the ra physiolab platform is advancing our understanding of the mechanisms underlying effective b cell-targeting ra therapies, and may guide the development of improved second-generation therapeutic approaches. methods: the hmscs were obtained at the operation.the mononuclear cells were extracted and the colony forming assay were performed after weeks. the hmscs were cultured and in passage ,the cell surface antigens of both groups were analyzed by flow cytometory.in passage , in control group, the cells were cultured with beta glycerophosphate(bgp) and in osteogenic group, the cells were cultured with bgp and ascorbic acid(aa) and dexamethasone(dex).after weeks, alp staining and activity were measured in each group.after weeks, alizarin red s assays were performed.rna was extracted from the cells cultured with bgp and aa and dex for weeks and weeks and the gene expressions of bone formation markers were examined by real time pcr. the mann-whitney test was used for the statistical analyses. the colony forming assays showed no significant differences in oa and ra. in flow cytometory, the cell surface antigens in oa and ra were almost same.in alp activity,there were no significant differences in oa and ra.in alizarin red s, there were significant differences.in real time pcr,the gene expressions showed no significant differences in oa and ra. conclusions: the hmscs of ra will be able to use for regenerative therapy. silje vermedal høgh ( ), hm lindegaard ( ), gl sorensen ( ), a høj ( ), c bendixen ( ), p junker ( ), u holmskov ( ) ( cytosolic phospholipase a (cpla ) plays a crucial role in eicosanoid production, by releasing an arachidonic acid from membrane phospholipids. in addition, cpla regulates the phagocyte nadph oxidase-releasing superoxides and that is the only isozyme responsible for the production of eicosanoid in phagocytic cells. collageninduced arthritis (cia) in mice is an experimental model of auto-immune diesease with several clinical and histological features resembling rheumatoid arthritis in humans. previous studies show that cpla -deficient mice are resistant to cia. thus we aimed to study whether cpla is up-regulated during the development of cia and to detect its exact location in the inflamed joints. immunoblot analysis revealed an increase in the level of joint cpla protein during the development of the disease which correlates with the severity of the inflammation, as examined by paw thickness. immunohistochemistry with specific anti-cpla antibodies revealed low positive cpla protein levels in skeletal muscles, sebaceous glands and skin (epidermis, dermis) tissues of healthy paws. in the joints of the cia mice, large amounts of inflammatory infiltrate containing cpla were detected. in addition, robust cpla protein expression in the skeletal muscles surrounding the joints and strong cpla positive staining in sebaceous glands were detected. the high correlation between the severity of inflammation and the elevated cpla protein, due to an inflammatory infiltrate and increased cpla expression, suggests an important role of cpla in the development of arthritis. rheumatoid arthritis (ra) is the complex disease depending on environmental as well as genetic factors. in spite of the large research efforts we know in fact only small number of genes involved in this disease. animal models are useful tools for better understanding of the pathogenic mechanisms and genes leading to the disease process. the aim of the current project is to identify the genes and their functional role importance for arthritis. two loci associated with arthritis were identified using a cross between the b .q (intermediate susceptible) and nod.q strains (resistant to arthritis). one on chromosome a disease protective locus (cia ) and another on chromosome a disease-promoting locus (cia ). nod.q allele at cia promotes arthritis whereas cia , has a protective effect in contrast to the b .q allele on chromosome . a promising candidate gene in cia locus is complement factor (c ) as the nod.q allele produced defective c protein. cia locus contains several genes of potential importance for disease such as fc riib, fc riii, fc riv ncf , fh. the results of cia (collagen induced arthritis) and caia (collagen antibody induced arthritis) experiments using the subcongenic mice generated that contains fc r region showed a significant difference in incidence and severity of arthritis. the disease is controlled in a recessive pattern. the fragment devoid of fc r region seems to be protective. the subcongenic for the cia locus has been generated recently which will be tested for cia and caia susceptibility. the results from these experiments will be discussed in detail. angela pizzolla, ka gelderman, r holmdahl ncf is a component of the nadph oxidase complex, which produces reactive oxygen species (ros) upon activation into phagosomes and extracellularly. polymorphisms in the ncf gene that impair the capacity to produce ros, enhance susceptibility of both mice and rats to arthritis. activation of autoreactive t cells drives arthritis development but neither ncf expression nor oxidative burst have been detected in t cells. we hypothesize that antigen presenting cells influence t cell activity by producing ros during antigen presentation. we aimed to clarify the role of ros produced by dendritic cells (dc) on t cell activation. dc were grown from bone marrow from ncf mutated and wildtype mice. we could show that ncf mutated dc proliferated and differentiated better, had higher expression levels of costimulatory molecules and mhc ii upon stimulation as compared to ncf wildtype dc. in addition, ncf mutated dc induced higher levels of il- production by hybridoma t cells. to analyze the role of ncf in dc on arthritis, mice were developed expressing functional ncf restricted to dc (b .qdcn). these mice are characterized for burst, ncf expression and ability to present antigen. we published that immunization with myelin oligodendrocyte glycoprotein (mog) protein resulted in higher disease severity than immunization with mog peptide in ncf deficient mice. this suggests that ncf plays a role in the uptake and processing of posters inflamm. res., supplement ( ) s antigens, probably by dc. this will be further investigated with the b .qdcn mice using in vitro assays as well as in vivo models for arthritis and multiple sclerosis. purpose: macrophage migration inhibitory factor (mif) is a pro-inflammatory cytokine involved in both innate and adaptive immune responses. it is expressed in human ra synovial tissue and its suppression inhibits t or b cell dependent animal models of ra. we investigated the role of mif in k/bxn serum transfer arthritis. methods: arthritis was induced by injection of k/bxn serum on days and in littermate wt and mif-/-mice. arthritis was scored clinically, ankle thickness was measured using microcallipers, and joints collected for histology. sections were scored for synovitis, synovial fluid exudate, cartilage degradation and bone damage. results: wt mice exhibited arthritis as early as day and % incidence was observed on day . mif -/-mice exhibited delayed arthritis, with onset on day and % incidence on day . mif -/-mice exhibited significantly reduced disease severity as measured by clinical disease score ( methods: osteoarthritis was induced by bilateral transection of the medial meniscus in dunkin-hartley guinea pigs using minimal invasive surgery to avoid cartilage damage due to inflammation and/or intra-articular bleeding. results: the first signs of osteoarthritis development were macroscopically observed four weeks after meniscal transection. twelve weeks after surgery the lesions were still restricted to the medial side of the joint and did not reach into the subchondral bone. cartilage destruction due to meniscal transection was also histologically detected. however, biomarkers for cartilage destruction (ctx-ii, hp/lp ratio, comp) were not increased. treatments aiming at different processes in osteoarthritis, such as bone destruction (risedronate), inflammation (pioglitazone and anakinra), and cartilage destruction (galardin) were not effective in this model. the early degenerative changes in this transection model are probably too mild to be measured in the systemic circulation using classic biomarkers. further research into new biomarkers is needed to detect and monitor the early stages of osteoarthritis. the ineffectiveness of the compounds tested in this model underscores the urgent need for new strategies to treat the disease. the meniscal transection model might prove to be useful tool for identifying new biomarkers and treatments. livia l camargo ( ), a denadai-souza ( ), lm yshii ( ), a schenka ( ), ma barreto ( ), d boletini-santos ( ), c lima ( ), v rioli ( ), mn muscar ( ), e ferro ( ), sk costa ( ) ( methods: aia was induced via intraarticular (i.a.) injection of methylated bovine serum albumin in immunized male rats. knee oedema and pain score were assessed daily in controls and animals treated with hemopressin ( or ìg/day; i.a.). histopathological changes, cell number and cytokines in the synovial fluid of aia rats were determined at day . results: aia rats developed a severe mono-arthritis characterized by a joint oedema and pain. at day , there was marked cellular infiltration, hyperplasia, pannus formation and destruction of bone and cartilage, but pro-inflammatory cytokines were undetectable by elisa. both doses of hemopressin significantly reduced the knee oedema, but only mg of hemopressin attenuated the pain score. acute joint inflammation was significantly reduced by hemopressin, but failed to significantly affect chronic histopathological signs (hyperplasia, pannus etc.). conclusions: hemopressin has potential for treating acute signs of aia by reducing synovial plasma protein extravasation, alleviating pain and reducing acute joint histopathological changes, thus providing an alternative strategy for treatment of oedema and pain in arthritis. calcitriol, the hormonal metabolite of vitamin d and it synthetic analogs exert an anti-inflammatory action on psoriatic skin lesions while eliciting mild inflammation on healthy skin. the map kinase erk plays an important role in the induction of chemokines, cytokines and adhesion molecules in keratinocytes that maintain the epidermal inflammatory response.we hypothesized that the dual effect of calcitriol may be partially attributed to differential effects on erk activation in the presence or absence of inflammatory mediators. our experimental model was immortalized non-tumorigenic human hacat keratinocytes cultured in the absence of exogenous growth factors or active mediators. inflammation was mimicked by exposure to tnf. level and activation of signaling molecules were determined by immunoblotting. by using the specific egf receptor (egfr) tyrosine kinase inhibitor, ag , we established that tnf activates erk in an egfr-dependent and egfrindependent modes. the egfr-dependent activation resulted in the induction of the transcription factor, c-fos, while the egfr-independent activation was of a shorter duration and did not affect c-fos expression. treatment with calcitriol alone increased erk activation and c-fos induction. pretreatment with calcitriol enhanced egfr-dependent erk activation and tyrosine phosphorylation of the egfr, but completely abolished egfr-independent tnf-induced erk activation. pretreatment with calcitriol increased the rate of de-phosphorylation of activated erk, accounting for the inhibition of egfr-independent erk activation by tnf. it is possible that effects on the erk cascade underlie the dual action of calcitriol and its synthetic analogs on cutaneous inflammation. christina barja-fidalgo ( ), r saldanha-gama ( ), ja moraes ( ), r zingali ( ), c marcienkewicz ( ) ( ) universidade do estado do rio de janeiro, rio de janeiro, brazil ( ) universidade federal do rio de janeiro, rio de janeiro, brazil ( ) temple university, philadelphia, usa neutrophils adhere on vascular endothelium and directly migrate toward inflamed tissue to exert their primary defense function. integrin are receptors that drive cell adhesion and motility and interfere with cell activation, functions and survival.acting as both anchoring molecules and signaling receptor, transducing signals outsidein and inside-out, integrins are potential targets for therapeutic and diagnostic opportunities. disintegrins are a family of cystein-rich low-molecular weight peptides that usually contain an rgd sequence, a cell attachment site of ecm and cell surface proteins recognized by integrins. they are considered selective and competitive antagonists of integrins, being potent inhibitors of platelet aggregation and cell-cell/cell-ecm interactions. we reported that rgd-disintegrins, selectively interact with integrin amb , a b and/or avb ) on human neutrophils, interfering with cell functions through the activation of integrin-coupled intracellular signaling pathways. recently showed that, a selective ligand of a /a b integrins, vlo , induces neutrophil chemotaxis, cytoskeleton mobilization and potently inhibiting neutrophil spontaneous apoptosis. these effects are mediated vlo interaction with a b integrin, activating the focal adhesion cascade. vlo effects on the delay of neutrophil is modulated by pi k, erk- mapk and nf-kb pathways that seems to interfere with the balance between anti-and pro-apoptotic bcl- family members and with mitochondrial membrane potential. data emphasize mechanistic details of the role of a b integrins interactions on human neutrophils and support the use of disintegrins as prototypes to develop logical combinations of drugs to optimize or minimize the susceptibility of a selected target cell population to apoptosis during therapeutic interventions. (faperj, cnpq, capes, ifs-sweeden) ( ), h serezani ( ), m peters-golden( ), sonia jancar ( ) '( ) university of s¼o paulo, brazil ( ) university of michigan, usa it is been shown that leukotriene (lt) b and cysteinyl lt (ltc , ltd and lte ) enhances fcr-mediated phagocytosis in alveolar macrophages (am), dependent on protein kinase c (pkc). in contrast, ltb but not cyslt effects are exclusive on syk activation. in the present study we investigated the role of specific pkc isoforms and its upstream and downstream targets involved in lt-enhanced fcr-mediated phagocytosis. to this purpose, ams were pretreated or not with inhibitors of pkc-d (rottlerin - um), pkc-a (ro- - - nm), pi k (ly - um and wortmannin- nm), erk / (pd - um), cpla (aacocf - um), p mapk (sb - um) and ca++ (bapta/am- um), before stimulation with ltb or ltd and addition of igg-opsonized red blood cells for min. activation (phosphorylation) of signaling molecules by lts were analyzed by western blot. our results demonstrate that ltb -enhanced phagocytosis is dependent on pkc-a, while ltd effects are mediated by pkc-d. cell proliferation and differentiation, adhesion, cell activation and apoptosis. while galectin- mainly acts as an anti-inflammatory and pro-apoptotic molecule, galectin- is known as a strong pro-inflammatory and anti-apoptotic signal. we have recently recognized galectin- as a new molecular target of immunomodulatory drugs in monocyte/macrophage-like cells. in this study we investigated the effects of immunomodulatory drugs (aspirin, indomethacin, hydrocortisone and dexamethasone), applied in therapeutic ranges on the expression of galectin- at gene and protein level in monocytic thp- cells. we have also tested the effects of these drugs on both galectins in the cells activated by lipopolysaccharide from e. coli (lps). the targeted mrna level was evaluated using quantitative rt-pcr technique and the expression of both galectins in cell homogenates was determined by western-immunoblot analysis. the results showed that immunomodulatory drugs affected the expression of galectin- on both, gene and protein level, and that the effects were dependent on drug type and applied concentration as well as time of the exposure. the modulatory effects of the applied drugs on galectin- and - expressions were also observed in the cells activated by lps. these findings represent important step in the understanding of the effects of immunomodulatory drugs on galectin- and- expressions, as well as the role of these lectins in the physiology of monocytes. introduction: pancreatitis-associated protein (pap) has been recently described as an endogenous mechanism involved in the regulation of inflammation. in the present study, we show some of the molecular mechanisms implicated in the intracellular signaling pathways modulated by pap. the pancreatic human cell line panc was incubated with pap ( ng/ml) and/or tnfa ( ng/ml). total rna was obtained and the expression of tnfa was examined by rt-pcr. in addition, the effect of pap on nfkb activation was measured by inmunofluorescence in cells. western blot analysis was used to determine the expression of nfkb mediators: phosphorylated ikk, ikba and p . results: we observed that pap administration to cells prevented nfkb translocation to the nucleus as well as the tnfa-induced tnfa gene expression. when tnfastimulated cells were treated with cycloheximide in order to block protein synthesis, the induction of tnfa gene expression was completely restored. on the other hand, pap had no effect on ikk phosphorylation or ikba degradation. conclusions:in this study we have provided evidence that pap modulates the inflammatory response by blocking nfkb translocation to the nucleus. this pap-induced nfkb inhibition requires a jak/stat-dependent de novo protein synthesis. objectives: this study investigated the inhibitory mechanism of hyaluronan (ha) on lipopolysaccharide (lps)stimulated production of proinflammatory cytokines in u macrophages. methods: ha was added to u macrophage cultures in the presence of lps, with or without pretreatment with anti-intercellular adhesion molecule- (icam- ) antibody. secreted levels of tumor necrosis factor a (tnfa), interleukin (il)- b, and il- were determined by enzyme-linked immunosorbent assay. the phosphorylation of nuclear factor (nf)-kb, ikba, and mitogenactivated protein kinases (mapks) was analyzed by immunoblotting. results: lps stimulated production of tnfa, il- b, and il- . in contrast to kda ha, kda ha at mg/ ml inhibited lps-induced cytokine production. anti-icam- antibody blocked the effects of ha on the lps actions on u cells. lps activated nf-kb and mapk pathways, whereas ha down-regulated p nf-kb and ikba phosphorylation by lps without affecting mapks. inhibition studies revealed the requirement of nf-kb for lps-stimulated cytokine production. anti-icam- antibody reversed the inhibitory effects of ha on phosphorylation of p nf-kb and ikba. conclusions: ha of intrinsic molecular weight suppresses lps-stimulated production of proinflammatory cytokines via icam- through down-regulation of nf-kb and ikb. exogenous ha into arthritic joints could act as an anti-nf-kb agent by the mechanism demonstrated in the present study. the principal eicosanoid product of endothelial cox- is prostacyclin (pgi ), which is a potent vascular patency factor. induction of endothelial cox- under hypoxic conditions is well documented. this response, along with associated pgi release, is likely an important protective homeostatic response. in order to explore the role of candidate signalling agents in cox- expression in response to hypoxia, studies were undertaken using luciferase reporter constructs of the cox- promoter region in huvec. cox- induction under hypoxic conditions was confirmed with the wild type construct. strategic mutations of transcription factor binding sites showed that sites for hypoxia inducible factors (hifs) were more important for cox- expression than those for nfkb. furthermore, expression of cox- was increased under normoxic conditions by transfection of huvec with normoxia-stable hif mutants. emsa showed hif binding in nuclear extracts from untransfected hypoxic huvec. under these hypoxic conditions, increased release of pgi but not vaso-occlusive thromboxane a was seen. thus the putative protective induction of cox- in endothelial cells in response to hypoxia involves signalling by hifs. crescentic glomerulonephritis is characterized by crescent formation and rapid progress to renal failure, where the predominance of th immune response plays a crucial role. however, the therapeutic efficacy of the regulation of th -predominant immune responses remains to be investigated. therefore, the effects of a th selective inhibitor tak- were investigated in a model of crescentic glomerulonephritis in wky rats. methods: tak- was administered orally, starting at the time of induction of glomerulonephritis. in group , the drug was administered daily for the initial days. tak- was administered on day only in group , and from day to in group . in each group, nephritic rats were killed on days and . results: in group , glomerular damage, including crescent formation, was improved on day , with reductions in the numbers of cd , cd and ed- positive cells, as well as in urinary protein excretion. protein and transcript levels of th cytokines in the diseased kidneys were markedly decreased by tak- treatment. renal pathology, including glomerulosclerosis and interstitial fibrosis, was ameliorated and proteinuria was markedly decreased. elevated levels of serum creatinine showed concomitant improvement. in group , in which treatment was initiated shortly after the appearance of glomerular abnormalities, glomerular damage was also diminished, resulting in a decrease in urinary protein excretion. treatment only on the first day in group , partially rescued renal dysfunction. conclusions: these results suggest that the initial inhibition of th immune response has an appealing therapeutic potential for crescentic glomerulonephritis. parasitic nematodes and their hosts are now known to produce a wide range of galectins. whilst host galectins have been shown to modulate the recruitment and effector function of inflammatory cells including mast cells, neutrophils and eosinophils, the role of secreted parasitic galectins is less well defined. studies at moredun have demonstrated that both the endoparasitic helminth, haemonchus contortus, and the ectoparasitic mite, psoroptes ovis, produce galectin-like factors which, in vitro, directly influence the migration and survival of eosinophils from their natural sheep host. excretory-secretory extracts from both parasites contained potent chemokinetic activity and were also able to promote eosinophil survival in the presence of dexamethasone. separation by affinity chromatography, as well as specific sugar inhibition and mass spectrometric profiling, revealed the active components to be galectins. in the case of h. contortus, there was homolgy with known est sequences, which allowed subsequent cloning and expression studies to be undertaken. a functional in vivo role for these parasitederived galectins awaits confirmation, but the possibility is raised that they could directly influence the host immune response following infection. this may have particular significance in mite infections in which exudates from the associated eosinophilc lesion appear s inflamm. res., supplement ( ) posters to provide the primary nutrient source for their survival. moreover, the observation that two very different parasites may have evolved similar mechanisms for manipulating the host inflammatory response to their benefit, raises the further possibility that parasite galectins may provide potentially novel therapeutic targets. the aim of the study was to investigate the time course of cytokine gene expression in liver and lungs of mice with lipopolysaccharide (lps)-induced septic shock and to assess the effect of three different immunomodulatory agents on cytokine mrna levels in these tissues. male cd- mice were injected intraperitoneally with mg/kg lps alone or concomitantly with an intravenous dose of pentoxifylline ( mg/kg), lisofylline ( mg/kg) or prednisolone ( mg/kg). the tissues were harvested , , , , and h following lps administration and stored at - c. relative quantification of tumor necrosis factoralpha (tnf-alpha), interleukin- beta (il- beta), interleukin- (il- ), and interferon-gamma (ifn-gamma) mrna levels was performed by real-time rt-pcr. the highest levels of cytokine mrna were observed at or h after lps administration, whereas the expression of tnf-alpha and il- beta in lungs and il- in liver reached the peak values at h and then decreased gradually. in addition, the lps effects on cytokine mrna were more pronounced in liver when compared to lungs. all administered compounds inhibited the lpsinduced tnf-alpha mrna expression (by up to approximately %), whereas lisofylline significantly increased ifn-gamma mrna levels in both tissues at most investigated time points. for other cytokines, the observed differences did not reached statistical significance. in conclusion, with the exception of ifn-gamma, the time course of cytokine mrnas differed considerably depending on the type of tissue. in the murine model of lps-induced septic shock, only tnf-alpha gene expression was suppressed by all compounds under investigation. maria sanz( ), m losada ( ), c company ( ), c lope-gines( ), l piqueras( ), j cortijo ( ) the migration of leukocytes into inflamed tissues involves a cascade of molecular events finely regulated by cell adhesion molecules and chemokines. fractalkine/ cx cl (fr) is a membrane-bound chemokine that functions as a mononuclear leukocyte chemoattractant and as an adhesion molecule. clinical studies and animal disease models have shown that fr is also involved in the pathogenesis atherosclerosis. we have demonstrated that angiotensin-ii (aii) has proinflammatory actions inducing the initial attachment of mononuclear cells to the arteriolar endothelium. in the present study we have investigated whether aii can cause the synthesis and expression of fr on human umbilical arterial endothelial cells (huaecs). huaecs were stimulated with ang-ii microm or with tnfalpha ( ng/ml) for , and h. fr was determined in the culture supernatants by conventional sandwich elisa. fr was only detected after h and h stimulation with tnfalphafnwhereas aii was unable to provoke the cleavage of the chemokine. semiquantitative rt-pcr analysis of huaecs showed increased fr mrna expression in aii-stimulated cells for and h. these effects were caused by the interaction of aii with its at receptor since they were abolished by losartan (at receptor antagonist). tnfalpha also increased fr mrna. immunohistochemical analysis of the cultured endothelial cells showed a clear expression of fr in huaecs stimulated with aii or tnfalpha for and h. these results suggest that fr could be a key chemokine in the selective adhesion of mononuclear leukocytes to the arterial endothelium elicited by aii. the lipophilic yeast malassezia is an exacerbating factor in atopic dermatitis (ad).among organisms of the malassezia species, m. globosa and m. restricta are particularly dominant on the skin of ad patients. our previous study has demonstrated that human keratinocytes responded to the two malassezia species with different th -type cytokine profiles, i.e. m. globosa induced il- , il- , and il- secretion from the keratinocytes, whereas m. restricta induced il- secretion.these findings suggest that m. globosa and m. restricta play a synergistic role in triggering or exacerbating ad by stimulating the th immune response. pattern recognition receptors (prrs) of human keratinocytes play an important role in the induction of inflammatory and innate immune responses. in this study, we assessed the role of prrs for cytokine production by human keratinocytes in response to malassezia species. human keratinocytes were pretreated with various anti-prr monoclonal antibodies (mabs) and stimulated with m. globosa or m. restricta. cytokine secretion from keratinocytes was measured by using fast quant elisa kit. exposure of human keratinocytes to m. globosa and m. restricta resulted in enhanced secretion of il- and il- , respectively. the m. globosainduced increase in il- secretion was inhibited by mabs against cd and cd . in case of m. restricta, mabs against toll-like receptor (tlr ) and cd suppressed significantly il- secretion from keratinocytes. these findings suggest that the distinct prrs interactions with fungal pathogen-associated molecular patterns (pamps) are key factors in differential cytokine secretion from keratinocytes stimulated with malassezia species. atopic dermatitis (ad) is a chronic, relapsing inflammatory skin disease associated with allergy. mdc (macrophage-derived chemokine/ccl ) and tarc (thymus and activation-regulated chemokine/ccl ) are th type cytokines, and it has been reported that serum mdc and tarc levels are associated with ad disease. in present study, we investigated the effect of prunus yedoensis matsum barks on the inflammatory chemokines (mdc and tarc) and jak-stat pathway in hacat keratinocytes. as a result, etoac fraction and e sub-fraction inhibited the mrna expression and protein level of mdc and tarc in a dose-dependent manner. also, e sub-fraction showed inhibitory activity on the stat protein level. these results suggest that p. yedoensis may have an anti-atopic activity by suppressing the inflammatory chemokines (mdc & tarc). the il- family now consists of members, most of which have assigned functions.there are members of the il- receptor family (including the decoy receptor type ii il- r).many of the il- family members possess neither a signal peptide nor an apparent prodomain, but nevertheless manage to exit the cell.the il- family members il- f , f and f signal through a complex of the il- r family member rp in association with il- r acp to activate common inflammatory pathways.the specific activity is is low, on the order of - ug/ml ec .we have found that removal of a few n-terminal amino acids from il- f , f , f and f can increase their bioactivity approximately -fold. the location of the n-terminus leading to increased specific activity is quite specific; removal of one more or one fewer amino acid eliminates the effect.in addition, n-terminally truncated il- f is capable of antagonizing signaling via il- rrp , but full-length f is inactive. ( ) university of ulsan, japan kyoto university, japan inflammation plays a pathogenic role in the development of obesity-related complications such as type ii diabetes and atherosclerosis. tumor necrosis factor alpha (tnfa) is closely associated with the enhanced inflammatory responses in obesity and the obesity-related pathologies. tr (hvem/tnfrsf ), which is a member of the tnf receptor superfamily and the receptor for lymphotoxins-related inducible ligand that competes for glycoprotein d binding to herpesvirus entry mediator on t cells (light/tnfsf ), is a potent mediator of inflammatory responses. the purpose of this study is to examine the hypothesis that obesity-induced inflammatory responses can be attenuated by inhibiting tr pathway. c bl/ tr knockout mice and their wild-type control were fed a high-fat diet for weeks and the obesity phenotypes were determined in the obese tr knockout mice and the control. the obese tr mice fed a high-fat diet elicited the attenuation of body weight gain and insulin resistance relative to wild-type control mice. expression levels of inflammatory genes significantly decreased in the adipose tissue of the obese tr knockout mice compared with those of the control. our results demonstrate that obesity-induced inflammatory responses and insulin resistance can be attenuated in obese tr knockout mice fed a high-fat diet. objectives: the present study was undertaken to investigate the role of insulin on allergic airway inflammation. methods: diabetic male wistar rats (alloxan, mg/kg, i.v.) and controls were sensitized with ova ( ìg) and al(oh) ( mg, s.c.) days after alloxan or saline injection. the animals were challenged days later by the intratracheal instillation of ova ( mg/ . ml). the following analyses were performed h thereafter: (a) total and differential cell counts in bronchoalveolar lavage (bal) fluid; (b) quantification of tnf-alpha and il- beta in the bal by elisa; and (c) immunohistochemistry for p-and e-selectins on lung vessels. results: compared to the control animals, diabetic rats exhibited reduced number of neutrophils ( %) and mononuclear cells ( %); reduced levels of tnf-alpha ( %) and il- beta ( %), and reduced p-selectin expression ( %) in response to ova challenge. these abnormalities were corrected after treatment of diabetic rats with a single dose of nph insulin ( iu, s.c.), h before ova challenge. although we did not find differences in e-selectin expression between diabetic rats and controls, expression of this molecule was amplified by insulin. conclusions: data presented show that insulin controls neutrophils migration during allergic airway inflammatory possibly by modulation of tnf-alpha and il- beta production and selectin expression. supported by fapesp and pronex. hormonally active vitamin d derivatives are beneficial in the treatment of cutaneous inflammatory disorders, particularly in psoriasis. their anti-inflammatory effect is usually attributed to inhibition of the activity of infiltrating immune cells.we examined whether vitamin d also interferes with the pro-inflammatory action of the keratinocytes themselves. human hacat keratinocytes cultured in the absence of exogenous growth factors or active mediators were exposed to tnf to simulate an inflammatory challenge and their response was monitored by assessing mrna levels of the cytokine tnf, the chemokine il- and the adhesion molecule icam- by real-time pcr. icam and il- were induced rapidly peaking after h, their mrna levels increased again from h to reach a plateau between h to h after exposure to tnf, whereas tnf mrna levels increased steadily between h and h. h pretreatment with calcitriol, the hormonal form of vitamin d, inhibited induction of il- but did not affect that of icam- or tnf h following exposure while calcitriol markedly inhibited the induction of all pro-inflammatory genes h after the tnf challenge.calcitriol inhibits the activation of jun kinase (jnk) and p by tnf. this action was mimicked by the posters inflamm. res., supplement ( ) s jnk inhibitor sp and the p inhibitor sb .the combination of the two inhibitors fully reproduced the time and gene dependent modulatory effect of calcitriol. we conclude that vitamin d attenuates the active contribution of keratinocytes to cutaneous inflammation and that this modulatory effect is explained by inhibition of the jnk and p cascades. ( ), cm lotufo ( ), p borelli ( ), zs ferreira ( ), rp markus ( ), shp farsky ( ) ( ) department of clinical and toxicological analyses, school of pharmaceutical sciences, university of s¼o paulo, brazil ( ) department of physiology, bioscience institute,university of s¼o paulo, braziil introduction: we showed that endogenous glucocorticoids (eg) control neutrophil mobilizations from the bone marrow and peripheral compartment by modulating the expression of l-selectin on segmented cells. aims: we evaluated the role of eg on endothelial cells (ec) and the molecular mechanisms responsible for hormonal actions in neutrophils and ec. methods: neutrophils were collected from blood, segmented leukocytes from femoral bone marrow and ec were cultured from testis vessels. cells were obtained from adrenalectomized (adx), ru -treated, shamoperated, vehicle-treated and non-manipulated (nm) wistar rats. results: circulating neutrophils and segmented cells from ru -treated rats presented elevated and decreased expressions of l-selectin vs cells from control animals, respectively. the effects were not dependent on alterations of l-selectin mrna levels. ec from adx animals presented more ability to adhere neutrophils from nm rats and enhanced mrna levels and membrane expressions of icam- , vcam- and pecam- . participation of the glucocorticoid cytosolic receptor(gcr) on these effects was shown by similar results in cells from ru treated rats. nfkappab translocation in neutrophils was equivalent in all groups of animals, but it was enhanced in ec from adx or ru -treated rats. conclusions: data show the participation of the gcr on events involved in neutrophil mobilizations, but nfkappab transcription is only involved on ec. ( ), y naito( ), t okuda ( ), k mizushima ( ), t okayama ( ), i hirata ( ), h tsuboi ( ), t suzuki ( ), o handa ( ) background: despite the inhalation of co at high concentrations had been considered as a toxic gas, the inhalation of co at low concentration has recently been shown the cytoprotective and anti-inflammatory effect against various animal models. however, it is unclear whether the direct exposure of co to the intestinal inflamed mucosa is effective or not. in this study, we investigated the therapeutic efficacy of the rectal co administration for rat colitis model. acute colitis was induced with trinitrobenzene sulfonic acid (tnbs) in male wistar rats. co( ppm- ml) was intrarectally administrated twice a day after the induction of colitis. rats were sacrificed at days after the administration of tnbs. the distal colon was removed, and the ulcer lesions were measured. thiobarbituric acid (tba)-reactive substances and tissueassociated myeloperoxidase (mpo) activity were measured in the colonic mucosa as indices of lipid peroxidation and neutrophil infiltration, respectively. moreover, we evaluated the expressions of cinc- mrna/protein and p-p mapk protein. the intracolonic administration of co ameliorated tnbs-induced colonic ulceration. the increases in tba-reactive substances and mpo activity after tnbs administration were both significantly inhibited by treatment with co. moreover, the rectal administration of co significantly inhibited the increased expression of cinc- mrna/protein and p-p mapk protein after the induction of tnbs-induced colitis. the rectal administration of co protected from the intestinal inflammation in rats. based on these data, the beneficial effects of co on the intestinal mucosal injury may be attributed to its anti-inflammatory properties. alessandra gambero ( ), m maróstica ( ), m saad( ), j pedrazzoli jr ( ) ( ) s¼o francisco university medical school, brazil ( ) state university of campinas, brazil recent studies have shown that adipocytes produce and secrete several bioactive molecules like adipocytokines. the adipose tissue can also present short and long-term changes during inflammation and infectious pathologies. in this study, the alterations of mesenteric and perinodal mesenteric adipose tissue during experimental colitis induced by repeated intracolonic tnbs instillations were evaluated. the adipocyte size was measured after collagenase digestion. the basal lipolysis (glycerol release) and adipocytokine production was monitored after short time culture of adipose tissue. the colitis animals showed higher mesenteric fat mass ( . +- . and . +- . % of body weight for colitis and control, respectively; p< . ) in despite of the lower body weight. the mesenteric adipocytes from colitis animals presented reduced diameter ( . +- . and . +- . um for colitis and control, respectively; p< . ), higher basal lipolysis ( . +- . and . +- . ug.mg- for colitis and control, respectively; p< . ) and tnf-alpha production ( . +- . and . +- . ng.mg- for colitis and control, respectively; p< . ). perinodal mesenteric adipocytes presented normal diameters, higher basal lipolysis ( . +- . and . . +- . ug.mg- for colitis and control, respectively; p< . ), increased tnfalpha ( . +- . and . +- . ng.ml- for colitis and control, respectively; p< . ), leptin ( . +- . and . +- . pg.ml- for colitis and control, respectively; p< . ) and adiponectin production ( +- and +- ng.ml- for colitis and control, respectively; p< . ). the mesenteric adipose tissue was modified during the experimental inflammation, but some alterations were site specific. perinodal adipose tissue retained the ability to produce anti-inflammatory and pro-inflammatory cytokines, while mesenteric adipose tissue only the pro-inflammatory one. this work was financially supported by fapesp. inflammatory bowel disease (ibd) is a group of chronic inflammatory disorders of the intestine. the role of the pro-inflammatory p mapk signalling cascade in the pathogenesis of ibd is highly controversial. we therefore aimed to investigate the role of p mapk in chronic dextran sodium sulfate (dss) induced colitis, an experimental model of ibd. chronic intestinal inflammation was induced by oral cyclic administration of % dss in sjl mice. clinically, the dss treatment produced episodes of colitis manifested by diarrhoea, gross intestinal bleeding, marked loss of body weight, and shortening of the colon. at the molecular level, this was accompanied by an up-regulation of tnfa, il- â, il- , il- , kc, cox- , igg heavy chain, and phospho-stat in the dss treated mice.the clinical and molecular features described above recapitulate findings reported in human ibd. in order to assess the role of p mapk, the activation of the p mapk signalling cascade was analysed by western blot analysis. the expression and phosphorylation levels of both p mapk and of mk and hsp , two down-stream targets, were not increased in dss treated animals compared to controls. leo , a potent inhibitor of p activity in vivo, was dosed as pretreatment and after completion of dss treatment. pretreatment had a deteriorating effect on all measured cytokines, whereas treatment after disease induction had no effect on any measured parameters. collectively, these results strongly suggest that the p kinase pathway only plays a minor role, if any, in the dss model. (sp) were gmcsf differentiated, dcs purified through gr + cell depletion, and spleen tcells isolated by pan tcell negative selection. spdcs or bmdcs were stimulated +/- ng/ml lps. for mlr, balb/c tcells were added for days. cells were incubated with sb ( -( -fluorophenyl)- -( -ethylsulfinylphenyl)- -( -pyridyl) h-imidazole, sb) or ml ((rs)-{ -[ -( -fluorophenyl)- -methylsulfanyl- h-imidazol- -yl]pyridine- -yl}-( -phenylethyl)amine, ml) and washed prior to lps stimulation (bmdcs) or cell mixing (t cells). hthymidine incorporation was measured, cell viability by mtt assay, tnfa and il- production by elisa. mlr tcell proliferation inspdcs or bmdcs was inhibited by sb (ic spdc . mm, bmdc . mm) and ml (ic spdc . mm, bmdc . mm). preincubation with dcs had no effect, despite reduced lps stimulated il- and tnfa synthesis by sb (ic il- . mm, tnf . mm) and ml (ic il- . mm, tnf . mm). preliminary data shows that preincubation of t cells with sb and ml modifies the mlr response. p plays a role in the interaction of dcs and t cells in antigen recognition. however, pre-incubation of drugs with dcs was ineffective. the role of t cell p mapk in the mlr is under investigation. p inhibitors may possess disease modifying properties because of reduced tcell antigen reactivity to dc antigen presentation. ( ), s luik( ), s laufer( ), m seed ( ), v holan( ), s fiorucci ( ) ( ) synovo gmbh, tübingen, germany ( ) university of tübingen, germany in vivo anti-inflammatory activity of certain p kinase inhibitors is limited by bioavailability. however, it is possible that they may be useful in the therapy of ibd should it be possible to mediate there uptake in and around bowel lesions. we reasoned that activity could be especially increased if drug physical properties were altered to allow preferential partition into macrophages and neutrophils (wbc) associated with lesions. we prepared prodrugs of p inhibitors and screened them using whole human blood, murine spleenocytes and peritoneal macrophage. pharmacologically inert macrocycle (azilide) conjugates were assessed for enhanced efficacy in murine collagen induced arthritis either therapeutically (after onset of signs) or prophylactically ( d post boost) or in a dss or tnbs model of ibd in the mouse. in both types of models, the prodrugs achieved improved suppression of arthritis and inflammatory score in colon sections at tolerated doses with optimal activity at mmolkg- d- . we propose that the prodrugs increase efficacy via improved pharmacokinetics partly related to biased disposition of the prodrug toward immune cells. despite the potent anti-inflammatory and immunosuppressive properties of glucocorticoids its applying in the management of severe necrotizing pancreatitis is still controversial. the plasma levels of interleukins (il- , il- ) and adhesion molecules (e-selectin and icam- ) were measured in patients with necrotizing pancreatitis. the measurement was performed immediately after admission, at the , , and day. all patients were divided on two groups: first group compiled patients, in which dexamethasone ( mg/day during - days) was applied in the complex management of acute pancreatitis, and control group - patients that did not receive corticosteroids. all patients received the initial therapy. the increased levels of il- , il- , il- , icam- , and eselectin were noted in both groups of patients at the time of admission. the gradually increase of all proinflammatory mediators plasma levels up to seventh day was noted in patients of the control group. its levels clear correlated with the severity of mods and spreading of necrotic processes confirmed by ct. starting from the third day the gradually decrease of mediators levels were noted in the patients of the first group. the incidence of contamination of necrotic foci had no difference in both groups of patients. the ability of glucocorticoids to inhibit expression of proinflammatory mediators due to the glucocorticoids-mediated repression of nf-kappa b pathway provide the pathogenetic substantiation for the applying of glucocorticoids in the complex management of necrotizing pancreatitis. the objective of this study was to examine whether t cell specific overexpression of the th transcription factor gata- can inhibit th /th cell mediated experimental mbsa arthritis. mbsa-immunized wild type mice developed joint inflammation which gradually increased in time with a maximum at day . at day , t cell specific gata- tg mice did not show any difference in arthritis score compared to wild type mice. however, at day , wild type mice had developed severe joint inflammation having the maximum arthritis score. in contrast, gata- tg mice showed only mild joint inflammation, suggesting that t cell specific overexpression of gata- protects against development of severe joint inflammation. facs analysis revealed low levels of il- +/ifn-gammacells in wild type as well as in gata- tg mice at day . as expected, il- positive cells were higher in gata- tg mice compared with wild type mice. interestingly, at day , the percentage of il- +/ ifn-gamma-cells were markedly increased in wild type mice but not in gata- tg mice, suggesting prevention of th expansion under gata- overexpression in vivo. these data revealed that t cell specific overexpression of the th transcription factor gata- protects against progression of severe joint inflammation during mbsainduced arthritis. furthermore, il- +/ifn-gammacells play a critical role in the progression of joint inflammation in this model and gata- overexpression in t cells prevents expansion of the il- +/ifn-gamma-t cell subset. pingping jiang ( ), pt sangild( ), t thymann ( ), hh-y ngai ( ), w-h sit ( ), k-l chan ( ), jm-f wan ( ) ( necrotizing enterocolitis (nec) is a severe intestinal inflammatory disease for which the disease etiology and progression remain unclear. preterm delivery and enteral milk formula feeding are factors predisposing to nec. to understand the pathophysiology of nec, two-dimensional gel electrophoresis ( d page) proteomic approach was applied in studying changes in intestinal protein pattern in preterm piglets with spontaneous nec occurring in response to days of parenteral feeding followed by day of enteral formula feeding. the intestinal proteomes of pigs with clinical symptoms of nec (n = ) were compared with corresponding pigs remained healthy (n = ). syproruby staining was used and differently expressed proteins were identified by maldi-tof ms or maldi-tof/tof ms. the proteins with significantly different expression between nec and healthy pigs involve in energy metabolism (sorbitol dehydrogenase, mitochondrial aldehyde dehydrogenase and chain a, medium-chain acyl-coa dehydrogenase with -thiaoctanoyl-coa etc.), inflammation (peptide-binding protein (pbp ) and snail homolog ), signal transduction proteins (thyroid hormone binding protein precursor, park protein and chain b, structure of the rho family gtp-binding protein cdc in complex with the multifunctional regulator rhogdi etc.) and anti-oxidation (manganese-containing superoxide dismutase(sod)). these data underscore the significant impact of intestinal proteomics in unraveling nec pathophysiology and biomarker discovery. blood are used to monitor the progression of inflammation. the aim of our study were to investigate systemic markers of disease in a rat model of lps induced pulmonary inflammation to provide a link between preclinical in vivo research and early clinical research. animals were exposed to bacterial lipopolysacharide (e.coli :b ) by inhalation. the lungs were lavaged hours post provocation and the level of cell influx was determined. relevant mediators of acute pulmonary inflammation were analysed with standard elisa technology in bronchoalveolar lavage fluid and in blood. in addition, measurement of changes in body temperature were performed at different time-points post provocation in order to monitor the systemic inflammatory responses to the local pulmonary inflammation manifested as alteration in body-temperature. results showing the effects of lps challenge on local and systemic parameters will be presented and the possible link to lps responses in man discussed. pulmonary inflammation models are widely used in pharmacological research. however, provocations and treatments aimed directly at the lung are often invasive which limits the possibility to perform repeated administrations of test agents and compounds. also, results derived from bronchioalvelar (bal) fluid are subject to variability if the retrieval techniques are non standardized. here we describe a non-invasive standardized method for retrieval of bal fluid to be used in mice. we present the characterisation of these techniques using the inflammatory response to lps and propose that this non invasive method can be used to refine lps and other challenge models. the objectives were to evaluate the dynamic response after a single intra-tracheal administration of of mg ( ml/animal) of lps (p.aeruginosa) to c bl/ j mice. control animals received a single dose of sterile ml . % saline/animal. the mice were terminated , , , and h after instillation using a non invasive and operator independent lavage technique. results showing the effects of single lps challenge on bal parameters, excised lung gas volume and lung weight will be presented showing reliable dynamic responses. these techniques open the possibility to run repeated treatments and chronic provocations and are not subject to variability from bal fluid retrieval. the human psoriasis xenograft scid mouse model is one of the most accepted and well characterized models for screening of novel anti-psoriatic compounds. the model has primarily been applied for testing novel treatment principles via systemic or intradermal administration routes. in order to evaluate the model for topical treatment, psoriatic keratome biopsies were grafted to immune-deficient scid mice. transplanted mice were treated with daivonex /dovobet (calcipotriol) and bms (betamethasone). the results show a strong antipsoriatic efficacy after treatment with bms (epidermal thickness reduced by %). treatment with daivonex / dovobet also showed an anti psoriatic effect with a % reduction in epidermal thickness. serum did not contain test compounds, indicating that the observed effect were not due to systemic exposure. the observed effects are in concordance with clinical results of treatment of psoriasis. it is concluded that the model is useful for testing topical treatments. we have demonstrated that adult rats offspring of dams submitted to protein restriction during early lactation, presented impaired acute immune responses probably related to an imbalance in glucocorticoids and insulin secretion (barja-fidalgo; inflamm res ( ): ) . here, we evaluated the innate immunity mediated by neutrophils and host defense against infection in adult rats offspring of dams fed with either a protein free diet (un-group) or % protein diet (c-group) during the first days of lactation. un rats showed lower number of blood pmn, though no difference in bone-marrow neutrophils number was observed. blood neutrophils from un-group presented a significantly reduced phagocytic activity against opsonized zymosan, constitutively expressed inos and spontaneously produced o -, no and tnf-alpha. in vivo treatment with lps, at non-lethal doses, significantly increases tnf-alpha and superoxide production by neutrophils, compared with controls. lps increased no production by neutrophils from both groups, inducing inos expression in control cells, but no further increase in inos expression in un rats. nucleare nf-kb is constitutively augmented in un rats. un animals presented a higher survival rate in a model of clp-induced severe sepsis. these results indicate that a metabolic programming induced during early lactation affects the innate immune responses in adult rats, which are unable to properly mount an inflammatory response, may predispose to chronic diseases in adult life. transgenic mice over-expressing vascular endothelial growth factor (vegf) in the epidermal basal layer under the human keratin (k ) promoter have previously been reported to develop a psoriasis-like inflammatory condition in the skin. important hallmarks of psoriasis are epidermal hyperplasia in association with infiltration of t-cells in the dermis and epidermis and also increased dermal angiogenesis. the aim of this study was to describe the epidermal hyperplasia and the infiltration of the skin with t-cells in transgenic k /vegf mice. we induced a cutaneous inflammation in the ear skin by repeated topical treatments with -o-tetradecanoylphorbol- -acetate (tpa), in order to investigate the inflammatory response. the in vivo pharmacological effect of topical treatment with a number of reference compounds, including betamethasone- -valerate, was also investigated. the ear thickness was significantly increased in transgenic animals compared to wild type animals following tpa-induction. the epidermal thickness measured in histological sections of biopsies from the ear skin was also significantly increased in transgenic animals. furthermore, increased dermal vascularisation was observed in the histological sections of the ear skin. a marked infiltration with cd -positive cells was observed in both dermis and epidermis, and this was highly correlated with the increase in epidermal thickness. finally, topical treatment with betamethasone- -valerate significantly reduced the ear swelling and epidermal thickness. we conclude that over-expression of vegf in the epidermal basal layer plays an important role in skin inflammation and for the development of important psoriatic hallmarks. the model may furthermore be used as an in vivo screening tool for novel anti-psoriatic compounds. background and aims: the diabetes-prone bb (bbdp) rat spontaneously develops insulin-dependent diabetes resembling type diabetes (t d) in man. the bbdp rat is t-cell lymphopenic with a profound lack of regulatory t cells. the recent thymic emigrants in bbdp rapidly undergo apoptosis unless rescued from apoptosis by tcr stimulation. the increase in apoptosis is due to a frameshift mutation in gimap which causes a severe truncation of the protein. the mutation is the strongest genetic factor for rat t d. we aim to detect how gimap affects the lifespan of t cells. results: overexpression in c cells of both wt gimap and gimap with the bbdp mutation causes an increase in apoptosis -the latter with a very rapid onset. reduction of human gimap by rna-interference in jurkat cells did not affect the number of apoptotic cells. overexpression of human gimap causes apoptosis in jurkat cells and primary naïve t cells but not in activated t cells. finally, gimap -mrna is upregulated in in vitro activated human primary t-cells (detected by rt-pcr). conclusions: based on the phenotype of the bbdp, rat gimap was expected to be anti-apoptotic. however, we report here that overexpression of both mutated and wt gimap causes rapid death of the cells. this suggests that gimap is pro-apoptotic. the results with human wt gimap support this conclusion: recently, much focus has been on the cellular cd / cadpr signaling system during inflammatory processes. the cd /cadpr system has been shown to be regulated by interferon, estrogen and the proinflammatory cytokine il- . to our knowledge, the expression and function of the cd /cadpr signaling system in the human detrusor muscle have not been described. cd protein expression in cultured (explant technique) human detrusor smooth muscle cells (hdsmc) was demonstrated by western blot (wb) and confocal microscopy (cm). cytosolic free ca + concentration ([ca +] i) in hdsmc and isometric force in human detrusor strips were measured by spectrofluorometry and myograph technique, respectively. wb and cm showed that hdsmc expressed cell surface cd which could be upregulated by il- ( ng/ml). in hdsmc briefly activated with il- ( ng/ml) cadpr induced a rapid, transient dose-dependent increase in [ca +]i. cyclic adpr-mediated ca + increase was greatly reduced in ca + free medium suggesting ca + entry as well as ca + release. cyclic adpr -elicited ca + increase was mimicked by -deaza-cadpr, and blocked by -bromo-cadpr, a cadpr antagonist, but not by nifedipine or verapamil. in the presence of il- , cadpr caused concentration-dependent relaxations of detrusor muscle. we report for the first time that ) hdsmc express cell surface cd , ) the expression and function of cd are augmented by il- , ) externally added cadpr elicited a rapid, il- -dependent, and -bromo-cadpr-inhibitable ca + mobilization, ) cadpr induces relaxation of human detrusor muscle. the study indicates a role of cd /cadpr in human urinary bladder inflammation. miao lin is a formulation of sen miao san and lingzhi that consists of cortex phellodendri, atractylodisa rhizoma, radix achyranthis bidentatae, and ganoderma lucidum. these ingredients are reported to have anti-inflammatory and analgesic effects. in this study, we have investigated the anti-arthritic property of miao lin in an animal model of arthritis induced by unilateral injection of freunds complete adjuvant (fca) into rat knees. contents of the miao lin capsules were dissolved in saline and administered to the rats daily by intraperitoneal or oral route for days before induction of arthritis and days after. extension angle, size and blood flow of the rat knee joints were measured to give indexes of algesia, oedema, and hyperaemia, respectively. assessments of the extent of cell infiltration, tissue proliferation, and erosions of cartilage and bone provided additional indexes of the arthritis condition. single unilateral injection of fca into rat knees produced significant oedema, algesia, hyperaemia, immune cell infiltration, synovial tissue proliferation, and erosions of cartilage and bone in the ipsilateral knees compared with the contralateral saline-injected knees. intra-peritoneal injection of miao lin ( mg/kg/day) suppressed oedema, pain and hyperaemia in the inflamed knees, and oral administration ( mg/kg/day) suppressed oedema and hyperaemia. histological examination showed that both routes of administrations of miao lin reduced immune cell infiltration and erosions of cartilage and bone, and intraperitoneally administered miao lin also attenuated synovial tissue proliferation. these findings suggest treatment with intra-peritoneal or oral miao lin could provide significant anti-arthritic effects. an extract of the anti-arthritic thermalife cream contains trace elements. diffusion studies were undertaken to assess the permeability of human epidermis to the trace elements. non-penetrating trace elements were discarded from the test formula (t ), and compared with the original formula (t ) for in vitro anti-inflammatory efficacy (tnf-a secretion in lps-challenged human monocytes). methods: human epidermis was mounted in vertical franz type diffusion cells (stratum corneum facing up). t cream (n= ) or no cream (n= ) was applied to the donor compartment of diffusion cells, with pbs in the receptor compartment ( . ml ; stirred continuously at c). min after administration the receptor fluid was analysed for presence of metal ions by icp-ms. a replication study used a different skin donor. subsequently, human monocyte cultures ( % fcs, % co ) were either stimulated with ng/ml lps (e.coli :b ,) or not in the presence of % t , % t , or no treatment. hours after incubation, culture media were collected, centrifuged, and assayed (cytokine elisa). statistical analyses used a treat by lps anova (p < . ). results: zinc was the only trace element to penetrate the human epidermis significantly. both formulations strongly suppressed lps-induced tnf-a secretion. t with zinc only was more effective than t (treat:f , = . , p < . ; lps:f , = . , p < . ; treat by lps:f , = . , p < . ). conclusions: anti-tnf efficacy from thermalife extracts was retained with zinc chloride as the only trace element. ( ) ( ) osprey pharmaceuticals limited, canada ( ) probetex, inc., texas, usa the ccl /ccr chemokine/receptor axis, infiltrating monocytes/macrophages (m/m), th cells and mast cells play a pathological role in tissue damage and fibrosis in kidney diseases. the eradication of the supernumerary activated leukocytes should curb the production of inflammatory mediators and modulate chemokine communications, thus ameliorating disease. a recombinant fusion protein comprised of the human ccl chemokine fused to a truncated form of the enzymatically active a domain of shiga toxin has been developed. the ccl portion binds specifically to ccr -bearing leukocytes and enters the cells, where the sa portion inhibits protein synthesis. the compound was tested in a model of anti-thymocyte serum (ats)-induced mesangioproliferative glomerulonephritis. male rats were injected with ats on day and treated intravenously with vehicle, or mg/kg of the recombinant protein q d from day until day . urine and blood collections were made prior to ats injection and on days and . animals were sacrificed on day . no treatment related effects on body weight or signs of clinical toxicity were observed. urine protein levels were decreased in treated animals. histopathological analyses of kidney sections revealed maximum reductions of , , , and % for glomerular lesions, m/m count, fibronectin and µ-smooth muscle actin, respectively. the latter two proteins are markers for extracellular matrix synthesis and mesangial cell activation, respectively. these results indicate a significant renal-protective effect in this model of nephritis. further observations suggest that different chemokine-ligand toxins may be used in the treatment of diseases modulated by other chemokine/receptor axes. inflamm. res., supplement ( ) posters immuno-depletion followed by fluorescence-activated cell sorting based on the cell surface expression of the sca- antigen. such isolated cells can subsequently be cultured and differentiate towards the osteogenic, adipogenic or chondrogenic linage in vitro. using this model we investigated the influence of the proinflammatory cytokines, tnfa or il- b, on early osteogenesis in vitro. under osteogenic conditions, il- b was found to inhibit cell proliferation in a dose dependent manner, whereas tnfa exhibited no effect. histochemical examination revealed the presence of either tnfa or il- b to dramatically decreased mineralization in a dose dependent manner. q-pcr analysis indicated that in the presence of il- b, despite increased expression of bone-specific alkaline phosphatase (akp ) mrna, levels of other osteogenesis markers (runx , col a and sp ) were decreased. in the presence of tnfa, levels of akp , runx and sp were all decreased. our findings indicate that the influence of early mesenchymal progenitor cells on bone remodelling may be substantially altered in the presence of proinflammatory cytokines. coronary artery disease (cad) is characterized by enrichment of inflammatory cells in the vessel wall. we hypothesized that an altered transmigration and activation of monocytes may contribute to plaque build up. in vivo transmigration was studied by use of a skin blister model. blisters are raised by suction and cells are analysed the following morning ( h blister) and after additional ten hours of incubation with pbs or autologous serum, corresponding to intermediate and intense blister. monocytes were analysed by flow cytometry for the expression of cd b, before and after in vitro fmlp stimulation. chemokines in serum and blister fluid was analysed in parallel. cd b expression on resting monocytes harvested from h blister was lower in patients as compared to controls (p= . ). lower expression of cd b in patients was also observed in the intermediate and intense blisters after stimulation with fmlp (p= . and p= . , respectively). the number of transmigrated cells was similar in both groups and increased with the intensity of inflammation. serum concentration of mip- µ was higher among patients (p= . ) and similar levels were seen in blister fluids. concentration of mcp- was similar in both serum and blister fluid. we demonstrate that monocytes from patients with cad have a reduced expression and ability to up-regulate the adhesion molecule cd b at sites of inflammation. these differences may modulate events related to the transmigration process and indicate a changed activation pattern. to which extent this feature might contribute to monocyte entrapment at the atherosclerotic site needs further studies to be delineated. in inflammation, nitric oxide (no) is produced by inducible nitric oxide synthase (inos) induced by bacterial products and cytokines, and no acts as a regulatory and proinflammatory mediator. one of the anti-inflammatory mechanisms of glucocorticoids is the inhibition of no production. the aim of the present study was to investigate the mechanisms how glucocorticoids inhibit inos expression and no production. dexamethasone and a dissociated glucocorticoid ru inhibited no production, and inos protein and mrna expression in murine j macrophages exposed to bacterial lipopolysaccharide (lps). in the presence of a glucocorticoid receptor (gr) antagonist mifepristone, the effects of dexamethasone and ru on no production were reduced. the role of histone deacetylation in the glucocorticoid effect was studied by using three inhibitors of histone deacetylases (hdacs); non-selective trichostatin a and apicidin, and hdac selective mc . hdac inhibitors reversed the effects of dexamethasone and ru on inos expression or no production. stably transfected a / cells containing human inos promoter were used in promoter-activity studies. cytokine-induced inos promoter activity was inhibited by dexamethasone and the inhibitory effect was reversed by trichostatin a. these results suggest that glucocorticoids inhibit inos expression and no production by a gr-mediated and gre-independent manner possibly through histone deacetylation and transcriptional silencing. we are investigating mechanisms involved in tnfainduced hyperalgesia in the mouse paw. previously, we have seen that tnfa causes a trpv -dependent bilateral hyperalgesia. here we investigate the role of cox in this process. female cd mice ( - g) were given intraplantar injections (i.pl.) of tnfa ( pmol/ microl) and tyrode (as vehicle, contralateral paw; microl). thermal hyperalgesic thresholds were measured using the hargreaves technique before and h after injection. indomethacin ( mg/kg) was co-injected with tnfa whilst contralateral paw was injected with tyrode and corresponding amounts of indomethacin vehicle ( % nahco ). another group of mice were injected with tnfa i.pl. plus % nahco with the contralateral paw injected with tyrode plus indomethacin ( mg/kg). results are expressed as mean ae s.e.m and statistical analysis performed using students t-test. tnfa ( pmol) leads to significantly reduced (p< . compared to baseline values) paw withdrawal latency in both paws h after injection i.e bilateral hyperalgesia. however, local injection of indomethacin ( mg/kg) with tnfa prevented this reduction in paw withdrawal latency in both paws suggesting that prostaglandins are important in the development of hyperalgesia. interestingly, indomethacin co-injected with tyrode in the contralateral paw did not prevent the reduction in paw withdrawal latency in both paws. the same results were seen using the selective cox- inhibitor, nimesulide. in conclusion, cox- derived prostaglandins are important in the development of hyperalgesia. local cox- inhibition at the site of tnfa-induced inflammation prevents the bilateral hyperalgesia suggesting that local prostaglandin production is sufficient to cause hyperalgesia in the contralateral paw. hydrogen sulfide (h s) is synthesized naturally in the body from cysteine by cystathionine g lyase (cse). h s has been variously reported to exhibit both pro-and antiinflammatory activity. in an attempt to obtain further information about the role of h s in inflammation we examined the effect of dexamethasone on lipopolysaccharide (lps)-mediated endotoxic shock. male sprague dawley rats ( - g) were administered dexamathasone ( mg/kg, i.p.) either h before or h after lps ( mg/kg, i.p.) injection. animals were killed h after lps administration and plasma and tissues harvested. as expected, lps injection significantly increased plasma tnfa and il- b as well as liver and lung myeloperoxidase (mpo) activity. lps also increased plasma nitrate/ nitrite (nox), h s concentration and liver and kidney h s synthesis from exogenous cysteine indicative of upregulation of cse in these tissues. either pre-or post treatment of animals with dexamethasone reduced signs of inflammation and also reduced the increase in plasma h s and tissue h s synthesizing activity. in separate in vitro experiments, exposure of rat peripheral leucocytes to lps ( ng/ml, h, oc) resulted in upregulation of both cse and inos (measured by western blot). dexamethasone ( nm) significantly (p< . ) reduced expression of both cse and inos. these data provide further evidence that h s is synthesised during endotoxic shock and suggest, for the first time, that at least part of the anti-inflammatory effect of dexamethsone may be related to inhibition of h s production. ( ), u jalonen ( ), h kankaanranta ( ), r tuominen( ), e moilanen ( ) ( ) the immunopharmacology research group, medical school, university of tampere and tampere university hospital, tampere, finland ( ) the division of pharmacology and toxicology, faculty of pharmacy, university of helsinki, helsinki, finland tristetraprolin (ttp), also known as nup , tis , g s and zfp , is a factor that binds to utr of mrna of some transiently expressed inflammatory genes and regulates mrna stability. ttp has been implicated in the posttranscriptional regulation of e.g. tumor necrosis factor a and inducible nitric oxide synthase. however, the regulation of the expression of ttp itself is largely unknown. in the present study, we investigated the role of classical protein kinase c (cpkc) isoenzymes in the regulation of ttp expression. in j macrophages ttp expression is induced by lipopolysaccharide (lps) and this can be further enhanced by addition of nm phorbol myristate acetate (pma). this additive effect of pma on ttp was abolished by a prolonged preincubation with a higher s inflamm. res., supplement ( ) posters concentration of pma for h, which also downregulated the expression of pkca, pkcbi and pkcbii isoenzymes. pkc inhibitors ro (inhibits pkcb, & and e), gÖ (inhibits pkca, b and &) and cgp (inhibits pkcbii) reduced lps + pma -induced ttp protein and ttp mrna expression. pkcbii inhibitor cgp did not affect ttp mrna half-life and therefore we measured the effects of cgp on the activation of transcription factors involved in ttp expression. cgp had no effect on the activation of nf-kb, egr or sp . in contrast, cgp reduced the activation of transcription factor ap- , which may explain its inhibitory action on ttp expression. the results suggest that pkcbii is involved in the regulation of ttp expression in activated macrophages, possibly through the activation of transcription factor ap- . the most widespread gracilaria verrucosa in the sea of korea is the attached form of red algae growing on a rockly substrate. in this study, we isolated fourteen compounds from g. verrucosa and investigated their inhibitory effect on the production of inflammatory markers (tnf-a il- , il- and no) in raw . cells. among them, -oxooctadec- -enoic acid and -oxooctadec- -enoic acid inhibited the production of tnf-a, il- , il- and no at the concentration of mg. also, these two compounds showed inhibitory activity on the mrna expression and protein level of inflammatory markers (tnf-a il- , il- and inos) in a dose-dependent manner. these results suggest that g. verrucosa may have anti-inflammatory activity through the inhibition of inflammatory cytokines and inos. lene jensen( ), p hjarnaa ( ), j fensholdt ( ), p-p elena( ), k abell ( ), tk petersen ( ) ( ) discovery, leo pharma, ballerup, denmark ( ) iris pharma, la gaude, france angiogenesis is known to play an important role in many inflammatory diseases including arthritis. additionally, inflammation is known to play a role in the angiogenesisdriven disease age-related macular degeneration (amd). we have synthesized a potent angiogenesis inhibitor, leo-a, targeting kinases related to angiogenesis, e.g. vegfr- . additionally, leo-a has potent effects on a broad panel of other kinases, whose normal functions are related to inflammation and immunity. the compound was tested systemically in inflammatory in vivo models in mice and rats. the in vivo models selected include the cia arthritis model (mice and rats), the local gvh rat model, the lps induced tnfa model (mice and rats), the anti-cd induced il- response mouse model and the rat argon laser-induced choroidal neovascularisation (chnv) model, a model for amd. the following results were obtained after systemic treatment with doses of up to mg/kg i.p. or mg/kg p.o. once daily: in the local gvh model, leo-a significantly inhibited the growth of the local lymph node by %. in the cia model, leo-a had a significant inhibitory effect on the progress of arthritis both in mice and in rats when dosed early (pretreatment). in the lps induced tnfa model in mice, high doses of leo-a were found to inhibit the tnfa release. in the chnv model, a significant effect was obtained following systemic treatment. in conclusion, leo-a has an interesting profile for the treatment of diseases in which inflammation and angiogenesis are involved. mice lacking pi kg and d isoforms display severe impairment of thymocyte development, but the outcome of this developmental defect has not been investigated. we show here that mice harbor pi kg gene depletion and pi kd kinase-inactive mutation, pik cgd koi, exhibited thymus atrophy, similar to previously reported pi kg and d double knockout (p g/d-/-) mice, and profound peripheral lymphoid depletion, markedly reduced lamda chain production and seemingly lymphopenia-provoked effector/memory t cell activity. in particular, serum igg / igg a ratio and ige level were elevated in pik cgd koi mice corresponding to a skewed th profile in vitro. histological analysis revealed eosionophil-and t celldominated inflammation in stomach and salivary gland as well as occasionally other organs of pik cgd koi mice, but organ-specific auto-antibody was not detected in circulation. on the contrary, when mature wt t cells were treated with pi k d or together with pi k g selective inhibitors, while th cytokines were suppressed th cytokines were not augmented in vitro. thus, t cell development, but not peripheral t cell proliferation or cytokine production, requires cooperativity of pi kg and d. genetic inactivation of these two isoforms leads to the development of severe lymphopenia, skewed type ig and t cell response, and increased susceptibility to eosinophilic multiple organ inflammation; whereas pharmacological inhibition at the adult stage would probably not promote th reaction but attenuate th medicated disorders. platelet activating factor (paf) is an important mediator in several pathophysiological processes. paf receptor activation can causes a series of cellular and tissue modifications and can lead to the production and/or release of diverse molecules, including cytokines, chemokines and receptors, amongst others, which are capable of amplifying the inflammation. paf can up-regulate kinin b receptor expression by various mechanisms. our aim was to investigate the role for kinases in paf-induced kinin b receptor up-regulation. wistar rats were treated with paf, or left untreated as controls, h before i.d. injection of . ml pbs containing des-arg -bradykinin (dapk, nmol right hind paw) and . ml pbs (for control, left paw). various kinase inhibitors were administered to the rats after paf treatment and oedema was measured by the use of a plethysmometer (ugo basile) - minutes after dapk-injection. oedema was expressed in ml as difference between right and left paws.additionally paw samples were taken for western blot analysis for total and phosphorylated forms of jnk and erk / . dabk-induced paw oedema after pafinjection is significantly inhibited by the selective jnk sp and erk / pd inhibitors. western blot analysis shows that phosphorylation of jnk and erk / is important in the up-regulation of b receptors. our results clearly show that the phosphorylation of both erk / and jnk mapkinases is an important step for the in vivo up-regulation of b receptors by paf. however, the exact mechanisms (transcriptional and post-transcriptional) by which paf can trigger kinase phosphorylation and then up-regulate the b receptor require further investigation. continued interest in development of small molecule inhibitors of p mitogen-activated protein (map) kinase is based on the central role this enzyme plays in inflammatory cell signaling. activation of p leads to increase production of pro-inflammatory cytokines such as tnf-a and il- b making it an prominent target for antiinflammatory drug discovery. a virtuell screening approach identified -( -chlorophenyl)- -(( -methoxyphenoxy)methyl)- [ , , ] triazolo [ , -b] [ , , ] thi adiazole as a potential hit. this was confirmed by synthesis and testing. to explore further sar, a first set of derivates was prepared by cyclization of the -substituted- -amino- -mercapto- h- , , -triazoles with carboxylic acids in presence of phosphorus oxychloride. the synthetic strategy used allows both variation at position and . synthesis and sar will be presented. cytokines like il- b and tnfa play central roles in inflammatory diseases like rheumatoid arthritis. production of cytokines in monocytes, macrophages and other cells is triggered by factors such as lps, uv-light, osmotic and cellular stress or physical and chemical attraction. in particular il- b and tnfa are key regulators as they amplify inflammatory stimuli in cells by induction and upregulation of further cytokines. involved in this signal pathway, p mapk as a pivotal enzyme is considered to be a validated drug target and therefore, p mapkinhibitors are of therapeutical interest. in this study, we developed and validated an economic in vivo whole blood assay for optimization and characterization of small molecule p mapk-inhibitors with promising in vitro activity. the assay procedure involves defined blood cell stimulation by lps and isolation of tnfa or il- b, which are subsequently quantified by tmb-elisa technique via photometric measurement. the validation of the assay conditions involved well characterized p mapk inhibitor sb and a highly active compound developed in our lab. a data set was generated by determining whole blood samples consisting of in each case three male and female individuals on three different days. statistical methods were used to analyze specificity, baseline-peak correlations, repeatability, robustness as well as gender specific intra-and interindividual differences. p mitogen-activated protein (map) kinase is required for the biosynthesis and release of pro-inflammatory cytokines il- and tnf a. inhibition of p map kinase could reduce the expression of these cytokines and is therefore a promising target for the treatment of many inflammatory disorders, like rheumatoid arthritis and inflammatory bowel disease. trisubstituted pyridinylimidazoles are potent inhibitors of the p map kinase. scope of this work was to investigate -thio-ether moiety as a position to link the inhibitors to macrocyclic drug carriers. we synthesised -alkylsulfanyl, -( -fluorophenyl), -( -aminopyridin- -yl) substituted imidazoles as p map kinase inhibitors. as substitution at this pyridinyl moiety allows both increase the anti-inflammatory activity as well as selectivity. the synthesis and biological testing of effective the -aminopyridin- -yl imidazoles with low inhibitory concentrations are described. biological data demonstrate both the imidazole derviates and the linked imidazoles lead to highly efficient inhibitors.variation at the -thio-ether moietywhich interacts in the phosphate binding region of the enzyme -with polar groups shows no loss of activity. studies underscored the importance of hydrogen bonding with the backbone nh group of met , for inhibitory activity. less clear is the importance of the hydrogen bond between n of the imidazole ring and lys of p map kinase.to investigate the role of lys in interacting with the scaffold we prepared two sets of , diaryl-substituted isoxazoles. these data suggest a dynamic interaction of the core heterocycle with lys , contrary to the observation on the compound vk- and p map kinase, that a nitrogen atom bearing a lone pair in position of the imidazole ring could be necessary to avoid a repulsive interaction with the positively charged side chain of lys rather than to form an attractive interaction with p map kinase. to complete our study, we focused on the interdependency of biological effects exerted by substitution at the pyridine ring for a series of -substituted and unsubstituted , -diarylisoxazoles investigating the interaction with the hydrophobic pocket ii of p . these data indicate that the isoxazole has better scaffold properties compared with imidazoles, suggesting that heterocycles that are stable as regioisomers, such as isoxazole (in contrast to tautomeric imidazoles), are worthy of further investigations. despite of the intensive research effort, sepsis is still the leading cause of death in critically ill patients. it is a consequence of acute inflammatory response to lipopolysaccharide (lps), a major component of the outer membrane of gram-negative bacteria. natural products are known sources of bioactive components exerting antioxidative and anti-inflammatory effects. in this study, we investigated the effect of ferulaldehyde (fa), a natural compound of red wine, on lps-induced endotoxic shock in mice and on lps-stimulated murine macrophage-like raw . cells. treatment of c bl/ mice with fa significantly attenuated the lps-induced inflammatory response in the gastrointestinal tract, and decreased the level of the two major pro-inflammatory cytokines tnf-a and il- b in the serum. the serum level of the anti-inflammatory cytokine il- was higher in mice treated with fa and lps compared to lps treatment alone. lps-induced phosphorylation and thereby activation of akt, and jnk was also strongly inhibited by fa treatment whereas the phosphorylation level of erk / and p mapks remained unaltered. activation of nuclear factor-kappab (nf-kb) in liver of fa-treated mice were significantly suppressed. although fa had no effect on the production of inflammatory cytokines, or on inhibition of signal transduction pathways in raw . cells either, it decreased the lps-induced ros and nitrite production in a dose-dependent manner. our results suggest that fa has antioxidative and anti-inflammatory activities by enhancing antioxidative defense systems, which in turn decrease inflammatory cytokine response and suppress nf-kb activity via the down-regulation of akt and jnk. myeloperoxidase (mpo), stored in the azurophilic granules of the neutrophil granulocyte, is a heme enzyme with the unique property of oxidising chloride ion to the powerful reactant hypochlorous acid in the presence of hydrogen peroxide. therefore, it plays an important role at inflammatory loci in killing invading micro-organisms. on the other hand, hypochlorous acid reacts with a variety of biomolecules as amino acids or membrane lipids and causes therefore host tissue damage resulting in widespread diseases like atherosclerosis or rheumatoid arthritis, e.g. the formation of chloramines from taurine or ammonium ions is one possibility to reduce tissue toxicity while maintaining bactericidal properties. membrane charge alterations during apoptosis provide docking sites for the kationic enzyme myeloperoxidase and this close contact to the membrane lipids opens the possibility for lipid alteration pathways even though these reactions will normally not take place because of their slowness. we investigated alterations in phospholipids after reaction with hypochlorous acid or the myeloperoxidase-hydrogen peroxide-chloride system by matrixassisted laser desorption and ionisation time-of-flight mass spectrometry (maldi tof ms). specific reaction products play an important role in the modulation of the immune response. comparative pathobiology of the disease is also discussed within the context of current human and animal reoviral disease models. objectives: to study the safety and efficacy of infliximab plus leflunomide combination therapy in adult rheumatoid arthritis (ra). methods: twenty patients with active ra received leflunomide mg for days followed by mg daily for weeks. at week all patients started infliximab mg/kg, and received a further four infusions at weeks , , and . results: the commonest adverse event was pruritis associated with an eczematous rash. there was no relationship between the serum concentration of a , the active metabolite of leflunomide, and adverse events. the mean disease activity score (das ) fell from . at week to . (p< . ) at week and remained between . and . up to week . in those patients remaining on treatment, more than % achieved an acr response from week to week , and up to % achieved an acr response. conclusions: infliximab plus leflunomide combination therapy appears to be highly efficacious in the treatment of adult ra. however, widespread use may be limited by adverse events, which were common and in some cases severe. objective: the transcription factor nuclear factor-kb (nfkb) regulates the expression of proinflammatory cytokines such as tnfa and il- those play pivotal roles in pathogenesis in rheumatoid arthritis. parthenolide, a sesquiterpene lactone, was reported to inhibit the dna-binding of nfkb. the objective of this study is to investigate the potential of parathenolid to inhibit the pathogenesis of collagen-induced arthritis. methods: mice were injected i.p. with a cocktail of anticollagentype ii mabs on day , followed by i.p. injection of lps on day to induce anti-collagen mab-induced arthritis. the mice were orally administrated with parathenolide ( mg/kg/day) starting on the day of first immunization (day ) in prophylactic treatment group and after the onset of arthritis (day ) in the therapeutic treatment group. clinical disease score, radiographic and histological scores were evaluated. mrna expression of il- b and tnfa in the affected joints were measured by real-time pcr. results: clinical disease scores were significantly reduced both in prophylactic treatment group ( . ae . ) and therapeutic treatment group ( . ae . ) compared to untreated group ( . ae . , p = . and p = . respectively). histological scores of joint destruction were significantly reduced in prophylactic treatment group compared to untreated mice (p< . ). steady state mrna levels of il- b and tnfa in isolated joints were significantly decreased in prophylactic treatment group compared to untreated mice (p< . ). the results in this study suggest that nfkb is an important therapeutic molecular target for treatment of inflammatory arthritis. fibrinogen is a soluble plasma glycoprotein, multifunctional, that participates in haemostasis and has adhesive and inflammatory functions through specific interactions with other cells. the concentration of this glycoprotein increase in inflammatory conditions. a fundamental paradigm involved in the acute inflammatory response is neutrophil migration to the affected tissues to mount an initial innate response to the aggression. the objective of this study is to characterize how fibrinogen modulates the pattern of neutrophil activation. neutrophils from healthy donors were isolated from peripheral venous blood and loaded with the fluorescent probe dihydrorhodamine ( ìm) to detect oxygen free radical production. the cells ( , x cell/ml) were then incubated with a range of concentrations of fibrinogen ( - mg/dl) for minutes. our results show that posters inflamm. res., supplement ( ) s fibrinogen leads to an increase in neutrophil activation as measured by free radical production. this effect becomes evident at borderline-high concentrations ( - mg/ dl), and in some of the individuals it was possible to differentiate two subpopulations of low-responsive and high responsive neutrophils to activation by fibrinogen. we hypothesize that, in this regard, the concentrations of fibrinogen identified as a risk factor might promote the setting of an inflammatory microenvironment in the circulation and facilitate cardiovascular disease progression. cyclooxygenases (cox- and cox- ) are isoenzymes involved in the first steps of the biosynthesis of prostanoids. the constitutively expressed isoform cox- is mainly involved in homeostatic processes, while the inducible isoform (cox- ) is associated with inflammatory reactions. various in vitro assays have been developed in order to define the selectivity against cox- and cox- of nonsteroidal anti-inflammatory drugs (nsaids). however, these in vitro assays can give discordant results related to several parameters. the aim of this study was to optimize and standardize two distinct in vitro methodologies to evaluate new nsaid candidates. first, in an enzymatic cox assay, the arachidonic acid concentration (aa; cox substrate), and the species of cox enzymes tested (ovine vs. human), two factors able to conceal the anti-cox activity of nsaids, have been evaluated and optimized. next, we developed an in vitro cell-based assay using human whole blood depleted from plasma and reconstituted in saline solution. this cell-based assay allows concomitant measurement of anti-cox- and anti-cox- effects by prostaglandin e (pge ) measurement after a (calcimycin) and bacterial lipopolysaccharide (lps) stimulations, respectively. both assays have been calibrated and compared by testing reference nsaids, selective or not for cox- or cox- . fifty % inhibitory concentration (ic ) values against cox- and cox- and cox- :cox- ratios obtained were in accordance with the previously described nsaid specificity and coherent between both assays (r= . ). in conclusion, both in vitro assays are optimized to determine the efficiency and the selectivity of new nsaid candidates against human cox- and cox- . for increasing the success of preclinical drug candidate molecules, there is a need for translatable animal models. the human serum skin chamber technique and the rodent carrageenan induced air pouch model are two wellestablished methods for measuring interstitial inflammation in respective species. we aimed to study the translational aspects of these models. material and methods: in humans, epidermal skin chambers were stimulated with autologous serum for hours. in rats, a dorsal subcutaneous air pouch was stimulated with autologous serum on day . the inflammatory response was measured after , and hours. the cellular distribution of in vivo transmigrated cells, the expression of cytokine receptors, adhesion molecules and inflammatory mediators was investigated. results: at / hours the cellular distribution was similar in air pouch and skin chambers. the major population constituted of granulocytes, followed by monocytes/macrophages and lymphocytes. both in human and rats the concentrations of mpo and mcp- were increased. furthermore, transmigrated cells displayed a different chemokine receptor pattern. in rats transmigrated cells expressed cd b, were cd lo, ssclo and rp- + (granulocyte marker). in humans, transmigrated granulocytes expressed cd and cd b. these cells had a significantly higher cd b expression compared to corresponding cells in peripheral circulation. our results indicate that the serum induced human skin chamber technique and rodent air pouch model translate well to each other. these models may be useful for bridgingpreclinical and clinical drug discovery. furthermore, they may work as translatable proof of mechanism (pom) models for drug candidates targeting different inflammatory components. objectives: to analyse if neopterin (a by-product of activated macrophage metabolism) is elevated in patients with systemic inflammatory insult at the time of ischemic stroke. material and methods: we investigated consecutive patients with mean age ae . years who were admitted within h after ischemic stroke. a control group of patients with mean age ae . years without ischemic stroke was also tested. measurement of serum neopterin levels were performed using enzyme linked immunosorbent assay. results: patients with acute ischemic stroke had significantly higher serum levels (mean value+sd) of neopterin than those without acute ischemic stroke: . ae . and ae . nmol/l. correlation analysis revealed p< . . discussion: immune mechanisms contribute to cerebral ischemic injury. the finding of higher serum levels of neopterin, which is regarded as a humoral component of the immune-mediated inflammatory response sustains the hypothesis that patients with ischemic stroke may show higher levels of inflammatory markers like neopterin. our results indicate increased macrophage activation after ischemic stroke. in patients with stroke it has been shown that neopterin was determinant of endothelium-dependent vascular dysfunction. however, these preliminary results need be confirmed by controlled studies. produced a marked (p< . ) reduction in the number and duration of ventricular tachycardia (vt) during both ischemic and reperfusion phases. the total number of ischemic ventricular ectopic beats (vebs) reduced from ffae in the control to ffae at the concentration of ffmg/ml (p< . ). in the ischemic phase, cynodon dactylon ( ffmg/ml) also decreased the incidence of vt from % (control) to %. in addition, incidences of reperfusion-induced vt, total vf and reversible vf duration were significantly lowered by the same concentration (p< . for all). the results show that cynodon dactylon has a protective effect against i/r-induced cardiac arrhythmias in isolated rat hearts. regarding the presence of flavonoid glycosides confirmed during phytochemical screening of the extract and their potential role in the scavenging of oxygen free radicals, it seems that the cardioprotective effects of cynodon dactylon probably is due to its anti-inflammatory properties.key words: cynodon dactylon; arrhythmias; anti-inflammatory; isolated heart; rat objectives: intestinal ischemia-reperfusion (iri) is well known to be associated with distant organ dysfunction; but no evidences to date have focused either the brain or skeletal muscle. we thus decided to investigate the effects of iri on nos and cox isoforms, neutrophil infiltration (mpo), lipoperoxidation (tbars) and protein tyrosine nitration (nt) in different brain areas and diaphragm muscle of wistar rats. methods: iri comprised the occlusion of superior mesenteric artery during min followed by h of reperfusion. sham animals were submitted to the surgical procedure with no interference on the blood flow. results: iri resulted in increased expression of mrna for nnos (cortex) and cox- (hypothalamus) associated to a marked reduction of ca +-dependent nos activity in cortex, hypothalamus and hippocampus (but not in cerebellum). tbars contents were also reduced in cortex and hypothalamus. neither mpo activity nor nt was altered by iri in the brain. diaphragms from animals with iri exhibited increased mpo and ca +-dependent nos activities, as well as tbars content and nt. in contrast, enos protein expression and both gene and protein nnos expression were reduced. no effects were observed on cox isoforms or enos gene expression. conclusions: these findings suggest that, within the first h of reperfusion following intestinal ischemia, an oxidative response is observed in diaphragm, involving both lipid and protein modifications. in the cns, distinctive susceptibility to the iri seems to occur in the different areas, probably as a defensive strategy aimed to counteract the iri-mediated systemic injury. anne-sofie johansson ( ), h qui ( ), m wang ( ), i vedin ( ), jz haeggstrçm ( ), j palmblad ( ) ( ( ), r carnuccio( ), p romagnoli ( ), f rossi ( ) ( ) second university of naples, italy ( ) university of naples, italy ( ) university of florence, italy we previously found that several inflammatory markers, e.g., nuclear factor-kb (nf-kb), were increased and a neointima was formed in a model of carotid surgical injury ( ) . the purpose of the present study was to determine if chronic treatment with rosiglitazone protects rat carotid artery from surgical injury induced by an incision of the vascular wall. to this aim we measured cox- , nf-kb, platelet aggregation and neointima formation in rats administered rosiglitazone ( mg/kg/ die, by gavage) for days before carotid injury and days after injury. control rats received physiological solution. days after injury cox- expression, evaluated by western blot, was significantly lower in the treated carotid versus controls (p< . ). rosiglitazone also caused a significant decrease of nf-kb/dna binding activity, evaluated by electrophoretic mobility shift assay, in nuclear extracts of treated carotids at all time points considered. platelet aggregation was reduced by % in treated versus control carotids (p< . ). the influx of inflammatory cells in response to injury, monitored by electron microscopy and immunohistochemistry, was lower in treated than in control carotids starting days after rosiglitazone treatment. the results indicate that rosiglitazone inhibits molecular and cellular inflammatory events induced by vascular injury. the aim of the present study was to investigate the relevance of peripheral macrophage activity for the susceptibility to the induction of experimental allergic encephalomyelitis (eae). rats of eae-susceptible dark agouti and eae-resistant albino oxford strain were immunized with guinea pig spinal cord homogenate (dagpsc and aogpsc), while non-immunized rats served as controls (danim, aonim). on day after immunization rat peritoneal macrophages were tested for adherence capacity, zymosan phagocytosis and respiratory burst. macrophages from aonim rats exhibited lower adherence capacity and higher phagocytosis and h o production then macrophages from danim rats. immunization decreased adherence and phagocytosis and increased h o production in macrophages from ao rats, but did not influence these activities in macrophages from da rats. our results suggest that inflammatory activities of macrophages from ao rats could be considered as regulatory mechanisms connected with the resistance to eae induction ( ( ), b sehnert ( ), h lanig( ), s päßler( ), r holmdahl ( ), h burkhardt ( ) ( ) johann wolfgang goethe university, frankfurt, germany ( ) friedrich-alexander university of erlangen-nuremberg, germany ( ) lund university, sweden objectives: the aim of the present study was to characterize the interaction sites between the prototypic arthritogenic murine igg mab ciic that is highly somatically mutated and its epitope on type ii collagen (cii, aa - ). methods: the establishment of a dynamic simulation modelling of a ciic single-chain fragment (scfv) in complex with the triple helical ciic epitope permitted structural insights into immune complex formation. the computer-based data were experimentally tested by mutations of predicted critical residues into alanine in the c scfvs and the respective ciic epitope that were produced as recombinant constructs. the binding affinities of the mutated scfvs were determined by elisa and surface plasmon resonance measurements. the mutation experiments confirmed the predicted interaction sites of cii in the cdr and cdr regions of both heavy andlight chain. surprinsingly also the model prediction, that the conversion of the c scfv sequence into the respective germline does not affect cii binding affinity (kd x - ) could be confirmed experimentally by the mutagenesis of (!) positions. our data indicate that potentially harmful cartilage specific humoral autoimmunity is germline encoded. the molecular modeling further demonstrate that the rigid collagen triple helix restricts the likelihood of molecular interactions with the corresponding cdrregions of the antibody considerably compared to globular antigens. these sterical constraints might provide an explanation why somatic mutations have no obvious impact on cii recognition by the arthritogenic autoantibody. moreover, the structural insights into cii-autoantibody interaction might be useful in future developments of collagenomimetic ligands for therapeutic and diagnosistic purposes. we observed a significant association between the mbp-elicited cd + t-cell proliferation and active brain lesions, on the one hand, and il- , il- and ifn-gamma, on the other. when grown in the presence of standard serum from a healthy donor, pbmc from healthy individuals responded to mbp with a higher il- production than pbmc from ms patients. thus, normal pbmc respond to mbp with production of tnf-alpha, ifn-gamma and il- , but ms is associated with enhanced tnf-alpha-, ifn-gammaand decreased il- responses, and disease activity is associated with mbp-induced proliferation of cd + t cells. ( ), k goula ( ), p georgakopoulos ( ) ( ) renal unit, st. anrdew hospital, patras, greece ( ) intensive care unit, st. anrdew hospital, patras, greece background: urethritis is an infection of the urethra. most cases are sexually transmitted. haemodialysedpeople seem more prone to all kinds of urinary tract infectionsthan others. patients with underlying diabetes are also a specific population at risk. urethritis may be caused by some sexually transmitted diseases (chlamydia, gonorrhea, and ureaplasma urealyticum infections) and by the same organisms that cause urinary tract infections (e. coli or klebsiella). viral causes of urethritis include herpes simplex virus and cytomegalovirus. neisseria gonorrhoeae and c trachomatis account for most cases of urethritis in men ( %). the aim of our study was to determine all cases of urethritis of haemodialysed patients at our unit during the last five years. we also determined diabetes as a coexisting factor in the infected patients. we retrospectively reviewed all cases of urethritis of maintenance haemodialysis patients at our center over the past years. the diagnosis was made according to patients symptoms and signs but also using urine specimens for culture. patients ( . %) from the study group were diabetic. results: cases of urethritis were determined. all infected patients were diabetic. isolated microorganisms were e. coli ( cases), enterobacter aerogenes ( case objectives: to explore the ability to use paquinimod as a steroid sparing drug in an animal model for sle. methods: mice were initially treated with a high dose of prednisolone ( mg/kg/day). thereafter the amount of steroid was reduced to . mg/kg/day and a low dose of paquinimod ( . mg/kg/day) was added. the development of glomerulonephritis was measured as hematuria during the experimental period. serum was collected for analysis of anti-dsdna antibodies. kidneys were collected and histopathological observations were performed. organ weight and lymphocyte sub-populations were assessed in the spleen. results: when treatment with high dose prednisolone was replaced by low dose prednisolone andpaquinimod a steroid sparing effect was seen in a number of variables. a significant reduction in the level of hematuria, in spleen enlargement and in the total number of cd +, cd + and on cd -cd -t cells was observed in mice treated with paquinimod and low dose of prednisolone compared to mice treated with high dose prednisolone alone. the development of glomerulonephritis was also significantly reduced in these mice. an almost complete inhibition of anti-dsdna in serum was seen in all treated groups. conclusions: when high dose prednisolone was replaced by low dose prednisolone and paquinimod a steroid sparing effect was seen when a number of variables e.g., hematuria, t-cell sub-populations and development of glomerulonephritis were examined. this setting could be of great importance in future treatment of human sle in order to reduce the steroid dose needed in the treatment of this disease. and la(ssb). the purpose of this study was to screen for novel antibodies against cell surface antigens in primary sjs. proteins (mp) were isolated from cell membranes (hela cells), and were tested with sera from sjs patients or healthy blood donors individually in western blot (wb) at : . mp were separated on -d gels and tested in wb ( : ) to locate the appropriate spots for mass spectrometry (ms) analysis. paraformaldehyde fixed hela cells were incubated with sera from patients or blood donors and examined by fluorescence microscopy. antigens were isolated at around , , , kda ( total positive/ tested patients). the dominant antigen was at kda. large quantities of endogenous proteins were obtained and the membrane fraction was enriched. one of the main obstacles to further study possible surface antigens as m muscarinic receptor was overcome. proteins were separated on d-gels and tested in wb to locate the relative spots for ms. the correct localization of the patients antibodies on the cell surface was confirmed by fluorescence microscopy. in conclusion, membrane or membrane-associated antigens were recognised by sera from sjs patients. one of them might correspond to m muscarinic receptor. this identification might help in developing a diagnostic assay for sjs. osamu handa, s kokura, k mizuahima, s akagiri, t takagi, y naito, n yoshida, t yoshikawa aim: various additives and preservatives are used in cosmetics, foods and medicines in order to prevent deterioration. however the precise mechanism of cytotoxicity of these additives are not known. in this study, we investigated the effects of ultraviolet-b (uvb) exposure on additives-treated human normal skin keratinocytes (hacat). most popularly used additives in cosmetics such as methylparaben (mp), octandiol (od) and phenoxyethanol (pe) were used. hacat keratinocyte was cultured in mp-containing medium for h, exposed to uvb and further cultured for another h. subsequent cellular viability was evaluated by fluorescent microscopy and flow cytometry using double staining method with hoechst and propidium iodide or annexin-v. same experiments were done using od and pe respectively instead of mp under same condition. in addition, gene chip analysis was performed in each group. results: uvb exposure enhances cytotoxicity of these additives even at low concentration. gene chip analysis showed that the expression of apoptosis-related genes, oxidative stress-related genes and transcription related genes were significantly upregulated in each group. these results indicate that some additives, which have been considered safe preservatives in cosmetics, may have harmful effects on human skin when exposed to sunlight. these kinases in the pathogenesis of psoriasis. recently, increased focal activation of the downstream target mitogen-and stress-activated protein kinase (msk ) was demonstrated in psoriatic epidermis. the purpose of this study was to investigate msk and the transcription factor camp-response-element-binding protein (creb) in psoriatic skin and in cultured normal human keratinocytes. keratome and punch biopsies were taken from patients with plaque-type psoriasis. normal human keratinocytes were cultured and stimulated by interleukin- â (il- ß) or anisomycin. some of anti-inflammatory plant flavonoids as a form of whole plant extracts have been used topically for skin inflammatory disorders. on human skin inflammation, matrix metalloproteinase- (mmp- ) plays a pivotal role on unbalanced turn-over or rapid breakdown of collagen molecules. in the present study, for establishing a therapeutic potential against skin inflammatory disorders, the effects of natural flavonoids on mmp- activity and mmp- expression were examined. from the results, the flavonols including quercetin and kaempferol were revealed to be strong inhibitors of human recombinant mmp- with the ic s of . - . ìm, while the flavones such as apigenin and wogonin showed weak inhibition. when the effects of flavonoids on mmp- induction were studied, it was found that quercetin, kaempferol, apigenin and wogonin ( . - . ìm) strongly inhibited mmp- induction from tpa-treated human dermal fibroblasts, but naringenin (flavanone) did not. by gel shift assay, these flavonoids were also found to inhibit the activation of the transcription factor, ap- , whereas naringenin did not. among mapks, quercetin inhibited the extracellular signal-regulated protein kinase (erk) and p mapk activation, and kaempferol inhibited the p mapk and c-jun n-terminal kinase (jnk) activation. on the contrary, the flavones and naringenin did not inhibit the activation of these three mapks. all these results indicate that the capacity of mmp- inhibition and mmp- down-regulation of flavonoids may block collagen breakdown in certain pathological conditions and certain flavonoids are useful to treat skin inflammation, especially by topical application. ( ) ( ) university of valencia, spain ( ) istituto di chimica biomolecolare cnr, napoli, italy avarol is a marine sesquiterpenoid hydroquinone with several pharmacological properties including antioxidant, anti-inflammatory, and antipsoriatic effects. recently, its derivative avarol- -thiosalicylate (ta) also demonstrated interesting perspectives as anti-inflammatory drug in vitro and in vivo.it is interesting to note that avarol and ta inhibited nf-Þb activation in hacat keratinocytes. now, the effect of avarol and ta was investigated in the tpa-induced hyperplasia murine skin model, which presents some similarities with psoriatic lesions. topical treatment with ta ( mg/ml) produced a % inhibition of oedema and a strong reduction of pge ( %), ltb ( %) and mpo activity ( %) in skin homogenates. the inhibitory effect of avarol at the same dose was % for oedema, % for pge , and % for ltb and mpo activity. histological study for both compounds showed a decrease in epidermal hyperplasia as well as leukocyte infiltration respect to tpa treatment. besides, the reduction of cutaneous tnf-a by avarol and ta was also detected by immunohistochemical analysis. these compounds were also capable of suppressing nf-Þb nuclear translocation in mouse skin. in summary, our results suggest that inhibition of proinflammatory metabolites by ta and avarol might be beneficial for the treatment of the inflammatory component of psoriasis. its mechanism of action is related to the inhibition of nf-Þb activation and can be mediated by the downregulation of intracellular signal-transduction ( ), ams silva( ), cmm santos( ), dcga pinto( ), jas cavaleiro( ), jlfc lima ( ) ( ) requimte, departamento de química-física, faculdade de farmµcia da universidade do porto, porto, portugal ( ) departamento de química, universidade de aveiro, aveiro, portugal -styrylchromones are a novel class of chromones, vinylogues of flavones ( -phenylchromones), which have recently been found in nature. several natural and synthetic chromones have demonstrated to possess biological effects of potential therapeutic applications. however, the anti-inflammatory potential of -styrylchromones has not been explored so far. thus, the aim of this work was to evaluate the putative anti-inflammatory properties of several synthetic -styrylchromones by studding their influence on different systems that are related to the inflammatory process. the putative inhibitory effects of several -styrylchromones on the proinflammatory enzymes cyclooxygenase (cox- ), cyclooxygenase (cox- ) and -lipoxygenase ( -lox) was evaluated in vitro and compared with structurally related flavonoids. the capacity of the studied -styrylchromones to scavenge reactive oxygen (ros) and nitrogen species (rns) was also assessed by different in vitro assays, which allowed to identify the influence of those compounds in each reactive species, separately. from the tested -styrylchromones, those having a catecholic bring where shown to be the most effective scavengers of ros and rns, being, in some cases, more active than flavonoids. no considerable correlation was found between the scavenging profile of these compounds and their interactions with pro-inflammatory enzymes. the results obtained from the present study indicate that some of the tested compounds are promising molecules with potential therapeutic value. the usefulness of -styrylchromones in the prevention or control of inflammation can only be clarified with additional studies concerning their influence on other relevant mechanisms of this pathology. the importance of tumor-associated inflammatory cells, able to affect different aspects of neoplastic tissue, is a current matter of debate. primarily monocytes are recruited from the circulation into solid tumors and metastases where they differentiate into macrophages with several phenotypes and, e.g., may significantly contribute to uptake of certain radiotracers. we therefore sought to characterize the uptake of various radiotracers used for positron emission tomography (pet) in a well characterized in vitro model of human monocytes/macrophages in comparison with that in various human tumor cells. uptake of radiotracers f-fluorodeoxyglucose (fdg), -o-methyl- - f-fluoro-l-dopa (omfd), and f-labeled native/oxidized low density lipoproteins (nldl, oxldl) in single-or cocultivated human myeloid (monocytic) leukemia cell line thp- was compared with that by squamous cell carcinoma (fadu), mamma (mcf- ) and colorectal adenocarcinoma (ht ) cell lines (without or in the presence of specific inhibitiors). several thp- phenotypes along the monocytic pathway (monocytes, differentiated macrophages, retrodifferentiated cells) were studied before, during and after incubation with phorbol myristate acetate. differentiated thp- cells show, when compared with tumor cells, a comparable fdg accumulation, a considerably lower omfd uptake, and a significantly higher oxldl uptake. on the other hand, during differentiation and retrodifferentiation thp- cells obviously establish a distinct sequence of biological processes also reflected by considerable alterations in radiotracer uptake. the observed differences in uptake of several radiotracers in vitro in-between thp- phenotypes and between thp- phenotypes and tumor cells, respectively, stimulate studies on the contribution of macrophage radiotracer uptake to the overall uptake in neoplastic or inflammatory lesions in vivo. genomic and full-length cdna sequences provide opportunities for understanding human gene expression. determination of the mrna start sites would be the first step in identifying the promoter region, which pivotally regulates transcription of the gene. although the mrna start sites of most genes show heterogeneity, this may reflect physiological, developmental, and pathological states of the particular cells or tissues. recently, we have developed a -end sage ( sage) that can be used to globally identify the transcriptional start sites and frequency of individual mrnas. a strong association exists between states of chronic inflammation and cancer, and it is believed that mediators of inflammation may be responsible for this phenomenon. another important factor in tumor development seems to be the epigenetic effects on tumor suppressor genes. because of its ability to suppress tumor cell proliferation, angiogenesis, and inflammation, the epigenetic drug such as histone deacetylase (hdac) inhibitor is currently in clinical trials. however, how epigenetic drugs mediate its effects is poorly understood. to assess the effects of epigenetic drugs, the gene expression by sage in colon cancer cell lines treated with epigenetically affecting agents, -aza- deoxycytidine, a potent inhibitor of genomic and promoter-specific dna methylation and trichostatin a, a hdac inhibitor was investigated. epigenetic modification induced not only the change of expression of several inflammation-associated genes and the cell cycle progression-associated genes in human colon cancer cells but also the gene expression with aberrant start sites. colon cancer is one of the most frequently diagnosed cancers in western societies. interleukin- (il- ) is a potent, pleiotropic, inflammatory cytokine that contributes to a multitude of physiological and pathophysiological processes. il- is produced by many different cell types. the main sources in vivo are stimulated monocytes, fibroblasts, and endothelial cells. a variety of studies have demonstrated that over expression of il- contributes to the pathogenesis of various inflammatory diseases as well as cancer. it has been reported that human colorectal cancer cells display a wide heterogeneity in their potential to express and produce il- . serum levels of il- are elevated in patients with colorectal cancer, however serum levels of il- were found to be independent of il- mrna expression in tumor tissue. in this study we analyzed il- mrna expression by real-time pcr in sporadic colon cancer tissue as well as corresponding normal mucous tissue. il- mrna expression in tumor tissue was lower than in the corresponding normal mucous tissue (p= , ). there was no correlation betweenil- mrna expression and tumor grade or stage. thus we can conclude that il- produced at the tumor site is not involved in sporadic colon cancer progression. ( ), t aiamsa-ard ( ), v chinswangwatanakul ( ), ki techatraisak ( ), s chotewuttakorn ( ), a thaworn ( ) ( material and methods: huvec were cultured as standard techniques and grown to confluence until use. serum was obtained from cholangiocarcinoma patients and normal healthy subjects. huvec were treated with % of serum and incubated for hours. cells were analyzed by using [ h] thymidine and immunoblotting assay for cell proliferation and cox- /nos- protein expression, respectively. results: serum of cca patients trend to have more effect on proliferation of endothelial cell than healthy control subjects. on the protein expression, cca serum significantly increased the expression of cox- but not nos- in hevec. however, the proliferate effect on endothelial cells by cca sera did not correlate with the expression of cox- . conclusions: this result suggested that some factors in serum of cancer patients could induce cox- protein expression in huvec. the increasing of cox- might be one of various factors involve in the proliferation process. aim: superoxide is responsible for the neutrophil-mediated tumoricidal activity. the aim of our work was to monitor the changes of superoxide production from neutrophil attributed to tumor development from the early phase to the advanced stage, and to investigate the effects of ok- @on neutrophil-derived superoxide production and tumor growth. methods: ah a rat hepatocellular carcinoma cells were implanted into the hind leg of male donryu rats. pmns were harvested from rat peritoneal cavity h after intraperitoneal injection of oyster glycogen. superoxide production were measured by the method of cladependent chemiluminescence, which has high sensitivity and specificity to superoxide. the counts of peripheral leukocytes were significantly increased during tumor progression, and there are significant difference between that of controls and tumor-bearing rats after days of tumor inoculation. both pma and oz-induced superoxide generation derived from neutrophils became significantly reduced in the advanced stage of cancer. the suppression of neutrophil-derived superoxide generation was accompanied with tumor progression and an increased number of neutrophils in the peripheral blood. the subcutaneous administration of ok- , a biological response modifier, prevented the suppression of neutrophil-derived superoxide generation during tumor progression, which might induce the tendency of tumor growth suppression. our results suggested that the decreased superoxide generation as well as the high leukocytes concentration in the peripheral blood could be considered as indicators of an advanced stage of cancer. furthermore, the effect of ok- on neutrophil-derived superoxide production in cancer-bearing rats may provide pharmacological evidence to the therapeutic effects of ok- . ( ), m jokic ( ), v zjacic-rotkvic( ), s kapitanovic ( ) ( ) university hospital sestre milosrdnice, bucharest, romania ( ) division of molecular medicine rudjer boskovic institute, bucharest, romania introduction: il- is a pleiotropic cytokine mapped to chromosome p - . its promoter snp - g/c is associated with high serum cytokine production, and according to current investigation can play a role in the development and progression of different gastrointestinal malignancies. we tested its genotypes in the gastrointestinal and pancreatic neuroendocrine tumors (gep-nets). patients and methods: dnas from patients diagnosed with gep-net and age and sex-matched volunteers were analyzed for - g/c snp of the il- gene. to analyze il - c/g polymorphism we used pcr -nlaiii rflp method. for statistical analysis Ä test and fishers exact test were used. the level of significance was . . results: there were no differences observed in the frequencies of the - high expression (gc and gg) genotypes between the patients and healthy volunteers (p= . ), as well as between patients with gastrointestinal or pancreatic endocrine tumors (p= . ). - g/c genotype was statistically more frequent among patients with non-functional pancreatic endocrine tumors (pets) than in those with functional pets (p= . ). conclusions: high expression genotypes of il- - snp are more frequent in non-functional pets and may be a marker for the mentioned malignancies. are important in inflammation, are found around and within a variety of human tumors. their number correlates with tumor vascularity and aggressiveness and is a negative indicator for patient survival. how mast cells influence tumor growth is not well understood. the neuroendocrine peptide, neurotensin (nt) is a potent secretagogue of mc that has tumor-promoting effects in animals and promotes the growth of a variety of human cancer cells, acting via its gpcr nt-type receptor (nts ). here we show that hmc- human mc express nt-precursor (pront) mrna and protein, and secrete immunoreactive nt when stimulated. rt-pcr on hmc- cell rna yielded a band with % sequence identity to pront and a band corresponding to the pront processing enzyme, pc a.immunocytochemistry on hmc- cells showed specific staining for pront. stimulation of hmc- cells with a + pma, pge , c / or mastoparan released immunoreactive nt.rt-pcr on hmc- cell rna yielded a band with % sequence identity to human nts . western blotting gave bands corresponding to unglycosylated ( kda) and glycosylated ( kda) nts .immunocytotochemistry on hmc- cells showed specific staining for nts . these finding have significance for the role of mast cells in tumor growth. ( ), j buddenkotte ( ), mp schçn( ), m steinhoff ( ) ( ) university hospital münster, germany ( ) university hospital würzburg, germany the proteinase-activated receptor par- has been demonstrated to modulate tumor growth, invasion and metastasis in various tissues. however, the role of par- in cutaneous cancerogenesis is still unknown. here we could show a protective role of par- in the development of epidermal skin tumors: we established a mouse skin tumor model using chemically induced carcinogenesis. to this end, par- -deficient and wild-type mice were painted once with dmba ( , -dimethylbenz[a]anthracene) for sensibilization, followed by topical application of the phorbol ester pma (phorbol myristate acetate ( -o-tetradecanoylphorbol- -acetate)) twice per week at the same sites. tumors started to appear after eight weeks. after weeks, par- -deficient mice showed a significantly increased number of skin tumors ( per animal on the average) in contrast to the wild-type (eight tumors per mouse). analysis of possible signal transduction pathways activated upon par- stimulation in hacat keratinocytes showed an involvement of extracellular signal regulated kinase / (erk / ) and profound epidermal growth factor receptor (egfr) transactivation, leading to secretion of the tumor-suppressing factor transforming growth factor-beta (tgf-â ). thus, our results provide the first experimental evidence for a tumor-protective role of par- . ( ), ma arbós ( ), a fraga ( ), i de torres( ), j reventós ( ), j morote ( ) ( pathogenesis of benign prostatic hyperplasia (bph) and prostate cancer (pca) is still unresolved, although chronic inflammation may play a significant role in disease progression. prostate stromal fibroblasts may be contributing to the inflammatory process through the expression and secretion of pro-inflammatory mediators, in particular proteoglycan-bound chemokines and other chemoatractants, and the interaction with inflammatory cells such as monocytes. to better understand molecular mechanisms underlying functional differences among prostate fibroblast populations, our primary objective was to characterize proteoglycan and chemokine gene expression in human fibroblasts of different histological/ pathological origin cultured in normal and monocyteconditioned media. we analysed primary human fibroblast cultures from normal transition zone (tz), normal peripheral zone (pz), benign prostatic hyperplasia (bph), and pathologically confirmed prostate cancer (ca). cells of different origin displayed distinct mrna expression profiles for the core proteins of proteoglycans and both sdf /cxcr and mcp /ccr chemokine axis. when incubated with monocyte-conditioned medium all four cell types significantly changed sdf /cxcr and mcp /ccr expression in a fibroblast population dependent manner. monocyte-fibroblast cell adhesion and the chemotactic response of fibroblasts to human peripheral blood monocytes were investigated in a coculture system. monocytes adhered rapidly to fibroblasts and preferentially to bph and pz cells. in addition, chemotaxis was significantly induced in both fibroblast cultures after incubation with monocytes. our results suggest that prostatic fibroblasts have a key inflammatory role associated to a distinctive proteoglycan gene expression and chemokine induction, which is dependent on their histological and pathological source. supported by the spanish urology society (madrid, spain). we have recently shown that paf-receptor is involved in phagocytosis of apoptotic and necrotic but not viable cells, possibly through its interaction with paf-like molecules present on the surface of these cells. removal of altered cells by macrophages could modify the microenvironment at an inflammatory site, and thus influence tumor growth. in the present study we investigated the impact of apoptotic cells or treatment with paf-r antagonist on ehrlich ascitic tumor (eat, ip) and melanoma b f (sc). paf-r antagonist, web ( mg/kg, ip) was given daily for days. we found that eat growth was significantly reduced by pretreatment with web , and that inoculation of apoptotic cells (thymocytes) before tumor implantation stimulated tumor growth, an effect reversed by web pretreatment. eat growth was accompanied by increased production of prostaglandin e , vegf and no which was reduced significantly by web treatment. in b f melanoma, web , alone or in association with an apoptosis inducer chemotherapeutic agent, dacarbazin (dcb, ug/kg,ip) significantly reduced tumor mass volume and the number of intratumoral small vessels. in association with dcb, web- reducedactive caspase- expression in the tumor andmarkedly increased the survival of tumor-bearing mice. the data obtained here show that during tumor growth, activation of paf-r by molecules present in the surface of apoptotic/necrotic cells, or by paf produced in the milieu, favors tumor growth and suggests that pafantagonists could be useful in tumor treatment, particularly when in association with chemotherapy. financial suport by fapesp and cnpq. ( ), mt quiles ( ), a figueras( ), r mangues( ), f vinals( ), jr germa( ), g capella ( ) ( ) institut de recerca vall de hebron, barcelona, spain ( ) translational research laboratory, idibell -institut cataladoncologia, spain the malignant potential of tumor cells may be influenced by the molecular nature of k-ras mutations. we have previously shown that codon mutations associate with an increased resistance to apoptosis. we hypothesized that their different malignant potential in vivo could be also related to the generation of a distinct angiogenic and inflammatory profile including vascular structure, macrophage infiltration and expression of angiogenic modulators, proteolytic mediators and the cxcl (sdf- )/ cxcr chemokine axis. to do so we have combined in vitro and in vivo studies using stable cys and asp nih t transfectants. cys tumors showed a higher microvessel density associated with shorter latency period. prominent vessels with µ-smooth muscle actin positives cells surrounded by f / macrophages were only observed in asp tumors associated with a shorter growth period. asp tumors displayed increased vegf expression both at the rna and protein levels, mainly produced by tumor cells. tsp- protein levels were similarly diminished in both transfectants. higher mmp and mmp activities and expression were observed in asp tumors probably produced by macrophages or stromal cells. total and active mmp levels were higher in cys tumors. the expression of sdf- and cxcr remained unchanged while sdf- g isoform was selectively induced in cys tumors, suggesting sdf- a or b are induced in asp tumors. these results show distinct k-ras mutations induce specific angiogenic phenotypes. the differential stimulation of vegf expression, metalloprotease activities and sdf- expression observed is the result of the joint action of tumor cells and the local microenvironment. contact information: dr maria a arbos via, institut de recerca vall de hebron, unitat de recerca biomedica, barcelona, spain e-mail: maarbos@ir.vhebron.net incisional hernias (ihs) represent a common complication of laparotomies, involving remarkable healthcare costs. representative ih animal models are lacking and characterization of human tissue resources is scant. this limited understanding of fundamental mechanisms regulating the destruction of the abdominal wall currently limits the prevention and treatment of ihs. here, we compared tissue specimens (carefully obtained > cm of the defect) and primary fibroblasts cultures from fascia and skeletal muscle of subjects with/without ih hernia. the most prominent morphologic characteristics of ih tissue were: alterations of the microstructure of the connective tissue and loss of extracellular matrix (ecm), and a paucity of fibroblasts. in ih muscles, inflammatory infiltrates were observed. other significant changes were: decreased collagen type i/iii ratio; differential proteoglycans mrna expression; enhanced metalloproteinases/ endogenous inhibitors ratio (mmps/timps); and upregulation of apoptosis effectors (caspase- and substrates; tnf-alpha; il- ). in vitro, hernia fibroblasts (ihfs) exhibited significantly higher ( -fold) cellular proliferation and migration rates and decreased strength of adhesion as compared to control fibroblasts, even after several passages. moreover, ihfs ultrastructure analysis revealed accumulation of autophagic vacuoles, autophagolysomes-like structures and multilayered lamellar and fingerprint profiles, as well as mitochondrial swelling. based on these descriptive results in human tissues, a novel hypothesis emerges regarding ih formation. specifically we propose that inflammation-related mechanisms triggering proteolytic and apoptotic effectors regulate cell turnover and eventually contribute to atrophy and progressive tissue insuffiency. overall, this may be causally involved in the mechanisms of ecm destruction yielding ih (supported by fis pi_ and gencat_agaur_ xt_ ). ( ), m spinola( ), c pignatiello( ), w cabrera ( ), og ribeiro ( ), n starobinas( ), t dragani ( ) ( ) butantan institute, sao paulo, brazil ( ) istituto nazionale tumori, milan, italy airmax and airmin mice are phenotypically selected for maximal or minimal subcutaneous acute inflammatory response, respectively, and display high inter-line differences in protein exudates and neutrophil infiltration, as well as in bone marrow granulopoiesis, inflammatory cytokines, and neutrophil apoptosis. in a combined experiment of urethane-induced lung inflammatory response and lung tumorigenesis, airmin mice developed a persistent subacute lung inflammation and a fold higher lung tumor multiplicity than airmax mice, which showed a transient lung inflammatory response. we have analyzed gene expression profiles of these outbred lines in comparison to the lung cancer resistant c bl/ and lung cancer susceptible a/j mouse strains. gene expression profile analysis of urethane-treated and untreated animals was performed using the applied biosystems mouse genome survey microarray containing , mouse transcripts. mrna expression of candidate differentially expressed genes was validated by quantitative real-time pcr and the over-represented biological themes were analyzed with the ease software. urethane treatment modulated the gene expression profile in all four lines. among the confirmed genes, vanin (vnn ) and major histocompatibility antigen e alpha (h -ea) resulted common to both mouse models. the most represented gene categories in air model were acute phase response, immunoresponse, electron and lipid transport, complement activation and tissue repair. mhc/antigen process and presentation and immunoresponse were the major themes in the inbred model. moreover, a gene cluster in chromosome ( . cm) was observed. the study suggests that the expression of a subset of genes may show a strain-and line-specific modulation pattern during inflammatory response and lung tumorigenesis. inhibition of tumour induced angiogenesis constitutes very attractive anti-cancer therapeutic approach.it is well established that the vegf signal transduction pathway is one of the key drivers of deregulated angiogenesis and selective inhibition can lead to inhibition of tumour growth. however, multiple angiogenic growth factors and pathways are involved, leading to a phenomenon of redundancy and overcoming of an inhibition of vegf signalling only. we have developed a nanomolar inhibitor (compound a) of the receptor tyrosine kinase vegfr-r (kdr), which was subsequently shown to be a potent inhibitor of closely related kinases (vegfr- and - , pdgfr, kit, csf- r) but also unrelated soluble tyrosine kinases (src-familily of kinases and raf). compound a potently inhibits vegf stimulated endothelial cell proliferation but has no effect on non-ec proliferation, which is suggestive of a selective antiangiogenic potential. the unique kinase inhibitory profile of compound a combined with excellent oral bioavailability ( %) has translated into superior in vivo anti- inflamm. res., supplement ( ) posters tumour efficacy when compared to the relatively selective kdr inhibitor ptk . thus, treatment of nude mice implanted with either commercial atcc derived tumour cells (a and du- ) or low passage patient derived tumors (cxf ; colon cancer, rxf ; renal cancer) with compound a resulted in inhibition of tumour growth which was significantly better than for ptk treated mice. compound a is fairly well tolerated by rodents and extended toxicological studies have been initiated to determine the therapeutic index, which also may allow for exploration of other non-cancer indications. ( ), p bobrowski( ), m shukla ( ), t haqqi ( ) ( ) albany medical college, usa ( ) rainforest nutritionals, inc, usa ( ) case western reserve university school of medicine, usa background: the amazonian medicinal plant sangre de grado (croton palanostigma) has traditional applications for wound healing and inflammation. we sought to characterize an extract (progrado) in terms of safety, proanthocyanidin profile, antioxidant activity and anabolic/catabolic actions in human cartilage explants. methods: acute oral safety and toxicity was tested in rats according under oecd protocol # . proanthocyanidin oligomers were quantified by hplc and progrados antioxidant activity assessed by the orac, norac and horac assays. human cartilage explants, obtained from surgical specimens, were treated with il- b ( ng/ ml) to induce matrix degradation and glycosaminoglycans (gag) release. progrado ( - mg/ml) was tested for its ability to maintain optimal igf- transcription and translation in cartilage explants and cultured chondrocytes. results: progrado displayed no evidence of toxicity ( mg/kg po) leading to gsh safety rating of /unclassifiable. oligomeric proanthocyanidin content was high ( mg/kg) with the majority of oligomers > mers.progrado was a remarkably potent antioxidant and in an ex vivo model of inflammation-induced cartilage breakdown, progrado was exceptionally effective in reducing both basal and il- b induced glycosaminoglycan release from human cartilage explants. progrado prevented il- b induced suppression of igf- production from human cartilage explants as well as stimulating basal igf- production (p< . ). comparable changes in igf- gene expression were noted in cultured human chondrocytes. conclusions: progrado has a promising safety profile, significant chondroprotective and antioxidant actions, and promotes the production of the cartilage repair factor, igf- . this suggests that progrado may offer therapeutic benefits in joint health, wound healing and inflammation. the solvent extracts from korean fermented soybean (chungkukjang) were evaluated for their protective effects against the generation of free radicals and lipid peroxidation. the activities of chungkukjang were compared with several antioxidants and soybean isoflavones including genistein and daidzein. in addition, the protective effects against h o -induced cytotoxicity and oxidative dna damage in the nih/ t fibroblasts line were examined. the extracts from chungkukjang and soy isoflavones inhibited the generation of , -diphenyl- picryl hydrazine (dpph) radicals, and had an inhibitory effect on ldl oxidation. the extracts from chungkukjang and soy isoflavones strongly inhibited h o -induced dna damage in the presence or absence of endonuclease iii and fpg. furthermore, they showed cytoprotective effects against h o , without cytotoxicity except for the hexane extract at high concentrations (> mg/ml). the ethanol and n-butanol extracts appeared to have most potent antioxidant activities. these in vitro results show that the extracts of chungkukjang may be a useful antigenotoxic antioxidant by scavenging free radicals, inhibiting lipid peroxidation and protecting against oxidative dna damage without having cytotoxic effect. moreover, the extracts of chungkukjang inhibited mda formation in the liver, dna damage assessed by comet assay and the microucleated reticulocyte formation of peripheral blood in kbro -treated mice. these in vivo results were similar to those of in vitro experiments. therefore, chungkukjang containing soy isoflavones is a promising functional food that can prevent oxidative stress. (supported by bk project from korea research foundation). sirt is a histone deacetylase, involved in oxidative stress and aging. because the role of aging and exercise on sirtuins activity in rats is unknown, we investigated the effects of exercise on age-related changes in the sirt activity, comparing heart (h) and adipose (a) tissue of sedentary young (n ), sedentary old (n ) and trained old (n ) rats. the trained old rats performed a -weeks moderate training on treadmill. on h and a tissue of all rats sirt activity was evaluated by assay kit, peroxidative damage measuring malondialdehyde (mda) and protein-aldehyde adducts -hydroxynonenal ( -hne), mnsod, catalase and foxo a by western blot, and gadd a, cyclin d and foxo a mrna by rt-pcr. aging reduced sirt activity in h (p< . ) without effects in a, producing an increase of mda (h, p< . ; a, p< . ) and -hne (h, p< . ; a, p< . ), and a decrease of mn-sod (p< . ) and catalase (p< . ) expression in both h and a. aging did not affect foxo a protein expression in h, and foxo a mrna in a. exercise produced an increase in h foxo a protein expression (p< . ) and in a foxo a mrna, associated to higher mn-sod (h, p< . ; a, p< . ) and catalase (h, p< . ; a, p= . ) levels in both h and a of aged rats. in heart exercise-induced higher sirt activity bring on decrease in cyclin d and increase in gadd a mrna expression. in a we found a similar decrease in cyclin d , without changes in gadd a mrna expression. these findings suggest that exercise is able to increase sirt activity in aged rats. ( ), t horiguchi( ), k abe( ), h inoue( ), t noma ( ) ( ) institute of health biosciences, the university of tokushima graduate school, tokushima, japan ( ) minophagen pharmaceutical co. ltd, japan objectives: glycyrrhizin (gl) is a major component of glycyrrhizae radix (licorice) that is generally used for treatment of hepatitis. gl has a regulatory activity on arachidonic metabolism, immunological function, and anti-viral effects. however, the molecular mechanisms of the effects remain unclear. to analyze the molecular basis of gl signaling, we performed the microarray analysis using ccl -induced mouse hepatitis models. methods: eight-week-old icr male mice were treated intraperitoneally with f×l/ kg bw of ccl w/wo mg/ kg bw of gl. after hours and hours, livers and serum were collected and analyzed. for microarray analysis, the expression patterns of genes between hour-treated-livers (ccl or ccl and gl) and no treated-livers were compared. results: gl-treatment dramatically decreased the gpt activity in plasma at hours compared to that in ccl treated plasma. however, the levels of mrna expression of inflammatory genes such as phospholipase a , hsp , and procollagen were still very high in gl-treated liver. after hours, the mrna levels of them were significantly reduced in gl-treated mice compared to those of ccl -treated liver. then, we screened , genes by microarray and found that genes were up-regulated and genes were down regulated in ccl +gl compared to ccl treatment. interestingly, ros scavenger-related genes were significantly up-regulated in ccl + gl. detail analysis is currently ongoing. we found the unique relationship between gl activity and ros regulation. this finding suggests a novel way to treat inflammatory diseases including hepatitis. objectives: experimental autoimmune encephalomyelitis (eae) is a demyelinating autoimmune disease that results from an immunological reaction against different myelin components at the cns. it is widely employed as an animal model of human multiple sclerosis. interestingly, the number of studies relating these diseases with peripheral organs is limited. we thus investigated the consequences of eae on the degree of lipoperoxidation (tbars) and mpo activity in different rat peripheral organs (eg. lung, spleen, liver, stomach, duodenum, colon, ileum, kidney and bladder). university of waikato, hamilton, new zealand mitochondria play a fundamental role in the life and death of all eukaryotic cells. cells with dysfunctional mitochondria are known to have higher levels of a molecular stress protein (cpn ). this protein is increasingly being implicated to play a role in modulating cellular inflammation. we have developed an in vitro model cell system using thp- monocyte cells with compromised mitochondrial bioenergetic functions to investigate the relationships between mitochondrial dysfunction, cpn expression and modulation of proinflammatory cytokine responses. we have found that the ability of cpn to modulate tnf-a expression was strongly correlated with the loss of mitochondrial bioenergetic functions in our thp- cells. we also demonstrate that such modulation involves both erk / and p mapk pathways. the significance of these results in relation to the role of mitochondria as modulators of inflammation will be discussed. ( ), b arnold( ), g opdenakker ( ) ( ) jagiellonian university, department of evolutionary immunobiology, krakow, poland ( ) german cancer research center, department of molecular immunology, heidelberg, germany ( ) rega institute for medical research, university of leuven, laboratory of immunobiology, leuven, belgium we showed that in mice genetically deprived of metalloproteinase (mmp- -/-) at least one compensatory mechanism operates as there are elevated levels of pge of cox- origin expressed by peritoneal macrophages during zymosan peritonitis; and this leads to increased early vascular permeability observed in those animals. also infiltration of peritoneal cavity by inflammatory neutrophils is changed in mmp- -/-mice as at hrs of inflammation, when otherwise highest numbers of neutrophils are detected in peritoneum, the cell numbers are significantly lower in the mice in comparison to their controls. in contrary, at hrs of peritonitis, when normally resolution of peritonitis takes place, no decrease in neutrophil counts is observed. thus the aim of the present study was to evaluate if impairment of neutrophil apoptosis could account for this latter phenomenon in mmp- -/-mice. for this numbers of apoptotic (annexin v) and necrotic ( -aad) peritoneal leukocytes were evaluated and levels of active caspases were tested by application of either caspase detecting antibodies or fluorochrome-labelled inhibitors; all analyses were performed by flow cytometry. the results revealed that both, numbers of apoptotic cells and levels of active caspase were significantly lowered in mmp- -/-mice while levels of caspase , and were significantly elevated in comparison to control animals. we conclude that an impairment of apoptosis is observed in mmp- -/mice during zymosan peritonitis and it is due to the decreased levels of active caspase . the increased activity of other examined caspases is most probably independent of apoptosis. ( ), h james ( ) the selective inhibition of nitric oxide generation by inhibiting the activity of nitric oxide synthase(nos) isoforms represents a novel therapeutic target for the development of anti-inflammatory agents. the aim of this study was to evaluate the activity of nos inhibitors in experimentally induced inflammation, pain and hyperalgesia. the effect on acute inflammation was studied in carrageenan-induced paw edema in rats. the effects on carrageenan-induced hyperalgesia, tail flick response to radiant heat and acetic acid-induced writhing were also studied. nos inhibitor ng-nitro-l-arginine methylester (l-name), and mg/kg produced a dose-dependent inhibition of paw edema ( % and % at h; % and % at h). a marked reduction in paw edema was observed with ng-monomethyl-l-arginine acetate (l-nmma), mg/kg( % at h; % at h). selective inducible nos(inos) inhibitor aminoguanidine hemisulfate inhibited the paw edema at a dose of mg/ kg( % at h; % at h) but not with a dose of mg/kg . the effects were comparable to nonselective cox inhibitor indomethacin mg and mg/kg ( % and % at h; % and % at h respectively) and selective cox- inhibitor rofecoxib, mg/kg ( % and % respectively). nos and inos inhibitors significantly increased the pain threshold latency in the tail-flick test. these inhibited the acetic acid-induced writhes, the effect being comparable to indomethacin. however, carrageenan-induced paw hyperalgesia was not inhibited. the results suggest that nitric oxide plays a role in carrageenan-induced acute inflammation and both nosand inos inhibitors have a potential anti-inflammatoryand anti-nociceptive activity. ( ), p hart( ), j edwards ( ), c quirk ( ) ( ) molecular pharmacology limited (usa), australian division, perth, western australia ( ) telethon institute for child health research, perth, western australia thermalife cream, an anti-arthritic biological product, has been successfully used off-label for sun burn recovery. a novel product, derived from thermalife, was assessed on its therapeutic potential in oxsoralen-uvb burns. as a possible mechanism for the sunburn efficacy, suppression of tnf-a and il- â production by human monocytes was assessed in vitro. methods: sunburn: four sites were marked on the arm of the subject. three sites were exposed to oxsoralen ( %) plus uva/uvb light, one site was exposed to oxsoralen only. cream was applied at min, or at hrs after injury. a third injury site was not treated. photographs were taken before, hrs, and weeks after injury. cytokines: human monocyte cultures ( % fcs, % co ) were either stimulated with ng/ml lps (e.coli :b ) or not in the presence of % or % active ingredient. hrs after incubation, culture media was collected, centrifuged, and assayed (cytokine elisa). results: at hrs after oxsoralen-uv, the min treatment site showed slight erythema, the hr treatment site had pronounced erythema and slight blister formation, whereas the untreated site had pronounced erythema and strong blister formation. weeks after injury, the min site was normal, the hr site was a dark colour, whereas the untreated site had a significant scar. oxsoralen alone had no effect on the skin. the novel product suppressed lps-induced tnf-a and il â secretion by . % and . %, respectively. conclusions: a novel thermalife-derived product reduced total injury after oxsoralen enhanced uva/ uvb burns, which is possibly related to cytokine suppression. ( ), p hart( ), j snowden ( ), maud eijkenboom ( ) ( ) molecular pharmacology limited (usa), australian division,perth, western australia ( ) telethon institute for child health research, perth, western australia a mixture of bovine plasma protein fractions and zinc chloride (bov-zn) was assessed for its ability to regulate cytokine production by lps-stimulated monocytes. dosereponse curves for tnf-a suppression were generated. further, competition with fcs in the culture medium and the metabolism of monocytes under influence of bov-zn were assessed. in all experiments the culture medium environment was similar. human monocyte cultures ( % fcs, % co ) were either stimulated with ng/ml lps (e.coli :b ) or not in the presence of %, . %, %, . %, %, % or % bov-zn (two pooled experiments). hours after incubation, culture media were collected, centrifuged, and assayed (cytokine elisa). a competitive inhibition design for the standard tnf-a assay was set up for %, %, %, % fcs against %, . %, %, % bov-zn. the culture media were treated as above. metabolism of non-proliferating monocytes was measured via accumulation of bioreduced formazan (promega celltiter ) in treated and untreated cell cultures over - hrs at intervals. the ic for tnf-a suppression was reached at . % bov-zn in each of two experiments. fcs did not compete with bov-zn in suppressing tnf-a in lpsstimulated monocytes. at low fcs concentrations bov-zn stimulated tnf-a production in the absence of lps. this tnf-a increase was countered with increasing concentrations of fcs. metabolism of cells was not affected by % bov-zn. conclusions: bov-zn could reliably and effectively reduce tnf-a secretion in vitro, without competing with the fcs in the culture medium, and without disturbing the metabolism of monocytes. inflammatory diseases such as rheumatoid arthritis (ra) result from overproduction of cytokines including tnf-£\ and il- fÒ. these cytokines are known to be regulated by the stress-activated p fnfnmap kinase pathway. because of this, inhibition of p map kinase has been one of the most compelling targets for the treatment of inflammatory disease. over the last years, numerous groups have reported on the development of p map kinase inhibitors. x-ray co-crystallization with the enzyme suggests a propensity to accommodate structurally diverse molecules. regions of the binding site are known to be unique to p vs other kinases, enabling the development of p selective molecules. inflamm. res., supplement ( ) posters anti-inflammatory activities. a series of labdane-type diterpenoids ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) with various patterns of substitution were tested for potential anti-inflammatory activity.of these compounds, and were selected to evaluate their influence in targets relevant to the regulation of the inflammatory response. these derivatives displayed good in vivo anti-inflammatory activity, and maximum inhibitions of and % were noted in the -o-tetradecanoyl-phorbol- -acetate (tpa)-induced ear oedema in mice. in addition, inhibition of myeloperoxidase activity, an index of cellular infiltration, was also observed. the diterpenoids also reduced the production of nitric oxide, prostaglandin e , and tumour necrosis factor-alpha in bacterial endotoxin-activated raw . macrophage cells with ic in the range - mm. inhibition of these inflammatory mediators was related to reductions in the expression of inducible nitric oxide synthase, and cyclooxygenase- , as determined by western-blot analysis and rt-pcr. since nuclear factor-kappab (nf-kb) plays a central role in the transcriptional regulation of these proteins, we investigated the effects of these diterpenoids on this signalling pathway. our results indicate that both compounds interfere with the phosphorilation of ikbalpha and ikbß, resulting in inhibition of their degradation. in summary, the anti-inflammatory effects of these labdane diterpenoids are related to the inhibition of inflammatory mediators by blocking nf-kb activation and provide potentially therapeutic perspectives in inflammatory conditions. the aim of the study was a research of mechanisms of inflammatory action of a new drug fepolen at a dosage of mg/kg prepared from bee products (bee pollen and phenolic hydrophobic extract of propolis) for prostatitis treatment. to fulfill above mentioned task a model of zimozan induced oedema whose dynamics gives a possibility to estimate influence of a drug on both routs of arachidonic acid metabolism -via cyclooxiginase and lipooxigenase was used. a comparison with diclofenacum at a dosage of mg/kg and substance ffw- Ñ (inhibitor of cyclooxygenase and lipooxygenase and has a high antiinflammatory effect ( %) at a dosage of mg/kg) was made. the results of influence of drugs dynamics of zimozan inflammation show that anti-inflammatory action of fepolen is based on decrease of release of biogenic amines and activity of lipooxigenase and is higher then effect of diclofenacum. fepolen revealed the highest effect during first min and hour of inflammation that was higher then action of diclofenacum. these data proves that fepolen decreases lipooxygenase activity that is in charge for the inflammation during this period. during next hours therapeutic effects of fepolen and diclofenacum were at the same level. fepolen showed the same dynamics of anti-inflammatory action as ffw- Ñ that demonstrates a property to influence both routs of arachidonic acid metabolism. in summary with previous results in conditions of carageninic inflammation we can conclude that anti-inflammatory action of fepolen is based mostly on influence on lipooxygenase then cyclooxygenase. the aim of this study was to establish a method by which probiotic bacteria can be selected for their immuno modulatory properties, especifically the ability of certain strains to suppress an inflammatory response. the gastrointestinal inflammatory condition crohns disease involves a th -response with increased levels of proinflammatory cytokines like tnfa and il , and mouse models of crohns disease show that the balance of il / is crucial for disease progression. we have used mouse bone marrow derived dendritic cells (bmdc) to model a proinflammatory crohns disease like condition in vitro with cocktail-induced bmdc secretion of il , il and tnfaf jthe model was validated using anti-inflammatory molecules like dexamethasone and prostaglandin d , which were able to suppress the cocktail induced il secretion. further validation of the model is confirmed by the fact, that probiotic strains which are able to suppress tnbs induced colitis in mice in preventive studies, also show potent anti-inflammatory activity in our model. among clinically relevant as well as novel probiotic strains, we have selected strains with potent anti-inflammatory properties, and are currently investigating the possible mechanism of action of these strains. in summary, our established model is suitable for identification of anti-inflammatory activity of probiotic strains and potentially other immune suppressing components, for rational selection of candidates for further preclinical and clinical evaluation and development. p - inflammation and human incisional hernia pathophysiology maria antonia arbos via( ) eae was induced by immunization of female lewis rats with guinea-pig myelin basic protein (mbp) in complete freunds adjuvant (cfa) and the animals were studied at the stage iii of the disease (characterized by complete paralysis of the hind-limbs) compared to cfa rats, eae resulted in increased mpo activity (u/mg tissue) in kidney ( ae vs. ae ae ; p< . ), and higher tbars contents (nmol mda/mg tissue) in liver acknowledgements: capes, cnpq, fapesp. contact information: ms simone teixeira, university of s¼o paulo, department of pharmacology, campinas, brazil e-mail: mone@usp.br tolerability, investigator and subject global assessments and rescue medication consumption supported by bk project from korea research foundation) contact information: mr young hoon kim here we report on the development of the pge mimetic combination therapy (dp- ) that inhibits basal and lps/tlr induced tnf-?, il- ß, and mmp- , , , in human and murine synovial membranes and peripheral macrophages.in a murine model of chronic synovitis (dorsal skin air pouch), dp dramatically inhibited il- ß, tnf-?, mip , mcp- and il- expression, delayed the profile of leukocyte/neutrophil extravasation and reduced exudate volume.in a model of inflammatory arthritis (collagen induced arthritis, cia), dp markedly reversed the inflammatory pathology by reducing synovial hyperplasia, cartilage erosion and articular inflammation.in addition, tnf-?, il- ß, mmp- and to a lesser extent mmp- expression/synthesis levels were strongly suppressed as judged by rt-pcr and elisa measurements we have developed two rias, one for functional blood levels of the above mentioned anti-tnf-alpha constructs, and one for anti-abs (all isotypes), and we have used these methods to monitor patients treated with infliximab/remicade and etanercept/enbrel ; i shall present some of these data ( , anatomy-physiology, faculty of medicine, laval university, quebec, canada neutrophils, which are often the first leukocytes to migrate at inflamed sites, can generate ltb from the -lipoxygenase pathway, pge through the inducible cyclooxygenase (cox- ) pathway and cytokines/chemokines as tnf-alpha, il- beta, il- , mip- alpha, mip- beta, mip- alpha and mip- alpha. engagement of the adenosine a a receptor (a ar) blocks the in vitro synthesis of ltb while it potentiates the cox- pathway in fmlp-treated neutrophils. in addition, it selectively prevents the expression and release of tnfalpha, mip- alpha, mip- beta, mip- alpha and mip- alpha in toll-like receptor- -stimulated human neutrophils. little effect was observed on il- beta and il- . using the murine air pouch model of inflammation with a ar-knockout mice, we observed that the activation of a ar positively impacts the expression of cox- in vivo, with particular magnitude in inflammatory leukocytes. in mice lacking the a a receptor, neutrophils that migrated into the air pouch h following lps injection expressed higher mrna levels of tnf-alpha, mip- alpha and mip- beta than neutrophils from wild type mice. together, these results indicate that neutrophils are important mediators of adenosines protective effects. given the uncontrolled inflammatory phenotype observed in a ar-knockout mice and in view of the potent inhibitory actions of pge on inflammatory cells, an increased cox- expression and a prevented release of tnf-alpha, mip- alpha and mip- beta caused by a ar activation, observed particularly in neutrophils, may take part in an early modulatory mechanism promoting anti-inflammatory activities of adenosine. sepsis induced by endotoxins including lipopolysaccharide (lps) is a big problem in clinical medicine. for a better insight into the molecular pathways and to assess markers of endotoxin-induced sepsis, we applied thetwo dimensional gel electrophoresis ( d-page) and maldi-tof to follow the changes of significant proteins in a murine macrophage cell line -raw . after challenged with lps (escherichia coli :b ) for , and hours. we identified proteins from approximately detected protein spots with either increased or decreased in relative abundance as a result of lps treatment. the proteins identified with increased expression are the retinoblastoma binding protein , capg protein, poly(rc) binding protein , isocitrate dehydrogenase (nad+) alpha, lactate dehydrogenase , a chain, guanine nucleotide binding protein (g protein), beta polypeptide like , triosephosphate isomerase and proteasome alpha subunit); and ones with decreased expression are the acidic ribosomal phosphoprotein p , malate dehydrogenase, soluble, proliferating cell nuclear antigen, proteasome (prosome, macropain) subunit, alpha type and rho, gdp dissociation inhibitor (gdi) beta). many of these altered proteins have interesting functions in inflammation. with the information obtained with the proteomic approach, it is possible to improve current methods of monitoring endotoxemia and to identify new therapeutic targets. the ubiquitous mitogen-activated protein (map) kinases are important enzymes in signal-tranduction cascades which regulates diverse cellular events such as cell transformation, proliferation, differentation, and apoptosis. they are therefore potential drug targets for therapeutic intervention in the treatment of inflammation, cancer, and other immune diseases. based on a virtual screening approach we identified -amino- -benzyl- -( -bromophenyl)- -methyl- , -dihydropyrano[ , c] pyrazole- -c arbonitrile as a potential novel lead structure as p map kinase inhibitors. a set of compounds were prepared starting from different substituted pyrazol- ( h)-ones via a base-catalyzed condensation with aldehydes and ch acids, such as malononitrile, to provide them for biological tests in a p enzyme assay. first structure-activity relationship confirm the value of this novel lead. this study was conducted to determine the physiological c-reactive protein (crp) and alpha -acid glycoprotein (aag) levels for two groups of beagle dogs: healthy dogs of various ages and pregnant dogs. serum crp levels were measured by elisa and aag levels were measured in healthy beagles of various ages by tia, and then separately -in pregnant beagles -by srid. serum crp levels ranged from . to . ìg/ml in male, and from . to . ìg/ml in female dogs. no significant sex-related differences were observed in the values. further, there were no significant age-related differences either. serum crp levels increased during pregnancy and peaked at . - . ìg/ml or days after ovulation, demonstrating two characteristic features of crp levels change in pregnant dogs. serum aag levels ranged from to ìg/ml in male, and from to ìg/ml in female dogs, without any significant sex-or age-related variation. serum aag levels increased in all pregnant beagles and peaked in the middle of gestation at - , ìg/ml. despite a high value of , - , ìg/ml being observed for serum aag levels in pregnant beagles inoculated with staphylococcus aureus, its levels in umbilical cord blood were below the detection limit of srid ( ìg/ml). no significant sex-/-age related differences were observed in serum both crp and aag levels and these levels increased during pregnancy. the results of aag levels in umbilical cords were below the detection levels suggest aag is not transported to the placenta. polymorphonuclear neutrophils (pmns) play a key role in the inflammatory response against infectious agents.however, they can elicit significant tissue damage and in this respect, anti-inflammatory drugs are of interest.in this study, we examined the effect of pbi- , a low molecular weight immunomodulatory molecule, on pmn activation by lps both in vitro and in vivo. we measured by elisa the production of tnf-a by human lps-activated pmn in the presence or absence of pbi- .the ability of pbi to modulate pmn activation and recruitment in vivo was assessed using a rat air pouch model of inflammation.exudates from different groups of animals (controls and pbi- treated animals, n= ) were used to assess leukocyte infiltration and to measure by elisa tnf-µ, mcp- and pge production.in vitro, pbi- is able to significantly decrease by . % ae . % (p< . ), tnf-a production by human lpsactivated pmn.in vivo, pbi- significantly decreased the production of tnf-a ( . % ae . %; p< . ), mcp- ( . % ae . %; p< . ) and pge ( . % ae . %; p< . ) induced by lps injection.however, pbi- did not significantly inhibit leukocyte infiltration.these results show that pbi- is able to modulate pmn activation and inflammatory response and suggest potential use as anti-inflammatory agent. ( ), lj lowenstine,( ), aj norris( ), t spangler( ), lm woods ( ) ( ) zoological society of san diego, usa ( ) department of pathology, microbiology, and immunology, university of california, davis, usa this study investigated the role of a novel reovirus in a outbreak of necrotizing typhlocolitis in american crows in california. included is a detailed characterization of the necrotizing and inflammatory characteristics of the disease, as well as a discussion of the implications of these findings upon proposed mechanisms of pathogenesis. complete histopathology including stains for lesion characterization and potential concurrent etiologic agents was performed on all outbreak crows. feces and ceca were submitted for culture, parasitology, and negative contrast electron microscopy. two control groups (n= each) were selected for parasitology and em (group ), and gross and histopathology (group ). all outbreak cases and group controls tested negative for west nile virus by pcr.all outbreak crows had marked, necrotizing heterophilic typhlocolitis, fibrinonecrotizing splenitis, and variable intestinal lamina proprial necrosis and hemorrhage.two cases had multifocal hepatic necrosis. negative contrast em revealed reovirus particles in % ( / ) of outbreak cases and in % ( / ) of controls. supplemental tests failed to suggest other concurrent or confounding etiologic agents.overall, the findings suggest association between the reovirus and the outbreak of typhlocolitis, and the absence of reovirus in controls suggests that it is not ubiquitous in the crow population.there was a noteable absence of similar typhlocolitis in archived crows submitted to the vmth from - , suggesting an emerging corvid disease in california, which bears further investigation. mitogen-activated protein kinase (mapks) pathways play an important role in the signalling system activated by proinflammatory cytokines. among the most important cascades the activation of erk / by mek / is reported to be responsible for inflammatory responses and degradation of osteoarthritic cartilage. as , a selective mek inhibitor, demonstrated anti-inflammatory properties in reducing tnf-alpha production induced by lps injection (ic mg/kg). therefore, primary aim of the present study was to assess the therapeutic strength of the as in a mouse model of collagen-induced rheumatoid arthritis (cia) assessing the effect of the compound on structural changes related to the cartilage. cia is characterized by severe polyarthritis affecting peripheral joints, synovial hyperplasia with persistent inflammation and cartilage erosion. as treatment was initiated when signs of arthritis were clinically visible (in terms of paw swelling and redness) and was continued for days (twice daily), by oral route at the doses of , and mg/kg. as at and mg/kg significantly reduced clinical arthritic read-outs such as clinical score and paw swelling. at histology, vehicle-treated animals showed severe inflammation and joint surface erosion. administration of as significantly decreased inflammatory infiltrates and treated cartilage surfaces that presented normal levels of proteoglycan content. in conclusion, the results obtained in this study clearly demonstrate that the selective blockade of mek could be considered as an innovative therapeutic approach to treat rheumatoid arthritis. experimental evidences have shown that the toxicity of ni salts may involve inflammatory processes, with a subsequent overproduction of reactive oxygen species (ros) and carcinogenicity. neutrophils are the most abundant leukocytes of blood, and participate actively in the inflammatory innate host defense response. however, relatively little is known about the potential of nickel salts in activating human neutrophils.thus, the aim of the present study was to evaluate the putative stimulation of oxidative burst in isolated human neutrophils by nickel nitrate. the measurement of neutrophil burst was undertaken in vitro, by chemiluminescence, by monitoring the oxidation of luminol by neutrophil-generated ros and reactive nitrogen species (rns). enzymatic inhibitors and specific reactive species scavengers were used to evaluate which species were involved in neutrophils activation by nickel nitrate. the obtained results showed that nickel nitrate stimulates human neutrophils burst in a concentration-dependent manner, within levels that may be attained in vivo. in the present experimental conditions, the reactive species involved in neutrophils activation by nickel nitrate were superoxide radical (o -.), hydrogen peroxide (h o ), hydroxyl radical (ho.) and perchloric acic (hocl). the observed activation of isolated human neutrophils burst by nickel nitrate and subsequent tissue damage due to a sustained formation of reactive species may contribute for the deleterious effects attributed to this transition metal, though this assumption needs to be confirmed in vivo. ( ), h spalteholz ( ), u reibetanz ( ), p salavei ( ), m fischlechner ( ), h-j glander( ), j arnhold ( ) ( ) university of leipzig, medical faculty, institute for medical physics and biophysics, germany ( ) university of leipzig, derpartment of dermatology, andrology training centre of the european academy of andrology, germany unintentional childlessness often caused by common reasons as inflammation affects - % of german couples. inflammations of the male genital tract lead to an infiltration of polymorphonuclear granulocytes (pmn), respectively induce a restricted spermatozoa quality associated with early triggered acrosome reaction (ar) and apoptosis as well as changes in the lipid structure and reduced mobility. stimulated pmn release the strongly cationic heme protein myeloperoxidase (mpo), which is able to bind to negatively charged membrane surfaces, e.g. apoptotic cell membranes with externalized phosphatidylserine (ps). a population of freshly prepared spermatozoa shows only a very small amount of cells with mpo binding ability as well as externalization of ps. the number of spermatozoa able to bind mpo raises considerably in samples containing predamaged cells or introducing the ar as could be observed with rhodamine b isothiocyanate (ritc)labelled mpo and antibody techniques by fluorescence microscopy as well as flowcytometry. the activation ofmpo with its substrate hydrogen peroxide (h o ) in the presence of chloride ions generates the powerful oxidizing and chlorinating species hypochlorous acid (hocl) and enhanced markedly the number of annexin v positive and non-vital cells. components of seminal plasma as well as serum albumin can protect spermatozoa for the deleterious effects of mpo. the coincidence of ps externalization and mpo binding to spermatozoa surfaces indicates an up to now unknown role of this enzyme in recognition and removal of apoptotic cells during inflammation. recent findings suggest a crucial role of proteinaseactivated receptor- (par ) in inflammation and innate immunity. par is the second member of a novel g protein-coupled receptor subfamily with seven putative trans-membrane domains. this subfamily is characterized by a unique mechanism of receptor activation. accessible serine proteases cleave the receptor to expose a new, previously cryptic, n-terminal sequence ("tethered ligand") which further interacts with the same receptor and activates it. tryptase, trypsin, and bacterial serine proteases are capable of directly activating par . par is expressed by human neutrophils, however its functions on these cells remained unclear. the data of our present study indicate that par agonists enhance interferon gamma (ifna)-induced up-regulation of cell surface fca;ri, one of the key receptors involved in neutrophil phagocytic activity. moreover, par agonists (serine proteases as well as synthetic activating peptide) and their receptor represent an additional system which controls neutrophil transendothelial migration and apoptosis in vitro. additionally, there is a significant increase of par expression on the neutrophil cell surface in the case of septic patients as compared to cells from healthy volunteers. together, our results indicate that par may be involved in the pathophysiology of acute bacteria-induced human diseases (sepsis or septic shock, for example) potentially by regulating neutrophil apoptosis, transendothelial migration and fca-receptor expression. aim: to ascertain the role of macrophages as direct inducers of regeneration after renal ischemia/reperfusion, and to establish whether inflammatory conditions contribute to the process. we determined whether adoptive transfer of macrophages at different stages of kidney inflammation after mouse renal i/r could restore reparation and assessed the influence of inflammation in the process.results: i/r provoked the increases in renal regeneration, as evaluated by inmunohistochemistry and pcr mrna of stathmin and pcna. the cytokine profile revealed the influence of the inflammatory environment on kidney repair. regeneration was macrophage-dependent, decreasing when depletion was provoked, and increasing with adoptive transfer of macrophages; however, administration of resting macrophages did not induce repair at the time points in which tissue was inflamed, and was only able to promote regeneration in the absence of inflammation ( hours). pro-inflammatory cytokines increased at the early stages of reperfusion, coinciding with low regeneration, and anti-inflammatory cytokines increased during the longer periods of reperfusion when regeneration was more evident.conclusions: macrophages directly induce renal regeneration after ischemia/reperfusion in an inflammationdependent manner. ( ), k bendtzen ( ), f sellebjerg ( ), ch nielsen ( ) ( antibodies against myelin basic protein (mbp) are present in sera from patients with multiple sclerosis (ms), but the role of these antibodies is controversial. we collected sera from ms patients and healthy individuals and found that both groups contained igm anti-mbp antibodies, while ms sera contained small amounts of igg anti-mbp. however, the two groups of sera did not differ significantly with respect to the content of either antibody subclass. addition of mbp to the various sera and subsequent addition of the mixtures to normal peripheral blood mononuclear cells (pbmc) resulted in a significant deposition of igm on cd + monocytes, indicating that formation of mbp/igm complexes had occurred. this deposition was strongly inhibited by addition of mm edta to the sera, indicating that it was complement dependent. the pbmc produced significant amounts of il- , tnf-alpha and ifn-gamma upon stimulation with mbp, and the extent of the cytokine production did not depend upon whether sera from ms patients or from healthy controls were present. however, disruption of the tertiary structure of mbp by boiling significantly reduced the production of all three cytokines, supporting a role for antibodies in the induction of cytokine responses to mbp. we propose that natural igm autoantibodies may form complexes with mbp, facilitating the uptake of mbp by antigenpresenting cells (apc). since sera from ms patients did not enhance this uptake and the subsequent cytokine production, the mechanism may be part of an appropriate peripheral regulation of self-reactivity. we currently investigate this possibility. loredana postiglione( ), g tarantino( ), a spanò( ), p ladogana ( ), fl perrone( ), s padula( ), a riccio ( ) ( ) federico ii university medical school of naples, department of molecular and cellular biology and pathology l.califano, naples, italy ( ) federico ii university medical school of naples, departmentof clinical and experimental medicine, naples, italybackground: hepatitis c virus (hcv) infection can induce immunological disorders with different clinical expression such asarthritis, sjogren sindrome and various form of vasculitis.aim: to study the prevalence of anti-cyclic citrullinated peptides antibodies (anti-ccp) in a group of patients affected by hcv-related arthritis and the eventual correlations with rheumatoid factor (rf) and/orantinuclear antibodies (ana), and articular involvement. study design: patients with arthritis were selected in a population of subjects affected by hcv infection. each patients was evaluated by clinical examination ( denoted poliarticular and mono-oligoarticualr involvement), by x-graphic aspects of joint involvement ( patients presented join erosions), by ana, rf and anti-ccp positiveness.results: , % of patients presented positivenessfor anti-ccp, without significant correlation between suchparameter and ana, rf and articular involvement. anti ccp resulted positive in out of the patients with joint erosions, and only in out of the patients without joint erosions. such frequency analyzed by chi square ended up in no significant differences. our patients presented an interesting prevalence of the positiveness for anti-ccp. these data suggest a consideration about the specificity, commonly attributed to this parameter in the diagnosis of rheumatoid arthritis. expression of nkg d on cd + t cells is generally rare in both mice and humans, but has been reported in a number of inflammatory diseases, including rheumatoid arthritis, crohns disease and an animal model of type diabetes. the monoclonal antibody cx recognizes murine nkg d and has been shown to block ligandbinding and mediate internalization of nkg d. furthermore, cx can inhibit and/or ameliorate disease in animal models of type diabetes and inflammatory bowel disease. thus, it is very likely that nkg d plays an important role in the development of inflammatory and autoimmune diseases. since little is known about the pharmacokinetics and pharmacodynamics of the cx antibody, we decided to study this in both regular balb/ c mice and immunodeficient cb .scid mice. different doses of cx antibody was injected intraperitoneally and pk and pd was measured by elisa (anti-cx elisa in serum) and flow cytometry (down-regulation of nkg d on cd b+ nk cells) for up to two weeks after administration. we found that cx very efficiently down-regulate nkg d on cd b+ nk cells and that the effects of the antibody can be seen for more than two weeks after one single injection. finally, we propose a model which may be helpful in predicting the effects of different doses of cx antibody in vivo. ( ), k mehta( ), n deo( ), j chaudhary( ), p bobrowski ( ) ( ) albany medical college, usa ( ) vedic lifesciences, usa ( ) rainforest nutritionals, inc, us background: the efficacy and safety of reparagen, in treating osteoarthritis was compared to glucosamine sulfate in a mumbai-based multi-center, randomized, double-blind study.methods: subjects (n= ) were screened and randomized to receive glucosamine sulfate (n= , mg/day) or reparagen (n= , mg/day), a polyherbal consisting of vincaria (uncaria guianensis) and rni (lepidium meyenii) administered orally, twice daily. primary efficacy variables were womac scores, visual analog score (vas) for pain, and response to treatment defined as a % improvement in womac pain, with assessments at , , , and weeks. secondary variables were results: subject randomization was effective and both treatments showed significant benefits in primary outcomes within one week (p< . ), with a similar, progressive improvement over the course of the week treatment protocol ( - % reduction in total womac or vas scores). the response rate was substantial for both glucosamine ( %) and reparagen ( %), which exceeded placebo responses ( %, p < . ) and supported by investigator and subject assessments. tolerability was excellent and safety parameters were unchanged. rescue medication use was significantly lower in the reparagen group (p < . ), and serum igf- levels were unaltered.conclusions: both reparagen and glucosamine sulfate produced substantial improvements osteoarthritis symptoms. response rates were high and the safety profile was excellent, with significantly less rescue medication use with reparagen. we speculate that the high response rate to glucosamine sulfate may reflect higher baseline pain levels or synergy with dietary curcumin. inflammation accompanies and aggravates progression of all modern human chronic pathological conditions. growing evidence indicates the beneficial role of proper nutrition in controlling inflammation. we investigated the effects of selected essential nutrients in experimental inflammation and the molecular mechanisms involved. tested nutrient mixture (nm) consisted of green tea catechins, citrus flavonoids hesperidin, naringenin and quercetin, ascorbate, lysine, proline, arginine and cysteine. systemic inflammation in mice challenged with bacterial lipopolysaccharide (lps) was monitored by blood plasma levels of fourteen key inflammatory cytokines. two week supplementation with mg nm/kg body weight prior to lps challenge provided significantly greater protection than did supplementation with ibuprofen. induction of interleukin- (il- ) and monocyte chemoattracting protein- , two cytokines especially responsive to lps challenge, was reduced in nmsupplemented animals by % and %, respectively. corresponding reduction in ibuprofen group was % and %. protective mechanisms involved were assessed in human cultured u macrophages stimulated with lps.the cytokines most responsive were tumor necrosis factor alpha ( % and % reduction by supplementation with nm and ibuprofen, respectively) and il- ( % and % in corresponding reduction). nm supplementation dramatically reduced prostaglandin e secretion by stimulated macrophages along with cyclooxigenase- (cox ) cellular protein expression. mrna levels forcox and inflammatory cytokines were also dramatically reduced. quercetin was the most effective nutrient when tested individually. however, nm appeared to surplus the combined effect of individual components. we conclude that the tested combination of essential nutrients demonstrates strong beneficial effects in experimental inflammation by targeting responsible gene expression. ( ), hp kim ( ), kh son ( ) ( ) college of pharmacy, kangwon national university, south korea ( ) department of food and nutrition, andong university, south korea chalcones belong to flavonoid family from plant origin and some of them possess anti-inflammatory activity. recently, several natural and synthetic chalcones were reported to inhibit inducible nitric oxide synthase (inos)-catalyzed no production in cell cultures. in the present study, to find the optimal chemical structures and to elucidate their action mechanisms, synthetic chalcones having the substituent(s) on a-and b-rings were prepared and their effects on inos-catalyzed no production were evaluated using lps-treated raw . cells. among the tested compounds, -methoxy- , -dichlorochalcone (ch ), -hydroxy- -methoxychalcone (ch ), -hydroxy- -bromo- -methoxychalcone (ch ) and -hydroxy- , -dimethoxychalcone (ch ) potently inhibited no production (ic s, . - . mm). the favorable chemical structures were found to be a methoxyl substitution in a-ring at adjacent position ( or ) to carbonyl moiety with/without -(or -)hydroxyl group and -halogen substitution in b-ring. when the cellular action mechanisms of ch , ch and ch were further examined, it was revealed that ch and ch clearly down-regulated inos expression while ch did not. moreover, ch and ch were proved to suppress the nuclear transcription factor-kb activation. from the results, it is suggested that certain chalcone derivatives potently inhibit inos-catalyzed no production by the different cellular mechanisms, inos down-regulation or inos inhibition, depending on their chemical structures. these chalcone derivatives may be possibly used as lead compounds for developing new anti-inflammatory agents. an oligomeric stilbene alpha-viniferin (avf) was isolated from root of carex humilis (cyperaceae) as an inhibitor of cyclooxygenase (cox)- activity by bioassayguided fractionation. the avf was later found to downregulate lipopolysaccharide (lps)-induced cox- expression as well as to inhibit nuclear factor (nf)-kb activation, in addition to its inhibitory effect on cox- activity. furthermore, the compound exhibited antiarthritic effect in vivo. avf is a trimer of resveratrol and contains benzofuran moieties in its central part. starting from benzofuran and its related chemicals, cyclohexylimino- -methyl- , -dihydro- h-benzo [ , ] oxathiol- -one (lyr- ) was discovered to inhibit lpsinduced nf-kb transcriptional activity in macrophages raw . . the lyr- reduced lps-induced dna binding activity and nuclear translocation of nf-kb as well as inhibited lps-induced degradation and phosphorylation of inhibitory kb (ikb) protein. these results suggest that lyr- could suppress lps signaling molecule, putatively ikb kinase (ikk) complex, upstream ikb degradation in nf-kb activating pathway. lyr- inhibited in vitro kinase activity, gst-ikb phosphorylation, of wild type ikkbeta or a constitutively active ikkbeta mutant (c/a, cys- to ala) but did not affect that of another constitutively active ikkbeta mutant (ss/ee, ser- and to glu). therefore, lyr- could inhibit lps-induced nf-kb activating pathway by targeting ser- and/or residues on the activation domain of ikkbeta. as pharmacological actions, lyr- prevented nf-kb-dependent expression of inducible nitric oxide synthase, cox- , or inflammatory cytokines at the transcription level in lps-stimulated macrophages raw . . furthermore, lyr- protected lpsinduced septic shock in vivo. faculty of medicine, institute of pharmacology, ljubljana, slovenia a part of anti-inflammatory action of antidepressants can arise from their effect on histamine elimination from the side of inflammation. in mammals histamine is mainly degraded by two enzymes: histamine-n-methyltransferase (hnmt) and diamine oxidase (dao). the aim of present investigation is to establish whether antidepressants amitriptyline and sertraline can affect histamine metabolism. their effects on enzyme activity and mrna expression were studied in guinea pig tissues. plasma and tissue homogenates were incubated with saline (control) and different antidepressant concentrations. specific enzymatic activities of dao and hnmt were determined by radiometric assay. in addition, guinea pigs were treated with saline or amitriptyline ( mg/kg, ip), afterwards dao and hnmt mrnas were detected by pcr in different tissues. results showed thatamitriptyline, nm, , and mm, increased guinea pig plasma dao activity by , , and %, respectively, while sertraline increased it at mm (by %). at higher concentrations ( and mm) sertraline decreased dao activity. in the guinea pig tissues hnmt activity changes were found only when incubated with amitriptyline; sertraline had no effect. at and nm amitriptyline the activity of hnmt increased by and %, respectively. in animals treated with amitriptyline an induction of dao and hnmt mrna expression was noticed in several tissues. our results suggest that in guinea pigs due to higher histamine metabolism antiinflammatory effects can be expected at lower concentrations of antidepressants. the effect might be the opposite with higher amitriptyline concentrations. steven hefeneider( , ), c macarthur ( ), d trune ( ), s mccoy ( ) ( ) oregon health and science university, portland, oregon, usa ( ) targeted gene delivery, inc., portland, oregon, usa engagement of toll-like receptors (tlrs) by bacterial components such as lps and dna initiates inflammation.the current study examines a novel anti-inflammatory peptide, termed p , for treatment of inflammation induced by either lps or bacteria.peptide p was derived from an immunoregulatory protein of vaccinia virus, and interferes with tlr signaling.in this study we examined the efficacy of p to limit inflammation in a mouse model of sepsis and a model of middle ear inflammation, termed acute otitis media (aom).we demonstrate in the sepsis model, that in vivo treatment of mice with p inhibited lps-induced production of serum inflammatory mediators.moreover, p treatment, administered after initiation of inflammation, significantly increased survival of mice injected with lps.in the aom model, peptide p significantly reduced in vivo middle ear inflammation and fluid accumulation initiated by h. influenza.assessment of route of administration and delayed treatment studies demonstrated the efficacy of peptide p .simultaneous injection of bacteria and peptide p resulted in a significant reduction in fluid accumulation, infiltrating cells, and tympanic membrane thickness.fluid accumulation within the eustachian tubes was also significantly reduced following p treatment.-subcutaneous and oral administrations of p , but not intravenous administration, were also efficacious in reducing inflammation. administration of p after initiation of an ongoing inflammatory response was effective at reducing inflammation and fluid development.taken together, these results demonstrate the therapeutic potential of peptide p to limit an inflammatory response and suggest a possible new treatment strategy for bacterial-induced inflammation. ( ), c zhou( ), y zhang( ), m sun( ), x wan ( ), h yu( ), x yang( ), rd ye ( ), j-k shen ( ) formyl peptide receptor-like (fprl ) is a structural homologue of fpr, which binds chemotactic peptides of as small as amino acids (e.g., fmet-leu-phe, fmlf) and activates potent bactericidal functions in neutrophils. in comparison, fprl ligands include peptides of - amino acids, such as trp-lys-tyr-met-val-[d]met (wkymvm) and other synthetic peptides. to determine the core peptide sequence required for fprl activation, we prepared various analogues based on wkymvm and evaluated their bioactivities in an fprl -transfected cell line. although substitution of d-met resulted in loss of activity, removal of val together with d-met produced a peptide that retained most of the bioactivities of the parent peptide. the resulting peptide, wkym, represents a core structure for an fprl ligand. further substitution of lys with nle slightly improved the potency of the tetrapeptide, which becomes a dual agonist for both fprl and fpr. based on these structure-activity studies, we propose a model in which the modified tetrapeptide trp-nle-tyr-met (wnleym) binds to fprl through aromatic interactions involving the side chains of trp and tyr , hydrophobic interaction of nle , and the thio-based hydrogen bonding of met , with the respective residues in fprl which have not been identified. the identification of the core sequence of a potent peptide agonist provides a structural basis for future design of peptidomimetics as potential therapeutic agents for fprl -related disorders.there is a growing awareness of the interaction of food constituents with the immune system. the present study aims to evaluate immunomodulatory effects of two of these nutritional components, i.e. glycine and lactoferrin. mice orally supplemented with glycine, lactoferrin or a combination were injected intradermal (in the ear) with zymosan. ear swelling, as a measure for inflammation, as well as il- , tnf-a and il- levels in the ear and the number of tnf-a producing spleen cells were analyzed.-glycine and lactoferrin were able to decrease the zymosan induced inflammatory response locally (decreased ear swelling and pro-inflammatory cytokine levels) as well as systemically (reduced number of tnf-a producing spleen cells).glycine effects ( , and mg/mouse/day) were concentration dependent whereas for lactoferrin only the lowest doses ( . and mg/mouse/ day) inhibited the inflammatory response significantly. surprisingly higher doses of lactoferrin ( and mg/ mouse/day) failed to influence the inflammatory reaction. a combination of both nutrients (lactoferrin . mg/ mouse/day in combination with glycine or mg/ mouse/day) inhibited the zymosan induced ear swelling synergistically. additionally an additive effect of both components was seen on the number of tnf-a producing spleen cells. the present data show anti-inflammatory activity of glycine and lactoferrin using the zymosan induced inflammation model.moreover a combination of both components demonstrated a synergistic effect on inflammation of the skin and an additive effect on the number of tnf-a producing spleen cells. ( ), p sambrook( ), k fukudome( ), m xue ( ) ( ) university of sydney, st leonards, nsw, australia ( ) saga medical school, saga, japan objectives: to investigate the i) expression of endothelial protein c receptor (epcr) in synovial membrane and peripheral blood monocytes from patients with rheumatoid arthritis (ra) and osteoarthritis (oa) and ii) role of epcr and its ligand, activated protein c (apc), on the function of monocytes from ra patients.methods: epcr, cd and pc/apc in synovial tissues were detected by immunostaining and in situ pcr. monocytes were isolated from peripheral blood of patients with ra and treated with apc, lipopolysaccharide (lps), and/or epcr blocking antibody, rcr . cells and supernatants were collected to analyze the expression/activation of epcr, nuclear factor nf-kb and tumour necrosis factor tnf-a.results: epcr was expressed by both oa and ra synovial tissues but was markedly increased in ra synovium. epcr was colocalized with pc/apc mostly on cd positive cells in synovium. in ra monocytes, apc upregulated epcr expression reduced monocyte chemoattractant protein- -induced chemotaxis of monocytes by approximately %. apc also completely suppressed lps-stimulated nf-kb activation and attenuated tnf-a protein by more than % in ra monocytes. the inhibitory effects of apc were reversed by rcr , indicating that epcr modulates the inhibitory effects of apc.conclusions: our results demonstrate for the first time that epcr is expressed by synovial tissues, particularly in ra, where it co-localizes with pc/apc on monocytes/ macrophages. in addition, apc inhibits the migration and activation of ra monocytes via epcr. these inhibitory effects on ra monocytes suggest that pc pathway may have a beneficial therapeutic effect in ra. key: cord- - p b authors: bohr, adam; tsapis, nicolas; foged, camilla; andreana, ilaria; yang, mingshi; fattal, elias title: treatment of acute lung inflammation by pulmonary delivery of anti-tnf-α sirna with pamam dendrimers in a murine model date: - - journal: european journal of pharmaceutics and biopharmaceutics doi: . /j.ejpb. . . sha: doc_id: cord_uid: p b abstract to improve the efficacy of nucleic acid-based therapeutics, e.g., small interfering rna (sirna), transfection agents are needed for efficient delivery into cells. several classes of dendrimers have been found useful as transfection agents for the delivery of sirna because their surface can readily be functionalized, and the size of the dendriplexes they form with sirna is within the range of conventional nanomedicine. in this study, commercially available generation poly(amidoamine) (pamam) dendrimer was investigated for pulmonary delivery of sirna directed against tumor necrosis factor (tnf) α for the treatment of acute lung inflammation. delivery efficiency was assessed in vitro in the raw . macrophage cell line activated with lipopolysaccharide (lps), and efficacy was evaluated in vivo in a murine model of lps-induced lung inflammation upon pre-treatment with tnf-α sirna. the pamam dendrimer-sirna complexes (dendriplexes) displayed strong sirna condensation and high cellular uptake in macrophages compared with non-complexed sirna. q-pcr analyses showed that the dendriplexes mediated efficient and specific tnf-α silencing in vitro, as compared to non-complexed sirna and dendriplexes with negative control sirna. also in vivo, the pamam dendriplexes induced efficacious tnf-α sirna inhibition, as compared to non-complexed sirna, upon pulmonary administration to mice with lps-induced lung inflammation. hence, these data suggest that pamam dendrimers are promising for the local delivery of tnf-α sirna in the treatment of lung inflammation via pulmonary administration. oligonucleotide-based therapeutics, including sirna, antisense oligonucleotides and mirna, are applied to intervene with the expression of specific target genes and are thereby thought to mediate more specifically disease treatment than therapeutics based on small molecules or peptides/proteins. inflammatory lung diseases are complex disorders that are usually treated with medications, which are relatively unspecific in their mode of action and are administered systemically, resulting in increased drug exposure but also undesired side effects [ , ] . here, local sirna-based treatment may provide a much more specific and safe therapy in which certain inflammatory pathways can be targeted [ ] . of the many pro-inflammatory cytokines involved in lung inflammation processes, tnfα is believed to play a central role in most inflammatory lung conditions, e.g., chronic obstructive pulmonary disease (copd), asthma, acute respiratory distress syndrome (ards) and acute lung injury (ali) [ ] . more recently, it was shown to be a major target in the treatment of inflammatory flares in covid- infection-related ards [ ] . hence, tnf-α is a promising target for sirna-based therapy against both acute and chronic lung inflammation. a sirna-based therapeutic targeting lung inflammation can be administered locally to the airways, either via inhalation or via nasal administration, where it can exert its effect directly in the inflamed tissue. local pulmonary delivery displays several therapeutic advantages, compared with systemic delivery, including (i) a quick onset of action, (ii) a reduced therapeutic dose required, and (iii) reduced side effects [ ] . besides, inhalation and nasal administration represent non-invasive routes of administration, which increases patient compliance, and the fast renal clearance of sirna, observed after systemic administration, is reduced after local delivery [ ] . although numerous promising therapeutic targets and oligonucleotide sequences have been identified during the past years, which have resulted in a handful of recently marketed or in advanced clinical trial products, there are still significant challenges associated with their delivery [ , ] . generally, sirna displays poor chemical stability against nucleases and exhibits low cellular uptake and transfection [ ] . hence, it is pertinent to identify efficient delivery systems that can protect the sirna from degradation, facilitate its transport across the cell membrane and mediate endosomal escape to achieve successful sirna delivery to the cytosol [ , ] . numerous transfection agents have been identified for nucleic acid delivery, and they include, amongst others, cationic polymerand lipid-based nanocarriers, which are very efficient for cellular delivery, but they are often associated with toxicity, even at relatively low doses [ , ] . dendrimers are synthetic globular polymers displaying a high degree of surface functionality and numerous possibilities for customizing their physicochemical properties, and they have shown great potential for pharmaceutical applications, including the delivery of nucleic acid-based therapeutics [ ] [ ] [ ] . cationic dendrimers like the commercially available polyamidoamine (pamam) dendrimers have been shown to mediate efficient cellular uptake and transfection of sirna in vitro in multiple studies [ ] . although widely used in vitro, there are only a few studies that have tested the ability of pamam dendrimers for sirna transfection in vivo [ , ] , and to date, no studies have been performed evaluating pulmonary delivery of pamam dendrimers in vivo. in this study, we investigated generation pamam dendrimers as transfection agents for pulmonary delivery of sirna targeting tnf-α and examined their efficacy and safety in a murine acute lung inflammation model. generation pamam dendrimers were selected because they display very good efficiency for dendriplex formation they were prepared at different dendrimer-sirna ratios and were characterized in vitro and in vivo concerning complexation, cellular uptake, cytotoxicity, in vitro transfection efficiency and in vivo therapeutic efficacy at the rna and protein levels, respectively. tnf-α sense '-pgucucagccucuucucauuccugct- ', and antisense '-agcaggaaugagaagaggcugagacau- ', where the underlined capital letters represent '-o-methylribonucleotides, lower-case letters represent deoxyribonucleotides and p represents a phosphate residue [ ] . a negative control sirna sequence was purchased from eurogentec (eurogentec, angers, france). the sequence of this negative control is not disclosed by the supplier. fluorescently labeled sirna with the dye tye™ was provided by idt with a sequence targeting luciferase: all additional chemicals used were obtained commercially and were of analytical grade. sense '-pgguuccuggaacaauugcuuuuaca- ', dendriplexes were prepared in mm hepes buffer at a nitrogen-to-phosphate (n/p) ratio of , , were used for upconcentrating the dendriplex suspensions by centrifugation of the samples at , g for min. the particle size distribution and polydispersity index (pdi) of the dendriplexes were determined by dynamic light scattering using the photon correlation spectroscopy technique, and their zeta potential was estimated by using laser-doppler micro-electrophoresis using a zetasizer nano zs (malvern instruments, worcestershire, uk) equipped with a nm laser and ° detection optics. the complexation of dendrimers and sirna into dendriplexes was assessed using gel electrophoresis. electrophoresis was carried out applying % (w/v) agarose gels (promega, city, usa) containing µl . mg/ml ethidium bromide solution, and samples consisting of . nmol sirna were loaded into each well of the gels. the gels were run for min at v in tris-borate-edta (tbe) buffer (ph . ). visualization of the sirna bands was performed using an mf-chemibis gel imaging system (dnr bio-imaging systems, neve yanim, israel). a murine macrophage cell line raw . was purchased from the american type culture collection (atcc, molsheim, france) and cells were maintained in dulbecco's modified eagle's medium (dmem) (aldrich, st quentin fallavier, france) supplemented with % (v/v) fetal bovine serum (paa laboratories, pasching, austria) and u/ml penicillin, μg/ml streptomycin. cells were grown under a controlled atmosphere with % co / % o at °c and were sub-cultured twice per week by washing and gently scraping the cells from the culture flask. cells were used between passages and . the cellular viability of the raw . cells was assessed by using the -( , -dimethylthiazol- -yl)- , -diphenyltetrazolium bromide (mtt) assay. cells were prepared in suspension, counted and seeded in -well plates at a density of × cells per well and left to attach overnight. the cells were subsequently incubated with dendriplexes (n/p ratio ) at different concentrations for a period of h. to each well with µl culture medium, µl of mtt-solution ( mg/ml in pbs, ph . ) was added and the plate was incubated for h at °c. subsequently, the medium was replaced with µl dimethylsulfoxide, and the absorbance was measured at nm, using a microplate reader (fluostar optima, bmg labtech, germany). cellular uptake of sirna was assessed in raw . cells using fluorescence microscopy and flow cytometry, respectively. for fluorescence microscopy, the cells were seeded in chambered cover slides µ-slide well (ibidi, planegg, germany) at a density of × cells per well and cultured for h. the cells were then treated with fluorescently labeled sirna for h (at an n/p ratio of for dendriplexes and a concentration of nm sirna), washed with pbs and fixed using % (v/v) paraformaldehyde. microscopy was performed using a zeiss lsm (carl zeiss, jena, de) fluorescence microscope equipped with a mw helium-neon laser and a plan-apochromat x objective lens (numerical aperture . , oil immersion). images were captured at a x magnification and overlayed with differential interferential contrast (dic) images. for flow cytometry, the cells were cultured in -well plates at a density of × cells per well. the cells were treated with fluorescently labeled sirna for h (at an n/p ratio of for dendriplexes and a concentration of nm sirna) and subsequently resuspended and analyzed using an accuri c (bd biosciences, franklin lakes, nj, usa) flow cytometer. the mean fluorescence intensity (mfi) was used to determine the relative cell uptake (the ratio between treated and non-treated samples), expressed in arbitrary units. cell transfection studies were performed as described previously [ ] [ ] . briefly, raw . cells were seeded at a density of × cells per well in -well culture plates. dendriplexes prepared at an n/p ratio of were transferred to culture plates to achieve a final sirna concentration of nm, and they were incubated for h. three hours before collection, the cells were exposed to lipopolysaccharide (lps) dispersed in pbs to obtain an lps concentration of ng/ml. negative controls were only given pbs, and positive controls only received lps, but were not treated with sirna. for collection, the culture medium was removed from the wells, ml ice-cold trizol (thermo fisher, villebon-sur-yvette, france) was added to each well, and the cells were scraped and homogenized by pipetting. rna extraction was performed following the stepwise instructions provided with the trizol reagent, and the total rna content of the extracts was assessed using a biomate uv spectrophotometer (thermo fisher) with a traycell ultra-micro cell (hellma analytics, paris, france). additional quality control of the extracts was performed with an rna labchip, using an agilent bioanalyzer (agilent, santa clara, ca, usa). reverse transcription was performed for µg rna extracts using a mix of primers [ ] and an iscript cdna synthesis kit (bio-rad laboratories, (hercules, ca, usa). the cdna was diluted : (v:v) in pcr-grade water and stored at − °c until further use. tnf-α mrna silencing was assessed by real-time reverse transcription-polymerase chain reaction (rt-pcr), essentially as previously described [ ] . real-time pcr was performed using a c thermal cycler instrument with a cfx real-time system (biorad) with the following cycling conditions: initial denaturation step at °c for min, followed by cycles including (i) denaturation at °c for s, (ii) annealing at °c for s, and (iii) elongation at °c for s. the cfx manager software . (biorad) was used for crossing point (cp) analysis, and the values were normalized against the average of the two reference genes ribosomal protein, large p (rplp ) and glucoronidase b (gusb). the reference genes were selected based on a screening study of eight reference candidates [ ] . all animal experiments were conducted following the european rules ( / /eec and / /eu) and the principles of laboratory animal care and the national french legislation (decree no. - and minimal stress to the animals. female swiss cd- outbred mice were purchased from envigo (gannat, france), and all experiments were performed using mice at the age of to weeks. the mice were housed in groups of four with access to water and food ad libitum and kept at a constant temperature ( - °c) and relative humidity ( - %). a well-known procedure for lps-induced lung inflammation [ ] [ ] [ ] was modified using swiss cd- injection of µl mg/ml pentobarbital solution). subsequently, the trachea was cannulated with a catheter, and the lungs were flushed twice with . ml pbs. the bal was centrifuged at g for min, the supernatant was stored at - °c for protein quantification and tnf-α determination. mice were dosed with sirna as a prophylactic treatment before inducing lung inflammation ( figure ). non-complexed sirna and dendriplexes (n/p ratio of ) were administered via intranasal administration similar to lps administration at a sirna concentration of . mg/kg using an average volume of µl sirna solution h before lps challenge. the bal was extracted and h, respectively, after the lps challenge, and a minimum of five mice was included in each sample group. negative controls were dosed twice with pbs, and positive control mice were first dosed with pbs and subsequently treated with lps. figure . schematic illustration of the in vivo experimental procedure. mice were prophylactically administered with treatments h before inflammation induction by lps. bal was collected either at or h for tnf-α determination. the tnf-α protein levels were assessed by using the cytometric bead array -mouse inflammation kit (bd biosciences). the supernatants of bal samples were diluted in assay diluent from the kit, and the samples were prepared following the manufacturer´s instructions. the bal tnf-α levels were quantified using an accuri c flow cytometer, where roi counts were measured for each sample. all samples were prepared in duplicates and analyzed using the bd accuri c software. the data of the in vitro studies are reported as mean values ± sd, and the data for the in vivo studies are reported as mean values ± sem. the statistical significance was determined using either a twotailed, unpaired student's t-test or anova, with the statistical significance set at * p < . , ** p < . . all dendriplexes displayed an average size between - nm and a pdi between . - . (table ). there was no clear correlation between the n/p ratio of the dendriplexes and their resulting sizes and pdis although it has previously been shown that the sizes of pamam dendriplexes decrease with an increase in n/p ratio [ ] . positive zeta potential values were observed for all n/p ratios, indicating a net positive surface charge and condensation of sirna. the zeta potential increased significantly (p < . , anova) as a function of the n/p ratio, which can be attributed to the increase in charge ratio, and it also indicates improved sirna condensation at higher n/p ratios. gel electrophoresis studies using etbr to stain the sirna were performed to assess qualitatively the binding between sirna and dendrimer at different n/p ratios. these studies show binding between sirna and dendrimers at all n/p ratios (figure ). in all cases, the major part of the sirna was retained inside the dendriplexes in the presence of etbr, as compared to free, non-complexed sirna. based on the size and binding results of the dendriplexes, subsequent experiments were performed at an n/p ratio of as a compromise between the complexation and high sirna loading as shown by gel retardation assay. moreover, this ratio was selected to reduce the cytotoxicity as much as possible. cell viability studies using the mtt assay showed that the dendriplexes were well tolerated up to a sirna concentration of nm (n/p ratio ) ( figure a) . a similar profile was observed for noncomplexed dendrimers (not shown). the cytotoxicity measured for the dendriplexes is relatively low, as compared to other cationic transfection agents, e.g., polyethyleneimine (pei) and poly-l-lysine [ ] , which indicates that pamam dendriplexes are well tolerated by raw . cells. cellular uptake assessed at a subtoxic concentration of nm sirna using flow cytometry showed that the uptake of non-complexed sirna was low and did not increase over time ( figure b ). in contrast, the cellular uptake of the dendriplexes increased gradually with time, resulting in a six-fold higher uptake transfection using tnf-α sirna and negative control sirna in macrophages activated with lps was evaluated at the mrna level using qpcr ( figure ). an sirna containing '-o-methylated nucleotides in the antisense strand was selected to minimize the innate immune response that occurs with the administration of sirna [ ] . another reason is provided by the greater stability of this chemically modified sirna [ ] . non-complexed tnf-α sirna mediated little but statistically significant inhibition ( %) of the tnf-α mrna expression (p < . ), as compared with the negative control sirna, and % inhibition, as compared to the lps-challenged positive control (p < . ). in contrast, the pamam dendriplexes with tnf-α sirna mediated % inhibition of tnf-α expression, as compared to the lps-challenged positive control (p < . ) and % inhibition compared with the pamam dendriplexes with negative control sirna (p < . ), which represents a substantially greater inhibition compared with non-complexed sirna. hence, pamam dendriplexes display high potential for tnf-α silencing, but some unspecific silencing of pamam dendriplexes with negative control sirna was also observed. the transfection data support the observations from the cell uptake studies, indicating the improved performance of dendriplexes, as compared with non-complexed sirna. figure . tnf-α mrna silencing in raw . cells by dendriplexes. samples were normalized to the lps-treated cells (control +) without sirna, and the results denote the tnf-α mrna expression level of cells treated with tnf-α sirna, relative to transfection with negative control sirna. the samples correspond to a sirna concentration of nm and an n/p ratio of . results denote mean values ± sd (n ≥ ). statistical significance: * p < . , ** p < . . to induce gene silencing in the lung, sirnas were delivered using pamam dendrimers. many studies have examined the effect of pamam dendrimers on the lung. at first, it was shown that % of the pamam dendrimers dosed were still remained in the lung . h post pulmonary administration to mice [ ] . several studies have stressed the lung biocompatibility of pamam dendrimers and the absence of any inflammatory response in the lung [ , ] . in several studies, it has been shown that intranasal challenge with lps results in lung inflammation characterized by an increase in the levels of tnf-α as well as other pro-inflammatory cytokines in the lungs [ ] . besides, the massive recruitment of macrophages and neutrophils takes place rapidly, and the inflammatory process lasts for several days [ , ] . chronic inflammation is usually characterized by occasional flare-ups, including copd, asthma, and cystic fibrosis, which represent the most common reason for contact between the patient and the health care system [ ] . these flareups can be triggered by both external and internal stimuli, e.g., exposure to pollutants and increased stress levels. hence, the optimal treatment strategy may be a prophylactic measure taken to reduce the response to such stimuli. therefore, in this study, we investigated the therapeutic effect of a prophylactic treatment with sirna given h before inducing an inflammatory response with lps. tnf-α levels were compared for mice treated with lps (control +) as well as untreated mice (control -) and were evaluated to assess the potential inflammatory response of the non-complexed sirna and the dendriplexes. dendriplexes were administered at a sirna dose of mg/kg, based on previous sirna studies on pulmonary delivery, where doses ranging between . - mg/kg were administered [ ] [ ] [ ] . the tnf-α concentrations in the bal were measured h and h after exposing the mice to lps. the tnf-α levels measured after h ( figure a macrophages. yet, based on in vivo studies it was demonstrated that dendriplexes with tnf-α sirna resulted in improved performance compared with non-complexed tnf-α sirna at early time e.g h after lps challenge and lower performance compared with non-complexed tnf-α sirna h after lps challenge, meaning that more frequent administrations are needed in patients displaying strong lung inflammation. in this study, tnf-α was selected as a target because this proinflammatory cytokine plays a central role in lung diseases and its actions are numerous and quite diverse being linked to many lung diseases including asthma, copd, ali/ards, sarcoidosis, and interstitial pulmonary fibrosis [ ] . the data show that a quick response occurs not lasting long which might be related to a very quick dissociation in the lungs. thanki et al. [ ] demonstrated that although part of complexed sirna was staying in the lungs, around % was permeating across the air-blood barrier within h and subsequently excreted via the kidneys [ ] . the low stability as well might explain why the efficacy does not stand for longer times. in a previous work [ , ] involving phosphated dendrimers, we compared two dendrimers in a lung injury model using the same anti-tnf-α sirna. it was clear from these studies that dendriplexes with the lowest kd were the more active with a long-lasting inhibition effect not only on tnf-α but other cytokines as well. this could be achieved with more frequent lung administration but would also raise the question of chronic toxicity which needs to be further evaluated. applying higher-generation pamam denderimers would have increased the stability and efficacy but again could have induced much higher toxicity. finally, two factors might explain the lower efficacy of dendriplexes at -hour. the first is the high stability of methylated sirna and the second is the fast elimination of dendriplexes due to the mucociliary clearance, as compared to the clearance of non-complexed molecules that can penetrate in the deep lung without undergoing this elimination process. in the current study, the performance of pamam dendrimers was investigated as a delivery system for sirna transfection, specifically for the local treatment of lung inflammation. we demonstrate a good ability to condensate sirna, high cellular internalization rate and a specific and efficient gene silencing of tnf-α in vitro in macrophages for pamam-based dendriplexes with tnf-α targeting sirna. in vivo studies in a murine acute lung inflammation model showed silencing of tnf-α for the dendriplexes although less pronounced compared to in vitro performance. the findings suggest that tnf-α targeting sirna can be used as a local treatment for overall suppression of lung inflammation and can be used as a prophylactic treatment administered one day before an inflammatory trigger. the copd pipeline global strategy for the diagnosis, management, and prevention of chronic obstructive lung disease: the gold science committee report cytokine inhibition in the treatment of copd role of tnfα in pulmonary pathophysiology trials of anti-tumour necrosis factor therapy for covid- are urgently needed kissel, sirna delivery to the lung: what's new? mechanistic profiling of the release kinetics of sirna from lipidoidpolymer hybrid nanoparticles in vitro and in vivo after pulmonary administration overcoming the challenges of sirna delivery: nanoparticle strategies nanoscale particles for lung delivery of sirna knocking down barriers: advances in sirna delivery rational design of cationic lipids for sirna delivery current progress in gene delivery technology based on chemical methods and nano-carriers non-viral methods for sirna delivery dendrimers and hyperbranched polymers nanomaterials based on phosphorus dendrimers anti-inflammatory effect of anti-tnf-α sirna cationic phosphorus dendrimer nanocomplexes administered intranasally in a murine acute lung injury model dendrimers for sirna delivery delivering sirna with dendrimers: in vivo applications poly (amidoamine)(pamam) dendrimer mediated delivery of drug and pdna/sirna for cancer therapy chitosan/sirna nanoparticle-mediated tnf-α knockdown in peritoneal macrophages for antiinflammatory treatment in a murine arthritis model comparison of polymeric sirna nanocarriers in a murine lps-activated macrophage cell line: gene silencing, toxicity and off-target gene expression il- , produced by lymphocytes and neutrophils, is necessary for lipopolysaccharide-induced airway neutrophilia: il- as a possible trigger local stimulation of alpha cholinergic receptors inhibits lps-induced tnf-alpha release in the mouse lung van der poll, the lps-induced lung inflammation in vivo elucidating the molecular mechanism of pamam-sirna dendriplex self-assembly: effect of dendrimer charge density polyamidoamine dendrimers with a modified pentaerythritol core having high efficiency and low cytotoxicity as gene carriers overcoming the innate immune response to small interfering rna, hum chemical modification of sirnas to improve serum stability without loss of efficacy effect of the route of administration and pegylation of poly(amidoamine) dendrimers on their systemic and lung cellular biodistribution polyamidoamine dendrimers can improve the pulmonary absorption of insulin and calcitonin in rats pamam dendrimers as nano carriers to investigate inflammatory responses induced by pulmonary exposure of pcb metabolites in sprague-dawley rats nasal lipopolysaccharide challenge and cytokine measurement reflects innate mucosal immune responsiveness a prominent role for airway epithelial nf-κb activation in lipopolysaccharide-induced airway inflammation acute lung injury: prevention may be the best medicine living with chronic obstructive pulmonary disease: a survey of patients' knowledge and attitudes in vivo tumor targeting via nanoparticle-mediated therapeutic sirna coupled to inflammatory response in lung cancer mouse models in vivo endothelial sirna delivery using polymeric nanoparticles with low molecular weight lipid envelope-type nanoparticle incorporating a multifunctional peptide for systemic sirna delivery to the pulmonary endothelium anti-tnfα therapy in inflammatory lung diseases elucidating the role of surface chemistry on cationic phosphorus dendrimer-sirna complexation the authors would like to thank the danish council for independent research key: cord- - vjh mq authors: selwyn, david; yang, ding; heward, elliot; kerai, ashwin; thompson, elinor; shommakhi, abulgasem; faulkner, scott; siau, richard; walijee, hussein; hampton, tom; chudek, dorota; singhera, supriya; din, waqas; lau, andrew s. title: a prospective multicentre external validation study of the liverpool peritonsillar abscess score (lps) with a no‐examination covid‐ modification date: - - journal: clin otolaryngol doi: . /coa. sha: doc_id: cord_uid: vjh mq objectives: our primary aim was to validate the liverpool peritonsillar abscess score (lps) externally in a new patient cohort. our secondary aim was to modify the lps in the light of the covid‐ pandemic to produce a no‐examination variant for use in this instance. design: prospective multicentre external validation study. setting: six different secondary care institutions across the united kingdom. participants: patients over years old who were referred to ent with any uncomplicated sore throat such a tonsillitis or peritonsillar abscess (pta). main outcome measures: sensitivity, specificity, positive predictive value and negative predictive value for both the original lps model and the modified model for covid‐ . results: the lps model had sensitivity and specificity calculated at % and %, respectively. the lps has a high negative predictive value (npv) of %. the positive predictive value (ppv) was slightly lower at %. receiver operating characteristic (roc) curve, including the area under the curve (auroc), was . which indicates very good accuracy. conclusions: external validation of the lps against an independent geographically diverse population yields high npv. this may support non‐specialist colleagues who may have concerns about mis‐diagnosing a pta. the covid‐ modification of the lps has a similar npv, which may be of use where routine oral examination is to be avoided during the covid‐ pandemic. reports suggest that there are increasing numbers of inpatient admissions to secondary care with pta and dnsi. it is therefore important for both non-specialists and specialists to arrive at the correct diagnosis in order to facilitate early and appropriate treatment. we previously reported the development of a predictive score for peritonsillar abscess through a prospective multicentre observational study (liverpool peritonsillar abscess score, lps). the lps is an additive threshold score consisting of five variables with the aim of predicting the likelihood of pta in adult patients. internal validation of the lps from the initial study sample produced a high sensitivity and negative predictive value (npv; % for both). the lps functions very much like the wells score (which estimates the probability of venous thromboembolism), and its strength lies in its npv. we theorise that the ppv is lower because a proportion of patients with tonsillitis will have trismus, and because the lps was developed using aspirate-proven pta (rather than peritonsillar cellulitis). our aim in developing the lps was to support non-specialists in preventing a missed diagnosis of pta and to facilitate remote triage by secondary care services and to avoid any need for significant extra resources or funding in the process. during the current covid- pandemic, ent uk has issued updated guidance, reserving oral examination only for severe cases of sore throat. a validated predictive score not reliant on oral examination may therefore be useful to the non-specialist when triaging or referring a suspected pta. our primary aim was to validate the liverpool peritonsillar abscess score (lps) externally against a separate cohort of patients through a multicentre prospective observational study. our secondary aim was to modify the lps in the light of the covid- pandemic to produce a no-examination variant for use in this instance. this study and manuscript adhere to the strobe reporting guidelines for observational studies. when developing the lps, we based our previous sample size estimate on the need for a certain number of events per variable (epv). however, in the external validation of a multivariable predictive model, statistical modelling suggested the need for "at least events and ideally (or more) events". we therefore set our sample size at . in line with our previous work, we defined a pta as clinical suspicion based on typical symptoms and signs with positive aspiration of pus from the peritonsillar space. non-pta sore throat, including tonsillitis and peritonsillar cellulitis, was defined either on senior review (ent specialty registrar st or more senior) or when there had been suspicion of pta but a negative aspirate. data were collected prospectively from august onwards until we met our sample size requirement. we included consecutive patients over years old who were referred to the ent service for assessment of sore throat such as tonsillitis or pta. patients were assessed in the local emergency department or surgical assessment unit according to standard local protocol. we did not apply any length of stay criteria. • there are increasing numbers of inpatient admissions with peritonsillar abscesses (pta) and deep neck space infections • the liverpool peritonsillar abscess score (lps) is a predictive quantitative score of five variables to be used as an adjunct in assessing patients with sore throats • the lps model has a high sensitivity and negative predictive value ( % and %) • the lps may be used as a tool to assist non-specialist colleagues in recognising pta • a modified lps which removes the need for oral examination still has high sensitivity ( %) and a negative predictive value ( %) which may be of particular use during the covid- pandemic | we excluded any patients under years old. we also excluded any patients referred with, or who were subsequently proved to have, supra-or epiglottitis or a dnsi. data quality and uniformity were ensured through the use of a single data collection proforma across all sites. investigators were briefed on the use of the proforma. data were collated locally before being pooled using unique study numbers. investigators scored patients both at the point of referral from either general practitioners or non-specialists in the local emergency department, and on assessment by an ent doctor. the final diagnosis was confirmed both by the presence of pus on aspiration and senior review. the lps covid- modification was developed to assist in risk stratification of pta during the covid- pandemic. since uvular deviation was the only variable requiring oral examination with a tongue depressor, we removed it from the parent lps and examined the classification function of the modified score. data from each collection site were collated prospectively and entered into excel workbooks (microsoft office , microsoft, redmond, wa, usa). we analysed sample demographics and reported summary measures. we also calculated the classification function and area under the receiver operating characteristics curve (auroc). statistical associations were determined using the mann-whitney u test for skewed continuous data, and the significance level (α) was set at . . in this study, we used the previously reported final iteration of the lps (figure ). we included patients referred with sore throat (table ) . of these, patients were diagnosed with pta based on the presence of aspirated pus. there was no significant difference in the median age of patient with or without a pta (p = . ).overall, % of patients were men and were significantly more likely to have pta (p < . ). in this external validation study, the lps continues to have a high negative predictive value (npv; %) and sensitivity ( %). its speci- the lps is validated for assessing the risk of peritonsillar abscess in paƟents ≥ years. it does not replace clinical judgement and appropriate ppe should be used. unilateral sore throat (rated : or more by paƟent) trismus (inter-incisor distance < cm) pharyngeal voice chance (hot potato voice) covid- modificaƟon: clinicians may remove the uvular deviaƟon component to avoid examining the mouth; there is no change to the cut-off value (see results for classificaƟon funcƟon). when tested against an independent geographically diverse population, the lps demonstrates high npv and sensitivity, as well as overall accuracy (auroc). we hypothesise that a lower specificity and ppv are because some patients with tonsillitis will have trismus, and because we defined pta using definite aspiration of pus in order to reduce subjectivity (table ) . since statistical analysis of the lps has yielded consistent findings across both internal and external validation, further iterative development of the lps is not indicated. we have developed a covid- modification to the lps, which may be of use where clinicians are trying to reserve oral examination for severe cases during the covid- pandemic. this modified score also demonstrates high npv and sensitivity. this observational study benefits from collection of prospective data over a broad geographical area with a pre-determined sample size. cohort characteristics are comparable to many other studies of patients with sore throats, and age and gender distributions are similar to the original observational study used to develop the lps. , the classification function and auroc yield high degrees of negative prediction and accuracy, respectively. in developing the lps, we have tried to use objective measures to ensure that the resulting score is highly reproducible in the hands of many different clinicians, including non-specialists. in this study, we have categorised diagnosis into discrete categories of "pta" or "non-pta" sore throat to ensure reproducibility and to reduce subjectivity: this is in line with our previous methodology. this is likely to mean that the lps does not differentiate between peritonsillar cellulitis and tonsillitis (in fact, it is likely that this is also reflected in the relatively lower specificity and ppv of the lps). the stated aim of the lps, however, is to avoid a missed diagnosis of pta rather than to differentiate between tonsillitis and peritonsillar inflammatory disease. in the uk, most pta is treated with a combination of drainage and antibiotics. by contrast, both peritonsillar cellulitis and tonsillitis can be treated medically with antibiotics and supportive advice. it is also possible that some of the patients diagnosed as having non-pta sore throat had ptas which did not yield an aspirate, either due to small size or due to inexperience on the part of the admitting clinician. however, no patient, once formally diagnosed with non-pta sore throat, required a repeat trial of aspiration. these factors may therefore affect relatively small number of patients and are unlikely to affect the reliability of the score. almost in parallel with our original study, spiekermannet al reported a pta predictive score which included six variables. of an inflammatory biomarker in both serum and saliva (s a / a complex). the study was retrospective in nature and included a smaller sample of patients ( acute tonsillitis; peritonsillar inflammation). the authors aimed to determine whether they could predict if a patient with peritonsillar inflammation would benefit from medical treatment or "abscess relief" with the primary aim being able to differentiate between peritonsillar abscess from cellulitis. demographics and key definitions were similar between that study and our own. the medical context, however, appeared to be different in that a significant number of patients underwent abscess tonsillectomy, which is now rarely performed in the uk. in addition, the use of s a /a complex as a biomarker for peritonsillar inflammation is not in widespread use at present, is limited to the research sphere and requires additional laboratory resources and funding. external validation now provides a measure of confidence in the reliability and utility of the lps. the lps does not seek to alter the threshold for admitting patients with sore throat (when they cannot swallow enough to remain hydrated and to take medication). instead, its clinical applicability lies in the referral interface between primary or emergency care and ent, and in stratification of a patient's pta risk. the primary method of transmission of covid- is thought to be via droplet spread: clinicians may be at greater risk when examining the oropharynx. updated ent uk guidance attempts to limit clinician exposure during patient assessment, and the covid- modification of the lps could be used to complement this guidance. in this study, we have validated the lps externally in a new patient cohort, yielding findings consistent with our original work in which this score was developed. we have demonstrated that lps is a robust and easy-to-use tool for colleagues seeking to stratify the risk of pta in their patients. none. the data supporting this study are available from the corresponding author upon reasonable request. a -year observational cohort of a million patient populationtonsillectomy rates in the context of two national policy changes prognostic factors and effect of early surgical drainage in patients with peritonsillar abscess the rising rate of admission for tonsillitis and neck space abscesses in england the liverpool peritonsillar abscess score: development of a predictive score through a prospective multicentre observational study evaluation of d-dimer in the diagnosis of suspected deep-vein thrombosis ent uk covid- adult tonsillitis & quinsy guidelines the strengthening the reporting of observational studies in epidemiology (strobe) statement: guidelines for reporting observational studies sample size considerations for the external validation of a multivariable prognostic model: a resampling study potential of the novel pta score to identify patients with peritonsillar inflammation profiting from medical treatment covid- and the otolaryngologist -preliminary evidence-based review key: cord- -abayh authors: graham, r. s.; zachs, d. p.; cotero, v.; dagostino, c.; ntiloudi, d.; kaiser, c. r.; graf, j.; wallace, k.; coleman, t. r.; ashe, j.; pellerito, j.; tracey, k. j.; binstadt, b.; chavan, s. s.; zanos, s.; puleo, c.; peterson, e.; lim, h. h. title: calming the cytokine storm - splenic ultrasound for treating inflammatory disorders and potentially covid- date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: abayh hyperinflammation and uncontrolled cytokine release, which can be seen in severe cases of covid- , require therapy to reduce the innate immune response without hindering necessary adaptive immune mechanisms. here, we show results from the first in-human trials using non-invasive ultrasound stimulation of the spleen to reduce cytokine release in the context of both an acute response in healthy subjects and a chronic inflammatory condition in rheumatoid arthritis patients. splenic ultrasound results in a reduction in tnf serum levels, as well as il- b; and il- transcript levels in monocytes. there is also a down regulation of pathways involved in tnf and il- production, and ifngamma- and nfkb-regulated genes. many of these cytokines or pathways are upregulated in covid- patients. there is also a reduction in chemokine transcript levels and other components of the chemotactic response, suggesting that reduction of cellular migration may contribute to the therapeutic effects of ultrasound. there is no inhibition of the adaptive immune response with ultrasound treatment relating to antibody production. this is consistent with a pre-clinical animal model where enhanced antibody production was achieved with splenic ultrasound. therefore, this new splenic ultrasound approach has the potential to treat acute and chronic hyper-inflammatory diseases, as it lowers cytokine levels without disrupting the normal adaptive immune response. portable ultrasound technologies are currently being developed and translated to the clinic to treat various inflammatory disorders, with more recent efforts directed towards combatting the hyperinflammation or cytokine storm in covid- patients. cytokines release by cells of the innate immune system drive inflammation ( , ). inflammatory reactions are typical in response to microbial or viral infection but can lead to health problems or life-threatening conditions if there is a persistent hyperactive innate immune response, involving cytokine toxicity and tissue damage ( , ) . a recent and devastating example is the hyperinflammation caused by the coronavirus disease (covid- , disease associated with sars-cov- viral infection). it is estimated that % of covid- cases experience a 'cytokine storm' that leads to severe or fatal respiratory disease, known as acute respiratory distress syndrome (ards) ( ) ( ) ( ) . in addition to other acute conditions, such as sepsis and acute kidney injury ( , ) , hyperinflammation is an issue that occurs across multiple chronic inflammatory systemic diseases, such as rheumatoid arthritis (ra) and irritable bowel syndrome ( , ) . the current pharmacologic approaches to treating hyperinflammation are associated with multiple side effects and high costs. over the past years, researchers within the field of bioelectronic medicine have investigated an unconventional approach for treating inflammation through the use of vagus nerve stimulation ( ) ( ) ( ) . electrical stimulation of the vagus nerve activates the splenic nerve and cells within the spleen, leading to activation of the cholinergic anti-inflammatory pathway ( , ) . this pathway depends on splenic nerve release of norepinephrine that activates acetyltransferase -expressing t lymphocytes, which in turn modulates innate immune cells to decrease systemic levels of key proinflammatory cytokines, such as il- , il- and tnf ( , ) . furthermore, electrical vagus nerve stimulation has been shown in mice and humans to provide therapeutic anti-inflammatory effects for chronic inflammatory diseases (e.g., ra and irritable bowel syndrome) and for acute inflammation (e.g., sepsis, renal ischemia, trauma/hemorrhagic shock, and acute lung injury following trauma/hemorrhagic shock; ( , ( ) ( ) ( ) ( ) . direct vagus nerve stimulation requires invasive implantation procedures. several groups have pursued non-invasive ultrasound stimulation of the spleen as a safer, non-surgical approach for modulating the cholinergic anti-inflammatory pathway. since the vagus nerve projects to the brain and multiple organs throughout the body ( ) , targeting neurons or cells specifically within the spleen can reduce unintended activation or side effects. there has been a recent surge of research which demonstrates the ability to activate or modulate cells with ultrasound energy ( ) ( ) ( ) ( ) ( ) ( ) there are several studies in rodents showing the ability to modulate the splenic nerve or immune cells within the spleen with ultrasound, to reduce inflammation and cytokine levels ( ) ( ) ( ) ( ) ( ) . one of the initial reports was in a mouse model of reperfusion injury and kidney inflammation, where ultrasound stimulation of the spleen significantly reduced kidney damage ( , ) . recently, our research groups discovered that specific parameters of ultrasound stimulation of the spleen can drive significant anti-inflammatory effects in rodent models of both chronic inflammation (inflammatory arthritis) and acute inflammation (sepsis) ( , ) , which was shown to be mediated through a similar cholinergic anti-inflammatory pathway accessed through vagus nerve stimulation. ultrasound is a potentially impactful clinical solution to inflammation as it can be applied non-invasively to the body with a wearable device and with energy parameters already shown to be safe for the human body based on numerous ultrasound imaging applications ( , ) . here, we show the first in-human results of pro-inflammatory cytokine reduction with noninvasive ultrasound stimulation of the spleen. in healthy individuals, a single three-minute administration of splenic ultrasound stimulation significantly inhibits whole blood tnf production upon ex vivo exposure to endotoxin compared to sham controls. in ra patients, we observed that daily splenic ultrasound stimulation results in reduction of blood-borne transcripts encoding for pro-inflammatory markers il- β, il- , and nfκb, as well as suppresses pathways involved in il- and tnf production. ultrasound also reduces pathways involved with monocyte migration, contributing to its anti-inflammatory effect. from a safety perspective, circulating immune cell composition does not change with ultrasound treatment, and ultrasound does not inhibit the adaptive immune response in humans. our additional pre-clinical animal data further demonstrates that in addition to dampening cytokine output and circulating monocyte invasiveness, prophylactic ultrasound activation of the splenic neuroimmune pathway results in enhanced antibody response upon exposure to an inflammatory antigen. thus, activation of the splenic neuroimmune pathway may provide a low risk therapeutic approach for a broad range of health conditions, due to its pleotropic nature and physiological role in suppressing specific cytokines involved in innate immunity while enhancing the transition to and maintenance of the adaptive immune response ( , , ) . together, these data suggest great potential for splenic ultrasound as a low-risk clinical therapy for a range of acute or chronic inflammatory diseases without requiring surgery or intake of artificial substances with possible side effects. considering that many of the same pro-inflammatory molecules suppressed by ultrasound are implicated as molecular drivers in covid- patients with severe disease, ultrasound also has potential as a non-invasive therapy to combat this pandemic. drug based suppression of these cytokines for tissue protection comes with the risk of inhibiting viral clearance or increasing susceptibility to bacterial co-infections ( , ) . non-invasive ultrasound activation of the splenic neuroimmune pathway may provide an alternative method to combat the cytokine storm without compromising the adaptive immune response in covid- patients, ultimately reducing the high mortality and morbidity rates confronting this worldwide pandemic that currently has limited treatment options. to investigate how non-invasive ultrasound stimulation of the spleen affects cytokine levels directly in human subjects, healthy participants were recruited into a study designed to investigate effect of different ultrasound parameters and locations of stimulation in the spleen on various immune and metabolic responses during acute stimulation (feasibility study described at clinicaltrials.gov: nct ). due to the urgency of identifying potential treatment options for covid- patients, data pertaining specifically to ultrasound stimulation of the hilum of the spleen with the most effective intensity parameter in the study (corresponding to one cohort from the study) are presented in this paper. participants received pulsed ultrasound stimulation of the spleen for three minutes ( . mhz, hz pulse repetition frequency, . mw/cm ispta) using the ge logiq e ultrasound system with the c - probe. an ultrasonographer first identified and targeted the hilum of the spleen, then delivered the pulsed ultrasound stimulus using a modified elastography software setting in the system. sham stimulation control subjects underwent the same procedure as the stimulated group, including transducer placement and movement of the ultrasound probe to the proper splenic location trajectory, but with no ultrasound energy being transmitted to the spleen from the transducer. since healthy subjects have low serum tnf levels, we revealed changes in inflammatory status using whole blood challenged with lipopolysaccharide (lps) ex vivo. this ex vivo cytokine production assay has been previously used to verify splenic neuroimmune activation using implanted vagus nerve stimulators and is a well-established method for non-invasively assessing activation of neuroimmune pathways ( , ( ) ( ) ( ) . a reduction in lps-induced tnf production in blood sampled from ultrasound stimulated subjects was tested at multiple lps concentrations (fig. a ) and significant reduction is observed when comparing the ultrasound stimulated group to the non-stimulated controls ( fig. b ; p-value = . using wilcoxon unpaired rank-sum test) at the optimal lps concentration ( ng/ml; fig. a ). these data are consistent with earlier reports using implanted nerve stimulators or pharmaceutical activators of the splenic pathway ( , ) . they are also consistent with the magnitude of tnf reduction using similar ultrasound stimulation parameters in a previous study activating the neuroimmune pathway in a rodent model of endotoxemia ( ) . further supporting the human results, we also demonstrated that splenic ultrasound stimulation in a rodent model achieves a reduction across a number of proinflammatory cytokines beyond tnf, including il- , ifnγ, il- β, il- α, and il- in splenic lysates (fig. s ), which is consistent with previous reports using implanted vagus nerve stimulators ( , ) . tnf response after lps incubation is shown for one ultrasound stimulated subject from samples collected before ( hours baseline) and after ( hours post stimulation) ultrasound application. the optimal dose of lps for measuring the immunomodulatory response to ultrasound was found to be at ng/ml, and this is used for comparing the tnf response between the ultrasound stimulated versus control groups. b) ultrasound stimulation of the spleen decreases whole-blood lps-induced tnf release, where blood was obtained from healthy human subjects prior to and then two hours following splenic ultrasound application (stimulated: n= , control: n= ). the data shown were taken from the ng/ml lps concentration for each dose response curve for all subjects and samples. p-value was computed using the unpaired wilcoxon rank-sum test. previous animal research demonstrated that ultrasound can significantly reduce inflammation and improve clinical measures in a chronic inflammatory arthritis model ( ) . expanding upon the findings in healthy human subjects in the previous section, we initiated a controlled, randomized clinical trial of splenic ultrasound treatment in ra patients (clinical study described at clinicaltrials.gov: nct ). participants received ultrasound stimulation to the spleen with the goal of lowering the over-active inflammatory response that occurs during ra flare-ups. up to ra patients are to be recruited into the study (enrollment is ongoing). due to the urgency of identifying treatment options for this covid- pandemic, data from single-cell rna sequencing (scrna-seq) in peripheral blood mononuclear cells (pbmcs) that has been collected thus far in five ultrasound-treated patients have been analyzed ahead of completion of the study for inclusion into this paper. pbmcs were isolated from whole blood taken from each research participant before ultrasound treatment (day , pre-stimulation) and after two weeks of daily -minute sessions of ultrasound to the spleen (day , post-stimulation). from these samples (subjects = , timepoints = ), a total of , pbmcs were successfully sequenced from single cells (table s ). in silico analysis identified cell-types based on gene expression patterns of standardly used marker genes (fig. s ). participants received stimulation with a mhz transducer ( . w/cm ; soundcare plus, roscoe medical) that was moved continuously across a -inch by -inch square, centered on the spleen, as identified by an ultrasonographer. further details on the clinical study design is presented in the materials and methods section. we aimed to identify molecular changes that occur with splenic ultrasound in humans to better understand how this ultrasound treatment can drive an anti-inflammatory response. we observed reduced monocyte expression of key pro-inflammatory cytokine genes encoding for il- β and il- from all five subjects post-stimulation compared with pre-stimulation ( fig. a , p-values given for transcripts showing significance of p< . ). although this result was determined by analyzing all monocytes, reduction of these two markers appears primarily in cd + monocytes as this is the cell type showing predominant expression of those genes (fig. b) . the same analysis in cd + t cells show a decrease in transcripts that encode for ifnγ ( fig. a ). the changes in transcript level of these cytokines can only be detected when comparing within the same cell type before and after treatment, within the cell that expresses the cytokine (fig. s ). consistent with known expression patterns of il b and cxcl transcripts (encoding for il- β and il- ), we see that these transcripts are only suppressed in the monocytes where they are primarily detected. likewise, ifng transcripts (encoding for ifnγ) are detected and found to be reduced in cd + t cells (fig. s ). in our dataset, other cytokines like tnf and il- did not show a significant change in monocytes; however, il- transcripts were detected in only of the , cells. therefore, we cannot definitively conclude if there is or is not a change in transcript levels encoding il- with ultrasound treatment. since monocytes play a critical role in the innate immune system and the inflammatory response, we further focused our analyses on transcriptional changes that occur in circulating monocytes after ultrasound treatment. differential expression analysis of all monocytes across the five subjects, pre-and post-ultrasound stimulation, shows differentially expressed genes (p-value< . ) with of those genes being down-regulated. these results reveal an overall shift in reduction of gene transcripts in monocytes with ultrasound treatment. additionally, this reduction in transcript levels did not appear as a result of sequencing differences, as we observe no obvious difference in the number of cells sequenced, the number of sequencing reads, or number of average genes detected per cell between monocytes taken from day as compared to day (table s , 'cd mono' and 'cd mono'). although these gene transcripts are defined as differentially expressed when comparing monocytes from all subjects ( fig. a) , we also observe that for most genes, this down-regulation is consistent across subjects ( fig. c ; columns represent cell-type averages for cd + and cd + cells for each subject that are similar across columns for many of the genes). showing cd + and cd + monocytes represented by orange and green dots, respectively. il- β and cxcl expression on day and day are also mapped on these umap diagrams. c) heatmap displaying the average relative expression (for all cells in specified group) of downregulated transcripts (genes listed on the right, p-value < . ). color denotes relative expression for post-ultrasound levels compared to (divided by) pre-ultrasound levels for each subject (represented by columns, n = , participant a-e shown left to right) in cd + cells or cd + cells. genes encoding for cytokine receptors, components of the jak/stat pathway, map kinases, and transcription factor (tf) are grouped and labeled on the left of the plot. these results are quite consistent given the high variability or inhomogeneity in age, sex, physical characteristics, disease properties and past treatments across subjects (participants a-e in table s correspond to columns - in fig. c , respectively). within these down-regulated genes in monocytes, we also observe that many cytokine receptors, such as those for tnf, il- , il- , il- , and ifnγ were reduced with ultrasound treatment (fig. c) . furthermore, components of signaling pathways downstream of these cytokines are also down regulated, including components of the jak/stat pathway, map kinases, and pro-inflammatory transcription factor nfκb (fig. c ). together, these findings demonstrate a consistent and robust reduction in transcripts encoding key pro-inflammatory cytokines and cellular signaling molecules downstream of their receptors after days of daily splenic ultrasound treatment across all five subjects. cytokines trigger multiple cellular pathways, which then contribute to systemic inflammation. our transcriptomic data reveal that many genes known to be regulated by ifnγ and nfκb are subsequently downregulated with ultrasound treatment, further promoting a cascade of suppression across multiple proinflammatory pathways with ultrasound treatment (fig. a) . we sought to determine if the down-regulated genes in monocytes showed statistical enrichment for functional gene ontology (go)terms associated a) volcano plot showing all detected genes in monocytes, highlighting differentially expressed genes (p-value < . ) and labeled ifnγ-regulated genes that are down regulated, as well as nfκb-regulated genes that are down regulated as characterized by kegg pathway gene lists. b) gene ontology (go) analysis using bioconductor package 'topgo', determining functional go terms significantly enriched within downregulated genes in monocytes. enriched go terms associated with cytokine production are shown, where bars represent number of significant genes in a list relative to number of genes that would be expected by chance, and with number of genes contributing to each go term represented by the shade of blue. pvalues for each term shown is from fisher exact test. with inflammation. the go system of classification assigns genes (for differentially expressed gene list) into bins based on their functional characteristics. gene sets can then be analyzed for go terms that are statistically over-represented among that set, allowing for a better understanding of which biological processes (e.g., inflammation) may be changed with treatment. out of the top statistically enriched go terms for the down-regulated genes, we observe many that contribute to pro-inflammatory cellular processes. genes suppressed by ultrasound show enrichment of go terms including "inflammatory response" (p-value = . Ε- , table s ), "cytokine-mediated signaling pathway," and "cellular response to cytokine stimulus" (p-values = . e- and . e- , fig. b ). we also observe enriched go terms associated with positive regulation of tnf, il- , and il- among the down-regulated genes, suggesting that inducing signals for tnf or il- are also suppressed, despite the absences of significant change in tnf or il- transcript levels (fig. b ). these go term results show that many gene transcripts encoding for cellular inflammatory functions go down after ultrasound treatment. within circulating monocytes, we observe ultrasound-treatment associated go term enrichment of cellular components associated with il- functionality (fig. b) , as well as a robust reduction in il- across subjects (fig. a , columns represent subject cell type averages). il- is a chemokine (chemotactic factor), which stimulates neutrophil movement towards inflammatory sites. therefore, we further assessed if ultrasound influences other signaling molecules involved with cellular migration (e.g., to sites of inflammation). go term analysis of down-regulated genes in cd + monocytes show enrichment of "positive regulation of cell migration" among the top significantly enriched go terms. other go terms, such as "lamellipodium assembly" (facilitates cell mobility), "ephrin receptor signaling" (promotes cell adhesion), and "actin nucleation" (involved with cell locomotion), all suggest that ultrasound treatment induces down regulation of genes involved with cell migration (fig. b) . furthermore, genes that more specifically contribute to "monocyte chemotaxis" are down-regulated post-ultrasound stimulation in cd + monocytes and many of these genes are consistently reduced across all five subjects (fig. a) , even with quite variable or inhomogeneous patient characteristics (table s ) . we sought to determine how ultrasound affects the circulating immune cell repertoire. in comparing the transcriptional profiles (based on principal component analysis and umap dimensional reduction) of all circulating peripheral blood mononuclear cells pre-and post-ultrasound stimulation, (table s ) . c) percent down regulation range is shown after days of ultrasound stimulation for each gene (represented by dots) in each functional category described previously. we observe remarkable consistency (fig. a) . also, the calculated relative percent of each celltype for each subject is displayed, and based on a paired wilcoxon rank sum test for pre-and poststimulation for each cell type, we observe no significant difference in the relative proportions of percent contribution of any of the cell types (fig. b , statistics shown in table s ). additionally, when comparing the number of cells sequenced for each cell type per participant there is no significant difference between timepoints (i.e. no technical bias, table s ). we also determined what degree of suppression occurred after ultrasound stimulation for genes involved in inflammation (genes shown in fig. c, a and a) . most genes show a ~ - % reduction in transcript levels, suggesting a moderate degree of suppression, with il- and genes involved in cell migration showing the highest degree of suppression (> %, fig. c ). these findings suggest that spleen-targeted ultrasound can potentially provide a way to blunt the elevated release of circulating cytokines and chemokines in inflammatory disorders or viral infections, without disrupting the overall immune cell repertoire. one concern in treating acute hyperinflammation is that the therapy may suppress the adaptive immune response, and the ability of the immune system to provide pathogen clearance and/or protection from co-infection with a second pathogen. therefore, we assessed whether non-invasive splenic ultrasound treatment suppresses the adaptive immune response or more specifically antibody production. our transcriptomic data do not suggest a suppression of the adaptive immune response. in fact, we observe that among genes upregulated in b cells, there is an enrichment for go terms associated with "b cell activation" (fig. a ). genes associated with this go term include transcripts that encode for iga, igg, and igm antibodies. interestingly, these gene transcripts are upregulated in some of the ultrasound-treated subjects (fig. b) , though with greater variability in results across subjects than was observed for suppression of cytokines and chemokines in monocytes. for example, we observe that for average transcript levels across b cells, transcripts encoding igg are only upregulated in or subjects (ighg and ighg , respectively) out of the subjects. regardless of the variability across subjects, these data support that ultrasound treatment does not generally inhibit b cell activation and antibody production across patients, and can even potentially enhance antibody production; thus, ultrasound is not expected to compromise the adaptive immune response. to further investigate the effects of splenic ultrasound stimulation on adaptive immune response and antibody production, as well as the variability we observed across human subjects for transcript levels that encode for iga, igg, and igm antibodies, we performed additional experiments using prophylactic ultrasound stimulation (i.e., ultrasound stimulation prior to antigen exposure) in a rodent model of endotoxemia ( ) . we observe that splenic ultrasound stimulation prior to endotoxin exposure (three minutes daily, once a day for days followed by lps injection on the fourth day) results in a significant increase in igm and igg antibody production hours after lps exposure compared to sham and/or non-lps controls (p-values listed in fig. c description) . furthermore, this enhanced antibody output is dependent on the presence of the toxin/antigen, as there is no observed ultrasound effect on antibody production in the absence of lps exposure. the increase in antibody transcript levels we observed in some of the human subjects, and not in the others, may be attributed to those participants also having encountered an antigen, such as a cold or other infection that was not identified during the study. this explanation is further supported by a recent report demonstrating that brain-spleen neural pathways are capable of modulating splenic plasma cells differentiation upon presentation of novel t cell dependent antigens ( ) . for naïve animals (no lps + sham), animals exposed to splenic ultrasound stimulation with no endotoxin (no lps + ultrasound), animals exposed to the endotoxin but receiving sham stimulation (lps + sham), and animals receiving both lps exposure and splenic ultrasound stimulation (lps + ultrasound). animals receiving lps + ultrasound showed significant increases in antibody production when compared to each of the other groups using an unpaired wilcoxon ranked-sum test for igm (no lps + sham, p = . ; no lps + ultrasound, p = . ; lps + sham, p = . ) and for igg (no lps + sham, p = . ; no lps + ultrasound, p = . ; lps + sham, p = . ). no other comparisons using this test showed significance, p < . . by presenting the first in-human data from two independent studies using different devices and protocols, we have consistently demonstrated that non-invasive ultrasound stimulation of the spleen drives anti-inflammatory effects in the context of both an acute response in healthy subjects and a chronic inflammatory condition. in ra patients after two weeks of daily -minute ultrasound treatment to the spleen, there was a reduction in cytokine il- β and chemokine il- in circulating monocytes (fig. ) . in healthy subjects, we also observed a significant reduction in tnf levels produced by ex vivo lps-stimulated whole blood, in individuals stimulated with minutes of pulsed ultrasound (fig. b) . in addition to the reduction of cytokines, we observed a reduction in activation of multiple signaling pathways in circulating monocytes with ultrasound treatment. cytokines signal by binding to cell surface receptors to set off a cascade of intracellular events. important cytokines in the context of ra include tnf, il- , and il- β; their downstream intracellular pathways and necessary signaling molecules include cytokine receptors, components of the jak/stat pathway, map kinases and nfκb ( , ) . many of the transcripts that encode for these critical components are downregulated with ultrasound treatment in ra patients (fig. b) . this finding suggests that in addition to cytokines and their immediate receptors, there is broad suppression of cellular proinflammatory pathways. down regulation of these various pro-inflammatory transcripts in ra patients occurs in the context of chronic inflammation. however, these same pathways are also involved in acute inflammatory responses ( ) . genes induced by lps-stimulated human monocytes include tnf, il- , il- , and il- ( ) . nfκb and associated pathways are also upregulated, and nfκb is activated via phosphorylation during lps exposure, allowing for a rapid induction of genes so the system can respond in an acute inflammatory setting. these same cellular components are downregulated with ultrasound treatment in our ra patients, suggesting that non-invasive splenic ultrasound could be used clinically to suppress acute inflammation as well. the discovery that splenic ultrasound can reduce or calm an overactive innate immune response in humans is quite timely. many of the same key inflammatory cytokines that are reduced with ultrasound stimulation are elevated in covid- patients. recent studies have revealed that covid- patients consistently produce high levels of il- β, il- , il- , and tnf, which strongly suggests dysregulation in the innate immune response ( ) ( ) ( ) ( ) . more specifically, sars-cov- infected individuals exhibit significantly higher expression levels of il- β and il- transcripts in circulating monocytes, as well as ifnγ in cd + t cells, shown through pbmc single-cell rna sequencing of covid- patients ( ) . single cell rna sequencing has further shown that bronchoalveolar lavage fluid from patients with severe covid- disease have higher levels of il- , il- and il- β compared to patients with only moderate disease ( ) . these molecules are key components of the covid- cytokine storm that function in acute lung injury to recruit neutrophils to alveoli, inducing lung tissue damage ( ) . recently, sars-cov- infection in children has been associated with a novel multisystem inflammatory disease (mis-c) resembling toxic shock syndrome or atypical kawasaki disease ( ) . kawasaki disease also presents with elevated transcripts levels of genes encoding for il- , il- , il- , and il- ( ) ; and along with ards, is likely to be a condition caused by the covid- -induced cytokine storm ( ) . clinical therapies that can suppress these pro-inflammatory cytokines and reduce migration of circulating immune cells to the lungs may be capable of treating patients with moderate to severe cases of covid- to calm the cytokine storm and prevent morbidity and mortality (fig. a) . ultrasound has the potential to prevent or improve severe symptoms associated with the cytokine storm in covid- patients. in addition to demonstrating the ability to reduce key proinflammatory cytokines and signaling pathways involved with a hyperactive innate immune response, splenic ultrasound also reduces il- and many components associated with cell migration, specifically monocyte migration (fig. ) . these findings are consistent with previous animal studies showing that activation of the cholinergic anti-inflammatory pathway suppresses monocyte migration in mice ( ) . taken together, one proposed mechanism of action for ultrasound treatment is that ultrasound modulates circulating immune cells initially within the spleen via activation of the cholinergic antiinflammatory pathway, which suppresses proinflammatory cytokines and signaling pathways, as well as inhibiting cellular migration, such that there is reduced inflammation at the target site (e.g., at joints in ra or alveoli for covid- ; fig. a ). furthermore, data in humans and preclinical animal data show an increase in antibody production with splenic ultrasound. these results demonstrate an advantageous feature of splenic ultrasound compared to pharmaceutical options for immunosuppression, such as tnf, il- , or il- blocking biologics ( , ) , in that cytokine suppression does not inhibit other functions of the adaptive immune system, such as b cell activity or antibody production. indeed, splenic ultrasound may enhance the adaptive immune system through the recently discovered brain-spleen neuroimmune pathway that enhances differentiation of antibodyproducing splenic plasma cells (sppcs) upon antigen activation ( ). one limitation of the presented results in the context of covid- or a cytokine storm is that ultrasound stimulation of the spleen may not generate anti-inflammatory effects sufficient to suppress a severe infectious inflammatory response. the anti-inflammatory effects in this paper in response to splenic ultrasound were demonstrated acutely (single -minute stimulation) in healthy human subjects or to a moderate extent in ra patients. the question remains whether the antiinflammatory effects observed in healthy subjects or in patients with a chronic inflammation disorder, such as ra, will translate to patients harboring a systemic viral infection, as observed for covid- . this question will be answered through a clinical trial that is currently being set up at the university of minnesota together with ge research to test this ultrasound approach in covid- patients funded by the defense advanced research projects agency (darpa; department of defense). one key asset of the upcoming covid- study is that ge's cart-based ultrasound system (ge logiq e ) with enhanced targeting capabilities will be used on patients. in the ongoing ra study at the university of minnesota, a commercially available ultrasound device (soundcare plus) was used on patients that did not have targeting capabilities tailored for the spleen. at the onset of that ra study, soundcare plus was one of few devices that was already fda regulated for various stimulation applications; thus, this unit was selected for the ra study. the ge logiq e device can be used to better target and stimulate specific locations of the spleen, which the ge team has already demonstrated with strong anti-inflammatory effects in healthy human subjects (fig. b) . previous animal studies have also demonstrated that stronger anti-inflammatory effects are possible with better focusing of stimulation within the spleen or longer periods on a given target ( , ) . furthermore, sufficient activation of the cholinergic anti-inflammatory pathway has been shown to combat acute and deadly cytokine storm conditions in sepsis animal models, and the magnitude of cytokine suppression provided by ultrasound is consistent with the protection provided by implant-based or pharmaceutical methods in these studies ( , , ) . in parallel with the darpa-funded study, both ge and a start-up company, secondwave systems, are developing wearable or portable ultrasound systems for neuromodulation (fig. b ) that can be positioned over the rib to steer and focus ultrasound energy to different locations of the spleen or other organ targets (fig. c) . both ge and secondwave are currently organizing clinical studies at multiples sites worldwide to assess the safety and feasibility of their therapeutic ultrasound devices. success with these initial clinical studies will propel further development and scale-up efforts for splenic ultrasound technologies to meet the large-scale demand for covid- , as well as open up opportunities to treat future viral infections and other inflammatory disorders in combination with or in lieu of biologics. for the splenic ultrasound study involving healthy human subjects (clinicaltrials.gov: nct ), we provide the data from the two cohorts most relevant to the planned darpa sponsored covid study: the sham control cohort (i.e., no ultrasound, with only mechanical pressure over the spleen) and a splenic ultrasound stimulation cohort (i.e., . mw/cm ispta or . mechanical index (mi) using the shear wave elastography setting on ge logiq e ) that insonified a single site (i.e., the splenic hilum; as identified by a trained ultrasonographer performing the stimulation). the trial was carried out in accordance with international conference inclusion and exclusion criteria. to be eligible to participate in the study, an individual must have met the following criteria: aged between and years, be without physical disability or conditions that may make them incapable of undergoing the study procedures or otherwise place them at greater harm, be without significant past medical or surgical histories that would render them at a greater risk of harm, be considered english proficient due to the study requirement to follow verbal commands during the ultrasound session, be considered active as assessed by type of activity (e.g., walking or running) and number of hours a week performing the various activities, be able to attend all study visits at approximately the same time of day, be able to comprehend the study goals and procedures, and able to provide informed consent for participation. see table s for subject characteristics used in the cytokine analysis shown in fig. . study timeline. subjects underwent a screening visit to days prior to baseline visit to assess eligibility to participate in the study. eligible individuals that agreed to participate and provided written informed consent then underwent a physical and neurological examination. women of childbearing potential were asked to provide a urine sample for pregnancy testing, and approximately ml of blood was drawn. on the first protocol visit (day ), participants again underwent a physical and neurological examination, and medical history review. a baseline blood draw was then collected for cytokine and blood chemistry analysis (~ ml). the blood draw was performed under sterile conditions using standard venipuncture techniques. individuals then underwent the ultrasound procedure based on random assignment to one of groups, including the two groups reported herein (i.e., the sham control group, and a stimulated group that received % power stimulus applied at a single splenic target location). for ultrasound stimulus delivery, individuals were asked to lie in the right lateral recumbent position with their arms above their heads to expose the splenic region of the abdomen. after ultrasound gel was applied to the region, the ultrasound probe was placed on the participant's abdomen. this procedure was performed for both the sham control group (n= ) and ultrasound stimulated group (n= ). note that one subject from each group was excluded, in which subjects were initially recruited to the study for each group (see table s ). stimulation paradigm. in the ultrasound stimulated cohort, the subject had the dimensions of their spleen assessed (using the b mode imaging setting on the ge logiq e ), and the probe (c - ) was positioned over the splenic hilum. stimulation was performed using a modified elastography setting on the logiq e operated in two modes: an imaging/targeting mode with short ultrasound pulses (on the order of microsecond) and relatively low acoustic power levels (on the order of mw/cm ), and a stimulation mode (from the clinical shear wave elastography setting) using longer ultrasound pulses (on the order of ms) and higher acoustic power levels ( . mw/cm ). stimulation was performed in twelve -second long epochs separated by seconds each, and a total stimulation duration of minutes. the second epochs were performed during breath holds, during which an ultrasonographer was holding the probe in position above the spleen. before each stimulation period, the ultrasonographer checked the location of the probe using the imaging mode to ensure that all stimulation pulses were on target. the focal landmark used for targeting and stimulation was the hilum of the spleen in the group included in the presented analysis. for the sham control group, the movements and probe contact for the ultrasound procedure were performed by the ultrasonographer, and the total duration of the procedure remained the same as the stimulated group, except that no ultrasound power was applied (i.e., ultrasound output via the probe was turned off). a post-ultrasound blood draw was again performed, and the -hour blood draw time-point data is reported herein. blood analysis procedures. prior to the ex vivo cytokine test, the lps was sonicated for minutes. a ml blood tube (heparin; green cap) was brought into a tissue culture/sterile room and blood was transferred to a ml conical tube. a blinded researcher then diluted the lps to the test concentrations in pre-labeled tubes containing replicates of the different concentrations of lps used for blood exposure (dilutions were made to achieve , . , , and ng/ml of lps when mixed with blood). upon mixing the lps with blood, the final tubes were capped and placed on a rocker within an incubator and the samples were kept at degrees celsius for hours during incubation. after the incubation, the samples were removed from the incubator, centrifuged at rpm for minutes, and the plasma supernatant was transferred (via pipette) for storage (frozen prior to analysis). tnf analysis was performed in triplicate from each sample using duoset elisa kit (r&d systems), as per manufacturer's instructions. this clinical study is a controlled, randomized, double-blinded trial of short-term ( days) treatment and is listed at clinicaltrials.gov (nct ) for which a portion of the results are presented in this paper. the study is still ongoing, but the rnaseq data obtained thus far in five ultrasound stimulated patients is presented in this paper for its relevance in revealing the use of splenic ultrasound for potentially treating covid- patients. the protocol, informed consent form, recruitment materials, and all participant materials were submitted and approved by the university of minnesota's institutional review board (irb) and monitored by ctsi in accordance with its institutionally approved monitoring plan. the overall objective of the clinical trial is to demonstrate safety and efficacy of spleen ultrasound stimulation in the treatment of ra in participants. for this ongoing study, participants have been recruited, consented, and enrolled with all patients completing the full study. the th participant completed the study, however the last 'in-clinic' visit was performed using clinician video conferencing due to the covid- pandemic, while in-person blood sample collection was still able to be performed. further details for this study can be found on clincialtrials.gov and will be presented in a future publication with complete outcome analyses. the description below only includes relevant methods and rnaseq analyses presented specifically in this paper. inclusion and exclusion criteria. to be eligible to participate in the study, an individual must have met the following criteria: years of age or older, carried a diagnosis of seropositive (rheumatoid factor-or cyclic citrullinated peptide antibody-positive) rheumatoid arthritis (ra), and exhibited symptoms or signs of inadequate disease control (either modified haq score > . or das- -crp > . ). exclusion criteria included active bacterial or viral infection, pregnancy, malignancy, or inability to provide daily self-care. the patient medical history and characteristics for each of the five subjects presented in this paper is provided in table s . study timeline. participants were assessed during in-clinic visits on days (before treatment began), , , , , and . minor flexibility was allowed for scheduling clinic visits outside of holidays for special circumstances. on the first visit, the spleen was imaged by an ultrasonographer and the center of the spleen trajectory was marked on the participant's skin. participants were instructed on how to self-administer ultrasound treatment to the spleen. the procedure involved sweeping the ultrasound wand continuously across a -inch by -inch square centered on the spleen mark placed by the ultrasonographer and ensuring full contact was made with the skin during the daily minute stimulation period. researchers were able to visually confirm that daily treatments were administered through daily online video sessions with the patients. these video sessions took place at about the same time each day for the -day treatment period. with each inclinic visit, participants received a joint examination, peripheral joint ultrasound, and blood draw, as well as completing several physical evaluations and questionnaires. full study details including analysis of all patients and assessments will be presented in a future publication. treatment. after an in-clinic training session on day , transcutaneous ultrasound was administered to the spleen for minutes daily for weeks ( days total) via a portable device ( . w/cm ; soundcare plus, roscoe medical) in the patient's home. the patients were randomized ( : ) to a treatment group or a control (sham) group, in which the latter received the exact same evaluations and stimulation paradigm as the treatment group, except that the device did not deliver any energy from the ultrasound transducer. ra patients and clinical assessors were blinded as to which patients received ultrasound or sham treatment. rna-sequencing and statistical analyses. ml of blood was collected in a green top lihep tube from each ultrasound stimulated participant (n= ) on day (before the first splenic ultrasound stimulation was administered) and on day (after the final treatment session). the samples were processed the same day as collection. peripheral blood mononuclear cells were isolated from whole blood using density gradient centrifugation via sepmate tubes (stemcell technologies) and following manufacturer's instructions. pbmcs were then gently frozen and stored at - ºc as per x genomics demonstrated protocols gc . following this protocol, once all samples had been collected and frozen, samples were thawed quickly in % fbs in pbs, resuspended in % fbs, and strained before proceeding to x genomics single cell protocol. samples were sequenced using umi-based approach from x genomics, as previously described ( ) and mapped to the human genome. transcriptomic analysis was performed using r statistical software, and primary filtering and normalization of each sample was done using seurat package as previously described ( , , ) . samples included two timepoints from five subjects and these individual samples were merged for cell type assignment and differential expression analysis. cell types were assigned using standard marker genes (see fig. s ) as previously described ( ) . differential expression was determined between timepoints for all cells in each cell type using a negative binomial generalized linear model. go term analysis was performed using the org.hs.eg.db and topgo packages. go term enrichment was assessed using the 'elim' algorithm with a fisher exact test cutoff of . , in which the top significant go terms in monocytes are shown in table s ( ) . pre-clinical tests in rodents were performed as previously described ( ) . experiments were performed under protocols approved by the institutional animal care and use committee of ge research. briefly, a vivid e (ge) ultrasound system was used to perform an ultrasound scan and locate the spleen. a hifu transducer and system was then positioned on the target area, and a separate ultrasound scan performed using a smaller probe ( s, ge) that was placed within the opening of the hifu transducer was used to verify alignment of the ultrasound beam with the spleen. adult sprague-dawley rats that were - weeks old ( - g; charles river labs) were housed at degrees celsius on a -h light/dark cycle and acclimatized for week, with handling before experiments to minimize the potential confounding measures due to stress response. water and regular rodent chow were available ad libitum. lps ( :b , sigma aldrich) was used to produce a significant state of inflammation in the naïve adult rodents. lps was administered to animals in the amount of mg/kg, which corresponds to a ld dose via intraperitoneal injection. treatment. for ultrasound stimulation, animals were anesthetized with - % isoflurane, and the animals were laid on a water circulating warming pad to prevent hyperthermia during the procedures. the region designated for ultrasound stimulation was shaved with a disposable razor and animal clippers prior to stimulation. ultrasound was applied to the designated area above the spleen using the probe and system described and calibrated from a previous study ( ) . an ultrasound stimulus using the previously found optimal stimulation parameters ( . mhz, . ms pulse repetition frequency, . microsecond pulse length, mw/cm ispta) was then applied for minutes, and lps was immediately injected post-stimulation. animals were allowed to incubate under anesthesia for one hour, and after incubation the animal was euthanized, and tissue and blood samples were collected. for experiments testing antibody production, an ultrasound stimulus was applied daily (in the morning) for three days prior to lps injection. on the fourth day, following the three consecutive treatment days, lps ( mg/kg) was injected intraperitoneally. the animals were then incubated for minutes in accordance with previous studies, and tissue and blood were harvested for analysis of cytokines and antibody production. tissue collection. an incision was made starting at the base of the peritoneal cavity extending up and through to the pleural cavity. the spleen was rapidly removed and homogenized in a solution of phosphate-buffered saline, containing phosphatase ( . -mm phenylmethylsulfonyl fluoride, µg/ml aprotinin, -mm benzamidine, -mm sodium orthovanadate, and -µm cantharidin) and protease ( -µl to mg of tissue as per roche diagnostics) inhibitors. a targeted final concentration of . -g tissue per ml pbs solution was applied in all samples. after collection of the whole blood, we allowed the blood to clot undisturbed at room temperature for - minutes. samples were centrifuged at , - , x g for minutes in a refrigerated centrifuge with the resulting supernatant being collected for antibody testing. samples were then stored at − °c until analysis. splenic tissue lysate was analyzed for cytokine concentrations using elisa kits for quantifying tnf alpha (abcam, ab ), ifn gamma (abcam, ab ), il- (abcam, ab ), il- (lsbio, ls-f ), il- (abcam, ab ), il- (abcam, ab ), il- beta (abcam, ab ), il- alpha (abcam, ab ), il- (abcam, ab ) and il- (abcam, ab ) as per manufacturer's instructions for tissue samples. serum was tested using elisa kits for quantifying antibody production including igg (abcam, ab ), and igm (abcam, ab ) as per manufacturer's instructions. figure s . modulation of cytokines with splenic ultrasound in rats figure s . cellular transcriptional signatures . s c ), and also showing number of reads (counts) and average number of genes for each cell type. we observe that all that all cells have a relatively consistent read depth for the sequencing reaction performed. also shown are the number of genes out of the human genome that are annotated with that go term, number of genes from the list of significantly downregulated genes with ultrasound, and the number of genes that would be expected by random chance. column labeled 'classicfisher' shows p-values from the fisher's exact test and 'sig_exp' is the number of genes found to be significant in our gene list (decreased with ultrasound in monocytes) compared to the number that might be expected by chance. c d d c r e m h s p h s e l l g im a p c a c y b p g n l y n k g c c l c d a m s a c d a m ir h g n m e f c g r a v m o c c l s a h l a − d q a g p r innate immunity and inflammation signaling in innate immunity and inflammation innate immune response to viral infection mechanisms and therapeutic relevance of neuro-immune communication acute respiratory failure in covid- : is it "typical" ards? epidemiological and clinical characteristics of cases of novel coronavirus pneumonia in wuhan, china: a descriptive study the inflammatory response in sepsis inflammation in acute kidney injury rheumatoid arthritis classification criteria: an american college of rheumatology/european league against rheumatism collaborative initiative irritable bowel syndrome bioelectronic medicine: technology targeting molecular mechanisms for therapy bioelectronic medicines: a research roadmap the inflammatory reflex neural reflexes in inflammation and immunity molecular and functional neuroscience in immunity vagus nerve stimulation mediates protection from kidney ischemia-reperfusion injury through alpha nachr+ splenocytes splenic nerve is required for cholinergic antiinflammatory pathway control of tnf in endotoxemia vagus nerve stimulation inhibits cytokine production and attenuates disease severity in rheumatoid arthritis vagus nerve stimulation attenuates the systemic inflammatory response to endotoxin uncovering the neuroenteric-pulmonary axis: vagal nerve stimulation prevents acute lung injury following hemorrhagic shock vagus nerve stimulation protects against acute liver injury induced by renal ischemia reperfusion via antioxidant stress and anti-inflammation avoiding off-target effects in electrical stimulation of the cervical vagus nerve: neuroanatomical tracing techniques to study fascicular anatomy of the vagus nerve reversible neuroinhibition by focused ultrasound is mediated by a thermal mechanism transcranial ultrasonic stimulation modulates single-neuron discharge in macaques performing an antisaccade task remote, brain region-specific control of choice behavior with ultrasonic waves effects of focused ultrasonic radiation on peripheral nerve, with observations on local heating ultrasound produces extensive brain activation via a cochlear pathway single-unit recordings reveal increased peripheral nerve conduction velocity by focused pulsed ultrasound ultrasound modulates the splenic neuroimmune axis in attenuating aki peripheral focused ultrasound stimulation (pfus): new competitor in pharmaceutical markets? noninvasive sub-organ ultrasound stimulation for targeted neuromodulation noninvasive ultrasound stimulation of the spleen to treat inflammatory arthritis therapeutic ultrasound attenuates dss-induced colitis through the cholinergic antiinflammatory pathway ultrasound prevents renal ischemia-reperfusion injury by stimulating the splenic cholinergic anti-inflammatory pathway safety of transcranial focused ultrasound stimulation: a systematic review of the state of knowledge from both human and animal studies elastography: general principles and clincial applications brain-spleen link tunes immunity vagal regulation of group innate lymphoid cells and the immunoresolvent pctr controls infection resolution risk of infection associated with anti-tnf-alpha therapy risk of infections in rheumatoid arthritis patients treated with tocilizumab dopamine mediates vagal modulation of the immune system by electroacupuncture whole blood cytokine attenuation by cholinergic agonists ex vivo and relationship to vagus nerve activity in rheumatoid arthritis nicotine exposure alters in vivo human responses to endotoxin physiology and immunology of the cholinergic antiinflammatory pathway brain control of humoral immune responses amenable to behavioural modulation selected cytokine pathways in rheumatoid arthritis cytokines in acute and chronic inflammation lps induction of gene expression in human monocytes single-cell landscape of bronchoalveolar immune cells in patients with covid- acute heart failure in multisystem inflammatory syndrome in children (mis-c) in the context of global sars-cov- pandemic the role of immune complexes revisited storm, typhoon, cyclone or hurricane in patients with covid- ? beware of the same storm that has a different origin therapeutic potential and limitations of cholinergic anti-inflammatory pathway in sepsis cholinergic agonists inhibit hmgb release and improve survival in experimental sepsis integrating single-cell transcriptomic data across different conditions, technologies, and species multiplexed droplet single-cell rna-sequencing using natural genetic variation improved scoring of functional groups from gene expression data by decorrelating go graph structure we thank all study participants. we are grateful for the assistance of individuals at the university of minnesota genomics center, especially jerry daniel and emma stanley. additionally, we would like to thank stuart sealfon and frederique ruf-zamojski for their assistance and advice processing pbmc samples for single cell sequencing. this work was supported by the united states defense advanced research projects agency (darpa) electrical prescriptions (electrx) program under the guidance of eric van gieson and gretchen table s . splenic ultrasound in healthy participants: subject features. characteristics and demographic data of enrolled subjects for study of biological effects of ultrasound of the spleen. ten subjects received full-powered ultrasound treatment at the spleen hilum and another ten subjects were sham controls with the ultrasound device turned off. one of the subjects from the stimulated group was excluded due the selection of the wrong ultrasound imaging protocol by the clinician. one of the subjects from the control group was excluded because their baseline glucose measurement was < mg/dl that is considered hypoglycemic. key: cord- -y r el e authors: prantner, daniel; shirey, kari ann; lai, wendy; lu, wuyuan; cole, alexander m.; vogel, stefanie n.; garzino-demo, alfredo title: the θ-defensin retrocyclin inhibits tlr - and tlr -dependent signaling and protects mice against influenza infection date: - - journal: journal of leukocyte biology doi: . /jlb. a - rr sha: doc_id: cord_uid: y r el e despite widespread use of annual influenza vaccines, seasonal influenza-associated deaths number in the thousands each year, in part because of exacerbating bacterial superinfections. therefore, discovering additional therapeutic options would be a valuable aid to public health. recently, tlr inhibition has emerged as a possible mechanism for protection against influenza-associated lethality and acute lung injury. based on recent data showing that rhesus macaque θ-defensins could inhibit tlr -dependent gene expression, we tested the hypothesis that a novel θ-defensin, retrocyclin (rc)- , could disrupt tlr -dependent signaling and protect against viral infection. in this study, rc- , a variant of the humanized θ-defensin rc- , blocked tlr -mediated gene expression in mouse and human macrophages in response to lps, targeting both myd - and trif-dependent pathways. in a cell-free assay, rc- neutralized the biologic activity of lps at doses ranging from . to eu/ml, consistent with data showing that rc- binds biotinylated lps. the action of rc- was not limited to the tlr pathway because rc- treatment of macrophages also inhibited gene expression in response to a tlr agonist, pam csk , but failed to bind that biotinylated agonist. mouse macrophages infected in vitro with mouse-adapted a/pr/ / influenza a virus (pr ) also produced lower levels of proinflammatory cytokine gene products in a tlr -independent fashion when treated with rc- . finally, rc- decreased both the lethality and clinical severity associated with pr infection in mice. cumulatively, our data demonstrate that rc- exhibits therapeutic potential for the mitigation of influenza-related morbidity and mortality, potentially acting through tlr-dependent and tlr-independent mechanisms. influenza viruses a and b are negative-sense, single-strand rna viruses in the orthomyxoviridae family. to combat these pathogenic viruses, seasonal influenza vaccines are formulated, and annual vaccination is recommended by the cdc for everybody mo or older for the - flu season [ ] . during the - flu season, the cdc estimated that ; million doses of influenza vaccine were distributed in the united states [ ] . despite the availability of these seasonal vaccines, annual estimated mortality from to from influenza ranged from to , deaths [ ] , peaking when influenza a (h n ) circulated in the population [ ] . these vaccines are only protective when the circulating strains of influenza closely match the vaccine strains, and the fact that many people do not get immunized can explain these statistics. importantly, these seasonal influenza vaccines do not protect against newly emerging strains that could potentially cause large outbreaks or pandemics in the future. in addition to the mortality and morbidity associated with the viral infection, coinfections can exacerbate the outcome of influenza infection, as exemplified by the pandemic, in which most deaths were due to secondary bacterial pneumonia [ ] . secondary bacterial infection after influenza infection accounts for a significant number of deaths worldwide [ ] . therefore, it would be a valuable aid to public health to have therapeutic options for treating influenza-infected individuals. this was an unmet need in the - influenza season, in which the circulating influenza a (h n ) strain was a drift variant of the seasonal vaccine strain, resulting in a vaccine with reduced efficacy [ ] . in nonimmunized individuals, the first barriers to influenza infection are cells of the innate immune system. these cells are capable of recognizing the invading virus, and that innate recognition is essential for initiating an adaptive immune response. according to pattern-recognition theory [ ] , cells of the innate immune system identify pathogens through germlineencoded proteins called pattern-recognition receptors. these proteins identify pathogen-associated molecular patterns present in the invading microorganisms. in the case of influenza infection, the cytosolic rna helicase rig-i has an important role in recognition, triggering production of protective proinflammatory cytokines, such as tnf-a and antiviral type i ifns [ ] . it is theorized that the host cell can distinguish the viral rna from cellular rna because of a panhandle structure in its triphosphorylated terminus [ ] . however, the influenza protein ns is capable of antagonizing rig-i-dependent signaling [ ] , highlighting the need for redundancy in innate immune recognition. tlr can also recognize viral rna [ ] and has been shown to be activated in response to influenza infection in human lung epithelial cells to induce proinflammatory cytokines [ ] . in certain cell types, such as plasmacytoid dendritic cells, tlr can also recognize influenza genomic rna [ ] . innate immune recognition does not occur exclusively through recognition of pathogen substructures. influenza infection can induce cellular stress, leading to mitochondrial dysfunction, which can culminate in nlrp -dependent activation of caspase- , leading to release of the proinflammatory mediators il- b and il- [ ] . infected cells can also release damps to alert neighboring cells of the danger. s a is produced during in vitro infection with influenza a virus [ ] , whereas hmgb is a damp that is released during in vivo influenza infections in both mice and humans [ , ] . reactive oxygen species released by cells in the lungs of humans and animals during viral infection can cause oxidation of endogenous phospholipids to produce oxidized phosphatidylcholine [ , ] . importantly, oxidized phospholipids, hmgb , and s a have all been shown to signal through tlr [ , , ] . relevantly, we reported that tlr / mice were resistant to infection with mouse-adapted influenza virus strain a/pr/ / (pr ) [ , ] , as well as a more potent, mouse-adapted strain ma-ca/ [ ] . the tlr -specific antagonist eritoran (e ; eisai co., tokyo, japan) blocked lethality and clinical symptoms caused by pr infection when given starting d after infection [ ] . blocking tlr signaling on d or on d and d after pr infection with an anti-tlr -specific ab also conferred protection [ ] . these and other tlr antagonists identified by our group [ , ] indicate that tlr antagonists may represent a novel class of drugs for use in treating patients with influenza. defensins are small, cysteine-rich, cationic peptides that primarily serve as innate immune defense mechanisms against infectious microorganisms. unlike the aand b-defensins, u-defensins are circular, formed by head-to-tail ligation of truncated a-defensin-like precursor peptides and possess broad antimicrobial activity, targeting bacteria, viruses, and fungi [ ] [ ] [ ] [ ] [ ] . all human u-defensin (deft) pseudogenes contain a premature stop codon, preventing translation [ , ] . however, solid-phase peptide synthesis has allowed researchers to construct artificially humanized u-defensins, called rcs [ ] . the individual rcs vary slightly in the noncysteine amino residues but bind with high affinity to n-and o-linked glycosylated proteins, such as hiv gp [ ] , and to prevent fusion mediated by influenza hemagglutinin [ ] . consequently, rcs can effectively prevent in vitro infection of human cell lines with either hiv [ , ] or influenza a virus [ ] . similarly, rcs are protective in vitro against hsv- / through binding to its surface glycoprotein [ ] . in addition to their role in fighting microorganisms, defensins might also have a key regulatory role in limiting tissue-damaging inflammation. human b-defensin can inhibit tlr -dependent signaling in vitro and in vivo [ , ] . rtds prevent cytokine secretion from human blood leukocytes treated with multiple tlr ligands [ ] , including the tlr agonist lps [ ] , demonstrating that u-defensins can also regulate host responses mediated by cellular glycoproteins. this also suggests a role for the structurally similar rcs in tlr antagonism. clinically, rcs could represent a novel therapeutic against influenza infection, inhibiting the detrimental production of tlr -dependent cytokines and blocking infection of the virus or coinfecting bacteria. the aims of our study were to determine whether rc- could inhibit lps-induced tlr signaling and whether rc- could be used therapeutically against influenza-mediated disease. rc- , containing an arginine-to-lysine substitution from the parent rc peptide [ ] , was made by solid-phase synthesis. backbone cyclization of rc is achieved through intramolecular native chemical ligation [ , ] of a thioester-ending linear peptide synthesized on solid phase using boc chemistry. disulfide bonds were formed under oxidative conditions in the presence of dmso. the peptide was purified by reversed-phase hplc to homogeneity (. % purity) and verified by electrospray ionization mass spectrometry. the final product, shown in supplemental fig. , was analyzed by reversed-phase hplc on a waters (milford, ma, usa) xbridge c column ( . mm, . mm) running at °c with a -min gradient of - % acetonitrile containing . % trifluoroacetic acid at a flow rate of ml/min. the observed molecular mass of . da is within the experimental error of the expected value of . da, which was calculated based on the average isotopic compositions of the peptide. rc- was solubilized in pbs before use. were purchased from the indicated vendors. mouse mab were used to visualize tlr proteins with the tags flag (m clone) (sigma-aldrich) and ecfp (clone oti a ) (origene technologies, rockville, md, usa) by western blot analysis. an anti-biotin-hrp-linked ab (cell signaling technology, danvers, ma, usa) was used to visualize lps-biotin. rabbit-specific polyclonal ab against rc- has been previously described [ ] . all other primary and secondary abs used in the study have been previously described [ ] . the lal chromogenic endotoxin quantitation kit ( ; thermo fisher scientific, waltham, ma, usa) was used, according to the manufacturer's instructions to test the biologic activity of e. coli :b lps (included in the kit as a positive control). chromo-lal and control standard endotoxin (e. coli :h ) were purchased from associates of cape cod, inc. (east falmouth, ma, usa) and used according to the manufacturer's instructions to measure the ability of rc- to antagonize the biologic activity of lps at doses higher than eu/ml. [ ] . tlr / mice (n $ ) on a c bl/ background were originally obtained from dr. shizuo akira (osaka university, osaka, japan) for breeding at university of maryland (baltimore, md, usa). thioglycollate-elicited, mouse peritoneal mfs were isolated from wt c bl/ j (jackson laboratory, bar harbor, me, usa), tlr / , trif / , and irak kdki and were cultured as previously described [ ] . human monocyte-derived mfs were matured from elutriated pbmcs of healthy donors as previously described [ ] . mouse and human mfs were pretreated with either pbs control or rc- within s of stimulation. in vitro infection with pr was performed as previously described [ , ] . mf lysates were harvested at the indicated times in tripure (roche diagnostics, north america, indianapolis, in, usa) for subsequent gene expression analysis. cdna was synthesized from rna as previously described [ ] . qrt-pcr reactions were monitored with the ht fast real-time pcr system (thermo fisher scientific) using the power sybr green mix pcr master mix (thermo fisher scientific). individual genes were analyzed with cytokine gene-specific human [ ] and mouse [ ] primers, which have previously been published. the total amount of mrna was calculated using the comparative cycle threshold (ddct) method [ ] and expressed as foldinduction compared with untreated or uninfected cells. hek t cells were grown in dmem, which was supplemented with mm l-glutamine, u/ml penicillin, mg/ml streptomycin, and % fcs, as previously described [ ] . one night before transfection, cells were plated in a -well tissue-culture dish. the following day, those cells were transfected with expression plasmids using the transfection reagent superfect (qiagen, valencia, ca, usa), according to the manufacturer's supplied instructions. specifically, mg of total plasmid (empty vector pcdna . , tlr -flag [ ] , or tlr -cfp [ ] ) was mixed with superfect at a : ratio. for dual transfections, . mg of each plasmid was used, and the amount of superfect was kept constant. after transfection, the cells were allowed to incubate in growth medium for h before monitoring transgene expression. after h, cells were washed twice with ice-cold pbs and lysed ( mm hepes, . % triton x- , mm nacl, and mm pmsf). whole-cell lysates were incubated on ice for min and centrifuged at , rpm for min at °c to pellet cellular debris. supernatants were withdrawn and analyzed by western blot directly (input samples) or immunoprecipitated. in the latter case, mg of whole-cell lysates (in ml) were incubated overnight at °c with gentle rotation in the presence of mg of flag-specific ab (sigma-aldrich). protein g agarose (thermo fisher scientific) bead slurries ( ml) were added to mixtures and incubated for a further min with constant rotation at room temperature. beads were harvested by centrifugation and washed times with binding buffer ( mm tris, mm nacl; ph . ). to harvest the bound proteins, beads were resuspended in ml laemmli buffer, vortexed, boiled for min at °c, and centrifuged. supernatants were separated by sds-page on a % gel and transferred to a polyvinyl difluoride membrane. for lps pulldowns, lps-biotin and rc- were incubated for min at room temperature in the presence or absence of excess, unlabeled e. coli k lps. pierce streptavidin ultralink resin beads ( ml slurry; thermo fisher scientific) were added to the samples and incubated for a further min with constant rotation at room temperature. beads were harvested by centrifugation and washed times with binding buffer ( . m phosphate, . m nacl; ph . ). to harvest the bound proteins, beads were resuspended in ml laemmli buffer, vortexed, boiled for min at °c, and centrifuged. supernatants were withdrawn and serially diluted. samples ( ml) were dot-blotted onto nitrocellulose membrane ( . mm pore size), air dried, blocked, and incubated in parallel for h when probing for biotin or overnight when probing for rc- . all mouse protocols were approved by the institutional animal care and use committee at the university of maryland, baltimore. on d , female, - -wkold wt c bl/ j mice were infected intranasally with ; tcid pr , as we have described [ ] . mice were injected daily on d - postinfection i.v. with ml of saline or rc- ( mg/mouse). a similar dosing regimen with the tlr antagonist eritoran was previously shown to protect mice against pr infection [ ] . mice were monitored daily for survival, clinical score, and weight loss for d. clinical scores ranging from (no symptoms) to (moribund) included piloerection, hunched posture, ruffled fur, audible lung crackling, and lethargy, as previously detailed [ ] . mice were euthanized as soon as they became moribund to prevent suffering of the animal. experiments were performed in duplicate for analysis of gene expression. the numerical values obtained from those assays were analyzed with graphpad prism (graphpad software, la jolla, ca, usa) with either a -way or -way anova, depending on the number of experimental variables. significance was determined to be p , . . for the -way anova, a tukey's post hoc test was used to look for significance among experimental groups within the data set. survival curves of mice were analyzed for significance with a log-rank test. supplemental material to this article includes figure. supplemental fig. shows the peptide sequence of rc- and a representative example of the purity of rc- after peptide synthesis. rtds have been shown to decrease cytokine secretion in response to multiple tlr agonists [ ] . because rc- is structurally similar to those rtds, we hypothesized that rc- would antagonize tlr -mediated signaling, leading to cytokine induction. consistent with that prediction, rc- inhibited lpsinduced ifn-b in a dose-dependent manner (fig. a) . induction of ifn-b was decreased in the presence of rc- as early as min after lps treatment (fig. b) . signal transduction in response to lps is initiated by homodimerization of tlr [ ] . tlr signal transduction contains a unique bifurcation, using both myd and trif as adaptor proteins [ , ] . ifn-b is considered a myd -independent gene induced downstream of the tlr -trif pathway [ , ] . in contrast to ifn-b, tnf-a and il- b represent myd -dependent cytokines [ ] . to determine whether myd pathways were also affected by rc- treatment, expression of those myd -dependent genes were analyzed. both lps-dependent tnf-a and il- b were inhibited by rc- ( fig. c and d) , albeit to a lesser extent than was evident for ifn-b mrna. to determine whether rc- mediated tlr antagonism was a species-specific response, primary human monocyte-derived mfs were also stimulated with lps in the presence of rc- . similar to that observed in mouse mfs, rc- also inhibited trif-and myd -dependent cytokine gene expression in response to lps stimulation ( fig. e-h) . decreased lps-dependent cytokine gene expression from rc- treatment correlated with a rc- -mediated block in activation-induced phosphorylation of the tlr -proximal signaling elements tbk and nf-kb p at and min after lps stimulation (fig. ) . similarly, activation of mapk pathways, erk, jnk, and p , were also decreased at those time points in the presence of rc- (fig. ) . tbk phosphorylation remained decreased at min after lps treatment in the presence of rc- (fig. ) , whereas phosphorylation of mapk and nf-kb was increased at that time point (fig. ) , indicating that rc- blockade of tlr only delays signal transduction at that dose of lps and does not ablate it entirely. inhibition of tlr signaling by rc- in human and mouse cells could possibly be explained by sequestration of lps from the host tlr signaling complex, so we sought to test that possibility in a cell-free system. the biologic activity of lps was measured in the lal assay in the presence or absence of rc- to determine whether our cytokine expression results were likely due to a interaction between rc- and lps. the biologic activity of endotoxin u/ml (; . ng/ml) of lps was partially inhibited by mg/ml rc- and almost completely inhibited by mg/ml rc- (fig. a) . the dose of lps used in the cellfree lal assay represents a -fold lower dose of lps than used for mf tlr stimulation (e.g., see fig. ). to test morerelevant doses of lps, we used a different lal assay that had a linear range up to eu/ml and maintained the dose of rc- at mg/ml. under those conditions, rc- ( mg/ml) strongly inhibited the biologic activity of all lps doses tested, including eu/ml (fig. b) . rc- also inhibited the lal activity of biotinylated lps at doses ranging from to ng/ml (fig. c) , suggesting that it is likely that rc- inhibits tlr signaling, in part, by sequestration of lps. to examine that possibility further, we performed an in vitro pulldown assay with biotinylated lps in the presence or absence of rc- . rc- did not impair the ability of streptavidin beads to pull down lpsbiotin (fig. d) . additionally, rc- was recovered upon pulldown of biotinylated lps with streptavidin beads, suggesting direct binding between rc- and lps (fig. d) . to verify that this binding was specific, an excess of unlabeled lps e. coli k was also added to the mix before pulldown. excess unlabeled lps prevented the pulldown of rc- with biotinylated lps, suggesting that the binding of lps and rc- is specific (fig. d) . in contrast to its interaction with lps, rc- was not pulled down by the biotinylated tlr agonist pam csk (fig. e) . although our data show evidence that rc- may mediate inhibition of tlr -dependent signaling though sequestration of lps, it must be noted that even % inhibition of lps activity because of sequestration might not affect tlr -mediated cytokine expression in our system because the lps dose we used to stimulate mfs ( ng/ml) was not in the linear portion of the cytokine dose-response curve ( fig. a and b) . for that reason, we also sought to explore the capacity of rc- to disrupt tlr dimers, using a model of tlr oligomerization that occurs in the absence of ligands. our laboratory has previously shown that overexpression of flag-tagged tlr in hek t cells can facilitate tlr -dependent signaling in the absence of lps [ ] . cotransfection of tlr -flag and tlr -cfp induced a physical interaction between the tlr constructs, as evidenced by coimmunoprecipitation of the epitope tags (fig. c ). using that model, we examined whether rc- could destabilize the interaction. addition of rc- failed to inhibit the coimmunoprecipitation of tlr -cfp with tlr -flag (fig. d) . after describing the inhibitory role of rc- in lpsdependent signal transduction, we sought to test whether its role was similar in other innate immune signaling pathways. tlr dependent cytokine secretion has been shown to be regulated by rtds in human blood leukocytes [ ] , so tlr might be expected to be regulated by rc- , similar to tlr . in our laboratory, rc- also inhibited proinflammatory, tlr dependent gene induction in mouse mfs (fig. a and b) , demonstrating that rc- -mediated inhibition is not restricted to tlr ; however, rc- failed to bind to biotinylated pam csk (fig. e) . therefore, inhibition of signaling is not restricted to agonists that rc- can bind directly. to further determine the degree of pathway specificity of rc- -mediated inhibition, mouse mfs were also stimulated with non-tlr agonists in its absence or presence. rc- failed to inhibit cytokine gene induction in response to either recombinant tnfa or dmxaa (fig. c-f) . dmxaa is a potent inducer of tbk dependent ifn-b in mouse mfs through intracellular activation of sting [ ] , unlike tlr and tlr , which initiate signaling extracellularly. note that the apparent lack of induction of ifn-b in response to lps in fig. e was due to a suboptimal time point for its expression (see fig. ). collectively, these latter results demonstrate that rc- is not a universal inhibitor of innate immune signaling pathways. the recent discovery that eritoran protects mice from lethal infection with pr [ ] suggested that treatment of mice with other tlr antagonists, such as rc- , may also protect against lethal influenza infection. the effect of rc- on pr infection was first examined in an in vitro infection model. in wt mfs, infected for and h in vitro, rc- inhibited pr -induced expression of the proinflammatory genes encoding pro-il- b and tnf-a ( fig. a and b) . although tnf-a secretion by pr infected mfs has been shown to be partially tlr dependent [ ] , we observed that induction of pro-il- b and tnf-a mrna was not significantly impaired in pr -infected tlr / mfs at h postinfection (fig. c and d) . similarly, pr -infected trif / or irak kdki mfs induced similar amounts of il- b and tnf-a mrna in response to pr as wt controls ( fig. e and f) . irak is the first kinase recruited to myd during signal transduction, and irak kdki mfs behave comparably to myd / mfs [ ] . this suggests that the damps that activate tlr -dependent or other tlr-dependent signaling in vivo are likely not present in our in vitro model at the time points tested. this result also indicates that rc- influences cytokine induction during in vitro infection independent of tlr , possibly by inhibiting the signaling of some other non-tlr patternrecognition receptors, such as rig-i, which is also capable of recognizing pr [ ] . hmgb is a damp that has been shown to be released during influenza infection in mice and humans [ , ] . importantly, hmgb is also known to activate tlr -dependent signaling [ ] . rc- suppressed cytokine gene induction in response to hmgb (fig. g and h) , demonstrating that it can also block tlr -mediated gene expression mediated by damps. to determine whether rc- could be used as an antiviral and/or anti-inflammatory therapeutic in vivo, mice were infected with pr (;ld ) and given daily i.v. injections of rc- ( mg/mouse), starting h after pr infection and continuing until d postinfection. we had previously reported that eritoran protects - % of mice using this identical dosing regimen [ ] . in this study, rc- treatment of mice also protected from pr -induced lethality (fig. a) and significantly lessened pr -induced clinical symptoms (fig. b) . this latter significant decrease in clinical symptoms suggests that rc- is not merely delaying the time to death because the survivors were typically healthy at the end of wk. because of the timing of the drug administration, rc- is not hypothesized to mediate its effect by blocking initial uptake of the virus. together, these data suggest that rc- and other u-defensins are strong candidates for future human clinical trials in the search for novel viral therapeutics. although the innate immune system has a crucial role in initiating an immune response against an invading microorganism, overexuberant signaling may actually contribute to pathology during the same infection. this double-edged sword effect of inflammation in response to innate immune signaling is illustrated during infection with influenza virus. the tlr adaptor myd is required for mouse survival after infection with pr [ ] . further illustrating the beneficial role of innate signaling pathways, pretreatment of mice with tlr or tlr agonists, before in vivo infection with influenza, has been shown to be protective [ ] [ ] [ ] . however, the timing of those signaling pathways is crucial because oxidative stress in the lung during infection with pr has been linked to the development of oxidized phospholipids [ ] and other damps that mediate tlr signaling-dependent acute lung injury [ ] . the detrimental role of inflammation can be further exacerbated by bacterial coinfection [ ] . because of the role of tlr during pr infection, tlr / mice are refractory to lethality associated with pr infection [ , ] . in further support of the role of oxidized phospholipids in mediating tlr -dependent lethality, mortality can be prevented by inhibiting oxidized phospholipid formation by administering the antioxidant n-acetyl cysteine [ ] . additionally, we reported that mortality can be prevented by inhibiting tlr signaling after pr infection with the potent synthetic tlr antagonist eritoran [ ] , an anti-tlr ab [ ] , and other agents that block tlr signaling [ , ] . thus, we hypothesized that drugs that target host-cell, tlr -dependent signaling pathways could potentially function as novel anti-influenza therapies. in our study, rc- was fully capable of inhibiting lpsdependent signaling in both mouse and human mfs. the impairment of both myd -and trif-signaling pathways suggests that rc- could function by preventing the initial interaction of lps with tlr , either through sequestration of lps or blockade of the signaling event itself. based on our study, we cannot rule out the strong possibility that a significant part of the tlr inhibition by rc- is mediated by lps sequestration from the tlr signaling complex because rc- could both bind lps in in vitro pulldown assays and inhibit a broad range of lps bioactivity in cell-free lal assays. however, the dose of lps used to stimulate mfs in our study was in the saturation range, where only strong antagonists would be expected to have a significant effect on cytokine gene expression. signal transduction in response to lps is initiated at the plasma membrane by homodimerization of tlr molecules [ ] . the ectodomain of human tlr contains n-linked glycosylation sites that are required for cell-surface expression and function [ ] . removal of those sialic acid residues increases lps-dependent signaling [ ] , suggesting that those sialic residues may be at the interface of tlr dimerization. because rcs bind with high affinity to proteins exhibiting n-and o-linked glycosylation [ ] , we hypothesized that rc- could antagonize tlr -mediated signaling by binding to the glycans on tlr to prevent ligandinduced dimerization. in line with that theory, human b-defensin can also inhibit tlr -dependent signaling through direct binding to tlr [ ] . using a model of ligand-independent oligomerization of tlr in hek t cells, we tested whether rc- could prevent or disrupt homotypic tlr interactions. addition of rc- failed to disrupt oligomers of tlr in this assay, which could mean that rc- does not target tlr directly or that its binding sites were unavailable in this conformation. unfortunately, we could not decrease the input concentrations of the tlr plasmids used for transfection to a level that permitted ligand-dependent signaling but that would still permit detection of coimmunoprecipitated tlr -flag and tlr -cfp molecules. the md- protein is a crucial part of the lps recognition complex [ ] and is also glycosylated [ ] , so it is also possible that md- , and not tlr , might be the primary target of rc- in this pathway. in addition to inhibition of lps-dependent signaling, we also found that pam csk -dependent activation of tlr signaling was inhibited by rc- . in contrast to its direct interaction with lps, rc- was unable to interact with pam csk in vitro. this suggests that rc- has the capacity to act in more ways than solely by ligand sequestration. human tlr also contains glycosylation sites [ ] , which could be a putative target of rc- . alternatively, tlr-dependent signal transduction is sensitive to the architecture of lipid-membrane microdomains [ ] . addition of a phosphatidylserine species can impair both tlr and tlr signaling pathways [ ] . given that the positively charged rc- has been found to bind to anionic membranes, such as dipalmitoyl phosphatidylglycerol [ ] , it is tempting to speculate that the ability of rc- to block both tlr -and tlr -dependent signaling is caused by an effect on the cell membrane itself. however, such a mechanism seems less likely because tnf-a-mediated signaling, which also requires receptor oligomerization [ ] , was not perturbed by rc- . the fusion protein for respiratory syncytial virus can be recognized in a tlr -dependent manner [ ] , but no influenza protein has been identified that can function as a tlr ligand. in contrast, damps, such as hmgb , are released during in vivo infection, and they signal through tlr . it is less clear whether those damps are produced in vitro and how much of an effect they have on gene regulation. secretion of proinflammatory genes at h postinfection with pr has been shown to be myd -dependent in bone marrow-derived and peritoneal mfs [ , ] . however, tnf-a protein secretion at and h was only decreased by ; - % in the absence of tlr , exhibiting only a partial tlr dependence [ ] . our data, using the irak kdki mfs, indicate that the difference in cytokine production mediated by a lack of myd -dependent signaling is not due to a defect in transcriptional up-regulation of myd -dependent genes, such as tnf-a or il- b, at h postinfection. as part of its role as an innate immune adaptor protein, myd has also been shown to be involved in stabilization of mrna species containing adenosine-and uridine-rich elements, which are common to many cytokines [ ] . so, we surmise that the tlr -myd pathway may be mediating its effect on cytokine secretion in response to pr through alterations in mrna stability and not through altered transcription. rcs offer the potential to limit inflammation mediated by damps and also to target the virus itself. although rc- may act by blocking initial viral entry, as previously described [ ] , it has been shown to be less effective against the mouse-adapted pr than against other influenza strains [ ] . for instance, preincubating the virus with various different rcs for min achieved only an ; % decrease in viral infectivity, as measured by a fluorescent focus formation assay [ ] . importantly, none of our experiments featured preincubation of the virus with rc- , which would further limit the ability of rc- to block the virus directly. as a proof of concept that rcs can be used therapeutically, rc- was tested in a mouse model of influenza infection. treatment with rc- , starting d postinfection, was shown to lessen the severity of pr -associated disease, resulting in a highly significant increase in survival (fig. ) . our results on the inhibition of tlr -mediated cytokine expression are consistent with the possibility that antagonism of tlr signaling accounts, at least in part, for protection in pr -infected mice. that the protective, therapeutic effect of rc- against pr is similar to what we have previously reported for the tlr antagonist eritoran [ ] suggests that rc- may act in vivo, at least partially, through tlr antagonism. however, we cannot rule out the possibility of an additional direct effect on the virus itself, as has been observed by others [ , ] . although eritoran does not directly kill influenza in vitro [ ] , eritoran therapy was associated with decreased influenza titers in vivo, but not until d after infection [ ] , which highlights an important connection between inflammation and viral replication in this infection model. it is quite possible that the effect of rc- in this system is both antiviral and anti-inflammatory. overall, our results in the mouse model are especially encouraging because the mouse-adapted pr strain used in our study lacks n-linked glycosylation on its cell-envelope hemagglutinin protein [ ] , making it more resistant than other influenza strains to rc- in vitro [ ] . therefore, rc- would be predicted to be more effective against human strains with increased glycosylation [ ] . similar to the efficacy demonstrated by rc- in our study, rtds protected mice from a lethal lung infection with a mouse-adapted severe acute respiratory syndrome-coronavirus and limited the potentially overexuberant cytokine response occurring in the lung [ ] . together, these data suggest that u defensins, and specifically, rc- , are strong candidates for future human clinical trials in the search for novel viral therapeutics. prevention and control of seasonal influenza with vaccines seasonal influenza vaccine total doses distributed estimates of deaths associated with seasonal influenza-united states estimating influenzaassociated deaths in the united states predominant role of bacterial pneumonia as a cause of death in pandemic influenza: implications for pandemic influenza preparedness bacterial coinfection in influenza: a grand rounds review early data suggests potentially severe flu season approaching the asymptote? evolution and revolution in immunology differential roles of mda and rig-i helicases in the recognition of rna viruses incoming rna virus nucleocapsids containing a -triphosphorylated genome activate rig-i and antiviral signaling rig-i-mediated antiviral responses to singlestranded rna bearing -phosphates recognition of double-stranded rna and activation of nf-kb by toll-like receptor cutting edge: influenza a virus activates tlr -dependent inflammatory and rig-i-dependent antiviral responses in human lung epithelial cells innate antiviral responses by means of tlr -mediated recognition of single-stranded rna mitochondrial protein mitofusin is required for nlrp inflammasome activation after rna virus infection damp molecule s a acts as a molecular pattern to enhance inflammation during influenza a virus infection: role of ddx -trif-tlr -myd pathway systemic release of high mobility group box protein during severe murine influenza high mobility group box in patients with pandemic h n influenzaassociated encephalopathy identification of oxidative stress and toll-like receptor signaling as a key pathway of acute lung injury the tlr antagonist eritoran protects mice from lethal influenza infection receptors involved in the oxidized -palmitoyl- -arachidonoyl-sn-glycero- -phosphorylcholine-mediated synthesis of interleukin- : a role for toll-like receptor and a glycosylphosphatidylinositol-anchored protein a critical cysteine is required for hmgb binding to toll-like receptor and activation of macrophage cytokine release novel signaling interactions between proteinase-activated receptor and toll-like receptors in vitro and in vivo variations in the hemagglutinin of the h n pandemic virus: potential for strains with altered virulence phenotype? novel strategies for targeting innate immune responses to influenza a decoy peptide that disrupts tirap recruitment to tlrs is protective in a murine model of influenza tlr antagonist fp inhibits lps-induced cytokine production and glycolytic reprogramming in dendritic cells, and protects mice from lethal influenza infection homodimeric u-defensins from rhesus macaque leukocytes: isolation, synthesis, antimicrobial activities, and bacterial binding properties of the cyclic peptides a cyclic antimicrobial peptide produced in primate leukocytes by the ligation of two truncated a-defensins circular minidefensins and posttranslational generation of molecular diversity defensins enable macrophages to inhibit the intracellular proliferation of listeria monocytogenes inhibition of dengue ns b-ns protease and viral replication in vero cells by recombinant retrocyclin- evolution of primate u-defensins: a serpentine path to a sweet tooth reawakening retrocyclins: ancestral human defensins active against hiv- retrocyclin: a primate peptide that protects cells from infection by t-and m-tropic strains of hiv- retrocyclin, an antiretroviral u-defensin, is a lectin carbohydrate-binding molecules inhibit viral fusion and entry by crosslinking membrane glycoproteins rc- , a retrocyclin- analogue with enhanced activity against primary hiv type isolates interactions of a-, b-, and u-defensins with influenza a virus and surfactant protein d ) u defensins protect cells from infection by herpes simplex virus by inhibiting viral adhesion and entry human b-defensin affects the activity of pro-inflammatory pathways associated with myd and trif human b-defensin has immunosuppressive activity in vitro and in vivo rhesus macaque u defensins suppress inflammatory cytokines and enhance survival in mouse models of bacteremic sepsis defective lps signaling in c h/hej and c bl/ sccr mice: mutations in tlr gene synthesis of native proteins by chemical ligation synthesis of proteins by native chemical ligation chemical, physical, biological properties of a lipopolysaccharide from escherichia coli k- reprogramming of murine macrophages through tlr confers viral resistance via traf -mediated, enhanced interferon production complete dependence on irak kinase activity in tlr , but not tlr , signaling pathways underlies decreased cytokine production and increased susceptibility to streptococcus pneumoniae infection in irak kinase-inactive mice -dimethylxanthenone- -acetic acid (dmxaa) activates stimulator of interferon gene (sting)-dependent innate immune pathways and is regulated by mitochondrial membrane potential ccr ligands inhibit hiv by inducing apobec g analyzing real-time pcr data by the comparative c t method analysis of proteinaseactivated receptor and tlr signal transduction: a novel paradigm for receptor cooperativity targeting tlr signaling by tlr toll/il- receptor domain-derived decoy peptides: identification of the tlr toll/il- receptor domain dimerization interface lipid a antagonist, lipid iva, is distinct from lipid a in interaction with toll-like receptor (tlr )-md- and ligand-induced tlr oligomerization unresponsiveness of myd -deficient mice to endotoxin identification of lps as a key transducer of myd -independent tir signalling cutting edge: a novel toll/il- receptor domaincontaining adapter that preferentially activates the ifn-beta promoter in the toll-like receptor signaling myd signaling is indispensable for primary influenza a virus infection but dispensable for secondary infection toll-like receptor prestimulation protects mice against lethal infection with highly pathogenic influenza viruses the tlr -trif pathway protects against h n influenza virus infection intranasal administration of the tlr agonist pam cys provides rapid protection against influenza in mice potential role for alternatively activated macrophages in the secondary bacterial infection during recovery from influenza n-acetyl-l-cystine (nac) protects against h n swine influenza virus-induced acute lung injury md- and tlr n-linked glycosylations are important for a functional lipopolysaccharide receptor sialyl residues modulate lps-mediated signaling through the toll-like receptor complex hbd- regulation of the immune response and the lps/tlr -mediated signaling pathway essential role of md- in lps responsiveness and tlr distribution four n-linked glycosylation sites in human toll-like receptor cooperate to direct efficient biosynthesis and secretion a phosphatidylserine species inhibits a range of tlr-but not il- b-induced inflammatory responses by disruption of membrane microdomains the cyclic antimicrobial peptide rtd- induces stabilized lipid-peptide domains more efficiently than its open-chain analogue dual role of the p tumor necrosis factor (tnf) receptor in tnf cytotoxicity pattern recognition receptors tlr and cd mediate response to respiratory syncytial virus toll il- receptors differ in their ability to promote the stabilization of adenosine and uridine-rich elements containing mrna specific sites of n-linked glycosylation on the hemagglutinin of h n subtype influenza a virus determine sensitivity to inhibitors of the innate immune system and virulence in mice tracking global patterns of n-linked glycosylation site variation in highly variable viral glycoproteins: hiv, siv, and hcv envelopes and influenza hemagglutinin rhesus u-defensin prevents death in a mouse model of severe acute respiratory syndrome coronavirus pulmonary disease key words: innate immunity • inflammation • cytokine regulation • signal transduction key: cord- -d g toc authors: yu, feng; zhu, jing; lei, ming; wang, chuan‐jiang; xie, ke; xu, fang; lin, shi‐hui title: exploring the biomarkers associated with different host inflammation of acute respiratory distress syndrome (ards) from lung metabolomics in mice date: - - journal: rapid commun mass spectrom doi: . /rcm. sha: doc_id: cord_uid: d g toc rationale: the aim of this study was to analyze the metabolomics of lung with different host inflammation of acute respiratory distress syndrome (ards) for the identification of biomarkers for predicting severity under different inflammatory conditions. methods: cecal ligation and puncture (clp) and lipopolysaccharide (lps)‐intratracheal injection induced acute lung injury (ali). a mouse model was used to explore lung metabolomic biomarkers in ali/ards. the splenectomy model was used as an auxiliary method to distinguish between hyper‐ and hypo‐inflammatory subtypes. plasma, lung tissue and bronchoalveolar lavage fluid (balf) samples were collected from mice after clp/lps. the severity of lung injury was evaluated. expression of tumor necrosis factor‐α (tnf‐α) in mice serum and lung was tested by elisa and pcr. polymorphonuclear cells in balf were counted. the lung metabolites were detected by gc/ms, and the metabolic pathways predicted using the kegg database. results: the lps/clp‐splen group had more severe lung injury than the corresponding ali group; that in the clp‐splen group was more serious than in the lps‐splen group. tnf‐α expression was significantly elevated in the serum and lung tissue after lps or clp, and higher in the lps/clp‐splen group than in the corresponding ali group. the level of tnf‐α in the clp‐splen group was elevated significantly over that in the lps‐splen group. both these groups also showed significant neutrophil exudation within the lungs. during differential inflammation, more differential metabolites were detected in the lungs of the clp‐group ali mice than inthe lps group. a total of compounds were detected in the lungs of the clp and clp‐splen groups. contrastingly, compounds were detected in the lungs of the lps and lps‐splen groups. the lps‐splen and clp‐splen groups had significant neutrophil exudation in the lung. random forest analysis of lung‐targeted metabolomics data indicated ‐hydroxyphenylacetic acid, ‐aminocyclopentanecarboxylic acid (acpc), cis‐aconitic acid, and hydroxybenzoic acid as strong predictors of hyper‐inflammatory subgroup in the clp group. furthermore, with splenectomy, differential metabolic pathways between the clp and lps groups were revealed. conclusions: hyper‐inflammatory subgroups of ards have a greater inflammatory response and a more active lung metabolism. combined with host inflammation background, biomarkers from metabolomics could help evaluate the response severity of ards. acute respiratory distress syndrome (ards) is an acute inflammatory lung injury, associated with increased pulmonary vascular permeability, increased lung weight, and loss of aerated lung tissue [ ] . although years have passed since the first description of ards [ ] , the overall mortality is still more than % [ , ] . unfortunately, it is a clinical feature of very different mechanisms, with complex syndromes, and biological and clinical heterogeneity. because of the heterogeneity of the host response, it is difficult to found the key to curing every patient with ards. the clinical and biomarker characteristics of ards patients demonstrated hypo-inflammatory and hyper-inflammatory effects [ ] .specific subsets of critically ill patients have higher risk of disease-related outcome or differential responses to therapy [ , ] .therefore, the different inflammatory sub-phenotypes of ards may indicate varied risks related to the disease [ ] . metabolic phenotypes, which represent different pathways important to the pathophysiology of ards, could potentially be used to identify the subgroup that may benefit from certain targeted therapies [ ] . about one third of hyper-inflammatory ards patient have a higher plasma level of inflammatory biomarkers [ ] . some biological indicators, such as endocan [ ] , srage and ang- [ ] , are closely related to the hyper-inflammatory subphenotype of ards. tnf-α is an important inflammatory factor that can induce t cells to produce various inflammatory factors, and then promote the occurrence of inflammatory reactions. it has been reported that tnf-α is a potential biomarker for acute respiratory distress syndrome, as well as for mortality in patients with obesity and coronavirus (covid- ) [ ] . in addition, studies have found that -hydroxybenzoic acid has anti-catabolism and anti-inflammatory effects, and prevents the upregulation of pro-inflammatory markers, including metalloproteinases and cyclooxygenase [ ] . whether acps, as a small molecule, also has such potential needs further study. thus, classification of patients with ards into hyper-and hypoinflammatory subphenotypes using plasma biomarkers may facilitate more effective targeted therapy [ ] . metabolism has increasingly been acknowledged as a potential target for therapies aimed at modulating the immune system either to enhance or to suppress immunological responses [ ] . due to the coupling of inflammation with metabolism, the novel "inflammation-immunitymetabolism axis" may be another useful way to propose new therapeutic implications and deeper understanding for ards. metabolomics is a rapidly expanding field of systems biology that provides the ability to generate a "snapshot" measurement of all small molecules and this article is protected by copyright. all rights reserved. metabolites in a given sample [ , ] . the burgeoning field of metabolomics lies in its application to acute lung diseases, specifically pneumonia and ards [ ] . the application of untargeted metabolomics for biomarker discovery is well suited to the complexity of ards because metabolomics can detect several hundreds of metabolites, depending on the analytical platform, from a single sample, with minimal bias and no prior knowledge of the sample composition [ , , ] .we have found that specific compounds related to hypoxia may serve as early biomarkers for ards, while metabolites with significant correlations with the partial pressure of arterial oxygen (pao )/percentage of inspired oxygen (fio ) may play a role in determining its severity [ ] . however, it is difficult to find specific metabolic evidence in clinical samples related to differences in inflammatory hosts. the spleen is a site where immune responses that are deleterious to the host can be regulated [ ] . the white pulp of spleen is a secondary lymphoid organ with key functions in immune response initiation and regulation. various immune cells (macrophages, dendritic cells, subsets of b and t lymphocytes) of the white pulp trap antigens and generate an antigen specific response against invading pathogens (bacteria, viruses and fungi). patients without a spleen (resulting from traffic accidents, trauma, etc.) have severe inflammation and a high risk of death in sepsis. therefore, we think that patients without a spleen may form acute lung injury (ali) models with "all or nothing" different subtypes of inflammatory hosts. we have also confirmed that this ali lack-of-spleen model will not die quickly as a result of complete immunodeficiency [ ] . in order to explore differences of metabolism in host inflammation of ards, we established a special ali mode (with spleen or without spleen, induced by lipopolysaccharide (lps)-tracheal infusion, or cecal ligation and puncture (clp). gc/ms metabolomics was then used to determine the endogenous metabolites in the lung tissues. c bl/ mice (male, - weeks old) from the laboratory animal center of chongqing medical university (cqmu, chongqing, china) were usedthe . previous studies had shown that estrogen provides a protective effect in ards [ ] . as, however, the role of hormones is not the research aim in this study, we chose only male mice for the studies [ ] .the mice were acclimatized to the new environment for seven days at °c with a free access to water and food and with a h light/dark cycle before experiments. the study was performed according to international, national and institutional rules concerning animal experiments, clinical studies this article is protected by copyright. all rights reserved. and biodiversity rights. the study protocol was approved by the ethics committee of our institute. the abdominal incision was then closed. in a "sham" operation on mice, the abdominal wall was cut, the spleen was only slightly spit, and there was no resection. the mice were given subcutaneous injections of ceftriaxone ( ug/g) for three days after surgery. fourteen days later, these splenectomy mice were used to establish the model [ ] . lps-induced ali was performed to establish an ali mouse model, using the same anesthesia method as described above. a mg/ml solution of lps was injected into mice through intratracheal instillation, and the control group was injected with the same volume of sterile phosphate-buffered saline (pbs) [ ] . the mice were randomly divided into three groups: control group (n= ), lps group (lps, n= ), and splenectomy group (lps-splen, n= ). the nice were euthanized by carbon dioxide box anesthesia h, h, and h after being challenges with lps or pbs, and blood and lung tissues were harvested for analysis. mice were randomly divided into three groups: control group (n= ), clp group (clp, n= ), and splenectomy group (clp-splen, n= ) and the anesthesia method was followed as previously described. the mice were positioned in dorsal recumbency. after shaving and this article is protected by copyright. all rights reserved. aseptic preparation of the surgical site, a ventral midline incision ( cm) was made to allow exteriorization of the cecum. the cecum was identified and was penetrated through-andthrough with a -gauge needle with a - silk suture at % from the tip. after being punctured, the cecum was gently squeezed to extrude a small amount of feces and returned to the abdominal cavity. the abdominal incision was then closed. sham-operated control mice (sham group) were subjected to the same surgical laparotomy after anesthesia, where the cecum was exteriorized and manipulated as described but not ligated or punctured. immediately after surgery, the animals were resuscitated with ml/kg saline injected subcutaneously [ ] . at the end of the experiment, the mice were euthanized by carbon dioxide box anesthesia h, h, and h after the clp procedure to collect whole blood and lung tissues for analysis. lung tissue samples were collected h post challenge with lps or clp. the superior lobe of the right lungs was fixed with % formalin in pbs for h, dehydrated in a graded ethanol series, and embedded in paraffin. paraffin sections were then stained with hematoxylin and eosin (h&e) followed by microscopic assessment and photographic documentation. lung injury scores were estimated; the higher the score, the more severe the injury. the four following indicators of lung injury were used to arrive at this score: alveolar congestion; bleeding; gap or vascular wall neutrophil infiltration or aggregation; and alveolar septal thickening or transparent membrane formation. the marking system was: marks, no or very slight damage; mark, mild injury; marks, moderate injury; marks, severe injury; and marks, very severe damage. the cumulative increase in the number of lesions related to the total score yields the pathological score of lung injury. the blood vessels leading to the lungs and the left bronchus were ligated h post challenge with lps or clp. μl of pbs was injected into the right lung through the trachea and, seconds later, the pbs was removed and the bronchoalveolar lavage fluid (balf) was collected. these lavages were carried out twice, and a total of μl of balf was collected. the serum, balf, spleen, and lung tissues were harvested for analysis. blood and lung tissue samples were taken h post challenge with lps or clp. blood was drawn from mice and allowed to coagulate for h at room temperature. the serum was then obtained by centrifuging the blood samples at °c for min at , ×g. the lungs were this article is protected by copyright. all rights reserved. ground in pbs ( mg tissues/ μl pbs). the serum and lung homogenate were then aliquoted and kept frozen at - °c until analysis. the tumor necrosis factor-α (tnf-α) levels were measured by elisa. total cellular rna was extracted from lung tissue using trizol reagent accompanied by dnasei digestion. quantitative real-time polymerase chain reaction (qrt-pcr) for mouse tnf-α was performed using specific primers (designed and synthesized by takara for s. gene expression normalized to gapdh was used to determine relative target gene expression by the ΔΔc(t) method. twenty mg of prepared mice lung tissue was added to fresh tubes, followed by the addition of μl of the internal standard, l-alanine- , , , -d ( mm). after adding μl of cold methanol-water ( % v/v), the tissues were homogenized and centrifuged ( , × g, min) to collect the supernatant. the lung tissue was dried using a speedvac and stored at - °c prior to derivatization. the volatilities of extracted metabolites were lowered using methyl chloroformate (mcf) derivatization, based on the protocol of smart et al [ ] . in brief, μl of sodium hydroxide ( m) was added to the speedvac-dried samples. ml of methanol and μl of pyridine were also added as the methyl group donor and catalyst, respectively. the reaction was started by adding μl mcf, followed by seconds of vortexing and subsequently adding another μl of mcf, followed by seconds of vortexing. in order to isolate derivatized metabolites from the reactive mixture, μl of chloroform and μl of sodium bicarbonate ( mm) were added and vortexed for seconds. the chloroform phase was isolated, and excess water was removed by adding anhydrous sodium sulfate. this article is protected by copyright. all rights reserved. the derivatized samples were analyzed using a gas chromatograph (agilent, santa clara, ca, usa) fitted with a zb- capillary column ( m × μm id × . μm with a -m guard column; phenomenex, torrance, ca, usa) coupled to an agilent msd single quadrupole mass spectrometer operating in electron ionization mode at ev. the gc and ms procedures followed the protocol outlined by smart et al. [ ] the isolated chloroform phase was injected at °c in pulsed splitless mode with helium carrier gas at a flow rate of ml/min. the program temperature was as follows: initial temperature of °c, ramped at °c/min to °c, then at °c/min to °c, and finally at °c/min to °c. the auxiliary temperature, quadrupole mass analyzer temperature, and source temperature were set to , , and °c, respectively. the mass range was m/z - , the scan speed . m/z units/s and the solvent delay . min. compound deconvolution and identification were performed by the automated mass spectral deconvolution and identification system (amdis; nist, gaithersburg, md, usa) software, using our internal methyl chloroformate derivatization mass spectra library of metabolite standards. the compounds were identified based on two criteria: > % match with the library spectrum and within a -min bin of the respective chromatographic retention time. the relative abundance of the metabolites was extracted via our in-house massomics software, using the peak height of the highest reference ion mass. the metabolite values were normalized by the abundance of the internal standard (l-alanine- , , , -d ) and total ion count, in order to correct for experimental variability. the metabolomics data have been deposited to the embl-ebi metabolights database (doi: https://doi.org/ . /nar/gks .) [ ] with the identifier mtbls . the abundance of identified compounds was adjusted to a gaussian distribution via log transformation prior to statistical analysis. multivariate analysis of anova followed by tukey's hsd test was performed in r. the predicted metabolic activities were determined using our pathway activity profiling r package based on the kegg online database. the relative metabolic activities were transformed to have a mean of zero and a standard deviation of one (z-score). subsequently, the metabolic pathways were classified according to their cellular processes, and only the predicted metabolic pathways with p values and q values less than . were displayed. the metabolic activities were first normalized by log transformation and pareto scaling, followed by ranking of the metabolic pathways using a random forest model to capture their contribution to the classification accuracy demonstrated in a vip plot. the metabolic network was constructed according to a pathway-based framework provided by this article is protected by copyright. all rights reserved. metscape that connected the kegg human metabolic pathways with our identified metabolites via the kamada-kawai layout, which relates the layout of metabolites to minimize metabolic reactions between metabolites within a metabolic network. all the illustrations and figures displayed were plotted using the ggplot r package, graphpad prism (graphpad software, san diego, ca, usa), and spss . (ibm, amonk, ny, usa). mice (with or without splenectomy) were euthanized h post challenge with lps or clp. the administration of lps or clp led to severe lung injury, compared with the controls ( figure a ). the lung injury scores showed that splenectomy can aggravate lung injury directly in either the lps or the clp group, p < . ( figure a ). all parts of the lung injury degree index, such as thickened alveolar wall, hemorrhage in the alveolus, alveolar collapse, and inflammatory cell infiltration in the lps-splen group or clp-splen group, were more severe than in the corresponding ali group. moreover, the lung injury in the clp-splen group is more serious than in the lps-splen group, p < . ( figure a ; supplementary table , supporting information). the concentrations of tnf-α were significantly elevated in the serum and lung tissue after lps or clp compared with the controls, p < . ( figures b and c ). in addition, the expression of tnf-α in the lps-splen or clp-splen groups was higher than in the corresponding ali (lps or clp) groups. the levels of tnf-α in the clp-splen group were significantly elevated compared with the lps-splen group, p< . ( figures b and c ). subsequently, infiltration of neutrophils was confirmed with neutrophil numbers in balf ( figure d ). after splenectomy, the lps(splen) and clp(splen) groups have significant neutrophil exudation in the lungs ( figure d ). the pls-da and leave-one-out cross-validation results are shown in figure . the supervised pls-da showed that the four different groups were well-clustered, with specific metabolic profiles for each ( figure b ). after anova was performed for the clp and clp-splen groups, a total of compounds were detected in the lungs, including metabolites from organic acids ( / , . %), amino acids ( / , . %), amino acid derivatives ( / , . %), and others ( figure a; supplementary figure , supporting information). in the random forest (rf) analysis the "mean decrease accuracy" indicates how much a certain metabolite contributes to separation of the groups, and the overall "predictive accuracy" is this article is protected by copyright. all rights reserved. indicative of the accuracy of a set of metabolites in discriminating spleen status [ ] . rf analysis of lung-targeted metabolomics data defined a set of metabolites that constitute the best predictors of differences in host inflammation status: in particular, increased hydroxyphenylacetic acid, -aminocyclopentanecarboxylic acid (acpc), and cis-aconitic acid, tridecane and hydroxybenzoic acid were strong predictors of the hyper-inflammatory subgroup in clp-induced ali ( figure b ). after anova was performed for the lps and lps-splen groups, only compounds were detected in the lungs. the organic acids ( / , %) make up the largest category among the subgroups of different inflammation in ali, which was induced by lps ( figure c ). consequently, under differential inflammation (hyper-vs hypo-), the lungs of clp-induced ali will detect more differential metabolites than the lpsinduced ali lungs ( figures a and c ). detailed information on this is displayed in supplementary table (supporting information). when clustering the hyper-inflammatory and hypo-inflammatory classes separately, difference in the lung metabolites between clp and lps were found. under splenectomybased conditions, there were differential metabolites between the clp and lps groups; the organic acids ( / , . %) and tca cycle inter mediates ( / , . %) were the two largest categories ( figure d ). under no-splenectomy-based conditions, there were only differential metabolites between the clp and lps groups and the organic acids ( / , . %) formed the largest category ( figure e ). while identifying the linked metabolic pathways, the anova test was used to extract significant pathways from them. for the clp and clp-splen groups, the kegg alignment revealed pathways that were linked to the above-detected metabolites. amino acid metabolism ( / , . %), chemical structure transformation maps ( / , . %), biosynthesis of other secondary metabolites ( / , . %), metabolism of other amino acids ( / , . %), and carbohydrate metabolism ( / , . %) were the first five largest categories ( figure a ). for the lps and lps-splen groups, however, only pathways linked to the above-detected metabolites were revealed from kegg. the benzoic acid family, bisphenol degradation, and folate biosynthesis were revealed in the h-post-lps-intervention group. in addition, tropane, piperidine and pyridine alkaloid biosynthesis, biosynthesis of phenylpropanoids, and phenylalanine metabolism were revealed in the h-post-lpsintervention group ( figure b ). the hyper-inflammatory and hypo-inflammatory groups were this article is protected by copyright. all rights reserved. then clustered to predict the metabolic pathways separately. under splenectomy-based conditions, there were differential metabolic pathways between the clp groups and the lps groups ( figure c ). however, we cannot find differential metabolic pathways between the clp groups and the lps groups under no-splenectomy-based conditions. ards is a clinically and biologically heterogeneous disorder associated with effects such as trauma, shock, infection, and sepsis. failure of clinical therapeutic trials prompted the investigation and subsequent discovery of two distinct phenotypes of ards (hyperinflammatory and hypo-inflammatory) that have different biomarker profiles and clinical courses and respond differently to management strategies [ ] . the hyper-inflammatory subgroup (about one third of all) shows t higher mortality, higher severity of illness, and worse clinical outcomes [ ] . even in covid- , the hyper-inflammatory response is closely related to the ards of critical covid- pathogenesis [ ] . a major issue is that ards is such a heterogeneous, multi-factorial, end-stage condition that the strategies for "lumping and splitting" are critical [ ] . metabolic phenotypes, representing different pathways important in the pathophysiology of ards, can be used to identify the subgroups [ ] . they can also help distinguish the subphenotypes of ards (hypo-inflammatory and hyper-inflammatory) and identify the risk of developing ards, diagnosis, risk stratification and monitoring. the use of metabolomics as a possible diagnostic tool for ards has been investigated in several studies, including exhaled breath and oedema fluid analyses. we previously found that phenylalanine, aspartic acid, and carbamic acid levels were significantly different in the plasma samples of ards patients [ ] . four metabolites (ornithine, caprylic acid, azetidine, and iminodiacetic acid) could serve as metabolic phenotypes to potentially predict the severity of ards [ ] . due to the limitations of the research conditions, it is difficult to distinguish the subtypes of inflammation solely by metabolomics. the spleen performs vital hematological and immunological functions. removal of the spleen had already been established as a routine technique to treat splenic trauma and other diseases affecting the spleen [ ] . however, splenectomized (asplenic) or hyposplenic individuals have an increased risk of infections [ ] , and this can lead to severe sepsis known as overwhelming post-splenectomy infection (opsi), which has a very high mortality rate [ ] . a previous study showed that a higher charlson comorbidity index score was significantly associated with severe sepsis/septic shock post-splenectomy [ ] . moreover, splenectomy can this article is protected by copyright. all rights reserved. alter the serum cytokine profile, exacerbating the systematic inflammatory responses and injury to multiple organs [ ] . the spleen is necessary for the recruitment of classical monocytes and neutrophil extravasation into the injured lungs [ ] , and it can play an important role in intestinal ischemia-reperfusion (iir)-induced ali [ ] . furthermore, the spleen coordinates interleukin(il)- -dependent il- production, which reduces lung injury during experimental acute kidney injury(aki) [ ] . splenic factors also exacerbate sap-associated lung injury [ ] . in animal experiments, the splenectomy model can be used as an auxiliary method to distinguish high and low inflammatory subtypes. therefore, an ali animal model of host inflammation differentiation can be established after carrying out a splenectomy. the splenectomy model, which could demonstrate the significant involvement of autoimmunity, plays an important role in the experiment. however, it has a higher mortality rate under experimental conditions, and this mortality rate is significantly increased when combined with the clp model [ ] . in our study, this mortality rate was very high after conducting the splenectomy followed by clp and the passage of time (from to h). in order to ensure the homogeneity of experimental mice in metabolic analysis, the same batch of mice was used to establish models synchronously, such that they would have an effective cluster effect. it was difficult to achieve a greater number of animals at each time point and in each group because of the extremely high mortality rate, and because of the existing limitations of these special animal models. ideally, more mice (over ) in each group would be beneficial for repetitive data collection. we found that the clp-splen and lps-splen groups had more severe lung damage than the corresponding non-splenectomy ali group ( figure a) . moreover, the lps-splen and the clp-splen groups showed significant neutrophil exudation in the lungs after splenectomy; however, the changes in the clp-splen group were more significant ( figure d ). possible causes may be that splenectomy did not affect neutrophil extravasation in the lps models of lung injury, as was confirmed by rieg et al [ ] , or because of tnf-α-induced adhesion of monocytes to endothelial cells and leukocyte transmigration in ali. we found that the expression in lps-splen or clp-splen group was higher than in the corresponding ali group, and that the levels of tnf-α in the clp-splen group were significantly elevated, compared with the lps-splen group, p< . (figures b and c) . cd receptor [ ] .the sensitizing effect of lps stimulation aggravates lung damage [ ] . lbp may play an important role in augmenting tnf-α expression by alveolar macrophages in the lung [ ] : the duration of clp action is prolonged, the stimulatory effect persists, and tnfα expression is higher than with lps-intratracheal injection (figures b and c) . on the other hand, the cholinergic anti-inflammatory pathway is completely inhibited following splenectomy [ , , ] . although, splenectomy itself was not associated with increased serum il- or lung injury, the absence of a counter anti-inflammatory response by splenic il- production results in a high proinflammatory response and lung injury [ ] . based on the difference of inflammation (after splenectomy) in ali, the metabolomic differences of lung tissues could be identified. pca and opls-da were used for discriminant analysis, and univariate statistical analysis was used to screen important differential metabolites in untreated(hypo-inflammatory) or splenectomy-treated(hyper-inflammatory) ali mice, which had been subjected to different modeling methods (lps or clp). a total of compounds and pathways were found in lungs, differing between hyper-and hypo-inflammatory in clp groups ( figures a and a) . however, only compounds and pathways were found to differ in the lungs between hyperinflammatory and hypo-inflammatory in lps (figures c and b ). this suggests that nonpulmonary ards (such as clp) has more active lung metabolomics changes that are involved in inciting the differences of host inflammatory response. moreover, continuous stimulation of clp promoted inflammation and injury to the lungs. organic acids form the largest group of differential metabolites in clp and lps under differential inflammatory conditions, and can be attributed to defects in the intermediary metabolic pathways of carbohydrates, amino acids, and fatty acid oxidation. related physiological analysis had found that sepsis experienced a highly catabolic status. many proteins decompose into amino acids to supply energy, which seemed to be relevant to poor prognosis [ , ] . thus, the concentration of amino acids and its derivatives ( / , . %) demonstrated a notable upward tendency in the clp group ( figure a ). among the metabolic pathways, this category, including amino acid metabolism ( / , . %) and metabolism of other amino acid ( / , . %), also dominates ( figure a ). interestingly, rf analysis of lung-targeted metabolomics data showed that the metabolic biomarker group with products was a strong predictor of the hyper-inflammatory subgroup in clp-induced ali ( figure b ). -hpa, as one of the major metabolites in polyphenols, is a necessary adaptive response of microbiota to the stress-induced changes in inflammation [ ] . -hpa could be a biomarker for quantifying leukocyte-mediated damage [ ] , and it has been confirmed to participate in the intermediate step of tyrosine degradation [ ] . acpc is a this article is protected by copyright. all rights reserved. nonmetabolized amino acids. amino acid transport by system "a" is sodium-dependent and results in a high intracellular-to-extracellular gradient [ ] . apac was also shown to have a high affinity for the "a" transport system of endothelial cell membranes [ ] . sepsis specifically decreases cell membrane potential and inhibits the amino acid transport system a [ ] . this is probably because of the reduction in the pulmonary absorption of amino acids in ards. hypoxia cause an imbalance of the nadph/nadp+ and nadh/nad+ ratios, accompanied by the accumulation of intermediates of the tca cycle, such as cis-aconitic acid, as was found by rf analysis. ane may be a substance derived from environmental factors. exposure to fuels and heavy metabolites ( -tridecanone, -tridecanol, and -tetradecanol) was observed only in the lung tissues, possibly indicating that metabolism occurred in the lungs [ ] . however, the aliphatic compound n-tridecane showed no cytotoxic effects on chemoattractant protein- (mcp- ) and il- production [ ] . we therefore believe that tridecane cannot be a member of metabolic biomarker group. low-molecular-weight phenolic acids (phas) are the products of the degradation of aromatic amino acids and polyphenols by the intestinal microflora [ ] , and all phas have an impact on mitochondria and neutrophils. low-molecular weight phas of microbial origin participate in the regulation of the ros production in both the circulation and tissues, thereby affecting the level of oxidative stress [ ] . therefore, there may be a group of metabolic biomarkers related to inflammation, hypoxia, infection, etc. between the hyperinflammatory and hypo-inflammatory subgroups of ards. the etiology has also become a variable. when we clustered the hyper-inflammatory and hypo-inflammatory separately, there were differential metabolites and differential metabolic pathway between the clpgroups and the lps-group in hyper-inflammatory subgroup ( figures d and c ). in the hypoinflammatory subgroup, there were only differential metabolites between the clp and the lps groups ( figure e ), although no different metabolic pathways could be found in this study. overall, the hyper-inflammatory subgroups of ards were observed to exert more abundant metabolism changes in the lung. metabolomics has the potential to improve our understanding of ards biology. from the analysis of lung metabolomics, the difference of host inflammatory response is a key link in determining the severity of ards. hyper-inflammatory subgroups of ards have a heavier inflammatory response and a more active lung metabolism. combined with host inflammation this article is protected by copyright. all rights reserved. acute respiratory distress syndrome: the berlin definition acute respiratory distress in adults acute respiratory distress syndrome current incidence and outcome of the acute respiratory distress syndrome biomarkers for acute respiratory distress syndrome and prospects for personalised medicine toward smarter lumping and this article is protected by copyright smarter splitting: rethinking strategies for sepsis and acute respiratory distress syndrome clinical trial design acute respiratory distress syndrome (ards) phenotyping coronavirus disease (covid- ): cytokine storms, hyper-inflammatory phenotypes, and acute respiratory distress syndrome genes dis. . epub ahead of print heterogeneous phenotypes of acute respiratory distress syndrome after major trauma endocan levels in peripheral blood predict outcomes of acute respiratory distress syndrome il- a and tnf-α as potential biomarkers for acute respiratory distress syndrome and mortality in patients with obesity and covid- regulation of inflammatory response in human osteoarthritic chondrocytes by novel herbal small molecules host-response subphenotypes offer prognostic enrichment in patients with or at risk for acute respiratory distress syndrome 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heparin and splenectomy on survival and plasma fibronectin levels in rat peritonitis lipopolysaccharide binding protein enhances the responsiveness of alveolar macrophages to bacterial lipopolysaccharide. implications for cytokine production in normal and injured lungs the effect of splenectomy on endotoxin-induced acute lung injury and its potential mechanism in rats. the chinese journal of trauma splenectomy inactivates the cholinergic antiinflammatory pathway during lethal endotoxemia and polymicrobial sepsis the spleen: the forgotten organ in acute kidney injury of critical illness splenectomy exacerbates lung injury after ischemic acute kidney injury in mice reprogramming of basic metabolic pathways in microbial sepsis: therapeutic targets at last? metabolomic analysis of the effects of adipose-derived mesenchymal stem cell treatment on rats with sepsis-induced acute lung injury gut-brain axis: how the microbiome influences anxiety and depression the metabolism and de-bromination of bromotyrosine in vivo association of the tyrosine/nitrotyrosine pathway with death or icu admission within days for patients with community acquired pneumonia aminocyclopentane carboxylic acid and alpha-aminoisobutyric acid: comparison to fluorodeoxyglucose and diethylenetriaminepentaacetic acid in morphologically defined tumor regions effect of sepsis on amino acid transport system a and its response to insulin in incubated rat skeletal muscle metabolites from inhalation of aerosolized s- synthetic jet fuel in rats il- from lung epithelial cells exposed to volatile organic compounds toxic effects of microbial phenolic acids on the functions of mitochondria effect of phenolic acids of microbial origin on production of reactive oxygen species in mitochondria and neutrophils we are very grateful to the laboratory of lipid & glucose metabolism at the first affiliated hospital of chongqing medical university to provide laboratory facilities. the authors declare no conflict of interest. this article is protected by copyright. all rights reserved. lungs from each experimental group were processed for histological examination after h&e staining. lps-and clp-induced mice exhibited obvious lung injury, p < . . lung injury scores were estimated by the method of mikawa, based on the following four indicators of lung injury score: alveolar congestion; bleeding; gap or vascular wall neutrophil infiltration or aggregation; alveolar septal thickening or transparent membrane formation. these were scored as marks: no or very slight damage, mark: mild injury, marks: moderate injury, marks: severe injury, marks: very severe damage. the number of lesions of the total score is the pathological score of the ali. the lung injury of clp-splen group is more serious than that of the lps-splen group, p < . .(b)(c) tnf-α in serum or lung of mice was detected by elisa or pcr. tnf-α was significantly elevated in the serum and lung tissue after lps or clp, compared with controls, p < . . expression of tnf-α in the lps-splen group or clp-splen group, was higher than in the corresponding ali (lps or clp) group. the levels of tnf-α in the clp-splen group were elevated significantly, compared with the lps-splen group, p< . .(d) infiltration of neutrophils was confirmed with neutrophil numbers in balf. after splenectomy, the lps(splen) group and the clp(splen) group have significant neutrophil exudation in the lung. ****p < . ,*p < . , by two-way anova followed by a lsd multiple comparisons test. each group n = , experiments are repeatable and most representative one was shown.this article is protected by copyright. all rights reserved. key: cord- -qmbo dmo authors: kesherwani, rashmi; kumar, raushan; minhas, ujla; rizvi, syed ibrahim title: euglena tuba extract provides protection against lipopolysaccharide-induced inflammatory response and oxidative stress in mice date: - - journal: biologia (bratisl) doi: . /s - - - sha: doc_id: cord_uid: qmbo dmo lipopolysaccharide (lps), an endotoxin, is known to induce inflammatory response and oxidative stress in rodents. we evaluated the protective role of euglena tuba extract (etme) against lps induced inflammatory response and oxidative stress in male balb/c mice. male balb/c mice were divided into groups. group (control) were intraperitoneally administered . ml pbs. group , and were treated with a single dose of lps (i.p. mg/kg body weight). prior h, group and received orally mg/kg body weight and mg/kg body weight etme respectively. biomarkers of oxidative stress including tbars, sod, catalase, liver marker enzyme (sgpt and sgot), nitric oxide, and inflammatory cytokines including il- and tnf-α, were estimated in serum. oxidative stress and inflammatory markers were significantly increased in the lps treated group, whereas etme treated group at different concentrations protected mice from pro inflammatory cytokines and oxidative stress. our results indicate that % methanolic extract of euglena tuba can efficiently counteract free radical generation and increased level of inflammatory cytokine in an lps induced mice model. euglena tuba is an alga that is predominantly found in freshwater ponds, puddles, lakes, and river banks, forming seasonal algal bloom prominently at warm temperatures during conditions of lower dissolved oxygen and acidic environment. blooms fully cover the pond forming a deep red to greenish coloured continuous layer on the surface of the water (deb ) . various bioactive compounds like phenolics, flavonoids, alkaloids, tannins, terpenoids, saponins, carbohydrates, and ascorbic acid are present in euglena tuba which provide many documented medicinal properties . studies have shown that euglena tuba has antioxidant, hepatoprotective, iron-chelator, antitumor activities and can induce apoptosis through ros-mediated mapk regulation (panja et al. , . lipopolysaccharide (lps) , an endotoxin, is the key component of the outer membrane of gram-negative bacteria. the administration of lps to mice is a widely used strategy to induce an acute systemic inflammatory response. incorporation of lps to the body activates an intracellular signaling pathway and causes the production of inflammatory cytokines which are responsible for activating innate immune response by regulating inflammatory mediators such as tnf-α, il- , and no (buras et al. ; schulte et al. ) . in response to these cytokines, reactive oxygen species are generated from neutrophils and other phagocytic cells which triggers oxidative stress (sugino et al. ) . uncontrolled inflammation and excessive oxidative stress may be the leading symptoms of sepsis culminating in multiple organ failure and death (steven et al. ) . the symptoms of covid also show an abnormally high inflammatory response (zhang et al. ) . based on already available reports that euglena tuba has excellent anti-inflammatory and antioxidant property, we focus our study to investigate the ameliorative antiinflammatory and antioxidant effect of % methanolic extract of euglena tuba (etme) on lps induced inflammatory response and resultant severe oxidative stress in mice. the algal sample was collected in the month of october from a pond in district kangra of the state of himachal pradesh, india, situated at ° ′ to ° ´n, ° ′ to ° ′e and ° ′- ° ′ east longitudes and ° ′- ° ′ north latitudes. samples were preserved in % formalin for identification and observed under the light microscope. morphological features were taken in account and characterized (kumar et al. ). the collected samples were thoroughly cleaned under the sterile condition with distilled water - times to remove dirt and then centrifuged at x g to wash out contaminating bacteria. the collected pellet of the biomass of euglena tuba was dried in sunlight for seven days and then finely powdered. in ml solvent (methanol: water : ) the powder ( g) was soaked and stirred for h, then centrifuged at g. the process was repeated again with the obtained pellet by using a ml fresh solvent. the total obtained supernatant was concentrated under reduced pressure in a rotary evaporator. the concentrated solution was lyophilized and the % methanolic extract of euglena tuba (etme) was obtained which was kept at − °c for future use. animals and experimental design - weeks old male balb/c mice were purchased from the indian institute of toxicological research, lucknow, india. the animals were grouped and housed in polyacrylic cages with animals per cage and maintained under standard laboratory conditions. animals had free access to standard diet purchased from paramount techno company and water ad libitum. animals were randomly divided into four groups, each comprising six animals. group the controls were intraperitoneally administered . ml pbs. group , and were treated with a single dose of lps (o : b e.coli purchased from sigma aldrich, mumbai) (i.p. mg/kg body weight) (lee et al. ) . prior h group and received orally mg/kg body weight and mg/kg body weight etme respectively. blood samples were drawn h after lps treatment from orbital sinus and plasma was separated. the survival of the mice was monitored for days and sacrificed by cervical dislocation. all experiments using mice were done in accordance with the guidelines of the institutional ethical committee ( /go/re/ /cpcsea). nitric oxide levels was measured in the plasma by griess reaction (yamamoto et al. ). the samples were incubated with griess reagent ( . % naphthalene diamine hcl; % sulfanilamide in % phosphoric acid mixed as : ) and the pinkcolored product thus formed was measured at nm on a spectrophotometer. production of nitric oxide was calculated by comparing standard sodium nitrite conc. and the results are expressed as μmol/l no. total protein content was estimated in plasma samples by using the method of lowry et al. ( ) . cytokine levels were estimated following the directions given as per the manufacturer's manual (krishgen bio framework, india) as described previously in detail (kumar et al. ). the result is reported in pg/ ml. malondialdehyde (mda) in plasma was determined by reaction with thiobarbituric acid (tba) (buege and aust ) . briefly; . ml tris -hcl buffer, . ml ferrous sulphate and . ml ascorbic acid. . ml sample was added and the volume was made up to . ml with ddw. . ml tca and ml tba were added after incubation at °c for min. tubes were plugged and incubated for min. in a boiling water bath. after incubation, tubes were centrifuged at rpm for min. and the supernatant was read at nm. the concentration of mda was calculated using the extinction coefficient of . × m − cm − . results are expressed as nmol mda/mg protein. the activity of the enzyme superoxide dismutase was determined according to the method of kono ( ) . auto oxidation of hydroxylamine hcl generates superoxide anions. these anions bring about the reduction of nbt to blue formazone. sod inhibits the reduction of nbt induced by hydroxylamine hcl. the activity of the enzyme was expressed as units/mg protein, where one unit of enzyme is defined as the amount of enzyme inhibiting the rate of reaction by %. catalase activity was assayed by the method of luck ( ) . catalase is an enzyme that catalyzes the decomposition of hydrogen peroxide to oxygen. the rate of decomposition of hydrogen peroxide is assessed spectrophotometrically at nm. the activity of the enzyme is expressed as μmoles of hydrogen peroxide decomposed per min per mg protein, using the molar extinction coefficient of hydrogen peroxide ( m − cm- ). determination of serum sgpt, and sgot level was performed using reagent kits from span diagnostic and erba diagnostics and measurements were made on an erba mannheim chem.- analyzer. all data are expressed as mean ± sd of six measurements and statistical analysis was performed using graphpad prism . software. data were analyzed by one-way anova. a probability of p < . was considered significant. figure shows levels of no in plasma of all groups. the no levels were found to be significantly (p < . ) higher in the lps group with respect to control group while significant decreases (p < . ) were seen in the etme group as compared to the lps group. cytokine level (il- and tnf -α) in the serum of mice cytokine levels are represented in fig. a and b . the activity of both cytokines is significantly (p < . ) increased in lps treated groups of mice with respect to control. a significant reduction is seen in both etme groups of mice when compared with lps group. lipid peroxidation is one of the important oxidative stress markers measured in the form of mda and value is reported in the form of nmol/mg protein. according to variously reported studies lipid peroxidation increases with oxidative stress. our result also confirms increase in the level of lipid peroxidation in the lps group as compared to control whereas both etme groups show significant (p < . ) decrease in the mda level with respect to lps group fig. a . superoxide dismutase figure b , shows the sod activity. significantly increased (p < . ) value of sod are found in the lps group as compared to control, whereas both etme groups show significant (p < . ) decrease in the sod level with respect to lps group. catalase is one of the important antioxidant biomarkers markers. figure c , represents the level of catalase activity. in the lps group of mice, there was (p < . ) significantly decrease in the level of enzyme activity with respect to the control group of mice while significantly increased (p < . ) in the enzyme activity are found in & etme groups when compared to the lps group. etme supplementation provides a better result with respect to etme. sgot and sgpt level are shown in fig. a and b . the activity of both markers is significantly (p < . ) increased in lps treated groups of mice with respect to control. a significant reduction is seen in both etme groups of mice when compared with lps group. effect of different doses of etme on the level of no in lps induced oxidative stress. * p < . significant changes occurs between etme to the lps group, @ represent significant p < . change occurs between etme compared to the lps group microalgae are the best antioxidants used in different pathologies related to the generation of free radicals associated with several diseases (sathasivam et al. ) . antioxidant properties of frequently consumed food, vegetables, and other herbs have been confirmed as a good source of potent antioxidant due to their phytochemicals (proteggente et al. ) . polyphenols and flavonoids are strong antioxidants that can act as reducing agent (karaman et al. ) , free radical scavengers (kähkönen et al. ), inhibitors of lipid peroxidation (williams et al. ) and thereby preventing oxidative damage (ross and kasum ) . hplc analysis of euglena tuba extract corroborate the presence of bioactive compounds including tannic acid,reserpine, methyl gallate, catechin, ascorbic acid and rutin, the presence of these compounds validate the strong antioxidant property of etme . moreover gc-ms analysis of etme also showed the presence of compounds such as isovanillin, -hydroxy- methoxy benzaldehyde, α-d-glucopyranoside, methyl- , -dihydroxy- -methyl benzoate, methyl palmitate, , octadecadienoic acid, and viminalol which may contribute to antioxidant, antiperoxidative, anti-inflammatory and anticancer activities ). overproduction of free radicals increases the level of oxidants which is the underlying mechanism of oxidative damage. excessive oxidative stress is a distinguishing aspect of lps. therapies targeting redox abnormalities could be useful for improving the management of sepsis. studies have revealed that prolonged vascular inflammation and oxidative stress in response to infection or endotoxin (lps) induction is a distinct trait related to sepsis (steven et al. ). the fig. a effect of different doses of etme on the level of mda in lps induced oxidative stress. * p < . compared to the control group, # p < . significant changes occurs between etme to the lps group, @ represent significant p < . change occurs between etme compared to the lps group. b effect of different doses of etme on the sod enzyme in lps induced oxidative stress. * p < . compared to the control group, # p < . significant changes occurs between etme to the lps group, @ represent significant p < . change occurs between etme compared to the lps group. c effect of different doses of etme on the catalase enzyme in lps induced oxidative stress. * p < . significant changes occurs between etme to the lps group, @ represent significant p < . change occurs between etme compared to the lps group il- (pg/ml) fig. a and b represents the cytokine level in mice serum.*, # and @ represent the significant increase (p < . ) in the value of cytokines with respect to their control lps and different concentration of etme supplementation antioxidant defense system of the body involves a range of antioxidants and enzymes such as sod, cat, gst, and gsh. our results provide evidence that the administration of lps alters the level of antioxidant enzymes and that oral administration of the euglena tuba extract can provide protection against oxidative stress. etme shows in vitro hydroxyl radical scavenging activity that can eliminate hydroxyl radical and significantly decrease the level of lipid peroxidation, suggesting that may etme function as a good antioxidant that can effectively reduce the cellular toxicity of lps . a high level of sod and low level of catalase is an adaptive response to increased oxidative damage created due to the administration of endotoxin lps (portolés et al. ) . our result suggests that etme shows superoxide radical scavenging activity that might be due to the presence of flavonoids which effectively scavenge superoxide anions (sunil et al. ) . however, the level of catalase significantly increases by the action of phenolics linked to enzymatic and nonenzymatic reactions of antioxidants (nirmal et al. ) . the development of reactive oxygen and nitrogen species is an important part of the innate immune response. high level of no is generated by i-nos after induction by any pathogen, cytokines, growth factors and endotoxin which plays a significant role in the pathology of several diseases such as arthritis, diabetes, atherosclerosis, and sepsis (nathan ; nussler and billiar ) . the overproduction of no is the characteristic feature of endotoxin. lps induction enhances the expression of i-nos that consequently activates the nuclear factor (nf-κb) and increases the production of no (virdis et al. ) . however, etme reduces the toxicity of no due to the presence of anti-inflammatory compounds which might inhibit nitrite formation by directly competing with oxygen in the reaction with nitric oxide . the liver is the main organ of oxidative and detoxifying action. during stress conditions increase in the amount of ros and inflammation plays an important role in altering the liver function markers sgot and sgpt. our results show increase in the level of both marker enzymes in the lps treated group of mice whereas the different concentration of etme reversed this effect, higher conc. of etme provides better results, so our finding support that the etme supplement reduced ros and inflammation and also work as a hepatoprotective compound. our findings assume great significance in present times when science is trying to find ways to limit the mortality caused by covid . the major cause of multiple organ failure in patients of covid is due to the production of very high levels of pro-inflammatory cytokines which is referred to as 'cytokine storm' (sallard et al. ; stebbing et al. ) . our results show that etme may reduce the level of lps induced cytokines thus providing an intervention strategy for the management of covid . our finding demonstrates that % methanolic extract of euglena tuba can efficiently counteract free radical generation and increased level of pro-inflammatory cytokine in an lps induced mice model. the outcome of this study strongly suggests that the extract of euglena tuba can be used as a therapeutic agent to reduce the risk of systemic inflammation and oxidative stress in chronic diseases. the results may also provide a line of defense for covid . acknowledgments we would like to thank dr. rakesh kumar for collection of euglena samples. financial support this work was supported by a research grant from serb-dst, govt. of india (emr/ / ). data availability the information that helps the finding of this study is accessible from the corresponding author upon reasonable request. ethics approval all experiments using mice were done in accordance with the guidelines of the institutional ethical committee ( /go/re/ /cpcsea). the authors of this manuscript have no conflict of interest. fig. a and b the serum sgpt and sgot are represented are in fig. .*, # and @ represent the significant increase (p < . ) between control, lps and different concentration of etme supplementation microsomal lipid peroxidation animal models of sepsis: setting the stage assessment of the phytochemical constituents and antioxidant activity of a bloom forming microalgae euglena tuba morphology and biochemical study of a microalga euglena tuba reported from the aquatic ecosystem of cachar antioxidant activity of plant extracts containing phenolic compounds comparison of total antioxidant capacity and phenolic composition of some apple juices with combined hplc-cuprac assay generation of superoxide radical during autoxidation of hydroxylamine and an assay for superoxide dismutase quantitative analysis and first report of euglena tuba from himachal pradesh hesperidin attenuates altered redox homeostasis in an experimental hyperlipidaemic model of rat the aerial part of taraxacum coreanum extract has an antiinflammatory effect on peritoneal macrophages in vitro and increases survival in a mouse model of septic shock protein measurement with the folin phenol reagent catalase nitric oxide as a secretory product of mammalian cells evaluation of behavioural and antioxidant activity of cytisus scoparius link in rats exposed to chronic unpredictable mild stress inflammation, immunoregulation, and inducible nitric oxide synthase phytochemical profile of a microalgae euglena tuba and its hepatoprotective effect against iron-induced liver damage in swiss albino mice a microalga, euglena tuba induces apoptosis and suppresses metastasis in human lung and breast carcinoma cells through ros-mediated regulation of mapks hepatic response to the oxidative stress induced by e. coli endotoxin: glutathione as an index of the acute phase during the endotoxic shock the antioxidant activity of regularly consumed fruit and vegetables reflects their phenolic and vitamin c composition dietary flavonoids: bioavailability, metabolic effects, and safety type interferons as a potential treatment against covid- microalgae metabolites: a rich source for food and medicine cytokines in sepsis: potent immunoregulators and potential therapeutic targets-an updated view covid- : combining antiviral and antiinflammatory treatments time response of oxidative/nitrosative stress and inflammation in lps-induced endotoxaemia-a comparative study of mice and rats the role of lipid peroxidation in endotoxin-induced hepatic damage and the protective effect of antioxidants total oligomeric flavonoids of cyperus rotundus ameliorates neurological deficits, excitotoxicity and behavioral alterations induced by cerebral ischemic-reperfusion injury in rats cyclooxygenase- inhibition improves vascular endothelial dysfunction in a rat model of endotoxic shock: role of inducible nitricoxide synthase and oxidative stress flavonoids: antioxidants or signalling molecules? increased nitric oxide (no) production by antigen-presenting dendritic cells is responsible for low allogeneic mixed leucocyte reaction (mlr) in primary biliary cirrhosis (pbc) covid- : melatonin as a potential adjuvant treatment publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord- -xytmq f authors: wyganowska-swiatkowska, marzena; nohawica, michal; grocholewicz, katarzyna; nowak, gerard title: influence of herbal medicines on hmgb release, sars-cov- viral attachment, acute respiratory failure, and sepsis. a literature review date: - - journal: int j mol sci doi: . /ijms sha: doc_id: cord_uid: xytmq f by attaching to the angiotensin converting enzyme (ace ) protein on lung and intestinal cells, sudden acute respiratory syndrome (sars-cov- ) can cause respiratory and homeostatic difficulties leading to sepsis. the progression from acute respiratory failure to sepsis has been correlated with the release of high-mobility group box protein (hmgb ). lack of effective conventional treatment of this septic state has spiked an interest in alternative medicine. this review of herbal extracts has identified multiple candidates which can target the release of hmgb and potentially reduce mortality by preventing progression from respiratory distress to sepsis. some of the identified mixtures have also been shown to interfere with viral attachment. due to the wide variability in chemical superstructure of the components of assorted herbal extracts, common motifs have been identified. looking at the most active compounds in each extract it becomes evident that as a group, phenolic compounds have a broad enzyme inhibiting function. they have been shown to act against the priming of sars-cov- attachment proteins by host and viral enzymes, and the release of hmgb by host immune cells. an argument for the value in a nonspecific inhibitory action has been drawn. hopefully these findings can drive future drug development and clinical procedures. viruses are one of the oldest organisms on earth. they consist simply of a protein envelope and nucleic acids which renders them unable to replicate outside of a host [ ] . nevertheless, they are extremely adaptable and can rapidly alter their structure to suit the environment. while dna viral copies are near exact, rna viruses are more often dissimilar. genetically compatible viruses can undergo recombination and create very difficult-to-treat infections [ ] . by exchanging information, they can spread immunity against medical treatments, or the host immune system [ ] . orthomyxoviridae, an example of a family of rna viruses which cause a disease commonly known as influenza or flu, can both rearrange compatible genes and mutate on a regular basis in order to remain invisible [ ] . that is why a new flu vaccine is needed every year [ ] . due to quickly developing resistance to antiviral pharmaceuticals, vaccination is currently the most effective protection against viruses. however, there is building evidence that herbal medicines, sars-cov- entry has been successfully inhibited in vitro by direct ace inhibition [ ] . targeting ace would allow for specific intervention in the binding of sars-cov- [ ] . direct inhibition of ace functioning could however have poor long-term effects on the body due to its importance in controlling blood pressure, as well as lung, kidney and cardiovascular function [ ] [ ] [ ] . pharmacological ace inhibitors such as lisinopril are not effective in reducing ace expression and at only % homology between the two enzymes, which can be inferred from their opposing physiological function, ace inhibitors are not competitive enough at the ace- attachment site with the sars-cov- to prevent infection [ ] . epidemiological studies assessing chronic conditions such as cardiovascular disease have found an association between dietary habits and prevalence [ , ] . phenolic compounds from green tea, coffee and wine were identified as of specific preventative interest [ ] [ ] [ ] . all phenol extracts from these foods were found to have a concentration dependent effect on inhibiting ace activity; however, no data on ace- was present [ ] . polyphenols, including green tea phenolic compounds, have a reputation for enzymatic inhibition, and have been reported to inhibit sugar digesting enzymes as well as lipases and proteases [ ] [ ] [ ] . this has been noted well in the human digestive tract, which poses a barrier to their further phenolic absorption, but benefits from enzymatic inhibition in various, notably low-sugar, diets [ ] . this can be attributed to phenolic compound dampening saccharolytic enzyme, e.g., invertase activity by % [ ] . crystal structure modeling of both ace- and sars-cov- s protein has identified flavonoids, a subgroup of phenolic compounds, as suitable candidates for binding and inhibiting both molecules in silico [ ] . assessment has started on these compounds in vitro, and there are successes showing sars-cov- inhibition with phenolic compounds also effective against sars-cov [ , ] . studies show that in order to complete the endocytic entry in to the cell, the sars-cov- virus needs further priming or cleavage of its s protein. this has been reported to be viable by tmprss [ , ] or cathepsin l [ ] proteinases, with further studies supporting that their dual inhibition completely abolishes the coronavirus family mers infectivity [ ] . a broad spectrum, nontoxic protease inhibitor would be able to perform such a function. further analysis of phenolic compounds could give more pharmacological guidance in this matter. while long term proteinase and ace- inhibition could be detrimental to cellular function and bodily homeostasis, targeted treatment partially reducing the effectiveness of coronavirus s protein attachment to the ace or to the priming proteinase could have the potential to drop the sars-cov- viral load before a state of septic shock is reached at the peak of infection. hmgb might be a critical molecule to allow innate immune cells to respond to both infection and injury, and is necessary for mammalian survival as shown by a knockout murine model which displays lethal hypoglycemia [ ] . serum hmgb levels were consistently found to be significantly higher in septic patients who did not survive than those who did [ , , ] . intratracheal administration of hmgb has been shown to induce lung neutrophil infiltration, local production of proinflammatory cytokines (e.g., il- , and tnf), and acute lung injury [ ] . traditional medicine and hospitalization cannot offer much more against sars-cov- than symptomatic relief such as the use of oxygen. some hmgb inhibitors like anti-ifn-γ antibodies, intravenous immunoglobulin, and minocycline seem to be helpful [ ] . anti-hmgb antibodies have also been shown to dose-dependently protect mice against lethal endotoxemia [ ] , as well as endotoxin-induced acute lung injury [ , ] while normally corticosteroids reduce inflammation, they are helpless at fighting a hmgb induced cytokine storm [ ] . moreover, intravenous liquids containing nutrient solution and glucose may have serious side effects, such as significantly increased viral load or diabetic ketoacidosis [ ] . on the other hand, insulin has a benefit of lowering hmgb levels [ ] . interestingly, both antithrombin iii and thrombomodulin decrease hmgb in vitro [ ] . a search of hmgb inhibitors shows a number of results, many of which are herbs and herbal constituents which have direct suppressive actions against this proteins activity. counter intuitively, nicotine also significantly suppresses hmgb in the lungs [ ] . the low molecular weight fraction of an aqueous extract of the chinese herb angelica sinensis (dang gui) is protective against lethal experimental sepsis and endotoxemia in a dose dependent manner. during laboratory induced lethal endotoxemia, % of mice which had the extract administered daily survived, vs. % survival in the control group, and during laboratory induced lethal sepsis % of mice survived when the extract was given daily, vs. % control. in the sepsis model, the administration of the extract did not begin until h after the onset of sepsis, at which point some of the test subjects had already succumbed. this effect was in vitro shown to be non-cytotoxic to macrophages at any of the tested concentrations [ ] . hmgb secretion was attenuated by the extract due to reduced translocation from the nucleus to the cytoplasm of activated macrophages, showing that even late administration of the dang gui extract significantly reduces serum levels of hmgb , and can be attributed to the rescue of mice in part due to the attenuation of systemic hmgb accumulation. inhibition of hmgb is therefore likely a key element in preventing septic shock induced death, as it is the only high mobility group protein with a cytokine-like activity which is important to starting and maintaining the septic response [ , ] . ferulic acid and a. sinensis polysaccharide are the main components of dang gui extract, and the most biologically active. the polysaccharide has been extracted and shown to be inhibitory to viral replication in a murine leukemia virus in vivo model, also exhibiting anti-inflammatory properties [ ] . ferulic acid, while anti-inflammatory via inhibition of nitric oxide (no) production, was excluded from a. sinensis mediated hmgb inhibition [ ] , however it does show some ability to inhibit human immunodeficiency virus (hiv) proteases [ ] . salvia miltiorrhiza (danshen) is also a natural remedy from the world of chinese medicine which has proven experimentally to interact with hmgb . traditionally used to treat cardiovascular disorders, it was shown to be protective against lethal lps-induced endotoxemia and sepsis by decreasing hmgb levels in vivo in a murine model [ ] . experiments with the danshen extract (main bioactive ingredient: danshensu) also demonstrated successful administration h after the onset of sepsis. this proves that the hmgb inhibition by the herbal extract of danshen can be preventatively inhibitory to the septic state, and also aid to arrest and reverse it. as an in vitro enterovirus treatment, ethyl acetate extracts of danshen were shown to have the strongest antiviral effect when administered along with the virus, suggesting interference in enterovirus entry mechanisms [ ] . danshen extract also possesses direct anti-viral activity as has been found to inhibit the hepatitis b reverse transcriptase [ ] . just as dang gui and danshen, epigallocatechin gallate (egcg-the main catechin found in green tea extracts) is an active natural extract able to rescue mice even if it is administered after the onset of induced sepsis. camellia sinensis is a source of a polyphenolic group of compounds called catechins. the most prominent green tea catechins are egcg, epigallocatechin, and epicatechin [ ] . these molecules are well known for their antitumor, antioxidative, and antimicrobial activities [ , ] . out of the three catechins found in green tea, only egcg was found to inhibit lps and cecal ligation and puncture (clp) induced sepsis in a dose dependent manner, with up to % greater survival rate than control mice, even if treatment was delayed up to h after the onset of sepsis. the effect was shown to coincide with egcg ( mm) almost completely eliminating hmgb release and macrophage cell surface clustering [ ] . green tea leaf (camellia sinensis folium) extract also reduces endotoxin-induced release of hmgb and is therefore proposed to possess the ability to decrease mortality from sepsis if taken regularly. complete inhibition of hmgb in vivo was seen under green tea extract doses as low as µl/ml or ml/kg, with no in vitro cytotoxicity of egcg to macrophage cultures [ ] . the mechanism through which egcg interacts and modifies the kinetics of hmgb are still elusive. reports show that egcg can bind to lipid raft associated receptors [ ] [ ] [ ] [ ] . macrophages depend on lipid raft complexes to deliver lps and signal an inflammatory state [ ] . an analysis of structural motifs of egcg could present a solution as to why it would locate to these lipid rafts, and whether its phenolic, enzyme inhibitory, activity plays a part in stopping the release of hmgb . in addition, egcg was shown to inhibit neuraminidase activity and viral genome synthesis of influenza, resulting in less effective cellular infection and viral replication. this disruption is thought to be caused by egcg structural analogy to glycosidases which occupy cellular polysaccharides. this mimicry can inhibit haemagglutinin and neuraminidase attachment to cellular membrane polysaccharide targets and stop influenza virus invasion and release respectively [ ] [ ] [ ] [ ] . haemagglutinin proteins, while not explicitly present in sars-cov- , are present in the coronavirus family, and are utilized in cellular entry by the human coronavirus hku [ ] . other mechanisms have also been studied for different viral families. hepatitis b viral (hbv) entry is dependent on na+-taurocholate cotransporting polypeptide (ntcp) expressed at the membrane of human hepatocytes. a clathrin basket endocytosis, mediated by egcg, caused a drop in ntcp surface expression and reduced hbv infectivity [ ] . herpes simplex virus (hsv) attachment was shown to be inhibited by the broad-spectrum activity of egcg which successfully blocks attachment to heparan sulfate or sialic acid [ ] , while the speed of ebola virus infection was slowed by egcg inhibition of human cell-surface heat shock protein a and therefore ebola virus attachment [ , ] ginseng, rich in ginsenoside, is another herb with potent anti-inflammatory effects. the antiseptic activity of ginsenoside rh (one of the main active constituents of the ginseng root extract) has been noted in hmgb -activated human umbilical vein endothelial cells (huvecs) and mice. ginsenoside rh increased the survival rate in a mouse sepsis model. it also significantly reduced hmgb release in lps-activated huvecs. furthermore, it suppressed the production of tnf-α, il- , activation of nf-κb and extracellular signal-regulated kinase (erk- / ) by hmgb . ginsenoside rh also inhibited hmgb -mediated hyperpermeability and leukocyte migration in mice. in addition, treatment with ginsenoside rh reduced the clp-induced release of hmgb , sepsis-related mortality and tissue injury in vivo [ ] . an extract from korean red ginseng significantly protected mice in experimental sepsis by decreasing tnf, il- , il- , and ifn-γ production via inhibition of nf-κb activation. it is likely that korean ginseng will also reduce hmgb levels, because cytokines under the control of nf-kb, such as tnf and ifn-g, induce hmgb [ ] . in fact, ginsenoside has been shown to decrease hmgb levels in a human uterine fibroid cell model [ ] . its direct anti-viral function has also been proven against influenza strain h n , which was prevented from attaching to host α - ' sialic acid receptors by ginsenoside interaction with viral haemagglutinin when administered topically to the viral infection site intranasally [ ] . the main medicinal parts of licorice are its roots and rhizomes. numerous studies have shown licorice to be antiviral against hepatitis c and hsv [ , ] , anti-inflammatory [ , ] , antioncogenic and antimicrobial [ ] . glycyrrhizic acid (ga) and glycyrrhetinic acid (gta) are the specific chemical compounds that may be isolated from the licorice plant. it has been found that depletion of sirtuin (sirt ) suppressed the number of human nasal epithelial cell cilia, and dramatically induced hmgb translocation from nucleus to cytoplasm in an epithelial tumor cell line. gta has been shown to have anti-inflammatory and anti-allergic activity: directly binding to hmgb protein extra-cellularly to inhibits its cytokine activities through a scavenger mechanism. in vitro studies using the -β-stereoisomer of gta to enhance sirt expression levels have shown inhibited translocation of hmgb protein from nucleus to cytoplasm and reversing its extracellular accumulation stimulated by lps. [ ] ga was also found to significantly attenuate lung injury and decrease the production of inflammatory factors tnf-α, il- il- β, and hmgb , the release of which was stimulated with lps treatment. ga also induces autophagy by enhancing the number of autophagosomes, possibly helping to deal with any necrotic tissue [ ] . ga can efficiently block hmgb directly, and reduce its devastating effects [ ] . astragalus mongolicus polysaccharide (aps) pretreatment has been shown to effectively inhibit hmgb -induced increased permeability in lung endothelial cells (ecs). signal transduction study has shown that aps inhibition of hmgb also affected a small guanylate rho, and its downstream effector rho kinase, in ecs, suggesting a multi layered involvement of aps [ ] . rosmarinic acid (ra) extracted from perilla frutescens was shown to potently inhibit the release of hmgb and down-regulate hmgb -dependent inflammatory responses in human endothelial cells. ra also inhibited hmgb -mediated hyperpermeability and leukocyte migration in mice. furthermore, ra reduced clp-induced hmgb release and sepsis-related mortality [ ] . ra has also proven effective in inhibiting viral replication and infection induced inflammation in a mouse model of japanese encephalitis virus mediated japanese encephalitis [ ] . the ethanol extract of prunella vulgaris herb (eepv) contains polysaccharides, flavonoids, and other phenols [ , ] . it inhibited hmgb release in lps-activated macrophages in a pi k-sensitive manner and reduced serum hmgb level and lung hmgb expression in cecal ligation and clp-induced septic mice. eepv is an inducer of heme oxygenase , which in turn reduces hmgb under lps stimulus [ ] . eepv also has some antiseptic and anti-inflammatory potential [ ] . it has been shown to exhibit anti-hiv, as well as anti-hsv (type and ) activity [ ] . the most active fraction of eepv is rich in caffeic acid, hence termed caffeic acid-rich fraction [ ] . prunella vulgaris extracts have been proven to inhibit virus/cell interactions as well as host binding in hiv infection models [ ] . aspalathin and nothofagin extracted from rooibos have been shown to effectively inhibit lps-induced release of hmgb , and suppressed hmgb -mediated septic responses, such as hyperpermeability, adhesion and migration of leukocytes, and expression of cell adhesion molecules [ ] . these molecules, as part of ethanol and alkaline extracts of the rooibos plant, have also been shown to reduce influenza a viral load at a late stage of the infection in vitro [ ] . vicenin- and scolymoside derived from honeybush can also effectively inhibit lps-induced release of hmgb , and therefore suppress hmgb -mediated septic responses such as hyperpermeability, the adhesion and migration of leukocytes, and the expression of cell adhesion molecules. in addition, vicenin- and scolymoside suppress the production of tnf-α and il- , and activation of nf-κb and erk / by hmgb [ ] . . . . lonicera caprifolium l. intravenous treatment with lonicerae flos, the main bioactive molecule of which is the chlorogenic acid, rescued lps-intoxicated c bl/ j mice under septic conditions and decreased the levels of cytokines such as tnf-α, il- β, and hmgb- in the blood [ , ] . extract of inulae radix in lps-activated raw . cells not only inhibited nf-κb luciferase activity, phosphorylation of iκbα, and inos/no, cox- /pge , hmgb release, but also significantly suppressed expression of intracellular and vascular adhesion molecules in tnf-α activated human umbilical vein endothelial cells [ ] . the main active compound in inula helenium is alantolactone, showing significant suppression of il- , tnf-α, and il- β release [ ] . rhodiola radix is a source of salidroside. the effect and mechanism of salidroside on sepsis-induced acute lung injury is mediated by the inhibition of inflammatory responses and hmgb production in bacterial lps-treated macrophages and mice. salidroside can also reverse the decreased sirt protein expression in lps-treated macrophages and mice [ ] . furthermore, salidroside was shown to alleviate the sepsis-induced lung edema, lipid peroxidation, and histopathological changes and mortality. salidroside significantly decreases the serum tnf-α, il- , no, and hmgb production, pulmonary inducible no synthase and phosphorylated nf-κb-p protein expression, and pulmonary hmgb nuclear translocation in septic mice [ ] . boeravinone x, isolated from abronia nana, has antiseptic effects. it was shown to inhibit lps-induced release of hmgb , and suppressed hmgb -mediated septic responses, such as hyperpermeability, adhesion and migration of leukocytes, and expression of cell adhesion molecules. comp also suppressed the production of tnf-α and il- , and the activation of nf-κb and erk / by hmgb [ ] . cucurbitacin e (cue), a tetracyclic triterpene isolated from cucurbitaceae plants, has proven to exert anti-inflammatory and immunologically regulatory activities. recent findings highlight that cue can ameliorate human bronchial epithelial cell insult and inflammation under lps-stimulated asthmatic conditions by blocking the hmgb -tlr -nf-κb signaling [ ] . use of the aconitum carmichaelii tuber extract, of which the majority constituent is the c -diterpenoid improved the liver function, decreased the pathological scores, and inhibited the expression of tlr , nf-κb, hmgb , and caspase- in a model rat liver injury treatment [ , ] . plumbagin prominently hampered hmgb expression and subsequently quelled inflammatory cascades, as nf-κb, tnf-α and myeloperoxidase activity. it also interrupted the reactive oxygen species-hmgb loop as evident by restored liver function [ ] . brown algae have been recognized as a food ingredient and health food supplement in japan and korea, and phlorotannins are unique marine phenol compounds produced exclusively by brown algae. phlorotannin rich extracts of the edible brown alga ecklonia cava were investigated against the hyper-inflammatory response in lps-induced septic shock mouse model. e. cava extract significantly increased the survival rate and attenuated liver and kidney damage in mice. in addition, e. cava attenuated serum levels of no, pge , and hmgb- . in macrophages, treatment with e. cava extract down-regulated inos, cox- , tnf-α, il- , and hmgb- . in addition, e. cava suppressed the nik/tak /ikk/iκb/nfκb pathway. dieckol, a major compound in the extract, reduced mortality, tissue toxicity, and serum levels of the inflammatory factors in septic mice [ ] . in terms of sars viral infectivity, dieckol has been shown to inhibit the sars-cov clpro cysteine protease, therefore inhibiting the viral ability to replicate [ ] . sars-cov clpro shares . % sequence identity with sars-cov- clpro [ ]. kiwifruit peel extract is rich in polyphenols, the main of which are procyanidins representing % w/w of the total. a kiwifruit extract inhibited the production of inflammatory molecules such as il- , il- β, tnf-α pro-inflammatory cytokines, hmgb and granzyme b serine protease by activated monocytes [ ] . study has shown that pelargonidin (pel) had effectively inhibited lps-induced release of hmgb and suppressed hmgb -mediated septic responses, such as hyperpermeability, adhesion and migration of leukocytes, and expression of cell adhesion molecules. furthermore, pel inhibited the hmgb -mediated production of tnf-α and il- , as well as nf-κb and erk / activation [ ] . luteolin was demonstrated to reduce the release of hmgb through destabilizing c-jun and suppressing hmgb -induced aggravation of inflammatory cascade through reducing akt protein levels [ ] . quercetin reduced the lung permeability changes and neutrophil and macrophage recruitment to the bronchoalveolar fluid compared to the placebo mouse model. additionally, quercetin significantly reduced cox- , hmgb , inos expression, and nf-κb p phosphorylation. these results suggest that quercetin may be a promising potential therapeutic agent against sepsis [ ] . baicalein from root of scutellaria baicalensis also significantly down-regulated the expression of matrix metalloproteinase- / and attenuated hmgb translocation from the nucleus to the cytoplasm [ ] . quercetin, luteolin, and baicalein have all been found to inhibit the sars-cov clpro protease [ , ] . quercetin has also been identified to target the same enzyme in mers-cov, as it has a flavonoid structure with carbohydrate attachments which favor localization to the s and s sites on the s protein [ ] . resveratrol has also been shown to inhibit hmgb . hmgb migrates out of the nucleus during dengue virus infection. this migration is inhibited by resveratrol treatment and is mediated by induction of sirt which leads to the retention of hmgb in the nucleus. the enhanced transcription of interferon-stimulated genes by nuclear hmgb also contributes to the antiviral activity of resveratrol against dengue virus [ ] . in murine and human serum of septic subjects hmgb persists to be secreted for a long time. peak levels in cell cultures are only reached h after stimulation [ ] . in contrast to other cytokines such as tnf with a peak at min from initial stimulus, hmgb generates further waves of inflammatory cytokine production through rage, and toll-like receptors and [ ] [ ] [ ] [ ] . hmgb and other inflammatory cytokines are persistently elevated in sepsis [ , ] . all of the identified herbal extracts and flavonoids exert suppression of hmgb activity. this is often accompanied by a drop in nf-κb activation, and reduction of tnf-α, il- , il- , and ifn-γ, signifying a reduction in the inflammatory response. multiple compounds boast anti-proteinase activity. even when the extract's anti-inflammatory effect is not direct, in multiple cases there are indications that it is due to an inhibition of a surface protease, which is either an effector of the internal inflammatory changes or is in turn necessary to cause them. the anti-proteolytic activity of herbal extracts covers an anti-inflammatory, as well as anti-viral effect across a variety of different viruses. many herbal extracts contain large groups of closely related polyphenols, making it difficult to separate them, and even more difficult to attribute the effect of the extract to just one of them. however, there is a lot of similarity in the effects of all the different extracts, just as there is similarity in the chemical structure of their main constituent molecules table . luteolin was demonstrated to reduce the release of hmgb through destabilizing c-jun and suppressing hmgb -induced aggravation of inflammatory cascade through reducing akt protein levels [ ] . quercetin reduced the lung permeability changes and neutrophil and macrophage recruitment to the bronchoalveolar fluid compared to the placebo mouse model. additionally, quercetin significantly reduced cox- , hmgb , inos expression, and nf-κb p phosphorylation. these results suggest that quercetin may be a promising potential therapeutic agent against sepsis [ ] . baicalein from root of scutellaria baicalensis also significantly down-regulated the expression of matrix metalloproteinase- / and attenuated hmgb translocation from the nucleus to the cytoplasm [ ] . quercetin, luteolin, and baicalein have all been found to inhibit the sars-cov clpro protease [ , ] . quercetin has also been identified to target the same enzyme in mers-cov, as it has a flavonoid structure with carbohydrate attachments which favor localization to the s and s sites on the s protein [ ] . resveratrol has also been shown to inhibit hmgb . hmgb migrates out of the nucleus during dengue virus infection. this migration is inhibited by resveratrol treatment and is mediated by induction of sirt which leads to the retention of hmgb in the nucleus. the enhanced transcription of interferon-stimulated genes by nuclear hmgb also contributes to the antiviral activity of resveratrol against dengue virus [ ] . in murine and human serum of septic subjects hmgb persists to be secreted for a long time. peak levels in cell cultures are only reached h after stimulation [ ] . in contrast to other cytokines such as tnf with a peak at min from initial stimulus, hmgb generates further waves of inflammatory cytokine production through rage, and toll-like receptors and [ ] [ ] [ ] [ ] . hmgb and other inflammatory cytokines are persistently elevated in sepsis [ , ] . all of the identified herbal extracts and flavonoids exert suppression of hmgb activity. this is often accompanied by a drop in nf-κb activation, and reduction of tnf-α, il- , il- , and ifn-γ, signifying a reduction in the inflammatory response. multiple compounds boast anti-proteinase activity. even when the extract's anti-inflammatory effect is not direct, in multiple cases there are indications that it is due to an inhibition of a surface protease, which is either an effector of the internal inflammatory changes or is in turn necessary to cause them. the anti-proteolytic activity of herbal extracts covers an anti-inflammatory, as well as anti-viral effect across a variety of different viruses. many herbal extracts contain large groups of closely related polyphenols, making it difficult to separate them, and even more difficult to attribute the effect of the extract to just one of them. however, there is a lot of similarity in the effects of all the different extracts, just as there is similarity in the chemical structure of their main constituent molecules table . attenuates serum levels of no, pge , and hmgb- . down-regulates macrophage levels of inos, cox- , tnf-α, il- , and hmgb- . inhibits sars-cov cl pro . reduces hmgb release. inhibits nf-κb activation thus reducing tnf, il- , il- and ifn-γ production. inhibits hemagglutinin. inhibits production of il- , il- β, tnf-α pro-inflammatory cytokines, hmgb and granzyme b serine protease by activated monocytes. glycyrrhizic acid decreases tnf-α, il- β, and hmgb production. stimulates sirt expression, which leads to hmgb nuclear retention. blocks extracellular hmgb . directly binds hmgb . inhibits nf-κb, iκbα. suppresses il- , tnf-α and il- β, icam- and vcam- release. planch ex miq. glycyrrhizic acid decreases tnf-α, il- β, and hmgb production. stimulates sirt expression, which leads to hmgb nuclear retention. blocks extracellular hmgb . directly binds hmgb . inhibits production of il- , il- β, tnf-α pro-inflammatory cytokines, hmgb and granzyme b serine protease by activated monocytes. glycyrrhizic acid decreases tnf-α, il- β, and hmgb production. stimulates sirt expression, which leads to hmgb nuclear retention. blocks extracellular hmgb . directly binds hmgb . decreases tnf-α, il- β, and hmgb production. majority of the molecules presented above belong to the polyphenol family: rotenoids, diterpenes, phenolic acids, flavonoids, phlorotannin, triterpene saponins, stilbenes and phenylpropanoids. an exception is the sesquiterpene lactone, which has an active lactone ring with an exomethylene group, and a polysaccharide composed of simple sugars with hydroxyl groups which give the molecule polarity and an anti-inflammatory activity. images reproduced from pubchem database [ ] . the pathogenesis of sepsis is attributable, at least in part, to dysregulated systemic inflammatory inhibits hmgb expression. stimulates sirt expression. decreases serum tnf-α, il- , no, hmgb levels and inos, nf-κb-p expression. majority of the molecules presented above belong to the polyphenol family: rotenoids, diterpenes, phenolic acids, flavonoids, phlorotannin, triterpene saponins, stilbenes and phenylpropanoids. an exception is the sesquiterpene lactone, which has an active lactone ring with an exomethylene group, and a polysaccharide composed of simple sugars with hydroxyl groups which give the molecule polarity and an anti-inflammatory activity. images reproduced from pubchem database [ ] . the pathogenesis of sepsis is attributable, at least in part, to dysregulated systemic inflammatory responses characterized by excessive accumulation of various proinflammatory cytokines. a ubiquitous nuclear protein hmgb is released by activated macrophages/monocytes in late stages of sars-cov- infection, and functions as a late mediator of lethal endotoxaemia and sepsis. circulating hmgb levels are elevated in a delayed fashion (after - h) in endotoxaemic and septic animals. administration of recombinant hmgb to mice recapitulates many clinical signs of sepsis, including fever, derangement of intestinal barrier function, lung injury, and lethal multiple organ failure. anti-hmgb antibodies or inhibitors (e.g., ethyl pyruvate, nicotine, stearoyl lysophosphatidylcholine and chinese herbs such as angelica sinensis) protects mice against lethal endotoxaemia, and rescues them even when the first doses are given h after onset of sepsis. most of the active compounds from the selected herbal medicines discussed here have aromatic ring structures, some studded with hydroxyl groups, almost all having pentose or hexose sugars, and occasionally nitro, sulphate or acetoxy functional groups as illustrated in table . experimental data suggests the majority have the ability to interrupt hmgb release and function, while some have also been shown to directly interrupt viral attachment and release. data regarding the exact binding moieties of herbal extracts is, however, still missing. it is unclear whether these functions are possible due to their specific chemical configuration which targets only viral or host proteins responsible for attachment, invasion, replication or release of the virus particle, or weather instead their general ability to inhibit a variety of glyosidic processes. effective enzymatic inhibition, competitive or not, could be attributed to a couple of factors. these molecules are amphiphilic allowing them to effectively embed within lipid membranes and protein rafts. common among the molecules is a multi-benzene ring steroid-like adaptation which serves as the lipophilic element, while a close resemblance of motifs to human glycolipids and glycoproteins can serve as a substrate for human and viral proteinases. this mimicry could slow down a variety of inhibits hmgb levels. antiviral, hepatitis b virus reverse transcriptase inhibition. majority of the molecules presented above belong to the polyphenol family: rotenoids, diterpenes, phenolic acids, flavonoids, phlorotannin, triterpene saponins, stilbenes and phenylpropanoids. an exception is the sesquiterpene lactone, which has an active lactone ring with an exomethylene group, and a polysaccharide composed of simple sugars with hydroxyl groups which give the molecule polarity and an anti-inflammatory activity. images reproduced from pubchem database [ ] . the pathogenesis of sepsis is attributable, at least in part, to dysregulated systemic inflammatory responses characterized by excessive accumulation of various proinflammatory cytokines. a ubiquitous nuclear protein hmgb is released by activated macrophages/monocytes in late stages of sars-cov- infection, and functions as a late mediator of lethal endotoxaemia and sepsis. circulating hmgb levels are elevated in a delayed fashion (after - h) in endotoxaemic and septic animals. administration of recombinant hmgb to mice recapitulates many clinical signs of sepsis, including fever, derangement of intestinal barrier function, lung injury, and lethal multiple organ failure. anti-hmgb antibodies or inhibitors (e.g., ethyl pyruvate, nicotine, stearoyl lysophosphatidylcholine and chinese herbs such as angelica sinensis) protects mice against lethal endotoxaemia, and rescues them even when the first doses are given h after onset of sepsis. most of the active compounds from the selected herbal medicines discussed here have aromatic ring structures, some studded with hydroxyl groups, almost all having pentose or hexose sugars, and occasionally nitro, sulphate or acetoxy functional groups as illustrated in table . experimental data suggests the majority have the ability to interrupt hmgb release and function, while some have also been shown to directly interrupt viral attachment and release. data regarding the exact binding moieties of herbal extracts is, however, still missing. it is unclear whether these functions are possible due to their specific chemical configuration which targets only viral or host proteins responsible for attachment, invasion, replication or release of the virus particle, or weather instead their general ability to inhibit a variety of glyosidic processes. effective enzymatic inhibition, competitive or not, could be attributed to a couple of factors. these molecules are amphiphilic allowing them to effectively embed within lipid membranes and protein rafts. common among the molecules is a multi-benzene ring steroid-like adaptation which serves as the lipophilic element, while a close resemblance of motifs to human glycolipids and glycoproteins can serve as a substrate for human and viral proteinases. this mimicry could slow down a variety of processes, hypothetically even inhibiting a virus which is adapted to deal with host specific glycoproteins and lipids, while ill prepared to digest plant analogues. generally, phenols are capable of disrupting multiple enzymatic processes, both human and viral. as the human body is still the most effective weapon against infection, it is paramount to give the immune system enough time to mount an attack. a diffuse inhibition of viral and cellular surface proteolytic processes could limit the speed of viral infection and cellular damage, giving the immune system some extra time to fight. experimental data established hmgb as a late mediator of lethal endotoxemia and sepsis, with a wide-over h-therapeutic window, which allows for the clinical management of lethal systemic inflammatory diseases. multiple therapeutic agents from herbal medicines have been identified as candidate compounds for the task. an analysis of combinations of extracts with complimentary functions, or a pharmacological generation of an optimum molecule, could further this field. molecular studies on protease inhibitors with human-polysaccharide or viral-polysaccharide mimicking docking motifs and difficult to digest phenolic groups could prove to be the most effective remedy against a well-established viral infection. clearer proof of direct inhibition of target proteases, molecules such as hmgb , and receptors would provide a strong basis for pharmacological development. with many animal model studies already establishing a clear link in sepsis attenuation by hmgb inhibition, it is now time to assess if these findings can translate on to human models. author contributions: each author has made substantial contributions in the conception and design of this work, or acquisition, analysis, and interpretation of the data. further, all authors have aided in drafting this manuscript, approve this submitted 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nervous system andersson, u. rage is the major receptor for the proinflammatory activity of hmgb in rodent macrophages involvement of toll-like receptors and in cellular activation by high mobility group box persistent elevation of inflammatory cytokines predicts a poor outcome in ards persistent elevation of high mobility group box- protein (hmgb ) in patients with severe sepsis and septic shock this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord- -god qzw authors: mao, kaimin; geng, wei; liao, yuhan; luo, ping; zhong, hua; ma, pei; xu, juanjuan; zhang, shuai; tan, qi; jin, yang title: identification of robust genetic signatures associated with lipopolysaccharide-induced acute lung injury onset and astaxanthin therapeutic effects by integrative analysis of rna sequencing data and geo datasets date: - - journal: aging (albany ny) doi: . /aging. sha: doc_id: cord_uid: god qzw acute lung injury (ali) and acute respiratory distress syndrome (ards) are life-threatening clinical conditions predominantly arising from uncontrolled inflammatory reactions. it has been found that the administration of astaxanthin (ast) can exert protective effects against lipopolysaccharide (lps)-induced ali; however, the robust genetic signatures underlying lps induction and ast treatment remain obscure. here we performed a statistical meta-analysis of five publicly available gene expression datasets from lps-induced ali mouse models, conducted rna-sequencing (rna-seq) to screen differentially expressed genes (degs) in response to lps administration and ast treatment, and integrative analysis to determine robust genetic signatures associated with lps-induced ali onset and ast administration. both the meta-analyses and our experimental data identified a total of degs in response to lps administration, and core degs (timp , ly i, cxcl , irf , cxcl , ccl , isg , saa , saa , tgtp , and gbp ) were identified to be associated with ast therapeutic effects. further, the core degs were verified by quantitative real-time pcr (qrt-pcr) and immunohistochemistry (ihc), and functional enrichment analysis revealed that these genes are primarily associated with neutrophils and chemokines. collectively, these findings unearthed the robust genetic signatures underlying lps administration and the molecular targets of ast for ameliorating ali/ards which provide directions for further research. acute respiratory distress syndrome (ards) is an acute inflammatory lung injury, associated with increased pulmonary vascular permeability, increased lung weight, and loss of aerated lung tissue [ ] . its less severe form is acute lung injury (ali). most patients need mechanical ventilation for support. the initial acute or exudative phase of ali/ards is characterized by the rapid onset of dyspnea, hypoxemia, respiratory failure, and bilateral infiltrates on chest radiographs that are consistent with pulmonary edema [ ] . ali/ards is common and has been associated with several clinical disorders, such as sepsis; pneumonia; aspiration of gastric contents, aging saltwater, or freshwater; major trauma; transfusion of blood products; acute pancreatitis; and drug reactions (for example, reactions to lipopolysaccharide) [ ] . in the past years, considerable progress has been made in understanding the epidemiology, pathogenesis, and pathophysiology of ards. however, ards is being increasingly recognized as a heterogeneous syndrome, generating momentum for the identification of clinical and biological features for classifying patients into subphenotypes that might be more responsive to specific therapies. lipid a (endotoxin), the hydrophobic anchor of lipopolysaccharide (lps), is a glucosamine-based phospholipid that makes up the outer monolayer of the outer membranes of most gram-negative bacteria [ ] . in recent years, lps, which has been most widely used in drug-associated ali models, can effectively induce a neutrophilic inflammatory response accompanied by an increase in intrapulmonary cytokines. many studies have shown that oxidative stress plays a major role in the pathogenesis of lung injury in a murine model of ali induced by lipopolysaccharide (lps) [ ] [ ] [ ] . in response to the increased formation of reactive oxygen species (ros), thioredoxin interacting protein (txnip) detaches from thioredoxin (trx), binds to the nucleotide-binding domain-like receptor protein (nlrp ), and then activates nlrp inflammasome [ ] . the activation of the nlrp inflammasome results in the maturation and release of pro-inflammatory cytokines, such as interleukin- β (il- β), which further aggravates the production of inflammatory cytokines (tumor necrosis factor-α (tnf-α), il- , inducible nitric oxide synthase (inos), and cyclooxygenase- (cox )) and induces oxidative stress [ ] [ ] [ ] . astaxanthin (ast) is a lipid-soluble, red-orange-colored xanthophyll carotenoid synthesized by many microorganisms and various types of marine life. the main producers of natural ast are microalgae and fungi. aquatic animals such as salmon, red seabream, shrimp, lobster and crayfish, which feed on ast-producing organisms, are significant dietary sources of ast for humans [ ] [ ] [ ] . it has been revealed that ast can prevent inflammatory processes by blocking the expression of pro-inflammatory genes as a consequence of suppressing nuclear factor kappab (nf-κb) activation [ ] . some studies also suggested that ast has a dosedependent ocular anti-inflammatory effect, through the suppression of no, pge , and tnf-alpha production, which is achieved by directly blocking nos enzyme activity [ ] . furthermore, ast has great therapeutic value for lung disease, such as an antifibrotic effect against the promotion of myofibroblast apoptosis based on dynamin-related protein- (drp )-mediated mitochondrial fission in vivo and in vitro [ ] and anti-inflammatory effect against lps-induced ali, as mentioned above [ , ] . however, at the transcriptional level, the mechanism of action of ast in the treatment of ali-/ ards-remains unclear. therefore, we hope to explore the molecular targets of ast against ali-/ ards-through further research, with the purpose of providing a new alternative for the clinical treatment of this acute lung disease. to determine the common molecular signatures underlying lps-induced mouse ali initiation, five microarray datasets were obtained from corresponding independent studies. the characteristics of the studies composing the five gene expression compendiums are listed in table and supplementary table . we extracted and annotated the five microarrays, which yielded a collection of unique genes from samples, including control and lps-induced ali mice. before the meta-analysis study, we comprehensively analyzed the five datasets by identifying the differentially expressed genes in each data set and evaluated overlapping significant genes. the overlapping results were used to generate a venn diagram (figure ), and three genes (ccl , zbp , and cxcl ) were identified in the common region, suggesting that these three genes were significantly correlated with lps management in mice in the five datasets. then, we performed a meta-analysis using networkanalyst (http://www.networkanalyst.ca), which is a comprehensive web-based tool designed to perform meta-analyses of gene expression data [ ] . an overview outlining the procedure of the analysis is presented in figure a . using three meta-analysis approaches, namely fisher's method, fixed effect model and voting count, , and differentially expressed genes, respectively, were identified. among these genes, were identified by all three methods ( figure b ), with ( . %) genes being upregulated and ( . %) being downregulated in the lps group compared with the control group. a full list of the common genes identified by the three meta-analysis methods is presented in supplementary table . a heat map of the top common degs across the five datasets is displayed in figure c . of note, the top upregulated genes (p< . ) were junb, vcam , ehd , ifrd, adm, cd , nadk, litaf, tubb , and ctps. the most significantly downregulated genes (p< . ) among the top common degs were acss and abcd . the merged data from this meta-analysis are listed in supplementary data . to further identify the robust expression signatures in lps-induced ali and investigate the transcriptional changes resulting from treatment of ali with ast, we divided mice into three groups, including the control group, lps group, and ast group. rna-sequencing (rna-seq) was performed to profile differentially expressed genes (degs) associated with lps-induced ali initiation and ast treatment. a total of degs were identified in the lps-induced ali group compared with the control group. among these genes, were table . then, we compared these genes with the degs obtained from the above meta-analysis, and generated two heat-maps of the common degs across the meta-analysis results and our experimental aging data, which are displayed in figure c and supplementary figure . in total, degs were detected in both published data and our experimental data, including upregulated and downregulated degs. to explore the therapeutic effect of ast against ali at the genetic level, we also compared the gene expression profile of the lps-induced ali group with that of the ast treatment group. in total, degs were identified after ast treatment ( figure b table ). we subsequently integrated the rna-seq and microarray meta-analysis data, and core degs (timp , ly i, cxcl , irf , cxcl , ccl , isg , saa , saa , tgtp , and gbp ) that were upregulated in ali models and downregulated significantly after ast treatment were identified ( table ) . to understand the function of the core degs, go enrichment analysis including molecular function (mf), biological process (bp) and cellular component (cc) categories (supplementary table ) was performed using the 'clusterprofile' package in r [ ] . in bp terms, the upregulated genes were associated with "cell chemotaxis," the "chemokine−mediated signaling pathway," and "neutrophil migration" ( figure a ). several studies have shown that neutrophil migration and related chemokine network regulation in the lung play roles in the pathogenesis and development of ali/ards [ ] [ ] [ ] . in the mf category, the core degs were associated with "glycosaminoglycan binding," "chemokine activity," and "receptor-ligand activity" ( figure b ). since glycosaminoglycan-cytokine interactions have been reported to support cellular mechanisms that cause acute inflammation [ ] , ast may affect these interactions by downregulating the genes involved to exert an anti-inflammatory effect. moreover, degs were enriched in the cc category involved in "high−density lipoprotein particles," "symbiont−containing vacuole membranes," and "plasma lipoprotein particles" ( figure c ). to further confirm the differences in the expression of the core degs (timp , ly i, cxcl , irf , cxcl , ccl , isg , saa , saa , tgtp , and gbp ) among the control group, lps group, and ast group, we divided mice into three groups and conducted qrt-pcr and ihc verification ( figure a - k, supplementary figure ). the results demonstrate that the relative expression levels of all genes were significantly upregulated in the lps group compared to the control group. more importantly, the expression levels of the above degs, as analyzed by qrt-pcr, were significantly inhibited after the application of ast. of the core genes, were tested by ihc, and the results were consistent with the qrt-pcr results, which further verifying the data (supplementary figure ) . overall, the rt-qpcr and ihc results were consistent with our integrative rna-seq analysis and metaanalysis, suggesting the critical role that the core dges might play in the mechanism by which ast alleviates ali/ards. as a life-threatening condition, ali/ards is an underrecognized condition, and its treatment is an unmet medical need. it is thought that inflammatory storm is the key factor in the occurrence of ali [ ] , and anti-inflammatory and antioxidant therapy should be the primary objective in ali/ards [ ] . to find the conserved genes responsible for lps-induced ali initiation and the effects of ast treatment, we identified robust changes in gene expression related to ali by meta-analysis and rna-seq using the gene expression omnibus (geo) database and mice, respectively. moreover, we performed functional enrichment analysis of core genes using the gene ontology (go) database to explore the possible molecular mechanisms that mediate the therapeutic effect of ast. before the meta-analysis of the five microarray datasets, we compared the differentially expressed genes in each dataset, and common differentially expressed genes (degs) were found in all five datasets: cxcl , zbp , and ccl . cxcl is abnormally expressed in the lung tissues of patients with idiopathic pulmonary fibrosis (ipf), and its circulating concentration is also highly correlated with the clinical manifestations and disease progression of individual patients. in the lung tissues of patients with ipf, cxcl may promote focal infiltration of nonproliferating b cells through the cxcl -cxcr axis [ ] . zbp is a host protein that was shown to be an innate sensor of viral infection, regulating cell death, inflammasome activation, and proinflammatory responses in a variety of situations, including infection and embryonic development [ ] . a previous study indicated that zbp is abnormally expressed in h n induced pneumonia associated with acute respiratory distress syndrome in mice [ ] . ccl (mcp ), which is elevated in pulmonary fibrosis, has been reported to mediate fibroblast survival through il- [ ] . since fibroproliferation is initiated early in lung injury, it has been observed that ccl is highly expressed in ards statistical analysis of significant differences between groups was achieved with one-way anova using prism software. ****p < . , ***p < . , **p < . , and *p < . were considered statistically significant. aging induced by severe sepsis [ ] . to reduce the study bias and increase the statistical power of individual microarray data, we performed a meta-analysis of five microarray gene expression profiles to assess the differentially expressed genes between lps-induced and control groups. consequently, differentially expressed genes (degs) were identified using three meta-analysis approaches. to further identify the robust expression signature related to lps-induced ali and investigate the transcriptional changes in response to the treatment of ali by ast, we performed rna-seq on three groups of mice and integrated the data with the results of the above mentioned meta-analysis. ultimately, we identified core degs that were significantly associated with ast treatment. saa , ly i, saa , irf , cxcl , ccl , timp , isg , gbp , tgtp , and cxcl were found to be overexpressed in the lps group compared with the control group but relatively downregulated in the ast group. our qrt-pcr and ihc verification of the core genes in the mice suggested that these genes might be the key mediators of the therapeutic effect of ast in ali/ards. among the core genes that were differentially expressed in response to ast mediation, two genes are members of the serum amyloid a (saa) family. saa is a critical acute-phase protein that is often increased by infection, trauma, cancer, or other causes of inflammation and plays an important role in the regulation of inflammatory responses [ ] . recent studies have indicated that an increased level of saa is positively correlated with the disease progression of covid , and can thus be a sensitive indicator for assessing the severity and prognosis of covid- [ ] . in our study, saa was the most significantly inhibited gene by ast application in lps-induced ali mice, and its downregulation was further confirmed by qrt-pcr and ihc. saa , the one of three isoforms of saa expressed in mice, is stimulated intensely in lps-induced acute systemic inflammation, which is consistent with our findings [ ] . high expression of saa in response to acute inflammation may be repressed by an interaction with noncoding rnas. it has been confirmed that mir- b- p may target saa to protect against lps-induced ali [ ] . additionally, lncrna malat can also target saa directly or indirectly to cause many diseases such as inflammation, diabetes and septic cardiomyocytes [ , ] . saa , another member of the saa gene family, is believed to have a pro-inflammatory effect, and its expression may aggravate tissue inflammation and damage [ ] . removing the n-and c-terminal sequences of saa can switch the protein to an anti-inflammatory role [ ] . however, other research has suggested that mice induced to express genetically modified human saa have a partial protective effect against the inflammatory response and lung injury caused by lps [ ] . moreover, saa is the direct target of mir- , which can protect nucleus pulposus cells from tnfainduced apoptosis in intervertebral disc degeneration [ ] . considering that saa might act as a biomarker of inflammatory disease, it is possible, that its downregulation induced by ast may partly indicate the antiinflammatory effect of ast. the deeper molecular mechanism underlying saa action in response to ast application deserves further exploration. interferon regulatory factor (irf ) is considered the master regulator of ifn-α against pathogenic infections [ ] . the excessive activation of irf promotes the development of acute lung injury (ali) caused by influenza a virus (iav), and attenuating irf activity can significantly prevent the progression of iavinduced ali in model mice [ ] . thus, the present finding that irf was upregulated by lps and downregulated in response to ast treatment may suggest that of ast protects against ali. regarding how irf regulates ifn production, mirna may act as an important mediator. previous research has shown that mir- c can downregulate irf and irf expression to mediate influenza a virus-induced ifnβ expression [ ] . additionally, mir- was shown to reduces the antiviral response by attenuating the traf -irf pathway to alter the cellular antiviral transcriptional landscape [ ] . however, whether mirna-irf interactions are involved in the pharmacological mechanism of ast remains to be further investigated. tissue inhibitor of metalloproteinase- (timp ), a member of timp family, is primarily recognized to regulate the degradation of the extracellular matrix by inhibiting the activity of matrix metalloproteinases (mmps) [ ] . it has been reported that an imbalance between mmp and timp plays a pivotal role in the pathogenesis of ards mainly through participating in airway remodeling, thus indicating the function of the mmp /timp ratio in the evolution of pulmonary fibrosis in ards [ ] . indeed, increased systemic levels of timp were proven to be associated with increased -day mortality in ards patients according to a large, prospective, multicenter study [ ] . additionally, other research has demonstrated that increased timp expression promotes an immune response, has a pro-inflammatory effect in the lungs after influenza infection and facilitates an injurious phenotype [ ] . the above observations not only support our present results regarding timp but also provide a considerable explanation for the increase in timp expression after lps application. intriguingly, given that timps are highly expressed in liver fibrosis and that the imbalance of mmps/timps promotes the progression of fibrosis, shen et al. found that astaxanthin is able to repress the activation of hepatic stellate cells (hscs) to ameliorate liver fibrosis through downregulating the expression of nf-κb and tgf-β and preserving the balance between mmp and timp [ ] . hence, it is reasonable to further investigate whether there is a similar mechanism by which ast downregulates the expression of timp to mitigate lps-induced ali. interferon-stimulated gene (isg ), which encodes the ubiquitin-like protein isg , which is primarily induced by type i interferons, is an essential player in regulating host signaling pathways such as damage repair responses and immune responses. isg can be induced by various pathogenic stimuli such as viral and bacterial infections, lipopolysaccharide (lps), retinoic acid, or certain genotoxic stressors [ ] . in accordance with our findings, previous studies have observed increased levels of isg conjugates in macrophages in response to lps treatment [ ] . moreover, research has found that systemic isg (mx , isg , ifit , and ifit ) expression within the first days of ards onset is associated with disease severity and prognosis. this response should be considered along with other identified genetic, environmental, and complex demographic factors as the cause of heterogeneity in ards prognosis [ ] . nevertheless, no data has been reported on the association between isg and ast in the literature. since the excessive recruitment of leukocytes appears to be a central contributor to the pathogenesis of ali, the elevation of proinflammatory cytokines and chemokines is considered the most important factor [ ] . similarly, we found that the expression of chemokines such as ccl , cxcl , and cxcl were increased after lps instillation but decreased after ast treatment. previous reports have documented an increased level of ccl in a mouse model of acute lps-induced lung inflammation [ ] . moreover, the expression of cxcl is also rapidly induced in ali murine models after lps administration [ ] . however, no data has on cxcl expression in ali models has been reported. therefore, we report for the first time the induction of cxcl after lps administration, which provides insights into the role of cxcl in the pathogenesis of ali. furthermore, the observation of decreased expression of ccl , cxcl , and cxcl may hint at the antiinflammatory properties of ast. although the roles of other degs (ly i, gbp , and tgtp ) have been described in many other diseases in detail, their regulatory mechanisms in ali-/ ards-are not fully understood. our results show that these degs are overexpressed to varying degrees in the lps group and that ast can effectively prevent this overexpression. further studies on the roles of these three genes in ali initiation and progression are need. to determine the functional mechanisms of these degs, go enrichment analyses were further conducted. according to the results, terms in biological process category, in cellular component category and in the molecular function category were enriched. the most significantly enriched terms in the bp category were associated with chemokines and neutrophils, indicating the dominant role of neutrophils and related chemokines in the pathogenesis and progression of lps-induced ali. in ali, the excessive recruitment of inflammatory cells and their mediators results in injury to endothelial and epithelial barriers [ ] . thus, agents such as ast, which can exert robust anti-inflammatory effects, may provide potential treatment prospects. despite this, several limitations of the current study need to be addressed. first, our research did not use the ali mouse models induced by other agents; thus, it did not address heterogeneity of ali initiation. second, given that findings in animal models of lps-induced lung injury may depend on the time point at which samples are obtained and physiological data are captured, the dynamic changes in lps-induced ali models may have been ignored to a certain extent [ ] . finally, in-depth research into the underlying mechanisms using knockout-gene mice for each differentially expressed gene will help further our understanding of the role of ast in ameliorating ali/ards. in conclusion, many genes were dysregulated in ali/ards. we not only identified genes that consistently differed in expression in the lps group compared to the control group but also revealed that ast can alleviate the abnormal expression of these genes and thus confer a certain therapeutic effect against ali/ards, suggesting the potential for ast to become a novel treatment for ali/ards. to identify the genes related to lps-induced acute lung injury in mice, five datasets (gse , gse , gse , gse , and gse ) were obtained from geo (gene expression omnibus, http://www.ncbi.nlm.nih.gov/geo) [ ] [ ] [ ] [ ] [ ] . lps and control treatments were used in this study. the detailed information (experimental design, transcriptome analysis, array information, data processing, and platform id) for these datasets can be obtained from the geo repository, and this information is partly aging summarized in table and is described in more detail in supplementary table . then, we conducted a microarray meta-analysis using networkanalyst . (https://www.networkanalyst.ca) [ ] . networkanalyst is a visual analytics platform for comprehensive gene expression profiling and metaanalysis. all gene probes were converted to a common entrez id using the gene/probe conversion tool in networkanalyst. following quantile normalization, all datasets were preprocessed through a log transformation and variance stabilizing normalization (vsn). each dataset was visualized in box plots to ensure an identical distribution among the samples. differential expression analysis was performed independently for each dataset using networkanalyst, with an fdr of . and a significance of p < . . the moderated t-test was based on the limma algorithm. for the meta-analysis, we used fisher's method, the fixed-effect model, and vote counting (combined p < . or vote counts ≥ were considered significant) to identify the differentially expressed genes (degs) and we selected the common degs identified by these three methods as the final degs. male c bl/ j mice ( - g, ~ -weeks-old) were purchased from beijing vital river laboratory animal technology co., ltd. (beijing, china). the mice were housed per cage under a h light/dark cycle in a laboratory at ± °c and % humidity. all experiment protocols conformed to the guidelines of the china council on animal care and use. these animal studies were approved by the institutional animal research committee of union hospital. the mice were randomly allocated into three groups: ( ) the control group (n= ), which was exposed to pbs alone and received an intraperitoneal injection of sterile saline; ( ) the lps group (n= ), which was exposed to pbs containing . mg/ml lps; and ( ) the ast group (n= ), which was intraperitoneally injected with ast ( mg/ml, dissolved in pbs) at a dosage of mg/kg body weight every day before one week of exposure to lps to evaluate its preventive and protective effects [ , ] , and intraperitoneally injected with mg/kg ast ( mg/ml, dissolved in pbs) hours after lps exposure in order to confirm the therapeutic effect of ast [ ] . ast was obtained from sigma-aldrich (st louis, mo, usa). for acute lps exposure, mice were exposed to an aerosol of phosphate buffer saline (pbs) alone or pbs containing . mg/ml lps for h, in a custom-built cuboidal chamber. the lps solution was aerosolized with a constant output ultrasonic nebulizer (model: b, yuwell, china) at a flow rate of ml/h. lps was purchased from sigma-aldrich (extracted from escherichia coli o : b , l ). the chamber was cm long, cm wide and cm high. total rna was extracted from mouse lung tissue samples with trizol® reagent (invitrogen, ca) following the manufacturer's protocol. the concentration and purity of the rna were measured by a nanodrop spectrophotometer (nanodrop technologies, technologies, wilmington, de, usa), the rna integrity was detected by agarose gel electrophoresis, and the rin was determined using an agilent bioanalyzer (agilent technologies, santa clara, ca, usa). the construction of a single library required a total of μg rna with a concentration of ≥ ng/μl and an od / ratio between . and . . then, oligo (dt) magnetic beads were subjected to capture mrnas that contained poly-a tails from the total rna. the resulting mrnas were subsequently randomly broken into small fragments of approximately bp by adding fragmentation buffer. the mrna fragments functioned as the templates for double-stranded cdna (dscdna) synthesis using the superscript double-stranded cdna synthesis kit (invitrogen, ca, usa). under the action of reverse transcriptase, a strand of cdna was synthesized by using random primers with mrna as a template, which was followed by two-strand synthesis to form a stable double-stranded structure. since there was a cohesive terminus in the double-stranded cdna structure, end repair mix was added to patch it into a blunt end, and an a base was added at the 'end to connect the yshaped adaptor. to purify and enrich the dscdna, cycles of pcr were performed, and clean dna beads were used to screen - bp bands. after quantification by tbs (picogreen, invitrogen, ca usa), high-throughput sequencing of the resulting libraries was performed on the illumina hiseq xten/novaseq sequencing platform (san diego, ca, usa), and the sequencing read length was pairedend (pe) . to ensure the accuracy of the subsequent biological information analysis, the raw sequencing data generated from rna-seq was firstly filtered to obtain high-quality sequencing data (clean data) to ensure the smooth progress of the subsequent analysis. quality control of the raw reads was performed using seqprep (https://github.com/jstjohn/seqprep) and sickle (https://github.com/najoshi/sickle). the processes were as follows. the first step was to remove the adapter aging from the reads and the reads that did not insert the fragment due to the self-connection of the adapter. second, bases with a low quality (quality value less than ) at the end of the sequence ( ' end) were trimmed. if there was still a quality value of less than for the remaining sequence, the whole sequence was discarded; otherwise, it was retained. third, reads with n ratios over % and sequences with lengths less than bp after quality trimming were also removed. finally, the error rate (%), q and q values, gc-content (%), and sequence duplication levels of the generated clean reads were assessed [ ] . after filtering the raw data, the clean data were aligned to the mouse reference genome grcm by 'bowtie ' software [ ] . then, read summarization was calculated by the 'feature count' tool. differently expressed genes (degs) between the lps samples and control samples were identified by t-test using the 'deseq ' r package, as were degs between the ast samples and lps samples [ ] . the raw p-value was adjusted to the false discovery rate by the benjamini method, and a false discovery rate (fdr) ≤ . and |log fc|≥ was chosen as the threshold. based on the hypergeometric distribution algorithm, go (gene ontology, http://www.geneontology.org/) biological process (bp), molecular function (mf) and cell component (cc) pathway enrichment analyses were performed using the 'clusterprofler' r package [ ] . a p-value ≤ . was set as the cutoff criterion. to validate the combined findings from rna-seq and microarray meta-analysis, the expression of core degs in the three groups was confirmed. rnaiso plus reagent (takara, tokyo, japan) was employed to extract total rna from mouse lung tissues from each group, and reverse transcription was performed to obtain cdna using primescript™rt master mix (takara, tokyo, japan) along with the gdna eraser kit (takara, tokyo, japan). relative mrna expression levels were determined using rt-pcr performed on bio-rad cfx maestro (bio-rad, usa) with tb green® premix ex taq™ ii (takara, tokyo, japan). all the above experimental steps were performed according to the manufacturer's instructions for the corresponding kit. glyceraldehyde- -phosphate dehydrogenase (gapdh) was selected as the reference, and the primer sequences are presented in supplementary table . qrt-pcr was performed under the following conditions: °c for min, followed by cycles at °c for s, °c for s, and °c for s. each analysis was implemented in triplicate, and the relative expression levels of the target genes were calculated by employing the -ΔΔct method [ ] . inmex and networkanalyst were applied for the network-based microarray meta-analysis. for qpcr data, statistical analysis of differences between groups was achieved by one-way anova using prism software (graphpad software inc., san diego, ca, usa). a twotailed test was used for all data, and differences with a p-value < . were considered significant. first of all, we used the search formula of lps[all fields] and ("lung"[mesh terms] or lung[all fields]) and ("mus musculus"[organism] and "expression profiling by array" [filter] ) to obtain results in geo datasets. by eliminating datasets of mirna sequencings, datasets not related to acute lung injury, and datasets that only researching on rna sequencing of specific cells such as macrophages and type ii alveolar epithelial cells, there were articles remained (gse , gse , gse , gse , gse , gse , gse and gse ). continuing to check the specific description of the sample in articles, we found some datasets were grouped with sample of n< and some mainly studied ali or ards induced by excessive ventilation or non-lps chemicals. in the end, there were datasets (gse , gse , gse , gse and gse ) that met the requirements of integrated analysis. we conducted a microarray meta-analysis using networkanalyst . combined three well-established meta-analysis approaches --fisher's method, fixed effect model, and vote counting. the features and main characteristics are given below (https://www.networkanalyst.ca). ( ) fisher's method (- *∑log(p)) is known as a 'weight-free' method and combines p values from multiple studies for information integration. ( ) effect size is the difference between two group means divided by standard deviation, which are considered combinable and comparable across different studies. in the fixed effects models (fem), the estimated effect size in each study is assumed to come from an underlying true effect size plus measurement error. ( ) vote counting is the simplest method in metaanalysis. differentially expressed gene is first selected based on a threshold to obtain a list of de genes for each study. the vote for each gene can then be calculated as the total number of times it occurred in all de lists. the final de genes can be selected based on the minimal number of votes set by the user. after the mice were sacrificed, the lung tissues were collected. immediately, the tissue was fixed in % paraformaldehyde for hours and embedded in paraffin. the embedded tissue was sliced into µm sections for staining. after the tissue sections were deparaffinized and rehydrated, they were heated in citrate buffer at °c for minutes to restore antigen activity. the sections were then incubated with . % hydrogen peroxide in methanol for minutes to inhibit endogenous peroxidase activity. after blocking nonspecific reactions with % normal bovine serum, the sections were incubated with rabbit polyclonal antibodies specific for ccl ( : , abcam), saa ( : , ab , abcam), ly i ( : , abcam), saa ( : - : , thermo), lrf ( : , thermo), timp ( : , thermo), isg ( : , thermo) and cxcl ( : , abcam). the treated samples were placed at °c for hours. the sections were then washed with pbs and incubated with horseradish peroxidase-conjugated secondary antibodies at °c for hours. the stained sections were imaged under an inverted phase contrast microscope. acute respiratory distress syndrome acute respiratory distress in adults. the lancet, saturday the acute respiratory distress syndrome lipopolysaccharide endotoxins cordycepin inhibits lps-induced acute lung injury by inhibiting inflammation and oxidative stress xanthohumol ameliorates lipopolysaccharide (lps)-induced acute lung injury via induction of ampk/gsk β-nrf signal axis linarin prevents lps-induced acute lung injury by suppressing oxidative stress and inflammation via inhibition of txnip/nlrp and nf-κb pathways curcumin and allopurinol ameliorate fructose-induced hepatic inflammation in rats via mir- a-mediated txnip/nlrp inflammasome inhibition troxerutin protects kidney tissue against bde- -induced inflammatory damage through cxcr -txnip/nlrp signaling astaxanthin: a review of its chemistry and applications astaxanthin, a carotenoid with potential in human health and nutrition biorefinery approach and environmentfriendly extraction for sustainable production of astaxanthin from marine wastes astaxanthin inhibits nitric oxide production and inflammatory gene expression by suppressing i(kappa)b kinase-dependent nf-kappab activation effects of astaxanthin on lipopolysaccharide-induced inflammation in vitro and in vivo astaxanthin prevents pulmonary fibrosis by promoting myofibroblast apoptosis dependent on drp -mediated mitochondrial fission astaxanthin alleviated acute lung injury by inhibiting oxidative/nitrative stress and the inflammatory response in mice astaxanthin prevents against lipopolysaccharideinduced acute lung injury and sepsis via inhibiting activation of mapk/nf-κb networkanalyst . : a visual analytics platform for comprehensive gene expression profiling and metaanalysis clusterprofiler: an r package for comparing biological themes among gene clusters inflammatory cytokines in patients with persistence of the acute respiratory distress syndrome neutrophils in the initiation and resolution of acute pulmonary inflammation: understanding biological function and therapeutic potential evidence for chemokine synergy during neutrophil migration in ards fernández-botrán r. modulation of acute inflammation by targeting glycosaminoglycan-cytokine interactions contribution of neutrophils to acute lung injury antiinflammatory activity of a novel family of aryl ureas compounds in an endotoxin-induced airway epithelial cell injury model c-x-c motif chemokine (cxcl ) is a prognostic biomarker of idiopathic pulmonary fibrosis zbp : innate sensor regulating cell death and inflammation dynamic gene expression analysis in a h n influenza virus mouse pneumonia model the cc chemokine ligand (ccl ) mediates fibroblast survival through il- complement inhibition decreases early fibrogenic events in the lung of septic baboons the cytokine-serum amyloid achemokine network serum amyloid a is a biomarker of severe coronavirus disease and poor prognosis serum amyloid a is a high density lipoprotein-associated acute-phase protein exosomes derived from microrna- b- p-overexpressing mesenchymal stem cells protect against lipopolysaccharide-induced acute lung injury by inhibiting saa long non-coding rna malat regulates hyperglycaemia induced inflammatory process in the endothelial cells il- induced lncrna malat enhances tnf-α expression in lps-induced septic cardiomyocytes via activation of saa emerging functions of serum amyloid a in inflammation suppression of lipopolysaccharide-induced inflammatory response by fragments from serum amyloid a serum amyloid a promotes lps clearance and suppresses lps-induced inflammation and tissue injury knockdown of mir- protects nucleus pulposus cells from tnf-a-induced apoptosis by targeting serum amyloid a irf : activation, regulation, modification and function attenuation of interferon regulatory factor activity in local infectious sites of trachea and lung for preventing the development of acute lung injury caused by influenza a virus mir- c mediates influenza a virus-induced ifnβ expression by targeting nf-κb inducing kinase mir- attenuates the host response to influenza virus by targeting the traf -irf signaling axis cytokine functions of timp- imbalance between matrix metalloproteinases (mmp- and mmp- ) and tissue inhibitors of metalloproteinases (timp- and timp- ) in acute respiratory distress syndrome patients serum mmp- and timp- in critically ill patients with acute respiratory failure: timp- is associated with increased -day mortality timp- promotes the immune response in influenza-induced acute lung injury isg in antiviral immunity and beyond lipopolysaccharide activates the expression of isg -specific protease ubp via interferon regulatory factor extremes of interferon-stimulated gene expression associate with worse outcomes in the acute respiratory distress syndrome role of chemokines in the pathogenesis of acute lung injury proteinaseactivated receptor- , ccl , and ccl regulate acute neutrophilic lung inflammation extracellular atp mediates the late phase of neutrophil recruitment to the lung in murine models of acute lung injury molecular dynamics of lipopolysaccharide-induced lung injury in rodents modulation of lipopolysaccharideinduced gene transcription and promotion of lung injury by mechanical ventilation bpifa regulates lung neutrophil recruitment and interferon signaling during acute inflammation altered gene expression profiles in the lungs of benzo[a]pyreneexposed mice in the presence of lipopolysaccharideinduced pulmonary inflammation effects of age on the synergistic interactions between lipopolysaccharide and mechanical ventilation in mice clara cells attenuate the inflammatory response through regulation of macrophage behavior astaxanthin suppresses cigarette smokeinduced emphysema through nrf activation in mice fastp: an ultra-fast all-inone fastq preprocessor fast gapped-read alignment with bowtie moderated estimation of fold change and dispersion for rna-seq data with deseq analysis of relative gene expression data using real-time quantitative pcr and the (-delta delta c(t)) method the authors declare that they have no conflicts interest. please browse full text version to see the data of supplementary tables to key: cord- -z sb s authors: tallam, aravind; perumal, thaneer m.; antony, paul m.; jäger, christian; fritz, joëlle v.; vallar, laurent; balling, rudi; del sol, antonio; michelucci, alessandro title: gene regulatory network inference of immunoresponsive gene (irg ) identifies interferon regulatory factor (irf ) as its transcriptional regulator in mammalian macrophages date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: z sb s immunoresponsive gene (irg ) is one of the highest induced genes in macrophages under pro-inflammatory conditions. its function has been recently described: it codes for immune-responsive gene protein/cis-aconitic acid decarboxylase (irg /cad), an enzyme catalysing the production of itaconic acid from cis-aconitic acid, a tricarboxylic acid (tca) cycle intermediate. itaconic acid possesses specific antimicrobial properties inhibiting isocitrate lyase, the first enzyme of the glyoxylate shunt, an anaplerotic pathway that bypasses the tca cycle and enables bacteria to survive on limited carbon conditions. to elucidate the mechanisms underlying itaconic acid production through irg induction in macrophages, we examined the transcriptional regulation of irg . to this end, we studied irg expression in human immune cells under different inflammatory stimuli, such as tnfα and ifnγ, in addition to lipopolysaccharides. under these conditions, as previously shown in mouse macrophages, irg /cad accumulates in mitochondria. furthermore, using literature information and transcription factor prediction models, we re-constructed raw gene regulatory networks (grns) for irg in mouse and human macrophages. we further implemented a contextualization algorithm that relies on genome-wide gene expression data to infer putative cell type-specific gene regulatory interactions in mouse and human macrophages, which allowed us to predict potential transcriptional regulators of irg . among the computationally identified regulators, sirna-mediated gene silencing of interferon regulatory factor (irf ) in macrophages significantly decreased the expression of irg /cad at the gene and protein level, which correlated with a reduced production of itaconic acid. using a synergistic approach of both computational and experimental methods, we here shed more light on the transcriptional machinery of irg expression and could pave the way to therapeutic approaches targeting itaconic acid levels. immune cells specifically respond to various inflammatory environments based on the nature and type of external stimuli. macrophages trigger defensive pathways upon stimulation of pattern-recognition receptors, such as toll-like receptors (tlrs) [ , ] . previous studies have shown that mouse macrophages under pro-inflammatory conditions, such as bacterial infections or lipopolysaccharide (lps) stimulation, highly express immunoresponsive gene (irg ) [ , ] . recently, we demonstrated the active role of irg in antimicrobial responses linking metabolism to immunity [ ] : immune-responsive gene protein/cis-aconitic acid decarboxylase (irg /cad) catalyzes the decarboxylation of cis-aconitic acid to itaconic acid (also known as methylenesuccinic acid) during the tca cycle. itaconic acid is an organic compound that inhibits isocitrate lyase, the first enzyme of the glyoxylate shunt, a savior pathway for bacterial growth under nutrient-deprived conditions. for the first time, we also demonstrated irg expression and itaconic acid production in lps-treated human peripheral blood mononuclear cells (pbmcs)-derived macrophages [ ] . thus, irg /cad contributes to the host immune response against bacterial invasion providing an additional support to the innate immune system. in addition to our findings, irg was also previously shown to be expressed in mouse macrophages under different tlr ligand stimulations [ , ] and microbial infections [ , ] . high upregulation of irg was observed in the lungs of mice when infected with influenza a virus, thus showing its induction also under viral infections [ ] . in a different context, irg was found to be highly expressed in the uterine luminal epithelium of the mouse during the early stages of pregnancy due to the synergistic regulation by progesterone and estradiol mediated by the protein kinase c pathway [ , ] . irg was also reported to be highly expressed in pbmcs of septic patients where it fosters endotoxin tolerance by enhancing a expression via reactive oxygen species (ros) signaling [ ] . apart from mouse and human, irg is also expressed in other species under microbial infections. in zebrafish (danio rerio), when infected with salmonella species, irg is specifically expressed by macrophage-lineage cells and is cooperatively regulated by glucocorticoid and jak/stat signaling pathways [ ] . furthermore, it was shown that irg is a key component responsible for the production of mitochondrial ros augmenting the bactericidal activity of macrophages. hence, in zebrafish, irg /cad additionally contributes to immune responses by the production of ros [ ] . taken together, these findings demonstrate a pivotal role of irg /cad in the immune metabolism axis connecting the immune system with cellular metabolism through the production of itaconic acid and ros. despite the profound biological importance of irg /cad, the molecular mechanisms that induce irg expression have not yet been investigated. the regulation of irg expression was reported on several findings, which are rather contrasting, such as de novo protein synthesis independent [ ] and dependent [ ] , myd -independent [ ] and dependent [ ] , trif-independent [ ] , tlr -and tlr -independent [ ] . interestingly, irg was highly induced when stimulated with ifnγ alone or in combination with tnfα and it was shown that the vast majority of the murine irg /cad protein was found in the mitochondrial fraction [ ] . all the above experiments differ by cell types, the nature of the stimuli and perturbing compound concentrations. it is highly likely that irg is regulated by a complex transcriptional machinery responding to cellular environments and external stimuli. hence, it is important to unravel its transcriptional machinery to understand its role and expression under specific inflammatory conditions. gene regulatory networks (grns) capture the dependency between transcription factors, genes, proteins and small molecules underlying cellular processes [ , ] . inferring regulatory interactions linked to irg can contribute to identify the major regulatory elements involved in the induction of irg . there are three main grn inference approaches, which are widely used. the first is based on existing literature information which use natural language processing algorithms and manual curation to mine scientific articles [ ] [ ] [ ] . the second approach is purely data-driven without any prior information. these category of methods infer the regulatory networks directly from multi-omics data based on the underlying conditional dependency structures such as co-expression [ ] , mutual information [ ] or regression [ , ] among the genes. the third category of methods uses predictive modeling and experimental approaches, such as chip-seq, to infer transcription factor-dna (tf-dna) binding sites (tfbs) and motifs [ , ] . all the three category of methods have their own advantages and disadvantages, while some require high dimensional data (i.e. high number of replicates and/or perturbation conditions), others are skewed with most studied interactions or biological processes. however, due to the course of dimensionality, most of them suffer from a high number of false positive interactions, thus making the subsequent network analysis of grns more elusive. to this end, in this context, we used an integrated algorithm, which leverages on the information from all the three different category of methods (i.e. literature, tfbs and data-driven) to infer the regulatory network of irg . this algorithm, with some minor modifications from [ ] , is used to infer the grn of irg from literature and tfbs, and to prune inconsistent interactions by contextualizing the model to predict differential expression from genome-wide expression arrays. putative transcriptional regulators of irg were hypothesized from the resulting grn and tested using sirna-mediated gene silencing experiments in mouse and human macrophages under lps stimulation. the murine raw . macrophage cell line [ ] was cultured in dmem medium (invitrogen, life technologies, carlsbad, california) with % fetal bovine serum (fbs), . mg/ml streptomycin (invitrogen, life technologies, carlsbad, california) and u/ml penicillin (invitrogen, life technologies, carlsbad, california). for the experiments, lipopolysaccharide (lps from escherichia coli :b , sigma) was used at a final concentration of ng/ml. primary monocytes were extracted from the blood samples of anonymous healthy male donors, donated by the luxembourgish red cross (http://www.croix-rouge.lu/). human blood samples in the present study were obtained under a mutual agreement between the university of luxembourg and the luxembourgish red cross for blood donation to non-therapeutic purposes. the institutional review board waived the need for consent. the comité national d'ethique de recherche (cner) (http://www.cner.lu/) approved this study. the components of the blood (peripheral blood mononuclear cells-pbmcs -, plasma and erythrocytes) were separated by ficoll density gradient separation. for this purpose, the blood was diluted : with phosphate buffered saline (pbs, invitrogen, life technologies, carlsbad, california) in falcon tubes and was transferred to leucosep tubes (greiner bio one, kremsmünster, austria) filled with ml of ficoll (vwr, radnor, pennsylvania). after a minute centrifugation ( g at room temperature without break), the pbmcs layer was collected and the cd + monocytes were isolated by using the macs technology (magnetic separation) from miltenyi biotec (bergisch gladbach, germany). pbmcs were mixed with μl of cd microbeads and incubated for min at °c on a rotating platform followed by magnetic separation using lscolumn. isolated cd + monocytes were plated at x cells per well in -well plates (or x cells per well in -well plates, x cells per well in -well plates, . x cells per well in -well plates) and differentiated for days into macrophages using rpmi medium (vwr, radnor, pennsylvania) supplemented with % human serum, off the clot, type ab (a&e scientific, paa, pasching, austria, lot number: c - ), . mg/ml streptomycin, u/ml penicillin and . mm l-glutamine (invitrogen, life technologies, carlsbad, california). during the differentiation, the medium was replaced with fresh medium on day and day . alternatively, cd + monocytes were plated at x cells per well in -well plates and differentiated for days into dendritic cells using human serum supplemented with ng/ml of both granulocyte-macrophage colony-stimulating factor (gm-csf, r&d systems europe ltd., united kingdom) and interleukin- (il- , r&d systems europe ltd., united kingdom). for the experiments, lps was used at μg/ml, while tumor necrosis factor alpha (tnfα, r&d systems europe ltd., united kingdom) and interferon gamma (ifnγ, r&d systems europe ltd., united kingdom) were used at a final concentration of ng/ml. total rna was purified from cultured cells using the qiagen rneasy mini kit (qiagen) according to manufacturer's instructions. first-strand cdna was synthesized from . to μg of total rna using superscript iii (invitrogen) with μl ( μm)/reaction oligo(dt) as primer. individual μl sybr green real-time pcr reactions consisted of μl of diluted cdna, μl of ×iq sybr green supermix (bio-rad), and . μl of each μm optimized forward and reverse primers in μl rnase free water. primer sequences designed using beacon designer software (bio-rad), provided by eurogentec, or directly designed by thermo scientific and are shown in s table. for the human irg primers, the ncbi/primer-blast tool available at http://www.ncbi.nlm.nih.gov/tools/primer-blast/ was used. the pcr was carried out on a light cycler (roche diagnostics), using the following program: min at °c and cycles of s at °c, s at °c and s at °c followed by s - °melting curves. all experiments, including three no template controls, were performed in triplicates for each sample. for normalization, l was amplified simultaneously. the on-targetplus smartpool, containing four different sirna sequences specifically targeting each of murine irf (sirna irf , l- - - ), murine cebpb (sirna cebpb, l- - - ), human irf (sirna irf , l- - - ) and the corresponding non-targeting control (sirna neg, d- - - ), were designed and synthesized by thermo scientific dharmacon. murine raw . macrophages were transfected with amaxa d nucleofector device, xunit (lonza) using the amaxa sg cell line d nucleofector kit for thp- cells according to the manufacturer's instructions. briefly, transfection with sirna complexes was carried out from pelleted and resuspended cells ( x cells per condition). transfection reagent and sirna were prepared according to the manufacturer's instructions (amaxa). specific sirnas were added at a final concentration of nm. after nucleofection using the program "raw . (atcc)", the cells were seeded at a density of x cells per well in -well plates in dmem supplemented with % fbs and incubated for h. human pbmcs-derived macrophages were transfected using the amaxa d nucleofector device, y-unit, which was specifically designed for transfection of adherent cells. for these experiments, cells were seeded at x cells in -well plates for days and the medium was then replaced with nucleofection reagent and specific sirnas ( μm) according to the manufacturer's protocol. the reagent solution was removed and the medium was added to the cells which were then incubated for h and stimulated with lps. for immunofluorescence the cells were grown on cellcarrier well plates (perkin elmer), washed with pbs, and fixed with % paraformaldehyde (ph . ) for min at ambient temperature. after washing in pbs, the cells were permeabilized for min with . % triton x- in pbs. permeabilization was followed by x min washing steps in pbs. for blocking, samples were incubated with % goat serum in pbs for h at room temperature. primary antibodies against irg /cad (anti-irg antibody produced in rabbit, sigma, / ) and a mitochondrial surface antigen (mab , millipore, / ) were diluted in pbs + % bsa and bound for h at room temperature. after three washing steps in pbs, secondary antibodies (a- , a- , invitrogen, both / ) were added and incubated at dark for h at room temperature. for staining of nuclei and stabilization of fluorescent signals the samples were covered with fluoroshield mounting medium containing dapi (f , sigma). image acquisition started after min of incubation. image stacks with planes were acquired on a confocal opera qehs high content screening microscope (perkin elmer), using a x water immersion objective (na . ). the antibody-labeled mitochondrial channel was excited with a nm laser and detected with a / band-pass filter. antibody-labeled irg /cad channel was excited with a nm laser and detected with a / band-pass filter. dapi was excited with a nm laser and detected with a / band-pass filter. automated image analysis was performed in matlab a. nucleus segmentation was following the rule that pixels of low pass filtered dapi images, convolved with a gaussian filter of size and sigma have to be at least % brighter within nuclei as compared to local surroundings as defined by an average filter of size . the minimum size of nuclei was set to pixels. for the detection of cell covered regions, the three channels were summed up and low pass filtered with a gaussian filter of size and sigma . resulting images were thresholded by the first quartile of pixel values. to detect single cell areas, a watershed algorithm was applied to the euclidian distance transform of the nucleus mask. mitochondria were segmented via a combination of local thresholding and segmentation of non-uniformly corrected mitochondria images. according to the local thresholding algorithm, mitochondrial pixel assignment requires a raw mitochondrial image, low pass filtered with a gaussian filter of size and sigma to be brighter than the same image convolved with an average filter of size . for non-uniform correction mitochondria channel images average filtered with a structuring element of size were subtracted from original mitochondria images. for segmentation these corrected images were thresholded to a pixel intensity of . for connected components confirmed by both rules the minimum volume was set to pixels. irg /cad positive pixels were defined by the same algorithm as mitochondrial pixels, in this case applied to the irg /cad channel. subcellular localization of irg /cad was evaluated by computing single cell proportions of irg /cad positive pixels in nuclei and mitochondria. to lyse the cells. cells were then scraped out from the plates and the lysate was shaken at °c for min at maximum speed. the lysate was then centrifuged at °c for min at maximum speed. the supernatant was collected and stored at - °c. the extracted protein samples were then quantified using a bradford assay and the measurements were used for subsequent western blotting. heat-denatured protein samples ( μg) were separated on a % sds-polyacrylamide gel electrophoresis followed by transfer to nitrocellulose membranes . μm (sigma). affinity-purified goat anti-mouse irf antibody (catalogue #: af ) and rabbit anti-goat igg secondary antibody (catalogue #: haf ) were obtained from r&d systems europe ltd., united kingdom, while rabbit anti-human irg antibody (catalogue #: hpa ) and normal rabbit igg (catalogue #: sc- ) were purchased from sigma and santa cruz biotechnology, respectively. goat anti-β-actin (catalogue #: sc- ) and anti-goat secondary antibodies (catalogue #: rpn ) were purchased from santa cruz biotechnology and ge healthcare, respectively. after blocking with % (wt/vol) dry milk in pbs, the membrane was incubated overnight at °c with primary antibody in % bsa/pbs (dilution : for irf and irg ) on a rotating platform. after three washing steps with pbs containing . % tween- , the membrane was incubated with secondary antibodies (dilution : ) coupled to horseradish peroxidase and revealed by chemiluminescence using the amersham ecl detection reagents (ge healthcare) and odissey imaging system. cells seeded in -well plates were washed with ml of saline solution ( . % nacl) and quenched with . ml cold methanol. after adding an equal volume of cold water, cells were collected with a cell scraper and transferred in tubes containing . ml cold chloroform. the extracts were vortexed at rpm for min at °c and centrifuged at rpm for min at °c. a volume of . ml of the upper aqueous phase was collected in specific gc glass vials and evaporated under vacuum at - °c using a refrigerated centrivap concentrator. extractions of metabolites from cells grown in -well plates were performed using half of the volumes. the interphase was centrifuged with μl cold methanol at rpm for min at °c. when needed, the pellet was stored at - °c for subsequent rna isolation. metabolite derivatization was performed using a multi-purpose sampler (gerstel). dried samples were dissolved in μl pyridine, containing mg/ml methoxyamine hydrochloride, at °c for minutes by shaking. after adding μl n-methyl-n-trimethylsilyl-triflouroacetamide (mstfa), samples were incubated at °c for min with continuous shaking. gc-ms analysis was performed by using an agilent a gc coupled to an agilent c inert xl msd. a sample volume of μl was injected into a split/splitless inlet operating in splitless mode at °c. the gas chromatograph was equipped with a m agilent j&w db- ms capillary column + m duraguard capillary in front of the analytical column. helium was used as carrier gas with a constant flow rate of . ml/minute. the gc oven temperature was held at °c for min and then increased to °c at °c/minute. the final temperature was held for min. the transfer line temperature was set constantly to °c. the msd was operating under electron ionization at ev. the ms source was held at °c and the quadrupole at °c. the gc-ms was operated in selected ion monitoring (sim) mode (m/z . , m/z . , m/z . ). the total run time of one sample was . min. all gc-ms chromatograms were processed using metabolitedetector [ ] for targeted data analysis. the software package supports automatic deconvolution of all mass spectra. the obtained mass spectra were matched against a reference library (including the mass spectrum of the authentic standard "itaconic acid"). compounds were annotated by retention time and mass spectrum (overall similarity: . or higher). for quantification, fragment ion m/z was used. total rna from pbmcs-derived macrophages was extracted using trizol reagent (invitrogen) while total rna from raw . cells was harvested using the qiagen rneasy mini kit (qiagen), according to the manufacturer's protocols. rna purity and integrity were monitored using nanodrop nd- spectrophotometer and agilent bioanalyzer with rna nano assay kit. only rnas with no sign of contamination or marked degradation (rin > ) were considered good quality and used for further analysis. genechip human gene . st and genechip mouse gene . st arrays (affymetrix) were used to determine the genome-wide expression profiles of pbmcs-derived macrophages and raw . cells, respectively. total rnas ( ng) were processed using the affymetrix whole transcriptome (wt) expression kit according to the manufacturer's instructions (user manual p/n rev. c / ). microarrays were hybridized, washed and stained using the affymetrix genechip wt terminal labeling and hybridization kit following the microarrays were washed, stained and scanned according to manufacturer's standard procedures (user manual p/n rev. ). genechips were scanned using an affymetrix genechip scanner generating cel files containing hybridization raw signal intensities which were preprocessed and normalized using the genechip robust multiarray averaging (gcrma) algorithm [ ] from bioconductor in r. redundant probe sets were merged by considering mean values, resulting in a list of unique annotated genes mapped based on entrez gene identifiers. using the limma package and the ebayes function from affy library in bioconductor, genes whose expression values between any two conditions having a difference with a log fold change (logfc) ! +/- and a p-value< . were considered as differentially expressed genes (degs). microarray expression data are available at gene expression omnibus (geo) database (http://www.ncbi.nlm.nih.gov/geo/) under the accession number gse . grn inference was performed using three distinct approaches: (a) from transcription factor-dna (tf-dna) binding models: upstream regulation of irg was inferred using match™ algorithm of transfac database from the biobase international corporation [ , ] . match™ algorithm is a weight-matrix based algorithm which searches for potential transcription factor binding sites (tfbs) on any given genomic sequence using the position weight matrices (pwms) library from transfac database. the pwms for transcription factors (tf) in transfac are constructed based on the consensus of its dna binding sequences across the genome. each pwm consists of the nucleotides and their frequency at respective position on the binding sequence. for our analysis, immune cell specific profiles of match™ consisting of pwms of tf that are involved in the immune responses in t-cells, bcells, mast cells, myeloid cells, natural killer cells and macrophages were used. transcription start site (tss) information of genes from refseq and genome sequences of range bp upstream and bp downstream with respect to tss from hg (human), mm (mouse) from ucsc website were obtained. taking these sequences as input, match™ algorithm searches for potential binding sites of tfs on the sequence using its collection of pwms. the output consists of information about the transcription factor, binding position, genomic strand and the two quality scores (core and matrix similarity), sequence motif of each binding site found. only the transcription factor binding predictions that have both the similarity scores ! . were considered for further analysis (s table) . (b) from published literature: on the other hand, downstream regulation of lps stimulus in macrophages was inferred using pathway studio desktop from elsevier [ ] . pathway studio [ ] is the knowledge base of ontologies, taxonomies and biological relationships derived based on the text mining algorithms and the expert curation of published scientific literature. medscan text mining technology was used to scan all the pubmed abstracts (% million) and relevant sentences were collected based on the manually curated dictionaries with the synonyms of biological terms. for this network, the extraction of published literature was restricted as follows: species: human/mouse; cell type: macrophages; interactions: direct regulations in the network building settings of the pathway studio software. (c) combining and contextualizing literature and tfbs networks: by virtue, the grn inferred above are from different cell, tissue and/or organism. also, for some of the tf-dna predictions and literature inferences, the mode of action is unspecified (i.e. activation or inhibition). therefore, in order to find context specific interactions (i.e. that are specific to irg expression in mouse macrophages and/or human pbmcs), we prune the inconsistencies in the inferred grn with an improved version of the previously developed contextualization algorithm [ ] . in gist, using feedback regulations as basic building blocks, this algorithm tries to predict differential expression from genome-wide arrays, using a boolean modeling framework, by removing as well as assigning mode of actions to the interactions in the grn [ ] . this method was implemented in matlab using the genetic algorithm (ga) function with the assumption that each cell phenotype represents a stable steady state (attractor) of the network. the pbn-matlab-toolbox (http://code.google.com/p/pbn-matlab-toolbox/downloads/list) with the synchronous updating scheme was employed in this algorithm for the boolean simulation. in order to identify the transcriptional regulators for irg under lps stimulation, the contextualised grn was used to hypothesize experimentally testable predictions. all simple paths connecting lps stimulation to irg from the inferred grn were computed using the graph k shortest paths algorithm of matlab. these paths provide the basis set of putative regulators and pathways for translating the lps signal to induce irg . further, using the simple paths, the importance of tfs were established using a gene essentiality metric score. this score defines the importance of a particular gene in propagating the signal from the ligand stimulus to the target gene. the score ranges from to with being the most essential gene and being nonessential. however, the score cannot be for any gene as all the genes are at least participating in one of the paths between lps-irg . these genes were then ranked according to their essentiality metric in these paths. the score was calculated according to the formula: where em j denotes the essentiality metric for gene j, n tp denotes the number of total paths, n jp denotes the number of paths in which gene j is absent. hence, if the number of paths in which a particular gene is absent is low, its essentiality metric will be high. the top ranked tfs were then chosen to perform sirna-mediated gene silencing experiments to analyze the effect on irg expression. overall, the workflow from the inference of grns to validation studies is depicted in fig . fig . workflow for the identification of transcriptional regulators for a specific gene. the scheme shows the workflow of the different steps followed to identify potential transcriptional regulators for a given gene under lps exposure. the upstream network was initially constructed using the pwms from transfac and the prediction algorithm match™. the literature network downstream of lps was inferred using the pathway studio knowledge database. these networks were merged and the merged network was contextualised using the booleanised genome-wide expression data. finally, the ranking scheme with simple paths and essentiality metric resulted in a set of potential transcriptional regulators which were tested using sirna mediated gene silencing experiments. otherwise mentioned, p-values were calculated according to the student t-test with the twotailed distribution assuming two-sample equal variance. we first analysed irg expression in human pbmcs-derived monocytes (fig a) , pbmcsderived macrophages (fig b) and pbmcs-derived dendritic cells (fig c) under various inflammatory conditions induced either by lps ( μg/ml), tnfα ( ng/ml), ifnγ ( ng/ ml), lps with tnfα or ifnγ exposures. after hours, the expression levels of irg were highly increased following the different pro-inflammatory treatments in all the analysed immune cells compared to the untreated cells, with the highest levels obtained after their exposure to lps in combination with ifnγ (fig ) . in order to investigate irg expression over time following a pro-inflammatory stimulus, we analysed its expression levels at different time points following lps activation. the results show that irg , similarly to the corresponding mouse gene [ ] , is expressed at the highest levels at - hours in both monocytes and macrophages (s fig) and that its expression is already significantly induced after minutes in human pbmcs-derived macrophages as well as after minutes in the murine raw . macrophage cell line (s fig). taken together, these results show that irg is expressed by human monocytes, macrophages and dendritic cells and that it is rapidly induced in both human and murine macrophages under inflammatory conditions. it was previously shown that the murine irg /cad protein associates with mitochondria [ ] . however, the sub-cellular localization of the corresponding human protein was not described until now. thus, to elucidate irg /cad compartmentalization in human macrophages, we applied immunofluorescence in combination with confocal microscopy to human pbmcsderived macrophages under inflammatory conditions. results showed that, similarly to the murine protein, human irg /cad is accumulating in mitochondria (fig a and b) . indeed, treatments with lps alone or in combination with ifnγ caused significant mitochondrial accumulation of irg /cad (wilcoxon rank sum test, p < . ) (fig c) . following our results at the gene and protein level, we investigated in more details the transcriptional machinery, which is responsible for irg expression in both murine and human macrophages. using biobase resources, such as transfac and match tm , we inferred the tf-dna interactions (directed and unsigned) with irg as the starting node. we adopted a similar approach for both the murine and human genes. the mouse analysis resulted in an irg upstream network containing nodes and edges. in parallel, the interactions (signed and directed) related to the biological process of lps stimulation in macrophages were inferred from pathway studio and resulted in a lps downstream network with nodes and edges. as expected, we did not obtain irg as one of the nodes in this network reflecting the lack of specific information about the transcriptional regulation of irg following an lps treatment. taking the union of all the interactions, these two networks were then merged into a single network. the merged network with nodes and edges established the connection between lps and irg with common nodes from both networks. similarly to the analysis performed in mouse macrophages, the match tm algorithm with the human irg promoter sequence and the immune cell specific profile yielded an irg upstream network with nodes and edges, which passed the quality threshold. the literature network downstream of lps inferred from pathway studio resulted in a network of nodes and edges. as for the mouse analysis, these two networks were then merged with a total of nodes and edges. since the murine and human merged networks were obtained from heterogeneous data, we generated genome-wide gene expression data in the murine raw . macrophage cell line and human pbmcs-derived macrophages after hours of lps stimulation for network contextualization. the differentially expressed genes (degs) with their respective fold changes and pvalues are included in the supplementary material (s and s tables). from these genes, we identified the top degs (log fc!| | and p-value < . ) between untreated and lps-activated conditions for both mouse raw . macrophages (s fig) and human pbmcs-derived macrophages (s fig), respectively. the results show that both mouse and human cells are highly affected by lps displaying a classical pro-inflammatory signature were the classical proinflammatory markers, such as il β, ccl and il , are among the top degs in both the species. gene ontology (go) analysis reveals up-regulation of biological processes that are reflecting a pro-inflammatory response, such as "response to molecule of bacterial origin" or "regulation of cytokine production" in both mouse and human cells (s fig and s fig) . (fig a and b) . the classical pro-inflammatory-associated transcription factors, such as nfκb, junb and irfs, are highly up-regulated in lps conditions when compared to untreated cells. in human cells, genes were up-regulated in lps conditions when compared to control and, among them, were transcription factors, while a total of genes were down-regulated and were transcription factors (fig c) . in mouse macrophages, a total of genes were up-regulated and, among them, were transcription factors, while genes were down-regulated and were transcription factors (fig d) . the relevance of these genome-wide gene expression data to prune the networks allowed us to remove the interactions that were inconsistent with the gene expression data as well as to infer the signs to the previously unsigned interactions from irg upstream networks. from the contextualised networks, we then aimed to identify the potential transcriptional regulators of irg . for this, all the possible paths that connect lps and irg were calculated using the k shortest simple (loopless) paths-"graphkshortestpaths"-implementation from matlab and the representation of the resultant networks is shown in fig . in the mouse network, irg has direct connections with the transcriptional regulators irf , prdm , cebpd and stat , while it has indirect connections with junb and cebpb (fig a) . similarly, in the human network, irg is directly connected to the transcriptional regulators irf , vdr, stat , runx , rara and ets , with rara and ets be predicted to have an inhibitory effect on irg expression. moreover, irg has one indirect interaction with fos, which, according to the network, transcriptionally regulates irg in multiple ways (fig b) . given the topology of these simple paths networks, we then calculated the gene essentiality metric for all the transcriptional regulators to rank their importance in the network ( table ). the essentiality metric scores were calculated as described in materials and methods. the total paths represent the number of paths in the simple paths networks from lps to irg . the paths present after perturbation of transcriptional regulators is the number of paths where a specific regulator is not present and does not disturb the signal transduction between lps and irg . a higher essentiality metric score value corresponds to a higher importance of the specific node between lps and irg . based on these scores, we were then interested to investigate the effect of the transcriptional regulator irf on irg expression, since from our ranking, irf resulted to be the top scoring transcriptional regulator in both the mouse and human networks. gene interference mediated by sirnas is widely used to study the effects caused by gene silencing in functional genomics and therapeutic applications [ , ] were transfected with sirna targeting irf or with a non-targeting sirna as control. after hours of transfection, mouse cells were stimulated with lps and rna was extracted after hours. as a proof of concept, we first silenced cebpb, our second top scoring transcriptional regulator. indeed, cebpb was already previously described to be a transcription factor responsible for irg induction in zebrafish [ ] . silencing of cebpb resulted in % and % decrease of the cebpb mrna levels when compared to the non-targeting sirna in unstimulated and lps-treated cells, respectively (s fig). correspondingly, irg expression was decreased by % and % in cebpb silenced cells as compared to non-targeting sirna in unstimulated and lps treated cells, respectively (s fig). thus, these results confirm the previous findings in zebrafish, showing that cebpb is a transcriptional regulator of irg in mouse macrophages, as also predicted by our analysis. silencing of irf , our first top scoring transcription factor, resulted in % and % decrease of the irf mrna levels when compared to the non-targeting sirna in unstimulated and lpstreated cells, respectively (fig a) . correspondingly, irg expression was significantly decreased by % and % in irf silenced cells as compared to non-targeting sirna in unstimulated and lps treated cells, respectively (fig b) . in parallel, proteins were extracted from the cells hours after lps stimulation. as expected, irf and irg /cad proteins were not detectable in control conditions, while they were detected in lps-stimulated cells in both sirna irf and non-targeting sirna conditions. in accordance with gene expression results, irf and irg /cad expression levels both decreased in irf silenced cells as compared to non-targeting sirna transfected cells following lps stimulation ( fig c) . as irg /cad has been recently described to enzymatically convert cis-aconitic acid into itaconic acid [ ] , we next measured itaconic acid levels in irf silenced cells. indeed, itaconic acid amounts in sirna irf cells were decreased by % in unstimulated cells and by % in lps-treated cells when compared to non-targeting sirna treated cells (fig d and e) . similarly to the mouse macrophages, human pbmcs-derived macrophages were transfected for hours with sirna specifically targeting irf or a non-targeting sirna as control. transfected macrophages were then stimulated with lps and rna was extracted after hours. differently from mouse macrophages, human primary macrophages do not have a detectable basal level of irg expression, thus we only analysed gene expression levels following lps exposure. as a result of irf silencing, which held a decrease of its expression levels, as an average of four donors, by % (fig a) , irg expression levels were correspondingly reduced by approximately % under lps conditions (fig b) in sirna irf transfected cells when compared to non-targeting sirna treated cells. in order to gain additional support for irf as a direct regulator of the irg locus, we overlaid the putative top scoring irf binding motifs identified in our match™ analysis (s table) with open chromatin regions in the human irg locus in pbmcs and blood cd + monocytes using publicly available encode data on ucsc genome browser (s fig). the results show that, in the human monocyte lineage, irf binding sites in the irg locus are associated with active chromatin marks, such as h k me and h k ac, which mark active/poised enhancers and which could represent putative irf binding sites responsible for irg expression. taken together, these results show that irf is a transcriptional regulator of irg in both mouse and human macrophages and that our combination of computational and experimental approaches represents an efficient method to identify transcriptional regulators of a given inducible gene. we have recently revealed that irg codes for the enzyme irg /cad which catalyses the decarboxylation of cis-aconitate, a tca cycle intermediate, to itaconic acid [ ] . this metabolite inhibits isocitrate lyase, the key enzyme of the glyoxylate shunt [ , ] . we demonstrated that silencing irg in murine macrophages decreases itaconic acid levels resulting in a defective antimicrobial activity towards different bacterial infections. thus, we showed that irg /cad, by the production of itaconic acid from a tca cycle intermediate, plays an essential role in innate immune responses. here, we additionally show that irg is not only expressed by human macrophages, but also by monocytes as well as by dendritic cells. thus, due to the important role of irg /cad at the interface between the innate immune system and the central carbon metabolism, we aimed to shed more light into its expression and transcriptional regulation under inflammatory conditions. for this, we designed an integrative approach of computational and experimental methods to identify the transcriptional regulators of irg in mammalian macrophages, where the notion "co-expression implies co-regulation" is satisfied [ ] . transcription factors play a pivotal role in modulating gene expression and thus contribute to the overall regulation of biological processes. in order to identify the transcriptional regulators responsible for irg induction, we used a weight matrix-based tool for searching putative transcription factor binding sites in dna sequences: match tm [ ] . this tool uses the matrix library collected in transfac , thus providing the possibility to search for a large variety of different transcription factor binding sites [ ] . the main bottleneck in using these tools is the high amount of false positives generated due to the short and degenerate sequence motifs of transcription factors. a comprehensive method is hence needed to increase the specificity. with our algorithm, we eliminated non-cell type/condition specific transcription factors, thus rendering the biological validation appropriate. though our method could potentially identify the transcriptional regulators of any given gene, it presents some limitations. for instance, to contextualize our networks, we exclusively considered degs from microarrays data, although some transcription factors are phosphorylated in order to be activated and shuttled into the nucleus to subsequently act as transcription factors. thus, in follow-up studies it would be valuable to add an additional layer of information on top of the transcriptomic data, such as phosphoproteomics data. this could allow contextualizing the generated networks not only taking into account gene expression data, but also the phosphorylation data of various regulating proteins. from our contextualized murine and human networks, we identified potential transcriptional regulators of irg in macrophages. of interest, our mouse and human analysis provide insights for the identification of species-specific regulators for irg induction under lps activation. although the human data were obtained from primary cells, while the mouse analysis was conducted using the macrophage cell line raw . , it is tempting to speculate that the transcriptional machinery inducing irg expression, with the exception of irf , is mostly species-specific, as highlighted by the different transcriptional regulators identified in the two species (table ) . experimentally, we confirmed that cebpb in mouse and irf in both species represent positive targets which modulate irg expression. we used cebpb as a proof of concept for our method, since we predicted it in our ranking and it has been previously shown to be a transcriptional regulator of irg in zebrafish (danio rerio). cebpb, as a primary response gene, is known as a transcriptional regulator of the acute phase response [ ] . in our experimental conditions in murine macrophages, cebpb is highly up-regulated under lps stimulation and, when silenced, irg expression levels are decreased. these results are in agreement with those obtained in zebrafish, thus highlighting cebpb as a transcriptional regulator of irg in both the species. irf is referred to as a positive transcription factor as it induces several genes, which are important in regulating several immunological and physiological functions in mammalian cells. irf is also transcriptionally activated by several pro-inflammatory cytokines and pathogens [ , ] . irf selectively regulates specific sets of genes depending on the cell type and the appropriate response needed to counter the external stimuli, thus having its function driven by cell-type specific factors. irf mainly activates interferons (ifns) and ifn inducible genes. some of the targets of irf which are known to play a role in host defence are ifn (α, β), inducible nitric oxide synthase (inos), interleukin (il ), cyclooxygenase (cox ), il , caspase - and lysyl oxidase [ , ] . among these, inos is one of the most studied downstream targets of irf . the enzyme inos catalyses the production of nitric oxide (no) which is essential for the antimicrobial and anti-tumorigenic properties of macrophages. no is a biologically active intermediate produced by the cells using l-arginine, an urea cycle intermediate, as the substrate [ , ] . increased no production correlates with resistance towards invading pathogens [ ] . several studies showed that the silencing of irf diminishes the expression of inos and the production of no, thereby attenuating the antibacterial and antiviral activities and thus worsening the severity of the disease [ ] [ ] [ ] . inos is induced upon stimulation by il , il , tnf, ifnγ and lps via irf [ ] . nevertheless, ifnγ and lps are the major and necessary stimulants to induce inos expression and no production in murine macrophages [ ] . its transcriptional induction following lps stimulation occurs after the synthesis of ifns and the jak/stat signalling pathway [ ] . inos promoter requires the dimeric binding of irf for the full cytokine activation. hence, other transcription factors, like nfκb, that are induced by other cytokines, may cooperate with irf to induce the full transcriptional activity of inos [ ] . irf protein is highly unstable with a half-life of minutes [ ] and lpsinduced no production can be abrogated by inhibiting the translation of irf by -hydroxytrans- -decenoic acid, a medium chain fatty acid [ ] . irf is serine-phosphorylated by protein kinase a (pka) and protein kinase c (pkc). mutation of these tyrosine residues inhibit the induction of irf expression [ , ] . it was reported earlier that lps induction of irg in macrophages is mediated via the pkc pathway [ ] . thus the pkc pathway may be responsible for irg induction through irf . irg was shown to be co-regulated and co-expressed with inos when murine cells were either infected with mycobacterium [ ] or stimulated with lps and was also reported as a family member of ifn inducible genes [ , ] . of interest, mycobacteria infection studies in irf knock-out mice revealed that irf is required for the mycobacteria induced granuloma necrosis. gene expression analysis of infected mice resulted in the grouping of differentially expressed genes into different clusters based on the dependence of gene expression on ifnγ or irf . irg was classified to the cluster of genes whose expression is mostly dependent on irf , but less on ifnγ [ ] . in line with the results obtained with mycobacteria infections, our results show that irf expression is highly up-regulated under lps exposure and that irf silencing induces a significant decrease of irg expression levels in both human and murine macrophages. upon tlr activation by external stimuli, such as lps, irf was shown to interact with myd adaptor molecule to translocate into the nucleus and induce the expression of several genes to mediate immune responses [ ] . the analysis of transcription start site (tss) distributions of immune related genes by liang et al. classified irf as a gene dependent on myd pathway along with irg which was stated as an interferon stimulated gene (isg) element [ ] . however, irg was previously shown to be expressed independently from myd or trif adaptor molecules [ , ] , thus demonstrating that additional pathways regulating irg expression independent from irf and myd could exist. it is already known that irf has a significant role in antibacterial and antiviral responses, induction of apoptosis and tumorigenesis, with several of these processes known to be mediated by inos and no [ ] . thus, from our results, it is tempting to conclude that irf regulates these processes also through the induction of irg and itaconic acid production. to this end, inos and irg can be considered as the gene twins, regulated by irf , which contribute to the host protection towards pathogen invasion through the production of effector molecules, such as no and itaconic acid, respectively. of interest, in contrast to mouse, inos expression and no production in human cellular systems have been an argument of discussion by cellular biologists and immunologists since a long time [ ] . even though there were reports showing low levels of inos and no in activated human cells [ ] [ ] [ ] , it was argued that these results neither show the functional pathway of no production, as through l-arginine in mouse, nor describe the precise experimental details, such as cell types and culture conditions [ ] [ ] [ ] . in accordance with this argument, accordingly to our microarrays data, we did not detect inos expression in lps-stimulated human macrophages, while its expression was highly up-regulated in mouse cells. however, taking the side of the discussion that human macrophages do not express inos, irf plays a crucial role in mediating antimicrobial responses through irg gene regulation in human immune cells. finally, in a recent study irf and irg expression levels were correlated when neurons were infected with several viruses such as saint louis encephalitis virus (slev) and a coronavirus (mouse hepatitis virus, mhv). irg had higher basal and also ifn-β-induced expression levels in granule cell neurons when compared to cortical neurons. viral replication was increased in granule cell neurons when transduced with lentiviruses expressing shrna targeting irg [ ] , thus postulating the role of irg in inhibiting viral replication in neurons. irf induction by interferons is already known as an antiviral response against certain viruses [ ] . even though the role of irg in antiviral responses is not yet reported, future studies could reveal that irf mediates antiviral actions through the induction of irg . here we provide evidence for a method integrating computational and experimental approaches as a tool to successfully identify transcriptional regulators of a given inducible gene, thereby stepping towards the understanding of its gene regulatory network. improved knowledge about irg transcriptional regulation provides a better understanding of the molecular mechanisms that are involved in specific inflammatory and infectious diseases. to this end, we identified irf as a transcriptional regulator of irg expression in both human and mouse macrophages under inflammatory conditions. understanding the mechanisms of irg induction during immune responses could lead to novel therapeutic approaches, which aim to modulate the intrinsic host response. show the mean of technical replicates (± sem) of irg mrna levels measured by real-time pcr normalised with l as the housekeeping gene. ÃÃÃ p < . , Ã p < . . b) rna was extracted from raw . macrophages at , , , , , minutes after treatment with lps ( ng/ml). the bars show the mean of biological replicates (± sem) of irg mrna levels measured by real-time pcr normalised with l as the housekeeping gene. ÃÃÃ p < . , ÃÃ p < . , Ã p < . . toll-like receptor signaling combined chromatin and expression analysis reveals specific regulatory mechanisms within cytokine genes in the macrophage early immune response cloning and analysis of gene regulation of a novel lps-inducible cdna mycobacterium paratuberculosis, mycobacterium smegmatis, and lipopolysaccharide induce different transcriptional and post-transcriptional regulation of the irg gene in murine macrophages immune-responsive gene protein links metabolism to immunity by catalyzing itaconic acid production the proinflammatory cytokine-induced irg protein associates with mitochondria myd -dependent changes in the pulmonary transcriptome after infection with chlamydia pneumoniae infection-and procedure-dependent effects on pulmonary gene expression in the early phase of influenza a virus infection in mice progesterone regulation of the mammalian ortholog of methylcitrate dehydratase (immune response gene ) in the uterine epithelium during implantation through the protein kinase c pathway immune-responsive gene is a novel target of progesterone receptor and plays a critical role during implantation in the mouse immune responsive gene (irg ) promotes endotoxin tolerance by increasing a expression in macrophages through ros augments bactericidal activity of macrophage-lineage cells by regulating β-oxidation-dependent mitochondrial ros production expression of many immunologically important genes in mycobacterium tuberculosis-infected macrophages is independent of both tlr and tlr but dependent on ifn-alphabeta receptor and stat lipopolysaccharide stimulates the myd -independent pathway and results in activation of ifn-regulatory factor and the expression of a subset of lipopolysaccharide-inducible genes regulation of lipopolysaccharide-inducible genes by myd and toll/il- domain containing adaptor inducing ifn-beta advantages and limitations of current network inference methods irf family of transcription factors as regulators of host defense meke: discovering the functions of gene products from biomedical literature via sentence alignment plau inferred from a correlation network is critical for suppressor function of regulatory t cells pathway studio-the analysis and navigation of molecular networks aracne: an algorithm for the reconstruction of gene regulatory networks in a mammalian cellular context. bmc bioinformatics trustful inference of gene regulation using stability selection wisdom of crowds for robust gene network inference matinspector and beyond: promoter analysis based on transcription factor binding sites matchtm: a tool for searching transcription factor binding sites in dna sequences predicting missing expression values in gene regulatory networks using a discrete logic modeling optimization guided by network stable states functional macrophage cell lines transformed by abelson leukemia virus metabolitedetector: comprehensive analysis tool for targeted and nontargeted gc/ms based metabolome analysis a model-based background adjustment for oligonucleotide expression arrays transfac: transcriptional regulation, from patterns to profiles discrete logic modelling optimization to contextualize prior knowledge networks using prunet detecting cellular reprogramming determinants by differential stability analysis of gene regulatory networks sirnas: applications in functional genomics and potential as therapeutics sequence-dependent stimulation of the mammalian innate immune response by synthetic sirna mycobacterium tuberculosis: here today, and here tomorrow mycobacterium tuberculosis isocitrate lyases and are jointly required for in vivo growth and virulence context specific transcription factor prediction induction of the c/ebp beta gene by dexamethasone and glucagon in primary-cultured rat hepatocytes irf- : the transcription factor linking the interferon response and oncogenesis expression and dna binding activity of the recombinant interferon regulatory factor- (irf- ) of mouse identification of ifn regulatory factor- binding site in il- p gene promoter new insights into the regulation of inducible nitric oxide synthesis altered immune response of interferon regulatory factor -deficient mice against plasmodium berghei blood-stage malaria infection requirement for transcription factor irf- in no synthase induction in macrophages involvement of the irf- transcription factor in antiviral responses to interferons interferon regulatory factor- regulates the autophagic response in lps-stimulated macrophages through nitric oxide release of reactive nitrogen intermediates and reactive oxygen intermediates from mouse peritoneal macrophages. comparison of activating cytokines and evidence for independent production nonconventional initiation complex assembly by stat and nf-kappab transcription factors regulates nitric oxide synthase expression binding of the transcription factor interferon regulatory factor- to the inducible nitricoxide synthase promoter activation of ifn-beta element by irf- requires a posttranslational event in addition to irf- synthesis inhibitory effect of -hydroxydecanoic acid on lipopolysaccharide-induced nitric oxide production via translational downregulation of interferon regulatory factor- in raw murine macrophages a role for casein kinase ii phosphorylation in the regulation of irf- transcriptional activity mutational analysis of interferon (ifn) regulatory factors and . effects on the induction of ifn-beta gene expression -aminopurine inhibits lipopolysaccharide-induced nitric oxide production by preventing ifn-beta production mycobacteria-induced granuloma necrosis depends on irf- evidence for licensing of ifngamma-induced ifn regulatory factor transcription factor by myd in toll-like receptor-dependent gene induction program analysis of changes in transcription start site distribution by a classification approach effect of cycloheximide on the expression of lps-inducible inos, ifn-beta, and irf- genes in j macrophages of mice and not men: differences between mouse and human immunology evidences for inos expression and nitric oxide production in the human macrophages expression of the inducible no synthase in human monocytic u cells allows high output nitric oxide production human mononuclear phagocyte inducible nitric oxide synthase (inos): analysis of inos mrna, inos protein, biopterin, and nitric oxide production by blood monocytes and peritoneal macrophages macrophage biology and immunology: man is not a mouse species differences in macrophage no production are important nitric oxide production and nitric oxide synthase type expression by human mononuclear phagocytes: a review differential innate immune response programs in neuronal subtypes determine susceptibility to infection in the brain by positive-stranded rna viruses we thank dr. elisabeth john and dr. lasse sinkkonen for helping with human rna samples for microarrays experiments. we are grateful to françois bernardin and dr. tony kaoma for excellent assistance with microarrays platforms and analysis. key: cord- -p q f c authors: nan title: posters_monday_ october date: - - journal: intensive care med doi: . /s - - - sha: doc_id: cord_uid: p q f c nan a huge variability in excess risk of death, ranging from to %, from ventilator associated pneumonia (vap) have been reported in the literature [ ] . this large between-study variation can be attributed to difference in definitions but also to incorrect estimation by standard statistical methods, i.e. inappropriate adjustment for informative censoring and time dependent confounders. the aim of this study was to take into account above statistical shortcomings and to assess the excess risk of vap by using an extension of a recently developed techniques from the field of causal inference [ ] . materials and methods. data was retrieved from a large longitudinal, high quality multi-centric icu database from france (outcomerea). a random sample of consecutive patients ventilated [ h from icus over a year period were included. vap was defined as clinical suspicion plus at least one positive proximal or distal sampling with quantitative count using classical thresholds. only the first vap episode was taken into account. we considered discharge from the icu as a competing risk and estimated the attributable -day icu mortality of vap by comparing the counterfactual cumulative incidence of death for the entire population under different hypothetical infection paths. baseline characteristics indicating underlying co-morbidity and longitudinal (daily measured) severity of illness indicators together with all other known confounders until vap developed were taken into account through the use of a marginal structural model [ ] . results. a total of , icu patients were included. mean (sd) age and saps ii score were ( ) and ( ), respectively. seventeen and % were admitted after scheduled and emergency surgery respectively, % were medical patients. forty-two percent had an underlying chronic illness (knaus). nine percent received dialysis in the icu. a total of ( %) patients developed vap within days of admission ( and % within and days, respectively). crude icu-mortality rates in patients with and without vap were and %, respectively. when taking into account all the confounders, we found a . % increase ( % ci . - . , p = . ) in the hazard of -day icu-death per additional day since the development of vap. provided vap could have been prevented in the whole population -day mortality would have decreased by . %. conclusion. the excess risk of death from vap estimated by marginal structural models is lower than commonly reported in the literature. this indicates that underlying comorbidities and the evolution of severity of illness until vap are insufficiently taken into account by current standard statistical methods. reference (s) . . rello et al ( ) introduction. the intensive care society (uk) has recently published national guidelines regarding many aspects of percutaneous tracheostomy management [ ] , however, these guidelines do not make any recommendations regarding antibiotic prophylaxis during the procedure. we recently audited uk practice and have established that only % of the units give prophylactic antibiotics for patients known to be colonised with methicillin-resistant staphylococcus aureus. the low rate of antibiotic use is surprising given that pneumonia and/ or bacteraemia following pt is frequently caused by organisms (non-mrsa) that colonise the patients' skin and/or airway [ , ] . objective. to establish the incidence of mrsa positive sputum and/or blood cultures following pt in patients colonised with mrsa in their nose or throat. methods. we audited all the patients who had pt performed between and who were known to be colonised with mrsa in their nose or throat, (but who had negative sputum cultures) before pt was undertaken. we wanted to find how many of these patients developed mrsa positive sputum and/or blood cultures in the first week following their pt. results. from a total of patients admitted to critical care between and only seven mrsa colonised patients required pt. all of these patients had mrsa colonised throat or nose with negative sputum and blood cultures prior to the pt. no patients were given prophylactic antibiotics during the pt as this was our standard practice. four ( %) developed mrsa positive sputum cultures in the first week following the procedure. one ( %) developed mrsa bacteraemia on day following pt. over the same period there were no case of mrsa bacteraemia in the mrsa colonised patients who did not undergo pt. microbiology bacterial biofilm has been observed in the surface of the endotracheal tube (ett) in mechanically ventilated patients and recent studies relate the presence of biofilm with the incidence of ventilator-associated pneumonia (vap). acinetobacter baumanii is a gramnegative opportunistic nosocomial pathogen involved in the production of vap, and capable of biofilm formation on abiotic surfaces. objective. to analyse the presence of biofilm in the ett by using scanning electron microscopy (sem) and to identify the microorganisms contributing to its formation in patients admitted in an icu endemic for a. baumanii. methods. from march to september , consecutive patients admitted to our unit and mechanically ventilated were included in the study. etts after extubation were (a) sent to microbiological culture, and (b) fixed with % paraformaldehyde- % glutaraldehyde for one hour, dehydrated with increasing ethanol concentrations, and processed for sem. etts were observed under sem to assess the presence and extension of the biofilm, and to recognize bacterial or fungal forms. results. there were males ( %) and females ( %) with a median age of years (range - ).the median apache ii score in the first h was (range - ). the median duration of intubation was days (range . causes of intubation were coma in patients ( %), respiratory failure in ( %), and heart failure in ( %). the microbiological isolations showed: a. baumanii ( %), staphylococcus non aureus ( %), pseudomonas spp. ( %), streptococcus viridans ( %), staphylococcus aureus ( %), enterococcus faecalis ( %), candida albicans ( %), and others ( %). under the sem, biofilm was identified in the % of all cases and was abundant in ( %), regular in ( %), and scarce in ( %). morphological identification of microorganisms observed under sem showed: cocci in ( %), bacilli in ( %), and yeast in ( % introduction. hospital-acquired infection is often linked to the standard of ward cleaning however the impact of increased quality of cleaning and deep cleans are unknown. objectives. this study aimed to determine the effect of enhanced cleaning on local contamination rates of hospital pathogens and whether this results in a reduction in patient colonisation. a cross-over one-year study was performed in the intensive care units (icu) of two teaching hospitals, which screened patients weekly for mrsa. in randomized two-month periods and in addition to conventional cleaning using detergent and mops, high contact areas were cleaned twice daily by a team of trained hygiene technicians using microfibre cloths. using contact plates, samples were taken at nine sites around the bed area and ward over bed-days per week. hand hygiene was encouraged and compliance audited. results. only . % of the planned , local samples were missed and this was equal between study phases. average hand hygiene compliance was similar between enhanced and standard phases [hospital a . % ( / ) vs. % ( / ) and hospital b . % ( / ) vs. . % ( / )]. patient characteristics were similar during standard and enhanced cleaning periods. of the sites tested, samples taken from bed rails were most likely to be contaminated with mrsa (or = . ; % ci: . - . ) followed by nurses' hands (or = . ; % ci: . - . ). analysis of these site-samples also confirmed that enhanced cleaning significantly reduced environmental contamination (or = . ; % ci: . - . ; p \ . ). the effectiveness of enhanced cleaning in removing mrsa contamination did not vary with the sample site. a sub-group analysis of samples only taken from nurses' hands showed a non-significant reduction in mrsa hand contamination associated with enhanced cleaning although associated uncertainty was large (or = . ; % ci: . - . ; p = . conclusions. this is the first prospective controlled study examining the effectiveness of enhanced cleaning in preventing spread of multiresistant pathogens within icu. although both environmental and hand contamination were reduced, enhanced cleaning of high contact surfaces was not associated with a reduction in cross infection. conclusions. isd of secretions reduces the incidence of vap in patients receiving. css alone, or in combination with isd has no significant effect on incidence of vap. hence, isd may be recommended for vap prevention, considerations other than prevention of vap should determine the choice of the suction system in a mechanically ventilated patient. to show a mortality benefit, larger, multi-center trials may be required. decreasing incidence of ventilator-acquired pneumonia (vap) is increasingly regarded as a priority in icu quality programs. subglottic secretion suctioning (sss) has been associated with a decreased risk of vap. a previous metaanalysis concluded that sss reduces the risk of vap, but it included only five randomized controlled trials (rct), and sss is still underused, perhaps considering the available evidence is insufficient. we planned a systematic review and metaanalysis of sss for vap prevention. pubmed, embase and cdsr were searched for rct studying the influence of sss on vap incidence. additional outcomes were mortality (within icu or hospital), icu and hospital stay, mechanical ventilation duration and time from intubation to vap diagnosis. additional references and sources of information were searched, and authors were contacted as necessary. rct were found, but one of them was excluded for not having enough data for analysis. rct were analysed, including , patients. quality of the rct was only moderate. qualitative outcomes were homogeneous between studies, so were analysed by a fixed effects model; quantitative outcomes were very heterogeneous, and were analysed by a random effects model. compared to control, sss decreased vap incidence (rr . ; % ci . - . ), but not mortality (rr . ; % ic . - . ). sss delayed vap onset for . days ( % ci . - . ), shortened mechanical ventilation for . days ( % ci . - . ) and decreased icu length of stay for . days (ic % . - . ). in two rct, no differences were found in the hospital length of stay. conclusion. sss reduces vap incidence and delays vap onset, shortening mechanical ventilation and icu length of stay, but not decreases icu or hospital mortality. data from rct support the use of sss as an adjunctive tool to prevent vap. introduction. in comparison to ventilator-associated pneumonia (vap), less data are available on ventilator-associated tracheobronchitis (vat). however, vat may be associated with considerable morbidity [ ] . aim. to investigate prospectively the incidence and outcomes of vat. we studied prospectively all patients who received mechanical ventilation in the general intensive care unit of a tertiary hospital in greece between september-november . vat diagnosis required temperature ([ °c) or leukocyte count [ . per ml or leukopenia \ . per ml) (at least one of these) plus new onset/change of purulent endotracheal secretions. vap diagnosis required the aforementioned criteria plus appearance of new and persistent pulmonary infiltrates on chest radiography. microbiological documentation was based on the growth of microrganisms in bronchial aspirations ([ . cfu) or bal ([ . cfu) . results. forty-six patients were included, median (iqr) age was ( . years. eleven ( %) patients presented vat, presented vap and patients presented none of these two disorders (np). there were no significant differences between vat and vap cases in terms of baseline characteristics (diagnosis, respiratory compliance, apachee, murray score), occurrence of sepsis or ards and microbiology; pseudomonas aeruginosa, acinetobacter baumannii, staphylococcus aureus and klebsiella pneum. were the most common bacteria in both vat and vap. patients who presented vat or vap had significant longer hospitalization and mechanical ventilation duration (days) compared to , ( - ) vs. ( - ), (p = . )] and [ ( - ) , ( - ), ( - ) , (p = . ), respectively]. icu mortality was , , %, for patients with vap, vat and np, respectively (p = . ). conclusions. incidence and microbiological pattern was similar in vap and vap in these case series. both vat and vap were associated with longer hospitalizations and mechanical ventilation duration. further analysis with a larger cohort of patients is required to give conclusive remarks. reference (s) . . nseir s et al ( ) nosocomial tracheobronchitis in mechanically ventilated patients: incidence, aetiology and outcome. eur respir j : - . g. c. choutas , v. g. nolas , a. kalantzi , a. moutzouri , g. k. anthopoulos intensive care unit, \ \ [ [ general air force hospital, athens, greece introduction. endotracheal suctioning is an essential part of care for patients receiving mechanical ventilation, to keep the airways free from bronchial secretions, assuring ventilation and oxygenation. there are two types of suction systems. in the open system, endotracheal suctioning requires disconnecting the patient from the ventilator and introducing a single-use sterile suctioning catheter into the endotracheal tube. closed systems are changed every and h. to determine whether ventilated patients treated with css in an intensive care unit (icu) differ as to airway bacterial colonization and colonization of the suction system based on cultivation of both bronchial secretion and suction catheter tip and if cultivation of suction catheter tip is adequate in place of bronchial aspirate cultivation. methods. patient, incubated and ventilated in the icu ward were studied in a period of one year ( to ) , on admission to the icu a css (trach care mac) was connected. closed multi-use catheters were changed daily. two-pass endotracheal suctioning was performed as needed. ba cultures were obtained on admission and the next day. radiographs taken before, during, and after ba and css cultures were graded for pneumonia and a modified score for vap. of the patients css samples ( . %) and ba samples ( . %) were sterile. airway colonization with gram-negative bacteria and fungi occurred in the majority of the patient . % and gram-positive bacteria in %. cultivation of css revealed gramnegative bacteria and fungi occurrence . % and gram-positive bacteria in . %. with the current sample no significant difference was found between the positive results of trach care tip cultivation and ba cultivation p = . . objectives. to reduce the use of sedatives and to decrease the amount of time spend on a ventilator by specific ramsay-instructions and checks for sedation-protocol-adherence. methods. in april , after introductional lessons to doctors and nurses, we started and collected data for months. a yellow reminder was attached to the medical-instructions-form and doctors were requested to fill in the ramsay-score on a daily basis. once in a week patients and records were screened to assess protocol-adherence. each nurse and each intensive care-unit received feedback on their compliance to the bundle-elements. the total amount of sedatives per month was divided by the number of ventilator days, resulting in an average dose midazolam/propofol per ventilator day. the median and interquartile range of ventilator days/patient was also calculated and all data were compared with the same period in . we accomplished a reduction in the use of sedatives and costs. introduction. ventilator associated pneumonia (vap) often occurs in patients who are mechanically ventilated. the incidence rate varies between and % for patients in the intensive care unit. it has been the second most common hospital-associated infection after that of the urinary tract. the diagnosis of vap is difficult because of different existing definitions. hypothesis. our hypothesis was, that lowering vap incidence rate, could be done by a bundle of five interventions. the purpose of introducing multiple ventilatory interventions as a bundle, was to lower vap incidence rate by %. methods. during the last months of all patients who were ventilated [ h, were investigated for vap. the diagnosis of vap was done according to the criteria supposed by the dutch working group on infection prevention. a new infiltrate on chest x-ray after h ventilatory support in combination with fever, leucocytosis, increased need for oxygen and culture of blind bronchial secretion c cfu/ml. a ventilator bundle was introduced on all icu wards as inspired by the institute of healthcare improvement. five interventions were introduced: head of bed [ °, reduction of sedatives as low as possible according to prescribed ramsay score, assessment of readiness to extubate, cuff-pressure measurement times a day with application of cuff pressures between and cm h o, and oral care with chlorhexidine . % times a day. icu nurses were trained in the first months of . the last months of were used to evaluate the bundle intervention in comparison with the last months of . results. patients were included in and in . after introduction of the ventilator bundle, the incidence per , ventilator days decreased from . % to . % per , ventilator days. introduction. continuous positive airway pressure (cpap) may improve oxygenation in patients with mild to moderate acute hypoxemic respiratory failure (ahrf) and avert further deterioration and need for intubation. objectives. aim of our study was to assess the physiologic effects produced by the addition of periodic hyperinflations (sigh) to cpap in patients with ahrf. we studied patients with non-cardiogenic ahrf. four trials of one hour each were performed at a constant fio % during ( ) spontaneous breathing (sb) via a venturi mask, ( ) cpap , ( ) cpap ? sigh/min of cm h o for s (cpap sigh ), ( ) cpap . cpap, via helmet, was maintained at cm h o troughout the whole study period. pao /fio ratio (p/f), paco , ph, respiratory rate (rr), arterial blood pressure (abp), heart rate (hr), dyspnea and patient comfort (by means of separate visual analog scales) were measured at the end of each trial. results. overall, p/f was significantly (p \ . ) improved by cpap (cpap ± mmhg, cpap sigh ± mmhg, cpap ± mmhg), as opposed to sb ( ± mmhg). overall, the sigh did not significantly improved p/f. in the six patients with bilateral infiltrates, however, the rate of improvement in p/f significantly (p \ . ) augmented with the introduction of a sigh as compared with those with monolateral infiltration ( vs. % respectively, being % the increase from venturi to cpap ). paco , ph, rr, hr, abp, dyspnea and comfort were not significantly different between trials. conclusions. in patients with ahrf, cpap improves oxygenation without affecting hemodynamics. the addition of a sigh to cpap further improved oxygenation only in patients with bilateral pulmonary infiltrates. background. adaptive support ventilation (asv) is a novel electronic ventilator protocol that incorporates the recent and sophisticated measurement tools and algorithms. the target tidal volume and respiratory rate are continually adapted to patient's respiratory physics and varying medical conditions. in injured lung, the asv should actively adjust ventilatory parameters achieving minimal work of breathing to meet the lung protective strategies. but there were little literatures describing its efficacy when applied to korean population. methods. from may to january , we observed initial mechanical ventilation parameters in patients receiving asv due to various causes ( lung injuries including community acquired pneumonia, hospital acquired pneumonia, interstitial lung diseases, pulmonary tuberculosis and idiopathic cases; without lung injury which comprise trauma cases, strokes, suicidal attempts and other cases). the mean age of studied population was . years (male:female = : ). the data were collected within the first h of mechanical ventilation. conclusion. as expected, adaptive support ventilation delivered smaller tidal volume and higher respiratory rates for injured lungs. asv efficiently operated in korean ali patients without any serious drawbacks and favorably adjusted the tidal volume and respiratory rates combination in relation with rcexp to meet lung protective strategies. introduction. neurally adjusted ventilatory assist (nava) is a mode of mechanical ventilation that uses the electrical activity of the diaphragm to control the ventilator obtaining an improved patient-ventilator synchrony and an efficient unloading of the respiratory muscles. nava is characterised by a variability of the breathing pattern and the absence of a constant flow, which makes impossible the determination of reliable data of respiratory mechanics by using the rapid occlusion method. we have previously demonstrated that the least squares fitting method (lsf) could be used during pressure support ventilation (psv), provided that the level of ps is sufficiently high to unload the inspiratory muscle. hence we made the hypothesis that ( ) reliable data of respiratory mechanics can be obtained by applying the lsf method during nava and ( ) the lsf method should work better during nava because of the characteristics of the flow and pressure traces. methods. ten patients undergoing mechanical ventilation for acute respiratory failure were enrolled. they were ventilated using randomly either psv or nava with the the same peepe and tidal volume (v t ). data of resistance (r rs ), elastance (e rs ) and total positive end expiratory pressure (peep tot ) were obtained by fitting the equation paw = r rs v ? v t / c rs ? peep tot during inspiration, because of the possible presence of expiratory flow limitation. the coefficient of determination (cd) of the applied equation was used to compare data obtained during nava and psv, the higher being the cd, the better the quality of the data. moreover patients were sedated and ventilated in volume controlled ventilation (acv) with the aim of calculating data based on rapid occlusion method and compare them with those obtained with lsf method by using the bland-altman analysis. ( ) data obtained with lsf were statistically more reliable during nava (mean cd: . ± . ) than during psv (mean cd: . ± . ; p \ . ). ( ) the cd obtained at every level of nava was always higher then . . on the contrary, the cd obtained at low level of psv was less than . due to the presence of inspiratory muscle activity. ( ) the bland-altman analysis demonstrated lower bias and higher precision between traditional data and those calculated during nava (bias: . ; limit of agreements: - . / . ) compared to psv (bias: . ; limit of agreements: - / . ). conclusion. the application of the lsf method during nava allows calculation of reliable data of rrs, ers and peeptot, which are independent of the level of nava applied. this is of clinical relevance since psv allows calculation of reliable data only at high level of pressure support. it appears that the influence of inspiratory muscle activity on respiratory mechanics is less relevant during nava ventilation, suggesting a more physiological ventilation during nava both in terms of timing and of delivering adequate level of assist throughout each breath. introduction. nava is a new spontaneous-assisted ventilatory mode based on the detection of diaphragmatic electrical activity (eadi) and its feedback to adjust ventilator settings. nava uses the eadi, an expression of the respiratory center's activity, to initiate pressurization, set the level of pressure support and cycle the ventilator into exhalation. therefore, nava should theoretically allow near-perfect synchronization between the patient and the ventilator. however there are few data documenting these effects in intensive care patients. to determine whether nava can improve patient-ventilator synchrony compared to standard pressure support (ps) in intubated intensive care patients. comparative study of patient-ventilator interaction during ps with clinician determined ventilator settings and nava with nava gain (proportionality factor between eadi and the amount of delivered inspiratory pressure) set as to obtain the same peak airway pressure as the total pressure obtained in ps. a min continuous recording with each ventilatory mode was performed allowing determination of trigger delay (t d ), patient neural inspiratory time (t in ), duration of pressurization by the ventilator (t iv ), excess duration of pressurization (t i excess = t iv -t in /t in ) and number of asynchrony events by minute: non-triggering breaths, auto-triggering, double triggering, premature and delayed cycling. results are given in mean ± sd. p is considered significant if . . . ± . . ± . . ± . n asynchrony/minute . ± . . ± . * . ± . respiratory rate (min - ) . ± . . ± . na * p \ . conclusion. compared to standard ps, nava improves patient ventilator interaction by reducing td and the overall incidence of asynchrony events. there is also a strong trend in reducing delayed cycling. this ongoing trial should provide evidence that nava can indeed improve patient-ventilator synchrony in intubated patients undergoing ps. introduction. high fio and hyperoxia may induce pulmonary injury and may increase oxidative stress. guidelines suggest a target arterial oxygen tension of kpa [ ] . a canadian questionnaire study found considerable variation in the attitudes, beliefs and practices of intensivists in the management of oxygen therapy [ ] . however, the actual response of intensivists to hyperoxia in patients has never been studied. in this retrospective database study we investigated adherence to guidelines concerning oxygen therapy in a dutch academic intensive care. all arterial blood gas (abg) data from mechanically ventilated patients from to were drawn from an electronic storage database (metavision) of a mixed -bed icu in a university hospital in amsterdam. mechanical ventilation settings at the time of the abg as well as the successive abg were retrieved. the statistical analysis was carried out with spss . . results. . abg's from mechanically ventilated patients were retrieved including corresponding ventilator settings. in . ( %) of the abg's po was[ kpa. initial ventilator settings and adjustments based on abg's of this group are shown in table [data represent median ( th/ th percentile)]. in % of the lowest fio group, fio was exactly %. in only % of cases with po [ kpa the fio was decreased. hyperoxia was accepted with no adjustments in ventilator settings if fio was % or lower. introduction. major patient ventilator asynchronies are frequent during non-invasive ventilation (niv) especially due to leaks.niv can be delivered using icu ventilators or specifically designed niv ventilators. although icu ventilators are traditionally used for invasive mechanical ventilation, specific niv modes have been recently implemented. the impact of these niv modes as well as niv ventilators on patient ventilator asynchrony is unknown. our objective was to compare the incidence of patient ventilator asynchrony between invasive or niv mode of icu ventilators and niv ventilators. patients and methods. icu patients with acute respiratory failure requiring niv were studied during three randomized consecutive min-periods of niv: icu ventilator with and without niv mode and niv ventilator. we used two icu ventilators: evita xl (dräger) and engström (general electrics) and niv ventilator: bipap vision (respironics). flow and airway pressure were continuously recorded to determine breathing pattern. to detect major patient ventilator asynchrony we used surface diaphragmatic and/or sternocleidomastoid electromyogram allowing to assess neural patient's inspiratory time and to define asynchronies: ineffective triggering, auto-triggering, double-triggering, prolonged and short cycles. asynchrony was quantified using an asynchrony index as previously described. results. these preliminary results concern ten patients ( males and female) with a mean age of ± and a saps of ± . reason for niv was acute exacerbation of copd (n = ), acute pulmonary edema (n = ) and post-extubation (n = ). at time of study, ph was . ± . , paco ± mmhg and pao ± mmhg. ventilatory settings were set by the clinician and kept constant during the three periods with a ps level of ± cm h o and a pressurization ramp of ± ms, a peep level of ± cm h o and a fio of ± %. using icu ventilators, inspiratory trigger was l/min and cycling off was % when adjustable. median asynchrony index was . % ( . - . ) using icu ventilators versus . % ( . - . ) using niv mode and . % ( . - . ) using bipap vision (p = . between invasive and niv mode, p \ . between niv mode and bipap vision). asynchrony index was greater than % in three patients using invasive mode, two patients using niv mode and no patient using bipap vision. auto-triggering was the main asynchrony. conclusion. niv ventilator (bipap vision) allowed a marked reduction in patient ventilator asynchrony during niv as compared to niv mode currently available on new generation of icu ventilators. grant acknowledgement. this study was supported by a research grant from respironics. a. armaganidis , k. stavrakaki-kallergi , c. sotiropoulou , a. koutsoukou , j. milic-emili , c. roussos athens university, athens, greece prevalence of expiratory flow limitation (efl) was estimated using the negative expiratory pressure method (nep) in anesthetized, paralyzed mechanically ventilated patients in icu. patients were studied in supine position at zero positive end-expiratory pressure (peep). a nep device especially designed and in build in an evita -draeger respirator, allowed the application of a pressure equal to- cm h o, starting at ms after the onset of expiratory flow and sustained throughout the end of the expiratory time set on the ventilator. patients were categorized in two groups: . non efl ( patients without flow limitation), in whom nep elicited an increase of expiratory flow over the entire expiratory flow-volume (v -v) curve. . efl ( patients with efl), in whom part or the expiratory v -v curve during nep was superimposed on the baseline v -v curve. half of our patients ( %) were flow-limited. no patient without pulmonary disease was found flow-limited, except of a percentage of morbidly obese patients ( %). efl was recorded in % of icu patients with pulmonary diseases: % of ards patients, % of patients with respiratory infection, % of asthmatics and % of patients with copd. time constant (s) and inspiratory flow (v insp) were found to predict the severity of flowlimitation expressed as efl % v t . objective. tidal volume (v t ) administered to ards patients can be adjusted depending on body weight. body weight may be estimated, measured or calculated for an ideal or a predicted value based on different formulas [ , ] . besides, those formulas require the measurement of height and may differ depending on gender. we hypothesized that v t value (ml/kg of body weight) may be different and show intrameasure variability depending on the method used. methods. ards patients were included prospectively in the first h after icu admission. the ventilatory parameters were selected by the attending physicians that were foreign to the study. all patients were ventilated by volume controlled-assisted mode. five independent observers estimated the weight (estw) of each patient. they also measured height with a metric tape for calculate the predicted body weight (pbw) [ ] and the ideal body weight (ibw) [ ] . after previous measurements, patients were weighed once with a calibrated scale (scaw). results were compared using analysis of variance. results. patients were studied, % women (age . ± . , saps ii . ± . ; apache ii . ± . ). ventilation parameters at inclusion: v t /scaw (ml/kg) mean ± sd . ± . . ± . . ± . . ± . . ** mean ± sd mean ± standard deviation, min minimum, max, maximum, pbw (men) or . (female) ? . (height in centimetres - . ), ibw (height in meters) , mean diff average of the intraindividual differences of calculated/estimated weight, range intraindividual difference in weight (estimated/calculated) expressed as minimum and maximum * estw versus pbw p \ . , pbw versus ibw p \ . , pbw versus scaw p = . ** v t /estw versus v t /pbw p \ . , v t /pbw versus v t /ibw p \ . , v t /pbw versus v t /scaw p \ . conclusions. our data show that there is no gold standard method for estimate or calculate body weight to adjust tidal volume in ards patients. recommendations based on pwb and ibw not guarantee that tidal volume administered is really those that we want to administrate. reference (s) . . ardsnet. nejm ; : - . . stewart te et al ( hôpital raymond poincaré ap-hp, service de réanimation médicale, garches, france, hôpital saint-louis, paris diderot university, paris, france, centre hospitalier d'etampes, etampes, france, université versailles saint quentin en yvelines, versailles, france rationale-objectives. dyspnea is a major respiratory symptom, which can reveal a severe disease. additionally, it can also result from an inappropriate ventilator setting in mechanically ventilated patients. if these patients are nowadays more and more conscious, prevalence of dyspnea and its clinical, biological and radiological correlates has never been assessed in this population. prospective cohort study conducted in two medical intensive care units (icu) during months. all patients intubated more than h and conscious have been included. the first day when the patient regained consciousness, dyspnea, anxiety and pain were assessed using a visual analogic scale (vas). if dyspnea was found, patient was asked if he experienced ''air hunger'', and/or ''excessive respiratory effort'' and if dyspnea vas was improved after ventilator setting has been changed. demographic, clinical, biological and chest x-ray data and ventilator settings have been collected. results. patients were included (age: ± years; simplified acute physiology score ii (saps ii): (iqr - ). reasons for mechanical ventilation included acute respiratory failure (n = , %), neuromuscular diseases (n = , %), coma (n = , %), and exacerbation of chronic obstructive pulmonary disease (n = , %). dyspnea was present in ( %) patients and was qualified as ''air hunger'' in patients ( %), ''excessive effort'' in ( %) and both in ( %). age, saps ii, reason for mechanical ventilation, respiratory rate, clinical examination, x-ray chest, pao /fio ratio, paco were not statistically different between patients with and without dyspnea. anxiety . ); p \ . ), assist controlled ventilation [ . ( . - . ) ] and diastolic blood pressure ; p = . ) were independently associated with dyspnea in multivariate analysis. ''air hunger'' tended to be associated with controlled ventilation (p = . ) whereas ''inspiratory excessive effort'' was significantly associated with low inspiratory flow, severe hypoxemia (median pao /fio ratio: , p = . ) and marked hypercapnia (median paco : mmhg, p = . ). in % of breathless patients, of ventilator resetting decreased dyspnea. length of icu stay was greater in patients with dyspnea (p = . ) whereas extubation within three days and icu mortality did not differ between the two groups. conclusions. dyspnea is frequent in mechanically ventilated patients and is strongly associated with anxiety, more frequently when controlled ventilation is used and is often reduced after ventilator resetting. assessment of dyspnea in conscious mechanically ventilated patients should be routinely performed in order to improve patients' comfort. methods. an experimental study design was used with a group of first year health care provider students. the students were divided into two groups related to familiarity of the location of exam. a part of students (n = ) were examined in demonstration room (dr) and the other part of students (n = ) in public place (pp) . every student received the same number of training hours ( h) and the same training method in demonstration room. during this exam the students performed a min long, single person cpr related to erc guideline. their performance was measured with calibrated ambu cpr software and the adapted point system of brendan b. spooner's scale. v and t test were used for comparison. p values less than . were considered statistically significant. we did not find difference between dr and pp groups in the correct sequence of bls steps, hand position, adequate frequency and depth of chest compression. between groups of characteristics of ventilation were not significant differences observed. it is first critical point in bls process to assess the quality of patients' spontaneous breathing; therefore it is crucial that the duration of check breathing may be sufficient long. the duration of checking for breathing was significantly (p = . ) shorter in dr groups than pr groups. in the pp groups time interval between chest-compression cycles were significantly (p = . ) longer-more than s-than in dr group. conclusions. the altered location of bls final exam shortens duration of checking for breathing which determines bls providers' decision making on starting chest compressions. the students may be full of confident in the well-known place represented by the shorter time of checking for breathing. the changed place of exam extended time interval between chestcompression cycles, therefore weaken the continuity of chest compressions, and decrease the chance of return of spontaneous circulation. aims. this paper reports an evaluation of the student experience of using a clinical competence assessment tool (ccat) in postgraduate critical care nursing education. the focus is on the perceptions of students in relation to the validity, reliability and usability of the assessment tool. the domains of competence assessed are based on five domains outlined by an bord altranais ( ) . they are: professional/ethical practice, interpersonal relationships, practical and technical skills, utilising a holistic approach to care, clinical decision making and critical thinking skills and organisation and management of care. the assessment process encompasses three clinical assessments and clinical competence is measured using the developmental process of novice, advanced beginner, and competent as described by benner ( ) . students are asked to reflect on their own learning needs prior to each assessment. the assessment includes a discussion on the knowledge that underpins practice thereby showing the integration of theoretical and practical knowledge. questionnaire was administered to all students who recently completed a graduate diploma in nursing studies (critical care) at a specific third level institution. results. the evaluation of the ccat as a mode of competence assessment in postgraduate critical care nursing education was generally positive from the students' perspective. some students considered the holistic nature of the ccat document to be a limitation, suggesting that their level of competency could have been better addressed with a tool that was more oriented toward critical care rather than being so 'broad' in nature. overall respondents considered that the ccat helped them to identify learning needs and found the use of the tool to be a positive experience and easy to use although some respondents considered that the wording of some of the sub-domains and indicators was difficult to interpret. competence assessment is about ensuring the delivery of safe and competent patient care. in order to determine competence a valid and reliable tool is needed. this small scale study presents the views of post registration critical care nursing students on using a competence assessment tool. the findings of this study cannot be generalised, however they do provide insight for educators and students using competence assessment tools in programmes preparing registered nurses for specialist nursing practice. the use of a holistic assessment process needs further explanation. students need to be encouraged to move away from the reductionist approach, which is focussed on tasks and move towards a broader understanding of competent practice. reference (s) . . an bord altranais ( ) requirements and standards for nurses registration education programmes, nd edn. . benner p ( ) from novice to expert excellence and power in clinical nursing practice. addison-wesley, california. to assess the usefulness of a web-based interactive learning package designed to supplement an undergraduate acute care course (very basic) taught to final year medical students. a web-based interactive learning package was developed to supplement a highly rated traditionally taught -day acute/critical care course consisting of pre-course reading, lectures, skill stations and interactive tutorials [ ] . the additional web-based package consisted of narrated lectures, interactive lessons, videos and animations to demonstrate practical procedures and clinical signs, self assessment quizzes and a question and answer forum. topics covered included arterial blood gas sampling and interpretation, acute metabolic disturbances, non-traumatic coma, acute respiratory failure and sepsis. both the package and the course are available to other medical schools free of charge. usefulness of the package was assessed by examining activity logs, a student questionnaire, formal focus group (conducted by an investigator not involved in course preparation or teaching), comparing the results of a post-course mcq based summative assessment with historical controls and comparing results of formative assessment included in the package with the summative assessment. results. over , student-activities were logged by students during the two week course. students completed the questionnaire. with regard to usability, [ % agreed or strongly agreed that interactive lessons and self assessment ran smoothly without faults, with a corresponding score of [ % for narrated lectures and ease of browsing. with regard to usefulness, c % agreed or strongly agreed that the question and answer forum was useful in clarifying areas of doubt and narrated lectures improved understanding of the course material; [ % agreed or strongly agreed that the content as a whole was useful in preparing the respondent to work as a doctor, interactive lessons improved their understanding of how to apply their knowledge, and their understanding of arterial blood gas interpretation and self assessment exercises improved their understanding of the course material. participants in the focus group indicated that the resources provided in the website were useful for learning, specifically the animations, narrated lectures and the question and answer forum. suggestions for improvement included improving the quality of the video and animations, increasing the range of topics covered and ensuring consistency with the printed course manual. there was no correlation between formative and summative assessments but, compared to historical controls, performance in the summative assessment improved ( vs. %, p \ . ). conclusions. the package provided a useful supplement to a traditionally delivered acute care course. introduction. faculty development refers to that broad range of activities that institutions use to renew or assist faculty in their roles. it includes activities that improve an individual's knowledge, skills and attitudes in important areas in teaching, education, research, leadership, administration and career development. in this abstract we will introduce one of the most important methods of faculty development programs. a meeting by the authors ''organizing group'' was conducted to decide on a topic for our workshop and discussed the planning and designing process. we decided on conducting a workshop on clinical teaching methods. a scientific and organizing committee was established, and accordingly work loads and assignments were distributed among them. we gave this workshop a title of ''i am the best clinical educator…are you!? our target audience was acute care management providers, with a capacity of up to participants. we gave a specific time and location of this event. venue was arranged. computers for group work, audiovisual and other logistics were provided. after summarizing the main points for the workshop the organizing committee distributed an invitation letters throughout the higher management and educational leaderships. an address remark was done through invitation from the organizing committee. hot and cold beverages and break lunch meals were provided. posters on the workshop were distributed through out the institution. folders with educational materials were provided for each candidate. pre-course registration was done. once the program for the workshop was finalized a reminder was sent out to the participants on the date and venue for the workshop. participants attended on time, folders, badges with usbs were handed out. a questionnaire was distributed to the audience to estimate their learning experiences and approaches towards teaching styles and methods which were used in their practice. certificates of attendance with cme credit hours were distributed. results. candidates attended this faculty development workshop. % were nurses and % were physicians, during this workshop, three topics were distributed over three groups, one group on how to break bad news. second group about how to conduct microteaching and the third group about how to give feedback. each group was evaluated by three members of the organizing committee, each group was ranked accordingly. all were performed by role play. at the end of the workshop an evaluation form was filled % responders. a five performance scale was used. the strength of the workshop was innovativity and ranked as strongly agree. the only weakness was the place constraint. conclusion. we concluded that a well organized workshop using role play, interactive sessions are effective modality for faculty professional development programs among acute care providers with high satisfaction rate. only of the respondents ( %) indicated they did understand the statistics they encountered in journal articles and % felt it was important to understand these concepts and that they would like to access more easily to biostatistics training. a patient is referred to a higher centre when services are needed to maintain continuity of care.there are guidelines for the safe inter and intrahospital transport of critically ill patients but no guidelines are available for the minimal mandatory content of interhospital referral notes of critically ill patients.this problem is manifold in developing countries. objective. to educate the critical care physicians regarding the deficits in the physicians referral notes with which critically ill patients are referred from one centre to another. it is a prospective observational study on out of hospital referred patients transferred to our intensive care unit (icu) over a period of year. after permission from the institutes ethical committee we reviewed the referral summaries of these patients at the time of icu admission regarding the information available of clinical details, course in the previous hospital and therapeutic interventions. patients with more than h of hospitalization before transfer were included in the study. introduction. in japan, closed icus have been gradually increasing at university hospitals. a closed icu is necessary for a university hospital not only for the hospital activity but also the education of medical students and the training of fellows. they can learn how to manage the circulation and respiration status of severely ill patients in icu. it is indispensable for effective education to ensure sufficient proper icu staffs. but the present condition of our country is that there are not so many intensivists enough to perform both of clinical duties and education of students and fellows. each icu of university hospitals is endeavoring to increase the number of intensivists. one of the popular methods is the announcement on web site to promote interest of young fellows. regrettably, the homepage of the japanese society of intensive care medicine has no such specific pages. each icu of university hospitals has to create attractive its own pages in the homepages of the hospitals. [ ] . most subjects are taught using lectures and group tutorials and the theory is applied in clinical areas to facilitate greater understanding of the newly acquired knowledge. there is no reported best practice mechanism for teaching medical ethics in a practical setting to medical students. objectives. the routine use of an ethical checklist has been proposed as a tool for the medical team to consider ethical issues on critical care [ ] . its use as a tool for teaching medical ethics within critical care has not previously been reported. the aim was to use this checklist to facilitate learning providing clinical case material for discussion in daily tutorials. one medical student (sm) undertook a one week period of study to learn about ethics in critical care practice. the checklist was used to review patient notes, guide further discussion with patients, when observing the professional behaviours and communication of the multidisciplinary team, and as a guide for case based discussions. results. the complexity and severity of patient conditions in critical care makes it the ideal setting for learning about ethics. sm considered more ethical dilemmas in this practical attachment than in the previous years of clinical placements. the checklist allowed identification of possible ethical issues relating to each patient and a deeper understanding of the patient's health care needs. it was used for daily tutorials to discuss the ethical principles and observed professional behaviours in a similar way to a discussion of clinical diagnosis and management of a patient case with a supervising doctor on a normal clinical attachment. complex issues such as capacity to consent, end of life treatments or resource allocation were seen in relation to ongoing care. on ward rounds it was observed that their conduct in an open environment could at times potentially compromise patient confidentiality. there was also a benefit from the consideration of ethics issues in a real time basis which allowed exploration and reflection on personal moral or spiritual beliefs and how they may differ from those of the patients and other medical professionals. conclusions. using an ethical checklist allowed application of theoretical lecture and workshop material to real life situations. by discussing the cases and observed behaviours with a senior critical care doctor it is possible for trainee staff to appreciate how difficult medical management decisions are made, and to improve the acquisition of the skills necessary to start to assess and discuss ethical issues surrounding a patient's care confidently. introduction. accurate data on patient's weight and height are important for management in intensive care units (icus). unfortunately, weight beds or bed scales are not available in a significant number of icus and these variables are often estimated by health care personnel. the accuracy of such estimations is poorly described. objective. to investigate the accuracy of visual estimation of weight and height in critically ill patients. methods. prospective study conducted in a -bed mixed medical and surgical icu. patients were consecutively weighed by an unblinded physician with a stretcher scale (t metric), and measured by a physical therapist using a measuring tape. the ideal weight was calculated using the ardsnet's formulas for predicted body weight. medical staff (ms), internal medicine resident (imr), nursing staff (ns), physiotherapist (pt) and nutritionist (nu) were asked to estimate patient actual weight, ideal weight and height. they were blind to the estimations during all the protocol. estimations in each healthcare group were computed as means, medians and percentage of error from actual and ideal weight and height, respectively. anova test was used to compare mean estimations between the groups. there were no significant differences between the groups in estimation of either weight (p = . ) or height (p = . ). conclusion. weight estimations from healthcare personnel are often inaccurate. there are no significant differences in accuracy between the estimations of weight and height in different healthcare groups. an effort should be made to weigh all critically ill patients. intraabdominal hypertension (iah) is often diagnosed in icu and it can lead to abdominal compartment syndrome, multiple organ failure and death [ ] . in clinical setting biochemical signals based on which iah is considered severe or detrimental on visceral tissues are scarce. currently, the only clinically relevant signal is decreasing hourly diuresis. in an attempt to find an early sign of metabolically relevant signal on clinically marked iah we investigated abdominal wall metabolite concentrations. previously high lactate/pyruvate has been detected in dialysate from rectus abdominis muscle (ram) in animal models of iah [ ] . in the present experiment we hypothesized that laparoscopic surgery which induces iah could lead to clinically significant increase of l/p ratio as a signal of anaerobic metabolism caused by iah and insufficient tissue perfusion. introduction. among the techniques proposed to assess microperfusion and oxygenation, nirs sounds to be convenient [ ] . if baseline measurements do not provide useful information for outcome of micro-vascular impairment, functional evaluation using vascular occlusion test (vot) seems to be promising [ ] . technological development of the nirs device (inspectra models and , hutchinson technology, hutchinson, minn) proposes to use a new probe measuring hemoglobin saturation at less depth than previously ( vs. mm between fiberoptic) with more data output ( value/ s vs. value/ . s) associated with an automated software to compute occlusion and reperfusion slopes. objective. to compare nirs results obtained, using the two different probes, at day of septic shock (ss) in two groups of patients having similar clinical characteristics. methods. patients (g ) and patients (g ) were included within the first h of ss. macrohemodynamic: heart rate (hr), mean arterial pressure (map), central venous pressure (cvp), cardiac output (co) and svo (mixed venous o saturation), ph, base excess, and lactate were collected as saps ii and sofa scores. baseline sto at thenar eminence was continuously monitored and a min upper arm(brachial artery) vot was performed. sto occlusion and reperfusion slopes were calculated manually in g (probe mm) using linear adjustment (r c . to be valid) or calculated by the software in g (probe mm) using the same method, p \ . was considered significant. results. median ± iqr. the two groups did not differ for macrohemodynamic nor for metabolic data (table ) . nirs data surprisingly were largely significantly different between the two groups for both baseline and slopes ( background. hypovolemia and hypovolemic shock are life-threatening conditions that occur in numerous clinical scenarios. near-infrared spectroscopy (nirs) has been widely explored, successfully and unsuccessfully, in attempt to function as an early detector of hypovolemia by measuring tissue oxygen saturation (sto ). in order to investigate the measurement site-and probe-dependence of nirs in response to hemodynamic changes, such as hypovolemia, we applied a simple cardiovascular challenge; a posture change from supine to upright, causing a decrease in stroke volume (as in hypovolemia) and a heart rate increase in combination with peripheral vasoconstriction to maintain adequate blood pressure. methods. multi-depth nirs was used in nine healthy volunteers to assess changes in peripheral vascular tone in the thenar and forearm in response to the hemodynamic changes associated with a posture change from supine to upright. a posture change from supine to upright resulted in a significant increase (***) in heart rate. thenar sto did not respond to the hemodynamic changes following the posture change, whereas forearm sto did. in the forearm, sto was significantly lower (***) in the upright position with respect to the supine position. conclusion. the primary findings in this study were that ( ) forearm sto is a more sensitive parameter to hemodynamic changes than thenar sto and ( ) cerebral hyperperfusion syndrome, caused by inflow at normal blood pressure into maximally dilated fine vessels, is a recognized complication of carotid endarterectomy (cea) strict blood pressure control in the early postoperative period can minimize the risk of cerebral hyperperfusion. until yet, diagnosis of cerebral hyperperfusion mainly relies on intermittent postoperative examinations (spect; ct angiography). non-invasive absolute cerebral oxygen saturation (scto by fore-sight technology) was validated to jugular bulb saturation (sjo ) monitoring with a constant difference of % higher for scto values. previously, sjo monitoring after severe head injury indicated cerebral hyperemia. in this study, we evaluated scto monitoring after carotid surgery as possible continuous on-line monitoring of cerebral hyperperfusion. fourteen pts scheduled for cea were monitored for h postoperatively after cea. bilateral scto monitoring was started before induction of anesthesia and maintained until h postoperatively. intra-operative eeg monitoring guided the decision to intraluminal shunt insertion. strict blood pressure control was applied at maintaining normotensive levels throughout the clamping procedure. early postoperative care focussed on strict maintenance of normotensive blood pressure. in no pt, any change in eeg was observed after carotid clamping. in all pts, ipsilateral scto significantly decreased after carotid clamping, without any scto value below %. we observed no changes in contralateral scto . mean clamping time was min ( - min). in all pts, clamp release restored ipsilateral scto to baseline values. in all pts, emergence from anesthesia was uneventful, without any new neurological deficit. in of pts, significant increases (scto [ %) in ipsilateral scto were observed in the postoperative period (m scto . %), without any changes in contralateral scto . this increase occurred at a mean of . h after carotid declamping with a mean duration . h. in these pts, we could not make any significant correlation to arterial blood pressure, as none of these pts needed more aggressive antihypertensive control. we noted that of these pts suffered from diabetes mellitus, while of pts revealed high ([ %) contralateral stenosis. further data will have to reveal the importance of these comorbide factors. non-invasive cerebral oximetry, enabling absolute cerebral oxygen saturation monitoring, could provide on-line estimation of cerebral perfusion state after cea. this could allow bedside detection (and eventual therapeutic interventions) of cerebral hyperperfusion after cea. introduction. analysis of microcirculatory alterations obtained by side-stream dark field (sdf) is time consuming. automated analysis with modern softwares could accelerate this process and help to quantify blood flow velocity. however, perfusion detection is based on the contrast between pixels and this may be influenced by image settings. objective. we aimed to compare data obtained with a new software to the traditional semi-quantitative analysis of sdf images. methods. we selected from our database six images of poor sublingual microcirculatory perfusion and six images of good microcirculatory perfusion registered by the sdf technique (microscan; microvision medical, amsterdam, the netherlands). the proportion of perfused vessels [ppv = (number of vessels with continuous flow/number of all vessels) ] \ % was used to define microcirculatory perfusion. total vessel density (tvd) was determined automatically by the software ava . (microvision medical) and also by the semi-quantitative technique, considered as the gold-standard (number of capillary crossing three equidistant vertical and horizontal lines divided by the total length of these lines). ava software was also used as default definitions or set to optimize analyses according to manufacturer instructions. vessels falsely detected (false positive = fp) or missed (false negative = fn) by the software, in comparison to the semi-quantitative evaluation, were also counted. results. tvd was significantly higher by the ava software either on default or on optimized mode than by the semi-quantitative method, and these differences were present with good or poor perfusion images (table ) . overall fp rate was %, and it was greater in poor perfusion images ( %). optimization of the ava set parameters attenuated fp rates both in poor and good perfusion images, at the expense of increasing fn rates (table) . due to intrinsic characteristics of the software, the mean total grid length was significantly lower in the ava than in the semi-quantitative analysis ( . vs. . introduction. perfusion index (pi) is the proportion of constant absorbed light compared to pulsatile absorbed light emitted from a pulse oxymeter. it ranges from a value below up to depedant of peripheral perfusion. it is measured primarily to evaluate the signal quality for the pulse oxymeter and is displayed by some pulse oxymeters to be acknowledged by the clinician. the pi changes with vasodilation and vasoconstriction. however, intubation is a stimulus able to increase endogenous catecholamines and thus leading to vasoconstriction possibly declining the perfusion index. therefore we found intubation with a double lume tube in a thoracic surgery setting as a suitable setting to evaluate changes in perfusion index as a reaction to intubation. after informed consent, we enrolled seven patients undergoing lung surgery requiring an double lume tube. they were monitored as it is standard of care in our institution with invasive blood pressure, ecg, and a pulse oxymeter displaying the pi. (radical , masimo, irvine, ca) the patients received the medication to induce anesthesia calculated adequately to their body weight. midazolam, propofol and fentanyl where used to anesthetize the patient, cisatracurium was used for muscle relaxation to facilitate intubation. pi, pulse and arterial saturation were recorded every minute from prior to induction until after successful intubation. a baseline value was recorded prior to induction and compared to the value minutes after induction. then the pi measured next to intubation was compared to the pi after induction and analysed using students t test. introduction. anticoagulation strategies for albumin dialysis suppose a difficult compromise between risk for bleeding and a high tendency to clot in the circuit. even thought the sessions are short, a premature clotting is a serious event because the lost of blood (high priming volume) and a high cost of the systems. we intended to demonstrate that the classical approach based in heparin is not adequate in these patients and should be substituted for a different strategy (mixed low dose of heparin plus epoprostenol). methods. data of a prospective registry of all cases treated in our centre (a third level, teaching hospital) with albumin dialysis (mars system). initially we used non-fractionated heparin at - u/(kg h) in patients without coagulation problems, epoprostenol [ - ng/ (kg min)] in cases with risk or thrombocytopenia and no anticoagulation when high risk for bleeding or contraindication for anticoagulation. after an intermediate analysis of our registry we detected a high number of filters clotted when heparin was used and changed our approach to use as first indication a mixed protocol with non-fractionated heparin [ u/(kg h) ] plus epoprostenol [ ng/(kg min) ]. data are presented as percentages. analysis was performed with chi-square test. to detect variables related to coagulation a stepwise backward logistic regression analysis was performed. we registered patients with a total of sessions. selecting only the first session for each patient to validate the first choice for anticoagulation, we used heparin in cases and detected the loss of filters ( . %) because clotting. after the change to mixed anticoagulation we used this as first indication in patients and in only ( . %) the sessions were prematurely ended because clotting (p ns). the rest of patients received isolated epoprostenol in cases (with - %-cases of premature clotting) and no anticoagulant in five cases (with - %-premature clotting). between the cases with heparin as first choice, three episodes of mild and one episode of severe bleeding were detected while no patients in the mixed group presented bleeding complications (p ns). in a logistic regression analysis over all registered sessions using coagulation of filters as dependent variable and type of patient, anticoagulant, arterial pressure, inr, tpta, platelets, haematocrit or bilirubin as independent variables, none of these was included in the regression model. even though more studies are necessary to validate this conclusion, a mixed protocol based in low dose heparin plus epoprostenol could be adequate as first indication for non-complicated patients submitted to a mars treatment with lower risk for bleeding than the classical approach of isolated non-fractionated heparin. optimizing oxygen delivery in critically ill patients is vital for the promotion of aerobic cellular metabolism. current practice includes the measurement of variables such as partial pressure of arterial oxygenation (pao ), cardiac index (ci) and percentage of oxygenated haemoglobin in arterial blood (sao ). these parameters reflect global oxygen delivery. the real point of interest is the end point of the oxygen cascade; oxygen utilisation in tissue mitochondria. near infrared spectroscopy (nirs) has been developed in an attempt to measure tissue oxygen saturation (sto ) in peripheral muscle microcirculation. manufacturers state normal values as ± %. it uses four wavelengths near the infrared spectrum ( - nm) to measure sto , a ratio of oxygenated haemoglobin to total haemoglobin. it is continuous and non-invasive. sto has proven efficacious in predicting oxygen delivery in trauma patients and claims to have been successfully used to guide early resuscitation [ ] . objectives. we were interested in assessing whether sto had a role in measurement of oxygen delivery in the intensive care population, and how it compared to the parameters currently used to predict oxygen delivery. we had particular interest in the usefulness of nirs in septic patients, where the pathophysiology of tissue oxygen utilization is disrupted. patients from a general, adult intensive care unit were enrolled over an month period. all patients had lidco monitoring. exclusion criteria were gtn, atrial fibrillation and patient refusal. mm sto probes were sited on the thenar eminence. serial recordings of sto , cardiac index, hr, sao , map, and pao were recorded. sto results were compared to more traditional parameters of oxygen delivery. sixteen patients were recruited, all met criteria for sirs and shock. four were excluded with incomplete data. results were analysed for individual patients and as a collective series. we found: • no statistical correlation between nirs and sao or pao . • a weak and clinically insignificant correlation between cardiac index and nirs (p \ . ). • supra normal nirs readings (normal [ %) were not infrequently gained in patients where all other parameters were indicating severe shock and poor oxygen delivery. conclusion. theoretically nirs has potential to be beneficial in measuring oxygen delivery. our results demonstrate that nirs is not accurate for our septic population. we found poor correlation with current methods used to predict oxygen delivery and it may well be more misleading than beneficial. more traditional methods of intensive care monitoring, although sometimes invasive, appear to provide a more accurate representation of a patient's oxygen delivery. background. urine output is a crucial parameter of renal function and fluid balance. conservative urine output monitoring harbors problems such as subjective reading, sampling time errors and nursing workload. an electronic urine collection device was introduced into the icu and connected to a computerized information system. this created a more reliable and accurate means for urine output monitoring and the ability to develop new calculated parameters. . to evaluate the effects of introducing an electronical urine collection device into a fully computerized icu. . to evaluate new parameters that were created by the combination of the device and a computerized data management system. patients included were all admitted to the icu at rambam medical center, haifa, israel, during the years - . urine production and flow were monitored continuously by the urinfo Ò device (med-dynamix, israel), a novel electronic urinometer, connected to a patient data management system (imdsoft, israel). graphical analysis of urine production was done and derived parameters continuously calculated. comparison was done to the conventional mechanical urine collection system. variables studied were: measurement accuracy, sampling time accuracy, nursing workload before and after the implementation process. correlation between derived parameters and conventional renal function measurements such as plasma creatinine and creatinine clearance time. results. the conservative urine output measuring system demonstrated percentage error span in range of - %, compared to a range of - % percentage error in measurement after implementation of the computerized system. before implementation, sampling time error span was found to be - min, while no sampling time error was present after implementation due to the automated recording system. time consumed by the workload of the conservative urine output monitoring system was measured at - min per nursing shift ( h). the computerized system eliminated this workload completely. derived parameters evaluated were continuous urine flow (in cc/min or cc/h), urine production acceleration rate (calculated via the slope of the ''up-rise'' in cc/min ) and the peak urine production rate (cc/min). these parameters were able to demonstrate immediate changes in renal function, hours before conventional measurements and calculations would show them. conclusion. implementation of a computerized urine monitoring system can lead to improved accuracy in renal function monitoring and eliminate a significant amount nursing workload. use of derived calculated parameters may lead to earlier detection of renal malfunction and thus lead to earlier intervention. ( g/ , ml), cica dialysate k tm (na mmol/l, k . mmol/l, mg . mmol/l, cl . mmol/l, hco mmol/l, glucose anhydrous . g/l, ph * , ) and calcium chloride mayrhofer tm cacl , mol/l. the filter was an ultraflux av s tm , the material of the bloodline tubing system was medical grade soft pvc. in three circuits used in two different patients we found an opaque white precipitation starting at the cacl side port growing along the line with the direction of the bloodflow up to a maximal mm wide and mm long stripe. to identify the composition of the white stripes we included histological examination of hematoxylin-eosin stained sections and lyophilisation with wet chemical analysis. blood samples were simultaneously taken from the venous port of the cvvhd circuit and the arterial line of one patient. results. histology showed **an organic material in form of calcific deposit, covered with coagulated blood. chemical analysis identified this deposit as calcium phosphate. the results of the blood samples are shown in table . calcium phosphate precipitates may have reached patient circulation and been deposited in the capillary bed of the lungs or other organs. no histological examinations of tissue were taken and adverse events could not be attributed to the described phenomenon. citrate anticoagulation was stopped and switched to combined heparin-epoprostenol sodium anticoagulation. conclusions. the combination of the fluids and materials used in this specific cvvhd circuit with citrate anticoagulation resulted in some patients in a detectable calcium phosphate formation in the circuit. physicians using the described setting should be aware of the phenomenon and stop citrate anticoagulation as soon as a deposit occurs. in vitro studies, using different compositions and concentrations of dialysate and substitution fluids and simulating different patient conditions (ph, ph, hb, alb,…) should clarify, which solutions could safely be used. in addition the material of the circuit should be investigated, since surface characteristics have been identified to influence the formation of a calcium phosphate layer [ ] . reference (s) objective. the aim of this study was to assess, in a medical population of critically ill patients, whether intraabdominal pressure at admission was an independent predictor for mortality and to evaluate the effects of intraabdominal hypertension on organ functions. all patients admitted to the medical icu of the hgu gregorio marañón over a period of days were studied prospectively. patients who fulfilled two or more risk factors for wsacs (diminished abdominal wall compliance, increased intra-luminal contents, increased abdominal contents and/or capillary leak /fluid resuscitation.) were included. iap was measured via a foley bladder catheter, according to the modified kron technique. data recorded on admission were the patient demographics with, acute physiology and chronic health evaluation ii score (apache ii), and type of admission; during intensive care stay, sepsis-related organ failure assessment score (sofa) and clinical concomitant factors and conditions. intraabdominal pressure were measured at least daily together with fluid balance. patients were followed throughout their hospital stay. forty-four patients were included in the study (age - , apache ii . . half were admitted for cardiopulmonary disease. twelve ( %) had pancreatic or gastrointestinal disease. twenty-two ( %) had severe sepsis or septic shock. the incidence of iah was %. mortality was %. the cause of the iah was capillary leak syndrome/fluid resuscitation in % of cases. there was no relationship between the presence of iah and the number of organ failure during admission. the only variables associated with mortality of the patients were sofa and apache ii. the presence of iah was not a factor associated with increased mortality, although these results may be confounded by sample size. conclusions. there is an unusually high incidence of iah in the population of critically ill medical patients with two or more medical risk factors for wsacs. however, unlike in other populations, our study does not demonstrate that the iap monitoring allow detecting a group at higher risk of developing multi-organ failure or death. background. drainage of ascitic fluid is a common practice in order to relief the respiratory discomfort of patients. the aim of the present study was to determine abdominal compliance after ascitic fluid removal by transcutaneous drainage. methods. twelve patients presenting with ascitic fluid were included. all patients had transcutaneous blind drainage with a wide catheter. the ascitic fluid removed was recorded, while the intraabdominal pressure (iap) was measured as proposed by wsacs. iap was measured before and min after the puncture. abdominal compliance (cabd) was calculated. results. the pre-drainage iap was . mmhg (ranging from . to . mmhg, sd . mmhg), while the post-drainage was . mmhg (ranging from . to . mmhg, sd . mmhg). the mean volume of ascitic fluid removed was ml (ranging from to , ml, sd ml). cabd after drainage was ml/mmhg (ranging from to ml/ mmhg, sd ml/mmhg). a linear correlation was found between ascitic fluid removal and iap variations. conclusion. the drainage of ascitic fluid reduces iap, facilitating in this way respiration. moreover, iap variation seems be in linear relation with the volume of ascitic fluid removed. this linear relation between iap and volume may probably predict the cabd quite accurately and vice versa. however, larger studies are necessaries in order to safely draw predicting diap-dv (cabd) diagrams, and determine the optimal ascitic fluid removal in order to achieve best comforting of the patient and slower fluid reformation. introduction. use of stroke volume variation (svv) to guide fluid therapy in preload responsive state has been studied well in patients undergoing cardiac or neurosurgery during anaesthesia. use of this dynamic monitoring variable has not been studied much in septic shock. we undertook this prospective study to evaluate utility of svv to optimize preload in patients with septic shock and ards. setting. bedded medical surgical icu of a bedded tertiary care centre in pune, india. inclusion criteria: ( ) patients with ards (po /fio b ), svv readings were taken every h with flotrac-vigileo system after confirming abolishment of spontaneous breaths by sedation or paralysis and increasing tidal volume transiently to ml/kg. fluid boluses were given to keep svv \ % for h after enrollment. attempts were made to reduce vasopressor doses keeping map c mmhg. results. patients with average age . ± . years and apache ii score . ± . were studied. each patient received an average . ± . l fluid in h after enrollment to keep svv below %. svv at h after enrollment was . ± . % improvement in microcirculation was evident as plasma lactate reduced from . ± . (at h) to . ± . mmol/l (at h) there was no worsening in pulmonary edema as po / fio increased from . ± . (at h) to ± . (at h) only out of patients needed renal replacement therapy. in patients, vasopressors could be stopped completely in . ± . h. of them survived till discharge from the icu and died of ards. in patients, vasopressors could not be weaned off completely and all of them succumbed. overall survival rate was %. conclusion. svv guided fluid therapy is a promising modality for pre load optimization in mechanically ventilated patients with septic shock and ards. introduction. cardiovascular function is an important determinant of outcome in sepsis, and heart rate (hr) has been associated with cardiovascular risk and mortality in large patient cohorts [ ] . to investigate the association between hr and or day mortality in septic shock. methods. this study is a post hoc analysis of septic shock patients who were included in the control group of a multicenter trial [ ] . demographic and clinical data, average hr and catecholamine requirements during septic shock, occurrence of acute circulatory failure, and day mortality were documented. a binary logistic regression model adjusted for the simplified acute physiology score ii (excluding hr) was used to investigate the association between mean hr and acute circulatory failure or / day mortality. a multiple logistic regression model was applied to identify independent risk factors for developing hr critical for outcome. conclusions. hr is associated with and days mortality in septic shock. hr persistently exceeding bpm during septic shock seems associated with a significant risk of death. introduction. different colloids can be used for treatment of hypovolaemia in septic pts. recently, small-volume resuscitation was introduced for initial therapy of severe hypovolaemia and shock. the concept of small-volume resuscitation encompasses the rapid infusion of a small dose of . % nacl/colloid solution [ ] . however, in septic pts hypovolaemia often associates with acute lung injury (ali). therefore in these pts great importance has influence of colloids on oxygen transport. objectives. the aim of the study was to evaluate and compare the effects of hhes and hes on oxygen transport in pts with sepsis and ss. methods. hypovolaemic pts with sepsis and ss were enrolled in the study. pts received - ml/kg ( ml) hhes ( . % nacl ? % hes) (fresenius kabi) within min and pts received hes / (voluven, fresenius kabi) ml/kg. in all pts before and after infusion the parameters of oxygen transport was measured by pulmonary arterial catheter and transpulmonary thermodilution (pulsion medical system). after infusion of hhes oxygen delivery index (ido ) increased because of increase of cardiac index (ci) despite of decrease of hemoglobin (hb) levels and absence of changes of arterial oxygen content. extravascular lung water (evlw) and shunt increased significantly immediately after hhes infusion, but this increase was not accompanied by deterioration of pao /fio . introduction. severe sepsis is characterised by a wide array of haemodynamic changes including increased capillary leak, vasodilatation, vascular hyporeactivity and myocardial depression. the resultant tissue hypoperfusion is an important catalyst of multi-organ failure [ ] . to further develop our understanding of the underlying mechanisms, we have developed and characterised a fluid-resuscitated mouse model of intraperitoneal polymicrobial sepsis. objectives. to assess alterations in cardiac performance in mice at , , and h following faecal peritonitis. methods. sepsis was induced in week old male mice (n = ) by intraperitoneal (i/p) injection of dilute faecal slurry. sham animals (n = ) received n-saline i/p. animals were fluid resuscitated at time ( ml/kg . % saline), and at and h ( ml/kg . % saline- % dextrose each time). under a minimum concentration of isoflurane to achieve light anaesthesia, peak velocity, stroke distance, heart rate and fractional shortening were measured in the short axis plane by echocardiography at the , and h timepoints. in separate sham and severe septic mice (n = per group) the cardiac response to intravenous colloid boluses was assessed at and h. results. we clinically characterised septic animals into 'mild' and 'severe'. mice with severe sepsis showed a % drop in peak velocity and cardiac output at h (vs. and % falls in the mild septic and sham-operated animals, respectively, p \ . ). while mild septic animals showed recovery by hr, cardiac output in severely ill mice remained significantly depressed (due to both low heart rate and stroke volume) compared to mild septic and sham animals [*p \ . ( fig. ) ]. stepwise . ml boluses of intravenous fluid at h in severe septic animals led to restoration of cardiac output to baseline ( h) values. however, in the h septic animals, fluid challenge produced an initial improvement in cardiac output followed by deterioration [ fig. purpose. myocardial dysfunction has been well-documented in sepsis even in hyperdynamic state, and may develop and contribute to morbidity and mortality. nicaraven, a radical scavenger, has been shown to protect the coronary endothelial and myocardial function from ischemia and reperfusion injury due to hydroxyl radical scavenging activity. the purposes of present study were to determine the effects of nicaraven on cardiac function and cytokine production in lipopolysaccharide (lps) induced sepsis. methods. this protocol was approved by our institutional committee. following arterial and venous cannulation and tracheostomy, rats ( - g) were anesthetized with pentobarbital, and mechanically ventilated with a control mode (v t = ml/kg, rr = rpm). after baseline measurements, rats (n = ) were administrated with lps ( mg/kg, intravenously) and randomly assigned to following two groups: the nicaraven group treated with nicaraven [ mg/(kg min), intravenously] and the control group treated with saline. the left ventricular pressure and volume were measured with the pressure and conductance catheter every one hour. cardiac function, including cardiac output (co), ejection fraction (ef), and maximal elastance of left ventricle (e max ) were analyzed with a computer soft. blood was collected, centrifuged ( , g, min, ) , and stored (- °c) from rats every h after operation to measure plasma concentration of tnf-a, il -b and macrophage migration inhibitory factor (mif) using enzyme-linked immunosorbent assays kits. blood lactate concentration was also measured. data were analyzed by repeated measure anova. results. the co in the nicaraven group was kept significantly higher than the control group (p \ . ). the ef and e max in the nicaraven group were also kept significantly higher than the control group (p \ . ). arterial lactate, tnf-a, il -b and mif were significantly lower in the nicaraven group versus the control group (p \ . ). conclusion. the current study indicates that the treatment with nicaraven improved cardiac dysfunction and reduced plasma concentration of cytokines, and improved lactic acidosis in septic model. methods. this protocol was approved by our institutional committee. following arterial and venous cannulation and tracheostomy, rats ( - g) were anesthetized with pentobarbital, and mechanically ventilated with a control mode (v t = ml/kg, rr = rpm). after baseline measurements, rats (n = ) were administrated with lps ( mg/kg, intravenously) and randomly assigned to following two groups: the oxytocin group treated with oxytocin ( iu/kg iv and followed by the continuous infusion of mg/(kg min), intravenously) and the control group treated with saline. the left ventricular pressure and volume were measured with the pressure and conductance catheter every h. cardiac function, including cardiac output (co), left ventricular peak pressure (lvpp), and cardiac work (cw) were analyzed with a computer soft. blood was collected from rats every h after operation to measure plasma concentration of blood lactate. data were analyzed by repeated measure anova. results. the co in the oxytocin group was kept higher than the control group but there is no significance (p \ . ). the lvpp and cw in the oxytocin group were kept significantly higher than the control group (p \ . ). arterial lactate was significantly lower in the oxytocin group versus the control group (p \ . ). conclusion. the present study indicates that the treatment with oxytocin improved cardiac dysfunction and reduced plasma concentration of lactate in septic model. introduction. conventional hemodynamic monitoring parameters like heart rate, mean arterial pressure (map), and central venous pressure may be misleading in assessment of circulating blood volume in severely septic patients. inadequate blood volume may compromise renal blood flow leading to acute kidney injury (aki). stroke volume variation (svv) is a sensitive indicator of relative preload responsiveness and has high sensitivity and specificity when compared to conventional indicators of volume status and their ability to determine fluid responsiveness. to assess the efficacy of svv guided fluid therapy in preventing aki in patients with severe sepsis on ventilatory support. mechanically ventilated patients with septic shock who had undergone resuscitation based on surviving sepsis campaign guidelines and still requiring vasopressor support were enrolled. patients with pre-existing renal failure were excluded. a total of patients were randomized to receive fluid therapy according to conventional indices or svv, in the first h after mechanical ventilation. svv was measured with flotrac vigelio after abolishing spontaneous ventilation by sedation and paralysis if required. fluid boluses were given to keep svv less than %. vasopressor therapy was optimised to maintain map [ mm hg. patients were followed during their icu course with respect to development of aki, need for renal replacement therapy (rrt), length of icu stay and icu mortality. aki was diagnosed as per the rifle criteria. primary outcome measure was development of aki. results. patients in both groups were similar with respect to age (p = . ), sex (p = . ), and admission apache ii score (p = . ). incidence of aki was / ( . %) and / ( . %) in conventional and svv groups, respectively (p = . ). there was no statistically significant difference in terms of need for rrt, icu length of stay and icu mortality ( [ ] . moreover, pnu- a (pnu), an inhibitor acting through the poreforming subunit of the channel, did not affect bp in our awake peritonitis rat model [ ] . given that vasoconstrictors, including ne, inhibit k atp channel activity [ ] , we speculate that the high sympathetic tone seen in sepsis [ ] objectives. the goal of this study was to determine if hfav improves microcirculatory alterations in ss patients. methods. by using side dark field videomicroscopy (microscan Ò , microvision medical) we evaluated sublingual microcirculation in ss patients who according to our local protocol care [ ] underwent a h-hfav as rescue therapy for refractory septic shock. hemodynamic parameters and microcirculation were assessed at baseline, after h of hfav, and h after stopping hfav. microcirculation assessments were performed at to different sublingual areas ( - s/image). images were analyzed according to recent consensus [ ] by semiquantitative scores of flow (mfi, mean flow index and ppv, proportion of perfused vessels), density (tvd, total vascular density; pvd, perfused vascular density), and heterogeneity (het mfi) of small vessels (\ lm introduction. disturbances within the microcirculation represent an important factor in the pathogenesis of multiple organ dysfunction in sepsis and septic shock [ ] . gender-specific effects may modulate the septic pathophysiology [ ] . therefore, we studied sepsis-induced changes within the intestinal microcirculation in randomly cycling and ovariectomized female rats. objectives. we hypothesized that estradiol (e ) and dehydroepiandrosterone (dhea) may have a beneficial effect on the microcirculation during experimental sepsis and resubstituted these hormones in the ovariectomized animals. methods. fifty female rats were divided in to five groups of ten animals. group received sham laparotomy without further treatment. in group - we induced experimental sepsis (colon ascendens stent peritonitis-casp model). animals of groups - were additionally ovariectomized weeks before sepsis induction. in group we administered mg/kg estradiol immediately after and h following casp surgery. the animals of group received mg/kg dhea immediately after sepsis induction. twenty-four hours after casp surgery intravital microscopy was performed to study leukocyte-endothelial interactions and functional capillary density. blood samples were taken for the measurement of estradiol, dhea and inflammatory cytokines. results. in ovariectomized rats subjected to casp the number of activated leukocytes was significantly increased in comparison to sham and not ovariectomized casp animals (p \ . ). in ovariectomized rats treated with e leukocyte adhesion was significantly reduced in comparison to untreated ovariectomized rats subjected to casp (p \ . ). the same observation was made in ovariectomized rats treated with dhea. in addition, in ovariectomized rats subjected to casp the functional capillary density was significantly decreased in comparison to sham and casp groups (p \ . ). in ovariectomized rats treated with e or dhea functional capillary density was completely restored. the results demonstrate the role of e and dhea in the sepsis-induced changes within the microcirculation. a rapid, non-genomic effect of both e and dhea is suggested [ ] . dhea may play a role through conversion to e or through direct acting on the e receptor. further investigations should be done to elucidate the underlying mechanisms. both e and dhea appear to be a promising adjunct for the prevention and treatment of sepsis-induced multiorgan failure. liver is involved in the production of no. the aim of this experimental study was to evaluate the time course of hepatic no production at the onset of hypotension occurring during septic shock. methods. male wistar rats were anesthetized with isoflurane Ò , and mechanically ventilated. a first group (sepsis group) underwent a cecal ligature and puncture (clp) peritonitis, the second one (control group) a laparotomy only. animals were euthanized at different times: h after surgery, at shock onset, and h after shock. shock was defined by systolic blood pressure lower than mmhg. each rat of sepsis group was matched with rat of control group. liver perfusion was measured using a direct laser doppler flowmetry probe. no generated in the liver was measured using a pulse voltametric method. results. rats were studied ( in each group). in sepsis group, shock occurred at ± min after clp. in sepsis group, a significant decrease of hepatic perfusion was identified h after clp ( fig. ) whereas hepatic no production was increased only at the time of shock onset (fig. ). intra hepatic no production conclusion. this study shows a time shift between hepatic perfusion disturbance, hepatic no production and shock onset in a septic animal model. introduction. microvascular blood flow alteration is a key element of severe sepsis and septic shock [ ] . one study show that microvascular alterations in septic patients could be improved with a nitric oxide donor nitroglycerin [ ] . studies in human have shown that infusion of magnesium sulphate has endothelium dependent and independent vasodilation properties, increase of red blood cells deformability in specific conditions. we hypothesized that combination of nitroglycerin with magnesium sulphate and order of priority influence microvascular improvement in patients with severe sepsis and septic shock. methods. ten septic patients who had already been fluid resuscitated randomly assigned to one of two groups. one group received magnesium sulphate infusion g/h with nitroglycerin . mg/h infusion added after min. another group received nitroglycerin . mg/h infusion with additional magnesium sulphate g/h infusion after min. if required we added crystalloids and use norepinephrine. sublingual microcirculation was evaluated using side dark field videomicroscopy (microscanÒ, microvisionmedical) . each patient's microcirculation was evaluated by examining different sublingual areas ( - s/image). in all patients measurements were obtained at baseline, at and min. images were analyzed by semiquantitative scores of flow (mfi, mean flow index; ppv, proportion of perfused vessels) and density (tvd, total vascular density; pvd, perfused vascular density). capillaries were defined as microvessels with a diameter \ lm. data are presented as median values (percentiles ; ). . the median age of the patients was ( ; ) years. in both groups we see tendency progressively increase of pvd, ppv and mfi after drug alone and combination after min, but pvd has tendency to be higher [ . ( ; . ) n/mm vs. . ( . ; . ) n/mm , p = . ] after min. in group, where magnesium sulphate infusion was given first. combination of magnesium sulphate with nitroglycerin, when magnesium sulphate is given first, has tendency to higher potential for improving of microcirculation in severe sepsis and septic shock patients, but further studies are needed to obtain more detailed results. severe sepsis remains one of the leading causes of death in critical care, with around % of patients dying within one month of diagnosis. rapid diagnosis and therapy of sepsis improves survival [ ] . in november the whittington hospital introduced a hospital severe sepsis guideline, based on the first surviving sepsis campaign guideline [ ] . the sepsis guideline was published on the hospital intranet and specified actions to be completed within the first h of the diagnosis of severe sepsis or septic shock [ ] . objectives. to assess whether publication of a sepsis guideline on the hospital intranet, coupled with departmental educational campaigns, improved the management of severe sepsis. the whittington hospital is a university associated general hospital in london. we audited the early management of severe sepsis and septic shock before and after the introduction of the new hospital sepsis guideline. the 'before' phase comprised patients with severe sepsis or septic shock admitted to critical care between november and november . the 'after' phase comprised patients with severe sepsis or septic shock admitted to critical care between january and november , after introduction of the guideline. data was retrospectively collected from case notes and observation charts. the audit tool compared immediate, , and h actions following diagnosis against the hospital guideline. the main outcome measures were compliance and day mortality. compliance was defined as the average of the percentage compliance with each of the items specified in the guideline. results were compared by chi squared. compliance with the severe sepsis guidelines was only % after publication of the hospital sepsis guideline, compared with % before publication (p. )! there was similarly no significant difference in day mortality (before %, after %, p. ). publication of a sepsis guideline on the hospital intranet, coupled with departmental teaching sessions, failed to improve compliance with surviving sepsis recommendations, perhaps because the guideline competes for attention with over other guidelines on the intranet. next we will implement an interdepartmental educational programme to try and improve guideline compliance. as guidelines proliferate it is difficult to ensure they are followed, but failure to implement a published hospital guideline may represent a significant clinical and medicolegal risk. methods. the icu is an intensivist-led bed intensive care in a bed non-academic teaching hospital. hospital mortality from sepsis in icu-patients was . % in . patients are treated under modern icu conditioning, including continuous venovenous hemofiltration and a lung-protective ventilation strategy including prone position. an intensive insulin therapy protocol for glycemic control is used. in the period between march until june , we prospectively screened all patients admitted to the icu for (severe) sepsis, without the knowledge of the nurses and most of the doctors. all severe septic patients were included in our surviving sepsis database. after h, we examined how many targets of the resuscitation and management bundles were applicable and reached. in the period between march until june , patients were admitted to the icu. twenty-two of them were suffering from severe sepsis ( . %), of which had a septic shock. focus of the sepsis was abdominal in patients ( %), pulmonary in five patients ( %), urogenital tract in five patients ( %), meningitis in one patient ( %) and catheterrelated in one patient ( %). table shows us the applicability and achievement of the bundle elements. only in one of the patients all targets were reached. however, mean individual bundle element performance was . % (sd . ). all patients received fluid resuscitation when indicated, and all patients on mechanical ventilation were ventilated in a lung-protective manner with plateau pressures \ cm h o. only percent of patients had glucose levels within the target range. scvo was never measured, though it was indicated in patients. one patient had an apache iiscore c and had no contraindications for administration of activated protein c. treatment was not considered for this patient by the attending physician. of these patients suffering from severe sepsis, three died within days after the diagnosis ( . %). introduction. the surviving sepsis campaign (ssc) guidelines give a group of interventions (''sepsis bundles'') expected to improve the outcome of patients with severe sepsis [ ] . objetives: the aim of this study was to evaluate the impact of the implementation of the ssc guidelines on the mortality in our intensive care unit (icu). methods. prospective, observational study. during one year period (january -january ) the sepsis bundles were applied to each patient with severe sepsis-septic shock and they were followed up until discharge. we considered as ''time o'' (the time of delay of the implementation of the sepsis bundles) the time of admission of the patients in the icu. for each severe septic patient the following data was registered: time delay, apache ii and sofa scores at icu admission, diagnosis, the rate of compliance with the resucitation and management bundles, microbiological data, evolution of levels of serum lactate, empiric antibiotic therapy, length of stay and mortality in icu. the application of guidelines impact on mortality was compared with historical data years before implementation in our icu ( . %) and spanish icu ( . %) [ ] . a total of severe septic patients were included in the study. ( . %) patients had severe sepsis and ( . %) septic shock. the median age was years. the mean apache ii was . (± ) and sofa was . (± . ). the main sources of infection were abdomen ( %), lungs ( %), urinary tract ( . %) and soft tissues ( . %). the most common clinical diagnosis related to an episode of severe sepsis was peritonitis ( %). a microbiological diagnosis of the infection was reached in . % and the infections were mostly caused by gram-bacilli. once the antibiogram was obtained, the initial treatment was considered appropriate in . % patients. the rate of compliance with sepsis bundles was %. the length of icu stay was . days. mortality was . %. the implementation of the sepsis bundles decreased icu mortality significantly ( . % before implementation vs. . % after implementation). non survivors were older (median age ± . ), had higher apache ii (mean . ± ) and sofa (mean ± . ), % had septic shock, . % had negative cultures and an increased on the levels of serum lactate in h. age, apache ii and sofa scores and the increased on the levels of the serum lactate were useful tools to predict mortality. conclusion. implementation of the surviving sepsis campaign guidelines was associated with a reduction in icu mortality. introduction. the objective of this before-after study is to assess the impact of a protocol of care for severe sepsis in a french emergency setting. methods. two months periods were surveyed before and after the initiation of a protocol of care for severe sepsis and septic shock. after the control period (p : november -february ), a procedure for early recognition, aggressive treatment and standardized antibiotherapy of severe sepsis was initiated. a campaign to raise medical physicians and nurses awareness concerning this new strategy of care was performed. the intervention period (p : november -february ) assessed the impact of these actions. . patients with severe sepsis or septic shock were included during p ( % of patients with a suspected infection and . % of all non trauma admissions) and during p ( . % of patients with a suspected infection and . % of admissions). the age and the proportion of patients with co-morbidities were similar during the p and the p periods ( years in median versus years, and vs. %, respectively). and % of the patients lived in long term care facilities. severe sepsis and septic shock were correctly identified by the emergency team in / ( . %) during p and in / ( . %) in p (p = . ). the delay between the admission in the emergency department and the administration of antibiotics was in median equal to h min in p and h min in p (p = . ). adequate iv fluid resuscitation was administered to % of patients in p and % of patients in p (p = . ). during p , % of patients did not qualify for admission to the intensive care unit compared to . % in p . hospital mortality did not change from . % ( / ) in p to . % ( / ) in p (p = . ). conclusion. the introduction of a standardized treatment protocol in an emergency department allowed a better recognition of severe sepsis with earlier adapted treatment . the study was not powered to demonstrate a reduction of the mortality in this elderly population. multiple studies have shown that early detection and therapy is crucial for the prognosis of a severe septic patient. many hospitals have joined the surviving sepsis campaign and its fight for the decrease of mortality in severe sepsis and have implemented the severe sepsis bundles into their daily practice. other institutions such as ours had so far not taken this step, perhaps because the process is hard and time consuming. we have tried to find an easy way to audit the implementation of severe sepsis bundles and its change in time in an institution without a set system and database for the implementation of severe sepsis bundles to help us prove, that a systemic change in clinical practice is essential. we have decided to use the first step of the resuscitation bundle-the measurement of lactate and audit the lactate requests in blood samples with elevated inflammatory markers in our hospital laboratory information system. we retrospectively audited the number of lactate requests in blood samples with c-reactive protein (crp) c mg/l and its evolvement in time between and before and after the introduction of surviving sepsis guidelines and severe sepsis bundles in our regional hospital with beds. we compared the total number of blood samples with elevated crp c over mg/l with or without procalcitonin request in our institution with the number of blood samples with crp c mg/l and lactate request (both arterial and venous) in the hospital laboratory information system. . the total number of lactate requests in samples with crp c mg/l had increased in time, the incidence widely differed between departments. the main increase was in patiens from intensive care units, the number of lactate requests in samples from general wards, emergency department and intermediate (step down) units had also increased ( lactate requests in samples with crp c mg/l- , % in and lactate requests in samples- . % in ) but still remains insufficient. surprising was that procalcitonin was in non icu patiens with crp c mg/l requested more often than lactate. although many lectures and seminars on severe sepsis bundles and the guidelines for the management of severe sepsis were organised in our intitution between the year and , it was not sufficiently effective. conclusion. retrospective audit in the hospital laboratory information system of the number of lactate requests in samples with elevated inflammatory markers appears to be a fast and a very easy first step for auditing how the surviving sepsis guidelines and severe sepsis bundles are implemented in your institution. the results help to quantify the present state, its change in time and may serve as an impulse to make systemic changes in the system of early detection and therapy of septic patients. introduction. early goal-directed therapy (egdt) is the accepted gold standard for resuscitation in septic shock [ , ] . international guidelines for the treatment of septic shock [ ] set an initial h limit to accomplish this goal. to test the hypothesis that egdt with fluids and vasopressors has better patient outcomes if each intervention is completed within h. thirty septic shock patients from the spring of and from spring were reviewed prospectively (n = ). septic shock was defined as a lactic acid c mmol/l and/or hypotension unresponsive to fluids. apache ii and sofa scores were calculated. patients were subjected to the hospital septic shock protocol according to guidelines [ ] . firstly, egdt compliance was met if the following interventions were achieved within h: lactate levels drawn, map c mmhg and cvp c mmhg; and secondly, if antibiotics were given\ h, blood cultures were taken before antibiotics and if ml/kg fluid bolus was administered prior to vasopressors. in patients / interventions were performed in time (''egdt-compliant''). the other were deemed ''egdt-noncompliant''. outcomes were mortality rate and discharge destination. fisher test was used in statistical analysis. . mr was % amongst the compliant and % amongst the noncompliant and admission to long-term care facilities (ltcf) was and %, respectively. neither one of these differences was statistically significant. a power analysis revealed that patients are required to attain statistical significance for mortality. discharge home was the same in both groups. there was no difference between groups in the number of new tracheostomies or new hemodialysis. conclusions. in a us community teaching hospital, compliance with guidelines in the treatment of septic shock had a trend towards lower mortality and higher discharge rates to ltcf but the difference was not statistically significant. larger numbers are needed for the benefits/effects of egdt-compliant therapy to reach statistical significance in the treatment of septic shock in this hospital setting. improve the survival rate of septic patients by means of education and implementation of a sepsis operative protocol including the activation of a specific consultation by an intensivist and an infectious disease specialist (i.e. sepsis team, st). aim of this study was to describe the first months activity of st, with a focus on the patients not admitted in intensive care unit (noicu). methods. the sepsis operative protocol, introduced in clinical practice in june , provides for specific instructions for the early identification and management of septic patients and for the early activation of the st for patients with severe sepsis or septic shock admitted in non-intensive departments. the st consultation ought to support the departmental health personnel in the management of septic patient and allows an early intensive care admission in case of shock or if mechanical ventilation is needed. to assess st activity, we evaluated in noicu patients the correct st activation rate, the number of st activations for each patient, the rate of central venous catheter insertion (cvc) and the days mortality. results. from june to december , the st was activated for patients ( . patients per month) whose ( %) were admitted to icu and ( %) were considered too sick to benefit. in ( %) of the remaining patients, st was properly activated: patients with severe sepsis and with septic shock. thirteen patients ( %) had no sepsis and ( %) had sepsis without organ dysfunction. % of st activations originated from medical departments (including emergency department) and % from surgical departments. the number of st activations for each single patient was ± . the days mortality was . % in patients with sepsis, % in patients with severe sepsis and % in patients with septic shock. conclusion. the rate of correct activation of st and the number of activations for each patient were acceptable considering that more than % of the activations refers to septic patients and that a mean of activations was sufficient for patient management. mortality rates observed are slightly lower than those reported by others, but further data are needed to evaluate the impact of st on patient outcome. a. estella , l. pérez fontaiña , j. i. sanchez angulo , e. moreno hospital of jerez, emergency and critical care unit, jerez, spain clinical evidence suggests that an early diagnosis and treatment of severe sepsis has been shown to improve outcome. frequently the initial management of septic patients occurs outside of the icu. objective. to describe clinical characteristics and outcome of septic shock patients admitted in icu and to compare mortality according origin prior admission in the icu (emergency department versus medical or surgical wards). consecutive patients with septic shock admitted in icu from july to november were registered. age, icu length of stay, source of infection, isolated bacteria, blood lactate concentration, apache ii score and mortality were collected. patients were classified according the origin prior admission in icu. . consecutive septic patients were admitted in icu during the time of study, global mortality was %. patients were admitted from medical or surgical wards and patients from the emergency department. mean age was years, male and female, icu length of stay was . ± . days, the mean apache ii score at admission in icu was . ± . abdominal infection, . %, was the commonest source of infection followed by pulmonary and urinary infection, . and . % respectively. patients ( %) had a positive bacterial culture, the mean baseline lactate level was . ± . mmol/l p \ . ( . ± mmol/l in the medical and surgical wards group versus . ± mmol/l in the emergency department group).there were not differences in clinical characteristics according origin prior admission in the icu except for lactate level, and mortality, . % in the medical and surgical wards group and . % in the emergency department group (p \ . ). conclusion. there were not differences in clinical characteristics, icu length of stay, source of infection, isolated bacteria and apache ii score between groups. mortality was lower in the group of patients admitted in icu from the emergency department than the group admitted from medical and surgical wards. although very high circulating concentrations are detectable in plasma, it is not known which organs actually produce the cytokines. we hypothesized that key abdominal organs affected by sepsis such as the kidney and liver produce cytokines and tested this hypothesis by measuring cytokine flux. materials and methods. pigs ( - kg) were randomised to control (n = ) and endotoxin (n = ) groups. hemodynamic measurements using picco and pulmonary arterial catheters and arterial blood gases were collected hourly. portal, hepatic and renal arterial blood flows were measured with transit time probes. arterial and venous cytokine concentrations (tnfa, il- b, il- and il- ) were measured from samples taken from each respective organ. cytokine flux was calculated as: organ blood flow (venous-arterial cytokine concentration difference). endotoxemic pigs had significant increases in heart rate (p \ . ) and mean pulmonary arterial pressure (p = . ) and decreases in cardiac output (p = . ). in contrast, these hemodynamic variables remained stable in the control animals. renal, hepatic and portal vein flows decreased significantly in all endotoxemic animals but remained stable in the control group. renal [ml/(kg min)]:control . ± . , . ± . , . ± . , . ± . versus endotoxin . ± . , . ± . , . ± . , . ± . for baseline, t = , , , respectively. portal [ml/(kg min)]: control . ± . , . ± . . . ± . , ± . versus endotoxin . ± . , . ± . , . ± . , . ± . for baseline, t = , , , respectively. hepatic [ml/(kg min)]: control . ± . , . ± . , . ± . , . ± . versus endotoxin . ± . , . ± . , . ± . , . ± . for baseline, t = , , , respectively plasma cytokines tnfa was detectable in very low concentrations (\ pg/ml) in of the endotoxemic animals, and none of the control animals. il- b, il- and il- increased significantly with time peaking at t = , and respectively in the endotoxin group. in the control group only few animals showed a cytokine response, in numbers insufficient for statistical analysis. in the endotoxin group there was a negative cytokine flux in the renal circulation, maximal at t = [- . ± . for il- b and - . ± . for il- (pg/ml), respectively]. there was a positive cytokine flux for il- reaching its peak at t = ( . ± . pg/ml). a similar pattern was seen in the hepatic ? portal circulation with maximal flux for il- b and at t = (- . ± and - . ± . pg/ml, respectively). for il- there was a positive flux peaking at -= ( . ± . pg/ml). although there was a negative il- b and il- cytokine flux in the renal, portal and hepatic circulations indicating net uptake, and vice versa for il- , none of these values reached statistical significance. conclusions. these data do not support that cytokines are produced nor consumed in the kidney and liver during endotoxemia. discussion. non-survivors show more severity at the beginning and during their icu stay, more altered biological markers and a higher mean glycemia, but do not show significant difference either at initial glycemia, history of diabetes, hypoglycemia event or insulin treatment. elevated mean glycemia appears to be a factor independently associated with higher mortality. hyperglycemia prevalence in critically ill patients is very high and the controversy whether it is a mortality marker or a mediator still remains. our results would justify starting an intensive insulin protocol and its subsequent analysis. the interest of continuous scvo was proven in the management of severe septic patients [ ] , but the place of discontinuous scvo remains unclear. objectives. to compare continuous scvo to discontinuous scvo concerning the number of therapeutic interventions in the management of severe sepsis (ss) and septic shock (ssc). methods. prospective randomized comparative study. inclusion criteria: age [ years, ss or ssc [ ] . two groups were defined: continuous scvo (c group) monitored by a central venous oximetry catheter (edwards lifescience x hs, irvine, usa), and discontinuous scvo (d group) measured on blood samples drawn every h and at the request of the treating physician. the hemodynamic management of these patients was based on the algorithm established by rivers [ ] . the primary endpoint was the number of therapeutic interventions (fluids, transfusions, inotropic drugs) triggered by a scvo \ %. non parametric tests (chi-square and mann whitney) and repeated-measures anova were used in statistical analysis (p \ . was considered significant). results. patients were included in a polyvalent intensive care unit (icu). the two groups were comparable concerning age, sex, weight, height, apache ii score, mods on admission and mechanical ventilation (mv). there were no statistical differences between the two groups concerning: mortality, duration of icu stay, duration of mv and the evolution of mods and plasma levels of lactate from day to day . the therapeutic interventions data are shown in table . introduction. the calcium activated potassium channel (bkca) exists in smooth muscle cells in most vascular beds and is believed to be important in sepsis induced hypotension and vascular hyporeactivity [ ] and also in neutrophil killing and macrophage production of proinflamatory cytokines. however the latter two roles have been disputed [ ] and we have found that bkca expression is not upregulated in aorta from septic mice using real time polymerase chain reaction. as its role in sepsis remains uncertain we sought to determine whether null mice for the bkca channel were (a) resistant to hypotension and (b) showed improved survival in a clinically relevant model of fecal peritonitis. methods. bkca null mice (based on balc) were obtained from jax Ò mice. agematched litter mates homozygous for bkca were wild types (wt). mice (age - weeks) had tethered arterial and venous lines inserted under isoflurane anesthesia. the tether enabled mice to roam cages freely whilst continuous blood pressure (bp) traces were obtained. h post surgery, echocardiogram and intraperitoneal injection of rat slurry was administered under anesthesia. fluid resuscitation of . ml/h voluven/ % dextrose ( : ) was given. at and h echo was recorded and mice culled with mesenteric arteries dissected for myography. data expressed as mean(sem) and statistical analysis anova. results. genotypic study and whole cell patch clamp recording in aortic smooth muscle cells confirmed bkca current was absent in null mice. fecal peritonitis induced equivalent hypotension in both wt (n = ) and bkca null mice (n = ) at - h (fig. a ). echocardiography at h post slurry showed no difference in cardiac output between wt- . ( . ) and bkca null mice- . ( . ) ml/min and no difference or improvement cf time (fig. b) . thus this fall in bp is due to reduction in total peripheral resistance not myocardial depression. in addition / of the bkca null mice died prior to h as opposed to / wt. hence myography was only performed on wt mesenteric arteries which were hyporeactive to norepinephrine (p = . , fig. c ). conclusion. there is no evidence from this transgenic mice study of fecal peritonitis that inhibition of the bkca channel would be beneficial for the treatment of hypotension in septic shock or would improve survival. reference(s). introduction. pro-and anti-inflammatory responses play a key role in the pathophysiology of sepsis [ ] . phosphodiesterase (pde) inhibition could play an anti-inflammatory role in this setting [ ] . previously, it was shown that among the three inhibitors of pde five currently available (sildenafil, vardenafil, tadalafil), only tadalafil could exhibit anti-inflammatory properties on endothelial cells (ec) stimulated by modified oxidized ldl or tnf alpha [ ] . to assess the potential anti-inflammatory role of tadalafil in ec stimulated by lps. methods. thp- cells ( . /ml in rpmi) were incubated alone (control group) or in the presence of either tadalafil ( lm; eli lilly, in, usa), lps from e.coli :b ( ng/ml; sigma-aldrich, inc.) or both. tnfa production, as a marker of inflammation, was measured in the supernatant (elisa assay; roche, mannheim, germany) after h of incubation ( independent experiments in quadruplet). comparisons were made by one-way anova, with bonferroni's post hoc test (mean ± sem). results. production of tnfa increased significantly after stimulation by lps alone compared to control ( . ± . -fold over the control, p \ . ) or tadalafil ( . ± . vs. . ± . -fold over control, p \ . ). levels of tnfa were significantly reduced in the lps ? tadalafil group, compared with the lps group ( . ± . vs. . ± . -fold over the control, respectively; p \ . ) (graph ). we hypothesized that daa provides varying protective effects in different organs as indicated by higher amounts of epcr in early murine sepsis. methods. sepsis was induced by cecal ligation and puncture (clp) in male nmri-mice (n = , body weight ± g). animals were randomly assigned to vehicle infusion (control), or clp sepsis with daa infusion [daa; lg/(kg hr)]. a third group received only sham operation and vehicle infusion (sham). h prior to clp all mice were given a permanent central i.v.-line and an arterial transmitter (pa-c , st. paul, mn, usa) to measure heart rate (hr) and mean arterial pressure (map). clp was adjusted to survive h. after h hearts, livers and kidneys were fixed in formalin and embedded in paraffin. immunohistochemical analysis of the paraffin sections was performed using the avidinbiotin-peroxidase complex (abc) method. for analysis an anti-mouse epcr antibody (clone , natutec, frankfurt, germany) was used (dilution : ) after heat pretreatment. anti-epcr positive cells were counted in fields in light microscopy (original magnification: . ) of each tissue and the average was recorded. data are presented as mean ± sd. *p \ . was considered significant. results. there were no significant differences in hr between the groups (sham ± per min; daa ± per min; control ± /min). map was significantly higher in sham group ( ± mmhg; p = . ) and non-significantly higher in daa group ( ± mmhg) when compared to control ( ± mmhg). anti-epcr positive cell count in heart tissue was significantly higher in sham-treated mice ( . ± . cells; p \ . ) and daa mice ( . ± . cells, p = . ) compared to controls ( . ± . cells). in kidney tissue epcr positive cells were significantly more in sham group ( . ± . ; p = . ) compared to control, but not in daa group ( . ± . ). liver samples showed no significant differences (sham . ± . ; daa . ± . ; control . ± . ). conclusion. our data showed higher amounts of epcr in murine sepsis undergoing daa therapy in heart and kidney tissues, but not in the liver when compared with control animals. this suggests that daa provides different effects in early experimental sepsis. background. caspofungin treatment is often initiated in hypovolemic shock patients, what could affect its pharmacokinetics and efficacy. the present study investigated the influence of hypovolemic shock and fluid loading on the plasma pharmacokinetic parameters and the pulmonary penetration of caspofungin in a pig model. after anesthesia and mechanical ventilation, pigs ( ± kg) were bled to induce a -h deep shock and resuscitated for h using normal saline based on hemodynamic goals. a -h perfusion of mg caspofungin was started at the beginning of the resuscitation period. lungs were removed h after the initiation of hemorrhage. sixteen animals were used as controls without hemorrhage. caspofungin concentrations were measured using high performance liquid chromatography method. in the shock group, the volume of removed blood was ± ml/kg and a volume of ± ml/kg of saline was infused through the resuscitation period. conclusion. hypovolemic shock followed by fluid loading in pig results in a significant decrease in plasma caspofungin exposition. it resulted in a decrease in the pulmonary concentration of caspofungin without affecting its diffusion to the lung. future investigations should focus on the interest for monitoring of plasma caspofungin concentrations in icu patients and on optimal dosing in these patients. objectives. the present study was designed to assess the effects of mps from septic origin on systemic hemodynamics as well as on the inflammatory, oxidative and nitrosative stresses. methods. forty healthy rats were randomly allocated to three groups: animals inoculated with mps isolated from control rats (cmps), animals inoculated with mps isolated from sham rats (shmps) and animals inoculated with mps isolated from rats with peritonitis (smps). rats were anesthetized, mechanically ventilated and were infused with the same amount of cmps or shmps or smps. we measured heart rate (hr), mean arterial pressure (map), carotid artery blood flow (cbf) and portal vein blood flow (pbf). hemodynamic parameters were recorded during h, and then animals were sacrificed. aorta and heart were harvested for further in vitro tissue analyzes. . the cellular origin (phenotype) but not the circulating concentration of mps was different in septic rats, characterized particularly by a significant increase in leukocyte derived mps. . smps but not cmps or shmps decreased mean arterial pressure without any effect on carotid artery and portal vein blood flows. all rats survived in the cmps and shmps groups whereas three rats died before the end of the experiment in the smps group. . rats inoculated with smps exhibited an increase in superoxide ion production and nf-kb activity, over-expression of inos with subsequent no overproduction and decrease in enos activation. pulse blood pressure recordings conclusions. rats with sepsis induced by peritonitis exhibited a specific phenotype of mps which could play a detrimental hemodynamic effect as a systemic vasodilatation. inoculation of smps in healthy rats decreased map likely by up-regulating nf-kb activity with subsequent inos, no and superoxide anion overproduction. these data confirm a proinflammatory detrimental role of mps in the vascular pathophysiology of septic shock. introduction. heat shock proteins (hsps) play an active part in modulating intracellular responses to stress. in the classical model for their activation de-repression of heat shock transcription factor (hsf ) occurs as a result of the titration of hsps away from hsf by misfolded proteins [ ] . however, hsps may change in many diseases without any changes in the levels of denatured proteins [ ] . objective. we propose that hsps are activated, in part, by a membrane dependent calcium channel receptor, possibly transient receptor potential vanilloid type- (trpv ). capsaicin, a known inducer of trpv , and capsazepine, a selective antagonist, were used on different mammalian epithelial cell lines. cells were pre-treated with micromolar concentrations of capsaicin or heat shock (hs) followed by treatment with capsazepine. results. capsaicin or hs induced hsf activation and the consequent accumulation of hsp , and chaperones. pre-treatment with capsazepine prior to hs or capsaicin abolished the heat shock response (hsr). capsazepine treatment prevented capsaicininduced stabilization of ikb and cell to cell adhesion and induced apoptosis. capsazepinemediated blockage of the heat shock response was reproduced with egta. moreover, treatment with trpv sirna resulted in a similar response to capsazepine. conclusion. hsr-sensing and signaling in mammalian cells depends, in part, on the transient entry of calcium by way of membrane dependent calcium channel receptor. these hsr modulators may hold promise in treating inflammation in the future. introduction. hydrogen sulphide gas, or its intravenous donor-sodium hydrogen sulphide (nahs), are promising therapeutic agents in ischaemia-reperfusion and haemorrhagic shock [ ] . we studied nahs in a short-term endotoxaemia model as relatively little is known about its effects during sepsis. methods. under isoflurane anaesthesia, male wistar rats (approx g weight) underwent left common carotid and right jugular venous cannulation for blood sampling/continuous bp monitoring and fluid administration, respectively. animals were kept normothermic on a heating mat. tissue oxygen tension (tpo ) was monitored using oxylite probes (oxford optronix, oxford uk) placed in thigh muscle. after a -min stabilization period, fluidresuscitated rats [ ml/(kg h)] were subjected to iv lps ( mg/kg over min). comparisons were made against animals receiving nahs ( . mg/kg bolus given immediately after lps, followed by a mg/(kg h) infusion). echocardiography (vivid , ge healthcare, bedford) and blood gas analysis were sequentially performed. sham-operated, non-septic animals also received nahs (n = ) or placebo (n = ). at the doses given, nahs had no effect on either sham-operated animals (data not shown), nor on the endotoxic rats (table ) . data shown as mean (±se). timepoints chosen reflect the biphasic response to endotoxin: = baseline, = initial hypotensive phase, = maximal recovery, = end of experiment. conclusion. nahs does not improve haemodynamics, tissue oxygenation nor shockrelated biochemical parameters in a severe model of fluid-resuscitated endotoxaemia. we will further investigate the effects of dose and time of therapeutic intervention in this model, in addition to testing it in a long-term septic model. intestinal endothelial and epithelial barrier dysfunction remain severe clinical problems as they may contribute to the development of sepsis and multiorgan failure. we have recently established an isolated rat small intestine model with access to vasculature, lumen and lymphatics for study of inflammatory changes in fluid balance [ ] stable for min, rendering it less suitable for examination of changes in gene and protein expression profile. the aim of this study was to assess the long term functional and metabolic stability of this model. adult female wistar rats were anaesthetized, small intestines cannulated and perfused vascularly ( . ml/min) and luminally ( . ml/min) and placed in a warm humidified chamber for up to h. arterial, venous and luminal pressures as well as venous, luminal and lymphatic effluent flows and intestinal weight were recorded continuously. as measures of metabolic integrity, oxygen consumption, lactate/pyruvate ratio and galactose uptake from luminally administered lactose were analysed every min. structural and barrier integrity were assessed as histostability score (mesenteric and antimesenteric fraction of fully epitheliated villi), wet/dry weight ratio and translocation of vascularly applied fitc albumin to lumen and lymphatics. data were compared using paired t tests. ± . / . ± . ml/(min g) dry weight (**)) as well as galactose uptake ( . ± . / . ± . mg/(min g) dry weight (n.s.)) were very stable with time pointing towards high metabolic stability. during the whole experiment, luminal effluent flow was slightly lower than applied ( . ± . ml/min, min) resulting in net liquid absorption over the whole time period ( . ± . / . ± . ml/min (n.s.)), and lymph production stayed in the physiologic range ( . ± . / . ± . ml/min (n.s.)). the organ weight did not change with time which, together with the balanced luminal fluid flow and end experimental wet/dry weight ratio of . ± . (compared to . ± . at the beginning of the experiment (**)), indicate absence of edema. minimal leakage of vascular fitc albumin to the lumen ( . ± . %) and a histostability score of . ± . show integrity of the vascular-luminal barrier until the end of the experiment. the isolated small intestine model presented earlier [ ] displays excellent long term physiologic, metabolic and histologic stability and opens up a wide field of applications including inflammatory gene transcription and protein expression. introduction. mitochondria play a major role during ischemia-reperfusion as well on cytotoxic pathways as protective such as ischemic preconditioning. the aim of this study is a better understanding of the mitochondrial pathophysiologic response to several oxygen regimens in an isolated mitochondria model. mitochondria were isolated from rat heart. enriched mitochondrial pellets were conditioned in presence of glutamate ( mm) and malate ( mm) inside the oxygraph chamber during min. oxygen partial pressures were: mmhg for control group; to mmhg for hypoxia group and mmhg for anoxia group. then, after a min oxygenation period, several measurements were realized: oxygen consumption (vo ) were measured with or without adp ( mm) (state and of mitochondrial respiration); calcium retention capacity (crc); mitochondrial membrane potentiel (dwm). to explore the involvement of reactive oxygen species (ros), mitochondrial vo were measured in presence of a specific mitochondrial antioxidant drugs (xbj). all results were expressed in percent of variation in comparison to control group [median (minimum-maximum)]. the different groups were analyzed using a kruskal-wallis, a mann-whitney with a bonferroni correction or a sign test when necessary. after hypoxia and reoxygenation the mitochondrial function was altered. this impairment of mitochondrial function was not found after anoxia and reoxygenation. this difference in mitochondrial function between hypoxia and anoxia suggests the involvement of ros. this hypothesis was confirmed by the effect of the antioxidant xbj that reestablished after hypoxia the same level of vo than after anoxia. [ ] . superoxide dismutase (sod) catalyses the dismutation of superoxide oxygen free radicals to oxygen and hydrogen peroxide (h o ). the therapeutic potential of exogenous sod administration in ards is evidenced by demonstrations of efficacy in acute lung injury models [ ] . anti-oxidant defenses, particularly the extracellular sod isoform, extracellular sod (ec-sod), are downregulated by endotoxin [ ] . we proposed that ec-sod delivered via a novel viral vector would ameliorate lung injury caused by lipo-polysaccharide (lps) pulmonary instillation. methods. three groups with nine rats per group were randomised to receive either adenoassociated virus expressing ec-sod (aav-ec-sod), adeno-associated virus coding for no product (aav-null), or vehicle control, days prior to planned lps instillation. a model of lipo-polysaccharide (lps) induced acute lung injury by pulmonary instillation was established in male sprague dawley rats. twenty-four hours following lps delivery, animals were anaesthetized and mechanically ventilated and their baseline compliance and oxygenation recorded. there was a statistically significant improvement in the oxygenation of animals recieving aav-ec sod as compared to aav-null or vehicle control (mean pao = . vs. . and . , respectively). there was a significant increase in amount of ec-sod as determined by real time pcr in the group who were administered aav-ec sod. no significant differences in static compliance or bronchoalveolar lavage cells counted were noted. conclusion. aav delivered ecsod is protective in a animal model of lps induced acute lung injury. the down regulation of the ec sod system seen in the systemic inflammatory response [ ] and its subsequent replacement exogenously may explain our findings. further work will focus on other components of cellular anti-oxidant pathways and confirmation of down regulation of ec sod in our injury model. aims. the endothelial specific angiopoietin (ang)-tie ligand-receptor system has been identified as a non-redundant mediator of endothelial activation in experimental sepsis. binding of circulating ang- to the tie receptor physiologically protects the vasculature from leakage, whereas binding of ang- antagonizes tie signaling and disrupts endothelial barrier function. we tested whether administration of exogenous recombinant ang- improves survival and attenuates multi organ failure in a lethal murine sepsis model. to induce septic acute kidney injury and to evaluate survival time cecal ligation and puncture (clp) was performed in twenty sv mice. half of the mice received an intravenous application of recombinant human ang- ( lg) immediately before clp and every h thereafter. in the other half, saline was administered in the same fashion. for tissue assessment (western blot, immunohistological) clp was induced in versus (ang- vs. saline) additional mice; animals were sacrificed after h. laparotomy served as sham control (n = ). further, a panel of cytokines has been assessed with a cytometric bead array (cba) system after h. . ± . mmol/l, p \ . ) were lower in ang- treated septic mice compared to controls. similar results were obtained at h after clp. renal tissue revealed that saline treated mice exhibit a marked loss of expression of vascular endothelial (ve)-cadherin, a major component of endothelial adherens junctions. in contrast, loss of ve-cadherin expression was prevented by ang- (pre-) treatment (wb densitometry: ang- : . ± . ; saline: . ± . ; p = . ). however, contrary to previous reports, intravenous injection of exogenous ang- enhanced not only the expression of adhesion molecules (icam- , vcam- ) in renal vasculature, but also circulating cytokine levels (tnfa, mcp- , il- , il- ). conclusions. our study demonstrates that administration of exogenous recombinant ang- improves survival time in a lethal experimental sepsis model. enhanced survival was accompanied by an improvement in microcirculatory function, probably via stabilization of adherens junctions. however, ang- injection deteriorated expression of vascular adhesion molecules and raised plasma cytokine levels. although ang- may have utility as an adjunctive agent for the treatment of septic multi-organ failure, additional dose-finding and efficacy studies are required. adaptive immune responses to infection. in contrast to neutrophils, macrophages or lymphocytes, there are virtually no data on the time course of circulating dcs in septic shock (ss). using a novel specific and sensitive assay, we analyzed the evolution of circulating myeloid (mdcs) and plasmacytoid (pdcs) dcs in ss. we enrolled immunocompetent adult patients with ss (n = ), shock from other etiologies (nss, n = ) and with sepsis without organ dysfunction (s, n = ). age-matched healthy controls (hc) served as reference for mdcs and pdcs. blood samples ( ll) were drawn on the day of shock, then after and days. dcs were counted using the dc-labelling kit trucount Ò assay (bd biosciences). cd c? cd -(mdc) and cd c-cd ? (pdc) cells were selected by flow cytometry (facscanto tm , bd biosciences). hla-dr mean fluorescence index (mfi) was measured. age, sex ratio, saps ii, sofa score, nosocomial infection (ni) and mortality rates did not statistically differ between ss and nss pts. at day , mdcs and pdcs counts were significantly lower in ss and nss pts as compared to hc and s ( fig. ). pts with ss had significantly lower mdcs and pdc counts than nss at days and . hla-dr mfi of mdcs and pdcs was lower in ss pts compared to hc (p = . and . , respectively). interestingly, of the ss pts developed ni after a median time of ( . - ) days in the icu. whereas mdcs increased in pts without ni, mdcs counts remained low at day in pts who developed ni: mdcs counts and their relative variation between day and were significantly lower in pts who developed ni than in those who did not (p \ . ). logistic regression analysis indicate that a negative mdcs relative variation is associated with an increased risk of nosocomial infection with an or ( . - ) (p = . ). figure conclusion . ss is associated with quantitative and qualitative abnormalities of circulating mdcs and pdcs as early as day , independently of the haemodynamic injury. the persistence of low counts of mdcs after ss is associated with the advent of nosocomial infection during the icu stay, suggesting that dcs play a role in the development of sepsisinduced immunosupression. introduction. liver dysfunction is common in sepsis but its mechanisms are unclear. the aim of the study was to evaluate the effects of lps on cultured primary human hepatocyte respiration over time. methods. human hepatocytes were isolated and cultivated from human liver resection specimens. cultivated cells were exposed to lps ( lg/ml) for , and h. after incubation, cells were trypsinized and respiration rates were measured using a high-resolution oxygraph (oxygraph- k, oroboros instruments, innsbruck, austria). glutamate ? malate (g ? m), succinate (s) or ascorbate/tmpd (a/t) were used as substrates to test the function of complex i, ii and iv, respectively. human hepatocyte mitochondrial function in the cells treated with lps for h exhibited a significant reduction in the maximal complex ii-dependent mitochondrial respiration [control: ± vs. lps: ± pmol/(s million cells) ( table ) ]. after and h of lps incubation no significant reduction in cellular respiration was observed ( and h: n = and h: n = ). statistics: paired t test, *p = . control vs. lps ( h incubation). introduction. acute kidney injury (aki) in critically ill patients is a frequent clinical problem and a rising incidence has been reported over the past several years. recently two consensus definition for aki have been developed: rifle [ ] in by the acute dialysis quality initiative workgroup (second conference) and akin [ ] in . insofar akin and rifle criteria have been applied in large retrospective studies, limited to the initial days of icu. nefroint is an italian initiative for an observational prospective multicenter study to evaluate epidemiology of aki in italian icus employing rifle and akin classifications. a pilot study has been performed in one of the centers enrolled. objectives. primary endpoints of nefroint are: application and comparison of rifle and akin criteria for aki definition in a prospective observational study; estimate, along such criteria, of aki incidence in critically ill patients; correlation of aki stages with prognosis. method. an observational prospective multicenter study has been designed, in italian adult icus (medical and surgical). all incident icu patients have been enrolled over a month period. exclusion criteria was age \ years, or icu stay \ h. data collection about patients was performed on a web-based electronic case report form. data included icu admission diagnosis, daily urine output ( h interval), daily laboratory data. sepsis events diagnosed on clinical and/or microbiological basis where as well marked for each patient. severity scores have been calculated at admission and daily. aki patients had higher severity of illness scores and higher serum creatinine values on admission. they also were older and more likely to have a respiratory diagnosis as reason for icu admission. conclusions. nefroint is an initiative aimed at comparing rifle and akin scores to promote a uniform use of a single definition of aki that will render subsequent studies comparable. early aki recognition could potentially allow implementation of timely corrective interventions, and hopefully prevent progression to more severe stages. aim. sepsis and septic shock remain the most important causes of acute kidney injury (aki) in critically ill patients and account for more than % of cases of acute renal failure (arf) in intensive care units (icu). its mortality varies with the severity of sepsis from % to %. the aim of this preliminary study was to investigate the differences in the course and prognosis of aki that was induced by community and hospital acquired sepsis. method. patients with sepsis induced aki were included in the study. rifle criteria were used to define aki. clinical and laboratory characteristics of the patients were compared with student t test and chi square tests. results. forty-one patients were included in the study and of them had community acquired septic aki (akic). ninety percent of the patients received mechanical ventilation (mv). etiologies of sepsis were mostly community acquired pneumonia and ventilator associated pneumonia. age, gender, admission apache ii scores and sofa scores at the time of aki diagnosis were similar across the groups (p [ . ). hospital acquired septic aki (akih) developed later when compared to community acquired septic aki ( th and rd days of sepsis respectively, p . ). akih was significantly and more frequently associated with oliguria ( vs. %, p . ), bacteremia ( vs. %, p . ), nephrotoxic antibiotic usage ( vs. %, p . ) and tend to progress more frequently to acute renal failure( vs. %, p . ) compared to akic. akic episodes were more frequently ( vs. %, p . ) and rapidly ( vs. days, p . ) reversible. mean blood pressure and scvo % were significantly lower and more vasopressor and steroid therapies were required during akih episodes compared to akic (p \ . ). while length of mv and mortality rates were similar, duration of hospitalization was significantly longer in the akih group ( vs. days, p . ). conclusion. these results suggest that, akih has worse clinic and prognosis than the akic so further and larger studies are necessary to investigate the preventive and therapeutic approaches. introduction. severity-of-illness or organ dysfunction scores are inaccurate to predict outcomes in patients with acute kidney injury (aki), even when specific aki scores are used. in recent years, the third versions of simplified acute and physiology score (saps ) [ ] and of mortality probability model (mpm -iii) [ ] scores were developed, and information on their use in patients with aki is scarce. objectives. to validate the use of saps and mpm -iii at the start of renal replacement therapy (rrt) in patients with aki. prospective cohort study conducted in the icus of three tertiary-care hospitals. data used to calculate the scores were collected at start of rrt. discrimination was assessed by area under receiver operating characteristic (aroc) curves and calibration by hosmer-lemeshow goodness-of-fit test. a total of consecutive patients were included between january and july . the mean age was . ± . years. the main contributing factors for aki were ischemia/shock ( %), sepsis ( %), contrast/nephrotoxins ( %), rhabdomyolysis ( %) and urinary tract obstruction ( %) (a patient could have more than one contributing factor). eightnine ( %) patients received rrt on the first day of rrt and ( %) thereafter; continuous rrt was used as first indication in ( %) patients. the icu and hospital mortality rates were and %, respectively. the mean saps score at the start of rrt was . ± . points. both the standard equation of saps and mpm -iii scores tended to underestimate mortality. discrimination was better for saps [aroc = . ( % ci, . - . )] than for mpm -iii [aroc = . ( % ci, . - . )], as was the calibration. however, mortality prediction and calibration improved when the customized equation of saps for countries from central and south america was used. in multivariate analyses, both higher prognostic scores and length of icu stay prior to rrt were the main predictive factors for hospital mortality. conclusions. the saps score at the start of rrt was accurate in our cohort of patients and seems a promising instrument for predicting hospital mortality critically ill patients with aki. objectives. the aim of this study was to investigate the effect of hes administration on kidney function compared with other colloids or crystalloids. methods. systematic review and meta-analysis of the effects of hes administration on kidney function. inclusion criteria for the study were prospective randomized trials comparing hes to control with reporting on variables of kidney function. aims. during the initiation phase of experimental acute kidney injury (aki), subtle but devastating changes, such as loss of brush borders, disruption of tubular cell polarity and cytoskeletal changes are detectable only to a certain extent by routine histologic methods. for this reason, subjective and moderate reproducible semi-quantitative scoring of tubular changes (e.g. vacuolization, detachment, cast formation, and necrosis) still remains the method of choice to quantify the extent of experimental aki. lectins are glycoproteins which are able to bind carbohydrate structures specifically. it has previously been shown that immunolabeling of the lectin phaseolus vulgaris erythroagglutinin (pha-e) is highly specific to the brush border of proximal tubular epithelial cells of rats, mice, and humans. the aim of this study was to ( ) develop a simple and fast lectin (pha-e) based staining protocol ( ) to objectively quantify, and ( ) to analyze brush border loss in a murine model of septic aki. methods. septic aki in mice (n = ) was induced by cecal ligation and puncture (clp). animals were sacrificed h after clp.sham operated (n = ) and healthy animals (n = ) served as controls. in order to specifically stain the tubular brush border, binding of biotinylated lectin pha-e was visualized by the biotin-avidin-complex (abc) glucose-oxidase (go) method coupled to tetranitroblue tetrazolium (tnbt) in -lm paraffin sections of renal tissue. the mean brush border area of five randomly chosen, non-overlapping cortical highpower fields was analyzed by planimetric software. lectin pha-e staining was highly selective for brush border of proximal tubules (black colour). virtually no staining was present in glomeroli and medulla. the xx software reliably identified lectin-positive areas, as confirmed by image overlay controls. we found a significant difference between sepsis induced aki, sham operated animals, and healthy mice (clp: . ± . ; sham: . ± . ; healthy controls: . ± . pixel ratio; p \ . ). our findings with the pha-e staining protocol correlated significantly with the conventional semi-quantitative scoring system (r = . , p \ . ). conclusion. the here presented lectin pha-e staining method followed by computerassisted planimetric quantification of brush border area is a highly reproducible and objective tool to analyze early histological changes during septic aki in mice. when an imbalance between oxygen supply and demand exist, anaerobic respiration commences and a metabolic acidosis develops. base excess and lactate have been used to identify a higher risk group of patients who should be admitted in icu prior to development of multiple organ failure. and at a time when appropiated therapy may previne the decline to death. acute kidney injury failure is a common complication in critically ill patients and it always difficult separate the acid base effects of critical illness per se from those of aki. the aim of this study was to examine wheter values of base excess or lactate taken on admission of patients with aki to a intensive care unit indicate prognosis and if wheter this can be used as screening tool for future intensive care admissions. we restropectively examinated data from patients with aki. to define the unique acid base characteristics of aki patients, we used a control group. the matched group consisted of icu patients wihtout aki matched for apache ii score. the base excess and lactate were collected at admission and then at h. a total of patients were enrolled at study over a month-period. there were no difference with respect age, sex and apache score between groups. the icu survival rates were % to the aki group and % to control group. the value of base excess with the best predictive prognosis ability was - mmol/l to the aki group and- . (p \ . ) to the matched group and the corresponding value for lactate was higher than . to both groups. the combination of these two markers on admission to the intensive care unit led to a sensitivity of % and specifity of % for mortality. conclusion. both base excess and lactate, or the combination of the two, can be used to predict day mortality in patients admitted to the intensive care unit. in patients with aki a different cut off of base excess should be used.these variables could be utilized to identify patients who have a higher risk for mortality to whom resources could be better directed. nonthyroidal disease (ntd) is a common finding in patients who are critically ill or on dialysis or with cardiovascular disease. its presence has been associated with inflamatory conditions. the aim of this study was to analyse the posible association of ntd with the development of acute kidney injury (aki). secondary targets where to estimate the incidence of ntd in a polyvalent icu and observe the realationship between the levels of t and some inflamatory markers: c reactive protein (crp), albumin and cortisol. during months in , after approval of the local ethical committee, we prospectively determined the following parameters in every patient admited to the icu: t , t , tsh, serum creatinine (scr), crp, albumin and cortisol. after excluding patients who died or were discharged before h, patients were studied. the degree of aki was calculated using the rifle scale. at admission the values of the analysed parameters were (mean ± sd): t . ± . pg/ml; t . ± . ng/dl; tsh . ± . liu/dl; scr [ ] . its incidence ( - %) is rising due to increasing numbers of ct scans and contrast studies conducted, and the higher prevalence of risk factors such as chronic renal impairment, diabetes mellitus and old age. although usually selflimiting, cin can be associated with a need for ongoing dialysis or increased mortality [ ] . to highlight the problem of contrast induced nephropathy and the difficulties in interpreting the current evidence for possible prevention strategies. we present the case of a year old man admitted to intensive care with acute pancreatitis. he underwent eight contrast-enhanced abdominal ct scans and received nacetylcysteine (nac) for all but one of these, after which he developed acute renal failure which did not recover. we also present a review of evidence for various proposed strategies. results. several studies have examined possible renal protective strategies around contrast administration. saline and bicarbonate have been shown to be beneficial when given pre-contrast [ , ] . theophylline has been shown in meta-analysis to have a significant beneficial effect, but heterogeneity of methodology between studies makes it difficult to clarify the degree of benefit achieved [ ] . nac has shown benefit in of trials. twelve meta-analyses showed inconsistent results, with showing nac to be beneficial. none showed harm. we analysed the heterogeneity of methods, endpoints and patient groups that makes these studies difficult to compare. critically ill patients may be considered at even greater risk of cin. only one study has specifically looked at this group. strategies such as volume loading may be inappropriate in some patients and there may not be time for nac for h pre-contrast. we were unable to find specific guidelines for the prevention of cin in critically ill patients. conclusion. the evidence for strategies to prevent cin specifically in critically ill patients is unclear. we review the current literature and propose renal protective strategies including hydration, nac and theophylline for this patient group based on the evidence available. objectives. the present study addresses the issue of how the different modes of rrt are currently used and performed. we conducted a prospective observational study in three portuguese intensive care units (icu). patient demographics, type of rrt used and outcomes were collected. we studied patients who were treated with rrt for rf, with a median age of years and a saps-ii score of . ± . , a sofa score of . ± . at admission; patients ( . %) were treated with continuous replacement therapy (crrt), patients ( . %) with sustained low-efficiency dialysis (sled)and patients ( . %) were initially treated with crrt and latter with sled. using the rifle criteria for the stratification of acute renal dysfunction at the beginning of the rrt we observed: risk- ( . %), injury- ( . %), failure ( . %), loss- ( . %), esrd- ( . %). we used anticoagulation in almost all patients ( . %). among patients who received anticoagulation, heparin was the most common choice ( . %), followed by low molecular weight heparin ( . %), and by sodium citrate ( introduction. in the intensive care unit (icu), severe sepsis and multiple organ failure are frequently associated with renal failure. continuous veno venous hemofiltration (cvvh), which is used as renal replacement therapy, also removes circulating inflammatory mediators. standard cvvh is currently prescribed with a substitution flow of ml/(kg min). theoretically, when hemofiltration is performed with higher volumes, buffer balance will be restored more rapidly, while also more inflammatory mediators will be removed. this may result in faster stabilisation from septic shock. indeed, animal-and some human studies show promising results, but have several (methodical) limitations. to evaluate hemodynamic and metabolic changes during hv-cvvh in patients with septic shock in comparison to (standard) cvvh. we performed a retrospective, observational, single-center study. all patients admitted with septic shock who were treated with cvvh in the period until were included. cvvh was defined as a substitution-flow b , ml/h, hv-cvvh as[ , ml/h. the decision to start with lv-cvvh or hv-cvvh was made by the attending icu-physician on an intention-to-treat basis. statistical analyses were performed with spss . introduction. haemostatic changes in critically ill patients are complex due to simultaneous pro-and anticoagulant processes. routine ptt and aptt assays monitoring clot formation poorly reflect hypo-or hypercoagulant state, especially during anticoagulation. endogenous thrombin potential (etp) comprises an in-vitro system for measuring thrombin generation beyond clot formation and may be more informative. objective. to assess whether etp has a role in monitoring systemic anticoagulation and predicting circuit clotting in critically ill patients receiving cvvh. methods. in a prospective study in an -bed general icu, we included patients with acute renal failure (arf) requiring cvvh (postdilution, - l/u). patients received a bolus of , iu of nadroparin followed by iu/h. samples of arterial and postfilter blood were taken at baseline and , , and h after start of cvvh to measure aptt, ptt, anti-xa and etp. we compared patients with early circuit clotting (circuit life £ lower quartile) and those with normal circuit life. median baseline arterial etp-area under the curve (auc) was ma (iqr - ma) (normal values - ma). baseline etp-auc was positively related to antithrombin and inversely to ptt, aptt, anti-xa (p \ . ) and sofa score (p = . ). median circuit life was . h (iqr - h). at baseline, the four patients with early filter clotting (£ h) had prolonged ptt and aptt, higher sofa score and a tendency to lower etp (table ) . during cvvh and nadroparin infusion, arterial and postfilter ptt and aptt were prolonged (p \ . ), antixa lower (p = . ) and etp-maximal concentration (cmax) lower (p \ . ) when circuits clotted early. while arterial etp-auc tended to be lower (p = . ), postfilter etp-auc was not different between groups. in critically ill patients with arf requiring cvvh with nadroparin anticoagulation, baseline etp is lower than normal and inversely related to organ failure and (a)ptt, probably reflecting consumption of coagulation factors. within the cvvh circuit, etp-auc and anti-xa show opposing patterns. the concurrence of early filter clotting with prolonged (a)ptt, lower antixa, lower etp and higher sofa score emphasizes the role of severity of disease and associated coagulation activation and heparin resistance in circuit clotting. introduction. nadroparin is a low-molecular-weight heparin (lmwh) used to prevent clotting in the extracorporeal circuit during cvvh. in renal failure lmwh accumulates and is associated with more bleeding (ref) . whether nadroparin is removed by hemofiltration and whether the anticoagulant activity accumulates during continuous infusion is controversial. objective. to study the kinetics and removal of anti-xa activity during continuous infusion of nadroparin in patients requiring cvvh using a cellulose tri-acetate filter. methods. in a randomized crossover trial in an -bed general icu, patients with acute renal failure (arf) were randomized. in group , postdilution cvvh was initiated at filtrate flow of l/h (blood flow (bf) ml/min), which was converted to l/h (bf ml/min) after min; in group , l/h was converted to l/h. patients (\ kg) received a bolus of , iu nadoparin followed by iu/h. samples of arterial blood, postfilter blood and ultrafiltrate were taken at baseline, h after the start and min, , and h after the conversion to measure anti-xa activity. results. fourteen patients with arf were equally randomized. patients in group had higher median sofa scores ( vs. , p = . ), baseline coagulation markers were not significantly different. arterial and postfilter anti-xa values are presented in fig. . during cvvh arterial anti-xa tended to decrease in time (p = . ). the median ratio of postfilter to arterial anti-xa was . (iqr . to . ). there were large differences between patients; differences between groups were not significant, except for postfilter anti-xa at h, which was significantly higher in group ( l/h) (p = . ) . anti-xa activity was not detectable in the ultrafiltrate. conclusions. critically ill patients receiving nadroparin during cvvh showed no signs of accumulation of anticoagulant activity, although extracorporeal removal of anticoagulant activity could not be demonstrated. apparantly, nadroparin is cleared by these patients despite renal failure. the differences in anti-xa between patients may be related to severity of disease. introduction. unfractionated heparin (ufh) is used as the first-line agent for anticoagulation of the extracorporeal circuit during continuous renal replacement therapy (crrt) in % of icus in the uk (uk) [ ] . its use is monitored with serial measurements of activated partial thromboplastin time (aptt) or its ratio (apttr) in % of icus [ ] . there is, however, considerable variation in practice [ ] . anticoagulation is useful for prolonging haemofilter life and facilitates the provision of continuous therapy, but must be balanced against the risk of haemorrhage, which has been correlated with increasing apttr [ ] . most icus in the uk use an apttr target of . - . [ ] , despite recent guidance that a target range of . - . provides adequate filter life with less risk of bleeding [ ] . objectives. to investigate the adherence to our local target range for ufh therapy (apttr . - . ) and the occurrence of over-anticoagulation in our patients. ]. there were apttrs ( %) which were above our target range, and incidences ( %) where the apttr was greater than or equal to . . the apttr was greater than . on occasions ( %). conclusions. this study was conducted in an icu which delivers crrt at a higher than average frequency [ ] , and which consistently has a standardized mortality rate below the national average. despite this, there was wide deviation from our target apttr range and a considerable incidence of significant over-anticoagulation, which may place our patients at risk of haemorrhage. the vast majority of apttrs were in excess of recent guidance [ ] . regional citrate anticoagulation (rca) may provide longer filter life with a lower incidence of bleeding [ ] . its use is increasing worldwide [ ] , though it is not commonly used in the uk [ ] . we are investigating the possibility of introducing rca in our icu. in the meantime, we will set a lower apttr target for our patients. we prospectively studied patients who received cvvh from july to december . age, gender, admission diagnosis, and apache-ii were obtained and the patients were divided into three groups: low dose heparin group, low molecular weight heparin group (lmwh), and no anticoagulation group (normal saline washing) based on assessment of coagulation status. for each circuit, circuit life, bleeding, platelet count, pt, inr, aptt, creatinine and urea were collected before and after crrt. results. seventy-seven critically ill patients with acute renal failure were treated with crrt and circuits were observed. among these circuits, received unfractionated heparin (ufh) anticoagulation, received lmwh anticoagulation and received no anticoagulation. the mean circuit life ( . ± . h) in low dose ufh group, was significantly longer than in lmwh ( . ± . h) and in no anticoagulation group ( . ± . h). there was no significant difference in baseline patient pre-crrt hb, creatinine and urea among three groups. the inr and pt and aptt in baseline were significantly higher in no anticoagulation group compared to the other two groups (p \ . ). the platelet count was significantly lower in the no anticoagulation group compared to ufh group and lmwh group in baseline and during crrt. there was no significant difference in the filter pt, aptt, among the three groups during crrt. the clearance of creatinine and urea during crrt were no significant difference among the three groups. bleeding complication secondary to crrt were no significant difference among the three groups. objectives. the purpose of the study was to assess the duration of time spent off therapy during the first five days of crrt in post-traumatic arf, and to identify the reasons for this. ullevaal between january and december , were retrospectively reviewed. the hospital is the regional trauma referral centre for approximately . million adult ([ years) persons. according to the local treatment protocol, dialysis filters were routinely changed after h due to time-out. individuals were identified and data collected using several institutional registries. patients were grouped according to presence of rhabdomyolysis based on peak serum creatine kinase levels exceeding , u/l or not. categorical data were compared employing two-sided pearson chi-square test, whereas continuous data were analyzed utilizing two-tailed mann-whitney u test. results. patients were included during the study period. during the first five days of therapy there was a total of dialysis days, and the total number of pauses was . the median duration of crrt was . h per day, giving a downtime of . h per day. the number of pauses per day was significantly larger in patients with rhabdomyolysis compared to patients without rhabdomyolysis ( pauses in dialysis days vs. pauses in dialysis days, p \ . ). this resulted in a shorter duration of crrt in rhabdomyolytic compared to non-rhabdomyolytic persons ( . vs. . h per day, p \ . ). overall the reasons for pauses during crrt were filter clotting ( %), therapeutic procedures ( %), catheter problems ( %), filter time-out ( %) and diagnostic examinations ( %). patients with rhabdomyolysis had more pauses due to therapeutic procedures ( vs. %, p = . ), whereas non-rhabdomyolytic persons had more pauses due to catheter problems ( vs. %, p = . ) and filter time-out ( vs. %, p \ . ). the number of pauses per day stayed relatively stable during the first five days of crrt, but the reasons for pauses changed during the study period. conclusions. this study indicates that trauma patients with rhabdomyolysis had more frequent dialysis pauses during the first days of crrt than those without rhabdomyolysis, resulting in shorter duration of dialysis therapy. the reason for this was more frequent use of therapeutic procedures, i.e. surgery and radiological interventions, in rhabdomyolytic compared to non-rhabdomyolytic persons. grant acknowledgement. the author is supported by institutional grants. introduction. treatment of acute pancreatitis is aimed at correcting any underlying predisposing factor and at the pancreatic inflammation itself. hypertriglyceridemia is an uncommon cause of pancreatitis. a serum triglyceride level of more then , to , mg/ dl is an identifiable risk factor. interestingly, serum pancreatic enzyme levels may be normal or only minimally elevated in such cases. severe necrotizing pancreatitis is associated with a high rate of complications and significant mortality. the reduction of triglyceride level to below , mg/dl effectively prevents further episodes of pancreatitis. this study aimed to determine the effectiveness of plasma exchange (pe) in reducing triglyceride levels during an acute attack of hyperlipidemic pancreatitis (hlp). methods. prospective, observational study including six patients hospitalized with hyperlipemic pancreatitis treated with plasmapheresis between and in the medical icu of a teaching hospital in malaga. demographic data, apache ii score, organ support needed and prognosis were prospectively collected. a total of hypertriglyceridemic patients with the complication of acute pancreatitis received one or two consecutive sessions. mean age was ± years and mean apache ii was ± . icu mortality was %. we performed sessions. the development of multiorgan failure in patients with hyperlipemic necrotizing pancreatitis was associated with grave prognosis ( %), needed mechanical ventilation, vasoactive agent and renal replacement therapy. however, we had a good outcome in the majority ( %) with a effective reduction of triglycerides after the session of plasmapheresis (pe). four of six patients ( %) recovered completely in a single session. two patients developed intraabdominal abscess, requiring more than one consecutive session and surgical debridement of infected necrosis and died due to both septic shock and multi-organ failure. the respective mean removal rates during a single pe for triglyceride were %. conclusions. the best treatment of hypertriglyceridemic pa is a drastic reduction of tg-s to normal. experiences with plasmapheresis are limited. we report six patients of hypertriglyceridemic necrotizing pancreatitis with mildly elevated amylase and lipase, treated successfully with plasmapheresis. in summary, pe treatment is an effective method to clear lipids and enzymes from plasma in a single session for most hlp patients. the presence of multisystem organ failure appears to be a more important indicator of outcome than does the presence of infection. results. sixteen ( %) patients were female and ten ( %) were male. the median age was years old. the median apache ii score was . . mechanical ventilation ( %), vasoactive agents ( %) and renal replacement therapy ( %) were the most common forms of organ support needed. sessions of plasmapheresis were performed. ( %) patients had been diagnosed with thrombotic thrombocytopenic purpura (ttp), six ( %) patients had hyperlipemic pancreatitis, five ( %) patients had pulmonary-renal syndrome (prs), three ( %) patients had guillain-barré syndrome (gbs) and two ( %) had myasthenia gravis. we obtained a decreased in the values of apache ii score following the plasmapheresis performed. there were six death ( % mortality) due of the severity of the disease. the number of complications were minimal and commonly described in the literature and there was a low mortality as a result. conclusion. results indicate that the performance of plasmapheresis was on a heterogeneous sample of patients with neuroimmunological diseases, rheumatology diseases and hyperlipemic pancreatitis. we conclude that plasmapheresis is a safe treatment which can be made by the staff trained in intensive care in any moment with a wide spectrum of clinical indications and with a minimum adverse effect. the aim of the study is to evaluate that early treatment of septic shock with cpfa may improve patient outcome. methods. twenty septic patients who were admitted to the icu have been enrolled in this study. cpfa treatment was performed immediately after septic shock was diagnosed (early group h after diagnosis). every patient had - cpfa treatments for h with q blood = ml/h, q ultrafiltration = ml/(kg h) and q plasma = % of q blood. we measured the plasma concentration of procalcytonin (pct), blood lactic acid levels, crp, serum creatynine, wbc and pao /fio ratio. the apache ii score, hemodynamic parameters, norepinephrine dosage were evaluated before cpfa (t ), t (after first cycle), t (after second), t (after third cycle) and t (after h). introduction. the development of electrolyte disturbances in intensive care patients could be prevented by the use of better adapted dialysis fluids. a common problem is hypophosphatemia which has been shown to occur in up to % of the patients. correction by intravenous phosphate supplementation is known to improve respiratory muscles, cardiac index, oxygen delivery to tissues and insulin resistance. lately it has been reported that phosphate can be added directly to the dialysis fluid. this facilitates phosphate handling, but there is a risk of precipitation with calcium. an additional problem is that the amount of phosphate required to correct total body deficit varies and repeated serum measurements are needed to establish phosphate insufficiency. the process is time consuming and leads to treatment delay and excessive cost. objectives. this study evaluated the possibility to achieve and maintain normal phosphate balance over time by using a new phosphate-containing dialysis fluid. objective. the purpose of this study was to evaluate the impact of different dialysate and replacement flows in the acid-base balance of the blood. furthermore we tried to assess the way partial pressure of oxygen (po ) in the blood is affected by high flow crrt. methods. this was a prospective observational study. thirty consecutive critically ill patients that were admitted in our icu and required crrt during their course were enrolled in the study. for each patient, blood flow, dialysate and replacement flow as well as ultrafiltration adjustments were performed by the responsible intensivist. any time that the clinical condition required a modification in any of these parameters, and after a period of time of no less than h, a simultaneous blood sample was drawn from both the arterial and the venous part of the circuit and the samples were analysed by a blood gas analyzer. arterial and venus samples were then compared for differences in ph, po and pco concentration. results. in total we performed measurements in patients. mean patient age was . years, mean apache ii score was , mean icu stay was days and mean crrt days was days. overall, ph in the venous line of the circuit was higher, pco was lower and po was lower as well compared to the respective values in the arterial line of the circuit, with no difference reaching a statistical significance. concerning the blood flow, we observed that when using high hemodiafiltration flows the difference in oxygen partial pressure between the arterial and the venous line of the circuit was greater, but again it did not reach statistical significance. conclusion. the use of crrt may influence the po in the returning blood. although we did not reach statistical significance in our study, there was a definite trend towards lower po in the venous line of the circuit when high flow crrt was applied. introduction. renal failure (rf) is a common complication in critically ill patient and is associated with high mortality and has a separate independent effect on risk of death. the continuous renal replacement therapy (crrt) is physiologically superior; however, there is lack of strong evidence to prove a clinical benefit. hybrid therapies (sled) that combine the benefits of intermittent haemodialysis and continuous therapies have emerged in the past few years. objectives. the aim of this study was to assess what type of renal replacement therapy (rrt) used and relate them to severity of the illness and outcome we conducted a prospective observational study in three portuguese intensive care units (icu). patient demographics, type of rrt used, saps ii and sofa score at admission and when we started the rrt and outcomes were collected. we studied patients who were treated with rrt for rf, with a median age of years and a median saps-ii score of ; patients ( . %) were treated with continuous replacement therapy (crrt), patients ( . %) with sustained low-efficiency dialysis (sled) and patients ( . %) were initially treated with crrt and latter with sled. aim. tetanus is traditionally treated with very high doses of diazepam and morphine. it often required prolonged periods of paralysis and was associated with very high mortality and prolonged periods of ventilation. magnesium sulphate (mgso ), due to its effects on neuromuscular and autonomic system should be effective in controlling muscle rigidity, spasm and autonomic instability in patients affected with tetanus. we introduced an icu protocol using mgso as first line treatment. we wanted to evaluate our patient outcome following the introduction of our protocol. we retrospectively analysed the effects of introduction of mgso in our intensive care for management of tetanus. aim. electrolyte disturbances were often seen in patients in intensive care unit (icu). hypomagnesemia is not enough described but can be contributed in icu mortality. the aim of this study was to define the prevalence of hypomagnesemia in critically ill patients and to evaluate its relationship with duration of mechanical ventilation day, length of icu stay and mortality. a prospective study was done on patients with respiratory failure admitted to the icu between . . and . . . total serum magnesium level, electrolyte levels, albumin, total protein, and lactate levels were evaluated at the admission. patients demographic features, accompanying neurological and cardiac diseases, apache ii score, duration of mechanical ventilation, and the length of icu stay and mortality were recorded. at admission % of patients had hypomagnesemia. a positive correlation was found between serum magnesium and calcium level (p = . ), but there was no relationship between other laboratory tests. also there was no relationship determined between hypomagnesemia and duration of mechanical ventilation, and the length of icu stay and mortality (p [ . ). conclusion. electrolyte levels are important in critically ill patients. however routine monitoring of serum magnesium level is not necessary. so we should increase the case number and also evaluate the serum magnesium level with urine magnesium level to see the effects of hypomagnesemia. method. medical records of copd patients who underwent invasive mechanical ventilation (imv) were reviewed. the patients' age, sex, body mass index (bmi), apache ii scores at admission, previous diagnosis of hypothyroidism or hyperthyroidism, history of thyroid replacement therapy or antithyroid medications, and the serum thyroid stimulating hormone (tsh), free triiodothyronine (ft ), and free thyroxine (ft ) at admission were recorded. the primary outcome measure was prolonged mv (pmv), which was defined as dependence on mv for [ days. the outcome and the relation between the serum thyroid levels were evaluated. results. ninety-five copd patients were included, % were male, with a mean age of . ± . years. bmi's of the patients were . ± . and the mean value of apache ii score was . ± . . only two patients ( %) had a history of hypothyroidism. two more patient were diagnosed hypothyroidism at admission and treated with thyroid medications. the patients treated with thyroid replacement therapy were liberated from mv successfully. patients ( . %) could not be weaned. serum ft level ( . ± . ) of the patients, who could not be weaned, was statistically lower than other group who could be liberated (p = . ).however there was no statistical difference between serum ft and tsh levels and two groups. hypothyroidism is an uncommon cause of ventilator dependent respiratory failure with an incidence of %, but it is treatable, so it should be considered in patients who can not be liberated.more prospective studies are also needed to evaluate the significance of hypothyroidism in patients with respiratory failure and failure to wean. smoke inhalation injury represents an important prognostic factor in patients admitted in the hospital after smoke exposition. objectives. we determined whether initial antithrombin (at) levels help in diagnosis and prognosis of sepsis after smoke inhalation. smoke inhalation was diagnosed according to classical clinical and laboratory findings in patients admitted in the hospital with suspected inhalation after smoke exposition. at levels, coagulation parameters (fibrinogen levels, prothrombin time (pt), activated partial thromboplastin time (aptt) and liver function tests were determined on admission and correlated each other and with outcome of the patients. . initial at and fibrinogen levels were significantly lower in patients with severe smoke inhalation compared to control (p \ . ). initial at levels were lower in the ones who developed septic complications with disseminated intravascular coagulation (dic) compared to those without dic (p \ . ). initial at levels were significantly lower in patients who died as compared to survivors (p \ . introduction. x-ray finding of pleural effusion is fairly common in icus. this may vary from mild to massive effusions and of different etiologies. epidemiological and outcome data for this icu problem are scarce in literature. the objective of this study was to find how common this finding is in our icu, their respective etiologies and any bearing on icu mortality. a single centre, prospective, observational study conducted in two mixed medical and surgical icus in kolkata, india. over six month period (october to march ) all consecutive patient admissions to these two icus were screened for a x-ray evidence of pleural effusion, either on admission or during their icu stay. as per icu protocol apache ii scoring were done in all patients. those with effusions were grouped according to etiology. finally in icu mortality were observed for those with or without an effusion. a total of icu admissions were studied. among these patients were found to have x-ray evidence of pleural effusion. median apache ii score was (iqr - ) among the study population with predominant ( . %) medical admissions. incidence of bilateral effusions were a total of ( %). the common causes of pleural effusion include chronic kidney disease (n - %), heart failure (n - %), pneumonia (n - %), post operative (n - %), chronic liver disease (n - %) and rest others (e.g. trauma, pancreatitis, pte, malignancy). the overall icu mortality was ( . %) and ( . %) in groups with and without effusion respectively with a p value of . , showing number of deaths in pleural effusion group were significantly higher. our study showed x-ray finding of pleural effusion quite common in icu patient population even many a times being bilateral. in this small study the overall icu mortality were also higher in pleural effusion group, but a wider multicentric study is needed. introduction. acute lung injury (ali) is a clinical manifestation of respiratory failure caused by lung inflammation and the disruption of the alveolar-capillary barrier. to prevent alveolar edema, it is of critical importance to preserve the physical integrity of the alveolar epithelial monolayer which is regulated by the balance between centripetal forces arising from cytoskeletal tension and cell-cell and cell-matrix tethering forces [ ] . intercellular junctions, such as tight junctions are closely related to actin cytoskeleton-related barrier regulation. proteins of the coagulation cascade such as thrombin (thr)-that stiffens [ ] and contracts [ ] alveolar epithelial cells (aec)-or activated protein c (apc)-an endothelial barrierprotective agent [ ] -could modulate this balance of forces in the epithelial monolayer. to study the combined effects of thr and apc on the barrier integrity through the tight junction zo- of aec by western blotting and immunofluorescence. methods. aec (a ) were incubated for h with apc ( lg/ml) or vehicle (control). subsequently, thr ( nm) or medium was added to the cell culture. for zo- western blotting, cell lysates were first ultracentrifuged ( , g, min, °c) to obtain membrane and cytosol fractions. then the samples were subjected to western blotting and the amount of zo- fractions was calculated by densitometry. for zo- immunofluorescence, aec were grown on glass coverslips and fixed in . % formaldehyde solution. zo- antibody was used to localize the tight junction and the zo- integrated optical intensity was then measured. . treatment with apc did not induce significant changes in any zo- amount of fraction protein analyzed by western blot. thr induced a *fivefold increase ( ± % of control values) in zo- membrane fraction while no changes were detected in zo- cytoplasm protein content ( ± % of control values). by contrast, apc concentration of lg/ml showed a clear tendency to reduce the effects induced by thr on zo- membrane fraction ( ± % of control values). for zo- inmunofluorescence, apc and thr treatments resulted in different patterns of zo- in the cell-cell contacts. after thr challenge cells showed discontinuous staining of zo- compared to untreated cells indicating a disruption of alveolar monolayer. conclusions. the increase in zo- amount of membrane fraction after thr challenge lends support to a protective mechanism avoiding cell-cell contacts disruption. treatment with apc reduced the increased zo- amount of membrane protein induced by thr suggesting an improvement of the barrier integrity in this model. ( ) interleukin- (il- ) is said to be involved in organ injury. we investigated the il- values of septic acute lung injury (ali) and acute respiratory distress syndrome (ards) patients. the subjects were patients during the -year period from to from whom it was possible to collect a blood specimen within approximately h of the onset of septic ali or ards. their mean age was years, and their mean apache ii score was . their sofa score was , and their mean pao /fio (p/f) ratio was . the p/f ratio was in the ali group and in the ards group. there were cases ( . %) in the -day mortality group, and cases ( . %) in the -day mortality group. the value of il- in died group was significantly higher than in survived group ( , ± , vs. , ± , pg/ml; p \ . ), and in the ards group also significantly higher than in ali group ( , ± , vs. , ± pg/ml; p \ . ). these results suggested that il- may play an major role in progression of ards in respiratory disorder as multiple organ failure (mof). [ ] . it is well-known that the pathophysiological mechanisms and factors involved in the liberation of no and the activation of inflammatory responses differ between aud and non-aud patients. objectives. the main hypothesis of this study is that ards patients with aud and non-aud differ in their response to the application of evidence based algorithms with respect to no response (aud patients are more frequent non-responders). patients with ards (meeting aecc criteria) were included in this ethically approved study. patients with severe chronic lung fibrosis and/or bridging for lung transplant were not included. patients were allocated to aud and non-aud patients. the auddetection was performed by the published algorithm [ ] . statistical analysis: wilcoxon-mann-whitney and chi-quadrat test was used. results. so far, patients with ards were included. prevalence of aud was % in our ards patients. baseline characteristics are given in table . frequencies of no nonresponse, extracorporeal lung support and mortality are given in table . frequency of no non-response was in tendency different: % in aud patients versus % in non-aud. overall mortality was % in aud patients versus % in non-aud patients. introduction. acute lung injury (ali) is a critical illness characterized by increased vascular permeability and impaired gas exchange leading to death in some cases. inflammation plays a pivotal role in the induction and maintenance of ali and is therefore therapeutic target to treat ali. rho, a small gtpase, is involved in the regulation of inflammation through the activation of recruitment of neutrophils to the site of inflammation and through activation of transcription factors such as nf-kb. we hypothesized that a rho kinase (rock) inhibitor, y- may be beneficial to dampen the inflammatory response in ali. male sd rats were intravenously pre-treated with either saline or rock inhibitor (y- , mg/kg). ali was induced by intratracheal instillation of mg/kg e. coli lipopolysaccharide (lps). control rats received saline intratracheally. h after the induction of ali, lungs were harvested and analyzed for myeloperoxidase (mpo) activity and expression of the proteins ijb, inos and enos. bronchoalveolar lavage fluid (balf) was used to assess total protein concentration as a measure of vascular permeability. pre-treatment with the rock-inhibitor resulted in significantly decreased levels of lps-induced mpo expression and prevented the upregulation of both lps-induced inos and enos expression. furthermore, lps-induced degradation of ikb was attenuated by pretreatment with y- . finally, y- improved vascular permeability by decreasing the lps-induced protein concentration in the balf. conclusion. inhibition of rho-kinase decreases lung inflammation and vascular permeability in acute lung injury and may therefore be a good approach to treat patients suffering from ali. we hypothesized that due to the cyclic changes of pulmonary air content there are po oscillations also in the mixed venous blood (pvo ), potentially influencing pao oscillations. in each of three healthy pigs of kg, anesthetized and ventilated with constant minute volume we studied three different tidal volume settings ( , and ml/kg) resulting in different respiratory rates. a calibrated oxygen probe (fiber optic, fluorescence-quenching probe, foxy-al ; ocean optics, dunedin, fl, usa) was inserted into the pulmonary artery through a fr catheter. the catheter position was previously controlled by pressure tracing. pvo was sampled with temperature compensation at hz with a multi frequency phase fluorometer (mfpf , tau theta, fort collins, co, usa) after a generated timestamp to synchronize with the electric impedance tomography (eit) signal (goettingen goemf ii, viasys healthcare, the netherlands) sampled at hz. eit and pvo were simultaneously recorded for min during each tidal volume setting and analysed with and without low pass filtering at the heart rate. we obtained pvo oscillations with amplitudes between to mmhg with the main frequencies matching the respiratory rate. ventilation with tidal volumes of ml/kg provided higher pvo amplitudes than ventilation with ml/kg. these results are preliminary and the source of the measured pvo oscillations is not clear. alternate backflow from the superior and inferior vena cava due to changes in intrathoracic pressures during mechanical ventilation may be responsible for these oxygen partial pressure oscillations in the mixed venous blood. conclusion. mixed venous oxygen partial pressure oscillates in accordance to the respiratory rate. whether arterial po oscillations are due to cyclic recruitment and derecruitment of the lung or to corresponding mixed venous oscillations remains to be evaluated. [ ] and in neonates [ ] . to our knowledge this is the first validation of the model using a large cohort of samples from intensive care patients. aim. to assess the ability of the severinghaus equations [ ] to estimate values for po and so in critically ill adult patients. methods. , sequential blood gas samples were analysed to validate the severinghaus oxygen dissociation curve, of these , measurements had a so b . % and were included in subsequent analyses. bland-altman plots were used to examine the agreement between measured po and that calculated from the severinghaus equations, and between measured and calculated so , both with and without correction for ph. the differences between measured and estimated values were analysed using paired t tests with a p value \ . considered significant. results. the severinghaus oxygen dissociation model accurately reflects the relationship between po and so observed in clinical samples. there is reasonable agreement between the measured and calculated values for po and so , with the majority of values falling between the lines of % agreement. there was a statistically significant difference between observed and calculated values of po even when adjustment for ph was made (p \ . ), however the mean difference between the groups was not clinical significant ( . mmhg when ph adjusted). there was also a statistical difference between measured and calculated values of so (p \ . ), again, however, this difference may not be considered clinically significant ( . %). patient data and severinghaus oxygen dissociation conclusions. the severinghaus equations accurately reflect the oxygen dissociation curve in critically ill adult patients and whilst they provide values for po introduction. zinc (zn) is an essential trace element, which plays a role in many biological functions including immune function. development of respiratory infections and changes in respiratory tract cells may be affected by low zn levels. in critically ill children mortality of septic shock and degree of organ dysfunction were associated to low blood zn levels , . our aim was to study serum zn in the beginning of acute respiratory failure (arf) and its association to development of organ failures and day mortality. during an -week study period (from april to june ) adult patients with arf were treated in intensive care units (= finnali-cohort). after consent blood sample for zn analysis was drawn at baseline). samples were taken in zn-free tubes, freezen and stored in - °c for analysis. all samples were analyzed with an atomic absorption spectrophotometry in the oulu university hospital laboratory. the range of normal values is - lmol/l. organ failures were assessed by daily maximal sequential organ failure assessment (sofamax) score. results. serum zn samples were obtained during h after the baseline with median time of h. only zn values were within and two over the normal range. median (iqr) serum zn levels were . ( . - . ) and . ( . - . ) lmol/l for survivors (n = ) and nonsurvivors (n = ), respectively, with no significant difference (p = . ). in patients with or without infection (pneumonia, respiratory infection or sepsis) during h prior to arf, zn levels were . ( . - . ) and . ( . - . ) lmol/l, respectively (p = . ). zn levels were significantly lower (p \ . ) in patients with cardiovascular sofa - than - , . ( . - . ) and . ( . - . ) lmol/l, respectively. a significant correlation of zn level and daily sofamax (spearman's q - . , p \ . ) was found (fig. ) . conclusions. low serum zn levels were detected in almost all patients with arf. no association to day mortality was detected to support the earlier findings with pediatric critically ill patients. however, we found a significant correlation to organ failure development in adult patients with arf. mountaineering is closely related to a range of adverse influences. the overriding factor that affects a climber may be the hypobaric hypoxia, which is compensated by hyperventilation and other adaptive changes in the pulmonary and systemic circulation. west ( ) theoretically predicted hypoxemia combined with respiratory alkalosis [ ] , and low oxygen saturation ( … %) has been observed on peak broad, karakorum [ ] . lack of adaptation is known as mountain sickness (occurrence … % [ , ] ), which may be alleviated by acetazolamide. the importance of understanding pathophysiology of mountaineering is dictated by the gradual expansion of western consumer-oriented society to higher altitudes. the goal of our study was to obtain precise information on changes in arterial blood gas composition, acid-base status, and degree of hemoglobin desaturation relative to altitude. materials and methods. experienced athletes-four males between and years and female years attempted to ascend mt. makalu ( , m) in april-may . acetazolamide , bid was used from april till may . femoral arterial blood rather than radial arterial blood was analyzed before reaching base camp ( background. ventilator associated lung injury is a complication of mechanically ventilated patients. knowledge about pathological pathways comes from animal studies, which are necessary to generate hypotheses to be tested in humans. various experimental methods of inducing acute lung injury (ali) have been used in animal models. the results of animal studies and human research appear to be conflicting; however, this may be a consequence from the different animal models used as such for comparison. we hypothesized that effects on gas exchange, respiratory mechanics, histo-pathologic lung damage and systemic inflammation are depending on the model of ali used. in five groups of pentothal anesthetized rats acute lung injury was induced by either lung lavage or hydrocloric acid aspiration. rats were then ventilated with lung protective settings in pressure controlled mode with positive endexpiratory pressure (peep) of cm h o or breathing spontaneously with continuous positive airway pressure (cpap) = cm h o for h. blood pressures, cardiac output, pulmonary mechanics and gas exchange were measured. results. the tidal volume was . ± . ml/kg in ventilated and . ± . ml/kg in cpap groups. respiratory rate and minute ventilation were constant in ali animals and controls, but showed variability in spontaneous breathing animals. only half of the cpap animals with ali survived [ h. no significant differences were found for pco , cardiac output or blood pressure between models, but mean arterial pressure decreased in ali. in the lavage and aspiration model, pao was lower after induction of ali ( ± and ± mmhg, respectively) than controls, and increased in lavage ( ± mmhg) but not the aspiration model ( ± mmhg) after h (p \ . ). dynamic compliance of the respiratory system decreased permanently after induction of ali to . ± . ml/cm h o (lavage) and . ± . ml/cm h o (aspiration) as compared to controls, which maintained at . ± . ml/cm h o after h. the lungs from five additional anesthetized, unassisted breathing animals, taken directly after induction, showed significant atelectasis, neutrophil infiltration and interstitial and alveolar edema (diffuse alveolar damage (dad) score . ± . ), as compared to control animals without ali (dad . ± . in ventilated, . ± . in cpap, respectively). the dad was higher in aspiration ( . ± . ) than in lavage ( . ± . ) induced ali, with no significant differences between ventilated and cpap animals. no hyaline membranes were observed. conclusions. anesthesia induces significant alveolar inflammation, which is partially reversible by use of peep. the ali model of acid aspiration induces persistent changes in gas exchange, respiratory mechanics and alveolar damage, which are more severe and consistent than those induced by the lavage model. background. acute lung injury (ali) is characterized by exaggerated inflammation and a high metabolic demand. mechanical ventilation can contribute to ali, resulting in ventilator induced lung injury (vili). a suspended animation-like state induced by hydrogen sulfide (h s) may reduce metabolism and co production, allowing for a lower minute ventilation to maintain gas exchange, thereby decreasing vili. h s may also limit lung injury via reduction of inflammation. the effect of h s-induced suspended animation on myocardial function is unknown. methods. in rats, vili was induced using a peak inspiratory pressure (pip) of mmhg and zero peep. controls were ventilated with a pip of and peep of mmhg. respiratory rate was adjusted to maintain normocapnia. suspended animation was induced by infusion of a h s donor, controls received saline. blood gases were drawn, bronchoalveolar lavage fluid (balf) was collected, lungs were removed. aortic flow was measured. statistics include kruskal-wallis and mann-whitney u. introduction. alveolar oedema is a hallmark of ards and ali. fluid clearance and the influence of anaesthetics on oedema resolution are poorly understood on a molecular level in the injured lung. oedema resolution is mediated by osmotic water reabsorption, following active sodium reabsorption via the apically located epithelial sodium channel (enac), driven by sodium-potassium-adenosin-triphosphatase (na ? /k ? -atpase). objectives. our aim was to investigate the influence of mac (= . vol%) sevoflurane on mrna and protein levels of enac and na ? /k ? -atpase in injured alveolar epithelial cells (aec). methods. primary culture of aec was stimulated with lipopolysaccharide (lps, lg/ ml) and exposed to normal air containing % co with or without sevoflurane. mrna levels were measured at h using the taq-man real-time pcr method. additionally, proteins for western blotting were analyzed at , and h (n = ). in the presence of sevoflurane mrna level of the a -subunit mrna of na ? /k ? -atpase in control cells was downregulated by % (p \ . ). a-subunit na ? /k ? -atpase protein expression, however, was not influenced by lps or sevoflurane at all time points. mrna of c-enac was decreased by % in the presence of sevoflurane and by % upon stimulation with lps. in the lps-sevoflurane group downregulation was even more pronounced with % (p \ . ) after h, but not statistically different from the lps group. on the protein level of c-enac protein expression a first change was observed at h with a downregulation of % upon lps exposure (p \ . ). sevoflurane did not have an effect of this transporter protein. previous studies have shown that halothane decreases na ? /k ? -atpaseand sodium channel activities in alveolar epithelial type ii cells [ ] . despite this finding for halothane, we could not see similar effects for the volatile anaesthetic sevoflurane. our results suggest that neither the driving force of alveolar oedema resolution, the sodium potassium atpase, nor c-enac, which is considered the rate limiting step in sodium coupled water reabsorption are influenced by sevoflurane and lps in an in vitro model of ards. to further characterize the impact of sevoflurane on water transport, functional analysis of these two transporters have to be performed. grant acknowledgement. objectives. we evaluated the effects of two nebulised sfa perfluorohexyloctane (f h ) and perfluorobutylpentane (f h ) at different dosages ( ml/kg vs. . ml/kg) on pulmonary mechanics and gas exchange in healthy lungs. design. after approval by the local animal care committee, prospective, randomized animal study. subjects. thirty-five new zealand white rabbits. interventions: tracheotomised and ventilated juvenile rabbits were nebulised intratracheally with either a high or a low dose of two different sfa (f h low/high and f h low/high ) or saline (nacl). ventilated healthy animals served as controls (sham). arterial blood gases, lung mechanics, heart rate and blood pressure were recorded prior to nebulisation and in min intervals during the -h-study period. results. immediately after starting aerosol therapy p a o /f i o -ratio and dynamic lung compliance decreased in all groups, with the exception of the f h low group which behaved like the sham group. although p a o /f i o -ratio showed a continuous improvement in the other groups over time respiratory mechanics still remained impaired. high dose groups with nebulisation of liquid perfluorohexyloctane (f h high ), perfluorobutylpentane (f h high ) or saline (nacl) showed no significant differences neither in oxygenation, blood pressure nor in pulmonary compliance and resistance. in contrast to f h high , there were no residues of f h high detectable in bronchoalveolar lavage. regarding f h low we were not able to detect any adverse effects on gas exchange or pulmonary mechanics. additionally, wet-dry-ratio of apical lung tissue samples revealed no significant edema. conclusions. high dose aerosolized sfa ( ml/kg), either f h or f h , equals effects of high dose inhalation of saline. when comparing the low-dose sfa-groups, there is a convincing discrepancy in favour of f h . f h low impairs pulmonary function, whereas a low dose application of f h (low) shows no interference. this may be due to the faster evaporation of f h . a new sfa-based pulmonary drug delivery system for lipophilic or water-insoluble substances could be developed on the basis of a low-dose application of f h . objects. hypertonic exposure reduces cell volume and thereby creates a relative excess of plasma membrane (pm). as a result the lipid bilayer of the pm can simply unfold with a minimal increase in lateral tension when an externally imposed shape change demands it. to test this hypothesis, we determined the effects of osmotic pressure on the susceptibility of deformation injury and pm wound repair. we measured deformation injury and repair responses of a . cell culture media were consisted of x hmem and mannitol (v/v / ) with osmolarity of (iso), (hyper) and mosm (hypo). cells conditioned with media were either stretched or deliberately injured with a scalpel. the fraction of wounded and healed cells was measured using a dual label method. . ( ) exposure to a hypertonic environment tends to lower the susceptibility of a to deformation injury ( . ± . % for iso, . ± . % for hyper), while exposure to a hypotonic environment uniformly increases it ( . ± . % for hypo) in stretch injury. introduction. the migration of polymorphonuclear leukocytes (pmns) into the lung plays a critical role in the development of acute lung injury (ali). adenosine receptor a (a ar) is one of four g protein-coupled adenosine receptors that has been demonstrated to modulate pmn trafficking in various models of inflammatory disorders including sepsis and asthma. however, the role of a ar in ali has not been investigated systematically yet. the objective of this study was to determine the role of the a ar in a murine model of lpsinduced lung injury and in an in vitro transmigration system with human cells. methods. the migration of pmns into the different compartments of the lung was determined by flow cytometry in adult male c bl/ mice (wildtype [wt] ) and homozygous a receptor knockout (a ko) mice. we used chimeric mice that were generated by transferring bone marrow between wild-type and a ko mice to differentiate the role of a on hemopoietic and nonhemopoietic cells. furthermore, microvascular permeability was assessed by the extravasation of evans blue and the release of chemotactic cytokines into the alveolar airspace was determined by elisa. paraffin-embedded sections of the lung were stained for pmns after lps inhalation to illustrate their accumulation in the lung. in a human in vitro assay, we quantified neutrophil transmigration across an epithelial monolayer (a cell line). in all murine in vivo experiments and in the in vitro transmigration assay, we assessed the effectivity of the specific a -agonist cl-ib-meca. all statistical analyses were performed by using anova. p \ . was considered statistically significant. results. inhalation of lps significantly increased the number of pmns in wt and a ko mice in all lung compartments. no differences in pmn counts were observed between wt, a ko, and chimeric mice. pretreatment with cl-ib-meca led to a significant decrease of pmns in all lung compartments of wt mice but not in a ko mice. pharmacological activation of a ar diminished the lps-induced microvascular permeability in wt mice but not in a ko mice. upon lps-inhalation, a ko mice exhibited significantly higher levels of the cytokines cxcl und cxcl / in the alveolar airspace than wt mice. in wt mice, pretreatment with cl-ib-meca reduced levels of tnfa and il- significantly. transmigratory activity of human pmns across an epithelial monolayer was reduced when a was activated in pmns. in contrast, pretreatment of the epithelial cells did not inhibit migration of pmns. introduction. lung overdistention during mechanical ventilation causes an increase in pulmonary vascular permeability, which is characterized by interstitial and alveolar edema secondary to a diffuse endothelial and epithelial injury. thrombopoietin (tpo), a humoral growth factor that stimulates the proliferation of megakaryocytes, has also been identified as a pro-inflammatory mediator in various clinical conditions. the receptor of tpo, c-mpl, is constitutively expressed on endothelial cells and may modulate the permeability of the endothelium. we investigated the contribution of tpo in the development of acute alveolar edema formation by mechanical stretch. in an ex-vivo model of mechanical ventilation (mv), lungs of c bl mice were ventilated for h with high stress pressure cycled ventilation (end inspiratory pressure = cm h o, peep = cm h o, i:e ratio = : ) and perfused with % bovine serum albumin rpmi medium at a rate of ml/min, in the presence or absence of tpo ( mg/ml). following ventilation, lung elastance was measured and protein concentration was analyzed in the bronchoalveolar lavage. data are mean ± se. mechanical ventilation (mv) to treat patients with ards or acute lung injury (ali) has the end objective to increase the dynamic functional residual capacity (dfrc), thus increasing overall functional residual capacity (frc). simple methods to estimate dfrc at the end of expiration for a given positive end expiratory pressure (peep) would provide a valuable metric to track and modulate therapy. however, such methods do not exist and current methods are time-consuming and relatively invasive. methods. this study utilizes a constant stress strain ratio for an individual patient's volume responsiveness to peep to estimate dfrc at any peep. the estimation model identifies two population parameters from clinical data to estimate a patient-specific dfrc, b and mb, where b captures physiological parameters of frc, lung and respiratory elastance and varies depending on the peep level used, and mb is the gradient of b versus peep. dfrc was estimated at different peep values ( , , , , ) cm h o, and compared to the measured dfrc for ali/ards patients to validate the model. patients and methods. in a years period patients ( males, females) with haematological malignancies were admitted in icu. malignancy type, reason for admission, haematological profile, requirement for invasive ventilation, bronchial and blood cultures and survival rate were recorded. results. patients suffered from: hodgkin's lymphoma ( ), non-hodgkin's lymphoma ( ), chronical lymphocytic leukaemia ( ), acute myelogenous leukaemia ( ) and multiple myeloma ( ) . admission to icu was precipitated by: emergency surgical procedure ( ), respiratory failure ( ), sepsis ( ), pulmonary oedema ( ) and coma ( ) . pulmonary infiltrates was the main finding in chest x-ray. bronchial secretions cultures were positives in patients while blood cultures were positives in patients. apache ii score ranged from to (average . ) and the icu days ranged from to (average . ). all the patients required invasive ventilation. all the patients with sepsis and serious neutropenia were died, while the total mortality was / ( . %). conclusion. the admission of patients suffering from haematological malignancies in icu is associated with high mortality. immunosupression that renders them susceptible to infections, thrombocytopenia, and invasive ventilation are factors that contribute to this. early recovery of bone marrow and non invasive ventilation could improve the outcome in these patients. in liver transplanted patients, immunosuppressive therapy can increase the risk of infections and post-operative arf. nimv has been proposed as an alternative technique to reduce complications related to endotracheal intubation. the aim of our study was to evaluate nimv in liver transplanted patients, developing arf in the post-operative period. materials and methods. in this study we evaluated liver transplanted patients, developing postoperative arf. measurements of respiratory and haemodynamic parameters were performed at baseline, after h and at the end of the treatment. we evaluated intubation rate, nimv tolerance, length of stay in the icu (los), icu and hospital mortality. results. ( %) out of patients were successfully treated with nimv, while ( %) failed and were intubated. we observed no significant differences among groups in gas exchange, but rr was significantly reduced in the success group during treatment (p \ . ). in both groups we found no significant differences in pao /fio initial improvement, but the success group showed a significantly higher rate of pao /fio sustained improvement (p \ . ). no significant differences between the two groups were found in terms of hours and days of nimv. success and failure groups were significantly different in saps ii (p \ . ) los (p \ . ), icu and hospital mortality ( vs. %, p \ . , vs. %, p \ . ). reasons for nimv failure were not related to respiratory causes, but acute systemic causes such as septic shock and mods. conclusions. nimv can represent a valid alternative to invasive mechanical ventilation for the treatment of postoperative arf in liver transplanted patients; in nimv success patients reduced los and mortality can be expected. the influence of body posture on expiratory flow-limitation (efl) was estimated in flowlimited, mechanically ventilated patients using the negative expiratory pressure (nep) method. a device especially designed and in build in an evita -draeger respirator allowed the application of a pressure equal to- cm h o, starting at ms after the onset of expiratory flow and sustained throughout the end of expiration. patients were considered flowlimited, if despite the application of nep part or the expiratory flow-volume curve was superimposed on the baseline curve. patients were studied in supine and in semi-seated position ( ) at baseline and then min after administration of bronchodilators ( mg of inhaled salbutamol) with a nebulizer connected to the inspiratory port of the ventilator. supine position was significantly related to the occurrence of efl (p = . ). efl was abolished in % of our patients when changing from supine to semi-seated position, while in general a significant improvement of efl was noticed (from to % of v t , p = . ). significant improvement of efl was achieved as well (p = . ) after bronchodilative therapy. peepi was the only variable significantly related to efl improvement when changing body posture from supine to semi-seated, while for bronchodilative therapy, none of the variables studied was significantly related to efl improvement. l. c. woodson , university of texas medical branch, anesthesiology, galveston, usa, shriners hospital for children, anesthesiology, galveston, usa aims. laryngeal injuries are common among burn patients and can result in long term functional deficits. we have included careful laryngeal examination with our initial fiberoptic bronchoscopic evaluation of burn patients. the goal has been to allow early identification of laryngeal injuries and to facilitate laryngology consultations. methods. digital video recordings were made of upper airway endoscopies performed during airway management on admission or at the time of anesthesia for initial wound excision. these recordings were used to identify laryngeal injuries and to facilitate laryngology consultations. a wide variety of laryngeal injuries were identified and the digital recordings (which can be communicated by email) greatly facilitated laryngology consultations. in many cases these recordings guided therapeutic interventions and were often sufficient to avoid a separate exam under anesthesia. diagnosis of thermal necrosis provided an indication for early tracheostomy. identification of the mechanism of mechanical airway obstruction (e.g. supraglottic edema, fibrinous exudates, granulomas, vocal fold dysmotility) resulting in failure of a trial of extubation frequently guided therapy. early identification of posterior glottic damage provided more timely corrective laryngological interventions. educational use of these videos helps increase awareness of risks of laryngeal injury in thermally injured patients. aims. the most used weaning predictor f/v t ratio, is not a consensual predictor. when it was reported on the first time, this ratio was considered highly sensitive and specific. but others papers seems to disagree with it, suggesting other cutoff values to determine weaning failure in specific populations, as the elders. advanced age is thought to be an import associated factor in the intensive care unit (icu), but its effect on the weaning process is unclear. no studies have found strong evidence that conventional weaning parameters are reliable for this population. the widest used weaning criteria, f/v t ratio, does not seems to keep the same performance in this kind of population. the main purposes of this study were to identify the possible differences of the f/v t ratio measured in a spontaneous breathing trial, between an adult and an elderly group. we designed a protocol to study the variation, sensibility and specificity of the frequency-to-tidal volume ratio between an adult group (ag; up to years) and an elderly group (eg; older than years) in a daily weaning screening trial. methods. the study cohort comprised patients ready to undergo weaning trial. the parameters studied were: weaning success ( h of spontaneous ventilation after extubation), respiratory rate (f), tidal volume (v t ), frequency/tidal volume ratio (f/v t ), gasometric and ventilatory parameters. the weaning method was spontaneous breathing trial (sbt). measurements were made in the beginning of sbt (t ) and min after (t ). we analyze possible differences in the sensibility and specificity of the f/v t ratio between elderly and adults and compare with previous values already published. the chi-square test, anova and the t test were used in the statistical analysis. weaning success was . % in eg and . % in ag (p = . ). the baseline characteristics were similar. comparisons of ag and eg at t and t showed statistical differences in weaning criteria: f, v t and f/v t ratio. conclusion. weaning success in our study was low, but similar to the described in other trials. elderly patients showed higher f and lower v t . consequently, f/v t ratio was lower too. the area under the roc curve for f/v t ratio was smaller than already published. results. / ( %) were successfully extubated and patients required re-intubation. the demographic data showed no differences in age, bmi, apache ii, icu admission diagnosis or sex distribution between groups (table ) . patients who failed extubation had small but statistically significant differences in vital capacity (vc), peak negative inspiratory pressures (pnip), pao /fio ratios (pf) and were ventilated longer prior to extubation. paradoxically, patients failing extubation had positive end expiratory pressures that were statistically but not clinically significant higher. the ratio of respiratory rate to tidal volume (f/vt) was not significantly different. patients failing extubation were also more likely to have weaker cough, gag, level of consciousness as measured by glasgow coma scale and more secretions ( table ). having no cuff leak did not predict failure of extubation. the most common reasons for reintubation were secretion retention and/or absence of cough ( %). pressure support ventilation (psv), a widely used assisted mode, has the purpose to avoid diaphragm disuse allowing the patient to generate spontaneous inspiratory efforts optimizing comfort and work of breathing. however, still little is known about the individual response of respiratory muscles under these conditions. we hypothesized that respiratory muscles of patients ventilated with clinic psv might result, at least sometimes, excessively unloaded. we performed an observational study in the intensive care unit on patients ventilated with psv set by the clinician in charge. twenty intubated, mechanically ventilated patients ( ± years old) during the weaning phase entered the study. the patients had no sedation at least for the last h. respiratory timing, tidal volume (v t ), peak airway pressure (paw peak ), electrical activity of diaphragm expressed as percentage of its maximum (edi/ edi max ), inspiratory (ptpes) and diaphragm (ptpdi) muscle effort were measured during min of clinic psv. results. we found that seven out of twenty patients generated a negative pes swing only during the psv inspiratory triggering phase (psv t ) in comparison with the remaining patients in whom pes was negative throughout most of the mechanical breath (psv n ). in the psv t group, pes swing was either flat or positive after inspiratory triggering. therefore, in the psv t group both ptpes /min and ptpdi /min were fivefold lower than normal values. v t / predicted body weight (pbw) was significantly higher in the psv t versus psv n group (see table ). during weaning with psv: ( ) a significant number of patients ( %) showed a pes shape similar to that observed during pressure assist/control modes, and inspiratory muscle effort abundantly lower than normal, both indicating excessive inspiratory muscle unloading; ( ) among the variables used to set psv, only a high v t /pbw (higher than ml/kg) hallmarked excessive unloading; ( ) due to the ample prevalence of the phenomenon, the question whether high levels of inspiratory muscle unloading can cause detraining and prolonged mechanical ventilation merits an answer from further research. introduction. the liberation from mechanical ventilation (mv) should be done as soon as possible in order to avoid complications and the risks associated with prolonged unnecessary mv, such as ventilator-associated pneumonia, ventilator induced lung injury, and increased icu and hospital stay. this procedure should be carried out properly and safely. objective. evaluate the extubation success rate, mv time and weaning time using a daily weaning screen followed by a spontaneous breathing trial (sbt). patients who were ventilated for more than h were subject to this procedure, which was carried out by respiratory therapists. methods. in our icu, between february and august of , all intubated patients who were ventilated for more than h underwent a daily weaning screen, which contained variables such as hemodynamic, gas exchange, consciousness and resolving the need for mv. if these variables were stable, these patients were submitted to a sbt and were extubated if they did not show any signs of respiratory discomfort or hemodynamic changes for at least min. conclusion. the use of a daily weaning screen followed by a sbt was associated with a high extubation success rate and a very short weaning duration with % of unsuccessfully extubations. c. chatt , d. pandit , g. raghuraman birmingham heartlands hospital, critical care, birmingham, uk, birmingham heartlands hospital, birmingham, uk aim. the aim of the study was to assess the impact of pmv on the course of weaning in mechanically ventilated patients. we wanted to assess the optimal pressure support at which pmv can be initiated which would enable prolonged use of pmv without affecting the duration of respiratory support. method. data on all patients who were mechanically ventilated for greater than eight days were obtained from icnarc database, who underwent tracheostomy as part of their weaning process. satisfactory level of pressure support was achieved (between and cm h o) pmv was introduced into the patient's breathing circuit and spontaneous ventilation was attempted. we applied mann whitney u tests for parametric data, fisher exact tests for non-parametric data and anova was used to compare the three groups with different pressure supports at initiation of pmv. a p value \ . be statistically significant. results. patients who were ventilated for greater than eight days identified. of these, patients were excluded because they did not have a tracheostomy during their period of ventilation. of the remaining patients, pmv was used in patients ( %). there were no significant differences between the demographic data (sex, age) or the data on admission to intensive care (apache ii score, ratio of medical to surgical patients) and duration of mechanical ventilation between the two groups. however, there were significant differences in the mortality, total respiratory support days after tracheostomy and length of stay in intensive care and length of hospital stay between the two groups. in the group on pmv, no record of aspirations was found documented on the intensive care charts. in patients in whom pmv was used (n = ), pmv was initiated at cpap (continuous positive airway pressure) in patients ( %). patients ( %) had pmv initiated at a pressure support of b cm h o and in patients ( %) pmv was initiated at a pressure support of [ cm h o. there was a significant difference in the duration of mechanical ventilation post tracheostomy (p = . ) and the length of hospital stay (p = . ) between the two groups, with the cpap group being ventilated for a shorter duration but with a longer stay in hospital the same difference was shown when comparing three groups of pressure support when pmv was commenced (cpap, pressure support b cm h o and pressure support [ cm h o). however, in the pressure support [ cm h o group we observed that the duration of use of pmv was lower than in the other two groups despite longer duration of mechanical ventilation and total respiratory support days. although this was not statistically significant, it could be clinically significant. our study suggests that use of pmv at pressure support b cm h o could increase the duration of its use without affecting the length of mechanical ventilation. we would therefore recommend weaning to a pressure support b cm h o before pmv is commenced in the acute setting. no data are available concerning the oxygenation target to aim during the weaning phase from mechanical ventilation. also, in opposition to ards patients there is no clear recommendation for the upper limit of spo to maintain during weaning. this study is part of a research project on peep and fio settings automation during mechanical ventilation. methods. this observational study was designed to assess the spo target aimed during the weaning phase of invasive mechanical ventilation (fio b . and peep b cm h o). patients were recruited in icus from several countries (canada, france, italy, tunisia, argentina). the following data were prospectively collected by the respiratory therapists at each round during a months period: spo , fio , peep level, ventilatory mode, anatomic site of the pulse oxymetry sensor, quality of the spo signal. results. data from centers ( icus) from quebec city, canada, and center from créteil, france are available. patients were prospectively included. , observations were performed. the mean level of fio was . ± . with fio c . and . in . % and . % of observation times respectively. the mean level of peep was . ± . cm h o and was below, equal or above cm h o in . , . and . % of the cases respectively. the most frequent ventilatory modes were pressure support ( %), simv ( %) and acv ( %). the pulse oxymetry sensor was applied on a finger of the hand in . % of the cases and was deemed of good quality in % of the time. the mean spo was . ± . % for the whole population and was . ± . % for patients with fio c . . spo was higher than % in % the observations. desaturation with spo below % were recorded in . %. the spo signal was deemed available by the bedside nurse in . ± . % of the time. conclusion. this study demonstrates that spo levels may be maintained at high levels unduly. this may have an impact on the weaning phase of mechanical ventilation. this study also shows that the spo signal availability was high enough to be used in a closed-loop oxygenation system. introduction. automated weaning systems are viewed as a challenge to weaning decision-making autonomy by some clinicians. clinician perceptions of the utility of such systems may influence uptake in to practice. to assess the perceived utility of the automated weaning system, smartcare/ ps. a survey was generated based on comprehensive literature review and -year's experience using smartcare/ps. survey pilot testing was conducted with senior clinicians experienced in smartcare/ps weaning in an independent icu. questions addressed perceived system usability and appropriateness of automated weaning, system benefits and disadvantages, as well as patient indications deemed suitable and unsuitable for smartcare/ps weaning. participants were also asked to indicate if they would continue using smartcare/ps on trial completion. the survey was administered to clinicians on completion of a randomized controlled trial conducted to compare smartcare/ps to non-protocolized weaning . of staff surveyed, surveys were returned by nurses and doctors (response rate %). eight respondents had no experience with smartcare/ps despite the year trial duration, leaving surveys with evaluable responses. the majority of respondents perceived smartcare/ps was easy to activate ( / , %) and to use once activated ( / , %). the system was observed to wean appropriately by / ( %) respondents; experienced smartcare/ps to wean inappropriately. comments on inappropriate weaning identified clinically unacceptably increases of pressure support (ps) for patients with profound tachypnea and complicated lung pathology. smartcare/ps' ability to reduce the overall duration of weaning was questioned by all but / ( %) respondents. ps adjustment according to patient requirements was the most frequently perceived benefit ( / , %). most respondents did not perceive any advantage of smartcare/ps for patient comfort / ( %), assessment frequency ( / , %) and automated control of weaning ( / , %). less control over weaning was the most regularly cited disadvantage of smartcare/ps ( / , %). system issues such as program abortion without identifiable reason and mandatory peep reduction prior to a spontaneous breathing trial to assess readiness for separation were less frequently cited disadvantages [ / ( %) and / ( %) respondents respectively]. most respondents ( / , %) felt smartcare/ps was best suited for weaning postoperative patients and should be avoided for patients with neurological dysfunction ( / , %). only / ( %) respondents stated they would not continue to use smartcare/ps. clinicians demonstrated moderate acceptance of smartcare/ps. more work is needed to identify those patients more likely to benefit and confirm the overall utility of smartcare/ps as a weaning tool. introduction. physician approaches to ventilation withdrawn varies among physicians whereas the prompt recognition of respiratory failure reversal and usefulness of weaning protocols in reducing duration of mechanical ventilation (mv) have been largely demostrated as nursing staff attend patients h a day its leadership in this process can be effective and safe. objective. to demonstrate that a nurse-directed protocol to withdraw mv could reduce a % its duration. prospective sequential study performed in two periods. during de first period ( months) data concerning weaning definite criteria appearance, duration of mv, reintubation or need for nonninvasive ventilation (nimv) and demographic data were collected to all mechanically ventilated patients blinded by attending nurses and physician. after a three months phase of staff training there was a second months period where weaning criteria were checked al each nurse working shift during the first days of mv. when criteria were fullfilled a min of spontaneous breathing trial was perfomed and tracheal tube removed if there were no intolerance criteria. same data as the first phase were collected. we used mann-whitney s u test to compare mv duration, time to reach weaning criteria (trwc) and extubation delay (mv duration minus trwc). weaning failure was compared using x square. data are presented as median ( - percentile). results. patients were screened ( in the first period and in the second) but only patient reached weaning criteria in the first days ( in the first period in the second), . % men, aged ( - ) years. aim of this study is present our experience about elective bedside pdt with the blue-rhino kit over an year period, in order evaluate its efficacy in terms of intraoperative and postoperative complications. patients and methods. the study included a total of consecutive icu patients requiring tracheostomy. all pdt were performed by icu staff physicians at patients' bedsides, using a blue rhino kit. the following data were recorded: age, sex, simplified acute physiology score (saps) ii, fraction of inspired oxygen (fio ) before the tracheostomy, days on mechanical ventilation before the tracheostomy, bleeding, tracheal tear, subcutaneous emphysema, pneumothorax, wound infection, hypotension, lowering sao during the procedure, inability to complete the procedure, and procedural mortality. distance follow-up included fiberoptic bronchoscopy to evaluate tracheal stenosis. results. there were a total of ( . %) complications (tracheo oesophagel fistula and bleeding). forty -one patients died in the icu ( %), although none of these deaths were related to technique complications. mean duration of the procedure was . ± . minutes. the pdt performed at bedside in the icu, using the blue rhino kit is a simple and safe procedure that offers many advantages in terms of safety and efficacy. objectives. questioning the need for several specialized physicians or extra assistance to perform a single percutaneous tracheostomy using fibrescopic tracheoscopy, we performed a prospective study into the complication rate of percutaneous tracheostomy without tracheoscopy on our mixed medical and surgical icu. , consecutive patients were included after having received a percutaneous tracheostomy. indication for tracheostomy was always a long anticipated duration of mechanical ventilation. if no contra indications were present, percutaneous tracheostomy was performed. if contra indications against the use of percutaneous tracheostomy without tracheoscopic control were present, tracheoscopy was performed to ensure maximum patient safety. the mean age at the time of receiving a tracheostomy was . ( - ) years. the cohort consists of male patients en female patients. only ttwo percutaneous tracheostomy were performed under fibrescopic control due to contra-indications for an uncontrolled procedure. in procedure, sixteen minor, and no major complications were encountered. this resulted in a . % minor complication rate. conclusions. the number of complications in our group is approximately the same as those which are suggested in international literature where tracheoscopy was performed during percutaneous tracheostomy. none of the complications encountered could have been prevented by the use of tracheoscopy. therefore we postulate that in the hands of an experienced team and in adherence to strict guidelines, percutaneous tracheostomy can safely and successfully be performed without tracheoscopy. objective. to assess the risks and complications associated with the bedside pdt in our years experience of over pdts in icu. pdt is a relatively newer technique and has been introduced as an alternative to open tracheostomy as a safer and convenient procedure. however, the risks and complications of the pdt have not been highlighted in the icu of a developing country. a retrospective analysis of the data gathered from patients undergoing pdt was done in a -bedded tertiary level multidisciplinary icu of a teaching hospital. the data was collected between april and march . all intubated patients with indications for elective tracheostomy, as well as patients who required emergency tracheostomy were included in the study. demographic and other clinical details of the patients who underwent pdt were collected. griggs [ ] technique was most commonly adopted while other adopted techniques were ciaglia [ ] , white tusk/blue rhino tapered dilator and percutwist technique. a total of , pdts were done in , patients, over a period of years. of the , patients ( %) were males and ( %) were females. the mean age of patients was . years. the average duration of intubation before pdt was . days. ( %) pdt were done bedside in icu while ( %) were done in wards, coronary care unit, high-dependancy unit and liver transplant unit. griggs technique was adopted in ( . %), ciaglia in ( . %), white tusk/blue rhino tapered dilator technique in ( . %) and percutwist technique in ( . %) patients. long-term ventilation was the most common indication in ( . %) followed by airway protection in ( . %), facilitation of weaning in ( . %) while airway obstruction/difficult intubation was observed in ( . %) patients. pre-procedure coagulopathy was observed in ( . %) patients, ( . %) were morbidly obese while ( . %) required emergency tracheostomy. no complications were observed in ( . %) patients. procedural complications were seen in ( . %) patients. bleeding from the site was the leading complication affecting ( . %) patients. difficult tube placement was seen in ( . %) patients, premature extubation in ( . %), false passage in ( . %), guidewire dislodgement in ( . %), subcutaneous emphysema in ( . %), arrhythmia in ( . %) and bleeding requiring transfusion was seen in ( . %) patients. no procedure related mortality was observed. conclusion. on the basis of this large single centric study we found that pdt is a safe, reliable and convenient procedure which can be easily performed bedside by experienced intensivists. results. of the recipients who underwent pdt, were liver, kidney and heart transplant recipients. the respective mean values for age, weight and apache ii score were . ± . years, . ± . kg, and ± . all pdts were performed at bedside by experienced staff anesthesiologists with direct bronchoscopic guidance. in all cases, the indication for pdt was prolonged mechanical ventilation due to acute respiratory failure. the mean time from transplant to pdt was ± months and the mean duration of endotracheal intubation before pdt was ± days. twelve patients had coagulopathies. the calculated lung compliance and pao :fio ratio improved after pdt ( . ± . vs. . ± . ml/cm h o, p = . and ± vs. ± , p = . respectively). transient hypoxemia (n = ) and mild extratracheal bleeding (n = ) were the only early complications. there were no procedural failures and no pdt-related late complications and deaths. conclusion. the results suggest that percutaneous dilational tracheotomy is an efficacious and safe technique for prolonged airway management with improved ventilatory mechanics in solid organ transplant recipients. a. vianna , g. cabral , r. azambuja , g. carleti , t. balbi , g. pereira clinica são vicente, rio de janeiro, brazil aims. we studied diferent aspects of tracheostomy procedures performed in intensive care units (icus) located in the municipality of rio de janeiro and compared them with the medical literature. a questionnaire was elaborated and sent through email to the coordinators of every icu in the city of rio de janeiro in the period of july to august . the questionnaire was sent to the coordinators of the icus located in rio, and was answered by ( . %) of them. among the studied icus, ( . %) are public, ( . %) are private, and ( . %) are part of university hospitals. ( . %) are medical/ surgical, ( . %) are medical, and ( . %) is a surgical unit. the average number of beds is ± . . the decision to perform the procedure is taken by the icu team in ( . %), by the patient's primary team in ( . %), and by both in ( . %). tracheostomy is performed by a surgeon in ( . %) units, by an intensivist in ( . %), and by both in ( . %). the procedure is performed at the bedside in ( . %) of the icus. the most frequent indications for tracheostomy are: prospect of prolonged mechanical ventilation, coma, and airway protection. . % of are performed between the first and second week of mechanical ventilation, and % between the second and third week. control chest x-ray is performed in . % of the units. surgical tracheostomy is available in all the studied units. only ( . %) units perform percutaneous tracheostomy. the reasons given for the preference for surgical tracheostomy were the lack of a qualified team for performing the percutaneous tracheostomy or material needed for this procedure. all icus that perform the percutaneous procedure use the ciaglia technique with bronchoscopic guidance. late followup is performed in ( . %) of the studied units. the study showed great differences between the tracheostomy protocols used in the hospitals of rio de janeiro and those found in the medical literature. in particular, the use of percutaneous tracheostomy is still infrequent in the icus of rio. on all the patients, aged at least years or more, admitted to our postoperative icu since january through december , we collected demographic profiles, operative data and short and long-term outcomes. spss . was used for statistical analysis and p \ . was considered the level of significance. a total of patients ( . %), . % males and with a median (iqr) age of ( - ) were admitted to our post-operative icu over the study period. iddm was recorded in the . % of the population, copd in the . %, hypertension in the . %, chronic renal failure in the . % and arteriopathy in the . %. out the total population, . % of patients, with a median (iqr) pre-operative crs of ( - ) underwent a coronary-artery bypass grafting (cabg) surgery, whereas . % of them, with a preoperative median (iqr) nyha of ( . - ) needed a valve replacement (vr) and . % of them combined (cabg ? vr) operations; moreover, . % of patients underwent other type of cardiac and aortic surgery. overall median (iqr) post-operative mechanical ventilation length was ( . - ) hrs. while no statistically significant difference was recorded in terms of mv duration among the four surgical groups. overall recorded mortality rate was %, with the lower . % for cabg and the higher . % for vr (p = . ). kaplan meier curves showed no differences in survival likelihood at th (log rank = . , p = . ) th (log rank = . , p = . ) and th (log rank = . , p = . ) days after surgery among the different surgical groups. conclusions. the outcome after heart surgery in octogenarians is excellent; the operative risk is acceptable and the late survival rate is good. therefore, cardiac surgery should not be withheld on the basis of age alone. introduction. re-do cardiac operations have been reported to be increasing in incidence and are associated with a higher operative risk [ ] . this study aimed to determine the impact on intensive care provision. methods. data from , procedures spanning twelve years (april -march ) was examined. the re-do operations were further analysed by gender, age, pathology (new, progressive, combined) , duration between procedure, theatre time, length of stay, complications and mortality. as the number of cardiac operations performed has increased over the twelve year period, the relative incidence of re-do procedures have remained stable at . %. operative length at re-do was significantly longer (mean min vs. ) however anaesthetic time pre surgical incision was not significantly increased. subsequent length of stay on the intensive care or high dependency unit increased by % (mean . vs. . days), with higher complication rates affecting all systems (except post operative myocardial infarction). renal and pulmonary complications showed the most significant increases. renal related complications occurred in % and pulmonary in . % of cases which represents an and % increase on first operation rates. infection rates were also significantly increased at double that of the initial procedure. the total hospital stay was found to be % longer (by . vs. . days, respectively) while in hospital mortality increased from . % at initial procedure to . % at re-do. mortality rates were further elevated in the presence of renal failure post operatively, as re-do valve mortality increased from . to % and re-do cabg from . to % in this subgroup. conclusion. these results, combined with the stability of percentage re-do surgery over the twelve year period, enable specific planning and management of intensive care provisions. the knowledge of extended theatre times and subsequent stay in intensive care/high dependency units has a further impact on the throughput of routine cases. the data also highlights the increased costs associated with these patients, as they not only require longer hospital stays but also suffer increased complications requiring more investigations and interventions. specific costing therefore applies to this subgroup of intensive care patients. objectives. we aimed to assess hrqol at days after surgery in relation to preoperative hrqol. we compared patients with decreased hrqol to patients with unchanged or increased hrqol to identify disparities between these two groups. a prospective cohort study including patient scheduled for cardiac, vascular, abdominal and orthopedic surgeries in a tertiary hospital was performed. patients filled-out a hrqol questionnaire (sf- ) the day before surgery and days after. preoperative, intraoperative, postoperative data were collected. changes of pre-and postoperative physical component summary ( simultaneus kidney-pancreas transplantation is the best treatment option for type diabetic patients with chronic kidney disease. currently, the medical and surgical complications have decreased significantly, although these represent a high risk of morbidity and mortality in the short term. objectives. this study sought to investigate the incidence of medical and surgical complications, the clinical characteristics and prognostic factors influencing graft and patient's survival in a recent cohort of pancreas-kidney recipients. patients and methods. the present study included patients who received simultaneous pancreas-kidney transplantation in our center from january to february . we studied demographic, clinical and immunological characteristics of patients, and surgical and medical complications during his admission to intensive care unit. results. the average age of recipients was . years and mean age of donors was . years. the median cold ischemia time was . h ( % confidence interval - ). the average stays on the waiting list was . days. % of patients were extubated within the first h. % of patients required transfusion during their icu admission, amine infusion was started at % patients in the early hours. during follow-up, surgical reintervention in the immediate postoperative occurred in % of the patients. major surgery complications reported in the literature are graft thrombosis, although in our serie there have been only kidney graft thrombosis and pancreas graft thrombosis. only % of patients died within the first months posttransplantation surgery. conclusion. surgical complications after pancreatic transplantation remain a significant concern. hence we our results add further evidence to support the notion that the double and simultaneous pancreas-kidney transplant is in fact the treatment of choice in selected patients with end-stage renal failure due to type diabetes mellitus. a. shono , t. mihara , y. murakami , j. ota , f. kono , y. saito shimane universiy hospital, anesthesiology, izumo, japan pulmonary catheter is widely used for cardiac surgery. the complications of indwelling pulmonary catheter, such as perforation of pulmonary artery, pulmonary embolism, are well known. however, the thrombosis associated with introducer sheaths has received much less attention. we evaluated the incidence and risk factors for internal jugular vein thrombosis (ijvt) associated with introducer sheaths for pulmonary catheter after cardiac surgery. methods. the patients who underwent cardiac surgery and insertion of introducer sheaths ( . f) at right internal jugular vein (ijv) were included. ultrasonographic evaluations of ijvt were performed prior to insertion and daily until introducer sheaths removal. we investigated demographic data, underlying disease, length of surgery, use of cpb and iabp, complications during cannulation and duration of catheterization. coagulation status (pt, aptt, platelet count, d-dimer) and cardiac index at before and after surgery were also recorded. the student's t test, v test, and fisher's exact test were used for statistics and p value of . was considered significant. results. patients were included in this study. mean age of patients was ± years (range - ), mean duration of catheterization was ± days. ( . %) patients developed ijvt which occurred only one day after insertion. the incidence of ijvt was related to presence of underlying disease (relative risk, . ; % confidence interval, . to . ) and was unrelated to emergency operation, the use of iabp and cpb, number of insertion attempts. there were significant differences between patients with or without ijvt in duration of catheterization ( . ± . vs. . ± . days, p = . ), cardiac index at day after surgery ( . ± . l/(min m ) vs. . ± . l/(min m ), p = . ), the value of d-dimer at day after surgery ( . ± . vs. . ± . lg/ml, p = . ). no clinical symptoms related to ijvt were found in observation period. our results demonstrated that ijvt associated with introducer sheaths was a frequent complication and cardiac index was significantly lower in patients with ijvt. though the incidence of ijvt was higher in patients with prolonged catheterization, it developed even on day after surgery and was usually asymptomatic. this risk should be carefully considered when the insertion of pulmonary catheter is chosen for cardiac surgery. methodology. it is a prospective study of patients admitted to our icu after undergoing robotic radical prostatectomy (da vinci) in the time interval going from january up to april . we analyzed clinical and demographic data, the length of stay in the icu and hospital, the need for blood transfusion, surgical times and the complications suffered during hospital stay. data are expressed as mean, median or percentage, using the student's t test and chi-square to compare averages and detect possible associations between variables. results. seventy three patients underwent surgery with a median of years. mean surgical time was min. in recent months this time is reduced to min. the mean haemoglobin at admission ( . g/dl) was significantly higher than when dismissed ( . g/ dl), p \ . . an average of two units of concentrated red blood cells was transfused in the surgery room in . % of patients. only one patient required transfusion at the icu. cardiac or renal mild complications appeared in . % of patients. this could not be associated with age. the median of mechanical ventilation length was h. one patient required conversion to open surgery due to profuse bleeding. there was no hospital mortality and no need for reoperation. mean stay at icu was day, significantly less than those patients who suffered complications (p \ . ). the median stay in ward was days. conclusion. robotic radical prostatectomy (da vinci) has a very low associated morbidity, minimal blood transfusion requirement and short is the stay at the icu and hospital, in contrast to published data with open surgery. there was no hospital mortality. objectives. to evaluate short-and long-term outcome in patients undergoing coronary artery bypass grafting (cabg), who received an intra-aortic balloon pump (iabp) prior to surgery. methods. between january and june , all patients (n = ) who received an iabp prior to on-pump cabg in our center were included. patients received the intra-aortic balloonpump for vital indications (i.e. either unstable angina refractory to medical therapy or cardiogenic shock; group ; n = ) or for prophylactic reasons (group ; n = ). a cox proportional hazards model was used to identify predictors of long-term all-cause mortality. compared with the euroscore predictive model, observed -day mortality in group ( . %) was not significantly higher than predicted ( . %). a dramatic decrease in -day mortality occurred in group (median predicted mortality was . % and observed was %, p \ . ; fig. introduction. physiological abnormality is associated with adverse outcome and a high percentage of patients admitted to icu have abnormal physiology in the hours prior to admission [ ] . track and trigger systems are used to enable timely intervention to a deteriorating patient. the mews system is used in our level care general wards with more intensive monitoring in our level care facilities. objectives. we hypothesised that the mews for patients admitted from the general wards were being inadequately documented and that this be may influencing outcome. we therefore also hypothesised that patients admitted from the level care general surgical and medical wards in our hospital had a poorer outcome following icu admission than those admitted from sites of level care, such as the high dependency unit (hdu), emergency department and theatre recovery. we undertook a prospective week audit of all patients admitted to our district general icu. all patients' case notes and monitoring charts were reviewed by an icu consultant with additional data (e.g. apache ii scores) obtained from the ward watcher icu database. day mortality data was obtained from the hospital's sci patient database. we then compared the data with that available for patients admitted over the previous year ( ) background and aim of study. aorto-femoral bypass (afb) is widely used for the patients with peripheral vascular disease (pvd). nevertheless, there is no consensus about the type of anesthesia for this difficult group of patients. we hypothesized that continuous spinal anesthesia (csa) will be more secure and suitable for the patients with pvd combined with pulmonary and cardio-vascular co-morbidities. the aim of our study was to compare alterations of the mean arterial pressure (map) and delivery of oxygen (do ) during afb and in the early postoperative period under the influence of ga and csa. after approval of our hospital helsinki committee prospective randomized study was performed between and : male patients with pvd were included in our work. risk of anesthesia was equal to the third degree according to asa scale. in the first group of patients (n = ) ga with mechanical ventilation was employed. in the second group of patients (n = ) csa was used. both groups of patients were similar with respect to age and co-morbidities (copd % in both groups, ischemic heart disease and arterial hypertension). for ga we used propofol, midazolam, fentanyl, and isoflurane. bupivacaine was used for csa. in combination with mental sedation by intravenous midazolam. map was measured directly through radial artery catheter and do with the help of tetrapolar rheovasography. both parameters were measured during fixed points: before the operation, at the end of induction of anesthesia, after cross clamping of the aorta, after release of aortic clamp, first hour after operation, h after operation and h after operation. mann-whitney test was used for statistical analysis of our results. in the patients with csa during the operation and in the early postoperative period, map was lower but statistically non significant. map was lower statistically significant only during the cross clamping of the aorta and in the first postoperative hour, most probably due to the influence of the sympathetic block. at the same time do had almost no difference in both groups. only in induction stage it was lower in the ga group that most probably was connected with negative influence of propofol on cardiac output. conclusions. both methods of anesthesia ga and csa gave us opportunity to preserve stable map and do during afb that we performed in this difficult pvd patients with copd and cardiovascular co morbidities. over the last few decades, several scoring system have been developed for use in critically ill patients, not only to assist therapeutic decision making but also to guide resource allocation and quality of care. to evaluate the tiss- in surgical intensive care unit (icu) patients and the possible relationship between tiss- and the type of surgery, severity of illness, and outcome in these patients. prospectively collected data from all patients admitted to a postoperative icu between st march and th june were analyzed retrospectively. a-priori subgroups were defined according to gender, age, saps ii score, sofa score, surgical procedures, and the occurrence of major morbidity or death in the icu or in-hospital. a total of , patients were admitted during the study period ( . % male, mean age . years) constituting , observation days. the highest tiss- scores were observed on the day of admission. the highest tiss- was observed in patients who underwent cardiothoracic surgery, the lowest in neurosurgical patients. during the first week in the icu, tiss- was correlated to the severity of sepsis syndrome; however tiss- scores remained elevated only in patients with severe sepsis/septic shock. tiss- score was correlated to saps ii (r = . , p \ . ) and sofa score (r = . , p \ . ) throughout the icu stay and was consistently higher in non-survivors than survivors during the first weeks in the icu. conclusions. the highest tiss- scores are observed on the day of admission to the icu with marked variations according to the type of surgery. tiss- correlates well with the severity of sepsis syndrome and outcome in these patients. our data could be helpful in icu planning, risk stratification, and resource allocation in the surgical icu setting. introduction. pain and opioids for treatment of pain can affect immune function in cancer patients, which may in turn influence metastatic capability of a primary tumour during and after surgical excision. it is also been shown morphine has a direct effect on cancer cells, but results of these studies have been conflicting. we therefore aimed to determine effect of morphine, commonly used in anaestehsia and intensive care, on in vitro breast cancer cell migration using two breast cancer cell lines. we used two cell lines: mcf is er positive breast cancer cell line while mda-mb- is er negative, less differentiated and more invasive. cells were incubated with or without morphine (concentrations - ng/ml) for , and h, corresponding to clinically relevant concentrations and exposure times. cell proliferation was determined using an mts (promega inc.). h cell migration was determined using a -well flourescent kit (chemicon). results were compared using independent sample t test for differences between the groups. morphine had positive effect on cell proliferation, which was greater in mda-mb- cells. proliferation of mda-mb- was increased the most at h incubation and higher concentrations ( and ng/ml caused and % increase in proliferation at h incubation and up to % increase at h incubation). proliferation of mcf cells was increased by % in and h incubation periods. morphine caused an increase in migration of both cell lines, which was again more evident with mda-mb- cells at higher concentrations of morphine ( , % and % increase with , and ng/ml respectively). our experiments have shown morphine has potential to directly stimulate breast cancer cell proliferation and migration in vitro, especially in less differentiated breast cancer cell line. further studies are needed to determine its effect on other metastatic mechanisms such as invasion and gene expression as well as the implication of these results for clinical practice. objectives. aim of this study was to evaluate the predictive value of nt-probnp levels and inflammatory markers (crp, il- , tnf) on late mortality in patients who underwent thoracic surgery for lung cancer. methods. patients median age ( ± years) without history of heart disease or renal failure. the blood tests for nt-probnp, crp, il- and tnf analyses were drawn one day preoperatively, h, h and days postoperatively. patients' demographic data, laboratory results and mortality were collected and assessed. results. nt-probnp at h was significant higher in non-survivors ( , ± , pg/ ml) compared to survivors ( ± pg/ml, p = , ). furthermore, nt-probnp at h was associated with survival in the cox-regression analysis (p = . , hr = , , % ci: , - , , units: pg/ml). crp preoperatively was significant higher in non-survivors ( ± mg/dl) versus survivors ( ± mg/dl, p = , ). il- preoperatively was significant higher in non-survivors ( ± pg/dl) compared to survivors ( ± pg/dl, p = . ). conclusions. high nt-probnp levels at h postoperatively, associated with increased mortality in patients undergoing thoracic surgery but there was no relation ship between crp, il- and mortality. introduction. the occurrence of post-operative delirium in elderly orthopaedic patients is associated with neurological complications and cognitive decline [ ] . although the etiology of the decline is less understood, cerebral ischemic events may be involved [ ] . high plasma concentration of n-methyl-d-aspartate (nmda) receptor antibodies (nr ab) has been proven highly predictable for occurrence of the postoperative neurological events in cardiac surgery [ ] . objectives. the aim of the present study was to investigate the predictive value of blood levels of nr ab for postoperative delirium, cognitive dysfunction or any other neurological complications after hip and knee replacement surgery. methods. the study enrolled consecutive patients, aged over , requiring acute or elective knee or hip replacement surgery. cognitive impairment was evaluated by minimental state evaluation (mmse) test administered before and after surgery. daily postoperative delirium was evaluated by confusion assessment method for intensive care unit (cam-icu). plasma levels of nr ab were recorded before surgery, at the moment of hospital discharge ( - days postoperative) and weeks after discharge. all other possible risk factors for postoperative delirium were also recorded. cognitive decline was present in patients ( %) before surgery and in patients ( %) at the moment of hospital discharge (p \ . ). plasma levels of nr ab were . ± . ng/ml preoperatively, . ± . ng/ml at the moment of hospital discharge and . ± . ng/ml weeks postoperatively, with no significant differences. conclusions. the incidence of cognitive decline in elderly patients after othopaedic surgery was significantly higher when compared with the preoperative status but there was no correlation between the cognitive decline and the plasma levels of nr ab. methods. patients older than years, whose performed endoscopic, colonoscopic or both procedures, under sedation performed by the intensive care deparment of the hospital del tajo. data were collected for months. demographic characteristics, medical history, asa (american society of anesthesiologists classification), drugs bolus and total dosages, respiratory and hemodynamic data, the length of procedure and recovery, and complications were collected. tolerance was assessed by endoscopist, with a (very bad) to (very good) scale. quantitative data are expressed with mean and standar desviation, and qualitative data with percentage. results. procedures were included. table shows main characteristics. tolerance and complications are referred in table . the . % of the procedures were appropriate ( or ) . the main complications were vomiting ( . %) and hallucinations ( . %). there were only incidences of respiratory depression and of hypotension. background. the authors hypothesized that the efficacy and quality of a remifentanil (r)-based regimen versus a piritramide (p)-based analgosedation in major post-surgical patients with renal and hepatic impairments still more potent even if prevention of narcotic induced hyperalgesia (nih) [ ] was done. the nih was made by sulphate magnesium (m), ketamine(k) or clonidine(c). methods. patients were randomly allocated to receive a blinded infusion of either r at a rate of . l/(kg min) (± . ) (g : n = ) or p at . mg/(kg h) (± . ) (g : n = ) coupled to an hypnotic sedation of propofol. r and p were titrated in icu after surgery, to achieve an optimal sedation as defined by a sedation conclusions. the remifentanil-based regimen allowed a more rapid emergence from sedation and facilitated earlier extubation diminishing total icu hospitalisation time and cost. even if we prevent the narcotic induced hyperalgesia by used of magnesium, ketamine or clonidine, needs of tramadol in rescue still lower in the remifentanil group due to its high power coupled with its high fexibility compared to piritramide. its reducing, by the way, risks of tramadol's metabolites accumulation in case of renal or leaver impairment. a ''fast track'' approach to cardiac surgery has significantly shortened the length of icu stay. however, quick awakening from anesthesia and subsequent extubation after discontinuation of sedative drug sometimes cause instability of hemodynamics, such as increase of bp or hr. dexmedetomidine (dex), a agonist, is a sedative drug that can be continuously infused during weaning and extubation. the aim of this double-blind study was to evaluate the effect of dex on time to extubation and hemodynamics during weaning from mechanical ventilation after cardiac surgery. with irb approval and informed consent, the patients undergoing cardiac surgery were randomly divided into two groups, dex group [infusion of . lg/(kg h) of dex] and saline group. drug administration was started at sternal closure and continued h. ramsay sedation score, times to extubation, systolic blood pressure, heart rate, respiratory rate, pulmonary artery pressure, central venous pressure, cardiac index were examined. we analyzed these parameters on icu admission, when the patients opened their eyes on order, at immediately before extubation, min, h, and h after extubation. unpaired t test was used for statistics and p value less than . was considered significant. results. patients were included in this study (n = in the dex group, n = in the saline group). there were no significant differences between two groups in age, length of surgery, length of anesthesia, and total dose of propofol and fentanyl. time to extubation was ± min in the dex group and ± min in the saline group (mean ± sd), which were also no significant differences. ramsay sedation score were maintained and no patients needed additional sedative drug during assisted ventilation in the dex group. althought mean systolic bp and mean pa, mean cvp, rr were similar in both groups during infusion. hr at eye opening, immediately before and after extubation were significantly lower in the dex group than in the saline group. conclusion. our results demonstrated that the infusion of . lg/(kg h) of dex decreased hr during weaning from mechanical ventilation. dex could not only provide adequate sedation but also suppress the stress response after cardiac surgery. dex is a useful sedative drug for preventing instability of hemodynamics on fast track approach. however, most anxiolytics impair intellectual function. dexmedetomidine (dex) is an alpha agonist that may offer sedation without overt cognitive decline. we performed a comparison between dex and propofol (pro), a drug often used for icu sedation. methods. prospective, randomized, double-blinded, cross-over study of awake and intubated brain-injured (bi, n = ) and non-bi ( ) icu patients, each receiving pro and dex using a cross-over design with periods of baseline (analgesic use fentanyl only), drug a, interval washout (fentanyl only), drug b. sedation was titrated to a score of or - (calm, cooperative) on the rass and hopkins nics scale. cognitive testing was performed at each study period using the validated -pt hopkins ace cognitive battery. objectives. we evaluated the effect on final outcome of a treatment regimen with lowdose haloperidol initiated when a positive cam-icu occurred as a quality improvement project. methods. the cam-icu was previously implemented in daily care in all patients who stayed[ h in our bed medical-surgical icu. in the first months of the study (period ), the cam-icu was used as an adjunct to daily care and the treatment of delirium was left to the physician on an intention to treat basis. subsequently, a month study pause was defined. thereafter, in the second months of the study (period ), the cam-icu was judged to indicate the presence of delirium and haloperidol was directly started with a loading dose of mg iv with subsequent daily dosing of . - . - mg iv (age \ ) or - oversedation is still common in many intensive care units (icu) despite the demonstrated benefits associated with sedation sparing strategies, including shorter duration of mechanical ventilation, and shorter length of stay in the icu and hospital. our aim was to observe whether a minimal sedation policy could be feasible in a multidisciplinary department of intensive care. prospective observational study over a two month period (december , to january , ) in a -bed medico-surgical department of intensive care of a university tertiary hospital. all adult patients who stayed in the icu for more than h were included. data were collected on duration, type, dose, and indication for sedative and opiate analgesic agents. self extubation was used as a safety surrogate. disease severity was assessed by the apache ii score within the first h of admission. statistical analysis was performed with spss software (spss incorporation, chicago, il, usa). a total of patients (male %) with a median age of years were included; ( . %) received some sedation, the majority [ ( . %)] during mechanical ventilation. midazolam ( %) and propofol ( %) were the most frequently used sedative agents. the most common indications for sedation were: early postoperative ( %), severe respiratory failure ( . %), short term procedures ( . %), and withdrawal syndrome ( . %). the median percentage of time during which patients received mechanical ventilation without sedation was . %, and was not related to severity as assessed by the apache ii score (rho . -p = . ). in the group of patients who required sedation for longer than just short procedures or uncomplicated postoperative care ([ h), the median percentage of time during which patients received mechanical ventilation without sedation was . %. analgesic opiates were often required ( %), predominantly by continuous infusion ( %). morphine was the most frequently used agent ( %). self-extubation occurred in patients, but only needed re-intubation. conclusion. in a mixed medical-surgical population of critically ill patients, a strategy of minimal or no sedation (''sedationless'') is feasible and without major adverse effects. we propose that comfort, hemodynamic instability, and mechanical ventilation should be abandoned as usual indications for sedation. grant acknowledgement. drs received grants from the doctoral fellowship program of capes/ brazilian ministry of education and from the federal university of rio de janeiro. r. russai the royal free hospital, anesthetics, northwood, uk postoperative cognitive dysfunction (pocd) is reported to occur frequently after cardiac surgery, even in low-risk patients. predictors of neurocognitive deficits can suggest the potential etiology and outcome of patient that has developed pocd. there is a wide range of neurological manifestations from subtle cognitive impairment to deadly stroke. over a period of weeks a population of patients underwent cardiac surgery in our hospital. we have looked for any signs of pocd in correlation with the possible etiology. data have been collected prospectively focusing on past medical history (pmh), possible contributors, manifestation, complications and treatment. pocd has occurred in patients ( male, female) with age range of to years (median age years), of whom % has had pmh of neurological impairment (predementia; cerebrovascular disease). multifactorial etiology was found: respiratory failure %, morphine %, tramadol %, renal failure %, remifentanyl %. in patients no related causes were recognised. all patients showed confusion as leading manifestation, although in patients confusion presented in combination with aggressive behaviour ( ), cognitive dysfunction ( ), paranoia ( ). in occasions pocd resulted in major complications such as difficulties in airway management ( ), removal of cvp line ( ), removal of arterial line ( ). the majority of patients ( ) required pharmacological treatment with single or multiple drugs therapy, the most common used was haloperidol ( % pts). the average length of stay in itu was . days, and the average length of hospital stay was . ( - ) days. conclusion. pocd is a common and potentially devastating complication with a complex and broad etiology, which may affect the rehabilitation process and the final outcome. early diagnose is essential for personalise treatments and therefore preserve in both life quality and life expectancy. introduction. sedation and analgesia is given to the icu patient for adaptation to the intensive care environment. however, side-effects of drugs used are increasingly acknowledged as negative factors increasing the risk of delirium, vasopressor therapy, prolonged ventilation and length of stay. objectives. the purpose of the proposed study was to study the practice of sedation in norwegian icu's and the challenges experienced by nurses and physicians. a national postal survey for clinicians in all norwegian icu's was conducted in september and october . all intensive care units treating mechanically ventilated patients for more than h (n = ) were included. two respondents from each unit ( intensive care nurse and icu physician) were invited to answer the questionnaire. the survey was based on two previous danish surveys. questions on practice and perceived problems were scored on a numeric rating scale and a lickert scale, as well as a few questions with response categories based on theme. results. the response rate was % (n = ). all icus were represented with nurses with formal education in intensive care and physicians specialized in anesthesiology as respondents. written protocols are not routine in norwegian icus, but half of the departments titrated sedation according to a scoring system, most commonly maas. the most commonly used sedatives were propofol and midzolam, while fentanyl and morphine were the most used analgesics. the main indication for sedation was to achieve tolerance to ventilation and to treat other bothersome symptoms. side-effects were reported to be frequent with both sedatives and analgesics. the most frequent side effects (% reported as often present or always present) with sedative agents were circulatory instability ( %), delayed awakening ( %) and sleep disturbances ( %), while the most frequent side effects experienced with analgesics were gastrointestinal problems ( %), circulatory instability ( %) and delayed awakening ( %). the main indication for tracheostomy was reported as longterm ventilation and the wish to reduce sedation. discussion/conclusion. written protocols were not routinely used. side-effects of sedation are perceived as a problem by the majority of clinicians, leading to circulatory instability and delayed awakening. tracheostomy is used first of all to be able to reduce longterm ventilation and sedation. these results indicate a potential for new sedative agents and analgesics with fewer side-effects and more focus on the use of sedation protocols. [ ] and has been associated with gaba agonist use [ ] . delirious patients may not be overtly agitated, so signs of delirium must be actively sought. the confusion assessment method for the intensive care unit (cam-icu) is a validated and easy to use screening tool [ ] . in a recent study on this bed medical and surgical intensive care unit (icu), more patients who had received gaba agonists were delirious compared with those who were sedative free [ ] . however, the percentage of patients having at least one episode of delirium was lower than expected ( %), perhaps because they were screened only once daily and in the daytime. we repeated this study with twice daily assessments (morning and after dark) and a larger number of patients in order to obtain a more accurate prevalence of delirium and confirm an association with gaba agonist use. methods. two doctors attached to the icu received a min tutorial on using the richmond agitation and sedation score (rass) and the cam-icu assessment tools. the cam-icu was performed on all rousable patients (rass score [ - ) twice daily (morning and after dark). the following information was also noted: ( conclusion. this study shows that the prevalence of delirium on this unit is comparable with published research [ , ] and higher ( . vs. %) [ ] when patients were screened after dark as well as in the daytime. the study shows that any sedating drug was associated with significantly increased prevalence of delirium. the unexpected higher prevalence of delirium in the patients receiving non-gaba agonists versus gaba agonists cannot be explained by haloperidol use to treat delirium. results. there were patients in the icus. the mean age was . years old with a predominance of chinese ( . %) and a slight male predominance of . %. forty-six per cent of the patients were mechanically ventilated and . % had tracheostomy done. there were an average number of devices per patient. sedation was administered in . % of the patients with no sedation scales used in a quarter of these patients. only . % of the sedated patients were on sedation protocol. the majority of patients ( . %) were monitored hourly and on propofol ( %) and midazolam ( . %). up to . % of sedated patients did not have daily interruption of sedation. there were no significant difference noted in the use of sedation between medical and surgical icus. slightly more than a third of patients were given analgesia (n = ) with no analgesia scales used in a third of these patients. one third of them were administered with paracetamol and about a third with morphine. patients in surgical icus were more likely to receive analgesia compared to medical icus patients. most of these patients ( . %) were monitored hourly. only patients were on neuromuscular blockade. there was no usage of any formal delirium assessment tools at all with . % of the patients being assessed for delirium based on clinical judgement of the caring team. only % of the patients had some form of sleep promotion in the icu. conclusions. this national multi-center study reveals several deficits in the adult icu with regards to sedation, analgesia and delirium assessment and management. several initiatives should be implemented to improve patients' safety and quality of care in the icu. methods. this study was conducted in patients who visited emergency care center following caustic ingestion during a period ranging from january of and august of , in whom a retrospective analysis of medical records was performed. findings for the esophageal lesion were classified according to the change of the esophageal wall and the infiltration of periesophageal soft tissue. also, clinical, laboratory, and endoscopic data from this patients were reviewed. the correlation between the degree of esophageal damage seen on ct scans and esophageal constriction seen on esophagography were then evaluated. a total of cases of caustic ingestion were identified (age range, - years). the most common caustic agent ingested was acid ( %). the most frequent cause for ingestion was attempt of suicide ( %) as opposed to accidental ( %). the findings of thoracic ct in patients were follows: first-degree esophageal injury in ( . %), seconddegree in ( . %), third-degree in ( . %), fourth-degree in ( . %). fourteen patients ( . %) developed caustic esophageal stricture. the degree of esophageal damage got closer to grade iv, the more prevalent esophageal constriction became. this correlation was statistically significant (p \ . ). of the total patients, underwent endoscopy in the early stage after they visited emergency care center. an analysis of the correlation between the degree of esophageal damage seen on endoscopy and that seen on ct scans was performed. this revealed a significant correlation (p = . , r = . ). conclusions. thoracic ct grading suggesting periesophageal soft tissue infiltration and fluid collection (grade iii to iv) rather than only edema (grade i) may be associated with stricture formation. early ct grading was very safe and useful for predicting the development of stricture induced by caustic ingestion. conclusion. in our area critical care transport teams provided safe transfers for critically ill patients.adequate preparation, strict adherence to checklists and adequate personal are the key of optimal solving of problems. introduction. although endovascular repair of traumatic aortic injury (ertai) has revolutionized the practice of vascular surgery, many questions still remain unanswered. endoleaks, coverage of the left subclavian artery, stent fold/collapse, access complications and durability are the most important complications associated with the procedure. we describe our experience with stent fold/collapse after endovascular repair of blunt aortic injury in otherwise healthy and young patients. from january to december , patients (mean age years, mean apache score , mean length of stay days) who underwent endovascular repair of a blunt aortic injury were admitted in our icu. every day clinical examination and invasive blood pressure monitoring were employed for all our patients. when persistent hypertension was detected, transthoracic (tte) and transoesophageal echocardiography (tee) were initially used, followed by spiral computed tomography (ct) and angiography as confirmatory methods. of the patients, ( %) developed a pressure gradient of [ mmhg at the level of the stent that was initially investigated with continuous wave doppler at the descending thoracic aorta (suprasternal view). the complication presented with refractory hypertension (requiring more than two classes of antihypertensives in high doses) and difficult weaning. the cause of hypertension in of those patients was a stent collapse, while in the other patients the stent appeared folded but not collapsed. endograft revision by open surgery was necessary in of the patients. conclusion. the absence of especially designed grafts for the treatment of blunt aortic injury and the subsequent use of oversized grafts are associated with severe complications. refractory hypertension after ertai can be a manifestation of poor stent alignment and/or stent collapse. echocardiographic monitoring proved to be a useful tool in the early diagnosis of this kind of stent-graft complication. as far as we know, it is the first time that echocardiography is described in the relative literature as an early diagnostic technique for this serious complicationction. facing an aging population, the number of interventions of the french emergency medical service (ems) among very elderly is increasing. a previous retrospective study showed that except from out-of-hospital cardiac arrest survival to discharge was remarkably high after ems intervention for life-threatening pathology [ ] . the aim of the present study was to evaluate prospectively outcomes of very elderly patients managed by ems. methods. after irb approval, we conducted a prospective study over year, including all patients aged years or more managed by our physician staffed ems department. characteristics of patients including previous medical status (mccabe and knaus scoring systems), functional independence (katz adl scale), clinical conditions, the index de gravité simplifié ambulatoire (igsa) severity score were recorded. patients were followed until their hospital discharge. the -month mortality was recorded as well as the adl score. data are expressed as mean ± sd, median [iqr] or percentage of patients and compared using univariate and multivariate analysis. a p \ . was considered the threshold for significance (*). results. of the patients included, died on-scene, were transferred to the hospital and patients were left on scene because of significant improvement in medical status making hospitalization unnecessary, or on the contrary in near-death situations. mean age was ± years ( men). their adl was ( - ) and % of patients were living at home. main conditions were pneumonia (n = ), acute coronary syndrome or chest pain (n = ) and acute pulmonary oedema (n = ). at months, survival rate was % (n = ). the proportion of patients living at home was % and adl among survivors was ( - ) (vs. ( - ) initially for this subpopulation, p = . ]. when compared with deceased patients, survivors were significantly younger ( ± vs. ± years*), had lower adl penetrating anterior chest wounds causing cardiac injury are typically fatal, with only % of patients surviving to reach hospital. while the majority of patients with thoracic trauma can be managed conservatively or with simple intercostals catheter, a small but significant number of blunt ( %) and penetrating ( - %) injuries, require emergency resuscitative mediam sternotomy as a component of initial resuscitation. case report. a years old men, fall from meters high, while working in a truss. he was immobilized with semirigid cervical collar and backboard in the scene and transport to our trauma center, witch was a h car-distance. anesthesiologist team was present since the initial management in emergengy room (er) and act according to advanced trauma life support principles. in primary survey, patient was paraplegic, had a gcs of / , a normal respiratory rate, a slight hypotension and a slight tachycardia. when surgeon places a chest drainage, it drains immediately more than , ml blood, and the patient vital signs started to fade, to extreme bradicardia. the patient was then intubated with a rapid sequence intubation, with a single lumen endobronquial tube, and ventilated with protective lung ventilation. hypotension postinduction was promptly treated with vasoactive drugs (nor-adrenaline and dobutamine) and ongoing volume resuscitation. an emergency resuscitative mediam sternotomy incision was perfomed in the er and reveled a clavicule and sternum fracture and laceration in the braquiocephalic artery which has repaired. maintenance was performed with total intravenous anesthesia with fentanyl and nondepolarizing muscle relaxant. monitoring include standard monitoring plus direct arterial and central venous pressures. during the surgical procedure we treat massive blood loss, with multiple transfusions of units of red blood cells (unmatched type-specific), seven units fresh plasma, and pools of plaquets, fibrinogen and cryoprecipitate. at the end of the surgery, still ongoing blood loses, made us suspect of coagulopathy, and to use octaplex Ò . it was also performed a nasal tamponade, to stop severe epistaxis and suture a major scalp wound with evidence of basal skull fracture. patient was transferred to an intensive care unit (icu) ventilated.we was extubated at the th day post-operative. after days, he still remains in icu, because he is recovering from lumbar spine fixation for a total fracture-dislocation of d -d . discussion. although he remains paraplegic, we think emergency sternotomy have had a significantly impact in this life-threating situation. the use of cell-saver Ò would have been beneficial, but it was unavailable in er. we included only patients attended in this unit by icu personnel. these patients belonged to the area assigned to cruces which has been reference centre of the northern area of spain until december . we collected all the information needed from the clinical history and the treatment sheet, and used the spss . programme to perform the statistic analysis. results. we found patients that meet the severely burned patients criteria and that were attended by the icu personnel in colaboration with the plastic surgey unit. their medium age was . ± . years, % of those patients were men, and the medium burned body surface was ± . %. these patients remained hospitalized in this unit during a medium time of . ± . days. during their stay, the % of them needed mechanical ventilation, % presented acute renal failure, % had a pao /fio less than , and . % suffered some kind of infection. methods. prospective and observational study developed in a burn unit, in which were included all patients with total surface body area burn (tsba) [ % and/or inhalation syndrome who were admitted in our unit from march to december . we used transpulmonar thermodilution by means of monitor picco Ò in a total of measurements per patient (admission and every h). we collected measurements of cardiac index (ci), intrathoracic blood volume (itbv), extravascular lung water (evlw), inhalation syndrome or not (it was diagnosed by broncoschopy), percentage of tsba and abbreviated burn severity injury score (absi) in a total of patients.the average change of measurements of ci, itbv and evlw was studied in the following determinations and their association with few factors with a general and lineal model of mixed effects longitudinal data unbalanced. results. the evolution of thermodilution measurements was the following (graphic ) cardiac index: ci = . , ci = . , ci = . , ci = . , ci = . , ci = . , ci = . , ci = . , ci = . , ci = . . intrathoracic blood volume: itbv = , itbv = , itbv = , itbv = , itbv = , itbv = , itbv = , itbv = , itbv = , itbv = . extravascular lung water: evlw = . , evlw = . , evlw = . , evlw = . , evlw = . , evlw = . , evlw = . , evlw = . , evlw = . , evlw = . . in our serie, % of patients were male and the average age was . ( - ). nine out of all the patients ( . %) suffered inhalation syndrome, the average absi was . ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) and the average of tsba was % ( - %). mortality in our serie was % ( patients).ci and itbv measurements increased significantly while the reanimation advanced (ci p . ) (vsit p . ). in evlw we only find significantly differences in post hoc study between first measurements and fourth one (p . ), th (p . ), th (p . ), th (p . ), th (p . ) and th (p . ).in the evlw/itbv ratio (permeability index = pi) we did not observe significantly changes in the evolution.the inhalation factor did not change ci outcomes neither magnitude nor in the measurements evolution (p . and p . respectively), the same form, itbv (p . and p . respectively), but inhalation modified evlw (p . ) and the permeability index was in the signification stadistic limit (p . ).mortality was higher in patients who ci was lower and evlw was in higher values. conclusions. thermodilution in the reanimation period in critically ill burn patient shows significantly haemodynamic changes in the evolution that can help to adapt the treatment.the inhalation syndrome only modified the measurements of evlw significantly in this period but it influenced neither ci, itbv nor pi. introduction. one major issue in trauma management is to get every patient directly from the scene to the appropriate hospital for the injury he sustained. patterns of interfacility transfers have been thouroughly investigated in trauma system settings, but scarce data are available about transfers in non trauma system settings. objectives. this study aims to assess interfacility transfers that eventuate in the abscence of a formal trauma system and to estimate the potential benefits from the implementation of a more organized process. the 'report of the epidemiology and management of trauma in greece' is a one year project of trauma patient reporting throughout the country. it provided data concerning the patterns of interfacility transfers. in greece there is no formal trauma system employed and to our knowledge, all available data concerning the epidemiology of trauma in the country are either extrapolations of relevant data from other countries or based on police reports and individual hospital reports. in this study, we attempted to evaluate the paterns of interfacility transfers, information reviewed included patient and injury characteristics, need for an operation, intensive care unit (icu) admittance and mortality. trauma patients were devided in two groups, the transfer group was compared to the non-transfer group. analysis employed descriptive statistics and chi-square test. interfacility transfers were furthermore assessed according to each health care facility's availability of five requirements; computed tomography scanner, icu, neurosurgeon, orthopedic and vascular surgeon. results. data on , patients were analyzed; . % were treated at the same facility, whereas . % were transferred. in transferred group there were more male, the mean age was lower than that of the non transferred group and the injury severity score was higher. transferred patients were admitted to icu more often, had a higher mortality rate but were less operated on compared to non-transferred. the transfer rate from facilities with none of the five requirements was . %, whereas the rate of those with at least one requirement was . %. facilities with at least three requirements transferred . % of their transfer volume to units of equal resources. conclusions. the assessment of interfacility transfers can reflect current trends in a nontrauma system setting and could indicate points for substantial improvement. results. , patients included, , injuries analyzed. average age was . , . % men. . % were car accidents, % falls, . % motorcycle, . % run over and . % bicycle. . % had one injury, . % two and . % three. most frequent injury was tbi ( . %), thoracic traumatism ( . %) and ortophaedic ( . %); severe tbi was . %. ctrate according to marshall classification was . % ii, . % v and . % iii. iss averaged , higher in dying patients than in the survivors ( ± . vs. . ± . ; p \ . ). of the non mechanical-ventilated patients, . % were so in the first h following admittance. during this, . % patients were given blood transfusions, platelets . %, plasma . % and prothrombinic complexes . %. in the first h . % underwent surgery, most frequent was neurosurgery ( . %). complications: nosocomial pneumonia ( . %), catheter related bloodstream infection ( . %), acute kidney injury ( . %), ards ( . %), cns infection ( . %). . % renal replacement therapy. invasive ventilation was used in . % with . ± . days, non invasive ventilation in . %. average stay in the icu was days. . % of the patients were transferred to a ward. . % were transferred to another hospital. gos on discharge was higher than on . %. % died in icu, % brain death. tbi as a main injury showed a . % mortality rate. depending on trauma type, mortality was higher in fall ( %) and run over ( . %). if due to car accident ( . %), motorcycle ( . %) or bicycle ( . %), mortality in icu was lower (p \ . ). prehospitalary variables related to mortality were age, gcs \ and a motor component \ , pupil alteration, shock, respiratory failure, prehospitalisation intubation and iss (p \ . ). on arrival to hospital, the variables were: haemodynamic instability in the first h, transfusions need and number, marshall iv-vi, mechanical ventilation (p \ . ) and initial fibrinogen (p \ . ). evolutive variables related to a higher mortality rate were days of stay, invasive ventilation, tracheostomy and the show up of complications (catheter related sepsis -p \ . -, nosocomial pneumonia, acute kidney injury, ards, renal replacement therapy (p \ . ). in a logistic regression model, prehospitalisation variables having an influence on icu mortality rate were age (or . ; p \ . ), mydriasis (or . ; p \ . ), gcs-motor component (or . ; p \ . ), shock (or ; p \ . ) and iss (or . ; p \ . ). conclusions. multiple trauma patients show a high need of resources, with many peaks of treatment involving a high monitorization and handling. many of the variables are related with a higher mortality rate in icu: iss, mydriasis, gcs motor component and shock. introduction. trauma systems are multifactorial modules that incorporate any aspect of traumatic injury from the very moment of the injury to the final outcome. a significant prerequisite for the development of a trauma system is the trauma registry. trauma registries are the actual core of any trauma system since they provide valuable information about the standard of care offered to the patients and are amenable to quality control and statistical evaluations, which in turn allow improvements and amendments in the definite care. contrary to what is common practice in the usa, trauma registries in european countries are in embryonic stage. in our country with no actual trauma system, the epidemiology of trauma and the reports on care outcomes are based on police reports and national emergency service reports. objectives. the purpose of this study was to assess the possibility of a national trauma registry in greece and to provide accurate data on the epidemiology of trauma in the country. methods. the project, entitled ''report of the epidemiology and management of trauma in greece'', was initiated in october and lasted for twelve months. all the national representatives of the hellenic society of trauma were invited to participate. the representatives are certified surgeons employed in hospitals receiving trauma. data presented here are those reported from two tertiary care facilities in athens and twenty eight other primary and secondary hospitals around the country. inclusion criteria were defined as trauma patients with documented need for admission in the hospital, patients that arrived dead or died in the emergency department of the receiving hospital and patients that required transfer to a higher level center. in total . trauma patients were included in the study in twelve months time. of them . % (n = , ) were male, aged . ± . (mean ± sd) and . % were female (n = ), aged . ± . (mean ± sd). as expected and reported in most trauma registries, males are leading in all subcategories of iss. the age group - years incorporates . % of the total injuries, in accordance to the axiom that trauma is the disease of the young. conclusions. trauma registries are the cornerstone of any trauma system. even in a non-trauma system setting, registries are a valuable tool for quality control of the provided health care and for further development of the health care system. objectives. determine the impact of rurality in epidemiology, injury severity, health care facilities, length of stay (los), mortality, functional outcome and quality of life in trauma patients. retrospective study in trauma patients that were admitted in our emergency room(er) between and . data was collected from the prospective trauma registry and follow-up registry months after the accident. we classified patients according to statistical national institute classification: urban areas-areas with more than inhab/km or have a place with more than , inhabitants; semi-urban areas-areas with more than inhab/km and less than inhab/km or have a place with more than , inhabitants and less than , inhabitants; rural areas-areas that were not classified as semi-urban or urban areas.patients were divided in three groups according to residence area: r (rural), su (semi-urban), u (urban). we studied several variables in order to find a relation with rurality: sex, age, type of injury, los in hospital and intensive care (icu), anatomic severity (ais), politrauma severity (iss), physiologic severity (rts), surveillance probability (triss index), pre-hospital care, previous admission in other hospital, icu admission and mortality. we report two outcome measures: euroqol to evaluate quality of life and extended glasgow outcome scale for functional outcome. we used qui-square test, t test, mann-whitney test, kruskal-wallis test for statistic analysis. results. , patients were admitted in the er. patients ( . %) were excluded with missing data related to residence area. we studied patients, where patients were from rural areas ( . %), from semi-urban areas ( %) and from urban areas ( . %). we find a statistical significant relation between rurality and pre-hospital care, previous admission in other hospital and icu admission. urban area patients had a higher incidence of pre-hospital care(r: . %; su: . %; u: . %, p \ . ). semi-urban and rural patients were admitted more frequently in other hospitals before admission in er (r: . %; su: . %; u: . %, p \ . ) and also had higher admissions in icu (r: . %; su: . %; u: . %, p \ . ). there were no statistical differences in the other variables studied. conclusions. rural trauma patients are similar from those that live in urban areas concerning epidemiology, injury severity and outcome. despite lack of pre-hospital care and higher previous admission in other hospital in rural patients, mortality between groups did not differed in our trauma centre. introduction. metformin-associated lactic acidosis (malta) is a rare but severe complication ( . / , patients/year) of metformin treatment in type-ii diabetes. metformin impairs neoglucogenesis and liver lactate clearance in the presence of a disease that enhances its production. although frequently used, there is no recommendations regarding hemodialysis in this poisoning. to study the prognostic factors of malta and the interests of blood metformin measurement. . on admission, patients presented profound lactic acidosis with arterial ph . ( . - . ), serum bicarbonate . mmol/l ( . - . ) and plasma lactate . mmol/l ( . - . ). early symptoms associated coma ( %), asthenia ( %), vomiting ( %), abdominal pain ( %), and diarrhoea ( %). renal function was significantly altered [creatinine clearance: ml/ min ( - ); p \ . ). all patients received massive alkalinization, / ( %) were hemodialyzed while / ( %) were mechanically ventilated and received catecholamines. six patients ( %) died in the icu. duration of icu stay was days [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . there was no significant differences regarding malta severity and treatments between suicidal and accidental poisonings. neither lactic acidosis severity nor acute renal failure were predictive of death. there was no correlation between prognosis and the time-course of plasma metformin concentrations, with or without dialysis. toxicokinetics showed significant tissue distribution when the patient was early admitted or plateaued concentrations if he was later admitted and survived, even though his situation improved and his lactates decreased. metformin dialysance suggested an interest for extra-renal elimination enhancement although its impact on survival could not be analysed based on this limited study. our study showed that malta is severe with elevated mortality in icu whatever the poisoning is accidental or intentional. metformin toxicokinetics are useful case by case to better understand the patient's outcome. the most important guidelines [ ] for trauma care recommend (us)-fast as the first step investigation to rule out major bleedings. however, us is less sensitive and accurate than multislice computed tomography (ct). the spreading concept that ''moderrn cts require little time'' often brings surgeons to ask for total body ct also in haemodynamically unstable patients. to understand how long it really takes to perform a total-body ct in patients suffering from major trauma (mt) we analysed the data of the ritg project, a pilot multicenter study to define the national standards for trauma care and establish a national trauma registry. methods. italian level trauma centers were involved into the first stage of the ritg project. data of all major trauma patients (iss [ ) who were admitted to either one of the three hospitals during a months period of time ( july - june ) were prospectively entered into the ritg database. time between hospital admission and the first scan were recorded for all patients. patients who, for any reason, were submitted to a ct with a delay of °or more were excluded. the time elapsed between the first scan and the patient's exit from the ct room was measured in a subset of pts from a single center equipped with a new generation ct next to the emergency room. during the study period mt patients were admitted to the three trauma center. patients were submitted to an emergency total body ct scan within . patients died before arrival in the ed. more died soon after admission and before the secondary survey. the interval times are shown in table . seven patients died in the ct room. the average interval between hospital admission and the first available scan was °. however, even where a new generation ct next to the er was used, the average time needed to stabilize the patients, get a correct position on the ct and start the scanning process was °as an average. in the most severely injured patients, who are frequently artificially ventilated, the time required to stabilize the patient and perform a total body ct scan is longer than expected and to a certain extent, independent from the ct scanner itself unless the very last technology as the sliding ct scanners are employed [ ] , thus ct should be considered with extreme caution in the unstable patients. in univariate analyses, survival to discharge was significantly lower with two of acute conditions (acute coronary syndrome and acute inflammation), and with five of chronic conditions (chronic heart failure, diabetes mellitus, kidney failure, hepatic cirrhosis and malignancy). recent surgery was strongly associated with higher odds of survival. the most consistent multivariate predictors of survival to discharge were liver cirrhosis (or . ; % confidence interval . - . ) and malignancy (or . ; . - . ). malignancy was not predictive for outcome after cpr attempts, whereas liver cirrhosis was predictive both in all dispatches and in dispatches involving cpr. recent surgery was strongly associated with higher multivariate odds of survival (or . ; . - . ) after cardiac arrest. in dispatches without cpr, absence of chronic conditions was associated with higher likelihood of survival (hr . ; % ci . - . ). increasing numbers of chronic conditions were significantly and continuously associated with lower survival (p for trend \ . ). advanced age only weakly predicted survival, but age c years was, along with malignancy, the strongest predictor of not attempting cpr in patients with cardiac arrest. conclusions. comorbidities are important determinants of survival after in-hospital met dispatches, with and without cardiac arrest. survival odds are lowest with malignancy and liver cirrhosis. recent surgery increases odds of survival by exclusion of those most severely ill. advanced age at best weakly predicts worse survival, but strongly predicts not attempting cpr. design. retrospective, cohort study. setting. emergency department (ed) and intensive care unit in a university hospital. measurements and main results. the study subjects included of consecutive severe trauma patients. a systematic review of the computer-based medical records of the patients was conducted to provide the base line characteristics and dic-related variables. the worst data of these variables were obtained at time points within h after arrival to the ed; time point , immediately after arrival at the ed to h after the arrival; time point , to h after the arrival; time point , to h after the arrival; time point , to h after the arrival. one hundred and forty one patients ( / , . %) diagnosed as jaam dic showed significant differences in the prevalence of multiple organ dysfunction syndrome (mods) and the outcome in comparison to the non-dic patients. a stepwise logistic regression analysis showed that the maximum jaam dic scores during the study period independently predicted the patient death (odds ratio . , % confidence interval . - . ). all of the patients who developed isth overt dic during the study period could be identified by the jaam dic criteria at early time points. the mortality rate and the incidence of mods of the patients with the isth overt dic were higher than those only met the jaam dic criteria. stepwise increases in the isth overt dic scores and the incidence of the isth overt dic were observed in accordance with the increases in the jaam dic scores. while the mortality rates were identical, there were marked differences in the incidence of mods and sequential organ failure assessment scores between the dic patients associated with trauma and sepsis. conclusions. the jaam scoring system has acceptable validity for the diagnosis of dic at an early phase of trauma. the jaam dic further exists in a dependent continuum to the isth overt dic. in addition to mods, other factors may affect the prognosis of the trauma patients associated with dic. introduction. in the uk, more than , units of fresh frozen plasma (ffp) are transfused every year. since there has been a reduction of % in its use, but it has been suggested that as many as % of transfusions in critical care may be inappropriate. there is significant morbidity associated with the transfusion of ffp. guidelines for the use of ffp do exist, but the indications for its use are limited. coagulation studies, such as prothrombin time (pt), abnormalities are often assumed to be a risk factor for bleeding prior to invasive procedures, but evidence suggests that ffp may not have a prophylactic role. in addition to this the pt or international normalised ratio (inr) were not intended to assess haemostasis in patients without a history of bleeding. review of the blood bank database between st january and st december revealed all prescriptions of ffp for patients on intensive care (itu). the case notes were analysed to find the indication and timings of administration and weight of the patient. the pathology database was examined to find the clotting studies immediately before transfusion. in patients received ffp; this was only . % of the total admissions to the itu. there were prescriptions and a total of units transfused. the mean prescription of ffp was . units and overall each patient received a mean of . units. the mean dosage of ffp was . ml/kg. the pt pre-transfusion mean was . ± . s with a median of . s. the aptt pre-transfusion mean was . ± . s with a median of . s. only . % of transfusions had not had a clotting screen done prior to administration of ffp. % of administrations were given prior to procedures being undertaken on the itu and a further % were given in preparation of the patient for the operating theatre. a significant number of patients are receiving ffp outside international guidelines. a third of transfusions were given for prophylactic correction of coagulopathy prior to an invasive procedure where there is least evidence for using ffp. most patients received a sub-therapeutic dose of ffp; there is ongoing debate on the correct dosage required to normalise coagulopathy, but it is likely to be greater than ml/kg. [ ] . little data exists on the demographics of mt and subsequent demand on a hospital's blood bank. we examined the mt requirements of a bedded tertiary referral hospital over a month period. objectives. to establish the mt demographic, speciality distribution, prbc demand and associated mortality; within a tertiary referral hospital over a month period. to assist with future mt logistics, planning, implementation and audit. methods. the hospital blood bank database was reviewed for cases of mt from jan to aug . inclusion criteria were the administration of c units of (prbc) within a h period. cross referencing with the laboratory records and medical notes was undertaken to establish patient demographics, hospital specialty, diagnosis, outcome and number of prbc transfused. results. patients received mt over a -month period; male ( . %) female ( . %). median age years (range - ). median mt of prbc was (range - ) units. units of prbc were transfused in the treatment of mt during the study period, accounting for a hospital expenditure of over € , . the main specialties associated with mt were the: emergency department ( patients, . %), cardiothoracic surgery ( patients, . %), and general surgery ( patients, . %). of the patients receiving massive transfusions ( . %), did not survive to hospital discharge. % of those patients who died, did so in the first h with a further % dying in the next h. % of the further deaths occurred within and % after thirty days. conclusions. mt in our establishment is associated with a high mortality and predominantly early deaths. recipients were generally elderly with significant co-morbidity. provision of prbcs and blood components for massive transfusion recipients, is challenging for blood bank services [ ] . the demand of mt, within our establishment, was predominantly within the acute specialties; emphasizing the need for close communication between them and the laboratory services. in light of this data we propose the implementation of a mt protocol together with continuous audit, to assess its effect on outcome. in the department of health updated the 'better blood transfusion' circular, a drive to decrease the use of blood products which have become increasingly scarce and expensive [ ] . there is evidence that blood product transfusions in icu patients are associated with an increase in morbidity, length of stay and mortality. there has been concern over the increasing use blood products and despite guidelines [ ] for their use, both national and local audits have demonstrated a high degree of inappropriate transfusion [ ] . derriford hospital is a -bedded tertiary referral centre. the blood bank database was examined for the use of blood products on icu from st january to st december . there was a steady rise in icu admissions from , patients in to , patients in . during this time there was a marked decline in both the transfusion of fresh frozen plasma (ffp) and cryoprecipitate. the decreased use of these blood products has occurred with only a very modest introduction of new pro-coagulant therapies, prothrombin complex concentrate (pcc) and recombinant factor viia (rfviia). usage of rfviia and pcc conclusions. our usage of blood products does not reflect the national trends of increasing use of cryoprecipitate and pcc with a small reduction in ffp transfusion, and is more in line with the hsc requirements for better use of blood products. this has been achieved with little use of the newer rfviia and pcc. the evidence for the use of all these blood products is not strong, particularly in critically ill patients. national guidelines exist for their use, but these are poorly adhered to. to test the hypothesis that transfusion of prbcs has a deleterious effect on clinically relevant outcomes in patients with septic shock receiving early goal directed therapy (egdt). retrospective cohort study of patients who presented in an academic center in septic shock and received egdt. data was collected on patients identified via the surviving sepsis campaign chart review database and linked to the project impact database. pearson chi square and fisher's exact test were used to test for clinical significance. primary outcome was mortality and secondary outcomes included mechanical ventilation days, intensive care unit (icu) length of stay, and hospital length of stay. . / patients presented via the emergency department. / patients received at least one prbc transfusion during their hospital stay. the two groups were balanced with respect to age, gender, apache ii, and baseline lactate levels. the prbc group had a mortality of . vs. . % in the no prbc transfusion group (p = ns). the prbc group also had more mechanical ventilation days ( . vs. . days, p = \ . ), longer hospital length of stay ( . vs. . days, p = \ . ), and longer icu length of stay ( . vs. . days, p = \ . ). conclusions. in this study, transfusion of prbc was associated with worsened clinical outcomes in patients with septic shock treated with egdt. this trial is limited by its small sample size and retrospective nature. however, the results are consistent with data from previous trials pointing to a deleterious effect associated with prbc transfusion. further studies are needed to determine the impact of transfusion of prbc within the context of early resuscitation of patients with septic shock, as the beneficial effects gained by an early and goal oriented approach to resuscitation may be lost by the negative effects associated with prbc transfusion. introduction. blood transfusion therapy (btt) is thought as one of transplantation of living cell, that means btt includes several risk such as infection and btt should be thought to derived from precious material by courtesy of donors. patients with traumatic cardiopulmonary arrest on arrival on the hospital (t-cpa) usually suffered from lethal hemorrhage and required rapid supplement of red blood cells for resuscitation of circulation and oxygen transport, that is to say btt. however, the prognosis of t-cpa patients is well known hopeless. the aim of this study is to evaluate the propriety of our strategy concerning btt for t-cpa patients. we retrospectively examined the medical records of t-cpa patients for the past years. we do btt until (the first period) for t-cpa patients regardless of rosc without any restriction. after then (the second period), we do btt case by case but only after rosc in principle. the rate of rosc, admission to icu, survive to discharge were compared between these two period, and were compared within the first period between the patients group who underwent btt (btt group) and the group who did not underwent btt (non-btt group). in blunt t-cpa and penetrating t-cpa patients, and % achieved rosc, and % admitted to icu, and and % were survive to discharge. in penetrating t-cpa in the first period, units of packed red cells (prc) were used before rosc for non-survivors. in the second period, no prc was used for non-survivor before rosc. in blunt t-cpa in the first period, prcs were used for non-survivors before rosc. in the second period no prc was used for non-survivors before rosc. concerning the effect of btt on the prognosis of t-cpa in all cases, the rate of rosc and admission to icu were statistically higher in the first period than in the second period (p = . and . ). however, there was no statistical difference in the rate of survive-to-discharge between these periods. there was a same tendency in witnessed cases. in cases with electrical rhythm on the scene, only the rate of rosc were higher in the first period (p = . ). restricted in the first period, only the rate of rosc was statistically higher in non-btt group than btt group in all cases, in witnessed cases, and in cases with electrical rhythm on the scene (p = . , . , and . ). however, there was no statistical difference in the rate of admission to icu and survive-to-discharge between these groups.. our retrospective serial study showed a possibility that btt before rosc for t-cpa improves the success rate of rosc but add no effect on the improvement of survival rate. btt is thought to be futile for t-cpa before rosc. management of refractory coagulopathy due to adult onset acquired autoimmune haemophilia. d. hendron , g. allen , m. brady , g. benson belfast city hospital, intensive care, belfast, uk, belfast city hospital, department of haematology, belfast, uk we report a case of life-threatening haemorrhage occurring as a result of a rare acquired condition caused by the production of an antibody to clotting factor viii. this necessitated administration of recombinant activated factor viia (novoseven) to bypass this step of the clotting cascade. a -year-old man presented to intensive care following ogd for acute upper gastro-intestinal haemorrhage, with recent haemoptysis and haematuria. ogd had demonstrated a large clot obstructing the oesophagus and extending through stomach into duodenum. this could not be removed and no bleeding points were identified. a coagulopathy was detected which failed to correct with administration of appropriate amounts of fresh frozen plasma, cryoprecipitate and activated prothrombin complex concentrate (apcc), necessitating clotting factor studies. this demonstrated a factor viii level of % with a detectable antibody inhibitor. acquired haemophilia was diagnosed and activated factor viia was administered resulting in rapid correction of coagulation studies and arrest of haemorrhage. it was necessary to continue daily activated factor viia at a dose of mg a day in addition to anti-inhibitor coagulant complex (feiba-vh)-an activated prothrombin complex with factor viii inhibitor bypassing activity. definitive treatment of the coagulopathy was chemotherapy with cyclophosphamide, vincristine and rituximab. this destroyed the factor viii inhibitor and returned his factor viii levels to almost %. laparotomy and gastrotomy were required to relieve the oesophageal obstruction from the accumulated clot. he was eventually discharged from hospital and remains well. acquired haemophilia is a rare haematological condition that presents with refractory haemorrhage and coagulopathy and these patients are likely to be referred to critical care services for ongoing support and management. it has an incidence of approximately . cases per million per year [ ] . underlying medical conditions can be identified in up to % of patients and include autoimmune disease, solid tumours, lymphoproliferative malignancies and pregnancy [ ] . international recommendations on the diagnosis and treatment of patients with this condition have recently been published and advise recombinant activated factor viia to control bleeding followed by a combination of corticosteroid and chemotherapy [ ] . the paucity of cases presents an obstacle for randomised controlled trials and therefore these recommendations are based on anecdotal evidence and expert opinion. reference (s) objective. to analyze the application of blood transfusion in critically ill trauma patients after wenchuan earthquake. a retrospective study was made in icu of huaxi hospital on patients who had received transfusion at least once during month after the earthquake. their primary diagnosis and clinical features and apacheii score were obtained at admission. non-active bleeding patients were classified into s group if operation was done during his icu stay, otherwise n group. the function of liver and kidney, and the state of circulation and oxgenation were compared between groups, as well as the hemoglobin level before each transfusion were investigated. a total of patients ( . %) had received transfusion at least once, among which were non-active bleeding. the average frequency was . ± . and . ± . , amount was . ± . ml and . ± . ml, the incidence of transfusion-related complication was . %( / ) and . %( / ) in active and non-active bleeding patients respectively. the apacheii score, mean arterial pressure, ast, serum creatinine, oxgenation index and hemoglobin level on day , , after admission to icu showed no statistically significant difference between s and n group. the frequency and amount of transfusion were similar also, while the hb level before each transfusion was significantly lower in n group ( . ± . g/l)than in s group ( . ± . g/l) (p \ . ). the incidence of transfusion-related and infectious complications, time with ventilator and the -day mortality were similar. conclusion. transfusion strategy is more strict in icu doctors than surgeons, while the similar result on organ function, incidence of complications and outcome raises the need for a more wide-accepted transfusion trigger. keywords. earthquake trauma transfusion trigger. extracorporeal life support (ecls) represents an ultimate rescue technique in poisonings. the optimal anticoagulation protocol remains unclear. objectives. we aimed to investigate the coagulation status at ecls decision in order to validate the best heparin protocol to administer. [ packs ( - ) ] and fresh plasma [ packs ( - ) ] transfusions were required within the first h. hemorrhages ( / ), thrombosis ( / ) or lower limb ischemia ( / ) seemed equivalent to previous series using more complicated anticoagulation protocols. conclusions. poisoned patients present at ecls time with important alteration in their clotting tests, associated with various degrees of hepatocellular failure, dic, defibrination, as well as dilution. a simple heparin protocol appears optimal to reduce complications in these critical situations. henna is the dried and powdered leaves of the henna plant. the plant is lawsonia alba and the powdered leaves are used to apply decorative designs over the skin. henna application is widely practiced in the arab and asian communities. they create fascinating designs over the skin, especially over the hands and feet. it is widely practiced during wedding ceremonies and at childbirth. g pd deficiency is common in the community of the arab world. lawsone, the chemical compound in the henna leaves, is capable of inducing severe acute hemolysis in g pd deficient cases. the compound is chemically related to naphtha. we report a case of acute sever hemolysis in a young girl who presented with dizziness and jaundice and diagnosed to have acute severe hemolysis. her symptoms had started while preparing for her wedding by henna application. the girl was g pd deficient, and found to have severe hemolysis resulting from henna application on her skin. very few cases have been reported of similar nature. the matter is also of tremendous practical implication in areas of g pd deficiency. the relevant literature is reviewed as well. background. lactate has prognostic use in critically ill medical and trauma patients, and is a core component in identification of early sepsis. elevated lactate levels in these patients prior to icu admission, e.g. in an a&e setting or pre hospital setting identify patients at risk of death and can trigger an earlier optimization of triage decisions and earlier targeted treatment. a range of poc methodologies for lactate measurement are available but there is little standardization between methodologies. stat sensor lactate is a new poc lactate meter based on a patented multiwell and multilayer electrochemical technology that incorporates control wells that measure and correct for common interfering substances. the electrochemistry technology is layered onto a gold platform providing a stable and robust surface for the electrochemical reaction kinetics. the aim of this study was to assess the performance and functionality of stat sensor lactate. whole blood venous samples were collected from adult patients admitted to a&e. samples were tested for lactate using statsensor lactate (nova biomedical) and the omni b bga (roche) routinely used for lactate measurement. precision was assessed using donated whole blood and spiked with a concentrated lactate solution. results. within run precision was acceptable at all levels tested. for the lowest level sample (mean . mmol/l) %cv for the two meters tested was ( . and . %) at the three other levels tested (mean . , . , . mmol/l) % cv precision was \ %. lactate values during the method validation ranged from . to . mmol/l by the reference method (nova . to . mmol/l background and objectives. this research work's intention is to describe the epidemiology in patients suffering from anemia who were interned into emergency room (er). a preliminary work will be conducted in which three days in june will be randomly chosen. during these days, all patients satisfying certain criteria will be registered. the criteria fitted to this work are the following: be using the emergency channel of the hospital, score any diagnostic and be over years old. paediatric, gynaecologic and traumatic cases fall out of this research. anemia was diagnosed according to who criteria. outcome. patients were interned through the aforementioned er channel. . % were subject of blood analysis using classification proceedings. from the latter, . % were diagnosed with anemia. age, intake of clopidogrel and/or aspirin, admission and place of admission resulted statistically significant among anemia patients versus non-anemia. anemia was to be found in . % of patients younger than years old, % of the times in patients between and and . % among patients above years old. according to vcm, . % were microcitic-anemia, % were normocític-anemia and . % resulted macrocític ones. conclusion. anemia is among a large share of patients coming into the hospital through emergency proceedings. its likelihood increases accordingly to the risk group analysed and dominating among the elderly population and among patients suffering from renal disfunction and non aggregated. most common are normocitic and macrocitic anemia-types. early identification and valuation could bear prognostic consequences. a. s. omar tawam hospital, critical care medicine, al ain, united arab emirates introduction. an elevated serum creatinin phosphokinae (cpk) and the presence of myoglobin in the urine characterize rhabdomyolysis. rhabdomyolysis had been described in various traumatic and non-traumatic conditions [ ] , there are few reports of its association with anaphylaxis. in this paper, we report cases of anaphylaxis both complicated with rhabdomyolysis. aim of the work. to discus the association between rhabdomyolysis and anaphylaxis and the value of early screening of cpk in such cases. setting. two patients were included in this review in multidisciplinary intensive care unit of tawam hospital/uae. the two patients survived, both developed rhabdomyolysis shortly after admission, evidenced by fivefold or greater increase in serum cpk [ ] . both patients had transient hypotension through the presentation, but none of them had persistent shock requiring vasopressors or complicated with acute renal failure. conclusion. we observed rapid increase in serum cpk in our two cases suggesting the potential benefits of early assessment of cpk in such patients which may amplify early goal guided management and avoiding logistic organ dysfunction. keywords. rhabdomyolysis, anaphylaxis. the blood oxygen and carbon dioxide levels are a direct measure of the effectiveness of ventilatory support in patients on mechanical ventilation. head injury patients require strict control of the cerebral homeostatic state. these patients also need careful management of sedation, maintaining a fine balance between patient comfort, hemodynamic instability and ability to assess conscious levels. biphasic intermittent positive airway pressure (bipap) ventilation is thought to be better tolerated by the patient allowing for spontaneous breathing at any point, thus reducing the amount of sedatives and muscle relaxants used. but the effectiveness of this ventilatory mode in achieving stable blood oxygen and carbon dioxide levels in this group of patients is not known. we hypothesised that bipap is more labour intensive to adapt to the target blood gas parameters as the volume delivery is not constant and that the blood gases may be more unstable in the initial resuscitation phase of head injury patients without conferring much advantages in terms of usage of sedatives and muscle relaxants. retrospective data collected from case record review of head injury patients with no primary respiratory insult, requiring mechanical ventilation with volume controlled synchronised intermittent mandatory ventilation (simv) was compared to the data from similar patients treated with bipap ventilation. both the data groups specifically looked at two time periods, the first h and - h after intensive care admission. blood gas parameters classified as hypocarbic, hypercarbic and/or hypoxic, use of muscle relaxants, number of episodes of raised intracranial pressure (icp) above mmhg as recorded in the intensive care chart every hour, number of episodes of cerebral perfusion pressure (cpp) below mmhg as recorded in the chart every hour was noted. need for muscle relaxant in the first h of admission was noted. the outcome was recorded as either ''alive'' or ''dead'' at the end of itu stay. the data was checked for normality of distribution and compared using non parametric tests (spss for windows). results. baseline characters were comparable between the groups. increased episodes of hypoxia ( . ± . vs. . ± % p = . ) and hypocarbia ( . ± . % vs. . ± . % p = . ) in bipap mode, compared to simv volume control mode. all measurements being percentages of total blood gases for that patient in the first h. there was no difference in the usage of muscle relaxant ( . vs. . % p = . ), raised icp, reduced cpp or mortality between the groups. conclusion. bipap mode of ventilation requires more intensive monitoring and changes in ventilatory settings before adapting to the target blood gas parameters in the first h of admission. at the same time the quoted advantage of using less sedatives and muscle relaxants is not significant. acute post-traumatic brain swelling is one variety of the pathological forms, which needs emergent treatment following traumatic brain injuries. we investigated the effects of clinical effects of decompressive craniectomy (dc) in patients with acute post-traumatic brain swelling (bs). seventy-four patients of acute post-traumatic bs with midline shifting more than mm were divided randomly into two groups: dc group (n = ) and routine temporoparietal craniectomy group (control group, n = ). the vital sign, the intracranial pressure (icp), the glasgow outcome scale (gos), the mortality rate and the complications were prospectively analysed. the mean icp values of patients in dc group at , , and h after injury were much lower than those of routine temporoparietal craniectomy group ( . ± . , . ± . , . ± . and . ± . mmhg vs. . ± . , . ± . , . ± . and . ± . mmhg, respectively). the mortality rates at month after treatment were % in the dc group and % in the control group (p \ . ). good neurological outcome (gos score of to ) rates year after injury for the groups were . and . %, respectively (p = . ). the incidences of delayed intracranial hematoma and subdural effusion were and %, respectively (p \ . ). in conclusion, dc has superiority in lowering icp, reducing the mortality rate and improving neurological outcomes over routine temporoparietal craniectomy. however, it increases the incidence of delayed intracranial hematomas and subdural effusion, some of which need secondary surgical intervention. therefore, the effects of dc in patients with acute post-traumatic bs should be further evaluated. we analyze among others variables: age, injury severity score (iss), abbreviated injury score (ais); admission and discharge glasgow coma score (gcs), extended glasgow outcome score (gose), complications, icu and hospital mortality. differences between groups were tested with students t test and v testing for statistical analysis. results. fourteen patients with intracranial hypertension were treated with decompressive craniectomy . compared with control group, patients with dc had a better gcs ( ± g ; ± g p = , ) and gose index not only at icu discharge ( ± g ; ± g p = , ) but also at hospital discharge ( ± g ; ± g p = . ). the mortality rate was lower in the craniectomy group (g : %, g ; % p = . ). conclusions. in our center, the use of dc for treat patients with severe tbi and refractory cranial hypertension (gcs b and pic c ) improved outcome and mortality significantly compared with medical conventional approach. method. in this retrospective study we present patients who underwent decompressive craniectomy following traumatic brain injury at king's college hospital between and . results. % of these patients presented at a&e with a glasgow coma scale of or below whereas the remaining % presented with gcs above and deteriorated following admission. the patients underwent decompressive craniectomy to reduce raised icp resistant to medical treatment (barbiturate coma excluded). the procedure resulted in significant decrease in icp. out of patients had the operation within h following their injury. we also found that dc in younger patients (\ years) was correlated with lower icp following the operation compared to older patients ([ ) . our study also showed that early dc (\ h) is correlated with a shorter stay in itu. conclusions. the findings of the present study are limited by its retrospective nature and small sample size which does not permit any definitive conclusions from these results. however, they form the basis for further investigation. we present the study with a review of the recent literature. introduction. the objective is to study the correlation of secondary icp indices with ct findings and outcome in tbi. a cerebrovascular pressure reactivity index (prx) can be determined as the moving correlation coefficient between mean icp and mean arterial blood pressure . it is a surrogate marker of cerebrovascular reactivity. the rap coefficient was calculated as the running correlation coefficient (r) between slow changes in pulse amplitude (a) and mean icp (p). it is a surrogate marker of pressure-volume compensatory reserve. all components of the icp waveform that have a spectral representation within the frequency limits of . to . hz can be classified as slow waves. methods. prospective observational study of patients with tbi at the royal london hospital. all patients were managed according to the local guidelines for the management of tbi . secondary indices derived from the icp waveform were analyzed by icm ? software. an initial ct was performed in all patients before admission to icu. marshall classification has been shown to predict mortality in tbi. we found a strong association between all these secondary indices and the initial ct findings. all these markers of cerebral haemodynamics correlate significantly with outcome in headinjured patients. conclusions. surrogate markers of cerebrovascular reactivity and pressure-volume compensatory reserve correlates with ct findings and outcome in tbi. these secondary icp indices may be used in the management of tbi. introduction. following the introduction of national guidance [ ] on the management of patients with head injury, the use computed tomography (ct) imaging of the head has increased markedly. the impact on anaesthetic and critical care services is unknown. . determine the impact of national guidelines on ct scanning in the head injured patient upon anaesthetic and critical care services in a university teaching hospital. . determine the incidence of acutely abnormal ct appearances in patients referred for ct scanning under the guidelines. . estimate in-hospital mortality in this population and its sub-groups. a case-note analysis was performed in october of consecutive emergency department (ed) patients who were recorded as having a ct head. of the cases, did not actually have ct head. details of the analysed subjects, indications for the scan and day mortality rates are reported in table . in patients with severe traumatic brain injury pro-and anti-inflammatory mediators are released into the systemic circulation. however, the relationship between the inflammatory response and the kind and duration of secondary insults remains unclear. objectives. the aim of this study was to investigate in severe traumatic brain injured patients the relationship between the systemic concentrations of pro-and anti-inflammatory mediators and the total duration of secondary insults occurring during the icu stay. methods. ten consecutive traumatic brain injury patients admitted to the icu were included. physiological variables were continuously recorded and analyzed minute-by-minute to identify the occurrence of secondary insults (intracranial hypertension, systemic hypotension, hypoxemia and hyperthermia) according to the edinburgh university secondary insult grading scale. serum samples were obtained at admission, , and h, in which pro-and anti-inflammatory mediators were analyzed by a bioplex assay. results. ten male patients were enrolled, mean age ± , gcs ± , apache ii ± , iss ± . patients were monitored for . days (median value, range - ; , total minutes recorded); intracranial hypertension occurred for , min ( . % of total period recorded, range . - %), hypotension occurred for , min ( . % of total period recorded, range . - %), hypoxemia occurred for min ( . % of total period recorded), not enough data were validated for fever. interleukin (il)- , il- beta, il- , il- and il- ra were in the detectable range. a significant correlation was found between the total duration of intracranial hypertension and the median value of il- (p \ . , r = . ), il- beta (p \ . , r = . ), il- (p \ . , r = . ), il- (p \ . , r = . ), and il- ra (p \ . , r = . ) measured during the period of observation. no correlation was found between these inflammatory mediators and the occurrence of hypotension or hypoxemia. no significant correlation was present between the baseline values of these inflammatory mediators and the severity indexes (gcs, iss and apache ii). conclusions. these results suggest that the duration of secondary insults such as intracranial hypertension was associated with a systemic inflammatory reaction, while the severity of injury on admission was not related to the initial concentrations of these inflammatory mediators. grant acknowledgement. aim. assessing behavioral responses to pain is difficult in severely brain-injured patients recovering from coma. we here propose a new scale developed for assessing pain in vegetative (vs) and minimally conscious (mcs) coma survivors: the coma pain scale (cps) and explore its concurrent validity, inter-rater agreement and sensitivity. methods. concurrent validity was assessed by analyzing behavioral responses of postcoma patients to a noxious stimulation (pressure applied to the fingernail) ( vs. and mcs; age range to years; non-traumatic and of traumatic origin). patients' were assessed using the cps and four other 'pain scales' employed in non-communicative patients: the 'neonatal infant pain scale' (nips) and the 'faces, legs, activity, cry, consolability' (flacc) used in newborns; and the 'pain assessment in advanced dementia scale' (pa-inad) and the 'checklist of nonverbal pain indicators' (cnpi) used in dementia. for the establishment of inter-rater agreement, fifteen patients were concurrently assessed by two examiners. results. concurrent validity assessed by spearman rank order correlations between the cps and the four other validated pain scales was good. cohen's kappa analyses revealed a good to excellent inter-rater reliability for the cps total and subscore measures, indicating that the scale yields reproducible findings across examiners. finally, a significant difference between cps total scores was observed as a function of diagnosis (i.e., vs or mcs). conclusion. the cps constitutes a sensitive clinical tool for assessing pain in severely brain injured patients with disorders of consciousness. this scale constitutes the first step to a better management and understanding of pain in patients recovering from coma. methods. study group: consecutive patients with cervical spinal cord injury admitted to icu. mean age: , years. patients asia a, asia b, asia c. the more frequent neurological level was c ( %). the requirement of mechanical ventilation was considered the key sign for establishing the diagnosis of severe respiratory failure. the blood gas values (po , pco , and pao /fio ) before and after connection to mechanical ventilation [mv(if needed)], were used to estimate the more probably mechanism of respiratory insufficiency. the increase of pco levels was considerate as a sign of neuromuscular weakness; the low po level before ventilation, and the persistence of pao /fio below normal values was considered a sign of v/q mismatch. for this purpose statistic analysis (mean values comparison using student t test) comparing blood gases before and after mechanical ventilation treatment was performed. results. ( %) patients developed severe respiratory failure. mean delay between admission and mechanical ventilation was h. previously to mechanical ventilation patients developed pulmonary atelectasis, and four pneumonia. the incidence en respiratory failure was significantly higher in patients with neurological level above c (p \ . ). conclusions. the incidence of respiratory failure is related with the severity of neurological deficit (relationship between incidence of respiratory failure and neurological deficit level). in addition, our data support that, besides the neuromuscular weakness (moderate increase of co levels), a significant v/q mismatch with shunting phenomena associated (significant hypoxemia no completely solved after mv) is involved in the respiratory failure of cervical spinal cord injured patients. . moderate and severe traumatic brain injury is more likely in middle aged men; more than one third present other major trauma and intensive first level medical treatment is required in most of them. . the most frequent complications found were infectious diseases like ventriculitis and vap. . independent mortality risk factors in moderate and severe trauma brain injury were age, high apache ii score, neuromuscular blocking drugs and icu los. . outcome was significantly improved after six months, and most of the patients only present mild disability and good recovery. nosocomial infections are leading causes of increased morbidity and mortality of severe brain injured patients [ ] . the mechanism underlying the susceptibility to the infections is a subject of great scientific interest and still to be clarified [ ] . it has been recently recognized that injury of brain induces a disturbance of balance between the central nervous and immune system [ ] . objective. the aim of this study was to investigate changes in frequency of lymphocytes subpopulation in peripheral blood of patients with severe brain injury during the course of intensive care treatment. human peripheral blood samples were taken from the severe brain-injured patients at day , and and peripheral blood mononuclear cells (pbmc) were immediately isolated by gradient density centrifugation. the percentage of lymphocytes subpopulation were analyzed by simultaneous detection of surface antigens using fluorochrome conjugated monoclonal antibodies directed toward cd , cd , cd , cd , cd , cd , cd . t lymphocytes were distinguished from the other lymphocyte subpopulation as cells labeled with anti-cd monoclonal antibody but negative for cd staining (cd ? cd - patients. eighty-seven patients with head injury, glasgow coma scale \ . measurements and main results. clinical and demographic data, and head ct scan were taken at admission. patients underwent advanced neuromonitoring and were treated according to brain trauma foundation guidelines. s b concentration was quantified at admission and , and h post-tbi (days , , and ). outcome was assessed months after discharge using glasgow outcome score. significant negative correlations were found between -year gos and s b concentrations on days - , but not on day (day , p = . ; day , p = . ; day , p \ . ; day , p \ . ). patients who deceased showed higher s b concentration than survivals for all the samples. good versus poor outcome (gos = - ) differed significantly on days and . logistic regression analysis showed that samples , and h post-tbi sample predicted death outcome. roc curve analysis showed -h sample was the strongest predictor for decease. poor outcome was only predicted by the -h sample. conclusions. s b levels h post-tbi was the strongest predictor for poor and fatal -year outcome, whereas levels at admission do not. a temporal profile of s b release from admission to h post-tbi is strongly recommended for use in identifying the subset of patients liable of developing a worse outcome. according to our results, s b protein might be an early, sensitive, accurate and useful biomarker for predicting long-term outcome in patients with acute severe tbi. grant acknowledgement. this research was made possible in part by the generous donation of protein s b electrochemiluminescence assay kits by roche diagnostics, mannheim, germany. introduction. brain intercellular fluid glycerol concentration as measured by microdialysis catheters has been recognized as an index of glial and neuronal cellular destruction. we present a data analysis correlating glycerol levels with intracranial pressure (icp), cerebral perfusion pressure (cpp), brain tissue oxygen partial pressure (pbtio ), lactate to pyruvate concentration ratio (l/p) and outcome. methods. data of head injured patients is presented. all had simultaneous monitoring of icp, pbtio and metabolic biochemistry by three brain intraparenchymal bolt catheters inserted via the same one burrhole (icp codman or camino, pbtio licox and microdialysis-cma). there was not a clear straight correlation of raised glycerol levels with bad outcome. however, glycerol elevation seemed to be a predictor of intracranial hypertension together with l/p raise. in subarachnoid hemorrhage patients glycerol elevation was an early sign of secondary ischemic insult. conclusion. multimodal monitoring with intracranial catheters is a useful clinical tool for management of critical neurosurgical patients. metabolic biochemistry as measured by microdialysis, and specially l/p and glycerol levels, can early predict incoming intracranial hypertension as well as secondary ischemia. the pulsatility index (pi), a parameter derived from the blood velocities along the cardiac cycle, has been used as an indirect way to evaluate intracranial pressure. the aim of this research has been to evaluate the accuracy of transcranial doppler sonography (through pulsatility index) in the inference of intracranial pressure. methods. population of the study group (high-pi-group): severe head injured patients (gcs at admission \ ; mean age . years; patients with diffuse injury (traumatic coma data bank) type ii ( %) and iii ( %)) who presented episodes of increase of pulsatility index (pi [ . ) in the acute phase of head injury. control group (normal-pi-group): severe head injured patients, with tcd recordings of normal pi (pi b . ). in all the patients the intracranial pressure (icp) was continuously monitored using a intraparenchymal device. all the tcd recordings are referred to the middle cerebral artery of the cerebral hemisphere were icp catheter was inserted. in the transcranial doppler recording, the pulsatility index was automatically calculated derived from the formulae: pulsatility index = (systolic velocity -diastolic velocity)/mean velocity. transcranial doppler sonography recordings of with pulsatility index c . (high normal value of pulsatility index) were correlated with the simultaneous icp value. the incidence of intracranial hypertension (icp [ mmhg) was analyzed in the high-pi-group, and compared with the incidence of intracranial hypertension in the normal-pi-group. methods. in a double-blind, randomized, placebo-controlled clinical trial, patients scheduled for elective cabg was randomly assigned into two groups. after matching inclusion and exclusion criteria and induction of general anesthesia, one group received intrathecal sufentanil (s) and the other group received the same dose of sufentanil plus supplemental bupivacaine (sb). except for this, all the cases were similar regarding anesthesia and surgery. mean arterial blood pressures were measured before and after induction of anesthesia, during the bypass time and after weaning from bypass were checked. also, the need of the patients for administration of inotropic agents after weaning was compared. results. there was more stable mean arterial blood pressure and less inotropic need after weaning from cardiopulmonary bypass in the sb group. also, the sb patients had a more stable hemodynamic profile during the bypass period; especially after the initiation of the bypass. less inotropic agents were needed after weaning in the sb patients. there was no difference between the two groups regarding the extubation time. discussion. the administration of intrathecal sufentanil plus bupivacaine seems to keep the hemodynamic status of the patients more stable than intrathecal sufentanil alone. methods. this study was approved by the hospital s ethics committee. prospective observational study including consecutive patients. preoperatory and postoperatory data were collected. interventions included blood samples for nt-pro bnp taken prior to operation, and and h in postoperative. troponin-i was taken and h postoperatively. blood obtained was processed for nt-probnp with cobas h system Ò point of care (poc) by roche diagnostics, with range from to , pg/ml. the serum nt-probnp level was also correlated with the logistic euroscore and ejection fraction (ef). serum ntpro-bnp and troponin i values were compared between patients with and without postoperative length of stay in the intensive cardiac unit (icu) [ h. and hospital [ days. all results are in median ± sd * p \ . , **p \ . tables ??? and ??? conclusions. preoperative euroscore and nt-probnp levels were higher in patients with ef \ %. the troponin i after surgery increased more in patients whose length stay in icu was longer. after surgery nt-probnp levels increased significantly,and they differ significantly between patients with length stay in icu for more than h and days at hospital. our data collection confirmed that measurement of nt-probnp is useful and helpful during postoperative period and it also predicted a higher possibility for a long stay in icu and a later hospital discharge. however, owing to the small size sample, these results must be regarded as preliminary. conclusions. in spite of the limitations of our trial, percutaneous aortic valve implantation appears to be safety. a high rate of maccv events were observed, essentially due to a disruption of the a-v conduction, in most cases transitional. despite the definition of ''inoperability'' is difficult, less-invasive aortic valve procedures will undoubtedly find a place within current cardiac surgical practice. objective. to describe the evolution of cardiac transplant patients, presenting clinical low cardiac output in the immediate postoperative period, and after handling routine, they are treated with levosimendán (lv). descriptive, prospective and observational in a postoperative care unit for cardiac surgery from a terciary hospital. study period: january -december . lv was used when the patient had inotropic dependence over h, to try to remove the amines or added to them in those cases that do not get these drugs with an adequate hemodynamics. bolus was used in occasions and then infusion of . - . mcg/ (kg min). we analyzed demographic variables, hemodynamic response to the input of the drug if you can reduce or discontinue other medications, clinical tolerance and side effects, overall development, the icu and hospital stay. we studied patients ( women and men). presented a mean age of . . before surgery, all of whom were in nyha functional class iii-iv. three patients were transplanted in emergency. in this series, there is a case without pulmonary hypertension (pah) pre-transplant, patients with mild htp and htp moderate to severe, with a transpulmonary gradient(gtp) between and mmhg. the patients with gtp [ mmhg had a positive reversibility test with sildenafil. ischemia time of surgery was . . in the immediate post, all the patients studied had low cardiac output syndrome by graft postoperative ventricular dysfunction, cardiac index measured by pulmonary artery catheter. in all patients echocardiography was performed to rule out a pericardial effusion with hemodynamic deterioration in cardiac cavities and showed ventricular dysfunction, right dominance in patients. in all patients we observed a good tolerance to the drug. in lv cases facilitated the withdrawal of the remaining. patients were used lv only after the withdrawal of treatment with inotropic dependence on it. in the remaining cases to be associated with other drugs. only two cases could not withdraw inotropic treatment after the lv infusion. in five patients with pulmonary arterial hypertension and prevalence of right ventricular failure, to reduce poscarga also added pulmonary arterial vasodilators. patients have a stay in icu between and days. one patient mortality. . the primary graft failure is a severe potential complication of post-cardiac, which is associated with a worse prognosis. . lv shows good tolerance, without serious adverse effects attributable to the drug, and facilitated the removal of amines and clinical recovery. . it is necessary to expand the case to confirm the results, and to establish the most appropriate indications and patterns of use of this drug. post-infarction ventricular septal defect (infarctvsd) is a rare but serious complication of myocardial infarction, usually quickly followed by low cardiac output. repair of infarctvsd is still a challenging procedure with a high risk of mortality. improvement of surgical outcome depends on results of large studies in this setting. the aim of this retrospective study was the evaluation of preoperative and surgical parameters influencing the -day mortality following surgical repair. conclusions. in this large study, pre-operative left ventricular function and troponin level were found to be the best predictors identifying patients at high risk for -day mortality following surgical closure of infarctvsd. both parameters may be helpful in deciding on the time of the operation and preoperative preparation. in contrast to other findings, in our cohort the location of the vsd (anterior vs. posterior) did not affect mortality. this may be due to improvement of surgical technique and perioperative management over time. adequate fluid therapy is the first step of hemodynamic optimization after cardiac surgery [ ] . cardiac surgery exposes patients to ischemia and reperfusion, which are well known risk factors for a systemic inflammatory response and increased capillary permeability in the lungs [ ] . it is still unclear what type of fluid should be given in the presence of increased pulmonary vascular permeability at hypovolemic status. objectives. aim of this study was estimate the optimal type of fluid for intravascular volume deficit treating without evoking pulmonary oedema. a prospective clinical study at the intensive care unit was performed on mechanically ventilated patients within h after elective cardiac surgery involving cardiopulmonary bypass. patients, divided into four groups, were subjected to fluid challenge according to the global end-diastolic volume index (gedvi) measurements with normal saline , ml or the colloids % gelatin, % hes / . or % albumin ml in min. hemodynamic and extravascular lung water index (evlwi), gedvi measurements were performed exactly before fluid challenge, afterwards and min after challenge. results. the change in evlwi did not differ between saline and colloid fluid challenge. gedvi increased by % in saline group, by % in % gelatine, in % hes / . and in % albumin. conclusions. all colloid fluid infusion leads to the greater increase in cardiac preload compare to normal saline (saline in four times larger volume). the change in evlwi did not differ between saline and colloid fluid groups and did not increase pulmonary oedema despite in the presence of increased pulmonary vascular permeability, when fluid overloading is prevented. introduction. the annual incidence of prosthetic valve thrombosis is up to - % (patients-year) despite the anticoagulant therapy. conventionally, the treatment of choice for this event was the surgical valve replacement. however, fibrinolytic therapy has become a valid alternative for the treatment of this serious complication, especially in high-risk surgery patients. to analyze the clinical factors, diagnosis and treatment management of patients with prosthetic valve thrombosis admitted to the acute cardiac care unit. we designed an observational-descriptive study, including patients admitted between and . clinical factors were analyzed: sex, age, prosthetic valve position, time from valve replacement, inr at admission, clinical features, diagnostic technique and treatment used. results. patients were included. . % were women, . % men. mean age was . ± . years. the highest incidence was at the tricuspid prosthetic valve position ( . %), followed by the mitral ( . %) and the aortic position ( . %). when a triple valve replacement was performed, the tricuspid position was the most often affected. mean time from the first valve replacement surgery was . ± . years. clinical features which led to the diagnostic were: acute heart failure ( . %), peripheral embolization ( . %), chest pain ( . %) and syncope ( . %). the diagnostic techniques used were transesophageal echocardiography (tee) and cinefluoroscopy in all the patients. inr at admission time was lower than adecuate anticoagulation recommendations in . % of patients. the most widely used treatment was the systemic fibrinolytic therapy ( . %), followed by surgery ( . %) and conservative treatment with heparin alone ( . %). the most widely used thrombolytic was rtpa in . % of patients, with a mean dosage of . ± . mg. one patient was treated with . mil. ui of streptokinase. unfractionated heparin was added to all patients whom received fibrinolytic therapy, with a mean dose of ± ui/h. a . % incidence of minor bleeding was found in the fibrinolytic group. there were no major complications due to fibrinolytic. total mortality rate was . %. our experience, suggests that systemic fibrinolytic therapy is safe and effective in patients with prosthetic valve thrombosis. objective. to describe the outcomes of patients with acute, refractory, non-ischaemic and not postcardiotomy, cardiogenic shock treated with extracorporeal membrane oxygenation (ecmo) and to evaluate whether survivors and non-survivors differed with respect to clinical characteristics, pre-ecmo treatment and laboratory values. design. in this retrospective cohort study, information is collected from a database with additional review of medical records. patients. consecutive adult patients, males, mean age . ± . year, presenting to hospital with non-ischaemic acute severe, refractory cardiogenic shock, supported by central or peripheral venoarterial (va) ecmo. measurements and main results. characteristics of survivors and non-survivors were compared using chi square test. twelve patients ( %) were transported to our institution on ecmo. eleven patients ( %) were weaned from ecmo, seven ( %) bridged to ventricular assist devices. in two patients ( %) ecmo support was withdrawn. mean duration of ecmo support was . ± . h. overall survival was %, and did not differ between patients with myocarditis (n = ), cardiomyopathy (n = ) and acute on chronic non-ischaemic cardiogenic shock (n = ). a larger proportion of the three patients with or more complications died as compared to the seventeen patients with less than complications ( % versus %, p = . ). pre-ecmo intra-aortic balloon counterpulsation (iabp) was used in patients, % survived, as compared to % of those who did not receive iabp (p = . ). we have not identified any other significant differences between survivors and non-survivors. conclusion. the survival of patients on ecmo in this unique heterogeneous patient cohort is similar to the survival of ecmo support for fulminant myocarditis in the literature. we recommend to institute ecmo early in all medical patients with acute non-ischaemic cardiogenic shock, refractory to conventional therapy, or to refer these patients in time to an ecmo centre. introduction. human parvo b virus is associated with a broad spectrum of clinical manifestations, mostly in children or immune-compromised patients. in adults, severe myocarditis due to this viral agent is a rare disorder, presenting as acute congestive heart failure. we describe a patient with rapidly progressive heart failure, needing circulatory support by extracorporeal membrane oxygenation (ecmo). methods. case report. a -year-old previous healthy female was admitted to our icu with nausea, vomiting, bradycardia and hypotension with a blood pressure of / mmhg. two weeks before admission, patient had signs of erythema infectiosum. on physical examination the patient was pale, with venous congestion, third heart sound and hepatomegaly. the initial electrocardiogram showed a slow, regular, ventricular rhythm. admission chest x-ray showed normal heart size with bilateral pleural effusion. echocardiography revealed dilated ventricles (rv and lv) with depressed systolic function and a thrombus in the rv apex. patient was initially treated with intravenous medical therapy, but unfortunately developed progressive cardiogenic shock. troponin levels, serum transaminases and bun were extremely elevated. it was therefore decided to implant a percutaneous ecmo by femoro-femoral cannulation which permitted to stabilize hemodynamic conditions while peripheral organ functions returned to normal range. progressive cardiac recovery was observed after days with a circulatory assistance with a mean flow rate of . l/min. as myocardial function improved, ecmo was gradually weaned and removed after days of support. however, atrioventricular conduction did not recover, necessitating implantation of temporary vvi-pacemaker, which was later replaced by a permanent ddd pacemaker system. pathology of the endomyocardial biopsy showed extensive lymphocytary infiltration with destruction of myocytes. parvo b dna-pcr was positive in both the biopsy and serum. these findings suggest that this patient developed severe myocarditis induced by parvo b viral infection. to our knowledge, parvo b viral infection is an uncommon cause of severe myocarditis in adult patients. sparse literature is available describing the use of ecmo in these adult patients. conclusion. this case report shows that parvo b virus should be recognised as a potential infective agent in adult patients presenting with severe myocarditis. furthermore, ecmo can be safely used to stabilize hemodynamics and peripheral organ perfusion in expectation of myocardial recovery in these patients. copeptin is easier to measure than vasopressin, and could be used as a marker of vasopressin release [ ] . the aim of the study was to compare plasma concentration of avp and cop during cardiac surgery, and specifically during post cardiac surgery vasodilatory syndrome (pcsvs). methods. two-month consecutive patients scheduled for cardiac surgery with cardiopulmonary bypass (cpb) were included in the study except patients suffering from chronic renal failure and under dialysis. blood samples were obtained from blood withdrawals routinely operated before cpb, during cpb and after surgery, at the postoperative hour (h ). these samples were used for avp and cop measurements. pcsvs, assessed as hypotension unresponsive to volume replacement therapy and without cardiogenic shock features, was treated with norepinephrine (ne). patients treated with ne have been compared to the others. statistical test consisted of variance analysis, non parametric test (mann whitney or wilcoxon) and linear regression. a p value of less than . (p \ . ) was considered statistically significant. results. patients have been included, out of which have been treated with ne. correlation between avp and cop plasma concentrations is significant (r = . , p \ . ). avp and copt concentrations increased significantly at h but the increase is less pronounced in ne-treated patients (fig. ) . ne-treated patients had lower preoperative left ventricle ef ( . ± . vs. . ± . %, p = . ), longer cpb ( . ± . vs. . ± . min, p \ . ) and clamping times ( . ± . vs. . ± . min, p \ . ), higher incidence of low output syndrome ( / vs. / , p \ . ) longer extubation time ( . ± . vs. . ± . h, p \ . ) and higher plasma cop before (t ) and during cpb (fig. ) . avp (ng/ml) et copeptin (cop, pmol/l), in patients. *p \ . ne versus others discussion. correlation between avp and cop is similar to that observed in other studies [ ] . the correlation coefficient is rather weak that is possibly related to avp dosage limitations (binding of avp to blood platelets, lack of antibody-specificity). increased cop plasma concentrations before and during cpb is observed in sicker patients undergoing more complex surgery, which seems to expose them to relative postoperative vasopressin deficiency and pcsvs. background. waiting list for heart transplantation has been growing up. high doses of cathecolamines has been an exclusion criterion for heart donation and norepinephrine use is still controversial. to assess if norepinephrine used on heart donors modify receptors outcome. methods. historical cohorts study from april to march . patients were divided in two groups: group : patients with local donors treated with norepinephrine (n = ). group : patients with local donors managed with other cathecolamines (n = ).cathecolamines were used at least for h and doses were between . and mcg/(kg min) if norepinephrine and between and mcg/(kg min) if dopamine or dobutamine. mortality risk factors published on the last international society for lung and heart transplantation guidelines were recorded. graft dysfunction risk factors were also collected. heart transplant outcome was measured by -day mortality, mortality rate at first, second, fifth and tenth years; and graft dysfunction incidence. chi-squared and t student test was used. multivariate logistic regression was used to evaluate norepinephrine impact on the outcome. mortality in group was . and % in group . no differences in mortality or graft dysfunction incidence were found in multivariate analysis. conclusions. norepinephrine used for donors management compared with dopamine and dobutamine does not increase mortality or graft rejection incidence in heart transplantation. groups were not uniform so further studies may be made to determine this association. introduction. coronary artery bypass surgery on cardiopulmonary bypass is associated with significant morbidity and mortality. with present technology, all arteries on the heart can be bypassed off-pump. the benefit of this technique is higher for patients whom are at increased risk of complications from cardiopulmonary bypass, such as those who have heavy aortic calcification, carotid artery stenosis, prior stroke, and compromised pulmonary or renal function. to evaluate the short-term follow up results of off-pump coronary artery bypass (opcab) and postoperative management of these patients admitted to our coronary care unit. we designed an observational study that included patients who underwent opcab from july to december . data were collected on preoperative age, sex, major cardiovascular risk factors, history of prior ami, number of affected vessels and ventricular function. after the surgery we evaluated: the extubation time, postoperative bleeding, troponin maximum level, need for blood transfusion, use of vasoactive drugs and intra-aortic balloon pump, development of renal failure, atrial fibrillation, neurological complications and reintervention. results. patients were included. . % were men and . % women. mean age was . ± . years. % of patients had one or more cardiovascular risk factor: hypertension was present in . %, smoking . %; diabetes mellitus . % and dyslipidemia in . %. there was prior myocardial infarction in . % of patients. prior coronary angiography showed . % of patients with vessels disease and . % of vessels disease. mean lvef was %. mean number of grafts was . . mean extubation time was . h. mean postoperative bleeding was estimated in cc. . % of patients needed blood transfusion; . % vasoactive drugs; and . % needed an intra-aortic balloon pump. . % of patients developed troponin t elevation with a mean level of . ng/ml. . % of patients developed atrial fibrilation, and . % renal dysfunction (two patients needed hemodialysis). there was no neurological complications. patient needed a reintervention. mean of intensive care unit stay was . ± . days. total mortality rate was . %. our experience shows that the off-pump coronary artery bypass graft surgery is a safe and effective technique for coronary revascularization, with low mortality and morbidity rates and reduced postoperative complications. objectives. to assess if deterioration of left atrial function in patients with severe sepsis and septic shock could predict mortality. we studied patients with severe sepsis or septic shock with mean age of . ± . . underlying echocardiographic parameters were measured on admission, th and th day, which comprised left ventricular ejection fraction (ef), and atrial function which is expressed as atrial ejection force (aef), with aef defined as the force that the atrium exerts to propel blood into the left ventricle (lv). all patients were subjected to bnp assay well. multivariate analyses adjusted for acute physiology and chronic health evaluation score ii (apache ii score) was used for mortality prediction. results. underlying source of sepsis was lung in patient ( %), blood in seven patient ( . %), abdomen in seven patients ( . %), while three patient ( %) had urinary tract infection (uti) as a cause of sepsis. only one patient had cns infection. severe sepsis was admission diagnosis for patients, patients were labeled as septic shock. look for days mortality. in-hospital mortality was . % ( patients) . admission ef showed significant difference between survivors and non-survivors . ± . versus . ± . % (p \ . ), on the other hand admission aef showed insignificant changes between the same groups . ± . versus . ± . k/dynes p = . , while bnp was significantly higher in the non-survivors , ± . versus . ± . pg/ml (p \ . ). multivariate logistic regression, the predictable variables for mortality was apache ii score, bnp then ef. conclusion. in septic patients, left atrial function unlike the ventricular function and bnp levels cannot be used as independent predictor of mortality. objectives. to analyse the relationship between plasma levels of nt-probnp and lcd diagnosed by echocardiograph during ss. methods. prospective observational cohort study. inclusion criteria: consecutive patients with ss [ ] . non inclusion criteria: creatinine clearance \ ml/min, years \ age \ years, cardiac surgery patients, pre existing coronary or cardiac insufficiency, neoplasia and systemic diseases. the evaluation of the left ventricular function was realised by a trans-thoracic or a trans-oesophageal echocardiograph on day . the lcd was defined by a left ventricular fraction of ejection \ % evaluated by teicholtz. the blood tests for nt-probnp analyses were drawn on days , , and . serum nt-probnp measurements were made automatically by elecsys analyser with the truss nt-probnp (roche diagnostics, myelan, france) by the electrochemiluminescence immunoassay method (eclia). data are expressed as mean ± sd and percentages. statistical analysis was performed by repeatedmeasures anova and roc curves (p \ . indicated statistical significance). . patients were included in a period of months (medical patients n = , surgical patients n = and trauma patients n = ), age = ± years, bmi = ± kg/m , apache ii = ± , igs ii = ± , duration of intensive care unit stay = ± days, mortality = %. lcd was observed in patients. the statistical analysis showed a significant elevation of nt-probnp in patients with lcd (table ) . on day , the area under roc curve was . , and the cut off value of nt-probnp predictive of lcd was , pg/ml (sensibility = %, specificity = %). introduction. fluid responsiveness can be predicted by the respiratory variation of arterial pulse pressure (ppv) or of pulse contour-derived stroke volume (svv) as well as by the changes in pulse contour-derived cardiac index during a passive leg raising manoeuvre (plr) or a tele-expiratory occlusion (teo). we evaluated the ability of an infrared photoplethysmography arterial waveform (cnap device) to estimate ppv. we also tested the ability of this non invasive estimate of ppv to predict fluid responsiveness compared to the invasive measure of ppv, to svv and to the plr and teo tests. in patients with septic shock ( ± years of age, receiving norepinephrine, saps = ± , lactate = . ± . mmol/l), we measured the response of cardiac index (pulse contour analysis, picco device) to fluid administration ( ml saline over min). before fluid administration, we recorded the ppv directly calculated from the non invasive arterial pressure signal (ppv ni ), the ppv directly calculated from the invasive arterial pressure signal (ppv i ), the ppv automatically provided by the picco device (ppv picco ), the svv automatically provided by the picco device, the changes in cardiac index induced by a plr test and the changes in cardiac index induced by a -s teo. results. five patients were excluded because the arterial curve could not be obtained by the cnap device due to excessive vasoconstriction. in the remaining patients, fluid administration increased cardiac index by more than % ( ± %) in ''responders''. the fluid-induced changes in invasive (? ± %) and non invasive (? ± %) mean arterial pressure were correlated (r = . , p \ . ). at bland-altman analysis, ppvni accurately reflected ppvi (bias %, limits of agreement ± %). for predicting fluid responsiveness in the patients, the receiver operating characteristics (roc) curves for ppv ni , ppv i , ppv picco , svv, plr and teo were . ± . , . ± . , . ± . , . ± . , . ± . , . ± . (all non significantly different). when considering only the patients ventilated with a tidal volume b ml/kg predicted body weight, were falsely classified as non responders by ppv ni , ppv i and two others by ppv picco and svv, but all four were well classified by plr or teo. in septic shock patients, provided that vasoconstriction is not excessive, the non invasive assessment of arterial pulse pressure seems valuable for predicting fluid responsiveness. introduction. mechanical ventilated patients often require inotropic support. however, the role of mechanical ventilation (mv) in myocardial depression is not well understood. septic patients often have impaired cardiac function and are in need of mechanical ventilation. we hypothesized that mv enhances sepsis-induced myocardial depression. objectives. in this study we investigated the influence of mechanical ventilation on cardiac function in an acute sepsis model. sepsis was induced in male wistar rats using ip injection of lps. healthy and septic rats were randomized to one of three ventilation groups; ( ) non-injurious ventilation with a tidal volume of ml/kg and cm h o peep (low tidal volume, ltv), ( ) injurious ventilation with a tidal volume of ml/kg and cm h o peep (high tidal volume, htv) and ( ) spontaneous breathing. arterial pressure was kept at least at mm hg. cardiac output (co, thermodilution method), central venous pressure (cvp) and mean airway pressure were measured in vivo. after h of ventilation, animals were sacrificed and cardiac function was measured ex vivo in a langendorff setup and expressed as developed pressure and ?dp/dt. cardiac wet to dry weight ratio was calculated. results. cardiac output in vivo was lower during htv ventilation than during ltv ventilation (p \ . ). cvp did not differ between ventilation strategies while mean airway pressure was higher in htv ventilation than in ltv ventilation (p \ . ). ex vivo, cardiac function of septic animals was depressed compared to healthy controls (p \ . ) in septic animals, cardiac function was better in htv ventilated animals than in non ventilated animals (p \ . ). ventilation lowered cardiac wet/dry ratio (p \ . ). developed pressure (p \ . ) and ?dp/dt (p \ . ) correlated inversely with cardiac wet/dry ratio. [ ] . perfusion may be also evaluated by other parameters such as lactate or venous-arterial pco gradient (delta pco ). objectives. to evaluate if early normalization of scvo after emergency intubation in septic patients persists over time and if it is associated with similar trends in lactate and delta pco . methods. ten septic patients subjected to emergency intubation for respiratory or circulatory failure and in whom scvo increased to [ % after the procedure. these patients were included in a large prospective study published elsewhere [ ] . patients used a common intubation protocol and we evaluated several perfusion related parameters before, min and h after emergency intubation. statistical analysis included friedman and wilcoxon tests. results. evolution of perfusion parameters after intubation is presented in table . five patients died during icu stay. as a whole, scvo remained stable in pts and decreased dramatically at h by[ % in non-survivor patients (lowest %). only pts had a high lactate before intubation that did not normalize at h (both non-survivors). delta pco exhibited erratic changes over time with no correlation with scvo changes and with mortality ( fig. ). introduction. venous to arterial carbon dioxide difference (pv-aco ) could reflect the sufficiency of blood flow in shock states. time evolution of pv-aco during early phases of resuscitation in septic shock has not been widely characterized. we proposed to describe the association between time course of pv-aco during the initial resuscitation and outcomes in septic shock. methods. patients with a new septic shock episode admitted to icu were included. general management was guided according surviving sepsis campaign recommendations. time (t ) was set when a central venous catheter was inserted to guide reanimation. simultaneous measurements of lactate and arterial-venous gases were obtained at t and h after (t ). pv-aco was calculated as the difference between venous co (blood samples drawn from a central catheter) and arterial co . a value of pv-aco [ was considered as high. survival at day was described for four groups: persisting high pv-aco (high at t and t ), increasing pv-aco (normal at t , high at t ), decreasing pv-aco (high at t , normal at t ) and persistently low (normal at t and t ). survival probabilities were estimated using kaplan-meier method. log-rank test was use to estimate a two-tailed p value for the differences in survival among groups. results. sixty septic shock patients were analyzed. mortality rate was . %. no demographic differences at baseline between survivor (s) and non-survivors (ns) were found. there were no differences in the amount of fluids administered at t and t . no significant differences in scvo at t for s introduction. septic shock (ss) has been defined as sepsis related hypotension despite adequate fluid resuscitation ? perfusion abnormalities such as lactic acidosis [ ] . despite this, an operationally simplified definition overlooking perfusion parameters, has been utilized in several landmark studies during the last decades [ ] [ ] [ ] [ ] . more recently, a new consensus reemphasized the pivotal role of hypoperfusion in ss definition and added low svo as a surrogate [ ] . several problems emerge from these apparently interchangeable definitions, including pathophysiologic and epidemiologic (incidence, outcome) issues. objectives. our aim was to evaluate if applicating different commonly used ss definitions to vasopressor-requiring septic patients leads to distinct outcomes. methods. we applied the two most utilized ss definitions to hypotensive septic patients managed with a ne-based algorithm [ ] for years, generating two major subgroups for analysis (fig. ) . statistical analysis included chi-square test. (fig. ) . pts of subgroup , exhibited persistent normal lactate levels with a mortality of . % which was similar regardless of svo [ or \ : p = . . (fig. ) . conclusions. commonly used ss definitions are not interchangeable and when applied to the same vasopressor requiring septic patients lead to statistically different mortalities. our data suggest that lactate and svo cannot be used indistinctly to define shock condition. a reappraisal of clinical septic shock definition appears to be necessary. objectives. to assess intra-and inter-observer agreement of ecg interpretation in adults with septic shock (vasst, nejm ; : ) . methods. patients were randomised to receive a blinded infusion of low-dose vasopressin or norepinephrine in addition to open-label vasopressors. eight icus participated in this ecg sub-study; and -lead ecgs were recorded at baseline (prior to study drug infusion), and h, and days after initiation of study drug. an intensivist (reader ) and a cardiologist (reader ), blinded to patient data and randomization group, interpreted all of the ecgs in duplicate, using a checklist. prior to ecg interpretation, a calibration exercise was performed to refine definitions and maximize inter-observer agreement; both readers reviewed ecgs (from the current study) representing the spectrum of normal to abnormal. cohen s kappa statistic was used to assess intra-and inter-rater reliability. methods. the model consists of eight elastic chambers including the heart and circulations. identification of the parameters is made only from measured pressures in the aorta and pulmonary artery, and the volume in the right ventricle. septic shock was induced in (n = ) healthy pigs with endotoxin infusion over min. right ventricular pressure-volume loops were recorded by conductance catheter and end-systolic ventricular elastance was assessed by varying right ventricular preload. consent was obtained from the university of liege medical ethics committee. errors for the identified model are within % when the model is identified from data, re-simulated and then compared to the clinically measured data. even with a limited amount of available experimental data to identify the parameters of the model, all simulated parameters trends match physiologically expected changes during endotoxic shock. in particular, a close match of the trends of the right ventricular end-systolic elastances are obtained, when compared to previously reported experimental results [ ] , including capturing of the peak after min and a decaying oscillation after min. conclusions. pig-specific parameters for the cvs model were accurately identified using a significantly reduced data set. this research shows the ability of the model to adequately and realistically capture the impact of pressure-volume changes during endotoxic shock. in particular, the model is able to aggregate diverse measured data into a clear, clinically and physiologically relevant diagnostic picture as the condition develops. this research thus increases confidence in the clinical applicability and validity of this overall diagnostic monitoring approach. background. conflicting data exist concerning the effects on the microcirculation of increasing mean arterial pressure (map) with norepinephrine (ne) in septic shock. nearinfrared spectroscopy (nirs) has been proposed as a tool to quantify microvascular dysfunction in patients with sepsis. by inducing a vaso-occlusive test (vot), a variety of nirsderived variables can be measured to assess local metabolic demand and microvascular dysfunction. this trial was conducted to test the effects of increasing map by ne on microvascular reactivity in patients with septic shock. after local ethical committee approval and informed consent, we enrolled patients in septic shock with an arterial pressure stabilized by ne. in addition to hemodynamic measurements, svo and blood lactate level, we measured thenar muscle oxygen saturation (sto ) and muscle tissue hemoglobin index (thi) by a tissue spectrometer (inspectra tm model , hutchinson technology inc, mn). serial vot (upper limb ischemia induced by a rapid pneumatic cuff inflation around the upper arm) were performed. we also recorded during the vot: basal sto , thi, the slope of the decrease in sto during the occlusion (desc slope; %/min) and the slope of the increase in sto following the ischemic period (asc slope; %/s). muscle oxygen consumption (nirvo i) was calculated as the product of the inverse of the slope value by the mean of thi over the first minute of arterial occlusion and is expressed in arbitrary units (u) (skarda shock ). all these data were obtained at different times: baseline and with map of mmhg, then at mmhg and mmhg of map by increasing the ne doses and finally to baseline . we report here data corresponding to the mean and sd of baseline and versus map mmhg analyzed by repeated measures analysis of variance (at % level) with bonferroni adjustment to account for multiple comparisons. increasing ne dose induced an increase in cardiac output (from . ± . to . ± . l/min, p \ . ) without any changes in heart rate and an increase of svo (from . ± . to . ± . %, p \ . objectives. to investigate: . the effects of ''successful'' protocolised resuscitation (egdt) on microvessel perfusion (particularly density). . whether there is different effects of egdt on the microcirculation of septic compared to critically ill non-septic patients and . whether there is a difference in the behaviour of ''true'' capillaries (i.e - lm) compared to larger microvessels ( - lm) at baseline or after resuscitation. prospective observational study in the emergency and intensive care departments of an urban teaching hospital. subjects: septic and critically ill control patients requiring shock resuscitation (map less than mmhg, ±cvp less than mmhg, ±central venous saturations less than %). all patients had invasive monitoring and identical cardiovascular targets. patients with known cardiogenic shock or pre-stabilised trauma were excluded. we performed sidestream dark field (sdf) videomicroscopy of sublingual microcirculation at the point of egdt initiation and again on attainment of at least out of cardiovascular goals. three sites were imaged for s and the clips were analysed randomly off-line to provide an average value for capillary density (total length and count per mm) and a semi-quantitative description of microvessel flow (continuous, intermittent or stopped) as previously described. vessels were grouped according to diameter as small ( - lm) and medium ( - lm). non parametric analysis was used for all within or between group comparisons, data is displayed as median values with [range]. *p \ . was considered significant. ( ) ( ) ( ) ( ) duration of occlusion (min), mean ± sd . ± . . ± . . ± . . ± . minimal sto (%), mean ± sd ± ± ± ± as expected, all septic shock patients, except one (for the vot fa % ) and two (for the vot a % ) had a recovery slope lower than normal when sto decreased to % during arterial occlusion. by contrast, when occlusion lasted min, many patients including patients who eventually died, were misclassified since their recovery slopes were in the normal range. these results could be due to the smaller decrease of sto and in turn a less strong hyperemic response when ischemia lasted only min. additionally, a significantly (p \ . ) shorter time to reach % was required when arm (compared to forearm) occlusion was performed. conclusion. when a vot is required for assessing microcirculatory disturbances in septic shock, we recommend performing it using an arm occlusion until sto reach %. aims. to analyze the correlation between sto (and its changes derived from a transient ischemic challenge) and global oxygen delivery (do ) parameters measured invasively using a pulmonary artery catheter (pac). observational study, performed in a -bed medical-surgical icu, at a university hospital. we recruited adult patients with cardiovascular insufficiency that required a pac placement for hemodynamic monitoring and resuscitation. we collected demographic data, and hemodynamic and oxymetric data derived from the pac. simultaneously, we measured sto and its changes derived from a vascular occlusion test (vot). results. twenty-two patients were studied. all the patients had a mean arterial pressure (map) above mmhg. the do index (ido ) range in the studied population was - mlo /(min m ). the mean svo value was ± %, mean cardiac index (ci) . ± l/ (min m ), and blood lactate . ± . mmol/l. the correlations found between sto and invasive oxygen delivery-related variables are shown in table . the sto -deoxygenation slope (deox) during the vot showed a significant correlation with svo (r . , p . ). we did not find any correlation between sto and global flow measurements, such as cardiac index (ci), but we found a correlation between sto and ido . this correlation seems related to the arterial oxygen content, and not to global flow. normal sto values could not rule out low ido and low ic states. therefore, sto seems to be poorly sensitive to exclude hypoperfusion states. in clinical practice there remains issues over the appropriate prescribing of antibiotics in patients with unproven sepsis. the prescribing of antibiotics is not without risk and creates a selective pressure on existing bacterial flora resulting in the emergence of virulent and resistant organisms [ ] . there is also a cost issue from the inappropriate prescription of antimicrobials [ ] . the diagnosis of sirs can be made with confidence [ ] , sepsis cannot and requires confirmation from microbial tests. empirical usage of antibiotic therapy is commonplace but not ideal. rapidly detectable, reliable markers of sepsis would help in directing antimicrobial therapy. objectives. the aims of this study are to determine the significance of % band forms in sirs patients suspected to have sepsis. can they be used as a diagnostic tool in conjunction with procalcitonin in order to direct antimicrobial therapy? methods. this is an observational study aiming to assess the ability of serum procalcitonin and percentage band forms in identifying nosocomial sepsis in patients with sirs. patients were recruited over an month period in a mixed medical-surgical university teaching icu. all patients had suspected sepsis arising 'de novo' and had not received prior antimicrobial therapy. patients had a septic screen performed along with baseline, and hpct and % band form count. introduction. pneumonia is the most frequent infectious complication after successfully resuscitated cardiac arrest (ca). however, diagnosis is difficult because of many clinical, biological and radiological confounding factors as well as the widespread use of therapeutic hypothermia. this could lead to a broad antibiotic prescription. to assess the utility of plasma procalcitonin (pct) measurements for diagnosis of early-onset pneumonia in successfully resuscitated ca. monocentric study (july -march with retrospective review of a prospectively acquired icu database focusing on all consecutive patients admitted for ca and surviving more than h. patients with an infection prior to ca or with an extra-pulmonary infection developing within days following admission were not studied. all files were reviewed to assess the diagnosis of early-onset pneumonia p(?), or not p(-) during the first days of icu stay. p(?) was defined by the presence of a new pulmonary infiltrate on chest radiography, persistent for at least h, associated with either positive quantitative culture of the endotracheal aspirates, either, in case of lack of bacteriological sample, conjunction of purulent sputum and hypoxemia (p/f \ ). pct was measured at admission, days (d) , and (brahms kryptor Ò ). among patients admitted for ca, were studied ( death before h, evolutive infections and incomplete samples). pneumonia was diagnosed in patients ( %), and antibiotics were prescribed in during the first days of icu stay. characteristics of p(?) and p(-) patients were (median, iqr): age ( - ) versus ( - ) (p = . ), ''no flow'' ( - ) versus ( - ) min (p = . ), ''low flow '' ( - ) versus ( - ) min (p = . ), shockable rhythm versus % (p = . ), cardiac etiology versus % (p = . ), therapeutic hypothermia versus % (p = . ), post-resuscitation shock versus % (p = . ) and icu mortality versus % (p = . ). using a threshold value of . ng/ml, negative predictive values were % at admission, % at d , % at d , whereas positive predictive values were , and %, respectively. patients with post-resuscitation shock had higher pct levels than those that did not require vasopressors: . versus . ng/ml at d (p \ . ), . versus . at d (p \ . ) and . versus . at d (p = . ). conclusion. diagnosic value of pct is poor in survivors of ca and pct should not be recommended to assess early-onset pneumonia. post-resuscitation disease could play a major role in the lack of specificity and predictive values. in acute community respiratory infection, low levels of procalcitonin (pct) have been shown to allow a marked reduction of antibiotic use. the aim of the study was to look for the same efficacy in case of suspicion of infection during icu stay. method. from april to december , patients hospitalized in the five intensive care units (icu) of the university hospital of liège in belgium, were prospectively randomized to either a procalcitonin guided approach to antibiotic therapy (pct group, n = ) or to a standard approach (ctrl group, n = ) when they were suspected of developing an infection. for pct group guided therapy only, the use of antibiotics was more or less strongly discouraged (pct level . or. lg/ml, respectively) and more or less recommended (pct level [ or[ . lg/ml, respectively) . number and duration of antibiotic treatments were recorded. diagnosis and treatment decisions were reviewed by infectious disease (id) specialists at the end of icu stay. results. there were no differences between groups in terms of age ( vs. ), saps ii score ( . ± . vs. . ± . ), type of patients (medical: vs. %, scheduled surgery: vs. %, emergency surgery: vs. %, trauma: vs. %), icu length of stay [ (iqr - ) vs. days (iqr - )] for pct and ctrl group respectively. suspicion of infection was either evoked on admission (in and %) or during icu stay (in and %) in pct group and ctrl group respectively. at the time of suspicion, pct levels was. lg/ l in . % of the infectious episodes in pct group and . % in ctrl group. episodes of suspected infection with pct level . lg/ml were recorded. clinicians decided not to treat % of these episodes (n = ). the remaining episodes were treated, of which % were eventually considered as probable or confirmed infections by id specialist (n = ). at the end of icu stay, id specialists classified infectious episodes of both groups as confirmed (n = ; . %), probable (n = ; %), possible (n = ; %) or absent (n = ; . %). for confirmed episodes of infection, pct levels were . lg/ml in as much as . % and above lg/ml in . %; for absence of infection, pct levels were . lg/ml in only . % and above lg/ml in . %. the ability of pct to discriminate between confirmed and probable infections on the one hand and possible or absent infection on the other hand, was tested by the measurement of the surface under the roc curve, which was . , which is too low to recognize pct as a valuable marker of infection. there were no difference in the number of treated patients ( vs. %) nor in the number of antibiotic days ( vs. %) between pct and ctrl group respectively. conclusions. procalcitonin level as an aid for the decision to treat infection in icu patients appeared not to be helpful. antibiotic consumption was not reduced using this tool in our study. introduction. respiratory infections, pneumonias in particular are a common cause of mortality in the intensive care unit (icu) patients worldwide. early identification and prompt management of these patients especially with associated sepsis is crucial in reducing the mortality. many clinical and laboratory markers have been studied extensively to predict the outcomes in them. there have been numerous studies on the clinical utility of serum procalcitonin (pct) in the past decade, in systemic inflammation, infection and sepsis. objective. to evaluate the role of serum procalcitonin, in predicting the outcomes of patients admitted in the icu with respiratory infections associated with sepsis. setting: bedded icu of a tertiary referral hospital. study design: prospective observational study. subjects: adult ([ years) patients admitted in the icu with lower respiratory tract infections with associated sepsis during the period july to january were prospectively followed up. primary outcome measure: day mortality. we measured pct levels using the brahms immunochromatographic technique(semiquantitative estimation) on the first day of admission into the icu . normal pct was taken as . ng/ml. patients were grouped into four groups-group a (pct \ . ng/ml), group b (pct [ . - ng/ml), group c (pct [ - ng/ml),group d (pct [ ng/ml). sepsis, severe sepsis, septic shock are defined according to the accp/sccm criteria. results. the overall mortality was . % with mortality of . , . , , and % in groups a, b, c and d, respectively. there is a statistically significant difference (p \ . ) in the mortality rates of groups c and d as compared with group a and b, but no difference was observed in the mortality rates between groups a and b and groups c and d .also significant statistically are the apache ii scores, septic shock and multiorgan failure incidence in the groups c and d as compared to groups a and b. conclusions. serum procalcitonin level [ ng/ml on the first day of admission in icu appears to be a good predictor of mortality in patients admitted with lower respiratory tract infections and associated sepsis. methods. in a retrospective study we assessed acutely ill patients investigated for pct and treated by a physician blinded for pct value. for each patient we also calculated new simplified acute physiology score (saps ii). we evaluated many clinical and instrumental parameters and diagnosis was done upon our usually clinical practice results. the mean age of patients (pt) was . yeats, shock was found in patients ( . %),median value of saps ii score was (iqr - ), and median estimated mortality from saps ii was % (iqr - ). bacterial infection was found in . % (septic shock . %, pneumonia . %, cholecystitis . %, pleural empyema . %, other infections . %) non infective disease in . % (pulmonary embolism . %, acute coronary syndrome . % heart failure . % other disease . %. a pct value [ . ng/ml was considered positive: so pct was elevated in . % of bacterial infection patients and in . % of non infective disease patients. we also compared pct values with antibiotic therapy and considered appropriate the administration if pct [ . ng/ml: there was discrepancy in . %. the review of these cases found medical decision wrong in cases versus ( . %); pt with pct \ . ng/ml had antibiotic therapy without bi and cases with pct [ . ng/ml did not have antibiotic therapy but had a bacterial infection. subsequent to this review discrepancy felt to . % (ci % . - . ) and was found especially in pt with pct \ . ng/ml. at cut off point of . the sensitivity was . (ci %: . - . ) specificity . (ci %: . - . ) or . and at point . the sensitivity was . (ci %: . - . ) specificity . (ci %: . - . ) or . , with high predictive positive value. all-causes mortality was . %. mortality if pct \ . ng/ml was . %, if pct . - . ng/ml was . %; if pct . - . lg/ml was . % and if pct [ ng/ml was . % without significant difference between bacterial infection and non infective disease group. comparing pct with saps ii score, area under roc-curve was not significantly different (pct . -ci %: . - . ) (saps ii . -ci %: . - . ). conclusions. pct in acutely ill patients is a useful marker to discriminate bacterial infections with high sensibility but low specificity and it may be useful to guide the therapy also with values higher than . ng/ml. our data suggest a real prognostic utility of pct in these patients, regardless of bacterial infections, but our efforts to elaborate a mathematical predictive model aren't still satisfying and further data are required in this setting. h. taniuchi , t. ikeda , k. ikeda , s. suda tokyo medical university, hachioji medical center, division of critical care medicine, tokyo, japan introduction. its apparent that detection of the causative bacteria is useful for the therapeutic strategy. however, conventional tests for the detection of the causative bacteria are not high sensibility. in order to diagnose sepsis or septic shock and start appropriate therapy rapidly, it's also important to know whether the infection is cause of gram negative bacteria, that is to say, whether the infection is cause of endotoxin. in this study, we investigate the severity level of sepsis and initiation criteria of direct hemoperfusion with polymixin b immobilized fiber column (pmx-dhp) treatment from the result of severity level by using endotoxin activity assay (eaa) and using measurement of procalcitonin (pct). subjects and methods. patients who developed severe sepsis or septic shock and admitted to icu were included. on the day of icu admission, a general blood biochemistry, eaa and pct levels, and apache ii and sofa score were measured. patients were evaluated retrospectively the relationship between the severity of sepsis and each measurements and investigated the relationship between the measurements and pmx-dhp. serum eaa level was measured using smart line eaa luminometers. serum pct level was measured using immune luminometric assay. results. the average age of the patients is ± , apacheii score was . ± . , sofa score was . ± . , the median pct was . ng/ml (range - ), eaa was . ± . . the underlying diseases of the enrolled patients were the abdominal infection ( patients), the urinary tract infection ( ), pneumonia ( ), the meningitis ( ), the soft tissue infection ( ) and other infection ( ) . the causative bacteria were gram positive bacteria ( ), gram negative bacteria ( ), virus ( ), and unknown ( ). there was no statistical correlations between eaa or pct level and apacheiiscore. there was no statistical correlations between eaa level and sofa score. although there was no statistical correlation between pct level and sofa score, the pct level tended to rise as pct level rises. we investigated the relationship between eaa and pct levels. there was also no statistical correlations between eaa and pct. we investigated the relationship between the causative bacteria (gram positive bacteria, gram negative bacteria and the others) and eaa or pct level. there was no statistical correlations between the causative bacteria and eaa level nor pct, that was contrary to our expectation that eaa level should be high for gram negative bacterial infection. we further investigated the relationship between whether or not the pmx-dhp was implemented and eaa or pct level. there was no statistical relationships. conclusion. high levels of the eaa and pct would not indicate the severe infection with gram negative bacteria, and the initiation of pmx-dhp. further study is needed, in which more patients will be enrolled and evaluated. introduction. sepsis still the major cause of death in the late post traumatic period in patients with major burns. early diagnosis of sepsis is crucial for management and outcome of critically burn patients. attempted in this study to assess whether plasma procalcitonin (pct) level was related to diagnostic and prognostic of sepsis in burned patients. patients and methods. pct was measured over the entire course of stay in patients with predictive signs of sepsis according to american college of chest physician. the patients were assigned to two groups depending on the clinical course and outcome: a = no septic patients, b = septic patients. optimum sensitivity, predictive values, and area under the receiver operating characteristic (roc) curve were evaluated. results. over a month period starting from july to december , patients were admitted. were investigated. in group a et in group b. procalcitonin was significantly higher in septic group . ± ng/ml compared to no septic group . ± . ng/ml. area under the curve was . on the day of sepsis diagnostic. pct cut-off value of . ng/ ml was associated with the optimal combination of sensitivity ( %), specificity ( %), positive predictive value ( %), and negative predictive value ( %). in survived septic patient the pct value was significantly lower than in deceased septic patients . ± . versus . ± . ng/ml. pct cut-off value for optimum prediction of outcome in septic patients was . ng/ml with sensitivity ( %), specificity ( %), positive predictive value ( %), and negative predictive value ( %). conclusion. procalcitonin appears to be a powerful marker of sepsis in burn patients. it is sensitive, specific, reliable and easy to measure. a high pct concentration ([ . ng/ml) would indicate poor outcome in septic patients. n. v. beloborodova , a. s. khodakova , a. y. olenin , s. t. ovseenko bakulev scientific center for cardiovascular surgery, moscow, russian federation objectives. accurate and timely diagnosis of sepsis remains challenging for clinicians. the diagnosis of sepsis is defined as typical symptoms of systemic inflammation (temperature, tachycardia, respiratory rate, leukocytosis) with clinical evidence of an infection site, but the criteria are met by a large number of intensive care unit (icu) patients. among studied biomarkers, serum procalcitonin (pct) has been described as one of the most promising predictors of bacterial sepsis, but in some clinical situations it is not enough. the search of reliable markers of sepsis is still in progress. in present study the significance of raised levels microbial phenylcarboxylic acids in serum of patients with sepsis are assessed. methods. the present study evaluated serum samples of patients (pts) with documentary sepsis, according to well known consensus criteria. the comparison groups were: no. - clinically healthy volunteers, no. - pts. with acquired heart diseases, no. - pts with ventilator-associated pneumonia. blood concentrations of phenylcarboxylic acids were determined by gas chromatography-mass spectrometry (gc-ms). results are presented as median and range of th and th percentiles. the statistically significant differences between the various groups were calculated using mann-whitney test. results. increased levels of phenyllactic (pla), p-hydroxyphenylacetic (hpaa), p-hydroxyphenyllactic (hpla) acids were observed in group of pts with sepsis. the level of hpaa was increased up to two orders in comparison with groups no. and [ . ( . - . ) vs. . ( . - . ) and . ( . - . ) lm, p \ . ). the levels of hpla and pla were increased up to one order [( . [ . - . ] table for illustration of importance of phenylcarboxylic acids blood level monitoring. introduction. acute kidney injury (aki) is a frequent complication of sepsis, and is associated with high mortality and morbidity rates. routinely used measures of renal function, such as levels of blood urea nitrogen (bun) and serum creatinine, increase only after substantial kidney injury occurs, resulting in delayed diagnosis of aki. therefore biomarkers, which enable early diagnosis, are needed. objectives. this clinical study was designed to investigate whether human interleukin- (il- ) and neutrophil gelatinase-associated lipocalin (ngal) are early predictive markers for sepsis-induced aki. urine and blood samples have been collected prospectively from icu patients, who met defined clinical criteria of severe sepsis. aki was defined by rifle criteria. urinary and serum levels of n-gal and il- have been quantified by elisa in patients with sepsis without aki (n = ) and in patients with sepsis induced aki (n = ). results. both, urinary il- and serum il- considerably increased (respectively, . and . -fold over the baseline) two days before the patients reached rifle risk. urinary ngal raised significantly ( . -fold over the baseline) one day before occurrence of aki, whereas serum ngal did not show any prior elevation. no increase in the levels of any of these markers could be found in patients who did not develop aki. conclusions. both urinary and serum il- seem to be sensitive early biomarkers for sepsis associated aki, while urinary ngal has less accuracy for aki prediction. objectives. to define a biomarker panel able to predict infection in case of severe acute dyspnea in emergency situations. we designed a prospective observational study of patients admitted in the emergency department (ed) and in medical polyvalent intensive care unit (icu) in a university hospital. inclusion criteria were acute dyspnea with spo b % and/or respiratory rate (rr) c b/min. patients with an immediate need of coronarography or with obvious spontaneous pneumothorax were excluded. five biomarkers were measured from blood sample at admission on ed or icu: nt b type natriuretic peptide (nt probnp), cardiac troponin i (ctni), ddimeres (dd), c-reactive protein (crp) and procalcitonin (pct). all clinical and biological data were recorded. an independent blinded data monitoring committee classified the patients according to all the available data including response to treatment and outcomes but blindly to biomarkers. the roles of biomarkers were assessed quantitatively and then using terciles of the distribution. the contribution of the biomarkers in the diagnosis was assessed using multiple logistic regression taking into account other clinical and biological explanatory variables. . patients were enrolled consecutively. the final diagnosis was: severe sepsis (n = ), acute heart failure (n = ), pulmonary embolism (n = ), copd (n = ), other causes (n = ). the days mortality was %. there was no significant association between infection diagnosis and dd, ctni, nt probnp. interestingly, a crp value of less than mg/l was not discriminant in predicting infection. adjusted on clinico-biological covariates selected, both pct with cutpoints of . and . ng/ ml (discrimination auc . ; p = . ) and crp with cutpoints of and mg/l (discrimination auc . ; p . ) were significantly associated with the diagnosis of sepsis. both biomarkers used simultaneously lead to a discrimination of the model (auc . ). conclusion. both crp and pct are able to predict the diagnosis of infection in case of severe acute dyspnea independently of clinico-biological variables. in this particular subpopulation, the best threshold for crp is higher than the standard one. an external validation is needed to prospectively validate the clinical utility of these findings. t. trefzer , i. nachtigall , a. weimann , c. de grahl , c. spies charite universitaetsmedizin berlin, campus virchow, department of anesthesiology and operative intensive care medicine, berlin, germany, charite universitaetsmedizin berlin, campus virchow, zentralinstitut für laboratoriumsmedizin und pathobiochemie, berlin, germany aims. infections are the most relevant icu-admission complication. crp and pct are labvalues used for diagnosis of infections. however, their use is often not evidence based. this study aimed to access whether the adherence rate increased after introducing an evidencebased standard operating procedure (sop). in an evidence-based sop was approved by experts of our department. in july it was made available to icu-physicians via intranet, which is accessible from every work station. altogether, we assessed sop-adherence rates of patients: in june (pre-sop), patients in august (one month post-sop) and in january ( months post sop). every crp and pct measurement was assessed for adherence to the standard operating procedure (sop). at first, the three periods were assessed for significant differences concerning the adherence. according to the percentage of sop-conform measurements the patients were then divided into two groups: the sop-group (c % of measurements sop conform) and the non-sop (nsop) group (\ % conform) in a second step, patients in the sop-and nsop-group were compared concerning icu scores (sofa, tiss, apacheii, saps) and outcome parameters (length of icu-stay, length of hospital stay, duration of mechanical ventilation, hospital mortality). statistics: p b . was considered as statistically significant; hospital mortality was assessed by a v test, icu scores and outcome parameters were compared using the mann-whitney u test. all parameters with p \ . were included into a logistic regression analysis. no change was observed concerning the implementation of the sop pre and postintroduction: . % in june , . % in august and . % in january . the non-conform pct-and crp-measurements resulted in additional costs of approximately . euros/year. the univariate analysis revealed significant differences in the sop-and nsop-group: the nsop-group had higher saps-, sofa-and tiss-scores, as well as increased length of icu-stay, length of hospital stay and duration of mechanical ventilation. logistic regression analysis revealed tiss score and length of hospital stay as an independent predictor for low sop adherence. conclusion. distribution of an evidence based sop without further education did not lead to a significant increase in adherence rates, but tiss score and length of hospital stay have shown to be independent predictors for low adherence to the sop. the significant higher tiss-scores in the nsop group might be a indicator for actionism of clinicians in the face of more severely ill patients. objetives. to asses the evolution of the risk-adjusted mortality rates of sepsis and septic shock in our icu in a ten years period. patients and method. analisys of prospectively recorded data of all pacients admitted with severe sepsis and septic shock in a bed icu during a period of years. patients were followed up until death or discharge from the hospital, excluding those with unknown outcome. mortality prediction was made using apache ii model with % confidence intervals. statistical analisys was made with spss . using anova test or t test to compare means and chi square test to compare categorical variables. results. from january to december a total of patients with sepsis were admitted, with an anual increase to reach % of all icu admissions. age and severity of illness increased anually as did sofa in the first h (sofa ) thus rising up calculated risk of death. from to mortality rate was between % ics of calculated risk of death, falling below inferior ic from and after . (fig. ). hospital mortality versus risk of death per year mortality was . % in the pre- period and . % from and on (p = . ) with non significant differences in apache ii, risk of death nor sofa , but with significantly greater age in the post- period ( . vs. . years p = . ). this non significant difference between the two periods of the study became significant when we analized the outcome in both sex. being significant in women (mortality . % in pre- period vs. . % in post- p = . ) but not in men ( . vs. . % p = . ). overall sepsis moratlity is lower in female without significant differences in age, apache ii score nor risk of death (table ) , being the only signifficant difference found in sofa ( . in male vs. . in female p = . ). introduction. low-grade systemic inflammation has been shown to play a key role in the pathophysiology of several chronic noncommunicable diseases [ , ] and may be attenuated by anti-inflammatory treatments such as administration of statins [ ] . so far, the association between acute systemic inflammation experienced during critical illness and long-term mortality after hospital discharge has not been investigated in intensive care unit (icu) patients. objectives. to assess the association between acute systemic inflammation, assessed by crp levels, and post-hospital mortality in non-surgical icu patients. methods. the study was performed as a prospective, observational follow-up study and included non-surgical critically ill patients with an icu length of stay [ h. patients who died during the icu or hospital stay, were \ years or pregnant, as well as patients discharged from the hospital with the plan to limit life support were excluded. demographics, chronic diseases, admission diagnosis, the simplified acute physiology score ii, length of icu stay, maximum crp levels during the icu stay (crpmax) and crp levels at icu discharge (crpdis) were documented. after a mean ± sd follow-up time of . ± . years, mortality and causes of death were determined. adjusted cox models were calculated to investigate the association of crpmax and crpdis with post-hospital mortality. a receiver operating characteristic analysis was used to identify optimal cut-off levels to predict post-hospital mortality. background. the prevalence of hiv infection is increasing worldwide as a public health problem. survival of hiv/aids patients has improved since highly active antiretroviral therapy, but sepsis has grown as an important cause of icu admission in this population. an international conference has set a system composed of specific risk factors, site and microbiology of severe infections and host response and organ dysfunctions (piro) to help identify patients at risk for sepsis. piro factors have not been classified for hiv/aids population yet. objectives. to identify predisposing factors, microbiology of infections, host clinical response and incidence of early organ dysfunctions of severe sepsis on hiv/aids patients, admitted to a specialized infectious diseases icu; to analyze long-term survival of hiv/aids critically ill patients. a prospective case-control study of septic and non-septic hiv/aids patients admitted between june and may was performed. demographic data, causes of admission, time since aids defining condition, cd cell count, and opportunistic infections were evaluated as predisposing factors to sepsis. microbiology and site of infections were registered. clinical response to severe infections was evaluated by ali/ards and shock incidence on day of icu admission. organ dysfunctions (sofa score) were reported soon after icu admission. icu length of stay, hospital and -month mortality were compared between septic and non-septic groups. a multivariate regression analysis was done to identify risk factors for icu mortality. kaplan-meyer survival curve was built. . icu admissions of hiv-infected patients were studied. half ( ) fulfilled criteria for severe sepsis diagnosis. septic group was younger ( . ± . vs. . ± . years, p \ . ) and had more female patients ( vs. %, p \ . ). time since aids diagnosis, cd cell count and opportunistic infections prevalence were not different. sites of infection were predominantly pulmonary ( %) and catheter-related ( %). ninety percent of infections were nosocomial. forty-three percent of septic patients presented bacteremia. pseudomonas sp, s aureus and enterobacteriacae were commonly identified, but five patients had mycobacterium tuberculosis isolated ( on blood cultures). multiple organ dysfunction syndrome was frequent, and incidence of cardiovascular, respiratory and hematological dysfunctions was significantly higher in septic group. longer length of icu stay ( . ± . vs. . ± . days, p \ . ) and icu mortality ( vs. %, p \ . ) was observed for septic patients. severe sepsis also influenced long-term survival, as mortality continues significantly higher after months (log rank . , p \ . ). conclusions. piro system is applied to septic hiv/aids patients. shock, ali/ards and hematological dysfunctions are prominent for septic hiv/aids population. septic hiv/ aids patients are at severe risk of short and long-term mortality. international guidelines for management of severe sepsis and septic shock suggest the use of recombinant human activated protein c (rhapc) in adult patients with high risk of death (apache ii c or multiple organ failure). the objective of this study is to analyse the characteristics and outcome of patients treated with rhapc in our medical intensive care unit. retrospective study of patients with severe sepsis/septic shock treated with rhapc between january to december . all of them were c years, with apache ii c and two or more organ dysfunction, and were treated on basis of a bundle for severe sepsis management: complete early goal-directed therapy, early administration of broadspectrum antibiotics; corticosteroids in vasopressors unresponsive patients and monitor for lactate clearance. chi-square analysis were used to compare categorical data. continuous data were compared using student's t test. prognostic factors of mortality were studied by means of multivariable logistic regression analysis. results. forty-one patients were studied. % were male. their mean age was ± years. % had comorbidities ( % immune pathology). severity scores. apache ii ± , sofa ± , % of patients had three o more organ dysfunction. % had septic shock. serum lactate level was . ± . mmol/l. the primary location of infections was: respiratory %, abdominal %, urinary %. . % were positive blood culture. % of patients needed mechanical ventilation ( ± days). % of rhapc infusions were not completed, mainly for bleeding risk ( %) and death ( %). . % of patients had bleeding event. at the end of the infusion % of patients remained with two or more organ dysfunction and % were vasopressors dependent. mean hospital stay was days and days in icu . days mortality was %, icu mortality . % and hospital mortality . %. analyzed data included age, comorbidities, primary location of infections, severity scores and serum lactate level. univariable analysis showed that statistically significant factors related to mortality were: apache ii ( ± vs. ± , p = . ), organ dysfunction number: vs. [ ( vs. %, p = . ) and primary location of infections: pneumonia versus others ( vs. %, p \ . ). a multivariable logistic regression analysis showed that age (or . , % ci . - . , p = . ), organ dysfunction number (or . , % ci . - . , p = . ) and serum lactate levels (or . , % ci . - . , p = . ) had statistically significant relationship to mortality. conclusion. in our study the patients with severe sepsis and septic shock remained with high vasopressors dependency and organ dysfunction at the end of the rhapc infusion. despite of rhapc therapy the mortality of patients was very high. the age and the severity at icu admission were independent prognostic factors of mortality. a higher incidence of severe sepsis in blacks compared to whites is well documented, however prior analyses do not discriminate whether this is due to a higher incidence of infections, a higher risk of developing organ dysfunction once infected, or both. objectives. we sought to understand whether higher severe sepsis incidence in blacks is due to higher infection susceptibility, higher risk of organ dysfunction once infected, or a combination of both. we analyzed , , hospitalizations from hospital discharge records of us states ( % of us population). we linked these records to us census data to generate age and sex-standardized incidence rates. we identified infections of bacterial and fungal etiology based on icd- cm criteria, including characterization by site and type of infection (gram negative vs. gram positive). we defined severe sepsis as documented infection plus acute organ dysfunction based on previous work by angus et al we estimated the risk of organ dysfunction among those hospitalized with infections using logistic regression, adjusting for age, sex and comorbidities (charlson score). fig. b ]. the combination of both events led to a % higher severe sepsis hospitalization rate for blacks ( . vs. . per , population, irr: . - . ). these differences persisted when stratified by sex, comorbidities, site and type of infection. infection incidence and severe sepsis risk conclusion. the higher incidence of severe sepsis among blacks is due to a higher hospitalization rate for infections, as well as a greater likelihood of organ dysfunction once infected. future interventions to reduce racial disparities in severe sepsis incidence should target both distinct events. grant acknowledgement. dr. mayr was supported by t hl - . objective. to describe recent epidemiological data and mortality risk factors of patients admitted to icu for severe pneumococcal pneumonia (pp). multicentric retrospective study (january -june ). prospective acquired data from patients admitted in french medical icu for severe pp were considered. patients with concurrent meningitis, severe copd with known sp colonization, hiv or aspiration pneumonia were not included. pp was defined by the combination of a suggestive clinical context, the presence of a new pulmonary infiltrate on chest radiography and a s.pneumoniae positive bacteriological sample (pulmonary quantitative culture, pleural fluid, blood culture or urinary antigen assay). all files were reviewed and approved by two independent investigators (nm, am). . patients were included. median age was ± . hospital survivors were significantly younger ( ± vs. ± , p = . ). sex ratio m/f was / , but male sex was associated with higher risk of death (male: vs. %, p = . ). active tabagism ( %) or alcohol abuse ( %) were more common than asplenia ( %). organ dysfunctions were mainly respiratory ( %), haemodynamic ( %) and renal failures ( %). low doses steroids were prescribed in % of patients with septic shock. icu mortality rate reached % ( % in the first days); hospital mortality rate was %. univariate analysis demonstrated that age, male sex, cirrhosis and organ failure support were strong predictors for icu mortality. multivariate analysis only highlighted age [or . ( . - . )], cirrhosis [ . ( . - . ) ] and renal replacement therapy [ . ( . - . )] as independent mortality predictors. activated protein c treatment was associated with decreased mortality [or . ( . - . )]. bacteremia had no impact on outcome. conclusion. this is the most important cohort of pp requiring icu admission. despite adequate antibiotherapy, mortality is still preoccupant. determination of factors related to the bacteria (virulence) or to the host (genetic susceptibility) could allow a better understanding of this important health problem. introduction. to identify the risk factors of mortality for patients with severe community-acquired bacteremic pneumococcal pneumonia. retrospective study realised in the intensive care units of two hospital medical centers. the studied population was patients with serious community-acquired bacteremic pneumococcal pneumonia. all the patients entered the intensive care units between january of and december of . study variables were: age, sex, concomitant pathology, toxic habits, pre-vaccinal ( - ) and postvaccinal periods ( ) ( ) ( ) ( ) ( ) ( ) ( ) , serotype and sensitivity of streptococcus pneumoniae to penicillin, the initial use of the non-invasive mechanical ventilation, the development of empyema pleural, apache ii and sofa scores during the first h after admission. results. the age average was of years. forty one percent of our patients required mechanical ventilation, and % had acute renal failure that required hemofiltration. average values of apache ii and sofa were . and . respectively. in hospital mortality of the series was of %. in patients with severe community-acquired bacteremic pneumococcal pneumonia: ( ) the presence of empyema pleural is an independent risk factor for mortality. introduction. procalcitonin (pct) is an interesting marker of pulmonary infection [ ] . it is useful as an help for infection diagnosis but also for treatment follow-up [ ] . besides, initiation of effective antimicrobial therapy is the strongest predictor of outcome in patients with septic shock [ ] . the aim of the study was to analyse whether kinetics of pct decline may reflect sensitivity of identified infectious agents to initial antimicrobial therapy (at). patients with diagnosis of severe pneumonia following major cardio-thoracic or vascular surgery were retrospectively included in the study. severe pneumonia was suspected as a combination of several manifestations including fever or hypothermia, hyperleucocytosis or leucopenia, new radiological infiltrate, and/or a clinical pulmonary infection score [ , pct [ ng/ml and pao /fio \ . initial antimicrobial treatment was chosen according to the guidelines in use in our institution for community-acquired or nosocomial infections. microorganism identification from endotracheal aspiration or bronchoalveolar lavage, and antibiotic susceptibility testing, allowed to classify patients according to appropriate (aat) versus inappropriate initial at(iat). pct was measured daily over days and its kinetics compared between both groups. data are expressed as median (extremes) or mean ± sd (decrease rate). results. patients aged ± ( - ), operated on vascular (n = ), thoracic (n = ), or cardiac surgery (n = ) have been studied from october to july . pneumonia occurred within the st to the st postoperative day (median . days), with a septic shock in cases and deaths at day . initial at was appropriate in % ( / ) patients. pct peak was not statistically different between aat versus iat patients ( . ± . ng/ml vs. . ± . , respectively) but pct decrease was significantly steeper and constant in iat patients (fig. ). pct decrease (%) from peak value over days discussion: the results suggest that absence of early decrease in pct within days may reflect failure of the at. conversely, an average decrease in pct plasma concentration of % in days seems to be a good marker of sensitivity of the causative infectious agent to the initial at. in case of unchanged pct within days at change should be considered. icu mortality was % for pts with rb early vap while it was % ( of cases) in those with sb or negative cultures (p = . ). mortality was higher than the predicted according to apache ii score in pts with rb vap ( vs. ± %, p \ . ). however, it was lower than predicted in those with negative or sensible isolates ( vs. ± %, p = . ). conclusions. rb were the most common cause of early vap among our patients. the burden of illness, los in icu before intubation and previous use of antibiotics were associated with early vap due to rb. inappropriate empiric therapy and mortality were higher among patients with early vap due to rb. to evaluate the performance of the saps piro model in patients with severe community acquired pneumonia (cap), over a period of years ( - ) , in a general icu in a central hospital. material and methods. we analysed data prospectively registered in an informatic data base, which contains information referring to all patients admitted in this unit. analysed were patients. discrimination was accessed by the area under the roc curve (aroc). calibration was evaluated by the by the hosmer-lemeshow Ĉ test. conclusion. implementation of a sedation protocol requires constant follow up and regular adaptation to prove efficient over time. constant feed back information to both the medical and nursing staff is mandatory. treatment of hyperactive delirium. hal haloperidol, bzd benzodiazepine, pro propofol, aa atypical antipsychotic, nd no drugs, na not answered discussion. there tends to be a general pessimism regarding obese patients within the intensive care community. our data indicates that this opinion could be misplaced. reduced ventilator days may reflect a reluctance to invasively ventilate obese patients. the apache ii scoring does not take into account the bmi which would eliminate any severity scoring bias. high bmi alone should not be a consideration in the decision regarding suitability for admission to critical care. during the last decades, a growing medical knowledges have changed the clinical approach to elderly patient diseases. they receive major surgery or intensive treatment for acute medical illness but often the recover is condictioned by the previous chonical diseases. this determines a long period to stay in intensive care unit (icu) because the slow improvement and cause an occupation of bed places. in our hospital, after a period of training performed by an intensivist (bc) and an internist (ag), icu patients who need a non invasive ventilation (niv) or tracheostomized elderly patients who have difficult weaning were admitted in a dedicated area in a medical department (md). this study desribes the results of one year of observation. in the last year, forty nine patients (age . ± . ; m f ) were transferred from icu to md. twenty three patients were treated with niv (age . ± . ; m f ), fourteen tracheostomized patients (age . ± . ; m f ) receive positive pressure ventilation because the difficult weaning in icu while twelve don't need any respiratory support. at the admission was performed a multidisciplinary plan and many specialists were involved (dietist,physiotherapist, pneumologist) and in invasively ventilated patients (ivp) was done a program of weaning. we follow all the patients until the discharge at home where someone need oxigenotheraphy, niv or mechanical ventilation. for the invasive ventilated patients we try to identificate significative differences beetween patients discharged at home and patients who died in hospital. data are given as mean ± sd and statistical analisis t test was performed results. patients underwent niv stay in hospital for . ± . days ( . ± . days in icu- . ± . in md) and ventilation was performed for the entire period in icu while for . ± . days in md. all the patients were discharged at home: twelve with niv, fourteen with oxygen. the lengh to stay in hospital for the ivp in wich weaning was failed in icu was . ± . days ( . ± . days in icu- . ± . in md). in md they continue the invasive ventilation for . ± . days. seven were weaned from ventilation after . ± . days, one was discharged at home with the ventilator while six died in hospital. patients who died were older ( . ± . vs. . ± . years-p . ), have more chronical diseases ( . ± . vs. . ± . -p . ), longer hospitalization ( . ± . vs. . ± . days-p . ), glascow coma scale ( . ± . vs. ). elderly patients often require a long period of recovery from acute ilness. in selected patients md could be a useful place where continue the treatment started in icu. in our study ivp who died had more chronical diseases and a more significative cognitive compromission. aim. documenting the qualitative and quantitative properties of administered and lost fluids is a common critical care monitoring practice. these nurse-registered fluid balances (fb) are used to optimize patient care and in clinical decision-making. this ''good clinical practice'' has also found application in research: recent studies reporting superior outcomes expressly refer to (negative) fb. we prospectively assessed the accuracy (review of all fluid balance charts and correction of arithmetic errors) and consistency (gold standard: body weight changes [bwc] registered with standardized measurements of body weight on admission and discharge [precision ± g]) of nurse-registered cumulative fb. total (tfb) and daily fb (dfb = total fb/los) were calculated. we analysed the unadjusted cumulative fb (unafb: without considering additional losses, i.e. perspiration/fever/liquid faeces) and the adjusted cumulative fb (adjfb: considering the above as proposed in the literature) in all patients (all) and in three subgroups (cardiaccerebral:card; septic:septic; others). exclusion criteria: lack of admission/discharge weight, incomplete fb data. we calculated l = kg. among patients admitted during the study period were eligible and analyzed. fb were inaccurate in cases ( %) (error range: - . to ? . l, mean arithmetic error ± sd: ? . ± . l, mean absolute error: . ± . l). the body weights at admission and discharge were . ± . kg and . ± . kg, with a bwc of . ± . kg ( . ± . kg per day). unatfb were . ± . l, unadfb . ± . l. adjtfb was . ± . l, adjdfb . ± . l. correlation (r ) and bland and altman was poor between bwc and unatfb ( . and - . ± . kg) and slightly better between bwc and adjtfb ( . and ? . ± . kg). the sd of the difference between bwc and fb per day of the icu stay was always [ kg. a multiple regression model including unatfb, duration of intubation, maximum temperature, estimation of liquid faeces, age and the calculated caloric deficit during the icu stay, only modestly improved correlation (r . ). compared to the two other groups, septic were significantly more severely ill, had a higher and longer fever, a longer los, larger bwc and cumulative fb, and presented larger differences between bwc and cumulative fb (poor correlation and bland and altman). though, consistency betwenn bwc and cumulative fb in card and other was still scarce. conversely, another multiple regression model (including only unatfb and the maximal temperature) in septic yielded an r of . . conclusion. fb are often inaccurate and they are not consistent with the gold standard of bwc. the correlation and the agreement with bwc of both adjtfb and unatfb are poor, with sd per icu day-stay[ kg or l. multiple regression models including several variables slightly improve correlation, yet remaining disappointing. consequently, clinical decisions should rather be based on other methods than fb. a prolonged hdu los was associated with a high sofa score for respiratory, hepatic and coagulation variables, preoperative ecg alterations, an increased urea and bmi and important bleeding. sofa score should be use in the first h to assess organ failure and a possible icu transfer for patients with an elevated score. evaluation of procalcitonin, neopterin, c-reactive protein, il- and il- as a diagnostic marker of infection in patients with febrile neutropenia financed by the following fellowships: rd / / from retics, fiss pi and fijc p-ef- reference(s). . bohoun c ( ) a brief history of procalcitonin biomarkers of sepsis: is procalcitonin ready for prime time? definitions for sepsis and organ failure and guidelines for the use of innovative therapies in sepsis procalcitonin assay in systemic inflammation, infection, and sepsis. clinical utility and limitations usefulness of procalcitonin for diagnosing complicating sepsis in patients with cardiogenic shock patients at our er, icu and wards of internal medicine and surgery meeting the ssc criteria were included. blood cultures were taken before administration of antibiotics, other cultures when appropriate. laboratory tests included wbc, crp, lactate, and pct. we categorized patients using bacteriological criteria group bacteriological proven infection (negative blood cultures but any other culture(s) positive) using chi square test there was no difference in survival, pct and findings on chest x-ray between the groups. with the mann whitney u test we found no differences in wbc, crp and lactate between survivors en nonsurvivors in a cohort of patients meeting the ssc criteria only % met bacteriological criteria for sepsis. wbc, crp, lactate and pct did not differ between patients with and without bacteriological proof of infection nor between survivors and non-survivors il rn and tnfr in the severity and outcome of community-acquired pneumonia to investigate whether polymorphisms within genes encoding for inflammatory or anti-inflammatory molecules are associated with susceptibility design. prospective observational, cohort study a cohort of , spanish caucasians with cap and subjects were genotyped for the following polymorphisms: tnfa - and - , lta ? sequential kaplan-meier survival analysis of tnfrsf b ? g/t polymorphism showed a protective role of the gt genotype. cox regression analysis adjusted for age, gender, hospital of origin and comorbidities showed that patients with gt genotypes had lower mortality rates compared with those patients with gg or tt genotypes (p = . our study does not support a role for the studied polymorphisms of the tnfa, lta, il and il rn genes in the susceptibility or outcome of cap. a protective role of heterozygousity for the functionally relevant tnfrsf b ? variability in genes involved in the inflammatory response in patients with community-acquired pneumonia to investigate whether polymorphisms within genes encoding for inflammatory or anti-inflammatory molecules are associated with susceptibility design. prospective observational, cohort study a cohort of , spanish caucasians with cap and , controlsinterventions: subjects were genotyped for the following polymorphisms: tnfa - and - , lta ? sequential kaplan-meier survival analysis of tnfrsf b ? tt versus gg/gt genotypes suggested a detrimental role of the tt genotype. longrank c tests at and days yielded p = . and . , respectively; cox regression for -and -day survival, adjusted for age, gender ghent university hospital, intensive care we aimed to describe the incidence and characteristics of healthcare-associated pneumonia (hcap) diagnosed at the emergency department of our university hospital compared to cap, hcap occurs in more debilitated or at-risk patients, is more frequently caused by nosocomial pathogens, and has worse outcome. nursing-home pneumonia is included within the definition of hcap but could have characteristics different from the other categories of hcap ) with a diagnosis of 'pneumonia'. episodes were categorized in cap and hcap according to the definition of the american thoracic society/infectious diseases society of america. within hcap, distinction was made between nursing-home pneumonia (nhp) and non-nhp hcap. severity of the pneumonia was assessed using curb- during the study period, episodes of pneumonia were diagnosed in patients; episodes ( %) were categorized as cap, and ( %) as hcap. within hcap, ( %), respectively ( %) episodes were further classified as respectively nhp and non-nhp hcap. median age of the patients was years ( - ) and % of patients were male median curb- pneumonia severity score in patients with cap and hcap was ( - ) and ( - ) respectively (p = . ); in nhp and non-nhp hcap, median curb in bivariate logistic regression analysis, both increasing curb- (or . , ci . - . ) and categorization as nhp (or . , ci . - half of the episodes of pneumonia diagnosed at our emergency department could be classified as hcap. severity of the pneumonia was higher in patients with hcap as compared to cap. categorization as nhp, but not as non-nhp hcap was independently associated with hospital mortality after adjustment for severity of the pneumonia medical intensive care unit community-acquired pneumonia (cap) is the leading cause of infectious death, severe sepsis and the seventh leading cause of overall death. severe cap (scap) is defined as need of aggresive intensive care unit (icu) management due to shock to describe the episodes of severe community-acquired pneumonia (scap) in a multicentric european study and to assess management practices and outcome of scap patients admitted to icu observational, prospective, multi-centre study conducted in icus of consecutive patients requiring invasive mechanical ventilation for an admission diagnosis of pneumonia or mv for [ h were recruited in each icu. statistic analysis was performed using spss years p \ . ) and presented a higher saps ii score at admission ( . sd . vs. . sd . p \ . ). patients were treated with monotherapy in . % and combination therapy . %. empirical antibiotic treatment was in accordance with idsa guidelines in ( . %) patients. combination was prescribed with macrolides in . % and quinolones in . %. in patients receiving combination therapy in accordance with idsa guidelines, a cox regression analysis adjusted by saps ii and age identified that macrolides use was associated with lower icu mortality when compared to the use of quinolones in patients with severe community acquired pneumonia who had therapy in accordance with the idsa guidelines, only combination therapy with macrolides was associated with better outcomes the eu-vap/cap project was endorsed by eccrn hospital de mataró, critical care, mataró, spain, hospital universitario germans trias i pujol, microbiology, badalona, spain, hospital de mataró, microbiology, mataró, spain, hospital universitario germans trias i pujol this leads to improved survival in a sterile acute lung injury model in wild-type mice, compared to mmp- knockout mice. we recently showed that mmp- deficient mice had better survival in a cecal ligation and puncture (clp) sepsis model than wild-type mice. in humans, functional genetic variations of the mmp- gene exist, but their relation to outcomes of severe infections, such as cap, is unknown. objectives. we hypothesized that functional human single nucleotide polymorphisms (snps) leading to increased mmp- levels are associated with worse survival and higher incidence of severe sepsis in patients with cap. methods. we examined data from genims, a multicenter prospective cohort study of patients with cap and analyzed potentially functional snps (rs , rs , rs ) in the mmp- gene in caucasians by polymerase chain reaction (pcr) the overall incidence of severe sepsis was . % (n = ), and . % of patients (n = ) died within days. the rs genotype distribution was significantly associated with -day mortality by armitage's trend test failure plot for rs conclusions. the non-synonymous rs snp is associated with -day survival in patients with cap. our findings suggests a trend towards a transcriptome analysis of ventilator associated pneumonia in trauma patients the sepsichip project the diagnosis of acute infection in the critically ill remains a challenge. transcriptional profiles (tp) of circulating leukocyte can be used to monitor the host response to infection. ventilator-associated pneumonia (vap) is a frequent complication of major trauma, raising morbidity and mortality data files were analyzed with r and bioconductor. unsupervised analysis was conducted using the dbf-mcl algorithm. supervised analysis was conducted using the significance analysis of microarray algorithm, using siggenes library. all statistical analysis used corrections for multiple comparisons. results. vap occurred in of the trauma patients ( . %). one hundred and fifteen samples were hybridized on husg k microarray ( sirs and sepsis samples for the patients who developed a vap, and sirs samples for those who did not). whereas clinical parameters (iss, chest trauma) discriminated trauma patients with or without vap, admission samples transcriptome analysis did not lead to the identification of prognostic markers. analysis of paired samples of the patients who developed a vap identified a transcriptional signature. these genes were involved in transcriptional regulation, cell survival, hemostasis and endocrine regulation. conclusions. by comparing whole blood admission samples, we were not able to identify transcriptional prognosis markers in trauma patients who did or did not develop vap. however, using vap as a model of sepsis in trauma patients, we identified a set of genes which may serve to diagnose vap in trauma patients to investigate the correlation between transfusion practice and the development of ventilator associated pneumonia (vap) in patients with traumatic brain injury (tbi) we analyzed which tbi individuals developed vap in regard to the transfusion practice and if the number of transfused prbcs increases the risk of pneumonia development. we counted the total amount of prbcs units received by each patient during icu stay, as well as those given before vap development. patient's data included: demographics, apache ii, iss, gcs, vap characteristics, duration of mechanical ventilation (mv), length of stay (los) and outcome. cpis and mods were calculated on the day of vap detection.statistical evaluation was performed using univariate and multivariate logistic regression, students t-test and pearson's chi square test %) tbi patients who developed vap received on average units of prbcs during icu stay, compared with non vap individuals who were transfused on average with two units of prbcs (p \ . ). vap patients received on average four units prbcs before vap development. after correcting for age, apache ii, gcs and iss, transfusion was independently associated with vap. the odds ratio for vap aspiration pneumonia (ap) in comatose patients (pts): clinical and microbiological findings ap is a common complication in comatose pts. we aimed to update data on their incidence standard guidelines were used for diagnosis and treatment of ap. daily chest x-ray were retrospectively reviewed. ap was diagnosed if pts met following criteria: persistent radiographic infiltrate within days following ti, and at least two of the following: purulent sputum, fever/hypothermia duration of mv was days ( - ) and length of stay in icu days ( - ) on the day of ap diagnosis, main pts characteristics were: saps ii %) received empirical antimicrobial therapy. main empirical antibiotics were coamoxiclav ( %) and third generation cephalosporin ( %) ) and mv duration ( vs. days, p \ . ), even considering only non-cardiac arrest pts. gcs h after ti and ap were associated with a [ days duration of mv in multivariate analysis ap was associated with higher overall -day mortality in univariate analysis ( vs. %, p = . ) but no longer in multivariate analysis mortality in the icu was . % with a corresponding hospital mortality of . %. a microbiological documentation was obtained in . % of the patients, with streptococus pneumonia being the most frequent ( % of the isolates). the cap was classified as localized in . %, unilateral mean (±sd) saps piro score was . ± . points, with a corresponding predicted mortality of . ± . % (standardized mortality ratio . ). the aroc was . ( . - . ). the value of the hosmer-lemeshow Ĉ test was saps piro presented a discrimination similar to the originally described. however, it significantly overestimated mortality sepsis mortality prediction based on predisposition, infection and response the piro-cap score was proposed earlier this year to stratify patients with severe community acquired pneumonia (cap) to evaluate piro-cap score in patients with severe cap, over a period of years we analysed data prospectively registered in an informatic data base, which contains information referring to all patients admitted in this unit survival curves were built as proposed by the original authors. outcome was evaluated at icu discharge overall, it was a severe population: . % of the patients presented at least one chronic disease, saps : . ± . points (predicted mortality . % ± . ), length of stay in the icu . ± . , icu mortality was . %. a microbiological documentation was obtained in . % of the patients, with s. pneumoniae being the most frequent ( % of the isolates). the cap was classified as localized in . %, unilateral icu mortality was piro-cap presented an excellent discrimination. however, mortality rates were greater than the ones described by the original authors in all groups (except group ), with the system significantly under-predicting mortality. consequently, we recommend caution in their widespread use. cumulative survival reference(s). clinical and biological assessments in icu patients delirium is a life-threatening, acute organ dysfunction with an incidence of % in uk mechanically haloperidol is recommended as treatment [ ] despite limited evidence base. objective. a national postal survey of consultant members of the uk intensive care society (ics) was performed to determine the current management of delirium in the ) drug treatment of hypoactive and hyperactive delirium as described by two clinical vignettes and ( ) level of agreement with five statements regarding delirium. results. six hundred and eighty one replies were received from , questionnaires senta response rate of %. twenty five percent of respondents routinely screen for delirium. only % use a validated screening tool, most ( %) of whom use the confusion assessment method, icu. hyperactive delirium is treated pharmacologically by %, the majority using haloperidol. hypoactive delirium is treated pharmacologically by %, with haloperidol again the most common treatment ( %) grant acknowledgement. intensive care foundation, intensive care society a practical algorithm to diagnose delirium in critical care-validity and reliability delirium occurs in up to % of critical care patients [ ], but often remains undiagnosed because standardized delirium monitoring is often dismissed as being too time-consuming or too complicated [ ]. the 'harvard flowsheet', derived from the 'confusion assessment method for intensive care unit' (cam-icu) [ ], provides a practical, algorithm-type handling advice to assess the four dsm-iv delirium criteria in a standardized fashion in intubated patients. it mostly allows for truncation of assessments to save time after approval from our institution's ethics committee, patients of a -bed surgical icu-department were screened in five sessions for delirium ( ) by a psychiatrist as the reference rater using the dsm-iv delirium criteria, and ( ) by two 'harvard flowsheet'-investigators, each unaware of other's ratings. motoric delirium subtypes were classified according to the richmond agitation sedation scale (rass) [ ], which was rated for the feature ('altered level of consciousness') of the 'harvard flowsheet'. patients were deemed as having hypoactive delirium if they were dsm positive by the reference rater and had rass - to , or having hyperactive delirium if their rass was between ? and ? . for interrater reliability the median time to complete the 'harvard flowsheet' in delirious patients was s (iqr, - s) vs. s ( - s)] in non-delirious patients. conclusions. the 'harvard flowsheet' has high sensitivity, high specificity and very high interrater reliability. false-negative ratings can occur infrequently and likely reflect the fluctuating course of delirium with intermittent lucid states a decrease of the overall cost of sedation and of sedation/day of mv followed protocol implementation and has been pursued each year: sedation cost which was greater than €/day in / has decreased to less than €/day in acute renal failure in critically ill patients: a multinational, multicenter study to evaluate current transfusion practice and the association between the age of red blood cells (rbcs) and outcome in critically ill patients design. prospective, multicenter observational study patients: critically ill adult patients receiving at least one unit of rbcs %)] revealed an unadjusted absolute reduction rate (arr) in mortality of % ( % ci - %). after adjustment for disease severity, patient age, other product transfusions, number of transfusions, pre-transfusion haemoglobin concentration, pre-icu transfusions, and cardiac surgery the odds ratio (or) for hospital mortality in critically ill patients in australia and new zealand transfusion of rbcs is delivered within current international recommendations. however, within such a practice patients enrolled included men and women, with mean age of ± years. there were ( %) liver transplantation, ( . %) renal transplantation and ( . %) pulmonary transplantation and ( . %) renal-pancreas transplantation. the days mortality for liver, renal, pulmonary and renal-pancreas was: ( . %), ( . %), ( . %) and ( . %). the mean saps score for liver, renal ic apache ii auc . % . - . , ic % . - . . conclusions. in these study, no differences were observed comparing saps and apache ii in the mortality prediction from liver, renal and pulmonary transplantation current opinion in critical care introduction of a rapid response team: why we are glad we met dew ma and members of the medical emergency response improvement team (merit) committee ( ) mature rapid response system and potentially avoidable cardiopulmonary arrest in hospital the effect of a rapid response team implementation in a private hospital adult patients often exhibit physiological deterioration hours before cardiopulmonary arrest to determine the effect of a rapid response team on the rate of in-hospital cardiac arrests, total and unplanned intensive care unit admissions, and icu and hospital mortality before and after implementation of a rapid response team standard criteria were used to activate the rrt and included acute changes in the patient's mental status, respiratory rate, heart rate, oxygenation, or blood pressure and hypoxia, chest pain, or worry from clinical staff. we measured: admitting diagnosis after rrt were a total the activations. the most common reasons for rrt activation were ventilator dysfunction ( %), cardiac changes ( %) and acute neurological changes ( %). % were transferred to icu and the main reasons were cardiac changes ( %), ventilatory dysfunction ( %) and acute neurological changes ( %) the rrt implementation was associated with decreases in rates of inhospital cardiac arrest, but was not associated with reductions in hospital or icu mortality the . lives campaign: setting a goal and a deadline for improving health care quality gender difference in critical care response team activations impact on outcome saudi arabia, king saud bin abdulaziz university for health sciences, riyadh, saudi arabia introduction. gender-related differences in outcome of ccrt intervention has been documented in the literature indicating that more men were admitted to the intensive care unit our center is the only center in the kingdome of saudi arabia which implements an intensivest-lead ccrt services h/ . the team is leaded by in house board certified in critical care medicine. ccrt services started in chest pain unit-viable option when risk of cardiac etiology is modest. multitudinous patient population brought together for structured survey and care at appropriate level what alters physicians' decisions to admit to the coronary care unit? m. camara , g. silva , s. silva , c. dias , j. nóbrega , e. maul hospital central do funchal, funchal, portugal introduction. procalcitonin (pct) and c reactive protein (crp) are markers of sepsis and the levels correlate with the severity of illness.aims. to evaluate the relationship of procalcitonin (pct) and c-reactive protein (crp) kinetics within the first days of sepsis with the appropriateness of antibiotic therapy and the outcome. a prospective cohort study, over months including patients with documented sepsis in our -bed intensive care unit. crp and pct were simultaneously measured four times (m -m ) during the first days of antimicrobial treatment. the pct and crp time course were analysed according to the appropriateness of the empirical antibiotic therapy as well as according to the patient outcome.results. between january and march of , patients were admitted to the icu. patients presented with sepsis on admission or during their stay. the most common infection site was the lung ( . %) followed by primary bacteraemia ( . %). gram-negative and gram-positive bacteria were isolated in the following proportion: . and . %, respectively. enterobacter, acinetobacter and escherichia were the most frequently isolated ( . % each). gram-positive sepsis was mainly caused by haemophylus influenzae ( . %). sepsis was polimicrobial in . % of cases. . % of the patients were given inappropriate antibiotics. the proportion of gram-negative bacteria isolated was significantly higher in patients who did not receive appropriate antibiotics. the magnitude of the pct and crp elevation was not associated with the appropriateness of antibiotic therapy. logistical regression analysis showed that infection without agent was an independent predictor of inappropriateness of antibiotic therapy.age, saps ii, apache ii and sofa were not associated with an unsuccessful treatment. regarding the absolute value of crp and pct there was no significant difference between successful or unsuccessful. multivariate analysis showed that dpct was not associated with antibiotic appropriateness and mortality.conclusions. although the sample is small, our study suggests that crp and pct kinetics are not associated with the appropriateness of antibiotic therapy and outcome. introduction. patients with hematological malignancy who need advanced life support in the icu because of a life-threatening complication may have a poor prognosis. that's why it is necessary to identify clinical, analytical and biological factors that can help doctors with the decision to admit these patients into the icu.objective. the aim of this study was to assess the utility of procalcitonin serum levels (pct) in predicting the outcome of patients with hematological malignancies admitted to the icu. a total of patients with hematological malignancy were admitted to the icu from january until march . epidemiological data were collected before admission, and patients were followed up clinically and analytically during icu stay. serum samples were collected from icu admission until a maximum period of days. pct values were measured by an immunofluorescent assay based on trace (time-resolved amplified cryptate emission) technology (kryptor pct, brahms ag, hennigsdorf, germany). mean age: (sd ); men/ women. among the patients included, hematological diseases were: non-hodgkin lymphoma ( patients), acute myeloblastic leukemia ( ), acute lymphoblastic leukemia ( ) , multiple myeloma ( ), chronic lymphoproliferative disorder ( ) , others ( ). twenty patients ( %) had previously received hematopoietic stem cell transplantation. thirty patients ( %) presented neutropenia at the moment of icu admission. the main causes for icu admission were respiratory failure in patients ( %) and septic shock in ( %). pct levels were not significantly higher in those patients that required mechanical ventilation. pct levels were significantly higher (p = , ) in those patients admitted because of septic shock. pct levels were lower in days , and in the patients who survived with respect to those who died: day : . ng/ml (sd . ) versus . (sd ); day : . (sd . ) versus . (sd . ) ; day : . (sd . ) versus . (sd . ). the differences were significant in days (p = . ) and (p = , ). there was a trend to have higher pct levels in those patients who had microbiologically documented infection respect to the rest; day : . ng/ml (sd . ) versus . (sd: . ); day : . (sd . ) versus . (sd . ) and day : . (sd . ) versus . (sd . ) .conclusions. serum pct levels are higher in patients with septic shock. serum pct measurement might be useful for predicting mortality in patients with hematological malignancy who require advanced life support. introduction. sepsis is a major cause of mortality in the intensive care unit (icu). efforts have been made to reduce the time needed to diagnose sepsis in order to reduce mortality from sepsis-related multiple organ dysfunction. procalcitonin (pct) has been reported elevated levels at the onset of bacterial infections and seemingly correlated to severity of infection. several clinical trials have detected a high pct level in patients with evidence of systemic bacterial infections, whereas relatively low pct levels occur in patients with only localized bacterial infections.objective. the aim of the present study was to assessed the ability of pct through sensitivity, specificity, positive and negative predictive value (ppv, npv) in patients with suspected sepsis, septic shock, inflammatory systemic response syndrome (sirs) and compared it with variables like crp, mortality, band%, renal failure, active cancer and an isolated bacterial cultures. finally we wanted to evaluate if exists a no infectious correlation in patients who received blood transfusions. we conducted an observational study including all patients admitted to the multidisciplinary icu of the abc medical center (tertiary reference hospital) to whom requested pct at admission in the suspect of sepsis and we followed their outcomes. total populations was patients (p). % were females and % were males. median age was years. of the total of pct sample % were positive and % were negative. the sensitivity and specificity in septic patients were and %. ppv and npv were and %, respectively. we did not found any statistical difference between positive value of pct and sepsis, septic shock, sirs, mortality, crp, band%, acute renal failure, acute lung injury, ards (acute respiratory distress syndrome), blood transfusions and active cancer. the mortality in the populations was %.conclusions. the pct has a wide range of diagnostic in the septic patients. in our study the rate of false positive was % and limited the use for sepsis diagnosis. we suggest that the better utility is for outcome biomarkers more than diagnosis biomarkers of sepsis. y. jin , c. guolong , iit study group of zhejiang province in china zhejiang hospital, hangzhou, china introduction. the use of intensive insulin therapy (iit) in severe sepsis and septic shock has been shown to decrease morbidity and mortality rates significantly when given to high risk surgical patients.objectives. the aim of this study was to assess the efficacy of iit in severe sepsis and septic shock patients in intensive care unit.methods. this is a muticentre, prospective, randomized and controlled study. we randomly assigned patients who admission to icu with severe sepsis or septic shock into three groups: a group (target range for blood glucose is - mg/dl); b group (target range for blood glucose is - mg/dl); c group (target range for blood glucose is - mg/dl as a control). primary end point ( -day mortality for any cause) and secondary end points (icu stay days, mv duration, apacheii scores and mods scores) were obtained serially for days and compared between the three groups. of the enrolled patients, were randomly assigned to group a and to group b and to group c; there were no significant differences between the groups with respect to base-line characteristics. -day mortality was percent in the group a and . percent in the group b assigned to iit, as compared with . percent in the group c assigned to conventional therapy (p = . ).during the interval from first hour to -day stay in icu, the patients assigned to group a and group b had a significantly lower apache ii scores( . ± . and . ± . vs. . ± . , p = . ) and mods scores( . ± . and . ± . vs. . ± . , p = . ) than those assigned to conventional therapy, there were no differences in icu stay days( . ± . , . ± . , . ± . , p = . )and mv duration( . ± . , . ± . , . ± . , p = . ) between the three groups. compared with the conventional therapy group, the group a had a higher rate of severe hypoglycemia [blood glucose level b mg/dl ( . mmol/l); . . vs. . %; p \ . ]. intensive insulin therapy provides significant benefits with respect to outcome and scores in patients with severe sepsis and septic shock in icu, on the other hand, intensive insulin therapy brings a higher rate of severe hypoglycemia. to determine the prognosis factors in elderly patients (c years) with severe sepsis admitted to an intensive care unit (icu).method. an observational, prospective and multicenter study was realized. it includes all the patients of the database edusepsis study (adults with severe sepsis admitted to spanish medical-surgical icus). the clinical and demographic characteristics of all patients including age, sex, origin of the infection, location of the patient at the moment of diagnosis of sepsis, apache ii modified score (apache ii score age excluded), number of organic failures, initial therapeutic strategy (measures of resucitación and measures of treatment), icu length of stay and hospital mortality were registered. the patient were classified in young cohort (\ years) and elderly cohort (c years). elderly cohort patients were also classified in young-old patients ( - years) and very-old patients (c years). descriptive comparative study of both cohorts was realized and multivariate logistic regression for the two subgroups of elderly patients was performed to study the risk factors of hospital mortality. a total of , patients wer enrolled ( . ± . years, apache ii modified score of . ± . , . ± . organic failures, hospital mortality . %). the elderly cohort (n = ; . %) presented a lower apache ii modified score ( . ± . vs. . ± . , p . ), higher abdominal infection as origin of the sepsis ( . vs. . %, p \ . ), higher nosocomial infection ( . vs. . %, p . ) and a lower application of measures at initial treatment ( . vs. . %, p . ) than the young cohort. there were not significant differences in the number of organic failures and days of stay in uci between both cohorts. the apache ii modified score (or . ; % ic . - . ; p \ . ), the nosocomial infection (or . ; % ic . - . ; p \ . ), the thrombocytopenia (or . ; % ic . - . ; p . ) and the acute renal failure (or . ; % ic . - . ; p . ) were associated independently to mortality in the subgroup of young-old patients. in the very-old patients only the apache ii modified score (or . ; % ic . - . ; p \ . ) was independently associated with higher mortality and in this population subgroup the application of measures of initial resuscitation was a protective factor (or . ; % ic . - . ; p . ).conclusions. the elderly patients (c years) admitted in the icu whith severe sepsis have higher mortality, more abdominal infections as origin of the sepsis and fewer application of measures of initial treatment than the young patients (\ years). nevertheless, in the subgroup of very-old patients (c years) the aggressive initial treatment decreases the mortality. objectives. the aims of this study were to determine the crude and related to bacteremia mortality rates in icu patients with bacteremia who receive appropriate empirical antibiotic therapy and to describe the factors associated to mortality in this appropriated treated patients material and methods. during a twelve years and a half period, from to , icu-patients with clinically significant bacteremia were prospectively evaluated. for purposes of this investigation, appropriate empirical antimicrobial treatment of a bloodstream infection (aeat) was defined as the microbiological documentation of infection that was effectively treated based on its antibiotic susceptibility at the time the causative microorganism were suspected. clinical and microbiological variables were recorded. logistic regression analysis was performed to determine the risk factors associated to global and associated to infection mortality. results. among icu-bacteremic patients, aeat was applied in patients ( . %). apache ii and sofa score were . ± . and . ± . , respectively and the incidence of septic shock was . % in this aeat patients. global and associated to infection mortality rates were . and . %, respectively in aeat patients. logistic regression analysis confirmed copd (or . ; % ci: . - . ) and age (or . ; % ci: . - ) as factors independently associated to global mortality and diabetes mellitus (or . ; % ci: . - . ) presentation as septic shock (or . ; % ci: . - . ) and serum levels of albumin (or . ; % ci: . - . ) as a protective factors for global mortality whereas factors as nosocomial origin (or . ; % ci: . - . ) and again serum levels of albumin (or . ; % ci: . - . ) were considered protective for related mortality to bacteremia conclusions. mortality rates remains excessively high in aeat bacteremic-icu patients. different factors were identified as predictive factors for global and associated to mortality in aeat patients. only serum levels of albumin seems to be an independent protective factor for both global and associated to infection mortalities. introduction. severe sepsis is hallmarked by organ hypoperfusion or dysfunction. the transition from severe sepsis to septic shock carries with it an increase not only in morbidity but also in mortality [ , ] . objectives. the aim of the study was to demonstrate the effect of shock at admission in sepsis comparing severe sepsis and septic shock admission diagnoses.methods. single center retrospective study in a bed mixed icu of a tertiary university hospital. during a -years period of study patients were unplanned admitted in the unit: the median was age of ( - ), the males were . % and the mean of sapsii was ± . we randomly select two groups: severe sepsis ( patients) or septic shock ( patients) at admission. statistical analysis of variables: v , mann-whitney test, unpaired t student test, cox regression. no statistical significant differences were found about age and sex between groups. about the origin of infection no statistical significant differences were found between groups, meanwhile the diagnosis respiratory infection appears to be more frequent in the severe sepsis group ( . vs. . %, p . ). the proportion of post-operative admissions (in surgical related conditions) was not different between groups. the sapss ii and sofa at h were higher in the septic shock group [ ( - ) vs. ( - ) , p \ . ]; ( - ) vs. ( ) ( ) ( ) ( ) ( ) , p \ , , respectively]. sofa at discharge appears to be higher in the shock septic group (excluding deaths) [ ( - ) vs. ( - ) (p . )]. the mortality and length of stay (excluding deaths) was higher in the shock septic [ . vs. . % (p \ . ); ( - ) vs. ( - ) (. ), respectively]. the ventilator associated pneumonia was not significantly different between groups. the probability of discharge, across an initial period of days, was lower in the septic shock group [hazard ratio . ( % ci: . - . )], mainly between the th and th days, as shown in the kaplan-meier plot (see graph ) .admission diagnosis: probability of discharge conclusions. septic shock at admission patients had a poorer outcome. the difference in the probability of discharge between groups was higher when mechanical ventilation related events are likely to occur [ , ] . we emphasize the importance of the institution of early goal-directed therapy in the wards and emergency departments prior to admission in an intensive care unit [ , ] .introduction. it is not clear whether patients with community acquired severe sepsis (cass) or hospital acquired severe sepsis (hass) have a same presentation. objectives. to evaluate the characteristics of a severe sepsis (ss) population admitted through the er (cass) and those coming from the ward (hass). all patients were treated by the same team of intensivists and er doctors in a shock room, so we could minimise the differences due to management. methods. all adult patients admitted to the medical icu were eligible if they met the criteria for ss. we collected demographic characteristics, apache ii and sofa score, comorbidities and immuno-compromised conditions. scvo or svo (if possible), lactate concentrations. the milestones of the surviving sepsis campaign (ssc) were measured regularly during the first h of treatment. the data collection went on in the icu stay too. treatment for septic shock was conformed to the recommendations of ssc. results. we enrolled pts with ss, including with cass and with hass. there was no difference in demographic features and comorbidities, including immuno-compromised conditions, haematological malignancy and chronic respiratory diseases .there were no significant differences in hemodynamic variables or indices of tissue perfusion like scvo (or svo ) and blood lactate levels, or in amounts of fluids infused or needs of vasopressor agents. the need for mechanical ventilation (mv) after the first has greater for hass than for cass patients, but during the icu stay the need for mv was the same for both groups; similarly, during the icu stay there was no difference in the need for extracorporeal renal support or need for adrenergic agents. at the beginning the scvo was around % for the entire population. after the first h both groups reached the target of %. at the admission % of patients had a scvo less than % ( . % for hass patients and . % for cass, without any difference between groups) and % of patients had a scvo higher than %. the mean svo for both groups was higher than % already at the beginning of the observational period. conclusion. only a half of pts with ss or sho had fever. the presence of fever is often associated with a positive microbiological diagnosis, but better prognosis. while hypothermia was often viewed in severe ill pts and was associated with a worse prognosis. to investigate the possible differences in characteristics and outcome between early and late-onset severe sepsis in surgical intensive care unit (icu) patients. we conducted a retrospective analysis of prospectively collected data from all adult patients ([ years) admitted to our -bed surgical icu between st march and th july .results. of , patients admitted to our icu during the study period, patients ( . %) had severe sepsis; ( . %) had early-onset and ( . %) late-onset severe sepsis. respiratory infections ( . vs. . %, p = . ) and infections of unknown origin ( . vs. . %, p = . ) were more frequently recorded in patients with late-onset than those with early-onset severe sepsis, whereas abdominal infections were more frequent in early-onset than in late-onset severe sepsis ( . vs. . %, p = . ). gram-positive infections were more frequent in late-onset than in early-onset severe sepsis ( . vs. . %, p = . ). the time of onset of severe sepsis was not independently associated with an increased risk of in-hospital death (early vs. late: or . % ci . - . , p = . ).conclusions. respiratory infections and infections of unknown origin were more frequently recorded in patients with late-onset than in those with early-onset severe sepsis, whereas abdominal infections were more frequent in early-onset than in late-onset severe sepsis. the time of onset of severe sepsis has no impact on mortality. objectives. to describe the causes, microbia spectrum, and prognosis of pregnancyassociated sepsis treated in icu in france along the last years. we conducted a retrospective study in a medico-surgical icu of beds in a non-teaching hospital in france where a high risk maternity unit was opened in . patients admitted between and for sepsis occurring during pregnancy or the post-partum period were included. the patients were excluded if the sepsis was due to a nosocomial icuacquired infection. charts were reviewed to collect data on sources of infection, microbia, maternal and fetal prognosis. data are shown as median (extremes) or percentage. data before and after were compared using non parametric tests.results. patients were admitted for pregnancy-associated sepsis ( % of total pregnancy-related icu admissions). included patients had the following characteristics on admission: age: ( - ) years, gravidity: ( - ) pregnancies, parity: ( - ) children, , . vasopressors, mechanical ventilation, and hemodialysis were required in respectively , , and % of cases.characteristics of infections are shown in table . microbiological data about bacterial infections, and specially infections of pelvic origin (chorioamniotitis, endometritis, septic thrombophlebitis), are shown in table .all urinary infections were due to e. coli. lung infections were most often documented clinically but not microbiologically.maternal mortality rate was % ( deaths before and deaths after ). for those infections that occurred in the pre-partum period, fetal mortality was %. after exclusion of fetal deaths that had occurred before icu admission, pregnancy was interrupted during icu stay in % of cases, resulting in fetal mortality of %.conclusions. despite of disappearance of post-abortum sepsis in france, sepsis remains a significant cause of icu admission during or after pregnancy, and a significant cause of maternal and fetal mortality.grant acknowledgement. none. introduction. the glasgow coma scale (gcs), universally used for assessing comatose states, has the advantage of ease of use making it accessible to all levels of clinical competence. there are, however, significant drawbacks. its relative subjectivity in the interpretation of verbal and eye responses and its mesencephalic limit in the rostro-caudal assessment of brain vitality. these two drawbacks limit its use particularly in icu intubated patients. we propose a new score, the sousse coma score (scs) that overcome the eye and verbal responses and explore the brain vitality up to brain death. the score ranges from (normal consciousness) to (brain death). the performance of this score was compared to a modified gcs (gcsm; gcs reduced to its only eye and motor components) and four score. our study interested prospective and consecutive comatose patients who were admitted to a medical icu, intubated and under ventilatory support. the level of consciousness was assessed at admission by physicians of different levels of competence. the inter-observer reliability was assessed by measuring the spearman correlation between the responses of different observers to the scs, gcsm, four score and their respective components. the prognostic predictive value of the three studied scores was assessed by the analysis of possible correlation with mortality and the roc curves. inter-observer reliability was excellent (spearman rho [ . ) for the three studied scores, but with better performance for the scs ( . , p \ . ) compared to the gcs ( . , p \ . ) and four ( . , p \ . ). the level of overall inter-observer reliability for gcs and four was paradoxically higher than that of their respective motor components. this is probably the result of a summation effect of their respective components.regarding the relationship between mortality and the studied coma scores, there was for all three scores a threshold below which mortality was %; / for scs, / for the gcs and / for the four. beyond this threshold, only the scs provides a highly significant correlation with the risk of death (spearman = - . , p = . ). a similar correlation was observed with the motor components of the gcs and four. a better correlation was found between mortality and the scs. the area under the roc curve, however, was poor for all three scores. in evaluating the minimally consciousness states and from eight value of scs, the gcs and four scores provided a wider and more subtle level of consciousness assessment. the scs provides a better inter-observer reliability and a better prediction of death while being easier to achieve. the apparently good inter-observer reliability of gcsm and four was simply the result of summation effect. however, scs was not very sensitive in detecting variations of the minimal consciousness states. the results of our study should be confirmed in larger multicenter studies for the external validation. introduction. the use of a sedation assessment scale and a sedation goal is recommended in critically ill adults [ ] . several studies have found a reduction of icu length of stay (los), of mechanical ventilation (mv) duration, and of cost [ , ] . but, durability and efficiency of such procedures over time have not been evaluated. a -bed medical icu in a university hospital. a sedation and analgesia protocol has been implemented since - . the sedation goal is prescribed each day by the intensivist; the ramsay sedation score is evaluated by the nurses every h and doses of drugs are adapted accordingly. we conducted an annual survey from to to evaluate the quantity of sedative drugs utilized, the impact on icu los and on mv duration, and cost of sedation; both the medical and nurse staff were regularly informed and the protocol was modified if necessary. u. guenther , j. weykam , u. andorfer , t. muders , h. wrigge , c. putensen university of bonn, anaesthesiology and intensive care, bonn, germany background. acute brain dysfunction (delirium and coma) is reported to occur in up to % [ ] , and to be associated with longer mechanical ventilation and stay in the icu, and increased -months mortality rates up to % [ ] . such outcome data, to the best of our knowledge, are predominantly given on medical patients with delirium incidences and mortalities much higher than we expected in surgical patients. this study assessed incidence and impact of acute brain dysfunction on length of stay on the ventilator and in the icu, and mortality in cardiac surgery patients. after approval from our local ethics committee, every patient admitted to our cardiac surgery -beds icu from october through november was daily monitored for delirium with the ''confusion assessment method for the intensive care unit (cam-icu)'' [ ] , level of consciousness was assessed with the richmond-agitation-sedation scale (rass) [ ] . acute brain dysfunction was diagnosed if patients were comatose without sedative medication or delirious. patients were contacted months later to obtain information about their further clinical course.results. patients were eligible for analysis [male , female , age, mean (iqr), ( - ) years]. % had acute brain dysfunction while in icu, these had significantly higher apache-scores on admission, higher tiss-and saps-scores, were longer mechanically ventilated [ ( - ) vs. ( - ) days, p \ . , mann-whitney test) and had longer stay in icu [ ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) vs. ( - ) days, p = . , mann-whitney test). patients were lost to follow-up; -months mortality in patients with acute brain dysfunction in icu was vs. % (p = . , log-rank test).discussion. incidence of acute brain dysfunction and mortality found here in cardiac surgery patients are lower compared to reports on medical patients. even though, duration of mechanical ventilation, length of stay in icu, and -months mortality were increased in patients with acute brain dysfunction in our icu.conclusion. these data emphasize the need for routinely monitoring of consciousness and delirium and to develop strategies to reduce incidence of acute brain dysfunction. introduction. the incidence of obesity is increasing globally, with billion overweight people (body mass index (bmi) [ kg/m ), and million of them obese [ ] (bmi [ kg/m ). the number of obese individuals presenting to critical care is likely to increase, but data on effect of obesity on outcome is conflicting [ ] . there is a general perception that obese patients are likely to have a higher incidence of adverse outcome in critical care. to investigate the effect of bmi on length of stay and mortality in patients admitted to critical care in city hospital, birmingham, uk.methods. an observational study was performed over a month period from february-april . pre-morbid data on bmi was collected from medical records and direct questioning of patients. apache ii score at h, critical care length of stay (los) and survival data to hospital discharge were collected. the figures were compared against their predicted mortality. readmissions, patients with a los \ h, patients \ years old and those without complete apache ii data were excluded. a total of patients were admitted. met the exclusion criteria, bmi data was unavailable for patients and apache data was unavailable for patients. patients were included in the final analysis. . % were female and . % were male. % of patients were above their ideal body weight, and % were obese. the obese cohort had a mean apache ii score of . with a mean los of . days and a mean hospital mortality rate of . %. the corresponding figures for the non-obese group (bmi \ ) was . , . days and . % (table ) . obese patients had reduced hospital mortality in comparison to predicted rates from apache ii scores. statistically, there was no increase in mortality of obese patients. the los and ventilated days were also comparable to the nonobese patients. introduction. managing glucose levels in critically ill hospitalised patients has been shown to play a role in improving clinical outcomes. as a result glycaemic control protocols are widely used in critical care settings and require rapid and frequent testing of patient glucose levels. poc glucose meters have migrated from ambulatory testing into hospital.there is increasing recognition that the clinical accuracy of nearly all commonly used glucose meters are affected by components or substances often present in the blood matrix of critical care patients giving rise to an increase risk of adverse incident. the aim of this study was to challenge the accuracy of a glucose meter designed to correct for these interferences.methods. paired random arterial whole blood samples were collected from icu patients admitted for [ h, samples were tested for glucose using stat strip glucose (nova biomedical) and the omni b bga (roche) routinely used for blood gas analysis.statistical methods: spearman rank correlation/regression, bland-altman analysis. results were compared to iso standard for glucose measurements. omni bga: correlation coefficient r = . , slope = . with intercept - . .bland altman plot for absolute glucose concentration showed that mean bias compared to reference was - . ± . mmol/l with limits of agreement - . - . mmol/l. study aim. to assess predictors of t and its impact on icu-, hospital-length of stay and costs in a cardiac surgical patient cohort admitted to our eight bedded icu, since through june .methods. all the pre-, intra-and post-operative variables were prospectively put into an electronic database. patients were divided into: ( ) ntg group, not needing a tracheostomy;( ) tg group, undergoing a tracheostomy. p values \ . were considered significant. out of a total of , patients with a median (iqr) age of years ( - ) ( ) from post-operative icu to the cardiac surgical ward and ( ) from the cardiac surgical ward to the rehabilitative one, increased significantly higher in the ntg group than in tg group (respectively: log-rank = . , p = . and log-rank = . , p = . ,). tg group showed a lower mortality ( . vs. . %, p = . ) than ntg one.conclusion. this study allowed us ( ) to define a predictive model for identifying patients that are likely to undergo a tracheostomy ( ) introduction. patients admitted to itu increasingly have significant medical comorbidities that require chronic therapy for adequate control . on admission to itu, acute medical problems take precedence and many long term medications, such as thyroxine, may be withheld or ceased. in chronic hypothroidism the patient is physiologically dependent upon ongoing administration of thyroxine. the optimal management of chronic medical conditions such as hypothyroidism within the itu may be essential to patient recovery and should be a quality assurance issue. to assess the prescription of thyroxine and the thyroid function in patients admitted to itu with previously diagnosed chronic hypothyroidism. a six year retrospective review of the electronic records of patients with hypothyroidism who were admitted to a bed tertiary referral hospital itu from to was performed. patients were included if they were admitted to the itu for a period of more than days and were on thyroid replacement therapy prior to itu admission. patient demographics, daily thyroid replacement dose/route, thyroid function tests (tsh and free t ) and rate and type of nutrition were obtained. patients were grouped by their worst recorded tsh according to predefined ranges . conclusions. patients did not receive their thyroid replacement for a significant proportion of their admission. this was predominantly due to either lack of prescription or lack of tolerance of enteral feed. the tsh was appropriate for the free t level. a significant proportion of patients ( %) did not have their tsh measured at all. of those that did, abnormal tests were inconsistently repeated or acted upon. having processes in place to ensure the appropriate prescription and adjustment of relevant chronic medications is essential in the provision of high quality care in itu.background. cell-free dna has been investigated as a diagnostic marker in many diseases, including acute conditions such as stroke, myocardial infarction, burns, sepsis etc. its serum and plasma levels have been shown to correlate with disease severity in all those. free circulating dna is released from dead cells (necrotic or apoptotic) and activated inflammatory cells. our hypothesis was that in acute pancreatitis free serum dna correlates with the extent of pancreatic necrosis and that it may be an early marker of severity. free dna was measured in sera from patients with acute pancreatitis at admission, on the first, fourth and seventh day following admission. severetiy of illness was assessed with atlanta criteria. on the first day following admission patients who would develop severe pancreatitis had significantly higher serum dna levels than those with mild disease (median . vs. . ng/ml respectively; p \ . ). this parameter showed very good characteristics as a potential predictor (area under roc curve . ). free serum dna was in correlation with the extent of pancreatic necrosis.conclusions. free serum dna correlates with the extent of pancreatic necrosis and is a potential early marker of severe acute pancreatitis.keywords. acute pancreatitis, cell-free dna, prognostic marker, pancreatic necrosis. introduction. the performance of general prognostic models in patients with transplantation in need for intensive care unit (icu) admission is poor, showing a tendency towards significant underestimation of the risk of dying. the objective of our study is to evaluate the acute physiology and chronic health score ii (apache ii) and simplified acute physiology score (saps ) and their days mortality prediction after liver, renal and pulmonary transplantation [ ] [ ] [ ] .methods. this is a prospective cohort study in a transplantation icu in porto alegre, brazil, during the period of may -december . clinical data of pos transplantation patients admitted at icu were collected at admission and saps and apache ii calculated with respective estimated mortality rates. the area under receiver operating characteristic curve (auroc) was obtained for both scores. objectives. to validate the saps model, over a period of one year, in a general icu. material and methods. we analysed data prospectively registered in an informatic data base (icdoc), which contain information referring to all patients admitted in this unit.we studied all the patients admitted in the year ( patients). excluded from the analysis were readmissions and one patient still in the hospital. analysed patients.results. discrimination was accessed by the area under the roc curve (aroc) and calibration by the hosmer-lemeshow ĉ test for the general equation and for regional equations (southern europe and mediterranean countries).the mean age of the patients was . ± . years. from the total of the patients, were medical ( . %), were scheduled surgical ( . %) and were emergency surgical ( . %). the mean icu and hospital mortality was . and . %. the mean saps score was . points ( - ). discrimination was good with an aroc of . ( . - . ).there was a statistical significant difference between the mortality predicted by the general equation and the observed mortality (ĉ = . ; p = . ); this discrepancy was not significant by using the regional equation (ĉ = . and p = . ). the saps overestimated hospital mortality with the predicted mortality by the regional equation getting closer to the observed mortality [standardized mortality ratio (smr) = . ] than the predicted by the general equation (smr = . ).conclusion. the saps model, particularly using the regional equation introduction. saps has been previously validated in our icu and it has been routinely used in hospital mortality prediction. as we have shown before, saps had a good accuracy regarding discrimination and calibration, with better predictions done by north american and western europe customized equations than the south american one [ ] . in a larger sample we have been observed deterioration in calibration model, especially among groups of lower probability of death, regardless of the equation in use. therefore we tested saps accuracy considering days mortality in comparison to hospital mortality. we considered consecutive admissions in a medical-surgical icu in a private tertiary hospital in sao paulo -brazil, in the period from january to november of . probability of death was derived from given equations of the original study [ ] . hospital and days mortality were considered as end point. discrimination was performed by the area under the roc curve (auroc) and calibration by the hosmer-lemeshow (hl) statistic. observed to expected (o/e) mortality ratio was also calculated. to assess factors concerning prognosis of patients older than years admitted to the icu: group a, to years old and group b, older than years. both groups were compared for the apache ii, admission group, lenght of stay, mortality and usual intensive care procedures (arterial and venous catheters, mechanical ventilation). statistical anlysis: quantitaive variables were expressed as mean and standard deviation (sd). student t test was employed for these variables. categorical variables were compared by the chi-square. p \ . was considered statistically significant. a total of patients were included in group a (mean age . , sd . ) and in group b (mean age . , sd . ). apache ii score was . for group a and . for group b (p = , ); predicted mortality was . and . % respectively (p = . ). ther were no differences for admission group or procedures among groups. mortality was significantly higher in group b ( . vs. . %, p = . ). when mortality was analyzed for admission groups, it was higher just in cardiological group, wich included ischemic cardiopathy, cardiac failure and arrhythmia ( . vs. . %, p \ . ). the investigation of the association between a differential access to intensive care services and patient or hospital outcomes is increasing markedly [ ] [ ] [ ] [ ] . objectives. the aim of this study was to compare demographic, clinical characteristics, and outcomes of patients admitted to tertiary-level intensive care units from a tertiary hospital ward (intrahospital transfer) to patients transferred from a secondary hospital ward (interhospital transfer). single centre retrospective study in a bed mixed icu of a tertiary university hospital. during the study period ( ) ( ) conclusions. the interhospital transferred patients are younger, but at admission severity of the disease is comparable.these findings, within this case mix of patients, suggest there are not significant differences in mortality, length of stay, icu-nosocomial respiratory infection or physiological disability at discharge between intrahospital and interhospital transferred patients to our unit. in this study we did not find a different impact in outcome considering these differential sources of admission. we prospectively analysed data of all patients (pts) undergoing cardiac surgery between january and june , and discharged from our icu by h from surgery. on all patients the following was collected:(i) demographics, risk factors and gravity scores anamnestic illnesses (ii) intra-operative variables [i.e. type of operation, cardiopulmonary by-pass (cpb) and aortic cross clamp (acc) times] (iii) icu-related variables. one-way anova test was used for continuous variables whereas, differences in proportions were compared using chi-squared test.a binary logistic regression model was used to estimate the effect of each considered risk factor on discharging from cardiac surgical to rehabilitative ward, considered as a dycotomous outcome (yes = early b days/no = late [ days). statistic analyses were performed using spss software. p values less than . were considered significant. on all the patients, aged c years, admitted to our post-operative icu since january through december , we collected demographic profiles, operative data and outcomes. a logistic regression model was set up to assess predictors of hospital outcome. a total of patients ( . %), . % males and with a median (iqr) age of ( - ) were admitted. the below table shows the outcome predictors (see table ). objectives. the purpose of this study was to evaluate prospectively in our medium the capacity of apache iii score to stratify prognostically critically-ill-patients upon their admission to the icu, not only with regard to hospital mortality, but also to hospital length of stay. study aim. to assess if cardiopulmonary by-pass (cpb), aortic cross clamp (acc) time and duration of mechanical ventilation (mv) may impact on icu and hospital length of stay in a cardiac surgical patient cohort admitted to our bedded icu, since through june . all the patient pre-, intra-and post-operative variable were prospectively put into an electronic database. on all patients the following was collected:(i) demographics, risk factors and gravity scores anamnestic illnesses (ii) intra-operative variables [i.e. type of operation, (cpb) and (acc) times] (iii) icu-related variables (i.e. duration of mechanical ventilation, use and type of inotropes. statistic analyses were performed using spss software. p values \ . were considered statistically significant. a total of , patients with a median (iqr) age of years ( - ) were admitted through the study period. . % underwent a cabg operation, whereas . % valve surgery and . % aortic and lung surgery. a bivariate analysis was performed considering as independents variables respectively the natural logarytm (nl) of ( ) cpb time, ( ) acc time, ( ) mv duration, whereas dependent variable was considered the nl of the total hospital stay. we showed that a linear correlation exists between total hospital stay (ln) and ( ) conclusion. this audit allowed us to assess that the longer is the cpb and acc time and mv duration the longer is likely to be the total hospital length of stay of the patients undergoing heart surgery. introduction. high-dependency units (hdu) were designed as a bridge between the operating theatre and the surgical ward for postoperative patients demanding a higher than standard level of care. the aim of our study was to determine the risk factors as well as the predictive value of four severity scores for a prolonged hdu length of stay (los). three hundred fifty-eight consecutive adult patients were included in the study for a period of months. asa, saps ii, possum and sofa scores were calculated for the first h following admission. the demographic and the scores variables were subjected to a univariate and, consecutively, a multivariate logistic regression analysis. a receiver operating curve (roc) model was used to determine the predictive value of the scores for a prolonged los. the presence of a patient for three or more days in the hdu was defined as prolonged stay.results. the median los was ( - ) days, patients were transferred to the intensive care unit and the in-hospital mortality was . % ( patients). the univariate logistic regression revealed the following variables as significant for a prolonged los (p \ . ): asa, possum preoperative, possum postoperative, possum total, possum cardiac, possum ecg, possum type of surgery, possum blood loss, sofa, sofa respiratory, sofa cardiovascular, sofa liver, sofa coagulation, igs ii, igs respiratory, igs urinary output, igs urea, igs potassium, igs bicarbonate, and bmi. seven variables were identified as having a statistically significant association with the los (table ) . according to the roc model, sofa score was the best predictor for a prolonged los, with an area under the roc (auroc) of . .warning systems and rapid response team: - s. saxena , s. jafrey , j. zwaal kingston hospital, anaesthetics, kingston, uk, kingston hospital, kingston, uk background. published data suggests that the patient group with the highest mortality in icus comprises those patients admitted from the hospital wards [ ] . studies have shown that in-hospital cardiac arrests are commonly preceded by physiological abnormalities [ ] . if admission to icu, is preceded by specific physiological derangement, then early identification of these high risk hospital in-patients may be possible. this may improve survival of patients. objectives. to determine . the effectiveness of new track and trigger pathway in identifying patients requiring icu admissions. . the impact of new system on outcome of icu admissions method. . retrospective case notes survey of all icu admissions from the ward over a month period. . the pathway is triggered when abnormalities are present in two or more of the following parameters: response to painful stimuli, respiratory rate, oxygen saturation, systolic blood pressure, and heart rate. . .triggering steps progress through involvement of junior medical staff and outreach teams at step , to more senior staff at step , to consultant involvement at step , depending on the level of deterioration of the patient. . forms were collected over a period of months. icu mortality: patients with abnormality at any time prior to icu admission: / ( %) icu mortality: patients with c abnormalities any time prior to icu admission: / ( . %) mortality of patients who were pathway followers: / ( %) mortality of patients who were pathway non-followers: / ( . %) average length of stay in icu who were survivors from pathway followers: days average length of stay in icu who were survivors from pathway non-followers: . days discussion. . there was low sensitivity of pathway for identifying icu admissions. . poor documentation of triggering events . pathway followed inadequately in majority of patients due to combinations of delay in, or absence, of triggering when indicated . lack of consultant involvement at step . no patients with chronic kidney disease admitted to intensive care have poor outcomes [ , ] . in % of cardiac arrest calls in our hospital were from the renal unit (personal communication.) at this time critical care outreach teams were recommended as means of improving intensive care outcomes through earlier ward assessment of critically ill patients [ ] [ ] [ ] . in modified early warning system (mews) charts to wards and a dedicated seven-day ward-based consultant led service ( - ) were introduced on our renal unit [ , ] . aims and methods. the impact of these change interventions was analysed. primary outcomes were the incidence of cardiac arrests calls to the renal unit, admission apache ii scores and icu mortality. secondary outcomes were age, sex, intensive care and hospital length of stay, in-hospital mortality, cpr prior to icu admission and emergency admissions to the renal unit. cardiac arrests, mortality rates and emergency admissions were compared with the v test; other outcomes via mann-whitney u test. a p value \ . was regarded as significant.results. the results are outlined in the table [ ] ; this group has a high mortality [ ] . deterioration in vital signs often precedes referral to critical care and this is evidenced by a rise in the patient at risk score (pars). higher pars may be associated with worse patient outcomes [ ] . most pars systems have a trigger value at which critical care input should be sought. we hypothesised that the duration of physiological deterioration prior to critical care admission would be associated with mortality and used the delay between pars trigger and admission as an estimate of this.methods. we collected data on over consecutive patients that had deteriorated on the ward and required admission to general critical care (hdu and icu) at both acute hospitals in sheffield. patients admitted to specialist facilities such as cardiac and neurosurgical units were excluded. those already triggering at time of hospital admission were also excluded. to assess if any of the ccrt activation criteria was associated with higher incidence of icu or hospital mortality. our hospital is bed tertiary care center. cohort analysis of prospectively collected data of each ccrt activation including demographic data of the patients and their outcome in terms of icu and hospital mortality and ccrt activation criteria. ccrt activation from st january to th september .the activation criteria for ccrt includes: threatened airway,tachypnea defined as respiratory rate more than or less than breath/min, hypoxemia defined as oxygen saturation less than % on oxygen flow l/min, arrhythmias defined as heart rate less than or more than beat per minute, hemodynamic instability if systolic blood pressure less than mmhg or more than mmhg, decrease level of consciousness defined as drop of gcs = or more points from baseline, seizure and serious concern about the patient.we analyzed each factor separately as independent predictor of icu and hospital mortality. [ ] . with this in mind efforts have been made to develop physiological early warning scoring systems which have been shown to predict subsequent outcome [ ] . we have recently introduced an early warning system (ews) chart for all patients in our hospital and we wanted to assess its impact on our icu admissions. to assess the calculation of the ews, the scores of patients admitted to icu and the compliance with guidelines regarding further intervention for patients who were ultimately admitted to icu.methods. chart review of twenty five consecutive emergency icu admissions, examining the ews in the h prior to admsission.results. ews charts were completed for % of emergency icu admissions; of these % of scores were calculated correctly. only % of ews had all parameters completed for all set of observations. the mean peak ews prior to icu admission was . with a range from to . higher peak ews was strongly associated with increased icu mortality: a ews of - was associated with mortality of . %, whereas a ews of - was associated with a mortality of % (see below). for each ews recorded specific action was required to be triggered according to the protocol. in % of cases appropriate action was taken, however, in % the required action was not taken and a number of patients were thought to have delayed referral to critical care as a result of this.conclusions. following this audit we have introduced a critical care outreach team and have embarked on an educational programme for staff with emphasis both on the complete and accurate recording of early warning scores and the necessity for appropriate action to be taken on the basis of these scores. aim. the quality of care prior to icu admission has been a focus of attention [ ] . mews had been chosen by the trust as a trigger device to identify deteriorating (sick) patients in the general wards. this retrospective study looked at the clinical characteristics of unplanned admissions to our icu and assessed the mews as a predictive tool to trigger early intervention in such cases.methodology. all patients who were non-electively admitted to our icu from the medical wards were included in the study (january-march [ ] score and standardised mortality ratio(smr) had occurred since the introduction of our nurse lead outreach serrvice and high dependency unit. this seemed counterintuitive so we decided to look at in futher detail and whether the phenomena of lead time bias occurred.objectives. primary end point was to assess if the apache ii scores and smr were different if assessed from the point of contact with outreach or hdu for patients admiitted to general intensive care. secondary endpoints looked at which physiological scores were most altered by these systems. a cohort prospective study was setup with ethics committee approval. all patients seen by outreach (group ) or on hdu (group ) prior to admission to general icu were included over a six month period. two sets of apache ii scores and mortality prediction were generated for each group, a 'pre' and 'post' score. the pre score was a h scoring period started from up to h prior to admission to icu on point of contact on hdu or by outreach. the post score was a period for scoring taken h from the point of admission to icu, ie the conventional apache ii score .therefore each patient had two sets of scores for apache ii and predicted mortality. the apache ii and predicted mortality scores were then compared using a two tail paired t test, the individual physiology scores were compared via a wilcoxon rank sum score. in total patients from hdu were included and patients from outreach were included.the primary question was answered as a significant difference in apache ii and smr was found in both groups. introduction. unplanned admissions to intensive care units (icu) are associated with an increased mortality. in order to identify in-hospital patients at risk of deterioration, several scores based on physiological parameters have been published. however, routine application of these parameters is not common in all european hospitals yet. the goal of this prospective study was to evaluate the efficiency of the current practice of handing over ward patients at risk for decompensation by physicians and nurses. furthermore, factors associated with admission to the icu or alarming of the physician on duty should be identified. the study was conducted at the university hospital of regensburg, germany on wards with predominantly gastroenterological and general medical patients ( beds). over a time period of months, the daily routine report of patients at risk to the physician on call after hours was recorded. in addition, the nurse assessment of patients at risk and the documentation of the decompensation defined by calling the physician on duty during the night were registered.results. patients were treated during the surveillance period. in total, patients ( women, men) with a mean age of ± years were either judged by the attending physicians or the nurses at risk for deterioration. patients suffered from decompensation during the night shift. of those, patients were correctly identified by physicians and patients by nurses, respectively. in patients ( %), an icu admission was necessary.discussion. only a small portion of patients reported at risk experienced a severe decompensation at night, defined as icu admission. interestingly, those were only in part correctly identified by the physician and nurse reports. a further evaluation of the correlation of those reports with the previously published ''early warning score'', and physiological parameters associated with decompensation are currently being performed in order to estimate the value of standardized patient assessment, and will be presented at the meeting. introduction. the need to implement a patient follow-up program after icu discharge arises from several facts: ( ) at icu discharge patients are now more fragile (aged, chronic comorbidities, complex). ( ) the demand for intensive care exceeds its availability. as much as % of patients die after discharge from the icu, many of them in spite of a low predicted mortality, perhaps due to premature icu discharge. ( ) compared to nursing care in the icu, the level of that received upon transfer to the floor, as measured by tiss- , may be reduced up to more than %. we believe that, in order to change icu behavior towards focus on long term outcomes, we need to increase global awareness of disability post-icu discharges, and expand the involvement of the icu team in key decision management outside the icu. we propose an alternative model of care for the critically ill patient. this involves an expanded role for clinicians with expertise in critical illness at several points along the continuum of care.objectives. due to lack of adequate clinical resources to care for some recoverable patients when are discharged to hospital wards after a long time in icu, we have planned a follow-up program focused in detecting risk factors associated to bad prognosis and, decreasing adverse events in general hospital wards. qualitative, prospective and interventional study realised during seven months (from march to september ), in the medical uci of a teaching hospital in malaga. we determined demographic data, icu admission reason, comorbidity index (charlson scale), follow-up reason (polineuropathy, tracheostomy, analgesia), family support in ward, difference in nursing activities score between icu and ward (tiss- ), intervention done out of icu with patient; satisfaction of patient, icu readmissions, reason to end follow-up and mortality at day after icu discharge. we enrolled patients in this analysis. comorbidity was charlson scale (very high) in . % of patients, apache-ii mean score points and mean expected mortality rate %. more than % of patients had five or more risk factors (age [ years, icu stay [ days, transfusions, inotropic drugs, mechanical ventilation, tracheostomy, kidney failure, parenteral nutrition, polineuropathy). nursing activities score in icu was . before discharge versus . in ward ( . % decreasement). mean follow-up were . (range - ).in hospital mortality rate was . %, rest of the patients were discharged at home. our study found the implementation of continue follow-up program from icu staff is associated with an important decreased of the mortality. encouraging clinical results and a non excesive workload for icu staff justify to continue this follow-up program in cases in which is going to be an important decrease in nursing care after icu discharge, and have bad prognosis risk factors. objectives. we examined the prevalence of adverse events (ae), suboptimal assessments of vital signs and whether there were advance directives prior to icu admission from the general wards among patients who died within days of icu admission. the patients were those admitted to the general icu from the general wards at the university hospital, lund in and who died within days after icu admission. there were patients with a mean age of years and a mean apache ii score of . we used the global trigger tool model for measuring ae (http://www.ihi.org). the frequency of vital functions assessments, and which parameters were controlled were studied in relation to patient status and the local routine for frequency of modified early warning scoring (mews).the patient records were also controlled for descisions to forgo treatment before admission to the icu. . patients ( %) suffered from at least one ae prior to icu admission. patients had an ae contributing to death, among those patients suffered from an ae that with a probability greater than % was deemed avoidable. seven of those patients suffered from a most likely avoidable ae contributing to death.vital signs were recorded inadequately in % of the patients in the h before admission to the icu. the vital signs most often recorded were blood pressure, heart rate and oxygen saturation, whereas consciousness and breathing frequency were the least recorded parameters.descisions to forgo resuscitation, or some other limitations due to ethical considerations were found only in % of the patients before admission to the icu.conclusions. patients admitted to the icu who died within days suffer from a considerable proportion of avoidable ae contributing to death. vital signs are not recorded in a satisfactory way during the h before admission to the icu in this most severely ill population. there are very rarely documented descisions to forgo treatment in this group of patients before icu admission. thus, poor control of vital signs in the general wards leads to severe and avoidable ae contributing to death. the lack of descisions to forgo treatment before icu admission in this group of patients most probably contributes to prolonged dying and suffering, and unnecessary intensive care. results. a significant correlation (p \ . ) was detected between the type of prehospital care and the early outcome among the patients. the majority of the patients was transported by ambulance services ( . %) from which half of the patients ( . % of the total) were seen by a paramedic and the rest by a physician beforehand. a relevant proportion of the patients visited the cpu without having been seen by medical personnel ( . %) before. a smaller group of patients was referred by an attending hospital physician to the cpu ( . %). . % of all patients were admitted to a cardiologic ward, . % to icu and . % underwent immediate cardiac catheterization. the rest was referred to other departments within the hospital, other hospitals or was discharged and no one died within h after admission to the cpu. almost half of the patients ( . %) who underwent immediate cardiac catheterization was transported by emergency physician car whereas half of the rest ( . %) visited the cpu as out-patient (p = . ). this very similar ratio can be seen within the patient admitted to icu ( . %). conclusion. the detection of the early symptoms of chest pain is an important prevention strategy for lay people because they can immediately turn to a chest pain unit ( . %) where almost half of them might have life threatening situation ( . %) requiring acute intervention (cardiac catherization or icu-treatment). the adequate in-time treatment can reduce the length of hospitalization and secure quicker recovery. key: cord- -li pwigg authors: nan title: esicm monday sessions october date: - - journal: intensive care med doi: . /s - - -x sha: doc_id: cord_uid: li pwigg nan methods. for the present investigation, healthy male volunteers with a mean age of ± . years were recruited for a cardiovascular screening exercise stress test prior to inclusion for the study. during the lbnp protocol, the subjects were exposed to sequential increasing negative pressures of - , - , and - mmhg while resting in a supine position with their legs sealed in the lbnp chamber at the level of the iliac crest. in addition to continuous registration of cardiac output (co) and mean arterial pressure (map), sublingual perfused vessel density (pvd) ( ) and microvascular flow indices (mfi) ( ) were measured using sidestream dark-field (sdf) imaging before (t ), during (t ; - mmhg), and after (t ) lbnp. results. there were no significant differences in mean co and map in our subjects. introduction. fever management remains controversial in sepsis. control of thermal balance might improve vascular tone but fever could play a role in host defence. objectives. the aim of this multicentre randomised controlled trial was to determine primarily whether external cooling might accelerate the weaning of vasopressors in patients with septic shock. patients with septic shock treated with epi/norepinephrine infusion and fever over . °c were enrolled in centres when also requiring mechanical ventilation and sedation. patients received external cooling to reach normothermia ( . - °c) during h (n = ) or had fever respected (n = ). a goal of mmhg for mean arterial pressure was used in the two groups. a similar algorithm was used for weaning of vasopressors. the main end point was the number of patients achieving a % decrease in the initial dose of vasopressor in the two groups. shock reversal was defined by vasopressor withdrawal for at least h. at inclusion the two groups (cooling/respect of fever) were similar for age ± versus ± years, saps iii ( ± vs. ± ), sofa score ( ± vs. ± ), and body core temperature ( . ± . vs. . ± . °c). a similar number of patients received steroids and a pc before enrolment. body temperature became significantly lower in the cooling group within the h of treatment: . ± . vs. . ± . at h and . ± . vs. . ± . °c at h (p \ . ). the decrease in vasopressor was more rapid in the cooling group (fig. ). shock reversal was vs. %, p = . and in-hospital mortality was vs. % in the cooling and the respect of fever groups respectively. conclusions. these preliminary results show that treating fever using external cooling in septic shock patients allows a more rapid decrease in the dose of vasopressor without apparent adverse effect. grant acknowledgment. aphp-scr . we set up to describe the antibiotic treatment regimens prescribed for patients with severe sepsis in spanish icus and to analyze the potential therapeutic benefit of combination therapy. methods. edusepsis subanalysis, including all patients with severe sepsis admitted to the participating icus during months, in three periods between november and june . there was analyzed the time between the presentation of sepsis and the initiation of antibiotic treatment and empirical antibiotic used in terms of focus and origin of sepsis (community/nosocomial). we also studied the combination therapy compared to monotherapy, assessing the impact on outcomes of combination therapy in particular. the results are presented as frequencies (percentage) or mean ± standard deviation. results. there were included , patients with severe sepsis (age . conclusions. combination therapy is not associated with a better outcome in this large cohort of patients with severe sepsis. nevertheless, there is room for improvement since % of patients did not receive antibiotic therapy within the first h from admission, as recommended by the ssc. introduction. tracheostomies are increasingly common in hospital wards and can lead to significant patient harm. this is partly due to bed pressures in uk critical care units and the increasing use of percutaneous and surgical tracheostomies for critical care patients. commonly, hospital wards lack the infrastructure to care for tracheostomies safely. objectives. analyse tracheostomy-related critical incidents reported in the uk over a year period. we wished to identify themes and make recommendations to improve patient safety. methods. the search was conducted from st october to th september and was conducted in february to allow time for incidents to be submitted. the selected incidents were then incorporated into an access database (microsoft office ) and the description of each incident was read and reviewed. we analysed tracheostomy-related critical incidents reported to the uk national patient safety agency over a year period, identified by key letter searches. we categorized the records to identify recurring themes and then performed root cause analysis where possible. results. we identified , incidents from the npsa incident database originating from hospital wards during the study period having the defined letter sequences. of these incidents, were associated with tracheostomies; directly affecting patients with the remaining not directly affecting individual patients. in the incidents where patients were directly affected ( %) were associated with some identifiable patient harm of which ( %) were associated with more than temporary harm. in incidents ( %) some intervention was required to maintain life and in cases the incident may have contributed to the patient's death. there were cardiac arrests and respiratory arrests described in these incidents. of the incidents, involved equipment and there were blocked or displaced tracheostomy tubes described. note: an individual incident could be classified in multiple fields conclusions. we were able to identify themes in incident reports associated with tracheostomies and identify areas where care could be improved to reduce risks to patients. there were a number of recurrent problems that contributed to incident evolution or severity that would be potentially avoidable. these include: introduction. the study of computerized thoracic tomography patterns can be of great help in the diagnosis of the causes of acute respiratory failure in the icu patients. we hypothesized that the consecutive analysis of a series of thoracic cts will contribute to the management of these critically ill patients. objectives. to study, over a three-month period, the thoracic cts performed in the adult icu in the albert einstein hospital in são paulo, brazil. methods. from may st to august st, , all the thoracic cts were analyzed by two radiologists from the albert einstein hospital staff according to a pre-established protocol: ( ) presence of parenchymal consolidations; ( ) ground-glass opacities; ( ) septal thickening; ( ) atelectasis ( ) pleural effusions; ( ) pneumothorax ( ) pneumomediastinum; ( ) subcutaneous emphysema; ( ) presence of nodules; ( ) presence of masses; ( ) presence of cysts; ( ) emphysema; ( ) bronchial thickening. results. hundred and sixteen thoracic cts were performed and analyzed over the study period, from ( . %) males and ( . %) females. the mean age of the patients was . ± . years. thoracic ct analysis revealed: ( ) parenchyma consolidations: ( . %); ( ) ground-glass opacities: ( %); ( ) septal thickening: ( . %); ( ) atelectasis: ( . %); ( ) pleural effusions: ( %) ( ) presence of pneumothorax: ( . %); ( ) pneumomediastinum: ( . %); ( ) subcutaneous emphysema: ( . %); ( ) nodules: ( . %); ( ) presence of masses: ( . %); ( ) presence of cysts: ( . %); ( ) emphysema: ( . %); ( ) bronchial thickening: ( . %). conclusions. thoracic ct is a useful tool for a detailed analysis of the lung parenchyma, specially in the detection of ground-glass opacities, consolidations and atelectasis, improving the diagnostic possibilities and management of acute respiratory failure. s. wolf , , a. rieß , j.f. landscheidt , c.b. lumenta , l. schürer , p. friederich charite campus virchow, department of neurosurgery, berlin, germany, klinikum bogenhausen, neurosurgery, muenchen, germany, klinikum bogenhausen, anesthesiology, muenchen, germany introduction. extravascular lung water index (evlwi) may present a valuable marker for the severity and treatment of acute lung injury and acute respiratory distress syndrome. measured by single indicator transpulmonary thermodilution and indexed to predicted body weight, a threshold of ml/kg is currently regarded as the upper limit of normality. however, so far only critically ill patients were studied and data from subjects with normal cardiovascular function is lacking. objectives. to prospectively investigate evlwi in patients without cardiopulmonary compromise. methods. patients requiring elective brain tumor surgery were equipped with a transpulmonary thermodilution device (picco . , pulsion medical systems ag, munich, germany). triplicate evlwi measurements were performed after induction of anesthesia (time point ), before (time point ), during (time points and ) and after surgery (time point ) as well as after extubation (time point ) and before discharge from the neurosurgical icu (time point ) . data were recorded electronically and investigated with a random effect model to cope for multiple measurements per individual. results. valid measurements were performed in patients ( female/ male, fig. ). no patient showed clinical signs of over-hydration or cardiopulmonary failure and all were discharged regularly from the icu on postoperative day one. indexed to predicted body weight, females had a mean evlwi of . (sd . , range - ) ml/kg and males had a mean evlwi of . (sd . , range - ) ml/kg (p \ . ). % of the measurements in females and % in males exceeded the threshold of ml/kg. no significant differences were between the different time points of measurement (p = . ) or during anesthesia and after extubation (p = . ). conclusions. measured with single indicator transpulmonary thermodilution and indexed to predicted body weight, evlwi frequently shows values above the previously established normality threshold of ml/kg in patients without cardiopulmonary compromise. females present significantly higher values than male patients. as we are not aware of any abnormal hemodynamic profile for brain tumor patients, we propose our findings as a close approximation to normal values for evlwi. introduction. cardiovascular dysfunction is though to be common during weaning from mechanical ventilation. however, its precise incidence is unknown in this setting. in addition, the respective impact of systolic and diastolic dysfunctions on the weaning process have not been studied. objectives and methods. this is an ancillary study of the ''bnp for the management of weaning'' clinical trial. patients were ventilated with an automated weaning system as soon as they tolerated pressure support ventilation with an fio b %, a peep level b cmh o, and a total inspiratory pressure b cmh o. a total of patients underwent transthoracic echocardiography (tte) at day (initiation of weaning). in addition, serial tte were performed in a subgroup of patients to explore left ventricle filling pressures variations during daily weaning trials (low-pressure support with zero end-expiratory pressure). filling pressures were assessed using the ratio of early transmitral peak velocity (e) over early diastolic mitral annular velocity (e ). results. day tte revealed a systolic (ejection fraction \ %) or diastolic dysfunction (defined as e \ . cm/s) in half of patients. treatment during weaning included diuretics ( % of patients), vasodilators ( %) , dobutamine ( %), amiodarone ( %) and betablockers ( %). diastolic dysfunction was more prevalent in patients with difficult or prolonged weaning as compared to those with simple weaning (weaning duration \ days). serial tte revealed a greater increase in e/e ratio during failed weaning trials as compared to successful trials. conclusions. when treated, systolic dysfunction does not seem to jeopardize weaning. in contrast, diastolic dysfunction is associated with difficult/prolonged weaning. during failed weaning trials, there is a more pronounced increase in filling pressures as compared to successful trials. introduction. monitoring and determination of fluid responsiveness in a critically ill patient who presents with circulatory compromise and septic shock is essential but often, challenging and difficult. continuous haemodynamic monitoring using arterial pulse contour analysis is less invasive compared to the thermodilution method using the pulmonary artery catheter. objectives. we aim to assess the utility of stroke volume variation as measured by the flotrac Ò device (edwards lifesciences, irwine, usa) as a predictor of fluid responsiveness in patients with septic shock. we studied mechanically ventilated adult patients with septic shock in the medical intensive care unit (icu) of a university hospital. haemodynamic parameters including stroke volume variation (svv) and stroke volume (sv) were recorded using radial arterial pulse contour analysis (flotrac Ò pressure sensor versions . and . ) before and after a crystalloid fluid challenge. fluid responsiveness was defined as an increase of c % in sv after the fluid challenge. results. the sensitivity, specificity, positive predictive value and negative predictive value of a svv of c % to predict fluid responsiveness were respectively . , . , . and . %. the area under the receiver operator characteristic curve for the prediction of fluid responsiveness using svv (pre) was only . . similarly, there was no correlation between svv (pre) and the absolute change in stroke volume (spearman's rho - . , p = . ). conclusions. our study's findings call for caution with the use of svv measured via versions . and . of the flotrac Ò device to predict fluid responsiveness in patients with septic shock. further studies are now required to assess if recent software upgrades may provide more accurate svv measurements in severely septic patients. objective. our objective was to assess the recent literature with respect to cco monitor validation. in particular we wished to determine if study protocols reflected the dimension of time. we looked at four different cco monitors: vigileo tm , picco tm , pulseco tm , and oesophageal doppler (odm). human validation studies of cco monitors were sought through the ovid interface, generating over , hits. manufacturers' websites were also searched. case reports were excluded, as were abstract-only publications, letters, and studies over years old. ultimately, studies were included. a full reference list and search strategy is available from the authors. a recent article provided suggested criteria for assessment of cco monitors [ ] : this was used to generate a proforma. to check for interobserver bias, a subselection of five studies was assessed by the three authors independently; no differences were found. the authors summarised the remaining studies individually. results. results are summarised in table . w rows do not add up because some studies evaluated more than one monitor researchers have yet to address the necessity of validating cco monitors with respect to their realtime functionality. while most studies give an assessment of bias based on essentially static measurements, fewer than half document sampling time or directional change reliability. response time and response amplitude to a step change in cardiac output are important variables which may influence patient treatment; in the vast majority of studies, these have not been assessed. in this respect, all four monitors have yet to be validated. this study offers two perspectives: one, for clinicians to realise that the cco monitor in their intensive care unit may not have been as extensively validated as they think; another, for researchers, to realise that work is still to be done. initial distribution volume of glucose rather than right ventricular end-diastolic volume is correlated with cardiac output following cardiac surgery j. saito , h. ishihara , e. hashiba , h. okawa , t. tsubo , k. hirota hirosaki university graduate school of medicine, anesthesiology, hirosaki, japan, hirosaki university graduate school of medicine, division of intensive care, hirosaki, japan introduction and objectives. rational decision making for cardiovascular and fluid management in critically ill patients requires reliable assessment of cardiac preload. we have reported that initial distribution volume of glucose (idvg) measures the central extracellular fluid volume and has potential as an alternative preload variable ) . idvg can be approximated rapidly and simply in any icu using a conventional blood glucose analyzer ) . right ventricular end-diastolic volume (rvedv) has been shown to be a better indicator of cardiac preload than cardiac filling pressure ) . this study was intended to determine whether idvg, rvedv, pulmonary artery wedge pressure (pawp) or central venous pressure (cvp) are correlated with cardiac output (co) during the early postoperative days following cardiac surgery in the absence of apparent congestive heart failure. methods. twenty-nine consecutive patients who underwent cardiac surgery such as coronary artery bypass grafting (either off-pump or on-pump: n = ), valve surgery (n = ) and aortic arch replacement (either hemi or total: n = ) were studied. patients associated with excess hyperglycemia ([ mmol/l), arrhythmias or mechanical cardiovascular support were excluded from the study. a volumetric thermodilution pulmonary artery catheter for continuous monitoring of co and rvedv was placed in the operating room. immediately after cardiovascular variables were recorded, idvg was determined using the incremental plasma concentration at min after administration of glucose ( g) as described previously ) . three sets of measurements were performed; on admission to the icu and daily at a.m. on the first postoperative days. the relationship between either volumetric or static variables and cardiac index (ci) was evaluated throughout the study period. a p value. was considered statistically significant. results. all but one patients required vasoactive drugs during study period. indexed idvg (idvgi) had a moderate correlation with ci (r = . , n = , p \ . ), even though indexed rvedv (rvedvi) had a slight correlation with ci (r = . , n = , p = . ). a linear correlation was also obtained between changes in idvgi and those in ci (r = . , n = , p \ . ). however, changes in rvedvi had not a correlation with those in ci (r = . , n = , p = . ). neither pawp nor cvp had a correlation with ci (r = - . , n = and r = - . , n = , respectively). although cardiac dysfunction has a significant impact on determining co early after cardiac surgery, our results demonstrate that idvg rather than rvedv is correlated with co. idvg has potential as being an alternative indicator of cardiac preload following cardiac surgery. (spv) are reliable predictors of fluid responsiveness in controlled mechanically ventilated patients [ ] . ppv and spv are calculated using an intra-arterial catheter. it is unknown whether an arterial pressure signal obtained with the nexfin tm system [ ] using only a finger cuff can be used to calculate ppv and spv. objectives. to validate ppv and spv measured with a finger cuff. methods. after their arrival on the icu, sedated and mechanically ventilated patients after coronary artery bypass graft surgery (cabg) were included. intra arterial pressure (iap) was measured using an arterial catheter inserted in the radial artery, and non-invasively, using the finger cuff of the nexfin tm monitor (bmeye, the netherlands). we took the mean value of ppv and svv in a -min time interval before and after the administration of a fluid challenge. agreement of the ppv and spv measured by the finger cuff and from the iap signal were assessed using the method described by bland and altman. results. nineteen patients were included and twenty-eight volume challenges were analyzed, resulting in simultaneous measurements. ppv and spv measured by the finger cuff correlated with ppv and spv from iap (r = . , p \ . and r = . , p \ . , respectively), see figure . the mean bias was - . and - . % for ppv and spv respectively, and limits of agreement were - . and . % for ppv and - . and . % for spv (see figure ). there was no correlation between the bias and the mean value of the two measurement methods. the correlation between changes in ppv and spv measured by the two different methods was r = . (p \ . ) for ppv and r = . (p \ . ) for spv. conclusions. in ventilated icu patients, ppv and spv can be reliably calculated using the nexfin tm monitor. reference(s). ( ) kramer, a., et al., chest, . ( ) . ( ) eeftinck schattenkerk, d.w., et al., am j hypertens, . ( ) . introduction. the transpulmonary thermodilution (tptd) technique with integrated pulse contour analysis (picco Ò -system) enables continuous monitoring of cardiac index (ci) after calibration by tptd [ ] . this monitoring technique is applied in patients with lung failure who undergo prone positioning (pp) which has been shown to potentially improve pulmonary gas exchange [ ] . objective. we sought to determine the influence of a modified pp ( °) on the accuracy of pulse contour derived ci (pcci) without recalibration by tptd. patients: after approval by our institutional review board and written informed consent by a legal surrogate we studied critically ill patients ( #, $, age - years) who were mechanically ventilated due to acute lung injury following lung contusions or acute respiratory distress syndrome. methods. all patients were prone positioned and had received an extended haemodynamic monitoring (picco Ò , pulsion medical systems ag, munich, germany). before turning from supine position (sp) to pp, ci was measured by tptd (tptdci) and pcci was calibrated. ten minutes after positioning, pcci was read from the monitor and then recalibrated by tptd. after - h, pp was ended and measurements were performed analogously to prone positioning. volume management between the respective time points remained unchanged. linear regression analysis and bland-altman plots were used for statistical analysis. all data are given as mean ± standard deviation, range in brackets. results. the tptdci in sp was . ± . ( . - . ) l/min/m . after proning, a pcci of . ± . ( . - . ) l/min/m and a tptdci of . ± . ( . - . ) l/min/m were measured. linear regression analysis revealed a correlation coefficient of r = . (p \ . ). mean bias (tptdci-pcci) was . ± . l/min/m . immediately prior to turning back to sp, tptdci was . ± . ( . - . ) l/min/m . after re-positioning, the pcci was . ± . ( . - . ) l/min/m and tptdci was . ± . ( . - . ) l/min/m , with a mean bias of . ± . l/min/m . the correlation coefficient was r = . (p \ . ). conclusion. pcci is only marginally influenced by prone positioning and is reliable without recalibration by tptd. however, in case of greater differences a recalibration by tptd is nevertheless recommended. objectives. the aim of this study was to analyze the clinical agreement between the intermittent bolus thermodilution technique (tdco) and apco in patients with non-traumatic intracranial hemorrhage requiring intensive care. methods. this was a prospective observational clinical study in a university level icu. we studied adult patients with non-traumatic intracranial hemorrhage, who for clinical indication underwent co monitoring by the tdco (pac, . fr, criticath tm sp h td catheter, becton-dickinson, singapore). in parallel, arterial pressure waveform was applied using the radial arterial pressure curve (flotrac/vigileo tm , version . and . , edwards lifesciences, ca, usa). tdco measurements were done approximately every h and when needed. the length of data recording was depending on the need for tdco monitoring and icu stays but was no longer than days. every tdco measurements and the simultaneous apco values were recorded and included into the analysis. results. data pairs were obtained. overall, mean co was . (sd . ) l/min for tdco and . (sd . ) l/min for apco. mean bias between tdco and apco was . l/min ( fig. ), % limits of agreement . to . l/min and the percentage error %. there was a large interindividual variation in mean bias and percentage error (minimum to maximum, - . to . l/min and - %, respectively). the bias was significantly greater if patient received norepinephrine ( . vs. . l/min, p = . ) but not if patient received dobutamine ( . vs. . l/min, p = . ) . only a small correlation between the bias and the rate of norepinephrine infusion was detected (q = . ). when cardiac index of . (l/min/m ) was used as a cut off value for need for intervention, the sensitivity and specificity for apco were . ( % ci . to . ) and . ( % ci . - . ), respectively. conclusions. according to our results the second generation of flotrac Ò /vigileo Ò monitoring system underestimates the tpco and the sensitivity is poor. there is also a large interindividual variation in bias. the use of norepinephrine may provoke the error. objectives. to compare cardiac output techniques to the reference tte method, which allows accurate measurement of the aortic flow section and of velocity time integral of aortic pulsed wave doppler signal to measure co. methods. monocentric prospective study included patients requiring invasive blood pressure and hemodynamic therapeutic intervention. tte co measurement was performed with aortic diameter measured in parasternal long axis view at the the aortic leflets, and velocity time integral measured using five apical view averaged on cardiac cycles. tod co was measured only when the pac insertion was decided. tte, uscom Ò , mostcare Ò and vigileo Ò were performed in all patients. each value was the average of successive cardiac cycles with consecutive measurements. each patient could have several measures. results. mechanically ventilated patients ( ± years; sofa ± . ) were investigated allowing to obtain measurements ( under norephinephrine). diagnostics: brain injury (n = ), sepsis(n = ) and others (n = ). patient had the methods ( measurements), patients had techniques ( measurements), patients had techniques ( measurements), conclusions. all methods correlated more or less with tte co, with a slope close to identity, and a low intercept. the best correlation was obtained between mostcare Ò and tod. agreement for almost all methods was large, within an acceptable range. for the pulse contour method, mostcare is correlated better than vigileo with tte co. the arterial signal has to be accurate as possible and requires a high quality chain for measurement avoiding overdamping or underdamping to allow effective signal digitalization. introduction. the lithium indicator dilution technique is attractive in paediatric intensive care because it is non-invasive. however, it requires calibration. the reliability of cardiac output measurement data rests on the reproducibility of the calibration factor (cf). objectives. to establish the number of calibrations (= x) that are required in a paediatric patient material, if the coefficient of variation for the calibration factor does not increase by % or more by (x + ) calibrations. to establish x it is also required that % of the patients do not show an increase in cv by % or more and that % of the patients show an increase in cv by % or more at (x + ). hemodynamically stable sedated and ventilator treated children under intensive care with a body weight of - kg were included. to perform calibration, . mmol/kg of lithium chloride was injected intravenously and the concentration of lithium ions in arterial blood was analyzed by a lithium selective electrode. the calibration process was repeated times and the cf as well as lithium indicator cardiac output (lidco) were calculated. results. results from children with a mean body weight of . kg are presented below. cv was below % throughout the investigation. introduction. stroke volume variation has been shown to be a better indicator of fluid responsiveness than static indices such as cvp or paop. a limitation of dynamic parameters is arrhythmias which produces abnormal svv. beat-to-beat variations reflect altered cardiac filling times not the effects of mechanical ventilation in fluid responsive conditions. a recently developed enhanced algorithm (newsvv) helps eliminate this limitation. newsvv rejects ectopic beats using multi-parameter signal recognition and restores the respiratory variation of the signal using spline-based interpolation. objectives. to evaluate the performance of the new arrhythmia rejection svv algorithm to predict fluid responsive from patient data with frequent arrhythmias. methods. newsvv was developed from data collected in a porcine model to limit the impact arrhythmias had on svv. comparing the current standard svv (svvstd) algorithm (flotrac-vigileo system edwards lifesciences, usa) with the newsvv showed a significantly improved sensitivity and specificity. ( ) in this preliminary study sets of patient data with frequent pvcs and atrial fibrillation (afib) were ran through the new algorithm and compared to the data from svvstd. in one patient fluid boluses ( - cc platelets and packed red blood) during a period of afib caused newsvv to decrease from to % and co to increase from . to . l/min, while svvstd algorithm did not show a significant change (varying randomly between and %). a second patient had non-paroxysmal afib. svvstd showed abnormally high values ranging between and %. patient was a non-responder to fluid and had a co ranging between and l/min. newsvv showed more realistic value of % depicting a non-responder range. the third patient had periods of afib followed by normal sinus rhythm (nsr). svvstd algorithm had abnormally high svv values ([ %) during the afib. during nsr, both algorithms correlated well with svv of %. (fig. .) conclusions. the newsvv algorithm improved svv with ectopics and afib and shows promise in eliminating a limitation of svv in those conditions. further studies are needed to fully evaluate the performance in patients with arrhythmia receiving fluid challenges. rd esicm annual congress -barcelona, spain - - october s methods. mechanically ventilated pigs (median weight kg) under general anesthesia were investigated. after instrumentation, baseline values were obtained after at least h of stabilization. ''shock'' phase (simulation of aaa rupture): ( - ) ml/kg of blood was gradually withdrawn and hemorrhagic shock maintained for h. abdominal cavity was filled with warmed saline to abdominal pressure of mmhg. ''clamp'' phase: infrarenal aorta was cross clamped for min and hemodynamics was resuscitated with shed blood and fluids. ''post-surgery'' phase lasted h and pigs were subsequently sacrificed. hemodynamics was obtained at baseline, every min for first h of hemorrhage, every h until postoperative phase and every h till the end of the study. data are presented as median (iqr), appropriate non-parametric tests were used for statistical analysis. results. baseline co measured by pac was ( - ) ml/kg/min. both vigileo ( - ) ml/kg/min (p = . ) and lidco ( - ) ml/kg/min (p = . ) differed significantly. the course of co is shown in fig. , all values are presented as a difference to baseline. the median difference between pac and vigileo was ( - )% and for lidco ( - )%. study limitations: both devices were designed for co estimation in humans but we do not expect huge differences in arterial system properties in pigs. young pigs reacted to hemorrhage by severe sinus tachycardia which caused failure in some co measurements but at least pigs are presented at every timepoint. conclusions. absolute co values obtained by both vigileo and lidco differ significantly from pac. unlike lidco rapid, flotrac/vigileo was able to track changes in co during severe hemorrhage. grant acknowledgment. iga mzcr ns - and vz msm . introduction. most important role of postoperative sedation is suppressing stress of the patients in icu. urinary -hydroxy- -deoxyguanosine ( -ohdg) can be a good biomarker for oxidative stress in clinical research. the aim of this study is to assess the free radical production under sedation in icu and compare the production between with midazolam and dexmedetomidine. subjects and methods. subjects were twenty-five patients with sedation after neck malignant tumor operation and ventilated for h in icu. patients with renal failure were excluded from this study. all patients received fentanyl ( lg/kg/day), fifteen patients were with midazolam ( . mg/kg/h: m-group) and ten patients were with dexmedetomidine ( . - . lg/kg/h: d-group) we examined the concentration of urinary -ohdg by high performance liquid chromatography (hplc) method with coolaray system every morning in icu. results. the average value of urinary -ohdg of healthy human volunteer is ng/ml. the values of urinary -ohdg were less than ng/ml in the both groups and no significant differences were observed between the groups in this study. conclusions. postoperative sedation with both midazolam and dexmedetomidine were effective in suppressing oxidative stress in icu patients. poorly controlled pain in the postoperative period can lead to slow recovery and life threatening complications, especially in elderly patients. it has also been suggested that the quality of postoperative analgesia could decrease delirium incidence and reduce duration of hospital stay in the elderly patients. however, the ideal postoperative analgesia management of elderly surgical patients in intensive care units remains to be determined. since, continuous epidural analgesia provides the required level of analgesia to support early mobilization and significant reduction in pulmonary and cardiovascular morbidity in the early postoperative period, we postulated that the use of low dose of continuous epidural morphine might improve postoperative analgesia and reduce undesirable side effects in elderly patientstherefore, the present study was designed to evaluate the effects of morphine administered via epidural patients controlled analgesia and intravenous tramadol + metamizol on postoperative pain control and side effects in elderly patients after major abdominal surgery. objectives. the purpose of this study was to compare the analgesic efficacy of morphine administered via epidural patients controlled analgesia (epca) with our standard analgesic for postoperative pain treatment, intravenous tramadol + metamizol in eldery patients undergoing major abdominal surgery. methods. forty patients older than years undergoing major abdominal surgery were randomly assigned to two groups. group i received epidural morphine mg at the end of surgery and used a patients controlled analgesia device programmed to deliver morphine . mg/h, . mg per bolus. group ii received intravenous infusion of mg tramadol plus mg metamizol in ml electrolyte infusion. the patients in group ii received ml of the infusion solution as a loading dose over min (corresponding to mg tramadol plus . mg metamizol) postoperative analgesia was tested at rest on a visual analogue pain scale ( = no pain, = worst possible pain) at , , , and h after surgery. patients' satisfaction, arterial oxygen saturation, respiratory rate, episodes of nausea, vomiting, pruritus and dizziness were also noted. results. both groups obtained adequate pain relief, and there were no between-group differences in pain scores. there were no significant respiratory differences but the patients in the epidural group were more sedated. in the tramadol metamizol group patients were treated for ponv while of the patients in the morphine group showed ponv. we conclude that combination of tramadol and metamizole provided postoperative analgesia equivalent to that provided by epidural morphine in early postoperative period. the both analgesic regimens were safe and suitable for the management of postoperative pain in elderly patients. h. poon , j. hulme sandwell and west birmingham hospitals nhs trust, birmingham, uk, sandwell and west birmingham hospitals nhs trust, intensive care medicine and anaesthesia, birmingham, uk a substantial amount of patients in intensive care units (itu) receive an inappropriate level of sedation with a tendency for over-sedation. although the ideal itu sedation practice is not known, many units use a protocol-based approach incorporating best practice consensus. the use of daily interruption of sedation infusions can reduce oversedation and is included in our current guidelines. objectives. the audit assesses compliance to our current protocols for sedation scoring and adjustment of sedation infusions. provision of sedation breaks in patients sedated more than -day is evaluated. methods. retrospective review of itu inpatients' daily record charts during june and july at sandwell and west birmingham hospitals nhs trust in two -bedded itu. charts were reviewed. results. our guidelines recommend hourly sedation scoring from h to midnight and hourly scoring from midnight till h; at , and h. % of the reviewed charts did not have a recorded hourly score. % did not have hourly scores overnight. % of the charts contain ramsay score - or - ; % of the charts contain ramsay score or . per protocol, sedation infusion is stopped at ramsay score - or - but in % of cases this did not occur. a sedation bolus should be given at a score of or . % did not receive a recorded bolus. our guidelines advocate restarting a sedation infusion at a lower rate after it has stopped and the patient is less sedated and increasing the rate after a bolus is given. however, correct infusion rate adjustments were only performed in % of cases. often, rates were changed but without first stopping, or administering a drug bolus. out of sedation days, there were only true daily breaks. conclusions. there is a tendency to over-sedation in our itu; less so under-sedation. common practice deviates from protocol and there is poor documentation. scores are recorded less often than protocol: there may continuous assessment by nursing staff that is not recorded. boluses are given more often than documented on patient charts. review of the current guidance is required to address appropriate frequency of formal scoring, nurse-led compulsory sedation breaks and pharmacokinetic education about the necessity to stop or bolus before infusion rate alterations. introduction. daily interruption of sedation has been shown to decrease length of stay in icu . recent national guidance in scotland promotes dis as part of a care bundle to decrease ventilator associated pneumonia. a week retrospective first round audit was completed prior to the introduction of dis and demonstrated % of patient hours where sedation could be improved. a second round audit of current practice was therefore undertaken to assess the impact of dis on sedation levels. our aim was to identify the frequency of periods where sedation levels were undesirable and investigate any clinical reason behind these. objectives. to assess whether dis would improve sedations levels of our patients already managed with a simple sedation algorithm (sa) aiming at a ramsay sedation score level - . methods. six months following the introduction of dis, a second period of weeks was studied retrospectively where the icu daily charts were examined to look at: demographic data, sedation score, and whether dis had been successfully carried out. results. cases were identified: males and females with an average age years and similar illness severity scores to the first group. dis was carried out on patient days, omitted on days and contraindicated on a further . , patient hours were examined. ( %) were excluded due to contraindications such as refractory hypoxia, drug overdose, and end of life care. the remaining patient hours were compared to the first round group [ , patient hours with contraindications ( %)] using the mann-whitney test as shown in fig. . conclusions. we could not demonstrate any difference in sedation levels following introduction of dis on our unit. we found a large proportion of patients where dis was unsuitable. introduction. delirium, an acute and fluctuating disturbance of consciousness and cognition, is a common manifestation of acute brain dysfunction in critically ill patients, occurring in up to % of ventilated patients in the united kingdom . delirium is independently associated with more deaths, longer hospital stay, and higher overall cost . hypoactive delirium is much more common, is easily missed and is associated with a worse prognosis than hyperactive delirium . objectives. the aim of our project was to quantify the presence of delirium on our critical care unit and to survey the practice of delirium assessment within our region. we conducted a prospective audit at the critical care unit, birmingham city hospital, uk. we used the confusion assessment method for the intensive care unit (cam-icu) to assess for delirium daily on all our patients for a period of weeks. simultaneously, we conducted a telephone survey to elicit the delirium assessment practice of the other units within the west midlands region of the unite kingdom. results. we carried out our assessment on patients over a -week period, which resulted in assessments ( were on non ventilated patients and on ventilated patients). assessments could not be completed because of deep sedation or language barrier. in total we had assessments positive for delirium giving an incidence of % for those patients we could fully assess. all of the patients with a positive assessment had hypoactive delirium. the telephone survey revealed only three of the twenty units in our region routinely assessed for delirium. none of the units had a formal management protocol/guideline for delirium. conclusions. delirium is a significant problem on our critical care unit. the incidence was lower than that reported in the literature this might be because firstly we were unable to assess a number of patients due to the patients being deeply sedated, and secondly we noted many of the patients during the assessment period were high dependency patients with minimal organ dysfunction. despite a large number of recent publications on delirium and increasing awareness amongst critical care professionals most of the units in our region do not routinely assess for delirium. without regular assessment most delirium will go unrecognised and thus opportunities to instigate preventative measures and early management will be missed. methods. six patients aged - years old ( females) who all sustained brain injury with cerebral oedema alongside other traumatic damage (i.e., fractures, abdominal trauma) are hereby presented. upon admission to the icu all patients received large propofol doses ( - ml/h of propofol infusion %), in an attempt to lower cerebral metabolic demand along with vasopressors to maintain a normal mean arterial pressure. three to days following admission all patients developed metabolic acidosis (base deficit ranged from - to - mmol/l), hyperkalemia (potassium concentration ranged from . to . mmol/l), evidence of muscle cell degradation (creatine kinase and myoglobin concentrations ranged from , to , u/l and from , to , lg/l, respectively) and lipaemia (triglyceride concentrations ranged from to . mmol/l). at that time all patients were clinically stable and usual laboratory tests as well as cultures were inconclusive, hence a diagnosis of pris was suggested. results. three out of six patients developed global left ventricular dysfunction, which was documented by echocardiographic evaluation, with normal cardiac enzymes, while all patients developed acute renal failure. cardiac and renal failure were observed within - h following the manifestation of the above abnormal laboratory findings. continuous hemofiltration was initiated promptly on a daily basis in all cases, while the administration of propofol was discontinued. abnormal laboratory findings normalized within - days, while cardiac and renal function gradually ameliorated within a week, following therapy in all cases. no deaths were recorded. conclusions. the initiation of continuous hemofiltration therapy is a crucial therapeutic tool for the elimination of propofol and its potentially toxic metabolites in cases of pris and may have a beneficial effect upon survival in these cases. l. zurong , w. yichun intensive care unit of hunan province tumor hospital, changsha, china objectives. our purpose was to compare the analgesic properties, effect, and side effects of intravenous butorphanol and fentanyl during chest tube removal in cardiac surgery patients. seventy-four patients with cardiac surgery were enrolled before chest tube removal. each patient received standard doses of either fentanyl ( lg) or butorphanol ( mg) before chest tube removal in a double-blind manner. pain intensity and pain distress were measured before analgesic administration, immediately after chest tube removal, and min later pain quality was measured immediately after chest tube removal. level of sedation was measured before and min after chest tube removal. results. the fentanyl (n = ) and butorphanol (n = ) groups were identical with respect to age, race, sex, and weight. pain intensity, pain distress, and sedation levels did not differ significantly between groups. however, procedural pain intensity (mean . , sd . ) and pain distress (mean . , sd . ) scores for all were low. patients remained alert, regardless of which analgesic was administered. conclusions. if used correctly, either fentanyl or butorphanol can substantially reduce pain during chest tube removal without causing adverse sedative effects. thus, clinicians may choose either safe and effective analgesic interventions during chest tube removal. introduction. delirium is defined as an acute alteration of mental status, with either a disturbance of consciousness or a change in cognition which develops over a short period of time and fluctuates during the course of the day. reported prevalence of delirium in critical care varies widely from to %. despite this, delirium remains grossly under-recognised and is often thought to be temporary and of little consequence in critical care. it is however one of the most frequent complications and, after adjusting for age, gender and severity of illness is an independent risk factor for prolonged length of stay and mortality ( ) . current recommendations are for the assessment and diagnosis of delirium using simple validated tools ( ) . pharmacological intervention should be considered when reversible precipitating factors have been corrected. haloperidol is considered the drug of choice. objectives. the purpose of this study was to detect knowledge and awareness of delirium, attitudes and behaviours towards its assessment and pharmacological management in critical care units of two large uk teaching hospitals a -point survey was distributed to all senior medical and nursing staff employed in critical care at the leeds general infirmary and the james cook university hospital, middlesbrough. a total of questionnaires were collected after four follow up rounds via web-based survey tool ''survey monkey''. results. the survey detected a significant awareness of the problem delirium poses in icu. the vast majority ( %) of practitioners did not screen for delirium routinely. of the % who screened only in used screening tools that were appropriate, the majority lacked the knowledge of suitable methods to do so. % of respondents felt that they required tools to aid diagnosis of delirium in their unit. the pharmacological management of delirium varied significantly, with a wide range of drugs used, suggesting the need for guidance. the majority of respondents ( %) felt that they needed guidelines for treatment of delirium, % felt that guidelines would change their practice. despite an awareness of the problem delirium poses in icu, data from this survey shows a lack of knowledge of assessment and treatment. given that most respondents needed guidelines, we have developed a delirium treatment protocol and implemented the confusion assessment method for icu (cam-icu) training package for all staff in the james cook icu. following its implementation we plan to re-evaluate in the hope that the awareness of delirium can be met with the appropriate knowledge to implement a sustained change in practice. results. patients were included, male ( %), mean apache ii score ± . midazolam was suspended in % after h. maximum dose of sufentanyl was mcg/kg/h. bilirrubin and creatinin did not change from initial values and there was no effect in enteral nutrition tolerance. bis values improved % from ± to ± (p = ns) and there were less hemodynamic effects as well as less necessity of amines and sedatives. results are shown in table . conclusions. sufentanyl is an efficient and safe sedative that reduces necessity of more sedatives, amines and generates adequate sedation without renal or hepatic effects. a.s. puxty , j. kinsella , k. anderson glasgow royal infirmary, department of anaesthetics, glasgow, uk etomidate is a sedative agent often used for the induction of critically ill patients. it is, however, a controversial drug with effects on the steroid axis that have been suggested may lead to a poor outcome. so far this has only been proven in infusions of the drug. despite this some have called for its withdrawal altogether objectives. to determine the attitude of anaesthetists and icu consultants in five hospitals in a major uk city towards the use of etomidate. methods. an online questionnaire was constructed using surveymonkey (portland, oregon, usa). this was then sent out to all anaesthetists in glasgow via an e-mail link. a reminder was sent after weeks. trainees from one of the hospitals were unable to be contacted via e-mail and so hard copies of the questionnaire were sent to them. of a total of anaesthetists in glasgow (trainees and consultants), ( %) completed the questionnaire successfully. of those answering the questionnaire, . % were sho level, . % spr, . % consultants and . % sas grade. these respondents sub-specialities/specialist interest were: general ( %), pain ( %), icu ( . %), obstetrics ( . %) and cardiac ( . %). overall ( % ci - )% of respondents were concerned about etomidate's effect on steroid synthesis, although when asked about induction of an emergency laparotomy ( - )% would still use it [with ( - )% avoiding it and ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) % responding that they were never involved in emergency laparotomies]. of those using etomidate, ( - )% would avoid it in a septic patient, and a further ( - )% would give steroid cover. the most common reason for using etomidate was cardiovascular stability [ ( - )%]. other reasons given were simple dosing ( [ - ] )% and habit ( [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] )%. more than one reason was allowed. looking for differences between groups the following was found: while icu consultants reported more concern over etomidate ( vs. %, p = . ), there was no difference in etomidate usage in emergency laparotomy ( % of general vs. % of icu consultants, p = . ). trainees were more likely to have their habit changed by the recent literature on etomidate ( vs. %, p = . ). conclusions. there is concern among anaesthetists and icu consultants regarding the use of etomidate but usage has not changed markedly by most. further evidence of harm would likely need to be demonstrated before abandonment of the drug. introduction. nociception and requirement of additional analgesia before painful procedures in icu are difficult to anticipate. under anaesthesia, the direct pupil light reflex reflects the sympatho-vagal balance and remains altered by pain and sedative drugs. the pupillometry is a reproductive, non invasive method based on automated flash light device assessing pupillary variations [ ] . to study pupillometric parameters to anticipate the tolerance to painful stimuli (surgical debridement). methods. eligibility criteria: sedated patients (morphinomimetics + benzodiazepines, bps = ) in days after invasive surgery for cervical necrotizing fasciitis (cnf), complicated or not with mediastinitis, requiring a surgical debridement three times a day. clinical evaluation performed before and during debridement: heart rate (hr), mean arterial pressure (map) and behavioral pain score (bps) [ ] . pupillometric test before debridement (calibrated bright flash of one-second at lux, (neurolight, id med) with recording of pupillary parameters: minimum and maximum diameter, variation rate, latency, velocity. noxious procedure was defined as an increase of at least one point of the bps (change in facial expression, upper limb movement or ventilator synchrony). the additional analgesia was decided blindly from pupillometric values. analysis compared two groups defined on bps variation induced by the procedure: group with dbps c and group with dbps = . comparison of pupillometric parameters before procedure between the groups. results expressed as median (interquartile range, iqr), mann-whitney test, significance at p \ . . . patients with cervical cellulitis of which complicated by mediastinitis, h/ fratio = / , age years ( ), igs ( ). in this population, during the procedure, hr and map were unchanged, the bps increased significantly [from ( ) to ( ) , p = . ] but remained unchanged in patients ( %) (group = high tolerance). pupillometric parameters before procedure before procedure group (n = ) group (n = ) p conclusions. all pupillometric parameters, except latency, were discriminant for subsequent debridement tolerance with significantly lower values in the group without pain experience. the pupillometric test seemed adapted to this clinical practice for evaluation of nociception status and may help for rationalizing analgesia for short noxious procedures. introduction. delirium is an acute and fluctuating change in mental status, with inattention and altered levels of consciousness. the incidence of delirium in orthopedic patients was ranges from . to %. delirium may present before or after the patient undergoing surgical procedure and has demonstrate increasing risk, including mortality. objectives. the purpose of this study was to compare the effectiveness and tolerability of intravenous propofol versus midazolam infusions in postoperative agitation/delirium therapy with using an epidural infusion for postoperative analgesia after major orthopedic surgeries in high-risk patients with chronic renal failure. with the institutional ethic committee approval; eighty-two high-risk chronic renal failure patients (asa iii, bun [ mg/dl and serum creatinin value [ ) after a major hip or knee operations with a diagnosis of agitation/delirium were eligible for the study in the postoperative period. richmond agitation sedation scale (rass) or confusion assessment method for the icu (cam-icu) were used for sedation/orientation levels. all agitated patients had an lumbar epidural catheter was inserted preoperatively in combination with spinal anesthesia intraoperatively. after the surgery the catheter was loaded with . % mg bupivacaine at the t to l sensory levels and a continuous infusion of . % bupivacaine was commenced at - ml/h in combination with patient controlled analgesia of meperidine ( mg/bolus). for agitation/delirium therapy in group p (n = ) propofol was used with intravenous loading dose of - mg/kg in a bolus and followed by continuous infusion at - mg/kg/h and group m (n = ) midazolam was used with intravenous loading dose of . - . mg/kg in a bolus and followed by continuous infusion at . - . mg/kg/h to ensure a target of rass in + and - values. hemodynamic parameters (heart rates, systolic and diastolic blood pressures), oxygen saturation with sedation-agitation scales were monitored periodically for h. adverse events were recorded. p \ . showed statistically significant. results. the groups were demographical comparable (p [ . ). group p patients reached the target rass scores earlier in group p (p \ . ). hemodynamic values were significantly higher in group m (p \ . ). the epidural bupivacaine consumption was significantly lower in group p with a limited analgesic requirement (p \ . ) and n treatment required adverse events was seen in group p (p [ . ). conclusions. intravenous propofol infusion may be an effective and safe approach adjunct to epidural analgesia for possible postoperative agitation/delirium treatment after major orthopedic surgeries in high-risk patients with chronic renal failure. introduction. ventilator-associated pneumonia (vap) is the most common nosocomial infection among the critically ill patients admitted in the intensive care units (icus). implementation of the available international evidence-based guidelines and recommendations to prevent and manage vap into the clinical setting may not be adequate leading to suboptimal patient care and increased vap rates. objectives. to assess the implementation of selected vap prevention strategies, and to learn how vap is managed by the intensivists practicing in the indian subcontinent. methods. three hundred -point questionnaires were distributed during an international critical care conference. ( . %) were returned and ( %) questionnaires of delegates from india, nepal and sri lanka were analyzed. most of the intensivists ( . %), reported using vap bundles in their icus with a high proportion including head elevation ( . %), chlorhexidine mouthcare ( . %), stress ulcer prophylaxis ( . %), heat and moisture exchangers (hme, . %), early weaning ( . %), and hand washing ( . %) as part of their vap bundle. use of subglottic secretion drainage (ssd, . %) and closed suction systems (css, . %) was also reported by many intensivists, whereas, use of selective gut decontamination was reported by only . % of respondents. most common method for sampling used for diagnosis of vap was endotracheal suction by ( . %) intensivists, and only . % intensivists reported using protectedsample brush. gram negative organisms (pseudomonas, acinetobacter) were reported to be the most commonly isolated organisms. majority of respondents ( . %) reported using proton pump inhibitors for stress ulcer prophylaxis. majority ( . %) believed that vap contributed to increased mortality in their icus with . % treating vap with an antibiotic course lasting for - days. de-escalating therapy was considered, in patients responding to treatment, by . and . % considered adding empirical mrsa coverage and . % considered adding nebulised antibiotics in certain high risk patients. overall there was good concordance regarding vap prophylaxis among the intensivists with a majority adhering to evidence based recommendations and guidelines. even though the gap between recommended guidelines and the actual clinical practice is closing, we could identify certain issues like the choice of agent for stress ulcer prophylaxis, use of hme, ssd and css, where there still exists some practice variability and opportunities for improvement to provide better patient care. objectives. to reassess the value of individual levels and dynamic alteration of procalcitonin (pct) in predicting the outcome of ventilator-associated pneumonia(vap) patient. methods. forty adult patients with vap were studied and divided into two groups according to their outcome at day after diagnosis of vap: death and survival. serum pct levels were measured on days , and (d , d , and d , respectively) and their alteration between different days (kinetics, pct) were calculated. to control the study, acute physiology and chronic health evaluation ii (apache ii), sequential organ failure assessment (sofa), and clinical pulmonary infection score (cpis) and c-reactive protein (crp) were also recorded and analyzed. all parameters have been investigated as independent variables in relation to -day death as dependent variable. results. the increase of pct levels on day , and were significantly predictive of death in univariate analysis with area under curve (auc) and % ci of . ( . , . ), . ( . , . ) and . ( . , . ), respectively. however, kinetics of pct levels among day , and did not show a significant difference between favorable and unfavorable outcomes (p [ . ). multivariate analysis revealed that only sofa ( ) conclusions. neither pct individual levels nor their kinetics during vap course can predict patient outcome. comprehensive evaluation of patients with multiple methods, in combination with pct levels, may increase predictive accuracy in the future. grant acknowledgment. we have no competing interests. introduction. the presence of new or progressive radiological infiltrates, pyrexia, leukopenia or leukocytosis, and the presence of purulent tracheal aspirates are key features defining the presence or absence of vap. the incidence of vap is dependent upon the number of diagnostic criteria applied, and the demographics of the critical care population to which it refers. a uk government directed ''patient safety first'' campaign in the uk, has highlighted vap as a complication related to mechanical ventilation that can be reduced through the introduction of a ''care bundle''. however, benchmarking between different units has practical limitations. objectives. selective data presentation can distort the perceived occurrence of critical events in order to deliver performance targets. we compared the incidence of vap between a neurosurgical and general intensive care unit in the same hospital. a variety of denominators were applied to explore the potential for data manipulation to realise performance targets a local database of neurosurgical and general intensive care admissions, covering a month period, was analysed retrospectively results. and subjects were admitted to gitu and nitu respectively in the month period. ( %) and ( %) subjects were ventilated for more than days. table summarises the demographics and measures of performance between the two units. a p value . was considered significant. parametric and non-parametric tests were applied as appropriate. . vap is associated with not only mortality but also considerable morbidity, prolonged duration of ventilation and increased cost of hospitalisation. to determine the incidence of vap in patients ventilated at the qensiu, to calculate mv resource utilisation, and to ascertain whether vap relates to outcome in acute sci. we undertook a retrospective case note review of all patients ventilated at the qensiu during a year period. patients were identified using a local computerised database. the qensiu receives referral of patients with sci from the whole country (pop * . m). vap was defined pragmatically as deteriorating gas exchange more than h following mv, coupled with a raised crp or wcc, together with either a positive sputum sample or new infiltrates on cxr. statistics: descriptive, median (range); analytical, mann-whitney u and fisher's exact test as appropriate. death occurred in % of patients, and was significantly greater in the group who were retrospectively assigned to the vap cohort: vap vs survival of the deaths, occurred following discharge from the unit ( of whom developed a subsequent vap). conclusions. vap incidence was apparently high in this patient population ( %), particularly in comparison to the reported general icu population incidence ( - %) . this may partially reflect our pragmatic diagnostic criteria, applied retrospectively. vap was also associated with an increase in mortality following discharge in patients with sci. a modified vap bundle adapted to sci patients is being developed locally, based on recent recommendations . introduction. the implementation of a prevention programme with feedback to nurses and healthcare workers (hcw) was associated with better outcomes and increased compliance [ ] . to assess the effects of feed-back in a study based on implementing a variables care bundle to prevent vap. a multicenter and prospective study was carried out in icus. the element care bundle consisted in hands hygiene, oral hygiene, monitoring cuff pressure, sedation vacation/adjustment and avoid ventilator circuits changing. beweekly feed-back displaying posters with information was carried out to update hcw in the compliance of the prevention measures and vap incidence. a questions with open answer questionnaire was performed months after the implementation of the feed-back in all icus to assess the knowledge of the compliance measures, vap incidence, opinion of the study and improvement suggestions. results. questionnaires were obtained. the median experience in intensive care was . years (sd . - . ). % of the staff was aware of the study: . % agree that was useful, . % disagree and . % didn't know. regarding if they thought that in their units they had a high vap incidence, . % answered that they did, . % answered negatively and . % didn't know. asking about the knowledge of the prevention measures' compliance . % answered right, . % said they knew but didn't specify the percentage, . % didn't know and . % didn't answer the question. finally, about the vap incidence, . % answered right, . % said they knew but didn't specify it, . % didn't know and . % didn't answer the question. . % added comments, the most relevant was: . % of the respondents wanted more verbal information. conclusions. poster feed-back failed to improve compliance on vap care bundle due to lack of information. our survey suggests to implement other communication strategies based on direct verbal interaction to improve implementation. objectives. to compare the effectiveness of two different methods of oral dental hygiene on ventilator-associated pneumonia (vap) prevention using the cpis (clinical pulmonary infection score). methods. twenty-seven critically ill patients, aged - years were studied. patients were randomly separated in groups: group (n = ) received oral care with tooth brushing using the bass technique followed by lavage with chlorhexidine . % (chx) solution in nacl . % (chx:nacl . % = : ). hexetidine . % (hex) was used for oral lavage alone in group (n = ). demographics, sofa, apache ii, gcs and cpis scores were recorded upon patient admission. on days , , and , bal and tongue surface cultures were obtained. the endpoints taken into account were the cpis on days , , , and as well as the number and species of bacteria cultivated. a cpis c was considered positive for vap. two-way anova, t test for independent samples, mann-whitney u, pearson correlation and kruskal-wallis tests were used for statistical analysis. p \ . was considered statistically significant. patient data analysis showed that both groups were homogeneous. on days , and there were more negative tongue surface cultures in the chx group than in the hex group (p = . ). even though the cpis did not differ significantly between the groups, a strong tendency of faster and greater reduction in the chx group was observed. moreover, a continuous gradual improvement of the cpis score was recorded in both groups on days , and . the overall incidence of vap was similar in both groups: group , . % (n = ) and group , . % (n = ). the same bacteria (p \ . ) developed in both bal and tongue cultures on days (p \ . ) and (p \ . ). nevertheless, the cpis was positive for vap only in the above patients on day , while day was totally free of vap. conclusions. even though the cpis improvement was more remarkable in group , both group scores showed parallel improvement overtime, the chx group being in a slightly more propitious position. diligence in patient oral care seems to be an additional effective practice for vap prevention, independently of the method used. introduction. specific patterns of cytokine gene expression are reported to be associated with the occurrence of infection in humans [ , ] . it is unclear whether these characteristic profiles are a consequence of an established disease process or precede the infective process. objectives. our primary endpoint was to determine whether hospital acquired pneumonia was associated with differential gene expression of ifn-c, tnf a and il- family of cytokines. secondary endpoint was to identify whether alteration in gene expression preceded the clinical onset of infection. methods. consecutive patients undergoing elective thoracic surgery were recruited. hospital acquired pneumonia was diagnosed as per nnis guidelines, independent of study investigators. mrna and protein levels were analysed pre operatively, h and days post operatively. . patients had an uncomplicated recovery. patients developed hospital acquired pneumonia. il- , il- , il -p , il- -p , il- -p , tnf-a, and ifn-c mrna and protein levels of il- , il- , and ifn-c in peripheral blood were analysed before surgery, h and day post surgery. il- p mrna levels were reduced in the pneumonia group ( , ; , - , ) compared to non pneumonia group ( , ; , - , ) day post surgery (p = . ). ifn-c mrna levels were reduced in the pneumonia group ( ; - , ) compared to non pneumonia group ( ; - , ) (p = . ) day post surgery. absolute copy numbers of mrna per million copy numbers of b-actin are quoted. all values are quotes as median and th to th centile range. patients with postoperative hospital acquired pneumonia exhibit distinct patterns of cytokine gene expression. these distinctive patterns manifest before the clinical onset of pneumonia. objectives. the aim of this study was to evaluate the impact of the implementation of a ventilator bundle on the incidence of vap in our intensive care unit (icu). methods. prospective, observational study. during a year period (january - ) a ventilator care bundle based on the cdc and canadian guidelines , was applied to each patient requiring more than h of mechanical ventilation (mv). the ventilator care bundle consisted in:hand hygiene. use of barrier precautions. preference of noninvasive ventilation (niv). orotracheal intubation. semirecumbent position. continuous aspiration of subglottic secretions. manteinance of the endotracheal cuff pressure. oral care with chlorhexidine. daily sedation vacation and assessment of readiness for weaning. no scheduled ventilator circuit changes. ventilation circuit maintenance. stress bleeding profilaxis. we followed general measures for infection control:single patient room. microbiologic surveillance. monitoring and early removal of invasive devices. program to reduce antimicrobial prescriptions. for each ventilated patient the following data was registered:age, apache ii, the reason of admission, risk factors, use niv, mv duration, timing of tracheostomy, time of diagnosis of vap, microbiological data, length of stay and mortality in icu. the compliance with the bundle was registered in a check list. the application of the ventilator care bundle impact was compared with historical data. . ventilated patients were included. the median age was years. the mean apache ii was . ± . . the reason of admission was in a % medical and % surgical. the most frequent risk factors were: age [ years, emergency surgery, pulmonary disease and immunosuppression. niv was used in %. the median duration of mv was days. tracheostomy was done in . % patients with a delay of ± days. there were late-onset vap episodes and the rate of vap per , ventilator days was . . the vap were caused by candida albicans, enterobacter aerogenes and pseudomona aeruginosa. the length of icu stay was . days. mortality was % (only one patient with vap). the rate of compliance with ventilator care bundle was %. the rate of vap per , ventilator days after implementation of the bundle decreased significantly (table ) . there is currently no consensus on the diagnostic criteria for ventilator associated pneumonia (vap) which has led to significant variation in the reported incidence ( - %) . icus need a reliable and accurate diagnostic system so that the efficacy of measures to reduce the incidence of vap can be assessed, and if vap is to be considered as a quality indicator of performance. objective. to establish a process to diagnose vap that can be used for continuous surveillance and to assess the effect of new therapeutic interventions. method. potential diagnoses of vap were identified prospectively by a senior intensive care doctor and clinical data was collected to calculate a modified clinical pulmonary infection score (cpis) . each case was subsequently reviewed at a multidisciplinary team (mdt) review forum with intensive care and microbiology clinicians. the case details, modified cpis and semiquantitative microbiological culture results were used to reach a consensus view on the diagnosis. a pilot audit performed over a week period in , gave a vap incidence of % ( vaps; ventilated patients; ventilator days) . a change to our ventilator care bundle was subsequently instigated, with the addition of chlorhexidine paste to daily mouth care before repeat surveillance. result: patients were admitted to the critical care unit over days. patients were ventilated during a total ventilator days. patient episodes were identified as potential vap and discussed at the mdt forum. cases were diagnosed as vap (incidence . %) and patient diagnosed as tube associated pneumonia. the other cases were excluded either due to clinical circumstances (n = ) or absence of positive microbiology (n = ). conclusion. we have developed a process for identifying and diagnosing vap within our critical care unit that continues to be used for ongoing surveillance. collaboration between the intensive care team and the microbiology department, along with a mdt forum, has been fundamental to this process. our data highlights the difficulty in diagnosing vap and the need for multidisciplinary expertise. of the patient episodes prospectively identified as potential vap, were deemed not to be vap when clinical data and results were available for review at the mdt forum. the reduction of vap incidence between the pilot study and subsequent screening suggests benefit from the introduction of oral chlorhexidine, however we recognise other contributing factors. controversially, icus in the uk are increasingly being asked to provide data on vap incidence as a quality indicator. we believe that we have a process that is robust and allows us to accurately monitor the incidence of vap. introduction. bacterial biofilm within the internal surface of the endotracheal tube (ett) may contribute to ventilator-associated pneumonia. the gene expression pattern of those bacteria, embedded within biofilm matrix, greatly differs from their planktonic counterpart and allows survival benefits and increased virulence. to compare biofilm production capability of planktonic versus sessile methicillin-resistant staphylococcus aureus (mrsa) retrieved from within etts, and to assess whether antimicrobials can hinder those processes. ultimately, to study any change in planktonic versus sessile bacterial dna. we previously developed a model of pneumonia in pigs mechanically ventilated up to h and challenged into both lungs with mrsa. from those studies, etts of pigs, treated with placebo (n = ), linezolid (n = ) or vancomycin (n = ) were retrieved. distal and medial parts of each ett were processed ( samples). the planktonic mrsa strain, inoculated to the pigs, was compared with sessile mrsa strains, retrieved from the internal surface of the etts, in capability to form biofilm, assessed through the adhesion-to-aplaque method for gram-positive bacteria. the optical density of biofilm was measured using a microplate reader at wave length k = nm and results are proportional to the planktonic control strain (value = ). pulsed-field gel electrophoresis (pfge) was used to determine potential bacterial dna recombination. introduction. ventilator-associated pneumonia (vap) is a common complication in mechanically ventilated patients and is associated with increased morbidity, mortality and costs. treatment-related risk factors associated with vap, include prolonged duration of mechanical ventilation, lack of antiseptic techniques, improper patient's position, inappropriate cuff pressure, peptic ulcer prophylaxis, and deep sedation. objectives. the aim of this study is to evaluate the presence of these risk factors and their association with vap development in a multidisciplinary icu. one hundred and fifty-seven critically ill patients consecutively admitted to a multidisciplinary icu, were prospectively studied. forty-one of them, developed vap ( m/ f age: ± years, apache ii score on admission ± , sofa score on admission ± ). inclusion criteria were intubation \ h prior to icu admission or after h of admission. exclusion criteria included: prior hospitalization in another icu, icu stay\ h, brain death, age \ years old and pregnancy. bronchial secretions, samples from the pharynx and gastric secretions were collected from each patient times weekly (on days , and ). we also documented the known risk factors for vap; sedation depth (using the ramsay scale), cuff pressure, head-bed elevation, reintubation, ventilator circuit changes, tracheostomy, the presence of levin, deep venous thrombosis and stress ulcer prophylaxis, three times weekly per patient results. in this selected sample of patients, the incidence of vap was per , ventilation days. out of this sample, % underwent at least one endotracheal tube change, % had at least once their ventilator circuit changed, % underwent a tracheostomy, % had levin, % did not have head-bed elevation above °, % had ramsay scale or , % had cuff pressure b cmh o, % did not received deep venous thrombosis prophylaxis and all of them received h -receptors antagonists, as gastric ulcer prophylaxis. these results indicate that several risk factors for vap, do indeed apply for the cases under study and that the incidence of vap is relatively high. therefore, the implementation of preventive strategies and the reinforcement of vap bundles can result in beneficial effects on the appearance of vap, in the monitored icu. introduction. pulmonary inflammatory response and progressive ards may complicate posttraumatic ali. clrt by a motor-driven bed is used in trauma patients to improve gas exchange and to prevent from ventilation-associated complications. we investigated the effect of clrt on the inflammatory response in the posttraumatic course. to assess systemic and pulmonary cytokines (interleukin (il- , il- ), intercellular adhesion molecule- (icam- ) before and days after begin of clrt. conventionally positioned patients served as a control group. after approval by our institutional review board trauma patients presenting with ali (pao /fio \ ) were prospectively randomized in clrt (n = ) and control group (n = ). cytokines were assessed from serum (s) and broncho-alveolar lavage (bal) after admission at the icu and on day , respectively. results. the mean age was ± years and mean injury severity score was ± . pulmonary gas exchange improved significantly in clrt in comparison with conventional positioning on day . changes in cytokine levels are presented in table (median and / percentile values, * = p \ . within groups and $ = p \ . between groups, wilcoxon and mann-whitney-u test). serum il- and il- levels were reduced statistically significant on day in both groups, but this effect was significantly more pronounced in clrt-group. in bal cytokines tended to be increased in clrt and control group on day . icam- levels were increased in s and bal after days treatment. conclusions. the early use of clrt reduces the systemic inflammatory response (il- , il- ), but has no influence on regional pulmonary cytokine expression. clrt might be a helpful tool for stabilization in trauma patients with ali. objectives. the aim of this study was to compare the performance of adaptive support ventilation (asv) with and without closed loop control by end tidal co (etco ) (asvco ) to that of pressure control ventilation (pcv) and volume control ventilation (vcv) during simulated ards and to compare the ability of all modes to manage etco , tidal volume (v t ), and plateau pressure (pp) as respiratory mechanics, peep and minute volume changed. methods. asv and asvco were compared to a v t of ml/kg in vcv and pcv using the michigan instruments test lung set up with co titrated into one of the test lung chambers. the compliance of the system was set at and ml/cmh o and resistance at . cmh o/l/min. at baseline ventilation co was titrated to establish a co production of ml/kg pbw/min ( kg) or ml/min. after stabilization and data collection co production was increased to ml/ min and then to ml/min then decreased to ml/min. after each co adjustment and stabilization period data was collected. statistical analyses were performed by anova or kruskal-wallis tests where appropriate, a p value. was considered significant. overall etco level in asvco ( . ± . mmhg) was higher than in other modes (asv . ± . , vcv . ± . , pcv . ± . mmhg) (p \ . ). at the lowest compliance etco in asvco ( . ± . mmhg) was higher than in vcv ( . ± . mmhg) and pcv ( . ± . mmhg). overall v t was similar in all modes except that v t in asvco ( . ± . ml/kg) was lower than in vcv ( . ± . ml/kg) (p = . ). at the lowest compliance v t in asv ( . ± . ml/kg) and asvco ( . ± . ml/kg) were lower than in vcv ( . ± . ml/ kg) and pcv ( . ± . ml/kg) (p \ . ). overall, pp in asv ( ± . cmh o) and asvco ( . ± cmh o) were lower than in vcv ( conclusions. our high mortality in this group of patients despite improved oxygenation concurs with published data. however, potential improvements with adherence to ph and oxygenation targets could be made to ensure optimal use of hfov as a lung protective strategy. objectives. this randomized cross-over controlled study was designed to assess safety, gas exchanges, and ventilator outputs obtained with intellivent Ò as compared to adaptive support ventilation (asv) ( ) in icu ventilated patients with acute respiratory failure. methods. the study was approved by ethic committee and inform consents were obtained by next-of-kin. intubated, sedated and ventilated patients were included (age = ± years, saps ii = ± , and ali/ards and normal lungs patients, respectively). patients were ventilated using a g (hamilton medical, switzerland) with a dedicated software. asv and intellivent Ò were delivered in random order for two periods of h with half an hour of washout in between. tidal volume (v t ), peak pressure (ppeak), spo , and etco were continuously recorded. blood gas analysis and plateau pressure (pplat) were measured at the end of each period. a paired t test was used to compare ventilation and oxygenation parameters between asv and intellivent Ò period. no patient was removed from intellivent Ò for major safety issue. intellivent Ò delivered a lower mv ( . ± . vs. . ± . l/min, p = . ), peep ( ± vs. ± cmh o, p = . ), fio ( ± vs. ± %, p \ . ), vt/pbw ( . ± . vs. . ± . ml/kg pbw, p = . ), and pplat ( ± vs. ± cmh o, p = . ) than asv. static compliance, pao /fio ratio and paco were not different between asv and intellivent Ò . with both asv and intellivent Ò , v t was between and ml/kg pbw in all the patients. figure is showing the distribution of mean of individual breath by breath peak pressure during the two recording periods. conclusions. intellivent Ò delivered lower volumes and pressures than asv for equivalent results on gas exchange suggesting more efficient ventilation. introduction. during neurally adjusted ventilatory assist (nava) pressure applied to the airways by the ventilator is moment-by-moment proportional (according to a proportionality factor called ''nava gain'', unit: cmh o/lv) to the electrical activity of the diaphragm (eadi, unit: lv), as measured through a modified nasogastric tube with a multiple array of esophageal electrodes. independently from its physiological correlation with real diaphragm activity, eadi and its variations are the only variables through which different nava gains may affect patient respiratory pattern. objectives. we explored how eadi influence the effect of varying the nava gain on tidal volume and peak airway pressure. we included twelve patients recovering from acute respiratory failure. all patients, connected to a servo-i ventilator (maquet critical care, solna, sweden) able of delivering both psv and nava, underwent to a random application of increasing gains ( . - - . - - . - - cmh o/lv) during nava and increasing pressure support ( - - - cmh o) during psv. each level was maintained at least min. all changes in eadi, tidal volume (vt), and peak airway pressure (paw peak ) were referenced respect to their respective values at nava gain . . introduction. lung protective ventilation strategies is considered 'gold standard' for patients with acute respiratory distress syndrome (ards). high-frequency oscillation (hfo) has been used as a rescue measure and claimed to protect the lungs from ventilator induced lung injury ( ) . theoretically hfo should be considered early in the course of ards. objectives. we were interested to determine the time course to achieve optimal gas exchange with early hfo and its impact on the use of other adjunctive therapies; inhaled nitric oxide, prone positioning and extracorporeal oxygenation or carbon dioxide removal. after ethical approval, a total of adult patients with ards were enrolled in this prospective observational study. the oscillation (metran co. ltd, - , -chome, kawaguchi, saitama, japan) was started when fio [ . or level of peep [ cmh o were required on a conventional ventilator. hfo frequencies, cycle volume and bias flow were altered until optimal gas exchange achieved. the pattern of gas exchange was observed for the first h and other adjunctive therapies were used when required. the following parameters were recorded: duration of hfo, arterial blood gases during the first h, frequency of need for other therapies, incidence of barotraumas, and re-requirement of hfo once weaned from. a total of patients (age between - years, male:female ratio = : ) were ventilated - days with conventional ventilator before start of hfo. duration of hfo varied from to days ( median). there was a significant improvement in pao at - h (p = . ) following the onset of hfo (fig. ) . the number of patients requiring other therapies are very low; inhaled nitric oxide n = , prone positioning n = , both prone positioning and nitric oxide n = , ecmo/novalung n = . seven patients required re-hfo once weaned and another two developed pneumothoraces requiring chest drainage. conclusions. the data of this study demonstrates that significant improvement in gas exchange occurs in patients with ards following - h of hfo. early oscillation also appears to reduce the need for other adjunctive therapies. further studies of early hfo strategy are warranted. introduction. neurally adjusted ventilatory assist (nava) is a novel method for patient triggering of pressure support. instead of relying on conventional pressure or flow triggering, the peak diaphragmatic emg signal (edi) is used to precisely synchronise patient demand with ventilatory assist. although, the physiology and science underpinning nava has been described, less is known about its clinical application and efficacy ( ) . in particular, clinical superiority over conventional pressure support has not been established. objectives. here, we describe the clinical, nursing and operational lessons learnt from the introduction of nava platforms (servo-i, maquet) into a -bedded central london intensive care unit over years. in parallel we present some novel data from an observational cohort study examining the effect of nava on sedation use and ventilator days. a protocol for the safe measurement of edi in critically ill ventilated patients was developed through multidisciplinary review and discussion. a year registry of clinical lessons and adverse events from the introduction of nava was kept. in a parallel observational cohort study we examined three populations of ventilated patients (n = ) matched for apache ii, oxygenation index and compliance. group had no nava catheter; group had edi signal measured during conventional pressure support; and group utilised the edi signal to act as a neural trigger to drive ventilatory support. results. the measurement of edi was not associated with a difference in hospital mortality. there was a significant reduction in sedation use, muscle relaxation, ventilator days and icu days in patients with a nava catheter (fig. ) . however, there was no significant difference in any of these parameters with edi measurement guiding conventional pressure support versus nava. ventilator days in nava versus pressure support conclusions. in this study we show that neurally adjusted ventilatory assist can be safely and effectively be introduced into a 'non-expert' intensive care unit. we further show that reductions in sedation use and ventilator days appear to be related to the earlier introduction of sedation holds and reductions in ventilatory assist (with consequent earlier spontaneous breathing trials) in order to acquire an edi signal rather than due to an intrinsic advantage gained by improved patient-ventilator synchronisation with nava. the causes of the respiratory failure were: infections n = ; . % (immunodeficiency n = , tuberculosis n = , aplastic anemia n = , congenital cardiomyopathy n = , sepsis n = ), rsv infection-bronchiolitis n = ; . %, bronchopulmonary dysplasias n = ; . %, leukemias n = ; . %, burn-ards n = ; . %. the mean duration of hfov was . days ( h to days). the parameters of ventillation ranged: fio : - %, mean pressure: - cmh o, dp: - cmh o. the i time and hertzs ranged within the acceptable for age limits. closed suction circuit was used. we didn't notice any side effects from cardiovascular system or distension of the thorax. eadi and respiratory effort indices decrease when assistance increases in nava. nava should also theoretically allow perfect synchronization between the patient and the ventilator, and no asynchrony has been described with this mode. nava level adjustment at bedside, however, is still a matter of research. we assessed breathing pattern, respiratory effort indices, and patient-ventilator synchronization throughout a titration of nava and of pressure support ventilation (psv). we assessed breathing pattern, respiratory effort indices, and patient-ventilator synchronization throughout a titration of nava and of pressure support ventilation (psv). methods. physiological study conducted in seven patients (preliminary results) during the weaning. airway pressure (paw) and flow, esophageal and gastric pressures and the eadi were continuously recorded. for each patient, the following levels of assistance were consecutively applied: in psv: - - - - cmh o; in nava: . - - . - - . - - - - cmh o/mvolt. results are given as median and [ th- th percentiles], and tidal volume (vt) in ml/kg of predicted body weight. results. the increase from the lower to the higher level of assistance was associated with an increase in vt from . [ . - . ] to . [ . - . ] ml/kg in psv and from . [ . - . ] to . [ . - . ] objectives. we describe technical possibilities, tolerance and gas exchange with combination of oscillation device with nimv. a specific oscillator device (sensor medics, vyasis) was selected to change conventional airflow to oscillatory airflow. connected by ''t'' piece with oscillatory devicerespiratory circuit-face mask. we selected bipap ventilator (vision resp inc), facial mask. bipap mode (iap/epap cmh o) to achieve stable mechanical ventilation (t ). a progressive increment in rate of oscillation (hz) and power oscillation to achieve airflow change (t ). to evaluate the differences of peepi during the release phase, referred to mechanical pressure release from p high to p low , and the inspiratory work of breathing (wob) during p high among currently available aprv ventilators, using a lung model (ttl , mi). methods. the six aprv ventilators were evaluated in this study: ) nellcor puritan-bennett (puritan-bennett), ) evita xl (drager), ) servo i (maquet), ) avea (viasys), ) g (hamilton) and ) engstrom (ge healthcare). ventilator settings in aprv ventilators were set as follows: p high /p low cmh o/ cmh o, t high /t low . s/ . s. when evaluated peepi during the release phase, t low was diminished by steps of . s from . to . s. the inspiratory pressure-time product (ptp), as an index of wob, was directly measured under with tidal volume/respiratory rate of ml and /min. measurement for each setting was performed in a random order. three breaths were analyzed and averaged for each measurement. the results are shown in fig. objectives. to perform bench study to test the hypothesis that ett or trach can reduce the insufflated (vti) and/or exsufflated (vte) volume as provided by coflator Ò . methods. coflator Ò is connected to a pneumatic model (ttl, michigan instruments) via ett mallinckrodt . , . , . , . , . ) or trach (mallinckrodt . , . , . ) . the set up is tested in conditions of compliance (c ml/cmh o) and resistance (r cmh o/l/s): c r , c r , c r , c r . the device is set in manual mode, at the highest inspiratory flow, inspiratory/expiratory time / , s pause after expiration. three pressures are used, , and cmh o in both inflation and deflation. a baseline condition without any tracheal prothesis nor r served as reference. airflow and pressure upstream ett or trach are measured (biopac mp ). five insufflations followed each by an exsufflation are performed in each condition. the data are analyzed by using a linear mixed effects model where ett or trach, and pressure have fixed effects and c or r a random effect. the dependent variable is vti and vte. the main end-point is the slope of the relationships between vti or vte and pressure. results. the vti-pressure slope was significantly lower with any ett than baseline at both c ( fig. a and b). the same was true for trach. even though these differences were statistically significant the reduction in vti from the baseline averaged ml with etts as a whole. the vte-pressure relationships were similar for ett and trach and showed no difference from baseline at c ( figure d ). however, at c , the relationships are scattered ( figure c ). moreover, expiratory pressure of - and - cmh o were not reached for ett . , . and . and trach . and . . conclusions. ett and trach slightly but significantly change the performance of the device. however, small sized ett or trach in c condition substantially modify the efficacy of the exsufflation. these findings desserves assessment in patients for relevance. rd esicm annual congress -barcelona, spain - - october s though humidified high flow nasal canula oxygen (hfnc) has been widely studied in pediatric population, few data in adult is available and its and its precise indications and actual benefits in these patients remains unknown. two studies have shown that hfnc generates a low level of positive airway pressure contributing to decrease the work of breathing. one study has reported a favourable effect on comfort and oxygenation of hfnc as compared to venturi mask and in another preliminary one, fewer patients using hfnc went on requiring non invasive ventilation. objectives. to evaluate the efficiency of hfnc (optiflow, fisher and paykel, auckland) in critical care patients with acute respiratory failure. methods. prospective single centre study in a university hospital intensive care unit. all patients exhibiting acute respiratory failure (arf) as defined by clinical signs and/or failure of haematosis, regardless of the aetiology, were eligible. hfnc was used at a mean output of ± l/min and a mean fio of ± % throughout the study period. results. thirty-five patients were included. the mean age was . ± . years old and mean saps ii is ± . pulmonary infection was the most common aetiology of arf. hfnc was used ± days. all but one patient were discharged alive from icu. optiflow significantly reduced respiratory rate (p \ . ), heart rate (p \ . ), dyspnea score (p = . ), sus clavicular recession and thoraco abdominal asynchrony, and improved pulse oxymetry (p = . ). there was no significant difference in ph, pao , paco on arterial blood gazes performed before and , and h after hfnc use. the pao /fio ratio at and h after hfnc initiation was higher than before use of hfnc (p = . ). patients ultimately intubated exhibited a higher respiratory rate min ( . ± . vs. . ± bpm, p = . ) and min ( with an incidence of up to % in routine sonography [ ] , pleural effusion is one of the most common complications in intensive care patients. the use of bedside d ultra-sound to detect pleural effusion is fast, practicable and minimises radiation exposure [ ] . however, sonographical volumetry tends to be imprecise, and ct imagingbased volumetry is the more accurate method to quantify pleural fluid in intensive care patients [ ] . objectives. the aim of this study was to compare the accuracy of d and d ultra-sound volumetry of pleural effusion in intensive care patients. methods. icu patients designated for thoracentesis because of radiological support for pleural effusion were examined both with d and d ultra-sound \ h before and after the intervention. effusion volume was calculated with the formula ''volume [ml] = sep [mm] '' [ ] for d measurements and compared to the d volumetry by the ge d view software. the difference of volumetry before and after intervention was compared to punctured volume (gold standard). the device used for u-sound measurements was the ge voluson i. . within the group under survey (n = ), eight patients were on the respirator. mean punctured volume was ml ( ml; ml). mean absolute value of deviation from punctured volume (gold standard) was ml ( . %) for the d measurements and ml ( %) for d measurements. biggest difference between methods was found between ml and ml punctured volume. none of the methods generally measures higher volumes than the other. conclusions. the size of deviations from gold standard in both directions in d measurements suggests that the method is not reliable in predicting pleural effusion volume. d measurements are more likely to predict the right effusion volume, making the method a better diagnostic tool than d u-sound measurements. although thoracocentesis was found to be safe for mechanically ventilated patients [ ] and d volumetry may not change the therapeutic decision, which depends on high-risk patients' overall clinical condition, it could nevertheless help to avoid unnecessary interventions and thus improve patient safety. ongoing work analyses results for a group of patients with ct imaging-based volumetry of pleural effusion as gold standard. we study the utility of deltacvp changes during pressure support ventilation (psv) as an indication of respiratory effort. patients after several t-trials failures were on psv. the level of ventilatory assistance was changed in order to set the psv. no aditional interventión was used in the management of these patients. we registered data from the ventilator and beside monitor (vt, rr, p , deltacvp) in two levels of ps. optimal-ps, as the lowest support without respiratory distress and with the middle of this level -psv. the inspiratory trigger was used with the highest sensibility without autotrigger, the inspiratory ramp was changed in every patient and expiratory was flow-cycled at % of the peak inspiratory flow. results. patients n, acute on chronic respiratory failure, n, heart failure, n, recovery of acute respiratory failure. age ± years, admission apache ii ± and they needed weaning with ps after ± days with control mechanical ventilation. the respiratory mechanics in this time were cst.rs ± ml/cmh o and raw.rs ± cmh o/l/s. the ramsay score was ± ( ) ( ) ( ) ( ) ( ) . when the optimal level of ps was decreased from ± to ± cmh o, all patients showed respiratory distress, and the variables studied changed significantly: p : . ± . to . ± . cmh o, deltacvp . ± . to . ± . cmh o, vt decreased from . ± . to . ± . l and rr increased from ± to ± bpm (p \ . ). the correlation between p and del-tacvp was . (p = . ). central venous pressure swing provided information about the respiratory effort and may be useful during pressure support ventilation. introduction. the use of a tidal volume of ml/kg of predicted body weight is part of the management of patients presenting with ards ( ) and prevents ventilator induced lung injury in icu patients undergoing mechanical ventilation ( ) . the setting of tidal volume is based on patient height. height seems to be more often estimated by the icu staff than actually measured ( ). objectives. our study aimed to assess the accuracy of the visual estimation of patient height and its impact on ventilator settings. thirty-two patients admitted to a surgical icu were prospectively included in this observational study. patients had their height visually estimated by icu staff members ( doctors, nurses and nurse-assistants) and then measured. we also measured the knee height from which height can be extrapolated. we then compared the mean estimated height for each patient to the actual measured height with the use of bland and altman plots and the consequences of the measurement error on the tidal volume setting. in most patients visual estimation overestimates the actual height. the measurement errors result in an increase in the tidal volume up to + ml/kg. this remains true whatever the subgroup studied: all patients (bias: . ± . , % ci: - . ; . ), male patients (bias: . ± . , % ci - . ; . ), female patients (bias: . ± . , % ci . - . ) and whatever the type of assessor. extrapolated height from knee height results invariably in an underestimation of actual height (bias - ± . , % ci - . ; . ). our results prove that neither visual estimation nor knee height measurement are reliable surrogates for measured height. therefore, measuring patient height should be mandatory in critically ill patients in order to minimise ventilator-induced lung injury. methods. , respiratory acts were recorded in icu patients selected because of severe asynchrony with the ventilator (g , hamilton medical) during psv. by visual analysis (va) of airway pressure and flow trajectory, ineffective efforts and inspiratory/expiratory delays were detected. the results of va were compared with those provided by a new algorithm based on the automatic analysis (aa) of flow trajectory. results. va identified ineffective efforts ( % of patients acts). among , assisted acts, average inspiratory and expiratory delay was ± ms and ± ms, respectively. significant inspiratory and expiratory delay ([ ms) occurred in , ( %) and in acts ( %), respectively. automatic analysis was able to identify , of , patients acts ( %). in , cases ( %), both inspiratory muscles contraction and relaxation were detected. in acts ( %) only relaxation was identified. compared with va, inspiratory and expiratory delay of aa was ± and ± ms, respectively. aa recognized the start and the end of patient's effort before the ventilator in , ( %) and , ( %) of assisted acts. sensitivity and specificity of aa in detecting ineffective efforts, inspiratory delays [ ms, expiratory delays [ ms were and %, and %, and % respectively. the new algorithm proved to be efficient as a real-time, continuous monitoring system of patient-ventilator interaction. the advantage over traditional flow and pressure based triggers has to be tested. introduction. mechanical ventilation remains as one of the most difficult safety issues in the icu, but parameters commonly monitored by ventilators only depict the most extreme risks for patients. new computerized approaches may manage exhaustive data and may include ''intelligent'' software that mirrors expert decision making. to test the clinical usefulness of a new computerized system as a herald for clinically significant alarms and its possible impact in outcome. methods. twenty-five mechanically ventilated patients were continuously monitored in a single mixed icu in a university-affiliated hospital. we recorded age, diagnosis, pao /fio , apache ii and sofa on admission. the computerized system grabs and process data from different devices, usually a monitor and a respirator, and evaluates the most relevant events in a ventilated patient. all the algorithms were designed and validated with the clinical staff. data of ventilated patients were recorded at the icu during months and a total of , , breaths from different patients, each of them with a register of at least % of total ventilation time, were collected. data include biomedical signals (waves and trends) as well as all the clinical events detected by the system, including trapped gas at end-expiration, presence of secretions, double-cycling, asynchronies during expiration, pulse pressure variation in patients not triggering the ventilator and stress index ([ . or . ) in those ventilated with square airflow. outcome variables were icu length of stay, hospital stay and mortality. statistical analysis included multiple regression models for length of stay and mortality. [ ] [ ] [ ] [ ] [ ] [ ] [ ] , icu length of stay . days [ . - ] , hospital length of stay days . the frequency of alarms were: trapped gas at end-expiration: . %, presence of secretions . %, double-cycling . %, asynchronies during expiration . %, stress index [ . . %, stress index . . %, and pulse pressure variation . %. multiple regression analysis found pao /fio associated with length of mv and close to significance with hospital stay and mortality, but any of the computerized alarms reached yet the level of significance. conclusions. the computerized system is able to detect and review more clinically significant problems than clinical routine. however, its impact to define patient outcomes warrants further investigation. objectives. we have assessed the ability of the ventilator t-bird vs and ltv- to deliver to a lung model with ards a set tidal volume (vt) at different simulated altitudes. we used a decompression chamber to mimic the hypobaric environment at a range of simulated cabin altitudes of , , , and , m ( , , , , , feet). ventilators were tested with realistic parameters. vt was set at and ml in an ards lung model. the positive end expiratory pressures (peep) were set at and cmh o. pressure drop across the pneumotachograph was measured by a differential pressure transducer (enertec tm ). the spirometer was checked at each altitude using a calibration syringe. the inspired oxygen content (fio ) was %. respiratory rate was breaths/min. the ratio inspiratory time/expiratory time was / . the protocol included three measurements for each simulated altitude. comparisons of preset to actual measured values were accomplished using a t test for each altitude. a significant difference was defined by p \ . . the standard deviation for the three measurements obtained at each altitude was consistently less than ml. respiratory rate delivered was breaths/min in all cases. variation of peep did not change the volume delivered. the t-bird vs showed a decrease in volume delivered. comparisons of actual delivered vt and set vt demonstrated a significant difference starting at , m for a vt set of ml, at , m for vt set of ml. at these altitudes, the variations between vt set and delivered were more than %. with decreasing barometric pressure, the ltv- showed mostly an increase in volume delivered. comparisons of actual delivered vt and set vt demonstrated a significant difference at , m for a vt set of ml, at , m for vt set of ml. the delivered tidal volume remained within % of the set vt. assuming that the patient is ventilated at sea level and gas exchange is normal, the movement to altitude would result in an increase in tidal volume which might in fact represent a clinical event. conclusions. the ltv- met the trial targets in all settings, whereas the t-bird-vso did not compensate well for altitude and progressively delivered lower volumes as barometric pressure decreased. such variations between delivered and set vt suggest lack of efficacy of altimetric correction in hypobaric conditions in some devices. the ltv showed a moderate increase in volume delivered for ards lung model with increasing altitude, but maintained the delivered volume within % of the set vt up to , m. the accuracy of the vt delivery was superior with the ltv- than with the t-birdvso . oxygen therapy is commonly used to correct residual oxygenation impairment in the post-extubation period. this is usually done through a venturi mask which allows to deliver predetermined fractions of oxygen humidified with bubble humidifiers. the low humidity delivered by such devices and the use of an oro-nasal mask may, however, reduce patient's comfort, possibly resulting in mask displacement or removal and consequent oxygen desaturations. nasal high-flow (nhf) oxygen therapy allows to deliver high-flow oxygen, humidified with heated humidifiers and delivered through nasal cannulae, with the potential to improve comfort and efficacy. objectives. in patients requiring oxygen therapy after extubation, we compared nhf vs. venturi mask in terms of oxygenation and comfort. patients who were mechanically ventilated for more than h, passed a spontaneous breathing trial, and had pao /fio \ at the end of the trial were randomized to receive oxygen with nhf or venturi mask after extubation. exclusion criteria were: tracheostomy, age \ , pregnancy, or anticipated need for noninvasive ventilation after extubation. in both groups, fio was set to obtain spo between and % ( - % in copd patients). with nhf, flow rate was set at l/min. arterial blood gases, respiratory rate, and discomfort were assessed at , , , , , , and h. discomfort was assessed by asking patients to rate their discomfort with the used device by using a numerical scale from (no discomfort) to (maximum imaginable discomfort). discomfort symptoms were also assessed for the dryness of the delivered oxygen (dryness of the mouth, throat, nose, difficulty to swallow and throat pain). incidence of desaturations and interface displacement was also assessed. objectives. to evaluate the optimal humidifier water temperature when using a helmet for noninvasive positive pressure ventilation. oxygen. each was sequentially tested in the following order: using the helmet without humidification at ambient temperature, with humidification with unheated chamber water, and with humidification with the chamber water at , , and °c. at each setting, after a min stabilization period, measurements were taken. comfort level at each setting was evaluated using a visual analog scale (vas) rated zero (most comfortable) to ten (least comfortable). temperature and relative and absolute humidity inside the helmet, and vas scores statistically significantly increased as the humidification chamber water temperature increased. the lowest vas, . ± . , was obtained when water in the humidifier chamber was at ambient temperature. conclusions. for patient comfort during cpap using a helmet, the most desirable conditions are likely to obtained by humidifying without heating, that is by leaving the water in the humidifier chamber at room temperature. introduction. the physiological and clinical effects of non-invasive ventilation (niv) on acute post-operative respiratory failure are relatively unknown. the aim of this study was to determine the prediction factors for failure in the use of niv with a helmet in this context. the use of niv was assessed for a period of years, in a post-operative intensive care unit (icu). demographic data was collected, as well as arf and arterial gas readings. haemodynamic changes were assessed using picco tm technology and the clinical development of patients was recorded. all patients who developed acute respiratory failure (arf) were treated using niv as their primary care, and the two groups for the study were determined in this way, depending on whether the technique was successful, or the patients required intubation. the risk factors that determined failure in the application of niv were subsequently determined. of the patients presenting with post-operative arf treated with niv using a helmet, did not require intubation ( . %). following a multivariate analysis using logistic regression, we determined that there are four independent risk factors for the failure of niv. the primary causes of respiratory failure are as follows: acute respiratory distress syndrome (ards) and pneumonia, and in second place, the high initial evlwi (extravascular lung water index) value, as a protective factor, is the increase in the po /fio ratio after the first hour of niv application. conclusions. niv using a helmet could provide an effective alternative to conventional ventilation in selected patients with post-operative arf. -jaber s, delay objectives. analyze niv practice in emergency departments. we have development an international epidemiology survey (march ) by electronic questionnaire to know niv organization, equipment and training in emergency departments (phase i). design. international multicenter prospective. we have enrolled information from hospitals: spain ( ); italy ( ); india ( ); usa ( ); slovakia ( ); turkey ( ); germany ( ); australia( ); chile ( ); singapore ( ); finland ( ) during to analyze noninvasive practice in prehospital and emergency medicine, equipment, interfase, ventilatory modes and common clinical applications. major results during month period analysis were: noninvasive mechanical ventilation were applied in prehospital: ( ) ( . %). nimv commonly was applied follow a objective pprotocol of nimv: ( ) ( ) ( . %). global rate indication of niv were copd exacerbation ( %) and cardiac pulmonary edema ( - %). all niv applications were successful applications in emergency departments (avoid eti %) with minor complications ( . %) (skin nose lesion). equipment more relavant were (cpap devices ( %) and facial mask ( %) was more frequent, follow total face( %); nasal mask ( %); helmet ( %); type ventilary mode: cpap ventilatory mode was frequent used as first line ( % ( ) suggest niv should be started within h of not responding to the maximal medical therapy for acute type respiratory failure patients. following an earlier audit presented as an abstract at esicm ( ) , which looked at possible delays in starting niv, protocols were implemented to start niv earlier at russells hall hospital acute admissions unit. objective. the aim was to assess the delays in starting the appropriate patients on niv at admission looking at impact on adverse outcomes. method. data was collected retrospectively using bts niv audit tool. cases admitted with type respiratory failure in our hospital between january and march who needed niv were included in the audit. delay was subdivided into a) door to first arterial blood gas (abg) sampling b) abg to decision for niv and c) from decision to actual starting of niv. results. the mean time to get an abg from admission was min. cases with delay more than h skewed the data. the median time, which was more representative of the usual delay between admission and first abg, was min. our audit showed that out of ( %) patients had abg within an hour compared to out of ( %) in the last audit. although noninvasive ventilation (niv) has been widely used in patients with acute on chronic respiratory failure (acrf) due to chronic obstructive pulmonary disease (copd), series studying patients with pulmonary restriction due to morbid obesity (mo) are rare ( ) , despite the disease is highly prevalent in our environment. objectives. the aim of our study is to analyze and compare the effectiveness of niv in patients with copd and om. we analyzed all patients admitted to icu for a period of years with diagnosis of acrf due to copd or mo and treated with noninvasive ventilation. niv success was defined as the avoidance of endotracheal intubation, survival in icu and at least h on a medical ward with no signs or symptoms of respiratory failure. variables are expressed as means ± standard deviation and percentages. comparison between variables by pearson's v test and student t. we analyzed survival and hospital readmission per year (log rank test). during the study period, patients were admitted with exacerbation of copd and with mo. all patients were treated with two levels of pressure. age differs between copd and om, ± and ± years, respectively (p = . ), as well as the percentage of men, . adaptive support ventilation (asv) Ò (hamilton galileo) has been shown to result in better patient synchrony, reduced weaning times and reduced work load for the icu staff ( , ) . but, data is lacking on its efficacy, especially as non invasive ventilation (niv) due to the concern of being closed loop ventilation. also, little is known about the risk factors of late niv failure in patients who improve initially ( objectives. to assess end tidal co monitoring in patients with hypercapnic exacerbations of copd requiring niv methods. simultaneous measurement of paco and petco was performed in groups of patients. paco was measured using arterial blood gas analysis and petco was measured using non-invasive capnography. the groups were; phase a: mechanically ventilated patients post coronary artery bypass graft, used to establish the reliability of the end tidal carbon dioxide monitor in a homogenous group of previously well patients. phase b: patients with copd who did not have symptoms associated with an exacerbation, used to assess the use of a non invasive sampling device and assess the sampling method in stable copd patients. phase : patients with a hypercapnic exacerbation of copd requiring niv. capnography was monitored continuously in this group and petco values were calculated based on a mean value min before and after the arterial blood gas sample. this was to avoid any sampling error as it was impossible to isolate the exact moment of arterial puncture. agreement between the sampling methods was assessed using the bland-altman method. phase a objectives. we aimed to evaluate the possible harm of niv failure in routine practice among spanish icus. methods. we extracted patients with acute respiratory failure requiring either invasive or noninvasive mechanical ventilation in spanish icus during the -month period of the validation of the sabadell score ( ). we recorded demographic parameters and treatments received during the icu stay. patients were followed until hospital discharge or death. results. we analyzed , patients, of whom , ( %) received only invasive mechanical ventilation (imv) and ( %) received niv. niv succeeded in % of patients, but the other % required intubation. niv failure was more common in neurologic ( %) and post operatory ( %) and less frequent in coronary patients ( %). mortality was lower than predicted in niv patients ( vs. %) and similar to predicted in imv patients ( vs. %). mortality was lower than predicted in patients in whom niv was successful ( vs. %) and (similar or slightly lower than to predicted) in those in whom niv failed ( vs. %). conclusions. routine use of niv seems to confer a benefit, even when it fails and intubation is needed. reference(s tables and shows the parameters on respiratory muscles. introduction. the effectiveness of non-invasive ventilation (niv) in the setting of hypoxemia de novo remains controversial. it has been detected that patients in whom niv fails and intubation is required have a high mortality. otherwise, in patients in whom niv avoids intubation, survival rate is also high. to identify the factors involved in success or failure of niv in critically ill patients with hypoxemia de novo. we retrospectively studied all the patients admitted in our -bed intensive care unit (icu) from january to december with the diagnosis of hypoxemia de novo. do-notintubate patients were excluded. the indication of niv was at medical discretion, as well as intubation criteria. we defined the hypoxemia de novo as acute non hypercapnic respiratory failure due to a different cause from cardiogenic pulmonary edema. we defined two groups of patients: ) niv failure, patients who required intubation, and ) niv success, patients who did not require intubation. we collected demographical variables (age and gender), etiology of the hypoxemia, severity scores on admission (saps ii and sofa), glasgow coma scale (gcs), respiratory rate (rr), pulsioximetry (spo ), ph and fio , before and h after starting niv, episodes of nosocomial respiratory infection, length of stay (los) in icu and icu mortality. we compared both groups using the mann-whitney non-parametric test. p \ . was statistically significant. we studied patients ( women and men) with a mean age of ± years. the etiology of respiratory failure was: ards (n = ), pneumonia (n = ) and others (n = ). there were patients in the niv failure group ( %), and in the niv success group ( %). niv failure rate was higher when hypoxemia was due to ards (p = \ . ). objectives. this retrospective analysis aimed to assess outcomes following instigations of niv in a variety of clinical conditions. outcome data for copd/apo and non-copd/apo groups were compared. we assessed whether outcomes differed between these groups. in addition we wished to assess how outcomes varied across non-copd, non-apo conditions. objectives. the aim of this study was to compare patient's respiratory effort with three different noninvasive ventilators currently used on critical care patients and selected from a bench study. six patients treated by niv to prevent respiratory failure after extubation were included. each subject was successively submitted to a randomly assigned min-period of ps-niv with three different ventilators: bipap vision (respironics), elisée (resmed) and oxylog (dräger medical). these ventilators have different performances in a bench comparison. ventilatory settings were adjusted for the first ventilator and maintained for the followings. ps level was increased in order to obtain a tidal volume of - ml/kg of body weight (ps ± cmh o). flow, airway and oesophageal pressures were recorded. the oesophageal pressure time product (ptpoes) and tidal oesophageal swing (dpoes) were measured to evaluate patient's respiratory effort. results. no significant differences in tidal volume, respiratory rate and autopeep were found between ventilators. the dpoes and ptpoes, however, were significantly higher with oxylog as compared to bipap vision and elisée , as expected from the bench comparison. there are limited data on niv s efficacy in hypoxic respiratory failure. objective. to investigate the epidemiology and outcomes of patients administered niv as first line respiratory support in a mixed medical-surgical icu over a year period (jan -dec ), in an academic medical center. methodology. data abstraction from icu database, clinical care manager and chart review. results. surgical patients (sp) and medical patients (mp) were administered niv. the sp were % male, and had a median age of years. the mp were % male, and had a median age of years. % of sp were admitted with type respiratory failure (t rf pao \ kpa), % were admitted with type respiratory failure (t rf paco [ kpa) and the remainder were admitted with respiratory distress (rd). % of mp were admitted with t rf (pao \ kpa), % were admitted with t rf (paco [ kpa) and the remainder were admitted with rd. the median length of stay (mlos) was days for sp (range - ); the mlos for mp was days (range - ). sp were commenced on niv on average . h after admission (range - h), and remained on niv for a median of . (range - ) h. % of surgical patients required intubation, and the mortality rate was . %. mp were commenced on niv on average h after admission (range - h), and remained on niv for a median of (range - ) h. % of medical patients required intubation, and the mortality rate was %. logistic regression was applied to all datasets. among medical and surgical patients there was no correlation between the type of respiratory failure, initial blood gas or ph and the need for subsequent intubation, or risk of death. hematology patients had a mortality rate of % and accounted for % of overall deaths. oncology patients also had a % mortality rate, and accounted for % of overall deaths. amongst the mp that presented with hypoxemia, the intubation rate was % and the mortality rate was % (although not all patients that died were intubated). amongst the mp that presented with hypercarbia, the intubation rate was % and the mortality rate %. summary. niv successfully prevented intubation in more than % of patients. patients presenting with hypoxic respiratory failure were no more likely to be intubated than those presenting with hypercarbia. two-thirds of hematology and oncology patients treated initially with niv subsequently died. a microdialysis system was composed and the time delay of the system, recovery time, was introduced and tested with a fluids switching method. twelve sd rats were divided into ir or control group. myocardial ir was induced by ligating ( min) or releasing ( min) the suture underlying lad. mycrodialyisis probe was implanted into the left ventricular myocardium perfusion area to be occluded. dialysate samples were collected every min. blood samples were drawn at the beginning and at the end of the procedures. dialysate calcium concentration ([ca++]i) was detected with an atomic absorption spectrophotometer. serum calcium and ctnt were detected. recovery time for the microdialysis system was min, recovery rate was %. [ca++]i showed no changes during ischemia and descended immediately after reperfusion,reached the lowest level at min after reperfusion, then escalated slowly while keeping lower than control with significant difference. there was no difference in serum calcium at the beginning ( objectives. to evaluate the causes, incidence and impact on outcome of admission hyperlactatemia in patients admitted to a general micu. methods. data were retrospectively collected from the patient records for all adult patients admitted in the micu during the -months period. data regarding patient demographics, probable cause of hyperlactatemia, presence of shock on admission, need for organ support and icu outcome were recorded. patients were divided into two groups based on admission lactate levels: high lactate, with levels of mmol/l or more and normal lactate, with levels less than mmol/l. patients in these two groups were compared in terms of need for organ support and icu mortality. the efficacy to discriminate between survivors and non-survivors was assessed by area under the receiver operating characteristic curve (auroc). introduction. during critical illness alterations in blood flow are thought to predispose to organ dysfunction and hemodynamic therapy is often targeted at maintaining organ perfusion. however, abnormal blood flow distribution during critical illness may cause regional blood flows to correlate poorly with systemic haemodynamics ( ) . currently, our understanding of blood flow distribution during critical illness in humans has been limited by the invasiveness of established techniques for its measurement. objectives. phase-contrast mri (pc mri) represents an entirely non-invasive, contrastfree, method of measuring blood flow in major blood vessels ( , ) . we sought to apply this technique to technique to the measurement of organ blood flow in the critically ill. in a pilot proof of concept study, we measured renal and portal blood flow by pc mri critically ill humans with sepsis, multi-organ dysfunction and acute kidney injury (aki). in individuals cardiac output was measured by thermo-dilution in the icu, in the remaining patients we measured cardiac output (ascending aortic flow) and also descending thoracic aortic blood flow using pc mri techniques. we studied critically ill individuals with severe sepsis and aki. when studied, were mechanically ventilated, were on continuous haemofiltration and required vasopressors. transport and mri examinations were carried out without complication. in these patients, median cardiac index was . l/min/m (range . - . ), median renal blood flow ml/min ( - , ) and median renal fraction of cardiac output . % ( . - . ). median portal blood flow was ml/min ( - , ). descending aortic blood flow (measured in patients) ranged between and % of cardiac output (median %). conclusions. phase-contrast mri can efficiently and safely assess organ perfusion during critical illness in man. near simultaneous measurement of cardiac output enables organ blood flow to be assessed in the context of the global circulation. preliminary observations suggest renal blood flow is consistently reduced as a fraction of cardiac output in established aki. pc mri may be valuable to future investigation of organ dysfunction and vasoactive therapies in sepsis and critical illness. objectives. we were interested in the effects of the higher pco -levels on the microcirculation of infants with birh weights \ , g. data were collected from infants, who were randomized either to treatment with permissive hypercapnia or normocapnia. inclusion criteria were a birth weight between and , g, a gestational age from rd to th+ weeks, intubation during the first h of life and no malformations. the pco target range was increased stepwise and was mmhg higher in the intervention group. skin microvascular parameters were assessed noninvasively with sdf on the right arm every h during the first week of life and on the th day. results. pco (auc: ± vs. ± ) differed significantly between the two groups (p = . ). functional vessel density (fvd) was significantly lower in the intervention group on the th day of life ( ± vs. ± cm/cm ; p = . ). the proportion of small vessels increased in the control group whereas they decreased slightly in the intervention group, but did not reach stat. sig. increasing target pco lead to a temporary hyperdynamic flow in both groups. conclusions. pco -levels influence significantly the microcirculation in preterm infants. elevation of pco -levels leads to a decrease in fvd, presumably due to shunting and vasoconstriction and might cause temporarily hyperdynamic flow. methods. blood from healthy volunteers were diluted with hes, albumin %, rl or autologous plasma to obtain a final hematocrit of %. in vitro wbv measurements were made by the rheolog tm device (rheologics, exton, pa), a new viscometer with a u-formed capillary. the flow rate (determined by the rate of change in height of the columns of blood) is directly related to the pressure drop across the capillary tube. the shear rate (from , to s - ) and viscosity of the sample can be mathematically derived. results were expressed as median values (with - % intervals) and compared by anova with bonferroni correction. a p value . was considered as statistically significant. hemodilution with rl and albumin decreased significantly the wbv for all shear rate compared with autologous plasma and hes ( fig. ). conclusions. in contrast to albumin and ringer's lactate, hes and autologous plasma increased the whole blood viscosity, suggesting that these solutions may be preferred in severe hemorrhagic shock to better preserve plasma viscosity and microcirculation. we divided into two groups the randomly selected sample from the scope of patients come through open-heart operation assisted with extracorporeal support at the university of pécs: therapeutic (continuous blood gas monitoring/cdi- ) and control (intermittent sampling) group. after the retrospective data collection we carry out the analysis with (prevalence) frequency and confidence interval calculation and khi square test. results. the following accompanying diseases occurred significantly higher rate in the therapeutic group: ami (p = . ), kidney disease (p = . ), chronic pulmonary disease (p = . ), and the aggregation of the accompanying diseases showed also significantly high degree (p = . ). the long interval operations occurred significantly higher rate (p = . ) in the therapeutic group, and the times of the aorta clinch (p = . ) and the perfusion (p = . ) was also significantly longer. despite of that during the perfusion in a significantly more cases remained the rates in the normal range concerning to the therapeutic group (ph: p \ . ; be: p \ . ; pco : p \ . ), and the prevalence of the restart of the heart showed also significantly higher rate (p = . ). the continuous blood gas analyses assure reliable and the postoperative recovery assisted ecc circulation support. this assists considerably for keeping the parameters in the physiological limits even in the higher rate of the incidences of complex operations and accompanying diseases. this could contribute to lower incidence of side effects, preventing the causeless elevation of the postoperative hospital charges. objectives. describe the changes in capillary perfusion after erythrocytapheresis during severe falciparum malaria. we report two cases of severe falciparum malaria and describe the evolution of the sublingual capillary perfusion after erytrocytapheresis. the sublingual microcirculation has been studied with sidestream dark-field imaging (microscan; microvisonmedical tm , amsterdam). the device was applied on the lateral side of the tongue and the video images ( - captures of - s.) of capillary perfusion were recorded. the microcirculatory scores were analysed offline: small vessels (\ lm) density (number of vessels/mm), percent of continuously perfused small vessels (ppv%) and mean flow index (mfi). mmol/l. the capillary perfusion has improved: capillary density increased ( . /mm), the proportion of perfused vessel increased ( %) and flow was continuous in most vessels (mfi: ). clinical evolution was rapidly favourable and the patient was discharged from the intensive care unit. case . severe falciparum malaria with high parasitemia ( %) and acute renal failure. before erythracytapheresis: macrohemodynamic parameters were normal but microcirculation was reduced: vessels density ( . /mm) with % of small vessels perfused and the flow was slow in most vessels (mfi: . ). after erythracytapheresis: parasitemia decreased ( . %). sublingual microcirculation has improved with an increase in small vessels density ( . /mm) among which . % were perfused with a continuous flow (mfi: ). the patient had a good outcome. conclusions. microcirculation monitoring should be assessed specifically in some critically ill patients, even if macrocirculatory parameters are in the normal range. during severe plasmodium falciparum malaria, this monitoring could be specifically important to assess the effect of erytrocytapheresis therapy on tissue perfusion. rd esicm annual congress -barcelona, spain - - october s objective. perioperative myocardial infarction (pomi) is associated with significant mortality and morbidity in cardiac surgery. the primary objective of this prospective multicenter study is to investigate whether monitoring of coronary sinus metabolic markers can reliably predict ischemia and pomi faster than conventional monitoring. method. patients undergoing cardiac surgery were monitored perioperatively using a transjugular implanted microdialysis catheter (cma microdialysis) to study the metabolic changes of the heart. coronary sinus (sc) samples of lactate, pyruvate and glycerol were obtained continuously through -h post-operatively. pomi was defined by ckmb c u/ l and troponin t c . lg/l. a total of patients met the criteria for pomi. patients showed at least one adverse event during the postoperative course. lactate, lactate-pyruvate-ratio and glycerol levels in the sc sharply increased up to h before rise of cardiac enzymes. analyses of regression and discriminate analyses showed statistically significant (p \ . ) relationships between elevated metabolite values and the occurence of pomi. roc analysis revealed that lactate, lp-ratio and glycerol from the sc are sensitive markers to predict pomi and postoperative clinical events. conclusions. coronary sinus metabolic markers are sensitive and early predictors for the detection of perioperative myocardial infarction and severe complications in patients undergoing cardiac surgery. beginning disorder can be detected far earlier than with any existing monitoring device. perioperative red blood cell transfusions (btx) are commonly used in patients undergoing cardiac surgery to correct for anemic conditions caused by blood loss and hemodilution associated with cardiopulmonary bypass circulation and anesthesiological procedures. however, several studies have shown btx might have adverse effects on patient outcome. the goal of btx is to correct anemia and to ensure an improvement in the oxygen delivery to the parenchymal cells by the increased presence of red blood cells in the microcirculation. the aim of this investigation was to test the hypothesis that btx during onpump cardiac surgery have a beneficial effect on sublingual microcirculatory perfused vessel density, and oxygenation. methods. adult patients undergoing on-pump cardiac surgery were selected for this study. sublingual microvascular flow index (mfi), detected vessel length (dvl), and functional capillary density (fcd) were assessed using sidestream dark-field (sdf) imaging in patients. sublingual reflectance spectrophotometry was applied in patients to monitor sublingual tissue oxygen saturation. in group a, btx resulted in increased fcd and dvl as depicted in fig. . mfi for small and medium microvessels was not affected by btx (fig. ). in group b, reflectance spectrophotometry demonstrated increases in microcirculatory hemoglobin and oxygen saturation ( fig. ). the main findings suggest that leukoreduced btx improves the systemic circulation and oxygen carrying capacity of the microcirculation by increasing fcd and thereby reducing diffusion distances without increasing significantly the convection of red blood cells. this reduction in diffusion distances causes an increase in microcirculatory oxygen saturation. d.m.j. milstein , k. yürük , r. bezemer , c. ince academic medical center at the university of amsterdam, translational physiology, amsterdam, netherlands aims. anemia is a common adverse effect of oncologic diseases as is the therapeutic options required for their treatment. however, as blood transfusions are directed at correcting for anemia and intrinsic hypoxic conditions, little evidence exists claiming that blood transfusions have successfully resolved anemic challenges as storage can significantly deteriorate rbc function. the aim of this study was to investigate the influence of rbc transfusions on sublingual microcirculatory perfusion and tissue oxygenation in anemic oncology patients. methods. eight consecutive ambulatory patients scheduled to receive packed rbc transfusion bags were selected for this study. baseline sublingual microcirculation functional capillary density (fcd) was measured using sidestream dark-field (sdf) imaging prior to and after min of the completion of the last infused blood bag. sublingual mucosal oxygen saturation (sto ) was measured at the same anatomical location and time points using near-infrared spectroscopy (nirs). results. figures and capillary refill time (crt) is a generally accepted method of assessing the circulatory status of a patient. we have previously showed that using . s as the upper limit of normality in critically ill patients could discriminate patients with a more unfavourable outcome . however, this upper limit of normality was defined based on variation of crt in an adult healthy population . the best crt in critically ill patients, therefore, should still be redefined. objectives. we aimed to define the best crt as predictor of organic and metabolic dysfunction in an intensive care unit (icu) population. methods. capillary refill time was measured by applying firm pressure to the distal phalanx of the index finger for s, and a chronometer recorded the time of returning to normal colour. we performed receiver operating characteristic curve (auc) to detect the best crt consistent with severe organ and metabolic dysfunction, as evaluated by sequential organ failure assessment (sofa) [ and acidosis (lactate [ mmol/l and be\ - meq/ l), respectively. in addition, we performed logistic regression analysis using the cutoff crt as binary to investigate its estimated odds ratio (exp(b)). of patients included in the study (age ± ; male), had circulatory shock, of whom had septic shock. mean crt in all patients was . ± . . figures and show the roc curve for sofa score[ and metabolic acidosis, respectively. using the best crt value, logistic analysis revelled the following estimated odds ratio: for sofa score[ : exp(b) = . ; p = . ); for metabolic acidosis (exp(b) = . ; p = . ). roc curve for crt relative to sofa score [ roc curve for crt relative to acidosis conclusions. we found that . s is the best time to define prolonged crt in critically ill patients, and that using this crt cutoff value could discriminate patients with a more severe organ and metabolic dysfunction. introduction. impairment of microcirculation in acute situations is associated with organ failure and depends on macrocirculation but also on specific factors ( ) . micro-perfusion, assessed by tissue hemoglobin saturation (sto measurement) or micro-blood flow (laser doppler, ld) are easy to use and non invasive methods. the obtained data could be an end point in critical care resuscitation or optimization. objectives. to assess the impact on microcirculation of cardiovascular (cv) support on the basis of mean arterial pressure (map) and cardiac output (co), to evaluate when microcirculatory parameters improved or not the modifications observed in map and co. methods. observational study: measure of co, map, svco , and lactate, thenar nirs (inspectra ; hutchinson technology) baseline sto , with performance of an arterial occlusion test ( mn, mmhg) so calculate occlusion-os and reperfusion slopes-rs ( ). similarly, forearm skin blood flow velocity (ld, blf d, transonic systems) basal ld, and post-ischemic peak velocity ldmax) ( ) were measured. data were collected before and after cv optimization (fluid loading, vasoactive or inotropic drugs). patients were defined: macrocirculatory responders (r) when co increased more than % versus nonresponders (nr); microcirculatory responders (rs+) when rs increased more than % versus nonresponders (rs-). statistical analysis: nonparametric tests (wilcoxon and mann-whitney test). results. patients ( % in shock) were studied. had sepsis ( %), hemorrhage ( %), pulmonary oedema ( %), or other ( %). therapeutic optimization challenges were performed: fluid challenges ( ml, . % nacl), dobutamine c/kg/min, nitrates, diuretic, electric shock and an increase in dosage of norepinephrine. in r group (n= , %), co was increased associated with map (p \ . ), svco (p = . ) and decreased lactate (p = . ). the micro-oxygenation improved with an increase of rs ( . [ . - . ] vs. . [ . - . ]%/s, p = . ) as microperfusion did: increase in ldmax ( . [ . - . ] vs. . [ . - . ] tpu, p = . ). in the nr group, both the macro or the microcirculation did not change. since no microcirculatory differences between r and nr were observed, patients with good or poor microcirculation could not be detected. the study based on microcirculatory responses showed % of responders (rs+). in this group, baseline sto (p = . ), basal ld (p = . ) and ldmax (p = . ) increased in a large amount in association with an improved co and map (p = . and p = . ). in the rs-group, co and map were also improved (p = . and p = . ). conclusions. improvement of macrocirculatory parameters can improve microcirculation but not in all patients. improvement in microcirculation may also be a target, regardless the effects on macrocirculatory parameters. this concept has to be tested prospectively. introduction. hypothermia is regularly used for brain protection after resuscitation from cardiac arrest but its impact on cardiovascular function, however, is not well defined. objectives. the aim of this study was to evaluate the cardiovascular response to mild therapeutic hypothermia and rewarming in a large animal model. seven anesthetized, mechanically ventilated and invasively monitored sheep were cooled with a cold intravenous saline infusion, ice packs and nasal cooling (rhinochill system, benechill, ca) to achieve a core temperature of - °c (the basal temperature in sheep is around °c). after maintenance of this temperature for h, sheep were progressively rewarmed to baseline temperature. a positive fluid balance was maintained during the entire study period to avoid any hypovolemia. the sublingual microcirculation was observed using sidestream dark-field (sdf) videomicroscopy and the proportion of perfused vessels (ppv) and perfused vessel density (pvd) evaluated using a semi-quantitative method. results. during cooling, systemic and pulmonary artery pressures did not change, but cardiac output decreased significantly along with the increase in vascular resistance. left and right ventricular stroke work index decreased reflecting altered ventricular function. nevertheless, there was an increase in mixed venous oxygen saturation (svo ), reflecting a decrease in oxygen extraction. sublingual microcirculation analysis showed a significant decrease in ppv and pvd. all the variables returned gradually to baseline during the rewarming phase. conclusions. in this intact healthy large animal model, the alteration in cardiac function during hypothermia was well tolerated because of the simultaneous decrease in oxygen requirements. arterial pressure was maintained by an increase in systemic vascular resistance associated with a reduction in peripheral microcirculatory density. grant acknowledgment. *rhinochill system was supplied by benechill, inc. objectives. to evaluate consequences of hypoxemia occurence on intestinal microcirculatory perfusion in mice submitted to controlled hemorrhage. tracheotomized and ventilated balb/c mice were submitted to systemic hypoxemia (pao = mmhg) during h. controlled hemorrhage to mean arterial pressure of mmhg was associated (from th to th min). groups were constituted: hh = hypoxia and hemorrhage, hr = hemorrhage, hx = hypoxia, cl = control (neither hypoxia nor hemorrhage). a segment of ileon was exteriorized through an abdominal midline incision. it was opened along the antimesenteric border and placed on a specially designed piedestal to facilitate observation of the villi with transilluminating and epifluorescent microscopy. the bowel segment was superfused with krebs solution maintained at °c. villous perfused density (dvp), red blood cell velocity in villous tip arteriole (vart) and villous capillaries (vcap) were observed after fitc-labeled erythrocytes were intravenously administered. mice were included in each group. leucocytes adhesion to intestinal wall venules ( - lm) was observed in a separated set of experiments including also mice per group. number of adherent leucocytes (l adh ) and leucocytes flux (l fl ) were observed in each group. measurements and arterial blood gases were collected at , , min (t ). data were expressed as mean ± sem and were compared by analysis of variance (anova). introduction. despite remarkable progress in hemodynamic monitoring, clinical examination, assessment of peripheral perfusion and comparison of surface and body core temperature still are diagnostic cornerstones of critical care. infrared non contact thermometers provide accurate measurement of body surface temperatures. the picco device using an arterial line with a thermistor tip in the distal aorta-in addition to transpulmonary thermodilution (tptd)-provides continuous body core temperature. objectives. therefore, it was the aim of our study to evaluate the predictive capabilities of surface temperatures and their differences to body core temperature regarding ci, svri and parameters of microcirculation. in icu-patients body core temperature was measured four times per day using a picco-catheter (tp), a thermistor-tipped urinary catheter (tu) and an ear thermometer (te) (thermoscan; braun). additionally, surface temperatures were determined on the great toe, finger pad, forearm and forehead using an infrared non contact thermometer (thermofocus; tecnimed). furthermore capillary refill time (crt), lactate and scvo were measured and peripheral perfusion was clinically assessed (normal, pale, mottled). immediately afterwards tptd was performed to obtain ci and svri. statistics: spss . . spearman correlation. compared to tp, t forehead (- . ± . °), t forearm (- . ± . °), t finger pad (- . ± . °) and t toe (- . ± . °) were significantly lower (p \ . for all comparisons). in multivariate analysis tptd-derived ci ( . ± . l/min sqm) was significantly correlated (r = . ) to the difference ''tp-t forearm '' (p \ . ), ''tp-t finger pad '' (p = . ), crt (p = . ), scvo (p = . ) and map (p = . ). tptd-derived svri was multivariately associated (r = . ) with ''tp-t forearm '' (p \ . ) and map (p \ . ). scvo was independently correlated to the difference ''tp-t finger pad '' (r = . ; p \ . ). lactate was independently correlated (r = . ) to crt (p \ . ). the roc areas were . and . for (tp-t forearm ) and (tp-t finger pad ) to predict ''ci \ . '' and ''scvo \ '', respectively. the sensitivity, specificity and negative predictive value of ''tp-t forearm [ . °'' were , and % regarding a ci \ . l/min/sqm. .) measurement of surface temperatures using non contact infrared thermometers and comparison to body core temperature provides useful data on macro-and microcirculation. .) the differences (tp-t forearm ) and (tp-t finger pad ) were independently associated to tptd-derived ci and svri, and ci and scvo , respectively. .) crt was independently associated to lactate level. v. shilov , a. astakhov ural state postgraduate medical academy, chelyabinsk, russian federation introduction. actuality of this problem consists of different disturbances of heart rhythm and heart conductivity (from sinual bradycardia and ventricular extrasystolia till sinuatrial arrest and fibrillation of ventricles) provoked by traction of oculomotorial muscles and pressure on eyeball. this reaction is called oculocardial reflex (ocr). it is necessary to note there is no definite strategy of ocr prevention. objectives. this study was conducted to estimate the possibility of the control of haemodynamic effects of ocr. the haemodynamics and hydrobalance were investigated with electric current probe ( and khz) using monitoring complex of cardiorespiratory system and hydratation of tissues -km-ar- «diamant». data documentation was carried out at stages of evisceroenucleation: . before anesthesia and surgery; . at induction; . during the intubation; . at eyeball mobilization and oculomotorial muscles traction; . while deepening of endotracheal anesthesia by inhalative anesthetics during - min after preceding stage; . at the end of surgery, after the extubation. results. the study confirmed ocr reflex, to appear at eyeball extraction and to manifest as bradycardia, cardiac output decreasing heart productivity, but peripheric vessel resistansce does not change. monitoring-controlled gradual deepening of inhalative anesthesia during - min has restored the haemodynamic data to normal eliminated ocr vessel reactions. hydrostatic changes took place only at the end of the operation, after the extubation. it manifested ad increasing of extracellular liquid confirmed by decreasing of low-frequent impedance. intracellular liquid remained intact. it seems the most possible, hydrostatic changes of extracellular liquid to depend on crystalloid infusion in blood vessels up to , ml during anesthesia and they eliminate with hypovolemia. conclusions. thus we can conclude that vascular manifestations of hemodynamics in ocr at eyeball extraction or active oculomotorial tractions may be eliminated with gradual deepening of inhalative anesthesia and monitoring of registed date of haemodynamic and hydrobalance. probably it's necessary to optimige the anesthesia using of pterygopalatal and pterygoorbital blockade to prevent ocr before the induction as retrobulbal anesthesia may be an ocr trigger. f. corradi , c. brusasco , a. vezzani , f. altomonte , p. moscatelli university of genoa, anesthesia and intensive care, genoa, italy, ospedale maggiore di parma, anesthesia and intensive care, parma, italy, azienda ospedaliera universitaria san martino, emergency medicine, genoa, italy introduction. despite improvements in trauma care, uncontrolled bleeding is the leading cause of potentially preventable early in-hospital deaths contributing to to % of trauma-related deaths ( ) ( ) . about % more deaths occur within the second/third hour after injury due to occult major internal haemorrhage. failure to recognize this situation may in part be due to lack of sensitivity of hb/hct levels, arterial blood pressure, heart rate, respiratory rate, injury severity score and markers of hypoperfusion (lactate and base excess) in initial assessment of blood loss. to study if early changes in spleno-vascular resistance index predict the development of hypovolemic shock after trauma. a prospective observational study conducted in adult haemodinamically stable patients admitted to the emergency department because of suspected or definite severe trauma and retrospectively divided into groups depending on whether or not they developed haemorragic shock requiring blood transfusion. doppler ultrasound measurements of splenic arterial branches at ilum were obtained and splenic doppler resistance index (sdri) was recorded at admittance (within h from trauma) and related to arterial blood gas analysis (haemoglobin, base deficit, lactate, co , ph), heart rate, and outcome in the first h (intensive care unit admittance, blood transfusion, sepsis, mortality). results. statistically significant differences between patients who developed shock within h and those who did not were the following: higher sdri ( . ± . vs. . ± . , p \ . ), lower base deficit (- . ± vs. . ± meq/l, p = . ) and higher lactate ( . ± . mmol/l vs. ± mmol/l p = . ). auc's of roc analysis were significant for sdri (auc = . , ci . - . , p \ . ) and lactate (auc = . , ci = . - . , p = . ), and borderline for bd, hr, hb, and ph. by multivariate analysis, sdri at admittance resulted to be the only good independent predictor of hypovolemic shock and bleeding (p \ . ), whereas haemoglobin, base deficit, heart rate, lactate and ph were not significant. in trauma patients with stable haemodynamic conditions at admittance spleen constriction occurs very early under heavy adrenergic stimulation in response to occult bleeding and can be non-invasively detected by sdri. the present study proposes sdri as a non-invasive measurement of changes in splanchnic circulation to detect blood loss and occult hypovolemia, which may help activate early surgical or radiological intervention for patients with major trauma and guide therapy to optimize splanchnic perfusion. introduction. approximately % of patients require temporary circulatory support due to cardiogenic shock following cardiac surgery. these patients are at risk of a mismatch between oxygen delivery and demand and carry a substantial mortality and morbidity risk. mixed venous oxygen saturation (svo ) is the still the ''gold standard'' for the determination of the ratio between systemic oxygen delivery and consumption (do /vo ratio) in cardiac surgery patients. a nonivasive technique is thought to be cerebral near-infrared spectroscopy determining cerebral oxygen saturation (rso ). purpose. the present analysis aims to compare rso and svo levels in adult patients undergoing ecmo therapy for postoperative cardiogenic shock. methods. data were collected hourly for the first h post operatively. each patient was equipped with a pulmonary artery catheter (pac) for continuous determination of svo connected to a vigilance ii-monitor (edwards lifesciences, irvine, usa) and an invos monitoring system (somanetics, troy, usa) to determine rso . data were analyzed by parametric testing and bland-altman analysis. a total of patients were enclosed. all svo values were in a range between and %. in this range, the linear correlation coefficient between svo and rso was r = . (p \ . ). the correlation coefficient for svo values below % was r = . (p \ . ) and r = . (p \ . ) for svo levels equal or higher than %. bland-altmann analyses of all collected oxygenation data (n = ) revealed a bias of . % (mean % ci: . to . ) and limits of agreement ( . standard derivation) of . to - . % (upper % ci: . to . ; lower % ci - . to - . ) for the raw data of the whole group ( figure ). bland-altmann analyses of svo values below % (n = ) showed a bias of . % (mean % ci: . to . ) and limits of agreement ( . standard derivation) of . to - . % (upper % ci: . to . ; lower % ci - . to - . ). bland-altmann analyses of svo values equal or higher than % (n = ) revealed a bias of - . % (mean % ci: . to . ) and limits of agreement ( . standard derivation) of . to - . % (upper % ci: . to . ; lower % ci - . to - . ). interestingly, despite svo values [ %, we noticed events in patients with rso values less than % for more than min. all events had been associated with arterial co levels below mmhg, whereas no other changes in hemodynamic or oxygenation parameters could be determined. conclusions. this pilot study suggest for the first time that rso highly correlates with svo in patients undergoing ecmo therapy due to refractory cardiac and/or pulmonary dysfunction. therefore determining rso may be a noninvasive alternative to monitor global tissue oxygenation under this condition. additionally, it was noted that cerebral hypoxia may be present despite a svo c mmhg. rd esicm annual congress -barcelona, spain - - october results : during severe hypothermia ( °c) cardiac index (ci), stroke index, mean arterial pressure and indexes of lv contractility (prsw and dp/dtmax) were reduced. after rewarming all variables remained reduced, except for ci that returned to prehypothermic values due to increased heart rate. systemic vascular resistance (svr), lv isovolumetric relaxation time (tau) and oxygen content in arterial and mixed venous blood increased during °c, while lv end diastolic pressure (lvedp) was constant. after rewarming svr and lvedp were reduced, while tau and the blood oxygen contents normalized. troponin-t and tnf-a were constant during °c but increased after rewarming. albumine plasma concentration was reduced during °c and remained so after rewarming. conclusions. surface cooling to °c followed by rewarming caused reduction of systolic, but not diastolic lv function. there were no signs of inadequate global oxygenation throughout experiments. the posthypothermic increase in troponin-t may reflect degradation of myocyte troponins secondary to a hypothermia-induced calcium overload. the increase in tumour necrosis factor alpha together with a posthypothermic reduction of plasma albumin concentration may indicate that the cooling and rewarming initiated an inflammatory response. we studied patients, mean age . ± . years, % male. the etiology of cardiogenic shock was: % (n = ) dilated cardiomyopathy, % (n = ) acute myocardial infarction, % (n = ) acute cardiac allograft rejection and % (n = ) acute myocarditis. the duration of ecmo support was . ± . h. weaning was possible in % (n = ) and the ecmo was used as a bridge to transplantation in % (n = ). -day survival was and . % of our serie were discharged from the hospital. in cases the ecmo was withdrown as a result of a limiting treatment decision. objectives. to describe the characteristics of patients with ca and its management with moderate hypothermia using arctic sun Ò device with hydrogel patches. descriptive, observational and retrospective study of patients who suffered ca and received moderate therapeutic hypothermia ( °c) according to the protocol implemented in a coronary intensive care unit of a tertiary hospital. we collected patients from june to april , first months of this therapy in our hospital. moderate therapeutic hypothermia is applied using the arctic sun Ò device consisting of hydrogel patches applied to the skin covering % of the body surface. the device is connected to a temperature control console, measuring core temperature with an urinary catheter. we analyzed demographic characteristics, cardiovascular risk factors and other relevant comorbidities. we collected data about the ca, its initial treatment and its icu management with moderate hypothermia, analyzing length of events and systemic and neurological outcome at discharge from icu. we also collected data about the infectious complications during the icu stay. results. during this period, moderate therapeutic hypothermia was applied to patients with a mean age of ± years. . % were male. the most frequent cardiovascular risk factor was cigarette smoking, present in % of individuals. the ca cause was an ami by % of cases; however, myocardial infarction or angina was documented before the event only in . % of patients. the ca event was outside the hospital in . % of cases and the initial heart rate recorded was ventricular fibrillation in . % of cases. the average ca length was . ± min. obtaining a temperature of °c took between and h from the ca in most cases; and this temperature was maintained for an average of ± h. the average time of induction of hypothermia was . h. the re-heating was performed between . to . °c per hour, averaging h to reach temperatures of . °c. midazolam sedation was performed in all patients and severe chills required muscle relaxation with cisatracurium in . % of patients. infectious complications occurred in . % of patients, the most common site of infection was respiratory. the average stay was days. at the time of icu discharge, average gcs was and the average gos was . mortality was . % ( patients). -implementation of a therapeutic hypothermia protocol is feasible. -infectious complications are common, being respiratory ones the most observed. -the arctic sun Ò device is quick and safe for induction of moderate therapeutic hypothermia. rd esicm annual congress -barcelona, spain - - october s objectives. up to now, it is not clear, however, whether mild hypothermia influences also markers of oxidative stress and nitric oxide production. methods. eleven patients after out-of-hospital cardiac arrest were included into this study, all were treated with mild hypothermia using endovascular system thermodard xp. target core temperature °c was maintained for h, re-warming rate was set at . °c per hour, followed by normothermia of . °c. blood samples for measurement of nitrotyrosine and nitrates/nitrites were taken at admission and then every h for days. during hypothermia the levels of nitrotyrosine and nitrates/nitrites were comparable with baseline values. in re-warming period serum levels of both parameters gradually increased and in normothermia the levels were significantly higher as compared with hypothermia: nitrotyrosine . ± . vs. . ± . lm/l, p = . ; nitrates/nitrites . ± . vs. . ± . lm/l, p = . . our results revealed that during mild hypothermia in cardiac arrest survivors the levels of nitrotyrosine and nitrates/nitrites are significantly lower. these data indicate that the reduction of oxidative stress and suppressed nitric oxide production may be involved in the protective effect of hypothermia. grant acknowledgment. this study was supported by the grant of the czech ministry of health, nr. . new volumetric variables of preload, such as total end-diastolic volume index (tedvi) and active circulation volume index (acvi) and central blood volume index (cbvi), have been shown to be good predictors of fluid responsiveness. during acute changes of intravascular volume, such as hemorrhagic shock, these variables allow a more accurate intervention. objectives. the aim of our study was to investigate the changes in tedvi, acvi, cbvi in a juvenile model of hemorrhagic shock. seven anesthetized ponies ( - months of age) were studied at normovolemia (base), after blood withdrawal to mean arterial pressure (map) of mmhg (hemo), after infusion of norepinephrine to a map of mmhg (ne), and after retransfusion (resu). tedvi, acvi, cbvi were measured by ultrasound dilution (ud) technology with costatus device. data were analyzed using kruskal-wallis analysis and dunn's t test. comparison of fluid load agreement by blant altman. results. tedvi and acvi had significant change during hemo and resu status. percentage of tedvi and acvi changes agreed with percentage of blood volume removed/ infused with bias and limits of agreement (loa) % (- . , . ) and - . (- . . %) respectively. ne administration induced map and cvp significant changes, whereas tedvi and acvi remained unchanged. cbvi showed high variability and seemed to be inconsistent on the identification of the volume status. conclusions. in this animal model, tedvi and acvi were superior to cbvi in accurately reflecting hemorrhage and were also suitable to predict fluid responsiveness. ne administration did not affect the volumetric variables tedvi and acvi. ( ). objectives. we sought to identify independent predictors of post-arrest neurological recovery, and of survival to hospital discharge with neurological recovery. in the course of a pre-planned interim analysis, we analyzed the data from participants of nct . this three-center, double blind, placebo-controlled, clinical trial is ongoing (estimated enrollment = patients) and aims to asses the efficacy of combined vasopressin and epinephrine during cardiopulmonary resuscitation (cpr) and of steroid administration during and after cpr. post-arrest neurological recovery was defined as glasgow coma scale score[ documented at least once by study-independent physicians in patients not receiving sedation for at least h. we identified a total of patients who were subjected to at least one post-arrest assessment of their neurological status. subsequently, we used backward stepwise logistic regression, and assessed the following potential predictors: cause of cardiac arrest (cardiac vs. non-cardiac); area of cardiac arrest occurrence (monitored vs. non-monitored); use of therapeutic hypothermia; number of cpr cycles; mean arterial pressure and serum lactate at min following resuscitation; and patient group allocation. results. the sole independent predictor of post-arrest neurological recovery was the occurrence of the cardiac arrest in an area of monitored patient care (i.e., intensive or coronary care unit, and operating or emergency room): odds ratio: . , % confidence interval = . - . ; p = . . the sole independent predictor of survival to hospital discharge with neurological recovery was the serum lactate concentration at min after resuscitation: odds ratio: . ; % confidence interval = . - . . conclusions. the results of this preliminary analysis suggest that post-arrest neurological recovery seems to depend more on the use of pre-arrest patient monitoring rather than the employed cpr protocol. also, patients with lower, early post-arrest serum lactate concentration seem to have a better chance of surviving to hospital discharge without concurrent, severe neurological deficits. reference(s). to quantify the attribution of intra-operative defibrillation on markers of myocardial injury (ck, ck-mb, tnt and hfabp). methods. single centre prospective study in which elective cabg patients were included in a month period in . patients with valve, emergency, off-pump surgery or rethoracotomies were excluded. patients were grouped as having had defibrillation or no defibrillation during surgery. serum levels of ck, ck-mb, tnt and hfabp were analyzed in blood samples taken at arrival on the icu and at , and h after admission to the icu. levels of these biochemical markers were compared using a paired t test. results. all data presented as mean ± standrad deviation conclusions. atrial fibrillation is a common problem associated with morbidity and mortality in critically ill patients; however, evidence-based recommendations are lacking leading to variability in treatment. our audit confirmed variability and low compliance to nice in treating new af. inconsistency in using appropriate first line drugs for rate control and inadequate thromboprophylaxis reflects lack of familiarity with nice guidelines. educating itu medical staff and promoting the use of well validated, easy to remember chads scoring system might improve compliance with nice guidance. also,promoting hemorr hages scoring system for assessing risk of bleeding and carat tool to guide prescribing antithrombotics may allow itu physicians to anticoagulate more patients with af with less fear of bleeding complications. in patients with acute coronary syndromes (acs) combined antiplatelet and anticoagulant therapy is recommended in addition to percutaneous coronary revascularization. heparins and glycoprotein iib/iiia receptor inhibition can be associated with immune-mediated thrombocytopenia of clinical significance in less than %, resulting in major bleedings and increased mortality rate. to evaluate the incidence of thrombocytopenia and its impact on in-hospital complications-bleedings, reinfarctions, in-hospital heart failure and mortality in patients with acs. retrospective evaluation of patients admitted during months, fulfilling the criteria for acs: rest chest pain up to h, changes in standard ecg with or without st-elevation with or without elevated serum troponin i. serum troponin i was estimated by immunochemical method (boehringer, mannheim, germany, normal levels . lg/l). patients were treated by combined antiplatelet therapy, heparins and percutaneous coronary revascularization. platelets were estimated by automatic analyzer sysmex xe , kobe, japan (normal levels - /l). thrombocytopenia was defined as platelet count less than /l or a drop in platelet count of more than % during inhospital stay. we registered demographic, laboratory, clinical data and in-hospital mortality. we included acs patients, . % ( / ) with and . ( / ) without stelevation ( . % men, mean age . ± . years). mean admission troponin i was . ± . lg/l, platelet count . ± . /l. in-hospital thrombocytopenia was observed in . % of patients. in thrombocytopenic patients in comparison to non-thrombocytopenic ones we observed significantly increased mean age ( . ± . vs. . ± . years, p = . ) and admission serum creatinine ( . ± . vs. . ± . lmol/l, p = . ), significantly decreased admission systolic blood pressure ( . ± . vs. . ± . mmhg, p = . ) and hdl-cholesterol ( . ± . mmol/l vs. . ± . mmol/l, p = . ), significantly increased bleedings ( . vs. . %, p = . ), in-hospital heart failure ( . vs. %, p = . ), but nonsignificantly increased reinfarctions ( . vs. . %), arrhythmias ( . vs. . %) and in in-hospital mortality ( vs. . %). thrombocytopenic patients were less likely treated by percutaneous coronary revascularization ( . vs. . %, p = . ). admission thrombocytopenia in comparison to normal admission platelet count was associated with significant increase in inhospital mortality ( vs. %, p = . ) and icu-mortality ( . vs. . %, p = . ). conclusions. thrombocytopenia, observed in more than % of acs patients, was associated with in-hospital complications and mortality, especially thrombocytopenia on admission. introduction. stress cardiomyopathy, also known apical ballooning or takotsubo cardiomyopathy (tts), has been recognized for several years. this syndrome is characterized by transient systolic dysfunction of the apex or mid segments of the left ventricle (lv) in the absence of coronary artery disease. several forms of mostly physical stress may evoke this syndrome. in this case we describe a very uncommon cause for tts in an unusual situation. a -year-old woman without cardiovascular history found her husband non-responsive in bed. after resuscitation he was admitted to icu. visiting her husband, she complained of chest pains, shortness of breath and hyperventilation. physical examination revealed no abnormalities but her ecg showed deep negative t-waves in leads i, ii, iii, avf, v -v . her troponin t level was . lg/l (ref \ . ), nt-pro-bnp was , ng/l (ref \ ). ck was ng/l with ckmb of ng/l. echocardiography showed very poor lv function with the typical apical ballooning of the lv along with hyperkinesis of the basal ring ( fig. ). there was no coronary artery disease. she was admitted and treated with beta-blockers. within days, the enzymatic changes normalized and echocardiography showed improved lv function with and normalization of the apical segments. she made full recovery within weeks. discussion. icu admittance has significant impact on family members. in the acute phase of the illness, most medical attention goes to the admitted patient. especially when prognosis is poor, stress to the family may be considerable. mostly spouses and relatives with female gender are at the highest risk for depression and anxiety disorders . in contrast, little is known about the occurrence in relatives of broader physical symptoms like pain and nausea or even acute onset severe medical conditions requiring treatment. in our case the wife experienced pain, anxiety and nausea along with hyperventilation. however, the underlying disease was a severe cardiomyopathy requiring admittance and treatment. the tts cardiomyopathy is known to icu physicians in relation to subarachnoid hemorrhage, but most likely not in the context of severe emotional stress. in summary, we stress the importance for intensive care physicians to be alert to the fact that despite many diverse symptoms related to stress and anxiety, relatives can develop acute medical conditions as well. a retrospective observation study. demographic profiles, operative data and short term outcomes in the icu were reviewed in the patients who underwent beating-heart (b-h) operation. we also compared b-h operation group ( - ) and conventional cardiac arrest (c-a) operation group (before ). both groups of patients were similar with respect to preoperative demographics (age, co-morbidities, lv function). in the b-h operation group, mean age was years ( - ). preoperative mean nyha functional class was . . and the mean lvef was . %. patients underwent single valve operation, and the rests needed combined valve operation or cabg. patients were included in the c-a operation group, with mean age of years ( - ), nyha functional class of . and mean lvef of . %. in the b-h operation group, no dc shock was needed, whereas % of the patient with c-a operation needed dc shock after aortic unclamp. in the single aortic valve replacement, b-h operation group had a tendency of shorter assist perfusion time after intracardiac procedure ( . vs. . min). in the icu, inotropic support (maximum dose of dopamine) was much less ( . vs. . r) than conventional c-a operation (p = . ) and additional iabp support was not required ( vs. % in c-a operation). low cardiac output syndrome was not encountered in the b-h operation group ( vs. % in c-a operation). no major postoperative complication was encountered except ventricular tachycardia in one patient. there was no day mortality ( vs. % in c-a operation). conclusions. in our series, valve surgery on the beating-heart had a superior postoperative hemodynamics and lower associated morbidity compared to conventional cardiac-arrest operation. this procedure is recommended especially in the patients with impaired lv function. ( ) objectives. does hrt measured during daytime or nighttime predict: one-year all-cause mortality in acs?; hospital readmission within one-year? methods. secondary analysis of the immediate aim study, prospective clinical trial of patients presenting to the emergency department (ed) with symptoms of acs (n = , ): holter recordings of patients, positive for acs and admitted to the hospital, started min (median time) after arrival in the ed; -year follow up after hospital discharge in % of the sample; recordings scanned to exclude artifact and non-sinus rhythm. hrt analysis performed using research software at the washington university heart rate variability lab; hrt parameters measured: ) turbulence onset (to), which characterizes the initial rate acceleration after a ventricular premature contraction (vpcs); and ) turbulence slope (ts), which characterizes the subsequent oscillation in heart rate. results. holter recordings eligible for hrt analysis; eliminated due to unanalyzable rhythm, \ vpcs needed to calculate hrt, or recording time \ -h. patients were diagnosed with ua, with nstemi, and with stemi. patients died and were re-hospitalized during follow up. hrt measures were dichotomized into low and high-risk groups based on previously reported cutpoints: to \ % normal, to c % abnormal; ts [ . ms/beat = normal, ts b . abnormal. chi square statistics calculated. findings include: abnormal -h ts significantly associated with -year mortality [odds ratio (or) . (p = . )]; re-hospitalization significantly associated with both abnormal -h to (or . , p = . ), and -h ts (or . , p = . ); abnormal night ( - ) to and day ( - ) ts also significantly associated with -year mortality (or . , p \ . for both); abnormal daytime to (or . , p = . ) and ts (or . , p = . ) each significantly associated with re-hospitalization. conclusions. patients with acs who have a ts \ . measured over h or during the daytime are at higher risk of dying within year after hospitalization. those who either have to c % or ts b . have a greater risk of re-hospitalization. assessment during the daytime only might provide sufficient information for risk stratification. hrt measured close to acs symptom onset may aid in risk stratification. objectives. we tried to find a correlation between trs and the severity of coronary artery disease (cad) found in coronary angiography. we analyzed all consecutive patients with nsteacs admitted to intensive care unit from june to december . all patients were stratified at admission with trs. pci were performed when it were indicated. for the study we grouped patients according to trs and the severity and extend of cad. considering the trs the patients were classified into three categories: trs - , trs - and trs - and considering the results of the coronary angiography were grouped into three categories: normal angiogram, one or two vessel disease and three vessel or left main disease. we excluded patients without pci. qualitative variables are expressed as absolute value and percentage and quantitative variables are expressed as means ± standard deviation or median ± interquartile range when correspond. comparisons between groups were made with the v or fisher's exact test for categorical variables and mann-whitney test for quantitative variables. a total of patients were admitted with nsteacs during the period of the study and underwent to pci. age median were higher in patients with trs - than other groups ( . years ± . p \ . ). men percentage and in-hospital mortality were similar in all groups (pns). between groups there weren't significant differences in prevalence of diabetes, hypertension, dyslipidemia, smoking, mean first troponin i and mean highest troponin i (pns). the v for all comparisons were . (p \ . ). normal angiogram were most likely found in patients with trs - than in those with trs [ (p \ . or , % ci . - ). one or two vessel disease were found more often in those with trs - than in those with trs\ o [ (p \ . or . , . three vessel or left main disease were found more often in those with trs - (p \ . or . , % ci . - . ). conclusions. the relationship between trs and clinical outcomes (recurrent angina, acute myocardial infarction and death) is well known but its relation with the extent and the severity of cad is not well determined. in our study we found a correlation of trs with the number of vessels affected in coronary angiography, making the trs as a good predictor of the extent and the severity of cad. a.b. ratnaparkhi , j. walton freeman hospital, anaesthetics, newcastle upon tyne, uk introduction. acute onset atrial fibrillation (af) is common phenomenon in the intensive care unit. atrial fibrillation poses risk for thromboembolism. practice of commencing anticoagulation after acute onset af varies in different intensive care units. anticoagulation comes with its own side effects in the already compromised patients in the intensive care unit. this regional audit was carried out in intensive care units of the north east region of the uk. to assess the practice of use of anticoagulation after acute onset of atrial fibrillation in the intensive care units. postal questionnaire were sent to the intensive care units of the north east region of the uk including two cardiac surgical intensive care units. the questions asked were; is there a protocol in your unit? are you aware of any guidelines? if yes, which guidelines? do you commence anticoagulation for acute onset af? what do you use for anticoagulation and in what dose? after what duration of onset of af you consider starting anticoagulation? how long do you continue anticoagulation? do you commence anti platelet therapy? we also put six clinical scenarios with acute onset atrial fibrillation. the aim was to assess if the units consider stroke risk stratification for commencing the anticoagulation. one example is; how would you manage anticoagulation for a year old patient with hypertension and diabetes, presented with sepsis following pneumonia. results. we received responses from out of intensive care units. the management of anticoagulation strategy was different in different unit. two units were aware of the nice guidelines, one unit was aware of the accp guidelines and two units were aware of the other guidelines. ten units responded that they commence anticoagulation for acute onset af. commonly used anticoagulation was low molecular weight heparin. four units use anticoagulation within less than h of the onset of af. there was no fixed duration for the continuation of the anticoagulation. different units consider various factors before commencing anticoagulation. conclusions. use of anticoagulation in acute onset af varies in the different units. each unit takes into account different factors for the commencement of anticoagulation. this audit highlights the possible need for the evidence based protocol for the use of anticoagulation in acute onset af in intensive care units. objectives. to study of the clinical features and analytical features of those patients with dilated cardiomyopathy treated with ecmo as a bridge to cardiac transplantation in order to determine which parameters are useful to predict the outcome methods. a retrospective study from december to december . all patients were divided into two groups: the a group: patients who died before transplantation; the b group patients who got transplantation. several clinical and analytical characteristics are compared before starting ecmo, at and h after the onset and immediately before withdrawing (''end time'') ecmo treatment (either for transplantation or for death). qualitative variables are expressed as % and quantitative ones a mean and standard deviation (sd). chi square and t student test are used as appropriated. a p \ . denotes statics significance. there are statistically significant differences between patients who died and patients who survived to be transplantated. the presence of multiorgan failure and severe tissue oxygen hypoperfusion, and its persistence after initiated treatment, denotes a worse prognose. the study of this differences could be useful to decide which patients benefit of ecmo treatment. objectives. to measure the diagnostic contributions of routinely used (nt b type natriuretic peptide (nt probnp), cardiac troponin i (t), ddimeres (dd), c-reactive protein (crp) and procalcitonin (pct)) and new biomarkers(mid-regional pro-atrial natriuretic peptide-(mr-proanp), pro adrenomedullin (pro adm), pro endothelin (pro et) and copeptin [pro vasopressin (cp)] for diagnosing infection in patients with severe acute dyspnea. we designed a prospective study of patients admitted in the emergency department and in medical intensive care unit in a university hospital. inclusion criteria were acute dyspnea with spo b % and/or respiratory rate (rr) c b/min. patients with obvious myocardial infarction or pneumothorax were excluded. clinical-biological data were recorded and biomarkers sampled. an independent blinded expert panel classified the patients according to all the data including response to treatment and outcomes blindly to biomarkers' results. the roles of biomarkers were assessed quantitatively and then using terciles of the distribution. the contribution of the biomarkers in the diagnosis was assessed using auc-roc curves and by multiple logistic regression taking into account other clinical and biological explanatory variables. objectives. to compare differences between a group of patients with lmca treated with percutaneous coronary intervention (pci) and others with cabg. to evaluate direct results and make a long term prognosis analyzing mayor cardiovascular complications (mcc) rate. observational retrospective study that includes a total of patients with lmca submitted to ca between january and december : patients ( %) were treated with pci and compared to patients ( %) treated with cabg. in the total of the pci cases drug-eluting stents were used. we exclude patients in cardiogenic shock and those with protected left main coronary artery. results. average age of the patients was . ± . . in the pci group most of the patients were older than years. in the cabg group there was a majority of male patients ( . vs. . %, p = . ) without significant differences in the rest of demographic information. in the pci group (p = . ) there were more previous record of acute myocardial infarction (ami) and pci found, and also a greater percentage of patients with lvef\ % (p = . ). average euroscore of patients from the pci group were greater than those from the cabg group. complete revascularization was obtained more frequently in the cabg group. in the cabg group (p = . ) the number of days between diagnosis and therapeutic strategy as well as the days hospitalized were greater. in the multivariate analysis, the type of therapeutic strategy wasn t associated to mortality when hospitalize. the median follow-up period was months. according to the classification ccs (p = . ), there was no significant difference in the grade of angina. tendency to a greater restenosis of stent, greater mortality during follow-up and greater mcc without statistically significant. in the multivariate analysis surgical strategy was associated to a lower mortality during follow-up (or . objectives. our objectives were to analyze the characteristics of the patients who were done a cardiac catheterization, the differences of the procedure and the incidence of complications. methods. we randomized consecutive patients referred to the hospital for cardiac catheterization since august until october . results. among patients, the age (mean ± sd) was ± . years and more frequently male ( . %). . % were angioplasty. the radial approach was used in patients ( . %; . % with f arterial sheaths and . % with f), and the femoral approach in patients ( . %). there was no difference in the baseline characteristics of the patients. the time required for the procedure and the fluoroscopy time were longer in the radial group (p = . ). a cross over was more often necessary in the radial group ( patients, . %) due to radial artery spasm, deviousness, loop, unstable catheter or artery dissection. only one patient required cross over from femoral to radial approach ( . %) due to serious deviousness in iliac artery. the intravascular ultrasound (ivus) and rotablator always were done by femoral approach. the incidence of complications was higher in the femoral approach group ( . vs. . %, p = . ). in the radial approach group, the most important complication was wrist haematoma ( % radial artery occlusion checked with allen test), however the femoral approach complications were: inguinal haematomas ( . %), big haematomas required blood transfusions ( . %), femoral artery pseudoaneurysms ( . %), arteriovenous fistulas ( . %), retroperitoneal haemorrhages ( . %), strokes ( . %). these complications increased the hospital stay ( . ± vs. . ± . days, p = . ). conclusions. the radial approach reduces peripheral arterial complication rates and allowed earlier ambulation, so also reduces the hospital stay. however, needs higher learned time, and the size of the artery can limit several procedures (ivus/rotablator on the other hand, the development of bundle branch block after that procedure has been associated with higher rates of complete av block, syncope, and sudden cardiac arrest at long term. objectives: our aim is to describe the incidence of cardiac conduction problems after pavi and to identify possible risk factors associated with these conduction problems. patients and methods. a total of consecutive patients who underwent a pavi were included in our analysis. the indication for pavi was a severe symptomatic aortic valve stenosis in patients who were rejected or had a high risk for conventional savr. permanent pacemaker implantation was performed in case of the presence of complete heart block or symptomatic bradycardia, persisting after at least the second postprocedural day. data are expressed as mean value ± sd for continuous variables and as numbers with percentage for categorical variables. between the variables selected for predicting av block after pavi (basal valvular area, annulus diameter, valsalva sinus diameter, left and right bundle branch block), the only independent predictor was the last one (or . , % ci . ( ). implementation of care bundles have been advocated to reduce the infection rate ( ). objectives. the aim of the study was to identify the effect of the introduction of the central venous catheter (cvc) bundle on crbsi rate on our critical care unit over a threeyear period. retrospective audit on the rate of crbsi for a months period before the implementation of the cvc bundle provided baseline data. prospective audits for the corresponding months were carried out after the cvc bundle was firmly embedded in clinical practice. the data was collected based on the information recorded in our clinical information system (cis). the cvc bundle consisted hand hygiene, barrier precautions on insertion, % chlorhexidine skin preparation, using femoral site as last resort, daily review of necessity of central access, daily inspection of insertion site, use of tpn on a dedicated port and maintaining asepsis when accessing the line. robust educational program was rolled out during the implementation phase for medical and nursing staff. compulsory elements of the care bundle were recorded in our cis. we collected data on overall compliance with the bundle, mean dwell time, number of crbsis, site of infection and whether the patient left the unit with a cvc line in situ. for statistical analysis chi-square test and wilcoxon test were used. our main results are summarised in table . lines removed prior to transfer (n) we have seen a significant increase in the compliance with the bundle and it resulted a significant and sustained reduction in mean dwell time, cvc related infection rate and number of patients transferred to the ward with cvc lines (all p \ . ). the bundle resulted in bigger scrutiny for cvcs, hence the reduction in the number of lines inserted. conclusions. our data shows that implementation of care bundles can significantly and sustainably reduce the rate of crbsi on the icu in a real life setting. our previously unacceptable infection rates were reduced and now are comparable with the recently published data ( ) . evidence-based catheter-care procedures, guided by healthcare workers perceptions and including bedside teaching, reduce significantly the crbsi rate and demonstrate that improving catheter care has a major impact on its prevention. to evaluate the incidence of catheter-related bloodstream infection (cr-bsi) and of the use of central venous catheters (cvc) after an intensive improvement program aimed at reducing cr-bsi. before-and-after study in patients admitted to a -bed medical-surgical icu from january through december . in we implemented an improvement program (analysis of barriers, creation of a working group, review of protocols, and implementation of an educational program and checklist) and a set of measures to reduce cr-bsi during cvc insertion and maintenance based on provonost et al.'s model ( ) . in the postintervention period, we suspended the use of the checklist and evaluated the degree of completion of the online training module ''bacteremia zero program'' and analyzed the staff turnover rate. we have monitored cr-bsi using the ''estudio nacional de vigilancia de infección nosocomial en uci'' (envin-uci) criteria since . we calculated the incidence rate ratio of cr-bsi and cvc utilization ratio for , , and . we compared the incidence rate ratios using the epitab module from the stata program and utilization ratios using chi-square tests. results. nine cr-bsi were diagnosed in , one in , and five in . the incidence rate ratio of cr-bsi in these periods was . , . , and . %, respectively. the incidence rate ratio in the postintervention period ( . %) was significantly lower than in the preintervention period ( . %) ( . : % ci . - . , p = . .) the increase in incidence rate ratio between and was not statistically significant ( . vs. . %, p = . ). the pre-and post-intervention cvc utilization ratios were . and . , respectively (no significant differences). during the year , and for existing staff in , rotating residents, nurses (turnover rate %), and nurse's aides (turnover rate %) joined the icu. the training module was completed by % of the new nurses and none of the physicians or nurse's aides. conclusions. the program was effective; its effectiveness may be related to the intensity of the measures. a low preintervention incidence rate ratio does not preclude the usefulness of an improvement program. introduction. in the intensive care unit (icu) the bloodstream infections (bsi) related to the central venous catheters (cvcs) represent a serious clinical complication and are a substantial economic burden. although the data are still somewhat controversial, the use of antibiotic impregnated cvcs is one of the generally accepted approaches in reducing the risk of bsi [ , ] . objectives. in order to determine the efficacy of antibiotic impregnated cvcs in our clinic we evaluated retrospectively the data of the cultures of cvcs and blood obtained from patients during their stay at icu within the last years (january till august ). conclusions. surprisingly, there was no difference in the incidence of the cvc and bloodstream infections in both groups. we can conclude that the strategy of using mrimpregnated cvcs did not reduce the incidence of catheter related bsi. although earlier studies have indicated that mr-impregnated cvcs are cost saving [ ] , our data add further proof to the suggestion that the cost effectiveness of these catheters is at least uncertain. results. from all patients, ( . %) developed infection from any reason during the icu stay. patients developed crbsi, . % of the total patient number and . % of the patients who developed any infection. we recorded episodes of bacteremia due to cvc during days of cvc placement stay, . % while the standard limit is four episodes of crbsi per , days cvc placement. during the year , we chanced our practice in order to avoid as risk factors as we can, using only antimicrobial/antiseptic impregnated catheters, improving our hand hygiene and aseptic technique, using only chlorhexidine and semipermeable polyurethane dressings and making catheter replacement at scheduled time intervals as a method to reduce crbsi. the previous year the recorded crbsi incidence was . % respectively. conclusions. the incidence of intravascular catheter related infection is recorded above the standard limits for second consecutive year assuming that we have to improve further our surveillance policy. on the other hand, the incidence is recorded smaller than the incidence of the previous year according to the change to our practice, assuming that our reforming policy, although not fully effective, still is better for the prevention of intravascular catheter related infections. introduction. intravenous catheter related blood stream infection is a major factor contributing to in hospital morbidity and mortality and extending hospital stay by days and expenditure by , to , lb . the incidence of central line associated blood stream infections (cr-bsi) in our unit was audited in and a comprehensive infection prevention program that included staff education, hand hygiene, maximal sterile barrier precautions and daily assessment of the need for a central line was introduced. we are also taking part in the national audit project matching michigan. objectives. assess the effectiveness of the infection prevention programme and re-audit the incidence of cr-bsi methods. data was collected daily for a period of months. this included the number of patients with central venous catheters in the unit, the number of lines removed or re-sited, the indications for line change, the site of line insertion and incidence of line infection. the lines were reviewed daily and removed if indicated clinically (pyrexia or raised white cell count) or if not required. results. over a period of months central lines were used amounting to line days. the lines inserted were subclavian (sc)- ( . %), femoral (f)- ( . %) and internal jugular (ij)- ( . %). the percentage of lines removed for clinically suspected cr-bsi reduced in this period from to . %. the average duration of stay for the lines were sc . days, ij . days and f days which was shorter than our previous audit showed. the percentage of microbiologically proven cr-bsi also dropped from . to . % ( from internal jugular lines and one from a femoral line). conclusions. introduction of simple and cost effective practices decreased the prevalence of cr-bsi in our unit by a factor of five. daily review of lines led to earlier removal of central lines once they were no longer required. the unit being a neurointensive care unit has a greater proportion of patients in whom femoral lines are often the only option. our survey proves that with strict adherence to guidelines and following infection control protocols diligently the risk of cr-bsi from all line types can be reduced. conclusion. this study implies that the scale of crbsi may be higher than is currently recognised and that the blood culture positivity rate for crbsi is %( / ). as concurrent antibiotic therapy may reduce blood culture and cvc tip positivity, the blood culture rate of % suggests that crbsi has an inherently high blood culture positivity rate despite concurrent antimicrobial therapy. ( ). in this context, we tested the introduction of chlorhexidine(chx)-impregnated sponges ( ) ( ), acinetobacter baumannii . % ( ), serratia marcescens . % ( ), stenotrophomonas maltophilia . % ( ), escherichia coli . % ( ) jai salmonella enteritidis . % ( ) . production of extended-spectrum beta-lactamases (esbls) was detected in % of klebsiella spp. and e. coli strains, overproduction of ampc beta-lactamases was recognized in . % of enterobacter spp., while only one k. pneumoniae strain was found to produce metalloenzyme. all eight strains of p. aeruginosa were susceptible to aminoglycosides, ciprofloxacin and carbapenemaces, both strains of s. maltophilia were susceptible to ticarcillin/clavulanate and trimethoprim/sulfamethoxazole. among a. baumannii isolates, . % were susceptible only to colistin. in total, . % of isolates were susceptible to imipenem and ciprofloxacin. conclusions. gram-negative bacteremia, in particular in the critically ill, is associated with significant morbidity and mortality. significant susceptibility to ciprofloxacin and imipenem was demonstrated. empiric treatment regimens should be based on unit-specific data. ben objective. to assess whether implementation of a national safety program to prevent cvc-related bacteremia had an impact on rates of devices-associated infections acquired in icu. methods. prospective, multicenter, incidence, surveillance study of vap, crb and uti carried out from - - to - - . simultaneously, a bundle for prevention of cvcrelated bacteremia and a comprehensive safety program were introduced at the national level. infections were diagnosed according helics definitions. the follow-up was carried out until discharge from the icu or to a maximum of days. the severity was assessed by the apache ii score. the rates are expressed as incidence density (id) per , days of risk factor. rates are compared with those of previous years ( ) ( ) . introduction. acute kidney injury (aki) is one of the most dreaded complications of severe malaria. occurs as a complication of plasmodium falciparum malaria in less than % of cases, but the mortality rate in these cases may be up to % [ ] . to evaluate the incidence of aki and compare akin and rifle classification systems with regard to hospital mortality. a retrospective analysis based on medical records of adult patients with severe plasmodium falciparum malaria admitted in the general icu of clínica sagrada esperança, in luanda, angola, from january to december . criteria for diagnosis included the standard who definition for severe malaria. only changes in serum creatinine were used to define the presence of aki by both criteria. logistic regression was used to access the association of each rifle and akin with hospital mortality. data are presented as odds ratios with % confidence intervals (ci). we enrolled patients. thirty-nine ( . %) were males. the mean age recorded was . ± . . the mean apache ii score was . ± . , with a mean predicted dead rate of . %. the mean sofa score on admission was . ± . . the mean length of stay in the icu was . ± . days. rifle allowed the identification of more patents than akin as having aki ( . vs. . % there was no statistic association between corticosteroids therapy and length of icu stay less than days (p = . ), duration of mechanical ventilation less than days (p = . ), severe infection (p = . ), re-intubation (p = . ), tracheotomy (p = . ), nosocomial infections (p = . ), myopathy (p = . ) or mortality (p = . ). although there is a tendency for a higher prescription of corticosteroids in dni patients with severe infection, the difference did not reach statistical significance. the use of steroids is neither associated with a better outcome nor with a higher frequency of adverse events or side effects, namely critically illness myopathy or nosocomial infections. ozbek introduction. q fever, a zoonosis due to coxiella burnetii, is more frequent and severe in men than women, despite a similar exposure. here we explore whether the severity of c. burnetii infection in mice is related to sex differences in gene expression profiles. methods. experimental study analyzing the transcriptome of c bl/ j mice. ten females and males were sterilized at weeks of age. after weeks, males and females ( intact and castrated animals of each gender) were killed. the other series of mice were injected intraperitoneally with c. burnetii organisms and sacrificed at day one after infection. organs were aseptically excised and stabilized in rnalater. total liver rna was retrotranscribed and labelled with cy . labelled cdna were hybridized onto whole mouse genome oligo microarray k (agilent). raw signal data were normalized with the quantile method. the significance analysis of microarrays test was used to study the gene expression in uninfected and infected mice. supervised analyses were carried out with r with the library bioconductor. pca was used to visually explore global effects for genome wide trends, unexpected effects and outliers in the expression data (library made ). in another set of experiments, mice ( intact males, castrated males, intact females and castrated females) were killed at , , and days after c. burnetii infection (same protocol). liver rna was analyzed by rt-pcr to confirm microarray results. results. multiclass analysis (sex and infection) identified , modulated genes (fdr = %, |fold change| [ . ) . we found that % of the genes are specifically modulated in males or females. only % of the genes are sexindependent. castration showed that sexual hormones are responsible for more than % of this sex-specific differential expression. the reduction of gene expression modulation upon castration is seen almost exclusively in males. functional annotation of male specific signature identified groups of keywords linked to cellular adhesion, signal transduction, defensins and cytokines and jak/stat pathway. functional annotation of female specific signature identified two group of keywords linked to intracellular metabolism and circadian rhythm. these results were confirmed by rt-pcr. the increased susceptibility to infection in males may be related to the overexpression of il and stat . the modulation of the circadian rhythm in female is linked to a more efficient bacterial clearance. conclusions. this study showed for the first time that the sexual dimorphism observed in q fever is reflected by sex related gene modulation, and is under the control of sexual hormones. this study also showed that the circadian rhythm seems to play an important role in infection in mice. this work open the way for deciphering the role of sex and circadian rhythm in human infections. the author's report a p.aeruginosa sepsis with skin and heart involvement in a previous healthy woman. a years old woman without a pertinent medical history came to the hospital after days with high fever ([ . °c), vomiting and diarrhea. at admission she was in septic shock with multiple organ disfunction (hemodynamic, cardio respiratory and renal) and presented genital skin lesions (round, ulcerated, painless lesions with necrotic black eschar and erythematous margin-ecthyma gangrenosum). the laboratory tests showed bicytopenia (leucocytes and platelets), hepatic necrolysis and elevated troponin t, associated with t wave inversion in anterior leads in the ecg. the ecocardiogram showed apical dyskinesis with normal systolic function suggesting tako-tsubo cardiomyopathy. hemocultures ( ) were positive to pseudomonas aeruginosa and skin lesions biopsy showed vascular ulcers with local p. aeruginosa inflamation. results. besides the fluid challenge and supportive therapy she began empirically piperaciline-tazobactam with rapid improvement of the clinical picture. she needed vasopressors (norephynefrine and dopamine) for h. the skin lesions have resolved in days and cardiac treatment was conservative and symptomatic. the patient was discharged from the intensive care after days. conclusions. ecthyma gangrenosum although relatively uncommon, was first considered a pathognomonic sign of p. aeruginosa sepsis, but now we known that other bacterias can have the same presentation. tako-tsubo cardiomiopathy or broken heart syndrome is a stress-induced cardiomiopathy characterized by transient systolic dysfunction that mimics myocardial infarction but without coronary disease. although the unusual p. aeruginosa clinical presentation sepsis should be treated with prompt supportive measures and the most adequate antibiotic. objective. we undertook this study to determine the relative frequency of meningitis and sepsis in a paediatric intensive care and to define the clinical and laboratory features at the time of admission and the outcome of these children. we reviewed the medical records of patients with sepsis and meningitis, in our paediatric intensive care, from to . results. among these patients % had meningitis, % had sepsis, % patients had bacteraemia, and % had meningitis and sepsisage ranged from month to years old ( % were - years old. boys %, girls %. temperature at the moment of admission was in % patients greater than °c. leucocytosis was noted in % (from , to , / mm ) and leucopenia % ( , - , /mm ) % of the patients had petechiaes, % had a positive lumbar puncture and % who did not have lumbar punctures had diffuse intravascular coagulationthe species of microorganism were in % meningococcus group b, in % no organism was found, in % were pneumococcus, and meninococcus group d in %. on admission, % of our patients had seizures. the duration of hospitalization in our picu was % (average length of stay from to days) % had hemodynamic instability, and % had a normal arterial pressure. from the patients who had hemodynamic instability, needed only fluids %, and % needed fluids and inotropes % of the patients received intravenous ceftriaxonewe had no mortality. conclusion. meningitis and sepsis remain a serious problem in picu. with the existing guidelines of therapy and prognostic signs at the moment of admission in picu we have a better outcome. howitz introduction. community acute bacterial meningitis is a relatively common disease. three bacteria are responsible in most cases: streptococcus pneumoniae (adults), neisseria meningitidis (older children and young adults) and listeria monocytogenes (in the elderly, alcoholics and immunosuppressed). the mortality rate ranges between and % and is higher in case of pneumococcal meningitis ( %) due mainly to increased intracranial pressure and the intense inflammatory reaction that produces pneumococcus in the cerebrospinal fluid. objectives. to study epidemiology, aetiology, clinical and evolution in acute meningitis in the adult community in our icu. methods. retrospective and descriptive study of patients admitted to a tertiary icu with beds from january to december . a total of patients of whom were males ( . %) and women ( . %). the mean age was . years (range: - ). the apache ii at admission was determined in of the patients with an average of points, which is associated initially with a good prognosis. the glasgow coma scale was found in % of the cases the majority ranging between and with a range: - . the average stay in icu was . days. patients died ( . %). risk factors include: infections in otolaryngology: cases ( . %) alcohol: case ( . %) and states of immunosuppression: ( . %) of which: there were two diabetic patients ( . %); hiv: ( . %) were chronic treatment with corticosteroids in one case ( . %); advp: case ( . %) liver transplantation: one case ( . %) and other case cerebrospinal fluid leak ( . %). in patients (% . ) found no risk factor. the most common complication was the need for endotracheal intubation and mechanical ventilation in a . % of patients, hydrocephalus followed by . %. hearing sequelae were found in patients ( . %) and persistent vegetative state in case ( . %). the outcome was favorable and without sequelae in cases ( . %). the etiology was bacterial germs and virus in cases in . bacteria, not unknown in cases ( %). filiated of the causative agent in the majority ( . %) were streptococcus pneumoniae, followed by neisseria meningitidis in cases ( . %), listeria, staphylococcus aureus, e. coli and mycobacterium tuberculosis were isolated in one case each ( . %) . in relation to the vhs virus was found in one case ( . %) and unable to filial the rest. conclusions. community acute meningitis is a disease with low prevalence and mortality in our environment. the agent most commonly streptococcus pneumoniae, clearly associated with increased morbidity. the most frequent complication was the need for endotracheal intubation and secondly hydrocephalus. mortality was associated with longer hospital stays and lower glasgow at the beginning, but not with age. rd esicm annual congress -barcelona, spain - - october s evaluated factors: patient characteristics, signs, symptoms, abscess location, time between symptoms and hospital admission and surgery, lab results, microbiology, antibiotic therapy, apache , saps , sofa, length of icu stay, surgical re-intervention, duration of mechanical ventilation, infectious complications, critical illness myopathy (cim), renal replacement therapy (rrt), re-intubation, tracheotomy, mortality. descriptive statistics were used to analyze data. objectives. to assess ventriculitis (vg) and to study outcome and disability indices in patients admitted in icu due to cerebral hemorrhage (spontaneous or traumatic). we prospectively studied patients hospitalized due to cerebral hemorrhage in the icu of university hospital of thessaly, between and . patients were followed for median follow up of ( - ) days. on admission, the neurological status of patients was described by the glasgow coma scale; disability was evaluated at months by the rapid disability rating scale (rdrs). results. one hundred twenty-one patients ( male) were studied; median (iqr) age was ( - ) years, gcs before intubation ( ) ( ) ( ) ( ) ( ) ( ) objectives. to analyze characteristics of patients diagnosed with infective endocarditis in a third-level hospital from january until december , evaluating the echocardiography findings, the therapeutic strategy used, and both morbidity and mortality rates in hospital and during long term follow-up. observational retrospective study of consecutive patients with following duke criteria with a mean follow-up of ± months. conclusions. this study showed a low mortality of sepsis and its sequential stages in children with meningococcal disease admitted to the picu, which was probably associated with the early use of antibiotics (up to the sixth hour) and aggressive fluid esuscitation. diagnosis and treatment of infections in critically ill patients: - background. about one-third of hospital mortality in critically ill patients occurs after intensive care unit (icu) discharge. post icu deaths may arise from incomplete resolution of the primary condition or from the development of new complications. some authors have recently hypothesized that unresolved or latent inflammation and sepsis may be an important factor that contributes to death following successful discharge from the icu. aim. the aim of our study was to determine the ability of the clinical and inflammatory markers at icu discharge to predict post-icu mortality. a prospective observational cohort study was conducted during a -month period in an bed polyvalent icu. acute physiology and chronic health evaluation (apache) ii score, simplified acute physiology score (saps) ii, sequential organ failure assessment (sofa) score, c-reactive protein (crp), body temperature and white cell count (wcc) of the day of icu discharge were collected from patients who survived their first icu admission. results. during this period patients were discharged alive from the icu. a total of patients ( . %) died in hospital after icu discharge. there were no differences in clinical and demographic characteristics between survivors and nonsurvivors. c-reactive protein levels at icu discharge were not associated with hospital mortality (mean crp concentration of survivors = . introduction. early diagnosis of bacterial infection can be challenging in critically ill patients, however prompt recognition and initiation of antibiotics improves outcome. serum procalcitonin (pct) has been proposed as a more reliable marker of bacterial sepsis than white cell count (wcc) or c-reactive protein (crp), however there is no consensus in how it should be used and pct measurement has not disseminated widely into critical care practice in the uk. to identify if clinical recommendations based on procalcitonin levels are being followed. we retrospectively studied pct use between october and december . assay results were interpreted as: . ng/ml, possible local bacterial infection . - . ng/ml, possible bacterial systemic infection, moderate risk of severe sepsis . - ng/ml, likely systemic bacterial infection, high risk of severe sepsis \ ng/ml, almost exclusively severe bacterial sepsis or septic shock. , this was compared to changes in antibiotic prescribing which were identified from our local audit database and used as a surrogate marker for clinical suspicion of sepsis. to provide context we also compared this to crp and wcc trends in a larger sample from june to december . results. forty-four episodes had matched antibiotic prescribing data and pct results. a further episodes provided synchronous wcc and crp data. pct assays were typically requested on day (median, interquartile range - ). distribution of pct results pct value (ng/ml) . . - . - [ number (%) ( ) ( ) ( ) ( ) there was poor concordance between pct and both wcc and crp trends and also when wcc and crp trends were compared. pct assays did not have significant correlation with antibiotic prescribing. pct conclusions. our study suggests pct results did not influence clinical practice. pct testing may be of greater benefit if used with a protocol with guidance for clinicians based on assay levels. routine and serial quantitative pct testing protocols may also be useful to guide antibiotic initiation and duration, particularly in cases of greater diagnostic uncertainty, for example traumatic brain injury. , references. introduction. procalcitonin (pct) is a reliable marker for diagnosis of infection after cardiac surgery, except in patients who previously received antibiotics, but its diagnostic role in patients with post-sternotomy pre-sternal wound infection and mediastinitis has not been studied in detail. ( ) . objectives. this retrospective study focused on the role of pct in the differentiation between poststernotomy pre-sternal wound infection and mediastinitis. methods. all patients (n = ; age: median , - years) who underwent surgical treatment due to poststernotomy superficial pre-sternal wound infection and mediastinitis between january and september were included in the study. procalcitonin (pct), c-reactive protein (crp) and leukocyte counts were routinely measured within the last h before surgical wound revision. body temperature and hemodynamic parameters were evaluated immediately before operation. bacteriologic samples were also routinely taken intraoperatively. results. the primary cardiac operation was cabg (n = ), cabg and valve procedure (n = ) and others (n = ). time between primary operation and wound revision was in median days (range sensitivity, specificity, positive and negative predictive value for diagnosing sepsis are presented in table . roc curves and auc are presented in figure . roc curves and auc conclusions. patients with sepsis have significantly higher levels of crp, pct, il- and lbp on admission to the icu as compared to patients with sirs. pct is more usefull in differentiating between sepsis and sirs than crp, il- and lbp. b. ergan arsava , s. bilekli , n. alayvaz aslan , e. er , a. topeli hacettepe university, ankara, turkey introduction and objective. the incidence and mortality of bacterial infections significantly increase with age. aging is associated with an impaired immune response, which causes not only an increased susceptibility for infections, but also a poor inflammatory response against them. procalcitonin (pct) is an inflammatory biomarker used as a diagnostic and prognostic tool in bacterial infections. there is no data regarding the diagnostic yield of pct in elderly patient populations. in this study we sought to identify the relationship between age and the magnitude of pct response in patients admitted to the intensive care unit (icu). methods. patients, who were admitted to icu between january and december , with the diagnoses of severe sepsis, pneumonia and chronic obstructive pulmonary disease exacerbation (copde) were included into the study. patients' demographics, apache- scores, admission pct values, intensive care and hospital outcomes were extracted from a database of prospectively collected clinical data. results. we studied a total of admissions ( female/ male, mean age ± standart deviation . ± . years). median (interquartile range-iqr) apache score was ( - ); the icu and hospital mortalities were . and . %, respectively. median (iqr) admission pct levels were . ( . - . ) ng/ml in patients with severe sepsis (n = ), . ( . - . ) ng/ml in patients with pneumonia (n = ) and . ( . - . ) ng/ml in patients with copde (n = ). there was a negative correlation between age and pct levels (spearman correlation coefficient: r = - . , p = . ); the median (iqr) pct levels were . ( . - . ) ng/ml in patients\ years-old and . ( . - . ) ng/ ml in patients c years-old (p = . ). in subgroup analyses it was found that the inverse correlation between age and pct levels mainly arised from patients with severe sepsis (r = - . , objectives. we intend to define the role of pct in the initial evaluation of the patient with a suspected sepsis admitted to the icu. preliminary data from a prospective observational single centre study (polyvalent icu in a third level university hospital). the ethics committee of the centre has approved this study. we included all patients admitted to our unit with diagnosis of sepsis since june- . we measured pct (lgr/ml) at admission and at the second and third day of stay beside the routine screening for the source of infection. then we analyzed the relation of pct with culture results. chi-square and anova have been used for the analysis. (fig. ). pct at admission showed an auc of . ( . - . ) for discriminating bacterial infection. we detected higher mortality in those patients with bacterial infections and sustained high levels of pct the third day ( fig. ) (p \ . ). figure , conclusions. in the initial approach to the septic patient, pct does not seem in our experience useful as an aid in decision-making but an early decrement of serum levels can be a marker for response and for a better outcome of patients with bacterial infections. ( - ) b-d-glucan (bg) assay in early detecting ici in critically ill pts and assess its reliability on arterial blood specimens compared to venous blood specimens. methods. all pts admitted to the -bed icu of our university hospital, between th of february and th of march , harboring an arterial line for more than days and suspected to have an ici, were prospectively enrolled. from all the pts, two blood samples drawn from the arterial line and direct femoral site were simultaneously obtained and subjected to both conventional cultures and bg assay determinations. candida colonization index (cci) and candida score (cs) were also calculated. results. during the study period, from admissions, pts were enrolled. bg assays, cutaneous and mucosal swabs and urine cultures were collected. in pts bg assays resulted negative from either arterial line and femoral site. all but one did not develop ici. in pts bg assays resulted positive. four of these pts did not develop ici, whereas the other six developed ici ( fungemias and mediastinitis). the positive and negative predictive values (ppv, npv), sensitivity (se) and specificity (sp) of bg assay, cci, and cs are shown in table . among pts with ici, (median sofa score value ± . ; median saps ii value . ± . ) had at the diagnosis bg levels c pg/ml and developed septic shock; two of them died within few days. in contrast, the clinical course of pts with bg assay below pg/ml was not complicated by septic shock (median sofa score . ± ; median saps ii value ± . ) and a rapid clearance of bg levels was observed. in addition, we observed a % agreement between arterial line and femoral site bg assays (positive and negative). in particular, we detected bg levels from arterial site specimens that did not significantly differ by those obtained from femoral site specimens (p = . ). conclusions. although conventional mycological culture remains the gold standard for ici diagnosis, we showed that bg assay seems to be a diagnostic tool that may help physicians in early detecting ici. sampling blood from the arterial line was shown to give a simple and adequate specimen to be used for bg assay. further studies are in progress in order to define the role of bg as a surrogate marker for an early diagnosis of ici. objectives. to assess which marker, if any, and at which cut-off value could add diagnostic information to enhance clinical assessment in the difficult context of long-term icu-patients. methods. long-term ([ days) critically ill patients prospectively enrolled in the general icu of a university-hospital. all patients were daily assessed by the attending physician for the accp-sccm classification. c-reactive protein (crp, mg/dl), procalcitonin (pct, ng/ ml), and interleukin- (il- , pg/ml) were measured after patient's discharge on daily stored sera. an independent overall clinical evaluation after patient's discharge, aware of the clinical course but blinded against biomarker's measurement, an ''a posteriori'' accp-sccm classification, was chosen as reference standard for all comparisons. results. we studied clinical variables and biomarkers in patient-days ( patients). the day by day accp-sccm classification of the attending physician overestimated the severity of the inflammatory response to infection. discriminative ability of each biomarker for diagnosis of sepsis is shown in table . methods. icu patients ( males and females) with new onset of fever and leukocytosis within the first days of icu admission were prospectively included in the study. exclusion criteria: age \ or [ years old, heart or renal failure, hypertension, copd, pregnancy and head trauma. serial plasma samples were taken on days , and after the onset of fever for procalcitonin and bnp levels measurement. procalcitonin and bnp values were correlated with severity scores (apache ii and sofa), progression to septic shock and final outcome. statistical analysis was performed. results. patients included in the study were divided in three groups according to clinical and laboratory findings: sirs, sepsis and septic shock. procalcitonin value on days and and bnp value on days , and was significantly associated with sofa max value and with sofa value at the first day of fever, but not with apache ii. there was found no correlation between procalcitonin value on days , and and final outcome. bnp value on days and was significantly associated with final outcome (p = . and . respectively). the optimal cut-off bnp value on day was estimated to be pg/ml (sensitivity = %, specificity = %). the optimal cut-off bnp value on day was estimated to be pg/ml (sensitivity = %, specificity = %). procalcitonin value on days , and was not able to differentiate between patients with sirs and those with sepsis. also procalcitonin value on days , and was not significantly associated with progression to septic shock. bnp value on day was useful in differentiating between patients with sirs and those with sepsis (p = . ). the optimal cut-off bnp value was estimated to be pg/ml (sensitivity = %, specificity = %). bnp value on days and was significantly associated with progression to septic shock (p = . ). the optimal cut-off bnp value on day was estimated to be pg/ml (sensitivity = %, specificity = %). the optimal cut-off bnp value on day was estimated to be pg/ml (sensitivity = %, specificity = %). conclusions. in icu patients with new onset of fever during the first days of icu hospitalization, bnp value on days and seems to be a good predictor of icu mortality and progression to septic shock. also bnp value on day may be useful in differentiating between patients with sirs and those with sepsis. in our study procalcitonin value on days , and was not found useful in predicting progression to septic shock nor the final outcome. due to the small number of patients included in our research, further studies are needed to confirm these findings. objectives. as c-reactive protein (crp) is regarded a marker for both inflammation and infection we decided to analyse the pct and eo status next to every crp request of critically ill patients, during month. a two-side immunoassay (sandwich principle) for procalcitonin, using both anti-katacalcin and anti-calcitonin (see fig. a ) was used for quantitative analysis of procalcitonin with the roche modular e . pct concentrations [ . lg/l were regarded as positive. crp was measured by immunoturbidimetric analysis using the roche modular p. a positive blood culture was regarded as infection, with exclusion of the coagulase negative staphylococcus aureus since this organism doesn't induce pct expression. every crp request of the icu during month was accompanied by a pct, wbc, and eosinophil count. conclusions. pct at randomly measured at the icu doesn't seem to contribute to an earlier diagnosis of sepsis. pct measurement seems to be useful only when sepsis is suspected and a blood culture request has been summoned. however, its non-specificity for infection, as demonstrated by the high number of pct-positive, no blood-culture requested patients (concerning mostly post-cardiac arrest and post-heart surgery patients), makes it difficult to apply routinely. the recently displayed effort of various companies to market pct in combination with cd and neopterin (other potential markers of infection) supports the conclusion that not one marker by itself can substitute the golden standard of blood culture today. objectives. in this prospective observational study we sought to investigate the role of serum c-reactive protein (crp) and procalcitonin (pct) in the diagnosis and prognosis of patients admitted to the icu with suspected h n infection. all patients older than year-old, presenting with severe acute respiratory disease (cough, fever and respiratory distress) admitted to the icus of an university hospital in southeast brazil were included in this study. serum levels of crp and pct were measured at inclusion and at day , and . were also were also significantly higher (p \ . and p = . , respectively) independently from the presence or not of a co-infection. conclusions. as a conclusion, in our experience, some severe forms of influenza a/ h n with respiratory failure had elevated levels of pct and/or crp in the absence of proven bacterial co-infection. low values were unusual in the presence of co-infection but high values are not synonymous of co-infection and may be related to the severity of the disease. a large prospective randomized study is needed to assess the clinical interest of these biomarkers during the next pandemic of influenza. methods. descriptive study of pregnant with influenza a admitted in the obstetric section icu. the study period runs from september to january . during this period patients were admitted. entry criteria (%): gestosis %, complicated postoperative gynecology and obstetrics , postpartum hemorrhage , acute respiratory failure in influenza pneumonia: . , sepsis and others respiratory failure respectively, others (pregnant myocardial infarction, trauma, renal failure, arrhythmias and heart failure) . acute respiratory failure due to influenza pneumonia was assessed using severity criteria the ats/idsa (major criteria ( ) were admitted in icu cases of severe influenza pneumonia, nasopharyngeal specimens confirmed with rt-pcr positive for influenza a (h n ): pregnant in icu og, and women and men general icu. the average age of pregnant was ± years, average stay days. % were in the rd trimester and one in the nd trimester (week ). two-third pregnancy and two primiparous. the apache ii on admission ranged between and . only one patient with pre-existing disease (diabetes type ). admission due: acute respiratory failure complicating pneumonia multilobar in %, with more than days of typical symptoms (fever [ °c, malaise, myalgia, headache and respiratory symptoms), no starting oseltamivir within h symptoms. caesarean was performed at %; % in the first h of admission and one after days (week ) intrauci, posterior cerebral hemorrhage fetal death. all newborns free of viral disease. invasive mechanical ventilation (mv) in the first h in patients and did not require. required aggressive parameters %: bipap, alveolar recruitment and prone. a percutaneous tracheostomy for weaning. the average duration of mechanical ventilation: ± days complications: barotrauma (pneumothorax and a pneumomediastinum ). % required vasoactive drugs. one patient with acute renal failure that required extracorporeal clearance techniques (hdfvvc), recovering renal function, deep vein thrombosis complicated with shaldon catheter. one brain death by massive subarachnoid hemorrhage. nosocomial infections in patients, most common germ staphylococcus epidermidis catheter and candida sp in urine. initial empiric coverage with ceftriaxone and clarithromycin, as well as oseltamivir. conclusions. during pregnancy, especially in the second and third quarters, there is an increased risk of complications associated with infection by influenza a virus h n , highlighting pneumonia, with more rapid progression to severe respiratory complications. objectives. the aim of this study was to describe baseline characteristics, management and outcomes of critically ill patients with influenza a (h n ) infection who were treated at icu. we performed a retrospective observational study which included critically ill patients with influenza a (h n ) infection admitted to icu between rd november and th march . the primary outcome measure was mortality. secondary outcomes included the rate of influenza a (h n )-related critical illness and introduction of mechanical ventilation as well as intensive care unit (icu) length of stay and hospital length of stay. in the early th century, burns patients were dying of multi-organ failure due to dehydration and hypovolaemia [ , ] . the parklands formula was devised to guide fluid resuscitation and prevent multi-organ failure from occurring. however, over enthusiastic fluid resuscitation will lead to other complications [ ] . objectives. we aimed to assess the adequacy and complications of fluid resuscitation in the st h of a burns patient admitted to our icu, a tertiary centre for burns intensive care. • a retrospective medical case notes audit on all patients admitted to our icu in with [ % tbsa (total burns surface area). • st h of burns resuscitation initiated by the referring hospital and our icu compared to the parkland's formula. • statistical analysis by spss . results. patients audited. • mean duration of transfer from burn injury to our unit = . h • mean age = years, burn area %, length of stay . days • day mortality = % • % had an inhalational injury • no statistical difference between the emergency department (ed) estimation of tbsa and whiston icu. • mean iv fluids given . l but the actual predicted requirement is . l, therefore an excess of . l (p \ . ) • average urine output during this period was . ml/kg/h suggesting that this amount was adequate. iv fluids in the st h conclusions. excessive amount of iv fluids in the st h is associated with prolonged ventilation and length of stay but is not associated with increased day mortality. the mean amount of fluid required in the st h is approximately ml/kg/h which is consistent with other studies [ ] . urine output is still an accurate marker of resuscitation. there was no statistical difference between the determination of tbsa by the ed and burns surgeons, contrary to other studies [ ] . introduction. icu-acquired hypernatremia appears to be associated with mortality in the icu . to reduce iatrogenic rise of serum sodium the use of balanced colloids has been advocated. objectives. aim of this study was to establish the incidence of clinically relevant hypernatremia on our icu and to evaluate the change in incidence of hypernatremia due to the introduction of a natriumacetate based colloid solution. we performed a single centre retrospective study in a -bed mixed icu with all available medical specialities except neurosurgery. sodium measurements of all patients were analyzed during a -month period prior to and after a switch from sodium-based (s) to natriumacetate -based (na) colloids. s contains a % kda polystarch with a mmol/l na and mmol/l cl concentrations (voluven Ò ). na contains a % kda hydroxyeethylstarch with a mmol/l na and mmol/l cl concentrations (volulyt Ò ). serum sodium measurements were routinely performed -hourly. by protocol colloids were provided to a maximum of l/day, independent of bodyweight. patients with hypernatremia at icu admission were excluded. data are expressed as mean ± sd. comparison of na concentrations between groups was performed with a t test for independent samples and comparison of incidence of hypernatremia with a pearson chi-square test. results. patient characteristics and number of samples are summarized in table . after the introduction of na mean serum sodium concentration in the total icu population decreased significantly from . ± . to . ± . , p = . . the incidence of serum na[ mmol/ l decreased from . to . %, p = . . the percentage of patients with at least one na measurement[ mmol/l did not change significantly: . % (s) versus . % (na), p = . . introduction of a natriumacetate based colloid solution in stead of a sodium-based colloid solution reduces the overall incidence of clinically relevant hypernatremia; however, the number of patients with such hypernatremia did not change significantly. patients admitted to intensive care frequently have a metabolic acidosis with previous studies demonstrating an association between the degree of acidosis and outcome. hyperchloraemia is a significant cause of metabolic acidosis and there is increasing interest in the adverse consequences associated with it, which include hypotension, renal dysfunction, impaired gut perfusion and increases in inflammatory cytokines. previous studies, however, have failed to show that hyperchloraemic metabolic acidosis (hcla) has a significant effect on survival. to assess whether patients with hcla had a worse prognosis than our general intensive care unit (icu) population, and the factors associated with the development of hcla. consecutive admissions to the intensive care unit over a month period were studied. patients with a base deficit[ mmol/l and a serum chloride[ mmol/l on the same arterial blood sample during their icu admission were classified as having an episode of hcla. apache ii scores on admission, length of stay on the unit, mortality rates and reason for admission were collected. hospital survival was investigated by logistic regression analysis, controlling for illness severity (apache ii) and admission category, and displayed as a kaplan-meier curve. of patients entering the unit during the retrospective study period, had an episode of hcla, with an odds ratio of death of . ( % ci . , . ) compared with those without hcla. after controlling for apache ii score on admission, and admission category the odds ratio reduced to . but was still statistically significant (p = . , % ci . , . ). the development of hcla was significantly more associated with medical than surgical admissions with an odds ratio of . ( % ci . , . ). within the surgical admissions, the occurrence of hcla differed significantly with the urgency of surgery, with an odds ratio of . ( % ci . , . ) for emergency surgery versus elective surgery. those with hcla had a longer median duration of stay and were overrepresented in the group of patients whose length of stay was c days. kaplan-meier graph showing survival to days conclusions. the results demonstrate that hcla occurs frequently in a general icu population and is associated with a significantly worse outcome. this is in contrast to previous studies which have demonstrated acidosis secondary to lactate and an elevated strong ion gap are associated with poorer outcomes, but not hyperchloraemia. a. dumoulin , a. janssen , j.j. de waele , j. decruyenaere , e.a. hoste universitair ziekenhuis gent, gent, belgium increased creatinine clearance or hyperfiltration has been reported in icu patients. enhanced renal clearance of antibiotics in patients with hyperfiltration may result in suboptimal antibiotic serum concentrations, and so affect patient outcomes. there are only limited data on the incidence of hyperfiltration in icu patients. objectives. assess the epidemiology of hyperfiltration in a cohort of icu patients. single center retrospective cohort study, including adult patients hospitalized during the period / - / in the bed icu of the ghent university hospital, a tertiary care center. data were retrieved by a specially designed electronic alert from the electronic icu database. urinary creatinine clearance (ccr) was calculated as ( h urine volume) (urinary creatinine)/([creatinine day - + day ]/ )/time. we retrieved the initial ccr, and the minimum and the maximum ccr. hyperfiltration was defined as a ccr c ml/min. patient days with anuria were excluded from the analysis. data are reported as median (interquatile range). patients with neurological disease. several factors may interfere with water and sodium homeostasis in these patients, including factors that are also present in other icu patients. in addition, these patients may develop a syndrome of antidiuresis (siad), or salt wasting syndrome (sw). the latter by secretion of brain natriuretic peptide (cerebral salt wasting syndrome (csw)), release of atrial natriuretic peptide in volume overload, or renal salt wasting. objectives. assess the epidemiology of hypona in icu patients with neurological disease. methods. retrospective single center study. data were retrieved from electronic icu databases. inclusion criteria were age c years, hypona (\ mmol/l), and neurological disease. only the first episode of hypona was considered. siad and sw was assessed with tonicity balance on data of the preceding h in patients with urinary sodium[ mmol/l, in whom other etiologies were excluded. sw was defined as negative salt balance and negative fluid balance, and siad as positive fluid balance. to evaluate the prevalence of anaemia among patients attended at the emergency room (er) and to estimate the need of an early diagnose and efficient treatment. observational transversal trial in which days in june were randomly chosen. all patient attending to the er is included. paediatric, gynaecologic and traumatic cases fall out of this research. anaemia was diagnosed according to who criteria. comparison means statistics methods for quantitative variables and chi square for categorical variables were used. prevalence data for the entire cohort, for men and women separately and for different age bands, medical history, anaemia related medication and red blood cells data were extracted. results. patients were interned through the er channel. % men, mean age . ± . years old (median ), and , % were subject of blood analysis using classification proceedings. from the analysed . % were anaemic. . % of them were under years old, % aged from to and . % elderly patients (over years old). most frequent co-morbidity was chronic obstructive pulmonary disease (copd) (n = , . %) and most related drug aspirin (n = , . %). % of the sample had a bleed but only . % needed red blood cell transfusion. we found statistic difference in mean age, antiplatelets therapy use, bleeding episode, need for transfusion, creatinin value and hospitalisation. anaemia classification according to vcm: microcytic . %, normocytic %, macrocytic . %. conclusion. unknown anaemia detection in the er and its following treatment could be a strategy to further reduce allogeneic blood transfusion. the presence of disorders of sodium balance on icu admission could be independently associated with mortality. we decided to study if the existence of dysnatremias at the time of icu admission could be related to mortality in critically ill patients. we conducted a retrospective study in a mixed icu with a database of , adults admitted consecutively over a period of years ( - ) . most patients ( . %) had normal sodium levels ( b na b mmol/l) on icu admission. the frequencies of borderline ( b na b mmol/l), mild ( b na \ mmol/l), and severe hyponatremia (na \ mmol/l) were . , . %, and . %, respectively. the frequencies of borderline ( \ na b mmol/l), mild ( \ na b mmol/l), and severe hypernatremia (na [ mmol/l) were . , . , and . %, respectively. all types and grades of dysnatremia were associated with increased raw and risk-adjusted hospital mortality ratios. multiple logistic regression analysis showed an independent mortality risk rising with increasing severity of both hyponatremia and hypernatremia. odds ratios and % confidence interval (ci) for borderline, mild, and severe hyponatremia were . , . and . , respectively. odds ratios and % ci for borderline, mild, and severe hypernatremia were . , . , and . respectively. conclusions. this observation suggests the possible correlation of natremia on icu admission with hospital mortality and confirms that both hypo-and hypernatremia present on admission to the icu could be independent risk factors for poor prognosis in icu populations. ( ) . a relationship between mortality and delay from time of pars trigger to critical care admission for patients not requiring surgery has recently been described ( ) . objectives. this study was to test the hypothesis that in cases of emergency laparotomy, prolonged physiological deterioration pre-operatively is associated with higher hospital mortality. we reviewed notes of patients that underwent emergency laparotomy between july and february at the northern general hospital, sheffield, uk. time at which patients triggered (pars c ), time of arrival in theatre and hospital mortality were recorded. two-tailed fisher's exact test was used to test null hypotheses that a delay of more than h after pars trigger does not affect hospital mortality. . patients had an emergency laparotomy during this period. of notes retrieved by our patient record service, were incomplete. of remaining patients patients did not trigger, of whom died ( . % mortality). patients triggered, died ( . %). amongst patients that triggered, arrived in theatre within h, of whom died ( . % mortality); of the patients that arrived in theatre after h died ( . % mortality). the odds ratio of death for those with a prolonged pre-operative deterioration (n = ) compared to those without (n = ) was . ( % ci . - . , p = . ). the number needed to treat early to save one life was . conclusions. our data suggest that in cases of emergency laparotomy, those who trigger pars pre-operatively have higher hospital mortality than those who do not. specifically, our data indicate that patients triggering pars c should arrive in theatre within h of first triggering. nothing is known about the effect of the duration of icu-related therapies on acute outcome. to identify the importance of the duration of invasive ventilation and of renal replacement therapy for acute prognosis of surgical patients treated in an intensive care unit (icu). we performed a retrospective analysis of prospectively collected data of an icu patient cohort linked to a local database. adult patients (n = , ) admitted to a -bed icu at a university hospital in munich, germany, between and who had an icu length of stay of more than days and who were followed-up until the end of the acute phase after icu admission. cox-type additive hazard regression models were used to analyse linear, nonlinear or time-varying associations of therapeutic variables with survival time. duration of different invasive therapies was evaluated by constructing specific vectors, which tested potential effects of time-dependant variables on outcome after a lag time of days. . patients ( . %) were still alive at the end of the acute phase after icu admission. during the acute phase, . % of the patients required invasive ventilation, and . % a continuous renal replacement therapy. besides the underlying disease and disease severity at icu admission, the need for invasive ventilation or renal replacement therapy was associated with poorer outcome. duration of invasive ventilation shortened survival (with a lag of week), if treatment lasted for more than days (non-linear association). in contrast, duration of renal replacement therapy was unimportant for acute prognosis. conclusion. prolonged duration of invasive ventilation, but not of renal replacement therapy is inversely related to acute survival. objectives. to identify the prognostic importance of preceding invasive ventilation, renal replacement therapy and catecholamine therapy for long-term survivors after surgical critical illness. we performed a retrospective analysis of prospectively collected data of an icu patient cohort linked to a local database. adult patients (n = , ) admitted to a -bed icu between and , who had an icu length of stay of more than days, were followedup until the end of the second year after icu admission. hazard function was explored by weibull modelling and likelihood ratio tests. cox-type structured hazard regression models were used to analyse linear, non-linear or time-varying associations of therapeutic variables with -year survival time of a patient subgroup, which had survived the period of high hazard. hazard rate declined exponentially up to day after icu admission, and became constant thereafter. patients reached this stable stage of their disease forming the study population. of these patients ( . %) were still alive at the end of the second year after icu admission. underlying diseases were major determinants for long-term outcome. long-term mortality was significantly associated with the acute extent of physiological derangement during icu stay (maximum apache ii score), but was independent from the duration of preceding invasive organ support. in surgical patients with a prolonged icu length of stay, an exorbitant mortality exists for about half a year after icu admission. later on, life expectancy of surviving patients is largely determined by the underlying disease and, to a minor degree, by the acute extent of homeostatic disturbance during icu stay. the duration of preceding invasive therapies does not limit long-term survival. b. rozec , a. desdoits , k. asehnoune , c. lejus , y. blanloeil chu nantes, hôpital laënnec, anesthesia and intensive care, nantes, france, chu nantes, hotel-dieu, anesthesia and intensive care, nantes, france postoperative stroke could be an endpoint in non-cardiac surgery morbidity studies [ ] . therefore, its frequency established in old studies and considered higher than in the non surgical population remains to be estimated more precisely. objectives. the aim of this evaluation was to calculate the frequency of stroke, firstly in a prospective study, and secondly in a review of the literature. strokes diagnosed in the prospective follow-up ( days) of surgery for hip fracture was confirmed by a neurologist and a ct-scan. retro and prospective studies (except abstract) published in journals (pubmed, ovid) from to were included in the analysis. statistics:% ic , multivariate analysis (effects of population size, date of publication, type of surgery, patient age, prospective vs. retrospective studies were evaluated). results. mean pre-operative possum scores between the two groups showed no significant differences. continuous measurements taken by the odm showed a mean stroke volume increase of mls at the end of surgery (paired t test, p-value = . ). ( . %) patients following implementation compared to ( . %) prior to implementation required post-operative critical care admission. following odm implementation, critical care los was reduced from . to . days and post-operative length of stay within hospital was also significantly reduced by . days. introduction. local (skeletal muscle necrosis) and remote (lung neutrophil infiltration) ischemia-reperfusion injury (ir) have been described in animals [ ] and humans after aortic surgery. postconditioning with cyclosporina (csa) was recently shown to prevent skeletal muscle infarction in pig latissimus dorsi muscle flaps [ ] . objectives. the aim of this study was to investigate local (gastrocnemius muscle, gc) and remote (lung and liver) ir in rats exposed to aortic cross-clamping with special focus on mitochondrial respiratory chain complexes activities and reactive oxygen species (ros) production. we also investigated whether pharmacological post-conditioning with csa would be protective in this setting. methods. anaesthetized (isoflurane) and mechanically ventilated wistar rats underwent laparotomy and were randomly assigned to one of the following groups: sham (n = ), ir (n = , clamping of the infrarenal aorta for h followed by h of reperfusion), ir+csa (n = , mg/kg csa administered intraperitoneally and min prior to reperfusion). maximal oxidative capacities (vmax) and complexes i, ii and iv of the mitochondrial respiratory chain were determined using glutamate-malate (vmax) and succinate (vsucc) as substrates in the gc permeabilized fibers and freshly harvested liver and lungs isolated mitochondria. tissue superoxide anion production was assessed with dihydroethidium (dhe) in thin sections of frozen gc. data are expressed as mean±sem and analyzed with anova followed by newman-keuls post-hoc test; a p value. was considered significant. esophagectomy with gastric tube reconstruction is associated with frequent postoperative complications due to a (surgery induced) systemic stress response, provoked by the overproduction of proinflammatory cytokines. in elective postoperative esophagectomy patients, we previously showed that levels of serum crp are associated with the occurrence of postoperative complications and reduced survival. plasma ngal (pngal) and urine ngal (ungal) are early predictors of acute kidney injury, however sepsis/sirs seems to accelerate their production even in the absence of aki. objectives. we examined the role of pngal and ungal as early indicators of postoperative complications at the icu in patients undergoing elective esophagectomy surgery methods. in a prospective follow-up cohort study, data of a total of patients admitted to the icu following elective esophagectomy with gastric tube reconstruction were collected in the period from september to april . patients who developed aki at the icu were not included in this study. postoperative pngal and ungal levels were determined at consecutive time points and the relation between the course of postoperative serum pngal and ungal, development of complications and outcome of the patients was investigated. in our experience, assisted by dv robot radical prostatectomy, despite requiring longer surgery time, was shown to be safer than conventional radical prostatectomy, with a significant less blood loss during surgery, less need for blood transfusion, fewer postoperative complications, included need to reoperate, and also a shorter length of hospital stay than conventional radical prostatectomy. objectives. the aim of the study was to evaluate safety and effectiveness of m infusion, impact on fluid balance and urine output (uo) and also whether we can avoid cvc line insertion. we conducted a prospective analysis of patients (pts) treated with m who were admitted to the shdu between oct and aug . demographic data and vital parameters were collected through the individual questionnaires before ( ) introduction. an ever increasing number of patients over the age of year are being admitted to critical care units [ ] . with no marked expansion in the number of critical care beds in our hospital and in the health region as a whole, this may lead to a huge strain on the service provision, with less availability of beds to treat elective and other emergency admissions. to determine the factors that affect outcome following admission to critical care of patients aged years and above(medical and surgical). methods. ethical approval was sought but deemed unnecessary as our study was observational (non-interventional). we prospectively looked at the number of patients (age year and above) who were admitted to icu/warrington general hospital over the period of year. our unit is a modern, -bedded general icu with an annual admission rate of approximately ( level and level care). we examined data that was related to the source of admission, gender, apache scores, the use of vasopressors (including inotropes) and the need for ippv in the first h of admission. we analysed the effect that each factor had on patient mortality (applying chi-square and z tests). we followed the patients up for months post-discharge from icu. the final report produced results that showed icu, hospital and -month mortality. results. there were admissions during the period st may - th april . last set of mortality data was obtained in september . female: male ratio was : . the overall -month mortality was %. in our study, patients admitted through a&e, theatre and ward had mortality rates of , and % respectively. patients who received vasopressors (including inotropes) in the first h had a significantly lower mortality than patients who did not receive any vasopressor support ( vs. %). invasive ventilation in the first h of admission was associated with significantly higher mortality rates ( vs. %). in this patient cohort, the overall -month mortality is higher than the general icu population. factors that determine mortality include the source of admission to icu, the need for vasopressor support and invasive ventilation in the first h of admission. introduction. the optimization of oxygen delivery (do ) is an intervention with fluids and inotropics to achieve supranormal goals of do during surgery, before disturbances of perfusion occur and oxygen debt accumulates. oxygen debt is directly linked to multiple organ failure and death. we aimed to evaluate the temporal pattern of oxygen debt in the intraoperative period in patients included in two studies of goal directed therapy to supranormal values of do . oxygen debt was calculated from data obtained from high risk surgical patients included in two randomized controlled trials were analysed. , the oxygen deficit was calculated by subtraction of the basal vo value from subsequential values of vo obtained during surgery and after the icu admission. the oxygen debt was calculated by the product of oxygen deficit and time (minutes) between measurements. patients treated with supranormal goals of do ([ ml/min/m ) using fluids and dobutamine showed lower levels of oxygen debt during icu stay. the peak of oxygen debt was , ml/m at min of surgery in the control group in comparison to , ml/ m in the protocol group. in the second study, the peak of oxygen debt occurred at min in the volume group ( , ml/m ) which was significantly higher than in the dobutamine group ( , ml/m ). higher oxygen debt during peroperative period correlated with poor outcome as shown on the original studies. conclusion. the use of supranormal goals of do with dobutamine and fluids in the peroperative period results in lower oxygen debt. post-operative nausea and vomiting (ponv) is a common problem in the patients undergoing laparoscopic surgery. the release of serotonin during surgical procedure may induce ponv. we investigated if postoperative increase in plasma serotonin metabolite was associated with ponv after gynecologic laparoscopic surgery. objectives. the patients who experienced nausea after gynecologic laparoscopic surgery (ponv group, n = ) and patients who had no or mild nausea (control group, n = ) were enrolled into this study. postoperative nausea was assessed during h in post-anesthetic care unit and ondansetron was administered if needed. blood samples were obtained before anesthesia and h after surgery. plasma serotonin metabolite ( -hydroxy indole acetic acid, -hiaa) was analyzed using high performance liquid chromatography (hplc) assay. perioperative change of plasma -hiaa and the degree of nausea were compared between groups. results. the degree of post-operative nausea varied from to ( mm visual analogue scale, vas) and median value was ( - ) in control group and ( - ) in ponv group (p \ . ). average -hiaa concentration of all patients increased after surgery ( . ± . to . ± . ng/ml, p \ . ). baseline plasma -hiaa concentrations were similar between groups, however, -hiaa of ponv group increased higher after laparoscopic surgery compared with control group ( . ± . to . ± . ng/ml vs. . ± . to . ± . ng/ml, p = . ). conclusions. the patients who experienced post-operative nausea showed more increase in -hiaa concentration. ponv after gynecologic laparoscopic surgery may be associated with a peripheral release of serotonin. introduction. the intracavitary ecg method is an easy, accurate and inexpensive methodology for real time positioning of the tip of central venous catheters. in particular, when the ecg method is performed using not the guidewire but the saline-filled catheter as electrode, the methodology is completely safe and can be applied to any central venous access device (vad). we have tested a new specific device (sapiens tls, romedex) which simplifies and standardizes the ecg method; it consists of a hardware (a small box with cables connecting it to a pc and to ecg electrodes) plus a software (which can be used on any pc). we tested the sapiens tls in patients who underwent positioning of central vads ( totally implantable ports, piccs and tunnelled catheters, all inserted by ultrasound guided venipuncture). our goal was to position the tip of the catheter at the cavoatrial junction: the length of the catheter was estimated by anthropometric measurements and the correct positioning was achieved by the intracavitary ecg method during the procedure. the final position was checked by a post-procedural chest x-ray. there was no insertion-related complication. the intracavitary ecg method was easily performed in all cases. at the final x-ray control, % of all tips were correctly positioned at the cavo-atrial junction (± cm), confirming the accuracy of the intracavitary ecg method. the anthropometric measurement tended to overestimate the length of the catheter both in port insertions ([ cm in % of cases) and in picc insertions ([ cm in % and [ cm in % of cases). conclusions. the intracavitary ecg method as performed with the sapiens tls was more accurate than the anthropometric measurement in terms of correct positioning the tip of the catheter during the procedure. the sapiens tls simplified the method, by standardizing the ecg tracking, and making it easy (no need of ecg monitor, no need of ecg commuter since the sapiens tls displays simultaneously the surface ecg and the intracavitary ecg). also, the sapiens tls allows the print-out of the intracavitary ecg reading for documentation, as well as the storing of the ecg reading in a computer-based database. previous studies have shown that hypernatremia impact graft function after orthotopic liver transplant (olt). the purpose of this retrospective investigation was to determine whether differences in serum sodium values after olt influenced postoperative short-term patient outcomes. objectives. the study was aimed at exploring the incidence of hypernatremia after orthotopic liver transplantation (olt) in order to provide critical monitoring and intensive care services. clinical and sicu laboratory data were collected; serum sodium was assessed an average of three times per day. hypernatremia was defined as two daily values of serum sodium above mmol/l. from to , we analyzed patients with hypernatremia after olt. the major outcome was death in the icu after days. conclusions. in sicu, olt patients are easy to suffer from hypernatremia ( . %) and have high mortality ( . %). hypernatremia is associated with an increased risk of death in patients with olt. early active fluid infusion is crucial, besides optional continue venovenous hemofiltration (cvvh). cywinski objectives. the aim of this study was to determine the value of blood lactate sequential dosages during the first postoperative hours for the diagnosis of gd. we conducted a retrospective study on consecutive patients admitted in icu after lt, between july and june . lt with auxiliary or splited grafts were excluded so were patients with septic or cardiogenic shock occurring during the first h after lt. criteria for gd diagnosis were: sgot [ , u/l with pt \ % between d and d , or re transplantation or death between d and d . demographics and biological data (transaminases, pt, serum bilirubin) were recorded between d and d . hopital bicêtre, kremlin-bicêtre, france, hôpital saint-louis, paris diderot university, biostatistics, paris, france, hôpital hôtel-dieu, medical intensive care unit, nantes, france, hôpital gabriel montpied, nephrology and transplantation, clermont-ferrand, france, hôpital edouard herriot, medical intensive care unit, lyon, france, conclusions. in kidney transplant recipients, arf is associated with high mortality and graft loss rates. increased pneumocystis and bacterial prophylaxis might improve these outcomes. early icu admission might prevent graft loss. a. umgelter , k. lange , p. büchler , h. friess , r.m. schmid technical university of munich, transplantationszentrum münchen rechts der isar, ii. medizinische klinik, münchen, germany, technical university of munich, transplantationszentrum münchen rechts der isar, chirurgische klinik, münchen, germany introduction. the shortage of donor organs in the eurotransplant region results in late allocation at a time when liver disease is already very advanced. the severe condition of patients at that stage negatively affects outcomes of orthotopic liver transplantation (olt). to support decision-making in these situations, clinical data are urgently needed. objectives. to evaluate outcomes of liver transplantation (olt) in icu-patients with multi-organ failure due to advanced acute on chronic liver failure (aclf). methods. in our centre, patients on the waiting list for olt are not automatically excluded from the procedure, if their condition deteriorates to multi-organ failure. a consensus of the team in each individual case is based on criteria such as the previously manifested will of the patient, exclusion of current infection, absence of neurologic damage or other organ damage expected to impair the possibility of rehabilitation. we retrospectively analyzed a database comprising data from evaluation for the waiting list of all patients transplanted in our center since implementation of the meld-score for allocation. only cirrhotic patients treated on our intensive care unit before transplantation were included. patients treated on the icu before retransplant for primary graft failure were excluded from analysis. data are presented as median ( th - th percentile). wilcoxon or mann-whitney u tests were used for comparisons of paired and unpaired data, respectively. results. from january until september patients ( m, f; age ( - ) years) fulfilled inclusion criteria. cirrhosis was due to alcohol (n = ), hcv (n = ), alcohol+hcv (n = ), alpha- -antitrypsin deficiency (n = ), budd-chiari-syndrome (n = ) or cryptogenic (n = ). upon icu admission icu, apache ii scores were ( - ), sofa scores ( - ); meld ( - ). after ( - ) days on the icu, directly before transplantation, sofa scores had deteriorated in all patients to ( - ) and meld scores to ( - ). patients had renal replacement therapy and patients were on single-pass albumin dialysis. the -day-mortality was %, hospital mortality % and -year mortality %. in hospital survivors, surprisingly, sofa scores ( ( - ) vs. ( - ); p = . ) and inr ( . ( . - . ) vs. . ( . - . ); p = . ) upon admission to the icu were significantly higher than in non-survivors. there were no significant differences in age, gender, meld scores or use of extracorporeal treatment in survivors vs. non-survivors. conclusions. liver transplantation in selected cirrhotic icu-patients with multi-organ failure is not medically futile. outcome, however, is much worse than usually considered acceptable. objectives. this work tries to study the clinical profile and the results with the immediate postoperative outcome of patients suffered from pancreas-kidney transplantation (pkt) and only pancreas transplantation (pt) admitted in our intensive care unit (icu) setting. methods. prospective study during years (from to ). we recorded epidemiological, demographical and clinical data, surgical and postsurgical complications, therapy, morbidity and mortality rate, length of stay in icu, organs survival, etc. the data were expressed in mean±typical deviation, median and percentages. results. we recorded patients. table expresses some of the data. the mean age was . ± . years old male . %. pkt from unic deceased donor: %. pt: %. the mean ischemia time was . ± . h for the kidney and . ± . for the pancreas. the most frequent surgical complications were bleeding ( . %), technical difficulties ( . %) and anesthetic complications ( . %). postoperative immunosuppression consists in methyl-prednisolone, tacrolimus, mycophenolate mofetil and thymoglobulin (as our protocol recommends) and was administered to the % of the patients. prophylactic antibiotic and antiviral therapy (ampicillin, ceftriaxone, fluconazole and gancyclovir) was given to almost the % of the patients. table expresses the blood test results. the mean insulin requirements per day during the stay in icu was ( - . ) iu. table represents the complications in the icu. the first leading cause of reoperation was vascular thrombosis ( %) followed by intraabdominal bleeding ( %). conclusion. the clinical profile of this patient in our setting is a years old man, with high blood pressure, retinopathy, dialysis, pancreas-kidney transplant recipient, with unic decesed-donor. he needs no insulin or minimal requirements. the principal complication is pancreatic vascular thrombosis that frecuently leads to removal of the graft. ( ) . in a clinical practice, a specific marker to evaluate and predict ischemic-reperfusion injury in liver transplantation (olt) is not available. poor organ perfusion and high pct levels appeared to predict early graft failure only in the cardiac donor ( ) . objectives. we evaluated pct as a predictor of ischemic-reperfusion injury in liver transplantation and pdr, as a predictor of complication and graft outcome. methods. prospective study. patients (age, child-pugh score, aetiology of liver cirrhosis) undergo liver transplant. bilirubin levels and pdr ( . mg/kg of ig in a central catheter with limon system Ò , pulsion medical system, munchen, germany) was measured once a day for postoperative days (pod). on the same day, aspartate aminotransferase (ast/gpt) and alanine aminotransferase (alt/got) were measured. sofa score was as a patient severity score. serum level of pct, c-reactive protein (crp) was collected at the liver reperfusion time and from the st to the rd pod. warm and cold ischemia time was collected. statistical analysis was performed with wilcoxon, spearman tests (p \ . ). linear correlation was performed too. a small rise on pct levels were observed early after olt, with a peak in the st day after olt. it was associated neither with hepatic post-olt dysfunction nor with other non infective complications. pct increased significantly after liver reperfusion (p \ . ) and correlate with pdr on the nd pod, but not correlation were found with crp, white blood cells, or liver enzymes after olt. crp levels increased rapidly after olt. pct increasing after liver reperfusion correlated with child-pugh olt (r = . at nd pod) in the recipients. the cold ischemia time did not correlate with pct serum levels after liver reperfusion as well as the warm ischemia time. a negative correlation was found between pdr and liver function in the recipient. pdr did not correlate with child-pugh score. the cold ischemia time well correlated with ast and alt on the first day after transplant (r = . ). it negatively correlate with pdr (r = - . ) at the same time. the warm ischemia time did not correlate with pdr, ast and alt. the same results were found between pdr and liver enzymes and lactates. no correlation was found between pdr and sofa score. conclusions. pct peak in the recipient at reperfusion and early post operative was not predictive of graft dysfunction or other non infective complication. it may represent a marker of ischemia-reperfusion injury. crp levels increased rapidly after olt and it could be an expression of surgical procedure, and it doesn't correlate with pct. objectives. the aim of this prospective observational study was to describe the kinetics of ngal following renal transplantation and to assess its ability to predict delayed graft function. introduction. lung transplantation is the recognized therapy for end-stage respiratory failure. many serious medical complications have been described occurring from months to years after lung transplantation, often necessitating admission to an intensive care unit (icu), which has been associated with a high mortality. we examined the factors associated with mortality. methods. all patients admitted to the general intensive care unit between and following lung transplantation were included in this retrospective study. data was collected regarding demographic parameters, intensive care unit stay and outcome. over the study period, forty patients were admitted to the icu. the main pretransplant diagnosis was idiopathic pulmonary fibrosis followed by chronic obstructive pulmonary disease. the majority ( %) of patients required mechanical ventilation during their icu stay. the main reason for icu admission was septic shock in patients ( %) of cases. an organism was isolated from of these patients; in cases, the organism was shown to be multi-drug resistant. the icu mortality was . %. non-survivors were characterized by a higher admission sofa score (p = . ), an admission diagnosis of sepsis ( . vs. . % for all other diagnoses, p \ . ), and a requirement for mechanical ventilation (p = . ). in addition, the incidence of chronic rejection was significantly higher in the non-survivors (p = . ). conclusions. severe sepsis remains the most important factor associated with a poor outcome. new strategies are required to alter the course of this common complication of lung transplantation. (the % of the infections were respiratory) and ( %) patients presented pulmonary allograft rejection. according to our immunosuppressive protocol, we started with methylprednisolone and tacrolimus and we added mycophenolate later. the ccd length of stay was ( - ) days and the median days of mechanical ventilation were ( - ). thirteen ( %) patients died, basically due to refractory respiratory failure, multiple organ dysfunction syndrome and haemorragic shock. conclusions. in our large series of lung transplantation a remarkable incidence of complications has been observed. despite this complications, lung transplanted patients presented an excellent short term outcomes. introduction. acute kidney injury (aki) poses a massive challenge after kidney transplantation, especially when kidneys from brain dead adult donors are transplanted into small paediatric recipients. inflammation mediated by cytokines is a key event in experimental models of ischaemic aki. objectives. the aim of this study was to investigate whether remote ischaemic preconditioning (ripc) reduced the inflammatory cytokine load and apoptosis in the kidney after transplantation. methods. kidneys were harvested from eight -kg brain dead donor pigs and transplanted into two groups of -kg recipient pigs after h of cold ischaemia. in one group (+ripc, n = ) ripc was performed before the -h reperfusion period, while no ripc was performed in the other group (-ripc, n = ). non transplanted kidneys from brain dead pigs served as controls. concentrations of tnf-a, il- , il- , and il- in renal tissue were determined by an immuofluorometric assay. renal apoptosis was quantified by immunohistochemistry for activated caspase- . high concentrations of tnf-a, il- , il- , and il- were detected in renal cortex in all three groups. no statistical differences between the two transplanted groups were found for any of the cytokines. compared to controls higher cortical levels of il- (control vs. -ripc, p = . , control vs. +ripc, p = . ) and lower levels of il- (control vs. -ripc, p = . , control vs. +ripc, p = . ) were found in transplanted kidneys. no differences were detected for tnf-a or il- . transplantation significantly increased the number of apoptotic cells in both glomeruli and tubuli (control vs. -ripc, p = . , control vs. +ripc, p = . ). no difference was found between recipients, (p = . ). conclusions. in transplanted kidneys from brain dead donors exposed to h of cold ischemia and ±ripc, we found increased tubular and glomerular apoptosis, but no increase in pro-inflammatory cytokines. the levels of il- were higher in transplanted kidneys compared to controls. remote ischaemic preconditioning did not modify cytokine load or apoptosis in the kidney graft. objectives. we present the case of a patient with confirmed hit and the management of its status during the perioperative period of the cardiac transplantation. a years old patient with a cardiac myxoma was operated under heparin anticoagulation. thrombocytopenia is noted at day after surgery. an enzymelinked immunosorbent assay (elisa) was performed and since the result was positive, the treatment was changed to lepirudin. the hit was confirmed by a heparin-induced platelet activation (hipa) test. the internal jugular vein thrombosis was observed. the post operative evolution was marked by the necessity of the implantation of a ventricular assisted device. the patient was submitted to two sessions of plasmapheresis which turned the antibodies negative. the patient underwent heparin anticoagulation during the surgery time and bivalirudin as the post operative treatment. the antibodies remained negative. two months later, the cardiac transplantation was performed; heparin was used for anticoagulation during surgery. due to a restored renal function, danaparoid was used postoperatively. conclusions. hit is a serious complication of heparin therapy. the diagnosis is difficult. when hit is strongly suspected, a non-heparin anticoagulant is recommended. the choice of the anticoagulant depends on the hepatic and renal function. plasmapheresis is a solution for the antibody purging prior to cardiac surgery. objectives and methods. a nationwide qualitative study investigating their perception of the meaning of professionalism, and how they learn to behave professionally was performed. all eight dutch icm training centres participated. the moderator asked participants to clarify the terms professionalism and professional behaviour. next, participants were asked to explore the questions 'how do you learn the mentioned items?' and 'what ways of learning do you find useful or superfluous?' qualitative data analysis software (maxqda ) facilitated analysis: an inductive approach applying open, axial and selective coding principles was used. results. fellows across eight groups participated. results relating to the subtopics 'elements of professionalism' and 'teaching and learning of professionalism' are described consecutively. elements of professionalism relevant to intensivists: the elements most frequently addressed were communication, keeping distance and boundaries, medical knowledge and expertise, respect, teamwork, leadership and organization and management. medical knowledge, expertise and technical skills seem to become more tacit when training progresses, and relate to ethical, cultural and legal dilemmas originating in the specific icu context, and working as a multidisciplinary icu team member. teaching and learning professionalism: topics can be categorised into the themes workplacebased learning, by gathering practical experience, by following examples and receiving feedback on their actions, including learning from own and others' mistakes. formal teaching courses (e.g. communication) and scheduled sessions addressing professionalism aspects were also valued. conclusions. the emerging elements considered most relevant for intensivists were adequate communication skills, and keeping boundaries with patients and relatives. the specific icm context, and working as multidisciplinary icu team member substantially influenced the icm fellows' perception of professionalism. whereas medical knowledge, expertise and technical skills seem to become more tacit when training progresses, professionalism issues continue to be learned during icm training. professionalism is herein mainly learned 'on the job' from role models. formal teaching courses and sessions addressing professionalism aspects were nevertheless valued, and learning from own and others' mistakes was considered especially useful. selfreflection as a starting point for learning professionalism was stressed. the latter can e.g. be stimulated by means of assessment, structured feedback and use of portfolios, for which guidelines are now being developed within the cobatrice project. introduction: during the past decades there has been an increase in mass casualty events with changing geopolitical and climate situations. in a mass casualty event comprehensive care for the individual is expected [ ] . to meet these obligations further education in disaster medicine seems obligate [ ] . therefore the german home department responsible for mass casualties passed a concept for student education in disaster medicine [ ] . objectives. the introduction of a summer academy ''disaster medicine'' (sadm) is a first approach at charité university of berlin to establish a curriculum for disaster medicine. the sadm is sponsored by the german academic exchange service (daad) for years. the enhancement of student education in disaster medicine is supposed to raise the level of skills and knowledge of future physicians in the face of mass casualties [ ] . international participants and an interdisciplinary approach are keystones of the sadm concepts. in a globalized world international networking should enable students to exchange knowledge about the handling of mass casualties in different parts of the world. disaster medicine needs an interdisciplinary approach [ , ] . psychological aspects are always a key factor in the successful handling of mass casualties. the teaching concept of sadm consists of four parts: e-learning ahead of a week training session, emergency medicine training, disaster medicine training and excursions to evaluate already existing disaster concepts. the concept will be evaluated [ ] using a knowledge test, a skills test and a structured written interview concerning motivation and satisfaction of the students. conclusion. the support of the daad for three consecutive years allows a further evolution of this concept by integrating the evaluation results. the sadm should enable future physicians to meet the challenges of mass casualties with greater confidence and skills. educational programs are being set up to provide training and skills in these core subjects for dental care professionals. objectives. to evaluate dental practitioner (dp) skills and knowledge prior to a day continuous medical education (cme) training session, and assess training efficacy at the end of the session. methods. nine ( ) multiple choice questions concerning medical emergencies and pain treatment were handed out to dps at the beginning of cme training sessions over a year period in metropolitan france. after the day training session, the same multiple choice was taken again and collected for statistical analysis (kruskall-wallis test). examination before and after cme, p \ . we evaluated dps and obtained a % answering rate. before the cme session, the correct answer rate was below % for several items, like the european emergency telephone number or performing back blows before the heimlich manoeuver for severe choking, and below % for identifying vasovagal malaise by bradycardia, giving insulin for diabetic malaise or treating anaphylactic shock by epinephrine. more worrisome still is the fact that nearly out of dps would prescribe non steroidal anti inflammatory drugs (nsaids) during late pregnancy. the overall impact of cme was highly significant (p \ . ), showing real efficacy but correct answering rates after cme still remained between and %, which leaves room for improvement. further studies are under way to evaluate long term memorization of cme sessions in order to determine their optimal frequency. conclusions. medical skill and proficiency evaluation before cme training sessions for dps allows to target the training sessions and to evaluate their efficacy in the short run. introduction. the positive impact of immediate bedside echocardiography for rapid diagnosis and management of acute hemodynamic disturbances in the critically ill patient is well established. it is advocated that peri-resuscitation echocardiography should be an integral part of training for all intensivists. however, a major challenge for the intensive care clinician is access to appropriate echocardiography training outside of specific fellowship programmes. objectives. one suggestion to meet this training need is to combine supervised practical instruction with self-learning through the use of on-line educational tools. the internet is ideally suited to studying echocardiography as e-tutorials serve to convey theoretical principles whilst stills/video clips aid image recognition and interpretation. here we review currently available web based learning resources. methods. an online search was performed using google Ò and yahoo Ò search engines with the following key words: echocardiography, tte, toe, education, training, programme, courses, on-line, web-based, critical care. the resulting hits were screened to identify relevant sites and these were then evaluated independently by each author before an overall consensus was reached. one author had no previous echocardiography training whilst the other had passed the american national board of echocardiography perioperative transesophageal echocardiography examination. a total of sites were identified for evaluation (see table ); these are listed below with a brief description. conclusions. our search demonstrated a number of sites dedicated to facilitating echocardiography training. these varied from those which were essentially atlases, to those with a modular learning programme supported by interactive discussions and self assessment. some were targeted at the beginner seeking a basic understanding of echo whilst others were aimed at the enthusiast preparing for examinations. with growing interest in critical care ultrasound it is likely that we will see the use of such resources increasing. however echocardiography is a practical skill and it is essential that on-line learning is conducted in parallel with supervised bedside training in a process of 'blended learning'. . to determine level of supervision for trainees in the elective mri setting as compared with critical care transfers to mri. . to gain insight into the learning resources used by medical staff on mri to allow existing training to be improved. methods. two online surveys were conducted in february , with invitations to participate via e-mail. the survey population included all anaesthesia and intensive care medicine consultants in the local tertiary neurosciences centre and all trainees for these specialties in the northern ireland deanery. first year trainees were excluded. results. the response rate was % for consultants and % for trainees. in total, consultants responded with over % having no experience of mri at consultant level, even though % worked in areas where mri skills could be required. trainees completed the survey, with % having experience of mri in the elective setting, all of whom had been directly supervised by a consultant. % of trainees had experience of critical care transfers for mri, but this was in an unsupervised capacity more than % of the time. despite this, % of trainees did not feel competent to work in mri unsupervised. web based learning was found to be a poorly utilised mri training tool, particularly among consultants. conclusion. we have demonstrated a need to formalize training for mri in our institution and for trainees in the local deanery. we propose to meet this need by a combination of e-learning and experiential sessions with defined competencies. this should increase the cohort of physicians who can provide optimal care , in this unique environment and subsequently improve both service delivery and patient safety. was not a priority in health systems. following the report: ''to err is human. building a safer health system'', by the institute of medicine, which had a great impact on the media, ''patient safety'' is included as an strategic line in most health systems. training in patient safety is essential to implement safety culture and as a result improve it. for that reason we developed a training program ( courses) in for physicians and nurses from our icu. objetive. patient safety training program assessment. methods. we designed a h course ( % practical), using simulated scenarios common in icu clinical practice. we pointed out the relevance of human factors such as teamwork and communication, and its leading role in the genesis of error. we discussed a ''sentinel case'', using the root-cause analysis method, and analysed an icu process through failure mode and effects technique. adverse events reported to the department website were reviewed. participants and instructors discussed specific aspects about insertion of central venous catheter, prevention of nosocomial infection and improvement of security in the different groups of icu patients, highlighting the need for fidelity to the established protocols for this purpose. finally, participants completed a survey that assessed various aspects in a score from to . results indicated the most and least interesting aspects and suggestions for improvement were included. results. assessment surveys were analysed. participation rate was %. overall results: appropriate and clear targets, accomplished goals and utility ( . ) , appropriate content objectives and organization ( . ) , time invested in development activity and oral presentations ( . ), faculty competence ( . ) , interest and faculty adaptability to the group needs ( . ), degree of satisfaction and practices ( . ) . most interesting comments: practice of root-cause analysis ( . %), continued participation and motivation ( . %), practices with hps and group discussions ( . %), importance of human factors ( %), theory and practice good balance ( . %). least interesting comments: too condensed contents ( . %), few scenarios ( . %)suggestions: do it again( . %), enhance preventive medicine sessions ( . %), increase course duration ( . %). conclusions. overall assessment was positive. adaptability and competence of teaching staff have been the most valued aspects; too condensed contents and oral presentations were the least valued. practice of root-cause analysis ease of participation, ongoing motivation, hps scenarios and group discussions are the most appreciated activities. final comment: good acceptance has encouraged us to continue in to complete participation of all interested professionals. introduction. our intensive care unit (icu) was one of the first to initiate a humanization program in daily routine in . since then, the program suffered changes, the icu grew up in number of beds and complexity and had great renewal of the members of our interdisciplinary group. objectives. to improve our knowledge we continually re-evaluated the stress factors for the patients from our staff members' perspective, putting them in the patients place. methods. between january and march of , a research form was used with the interdisciplinary icu team. the following items were analyzed: profile of the interviewed, evaluation of the environment of the icu and the stress factors for the patients. the results were compared with the questionnaire form filled by the patients after icu discharge, as a part of our quality improvement program. results. about . % of our icu team answered the research (n = ). the mean age is . years (sd . ), . % of female, . % married, . % protestants and . % catholics and icu professional experience of . years (sd . ). our icu is noisy for . %, very illuminated for . %, easy-going for . %, organized for . %. in a preview research we found closed results. according to the team, factors that bother the patients are: noise ( . %), bed bath ( . %), loneliness ( . %), lack of privacy ( . %), anxiety ( . %), distortion of time perceptions ( . %) and fear ( . %). the patients (n = ) described as main complaints after icu discharged: distortion of perceptions of time ( . %), anxiety ( . %), sleeplessness ( . %), noise ( . %), loneliness ( . %), fear ( . %), pain ( . %)bed bath ( . %) and lack of privacy ( . %). the study showed differences of icu team opinions and the patients' complaints. when the team is placed in the patient's perspective they may experience a better view of how harmful is an icu and how much we can do to improve it. this is our daily challenge: take care with quality, respect, affection and always search for improvement. introduction. endotracheal intubation is a routine procedure to protect the airway in critical care, that is performed by a wide variety of clinicians from different specialities with different levels of experience in airway management. serious complications can result from misplacement of an endotracheal tube (ett) in a main stem bronchus. a widely recommended method for the prevention of this complication is bilateral auscultation of the lungs; but this method frequently provides only inconclusive results ( ) . other routinely used tests to verify correct endotracheal tube placement include observation of symmetric chest movements, and inserting the ett to a specific depth, but it remains unclear which of these tests detects endobronchial intubation best. objectives. we therefore designed this study to determine which bedside method has the highest sensitivity and specificity for detecting endobronchial intubation in adults and whether sensitivity and specificity increases as a function of the anesthesiologist's experience. methods. surgical patients were randomized to two study groups. in the first, the ett was fiberoptically positioned . - -cm above the carina, whereas in the second group the tube was positioned in the right main stem bronchus. first year residents and experienced anesthesiologists randomly performed only one of the following tests to verify the position of the tube: ) bilateral auscultation of the chest (auscultation); ) observation and palpation of symmetric chest movements (observation); ) estimating the position of the ett by the insertion depth (tube depth); and, ) a combination of all three mentioned tests (all three). results. patients ( female/ male) with observations by experienced and inexperienced anesthesiologists were included in the study. tube depth and all three had a higher sensitivity ( . and ) in detecting endobronchial intubation than auscultation ( . ) and observation ( . ) (p \ . ). experience increased the sensitivity only for auscultation, with % of first year residents versus % of experienced anesthesiologists detecting endobronchial intubation by auscultation correctly. the optimal ett insertion depth was found to be cm in women and cm in men. we conclude that auscultation alone is inadequate for assessment of correct ett insertion depth, and that checking for symmetric chest movements is of little use. our results suggest that the hierarchy of the methods used to assess the correct ett insertion depth should be changed and that clinicians should rely more on depth of ett insertion than on auscultation. this is especially true for physicians with less experience in airway management and in situations where auscultation is difficult or impossible. min usa) , uses a new probe measuring hemoglobin saturation at a lesser depth ( vs. mm before), with more data output ( value/ s vs. value/ . s). the new device contains automated software to compute parameters such as occlusion and reperfusion slopes of sto obtained during and after a vascular occlusion test (vot). objectives. to compare nirs parameters obtained with the devices used simultaneously in healthy volunteers and critical care patients to test if the new device gave similar results than the older one. methods. micro-oxygenation parameters were collected simultaneously with the different nirs models, one on each thenar eminence, before (baseline) and during a min upper arm (brachial artery) vot in patients ( septic shock (g ), trauma (g )), compared to healthy volunteers (hv)(g ). nirs probes were then shifted to the contra lateral thenar eminence and a second vot was performed. sto occlusion and reperfusion slopes from both devices were calculated in all groups by the same software, using linear adjustment (r c . to be valid); p \ . was considered significant. following parameters were collected in patients: saps ii and sofa scores, macrohemodynamic (heart rate (hr), mean arterial pressure (map), central venous pressure (cvp), cardiac output (co) and svo (mixed venous o saturation) or scvo ), and metabolic parameters (ph, base excess, and lactate). results. median ± iqr. patients (g and g ) did not differ for macrohemodynamic or metabolic data, except map ( ( - )mmhg vs. ( - )mmhg; p = . ). baseline nirs sto values were similar for both groups and for both devices, but were lower than in hv. during vot, reperfusion slopes were also lower in patients than in hv regardless the device used. the minimum sto during vot, occlusion and reperfusion slopes were significantly different between the devices: intraclass correlation coefficient (icc) . , . and . , respectively, and bland and altman poor agreement and large bias. conclusions. data obtained with model largely differ from those obtained with model , regardless of the studied population for both sto baseline and slopes. these differences appeared more pronounced in hv than in patients. such differences may result from muscle depth, number of data output allowing to more precise linear adjustment, or the minimum value reached during occlusion. it becomes hazardous to compare data obtained with these devices either in hv or in critically ill patients. crrt is used increasingly for the management of acute renal failure in critically ill patients. one major problem with crrt is coagulation of the filters, leading to decreased efficacy and increased costs. regional anticoagulation with citrate is an effective and established form of anticoagulation during crrt in critically ill patients ( , ) . objectives. the aim of this study was to investigate the filter life span during regional anticoagulation with citrate and regarding cost effectiveness. methods. this observational, retrospective study was performed in a mixed surgical and trauma icu in a university hospital. clinical characteristics are shown in table . citrate crrt was performed using commercially available equipment and fluid solutions (multifiltrate Ò with integrated cica Ò -system; fresenius medical care; germany). to maintain stable metabolic and hemodynamic conditions we used an internal standard protocol for citrate crrt. reimbursement for crrt is calculated on procedure related rates (according to german drg). data are shown as mean or median and standard deviation. results. f patients treated with citrate crrt from april through december were evaluated ( table ). the mean circuit lifetime of crrt for all patients was ± h (fig. ) . mean daily costs per patient were calculated as eur and mean benefit for crrt as eur (table ) . commercially available interstitial glucose sensors have already been evaluated for this purpose with promising results. however, because of the range of medications administered in the icu, potential interference with sensor performance must be characterized. to minimize the undesired offset caused by these medications, an interference rejection membrane (irm) was uniquely developed for a new subcutaneous glucose sensor for in-hospital monitoring. the novel irm was studied within the icu setting to gain a realistic picture of its performance in clinical use. objectives. acetaminophen is known to be an interfering agent for electrochemical sensors. to study the functionality of the new irm, the effect of acetaminophen on sensors worn by critically ill patients was assessed. sensor signals were characterized to identify any undesired response from the medication. methods. icu patients simultaneously wore - -day sensors that were connected to ipro tm (medtronic diabetes, northridge, ca) recorders to gather blinded sensor glucose values. patients were given acetaminophen or a mix of hydrocodone and acetaminophen during their icu stays; staff charted the exact time of each medication administration. to assess whether a signal offset would be introduced by the acetaminophen, min of sensor signals before and after medication administration were compared. the period of min was chosen based on acetaminophen's pharmacokinetic profile and the time to reach maximal plasma concentration. the normalized medians for the signal segments before and after each acetaminophen delivery were calculated. the medians formed vectors, each with elements representing signal characteristics before and after the medication deliveries. a paired t test was used to compare the vectors and assess for any effect (p \ . ) on sensor performance. across the patients evaluated for this study, acetaminophen and a mix of hydrocodone and acetaminophen were administered a total of times. one sensor was not available during a medication delivery; thus, occurrences of acetaminophen administration were analyzed. no effect on sensor signals could be identified in the instances of acetaminophen delivery. no statistically significant difference was observed between the signal segments before and after administration (p [ . ). conclusions. this study demonstrates that a novel irm effectively reduces the undesired interference of acetaminophen on a continuous glucose sensor signal during clinical use. although this analysis was focused on acetaminophen, the outcome suggests that the irm may also effectively suppress interferences from other medications administered in the icu. [ ] . however, assessing elastance requires a highly invasive vena-cava occlusion maneuver and left and right ventricle pressure/volume waveforms, which are not typically available in an intensive care unit (icu) and may raise ethical issues in regular use. a validated, lumped-parameter chamber cardiovascular system (cvs) model is used to evaluate a time-varying elastance estimate at the bedside using standard clinical measurements. objectives. to assess time-varying elastance at the bedside for the left and right ventricles using available icu data, and prove the concept on a porcine model of pulmonary embolism. five pigs had pulmonary embolism (pe) induced via injection of blood clots over h, developing full pe in stages from a healthy state. at each state several data sets were taken ( in total over pigs), measuring aortic and pulmonary artery pressure waveforms (p ao (t), p pa (t)), left and right ventricular volume and pressure waveforms (vlv(t), vrv(t), plv(t), prv(t)). at each cardiac state in inducing pe, the time-varying elastances are estimated as elv = plv/ vlv and erv = prv/vrv. these values are correlated to readily measured quantities (pao and gedv). these correlations are used to approximate time-varying elastances erv* and elv* for use in a clinically validated -chamber cvs model. note these approximations are load dependent and thus change with cardiac state. a fivefold cross validation was used to validate the model. a time-varying elastance is generated from data from pigs and used to simulate the fifth pig. simulated pv loops are compared to the originally measured pv loops to validate the approach. results. p ao (t) and elv were highly correlated over the data sets (r = . to r = . ). p ao (t) and elv, gedv and erv are also well correlated (r = . to r = . in this case we report the worldwide first use of the novel deltastream-dp -system (medos corp.) in a patient suffering from acute right heart failure due to pulmonary embolism following cardiac surgery. in a year-old male patient days after mitral valve reconstruction cardiac arrest occurred during physiotherapy treatment. after failure of restoring circulation, a short-term ecls system (lifebridge Ò ) was implanted under cardiopulmonary resuscitation by inserting cannulas in venous and arterial femoral vessels and then switched immediately to the dp -deltastream system. ct-scan revealed the diagnosis of a massive central pulmonary embolism. transesophageal echocardiography showed a dilated failing right ventricle. a thrombolytic therapy was carried out by administering mg alteplase. following ct-scan showed reduced thrombus burden. the deltastream system was carried out for h. ptt was maintained to . -fold under i.v. heparine therapy. pump blood flow was held at a maximum of . l/min ( , - , r/min) for days. despite transesophageal echocardiography showing improved left ventricular function the ecls flow was maintained for further days focussed on the improvement of right heart. further on the pump flow was reduced every h and the system could be explanted after days in now stable cardiolpulmonary situation. the patient was discharged on day at home in good state without any neurological dysfunction. overall duration of the ecls deltastream therapy was h. no system related major complication occurred during the time. even in maximum blood flow of . l/min no relevant hemolysis was measured. ldh level was only slightly elevated to - u/l. introduction. airway management has progressed dramatically in the last years but the most significant advance has been video laryngoscopy. several devices have been introduced since, the most important currently available are the glidescope Ò , c-mac Ò , mcgrath Ò , pentax airway scope Ò , airtraq Ò , among others. a common practice has been to abandon direct laryngoscope intubation (dli) after attempts and move onto advance airway devices such as video laryngoscopy which is becoming the first choice when available. although dli is successful in the majority of patients, poor glottic exposure is more likely to require prolonged or multiple intubations attempts and therefore be associated with complications such as oxygen desaturation or airway and dental injuries. in the intensive care environment an airway should always be considered a difficult airway due to scarce time to perform assessment, to make decisions and to act. should the use of video laryngoscopy be implemented as a routine for airway management in a critical care setting ? objectives. the purpose in this study was to describe for the first time the use of video laryngoscopy, specifically the vel , as a routine choice for airway management in the intensive care environment. single center, prospective observational study, from november to february , was conducted in our intensive care facility, which involved utilization of the vel for all tracheal intubations, no exclusion criteria, rapid sequence intubation (rsi) was the standard procedure. information was recorded by the operator assistant on the same day identifying timings of intubation, number of attempts, success or failure and the difficulties encountered. vel was developed in our institution in and later adopted as a standard airway management by the department of anesthesia. the device has an original mccoy blade with an attached port that holds a channel for the displacement of an optical shaft mm long, . mm in diameter with a °angle view (tekno-medical Ò germany) which is assemble to a video camera (telecandx ii, karl storz, germany), an external light source and to a -in monitor. results. there were tracheal intubations performed by operators, crash intubations and rapid sequence intubations. all intubation attempts were successful, mean number of attempts . the median time to successful intubation was s with no complications. subjective assessment post intubation showed that in all cases vocal cords were view in full, all operators manifested to feel comfortable with the handling of the apparatus but felt dependent on a assistant specially to maintain view while maneuvering the endotracheal tube. conclusions. routine airway management with vel in critical care setting is effective with a high rate of success and most important, with a positive impact for patient safety. introduction. hypovolemia is a common complication in many clinical scenarios and its detection is considered of prime importance. in previous clinical studies, tissue oxygen saturation (sto ) measured by near-infrared spectroscopy (nirs) has been explored for this purpose; however, results are disappointing. it has been suggested that the sensitivity of nirs for detection of hypovolemia might be improved when nirs is applied in combination with a vascular occlusion test (vot). nirs in combination with a vot, consisting of a -min period of arterial occlusion followed by reperfusion, allows quantification of muscle deoxygenation during ischemia (sto downslope; a measure of muscle oxygen consumption rate) and muscle reoxygenation after ischemia (sto upslope; a measure of microvascular reperfusion rate). objectives. in the present study we applied multi-site and multi-depth nirs in combination with a vot in a model of simulated central hypovolemia; lower body negative pressure (lbnp). eight healthy male subjects, with a mean ± sd age of ± years, participated in this study. the lbnp protocol consisted of a stepwise increase of lbnp from to - mmhg. stroke volume (sv), heart rate (hr), cardiac output (co), and mean arterial pressure (map) were continuously measured using near-infrared finger plethysmography (nexfin). multi-depth nirs, with probing depths * and * mm, was performed on forearm and thenar for the measurement of sto . three-min vots were performed by rapidly inflating a pneumatic cuff around the left upper arm before application of lbnp and at lbnp = - mmhg. vot-derived sto traces were analyzed for baseline, downslope, and upslope. . from baseline to lbnp = - mmhg, sv decreased from ± to ± ml (p \ . ), hr increased from ± to ± bpm (p \ . ) and co and map were maintained around baseline level. forearm sto baseline decreased significantly from ± to ± (p \ . ) and ± to ± % (p \ . ) for the and mm probing depth, respectively. forearms sto downslope, measured with the and mm probe, decreased from - . ± . to - . ± . %/min (p \ . ) and - . ± . to - . ± . (p \ . ), respectively. forearm sto upslopes remained unchanged during lbnp. vot-derived sto parameters measured on the thenar did not shown any changes as a result of lbnp. conclusions. vot-derived sto parameters measured on the forearm seem to be more sensitive to the hemodynamic changes associated with lbnp compared to sto parameters measured at the thenar. grant acknowledgment. this project was supported in part by hutchinson technologies inc. introduction. hypovolemia is a common complication in many clinical scenarios and its detection is considered of prime importance. in previous clinical studies, near-infrared spectroscopy (nirs) has been explored for this purpose; however results are conflicting due to inconsistencies in methodology with respect to nirs probing depth and site. objectives. in the present study we applied multi-site and multi-depth nirs in a model of simulated central hypovolemia; lower body negative pressure (lbnp). fifteen healthy male subjects, with a mean ± sd age of ± years, participated in this study. the lbnp protocol consisted of a stepwise increase of lbnp from to - mmhg. stroke volume (sv), heart rate (hr), cardiac output (co), and mean arterial pressure (map) were continuously measured using near-infrared finger plethysmography (nexfin). multi-depth nirs, with probing depths * and * mm, was performed on forearm and thenar for the measurement of tissue oxygen saturation (sto ). . from baseline to lbnp = - mmhg, sv decreased from ± to ± ml (p\ . ), hr increased from ± to ± bpm (p \ . ), and co and map were maintained around baseline level. forearm sto decreased significantly from ± . to ± . % (p \ . ) and ± . to ± . % (p \ . ) for the and mm probing depth, respectively. thenar sto measured with the mm probe remained unchanged, but measured with the mm probe, a decrease from ± . to ± . % (p \ . ) could be observed. conclusions. forearm sto seems to be more sensitive to (simulated) hypovolemia compared to thenar sto and the sensitivity of nirs seems to increase for increasing probing depth. grant acknowledgment. this project was supported in part by hutchinson technologies inc. introduction. sidestream dark field (sdf) is a microcirculatory imaging modality implemented in a hand-held microscope for the non-invasive bed-side visualization of the human microcirculation. despite the many studies showing the importance of microcirculatory imaging in intensive care patients the introduction of sdf imaging into routine clinical practice remains cumbersome. one of the challenges is the need for automatic analysis of the images which currently is subjective and time consuming. objectives. in the present study, we introduce a rapid automated software method for automatic quantification of microvascular density, a key microcirculatory parameter, based on sdf image contrast analysis. methods. twenty-five sequential sdf images (duration = s, resolution = pixels) were isolated from an sdf movie clip, stabilized, and averaged. subsequently, the mean ± sd gray scale intensity in a sliding pixel window was calculated and the sdvalue was assigned to the window center pixel, creating an sdf contrast image. this is a simple and rapid algorithm for vessel wall detection as a pixel window at a tissue-vessel junction will have a high sd-value due to the presence of both light tissue cells and dark red blood cells. conclusions. here, we introduce and validate a rapid automated method for quantification of microvascular density in sdf images. as this algorithm detects vessel walls rather than vessel lumen, smaller and larger vessels have similar contribution to the microvascular density assessment. a limitation, however, is that vessel diameters cannot be detected with this algorithm. the preliminary results confirm the proof of concept of the sdf image contrast analysis software, however, further research is required for its optimization. the criteria believed to be necessary for the implementation of hcs in practice were that his name would be written{ ( )}, the document dated{ ( )} and signed{ ( )}. physicians in private practice wanted date(p = . ) and signature(p = . ) more often than in institution. ( ) physicians thought that the patient must be competent at the designation' time of hcs, especially those who possess advances directives(p = . ) and a hcs(p = . ) themselves. ( ) thought the hcs should know about the patient's wishes regarding treatment and care objectives. conclusions: more than / of physicians did not know who the hcs is. more than / thought hcs useful and at least / would encourage a patient to designate one before heart surgery. about % thought that being a hcs is a too high responsibility and that the hcs could not be the best representative when needed. the potential fear this topic might induce is a barrier for this minority. introduction. the use of a daily goals chart has been shown to improve communication between the multi-disciplinary team leading to an increase in understanding of daily patient goals and a decrease in length of patient stay on the intensive care unit (icu) [ ] . we have used a daily goals chart on our icu since . we wanted to assess the value of this initiative in a general adult icu. methods. the royal cornwall hospital is a large uk district general hospital. we conducted the survey over a week period in the icu. each day, after the morning multidisciplinary ward round, the consultant in charge was asked to give the main goals for each patient. these were compared with those written on the daily goal chart, or stated by the house medical and nursing staff. they were graded as complete match ( % of consultant goals matched), partial match ( - % matched) or non match (\ % matched). results. surveys were conducted. the daily goals sheet matched the consultant completely on ( %) occasions and partially on ( %) occasions. in comparison, the combination of house medical and nursing staff had complete match on ( %) occasions and partial match on ( %) occasions. house medical staff had a % complete or partial match, house nursing staff had a % complete or partial match. overall house staff understanding of the goals set on the ward round is far better than that recorded on the goals chart. the goals related by medical and nursing staff showed differences that reflected their differing clinical priorities. combining results of all staff led to higher levels of complete match than either group independently. low levels of non-matches indicate that there is good overall understanding and communication within the team. use of daily goals charts is an effective aid to augment communication on the icu multidisciplinary ward round. objectives. to assess the effectiveness of an icu diary on post-icu psychological symptoms of patients (pts) and their families. single centre prospective study. three periods: = control ( to / ), = diary ( / to / ), = control ( / to / ). all the pts admitted c days for the first time to our medical-surgical icu were included. during the intervention period, the diary was filled both by the caregivers and the pts' relatives, without directives except for the first (medical summary) and the last (recovery wishes) ones. at icu discharge, their families were asked to fill a satisfaction questionnaire (ccfni) and the hospital anxiety and depression scale (hads), and to be contacted by phone to assess peri traumatic stress disorders [dissociation and impact of event scale-revised (ies-r)], hads at months and year after icu discharge. we excluded pts if they or their family refused to participate, were not fluent in french, or if their family was not present around the day of icu discharge. the optimal theoretical content of the diary was determined by a delphi technique involving a panel of icu and non-icu caregivers and a voluntary visitor. the content of the diaries was analysed and linked to the outcome measurements. of the admitted patients, were included. after exclusion of pts, formed the basis of the study. the content of diaries and the results of ies-r are under analysis. the year data is not yet available the saps ii at admission, icu and months post icu mortality were not significantly different between the three periods. the family satisfaction score was high and was not significantly different between the three periods. included: patients with failure of two or more organs in the first h, admitted to icu during . excluded: neurocritical and politrauma patients. contact year following discharge from; questions were asked about the patients' different perceptions during their stay in icu. if it was not possible to contact the patient, the next of kin was asked. results. patients included. general characteristics during admittance to icu: % male; age . ± . ; sofa * ± . ; apache ** ii . ± . ; apache ** iv ± . ; length of stay in icu: . ± . days; . % on invasive mechanical ventilation and . % on non-invasive mechanical ventilation. data collection was carried out over a period of ± . months, on average months (range: - months). . % ( patients) had died at the time of contact. the person interviewed was the patient in . % of the cases, the spouse in . % and immediate family (patient s parent/child/sibling) in . % of the cases. overall, . % do not have any memory of their stay in icu. for . %, the experience was unpleasant and for . % of patients the memory is very unpleasant. . % experienced fear, . % disorientation, . % a feeling of lack of hygiene, . % a feeling of suffocation/drowning (with the endotraqueal tubes, etc.), . % a lack of privacy (nudity, etc.) and . % pain during procedures. . % were very grateful for our phone interview. . % were satisfied with the staff. conclusions. in patients with high severity scores during their time in icu, less than half have memory of their stay after year. in those who do, the feeling of fear and disorientation predominates. to determine the occurrence of communication failures in clinical icus, identifying their main detection tools and disclosing their effects on patient condition. a prospective cohort was conducted in four icus of a -bed academic, tertiary-care urban hospital in sao paulo, brazil, enrolling critical ill patients older than years from july to august . communication failures were identified by daily direct observation of medical and nursing rounds and also by chart reviews. the association between communication failures and adverse event occurence was determined using multivariate logistic regression. results. among the enrolled admissions, as much as admissions ( %) were affected by communication failures, with occurrences. the vast majority of the communication problems was not registered in patient charts, and could only be identified during the medical and nursing direct monitoring. none of the identified communication failures caused patient harm. nine out of ten communication issues involved exclusively members of the multidisciplinary icu health team, patients and their relatives being seldom included in this scenario. despite communication failures are considered important adverse event risk factors, no association was identified between these two variables. conclusions. the incorporation of direct observation as a research tool for identifying untoward events was essential to the detection of communication failures in our study. almost half of the studied admissions was affected by communication flaws, most of them involving exclusively the healthcare team. nevertheless, these figures are underestimated, since the research team remained in the studied icus for no more than h a day. although patients were not harmed fortunately, the presence of these communication issues suggests the existence of important gaps in the provision of critical care. the issue regarding communication deficiencies in icus setting affecting patient safety deserves attention. relatives of patients in the intensive care unit (icu) are exposed to considerable stress . effective communication with relatives has been shown to provide support and minimise stress whilst improving their wellbeing and decision making for critically ill patients . furthermore, satisfaction is dependent on communication by a senior caregiver . no published guideline or recommendation exists for when relatives should be first spoken to, how often they should be updated, or how these conversations should be documented. to determine how well relatives of patients in the icu are kept informed and to assess the quality of documentation. we retrospectively analysed data from the metavision Ò clinical information system of patients staying over days during / / - / / on the -bed icu at the nnuh. data obtained from the 'relatives communication' page included: when relatives were first spoken to, how often they were spoken to (according to the number of entries made) and the members of staff involved in the conversations. these variables were analysed in relation to patient outcome and length of stay on the icu. . patients were analysed. communication with relatives was not documented in % of patients. % of communication was carried out by a consultant. discussions were more likely to occur with relatives of patients who died; % compared to % of patients discharged to the ward. similarly, relatives of patients who died were spoken to more frequently; % were talked to on more than one occasion compared to % of patients surviving to discharge. relatives were more likely to be spoken to with an increased duration of admission on the icu; communication occurred with only half the relatives of patients staying - days, compared to % of those staying more than days. two-thirds of relatives of patients staying more than days were not communicated with until after the fourth day of admission, although the majority of these were spoken to on numerous occasions and all were seen by a consultant. relatives of patients dying on the icu are more likely to be communicated with, and are updated more often than those of patients surviving to discharge. a delay in communication with relatives of patients staying more than days on the icu was noted, but conversations occurred more regularly and involved a consultant. we suspect that our results demonstrate a lack of documentation rather than actual communication; auditing relatives' satisfaction with communication on the icu may help clarify areas for improvement. assessment of satisfaction with the quality of care provided to patients hospitalized in intensive care units, in most cases, is transferred to the relatives of the same, given the context of the patient himself unable to speak. the diagnosis of the needs of families of critically ill patients has been the subject of several studies. aiming to assess the needs of relatives of patients admitted to the picu (polivalent intensive care unit), we conducted studies in particular through an adapted version of the questionnaire ccfni (critical care family needs inventory) developed from the adaptation made by johnson and col. ( ) and focus group. one of the needs identified in these studies was to improve information about what happens in the picu. with this in mind we designed a manual to support relatives in order to improve communication and understanding in the context of the intensive care unit. objectives. this study aims to assess the impact on the level of family satisfaction of a manual we've created. the manual is available from january to all visitors at the entrance of that unit. the questionnaire was mailed to all families who had a family member hospitalized in the picu during the year following the introduction of the manual. together followed a letter to present the study and a stamped and addressed envelope for their return. beyond the satisfaction and access to the manual or not, were collected socio-demographic data from relatives and socio-demographic and clinical data of patients. we obtained responses, representing % of all potential families. questionnaires were returned because of address failure ( . %). statistical analysis was performed using spss Ò v. . results. the satisfaction of family members who had access to the manual was better in all dimensions tested (support, comfort, information, access, trust), and with a statistically significant difference (p \ . ). this difference was clearer in the fields support (med , / . ) and information ( . / . ). conclusions. the impact of the manual on the improvement of family satisfaction was positive in the various dimensions assessed. the questionnaire of family satisfaction monitoring and understanding of information given through a manual created by us can contribute to a better understanding of the needs of families and hence for the continued improvement of service quality. johnson introduction. the burnout can be defined in its multidimensionality: emotional exhaustion, understood as a feeling of exhaustion and failure of the person to give more of herself; depersonalization, in which the person's relationship with patients and with colleagues becomes cold, distant and guided by some cynicism, lack of personal and professional completion, which may manifest itself, on one hand, by the sense of incompetence and inability to respond to requests or, on the other hand, by the sense of omnipotence. the provision of intensive care can lead to health care provider's physical, psychological and emotional exhaustion, which may develop to burnout. we notice the absence of specific studies on this syndrome, in portuguese intensive care units. objectives. the study here presented intend to identify the levels of burnout of physicians and nurses working in portuguese intensive care (adult polyvalent units in the north of the country), and to identify factors that can lead to the development of burnout in the portuguese physicians and nurses working in that setting. the methodology presented consist of application of a questionnaire for self fulfilment with items: , socio-demographic data of the study population; , experiences in the workplace; , maslach burnout inventory-general survey. for the application of methodological tools, we requested the authorization by the competent institutional bodies: the board, ethics committee and directors of services. the professionals who participated in the study were asked informed consent, whether in formal or informal. in addition, each instrument was accompanied by a cover sheet of the same. we have also done observation of the work contexts, and interviews. in this study we will focus on the results of the questionnaire. . sample: hospitals with a total of intensive care units. professionals participants in the study , physicians nurses. the mean ages of respondents , of professional experience years and of experience in intensive care were years. mbi preliminary results:distribution of levels of burnout by occupational category: at the moment, portuguese physicians and nurses who work in intensive care units seam to have medium levels of burnout, obtained through the mbi. results show higher levels at emotional exhaustion in nurses never less in general they showed higher personal and professional completion than physicians. depersonalization were higher in physicians. the results presented here underline the importance of promoting the prevention of burnout at intensive care. the development of the burnout syndrome in physicians and nurses in intensive care has serious consequences, both for themselves, or the consequences that entails for patients and their families. introduction. the hospitalization of a member of the family in the intensive care unit (icu) usually occurs in an acutely and inadvertent way, leaving little time for a family adjustment. facing the stressful situation, the family may feel disorganized, helpless and with difficulties to mobilize themselves, enabling the rise of different types of needs. the scope of those needs leads to the alleviation of tension and uncertainties that could provide to the family the stability needed to cope with the situation disease . to identify the needs of care of family members with persons admitted to the icu. methods. this is a transversal study, held in two icus (a public one and a private one) in the city of feira de santana, bahia, brazil, after approval by ethics and research committees. the relative person is understood by the person who had consanguinity ties or who was closest to the patient, who lived with him and had close relationships. relatives were interviewed when his relative was over h of hospitalization. the the brazilian adaptation of the critical care family need inventory (inefti) was used for measuring the degree of importance, once it has items distributed in five dimensions. descriptive statistics were used for analysis. the inefti reliability was satisfactory (cronbach a = . ). results. the needs of care considered most important by family members were those related to the security dimension, expressed by the items ''to know what are the chances of improvement of the patient'' ( . ± . ), ''to be informed about everything that relates to the evolution of the patient'' ( . ± . ) and ''to feel that hospital people care about the patient'' ( . ± . ). in the category information, the item ''be able to talk to the doctor everyday'' ( . ± . ) obtained more average. in the category proximity was consider more important to ''see the patient frequently'' ( . ± . ). the needs of the categories support and comfort categories showed lower scores. these results are similar to those presented by literature , , what confirms the appreciation of the family to the aspects related to the recovery of the hospitalized relative, in detriment of their own needs. conclusions. having security, information and being around its ill relative is what the families need. the security is provided by the conviction that the person receives the best care in the pharmacological, technological and human aspects, and can be perceived by the information transmitted by the team and by the proximity established in the interaction with the sick relative. a collaborative project was developed between the itu clinical staff of a large, inner city teaching hospital, palliative care clinicians and an academic department of palliative care. qualitative data collection included: (i) semi-structured interviews with staff and relatives of patients thought to be at the end of life; (ii) focus groups with staff (iii) observation of care and (iv) clinical note review. data was analysed using the framework approach to identify key themes. results. semi-structured interviews were carried out with staff and focus groups took place. a total of relatives, representing patients thought to be at the end of life, were interviewed. half the patients represented were female, with diagnoses including infection, hypoxic brain injury, malignancy and liver failure. the participants were aged - and included a range of ethnic groups and religious affiliations. non-participant observations of care took place for and clinical note review for of these patients. data from the interviews with staff describe that an existing withdrawal of treatment document was working well but could be developed further along with suggestions for amendments. the interviews with relatives, observations and review of clinical notes show key themes: communication, decision-making, patient and family needs, and symptoms and their management. through discussion at itu end of life group meetings, a consensus was reached to pilot a complex intervention comprising an amended withdrawal document; a psychosocial assessment; education and awareness-raising; palliative care team input and increased psychosocial support. the psychosocial assessment document was deemed valuable to all patients and was rolled out for all patients admitted to itu. initial evaluation shows greater staff awareness. documentation of end of life issues and the collaborative research process has improved communication between itu and palliative care staff. introduction. consumer-centric healthcare is a key component of nhs policy. when patients are critically ill, family members act as surrogates. family members alone may inform patients of events that occurred, and provide physical, emotional and socioeconomic support during rehabilitation. thus, high family satisfaction (fs) is important. the fs-icu instrument was developed in canada to quantify family satisfaction and benchmark intensive care units (icus). we have piloted and validated previously an adaptation of the fs-icu such that its language was appropriate for the uk . to date, no intervention has demonstrated improvement in the fs-icu for a critical care unit. we hypothesise that provider-driven interventions fail to recognise central issues. co-production is a framework that enables creation of parity between providers and consumers by validating both individual worth and specialised knowledge . there are no published data on the use of co-production in intensive care. we undertook to co-produce interventions targeted to improve family satisfaction. the fs-icu instrument will be used as an objective measure of their efficacy. objectives. to co-produce some interventions targeted to improve family satisfaction and to use the fs-icu instrument as an objective measure of their efficacy. methods. fs-icu questionnaire responses were used to highlight potential areas for service development. focused interviews with families provided detailed descriptions of the ''the way the icu works''. these data were used to build exercises for a workshop of service users and providers which aimed to co-produce service developments. results. fs-icu questionnaires were received over the months to april ( % response). quality and consistency of communication between icu doctors and relatives; the level of relatives' inclusion in decision-making processes; and the icu waiting room atmosphere were identified as needing improvement. four families were interviewed in detail. workshop participants included trust directors, managers, clinicians, nurses, patients and their families. proposed interventions from the workshop included: development of a non-clinical family liaison officer role with a dedicated contact number; increasing focus on managing patients' and relatives' expectations of care delivery; specific improvements to the waiting room area. intensive care patients' relatives provided a unique insight into the icu functioning that should be utilised as a resource. co-production was used to design service improvements that may not have been obvious from a provider perspective. workshop transactions were empowering for both staff members and patients' families, generating social capital that creates and improves social provider-consumer networks, now and in the future. to evaluate the degree of satisfaction of icu patients regarding their icu stay. as the result of a fund sponsored by former patients and their relatives, our dept of intensive care is able to provide a small team of assistants to welcome and accompany the relatives of icu patients. one role of this team is to collect and evaluate impressions and criticisms from patients and relatives shortly after the icu stay. we studied a convenience sample of icu patients who stayed in our multidisciplinary dept of intensive care between september and april . the evaluation included simple questions about the welcome (friendliness of the personnel, explanations), quality of care (including pain control, attention to patient needs, availability of nurses and speed of response) and comfort (temperature, light, noise). data were analyzed using non-parametric (mann-whitney) and chi tests. we collected answers from of patients ( were incapacitated, had died and declined), including unplanned admissions and patients after major surgery. more than % of the patients were very satisfied with all items, except for information provided by the attending physician ( % of patients) and the room temperature ( % of patients) (figure ). post-icu enquiries can provide valuable feed-back information that could improve the quality of care in the icu. introduction. previous research suggests that family members of critically ill patients hospitalized in the icu frequently suffer from severe anxiety. a survey conducted in our unit-a -bed, university-affiliated tertiary-care, closed, general icu with restricted visiting hours-revealed a willingness of family members to participate in a support group. such a group was recently introduced and we report on our initial experience over the last year. methods. the purpose of the support group was to provide a forum where family members could freely raise any topic related to the care of their loved one as well as to family-related issues. the meetings were held weekly in the icu and chaired by a senior nurse and the unit social worker. family members were informed of the meetings when the patient was admitted to the icu and notifications were placed in the family waiting room. all family members were encouraged to take part and to raise any topic they felt was relevant. results. since its introduction in , there has been an increase in the percentage of a family representative attending the meetings from to %. the most frequently raised issues included staff-family interaction (especially lack of empathy), lack of information regarding the patient's status and prognosis, and the lack of adequate visiting hours. in addition, other issues included technical aspect related directly to the family, in particular, overcrowding and lack of privacy in the waiting room. finally, participants wanted to learn skills in order to cope with their new and uncertain circumstance. we have noted an ongoing readiness of family members to take part in the support group. the issues raised have and will allow us to make appropriate changes and to improve the current situation. in particular, the meetings help us to identify family members at risk who require more immediate and personal attention. introduction. the soap study suggested outcomes of cancer patients admitted to icu are similar to those without cancer in contrast to other reports . we wanted to compare this with our own experience. ( ) to determine critical care and hospital outcome of patients with malignancy referred to critical care in the previous years. ( ) to identify any factors influencing treatment decisions and survival after admission. retrospective chart review of patients undergoing treatment for malignancy admitted to icu for medical or surgical reasons from may to feb . leukaemia patients were not included as they are treated at a different hospital by a different group of clinicians. demographic information, tumour/treatment related factors e.g neutropenia, preadmission status and critical care diagnoses e.g. sepsis, were collected in addition to patient outcomes. results. patients were identified ( . % of all critical care admissions). itu mortality was . % (n = ), however only . % (n = ) survived to hospital discharge (comparable overall unit mortality: - %, hospital mortality: %). hospital survivors were younger (median . vs. years), and more non-survivors had pre-existing comorbidities, sepsis, ali and required more organ support (all ns). there was no difference between the groups regarding cancer treatment. non-survivors had a longer stay in critical care and treatment withdrawal/limitation decisions were more common suggesting these were often based on lack of medical progress whilst on icu rather than diagnostic nihilism. hospital mortality in patients with malignancy is higher in our specialist centre than reported for a europe wide cohort ( . vs. % overall and % in the medical subgroup). the majority of our patients were medical and not post-surgical unlike in the soap study. this may account for the greater mortality as a larger proportion of our patients had ali, sepsis, neutropenia and required inotropes. numbers admitted to critical care are much smaller than the . % reported in the soap study, suggesting some referral and admission triaging by the oncologists and the icu team. our results are similar to single centre french (hospital survival rate . vs. . %) and brazilian studies ( . vs. %), although a majority of our patients did not receive mechanical ventilation. , a diagnosis of cancer or active treatment for it should not be the major determinant of critical care support, but the patient's general premorbid status and the extent of organ failures appear to be important factors in decision making as for any other critical care patient. figures from the soap study for non specialist centres do not appear to reflect the experience of specialist oncology centres. historically there has been a negative perception of the prognosis for patients with haematological malignancies requiring admission to the intensive care unit (icu). however, advances in chemotherapeutic regimes and haematopoietic stem cell transplantation (hsct), along with improved monitoring and supportive measures have suggested that outcomes for these patients have improved [ ] . establishing key prognostic indicators predictive of outcome may be useful in identifying patients most likely to benefit from icu therapy. the aim of this study was to describe clinical outcomes and identify prognostic factors in patients with haematological malignancy requiring admission to icu. following research approval, a retrospective cohort study was undertaken in a -bedded specialist cancer icu over a -year period (october -september . recorded patient variables included demographics, haematological diagnosis, reason for icu admission, hsct, apache ii, admission laboratory data, number of organ failure, use of invasive mechanical ventilation, renal replacement therapy (rrt) and vasopressors. the primary outcome was in-hospital mortality. key prognostic variables in determining inhospital mortality were identified using univariate and multivariate analysis. results. patients with haematological malignancies were admitted to the icu during the study period: mean age . (sd . ); . % female; haematological diagnosis ( . % leukaemia, . % lymphoma, and . % myeloma); . % emergency admissions and . % were post-hsct. mean apache ii was . (sd . ), mean number of organ failures . (sd . ), % required invasive mechanical ventilation, . % rrt and . % vasopressor therapy in the first h of icu admission. icu, in-hospital and -month mortality were . , . and % respectively. significantly higher mortalities were seen in patients who were mechanically ventilated ( vs. % non-ventilated patients p \ . ), on vasopressor support ( vs. % no vasopressor support p \ . ), neutropenic ( vs. % non-neutropenic p \ . ) and in multi-organ failure defined as c organ failures ( deaths vs. deaths in patients with b organ failure, p \ . ). univariate analysis revealed mechanical ventilation, vasopressor support, albumin \ g/l, neutropenia, platelet count \ /l and multi-organ failure were all significant with p values . , . , . , . , . and. respectively. multivariate analysis revealed that multi-organ failure was the only independent prognostic predictor of in-hospital mortality. conclusion. mechanical ventilation, apache ii, vasopressor support, albumin\ g/l, neutropenia, platelets \ /l and multi-organ failure all had a significant association with mortality; however multi-organ failure was the only independent factor that predicted poor outcome. c.y.c. michael , a. vasu , s. eillyne tan tock seng hospital, emergency department, singapore, singapore introduction. coronary heart disease is the leading cause of mortality and morbidity for both women and men. although men are affected in greater numbers, women have been shown to have worse outcomes and higher mortality. objectives. this study aims to examine gender differences in risk factors, angiographic severity, treatment and in-hospital mortality after stemi. methods. in this retrospective study, the medical records of patients with an admitting diagnosis of stemi from tan tock seng hospital, emergency department (ttsh ed) between st january and st december were reviewed. we extracted the data from the electronic records of the emergency case notes and inpatient discharge summaries. results. of the patients studied, ( . %) were women and ( . %) men. four hundred and forty-nine ( . %) patients underwent coronary angiography. one hundred and seventy ( . %) patients did not undergo coronary angiography, majority ( . %) were elderly aged c years (men . % and women . %). between women and men, there was no significant difference between the number and distribution of diseased coronary vessels (including triple vessel and left main stem diseases). regardless of age, men were frequently treated with a coronary artery stent ( . %). elderly women (aged c years) were more often treated conservatively ( %) while those younger women (aged b years) were frequently treated with a coronary artery stent ( . %). in-hospital mortality rate was significantly higher for women than men ( . vs. . %, p = . ). amongst the patients treated conservatively, elderly women had the highest in-hospital mortality when compared to the other patients (women c years . vs. women b years . %; men c years vs. men b years . %). compared to men, women were significantly older (p \ . ; % ci . - . ) , more likely to have a history of hypertension ( . vs. . %; p \ . ), diabetes ( . vs. . %; p \ . ), hyperlipidemia ( . vs. . %; p = . ), peripheral vascular ( . vs. . %; p = . ) or ischemic heart diseases ( . vs. . %; p = . ) and less likely to be smokers ( . vs. . %; p \ . ) or consume alcohol ( vs. . %; p \ . ). conclusions. elderly women who were treated conservatively had the highest in-hospital mortality during the early management of stemi. hôpital saint-louis, ap-hp, paris diderot university, hematology department, paris, france introduction. aml is considered as an oncology emergency as a proportion of patients experience life threatening complications within the first hours or days after diagnosis. early death had been shown to be statistically related to high white blood cell (wbc) and monoblastic leukemia - , with leukostasis and lysis syndrome as the most deadful events. objectives. to evaluate the relationship between timing of icu admission and outcomes in high risk aml patients at the earliest phase of the malignancy (before any chemotherapy) methods. retrospective study in a tertiary care teaching hospital. adult patients with newly diagnoses aml from to were included. patients admitted for an immediate life sustaining therapy (ventilation, vasopressors or renal replacement therapy) were excluded. patients admitted directly to the icu (early admission) were matched for age, wbc and fab subtype with patients primarily admitted in hematology ward. datasets were extracted from medical charts. results. patients were included ( early admitted to the icu and admitted first to the wards). median follow up was . months. median age was . years ( - ). fab m or m was retrieved in % of the patients. karyotype was favorable for % and poor for %. median wbc was l - . no statistical difference was seen for demographic and hematological parameters between early admitted patients and matched controls. among the patients admitted first to the wards (controls), were subsequently admitted to the icu (lately admitted) and remained in ward during the entire treatment course (never admitted). the median time between diagnostic and icu admission of this last group was ( - ) days. strikingly, patients lately admitted had more frequently dyspnea,oxygen requirement, high respiratory rate, low diastolic arterial pressure and lower first h urine output. lately admitted patients were less likely to receive the complete dose of induction chemotherapy ( vs. %) furthermore, late admission resulted in increased use of invasive mechanical ventilation ( vs. %) and vaso-active drugs ( vs. %). these differences resulted in longer stay in icu and decreased survival. conclusion. patients at the earliest phase of high risk aml who are lately admitted to the icu experience worse outcomes, with increased use of life-sustaining therapies and higher mortality, compared to patients early admitted to the icu. physiologic parameters at the time of aml diagnosis such as respiratory rate, diastolic blood pressure, spo , or oxygen need are likely to help clinicians distinguish those patients at risk of late icu admission and subsequent adverse outcomes. studies are needed to assess the right place for newly diagnosed aml with physiological abnormalities but no organ dysfunction. atrial fibrillation (af) is the most common sustained tachyarrhythmia in the community. it has a prevalence of * % in those over years of age ( ) . the chronic health consequences of chronic af are significant. it can cause impaired cardiac function, a fivefold increased risk of stroke and decreased life expectancy ( ) . af is also the commonest arrhythmia in the critically ill, though a recent systematic review ( ) was unable to recommend evidence based standards due to the heterogeneity of the studies. objectives. a retrospective cohort study to assess the impact that chronic af has on the outcome from critical illness. methods. all patients admitted with chronic af between / / and / / were identified. we recorded age, apache ii and predicted hospital mortality, actual icu and hospital mortality, past medical history, admitting diagnosis, medication, echo findings, anticoagulants given, therapy instituted, and any further events between icu and hospital discharge. the only data collected for the patients who did not develop af was their age, apache ii and predicted hospital mortality and actual icu and hospital mortality. data analysis using chi square test and mann-whitney u test were used where appropriate. results. patients were admitted to the icu over the study period, of which had a history of chronic af ( . %), the remaining results are shown in table . chronic af had a prevalence of . %, in keeping with previous studies, and the mean age in the chronic af group was significantly higher. interestingly, there was no difference in icu and hospital mortality between the groups. despite the chronic af group being older with significantly worse apache ii scores. indeed the hospital mortality ( . %) of those patients admitted with chronic af was over % less than predicted hospital mortality ( . %). why patients with chronic af are outperforming expectation is not clear. it could be that apache ii is over estimating the severity of illness in these individuals, or is there something about the way chronic af is treated that affects the response to critical illness, for example, anticoagulation therapy? one of the major outcome measurements in burns centers is still mortality after severe burns. there are many predictive factors in admission as well as factors that are related with all the course of the disease responsible for survival after severe burn. many centers have a minimum standard of burn survival or la (the body surface area that kills % of people) and also have generated computer models of death probabilities based on age and tbsa (total body surface area) burned. objectives. to evaluate the outcome of the severely burned patients treated in the burn center and to develop a predictive model for survival from major burns in albania. the medical records of all acute burn patients admitted to the burn center of the university hospital center ''mother teresa'' in tirana, albania are reviewed retrospectively. statistical analyses are conducted using spss version . logistic regression is used for the prediction of death probability for two risk variables, tbsa burned and age. based on the index of evidence the variables are grouped in significant strata, from to for each variable. logistic regression equation is: where z = , - , age - , age - , age - , age + , tbsa - , tbsa - , tbsa - , tbsa after calculating the probability of death for each record, we have done respective grouping according the mortality from - %. results. during - are admitted altogether , patients in the burn center. overall mortality in icu is . % with a significant reduction during the years, up to . in . row burn mortality is . for , persons per year. la for children is % tbsa; for adults % tbsa and for aged % tbsa. based on probability of death, we notice that older age and larger burn size are associated with a higher like hood of mortality. figure gives an overview of death probability in our burn center. conclusions. the mortality reduction speaks up for a better work of our staff toward the patients. the predictive model may assist all the burn team to identify the crucial determinants of clinical outcome to establish a real basis for treatment standards and to allow future comparisons of new treatment strategies. ( ) in mechanically ventilated (mv) critically ill patients. methods. prospective observational multicenter study during weeks in november . consecutive patients admitted to the participating icus and requiring mv for at least h were included. maximal, minimal and mean intra-abdominal pressure (iap), were recorded on day , , and . iah was defined as mean iap c mmhg/ h at least day. following risk factors were recorded if evident during the first icu day or immediately before: respiratory failure, abdominal surgery with fascial closure, damage control laparatomy, major trauma/burns, prone positioning, gastroparesis, ileus, colonic pseudo-obstruction, ascites, hemo/pneumoperitoneum, intra-abdominal fluid collection, acidosis (ph \ . ), hypothermia (core t°\ °c), massive transfusion ([ u of packed red cells/ h), massive fluid resuscitation ([ l/ h), coagulopathy, oliguria and sepsis. results. patients from icus were included; mean apache ii score on admission was . ( . ) and -day mortality %. mean number of iap measurements was . per day. iah occurred in patients ( . %). only pt ( . %) had none of the studied risk factors, nevertheless % of them still developed iah. of the patients with or more risk factors, only . % developed iah (table ) . objectives. to describe the icu admission of our hospital for serious complications of hematology patients in the last years. compare the characteristics of these patients throughout the study period. analyze mortality and their evolution from their admission to the icu. the evolution of hematologic patients has improved in recent years due to better supportive treatment, sometimes involving the use of specific treatments in the icu. a retrospective study of medical records of all patients with hematologic diseases were admitted to our icu from april until may . we excluded patients admitted for channeling central catheter, diagnostic tests and bone marrow transplants. we selected a total of patients ( % male) with a mean age of years (range - ). the main hematological diagnoses were the most common aml ( %), acute lymphatic leukemia ( %), lymphoma (non-hodgkin's lymphoma) ( %), coagulopathy ( %), myelodysplastic syndrome ( %) and myeloma multiple ( %). the principal reason for admission in the unit were: acute respiratory failure ( %), followed by sepsis ( %) and less cns and cardiac problems ( and %) respectively. as important risk factors of neutropenia and peripheral blood stem cells after transplantation. the icu mortality reached . %. the average stay was . days. conclusions. the transfer to the icu allows a high percentage of hematological patients survive severe complications and the benefit continues after discharge. the mortality of icu patients in our series has not changed over the past years, keeping both the characteristics of patients transferred. the consensus among the services of hematology and intensive care is essential to select and treat the best candidates to benefit from support in the icu and to improve current survival results. a retrospective (from to ) and prospective (from to ) analysis of obstetric patients (pregnant or postpartum admissions) admitted in our ccd was performed. results are expressed as mean (standard deviation) or frequency (percentage). chi and t student tests were used for statistical analysis according to the different variables (spss . , inc. chicago, il), accepting a p-value . as significant. results. obstetric patients were included. mean maternal age was . ( . ) years and mean gestational age was . ( ) weeks. apache ii score was . ( ) . ( . %) patients were admitted to ccd due to an obstetric cause. the main diagnosis of this group were thrombotic microangiopathies ( . %) and hemorrhagic shock ( . %). thrombotic microangiopathy included ( %) eclampsia-preeclampsia, ( %) acute fatty liver, ( %) hellp syndrome and ( %) ptt-shu. in the remaining . % ( patients) the main reason for ccd admission not related to the pregnancy was respiratory failure ( . %). from the whole population included, patients ( . %) required mechanical ventilation (mv) with a mean duration of . ( . ) days. furthermore, ( . %) patients required surgical intervention ( . % hysterectomy). the ending of pregnancy was made in patients ( . %), most cases by caesarean . % ( patients). mean length of stay in ccd was . ( . ) days. maternal mortality was . % ( patients), basically in the non-obstetric group ( vs. ) . conclusions. this is a large series of young obstetric critically ill patients with a low mortality. however, a non-depreciable part of the population included presented important morbidity. objectives. to identify the association of co-morbidities with mortality. methods. retrospective analysis of clinical process of diagnosing patients with severe sepsis/septic shock admitted to the intensive care unit (icu) in the period of november to october . we collected demographic data, co-morbidities, and mortality in the icu hospitalization. statistical tests used were student's t and chi-square. we analyzed patients admitted with this diagnosis, median age of years and females . %. in . % ( patients) appear co-morbidities, distributed as follows: hypertension . %, . % diabetes mellitus, cerebrovascular disease . %, . % chronic kidney disease; . % neoplasic disease and chronic obstructive pulmonary disease . %. the mean age ( . , p \ . ) was higher in this group. the overall mortality in the icu was . % that has not increased significantly to . % in the group with comorbidities, and the overall in-hospital mortality was . % and rise significantly to . % (p \ . ). conclusions. in our study, around - patients had co-morbidities and these facts and the age were those who contributed to higher mortality. the factors of greatest weight are those related to metabolic disease. the characterization of chronic illness in the icu is important in future larger epidemiological studies to better characterize this group of patients and the factors predictive of mortality to decrease the suffering of the patient and plan for admission to intensive care units. one year mortality of patients treated with an emergency department based early goal directed therapy protocol for severe sepsis and septic shock: a before and after study. introduction. despite the advances in respect to the development of objective criteria for admission of patients with hematologic malignancies to intensive care unit (icu), no evidence exists that they contributed to a reduction in the mortality, which depends from the aggressiveness of the cancer itself, its complications and even as a consequence of therapy. since the decision to admit one of these patients in icus involves a complex decision-making process, it becomes imperative to identify predictors that may help the clinician to discriminate the patients who may benefit from intensive care than those in which intensive care will be associated with just a prolongation of an agony. objectives.: to identify early prognostic factors in the admission of patients with hematological malignancy, admitted in the icu of a central university hospital. analysis of data prospectively collected and registered in a database of patients with hematological malignancy, admitted to the icu between january and december . we collected for each patient demographic and clinical data (age, sex, length of stay, origin, previous treatment, stage of disease at admission, type of malignancy and aggressiveness, organ dysfunction at admission, co-morbidities, reason of admission), general severity scores (saps ii and apache ii) and organ dysfunction scores (sofa at admission to the icu, maximum sofa score and delta sofa). specific variables were correlated with mortality at the icu and hospital discharge. results. patients ( males and females) fulfilled the inclusion criteria. the average age was ± . years ( - years). the type of hematological malignancy was acute leukemia ( . %), multiple myeloma ( . %), myelodysplastic syndrome ( . %), chronic leukemia ( . %), low grade non-hodgkin lymphoma ( . %), high grade non-hodgkin lymphoma ( . %). the average length of stay in the hospital was . ± . days. most patients were admitted from the department of hemato-oncology ward of the hospital ( . %), . % of the emergency department and . % of another hospital. the icu mortality was . %, with a corresponding hospital mortality of . %. the discriminative capacity of the severity scores, as assessed by the area under the roc curve (aroc) was . for saps ii and . for apache ii. for the delta sofa calculated for each organ dysfunction, progression of respiratory dysfunction/failure and cardiovascular failure demonstrated the best discriminative power (aroc of . ). conclusions. none of the variables showed a statistically acceptable relationship with icu or hospital mortality. the general severity indices saps ii and apache ii demonstrated a better discriminative power than the multiple organ failure scores. however, in this group of patients,it is still difficult to know objectively what factor or combination of factors may be useful in deciding the admission of the patient in an icu. recently due to new developments in interventional gastroenterology and new therapeutic options for treatment, gastroenterological and hepatological (geh) admissions to acute care settings has been decreased. for general intensive care units (icu) gastroenterological and hepatological (geh) diseases consititutes the minority of icu admissions. so we planned to find the incidence and clinical course of admissions due to geh complaints in a medical icu. objectives. main objective is to analyze clinical and epidemiological features of patients admitted to icu with geh disorders. other objectives are to analyze the mortality rate and the factors contributing mortality in these patients. and who stayed for more than h were included. the prospectively developed data including demographics, prognostic scores and clinical features of patients were analyzed retrospectively. patients with geh disorders consituted % of patients admitted to icu. one hundred thirthythree patients with an age of [ - ] years and gender of % male were included. more than half of these patients ( %) did not have any chronic geh disease. the patients were admitted most often from the emergency department ( %). the most frequent admission diagnosis was gastrointestinal bleeding ( %) followed by hepatic diseases including hepatic failure and acute hepatic encepahalopathy, biliary tract infection ( %), pancreatitis ( %) and enteric diseases including massive diarrhea and bowel obstruction ( %). on admission median apache ii and glasgow coma scores were [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] and [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , respectively. acute kidney injury (defined by rifle criteria risc, injury or failure) was found in (% ) patients. the most common rifle class was class failure ( %). during icu stay patients ( %) needed renal replacement therapy and patients ( %) received mechanical ventilation. nosocomial infection developed in ( %) patients and icu aqıired severe sepsis occured in ( %) patients. icu and hospital mortality were % and % respectively. length of icu and hospital stays were [ ] [ ] [ ] [ ] [ ] [ ] and [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] days respectively. respiratory failure requiring mechanical ventilation, acute renal failure on admission and severe sepsis in the icu were found to be the independent factors determining mortality in these patients (p = . , p = . and p = . respectively). patients with geh constitued % of patients admitted to icu. they usually do not have any chronic geh disease. gastrointestinal bleeding is the most frequent admission diagnosis. respiratuar and renal failure on admission and severe sepsis occured in the icu are the major determinats of mortality in these patients. introduction. in recent years described series with hematological patients in icu, but these studies are often limited because they are retrospective, single center in a few patients divided over many years. to determine the characteristics of mortality in this group is very important to assess their management in the icu. objective. to analyze prognostic factors associated with icu mortality of patients (pts) with hematologic malignancies admitted to the intensive care unit (ptu). method. an observational, transversal, prospective, multicenter oct conducted between june and october . we conducted a descriptive analysis, chi-square, bivariate and logistic regression including variables with a value of p \ . with the sas statistical pauet to assess the factors influencing mortality in icu. we included patients from icus. the mean age was years. the apache ii at admission was . ± . ) and the first day sofa ± . . ( . - . ). the crude mortality icu was . % ( pts). we divide related mortality by infectious etiology ( %) versus other causes ( %). in univariate analysis the variables significantly associated with mortality were: males p \ . , hematology plant from . , . multiple myeloma, respiratory failure at admission . , tachycardia, . , . hypothermia, tachypnea , apache ii c , more than two organ failures . , presence of ards \ . , invasive mechanical ventilation (imv) \ . , niv . , . transfusion (of all products: red cells, platelets and plasma), acquisition intrauci infection . , days longer stay in icu . . the presence of neutropenia was not associated (p = . ) at a significantly higher mortality, or personal history, septic shock, bone marrow transplant or other reasons for admission to the icu. table describe the independent factors associated with mortality in logistic regression analysis. variables classics such as septic shock or neutropenia not associated with mortality. and the independent variables associated with increased icu mortality were: vm, ards, severity and need for transfusion of blood products. results. patients were identified. major haematology diagnoses were acute leukaemia %, nhl % and lymphoma %. mean time to icu admission was . days with % admitted within h. the commonest reasons for icu admission were respiratory failure %, sepsis % and acute renal failure %. the mean number of organs supported was . . % of patients had c organ failure. mean apache ii score was . . increasing organ failure correlated with increasing mortality. patients with or organ failure had % mortality. mean icu stay was days with % having an icu stay of less than h. icu mortality rate was . %. . % received invasive ventilation, . % failed non-invasive ventilation (niv) and required invasive ventilation, . % had niv only and % received no respiratory support. vasoactive support was given to . % and rrt to . %. invasive ventilation and niv were associated with a higher mortality; and % versus % in spontaneously ventilating patients. vasoactive support was associated with more organ failure, longer icu stay and higher mortality. rrt was associated with a higher mortality versus %. patients with a documented poor haematological prognosis had a higher mortality but also more organs supported. conclusions. from this study invasive ventilation, cardiovascular support and multiorgan failure are strongly associated with increased mortality. the need for rrt was not an independent predictor of mortality. close collaboration is needed between the specialities to allow early resuscitation and critical care support to avoid delayed admissions with multi organ failure. introduction. the prescription of stress ulcer prophylaxis (sup) in critically ill patients is relatively commonplace due to the association between physiological stress and gastrointestinal (g.i.) bleeding. however, recent guidelines recommend that only patients who are mechanically ventilated, and/or have a coagulopathy warrant prophylaxis. they also state that histamine receptor blockers (h rb's) can be used primarily for sup ( ). objectives. the aim of this audit was to compare prescription practice of sup at the itu/hdu at mayday hospital with those set out in the guidelines and to calculate the potential cost savings resulting from following these guidelines. methods. data prospectively collected from consecutive admissions to mayday hospital itu/hdu between october and november . . data was collected on patients, had a g.i. bleed on admission, and were already on sup so were excluded. of ( %) patients with major risk factors were prescribed sup, compared with of ( %) patients with no major risk factors. proton pump inhibitors (ppi's) were prescribed preferentially to h rb's; versus . the cost of sup during the audit period was £ , . if we had only prescribed it to those at high risk the cost would have been £ , . , and if we had only used h rb's the cost would have been £ . . prescribing of sup in our unit does not reflect quenot's guidelines. this not only represents an increased cost but there are increased rates of nosocomial pneumonia and c. difficile diarrhoea associated with sup ( , ) . there is little evidence showing the superiority of ppi's over h rbs in the prophylaxis of bleeds; and there is evidence of an increased rate of the aforementioned infections with ppi's as compared to h rb's ( ). we prescribed ppi's to a significant majority of our patients; however, it is our opinion that our current unit practice is not dissimilar to that of the rest of the uk. we would encourage all critical care units to review their sup prescribing as our results show that significant savings can be made with judicious prescription of these drugs. . surprisingly little is published on the cost of drug treatment for critically ill patients. critical care is expensive, mainly due to the high staff ratio, expensive equipment but also due to a significant reliance on pharmacological management, which is usually funded with a limited drug budget. objectives. to explore the relationship between drug expenditure, patient acuity and outcome. methods. data was generated by retrospective analysis of consecutive patients admitted to our bedded general adult icu/hdu in a london teaching hospital, during february . patients were excluded from analysis if they were present in icu for less than h. the first and final ccu days stay were not included so that only full days were analysed. daily drug-use per patient was manually extracted from the computerized icu management system (cis, qs, ge medical). costs of prescribed drugs, fluids and parenteral nutrition (pn) were calculated from the pharmacy computer system and analyzed using regression analysis (spss ver ). results. the patient characteristics and outcomes of the patients are described in table table patient characteristics and outcomes age ( - )* gender (male n and %) ( %) apache ii score . ( . )** tiss score/patient . ( . )** length of ccu stay (days) ( - )* ccu survival (n and %) ( . %) daily drug cost for each patient's stay £ . (£ . - . )* daily drug cost for ccu and hospital survivors n = £ . ( . - . )* daily drug cost for ccu survivors who died in hospital n = £ . ( . - . )* daily drug cost for ccu non-survivors n = £ . ( . - . )* *median (interquartile range), **mean (standard deviation) the median daily drug cost was £ . . of note, the drug cost was highest for ccu nonsurvivors compared with survivors and also compared with patients who died in hospital after ccu discharge (p = . ). multivariate regression analysis demonstrated that median daily drug cost/patient = - . + . (mean tiss score) + . (apache ii score), r = . %; i.e median daily drug cost/patient was positively associated with tiss and apache ii score explaining % of the variation in cost seen. conclusions. this is the first study to show that daily drug expenditure in all general ccu adult patients correlates with patient acuity. median daily drug costs/patient were found to be £ . . this parameter would make an interesting comparison with other units both nationally and internationally. daily drug costs can be predicted on the basis of apache ii and tiss scores. furthermore, this may be further refined to develop a quality marker of daily drug cost in relation to survivors and non-survivors. material, methods and results. all patients admitted to the icu of neurotrauma, which underwent a tracheostomy after admission. data were collected: affiliation, cause of admission, average stay, indication of tracheostomy, tracheostomy time delay from its indication, place of performance of the procedure (icu or operating room), perioperative complications (event during transfer to operating room, event during surgery: hypoxia, hypotension, arrhythmia, bleeding, premature extubation, false cannulation, cardiac arrest, pneumothorax or death), and postoperative complications in the first week (bleeding, difficulty in changing cannula, stomal infection, pneumothorax, death conclusions. tracheostomie is a simple surgical technique and . % of tracheostomíes could be safely performed in the icu, saving hours of scheduled interventions in the operating room. there were no serious event during the transfer to the operating room or during the performance of tracheostomy. tracheostomized patients in icu, had a higher incidence of hypotension during surgery, although this complication in any case was serious or required treatment with vasoactive amines. when the tracheostomy is performed in the operating room, the delay shows a tendency to be higher, although this difference is not ss introduction. the concept of tight glyceamic control in critically ill has led to the rise of number of insulin infusion protocols designed to keep the blood sugar (bs) in predefined range. at the same time monitoring practices and patients populations vary greatly between intensive care units and thus so do the results. the matter is complicated by the absence of a widely agreed common glyceamic control indicators against which protocols can be evaluated and compared ( ). to establish the quality of glyceamic control in two different intensive care units. to compare the quality of glyceamic control between two intensive care units, with different glyceamic protocols and blood sugar measurement practices. we conducted retrospective non-randomized population study comparing quality of glyceamic control in two independent and non-related intensive care units. time spend in pre-defined glyceamic range was chosen as a quality indicator for both units ( , ) . data was collected from the electronic database and point of care bs measuring devices. the frequency distribution was analyzed to establish the patient-to-patient variability and a degree of bs deviation from the target value. results. units were different in method of sampling, frequency of sampling, target for optimal glyceamic range and instigated insulin protocol. data was collected on patients ( , bs measurement) in itu and patients ( , bs measurements) in itu . mean bs was . (sd = . ) mmol/l in itu and . (sd = . ) mmol/l in itu . conclusions. the performance of both protocols were satisfactory- . and . % of the time patients spend with bs less than mmol/l in itu and itu respectively. the quality of glyceamic control on both itus is similar in terms of proportion of time spend in different glyceamic bands, with the exception of the longer time spend in a hyperglyceamic state in itu . this study confirms the notion for a need of unified approach for evaluating quality of glyceamic control for in-patient populations. with an icu mortality of . % and in-hospital mortality of . %. the median transfusion threshold was a platelet count of /l with yearly medians ranging from . /l to /l. the % of platelet transfusions complying with bcsh guidelines increased from . to . % during the year study period. the specialties with the highest platelet requirement were general surgery ( . %), haematology ( . %) and general medicine ( . %) as a % of total units transfused. the yearly median threshold for haematology patients fell from . /l in to x /l in , increasing guideline compliance from . introduction. numerous protocols (e.g. glycaemic control, hhh, renal rescue) have been introduced into icu. these protocols involve blood sampling to assess gases, haemoglobin, glucose and electrolytes. this may result in anaemia and subsequent transfusion and adverse clinical outcome ( , ) . reducing blood loss due to sampling is an important blood conservation strategy ( ) . currently, our icu processes over , blood samples per month. objectives. to study the indications for blood gas sampling in our icu and identify strategies to reduce sampling. methods. we performed a prospective, observational study over week in . the nurses completed a questionnaire per shift per patient to assess the primary and any secondary reasons for each sample. subsequent management changes, haemoglobin levels, active bleeding, and transfusion were also recorded. results. blood samples from patients and questionnaires were analysed ( % of the nursing shifts). the range was - samples per patient per h shift with a mean of . the secondary reasons showed that many samples were also being used for potassium ( %) and glucose ( %) monitoring. only % of samples changed management (potassium %, ventilatory settings %, glucose %). haemoglobin levels dropped by an average of g/dl per week per patient with no active bleeding. units of blood were transfused during the study period. conclusions. our study shows that reasons for sampling are often relatively weak and sampling is promoted by icu protocols. frequent sampling does not change management for a large proportion of samples and may cause anaemia. there are financial implications to frequent sampling-at the time of the study each sample cost £ . (€ . ) to process. each unit of blood transfused cost £ (€ ). we have considered ways to reduce sampling including changing the glucose protocol to capillary sampling, using ml syringes, increased use of end tidal co monitoring, protocol redesign and education of staff. reference(s it has been reported that tight glyceamic control is associated with net savings in terms of length of stay on the itu and critical care bed occupancy ( ) . whilst it might be true for the overall length of stay, there is as yet, an un-quantified effect of frequent blood sugar measurement on the overall available nurse-patient time ( ) . there is finite amount of nurse-patient time within any given shift and so prioritizing nursing care will be an important factor in critically ill and high dependency patients. this is specifically important for any saving to be realized from the introduction of automated blood sugar measurement devices ( ). objectives. to quantify the amount of nursing time devoted to glyceamic control on itu or post-operative critical care environment based on data from four cohort studies on quality of glyceamic control in three intensive care units. a mathematical model, which takes into account frequency of blood sugar measurements and time to take each measurement was developed. stochastic analysis was used to calculate interdependency between quality of glyceamic control and the frequency of blood sugar measurements. introduction. introduction of trans-catheter aortic valve implantation (tavi) has been the latest technological advance in minimizing surgical stress and improving the chances of high-risk patient undergoing a successful aortic valve intervention ( ) . the latest technology comes at considerable cost, which relates to both-the cost of the tavi valve and to it's delivery system. currently, there are no randomized controlled trails addressing the issue of cost-effectiveness of tavi versus surgical avr ( ) . to build a cost-effectiveness model for patients undergoing either a tavi procedure or a surgical avr based on the level of care: level (ward based care), level (high dependency unit) and level (intensive care unit) during in-hospital stay, taking into account the rate of post-operative complications in both groups. methods. tavi patients were matched against patients who had previously undergone surgical avr. the groups were matched for demographic and physiological risk factors, as well as euroscore. a decision analytical tree was constructed based on the length of stay in hospital and post-operative complications. a markov model was built and the effectiveness was measured in terms of improvement in nyha class, which was translated into the quality adjusted life years (qaly) ( ) . results. the average in-hospital cost for tavi was £ , versus £ , for the surgical avr. the cost did not include the theatre time cost. in the surgical avr group the in-patient mean cost was greater than respective cost of the tavi group due to longer overall length of stay as in-patients. patients in the avr group spent more days in level and level care as compared to the tavi group. conclusions. the shorter length of stay and reduced rate of post-operative complications in the tavi group has got the potential to substantially reduce the overall in-patient cost and offset high cost of the valve. the effectiveness arm of the models did not differ for both groups, due to the lack of published literature, and raises a need for a qaly assessment for the effectiveness of tavi. the rate of post-operative complications in surgical avr group (higher rate of stroke and need for cardiac pacemaker) substantially affected the projected long-term cost. objectives. to determine if interventions for permanent pacing (ppm) and change of generator are more efficient in small hospitals. retrospective, transversal, observational study, measured through five diagnosis related groups (drg) that make up the casemix of pacemakers from the spanish minimum basic data set in , descriptively analyzing demographic variables (age, gender), clinical (number of secondary diagnoses (nsd) and procedures (np), mortality) and management (total, preoperative length of stay, access, discharge, hospital size), defining inefficient stays exceeding days the average. a bivariate study contrasting quantitative variables and comparisons between nominal and categorical, evaluating the independent association between short stay and different covariates studied building a binary logistic regression model, introducing as independent variables those that were significant in the bivariate as well as those considered that might be associated with the dependent variable. introduction. blood products are in short supply and with an ageing population the demand is likely to increase. blood use has been shown to be declining within the surgical specialties and intensive care, however overall use has remained unchanged. this audit looks at the use of packed red cells amonsgst medical inpatients to determine appropriateness. to determine if red cell use is appropraite among medical inpatients methods. medical blood transfusions were examined between august and august . patients were selected and pre and post transfusion haemoglobins were determined along with chronicity of anaemia. transfusions with haemoglobins of c . g/dl triggered a case note review. over months , patients were transfused , units. , units ( %) were given to medical patients ( %), of which patients were reviewed receiving transfusions. average age was . in patients pre transfusion haemoglobin was b g/dl ( %) and in patients c . g/dl ( %). in the group b g/dl patients had acute anaemia and had chronic anaemia. in the group c . g/dl patients had acute anaemia and had chronic anaemia. patients were not transfused and had absent data. out of case notes only were available. patients were transfused for acute anaemia, for chronic anaemia of which patients had cardiac disease, had haematological disorders, patients had iron deficiency anaemia and patient was folate deficient. conclusions. chronic anaemia in the over s accounted for the majority of transfusions. documentation was substandard. transfusions in chronic anaemia may be reduced by up-to-date guidance on transfusion triggers and alternative strategies to the use of blood products. ( ) results. the commonest indication for pct was long-term mechanical ventilation ( %) followed by airway protection ( . %). . % patients had platelets count\ lac while . % had severe thrombocytopenia (\ , ). . % patients had an additional coagulopathy (hepatic failure and multiple organ failure), with inr [ . was present in . % and deranged aptt in . % patients. pct was safely performed in all these patients. the patients received platelets or fresh frozen plasma(ffp) before the procedure to optimize coagulation. only . % had minor bleed through stoma, which was stopped in - min requiring gauze compression. conclusions. pct under videobronchoscopic guidance has low haemorrhagic complication rate in patients with deranged coagulation profile. platelets/ffp should be transfused before the procedure in these patients. introduction. blood components transfusion is common in the critically ill patient, as in the acute bleeding or the acute illness with multiorganic failure context. as any medical intervention, it has clinical indications and associated risks. clinical guidelines have evolved in a restrictive direction, suggesting that decision should be based on particular clinical situation and not only on analytical results. objectives. understand our transfusional practice and how close it is to clinical recommendations, as a quality indicator of our intensive care unit (icu). retrospective study using the icu patients data base. the population consists of patients with more than h icu stay in . the variables analysed are sex, age, diagnostic class (medical, surgical, trauma), saps ii score, mortality, number of transfusional events (erithrocyte concentrate, platelets, fresh frozen plasma and albumin) and the concordance to our hospital clinical guidelines. results. the population is of patients, % of male gender, with an average age of years-old. the admission diagnostic is medical in % of patients, with an average saps ii score of , median icu stay of days and a mortality rate of %. % (n = ) of patients received any kind of blood component transfusion, mostly erithrocyte concentrate ( % of patients), followed by albumin ( %). the populations of transfused patients is older ( vs. years-old), has a longer icu stay ( vs. days), higher saps ii score ( vs. ) and mortality rate ( . vs. . %) . pretransfusional values are hemoglobin of . g/dl, , platelets/ul, and albumin of . g/dl. the level of concordance with recommendations is high for erithrocyte concentrate ( %), platelets ( %) and fresh frozen plasma ( %) but not for albumin ( %). conclusions. the level of transfusion is high in icu patients. the population who received transfusion has a more severe clinical condition and higher mortality rate. the level of concordance with recommendations is high with the exception of albumin, which use is still less standardized. with increasing acuity due to escalating icu bed demand, but the impact on patient safety is unclear. sdu continuous non-invasive physiologic monitoring of hr, rr, bp and spo identifies cardio-respiratory instability often unnoticed by caregivers. causes may be alarm fatigue and/or high sdu nurse-to-patient ratios which make bedside monitoring insensitive. instability may become more resistant to intervention the longer it occurs. the impact of instability duration upon sdu patient outcomes is understudied. objectives. the study purpose was to determine the impact of cardiorespiratory instability duration experienced by in-patients being cared for on a monitored sdu upon hospital length of stay (los) and hospital charges. prospective study of monitored patients on a -bed trauma sdu over weeks. noninvasive continuous monitoring data were downloaded from bedside monitors and analyzed for vital signs (vs) beyond local instability criteria: hr\ or [ , rr\ or [ , systolic bp \ or [ , diastolic bp [ , spo \ % . vs time plots of unstable patients were further assessed to judge instability as mild or serious. instability duration categorized as: none, [ ] [ ] [ ] [ ] [ ] [$ k) . relationships between instability duration and outcomes analyzed with chi-square for mild and serious instability. conclusions. there has been a marked improvement in the overall recording of sews since the previous study. it is of concern that respiratory rate was again the least well recorded parameter as this has been shown to be the best physiological predictor of impending cardiopulmonary arrest , . this may be because respiratory rate is not provided by the automated monitoring devices available on the general wards in our hospital, and must be calculated manually. it demonstrated an increase in mortality even when tiss scores were taken into account as an independent risk factor. since these publications critical care outreach and the use of early warning scores have become common place; however it was felt that time of discharge was still impacting on patient outcome. to review our post-unit mortality and readmission rate, with particular focus on the time of discharge. conclusions. our mortality and readmission data compare favourably with a recent publication. there is a clear difference in mortality related to time of discharge; however this is for evening discharges as compared to night discharges in previous papers. [ ] [ ] [ ] [ ] the time of discharge may represent logistical issues of planned discharges or early discharge decisions due to pressure for beds. overnight discharge is an uncommon occurrence in our unit; this evidence suggests that previous concern about night discharges should be extended to evening discharges. transferring critically ill patients is a challenging task in the day to day activities of the critical care team. safe accomplishment of these transfers relies on skills of the persons accompanying and the resources available. guidelines have been produced by various professional bodies [ , ] to safely accomplish these transfers. the competency document released by the royal college of anaesthetists, uk requires that junior trainees have appropriate knowledge, skills, attitude and behaviour in the principles of safe transfer of critically ill patients [ , ] . to obtain information about trainee's perspective, experience and knowledge in transfer of critically ill. a web based online survey was sent to all the anaesthetic/itu trainees in the west midlands region of the uk. results. total number of respondents were . of these, . % had less than months of anaesthetic training before undertaking a transfer. only % had formal training on transfer of critically ill patients. % of the trainee's didn't have any competency based formal assessment of their skills, attitudes and behaviour in transfer of critically ill patients. majority of them ( %) felt that every one should undergo formal training before undertaking transfers. while % of the respondents have undertaken transfers during their training, only % have experienced some form of critical incident during these transfers. more than % of these adverse events were related to equipment failures while % were due to patient deterioration. nearly % of the trainees were not aware of terms and conditions of the insurance cover for these transfers. conclusions. this survey highlights the deficiencies involved in training the trainee's for transfer and the transfer itself. the results demonstrates that majority of the trainees would prefer to attend specific transfer courses before venturing out on an actual transfer. we hence recommended the following for implementation: improvement of training process for those undertaking transfers; regular monitoring of this process; regular analysis of critical incidents and acting upon it; making the insurance compulsory for those undertaking the transfers. greek hospitals, including initial management of critically ill patients and primary care for a growing proportion of the population. the impact of ed length of stay (los) on patient outcome has not been covered adequately by existing surveys so far. objectives. the aim of this study was to determine the association between ed overcrowding and outcomes for critically ill patients. in the present study, we included medical and surgical pts that all of them were intubated promptly to ed of general hospitals of athens gr, for months. pts survived [ h were divided into groups: ed boarding \ h (group a) and ed boarding c h (group b). demographics, apache ii, diagnosis, los, and icu and hospital mortality were recorded. ed boarding time was measured in min. groups were compared using chi-square, mann-whitney, unpaired student's t tests and stepwise regression analysis. the collection of data lasted months. results. in the ed, critically ill patients with a mean age . ± . years and apache ii score . ± . were intubated. pts were males and were females with a mean age . ± . and . ± . years, and apache ii score . ± . and . ± . respectively. main diagnosis was multi trauma ( ) objectives. we sought to assess the baseline characteristics and outcomes of the patients presenting af as a cause of met call activation. using the met database of one tertiary teaching hospital, we retrospectively reviewed all patients for which the met diagnosis was atrial fibrillation. we reviewed their clinical history, immediate treatment and outcome. these data were compared to those of a control group of randomly selected met calls with patients being matched for age, gender and ward of origin (surgical or medical). objectives. to ascertain the proportion of preventable in-hospital cardiac arrests occurring at university hospital lewisham. furthermore, to identify any common predictors of poor outcome that were apparent prior to those arrests and whether these are potentially modifiable. a case note review was performed on the cohort of patients who suffered inpatient cardiac arrests and who were admitted for icu (level- ) care post-resuscitation. these patients were identified using our quarterly feedback from the intensive care national audit and research centre (icnarc) case mix program dataset between april and september . we found that half ( out of ) of our in hospital cardiac arrests resulted in death despite level- care post arrest within the audit period. of these in-hospital arrests were deemed preventable from case note review and trust cardiac arrest call audit forms when available. in addition, in the preventable sub-group an arterial blood gas sample was not obtained in out of , %. in all of these cases, the icu outreach team was not aware of the patient prior to the arrest. conclusions. in keeping with widely published data regarding survival to discharge after in-hospital cardiac arrest, the high mortality rate of % for this cohort of patients emphasises the importance of early recognition of abnormal physiology and timely intervention. with the sensitivity, specificity and validity of ews yet to be validated and no clear benefit proved from the introduction of met/outreach teams, an alternative strategy for earlier recognition of critically ill patients is needed. our data suggests that arterial blood gas sampling, an essential investigation central to the recognition of critically ill patients is being consistently overlooked and is an important factor influencing outcome. results. attended patients were , , with mean age of , years, and women represent , % of them. most demanding services were internal medicine ( %) followed by general surgery, haematology and nephrology. global data may be seen in table . with regards to admissions to the icu of these patients, table depicts the proportion between requested admissions, and refusals. introduction. tradicionally, critical care interventions are highly intensive, expensive and brief. critical illnesses and interventions that we use, can both contribute to posticu disability: catheter-related bacteraemia, polineuropathy, resistant organism, nutricional problems, complications of tracheostomy, prolonged analgesic. all these factors and a premature discharge from an ever full icu, can even have an impact on occult mortality after discharge from icu (between and %). in our unit a follow up program have been implanted. when patients are about to be discharged from icu, icu clinicians selected those considered to be recoverable but fragile enough to have poor prognosis. objectives. to quantify the workload that a after icu follow-up entails, and to determine if this program impacts on mortality posticu. prospective and interventional study carried out during a months period. at a beds medical uci of a teaching hospital in malaga icu, patients were enrolled in the follow up program. we assessed prognosis with sabadell score and severity of illness with apache ii score; and registered our interventions after discharge from icu. the final endpoint was status at hospital discharge: survivant or dead. we did interventions in patients: we changed a venous catheter ocasions ( % of patients), changed analgesic schedule times ( . %), stopped antibiotics times ( %), modified parenteral nutrition times ( %) . we searched and treated sources of sleep deprivation (delirium, anxiety or insomnio) in patients ( %); treated tracheostomy complications in patients. mortality of patients enrolled in this program was . % ( patients) even if the mean expected mortality by apache ii score was [ %. conclusions. in our study, implementation of a continued follow-up program after icu discharge in selected patients, carried out by icu staff, was associated with an important decrease of mortality. encouraging clinical results and a non-excesive workload for icu staff justify continuing this follow-up. objectives. various therapeutic protocols were used for the management of sepsis including hyperbaric oxygene (hbo) therapy. it has been shown that ozone therapy (ot) reduced inflammation in several entities and exhibits some similarity with hbo in regard to mechanisms of action. thus, we designed a study to evaluate the efficacy of ot in an experimental rat model of sepsis and to compare these effects with hbo. methods. forty male wistar albino rats were divided into sham, sepsis+cefepime (control), sepsis+cefepime+hbo (hbo), and sepsis+cefepime+ot (ot) groups. sepsis was induced by an intraperitoneal injection of . cfu escherichia coli; hbo was administered twice daily at . -atm pressure for min; ot was set as intraperitoneal injections of . -mg/kg ozone/oxygen gas mixture once a day. the treatments were continued for days after the induction of sepsis. at the end of experiment the lung tissues and blood samples of the study animals were harvested for biochemical and histopathologic analyses. results. lung tissue myeleperoxidase activities and oxidative stress parameters, and serum proinflammatory cytokine levels, il- b and tnf-a, were found to be ameliorated by the adjuvant use of hbo and ot when compared with the antibiotherapy alone group. histopathologic evaluation of the lung tissue samples confirmed the biochemical outcome. some measures indicated significantly more efficacy of ot than hbo. conclusions. our data presented that both hbo and ot reduced inflammation and injury in the septic rats' lungs; a greater benefit was obtained for ot. these findings suggest that it may be possible to improve the outcome of sepsis by using ot as an adjuvant therapy. objectives. to investigate the regularity for change of paf, tm and vwf in septic rat, and the protective effects of statins on vascular endothelium. methods. fifty-four male sd rats were randomized into simvastatin with lps group (group a, n = ) and lps group (group b, n = ) and control group(n = ). they were respectively accepted ml/kg normal saline (ns) abdominal injection for both control group and group b, ml/kg simvastatin abdominal injection for group a, then h later, total male sd rats from group a and group b were respectively accepted lps ( mg/kg weight) abdominal injection to establish sepsis model and ml/kg ns abdominal injection for control group. thereafter, detected the serum concentration of von willebrand factor (vwf), thrombomodulin(tm) and antithrombin (at-iii) at different point of time ( , , and h after lps abdominal injection) in both group a and group b by elisa, the endothelial cells from thoracic aorta was observed with electron microscope. under electron microscope scanning, endothelial cells in septic rats from group b were found disarranged. under transmission electron microscope, endothelial cells were found to be in prophase of apoptosis characterized by unclear cell membrane, thickened cellcell conjunction, disappeared desmosome and microfilament, dissolved or vacuolized organelles and agglutinated and evaporated chromatin gathering under the karyolemma, but the karyorrhexis were not found. no similar changes were found in group a. ( ) introduction. sepsis induced lymphocyte apoptosis is believed to play an important role in the pathogenesis of sepsis and in the development of the immunesuppresion observed in septic patients. lymphocyte apoptosis not only decreases the number of functional lymphocytes but may also modify the immune response towards an anti-inflammatory state. erythropoietin (epo) has recently been recognized as a multifunctional cytokine with antiinflammatory, antioxidative, and antiapoptotic properties. objectives. this study aimed to test whether epo could mitigate peripheral blood mononuclear cell (pbmc) apoptosis and whether epo could modify the dynamic changes in lymphocyte-subsets in a porcine model of acute endotoxemia. methods. twenty-eight anesthetized and mechanical ventilated pigs were randomized to one of three groups: ) epo group, epo administered h prior to endotoxemia (n = ); ) placebo group, vehicle administered h prior to endotoxemia (n = ); ) sham group, animals only anesthetized and mechanical ventilated. endotoxemia was induced by an infusion of lipopolysaccharide (lps). after h the lps infusion was reduced to a maintenance dose and the animals were fluid resuscitated. pbmc were isolated at time , , , and min of endotoxemia. apoptosis in pbmc and relevant lymphocyte subsets were assessed by staining with -amino-actinomycin d ( aad) and annexin v using multicolor flow cytometry. apoptotic lymphocytes in spleen were quantified by immunohistochemical staining for activated caspase- . endotoxemia increased the number of apoptotic mononuclear cells in both blood (p = . ) and in spleen (p = . ), but with no significant modifying effects of epo. the numbers of both cd + (t-helper) and cd + (cytotoxic) t-cells declined during endotoxemia. cd + cells, defining b-lymphocytes, demonstrated a biphasic response with an immediate decline followed by an increase in number of b-cells. the dynamic changes in the lymphocyte subsets were not modified by epo. , and reduced the number of circulating leucocytes. epo had no modifying effects on these dynamic changes. furthermore, epo did not mitigate apoptosis in pbmcs analyzed by flow cytometry or in spleen lymphocytes analyzed by immunohistochemistry. this study does not support that epo confer protection against lymphocyte apoptosis. objectives. aim of this study was to investigate the effects of combined, recombinant human activated protein c (rhapc) and ceftazidime (cef) in our established model of acute respiratory distress syndrome (ards) and septic shock methods. thirty sheep ( - kg) were operatively prepared for chronic study, and were randomly allocated either to sham, control, rhapc, cef, or rhapc/cef groups (n = each). after tracheostomy, acute lung injury and sepsis was produced in all groups, following an established protocol ( , ) , except the sham group that received the vehicle. the sheep were studied for h in an awake state and were ventilated with % oxygen. pao /fio ratio was determined intermittently. cef ( g) was administered intravenously and h post injury. rhapc was given as a continuous infusion ( mcg/kg/h), starting h post injury. the animals were resuscitated with ringer's lactate solution to maintain filling pressures and hematocrit. lung tissue was obtained during necropsy and analyzed for myeloperoxidase (mpo) using a commercially available kit. statistical analysis: two-way anova and student-newman-keuls post hoc comparison. data are expressed as mean ± sem. significance p \ . . . mpo levels (mu/mg protein) were ± in sham and significantly increased in the control group ( ± *). the rhapc ( ± *) and cef group ( ± *) increased significantly vs. sham and tended to be lower than controls, but not statistically significant. mpo levels of combined rhapc/cef ( ± *) showed no difference to sham, but were significantly lower than controls or rhapc or cef alone. conclusions. combined administration of rhapc and ceftazidime in ards associated with septic shock improved oxygenation more than cef or rhapc alone, and prevented the onset of ards. seleno-compounds, such as sodium selenite (na seo ) show conflicting clinical results in the treatment of sepsis. efficacy, as well as mechanism of action of na seo , are unclear, with prevailing opinion that it acts as an anti-oxidant. however, na seo has also oxidant properties that could have a paradoxical therapeutic role in septic shock by reducing over-activated phagocytic cells. indeed, in septic sheep, high dose na seo injection as bolus rather than continuous administration resulted in a beneficial effect on survival time, macro and microcirculation ( ). objectives. to investigate at the endothelial level the mechanism of action of a bolus injection of a high oxidative dose of na seo . in male wistar rats, lipopolysaccharide (lps, mg/kg) or normal saline were injected intraperitoneally, followed h later by either an intravenous bolus injection of na seo (corresponding to . mg/kg se) or normal saline. after h of lps, extravasation of fluoroisothiocyanate-dextran and leukocyte-endothelium interaction in venules of the cremaster muscle were quantified by intravital microscopy. results. na seo did not alter systemic haemodynamic variables as compared to lps rats. there were no intergroup differences in fluoroisothiocyanate-dextran extravasation. lps significantly decreased leukocyte rolling when compared to control animals (p \ . ). bolus injection of na seo did not alter leukocyte rolling but decreased leukocyte adhesion and extravasation levels to control values. our results in endotoxemic rats suggest that a toxic dose of na seo may have a beneficial effect of on leukocyte-endothelium interaction without a significant effect on plasma extravasation. objectives. to design a model of sepsis in pigs characterized by an unchanged q t over time. methods. after a h fasting, pigs (weight - kg) were sedated with ketamine ( mg/ kg) and midazolam ( . mg/kg) i.m. animals were tracheostomized and anesthetized (propofol mg/kg iv bolus, followed by mg/kg/h), atracurium ( . mg/kg/h) and fentanil ( lg/kg/ h). the internal jugular vein, carotid artery and pulmonary artery were catheterized for iv fluid administration and monitoring. a lumbotomy was performed and an ultrasonic blood flow and a laser-doppler microvascular flow probes were placed in the left renal artery and on the kidney surface to measure renal artery blood flow (rabf) and renal cortical blood flow (rcbf), respectively. a cystostomy was performed to collect and measure urine output (uo). sepsis was induced by the iv administration of live e. coli ( . our previous study showed that citrulline (cit) supplementation during endotoxemia improved microcirculatory flow and endothelial function, and prevented glycocalyx degradation as a consequence of increased arginine (arg)-dependent vascular nitric oxide (no) production. during sepsis the availability of arg, the substrate for endothelial no production, is tempered as a consequence of increased inflammatory no synthase (inos) activity. the reduced endothelial nos (enos) activity and vascular no production is believed to result in endothelial and vascular dysfunction. a shortage of arg availability for enos is considered the main cause of the dysfunction. previous studies have indicated cit as an important, if not exclusive, mediator for enos-derived no production. cit is a substrate for argininosuccinate synthetase, an arg-producing enzyme that co-localizes with enos in the caveolae, thus directly and exclusively supplying arg to enos. objectives. we investigated whether cit supplementation during an ongoing endotoxemia rescues the enos-derived no production in endothelial cells, thereby providing a mechanistic explanation for its positive in vivo effects. mice received a continuous intravenous endotoxin (lps, lg total) infusion for h alone or an h lps infusion with cit ( . mg total) during the last h of endotoxin infusion. after the h infusion, the mice were sacrificed, arterial blood was sampled and the carotid arteries were removed. no production in the carotid arteries was measured ex vivo with -photon fluorescence microscopy, using a fluorescent copper-based no probe. amino-acid concentrations in plasma were measured by hplc. results. both cit and arg plasma concentrations were significantly increased in the lps-cit group compared with mice treated with lps alone (p \ . ). in vivo cit supplementation led to detectable levels of no production ex vivo in carotid smooth muscle cells (smc) and endothelial cells (ec) by using the no-probe with -photon fluorescence microscopy. while ec-derived no production was absent in the carotid arteries of mice treated with only lps, the smc-related no signal was undisturbed. no production in the ec of the lps-cit group was not blocked by the inos inhibitor , w, suggesting enos to be responsible for the observed effect. furthermore, ex vivo incubation of the carotid arteries of the lps-cit mice for min with extra cit ( mg/ml) resulted in prominently increased no production in the carotid ec, whilst this effect was not observed in the carotid arteries of lps without cit treated mice. conclusions. cit supplementation during murine sepsis rescues the enos-derived no production in carotid artery endothelial cells, providing a mechanistic base for the positive effect of cit supplementation on endothelial no synthase during endotoxemia. grant acknowledgment. objectives. investigated the mechanism involved in the clearance of bacteria observed after rpaf-ah treatment in sepsis model. mice were subjected to clp model, after min, the mice were treated with rpaf-ah. the cfu counts and measured of mediators were determined. results. the numbers of bacteria (cfu) recovered in the peritoneal fluid was inhibited in rpaf-ah treated group ( . / . ), suggesting a more efficient clearance of bacteria after rpaf-ah treatment. direct incubation of s. typhimurium, e. coli and s. aureus failed to affect bacterial growth indicating lack of a direct effect of paf-ah on bacteria. administration of rpaf-ah in ccr (receptor for mcp- /ccl ) deficient mice failed to increase bacterial clearance after clp, suggesting that mcp- signaling is involved in this phenomenon. rpaf-ah treatment also failed to increase bacterial clearance in inos deficient mice and no levels were found to be elevated ( . ± . / . ± . ) in peritoneal fluid of the mice treated with rpaf-ah after clp surgery. synergism for no production was also seen when macrophages stimulated with e. coli were treated with rpaf-ah+mcp- and correlated with better bacterial killing by macrophages. peritoneal macrophages from knockout mice for mcp- , stimulated from lps+ifn inhibited no levels when compared to wt mice ( . ± . / . ± . ). this results indicating that, excessive mcp- favors macrophage production of no and hence the ability of macrophages to deal with invading bacteria. conclusions. we conclude that the increase in bacterial clearance is important for the protective effect of rpaf-ah in sepsis and that exist a signaling involving mcp- /ccl and no in this system. introduction. disturbances within the microcirculation represent an important factor in the pathogenesis of multiple organ dysfunction during systemic inflammation and sepsis [ ] . dehydroepiandosterone (dhea) has immunomodulatory effects and improves survival in several animal models of trauma, hemorrhage and sepsis but also causes potent vasodilatation [ ] . to maintain efficient microcirculation we combined dhea with sodium orthovanadate (sov), which augments vascular contraction. furthermore, sov has been identified to attenuate tissue injury and improve survival related to inflammatory response [ ] . objectives. we investigated whether the combined administration of dhea and sov has beneficial effects to microcirculation in experimental sepsis. we divided sixty male lewis rats into six groups: control group; ethanol (solvent) treated control group; dhea ( mg/kg) + ( . mg/kg) treated control group; endotoxemic group (lps mg/kg); dhea + sov treated endotoxemic group; dhea ( mg/ kg) + sov treated endotoxemic group. two hours after lps challenge we performed intravital fluorescence microscopy of the intestinal wall in order to study leukocyte adhesion and functional capillary density (fcd). tnf-a, il- a, il- and infc, gm-csf and mcp were measured at baseline and following h of endotoxemia in all experimental groups. in comparison to untreated rats subjected to endotoxemia the treatment with dhea (both dosages) and sov resulted in a significant reduced number of adhering leukocytes in intestinal submucosal venules. furthermore, the mucosal functional capillary density was significantly improved. we did not identify any changes in cytokine plasma levels. conclusions. the study demonstrated beneficial effects of combined treatment with dhea and sov within the intestinal microcirculation in experimental endotoxemia. concomitant administration of sov permitted to reduce dhea dosage and prevent potential vasodilation without affecting anti-inflammatory dhea action. . spronk pe, zandstra df, ince c: bench-to-bedside review: sepsis is a disease of the microcirculation. crit introduction. sepsis is a disease of the microcirculation and impairment of the intestinal microcirculation during sepsis may cause a breakdown of gut barrier function thus releasing bacteria and their toxins into the systemic circulation [ ] . consequently, the protection of the intestinal microcirculation represents a pivotal therapeutic target in severe systemic inflammation. cannabinoids that interact with cannabinoid receptors (cb r and cb r) have been shown to have immunomodulatory properties in in vivo and in vitro studies and the endocannabinoid system has been shown to be involved during systemic inflammation [ ] . objectives. the aim of the present study was to examine the effects of cb receptor modulation on the intestinal microcirculation in experimental sepsis (endotoxemia) using intravital microscopy (ivm). we studied four groups of animals (lewis rats, n = per group): healthy controls (con), endotoxemic animals ( mg/kg lipopolysaccharide; lps), endotoxemic animals treated with cb agonist, hu ( mg/kg iv), and endotoxemic animals treated with cb antagonist, am ( . mg/kg iv). intravital microscopy of the intestinal microcirculation was performed following h lps/placebo administration. leukocyte adhesion and functional capillary density (fcd) were measured offline in a blinded fashion. results. following h of endotoxemia, a significant increase of leukocyte adhesion in the intestinal submucosal venules (e.g., v venules: con . ± . n/mm , lps . ± . n/mm , p\ . ) was observed. capillary perfusion of the muscular and mucosal layers of the intestinal wall was significantly reduced (e.g., circular muscular layer: con . ± . cm/cm , lps . ± . cm/cm ). treatment of endotoxemic animals with the cb receptor agonist, hu , further increased leukocyte adhesion (v venules: . ± . n/mm ), whereas cb receptor inhibition by am significantly reduced leukocyte activation (v venules: . ± . n/mm ) and restored capillary perfusion (circular muscular layer: . ± . cm/cm ). conclusions. the data support the hypothesis, that cb receptor signalling is involved in the impairment of the intestinal microcirculation during sepsis. blocking cb receptor signalling reduces leukocyte activation and improves capillary perfusion in acute endotoxemia in rats. the long-term effect of modulating cb receptors in more clinical sepsis models needs further investigation. [ ] . this study compares dobutamine and levosimendan for the treatment of circulatory failure in septic shock and assesses survival benefits. objectives. in this controlled randomized doubleblinded study anaesthetized and ventilated pigs ( . ± . kg) were enrolled after approval by the local governmental commission. methods. by continuous infusion of endotoxin (escherichia coli serotype :b , sigma-aldrich; . ± . lg/kg/h) over a time period of . ± . h, septic shock was induced. hemodynamic stabilization was performed by either use of the vasopressor norepinephrine alone (control group; n = ) or in combination with levosimendan ( . lg/kg/min; n = ) or dobutamine ( . lg/ kg/min; n = ). in a setting of h of measurements and treatment heart rate (hr), map, central venous pressure (cvp), pulmonary artery pressure (mpap) and cardiac output (co) were recorded continuously and evaluated hourly. beside norepinephrine requirement and mixed venous oxygen saturation (svo ) mean survival time and survival rate within the measurement period were analysed. results. after endotoxinemia septic shock was marked by reduction of co and svo [p \ . ]. mean survival time and survival rate were superior in levosimendan treated animals ( table ). norepinephrine consumption was lowest in the levosimendan group. after h, co of surviving animals was highest in the levosimendan group and statistically different compared with the control group. comparison of parameters hr, map, cvp and mpap showed no differences between treatments. conclusions. the complementary use of the calcium sensitizer levosimendan provides potential survival advantage in endotoxemic septic shock. beside an increase in co, improvement of regional organ perfusion or protection could be an explanation and has to be shown by further analysis. reference(s methods. the study group consisted of patients with shock on vasopressor support and control group had normotensive patients. arterial and capillary samples were taken simultaneously and were tested immediately at the bedside. the results of the paired measurements were analysed as a scatter plot by bland and altman method and were expressed as a correlation coefficient. values were considered to disagree significantly when the difference exceeded %. results. mean arterial and capillary sugars (mg/dl) in study and control groups were . ± and . ± . , and . ± . and . ± , respectively. on bland-altman analysis, % in study group and % in control group were out of range (acceptable limit \ %) [ figures , ] . correlation between capillary and arterial values was less in the study group (r = . , p . vs. r = . , p \ . ). in addition, the disagreement between capillary and arterial values was more than % in % of the patients in the study group vs. % in control group (p = . ) (iso standard \ %). conclusions. capillary blood glucose monitoring can be applied reliably to patients in icu. however, caution must be exercised in patients with shock in whom arterial blood may be preferred. rd esicm annual congress -barcelona, spain - - october objectives. our primary objective was to evaluate the safety and efficacy of a single oral high dose vitamin d supplementation in an intensive care setting over a one-week observation period. methods. , iu (corresponding to . mg) of cholecalciferol (d) dissolved in ml herbal oil or matched placebo (pbo) were given enterally (via nasogastric feeding tube or swallowed) to patients with vitamin d deficiency [ (oh)d b ng/ml] in the medical icu. results. baseline characteristics including age, sex and saps ii were balanced between the two groups (mean age ± years, % male, saps ii ± ). mean serum (oh)d levels at baseline were ± ng/ml in both groups. the mean serum (oh)d increase in the intervention group was ng/ml (range - ng/ml). two patients showed a small ( ng/ml) or no response ( ng/ml) attributable to gastrointestinal dysfunction after prolonged hypoxia and gastrointestinal gvhd after allogeneic stem cell transplantation. the time course of the (oh)d response is given in figure . introduction. considerable controversy has emerged as to whether tight glucose control (tgc) is warranted in all critically ill adult patients. recently, a new blood glucose upper limit ( mmol/l) has been assessed as more appropriate. rather than blood glucose target ranges, algorithms used to achieve tgc should be numerically evaluated before initiating clinical trials (preclinical validation test). our purpose was to assess performances of tgc algorithms in realistic virtual icu patients. we compared numerically the nice-sugar algorithm (n-s) and the cgao system (cgao) used in the ongoing cgao-rea study [clinicaltrials.gov, id:nct ] . a set of virtual patients constituting the test bench was built with ) real data coming from patients controlled with cgao before starting cgao-rea and ) a non-linear pharmaco-dynamic glucoseinsulin system model where patient endogenous glucose clearance and insulin-sensitivity were time varying parameters. in order to anticipate how algorithms would manage glycaemic control in clinical settings, delayed controls and inaccuracy of glucometers were implemented. the overall performance of each algorithm over the whole stay was assessed according to standard scores. results. the percentage of time in the target range [ . - . mmol/l] with n-s was less than % for almost all patients. in insulin-sensitive patients, glycemic fluctuations and sometimes severe hypoglycemia are induced by n-s (fig. ). the mean time in the target range with cgao was about % and variability scores were significantly lower than with n-s. mean glucose and standard deviations were always lower with cgao than with n-s. a numerical test bench constituted of realistic virtual icu patients, whose features were defined from real data obtained in patients under glycemic control, enabled to determine the best algorithms candidate for further evaluation in clinical settings. according to this approach, the algorithm used to achieve tgc in nice-sugar would not have been selected for such a large clinical trial while cgao reached the first validation step in simulation. we recommend that further glucose control studies focus not only on the target range but also on the algorithmic properties. introduction. there has been much debate in recent years about the appropriate level of blood glucose for intensive care patients with proposals of different levels of glucose control using insulin infusions. one risk of intensive glucose control is hypoglycaemia and this has been proposed as a measure of quality of care given by delivering the protocol safely. the nice-sugar trial found that intensive glucose control increased mortality among adults in intensive care. objectives. the aim of our study was to record hypoglycaemia and study it's relation to insulin therapy. insulin therapy on our unit follows the recommendations of the nice-sugar trial. methods. hypoglycaemia was recorded as a blood glucose level\ mmol/l. levels were detected using the blood gas analyser (radiometer m). data was recorded at the time of hypoglycaemia to provide an explanation using the innovian system which is the paperless patient record system on our unit. data was obtained over a period of months between october and december . data recorded included adverse events which were defined as worsening shock and/or increasing inotropic support. feeding status at the time of hypoglycaemia was recorded. results. there were a total of admissions over this period and there were a total of , blood glucose measurements. incidents of hypoglycaemia were recorded, of which patients were on insulin and were not. of the patients who were on insulin, had adverse events at the time of hypoglycaemia. all these patients died within h of the adverse event. all except one was on full feed. the others had minimal feed due to poor absorption. of the patients who did not have adverse events, were discharged and one died days after the hypoglycaemic event due to worsening sepsis. of the patients on insulin, there were iatrogenic errors where feeding was stopped and the insulin was left on. none resulted in any adverse outcome for the patients. of the patients who were not on insulin therapy, had adverse events at the time. died within h of the adverse event and died days later. the remaining patients were discharged. none of the patients were on full feeding protocol. conclusions. our findings suggest that hypoglycaemia in our unit is not primarily related to insulin therapy. it is related to adverse events and possibly inappropriate feeding at the time of hypoglycaemia. hypoglycaemia, in the absence of insulin therapy, is associated with a poor outcome. use of hypoglycaemia as a quality indicator should be interpreted with caution. introduction. vitamin d deficiency seems increasingly prevalent. pleiotropic effects of vitamin d like immunomodulation and effects on muscle strength may be of special importance to critically ill patients [ ] . however, vitamin d deficiency has only been studied in small and selected groups of icu patients [ ] . objectives. to prospectively determine the prevalence of vitamin d deficiency in winter and summer and relate vitamin d status to outcome in cohorts of critically ill patients. results. vit d was measured in patients admitted in winter and patients admitted in summer (table ). mean vit d was significantly lower in winter than in summer. in winter, % was deficient, % severely deficient. in summer, % was deficient, % severely deficient. predicted mortality was higher in winter and higher in vit d deficient patients. observed mortality was lower than predicted in all groups, but not different between groups. including both vit d and season in a multiple regression analysis, winter (p = . ) and not vit d (p = . ) was related to predicted mortality. introduction. glucagon-like peptide- (glp- ) lowers blood glucose via stimulation of insulin and suppression of glucagon secretion, as well as slowing gastric emptying. we have previously shown that exogenous glp- attenuates hyperglycaemia in non-diabetic critically ill patients [ , ] . however, islet cell function in critically ill diabetic patients may be so disturbed that pharmacological doses of glp- have no effect in this group. objectives. the aim of this study was to evaluate the effect of exogenous glp- on glycaemic excursions during intraduodenal nutrient infusion in critically ill patients with preexisting type- diabetes mellitus. methods. nine critically ill, mechanically ventilated, patients with pre-existing type- diabetes ( m: f, age ± years, hba c . % ± . %, bmi ± kg/m , apache ii on day of study ± , days in icu on day of study ± ) received iv infusions of glp- ( . pmol/kg/min), and placebo, from t = - min on separate days in a randomised, double-blind, fashion. between t = - min a liquid nutrient (ensure) was infused intraduodenally at a rate of kcal/min via a naso-enteric feeding catheter. blood glucose concentrations were measured by glucometer at min intervals. data are mean±sem and comparisons are using student's t test. results. prior to the commencement of iv infusions there was no difference in blood glucose between the groups (at t- min: glp- : . ± . mmol/l vs. placebo: . ± . mmol/ l; p = . ). during fasting, glp- had no effect on glycaemia (at t = min: glp- : . ± . mmol/l vs. placebo: . ± . mmol/l; p = . ). however, glp- attenuated the overall glycaemic response to the nutrient (auc - min : glp- : , ± mmol/l.min vs. placebo: , ± mmol/l.min; p \ . ), as well as the peak blood glucose (glp- : . ± . mmol/l vs. placebo: . ± . mmol/l; p \ . ) conclusions. exogenous glp- is effective in reducing the glycaemic excursions that occur with enteral nutrient critically ill patients with pre-existing type diabetes mellitus. these data indicating that further studies using glp- , or its analogues, are warranted in this group. , , , , whilst raising concerns regarding an increased risk of hypoglycaemia. , , . locally most units adopt a protocol that reflects the practice of the original study. objectives. this study was conceived due to concerns around the safety of tight glycaemic control (tgc). our objectives were to measure adherence to our local policies and ascertain our true rates of hypoglycaemia. methods. this study was designed as a retrospective audit on four critical care units in the cheshire and mersey critical care network. each site used the same audit tool but adapted it to allow for differences in local practice and protocols. data pertaining to the prescribing and administration of insulin was collected daily over a week period (the time of data collection varied from day to day). the doctors and nursing staff were unaware of the audit and the data was collected by the ward pharmacist who suggested modifications to therapy if it was deemed inappropriate or unsafe. results. patient days worth of data was collected with blood glucoses checked in this period. % of patients receiving insulin had insulin prescribed. only % of blood glucoses were within the target range set by the local protocol. however, of all the results only . % were ''low'' as defined by the local protocol, and only . % ( / ) were hypoglycaemic episodes as defined in the greet van den berghe paper of (\ . mmol/l). conversely, . % were above the target range. in the trusts that recorded how many of these levels were[ mmol/l (a proposed alternative upper limit), the rates were and %. in response to a blood glucose the policies suggest dosage adjustments/maintenance. on only % of occasions were the adjustments made correct. insulin infusions appeared to be managed safely by nursing staff. insulin, if given, was always prescribed and hypoglycaemia (blood glucose \ . mmol/l) occurred on only one occasion. although safe, adustments often didn't follow the protocols and the patients' blood glucose were within the target range only % of the time, potentially negating many of the perceived benefits of tgc. reasons for non-compliance with the protocols was difficult to objectively establish reference(s). introduction. diabetes mellitus has been associated with an increased risk of adverse outcomes after coronary artery bypass grafting. hemoglobin a c is a reliable measure of long-term glucose control. it is unknown whether adequacy of diabetic control, measured by hemoglobin a c, is a predictor of adverse outcomes after coronary artery bypass grafting. material and method. we evaluated consecutive diabetic patients who underwent primary, elective coronary artery bypass grafting at the anadolu medical center. hba c levels of all patients with diabetes mellitus were measured and value of % or greater was used as a threshold for uncontrolled hyperglycemia. all the peroperative variables were recorded and then, statistically evaluated. the statistical analysis was realised by t test for parametric variables and chi-square test for nonparametric variables. results. there were consecutive patients that underwent elective coronary artery bypass graft surgery between january and april . among them, patients had diabetes mellitus and others not. there were no significant differences between groups regarding each adverse outcomes (table ) . although, ( . %) of total surgical site infection in patients had been seen in diabetic patients, there were also no significant differences between groups regarding the rate of infections (table ). there was no early postoperative mortality in diabetic patients. insuline treatment (iit) , by implementing a completely nurse driven protocol as in the leuven i study, to achieve tight glucose control in our -bed medical(cardio-)surgical icu and non-ventilator beds. in the last year, the benefit of iit and the possible detrimental effects of hypoglycemia on survival have been heavily debated. objectives. the goal is to analyze our daily practice in all icu patients and compare this with the intensive treated groups from the leuven , visep en nice-sugar trial. methods. we compared mean morning blood glucose levels and the percentage of patients who had a hypoglycaemia, defined as glucose below . mmol/l, from to . the frequency of control and the insulin dosage was comparable to the leuven study. enteral or parenteral feeding was started at admission. no standard intravenous glucose was used. glucose was measured with arterial blood samples on the abl flex radiometer as poct. results. in our patients, the mean morning blood glucose was higher than in the leuven study and comparable to the visep and nice-sugar. the percentage of hypoglycemia on our icu was lower in comparison with the visep and nice-sugar. this may be explained by the availability of a poct on our icu which allows quick adjustments of the insulin dosage. conclusions. effective tgc with sprint resolved organ failure faster, and for a greater percentage of patients who had similar admission and maximum sofa scores, compared to a matched retrospective conventional control cohort. these morbidity reductions mirror the reduced mortality seen with sprint. these results suggest that reduced organ failure, assessed by sofa, is a fundamental element in reduced mortality when tgc is implemented effectively. introduction. tight glycaemic control was reported to reduce mortality in selected surgical critically ill patients and lowering of blood glucose (bg) levels was recommended as a means of improving patient outcomes ( ) . however, this approach has been linked with significant risk of hypoglycaemia. recently, several studies have confirmed significant associations between variability of bg levels and patient outcomes ( ). objectives. to evaluate the association between bg variability and hypoglycaemia in a mixed adult icu. methods. retrospective analysis of the prospectively collected and stored bg measurements over a year period, during which tight glycaemic control was targeted in all patients. every day we have calculated the bg coefficient variation as expressed by sd/mean bg level. we have divided the patients into low, medium and high variability groups ( - , - and [ , respectively) . hypoglycaemia was determined if bg was below . mmol/l. for statistical analysis chi-square test and pearsons correlation test was used. results. patients were admitted over the -year period, providing daily data points. bg variability was high in daily measurements ( . %), medium in ( . %) and low in ( . %). hypoglycaemia occurred in measurement points ( . %). hypoglycaemia was observed at all points ( %) when bg variability was high vs. . % when bg variability was medium and . % when bg variability was low and this difference was statistically significant (p = . ). we observed a significant correlation between increased bg variability and hypoglycaemia (r = . , p = . ). conclusions. increased bg variability as expressed by coefficient variation is associated with hypoglycaemia, when measured daily in a mixed icu population employing tight glucose control. decreasing the variability of the bg concentration may be an important dimension of glucose management. if reducing swings in the bg concentration is a major mechanism behind the beneficial effects of glucose control, it may not be necessary to pursue lower glucose levels with the associated risk of hypoglycemia. ). there were two major outliers which may skew the results in favour of the hypothesis. if these two results are removed (fig. ) the statistical significance remains strong (n v * p \ . ; **p = . ; ***p = . )). standard multiple regression analysis found the most useful predictors of t [mid] were 'time with aki' and 'serum urea' (beta coefficient . and . (p \ . ) respectively). crcl, serum creatinine and urine output did not add further predictive statistical power. conclusions. this study demonstrates a reduction in the hepatic metabolism of midazolam associated with aki. this effect is related most strongly to the length of time the patient has suffered with aki. our results are similar to the ncepod report. even with multiple recommendations by ncepod and the national institute for clinical excellence (nice) recognition of the critically ill remains poor. detection of organ failure risk is vital to implement preventative strategies. we found a delay in aki recognition and a lack of risk assessment. observations, included in admission protocols, were recorded, but investigations outside of these, were often absent. nice suggest management should be physiologically and not diagnosis based but few patients had a documented physiological plan. we suggest improving under and postgraduate education to increase awareness of aki. this could occur as an extension to the national, acute life-threatening events recognition and treatment course. an aki admission protocol may allow identification of at risk patients and instigate appropriate monitoring, investigation and management. improved ward based fluid monitoring and management would reduce deterioration. incorporation of a physiological monitoring plan on the icu observation chart may reduce preventable aki. there was no effect in patients with extensive stroke and high severity of a glasgow score ( - points in an observational prospective study, a total of patients who admitted during months in a medical and surgical intensive care unit and didn t have any recent history of renal replacement therapy were included in the study. ( %) of all patients was in aki (acute kidney injury) group according to the akin (acute kidney injury network) definition. the mean of age in aki group was more than non-aki ( . ± . , . ± . respectively; p \ . ); and had worse condition according to apache ii (acute physiology and chronic health evaluation ii) score ( . ± . vs. . ± . ; p \ . ). the aki patients stayed longer in icu rather than non-aki patients ( . ± . vs. . ± . days respectively; p \ . ); with more mortality rate ( . vs. %; p \ . ). also the mechanical ventilation days, time of vasoacive drugs and the use of dobutamin were more in aki group (p \ . ; p = . and p = . respectively). the aki was a significant predictor for mortality using the multivariate logistic regression (or adj = . ; %ci: . - . ); and had the same sensitivity as the apache ii score in prediction of mortality (sen. = . ). objectives. the purpose of this study was to evaluate renal function in children with congenital heart disease (chd) undergoing cardiac surgery with cpb. we conducted prospective, non randomized observational study at the tertiary care university children's hospital -bed surgical icu. study protocol was approved by hospital ethics commission. the study included patients with chd with body weight from . to kg (mean . ± . kg) and age from days to years (mean age months). there were patients with ventricular septal defect (vsd), patients had atrioventricular septal defect (avsd), two had total anomalous pulmonary venous drainage (tapvd), one had tetrology of fallot (tof), one had transposition of great arteries (tga), and one had aortic stenosis, requiring ross operation. urine was collected in the postoperative period during the first h after surgery for determination of clcr. the serum creatinine (scr) level was determined by jaffé s method (cobas analyzer, roche). harrison am et al. [ ] shows that estimated creatinine clearence (clcr) using schwartz formula does not accurately predict clcr. therefore we used standard formula for clcr calculations. urine output, inotrope score, duration of aortic cross clamping and cardiopulmonary bypass was recorded. we applied rifle criteria to assess renal functions, using clcr as a variable reflecting glomerular filtration rate (gfr). objectives. evaluate whether a real-time alert of worsening of rifle class, through the physicians' dect telephone system, would affect therapeutic interventions for aki and progression of rifle class. single centre, prospective intervention study during a -month period in our bed surgical and medical icu. three study phases were compared: a . -month control phase (con ) where physicians were blinded for the electronic alerts, a -month intervention phase where electronic alerts of worsening rifle class were made available to the physicians through the dect telephone system (int), followed by a second . month control period (con ). pasw statistics was used for statistical analysis and a double sided p value of . was considered as significant. at study entry, before and after to receive antioxidant or placebo concentration the blood was drawn, to posterior determination of thiobarbituric acid reactive species (tbars), protein carbonyls, total nitrite concentration and il- . results. the use of nac+dfx decreases oxidative damage parameters. patients at antioxidant arm have, despite not reaching statistical significance, a decrease on plasma il- levels h after the start of treatment. as observed to oxidative damage parameters, il- returned to the placebo levels after the end of antioxidant administration. the nitrite levels increased h after nac+dfx, returning to placebo levels and h. the incidence of arf using the rifle criteria was not significantly different in the two arms, and this was also true to a number of secondary end points, none of which showed significant differences between the treatment arms. analyzing the subgroups of the sofa score we observed at day a worse cardiovascular sofa in the nac+dfx arm ( . ± . vs. . ± . , p = . ) and a better renal sofa in antioxidant treated patients ( . ± . vs. . ± . , p = . ). conclusions. we demonstrated that nac + dfx administration was able to decrease plasma markers of oxidative damage and to a minor extend il- plasma levels. we believe that the use of antioxidants could be an alternative adjuvant therapy to prevent arf in critical ill patients with hypothension. table were found to be independent risk factors for postoperative aki: objectives. we aimed to access prospectively whether the use of antioxindants has beneficial effects in renal function of critical-ill patients undergoing imaging studies with intravenous radio-opaque agents (ivca). patients were recruited from those hospitalized in a tertiary intensive care unit between and . inclusion criteria were: a) requirement for imaging studies with ivca b) no use of renal replacement therapy. patients were randomized to receive before and after imaging, either antioxidants (n-acetyl-cysteine , mg and ascorbic acid g and ml ns . %) (sg) or cc ns . % (cg). renal function was assessed by serum levels of creatinine and cystatin c assessed before and at , h following administration of ivca. patients were followed until discharge. systatin c was measured by elisa. conclusions. the results of this study suggest that the use of preventive antioxidant therapy may protect critical-ill patients from contrast-induced nephropathy. our preliminary results have to be confirmed in larger cohorts. acute coronary occlusion is the leading cause of cardiac arrest. because of limited data, the indications and timing of coronary angiography and angioplasty in survivors of out of hospital cardiac arrest are controversial. objectives. using data from the parisian region out of hospital cardiac arrest (procat) prospective registry, we performed an analysis to assess the impact of an invasive strategy on hospital survival. between january and december , survivors of out of hospital cardiac arrest were referred to a tertiary center in paris, france. in survivors with no obvious extra-cardiac cause of arrest, an immediate coronary angiogram followed if indicated by coronary angioplasty was performed at admission. the prognostic value of pre-hospital and in-hospital characteristics on in-hospital mortality was evaluated using logistic regression analysis. results. at least one significant coronary artery lesion was found in ( %) therapeutic hypothermia has been shown to improve survival and neurological outcome in patients who have suffered out-of-hospital cardiac arrest and in whom the initial rhythm was ventricular fibrillation (vf) [ , ] . international guidelines now recommend the use of therapeutic hypothermia as part of post-resuscitation care in patients fulfilling the above criteria [ ] . objectives. we surveyed current practice regarding the use of therapeutic hypothermia for post resuscitation care in northern ireland (ni) intensive care units. a questionnaire was devised, reviewed and agreed by each author prior to posting to the lead clinician in each of northern ireland's adult intensive care units. a % response rate was obtained. we asked about the existence of a protocol for cooling, which patients were cooled, duration of cooling, by what particular method(s) cooling was achieved and how temperature was monitored during cooling. results. out of ( %) adult icus in ni institute therapeutic hypothermia routinely as part of their post-resuscitation care. only out of the units ( %) have a protocol for institution and maintenance of hypothermia. all units that utilise hypothermia do so regardless of the initial cardiac rhythm. out of ( . %) icus target a temperature of - °c with out of ( . %) targeting a temperature of - °c. all units utilise surface cooling methods with out of ( %) also using cold intravenous fluids occasionally. out of ( %) units cool for - h, ( . %) unit - h and unit - h. all units use more than one method of temperature monitoring during cooling. all units sedate patients during cooling and out of ( . %) also routinely curarise patients during cooling. the units that do not currently use therapeutic hypothermia cited lack of resources/funding as the main obstacle to adopting this evidence based practice. conclusions. the practice of therapeutic hypothermia post cardiac arrest has been embraced by the majority of icus in ni. there appears however to be variation in the target temperature and duration of hypothermia once instituted. icus that cool patients appear to do so regardless of initial cardiac rhythm. regional protocolisation of this therapeutic modality may help standardise practice across ni icus. reference(s we compared our data with those from retrospective audit [ ] . the method of th was via surface cooling technique together with cold intravenous saline infusion but not ivcd. total patients presented with cardiac arrest: underwent th ( ooh vf/vt and ooh non-vf/vt). in , there was an overall improvement in adherence to the audit standards, as shown in table : table : table hospital survival rate of th % gcs of the survivors at icu discharge / ( %) / ( %) / ( %) / ( %) conclusions. introduction of ivcd has led to an improved compliance with local and ilcor th guidelines. although the total numbers are small, there has been an increase in the patients discharged with gcs from our icu using ivcd. there are areas that require further improvement, notably the time to reach target temperature and prevention of rebound hyperthermia. work continues on protocolised evaluation of neurologically damaged survivors. rd esicm annual congress -barcelona, spain - - october s target temperature management after out-of-hospital car-diac arrest, an international, multi-centre, randomised, parallel groups, assessor blinded clinical trial-rationale and design of the ttm-trial n. nielsen , , and the ttm-trial study group helsingborg hospital, department of anesthesia and intensive care medicine, helsingborg, sweden, lund university, department of clinical sciences, section of anesthesia and intensive care medicine, lund, sweden introduction. experimental studies and previous clinical trials suggest an improvement in mortality and neurological function with induced hypothermia after out-of-hospital cardiac arrest (ohca). previous trials have included highly selected populations and the optimal target temperature is not known. objectives. to evaluate differences in efficacy and safety with target temperature management at and °c for h after ohca of presumed cardiac cause. methods. intervention: patients will be managed with h of temperature control at versus °c according to randomisation. temperature control will be delivered with temperature management equipment at the discretion of the trial sites. to facilitate cooling, when applicable, and to stabilise the circulation all patients will be treated with ml/kg of crystalloid infusion ( °c or room temperature according to treatment arm). design. randomised trial with : concealed allocation of ohca patients to temperature control for h at versus °c with blinded outcome assessment. sample size is based on a relative risk reduction of % with a risk of type- error of % and a power of % with a % loss to follow-up. conclusion. this study demonstrated that health care professionals, despite guidelines, are hyperventilating simulated cardiac arrests patients. suboptimal ventilation was a problem across all the backgrounds investigated; although doctors performed best here, they were still found to be hyperventilating to an unacceptable level. hyperventilation has a number of deleterious physiological effects and is associated with poor outcomes. increased training, awareness and recertification may be the answer, and certainly improves short term compliance with guidelines. however, these effects may be short lived and other changes may be needed. a reasonable course of action may be the use of paediatric ( l) self inflating reservoir bags as a first line device. this simple measure may ensure delivery of more guideline consistent ventilation, independent of the level of experience. extracorporeal life support (ecls) has been proposed as the ultimate heroic rescue measure in prolonged cardiac arrest unresponsive to conventional cardiopulmonary resuscitation. ecls effectiveness in out-of-hospital cardiac arrest remains to be addressed. decision to discontinue cpr due to medical futility is based upon presumed prolonged anoxia, with existing guidelines for termination. however, even when ecls is implemented, failure to maintain stable hemodynamic conditions due to marked capillary leak frequently results in patient's death. to evaluate the usefulness of routine laboratory parameters in the decision to treat refractory cardiac arrest patients with ecls . methods. sixty-six adults with witnessed cardiac arrest of cardiac origin unrelated to poisoning or hypothermia undergoing cardiopulmonary resuscitation without return of spontaneous circulation (duration: min [ - ], median, [ - %-percentiles]) were included in a prospective cohort-study. ecls was implanted under cardiac massage, using a centrifugal pump connected to a hollow-fiber membrane-oxygenator, aiming to maintain ecls flow c . l/min and mean arterial pressure c mmhg. introduction. due to the human lifespan increasing, people are living longer. cardiac arrest (ca) in old people could be seen as a natural end of life process and cardio-pulmonary resuscitation (cpr) in this setting as a disturbance. therefore, the question of prognosis in patients has been raised when performing cpr in the elderly. data from the s found month mortality in % of patients over suggesting that cpr in elderly people could be futile ( ) . the recent progresses in the management of resuscitated patients, such as mild hypothermia, were not evaluated in patients older than years ( , ) . in a recent study, we found that age [ years was an independent pejorative prognostic factor ( ) . hence there is virtually no data of prognosis factor of elderly patients after ca. our aim was to determine the prognosis factors in patients older than years successfully resuscitated. methods. all patients admitted to icu for ca with successful rosc were consecutively included between and . ca data were prospectively entered in a registry according to utstein recommendations. patients were managed following standardized procedures. good prognosis was defined as cpc or at icu discharge. factor associated with a good outcome were identified using multivariate analysis. results. among , patients admitted for ca, were older than years. median age was . years ( - ), ca was from cardiac origin in % of patients and . % had a vt/vf initial rhythm. mean no flow (nf) and low flow (lf) were . (± . ) and . min (± . ). mean blood lactate and creatinine level at admission were . mmol/l (± . ) and lmol/l (± ). . % of patients presented post-resuscitation shock (prs) and . % were treated with hypothermia. conclusions. ca in elderly patients is associated with an in-icu % good outcome rate. this should promote the cpr in non-severely disable elderly patients with ca regardless of their age. we plan to collect the -month mortality and the functional status of survivors. introduction. post-resuscitation phase is often characterized by a ''sepsis-like'' syndrome, which may be associated with the development of organ dysfunction. microcirculatory abnormalities play a key role in sepsis-related organ failure; however no data are available on microvascular function after cardiac arrest (ca). objectives. the aim of this study was to investigate peripheral microcirculation during and after therapeutic hypothermia (th) in ca patients. methods. this prospective, observational study included patients treated by th after ca. sublingual microcirculation was evaluated using sidestream dark-field (sdf, microscan, the netherlands) videomicroscopy at hypothermia and normothermia in all patients. at least images of s each from separate areas were recorded at each time point and stored under a random number to be analyzed, using a semi-quantitative method, by an investigator blinded to time and condition. thenar oxygen saturation (sto ) was measured using a tissue spectrometer (inspectra ; hutchinson, usa). a vaso-occlusive test was performed at hypothermia and normothermia by rapid inflation of a pneumatic cuff around the arm to evaluate sto reperfusion rate, reflecting microvascular reactivity. results. compared to hypothermia, measurements at normothermia showed a significant increase in functional capillary density (fcd) from . ± . to . ± . n/mm (p = . ), the proportion of small perfused vessels (ppv) from ± to ± % (p = . ) and mean flow index (mfi) from . ± . to . ± . (p = . ). fcd and ppv values were significantly correlated with body temperature. sto reperfusion rate was largely decreased when compared to healthy volunteers, but it did not change over the study period (from . ± . to . ± . %/sec) and showed large inter-individual variability. the same was found for sto (from ± to ± %). conclusions. mild hypothermia is associated with decreased fcd and ppv in the sublingual area when compared to normothermia. microvascular reactivity is decreased but changes are unpredictable. introduction. acute posthypoxic myoclonus (phm) occurs in deeply comatose patients, soon after a hypoxic episode. it is characterized by generalized, severe body jerks with violent flexor movements, but more focal myoclonus is reported too ( ) . acute phm and status myoclonus are considered to have a poor prognostic outcome ( , ) . although the cerebral cortex is known to be the most common origin of myoclonus in ambulant patients ( ) , the origin of acute phm is uncertain ( ) . to determine whether acute phm originates from damage in cortical or subcortical structures. for this study patients with myoclonus in the first h after admission were selected from the propac ii study, a prospective cohort study including patients admitted after cpr and treated with hypothermia. exclusion criteria: pre-existing disease with life expectancy\ months and severely disability before cpr. baseline characteristics were used from the main database. additional data of eeg and ssep recordings made after rewarming were collected. eegs were evaluated for presence of epileptic activity, status epilepticus, generalized periodic discharges, burst suppression pattern, iso-electric or low voltage amplitudes and reactivity of the background pattern. data collected from sseps: n potential, giant potential (defined as a potential five times the size of a normal potential) and p /n amplitudes (done by jhk). the glasgow outcome scale (gos) was used to assess outcome after months, poor outcome was defined as a gos of - (death, vegetative state, severe disability), good outcome as a gos of - (moderate disability, good recovery). . from a total of patients included in the propacii study, ( %) patients developed myoclonus. baseline characteristics of this group: age , % male, time to rosc min, primary cardiac arrest in patients, hypoxic arrest in . ssep recordings were available from patients. n potentials were present bilaterally in % ( ) and giant potentials were seen in % ( ) of the patients with a present n potential. eegs were made, epileptic activity was seen in % ( ) and a status epilepticus in % ( ), thus % of the eegs did not show any type of epileptic activity. good outcome was seen in % of the patients, poor outcome in %. mortality was %. conclusions. the results of this study show that acute phm is found in % of patients admitted after cpr and treated with hypothermia. it did not necessarily lead to a poor outcome, but we did not have information about the type of myoclonus. the available data seem to support the idea that the myoclonus originates mainly from subcortical structures, given the low number of patients with eegs showing epileptiform activity and sseps with giant potentials, which can be seen in cortical myoclonus ( ). introduction. the international liaison committee on resuscitation, the american heart association and the european resuscitation council recommend that mild therapeutic hypothermia improve neurological outcome in unconscious adult patients with return of spontaneous circulation (rosc) after out-of-hospital cardiac arrest (ohca) due to ventricular fibrillation (vf) or ventricular tachycardia (vt). in our intensive care unit (icu) we use mild hypothermia in all patients following cpr with successful rosc regardless of initial rhythm. in this study we compared the effect of mild therapeutic hypothermia at neurological outcome and mortality between the patients who had ohca due to vf or vt and them who had ohca due to a different initial cardiac rhythm as asystole or pulseless electrical activity. the study protocol was approved by the local ethics committee on human research. a total of patients were admitted to our icu with rosc after ohca between may and december . therapeutic hypothermia was initiated after admission in icu by intravenous infusion of cold saline ( °c , ml bolus) followed by intravenous cooling device (coolline catheter, coolgard alsius corporation irvine, ca, usa). the target temperature was °c maintained for h followed by slow active re-warming over a minimum period of h ( . °c per hour). intravenous anesthesia was induced in all patients by a combination of propofol and remifentanyl with dose adjustment as needed. to prevent shivering, patients received muscle relaxation by iv administration of sisatracurium every h. the primary end point was the neurological outcome at months according to the pittsburgh cerebral performance category (cpc). secondary end point was mortality at months. prehostital cooling procedures were not applied. nine of the patients ( %) of the group of the patients who had ohca due to vf or vt had favourable neurological outcome cpc or as compared with of ( %) of the group of the patients who had ohca due to a different initial cardiac rhythm. mortality at months was % ( of patients died) in the group of the patients who had ohca due to vf or vt as compared with % ( of patients died) in the group of the patients who had ohca due to a different initial cardiac rhythm. in patients who had ohca due to vf or vt mild therapeutic hypothermia inproves the neurological outcome and reduces mortality as compared with the patients who had ohca due to a different initial cardiac rhythm. objectives. we aimed to compare therapeutic hypothermia using either surface or endovascular techniques in terms of efficacy, complications and outcome at our institution, a bedded tertiary referral icu. a local research ethics committee reviewed the proposed study and waived the need for a full ethics submission, as the study met the national criteria for service evaluation. data were collected from patients undergoing therapeutic hypothermia following cardiac arrest over a . year period by retrospective casenote review and interrrogation of the carevue (phillips uk) database. therapeutic hypothermia was initiated in the icu using iced hartmann's solution, followed by either surface (n = ) or endovascular (n = ) cooling; choice of technique was based upon endovascular device availability. the target temperature was - °c for to h, followed by rewarming at a rate of . deg h - . the mean age was ± years; % of arrests occurred out of hospital, and % were ventricular fibrillation/tachycardia. endovascular cooling provided a longer time within the target temperature range (p = . ), less temperature fluctuation (p = . ), better control during rewarming ( . ), and a lower -h temperature load (p = . ). endovascular cooling also produced less cooling-associated complications in terms of both overcooling (p = . ) and failure to reach the target temperature (p = . ). after adjustment for known confounders, there were no differences in outcome between the groups in terms of icu or hospital mortality, ventilator free days and neurological outcome. conclusions. endovascular cooling provides better temperature management than surface cooling, as well as a more favorable complication profile. the equivalence in outcome suggested by this small study requires confirmation in a randomized trial. introduction. ventricular assist devices (vads) are successfully used in patients with end stage heart failure, usually as a bridge to transplantation or recovery, but increasingly as destination therapy as well. a major threat for patients with a vad is the frequent occurrence of, mainly thromboembolic, stroke, with a reported incidence of up to %. manufacture guidelines for anticoagulation therapy are based on relatively small observational studies and common sense rather than evidence, and as a consequence anticoagulation protocols vary widely between centers. objectives. the aim of this systematic review was to provide more evidence in order to determine the optimal anticoagulation protocol to prevent stroke in patients supported with a vad. a systematic search in pubmed and embase was performed in which we included all types of vads. all types of anticoagulation drugs applied to prevent thromboembolism were included and divided in three categories; heparin, coumarins and antiplatelets. we included references with a full text available, written in english, dutch, german or french, and which described patients with a stroke or tia. our primary outcome measure was defined as the onset of any type of stroke. two authors evaluated independently the results of this search; doubtful references were evaluated by two other authors. after critical appraisal articles were selected as relevant, which include cohort studies, case-control studies and trials, totaling patients with vad support between and . the mean age was years (range - ) and the mean duration of support was days ( - ). stroke occurred in - % patients supported by vad, with an incidence of - /patient-year. the majority of strokes occurred within the first year. six types of anticoagulation protocols were used that combines drugs from one or all three categories. most protocols used a combination of all categories ( , patients with a total follow up of , days) and had an average stroke incidence of . events/patient-years. the lowest average stroke incidence was reported in studies that used only antiplatelets ( . events/patient-years) and the highest in which only heparin was used ( events/patientyears). we could not detect a decreased risk for stroke in patients with vad support when coumarines or heparin were used instead of or in addition to antiplatelets. antiplatelets should be part of an anticoagulation protocol to prevent stroke in patients supported by vad. a.m. de la torre , c. marco , d.j. palacios , a. pedrosa , i. lopez de toro , v.a. hortigüela hospital virgen de la salud, toledo, spain objectives. to analyze the difference between two groups of patients with ich considering severity, treatment, evolution and mortality. methods. description retrospective study of admitted patients between st of january of to st of december and st of january of to st of december in the icu of virgen de la salud hospital (toledo) with the diagnosis of ich. there are patients in the group of - and patients in the groups of - without any difference of age. regarding comorbidity between the two groups no differences can be found regarding the previous presence of hta, but regarding diabetes and dislipemy, we do find a higher prevalence on the second group ( . vs. . %, p \ . and . vs. . %, p \ . respectively). reviewing the presence of anticoagulated patients, no differences of significance can be found, but a trend ( . vs. . %, p \ . ). regarding the location of the hemorrhage, the most frequent is the basal ganglia ( . vs. %), existing no differences amongst the two groups. we don't find differences either in the presence of a intraventricular component, whether it is neither supratentorial nor infratentorial. there are differences between the ich score of both groups with p \ . : with a score of ( . vs. conclusions. an increase in the comorbidity can be observed in the included patients, which can be due to a better screening of these pathologies. we also find that the ich score is higher than in the - sample, which is attributed to the admittance in the icu of patients with the ultimate goal of organ donations. in the evolution, it can only be observed a longer stay in patients from the second sample, likely because they are more serious patients with a higher ich score. the analysis of the two groups has not been conclusive when it comes to assessing the improvements that might have come out in these last years, although it is necessary a deeper analysis of the data. introduction. sodium dysbalances are frequent medical complications in patients with subarachnoid hemorrhage (sah). hyponatremia is more frequent but it is associated with better outcome than hypernatremia. the aim of this study was to observe differences in outcome between hyponatremic and hypernatremic patients with sah. we performed the prospective years study in incidence of hyponatremia (serum sodium \ mmol/l) and hypernatremia (serum sodium [ mmol/l) in patients (pts) with sah. we compared the incidence of cerebral complications, glasgow outcome scale (gos) upon discharge from the neurointensive care unit (nicu) and in mortality nicu. results. there were ( %) pts with dysnatremia, more patients had hyponatremia ( , %), less hypernatremia ( , %). between these groups there were no diferences in stay in nicu (p = . ), duration of dysnatremia (p = . ), fluid intake (p = . , ml/day), daily sodium intake (p = . , mmol/day) and fluid output (p = . , ml/day). hyponatremia was more frequent on admission (p \ . ) and connected with higher diuresis (p \ . ). hypernatremic pts received more antiedematic therapy (p \ . ). hypernatremia was arised in pts with significantly lower glasgow coma scale (p = . ). these pts had more cerebral complications (p = . ), worse glasgow outcome scale upon discharge from nicu (p \ . ) and higher mortality in the nicu (p = . ). conclusion. dysnatremia is frequent in patients with sah ( %). hyponatremia occurs more often, but hypernatremia is connected with worse outcome. objectives. to analyze clinical, epidemiologic and outcome differences and to identify predictor factors of mortality at discharge from icu of patients admitted after craneoencephalic trauma (cet) according to glasgow coma scale (gcs) score. observational prospective study of patients admitted in the intensive care unit (icu) after cet. we classified the traumatic brain injury, according to gcs score of b or c points groups. we analyzed clinical and demographic data during icu stay, as well as physiological, functional and emotional data measured with paecc scale (project for the epidemiological analysis of critical patients) at discharge from icu. qualitative variables are expressed as a percentage and quantitative variables as mean and standard deviation. we used chi test, t-student and multivariate analysis as required with a maximum alpha error of %. we analyzed patients, . % male. at admission . % had gcs b . traffic accidents ( . % in gcs c and . % in gcs b ) were the most frequent cause of cet. there was a higher rate of out hospital hypotension in the gcs b group (p = . , or . , % ci . - . ). cranial computed tomography (ct) scan findings in the gcs c group were diffuse injury i and ii ( . %) after marshall classification versus . % in the gcs b group (p = . ). fewer complications were detected in patients in the gcs c vs. gcs b group ( . vs. . %, p = . , or . , % ci . - . ). icu mortality rate was significantly lower in the gcs c group than in the gcs b group ( . vs. . , p \ . ). predictors of mortality were gcs at admission (p = . ), ct findings type iii, iv and v (p = . , . and . respectively), complications (p = . ), tracheotomy (p = . ), days on ventilator (p = . ), apache ii (p = . ) and the length of icu stay (p = . ). best overall score in paecc questionnaire at discharge from icu was better in the gcs c vs. gcs b group. ( . ± . vs. . ± . , p = . ). conclusions. patients with lower gcs at admission presented higher rate of prehospital hypotension, more severe ct findings, more complications at icu, worse physiologic, functional and emotional outcome, and higher rate of mortality, than patients with gcs c . factors associated with increased mortality were coma level, type of findings in ct, complications, prolonged mechanical ventilation, length of icu stay, apache ii, and the need for a tracheotomy. objectives. the aim of our study was to estimate the influence of hyperhaes infusion on haemodynamic regulation. we examined patients with severe brain trauma (gcs score \ , sedation, artificial ventilation). the bp, stroke volume (sv) and their variability (bpv, svv) were determined by the bioimpedance method. additionally we determined the peripheral pulse (pp) and its variability (ppv) using plethysmography curve registration. special attention were paid to the p ( . - . hz) and p ( . - . hz) bands of bpv, svv and ppv, connected with volume state, breathing, and autonomic regulation, especially baroregulation. all the comparisons were made before and after min. of the infusion of hyperhaes ( ml). all patients had the autonomic dysfunction: the waves of baroregulation (p ) had low power compared with healthy. hypovolemia was moderate: sv ( ± . ) and pp ( ± . ) were slightly decreased, but bp were ± . mmhg. the bp increasing was registered as the first reaction on the infusion of hyperhaes ( ± . mmhg). the same time pp increased more then twice and became ± . , and p of ppv decreased from ± . to ± . , that reflected the improving of the volume state. although sv did not rise significantly, the increasing of p were estimated in svv, as a marker of the baroregulation restoration. conclusions. the infusion of hyperhaes in severe brain trauma not only decrease the range of hypovolemia, but restore the baroregulation as a significant part of autonomic regulation, needed for cerebral perfusion support. introduction. continuous measurement of intracranial pressure (icp) using intraparenchymal sensors has become part of the standard management of patients at risk of developing intracranial hypertension. whilst reliability has been explored previously little is published on the accuracy of depth to which the sensor is placed. a difference in sensor location has been shown to affect the reliability of icp readings and could impact negatively on patient management ( ). to determine whether icp sensors (codman, j&j) placed by neurosurgical staff were within the optimum depth of cm from the cortex. methods. consecutive patients were identified from a prospectively collected neuro icu database who had had a ct of the head (cth) performed whilst an icp sensor was in situ during . the depth of the sensor tip on cth was measured and patients were stratified according to whether they had surgery or no surgery to determine any differences between open and percutaneous placement. of the icp sensors ( %) were placed deeper than cm. the greatest incidence of deep placement was in surgical patients / ( %) and in craniotomy patients % of these were deeper than cm conclusions. this investigation has shown that the majority of patients admitted to our neuroicu have less than optimally placed icp sensors. inappropriately deep sensors appear more common in surgical patients, particularly following craniotomy. the impact of this on patient outcome is unknown and would require further study. this study has highlighted we need to implement new methods to improve the accuracy of our icp sensor placement. graduated marks on the sensor sheath during manufacture may assist accurate placement. conclusions. if icp is largely used, it remains below %. % of the cases have been monitored with at least techniques suggesting an acceptance tendency for a multimodal monitoring. among these techniques, the control of pco seemed considered important as the use of tcd to assess perfusion and vessel tone. on-going analysis of these data will provide information on the therapeutic strategy performed and the impact on outcome. introduction. raised intracranial pressure (ricp) can be evaluated sonographically by measuring the optic nerve sheath diameter (onsd). a wide variation in the threshold measurement of onsd for ricp has been reported in literature. it is likely that exaggeration of the hypoechoic edge artifact around the dura by high frequency ([ mhz) linear transducers and uncertainty over whether to measure -mm behind the papilla or behind the globe (posterior margin of sclera) could have resulted in such differences. to determine the optimal site of measurement of onsd by correlating it with ricp determined clinico-radiologically. we also evaluated if different sonographic appearances of the onsd could be used qualitatively to determine the presence or absence of ricp. methods. initially, in order to precisely delineate the anatomical dura sonographically and assess optimal cursor placement, cadaver orbital preparations were studied before and after subarachnoid fluid insufflation. scans were then done by a single sonographer using a - mhz linear transducer on healthy volunteers and patients admitted to the medical/ neurosurgical intensive care units. onsd was measured at locations; -mm behind the papillae and -mm behind the globe. in each location measurement was made within the anatomical dura and another between the echogenic margins of retrobulbar fat as described in literature. four patterns (fig. ) of the nerve sheath were identified based on the appearance of the csf space and edge artifact. ricp was diagnosed by clinically correlated computed tomography of the brain. an independent sample t test was done to correlate measurements with ricp. classification of optic nerve sheath appearances results. / ( %) scans were done on patients with ricp. all measurements independently correlated with ricp (p \ . ); albeit cut-offs differed substantially ( table ) . the presence of a type pattern (fig.) in both eyes strongly suggested ricp ( % positive predictive value) whilst its absence in both eyes ruled out ricp ( % negative predictive value). methods. three years after the introduction of the concept of bs in our icu, a questionnaire was sent to people directly involved in the nursing of critically ill patients to study the current practice during the previous month. responses were received from persons, a % response rate. ( %) persons had practiced bs. only ( %) employees used calming bath and ( %) an invigorating bath. guided oral care and guided suction was performed respectively by ( %) and ( %) nursing persons. orientative positioning was used by ( %) nurses. everybody is very satisfied with the instruction manual which can be found in each room. ( %) persons estimated to be well informed about bs. others wanted more practice course and training at the bedside. conclusion. pattern suggested an increasing interest in bs since its introduction. initial touch is well implemented. more effort is needed for continuous education and training. introduction. admittance to hospital for surgical treatment, is often linked with insecurity and anxiety for many patients. to most patients, the postoperative care unit constitutes an unknown environment, and can represent a frightening experience. research has shown that preoperative information leads to subjective outcome as anxiety reduction, and objective outcome as shorter hospital stay and less intake of pain medication. few studies, however, have addressed patients' experiences with preoperative information about the early postoperative phase. objectives. the purpose of this study was to describe patients' experiences with preoperative information about events they may experience during their stay in the postoperative unit. patients' experiences may contribute to increased knowledge about this topic. the study design was exploratory-descriptive, and a semi-structured interview based on thematic guide was used. nine patients met the inclusion criteria, and they had an average age of years. they were admitted to elective surgery for cancer and their stay in the postoperative unit varied from to . days. the interviews were conducted - days after surgery and transcribed verbatim. the data material was subjected to qualitative content analysis. results. experience with information before surgery and in the early postoperative phase, was categorized into four themes: being prepared before surgery, reactions to differing experience, discomfort and pain, management of some self-care activities and experiences with the environment of the postoperative unit. conclusions. the patients received a fair amount of information before surgery, but only limited information concerning what to expect while in the postoperative unit. the patients' information needs differed and patients with former experience with surgery were more prepared for what to expect. the patients got mainly verbal information and most of this was given the day before surgery. o.m. peters-polman , m. van roosmalen , j.e. tulleken , j.g. zijlstra umc groningen, intensive care, groningen, netherlands, arup nederland, amsterdam, netherlands, umc groningen, groningen, netherlands background. who guidelines concerning sound levels in hospitals requires a maximum of db in daytime, a nighttime sound level below db to allow good sleep quality and peak levels that do not exceed db at all times. in an icu environment, apart from being critically ill, patients are exposed to typical icu environmental noise. sleeping cycles disturbed by illness are further disrupted by care providers performing procedures and taking vital signs and alarms with delirium as a common result. we describe the acoustic environment in our mixed icu. we conducted an observational study in which we continuously measured the sound level (db), frequency and repetition of sound in our icu. we used a splnet microphone and noise monitoring system which was placed, after informed consent, at the ear level of the patient. results. noise sources are numerous and consist of human voices, doors slamming, pagers, telephones, shoes and equipment alarms. there is a round the clock continuous background noise of - db, staff conversation with levels of - db and peak levels up to - db, mainly due to alarms. these levels of sound are comparable to loud conversation at meter distance ( - db), walking along a motorway ( - db) or standing next to a roaring engine ( db). conclusion. who guidelines clearly state maximum sound levels of db in daytime, db at night and peak levels of db. the sound level in our unit is exceptionally and unacceptably high throughout day and night and requires a behavioral intervention. further research on the influence of these noise levels on our patients is necessary. objectives. this study has been achieved in order to realize the comparison of efficiency of manual and mechanical compression techniques used for the maintenance of haemostasis after femoral sheath removal. methods. this study was planned and applied as a randomized controlled trial. the study was executed at a military education and research hospital in turkey between january and march . data collecting form was prepared by the investigators after the literature examination. the form consists of questions which are evaluating the demographic data of patients, the compression time, the pain level (before sheath removal, during the manual/ mechanical compression and after the sheath removal), the complications occurring in femoral zone, the mobilization/discharge period and the problems (bruise, oedema, hemorrhage and etc.) occurring the th day after the discharge. the patients have been called up in order to evaluate the problems occurring on the th day. the persons that were applied mechanical compression have constituted the experimental group and the patients that were applied manual compression have formed the control group. the patients volunteer to participate to the study have been informed in regard with the implementing procedures before the application. descriptive statistics were shown in numbers and percentages for the variables obtained by counting and in mean ± standard deviation for variables obtained by measurement. results. the average of age is . ± . in the experimental group and is . ± . in the control group. the average of compression time is . ± . min in the experimental group and is . ± . min in the control group (p \ . ). the average of mobilization time after sheath removal is . ± . min in the experimental group and is . ± . min in the control group (p \ . ). it has been observed a lower pain level during compression and after sheath removal in patient that were applied mechanical compression in comparison to the patients that were applied manual compression(p \ . ). when the groups were compared in terms of femoral zone complications while no haematoma was observed in the experimental group, haematoma has been occurred in the . % (n = ) of the control group. conclusions. the mechanical compression provides an earlier mobilization and earlier discharge of the patient. this study shows that mechanical compression is a method as safety as the manual method in order to obtain a haemostasis a correlational survey was conducted in public hospitals located in athens. critical care nurses completed anonymous questionnaires , , yielding a response rate of %. greek critical care nurses believe that open visiting increases family's satisfaction ( . %), exhausts family members ( . %) and provides emotional support to the patient ( . %); nevertheless the effects of visiting depend both on patient and family ( . %). furthermore open visiting hampers the planning of adequate nursing care ( . %) and is not a helpful support for the caregivers ( . %) while increases their physical and psychological burden ( . %). critical care nurses' attitudes toward visiting hours were rather negative and they didn't want to liberalize the visiting policy of their unit ( . %). there was a positive correlation between nurses' beliefs and attitudes regarding visiting (r = . , p \ . , r = . , p \ . ). the factors ''working experience'', ''adequacy in staff'' and ''the number of shifts'' were found to be independently correlated and they predicted the score of the scales of the questionnaire. greek icu nurses have rather negative beliefs and attitudes toward visiting and open visiting policy. this will be a challenging barrier to overcome when imposing new flexible policies in icus . objectives. our aim was to elucidate potential mechanisms for the beneficial effects of rhapc on ali, as assessed by microarray analysis of lung tissue after sepsis induced by clp in rats. methods. sepsis (n = ) was induced in rats by clp. a sham-operated group (n = ) underwent laparotomy and closure without clp. a clp group (n = ) received subcutaneous saline ( ml/kg) and a clp + apc group (n = ) additionally received mg/kg rhapc. twelve hours postoperatively, lung tissue was preserved in mrna later until mrna isolation by promega total rna kit and analyzed using illumina beadarray. data were log variance stabilized and quantile normalized using the lumi package in r and the limma package was used for group comparisons and false discovery rate correction. data were further analyzed using panther and david. the clinical outcomes of this study showed a marked attenuation of the sepsisinduced increase in lung permeability in rats treated with rhapc . although no formal statistical significance was reached for the gene expression changes between the clp and the clp + apc groups, there was a clear attenuating effect of rhapc on the changes in gene expression caused by sepsis reflected in generally lower fold change values and fewer significantly differentially regulated genes in the clp + apc versus sham group compared to the non-treated clp vs. sham group ( vs. genes). nevertheless, there were only genes of which the fold change difference between clp versus sham and clp + apc versus sham was more than one, indicating that although there was a large difference in the number of differentially expressed genes, the difference in fold change between these genes was small. conclusions. these data suggest that the rhapc treatment of septicemic rats does not only cause a down regulation of specific pathways as argued by previous investigators, but leads to a global reduction in the inflammatory response at a mrna level. introduction. septic shock guidelines recommends the use of recombinant human activated c protein (acp) in high risk mortality patients. the aim of our study is to describe the clinical characteristics, and the outcome of patients treated with acp in our hospital. methods. retrospective and descriptive study which includes patients with severe sepsis/ septic shock treated with pca in a tertiary hospital intensive care unit (icu) over years ( - ) . we analyze epidemiological data, reason for admission, infectious focus and agent, severity scores, organ failure, complications, stay and mortality. we used chi-square analysis to compare categorical data and student's t test to compare continuous variables. conclusions. in our study most patients were admitted from emergency department, with organic failure caused by pneumonia. we haven't detected deaths related with acp complications, even in patients undergoing surgery. we found as prognostic factors for mortality: organ dysfunction, acp indication, renal failure, pao /fio relation, amount of vasopressors, bicarbonate and base deficit levels, apacheii and icu stay. objectives. to analyze changes on hemodynamics in patients with severe sepsis treated with high volume hemofiltration ( ml/kg/h) vs. patients treated with very high volume hemofiltration ([ ml/kg/h). we conducted a prospective randomized trial from january to november in patients admitted into icu with a diagnosis of septic shock in which hf was indicated. patients were randomized to one of each group of therapy. the control group received high volume hemofiltration therapy and the experimental group received very high volume hemofiltration. the hemodynamic parameters were measured at the admittance in icu and every h onwards. results. data of patients were collected ( men and women) mean age ± years old. the hemodynamic parameters registered at the admittance and during the therapy had no significant difference between the groups. the control group received hf therapy during . ± . days and the intervention group . ± . days. there wasn't any difference either in the administration of vasoactive drugs between both groups. the most significant difference between the groups was the -day survival rate, . % of the experimental group against . % of the control group (p = . ). from these results we can conclude that very high volume hemofiltration therapy should be the therapy of election because it improves the survival of patients with severe sepsis without impairing the hemodynamic parameters. we have to point out the importance of the nursing staff in the assembly and management of the equipment as well as in the patient care and thus avoiding potential complications. introduction. the use of herbal products is increasing, and may result in increased drug-herb interactions or form a potential for adverse reactions in cardiovascular surgery patients. objectives. the aim of this study was to describe the utilization patterns for herbal products in patients with cardiovascular disease. methods. this was a descriptive study which was carried out among adult patients presenting to cardiovascular surgery department for elective cardiac surgery between september and april in a research and training hospital. after giving informed consent, patients were interviewed by researchers using a structured survey instrument in the preoperative period. results. interviews were conducted with patients (mean age . ± . , range: - years), % of them were married, % were men, and % of the patients had high school or university education. the majority ( %) had coronary artery disease and most of the patients ( %) had concomitant diabetes mellitus and hypertension. the most common used drugs were anti-hypertensives, nsai's, and anti-aggregants. most patients ( %) reported the regular use of drugs. eighty-nine ( %) among the surveyed patients reported the use of herbal products. the most common used herbal products were garlic ( %), apple vinegar ( %), lavandula stoechas ( %), cratageus ( %) and ginger-honey mixture ( %). the average educational degree of herbal product users was found to be higher when compared with the others. many patients report being informed about those products from television, internet, newspapers, herbal-stores personal communications, and report the use of those products after the diagnosis is made. none of those patients have informed physicians or nursing staff about the use of those herbs. the demographic variables of patients and the herbal-product usage has failed to show a statistically significant difference (p [ . ). conclusions. the use of herbal products is common among the patients with cardiovascular diseases. health professionals should be aware of the usage of those products in order to prevent possible adverse reactions and drug-herb interactions. introduction. nurses employed in the icu operate in a complex environment, under time pressure, and with limited information available. thus, errors are inevitable and, besides their adverse consequences, they offer the potential for learning . in this concept, properly copying with errors is a prerequisite for avoiding their recurrence and improving nursing practice. objectives. our aim was to investigate how error-copying strategies are associated with constructive and defensive changes in icu nursing practice. methods. questionnaires were completed (a % response rate) from nurses employed in the icus of adults, children, and the coronary care unit of two greek hospitals, between january-june . ''ways of copying'' scale as revised by wu et al. was used for evaluating copying strategies. this includes copying subscales, each ranging between and . constructive changes in response to error included items (paying more attention to detail, keeping better patient records, reading patient notes more carefully, seeking advice, discussing with colleagues about similar situations, devoting more observation on patients, reading for covering knowledge deficiencies), while defensive changes included items (getting more worried, feeling less confident at work, being more likely not to discuss errors, being less trusting of others, thinking to leave profession.). a four-point likert scale ( - ) was used for evaluating each item, and points were summed to estimate total scores of constructive and defensive changes. . . % of participants were female and . % were registered nurses. mean (±se) age was . ± . years and mean icu experience was . ± . years. multiple linear regressions with constructive and defensive changes in practice as dependent variables and copying strategies as independent variables are summarized in table . participants were more likely to make constructive changes if they coped by seeking social support (p = . ) and accepting responsibility (p = . ). at the same time, they were more likely to make defensive changes if they coped by escape-avoidance (p = . ). reasons for high risks are e.g. sedation and analgesia, immobility, malnutrition and hemodynamic or oxygenation problems as well as poor identification or unsystematic assessment of the risks. in our unit, we performed a months retrospective review of pressure ulcer risks for all patients (n = ) using jackson-cubbin calculator. the risk point level in this instrument is and below. patients of ( %) exceeded the risk limit already in the icu admission phase. objectives. to change care practices of pressure ulcer prevention and care with action research. with the measurement tool nurses are able to identify patients at risk of developing pressure ulcers during the whole icu in-patient time, and to prevent risks of pressure ulcer in an early stage. with the assessment tool, the measurement is systematic which enables the benchmarking and have effects on material recourse planning and cost caused by pressure ulcers. the study unit is a -bed icu for adults taking care annually circa , patients with multiple disorders. firstly, the jackson-cubbin pressure area risk calculator was translated into finnish. a few changes which have an effect on pressure ulcer risk were added; weight limits were also defined with bmi values, hyperbaric oxygen therapy was added in deducted points as well as h limit for blood transfusions and limits for hypothermia from celsius or under. secondly, standardized guidelines for different risk levels were developed; if patients have a high risk specialized mattresses should be used and changes of positions and beds should be stressed. thirdly, an electronic evolution form for patient information system was planned and implemented. finally, a systematic education program for icu personnel including special lectures, material demonstrations and familiarization by the nurse responsible for wound care was started. results. after the development project and systematic education the knowledge of personnel about pressure ulcer risks and care has increased. risk points are counted once a day for every patient using the electronic form. all the mattresses at the icu have been changed to medium and high risk mattresses and are chosen for a patient related to the risk assessment points. conclusions. a reliable assessment scale, systematic measurement and continuous evaluation and education are crucial for identification of high risk pressure ulcer patient at the icus. with a systematic measurement and recognizing high risk patients we can also improve patients' quality of life and reduce the cost caused by pressure ulcers. reference(s ) is considered to be one of the main agents in gram negative sepsis. in recent years several adsorption dispositives have been designed in order to achieve low blood endotoxin levels with a theorical clinical improvement. toramyxin (polymixine b fixed to polyesthirene fibers) and alteco lps adsorber (polyethylene discs) are two of these dispositives. both are used with h sessions for several days until patient clinical improvement. • design a nursery prothocol with the most important procedures in gram negative septic shock patients with acute renal failure that undergo adsorption cartridge therapy. • evaluate initial experience in the use of endotoxin adsorption cartridges. methods. nursery prothocol with special attention to the settlement and management of the cartridge therapy, based on library references and practice guidelines. once prothocol was stablised, prospective observational study was started from january till january . inclusion criteria were: patients admitted to our intensive care department with gram negative confirmed septic shock and acute renal dysfunction requiring continuous renal replacement therapies (crrt). adsorptive cartridge were added and several parameters were studied: heart rate, mean blood pressure, vasopressor support (norepinephrine), pao / fio , and lactate. the information was analyzed in excel. results. nursery protocol was correctly applied in all patients and showed to be basic in the maintenance and early complication detection. hemodynamic improvement that allowed norepinephrine lowering dose and normalized lactate levels with no changes in pao /fio . patient (toraymyxin): years man, acute pancreatitis with septic shock. patient (toraymyxin): years man, pneumococic pneumonia and klebsiella septic shock. patient (alteco): years man, acute pancreatitis with enterobacter cloacae septic shock. patient (alteco): years woman, acute peritonitis with e. coli septic shock. results of a program to reduce catheter-related blood-stream infection in the icu: two years' follow-up a systematic review comparing the relative effectiveness of antimicrobial-coated catheters in intensive care units benefits of minocycline and rifampicin-impregnated central venous catheters which antimicrobial impregnated central venous catheter should we use? modeling the costs and outcomes of antimicrobial catheter use when is hit really hit? anticoagulative management of patients requiring left ventricular assist device implantation and suffering from heparin-induced thrombocytopenia type ii -hit happens: diagnosis and evaluating the patient with heparininduced thrombocytopenia informe del registro mami - grupo de trabajo de la sociedad europea de cardiología (esc) sobre marcapasos y terapia de resincronización cardíaca moreno millán e. variación de la estancia preoperatoria en españa según grupos de edad, sexo y modo de acceso hospitalario reference(s). . association of anaesthetists of great britain & ireland. safety guideline on interhospital transfer intensive care society: guidelines for the transport of the critically ill adult competency-based st st training and assessment. a manual for trainees and trainers. royal college of anaesthetists training and assessment of competency of trainees in the transfer of critically ill patients adverse events experienced while transferring the critically ill patient from ed to the icu a modified mccabe score for stratification of patients after intensive care unit discharge: the sabadell score surviving intensive care. edicion taurine and niacin block lung injury and fibrosis by down-regulating bleomycin-induced activation of transcription nuclear factor-kappab in mice antioxidants and sepsis: can we find the ideal approach? post-cardiac arrest syndrome: epidemiology, pathophysiology, treatment, and prognostication. a scientific statement from the international liaison committee on resuscitation therapeutic hypothermia after cardiac arrest: unintentional overcooling is common using ice packs and conventional cooling blankets it's well known that normobaric hyperoxia (nh) increases arterial and brain oxygen tension. however the influence of nh on intracranial pressure (icp) and cerebral metabolism in patients to investigate the dynamics of icp, jugular bulb saturation (svjo ), brain oxygen tension (pbro ) and cerebral metabolism during nh in patients with ich icp monitoring and tissue microdialysis were used in all patients, svjo monitoring -in , pbro -in . microdialysis and pbro catheters were placed in lesioned (les) and intact (int) brain tissue. icp, svjo , pbro , glucose and glycerol levels, lactate/pyruvate ratio dynamics during fio ) to ± mmhg (ð \ . ) (fio . ) and ± mmhg (ð \ . ) (fio . ) cerebral metabolism didn t change significantly, except glycerol level increase in lesioned brain tissue from ( ; ) mlmol/l (fio . ) to ( ; ) mlmol/l (fio . ) and ( ; ) mlmol/l (fio . ). fio . : glucose (int) - . ( . ; . ) mmol/l, glucose (les) - . ( . ; . ) mmol/l, lactat/piruvat (int) - . ( . icp was stable during investigation ( . ± . mmhg nh is accompanied by pao and pbro increase and doesn't influence icp and cerebral metabolism in ich patients with normal vo /do relationships effects of anesthetics oc cerebral blood flow and cerebral metabolic rate isoflurane preconditioning improves long-term neurologic outcome after hypoxic-ischemic brain injury in neonatal rats this no-profit trial has been supported by depuy/ hemedex, providing the probes. no other economical support has been received effect of equiosmolar solutions of mannitol versus hypertonic saline on intraoperative brain relaxation and electrolyte balance a comparison of % hypertonic saline and mannitol for brain relaxation during elective supratentorial brain tumor surgery hypertonic resuscitation and blood coagulation: in vitro comparison of several hypertonic solutions for their action on platelets and plasma coagulation effect of the combination of mannitol and ringer acetate or hydroxyethyl starch on whole blood coagulation in vitro no well defined threshold for transfusion or target hemoglobin (hb) level for these patients exists. objectives. to examine associations of anemia and transfusion with adverse outcomes in patients with sah, and better define hb level thresholds associated with these. methods. retrospective, observational study of consecutive patients with sah admitted to icus at mayo clinic in jacksonville and rochester, usa, over -year period. data included demographics, nadir hb, blood transfusion, ali/ards, vasospasm, radiographically confirmed cerebral infarction, apache and wfns. primary outcome was association of anemia and transfusion with death and secondary outcomes were vasospasm, cerebral infarction and ali/ards. results. we identified patients, mean age was (± ), ( %) were females. mortality was %. seventy-two ( %) of patients were transfused. ( %) patients had vasospasm and ( %) cerebral infarction. there was a strong association between transfusion and increased mortality (p = . ), vasospasm (p = . ), cerebral infarction (p \ . ) and ali/ards (p \ . ) outcome prediction in mild traumatic brain injury: age and clinical variables are stronger predictors than ct abnormalities comparison of simultaneous continuous intracranial pressure (icp) signals from a codman and a camino icp sensor therapeutic actions may correct abnormal values and perform potentially cerebral blood flow/oxygen extraction coupling. objectives. aim of this study was to describe the incidence and the type of bedside icu monitoring devices used in the management of patients with severe tbi in french icu's. methods. multicentric observational study including patients having severe tbi (glasgow coma scale (gcs) \ ) picked on scene by a mobile medical unit for pre-hospital care (samu). inclusion period: months general hospital of athens, department of intensive care unit o poder na relação enfermeiro-utente relacionamento enfermeiro, paciente e família: factores comportamentais associados à qualidade da assistência investigação qualitativa em enfermagem: avançando o imperativo humanista the experiences of families of critically ill patients in greece: a social constructionist grounded theory study. intensive and critical care nursing visiting hours policies in new england intensive care units: strategies for improvement this study was supported by a post graduate programme in nursing surviving the nursing shortage: developing a nursing orientation program to prepare and retain intensive care unit nurses clinical utility of apc-pci (activated protein c-protein c inhibitor) complex as predictors for severity and prognosis in sepsis: preliminary study republic of korea introduction. recently human recombinant activated protein c (drotrecogin alfa [activated]) has been shown to reduce mortality in severe sepsis. in severe sepsis, conversion of protein c to apc (activated protein c) is impaired due to endothelial dysfunction. apc level is related to coagulation cascade known to be important in sepsis pathophysiology. objectives. in this preliminary study, we checked apc-pci (activated protein c-protein c inhibitor) complex level to evaluate protein c activation using apc-pci elisa kit they were admitted asan medical center (seoul, korea) medical intensive care unit (icu) between sep and . , respectively. apc-pci level had no statistically difference among sepsis, severe sepsis, septic shock ( . , . , . ng/ml, p = . ). between hospital survivor group and non-survivor group there was also no difference in apc-pci level ( . , . ng/ml but apc-pci level was tended to decrease in severe sepsis, septic shock group as compared to sepsis group activated protein c versus protein c in severe sepsis endogenous protein c activation in patients with severe sepsis efficacy and safety of recombinant human activated protein c for severe sepsis septic critical patients to evaluate the experience with drotrecogine alfa (da) in septic critical patients (scp) in a analysis of scp admitted in an icu unit between december and which received treatment with da. patients were included or excluded on the approved specifications fda and emea. inclusion criteria were septic patients and apache ii score c and/or two or more organ dysfunction (od) thirty-eight patients were eligible od: respiratory %, renal %, cardiovascular %, metabolic %, hematologic %; sdra . , vmc . , vni, . , all patients received empirical antibiotic treatment in the first h according to epidemiologic characteristics. microbiology: some germens were isolated in patients ( %) mortality (%) by presumed site of infection day /day : respiratory / , abdominal / , urologic / ; skin / . average hours drug administration was h. da was administrated at % of the patients between hour and hour , the other % between hour y critical septic patient treated early with empirical antibiotics, the best standard care and da have a low mortality rate. more early da administration was associated to lower mortality the treatment of severe sepsis in france: overview of a -year survey period bichat hospital, surgical intensive care unit lariboisière university hospital, medical intensive care unit cochrane database leptin is an adipocyte-derived cytokine regulating energy homeostasis, metabolism as well as immune-inflammatory processes. leptin also has thermogenic actions and regulates enzymes of fatty acid oxidation. leptin is significantly increased in response to acute infection and sepsis and exerts direct effects on cd + t-lymphocyte proliferation, macrophage phagocytosis, and secretion of inflammatory cytokines such as il- and tumor necrosis factor (tnf) a. we measured leptin in blood of septic patients we measured leptin serum levels in septic patients leptin levels in septic patients (mw = , . ng/ml ± sem = , . ng/ ml) are significantly higher compared to healthy controls (mw = , . ng/ ml ± sem = , . ng/ml, p \ . ). serum levels of leptin were significantly higher on day (mw = , . ng/ml ±sem = , . ng/ml) and day (mw = , . ng/ ml ± sem = the aim of this multi-centre retrospective observational study was to resuscitation bundle the above preliminary data indicated that an experienced use of rhapc, when compared to other survey ( ), was associated with a reduction of serious bleeding events, a more frequent off-label use and a similar mortality rate. the concomitant adherence to evidence-based guidelines improved significantly the patient survival ) macias et al. sources of variability on the estimate of treatment effect in the prowess trial: implications for the design and conduct of future studies in severe sepsis use of drotrecogin alfa (activated) in italian intensive care units: the results of a nationwide survey acute lung injury (ali) is one of the most frequent complications of sepsis. although coecum ligation and puncture (clp)-induced sepsis is a frequently used model, we found only three microarray studies of clp-induced sepsis - , of which one looked at lung tissue . none of them had examined the effects of recombinant human myocardial transcriptional profiles in a murine model of sepsis: evidence for the importance of age molecular signatures of sepsis: multiorgan gene expression profiles of systemic inflammation sepsis gene expression profiling: murine splenic compared with hepatic responses determined by using complementary dna microarrays endothelin- in endotoxin-and sepsis-induced lung injury activated) in real-life clinical practice for management of severe sepsis in surgical patients in patients with severe sepsis and multiple organ dysfunction, major surgery is not a contraindication for early ( - h after surgery) daa administration retrospective, observational, descriptive cohort study in patients with severe sepsis and multiple organ dysfunction treated with daa the principal focus of sepsis differed between the two groups (p \ . ): in the surgical group it was the abdomen ( . %) followed by the skin and soft tissues %) followed by the urinary tract ( . %) in the perfusion period, the distribution of severe/ moderate hemorrhages in the surgical group was %/ . vs. . %/ . % in the medical group. we found no differences between groups in death from any cause at days ( . vs. . %; rr ; % ci . - . ; p = . ). nevertheless, at days overall mortality had increased in both groups, although this difference was not statistically significant: the percentage of increase with respect to the -day mortality was % in the surgical group and % in the medical group in patients with severe sepsis and multiple organ dysfunction, major surgery is not a contraindication for early ( - h after surgery) daa administration. given our findings at days, studies including a wider period from the initiation of daa administration are necessary to evaluate the cost-efficacy efficacy and safety of recombinant human activated protein c for severe sepsis recombinant human activated protein c, package labeling and hemorrhage risk reference(s). iom. to err is human incidence of adverse events and negligence in hospitalized patients matrix metalloproteinase- promotes repair after ventila-tor-induced lung injury supported by the parker b. francis foundation, physician services incorporate, ontario thoracic society and canadian lung association sepsis-induced immuno-endocrine dysfunction impact of arginine-vasopressin (avp) and apelin (apl) exogenous administrations in a rodent model because the corticotrophic pathway is disturbed during circi, with acth-cortisol dissociation, alternative physiologically nondominant pathways such as the vasopressinergic/apelinergic axis, become essential to the hpa adaptation to stress. objectives. seeking the impact of arginine-vasopressin (avp) and apelin (apl) exogenous administrations on: acth & corticosterone blood contents, and respective pituitary and adrenal gland receptor expression (v b, apj) in a rodent model of endotoxin challenge. methods. a rodent model of endotoxin (lps e. coli :b , mg/kg i/p)-induced hpa axis has been selected to study the committment of avp/apl and related receptors (v b/ apj). rats (n = ) were equipped with subcutaneous osmotic minipumps (alzet, ) containing: saline, apl- ( , , , lg), or avp ( . , lg) for h. results. without lps challenge, exogenous apl administration did not alter acth & corticosterone blood contents whereas high dosage of exogenous avp significantly increased corticosterone blood content (p \ . vs. control). lps i/p challenge induced a huge increased of blood acth & corticosterone, both culminating at . h ( -and -fold increases respectively, p \ . vs. baseline), which was normalizing at h for acth whereas corticosterone remained high. this dissociation validates the model, matchs with human observations, and suggests non acth-dependent corticosterone release after lps challenge apl administration completely reversed the above down-regulation while avp also partially restored apj pituitary expression by almost % in a dose-dependent manner, and to a lesser degree in the adrenal gland (p \ . vs. lps). selective non-peptide v b (ssr ) and peptide apl antagonists (f a) substitutions confirmed the above effects were directely mediated. conclusions. apl and avp pituitary neuronal and bloodstream contents behave differently, and blood acth & corticosterone contents were dissociated, after acute lps challenge. apl as well as avp exogenous administrations were able to reverse (partially for the later) the lps-induced apj down-regulation in both pituitary and adrenal glands. reference(s) supported by the esicm young investigator award from infection diagnosis to therapy an antimicrobial stewardship program (asp) is a method of optimizing antimicrobial prescribing, altering antimicrobial resistance, reducing costs, and improving patient care. the intensive care unit (icu) is an ideal environment for the application of an asp given the complexity of the patient population, the ecology of resistant organisms, and the fact that selection of inappropriate antimicrobials or delays to determine the impact of the introduction of an icu asp on prescribing, patient outcomes, resistance, and costs we implemented an asp in a -bed medical surgical icu of a university-affiliated hospital. the asp team used prospective audit with interaction and feedback providing suggested changes in therapy (e.g. antibiotic choice, dose, duration) on a daily basis. asp provided consultation on all icu patients not followed by the infectious diseases consult service. parameters collected included demographic data, details of antimicrobial regimens, culture results, defined daily doses (ddd)/ patient days and antimicrobial costs mean monthly antibacterial ddd/ patient-days post-asp was reduced by . % ( . vs. . , p \ . ). the implementation of the asp was associated with a . % decrease in mean monthly antibiotic costs/ patient-days ($ vs. $ ) for a total cost reduction of $ , . in terms of prescribing and resistance, the introduction of asp was associated with a reduction in the prescribing of anti-pseudomonal agents (compared with antibiotics not covering pseudomonas) (mean monthly ratio . vs. . ), mrsa vs. mssa covering antibiotics (mean monthly ratio . vs. . ) and improved susceptibility pattern for pseudomonas aeruginosa as demonstrated by increases of , and % to tobramycin, meropenem, and piperacillin-tazobactam susceptibilities, respectively. there was no difference in icu mortality rate the asp team worked collaboratively with the icu team to improve antimicrobial therapy and as a result improved overall antibiotic usage and resistance patterns of important icu microorganisms, and decreased antimicrobial costs. appropriate and judicious antimicrobial use guided by an asp is beneficial to the icu can pct and/or crp help identify associated bacterial infection in patients with influenza pneumonia? procalcitonin (pct) is a recognized marker of bacterial infection and might be a prognostic marker in lower respiratory tract infections. objectives. to determine if pct and/or c-reactive protein (crp) levels at admission in intensive care unit can help identify associated bacterial infection in patients with influenzae pneumonia. methods. a nationwide registry (reva) was set up in france during the h n influenza pandemic. levels of pct and crp at icu admission were compared between patients presenting with influenzae pneumonia associated or not with a bacterial coinfection. results. patients were included, of whom received antibiotics prior to hospitalization. of the remaining patients, ( %) had documented bacterial co-infection. the bacteria involved in the co-infections were streptococcus pneumoniae (n = , . %), staphylococcus aureus (n = , . %), streptococcus a group (n = , . %). figure shows the initial values of procalcitonin and crp. median values for both pct ( . vs. . ng/ml, p \ . ) and crp ( vs. mg/l, p = . ) were significantly higher in patients with bacterial coinfection. the area under the roc curve was . and . , respectively for pct and crp. a cutoff of[ . ng/ml for pct (sensitivity % and specificity %) best identified patients with bacterial co-infection. for crp, a cutoff of[ mg/l (sensitivity %, specificity %) best identified patients with bacterial co-infections. comparison of the h n pneumonia and bacterial pneumonia group revealed no differences except for a higher saps in the latter pro endothelin (pro et) and copeptin (pro vasopressin cp)) for diagnosing infection in patients with severe acute dyspnea. methods. we designed a prospective study of patients admitted in emergency department (ed) and medical intensive care unit (icu) in a university hospital. inclusion criteria were acute dyspnea with spo b % and/or respiratory rate c b/min. patients with obvious myocardial infarction or pneumothorax were excluded. all clinical and biological data were recorded and biomarkers sampled. an independent blinded expert panel classified the patients according to all the available data including response to treatment and outcomes blindly to biomarkers' results. the roles of biomarkers were assessed quantitatively and then using terciles of the distribution. the contribution of the biomarkers in the diagnosis was assessed using auc-roc curves and by multiple logistic regression taking into account other clinical and biological explanatory variables. results. consecutive patients ( % male, med age years, day mortality %) were enrolled. the final diagnosis was severe sepsis for ( . %) (pulmonary: n = , non-pulmonary n = ). the parameters independently associated with infection lead to a clinico-biological model with an auc = . and a good calibration (p (hlchi ) = . ) and included temperature, arterial pressure, cyanosis, stupor and coma, orthopnea, localized chest sound abnormalities, pao /fio ratio and localized infiltrate on chest x ray although new biomarkers were different between septic and non septic patients with severe acute dyspnea, only mid pro-anp may add a significant contribution. further analysis about the prognostic value of these biomarkers is ongoing grenoble university hospital and brahms diagnostic czech republic, st faculty of medicine charles university and thomayers' hospital, anesthesiology and intensive care medicine fluid management with cvp is still common despite lack of efficacy. objectives. implement stroke volume maximisation during major surgery using odm guided fluid challenges. assess impact of odm use on central venous line insertion rates. identify and overcome barriers to adoption of odm technology. methods. nhs technology adoption centre project (ntac) supported an implementation project at hospitals. the audit into central catheter use during major surgery was undertaken at manchester royal infirmary. fourteen anaesthetic consultants volunteered to champion odm use to guide targeted fluid challenges with hes / . (voluven, fresenius kabi) within a range of major surgical procedures (colo-rectal, hepatic, pancreaticobiliary, urological, reno-pancreas transplant and emergency surgery). prospective data was collected for patients who underwent major surgery between with no significant differences in preoperative risk, intervention patients had enhanced post-operative outcomes. cvc insertion reduced after anaesthetists had the opportunity to use odm to guide fluid therapy. reference(s) clinically used fluids modify in vitro phenotype and function of circulating immune cells peri-operative fluid loading, i.e. ''hemodynamic optimization'', reduces post-operative complications and hospital length of stay. mechanisms involving perfusion have been studied, but fluid could also alter phenotype and function of circulating immune cells, and consequently the systemic inflammatory response induced by surgery blood from control donors has been diluted in crystalloid fluid (isotonic saline, wsio), colloids (hydroxyethyl starch (hes kd, . %) and % albumin solution (alb)), or autologous plasma, which corresponds to clinical situations in programmed anesthesia and surgery. two dilution levels have been studied, to achieve - g/dl hemoglobin (dilution ) and - g/dl (dilution ) )* cd mono (sites/cell) , ( , ) , ( , )* , ( , ) , ( , )* , ( , )* cd b pmn (sites/cell) alb only increased ord, but not activation and adhesion markers. conclusions. wsio had clear anti-inflammatory properties, whereas colloids were more inflammatory, with a dissociation of the effects between different types of fluids. mechanisms have to be precised, especially regarding physico-chemical, immuno-inflammatory and metabolic regulations. reference(s). none. grant acknowledgment. plan quadriennal outcome-related factors federacion panamericana e iberica de sociedades de medicina critica y terapia intensiva (fpimcti) our aim was to conduct a multicenter study to evaluate the epidemiology of delirium in intensive care units (icu) a -day point-prevalence study was performed with the aim of describing in icus from countries in south and north america and spain % were admitted to the icu due to medical causes and sepsis was the main diagnosis (n = , . %). patients were sedated and only ( . %) patients could be evaluated with the cam-icu. the prevalence of delirium was . % (n = ). as compared to patients without delirium, those with the diagnosis of delirium had a higher severity of illness at admission as demonstrated by higher sofa increased use of invasive devices such as central venous catheter (p \ . ), arterial catheter (p = . ) and urinary catheter (p = . ) were more frequent in patients with delirium. on multivariate analysis, delirium was independently associated with increased icu mortality in this one-day international study, delirium was frequent in icu patients and associated with increased mortality and icu los. the main modifiable risk factors associated with the diagnosis of delirium were the use of invasive devices and sedatives the study was funded by the federacion panamericana e iberica de sociedades de medicina critica y terapia intensiva (fpimcti). the fpimcti has used in part an educational grant from hospira recent studies suggest that increased blood glucose variability (bgv) is associated with icu mortality . hypothermia is known to induce insulin resistance, thus potentially increasing bgv. no studies however have examined the effect of therapeutic hypothermia (th) on insulin requirements and bgv. objectives. to examine the effect of th on bgv and its relationship to all patients were treated with intravenous insulin (blood glucose target - mm), according to a written algorithm, with nurse-driven adjustment of insulin dose. for each patient, standard deviation of repeated blood glucose samples was used to calculate bgv. two time-points, comparable in duration, were studied: th (stable maintenance phase, i.e. - h, core temp ± °c) vs. normothermia (nt, i.e. after rewarming, stable normothermic phase, core temp ± °c). mortality and neurological recovery (glasgow-pittsburgh cerebral performance categories, cpc, dichotomized as good = cpc - vs. poor = cpc - ) were assessed at hospital discharge. statistical analysis was performed with anova for repeated measures therapeutic hypothermia is associated with increased insulin requirements and higher blood glucose variability, which in turn correlates with worse prognosis in patients with post-ca coma. strategies aimed to maintain stable glycemic profile and avoid blood glucose variability might contribute to optimize the management of th and may translate into better outcome glucose variability is associated with intensive care unit mortality therapeutic hypothermia post cardiac arrest has been shown to improve survival and neurological outcome . there are approximately , treated cardiac arrests in the uk each year with one-eighth we aimed to establish if implementation of an agreed care bundle including therapeutic hypothermia reduced mortality and improved neurological outcome patients were categorized according to initial cardiac rhythm, ventricular fibrillation/tachycardia (vf/vt) or non-vf/vt. we recorded the degree of implementation of the post cardiac arrest care bundle, comprising; coronary reperfusion, haemodynamic optimisation, control of ventilation, blood glucose, temperature and seizures. data was compared with survival to discharge and neurological function there were ihas, male (mean age . years) and female ( . years) and oohas, male ( . years) and female ( . years). the predominant presenting rhythm in oohas was vf/vt ( . %) compared to ihas which was non vf/vt ( . %). underlying co-morbidities included - °c) was achieved in % of vf/vt oohas compared to . % of vf/vt ihas. the complete care bundle was delivered to . % of oohas and . % of ihas survival rates were higher in all patients with complete bundle of care versus those with an incomplete bundle independent of location or rhythm ( . vs. . % p = . ). this improval in survival was also demonstrated in vf/vt arrests receiving the complete bundle of those patients who had a vf/vt arrest, survival with full neurological recovery (gcs on discharge) was higher in those receiving therapeutic hypothermia . versus % where therapeutic hypothermia was not achieved adherence to the post cardiac arrest care bundle led to significantly improved outcome following both vf/vt and non vf/vt arrests. there was a trend towards therapeutic hypothermia improving neurological outcome on discharge reference(s). . the hypothermia after cardiac arrest study group. mild therapeutic hypothermia to improve the neurological outcome after cardiac arrest royal sussex county hospital intensive care unit, brighton, uk for patient data h. altemimi , s. altaf , j. brown , s. al-juboori , v. jadhav queen elizabeth hospital, kings lynn, medical assessment unit, kings lynn, uk introduction. the medical assessment unit (mau) in our district general hospital (with beds) provides assessment and treatment of acute medical patients from general practice (gp) referals and the emergency department (ed). patients arriving in mau are first triaged by the nurses before assessment by junior doctors. the early warning score (ews) is an indepenently verified scoring systems used to prioritise patient assessment. appropriate referal to critical care outreach teams and intensive care units can be triggered by nurses or doctors according to the ews score. clear and seemless referal pathways and communication between those involved with acutely unwell patients is essential. objectives. to measure key performance indicators in the mau and identify specific areas for improvement. methods. retrospective audit of all admissions to medical assessment unit during week in . the following parameters were recorded for patients admitted. patient ages ranged from to : [ . of these, only were reviewed within h. . % of patients reviewed within min. conclusions. in the uk, early warning scores have been developed to trigger early review. the most sophisticated intensive care becomes unnecessarily expensive terminal care when the pre-icu system fails to refer in a timely manner. our results showed that when a patient was recognised as being unwell, they were seen rapidly by the outreach team. however, not all new admissions had their ews documented, overlooking an important opportunity to risk stratify patients before formal medical clerking. our critical care outreach team have prominent role in education to identify abnormal physiology early, and take action as appropriate. immunological host reactions are primarily believed to determine the clinical course of this disease. an overwhelming inflammatory response to microbial invasion may be involved in the pathogenesis of sirs, sepsis and multiple organ failure. it is important to block the inflammatory response and stop or alleviate sepsis injuries. immunotherapy is regarded as effective approach to improve the immunological function. objectives. immunomodulation in the critically ill is an appealing notion because of the abnormal immune responses. the aim of this study is to evaluate the immunomodulation and its mechanism to improve immune function and prognosis in sepsis. methods. experimental part:clp model were divided into three groups including sham group, control group and experimental group. control group only used antibiotic and experimental group used antibiotic plus immunomodulation. blood collection were made after clp model in , , and h. lymphocyte counting, cd +, cd + t lymphocyte and cd /cd ratio were checked. the apoptosis of lymphocyte in thymus and spleen and survival rate were checked. clinical part: prospective analysis seventy patients conformed to the enrolled standard. it was divided into two groups at random. one was control group with regular therapy, the therapy group with ulinastatin plus thymosin-a in a week. the immune index before and after therapy in the , st, rd, thday was observed. results. experimental part: lymphocyte, cd + t lymphocyte and cd /cd ratio in experimental group increased more significant than in control group (p \ . ). lymphocyte apoptosis index of thymus and spleen in control group increased more significant than in experimental group (p \ . ). h-survival rate in experimental group was higher than in control group. clinical part: cd + t lymphocyte, lymphocytes and hla-dr cd + had more significant increase in therapy group than in control group (p \ . ). twenty patients died in the control group and thirteen patients died in the therapy group (p \ . ). conclusions. immunomodulation in sepsis can improve immune function and alleviate the lymphocyte apoptosis and extend the survival time. in recent years, the antiinflammatory effects of niacin by reducing nuclear factor jb (nf-jb) activation have attracted attention. however, the protective effects of niacin on the development of acute lung injury and systemic inflammatory response during sepsis have not been studied.objectives. we performed this study to determine whether niacin attenuates acute lung injury (ali) and improves survival during sepsis and the beneficial effects of niacin are associated with the down-regulation of nf-jb pathway.methods. lipopolysaccharide (lps) at a dosage of mg/kg was injected into the tail vein to induce endotoxemia in rats. then, vehicle, low dose niacin ( mg/kg), or high dose niacin ( mg/kg) diluted in distilled water with same volume ( ml/kg) was administered once to the rats through orogastric tube, respectively. we observed the survival of the subjects. at h post-lps injection, we measured serum tumor necrosis factor-a (tnf-a), interleukin- (il- ) levels, lung cytoplasmic phosphorylated inhibitor jb-a (p-ijb-a), ijb-a, nuclear nf-jb p expressions, nf-jb p dna-binding activity, and ali occurrence.results. high dose niacin improved survival during sepsis. niacin induced dose-dependent reduction of serum tnf-a, il- levels, lung cytoplasmic p-ijb-a, nuclear nf-jb p expressions, nf-jb p dna-binding activity, and ali occurrence in endotoxemic rats. in contrast, niacin preserved lung cytoplasmic ijb-a expression dose-dependently in endotoxemic rats.conclusions. niacin attenuated acute lung injury and improved survival during sepsis in rats. the protective effects of niacin were associated with the down-regulation of nf-jb pathway. niacin can be considered as a therapeutic agent for sepsis. f. barbani , m. boddi , r. cammelli , a. cecchi , e. spinelli , m. bonizzoli , a. peris university of florence, postgraduate school of anesthesia and intensive care, florence, italy, university of florence, department of critical care medicine and surgery, florence, italy, careggi teaching hospital, anesthesia and intensive care unit of emergency department, florence, italy introduction. acute kidney injury (aki) is a frequent complication in critically ill patients. there is emerging evidence that aki can lead to chronic kidney disease and that even only partial renal recovery after aki is associated with a higher long-term mortality.objectives. we investigated whether measurement of renal resistive index (rri) by ultrasound could predict renal function recovery after aki.methods. rri has been determined in patients who had been admitted (jan -feb to our mixed intensive care unit (icu) and referral trauma center (careggi teaching hospital, florence, it) and developed aki. rri measurements were performed within h from aki diagnosis, according to rifle criteria. renal recovery was defined as the return to the normal renal function parameters.results. patients studied were ( male vs. female) with a mean age of . ± . years. aki was due to sepsis (n = ), shock (n = ), rhabdomyolysis (n = ), abdominal compartment syndrome (n = ) and major surgery (n = ). mean length of icu stay was ± days. at discharge patients showed a complete recovery of renal function, while patients had persistent altered renal function parameters or needed renal replacement therapy (rrt); mortality rate was . % ( / ) . rri measured at aki onset was significantly higher in patients with persistence of renal failure than in patients with complete renal recovery ( . ± . vs. . ± . , p \ . ). rri [ . had a sensitivity of % ( % ci - %), a specificity of % ( % ci - %) and a positive likelihood ratio of . ( % ) for persistent renal dysfunction at the discharge.conclusions. our findings suggest that doppler-based determination of rri at the onset of aki can identify those patients who are at greater risk for no complete recovery of renal function. further studies on larger populations are required to confirm these preliminary results so to promote therapies dedicated to this high risk population.conclusions. apache score on admission, hypothermia and mean arterial pressure are not statistically significant, but age approached significance at % level in ccu survival. blood sugar control is not statistically significant at % level but approached significance as p value was less than . for ccu survival. for hospital survival,age is significant at % level; apache score on admission and mean arterial pressure are significant at % level while cardiac index, hypothermia and glycaemic control are not statistically significant . in the intensive care society (ics) published a care bundle for the management of patients following cardiac arrest . the american heart association now recommends regionalisation of post-resuscitation care following out of hospital cardiac arrest (ohca), in a centre capable of providing rapid therapeutic hypothermia, h emergency angiography and percutaneous coronary intervention (pci) as necessary . objectives. the audit's purpose was to evaluate work load, treatment and outcomes of ohca on the icu following publication of the ics care bundle and inception of a h pci service in our hospital. methods. retrospective audit of ohca patients admitted to icu from / to / to examine number admitted, cause of arrest, treatment and mortality. predicted mortality from the icnarc model was used to calculated standardised mortality ratio (smr). comparison was made with an audit of ohca patients between / and / . the sample was subdivided into subgroups with cardiac and non-cardiac cause for ohca. the chi-square test was used in analysis. results. comparison conclusions. we demonstrated: . a significant increase both in the absolute number of ohca and the proportion due to cardiac causes, due in part to in-region transfers for emergency angiography/pci. . a significant increase in the use of angiography and pci, increasing use of therapeutic hypothermia and iabp. . improved survival from previous audit. . better than predicted survival, particularly in the 'cardiac' group. this audit lends support for the protocolised icu care of post-ohca patients in a regional centre able to offer h emergency pci. there is scope for increased use of cooling for arrests of all causes, and extending provision of angiography/pci to vf arrests and those of unknown cause . reference(s). . despite all the research, education and training that has gone into the field of cpr during the last years, survival rates remain bleak for the majority of patients . so, what is lost in translation? much of the problem is that what the medical community is being trained to do is not being replicated in clinical practice . given this discrepancy, along with the deleterious outcomes, we conducted a manikin-based study to assess the quality of ventilation delivered during simulated cpr, in a large uk centre. objectives. to demonstrate whether uk-based medical personal were adhering to ventilation guidelines and to see how this result varied across specialities. methods. a simulated cardiac arrest scenario was undertaken by (a- . , power- %) participants from a range of medical specialties. participants were asked to asynchronously ventilate our manikin for a period of min during which time tidal volumes (tv), minute volume (mv) and ventilation rates (vr) were recorded. to help limit outside influence, at no point during the trial was any feedback about ventilation technique or als principals discussed with the participant. results. the mean and sd across the sample population for the mv, vr and tv were . l min - (sd . ), . min - (sd . ) and ml (sd ) respectively. comparison of groups can be seen in table introduction. mild hypothermia reduces cerebral blood flow (cbf) without concurrent increase of cerebral oxygen extraction rate in the first h after cardiac arrest, indicating a lower cerebral metabolic activity with a preserved metabolic coupling.objectives. the aim of this study was to assess the cerebral blood flow and cerebral oxygen extraction in patients treated with prolonged hypothermia, as previously was performed in newborn infants with perinatal asphyxial encephalopathy . patients were included after restoration of spontaneous circulation (rosc) after asystole, pulseless electrical activity based circulatory arrest or after rosc after ventricular fibrillation based prolonged resuscitation. in this prospective observational study comatose patients after cardiac arrest were treated with prolonged hypothermia for h. after h patients were passively rewarmed. mean flow velocity in the middle cerebral artery (mfv mca ), reflecting cbf, was measured after , , , , , , , , and h after admission to the icu by transcranial doppler (tcd). jugular bulb oxygenation (sjbo ) and arterial oxygenation were measured at intervals of h. introduction. isoflurane is a volatile anesthetic known for its direct vasodilating effect on cerebral vessels, producing a cbf increase. moreover, it has been shown in animal studies that isoflurane has neuroprotective properties, inducing tolerance to ischemia when used as a preconditioning agent. at the present time, isoflurane is not used as a sedative agent in neuroicu because of the fear of an increase in intracranial pressure (icp) caused by the increase in cbf. however, sah patient at risk for vasospasm may benefit from an increase in cbf. the ischemic risk will peak at day - , leaving a window of opportunity for neuroprotection. objectives. we decided to measure rcbf during traditional intravenous sedation with propofol and during volatile sedation with isoflurane in sah patients not having intracranial hypertension. the clinical trial was approved by the hospital irb and registered on trial.gov (nct ). we enrolled sah (fisher - , wfns b ) patients, monitored with an intraparenchimal icp device and a thermal diffusion probe (tdp, hemedex) for the assessment of rcbf. an icp [ mmhg was an exclusion criteria. cerebral and haemodynamic parameters were assessed at steps:step during sedation with propofol - mg/kg/ h; step : after h of sedation with isoflurane . - % mac administered through an anesthetic conserving device; step after h of propofol re-started at the same infusion rate. in all patients sedation with isoflurane produced an increase of rcbf although not yet significant in the population (step : ± ml/ g/min; step : ± step : ± ). jugular vein oxygen saturation, sjo , was significantly higher at the end of the isoflurane step (step : ± %; step : ± ; step : ± ). icp did not change significantly and remained below the pathological threshold (step : . ± ; step : . ± ; step : ± mmhg).conclusions. the small population of this pilot study phase causes a lack in statistical significance, however our data already suggest that isoflurane induces a marked cerebral vasodilatation and an increase in rcbf compared to propofol. patients with a normal icp did not develop an intracranial hypertension. at short and long term and its correlation with severity scales, scales of quality of life and endovascular treatment m there is not a consensus about which scales should be used to define the outcome after aneurysmal subarachnoid hemorrhage. objectives. the purpose of this study was to determine the risk factors and impact on functional outcome and quality of life months after spontaneous sah due to ruptured intracranial aneurysms. methods. we performed a prospective study of patients with aneurysmal spontaneous sah admitted to our centre from july to august . diagnosis of sah was done by ct and ethiological diagnosis by brain angiography. we paid attention to previous pathological history, clinical characteristics at admission and radiological characteristics. the severity was measured by the hunt-hess (hh), wfns, graeb and fisher scales. the months outcome was assessed by the glasgow outcome scale (gos) and modified rankin scale. the basic activities of daily living were evaluated with the barthel index. moreover, patients were asked about their subsequent incorporation to their previous occupation. we divided our population into two groups depending on the clinical grade at admission (hunt-hess scale): group i, hh , or ; and group , hh or . long-term followed up continues. results. during the year of study a total of patients have been included. the mean age of patients was years with a prevalence of % in women, being arterial hypertension and smoking history the main factors of related risk. the angiography was performed in . % of the patients. an aneurysm was confirmed as the origin of the bleeding in . %. poor clinical grade at admission (hh or ) was associated with apache ii, sofa, glucose, more sah computed tomography on admission, and infection and icu stay. there is % mortality in the series. after a months period, group patients (hh or grade) had a severe disability and functional dependence to perform instrumental and basic activities of daily living (p = . ). only % of patients were able to return to their previous occupation months after the initial bleeding. scales and outcome by clinical grade at admission introduction. aneurysmal subarachnoid hemorrhage (asah) remains a therapeutic challenge due to unacceptably high levels of mortality and morbidity. for survivors of the initial insult, cerebral vasospasm and related delayed ischemic deficits are the major determinants of final outcomes. traumatic sah (tsah) occurs in as high as % of patients with tbi, and is associated with a twofold increase in risk of death. statins may be an alternative for conventional treatments of vasospasm due to their beneficial pleiotropic effects on cerebral vasomotor reactivity as well as absence of negative haemodynamic influence.objectives. the goal of our study was to examine the effects atorvastatin therapy on cerebral vasospasm after asah and tsah as well as on short term outcomes of icu patients (length of stay and severity of condition upon discharge from icu). hypertonic saline infusion is an alternative to mannitol to decrease intracranial pressure before craniotomy [ , ] . previous studies have demonstrated that both hypertonic saline and mannitol interfere negatively with various components of blood coagulation [ , ] . normal blood coagulation capacity is essential during craniotomy and, therefore, we created an in vitro model to examine the effects of hypertonic sodium chloride and mannitol solution on whole blood coagulation. fresh citrated whole blood, withdrawn from volunteers, was diluted with . , . or . % sodium chloride solution or % mannitol to make vol.% and vol.% hemodilution in vitro. the diluted blood and undiluted control samples were analyzed with thromboelastometry (rotem Ò ) using two activators, tissue thromboplastin without (extem Ò ) or with cytochalasin (fibtem Ò ). all the study solutions with the same vol.% hemodilution induced comparable decrease in hematocrit. in extem Ò analysis, alpha-angle was smaller in the mannitol group than in the . % or . % sodium chloride group after vol.% dilution (p. ). in vol.% hemodilution, alpha-angle was also more decreased, and clot formation time more delayed in the mannitol group than in the . , . or . % sodium chloride groups in extem Ò analyses (p \ . ). maximum clot firmness (mcf) in extem Ò analysis was similar with all the study solutions after vol.% dilution, but after vol.% dilution mcf was weaker in the mannitol group than in the other groups. mcf was also weaker in . % than in . % sodium chloride group after vol.% dilution. in fibtem Ò analysis, mcf was stronger in the . % sodium chloride group than in the mannitol group after both dilutions (p \ . ). a or vol.% dilution of % mannitol disturbs whole blood coagulation more than equiosmolar . % sodium chloride. this disturbance seems to be attributed to overall clot formation and strength but also to pure fibrin clot firmness. . % sodium chloride might be more favorable than mannitol before craniotomy in patients with high bleeding risk. introduction. delayed cerebral ischemia (dci) is a major complication after aneurysmal subarachnoid haemorrhage (sah), occurring in around % of patients and increasing case fatality . - -fold. induced hypertension, alone or in combination with haemodilution and hypervolemia (triple-h), is used around the world as a therapy in the treatment of dci, but its efficacy in improving outcome is not based on a randomised clinical trial. objectives. to investigate the outcome of induced hypertension versus no induced hypertension in patients with dci after aneurysmal sah. study design a multi-centre, single blinded, randomized controlled trial. study population patients admitted after recent sah with a treated aneurysm and dci based on the onset of a new focal deficit and/or a decrease of the level of consciousness of at least point on the glasgow coma scale with exclusion of other causes of deterioration, will be randomized to either hypertension (n = ) or no hypertension (n = ). interventions in the intervention group, blood pressure will be raised with norepinephrine or additional dobutamin in case of low cardiac output. when clinical improvement occurs, hypertension will be continued for h, after which the dose norepinephrine will be tapered daily, but resumed in case of clinical deterioration. when no clinical improvement occurs within h, induced hypertension will not be continued. patients in the reference group will be treated according to the standardised sah treatment protocol of the participating centre including oral nimodipine and normovolaemia without haemodilution. main outcome measurement the modified rankin scale at months after the sah, will be compared between patients who were randomized to induced hypertension and patients who were randomized to no induced hypertension. objectives. to investigate brain metabolism at different serum glucose levels.methods. six patients with aneurismal sah and vasospasm were enrolled in the study (age . ± . ; male/female / ; . cerebral microdialysis was used in all patients. microdialysis catheters were placed into ''lesioned'' (brain tissue perfused by involved artery) and ''intact'' brain tissue. glucose levels in arterial blood (glu art ) and in brain interstitial fluid were compared. we analyzed brain glucose levels, intracranial pressure (icp), lactate/pyruvate (l/p) ratio, pao , paco and cerebral perfusion pressure (cpp) at blood glucose levels b mmol/l (group i, n = ), . - mmol/l (group ii, n = ), . - mmol/l (group iii, n = ), c mmol/l (group iv, n = ). we found out tight correlation between glucose levels in arterial blood and in ''lesioned'' brain tissue (n = ; r = . ; p \ . ) and weak correlation between gluart and glucose levels in ''intact'' brain tissue (n = ; r = . ; p \ . ). pao , paco , icp and cpp were comparable between groups. glu art was . ± . mmol/l in group i, . ± . mmol/l in group ii, . ± . mmol/l in group iii and . ± . mmol/l in group iv. normal glucose levels in arterial blood (group i) were accompanied by low glucose levels in ''intact'' ( . ± . mmol/l) and ''lesioned'' ( . ± . mmol/l) brain tissue. arterial blood glucose elevation has led to significant increase in brain glucose levels. but brain glucose levels were in normal ranges during hyperglycemia. glucose levels in ''intact'' brain tissue: . ± . mmol/l (group ii), . ± mmol/l (group iii), . ± . mmol/l (group iv). glucose levels in ''lesioned'' brain tissue: . ± . mmol/l (group ii), . ± . (group iii), . ± . (group iv). we didn't find out any significant differences in l/p ratio at different blood glucose levels. however, low glucose levels in ''intact'' brain tissue (b . mmol/l; . ± . mmol/l; n = ) were accompanied by significant increase of l/p ratio ( . ± . vs. . ± . at normal brain glucose levels (c . mmol/l; . ± . mmol/l; n = )). we didn't notice any significant differences in l/p ratio in ''lesioned'' brain tissue at different brain glucose levels, opposite to intact brain tissue. l/p ratio was . ( . ; . ) at glucose levels in ''lesioned'' brain tissue b . mmol/l ( . ± . mmol/l; n = ) vs. ( . ; ) at glucose levels in ''lesioned'' brain tissue c . mmol/l ( . ± . ; n = ).conclusions. hyperglycemia is not accompanied by high glucose levels and brain metabolism impairment in ''intact'' and ''lesioned'' brain tissue in severe patients with aneurismal sah. low glucose levels in ''intact'' brain tissue are related with significant increase of l/p ratio. introduction. the national confidential enquiry into patient outcome and death report found that less than % of patients with a severe head injury received a standard of care that was judged to be good [ ] . our intensive care unit (icu) at the royal cornwall hospital, a large district general hospital, is one of the few non-neurosurgical centres in the uk to use intracranial pressure (icp) monitoring. we aimed to assess if the practice of icp monitoring in our non-neurosurgical centre was valuable and safe. retrospective audit of case notes and charts of all patients admitted with traumatic brain injury receiving icp monitoring over a year period from st january until st january . a total of patients who had icp monitoring for traumatic brain injury were identified. the codman tm strain-gauge catheter was used in all cases, ( %) were male, ( %) were female. mean age was years (range - ). median reported gcs at the scene of injury was (range - ), median gcs prior to intubation was (range - ). median apache score was (range - ). all patients were discussed with the neurosurgical referral centre. patients were monitored for a median of days (range [ ] [ ] [ ] [ ] . icp was raised in patients ( %). elevation of icp triggered the following interventions: patients ( %) received hyperosmolar agents, patients ( %) were treated with therapeutic hypothermia, patients ( %) with barbiturate coma and none of the patients received steroids. patients were transferred to a neurosurgical centre ( % of patients with elevated icp, % of total). complications comprised one minor intracranial haematoma and one monitor failure. review of documented care bundles for head-injury patients revealed: head-up tilt in %, gastric protection in %, normoglycaemia in %, early enteral nutrition in %, appropriate thromboprophylaxis in %, appropriate sedation in % and appropriate analgesia in %. % survived to hospital discharge.conclusions. in our audit the majority of patients received specific treatment for raised icp, which might have gone undetected without invasive icp monitoring. at the same time most patients were managed without neurosurgical intervention. in light of the paucity of neurocritical care beds [ ] this approach appears to help to select a suitable subgroup of patients needing transfer to a specialist centre. our data adds weight to the evidence that icp monitoring is a valuable and safe monitoring modality in a non-neurosurgical icu if used appropriately within established guidelines and in collaboration with a neurosurgical referral centre. introduction. assessment of injury severity and prediction of outcome represent major challenges following severe traumatic brain injury (tbi). objectives. this study was aimed to evaluate relationships between cerebrospinal fluid (csf) levels of purported biomarkers of tbi including glial fibrillary acidic protein (gfap), ubiquitin c-terminal hydrolase (uch-l ) and alpha-ii spectrin breakdown product (sbdp ), and partial pressure of brain tissue oxygen (ptio ) as well as brain temperature during the first h and up to days post-injury.methods. twenty-seven severe tbi patients with csf drainage and invasive monitoring (licox probe) have been studied with quantitative csf-elisa for sbdp , uch-l and gfap on admission and every h up to days. in the first h, biomarker levels decreased while those of ptio increased. all three biomarkers correlated with ptio (p \ . , p = . and p = . , respectively). after the first h, there were statistically significant changes in levels of the three markers as well as in levels of ptio (p = . , p \ . , p = . , p \ . , respectively). however, the correlation between biomarkers and brain tissue oxygenation was sustained and, for uch-l improved (p \ . ). no significant correlations between biomarker levels and brain temperature were found. the results indicate that cfs levels of sbdp , uch-l and gfap are related to ptio following severe tbi. future studies are on their way to unravel whether these biochemical markers and ptio measurement could serve the better care of the head injured. introduction. this was a pilot study to compare the cerebral neurochemical changes in patients with traumatic brain injury (tbi) who underwent conventional blood glucose level (bgl) control and intensive bgl control with continuous titrated insulin.objectives. to demostrate that intensive glycaemic control using insulin induced a decrease of cerebral glucose.methods. this prospective, randomized study was conducted in traumatic brain injury patients in a surgical and trauma intensive care unit. patients admitted over an -month period with tbi were prospectively divided into two groups according to the method used for bgl control: the intensive group consisted of patients who underwent continuous titrated insulin infusion to maintain a lower normoglycemic level of - mmol/l, and the conventional group consisted of patients whose bgl was maintained at between . and . mmol/ l using conventional sliding scale bolus subcutaneous insulin administration. data on cerebral haemodynamics, interstitial brain oxygenation (ptio ( )) and neurochemical monitoring were collected via microcatheters inserted in the penumbral region. we analyzed , cerebral microdialysis samples. in patients treated with intensive insulin therapy, there was a reduction in microdialysis glucose by % of baseline concentration compared with a % reduction in patients treated with a conventional blood glucose level control. intensive insulin therapy was associated with increased incidence of microdialysis markers of cellular distress, elevated glutamate ( ± vs. ± %, p \ . ), elevated lactate/pyruvate ratio ( ± vs. ± %, p \ . ) and low glucose ( ± vs. ± %, p \ . ), and increased global oxygen extraction fraction. cerebral microdialysis glucose was lower in nonsurvivors than in survivors ( . ± . vs. . ± . mmol/l, p \ . ).conclusions. intensive glycaemic control using insulin induced a decrease of cerebral glucose and an increase in microdialysis markers of cellular distress. in patients with severe brain injury, tight systemic glucose control is associated with increased mortality. introduction. we have previously used c-flumazenil positron emission tomography to show that selective neuronal loss in the thalamus is pervasive after traumatic brain injury (tbi) and correlates with functional outcome, findings that are concordant with previous post-mortem data. the mechanisms responsible are unclear, but may involved global hypoxia/ischaemia as well as retrograde degeneration.objectives. we hypothesised that early brain tissue oxygenation would correlate with late diffusion tensor imaging (dti) abnormalities in the thalamus, and therefore, help to provide an explanation for late neuronal loss. nine patients underwent brain tissue oximetry (pbo ) following acute tbi, using a licox pbo probe, sited in structurally normal frontal white matter. mean pbo was calculated for the duration of their intensive care admission. at a median of . months (range - days) they underwent magnetic resonance imaging, including dti. apparent diffusion coefficient adc (maps) were created, adc calculated in regions of interest in the frontal lobes, splenium of the corpus callosum and thalami, and correlated with mean pbo using spearman's rho. ethical approval was obtained from the local research ethics committee, and assent from next-of-kin was obtained in all cases.results. mean pbo was inversely related to adc in both frontal lobes (r = - . and - . ; p = . and . ), and with the adc in the thalamus bilaterally (r = - . and - . ; p = . and p = . ). in contrast, no correlation was seen between mean pbo and adc in the splenium of the corpus callosum, a common site of traumatic axonal injury (tai; p = . ).conclusions. the inverse correlation of mean pbo associated with adcs in the monitored brain region is unsurprising, but the correlations observed with contralateral regions and deep grey matter suggest that the burden of tissue hypoxia has a significant impact on secondary neuronal loss across the brain. in contrast, the lack of correlation with adc changes in an area at risk of tai suggests a less significant impact of hypoxia on the progression or maturation of tai. the correlations with measures of thalamic microstructural injury are particularly significant, since they establish a clear link between acute physiology, tissue fate in key brain regions, and clinical outcome. introduction. earlier studies have suggested that autonomic dysfunction is associated with poor outcome in traumatic brain injury (tbi).objectives. this study was performed to assess changes in baroreceptor sensitivity and heart rate variability as indices of autonomic dysfunction in relation to icu management variables during early tbi.methods. ten patients ( females/ males) with a median age of (interquartile range, iqr, - ) admitted to icu following tbi were prospectively studied for the first consecutive days. all patients were sedated and mechanically ventilated with icp monitoring in place. high fidelity signals at hz were sampled for ecg and invasive arterial pressure (radial) to monitor baroreceptor sensitivity (brs) based on three or more consecutive beats in which successive systolic pressure and rr intervals increased or decreased with the threshold set at mmhg and ms, respectively. heart rate variability (hrv) was analysed by fourier transformation in the low (lf, . - . hz) and high frequency (hf, . - . hz) domains. the lf/hf ratio and total power ( . - . hz) were also calculated for hrv. management variables included use of inotropes, vasopressors, icp, use of decompressive craniectomy, icu length of stay (los) and mortality. statistical significance was set at p \ . using mann-whitney one-way anova and spearman correlation tests. median days (and iqr) were . ( - . ) for inotropes, . ( - ) for vasopressors and . ( - . ) for icu los. brs and lf, hf, lf/hf ratio and total power were all depressed throughout early icu management with no significant changes in the first week. no significant correlations were found between brs/hrv and days on inotropes/vasopressors or icu los. four patients underwent decompressive craniectomy and one patient died while in icu. no correlation was found between these events and brs or hrv changes brs, hrv and icp data conclusions. both brs and hrv were depressed early following tbi but did not correlate to icu management variables. while autonomic dysfunction is evident early in the icu treatment of tbi, no evidence to support an association between severity of impairment and icu outcome was found in this pilot study. introduction. in neurocritical care, raised intracranial pressure (icp) is associated to a poor outcome and its detection still leads the therapeutic management of the patients suffering from head pathologies.objectives. although invasive devices are recommended to detect intracranial pressure (icp), we investigated the correlation and the reliability with non-invasive ultrasound techniques as the optic nerve sheath diameter (onsd) assessed by ultrasonography and the pulsatility index (ip) measured by transcranial doppler sonography (tcd). we included patients suffering from intracranial hypertension, sedated and mechanically ventilated and control individuals, chosen among healthy people. all the patients had icp measured invasively either by external ventricular drains (evd) or intraparenchymal catheter. everyone underwent non-invasive measurements of onsd bilaterally and simultaneous medial cerebral artery ip assessment at the side of the best window.results. onsd had a significant difference between volunteers ( . ± . mm) and patients ( . ± . mm). ip's were . ± . for the control individuals and . ± . for the patients (media icp = ± mmhg) and also revealed a significant difference. onsd strongly correlated with icp (y = . x + . , p \ . , r = . ) whereas ip had some correlation but without statistical significance (y = . x + . , p = . , r = . ). onsd was found wider (p = . ) within patients with both icp and ip abnormal. a strong correlation was found between onsd and ip (y = . x + . , p = . , r = . ). however, we could not find the best cut-off values of onsd and ip for predicting an icp [ mmhg.conclusions. ip and onsd correlated with icp with a stronger reliability results of the latter, suggesting the possibility to integrate their use in the case of icp invasive monitoring would be contraindicated or not available. we studied the effects of normothermic therapy during the acute phase of traumatic brain injury.methods. twenty patients ( males, ± years old) who were admitted in the intensive care unit due to traumatic brain injury (glasgow coma scale upon admission - , marshall scale ii to iv) were studied. if patients' core temperature was above . °c, a cool line Ò , alsius corporation, irvine, ca. usa intravascular heat exchange central venous catheter (coolgard Ò device) was inserted via the femoral or the subclavian route, in order to achieve and maintain a target temperature of °c. intracranial pressure (icp) was measured (a) invasively by means of a intraparenchymal catheter (camino, camino laboratories, san diego, ca, usa) and (b) noninvasively by means of transcranial doppler sonography (tcd), using a philips hd xe (philips medical systems; bothell, wa, usa) equipped with a mhz transducer and a mhz linear transducer. estimated icp was calculated by tcd using the equation proposed by czosnyka and colleagues and the pulsatility index (pi = vs -vd/vm) was assessed in the middle cerebral artery bilaterally. all measurements were conducted at baseline and repeatedly on a daily basis for the next days and the average values were used in the statistical analysis.results. target temperature (t: . ± . °c) was achieved within the next h of the catheter insertion. at baseline (t: . °c) pulsatility index (pimean) was . ± . , icpmean/invasive was ± . mmhg and icpmean/noninvasive was . ± . mmhg. invasive and noninvasive icp values correlated significantly (r = . , p \ . ). following ± h from the insertion of the catheter the above parameters were significantly decreased as compared to baseline values (pimean = . ± . , icpmean/invasive = ± . mmhg, icpmean/noninvasive = . ± . mmhg, respectively; all p \ . ). out of patients, progressed towards brain tamponade, remained in a persistent vegetative state and were discharged with normal consciousness and motor deficits. however, due to the small number of patients no analysis could be performed to estimate the possible impact of normothermic therapy on the survival of these patients. these preliminary results suggest that normothermia during the acute phase of traumatic brain injury may decrease icp and ameliorate cerebral blood flow. introduction. extracranial organ dysfunctions are extremely common in patients with severe traumatic brain injury (tbi). although it has been shown that development of tbiinduced multiple organ failure (mof) is associated with a poor outcome, the underlying mechanisms leading to mof after tbi have not been investigated.objectives. to investigate in vitro the effect of plasma from tbi patients developing different degrees of organ failure on human endothelial cells.methods. ten consecutive severe tbi patients were included. gcs, iss and sofa score were recorded at admission; sofa was also recorded after days. plasma samples were obtained at the same time points. adhesion of freshly isolated human neutrophils on spontaneously transformed human umbelical vein endothelial cells (ecv ) was evaluated after h in vitro stimulation with % of plasma collected from tbi patients days after admission. expression of intercellular adhesion molecule- (icam- ) was assessed by immunofluorescence. to determine the impairment of the endothelial cell barrier function, trans-endothelial electrical resistance (teer) and permeability to fitc-dextran were measured. plasma from healthy volunteers were used as control. data are expressed as mean ± sd. results from different experiments were compared by unpaired t test.results. ten male patients were enrolled, mean age ± , gcs ± , iss ± , sofa was . ± . on admission and ± . on day . plasma derived from tbi patients increased the adhesion of neutrophils on ecv cells ( ± vs. ± cells/field, p \ . ), induced a significant reduction of teer ( . ± . ohm/cm vs. . ± . ohm/cm , p \ . ) and caused a concomitant increase of endothelial permeability to dextran ( . ± . vs. . ± . %, p \ . ) compared to control (healthy plasma). a visual up-regulation of icam- expression was also observed. there was a significant correlation between delta sofa score (day -day ), calculated excluding gcs, and neutrophil adhesion on endothelial cells exposed to plasma from tbi patients (p = . , r = . ).conclusions. plasma from patients with tbi causes the increase of endothelial permeability and neutrophil adhesion, which correlates with the development of extracranial organ dysfunction expressed by sofa. these results suggest a mechanism potentially responsible for the development of mof after traumatic brain injury.grant acknowledgment. fondi universitari ex- %, regione piemonte-ricerca sanitaria finalizzata. objectives. the aim of this study is to describe indications of dc and outcomes in our unit.methods. it is a retrospective revue collecting patients who had a dc for severe icht between january and july . inclusion criteria were: icht refractory to medical management due to cerebral oedema. we define intractable icht as intracranial pression (icp) over cmh o, pupil modification, neurologic deterioration persisting more than min despite medical management or ct scan findings. exclusion criteria were imminent cerebral death, icht due to an acute hematoma, tumour, hydrocephalus and prediction of short life expectancy. objectives. historically, research has targeted problems experienced several months post discharge from critical care, namely using data from follow up clinics \ months after the critical illness. in contrast, this study aimed to review data from patients recently transferred from critical care to the wards. this data can then be compared with patients' progress after earlier rehabilitation. methods. this was a prospective, longitudinal study, involving long stay critical care patients ([ days), between april - . this cohort included all patients nursed within our itu/hdu, regardless of speciality, age or gender. all reviews took place whilst the patients were still hospitalised. the study used qualitative data, using specifically designed questionnaires which highlighted the type of long term problems our patients suffered. these were developed using data from previous informal patient interviews and the had (hospital anxiety and depression) questionnaire. the latter was considered too intrusive for the ward environment. a data base was used to list the frequency of these problems to provide quantitative data. a total of patients were reviewed over years. the qualitative data highlighted different, common morbidities, in addition to disease specific morbidities. between, april - , % of patients demonstrated at least physical or psychological problems and % displayed or more. the quantitative data showed the most prevalent morbidities were: poor mobility at %, nightmares %, loss of appetite % and insomnia %. year showed similar findings, plus global weakness at %.conclusions. this research showed significant post critical illness morbidities in this cohort which appeared to impact on the patients' recovery on the wards. the challenge for our follow up is to target support and rehabilitation whilst the patients are in critical care and then re-evaluate the results. if this expedites a return to a near normal quality of life, it will have improved the efficacy of our service and may impact on follow up care in general. . it may affect the balance of family and change their roles and responsibilities, mainly due to the separation from their relative imposed by hospitalization. this admission may also generate other discomforts to the family, as relationship problems, emergence of diseases, lack of financial resources for expenditures now installed, anxiety, depression, fear and irritability . objective. to get to know the discomfort that characterizes the changes experienced in the daily life of families who have a relative in icu. method. this is a qualitative study, conducted in a general icu of a large hospital in the city of salvador, bahia, brazil, from august to october of . nine relatives of people hospitalized for at least h in icu were interviewed. the data were analyzed by the use of procedures of analysis of grounded theory. results. the hospitalization of a family member in icu produced discomfort for her, as the uncertainty of recovery, the fear of loss and the sudden physical separation of the relative. the interaction between the huge discomfort of life threat and the separation from the relative, in turn, spawned other discomforts in the daily lives of families, expressed by two categories. the first one, having difficulties to answer psychobiological needs, meant for the family member the experience of sleep deprivation, loss of appetite and constant concern with the relative. the second one, suffering a discontinuity in the daily life, meant to the family the disruption of daily activities with the relative, irritability front other family members, removal of the routine of home, loss on work performance and studies and difficulties to enjoy the leisure and recreation.conclusion. the coping of illness and hospitalization of a relative in intensive care produced discomfort for family members, characterized by changing of the routine of the daily life and care for oneself. the family's attention is primarily directed toward the ill relative. it starts to experience the routine of a hospital and disrupts the organization of familiar and personal life, it finds itself suffering a break from daily life. the discomfort experienced by the family can be minimized by practices of the healthcare team, which provide care, information, support, safety and convenience. (fig. ). this is a unique opportunity to study the effects of nursing environment on sleep quality and quantity in icu patients. to study the effects of nursing environment on sleep quality and quantity in icu patients. a total of patients will be included in this ongoing study: ten subjects who were admitted to the old, ward-like icu (fig. ) , and ten patients who will be admitted to the new, single-room icu (fig. ) . objectives. in order to understand the family's perception of nursing care, the authors undertook this study, assessing the family satisfaction and use the results to increase the quality of care.methods. this is a qualitative study of inductive nature. data was collected along the time of the study by in-depth interviews, to six relatives of each patient, after discharge the intensive care unit. for data analysis we followed the steps of the phenomenological method, according to max van manen ( ) . results. from data analysis, the results were divided in categories: ) relative needs; ) icu environment; ) relative's feeling; ) nurses role and ) suggestions. all this categories were grouped in a central theme called ''being a relative in a general intensive care unit''. the results show that relatives of patients admitted to the intensive care unit often require complete knowledge of the medical condition of the patient, a specific area for that purpose and a comfortable waiting room. they express the need to be near the patient and participate in care. they feel fear and anger with the situation that inevitably are forced to live and think, mostly, that nurses are effective and efficient in meeting the needs of the patient and family. conclusions. it is essential to promote attitudes and behaviors that provide comfort, safety and privacy for relatives, and acknowledge that they have a role in the process of care, reinforcing their importance in decision making process.introduction. heart failure (hf) is a complex syndrome that commonly affects elderly patients in whom it has a major impact upon longevity and quality of life. it is usually associated with symptoms such as dyspnoea, fatigue, and fluid retention, and results in frequent episodes of hospitalisation.objectives. this study was planned to examine the relationship between self-care behaviors and quality of life in patients with hf.methods. this study was planned and applied as a descriptive and a cross-sectional study. introduction. the hospitalization of a relative in icu, especially when it's unexpected, is considered a too stressful experience for the family, usually compounded by the disruption of daily life. from the relative's hospitalization at the icu, the family will necessarily interact with health practices, the rationality that underlie it and institutional objects that may be a source of comfort or discomfort , .objectives. to understand the situations defined as comfort to relatives of people in critical state of health and the sense of comfort in this situation.methods. this is a qualitative study conducted in the general icu of a large hospital in the city of salvador, bahia, brazil, from june to october of . fourteen family members of persons hospitalized for at least h in icu were interviewed by a specific questionnaire. all recorded interviews were transcribed and the data were analyzed by the use of procedures to encode the data of grounded theory. seven categories expressed the experience and sense of comfort for the relatives who had a person hospitalized in the icu: ) security: confidence of relatives in technical-scientific and humanistic team and in the possibility of recovery of the person who is hospitalized, ) reception: comfort experienced by the family by being treated as a person by the professional of icu when they interact with a supportive attitude, ) information: comfort experienced by the family when it feels conscious of the reality of the health condition of their relative and to receive guidance about the unit s routine, ) social and spiritual support: comfort experienced by the receiving of help and support of the family, friends and religion, ) proximity: the comfort of being close of the relative and being able to enjoy the interaction established between them, ) convenience: comfort experienced in interacting with pleasant elements and support of basic needs of the family, offered by the environment and physical structure of the hospital; ) integration with itself and the daily: the possibility of the family member to take care of himself, to help the relative and to give continuity to the family routine as it did before the hospitalization.conclusions. the comfort is a positive, subjective and dynamic experience, which changes in time and space, which is the result of the interactions that the individual sets with himself, with those around him and with the situations he faces, without losing view that every family is unique and can experience this process in a proper way. to ascertain the perceptions of icu nurses' about the needs of families of critically ill patients methods. this is a transversal study. the data were collected in four icus in the city of feira de santana, bahia, brazil, after approval by ethics and research committees. all clinical nurses of icus were interviewed. the brazilian adaptation of the critical care family need inventory (inefti) was used for measuring the degree of importance of needs of family members, valued at increasing levels from to . descriptive statistics were used for analysis, needs with a mean score [ were defined as having the greatest importance.results. the nurses identified % of needs as important for family members. the items related to security ''to be sure that the best possible treatment is being offered to the patient'' ( . ± . ), and the information ''to talk to the doctor every day'' ( . ± . ), were pointed out as the most important, with consistent results with the literature. some needs of support ''to have general guidelines on the icu at the first visit'' ( . ± . ) and comfort ''to have a bathroom near the waiting room'' ( . ± . ) were also identified as important. however, the needs of proximity, like being close to their relative was not identified as important to the family for all nurses.conclusions. the nurses identified the need for security and information as important, however the wish that the family has to be near their relative was not considered important, as described in the literature , , . a movement of nursing towards the family can be perceived, so nurses should plan their interventions based on knowledge of the demands of the family in order to promote care for the relief of immediate distress and anguish, which will consequently encourage the recovery of the ill relative reference(s). nurse educator, realized that with an ongoing critical care nursing shortage world wide, even when retention is high, some turnover is inevitable necessitating an orientation tool to guide charge nurses in assigning new hires to critically ill patients. impetus for this clinical orientation tool arose from observations that new hires were often overwhelmed or disengaged at the bedside, and patient assignments did not consistently foster the development of critical care skills. the orientation tool reflected a staged approach to patient assignments; gradually exposing the new hire to progressive levels of complexity. embedded within the tool were guidelines specifying performance competency expected of new hires at month intervals in their first year of critical care practice. evaluation at this stage involved on the spot interviews. use of the tool began in january . since its introduction, this tool has guided patient assignments for newly hired nurses. in six cases, nurses moved through the stages more quickly than anticipated. reports from staff nurses, clinical educator, patient care coordinator and nurse manager suggest that anxiety and stress of novice critical care nurses related to the complexity of patient assignments have decreased, and that the tool's structure provides clear goals and has enhanced satisfaction with the consolidation experience. our goal was to ensure the tool facilitated an optimal learning experience, structured around orientation standards and leading to the development of confident, competent practitioners. future plans for on-going evaluation include: formal surveys with present msicu staff, and exit interviews with nurses new to critical care who have left their msicu positions prior to the introduction of the orientation tool. ( ) has previously been claimed to show an association between improvement of score (or lack of) over time and survival status ( ) . severe sepsis in patients is associated with considerable mortality. activated protein c (apc) is a mediator of the inflammatory and coagulation systems, which has shown decreased mortality in severe sepsis ( ) beyond h sofa scores showed further distinct improvement over the apc infusion period in the survivor group, whereas minimal improvement was seen in the non survivors.change in daily sofa scores conclusions. our analysis appears to agree with levy's previous findings of increasing sofa (albeit modified) scores and mortality. we, however, looked at a different treatment period, (apc infusion rather than first day of sepsis) but, nonetheless, found the same association of increasing score and mortality and decreasing score and survival. the mean age in survivor group was considerably less than that in non survivors and this age discrepancy largely accounts for the difference in mean apache score ( points) between groups. the depicted trend in sofa scores is more apparent beyond the initial h of treatment, and suggests that improving sofa scores and outcome prediction is possible beyond the previously reported h. our numbers are small, but lend support to the predictive potential of repetitive sofa scores and outcome.objectives. this real-life registry was implemented to describe clinical characteristics of patients treated with drotrecogin alfa (activated) (daa) in france and the use of this drug.methods. this national multicenter observational study, proposed to intensive care units (icus) which used daa, was conducted by data abstraction from hospital files of patients admitted in icus and treated by daa. two sets of data were obtained: a) retrospective data collected between january st, and beginning of the prospective phase in each site; b) prospective data for patients treated until november . this current analysis aimed to describe the patients retrospectively enrolled and treated between january and april . statistical analysis was mainly descriptive. conclusions. this study showed a good concordance between the target population and population treated by daa in terms of treating patients with higher disease severity. patients treated in real-life had a particularly severe sepsis as shown by the saps ii score of and high number of organ dysfunctions at time of infusion initiation. the -month observed mortality was lower than the predicted hospital mortality of % with this level of saps ii. introduction. mortality in severe sepsis is variously described, but is often up to % ( ) . activated protein c (apc), a mediator of the inflammatory and coagulation system has shown a decrease of hospital mortality from . to . % ( ) at year post apc we found that patients ( %) out of were still alive ( patients are still less than year post apc and therefore not eligible for consideration). conclusions. this study is limited in size, but demonstrates further favourable evidence to support the administration of apc for patients with severe sepsis, and appears to contradict the cochrane findings ( ) . we have shown that our hospital survival has improved since initial report ( ) . we attribute this improvement in smr to better targeting of apc to the more severely ill septic patients (as evidenced by the increased apache score).longer term survival data was also encouraging, % of our patients were alive at year, post apc. this should be considered against an initial predicted survival to hospital discharge (never mind year) of . %. we find this result very promising, it would appear that initial survival advantage with apc is in fact sustained beyond hospital discharge. objectives. medication errors reported in a self reporting medical incident system were systematically analyzed to identify root causes and obtain preventive measures methods. all medication incidents received within year in a -bed mixed icu, were analyzed by trained persons in analyzing medical adverse events. the systematic approach consisted of five steps. . description of the incident in a causal tree. . all causes were classified into the main categories according to the prisma incident analysis tool (technical failure, organization failure, human failure, patient related and non classified). . all medication errors were categories into the broad stages of medication process (prescription, transcription, preparation, dispensation and administration). . the recovery phase of all near miss were analyzed. . development of an action matrix based on the most suitable solution (equipment, procedures, information/communication, training and motivation) for each root cause. . incidents/near miss were recorded. % were medication or fluid therapy related incidents/near miss. human intervention ( %), verification ( %) and organizational/ protocol ( %) were the most common causes of medication incidents/near miss. % of all errors occurred in the administration phase and % in the prescription phase. the most suitable solution for the recorded medication errors are shown in fig. . conclusions. this systematic approach reveals that introduction of new equipment, such as a patient data management system (pdms), and improvement of the procedures are the most important actions to reduce medication errors in our icu. objectives. this study is a descriptive study which is carried out in order to determine the perspectives of newly graduate and experienced nurses on medication errors working in a military education and research hospital. methods. this study was planned and applied as a descriptive and a cross-sectional study. study was executed at a military education and research hospital in turkey between july and august . totally nurses were involved, of those were newly graduate and were experienced nurses. data collection form which has been prepared by the researches in order to determine the perspectives of nurses on medication errors consists of two parts. the first part consists of questions prepared in order to determine the ages, departments, educational levels, experiences and some informative characteristics of the nurses. in the second part there is questionnaire form on perspectives of the nurses on medication errors which was prepared by gladstone in . the study was applied after written ethical approval of the ethical committee of the military education and research hospital and application permission of the nursing department. the application was realized by surveying on volunteer nurses after making necessary explanations about the aim of the study and the application procedures to the participants. the data were analyzed using percentages, mean ± standard deviation, chisquared test and independent-samples t test. conclusions. in this study among the causes of drug errors; tiredness and exhaustion of nurses is stated in the first place. it is thought that rearrangement of working hours of the nurses, reduction of long working hours by the nursing administration will be effective on preventing drug errors. ( ). to test the basic knowledge and practical implementation of picco measurements by icu personnel. descriptive trial in which (para)medical icu personnel were asked to participate in a written or online survey ( questions based on the information found in the manual of the picco system).results. so far, persons have participated: nurses and medical doctors ( were residents in training), all of them actively working in an icu. in total, % of the respondents knew that a picco co measurement is performed intermittently by transpulmonal thermodilution and on a continuous basis by arterial pulse contour analysis. about % is convinced that a picco measurement is an invasive procedure, while in fact it is considered minimally invasive. opinions are divided upon the indications for picco measurement. some participants do not know that the measurement of extravascular lung water provides valuable additional information in pts with acute hypoxic respiratory failure and some even believe that picco can also measure pulmonary capillary wedge pressures. the basic knowledge on co calibration is insufficient: % do not know that the temperature of the injection fluid should best be below °c and only % know that the volume of the calibration fluid depends on the patients' weight. % faulty believe that the patient has to be in the supine position to perform a measurement and % is not informed on the fact that the co obtained should not differ more than % from the mean co value. only % of the respondents carry out a rapid flush test before each picco measurement and only % know that the calibration fluid has to be injected in less than s to obtain a correct measurement. finally, % believe that it is necessary to input the cvp value to calculate a correct co, although % of the respondents correctly knew what to do in case the delta t°is too small and % could correctly interpret the thermodilution curves displayed in the survey.conclusions. from these data we can conclude that a big variation exists in the knowledge on the basic principles and the practical implementation of picco measurements. some confusion exists with regard to the terminology used. we conclude that (as with any new technique) high quality education on picco measurements is necessary for icu personnel. this education can be facilitated by a good protocol, that can be implemented by nurses and doctors at the bedside to avoid human errors. a.c. beers vu university medical center, intensive care, amsterdam, netherlands health insurance companies in the netherlands sign exclusive contracts with hospitals. patients are more critical and independent. they consciously choose a particular hospital or treatment. this is why our management gave high priority to the subject of customer service in their long term policy plan.objectives. in january a project group was launched, which aimed for a number of specific improvements but also by increasing awareness and enthusiasm for customer experience amongst employees. the project is focused on the experience or perception of patients and visitors.methods. the first step was a baseline measurement by hcg (hospitality consulting group). this measurement included interviews with employees from different icu locations and an online survey that was completed by employees, next of kin and other visitors. this resulted in a high score for commitment of staff towards patients and visitors. remarkably, employees thought that aspects for example reliability, professional care and privacy would be valued higher by customers. they attach more importance to how they are received, to empathy and sympathy. respondents also mentioned other things for improvement for example better signage, improved telephone access, better information about rules and procedures, unambiguity in approach, a better visibility of staff and a pleasant and hygienic department. . several improvements such as product, behavior and environment were achieved: we employed family counsellors, developed an information folder and started a pilot for an improved name badge. we can still make improvements in awareness, behavior and addressing each other on this subject. this year we plan to come to an agreement on standards, competencies and control by means of several management training sessions and workshops for employees. we can measure changes in patient satisfaction by family evaluation surveys or an instrument called netto promotor score. the netto promotor score (nps) indicates how many respondents will recommend our ward or hospital to their family and friends. to quote fred lee: ''if a service is provided as expected, patients or visitors will not remember it, they are merely satisfied. satisfied patients will forget a service quickly, have no story to tell to their family and friends and are not really loyal to your organisation. therefore you must create an unforgettable experience, because an experience that remains in memory, is told to others.'' reference(s). critically ill medical, post-surgical, and trauma patients are at greater risk for hyperglycemia with associated increase in mortality and morbidity. tight glycemic control (tgc) has been well documented as a method to control hyperglycemia by managing blood glucose fluctuations through carefully controlled continuous insulin infusion. in order to determine the amount of time it takes within practice for nurses to implement effectively a tgc protocol within the critical care unit, we conducted a pilot time-in-motion study to elucidate the effect on workload. a time-and-motion study was carried out at a hospital located in london, uk in order to document the time associated with tgc activities. a timing workflow, used to capture the key steps involved with effective tgc implementation when utilizing blood gas analyzers for the determination of whole blood glucose (bg) and the time required to complete each step, was designed and then validated by ccu staff. ccu staff was trained on the timing workflow and mechanism. independent observers shadowed ccu nurses, observing when a blood glucose measurement was taken, which steps were completed, and the length of time required to complete each step. other data such as time of the previous bg measurement and status of the last bg test was collected for analysis purposes. during the past few years, the increased incident rate of medical errors occurring in hospitals under governing of hong kong hospital authority has contributed significant attention from the public and health care policy makers. in such a situation, promote patient safety culture becomes paramount for all health care professionals and hospital settings. interdisciplinary teamwork is important in the intensive care units. the benefits of good teamwork have been well documented in the literatures. they included fewer delays, increase in working moral, increased in job satisfactory and decreased in medical errors. in relation to patient safety, fewer errors occur when there is strong teamwork because patient care activities are planned, well organized and standardized. therefore, substantial attention should be given to decrease of medical errors and nurture patient safety culture within high-risk areas such as icus. objectives.• to examine the perception of teamwork and patient safety culture of doctors and nurses between icus and within icu • to investigate the relationship between teamwork and patient safety culture of icu doctors and nurses methods. a cross-sectional surveyed of doctors and nurses in three intensive care units with various size, level of care and staff to patient ratio of hong kong hospitals. totally icu doctors and nurses have been included in this study. a modification of safety attitudes questionnaire developed by sexton and colleagues in was adopted. results. the overall response rate was . %. there were no significant difference of perception of teamwork and patient safety attitude among studied icu's doctors and nurses. however, icu (a) and icu (c)'s doctors demonstrated more positively and showed significant different in perceptions of teamwork (p = . ; . ) than nurses. regarding patient safety attitude, icu (a)'s doctor also showed significant difference (p = . ) and rated more positively than nurses working in the same clinical area. a highly statistically significant association between patient safety attitude and teamwork was shown in the spearman rho statistics with r s ( ) = . , p = . . conclusions. the rate of agreement on teamwork and patient safety attitude were higher in icu doctors. they were more likely to perceive effective teamwork and patient safety in the working area. nurses tended to rate both items lower. as teamwork has been shown to have strongest relationship with patient safety issues, more attention should be given to improve teamwork for icu nurses. tnf is upregulated within the alveolar space early in the course of ventilator-induced lung injury (vili), and plays a major role in the pathogenesis. we previously found in knockout mice that two tnf receptors play opposing roles during vili, with p promoting but p preventing pulmonary oedema. this suggests that specific blockade of the p receptor within the alveoli is a potential therapeutic strategy for vili. domain antibodies (dabs) are the smallest antigen-binding fragments of the igg molecule, which may have advantages over complete antibodies due to their small size (short half life, enabling regional delivery) and monovalent binding (no receptor cross-linking). objectives. we tested the effects of an intratracheally (i.t.) delivered dab that binds to and inhibits the mouse p receptor (biopharmaceutical r&d, glaxosmithkline), on pulmonary oedema and inflammation during vili. methods. c bl mice were ventilated with a high-stretch protocol (plateau pressure . - . cmh o, tidal volume - ml/kg, peep cmh o, o with - % co ). immediately after the start of high-stretch, mice were given an i.t. bolus of non-specific 'dummy' dab or p -specific dab ( lg in ll) and ventilated for up to h ( -hit model). as a -hit model, ng lps was included in the dab bolus. respiratory elastance (ers) and blood gases were monitored, and bal performed at termination. in the -hit model, lung cell suspension was analysed for intravascular margination of neutrophils (pmn), and bal fluid (balf) assessed for pmn infiltration and alveolar macrophage (am) activation using flow cytometry. results. high-stretch ventilation produced deteriorations in ers and po , and high balf protein in both models. treatment with the p -specific dab substantially attenuated all of these changes in the -hit model (table ). in the -hit model, p blockade prevented deteriorations in ers and po , and significantly decreased pmn margination, intraalveolar pmn infiltration and icam- expression on ams (table ) . introduction. ventilator-induced lung injury (vili) triggers a variety of molecular responses within the lungs. however, the contribution of these pathways to lung repair has not been identified.objective. to identify the molecular mechanisms involved in lung repair after vili.methods. vili was induced in mice by ventilation using high pressures ( cmh o) without peep for min. after this, pressure was decreased to cmh o and peep increased to cmh o for h more to promote lung repair. we quantified histological damage, protein content in alveolar lavage (balf) and different molecules in lung tissue (collagen, matrix metalloproteinases- and - , tnfa, ifnc, il- , il- , mip- and lix) in the different conditions (baseline, after injury and after repair). additionally, survivors and non-survivors to the repair phase were compared. the effects of the differentially released mediators were studied in a wound model using murine alveolar cells cultured in presence of balf obtained from ventilated animals, and human alveolar cells and balf from ventilated patients. results were compared using an anova, with a significance level of p = . . . mice were studied ( at baseline, after injury and after repair). high pressure ventilation caused lung tissue injury, with significant increases in balf protein content, mmp- , mmp- , tnfa and mip- , and a significant decrease in il- . during the repair phase, tissue injury was partially reverted, balf proteins and levels of tnfa decreased, but mmps and mip- persisted elevated. mortality during the repair phase was %. survivors showed lower levels of collagen and higher levels of mmp- ( ± vs. ± units, p \ . ) and mip- ( ± vs. ± pg/mg protein, p \ . ).blockade of mmp- , but not mip- , delayed wound closure in both murine and human alveolar cells cultured in presence of balf from ventilated mice or patients respectively.conclusions. vili is partially reversible by decreasing airway pressures and increasing peep. mmp- promotes epithelial repair.grant acknowledgment. universidad de oviedo (unov- -becdoc), ficyt (cof- - ). introduction. critically ill survivors present significant long-term brain-related morbidity. excessive end-inspiratory stretch during mechanical ventilation (mv), even in healthy lungs, may promote alterations in the local and the systemic inflammatory cascade. the effects of ventilator-induced systemic inflammation on brain structures are unknown. to characterize the role of the ventilatory pattern (high vs. low tidal volume (vt)) in the development of local or systemic inflammatory response and regional neuronal brain activation in rats. brains were processed for c-fos immunohistochemistry, as a cellular marker for activated neurons, in the following regions: thalamus, cerebral cortex, amygdala, hippocampus, hypothalamus, and caudal striatum. data were analyzed using one-way anova (p \ . ). results. map and lung compliance remained stable and in the normal range in both groups. pao decreased and paco increased at h in lvt. mv animals presented high levels of systemic and lung inflammatory mediators compared with baseline levels. hvt significanly increased tnfa and il- in plasma when compared with lvt group. in the lungs mv increased il- , il- , il- b and mip- proteins, irrespective of the vt level (lvt or hvt). mcp- only increased in hvt lungs, while tnfa lung levels are similar in ventilated and non-ventilated animals. a significant increase in the number of c-fos immunopositive neurons was only found in retrosplenial cortex and thalamus in hvt animals as a sign of neuronal activation of those areas. none of these two areas were activated in lvt or in control animals. mechanical ventilation produced a moderated systemic and lung inflammation in the context of a preserved lung function. high tidal volume ventilation promoted differential neuronal activation in the brain compared with lvt animals. these findings suggest a novel cross-talking mechanism between lung and brain in the context of experimental acute lung injury.grant acknowledgment. mec bfu - /bfi, fundació parc taulí. jl-a is senior researcher program i isciii, and ciberes. ( ).in an ex vivo perfused human lung preparation injured by e. coli endotoxin, allogeneic human mscs or the conditioned medium restored normal fluid balance ( ).objectives. we wished to evaluate the potential for mscs to modulate inflammation and enhance repair after ventilator induced lung injury (vili). adult male sprague-dawley rats were anaesthetised, orotracheally intubated and subjected to injurious mechanical ventilation. following the development of vili, animals were recovered and extubated. thereafter the animals received two intravenous injections of mscs ( . million cells) or vehicle immediately post injury and at h. the extent of the inflammatory response and recovery from vili, as measured by systemic oxygenation, respiratory static compliance, lung wet:dry ratio and lung lavage inflammatory cell infiltration, was assessed at h. mscs reduced inflammation and enhanced repair following vili. msc treatment improved respiratory static compliance, reduced total lung water as assessed by wet:dry ratio, and decreased bronchoalveolar lavage total inflammatory cell and neutrophil counts, from , cells/ml to , cells/ml (ci . - . ) (fig. ). there was a trend towards better oxygenation in the msc group.conclusions. these findings demonstrate the potential of mscs to modulate inflammation and enhance repair following vili. further analysis of our work, including bal cytokine assay and histological assessment of injury, will provide insight into the utility of mscs to enhance repair in the lung. to determine the role of vagus nerve signaling in vili and establish whether stimulation of the vagus reflex can mitigate lung injury from high volume ventilation.methods. first we demonstrate that disruption of the cap reflex by bilateral vagotomy results in worsening lung injury in a mouse model of high-volume-induced lung injury. in a clinically relevant rat model of injurious ventilation following hemorrhagic shock/resuscitation (hs; model of lung ischemia/reperfusion injury), we then tested the hypothesis that electrical and pharmacological stimulation of the vagus nerve can attenuate injurious effects of vili. finally, to determine the molecular mechanisms by which stimulation of the cholinergic response mitigates vili, we exposed human bronchial epithelial cells (beas b) to cyclic stretch ( cycles/min, pka) in the presence of specific agonist or antagonist of the subunit of the acetylcholine nicotine receptor. vagotomy exacerbates lung injury from high volume ventilation in mice as demonstrated by increased wet-to-dry ratio, infiltration of neutrophils in bronchoalveolar lavage fluid and lung tissues, and increased tissue levels of interleukin- . vagotomy exacerbated while vagus stimulation attenuates lung injury in rats after ischemia reperfusion injury ventilated with either high or low volume strategies. treatment of both mice and rats with the vagus mimetic drug, semapimod, resulted in decreased lung injury. vagotomy also increased pulmonary apoptosis whilst vagus stimulation (electrical and pharmacological) attenuated vili-induced apoptosis. in vitro studies suggest that vagus-dependent effects on inflammation and apoptosis are mediated via the a nachrc-dependent effects on cyclic stretch-dependent singling pathways c-jun n-terminal kinase (jnk) and fas (tnf receptor superfamily, member ).conclusions. stimulation of the cholinergic anti-inflammatory reflex may represent a promising alternative for the treatment of vili.introduction. so far, histological data on critical illness myopathy (cim) primarily refers to muscle biopsies taken during protracted critical illness (after weeks), repeatedly describing pronounced type-ii muscle fibre atrophy.objectives. we speculate that type-ii fibre atrophy develops during early critical illness in patients with non-excitable muscle membrane which predicts cim. ( ) methods. due to their elevated risk for cim, critically ill patients with sofa scores c on of consecutive days within the first days after icu admission were eligible for inclusion into this prospective, observational study. preexisting iddm or neuromuscular disorder, pregnancy, bmi c kg/m , age \ years, or pretreatment[ days on other icu constituted exclusion criteria. surgical muscle biopsies were taken from vastus lateralis muscles between day and after first sofa c and postprocessed according to standard procedures (isopentane, liquid nitrogen, atpase/toluidineblue staining). we assessed muscle membrane excitability after direct muscle stimulation, abnormal muscle membrane excitabilty indicating cim ( ). after quantifying fibre-type specific median cross sectional areas (csa) with imagej-software, we compared fibre-type specific csa in patients with and without non-excitable muscle membrane. nonparametric tests (mann-whitney u) were used for statistical analyses, results expressed as median and ( th/ th) percentiles for continuous variables. . patients were enrolled and subsequently biopsied. patients were evaluated for muscle membrane excitability, of whom % (n = ) showed non-excitable muscle membrane. reliable csa quantification was obtainable for patients.type-ii but not type-i muscle fibre csa during early critical illness was significantly decreased in patients with non-excitable muscle membrane ( , lm compared to , lm for type-ii, p = . ; , lm compared to , lm for type-i, p = . ; n = ). furthermore, non-excitable muscle membrane was associated with significantly lower mrc scores after end of sedation ( . ( . / . ) vs. . ( . / . ) , p = . , n = ). in patients showing non-excitable muscle membrane after direct muscle stimulation we could observe selective type-ii fibre atrophy as early as within the first days after icu admission (day - after st sofa c ). our findings demonstrate that nonexcitable muscle membrane indeed is associated with a histomorphological correlate previously linked to cim. these results highlight the need to focus on early critically illness in order to investigate pathophysiological aspects of cim. bacterial sepsis is a major threat in neonates born prematurely, and is associated with elevated morbidity and mortality. little is known on the innate immune response to bacteria among extremely premature infants. objectives. identify innate immune defect in premature infants as risk factor for the development of neonatal sepsis. methods. we compared innate immune functions to bacteria commonly causing sepsis in infants of less than wks of gestational age, infants born between and wks of gestational age, term newborns and healthy adults. levels of surface expression of innate immune receptors (cd , tlr , tlr , and md- ) for gram-positive and gramnegative bacteria were measured in cord blood leukocytes at the time of birth. the cytokine response to bacteria of those leukocytes as well as plasma-dependent opsonophagocytosis of bacteria by target leukocytes were also measured in the presence or absence of interferon-c. results. leukocytes from extremely premature infants expressed very low levels of receptors important for bacterial recognition. leukocyte inflammatory responses to bacteria and opsonophagocytic activity of plasma from premature infants were also severely impaired compared to term newborns or adults. these innate immune defects could be corrected when blood from premature infants was incubated ex vivo h with interferon-c. conclusions. premature infants display markedly impaired innate immune functions, which likely account for their propensity to develop bacterial sepsis during the neonatal period. maturation of the innate immune response to bacteria can be induced by interferon-c ex vivo and represents a promising strategy to prevent neonatal sepsis. the anaphylotoxin c a impairs neutrophil phagocytosis in animals and humans with sepsis. although dependency on the phosphoinositol -kinase delta (pi kd) pathway has been identified , , greater understanding of the mechanism will allow novel therapeutic options. objectives. to test the hypotheses that c a mediates its effect on phagocytosis by impairing rhoa activation, and that similar defects will be found in neutrophils from critically ill patients. methods. the mechanism was dissected using an in vitro model of c a-mediated neutrophil dysfunction, treating healthy human donor neutrophils with c a at concentrations found in sepsis ( nm). phagocytosis was assessed by zymosan particle uptake. neutrophils exposed to zymosan were assayed for rhoa activity, a key mediator of actin polymerisation in phagocytosis . phagocytosis by neutrophils from critically ill patients was investigated, looking for correlations with a marker of c a exposure (cd , the main c a receptor) , an examination of the rhoa and actin polymerisation response to zymosan, and the ability of gm-csf to restore phagocytosis ex-vivo. results. c a inhibited phagocytosis of zymosan by healthy donor neutrophils (reducing from to %, p \ . ) and also impaired rhoa activation (figure) . blocking pi kd, using inhibitor ic , prevented the inhibition of rhoa by c a, and prevented the reduction in phagocytosis. treatment with gm-csf restored phagocytosis and rhoa activation (fig. ). neutrophils from patients with critical illness showed a strong correlation between phagocytosis and surface cd expression (r = . , p \ . ), consistent with our previous findings . patient neutrophils failed to up regulate rhoa or polymerise actin in response to zymosan, in marked contrast to cells from healthy donors (p = . and p = . respectively). ex-vivo gm-csf was able to improve phagocytosis by patient neutrophils from to %, p \ . . conclusions. these data demonstrate that c a inhibits rhoa activity through pi kd, inhibiting phagocytosis. gm-csf is able to reverse this inhibition. similar effects are seen in neutrophils from critically ill patients, providing new avenues for therapeutic intervention in critical illness. host infection triggers an innate immune response leading to a systemic inflammatory response, often followed by an immune dysfunction which impairs the lung defence mechanisms in mice and increases susceptibility to secondary p. aeruginosa pneumonia. activation of the toll-like receptor (tlr)-dependent signalling pathways influences the magnitude of the initial pro-inflammatory phase of sepsis. contribution of tlr signaling to the subsequent development of post-infective immunosuppression has been poorly studied.objectives. to investigate the relative contribution of tlr and tlr in lung defence towards p. aeruginosa in the setting of sepsis-induced immune dysfunction. we used wild-type (wt) c bl /j mice and littermates deficient for tlr (tlr ko), tlr (tlr ko) or both tlr and tlr (tlr ko). these animals were subjected to a sublethal polymicrobial sepsis (cecal ligature and puncture, clp) followed by a secondary p. aeruginosa pneumonia at day post-clp . we evaluated -day survival and the lung response and h after instillation through lung histology, quantification of protein level, cell recruitment and myeloperoxydase (mpo) activity. lung expression of tlr , tlr and tlr was assessed through quantitative rt-pcr. bacterial lung clearance was evaluated through quantitative culture of bronchoalveolar lavage fluid (balf). bacteremic dissemination was assessed through quantitative blood cultures. finally, we measured cytokines in the balf and in the whole lung. post-septic wt and tlr ko mice displayed high susceptibility (mortality rate %) towards secondary pneumonia. in contrast, post-septic tlr -deficient mice (either tlr ko or tlr ko), survived the secondary pulmonary infection (mortality rate \ %). in wt mice, clp increased lung expression of tlr , but neither of tlr nor tlr . tlr ko mice displayed improvement in lung bacterial clearance and reduction in bacteremic dissemination as compared to wt mice. with regard to pulmonary inflammation, tlr ko mice displayed decreased alveolar damage. furthermore wt and tlr ko mice displayed differences in the pulmonary release of cytokines. thus tlr ko mice exhibited increased production of tnf-a and ifn-c and a decreased production of il- .conclusions. in a model of polymicrobial sepsis followed by p. aeruginosa pneumonia, tlr deficiency improves survival by promoting efficient bacterial clearance and decreasing pulmonary inflammation. tlr -dependent mechanisms that specifically contribute to lung defence in the setting of sepsis-induced immune dysfunction are currently investigated. infection is a serious complication in critically ill patients, who can be in a state of secondary immunodeficiency due to a severe illness. apart from common nosocomial pathogens, highly unusual microorganisms may be found in these patients, i.e. pathogens whose cultivation requires specific conditions, and/or agents which are difficult to cultivate. molecular biology-based methods (pathogen-specific probes with real-time pcr detection, or universal system pcr detection with subsequent sequenation) make diagnosis faster and more accurate.objectives. i) to assess an agreement of investigation results using classical microbiology techniques and molecular biology-based methods; ii)to evaluate the clinical effect of the diagnoses based on the frequency of changes in antibiotic therapy as a direct result of molecular detection of ''pathogens'' (mpd) and to assess the effect of this change by evaluating a -day trend of inflammatory parameter concentrations i.e. of procalcitonin (pct) and c-reactive protein (crp). a total of samples (blood, bal, tracheal aspirate, urine, cerebrospinal fluid and secretions from abdomen drains and thoracic punctures) were taken from icu patients (aged - ). these were investigated simultaneously, both by classical microbiology and microbial methods, using the system of pathogen-specific probes with real time pcr for agents. each sample was tested simultaneously with the universal pcr detection system for bacteria and fungi with the subsequent sequenation.results. an agreement between the two compared examination methods was found in % of samples and disagreement in % of the samples. % of the results were classified as ''not possible to interpret''. in % of the samples, mbm detected the presence of other agents, which were not confirmed by cultivation. in % of cases, the mdp results did not contribute to the decision to change the atb. in % of cases, a modification of atb treatment followed; a change, reduction or stopping the administration of the drug. in % of cases, atb was changed without any direct connection to the results. in % of patients who underwent the change in atb treatment, a decrease in inflammatory parameters occurred (pct and crp), however, in % there was an increase. the remaining % are divided equally between those ''without any change'' and ''data not available''.conclusions. the advantage of septic-state diagnostics using molecular biology techniques, as opposed to classical microbiology methods, is the fast availability of the examination results ( - h) , and its high sensitivity and specificity. proving the presence of agents in biological material does not necessarily signify its pathogenicity. however, in combination with a thorough assessment of the clinical progression, including laboratory indicators of inflammation, it is of considerable benefit in decisions about the efficacy of antibiotic therapy. common antibiotics and the number of patients on high or low intensity crrt recruited were: ciprofloxacin ( , ) , meropenem ( , ), piperacillin-tazobactam ( , ) , vancomycin ( , ). the clearance of individual antibiotics varied approximately -and fold within a single crrt regimen for high and low flow rates, clearance, estimated using ccrt extraction ratios for the two flow rates, differed significantly: meropenem ( vs. ml/min; p = . ) and vancomycin ( vs. ml/min; p = . ). using dialysate clearances, significant differences for vancomycin ( vs. ( ) objectives. to perform a meta-analysis on incidence and outcome of intra-abdominal hypertension (iah) in different icu populations, the evolution of iap over time and the correlation with organ failure and fluid balance (fb)methods. pts admitted to icu with iap measurements (gastric or bladder) were included. data from existing databases were collected on , pts from centers ( countries [ ] . recently, it was demonstrated that % hes / . induces increased inflammation and leads to more tubular damage compared to % hes / . in an ex-vivo kidney perfusion model [ ] . we investigated whether different hes solutions lead to disturbed cell proliferation or to increased apoptosis in murine kidney cells. we performed a large cohort study on prospectively collected data over a year period ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . data were extracted from the cub-réa network , a french regional database in which icus from paris and its suburb prospectively record data using standardized coding methods. cancer patients with septic shock were selected through keywords including the malignancy status on one hand (''hematological malignancy'', ''cancer'', ''cancer with metastasis''), and ''septic shock'' or the combination of ''septicemia'' and ''shock'' on the other hand. extracted variables included demographic characteristics, type of malignancy, requirements for organ failure supports (vasopressor therapy, mechanical ventilation, renal replacement therapy) and severity-of-illness score (saps , , , , however, some studies have suggested little effect on morbidity or mortality. , clarification of any differences would improve pre-operative risk assessment, providing more information for the clinician and patient. it would also aid resource planning in the critical care unit. we hypothesized a proportional increase in the extremes of bmi occurring over the -year study period. we analysed data collected prospectively between april and april on patients undergoing cardiac surgery in our unit using the patient analysis and tracking system (pats Ò ) database. the patients were grouped according to bmi. for each group we calculated organ specific complication rates, re-operation and readmission rates, itu and postoperative length of stay and overall mortality. we studied the change in mean bmi of patients over the year period. in comparison with normal bmi range ( . - . ), patients with bmi range \ . had significantly increased rates of peri-operative myocardial infarction (mi) ( conclusions. both the high and low extremes of bmi range show significant increases in complication rates compared to normal bmi patients undergoing cardiac surgery. the bmi group - . shows significant increase in re-operation, readmission and mortality rates.there is a year on rear increase in the bmi group [ . this suggests a greater demand for resources particularly in the intensive care unit as the population with a higher morbidity and mortality increases. we would welcome an opportunity to present a detailed analysis of our findings. with advances in critical care medicine, more patients are surviving intensive care units stays. patients admitted to critical care may experience morbidity that affects their life after discharge . in addition to any physical morbidity, treatment in critical care may also be stressful and psychologically traumatic for patients .objectives. to describe the psychological outcome of patients surviving icu admission at months. a second end-point was to find possible relationships between patients' background and intensive care variables, post-traumatic stress and depression disorders.methods. retrospective analysis of data from icu follow-up clinic. data were collected in questionnaires (ptss- and beck inventory) during an interview at months after discharge of icu in the last years. statistics analysis: pearson's chi-square or fisher's exact test, significance for p \ . . . patients were interviewed, mean age was ± , % were male and . % were trauma victims. mean icu length of stay was ± days and sapsii score ± . . % of patients had less than years of education. concerning previous health status . % were healthy and . % were dependent for daily living activities. . % were retired. more than % had new complaints after discharge. only . % of the previously professionally active patients resumed their work and only % of the retired were able to maintain their normal activity. almost % of patients had new psychiatrics symptoms and only . % were being followed by psychiatry: % were on benzodiazepines and . % on anti-depressants. . % of the patients had symptoms suggestive of diagnosis of post-traumatic stress disorder (ptsd) having a ptss- superior to . regarding beck inventory, % were considered to have a depression, with . % having moderate to severe depression. after the interview % were oriented to a psychiatric consultation. % of depressive patients had new symptoms (p \ . ). of the previously active patients who did not resume normal activities, a significant (p \ . ) part was depressed ( . %). the same is true for the retired patients ( . %), p = . . longer hospital and icu lengths of stay were related with development of depression (p = . and p = . , respectively). patients with higher sapsii were more prone to develop ptsd. women had more ptsd than men (p = . ). patients in risk of ptsd and depression were younger (p = . ; p = . , respectively). as with depression these patients also had more new complains and did not return to work. introduction. mild hypothermia improves outcome after cardiopulmonary resuscitation (cpr). modes of action for it are manifold, though one way might be reduction of basal metabolic rate (bmr). therapeutic hypothermia was able to reduce bmr in patients with traumatic brain injury and critically ill patients with fever.objectives. in the present study we investigated the metabolic effect of therapeutic hypothermia in patients after successful cpr.methods. patients after cpr were treated with therapeutic hypothermia ( °c) for h and subsequently rewarmed with a rate of . °c per hour until °c was reached. all patients received standardized sedoanalgetic medication and neuromuscular blockers. indirect calorimetry was performed at , . and °c, as well as between . and . °c and - h after cpr. for statistical analysis repeated measures anova, linear and logistic regression were used. a linear relation between bmr and temperature was detected ( kj/m / . °c; p \ . ). therapeutic hypothermia ( °c) was associated with a reduction of bmr by ± % compared to °c. in this regard no difference was found between patients with good and bad neurological outcome (good outcome vs. bad outcome: ± vs. ± %; p = . ). concerning substrate oxidation rates only fat oxidation rate showed a temperature dependency ( g/day/ . °c; p \ . ). in contrast to protein oxidation rate (good outcome vs. bad outcome: ± vs. ± g/day; p = . ) patients with good neurological outcome had a significantly higher fat oxidation rate (good outcome vs. bad outcome: ± vs. ± g/day; p = . ) and a significantly lower glucose oxidation rate (good outcome vs. bad outcome: ± vs. ± g/day; p = . ) as compared to patients with bad neurological outcome.conclusions. in patients after cpr mild therapeutic hypothermia ( °c) was associated with a reduction of bmr by %. a linear relation between temperature and bmr was detected. fat oxidation rate was temperature dependent in contrast to protein and glucose oxidation rate. a significant difference in glucose and fat oxidation rates was found between patients with good and bad neurological outcome. objectives. our goal was to determine whether its institution after resumption of spontaneous circulation (rosc) improves survival and neurological recovery in an experimental model of cardiac arrest in rabbits.methods. ventricular fibrillation was induced in anesthetized rabbits. after -min of untreated fibrillation, cardiopulmonary resuscitation was attempted using external massage, electric shocks and intravenous epinephrine. after rosc, rabbits randomly underwent either normothermic life support (control group with conventional ventilation until weaning) or hypothermic support with rapid cooling (tlv group). in this last group, a °c hypothermia was induced by -min of tlv using a perfluorocarbon. subsequently, the perfluorocarbon was removed from the lungs and rabbits were conventionally ventilated with maintenance of hypothermia during h. rabbits were further warmed and weaned from ventilation. in both groups, hemodynamic and biochemical parameters were monitored, as well as survival and neurological recovery. after days, survivors were finally euthanized for post-mortem analyses. neurological dysfunction was assessed by a - % scoring system evaluating reflexes, postural reactions and behaviour ( %: no dysfunction; %: brain death).results. ten rabbits were randomized to the control group and to the tlv one. defibrillation was obtained using . ± . and . ± . electric shocks, respectively. subsequent rosc was observed after . ± . and . ± . min, respectively. oesophageal and tympanic temperatures were rapidly reduced in the tlv group, achieving . ± . and . ± . °c within -min versus . ± . and . ± . °c in control, respectively. in the tlv group, rabbits returned to normothermia within - h after the hypothermic episode. throughout follow-up, no significant difference in blood pressure was observed between both groups (e.g., ± and ± mmhg in control vs. tlv at h after cardiac arrest, respectively) whereas heart rate was decreased throughout hypothermia in tlv vs. control (e.g., ± vs. ± beats/min at h following cardiac arrest, respectively). lactates's concentration and epinephrine dosages were not significantly different between groups. importantly, neurological dysfunction was significantly attenuated in tlv vs. control (e.g., ± vs. ± % after h). in control, / ( %) rabbits survived throughout the follow-up and the others died or should be euthanized earlier following severe disability. in the tlv group, survival was significantly increased as / rabbits survived to the entire follow-up ( %).conclusion. ultra-fast cooling induced by tlv after rosc improves survival and neurological recovery following -min of experimental cardiac arrest in rabbits.grant acknowledgment. (ca) . th involves at least h of induced hypothermia ( - °c), mechanical ventilation and sedation. th may affect sedation through changes in pharmacology. still, no clinical studies have investigated the use of sedation during th. objective. to compare the efficacy of two sedation protocols for patients treated with th. methods. open, randomised, controlled, population based study of patients treated with th ( - c for h) after ca in two norwegian university hospitals. patients were randomised to sedation with remifentanil + propofol (rp) or fentanyl + midazolam (fm). baseline characteristics (age, sex, bmi, saps-ii) and cardiovascular variables during study drug infusion (blood pressure, heart rate, use of fluids, vasopressors and inotropic drugs) were recorded. the primary end point was defined as time from stop of sedation to extubation. results. sixty patients were randomised. one patient was withdrawn by next of kin. baseline characteristics were similar in the two groups. for two patients in the rp group, and one in the fm group, study drugs were stopped shortly after allocation due to cardiovascular instability. the rp group had lower heart rates and more patients needed noradrenaline infusions than the fm group ( . ( . ) (mean(sd)) vs. . ( ) beats/min, p = . , and vs. patients, p = . , respectively). other circulatory variables were similar. reasons to not stop sedation or not extubate after stop of sedation were; cessation of icu treatment (n = ), need for mechanical ventilation (n = ), inadequate awakening (n = ), seizures after stop (n = ), and other (n = ). sedation was stopped according to protocol in of patients. median (range) time from stop of sedation to extubation for the patients who could be extubated according to protocol was . ( - . ) vs. . ( . - . ) h in the rp and fm group, respectively, p = . . ''cerebral performance category'' on day - was similar in the two groups. conclusions. time to extubation after cessation of sedation was significantly shorter in patients sedated with rp compared to fm. however, the benefit from a short time to extubation is limited by that only one-third of the patents can be extubated according to protocol. the rp group had lower heart rates and needed more noradrenaline. no major differences were observed for outcome.grant acknowledgment. ntnu. increased blood glucose variability during therapeutic hypo-thermia and neurological recovery after cardiac arrest key: cord- -w ysjf authors: nan title: th international symposium on intensive care & emergency medicine: brussels, belgium. - march date: - - journal: crit care doi: . /s - - - sha: doc_id: cord_uid: w ysjf nan ventriculostomy-related infection (vri) is a serious complication in patients with hemorrhagic stroke. in such patients, diagnosis of vris is complicated by blood contamination of csf following ventricular hemorrhage. we aimed to evaluate the diagnostic potential of white blood cells count (wbc), c-reactive protein (crp), and procalcitonin (pct) to identify vris in patients with hemorrhagic stroke during the time of external ventricular drain (edv) in situ. this retrospective study was conducted at the neurosurgical-icu, university hospital of zurich. a total of patients with hemorrhagic stroke and an external ventricular drain (evd) were admitted over a years period at the icu. of those, patients with vris ("vri"), defined by positive csf bacterial culture and increased wbc in csf (> /ul), and patients without vris and with serial csf sampling ("no-vri") were analyzed. patients with csfcontamination or suspected vri (negative csf cultures but antibiotic treatments) were excluded. wbc, crp, and pct were measured daily. csf was sampled routinely twice a week or by t> °c. for the analysis, mean peak values of wbc, crp, pct during the time of evd in situ were compared between groups (t test). data are expressed as mean with ci %. results: between groups, wbc and crp were similar (wbc: . g/l and . g/ l, p= . and crp: . mg/l and . mg/l, p= . in the group vri and no-vri, respectively) ( figure , panel a and b ). in the group vri, pct was low and significantly lower than in the group no-vri ( . ug/l and . ug/l, p= . in the group vri and no-vri, respectively) (panel c). wbc in csf were similar between groups ( . /ul and . /ul p= . in the group vri and no-vri, respectively). in this study, serum-inflammatory markers were not able to screen patients with vris. their routine measurement should be carefully evaluated. introduction: central nervous system (cns) infections constitute a potentially lifethreatening neurological emergency. patients admitted to the intensive care unit (icu) usually present with a severe disease and organ failure, leading to high mortality and morbidity. we have performed a retrospective analysis during a -year period of patients admitted to a polyvalent icu. clinical, demographic and outcome data were collected to evaluate its clinical impact on the outcome of patients with cns infections. we identified patients with the diagnosis of meningitis, meningoencephalitis and ventriculitis, where the median age was , years (range - ). upon clinical presentation, their most frequent signs were fever ( %), meningeal signs ( %), seizures ( %), and a glasgow coma scale score < ( %). all needed ventilation support and % needed cardiovascular support. a definitive microbiological diagnosis was achieved on patients and antibiotic therapy was adjusted on of them. most common microorganisms were streptococcus pneumoniae (n= ), listeria (n= ) and pseudomonas aeruginosa (n= ) (figure ). other gram negative microorganisms were detected and lead to more adverse outcomes. meningitis was the cause of admission on patients and on a minority (n= ) meningitis was considered to be a secondary diagnosis on patients admitted for other causes (traumatic brain injury, subarachnoid or intraparenchymal hemorrhage, postoperatively of neurosurgical tumor). patients that eventually died had at least one risk factor (age> , immunocompromised due to diabetes, corticotherapy, hiv or heart transplantation). patients admitted to the icu were not so aged, but had some comorbidities and risk factors leading to more uncommon microorganisms, increasing the risk of adverse outcomes. this lead to an increase of mortality: % in the icu and an overall of %. study of selenium levels in unresponsive wakefullness (uws) patients with systemic inflammatory response syndrome (sirs) e kondratyeva , s kondratyev , n dryagina the objective of this study was to evaluate the pharmacokinetics (pk) of levetiracetam (lev) in critically ill patients with normal and augmented renal clearance (arc), and determine if the recommended dosage regimen provides concentrations in the therapeutic range ( - mg/l) [ ] . a prospective observational study was conducted in a tertiary hospital. six blood samples were taken during a dose interval at steady state and lev was quantified by hplc. a population pk study was carried out. statistical analysis was conducted to evaluate the differences in pk between patients with and without arc. the suitability of drug concentrations was also assessed. results: seventeen patients were included, with normal creatinine clearance (crcl) ( - ml/min) and with crcl≥ ml/min (arc). ten patients received mg q h, one mg q h and two mg q h. the data were best fitted to a two-compartment model. figure shows lev concentrations during the dosing interval. mean clearance (cl) was l/h and mean volume of distribution of central compartment (v) was l. interindividual variability was and % for cl and v, respectively. no differences were identified between both groups (p> . ) in pk parameters. no correlation was found between lev cl and crcl. trough levels were below the minimum concentration (c min ) mg/l of the therapeutic range in all patients except . furthermore, between - h % of samples were below the c min . conclusions: administered doses were not able to maintain lev concentrations in the recommended therapeutic range. other dosage strategies, such the extension of infusion time with higher doses, could be evaluated in order to obtain a more favourable profile. no correlation between lev cl and crcl was found. the mechanical properties of muscles such as tone, elasticity, and stiffness are often affected in chronic critical ill (cci) patients. a hand-held device known as the myotonpro demonstrated acceptable relative and absolute reliability in a ward setting for patients with acute stroke [ ] . the technology works on the principle of applying multiple short impulses over the muscle bulk via the testing probe. the aim of our study is to assess the feasibility of objective measurement of muscle tone in cci patients with neurological dynamics and serum biomarkers. the study included cci patients with neurological disorders (stroke, traumatic brain injury, neurosurgical intervention for brain tumors) with more than a -weeks stay in icu. dynamic measurements of the muscle properties were taken on the deltoideus, brachioradialis, quadriceps femoris, gastrocnemius using the myo-tonpro. to identify the leading factor in impaired muscle tone also were measured neurological (s , nse), inflammatory (il- ), bacterial load (pct) biomarkers using elecsys immunoassay and the serum level of microbial metabolites using gc-ms (thermo scientific). results: all patients were divided into groups depending on positive and negative clinical dynamics. significant differences were obtained in parameters characterizing changes in muscle tone of lower limbs -f gastrocnemius (tone) - . vs . hz, r quadriceps femoris (the mechanical stress relaxation time) - . vs . ms (p < . , respectively). some significant correlations between five parameters of muscle tone biomarkers and microbial metabolites were revealed. the results of a quantitative measurement of muscle tone objectively reflect the dynamics of neurological status, which in the future may be promising technique for the personalized approach cci in patients. introduction: changes in hormonal status in patients with unresponsive wakefulness syndrome (uws) remains poorly understood. methods: patients in uws were examined at the period from to . patients ( men) with tbi and patients ( men) after hypoxia. acth, cortisol, tsh, free t and t , sth, prolactin and natriuretic peptide were studied in the period from to months uws. in men, the level of total testosterone, lh and fsh was additionally studied. the obtained data was compared with the uws outcome in - months (crs-r scale assessment). none of the studied hormones of the hypothalamic-pituitary-adrenal axis were a reliable criterion for predicting the outcome of uws. most often and consistently was revealed a tendency of disrupt the rhythm of cortisol secretion, with higher rates in the evening hours. the average value of sth was higher in men with the consequences of head injury who had recovered consciousness than in those who remained in uws. significant decrease in testosterone levels, regardless of age, was found in patients with a consequence of tbi. mean levels of lh were higher in patients with tbi and hypoxia who remained unconscious than in patients who later restored consciousness. the average level of fsh was higher in patients who had recovered consciousness . the increase of natriuretic peptide level was observed both in patients who remained in chronic uws and in those who restored consciousness. no certain endocrine background, characterising this category of patients was found. violations of some hormones secretion rhythms, in particular, cortisol can be considered usual for uws patients, especially in patients with tbi. therapeutic hypothermia has not been used before our research in chronically critically ill (cci) patients. temperature decrease in neuronal cells is a strong signal that triggers endogenic cytoprotection programs using early response genes expression. our goal is to determine influences of craniocerebral hypothermia (cch) on level of consciousness in cci patients. we examined patients with different types of brain injuries. males and females, mean age . ± . . patients were divided into groups: main group - patients (vegetative state (vs) - , minimally conscious state (mcs) - ), comparison group - patient (vs - , mcs - ), groups were equal on main parameters (severity, functional state, comorbidity). patients from main group received courses of cch, duration - minutes, scalp temperature - °С, cerebral cortex cooling up to - o c, session end was without slow reheating period, and session's amount was set -until signs of consciousness recovery. cortex temperature check done noninvasively by using detection of brain tissue emi in shf-range. consciousness recovery in vs and mcs patients controlled using crs-r scale. results: cch sessions significantly increased level of consciousness in vs and mcs patient groups. in vs patients vegetative state increased until minimally conscious state and mcs +, and in mcs group until lucid consciousness (p < . ) (figure ). craniocerebral hypothermia is used in chronically critically ill patients for the first time. our research results demonstrated effectiveness of cch as an additive treatment tool in such patients. this let us optimistically determine the perspective of inclusion of cch method in chronically critically ill patient's rehabilitation to increase level of consciousness. despite the clinical benefit of endovascular treatment (evt) for large vessel occlusion (lvo) in ischemic stroke, space-occupying brain edema (be) represents a common complication during the course of disease. routinely, ct imaging is used for monitoring of these patients, notably in the critical care setting, yet novel and easy bed-side techniques with the potential to reliably predict be without repetitive imaging would be valuable for a time and cost effective patient care. we assessed the significance of automated pupillometry for the identification of be patients after lvo-evt. we enrolled patients admitted to our neurocritical-care unit who received evt after anterior circulation large vessel occlusion. we monitored parameters of pupillary reactivity [light-reflex latency (lat; s), constriction and re-dilation velocities (cv, dv; mm/s), and percentage change of apertures (per-change; %)] using a portable pupilometer (neuroptics®) up to every minutes during the first hours of icu stay. be was defined as midline-shift ≥ mm on followup imaging within - days after evt. we assessed differences in pupillary reactivity between patients with and without be (u-test) and evaluated prognostic performance of pupillometry for development of be (roc analysis). in patients ( women, . ± . years) without be, , assessments were compared to assessments in patients ( women, . ± . years) with be. on day , day , and day after evt, patients with be had significantly lower cvs and dvs, and smaller perchanges than patients without be, whereas lat did not differ between both groups. roc-analyses revealed a significant negative association of cv, dv, and per-change with development of be. conclusions: automated pupillometry seems to identify patients at risk for be after evt. a prospective study should validate whether automated pupillometry harbors the potential to reduce unnecessary follow-up ct imaging. the aim of this preliminary analysis is to detect differences between the qualitative and quantitative evaluation of the pupillary function carried out by doctors and nurses of an intensive care unit (icu) of a tertiary level hospital. secondary purpose is to investigate new indications for the use of pupillometry in a population admitted in icu methods: the study has been conducted (currently in progress) at the intensive care unit and ecmo referral center at careggi teaching hospital (florence; italy). the enrolled patients are adult subjects (> years) with alteration of consciousness defined by a glasgow coma scale (gcs) < , following a primary brain injury and/or the use of sedative drugs. the studied parameters, obtained with neurolight pupillometer ® (id-med, marseille, france) are analyzed, integrated and visual/qualitative evaluation of the pupil function shows a lower reliability if compared to automated pupillometry. the estimated error in the proper determination of photomotor reflex is . % (p< . ). no significant difference is reported between quantitative and qualitative pupillometry in the detection of anisocoria. our preliminary results are compatible with previously reported data [ ] [ ] [ ] , even if there was no difference in anisocoria determination. interestingly, a longer latency period among patients treated with opioids has been observed. other results are still in progress. introduction: due to the dynamic of critical care disease, a rapid bedside, noninvasive and highly sensitive and specific method is required for diagnosis. in this study we set out our experience with trancranial color-coded duplex ultrasound (dxt) [ ] . the dxt study identifies cerebral arteries as well as hemorrhagic phenomenon, hydrocephalus, mass-occupying lesions and midline shift. this is the main difference between dxt and conventional transcranial doppler (dtc) which is a blind study and do not provide any image. descriptive, cross-sectional and observational study from december to june . patients were included. inclusion criteria: neurocritical patients. exclusion criteria: no acoustic window, presence of ultrasound artifacts. data collection was performed. it was used a lowfrequency transducer from . - . mhz with trancranial duplex preset ( figure) . the patterns were defined as normal, vasospasm, high resistance, hypermedia and cerebral circulatory arrest, depending on the cerebral flow velocity, lindegaard ratio (lr) and pulsatility index (ip). results: men ( . %) and women ( . %). average age . ( - ). patients diseases: subarachnoid hemorrhage , traumatic brain injury , av malformation , stroke , hemorrhagic cerebrovascular accident and mass occupying lesions . normal pattern: patients (rel. freq . ). vasospasm: patients (rel. freq . ). high resistance: patients (rel. freq . ). hyperemia: patient (real. freq . ). cerebral circulatory arrest: patient (rel. freq . ) conclusions: dxt should be part of the routine of neuromonitoring, it allows real time images especially useful in unstable conditions. although it will be needed a large amount of patients to be statistical significant, dxt is useful considering a non invasive study, bedside and it allows early identification of different clinic conditions. introduction: embolization of the draining vein during endovascular treatment of arteriovenous malformation (avm) may result in venous outflow obstruction and hemorrhage. anaesthesiologist can use deliberate hypotension to reduce blood flow through avm which may be somehow helpful to prevent this scenario. adenosine-induced cardiac arrest may facilitate the embolization too. the goal of our study was to improve the results of endovascular treatment of avm using adenosine-induced cardiac arrest. methods: after obtaining informed consent patients ( male, female) were selected for adenosine-induced cardiac arrest during endovascular avm embolization. main age was , ± years old. of them were evaluated as iii class asa, as iv. endovascular treatment in all cases was performed under general anaesthesia. propofol, fentanyl, rocuronium were used to induce anaesthesia, then all the patients were intubated and ventilated with parameters to keep etco - mm hg. sevoflurane , - , vol% ( cases) or desflurane vol% ( case) were used to maintain anaesthesia. hemodynamic monitoring consisted of ecg, pulsoximetry, non-invasive blood pressure measurement. onyx or/and squid were used as embolic agents. ct was performed to every patient just after procedure as well as neurological examination. results: adenosine dosage was . - . mg/kg. time of consequent cardiac arrest was - sec. there were cases we administered adenosine for time, in one case we had to administer it twice, in one fig. (abstract p ) . circle of willis and pulsed-wave doppler mode of middle cerebral artery - times and times in one more case as well. hemodynamic parameters recovered without any particular treatment in all the patients. embolization has been performed in all the cases uneventfully. postoperative ct showed no hemorrhage. nobody from investigated group had neurological deterioration in postoperative period. our study shows that adenosine-indused cardiac arrest is not very difficult to perform method and it can be useful during avm embolization. a major risk factor for stroke is atrial fibrillation (af). to treat af anticoagulation is needed. there are now several anticoagulants available. however, a lack of head to head data as well as the absence of accurate techniques makes it difficult to compare them and measure determine there efficacy. stroke is known to produce an abnormal clot microstructure which is a common factor in many thrombotic diseases. this pilot study aims to use a functional biomarker of clot microstructure (d f ) and clotting time (tgp) to investigate the therapeutic effects of different anticoagulants in stroke and af. we recruited patients ( af and stroke & af). two samples of blood were taken: before anticoagulation (baseline) and post anticoagulation ( - weeks) . patients were either given warfarin ( %) or axipaban ( %). d f and tgp were measured and compared before and after anticoagulation. results: warfarin increased t gp ( ± secs to ± secs (p< . )), and decreased d f ( . ± . to . ± . (p< . )). apixaban increased tgp ( ± sec to ± sec (p< . )) but did not change df ( . ± . & . ± . ). interestingly we found that in the apixaban group tgp significantly correlated (p= . ) with blood drug concentration levels. in this study we show that d f and tgp can quantify and differentiate between the therapeutic effects of two different oral anticoagulants. showing that warfarin prolongs clotting and weakens the ability of the blood to form stable clots. conversely apixaban prolongs clotting time but does not affect the bloods ability to form stable clots. this shows the utility of the d f and tgp biomarkers in comparing two different treatment options, something no other current marker has proven able to do. where d f and tgp may prove useful tools in a personalized approach to anticoagulation treatment and monitoring in an acute setting. hospital mortality compared to the model with the original hairscore. patients with poor-grade aneurysm subarachnoid hemorrhage (asah) world federation of neurological surgeons (wfns) grades iv and v, have commonly been considered to have a poor prognosis ( - % mortality). though early intervention and aggressive treatment in neuroicu has improved outcome in the past years, it is controversial because most of the patients left hospital severely disabled. the objective of this study was to investigate the clinical and social outcomes in intracranial aneurysm patients with poor-grade asah underwent different intervention therapies. a single center observational registry of poor-grade asah consecutive patients, defined as wfns grades iv and v, treated at tertiary chilean referral center from december to march were enrolled in this study. the clinical data including patient characteristics on admission and during treatment course, treatment modality, aneurysm size and location, radiologic features, signs of cerebral herniation (dilated pupils), and functional neurologic outcome were collected. clinical outcomes were assessed via gose and and sociooccupational outcome, both at discharge and at months. figure ). % mortality is less than previously reported, and survivors had a favorable recovery, confirmed with neuro psychological test. poor-grade asah patients in our study shows a more positive outcome than previously considered. prognosis of subarachnoid hemorrhage (sah) is scarce, indeed almost half patients die or become severely disable after sah. outcome is related to the severity of the initial bleeding and delayed cerebral infarction (dci). infection and more precisely pneumonia have been associated with poor outcome in sah. however, the interaction between the two pathologic events remains unclear. therefore, we hypothesized that dci may be associated to pneumonia in sah patients. thus the aim of our study was to analyze the association between delayed cerebral infarction and pneumonia in patients with sah. in this retrospective, observational, monocentric cohort study, patients included in the analysis were admitted in neurosurgical intensive care unit or surgical intensive care unit in the university hospital of brest (france) for non-traumatic sah. primary outcome was diagnosis of dci on ct scan or mri months after sah. multivariate analysis was used to identify factors independently associated with dci. a total of patients were included in the analysis (female male ratio / , median age [ - ] years). multivariate analysis was adjusted on sedation, intracranial surgery, fisher classification of sah severity, pneumonia occurrence and non-pneumonia infectious event occurrence ( figure ). pneumonia occurred in patients ( . %) and other causes of infections in patients ( . %). dci was found in patients ( . %). factors independently associated with dci were pneumonia (or . [ . - . ]; p= . ) and non-pneumonia infectious events (or . [ . - . ]; p= . ). interestingly severity table (abstract p ). correlation of safety and efficacy markers of thrombolysis and thrombolysis time with distance from stroke centre results expressed as odds ratio with % confidence interval of initial bleeding evaluated by fisher scale was not independently associated with dci. dci is independently associated with the occurrence of pneumonia or other cause of sepsis. those results may highlight the need for rigorous approach for prevention protocol, early diagnosis and treatment of hospital acquired infectious diseases in sah patients. introduction: traumatic brain injury (tbi) can have devastating neurological, psychological and social sequelae. increased psychiatric morbidity after tbi has been shown in both adult and the pediatric population. also, critical illness as such is a risk factor for psychiatric problems in youth. our aim was to assess risk factors for later being prescribed psychiatric medication in survivors of intensive care unit (icu)-treated pediatric tbi. we used the finnish intensive care consortium (ficc) database to identify patients - years of age, treated for tbi in four icu in finland during the years - . we examined electronic health records and ct scans and collected data on drug prescription after discharge. we used multivariable logistic regression models to find statistically significant risk factors for psychiatric drug reimbursement. we identified patients of which patients received psychiatric drug prescription ( %) during follow up. the median time to prescription was months after tbi (interquartile range [iqr] - months). patients received antidepressants, received stimulants and received antipsychotics. increasing age showed a positive association with all drug prescriptions except for stimulants, where an inverse relationship was observed (table ) . using multivariable analyses, we could not find any admission or treatment related factors that significantly associated with being prescribed psychiatric medications. teenage survivors with moderate disability (glasgow outcome scale [gos] ) showed high numbers of psychotropic drug utilization ( % received any medication, % received antidepressants, % received antipsychotics). our data suggests, that the risk of psychotropic drug prescription after tbi depends on factors other than those related to injury severity or treatment measures. the incidence of drug prescription is especially high in patients with moderate disability. the effects of -adamantylethyloxy- -morpholino- -propanol hydrochloride on the formation of steroid neurotoxicity in rats with brain injury a. semenenko , s. semenenko , a. solomonchuk , n. semenenko depending on the nature of the brain injury and the severity of the victims, mortality in traumatic brain injury (tbi) ranges from to % [ ] . one of the targets for pathogenetic influence on the course of tbi is the use of pharmacological agents that are able to counteract the negative effects of excess concentrations of glucocorticoids on brain. the therapeutic effect of new pharmacological derivative adamantylethyloxy- -morpholino- -propanol hydrochloride (ademol) in rats with tbi was evaluated for days. the pseudoperated animals and control group received . % nacl solution and the comparison group received amantadine sulfate. cortisol levels were used to determine the efficacy of the test drugs in tbi. in rats treated with ademol, the level of cortisol in the blood ranged from to ng/ml (p -p ) and was . -fold lower (p< . ) compared to control pathology group on the day of therapy. instead, the effect of amantadine sulfate on the level of cortisol in the blood was significantly less than that of ademol. the concentration of cortisol in rats with amantadine sulfate in the blood ranged from - ng/ml (p -p ), was . times lower (p< . ), compared with the control pathology group, and by . % (p< . ) exceeded the corresponding value in animals treated with ademol. therapeutic treatment of rats with severe tbi with a solution of ademol, preferably better than rats in the group with . % nacl and amantadine sulfate protect the brain from the formation of steroid neurotoxicity by cortisol (p< . ). although cerebrovascular pressure reactivity (prx) well correlate to patient's outcome [ ] , it requires continuous monitoring and mobile average calculation for its determination. we therefore hypothesized that a simplified model of variation between mean arterial pressure (map) and intracranial pressure icp over the first three days of admission would have been able to predict patient outcome: we call this new parameter cerebrovascular pressure correlation index (cpc). we performed a retrospective observational study of all adult patients with severe tbi admitted to icu from january to april inclusive. all consecutive patients with a clinical need for icp monitoring were included for analysis. both for icp and map data were mean value over -hours registration, for a total of observations/day, cpc was therefore calculated as the pearson correlation coefficient between icp values (x axis) and map values (y axis), obtaining one single value every hours. variables included in the model (i.e. cpc, cpp, icp, systemic glucose, arterial lactate, paco , icp, and internal body temperature) were collected for the first days since trauma. for the main outcome only the minimum value of cpc fit the regression analysis (p = . ). the correspondent roc curve showed an auc of . . the associated youden criterion was ≤ . (sensitivity = . ; specificity = . ). of all the variables considered for the secondary outcome only cpcmin fit the regression model (p = . ). table reports the median and iqr range for sg and nsg of all the variables considered in the model. this observational study suggests that cpc could be a simplified model of variation between map and intracranial pressure icp over the first three days of admission predicting patient outcome. introduction: impaired cerebrovascular reactivity (car) after traumatic brain injury (tbi) is a marker for disease severity and poor outcome. it is unclear how dynamic changes in body temperature and fever impact car and outcome. we calculated the pressure reactivity index (prx) using the center-tbi high-resolution intensive care unit cohort, as a moving correlation coefficient between intracranial pressure (icp) and mean arterial pressure (map). minute and hourly values of prx and temperature were averaged in patients with simultaneous recording of icp and abp. demographic data was based the core registry (v . ). linear mixed models were calculated based on minute-by-minute data using r with lme v . - and ggeffects v . . . generalized estimating equation models were used to analyze changes during effervescence (increase of temperature of > °c within hours). we assessed high frequency physiological data during days of patients admitted to the icu with predominantly a closed injury type (n= / ). median age was years (iqr - ), baseline gcs was (iqr - ), and % had at least one unreactive pupil. the main measurement site for temperature was the urinary bladder / ( %). half of the patients ( / ) developed fever(> h with mean t ≥ . °c) with a total of h fever and a median of h fever(iqr - ) per patient. of effervescence episodes ( %) reached the febrile threshold of . °c which was associated with an increase in prx from . (±sd . ) at baseline ( h before) to . (±sd . ) during the febrile peak (p= . ) (figure -a) . linear mixed models showed a quadratic relationship between prx and temperature (p< . ) with an increase in predicted prx with febrile and hypothermic temperatures ( figure b ). the association of increasing body temperature with worsening of car supports prevention of fever in severe tbi. prospective studies are needed to further differentiate between mechanisms involved (i.e. inflammation) and central autonomic dysregulation. fig. (abstract p ) . the patients with a good -month outcome (gose> ) after severe traumatic brain injury showed an increase in root mean square of successive differences between normal heartbeats (rmssd) (compared to baseline -minutes before tracheal succtioning) acute kidney injury (aki) is relatively common in patients with severe traumatic brain injury (stbi) and it can contribute to morbidity and mortality [ ] . nephrocheck is a point-of-care urine test that flags two biomarkers that indicate if a critically ill patient is at risk for aki. we investigated the incidence of subclinical aki in patients with stbi. we performed a prospective observational study of all adult patients with severe tbi admitted to icu from january to april inclusive. all consecutive patients with a clinical need for icp monitoring were included for analysis. urine samples of severe tbi patients was collected at icu admission from patients to measure nephrocheck (nc) test [igfbp ] x was performed using the nephrocheck® astute ™ meter. serum creatinine was collected at admission, during the first three days, at icu dismission and -days follow up to assess renal recovery. the diagnosis of aki was based on kdigo criteria. hemodynamics, electrolytes, peep, p/f, kind of fluid administered, fluid balance, % fluid overload, length of stay, the sequential organ failure assessment score, injury severity scores and mortality were collected. a total of patients ( %) presented a median nc higher values at icu admission. one patient with positive nc value experienced aki at hrs. the positive nc group had more plasma transfusion (p-value . ) and a lower median hematocrit at hrs (p-value . ), but similar hospital length of stay (p= . ) and mortality rate (p= . ) conclusions: nc at icu admission identifies subclinical aki in tbi patients and it maight be used to predictclinical aki. hemodilution (but not fluid overload) seems to be associated with development of subclinical aki. higher nc at icu admission is not associated with worst longterm outcome in tbi patients. severe traumatic brain injury (tbi) is considered a serious public health problem in europe. partly because of the heterogeneity of tbi, considerable uncertainty may exist in the expected outcome of patients. the international mission for prognosis and analysis of clinical trials in tbi (impact) and the corticosteroid randomization after significant head injury (crash) prediction models are considered the most widely validated prognostic models [ , ] . however, studies using these prediction models for benchmarking of outcomes have been scarce. we aimed to compare actual outcomes in a tbi cohort of critically ill tbi patients with predicted outcomes in a quality of care initiative in an academic hospital. in this retrospective cohort study, we included consecutively admitted tbi patients to the icu adults of erasmus mc, university medical center, rotterdam, the netherlands between january and february . we included patients with tbi. -day mortality was %, sixmonth mortality was % and six-month unfavourable outcome was %. the impact core+ct+lab model predicted % -month mortality (vs % actual, p= . ) and % unfavourable outcome (vs % actual, p= . ). the -day mortality prediction by crash prognosis calculator was % versus actual -day mortality of only % (p= . ), whereas -month unfavourable outcome prediction by crash was % (vs. % actual, p= . ) ( figure ). the impact model, although developed more than a decade ago, seemed appropriate for benchmarking purposes in this single center cohort in the netherlands, while crash predictions were less applicable to our setting. introduction: out of hospital cardiac arrest (ohca) continues to be associated with significant mortality and morbidity. centralisation of care has considerably improved patient survival but has resulted in increased morbidity in the form of neurological deficit. accurate neurological prognostication remains challenging incorporating repeated clinical examination and ancillary investigations [ , ] . data was collected retrospectively and analysed for patients admitted post ohca from october to october . patient arrest demographics were collected in conjunction with extensive inpatient investigation findings including ct, traditional pupil assessment, pupillometry and eeg. results: % of patients survived to hospital discharge. patients presenting in a shockable rhythm continue to have higher survival rates ( table ) . % of patients who received immediate cpr survived to hospital discharge in comparison to % of patients who did not receive immediate cpr. % of patients underwent non-contrast ct head. % of patients had traditional pupillary examination performed on arrival. pupillometry was introduced in december ; out of a possible patients had pupillometry during their inpatient stay. eeg was undertaken in % of cases. our data shows receiving immediate cpr and presenting with a shockable rhythm remain positive prognostic factors. ct head as a stand-alone prognostic modality is unreliable with % of patients who survived to discharge, with intact neurology, had an admission ct head reported as hypoxic brain injury. a new neuroprognostic strategy is required in our unit that adds further certainty to likely clinical outcome. this includes increased use of tests such as eeg and pupillometry and the introduction of biomarkers such as neuron specific enolase, somatosensory evoked potential testing and magnetic resonance imaging. introduction: post-resuscitation care of patients following an out-of-hospital cardiac arrest (oohca) is set out by the uk resuscitation council [ ] . this is in line with the european resuscitation council guideline [ ] . the aim of this audit was to review compliancy to this guideline at the intensive care unit at the bristol royal infirmary . a retrospective audit was performed over a six-month period in adults who were admitted to the intensive care unit at the bri following an oohca whom later died during that admission ( patients). the focus was on whether the neuroprognostication and end-of-life (eol) care received was as per the standards set by the uk resuscitation council. the main neuroloical examinations documented were pupillary reflex ( %), corneal reflex ( %) and motor response to pain ( %). . % of patients received an ssep analysis > hours post-rosc, . % underwent an eeg and . % had > serum neuron-specific enolase measurements recorded. all patients ( %) underwent a ct head during their admission. . % of patients were referred to palliative care during their admission. % of patients were prescribed all eol medications. most common prescriptions included alfentanil ( . %) and midazolam ( . %). finally, % of appropriate patients were referred to be potential organ donors. the audit reflected our local practice and that some parameters were not being maintained as set by uk resuscitation guideline. multiple introduction: the prognostication of neurological outcome in comatose out-ofhospital cardiac arrest (ohca) patients is an integral part of post cardiac arrest care. biochemical biomarkers released from cerebral cells after hypoxic-ischemic injury represent potential tools to increase accuracy in predicting outcome after ohca. currently, only neuronspecific enolase (nse) is recommended in european prognostication guidelines. in this study, we present the release dynamics of gfap and uch-l after ohca and evaluate their prognostic performance for long-term neurological outcome in ohca patients. serum gfap and uch-l were collected at , and h after ohca. the primary outcome was neurological function at -month follow-up assessed by cerebral performance category scale (cpc), dichotomized into good (cpc - ) and poor (cpc [ ] [ ] [ ] . outcome prognostic performance was investigated with receiver operating characteristics (roc) by calculating the area under the receiver operating curve (auroc) and compared to nse. results: of included patients had at least one serum gfap or uch-l value at , or h after ohca. gfap and uch-l levels were significantly elevated in patients with poor outcome. gfap and uch-l discriminated excellently between good and poor neurological outcome at all time-points (auroc gfap . - . ; uch-l . - . ) and overall predictive performance measured by auroc of gfap and uch-l was superior to nse (auroc . - . ) ( figure ). however, the roc at the highest specificities of uch-l and gfap overlap those of nse and comparing the sensitivities for uch-l and gfap with those of nse for the highest specificities (> %) revealed higher sensitivities for nse than for uch-l and gfap at and h. gfap and uch-l predict poor neurological outcome in patients after ohca excellently and with a higher overall accuracy than nse, but both biomarkers perform inferior to nse at specificities over % at and h limiting their clinical use to guide decisions on prognosis. blood pressure after cardiac arrest and severity of hypoxicischemic encephalopathy c endisch , s preuß , c storm introduction: blood pressure management in post cardiac arrest (ca) patients ensures sufficient cerebral perfusion to avoid secondary brain injury. in local chain-of-survival improvements affect p-ohca survival [ ] [ ] [ ] [ ] [ ] . also initial rhythm in p-ohca is an important predictor of survival [ , ] . little is known about the relationship between initial rhythm in p-ohca and long-term outcome [ ] [ ] [ ] . our aim was to establish the relation between shockable rhythm and favorable long-term outcome in pohca. all children aged day- years who experienced non-traumatic ohca between - and were admitted to the sophia children's hospital in rotterdam were included. long-term outcome was determined using a pediatric cerebral performance category score at the longest available follow-up interval. the primary outcome measure was survival with favorable neurologic outcome, defined as pcpc - or no difference between pre-and postarrest pcpc. the association between shockable rhythm and the primary outcome measure was calculated in a multivariable regression model, adjusted for the pre-defined variables. from the patients included in the year study period ( %) patients survived to hospital discharge of which patients ( %) had favorable neurologic outcome (median follow-up duration of months). the rate of favorable neurologic outcome rose from % in to % in (p < . for trend) (fig. ) the odds of favorable neurologic outcome at the longest follow-up duration were significantly higher after a shockable initial and unknown rhythm. secondly, trend analysis showed an increase in aed defibrillation and shorter cpr duration. this was followed, finally, by a rise in rosc, survival to hospital discharge and favorable neurologic outcome rate. low socioeconomic status is associated with worse outcome after cardiac arrest. this study aims to investigate if patients´socioeconomic status impacts the chance to receive early coronary angiography after cardiac arrest. in this nationwide retrospective cohort study, patients admitted alive after out-of-hospital cardiac arrest (ohca) and registered in the swedish registry for cardiopulmonary resuscitation were included. individual data on income and educational level, prehospital parameters, coronary angiography results and comorbidity were linked from other national registers. in the unadjusted model there was a strong correlation between income level and rate of early coronary angiography where % of patients in the highest income quartile received early angiography compared to % in the lowest income quartile. when adjusting for confounders (educational level, sex, age, comorbidity and hospital type) there were still higher chance of receiving early coronary angiography with increasing income, or . (ci . - . ) and . (ci . - . ) for the two highest income quartiles respectively compared to the lowest income quartile. when adding potential mediators to the model (initial rhythm, location, response time, bystander cardiopulmonary resuscitation and if the arrest was witnessed) no difference in early angiography related to income level where found. the main mediator was initial rhythm (figure ). higher income is strongly related to the rate of early coronary angiography after ohca. this finding is consistent when adjusting for known confounders. however, the association between income and early angiography seems to be mediated by initial rhythm. patients with low income more often presents with non-shockable rhythms which lowers the likelihood to undergo early coronary angiography. a. the total amount of mortality as a stacked bar: in light-red the number of patients who deceased at scene, in green the number of patients deceased during admission, in red patients who died after discharge. the grey line is the total number of inclusions. b. the rate of bystander aed use, rate of initial shockable rhythm, rate of less than minutes of cpr and rate of favorable neurologic outcome over time. p for trend significant for bystander aed use, less than minutes of cpr and favorable neurologic outcome. trend analysis performed using binary logistic regression for dichotomous data (and a kruskal-wallis test for non-normally distributed continuous data) effect of simulation teaching of cardiopulmonary resuscitation for nursing v spatenkova introduction: simulation teaching is a modern type of critical care (cc) education. the aim of this study was to assess the effect of simulation teaching of cc on a comparison of final examination in different model levels of cardiopulmonary resuscitation (cpr) after the first (cc ) and third, final cc . the success rate of cpr was tested in prospective study ( ) ( ) on two groups with a total of students in cc and cc at the faculty of health studies. three semester of undergraduate nursing simulation education (lectures and training) used the laerdal simman g. quality of cpr was evaluated according to parameters: compression depth, compression rate, chest release and time of correct frequency. we tested if cpr quality differed between the two groups. for the compression depth and compression rate parameters, first the conformity of variance was verified and then two-sample t-test. as the chest release and time of correct frequency are recorded as percentages, the wilcoxon rank-sum test was conducted for these parameters. to ensure good resuscitation, all recorded parameters must be properly performed during resuscitation. thus, pivot tables were used to generate statistics and test if the number of correctly performed resuscitation parameters for cc and cc differ. the compression depth parameter was statistically significantly higher for the cc than for the cc (p= . ). there were no differences in compression rate (p= . ), chest release (p= . ) and time of correct frequency (p= . ). it was also tested how many of the parameters were performed correctly by students at cpr. the chi-square test shows the relative frequency of cpr success is higher for the cc group than for the cc group. at least out of parameters were correctly performed by % of cc students compared to % of cc students. the study showed a significant improvement of cpr in the final cc and supported the three semester simulation education. changes in blood gases during intraoperative cardiac arrest jj wang, r borgstedt, s rehberg, g jansen protestant hospital of the bethel foundation, anaesthesiology, intensive care and emergency medicine, transfusion medicine and pain therapy, bielefeld, germany critical care , (suppl ):p introduction: blood gas analysis (bga) is a common approach for monitoring the homeostasis during surgery. while it is well known that cardiac arrest (ca) leads to circulatory collapse and disturbances in homeostasis, little is known about changes of blood gas during peri-operative ca. we retrospectively analysed patients ≥ years who suffered from peri-operative ca during non-cardiac surgery from / to / . peri-operative ca was defined as need for cardiac compression during anaesthesia care. collected data included ph, paco , pao , return of spontaneous circulation (rosc) and -day mortality after ca. within the study period, we observed peri-operative ca (m= , f= ; age ± ) during anaesthesia procedures (rosc occurred in patients ( %). days after ca, the mortality was % (n= ), % (n= ) were discharged, and % (n= ) still in hospital. % (n= ) of ca patients had an invasive blood pressure monitoring, % (n= ) had bga before and % (n= ) during peri-operative ca. prior to ca, the average values were: ph . ± . , paco ± and pao ± . during ca, the average values were ph . ± . , paco ± and pao ± . table shows the distributions of blood gas before and during ca. there were no statistical differences between the groups (ph: p= . ; paco : p= . ; pao : p= . ). hypercapnia and respiratory acidosis is common in peri-operative ca. these data suggests inadequate ventilation during peri-operative resuscitation. further studies should focus on its impact on the outcome. ]. comparing cases with and without rosc, there were significant more diagnostics done in the group without rosc but more therapeutic consequences seen in the rosc-group (table ) . icu-ca is frequent. diagnostics to detect reversible causes of ca were used rarely in icu-ca ( %), even in patients without rosc. notably, diagnostics often had therapeutic consequences particularly in rosc. further studies are required to define standardized diagnostic algorithms during icu-ca. continuous monitoring of cardiac patients on general ward were improved short term survival of in-hospital cardiac arrest uj go introduction: the importance of early detection in the in-hospital cardiac arrest (ihca) is emphasized. previous studies have reported that clinical outcomes are improved if ihca is witnessed, or if a patient admitted to a monitored location [ , ] . this study aimed to evaluate the association between continuous monitoring and survival of ihca on general ward. a retrospective cohort study of ihca in patients admitted to ward at an academic tertiary care hospital between january and december was performed. the primary outcome was return of spontaneous circulation (rosc). the secondary outcomes were hour survival and survival to hospital discharge. (table ) . cardiac patients with continuous monitoring on general ward showed improving rosc and -hour survival but not survival to hospital discharge in ihca. in-hospital cardiac arrest is associated with poor outcomes. although steroids are frequently used in patients with septic shock, it is unclear whether they are beneficial during cardiac arrest and after return of spontaneous circulation (rosc). of cardiac arrest patients evaluated, were enrolled. advanced life support was conducted according to the resuscitation guidelines. forty-six patients were randomly assigned to receive methylprednisolone mg during resuscitation, and to receive saline (placebo). after resuscitation, steroid-treated patients received hydrocortisone mg daily for up to days, followed by tapering . there was no significant difference between the two groups in scvo andall the secondary outcomes (p> . for all comparisons). the present study found no significant physiologic benefit of corticosteroid administration during and after resuscitation in hospitalized patients with cardiac arrest. the experiences of ems providers taking part in a large randomized trial of airway management during out of hospital cardiac arrest, and the impact on their views and practice. results of a survey and telephone interviews m thomas introduction: the aim is to explore ems experiences of participating in a large trial of airway management during out-of-hospital cardiac arrest (air-ways- ), specifically to explore: . any changes in views and practice as a result of trial participation. . experiences of trial training. . experiences of enrolling critically unwell patients without consent. . barriers and facilitators for out-of-hospital trial participation. an online questionnaire was distributed to ems providers who participated in the trial. in-depth telephone interviews explored the responses to the online questionnaire. quantitative data were collated and presented using simple descriptive statistics. qualitative data collected during the online survey were analysed using content analysis. an interpretive phenomenological analysis approach was used for analysis of qualitative interview data results: responses to the online questionnaire were received from % of airways- study paramedics and study paramedics were interviewed. paramedics described barriers and facilitators to trial participation and changes in their views and practice. the results are presented in five distinct themes: research process; changes in views and practice regardingairway management; engagement with research; professional identity; professional competence. conclusions: participation in the airways- trial was enjoyable and ems providers valued the training and study support. there was enhanced confidence in airway management as a result of taking part in the trial. study paramedics expressed preference for the method of airway management to which they had been randomized. there was support for the stepwise approach to airway management, but also concern regarding the potential to lose tracheal intubation from 'standard' paramedic practice. causes of medical care-associated cardiac arrest on the intensive care unit s entz introduction: cardiac arrest on intensive care unit (icuca) following therapeutic interventions is of imminent importance, because the interventions are comparatively predictable and precautions can potentially be taken. this study investigates medical care associated complications that led to icuca. intensive care database was screened for patients ≥ years who experienced icuca in a tertiary hospital with five icu (two medical, two surgical, one interdisciplinary, with a sum of icu beds) in germany from - . icuca was defined as receiving chest compression and/or defibrillation after admission on icu and classified as "medical care associated" if it was preceded by a therapeutic intervention (i.e. induced by medication, bedding procedures, iatrogenic injuries, procedure associated). subgroups included patients with recurrence of spontaneous circulation (rosc) vs. no-rosc and patients with vs. without vasopressor therapy before intervention. there were icuca in patients of totally , icu patients. medical care associated complications leading to icuca were detected in cases ( %) [incidence . / , (ci . - . )]. icuca following therapeutic interventions occurred because of circulatory insufficiency [n= ( %)], respiratory failure [n= ( %)] and airway associated problems [n= ( %)]. nine of the patients ( %) with care-associated icuca died. table demonstrates therapeutic interventions followed by icuca. care-associated complications were common reasons for icuca. most of events were induced by circulatory insufficiency due to induction of anaesthesia and bedding procedures. further investigations should focus on preventive strategies, such as vasopressor infusion before therapeutic interventions. in-hospital cardiac arrest (ihca) is a lethal event. however, ihca has received less attention than out-of-hospital cardiac arrest (ohca). there have been some studies on ihca; however, there is a lack of information on the evidence and clinical features of ihca compared with information for ohca. we therefore conducted this study to clarify important aspects of the epidemiology and prognosis of ihca in patients with code blue activation. we carried out a retrospective observational study of patients with code blue events in our hospital during the period from january to october . we obtained information on the characteristics of patients including age and gender, ihca characteristics including the time of cardiac arrest, event being witnessed, presence of bystander cardiopulmonary resuscitation (cpr), initial shockable rhythm, vital signs h or h before cardiac arrest, survival to hospital discharge (shd), and the cardiac arrest survival postresuscitation in-hospital (caspri) score. the primary endpoint was shd. we performed univariate and multivariate logistic regression analyses. a total of code blue events were activated during the study period. finally, patients were included in this study. overall, the shd rate was . %. the median time of cpr was min (interquartile range, - min). the rate of initial shockable rhythm was . %. there were significant differences in cpr duration, shockable rhythm, and caspri score between the shd group and non-shd group by univariate-logistic regression analysis. caspri score was found to be the most effective predictive factor for shd (or= . , p= . ) by multivariate-logistic regression analysis. our results demonstrated that caspri score is associated with shd in cpa patients with in-hospital code blue events. caspri score in ihca patients would be a simple and useful adjunctive tool for management of post-cardiac arrest syndrome (pcas). peri-operative cardiac arrest in prematurityincidence and causes at a tertiary care hospital between - g jansen, j popp, e lang, r borgstedt, b schmidt, s rehberg protestand hospital of the bethel foundation, anaesthesiology, intensive care and emergency medicine, bielefeld, germany critical care , (suppl ):p the peri-operative care of premature pediatric patients requires special expertise and is therefore reserved for specialized centers. although premature birth is described as a risk factor for peri-operative complications and cardiac arrest (poca) there are no data on its incidence and causality in this particular population [ ] . the present study investigates the incidence and causality of pediatric poca at a tertiary care hospital and level i perinatal center in germany. in the anesthesia database of the study center, all anaesthesiological procedures in patients < years of age were examined for poca in preterm infants (gestational age < th week of gestational age) between and . the peri-operative period was defined between the beginning of anesthesiological care up to minutes after anesthesia and/or sedation. we defined cardiac arrest as the necessity of chest compressions. the perioperative phase and the cause of the poca, gestational age and birth weight were recorded. between and , ( . %) of the , pediatric anesthesiological procedures were performed on premature infants. in total, poca occurred in of these patients (f= , m= ; average gestional age ± days; average birth weight ± g (incidence . %, ci . - . %). the time of occurrence and the causes of poca are shown in table . poca in premature babies is rare and has an incidence of . %, which is significantly higher than the non-premature babies. the main causes are problems or complications associated with the respiratory tract and its management, as well as massive hemorrhage. introduction: peri-operative cardiac arrest (poca) in children's anesthesia care is a dreaded event. depending on the country and population, studies describe incidences between . - . per , children's anesthetics. there are no data on the current incidence of pediatric poca in germany. the present study investigates the incidence of poca at a tertiary hospital and level i perinatal center in germany. in the anesthesia database of the study center, all anaesthesiological procedures in patients < years were examined for poca. the peri-operative period was defined between the beginning of anesthesia care up to minutes after anesthesia or sedation. cardiac arrest was defined as the necessity of chest compressions. age, weight, asa status, cause of death and survival after days were recorded. results: poca (median weight was g [q ;q ( )]) were observed in , anaesthesiological procedures (incidence . ± . per , [ci . - . ]). table shows the distribution of the individual age groups, incidences and mortalities of poca. peri-operative -day mortality was per , [ci [ ] [ ] [ ] [ ] [ ] . three children died intraoperatively as a result of hemorrhagic shock, one on the picu as a result of malignant hyperthermia. days after poca, more children had died on the icu due to their underlying disease. poca is a rare event. risk factors are an age < days and an asa status ≥ iii. the main cause of peri-operative death in patients < years of age is massive hemorrhage, the -day mortality is determined by the underlying disease. in-hospital cardiac arrest -predicting adverse outcomes t partington, j borkowski, j gross northwick park hospital, anaesthesia/critical care, london, united kingdom critical care , (suppl ):p introduction: cardiac arrest occurs in . per hospital admissions in the uk. return of spontaneous circulation (rosc) is achieved in approximately half of resuscitation attempts, but rate of survival to hospital discharge is substantially lower [ ] . in our centre, post-arrest care accounts for . % of icu admissions. premorbid social function is purported to affect outcomes, but comorbidity scores are more often used for risk stratification. using a novel social function score alongside an existing comorbidity scale, we aimed to identify trends to inform management of patients at risk of deterioration. a six-month prospective observational study was conducted in a major uk hospital from october to april . for all adult inpatient cardiac arrests, medical notes were reviewed and data collected on the following domains: patient demographics comorbidities and functional status admission details post-arrest events statistical analysis was performed using student's unpaired t-test. results: cardiac arrests occurred. % were in medical patients, with the majority male ( %) and aged over ( %). % were emergency admissions, with mean duration of hospital stay pre-arrest days. in cases ( %) sustained rosc was achieved. however, seven of these ( %) were not subsequently admitted to the icu. only six patients ( %) survived to hospital discharge. pre-admission function and comorbidity were worse in patients who did not survive to discharge ( fig. ), but these were not statistically significant in view of small survivor group size. in an increasingly frail inpatient population, a substantial proportion of patients in whom circulation is restored after cardiac arrest are subsequently considered unsuitable for icu admission. given our understanding of inferior outcomes in patients with poor physiological reserve, we encourage early discussion regarding the appropriateness of cpr in selected patients, guided by social function and comorbidity. references: . national cardiac arrest audit / introduction: there are studies that determine events related to poor outcome in cardiac arrest [ ] . in our study, following parametres were determined ohca patients; age median years, asian/europe/syrian, bystander cpr, bystander aed, ems defibrillation, initial cardiac rhythm, prehospital rosc, corneal and pupillary light reflex and day survival. we determineted poor prognostic sign with post-cardiac arrest patients. in this study, we identified the causes of poor outcome in patients with ohca. this was a single-centre, retrospective study. we determined incidence and epidemiological factors including: demographics, initial cardiac rhythm. our study population were non-traumatic ohca. our icu, all ohca patient were evaluated wtih echo, and fluid, inotrope and vazopressor were added according to cardiac performance. results: during our study, patients who were admitted to intensive care unit between - were screened. of these patients were out-of-hospital arrest and of them were in-hospital arrest. development of cerebral oedema during treatment in hospital remains a poor prognostic sign. the evaluation of initial cardiac ritm is useful to predict neurological outcome in post-cardiac arrest patients. survival after ohca remains low. the evaluation of initial cardiac ritm is useful to predict mortality and neurological outcome in postcardiac arrest patients. basic life support (bls) education and training for school children is active in japan. however, the bls action by schoolchildren may be limited by school rules. this study aimed to analyse the time factors for basic life support performance and outcome in classmatewitnessed out-of-hospital cardiac arrest (ohca) and to investigate how schoolchildren act when they detect ohca. methods: nation-wide database for , school children cases with ohca and local extended database for , ems-unwitnessed ohca, both of which were prospectively collected during the period of - , were retrospectively analysed. proportion of schoolchildren-detected ohca was low in classmate cases ( . %, / ) in nationwide database and extremely low in all ems-unwitnessed ohcas ( . %, / , ) in local database. nationwide database analyses revealed that both emergency call and bystander cpr were delayed when a classmate witnessed the ohca case: median, vs. min and vs. min, respectively. classmate-witnessed cases were associated with higher incidences of shockable initial rhythm, aed use and traumatic causes. the rate of neurologically favourable outcome was . % and . %, respectively in classmate-witnessed and other cases: adjusted or; % ci, . ; . - . . of cases detected by schoolchildren in our prefecture, ( %) cases had presumed cardiac aertiology and ( . %) cases were caused by suicide attempts (hanging and fall). school children placed emergency calls as the first action only in ( . %) cases. emergency calls were largely delayed when school children dialled other numbers or left the scene to seek adult help. school children were rarely involved in bystander cpr ( %) and aed placement ( %). school children are rarely involved in entire bls. emergency calls and bystander cpr are delayed when schoolchildren act to seek help. because schoolchildren detect suicide-related ohcas, psychological care to schoolchildren involved in bls may be necessary. prognostic value of neutrophil/lymphocyte and platelet/ lymphocyte predicting cardiopulmonary resuscitation with spontaneous circulation recovery c li the affiliated suzhou hospital of nanjing medical university, suzhou, china critical care , (suppl ):p to investigate the predictive value of peripheral blood neutrophil-tolymphocyte ratio (nlr) and platelet-to-lymphocyte ratio (plr) on inhospital mortality in patients with spontaneous circulation recovery after cardiac arrest. a retrospective analysis was made of patients who recovered from cardiac arrest in our hospital from april to november and were admitted to the intensive care unit for more than hours. they were divided into survival group and death group according to the outcome of discharge.the dynamic changes and differences of nlr and plr in hours and - hours after admission to icu between the two groups were analyzed and compared. multivariate analysis and roc curve were used to explore the predictive value of nlr and plr for in-patient mortality. compared with the survival group, plr in the dead group was significantly lower within hours of admission to the intensive care department (p < . ), while nlr in - hours was significantly higher (p < . ). the nlr of surviving group was significantly lower than that of hours (p < . ), while the nlr and plr of death group were not significantly different (p < . ) from that of hours (p < . ). multivariate logistic regression analysis and roc curve showed that nlr of - h in icu was an independent risk factor for predicting in-patient mortality, and had high sensitivity and specificity in predicting death outcomes. neutrophil to lymphocyte ratio, platelet to lymphocyte ratio can help to judge the outcome of patients with cardiac arrest and recovery of autonomic circulation after cardiopulmonary resuscitation. [ , ] patients with sofa score > (vs sofa score ≤ ) had a higher free iron level ( . μmol/l vs μmol/l, p = . ) ( figure ). we found a positive correlation between free iron level at h and changes of sofa score between h and h (r= . ic [ . ; . ]). out-of-hospital cardiac arrest is associated with a significant change of plasma free iron level. free iron level at admission is associated with short term outcome. further research is warranted to better determine the significance of such changes. the optimal level of arterial oxygen in the post-resuscitation period is unknown. recent studies show conflicting results in regard to hyperoxia and its association with survival after out-of-hospital cardiac arrest (ohca) [ ] . the aim of this trial is to study the association between early hyperoxia after ohca with return of spontaneous circulation (rosc) and -day survival. observational study using data from three swedish national registers (i.e. intensive care, cardiac arrest and national patient registries after a successful resuscitation, a systemic inflammatory response occurs, and the c-reactive protein (crp) level represents the degree of inflammation [ ] [ ] [ ] . this study examined the association between increased inflammation and early-onset pneumonia (eop) in patients treated with extracorporeal cardiopulmonary resuscitation (ecpr) after out-of-hospital cardiac arrest (ohca). this retrospective study included data of patients with ohca treated with ecpr admitted to st. luke's international hospital between april and april . the exclusion criteria were as follows: age < years, therapeutic hypothermia withdrawal due to death or circulatory failure, or sepsis as a suspected cause of cardiac arrest. patients were diagnosed with eop according to clinical signs and symptoms acquired after a hospitalization period of > h and within days of admission. the crp levels were measured daily from admission to day . we studied patients with a median age of years (interquartile range: - years). furthermore, ( %) patients were males, and the median time interval from collapse to adequate flow was ( - ) min. all patients received prophylactic antibiotics, and ( %) of them had favorable neurological outcomes (cpc, - ). eop occurred in ( %) patients, with a significantly higher crp level on day than that in those without eop ( . categorizing reasons for death after ecpr is important for comparing outcomes to other studies, assessing benefits of interventions, and better define this heterogeneous patient collective. a categorizing for death after cardiac arrest in both in-hospital (ihca) and outof-hospital (ohca) arrests has been proposed in non-ecpr patients by witten et al. here, we adopt this categorization to ecpr patients. single-center, retrospective, cohort study of patients without rosc after ihca or ohca and ecpr between and . patients with survival below hours were excluded. patients were allocated to one of five predefined reasons for death. results: va-ecmo patients were included (age . ± . , . % female, % ecpr, day survival . %). reasons for death for patients with va-ecmo for shock (survival %) and ecpr ( %) were: neurological withdrawal of care ( % vs %), comorbid withdrawal of care ( % vs %), refractory hemodynamic shock ( % vs %), respiratory failure ( % vs %), and withdrawal due to presumed patient will ( % vs %) ( figure ). the differences in reasons for death among the two groups were significant (p < . ), driven by withdrawal due to neuroprognostication, comorbidity and hemodynamic instability. categorizing death after va-ecmo into five categories is feasible. there are significant difference between patients with va-ecmo for shock and ecpr. interestingly, only a quarter of patients after ecpr died due to brain damage. introduction: scarcity of potential dead brain donors and the persistent mismatch between supply and demand of organs for transplantation has led the transplant community to reconsider donation after circulatory death (dcd) as a strategy to increase the donor pool. normothermic regional perfusion (nrp) by extracorporeal membrane oxygenation (ecmo) may be the most effective method for preserving abdominal organs in dcd, especially in liver transplantation [ , ] . a pitfall of this method is its complexity and the unavailability of this resource in some hospitals, especially in regional hospitals, where potential dcd donors may exist. aim of this study is to report the use of mobile ecmo team in controlled dcd. from june to november our group has worked as a mobile ecmo team for cdcd outside our center. portable equipment included cannulation material and the ecmo device. the transplant team consisted of transplant coordinator (anesthesiologist-intensivist, ecmo operator and organ extraction supervisor), cardiac surgeon (cannulation), interventional radiologist (cannulation) and one cardiovascular perfusionist (ecmo operator). twenty-five cdcd donations were performed. characteristics of donors and organs retrieved are summarized in figure . from cdcd, livers, lungs, kidneys were obtained. the evolution of grafts and receptors was favorable at day post-transplant. mobile ecmo teams may enable cdcd in hospitals without these resources, thereby increasing the pool of donors and optimizing graft outcomes. what is the useful coagulation and fibrinolysis marker for predicting extracorporeal membrane oxygenation circuit exchange due to intra-circuit thrombus? y izutani, k hoshino, s morimoto, k muranishi, j maruyama, y irie, y kawano, h ishikura fukuoka university hospital, emergency and critical care center, fukuoka-shi, japan critical care , (suppl ):p a thrombus formation is one of the most frequent and adverse complications during extracorporeal membrane oxygenation (ecmo) support. previous studies have reported that increased d-dimer is a useful predictor of thrombus formation within the ecmo circuit. the purpose of this study was to identify coagulation/fibrinolysis markers for predicting the replacement of ecmo circuit due to intra-circuit thrombus during ecmo support. fourteen patients who underwent veno-venous ecmo for acute respiratory failure between january and december were enrolled. these patients received a total of days of ecmo support. of these, days (times) on which the ecmo circuits were replaced was regarded as the replacement group, while the remaining days were considered as the non-replacement group. the several coagulation/fibrinolysis markers were routinely measured every day during ecmo support. we compared with the levels of these markers between two group to identify the most relevant marker for ecmo circuit replacement due to thrombus. the mean duration of ecmo support was ± days, and the mean number of ecmo circuit replacement was . ± . times per patient. ddimer, thrombin-antithrombin complex (tat), plasmin-α plasmin inhibitor complex (pic), and soluble fibrin (sf) were significantly higher in the replacement group rather than in the non-replacement group (p < . , respectively). according to a multivariate analysis, sf was the only independent predictor of ecmo circuit replacement due to thrombus. the odds ratio ( % confidence intervals) for sf ( μg/ml) was . ( . - . ). the area under the curve and optimal cut-off value were . and ng/ml for sf, respectively (sensitivity, %; specificity, %). from these results, we concluded that sf may be the useful marker rather than d-dimer for predicting the replacement of ecmo circuit due to intra-circuit thrombosis. inhomogeneity of lung elastance in patients who underwent venovenous extra corporeal membrane oxygenation (v-v ecmo)-a computed tomography scan study rd di mussi , ri iannuzziello , fm murgolo , fd de carlo , e caricola , na barrett , lc camporota , sg grasso università degli studi di bari "aldo moro", department of emergencies and organ transplant, bari, italy; università degli studi di bari "aldo moro", bari, italy; department of adult critical care, guy´s and st thomas´nhs foundation trust, king´s health partners, london, uk critical care , (suppl ):p in patients with acute respiratory distress syndrome (ards), nonaerated, poorly aerated, and normally aerated regions coexist to variable degrees in lung parenchyma. the recruitment maneuvers aim to reopen collapsed lung tissue. in a theoretical point view, this strategy may also prevent the normal aerated lung tissue hyperinflation [ ] . the objective of our study was to evaluate lung characteristics in terms of hounsfield units (hu), volume and elastance before and after a recruitment maneuver. in patients with severe ards who underwent v-v ecmo, computed tomography scans (ct-scans) at cmh o of continuous positive airway pressure (cpap) and cmh o were performed. the same ct image was selected at the two different levels of pressure. the distribution of lung opacities, in terms of hu, was classified using the "ucla" colour coding table (osirix image processing software, geneva, switzerland). correspondent lung regions of about voxels were selected. the quantitative analysis, in terms of volume air (vair) was performed with maluna software (version . ; maluna, goettingen, germany). elastance was calculated as the pressure(cmh o)/ vair (ml) ratio. results: see figure . lung inhomogeneity occurs also after recruiting maneuvers. our data confirm that the elastance of recruited lung regions is higher than the elastance of the normal aerated lung regions at low positive end-expiratory pressure (peep) (baby lung). on the contrary the "baby lung" frequently develops hyperinflation. the unpredictable pattern of distribution of volume after recruitment maneuverers may explain the controversial role of peep during the ards treatment. . formal recommendations on target, timing, and rate of at supplementation are lacking. we conceived this study to evaluate the effect of prolonged at supplementation in adult patients requiring veno-venous ecmo for respiratory failure on heparin dose, adequacy of anticoagulation and safety methods: before ecmo start patients were randomized to either receive at supplementation to maintain a functional at level between and % (at supplementation group) or not (control group) for the entire ecmo course. anticoagulation was provided with unfractionated heparin following a standardized protocol [ ] . the primary outcome was the dose of heparin required to maintain the ratio of activated partial thromboplastin time between . and . secondary outcomes were the adequacy of anticoagulation measured with anti-factor xa and the incidence of hemorrhagic and thrombotic complications and amount of blood products fig. b) . conclusions: this retrospective analysis was not able to show a survival benefit for additive pp to ecmo support in general. early initiation of pp could be an important factor for improving survival in this setting and should be considered in a randomized controlled trial for further evaluation. cause-specific mortality during extracorporeal membrane oxygenation, a single center review of medical records m panigada, d tubiolo, p properzi, g grasselli, a pesenti fondazione irccs ca´granda ospedale maggiore policlinico, intensive care unit, milano, italy critical care , (suppl ):p introduction: mortality during extracorporeal membrane oxygenation (ecmo) settles around % and the occurrence of bleeding during ecmo is associated with a high mortality rate. however, cause-specific mortality is rarely reported, probably due to the difficulty of its classification. the purpose of the study was to evaluate the agreement between two expert icu physician in the classification of the cause of death of patients supported with ecmo for either respiratory or cardiac support. methods: two intensive care unit (icu) expert staff physicians independently reviewed the entire medical records of all ecmo patients who died before icu discharge from january to september at fondazione irccs ca' granda, milan. they were asked to choose the cause of patient's death among six categories. in case of disagreement, a third expert adjudicated the case. the two reviewers were also asked whether, in their opinion, bleeding during the last hours contributed to death. elso definition of major bleeding [ ] during the last hours was also recorded for each patient. results: two-hundred and two patients were supported with ecmo of whom ( . %) died. most of these patients (n= , . %) died during ecmo. interrater agreement for cause-specific mortality between the two expert physicians was substantial (k . , se . , p< . ) of the discordant cases were categorized as refractory respiratory failure and as multiorgan failure and septic shock respectively. the distribution of cause-specific mortality is shown in figure . major bleeding (elso) was present in ( . %) patients, only in ( . %) of them bleeding contributed to death according to the reviewers. patients treated with early pp while ecmo showed a superior survival to patients treated with late pp or without pp while ecmo. optimal cut off value for duration of ecmo initiation to first pp was calculated using roc-analysis (auc = . ) and the youden-index. highest sensitivity and specificity for beneficial survival were achieved for a beginning of pp in < . days. (log rank= . ). pp: prone positioning p non-invasive mechanical ventilation in veno-venous extracorporeal membrane oxygenation j rilinger, v zotzmann, x bemtgen, pm biever, d duerschmied, c bode, dl staudacher, t wengenmayer heart center freiburg university, department of cardiology and angiology i, freiburg, germany critical care , (suppl ):p introduction: veno-venous extracorporeal membrane oxygenation (ecmo) support can be combined with a variety of different non-invasive ways to deliver oxygen to the patient's lung. several positive effects might be linked to this so called "awake ecmo". so far there is little evidence about indications and outcome of this approach. we report retrospective registry data on all ards patients treated with ecmo support at a university hospital between / and / . in a systematic review of medical records, we distinguished between patients with invasive mechanical ventilation (imv) from the initiation of ecmo therapy (imv group) and patients that received any kind of non-invasive oxygen supply (non-imv group). a total of patients could be analysed. ( . %) patients received non-imv ecmo support. patients receiving non-imv ecmo therapy showed severe underlying pulmonary disease and immunosuppression (fig. ) . these patients had higher rates of lung fibrosis, long-term oxygen therapy, pulmonary hypertension, renal insufficiency and immunosuppression (p< . ). of patients ( %) required imv during the hospital stay in average . ± . [ . - . ] days after ecmo initiation. reasons were hypoxia despite of ecmo, insufficient ecmo-flow, insufficient protective reflexes or patient agitation. patients with initially non-imv ecmo support showed a numerical but not significant lower icu and hospital survival ( . % vs. . %, p= . ). non-imv ecmo support was applied in patients with severe underlying pulmonary disease and/or immunosuppression. in a high proportion of patients the ventilation regime had to be switched from non-invasive to invasive. survival in this very selected cohort was low. in this retrospective analysis no evident benefit for a noninvasive ventilation strategy could be found. the high proportion of patients who switched from non-imv to imv therapy underlines the need for rigorous patient selection. intra-hospital transportation on extracorporeal membrane oxygenation (ecmo) -a single centre experience in ireland. z siddique, s o´brien, e carton, i conrick-martin mater misericordiae university hospital, department of critical care medicine, dublin, ireland critical care , (suppl ):p the objective of this study is to evaluate intra-hospital transportation of patients on extracorporeal membrane oxygenation (ecmo). it is a retrospective analysis of prospectively collected database, performed as part of ongoing quality improvement initiatives. the setting of this study is an -bed, combined surgical and medical adult intensive care unit (icu) located in a -bed hospital that serves as the national referral centre for cardiothoracic surgery, heart & lung transplantation and ecmo in ireland. we reviewed months of data (from to ) regarding patients admitted to our critical care unit who required intra-hospital transfer for diagnostic and/or therapeutic interventions. we also compared the data to available local guidelines. results: patients were transported on ecmo on a total of occasions; the most common indication being ct brain (table ) . ecmo cannulation sites were peripheral in patients, patients were centrally cannulated. median time from start of the transfer until the patient was returned to icu was minutes (range: - ). the ecmo console was placed on a dedicated ecmo trolley apart from two occasions where it was placed on the patient's bed. number of staff required for transport was between to ; with an icu consultant as team leader. ecmo specialist nurses were always present on the transport team. transfers were during normal working hours with happening on a weekend. a total of complications occurred during the transports, of underlying pulmonary disease or status of immunosuppression in ecmo patients without invasive mechanical ventilation which was significant and were not. the significant complication encountered was ventricular tachycardia in a v-a ecmo patient which required electrical defibrillation. no adverse events related to transport were seen following return to icu. in this single-centre study, we have demonstrated safe intra-hospital transport of ecmo patients. the use of local guidelines, appropriate personnel and performance during normal working hours is recommended. a novel approach for flow simulation in ecmo rotary blood pumps a supady , c benk , j cornelis , c bode , d duerschmied heart center freiburg university, cardiology and angiogiology i, freiburg, germany; heart center freiburg university, department of cardiovascular surgery, freiburg, germany; fifty technology gmbh, freiburg, germany critical care , (suppl ):p introduction: extracorporeal membrane oxygenation (ecmo) is used increasingly in critically ill patients suffering from acute respiratory failure, cardiogenic shock or cardiac arrest. however, this therapy can have deleterious side effects such as bleeding or clotting complications and hemolysis. these complications are particularly caused by physical stress acting upon the blood components while passing through the ecmo system, especially within the rotary pump. we here present a novel approach to simulate blood flows through rotary blood pumps used in current ecmo systems in order to better understand the genesis of these complications. geometries of the xenios dp (xenios ag, heilbronn, germany) rotary pump were reconstructed by ct-scans and manual measurements using computer-aided design (cad). the computational fluid dynamics (cfd) simulation was performed using the software preon-lab (fifty technology gmbh, freiburg, germany), which implements a mesh-free lagrangian method requiring minimal preprocessing of the cad data. the geometries are introduced to the simulation model as tessellated surfaces. five operating points have been specified by the rotation of the centrifugal fan and the corresponding inflow and outflow of blood. the blood is approximatively modelled as a newtonian fluid with a density of kg/m . preonlab allows detailed assessment of the blood flow while passing through the rotary pump including analysis of local flow rates, pressure gradients and shear stress acting upon the blood. dead zones in the fluid flow can be detected which gives reference points for optimizations of the pump design. for the first time, we demonstrate a novel approach for flow simulation in an ecmo rotary pump ( figure ). this approach may help better understand hemodynamics within the extracorporeal system to define optimal operating points or re-design components aiming to limit hemolysis, coagulation disorders and bleeding in seriously ill patients. one-year experience of bedside percutaneous va-ecmo decannulation in a territory ecmo center in hong kong km fong, sy au, pw leung, kc shek, hj yuen, sk yung, hl wu, so so, wy ng, kh leung queen elizabeth hospital, intensive care unit, hong kong critical care , (suppl ):p when veno-arterial extra-corporeal membrane oxygenation (va-ecmo) support can be terminated, arteriotomy wounds of the patients of are traditionally closed by open repair in the operation theaters. lots of manpower are involved and timeslots in operating theaters are scarce. transport of the critically-ill is risky. successful va-ecmo decannulation using percutaneous device called proglide has been reported and our group had adopted and modified this approach [ ] . methods: this is a retrospective study analyzing the one-year experience of bedside va-ecmo decannulation. our institution is a -bed tertiary ecmo referral center in hong kong. our first bedside decannulation was performed in november , and since then, this practice had replaced the traditional open repair, unless contraindicated. data from november to october were analyzed. in the study period, patients received va-ecmo. survived to decannulation and received bedside percutaneous decannulation. their median age was ( - ). the default arterial catheter size was fr, with fr in cases and fr in one. five ( %) failed percutaneous closure and they were subsequently surgically repaired without extra corporeal life support (ecls) continues to be associated with high mortality rates. our ability to predict outcome prior to initiation ecls remains limited. here we take a single cell rnaseq approach in an effort to identify novel immune cell types that are associated with-and may contribute to-survival on ecls. whole genome transcriptomic profiles were generated from~ , peripheral blood monocytes obtained from patients at the time of cannulation for veno-arterial ecls (va-ecls). within each subpopulation, differential gene expression analysis was performed to identify new markers associated with survival. findings were validated in a additional cohorts by flow cytometry. surviving patients had significantly higher proportions of cd + nkt cells (cd + /cd + /cd -/cd + ) that were cd + (p = . , fdr < . ) ( figure ). to validate this observation, we performed fc analysis of a second cohort of patients. for each patient, we quantified the proportion of cd + nkt cells that were cd + . using the median proportion as the cutoff, we again found that a high proportion of cd + cells among cd + nkt cells was predictive of hour survival (p= . ). we noted that while high levels of cd + cells among the cd + nkt cells was protective in this cohort of va-ecls patients, this relationship did not hold for patients with sepsis. as only a few the va-ecls patients were septic, we analyzed a third cohort of septic ecls patients. we observed that high levels of cd + cells among the cd + nkt populations was not protective in this population. the proportion of cd + nkt cells that are positive for cd is predictive of survival among patients undergoing va-ecls for noninfection related indications. introduction: the use of calcium sensitizers has grown enormously in the last decade, probably due to their interesting pharmacodynamic properties. levosimendan (ls) is frequently administered in patients under mechanical circulatory support. we performed a retrospective evaluation of patients treated with ls prior to weaning from mechanical support. this evaluation was combined with a review of the literature. a query of our icu patient data management system revealed patients receiving ls prior to or during vad/ecls support. outcome data were obtained from the patients medical records. of our patients, % was successfully weaned off ecls. fourteen patients ( %) died before being discharged of whom while on ecls support. of the weaned patients, died afterwards. of the converted patients needed subsequent veno-venous ecls support for right ventricular support after the implantation. survival to discharge ratio for the whole group was %. more detailed demographic results can be found in table . a pubmed search using the terms "(ecmo or ecls) and ls and weaning" resulted in publications which dealt specifically with weaning of ecls support. several weaning approaches are available, however poor outcome has remains a problem. some recent studies show a possible beneficial effect of ls infusion prior to weaning from ecls. however most of these studies are retrospective or observational at best. because ls is primarily reserved for the most severe cases, outcome interpretation is difficult. overall weaning success ranges from %- % and variation is very dependant of inclusion criteria. the calcium sensitizer ls can be used when weaning off patients from ecls, certainly given its low incidence of complications. future, large randomized trials are however needed in order to confirm this strategy. cardiogenic shock is well described in newly diagnosed pheochromocytoma, and crisis may be precipitated by hemorrhage into tumour. v-a ecmo represents a rescue therapy in a subset of these patients refractory to medical management, facilitating cardiac recovery and subsequent definitive surgery. consent to publish: written informed consent for publication was obtained from the patients. during a spontaneous breathing trial respiratory mechanics can worsen, and respiratory muscle effort can increase, leading to respiratory muscle fatigue, pump failure, hypercapnia and an unsuccessful weaning from mechanical ventilation. this case report discusses the possibility of applying extracorporeal co removal (ecco r) to reduce respiratory muscle effort in a liver transplant recipient who already failed three weaning attempts from mechanical ventilation. the ecco r membrane lung was integrated into a conventional renal replacement therapy circuit and blood flow was increased from to ml/min. measurements of respiratory mechanics (including esophageal pressure, as shown in fig. ) were used to assess the reduction of respiratory effort before and during the application of ecco r. was delivered through a fr-double-lumen-cannula; ml/min blood-flow with lt oxygen sweep-gas-flow and aptt . - baseline were maintained (iv-heparin). in all cases respiratory and metabolic parameters improved without complications ( figure ). ecco r-crrt facilitated extubation ( out imv pts). in out of pts at risk of niv failure, it avoided imv. treatment mean duration was ± hours, mean lenght of icu stay was ± days. all patients survived to the treatment, nevertheless patients died due to irreversible multiple mof. in our aecopd series prismalung®-prismaflex® facilitated weaning from imv and avoided intubation in patients at risk of niv failure without complications. these positive results may be related to minimal invasiveness of the low-flow device used and may constitute the rationale for a larger randomized controlled trial. consent: written informed consent for data publication has been obtained. extracorporeal the primary outcome findings from the supernova trial [ ] demonstrated that the use of extracorporeal carbon dioxide reamoval (ecco r) allows a reduction in tidal volume (tv) to ultraprotective levels (≈ ml/kg predicted body weight or pbw) during mechanical ventilation in ards patients without significant increases in the arterial partial pressure of carbon dioxide (paco ). unfortunately, it was not feasible to directly measure ecco r rates during the trial. we used a mathematical model of whole-body oxygen (o ) and carbon dioxide (co ) transport and biochemistry [ ] to calculate ecco r rates that permit a fit to the data reported for hemolung (alung technologies) and ila (novalung)/cardiohelp (getinge) devices in the supernova trial [ ] . the mathematical model was calibrated under baseline conditions where patients were mechanically ventilated at a tv of ml/kg pbw in the absence of an ecco r device; the o consumption rate, co production rate and pulmonary shunt fraction were adjusted to match the measured baseline arterial partial pressure of o and paco . assuming all baseline parameters were fixed, tv was then reduced to . ml/kg pbw and the mathematical model predicted the ecco r rate to the change in the paco level. model predictions for the devices are shown in table . these predictions suggest that ecco r rates for ila/cardiohelp devices were approximately twice those for hemolung devices during the supernova trial. these results may be useful to evaluate the expected performance of novel ecco r devices. efficiency and safety of a system crrt plus ecco r to allow ultraprotective ventilation protocol in patients with acute renal failure f maldarelli despite renal function replacement techniques (crrt), a patient who develops acute renal failure(aki) in intensive care unit (icu) has a mortality rate of - %. this risk is partly due to the adverse effect of aki on other organs than the kidney. respiratory complications are frequently associated with the development of aki. new machines combining crrt with a carbon dioxide removal membrane (ecco r) allows the setting up of an ultra-protective ventilation ( ml/kg of predicted boby weight (pbw)) to reduce any lung damage from mechanical ventilation (mv). the reduction in tidal volume (vt) is associated with a decrease in lung damage partly triggered by aki. we evaluated the efficacy of a combined system crrt+ecco r to reduce the vt to ultraprotective values in patients with acute respiratory failure and aki. ards is a syndrome with high morbidity and mortality. an emerging treatment option is ecco r, but the benefit its remains unclear. we assess different degrees of ecco r and varying dead space (ds) on ventilator settings in order to minimize mechanical power. we calculated mechanical power as ( ) power=rr*{Δ〖vt〗^ *[ / *el+rr*( +i:e)/( *i:e)*r]+ Δvt*peep} (el: system elastance, r: airway resistance, peep: positive end expiratory pressure, i:e: inspiratory to expiratory ratio). we calculated the combination of respiratory rate (rr) and tidal volume (vt) ("optimal rr" and *optimal vt*) leading to minimal applied power for a stable carbon dioxide elimination of ml/min (vco ) for two scenarios: ) variation of physiological ds from to % of vt at a fixed rate of eccor . ) variation of ecco r of either , , or ml/min at a fixed physiological ds of %. the alveolar ventilation (va) necessary to eliminate the vco was calculated as ( ) va= (-vco *σ_co *r*t*( +k_c ))/(vco /q-p_vco *σ_co *r*t*(( +k_c ))/ ) σco : co solubility in blood, r: gas constant, t: temperature. pvco : venous partial pressure, kc: function of ph ( . for a ph of . ), q: blood flow [ l/min]). increasing ds from to % increases the minimal mechanical power from . to . j/min, primarily caused by an increase of optimal vt ( - ml). optimal rr was only slightly increased ( . - . /min, figure panel a). for varying ecco r removal, necessary ventilation ranges from . to . l/min. this predicts a minimal power between . and . j/min with an unchanged optimal vt ( - ml) and an increasing optimal rr ( . to . /min ( figure panel b)). in order to minimize mechanical power, increasing shunt or co production should be met with increases in rr while increases in ds should be met with increases in vt. our results indicate that during ecco r, mechanical power and thus risk for lung injury can be minimized with higher vt compared to conservative ventilation strategies. validity of empirical estimates of physiological dead space in acute respiratory distress syndrome jd dianti, eg goligher, as slutsky university of toronto, interdepartmental division of critical care medicine, toronto, canada critical care , (suppl ):p increased physiological dead space fraction (v d /v t ) is a hallmark of the acute respiratory distress syndrome (ards) and has been shown to predict ards mortality. v d /v t is also important in estimating the reduction in tidal volume (v t ) and driving pressure (Δp) with extracorporeal co removal (ecco r). v d /v t can be measured with volumetric capnography but empirical formulae using the patient's age, weight, height, gender and paco have been proposed to estimate v d /v t based on estimates of co production (v co ). the accuracy of this approach in critically ill patients, however, is not clear. secondary analysis of a previously published trial [ ] in which v d /v t and v co were measured in ards patients. estimated dead space fraction (v d,est /v t ) was calculated using standard formulae. agreement between methods was evaluated by bland-altman analysis. the predicted change in Δp with ecco r was evaluated using both measured and estimated alveolar dead space fraction (v dalv /v t ). results: vd,est/vt was higher than measured vd/vt, with a low correlation between the (r = . ). vco was underestimated by the predicted approach (table ) , accounting for % of the error in estimating vd/vt. the expected reduction in Δp with ecco r using vdalv/ vt was in reasonable agreement with the expected reduction using introduction: acute respiratory distress syndrome (ards) is a common condition in critically ill patient. however neuromuscular blockers (nmb) result controvertial in early treatment of ards [ ] . we ought to search systematically and realize a meta-analysis on the matter. an electronic search of randomized clinical trials in adult patient treated with early neuromuscular blockers compared without neuromuscular blockers in ards. the primary objective of the analysis was the mortality at to days. secondary endpoints included mechanical ventilation free days, icu acquired weakness and barotrauma. the search obtained studies for the analysis [ ] [ ] [ ] [ ] [ ] [ ] (figure ). the early use of neuromuscular blockers in ards showed no increase in mortality, but the results should be taken with caution. there was no differences in mechanical ventilation free days. barotrauma is less with the use of nmb. ultrasound is fairly sensitive in the detection of lung infiltrates in patients with hematologic malignancies. in patients with pneumonia requiring intensive care (icu) admission, we hypothesise that abnormal right ventricular (rv) function is associated with an increased -day mortality. rv dysfunction in critically ill patients has a well-known association with adverse outcomes [ ] . however, its impact on mortality in patients with pneumonia has not been directly studied. patients admitted to the queen elizabeth hospital birmingham icu between april and july with a diagnosis of pneumonia who had a formal cardiologist tte were included. abnormal rv function was defined by either depressed function, dilated size or moderate to severe risk of pulmonary hypertension (phtn). abnormal lv function was defined by an lv ejection fraction £ % or grade ii or more diastolic dysfunction. patients with a clinical suspicion of pulmonary embolism were excluded. the primary outcome was -day mortality. continuous data is presented as median (iqr). categorical data is presented as % and analysed using a chi-squared test. results: patients were admitted to icu with pneumonia, of which ( %) had a tte. patients were % male, had a median age of ( - ) and -day mortality of %. abnormal rv function was present in % (n= ), with % depressed, % dilated and % with moderate to severe risk of phtn. rv dysfunction was associated with an increased -day mortality compared to normal rv patients ( % vs. %, p< . ). lv function was abnormal in % (n= ) and was not associated with a higher -day mortality compared to normal lv patients ( % vs %, p = . ). rv dysfunction was associated with a higher -day mortality than lv dysfunction ( % vs %, p = . ). conclusions: this is one of the first studies to demonstrate that abnormal rv function is associated with an increased mortality in icu patients with pneumonia. interestingly, abnormal lv function was not associated with an increased mortality. rakuno gakuen university, anesthesiology, hokkaido, japan critical care , (suppl ):p we previously reported a simple correction method of estimating pleural pressure (ppl) by using central venous pressure (cvp) and that it can be used to estimate ppl and transpulmonary pressure in pediatric patients with respiratory failure. however, it remains unknown that this method can be applied to patients with various levels of chest wall elastance and/or intravascular volume. the objective of this study is to investigate whether our method is accurate in various conditions of chest wall elastance and intravascular volume. the study was approved by the animal care and use committee of rakuno gakuen university. ten anesthetized and paralyzed pigs ( . ± . kg) were mechanically ventilated and subjected to lung injury by saline lung lavage. each pig was subjected to different intravascular volume and different intraabdominal pressures; in each condition, the accuracy of our method was tested. specifically, airway flow, airway pressure (paw), esophageal pressure (pes), and cvp were recorded in each condition, then changes in pes (Δpes) and Δppl calculated using a corrected Δcvp (cΔcvp-derived Δppl) were compared. cΔcvp-derived Δppl was calculated as κ × Δcvp, where κ was the ratio of the Δpaw to Δcvp during the occlusion test. means and standard deviations of the two variables that reflect Δppl (Δpes and cΔcvp-derived Δppl) in all pigs with all conditions were . ± . and . ± . cmh o. the bland-altman analysis for the agreement between Δpes and Δcvp showed a bias of - . the activity and functionality of the diaphragm are difficult to measure in patients ventilated in intensive care. ultrasound can be a useful tool for monitoring diaphragm muscle activity during different ventilation modes. few data currently exist on diaphragm muscle activity in critically ventilated patients [ ] . our goal is to evaluate the respiratory muscular work of the diaphragm with different settings of the respirator by means of an ultrasound scan. the ultrasound assessments of the diaphragm were performed with a mhz linear probe at the apposition zone. we measured the thickening of the diaphragm with the respiratory acts, through the thickening fraction (thickening fraction, tf), defined as:tf = (tdimax -tdimin / tdi min)% tdimax: diaphragm thickness at the end of inspiration (maximum thickness) tdimin: diaphragm thickness at the end of expiration (minimum thickness). ventilatory support was divided into classes: -spontaneous breathing (sb) or continous positive airway pressure (cpap); -pressure support ventilation (psv) with low pressure support ( - cmh o); -psv with high pressure support (> cmh o); -controlled mechanical ventilation (cmv). a total of assessments were performed in patients. the evaluations were all possible at the right hemidiaphragm, while on the left they were not possible in % of the cases. the median tf (iq range) of the ventilation classes was respectively: % ( - %) in sb / cpap; % ( - %) in low-psv; % ( - %) in high psv; and % ( - %) in cmv. the kruskal-wallis test confirms a significant difference between the groups (p < . ). the ultrasound of the diaphragm can be a valid tool for monitoring respiratory muscle activity during mechanical ventilation. introduction: extubation failure is defined as reintubation after hours of extubation in mechanically ventilated critically ill patients. it is associated with morbidity and mortality. the aim of our study was to assess reintubation rates in a busy district general hospital and evaluate the impact of high flow nasal oxygen therapy (hfno) on reintubation rates. we performed a retrospective observational study looking at patients admitted to our bedded level critical care unit ( patients a year) for a period of years between st november and st october . we included patients over years of age who were mechanically ventilated and length of stay was greater than hours. exclusions were age < years, tracheostomy and patients requiring ventilation for < hours. data was collected from ward watcher, a sicsag database and electronic patient records. our study failed to show any impact of hfno on reducing extubation failure. further work is needed to develop a standardized approach to weaning and to consider routine application of noninvasive ventilation to reduce reintubation rates [ ] . fig. (abstract p ) . the bland-altman analysis for the agreement between Δpes and cΔcvp-derived Δppl in various conditions. low: low intravascular volume, normal: normal intravascular volume, high: high intravascular volume, abd-: without an abdominal compression band, abd+: with an abdominal compression band oral endotracheal intubation is common to critically ill patients in intensive care unit. oral care for an intubated patient is important to maintain the moisture of oral mucosa. also, the securement method of oral endotracheal tube developed from cloth tape to commercial tube holder. training powerpoint and video for microteaching was prepared to train up icu nurses to perform the new practice. demonstration and re-demonstration was arranged to assess skills of every nurse. afterwards, each nurse answered a quiz to evaluate the understanding of oetth and its special techniques in application. questionnaire was designed to collect the feedback from all nurses too. the result showed there was nurses ( %) out of nurses achieved full marks in the post-quiz which demonstrated their full understanding of the use of oral ett holder and its nursing care. about the feedback from nurse, % of nurses claimed that they were confident in using the new oetth in clinical setting after training. % of nurses agreed in time-saving of nursing care routine with the use of an oetth. however, only % of nurses agreed that the oetth is effective in prevention of oral mucosa injuries and another % of nursing staff disagreed on its function in improving the patient's oral care. in conclusion, some of the nurses did not agree the prevention of oral mucosa injuries by the new securement method with oetth while some nurses welcomed the new oetth as more easy and effective in oral care to intubated patients. execution of percutaneous dilatational tracheostomy using the standard laryngeal mask airway for ventilation: a prospective survey study g gagliardi , v gagliardi , c chiani , g laccania , f michielan aulss -veneto, anesthesia and intensive care, adria, italy; aulss -veneto, university of padua, adria, italy; aulss -veneto, anaesthesia and intensive care, adria, italy; aulss -veneto, anaesthesia and intensive care, padua, italy critical care , (suppl ):p we fulfilled a survey study dealing with bronchoscope-guided percutaneous dilatational tracheostomies (pdt), using the classic laryngeal mask airway (lma) for the airway management [ ] . the aim was to verify the safety and the effectiveness of the aforementioned procedure methods: we performed an observational prospective survey study enrolling patients hospitalized in the intensive care unit. before performing the tracheostomy, the endotracheal tube has been replaced by the laryngeal mask airway. arterial blood gases, ventilation pressures and tidal volumes have been monitored, registered and compared. the median peak inspiratory pressure has been detected stable in all patients. furthermore, during the ventilation with the laryngeal mask, the tidal inspiratory and expiratory volume difference observed between before and after the bronchoscope positioning, has shown a statistically significant variation. finally, in all cases etco , spo . , pao , and blood ph values persisted within the normal range. the standard lma provides for a reliable airway management and allows an effective ventilation while performing the pdt. once positioned in the supraglottic zone, the lma does not need to be moved throughout all the pdt performance, avoiding risks of displacement, glottic harm and airway device damage, and permitting an easy handling of the bronchoscope, which gives an appropriated visualization of the trachea and a more efficient aspiration. in consequence to the large internal diameter of the lma tube, ppeak has continued to be stable in all patients, providing for minor resistance and inspiratory work. eventually, no late complications, such as tracheal stenosis and infections, have occurred. tracheostomies are the most common surgical procedure performed on critically ill patients. randomized control trials comparing tracheostomy timing in intensive care patients have been equivocal. in order to perform non-urgent tracheostomy in our icu, consent is required from the patient or a formal guardian appointed ad hoc by the courts. since tracheostomies are practically the only elective surgery performed in the critically ill, icu requested guardianship almost always indicates a clinical decision to perform tracheostomy. as appointing a guardian and arranging a tracheostomy takes about a week, the decision to appoint a guardian offers a unique "intention to treat" opportunity to evaluate outcomes in patients for whom tracheostomy is planned. we performed a retrospective analysis over years on patients for whom guardianship was sought excluding those requiring urgent tracheostomy and those with a do-not-resuscitate order. patients were divided according to outcome (tracheostomy, extubation or death prior to tracheostomy) and compared. guardianship was sought for ventilated patients. a decision to withhold tracheostomy was made for patients, who were excluded, leaving patients for analysis. tracheostomy was performed for / ( %) patients, / ( %) were extubated and / ( %) died while waiting for tracheostomy (from nonairway related reasons). tracheostomy was performed on mean ventilation day ± . comparing extubated patients to those who had tracheostomy (table) shows similar demographics, but significantly lower mortality and hospital length of stay. a significant proportion of patients initially planned for tracheostomy were successfully extubated. despite demographic similarities, mortality in this group was significantly lower than for patients undergoing tracheostomy. for a selected subgroup of possibly difficult to characterize patients, delaying tracheostomy may be beneficial. figure ). ptis were analysed by speciality and by outcome. complications occurred in cases (incidence . %). there were cases of subcutaenous emphysema, pneumothorax (occuring d post procedure) and case each of stoma and suture site infection. there was unplanned cannula change within days of insertion. % of cases had cuff inflated on discharge from icu. handover of care was suboptimal; follow up care plans were documented in % of cases. a supervising consultant was present for all ptis. there was a trend of increased insertion by consultant and increased reliance on theatre, with corresponding decrease in the number inserted by trainees. pti in our training icu appears safe with low incidence of complications and good senior support for tracheostomy insertion. emphasis must continue on training junior intensivists in pti. transition of care beyond icu requires further work where currently there is suboptimal handover of care and safety netting for non-icu colleagues. supplemental oxygen administration is ubiquitous in the critical care environment, yet evidence is mounting for the deleterious effects of hyperoxia [ ] . concerns over the adverse effects from hypoxaemia often exceed those of hyperoxaemia in developing world settings, and inconsistent availability of blood gas monitoring may limit judicious oxygen titration. the aim of this project was to audit oxygen delivery practice and introduce qi measures to avoid excess oxygen delivery in a tertiary icu in lusaka, zambia. a prospective snapshot of ventilatory parameters were recorded for critically ill patients over a -week period, including positive end expiratory pressure (peep), fio , and time-course spo . systematic education was provided through group and one to one tutorials to empower nursing and medical staff to titrate oxygen safely and appropriately. repeat data collection was then performed over weeks. initially / patients ( %) were over-oxygenated, as defined by fio > . and spo consistently > %. / patients with an fio of > . had peep ≤ cm ( %). no patient had a pao recorded in the past hours. education was provided as well as implementation of unit protocols above all patient beds documenting a stepwise approach to titration peep and fio . post intervention fewer patients were over-oxygenated: / ( %) had fio > . and spo consistently > %, and / with an fio > . ( %) had a peep ≤ cm. in addition, / ( . %) had a pao recorded within hours. this qi project has shown that nurse engagement and systematic education to titrate fio and peep can be achieved in a resource poor setting and may decrease the incidence of hyperoxia in critically ill patients. availability of blood gas monitoring and knowledge of interpretation was a major barrier to oxygen titration tracheal intubation (ti) in adult burn patients might be unnecessary in to % of cases [ , ] . in pediatric burn patients, there is little data on both the rate of ti and the rate of early extubation [ ] . it has been common practice for a child with a facial burn and/or a suspected airway injury to be intubated early due to the risk of losing airway patency. however this risk should be mitigated against the potential risks of ti and mechanical ventilation in children. therefore the aim of this study was to describe the airway status of child burn victims taken in charge of in our pediatric burn intensive care unit. focused on patients arriving with ti, we investigated the rate of early extubation. in addition we compared non intubated patients with those with prolonged ti. this retrospective study described a cohort of patients hospitalized between and . data was retrospectively recorded from the patient's paper clinical chart. the mean age of our patients was . ± . years [mean±sd] with an average burn area of ± %. % had scald burns and % had facial burns. % of the children were admitted in the burn icu with ti. for % of them, tracheal tube was removed within the first hours after admission. the probability of prolonged ti increased independently with the burned skin area (bsa) (p < . ), the presence of facial burns (p = . ), and in case of flame burns (p = . ) ( figure ). among patients with more than % bsa, % were intubated more than h. among patients with less than % bsa, . % were intubated more than h. according to our retrospective data, it seems appropriate to intubate children with % and more bsa, while for patient with less than % bsa, it might be relevant to seek guidance from physician of the nearest burn center. under % bsa, ti seems rarely required. an analysis of the predictive applicability of initial blood gas parameters for the need for intubation and the presence of inhalation injury in patients with suspected inhalation injury c pirrone , m chotalia , t mangham , r mullhi , k england , t introduction: we hypothesise that initial blood gas parameters have a good predictive applicability in detecting the need for intubation and the presence of inhalation injury in patients with suspected inhalation injury. to the best of our knowledge, this has not been directly studied in the literature. patients with suspected inhalation injury admitted to the icu at queen elizabeth hospital, birmingham between april and may were included. the initial blood gas parameters analysed were pao (kpa), paco (kpa), ph, carbon monoxide level (cohb; %) and pao /fio (pf) ratio. receiver operator characteristics (roc) for these parameters were plotted against the need for intubation for more than hours and the presence of inhalation injury as detected by bronchoscopy and laryngoscopy. area under the curve (auc) for each parameter was calculated. results: patients were admitted with suspected inhalation injury to the icu. % were intubated for more than hours. of patients who were intubated, % had inhalation injury as indicated by bronchoscopy or laryngoscopy. table outlines the auc for initial blood gas parameters in detecting the need for intubation for more than hours and the presence of inhalation injury. ph was the parameter with the most prominent auc, with reverse correlation indicating fair accuracy. no clear inflection point was identified, although all patients with ph < . required intubation and had inhalation injury. paco had a fair predictive applicability in detecting the need for intubation. pf ratio, pao and cohb had poor accuracy. conclusions: initial blood gas parameters had a broadly poor predictive applicability for the need for intubation and the presence of inhalation injury in patients with suspected inhalation injury. severe acidosis (ph < . ) was the most useful blood gas parameter. clinicians should be cautious in using blood gas parameters alone to inform intubation decisions. lung cancer surgery is associated with a high rate of pulmonary complications including ards and mandates lung protective ventilation strategies [ , ] . such strategies include non-intubated video assisted thoracic surgery (nivats) with spontaneous breathing [ ] . currently neither data on respirator settings nor on gas exchange have been reported for applying the latter. this data constitutes a prerequisite for meaningful evaluating the respiratory consequences of non-intubated spontaneous breathing during lung cancer surgery. the aim of this case series was for the first time providing such data from lung cancer surgery including pneumonectomy. during a month period patients without contraindications [ ] scheduled for video assisted thoracic surgery (vats) for non-anatomical and anatomical lung resection including one pneumonectomy (px) were offered non-intubated spontaneous breathing. all patients gave informed written consent to the procedure as well as for analysis and publication of data. anaesthetic management included target controlled infusion of propofol and remifentanil, laryngeal mask airway, and pressure support ventilation. we present early data that early trials of cuff deflation within hours of tracheostomy insertion can be achieved using a standardized protocol. its impact on length of stay, duration of ventilation and patient-centered outcomes needs to be investigated in larger multi-centre trials. preventing underinflation of the endotracheal tube cuff with a portable elastomeric device. a randomized controlled study je dauvergne , al geffray , k asehnoune , b rozec , k lakhal hopital laënnec -chu de nantes, service d´anesthésie-réanimation, nantes, france; hotel-dieu -chu de nantes, service d´anesthésieréanimation, nantes, france critical care , (suppl ):p the management of the endotracheal tube cuff pressure (p cuff ) is routine practice for critical care nursing staff. underinflation could lead to ventilator-associated pneumonia [ ] whereas overinflation exposes to tracheal damage [ ] . multi-daily check and adjustment is recommended to ensure that p cuff lies between and cmh o [ ] . to automate this task some devices exist but may be inconvenient, bulky and/or ineffective. their use is not supported by guidelines. a portable elastomeric device could be appealing for p cuff automated regulation. this prospective randomized controlled study tested whether the tracoe smart cuff manager tm reduced the rate of patients undergoing ≥ episode of underinflation (p cuff < cmh o), as compared with routine manual p cuff adjustment. monocentric, randomized controlled study. patients with acute brain injury and receiving mechanical ventilation were prospectively allocated to one of the two arms: manual reading and adjustment of p cuff at least every h (routine care) or adjunction of the smart cuff manager tm (intervention). this study was approuved by an institutional review board. among randomized patients (routine care in , smart cuff manager tm in ), measurements were performed in h. with routine care, a higher rate of patients experienced at least one episode of underinflation ( . vs. . %;p< . ). episodes of underinflation episodes ( % vs. %;p< . ) and manual adjustments ( % vs. %;p< . ) were more frequent with routine care. for overinflation, there was no between-arms difference (p> . ). the adjunction of continuous p cuff control with the tracoe smart cuff manager tm reduced the incidence of p cuff underinflation as compared with manual intermittent adjustments. overinflation was not promoted by this device. direct laryngoscopy as a technique for tracheal intubation is a potentially lifesaving procedure that healthcare professionals in a variety of fields are taught. however, this skill is challenging to acquire and difficult to maintain. poorly performed intubation technique can lead to potentially serious complications [ ] . the intersurgical iview video laryngoscope is a new intubation tool which may have advantages over direct laryngoscopes, such as the macintosh, in the hands of novice personnel. a prospective randomized counterbalanced trial of medical students, who did not have previous airway management experience, was conducted. each student received brief didactic teaching,following this, participants were directly supervised performing laryngoscopy and intubation using the macintosh and iview devices in an alternating pattern. students were permitted up to three attempts to successfully intubate under four conditions, three laryngoscopy conditions using alaerdal intubation trainer and one using a laerdal simman manikin. there was no significant difference in the success rate of intubation or time to intubation between the two devices. the iview outperformed the macintosh in time to intubation in the normal airway in the final scenario, once students gained experience with both devices. no significant difference was found in the number of optimisation manoeuvres, or intubation attempts between groups. areas where the iview outperformed the macintosh included severity of dental trauma and participants' perception regarding ease of use ofthe device. the iview may prove to be a useful teaching tool for novice personnel who are acquiring the skills of tracheal intubation. patients with a primary pulmonary pathology were more likely to respond to aprv. this association has not been described before and warrants further multi-centre exploration in a larger patient group. introduction: airway suctioning is common during mechanical ventilation, using either an open endotraqueal suctioning or closed endotracheal suctioning (ces). closed circuits were developed to prevent arterial desaturation and atelectasis associated to ventilator disconnection. however, ces may cause substantial loss of lung volume. the purpose of this study was to investigate the effects of a compensation method to prevent the loss in aeration during ces. the suctioning technique was performed for seconds, negative pressures limited at mmhg. closed suction catheters with fr (halyard health, georgia, eua) were used. electrical impedance tomography (eit) monitoring and arterial blood gas were collected. a nihonkoden mechanical ventilator (nkv , california, eua) was applied, having a newly developed algorithm for suctioning which overcomes any pressure loss during suctioning (inlinesuction-app). when activated, the app delivers pcv ventilation, adding cmh o of end-expiratory pressure above peep, and delivering driving pressures of cmh o. results: pigs ( ± . kg) with injured lungs and mechanically ventilated. we tested the aspiration procedures using low peep= cmh o, or high peep=± . cmh o with v t o), whereas maintenance of compliance was observed when the app was on (from . ± . ml/cmh o to . ± . ml/cmh o. blood gas in a representative animal showed a drop in pao when app was off (from , to mmhg after min, and to mmhg after min) ( figure ). with app on the pao changed from (pre-suction), to ( min), to mmhg ( min). the new nksoftware, delivering pcv ventilation during suctioning, could prevent atelectasis and functional loss associated to the procedure. tyrosine kinase inhibitor: an effective tool against lung cancer involvement responsible for acute respiratory failure in icu y tandjaoui-lambiotte patients with advanced-stage non-small-cell lung cancer have high mortality rates in the intensive care unit (icu). in the last two decades, targeted therapies have changed the prognostic of patients with lung cancer outside the icu. the fast efficacy of targeted therapies led some intensivists to use them as rescue therapy for icu patients. we performed a national multicentric retrospective study with the participation of the grrroh (groupe de recherche en réanimation respiratoire en onco-hématologie). all patients with non-small-cell lung cancer admitted to the icu for acute respiratory failure between and were included in the study if a tyrosine kinase inhibitor was initiated during icu stay. cases were identified using hospital-pharmacies records. the primary outcome was overall survival days after icu admission. results: thirty patients (age: +/- years old) admitted to a total of icus throughout france were included. seventeen patients ( %) were nonsmoker. adenocarcinoma was the most frequent histological type (n= , %). most patients had metastatic cancer (n= , %). epithelial growth factor receptor mutation was the most common oncologic driver identified (n= , %). during the icu stay, ( %) patients required invasive mechanical ventilation, ( %) catecholamine infusion, ( %) renal replacement therapy and one ( %) extracorporeal membrane oxygenation. eighteen patients ( %) were discharged alive from icu and ( %) were still alive after days (see figure) . moreover, patients ( %) were alive one year after icu discharge. despite a small sample size this study showed that, in the context of lung cancer involvement responsible for acute respiratory failure, the use of tyrosine kinase inhibitor should not be refrained in patients with severe condition in icu. the burned patient is one of the most complex patients whith a very high mortality. those patients with inhalation injury have a worst prognosis, typically associated with respiratory complications. the aim of our study is to evaluate the mortality of burn patientes with inalation injury in a critical burn unit. a prospective, observational and descriptive study was conducted over a period of years. inhalation injury was defined with these criteria (≥ ): history of injury in an enclosed space, facial burns with singed nasal hair, carbonaceus sputum and stridor. if they were intubated it was diagnosed by bronchoscopy. demographic data, tbsa, absi, baux score, apache ii, sofa, mechanical ventilation (mv), complications, length of stay, hospital course and mortality data were collected. results: burns patients were admitted. % ( patients) had inhalation injury. mortality among patients with inhalation injury was , % ( patients). most patients were men and those who died were older and with higher severity scores (fig. ) . we found no significant differences between groups in the need for mv ( % vs. %) or in the percentage of tracheostomy performed ( . vs. . ). however, patients who died had more respiratory complications like ards, and also shock, renal failure and need of renal replancement therapies although infectious complications were similar in both groups. there was no statistically significant difference in volume used during initial resuscitation in the different groups. patients with inhalation injury who died had higher severity scores at the begining. although there were no differences in the need for mv patients who died had more respiratory complications as well as shock, renal failure and need of rrt, but no infectious complications.the volume used during inicial resuscitation, that was always related to the prognosis, was similar in both groups. further studies are needed to see if this greater initial severity corresponds to the degree of inhalation. aerogen, medical affairs, galway, ireland; aerogen, science, galway, ireland critical care , (suppl ):p patients with acute exacerbations such as asthma are prescribed aerosol therapy from presentation in the emergency department to progression through to the intensive care unit. however, the variability in dose delivery to the lung across the possible patient interventions is not well characterized. here, we assess the predicted lung dose of a bronchodilator in a simulated spontaneously breathing adult patient via both facemask and nasal cannula, and via tracheostomy during mechanical ventilation. a standard dose of . mg in . ml salbutamol was aerosolized using the aerogen solo nebulizer (aerogen, ireland). for facemask testing, the nebulizer was used in combination with the aerogen ultra with lpm supplemental oxygen flow. for nasal cannula testing, the nebulizer was used in combination with the airvo system (fisher and paykel, nz) system at both and lpm gas flow rate. tracheostomy-mediated ventilation was assessed in combination with a hme, with the nebulizer placed between the hme and the tracheostomy tube. international standard iso adult breath settings (vt ml, bpm , i:e : ) were used across all tests, and generated using a breathing simulator (asl , ingmar medical, usa) or mechanical ventilator (servo-u, maquet, sweden). the dose delivered to the lung was assessed using a capture filter at the level of the trachea, with drug mass determined using uv spectrophotometry at nm and interpolation on a standard curve. the results of testing are illustrated in figure . the bronchodilator dose delivered to the simulated patient was seen to be relatively consistent between progressive interventions, except during high flow therapy, with the more clinically relevant lpm gas flow rate having a profound effect on the dose. these results may go some way towards explaining how different patient interventions can affect aerosol dose. the the mechanical ventilation (mv) have been identified as an independent factor indicating a worse prognosis for lung cancer patients [ ] . this study was conducted in order to assess the results of noninvasive mechanical ventilation (niv) and/or invasive mechanical ventilation (imv) modalities in lung cancer patients admitted to the icu with acute respiratory failure (arf). in this study, lung cancer patients with respiratory failure who were admitted to the icu between january and december were evaluated retrospectively. results: patients were included in the study. the mortality rate was . %. patients had niv. imv was applied to patients. in the first hours, of the patients who were initially treated with niv were administered imv. the duration of hospital stay, diagnosis of pneumonia and mortality rate were found to be significantly lower in patients treated with niv alone (p≤ . , p= . , p= . ), but glaskow coma score (gcs) was significantly higher in this group (p≤ . ). the mortality rate was similar between the patients who were initially treated with imv and those who were treated with imv in the first hours. charlson comorbidity index (cci) and mv duration were significantly higher in patients who died (p= . , p= . ), but gcs was significantly lower in this group (p= . ). in the linear regression model for the likelihood of mortality, ccl≥ and unsuccessful niv increased the mortality rate by . ( . - . ) and . times ( - . ) respectively (p= . , p= . ). niv has been an effective modality for respiratory support in most lung cancer patients presenting with arf. however, failed niv seems to be a factor for increased mortality. therefore, the choice of respiratory support modality to be applied in this patient group should be decided by considering the gcs, cci and etiology of arf. the interaction between ventilator settings and the occurrence of acute kidney injury is not fully elucidated. this study aimed at investigating the effect of stepwise increase in peep level on the risk of acute kidney injury as evaluated with the renal resistivity index (rri).the primary outcome is to investigate whether increased levels of peep could lead to increase rri and whether rri could predict the occurrence of aki. methods: patients mechanically ventilated for at least hours and without aki at admission were included in the study. rri was calculated at icu admission. posterolateral approach was used for kidney ultrasound. the peak systolic velocity (v max ) and the minimal diastolic velocity (v min ) were determined by pulse wave doppler, and the rri was calculated as (v max -v min )/v max . the exam was performed modifying the peep levels: , and cm h o in random order for minutes. occurrence of aki was defined within days according to kdigo criteria. sixty-four patients were enrolled in the study and incidence of aki was / ( %). demographical and clinical characteristics are reported in table . increase in peep showed a significant increase in rri from peep to peep (p< . ) and from peep to peep (p= . ) ( figure ). the area under the roc curve of rri to predict aki was . at peep , . at peep and . at peep (all p< . ). the youden index analysis showed an rri> . as the best cut off for aki with a sensibility of % and a specificity of %. patients with rri> . were / ( %), / ( %) and / ( %) at peep ,peep and peep respectively. patients ventilated with a peep value associated with rri> . had higher incidence of aki ( / vs / , p< . ). the application of peep can increase intrarenal vascular resistance,which is associated occurrence of aki; peep level should therefore be balanced taking into account the rri. the rri seems able to predict occurrence of aki in mechanically ventilated patients. alveolar and respiratory mechanics modifications produced by different concentrations of oxygen in healthy rats subjected to mechanical ventilation with protective ventilatory strategy d dominguez garcia , r hernandez bisshopp , jl martin barrasa , d viera camacho , a rodriguez gil , j arias marzan , s garcia hernandez high oxygen can damage tissues [ ] . in this study, we analyze the histological and pulmonary mechanics modifications that can occur when identifying different inspiratory oxygen fractions (fio ) in lungs of healthy rats during protective mechanical ventilation. we use sprague-dawley rat. groups were designed, each with animals, the tidal volume ( ml/kg), peep ( cmh o) and respiratory rate ( rpm) were kept constant, changing the fio between the groups. four groups were established: fio . , . , . and . after hours, the lungs were removed for histological study and obtaining the wet/dry index. the histological modifications studied were: alveolar septa (as), alveolar hemorrhages (ah), intraalvelolar fibrin (if) and inflammatory infiltrates (ii). each parameter was rated from to [ ] . peak pressure (pp) and pulmonary compliance were monitored every minutes. different statistical tests will be used to analyze the data. results: references to the damage produced in the as, ah, if, ii and the global histological pattern were identified in the groups with the highest fio and there was more damage (p < . ) ( figure ). the wet/dry index rose significantly as the oxygen concentration increased (p = . ). in the groups to which a fio of . and was administered, the pp selected specific values with respect to the baseline intake from the first minutes, an aspect that was not appreciated in the other groups (p < . ). regarding pulmonary compliance, it will be seen that, in the fio . and groups, it decreased from the first minutes, finding differences with respect to the other groups (p < . ). conclusions: mechanical ventilation applied for hours in healthy animals produces disorders that are more pronounced as oxygen concentration increase. fio greater than or equal to . should be avoided without clinical justification. introduction: patients requiring prolonged acute mechanical ventilation (pamv, defined as + days on mv) are sicker and incur disproportionate morbidity and costs relative to patients on short-term mv (stmv, < days of mv). we quantified specific clinical outcomes among patients requiring pamv vs. stmv in a contemporary database. we conducted a multicenter retrospective cohort study within~ hospitals in the premier database, - . using icd- -cm and icd- codes we identified pamv and stmv patients, and compared their baseline characteristics and hospital events. because of the large sample size, we omitted hypothesis testing. a total of , patients met the enrollment criteria, of whom , ( . %) received pamv. at baseline, patients on pamv were similar to stmv with regard to age (years: . ± . pamv vs. . ± . stmv), gender (males: . % pamv vs. . % stmv), and race (white: . % pamv vs. . % stmv). pamv group had a higher comorbidity burden than stmv (mean charlson score . + . vs. . + . ). the prevalence of each of the indicators of acute illness severityvasopressors ( . % vs. . %), dialysis ( . % vs. . %), severe sepsis ( . % vs. . %), and septic shock ( . % vs. . %)was higher in pamv than stmv, as were hospital mortality and combined mortality or discharge to hospice (figure ), extubation failure ( . % vs. . %), tracheostomy ( . % vs. . %), development of c. difficile ( . % vs. . %), and incidence density of ventilator-associated pneumonia ( . / , patient-days vs. . / , patient-days). conclusions: over / of all hospitalized patients on mv require it for days or longer. pamv patients exhibit a higher burden of both chronic and acute illness than those on stmv. commensurately, all clinical outcomes examined are substantially worse in association with pamv than stmv. identifying the readiness of patients recovering from critical illness for liberation from invasive mechanical ventilation (imv) is not always straightforward [ ] . the scottish intensive care society (sics) trainee audit conducted a scotland-wide study to understand current practices relating to liberation from imv. data were prospectively collected on patient demographics, indication for intubation, spontaneous breathing trial (sbt) practices, physiological markers, icu outcome and icu los. all patients > years ventilated with imv for > hrs from the st nov. - th nov. were eligible for inclusion. exclusion criteria included extubation for end-of-life, death whilst intubated and presence of tracheostomy. logistic regression was performed to detect factors associated with extubation failure (ef). results were analysed via excel and stata v. . . patient benefit and privacy panel approval was granted. total population of patients were included: ( %) male and median apache score (iqr - ). ef at first attempt occurred on occasions ( . %), median icu los of days (iqr - ), mortality rate . %. the cohort successfully extubated first time had a median icu length of stay of days (iqr - ) and mortality rate of . %. methods of sbt and extubation outcomes detailed in table . no sbt prior to extubation had higher odds of ef (or . , ci . - . , p= . ); patient ventilation for < days had a three times higher odds of ef (or . , ci . - . , p= . ). these were independently associated with ef on multivariate analysis conclusions: we found a reintubation rate of . % in scottish icus. type of sbt most commonly used is divergent from the methods advocated in the literature. the lack of sbt and early extubation attempt was associated with failure, which in turn was associated with longer icu los and higher mortality. in patients undergoing prolonged invasive ventilation we hypothesise that abnormal right ventricular (rv) and left ventricular (lv) function are associated with increased -day mortality. whether changes in lv or rv function could aid in the prognostication of these patients has not been directly studied. patients admitted to the queen elizabeth hospital birmingham icu between april and july who were intubated and ventilated for more than days and had a formal transthoracic echocardiogram (tte) whilst in icu were included. abnormal rv function was defined by the presence of depressed function, dilated size or moderate to severe risk of pulmonary hypertension. abnormal lv function was defined by the presence of lv depression (lv ejection fraction £ % or grade ii or more diastolic dysfunction) or a hyperdynamic lv (formally mentioned in tte report). patients who had a neurological cause for prolonged ventilation were excluded. the primary outcome was -day mortality. categorical data is presented as % and analysed using a chi-squared test. continuous data is presented as median (iqr). results: patients required prolonged ventilation, of which ( %) had a tte. patients were aged ( - ), were % male and had a % -day mortality. the median ventilator days were ( - ) and % required a tracheostomy. abnormal rv function was present in % (n= ) and was associated with an increased -day mortality compared to normal rv function ( % vs. %, rr . [ . - . ], p< . ). lv function was abnormal in % (n= ) and was associated with an increased -day mortality compared to normal lv function ( % vs %, rr . [ . - . ], p < . ). abnormal rv function had a trend towards an increased mortality compared to abnormal lv function ( % vs %, rr . [ . - . ], p = . ). in this study, abnormal rv and lv function were present in a quarter of patients undergoing prolonged ventilation and were associated with an increased mortality. introduction: tidal volume delivered by mechanical ventilation (mv) in sedated patients is distributed preferentially to ventral alveoli, causing overdistention and associated collapse in dorsal alveoli, driving volutrauma, atelectrauma and ventilator-induced lung injury [ ] . temporary transvenous diaphragm neurostimulation (ttdn) stimulates diaphragm contraction [ ] . when used in synchrony with mv, ttdn encourages increased dorsal ventilation due to the change in pressure gradients with diaphragm contraction, mimicking a more normal physiological pattern. this may improve gas exchange and reduce injury. a pilot study was conducted using kg pigs undergoing mv in a mock icu. deeply sedated subjects were provided lung-protective volume-control ventilation at ml/kg. ttdn diaphragm contractions were delivered in synchrony with inspiration on every second breath, reducing the ventilator pressure-time-product by - % during mv+ttdn breaths. tidal volume distribution was recorded in each condition using electrical impedance tomography, and compared to never-ventilated, spontaneously breathing subjects (nv). results: dorsal ventilation changed from % during mv breaths to % during mv+ttdn breaths, compared to % in the nv group (p= . ). ventral ventilation changed from % during mv breaths to % during mv+ttdn breaths, compared to % in the nv group (p= . , figure ). conclusions: ttdn diaphragm contraction used as an adjunct to mv yields a more physiological pattern of volume distribution. this translates into less overdistension in the ventral areas and less atelectrauma in the dorsal areas and reduces ventilator-induced lung injury. this technology introduction: by measuring the pes and its derivatives, we can measure the relationship that exist between the diaphragmatic excursion and the oscillation of the esophageal pressure curve: pswing (ps) so we infer that, just as with the pes, the variations of it might be related to a weaning failure [ , ] . however, no nominal value exists in the bibliography to predict the test result. patients who meet with the inclusion criteria start the weaning process through a test of minutes of spontaneous ventilation, t-tube (tt). and also the respiratory rate (rr) and the tidal volume (tv). from this analysis, an average ps (aps) is determined for each moment of the test (aps , initial and aps , final.).a quotient was obtained in relation to these variables using the value previously obtained (quotient dtv/dps x . a total of patients were included (n= ).regarding the evolution during tt, (n= ) ( %) were successful, while (n= ) ( . %) failed when analyzing a rate that relates the variables tv and ps, a quotient was obtained in relation to these variables using the value previously obtained (quotient dtv/dps) for patients who were successful and who failed, (dtv/dps)/ successful patients presented a value of . while those of the failure group presented a value of . , (or , - p= . ) ( table ) . when presenting the relationship between tv and ps through the quotient (dvt/dps)/ , it is observed a tendency to have a higher quotient among patients who failed versus those who did not fail. the process of weaning from mechanical ventilation imposes an additional workload on the cardiovascular system, which may result in impaired myocardial function, increase in left ventricular filling pressure and respiratory distress. among surgical patients, those undergoing heart surgery are particularly susceptible to cardiac dysfunction induced by weaning because of inadequate cardiovascular reserve. the aim of our study was to depict the pathophysiological changes assessed by echocardiography during the steps of weaning and to identify possible predictors of weaning failure (wf). we enrolled consecutive patients undergoing isolated coronary artery bypass grafting in our institution. data were obtained by intraoperative transesophageal echocardiography before sternotomy (t ) and by transthoracic echocardiography at the beginning of weaning (t ) and at the time of extubation (t ). wf was defined as deferral of planned extubation or respiratory failure needing reintubation or non-invasive mechanical ventilation within hours. results: wf occurred in patients ( . %) and involved manifestations of respiratory distress in ( . %). we found a significant association between left ventricle outflow tract-velocity time integral (lvot-vti) and ventricular-arterial coupling measured at t and wf, with lvot-vti emerging as the best predictor of wf with an area under roc curve of . ( figure ); an optimal cutoff value of cm provided % sensitivity and % specificity. significant increase in e/e' measured at t ( . vs . , p . ) suggested a cardiac etiology of respiratory distress in patients who failed the weaning trial. our study showed that serial assessment of hemodynamic parameters by means of echocardiography is feasible in cardiac surgical patients and can provide insight into pathophysiological changes during weaning. although these preliminary data need to be confirmed in a larger population sample, lvot-vti emerged as a promising predictor of subsequent wf. compliance with guidelines for respiratory therapy in preclinical emergency medicine g jansen, n kappelhoff, s rehberg protestand hospital of the bethel foundation, anaesthesiology, intensive care and emergency medicine, bielefeld, germany critical care , (suppl ):p introduction: current guidelines on pre-hospital emergency ventilation are based on the guidelines for lung protective ventilation in the intensive care unit. the present survey was designed to determine the accordance of actual pre-hospital emergency ventilation by german emergency physicians (gep) with these recommendations. recommendations include a respiratory rate (rr) between - /min, a tidal volume (vt) between - ml/kg, a maximum pressure (pmax) < mbar and a positive end-expiratory pressure (peep) of mbar. an anonymous web-based questionnaire encompassing questions was sent to gep from september to december of . gep were asked to specify their level of education, their preferred ventilation settings and the usually chosen parameters employed to guide mechanical ventilation. statistical analysis was performed using the ch²-test with a significance level ≤ . . % of the questionnaires were completed ( / ). % of the participants were trainees (tr), % consultants (co). as target parameters for guidance of ventilation, % of the tr and % of the co use capnometry. the vt controlled % of the tr and % of the co on the basis of body weight. % of the tr and % of the co reported to control oxygenation using spo . table shows our analysis of the given answers. there were no statistically significant differences between the groups. deviations from the guidelines of pre-hospital emergency ventilation settings are common and mainly concern the use of a guidelinecompliant peep. in addition, recommended target parameters for guidance of ventilation were not applied in a significant proportion of gep. prospective observational study including ltx recipients admitted to our icu from february to january , who underwent a spontaneous breathing trial (sbt) using a t-piece for minutes. clinical variables and arterial blood gas samples were recorded before starting sbt and after minutes on the t-piece. diaphragmatic excursion (de) and thickening fraction (dtf) were also assessed using ultrasound(us) after minutes on the tpiece. us-dd was defined as de< mm or dtf< . of at least one hemidiaphragm. patients who successfully completed a sbt, defined according to clinical criteria,were extubated. extubation failure was defined as the need for reintubation within h. results are expressed as medians (iqr) or frequencies (%). ltx recipients were admitted to the icu, of whom underwent an sbt. were male, and the median age was y. main indications for ltx were interstitial lung disease ( . %), copd and cystic fibrosis. were bilateral ltx, and and were left and right unilateral ltx respectively. patients were extubated after sbt and required reintubation within h. presented us-dd, though there were no differences between patients who succeeded and those needing reintubation. in contrast, patients who succeeded showed higher pao /fio after minutes on the t-piece (table ) . similarly, higher reductions in deltapao /fio after minutes on the t-piece were observed in patients who failed. oxygenation after sbt performed using a t-piece may predict extubation failure in ltx recipients with successful sbt. us-dd was not associated with the need of reintubation. descriptive study about the relationship between self-extubation episodes and patient-ventilator interaction s nogales , introduction: to evaluate the relationship between self-extubation and patientventilator interaction, among other physiological variables, in order to predict and to prevent these events. self-extubation (se) are quality indicators in patients under invasive mechanical ventilations (imv) and are related with mortality [ ] . planned secondary analysis of a prospective data base of clinical and physiologic signals of patients receiving imv. we included se episodes ( - ) with continuous record of ventilator and monitor signals (bclink bettercare®). we analysed demographic data, physiological parameters (peripheral oxygen saturation spo , heart rate hr, respiratory rate rr and media arterial pressure map) and patientventilator interaction (asynchrony index ai, ineffective efforts during expiration iee and double cycling dc). we studied a period of hours prior to the se episode. we used the wilcoxon non-parametric test and for a proper analysis a linear mixed effects model. we included episodes of se, mean age ± years, %men, apache ii at admission ± , , ± , days under imv until the episode, reintubation rate . %, icu stay , ± , days, icu mortality %. at the time of the se, % were under sedation, % with physical restraint. the % were in weaning. we observed a trend to increase in spo , rr, hr, map and asynchronies in the -hour period prior to se episode. we compared these variables from this period with a -hour period before and we observed a statistically the data presented in this study show that our results are in accordance with the literature with favorable mortality and early postoperative complication rates and support that this procedure is an excellent alternative for surgery in the elderly patients. it is reported that patients with pulmonary hypertension (ph; systolic pulmonary arterial pressure (spap)≥ mmhg)) have frequent cardiac complications after transcatheter aortic valve implantation (tavi). ph often gets worse in some patients despite the normal cardiac function after tavi. no studies have ever examined prognosis after tavi in patients with or without worsening of ph. therefore, we retrospectively examined the frequency of mid-to long-term heart failure and cardiac death in patients with and without deterioration of ph after tavi. among patients who underwent tavi at our hospital between february and march , we analysed patients with ph (spap≥ mmhg) before surgery. spap was measured in transthoracic echocardiography before and within week after tavi. patients were divided into two groups according to whether spap worsened/ did not change or improved after tavi. we examined the frequency of admission due to heart failure or cardiac death (death caused by heart failure, angina, or myocardial infarction) during the period of years after tavi. ph worsened or did not change after tavi in patients, while it improved in patients. the left ventricular ejection fraction measured within week after tavi showed no difference between the two groups ( . ± . % vs . ± . %, p= . ). the worsened/ no change group was higher in frequency of admission due to heart failure (logrank; p< . ) and cardiac death (logrank; p< . ). despite successful treatment for as by tavi, the frequency of heart failure and cardiac death was higher in patients who did not show improvement of ph after tavi, even in the absence of cardiac function decrease. vigorous intervention for ph worsening after tavi may be helpful to improve prognosis. the there are several different anti platelet drugs that can be used to treat acute cardiac events. currently there are no effective markers that can assess how these drugs modify coagulation profile and quality. a new functional biomarker that measures fractal dimension (df ) and clot formation time (tgp) has been developed [ ] . df quantifies clot microstructure whereas tgp is a real-time measure of clotting time. we aimed to validate df and tgp in st elevation myocardial infarction (stemi) and assess the effect of two p y inhibitors which have different pharmacological mechanisms: clopidogrel and ticagrelor. we prospectively recruited stemi patients in the emergency setting. venous blood samples were collected hours after admission, following treatment with either ticagrelor or clopidogrel, in accordance with the local guidelines at the time. the blood samples were tested using the df and tgp biomarker, platelet aggregometry, clot contraction and standard markers of coagulation. results: patients received clopidogrel and received ticagrelor. the df for clopidogrel was higher than ticagrelor ( . ± . vs . ± . , p= . which corresponds to a decrease in clot mass of % figure ) and the tgp was reduced ( ± sec vs ± sec, p= . a % reduction in time). the results of the study suggest that clopidogrel is less powerful in its effects on clotting characteristics compared to ticagrelor. blood from patients receiving clopidogrel formed quicker and denser clots. this would suggest the risk of secondary events or stent occlusion is lower in those patients on ticagrelor, highlighting that df and tgp may be important in identifying patients at risk of future thrombotic events, the study is ongoing and will investigate the long term outcome in these patients. introduction: new onset atrial fibrillation (noaf) during critical illness frequently resolves prior to discharge. however long-term risks of noaf (i.e. heart failure, ischemic stroke and death)remains high [ ] . previous studies noted that nearly half of noaf cases did not have diagnosis recorded [ ] . addressing this may reduce post critical illness mortality by increasing af surveillance post intensive care (icu) discharge. retrospective data was collected from an electronic health record for icu admissions over a month period from a biomarker is defined as a measurable indicator of some biological state or condition. combined with a good clinical evaluation, they can enable an early and safe diagnostic, thus a faster management for the patient. cardiac biomarker testing is not indicated in routine in the emergency department (ed) because of low utility and high possibility of false-positive results. however, current rates of testing are unknown. the aim of our study was to evaluate the importance of measuring cardiac biomarkers especially troponins, d-dimer, and btype natriuretic peptide in our daily practice, and to identify the latest recommendations for a better use of these biomarkers in the diagnostic and therapeutic approaches. we conducted a prospective observational study, over a months periods performed in the ed of the university hospital center ibn rochd, casablanca, morocco, including all patients admitted during our study period and having a blood test for at least one biological marker. the dataset was analyzed by spss statistics . . a total of patients was enrolled. troponins were tested in . % patients (high sensitive in . % and troponin i tni in . %), ddimer in . %, bnp % and nt pro bnp in . % of cases. the diagnostic impact was significant in . % of cases for troponins, . % of cases for d-dimer and . % for bnp. the therapeutic impact was considered important in . % cases for troponins, . % for ddimer and . % for bnp. cardiac biomarkers have an important role in the ed, not only do they confirm the diagnosis (including the role of troponins in acs) but also eliminate others (with a strong negative predictive value of d-dimer for thromboembolic disease) and prove the cardiopulmonary origin of acute dyspnea (the significant place of bnp in confirming the diagnosis of acute heart failure). a multicenter study on the comparison of inter-rater reliability of a new and the original heart score among emergency physicians from three italian emergency departments the heart (based on history,ecg,age,risk factors,troponin) score is a valid tool to stratify the acs in chest pain. but some reports suggest that its reliability could be low for heterogeneity in the assignment due to the subjective interpretation of the history. we used the chest pain score for the "history". in this study we compare the reliability of the new heartcps and original heart. this is a multicenter retrospective study conducted in italian ed between july and october using clinical scenarios. ten physicians were included after a course on heart and heartcps score. we used scenarios which included clinical and demographic data. each participant independently assigned scores to the scenarios using the heart and heartcps. we tested the interrater agreement using the kappa-statistic (k), the confidence intervals are bias corrected ; we used stata/se . statistical software . a p-value of < . defines statistical significance. the overall inter-rater reliability was good for heart and heartcps: kappa = . (ci %; . - . )and , (ci %; . - . ); with good agreement among all the class of risk for heartcps but moderate in the medium class for heart . we found significant differences of inter-rater reliability among the senior and junior physicians who used the heartcps:k= . (ci %; . - . )and . (ci %; . - . ). heartcps score increased its history inter-rater reliability specially among the junior physicians from k= . (ci %; . - . ) to k= . (ci %; . - . ).the junior physicians seem to be more reliable than senior with the heartcps:k= . ( . - . ) vs k= . (ci %; . - . ). the heartcps showed inter-rater reliability better than original heart among the medium class of risk and the junior group. it could be proposed to young doctors to stratify the acs risk of chest pain. limit: we used scenarios rather than real patients. a hybrid approach as treatment for coronary artery disease: endo-cabg or pci first, does it matter? introduction: the aim of this study is to discuss the short-term results of a hybrid approach combining minimally invasive endoscopic cabg (endo-cabg) with a percutaneous coronary intervention (pci). to bypass the disadvantages and potential complications of conventional cabg via median sternotomy, we developed the endocabg technique to treat patients with single-and multi-vessel coronary artery disease (cad). this procedure is performed with three -mm thoracic ports and a mini-thoracotomy utility port ( cm) through the intercostal space. this technique can be combined with pci: the hybrid approach. the sequence of the procedures (endocabg followed by pci or vice versa) may result in different outcomes. from / to / data from consecutive patients scheduled for a hybrid technique at jessa, belgium, were prospectively entered into a customized database. this database was retrospectively reviewed. subgroup analysis was performed to compare outcomes of patients who first received endocabg with patients who first received pci. a p-value < . is considered significant, a p-value < . is considered as a trend toward significance. four patients underwent revision surgery and patients died within the first days. in patients the left anterior descendens artery (lad) was grafted with the left internal mammary artery (lima), the right coronary artery (rca) was the most stented vessel using pci. patients first treated with pci received more units of fresh frozen plasma after endocabg compared to those who were first treated with endocabg (p= . ). there was also a trend toward significant more transfusion of packed cells in this small subgroup (p= . ). the hybrid approach is a feasible technique as a treatment option for patients with multi-vessel cad. if cabg follows the pci, patients are more likely to receive transfusion. a possible explanation could be the need for dual antiplatelet therapy prior to surgery in this group, but this needs further investigation. prognostic difference between troponin elevation meeting the mi criteria and troponin elevation due to myocardial injury in septic troponin t (ctnt) elevation in critically ill patients is common and is associated with poor outcome. using common assays, - % of patients in the icu will have elevated troponin level. our aim was to determine whether there is any prognostic difference between troponin elevation meeting the mi criteria (rise and fall more than % together with echo and ecg new abnormalities) and troponin elevation due to myocardial injury in septic patients. we enrolled patients with sepsis and mean sofa score , respectively in which ctnt level was measured more than once and analyzed there ecg and echo findings. patients were classified into three groups:definite mi (rise and fall ctnt ≥ % and contemporaneous changes on ecg and/or echo),possible mi (rise and fall ctnt ≥ % and no other findings),myocardial injury (ctnt rise less than %) results: data from patients were analyzed ( % female; mean age . (sd . )). a total of patients had at least one elevated ctnt more than . mkg/l. in ( %) of patients ctnt level rised more than % from the first elevated measurement. ( %) of patients met mi criteria considering new ecg and echo findings. the overall mortality rate in all patients was . %.the mortality rate didn't differ significantly in three groups: in the definite mi group . %, in the suspected mi group %, in the non mi ctnt elevation group , %, p= , . coronary angiography was performed in ( %) of patients from the definite mi group,pci was performed in ( %) of patients. the mortality rate in the invasive group was not significantly lower comparing to the nonivasive group % vs , %, p= , . bleeding complications were significantly more frequent in the definite mi group % vs % and % respectively conclusions: ctnt level elevation is associated with poor outcome regardless coronary or non coronary injury. myocardial revascularization may be beneficial in patients with sepsis and definite mi, but it is also associated with increased bleeding risk. diagnostic interest of "marburg heart score" in patient consulting the emergencies department for acute chest pain chest pain is a common reason for emergency department visits, although this primarily refers to acute coronary syndrome (acs), this symptom may be frequently related to other non-ischemic etiologies. the aim was to validate the marburg heart score as a tool to exclude coronary artery disease in emergency department patients with nontraumatic acute chest pain. methods: a prospective, observational, descriptive and analytic cohort study conducted in the emergency department, from february st to march st, , collecting patients consulting for nontraumatic acute chest pain, the "marburg heart" score was calculated for all these patients. telephone contact was made after weeks to look for an ischemic cardiovascular event. we included patients. the mean age was +/- years, the sex ratio was . . the majority of the patients ( . %) consulted directly to the emergency department, . % were referred by a primary care physician. the median time to consultation after the onset of chest pain was hours. high blood pressure was the most common risk factor ( . %), followed by smoking ( %), diabetes ( . %) and dyslipidemia ( . %). thirty-five patients ( . %) had already coronary heart disease, ecg was pathological in . % of patients, patients had an acs with st segment elevation. at six weeks, . % of the patients had an acute coronary event. according to the patients' answers on the questions of the marburg heart score. the area under the roc curve of this score was . with a negative predictive value of . %; the "marburg heart score" is a simple, valid and reproducible clinical score with a discriminatory power to rule out the diagnosis of coronary artery disease from the first contact with the patient presenting for chest pain in emergencies. the abdominal aortic aneurysm (aaa) surgery is a complex procedure in elderly patients with high cardiovascular risk. anesthesiological techniques should play special attention to the volume status during cross-clamping as well as to the blood loss. goal directed fluid therapies (gdt) in aaa surgery in elderly patients decrease the perioperative morbidity and mortality [ ] . aim of this study is to investigate administration of fluid-based on either a gdt approach or a control method (fluid administered based on static preload parameters and traditional hemodynamic) in all phases of aaa surgery and especially in the phase of clamping and de-clamping. a total of patients asa iii, randomly scheduled for elective, open aaa surgery were included in this clinical trial. they were randomly assigned to two groups i -gdt with targeting stroke volume variation (svv) and ii -control group where fluids were administered at the discretion of the attending anaesthesiologist. in both these groups hemodynamic parameters, central venous pressure (cvp), temperature, blood loss and diuresis were registered during the operation and hours postoperatively. each group was assessed for postoperative complications. gdt group received less fluids and had a higher cardiac index (ci) ( . ± . vs. . ± . l/minute per m , p < . ) and stroke volume index ( . ± . vs. . ± . ml/m , p < . ) than the control group. there were significantly fewer complications in the intervention than control group ( vs. , p = . ). gdt fluid administration enables less use of fluids, improved hemodynamic and fewer postoperative complications in elderly patients undergoing aaa surgery. ultrasonography is a valid diagnostic tool, used to measure changes of muscle mass. the aim of this study was to investigate the clinical value of ultrasound-assessed muscle mass, in patients undergoing cardiothoracic surgery that present muscle weakness postoperatively. for this study, consecutive patients were enrolled, following their admission in the cardiac surgery intensive care unit (icu) within hours of cardiac surgery. ultrasound scans, for the assessment of quadriceps muscle thickness, were performed every hours for days. muscle strength was also evaluated in parallel, using the medical research council (mrc) scale. of the patients enrolled, ultrasound scans and muscle strength assessment were performed in patients. the muscle thickness of rectus femoris (rf), was slightly decreased by . % ([ %ci: - . ; . ], n= ; p= . ) and the combined muscle thickness of the vastus intermedius (vi) and rf decreased by . % ([ % ci: - . ; . ], n= ; p= . ). patients whose combined vi and rf muscle thickness was below the recorded median values ( . cm) on day (n= ), stayed longer in the icu ( ± vs ± hours, p = . ). patients with mrc score ≤ on day (n= ), required prolonged mechanical ventilation support compared to patients with mrc score ≥ (n= ), ( ± vs ± hours, p = . ). the use of muscle ultrasound seems to be a valuable tool in assessing skeletal muscle mass in critically ill patients after cardiothoracic surgery. moreover, the results of this pilot study showed that muscle wasting of patients after cardiothoracic surgery is of clinical importance, affecting their stay in icu. prediction of cardiac risk after major abdominal surgery s musaeva, i tarovatov, a vorona, i zabolotskikh, n doinov kuban state medical university, anesthesiology and intensive care, krasnodar, russia critical care , (suppl ):p the aim is to assess the incidence of cardiovascular incidents in major abdominal surgery [ ] using the revised lee index. a study was conducted of elderly patients who underwent major abdominal surgery in the krasnodar regional clinical hospital no. under combined anesthesia. in the preoperative period, the risk of cardiovascular incidents was assessed using the revised lee index and the functional status was assessed by met. depending on the lee index, groups were identified: group (n = ) -low risk (index value - ), group (n = ) -intermediate risk (index value - ); group (n = ) -high risk (index value> ). we estimated the incidence of critical incidents in groups: hypo-, hypertension, arrhythmias, and bradycardia. in the general population, cardiac risk was . ± . points; functional status - . ± met. the greatest number of critical incidents was recorded in patients with high risk ( . %), the smallest -in patients with low risk ( . %), in patients with intermediate risk - . % (n < , between groups according to chi-square criterion). in the structure of critical incidents, hypotension was most often encounteredin ( %) patients, while some patients revealed several incidents from the circulatory system (n = ). overall, the lee scale showed good prognostic ability (auroc = . ) in predicting hemodynamic incidents. the revised lee index is a useful tool to help assess the risk of cardiovascular incidents and determine patient management tactics in the perioperative period. postoperative cognitive dysfunction (pocd) remains an unresolved problem due to lack of consensus on its etiology and pathogenesis. some believe that pocd is the result of the direct toxic effect of general anesthetics on the nervous system. others claim that surgical trauma activates proinflammatory factors that induce neuroinflammation. wistar rats were allocated into groups: -minor surgery (n= ), major surgery group (n= ). after days of handling and habituation rats undergone surgery under isoflurane general anesthesia ( vol.%). group rats underwent laparotomy with gentle gut massage followed by wound closure. rats in group undergone left side nephrectomy. starting from the th postoperative day spatial memory in rats was studied in morris water maze which is a cylinder metal pool with a diameter of . and a height of . m filled with water (temp. ± o c) up to half. it has a platform with a diameter of cm and a height of cm below the water level. testing was preceded by a training stage, which included sessions daily for days. thus, rats developed spatial memory to the location of the platform. on the th day of the study test stage was conducted to assess spatial memory: rats were launched from points into maze without platform and data were recorded for seconds at each session. time spent on the target quadrant (ttq) and the number of target area crossings (tac) were registered. a second test was conducted days after the first test to evaluate long-term spatial memory. the duration of surgery and anesthesia did not differ significantly between groups. there was a significant difference between groups in average ttq and tac in test (table ). in test minor surgery group showed better results but they were less significant. major surgery is associated with a more pronounced deterioration of spatial memory in rats in early postoperative period compared to minor surgery. cardiac inflammatory markers in icu patients with myocardiac ischemia after non cardiac surgery (a pilot study) p manthou , g lioliousis , p vasileiou , g fildissis national kapodistrian university of athens, athens, greece; national kapodistrian university of athens, general thoracic hospital´´sotiria´´, athens, greece; national kapodistrian university of athens, university of athens, athens, greece critical care , (suppl ):p patients with known coronary artery disease have higher perioperative risk for myocardial ischemia [ , ] . mortality is frequent following cardiac ischemia in the intensive care unit (icu) after non-cardiac surgery. the first group includes patients admitted to the intensive care unit for post-operative follow-up without myocardiac ischemia in the first hours. the second group includes patients with myocardiac ischemia postoperatively and needs intensive care monitoring. cardiac risk assessment was made with the lee index,hemorrhagic risk assessment with the has-bled bleeding score and thrombotic risk assessment with cha ds -vasc score. postoperatively, pathological test values such as bnp, troponin, crp, calcitonin were estimated. the sequential organ failure assessment (sofa) systeme was used to assess sepsis. the nursing activity score (nas) scale was used to measure the workload of various nursing activities in the icu. according to the pilot study, the sample consists of patients. . % had myocardial ischemia. the lee index was significantly higher in patients with myocardial ischemia. the duration of hospitalization, the high dose of vasoconstrictive drugs, the length of stay in the icu, the duration of mechanical stay and the nursing workload were higher in patients with myocardial ischemia. ck-mb and troponin levels differed significantly between the two groups. creatinine, bilirubin and bnp during the hours were significantly higher. patients with myocardial ischemia had significantly higher mortality. cardiac risk assessment, has-bled score and cha ds -vasc score in combination with cardiac enzymes such as troponin could predict myocardiac ischemia in severely ill icu patients. introduction: according to the literature an airway complication followed thyroid gland surgery are: difficult trachea intubation, tracheomalacia, postextubation stridor and bleeding [ , ] . most common cause of death was problem with respiration and airway obstruction [ ] . subsequent hypoxia could require emergency airway and even tracheostomy [ ] . aim of our study was to determine the most common of airway complications and their association with type of surgery in our region. the retrospective cohort study included pts., ( women, men) was performed in odessa regional hospital, oncology centre odessa. there were three types of patients: with euthyroid goiter - ( %), polynodos goiter - ( %) and thyroid cancer - ( %) ( table ) . airway complications were diagnosed after trachea extubation based on indirect laryngoscope, presence of stridor, desaturation. the pearson's criteria was calculated. the ratio of airway complications after thyroid surgery was . % ( pts). the main reasons of airway complications in thyroid surgery included: laryngeal edema - pts ( . %); recurrent laryngeal nerve injury - pts ( . %) and postoperative bleeding pts ( . %). thyroid gland cancer and polynodosal goiter associated with laryngeal edema and recurrent laryngeal nerve injury (pearsen criteria were . -moderate and . consequentially). it's may require more attention from the anesthetists after extubation and readiness for an urgent airway. serum iron level and development of multiple organ dysfunction syndrome in patients in the perioperative period s tachyla mogilev regional hospital, department of anesthesiology and intensive care, mogilev, belarus critical care , (suppl ):p recently there has been attention of researchers to the problem of perioperative anemia. it was found that it increases the risk of death and postoperative complications. threatening complication is multiple organ dysfunction syndrome (mods). the objective was to determine the level of serum iron in the perioperative period in patients with endoprosthetics of large joints, and with the presence of mods in abdominal surgery. a prospective cohort study was conducted in patients, including men and women, age . ± . years. two groups were identified: st (control) -patients after endoprosthetics of large joints (n = ), nd (main) -patients in abdominal surgery with the presence of mods (n = ). the presence of mods was established based on the criteria for the sccm / accp conference. serum iron was monitored using an au analyzer (usa). the study identified several stages: st -before surgery, nd - st day after surgery, rd - rd day, th - th day, th - th day. when studying the indicators of serum iron, its significant decrease (p < . ) in the postoperative period was established. in the st group: st stage - . ( - . ) mmol / l, nd stage - . ( . - . ) mmol / l, rd stage - . ( - . ) μmol / l, stage - . ( . - . ) μmol / l, stage - . ( . - ) μmol / l. in the nd group: st stage - . ( - ) mmol / l, nd stage - . ( . - . ) mmol / l, rd stage - , ( . - . ) μmol / l, stage - . ( . - . ) μmol / l, stage - . ( . - ) μmol / l. moreover, in both groups, iron increased at the th stage against the nd stage (p < . ). when comparing the level of iron between the groups, significant differences were found (p < . ) at the nd, rd and th stages. in patients in the postoperative period, a decrease in serum iron is observed, the level of which rises by the th day, but does not reach the initial values. this decrease is more pronounced in patients with the presence of mods after abdominal surgery. kidney and pancreatic graft thrombosis happened in . % and . %, respectively, and bleeding in . %. forty-one ( . %) developed at least one infection during hospital stay. infection during icu was found in . % and main pathogens were gram negative bacilli sensible to beta-lactam. after icu, the incidence of multi-drug resistant pathogen was . %, predominantly gram negative bacilli. fungal infection was lower %. all-cause hospital mortality rate was . %. infectious complications are the main cause of morbidity and mortality following spk transplantation. the administration of broadspectrum prophylactic antibiotics are leading to the appearance of multi-drug resistant pathogens. knowing local microbiological flora may be helpful, allowing more adequate antibiotic prophylaxis. introduction: cardiopulmonary bypass (cpb) is associated with thrombotic complications. occurrence of thrombosis after cpb is % which takes the third place between cpb-associated complications. our study determined preoperative predictors of thrombosis in children with congenital heart defects. patients with congenital heart diseases in age up to months days (median age - , months, youngest age - days after birth, oldest - months days), underwent cardiac surgery with cpb, were enrolled in this study. all patients were divided into two groups: st -without thrombosis, nd -with thrombosis. protein c, ddimer, von willebrand factor and plasminogen plasma levels were assessed directly before surgery. thrombotic cases were proven by performing doppler ultrasound or mri. thrombotic complications were diagnosed in children ( %). between all thrombotic complications ischemic strokes were diagnosed in % ( cases), arterial thrombosis in % ( cases), intracardiac thrombus in % ( cases) and mechanical mitral prosthetic valve thrombosis %( ). receiver operating characteristic (roc) curves are created for the listed indicators. area under the curve (auc) for protein c , (sensitivity(sn)- %, specificity(sp) - %), d-dimer is , (sn - %, sp %), for plasminogen activity - , (sn %, sp %) and for von willebrand factor level - , (sn %, sp %). an roc curve was created for all three indicators, the auc was . (sn - %, sp - %). these parameters can be recommended as predictors of thrombosis in children after cardiac surgery. cpb is related with a large number of life-threatening complications. in our work, preoperative predictors of thrombosis were identified. based on this data, it is possible to create thrombosis risk scale change the tactics of the anaesthetic approach, the prevention of thrombosis in the postoperative period. further studies are needed to identify other possible predictors of thrombosis. introduction: abdominal ischemia occurs in % of patients submitted to aortic aneurysm repair. its early diagnosis requires an elevated index of suspiction, particularly in more severe patients. we hypothesized that earlier increase and higher levels of c-reactive protein (crp) may help to predict intra-abdominal ischemia. we performed a retrospective study of patients admitted to the intensive care department (icd) after abdominal aorta aneurism surgery. we included all patients admitted during a two-year period, that survived for more than hours. primary outcome was splanchnic ischemia assessed by abdominal ct-scan. we also evaluated the presence of bacteremia, abdominal compartment syndrome and icd mortality. association between inflammatory parameters and ischemia was evaluated by multivariate logistic regression. introduction: crp (c-reactive protein) has been shown to be a useful biomarker in identifying complications after major abdominal surgery. gastrectomy is a high-risk surgical procedure that requires post-operative critical care support to monitor for complications which are predominantly infective in nature. the aims of this study were to determine whether there is a relationship between post-operative crp levels and patients who developed post-operative infective complications. a retrospective analysis was performed on patients undergoing elective gastrectomy for gastric cancer at a single centre between september and july . post-operative crp levels for each day following resection were analysed for all patients. roc curve analysis was used to determine which post-operative day (pod) gave the optimal cut-off. of patients included, the majority were male ( . %), mean age was . years and . % had node-negative disease. a total of patients ( . %) had an infective complication, which includes those who experienced an anastomotic leak. crp levels on post-operative day gave the greatest auc for the gastrectomy group ( . ). crp cut-off of mg/l was significantly associated with infective complications (or . , % ci . - . , p= < . ) and gave a sensitivity of % and specificity % (ppv %, npv %). more patients with a crp > on post-operative day experienced an infective complication ( % vs %, p = < . ) or a leak in particular ( % vs %, p = . ). a crp level of less than mg/l on pod may be useful to predict the development or exclude the likelihood of such infective complications in this group of patients prior to clinical signs (ppv %, npv %). this may prompt and facilitate decision-making regarding early investigation and intervention or prevent inappropriate early discharge from critical care, whilst providing more assurance in identifying those who could be stepped down to ward level care. vasoplegia is commonly observed after cardiopulmonary bypass surgery (cpb) and associated with high mortality. chronic use of reninangiotensin aldosterone system inhibitors (raasi) is associated with its incidence and ensuing need for vasopressor support after cpb. renin serves as marker of tissue perfusion [ ] . we examined the role of renin in the setting of raasi exposure and vasopressor needs in the peri-cpb period. prospective observational study of adult patients undergoing cpb, aged . ± . years ( men, women). blood was collected ) post induction, pre-cpb; ) min post cardioplegia, and ) immediately post bypass. vital signs and perioperative medications were recorded. as control, blood was collected from men and women aged . ± . , not diagnosed with lung disease and not prescribed any raasi. baseline plasma renin in cpb patients tended to be higher than in control subjects (mean= . pg/ml± . vs. . pg/ml ± . , respectively, p= . ). minutes into cpb, mean renin was increased from baseline ( . pg/ml± . , p= . ), and remained elevated immediately post cpb ( . pg/ml± . ). patients using raasi prior to cpb tended to have a larger increase in renin post cpb (delta= . pg/ ml± . ) vs. those not previously on raasi ( . pg/ml± . , p= . ). renin was elevated in patients requiring vasopressor support in the hours post cpb vs. those not requiring pressors ( . pg/ ml± . vs. . pg/ml± . p= . ). in those prescribed raasi and requiring pressors post cpb, there was a tendency toward greater renin increase than those not requiring pressors postoperatively ( . pg/ml± . vs. . pg/ml± . , p= . ). this study suggests a trend toward higher renin levels, particularly during cpb, in patients prescribed raasi, and a positive association between renin and postoperative vasopressor needs. we speculate that increased renin levels may predict postoperative vasoplegia. cardiac surgery is associated with perioperative blood loss and a high risk of allogenic blood transfusion. it has been recognized that high blood product transfusion requirement is associated with adverse clinical outcomes. guidelines on patient blood management therefor aim at reducing blood loss and blood transfusion requirements in cardiac surgery. as there remains controversy about the advantage of minimal invasive techniques on blood loss an transfusion requirements, we wanted to investigate if the average blood loss and transfusion requirement in minimal invasive endoscopic coronary artery bypass graft surgery (endo-cabg) differ from conventional technique. we assessed the influence of pre-operative anticoagulant medication for blood loss. estimated average blood loss after conventional cabg is ml (+/- ) and transfusion requirement , units packed red blood cells . we performed a retrospective cohort study of our cardiac surgical database. from / / to / / , we collected data from patients undergoing endo-cabg. we analyzed blood loss, transfusion as well as pre-operative use of anti-coagulants as a risk factor for blood loss. we found that mean total blood loss in endo-cabg does not differ from conventional cabg, nonetheless mean transfusion requirement was lower in our cohort. use of direct oral anticoagulant is aossciated with increased blood loss and transfusion requirements (table ) . total blood loss is not influenced by minimal invasive technique for cabg (endo-cabg). an explanation for the lower transfusion requirements is the use of a minimal extracorporeal circulation, which is known to reduce the risk of transfusion. another important factor is the implementation of a standardized transfusion-protocol based on available evidence. reducing transfusion requirements is an important component in improving patient outcome after cardiac surgery and is related to multiple factors in perioperative care of our patients. retinal microvascular damage associated with mean arterial pressure during cardiopulmonary bypass surgery v shipulin retinal perfusion corresponds to cerebral perfusion and it is very sensitive to hemodynamic disturbances [ , ] . we investigated the association between retinal microvascular damage and hemodynamic characteristics in patients undergoing coronary artery bypass grafting surgery (cabg) with cardiopulmonary bypass (cpb). methods: patients with coronary artery disease and systemic hypertension were examined. ophthalmoscopy and optical coherence tomography were performed before and - days after cabg. the hemodynamic parameters during cpb were analyzed. results: ( %) patients had changes in the retinal vessels and in the ganglionic fiber structure on - day after surgery: in % of patients the foci of ischemic retinal oedema appeared, in % the decrease of the thickness of ganglionic fiber were observed. these changes may be associated with intraoperative ischemia of the central retinal artery. in ( %) patients the mean arterial pressure (map) during cpb was increased up to mmhg. in ( %) of them the association between map and foci of ischemic retinal oedema were revealed. the ischemic retinal changes were observed significantly more often if the delta of map during cpb was over then mm hg compared with the patients where the delta of map was less than mm hg (p= . ). this is probably due to an intraoperative disorders of the myogenic mechanism of blood flow autoregulation in the retinal microvasculature in patients with coronary artery disease [ ] . the level of map up to mm hg during cpb is associated with retinal blood flow impairment and the foci of ischemic retinal oedema. delta of map more than mmhg was associated with the foci of ischemic retinal oedema and decreased ganglionic fiber thickness in % of cases. atrial fibrillation after cardiac surgery: implementation of a prevention care bundle on intensive care unit improves adherence to current perioperative guidelines and reduces incidence introduction: atrial fibrillation after cardiac surgery (afacs) is a very frequent complication affecting - % of all patients. it is associated with an increase in morbidity, mortality and hospital and intensive care unit (icu) length of stay. we aimed to implement an afacs prevention care bundle based on a recently published practice advisory [ ] , focusing on early postoperative (re)introduction of β-blockers. baseline afacs incidence and β-blocker administration practices in our centre were audited for all patients undergoing valve surgery or coronary artery bypass graft (cabg) during a weeks period. the afacs prevention care bundlean easy to follow graphical toolwas subsequently introduced to the cardiac icu by a multidisciplinary team and audited following a model of improvement approach. after exclusion of patients with preoperative af, differences between pre-and post-implementation groups were compared with chisquare and fisher's exact tests for categorical, and one-way anova for continuous variables, using spss. a total of patients were analysed. patient and surgery characteristics did not differ between groups. significantly more patients received postoperative β-blockers after bundle implementation ( . % pre-vs . % post-bundle, p= . ) with a higher proportion on day ( . % pre-vs % post-bundle, p< . , figure ). the incidence of afacs was significantly reduced from . % to . % (p= . ), with a particularly marked reduction in the age group - years and for isolated aortic valve and cabg surgery. there was no significant reduction in hospital length of stay for this cohort. introduction of an afacs prevention care bundle using a graphical tool improved adherence to current guidelines with regards to early β-blocker administration and significantly reduced afacs incidence. future care bundles should include preoperative interventions and might reduce hospital length of stay. in neonates with univentricular physiology, there is a delicate balance between pulmonary and systemic circulations, with a tendency towards generous pulmonary blood flow, and a risk of systemic underperfusion. preoperatively, the use of hypoxic gas mixture (hm) has been advocated as a therapy to increase pvr, with the aim of improving systemic oxygen delivery. it is a therapy which has been routinely initiated in our institution in the setting of signs of pulmonary overcirculation. we performed a retrospective analysis of all patients in our institution who underwent a norwood procedure and who received hm preoperatively. we compared peripheral saturations, arterial blood gas analysis, serum lactate, regional cerebral and renal saturations and invasive blood pressure, prior to, and then , and hours after hm was commenced. between and (inclusive), patients underwent the norwood procedure. patients received preoperative hm. average fio was % during administration of hm. average peripheral saturations were . % prior to hm, and dropped to . % at hours, and % at and hours after initiation (p < . ). there was no change in any of the measured markers of systemic oxygen delivery, including regional cerebral and renal saturations, lactate, urine output or blood pressure. there was an association between an extended period of hm (> hours) and the need for pulmonary vasodilator therapy post norwood procedure. hypoxic gas mixture in patients with parallel systemic and pulmonary cicrculations causes desaturation and hypoxia. it does not lead to an increase in systemic perfusion and thus an improvement in systemic oxygen delivery. its ongoing use in this fragile population should be considered. introduction: analgesia in the critical patient, and especially in the neurocritical patient, is a basic goal in all therapeutic practices. patients in the icu are frequently administered prolonged and/or high doses of opioids. multiple serious complications due to the use of infusion of opioids at large doses has been described. to reduce high doses of intravenous opioids, multimodal forms of analgesia can be used. prospective observational study of the use of tapentadol enteral and buprenorphine in transdermal patches, at low doses, for the control of pain and its effect on reducing the use of fentanyl infusion in high doses on patients admitted to neuro icu of indisa clinic during consecutive years ( - ). enteral tapentadol (through ng tube) mg/ hours, was considered in patients who required intravenous fentanyl in continuous administration. buprenorphine was also added at low doses ( ug/hr) in a weekly transdermal patch, in cases of neurosurgical spine patients, fractures and long-term neuropathic pain. pain was controlled on behavioral pain scale (bps) and visual analogical scale (vas) scores, according to the conditions of each patient. their hemodynamic, gastrointestinal complications and the appearance of delirium episodes according to cam-icu scale were recorded. results: patients received tapentadol. of them also received transdermal buprenorphine. all managed to maintain adequate level of analgesia, not requiring fentanyl at doses greater than . ug / kg / hr. distribution by diagnoses: neurotrauma patients, guillain barre , spine surgery , hsa , hice , malignant ischemic acv . complications: gastric retention patients ( %), hypotension ( %), acute hypoactive delirium ( . %), acute hyperactive delirium ( %). no drug interactions were found. the introduction of enteral tapentadol and buprenorphine patches in neurocritical patients was safe and resulted in a decrease in the use of endovenous opioids and its adverse effects. we hypothesized that changing the pain management for our post cardiac surgical patients to an assessment-driven, protocol-based approach using fast acting and easily titratable agents will significantly improve patient satisfaction by reducing pain intensity in the first h after surgery as suggested by society of critical care [ ] guideline. we prospectively assessed and ( . vs . ) consecutive patients before and after introducing our pain management protocol. the nursing and medical team received rigorous training on the guideline as well as the correct assessment using appropriate pain scores measured at least hourly (numeric pain score, ≥ is timing of beta-blocker (re)initiation versus incidence of afacs before and after prevention care bundle implementation, per post-operative day and for postoperative days - (insets) moderate to severe or critical care observation tool, > is moderate to severe). we introduced a multimodal approach with a combination of fast acting iv, long acting oral opiates, regular paracetamol and rescue iv boluses for difficult to control situations and we created a prescription bundle on our electronic prescribing record. among other variables we assessed hours spent in moderate to severe pain in the first h after surgery and compared to the data collected before the guideline was introduced. we analysed patients from and from . baseline characteristics were similar between the two groups. in only . % of the patients spent less than hours and . % spend more than hours in moderate to severe pain. the data showed significant improvement in that . % of patients spent less than hours and only % patients who spent more than hours in moderate or severe pain. (p < . , chi square) ( figure ). only % of the patient needed rescue medications. % of time was the protocol inadequate necessitating other approach. introducing an assessment driven, stepwise, protocolized pain management significantly improved patient satisfaction by reducing pain intensity in the first h on our cardiothoracic intensive care unit. introduction: proximal femur fractures are most common fractures in the elderly and associated with significant mortality and morbidity, with high economic and social impact. perioperative pain management influence outcomes and mortality after surgery with early mobilization being possible [ , ] . the goal of the study was to compare the efficacy and safety of the compartment psoas block for perioperative analgesia in elderly patients with proximal femur fractures. the randomized controlled study was held in medical center "into-sana" (odesa, ukraine) from january till july . patients with proximal femur fractures and older than years were included in the study. they were randomly allocated to groupscompartment psoas block group (bupivacaine analgesia was started as soon as possible before surgery and prolonged during and after surgery with additional ischiadicus block before surgery) and general (inhalational) anesthesia with systemic analgesia perioperatively. results: patients were included in this study. perioperative compartment psoas block was associated better pain control, decreased opioid consumption, better sleep quality, earlier mobilization after surgery, decreased incidence of opioid-associated vomiting/nausea and myocardial injury. there were no difference in the incidence of hospital acquired pneumonia and delirium. perioperative compartment psoas block is effective and safe for perioperative analgesia in elderly patients with proximal femur fractures, and is associated with better pain control and decreased complications incidence. parenteral olanzapine is frequently used in combination with parenteral benzodiazepines for hospitalized patients with severe agitation. the fda issued a warning for increased risk of excessive sedation and cardiorespiratory depression with this combination based on post-marketing case reports with overall limited quality of evidence [ ] . the purpose of this study is to evaluate the safety and efficacy of concomitant parenteral olanzapine and benzodiazepine for agitation. this retrospective chart review evaluated agitated patients who received concomitant parenteral olanzapine and benzodiazepine within minutes from / / to / / . the primary end points were rate of respiratory depression requiring mechanical ventilation and hypotension requiring vasopressors. the secondary end points were percentage of patients requiring additional sedatives for agitation during the same time frame, cumulative dose of olanzapine and benzodiazepine (midazolam equivalent) received, and rate of cardiac arrest and death. a total of patients were included with notable baseline characteristics: median age of years old, % with a history of substance abuse, and % with a history of psychiatric illness. for the primary outcomes, . % of patients required mechanical ventilation and % required vasopressors. additionally, . % patients received additional sedating agents to control agitation. refer to table for more details. no cardiac arrests or deaths were observed. concomitant use of parenteral olanzapine and benzodiazepine within minutes for the treatment of agitation appears to have a small risk of respiratory depression without significant hypotension. hip fracture is very common in the elderly,it causes moderate to severe pain often undertreated. ficb is a simple safe method, easy to learn and use. the aim of our study is to assess the efficacy and safety of preoperative ficb compared with intravenous analgesia for elderly patients with femoral fracture and hip surgery in terms of opioid consumption and perioperative morbidity methods: after informed consent obtained, patients - yo asa i-iii with hip fracture were randomized to receive either an us guided ficb( ml of ropivacaine , %) or a sham injection with normal saline ' before surgery. both groups were operated under general anesthesia. postoperative analgesia was done according to vas: vas - mm, paracetamol g iv at h, vas - mm, ketoprofen mg iv at h, vas> , morphine , mg/ kgbw iv. the primary outcome was the comparison of vas score at rest over the first 'following the procedure, at the end of the surgery and at h intervals for h. the secondary outcome were the incidence of the cardiovascular events, of the ponv and of the confusion episodes, the amount of morphine consumption for h results: at baseline, ficb group (a) had a lower mean pain score than the sham injection group (b). the same difference was observed over h of follow-up (p< . ). there was a significant difference between the two groups in total cumulative iv morphine consumption at h and in the incidence of ponv and confusion episodes ( figure ). ficb provides effective analgesia for elderly patients suffering from hip fractures, with lower morbidity and lower opioid consumption compared with intravenous analgesia. pain assessment in chronic disorders of consciousness patients with ani monitoring e kondratyeva, m aybazova, n dryagina almazov national medical reseach centre, minimally conscious research group, st petersburg, russia critical care , (suppl ):p pain and suffering controversies in doc to be debated by the scientific, legal and medical ethics communities. methods: ani (anti nociception index) monitor was used to assess pain in patients with chronic disordersof consciousness (doc) age range to years - in vegetative state/ unresponsive wakefulness syndrome (vs/uws) and minimal consciousness state (mcs). average age: in mcs group , ± , and , ± , in vs/uws group. neurological status was assessed using crs-r scale. the average score on the crs-r scale was ± . in vs/uws and . ± . in mcs. pressure on the nail phalanx was used as a pain impulse. ani and nociception coma scale was evaluated before the application of pain stimulus, immediately after and past minutes. prolactin level was measured before the pain stimulus application and minutes after. ani less than indicates pain, - hypoalgesia, severe pain. the mean value of the ani in mcs patients: before the pain stimulus . ± . , after the pain stimulus application ± . and minutes later . ± . . prolactin level in mcs patients before pain . ± . ng/ml; after pain . ± . ng/ml (p> . ). prolactin in vs/uws patients before pain . ± . ng /ml, after pain . ± . ng / ml (p> . ). conclusions: ani monitor revealed that vs/uws and mcs patients react equally to the pain impulse. prolactin dynamics showed poor statistical mean and can not be consider as a marker of nociception in this group of patients. it is possible that the level of pain impulse was insufficient neuroendocrine response activation or the increase of prolactin level occurs in the long term (more than minutes). in all patients the total hip arthroplasty tha is one of the most common major surgical procedures associated with significant postoperative pain that can adversely affect patient recovery and could increase morbidity. effective perioperative pain management allows an accelerated rehabilitation and improve the functional status of these patients. multimodal analgesia mma combines analgesics with different mechanism of action which by synergistic and additive effects enhance postoperative pain management and reduce complications. the aim of our study is to assess if perioperative association of very low dose of ketamine, a potent nmda antagonist and dexamethasone, by antiemetic and antiinflammatory properties could decrease opioid consumption and postoperative morbidity of patients with tha. after informed consent, patients scheduled for primary hip joint replacement surgery aged - yo asa i-iii were prospective randomized in two groups. both groups were operated under general anesthesia fentanyl/sevoflurane. supplementary, patients in group a received mg iv dexamethasone and mg at h and ketamine mg iv bolus at induction and mg/h iv during surgery. postoperative analgesia was done according to vas, - mm paracetamol g iv at h, - mm ketoprofen mg iv at h, vas> mm morhine , mg/kgbw iv. we recorded perioperative opioid consumption, the number of intraoperative cardiac events, vas score at the end of surgery and at h, the incidence of ponv and persistance of chronic pain at months. we obtain a significant less pain score at the end of surgery p< . in group a, no significant difference at h, a significant less chronic pain at months, a fewer npvo and cardiovascular events in group a, p< . ( figure ). a multimodal approach with very low doses of ketamine and dexamethasone could be efficent in the treatment of pain for elderly patients with hip arthroplasty, decreasing postoperative side-effects and reducing chronic pain persistance. introduction: treatment in an intensive care unit (icu) often necessitates uncomfortable and painful procedures for patients. chronic pain is becoming increasingly recognized as a long term problem for patients following an icu admission [ ] . throughout their admission patients are often exposed to high levels of opioids, however there is limited information available regarding analgesic prescribing in the post-icu period. this study sought to examine the analgesic usage of icu survivors pre and post icu admission. methods: patients enrolled in a post-intensive care programme between september and june . intensive care syndrome: promoting independence and return to employment (ins:pire), is a -week multicentre, multidisciplinary rehabilitation programme for icu survivors and their caregivers. patients' level of analgesia was recorded pre-admission and upon attending ins:pire, their level of prescribed analgesia was categorized using the word health organisation (who) analgesic ladder [ ] . results: . % of patients (n= ) were prescribed regular analgesia preadmission; this increased to . % (n= ) post-admission, representing a significant absolute increase of . % ( % ci: . % - . %, p< . ) in the proportion of patients who were prescribed regular analgesia pre and post icu. in addition, pre-admission, . % (n= ) of patients were prescribed a regular opioid (step and of the who ladder) compared to . % (n= ) post-admission, representing an absolute increase of . % ( % ci: . % - . %, p< . ). this study found a significant increase in analgesic usage including opioids in icu survivors. follow-up of this patient group is essential to review analgesic prescribing and to ensure a long term plan for pain management is in place. introduction: pain, agitation, and delirium (pad) are commonly encountered b patients in the intensive care unit (icu). delirium is associated with adverse outcomes, including increased mortality and morbidity. clinical guidelines suggest that routine assessment, treatment and prevention of pad is essential to improving patient outcomes. despite the well-established improvements on patient outcomes, adherence to clinical guidelines is poor in community hospitals. the aim of this quality improvement project is to evaluate the impact of a multifaceted and multidisciplinary intervention on pad management in a canadian community icu. a pad advisory committee was formed and involved in the development and implementation of the intervention. the -week intervention targeted nurses (educational modules, visual reminders), family members (interviews, educational pamphlet, educational video), physicians (multidisciplinary round script), and the multidisciplinary team (poster). an uncontrolled, before-and-after study methodology was used. adherence to pad guidelines in the assessment of pad by nurses was measured weeks pre-intervention and weeks post-intervention. data on patient-days (pd) and pd were available for analysis during the pre-and post-intervention, respectively. the intervention significantly improved the proportion of pd with assessment of pain and agitation at least times per -hour shift from . % to . % and from . % to . %, respectively ( figure ). proportion of pd with delirium assessment at least once per -hour shift did not significantly improve. a multifaceted and multidisciplinary pad intervention is feasible and can improve adherence to pad assessment guidelines in community icus. quality improvement methods that involve front-line staff can be an effective way to engage staff with pad. oversedation introduction: sedation is a significant part of medical treatment in icu patients. a too deep sedation is associated with a longer time of mechanical ventilation, lung injury, infections, neuromuscular disease and delirium, which can lead to a longer duration of icu hospitalization, as well as an increase of morbility and mortality. many patients spend a considerable amount of time in a non-optimal sedation level. a continuous monitoring system of the sedation level is therefore necessary to improve clinical evaluation. our goal was to evaluate the incidence of non-optimal sedation (under and over sedation) comparing the parameters expressed from ngsedline with clinical evaluations and to correlate oversedation and the incidence of delirium. we have studied a cohort of patients admitted to the icu of spedali civili of brescia university hospital requiring continuous sedation for more than hours. in addition to standard monitoring, the patients have been studied using next generation sedline (masimo). sedation depth was evaluated through rass scale and the presence of delirium was evaluated with cam-icu scale. we collected data from adult patients. our data showed high incidence of oversedation. of our patients had a sr> and had a psi level< . a logistic regression analysis was performed and it showed statistically significant association between incidence of delirium and the age of the patients (p . ). the association between delirium incidence and suppression rate time was at the limits of statistics significance (p . ) and was statistically significant for non neurocritical patients (p . ). our study didn't show an association between delirium and the total time of sedation. non-optimal sedation is an unsolved problem in icu, affecting lot of patients, with a major incidence of over-sedation compared to under-sedation. our study shows an association between sr levels and the incidence of delirium. predictors of delirium after myocardial infarction, insights from a retrospective registry m jäckel, v zotzmann, t wengenmayer, d dürschmied, c von zur mühlen, p stachon, c bode, dl staudacher heart center freiburg university, department of cardiology and angiology i, freiburg, germany critical care , (suppl ):p delirium is a common complication on intensive care units. data on incidence and especially on predictors of delirium in patients after acute myocardial infarction (mi) are rare. by analyzing all patients after acute mi, we aim to identify incidence and potential risk factors for delirium. in this retrospective study, all patients hospitalized for acute mi treated with coronary angiography in an university hospital in were included and analyzed. incidence of delirium within the first days of care attributed to the mi and was defined by a nudesc score ≥ , which is taken as part of daily care three times a day by especially trained nurses. this research is authorized by ethics committee file number / . results: patients with acute mi (age . ± . years, stemi, mortality . %) were analyzed. delirium occurred in ( . %) patients and was associated with a longer hospital stay ( ± . d vs . ± . d, p< . ). patients with delirium were significantly older than patients without ( . ± . vs. . ± . years, p< . ) and had more often preexisting neurological diseases ( . % vs. . %, p< . ) and dementia ( . % vs. . %, p< , ). multivariate logistic regression analysis suggested that odds ratio for delirium was higher in patients after resuscitation or . ( % ci . - . ), preexisting dementia or . (ci . - ) and in patients with alcohol abuse or (ci . - ). while maximum lactate was also connected to delirium or . (ci . - . ), infarct size or type had no effect on the incidence of delirium. in patients with mi, delirium is frequent. incidence is associated with clinical instability and preexisting neurological diseases rather than infarct size. incidence and risk factors of delirium in surgical intensive care unit ma ali, b saleem aga khan university, anaesthesia, karachi, pakistan critical care , (suppl ):p introduction: delirium in the critically ill patients is common and distressing. the incidence of delirium in the icu ranges from % to %. although delirium is highly common among intensive care patients, it is mostly underreported. to date, there have been limited data available related to prevalence of delirium in surgical patients. in a study published in , the risk was observed % in surgical and trauma patients [ ] . the purpose of this study was to find out the incidence and associated risk factors of delirium in surgical icu (sicu) of a tertiary care hospital. we conducted prospective observational study in patients with age more than years and who were admitted to the surgical icu for more than hours in aga khan university hospital from january to december . patients who had preexisting cognitive dysfunction or admitted to icu for less than hours were excluded. delirium was assessed by intensive care delirium screening checklist icdsc. incidence of delirium was computed and univariate and multivariable analyses were performed to observe the relationship between outcome and associated factors. delirium was observed in of patients with an incidence rate of . %. multivariable analysis showed that copd, pain > and . ] were also the strongest independent predictors of delirium while analgesics exposures was not statistically significant to predict delirium in multivariable analysis. delirium is significant risk factor of poor outcome in surgical intensive care unit. . there was an independent association between pain, sedation, copd, hypernatremia and fever in developing delirium delirium is an acute mental syndrome which may cause negative consequences if it is misdiagnosed [ , ] . the aim of this study was to determine the incidence of delirium in different intensive care units and reveal the risk factors. the study was performed with patients hospitalized in intensive care units of anesthesia, neurology and general surgery departments. written informed consent was obstained from patients or relatives. delirium screening test was performed twice daily with camicu (confusion assessment method for the icu). patients who met the study criterias, were evaluated for the possible risk factors of delirium and the data was recorded daily. patients were reevaluated after the treatment. the incidence of delirium was . %. delirium was found to increase with the length of stay (p < . ). the mean age of the patients with delirium was . . this was higher than the patients without delirium ( . ) (p< . ). visual impairment (p< . ), hearing impairment (p= . ), educational status (p= . ), hypertension (p= . ), mechanical ventilation (p = . ), oxygen demand (p= . ), midazolam infusion (p= . ), propofol infusion (p= . ), infection (p < . ), sofa (p = . ), apache ii (p < . ), nasogastric catheter (p= . ), aspiration (p < . ), number of aspirations (p< . ), enteral nutrition (p< . ), albumin (p= . ), steroid (p= . ), hypercarbia (p= . ) hypoxia (p= . ), sleep disturbance (p< . ) were found risk factors for delirium. oral nutrition (p< . ) and mobilization (p= . ) were found to prevent delirium development. various factors are important in the development of delirium. these risk factors should be considered in reducing the incidence of delirium in intensive care units. ). an unplanned and brutal stop of alcohol consumption, as it can occur during icu admission, may lead to an alcohol withdrawal syndrome (aws). the most severe clinical manifestation of aws is described as delirium tremens (dt). there are no current guidelines available for aws treatment in icu. the study's aim was to describe the clinician's practices for dt treatment and the outcome of dt in icu patients. observational retrospective cohort study in two icus of a universityaffiliated, community hospital in france. patient diagnosed for dt during their icu stay, as defined by dsm-v classification, were enrolled in the study. results: patients with dt were included between and . benzodiazepines was administered to % of the patients in order to prevent an aws. as associated measures, vitamin therapy was administered to % of the patients and % had an increased fluid intake (mean . l+/- . ). concerning the curative approach of aws, the treatment's heterogeneity was notable. there was a high frequency of treatment's association ( % of the patients), every patient had benzodiazepines and the use of second line treatments such as neuroleptic, alpha- agonist, propofol was variable ( figure ). complications of dt were the following: need for mechanical ventilation due to unmanageable agitation or acute respiratory distress ( % of the patients) self inflicted injuries such as pulling out of central lines, tubes, surgical drain ( %) falls ( %). seizures ( %). delirium tremens is a severe complication of an untreated aws, which can lead to serious adverse events in icu. the current lack of evidence concerning the management of aws in icu probably explains the heterogeneity of treatments. given the potential severity of aws in icu, further evidences are required to optimize care of aws in icu patients. the incidence and related risk factor of delirium in surgical stepdown unit s yoon , s yang , g cho , h park , k park , j ok , y jung asan medical center, nursing department, seoul, south korea; asan medical center, seoul, south korea critical care , (suppl ):p step down units (sdus) provide an intermediate level of care between the icu and the general medical-surgical wards. the critically ill patients who are in recovery after long-term intensive care or who require monitoring after acute abdominal surgery are admitted to sdus. delirium in critically ill patient is common and leads to poor clinical outcomes. it is, however, preventable if its risk factors are identified and modified accordingly. to determine risk factors associated with delirium in critically ill patients to admitted surgical sdu at asan medical center. this is retrospective study conducted on critically ill patients who were admitted to the sdu from september to april and able to express themselves verbally. delirium status was determined using the short-cam tool. data were analyzed by spss . software, using t-test, fisher's exact test and logistic regression. the incidence of delirium was . %( of patients) and hypoactive delirium( case, . %) was the most commonly assessed, followed by hyperactive delirium( case, . %), mixed type( case, . %). risk factors associated with developing delirium identified from univariate analysis were age(p= . ), admission via icu (p= . ), tracheostomy (p= . ), chronic heart failure (chf) (p= . ), invasive hemodynamic monitoring (p= . ), heart rate (p= . ). after adjusted in multivariate analysis; factors those remained statistically significant were old age (rr we identified risk factors consistently associated with incidence of delirium following admitted to surgical sdu. these factors help to focus on patients at risk of developing delirium, and to develop preventive interventions that are suitable for those patients. patients with sepsis frequently develop delirium during their intensive care unit (icu) stay, which is associated with increased morbidity and mortality. the prediction model for delirium in icu patients (pre-deliric model) was developed to facilitate the effective preventive strategy of delirium [ ] . however, the pre-deliric model has not yet been validated enough outside europe and australia. the aim of this study is to examine the external validity of the pre-deliric model to predict delirium using japanese cohort. this study is a post hoc subanalysis using the dataset from previous study in nine japanese icus, which have evaluated the sedative strategy with and without dexmedetomidine in adult mechanically ventilated patients with sepsis [ ] . these patients were assessed daily throughout icu stay using confusion assessment method-icu. we excluded patients who were delirious at the first day of icu, were under sustained coma throughout icu stay and stayed icu less than h. we evaluated the predictive ability of the pre-deliric model to measure the area under the operating characteristic curve. calibration was assessed graphically. of the patients enrolled in the original study, we analyzed patients in this study. the mean age was . ± . years and patients ( %) were male. delirium occurred at least once during their icu stay in patients ( %). to predict delirium, the area under the receiver operating characteristics curve of the pre-deliric model was . ( . to . ). graphically, the prediction model was not well-calibrated ( figure ). to predict delirium in japanese icus, we could not show the well discrimination and calibration of the pre-deliric model in mechanically ventilated patients with sepsis. introduction: delirium is a serious and common complication and in some cases it treatment is difficult. aim of the study was an evaluation of the prevalence, structure of delirium and efficacy of dexmedetomidine and haloperidol sedation in geriatric patients after femur fracture. after local ethic committee approval case-records of geriatric patients with femur fracture in the period from to in the institute of traumatology and orthopedics in astana were analyzed. patients was divided for groups: in dpatients with delirium treated by i/v dexmedetomidine ( . - . mkg/kg per hour), in g group patients with delirium treated by i/v galoperidol ( . - . mkg/kg). delirium was assessed by rass at day of permission and every day at a.m. the prevalence, structure of delirium and efficacy of sedation were analysed. results: by anthropometric and gender characteristics of the group did not differ. the average age in the d-group with delirium was . ± . years old, which was comparable to the g-group - . ± . years old (p = . ). all study participants had similar comorbidities. delirium in all patients debuted at . ± . days, with an average duration of . ± . days. the effect of dexmedetomidine was better and expressed in % decrease in the duration of delirium in compare to haloperidol (p < . ). dexmedetomidine provided a more controlled and safe sedation compared with haloperidol. the average consumption of narcotic analgesics in the subgroup with dexmedetomidine was two times less than in the subgroup with haloperidol. thus, the average consumption of trimeperidine hydrochloride in patients of group d was . mg versus . mg in group g (p = . ). in gerontological patients with femur fracture treatment delirium by dexmedetomidine was more effective in compare with haloperidol. when using dexmedetomidine, the consumption of narcotic analgesics in postoperative period was % less than with haloperidol. live music therapy in intensive care unit mc soccorsi , c tiberi , g melegari , j maccieri , f pellegrini , e guerra intensive care units (icu) are not comfortable for patients, relatives or next of kin. in the last years many news approaches were described to implement the humanization of medical treatments. the positive effect of music therapy in icu is well described, especially reducing delirium risk [ ] . the aim of this paper is describing the effect in patients and their family of a music live performance in icu. after ethical committee approval (procedure aou / , italy) for three months (november -january ) patients in icu were treated twice a week with live music therapy performed by coral vecchi-tonelli of modena, italy (fig. ). data were collected all awake and conscious patients. vitals parameters, gcs, raas and cam icu were collected before, during and after the treatment, at every performance. after the treatment a feedback questionnaire were given to patients and to next of kin. results: subjects were enrolled in the research with mean age of . years old, delirium rate before the treatment was . % later . %, raas does not show any difference. over % of patients were satisfied, and relatives felt less anxiety. we recorded also a satisfaction also in relatives not enrolled. the study does not demonstrate a delirium risk reduction for the small sample and the length treatment, anyway it was recorded a low delirium rate. the safety and the potential effect of music therapy are well known, surely the research underlines the feeling of patients and their next of kin: icu is the most stressful setting for admitted patients and its humanization is a current topic for medical literature. live performances could be an entertainment moment and probably create a moment of an interaction among patients, their family and medical and nurse: icu become more human. the high level of satisfaction push us to continue this experience. introduction: patients undergoing medical procedures benefit from distraction techniques to reduce the need for drugs alleviating pain and anxiety. this study investigates if medical hypnosis or virtual reality glasses (vrglasses) as adjuvant method reduces the need for additional drugs. in a prospective, randomized, interventional trial, patients undergoing procedures were stratified in four age groups, and randomly assigned into three arms by means of a closed envelope system. all patients received standard care for pain before the procedure; the control group received further drugs for pain and stress as indicated by the visual analog scale (vas; threshold / ) and comfortscore (threshold / ), two index groups received either medical hypnosis or vr glasses as a plus before and during the procedure. vas and comfort were scored continuously and analysed with the kruskal-wallis test. patients, parents and healthcare providers scored their satisfaction at the end. of included patients to years old, % were female. regardless of age, pain and comfort scores were similar before and at the start of the procedure (vas . - . ; comfort - . ), but as of one minute after starting the procedure, both vas and comfort reduced significantly more in both index groups compared to the control (p< . ), remaining far below the threshold for both pain and stress ( figure ). there was no advantage of one index group over the other (p= . ). there were no adverse effects. patients in the vr group were more satisfied than in the standard group (p= . ) or in the hypnosis group (p= . ). there was no significant difference in satisfaction of parents or healthcare providers. from the very start of the intervention, the application of either medical hypnosis or vr glasses significantly reduces pain and anxiety in patients undergoing medical procedures. more studies are needed but both are promising safe adjuvant tools to standard pharmacological treatment. music to reduce pain and distress due to emergency care: a randomized clinical trial ne nouira, i boussaid, d chtourou, s sfaxi, w bahria, d hamdi, m boussen, m ben cheikh mongi slim academic hospital, emergency department, tunis, tunisia critical care , (suppl ):p recent clinical studies have confirmed the benefits of music therapy in managing pain and improving quality of care in the emergency department. the aim wasto evaluate the impact of receptive music therapy on pain and anxiety induced by emergency care methods: a randomized controlled study in patients consulting the emergency department. two groups: the music therapy group; patients needed venous sampling, peripheral venous catheter or arterial catheter. will bless ten minutes music therapy by headphones and a second control group of patients with the same care without music therapy. consent was requested from all participants. the level of pain caused by the act of care was assessed by visual analogic scale. heart rate, blood pressure and the mood of the patient were assessed before and after emergency care. we assessed patient satisfaction, adverse events. patients admitted to the emergency room, patients with communication difficulties and non-consenting patients were not included results: two hundred and forty patients were included randomized in both groups, with music therapy and without music therapy, the results showed comparable characteristics between the two groups: demographic data, pathological history, and initial clinical presentation. after the session of music therapy a difference was noted in the evaluation of the mean vas who was in the group with music of . ± . versus . ± . in the control group p< . ci % [- . ; - . ], and the mean of diastolic blood pressure which was , mmhg in the first group against . mmhg for the control group p = . ci % [- . ; - . ]. as for the mood, the patients were more smiling after the act of care in the group music therapy. all patients were satisfied with their experience and % recommend this therapy to their relatives . music therapy may reduce pain and anxiety in patients during emergency care. the music therapy is the intervention of music and/or its elements to achieve individual goals within a therapeutic.the music has proved to have positive physiological and psychological effects on patients [ ] . patients admitted to the intensive care unit (icu) experience anxiety and stress even when sedated, negatively influencing recovery [ ] . methods: two groups are established, a music therapy group (mg) and a control group (cg). the first one undergoes music therapy interventions, it consists of -minutes sessions of live music. patients of the gc will receive the usual treatment established by the service protocol for weaning management and the data are collected during the same time interval. data collection includes mean arterial pressure (map), heart rate (hr), respiratory rate (rr), oxygen saturation (sao ) and temperature (t). a total of patients were recruited, of which patients had to be excluded for meeting any of the exclusion criteria (n= ). of which (n= ) were randomized in the gm and the rest to the gc (n= ) ic %. regarding delirium in gm ( . %) presented a positive cam-icu, while in the cg were ( . %) (p= . ). when analyzing the variables in the cg and gm, it was observed that there were no differences with respect to hr, rr and map variable ( figure ). according to the results, we can say that music therapy as a nonpharmacological strategy for management of anxiety and delirium in patients of critical care units, might be an useful tool for the management of patients in weaning of mechanical ventilation introduction: coagulopathy and basopenia are common features of anaphylaxis, but the role of coagulopathy in anaphylaxis remains uncertain. the aim of this study is to evaluate the association between coagulopathy and clinical severity or basopenia in patients with anaphylaxis. we conducted a single-center, retrospective study of patients with anaphylaxis about their coagulopathy. levels of fibrin degradation products (fdp) and d-dimer were analyzed with the cause of anaphylaxis, clinical symptoms, medications and outcomes. we also studied the levels of intracellular histamine as a biomarker of basophil degranulation in the peripheral blood in relation to fdp and ddimer. in total, sixty-nine patients were enrolled to the study, and the levels of intracellular histamine were analyzed in patients. the symptoms included respiratory failure (n= ), shock (n= ), abdominal impairment (n= ), and consciousness disturbance (n= ). thirty-two patients needed continuous intravenous vasopressors for refractory shock. the increase of fdp was significantly associated with consciousness disturbance (p= . ) and refractory shock (p< . ). the increase of d-dimer was also significantly associated with refractory shock (p= . ). there was no correlation between the levels of intracellular histamine and either of fdp or d-dimer (p= . and p= . , respectively). the increase of fdp and d-dimer were associated with severe symptoms of anaphylaxis, while they were not correlated with intracellular histamine. these results suggest that anaphylaxis is closely associated with coagulopathy in a mechanism which is different from basophile degranulation in anaphylaxis. cardiac manifestations of h n infection in a greek icu population e nanou , p vasiliou , e tsigou , v psallida , e boutzouka , v zidianakis , g fildissis agioi anargiroi hospital, attiki, greece; agioi anargiroi hospital, icu, attiki, greece critical care , (suppl ):p introduction: cardiovascular involvement in influenza infection occurs through direct effects on the myocardium or through exacerbation of pre-existing cardiovascular disease [ ] . the aim was to study cardiac manifestations in all pts admitted to the icu with severe influenza's attack. clinical, laboratory, electrocardiographic, echocardiographic and hemodynamic data were retrospectively recorded in all pts admitted to the icu due to influenza infection (winter -spring ). diagnosis was established by pcr on bronchial aspirates the next days after admission. myocardial injury was defined by troponin levels > pg/ml ( fold uln). left ventricular systolic dysfunction was defined as ef < % and was characterized as either global or regional. hemodynamic monitoring by fig. (abstract p ) . comparison between mg and cg transpulmonary thermodilution method (picco) was recorded in pts with shock (norepinephrine > . μg/kg/min). values are expressed as mean±sd or as median (ir). results: nine pts ( males) with a mean age . ± . years, apache ii ± . and sofa score . ± . were assessed. icu admission was due to ards ( ) and copd exacerbation ( ) . icu los was . ± . days and mortality rate was %. no history of vaccination or coronary heart disease was referred. results are shown in table . levosimendan was administered in pts with severe cardiogenic shock. in all survivors, shock and indices of myocardial dysfunction subsided till discharge. coronary angiography was performed in pt showing no abnormalities. mortality was attributed to septic shock and multi-organ failure. myocardial involvement, though common in influenza pts admitted to the icu, didn't contribute to a dismal prognosis. the cardioprotective effects of levosimendan could be related to the modulation of oxidative balance. we aimed to examine the effects of levosimendan in patients with cardiogenic shock or with ejection fraction (ef) lower than % on cardiac systo-diastolic function and plasma oxidants/antioxidants (glutathione, gsh; thiobarbituric acid reactive substances, tbars). in patients undergone coronary artery bypass grafting or angioplasty, cardiovascular parameters were measured at t (before the beginning of levosimendan, . mcg/kg/min), t ( h after the achievement of the therapeutic dosage of levosimendan), t (at the end of levosimendan infusion), t (at h after the end of levosimendan infusion), t (at the end of cardiogenic shock). the same time-course was followed for plasma gsh and tbars measurements. we found an improvement in cardiac output, cardiac index and systolic arterial blood pressure. ef increased from mean % to %. a reduction of central venous pressure and wedge pressure was also observed. moreover, indices of diastolic function were improved by levosimendan administration (e/e' from to ; e/a from > to < ) at early t . it is to note that an improvement of gsh and tbars was observed early after levosimendan administration (t ), as well ( figure ). the results obtained have shown that levosimendan administration can regulate oxidant/antioxidant balance as an early effect in low cardiac output patients. the modulation of oxidative condition could be speculated to play a role in exerting the cardio-protection exerted by levosimendan in those patients. table . early administration of vasopressors and their use in the emergency department was associated with survival in septic shock. this seemed to be independent of median map recorded in the ed. we excluded all the traumatic or post-myocardial infarction forms. out of patients, the tuberculous etiology was identified in cases ( , %), mean age was years, , % were men. patients reported a tb contact in their environment, had a medical history of pulmonary tb. after pericardiocentesis, the liquid was citrine yellow in cases and hematic in patients, no patient underwent surgical drainage in our serie. mycobacterium tuberculosis was found in the expectorations in cases and ada was positive in patients. hiv serology was negative in all our patients. a months anti bacillary therapy with isoniazid, rifampin, pyrazinamide, and ethambutol was initiated in all our patients with a good evolution in cases, deaths, chronic constrictive pericarditis, small pericardial effusion and lost to follow-up. althought cardiac tamponade is rarely caused by tuberculosis, this condition remains common in endemic countries such as morocco and affect younger population, hence the importance of a better knowledge of its prevalence and and multidisciplinary management and more importantly the treatment of the underlying cause using combined antibacillary medication that has shown satisfying results. . the main perceived limiting factor is the absence of a standardized didactic program, followed by mentor's availability in residents' perception and by mentor's experience in consultants' one. pocus teaching is present although not optimal and not homogenous in italian acc residency schools. standardisation of residents' ultrasound curriculum is suggested to improve ultrasound teaching. the study included a convenience sample of critically ill patients with supradiaphragmatic cvcs and a cxr for confirmation. us is used for direct confirmation of the guidewire in the internal jugular (ijv) or subclavian (scv) vein and visualizing the guidewire in the right atrium. to evaluate for pneumothorax, "sliding sign" of the pleura was noted on us of the anterior chest. results: patients have been included, % of the catheters have been placed in the scv and % in the ijv. it was possible to confirm the position of the cvc tip for . % ( correct, incorrect cxr) of (figure ). overall, it was not possible to identify the guide in the right atrium cases ( false negatives, of them due to the presence of defibrillator leads). regarding the case where an incorrect position was seen on cxr it was also detected on ultrasound: us of the inserted vein and a negative tte confirmation. in all cases it was possible to exclude a pneumothorax by us. these results show that bedside ultrasound might be a feasible technique to confirm the cvc positioning. it is important to note that the level of the operator's expertise is significant when assessing the feasibility of this method. we only had a limited sample size and the occurrence of only one misplaced catheter. these preliminary results need to be confirmed on a larger scale. central venous catheter (cvc) misplacement occurs more frequently after cannulation of the right subclavian vein compared to the other sites for central venous access. misplacement can be avoided with ultrasound guidance by using the right supraclavicular fossa view to confirm correct guidewire j-tip position in the lower part of the superior vena cava. however, retraction of the guidewire prior to the cvc insertion may dislocate the j-tip from its desired position, thereby increasing the risk of cvc misplacement. the aim of this study was to determine the minimal guidewire length needed to maintain correct guidewire j-tip position throughout an us-guided infraclavicular cvc placement in the right subclavian vein. methods: adult intensive care patients with a computed tomography scan of the chest were retrospectively and consecutively included in the study. the distance from the most plausible distal puncture site of the right subclavian/axillary vein to the junction of the right and left brachiocephalic veins (= vessel length) was measured using multiplanar reconstructions. in addition, measurements of the equipment provided in commonly used - cm cvc kits were performed. the minimal guidewire length was calculated for each cvc kit. the guidewires were up to mm too short to maintain correct j-tip position throughout the cvc insertion procedure in seven of nine commercial cvc kits. four of these are shown in table . when us guidance is used to confirm a correct guidewire j-tip position, retraction of the guidewire prior to the cvc insertion must be avoided to ensure correct cvc-tip positioning. this study shows that most of the commonly used - cm cvc kits contain guidewires that are too short for cvc placement in the right subclavian vein. the reliability of lung b-lines to assess fluid status in patients with long period of supine introduction: ultrasound-guided cannulation is usually done using either longitudinal or transverse approach. the oblique approach utilizes advantages of both these approaches allowing visualization of the entire course of needle including tip and lateral discrimination of artery from vein [ ] . the reported incidence of the complete overlap of femoral vein by the femoral artery is - percent [ , ] . we describe the use of the oblique approach for successful cannulation of such a femoral vein which is not possible by usual approaches (figure ). endothelial cells play a pivotal role in the atherogenic process. endothelial cell dysfunction (ed) is the main risk factor for cardiovascular diseases such as hypertension, coronary heart disease (chd) and peripheral occlusive disease (pod). these diseases significantly increase the risk for perioperative complications. therefore, identifying patients with ed is important and should influence our prospective perioperative strategy. however, sensitive tools to diagnose ed are still missing and do not belong to our standard of care. aim of this study was the validation of a new non-invasive method to detect ed and a correlation with a set of established an new endothelial biomarkers. the cohort includes preoperative patients without anamnestic relevant cardiovascular disease and patients with known peripheral occlusive disease (pod). we used non-invasive endopat® technology from itamar-medical to measure ed by changes in vascular tone before and after occlusion of the brachial artery and calculate a reactive hyperemia index (rhi). in addition, we measured established markers and alternative biomarkers potentially indicate vascular diseases such as substrates and products from the no-metabolism l-arginin, asymmetric/symmetric dimethylarginine (adma/sdma), von-willebrand factor (vwf) and sphingosine- -phosphate (s p). rhi was able to identify patients with pod. rhi was significant lower in patients with clinical signs and symptoms of pod (p< . ). among other markers adma was significant higher in pod patients compared to controls and correlates with rhi. the pad technology is a helpful non-invasive functional test to measure ed and seems able in identify patients with vascular disease. in future, a combination of anamnesis, new diagnostic tools and biomarkers may further increase our sensitivity in identifying risk-patients. single-lumen fr and triple-lumen fr peripherally inserted central catheters (piccs) for cardiac output assessment by transpulmonary thermodilution s d´arrigo achieving effective critical care in low-and middle-income countries is a global health goal [ ] , which includes the provision of effective point of care ultrasound [ ] . we sought to establish zambia's first focused critical care echocardiography training programme in a bedded icu at university teaching hospital, lusaka. the programme was accredited by the uk intensive care society fice programme, with teaching adapted for local disease patterns such as tuberculous pericardial effusions. parasternal, apical and subcostal windows were used to assess ventricular dysfunction, hypovolaemia, pleural effusion, alveolar interstitial syndrome and pneumothorax. zambian doctors working with critically ill patients received an intensive one-day course, followed by mentored scanning at the bedside. teaching was delivered by visiting fellows from the uk who are accredited in echocardiography and experienced ultrasound educators. patients with abnormal mean ci or hr suffer from increased hospital mortality. abnormality of mean svi was not associated with mortality. these data support accurate measurement of ci as a hemodynamic target and the normal range defined for ci. since ci also carries the hr information, ci seems to be the more important target than svi. our data cannot necessarily be interpolated to less invasive and less precise measurements of ci. an evaluative study of the novelty device with the function of auto-aspirating and pressure indicator for safety central venous catheterization ly lin, wf luo, cy tsao national taiwan university hospital, taipei, taiwan critical care , (suppl ):p previous studies have shown that . % of cvc attempts resulted in arterial punctures that were not recognized by blood color. to overcome the problem, our team has developed a concept of pressure detecting syringe that can indicate the artery puncture [ ] . based on previous research, different springs, the actuator of the design, have been evaluated to optimize the proposed device and reduce the risk of cvc procedure. tested devices -the inner-spring is set between the pressure indicator and plunger (fig. a ). three springs are tested. test condition -blood samples were simulated by glucose solution with absolute viscosities of and mpa-s. different blood pressures were applied to simulate the artery and vein (fig. b) . the response time (rt) is defined as the time required to show the indicating signal (is) which is the movement of the piston from the position in fig. b : a - to a - . the rt is strongly influenced by spring (fig. b) but every design can show the is when pressure is higher than mmhg, the assumed minimum artery pressure. the rt of s , the strongest spring design, is about s in the mmhg-pressure and high viscosity condition. during our tests we found the user can realize the is before the position be fully changed from fig. ib : a - to a - . thus, we believe the s rt, the worst case, is still acceptable. we also found the weak spring force may lead to difficulty to empty the syringe because the spring must to overcome the blood pressure and the friction between the piston and barrel. as a result, it was difficult for s to absolutely empty the syringe even if the blood pressure is only mmhg. the spring will be compressed as fig. b : a - and fail to push the piston when pushing the plunger forwardly, which is not acceptable in clinical use. the results indicate the feasibility of using the device to facilitate cvc and we believe the s or s are more suitable for the future application. introduction: models using standard statistical features of hemodynamic vital sign waveforms (vs) enable rapid detection of covert hemorrhage at a predetermined bleed rate [ ] . by featurizing interactions between vs we can train powerful hemorrhage detectors robust to unknown bleed rates. waveforms (arterial, central venous, pulmonary arterial pressures; peripheral and mixed venous oxygen saturation; photoplethysmograph; ecg) of healthy pigs were monitored min prior and during a controlled hemorrhage at ml/min (n= ) and ml/min (n= ). two sets of vs features were extracted: statistical features [ ] and maximal pairwise cross correlations between pairs of vs within a s lag over various time window sizes ( s, s, s, s); and normalized with pre-bleed data of each given animal. for each feature set, a tree-based (ert) model [ ] was trained and tested in a one-animal-out setting to mitigate overfitting on the ml/min cohort, and another trained on the ml/min and tested on the ml/min cohort. we evaluated models with activity monitoring operating characteristics curves [ ] that measure false alert rate as a function of time to detect bleeding. models using cross-correlations show no significant deterioration of performance when applied to detect bleeding at different rates than trained for, while standard models require s longer on average to detect hemorrhage at % false alert rate in the previously unknown setting ( figure ). correlations between vs data encode physiologic responses to hemorrhage in a way independent of the actual bleed rates. this enables training effective hemorrhage detectors using only limited experimental data, and using them in practice to detect bleeding that occurs at rates other than used in training. we validated a dataset of data lines containing hemodynamic variables and treatment options. we selected nine hemodynamic variables as inputs. furthermore, data were collected regarding underlying conditions: heart failure, septic shock, renal failure or respiratory failure or a combination. we applied datastories regression on the dataset (turnhout, belgium, www.datastories.com). six different interventions were analyzed as kpi: administration or removal of fluids, increasing or decreasing inotropes and increasing or decreasing vasopressors. finally, we elaborated and challenged predictive models to generate a decision algorithm to predict each kpi. we first looked at how each hemodynamic parameter impacts the prediction of each kpi individually and performed a standard correlation analysis as well as a more involved analysis of the mutual information content between each kpi and all other hemodynamic parameters individually. confusion matrix and variable importance was obtained for each kpi. the baseline hemodynamic parameters were: gedvi ± ml/m , evwli . ± . ml/kg pbw, svv . ± %, mbp . ± . mmhg, hr . ± . bpm, ci . ± . l/min.m . the results of the regression analysis identified the different variables of importance for each of the different interventions ( fig a) . based on these results the hemodynamic variables (hr, mbp, gedvi, elwi, ci, svv) were used to develop the final hemoguide prediction model ( fig b) . the hemoguide app can be used to advise physicians with respect to basic therapeutic decisions at the bedside or as an educational tool for students. with the collection of new data, the accuracy of the system may grow over time. the next step of the project is to develop a more-sophisticated suite: the icu cockpit. feedback function contributes to accurate measurement of capillary refill time r kawaguchi , ta nakada , m shinozaki , t nakaguchi , h haneishi , s oda chiba university, department of emergency and critical care medicine, chiba, japan; chiba university, chiba, japan critical care , (suppl ):p capillary refill time (crt) is well known as an indicator of peripheral perfusion. however, it has been reported to have an intra-observer variance, partly because of manual compression and naked-eye measurement of the nailbed color change. we hypothesized that a we developed a novel portable crt measurement device with an oled display that feedbacks weather the strength of the nailbed compression is enough and counts the time. we settled the target strength and time as n and seconds according to the study we reported before [ ] . examiners measured crt with and without the feedback function. the pressing strength and time during the measurement were evaluated. there was a significant difference among the pressing strength and time between the crt measurement using the device with and without the feedback function (strength: p< . ; time: p< . ). furthermore, intra-examiner variance was significantly reduced with the feedback function (strength: p< . ; time: p< . ). in all measurements without the feedback function, % was outside the optimal strength while the measurements with the feedback function % achieved the targeted range. without the feedback function, % could not reach the optimal time, while % with the feedback function did. in total, % of the measurements could not achieve the optimal pressing strength and time. the feedback function for crt measurements, guiding examiners to an optimal pressing strength and time, fulfilled the required measurement conditions and reduced intra-examiner variance. our novel portable device would assist an accurate crt measurement regardless of personal work experience. introduction: the aim of the study was to detect the difference of conjunctival microcirculation between septic patients and healthy subjects and evaluate the course of conjunctival microcirculatory changes in survivors and non-survivors over a hours period of time. this single-centre prospective observational study was performed in mixed icu in a tertiary teaching hospital. we included patients with sepsis or septic shock within the first hours after icu admission. conjunctival imaging using idf videomicroscope as well as systemic hemodynamic measurements were performed at three time points: at baseline, hours and hours later. baseline conjunctival microcirculatory parameters were compared with healthy control. a total of patients were included in the final assessment and analysis. median apache ii and sofa scores were ( - ) and ( - ) respectively. ( %) were in septic shock, ( %) required mechanical ventilation. patients were discharged alive from the intensive care unit. we found significant reductions in all microcirculatory parameters in the conjunctiva when comparing septic and healthy subjects. we found a significant lower proportion of perfused vessels and microvascular flow index (mfi) of small vessels during all three time points in non-survivors compared with survivors. in nonsurvivors we observed no significant changes in conjunctival microcirculatory parameters over time. however, survivors had significantly improved mfi of small vessels at second and third time points compared to first time point. microcirculatory perfusion in conjunctiva was altered in septic patients. over hours evaluation survivors in comparison with nonsurvivors had better microcirculatory flow with incremental improvement of microvascular flow index. healthy pigs were centrally cannulated for veno-arterial ecmo and precision flow probes were placed on the pulmonary artery main trunk for reference. ml boluses of iced . % saline chloride solution were injected into the ecmo circuit and right atrium at different ecmo flow settings ( , , , l/min). rapid response thermistors of standard pa-catheters in the ecmo circuit and pulmonary artery recorded the temperature change. after calibration of the catheter constants for different injection volumes in the ecmo circuit, the distribution of injection volumes passing each circuit was assessed and enabled calculation of pulmonary blood flow. analysis of the exponential decay of the signals allowed assessment of right ventricular function. calculated blood flow correlated well with true blood flow (r = . , p < . , figure panel a, individual measurements organ congestion is susceptible to be a mediator of adverse outcomes in critically ill patients. point-of-care ultrasound (pocus) is widely available and could enable clinicians to detect signs of venous congestion at the bedside. the aim of this study was to develop prototypes of congestion scores and to determine their respective ability to predict acute kidney injury (aki) after cardiac surgery. this is a post-hoc analysis of a prospective study in patients for which repeated daily measurements of hepatic, portal, intra-renal vein doppler and inferior vena cava (ivc) ultrasound were performed before surgery and during the first hours after cardiac surgery [ ] . five prototypes of venous excess ultrasound (vexus) scores combining multiple ultrasound markers were developed (figure ). the association between each score and aki was assessed using timedependant cox models as well as conventional performance measures of diagnostic testing. a total of ultrasound assessments were analyzed. we found that defining severe congestion as the presence of severe flow abnormalities in multiple doppler patterns with a dilated ivc (> cm), corresponding to grade of the vexus c score, showed the strongest association with the development of subsequent aki compared with other combinations of ultrasonographic features (hr: . there is an increasing awareness on the consequences of fluid administration in patients leading to the development of methods that evaluate the effects of fluids loading on the cardiocirculatory system. however, most of methods used in the clinical practice investigate the effects of fluids on the cardiac function, instead of investigating those on the determinants of venous return. besides volume of fluids, the determinants of fluid loading are the blood volume distribution and the availability of vascular bed. in this study we aimed to test non-invasively the effects of fluids administration on the venular compartment in the skeletal muscle. in addition to the mean systemic filling pressure (msfp), we calculated changes in the stressed and unstressed volumes (vs, vu) and the venular bed availability. we enrolled critically ill patients in our intensive care unit. we assessed volumes and pressures by the near infra-red spectroscopy on the forearm using graded venous occlusions in steps of mmhg from to mmhg. the msfp, vu and vs were measured as previously reported (microcirculation ; : - ). the vascular bed availability was measured by changes in the volume recruited from the occlusion maneuvers. all the measures were done at baseline and after a fluid load ranging from to ml. values were expressed as median and interquartile range. wilcoxon test was used to compare data and a p< . was considered as significant. introduction: hypotension is a common side effect of general anesthesia (ga) and is associated with organ hypoperfusion and poor perioperative outcome [ ] . post-induction hypotension (pih) is caused by the depressant cardiovascular effect of anesthetic drugs and could be amplified by hypovolemia. the aim of this study was to assess the ability of two echocardiographic fluid responsiveness markers to predict pih: the inferior vena cava collapsibility index (ivc-ci) and the velocity time integral change (Δvti) after passive leg raising. sixty patients > years of age and scheduled for elective surgery were included. ivc-ci and Δvti were measured before ga induction. anesthesia protocol, fluid infusion and vasopressor administration were standardized in all patients. pih was defined as a mean arterial pressure (map) < mmhg or a relative decline from pre-induction value of at least % within minutes of ga induction. receiver operating characteristic (roc) curve analysis was used. the optimal cutoff was selected to maximize the youden index (sensitivity + specificity − ). the measurement of ivc-ci and/or Δvti were unsuccessful in seven patients ( . %). pih occurred in patients (incidence %). the areas under the roc curves ( figure ) preload responsiveness might be detected by the changes of cardiac index (Δcimini) induced by a "mini-fluid challenge" (mini-fc) of ml or even by the changes (Δcimicro) in response to a "micro-fluid challenge" (micro-fc) of ml. however, the smaller the fluid challenge, the larger the "grey zone" of diagnostic uncertainty. we tested whether ( ) micro-and mini-fc monitored by calibrated pulse contour analysis detect preload responsiveness and ( ) adding ml when the result of a micro-fc is within the grey zone improves diagnostic accuracy. in patients with circulatory failure, we infused ml saline over s followed by ml over s. we measured Δcimicro and Δcimini by the pulse contour analysis (picco ). preload responsiveness was defined by an increase in ci (Δciplr) during a passive leg raising test ≥ %. diagnostic uncertainty was described by calculating the grey zone after bootstrapping. Δcimicro were larger in responders than in non-responders ( . for the micro-fc, the area under the receiver operating characteristic curve was . ± . (threshold %), while it was . ± . for the mini-fc (threshold %). for the micro-fc, the grey zone ranged from . % to . % and included ( %) patients. for the mini-fc, it ranged from . % to . % and included ( )% patients, among which were already in the grey zone of the micro-fc. when evaluated by pulse contour analysis, micro-and mini-fc reliably detect preload responsiveness but with a large diagnostic uncertainty. it seems that adding ml more fluid to a micro-fc when its result is within the grey zone does not improve the diagnostic accuracy. the study is ongoing. the starling-sv bioreactance device (cheetah medical) reliably detects passive leg raising (plr)-induced changes in cardiac index (Δci). we tested whether it can also track the small and short-time Δci induced by the end-expiratory occlusion (eexpo) test, and whether shortening the time over which it averages cardiac output ( s in the commercial version) improves the detection. in mechanically ventilated patients, during a -sec eexpo, we measured Δci (in absolute value and in percentage) through calibrated pulse contour analysis (ci pulse , picco device) and starling-sv. for the latter, we considered both ci starling- provided by the commercial version and ci starling- obtained by averaging the raw data over s. we calculated the correlation between Δci pulse and both Δci starling- and Δci starling- , and the area under the receiver operating characteristic curve (auroc) to detect preload responsiveness, defined by a plr test. when considering absolute values, the correlation coefficient r between Δci pulse and Δci starling- was . (p= . ), which was lower than the one between Δci pulse and Δci starling- (rr comparison). when considering percentage changes, no correlation was observed between Δci pulse and Δci starling- . conversely, the correlation coefficient between Δci pulse and Δci starling- was . (p= . ), but it was lower than the one obtained for absolute values (p= . for r comparison). eexpo-induced Δci starling- , both in absolute values and in percentage, detected preload responsiveness with aurocs of . (sensitivity %, specificity %) and . (sensitivity %, specificity %), respectively. shortening the averaging time of the bioreactance signal increases the reliability of the starling-sv device to detect eexpo-induced Δci. moreover, the accuracy of the method is increased when absolute rather than percentage changes of ci are considered. fluids are among the most prescribed drug in intensive care, particularly among patient with circulatory failure. yet, very little is known about their pharmacodynamic properties and this topic has been left largely unexplored. there is a lack of strong scientific evidence in current guidelines for fluid administration in shock. several factors may impact the hemodynamic efficacy of fluids among which the infusion rate. the aim of this study was to study the influence of fluids administration rate on their pharmacodynamics in particular by studying mean systemic pressure (p ms ). we conducted a prospective observational study in patients with circulatory failure to compare two volume expansion strategies. when a patient required a fluid bolus, ml of normal saline were administered and several hemodynamic parameters were recorded continuously: cardiac output (co), arterial pressure (ap), mean systemic pressure (p ms ). infusion rate was let to the discretion of the attending physician and a "slow" and a "fast" group were determined based on the median of the infusion time. fluids effect was measured by the area under the curve (auc), maximal effect (e max ) and time to maximal effect (t max ) for each hemodynamic variable. results: p ms auc was higher in the "fast" group compared to the "slow" group (p= . ). we observed a shorter t max and a higher e max for p ms in the "fast" group compared to the "slow" group (p= . and . respectively). regarding co, t max was also shorter in the "fast" group (p= . ). auc and e max were similar between the two groups. fluid effect dissipated within minutes following the end of fluid infusion for every patient in both groups. the decreasing slope from maximal effect was comparable in the groups, for p ms and co alike. the effect of a ml fluid bolus in septic shock patients vanished within one hour. a faster infusion rate increased maximal effect and shortened the delay to reach it. study is ongoing. fluid management in the control arm of sepsis trials aa anparasan, ac gordon, mk komorowski imperial college london, department of surgery and cancer, london, united kingdom critical care , (suppl ):p in the past, high-volume intravenous fluid resuscitation in severe sepsis and septic shock was common. more recently, concerns over the harmful effects of this practice have led some clinicians to adopt less liberal fluid strategies. we sought to analyse temporal trends in fluid administration in the control arms of recent adult sepsis trials and assess any correlation with patient severity and mortality. a literature search was conducted to identify relevant randomized controlled trials that reported fluid administration published post . we recorded outcomes: total amount of iv fluid administered in the control arms of these trials between hospital admission and hour and hour following trial enrolment, mortality rates at the latest reported time point and apache-ii score at admission. we computed the pearson correlation coefficient and linear regression between study dates and the outcomes. we identified relevant trials [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , which recruited a total of , patients in their control arms, from to . the temporal analysis revealed no obvious trend in the in the total volume of iv fluid given by hour following trial enrolment (correlation p= . ) ( figure ). however, the total volume of fluid given by hour decreased significantly over the period of interest (r=- . , p= . ). in parallel, we observed a decrease in mortality (r=- . , p= . ) but there was no evidence of decrease in illness severity over time (p= . ). we found that in published rcts over the last two decades, the amount of intravenous fluid given to patients with sepsis in the initial hours did not appear to change, however less intravenous fluid was given over the first three days. upcoming large rcts will test the safety and efficacy of restrictive fluid administration approaches in sepsis. clinical practice guidelines recommend prompt intravenous (iv) fluid resuscitation for pediatric sepsis, including an initial fluid bolus of ml/kg [ ] . however, recent evidence is conflicting as to the effectiveness, volume, and consequences of aggressive fluid resuscitation in septic children. therefore, we sought to determine the epidemiology of early iv fluid resuscitation in an integrated health system, specifically at community hospital emergency departments (ed). we studied a retrospective cohort of pediatric patients (ages > month to < years) with sepsis identified in electronic health record data at community eds in southwestern pennsylvania from to . sepsis was defined as ) suspected infection (combination of fluid culture collection and administration of antibiotics and ) organ dysfunction (pediatric sofa score ≥ ) within hours of suspected infection. fluid bolus therapy was defined as electronic documentation of administration of . % normal saline iv bolus within hour of the time of sepsis onset. results: among , patients with pediatric sepsis, ( %) received iv fluid bolus therapy within hour of time of sepsis onset. the volume of fluid administered ranged from ml/kg to ml/kg (figure , panel a), corresponding to a median volume of ml/kg (iqr - ml/kg). patients who received ≥ ml/kg of fluids (n = , %) were younger (mean age years, sd vs. years, sd ; p< . ), more often had blood cultures collected during evaluation ( % vs. %, p= . ), and were more often transferred to another facility ( % vs. %, p< . ) when compared to patients who received < ml/kg of fluids (n = , %). mean fluid bolus volume within hour of time of sepsis onset by hospital ranged from ml/kg to ml/kg (figure , panel b) . in a cohort of community emergency departments, % of septic children received intravenous fluid boluses within one hour, and of those, only one half received volumes concordant with guidelines. (figure ). a wide range of fluid balance exists in septic shock patients cared for in icu. trends of serum albumin in septic and non-septic critically ill introduction: the link between hypoalbuminaemia and poor outcomes in critical care is well established [ ] . limited data are available on serum albumin trends during critical illness [ ] . in this study we assessed trends in serum albumin for up to days in both septic and non-septic critically ill patients. we retrospectively examined the records of adult patients admitted to critical care at the royal liverpool university hospital between and . we then excluded patients who did not have albumin data available for the first days, leaving us with patients. patients ( . %) had sepsis, and of these patients had died by day . of the non-septic patients ( . %), patients had died by day . albumin levels were collected for days from admission to critical care, in addition to other demographic and biochemical data. statistical analysis was performed using repeated measures analysis. septic patients had lower serum albumin than non-septic patients throughout the day period (p< . ). we observed a decrease in albumin by day in all groups, with levels increasing over the subsequent days. there was no difference in daily serum albumin between non-septic patients who survived or died. this is the first study, to our knowledge, to compare albumin trends in septic and non-septic critically ill patients over days. further research is needed to elucidate the optimal recipients and timing of albumin therapy. introduction: burn injury is characterized by marked inflammation, capillary leakage, and profound hemodynamic alterations. early albumin resuscitation is avoided fearing a paradoxical fluid escape into the interstitium. on the other hand, administration of crystalloids in massive amounts causes tissue edema and fluid extravasation, which deteriorates tissue perfusion by increasing oxygen diffusion distance. albumin administration could reduce the amount required to maintain hemodynamic stability in this population. we investigated whether albumin improves tissue perfusion and microcirculation by reducing tissue edema. this is an observational study conducted in the burn unit of maasstad hospital, rotterdam. patients with burns higher than % of total body surface area (tbsa) were included in the study. sublingual microcirculation was measured at admission (t ), (t ), and (t ) hours after burn injury. total vessel density (tvd) and functional capillary density (fcd) were analyzed. fluid management was calculated according to the modified parkland formula. albumin ( %) infusion was started hours after the burn insult. a total of nine patients were recruited between january and december . patients were included in the study after . ± . hours of the insult with a mean tbsa of ± %. the amount of crystalloid infusion was ± ml and ± ml at t and t ,respectively. within the first h (t ) ± ml albumin was given. tvd decreased from . ± . at t to ± . at t (p< . ) (figure ) introduction: spontaneous bacterial peritonitis (sbp) accounts for ≥ % of the bacterial infections that occur in patients with cirrhosis, and sbp has a high mortality rate ( % to %). albumin infusion has been shown to improve the outcome of sbp. the aim of this study is to examine the impact of albumin infusion on hospital length of stay (los) for cirrhotic patients with sbp. we utilized a nationwide electronic health record data set (cerner health facts®) to extract real-world data on adult patients (≥ years old) with cirrhosis and sbp who received antibiotics and admitted between january , , and april , . international classification of diseases (icd- / ) codes were used to identify cirrhosis and sbp. we used laboratory data for calculation of the model for endstage liver disease sodium (meld-na) score and vital signs data for calculation of the quick sepsis related organ failure assessment (qsofa) score at baseline for each encounter. a generalized linear model was used to assess the relationship between albumin infusion and hospital los. results: there were , encounters that identified patients with sbp and cirrhosis, of which , survived hospitalization. albumin was infused within hours of admission ('early albumin') in % (n= ), after hours in % ('late albumin', n= ), and not administered in % ('no albumin', n= ). meld-na was higher at presentation in early albumin cases versus late-or no-albumin cases (mean . and . ). unadjusted los was lower in patients receiving early albumin ( . days versus . days). risk-adjusted analysis demonstrated that early albumin led to a . % reduction in los ( % ci . %- . %, p = < . ). in these real-world data, albumin infusion within hours of admission in patients with cirrhosis and sbp was associated with a shorter hospital stay despite more severe illness. early albumin may not only improve clinical outcomes but may also reduce the costs of hospitalization in cirrhotic patients with sbp. early albumin use in patients with septic shock is associated with a shorter hospital stay: real-world evidence in the united states introduction: septic shock is among the most common critical care illnesses and incidence is rising, with mortality in excess of %. septic shock predisposes patients to multiple organ failure. while albumin is effective in management of circulatory dysfunction in septic shock, its utilization in this population is understudied in the us. we evaluated the impact of albumin utilization on hospital length of stay (los) among septic shock patients. we used a nationwide electronic health record data set (cerner health facts®) to extract real-world data on adult patients (≥ years old) with severe sepsis or septic shock, admitted between january , , and april , , identified by international classification of disease (icd- / ) codes, and receipt of antibiotics and vasopressors. we calculated the charlson comorbidity index (cci) and the acute physiology score (aps) at baseline. a generalized linear model was used to examine the association between albumin and hospital los, especially accounting for the timing of albumin infusion. we identified , unique visits for septic shock patients that survived to discharge. albumin was infused within hours of admission ('early albumin') in %, after hours ('late albumin') in %, and not administered in %. both cci and aps were higher, at presentation, in early albumin cases than late-or no-albumin cases (mean: . and . , and . and . , respectively). unadjusted los was slightly lower in patients receiving early albumin ( . days versus . days). a risk-adjusted analysis demonstrated that early albumin was associated with . % shorter los ( % ci . %- . %, p = . ). albumin infusion within hours of admission was associated with a shorter length of hospital stay. early albumin infusion may lead to better outcomes and reduced costs in patients with septic shock. further research is being conducted to assess other potential benefits of early albumin administration in this patient population. every new septic event follows by hemodynamic instability may lead sequentially to decreased organ perfusion, multiple organ failure. acute renal failure is recognized clinical feature during sepsis (up to - % in all cases). furthermore, urine output close monitoring is a cornerstone diagnostic clinical tool in each septic critically ill patient. in present study, we analyzed the dynamic minute-to-minute changes in the urine flow rate (ufr) and also the changes in its minute-to-minute variability (ufrv) during new septic event in critically ill patients. demographic and clinical data were extracted from the of critically ill patients who were admitted to the icu and developed new septic event (followed by fever and leukocytosis) and analyzed. a foley catheter was inserted into the urinary bladder of each study patient. the catheter was then connected to electronic urinometer, a collecting and measurement system which employs an optical drop detector to measure urine flow. the urine flow rate variability (ufrv) is defined and calculated as the change in ufr from minute to minute. results: ufr and ufrv both decreased significantly immediate after new septic episode until beginning fluid resuscitation (ppvalues < . ) (figure ) . statistical analysis by the pearson method demonstrated a strong direct correlation between the decrease in ufr, ufrv and the decrease in the map (r= . , p= . ; r= . , p= . ) ( figure ), and heart rate (r= . ,p=< . ) since systemic pressure starts to drop. ufrv and ufr demonstrated good clinical response to fluid administration despite the fact that systemic blood pressure did not improve (figure ) . we consider that dynamic changes in ufrv and ufr could potentially serve as a more sensitive signals ofclinicaldeterioration during the new septic event in critically ill patients.we also suggest that those parameters mightbeable to identify the optimal end-point of fluid resuscitative measures in septic critically ill patients. diminished urinary output (uo) is largely used as marker of acute kidney injury (aki) in critically ill patients. we aimed to explore the role of urinary output on incidence and mortality of aki developed during icu admission. the study population consists of all patients admitted between and to one of the dutch icus included in the nice database with an icu length of stay of at least hours, having daily measurement of creatinine and uo. only patients without renal replacement therapy that have a serum creatinine lower than . mg/dl ( . μmol/l) or a uo above . ml/kg/h on the day of the index icu admission were considered at risk for aki. patients were followed during their icu stay and classified according to the highest kdigo criteria reached based on creatinine alone (model ) and creatinine plus uo (model ) using icu admission serum creatinine as baseline. in both models, patients were classified as: no aki, renal impairment at the first day of icu admission, aki stage , aki stage , and aki stage . we identified , patients ( % male, mean age years, median icu-los days). of those, . % of patients had renal impairment at the first day of icu admission. among the remaining patients, . % in model and . % in model were classified as having no aki, . % and . % as aki stage , . % and . % as aki stage , and . % and . % as aki stage , respectively. survival at -day markedly differed according to the aki classification model used (figure) . similarly, adjusted hrs for -day mortality differed among patients with and without aki compared to patients with renal impairment at the first day of icu admission ( figure ) . among patients admitted to the icu % had renal impairment at the first day of icu admission. our findings suggested that uo plays an important role both on aki incidence and mortality and should be carefully interpret in the clinical setting especially in aki stage classification. introduction: acute kidney injury (aki) mostly attributed to renal tubular damage, has a high morbidity and mortality outcome [ ] , so a sensitive tool to assess the degree of tubular affection is needed for early detection and management of this condition. we investigated the ability of furosemide stress test (fst) (one-time bolus dose of mg/kg or . mg/kg if on prior furosemide-intake) to predict progression to akin stage-iii in critically ill subjects with early aki. we studied subjects; consecutive patients in group i receiving fst and consecutive patients in group ii receiving standard medical management for aki; patients ( . %) and patients ( %) met the primary endpoint of progression to akin-iii in groups i and ii respectively. patients with progressive aki had significantly lower urine output following fst in the first hours (p< . ). the area under the roc curves for the total urine output over the first hours following fst to predict progression to akin-iii was . (p = . ). the ideal-cutoff for predicting aki progression during the first fig. (abstract p ) . thirty-day survival according to aki classification model and model . hazard ratios (hrs) for -day mortality adjusted by sex, age, type of admission, apache iv score, sofa score at day of admission (excluded renal sofa score) for patients with aki classified with model and model fig. (abstract p ) . clinical correlation between urine flow rate variability (ufrv) and ufr and mean arterial blood pressure over new septic event (black arrows) and and after initial fluid resuscitation (red arrows). note: the ufrv and ufr decreased progressively in parallel with the falling mean arterial blood pressure and, than, rose again after the administration of fluids hours was a urine volume of less than milliliters with a sensitivity of . % and specificity . % group receiving fst. on the other hand, statistically significant hypotension, hypo-(kalemia, phosphatemia and magnesemia) occurred in group i. the fst in patients with early aki could predict liability for progression of aki, however it should be performed under adequate monitoring. introduction: ischemia-reperfusion (ir) causes renal dysfunction and damage. ir induces renal tubular injury triggered by hypoxia and hyperoxia, mediated by oxidative stress and inflammation. furosemide inhibits na + -k + - clcotransporter in the thick ascending limb of the renal medulla to decrease na + reabsorption, reducing oxygen consumption. we investigated if furosemide could improve renal oxygenation, function and damage by reducing o consumption and oxidative stress after ir. methods: wistar albino rats were divided into groups, with in each group; sham-operated control (c), control + furosemide (c+f), ir and ir+f. after anaesthesia (bl), min supra-aortic occlusion was applied to ir and ir+f groups followed by min (t ) and hours of reperfusion (t ). furosemide μg/kg/h infusion was simultaneously administered to c+f and ir+f after ischemia. systemic hemodynamic, renal blood flow (rbf), renal vascular resistance (rvr), renal oxygen delivery (do ren ), renal oxygen consumption (vo ren ), creatinine clearance (ccr), sodium handling, urine output (uo), cortical (cμo ) and medullar (mμo ) microvascular oxygenation were measured. results: rbf was reduced in ir ( . ± ) and ir+f ( . ± ) at t (p< . ) but it was further reduced in ir+f ( . ± ) (p< . ) at t compared to c and c+f. rvr was increased in ir ( ± ) and ir+f ( ± ) at t compared to c. rvr was normalized in ir ( ± ) but not in ir+f ( ± ) at t compared to c (p< . ). cμo and mμo did not differ between groups after ir insults (figure ). tissue o was reduced at the medulla, but not at the cortex in ir+f group compared to ir. do ren and vo ren were reduced in ir ( ± and ± ml/ min) and ir+f ( ± and ± ) at t (p< . ). pc was higher in ir+f ( . ± . ) compared to ir . ± . (p< . ). vo / tna + was increased in ir+f compared to ir. no change in ccr and uo was observed. furosemide after ir causes further impairment of renal perfusion, energy utilization and renal oxygenation resulting in renal damage. acute renal failure induced by hypoxemia: incidence and correlation study a trifi , h fazzeni , a mehdi , c abdennebi , f daly , y touil , s abdellatif , s ben lakhal la rabta hopital, medical intensive care unit., tunis, tunisia; la rabta hopital, tunis, tunisia critical care , (suppl ):p introduction: acute renal failure (arr) is a common complication in icus and usually caused by hypoperfusion. arf induced by hypoxemia is a concept rarely reported in icu. its incidence and pathogenesis are not well understood. we aimed to study the relationship between hypoxemia and the occurrence of arf. retrospective cohort study including patients with hypoxemia whatever its etiology between january and august . patients with chronic renal failure were excluded. arf was defined and ranked according to the kdigo criteria . arterial blood gas, urea, creatinine and clearance were reordered on the first, third and seventh days of evolution. results: patients were included and groups were obtained: group of hypoxemic patients with arf (arf+, n= ): versus group of hypoxemic patients without arf (arf-, n= ). the incidence of hypoxemie-induced arf was therefore %. clinical characteristics were comparable in both groups with a mean age of ± and a sex ratio of . . the comparative study showed in arf+ group: a lower ph ( . . ], p = . ). the most significant correlation was showed with mdrd clearance at day and p/f ratio at day (rho = . , p = . ). multivariate analysis found that septic shock and non invasive ventilation in hypoxemic patients were the factors related to arf with respectively or= . , % ci= . - . , p= . and or= . , % ci= . - . , p= . . overall mortality was % (n= ) and arf was an independent factor of mortality: or= , and % ci= . - . , p = . . hypoxemia-induced arf is a common complication associated with excess mortality. our study suggests that renal function is correlated with the degree of hypoxemia and that this correlation is rather distinct hours from hypoxemia. in preclinical models of sepsis, we have previously demonstrated that activation of amp activated protein kinase (ampk) using metformin, improves survival and organ function. thus, ampk activation is a potential therapeutic target in sepsis, and we hypothesize that exposure to metformin during sepsis is associated with decreased aki and mortality methods: retrospective analysis of a -hospital cohort of adult icu patients with type diabetes mellitus (t dm) who presented sepsis. we investigated if exposure to metformin during the hospitalization was associated with reduced -day mortality and aki. we used : propensity score matching (psm), propensity score stratification (pss) and propensity score weighting (psw) based on the probability to be exposed to metformin using covariates. for psm an exact match for insulin, amputation, cardiovascular diseases, retinopathy, charlson index, egfr, hba c, and apache iii, were used. sepsis was defined using sepsis criteria, and aki as kdigo stage or . from , patients, we found diabetic adults exposed to metformin during hospitalization and , who were not. metformin exposure during hospitalization is associated with decreased -day mortality and aki in septic adult patients with t dm. these findings suggest that metformin may constitute a potential therapeutic strategy in sepsis, and the potential role of ampk activation as a protective mechanism. however, studies are needed to confirm this association and the specific mechanisms of action. introduction: acute kidney injury (aki) may occur up to % in the intensive care unit (icu). predicting aki recovery may allow for risk stratification of patients, patient and family counseling, and early post-discharge renal care planning. however, predicting aki recovery at an early stage remains a challenge. methods: this is a retrospective study of the epanic multicenter randomized controlled trial database [ ] , which was split into development (n= ) and validation (n= ) cohorts, and patients experiencing aki stage and/or renal replacement therapy (rrt) in the icu were included [ ] . aki recovery was defined as being alive, without any stage of aki, and without need of rrt at hospital discharge. a logistic regression model with backward feature elimination was developed. the model performance was assessed by discrimination, calibration, and net benefit analysis, and internally validated with ten-fold cross validation. only the results in the development cohort are reported. of the patients who developed aki , patients ( . %) recovered from aki. the multivariable model selected age, bilirubin, heart rate, mean arterial blood pressure, surgical diagnostic group on icu admission, mechanical hemodynamic support on icu admission, suspected sepsis on icu admission as aki recovery predictors. the model had a mean area under the receiver operating characteristic curve (auroc) of . (standard deviation (sd) . ), mean calibration slope of . (sd . ), and mean calibration-inthe-large of < . (sd . ) (figure ). at the classification threshold that maximized sensitivity and specificity, mean net benefit with respect to treat-none was . (sd . ) and mean net benefit with respect to treat-all was . (sd . ). by using the routinely collected clinical data, the developed prediction model can fairly identify patients with a higher chance of aki recovery at hospital discharge. introduction: acute kidney injury (aki) is a frequent complication in critically ill patients and is associated with increased morbidity and mortality. sepsis is one of the most common cause of aki. a prospective study was conducted over months (january -june , ).we included patients with septic shock at admission or at any time during hospitalization.the aki staging was based on kdigo criteria.patients were divided into two groups, a group with aki (aki+) and a group without aki (aki-).then we compared the baseline characteristics, laboratory and physiologic data. patients with aki (aki+) were subdivided according to their prognosis. were enrolled patients. the mean (sd) age was . (± ) years.sex ratio was . . fifty-two ( %) patients developed aki.sapsii and sofa score in admission were higher in patients with kidney injury [ vs points (p= . ), . vs points ;(p= . )] respectively.the serum lactate level was significantly higher in (aki +) group patients during the first day of septic shock [ . ± . mmol/l (aki+)vs . ± . mmol/l(aki-);(p= . ) ] and its clearance was lower [( ± . % (aki +)vs ± %(aki-);(p= . )]. a significant difference was observed in c reactive protein level [ ± mg/l (aki +) vs ± mg/l (aki-) ; (p= . )].among (aki+) patients, kadigo iii was observed in . % of cases.nineteen ( . %) patients received hemodialysis.a normal kidney function was recovered in . % of cases.aki+ patients had a higher occurrence in disseminated intravascular coagulation ( vs patients, p= . ),acute respiratory distress syndrome ( vs patients; p= . ) and cardiac dysfunction ( vs patient, p= . ).mortality was higher in aki group ( % vs %; p= . ). the development of septic aki was associated with poor outcomes and prognosis.a better understanding of sepsis induced aki pathway will enable us to develop targeted therapeutic protocols.newer tools,permitting aki early detection, may make these therapies more fruitful. this study aims to show that contrast procedures do not significantly increase the risk of renal injury and should not be deferred. traditionally ciaki is the most important cause of in-hospital renal failure after nephrotoxic drugs and shock. problem is also the non-uniform definition of ciaki proposed by three different initiatives (akin, esur and kdigo). akin, being the most rigorous, defines ciaki as an increase in serum creatinine > . mg/dl or > % of baseline within hours. a retrospective observational single-centre cohort study analyzed patients who underwent a contrast procedure with iomeron . the first group underwent a ct pulmonary angiography (ctpa), and the fig. (abstract p ). internally validated model performance: (top row) roc curve; (middle row) calibration curve; (bottom row) decision curve second a coronary angiography with pci. no patient was previously prepared (raas blockade removal, crystalloid administration etc). we studied demographics, history of ckd and comorbidities and their impact on the ciaki by the akin criteria. a total of patients were divided into two groups (ctpa and pci). ctpa group ( m, f) all had acute pe and the pci group ( m, f) were treated for acs. the mean age was and years respectively. ckd was more prevalent in the pci group ( pt vs. pt) possibly explained by the more advanced atherosclerotic disease. advanced chd (nyha iii/iv) was found in pt (pci) vs. pt (ctpa) while diabetes and shock were equally distributed ( pt and pt) in both groups. the mean amount of contrast was significantly higher in the pci group ( . ml vs. ml). the mean creatinine/egfr measured before and after contrast in the ctpa group was . the goal of this study was to determine whether changing the body mass (bm) with fat-free mass (ffm) in cockcroft-gault (cg) formula could provide a more accurate prediction of aki in obese patients undergoing cardiac surgery. in this retrospective study, we reviewed institutional data of patients who underwent elective cardiac surgery in a tertiary referral university hospital. baseline patient creatinine value was collected and gfr was estimated using the mdrd, ckd-epi and cg formulas. cg formula was further modified by replacing the bm with ffm derived from the bioelectrical impedance analysis. postoperative aki was defined by kdigo creatinine change definitions. accuracy of the egfr values to predict the aki was calculated with roc-auc analysis. all the calculations were performed in different categories of bmi. figure ). the egfr is a poor predictor of aki in obese patients undergoing cardiac surgery. the ffm modified cauckraft-gault formula yield more accuracy in this specific group. retroaki: a ten-year retrospective study of acute kidney injury in intensive and progressive care units introduction: acute kidney injury (aki) is a frequent condition in intensive care units (icu) and progressive care units (pcu), affecting % to % of the patients, depending on the studied population and aki definition. aki has been identified as an independent risk factor of icu mortality and development of chronic kidney desease. the objective of this study was to describe the incidence of each aki stages as defined by kdigo definition (with evaluation of urine output, serum creatinine and initiation of renal replacement therapy (rrt)), in a mixed medical and surgical population of patients hospitalized in icu and pcu over a -year period ( - ). we included all patients who stayed more than hours in icu or pcu of edouard herriot hospital from may to january . data used to classify the patients were the urine output over a sixhour period, serum creatinine and the need for rrt, according to kdigo classification results: , hospital stays were analyzed. median icu/pcu length of stay was days [iqr: . - . ]. among icu patients, % had at least one aki episode graded , or and % had at least one severe episode (stage or ). among pcu patients, % had at least one episode of aki and % a severe episode of aki. patients had an average of . episodes of aki per stay. table represents the incidence of maximal aki stage during one stay. we found that urine output was the more frequent criteria to make diagnosis of aki stage or whereas rrt was more frequent for aki stage . this retrospective study reports a more important aki incidence in our icu/pcu than in previous studies. the difference could be fig. (abstract p ) . when comparing auc in different categories of bmi, the mcg appeared to be the only statistically accurate formula in patients with bmi - . explained by the difficulty to collect urine output from conventional database. serum creatinine and the use of rrt are often the only two criteria used to define and classify aki. these results confirm the high incidence of aki in icu and pcu and the importance to make an early aki screening of patients for whom preventive nephroprotective actions are needed. introduction: icu-patients with acute kidney injury (aki) requiring renal replacement therapy (rrt) are at risk for infections [ , ] . in this study we evaluated the incidence of infection in icu patients with and without less severe aki. finally, impact on outcomes was explored. this is a retrospective study on the pdms (protection data management system) of the adult icus of a university hospital. aki was assessed on kdigo criteria (creatinine (scr) and urine output), during the first -d of icu stay. infection was validated in the pdms by a team of icu specialists. results: during a -year period, a total of subjects were enrolled. aki was diagnosed in . % of patients during icu stay. aki patients were older ( vs. y, p= . ), had higher saps ( vs. , p< . ), and had more urgent icu admission ( % vs. %, p< . ). more aki patients had mechanical ventilation ( % vs. %, p< . ) and vasopressors on d- ( % vs. %, p< . ). aki stage , , and was present in . %, . % and . % of patients. more aki patients had infection ( % vs. %, p< . ) and increasing aki stages were associated with higher infection rates (aki- : %; aki- : %, aki- : %, aki- : %, p< . ) (figure ). we observed - times higher mortality in aki patients with infection, and a stepwise increase of mortality with increasing aki stages. after correction for infection and other confounders we found that all aki stages were associated with in-hospital mortality (ors aki- : . , aki- : . , aki- : . , all p< . ). over half of aki patients experienced an episode of infection and increasing aki severity was associated with higher infection rate. aki patients with infection had marked higher mortality, suggesting that infection was an important driver of outcome. however, after adjustment, aki stages had strong association with hospital mortality. several new biomarkers have been introduced to improve early diagnosis of acute kidney injury (aki). "nephrocheck" (nc; astute medical, usa) is a bedside test calculating "akirisk" (product of urinary concentration of the cell cycle arrest-markers timp- and igfbp ). several studies suggest the usefulness of nc in selected populations. however, the value of early routine measurement of nc is unclear. methods: therefore, we compared the prediction of a combined endpoint (cep: death < days and/or requirement of renal replacement therapy rrt) by nc within h of icu admission (nc ) and h later (nc ) with admission values of serum-creatinine, bun, cystatin c, urinary ngal, apache ii and sofa (roc-analysis). as a secondary endpoint we investigated the additional value of pathological measurements of nc ≥ . critically ill patients showed increased relative uce in the first days of icu admission, which may be attributed to higher protein catabolism. increased relative uce was associated with arc and both had no effect on -day mortality. introduction: this study compared epidemiology, short-and long-term outcomes for patients with community-acquired (ca) and hospital-acquired (ha) acute kidney injury (aki). we retrospectively analyzed all episodes of aki over a period of . years ( - ) on the basis of routinely obtained serum creatinine measurements in , patients whose creatinine had been measured at least twice and who had been in the hospital for at least two days. we used the "kidney disease: improving global outcomes" (kdigo) criteria for aki and analyzed the first hospital admission. a total of were admitted in hospital and fulfilled the inclusion criteria. average observation period per patient was days. the incidence of ca-aki among included hospital admissions was . % compared with an incidence of . % of ha-aki, giving an overall aki incidence of . %. patients with ca-aki were younger than patients with ha-aki ( vs . y) and had significantly less comorbidities, including preexisting cardiac failure, ischemic heart disease, hypertension, diabetes. patients with ca-aki were more likely to have stage aki ( , vs , %, p< . ) and had significantly shorter lengths of hospital stay than patients with ha-aki ( vs d, p< . ). those with ca-aki had better survival than patients with ha-aki (figure ; p< the evidence base for management of fluid removal during renal replacement therapy (rrt) is limited. a recent international survey revealed the extent of practice variation worldwide [ ] . our aim was to summarise the responses from europe-based healthcare professionals who participated in the survey. the international self-administered, cross-sectional, internet-assisted, open survey was disseminated between january and january via website links and emails to members of different critical care societies. results: participants from european countries completed the survey of whom ( %) were intensivists and ( %) worked in university-based hospitals. persistent oliguria / anuria was the most common indication for fluid removal ( % responders). the parameters which guided fluid removal included hemodynamic status ( % responders), cumulative fluid balance since admission ( % responders), and -hour fluid balance ( % responders). % of participants reported using crrt with a median net ultrafiltration rate ml/hr (iqr - ml/hr) for hemodynamically unstable and a rate of ml/hr (iqr, - ml/hr) for hemodynamically stable patients. only % of practitioners checked net fluid balance hourly ( % nurses, % physicians). new hemodynamic instability, defined as new onset or worsening tachycardia, hypotension, or need to start or increase the dose of vasopressors was reported to occur in % fig. (abstract p ). long-term survival patients (iqr . - . ). different strategies to re-gain hemodynamic stability were used. (figure ) main barriers to fluid removal were patient intolerance ( % physicians, % nurses) and interruptions in fluid removal ( % physicians, % nurses). the majority of participants agreed that guidelines and protocols would be beneficial. the practice of fluid removal during rrt is very variable across european countries. nurses and doctors identified a need for evidencebased protocols and clear guidelines. introduction: kidney disease improving global outcomes (kdigo) guidelines suggest the use of anticoagulation in continuous renal replacement therapy (crrt) [ ] . the effectiveness of the anticoagulation is important because replacing the hemofilter and tube interrupts crrt and increases total therapy time. regional citrate anticoagulation (rca) and unfractionated heparin (ufh) are most commonly using methods for crrt anticoagulation [ ] . the aim of this study was to investigate the efficacy, safety and metabolic differences of the patients in icu who underwent crrt and anticoagulation method changed from ufh to rca for different reasons. after ethics committee approval ( - / ) patients who underwent crrt between - at bursa uludag university hospital icu have been investigated and patients who underwent crrt by both rca and ufh included in the study. we divided patients in two groups (rca, ufh), demographic data (sex, age), sofa score, creatinine, urea, mean filter life time (flt) and ultrafiltration flow (uf), platelets, electrolytes (na, k, ca, mg), lactate, nahco and ph of groups at beginning and ending of first rca and ufh hemodialysis collected. we used t-test and bootstraps statistic tests. in agreement with other studies [ , ] , flt and uf was statistically significant lower in ufh group (table ) . there was no statistically significant difference in efficiency (urea and creatinine decrease), ph, lactate, nahco level, platelets count and electrolytes between two groups. to our knowledge, there are no studies comparing these two anticoagulation methods in the same patients. small number of patients and retrospective evaluation are limitations of the study. our results suggest that the implementation of rca method is safe and effective as ufh method with longer flt and uf. regional citrate anticoagulation during crrt in liver failure mj jain, pk kumar g, dg govil, jk kn, sp patel, ms shafi, rh harne, dp pal, sm monanga medanta the medicity, critical care, gurugram, india critical care , (suppl ):p continuous renal replacement therapy (crrt) with regional citrate anti-coagulation (rca) is increasingly being used as a treatment modality in critically ill patients. there is limited experience of use of citrate anticoagulation patients with acute liver failure and acute on chronic liver failure who pose a tough challenge of being at a higher risk for bleeding. an institutional protocol was formulated for use of commercially available citrate solutions and the same was studied to assess filter life and safety of citrate in liver disease. the primary objective was to assess safety of citrate anticoagulation in liver disease. this study was a single centre, prospective, non-randomized, single arm, observational study. all adult patients, with acute liver failure and acute on chronic liver failure requiring crrt were included. blood ionized calcium levels of . to . mmol/l was targeted throughout the therapy and total to ionized calcium ratio of less than . was maintained. rca was stopped if the ratio was more than . for consecutive assessments. incidence of citrate accumulation and toxicity were assessed. average filter life was also assessed. metabolic parameters, electrolytes and strong ion gap were followed till hours after completion on crrt. a total of patients were included in the study. nineteen patients of acute on chronic liver failure and patients of acute liver failure underwent crrt with rca. baseline average serum bilirubin, lactate and inr were . mg/dl, . mmol/l and . respectively. the average filter life was hours minutes. citrate accumulation took place in (n= ) patients and rca had to be stopped for ( n= ) patients due to the same. none of the patients had evidence of citrate toxicity. citrate anticoagulation was well tolerated in patients with acute liver failure in patients with or without pre-existing chronic liver disease on crrt. introduction: the intention of this study is to highlight the levels of citrate load for the general population that increases the risk of citrate complications (insufficient trisodium citrate delivery; net citrate overload and citrate accumulation) [ ] . this was a prospective data collection between february and march in a fourteen bedded critical care unit. eleven consecutive episodes of crrt were collected (a new episode characterized if crrt was discontinued for hours and above). one episode was excluded due to short duration (less than hours). patients undergoing rca-crrt received either a fixed or ml/kg/h effluent dose protocol. median patient age was , male %. average time on crrt was . days ( - ). % of the patients had complications, although % were minor ( figure ). all of the patients with net citrate overload had citrate loads of . mmol/h or above. the main risk factors were found to be shock and liver impairment which occurred in % of cases of which % developed complications. a fixed dose effluent protocol to standardise practice can potentially lead to a higher risk of minor complications. in our experience this is likely due to a lack of appropriate monitoring for rca-crrt complications. despite this, our complication rate of citrate accumulation is in line with that reported in literature. citrate loads in our ml/kg/ hr protocol were . % higher than our ml/kg/hr protocol and strongly related to higher complication rate that worsened in patients with risk factors for poor citrate metabolism. introduction: there is no optimal timing of continuous renal replacement therapy (crrt) in acute kidney injury (aki); however, it is based on volume overload, azotemia, hyperkalemia and severe metabolic acidosis [ ] . an important reason for metabolic acidosis in aki is increased unmeasured anions (ua) [ ] . delta-ph-ua (Δph ua ) detects the degree of metabolic acidosis caused by ua and is calculated by using 'the partitioned ph model' [ ] . in this study, we investigated whether Δph ua was a predictor to start crrt in patients with aki. the study was designed as a multicentric, prospective, observational study in . patients who were ≥ years old and diagnosed with aki [ ] were included. the moment aki was diagnosed, arterial blood gas, albumin, magnesium, inorganic phosphorus, urea, creatinine and Δph ua values were recorded. all patients were divided into two groups as crrt(-) and crrt(+) which consists of patients performed crrt due to traditional criteria. fig. (abstract p ) . incidence of complications introduction: continuous renal replacement therapy (crrt) is labor intensive and requires advanced nursing knowledge and skills. however, % of registered nurses (rn) are less than -year post-registration experiences in our unit. also there is an increasing demand of crrt from crrt days in to crrt days in . the obstacles for crrt in our department, includes variation of regimen, complicated workflow and insufficient training of nurses. a continuous quality improvement project is carried out to standardize the regimen, enhance workflow and provide structured training to nurses in the intensive care unit, to enhance nursing competence. methods: introduction: sepsis and septic shock is a leading cause of mortality in the intensive care unit. we tried to evaluate a novel hemoperfusion cartridge through a retrospective evaluation of patient's data in our centre. we used it as an adjuvant therapy in our patients with sepsis and septic shock due to varied causes. the aim of this study was to evaluate the efficacy of therapeutic hemoperfusion cartridge (hc-foshan biosun medical ® ) in the management of patients with sepsis. we retrospectively analysed data of group (n= sepsis) and group (n= sepsis+hemoperfusison; sepsis treated with hemoperfusion cartridge) admitted between to . group had received hemoperfusion cartridge as adjuvant therapy along with standard of care. demographic data, procalcitonin [ ] and leukocyte levels before and after therapeutic cytokine removal and duration of hc were recorded. while the mean duration of cvvhdf was . hours, the duration of hemoperfusion cartridge (application was . ± . hours). among patients who survived patients were administered hemoperfusion cartridge within hours of icu admission. there was a significant reduction in scores like apache and sofa score post hemoperfusion cartridge therapy procalcitonin and leucocyte levels after therapeutic hemoperfusion cartridge were found significantly lower than the pretreatment values (respectively p= . , p= . ). retrospective analysis showed significant reduction of vasopressors, and improvement in map in group . therapeutic hemoperfusion cartridge with cytokine removal applied with cvvhdf in septic patients have positive contributions to provide survival advantage. removal of activated leukocytes and endotoxin from the blood is a complex therapeutic effect of the device for removing endotoxin. in the main group ( patients with abdominal septic shock) after surgery, the traditional treatment was supplemented with two sessions of endotoxin removal ( hours each with an interval of hours) using "alteco lps adsorber" (sweden). the control group consisted of patients with a similar diagnosis and only traditional treatment. results: % of white blood cells were adsorbed in lps adsorber. among them, granulocytes ( %) were maximally extracted, then cd + monocytes (cd + mo) ( %), hla-dr + mononuclear cells ( %), monocytes ( %). il- , il- , procalcitonin (pct) were not adsorbed. the -day mortality rate in the main group was % and was lower compared to the control group - %. during monitoring, in the main group hours after the first removal of endotoxin, a decrease in the initially increased amount of activated cd + mo by . times, as well as functionally mature defensin + granulocytes (def + gran) by . times was observed. il- , il- , and pct decreased by . ; . ; and . times, respectively. during this period, the control group showed an increase in cd + mo and def + gran, while il- , il- did not change, and pct increased . times. a day after the second removal of endotoxin and then days later, the main group of il- , il- , and pct continued to decline. in the control group, only il- decreased after days, the rest continued to grow. the cellular adsorption of endotoxin-bound cd + mo and mature def + gran is an important part of the mechanism of action of the endotoxin removal device. does the endotoxin adsorption of pmx column saturate in hours? preliminary study c yamashita in the euphrates trial, the polymyxin b-immobilized fiber column (pmx) hemoperfusion (hp) had no significant effect on -day mortality. endotoxin (lps) burden by endotoxin activity assay > . may exceed μg [ ] , so the dose and duration of pmx-hp could be insufficient to lower the lps burden. to confirm this issue, we experimented in a closed-circuit with h continuous lps addition, and pmx can adsorb > μg [ ] . further, lps concentration became constant within h in the single lps spike test for determining pmx-hp duration [ ] . to prove our hypothesis that the single lps spike test reflects the adsorption equilibrium, and not saturation, we added lps intermittently to reaction. methods: lps ( ng/ml) was mixed with ml deactivated fetal calf serum as a reflux solution, as previously described [ ] ; this concentration is much higher than that observed in septic patients. we created a closed circuit that incorporates pmx- r at / th the amount of an adult pmx and performed pmx-hp at ml/min for h. lps was added in two shots (post h: ng, ng/ml; post h: ng, ng/ml). lps was measured using the limulus amebocyte lysate test at , . , , , , and hr. after an initial decrease between and h, lps concentration did not decrease between and h after pmx-hp initiation. post lps pulse addition at h, it increased and then decreased till h. futher, it did not decrease between and h, but it increased and then decreased again after lps pulse addition post h (figure ). lps adsorption rates were . , . , and . % at , , and h, respectively. conclusions: lps adsorption capacity of pmx- r was maintained even after two additional shots of lps, suggesting that the constant lps concentration in the previously reported lps spike test might be indicative of adsorption equilibrium rather than saturation. a coohort study included patients admitted to three intensive care with sepsis / septic shock ( sepsis criteria ) and aki ( akin score). all patients were submitted to cvvhdf with the oxiris filter (baxter, usa) . the main clinical data, il , procalcitonin, endotoxin ( eaa ) and sofa score were evaluated at basal time ( t ) and at the end of the treatment ( t ). all data are expressed as mean ± sd or median and iqr . anova test was used to compare the changes in the time. results: patients were submitted to rrt with the oxiris filter for ± hours . patients had aki stage , patients aki stage and patients had aki stage. at t all groups had an high vasopressor fig. (abstract ) . lps concentration in lps pulse addition test support to maintain map ≥ mmhg. il , procalcitonin eaa and sofa total were also elevated with no difference between the groups. at t creatinine improved better in aki ( p< . vs. t ) and in aki ( p< . vs t ) then in aki group. map increased in aki ( p< . vs t ) and aki ( p < . vs t ) , but not in aki group. il , procalcitonin decreased more in aki ( p < . vs t ) then aki . at t sofa total was higher in aki then aki ( p< . ) and aki ( p< . ). conclusions: aki and aki stage patients submitted to bp with the filter oxiris respond better then aki stage patients . -this transalte in a better clinical course. -crrt with oxiris filter is useful in septic patients with aki, but aki stage septic patients represent an high risk group. a non-interventional, multicenter, non-randomized patient registry for multiple organ dialysis with the advos system multiple organ failure is a challenging problem in the icu. as an advanced dialysis system, the advos procedure can eliminate watersoluble and protein-bound substances, regulate the acid-base balance as well as fluid and temperature. in , a national registry was established to collect data under "real-life" conditions of patients treated with advos without any trial-specific interventions (drks id: drks ). methods: data from / to / from german hospitals (university hospitals in hamburg-eppendorf, mainz, essen, and klinikum weiden) were analyzed. clinical parameters, treatment settings and adverse events were documented. the -and -day mortality rates were compared with extrapolated rates based on the sofa score. results: patients with a median age of years (iqr - ), of whom ( %) were male, were evaluated. patients had a median sofa score of (iqr: - ) before the st advos treatment, which is associated with an expected mortality of %. the number of failing organs was (iqr - ): cardiovascular ( %), lungs ( %), liver ( %), kidneys ( %), coagulation ( %) and cns ( %). treatments with a median duration of (iqr: - ) hours were evaluated. were discontinued, of which ( %) were due to a device error. adverse events were documented, were related to the device (all due to clotting and recovered without sequelae). significant removal of protein-bound (bilirubin: . vs . mg/dl) and water-soluble toxins (bun vs and creatinine . vs . mg/dl). in addition, improvement in acid-base balance was observed: ph ( . vs. . ), bicarbonate ( . vs. . mmol/l) and base excess (- . vs. . mmol/l) ( table ) . -and -day mortality rates were % and %, respectively. in a cohort of patients with multiple organ failure, we observed an improvement in the expected mortality rate, especially if the advos procedure was applied early. adverse events are comparable to other dialysis therapies in intensive care patients. introduction: acute kidney injury (aki) due to ischemia-reperfusion affects onethird of the patients in cardiac surgery. we investigated the potential role of cyclosporine (csa) to prevent postoperative aki and mitigate inflammatory response to extracorporeal circulation (ecc). methods: double-blind, randomized, placebo-controlled single-center study. patients (n= ) scheduled for elective cardiac surgery were randomized to , mg/kg csa or placebo before the surgery. the primary objective was to assess the role of csa to reduce the incidence of postoperative aki. the secondary objective was to study csa induced changes in the inflammatory response to ecc. results: all enrolled patients were analyzed. postoperative aki was more pronounced in the cyclosporine group compared to placebo. or= . ( . - . ), % ci. the cytokine production in response to ecc was not affected by cyclosporine (figure ) . in patients undergoing cardiac surgery, a single preoperative dose of csa does not prevent the postoperative decrease in renal function. csa does not alter cytokine release in response to extracorporeal circulation. elevated post-ecc levels of pro-inflammatory cytokine il- are associated with kidney dysfunction and may be predictive. new generation adsorbent such as oxiris r was introduced as novel technique in renal support for critically ill patients [ ] . septic shock patients require decatecholaminization strategies emphasizing blood purification to remove catecholamine-producing mediators and evacuate overload fluid in interstitials. our -year-old female patient, admitted to icu after surgery with history of ovarium cancer. her septic shock was worsened with ards, hypercoagulable state and aki. vasopressors were set. patient was controlled with mode simv ,ps ,tv ml,peep ,fio %. renal support was implemented by diuretic and cvvh started on the second day. at first,regular adsorbent was used, post-filter mode was set, and periodic fluid removal target was ml/h. but after hours, no significant changes observed. oxiris r added and after hours passed, requirements of vasopressors reduced, tidal volume increased, hemodynamic parameters stabilized, urine production increased. it was continued for days and patient was recovered. our patient had fallen into inadequate cars stage in which not able to counter septic effects on vital organs (figure ). renal would be primary target for filtration and monitoring tool. adsorbent consisted of an and polyethyleneimine was useful to purify blood from endotoxins conjoined with slower filtration. continuous yet cautious process in cvvh evacuate fluid and mediators while maintain steady hemodynamics. biomarkers could not be evaluated due to limited resources, but improving parameters could be signs that showed recovery process had already took place. advanced hemofiltration is a privilege. implementing and enhancing it with new generation adsorbent would increase survivors by extracting unnecessary fluids and eliminating catastrophic endotoxins and mediators. consent to publish: written informed consent for publication was obtained from the patient. analysis of retrospective cohort study data of patients (pt) treated for dka at icu of kaunas clinics during - has been carried out. serum kalemia, glycemia; hypokalemia, hypoglycemia episodes; rate of insulin interruption for hypo-and normoglycemia during ketoacidosis; use of nah co for ketoacidosis, and los in icu were analysed. spss . was used for statistic calculations. traits evaluated as significant at p < . . at the beginning of dka treatment in totally hypokalemia ( . ± . mmol/l) was recorded in / pt ( . %). due to ignoring of blood ph ( . - . ( . ± . ) kalemia was falsely misinterpreted as "normo-" or "hyper-" . - . ( . ± . mmol/l) in / pt ( . %), thus disregarded so complicated by obvious hypokalemia additionally in / pt ( . %). in hypokalemia los in icu was . ± . vs . ± . h, p < . . insulin use has caused hypoglycemia ( . - . ( . ± . mmol/l)) in / pt ( . %), los in icu . ± . vs . ± . h, p < . .insulin use was interrupted in case of normoand hypoglycemia with still persisting ketoacidosis in / pt ( . %), los in icu was found to be . ± . vs . ± . hr, p < . . nah co was given for symptomatic treatment of ketoacidosis during first h of dka in / pt ( . %) with stable hemodynamic: hco - buffer has increased ( . ± . - . ± . mmol/l), p < . , but it didn't control ketoacidosis, and los in icu was . ± . . vs . ± . h, p < . . hypokalemia, hypoglycemia, precocious interruption of insulin use were recorded as complications of dka treatment. all of them have prolonged los in icu. symptomatic treatment of ketoacidosis with nah co had no effect on it, and prolonged los in icu as well. a growing interest exists about co derived parameters in shock management. central venous-arterial pco difference (p cv-a co ) is strictly related to cardiac output; central venous-arterial pco difference to arterial-central venous o content difference ratio, p cv-a co / c a-cv o , has been proposed as anaerobic metabolism when it's > . mmhg/ml [ ] . to evaluate p cv-a co /c a-cv o reliability in detecting anaerobic metabolism, we analyzed it in consecutive patients affected by mala admitted to our icu, considering these patients as a prevalent anaerobic metabolism model. we calculated, by douglas formula, central venous-arterial co content difference to arterial-central venous o content difference ratio, c cv-ca co /c a-ccv o , as a respiratory quotient surrogate. we performed arterial and central venous blood gas analysis simultaneously at admission, we calculated p cv-a co , p cv-a co /c a-cv o and c cv-a co /c a-cv o and we recorded scvo . we verified relationship between p cv-a co /c a-cv o and scvo and arterial ph, arterial lactates, sofa score at admission and c cv-a co /c a-cv o by linear regression analysis. pcv-aco /ca-cvo greatly increases in mala ( . ± . ). pcv-aco / ca-cvo (fig. ) shows significant co-variation with ph (r = . ; p= . ) and sofa score at admission (r = . ; p= . ). pcv-aco / ca-cvo has poor agreement with ccv-aco /ca-cvo (r = . ) and disagrees with it in identifying anaerobic metabolism, in our series, in fact, ccv-aco /ca-cvo is, in patients, < like an aerobic rq value. pcv-aco /ca-cvo shows better agreement with ph, sofa score and lactate level than scvo . in our series, p cv-a co /c a-cv o is good illness and acidosis severity marker, but it seems to be affected by ph value in accord with haldane effect [ ] . p cv-a co /c a-cv o , in our study, doesn't seem to be a reliable anaerobic metabolism marker nor a rq surrogate. it is thought that early administration of basal insulin to patients with diabetic ketoacidosis (dka) may improve outcomes. small studies have shown trends towards decreases in time to closure of anion gap (tcag), rates of rebound hyperglycemia following discontinuation of intravenous (iv) insulin, rates of hypoglycemia, intensive care unit (icu) length of stay (los), and hospital los [ ] [ ] [ ] [ ] . this was a single-center, retrospective chart review of our institution's dka protocol between january and august . patients that received early basal insulin within hours of initiation of iv insulin and before closure of the anion gap (ag) were compared to those that did not receive early basal insulin. the primary outcome was median tcag. secondary efficacy outcomes include: time on iv insulin infusion, time to de-escalation of level of care, hospital los, and re-elevation of ag. secondary safety outcomes included incidences of hyperglycemia, hypoglycemia, and hypokalemia. a total of patients were identified meeting inclusion and exclusion criteria. median tcag was longer in the experimental group ( vs. hours, p < . ). incidence of re-elevation of ag and incidence of hyperglycemia were lower in the experimental group. other outcomes were similar (figure ). early administration of basal insulin to patients with dka resulted in a longer tcag with a lower incidence of re-elevation of ag and hyperglycemia. early administration of basal insulin appears to be safe with respect to hypoglycemia and hypokalemia. glycaemic control continues to be a challenge in critically ill patients. stress induced hyperglycaemia has been associated with increased morbidity and mortality [ ] . conversely, patients receiving intensive glucose control have a higher risk of death [ ] . a quality improvement project was designed to develop a comprehensive insulin protocol that recognized pre-existing diabetes and reduced hypoglycaemia. data was collected prospectively in all adult patients admitted to the rah intensive care unit (icu) between october and august from the national icu audit database and electronic patient records. daily figures were collected for numbers of hypoglycaemic episodes (< mmol/l), "in range" ( - mmol/l) blood sugar measurements and patients with a pre-existing diagnosis of diabetes. data was collected and analysed using microsoft excel. results: patients were identified; patients ( . %) had pre-existing diabetes. a total of blood sugar measurements were reviewed; ( . %) were "in range" and hypoglycaemic episodes ( . %) occurred. there was no significant correlation between number of diabetic patients and measurements within range. of note, there was an increase in number of measurements per patient in the second half of the time period ( vs ). the development of this protocol has improved glycaemic control in our icu. there are considerably fewer episodes of hypoglycaemia and a large proportion of blood sugar measurements are in range. we hope to continue data collection and interrogate the prevalence of pre-existing diabetes further to reduce glycaemic variability. the optimal management of blood glucose levels for critically ill patients remains unclear. hypoglycemia, hyperglycemia and glycemic variability are associated with mortality. the time in targeted blood glucose range (tir) has been suggested to correlate with mortality depending on the status of antecedent glycemic control, but it has not been verified optimal tir and whether there is an optimal disease-specific tir. a retrospective observational study was performed at a single center. in the present study, we enrolled all critically ill patients admitted in intensive care unit from january to october. patients with diabetic ketoacidosis or hyperosmolar hyperglycemic syndrome and patients who had < blood glucose readings were excluded. gathered information included, in part, demographics, comorbidities, severity of illness scores, diagnosis at admission, length of icu stay and hospital discharge status. the primary outcome was -day mortality. we analyzed to find the optimal tir for critically ill patients. several tirs were each tested for correlation with mortality. a total of , patients, . % of whom had diabetes, were studied. tir to mg/dl (or, . ; %ci, . - . ), tir to mg/ dl (or, . ; %ci, . - . ) and tir to mg/dl (or, . ; %ci, . - . ) > % was independently associated with mortality in critically ill patients respectively. the optimal tir did not differ depending on diagnosis at admission. in this retrospective evaluation, tir to mg/dl > % was independently associated with mortality in critically ill patients, especially those with good antecedent glucose control. these findings have implications for the design of future trials of intensive insulin therapy. the prevalence of chronic dysglycemia (diabetes and prediabetes) in patients admitted to swedish intensive care units (icus) is unknown. we aimed to determine the prevalence of such chronic dysglycemia and asses its impact on blood glucose control and patient-centred outcomes in critically ill patients. in this retrospective, observational study, we obtained routine glycated hemoglobin a c (hba c) measured in patients admitted to four tertiary icus in sweden between march and august . based on previous diabetes history and hba c we determined the prevalence of chronic dysglycemia (prediabetes, undiagnosed diabetes and known diabetes). we compared indices of acute glycemic control in the icu and explored the association between chronic dysglycemia and icu-associated infections, mechanical ventilation, renal replacement therapy, vasopressor therapy, and mortality within days. of patients, ( %) had chronic dysglycemia. of these patients, ( %) had prediabetes or undiagnosed diabetes and fig. (abstract p ) . results ( %) had a known diabetes diagnosis. during icu stay, patients with chronic dysglycemia had higher average blood glucose, spent less time in target glucose range, had greater glucose variability, and were more likely to develop hypoglycemia than patients without chronic dysglycemia. chronic dysglycemia was associated with greater need for renal replacement therapy (odds ratio . , % ci . - . ) and increased -day mortality (hazard ratio . , % ci . - . ) after adjustment for simplified acute physiology score . in contrast, chronic dysglycemia was not associated with mechanical ventilation, vasopressor therapy, or icu-associated infections. in four tertiary swedish icus, measurement of hba c showed that / of patients had chronic dysglycemia (prediabetes or diabetes). chronic dysglycemia was associated with marked derangements in glycemic control during icu stay, greater need for renal replacement therapy and with increased mortality at days. case report: modern antidiabetic therapie causes ketoacidosis am heiden, m emmerich krankenhaus bad oeynhausen, institut für anästhesie, bad oeynhausen, germany critical care , (suppl ):p the modern antidiabetic class of sglt -inhibitors, that are known to reduce the risk for cardiac events [ ] , are increasingly used in the last few years. a -year old male patient with diabetes mellitus suffered days after colectomy surgery from abdominal pain and nausea. the patient had an antidiabetic therapy with empaglifozin that was paused until day after surgery (nutrition start on day , weaning on day ). methods: this is a case report of one male patient seen in the icu setting. daily blood values including arterial blood gases, vital parameters and clinical status of the patient were observed and evaluated. the blood gases showed this metabolic acidosis: ph . ; pco . mmhg, bicarbonate mmol/l, be - . mmol/l, lactate . mmol/l, glucose mmol/l. a ketonuria despite normal blood glucose values was noticed, so that the diagnosis of ketoacidosis was clear. after analyzing the possible causes we found out, that empaglifozin in times of catabolism and fasting can cause this severe symptomatic. we terminated the therapie with empaglifozin and under the treatment with insulin the symptoms disappeared within days and the patient could be discharged from the icu on day after surgery. after one episode of ketoacidosis the therapy with sglt -inhibitors should lifelong never be started again. we recommend that intensivists should be aware of the modern sglt -inhibitors because of the shown severe complications and the increased use of this medication. consent to publish: written informed consent for publication was obtained from the patient. while obesity confers an increased risk of death in the general population, numerous studies have reported an association between obesity and improved survival among critically ill patients. this contrary finding has been referred to as the obesity paradox. this retrospective study uses two causal inference approaches to address whether the survival of non-obese critically ill patients would have been improved if they had been obese. the study cohort comprises , adult critically ill patients hospitalized at the intensive care unit of the ghent university hospital between and . obesity is defined as a body mass index of ≥ kg/m . two causal inference approaches are used to estimate the average treatment effect in the untreated (atu): a naive approach that uses traditional regression adjustment for confounding and that assumes missingness completely at random, and a robust approach that uses super learning within the targeted maximum likelihood estimation framework and that uses multivariate imputation of missing values under the assumption of missingness at random. obesity is present in . % of patients. the in-hospital mortality is . % in non-obese patients and . % in obese patients. the marginal associational risk difference for in-hospital mortality between obese and non-obese patients is - . % ( % confidence interval (ci) - . % to . %, p= . ). the naive approach results in an atu of - . % ( % ci - . % to - . %, p= . ), whereas the robust approach yields an atu of - . % ( % ci - . % to . %, p= . ). a robust causal inference approach that may handle confounding bias due to model misspecification and selection bias due to missing data mitigates the obesity paradox, whereas a naive approach results in even more paradoxical findings. the robust approach does not provide evidence that the survival of non-obese critically ill patients would have been improved if they had been obese. bowel management within an icu environment is often difficult. recent data collection from an intensive care unit at the rvi identified either loose stool or constipation on > % of patient days. it was postulated this could be improved with a more tightly controlled bowel management regimen. to test this hypothesis a step-wise bowel protocol was created and introduced. data was collected in the month period following its implementation with the following aims: ) assess effectiveness of the protocol ) further observe the reasons for loose or constipated stool on an diarrhea is an important problem in each critically ill pateints [ ] . we aimed to investigate the frequency and management of diarrhea in our icu. in this study patient retrospectively reviewed, in our icu between . . - . . . patients were divided into two group as diarrhea "positive" and "negative". patients with diarrhea had fluid or loose stools or more times a day. each diarrhea period of the patients with diarrhea was examined separately and compared with the group without diarrhea. nutritional status, enteral product formulation, leukocyte, neutrophil, albumin values, gastric sparing, antibacterial and antimycotic use, los in hospital and in icu were compared. in diarrhea positive group, on the day of hospitalization, laxative and/or enema administration, toxin a in stool, nitrogen balance before and after diarrhea, enteral product change in diarrhea, probiotic, metronidazole or oral vancomycin use were examined. the incidence of diarrhea was . %. the most common diagnosis of icu admision was respiratory failure ( - %) in both groups. diarrhea occurred in two days after laxative and/or enema treatment. enteral nutrition was higher in both groups (≥ %). nasogastric tube feeding was significantly higher in the diarrhea group (p= . ). there was no difference between nutritional product formulation and diarrhea development (p> , ). antibacterial use was high in both groups ( %); however, teicoplanin use was significantly higher in the group diarrhea negative group (p= . ). the los in icu, and hospital was higher in diarrhea group (p< . ). no difference in mortality rates (p> . ). many factors may cause diarrhea in icu, and diarrhea may adversely affect patient treatment and increase morbidity. we think that preventive methods are as important as the treatment of diarrhea. the use of parenteral glutamine is studied in number of rcts and systemic reviews (heyland d , wischmeyer p ), while there is a lack of data about the use of enteral glutamine. the aim of our study was to determine the effect of enteral glutamine supplementation on the incidence of hospital infections and death. design: retrospective cohort study. inclusion criteria: males and females > years of age, tbsa burned %- %, nasogastric intubation.patients were divided in two groups: glutamine group (n= ) and control group (n= ). in the study group enteral glutamine was administered to the patients for days after admission to the icu. baseline characteristics were well balanced between groups. no significant difference was found between groups on patients' age, sex, tbsa, need for mechanical ventilation and rate of inhalation injury. primary outcome was all-cause mortality. secondary outcome was rate of nosocomial infections (skin and skin structure infections (sssi), lower respiratory tract infections, urinary tract infections, bacteremia, sepsis). mortality rate was ( %) and ( %) in the glutamine group and the control group, respectively, p= . . rate of nosocomial infections was ( %) in the glutamine group and ( %) in the control group, respectively, р= . . rates of sssi, lower respiratory tract infections, urinary tract infections and sepsis did not differ significantly between the groups: ( %) and ( %), p= . ; ( %) and ( %), р= . ; ( %) and ( %), р= . ; ( %) and ( %), р= . , respectively. rate of bacteremia was significantly different between the groups: ( %) in the glutamine group and ( %) in the control group, p= . . retrospective design is a significant limitation of our study. enteral glutamine supplementation may reduce the incidence of bacteremia in burn patients, but has no influence on the incidence of other nosocomial infections and mortality. further large clinical trials are needed. with outcomes were assessed with multivariable logistic regression and cox proportional hazard analyses, adjusted for baseline risk factors and randomization. in sensitivity analyses, models were further adjusted for key regulators of ketogenesis to assess whether any effect was direct or indirect. late pn increased plasma hb as compared with early pn, with maximal effect on day (p< . for day to and for the "maximal effect" day in the patients). adjusted for baseline risk and randomization, plasma hb associated with a higher likelihood of earlier live weaning from mechanical ventilation (p= . ) and of earlier live picu discharge (p= . ). as plasma hb replaced the effect of the randomization, the hb effect statistically explained these benefits of the randomization. further adjustment for key regulators of ketogenesis did not alter these findings. plasma hb did not independently associate with the risk of infections and mortality. withholding early pn increased ketogenesis in critically ill children, an effect that statistically mediated part of its clinical benefits. critical care patients are prone to frequent feeding interruptions for various reasons including feeding intolerance. these interruptions can lead to adverse outcomes. the aim of the study was to determine the reasons for and the duration of interruptions of enteral nutrition (en). single-center observational, cross-sectional study in a -bed mixed icu of a tertiary hospital. duration: months. patients, aged . years old (± . ), that stayed in the icu > hrs and were fed with en were included. anthropometric data, bmi, time of initiation of prescribed en, type of en formula, daily calories delivered were recorded. energy intake was calculated according to espen guidelines ( kcal/ kg bw/day). the causes for and duration of interruption were reviewed from the patient's chart. apache ii and mnutric score was calculated for all patients. mnutric score ≤ was used to diagnose malnutrition. all patients included in the study were endotracheally intubated. apache ii was . ± . . % of patients had increased risk of malnutrition. icu stay was . ( . ± . ) days, and the in-hospital mortality was %. there were episodes of en interruptions over a median icu stay of . days. median . interruptions/patient. the most common reason for en interruption was gastric residual volume monitoring followed by diagnostic and therapeutic procedures (figure ). other reasons include surgery, intolerance and/or delayed feeding and extubation. the median lost feeding time was . hours/ day ( . - . ) for all causes, while the mean loss of total energy intake was kcal/day (± )/day. average body weight of the patients was kg (± ). caloric deficit was calculated at kcal/day or % of the prescribed caloric goal. the results of this study showed that interruptions can lead to substantial caloric deficit, malnutrition and adverse events. an interruptionminimizing protocol could be useful in order to reduce the missing hours and to improve the clinical outcomes. relationship of goal-directed nutritional adequacy with clinical outcomes in critically ill patients pc tah there are controversies surrounding the effects of optimal nutritional intake on clinical outcomes in critically ill patients. this study aimed at investigating the relationship of goal-directed energy and protein adequacy on clinical outcomes which includes mortality, intensive care unit(icu) and hospital length of stay (los), and length of mechanical ventilation (lomv). this was a single centre prospective observational study. nutritional requirements were guided by indirect calorimetry and -h urinary urea.nutritional intake was recorded daily until death, discharge, or until day of icu stay. clinical outcomes were collected from patient's hospital record. the relationship between the two groups (< % and ≥ % of overall nutritional requirement) with mortality outcomes was examined by using logistic regression with adjustment for potential confounders. terlipressin, despite being one of the main treatments for acute variceal bleeding, may lead to severe hyponatremia due to its antidiuretic activity.we aimed to identify risk factors for development of hyponatremia during terlipressin treatment. retrospective study of patients admitted to acute intermediate care unit for hypertensive upper gastrointestinal bleeding due to chronic liver disease who received terlipressin(december -decem-ber ).hyponatremia was defined as a decrease in na serum levels ≥ meq and severe hyponatremia as > meq within days of treatment. we studied patients, . % male, mean age of . years (sd . ). alcohol-related liver disease was the most frequent etiology. hyponatremia occurred in patients ( . %). serum na Δbetween - and - meq and serum na Δ>- meq occurred in . and . %, respectively (table ) . severe hyponatremia occurred in patients ( . %) and symptoms were reported in two cases (status epilepticus and altered mental status). patients with higher baseline levels of na were more susceptible to terlipressin-induced hyponatremia and a longer length of stay was observed in patients with serum naΔ>- meq ( . vs . days, p< . ). the prevalence of hyponatremia in our study was lower than previously reported.higher serum na at admission and aih as etiology of cirrhosis were predictors of terlipressin-induced hyponatremia. neither the cumulative dose of terlipressin nor the duration of treatment appear to be related to the development of hyponatremia a Δ h-[na] > mmol/l was associated with larger hazards of mortality ( figure ). an increase in serum sodium in the first hours of icu admission is independently associated with a higher mortality in patients admitted with mild hyponatremia, normonatremia, and hypernatremia. based on our findings, it is possible that mild hyponatremia may be a protective mechanism in critical illness, which questions common practice of routinely correcting serum sodium when it is too low. introduction: acute liver failure (alf) represents a life-threatening organ dysfunction associated with increased mortality and liver transplantation represents the only definitive treatment. the aim of this study was to assess the effects of renal replacement therapy in combination with hemoadsorption in alf patients. twenty-nine patients with alf admitted to the intensive care unit (icu) of fundeni clinical institute were included in the study. after icu admission, consecutive session of hemoadsorption in combination with continuous veno-venous hemodiafiltration were applied. number of organ dysfunctions and sirs criteria were recorded at icu admission. the following data were recorded before and after the hemoadsorption therapies: glasgow coma scale, pao /fio , creatinine, -hours urine output, bilirubin, leucocyte and platelet count, heart rate, mean arterial pressure and vasopressor support, c-reactive protein and procalcitonine. clif-sofa score was calculated before and after the therapy. icu length of stay and -days outcome were noted. the mean age in the study group was ± years. the median number of sirs criteria was [ , ] and the median number of organ dysfunctions was [ , ] . the use of hemoadsorption was associated with a decrease in creatinine (from . ± . to . ± . mg/dl, p= . ), bilirubin (from . ± . to . ± . mg/dl, p= . ) and platelet count ( ± / ul to ± /ul, p= . ). we also observed a decrease in clif-sofa score from . ± . to . ± . (p= . ). overall mortality was . % (n= ). six patients ( . %) underwent liver transplantation with % -days survival. the use of hemoadsorption in patients with alf is associated with improvement in liver and kidney functional tests and may represent a new therapy in bridging these patients to liver transplantation. introduction: impairment of intestinal mucosal barrier function is the initiating factor of sepsis. in order to explore the effect of lactic acid bacteria on intestinal barrier function impaired by sepsis, it is necessary to establish sepsis and lactic acid bacteria ecological models. however, how to construct these models is still unclear. co-cultures with a gradient of lactic acid bacteria and caco- cells were constructed. the symbiotic state was observed under an inverted microscope and lactate dehydrogenase (ldh) toxicity tests, transepithelial electrical resistance(teer) tests and western blots were used to determine effective concentrations of lactic acid bacteria in monolayer cell models. lipopolysaccharide (lps) was used to treat cells, and cell counting kit- , quantitative reverse transcription pcr(rt-qpcr) and enzyme linked immunosorbent assays (elisa) were used to determine the appropriate concentration for sepsis models. the number of living cells decreased significantly when the moi(number of lactic acid bacteria/cell number) reached ( figure , panels a, b). the release of ldh indicated that damage to cells began to increase when the moi exceeded (panels a, b). at an moi of . , resistance values began to increase over time, whereas resistance values began to decrease when the moi reached (panel ). as the number of lactobacilli increased, the expression of tight junction protein increased and then decreased (panel a, b, c). in sepsis model experiments, the cell survival rate began to decrease once the concentration of lps exceeded ^ ng/ml (panel ). rt-qpcr results showed that ng/ml lps significantly increased inflammatory cytokines (panel ), and elisa results consistently showed that tnf-α and il- increased significantly when lps concentrations reached ng/ml (panel a, b). it is feasible to construct a cell monolayer model of lactic acid bacteria and lps. the appropriate moi of lactic acid bacteria is . and the optimal concentration of lps is ng/ml. introduction: sepsis is associated with high mortality and morbidity. as the severity increases, physiological parameters such as ph changes are one of the most notable features in metabolic acidosis secondary to high lactate. currently there is no point of care test other than blood gas measurement that could detect these ph changes. this is challenging especially in prehospital environment. the aim of this study is to develop a novel rapid point of care testing using a sensor to detect ph change in blood. sensors were produced by screen printing graphene and silver electrodes and functionalizing the graphene working electrode with an active layer of melanin. a preclinical sensor model was produced by adding lactic acid to a citrated plasma sample thus altering its ph over a clinically relevant range. the ph sensors were exposed to modified plasma, recording any changes in the voltage. the relationship between the voltage potential and plasma ph was established using weighted least squares regression. a ph dependent change in the measured voltage, with respect to the ph of the solution, was observed with a sensitivity of - . mv/ph +/- . over a physiologically relevant ph range between ph . and ph . . in this first phase proof of concept study a low cost, ph sensor was fabricated and demonstrated to be effective in measuring the ph of the plasma. this is the first time that such a sensor has been demonstrated and validated to work in this preclinical model of acidosis. the technology demonstrated here is a promising candidate for a point of care test whereby abnormal blood ph levels can be detected and monitored outside of a laboratory environment in a rapid manner. further studies are now underway to detect this change in whole blood. (figure ) . over one year only a small proportion of patients (n= , %) were classified as 'intermediate high' risk and potential candidates for reperfusion therapies. the revised national early warning score (news) with modified glasgow prognostic score (mgps) is superior to the news for predicting in-hospital mortality in elderly emergency patients t mitsunaga jikei university school of medicine, emergency medicine, tokyo, japan critical care , (suppl ):p the national early warning score (news) was developed in the ukto identify the risk of death. the previous study showed that the modified glasgow prognostic score (mgps) correlate with frailty in elderly patients [ ] . the aim of this study is to evaluate the predict value of the revised news with mgps for in-hospital mortality (in days) in elderly emergency patients. this study is secondary analysis and was carried out in jikei university kashiwa hospital, in japan, from april to march . the acute medical patients aged and older were included. the news was derived from seven physiological vital signs. the mgps was derived from c-reactive protein (crp) and albumin. discrimination was assessed by plotting the receiver operating characteristics (roc) curve and calculating the area under the roc curve (auc). the aucs for predicting in days in-hospital mortality were . for revised news with mgps and . for the original news. the auc of the revised news with mgps was significantly higher than that of the original news for predicting in-hospital mortality (p < . ) (figure ) . our single-centred study has demonstrated the utility of the revised news with mgps as a high predictor of acute phase in-hospital mortality in elderly emergency patients. the diagnostic performance of the five main emergency department (ed) triage systems has been shown to be poor in distinguishing acute coronary syndromes (acs) from mild severity diseases in chest pain patients. these ed triage systems are either clinically-based, being more sensitive or ecg-based, more specific [ ] . the goal of the study was to evaluate if incorporation of cardiovascular risk factors (cvrf) into ecgbased triage could increase his diagnostic performance. cecidoc is a prospective, observational, single-center study in an academic hospital. all consecutive adult patients admitted for acute chest pain were included. we compared the ecg-based french triage system [ ] to a modified system upgrading patients with a normal ecg but significant cardiovascular risk from a low acuity triage score (waiting period before medical assessment of max. min.) to a high acuity triage score (waiting period before medical assessment of max. min.). the final diagnosis was determined after a -day follow-up. we predefined as being adequate a high-acuity triage score (level or ) for acs and a low-acuity score (level , or ) for mild severity diseases. a total of patients was enrolled over a -month period (age . ± . ; m/f ratio . ). triage scores of patients ( . %) with acs were compared to patients ( . %) with mild severity diseases. taking into account cvrf, the sensitivity of the triage system increased from to % whereas the specificity decreased from to %. area under the roc curve (auc) went from . to . (fig. ) . for chest pain triage at ed, addition of cardiovascular risk factors into ecg-based triage increases his diagnostic performance. approximately % of patients presenting to hospital with an intentional overdose require admission to an intensive care unit (icu) [ ] . there are currently no uk guidelines regarding the optimal use of ct head scans (cth) in this patient cohort [ , ] . this study aims to determine whether we should be performing ct head scans in obtunded patients with suspected overdose requiring admission to intensive care. we performed a retrospective search of the icnarc database for plymouth university hospital trust, looking for patients admitted to the icu with overdose or self-poisoning as a primary diagnosis. patients were identified and of these patients required intubation due to obtundation(gcs< ). there were males and females with an average age of years old. the median length of stay on the unit was day. of the patients has a past medical history of mental illness, and overdosed on prescribed medications. the average gcs recorded on admission was . of the ( %) patients had a cth on admission, of which were part of a trauma scan. were known overdoses and were suspected overdose as per the cth request form. the main rationale behind those requests were to exclude additional intracranial injury. none of those cth showed any signs of acute pathology (figure ) . in this retrospective study, obtunded patients with suspected or known overdose with no history of apparent trauma or injury do not benefit from cth. in the absence of a history of trauma or focal neurological signs our conclusions are that cth provides limited value in the management of these patients. the audit was carried out to objectively investigate the problems associated with technique of folley catheterization in emergency department and indoor units of internal medicine wards [ ] . introduction: cellular and molecular mechanisms, epigenetic aspects of acute clozapine poisoning are studied insufficiently. the aim of this study was to identify morphological and epigenetic alteratons in brain neurons during acute exposure to clozapine combined wit ethanol. the experiments were carried out on male wistar rats weighting - g (n= ). group i (control) received . % nacl solution enterally; group iiclozapine mg/kg in . % nacl solution; group iiiclozapine mg/kg in % ethyl alcohol. after hours euthanasia was performed. autopsy included withdrawal of brain samples for histological examination (n = ) and for determination of global dna methylation level (n = ). the global dna methylation level ( -mc%) was determinated by fluorimetric method. inter-group comparisons were made by kruskal-wallis test. histological examination of paraffin sections of brains stained with hematoxylin and eosin was performed by light microscopy. in acute сlozapine poisoning and its combination with ethanol morphological changes in neurons of the cerebral cortex were detected. in acute сlozapine with alcohol poisoning an increase of global dna methylation level was observed. probably the identified changes have a common pathogenesis which will be clarified in our further studies. there is limited information available regarding the prevalence of adder bites and the complications of envenomation. nhs data suggests there are adder bites annually in the uk with the last fatality in [ ] . we performed an audit into adder bites in south west wales to identify the number attending our emergency departments, their management and clinical course as well as any environmental factors that predict increased likelihood of being bitten or the severity of the bite. a retrospective study of adder bites attending emergency departments in south west wales was undertaken (jan to aug ). measurements included were patient demographics, clinical presentation, type of treatment (conservative vs anti-venom) and outcome. results: patients were included, age range - years ( figure ). the majority of bites occurred in sand dunes ( . %) and all bites were on extremities. anti-venom was administered to . % ( / ) of patients. there was a significant positive association between the use of anti-venom and the length of hospital stay (r = . ; p= . ) and a significant negative correlation between the anti-venom use and both diastolic and systolic blood pressure (p= . and . respectively p= . ). all patients fully recovered. in this study, we demonstrated that with a full clinical assessment on presentation it is safe to decide whether anti-venom is required. the current guidelines are safe and effective in the treatment of adder bites. μmol/l, for pao < . kpa and > . kpa, platelets < * ^ /l and > * ^ /l, and bilirubin > μmol/l. in our population of adult ed patients, the thresholds of vital values associated with increased -day mortality were very close to routinely used values, and most of the thresholds were included in the lowest urgency level in triage and risk-stratification scoring systems. the workload in the emergency room: direct assessment by the therapeutic intervention scoring system- and indirect assessment by the nasa task introduction: the number of emergency room admissions continues to increase each year, which increases the care workload of the emergency department staff, who should to use its theoretical and practical knowledge in order to provide quality care in difficult working conditions. the aim of our study was to assess the emergency room staff workload its impact on health workers and patients and to suggest an improvement strategy to decrease this workload. a prospective, monocentric cohort study with descriptive and analytic approach over one month (december ) conducted at the emergency department of an academic hospital. the workload endured by the emergency room staff was evaluated by the nasa task load index and on patients by the therapeutic intervention scoring system- . there were cumulative days of hospitalization in consecutive patients admitted to the emergency room. the average age was ± years. the average length of stay at the emergency room was about ± h. the average tiss- score was . ± . . factors associated with important care workload were: age ≥ years, diabetes, more than comorbidities, the use of intravenous antibiotics; the use of vasoactive drugs and the use of mechanical ventilation; a high tiss score was predictive of emergency room mortality. in the indirect assessment of the care workload, medical and paramedical staff were interviewed, % of them were under years old with a sex ratio of . . a high level of mental and physical workload was expressed by ed staff with considerable level of frustration; the ed staff suggested mainly to improve the working conditions, communication and to redefine tasks "who does what". our study had shown a significant workload in the emergency room, a process to reduce this workload is being implemented medical simulation is a modern teaching tool increasingly used in specialties such as anesthesia, emergency medicine and obstetrics. however, it's not widely used in specialties like cardiology, althought cardiovascular emergencies are very frequent. the purpose of our study was to assess the effectiveness of simulation-based medical education in the management of cardiovascular emergencies among moroccan graduate students. we conducted a prospective, observational, multi-centrer study including the students of three moroccan universities from the th to the th year of medicine who underwent phases: first a pre-test, then a theoretical and practical training on cardiovascular emergencies after which the students were separated in two groups, one undergoing the medical simulation training (group ) and one who didn't (group ), followed by a theoretical then a practical post-test on resusci anne and simman®. at last, the students were asked to answer a satisfaction survey. the reform procedure in the tunisian army consists in repairing the physical damage and deciding on the applicant's ability to continue working. terrorism increases the impact of the co-morbidity generated and the socio-economic consequences that result from it. the purpose of this work was to study the epidemiological, clinical and evolutionary profile of terrorist injuries, to specify the rates of consequent partial permanent disability (ppi) and the possibilities of returning to work. descriptive retrospective cross-sectional study of reform files on military personnel injured during anti-terrorist operations from fig. (abstract ) . changes in total bcpr rate in family-and friends-witnessed ohca cases with dispatcher-assisted instruction during -week period after the day of disaster during three years january to september . the data collection was carried out on the basis of a collection form. our wounded were male, % of whom belonged to the army. the average age was years and months ± . . half of our wounded were troopers. infantry and special forces were the most exposed military units. half of the accidents were recorded in the kasserine region ( cases). chronic post-traumatic stress disorder (cptss) was found in injured, followed by amputations in injured. the after-effects were psychological in %, physical in % and mixed in % of our injured. the ppi rate ranged from % to % in . % of injuries.. more than half of the injured had returned to their professional activity, % were put on reform for health reasons. our results showed that the esptc was the most recorded sequel, and that the ppi rate was significant in a quarter of our injuries. in our series, a third of our wounded were put on reform for health reasons. to state the importance of initial care and adequate and rigorous follow-up to recover a greater number of war wounded. introduction: the rapid response system (rrs) has been shown to decrease hospital mortality [ ] . the japanese coalition for patient safety has set a major goal for hospitals to more widely implement the rrs. however, prevalence and actual circumstances of use in acute care hospitals (including small scale hospitals) in japan are as yet not well-known. web-based questionnaires were sent to acute care hospitals (of scale beds-or-larger) of prefectures in western japan. each participant hospital selected a certain department which answered the questionnaire. the rrs included the medical emergency team (met), the rapid response team (rrt), and the critical care outreach team (ccot). we investigated the presence and circumstances of in-hospital emergency calls, rrs and other systems, and then illuminated issues to be solved. our study suggests that delays in patient transfer to the icu after rrt activation in the wards were associated with slower physiological improvement.these findings support further and larger studies. blood and blood products use in intensive care unit m akcivan, s bozbay, o demirkiran istanbul university cerrahpasa, anesthesiology and intensive care, istanbul, turkey critical care , (suppl ):p blood and blood product (bp) transfusions are frequently used in intensive care units (icu) [ ] . it is important to know transfusion epidemiology and the effect of adverse transfusion reactions and their effect on mortality and morbidity.we aimed to investigate the blood and bp transfusions in the icu. blood and bp transfusions in icu, between - were reviewed retrospectively. we evaluated each transfusion as a data and examined the pre-and post-transfusion laboratory values, demographic data, cause of icu admission and comorbidities. results: patients who underwent transfusion in the icu, and transfusion data from these patients were included. the most frequent cause of hospitalizations were respiratory failure and sepsis. the rate of patients transfused in the five-year period decreased from . % to . %. the hemoglobin threshold before transfusion decreased from . g / dl to . g / dl. a total of transfusion reactions were observed and the most common transfusion reaction was febrile non-hemolytic reaction. the most commonly transfused product was red blood cell suspension. transfusion reactions were found to be slightly higher in men than women in young age group(< y) (p = . and p= . , respectively). transfusion reactions were found to be more frequent in emergency transfusions (p < . ). the number of transfusions was significantly lower in patients with apache ii score < (p < . ). the need for transfusion was found to be higher in patients with hematological malignancy (p < . ). it was observed that as the mean number of transfusions increased the mortality is also increased (p < . ). transfusion therapies are the treatments that are vital but have a serious mortality and morbidity risk. in particular, intensive care patients should be considered in detail because of their specific features. restrictive transfusion practices have positive results. association between anemia or red blood cell transfusion and outcome in oncologic surgical patients. figure a) . the association between rbc transfusion and adverse events also remained after adjustment (or . [ . - . ] ; p < . ) ( figure b) . in oncologic surgical critically ill patients, there was an independent association between anemia (even moderate anemia) or rbc transfusion and patient outcomes. our findings highlight the need for further research to determine the optimal transfusion strategy in surgical oncologic patients. transfusion impaired skin blood flow when initially high e cavalcante dos santos, w mongkolpun, p bakos, al alves da cunha, c woitexen campos, jl vincent, j creteur, fs taccone erasme hospital, intensive care department, brussels, belgium critical care , (suppl ):p red blood cell transfusion (rbct) increases global oxygen delivery (do ) and may improve microcirculation. however, the effects on blood flow have been found to be conflicting. we studied icu patients with stable hemodynamic status (mean arterial pressure (map) ≥ mmhg for at least hours) and without active bleeding, who received a rbct. skin blood flow (sbf) was determined (periflux system , perimed, index finger; perfusion unit, pu) together with map, heart rate (hr), hemoglobin (hb), lactate levels and scvo before and after rbct. sbf was measured before rbct (t ) and after (t ) for each min. according to previous data indicating the lowest sbf value found in noninfected icu patients was pu, all patients were analyzed according to the baseline sbf (i.e. < pu -low sbf vs. ≥ puhigh sbf). the relative change of sbf (Δsbf) was calculated after rbct and the responders were defined by the function of > %. results: icu patients were studied. rbct was associated with increases in map and scvo but no change in sbf. at baseline, scvo was lower in the responders than in the non-responders (p= . ) and lower in patients with low sbf than in the high sbf (p= . ). there was no difference in hb, map, and lactate, between the patients with low and high sbf. after rbct, map rose in the responders (p< . ) and in the non-responders (p= . ), sbf (p< . ) rose in patients with low sbf, and sbf (p= . ) decreased in patients with high sbf. there was a negative correlation between baseline scvo (r= - . , p< . ) or baseline sbf (r= - . , p< . ) and the relative increase in sbf after rbct. rbct increases skin blood flow only when it is impaired at baseline. severe immune dysregulation is associated with adverse outcomes and is common in intensive care unit (icu) patients [ ] . erythropoietin-stimulating agents (esas) have both anti-apoptotic and immune-modulating properties [ ] . despite potential benefit, both the safety and efficacy of these agents remains unclear [ ] . here we evaluate the impact of esas on morality at hospital discharge in critically unwell adult patients admitted to the icu. we conducted our search strategy in accordance with a predetermined protocol. the use of ffp is associated with an increased incidence of complications such as acute respiratory distress and infections, and the rate of complications increased with the quantities of ffp transfused [ ] . pcc contain several important coagulation factors and it has been suggested that they could replace ffp. this has been shown mainly in case reports or series in which coagulation factor deficit was detected by using poc viscoelastic tests in trauma [ ] or traditional hemostatic tests in obstetric patients [ ] . multicenter observational study of the safety and efficacy of the prothrombin complex concentrate. a survey of anesthetists was conducted in maternity hospitals at various levels of care in the russian federation. data has been collected and processed. as a result, patients were analyzed. pph was determined as a volume of blood loss more than ml during vaginal delivery or cs. the most significant risk factors for pph were: preeclampsia or arterial hypertension and a history of postpartum hemorrhage. . % had no risk factors for pph. it was determined that the use of prothromplex iu decreased the number of patients with transfusion ffp - ml/kg by . % and increased the number of patients without transfusion by . %, compared with patients without use of prothromplex iu (figure ). no complications were detected. the use of pcc safety and efficacy reduce use of ffp during pph. the full analysis included patients on either hfc (n= ) or cryoprecipitate (n= ). the intraoperative and postoperative changes in etp and fibrinogen concentration are shown in table . for fibtem a (intraoperatively) and fibrinogen concentration (intraoperatively and postoperatively), the mean numerical values appeared higher with hfc than cryoprecipitate. fxiii (hfc: . %, . %; cryoprecipitate: . %, . %, at baseline and hr after surgery start), fviii and vwf were maintained throughout surgery in both treatment groups. this was also the case for laboratory tests activated partial thromboplastin time, prothrombin time and platelet count. the forma- coagulation parameters analyses showed broad overlaps between hfc and cryoprecipitate, with satisfactory maintenance of the clot quality parameters, fxiii concentrations and thrombin generation parameters. the study group includes men and women with a mean age of , vs. . years (p= . ) admitted with the diagnosis of multiple trauma. we found a directly proportional and highly significant statistical correlation between base excess and fibrinogen level diagnosed using the mcf/fibtem parameter(r= . , p< . )and an inverse proportional correlation between lactate level and fibrinogen level (r= - . , p= . ). in the roc analysis that uses as a variable the level of base excess and as a criterion of classification the fibrinogen deficit (mcf/fibtem< mm) it can be observed that at a value of be<- mmol/l, we can diagnose a fibrinogen deficit with a sensitivity of . % and a specificity of . % (auc= . ,p< . ). lactate appears to be inferior to the excess base (figure ) , but still has a good diagnostic power, a value of . mmol/l has a sensitivity of . % and a specificity of % (auc= . ,p< . ). the difference between the two roc curves ( . ) is statistically significant (p = . ). both base excess and serum lactate can be used to diagnose fibrinogen deficiency with the mention that base excess appears to have a higher sensibility and specificity ability. based goal-directed algorithm. this approach requires further clinical validation. we conducted a retrospective study comparing transfusion strategies in patients with major trauma between and . we retrieved demographic data and blood products administered from patients with at least one red-blood cell (rbc) transfusion. primary outcome was a reduction of rbc administration. secondary outcomes were mortality, icu length of stay and acute kidney injury. we included patients admitted in the icu due to severe trauma (sapsii: . ± . ), and mainly after emergent surgery ( . %). they featured a mean age of . ± . y, were predominantly male ( . %) and % were in shock. in the first hours of hospital admission a mean of . ± . rbc units were administered. most patients received a fibrinogen-based protocol (fbp) ( %), with an average of ± g of fibrinogen and ± fresh-frozen plasma (ffp) units, versus ± g of fibrinogen and ± ffp units in the ffp group. the fbp was associated with a decrease administration of rbcs in the first hours (r = - . ; p < . ), even after adjustment for severity (p= . ) and for tranexamic acid use (p = . ). it was associated also with a decrease of platelet transfusion (p= . ). fibrinogen-based protocol was not associated with a decrease in mortality, acute kidney injury or noradrenaline dose. treatment of tic in past years has progressively changed to a goaldirected fibrinogen-based approach. in our population, the use of fbp lead to a reduction of rbc administration in severe trauma patients. prospective, multicenter, randomized study comparing administration of clotting factor concentrates with a standard massive hemorrhage protocol in severely bleeding trauma patients the objective of this study was to assess the ability of the quantra® qstat® system (hemosonics) to detect coagulopathies in trauma patients. many level trauma centers have adopted whole blood viscoelastic testing, such as rotational thromboelastometry (rotem®, fig. (abstract ) . study treatment plan instrumentation lab) for directing transfusion therapy in bleeding patients. the quantra qstat system is a cartridge-based point-of-care (poc) device that uses ultrasound to measure viscoelastic properties of whole blood. and provides measures of clot time, clot stiffness and a test of fibrinolytic function. methods: adult subjects were enrolled at two level trauma centers which use a rotem based protocol to guide transfusion decisions. study protocols were approved by the site's ethics committee. for each subject, whole blood samples were drawn upon arrival to the emergency department and again, in some cases, after administration of blood products or antifibrinolytics. samples were analyzed on the quantra (at poc) in parallel to rotem delta (in lab). a total of patients were analyzed. approximately % of samples had a low clot stiffness (cs) values suggestive of an hypocoagulable state. the low stiffness values could be attributed to either low platelet contribution (pcs), low fibrinogen contribution (fcs), or a combination ( figure ) . additionally, % of samples showed evidence of hyperfibrinolysis based on the quantra clot stability to lysis parameter. samples analyzed on standard rotem assays showed a lower prevalence of low clot stiffness and fibrinolysis based on extem, fib-tem results. the correlation of cs and fcs vs equivalent rotem parameters was strong with r-values of . and . , respectively. this first clinical experience with the quantra in trauma patients showed that the qstat cartridge detected coagulopathies associated with critical bleeding and may be useful for directing blood product transfusions in these patients. ability to perform testing at poc may provide additional clinical advantage. the objective of the study was to describe the conditions of use of fibryga® g, a new, highly purified, human fibrinogen (hf) recently granted a temporary import authorization for use in congenital and acquired fibrinogen deficiencies in france. observational, non-interventional, non-comparative, retrospective study conducted in french hospital centres using fibryga®. data from patients with fibrinogen deficiency having received fibryga® from december to july were retrieved from their medical files. indications, modalities, efficacy and safety outcomes were recorded. indications encompassed non-surgical bleeding (nsb) either spontaneous or traumatic, including post-partum hemorrhage (pph), bleeding during surgery (sb) or administration to prevent bleeding during planned surgery. treatment success was defined as control of the bleeding or hemoglobin loss < % for bleeding treatment and as absence of major perioperative hemorrhage for pre-surgical prevention. this analysis included patients aged , ± . years and % were male. all presented an acquired fibrinogen deficiency requiring administration of hf. indications were nsb (n= , . %) including ( . %) pph, sb (n= , . %), and prevention of sb (n= ; , %). cardiac surgeries were the main procedures associated with treatment and prevention of sb. mean total doses of fc were . ± . g, . ± . g and . ± . g for nsb, sb and prevention of sb. success rates were . % ( %ci . - . %), . % ( %ci . - %) and . % ( %ci . - %) respectively. for pph, mean dose of hf was . ± . g with a success rate of . % ( %ci . - %). overall, tolerance was good. fibrinogen concentrate fibryga® is mostly used for bleeding control. in one third of patients, hf was administered preventively to avoid bleeding during surgery. use of fibryga® was associated with favourable efficacy outcomes. functional testing for tranexamic acid effect duration using modified viscoelastometry t kammerer , p groene , s sappel , p scheiermann , st schaefer ruhr-university bochum, institute of anaesthesiology, heart and diabetes center nrw, bad oeynhausen, germany; ludwig-maximilans university, department of anaesthesiology, munich, germany critical care , (suppl ):p tranexamic acid (txa) is the gold standard to prevent or treat hyperfibrinolysis [ ] . effective plasma concentrations are still under discussion [ ] . in this prospective, observational trial using modified viscoelastometry we evaluated the time-course of the antifibrinolytic activity of txa in patients undergoing cardiac surgery. methods: patients were included. modified viscoelastometry (tpa-test) was performed and txa-plasma-concentration, plasminogen-activatorinhibitor- (pai- ) and pai-antigen-plasma-concentrations were measured over h. additionally, in vitro dose-effect-curves from blood of healthy volunteers were performed. data presented as median with interquartile range (q /q ). results: txa plasma-concentration was increased compared to baseline (t : μg ml - ) at every time-point with a peak concentration min (t ) after application (p< . ; see fig. a ). lysis was inhibited from min (lysistime tpa-test : p< . ; lysisonsettime tpa-test :p< . ). maximumlysis tpa-test was decreased at t (t : % ( / ) vs. t : % ( / ); p< . ). of note, after h some patients (n= ) had normalized lysis whereas others (n= ) had strong lysis inhibition (ml< %;p< . ) up to h. high and low lysis groups differed regarding kidney function (cystatin c: . mg l - ( . / . ) vs. . mg l - ( . / . );p= . ) and active pai- ( . ng ml - ( . / . ) vs. . ng ml - ( . / . );p= . ). in-vitro, txa concentrations > μg ml - were effective to inhibit fibrinolysis. in our trial, after h there was still completely blocked lysis in patients with moderate renal impairment. this could be critical with respect to postoperative thromboembolic events [ ] . here modified viscoelastometry could be helpful to detect the individual fibrinolytic capacity. introduction: peri-operative coagulopathy correction based on viscoelastic hemostatic assays (vhas) and single-factor coagulation products has changed the paradigm of bleeding management in cardiac surgery [ ] . in a retrospective study, we analysed patients with emergency surgery for thoracic acute aortic dissection (taad), before and after the introduction of fibrinogen concentrate in clinical practice. data were collected from paper and electronic records. the study was approved by the institutional ethical committee. patients were included in the analysis, operated in , before fibrinogen concentrate was approved for human use, and in - . therapy was guided by a rotational thrombo-elastometry (rotem) algorithm. exclusion criteria were non-compliance with the institutional protocol and intra-operative death. we investigated allogeneic blood transfusion (abt), fibrinogen use, peri-operative bleeding (pob), surgical reexploration and post-operative complications (poc). the groups were similar in gender, age, body weight, additive euro-score and aortic cross-clamp time. fresh frozen plasma, cryoprecipitate and red blood cell transfusion were lower in the fibrinogen group, but not platelet transfusion (table). , % of patients in the study group received fibrinogen concentrate and median dose was g (iqr - ). day postoperative chest tube drainage and surgical reexploration were significantly lower. there were no differences in stroke, renal replacement therapy, mechanical ventilation time and icu stay. in patients with taad surgery, rotem-guided algorithms which include fibrinogen concentrate are associated with less (pob), surgical re-exploration and abt. further research is needed to document the role of vhas and concentrated factors in reducing (poc). andexanet alfa (aa, portola pharmaceuticals, san francisco, ca) represents a modified factor xa agent which is approved antidote for apixaban and rivaroxaban. andexanet alfa may also neutralize the anti-xa effects of betrixaban and edoxaban. this study aims to compare the relative neutralization of these four anti-xa agents by andexanet alfa in different matrices. andexanet alfa was diluted at mg/ml. apixaban (a), betrixaban (b), edoxaban (e) and rivaroxaban (r) were diluted in ph . , . m tris buffer (tb), blood bank plasma (bbp) and in % albuminated buffer (ab) at . - . ug/ml. anti-xa activities of all four agents were measured in three systems and the reversibility indices of aa were profiled. the reversibility index (ri ) of anti-xa effects by aa was determined at - ug/ml. each of the four agents produced varying degrees of inhibition of anti-xa at . - . ug/ml, the ic ranged . - . ug/ml in bbp, . - . ug/ml in ab and . - . ug/ml in tb. andexanet alfa produced a concentration dependent reversal of all four anti-xa agents. in the bbp, the ri values for a ( ug/ml), b ( ug/ml), e ( ug/ml) and r ( ug/ml). in the ab, the ri values for a ( ug/ml), b ( ug/ml), e ( ug/ml) and r ( ug/ml). in the tb, the ri values for a ( ug/ml), b ( ug/ml), e (> ug/ml) and r ( ug/ml). each of the four anti-xa agents exhibit varying degrees of matrix independent anti-xa potencies in different systems, the collective order follows edoxaban > apixaban > betrixaban > rivaroxaban. andexanet alfa produced matrix dependent differential neutralization of the anti-xa effects of these agents. individualized dosing of andexanet alfa may be required to obtain desirable clinical results. the diagnostic and prognostic value of thromboelastogram (teg) in sepsis has not been determined. this study aimed to assess whether teg is an early predictor of coagulopathy [ , ] and is associated with mortality in patients with sepsis. in total, patients with sepsis on intensive care unit admission were prospectively evaluated. we measured teg and conventional coagulation tests(ccts)on preadmission and observed for development of , days and , , days respectively. multivariable logistic regression was utilized to determine odds of icu/hospital mortality. the parameter of teg (maximum amplitude, reaction time; ma/r ratio) was calculated to evaluate sepsis-induced coagulopathy. the admission patients were divided into three groupsma/r group(ma/r= - mm/min); ma/r group(ma/r> mm/min)and ma/r group(ma/r< mm/min). in our cohort of patients with severe sepsis, coagulopathy defined by ma/r ratio was associated with increased risk of icu/hospital mortality. introduction: blood sampling for coagulation assessment is often carried out in either arterial or venous samples in the intensive care unit (icu). there is controversy as to the accuracy of this method due to the inherent differences in physicochemical properties as well as the underlying effects of individual diseases in arterial and venous blood. clot microstructure has shown to be a new biomarker (fractal dimension-d f ) which encompasses the effects of diseases in all aspects of the coagulation system [ , ] . in this study, we compared the effect of all these factors in venous and arterial blood to see if there is a difference in the clot microstructure and quality. patients admitted to a tertiary intensive care unit and busy teaching hospital were recruited. arterial and venous blood was sampled from an arterial line and central venous catheter in situ from the same patient. standard markers of coagulation (pt, aptt, fibrinogen, full blood count), rotational thromboelastometry (rotem), whole blood impedance aggregometry and measured clot microstructure (d f ) were measured on both arterial and venous samples. no significant difference was observed in standard laboratory markers, rotem and platelet aggregation between arterial and venous blood. there were no differences in the fractal dimension (d f ) between the arterial and venous blood samples (d f . ± . vs . ± . respectively, p= . ). samples from patients with critical illness give comparable results from either arterial or venous blood despite their underlying pathophysiological process or treatment. this confirms blood for coagulation testing can be taken from arterial or venous blood. clinicians in the emergency setting use a wide range of hemostatic markers to diagnose and monitor disease and treatment. current methods rely on the anticoagulant effect of citrate on whole blood prior to laboratory analysis. despite the well-recognized modulatory effects of citrate on hemostasis, the use of anticoagulated blood has clear analytical advantages, including repeat sampling and storage. however by altering the physiological state of the blood reproducibility and accuracy of the test is affected. recent studies have shown the potential of a novel functional biomarker of clot formation: fractal dimension (d f ), that may give an improved diagnostic accuracy. in this study we assessed the potential of this new biomarker in scientifically measuring the effects of recalcification of citrated samples. methods: healthy volunteers were included. unadulterated and sodium citrate samples of blood were taken from each volunteer. citrated samples were recalcified using ( m cacl ). in the study we compared unadulterated whole blood d f results to citrated d f results and repeated the citrated d f experiments times for each sample over a hour period to ascertain reproducibility. the d f of citrated blood was significantly lower than that of unadulterated blood ( . ± . vs . ± . , p< . ). the results of the citrate samples when tested times over hrs gave a coefficient of variation of . %. for the first time we show that a functional biomarker of clot microstructure, d f , can precisely quantify and measure accurately the direct effect that the addition of the anticoagulant sodium citrate has on whole blood clot microstructure. the study also shows that the test is reproducible and has potential utility as a biomarker of acute disease in the emergency setting in citrated blood. this procedure now needs to be evaluated in a group of acute disease states. in this study, we analyzed the hematological abnormalities of dengue patients by thromboelastography (teg) at initial and -hour of fluid resuscitation. methods: this is a cross-sectional study evaluating teg readings of dengue patients with different severities presenting to the emergency department. laboratory confirmed dengue patient (positive ns antigen or igg/igm) was consecutively sampled. teg readings were taken at presentation and after -hour of fluid resuscitation. twenty dengue patients with varying severity had a median reaction time (r), α -angle, k time, maximum amplitude (ma) and lysis % (ly ) of . min, . ο , . min, . mm and . % respectively. mean fibrinogen was normal before and after fluid infusion. there is a non-significant reduction in ma with prolongation of other teg parameters between different dengue severities. there is a statistically significant reduction of α-angle and ma between pre and post -hour fluid resuscitation (p= . and p= . ). normal fibrinogen with low ma, which signifies a weak clot strength, may indicate either a platelet reduction, platelet dysfunction or both. reduction in ma and α-angle post fluid resuscitation is an alarming finding. this is in contrast with previous teg studies although none of it used normal saline exclusively, studied initial fluid resuscitation in emergency department settings or studied a subject with dengue. a bigger study, especially in severe dengue is needed to validate our findings. agreement between the thromboelastography reaction time parameter using fresh and citrated whole blood during extracorporeal membrane oxygenation with teg® and teg® s m panigada, s de falco, n bottino, p properzi, g grasselli, a pesenti fondazione irccs ca´granda ospedale maggiore policlinico, intensive care unit, milano, italy critical care , (suppl ):p the r (reaction time) parameter of kaolin-activated thromboelastography (teg) may be used to assess the degree of heparinization of blood during ecmo. a teg analysis is usually performed on two types of samples: fresh (f) or citrated-recalcified (c) whole blood. teg® can perform the analysis on c and f whole blood, the new teg® s (haemonetics corp., ma, usa) only on c whole blood. aim of the study was to compare the response of r to heparin using the two types of samples and two teg devices methods: during a three months period at fondazione irccs ca' granda -policlinico of milan, teg was performed (using teg ® and teg s® with and without heparinase, an enzyme that degrades heparin) on consecutive ecmo patients (as part of the gatra study, nct ) and in consecutive non-ecmo patients in whom a teg was requested for clinical purposes. bland altman analysis and lin's concordance correlation coefficient were used to assess agreement results: a total of paired samples were taken ( in-ecmo and off-ecmo). ecmo patients received . ( . - . ) iu/kg/h of heparin. among non-ecmo patients, of them did not receive any dose of heparin, two of them a very low prophylactic dose ( . and . iu/ kg/h, respectively), and one of them . iu/kg/h of heparin. using teg® , r was - . (- . ; . ) min shorter on c compared to f blood in patients receiving heparin (this difference disappeared using heparinase) and only - . (- . ; . ) min shorter in patients notreceiving heparin. r was - . (- . ; . ) min shorter using teg® s (which performs the analysis only on c blood) than teg® on f blood (figure ) . when evaluating the effect of heparin using teg, clinicians should be aware that results obtained using citrated-recalcified or fresh whole blood are not interchangeable. using citrated-recalcified blood to perform teg might lead to underestimation of the effect of heparin trauma patients are at high risk for venous thromboembolism (vte). the east guidelines recommend low molecular weight heparin (lmwh) for vte prevention and antixa monitoring after initiation of the medication or after adjusting doses in certain populations [ ] . studies have shown standard enoxaparin dosing of mg every hours may result in low antixa levels [ ] . this study aims to evaluate the efficacy of a pharmacist-lead protocol for adjusting enoxaparin dosing based on antixa levels in trauma patients. this single center retrospective chart review included adult trauma patients admitted from / / to / / . per protocol, patients with body mass index (bmi) ≤ kg/m were initiated on enoxaparin mg twice daily, and patients with bmi > kg/m were initiated on enoxaparin mg twice daily. peak antixa levels were drawn to hours after at least the third dose of enoxaparin with a goal therapeutic range of . - . iu/ml. the primary objective was time in days to goal peak antixa level. secondary objectives include vte occurrence, bleeding attributed to lmwh, and dosing regimens utilized. subgroups were analyzed based on body mass index (bmi). of patients identified, patients met inclusion criteria. median time to therapeutic antixa level was days (iqr - ). of patients fig. (abstract ) . agreement between teg® s and r teg® on citrated recalcified and fresh whole blood with bmi ≤ kg/m , patients ( . %) were dosed initially per protocol and / patients ( . %) met goal antixa level at first check (table ) . of patients with bmi > kg/m , patients ( . %) were dosed initially per protocol and / patients ( . %) met goal antixa level at first check. our results indicate the protocol is safe due to lack of bleeding attributed to enoxaparin, but less than % of patients achieved goal antixa level at first check. however, despite low rates of achieving goal antixa level, vte rates also remained low. introduction: most patients in the icu are given prophylactic anticoagulation with a fixed dose of mg once daily of enoxaparin (clexane) if cct is normal and mg if cct is low. studies on non icu patients have shown that afxa is below desired range for venous thromboembolism (vte) prevention. in the icu, many factors might influence afxa levels including weight, creatinine clearance (cct), shock and other medication. atxa activity was not yet reported in a big mixed icu population with variable morbidity. our study hypothesis is that enoxaparin is underdosed in most cases and routine afxa activity should be monitored in all icu patients. preventive enoxaparin ( mg qd) was given to all patients unless therapeutic dose was needed or contraindication existed. levels of afxa activity were taken hours after the rd dose. therapeutic vte preventive effect was defined as afxa activity of . - . . patient data was collected from medical files. the study is still ongoing, preliminary results were analyzed for patients. of patients ( %) had afxa activity below normal (subtherapeutic). weight and cct were negatively correlated with afxa activity (figure ). mean weight in the subtherapeutic afxa was significantly higher than the therapeutic group ( . vs. . respectively, p= . ). cct in the subtherapeutic afxa was significantly higher than the therapeutic group ( . vs. . respectively, p= . ). the normal cct group (> ) had significantly more patients with subtherapeutic afxa ( vs , p= . ). in our icu, % of the patients receive insufficient vte prophylaxis. overweight patients and patients with normal cct should probably receive higher enoxaprin dose. afxa activity should be routinely monitored in icu patients. in this study we use a new bedside biomarker to test its ability to measure anticoagulation effects on patients who present with acute first time deep vein thrombosis (dvt). dvt requires oral anticoagulants to prevent progression to potentially fatal pulmonary embolism and recurrence. therapeutic efficacy monitoring of direct oral anticoagulants (doac) including rivaroxaban is problematic as no reliable test is currently available. advances in hemorheological techniques have created a functional coagulation biomarker at the gel point (gp) which allows quantitative assessment of: time to the gel point (t gp ), fractal dimension (d f ) and elasticity (g') [ , ] . the prospective observational cohort study measured t gp , d f , g', standard coagulation and cellular markers in first time dvt patients at three sample points: pre-treatment and approximately and days following mg bd and mg od rivaroxaban respectively. strict inclusion and exclusion criteria applied. results: dvt patients (mean age years [sd± . ]; male, female) and non-dvt patients were well matched for age, gender and co-morbidities. mean t gp on admission was s (sd± . s) and . s (sd± . s) for dvt and non-dvt respectively. doac therapy significantly increased t gp to . s (sd± . s) after days, and subsequently increased to . s (sd± . s) at days as shown in table . d f , g' and standard hemostatic markers all remain within the normal range. conclusions: t gp demonstrates its utility in determining the anticoagulant effect of rivaroxaban. the significant difference in t gp between males and females needs further exploration. localized stasis as a result of transient provoking factors appears not to generate a systemic strength fig. (abstract p ) . correlation of anti factor xa activity with patient cct and weight. anti fxa activity value below . (red line), was considered "non-effective prevention" introduction: trauma remains the leading cause of death all over the world. to better exploit the trauma care system, precise diagnosis of the injury site and prompt control of bleeding are essential. here, we created a nursing protocol for initial medical care for trauma. the aim of this study was to evaluate the impact of protocoled nursing care for trauma on measures of quality performance. this was a retrospective historical control study, consisted of consecutive severe trauma patients (injury severity score > ). people were divided into two groups: protocoled group (from april to march ) and control group (from april to march ). we set the primary endpoint as mortality for bleeding. the secondary endpoints included time allotted from arrival to start of ct scan and surgery, administration rate of several drugs (sedations, painkillers, preoperative antibiotics, and tranexamic acid). for the statistical analysis, continuous variables were expressed as median (interquartile range) and were compared by wilcoxon rank sum tests given a nonnormal distribution of the data. we included patients in the study: in the control group before the introduction of the protocol, in the protocoled group. as a primary endpoint, the mortality for bleeding was similar between two groups ( % in the control group and % in the protocoled group). as a secondary endpoint, the time to ct initiation [group a ( - ) min vs group b ( - ) min; p < . ], and emergency procedure [group a ( - ) min vs group b ( - ); p < . ] were shortened by the protocol introduction. furthermore, the administration rates of sedations, painkillers, preoperative antibiotics, and tranexamic acid were increased in the protocoled group compared with the control group. although the mortality as a patient-oriented outcome was not affected, improved quality of medical care by nursing protocol introduction may be suggested in this analysis. this single-institutional prospective study included patients with uprf who were admitted to the trauma surgical intensive care unit (tsicu) and survived until discharge to home between and . we evaluated the activities of daily living after the discharge using physical and mental component scores of sf- ® and defined physical dysfunction (pd) as physical function (pf-n) score of or less. we divided the patients in the pd (n= ) and control (without pd, n= ) groups and compared the groups. the patients had experienced blunt injuries, including falls ( %) and pedestrian injuries ( %). the mean age was . years (men: . %); the median injury severity score was (interquartile range: - ); and the mean length of tsicu stay was . days. the average period from the injury until the survey was . months. there was no difference between the pd group and the control group in the patient characteristics, fracture type, pelvic fixation, and complications. at the time of the survey, the pd group had significantly more painful complaints than the control group (pd: . %, c: . %, p < . ), and had more physical and mental problems. the sf- ®subscale score showed a significant positive correlation between physical function and body pain, mental health respectively. the percentage of those who were able to return to work was not different in both groups (pd: . %, c: . %). in the multivariate analysis of pd, only age (odds ratio: . , % ci: . - . , p = . ) was relevant. long-term pd was observed in % of patients with uprf. the elderly were particularly prominent, and there was an association between pain and mental health. cells (rbc) this can lead to inhibition of oxygen transport function and development of hypoxia. currently used methods for analyzing the state of rbc either do not have sufficient accuracy or require lengthy analysis and expensive equipment. the use of a simpler and more informative electrochemical approach to assessing the state of rbc is very promising. electrochemical measurements in rbc suspensions (~ • cells / l) were carried out in a special electrochemical cell [ ] in the potentiodynamic mode in the potential range from - . to + . v using the ipc pro mf potentiostat (kronas, russia); optical measurements were performed using an eclipse ts inverted microscope (nikon, japan), a cfi s plan fluor elwd x / . lens (nikon, japan); rbc morphology was recorded in real time using a ds-fi digital camera (nikon, japan). when examining rbc of patients with severe multiple trauma a decrease in the ability of rbc to change their shape during electrochemical exposure was observed, indicating a decrease in deformability, which can lead to a disruption in the oxygen supply to tissues. at the same time, with the stabilization of the patient's condition a restoration of the ability of rbc to change morphology was detected which in turn could have a positive effect on the rheological characteristics of the blood (fig. ) . the results of the analysis of red blood cells using electrochemical changes in their morphology can be used as an additional method for the diagnosis of critical conditions. severe trauma should be treated immediately. whole-body ct (wbct) is widely accepted to improve the accuracy of detecting injuries. however, it remains the problem of time-consuming. therefore, we focused on the scout image taken in advance of wbct. detecting major traumatic injuries from a single scout image would reduce the time to start treatment. a previous study suggested that even specialists could not easily find chest and pelvic injuries using wbct scout image alone. in this study, we aimed to develop and validate deep neural network (dnn) models detecting pneumo/hemothorax and pelvic fracture from wbct scouts. we retrospectively collected anonymous wbct scouts together with their clinical reports at the osaka general medical center between january , , and december , . we excluded incomplete, younger than years old, postoperative, and poorly depicted images. the part of this dataset from january , , until december , , was used for validation and the rest for training dnn models. pneumo/hemothorax detection model and pelvic fracture detection model were trained respectively. accuracy, and areas under the receiver operating characteristic curves (aucs) were used to assess the models. the training dataset for pneumo/hemothorax contained images (mean age years; % female patients), and for pelvic fracture consisted of images ( years; %). the validation dataset for the former contained images ( years; %), and for the latter consisted of images ( years; %). the models achieved % accuracy and an auc of . for detecting pneumo/hemothorax, % and . for pelvic fracture. our results show that dnn models can potentially identify pneumo/ hemothorax and pelvic fracture from wbct scouts. increasing the number of samples, dnn model could accurately detect severe trauma injuries using wbct scout image. clinical information system (cis) is a computer system used in collecting, processing, and presenting data for patient care. it can reduce staff workload and errors; help in monitoring quality of care; track staff's compliance to care bundles; and provide data for research purpose. however, the transition from paper record format to electronic record involves changes in all kind of workflow in icu. therefore, an effective, efficient and evaluative rollout plan was required to minimize the risk that might arise from the new practice. methods: . small groups training were provided. a working station with different case scenarios were set up for practices. . individual tutorials were conducted to clarify questions. emphasis on patient care was always top priority. . contingency plans were available in case of server breakdown and power failure. downtime drills were conducted to prepare the staff in emergency situations. . step-by-step transition from paper record to electronic format was gradually carried out. a plan was discussed among cis team with clear dates and goals. . new items in cis were first reviewed and amended in team meeting until consensus was made; then were promulgated to all staffs during handover before implementation. fig. (abstract p ) . the effect of therapy on the electrochemically induced change in the morphology of red blood cells in patients with combined trauma . staff compliance and outcomes were then monitored; further review and amendment would be possible if necessary. cis roll-out plan was smooth. all staffs were able to integrate cis into the daily routine. the contingency plans were well acknowledged. new items were followed as planned. ongoing enhancement in cis was put forward on nursing orders, handover summary, and integration with inpatient medication order entry (ipmoe) system. with emerging benefits cis brings along, our staff has more time to devote to direct patient care. human input in data interpretation and clinical judgment on top of cis play an irreplaceable role in patient care. the daily request for laboratory tests in intensive care units is a common practice. although common, this strategy is not supported, since more than % of the exams requested with this rationale may be within the normal range [ ] . misconduct based on misleading results, anemia, delirium and unnecessary increase in costs may happen [ ] . we have developed a strategy to reduce laboratory tests without clinical rationale. observational retrospective study, from july to june . the number and type of laboratory orders requested, the epidemiological profile of hospitalized patients, the use of advanced supports, the average length of icu stay and the impact in outcomes such as mortality and hospital discharge at a private tertiary general hospital in the city of rio de janeiro / rj -brazil were analyzed. a strategy was implemented to reduce the request for exams considered unnecessary. approximately , patients underwent icu during this period. the epidemiological profile and severity of patients admitted to the unit were similar to those observed historically. there was a significant reduction (> %) in the request for laboratory tests and there was no negative impact on outcomes such as mortality, mean length of stay and no greater use of invasive resources. over the period evaluated, the estimated savings from reducing the need for unnecessary exams were approximately $ , per year. the rational use of resources in the icu should be increasingly prioritized and the request for routine laboratory tests reviewed. a strategy that avoids such waste, when properly implemented, enables proper care, reducing costs and ensuring quality without compromising safety. evaluating the medication reconciliation errors in icus after implementing a hospital-wide integrated electronic health record system a rosillette, r shulman, y jani university college hospital, centre for medicines optimisation research and education, london, united kingdom critical care , (suppl ):p introduction: medication errors in intensive care unit (icu) are frequent [ ] and can arise from a number of causes including transition of care. our aim was to investigate the impact of an integrated electronic health record system (ehrs) on medication reconciliation (mr) errors occurring at critical steps: during the transition from an icu to the hospital ward and from the ward to hospital discharge. the objective was to examine the influence of icu admission on long-term medication. we performed a monocentric study in icus of a university-affiliated hospital using drug chart and medical notes review to identify mr errors before, during and after icu admission. data were collected retrospectively from ehrs for consecutive patients discharged from the icu between june- july , and who were newly initiated on specific drugs of interest. results: drugs of interest were initiated in icu. many of these were continued after hospital discharge as shown in table . there was appropriate discontinuation of all the antipsychotics newly initiated in icu. other than anticoagulants, there was no reason documented for continuation of the initiated drugs. the planned durations were documented more often after hospital discharge than icu discharge for the following drug classes (% of patients with a plan after icu discharge to the ward; % after home discharge): antibiotics ( . %; . %), and steroids ( . %; . %), but less so for analgesics ( . %; . %), insomnia ( . %; . %), and gastroprotective drugs ( . %; . %). our study has shown that medications initiated in the icu can be inadvertently continued at icu and hospital discharge due to failure in documenting indication or duration. systems are required to deprescribe icu only drugs at discharge or communicate a plan for ongoing treatment. introduction: the surviving sepsis campaign advocates the use of care bundles to guide the management of sepsis and septic shock [ ] . our study aim was to assess compliance with a locally introduced sepsis pathway and to review intensive care unit admission outcomes. we carried out a prospective audit of patients admitted to the icu at royal surrey county hospital with a diagnosis of sepsis between / / and / / , assessing compliance with local sepsis bundle delivery, outcome of icu admission and degree of associated organ dysfunction. results: patients were identified, male ( . %), with a mean age of . ( - ). mean st hour sofa score on icu was . ( - ). % of patients required vasopressors, with % requiring noradrenaline > . mcg/kg/min, and % requiring an additional vasopressor/ inotrope. % required niv, % invasive ventilation and % rrt. icu mortality was %, in-hospital mortality %, mean icu stay days ( - ), and mean length of hospital stay days . in the presence of septic shock mortality was % with post-resuscitation lactate > , versus % in patients with no vasopressor requirement or lactate < (p< . ). the sepsis bundle was delivered in one hour to patients ( %). where the bundle wasn't completed, antibiotics were delayed in % of cases and blood cultures weren't taken in %. where the bundle was fully delivered, unit mortality was % vs. % where it was not (p< . ), but there was no significant difference in hospital mortality ( % vs. %, p> . ) or rates of vasopressor requirement, niv, ippv or rrt. there is room for improvement in timely delivery of the sepsis bundle in our hospital and various measures are being instituted. though there was no significant difference in hospital mortality, icu mortality was significantly lower in patients when the bundle was fully delivered. surviving sepsis campaign recommends h and h sepsis resuscitation bundle for sepsis. the study was done to assess the feasibility of the guideline and the compliance to sepsis- recommendations at an emergency department. prospective interventional study was conducted during one year. were involved in the study all sepsis cases with a qsofa ≥ . were assessed a composite of six components (measurement of serum lactate, obtaining blood culture before antibiotic administration and provision of broad-spectrum antibiotic before the end of h and provision of fluid bolus in hypotension, attainment of target central venous pressure assessed by cardiac ultrasonography, target lactate to normal level before the end of h ). time base line was the first medical contact at triage zone. secondary outcomes of study were the mortality rate and length of stay at intensive care unit (icu). were involved in the study, patients (mean age ± years, sex ration , ). pulmonary infections were the main cause of sepsis ( %) and urinary tracts infections ( %). at h components were achieved in % of cases [lactates ( %), blood culture ( %) and provision of antibiotics ( %)]. at h components were executed in % of cases (fluid provision achievement in %, ultrasonography assessment in % and normal lactate target achieved in %) (figure ). the reliability-adjusted rate for completion of the hours and hours bundle was at %. patients compliant to composite bundle got the mortality benefit (odds ratios = . , % [confidence interval, . - . ]). the study, however, did not show any benefits of mean intensive care unit (icu) length of stay. faisability of - h bundle ratio was at %. it has shown a significant improvement in adaptation and mortality benefit without reducing mean hospital/icu length of stay. more adapted procedures are needed to improve results targeting full compliance of patients to the - h bundle sepsis management. patterns and outcome of critical care admissions with sepsis in a resource limited setting m edirisooriya maddumage , y gunasekara , d priyankara national hospital of sri lanka, medical intensive care unit, colombo , sri lanka; sri jayawardenepura general hospital, department of critical care, nugegoda, sri lanka critical care , (suppl ):p introduction: paucity of epidemiological data is a major barrier in expansion of critical care services, especially in resource limited settings. we evaluated the patterns and the outcome of critically ill patients with sepsis admitted to a level medical intensive care unit in sri lanka. a retrospective cohort study was performed to describe the characteristics and outcome of patients with sepsis, admitted to a medical intensive care unit. sepsis is defined according to sepsis definition. we examined critically ill patients admitted over a period of months. sepsis was the commonest presentation, accounted for . % of all admissions. mean age was . ± . years. septic shock was present in . % on admission. pneumonia ( . %) was the commonest cause, while leptospirosis ( . %) and meningoencephalitis ( . %) accounted for fig. (abstract p ) . sepsis - h bundle components (% of goals achievment) second and third commonest causes of sepsis respectively. the sofa score on admission ( . ± . vs . ± . , p< . ), occurrence of aki ( % vs . %, p< . ) and the length of icu stay ( . days vs . days, p < . ), were significantly higher in sepsis than in patients without sepsis. icu mortality in sepsis (n= ) did not show a significant difference to nortality (n= ) in those without sepsis ( % vs %, p= . ). patients with leptospirosis had a mean sofa score of . , however the mortality ( . % vs %, p = . ) was similar to others with sepsis. in contrast, mortality related to sepsis was significantly high ( %, p< . ) in the packground of immunosuppression (n= ). respiratory failure secondary to pneumonia was the commonest cause of critical care admission with sepsis. sepsis related icu mortality was high in the background of immunosuppression. introduction: training in placement, and the subsequent safe confirmation of position, of a nasogastric (ng) tube, relies on clinicians completing an e-learning module at our trust. feeding through an incorrectly placed ng tube is a 'never event,' associated with significant morbidity and mortality [ ] . analysis of these incidents reveal that the misinterpretation of chest radiographs, by medical staff, who had not received competency-based training, is the most frequent cause [ ] . e-learning has revolutionized the delivery of medical education [ ] , however, there are barriers to its use [ ] . we hypothesized that, by taking e-learning content, and delivering it face-to-face, we would improve training rates, and thus patient safety. a questionnaire was completed by critical care doctors, concerning their knowledge of the existence of the e-learning module, whether they had completed formal training in ng tube placement, and how confident they were, on confirming correct positioning, using a point likert scale. all clinicians underwent training in the interpretation of ng placement, using chest radiographs. after the session they were asked to re-appraise how confident they felt. results were compared using paired t tests. confidence improved in all, rising from a pre-test average score of . (sd= . ), to post-session . (sd= . ), p=< . . prior to the intervention, % of the doctors were aware of the trust guidelines, but only % had completed the training. after the session, % were aware of the guidelines, and % had completed the training (figure ) . conclusions: e-learning is a useful tool, but has its limitations. by using course content, delivered with more traditional learning methods, we im-proved the number of appropriately trained clinicians, and thus the safe use of ng tubes in our unit. a systematic review of anticoagulation strategies for patients with atrial fibrillation in critical care a nelson, b johnston, a waite, i welters, g lemma university of liverpool, liverpool, united kingdom critical care , (suppl ):p there is a paucity of data assessing the impact on clinical outcomes of anticoagulation strategies for atrial fibrillation (af) in the critical care population. this review aims to assess the existing literature to evaluate the effectiveness of anticoagulation strategies used in critical care for atrial fibrillation. only studies contained analysable data. anticoagulated patients had a lower mortality at days and days post admission to critical care, however there was an increased incidence of major bleeding events compared to the non-anticoagulated population. thromboembolic events were comparable in both cohorts. data from current literature is scarce and inferences regarding the effectiveness of anticoagulation in patients in critical care with af requires further investigation and research. every new admission to the icu prompts a handover from the referring department to the icu staff. this step in the patient pathway provides an opportunity for information to be lost and for patient care to be compromised. mortality rates in intensive care have fallen over the last twenty years, however, % of patients admitted to an icu will die during their admission [ ] . communication errors contribute to approximately two-thirds of notable clinical incidents; over half of these are related to a handover [ ] . nice have concluded that structured handovers can result in reduced mortality, reduced length of hospital stay and improvements in senior clinical staff and nurse satisfaction [ ] . a checklist was created to review the information shared and to score the handover. this checklist was created with doctors and nurses and is relevant for handovers between all staff members. information was gathered prospectively by directly observing handovers on the icu. there is a notable discrepancy in the quality of handovers of new patients ( figure ). this is true of handovers between doctors, nurses and a combination of the two. it is also true of all staff grades. whilst a doctor may have reviewed the patient prior to their arrival, % (n= ) of patients weren't handed over to a doctor. the most commonly missed pieces of information were details of the patient's weight ( %, n= ), their height ( %, n= ), whether the patient has previously been admitted to an icu ( %, n= ) and whether the patient has any allergies ( %, n= ). the handover of new patients to the icu is often unstructured and important information is missed. this can be said for all staff members and grades, and for handovers from all hospital departments. post intensive care syndrome-family (pics-f) describes new or worsening psychological distress in family and caregivers after critical illness but remains poorly studied within specialist groups [ ] . we aim to define the degree of pics-f within our tertiary referral cardiothoracic centre and map change over the course of months. caregivers attended a -week multi-professional clinic alongside patients. peer support was facilitated through a café area and a caregiver group psychology session was offered with individual appointments if required. caregiver surveys were completed including: caregiver strain index; hospital anxiety and depression scale (hads); and insomnia severity index. patients also completed hads questionnaires. repeat surveys were completed at and months. results: over cohorts, caregivers attended, of which were spouses ( %), children ( %), and others ( %), with caregivers completing surveys at months. patients' median apache score was (iqr - . ) and median icu length of stay was days (iqr - . ). most admissions were from scheduled operations ( %). severe caregiver strain was present in / ( %) with changes to personal plans ( %) the most common sub category. hads demonstrated caregivers ( %) with anxiety and ( %) with depression. caregiver anxiety exceded that of patients', only reaching fig. (abstract p ) . each handover was scored according to the information accurately given to icu staff similar levels at months, while depression remained static ( figure ). median number of nights with 'bothered' sleep was (iqr - . ) and % of caregivers expressed problems with sleep. conclusions: significant psychological morbidity in caregivers from our tertiary cardiothoracic centre is in keeping with the general icu population [ ] . caregiver strain was reduced suggesting higher levels of resilience. future work should address mental wellbeing, particularly anxiety, to minimise the effects of pics-f. burnout syndrome is an illness that has increasingly affected health professionals. it is characterized by great emotional stress, physical and mental exhaustion and depersonalization of the individual. more serious cases can lead to job loss or even suicide. the described work identifies the burnout level of the multidisciplinary team through a specific questionnaireburnout syndrome is an illness that has increasingly affected health professionals. it is characterized by great emotional stress, physical and mental exhaustion and depersonalization of the individual. more serious cases can lead to job loss or even suicide. the described work identifies the burnout level of the multidisciplinary team through a specific questionnaire methods: application of a questionnaire suitable for the multidisciplinary group in november . the same was answered by professionals among physicians and nursing team. there was no identification of employees. after analysis of the results it is observed that % of the group presents initial burnout, % with the syndrome installed and about % with characteristics of greater severity. main factors found were: mental and physical exhaustion during the work day, the level of responsibility existing in the activity and the perception of disproportionate remuneration by work performed. all interviewees presented some degree of burnout or high risk to develop it. the most severe cases should be traced through occupational medicine and anti-stress measures with reorganization of work performance should be discussed in order to reduce the prevalence of this syndrome. introduction: burnout affecting the psychological and physical state of healthcare workers is recognized in the last years. burnout has been shown to affect the quality of care. whilst some risk factors have been identified, there are gaps within the literature related to mental health and burnout. the aim of this study is to measure levels of burnout across icu units in the metropolitan setting. to determine the level of burnout we used surveys, the maslach burnout inventory human services survey (mbi-hss) and the centre for epidemiologic studies depression scale (ces-d). with the mbi-hss we analysed different variables of burnout; exhaustion, cynicism and emotional exhaustion. basic demographic data and information regarding workout schedules were collected. we studied prevalence and contributing risk factors using and analysing the outcomes of the self-scoring questionnaires. analysis was performed using descriptive statistical analysis. there were respondents, % scored the threshold for depressive symptoms on the ces-d depression scale. interestingly, % (ci . - . %) of those meeting the score for depressive symptoms identified as having frequent restless sleep compared with % ( . - . %) from those not meeting. gender did not affect depressive symptoms % of females and % of males met the threshold. with the mbi-hss for exhaustion the mean was . (sd . ) which is a high level of exhaustion, the second variable cynicism the mean score was . (sd . ), which was considered high. the final variable was emotional exhaustion the mean was . (sd . ), this is considered moderate levels of emotional exhaustion. fig. (abstract p ) . hospital anxiety and depression scale (hads) scores for patients and caregivers at baseline, months, and months there was high prevalence of burnout in icu in all different categories as well as depressive symptoms. age and gender had no affect on burnout. interestingly, we identified that sleep and shift variables were linked to increased burnout. following the implementation of a fully integrated ehrs on march at our university-affiliated hospital we conducted a prospective study in icus by analysing pharmacists' contributions during data collection periods of days at , , and weeks post implementation. a pharmacists' contribution was defined as contacting the physician to make a recommendation in a change of therapy/ monitoring [ ] . the types of contribution were: a medication errorrectification of an error in the medication process; an optimizationproactive contribution that sought to enhance patient care, and a consult -reactive intervention in response to a request. a panel of experts composed of a senior pharmacist, a consultant, a nurse, and a pharmacy student assessed the impact of each contribution, scoring low impact, moderate impact or high impact. there were pharmacist contributions recorded in the periods. of these, ( . %) were medication errors, ( . %) were optimizations, and ( . %) was a consult ( table ) . % of the contributions were assessed as having medium impact, % as high impact and % as low impact. in general, the consultant assessed fewer contributions as having high impact compared to other members of the panel, with contributions assessed as high impact by the consultant versus by the senior pharmacist. implementing an ehrs in combination with contributions of clinical pharmacists can prevent medication related issues. interestingly the types of incident did not change over time. introduction: most icu's are noisy and may adversely affect patients outcomes and staff performance [ ] . who reports that the noise level in hospitals should not exceed db at daylight and db at night. the aim of this study is to evaluate the noise levels in intensive care unit, to apply awareness training to intensive care staff in terms of noise and to compare the noise levels before and after education. noise measurement areas are separated into points including patient bedsides, nurse desk, staff desk, wareroom, corridor and entrance of intensive care unite. measurements were performed times per day. after day, awareness training were given to staff in terms of harmful effects of noise. after the training, noise measurements were repeated during days. after total days the measurements were terminated. noise was measured with incubator analyzer (fluke model: bio-tek serial no: ). the mean noise values before and after the training were not statistically different from the mean average noise values (p> . ). when the time of measurement were compared, the noise levels were higher between - hours to other measurements before and after the training statistically (p= . ). seventeen different noise measurement areas were compared in terms of noise level, there was no statistically significant difference (p> . ). the differences were examined at the same hours between before and after training. contrary to expectations, noise levels were found to be higher after training statistically (p< . ). all of noise measurements were higher than the threshold values that who recommended. increased noise levels in critical care units may lead to harmful health effects for both patients and staff. our results suggest that much noise in the icu is largely attributable to environmental factors and behavior modifications due to education have not a meaningful effect. critical care medicine has focused on continuous, multidisciplinary care for patients with organ insufficiency in the face of lifethreatening illness. despite significant resource limitation low income countries carry a huge burden of critical illness. available data is insufficient to clearly show the burden and outcomes of intensive care units in these developing countries [ ] . the objective of our study is to evaluate the morbidity and outcomes of patients admitted to the intensive care unit of a tertiary university hospital in hawassa, ethiopia. this was a prospective observational study. data was registered and analysed starting from patient admission to discharge during a month period beginning september . data regarding demographics, sources of admission, diagnosis, length-of-stay and outcomes were analysed. the total number of patients admitted to the icu was , with patients dying over a one year period. the highest admission was from emergency medical unit, % and the lowest source was from pediatrics department, %. out of these, . % were males. the mean age was years ( - ). the most frequent aetiologies of morbidity in the admitted patients were traumatic brain injury ( . %), acute respiratory distress syndrome ( . %) and seizure disorder ( %). average median length of stay was . days (interquartile range: . - . ). the overall mortality rate was . %. the top four causes of death in the icu were respiratory illness at % followed by sepsis with multiorgan failure at %, trauma ( %) and central nervous system infection ( %). infection morbidity and mortality remains very high and needs institution of aggressive preventive strategies. the increase in frequency of trauma patients need to receive due attention. sepsis causes a high number of deaths, though overtaken by respiratory illnesses. improving the overall system of icu may achieve better outcomes in resource limited countries. introduction: icu mortality has been widely studied in the literature in relation to outcome index that primarily value organic failure [ ] . however, early mortality, in the first hours of admission has been little documented in the literature. the aim of this study is to analyze factors related to early mortality in icu. retrospective study at a second-level hospital. time of study was months. patients who died in icu were included, patients were classified according timing of dead, including those who died within the first hours of icu admission. the variables analyzed were age, sex, comorbidity, charlson index, apache ii, need for supportive treatments, more frequent admission diagnosis, origin and support treatment limitation decisions. the statistical study was carried out using the spss statistical program. patients were included during the study period, ( . %) died within the first hours of admission. no differences in the needs of support treatments were observed, more than % of patients received mechanical ventilation and vasoactive therapies. table shows characteristics of patients. half of icu deaths occur within the first hours of admission. severity at icu admisison was the main factor related with early mortality. severe stroke and coronary disease were the most frequent causes of early deaths in icu. in august the royal college of anaesthetists published guidelines on care of the critically ill woman in childbirth and enhanced maternal care [ ] . approximately babies are born across the area covered by leicester university hospitals that includes two large maternity units and is part of the uk ecmo network. this audit sets out to assess current practice and form a basis for future planning, which will likely be representative to most major obstetric centres. a retrospective audit of all patients admitted to 'intensive care units' in leicester over a month period following publication of the guidelines. the focus was on patients admitted to general adult intensive care and excludes all patients cared for in 'enhanced obstetric care' units. simple standards were proposed relating to accessibility, resuscitation, follow up and multi-disciplinary learning. in total women were identified with a broad range of diagnosis. the intensive care services are split across hospitals and we found this led to a number of problems. the presence of trained staff to resuscitate a newborn were easily accessible, no steps to provide necessary equipment pre-emptively were present in any centre. none of our critical care units had a plan for perimortem section. on-going reviews by the obstetric and midwifery teams were very variable. contact with the infant and breastfeeding support was also poor. despite the large number of deliveries significant work needs to be done in order to come in line with the new national guidelines for critically ill woman in childbirth. clearly defined pathways around escalation of care, resuscitation of both the mother and baby, integrating care of the mother and the infant in the first few days of life, and multidisciplinary learning events are being produced de novo in response to these guidelines, some of which will be illustrated in the associated poster. interprofessional collaboration scale [ ] . data were analyzed with ibm spss . results: it was found that cooperative attitudes with an average score of to are considered to be of average significance. interprofessional cooperation at an average score of , states that the level of cooperation is high and the quality of working life averages to , suggesting that it is very good. as far as professional satisfaction is concerned, nurses are happy, content and satisfied with their work, despite workload and burnout conclusions: interprofessional cooperation at the icu of the general hospital of larissa is high, but satisfaction from wages, resources, working environment and conditions is low. in addition, the results showed that improvements in hospital communication between staff, has a positive impact on the quality of professional life (table ) . contrasting with previous reports, decreased admissions per unit population in older and oldest age groups, and those with high comorbidity, suggest resource constraints may have influenced admission discussion and decision-making over the -year study period in wales. further investigation is warranted. icu discharge into weekends and public holidays: an observational study of mortality n mawhood, t campbell, s hollis-smith, k rooney bristol royal infirmary, general intensive care unit, bristol, united kingdom critical care , (suppl ):p introduction: up to a third of in-hospital deaths in icu patients occurs following ward stepdown [ ] . discharge time seems to be associated with in-hospital prognosis, but meta-analyses have not shown a difference in weekday compared to weekend discharge [ , ] . however, papers that examined discharge 'into' out-of-hours days, particularly on fridays, have found differences [ ] . our aim was to assess whether discharge from icu 'into' out-of-hours (ooh -weekends and public holidays) is associated with in-hospital mortality or re-admission to icu, and whether these patients were seen on the wards ooh by medical staff. all adults discharged from the general icu to a ward at the bristol royal infirmary in december - were included. in-hospital mortality rates were assessed for each day, with 'into weekdays' defined as sunday to thursday and 'into ooh' friday, saturday and the day before a public holiday. a subset of patients with data on readmission rate to icu was also examined. all available notes from patients discharged into ooh in were reviewed. the study included patients with a subset of with readmission data. sets of notes were reviewed from patients discharged into ooh (figure ). the in-hospital mortality was significantly higher in patients discharged into ooh ( . % vs . %, p= . ). within the subset, ooh was associated with in-hospital mortality or readmission to icu ( . % vs . %, p= . ), though readmission rate alone was not ( . % vs %, p= . ). of patients discharged into ooh, once on a ward % were reviewed by a specialty doctor but . % were not seen. this is the first study to examine icu discharge 'into' ooh days including public holidays. we found increased hospital mortality in ooh, similar to other studies [ ] . up to a fifth of high-risk icu stepdown patients were not reviewed by a doctor on ooh days. exploring the experiences of potential donors' family members (fm) in a follow up clinic is crucial to analyze the effects of organ procurement (op) on the bereavement process, to gain insight on the reasons of family refusals (fr), and to improve family care during op. a mixed-method study involving fm at and months after patients' death was developed and approved by local ethics committee. fm of potential donors after brain (dbd) and cardiac death (dcd) treated in careggi teaching hospital, florence (italy) were eligible if adult and consenting. invitation letters were sent to the entitled months after death and those who actively responded were involved in an encounter with a multidisciplinary group including a clinical psychologist, two nurses and two cultural anthropologists with expertise in op. organ replacement procedures such as ecmo (extracorporeal membrane oxygenation), lvad (left ventricular assist device) and dialysis are routinely used to treat multi-organ failure (mov). globally transplantation programs struggle with increasing organ shortage. patients (pts) with mov are a potential source for procurement. however, outcome data after kidney transplantation (ktx) from such donors are sparse. we retrospectively studied the cadaveric ktx at the charité berlin in and identified donors with ongoing organ replacement procedures. donor and recipient risk factors were assessed. overall patient and graft outcomes were analyzed at months post-transplant. a total of kidneys were transplanted. we identified ktx from donors with mov ( following cardio-pulmonary resuscitation, with acute renal failure - on dialysis) (figure ). in donors, a venoarterial ecmo was implanted during ecls-resuscitation. one donor needed a veno-venous ecmo due to ards, and donor had a lvad implanted due to cardiac failure. the donor age was ± . years (yrs). in addition, donors had at least one cardiac risk factor. the kidney donor risk index averaged . (sd ± . ) and s-creatinine prior to ktx was . (sd ± . one way to expand the potential donor pool is donation after circulatory death (dcd), and a strategy to reduce the complications related to the ischemic time is the use of normothermic regional perfusion (nrp) with extracorporeal membranous oxygenation (ecmo) [ , ] . we compare the use of standard nrp with an effective adsorption system inflammatory mediators (cytosorb®) in the regional normothermic reperfusion phase via regional ecmo, that involves a reduction in cellular oxidative damage, assessed as a reduction in levels of proinflammatory substances. we report a case series of dcd-maastricht iiia category donors, treated in ecmo with nrp, to maintain circulation before organ retrieval, in association with cytosorb® in patients. during perfusion, from starting nrp (t ), blood samples are collected times, every minutes (t , t , t ). during treatment with cytosorb®, lactate levels progressively decrease, ast and alt increase less than without cytosorb®, as sign of improvement in organs perfusion ( figure ). nrp with cytosorb® might help to successfully limit irreversible organ damages and improve transplantation outcome [ ] . development and implementation of uniform guidelines will be necessary to guarantee the clinical use of these donor pools. introduction: shock is a common complication of critical illness in patients in intensive care units (icus), who are undergoing major surgery. this condition is the most common cause of death in postsurgical icus. nowadays, there are different icu scoring systems for predicting the likelihood of mortality, such as apache or sofa. nevertheless, they are used rarely because they also depend on the reliability and predictions of physicians. in these sense, gene expression signatures can be used to evaluate the survival of patients with postsurgical shock. methods: mrna levels in the discovery cohort were evaluated by microarray to select the most differentially expressed genes (degs) between groups of those that survived and did not survive days after their operation. selected degs were evaluated by quantitative real time polymerase chain reactions (qpcr) for the validation cohort to determine the reliability of the expression data and compare their predictive capacity to that of established risk scales. introduction: this study evaluates the prognostic ability of frailty and comorbidity scores in patients with septic shock. the -day mortality rate of individual medical conditions are also compared. the burden of comorbid illness and frailty is increasing in the critical care patient population [ ] . outcomes from septic shock in patients with chronic ill-health is poorly understood. interstitial lung disease is a group of diseases associated with poor prognosis in the intensive care unit despite major improvement in respiratory care in the last decade. the aim of our study is to assess factors associated with hospital mortality in interstitial lung disease patients admitted in the intensive care unit and to investigate the long-term outcome of these patients. we performed a retrospective study in an intensive care unit of teaching hospital highly specialized in interstitial lung disease management between and . a total of interstitial lung disease patients were admitted in the intensive care unit during the study period. overall hospital mortality was %. two years after intensive care unit admission, / patients were still alive ( %). one hundred eight patients ( %) required invasive mechanical ventilation of whom % died in the hospital (figure ). acute exacerbation of interstitial lung disease was associated with hospital mortality (or= . [ . - . ] ), especially in case of acute exacerbation of idiopathic pulmonary fibrosis. multiorgan failure (invasive mechanical ventilation with vasopressor infusion and/or renal replacement therapy) was associated with very high hospital mortality ( / ; %). survival after intensive care unit stay of patients with interstitial lung disease is good enough for not denying them from invasive mechanical ventilation, except in case of acute exacerbation for idiopathic pulmonary fibrosis patients. if urgent lung transplantation or extracorporeal membrane oxygenation are ruled out, multiorgan failure should lead to consider withholding or withdrawal life support therapies. Αgi is a malfunctioning of the gi tract in icu patients associated with prolonged mechanical ventilation, enteral feeding failure and high mortality risk. the wgap of esicm proposed a grading system for agi. four grades of severity were identified: agi grade i, a selflimiting condition; agi grade ii (gi dysfunction), interventions are required to restore gi function; agi grade iii (gi failure); agi grade iv, gi failure that is immediately life threatening. the aim was to evaluate the feasibility of using agi grades i and ii as predictors of malnutrition and -year mortality in critically ill patients methods: single-center retrospective cohort study in a tertiary university hospital ( - ). agi grade iii and iv patients were excluded. Αnthropometric data, gi symptoms (vomiting,diarrhea), feeding intolerance, gastric residual volumes and abdominal hypertension were recorded. daily prescribed caloric intake was calculated using a standard protocol and daily achievement of caloric intake was recorded. mnutric score was calculated for all patients. a score ≤ was used to diagnose malnutrition. patients ( % men, mean age years) that stayed in the icu for > hours were included in the study. % were at high nutritional risk. -year mortality was %. the prevalence of agi ii was %. age, gender, bmi, mortality and energy intake did not differ significantly between patients with agi ii and those with agi i (table ) . logistic the study aimed to assess the effects of icu admission on frailty and activities of daily living in the ≥ 's population at -months. a prospective observational study with data used as a subset of the vip- trial [ ] . research ethics committee approval from the mater misercordiae university hospital (mmuh). inclusion criteria -≥ years of age and acute admission to icu from may to july . data collected on consecutive patients. frailty and activities of daily living (adl) were assessed using the clinical frailty score (cfs) and the katz index of independence in activities of daily living (katz). results: csf pre-admission frailty was present in % of patients, increasing to % at months ( figure ). % of survivors at -months had a cfs score increase by ≥ point. pre-frail and frail cfs patients suffered an average -point deterioration in their instrumental activities of daily living (iadl). % of katz patients were fully functional preadmission, deteriorating to % at months. % of patients declined by adl at months. % of the deceased were deemed fully functional initially. we demonstrate an association between an icu admission event and enduring functional decline at months. icu admission resulted in patients acquiring on average . new iadl limitations despite their initial cfs. this is echoed in a study by iwasyna et al. who also showed similar deteriorations in iadl and cognitive impairment [ ] . katz benefits may be best used in describing functional decline. % of patients developed at least one new limitation. however, the cfs takes into account iadl's and thus may be more sensitive in predicting the functional outcomes of an icu event at months. frailty: an independent factor in predicting length of stay for critically ill t chandler, r sarkar, a bowman, p hayden medway maritime hospital, critical care, gillingham, united kingdom critical care , (suppl ):p frailty has attracted attention in the healthcare community in recent years, as it is associated with worse outcomes and increased healthcare costs [ ] . our objective was to study the impact of frailty as recorded by clinical frailty scale(cfs) to prospectively evaluate the effect of frailty on hospital length of stay (los). a retrospective analysis of consecutively admitted critical care (cc) patients' data (jan' -oct' ) was performed. electronic health records were used to collect demographics, cfs and clinical outcomes. statistical analysis was performed using stata. students t-test, simple and multiple (adjusted for age, disease severity/icnarc score) linear regression were used for comparison between groups and to see group effect. we excluded extreme outliers (los> days; n= ). frailty was defined as cfs> . out of the patients (male %), ( %) were emergency admissions, the rest elective (table ) . ( %) were non-frail. the mean los were days (d) ± and d± (p< . ) in the frail and non-frail patients respectively. for emergency patients, los were d(± ) and d(± ) for the groups, (p< . ). for elective patients; los were d(± ) and los d(± ), (p= . ) for frail and nonfrail respectively. after adjusting, los was significantly higher in frail patients by days ( %ci , ; p< . ), by days ( %ci , ; p= . ) and by days ( %ci , ; p< . ) for total cohort, elective and emergency admissions respectively. the los was days higher in frail than non-frail (p< . ) for cc survivors. frailty was associated with significantly increased los in this cohort, independent of age and illness severity. hospital capacity planning should take this into consideration when modelling bed allocation fig. (abstract p ) . clinical frailty score -month trend robust clinical governance requires analysis of patient outcomes during an icu admission [ ] . on one adult icu weekly mortality meetings are used for this purpose and aid multidisciplinary reflections on individual patient deaths. however, such reviews run the risk of being subjective and fail to acknowledge themes which may relate to preceding or subsequent deaths. this paper describes a new mortality review process in which: a) reviews are structured using the structured judgement review (sjr) framework [ ] ; and b) themes are generated over an extended period of time to create longitudinal learning from death. the sjr framework has been developed by nhs improvement for the new medical examiner role, looking at inpatient deaths. we adapted this to better suit the icu creating a novel review structure. this involves explicit judgement comments being recorded, and the use of a scoring system to analyse the quality of care during the patient's stay with a focus on elements of care delivered on the icu. tabulation of this information allows analysis over time, identifying trends across all patients, and in specific subgroups. this framework has been rolled out at the st george's cardiothoracic icu weekly mortality meetings. themes that have emerged include parent team ownership, delayed palliative care referrals and inadequate documentation of mental capacity. this will continue as part of a three-month trial and following review of this trial may be extended to other critical care units in the trust. this system allows greater insight into patient deaths in a longitudinal fashion and facilitates local identification of problems at an early stage in a way that is not possible within the traditional mortality review format. the nature of the process means that key areas for change can be identified as a routine part of the clinical week. [ ] . in this study, we evaluated three distinct machine-learning methods for predicting possible patient deterioration after surgery. the data was collected retrospectively from the catharina hospital in eindhoven. this dataset contained all the surgeries conducted in the hospital from up to . the variables in this dataset were tested on their ability to differentiate between patients with a normal recovery versus patients with an unplanned icu admission after being admitted to the ward. the dataset contained variables related to either the preoperative screening, surgery or recovery room. all variables were tested for statistical significance using a univariate logistic regression (lr), from which a subset of statistically significant (p< . ) variables was created. these variables were used to train three different types of models, namely, the lr, support vector machine (svm) and bayesian network (bn). the network structure of the bn was designed using expert knowledge and the probabilities were inferred using the data. the three models were validated using five-fold cross-validation, resulting in the following areas under the receiver operating characteristic curve: . ( . - . ) for lr, . ( . - . ) for svm and . ( . - . ) for bn (fig. ) . the results indicate that machine learning is a promising tool for early prediction of patient deterioration. the bn was included because it permits incorporating clinical domain knowledge into the learning process. however, its performance resulted inferior to the lr and svm. in future work, we will investigate alternative domainaware methods, and compare the performance with that of the clinical experts. intensive care unit (icu) admission decisions of patients with a malignancy can be difficult as clinicians have concerns about unfavourable outcomes, such as mortality [ ] . a diagnosis of a malignancy is associated with an almost -fold increased likelihood of refusal of icu admission [ ] . recent large long-term mortality studies of patients with a malignancy admitted to the icu are scarce. therefore, our aim was to compare mortality of patients with either a hematological or a solid malignancy to the general icu population, all with an unplanned icu admission. all adult patients registered in a national intensive care evaluation registry with an unplanned icu admission from to were included. subsequently, we divided these patients into cohorts: cohort (all patients with a hematological malignancy), cohort (all patients with a solid malignancy), and cohort (a general icu population without malignancy). as primary outcome, we used -year mortality, and as secondary outcome, icu and hospital mortality. we included , ( . %) patients in cohort , , ( . %) patients in cohort and , ( . %) in cohort ( table ). the year mortality of patients of cohort , , and was . %, . % and . %, respectively (p< . ). age, comorbidities, organ failure, and type of admission (i.e. surgical or medical) were positively associated with -year mortality in all cohorts (p < . ). one-year mortality is higher in both patients with a hematological malignancy and patients with a solid malignancy compared to the general icu population. in addition, several factors were positively associated with -year mortality, i.e., age, comorbidities, medical icu admission, and organ failure. future research should focus on predictive modelling in order to identify patients with a malignancy that may benefit from icu admission. introduction: drug abuse is associated with immunosuppression in multiple mechanisms. despite that, the only study retrospectively reviewing drug abusers in the icu demonstrated less infections and better outcomes. we compared matched patient populations in order to fully understand whether drug abuse is a risk factor for infection and a predictor of poorer prognosis as is perceived by most physicians. we hypothesized that the drug abusers admitted to the icu will fare as good as or better than non-abuser icu patient populations. methods: this is a prospective study done between the years - on the entire patient population of the detroit medical center. after the drug abuse population was identified, controls were matched according to age and admission icu units. patients charts were reviewed and data regarding baseline demographics, infectious complication and outcome was extracted. data was retrospectively collected for drug abusers and matched controls. comorbidities and hospital admission diagnosis were significantly different between the two groups. disease severity scores were significantly higher in the drug abuser's patient group (dapg) on admission and during the icu stay. dapg had significantly more organ failure: more need for ventilation ( . % vs . % in the dapg (p< . )), more ards ( % vs . %, p= . ), more renal failure ( % vs . %, p= . ) and more need for renal replacement therapy ( . % vs . %, p< . ) .they had longer hospital length of stay (los). there was no difference in icu or hospital mortality. multivariable modeling did not find drug abuse to be an independent risk factor for hospital mortality, icu mortality (hosp: or = . , p = . ; icu: or= . , pp = . ), but was a risk factor for a longer hospital los (me= . , p < . ). drug abuse is not an independent risk factor for mortality or icu los. drug abusers should be evaluated like other patients based on baseline comorbidities and disease severity. this is a small audit which although it did not include general icu still reflects the need for encouraging clinicians and patients to speak freely regarding escalation plans. medical decsions is clinician led however this audit was carried by nursing staff as we have a duty to be advocate for our patients involvement in medical care [ ] . a retrospective analysis of independent risk factors of late death in septic shock survivors c sivakorn , c permpikul , s tongyoo (fig. ) . the pap and katz scales seem to be adequate for predicting mortality of critically ill patients admitted to a medical icu. this finding may help in the elaboration of future icu mortality scoring systems, as well as in more rational use of resources. however, further multicenter studies are needed to better elucidate these results. adherence this last group was chosen because of its experience and specific training in the field of bioethics as a control group or reference. a total of respondents participated in the study. . % were emergency physicians, . % intensivists, . % emergency nursing, . % icu nursing, . % resident doctors, . % medical students and . % other professions. we observed variability in the responses observed not only between different groups of professionals but even within the same group reflecting the difficulty in decision making. variability was observed regarding decisions in end of life ethics conflicts. a high degree of similarity with the group of master in bioethics was observed in the responses issued by medicine students. the barriers and facilitators to framing goals of patient care (gopc) and factors motivating decision making is relatively unexplored [ , , ] . a three part survey of physicians at an australian hospital in a culturally and linguistically diverse suburb ( table ) . identification of levels of confidence and barriers and facilitators to gopc discussion and decision making was the main outcome measure. factors influencing decision-making was analysed through scenarios. results: out of eligible participants responded; female, male, clinical experience - years. level of confidence was ranked between "somewhat confident and very confident." all but one respondent had six months of icu experience. no differences in the level of confidence among physician groups. barriers and facilitators were identified; poor prognosis and patient or family request were most common facilitators; conflict between treating teams and the patient/surrogate and language barriers were most common barriers. factors driving gopc decision-making included clinical, value judgement, communication, prognostication, justice and avoidance. numerous barriers and facilitators were identified. factors driving decision making did not just consider clinical factors; conflict and we aimed to investigate physician-related factors contributing to individual variability in end-of-life (eol) decision-making in the intensive care unit (icu). qualitative study with semi-structured interviews with specialists in critical care, (experience - years) from swedish icus. data was analyzed in accordance to principles of thematic analyses. most of the respondents felt that the intensivist's personality played a major role in eol decisions (table ) . individual variability was considered inevitable. views on acceptable outcome: respondents experienced that the possible outcome for patients was interpreted very differently and subjectively among colleagues, and what seemed an acceptable patient-outcome for one doctor, was not acceptable for another. values: most of the respondents were well aware that they might be affected by their own values and attitudes in the decision-making process. interestingly, several respondents mentioned that they thought that patients that were marginalized by society, especially drug-abusers could be at risk for receiving decisions to limit life sustaining treatments (lst) more often than others. none of the respondents thought that their own religious beliefs played any part in decision making. fear of criticism: among the less experienced respondents there was a clear sense of fear of making a questionable assessment of the patient's medical prognosis. there was a fear for criticism from colleagues that were not directly involved in the decision-making, and may have made another decision. this created a wish among younger respondents to defer or avoid participating in decision-making. physician-related, individual variability in eol decisions primarily consisted of differing views on acceptable outcome, values and fear of criticism. can (figure ). within each quartile of sofa score, mortality was highest in patients with pneumonia and peritonitis and lowest in patients with cellulitis (see figure ). the sepsis- consensus definition identified organ dysfunction as the hallmark feature of sepsis [ ] . in developing sepsis- , the sequential organ failure assessment (sofa) score was chosen for its prognostic value and relative ease of implementation clinically [ ] . we propose an update based on epidemiologic data from two intensive care databases that more effectively captures organ dysfunction in the context of sepsis- . using the mimic-iii (exploration) and e-icu (validation) databases, we extracted patients with suspicion of infection to form the study cohort. the predictive power of each sofa component was assessed using the area under the curve (auc) for in-hospital mortality. a logistic model with the lasso penalty was used to find an alternative statistically optimal score. results: by utilising alternate markers of organ dysfunction (e.g. lactate, ph, urea nitrogen) we demonstrated a significant improvement in auc for several versions of the new score, sofa . ( figure ). the sofa score can be updated to reflect current advances in clinical practice. using epidemiologic data, we have shown that substitution of existing components with more powerful measures of organ dysfunction may provide an improved score with greater predictive power. moreover, sofa . exhibits equivalent ease of implementation, but better reflects organ dysfunction in the context of sepsis- . introduction: risk of acute organ failure (aof) in cancer patients(pts) on systemic cancer treatment isunknown. however, % of non-hematologic and % of hematologic cancer pts will need admission to intensive care unit (icu). ipop-sci- / is a prospective cohort study designed to ascertain the cumulative incidence of aof in adult cancer pts. single centre prospective cohort study with consecutive sampling of adult cancer pts admitted for unscheduled inpatient care while on, or up to weeks after, systemic cancer treatment. primary endpoint was aof as defined by quick sofa. six months accrual expected an accrual of pts to infera population risk aof with a standard error of %. between / and / pts were on systemic anticancer treatment, had unscheduled inpatient care and were eligible for inclusion and were included. median age was years, % were male, % had adjusted charlson comorbidity index (cci) &gt; and hematologic cancers accounted for % of pts. the cumulative risk of aof on hospital admission was % ( %ci: - ); and of aof during hospital stay was % ( %ci: - ). aof was associated with older age, cci &gt; ,hematologic malignancy, shorter median time from diagnosis and &gt; prior line of therapy. on admission, % of pts were considered not eligible for artificial organ replacement therapy (noaort) and % of pts who developed aof while inhospital were judged noaort. overall, ( %) of aof pts wereadmitted to icu, . % for aort. median follow up . months (min ; max ). inpatient mortalitywas %, with icu mortality rate of %, with median cohort survival . months ( %ci: . - . ). on multivariate analysis, aof was an independent poor prognostic factor (hr . ; %ci . - . ). risk of aof in cancer pts admitted for unscheduled inpatient care while on systemictreatment is %, and risk of icu is %. aof in cancer pts was an independent poor prognostic factor. a severity-of-illness score in patients with tuberculosis requiring intensive care u lalla, e irusen, b allwood, j taljaard, c koegelenberg tygerberg academic hospital, internal medicine, division of pulmonology and icu, cape town, south africa critical care , (suppl ):p we previously retrospectively validated a -point severity-of-illness score aimed at identifying patients at risk of dying of tuberculosis (tb) in the intensive care unit (icu). parameters included septic shock, human immunodeficiency virus with cd < /mm , renal dysfunction, ratio of partial pressure of arterial oxygen to fraction of inspired oxygen (pao :fio ) < mmhg, diffuse parenchymal infiltrates and no tb treatment on admission. the aim of this study was to validate and refine the severity-of-illness score in patients with tuberculosis requiring intensive care. we performed a prospective observational study with a planned post-hoc retrospective analysis, enrolling all adult patients with confirmed tb admitted to the medical intensive care unit from february to july . descriptive statistics and chi-square or fisher's exact tests were performed on dichotomous categorical variables, and t-tests on continuous data. patients were categorized as hospital survivors or non-survivors. the -point score and the refined -point score were calculated from data obtained on icu admission. results: forty-one of patients ( . %) died. the -point scores of nonsurvivors were higher ( . +/- . vs . +/- . ; p= . ). a score ≥ vs. < was associated with increased mortality ( . % vs. . %; or . ; %ci, . - . ; p= . )( table ) . post-hoc, a pao :fio < mmhg and no tb treatment on admission failed to predict mortality whereas any immunosuppression did. a revised -point score (septic shock, any immunosuppression, acute kidney injury and lack of lobar consolidation) demonstrated higher scores in non-survivors ( . +/- . vs. . +/- . ; p< . ). a score ≥ vs. ≤ was associated with a higher mortality ( . % vs. . %; or . ; %ci, . - . ; p< . ) ( table ) . the -point severity-of-illness score identified patients at higher risk of death. we were able to derive and retrospectively validate a simplified -point score with a superior predictive power. chronic critical illness remains a scientific challenge, from its conceptualization to its impact on patient prognosis [ ] . we evaluated the long-term evolution of icu survivors by identifying the real burden of prolonged critical illness on survival, quality of life and hospital readmissions. we conducted a prospective cohort in brazilian hospitals including icu survivors with an icu stay > h. we compared the patients diagnosed with chronic critical illness with the other patients. telephone follow-up at and months. quality of life was measured by the sf- questionnaire. it was observed that % of patients had some definition of chronic critical illness. chronic critically ill patients had higher mortality at months (p= . ). this difference is mainly due to higher intrahospital mortality (p= . ). mortality after hospital discharge was similar between groups. there was no difference in hospital readmission rate at months. various scores are developed to predict pulmonary complications such as ariscat for patients at-risk of postoperative pulmonary complication [ ] and lips for patients at-risk of lung injury [ ] . the aim of this study was to compare these scores with ours for predicting pulmonary complications in mechanically ventilated patients in sicu. this prospective observational study was conducted in sicu at a university hospital. adult patients admitted to sicu and required mechanical ventilation > hours were included. primary endpoint was the composite of pulmonary complications including pneumonia, ards, atelectasis, reintubation, and tracheostomy. multivariate analysis was performed to identify risk factors of pulmonary complications and the predictive score was developed. the roc analysis was performed to compare power of ariscat, lips and our newly developed score for predicting pulmonary complications. outcomes in intensive care units have been reported to be better in higher-volume units [ , ] . we compared outcomes for high-risk patients between low and higher volume units. audit data from irish icus is analysed and reported by the intensive care national audit & research centre (icnarc) in london. icnarc report risk-adjusted mortality rates in all patients and in low-risk patients(predicted mortality rate < %) for each unit, using the icnarch- model to predict the risk of death. we used this data to calculate the proportion of high-risk patients(predicted mortality > %) in each unit, the mortality rate for high-risk patients, the riskadjusted mortality rate and we compared the overall risk-adjusted mortality between low and high volume units. the median number of annual new-patient admissions among participating units was ; units below this were defined as lowvolume and those above as high-volume units. the proportion of all admissions to each unit who were high-risk ranged from % to %(mean %). unit mortality rates for high-risk patients ranged from % to %. the ratio of observed to expected mortality(standardized mortality ratio -smr) for high risk admissions in each unit ranged from . to . (mean . ). in fig. introduction: adl weakening is often seen after intensive care and called postintensive-care syndrome (pics). this is also seen in even outside icu and proposed to be called post-acute-care syndrome (pacs), especially in elderly patients. in patients with infection, sofa score is famous for predicting in-hospital mortality, but there are no tools for predicting adl weakening during admission. to search for risk factors for adl weakening during admission other than the age, we conducted a retrospective observational study. the subjects were surviving patients with infection, aged from to who were admitted to our department from april , to may , . information of basic characteristics, laboratory data on admission and adjunctive therapies were extracted from our database. we use barthel index (bi) as adl evaluation, and the bi at discharge were evaluated by nurses. we stratified patients by bi at discharge of over or not, and investigated factors that predicted it. we compared each factor between groups, and perform a logistic regression analysis with those that had a significant effect clinically or statistically. despite improved outcomes of intensive care unit (icu) patients, sleep deprivation remains a major concern after icu discharge. multifaceted causes make it difficult to treat and understand [ ] . not many studies have explored sleep deprivation beyond icu. this is evidenced by findings from a recent systematic review [ ] which included studies with only one study [ ] reporting sleep deprivation beyond icu. the aim of this paper is to present findings of sleep deprivation beyond icu from a larger study that examined the experience of critical illness in icu and beyond in the context of daily sedation interruption. hermeneutic phenomenology was used to conduct the study. participants aged years and above who fulfilled the enrolment criteria were enrolled into the study. the cohort comprised male and female participants. in-depth face to face interviews at two weeks after discharge were conducted and repeated at six to eleven months. interviews were audio taped, transcribed and thematically analysed. significant statements were highlighted and categorized for emergent themes. six participants continued to experience sleep deprivation up to eleven months after icu. two cited dreams about icu, three could not explain why they continued to fail to sleep and one stated that he continued hearing icu alarms in the silence of the night. sleep deprivation continues beyond icu due to nightmares, delusional memories and unexplained reasons. further research is needed to establish causes of sleep deprivation and explore ways to promote sleep in critical illness survivors after icu discharge. frailty is being increasingly seen as an independent syndrome. frail patients now account for an increasing proportion of hospital and critical care admissions [ ] . we aimed to compare frailty and mortality in our intensive care unit. clinical frailty score (cfs) was incorporated within the electronic health record (ehr) . we performed this retrospective analysis on the data collected between jan' and oct' . the predictor and outcome for this study were frailty and hospital mortality respectively. all demographic data, acute physiology score, critical care and hospital outcome data were automatically collected in the ehr and recorded. we used a cut off of cfs> and above to define non-frail and frail respectively. chi-squared test, simple and multiple logistic regression were used. adjustment was done for icnarc score and age. total number of patients was , of which ( . %) died in hospital. within the patients< years (n= ), ( %) were recorded as frail or vulnerable. the number of elective and emergency admission were ( %) and ( %) respectively. in the frail and nonfrail, mortality rates were % and . % (p< . ) respectively, with odds ratio of . ( % ci . , ; p< . ) ( age is a well-known risk factor for critical care (cc) outcome and is incorporated into many prognostic tools; however, this has been criticized for assumption of normal physiology for young at baseline. in recent years, frailty in cc prognostication has been of interest, with meta-analysis correlating worsening outcomes with increasing frailty [ ] . in this study, we compared the effect of frailty versus age for determining hospital survival for critically ill patients. we conducted a prospective cohort in brazilian hospitals including survivors of an icu stay > h. we compared chronic critically ill patients (icu stay> days) and the other patients. we performed psychological and functional presential assessment in patients within hours of icu discharge and by telephone at and months. the prevalence of chronic critically ill patients was %. regarding outcomes, chronic critically ill patients had a higher incidence of depressive symptoms than other patients in the immediate post-icu discharge (p = . ), as well as a higher incidence of muscle weakness (p < . ). however, in subsequent evaluations, we found no difference between groups regarding psychological symptoms -depression, anxiety and post-traumatic stress. higher functional dependence was observed in critically ill patients, but without difference in the quality of life score, both in the physical (p = . ) and mental (p = . ) domains. chronic critically ill patients, when compared to patients with stay> h, have a higher incidence of depressive symptoms at icu discharge. this difference disappears in the follow up. chronic critically ill patients present higher levels of functional dependence but without repercussions on quality of life scores. introduction: activation of the inflammatory response after cardiac arrest (ca) is a welldocumented phenomenon that may lead to multi-organ failure and death. we hypothesized that white blood cell count (wbc), one marker of inflammation, is associated with one-year mortality in icu treated ca patients. we used a nationwide registry with data from five academic icus to identify adult ca patients treated between january st and december st . we evaluated the association between the most abnormal wbc within hours of hospital admission and one-year mortality. we accounted for baseline risk of death using multivariable logistic regression (adjusted for age, gender and h sequential organ failure assessment [sofa] score). a total of , patients were included in the analysis. of those patients , ( %) were alive one year after ca. we plotted wbc against baseline risk of death and through graphic examination of a locally weighted scatterplot smoothing (lowess) curve found the lowest risk of death to be associated with a wbc of (e /l) ( figure mrps were identified by a specialist icu pharmacist during this programme and classified by their significance on a scale of one to four. logistic regression was used to determine if demographic factors were associated with the occurrence of a clinically significant mrp -a significance score of two or above (figure ) . the adjusted model included age, icu los, hospital los, apache ii, number of days of renal replacement therapy, number of days of ventilation, the number of medications prescribed at icu discharge, and the who analgesia classification at ins:pire. there were increased odds of having a clinically significant mrp for hospital los (or results: · % (n= ) of patients required at least one pharmacy intervention. the median number of interventions required per patient was one (iqr - ); the maximum number was six. mrps were recorded in this cohort. the most common intervention was clarifying duration of treatment (n= ), followed by education (n= ), and correcting drug omissions (n= ). the bnf drug class most frequently associated with mrps was neurological (n= ), which comprises analgesics (n= ) and psychiatric medications (n= ) ( figure ). this was followed by cardiovascular medications (n= ), gastrointestinal medications (n= ), nutritional medications (n= ), and others (n= ). many icu survivors experience mrps. the most common class of mrp was neurological, reflecting the high incidence of chronic pain and psychiatric illness in this population following discussion with icu staff, ward staff and fy doctors, a formal standardized handover system was introduced. this involved a verbal handover to the appropriate fy by an icu doctor and the patient drug chart to be rewritten in icu at the time of handover. the next change was to display posters on the wards to alert staff that the medical team are to be contacted when a patient comes to the ward from icu and to ensure the drug chart is completed. the baseline data showed a median time delay of hours, with one patient waiting hours for a drug chart. following the interventions the median time delay has decreased to hours within months as demonstrated in figure . the changes have received positive feedback from icu staff, ward staff and fy doctors. the aim of reducing the time delay by % has been achieved with the median time delay now hours. this has improved patient safety by significantly reduced delays in medications and through the introduction of a standardized handover. this has also provided an opportunity for junior doctors on the wards to seek clarification regarding medications and the clinical management plan for the patient. this has established a communication channel between icu and the wards making patient care safer and more effective. telemonitoring outside the icu is scarce. but with innovative wearables measuring respiratory and heart rate wirelessly, culture on intrahospital telemonitoring should definitely change. however, culture has been known to be one of the most crucial success factors in innovation, especially in health care. human design thinking is a promising tool in health care innovation but rarely used in a multidisciplinary team to initiate an innovation culture and stimulate sustainable collaboration. the aim of this study was to initiate a pilot project with a multidisciplinary team to start using wearables for early warning score (ews) on a clinical ward. human design thinking was used to write a value proposition on wearables in clinically admitted neutropenic hematologic patients in an academic center. a multidisciplinary team was performed to cover all disciplines involved in the technical, clinical and administrative parts of the project. a vendor was chosen based on its product specifications in relation to the present hospital monitoring infrastructure. in design thinking sessions, critical appraisal of multiple telemonitoring factors was performed by sub teams and a canvas projectplan was constructed. the project team was formed of registered nurses, physicians, itspecialists, electronic health record consultants; a critical care physician was appointed as project leader. the main critical factors were: unseamlessly transmitting of both heart and respiratory rates including appropriate movements filtering to the nurse's smartphones direct uploading into electronic health record with automated ews calculation nurse driven protocol on ews follow up. philips healthcare with their intellivue guardian wearable biosensor was the chosen vendor ( figure ). design thinking in a multidisciplinary health care team could positively influence the innovation culture. scientific evaluation of this wearable will focus on both nurse's acceptance and data storage and is expected in the summer of . severity, readmission and lengh of stay were lower in patients receiving discharges directly to home. it seems like a safe way to discharge low-risk short stay patients. it seems to save resources and reduce costs, as well as the need for hospital beds. however, futher estudies are needed to actualy evaluate this safety. forty-four cultures were analyzed with eplex ( figure ). complete agreement with conventional diagnostics was observed in / cases. no false-positive results were observed, yielding a sensitivity and specificity of % and % respectively for target pathogens. time to result was, on average, . h faster with eplex compared to conventional diagnostics. antimicrobial therapy could have been optimized in patients based on the eplex result, but treatment was only changed in one case (e.coli ctx-m+) receiving meropenem . h before the antibiogram was available. the eplex blood culture panels provide high accuracy and significantly faster results. the current implementation offers substantial potential value at a minimal cost, and is a feasible approach to -h/ days blood culture diagnostics in many hospital settings. however, efforts to increase adherence are needed. the rapid increase of extended spectrum β-lactamases (esbl)-producing pathogens worldwide makes it difficult to choose appropriate antibiotics in patients with gram-negative bacterial infection. cica-beta reagent (kanto chemical, tokyo, japan) is a chromogenic test to detect beta-lactamases such as esbl from bacterial colonies. the purpose of the study was to reveal whether cica-beta reagent could detect esbl-producing pathogens directly from urine rather than bacterial colonies to make a rapid bedside diagnosis of the antibiotic susceptibility of gramnegative pathogens. we conducted a prospective observational study from july to october . patients were eligible if they were performed urinary culture tests and gram negative pathogens were detected at least + from their urine samples. the urine sample was centrifugated at x g for min. the supernatant of sample was re-centrifugated at x g for min and the pellet was mixed with cica-beta reagent. the test was considered positive when the enzymatic reaction turned from yellow to red or orange. (fig. ) . the bundle approach could be an effective strategy to prevent hospital-acquisition of drug-resistant pathogens in icus. fig. in the aspect-np trial, c/t was noninferior to mem for the treatment of habp/vabp. we evaluated outcomes from that study in the subgroup of pts failing current antibacterial therapy for habp/vabp at enrollment. methods: aspect-np was a randomized, controlled, double-blind, phase trial in which mechanically ventilated pts with habp/vabp received g c/t or g mem every h for - days. pts with > h of active gram-negative antibacterial therapy within h prior to first dose of study therapy were excluded, except those pts failing current treatment (i.e. signs/symptoms of the current habp/vabp were persisting/worsening despite ≥ h of antibiotic treatment). primary and key secondary endpoints, respectively, were -day all-cause mortality (acm) and clinical response at test of cure (toc; - days after end of therapy) in the intent to treat (itt) population. pts failing current antibacterial therapy for habp/vabp were prospectively categorized as a clinically relevant subgroup. at baseline, failing current therapy for habp/vabp was reported in / ( %) c/t and / ( %) mem itt pts, mostly piperacillin/ tazobactam ( %), rd/ th-generation cephalosporins ( %), fluoroquinolones ( %), and aminoglycosides ( %). baseline demographic and clinical characteristics in this subgroup, including prior therapy regimen, were generally similar between treatment arms. there were greater proportions of patients with esbl+ enterobacterales ( %) and pseudomonas aeruginosa ( %) in the c/t arm than the mem arm ( % and %, respectively). lower -day acm was seen with c/t than mem, as evidenced by % confidence intervals for treatment differences that excluded zero ( figure ); statistical significance cannot be assumed because subgroup analyses in this study were not corrected for multiplicity. conclusions: c/t was an effective treatment for habp/vabp pts who had failed initial therapy. catheter-related blood stream infection (crbsi) is common serious infections and associated with increased mortality in intensive care units (icu). one of the most important strategy to prevent crbsi is to minimize the duration of central venous catheterization. we built a medical team consisting of doctors, nurses and pharmacists in icu to discuss whether patients needed central venous catheter (cvc) in terms of monitoring hemodynamics and administering drugs, and recommend catheter removal to attending physicians every day in april . the purpose of this study is to evaluate whether our team-based approach could shorten the total duration of catheterization and reduce crbsi. this was a retrospective historical control study conducted from april to october in the icu of a tertiary care hospital in japan. every patient admitted to the icu during the study period was eligible if they were inserted cvc. patients were divided into groups: conventional (from april to march ) or intervention (from april to october ). we set the primary endpoint as onset of crbsi. the secondary endpoints included the duration of central venous catheterization, the length of icu stay and hospital mortality. crbsi was defined as bloodstream infection in patients with cvc, not related to another site. we included patients: in the conventional group and in the intervention group. the reduced, though nonsignificant, tendency of crbsi was observed in the intervention group [hazard ratio, . ( % confidence interval, . - . ; p = . )]. the intervention group was significantly associated with reduced duration of central venous catheterization ( days vs days; p < . ). no difference was observed in the length of icu stay and in-hospital mortality between groups. the team-based approach to assess cvc necessity could shorten the duration of central venous catheterization and might reduce crbsi. introduction: empiric antibiotic therapy decisions are based upon a combined prediction of infecting pathogen and local antibiotic susceptibility, adapted to patients' characteristics. the objective of this study was to describe the pathogen predominance and to evaluate the probability of covering the most common gram-negative pathogens in icu patients with respiratory infections. methods: data were collected from multiple us and european hospitals as part of the smart surveillance program ( ). mic (mg/l) testing was performed by broth microdilution, with susceptibility defined as follows for p. aeruginosa & enterobacterales: ceftolozane/tazobactam results: hospitals from countries provided gram-negative respiratory isolates from patients located in an icu in the us ( %), eastern europe ( %) and western europe ( %) in . the most common pathogens isolated were p. aeruginosa ( %), k. pneumoniae ( %), e. coli ( %), and a. baumannii ( %). among enterobacterales, % ( / ) were esbl positive. figure provides the probability of covering the most common respiratory gram-negative pathogens from icu patients. co-resistance between commonly prescribed first line β-lactam antibiotics is common: when nonsusceptibility (ns) of one agent was present, susceptibility to other βlactams was generally < %. ceftolozane/tazobactam provided the most reliable in vitro activity in both empiric and adjustment prescribing scenarios compared to other β-lactam antibiotics. ceftolozane/tazobactam ensured a wide coverage of the most common gram-negative respiratory pathogens demonstrating high susceptibility levels and provided the most reliable in vitro activity in both empiric and adjustment antibiotic prescribing scenarios. further studies are needed to define the clinical benefits that may translate from these findings. evaluation of compliance of icu staff for vap prevention strategies on the outcome of patients a kaur fortis hospital, critical care, mohali, india critical care , (suppl ):p ventilator-associated pneumonia is the most common nosocomial infection diagnosed in adult critical care units. it is associated with prolonged duration of mechanical ventilation, increased icu stay and increased mortality. it continues to be a major challenge to the critical care physicians despite advances in diagnostic and treatment modalities. the primary objective of the study was to determine the compliance of icu staff towards vap prevention bundle and secondary objective was to determine the incidence, risk factors and outcome of vap patients. single center, prospective, observational study carried out from february to july . patients mechanically ventilated for more than hours and satisfying the inclusion and exclusion criteria were enrolled in the study. vap was diagnosed using the cdc criteria and clinical pulmonary infection score. vap preventive strategies were employed and compliance of icu staff was assessed. a total of patients were admitted to icu over the set time period and out of them patients were ventilated for more than hours. among them only patients fulfilled the inclusion and exclusion criteria and were enrolled in the present study. excellent compliance was observed in head end elevation, sedation vacation, stress ulcer prophylaxis, and heat moist exchanger filter use, good compliance in oral care and hand hygiene and moderate to poor compliance in subglottic suctioning. the incidence of vap was . % with a vap rate of . / ventilator days. there was a significant correlation between primary diagnosis, hemodialysis, massive blood transfusion and development of vap (p< . )). mean duration of ventilation (p< . ) and mortality (p< . ) were highly significant in vap patients. conclusions: improvement in compliance towards vap bundle and reduction of risk factors can help decrease incidence of vap and related morbidity and mortality. preventive strategies are effective in reducing ventilation-associated pneumonia (vap) in adults [ , ] . in paediatric population there are no data about vap prevention, so we introduced a new bundle (vap-p) based on the available evidence for adults. this was designed as a before-after study. we enrolled all patients admitted to -bed medical-surgical paediatric icu at gemelli hospital in rome, requiring mechanical ventilation for at least hours. patients with pre-existing tracheostomy were excluded. vap-p has been introduced since in order to improve quality of assistance. our bundle consisted in twice a day oral hygiene with chlorhexidine swab, daily check of oral bacterial colonization and aspiration prevention. comparison was made with an historical group including patients admitted before vap-p introduction (since to ). all data about demographics, antimicrobial therapy, icu stay and treatments, were collected. results: patients were included ( after and before vap-p introduction). ( %) events of vap were recorded in vap-p group compared to ( %, p= . ) vap-p group had less vap per days of mechanical ventilation ( / compared to . / p= . ). multivariate analysis yielded an or of . ( %ci . - . ) for vap incidence after bundle introduction. mortality rate was slightly reduced in vap-p group ( . %vs . % p=ns). patients who developed vap required more days on mechanical ventilation and had higher mortality rate ( vs days p< . and %vs % p= . , respectively). our vap-p seems effective in reducing vap incidence in critically ill paediatric population. introduction: ceftolozane/tazobactam (c/t) is a new antibiotic against mdr gramnegative bacteria infections, whose target population are the critically ill patients. even though / g dose safety administered as a hour-infusion has been already assessed, these patients can be under renal replacement therapy (rrt) and suffer changes in their volume of distribution (vd) that may affect antibiotic concentrations. the objective was to determine concentration reached by g c/t ( hour infusion) in septic patients on rrt (cvvhdf) and interdose behavior. we have used rrt machine prismaflex with oxyris filter and m . hplc-uv method was used for simultaneous quantification of c/t. study population consisted of three obese critically ill patients with sepsis, on cvvhdf while receiving g c/t every hours. samples were taken of prefilter, post filter blood and effluent, min before infusion and , and hours after the end of it. we found great interpatient variability with the lowest cconcentration values in the patient with more hemodynamic instability using oxyris filter. even though cmax was less than reported in healthy subjects, we found similar values of auc and t ½ in comparison with healthy population studies. cmax of t was also compromised in comparison with values reported in healthy subjects, but with higher auc and t ½. cvvhdf contributes to c/t clearance. m filter showed the least clearance and higher values of auc and t ½. extraction rate was similar in all patients and filters (figure ) . cmax achieved may be impaired because of the varying vd caused by obesity and rrt, but not affecting the antibiotic characteristics and behaviour. we conclude that because of the variety of clinical conditions, c-concentration is compromised particularly in hemodynamically unstable patients. however, the small sample doesn´t let us extrapolate these results. the extended infusion seems to be adequate to achieve the interdose antibiotic concentration. the use of biomarkers in sepsis is useful for early diagnosis and prognosis. the desired marker should be sensitive, specific, fast and accurate. procalcitonin (pct) measurement is approved by the fda even its efficacy is still under question. the determination of alfatorquetenovirus (ttv) could be a useful marker [ ] . we analyzed samples from patients admitted to icu with clinical suspicion of sepsis. analytical data of c-reactive protein (crp), neutrophils and procalcitonin were collected. the sofa and apache ii scales were calculated and patients stratified according to these values in good and poor prognosis. ttv quantitative determination was carried by using a quantitative crp . we calculated area under the curve (auc) of ttv plasma levels as a function of time. the statistical analysis involved u-mann-whitney and spearman test, using chi for qualitative variables. results showed a not significant (ns) inverse relationship between the ttv auc and the patient proinflammatory level. a tendency (ns) was found between poor prognosis and the pct median values and crp being higher in the poor prognosis.group. a trend showed lower ttv dna count related to worse prognosis. an inverse relationship was found between pct and crp values and the ttv copies /ml plasma, ns correlation in the case of pct. there was a clear trend between the neutrophils´expansion and the regression line slope, obtained between ttv loads in the first two study steps. fig. (abstract p ) . patient pk/pd measurements value> . ), suggesting that the adsorptive mechanism wasn't primarily mediated by plasma protein. ha was saturated after adsorption of a total of . ± . mg of van. the adsorptive kinetics showed an exponential reduction of van mass that reached a plateau after minutes of circulation. in our study, simulating in vivo conditions of hp using ha during sepsis, a rapid and clinically relevant removal of van has been shown. after hours of hp, we suggest to assess van plasma concentration and a loading dose of van should be considered. however, not knowing the potential interactions with other drugs, further in vivo studies are warranted to confirm these findings. assessing the volume of blood taken for blood culture and culture positivitydo we need to take less blood? it is commonly accepted that larger blood culture (bc) volumes (bcv) increase the yield of true positive cultures, and optimally cc of blood should be obtained per set ( bottles). only scarce data exists on the matter of optimal bcv. it is unknown what is the minimal volume that is acceptable for bc. the objective of this study was to determine the association between bcv and the rate of positive bc. blood taken for cultures in bd bactec plus aerobic/f negative bottles was collected from icus and acute care floors at hospitals at the dmc over months. blood volume was estimated automatically from blood background signal data in the bd bactec fx instrument. cultures were analyzed for each bottle. data was summarized for every month as the average volume and number of cultures taken and rate of positive bc for every unit. units were classified according to unit type (icu, medicine, surgery, mixed, emergency department (ed), organ/bmt or "other" which did not fit the previous categories) and analyzed as a group. a total of cultures were taken in units. there is a positive association between bv and positive bc rate for ed and "other" units (irr= . , p= . for the ed, irr= . , p< . for "other" unit). all other units had no association between bv and positive bc rate (figure ). secondary analysis, excluding pediatric units, gave very similar results. when comparing bv between unit types, the ed and "other" unit had significantly lower bv ( . ml in the ed and . ml in "other" unit compared to . ml in the icu, . ml in surgery, . ml in mixed and . ml in bmt). the correlation between bv and positive bc rate is probably limited to units taking very low bv for cultures. units taking volumes above ml show no improvement in positive bc rate when higher volumes are taken. better prospective studies should be done to further establish the minimal bcv needed and spare unnecessary blood loss to hospitalized patients without compromising bc yield. de-escalating antibiotics in sepsis with the use of t mr in a bed greek university icu c vrettou, e douka, i papachatzakis, k sarri, e gavrielatou, e mizi, s zakynthinos st icu department, university of athens, evangelismos general hospital, icu, athens, greece critical care , (suppl ):p in septic patients, the early use of appropriate empiric antibiotic therapy reduces morbidity and mortality. de-escalation refers to narrowing the broad-spectrum antibiotics once the pathogen and sensitivities are known. t magnetic resonance (t mr) is a novel method of detecting eskape pathogens. we aim at investigating if using t mr technology can expedite de-escalation of broad spectrum antibiotics. this is a prospective observational study conducted in our -bed university icu. inclusion criteria were critically ill patients age> y.o., with newly diagnosed sepsis and clinical suspicion of eskape bloodstream infection. a sample for t mr and a blood culture (bc) sample were collected simultaneously from the patients enrolled. the t mr bacteria panel test was run according to the manufacturer's guidelines and the bcs were processed according to the hospital standard procedures. we recorded clinical data and administered antibiotics. results: patients were included in the study. mean time to culture positivity was hours while mean time to t mr result was . hours. in patients the results of t mr were in concordance with the bcs. in the remaining cases, the bcs were negative while the t mr detected one or more eskape pathogens. there were no false negative results. de-escalation in at least one drug was applied to patients ( . %). no escalation was applied to patients ( . %) and antibiotic escalation in ( . %). conclusions: t mr provides a quicker detection time that could shorten the time to targeted therapy. in our population this corresponded to early (within - h) antibiotic de-escalation in approximately / of the included patients. antibiotic stewardship in icu. a single experience l forcelledo , e garcía-prieto , l lópez-amor , e salgado , j fernández dominguez , m alaguero , e garcía-carús the increasing antibiotic resistance in microorganisms urged interventions such as the antibiotic stewardship programs in icu focused on reducing the inappropriate use of antibiotics by improving the antibiotic selection, the dosage, administration route and length as well as improving clinical outcomes and reducing antibiotic resistance. retrospective study where antibiotic consumption was analysed and measured in days of therapy (dots) between and in a medical-surgical icu of a university hospital where a multimodal educational program was established. specific training in infectious diseases in critically ill patients, periodic clinical and formative sessions fig. (abstract p ) . correlation of blood culture positivity rate with blood culture volume by unit type were performed for icu staff and specific leaders within the icu staff designated. results: patients were admitted to icu. there was a reduction of , % in dots (figure ), reduction in antimicrobial resistance rates ( , in , , in [days of resistant microorganism/ patientdays]) without an impact in icu global mortality ( , % in , , % in ). the resistant bacteria registered were acinetobacter baumannii, s. aureus mr, blee and carbapenemase-producing enterobacteriaceae, pseudomonas aeruginosa mr and clostridium difficile. the safe in antimicrobial consumption was € ( % reduction). the icu stay decreased from , days ( ) to , ( ) , with no variation in mean apache ii ( , ) . the bigger decrease in antibiotic consumption was in colistin related to the reduction in resistance bacteria, in special acinetobacter baumannii, in linezolid and in piperacilin/tazobactam, even more remarkable in due to shortage of supplies which meant an increase in meropenem. the application of an antibiotic stewardship program in icu succeeded in reducing antibiotic consumption, antibiotic resistance and costs without an impact in clinical outcomes like mortality or icu stay. clinical outcomes of isavuconazole versus voriconazole for the primary treatment of invasive aspergillosis: subset analysis of indian data from secure trial p kundu, s kamat, a mane pfizer limited, medical affairs, mumbai, india critical care , (suppl ):p the secure trial was designed to compare the safety and efficacy of isavuconazole (a) versus voriconazole (v) for primary treatment of invasive mould disease caused by aspergillus and other filamentous fungi. the present analysis is aimed at comparing the indian subset of patients with that of the overall trial population and to ascertain any similarity or difference in the primary efficacy endpoint and safety/tolerability in these two groups. in secure trial, patients in one group received (i) & another patients received (v). the indian subset had patients. we have done a qualitative analysis as the sample size of the indian subset was small. non-inferiority of (i) to (v) in terms of all cause mortality from first dose to day was assessed in overall patients. the treatment difference between (i) and (v) group in the indian subset of patients was analyzed. proportion of patients who had to discontinue treatment due to teaes was analyzed. the all-cause mortality in the overall trial population met noninferiority margin (table ). in the indian subset, it was higher for (i) than (v). there was a lower incidence of ocular, hepatobiliary, skin & subcutaneous tissue disorders in the (i) treated patients (see table ). in indian subset, the above adverse events were less in the (i) group, but statistical inference could not be done due to small sample size. however, similar trend of less number of patients discontinuing therapy due to teaes in the (i) treated patients was seen in the overall patients & the indian subset. the all-cause mortality in the indian subset was higher in the (i) patients. a trend similar to the overall population regarding safety parameters favoring (i) was seen in the indian patients. considering the significantly higher prevalence of ia in india, suitably powered study design is necessary to draw definitive conclusions on the non-inferior efficacy & better safety & tolerability of (i) over (v) in patients of ia. introduction: ventilator-associated pneumonia (vap) is one of the most frequent healthcare-associated infections, correlated with increased mortality,extended hospital stay and prolonged mechanical ventilation. considering the latest outbreak of multiresistant a. baumannii infections in the critically ill patients with vap, there is a growing concern regarding challenges of the antibiotherapy in these patients. although ceftazidim-avibactam is considered to have limited effects on a. baumannii, it is reported to have a synergic activity in combination with other antibiotics. we performed a retrospective, observational study which included icu patients diagnosed with vap(cpis > ). oxa a. baumannii was isolated from the tracheal secretions using a rapid molecular diagnostic platform(unyvero a system). patients were divided in two groups according to the antibiotherapy:group a meropenem + colistin and group b meropenem + colistin + ceftazidim-avibactam.statistical analysis was performed using graphpad applying t-test and kaplan-meier curves, having the in-hospital mortality as primary outcome and days of mechanical ventilation and hospital stay as secondary outcomes. mean age(y.o) in group a was and in group b and in both groups mean charlson comorbidity index was points. survival percent was higher in the group treated with ceftazidim-avibactam ( % vs %, p = . )- (fig. ) . length of stay was significantly decreased in group b ( . days vs days in group a, p = . ). number of days under mechanical ventilation was also decreased in the ceftazidim-avibactam group ( vs ) but the data was not statistically significant. in light of the important thread of multiresistant a. baumannii and the lack of therapeutic measures, the synergistic activity of ceftazidim-avibactam use in combination with other antibiotics may be a promising approach to lower the mortality and hospitalization in critically ill patients diagnosed with vap. impact of patient colonization on admission to intensive care on and days mortality g dabar , c harmouch , e nasser ayoub , y habli , g sleilaty , j infections caused by multi resistant bacteria are a major health problem, especially in icus, and it may be associated with high mortality rates. colonization precedes infection in most instances; therefore it may be a marker of a poor outcome. we tried to determine the impact of colonization on mortality at and days in a population of patients admitted to one medical and one surgical icu in the same institution. medical records review over three years - of all patients admitted to one surgical et one medical icu at hotel dieu de france hospital staying more than h. colonization to resistant bacteria was defined as mrsa, esbl, mdr, and vre. all patient received a nasal and rectal screen on icu admission, in intubated patients tracheal aspirate was considered as colonization in the absence of clinical respiratory tract infection. demographics, apache, sofa, immunosupression, charleston comorbidity index, length of stay, mechanical ventilation, hospitalization and antibiotic use in the previous month were collected. mortality at and days was assessed through medical records or phone call. pearson chi-square was calculated for the association of colonization and mortality at and days, and subsequently odd ratio was estimated. introduction: critically unwell patients have been observed to respond unpredictably to traditional intermittent dosing (id) schedules of vancomycin, likely due to the complex physiological derangements caused by critical illness. continuous infusion (ci) of vancomycin has been suggested to overcome such problems by allowing more regular therapeutic drug monitoring and subsequent effective dose titration [ ] . this study conducted at a tertiary intensive care unit, reports our experience following implementation of a continuous vancomycin infusion protocol. prospective data was collected over two consecuative periods of three months, initially capturing plasma levels for id (target level of - mg/l) followed by reviewing plasma concentration levels in a ci protocol (target level of - mg/l). patients recieving renal replacement therapy were excluded. a total of intermittent vancomycin prescriptions were administered and dosing levels observed. in the three month ci period, patients received ci vancomycin and levels subsequently checked. the ci protocol resulted in increased blood sampling ( samples in ci group vs. samples in id cohort). two non serious incidents were reported in the ci cohort relating to preparation of vancomycin. both groups had a comparable median time to therapeutic range ( hours). however, ci vancomycin group had a greater proportion of first samples outside the desired therapeutic range ( %vs %) (figure ). as the therapy continued, ci vancomycin demonstrated a greater propensity towards consistent therapeutic levels than that observed with id. % of patients on a ci regime achieve the desired target levels compared to % in the id cohort (fig. ) . it was positive for single or multiple microbes in ( . %) and ( . %) samples respectively. single or multiple resistance genes were detected in ( %) and ( %) samples respectively. bfpcr was positive only for bacteria in ( . %), virus in ( . %) and for both in ( . %) cases. influenza a was found in ( . %) cases. the most common organisms in community and hospital acquired pneumonia were streptococcus pneumoniae ( / ) and a. baumannii ( / ) respectively. bacterial cultures were concordant with bfpcr in / ( %) of positive cases. decisions to change antibiotics could be taken earlier based on bfpcr (p< . ) than if were based solely on culturesboth in culture positive ( . ± . vs . ± . hrs) and negative cases ( . ± . vs . + . hrs) where antibiotics would have remained unchanged. based on bfpcr antibiotics were escalated in ( %) patients and teicoplanin ( / ) was most often stopped. bal bfpcr were obtained significantly earlier, identified more organisms and bacterial resistance than culture reports and lead to more frequent and earlier antibiotic changes. severe community-acquired pneumonia (scap) is a frequent cause of hospitalization and mortality. ceftaroline is efficacious for treatment of cap (port risk class iii or iv). most severe patients were excluded from the clinical trials, so the efficacy of ceftaroline in these kind of patients is unknown methods: this is a health record-based retrospective before-after study in a tertiary care hospital. all scap patients admitted in icu between november and february receiving ceftaroline were included. control group included patients with same inclusion criteria but receiving ceftriaxone. propensity scores to adjust for potential baseline differences between groups were performed. levofloxacin or azythromicin were administered in both groups. primary outcome was the change in sofa score over the first h and secondary were days of mechanical ventilation, respiratory failure at h, need of rescue antibiotics, length of stay and mortality results: there were patients in ceftaroline group and in ceftriaxone group. baseline characteristics were similar except from more intubated patients in ceftaroline group (figure ). there were less respiratory failure at h in patients with ceftaroline treatment (- . % vs. - . %; p , ), but no differences in other organ failures, mortality, days of mechanical ventilation or los. there were more need of rescue antibiotics in ceftriaxone group ( . % vs . . %; p , ). we found more streptococcus pneumoniae isolation in ceftaroline group ( ( . %) vs ( . %); p = . ); more empiric use of oseltamir ( ( . %) vs ( . %); p = . ), but no more influenzae infections ( ( . %) vs ( . %); p = . ). s. aureus was detected in patient in ceftaroline group and in in ceftriaxone group. introduction: acute respiratory failure (arf) due to pulmonary infections is a usual cause of intensive care unit (icu) admission. immigration patterns and iatrogenic immune-suppression have made tuberculosis (tb) a common disease in western europe. severe tb requiring icu care is rare. nevertheless, mortality associated with active tb and arf is poor [ ] . adult patients with tb admitted to icu from - were identified retrospectively. diagnosis was based on: positive cultures of sputum, bronchial aspirates or bronchioalveolar lavage fluid. demographic characteristics, reasons for admission, hiv status, anti-tb treatment and mortality were recorded. total of patients with tb were admitted to icu. mean apache ii score was , ± , . sixteen were male. mean age , ± , years. eight ( %) were hiv-positive, ( %) diabetes mellitus type , ( %) chronic liver disease. six ( %) had other causes of immunesuppression. main causes for icu admission were arf due to non- mycobacterium tuberculosis pathogens in %, acute liver failure in %, septic shock due to non-respiratory cause in %. overall, % were on anti-tb treatment at time of admission. tb involved the lung parenchyma in all patients. pleural involvement was present in % and lymph node in %. extrapulmonary sites were present in %: urogenital, gastrointestinal, bone marrow. pathogens identified in over-infections: % gram positive coccus, % gram negative bacilli, % fungal, % mdr-pathogen. one patient hiv-positive suffered arf due to pneumocystis jiroveci. overall, % died during icu stay. besides its latent evolution, mortality of tb patients admitted to icu is extremely high. arf due to over-infection seems to be the main cause for icu admission and mortality. better preventive approach of these patients may improve their outcome. introduction: human african trypanosomiasis (hat) is rarely encountered by critical care clinicians, but is an important differential for fever in the returning tropical traveler. late disease is characterized by seizures, fever and multi-organ failure [ , ] . we present an anonymized case presenting from an endemic area in zambia referred for tertiary critical care management. the patient was too obtunded to give informed consent and his relatives could not be contacted despite extensive efforts. a middle-aged man with no past medical history from rural zambia presented to a local clinical officer post with fever and arthralgia. he was treated twice with anti-malarial medication without resolution of symptoms. two months later he was admitted febrile and obtunded to a local hospital with worsening confusion. he was transferred hours by ambulance to our facility in lusaka, which is the only public tertiary critical care unit in zambia results: gcs on arrival was e m v without localizing neurology. microbiology investigations were negative, including for toxoplasma, cryptococcus, hiv or malaria. the patient suffered a generalized seizure followed by a sustained gcs of and was admitted to the icu for invasive ventilation and seizure control. peripheral blood smears demonstrated trypanosomes consistent with hat secondary to trypanosoma brucei rhodesiense. he was commenced on melarsoprol but rapidly deteriorated, with signs of melarsoprol-induced arsenic encephalopathy and subsequent tonsillar herniation. his death was confirmed by neurological criteria. conclusions: icu management of fulminant hat involves supportive neurocritical care plus melarsoprol, a toxic arsenic compound with common side effects of hepatotoxicity and dysrhythmia. arsenic encephalopathy occurs in % of late hat, with a fatality rate of % [ ] . early diagnosis is associated with a % survival rate in developed world travelers repatriated from endemic areas [ ] . lithium chloride to prevent endothelial damage by serum from septic shock patients (in vitro study) a kuzovlev the aim of the study was to investigate into effectiveness of lithium chloride (licl) as agent that prevents damage to the monolayer of endothelial cells under the action of serum from multiple trauma patients with septic shock. methods: serum from pts with septic shock (sepsis- ) and healthy donors was withdrawn. monolayer of ea.hy endothelial cells were incubated for hrs at °c with healthy person's serum and with septic patient's serum without licl and with it at concentrations of . mmol, . mmol, mmol, mmol. licl was added hour before the change of serum. after incubation cells were washed and fixed with % paraform solution and permeabilized with % triton x- solution. fixed cells were stained with primary antibodies to vecadherin and then incubated with secondary antibodies conjugated with oregon green fluorescent dye as well as with phalloid red and hoechst dye . images were processed by fluorescence microscope and imagej . p and metavue . programs. western blotting was used to detect antibodies to ve-cadherin, claudin and gsk- beta. statistics included mann-whitney test and chi-square test. incubation of a monolayer of endothelial cells with % serum of septic shock patients led to loss of ve-cadherin contacts and decrease of claudine. preincubation with licl . mmol did not prevent dismantling of claudine, actin, ve-cadherins; . mmol licl prevented it (p> . ), but at higher concentrations ( mmol, mmol) almost completely protected endothelial monolayer from destruction of intercellular contacts (p< . ). serum had almost no effect on the phospho-gsk- β level after min, min, min and hr, but caused a significant ( %) decrease in its level after and hrs. licl ( mmol) caused a significant increase in phospho-gsk- β already mins and up to hrs after exposure. licl prevents septic damage to the monolayer of endothelial cells in vitro in a gsk- beta mediated way. introduction: the autonomic nervous system (ans) controls both heart rate and vascular tone, which are known to be impaired during septic shock (ss) . acute inflammation is presumed to increase arterial stiffness of large arteries in experimental studies [ ] . the objectives of this work are to verify if standard ss resuscitation modulate mechanical vascular properties and to verify if alterations in these vascular properties and ans activity are correlated. a protocol of fecal peritonitis septic shock and standard resuscitation (fluids and noradrenaline) was applied on pigs. the arterial blood pressure waveform was recorded in the central aorta and in the femoral and radial arteries. the characteristic arterial time constant tau was computed at the three arterial sites, based on the twoelement windkessel model [ ] . the total arterial compliance (ac) and the total peripheral resistance (tpr) were also estimated. baroreflex sensitivity (brs), low frequency (lf, . - . hz) spectral power of diastolic blood pressure, and indices of heart rate variability (hrv) were computed to assess ans functionality. results: septic shock induced a severe vascular disarray, decoupling the usual pressure wave propagation from central to peripheral sites, as shown by the inversion of pulse pressure (pp) amplification, with a higher pp in the central aorta than in the peripheral arteries during shock. the time constant tau together with ac and tpr were independently decreased. a decrease in brs, lf power, and hrv describe an ans dysfunction. after the administration of fluids and noradrenaline, both vascular and autonomic dysfunction persisted and these were found to be significantly correlated. measures of mechanical vascular function and ans activity could represent an useful end-point to guide further clinical investigations and refine our understanding of ss mechanisms, especially under medical treatment. introduction: lipopolysaccharide (lps), is a component of gram-negative bacteria known for its activation of the host immune system. the phospholipid transfer protein (pltp) has previously been shown to promote the binding of lps to lipoproteins, to limit inflammation and to lower mortality following injections of lps or bacterial infection. the aim of the present study was to investigate the role of pltp and lipoproteins in the detoxification of lps from the peritoneal cavity. injection of lps intra-peritoneally (ip) ( mg/kg) to wild type (wt) and pltp knocked-out mice (pltp-ko) (n = per group). mass concentration and activity of lps were quantitated by lcmsms analysis of -hydroxymyristate and lal bioassay, respectively. lipoprotein fractions in plasma were separated by ultracentrifugation (n= vs n = ). following intra-peritoneal injection, clearance of intra-abdominal lps was faster and plasma neutralization was more efficient in wt than in pltp-ko mice ( figure ) . indeed, lps found in plasma of wt mice was proportionally less active, sustaining a higher capacity for wt mice to neutralize lps (figure b) . quantitative dosage of lps in portal blood, minutes after ip injection, revealed that plasma lps associates rapidly with the lipoprotein fraction (hdl plus ldl), and in higher proportions as compared to pltp-ko mice ( [ - ] % vs [ - ] %, respectively; p < . ). in line with previous studies, these observations now indicate that, lps readily associates with lipoproteins in a neutralizing process pltp mediated. finally, even with a heavy lps load ( mg/kg), the bulk of lps was still found in the lipoprotein fraction ( [ - ] %), suggesting that lipoproteins plus pltp in wt mice have a high capacity to detoxify intraperitoneal lps. in a model of peritonitis, lipoproteins and pltp were found to constitute key playors for peritoneal clearance and neutralization of lps. it emerges as a key pathway for the resolution of the inflammatory response in peritonitis. introduction: autotaxin (atx, enpp ) is a secreted enzyme present in biological fluids that catalyses the production of lysophosphatidic acid (lpa). lpa is a bioactive phospholipid evoking various cellular responses in most cell types. upregulated atx levels have been reported in various chronic inflammatory diseases. given the established role of lpa in the inflammatory response, we investigated a possible role for the atx/lpa axis in lps-induced endotoxemia. methods: lps was injected intraperitoneally ( mg/kg) in mice producing % atx levels (atx df/+ , heterozygous null mutant mice), in mice producing - % reduced atx levels upon inducible inactivation (r creer t /enpp n/n mice) and in mice expressing - % increased atx levels (enpp -tg mice). kaplan-meier survival analysis was performed. atx activity was measured using the toos activity assay. results: atx df/+ mice that produce almost % reduced serum atx levels show increased survival compared to their littermate controls. for the inducible inactivation of atx, enpp n/n targeted mice were crossed with the r cre-er t mice and tamoxifen induction enabled temporal control of floxed gene expression. r creer t /enpp n/n mice were more protected against lps-induced endotoxemia compared to control mice. enpp -tg mice overexpressing autotaxin and showing a -fold increase in plasma levels do not display improved survival rates compared to control group. conclusions: atx participates in systemic inflammation, as reduced atx levels in circulation decrease lethality of mice from caused by lps. the excess amount of circulating atx does not exacerbate the systemic inflammatory response to lps. introduction: pneumonia (pn) is a prevalent and severe infectious lung disease. host genetics plays an essential role in the pathogenesis of infectious diseases including pn [ ] . the aim of the study was to analyze the variability of genes associated with neutrophil activation in pneumonia. to identify differential expressed genes (degs) in communityacquired (cap) and hospital-acquired pneumonia (hap) dataset «genome-wide blood transcriptional profiling in critically ill patients -mars consortium» (gse ) from gene expression omnibus was analyzed (logfc≥ . , fdr-corrected p-value< . ). degs associated with neutrophil activation were selected according to gene ontology go: («neutrophil activation»). with the use of gtex portal and blood eqtl browser, we searched for esnps (expression single nucleotide polymorphisms) in whole blood for neutrophil activation genes differentially expressed in cap/hap. these esnps were further analyzed for their association with pn via the global biobank engine (gbe). a total of degs from gse correspond to go: genes ( up-and down-regulated) of which genes were common to cap and hap. functional enrichment of degs based on disgenet detected top- diseases associated with these genes (fdr-corrected p-value< . ): myeloid leukemia, chronic; sepsis; asthma; lung diseases; allergic asthma. for these genes esnps common to gtex portal and blood eqtl browser were identified. more than half of all variants were located on the second chromosome and influenced the expression of tnfaip and il rap genes. among all esnps we identified variants associated with pn in the gbe (table ) . we identified genes related to neutrophil activation, genetic variability of which was associated with pneumonia. sepsis was induced in wild-type c bl mice (n= ) and cse knockout mice (n= ) by i.p. injection of cfu/mice mdr p. aeruginosa. similar experiments were repeated after cyclophosphamide induced neutropenia. survival was recorded for days. mice were sacrificed for determination of bacterial load and myeloperoxidase (mpo) activity as a surrogate marker of myeloid cell recruitment. cytokines were measured in serum by legendplex inflammatory panel. total leukocytes from mice spleens, with or without pretreatment with the h s donor gyy , were incubated with x cfu/ml mdr p. aeruginosa. bacterial clearance was recorded. we observed a significant decrease in survival of cse -/mice as compared to cse +/+ mice ( % vs. %; p: . ). this survival advantage was eliminated in neutropenic mice ( % for both groups, p: . ). cse -/mice had increased pathogen load in the liver ( . ± . vs . ± . , p: . ) and lung ( . ± . vs . ± . , p: . ). mpo activity was lower in cse -/mice in the liver ( ± vs ± , p: . ) and lung ( ± vs ± , p: . ). cse +/+ mice had increased serum levels of il- ( . ± . vs . ± . of cse -/-, p: . ); mcp- ( . ± . vs . ± . , p: . ) and gm-csf ( . ± . vs . ± . , p: . ). phagocytic activity of leukocytes from cse -/mice was reduced compared to cse +/+ mice. this deficit was eliminated after gyy pretreatment (fig. ) . deficiency of host-derived h s leads to increased susceptibility to mdr p. aeruginosa infection due to an inefficient neutrophil chemotaxis and neutrophil mediated phagocytosis. acknowledgement funded by the itn horizon marie-curie european sepsis academy introduction: neuroinflammation often develops in sepsis along with increasing permeability of the blood-brain barrier (bbb), which leads to septic encephalopathy [ ] . the barrier is formed by tight junction structures between the cerebral endothelial cells [ ] . we investigated the expression of tight junction proteins related to endothelial permeability in brain autopsy specimens in critically ill patients deceased with sepsis, and analyzed the relationship of bbb damage and measures systemic inflammation and systemic organ dysfunction. case series included all adult patients deceased with sepsis in the years - with brain specimens taken at autopsy available. specimens were categorized according to anatomical location (cerebrum, hippocampus, cerebellum). the immunohistochemical stainings were performed for occludin, zo- and claudin. patients were categorized as having bbb damage if there was no expression of occludin in the endothelium of cerebral microvessels. results: % ( / ) developed multiple organ failure before death. . % ( / ) had septic shock. the deceased with bbb damage had higher sofa maximum scores ( vs. , p= . ), and had more often procalcitonin levels above ( % vs. %, p= . ). bbb damage in cerebellum was more common in cases with c reactive protein above mg/l as compared with crp less than ( % vs. %, p= . ). absence of zo- expression in cerebral meningeal samples associated with bbb damage ( % vs. %, p= . ). positive blood cultures (n = ) were associated to absence of zo- expression in cerebellar glial cells ( % vs. %, p= . ). in fatal sepsis, damaged bbb defined as loss of cerebral endothelial expression of occludin ( figure ) is related with severe organ dysfunction and systemic inflammation. loss of zo- in endothelial cells associates with bbb damage, and sepsis contributes to zo- loss in cerebellar glial cells. oxylipins are oxidative breakdown products of cell membrane fatty acids. animal models have demonstrated that various vasoactive oxylipin pathways may be implicated in septic shock pathophysiology but these have been poorly studied in humans. oxylipin profiling was performed on serum samples collected on enrolment to the vanish (vasopressin vs. norepinephrine as initial therapy in septic shock) trial. samples were analysed with liquid chromatography-mass spectrometry. patients were followed up until days. results: samples were collected from of ( . %) patients on inclusion to the trial and ( . %) had died by days. non-survivors were found to have higher levels of a number of oxylipins including: , -dihydroxyeicosatrienoic acid (dhet) (p< . ), , -dhet (p= . ), (s)-hydroxyeicosatetraenoic acid (p= . ), -hydroxyoctadeca-pentaenoic acid (p= . ) but lower levels of the precursor eicosapentaenoic acid (p= . ). when corrected for multiple comparisons with the benjamini-hochberg test, only , -dhet remained significant (p= . ). although there was a difference in median , -dhet levels between survivors and non-survivors, many values were below the level of detection (n= / ( . %)). as such, we also analysed - -dhet as a binary variable (figure ). patients with detectable , -dhet were more likely to die (hr . [ % ci . - . ], p< . ) and have a higher median lactate (p = . ) and total sofa score (p< . ) than those patients where baseline , -dhet was undetectable. our study suggests the oxylipin , -dhet may be associated with septic shock severity and -day mortality. these results are consistent with the known vasodilatory actions of this class of oxylipin. more work is needed to confirm its exact role in septic shock and whether this pathway is amenable to therapeutic intervention. introduction: activation of neutrophils is a mandatory stage and a sensitive marker of systemic inflammatory conditions that can lead to the development of multiorgan failure. the aim of the study was to investigate into the antiinflammatory effects of lithium chloride on human neutrophils in vitro. study was carried out on neutrophils isolated from the blood of healthy donors. % of neutrophils were activated by mkm fmlp, % -by ng/ml lipopolysaccharide (lps); then their activity was evaluated by fluorescent antibodies to cd b and cd b degranulation markers. intact and activated neutrophils were treated with a solution of lithium chloride ( mmol). immunoblotting was used to assess gsk b activity in neutrophils. mann-whitney criterion and p< . were used for statistics. results: lithium chloride mmol decreased the level of expression of cd b on intact neutrophils by % (p= . ), cd b by % (p= . ). fmlp increased cd b expression on neutrophils by . times (p= . ), cd b by . times (p= , ). addition of lithium chloride solution to fmlp activated neutrophils reduced the expression of cd b (p= . ) and cd b (p= . ). lps increased cd b and cd b expression by . times (p= . , p= . , respectively); addition of lithium chloride reduced the expression of cd b (p= , ) and cd b (p= . ) on neutrophils. fmlp led to a dephosphorylation of gsk- b by % (p< . ), lithium chloride increased its phosphorylation by % (p < . ). adding lithium chloride to activated fmlp neutrophils restored the level of gsk- b phosphorylation by % compared to controls (p< . ). lithium chloride modulates the inflammatory activation of neutrophils by bacterial components through the phosphorylation of gsk b in neutrophils. human host immune responses to lipopolysaccharide: a comparison study between in vivo endotoxemia model and ex vivo lipopolysaccharide stimulations using an immune profiling panel dm tawfik introduction: sepsis, a leading cause of mortality among critically-ill patients in the icu, recently recognized by the who as a global health burden. patients that suffer from sepsis exhibit an early hyper-inflammatory immune response which can lead to organ failure and death. in our study, we assessed the immune modulations in the human in vivo endotoxemia model and compared it to ex vivo lipopolysaccharides (lps) stimulation using transcriptomic markers. methods: eight healthy volunteers were challenged with intravenous lps in vivo. in parallel, blood from another volunteers was challenged with lps ex vivo. blood was collected before and after hours of lps challenge and tested with the immune profiling panel (ipp) prototype using the filmarray® system. the use of ipp showed that markers from the innate immunity dominated the response to lps in vivo, mainly markers related to monocytes and neutrophils. comparing the two models, in vivo and ex vivo, revealed that most of the markers were modulated in a similar pattern ( %). some cytokine markers such as tnf, ifn-γ and il- β were under-expressed ex vivo compared to in vivo. t-cell markers were either unchanged or up-modulated ex vivo, compared to a down-modulation in vivo. interestingly, markers related to neutrophils were expressed in opposite directions, which might be due to the presence of cell recruitment and feedback loops in vivo. the majority of ipp markers showed similar patterns of expression post-lps challenge in both models, except for several markers related to neutrophils and t-cells. the ipp tool was able to capture the early immune response in the human in vivo endotoxemia model, which is a translational model mimicking immune host response in septic patients. introduction: serum levels of tyrosine kinase receptor mer and its ligand gas predict mortality in septic patients in the intensive care unit. however, whether their early measurement at emergency department (ed) presentation also predicts mortality and organ failure still needs to be clarified. in this multicentre observational study, septic patients admitted to italian eds were included [ ] . at ed presentation blood samples were taken for routine biochemical analyses and serum mer and gas measurement. urinalyses, blood gas analyses and chest x-ray were routinely performed. mortality at and days, as well as the presence of organ damage such as acute kidney injury (aki), thrombocytopenia, pt-inr derangement and sepsis-induced coagulopathy (sic) were evaluated according to baseline levels of mer and gas . in conclusion, neither mer nor gas are early predictors of mortality in septic patients at ed presentation. however, mer independently predicted the development of sic, thrombocytopenia and pt-inr derangement in this population. glycocalyx shedding correlates with positive fluid balance and respiratory failure in patients with septic shock n takeyama, y kajita, t terajima, h mori, t irahara, m tsuda, h kano aichi medical university, department of emergency and critical care medicine, aichi, japan critical care , (suppl ):p endothelial hyperpermeability would play a major role in septic shock related organ failure. the aim of this study is to clarify the relationship between glycocalyx shedding and respiratory failure, sofa score, plasma angiopoietin (ang)- level and patient survival. methods: plasma samples were collected from septic shock patients from admission to icu discharge and healthy volunteers. plasma syndecan (syn)- and ang- were measured and clinical data was also collected. septic shock patients were classified into groups according to the time-course change of syn- levels. excess syn- (> ng/ml) during to days and remaining high following to days were assigned to group i. excess ang- during to days and decreased following to days were assigned to group ii. moderate increase (< ng/ml) during to days were assigned to group iii. results: plasma syn- levels are positively associated with increased ang- levels (r = . , p= . ), suggesting that ang- is involved in endothelial hyperpermeability. fluid balance and ventilator-free days (vfd) are significantly increased in group i as compared with group iii. sofa score, apache ii and patient outcome does not show any differences between groups i, ii, and iii. the positive correlation between glycocalyx shedding and fluid balance indicates plasma syn- may be a valuable marker for endothelial hyperpermeability. the negative correlation between glycocalyx shedding and vfd indicates plasma syn- may be a valuable marker for respiratory failure. the plasma level of syn- for prognosis and organ failure excluding ards in patients with septic shock requires further investigation. serial procalcitonin measurements in the intensive care unit at hiroshima university hospital k hosokawa, s yamaga, m fujino, k ota, n shime hiroshima university hospital, department of emergency and critical care medicine, hiroshima, japan critical care , (suppl ):p introduction: serum procalcitonin (pct) is a promising biomarker for differentiating bacterial infections from other inflammatory states. moreover, including serial pct measurements in the management of acute respiratory infection reduces the duration of antibiotic therapy without increasing the mortality. however, limited real-world information is available regarding the use of pct in intensive care units (icus). we extracted and analysed data from january to december , from all the orders and results of pct measurements in the icu ( beds) at hiroshima university hospital. a total of , pct measurements from icu patients were included. in patients, pct was tested ≥ times during a single icu stay. serial pct measurements showed a fade-out pattern ( [ %] patients), a second day-peaked decrease pattern ( [ %] patients), and a series of negative patterns ( [ %] patients). compared to patients who demonstrated the fade-out pattern, those who demonstrated the second day-peaked decrease pattern had higher mortality rates ( % vs. %, p < . ). approximately one-third patients in the icu who had decreasing serial pct values demonstrated the second day-peaked decrease pattern. since this group of patients had poorer survival, further studies are needed to clarify the association between a late rise in pct levels and delayed therapeutic intervention. the research was performed on full-term newborns; no clinical signs of bacterial infection were diagnosed. on the , , days the plasmà concentration of il- ß, il- , il- , tnf-α, g-csf, sfas, fgf, no was determined by capture elisa; cd cd , cd cd , cd cd , cd , cd , cd , hla-dr, cd , cd , cd cd , lymphocytes in apoptosis -immunophenotype analysis. by applying the statistical cluster population analysis of the immunological criteria under study we have evaluated the feasibility of sepsis diagnostics at the admission to the intensive therapy unit. the diagnostic rule for sepsis has been formulated by applying the "decision tree" approach to the "r" statistic medium. the cluster analysis confirms the presence of two clusters (presence of absence of sepsis: these two components explain the . % of the point variability). the diagnostic rule for the early diagnostics of sepsis is as follows: disease develops providing during the first hours cd ≥ . %, no≤ . mkmol/l or cd ≤ . %, cd ≤ . %, cd ≥ . % or cd ≤ . %, cd ≤ . %, cd ≤ . % and lymphocytes annexinv-fitc+pi-≥ . %. newborns featured the confirmed sepsis development. the accuracy of this diagnostics amounts to . %; sensitivity to . %; specificity to . %; diagnostic false positive share to . %; diagnostic false positive share to . %; positive result accuracy to . %; negative result accuracy to . %. the aggregate determination of cd , cd , annexinv-fitc+ pi-, cd and the plasma concentration of no enables the pre-clinical diagnostics of sepsis development. efficacy of pancreatic stone protein in diagnosis of infection in adults: a systemic review and metaanalysis of raw patient data j prazak , p egimann , i irincheva , mj llewelyn , d stolz , lg de guadiana-romualdo , r graf , t reding , hj klein , ya que fig. (abstract p ) . impact of h lactate and bio-adm values in patients with elevated lactate level at admission. the green curve in the left km-plot illustrates data from patients with events; the red curve patients with events. the green curve in the right km-plot illustrates data from patients with events; the red curve patients with events. of note, differences in numbers between admission (n= ) and h (n= ) is related to initial mortality introduction: adrenomedullin (am) is a peptide synthesized in vascular endothelial cells and cleared by the lungs. the use of am as an inflammatory biomarker and his predictive value has been studied in critically ill patients, but not yet in veno-venous extracorporeal membrane oxygenation (ecmo). the purpose of this study was to describe the plasmatic levels of am in patients supported with ecmo for acute respiratory failure methods: am (normal values < . nmol/l) was measured at time points: immediately before (t ), -h (t ) and -h after (t ) ecmo initiation and immediately before (t ) and -h (t ) after ecmo removal, in consecutive patients with severe respiratory failure supported with ecmo enrolled in the gatra study (nct ) at fondazione irccs ca' granda -policlinico of milan. data are reported as median ( th - th percentile). statistical analysis was performed using logistic and random effects regression models (to account for repeated measurements within individuals) results: a total of measurements were taken in consecutive patients. am (nmol/l) decreased along the course of ecmo: t = . ( . - . ), t = . ( . - . ), t = . ( . - . ), t = . ( . - . ), t = . ( . - . ) (mean diff.= - . , %: ci - . , - . ). am was lower in patients with viral compared to bacterial ards (mean diff.= - . , %ci - . , - . ) (figure ). am was higher in more severe patients (sofa>= , n= ) compared to less severe patients (sofa< , n= ): . ± . vs . ± . nmol/l, respectively p< . . basal values of am could not predict mortality at days (or= . , %ci: . - . ) after conditioning for sofa score and respiratory failure etiology conclusions: am plasmatic values seem to be higher in more severe patients and in patients with bacterial ards. am decreased along the ecmo course but could not predict mortality in our group of patients fig. (abstract p ) . plasmatic adrenomedullin during ecmo heparin binding protein (hbp) is released from activated neutrophils upon stimulation of b integrins. this pro-inflammatory effect generates the hypothesis that it can be a sepsis biomarker for patients admitted at the emergency department (ed) methods: the prompt study (clinicaltrials.gov nct ) took place at the ed of six greek hospitals. participants were admitted with suspected acute infection and at least one vital sign change. hbp was measured by an enzyme immunosorbent assay in plasma. sepsis was diagnosed by the sepsis- criteria. the primary study endpoint was the sensitivity for the diagnosis of sepsis. outcome prediction was the secondary endpoint. a total of patients were enrolled; had sepsis. the most common infections among patients without and with sepsis were upper respiratory tract infections in . % and . %; community-acquired pneumonia in . % and . %; and acute pyelonephritis in . % and . %. median hbp was . and . ng/ml respectively (p: . ). following analysis of the area under the curve (auc) it was found that the best discriminatory cut-off for sepsis was . ng/ml. the comparative diagnostic performance of hbp versus qsofa score is shown in figure . the odds ratio for sepsis with hbp above . ng/ml was . (p: . ). at the same cut-off point the sensitivity, specificity, positive predictive value (ppv) and negative predictive value (npv) for the prediction of early death after hours was %, . %, . % and % respectively. hbp is more sensitive but less specific than qsofa for the diagnosis of sepsis in the ed. the rule-out prediction of early death seems the great merit. chronobiological and recurrence quantification analysis of temperature rhythmicity in critically ill patients introduction: rhythmicity and complexity of several circadian biomarkers, such as melatonin, cortisol and temperature have been found to be modified by critical illness. we examined the potential alterations of core body temperature (cbt) fluctuations and complexity in three groups (n= ): patients with septic shock upon icu admission (group a, n= ), patients who developed septic shock at icu hospitalization (group b, n= ) and controls (group c, n= ). the hourly, average cbt was computed for h upon icu admission and discharge in groups a and c, as well as during septic shock onset in group b. cosinor analysis of cbt curves was performed leading to the estimation of mesor (mean value), amplitude (the difference between peak and mean values) and acrophase (phase shift of maximum values in hours). complexity of cbt signals was evaluated with recurrence quantification analysis (rqa). no significant alterations in any circadian feature within groups were found, except for amplitude. controls exhibited increased entry cbt amplitude ( . ± . ) compared to groups a ( . ± . , p < . ) and b ( . ± . , p < . ). higher entry cbt amplitude in groups b and c was related with lower saps ii (r = - . and - . , p < . ) and apache ii scores (r = - . and - . , p < . ) respectively, reduced icu and hospital stay in group b (r = - . and - . , p < . ) and entry sofa score in group c (r = - . , p < . ). recovery cbt time series appeared more periodic in relation with icu entry, for all groups. a more random cbt signals pattern upon results: among . . individuals, . received inpatient treatment for sepsis. % had severe sepsis. % of sepsis and % of severe sepsis patients had an explicitly coded hai. the proportion of hai was higher in patients that received icu-treatment than in patients without icu-treatment ( % in icu/ % in non-icu sepsis, % in icu/ % in non-icu severe sepsis patients). tab. shows the foci of explicitly coded hai. nosocomial pneumonia was the most common hai in all patient groups. clabsi occurred more frequently in icutreated patients; % were affected. cauti and c. diff infections were more common among non-icu-treated sepsis patients. more than one quarter of non-icu-treated sepsis patients had a c. diff infection. hai are common causes of sepsis and pose a significant healthcare burden. the proportion of patients affected and the distribution of foci differ between non-icu-and icu-treated sepsis patients with important implications for sepsis management within hospitals. impact of sepsis protocol triggered by ramathibodi early warning score (rews) in ipd sepsis on clinical outcomes s matupumanon , y sutherasan , d junhasawasdikul , p theerawit sepsis is now early identified and managed during triage in the emergency department. however, there is less focus on the effect of patients' management at the ward level. we aim to evaluate the impact of the implementation of the sepsis protocol on clinical outcomes in in-patients with new-onset sepsis. we conducted a prospective observational cohort study among adult medical patients admitted to the general wards in a university hospital. a -month pre-protocol period (august to august ) was assigned to a control group, and a -month protocol period (september to october ) was allocated to a protocol group. an in-patient sepsis protocol comprised nurse-initiated sepsis protocol by ramathibodi early warning score (rews)≥ plus suspected infection, prompt antibiotic, lactate measurement, and fluid resuscitation was implemented. (table ) . the implementation of in-hospital sepsis protocol was associated with significant improvement in patients' outcomes, namely lactate measurement, starting antibiotic within hr, fluid management, and the shorter length of icu stay. icu routine nursing procedures interfere with cerebral hemodynamics in a prolonged porcine fecal peritonitis model sl liu , dc casoni , w z'graggen , d bervini , d berger , sj jakob routine nursing procedures (np) can interfere with blood pressure and cardiac output and may therefore alter cerebral hemodynamics in critical illness. this may be risk factor of sepsis-associated encephalopathy. methods: sedated and mechanically ventilated pigs were randomized to fecal peritonitis or controls (n= , each). after hours of untreated peritonitis, the animals were resuscitated for hours (resuscitation period). np [assessment of sedation (as), tracheal suctioning (ts), change in body position (cp), lung recruitment maneuver (rm)] were performed at baseline and h, h, h and h after start of rp. systemic and cerebral hemodynamics and o saturations were recorded continuously. shock is the most common cause of death in the postsurgical icu, including septic shock and hypovolemic shock, reaching the - % mortality in septic shock. the inadequate response of the immune system to the infection triggers a potent inflammatory cascade, where the c-reactive protein (crp) is an essential key in the amplification and maintenance of this cascade. the gene encoding to crp is located on the proximal long arm of human chromosome ( q ). the gt polymorphism in the promoter sequence of crp gene (rs ) has been associated with invasive pneumococcal disease. thus, we analyze the relationship between rs polymorphism and the risk of developing septic shock in postsurgical patients. an observational, retrospective and single-center study was conducted on a sample of caucasian patients undergoing major abdominal surgery, of which one part developed septic shock and another part developed systemic inflammatory response syndrome, who were used as control. the rs polymorphism was analyzed by vasoactive medications are commonly used in sepsis treatment but may correlate with peripheral ischemia and the well-publicized complication of limb and digit loss. yet, the association between limb and digit threat and the intensity, duration, and pattern of vasopressor exposure are unknown. we studied adults ( - ) at hospitals in an integrated health system who met criteria for sepsis- . we identified the time to clinically apparent limb or digit threat using clinical adjudication among those with vasopressor-dependent sepsis (i.e. > hour of vasopressors at sepsis onset) who had a surgical evaluation within -days of sepsis onset. we defined daily vasopressor intensity as to vasopressors administered. then, we created a time-dependent model for threat with mortality as a competing risk with a weight function to estimates the varying contribution of vasopressors over time. we determined the subdistribution hazard (sh) ratio of threat for various patterns of vasopressor exposure and intensity, adjusted for age, baseline risk factors, and sequential organ failure assessment (sofa) score at sepsis onset. of , adults with sepsis, , ( %) were vasopressordependent (age, [iqr, - ]; , [ %] males; max sofa score, [sd ] ). of these, , ( %) died and ( . %) had evaluations for limb or digit threat [iqr, - ] days after sepsis onset. the model-based weight function showed the contribution of vasopressors to threat was stable over time ( fig a) . overall, a unit increase in cumulative vasopressor exposure was associated with risk of threat (sh ratio, . [ %ci, . - . ], p<. ). for various patterns of vasopressor exposure, greater intensity associated with increased risk of threat ( fig b) . compared to constant exposure, an increasing and peak pattern associated with the greatest sh (fig c) . cumulative vasopressor exposure was associated with an increased risk-adjusted hazard of limb or digit threat following sepsis. fig. (abstract p ) . relationship between vasopressor exposure and limb or digit threat following vasopressor-dependent sepsis. panel a demonstrates the estimated contribution of daily vasopressor intensity prior to surgical evaluation for limb or digit threat, with mortality as a competing risk. panel b and c explore the relationship between threat and both cumulative vasopressor exposure and the pattern of exposure following sepsis onset. (b) the maximum cumulative vasopressor exposure was associated with the highest risk of limb or digit threat (shr . ) when compared to reference exposure pattern (shr . , reference). (c) increasing (shr . ) and peak (shr . ) patterns of cumulative exposure were associate with an increased sh of limb threat, while a decreasing pattern was associated with a lower risk (shr . ) when compared to constant intensity (shr . , reference). abbreviations: shr: subdistribution hazard ratio proportion of encounters transitioning from phenotype at presentation within hrs, by arrival phenotype assignment and probability of membership. (c) tsne plots for α-type, ß-type, y-type, and ∂-type, with core (dark), marginal (light), and non-members (grey) in plots on the left and core, marginal, non members, and transitioning members (black) on the right fig. (abstract p ). isolated microorganisms critical care references: . wertz et al. critical care explorations : e the process investigators choosing wisely guidelines for the provision of intensive care services, version . ics structured patient handovers references: . care of the critically ill woman in childbirth the proqol manual: the professional quality of life scale:compassion satisfaction, burnout & compassion fatigue/secondary trauma scales references: . shimabukuro-vornhagen a et al. ca the code: professional standards of practice and behaviour for nurses, midwives and nursing associates p introduction: the aim of this study was to compare factors associated with the icu mortality for vap due to multidrug-resistant (mdr) klebsiella spp. in case of monobacterial (mo) vs polibacterial (po) origin. methods: retrospective data analysis of patients treated in icu with mdr klebsiella spp. strains as pathogens of vap during three year period was carried out. results: data of patients were evaluated. mo vs po of mdr klebsiella spp. vap cases was found to be ( . %) vs ( . %), p = . . the icu mortality was / ( . %) in mo, and / ( . %) in po one, p = . . statistical significant differences of survivors vs non-survivors in mo and po vap due to mdr klebsiella spp. were found in medians of neutrophilosis p introduction: we study the population structure and resistome of mdr enterobacterales and pseudomonas aeruginosa isolates, c/t-susceptible or -resistant, recovered from low respiratory, intraabdominal and urinary tract infections of icu patients of portuguese hospitals (step study results: in e. coli, two vim- producers were found (st -b -h -o :h -ctx-m- and st -c-h -o :h ) (c/t-mic= . / - / mg/l). a kpc- -st -cladev-h -o :h ( / mg/l) was also detected. the most frequent esbl-e. coli clone was st cpr klebsiella pneumoniae ( patients), candida spp. ( patients). the comparison subgroup consisted of patients with bacteremia caused by non-escape pathogens. we evaluated the days of mechanical ventilation, duration of antibiotic therapy (amt), icu length of stay (los), hospital los and mortality (table ). results: mortality in patients with bacteremia caused by non-eskape pathogens was . %, candida spp vancomycin mass removal over minutes of hemoperfusion using ha . bars refer to vancomycin mass (mg): blue (experiment ) and red (experiment ) bars using blood while green (experiment ) bar using balanced solution. yellow dashes are mean mass values of the three experiments (with standard deviations) and yellow line represents the reduction curve over time table (abstract p ). results. * p-value versus non-eskape subgroup mechanical ventilation p translational value of the microbial profile in experimental sepsis studies sp tallósy , a rutai , l juhász , mz poles , k burián , d Érces , a szabó , m boros invasive hemodynamic monitoring and blood gas analyses were performed on anesthetized animals between - h of sepsis. the respiratory, cardiovascular, renal, hepatic and metabolic dysfunctions were evaluated with the species-specific sequential organ failure assessment (sssofa) score, the microbial profile was determined with selective media and maldi-tof ms in the initial inoculum and in the abdominal fluid taken h after sepsis induction. results: strong correlation was found between the initial dose of the inoculum (cfu) and the sssofa scores for organ dysfunction (rats: r = . , p= . ; pigs: r= . , p = . ) p introduction: pancreatic stone protein (psp) has shown promise as a biomarker of infection however, its diagnostic potential has not been systematically evaluated. we performed a systematic review and meta-analysis of available data on psp to evaluate its value for detecting infection in adults and determining a plasma or serum threshold value. methods: the pubmed and cochrane library database were searched for studies on psp in adult patients and their raw data were analyzed to estimate the best psp cut-off value that could detect infected patients using the youden's index. the cut-off sensitivity, specificity, positive predictive value (ppv) and negative predictive value (npv) were computed and compared to those for procalcitonin (pct) and c-reactive protein (crp). finally, we explored the potential value of a model combining all three biomarkers to detect infection. results: from a total of potentially eligible published studies, containing patients were included in quantitative analysis. among them, patients suffered from a clinically confirmed infection. the median appropriate statistical tests were used using spss . cd was expressed as % age of neutrophils expressing positivity. results: sixty patients were analyzed. all parameters were compared between survivors and non survivors. demographics were comparable. most common source of sepsis was lungs and majority were admitted due to medical reason. non-survivors had significantly increased number of days with septic shock. at day median values of all the biomarkers and the sofa score were significantly higher in the nonsurvivor group (p< . ). there was a decreasing trend of all biomarkers and sofa score amongst survivors. on multivariate logistic regression analysis, increased cd and crp levels between baseline and day , increased days with septic shock and increased sofa references: introduction: we characterized the association of c-reactive protein (crp) with extracellular vesicles (evs) in plasma from sepsis patients and assessed a commercial crp adsorbent (pentrasorb, pentracor, hennigsdorf, germany) to deplete free and ev-associated crp. in addition, we characterized the potential pro-inflammatory effects of ev-bound crp on monocytes and endothelial cells monocytes and human umbilical vein endothelial cells (huvecs) were stimulated with isolated evs ( , g, min) monocyte il- secretion was quantified by elisa; the activation of huvecs was assessed by their expression of icam- and e-selectin using confocal microscopy. results: septic plasma (n= ) contained . ± . mg/l crp vs. . ± . mg/ l for healthy controls (n= ). both, total evs and crp + evs were significantly elevated in septic plasma as incubation of septic plasma with pentrasorb resulted in depletion of free crp ( . ± . mg/l before vs. . ± . mg/l after adsorption) as well as in a significant reduction in crp evs from crp-depleted septic plasma induced significantly lower il- levels. huvec icam- or e-selectin expression, however, did not increase upon stimulation with septic evs. conclusions: treatment of septic plasma with pentrasorb efficiently removes free crp and detaches crp from the ev surface, resulting in reduced proinflammatory effects flow cytometry confirmed the association of monocytes with platelets and platelet-derived evs as well as the uptake of evs by monocytes. conclusions: storage of isolated monocytes induces a shift towards cd expressing proinflammatory monocytes, which seems to be mediated by residual platelets and platelet-derived evs. it remains to be clarified whether evs released from activated platelets can also trigger a shift towards proinflammatory, intermediate monocytes in vivo ethical approval was provided by ucl research ethics committee ( / ). paired parametric analyses were performed and data displayed as mean +/- % ci. results: plasma calprotectin concentration began to increase . hours after endotoxin administration, was significantly higher than baseline by hours ( . ng/ml vs. ng/ml, p < . ), peaked at hours (mean ng/ml, figure ) and normalized by hrs. calprotectin peaked earlier than comparator soluble mediators (procalcitonin hrs, crp, hrs) and exhibited % sensitivity; all participants demonstrating a minimum -fold increase from baseline (mean . x). calprotectin displayed greater baseline variability (sd . ng/ml) than either crp or procalcitonin. conclusions: our results indicate the potential of plasma calprotectin as a biomarker for bacterial infection. it increases earlier and peaks more rapidly than standard biomarkers. whilst higher baseline variability was observed p a multicenter randomized controlled study on landiolol for the treatment of sepsis-related tachyarrhythmia: subanalysis of the j-land s study o nishida kagoshima university graduate school of medical and dental sciences, department of emergency and intensive care medicine methods: we analyzed a retrospective cohort of electronic health records from adult sepsis patients at upmc hospitals from to . we defined sepsis- by i.) suspected infection (e.g., administration of antibiotics or body fluid culture) & ii.) organ dysfunction (e.g., or more sofa points) in the first hours of care. data were organized by hour and included vital signs, lab values, and treatments (e.g., total hourly iv fluids (ml) and norepinephrine equivalent dose). for each hour we describe, i.) available data elements, ii.) presence of sepsis- , and iii by hour , most patients had vital signs ( %; n= , ), basic labs ( %; n= , ), fluid cultures ( %, n= , ), while serum lactate was completed in % (n= , ) conclusions: early sepsis care patterns are variable. iv fluids were given during early hours, when uncertainty about sepsis was greatest, while vasopressors were administered after sepsis- elements were present. p effects of abdominal negative pressure treatment on splanchnic hemodynamics and liver and kidney function in a porcine fecal peritonitis model sl liu department of intensive care medicine splanchnic hemodynamics and laboratory parameters were measured at baseline (bl, start of rp), and h, h and h after start of rp. two/three-way rm-anova or mixed-effects analysis, and student t tests were performed. results: npt in controls had no effect. after sepsis induction, mean arterial pressure (map) decreased by ( - ) mmhg, cardiac output (co) by . ( . - . ) l/min, and arterial lactate increased by . ( . - . ) mmol/l. sepsis and resuscitation was associated with increasing hepatic and renal arterial flows (p≤ . , both), and increasing prothrombin time npt in sepsis resulted in numerically less noradrenaline administration ( . ± . ug/ min/kg in sepsis with npt vs. . ± . ug/min/kg without npt, p= . ) and positive fluid balance ( . ± . ml/h/kg with npt vs. . ± . ml/h/kg without, p= . ). conclusions: in our experimental fecal peritonitis model, npt did neither impair splanchnic hemodynamics nor abdominal organ function. whether npt helps to reduce noradrenaline and volume administration in abdominal sepsis should be evaluated in further studies. p association between a c-reactive protein gene polymorphism (rs ) with the risk of develop septic shock in postsurgical patients of major abdominal surgery p martínez-paz valladolid, spain; hospital of medina del campo notably, the three groups received a comparable pro kg dose of acetaminophen. no difference was found between groups in term of toxic effects. patients carrying the cyp a p showed a more pronounced effect on body temperature in respect of wt and ugt a p °c respectively, but it does not reach statistical significance (fig. b). only % of the patients reach a temperature < °c at t and only % < . °c. conclusions: polymorphisms in enzymes involved in the metabolism of acetaminophen are relatively common. cyp a p seems to lead to higher peak plasmatic concentration and a slightly increased efficacy in fever control panel a: variations of acetaminophen plasmatic levels after minutes (t ) and hours (t ) after administration of an iv dose of g of paracetamol in wt patients and patients carrying mutation; panel b: body temperature variations in wt patients and patients carrying mutations clinical research, investigation, and systems modeling of acute illness (crisma) center, department of biostatistics we determined phenotype cohesiveness using probability of assignment at presentation, defining core members as ≥ % and marginal as < % probability. we determined how members transitioned to other phenotypes over hrs using t-distributed stochastic neighbor embedding (tsne) plots and determined the odds ( %ci) of transition. results: we studied , adult sepsis encounters (median age c) the odds of ever transitioning from presenting phenotype increased significantly for marginal members vs publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we thank the department of education of the basque government (piba - ) and the university of the basque country upv/ehu (ppg / , giu / ) for their financial support. a great disaster affects the family-and friend-performance of bcpr by diminishing the willingness of family and friend bystanders to follow the instruction provided by dispatchers. the experimental method ifitem could be an alternative of fibtem in cases when internal coagulation pathways assessment is prioritized (i.e. heparinized patients on extracorporeal supports). patients undergoing limitation of life-sustaining therapy had lower karnofsky scale scores. therefore, this scale may be useful to guide end-of-life decisions in the future, but further studies with larger number of patients are needed. readmission after discharge home from critical care: a qualitative study c robinson , f nicolson , p mactavish , t quasim , jm mcpeake nhs greater glasgow and clyde, nhs greater glasgow and clyde, glasgow, united kingdom; university of glasgow, nhs greater glasgow and clyde, glasgow, united kingdom critical care , (suppl ):p readmissions to acute care occur in a high number of critically ill patients within days of hospital discharge [ ] . biomedical drivers such as frailty and pre-existing co-morbidities have been identified as drivers for readmission. however at present there is limited data on the influence of social problems on readmission. this study, using a grounded theory approach, sought to understand from a patient/caregiver perspective what the drivers for readmission to acute care were. ethical approval was granted from the west of scotland research ethics service ( /ws/ ). a grounded theory approach was used to explore from a patient and caregiver perspective what the drivers for readmission are [ ] . using a clinical database, we identified those patients who had an icu admission ≥ days who were readmitted to acute care within days of hospital discharge. the researcher attended the ward and after discussion with the direct care team conducted a semi-structured interview with patient and/or caregiver. the interview was recorded and transcribed verbatim. the transcripts were analysed to generate initial codes, followed by the development categories and sub-categories. theoretical sampling was undertaken. results: participants were interviewed. ( . %) were patients and ( . %) were caregivers. the themes that have emerged from the data were: pain and polypharmacy; lack of social support and/or isolation; strained relationships with primary care providers and information provision across the patient journey. subsequent theory development is underway to understand how this learning could help reduce readmissions in future. in conclusion, both social and biomedical drivers are likely to contribute to acute care readmission in this group. future interventional work is required in order to identify modifiable factors to reduce this burden for patients and the healthcare service. frailty has shown to have prognostic relevance for patients with critical illness. since a wide range of tools has been described to screen for frailty, we aimed to describe the association of two frailty screening tools, the clinical frailty scale (cfs) score and the modified frailty index (mfi) in critically ill patients. we performed a post-hoc analysis of a multicenter cohort of patients admitted to six canadian intensive care units (icu) between february and july . frailty was identified using the clinical frailty scale (cfs) and the modified frailty index (mfi). concordance of the frailty screening tools was evaluated with partial spearman rank correlation and intraclass correlation (icc). discrimination and predictive ability of the tools for hospital mortality, -year mortality, hospital readmission and adverse events were compared using concordance statistic (c-statistic) and calibration plot adjusting for age, sex, sequential organ failure assessment (sofa) score and icu admission source, respectively. the cohort included patients. prevalence of frailty was . % ( % confidence interval [ci] . %- . %) with the cfs and . % ( % ci . %- . %) with the mfi. concordance between the two tools was low [(icc of . ; % ci . - . ) and partial correlation coefficient of . ( % ci . - . )], even after adjustment. hospital and -year mortality were greater for frail compared to non-frail patients using of both tools. similarly, both tools found frail patients were less likely to be living independently after hospital discharge, and more likely to be rehospitalized when compared to non-frail patients. while the cfs and mfi show low concordance, both showed good discrimination and predictive validity for hospital mortality. both tools identify a subgroup of patients more likely to have worse clinical outcomes. the post-intensive care syndrome (pics) is a myriad of physical, psychiatric and cognitive disorders secondary to critical illness, leading to a decreased quality of life and an important socioeconomic burden. this study aimed to identify if the conformity to a pics prevention bundle was able to reduce the incidence of the syndrome at icu discharge. all patients admitted to the icu from january st to december st were included. the conformity to each of the ten components of the pics prevention bundle was assessed daily, and the patients were evaluated for anxiety, depression, cognitive dysfunction, muscular weakness, mobility impairment and nutritional risk at icu discharge and at a -to- -months follow-up consultation. the patient cohort was divided in terciles according to bundle conformity for the analysis. results: from the enrolled patients, ( %) were evaluated at icu discharge, and ( %) attended to the follow-up consultation. there was no difference in baseline characteristics between the cohorts. there was no correlation between the prevalence of pics at discharge and bundle conformity during icu stay ( % vs. % vs %, p . ), though there was a decrease in nutritional risk and days in mechanical ventilation (table ) . after to months there was a reduction on the prevalence of any kind of pics, mobility impairment, muscular weakness and nutritional risk. the patients that developed pics were older and had a higher simplified acute physiology score iii at icu admission. a higher adhesion to a pics prevention bundle was not able to prevent the occurrence of the syndrome. post intensive care syndrome (pics) is well recognized following general icu care [ ] . intensive care syndrome:promoting independence and return to employment (ins:pire) is a multidisciplinary complex intervention designed to address pics [ ] . with a paucity of evidence on pics after cardiothoracic intensive care, we aim to evaluate pics and the feasibility of the ins:pire intervention in this population. those attending the clinic received weeks of intervention including individual appointments with icm nurse, physician, pharmacist, and physiotherapist. a café area facilitated peer support alongside psychology group sessions. primary outcome was quality of life measured by eq- d- l. further surveys included: pain, mental health, and selfefficacy. questionnaires were taken at baseline, and months. results: over cohorts, patients attended, % male, median age years (iqr - ), median apache score of (iqr - . ), and median icu length of stay was days (iqr - ). a total of ( %) patients completed surveys at one year. scheduled admissions represented % of those attending. mean euroqol eq-vas score was / (sd +/- ) at baseline increasing to / (sd +/- ) by year (table ) . those with problems in at least one domain of eq- d- l fell from % at baseline to % at -year with the breakdown shown in table . severe problems were seen in % falling to % at year. hads demonstrated an anxiety or depression rate of %. brief pain inventory identified patients ( %) with ongoing chronic pain. mean self-efficacy was / (sd +/- ) at baseline and / (sd +/- ) at year. cardiothoracic intensive care patients have ongoing and persistent features of pics with significant effects on health-related quality of life. further, the ins:pire multi-professional complex intervention is feasible within this specialist group. screening approach might be implemented whenever screening of the total icu population is not deemed feasible. influenza is an acute viral illness with a significant financial burden. point of care testing for influenza is available and has demonstrated accuracy [ , ] , the current gap in knowledge is the question around the opportunity cost of influenza testing. if poct is financially a less costly test this could free up scarce resource. the study adopts a cost minimisation approach. the point of care test is the roche cobas® liat® machine which can detect flu a/b and is compared with the west of scotland specialist virology centre's established in house multiplex real time pcr assay.the model was developed using microsoft excel and has arms comparing analysis of the above mentioned tests. the model estimates that the total cost of poct per patient tested is £ . compared with £ . for lab testing ( figure ). this is a saving of £ . per patient when poct is used. the result swings in favour of the lab test when poct specificity falls to . %. if the lab could provide the result of influenza testing within hours the result would swing in favour of lab testing. zanamivir which will potentially be used increasingly in the intensive care setting can more than double the difference between the tests in favour of poct. this research suggests that poct offers potential cost savings in the icu setting. this is the case as long as poct specificity is higher than a threshold of . % and the lab take longer that hours to return the result. the sensitivity analysis should allow for external validity given the usual variations in icu practice. the aim of the present study is to describe the demographic, clinical, microbiological aspects and the outcome of patients with intensive care unit-related (icu-related) bacteremia. moreover, we aimed to study the patient outcome in association with colistin susceptibility. retrospective, single-center study in a -bed icu for months, from / / to / / . icu-related bacteremia was defined as bacteremia in patients with icu stay > hours or icu readmission (first admission ≥ month before). only the first episode of bacteremia was considered. the primary outcome was -day mortality. data regarding clinical, demographic and outcome characteristics were retrieved from the patient files. the hospital's ethics committee approved the present protocol. moreover, the patients with bacteremia due to colistin-resistant pathogens were compared with the patients affected by colistin sensitive microbes. forty episodes of gram-negative icu bacteremia were collected during the aforementioned period in patients ( . % male) with a mean age and apache ii of . ± . years and ± . , respectively. the event had taken place at an average of . days. the responsible isolates were resistant to carbapenems in . % of the episodes. the majority of the events were due to a single isolate ( %). acinetobacter baumannii and klebsiella pneumoniae presented the majority of the implicated microbes ( % and . %, respectively). the crude -day mortality was %. finally, we could not detect any difference in mortality between the colistin sensitive and the colistin-resistant pathogens ( figure ). the present study denotes that, in a setting of extremely drugresistant pathogens with limited treatment options, gram-negative bacteremia in the icu is associated with increased mortality. image : characterization of resistance mechanisms affecting ceftolozane/ tazobactam in enterobacterales and pseudomonas aeruginosa icu isolates using whole genome sequencing (step study) m hernández-garcia , cc chaves , jm melo-cristino , ds silva , ar vieira , mp f. pinto , jd diogo , eg gonçalves , jr romano , rc cantón hospital ramón y cajal-irycis, microbiology department, madrid, spain; introduction: clostridium difficile infection (cdi) is the main cause of hospital acquired diarrhoea [ ] . the aim of this study was to compare characteristics of cdi during yr and . a retrospective observational study was carried out in lithuanian university of health sciences hospital -the largest teaching facility of tertiary care in country. according to department of infection control records, patients (pt) with (w.) diarrhoea and the first positive stool test for c.difficile toxin a/b were included. age, charlson comorbidity index (cci) score, profile of hospital department (medical (md), surgical or icu) where cdi was diagnosed, type of cdi (healthcare-associated (ha), hospital or community-acquired) and rate of risk factors (rf) have been estimated in both and . ibm spss . ; pearson's chi-square, fisher's exact tests were used for statistics. p < . was statistically significant. results: in total pt from , from were enrolled. in n= ( %) pt were ≥ yr old, in -n= ( %), (p= . ). in cci> was estimated in n= ( %) pt in comparison of n= ( %) in , (p= . ). in n= ( %) of cdi cases were ha, in -n= ( %), (p= . ). in n= ( %) of cdi were diagnosed in md in comparison of n= ( %) in , (p= . ). in weeks prior to cdi n= ( %) pt have been admitted to hospitals, n= ( %) have been treated w. antibiotics, n= ( %) -w. ppis, n= ( %) -w. h antagonists, n= ( %) -w. immunosupressants in comparison of n= ( %), n= ( %), n= ( %), n= ( %) and n= ( %) in , respectively, (p> . ). overall rate of cdi cases among in-hospital patients increased tenfold by yr and . in , more elderly patients had cdi and severe comorbidities were less frequent in comparison with . in , more cases of cdi were hospital-acquired and have occured in medical departments. rate of risk factors of cdi remained unchanged.these results indicate a possible relationship between ttv dna count and immunological alteration. the ttv quantitative determination could be useful as a proinflammatory marker in sepsis, with some benefits: low cost, easy determination and good correlation with immune system functionalit. it will be necessary to perform a larger study to check our hypothesis and to establish a ttv level threshold that may allow to anticípate the disease prognosis. introduction: acute kidney injury (aki) is a serious complication in sepsis and associated with high morbidity and mortality. the combination antimicrobial regimens with vancomycin (vcm) and broad-spectrum betalactams (bsbl), such as piperacillin tazobactam and cefepime, have been identified as potentially nephrotoxic combinations, but existing studies have not provided sufficient evidence. the aim of this study was to evaluate detailed association between the combination antimicrobial therapy and the risk of aki in septic patients. this investigation was a post hoc analysis of prospective nationwide cohorts enrolling consecutive adult patients with sepsis in intensive care units in japan. in this study, progression of aki was defined as one or more elevation of renal sub-score in sequential organ failure assessment score from day to day . we regarded anti-pseudomonal penicillins, fourth generation cephalosporines, and carbapenems as bsbl. multivariable logistic regression analysis including a two-way interaction term (vcm x bsbl) was performed to assess the add-on effects of each antimicrobial agent on the progression of aki. the final study cohort comprised patients with sepsis. among them, received vcm without bsbl, received bsbl without vcm, received both vcm and bsbl, and received other type of antimicrobials. the administration of vcm was associated with an increased risk of aki in patients with bsbl [odds ratio (or), . ( . - . ); p= . ]. however, the tendency was not evident in patients without bsbl [or, . ( . - . ); p= . ]. the interaction effect on the progression of aki between vcm and bsbl were statistically significant (p for interaction= . ). the regression model including two-way interaction term suggested that the combination of vcm and bsbl might synergistically increase the risk of aki in patients with sepsis. increasing resistance to carbapenems due to carbapenemase productionone of main actual problems of antibacterial resistance in burn icu. production of several types of carbapenemases (kpc, ndm and oxa- ) is common in k. pneumoniae strains. carbapemenase production is a marker of extreme antibacterial resistance. the aim of our study was to investigate the epidemiology of nosocomial infections caused by producing kpc, ndm and oxa- k. pneumonia strains in burn icu. total of patients with nosocomial infections caused by carbapenem resistance strains of k. pneumoniae were included in the study, from whom had lower respiratory tract infection, had skin and skin structure infection. initial identification of isolates was performed in laboratory by automatic microbiological analyzer. for all of k. pneumoniae isolates presence of bla ndm , bla oxa- and bla kpcgenes were examined by pcr method. baseline characteristics of patients: me (iqr) of age - ( ; ) years, me (iqr) of tbsa - ( ; ) percent, me (iqr) of icu los - ( ; ) days. inhalation injury was diagnosed in ( . %) patients. total of patients died, mortality rate was . %. all patients were diagnosed with nosocomial infection caused by k. pneumoniae. from k. pneumonia strains ( . %) were found to be producing kpc, ( . %)producing ndm and ( . %) -producing oxa . only ( . %) carbapenem resistance k. pneumoniae isolates were not producing carbapenemases. from patients infected by oxa producing k. pneumoniae patients died, mortality rate was %. from patients infected by oxa or ndm producing k. pneumoniae patients died, mortality rate was . %. from patients infected by non-carbapenemase producing k. pneumonia no one died. carbapenemase producing strains are widely spread among carbapenem resistance strains of k. pneumoniae in burn icu. mortality of patients infected by producing oxa or ndm k. pneumoniae strains reaches . %. the rationale for blood purification as adjunctive therapy during sepsis involved the capacity in removing endogenous and exogenous toxins, but currently no recommendations exists [ ] . a critical point may be the potential interaction with antimicrobial therapy, which remains the mainstay of sepsis treatment. the aim of our study was to investigate the vancomycin (van) removal during blood purification using an in vitro model of hemoperfusion (hp) with ha cartridge (jafron, zhuhai city, china), most widely used in china and actually available in europe. this is an experimental study. three independent experiments were performed: we injected mg of van in ml of whole blood from healthy donors (experiment and ) or in ml of balanced solution (experiment ) in order to assess membrane saturation. a closed-circuit (blood flow of ml/min) simulating hp ran using ha . samples were collected from arterial line at , , , , , , , , minutes; van plasma concentrations were measured and removal was evaluated using mass balance analysis. differences in mass removal was assessed using kruskal-wallis test. results: figure shows van mass at each timepoints. we observed no difference between in blood and in balanced solution experiments (p- the aim of this study is to determine if routine bbv testing in the icu contributes to the discovery of undiagnosed bbv infections. icu patients may require renal replacement therapy (rrt). sharing rrt equipment carries a risk of bbv transmission, which mainly relates to hepatitis b (hbv), hepatitis c (hcv) and hiv. since , all glasgow royal infirmary icu patients undergo routine bbv screening, with rrt machines allocated for patients with specific bbv statuses. routine bbv testing is beneficial to both the individual and society. hcv is a pertinent health issue in scotland. the scottish government aims to eliminate hcv by and is researching innovative and costeffective methods to identify undiagnosed infections. this single-centre retrospective observational study examined prospectively collected clinical data from icu admissions. proportions were compared using a two-proportion z-test and a logistic regression model was carried out to determine if deprivation quintile was independently associated with the seroprevalence of bbvs. the bbv seroprevalence in the cohort studied: . % (hbv), . % (hcv), . % (hiv). the seroprevalence of hbv in the cohort studied was similar to that of scotland (p= . ), but the seroprevalence of hcv (p< . ) and hiv (p= . ) were statistically significantly higher than that of scotland. due to the small number of reactive test results for hbv and hiv, the relationship between deprivation and bbv seroprevalence was explored for hcv only. the only independent variable associated with a reactive anti-hcv test result was "current or previous illicit drug use" (adjusted odds ratio of . ; % confidence interval of . - . ; p< . ). this study shows that routine bbv testing in the icu is useful in discovering new bbv infections. this is the first observational study focusing on the value of routine bbv testing in an icu setting to our knowledge. continuous infusion vancomycin protocol is a safe, acceptable and effective alternative to intermittent dosing of vancomycin in critical care. ceftaroline is an efficacious treatment in patients with severe cap, admitted in icu. it relates to earlier resolution of respiratory failure and less rescue antibiotics. we need an adequately pragmatic trial to confirm our findings organ dysfunction in scrub typhus, incidence and risk factor a sarkar , a guha , r dey [ , , , , ] . its preads by bite of larval stageof thromboculid mites or chigger [ ] . clinical features may include fever, headache, myalgia, lymphadenopathy, eschar, skinrash. it may also cause pneumonia, renal failure, shock, meningoencephalitis, multiple organ failure [ , ] . our study aims to discuss the incidence of organ dysfunction in a comprehensive way taking the overall population of patients with identified scrub typhus infection. there is lack of data in eastern india regarding the incidence and risk factors of developing multiorgan dysfunction syndrome (mods) in scrub typhus. in this retrospective study we studied the incidence of various organ involvement and the risk factors associated with the development of mods in scrub typhus. we collected data from december to november in tertiary care hospital at kolkata. we have included all patients who are having fever, scrub typhus igm antibody positive, age more than years. sofa score was used in evaluating patients with mods. exclusion criteria involves patient who are having coinfectional ong with scrub typhus. in a cohort (n= ), patients with multiorgan dysfunction syndrome was seen in patients ( . %), the mean age in group of patients with mods was . +/- . years (mean+/-sd). in group of patients with mods, fever duration in days was of +/- . days (mean+/-sd), interval from treatment to defervescenc in days was . +/- . days (mean +/-sd). among patients with mods, hematologic involvement was seen in patients ( . %), hepatic involvement was seen in patients ( . %), renal involvement was seen in patients ( . %), neurologic involvement was seen in patients ( %), respiratory involvement was seen in patients ( . %), cardiovascular was seen in patients ( . %), icu shifting was necessary in patients ( . %), mechanical intubation was needed in patients ( . %) in multiorgan dysfunction syndrome patients. hospital mortality in patients with mods was patients ( . %). no mortality was seen in patients without mods. other parameters were evaluated among patients with mods. they include eschar in patient ( . %), seizure in patients ( . %), hepatoslenomegaly in patients ( . %), leucopenia in patients ( . %), leucocytosis in patients ( . %), thromnbocytopenia in patients ( . %),decreased hemoglobin in patients ( . %), transaminitis in patients ( . %). the risk factors associated with the development of mods are platelet counts, bilirubin, transaminitis, glasgow coma scale, time interval from treatment to defervescence, hemoglobin, total leucocyte count and fever duration. scrub typhus is an important cause of acute febrile illness in this part of the country and is frequently associated with organ dysfunction. however, the overall mortality is low which is similar to other studies done before [ ] . score at baseline were significant (p< . ) predictors of mortality.highest area under the roc curve was obtained for number of days with septic shock ( . ) followed by increased cd between baseline and day ( . ). though serial pct levels significantly increased amongst non-survivors, it did not predict mortality. serial level of biomarkers in icu patients may predict mortality. larger trials are needed to confirm the results. plasma strem- levels were retrospectively measured at day - , - and - in septic shock patients from the immunosepsis cohort (nct ), included between / and / , using a validated elisa method. the associations between strem- , mhla-dr, -day survival status, and occurrence of icu-acquired nosocomial infection (ni) were assessed. neither strem- nor mhla-dr levels at d / were associated with the occurrence of icu-acquired ni. however, -day mortality was significantly higher in patients with d - strem- value superior to the median ( . % vs . %, p= . ; median= pg/ml). a significant inverse correlation was found between mhla-dr at d - and strem- at d - (sp - . , p< . ) and at d - (sp - . , p< . ). at d - , when stratifying patients based on strem- ( pg/ml) and mhla-dr ( ab/c), patients combining elevated strem- and low mhla-dr presented with significantly higher day mortality ( . % vs . %, p = . , chi-squared test) and ni incidence ( . vs %, p= . ) compared with patients with low strem- / high mhla-dr. this study shows for the first time that trem- pathway activation is associated with septic shock-induced immunosuppression, as shown by an inverse correlation between strem- at baseline and mhla-dr expression at d - . persisting high strem- values and low mhla-dr expression in septic shock patients are significantly associated with higher rate of icu-acquired infection and mortality. introduction: sepsis mortality remains high [ ] . the surviving sepsis campaign (ssc) recommends to guide resuscitation on normalization of lactate levels [ ] , however this is debated [ ] . we have shown that plasma levels of bio-adrenomedullin (bio-adm) were associated with patient outcome during sepsis [ ] . we therefore aimed to evaluate the added value of bio-adm to lactate measurement in the adrenoss cohort. this is a post-hoc analysis of the adrenomedullin and outcome in severe sepsis and septic shock (adrenoss) cohort study. the adre-noss study is a prospective observational study conducted in twenty-four centers and included septic patients [ ] . we studied the relationship between the association of initial evolution of lactate plasma levels and bio-adm level at h and outcome in patients for whom both markers were available at admission and one day later (" h"). bio-adm levels below pg/ml were considered as low, and high if greater than pg/ml [ ] . in patients with high lactate levels (> mmol/l) at admission (n= ), lactate normalization (< mmol/l) at h was associated with better outcome than in patients with persistently high lactate at h ( day mortality . % vs . % respectively, hr . [ . - . ], p< . ) ( figure ). among patients with decreasing lactate, high and low bio-adm levels at h identified patients with different outcomes ( day mortality % vs % for low vs high bio-adm respectively, hr . [ . - . ], p< . ). high and low bio-adm levels at h also differentiated outcome of patients with persistently elevated lactate (hr . [ . - . ], p< . ). in patients with low initial lactate, neither lactate or bio-adm had no added prognostic. our data suggest that measurement of bio-adm in addition to lactate may help physicians to refine risk stratification and therefore to guide resuscitation during sepsis. the effect of fluid replacement in sepsis, severe sepsis and septic shock in first hrs in clot quality and microstructure s pillai , g davies the inflammatory response in sepsis can lead to a spectrum of coagulation system defects [ ] . sepsis and severe sepsis is associated with a hypercoagulable state where the clot microstructure is known to be a tight and highly elastic clot, which is potentially resistant to fibrinolysis ( figure ). conversely, septic shock is associated with a hypocoagulable state where the clot microstructure is loose and structurally weak. the study aim to investigate the effect of fluid resuscitation and replacement in clot microstructure over hours. methods: patients ( sepsis, severe sepsis and septic shock) were included in the study. all these patients received standard fluid replacement therapy with crystalloids. blood samples were collected at hours, hours and hours. clot microstructure, standard markers of coagulation and inflammatory markers were measured. in sepsis group following fluid administration, the d f reduced initially and then remained stable ( . - hours, . - hours, . - hours, normal d f range . ± . ). in severe sepsis group, the d f reduced initially, then increased ( . - hours, . - hours, . - hours) and in septic shock, the df was very low to start with and there were only slight increase with fluid administration ( . - hours, . - hours, . - hours). the hypercoagulable state and clot quality in both sepsis and severe sepsis group improved with fluid resuscitation, however despite an early improvement in clot quality, ongoing fluid resuscitation resulted in markedly reduced functional clot with very low clot strength and functionality. this study demonstrates that d f as a marker of clot quality and function may have potential in fluid and component replacement in critical illness and injury. this study analyses the prognostic ability of white blood cell count (wbc), neutrophil:lymphocyte ratio (nlr) and c-reactive protein (crp). hypo-and hyperimmune responses have been associated with increased mortality from septic shock [ ] . patients with septic shock (sepsis . ) admitted to queen elizabeth hospital birmingham, between december and july were included. the primary outcome was -day mortality. data was tested for normality and presented as median (iqr) and analysed using a mann whitney u test. categorical data was presented as % and analysed using a chi-squared test. a p value of < . was used to determine significance. a multivariate binary logistic regression analysis was conducted using age, apache ii, charlson comorbidity index, performance status, and initial lactate as covariates. a hosmer lemeshow test of > . indicated good fit. results: patients were admitted with septic shock. the majority ( %) were male, with a median age of ( - ) and a -day mortality of %. on day , wbc was lower in patients who died compared to patients who survived ( [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] patients who died of septic shock had a lower wbc, nlr and crp response early on compared to survivors. this may represent early immunoparesis that allows infection to propagate unchecked. however, this was not independently associated with mortality when confounding factors were accounted for. a specific metabolite of mitochondriaitaconic acid is formed upon proinflammatory activation. the attempts of various researches to find the itaconic acid in peripherical blood of patients with sepsis were unsuccessful [ ] . some phenylcarboxylic acids (phcas) are known to be microbial metabolites and sepsis biomarkers; they also affect the mitochondrial functions [ ] . concentrations of phcas (phenyllactic, p-hydroxyphenylacetic, phydroxyphenyllactic acids) and mitochondrial metabolites (succinic, itaconic acids) in serum samples from patients on the st day of diagnosis of sepsis and serum samples from patients with late stages of sepsis (sepsis- ) were measured by gas chromatographymass spectrometry; control group - donors. results: itaconic acid was found in low concentrations ( . - . μm) only at early stage of sepsis. the multiple increase in levels of phcas and mitochondrial metabolites were detected in patients with late stage of sepsis in comparison with early stage and donors, p< . . increased succinic acid (up to - μm) concentration is the result of succinate dehydrogenase inhibition by microbial metabolism intermediates (phcas), which was confirmed by in vitro experiments in isolated mitochondria (fig. ) . itaconic acid may be a promising marker in early stage of sepsis, which needs to be proved. prediction of severe events in clinical sepsis is challenging. for such prediction we aimed to compare the novel biomarker calprotectin in plasma, with routine biomarkers. in a prospective study, blood samples were collected from consecutive patients who triggered the sepsis alert in the emergency department in our hospital. c-reactive protein (crp), procalcitonin, neutrophils, and lymphocytes were analysed according to routine practice. p-calprotectin was analysed using a specific particle enhanced turbidimetric assay (gentian diagnostics as). the composite endpoint, which was termed severe event, was defined as death or admission to the intensive care unit (icu)/high dependency unit (hdu) within hours from arrival. the study included patients with written informed consent, of whom were considered to have infection (defined as obtained blood culture and subsequent antibiotic therapy for at least days or until discharge or death), and had no infection. seventy-four patients ( %) with infection developed a severe event. mean pcalprotectin was . mg/l (standard deviation (sd) . ) among patients with infection and . mg/l (sd . ) among patients without infection (p= . ). in patients with infection mean p-calprotectin was . mg/l (sd . ) among those with and . mg/l (sd . ) among those without a severe event (p= . ). analysis of area under the receiver-operating characteristic (roc) curve for prediction of severe events showed superiority for p-calprotectin compared with procalcitonin and neutrophil-lymphocyte-ratio, both regarding all sepsis alert cases and regarding the patients with infection (p< . for all comparisons), fig . in addition, there was a trend toward superior performance compared to crp (p= . and . ). in sepsis alert patients, p-calprotectin was elevated in those who subsequently developed severe events. p-calprotectin was superior to traditional biomarkers for prediction of severe events. introduction: rapid diagnosis of acute infections and sepsis is critical in emergency departments (eds). current tests have slow turnaround times, low sensitivities, and/or signals from contaminant or commensal organisms. empirical antimicrobial treatment may result in severe adverse events and contributes to antimicrobial resistance. diagnostics to distinguish bacterial from viral infections and noninfectious etiologies support clinicians in efforts toward antimicrobial stewardship. in a prospective, non-interventional study in the eds of sites in greece (prompt study nct ), we evaluated hostdx sepsis, a host response test for suspected acute infections and suspected sepsis. hostdx sepsis measures human mrna targets and employs advanced machine learning to differentiate patients with bacterial and viral infections, and noninfectious etiologies. adult patients presenting with suspected acute infection and at least one vital sign change were enrolled. whole blood rna was quantified using nano-string ncounter. predicted probabilities of bacterial and viral infection were calculated (bvn- algorithm). patients were adjudicated in a retrospective chart review by independent infectious disease specialists blinded to hostdx sepsis results. among patients adjudicated as bacterial ( ), viral ( ), noninfected ( ), or indeterminate ( ) the area under the receiver operating characteristics (auroc) of hostdx sepsis for predicting bacterial vs. viral/non-infected patients was . , and auroc for viral vs. bacterial/non-infected patients was . (fig. ) . our results indicate that hostdx sepsis distinguishes bacterial from viral infections and other etiologies with high accuracy. hostdx sepsis is currently developed as a rapid point-of-care device with a turnaround-time of less than minutes. hostdx sepsis may therefore assist ed doctors in making appropriate treatment decisions earlier, towards the ultimate goal of antimicrobial stewardship. we studied the diagnostic value of a leukocyte deformability assay that rapidly quantifies the immune activation signatures of sepsis in an undifferentiated population of adults presenting to the ed. ed clinicians must balance the benefits of early intervention against the risks of indiscriminate use of resource-intensive interventions. there are no currently available rapid diagnostics with acceptable performance to achieve this balance. we prospectively enrolled adult patients within hours of presentation with signs of suspicion of infection in two eds in the usa. edta-anticoagulated blood was drawn and analyzed using deformability cytometry [ ] . procalcitonin (pct) levels were also measured. patients were retrospectively adjudicated for sepsis- by physician committee using the entire medical record. diagnostic performance characteristics and receiver operating curves were used to examine the diagnostic performance of the assay as well as pct. of the patients enrolled, . % were adjudicated as septic. the leukocyte deformability assay demonstrated % sensitivity, % specificity, and % negative predictive value for a single cutoff. the auc was . ( figure ). pct with a cutoff of . ng/ml had % sensitivity, % specificity, and % negative predictive value. the auc for pct (as continuous variable) was . . the leukocyte deformability assay of immune activation signatures demonstrated superior diagnostic performance for sepsis when compared to pct. the assay's diagnostic performance and rapid turnaround time of minutes may positively impact patient outcomes while minimizing indiscriminate use of valuable resources in the ed. it is already known in literature that high levels of midregional proadrenomedullin (mrproadm) are related with organ disfunction in infections despite of source and pathogens [ ] . similarly, microcirculatory impairment has been reported in sepsis. we examine the correlation between microcirculatory disfunction and mrproadm as a sign of early organ failure. we included consecutive adult patients with suspected infection, sepsis or septic shock admitted to our intensive care unit (icu) as first hospital admission with an expected icu stay of > hours. mrproadm was measured daily during the first five consecutive days and sublingual microcirculation was assessed with incident dark field (idf) technology at t , t , and t . we collected information on saps ii, apache scores, and sofa score for each timepoint. results: ten patients had septic shock, sepsis and infection. three patients died during icu stay. a mrproadm clearance of % or more between t and t was found associated with the improvement of mfi (mann-whitney u test, median increase . % versus . %, p= . ) (figure ) . a mrproadm > . nmol/l at the icu admission was associated with a worse sofa score at all the timepoint. moreover, mrproadm levels at admission was found significantly related with icu mortality (auc . [ . - ]; p= . ). mrproadm shown no relation with absolute value of mfi. the study shows a good correlation between the clearance of the biomarker and the improvement in mfi. moreover, our results support previous findings on the prognostic value of mrproadm in terms of sofa and icu-mortality. clinical performance of a rapid sepsis test on a near-patient molecular testing platform r brandon , j kirk , t yager , s cermelli , r davis , d sampson , p sillekens , i keuleers , t vanhoey immunexpress, seattle, united states; immunexpress, immunexpress, seattle, united states; biocartis nv, biocartis, mechelen, belgium critical care , (suppl ):p the purpose of this study was to clinically validate a new, rapid version of the septicyte™ assay on a near-patient testing platform (biocartis idylla™). septicyte™ lab is the first-in-class sepsis diagnostic to gain fda-clearance but has a complex workflow and a turnaround time (tat) of~ hours. the assay in idylla™ cartridge format is called septicyte™ rapid. septicyte™ lab was translated to the biocartis idylla™ near-patient testing platform and analytically validated. for this study, . ml of peripheral blood paxgene tm solution from previously collected patient samples was pipetted directly into the cartridge and inserted into the idylla™ reader. patients were part of an independent cohort (n= ) from intensive care units located in the usa and europe. septicyte™ rapid results were reported as a septiscore™ between and with higher scores representing higher probability of sepsis. assay performance determined included technician hands-on-time (hot), assay tat, failure rates, and area under roc curve based on comparison to retrospective physician diagnosis. average hot was minutes, and average tat was minutes. clinical samples could be processed immediately with septicyte™ rapid and did not require hour pre-incubation of paxgene blood, greatly improving tat. correlation of septiscore™ values between lab and rapid, based upon a subset of samples run on both platforms, was very high (r > . ). estimated roc auc performance for discriminating sepsis from non-infectious systemic inflammation (nisi/sirs) was similar to that previously reported for septicyte™ lab. this is the first demonstration of a validated, fully-integrated, rapid, reproducible, near-patient, immune-response sepsis diagnostic, providing actionable results~ hr, to differentiate sepsis from non-infectious systemic inflammation / sirs. accuracy of septicyte™ for diagnosis of sepsis across a broad range of patients r brandon , k navalkar , d sampson , r davis , t yager immunexpress, seattle, united states; immunexpress, immunexpress, seattle, united states critical care , (suppl ):p the purpose of the study was to demonstrate sepsis diagnostic performance of the biomarkers of septicyte™ in subjects other than critically ill adults, and in hospital locations other than icu. septicyte™ lab was the first immune-response sepsis diagnostic assay to gain fda-clearance (k ) and, as part of gaining this clearance, clinical validation was performed on adult patients admitted to intensive care (icu) only [ ] . we therefore performed an in silico analysis across a broad range of patients using the septicyte™ host immune response biomarkers and algorithm. peripheral blood gene expression data, including public and private datasets, were chosen based on quality, annotation, and clinical context for the intended use of septicyte™. multiple comparisons were performed within datasets to better understand the diagnostic performance in certain cohorts including healthy subjects. diagnostic performance was determined using area under curve (auc). results: table shows some characteristics of the selected datasets and patients, including number of datasets (n= ) and comparisons (n= ), number of cases (n= ) and controls (n= ) used in comparisons, patient category and hospital location. septicyte™ aucs for the three groups of adults, adult / pediatric and pediatric / neonates were . , . , and . respectively, which is similar to that previously reported ( . - . ) [ ] . these results suggest that the septicyte™ signature has diagnostic utility beyond adults suspected of sepsis and admitted to icu. this signature has now been translated to the near-patient testing platform biocartis idylla™ (as septicyte™ rapid) which promises rapid (~ hour) diagnosis of sepsis in a broad patient population following further validation. introduction: especially extracorporeal cardio pulmonary bypass (cpb) is known to induce severe inflammation. postoperative inflammation is associated with a sepsis like syndrome including endothelial barrier disruption, volume depletion and hypotension. sphingosine- -phosphate (s p) is a signaling lipid regulating permeability and vascular tone. in septic humans decreased serum-s p levels could be identified as marker for sepsis severity. we addressed three main issues: ( ) are serum-s p levels affected by cardiac surgery? ( ) are potential alterations of serum-s p levels related to changes of acute-phase proteins, s p sources or carrier? ( ) is the invasiveness of the surgery a factor that may influence serum-s p levels? methods: elective major cardiac surgery patients were prospectively enrolled in this study. serum samples were drawn pre-, post-procedure and on day and day after surgery. we analyzed s pand its potential sources: red blood cells (rbc) and platelets. we further quantified levels of other inflammatory markers and documented other clinical parameters. median serum-s p levels in all patients before the procedure were . (iqr . - . ) nmol/ml. serum-s p levels decrease after surgery, whereas all other inflammatory markers increase. serum-s p levels dropped by % in the on-pump and % in the off-pump group. changes of serum-s p levels are associated with s p sources and carriers: albumin, hdl and vwf:ag activity. patients with a full recovery of their serum-s p levels after surgery compared to their individual baseline presented with a lower sofa score (p> . ) and shorter icu stay (p< . ). serum-s p levels are disrupted by open heart surgery and levels might be negatively affected by endothelial injury or loss of s p sources. low serum-s p levels may contribute to prolonged icu stay and worse clinical status. future studies may investigate the beneficial effects of s p administration during cardiac surgery. the aim of study is to measure and correlate the expression of ncd , mhla-dr, pct (procalcitonin) and qcrp (quantitative creactive protein) to predict development of sepsis and its outcome. in this tertiary centre based longitudinal cohort study, a total patients were enrolled in whom sepsis was suspected on the basis of clinical diagnosis and supported by lab investigations. they were divided into two groups sepsis/case and non-sepsis/control. disease severity in icu was assessed by sequential organ failure score (sofa). blood samples for routine lab investigations and biomarkers were taken at the time of admission in icu before administration of first dose of antibiotics at time d /d . assessment of biomarkers was done simultaneously with tlc at d /d , d and during follow up of patients till their final outcome. there was no significant (p> . ) mean change in pct, qcrp, sofa, ncd , mhla-dr from day to day , however, mean change was higher among cases than controls.on comparison of mhla-dr between the groups across time periods, mhla-dr was significantly (p= . ) lower among septic patients than controls at both day and day . all biomarker correctly predicted cases among different percentage of patients with different sensitivity and specificity. there was no significant (p> . ) association of mortality with the study biomarkers except for pct. in our study, diagnostic value of pct in differentiating sepsis from non-sepsis was similar to ncd among all biomarkers studied. no advantage of ncd or mhla-dr was found over pct in diagnosis and correlation with disease progression and mortality. introduction: aqp is a water channel protein contributing to astrocyte and immune cells migration, blood-brain barrier maintenance and cell survival [ ] [ ] . aqp genetic variants represent biomarkers associating with outcome after traumatic brain injury and intracerebral hemorrhage [ ] [ ] . linking aqp genetic polymorphism to the course of sepsis has not been studied. methods: study cohort included icu patients diagnosed according to sepsis- consensus. aqp rs polymorphism was studied by analyzing pcr products in a % agarose gel using an aqp specific polynucleotide tetraprimer set. data were analyzed by log rank test (medcalc . . ), and odds ratios/hazard ratios were computed. statistical significance was determined by fisher test (ft) or mann-whitney test. results: of sepsis patients had the minor mutation a for snp rs located within the regulatory ' region of the aqp gene. septic shock occurred more frequently in homozygotic carriers of aqp c allele vs. patients with aa or ca genotype: or= . ( %ci: . - . ), p= . (ft). lethality in septic shock patients, n= , significantly increased compared to sepsis patients with no shock, n= ( % vs. %, p= . , ft). maximum sofa values were significantly lower in patients with minor allele a compared to cc carriers of ( . vs. . , respectively, p= . ). in post-surgery group of patients, carriers of ac or aa genotypes had significantly increased survival compared to patients with cc genotypes: chi-square= . ; hr= . ( %ci: . - . ) for lethality; p= . (figure ) . association of minor allele a of aqp snp rs with survival in sepsis patients seems secondary to linking the snp to decreased development of multiorgan failure and septic shock that contribute to mortality. validation of presepsin as a biomarker of sepsis in comparison to procalcitonin, il- and il- v chantziara , f kaminari , c sklavou , s fortis , p kogionou , s perez , a efthymiou saint savvas hospital, icu, athens, greece; saint savvas hospital, cancer immunology and immunotherapy center, athens, greece critical care , (suppl ):p sepsis is an everyday challenge for the intensivist and biomarkers are useful tools for identification and treatment of this syndrome. we sought to validate presepsin as a biomarker of sepsis in comparison to pct(procalcitonin) and interleukins (il- ,il- ). we enrolled patients, men and women average age ( . - ) years old, apache ii ( . - . ), saps ii ( . - . ), sofa ( . - ). patients were septic on admission (according to surviving sepsis campaign: international guidelines for management of sepsis and septic shock: ), had a septic episode during their hospitalization in the icu while patients never endured sepsis. we measured presepsin, procalcitonin, il- , il- during sepsis and on remission. results: all septic patients had increased values of presepsin, pct, il- and il- during sepsis with a cutoff value for presepsin pg/ml, while the values of these biomarkers were significantly decreased during remission or in comparison to non-septic patients(presepsin p = . , pct p≤ . , il- p≤ . , il- p= . . all patients who were not septic survived while among septic patients died ( % mortality). presepsin correlated significantly with pct, il- and il- (p< . ). presepsin is a valid biomarker of sepsis and correlates significantly with all the other values of pct, il- and il- . clinical sepsis phenotypes are proposed at hospital presentation. these phenotypes, biomarker profiles, and outcomes are not yet reproduced in prospective data. even less is known about the biologic mechanism the drives these distinct groups. thus, we sought to validate clinical phenotypes and to determine markers of innate immunity, coagulation, tolerance and tissue damage in a prospective cohort. we prospectively studied patients with sepsis- criteria within hours of presentation at hospitals in pennsylvania ( - ) using automated electronic alerts. using clinical variables, we predicted phenotypes (α, β, γ, δ) for each patient using euclidean distance anchored to published seneca phenotype centroids. discarded blood was analyzed in a subset (n= ) for markers of innate immunity (e.g. il- , il- ), coagulation (e.g antithrombin iii, eselectin), tolerance (e.g. ho- , igfbp ), and tissue damage (e.g. serum lactate, bicarbonate) results: among patients, α-type was present in ( %), β-type in ( %), γ-type in ( %) and δ-type in ( %, figure a ). on average, β-type was older and more comorbid (mean , sd yrs; mean elixhauser . , sd . ) with renal dysfunction (median creatinine . [iqr . - . ] mg/dl, p< . all). the δ-type had more acidosis (mean hco - . , sd . meq/l), higher serum lactate (median . [iqr . - . ] mmol/l, p < . both) and inpatient mortality ( %, figure b) . the γand δ-type had greater markers of innate immunity and abnormal coagulation (e.g il- , icam p< . both), while markers of increased tissue damage (lactate) and poor tolerance (ho- ) were present in δ-type, compared to α-type (figure c) . the distribution and characteristics of clinical sepsis phenotypes were reproduced in a prospective validation cohort. similar to the seneca study, distinct biomarker profiles of tissue damage, innate immunity and poor tolerance were present for the δ-type. the effect that neoadjuvant chemotherapy and hyperthermic intraperitoneal chemotherapy (hipec) may have in the postoperative kinetics of biomarkers remains unknow. some studies demonstrate that neoadjuvant chemotherapy and hipec do not invalidate the use of inflammatory markers in postoperative patient monitoring, but none have compared biomarkers kinetics between patients who underwent hipec or only cytoreduction surgery. our main purpose was to identify a difference pattern in c-reactive protein (crp). we conducted a single-center observational study from january to november , including all patients who underwent cytoreductive surgery with or without hipec. crp was measured daily until seven post-operative day. we compared patients with and without hipec. a total of patients were included, were female. mean age was yrs ( - ). no clinical and demographical differences were observed between groups. no documented infection was found. after surgery crp increased markedly in both groups. crp time-course from the day of surgery onwards was significantly different in hipec patients ( . ± . mg/dl vs . ± . mg/dl; p= . ). multiple comparisons between hipec and non hipec patients were performed and crp concentration was significantly different on the th and th pod (figure ). no differences were found in other biomarkers (leucocytes and platelets) neither in body temperature. after a major elective surgical insult crp levels markedly increase independently of hipec. serum crp time-course showed a higher pattern in hipec patients despite no infection detected. decreased thrombin generation potential is associated with increased thrombin generation markers in sepsis associated coagulopathy d hoppensteadt , f siddiqui , e bontekoe , r laddu , r matthew , e brailovsky , j fareed. introduction: sepsis associated coagulopathy (sac) is commonly seen in patients which leads to dysfunctional hemostasis in which uncontrolled protease generation results in the consumption of clotting factors. the purpose of this study is to determine the thrombin generation potential of baseline blood samples obtained from sac patients and demonstrate their relevance to thrombin generation markers. baseline citrated blood samples were prospectively collected from patients with sac at the university of utah clinic. citrated normal controls (n= ) were obtained from george king biomedical (overland park, ks). thrombin generation studies were carried out using a flourogenic substrate method. tat and f . were measured using elisa methods (seimens, indianapolis, in) . functional antithrombin levels were measured using a chromogenic substrate method. the peak thrombin levels and auc levels were lower in the sac patients in comparison to higher levels observed in the normal plasma ( table ). the sac group showed much longer lag time in comparison to the normal group. wide variations in the results were observed in these parameters in the sac group. the f . and tat levels in the sac group were much higher in comparison to the normal. the functional antithrombin levels were decreased in the sac group. these results validate that thrombin generation markers such as f . and tat are elevated in patients with sac. however, thrombin generation parameters are significantly decreased in this group in comparison to normal. this may be due to the consumption of prothrombin due to the activation of the coagulation system. thus, persistent thrombin generation with simultaneous consumption of clotting factors such as prothrombin contributes to the consumption coagulopathy observed in sepsis patients. introduction: procalcitonin (pct) is used in the icu as an inflammatory marker to monitor bacterial infections and guide antibiotic therapy. whether pct can predict bacteremia and therefore could prevent expenses attached to bloodcultures is unknown . we investigated whether pct can predict the outcome of blood cultures in the icu and reduce expences. a single centre observational cohort study was performed in a dutch community teaching hospital . adult patients who were staying in the icu and were suspected of bacteremia were included. simultaneously with drawing of blood cultures, samples for pct measurement were obtained. expenses for pct measurement and bloodcultures were calculated. in the study period of one year, a total of patients were included. three patients were excluded because of incomplete data. out of the included patients, ten patients had positive blood cultures. there was a significant difference in pct levels between patients who had positive bloodcultures versus patients with negative bloodcultures ( . ng/ml vs . ng/ml) ( figure ). the negative predictive value for negative blood cultures is % when pct is below ng/ml, there was no difference in crp levels between the two groups ( mg/l vs mg/l, p= . ).a set of negative blood cultures in our centre costs euros. positive blood cultures however costs significantly more depending on the micro-organisms found. pct only costs . euros per measurement. so when blood cultures are omitted when the pct level is below ng/ml, a cost reduction of % can be achieved. a pct value below ng/ml is a good predictor of a negative blood cultures in icu patients suspected of bacteremia. pct guided bloodculture management in these patients could lead to a significant cost reduction introduction: level of cfdna in plasma is a promising prognostic candidate biomarker in critical illness [ ] . oxidized cfdna (ocfdna) have not been studied as a biomarker although its functional role in cellular stress have attracted attention of researches [ ] . the goal of our study was to assess the early prognostic value of plasma cfdna/ocfdna for sepsis in a nicu setting. the cohort included nicu patients diagnosed with stroke, intracerebral hemorrhage (ich), anoxia, encephalopathy. cfdna was isolated from day plasma and stained with picogreen. oxidized dna was determined using dna immunoblotting with anti- -oxo-desoxiguanosine antibodies. genotyping of allelic variants of the tlr rs gene was performed using a pcr and designed allele-specific tetraprimers followed by electrophoretic separation of the products statistics was performed by the fisher test and mann-whitney test. results: sepsis was diagnosed by sepsis- criteria in patients ( . %). average nisu staying was , ± , days. circulating dna plasma levels on day predicted the future sepsis development (figure ): or for cfdna was . ( %ci: . - . ), p< . ; or for ocfdna was . ( %ci: . - . ), p= . . power of both performed tests with alpha= . : . . log rank test demonstrated better predictive value of cfdna vs. ocfdna (figure) . concentrations of cfdna, but not ocfdna, on day significantly positively correlated with maximum sofa values during hospitalization, day and pre-outcome leukocyte count and neutrophil-to-lymphocyte ratios in a limited cohort of nisu patients with tlr rs cc genotype and not in other patients with genotype tlr ct+tt. increased level of plasma cfdna better then ocfdna predicts sepsis development in nisu. further studies are warranted to clarify the fig. (abstract p ) . pct values in patients with positive blood cultures and patients with negative blood cultures possible utility of tlr rs polymorphism determining for sepsis risk stratification early on nisu admittance. admission was related with higher severity of illness and extension of icu stay for all groups. reduced cbt fluctuations upon icu admission was found to more severely ill patients with worse clinical outcomes, while the more periodic cbt patterns were correlated with high cbt rhythmicity and better outcome. the impact of sex on sepsis incidence and mortality have been elucidated in previous studies, and sex is increasingly recognized as one key factor in sepsis [ ] . some studies indicate that women have better immunologic responses to infections [ ] . later investigations assume this advantage is linked to immune modulating genes located on the x-chromosome [ ] . the purpose of this study is to reveal sex differences in incidence of and mortality of sepsis in a large population-based cohort. methods: adult participants in the hunt study ( - ) were followed from inclusion through end of . incident bloodstream infections (bsi) from all local and regional hospitals in nord-trøndelag county were identified through linkage with the mid-norway sepsis register, which includes prospectively registered information on bsi used as a specific indicator of sepsis. we estimated age-adjusted cumulative incidence of first-time bsi and compared the risk of a first-time bsi and bsi mortality in men and women using age-adjusted cox proportional hazard regression. during a median follow-up of . years individuals experienced at least one episode of bsi, and died within days after a bsi. cumulative incidence and cumulative mortality curves are shown in fig. a introduction:the proportion of hospital-acquired infections (hai) among sepsis patients is unknown in germany. systematic differences in hai foci between sepsis patients with and without icu treatment are insufficiently described. retrospective cohort study based on nationwide health claims data of the german statutory health insurance aok. incident inpatient sepsis cases were identified in / among insured persons > y without preceding sepsis in months prior to index hospitalization. sepsis was defined according to explicit sepsis icd- -codes (incl. severe sepsis/septic shock). hai were defined based on specific icd- -codes for surgical site infection, catheter- introduction: elevated renin is associated with an increased risk of death in patients with vasodilatory shock (vs). recent data show that patients with vs and elevated renin levels have improved survival when treated with angiotensin ii (ang ii) + standard care (sc) vs placebo + sc. patients with acute respiratory distress syndrome (ards) can develop angiotensin-converting enzyme (ace) defects that can lead to elevated renin levels and insufficient endogenous ang ii production. we hypothesized that patients with severe ards and elevated renin shock would have improved survival when treated with ang ii + sc vs placebo + sc. in the randomized, placebo-controlled, double-blind athos- study, patients with severe vs receiving > . μg/kg/min of norepinephrine or the equivalent were randomized to intravenous ang ii (n= ) or placebo (n= ). in a post hoc analysis, we assessed the subset of patients with elevated renin (defined as a renin level greater than the median value of the overall athos- population) and ards (defined by a pao /fio ratio < ) at the time of randomization. survival to days was compared between the ang ii group (n= ) and the placebo group (n= ). in patients with elevated renin and ards, baseline age, acute physiology and chronic health evaluation ii score, and blood pressure were similar in the ang ii and placebo groups. the median serum renin level was . pg/ml (iqr: . - . ) compared to the normal range for serum renin: - pg/ml. a significantly higher proportion of patients receiving ang ii survived to day compared to those in the placebo group ( % vs %; p= . ). elevated renin identified patients with vs and ards who were most likely to gain a survival benefit from ang ii. elevated renin is likely caused by an ace defect and may describe an important subset of patients with a biotype that responds well to ang ii therapy. introduction: elevated renin levels have been shown to be associated with an increased risk of death and more severe acute kidney injury (aki) in patients with vasodilatory shock (vs). recent data show that patients with vs and elevated renin levels have improved survival when treated with angiotensin ii (ang ii) + standard care (sc) vs placebo (pbo) + sc. we hypothesized that vs patients with severe aki and elevated renin levels would have improved survival and enhanced renal recovery with ang ii treatment. in the randomized, pbo-controlled, double-blind athos- study, patients with severe vs received > . μg/kg/min of norepinephrine or the equivalent and were randomized to intravenous ang ii + sc (n= ) or pbo + sc (n= ). in a post hoc analysis, we assessed the subset of patients with elevated renin (defined as a renin level greater than the median value of the overall athos- population) and severe aki (defined as those with aki requiring renal replacement therapy [rrt] at baseline). survival and renal recovery were assessed in patients treated with ang ii + sc (n= ) and pbo + sc (n= ). in patients with elevated renin and severe aki, baseline age, acute physiology and chronic health evaluation ii score, and blood pressure were similar between ang ii + sc vs pbo + sc. the median baseline serum renin level in the whole group was . pg/ml (iqr: . - . ; normal range for serum renin: - pg/ml). a significantly higher proportion of patients receiving ang ii + sc vs pbo + sc survived to day ( % vs %, respectively; p= . ). ang ii recipients also had a higher rate of discontinuation from rrt by day ( % vs %; p= . ). in this study, elevated-renin shock patients with aki treated with ang ii + sc gained a survival benefit and earlier discontinuation from rrt compared to those receiving pbo + sc. elevated renin is likely caused by an angiotensin-converting enzyme defect and may identify those patients with a biotype that responds well to ang ii therapy. most clinical trials conclude the ineffective use of anticoagulation for sepsis-induced coagulopathy [ ] . however, post hoc analyses of randomized control trials report positive results [ ] , suggesting anticoagulation is effective in specific populations exhibiting coagulopathy. further, anticoagulants should be administered in the early phase [ ] ; however, methods for precisely predicting the progression of sepsis-induced coagulopathy are not established. this study aimed to create and evaluate a prediction model of coagulopathy progression using machine-learning techniques. we performed a subgroup analysis of data from a retrospective cohort study involving adult septic patients in japanese institutions from january to december and used the japanese association for acute medicine disseminated intravascular coagulation (dic) score as a dic severity index test. the predictive ability of Δdic ([dic score on day ] -[dic score on day ]) was evaluated using various statistical methods. using variables available at the outset, we compared the predictive ability of random forest (rf) and support vector machine (svm) with that of multiple linear regression analysis. a total of adults with sepsis were included in the analysis. the root mean square error in Δdic score for the multiple linear regression analysis model was . compared with values of . and . for rf and svm, respectively. thus, the rf method predicted the progression of sepsis-induced coagulopathy more accurately than multiple linear regression analysis. conclusions: rf, a machine-learning technique, was superior to multiple linear regression analysis in predicting the progression of sepsis-induced coagulopathy. this prediction model might enable us to use anticoagulation in an early phase. this study examined the efficacy and safety of landiolol, an ultrashort-acting β -blocker, for treating sepsis-related tachyarrhythmia, according to patient background characteristics. the j-land s study (japiccti- ) was conducted in patients with sepsis, diagnosed according to the sepsis- criteria, and tachyarrhythmia (atrial fibrillation, atrial flutter, or sinus tachyarrhythmia). the patients had a mean heart rate of ≥ beats/min and required catecholamine administration to maintain a mean blood pressure of ≥ mmhg. the efficacy endpoint was the percentage of patients whose heart rate could be controlled within - beats/min at h of registration. the safety endpoint was the incidence of adverse events within h of registration. subgroup analyses of efficacy and safety were performed after stratifying the patients according to various patient background characteristics. a total of patients were randomized, to landiolol and to the control group. the efficacy endpoint, percentage of patients with a heart rate of - beats/min at h of registration, was significantly higher in the landiolol group ( . % vs . %; mantel-haenszel test: p = . ). the incidence of adverse events was . % and . % in the landiolol and control groups, respectively, and there was no difference between the two groups. most adverse events were related to sepsis or septic shock. the subgroup analyses showed that no patient background characteristic clearly affected the efficacy and safety of landiolol. landiolol is a well tolerated and effective therapeutic agent for controlling heart rate in patients with sepsis-related tachyarrhythmias; its safety and efficacy were not affected by the patient background characteristics investigated. tissue oxygenation monitoring in sepsis r marinova, at temelkov umhat alexandrovska, anesthesiology and intensive care, sofia, bulgaria critical care , (suppl ):p near-infrared spectroscopy (nirs) was proposed as a concept in the end of th century. this method offers noninvasive monitoring of oxy-and deoxyhemoglobin in tissues.nirs could be measured on the thenar or forehead within few santimeters of the skin. it was first applied as a monitoring in cardiovascular surgery. patients with sepsis have changes in the microcirculation which are important target for therapy. invasive monitoring of oxygen delivery and consumption has been used in patients with sepsis but as every invasive technique such a monitoring hides risks. nirs offers a noninvasive method for tissue oxygenation monitoring (sto ) and could be useful in patients with sepsis and septic shock. the aim of the study is to compare noninvasive tissue oxygenation monitoring with hemodinamic monitoring and lactate values in patients with sepsis methods:the study includes critically ill patients in icu of umhat alexandrovska, sofia. of the patients fullfil the criteria for septic state. the other patients do not have sepsis. in both group of patients are measured tissue oxygenation with invios monitor, mean arterial pressure, oxygen saturation in mixed venous blood and lactate values during h after icu admission. patients with sepsis are reported with significantly lower values of tissue oxygenation, compared to patients without sepsis. the values of tissue oxygenation correlate well with the mixed venous blood oxygenation, mean arterial pressure and lactate values but not significantly with apache scores. conclusions: nirs when used for tissue oxygenation monitoring correlates well with the hemodinamic monitoring and lacate values in patients with sepsis and could be used as an noninvasive monitoring for guiding teurapeutic strategies. tissue oxygenation monitoring has no linear correlation with the severity of illness in patients with sepsis and could not be reccomended as a guidance in the early ressuscitating stage of sepsis. further investiganions in these field are needed.the sequenom´s massarray platform and a recessive inheritance model was selected (cc vs tt/ct). the possible association between the cc recessive form of the rs polymorphism and the septic shock risk was analyzed, demonstrating a statistically significant relationship (p= . ) between both conditions. among patients who developed septic shock, . % presented a recessive inheritance pattern while . % showed the ct/tt genotype. on the other hand, those patients with the recessive form of the rs polymorphism were selected and a statistical analysis was performed comparing those patients who developed septic shock from those who did not develop it, obtaining a statistically significant relationship (p= . ) between the presence of the recessive form of polymorphism and the likelihood of developing septic shock. the recessive form of rs polymorphism is a risk factor for septic shock in post-operative patients of major abdominal surgery. introduction: sepsis remains one of the major causes of morbidity with mortality rates as high as % worldwide, representing significant clinical challenge to confront highly intangible therapeutic needs. rnabased structures are emerging as versatile tools encompassing a variety of functions capable to bypass the current protein-and cellbased therapies. rna aptamers act as disease-associated protein antagonists. here, the effects of an aptamer, apta- , were evaluated in animal models that mimic systemic inflammation in humans. high dose of lps endotoxin was used to induce systemic inflammation in mice and in non-human primate animal models. apta- was administered intravenously in two doses post lps infection. animals were monitored and blood samples collected up to hours after apta- administration. healthy-and lps-only treated animals served as control groups. complex analyses of clinical parameters, hematology, serum biochemistry, inflammation and tissue damage markers were performed. results: apta- increased survival of endotoxin challenged animals up to % in a dose-dependent manner and exerted profound effects on wellbeing and recovery of healthy eating habits. administration of apta- led to delayed coagulation and enhanced fibrinolysis; maintained the complement cascade activated while preventing it from further amplification. expression of pro-inflammatory cytokines was reduced while anti-inflammatory increased. endogenous pro-inflammatory molecules (damps), secreted from injured cells, were preserved at healthy level in animals treated with apta- . systemic inflammation and sepsis lead to severe dysregulation of several arms/axis of innate immune response. our studies showed that apta- affects various components of this system and restores the organism's control over its dysregulated immune response. thus, apta- might be a promising potential therapeutic candidate to treat life-threatening conditions such sepsis. several preclinical studies demonstrated beneficial effects for methane (ch ) administration in various inflammatory conditions. our aim was to investigate the consequences of post-treatment with inhaled ch in a clinically relevant intra-abdominal sepsis model. anesthetized minipigs were subjected to fecal peritonitis ( . g/kg, - x cfu i.p.; n= ) or sham-operation (sterile saline i.p; n= ). invasive hemodynamic monitoring with blood gas analyses was started between - hours, organ dysfunction parameters (pao /fio ratio; mean arterial pressure; lactate, bilirubin, creatinine; urine output and platelet counts) were determined according to a modified porcinespecific sequential organ failure assessment (ps-sofa) score system, the perfusion rate (pr) of sublingual microcirculation was measured by incident dark field illumination imaging. the animals were divided into non-treated septic or septic shock groups (n= - ) and ch treated septic or septic shock (n= - ) subgroups, ch inhalation started from the th hr ( . % ch in normoxic air; ml/min). despite the standardized induction, heterogeneous severity of organ damage was evolved. in septic and septic shock groups the median values of ps-sofa score reached ( . - . ) and ( . - ), respectively. septic shock was characterized by significant elevations of creatinine and bilirubin levels, while the platelet count decreased (from to * /l). inhalation of ch increased the sublingual pr by % in the septic group, the creatinine and bilirubin levels were decreased by % and %, respectively. ch post-treatment significantly decreased the ps-sofa score (to ; . - . ) and resulted in lower values in septic shock group (to ; . - . ). methane post-treatment effectively influences sepsis-related end organ dysfunction. up to a severity threshold it may be a promising additional organ protective tool. evaluation of sepsis awareness among various groups in turkey: a survey study s erel, o ermis, Ö nadastepe, l karabıyık gazi university school of medicine, anesthesiology and intensive care, ankara, turkey critical care , (suppl ):p introduction: sepsis is a common life-threatening condition in critically ill patients [ ] . public awareness is important for early recognition of sepsis and improvement of outcomes [ ] . we aimed to evaluate sepsis awareness among different groups of people. methods: prospective paper-based surveys were issued between st july and st august to patients, the relatives of the patiens, hospital staff and general public who gave consent to participate in the study. the questionnaire included ten questions about demographic informations, occupational informations of hospital stuff and sepsis awareness. a total of participated in the survey. of these participants, ( . %) were patients, ( . %) were relatives of patients, ( . %) were physicians, ( . %) were medical students, ( . %) were nurses, ( . %) were other hospital stuff and (% . ) were other people. of these participants, ( . %) had heard of the word "sepsis". ( . %) responded correctly regarding the definition of sepsis. ( . %) of the participants heard the word "sepsis" during their education, but only ( %) heard it through the media. in the groups of high school graduates, university graduates and postgraduates, the rate of hearing the word sepsis and correctly identifying sepsis is significantly higher than the primary school graduates or illiterate groups. (p< . ). physicians, nurses and medical students were heard of the word "sepsis" significantly more than other groups (p< . ). physicians and medical students responded more accurately to the definition of sepsis than other groups (p< . ). public awareness of sepsis is limited compared to healthcare workers. increasing public knowledge of sepsis through education and through media may contribute to raising public awareness and improving outcomes. the association between clinical phenotype cohesiveness and sepsis transitions after presentation jn kennedy , eb brant , km demerle , ch chang , s wang , dc angus , cw seymour key: cord- - yoilsho authors: nan title: abstracts of the (nd) annual meeting of the german society for experimental and clinical pharmacology and toxicology (dgpt) and the (th) annual meeting of the network clinical pharmacology germany (vklipha) in cooperation with the arbeitsgemeinschaft für angewandte humanpharmakologie e.v. (agah) date: - - journal: naunyn schmiedebergs arch pharmacol doi: . /s - - -y sha: doc_id: cord_uid: yoilsho nan in vitro systems and mechanistic investigations i the demand of alternative test systems which closely mirror the in vivo situation is one of the main challenges in modern toxicity testing. the major goal is the development of in vitro systems that partly display the complexity of an organism and thus may mimick in vivo conditions. despite great efforts in the past no adequate in vitro systems are available yet. on the other hand, cell cultures from almost every organ are easily accessible and therefore may help to roughly assess the toxic potential of substances at target structures. nonetheless, the complex interactions which take place in vivo cannot be addressed in single cell cultures. in the liver, hepatocytes comprise % of the total liver volume while non-parenchymal cells -endothelial cells, stellate cells and kupffer cells (that is, liver resident macrophages) -contribute only . % of the volume, but % of the total cell number (kmiec ) . it has been increasingly recognized that in the liver neighboring non-parenchymal cells release molecules which contribute to the inflammatory damage and even aggravate it (adams et al. ) . in our project a human in vitro co-culture system was established by combining a hepatic and a monocytic cell line, the latter of which can be differentiated to a macrophage-like phenotype. in this system the hepatotoxicty of substances has been analyzed, and the results were compared to single cultures and to published data from in vivo studies. using ketoconazole, an antifungal, as a known hepatotoxic substance, inflammatory markers were studied and proved to be significantly upregulated only in coculture. conversely, cultures of hepatic cells only did not display this increase in inflammatory markers. at the same time, a negative substance, caffeine, failed to show any hepatotoxic potential in the co-culture system. our results demonstrate that this novel in vitro co-culture model represents a promising tool to evaluate the hepatotoxic potential of substances. in drug research, it might help to reduce animal testing as drugs with a high dili potential can be dropped early in the development phase. question: raman spectroscopy (rs) is a highly sensitive analytical method for markerfree and non-invasive identification and characterization of cells. here, we present rs as a novel tool for gentle yet precise cell analysis in three independent experiments, focusing on monitoring cellular reactions upon treatment. we could provide evidence that rs is a suitable tool to monitor cell differentiation, analyze cell modification and study cell apoptosis after drug application. methods: in a first experiment, mesenchymal stem cells (mscs) were treated with erythropoetin (epo) for certain time points and subsequently fixed with paraformaldehyde (pfa) for raman analysis. in addition, skbr breast cancer cells were exposed to the anti-cancer drug herceptin ( μg/ml). cells were then fixed in pfa for rs. in a last experiment, molm- cells were separately cultivated in microwells and treated with thymidine for different time points prior to raman analysis. results: raman spectroscopy was able to monitor differentiation of epo treated mscs and found that around % of treated cells showed fibroblast like raman profiles. in case of herceptin treated skbr cells, rs found internal changes of the cell´s metabolism as reaction on drug application. analyzing the most prominent differences in raman spectra revealed discrimination of cells to be mainly due to changes in amid i, lipid and protein content. in the last experiment, rs was able to follow apoptosis of molm- cells after thymidine application and discriminate early from late apoptotic states. discussion: rs is a photonic marker for gentle yet highly specific cell analysis, which allows monitoring of single cell reaction after drug treatment. thereby, rs provides information about changes within the entire metabolome on a single cell level. raman spectra are as characteristic as a "fingerprint". rs works label-free and non-invasive and thus does not impare cell viability. this allows to gain new insights in pharmacological development and toxicological survey. acknowledgement: this project received funding from the eu th health program grant agreement no . deutsches zentrum für herzinsuffizienz, würzburg, germany extracellular signal-regulated kinases and (erk / ) are essential for the regulation of cell growth and cell survival and their kinase activity is up-regulated for example in different types of cancers and pathological cardiac hypertrophy. while inhibition of erk / activity by kinase inhibitors prevents tumor growth, it can also lead to exacerbated cardiomyocyte death and impaired heart function. interestingly, we have previously identified an erk autophosphorylation at threonine as a prerequisite for nuclear erk / signaling and erk-mediated cardiac hypertrophy. here, we investigated an alternative strategy to interfere with erk / signaling: since activation of erk / triggers erk dimerization, a prerequisite for erk t autophosphorylation, we chose the erk dimer interface as a possible target to selectively interfere with erk t -phosphorylation. first, we investigated the impact of monomeric erk on cardiac function. to address this issue, we generated mice with cardiac overexpression of monomeric erk ∆ - and performed transverse aortic constriction (tac) to induce cardiac hypertrophy. compared to wild-type mice, erk ∆ - overexpression attenuated tac-induced cardiac hypertrophy, interstitial fibrosis and mrna expression levels of collagen and brain natriuretic peptide (bnp), while cardiomyocyte survival and cardiac function were largely preserved. because of the positive effects of monomeric erk ∆ - in the heart, we designed a peptide to interfere with endogenous erk dimerization. cross-linking and coimmunoprecipitation experiments showed that the peptide binds to erk and prevents its dimerization. moreover, the peptide effectively inhibited erk t -phosphorylation and nuclear translocation of yfp-tagged wild-type erk after phenylephrine stimulation. further, adenoviral or adeno-associated virus serotype (aav )-induced overexpression of the peptide in neonatal rat cardiomyocytes (nrcm) or mouse hearts resulted in a significantly reduced hypertrophic response to phenylephrine or tac, background: g i -proteins have been proposed to be cardioprotective. it's matter of debate whether this depends on the particular g i isoform and/or on the particular conditions (e.g. cardiac "stress") only. in our study we investigated effects of a gα i knockout on cardiac function and survival in a murine heart-failure model of cardiac β adrenoceptor overexpression. methods and results: β -adrenoceptor overexpressing mice lacking gα i (β -tg/gα i -/-) were compared to wild-type (c bl/ ) mice and littermates either overexpressing cardiac β -adrenoceptors (β -tg) or lacking gα i (gα i -/-). at days of age mortality of mice only lacking gα i was higher compared to wild-type or β -tg, but similar to β tg/gα i -/mice. beyond days mortality of β -tg/gα i -/mice was enhanced compared to all other genotypes (mean survival time: ± days). echocardiography revealed similar cardiac function of wild-type, β -tg and gα i -/mice, but significant impairment for β -tg/gα i -/mice (e.g. ejection fraction ± % versus ± % in wild-type mice). significantly increased ventricle-to body-weight ratio ( . ± . % versus . ± . % in wild types), left-ventricular size (length . ± . cm versus . ± . cm in wild types) and anp and bnp expression (mrna: % and % of wild type, respectively) clearly indicated hypertrophy. gα i was significantly upregulated in gα i -knockouts (protein compared to wild-type mice: ± % in gα i -/and ± % in β -tg/gα i -/-, respectively). radioligand binding experiments confirmed cardiac overexpression of β adrenoceptors in β -tg mice. of note, overexpression levels differed depending on the particular wild-type background. on an fvb/n background we found the overexpression level to be more than -fold higher (b max : ± fmol/mg) than on the otherwise used c bl/ background. accordingly, fvb/n-based β -tg mice showed a significantly impaired cardiac function at an age of d while c bl/ -based β -tg mice did not. conclusions: gα i -deficiency combined with cardiac β -adrenoceptor overexpression strongly impaired survival and cardiac function. on a c bl/ background β adrenoceptor overexpression alone had not induced cardiac hypertrophy or dysfunction at day while there was overt cardiomyopathy in mice additionally lacking gα i . we propose an enhanced effect of increased β -adrenergic drive by lack of protection via gα i . the observed gα i upregulation was not sufficient to compensate for gα i deficiency suggesting an isoform-specific and/or a concentration-dependent mechanism. the role of gα i is currently addressed in a subsequent study using β -tg and gα i deficient mice. heart failure is accompanied by morphological and functional alterations (e.g. hypertrophy, decreased contractility) which are summarized by the term "cardiac remodeling". while the β-adrenergic signaling pathway is essential for short-term modulation of cardiac performance, its chronic stimulation by elevated plasma catecholamines and the subsequent activation of camp-dependent signal transduction pathways is regarded as a fundamental factor in the pathogenesis of cardiac remodeling. however, the mechanisms mediating the transition of physiological conditions of short-term to detrimental remodeling under long-term β-adrenergic stimulation are not understood in detail so far. in this context, icer, an isoform of the camp dependent transcription factor crem (camp responsive element modulator), acts as an early response gene strongly induced by beta-adrenergic stimulation via camp responsive elements (cre) in its promoter. contrary to its cre-mediated induction, icer is a strong inhibitor of cre-mediated transcription by itself. here we study the role of icer induction in the catecholamine-induced cardiac remodeling in a time dependent manner by the use of icer deficient mice (iko) and wild type (wt) controls, which were treated with isoproterenol (iso; mg/kg per d) for and hours and days. overall days of iso stimulation resulted in an elevation of cardiomyocyte length in iko (in µm; d iso ± ) vs. wt cardiomyocytes ( d iso ± ). at this time point a % decrease of cardiac output and a % decrease of the maximal rate of rise of left ventricular pressure (dp/dt max ) in iko vs. wt animals was detectable. the maximum increase of icer mrna in wt cardiomyocytes already occurred after h ( -fold), and declined after h ( -fold) to . fold increase after days, while icer mrna was not detectable in iko mice. this raised the hypothesis, that the early induction of icer modulates transcriptional processes after beta-adrenergicstimulation, involved in cardiac remodeling of the heart. profiling of mrna expression levels between iko vs. wt cardiomyocytes at the different time points revealed: regulated genes (up-regulated: %) in untreated; altered genes (up %) after h; changed genes (up %) after h and altered genes (up %) after days of iso treatment. in summary, the absence of icer induction in myocytes resulted in an increase of cardiomyocytes length and a decrease of heart performance after days of betaadrenergic stimulation. this is preceded by upregulated mrna levels of several hundred genes at h, which is going along with the induction of transcriptional inhibitor icer after a few hours of beta-adrenergic stimulation. this suggested a protective role of icer by inhibiting the progression of cardiac remodeling after betaadrenergic stimulation in an early responsive manner. (supported by the dfg) the performance of the adult heart is tightly regulated by g protein-coupled receptors. adrenergic and angiotensin receptors efficiently control heart rate and contractility. muscarinic receptors, on the other hand serve as master regulators of the conduction system, which is often lost upon myocardial infarction. this function of muscarinic receptors has been well described in the adult or late embryonic heart. here we provide evidence that muscarinic receptors are crucial to constrain pacemaker cell identity. we applied subtype-specific inhibitors of muscarinic receptors to zebrafish embryos of different stages. we observed that both, early cardiac function as well as specification are specifically regulated by muscarinic m receptors, while m receptors appear to exert a heart-specific function only at later stages. continuous m blockage renders zebrafish with greatly altered cardiac morphology, particularly of the conduction system. furthermore, embryos with m inhibition display impaired ventricular function most likely due to an av-block and substantial arrhythmia in the atrium. importantly, to observe these phenotypes it was sufficient to block m receptors during stages of cardiac differentiation, which is long before a heart tube has formed. we corroborated our findings regarding these morphological changes using marker gene analysis. furthermore, we obtained evidence for m receptors preventing a transcriptional program towards the induction of pacemaker cells at the expense of av canal cells. importantly, this is not only true during heart development. a pacemaker program is also induced in adult hearts upon m inhibition. taken together, we postulate that muscarinic m receptors confine a pacemaker lineage during early steps of heart development as well as in the adult heart. our data suggests m receptors as potential new therapeutic targets for the regeneration of hearts with an injured sinoatrial node. systemic inhibition of mir- has proved effective against fibrosis of the myocardium and in other organs. mir- has been reported to exert detrimental effects in cardiac fibroblasts and protective roles in cardiac myocytes and other myocardial cell types. a better definition of the cell types that contribute to the beneficial effects of inhibiting mir- in vivo may aid the development of strategies with enhanced therapeutic efficacy. thus far, no approach to selectively manipulate micrornas in the non-myocyte population of cardiac cells in vivo has been available. in this study, we developed an icre-encoding mml virus for application in mir- fl/fl mice. delivery of this vector to neonates achieved targeted genetic ablation of mir- in non-myocyte cardiac cells. immunohistochemistry and flow cytometry confirmed that mmlv was highly selective and effective for cardiac fibroblasts and endothelial cells. in parallel, an aav -icre vector allowed for specific and almost complete deletion of mir- in cardiac myocytes. when tested in a model for chronic left ventricular pressure overload, mmlv-icremediated deletion of mir- in cardiac fibroblasts and endothelial cells significantly reduced cardiac fibrosis and hypertrophy and improved cardiac function. the benefit of this cell-type-specific inhibition exceeded that observed upon global genetic deletion of the mir- gene in mice. aav -mediated deletion of mir- , albeit lowering cardiac hypertrophy, had no effect on fibrosis or cardiac function. taken together, neonatal delivery of engineered icre-encoding viruses enabled for the first time a differential gene targeting in non-myocyte and myocyte cells in myocardium. non-myocyte deletion of mir- demonstrated that mir- exerts its cardiac profibrotic activity directly in cardiac fibroblasts and in endothelial cells. this novel finding should encourage tailoring of antimir- therapy towards cellular tropism. chronic inflammatory diseases, such as psoriasis or rheumatoid arthritis, are characterized by constant leukocyte infiltration and ongoing angiogenesis in the inflamed tissue. as current anti-inflammatory pharmacotherapy is not always satisfying, there is a great demand for the discovery of new drug leads as well as novel drug targets. the synthetic carbazole alkaloid derivative c acts as a multikinase inhibitor. results of a thermal shift assay revealed that c shows by far the highest binding affinity to the bmp- -inducible kinase (bmp k/bike). bmp k represents an as yet largely uncharacterized protein, which is not regulated by bmp- in endothelial cells. therefore, we aimed to analyze (i) the pharmacological potential of c and (ii) the role of bmp k in angiogenic and inflammatory processes in the vascular endothelium. initial experiments show that only high concentrations of c affected the viability of human umbilical vein endothelial cells (huvecs). both c and the knock-down of bm k (rnai) reduced the migratory capacity of a human microvascular endothelial cell line (hmec- ). also the proliferation of hmec- was reduced by c treatment (ic : µm). a tube formation assay on matrigel demonstrated that c significantly impaired the formation of capillary-like structures in a dose-dependent manner. interestingly, the analysis (western blot) of signaling molecules in huvecs that play a crucial role in cell proliferation (e.g. erk, akt) revealed that these pathways are not influenced, neither by c treatment nor by bmp k gene silencing. in regard to inflammatory processes, c treatment or bmp k silencing of huvecs decreased the adhesion of thp- cells, a monocytic cell line, onto the activated endothelial cells. as the interaction of leukocytes is mainly mediated by cell adhesion molecules (cams), the effect of c or bmp k silencing on their expression was analyzed (flow cytometry, qpcr). while the expression of cams was strongly decreased after c treatment, the knock-down of bmp k did not markedly affect their expression. furthermore, both approaches did not lead to the reduction of tnf-induced iκbα degradation (western blot) or p translocation into the nucleus (microscopy). our study provides first insights into the anti-inflammatory and anti-angiogenic potential of the carbazole alkaloid derivative c in vitro. the precise role of bmp k in angiogenic and inflammatory endothelial processes as well as the involved pathways during bmp k silencing and c treatment will be further elucidated. moreover, since the inhibition of bmp k seems not to be responsible for all actions of c , we will investigate the role of other kinases affected by the compound in these processes. the chemokine receptor cxcr is a multifunctional receptor which is activated by its natural ligand c-x-c motif chemokine (cxcl ). cxcr seems to be part of the lipopolysaccharide sensing complex, suggesting that an intervention with cxcr agonists or antagonists could result in reduced tlr signaling. however, the role of cxcr and the influence of different cxcr ligands in acute as well as chronic inflammatory diseases are still contradictious. therefore, we aimed to characterize the systemic effects of cxcr activation in severe systemic inflammation and to evaluate its impact on endotoxin induced organ damages by applying a sublethal lps dose ( mg/body weight) in mice. the plasma stable cxcl analog ctce- d was synthesized and administered subcutaneously shortly before lps treatment to ensure a delayed release and thereby a prolonged effect of the drug. hours following lps administration, mice were sacrificed and blood was obtained for tnf alpha, ifn gamma and blood glucose evaluation. additionally, histopathological changes and oxidative stress in the liver and spleen were assessed and liver biotransformation capacity was determined. finally, cxcr , cxcl and tlr expression patterns in liver, spleen and thymus tissue as well as the presence of different markers for oxidative stress and apoptosis were evaluated by means of immunohistochemistry. ctce- d improved the health status and distinctly reduced the lps mediated effects on tnf alpha, ifn gamma and blood glucose levels by approximately %, % or %, respectively. it attenuated oxidative stress in the liver and spleen tissue and enhanced liver biotransformation capacity unambiguously. ctce- d diminished the lps induced expression of cxcr , cxcl , tlr , nf-κb, cleaved caspase- and gp phox, whereas heme oxygenase expression and activity were induced above average. furthermore, tunel staining revealed anti-apoptotic effects of ctce- d in all organs. the cxcr is undoubtedly involved in inflammation. its activation was accompanied by anti-inflammatory, anti-oxidative and cytoprotective effects as ctce- d attenuated tlr signaling, induced heme oxygenase activity and mitigated apoptosis. thus, the administration of cxcl analogs seems to be a promising treatment option to control acute systemic inflammation, especially when accompanied by a hepatic dysfunction and an excessive production of free radicals. the neurodegenerative disease friedreich ataxia (frda) is caused by a gaa triplet repeat expansion in the first intron of the frataxin gene, which results in a reduction of the corresponding mitochondrial protein. despite several cellular and animal models the exact function of frataxin is still a matter of debate, but the role of frataxin in iron sulfur cluster biosynthesis is generally accepted. however, we still don't know which primary metabolic events are caused by a frataxin deficit and until now, there is no therapeutic option available. we developed a new cellular model for frda by using the cre/loxp recombination system in mouse embryonic fibroblasts (mef). c bl/ j mouse strains with a loxp flanked exon of the frataxin gene and an tamoxifen-inducible cre-recombinase (creer t ) were crossed and several mef cell lines isolated. after selection by genotype and growth manner the fx-mef - (fxn -/-) and fx-mef - (fxn +/-) cell lines were finally choosed. the generation of the homozygous or heterozygous frataxin knockout was successfully tested on rna and protein level. long maintenance of the frataxin depleted fibroblasts revealed a strong growth inhibition consistent to earlier observations in other cell systems. therefore, we established a pattern of treatment over days, with medium and substance changes at day and , which allows us to get a fully functional knockout and overcome the growth inhibition problem. endpoint measurements of known metabolic phenomena from mammalian and non-mammalian models were studied at day of our novel cell system. the induced total disruption of frataxin leads to a clearly reduced aconitase activity, cell division and oxygen consumption as well as an increase in ros production. in the heterozygous knockout with residual frataxin activity no such changes were observed. in addition, our pattern of treatment enables us to monitor the full and partial frataxin knockout in the course of time, to detect early and late metabolic events after frataxin disruption. therefore we analysed the mentioned parameters (with additional atp and iron content) in parallel at day , , and and could identify an initial event followed by secondary consequences and parameters, which seem to play only a minor role in the frda pathogenesis. on the contrary, a partial deficit of frataxin didn't result in any differences over time and suggests that there are only cellular alterations below a critical threshold. in conclusion, our new established mammalian cellular frda model mimics typical metabolic consequences of the human disease and seems to be qualified for frda research. the model shows for the first time six different metabolic events in the course of time in parallel and reveals insights into primary and secondary events of frda pathogenesis. these observations can be used to better understand the function of frataxin and can help to develop new therapeutic strategies to address the consequences of frataxin deficiency. moreover, the transfer of this cell model into well plates offers the possibility for a high-throughput screening of potential therapeutic substances. the disease diphtheria is caused by the diphtheria toxin (dt) which belongs to the group of single-chain ab-type bacterial protein toxins. receptor-binding of the b-domain on the target cell surface is followed by receptor-mediated endocytosis and internalization into early endosomal vesicles. endosomal acidification triggers membrane insertion and pore formation of the transmembrane (t) domain together with translocation of the (partially) unfolded catalytic (c) domain into the cytosol. herein, dta catalyzes adp-ribosylation of elongation factor which leads to disruption of protein synthesis and finally causes cell death [ ] . in hela cells, these events are related to cell-rounding functioning as a specific endpoint to monitor the uptake of dta into the cytosol of the host cell. as for other adp-ribosylating toxins such as c. botulinum c toxin, c. perfringens iota toxin and c. difficile cdt, also in case of native dt we demonstrated the involvement of several host cell factors during the translocation step of the catalytic domain across the endosomal membrane [ , ] . in detail, we confirmed the involvement of the host cell chaperone hsp and the thioredoxin reductase (trxr), the latter presumably responsible for the reduction of the interchain disulfide bond between the dta and dtb moieties [ , , ] . furthermore, we identified another group of protein folding helpers, the family of peptidyl-prolyl cis/trans isomerases (ppiases) including cyclophilin a (cypa), cyp and fk -binding protein (fkbp) as required cytosolic factors for dta translocation. to characterize the role of the protein folding helpers in more detail, we investigated their interaction with purified dta in vitro by performing dot blot analysis with immobilized recombinant host cell factors, co-precipitation of cellular factors with dta from hela lysate and isothermal titration calorimetry with purified proteins therewith determining the thermodynamic parameters of the individual binding events. thereby, we detected binding of dta to hsp , cypa, cyp , fkbp and fkbp . the data increase the knowledge of the molecular mechanisms underlying dt uptake and especially dta translocation which can be medically used to develop novel therapeutic strategies against the disease diphtheria. [ ] murphy ( ) toxins , - . [ ] barth ( ) naunyn-schmied arch pharmacol , - . [ ] kaiser et al. ( ) cell. microbiol. , - . [ ] dmochewitz et al. ( ) cell. microbiol. , - . [ ] ratts et al. ( ) j. cell biol. , - . [ ] schnell et al. ( ) novel afflictions as for example clostridium (c.) difficile associated diseases (cdad) caused by clostridium difficile are on the increase and challenging to treat. cdad most frequent occur in hospitalized patients after prolonged treatment with antibiotics. cdad includes among others diarrhea and the severe form of pseudomembranous colitis. not only the treatment of the infection, but also the treatment of the toxins has a high clinical significance. c. difficile secretes the exotoxins a (tcda) and b (tcdb), which glycosylate and thereby inactivate rho-gtpases in mammalian cells. tcda and tcdb are considered as the causative agents of cdad. in the last few years, more and more hypervirulent strains of c. difficile were described. in these hypervirulent strains, the adp-ribosyltransferase cdt was found as a third toxin in addition to tcda and tcdb. given the lack of agents effective against antibiotic-resistant bacterial strains and bacterial exotoxins, the development of novel pharmacological strategies is needed. the antimicrobial activity of naturally occurring substances is already known for a long time. one important mechanism of the innate immune system is the production of natural peptides showing antibiotic features. in recent years, it was shown that human antimicrobial peptides as important part of the native innate immune system plays a crucial role not only in inactivation of bacteria but also in inhibition of bacterial toxins ( ). prompted by these result, we found that only human α-defensin- (hnp- ) but not human β-defensin- (hbd- ), both important effectors of the innate immune system, protected cultured epithelial cells from intoxication with tcda and cdt when applied prior to the toxins to the cells. moreover, α-defensin- prevented also the cytotoxic effects of all three c. difficile toxins tcda, tcdb and cdt combined in the medium. the combined investigation of all three toxins might be even more suitable to mimic the situation after an infection with hypervirulent c. difficile. the inhibition of the toxins was monitored by cell rounding caused by each of the toxins. currently, the molecular mechanisms underlying the inhibitory effects are still unknown and will be investigated in different cell lines. in conclusion, our results demonstrate that hnp- causes a loss of cytotoxicity of the c. difficile toxins and may act as novel drugs to cure c. difficile infections that contribute to cdad. neurodegenerative diseases like parkinson´s disease (pd) are accompanied by altered gene expression levels in the brain. recent studies support a role of regulatory noncoding rnas, such as micrornas (mirnas), which silence a specific set of mrnas at the post-transcriptional level. upon their aberrant expression, they are likely involved in the pathophysiology of specific neuronal loss. manipulation of neuronal gene expression is pivotal for understanding the function of proteins and the development of new therapeutic strategies. rna interference (rnai) strategies can be employed through the administration of small interfering rnas (sirna), which mediate the specific knockdown of a selected target gene. however, the main challenge is the delivery of these rnas into the neurons of interest. in this pilot study, we present a method for delivering sirnas in polymeric nanoparticles based on low molecular weight polyethylenimines (peis). their intracerebroventricular (icv) injection leads to in vivo silencing of neuronal gene expression in the brain of mice overexpressing α-synuclein (thy -asyn mice). in a first step, pei-complexed sirna tagged with afluorescencedye were injected to track the localization and distribution after icv administration. five days later, fluorescent cells were visible throughout the brain, with the highest fluorescence intensity around the ventricles. fluorescence was also observed in large cells of the lumbar spinal cord. moreover, preliminaryresultsdemonstrate a . % knockdown (p< . student's t-test, n= ) of human α-synuclein (snca) in thetargetstructurestriatum upon a single icv injection of pei-complexed specific sirna compared to the control injection group (n= ). hence, our first results support the usability and efficacy of pei nanoparticle-mediated delivery of short rnas, namely sirnas, for rapidly and efficiently reducing the expression of a neuronal target gene of interest in the brain in vivo. this may allow the development of gene therapy strategies for the treatment of neurodegenerative diseases. is a propenylic alkenylbenzene found in several plants, e.g. acorus calamus. bacontaining plant materials are used to flavor foods, and are active ingredients in traditional plant medicines. thus, human exposure results primarily from the intake of bitters and teas, as well as from calamus-containing medicines and plant food supplements. although many (positive) pharmacological properties/effects of asarone isomers are described in the literature, ba was found to be carcinogenic in rodents (liver, duodenum) when given daily or in a single dosage. early experiments indicated that ba is not activated via hydroxylation and sulfonation as it is the case for known hepatocarcinogenic allylic alkenylbenzenes such as estragole or methyleugenol. because the mechanism of metabolic activation of ba in not known, we investigated the metabolism of ba in liver microsomes and human cytochrome p (cyp) enzymes, the mutagenicity of ba and its metabolites in the ames fluctuation assay and the dna adduct formation in primary rat hepatocytes. we found that side-chain epoxidation (leading to diols and a ketone) was by far the most dominating metabolic route of ba in liver microsomes and human cyp enzymes. ba was mutagenic in the ames test (+s mix), as was the synthesized ba-epoxide (-s mix). furthermore, we were able to synthesize and characterize a ba epoxide-derived dna adduct with deoxyguanosine. this dna adduct was formed in a concentration-dependent manner in rat hepatocytes incubated with ba. our results strongly indicate that ba is genotoxic with the side-chain epoxide being its ultimate carcinogen. morbid obesity is an independent risk factor for cardiovascular disease, type diabetes mellitus and certain types of cancer. bariatric surgery -with the roux-en-y gastric bypass (rygb) being the gold standard -has become the therapeutic option of choice as a sustained weight loss and improvement of associated morbidity is achieved in the majority of patients. there is, however, a lack of evidence focusing on bariatric surgery induced sustained weight loss and its possible impact on cancer risk. we investigated the association between obesity, oxidative stress and genomic damage after roux-en-y gastric bypass surgery (rygb) or caloric restriction induced weight loss in the obese zucker rat. obese male zucker fa/fa rats were divided into three groups: sham surgery (sham), rygb and caloric restriction (cr) and were compared with lean controls (lean; zucker fa/+ rats). shams showed impaired glucose tolerance and elevated plasma insulin levels, which were less severe in rygb and cr. oxidative stress was elevated in kidney, liver and colon tissue of sham and reduced again after weight loss induced by either rygb or bwm. urine-derived oxidization products of lipids, dna and rna increased in shams and decreased after weight loss (rygb and cr). dna double strand breaks were more frequent in shams than in the weight loss groups or lean. dna damage in zucker fa/fa rats correlated with their basal plasma insulin values. obese rats showed elevated oxidative stress and genomic damage in comparison to lean rats. after body weight loss, achieved by either rygb or caloric restriction alone, oxidative stress level and genomic damage were decreased. this may indicate a reduction of the elevated cancer risk in obesity. ) mice were treated with the nocrelated compound azoxymethane (aom) followed by the administration of dextran sodium sulfate to trigger crc. tumors were quantified by non-invasive mini-endoscopy, which revealed a non-linear increase in crc formation in wt and aag -/mice. in contrast, a linear dose-dependent increase in tumor frequency was observed in mgmt -/and mgmt -/-/aag -/mice. the data was corroborated by hockey stick modeling, which yielded similar carcinogenic threshold doses for wt and aag -/mice. o -meg levels and depletion of mgmt activity correlated well with the observed dose-response in crc formation. aom dose-dependently induced double strand breaks (dsbs) in colon crypts including in lgr -positive colon stem cells, which coincided with atr-chk -p signaling. intriguingly, mgmt-deficient mice displayed significantly enhanced levels of γ-h ax, suggesting the usefulness of γ-h ax as an early genotoxicity marker in the colorectum. this study demonstrates for the first time a non-linear dose-response for alkylation-induced carcinogenesis and reveals dna repair by mgmt, but not aag, as a key node in determining a carcinogenic threshold at low alkylation dose levels [ ] . obesity is characterized as a status where the excessive accumulation of fat in adipocytes leads to local inflammation and hypoxia; both contributing to severe obesity associated co-morbidities such as cardiovascular disease and type diabetes mellitus. local inflammation is mediated by macrophages, stromal vascular cells, preadipocytes, and adipocytes as well as by a number of proinflammatory cytokines and chemokines ( ). in particular, the chemokines monocyte chemoattractant protein (mcp- , ccl ), interleukin- (il- /cxcl ) and stromal cell-derived factor (sdf- a/cxcl ) secreted by stromal vascular cells, preadipocytes, and adipocytes exert paracrine effects by recruiting neutrophils, monocytes/macrophages, and t-and b-cells. interestingly, deficiency in cxcl was shown to attenuate obesity in mice ( ) . the cc chemokine ccl is known to stimulate ccr receptors, and the cxc chemokines cxcl and cxcl to activate cxcr and cxcr or cxcr and cxcr , respectively. the receptor(s) activated by cxcl is currently unknown. a role of cxcl in modulating cxcr signaling has been proposed. we initiated our studies to determine the presence and functional significance of chemotaxin receptors in human adipocytes and their precursor cells. to this end, the mrna expression pattern of cc chemokine receptors, cxc chemokine receptors formylated peptide receptor fpr and the related receptor fprl , and cc and cxc chemokines was analyzed during in vitro adipose differentiation of human simpson-golabi-behmel-syndrome (sgbs) preadipocytes, and under conditions mimicking an inflammatory response. in particular, we focused on the expression pattern of human ccr receptors, since previous reports indicated a role in adipogenic differentiation. however, our comprehensive analysis using different sources of adipocytes and their precursors indicated that ccr receptors were absent ( ) . yet, the analysis revealed appreciable levels of mrna encoding ccl , cxcl , and cxcl , and ccr , cxcr , cxcr , fpr , and fprl , and cxcr . of interest, cxcr -and cxcr -mrna were found to be up-regulated under the proinflammatory conditions. to analyze the responses of adipocytes and their precursors to chemokine receptor agonists, we used chemokine-mcherry fusion proteins purified from baculovirus-infected insect cells, e.g ccl , cxcl , cxcl . while sgbspreadipocytes and adipocytes did not accumulate ccl -mcherry upon stimulation, they showed a small accumulation of cxcl -mcherry, and a strong accumulation of cxcl -mcherry in the endosomal compartment. similar results were obtained in murine t l- preadipocytes. using mass spectrometry analysis, we set out to identify the cxcl -binding putative receptor protein(s) in murine t l- preadipocytes. ( ) makki, k. et al., ( ) in personalized medicine tumors are screened for several mutations in oncogenes or tumor suppressors. however, the cellular protein content not exclusively depends on the dna. we identified new rac variants generated on the mrna level in androgenindependent prostate cancer cells. all variants represent active forms of the gtpase. they are capable to suppress rhoa-induced apoptosis and additionally, mediate the synthesis of genes which are under the control of the androgen receptor. importantly, expression of the rac variants is sufficient to support tumor growth in mice. we prove the existence of the variants and verify their clinical appearance and relevance in tissue samples of a prostate cancer patient. dna analysis, however, revealed the wildtype sequence of rac. therefore, routine analysis of patient tumor tissue would miss the detection of active rac which precludes the success of therapy. the existence of active rac variants in prostate cancer tissue that promote resistance towards androgen deprivation suggest rac inhibition as an effective add on therapeutic strategy against prostate cancer. the bacterial effector protein exotoxin y (exoy) of pseudomonas aeruginosa is delivered into host cells via the bacterial type iii secretion system. once arrived in the host cell nucleotidyl cyclase activity of exoy is activated by a yet unknown cofactor and thus has a profound effect on concentrations of cyclic nucleotides: in addition to production of cyclic amp (camp) and cyclic gmp (cgmp) there is a massive synthesis of cyclic ', and to some extent of the corresponding cytidylyl analogue ccmp , . currently, the role of cump and ccmp during the pathogenesis of p. aeruginosa infection remains unknown . one of our hypotheses is that these cyclic nucleotides fulfil a role as first messengers, e.g. in the communication between individual bacteria or bacterial populations during establishment of acute or chronic infections. to test this hypothesis, the intra-and extrabacterial concentrations of cyclic nucleotides were measured via hplc-ms/ms at different time-points in liquid cultures of p. aeruginosa, either in a complete (lb medium) or a starving medium (vogel-bonner medium). additionally, we tested if supplementation of the media with extrinsic cump or ccmp had an effect on these measured concentrations. the influence of extrabacterial cyclic nucleotides on the bacterial metabolism and homeostasis was evaluated with a microarray of bacterial total cdna extracted at different time points of p. aeruginosa liquid culture with or without extrinsic cump/ ccmp. furthermore we investigated a potential function of the cyclic nucleotides in biofilm formation. cyclic ump and cyclic cmp have differential roles in bacterial metabolism and communication. for example, whereas ccmp is synthesized by p. aeruginosa when the bacteria are in a nutrient-rich environment, we could not detect bacterial cump under any tested circumstance. in our biofilm formation assays, only ccmp had a biofilmpromoting effect, but only in very high concentrations. the currently ongoing analysis of gene expression data in the presence or absence of cump may reveal a role of this cyclic nucleotide as first messenger, too. in further studies we will elucidate the signal transduction processes underlying the observed cump / ccmp effects, for example by identifying cump and ccmp binding proteins and their coupling mechanisms to intracellular signalling cascades. synapses are complex computational platforms that transmit information encoded in action potentials but also transform their functionality through synaptic plasticity. g protein-coupled receptors (gpcrs) play a major role in modulating the strength of the synapses via the second messenger camp . however the spatio-temporal dynamics of the mode of action of camp underlying synaptic plasticity are still controversial. the role of this study was to investigate the dynamics of camp signaling at the drosophila neuromuscular junction, where octopamine binding to its receptors has been shown to cause camp-dependent synaptic plasticity . for this purpose, we generated a transgenic drosophila expressing the camp sensor epac -camps in motor neuron. this allowed us to directly follow the octopamine-induced camp signals in real time by fluorescence resonance energy transfer (fret) in different compartments of the motor neuron (i.e. cell body, axon, boutons). we found that octopamine induces a steep camp gradient from the synaptic bouton (high camp) to the cell body (low camp), which was due by higher pde activity in the cell body. high octopamine concentrations evoked a response also in the soma. notably, these signals were independent and isolated form each other. moreover, application of octopamine by iontophoresis to single synaptic boutons induced bouton-confined camp signals. these data reveal that a motor neuron can posses multiple and largely independent camp signaling compartments, and provide new basis to explain how camp could control neurotransmission at a level of a single synapse. kandel, e.r., dudai, y. & mayford, m.r. the molecular and systems biology of memory. cell , - ( ) . koon, a.c., et al., autoregulatory and paracrine control of synaptic and behavioral plasticity by octopaminergic signaling. nat neurosci. ( ): p. - ( ) . nikolaev vo, bünemann m, hein l, hannawacker a, lohse mj novel single chain camp sensors for receptor-induced signal propagation. j. biol. chem. , - ( ) cardiovascular pharmacology hyaluronic acid deposition determines engineered heart muscle characteristics and can be pharmacologically targeted to enhance function s. schlick background: engineered human myocardium (ehm) can be generated from psc derived cardiomyocytes (cms) and primary fibroblasts suspended in a collagen i hydrogel ( %: %: . mg/ml). ehm development encompasses an early consolidation phase followed by functional maturation. the presence of fibroblasts is essential for consolidation into a force-generating ehm. here we assessed the hypothesis that fibroblasts of different origin support ehm formation differentially as a function of hyaluronic acid deposition. methods and results: oscillatory rheology ( % strain, hz) on cell-free and cell containing collagen i hydrogels directly after casting revealed enhanced consolidation in the presence of human foreskin fibroblasts (ffbs) compared to primary adult cardiac fibroblasts (cfbs) -change in storage modulus over time (pa/min): collagen . ; collagen + cms . ; collagen + cms + cfbs . , collagen + cms + ffbs . . we next generated ehm with cms and ffbs or cfbs. after weeks of culture under serum-free conditions, we assessed ehm function by contraction measurements. ffb-ehms developed a significantly (p< . ) higher force of contraction (foc) per cross sectional area (csa) than cfb-ehms (maximal foc/ csa are in mn/mm : . ± . , n= vs. . ± . , n= ). cross sectional area (csa) of tissues was greatly increased (p< . ) in cfb-ehms (csa in mm : . ± . , n= vs. . ± . , n= ) and nonmyocyte content was higher in cfb-ehms ( . ± . , n= vs. . ± . , n= ; x cells/ml). histological analysis revealed that cardiomyocytes were only poorly matured in cfb-ehms compared to ffb-ehms. extending ehm functional data, principal component analysis of rnaseq data revealed distinct expression patterns for ffbs and cfbs, in which hyaluronic acid synthase (has ) enzyme was significantly (p< . ) upregulated. based on these findings, we pharmacologically intervened with has mediated hyaluronic acid (ha) deposition by treating cfb-ehms with hyaluronidase during all weeks of culture. interestingly, ecm manipulation with low concentrations of enzyme significantly (p< . ) reduced csa (csa in mm : control . ± . , n= ; hyaluronidase of concentrations from . u to u . ± . , n= ) with a concurrent, statistically significant (p< . ), increase in contractile function and improved cardiomyocyte morphology on a histological level (maximal foc/csa in mn/mm : control . ± . , n= ; hyaluronidase of concentrations from . u to u . ± . , n= ). summary and conclusions: our data suggest that ehm consolidation is influenced differentially by fibroblasts of different tissue origin with hff-ehm being functionally superior to cfb-ehm. cfb-ehm could be rescued by hyaluronidase leading to reduced ha deposition. the latter demonstrates that extracellular matrix composition is centrally involved in ehm development. angiogenesis is the process of formation of new blood vessels from the pre-existing ones. vascular endothelial growth factor (vegf) is the most studied regulator of this process. by binding to its type receptor (vegfr ), it has been shown to activate a variety of different signaling-pathways leading to enhanced angiogenesis. camp, on the other hand, is a versatile second messenger which regulates various endothelial functions including barrier function. it directly activates protein kinase a (pka) or the exchange protein directly activated by camp (epac) which is a guanine exchange factor (gef) for the small monomeric gtpase rap. as human umbilical vein endothelial cells (huvec) express both camp effectors (epac and pka), we investigated the role of camp-signaling using a spheroid based sprouting assay as an in vitro model for angiogenesis. interestingly, the activation of β-adrenergic receptors with µm of isoproterenol significantly increased the cumulative sprout length. similarly, the selective activation of epac with µm of the camp analog -pcpt- '-o-camp ( ) significantly increased the basal and the vegf-induced cumulative sprout length. in accordance, sirna-mediated depletion of epac in huvec decreased the basal and vegf-induced sprouting. surprisingly, µm of forskolin increased basal and vegf-induced cumulative sprout length stronger than , indicating an additional role of pka. in accordance, µm of myristoylated pki, a membrane-permeable specific pka inhibitor, significantly attenuated the forskolin-induced increase in sprouting. in all conditions tested, ng/ml of vegf always showed an additive effect to the same extent on cumulative sprout length. therefore, our data indicate that the vegf-pathway is acting independently of the camp-pathway in the regulation of the sprouting angiogenesis. the β-adrenergic receptor-mediated activation of camp signaling in huvec induces angiogenic sprouting by activation of epac and pka. introduction: hypertension is one major risk factor for the development of chronic heart and kidney disease. mineralocorticoid receptor (mr) antagonists are a cornerstone in the therapy of heart failure and there is first evidence for a beneficial effect on the kidney as well. inflammation plays an important role in hypertensive organ injury. thus, this study was designed to evaluate and directly compare the effect of mr deletion in endothelial cells on blood pressure and cardiac vs. renal injury in a mouse model of deoxycorticosterone acetate-induced hypertension. methods and results: mice lacking the mineralocorticoid receptor in endothelial cells (mr cdh cre ) were created using the cre/loxp system. mr cdh cre and cre-negative littermates (mr wildtype ) underwent unilateral nephrectomy and received % nacl with drinking water for weeks. the mineralocorticoid deoxycorticosterone acetate (doca, . mg/d) was delivered by subcutaneous pellets. untreated mice served as controls (ctrl). ambulatory blood pressure was determined by implantable telemetry in awake mice. doca/salt treatment increased mean blood pressure in mr wildtype ( . ± . vs. ctrl . ± . mmhg, p< . ) and mr cdh cre ( . ± . vs. ctrl . ± . mmhg, p< . ) without differences between genotypes. cardiac hypertrophy after doca/salt treatment was ameliorated in mr cdh cre mice (ventricle weight . ± . mg vs. mr wildtype . ± . mg, p< . ). doca/salt significantly increased cardiac fibrosis and the expression of fibrotic marker genes in mr wildtype but not in mr cdh cre mice. this was accompanied by an increased expression of the vascular cellular adhesion molecule (vcam ) in mr wildtype cardiac endothelial cells. renal function was not altered by mr deletion in endothelial cells at baseline. doca/salt treatment lead to marked interstitial fibrosis in the kidneys of mr wildtype (sirius red fibrosis score: . ± . vs. ctrl . ± . , p< . ) and mr cdh cre ( . ± . vs. ctrl . ± . , p< . ) mice. mrna expression of the fibrosis marker gene col a (mr wildtype . ± . -fold; mr cdh cre . ± . -fold vs. ctrl) was similarly increased. periodic acid-schiff staining revealed glomerular injury in both genotypes. this was associated with a marked rise in urinary albumin / creatinine ratio (mr wildtype . ± . fold; mr cdh cre . ± . -fold vs. ctrl). in the kidney vcam mrna expression and the number of macrophages was increased by doca/salt treatment independently from endothelial mr deletion. conclusion: in conclusion, mr deletion from endothelial cells ameliorated doca/saltinduced cardiac but not renal inflammation and remodeling independently from blood pressure. these findings suggest different mechanisms for the beneficial effect of mr antagonists in hypertensive heart vs. kidney disease. platelets are relevant cells implicated in morbidity and mortality provoked by cardiovascular thrombosis. even with the actual antiplatelet therapy there is still a substantial incidence of arterial thrombosis. therefore, a better understanding of the mechanisms involved in platelet activation and aggregation is required to develop improved antiplatelet therapies. the increase in the intracellular ca + concentration due to ca + entry from the extracellular space is critical for platelet activation and aggregation. ca + entry follows activation of plasma membrane receptors including gqcoupled receptors for adp, thromboxane a (txa ) or thrombin, as well as the collagen receptor glycoprotein vi (gpvi). the cellular signalling pathways downstream these receptors involve activation of phospholipase c and second messengers that are known to mediate activation of trpc channels. trpc proteins form receptor-operated cation channels, but their regulation and permeability differ depending on the cell type. it has been proposed that trpc proteins might contribute to platelet function as constituents of agonist-activated ca + entry channels; however, the experimental approaches used so far and the lack of specific agonists or antagonists have not allowed to determine the individual contribution of trpc proteins for agonist-induced ca + entry in platelets, aggregation and thrombosis formation. we detected the expression of trpc and trpc in human and mouse platelets. we identified that those proteins together are essential components of a system of coincidence detection in cellular ca + signalling. this coincidence detection triggered by simultaneous stimulation of both thrombin and collagen receptors is required for the phosphatidylserine exposure in human and murine platelets, indicating the role of trpc /c proteins for procoagulant activity. in addition, we detected the expression of s trpc transcripts in mouse platelets. therefore, we tested trpc /c /c -deficient mice in an in vivo model of arterial thrombosis where they showed reduced thrombus formation. regardless of the protective effect of trpc /c /c inactivation observed in the thrombosis model, no differences were detected in tail bleeding. to evaluate the relevance of these trpc proteins in platelet aggregation we measured in vitro platelet aggregation in platelets from trpc /c /c -deficient mice and we observed that the aggregation was reduced after adp ( µm) or txa -analogue ( µm) stimulation, but not after collagen stimulation ( µg/ml). we are currently analyzing the in vitro aggregation and the agonist-evoked ca + response in platelets from different trpc-compound and single deficient mouse lines to understand the mechanisms behind this phenotype with regard to its complementarity to actual antiplatelet therapy. background: thrombin signaling initiates inflammatory events directly and through activation of platelets. endogenous and pharmacologic inhibitors of thrombin are therefore of relevance during atheroprogression and for therapeutic intervention. the small leucine-rich proteoglycan biglycan (bgn) is such an endogenous thrombin inhibitor that acts through activation of heparin cofactor ii (hcii). here, the effect of genetic deletion of bgn on thrombin activity, inflammation and atherosclerosis was addressed. methods and results: bgn concentrations were elevated in the plasma of patients with acute coronary syndrome. in apoe -/mice, bgn was detected in the plasma as well as in the glykokalyx of capillaries. additionally, bgn expression occurred in the subendothelial matrix of arterioles as well as in atherosclerotic plaques. in line with a role of bgn in balancing thrombin activity, apoe -/-/bgn -/ mice exhibited higher activity of circulating thrombin and increased numbers of activated platelets than did apoe -/mice. furthermore, higher concentrations of circulating cytokines in apoe -/-/bgn -/ mice suggested a pro-inflammatory phenotype. likewise, immunohistochemistry and facs analysis of the aorta demonstrated increased macrophage content in atherosclerotic lesions of these mice. in addition, apoe -/-/bgn -/ mice exhibited higher aortic plaque burden and larger atherosclerotic lesions at the aortic root. of note, apoe -/-/bgn -/ mice showed progressive dilatation of the aortic arch corresponding to a decrease in collagen fibril density suggestive of an outward remodelling in the absence of bgn. no differences were evident with respect to lipid content of the aortic root plaques or circulating plasma lipids. treatment with the thrombin inhibitor argatroban reversed platelet activation and aortic macrophage accumulation in apoe -/-/bgn -/ mice. conclusions: the present results strongly suggest a protective role of bgn during the progression of atherosclerosis by inhibiting thrombin activity and platelet activation, and ultimately macrophage-mediated plaque inflammation. the exposure to environmental or human-made xenobiotics including drugs induces the hepato-intestinal transcription of metabolizing enzymes and transporters. the time-span of induction is thought not to exceed xenobiotic exposure, in order to minimize disturbances of endobiotic metabolism. in contrast, we find cross-generational transmission of the induction of the phase i enzyme cyp b ( -fold in females, -fold in males) in -day old offspring of adult female mice exposed one week prior mating to tcpobop ( mg/kg i.p.) , the model ligand of the xenosensing nuclear receptor car. such cross-generational effects of xenobiotics are of great clinical interest as they could have profound consequences on the health status of the offspring, including interferences with drug therapies. the multigenerational transmission of tcpobop-driven induction could be mediated by pre-uterine/pre-conceptional epigenetic changes of oocytes. alternatively, they could be brought about by direct intrauterine/post-conceptional contact with tcpobop released from long-term depots. to discriminate between these mechanisms we conducted embryo transfer experiments. both donor mothers and foster mothers were injected with tcpobop ( mg/kg) prior to mating. the analysis of hepatic cyp b expression in -day-old offspring is clearly consistent with a post-conceptional onset of tcpobop effects. thus, offspring of solvent-injected donor mothers transferred to tcpobopexposed foster mothers display a -fold induction while offspring from the reciprocal experiment show no changes. cesarean sections on day e . followed by crossfostering proved transmission to be mediated predominantly via lactation (f hepatic cyp b induction -fold) and only to a minor part via intra-uterine exposure ( fold). this mechanism is consistent with the absence of induction transmission via the male germline. to analyze if tcpobop leads to functional consequences in drug metabolism of f and f generation, we conducted in vivo zoxazolamine paralysis assays taken as a functional test for cyp b catalytic activity. in both tcpobop-pretreated f and in their f descendants, the induction reduced the duration of paralysis evoked by zoxazolamine by > %. the characterization of cross-generational tcpobop-mediated effects on other processes controlled by car such as energy and bone metabolism is in progress. first tests indicate a transmission of anabolic effects on bone, as evidenced by the induction of serum osteocalcin expression by % in -weeks-old offspring. in summary, the car-mediated cyp b induction by tcpobop is transmitted to the offspring mainly via lactation, resulting in lasting phenotypic consequences in drug and bone metabolism. the effects of similarly lipophilic drugs and anthropogenic environmental pollutants are currently being investigated. such compounds could affect offspring despite discontinuation of intake or exposure well ahead of pregnancy. age-related cognitive decline can eventually lead to dementia, the most common mental illness in elderly people and an immense challenge for patients, their families and caregivers. cholinesterase inhibitors constitute the most commonly used antidementia prescription medication. the standardized ginkgo biloba leaf extract egb ® is approved for treating age-associated cognitive impairment and has been shown to improve the quality of life in patients suffering from mild dementia. a clinical trial with alzheimer´s disease patients indicated that the combined treatment with donepezil and egb ® had less side effects than donepezil alone (yancheva et al., ) . in an animal model of cognitive aging, we compared the effect of combined treatment with egb ® or donepezil monotherapy and vehicle. we compared the effect of chronic treatment ( days of pretreatment) with donepezil ( , mg/kg p. o.), egb ® ( mg/kg p. o.), or the combination of the two drugs, or vehicle in - month old male ofa rats. learning and memory performance were assessed by morris water maze testing, motor behavior in an open field paradigm. in addition to chronic treatment, the substances were administered orally minutes before testing. compared to the first day and to the control group, only the combination group showed a significant reduction in latency to reach the hidden platform on the second day of testing. moreover, from the second day of testing onwards, the donepezil, the egb ® and the combination group required less time to reach the hidden platform compared to the first day. the control group did not reach the same latency reduction until day three. there were no effects on motor behavior. these results suggest a superiority of the combined treatment of donepezil with egb ® compared to monotherapy. literature: yancheva, s., ihl, r., nikolova, g., panayotov, p., schlaefke, s., & hoerr, r. ( ) . ginkgo biloba extract egb (r), donepezil or both combined in the treatment of alzheimer's disease with neuropsychiatric features: a randomised, double-blind, exploratory trial. aging ment health, ( ) , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] institut für vegetative physiologie und pathophysiologie, göttingen, germany values are well below the plasma concentrations ( - µm; just et al. expert opin emerging drugs : - , ) observed in patients treated with dantrolene. although not yet proven directly, oat may be involved in renal secretion of dantrolene and -oh dantrolene by mediating the first step, i.e. the uptake across the basolateral membrane of proximal tubule cells. the second step, the release of these compounds into the urine across the luminal membrane, is possibly mediated by mrp . since oat was also detected in the cytoplasmic membrane of skeletal muscle cells (takeda et al. europ. j. pharmacol. : - , ) , dantrolene may reach its target, the intracellular ryanodine receptor, ryr , by influx through oat , where it inhibits calcium efflux by ryr thereby preventing severe muscle contraction and malignant hyperthermia. this assumption, however, awaits a direct demonstration of dantrolene transport by oat . in addition, we identified, besides dantrolene and -oh dantrolene, several further fdaapproved drugs such as tyrphostin ag , ceefourin , glafenine, nalidixic acid, and prazosine, as inhibitors of es uptake by oat . controls with other defects and controls with genetic disorders. all drugs used in the first trimester were identified from the database, and were cross-referenced against previously compiled lists of drugs with reactive intermediates and drugs with faa. drugs with reactive intermediates, with systemic absorption and with a daily dose ≥ mg were considered os-inducing drugs. when there was an association between os-inducing drugs and a group of birth defects, we further investigated two different faa exposure categories: concurrent exposure to both os-inducing drugs and faa drugs (os+/faa+) and exposure to os-inducing drugs only (os+/faa-). when the number of subjects allowed (at least five cases/controls were exposed), we examined the role of folic. odds ratios (ors) with % confidence intervals were adjusted for maternal smoking and alcohol use in the first trimester in controls and additionally adjusted for maternal age in controls . results: a total of nine groups of birth defects were investigated. only nervous system defects were associated with os-inducing drugs. exposure rates were / ( . %) for cases, / ( . %) for controls and / ( . %) for controls and adjusted ors ( %cis) were . ( . - . ) and . ( . - . ), respectively. this association was unchanged when we examined os+/faa+ and os+/faa-separately. the os+/faa+ category, however, had slightly higher or values than the os+/faa-( . vs. . for controls , and . vs. . for controls ). because of the low number of exposed subjects, we could only examine folic in relation to os+/faa-. using os-/faa-/folic+ as reference, we found the highest risk with os+/faa-/folicand a lesser magnitude with os+/faa-/folic+ (ors being and . times respectively for both controls). conclusion: our study suggests an increased risk of having a child with nervous system defects in mothers who were exposed to os-inducing drugs during pregnancy, and a potential risk reduction with folic. background: inhibition of rho-gtpases with statins as well as specific inhibition of the small gtpase rac protects non-transformed cells from topoisomerase ii-(top )-poisoninduced cleavable complex formation and thereof derived dna double-strand breaks. this effect rests at least partially on rac -mediated regulation of topoisomerase ii activity. however, the link between rac and top -poisoning is only poorly understood. furthermore, it is unclear whether mitochondrial or nuclear type ii topoisomerases are the most relevant target for top -poison-induced cytotoxicity. here, we investigated the relevance of rac -regulated actin cytoskeleton integrity as well as mitochondrial integrity in top -poison-induced dna damage responses as well as cytotoxicity under situation of rac inhibition. methods: since endothelial cells are the first barrier for any kind of systemically administered chemicals and cardiomyocytes are particular sensitive to anthracyclines, endothelial cells (h v) as well as cardiomyocytes (h c ) were chosen as in vitro model systems for top -poisoning. the cells were pre-treated with rac inhibitors, statins or actin cytoskeleton disruptors and were subsequently treated with the topoisomerase ii poisons doxorubicin or etoposide. to compare the levels of induced dna damage, γh ax foci quantifications as well as the comet assay were employed. actin disruption was visualized by phalloidin-fitc staining. to be able to detect relevant changes in mitochondrial mass or integrity, high doses of top -poisons had to be used in both cell lines. changes in mitochondrial homeostasis as well as integrity were detected by the jc -assay, mitotracker assay as well as atp-assay. additionally, pcr-and gelelectrophoresis-based methods were used for detecting mitochondrial dna damages. selected components of the dna damage response machinery as well as factors of mitochondrial homeostasis were detected by western blot. results: disruption of the integrity of the actin cytoskeleton attenuated the dna damage response to a similar extent as seen by rac inhibition, pointing to a role of actin filaments in the dna damage response after genotoxic insults. the actin cytoskeleton seems to participate in genotoxin-induced dna damage, -repair or in the dna damage response as reflected by reduced numbers of nuclear h ax-foci as well as the comet assay after treatment with doxorubicin. this was not related to nuclear import or export of doxorubicin. disturbance of mitochondrial homeostasis or integrity was only detectable at high doses of topoisomerase ii poisons. this was largely unaffected by pre-treatment with statins or rac -inhibitor. top -poison-induced raise in mitochondrial mass was slightly enhanced by the rac -inhibitor and statins. interestingly, inhibition of rac counteracted doxorubicin-induced phosphorylation of the amp-kinase in endothelial cells but not in cardiomyocytes. conclusion: mitochondrial toxicity seems to play only a minor role in top -poisoninduced cytotoxicity in h c and h v cells. the data point to a role of rac -regulated filamentous (nuclear?) actin in the dna repair and/or dna damage response after treatment with top -poisons. poly(adp-ribose) polymerase (parp ) and the recq helicase werner syndrome protein (wrn) are important caretakers of the genome. they physically interact with each other and are both localized in the nucleus and in particular in the nucleoli. both participate in various overlapping mechanisms of dna metabolism, in particular genotoxic stress response and dna repair [ ] . previously, we and others have shown in biochemical studies that enzymatic functions of wrn are regulated by parp as well as by non-covalent poly(adp-ribose)-wrn interaction [ ] [ ] [ ] . furthermore, pharmacological parp inhibition as well as a genetic parp ablation in hela cells alters the recruitment kinetics of wrn to sites of laser-induced dna damage [ ] . here we report a novel role for parp and poly(adp-ribosyl)ation in the regulation of wrn's subnuclear spatial distribution upon induction of oxidative stress. we could verify previous reports that wrn is transiently released from nucleoli upon induction of oxidative stress, camptothecin (cpt) treatment, and laser-induced dna damage in a time-dependent manner. while, cpt-induced translocation appears to be a parpindependent process, our results reveal that upon h o -induced oxidative stress, parp is essential for the translocation of wrn from the nucleoli to the nucleoplasm. parp activity only partially contributes to wrn release from nucleoli, underlining the importance of a direct wrn-parp interaction for subnuclear wrn redistribution. furthermore, we identified a novel par-binding motif within the wrn sequence that is located in its rqc domain, which also harbors the binding site for parp and is necessary for wrn's nucleolar localization under non-stress conditions. currently, we are testing corresponding wrn mutants to analyze if this region is responsible for the parp -dependent release of wrn from nucleoli to sites of dna damage. in conclusion, we provide novel insight into the role of parp in wrn's spatio-temporal regulation in the nucleus during the oxidative stress response. host-cell reactivation (hcr) is an assay used to determine dna repair capacity of cells. in its canonical layout, the test utilised a virus or a plasmid with a marker gene, inactivated by uv-damage [ , ] . among the infected or transfected host cells types, only those with functional dna repair pathway would re-activate the damaged dna, thus providing a rationale for identification of dna repair genes in the mutant screens. an obvious advantage of hcr is that repair can be measured in cells that have not been exposed to a damaging agent. however, because of multiple variables of the damage generation, transfection and interpretation of results, the assay has been hard to harmonise and develop into a widely accepted quantitative dna repair assay. over the last years, my team has developed and validated several major improvements of the mammalian hcr assay. exploiting sequence-specific nicking endonucleases and customised design of the reporter vectors, we proposed an innovative and very efficient technique for incorporation of synthetic oligonucleotides, containing single structurally defined dna base and backbone modifications, into desired gene elements [ ] . this ad hoc approach allows examination of the repair in a stand-specific manner and at single nucleotide resolution. we efficiently applied hcr in its new layout for measurement of the nucleotide excision repair of various dna adducts. moreover, we demonstrated that the enhanced hcr assay can differentiate between the transcription-coupled (tc-ner) and global genome (gg-ner) subpathways of ner [ ] . we further obtained new significant insights into the lesion-specific mechanisms of base excision repair of several endogenously occurring aberrant dna bases [ - ] and plan to adapt the assay to the detection of mismatch repair and translesion dna synthesis. in addition to the applications in the dna repair field, the enhanced hcr assay provides a tool for investigation of the dynamics and transcriptional impact of the regulatory dna bases methylcytosine and -hydroxymethylcytosine as well as their derivatives ( formylcytosine and -carboxycytosine a coordinated and faithful dna damage response is of central importance for maintaining genomic integrity and cell survival. transcriptional activation of dna repair genes is an important regulatory mechanism contributing to the adaptation of cells to genotoxic stress and protection against genotoxin-mediated cell death. here we show that exposure to a low dose of benzo(a)pyrene , , the active metabolite of benzo(a)pyrene (b[a]p), which represents the most important carcinogen formed by incomplete combustion during food preparation and smoking, causes upregulation of several dna repair genes. combined induction of the nucleotide excision repair (ner) genes ddb , xpc, xpf and xpg enhanced repair activity and protected cells against a subsequent bpde exposure. furthermore induction of the translesion polymerase polh was also involved in protection against bpde-induced apoptosis, however led to an enhanced mutation frequency in the surviving cells. activation of these dna repair pathways was also observed upon exposure to b[a]p and in vivo in buccal cells of male individuals upon smoking, indicating that this mechanism may be involved in the formation of smoking-related cancers. altogether, we could show that low-dose bpde exposure activates a complex network of transcriptional alterations, leading to protection against cell death, at the cost of increased mutation frequency, highlighting the danger of occasional smoking. poly(adp-ribosyl)ation (parylation) is an essential posttranslational modification with the biopolymer poly(adp-ribose) (par). the reaction is catalyzed by poly(adp-ribose) polymerases (parps) and plays key roles in cellular physiology and stress response by regulating physico-chemical properties of target proteins. of the members of the human parp gene family, at least four have been shown to exhibit par-forming capacity. upon dna damage parp is catalytically activated and is thought to contribute to the bulk of the cellular par formation. parp inhibitors are currently being tested in clinical cancer treatment, in combination therapy, or as monotherapeutic agents by inducing synthetic lethality (mangerich and bürkle , mangerich and bürkle ) . here we generated a genetic knock out of parp in one of the most widely used human cell systems, i.e. hela parp ko cells, via talen-mediated gene targeting and characterized these cells with regards to parylation metabolism and genotoxic stress response. furthermore, by reconstituting hela parp ko cells with a series of artificial and natural parp variants, we analyzed structure-function relationships of parp in a cellular environment without interfering with endogenously expressed wt-parp . we confirmed that the parp e k mutant exhibits mono-adp-ribosylation activity and extended previous reports by demonstrating that the parp l f mutant is constitutively active in a cellular environment, leading to high cellular par levels even in unchallenged cells. additionally, both mutants exhibited significantly altered recruitment and dissociation kinetics at sites of laser-induced dna-damage, which can partially be attributed to non-covalent parp -par interaction via at least one specific par binding motif located in zinc finger of parp . expression of both artificial mutants led to distinct cellular consequences, caused by the altered cellular biochemistry. while the expression of parp l f itself triggered apoptosis, parp e k expression led to a strong g -arrest during cell cycle and sensitized cells to camptothecin treatment. interestingly, pharmacological parp inhibition with abt mitigated effects of the e k mutant, suggesting distinct functions of mono-adp-ribosylation. finally, by reconstituting parp ko cells with a natural cancer-associated parp snp variant (v a), as well as a newly identified parp mutant present in a patient of pediatric colorectal carcinoma (f l-v a), we demonstrate, that these variants exhibit altered biochemical and cellular properties, potentially supporting carcinogenesis. together, this study establishes a novel model to study parp -dependent parylation during genotoxic stress response and reveals new insight into the structure-function relationships of artificial as well as natural parp variants in a cellular environment, with implications for parp research in general. mangerich, a. and a. bürkle ( ) . "how to kill tumor cells with inhibitors of poly(adpribosyl)ation." int j cancer ( ): - . mangerich, a. and a. bürkle ( ) . multitasking roles for poly (adp-ribosyl) ation in aging and longevity. parp inhibitors for cancer therapy, springer: - . leukemic cells frequently overexpress the transcription factor wilms tumor (wt ) and the persistence of wt expression after chemotherapy indicates remaining leukemic stem cells. hydroxyurea induces replicative stress by its ability to inhibit ribonucleotide reductase, an enzyme that catalyzes the synthesis of dntps from ntps. we demonstrate that the expression levels of wt determines the extent of dna damage and apoptosis in a panel of leukemic cells treated with hydroxyurea. accordingly, inhibiting apoptosis through chemical inhibition of caspases or by overexpression of mitochondrial anti-apoptotic bcl proteins prevents the hydroxyurea-induced depletion of wt and cell death. in addition, we show that an rna interference-mediated elimination of wt sensitizes leukemic cells to the pro-apoptotic and dna damaging effects of hydroxyurea. furthermore, such a loss of wt suppresses hydroxyurea-induced erythroid differentiation. pharmacological approaches that diminish wt also sensitize cells to hydroxyurea. these include the tyrosine kinase inhibitor (tki) imatinib or epigenetic modifiers belonging to the histone deacetylase inhibitor (hdaci) group. thus, an inhibition of wt is therapeutically exploitable for a targeting approach against leukemic cells undergoing replicative stress. our novel findings reveal that wt is a novel biological target of hydroxyurea and they suggest that wt has a previously unrecognized ability to prevent dna damage when replication forks halt and eventually collapse. fret (fluorescence resonance energy transfer)-based cell assays were developed to directly monitor receptor activation and receptor-stimulated camp response. mutant ß ar were generated by insertion of cyan and yellow fluorescent proteins (cfp and yfp) into the third intracellular loop and the c-terminus, respectively (bornholz et al., cardiovasc res : , ) and stably transfected to hek cells (hekß -fret). to monitor the camp response the epac -based fret sensor of camp, was stably transfected alone (hekwt-e ) and together with a moderate level of native ß ar to hek cells (hekß -e ; nikolaev et al. jacc : , ) . fret-activity was measured with recently developed fluorescence detectors ( channels) equipped with fast semiconductor technology, avoiding any movable optical and mechanical parts, using nm for excitation and / nm for the emmission ratio. cells were cultivated in -format -well strips, incubated in physiological hepes-buffered salt solution and treated with ßar agonists of different selectivity and affinity to determine their ßar-subtype preference. catecholamines tested in hekß -fret cells exhibited ec -values (-log, m) which matched k d -values (-log, m) known from native heart receptor membranes (isoprenaline, iso: . ± . , adrenaline . ± . , noradrenaline . ± . ). ßar-expression levels were controlled by radioligand binding with [ h]-(-)-cgp , resulting in different densities of ~ x and ~ . x receptors/cell in hekß -fret and hekß -e , respectively, whereas in hekwt-e cells only ~ ß ar were found. surprisingly, the low level of ßar in hekwt-e cells allowed the measurement of the action of ß -sympathomimetics (ß sym), e.g. fenoterol, thereby amplifying receptor binding (pk d~ . ) to an effective regulation of fret activity in the presence of . mm ibmx (pec ~ . ± . ), nearly matching the ~ -fold amplification of iso (pk d~ . ; pec ~ . ). in order to determine ß ar-mediated side effects of ß sym, hekß -e cells characterized by a -fold higher level of ß ar over ß ar were assayed for fret activity. fenoterol maximally inhibited fret activity with a pec ~ . whereas the high affinity ß sym salmeterol acted a partial agonist (~ % of iso maximum, pec ~ . ), both compounds being rather insensitive against the highly effective ß ar-blockade with µm cgp , a. for that reason hekß -fret cells were used characterizing fenoterol as partial agonist (~ %) whereas salmeterol activated less than % of maximum receptor activation by iso. thus, it has to be concluded that the low level of effectively coupled wt-ß ar present in hekß -e cells precludes the exact determination of ß ar-mediated side effects of ß sym, and that cfp/yfp labelled receptors have to be used for the determination of the subtype specific intrinsic activity of an agonist. they account for about one third of all drug targets. their regulation from desensitization to internalization and alternative signal transduction is largely dependent on phosphorylation of intracellular serine and threonine residues of the activated receptor. even though the β -adrenoceptor is of tremendous importance in a number of diseases its phosphorylation remains poorly understood. we addressed this question in a qualitative and quantitative way. by using radioactive phosphorylation assays and mass spectrometry, we were able to elucidate the phosphorylation pattern of the human β -adrenoceptor in vitro. we identified ten previously unknown phosphorylation sites in the third intracellular loop and the receptor's c-terminus. labeling hek cells with stable heavy isotopes (silac) lead to the discovery of a stimulation-dependent regulation of several of these phosphorylation sites. furthermore, mutagenesis studies in stably transfected hek cells revealed the impact of phosphorylation for arrestin binding and internalization of the receptor. fluorescence resonance energy transfer experiments with β -adrenoceptor variants carrying point mutations of putative phosphorylation sites identified two c-terminal phosphosites that determine arrestin recruitment. our current goal is to further investigate the functional implications of these newly identified phosphorylation sites on downstream signal transduction, with an emphasis on the map kinase pathway. a moderate increase in arrestin affinity to the β -adrenergic receptor is sufficient to induce arrestin internalization. the homologous desensitization of g-protein-coupled receptors is a two-step process. initially, g-protein-coupled receptor kinases phosphorylate agonist-occupied receptors which are subsequently bound by arrestins. in many cases, the resulting receptorarrestin complex is then internalized via clathrin-coated pits. dependent on the identity of the receptor and the ligand, the complex between receptor and arrestin may exist only in the proximity of the plasma membrane or internalize into the cell interior. we constructed mutants of the β -adrenergic receptor carrying three additional serine residues in various positions at the c-terminal tail. one of these mutants which carried the serine residues in close proximity to the endogenous grk phosphorylation sites (β ar-sss) showed increased isoprenaline-stimulated phosphorylation and differences in arrestin- affinity and trafficking. the affinity of arrestin- to the receptor was measured by fluorescence resonance energy transfer (fret) between the receptor and arrestin- and by two-color fluorescence recovery after photobleaching (frap). in the fret assay, arrestin- dissociation from the β ar-sss receptor upon agonist washout was prolonged approximately two-fold compared to the wild-type receptor. frap was performed with an n-terminally tagged receptor immobilized with an antibody against the n-terminal tag either in solution or on a micropatterned surface. in these assays, the recovery of arrestin- into the bleached region was prolonged between two-and fourfold for the β ar-sss receptor compared to the wild-type. even though this two-to fourfold increase in affinity seemed rather modest, it resulted in the trafficking of receptorarrestin complexes to the early endosome whereas the wild-type receptor interacted only transiently with arrestin at the plasma membrane. furthermore, the increased affinity of arrestin led to more efficient internalization of the β ar-sss compared to the wild-type receptor. however, recycling to the plasma membrane after agonist washout was very similar for both receptors. we conclude that even a modest change in affinity between a g-protein-coupled receptor and arrestin can lead to substantial alterations in arrestin trafficking which in turn may have effects on cellular signaling. despite recent structural research allow for better understanding of gpcr structure, the crucial aspects of the selectivity mechanism of receptor -g protein subtype coupling remain unresolved. based on the hypothesis that the affinity of the ternary complex (agonist/gpcr/g-protein) in the nucleotide-free state determines the selectivity of gpcr-g protein coupling, we set out to measure gpcr-g protein interaction in membranes of single cells. in order to quantify the affinity of gα-subunit towards gpcrs in single cell, we determined the lifetime of the receptor-g protein complex in living cells upon agonist withdrawal under conditions of gtp-depletion. therefore, we utilized förster resonance energy transfer (fret) based assays to study interactions between fluorescent muscarinic receptors and heterotrimeric g proteins in single permeabilized hek t cells transfected with the appropriate cdnas. here we focused on muscarinic m -, m -, and m -receptors and characterized the kinetics of agonist-induced binding of go/i -and gq/ -proteins to muscarinic receptors and their subsequent dissociation in the absence of nucleotides. as a measure of affinity we calculated the rate constant of g protein dissociation from the receptor after agonist withdrawal. the dissociation kinetics of go protein from m -and m -achrs was found to be -fold faster in comparison to gq. similarly, we observed a -fold right shift of the concentration-response curves of go proteins binding to m -achr in comparison to gq. in order to ensure, that the affinity of the ternary complex correlates with the efficiency of g protein activation, we performed experiments on the g protein activity in intact cells expressing non-fluorescent m -achr by using a fret-based assay. our results showed that gq activation required -fold lower agonist concentration compared to go activation, suggesting that indeed the stability of the ternary complex in the absence of nucleotides determines the selectivity of gpcr-g protein coupling. we further explored the subtype selectivity of m -achr for gi family members by comparison of dissociation kinetics of gi -, gi , gi -, and go-proteins from m -achr under nucleotide depleted conditions. k off of gi and gi were found to be two-fold higher in comparison to gi and go proteins, indicating the higher affinity of the latter ones to m -achr. our fret-based assay to study receptor-g-protein interactions in membranes of single cells has been proven to be a fast and reliable method to quantify the affinity of the ternary complex. the g protein subtype dependent differences in the affinity towards activated receptors correlate with the g protein coupling efficiency of this receptor. despite their tremendous pharmacological relevance and potential for the development of new drugs, our understanding of g protein-coupled receptor (gpcr) architecture and signaling mechanisms are still limited. major reasons for this are the low abundance and poor biophysical properties of gpcrs, which makes them one of the most challenging class of proteins for structural and biophysical studies. among the superfamily of gpcrs, the class b receptors comprising receptors are structurally least understood because to date it has not been possible to obtain a crystal structure of this receptor class. to overcome these limitations, we have developed a method for improving functional expression and simultaneous thermo-stabilization of gpcrs by directed evolution which is based on expression of receptors in saccharomyces cerevisiae and subsequent selection of highly expressing variants by flow cytometry with fluorescent ligands. by this strategy, key residues within a receptor sequence can be rapidly identified that are responsible for improved biophysical properties without greatly affecting the pharmacological features of the receptor. we have now applied this method to the human parathyroid hormone receptor, a member of the class b of gpcrs which is a major regulator of calcium homeostasis in the body and a key target for the treatment of osteoporosis. from two rounds of directed evolution in yeast we obtained several mutants of parathyroid hormone receptor that exhibit strongly improved expression levels and that remain stable after solubilization in detergents. these receptor variants are ideal candidates for subsequent structural and biophysical analysis. opioid drugs exert nearly all of their clinically relevant actions through stimulation of mors (μ-opioid receptors). the molecular biology of endogenous opioid peptides and their cognate receptors has been studied extensively in vitro. for mor, signaling efficiency is tightly regulated and ultimately limited by the coordinated phosphorylation of intracellular serine and threonine residues. morphine induces a selective phosphorylation of serine that is predominantly catalyzed by g protein-coupled receptor kinase . as a consequence, the selective morphine-induced s phosphorylation does not lead to a robust beta-arrestin mobilization and receptor internalization. by contrast, high-efficacy opioid agonists such as fentanyl or etonitazene not only induce phosphorylation of s but also drive higher order phosphorylation on the flanking residues threonine , threonine , and threonine in a hierarchical phosphorylation cascade that specifically requires grk and grk isoforms. as a consequence, multisite phosphorylation induced by potent agonist promotes both betaarrestin mobilization and a robust receptor internalization. however, little is known about agonist-selective phosphorylation patterns in vivo after acute and chronic drug administration. to learn more about mor regulation in vivo we have generated a new μopioid receptor knock in mouse with an n-terminal ha-tag. using these mice, we were able to study in vivo phosphorylation of an endogenous g protein-coupled receptor using both mass spectrometry and phosphosite-specific antibodies. we were also able to address the question which of the many putative mor splice variants detected on the mrna level are indeed expressed as functional receptors in mouse brain. ion channels hcn in thalamic relay neurons is necessary for oscillatory activity in the thalamocortical system institut für physiologie i, westfälische wilhelms-universität, münster, germany hcn channels underlie the i h current and are involved, among other functions, in the genesis of epilepsy. the significance of hcn and hcn isoforms for brain function and epilepsy has been demonstrated, however the role of hcn , the third major neuronal hcn subunit, is not known. here we show an unexpected role of hcn in controlling oscillations in the thalamocortical network. hcn is predominantly expressed in several thalamic relay nuclei, but not in the thalamic reticular nucleus and the cerebral cortex. hcn -deficient thalamocortical relay neurons showed a massive reduction of i h and strongly reduced intrinsic burst firing. evoked thalamic oscillations in a slice preparation were completely abolished. in vivo, brain-specific hcn null mutants were protected against induced spike-and-wave discharges (swd), the hallmark of absence seizures. our findings indicate that hcn is necessary for rhythmic intrathalamic oscillations and that the channels constitutes an important component of swd generation. ludwig-maximilians-universität, walther-straub-institut für pharmakologie und toxikologie, münchen, germany trpc and channels are members of the classical transient receptor potential (trpc) family whose activation mechanism downstream of phospholipase c (plc) largely remained elusive until now. while trpc / / channels are directly activated by diacylglycerol (dag), trpc and channels are commonly regarded as daginsensitive. in contrast to trpc / / channels, they contain a c-terminal pdz-binding motif allowing for binding of na + /h + exchanger regulatory factor (nherf) and . interestingly, performing electrophysiological measurements, co-immunoprecipitations and intermolecular dynamic fret experiments, we found that dissociation of nherf proteins from the c-terminus of trpc confers dag-sensitivity on trpc channels. trpc channels were dag-sensitive under the following experimental conditions: inhibition of protein kinase c, amino acid exchange in the c-terminal pdz-binding motif, pip depletion with and without involvement of plc, over-expression of g-protein coupled receptors, down-regulation of endogenous nherf and proteins and overexpression of a nherf mutant incapable of trpc binding. these findings strongly argue for nherf proteins as molecular determinants for channel activation. interestingly, pip depletion itself caused slight trpc current increases while during pip depletion, the membrane permeable dag analogue oag evoked even higher trpc currents suggesting that pip depletion induces an active and dag-sensitive channel conformation. receptor mediated pip depletion also resulted in dissociation of nherf and from the c-terminus of trpc thereby eliciting a dag-sensitive trpc channel conformation. thus, our findings suggest that dag-sensitivity of trpc is the result of an activation cascade starting with pip depletion and subsequent dynamic dissociation of nherf and from the c-terminus of trpc . altogether, dagsensitivity is a unifying functional hallmark of all trpc channels. the melastatin-related transient receptor potential channel trpm is a heat-activated nonselective cation channel expressed in sensory neurons of dorsal root ganglia. since trpm -deficient mice show impaired inflammatory thermal hyperalgesia, the pharmacological inhibition of trpm may exert antinociceptive properties. fluorometric ca + assays and a compound library containing approved drugs were used to identify trpm inhibitors and to characterize their potency and selectivity. biophysical properties of the block were assessed using electrophysiological patch-clamp methods. microfluorometry in fura- -loaded single cells was applied to monitor [ca + ] i signals in isolated dorsal root ganglion (drg) neurons. analgesic effects were assessed applying pregnenolone sulfate (pregs)-induced chemical pain and heat stimuli at mice. in the screening approach using stably transfected hek trpm cells we identified the nonsteroidal anti-inflammatory drug (nsaid) diclofenac, the tetracyclic antidepressant maprotiline and the anticonvulsant primidone as highly efficient trpm inhibitors. the compounds exhibited half-maximal inhibitory concentrations of . - µm. the selectivity profiles of maprotiline and primidone for trpm were promising with no inhibitory effects on trpm , trpm , trpa , trpv , trpc , trpc and p x receptor channels. primidone inhibited pregs-induced [ca + ] i signals in rat drg neurones, indicating a block of native trpm channels. consistently, primidone attenuated nocifensive responses of mice to paw-injected pregs. furthermore, intraplantar primidone reduced nociception in healthy and hyperalgesic cfa-inflamed paws in the hot plate test. the finding that an approved drug can inhibit trpm at concentrations that may be therapeutically relevant and thereby can act as an analgesic, provides a method to study trpm -related effects by acutely challenging the channel´s function. pharmacological interference with trpm applying an approved drug or optimised successor compounds may pave the way to better understanding of physiological functions of trpm in humans and may represent a novel concept for analgesic treatment. excitotoxicity, calcium deregulation, mitochondrial dysfunction and neuroinflammation contribute to progressive cell death in many neurodegenerative diseases. therefore, proteins that prevent deregulation of these pathways are considered as drug targets. potential therapeutic approaches may benefit from modulation of small-conductance calcium-activated potassium (sk) channels, since recent data supports the hypothesis that sk channel activity promotes neuronal survival against cellular stress via a dual mechanism of action: i) by controlling neuronal excitability and ii) by preventing mitochondrial dysfunction and inflammation. our previous studies showed that activation of sk channels in neurons exerted protective effects through inhibition of nmdarmediated excitotoxicity. further, we revealed recently that in a model of glutamate oxytosis, activation of sk channels attenuated mitochondrial fission, prevented the release of pro-apoptotic mitochondrial proteins, and reduced cell death. however, little is known about the function of sk channels in cell metabolism and neuroinflammatory processes in non-neuronal cells, such as microglial cells. in this study, we addressed the question whether sk channel activation affected primary mouse microglia activation upon lps and α-synuclein challenge. we found that activation of sk channels significantly reduced activation of microglia in a concentration-dependent manner, as detected by real-time xcelligence cell impedance measurements. further data on cytokine (tnf-alpha and il- ) analysis revealed that activation of sk channels attenuated α-synuclein-induced cytokine release. inhibition of glycolysis prevented microglial activation and cytokine release. although sk channel activation slightly reduced atp levels, it attenuated α-synuclein-induced no release. furthermore, glycolytic products and ampk signaling were evaluated. overall, our findings show that activation of sk channels attenuates microglial cell activation. thus, sk channels are promising therapeutic targets for neurodegenerative disorders, where neuroinflammation and cell metabolic deregulation are associated with progression of the disease. mutant of the residue pair e /k can be crosslinked efficiently in both states, the closed-and open state of the p x r. interestingly, oxidative crosslinking of cysteine substitution mutants of each individual residue pair significantly reduced the atpinduced current amplitudes. charge reversal or swapping mutagenesis and cysteine modification by charged mts-reagents indicated the electrostatic nature of the pairwise interactions in these four residue pairs. furthermore, preliminary data from triple, tetra and penta mutant cycle analysis indicated energetic coupling between the residue pairs e /r , e /k , e /r and e /k and thus indicates the cooperative interaction in a larger salt bridge network. together with the markedly reduced current amplitudes following disulfide crosslinking, our data suggest that the salt bridge network serves to stabilize the closed-state conformation of the p x r. the comparison of the closed-state and open-state model of the rat p x r showed that atp promotes a marked rearrangement of the side chains of the residues r and k to enable the strong ionic coordination of the γ-phosphate oxygen of atp. in summary, our data are in line with the concept that the electrostatic interaction of r and k with atp competitively releases e , e and e from their strong electrostatic coupling and thus initiates a destabilization of the closed-state, which favors channel opening. fig. in a similarity search using sequence motifs conserved amongst various members of the trp protein family we identified three non-annotated putative membrane proteins that we initially termed tmem , tmem and tmem . expression analysis using the nanostring ncounter system, northern blotting and rt-pcr showed that murine tmem is expressed in various tissues including heart, brain, lung, endothelium, colon, cardiac myocytes, cardiac fibroblasts, embryonic fibroblasts, mast cells and pancreatic acinar cells. hydropathy analysis predicts that tmem proteins exhibit to plasma-membrane spanning domains, but fluorescently labeled tmem fusion constructs expressed in mouse embryonic fibroblasts revealed a vesicular subcellular localization pattern. in contrast to the prediction by the psort ii algorithm, tmem -eyfp could not be identified in the plasma membrane of fibroblasts, cardiac myocytes, mast cells or pancreatic acinar cells but showed a significant colocalization with markers and fusion constructs specific for acidic compartments including lysosomes. in tmem -/mice, a marked elevation of amylase and lipase plasma levels was observed. we found that constitutive but not stimulated amylase secretion from tmem -deficient acinar cells is elevated indicating a cell autonomous defect. calcium (ca ++ ) is an important signaling molecule regulating stimulated as well as constitutive secretion from pancreatic acinar cells. microfluorimetric measurements using fura- or indo- indicate higher resting ca ++ concentrations in tmem -/-pancreatic acinar cells correlating with elevated basal enzyme secretion. in tmem -yfp-knock-add-on mice we identified tmem in organelles of the apical acinar cell pole and a partial colocalisation with lamp proteins. furthermore, largely increased elevations in cytoplasmic ca ++ concentration were observed upon osmotic lysis of lysosomes triggered by gly-phe β-naphthylamide (gpn) or by nh cl application. the role of tmem for ca ++ release was evaluated by stimulation with low concentration of cholecystokinin pm) in the absence of extracellular ca ++ using both microfluorimetric recording of cytosolic ca ++ transients as well as electrophysiological recordings of ca ++ -activated chloride currents. these measurements revealed a higher frequency of intracellular ca ++ oscillations and a larger area under the curve of ca ++ activated chloride currents upon cck- stimulation indicating that tmem inactivation leads to an enhancement of the globalization of cck- evoked ca + release from intracellular organelles. taken together, our study identifies tmem as a novel regulator of ca ++ release from intracellular organelles including endo-lysosomes and as a critical determinant of constitutive protein secretion in pancreatic acinar cells. while generally highlighting toxicology as a translational science that requires academic anchoring, gundert-remy and co-workers [ ] have called for efforts to improve the relevance of in vitro methodologies in predicting in vivo effects. against this background, the german society of toxicology working group on alternative approaches to animal testing proposes specific quality criteria (qc) for in vitro methods and for research work using in vitro methods. these qc may serve to evaluate in vitro methods that are developed or applied in-house or that are described in work plans, peer reviewed articles, etc. for the time being, the qc focus on in vitro cell or tissue culture methods that address human health endpoints in the context of substance-related regulatory toxicity testing. nevertheless, these qc are also generally applicable to in vitro research conducted for other toxicological purposes. relevant work from, e.g., the organisation for the economic co-operation and development has been taken into account in specifying the qc that cover the following aspects:  the rs impact of an in vitro method in replacing, reducing (and refining) a specific animal test for a specific toxicological endpoint. this aspect also includes scientific hurdles that, in the past, had impeded the successful development of in vitro methods for the given toxicological endpoint.  scientific relevance and reliability, i.e. which fundamental requirements should an in vitro method meet to ensure that its results are relevant and reliable.  practicability and applicability, i.e. what is the expected expenditure for the in vitro method, and have relevant authorities and industrial sectors been involved in the development of the in vitro method. qc related to the scientific relevance of research work using a specific in vitro method provide a tool to justify, e.g., the suitability of the selected test system and in vitro endpoint(s) for the given purpose; the selection of test substances, positive and negative controls; the setting and control of test concentrations; and the definition of acceptance criteria to determine the relevance of test results. the proposed qc may serve as a framework to assess the relevance of in vitro methods and in vitro research work. thereby, they aim at improving in vitro predictivity of in vivo toxicological effects, which in return contributes to reducing and replacing the need for animal testing. [ ] gundert-remy, u. et al. ( ) . toxicology: a discipline in need of academic anchoring -the point of view of the german society of toxicology. arch toxicol : - . during the past decades, considerable progress has been made in implementing r approaches in routine safety assessment. in spite of these achievements, animal tests still need to be conducted if legal requirements prescribe in vivo tests or if no reliable, accepted alternative method exists. efforts to foster r approaches and make them 'ready for use' focus on three levels: development and validation of scientific methods/strategies, regulatory acceptance of acknowledged approaches and global harmonization of standards. strong cooperation between toxicological experts from scientific bodies, national and international authorities and industry is needed to advance on all three levels for the benefit of animal welfare. r approaches that have already been implemented in routine safety assessment of consumer goods do not focus solely on replacement of animal testing by use of accepted alternative methods. in cases where reliable alternative approaches are not yet available, reduction in animal numbers and refinement of testing procedures can be achieved on a case-by-case basis. a tailor-made, tiered testing strategy is usually pursued that involves knowledge on specific characteristics of the test item and makes use of all available data, including details on exposure and results obtained with structural homologues. hurdles to apply alternative approaches can even occur for established methods. as legislations give different priority to alternative approaches, it remains challenging to fulfil conflicting legal requirements in different regions of the world, or even to address horizontal legislations of the same region. furthermore, successfully validated and legally implemented alternative approaches might not always provide the safety assessor with meaningful test results. with gaining experience, limitations of test systems can become evident that affect for example the applicability domain of the method, as has been the case both for some in vitro and in vivo methods. in these cases, the new information needs to be shared not only among safety assessors, but also with method developers and regulators to facilitate refinement of scientific approaches and/or amendments of regulations. leibniz-institut für arbeitsforschung (ifado), vistox, dortmund, germany two-photon microscopy facilitates imaging of biological processes in vivo. establishing this recent technique in mouse liver allowed us to record in a real-time the sequence of events during acetaminophen (apap) induced-liver damage. although apap is intensively studied and described in vivo imaging revealed so far unknown scenarios of cell death. the hepatocytes close to the central vein of a liver lobule went within hours into cell death as commonly described due to the toxic metabolite napqi. surprisingly, we observed a distinct way of cell killing at the outer border of the dead cell area which is accompanied by bile acid decompartmentalization. there, within an hour after apap administration dilatation of bile canaliculi was observed. subsequently, bile acids containing invaginations arouse from the apical side of a hepatocyte into the cytosol. these invaginations ballooned until the bile leaked into the hepatocyte volume and subsequently the plasma membrane of the affected hepatocytes lost its integrity leading to cell death. this mechanism emerged in an environment for hepatocytes where moderate napqi levels meet intracellular high bile salt concentrations of the midzonal region. in conclusion, establishing in vivo imaging in mouse liver enabled us to identify new cellular mechanisms which cannot be discovered by conventional methods. universitätsklinikum düsseldorf, institut für toxikologie, düsseldorf, germany introduction: lung inflammation and fibrosis are considered as major toxicities after thoracic cancer radiotherapy. up to now effective pharmacological interventions for normal tissue protection are largely missing. hmg-coa reductase inhibitors (statins), which are used in the clinic for lipid-lowering purpose, are reported to have multiple inhibitory effects on genotoxic stress responses. for this reason we aim to investigate the usefulness of statins to protect normal lung cells in vitro and lung tissue in vivo from damage provoked by ionizing radiation (ir). methods: according to clinically relevant anticancer radiation regimens, we used fractionated irradiation schemes ( x gy) for both in vitro as well as in vivo experiments. we analyzed the effect of lovastatin on ir-induced dna damage formation and repair, dna damage response (ddr) and cell death in non-proliferating human lung fibroblasts, epithelial as well as endothelial cells. furthermore, we established an irradiation device that is useful to selectively irradiate the right lung of mice and investigated the influence of lovastatin on lung damage following fractionated and selective irradiation of the lung in vivo (balb/c mice). results: compared to lung fibroblasts and epithelial cells, endothelial cells exhibited the highest radiosensitivity and underwent ir-induced apoptosis which was partly prevented by lovastatin. by contrast fibroblasts and epithelial cells did not undergo apoptosis upon irradiation. lovastatin did not affect initial dna damage formation in any of these cells. in all three lung cell types lovastatin enhanced the repair of dna double-strand breaks as analyzed h after the last irradiation by γh ax nuclear foci formation. depending on the cell type lovastatin affected various components of the ddr machinery in vitro. in vivo, lovastatin prevented ir-mediated increase in breathing frequency as determined two and four weeks after fractionated irradiation. moreover, statin treatment attenuated the level of residual dna damage and ir-induced apoptosis as analyzed four weeks after irradiation. these results were mimicked when eht , a small molecule inhibitor of the small rho-gtpase rac , was applied in vivo, pointing to an involvement of rac in statin-mediated radioprotective effects. conclusion: bearing in mind that statins are well tolerated in humans, we suggest the application of statins as a promising pharmacological strategy for the prevention of irradiation-induced damage of the lung. targeted genome engineering by crispr/cas is an evolving tool for generating specific knockout cell lines. co-expression of crispr/cas allows for efficient dna cleavage and introduction of so called indel mutations (insertion/deletion point mutations) that lead to either misfolded non-functional proteins or complete knockout. we exploited this tool to generate a bid (bh -interacting domain death agonist) knockout cell line in neuronal ht- cells. bid has been shown to be involved in regulated cell death pathways like oxytosis where its activation mediates mitochondrial demise, subsequent release of apoptosis inducing factor (aif) and cell death. in the cell death model of oxytosis the cystine/glutamate antiporter (x c -) is inhibited by high extracellular glutamate concentrations. following events such as increasing lipid peroxidation and ros production resemble major characteristics of another emerging cell death pathway, called ferroptosis. in this study we generated a bid crispr/cas -knockout cell line to elucidate the role of bid as a potential link of oxytosis and ferroptosis in the ht- cell line. in order to investigate the potential mechanistic overlap at the level of mitochondrial death pathways, we induced oxytosis with glutamate or ferroptosis with erastin in wild-type cells and analyzed the respective effects of the well-established inhibitors ferrostatin- and the bid inhibitor bi- c on cell death and mitochondrial paradigms. these results were then compared to the effects of glutamate or erastin in crispr/cas -bid-knockout cells. bi- c inhibited glutamate-induced morphological changes of ht- cells and also prevented cell death as assessed using the mtt assay and annexin v/pi staining. similar results were observed with ferrostatin- in the model of erastin-induced ferroptosis. subsequent facs analysis of lipid peroxidation by bodipy staining demonstrated that bi- c abolishes lipid peroxide formation in the erastin model and ferrostatin- in the model of oxytosis. facs analysis was further employed for the detection of mitochondrial ros formation. mitosox staining revealed a significantly decreased production of mitochondrial ros by bi- c and ferrostatin- in the respective model systems. investigating the crispr/cas -bid-knockout ht- cell line revealed that bid knockout prevented cell death, lipid peroxidation and mitochondrial toxicity in both model systems of cell death, oxytosis and ferroptosis. in conclusion, the present study exposes bid as a pivotal molecular link between the previously separated cell death pathways oxytosis and ferroptosis at the level of mitochondria. parkinson's disease is a common neurodegenerative movement disorder characterized by midbrain dopaminergic neuronal loss in the substantia nigra that has been linked to alpha-synuclein toxicity. however, the molecular mechanisms underlying alphasynuclein-mediated toxicity in human dopaminergic neuronal loss are not well defined. the goal of this study was to investigate the deleterious effects of alpha synuclein in particular mitochondrial toxicity in human dopaminergic cells. therefore, we have generated neuron specific, adeno associated virus type (aav ) expressing cytosolic as well as mitochondrial targeted alpha synuclein and egfp expressing viruses used as respective controls. overexpression of both, the cytosolic and the mitochondrial variants of alpha synuclein severely disrupted the dendritic network, induced loss of cellular atp, enhanced mitochondrial ros production, and was associated with activation of caspases and dopaminergic cell death in a time-dependent manner. in addition, real-time analysis of mitochondrial bioenergetics using the seahorse bioscience system following aav infection elicited a complete damage to mitochondrial respiration capacity in the dopaminergic neurons. our results suggested that mitochondrial targeted expression of alpha synuclein appeared to be more toxic than the cytosolic form of alpha synuclein. in addition, ultrastructural mitochondrial morphological analysis by transmission electron microscopy illustrated a number of deformed cristae in cells expressing the cytosolic alpha synuclein and a complete loss of cristae structure and massively swollen mitochondria following the expression of mitochondrial targeted alpha synuclein in the human dopaminergic neurons. in addition, we found that inhibition of caspases by the broad spectrum caspase inhibitor qvd significantly ameliorated alpha synuclein-induced dopaminergic neuronal death. interestingly, inhibition of caspases preserved neuronal network integrity, atp levels and mitochondrial respiration capacity in both paradigms of cytosolic and mitochondrial alpha synuclein overexpression. overall, our findings show that cytosolic as well as mitochondrial targeted expression of alpha synuclein is detrimental to human dopaminergic neurons, while inhibition of caspases amend alpha synuclein toxicity at the level of mitochondria. thus, caspase inhibitors provide promising therapeutic potential to prevent dopaminergic neuronal death in parkinson's syndromes that are associated with alpha synuclein toxicity. degradation of and adverse effects caused by tattoo and permanent make-up pigments upon sunlight exposure and laser removal have been occasionally reported in the last decades. until now, only the ban of certain azo-pigments has been addressed in the national legislation. the regulation was based on a number of studies showing the cleavage of azo-bonds by ultra violet light and laser-irradiation leading to the formation of carcinogenic aromatic amines. as a result, especially german tattoo ink manufactures switched to the use of more light-fast polycyclic pigments assuming these would be safer for this kind of application when compared to azo-pigments. to assess the potential risks of polycyclic pigments in terms of decomposition in the skin, we compared the photochemical cleavage of the widely used azo-pigment orange and the polycyclic pigment copper phthalocyanine blue. main decomposition products are qualitatively and quantitatively analyzed after q-switched laser irradiation of mg/ml aqueous suspensions and tattooed pig skin. irradiated specimen were extracted with ethyl acetate and analyzed with gas chromatography coupled to mass spectrometric detection (gc/ms) using liquid injection and head-space sampling techniques. we were able to confirm the cleavage of pigment orange at the azo-and other weak bonds in our experimental set-up (fig. a) . amongst other substances, the carcinogens aniline (max. conc. . ± . µg/ml) and , -dichlorobenzidine (max. conc. . ± . µg/ml) are formed. despite the lack of such weak bonds, the highly stable porphyrin-like structure of copper phthalocyanine blue is as well decomposed upon laser-irradiation (fig. b) . here, , -benzenedicarbonitrile (max. conc. . ± . µg/ml) were found as the main decomposition product in all experimental setups. concentrations of cleavage products were generally higher in aqueous suspensions compared to pig skin extracts with both pigments. additionally, the highly toxic gas hydrogen cyanide (max. conc. . ± . µg/ml) and the human carcinogen benzene (max. conc. . ± . µg/ml) were formed from both pigments, dependent on the laser wavelengths used. cyanide levels of ≥ µg/ml evolving upon ruby laser irradiation of > . mg/ml aqueous suspensions of phthalocyanine blue were proven to significantly reduce cell viability in human skin cells in vitro. reference schreiver, i., hutzler, c., laux, p., berlien, h. p. & luch, a. formation of highly toxic hydrogen cyanide upon ruby laser irradiation of the tattoo pigment phthalocyanine blue. sci rep , ( ) . understanding the interactions between nanoscaled objects and living cells is of great importance for risk assessment, due to rising application of nanomaterials in foodrelated products. several studies show that silver nanoparticles can reach the intestinal epithelia in nanoform in a human in vitro digestion model. nevertheless, only sparse data concerning the direct quantification of cellular uptake of silver nanoparticles are available. therefore, this study was focused on a systematical quantitative comparison of the cellular uptake of differently coated silver nanoparticles of comparable size. intracellular uptake was determined quantitatively via a transwell tm -system with subsequent elemental analysis (aas) and ion beam microscopy (ibm). silver nanoparticles were coated with poly (acrylic acid) and polyvinylpyrrolidone and characterized extensively by tem, dls, saxs, zetasizer and nanosight. agpure tm as a widely used reference nanoparticle coated with tween and tagat to v was also used for comparison. different intestinal cell models were applied to get closer to the complex in vivo situation: beside the widely used caco- model we also investigated particle uptake in a model which considered the enterocyte-covering mucus layer, as well as in a model specialized on particle uptake, the so-called m-cell model. our findings suggest that silver uptake is clearly a particle-and not an ion-related effect. the internalization of silver nanoparticles was enhanced in uptake-specialized m-cells, although no enhanced transport through the cells was observable. furthermore, the mucus did not providing a substantial additional barrier for nanoparticle internalization. rutherford backscattering spectrometry (rbs) via ibm allowed distinguishing between adsorbed an internalized material and the results were in accordance with the transwell tm -data. additionally, ibm investigations via particle-induced x-ray emission (pixe) showed intracellular association of silver with sulfur. the quantification of silver nanoparticle internalization revealed a clear particle-specific and a coating-related uptake. furthermore, a high amount of silver nanoparticles is taken up in cell models of higher complexity. thus, an underestimation of particle effects in vitro might be prevented by considering cell models with greater proximity to the in vivo situation. analyzing iron oxide nanoparticles for drug delivery -innovative investigation tools for nanotoxicology nanoparticles offer promising new possibilities for medical applications including therapy and diagnosis of various diseases. especially nanoparticle systems with magnetic cores provide a broad application spectrum as contrast agents, magnetic transporters, or heat carriers in hyperthermia treatment. for bench to bedside translation of superparamagnetic iron oxide nanoparticles (spions) for medical applications, safety issues have to be clarified. for that, reliable standards must be established on the basis of comprehensively validated physicochemical and biological characterization methods. spions consisting of maghemite and magnetite are usually of brown or black color. due to these special properties, spions and other metal oxide nanoparticles are prone to interfere with classical toxicological assays relying on optical detection of colorimetric, fluorescence or luminescence signals. particularly, nanoparticle concentration and cellular uptake are further influencing factors. consequently, for reliable analysis of nanoparticle mediated effects, alternative robust and interference-free readouts have to be established. based on long lasting experience working with spions, we suggest a combination of complementary methods to analyse nanoparticle-mediated effects: multiparameter analyses in flow cytometry deliver statistically relevant data and link uptake of nanoparticles (side scatter increase) with cellular effects in a high-content style. combination of noninvasive, label-free impedance measurements (xcelligence system) with real-time (fluorescence) microscopy enables us to monitor cellular proliferation and morphology over several days without interference by nanoparticles. additional experiments in multicellular tumor spheroids provide information about tissue infiltration and thus, more closely resemble the in vivo situation. using those complementary methods, several drug-loaded spion systems dedicated for medical applications have been successfully characterized previously. in sum, nanotoxicology is a complex and interdisciplinary challenge, where physicochemical parameters, as well as in vitro and in vivo behavior of nanoparticles have to be considered. to address these basic requirements, we are working on a stringent standardized road of characterization for iron oxide nanoparticles synthesized for medical applications. reference: lyer s, tietze r, unterweger h, zaloga j, singh r, matuszak j, poettler m, friedrich rp, duerr s, cicha i, janko c, alexiou c. nanomedical innovation: the seon concept for an improved cancer therapy with magnetic nanoparticles. nanomedicine (lond). ; ( ) acrylamide (aa) is an α,β-unsaturated compound, which is categorized as probably carcinogenic to humans [ , ] . aa is known to arise in foods by heat treatment in the course of the maillard reaction between reducing sugars and amino acids at processing temperatures > °c [ ] . dietary aa exposure has mainly been estimated on the basis of dietary recall, assessing consumption of foods with known aa contents. the use of human biomarkers of aa exposure, primarily haemoglobin adducts of aa and its genotoxic metabolite, glycidamide ( ga) in red blood cells, as well as mercapturic acids excreted in the urine, is a promising alternative. such biomarkers are to be validated by exact measurement of aa uptake in duplicates of food as consumed (duplicate diet studies) [ ] . we here present results of a nine-day human intervention study with healthy male volunteers. aa contents were determined in duplicates of servings as consumed and kinetics of aa-associated mercapturic acids (aama and gama) monitored in total urine [ ] . the study design included washout periods with an aa-minimized diet ( - ng /kg bw), a low aa intake day ( . - . µg /kg bw) as well as a high aa intake day ( . - . µg /kg bw). after a three-day washout period an aama baseline level of ± nmol/d was determined. low aa intake led to an aama excretion within h of ± nmol/d, high intake to ± nmol/d corresponding to an aama excretion rate of about % of the ingested aa dose within h, whereas aama output within h corresponded to % of the respective aa intake, the aama baseline after days washout corresponds to a net exposure level of . - . μg aa/kg bw/d. whether this represents a true baseline level is to be clarified in a follow-up study. in summary, this study provides important quantitative information on kinetics of urinary short-term exposure biomarkers validated by analytically verified dietary aa intake at present day food contamination levels. [ ] deutsche forschungsgemeinschaft (dfg), mak-und bat-werte-liste , doi: . iarc, iarc monographs on the evaluations of carcinogenic risks to humans , . [ ] tareke et al., j. agric. food chem. , , - . [ ] efsa panel on contaminants in the food chain (contam), efsa journal ; ( ): [ pp.] . [ ] ruenz et al., arch. toxicol. doi: . /s - - heinrich-heine-universität, institut für toxikologie, düsseldorf, germany objective: flavonoids are known to modulate distinct signaling pathways thereby causing different physiological effects. effects of the flavonoids baicalein and myricetin as well as several methylated derivatives were analyzed in the nematode caenorhabditis elegans and in in hct colon carcinoma cells and to get insights in molecular mechanisms modulated by these compounds. methods: radical-scavenging activity (teac, dcf), stress resistance (sytox, sodium arsenite), modulation of signaling pathways (nrf /skn- , daf ), life span. results: baicalein enhances the resistance of c. elegans against lethal thermal and sodium arsenite stress and dose-dependently prolongs the life span of the nematode (median life span: + %). using rna interference we were able to show that the induction of longevity and the enhanced stress-resistance were dependent on skn- (homolog to mammalian nrf ), but not daf- (homolog to mammalian foxo), another pivotal transcription factor. negletein was the only methylated derivative which was able to enhance the life span of the nematode. in hct cells, baicalein activates nrf ; the methylated derivatives oroxylin a and negletein showed a comparable redox-active potential in these cells, but only negletein was able to activate nrf . the dietary flavonoid myricetin as well as the methylated derivatives laricitrin, syringetin and myricetintrimethylether strongly enhance life span of c. elegans, decreased oxidative stress (dcf) and accumulation of lipofuscin. in contrast to myricetin, the methylated compounds strongly enhanced the resistance against thermal stress. furthermore, treatment with the derivatives induced a much stronger nuclear localization of the daf- transcription factor. conclusion: baicalein increases stress-resistance and life span in c. elegans via skn- but not daf- . experiments with methylated baicalein derivatives suggest that the redox-active potential has a minor impact on the nrf /skn- activation since only distinct derivates activate this pathway. in case of myricetin, the methylation increases the stress resistance of the flavonoid. methylation seems to enhance the biofunctionality of the flavonoids. our results may be useful to understand molecular mechanisms of flavonoids and methylated derivatives used as food supplements or pharmacological extracts. the loss of progesterone during menopause is linked to common sleep complaints of the affected women. consequently, a previous study of our laboratory demonstrated sleep promoting effects of oral progesterone replacement in postmenopausal women [ ] . the oral administration of progesterone, however, is compromised by individual differences in bioavailability and metabolism of the steroid. we therefore investigated the sleep-eeg effects after intranasal application of progesterone in healthy postmenopausal women ( - yrs).in a randomized doubleblind protocol each subject received four treatments, doses of intranasal progesterone ( . mg mpp ; . mg mpp ), mg of zolpidem and placebo. the conditions consisted of experimental nights (adaptation + examination) separated by at least one week. during each examination sleep eeg was recorded from : to : . simultaneously blood was collected every min between : and : by long catheter for later analysis of the hormones growth hormone (gh), cortisol, melatonin and progesterone. conventional sleep-eeg was statistically evaluated by multivariate analyses of variances (manovas) with repeated measures designs after removal of two outliers, which showed a low sleep efficiency index (sei) after . and . mg mpp . univariate f-tests in the manovas pointed to the following results (significant p-values at α= . ). sei was higher after zolpidem than after the other three treatments. after . mg mpp sei was elevated significantly in comparison to placebo. subjects spent more time in nonrem sleep and less time in intermittent wakefulness after . mg mpp and after zolpidem than after placebo. total sleep time was elevated and wake after sleep onset (waso) was reduced after . mg mpp and after zolpidem. after all active treatments with mpp and zolpidem the time spent in sleep stage was higher than after placebo. the amount of slow-wave sleep was higher after zolpidem than after placebo. in addition, the higher dose of mpp resulted in an increase of spindle and β frequencies combined with a decrease of δ oscillations during nonrem sleep. in comparison, administration of zolpidem resulted in strong increase of δ, spindle and high β frequencies as well as strong decrease in θ and α frequencies. nocturnal progesterone levels increased after . mg mpp . no other changes of hormone secretion were found. our study show sleep promoting effects of . mg mpp. as expected the sleep promoting effect of zolpidem was confirmed. the spectral signature of intranasal progesterone partly resembled the well-known sleep-eeg alterations induced by gaba active compounds. progestereone levels were elevated after . mg mpp . no other endocrine effects were observed. introduction: anticholinergic drugs or drugs with anticholinergic side effects are commonly used for the treatment of various diseases in the elderly population. elderly patients are particularly vulnerable to anticholinergic-related cognitive effects. moreover, there is a relationship between anticholinergic exposure and cognitive impairment. however, there is currently a lack of data on the anticholinergic burden in geriatric patients in germany. it was therefore the aim of this study to evaluate the anticholinergic burden in a large representative cohort of geriatric patients. materials and methods: in this retrospective cohort study, (co)-prescriptions of anticholinergic drugs as well as anti-dementia drugs were evaluated using the discharge medication of geriatric patients between january and june from the geriatrics in bavaria-database (gib-dat). anticholinergic drugs were classified according to the anticholinergic cognitive burden (acb) scale in three groups (definite anticholinergics with a score of or and possible anticholinergics with a score of ). the acb scale was modified by omitting trospium and by adding the three drugs biperiden, metixen and maprotilin, which are used in germany, with a score of . a patient's individual score of or higher is considered to be clinically relevant. in total, , geriatric patients (median age years, . % female, median no. of drugs ) were evaluated. of these, , ( . %) patients took at least one drug with anticholinergic properties. two or more anticholinergic drugs were coprescribed in , ( . % of the patients taking anticholinergic drugs) patients. , ( . % of the patients taking anticholinergic drugs) patients had a score of or higher. the most common anticholinergic drug combinations involving two definite anticholinergic drugs were amantadine/quetiapine ( ), amitriptyline/quetiapine ( ) and amitriptyline/carbamazepine ( ). , ( . %) patients received anticholinergic drugs in combination with anti-dementia drugs. conclusions: one third of patients in a large geriatric population were prescribed at least one anticholinergic drug. one quarter received a co-prescription of anticholinergic drugs. caution is advised prescribing anticholinergic drugs to elderly patients especially with dementia. the antiglaucoma agents brimonidine and timolol are novel substrates of the organic cation transporters oct and mate expressed in human eye c. neul purpose: glaucoma is a leading cause of visual loss in the world population. lowering intraocular pressure by topical administration of antiglaucoma agents is still the mainstay for glaucoma treatment. , although many effective drugs exist, the major challenge is their efficient intraocular delivery, which is estimated to amount to the involvement of membrane drug transporters in the intraocular delivery of the widely prescribed antiglaucoma prostanoid latanoprost has been described. however, it is currently unknown whether the cationic drugs brimonidine and timolol, which are also commonly used antiglaucoma agents, are similarly transported by drug transporters and whether these transporters are expressed in human eye. brimonidine is an α -adrenergic agonist, which inhibits the activity of the adenylate cyclase subsequently leading to a reduced production of aqueous humor. timolol is a β-adrenergic receptor antagonist, which blocks β-receptors on the ciliary epithelium also resulting in a reduced aqueous humor production. the aim of the present study was to determine whether brimonidine and timolol are substrates of the organic cation drug transporters oct (encoded by slc a ), oct (slc a ), oct (slc a ) and mate (slc a ). a further aim was to investigate whether these transporters are localized in different human eye substructures. experimental design: transport of brimonidine and timolol was studied using the mammalian cell line hek stably expressing the organic cation transporters oct , oct , oct or mate . intracellular accumulation of brimonidine and timolol was analyzed by mass spectrometry. immunohistochemistry and immunofluorescence experiments were performed to study the localization of these transporters in different substructures from glaucomatous and non-glaucomatous human eyes. results: uptake experiments revealed that brimonidine is transported by oct and mate in a time-and concentration-dependent manner, but not by oct or oct . timolol is only transported by mate , but not by the octs. as shown by immunolocalization studies, the oct and mate transporter proteins were expressed in all anterior eye substructures of non-glaucomatous and glaucomatous eyes, i.e. the cornea, the conjunctiva and the ciliary body. conclusion: our data demonstrate that oct and mate may play a role in the ocular disposition of the antiglaucoma drugs brimonidine and timolol and may contribute to interindividual variability of drug concentrations and effects. references: . zhang et al., nat rev drug discov. jun ; ( ) : - . . lavik et al., eye (lond). may; ( ): - . . gaudana et al., pharm res. may; ( ): - . . kraft et al., invest ophthalmol vis sci. ( ): - . . nies et al., plos one. ( ) :e . supported by the robert bosch foundation, stuttgart, germany. immature platelet count or immature platelet fraction as optimal predictor of antiplatelet response to thienopyridine therapy c. stratz , t. nuehrenberg background: previous data suggest that reticulated platelets impact significantly on antiplatelet response to thienopyridines. it is unknown which of the parameters describing reticulated platelets is the optimal predictor of antiplatelet response to thienopyridine therapy. methods: this study is a prespecified subanalysis of the excelsiorload trial that randomized elective patients undergoing coronary stenting to loading with clopidogrel mg, prasugrel mg or prasugrel mg (n= ). adp-induced platelet reactivity was assessed by impedance aggregometry before loading (=intrinsic platelet reactivity) and on day after loading. multiple parameters of reticulated platelets were assessed by an automated whole blood flow cytometer: immature platelet fraction (ipf, proportion of reticulated platelet of the whole platelet pool), highly immature platelet fraction (hipf), absolute immature platelet count (ipc). results: each parameter of reticulated platelets correlated significantly with adpinduced platelet reactivity: ipf (r s = . ; p= . ), hipf (r s = . ; p= . ), ipc r s = . ; p< . ). in a multivariable model including all three parameters, only ipc remained as significant predictor of platelet reactivity (p< . ). after adjustment to known predictors of on-clopidogrel platelet reactivity including cytochrome p c polymorphisms (* and * ), age, body mass index, diabetes, smoking and intrinsic platelet reactivity, ipc s was the strongest predictor of on-treatment platelet reactivity (partial η = . ; p< . ) followed by intrinsic platelet reactivity (partial η = . ; p < . ). these findings prevailed when analyzing subgroups of patients on clopidogrel or on prasugrel. conclusion: immature platelet count is the strongest platelet count derived predictor of antiplatelet response to thienopyridine treatment. given its easy availability together with its even stronger association with on-treatment platelet reactivity when compared to known predictors including the cyp c * polymorphism, immature platelet count might become the preferable predictor of antiplatelet response to thienopyridine treatment. cutaneous squamous cell carcinoma (cscc) is the second most common human cancer with continuously rising incidences worldwide. primarily caused by cumulative uvb exposure, cscc accounts for considerable costs for health care systems and poses a deadly risk especially to organ transplant recipients [ ] . current chemotherapy needs to be improved, because even the topical treatment for cscc's carcinoma in situ bears limited efficacy and painful adverse effects [ ] . however, animal-based approaches in preclinical development contribute to the frequent failure of investigational new drugs in clinical trials [ ] . herein, we characterized a human cell-based cscc model, normal reconstructed human skin (rhs) served as control. whereas rhs exhibited low proliferation, the co-culture with cscc increased ki- index -fold in the cscc model (p£ . ). while the presence of claudin- and occludin were distinctly reduced, zonula occludens protein- was more wide-spread, and claudin- was heterogeneously distributed within the cscc model compared with rhs. this is in accordance to the in vivo situation [ ] and likely contributes to the impaired barrier function of the cscc model, as demonstrated for . -fold increased caffeine permeation. finally, the ingenol mebutate effects in the cscc model and rhs closely mimic the anti-tumor effect and the adverse reactions in patients [ ] , both linked to the drug's inherent cytotoxicity. in conclusion, the thoroughly characterization of disease models fosters both advanced preclinical drug development and improved cscc treatment. funded by the german government, the berlin-brandenburg research platform bb r with integrated graduate education was launched in . the aim of this research platform, along with the associated graduate school, is to close substantial knowledge gaps in the fields of the rs and to find alternatives to animal experimentation within the next years. a panel of r experts has been set up to provide advice and assistance and to raise awareness in society for r-related issues. research in bb r investigates physiological functions on different levels to establish alternative methods for preclinical drug development and basic research. the principal investigators aim at facilitating research collaborations and sustainable research activities in the region berlin-brandenburg and abroad. an integrated bb r graduate program has been developed to offer structured training to graduate students in a specific, mandatory course program on r including modules on ethics and legislation. currently, phd students are qualified for management positions in professional areas related to the rs, and three junior research groups are now ready to expand their regional research activities nationwide. furthermore, the concept of a novel lecture series for master students and undergraduates has been designed and awarded the animal welfare research award for berlin-brandenburg in teaching and education. the state government of berlin supports the research platform bb r and will be funding an additional professorship at the fu berlin to further promote research on alternative testing. finally, co-operations with national and international partners are being built to facilitate the project-based exchange of scientists and joint research. currently, the identification and evaluation of skin sensitizers is mainly restricted to animal testing using the guinea pig maximization test, buehler test or the murine local lymph node assay. recently, an adverse outcome pathway of skin sensitization has been released by the oecd, identifying the key events leading to allergic contact dermatitis. in vitro tests address these key events and two assays are now regulatory adopted (oecd c and d). the use of the current in chemico and in vitro models is, however, limited since they do not reflect dermal penetration, complete biotransformation and cell cross-talk in an organotypic environment. in this study, we aimed to overcome these limitations by establishing reconstructed skin tissues containing langerhans cells (lcs). in vitro generated immature monocyte-derived (molcs) or mutz- -derived cells (mutz-lcs) cultivated with keratinocytes on a dermal compartment with fibroblasts form a stratified epidermis after days as indicated by the expression of epidermal differentiation markers. molcs or mutz-lcs were mainly localized in suprabasal layers of the epidermis and distributed homogeneously in accordance with native human skin. topical application of the extreme contact sensitizer , -dinitrochlorobenzene (dncb) induced il- and il- secretion in skin models with lc-like cells, whereas no change was observed in control rhs lacking immune cells. increased gene expression of cd and pd-l in the dermal compartment indicated lc maturation. we confirmed the enhanced mobility from epidermal to dermal compartments for mutz-lcs and molcs in the presence of dncb. in summary, we successfully integrated immature and functional lc-like cells into reconstructed human skin. this fosters the development of animal-free test systems for advanced and potentially individualized hazard assessment of skin sensitization. computational methods for prediction of in vitro activity of new chemical structures. background: with a constant increase in the number of new chemicals synthesized every year, it becomes highly important to employ the most reliable and fast in silico screening methods to predict their safety and activity profiles. in recent years, in silico prediction methods received great attention as alternatives to animal experiments for evaluation of various toxicological endpoints, complementing the theme of replace, reduce and refine ( rs). various computational approaches have been proposed for prediction of toxicity of chemicals ranging from quantitative structure activity relationship modeling to molecular similarity based methods and machine-learning methods. within the "toxicology in the st century" screening initiative, a crowdsourced platform was established for development and validation of computational models to predict the interference of chemical compounds in nuclear and stress receptor pathways based on a training set containing more than , compounds tested in high-throughput screening assays. methods: here we present the results of various molecular similarity-based and machine-learning-based methods over an independent evaluation set containing compounds. further, we compare the performance of these methods when applied individually and together. in retrospect we also discuss the reasons behind the superior performance of an ensemble approach, that combines a molecular similarity approach and a naïve bayes machine learning algorithm in achieving best prediction rates in comparison with other individual methods, explaining their intrinsic limitations. results and conclusions: our results suggest that, although prediction methods were optimized individually for each modeled target, an ensemble of similarity and machinelearning approaches provides promising performance indicating its broad applicability in toxicity prediction. charité -universitätsmedizin berlin, structural bioinformatics group, berlin, germany a multitude of drug candidates (approx. %) fail in the late drug development due to toxicity and adverse effects [ ] . immunotoxicity can be divided into four types of immune-mediated adverse effects: immunosuppression, immunostimulation, hypersensitivity and autoimmunity. current safety evaluations of drug candidates with respect to immunotoxicity are comprised of in vivo and in vitro assays. here, we present an in silico approach for predicting immunotoxic substances based on immune suppressive and not toxic compounds using the laplacian-modified naϊve baysian model as a supervised machine learning method. therefore, we examined the relations between about , compounds and cancer cell lines from the national cancer institute's (nci) nci- growth inhibition data [ ] with focus on five immune cell lines (rpmi- , ccrf-cem, . different fingerprints encoding the chemical structures have been evaluated for their predictive power (e. g. extended-connectivity fingerprints, substructure fingerprints) and showed good prediction rates in a retrospective analysis. acting in phase ii metabolism, sulfotransferases (sult) transform endo-and exogenous molecules such as drugs into more hydrophilic entities that are easily excreted from the human body [ ] . although serving detoxification, sult-mediated transformation of molecules has also been associated with the formation of chemically reactive metabolites that could promote adverse reactions [ ] . the development of a computerbased model that allows prediction of molecules susceptible to metabolism would improve drug development and drug safety [ ] , and encourage the reduction of in vivo testing according to the principles of the rs (replacement, reduction and refinement). in our study, we developed an in silico model to predict human sult subtype e activity acting in phase ii metabolism. structure-based molecular modelling of sult activity is challenging due to the broad and overlapping substrate spectrum of sult subtypes. this low substrate specificity can be attributed to the high degree of conformational flexibility of the enzyme, particularly in the active site. therefore, molecular dynamics simulations were performed to address enzyme flexibility and the broad substrate spectrum of sult ( figure ). based on a collection of selected enzyme conformations from molecular dynamics simulations and a dataset of active sult e ligands, ensemble docking was utilized to generate ligand-protein complexes that served as a basis for d pharmacophore development. eight specific d pharmacophores were created that allow identification of sult e substrates and inhibitors. for further refinement of the computer-based prediction, machine learning models and post-filtering steps were generated that allow classification of predicted hits. the final prediction model was used to screen the drugbank (a database comprising over , experimental and approved drugs). a major part of the predicted hits could be confirmed from literature. from the remaining hits, a representative selection of molecules was experimentally tested for sult e inhibition or biotransformation. experimental results were in agreement with our computer-based models and revealed previously-unknown biotransformation by or inhibition of sult e for compounds listed in the drugbank. introduction: vascularization of the dermal equivalent of full-thickness skin constructs by endothelial cells is highly desirable, because such constructs closely mimic the architecture of real skin. unfortunately, the realization of a capillary network in skin constructs is still difficult. in our study of full-thickness skin constructs, following the methodologies of küchler et al. ( ) , there were alterations in the epidermal differentiation after endothelialization of the dermal equivalent. the aim of this study was to characterize these changes on a morphological level. material and methods: non-endothelialized constructs (keratinocytes, fibroblasts) were prepared according to küchler et al. ( ) . to obtain endothelialized constructs, the dermal equivalent of the non-endothelialized constructs was enriched with endothelial cells. after two weeks of in vitro culture, the skin constructs were processed for quantitative as well as qualitative assessment by light and electron microscopy. results: both types of skin construct developed all strata, i.e., stratum basale, spinosum, granulosum, corneum of a stratified soft-cornified epidermis, although the two constructs displayed differences in every stratum: significantly more mitoses occurred in the epithelial germ layers of the endothelialized constructs (p= . ). in addition, significantly more keratohyalin granules were counted within their stratum granulosum (p= . ). % of the shapes of the spinous and the granulosum cells were irregular and these cells were separated by wide intercellular spaces. the typical epidermal lamellar bodies appeared in the endothelialized constructs more often than in the nonendothelialized ones. at the stratum granulosum -stratum corneum interface, no cohesion between the strata was present. background and novelty: during the last decade organ-on-a-chip technologies received increasing attention in the scientific community. the idea of combining different tissue types on a physiological-like system creates completely new options on how substances can be characterized without the use of animal experiments. animal models were used for the investigation of skin sensitization as a standard method until animal testing for cosmetic substances was banned in the e.u. in . by combining skin models with an immunological counterpart, new data will be presented to see if the multi-organ-chip add value to the current need of alternative methods regarding skin sensitization and immunotoxicity testing. experimental approach: the multi-organ-chip platform is designed to combine different human cell and tissue types like d-spheroids, barrier models or biopsies in one microfluidic system. a peristaltic on-chip micropump enables circulation of medium, allowing for a constant perfusion between the compartments. first experiments were performed using a dendritic-cell-only approach in the -organ-chip. in subsequent cocultivation experiments ex vivo human epidermis and dendritic cells were cultivated each in one culture compartment connected by the microfluidic channels. for analysis we measured the typical activation marker cd and the vitality of the dendritic cells by flow cytometry. functionally different sensitizers were selected to investigate their effects in our model. finally a more complex d matrices including different immunological cell types to emulate in vivo-like reactions like in the human lymph node were cultivated in the -organ-chip. results and discussion: our data show a strong influence of pump pressure and pumping frequency on the activation of dendritic cells. hence, we established an adequate set up by cultivating the dendritic cells in cell culture inserts, preventing cell activation due to shear stress. compared to existing sensitization assays, the main advantage of the perfused -organ-chip sensitization assay is the presence of an epidermis equivalent, partially integrating important parameters such as metabolism and skin barrier function. we compared our data with reference cd -values from the pbmdc (peripheral blood monocyte derived dendritic cells) skin sensitization assay. for identical substances, we observed differences in dendritic cell activation between the pbmdc assay and the -organ-chip perfused assay. here we present the first-time cultivation of primary derived immune cells cultivated on our microfluidic system which is a promising enhancement to integrate immunological reactions on further multi-organ combinations. due to growing social and political pressure, the interest in alternatives to animal testing has constantly increased during the past years stimulating the development and validation of new in vitro test systems including reconstructed skin models. additional to toxicological studies and permeability assays, skin models are of high interest for fundamental research to elucidate basic physiological and pathophysiological processes in human skin [ , ] . as for today, most of the in vitro skin models are grown from primary keratinocytes and fibroblasts that were either isolated from excised human skin or from juvenile foreskin following circumcision. in this project, we aimed for the generation of in vitro skin models using hair folliclederived cells. therefore, different methods to optimize cell isolation and expansion of outer root sheath (ors) cells from human hair follicles were systematically investigated. the best procedure for isolation of ors cells was the direct cell outgrowth on a cell culture insert which was co-cultured with a feeder layer of postmitotic human dermal fibroblasts. following outgrowth, the cells were either further cultivated with feeder cells in specific serum-enriched cell culture medium to obtain hair follicle-derived keratinocytes or using the same culture medium without feeder cells to obtain fibroblasts. afterwards, the generation of hair follicle-derived fibroblasts and keratinocytes was verified via the fibroblast-specific markers vimentin and desmin and the keratinocyte marker cytokeratin (ck) clearly showing that vimentin and desmin are expressed in hair follicle-derived fibroblasts and in dermal fibroblasts. as expected, these cells were negative for ck , which was abundantly expressed in hair folliclederived keratinocytes. moreover, the expression of collagen type i, iv, tgf-beta, alpha sma and il- alpha in hair follicle-derived fibroblasts and dermal fibroblasts showed no significant differences. ultimately, hair follicle-derived keratinocytes and fibroblasts were used to grow full-thickness skin models which were subsequently characterized with regard to epidermal differentiation, skin permeability and skin surface ph. again, no significant differences compared with skin models grown from skin-derived cells were detected showing the potential of using hair follicle-derived cells for generating in vitro skin models. [ ] vávrová, k., henkes, d., strüver, k., sochorová, m., Školová, b. et al. filaggrin deficiency leads to impaired lipid profile and altered acidification pathways in a d skin construct. the journal of investigative dermatology. , - ( bundesinstitut für risikobewertung, experimentelle toxikologie und zebet, berlin, germany background: the eu directive / has been drawn up with the aim of ultimately replacing animal testing. wherever animal experimentation is necessary, the -rprinciple of russel and burch (replace, reduce, refine) has to be observed. the primary goal of the -r-principle is to replace animal testing with alternative methods. if no alternative method can be applied, the total number of animals is supposed be reduced. consequently, some animals are used multiple times in the course of an experiment. for example, in imaging studies, rodents are exposed to anesthesia several times in order to control the progress of a disease. however, the directive claims that "the benefit of reusing animals should be balanced against any adverse effects on their welfare, taking into account the lifetime experience of the individual animal". objective: we are looking into whether multiple exposures to anesthesia cause more stress than a single exposure. methods: the most common mouse strain c /bl j and anesthetics isoflurane and the combination of ketamine/xylazine are used. with regard to recent studies, the animals are anesthetized six times for minutes over a period of three weeks. all parameters observed are compared between controls, animals with a single and repeated anesthesia. the interval between the administration of the anesthesias is three to four days. when the animals are under anesthesia, their vital parameters are continuously monitored and afterwards their general condition is examined. the grimace scale is scored and minutes after anesthesia. besides pain, the grimace scale can also assess anxiety, stress and malaise. the display of so-called luxury behaviors like nest building and burrowing behavior serves as an indicator of wellbeing. furthermore, activity, food and water intake are monitored for hours. a behavioral test battery including the free exploratory paradigm, open field, balance beam and rota rod test is performed one, seven and ten days after the last anesthesia. motor coordination and balance are assessed by the balance beam and rota rod. the open field is a test to investigate anxiety-related and exploratory behavior, the free exploratory paradigm estimates trait anxiety. moreover, corticosterone metabolites are measured in feces and fur in order to prove evidence of cumulative stress. results: the first results of our study will be presented at the nd meeting of dgpt. conclusion: we are confident that the results of our study will contribute to the assessment of the severity level caused by multiple exposures to anesthesia and in this way be a benefit for the welfare of laboratory rodents. bb r is funded by bmbf. in the r-principle was defined by the british scientists william russel and rex burch in their book 'the principles of human experimental techniques'. the r refer to replace, reduce, and refine. they set the goals to use alternative non-animal methods (replace), to reduce the number of laboratory animals (reduce) and to minimize the distress of laboratory animals and to refine their welfare (refine). the implementation of the r-concept is the overall goal of scientific animal welfare. article of the 'directive / /eu on the protection of animals used for scientific purposes' state that the research on refinement is as important as on replacement and reduction [ ] . according to the current statistics on laboratory animals, the mouse is the most commonly used animal in experiments with approximately % [ ] [ ] . effective pain treatment is crucial not only for ethical and legal considerations but also to achieve highquality results [ ] . pain in mice is only obvious if it is severe or acute pain. however, it is difficult to identify slight or moderate pain. the determination of pain levels and effective dosages of analgesics is therefore challenging. the most commonly used analgesics for postsurgical pain treatment in mice are opioids. however, the recommendations for their use show vast dosage ranges. for example, the recommended dose of buprenorphine ranges from . to . mg/kg per bodyweight [ ] [ ] [ ] depending on the pain model used. additionally, putative pharmacogenetic strain differences have to be considered. for example, the analgesic treatment with morphine shows mouse strain specific differences in pain sensitivity [ ] . the goal of the project is the refinement of the recommendations for effective dosage of opioid analgesics in laboratory animals for mouse inbred strains by incorporating strain specific differences. regarding the phenotype, we want to identify a putative inbred strain dependent pain threshold and measure the drug level in plasma and brain. additionally the metabolic capacity and mrna expression level will be investigated. genotype identification is based on a data bank analysis used for correlation with phenotypical parameters. [ ] rl / /eu. [ ] bmel ( ) . anzahl der für versuche und andere wissenschaftliche zwecke verwendeten wirbeltiere. [ ] eu commission ( ) . seventh report on the statistics on the number of animals used for experimental and other scientific purposes in the member states of the european union. [ ] carbone l ( ) pain in laboratory animals: the ethical and regulatory imperatives. plos one , e . [ ] gv-solas, a.f. a.d. ( ) . empfehlung zur schmerztherapie bei versuchstieren. [ ] carpenter, j.w., t.y. mashima, and d.j. rupiper ( ) . exotic animal formulary, nd edition, w.b. saunders co., phila. [ ] flecknell, p. ( ) . laboratory animal anaesthesia, nd edition, academic press, san diego, ca. introduction: göttingen minipigs™ are frequently used large animal models for orofacial research, for example dental implant surgery. requests from experimental surgeons for detailed anatomical information can not be answered because there is no existing data, especially not age-related. because of unavailable data and the false choice of animal age, surgical interventions fail or lead to enormous post-operative suffering. therefore the aim of this study is the acquisition of detailed anatomical data of the mandibula and other organs and structures without sacrificing pigs for this reason. animals, materials and methods: ct scans of a -slice scanner were collected from female minipigs, consisting of animals aged months (group , n= ) and animals (group ; n= ) examined at the age of and months. these minipigs were involved in experiments, approved by the regional office for health and social affairs, berlin. image analysis was performed using vitrea advanced® (vital images). more than parameters concerning teeth, the mandibular body, frame and canal, coronoid process and mandibular condyle were defined and measured. for now, we focused on the development of the mandibular canal and the distance between the dorsal borders of the mandibular canal to the alveolar ridge at the most posterior mental foramen, parameters immensely important for planning interventions when testing new dental implants. results: the measurements by computed tomography showed variations of several parameters between left and right ramus mandibulae and within the different age groups. the volume of the canalis mandibulae increases in time. animals of the same age show significant differences in volume, with a range of up to % where the largest volume was , ml and the lowest , ml. the distance between the dorsal borders of the mandibular canal to the alveolar ridge decreases between and months of age. comparing and months old minipigs, no significant difference in distance could be observed. from the age of months the position of the mandibular canal in relation to the alveolar ridge remains constant. conclusion: the decrease of the distance between the mandibular canal and the alveolar ridge between and months of age indicates ongoing anatomical changes of this parameter until the age of months. therefore animals should be older than months if included in long-term studies after orofacial experiments, like implant surgery of the mandibula. because of the described individual differences, the authors strongly suggest to support the planning of orofacial interventions by ct imaging or other radiographic techniques. background: laboratory housing conditions are standardized to a high level. under these conditions, the occurrence of stereotypic behaviour (sb) can be observed. stereotypies are commonly known as deviations from normal behaviour that are repetitive, invariant and without any obvious function or aim for the animal [ ]. worldwide it is estimated that over million animals perform sb, with the highest prevalence in laboratory animals and the agricultural sector. fvb/n is a typical inbred mouse-strain that shows different types of stereotyped movements. it is known that environmental enrichment decreases the incidence of sb, yet they still occur [ ] . since animal's behaviour highly influences its metabolism and immune system, differences in handling, caring and keeping can lead to varying results, even with an identical experimental setup [ ] . aim: of the study aim of the study is to observe different life stages of fvb/n-mice and immediately detect the development of sb. observations are linked to various behavioural tests and the characterisation of the metabolic and immunological phenotype. the results will lead to a better understanding of the mechanisms driving the development of sb and clarify its implication to animal welfare or to what extent the performance of stereotypies even reflect emotional suffering. the strain-related behaviour and sb are recorded with computer-assisted programmes. observational periods already begin with the parental generation. as possible indicators for later developing sb, data on reproductive success and maternal care are collected, such as different behavioural tests. the animals are characterized by a specific protocol for detecting the metabolic and immunological phenotype. finally, histological and molecular biological analyses follow. outlook: it is of paramount importance to take good care for the welfare of laboratory animals ( r-refinement). though, the knowledge about the ethological particularities of animals and the motivational base of animals performing sb are not enough to generally avoid stereotypies. therefore the meaning according to the character of sb has to be analysed more intensively to understand the needs of laboratory animals and evolve recommendations for optimizing the breeding and keeping such as for the assessment of possible distress in animals performing sb. objective: thermoregulation is a vital function in both humans and animals with the serotonin ( -ht) system, in particular the -ht a receptor, playing a major role. activating -ht a receptors by the -ht a receptor agonist -hydroxy- -(dipropylamino)tetralin ( -oh-dpat) leads to reduced body temperature. while there is consensus that hypothermia is induced by the stimulation of postsynaptic -ht a receptors in rats and humans, the regulatory mechanisms in mice are less clear. in our group, within phenotyping a transgenic mouse line permanently overexpressing the -ht a receptor in serotonergic projection areas, bert et al. ( , pmid: ) revealed exaggerated -oh-dpat-provoked hypothermic response. thus, the objective of the present study was to substantiate the contribution of postsynaptic -ht a receptors to thermoregulation, more precisely to the hypothermic effect of -oh-dpat, in mice. methods: we used radio telemetry technique to monitor the basal body temperature and the hypothermic effect of different doses of -oh-dpat ( . mg/kg - mg/kg i. p.) in male transgenic mice in comparison to nmri wild-type males. additionally, we investigated whether reduction of serotonergic activity by pretreatment with the -ht synthesis inhibitor parachlorophenylalanine (pcpa; mg/kg, i. p. on four consecutive days) would alter the effects of -oh-dpat on body temperature in transgenic mice postsynaptically overexpressing the -ht a receptor. results: -ht a overexpressing mice revealed lower levels of basal body temperature than wild types (transgenic mice: . °c; nmri wild-type mice: . °c). in both genotypes, systemic administration of -oh-dpat dose-dependently decreased body temperature, being significantly more pronounced in mutant mice (- . °c compared to - . °c in nmri wild types). dose response curves of -oh-dpat revealed an ed = . mg/kg in transgenic and an ed = . mg/kg in nmri wild-type mice. pcpa pretreatment did not alter the hypothermic response to -oh-dpat in mice. the dose-response curves indicate a higher potency of -oh-dpat in transgenic mice. the exaggerated hypothermic response to -oh-dpat in mutant mice implies that postsynaptic -ht a receptors could be involved in thermoregulatory function in mice. this assumption is further confirmed by the fact that -oh-dpatevoked thermal responses were not influenced by pretreatment with pcpa, most notably in transgenic mice overexpressing -ht a receptors postsynaptically. genetic variation within g protein-coupled receptors compromises the therapeutic application of drugs targeting these receptors. one of the most intensely studied variation is p.arg gly in the human beta -adrenoceptor (adrb ). the adrb carrying arginine at position in helix in the proximal carboxy terminus is more frequent among caucasians and is hyperfunctional. yet, the molecular basis for the differences between the beta -adrenoceptor variants arg -adrb and gly -adrb is poorly understood. despite its hyperfunctionality, we found the arg -variant of the adrb to be hyperphosphorylated upon continuous stimulation with norepinephrine when compared to the gly -variant. using adrb sensors to monitor activation kinetics by fluorescence resonance energy transfer (fret), the arg -adrb exerted faster activation speed and arrestin recruitment than the gly -variant. both depended on phosphorylation of the receptor as shown by knockdown of g protein-coupled receptor kinases and by fret experiments using phosphorylation-deficient adrb mutants. point mutation of single serines and threonines in the carboxy terminus of the adrb finally revealed a variant-specific phosphorylation pattern that determines arrestin recruitment. taken together, these findings suggest that differences in receptor phosphorylation determine the differences in activation speed, efficacy and arrestin recruitment of adrb variants. opioid drugs are the most potent analgesics, which are used in the clinic; however, by activating the μ-opioid receptor (mor) they also produce several adverse side effects including constipation, antinociceptive tolerance, and physical dependence. there is substantial evidence suggesting that g protein-coupled receptor kinases (grks) and βarrestins play key roles in regulating mor signaling and responsiveness. following phosphorylation by grks, β-arrestins bind to phosphorylated mors, which prevents further interactions between the receptor and g proteins even in the continued presence of agonist resulting in diminished g protein-mediated signaling. we have previously shown that agonist-induced phosphorylation of mor occurs at a conserved -residue sequence, trehpstant , in the carboxyl-terminal cytoplasmic tail. morphine induces a selective phosphorylation of serine (s ) in the middle of this sequence that is predominantly catalyzed by g protein-coupled receptor kinase (grk ). by contrast, high-efficacy opioids not only induce phosphorylation of s but also drive higher-order phosphorylation on the flanking residues threonine (t ), threonine (t ), and threonine (t ) in a hierarchical phosphorylation cascade that specifically requires grk / isoforms. to investigate this mechanism further, we have developed novel β-galactosidase complementation assays to monitor agonist-dependent recruitment of grk and grk to activated mors. using this assay, we were able to show that activation of mor by high-efficacy agonists such as damgo results in a robust translocation of grk / . in contrast, activation by low-efficacy agonists such as morphine results in a much less pronounced recruitment of grk / isoforms. interestingly, damgo-induced β-arrestin recruitment was strongly inhibited by sirna knock down of grk or grk . conversely, the morphine-induced β-arrestin recruitment was strongly enhanced by overexpression of grk or grk . mutation of s to alanine strongly inhibited both grk and β-arrestin recruitment. however, mutation of all carboxyl-terminal serine and threonine residues of mor was required to completely abolish its interaction with arrestins and grks resulting in a complete loss of mor internalization and desensitization. heterotrimeric g proteins are located at the inner leaflet of plasma membranes and are a major primary transducer of cell signaling initiated by g protein-coupled receptors (gpcrs). based on sequence similarity, heterotrimeric g proteins can be subdivided into four main classes, i.e. gi/o, gs, gq/ , and g / , which interact with distinct cellular effectors to shape the final cellular response . identification of new selective and cell-permeable g protein inhibitors is of great interest as these may be beneficial in complex pathologies that involve signaling of multiple gpcrs . mechanistically, small molecule g protein inhibitors may, for instance, block nucleotide exchange by inhibiting gdp release (i.e. guanyl nucleotide dissociation inhibitors, gdis) or allow gdp release but block gtp entry by stabilizing the g protein in an empty pocket conformation . here, we set out a new approach to classify g protein inhibitors regarding their mechanism of action in radioligand binding experiments. in particular, we investigated the influence of the specific gα q/ / inhibitor fr on agonist-radioantagonist binding experiments performed with membranes of cho cells stably expressing the muscarinic m acetylcholine receptor (cho-m ). agonistradioantagonist competition under these conditions is biphasic because agonists bind with higher affinity in the ternary complex consisting of agonist, receptor and nucleotidefree g protein compared with a g protein-free receptor , . we show that fr can be classified as a gdi as it significantly reduced high affinity binding of carbachol in cho-m membranes. in contrast to this, bim- , a pan g protein inhibitor with a cell-type-dependent preference for gq, did not influence high affinity agonist binding and thus stabilized gq in an empty pocket conformation . interestingly, inhibition of high affinity agonist binding by fr was incomplete in agonist-radioantagonist displacement studies and also when a radioagonist was applied. to fully prevent high affinity agonist binding by fr , combined uncoupling of both gi and gs proteins from m was required by pre-treatment with pertussis toxin and cholera toxin, respectively. these data demonstrate that not only gq, but also gi, and gs, contribute to the high affinity fraction of m receptors. taken together, our findings show that radioligand binding experiments are an attractive approach to classify new g protein inhibitors according to their mechanism of action. universität, würzburg, germany g protein-coupled receptors (gpcrs) are cell surface receptors which, upon a conformational change in the receptor protein induced by an extracellular stimulus, can transduce the signal onto intracellular adaptor proteins such as heterotrimeric g proteins [ ] . gpcr-induced cell signaling can be rather complex as several gpcrs may activate multiple different adaptor proteins and can additionally be activated via distinct binding sites, i.e. the orthosteric transmitter binding site and other "allosteric" binding sites [ ] . in the present work, we wanted to investigate the influence of an allosteric binding site on receptor activation of muscarinic acetylcholine receptors (machrs). to this end, we employed the orthosteric full agonists acetylcholine and iperoxo as well as several dualsteric compounds consisting of iperoxo linked to an allosteric phthalimide (phth) or naphthalimide (naph) moiety through alkyl chains of different length or through a diamide linker (fri). binding of the allosteric part to the receptor protein may restrict the conformational flexibility of the receptor protein and thus interfere with receptor activation [ ] . therefore, application of different linker length may control the signaling outcome. here, we applied the human m machr which preferentially activates g proteins of the g q/ type but can also promiscuously stimulate g s proteins. g q/ -and g sdependent signaling pathways were analyzed by application of cho cells stably transfected with the human m machr in ip and camp accumulation assays, respectively. in comparison to the orthosteric building block iperoxo, all dualsteric compounds under investigation showed a decrease in potency for both g q -mediated and g s -mediated signaling. our findings show that the bulkier allosteric naph residue impaired both signaling pathways to a greater extent than the smaller substituent phth. particularly, the compound iper- -naph completely lost intrinsic activity for both g q/ and g s activation at the m machr. moreover, g s -mediated pathway activation is more sensitive to spatial restriction in the allosteric vestibule than g q -signaling. interestingly, longer linker length led to improved signaling for both pathways (g q and g s ) in both hybrid series. iper- -phth seems to be an exception as it had a higher intrinsic efficacy for g s -dependent signaling than the other phth hybrids with longer linker chains. strikingly, only iper-fri-phth, which corresponds to iper- -phth in linker length, but is able to engage increased hydrogen bonding with the receptor protein, acted as a full agonist on m machr for both signaling pathways under investigation. taken together, these data strongly suggest that, in comparison to g q/ -mediated signaling, activation of the g s protein in m machr is more sensitive to spatial restriction in the allosteric vestibule. thus, it appears to be possible to control signaling of the m machr by allosteric constraint of the receptor's conformational flexibility. for more than three decades -( h-imidazol- -yl)propylguanidine (sk&f- , ( ) [ ] ) is known as the prototypic pharmacophore of highly potent histamine h -receptor (h r) agonists of the guanidine class of compounds including, e.g., impromidine and arpromidine. [ ] in order to get more insight into the structure-activity relationships of alkylated analogues of sk&f- , , we characterized newly synthesized derivatives including several bivalent compounds (e.g., ) by using different pharmacological in-vitro methods. [ ] the potential h r agonists were subjected to a broad screening procedure utilizing radioligand binding assays with membranes of sf cells [ ] (hh , , , r). compounds were also functionally characterized in the [ s]gtpgs assay (hh r, sf cell membranes). [ ] selectivity vs. hh , , r was determined for selected derivatives also using this technique. organ bath studies (gph r (ileum), gph r (right atrium)) yielded functional data in a more physiological environment. the major part of the new sk&f- , analogues displayed partial or full agonism via hh r and gph r, respectively. the most potent analogue, bivalent thiazole-type bisguanidine , was a partial agonist (e max = %) and -times as potent as histamine vis-à-vis the gph r. attempts to antagonize the positive chronotropic effect of (partial) agonists by preincubation with cimetidine, or by adding a cimetidine bolus at the end of the concentration-response curve, respectively, were successful and furnished pa values for the antagonist ( . - . ) which are in accordance with literature data. however, in the functional in-vitro assay on gph r, the positive chronotropic response evoked by sk&f- , was surprisingly resistant to antagonism by cimetidine and other typical h r antagonists (ranitidine, famotidine), although the compound so far has been unanimously classified by others as a weak partial h r agonist. this behaviour is unique within the large series of sk&f- , analogues studied so far under similar conditions. probably the positive chronotropic effect of the lead compound is -at least in part -the result of a second molecular interaction which has been overlooked so far. [ ] parsons, m.e. et al.; agents actions , , . [ ] buschauer, a.; j. med. chem. , , - [ ] pockes, s.; dissertation, univ. regensburg . [ ] seifert, r. ; j. pharmacol. exp. ther. , ( ) , - . the nociceptin/orphanin fq (n/ofq) peptide (nop) receptor is the fourth most recently discovered and least characterized member of the opioid receptor family (mor, kor and dor). nop receptor is widely distributed and modulates several physiological processes by its endogenous ligand nociceptin. the nop receptor is a potential target for the development of ligands with therapeutic use in several pathophysiological states such as chronic and neuropathic pain. consequently, there is increasing interest in understanding the molecular regulation of nop receptor. recently, we generated two phosphosite-specific antibodies directed against the carboxyl-terminal residues serine (s ) and threonine /serine (t /s ), which enabled us to selectively detect either the s -or the t /s -phosphorylated forms of the receptor. our results show that nociceptin, mcoppb, sch and ro - induce a stably phosphorylation at s and t /s followed by a profound internalization of the receptor. the nociceptin-induced s and t /s phosphorylation can be blocked by selective nop receptor antagonists such as j or sb , . nnc - , buprenorphine and norbuprenophine failed to induce a phosphorylation at these sites. in the presence of nociceptin, s phosphorylation occurred at a faster rate than phosphorylation at t /s indicating that s is the primary site of agonistdependent phosphorylation. activation of pkc by phorbol -myristate -acetate facilitated receptor phosphorylation only at s but not at t /s , indicating that s can also undergo heterologous phosphorylation. using nop-gfp knock in mice, we detected nop receptors in brain, spinal cord and dorsal root ganglia (drg). we were also able to demonstrate a dose-dependent nop receptor phosphorylation at t /s in mouse brain in vivo using western blot and mass spectrometry. in contrast, mcoppb and sch failed to induce phosphorylation in vivo. together, these data provide new insights into the molecular regulation of the nop receptor in vitro and in vivo. several findings indicate that inflammatory diseases are initiated or maintained by an imbalance of receptor baised signaling; the latter referring to the ability of distinct ligands to endue individual receptors with qualitatively different g-protein-and/or b-arrestindependent signaling ( ). chemokines and their receptors regulate a wide array of leukocyte functions, including chemotaxis, adhesion, and transendothelial migration, and thus play important roles in regulating inflammation ( ) . in man, two cc chemokine receptors ccr a and ccr b are present that only differ in their carboxyl terminal portions; the latter known to interact with multi-protein complexes made up of heterotrimeric g proteins (pertussis toxin sensitive and -insensitive) and non-g-protein components, including b-arrestin ( ) . interested in differential signaling of ccr a and ccr b we comparatively analyzed ligand-induced g-protein-regulated signaling pathways (e.g. activation of phospholipase c isoenzymes and rhogtpase-induced serum response factor [srf] activity) and b-arrestin-regulated pathways (e.g. internalization of receptors and phosphorylation of erk / ) in the presence of ccl , ccl , ccl , and ccl . all these chemokines have been shown to interact with human ccr receptors. in addition, the structural requirements of the carboxyl terminal portions involved in determining specificity in g-protein-dependent signaling was addressed by using ccr receptor mutants. the comparative analysis revealed that differences in ligand-induced activation of g-protein-dependent (pertussis-toxin-sensitive versus pertussis-toxin-insensitive) and/or b-arrestin-dependent signaling exist. for example, activation of ccr b receptors by ccl induced both rho-dependent srf activation and receptor internalization, while ccl -stimulation resulted in srf but little if any receptor internalization. in contrast, ccr a-expressing cells showed ccl dependent srf-activation but any receptor/ligand internalization. analysis of the structural requirements of ccr receptors for coupling to g proteins revealed that arginine within the putative 'eighth helix' of the carboxyl-terminal portions of ccr a and ccr b is not involved in ga i -mediated induction of erk / and plays a minor role in ccr b receptor internalization, but is specifically required for the ccr a/ and ccr b/ga q -mediated activation of srf. serotonin -ht c receptors ( -ht c r) are functional engaged with gq proteins and expressed in the central nervous system (cns). -ht c r significantly regulate emotion, feeding, reward or cognition and thus might serve as promising targets for drugs against psychiatric disorders or obesity. due to the technical difficulties in isolating cells from the cns and the hitherto lack of suitable cell lines that would endogenously express -ht c r, our knowledge about this receptor subtype is rather limited. recently established hypothalamic mhypoa - cells show some characteristics of appetite-regulating hypothalamic neurons of the paraventricular nucleus, where -ht c r in vivo expression has been detected. thus, we tested mhypoa - cells for putative -ht c r expression by performing single cell calcium imaging. we observed that serotonin and the specific -ht c r agonist, way , induced robust calcium transients, which were strongly inhibited by two unrelated -ht c r-selective antagonist (sb , , rs , ). serotonin and way , also activated a camp response element-dependent reporter gene construct and induced significant phosphorylation of extracellularregulated kinases- / in a sb , and rs , sensitive manner, providing further evidence for functional -ht c r expression in mhypoa- / cells. intrinsic activity of way , ranged between . and . compared to serotonin in all assays, defining way , as a partial -ht c r agonist. in conclusion, we provide convincing data that hypothalamic mhypoa- / cells endogenously express -ht c r and thus represent the first cell line to analyse -ht c r pharmacology, signaling and regulation in its natural environment. optical and electrophysiological methods allow detection and characterization of g i/o -protein coupled receptors j. straub walther-straub-institut für pharmakologie und toxikologie, münchen, germany g-protein mediated signaling pathways are essential components of basic cellular functions. of note, g-protein coupled receptors (gpcrs) constitute one of the major drug targets in modern medicine. however, despite their clinical importance, fundamental properties of these receptors remain unknown. in particular, regulation of the major second messenger camp by g s -and g i/o -protein coupled receptors is of special interest. the classical biochemical method to detect receptor-mediated camp level changes uses pre-labeling with h-adenine and calculation of the conversion rate to h-camp. although, this multi-cellular method is highly sensitive and reproducible, it lacks time resolved and spacial assessment of camp formation in single living cells. to measure g s -protein induced increases of intracellular camp levels in single living cells in a time resolved manner, the fret-based camp-sensor epac is commonly used. however, it was unknown whether this sensor might be suitable to detect g i/o -protein mediated decreases of intracellular camp levels as well. in this study, we show that fret-based camp sensors can be deployed to reliably monitor g i/o -protein mediated camp level decreases. fret experiments with adrenergic α a or µ opioid receptors in combination with different fret-based camp sensors showed a notable reduction of intracellular camp levels upon receptor activation which could be significantly reduced by selective receptor antagonists. of note, hek cells had to be pre-incubated with forskolin in submaximal concentration to increase basal camp levels. our findings suggest that fret based epac sensors are suitable to detect g i/o -protein activation similar to electrophysiological whole-cell measurements with g i/o -protein coupled receptors and trpc or heteromeric kir . /kir . or kir . /kir . channels coexpressing cells. hereby, agonist stimulations caused current increases with characteristic current-voltage relationships. altogether, our findings indicate that both optical and electrophysiological approaches allow time resolved detection and characterization of g i/o -protein coupled receptor activation in single living cells. histamine can exert positive inotropic and chronotropic effects in humans via histamine h -receptors. we have generated and partially characterized transgenic mice (tg) which overexpress the human histamine h -receptor specifically in cardiomyocytes via the α-myosin heavy chain promoter. in these mice, but not in wild type mice (wt), histamine increased heart rate and ejection fraction (ef) measured in vivo by echocardiography under isoflurane anesthesia. to investigate some aspects of the signaling pathway in these mice, we crossed the tg mice with pp a mice leading to double transgenic mice (h xpp a = dt). pp a mice (j biol chem ; : ) overexpress the catalytic subunit of protein phosphatase a (pp a) in cardiac myocytes and develop a cardiac hypertrophy. at an age of about days we noted reduced ef in pp a ( . ± . %, n= ) compared to wt ( . ± . %, n= ) and tg ( . ± . %, n= ). interestingly, in dt the ef ( . ± . %, n= ) was higher than in pp a at similar heart rates. e´/a´ was elevated in dt compared to wt. relative heart weights were unchanged between these groups. in summary, we demonstrated that pp a is involved in h -receptor signaling and we tentatively conclude that the h -receptor is able to ameliorate systolic but not diastolic cardiac function of pp a mice. serotonin ( -ht) can exert positive inotropic and chronotropic effects in humans via -ht -receptors. we have generated transgenic mice (tg) which overexpress the human -ht -receptor selectively in cardiomyocytes via the α-myosin heavy chain promoter. in these mice, but not in wild type mice (wt), serotonin induced increases in heart rate and ejection fraction. we treated the mice with µg lps (lipopolysaccharide, i.p.; a standard model of sepsis) per g body weight or isotonic sodium chloride solution (as solvent control). echocardiography with isoflurane anesthesia was performed before and , and hours after lps treatment. lps led to a time-dependent deterioration of cardiac function in both tg and wt. the deterioration included systolic function (left ventricular ejection fraction=ef) as well as diastolic function (height of a and e waves through the mitral valve: e/a). for instance, ef amounted to . ± . % seven hours after lps in wt and to . [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] p< . vs pre-lps value). however, hours after lps, diastolic function, measured as e/a, amounted to . ± . in p< . tg vs. wt). moreover, after hours a less pronounced decline in body temperature (probably due to superficial abdominal hyperemia) occurred in tg versus wt. in contrast, while all flow parameters declined after and hours of lps, they were not different between wt and tg. for instance, maximum flow (in mm/s) through the ascending aorta declined from . ± . to . ± . in wt and from . ± . to . ± . in tg (tg vs. wt, p> . , n= - ; after hours). we tentatively conclude: -ht -receptors overexpression protects the heart against sepsis, putatively by interference with the intracellular biochemical pathway of lps in cardiomyocytes. histamine can exert positive inotropic and chronotropic effects in humans via histamine h -receptors. we have generated transgenic mice (tg) which overexpress the human h -receptor specifically in cardiomyocytes via the α-myosin heavy chain promoter. in tg, but not in wild type mice (wt), histamine (ec = nm) or amthamine (ec = nm), a more selective and potent h -receptor agonist, induced positive inotropic effects (pie) and positive chronotropic effects (pce) in isolated left and right atrial preparations, respectively. in order to investigate the signal transduction for the pie in atrium, contractile studies using partially depolarized preparations were performed. therefore, left atrial preparations were equilibrated in the organ bath to mm potassium chloride. thereafter, histamine ( µm) induced a pie ( . ± . % of control, n= ) in tg but not in wt preparations whereas isoprenaline ( µm) increased force in both wt and tg. the pie of histamine in potassium treated tg atrium could be blocked by cimetidine. compound / , a releasing agent of endogenous histamine, increased force of contraction in tg left atria to a higher extent than in wt. furthermore, we tested whether analgetics known to release histamine were inotropically active in tg. however, morphine ( µm) was unable to affect contractility in wt or tg, whereas ketamine and fentanyl increased left atrial contractility in both tg and wt. in summary, we demonstrated an involvement of the l-type calcium channel current in the h -receptor mediated pie in tg atria. we failed to release inotropically active histamine using classical analgetics, arguing that a direct effect also in the human heart is unlikely to occur. the initial step in the homologous desensitization of g-protein-coupled receptors is their phosphorylation by one of the g-protein-coupled receptor kinases (grks). we demonstrate here measurement of the interaction of grk , a ubiquitously expressed grk, with the μ-opioid receptor (µor) by fret and its dependence on agonist efficacy. hek t cells transfected with yfp-tagged µor and mturquoise-tagged grk as well as non-fluorescent gα i , gβ and gγ subunits showed a robust increase in fret upon superfusion with µm [d-ala , n-mephe , gly-ol]-enkephalin (damgo) which was reversible upon agonist washout. the partial agonist morphine ( µm) also caused a fret increase but the amplitude of the fret signal was reduced to approximately - % of that of the corresponding damgo signal. grk binds g-protein βγ (gβγ) subunits, and therefore we aimed to find out how cotransfection of grk affected the interaction of gβγ with the µor. however, we could not measure any damgo-induced fret changes between yfp-tagged µor and mturquoise-tagged gβ in the presence of non-fluorescent gα i and gγ. this was unexpected because we had previously successfully determined interactions between gβγ and the α a -adrenergic receptor or the m muscarinic acetylcholine receptor. this lack of fret was not due to an inability of the tagged gβ to interact with the µor as we could measure damgo-induced fret changes between yfp-tagged gα i and gβγ (gβ tagged with mturquoise) in the presence of non-fluorescent µor. moreover, when we attempted to establish an effect of grk on the interaction between the µor and gβγ, we could pick up fret between the µor and gβγ. comparison of the on-and off-kinetics of the µor-grk interaction with that of the µor-gβγ interaction in the presence of grk revealed similar time constants both for the on-and off-kinetics (grk : k on . s ). this suggests that, in the absence of grk , the orientation of the two fluorophores on the µor and gβγ may be unfavorable or the interaction may be too short-lived to produce an appreciable fret signal. in the presence of grk , however, gβγ changes its position relative to the µor in a way that allows the interaction of the grk -gβγ complex with the µor to be detected by fret. similarly, we measured fret between gβγ and the α a -adrenergic receptor or the m muscarinic acetylcholine receptor in the absence and presence of grk and compared the kinetics with the kinetics of grk binding and unbinding to these receptors. in both cases, we found that grk and gβγ in the presence of grk associate and dissociate from these receptors with comparable kinetics. our results suggest that ligand efficacy for µors is already apparent on the level of receptor-grk interaction. institute of pharmacology, university medical center göttingen, ag lutz, göttingen, germany introduction: the monomeric gtpase rhoa is dysregulated in heart disease and in vivo models provide evidence of rhoa signaling being involved in the progression of cardiac fibrosis and hypertrophy. how rhoa is regulated within this context on a cellular level is not defined. objective: the goal of this study was to analyze rhoa activation in adult cardiomyocytes (amcm) from normal and diseased mouse hearts in response to g protein-coupled receptor activation. this project also aimed at providing new insight into the dependence of rhoa localization and activation on the signaling organizing caveolae in neonatal as well as adult cardiomyocytes and engineered heart muscles. methods: cardiomyocytes from sham-operated c bl/ mice, from mice subjected to transverse aortic constriction (tac) or from neonatal rats were either directly fixed after isolation or cultured in the presence or absence of methyl-b-cyclodextrin (mβcd). for analyses of rhoa activation cells were treated with endothelin- (et- ) for sec. cells were prepared for immunofluorescence analysis or lyzed for immunoblotting. imaging was performed using confocal microscopy. effects of mβcd were further studied in d engineered heart muscles (ehm) made from total neonatal rat cardiac cells. the contractile function of ehm was assessed in the organ bath and cells were studied in sections by immunofluorescence analysis. results: in amcm from sham mice active rhoa mainly localizes at the sarcolemma and is augmented in response to et- treatment. in tac-amcm the basal level of active rhoa is increased and surprisingly et- had no further effect. immunoblot analysis demonstrated that in tac-amcm rhoa expression was per se higher and the major caveolae protein caveolin- was reduced. to test the influence of caveolae on rhoa activation and expression, we treated nrcm with mβcd and found the expression of rhoa as well as of rhoa target genes ccn and ccn to be moderately up-regulated after h. in addition, an intensified longitudinal alignment of sarcomeric actin fibers was detectable, which could also be seen in mβcd-treated ehm. however, mβcd had no effect on ehm twitch tension but increased the resting tension compared to control. we further treated amcm with mβcd and found rhoa expression to be increased and its activity less sensitive to et- treatment. finally, we could show that the perinuclear localization of cav and rhoa was strongly reduced after mβcd treatment. whereas g-protein coupled receptors (gpcrs) have been long believed to signal through cyclic amp only at cell surface, our group has previously shown that gpcrs not only signal at the cell surface but can also continue doing so once internalized together with their ligands, leading to persistent camp production ( ). this phenomenon, which we originally described for the thyroid stimulating hormone receptor (tshr) in thyroid cells, has been observed also for other gpcrs ( ) ( ) ( ) . however, the intracellular compartment responsible for such persistent signaling was insufficiently characterized. the aim of this study was to follow by live-cell imaging the internalization and trafficking of tshr, tsh and effector proteins in thyroid cells. mouse primary thyroid cells were transfected with fluorescent-protein tagged tshr, g-proteins, nanobody specific for active g-proteins and/or subcellular markers by electroporation, stimulated with fluorescently labeled tsh and visualized using highly inclined thin illumination (hilo) microscopy. our results suggest that tsh is internalized in complex with its receptor and they traffic retrogradely via the trans golgi network (tgn). while we could not find any evidence of internalized tsh/tshr complexes activating g-proteins in early endosomes, we show that tsh/tshr complexes meet the intracellular pool of gαs in the tgn and activate it, as visualized in real-time by a nanobody specific for active gαs. upon acute brefeldin a-induced golgi collapse, the retrograde trafficking of tsh/tshr via tgn is hindered. bulk tsh stimulations in primary mouse thyroid cells isolated from transgenic mice expressing the camp sensor, epac -camps, also show a significantly reduced camp production in the presence of brefeldin-a. these data provide evidence that internalized tsh/tshr complexes meet and activate g-proteins at the tgn, which might serve as the main platform of persistent camp signaling after receptor internalization. objective: sphingosine -phosphate (s p) is generated by sphingosine kinase (sk)- and - and acts mainly as an extracellular ligand at five specific g protein-coupled receptors, denoted s p - . after activation, s p receptors regulate important processes in the progression of renal diseases, such as mesangial cell migration methods and results: here we demonstrate that dexamethasone treatment lowered s p mrna and protein expression levels in rat mesangial cells measured by taqman® and western blot analyses. this effect was abolished in the presence of the glucocorticoid receptor antagonist ru- . in addition, in vivo studies showed that dexamethasone downregulated s p expression in glomeruli isolated from c bl/ mice treated with dexamethasone ( mg/kg body weight). functionally, we identified s p as a key player mediating s p-induced mesangial cell migration. using boyden chamber assays, we could show that dexamethasone treatment significantly lowered s p-induced migration of mesangial cells. this effect was again reversed in the presence of ru- . conclusion: we suggest that dexamethasone inhibits s p-induced mesangial cell migration via downregulation of s p . overall, these results demonstrate that dexamethasone has functional important effects on sphingolipid metabolism and action in renal mesangial cells (koch et al., biol. chem. ; : - ) . the g protein-coupled receptor mrgd is a receptor for angiotensin-( - ) involving g alphas , camp, and phosphokinase a rationale: angiotensin (ang)-( - ) has cardiovascular protective effects and is the opponent of the often detrimental ang ii within the renin-angiotensin system. although it is well-accepted that the g-protein coupled receptor mas is a receptor for the heptapeptide, the lack in knowing initial signalling molecules stimulated by ang-( - ) prevented final verification of ligand/receptor interaction as well as the identification of further hypothesized receptors for the heptapeptide. objective: the study aimed to identify a second messenger stimulated by ang-( - ) allowing confirmation as well as discovery of the heptapeptide's receptors. we identified camp as the second messenger for ang-( - ). the heptapeptide elevates camp concentration in primary cells such as endothelial or mesangial cells. using camp as readout in receptor-transfected hek cells, we provided final pharmacological proof for mas to be a receptor for ang-( - ). more important, we identified the g-protein coupled receptor mrgd as a second receptor for ang-( - ). consequently, the heptapeptide failed to increase camp concentration in primary mesangial cells with genetic deficiency in both mas and mrgd. furthermore, we excluded the ang ii type receptor at as a receptor for the heptapeptide, but discovered that the at blocker pd can also block the mas and mrgd receptors. conclusions: our results lead to an expansion and partial revision of the reninangiotensin system, by identifying a second receptor for the protective ang-( - ) but excluding the at receptor, and by enforcing the revisit of such publications which concluded at function by using pd as a specific at blocker. members of the g protein-coupled receptor (gpcr) superfamily are integral membrane proteins that are activated by extracellular ligands and induce cell signaling via g proteins and other adaptor proteins. rhodopsin, the prototypical gpcr that mediates vision, is activated by photons that isomerize its covalent ligand. spectroscopic analyses of the cognate agonist retinal allow a detailed description of rhodopsin dynamics at submillisecond resolution. using rhodopsin as a model, it has been demonstrated that receptor activation, i.e. a switch from the fully inactive to the fully active state, occurs within ms . activation kinetics of other receptors have been studied mainly using fluorescence resonance energy transfer (fret) which allows kinetic studies with high resolution in living cells . in contrast to rhodopsin, activation constants of several gpcrs using agonist superfusion have been determined to range between - ms . however, it is unknown if all gpcrs with diffusible ligands are really activated on a longer timescale or if ligand diffusion to the binding site is rate limiting. in this study, we intend to overcome ligand diffusion by using photodestruction of caged ligands. we monitor activation-related conformational changes of homodimeric metabotropic glutamate receptor (mglur ) sensors by fret after uncaging of an inert glutamate derivative. -methoxy- -nitroindolinyl-l-glutamate (mni-glutamate) is a caged derivative of glutamate that does not activate mglur s. upon pre-incubation of hek-tsa cells expressing both cfp-and yfp-tagged mglur protomers with mniglutamate, a short uv laser pulse releases active l-glutamate close to the receptor binding site. we demonstrate very rapid mglur activation kinetics and this allows us to study the process of signal transduction of this homodimeric gpcrs opioids are still the mainstay of modern pain treatment. most of the clinically established substances primarily exert their effects via the µ-opioid receptor (mor). however, many side effects such as tolerance, constipation and respiratory depression limit their therapeutic use. the efficacy of mor agonists in the treatment of chronic pain is unsatisfactory. in general analgesic effects can be mediated by all four members of the opioid receptor family. the nociception receptor (nop) is the latest member of the opioid receptor family. there is a rapidly growing interest for the development of novel nop and combined mor/nop agonists. the aim of this developement is novel therapeutic agents with improved analgesic characteristics and less classical mor-mediated side effects. here we used buprenorphine as a clinically established opioid which exerts its effect on multiple opioid receptor subtypes. recently, nalfurafine, a potent kappa-opioid receptor (kor) agonist was granted by japanese authorities for the treatment of uremic pruritus. even though kor agonists are known to mediate dysphoria and hallucinations this has not been reported for nalfurafine. rudolf-virchow-zentrum für experimentelle biomedizin, würzburg, germany g protein-coupled receptors (gpcrs) belong to a superfamily of cell surface signaling proteins that mediate many physiological responses to hormones and neurotransmitters. they represent the prime targets for therapeutic drugs in healthcare. however, due to the limited knowledge about the pharmacology of the majority of gpcrs, only few of them are employed as therapeutic target. in our lab, the activation kinetics of the α aadrenergic receptor, among others receptors, has been extensively studied in single cell assays ( ) ( ) . the activation kinetics of the labeled α a -adrenergic were monitored by förster resonance energy transfer (fret). the goal of our study is to design a sensor to monitor receptor activation kinetics in high throughput screening assays. for the proof of concept, we used the α a -adrenergic receptor as a prototype. to optimize the fret efficiency we exchanged the previous acceptor (yfp) with the halotag technology ( ) . additionally, we used halotag in combination with the nanoluc ( ) to explore the possibility of using bret as a high-throughput approach to monitor receptor activation kinetics. the fret-based sensor α a -halo/cfp showed an increase in fret upon application of the full endogenous agonist norepinephrine with an ec value in accordance with the previously published data. this suggests the functionality of the fret-based α a -halo/cfp sensor. similar results were obtained with the α a -adrenergic receptor bret-based sensor. in contrast to the full agonist norepinephrine, the inverse agonist, yohimbine, decreased the ratio in both, fret as well as bret-based α aadrenergic receptor sensors. this suggests the sensitivity of the methods to discriminate among agonist (increased ratio) and antagonist (decreased ratio) induced receptor kinetics. our results show the feasibility of using halotag to monitor receptor activation via fret in a single cells format and halotag, in combination with nanoluc, can be used to monitor receptor activation in high-throughput format. , c. hoffmann the homologous visual arrestins) that has recently been solved by x-ray crystallography. here we investigated both the interaction with gpcrs and β-arrestin conformational changes in real time and in living cells with a series of fret-based β-arrestin biosensors. upon stimulation, β -adrenergic receptors bound β-arrestin with a time constant τ = . ± . s, indicating that β-arrestin binding rapidly terminates their gprotein signaling. we observed a subsequent receptor-mediated conformational change in β-arrestin with a τ = . ± . s. stimulation of β -adrenergic vs. m muscarinic or ffa receptors resulted in different patterns of conformational changes in the various β-arrestin sensors and also in downstream kinase signaling, revealing receptor-specificity in β-arrestin activation. upon agonist removal, first the interaction (delay = . ± . s) and only then the active state of β-arrestin (delay = . ± . s) were reversed. accordingly, β-arrestin localization at the cell membrane lasted much longer than the direct interaction with β -adrenergic receptors. our data indicate a rapid, receptorspecific, two step binding and activation process between gpcrs and β-arrestins; they further suggest that β-arrestins remain active following dissociation from receptors, allowing them to remain at the cell surface and presumably signal independently. thus, gpcrs trigger a rapid, receptor-specific activation/deactivation cycle of β-arrestins, which permits their active signaling. patghogenic clostridium difficile produce two large glucosyltransferases, tcda and tcdb, which are the main pathogenicity factors. the cytotoxin tcdb is about , fold more potent than tcda. tcda and tcdb are a/b structure toxins exhibiting an enzymatically active (a) domain and a binding/translocation domain (b) to deliver the active glucosyltransferase domain into the cytosol of host cells. beside its glucosyltransferase activity by which the substrate proteins mainly of the family of rho gtpases are inhibited, tcdb has additional cytotoxic effects that are independent of rho glucosylation. to investigate the mechanism by which tcdb induces early cell death, we applied chimeras of tcdb from different toxinotypes where different glucosyltransferase domains were combined with different translocation domains. to this end we cloned tcdb from strain vpi (historical strain), strain (serotype f, variant tcdbf), strain r (hypervirulent strain, ribotype o ), and strain r (hypervirulent strain with tcdbf characteristics). we were able to investigate the impact of the glucosyltransferase domains with different substrate specificity when translocated into the host cell by identical translocation domains. furthermore, we tested different translocation domains to deliver the same glucosyltransferase domain into host cells. we found that the glucosyltransferase domain of tcdbf (strain ) is less cytotoxic with respect to early cell death mediated by reactive oxygen species than that from reference strain vpi . in addition, the translocation domain also showed significant impact on cytotoxicity, probably by faster intracellular delivery of the gtd. by using glucosyltransferase deficient mutants where the highly conserved dxd-motif was changed to nxn, we were able to show that glucosylation of rho gtpases counteracts the cytotoxic effect, since the mutants were more cytotoxic than wildtype toxins. in conclusion, the cytotoxicity of tcdb mainly depends on the translocation efficiency into the host cell and on the kinetic of glucosylation of their substrate gtpases. thus, sensitivity of target cells towards cytotoxic effect also depends on receptor abundancy and intracellular status of rho gtpases, whereas the cytopathic effect, i.e. cell rounding, is predominatly determined by the substrate specificity. introduction: p rhogef activates the g protein-coupled receptor (gpcr)-mediated induction of rhoa in different cells. however, its role in cardiac fibroblasts (cf) is not defined yet. thus we studied its localization and function in cf in d and d culture experiments. methods: neonatal rat cardiac fibroblasts (nrcf) and adult ventricular fibroblasts (amcf) from wild type mice and p rhogef-knockout mice were adenovirally transduced for to h with recombinant adenoviruses or directly used. for d studies the cells were treated with angiotensin ii (ang ii). the location of the involved signaling components, rhoa activation and down-stream effects were studied by confocal microscopy and biochemical analyses. in addition, cf were used to prepare cf-containing engineered connective (ect) or muscle (ehm) tissues. results: we could show that p rhogef locates at the plasma membrane, adjacent to the golgi apparatus and at the base of primary cilia. in accordance, p rhogef regulates in response to ang ii the expression and secretion of the connective tissue growth factor (ctgf) in nrcf involving the serum response factor. in ect, p rhogef increases the stiffness of these tissues and in ehm containing cf expressing gain-and-loss-of-function p rhogef variants it influences the contractility. interestingly, the increase in ect stiffness was independent of p rhogef's regulatory function of ctgf, as overexpression of ctgf in cf had no impact on ect properties arguing for a more general role of p rhogef in auto-and paracrine signaling. moreover, our data on amcf with a genetic deletion of p rhogef implies that p rhogef is not only a transducer of gpcr-dependent rhoa activation as its loss led to an increase in rhoa expression accompanied by an increase in rhoa-dependent gene expression suggesting a role of p rhogef in the feedback regulation of this signaling cascade. conclusion: in summary, our data show that p rhogef regulates auto-and paracrine signaling in cardiac fibroblasts. the atrophic rhinitis is characterized by a drastic destruction of nasal turbinate bones in different animals. it leads to shortening and twisting of the snout and a growth retardation of young pigs. this bone degradation is induced by pasteuralla multocida toxin (pmt), a toxin produced by p. multocida serogroups a and d. this destructive effect indicates an interaction of pmt with bone cells like osteoclasts and osteoblasts. we demonstrated that pmt stimulates the differentiation of osteoclasts and inhibits the differentiation of osteoblasts in a gq-dependent mechanism. the underlying molecular mechanism of the toxin is the deamidation of an essential glutamine residue in the αsubunits of heterotrimeric g proteins, which results in the constitutive activation of the g protein. until now only the function and the pmt-dependent effects on osteoblasts and osteoclasts were studied in detail, but there is also a third important cell type in bone, the osteocytes. osteocytes are discussed as the regulator of the bone turn-over by interacting with osteoclasts and osteoblasts e.g. via secretion of several osteoclastogenic and osteoblastogenic cytokines. therefore, we studied the effects of pmt on the function of osteocytes in more detail. we utilized an osteocyte like cell line and primary osteocytes isolated from tibiae and femurs from mice. the susceptibility of primary osteocytes and the osteocyte like cell line towards pmt was demonstrated by detection of toxin-induced deamidation of g proteins. we also observed a pmt-induced secretion of different cytokines, like rankl, il- and tnf-α, which are known to induce osteoclastogenesis or inhibit osteoblastogenesis. furthermore, we studied the underlying signal transduction pathways and other pmtinduced effects on osteocytes, like morphological changes. in summary, we show that pmt acts on osteocytes by stimulating heterotrimeric g proteins. this might have impact on overall bone metabolism due to modulation of osteoblast and osteoclast activity. pasteurella multocida is an opportunistic pathogen often residing in the nasal pharyngeal space of animals. one virulence factor of p. multocida serogroups a and d is the protein toxin pmt (p. multocida toxin), which is the causative agent of atrophic rhinitis characterized by degradation of nasal turbinate bones in pigs and other animals. on the molecular level, pmt activates distinct members (g q/ , g / and g i ,) of heterotrimeric g proteins leading to a modulation of bone metabolism: the toxin stimulates osteoclastogenesis but blocks osteoblastogenesis which results in bone loss. this mechanism of action of pmt might be exploited to counteract the human disease fibrodysplasia ossificans progressiva (fop), a rare and highly disabling disorder of extensive heterotopic bone growth. the underlying cause of fop is a point mutation in the activation domain of acvr (r h), a bmp (bone morphogenic protein) type receptor. this mutation leads to an inflated bmp-signaling and heterotopic osteoblastogenesis. here, we report that c c cells, a mouse myoblast cell line often used as a fop model, are susceptible to pmt intoxication. pmt induces deamidation of g proteins in these cells. furthermore, pmt very efficiently inhibits bmp -induced osteoblast differentiation in c c cells. this has been shown by measuring alkaline phosphatase expression which is an early marker of osteoblast differentiation. additionally, the impact of pmt on acvr r h induced osteoblastogenesis will be investigated and the involved cellular signaling pathways will be characterized in detail. the data indicate that activation of heterotrimeric g-proteins might be a rationale for pharmacological therapy of fop. p rhogef is an activator of the monomeric gtpase rhoa and was shown to be expressed in the heart. in cardiac fibroblasts and smooth muscle cells, p rhogef regulates rhoa in response to angiotensin ii and controls the actin cytoskeleton as well as protein expression and secretion. its role in cardiomyocytes, however, has not been elucidated so far. cardiomyocytes were isolated from neonatal rat hearts (nrcm), wild type mouse hearts (wt-amcm) and homozygous (ko-amcm) p rhogef knockout mouse hearts. the cells were either directly fixed or adenovirally transduced for h in culture. for activation of the g q/ -signaling the cells were treated with endothelin- (et- ), angiotensin ii (ang ii) or phenylephrine (pe) for s. rhoa activation was assessed by affinity binding or with a specific active-rhoa antibody. other proteins were detected by immunoblot or immunofluorescence analysis with subsequent confocal imaging. in nrcm p rhogef is involved in the regulation of the et- -induced rhoa activity and thus increases the expression and secretion of the rhoa target gene ctgf. in accordance, p rhogef was found to be localized at the sarcolemma as well as in intracellular membrane compartments. the strongest co-localization was detected with the kdel-receptor (kdelr ) which resides in the endoplasmatic reticulum membrane. next, we analyzed rhoa activation in wt and ko-amcm and could show that a loss of p rhogef led to an increase in basal rhoa activity and an uncoupling from the gpcrs. interestingly, in the ko-amcm caveolin- the major component of caveolae, in which several gpcrs are clustered, was reduced in its expression and a shift in localization from transverse to longitudinal membrane tubules was found, arguing for a role of p rhogef in intracellular protein transport. in accordance, golgi apparatus particles, which were demonstrated to play role in caveolae formation, were reduced in size in ko-amcm. to further address the role of p rhogef in the transport of membrane proteins, we overexpressed p rhogef in wt-amcm and could show that this led to an increase in the expression of the kdelr and its co-localization with p rhogef in the perinuclear region and at the sarcolemma. no sarcolemmal localization of kdelr was found in control-transduced cells. further, p rhogef was localized adjacent to golgi apparatus particles which were similar reduced in size as detected in the ko-amcm. finally, we expressed the dominant negative construct p dn and detected similar changes with respect to kdelr localization and golgi structure suggesting that this regulatory function of p rhogef is not dependent on its activity. conclusion: p rhogef mediates the activation of rhoa from gpcrs coupled to g q/ proteins. moreover, it has a function in intracellular transport and distribution of membrane proteins independent of its activity. universität bonn, pharmacology and toxicology section, bonn, germany g protein signaling is a means allowing cells to quickly respond and adapt to environmental changes. four major g protein classes (gs, gi/o, gq/ , g / ) exist in mammals and these must suffice to convey signals from about g protein-coupled receptors to the cell interior. as such, g proteins receive, interpret, and finally route the gpcr signals to diverse sets of downstream target proteins and thereby permit cells to respond to their ever changing environment. understanding contribution of individual g protein classes or even isoforms to complex signaling networks in living cells requires capacity to activate or inactivate proteins with great precision and selectivity. one approach towards inactivation of g protein function is via chemical inhibition. however, "true specificity" of chemical inhibitors for their associated targets may often be debated. in this study we posit that fr , a cyclic depsipeptide isolated from the leaves of ardisia crenata, may clearly be designated as "truly specific" for inhibition of gq signaling. using a broad set of complementary methods based on label-free holistic cell sensing, classical endpoint assays, and bioluminescence resonance energy transferbased g protein biosensors we assign exceptional selectivity to fr for inhibition of gq/ / over all other mammalian isoforms ("on-target effects"). in holistic label-free recordings using hek cells that lack functional gq/ alleles by crispr-cas genome editing, bona-fide gq stimuli were undetectable. however, reintroduction of gq into the knockout background was required and sufficient to fully restore both, agonist responses and their inhibition by fr. moreover, fr was completely ineffective in cells lacking gq/ using phenotypic assays that examine basic cellular functions such as cell growth, viability, morphology and expression of housekeeping genes ("off-target effects") . from these results we conclude that fr is of outstanding value as molecular probe to unravel contribution of gq signaling in complex biological processes in vitro, ex vivo and in vivo. just as pertussis toxin, applied world-wide by numerous laboratories to diagnose signaling of gi/o proteins, we anticipate fr to stand out at least equally for investigations into the biological relevance of gq. binary actin adp-ribosylating toxins like c. perfringens iota toxin and c. difficile transferase cdt cause depolymerisation of the cortical actin cytoskeleton, induce the formation of microtubule-based cell membrane protrusions and redirect rab-dependent intracellular traffic (schwan et al. ). here, we employed the model of toxin-induced protrusions to study the formation of cilia. we found that toxin-induced microtubule-based protrusion formation at the cell membrane depends on recruitment of septins, which are highly conserved, small gtpbinding proteins. similar to toxin-caused protrusions, septins are also recruited to the site of ciliogenesis. inhibition of septins by shrna-based knockdown inhibits ciliogenesis as well toxin-induced protrusion formation. septins are suggested to be involved in exocytotic processes, which are important for ciliogenesis and also for toxin-induced protrusion formation. accordingly, translocation of septins is accompanied by a recruitment of rab proteins and proteins of the exocytotic machinery. the data indicate that septins function as a scaffold at the base of cellular processes like cilia and toxin-induced protrusions, organizing the cross-talk between the actin cytoskeleton and microtubules to regulate the vesicle traffic-and exocytotic machinery. hypervirulent clostridium difficile strains are associated with increased morbidity and mortality. these strains produce the actin-adp-ribosylating clostridium difficile toxin cdt. cdt depolymerizes the actin cytoskeleton, causes formation of microtubule-based protrusions and increases pathogen adherence. septins are essential for cdt-induced protrusion formation. sept , , and accumulate at predetermined protrusion sites and form collar-like structures at the base of protrusions. the septins are a prerequisite for protrusion formation. the inhibitor forchlorfenuron or knock-down of septins inhibit protrusion formation. septins colocalize with active cdc and its effector borg which act as up-stream regulators of septin polymerization. microtubules interact with septin structures. precipitation and surface plasmon resonance studies revealed high-affinity binding of septins to microtubule plus end tracking protein eb thereby guiding incoming microtubules. the data indicate that cdt hijacks conserved regulatory principles involved in microtubule-membrane interaction, depending on septins, cdc , borgs and restructuring of the actin cytoskeleton. the zebrafish danio rerio has become an important vertebrate model organism for a wide range of scientific questions [ ] . current studies are mainly focused on development, genetics and disease for which the zebrafish is particularly well suited due to its small size, rapid development, short generation time, optical transparency of embryos and larvae as well as conservation in functional domains [ ] . hitherto, nothing is known about the composition and endogenous level of different cnmps in various developmental stages and organs of danio rerio. therefore, we used the zebrafish in our study as a vertebrate model to characterize systematically the temporal-and organspecific occurrence(s) of all cnmps including cump in vivo. cyclic nucleotides were quantified by high performance liquid chromatography quadrupole tandem mass spectrometry. we observed specific cnmp patterns in developmental stages and different organs from adult zebrafish, which is in support of the hypothesis of a distinct cnmp signaling code [ ] . camp, cgmp and cump were present in tissue samples of both developmental stages (embryos at hours post fertilization, larvae at days post fertilization) and within all harvested organs. remarkably, these three cnmps were the only ones detected in the brain. camp concentration of entrails as well as camp and cgmp concentrations in the brain were similar to those previously described in mouse tissues [ ] . ccmp was detected throughout development and was present in all organs except the brain. the identity of ccmp and cump in the zebrafish was confirmed by high performance liquid chromatography quadrupole time-of-flight mass spectrometry (hplc-ms/tof). thus, we unequivocally show for the first time that cump occurs in vertebrates. furthermore, we detected cimp in several developmental stages of the zebrafish, and observed the highest concentrations in testes and heart, but we were unable to unequivocally identify cimp via hplc-ms/tof. in the zebrafish, sac is evolutionarily not conserved and absent, since a search in the ncbi gene data base revealed no entry for sac (also referred to as ac ). therefore, sac can be excluded as cump-and ccmp generator in this system and sgc remains as the only bona fide cump-and ccmp generator in the zebrafish. to test this hypothesis, the effects of no donors, sgc stimulators and sgc activators on cump levels in zebrafish should be examined in future studies. recently, induction of apoptosis in mouse lymphoma cells by ccmp-am has been described [ ] . thus, it would be interesting to examine the effect of ccmp-am on zebrafish embryos in future studies as well. [ ] seifert, r.: ccmp and cump: emerging second messengers, trends in biochemical sciences , - ( ) . ccmp, ', '-cump and ', '-cimp were ineffective. to further characterize the action of ', '-cgmp on hut- cells, we determined apoptosis (propidium iodide/annexin v staining) and proliferation (carboxyfluorescein succinimidylester staining). ', '-cgmp significantly increased apoptosis (ec = µm) and inhibited proliferation (ec = µm) of okt -activated hut- cells. interestingly, also ', '-cgmp exhibited comparable effects on apoptosis and proliferation with ec values of µm and µm, respectively, while ', '-camp, ', '-ccmp and ', '-cump were ineffective. this indicates that the pro-apoptotic and antiproliferative action of cgmp does not depend on the position of the phosphodiester bond. we also tested ', '-cgmp degradation products under the same experimental conditions and found that both '-gmp and guanosine increased apoptosis and inhibited proliferation with ec -values between and µm. by contrast, adenosine did not influence cell growth and viability, suggesting that adenosine receptors are not involved in the observed effects. our results suggest that the guanosine moiety is responsible for the pro-apoptotic and antiproliferative effects of ', '-cgmp, ', '-cgmp, '-gmp. it has been reported earlier that guanosine is toxic to jurkat cells, another t cell lymphoma cell line [ ]. ', '-cgmp may be hydrolyzed by an ekto-phosphodiesterase on the cell surface of hut- cells (e.g. enpp ), yielding '-gmp, which could be further degraded to guanosine by the '-ekto-nukleotidase cd . a similar pathway may lead from ', '-cgmp to guanosine. a previous analysis of phosphodiesterases (pdes) revealed that the dual-specific pde isoforms a and b as well as the cgmp-selective pde a also degrade the emerging second messenger cump [ , ] . we analyzed the enzyme kinetics of pde b-mediated cump-hydrolysis using recombinant gst-tagged pde b and a highly sensitive and specific hplc-ms/ms method. our data show that pde b is a low-affinity enzyme for cump with a cump k m -value of > µm. the pde -selective competitive inhibitor milrinone inhibited pde b-mediated cump degradation, suggesting that cump binds to the catalytic center. pde b is highly expressed in adipose tissue [ , ] . thus, we differentiated murine t -l -mbx-fibroblasts into adipocytes and analyzed differentiation-dependent alterations of pde b expression and basal cnmp-concentrations. in both differentiated and undifferentiated t -l cells cump and ccmp were detected in addition to the established second messengers camp and cgmp. differentiation to adipocytes reduced camp and cgmp by % and %, respectively, while ccmp was reduced by % and cump even by %. these findings suggest that cump plays a distinct role in adipocyte differentiation. the cump-hydrolyzing pde b was upregulated ~ -fold on mrna level after adipocyte differentiation, which may contribute to the observed reduction of basal cump concentrations. we currently investigate the potential biological role of cump in differentiation and lipolysis experiments, analyzing the effects of the membrane-permeant cumpacetoxymethyl ester cump-am. in future experiments, we will also analyze the enzyme kinetics of pde a-mediated cump hydrolysis. pde a is the first example of a cgmp-"specific" cump-hydrolyzing pde. background: cgmp and camp are cyclic nucleotide messengers relevant to many physiological and pathophysiological conditions. live-cell imaging with fret-based biosensors is a powerful method to study the spatiotemporal dynamics of cgmp and camp under close-to-native conditions. however, with the existing biosensors it is difficult to resolve potential membrane-associated cgmp microdomains and to monitor cgmp and camp signals in parallel in the same cell. we have generated novel versions of the "green" cfp/yfp-based cytosolic cgmp biosensor, cgi . they comprise a "green" membrane-targeted version (mcgi ) and a "red" variant (red cgi ) that contains the fluorophores tsapphire and dimer . methods: the sensors were expressed and characterised in primary vascular smooth muscle cells (vsmcs). intracellular cgmp was elevated in intact vsmcs by application of a nitric oxide donor or natriuretic peptides, and the sensor's sensitivity to each stimulation and its signal-to-noise ratio were determined. to test each sensor's sensitivity and specificity for cgmp versus camp, sensor-expressing cells were permeabilised with β-escin and exposed to defined concentrations of cyclic nucleotides. results: the original cgi sensor showed a good signal-to-noise ratio, an ec value of ≈ . µm for cgmp, and a high selectivity for cgmp over camp (> -fold). flincg , a non-fret-based cgmp sensor, showed similar properties as cgi . the new membrane-targeted mcgi and the new red cgi displayed ec values of ≈ . µm cgmp and a high selectivity for cgmp over camp (> -fold). in vsmcs, the red cgi showed a better signal-to-noise ratio than the previously described "red" cgmp sensor, red cges-de . the "green" fret-based camp sensor, epac -camps, showed a signal-to-noise ratio comparable to that of cgi , an ec value of ≈ µm for camp and a selectivity for camp over cgmp of ≈ -fold. finally, imaging of cells expressing both the epac -camps and the red cgi demonstrated the feasibility of combined visualisation of camp and cgmp signals in the same cell. the new cgmp biosensors should be useful for a broad spectrum of applications requiring real-time monitoring of cgmp signals. for example, mcgi would be useful to investigate membrane-associated cgmp compartments and red cgi to study the crosstalk between cgmp and camp signalling in living cells and tissues. the cgmp-system is a major regulator of blood pressure. cgmp-dependent protein kinases (cgks), located in the smooth muscle layer of vessels, enable cells to dilate and therefore cause a decrease in blood pressure (bp). to the contrary, the reninangiotensin-aldosteron-system (raas) acts as opponent and causes an increase in bp; furthermore, it influences fluid-electrolyte balance. renin, which is secreted from reninproducing cells located in the juxtaglomerular apparatus (jga), is the key regulator enzyme in this system. pharmacological inhibition of the raas, e.g. via ace-inhibitors or at -receptor-antagonists, is a powerful tool to treat hypertension, but chronically challenges this endocrine system, resulting in an enhancement of renin expression. this is caused by an increased number of renin-expressing cells (the so-called reninrecruitment), which derive from a reversible metaplastic retransformation of extraglomerular and smooth muscle cells of afferent arterioles. next to regulation of renin-function via camp/pka, it has been shown that enos-derived no supports this recruitment via activation of sgc and subsequent generation of cgmp [ ] . whether this causes an activation of cgks is not known. these enzymes exist in different isoforms, cgkiα, cgkiβ und cgkii. in contrast to the β-isoform, cgkiα (as well as cgkii) is highly expressed in the jga [ ] , [ ] . therefore, we analyzed, whether cgkiα also plays a role regarding renin synthesis, secretion or recruitment. to characterize the function of cgkiα in jga-cells, we generated renin-cell specific cgkiα-knockout mice (ren cre-cgki fl/fl) and stimulated renin recruitment via administration of a low salt diet ( . % na + ) and enalapril ( mg/kg/d) for weeks. we analyzed blood pressure, mrna and renal protein content of renin and cgkiα, plasma renin activity and renin recruitment. furthermore, we activated the cgmp-system in these mice using bay - , a sgcstimulator, and re-analyzed the above-mentioned parameters. our results indicate that cgkiα could be an additional system supporting renin recruitment but is not a crucial pre-requisite. in contrast, the basal renin concentration and activity appears to be downregulated in ren cre-cgki fl/fl-mice, thus, cgki could be an important regulator of renin synthesis. universität regensburg, pharmakologie und toxikologie, regensburg, germany jaw /lrmp (lymphoid-restricted membrane protein) is a type membrane protein, localised to the cytoplasmic face of the endoplasmic reticulum. it encodes a amino acid protein with a highly conserved coiled-coil domain in the middle third of the protein and a cooh-terminal transmembrane domain [ ] , [ ] . jaw and irag have a limited homology throughout the length of the protein. the coiled-coil domain and the putative transmembrane anchor at the c-terminus of jaw and irag share the highest homology [ ] , [ ] . the coiled coil domains of irag and jaw are important for the interaction with ip rs. as already known, irag forms a trimeric complex with cgkiβ and ip r and gets phosphorylated by cgkiβ [ ] . hence we examined if jaw is a new target protein of cgkiβ. the recognition site, where a substrate can be phosphorylated by cgki, is composed of the following amino acids: (k/r)(k/r)x(s/t). in the amino acid sequence of jaw possible phosphorylation sites can be found. our in vitro studies with jaw and cgki showed that jaw gets phosphorylated in a cgmp-dependent manner by cgkiβ. in contrast, jaw was not phosphorylated by cgkiα. furthermore, no stable interaction between jaw and cgkiβ was detected. to examine the importance of jaw in vivo, we generated a conditional knockout mouse. mating with a cmv cre mouse, resulted in an ubiquitous deletion of jaw . mrna analysis and western blot analysis approved the deletion. the expression pattern revealed high expression in the thymus and weaker expression in the lung, spleen, colon, pancreas and the tongue. as already published by shindo et al., jaw was found in sweet, bitter, and umami taste receptor-expressing cells of mouse circumvallate, foliate, and fungiform papillae. we confirmed these results by x-gal staining and mrna analysis. therefore, we decided to analyse if jaw influences taste perception. two bottle preference tests did not result in significant differences between wildtype and knockout mice, indicating that taste perception is not altered by jaw . hence, the function of jaw in taste receptor expressing cells has to be further examined in future studies. the cyclic purine nucleotides adenosine ', '-cyclic monophosphate (camp) and guanosine ', ' cyclic monophosphate (cgmp) are well-characterized second messengers. both are generated by nucleotidyl cyclases and degraded by phosphodiesterases. several binding partners of camp and cgmp were already identified and functionally analyzed, e.g. camp-dependent protein kinase (pka) and cgmp-dependent protein kinase (pkg) as well as exchange protein activated by camp and , hyperpolarization-activated cyclic nucleotide gated channels and phosphodiesterases. recent data indicate that the cyclic pyrimidine nucleotides cytosine ', '-cyclic monophosphate (ccmp) and uridine ', '-cyclic monophosphate (cump) also fulfill the criteria of second messengers [ , ] . the interaction of ccmp with the regulatory subunits of pka (pkariα and pkariiα) has already been shown by using ccmpagarose [ ] . additional ccmp-and cump-binding proteins such as calnexin (chaperone), myomegalin (phosphodiesterase-interacting protein) and akap (a-kinase anchoring protein) were identified by mass spectrometry analysis. to verify the interaction of ccmp and cump with these potential target proteins, ccmp and cump linked to biotin were used as another approach. the biotin-constructs exhibit lower steric interference than the ccmp-and cump-agarose matrices, which were previously used to confirm the binding of pkariα to ccmp and cump [ ] . flag-tagged calnexin, flag-tagged myomegalin and myc-tagged yotiao (smallest splice-variant of akap ) were examined as potential ccmp and cump target proteins. hek cells were transiently transfected with the cdna of the respective proteins. the lysates of the protein-overexpressing cells were then incubated with ccmp-and cumpbiotin matrices and bound proteins were purified using strep-tactin® beads (iba). afterwards, the interaction of ccmp and cump with the potential binding partners was analyzed by western-blotting. a pkariα antibody was used as a positive control. analogous experiments were also performed using ccmp-and cump-agaroses. once the interaction between the cyclic pyrimidine nucleotides and the potential binding partners has been confirmed, deletion mutants will be cloned to localize the ccmp-and cump-binding area of the target proteins in further studies. axonal branching is essential for the correct formation of neuronal circuits and enables the simultaneous transmission of information throughout the body. in mice, the bifurcation of axons of sensory neurons at the dorsal root entry zone of the developing spinal cord depends on a cgmp signaling cascade that includes c-type natriuretic peptide (cnp), natriuretic peptide receptor (npr , also termed gc-b), and cgmpdependent protein kinase iα. in this study it was investigated, if a disturbance of cgmp signaling induced by manipulation of cgmp breakdown or cnp scavenging affects axon bifurcation of murine embryonic dorsal root ganglion (drg) neurons. rt-pcr screens, in situ hybridization, and fret-based cgmp imaging in living neurons revealed phosphodiesterase a (pde a) as the major enzyme for degradation of cnp-induced cgmp in embryonic drg neurons. interestingly, cgmp measurements and dii labeling of pde a knockout embryos indicated that a strongly elevated concentration of cgmp does not impair sensory axon bifurcation of drg neurons in vivo. the natriuretic peptide receptor (npr ) was found to be expressed in the roof and floor plate of the spinal cord as well as in the dorsal roots of e . embryos. because npr binds natriuretic peptides, but does not generate cgmp, it is thought to act as a natriuretic peptide clearance receptor. by scavenging cnp, npr could lower the activity of the cnp-npr -cgmp signaling cascade in drg neurons. in the absence of npr , the majority of sensory axons showed normal bifurcation, but » % of the axons turned only in rostral or caudal direction. this study shows ( ) that pde a is important for the degradation of cgmp in embryonic drg neurons, ( ) that the bifurcation of sensory axons in the spinal cord can tolerate high levels of intracellular cgmp in the absence of pde a, and ( ) in the central nervous system, no-dependent cgmp signalling is associated with many different developmental processes and brain functions, and plays an important role in memory consolidation and cognition. to analyse cgmp signals in primary cells, a knock-in mouse was generated which stably and ubiquitously expresses a fret-based cgmp indicator (cgi ). cultured cortical and hippocampal neurons were found to respond to exogenously applied no (gsno). in these cell types, endogenous no is mainly generated by the neuronal no synthase (nnos) isoform which requires a rise in intracellular calcium for activation of the no/cgmp-signalling cascade. here, we show that ampa-type ionotropic glutamate receptors were capable to induce cgmp response in cultured cortical and hippocampal neurons. surprisingly, amparinduced cgmp signals were independent of nmdar activation, as inhibition of nmdars with the nmdar antagonist d-apv (d- -amino- -phosphonopentanoic acid) did not block ampar-induced cgmp response. however, cgmp accumulation depends on no synthase activation as the nos inhibitor l-nna (ng-nitro-l-arginine) completely abolished cgmp accumulation. whether ampar-induced nos activation depends on calcium influx via calcium permeable ampars, vgccs (voltage gated calcium channels) or calcium release from intracellular stores will further be investigated in detail. cyclic adenosine monophosphate (camp) is an important and ubiquitous cellular second messenger. a dogma in signaling is that camp is distributed homogenously in the cell and that its concentration changes equally upon stimulation. in contrast, a large body of evidence suggests the existence of concentration gradients (so-called microdomains) of camp. in this regard, phosphodiesterases (pdes), the only enzymes which can degrade camp, have been suspected to be responsible for maintaining those gradients. however, how pdes establish camp gradients is entirely unknown. here, we measure local camp levels in hek cells and cytosolic fractions thereof using the camp fretsensor epac -camps fused to a phosphodiesterase (pde a ). we demonstrate the existence of low camp concentrations in close proximity to pdes and show that this gradient is maintained by pde hydrolytic activity. further we establish that camp gradients cannot be maintained solely on the basis of pde activity as the camp turnover is very slow. we provide evidence that pdes are structurally organized in yet unspecified 'microstructures' in which camp diffusion must be considerably slowed down. taken together, we suggest that camp gradients are established by pde hydrolytic activity in cellular regions with slow diffusion of camp. our study sheds light on the organization and maintenance of signaling compartments in cells. the influence of pde hydrolytic activity on camp gradients universität würzburg, pharmakologie und toxikologie, würzburg, germany phosphodiesterases (pdes) are a family of enzymes that degrade cyclic amp (camp) and cyclic gmp (cgmp) to their respective monophosphates. although several pdes have been shown to play an important role in a wide variety of physiological and pathological processes, their complexity and function in cell signalling is only beginning to be understood. it is especially astonishing that eukaryotes express more than different pde isoforms while their single function is to degrade camp, cgmp or both. in recent years, a large body of evidence has suggested that pdes (especially isoforms of the pde families , and ) are key players in establishing signalling compartments. these so-called microdomains are yet unspecified regions in cells where the concentrations of camp and cgmp are higher or lower than in the bulk cytosol. in a companion abstract (bock & lohse) we provide evidence that camp nanodomains, i.e. regions of low camp concentrations, exist in cells in the direct vicinity of pde . however, the mechanisms by which pdes establish and maintain camp gradients are largely unknown. here we study if establishing camp gradients is a general role of pdes and, moreover, if the size of camp nanodomains mainly depends on pde hydrolytic activity. by fusing an ultra-fast pde a (v max = µmol/min/mg) to a camp fret sensor (epac -camps) we monitor camp concentrations in direct vicinity of pde a . in comparison to pde , we show that pde a , albeit displaying a high camp turnover, only establishes a small camp gradient in both cytosol preparations of transfected hek cells and in living cells. interestingly, this gradient can be increased by deleting the nterminal regulatory domains while maintaining fast camp turnover. biochemical mapping of the camp gradient gives an estimate of the size of nanodomains. taken together, our data suggest that establishing camp gradients does not exclusively depend on pde hydrolytic activity. arglabin is a plant-derived sesquiterpene lactone used for cancer therapy in kazakhstan and russia. signaling pathways targeted by arglabin are poorly understood. we have isolated arglabin by using high performance liquid chromatography from a methanolic extract of artemisia glabella, a plant endemic in kazakhstan. mass spectrometric analyses confirmed the chemical structure and the purity of the isolated arglabin. in j macrophages, arglabin strongly induced accumulation of lc type ii protein in the absence of inflammasome activators, and also in cells activated with lps and cholesterol crystals. in addition, arglabin induced clustering of lc -ii at autophagosomal membranes, as evidenced from its punctuated pattern in confocal microscopic images of arglabin-treated macrophages, which is a characteristic sign for autophagy. since autophagy activation leads to increased degradation of nlrp and pro-interleukin (il)- β, we further analyzed whether arglabin inhibits the nlrp inflammasome. arglabin reduced expression of nlrp and pro-il- β, inhibited activation of caspase- , and release of mature il- β by lps-and cholesterol-crystal-stimulated macrophages consistent with inhibition of the nlrp inflammasome. intraperitoneal injection of arglabin into female apoe .ki mice fed a high fat diet resulted in significantly decreased plasma levels of proinflammatory il- β. moreover, arglabin markedly reduced mean lesion areas in the sinus and whole aorta in mice. thus, arglabin may represent a new promising drug to treat diseases associated with inflammasome activation, e.g. atherosclerosis. this work was supported in part by a grant from the nouvelle société francophone d'athérosclérose (nsfa). recently, we found that activation of the proteinase-activated receptor (par) stimulates renin release in the isolated perfused kidney model. therefore, we determined in the current experiments the response of plasma renin concentration (prc) to acute intraperitoneal administration of the par activating peptide sligrl ( µg/kg), hydralazine ( mg/kg), isoproterenol ( mg/kg), losartan ( mg/kg) and furosemide ( mg/kg) in conscious wild-type (wt) and par -deficient mice. prc was measured in plasma obtained by tail vein puncture. renal renin expression was determined by quantitative rt-pcr. renal protein expression was measured by immunohistochemistry. on a control diet ( . % nacl), plasma renin concentration (in ng angiotensin i per ml per hour) was significantly lower in par -deficient mice than in wild-type mice ( ± versus ± ). renin mrna expression was ± % of wt. renin-expressing cells were located at the juxtaglomerular position and renal renin protein expression was lower in par -deficient mice. as measured by tail-cuff method, systolic blood pressure was not different between par (-/-) and wt mice. administration of sligrl increased renin secretion about -fold (p< . ). acute stimulation of renin release by furosemide, isoproterenol, losartan and hydralazine caused significant increases of plasma renin concentration in both par (-/-) and wt mice. the absolute changes (delta prc) were similar ( ± , ± , ± , ± in wt, and ± , ± , ± , ± in par (-/-)). in conclusion, chronic absence of par reduces basal renin expression and renin release. however, par -deficiency does not alter renin release in response to typical stimuli for renin secretion. therefore, par does not appear to be a mandatory and specific requirement for acute regulatory responsiveness. increased myofilament ca + sensitivity could be the underlying cause of diastolic dysfunction. we evaluated acute effects of epigallocatechin- -gallate (egcg), which has been shown to decrease myofilament ca + sensitivity, on cardiac myocyte contractility and force-ca + relationship of skinned cardiac muscle strips in an hcm mouse model with left ventricular hypertrophy and both systolic and diastolic dysfunction. methods: the hcm mouse model used in this study carries a point mutation in the cardiac myosin-binding protein c gene at the homozygous state (mybpc -targeted knock-in; ki). we isolated ventricular myocytes from adult ki and wt mice and analyzed sarcomere shortening and ca + transients at °c under hz pacing using the ionoptix system in the absence or presence of egcg ( . µm). furthermore, force-ca + relationships of skinned cardiac muscle strips of ki and wt mice were obtained ±egcg ( µm). results: in baseline settings and absence of fura- , ki cardiomyocytes displayed higher sarcomere shortening ( type- serine/threonine protein phosphatase (pp ) comprises a family of enzymes that dephosphorylate cardiac regulatory proteins, thereby modulating ca + handling and contractility. all pp heterodimers possess a catalytic subunit, which is selectively inhibited by inhibitor (i- ). it has been shown by our group that the heart-directed overexpression of a truncated, constitutively active form of i- resulted in an improved basal ca + handling and contractility. in contrast, chronic pressure overload by transverse aortic constriction exacerbated the progression of cardiac remodeling and heart failure in transgenic mice. in the present study, we tested whether the overexpression of a full-length form of i- , regulated by gsk -dependent phosphorylation at thr , resulted in comparable functional alterations using a model of induced heart failure. for this purpose transgenic (tg) and wild-type (wt) mice were subjected to chronic application of isoprenaline (iso, mg/kg/d) via osmotic minipumps. iso-stimulated mice were compared to mice treated with . % nacl (n= ). after one week of iso administration, cardiac hypertrophy was comparable in tg and wt. ca + transients were measured in isolated, indo- -loaded myocytes. the peak amplitude of [ca] i was reduced by % in tg nacl compared to wt nacl (p< . ), whereas chronic iso application was associated with comparable effects in tg and wt. [ca] i decay kinetics were comparable in nacl-treated groups but hastened by % in tg iso compared to wt iso (p< . ). consistently, sr ca + load was diminished by % in tg nacl compared to wt nacl (p< . ). chronic iso stimulation led to an unchanged sr ca + content in tg and wt myocytes. biochemical analyses revealed that chronic βadrenergic stimulation was accompanied by a more than -fold higher phospholamban phosphorylation at ser in tg (p< . ). thus, these findings suggest that overexpression of i- is able to reduce the progression of heart failure by an improvement of myocyte ca + handling. the ligand-activated farnesoid x receptor (fxr) is a nuclear receptor highly expressed in gastrointestinal and metabolic tissues, such as the duodenum, jejunum, ileum, colon and the liver, but also in lower amounts for instance in macrophages. the endogenous agonists for this receptor are bile acids with the primary bile acid chenodeoxycholic acid as the most active one. activation of fxr regulates the transcription of target genes relevant in bile acid homeostasis, glucose and lipid metabolism, liver protection, inflammation, and cancerogenesis. agonists for fxr have been discussed as possible therapeutic options for the treatment of obesity and the metabolic syndrome. atherosclerosis is the main pathology underlying cardiovasular diseases and often occurs side-by-side with the metabolic syndrome. cholesterol deposition and the formation of cholesterol-loaded foam cells from macrophages lead to the formation of atherosclerotic plaques. this can be prevented by stimulation of cholesterol efflux from macrophages. based on leoligin, a lignan-type secondary plant metabolite, naturally occuring in leontopodium alpinum cass., derivatives were synthesized and subjected to a fxr pharmacophore-based in silico screening. testing of virtual hits in a luciferase-based fxr transactivation assay yielded one compound with promising activity on fxr. moreover, the heterodimer partner of fxr, rxrα, was not activated by this leoligin derivative in a luciferase-based rxrα assay. in addition, this compound was able to increase cholesterol efflux in thp- macrophages without affecting cell viability. western blot experiments revealed an increase in atp-binding cassette transporter a (abca ) expression in human thp- macrophages by this leoligin derivative. the transporters abcg and scavenger receptor class b (sr-bi), which also play a key role in macrophage cholesterol efflux, will be investigated. moreover, the effect of the leoligin derivative on liver x receptor activation, the nuclear receptor responsible for upregulation of these transporters, has to be studied. based on this data, further characterization of the molecular mechanism underlying the described effects will provide valuable insights in a possible crosstalk between macrophage cholesterol efflux and fxr activation. background: rho-associated kinases rock and rock are serine/threonine kinases that are downstream targets of the small gtpases rhoa, rhob, and rhoc. rock and rock are known to play a pivotal role in the pathogenesis of myocardial fibrosis. however, their specific function in cardiac fibroblasts (cf) remains unclear. remodelling of the diseased heart results in the transition of fibroblasts to a myofibroblast phenotype exemplified by an increased proliferation, migration rate and synthesis of extracellular matrix (ecm) proteins. therefore, we sought to investigate whether rock protein signalling intermediates have an impact on cellular characteristics, intracellular protein expression and mechanical properties in cf and engineered tissues. methods: neonatal cardiac fibroblasts were isolated from wild type rats and downregulation of rock and rock by % was achieved by lentiviral transduction or transfection. wild type fibroblasts were treated with μm fasudil or µm h p for general rock inhibition and µm slx- for inhibition of rock . protein expression and modification was determined by immunoblot analysis, gene expression by qpcr analysis, cf morphology and the localisation of cytoskeletal proteins by immunofluorescence analysis, cell proliferation by automated nuclei counting, cell migration on a planar surface by life cell imaging, and rigidity of engineered tissues by rheological measurement. results: our results show that both rock and rock influence cf morphology, gene expression, proliferation and migration. the knockdown and inhibition of rocks was associated with changes in cf morphology accompanied by a disorganization of higher-order actin structures including stress fibers and geodesic domes. moreover, the knockdown of rock and rock in cf increased adhesion velocity, whereas proliferation was attenuated. interestingly, downregulation of rock , but not of rock led to a significantly decreased migration velocity and distance suggesting an isolated principle role for rock in cardiac fibroblast migratory behavior. analysis of a three dimensional engineered tissue model composed of cardiac fibroblasts (engineered connective tissue, ect) suggested that rocks are involved in the regulation and turnover of the extracellular matrix (ecm) and thus influence viscoelastic properties of engineered tissues. destructive tensile strength measurement in ect treated with rock inhibitors showed that rigidity was significantly reduced when compared to control tissues. rna sequencing of ect treated with the rock inhibitor h p and qpcr analysis of cf with a downregulation of rock and rock showed that both rocks are involved in the regulation of ecm proteins, such as collagens a , a, and a , biglycan, decorin, elastin and its respective degrading enzyme mmp . conclusion: this study demonstrates that rock signalling controls myofibroblast characteristics of cf via remodelling of the cytoskeleton and the ecm. background: regulation and fine-tuning of gene transcription in cardiomyocytes (cms) is a centerpiece of cardiac development, function, and disease. in order to obtain authentic data, cell type-specific analyses are indispensable. recently, high-purity isolation protocols for cm nuclei were established [bergmann, exp cell res, ] and employed for detailed genetic and epigenetic studies on cardiac gene transcription [gilsbach, nat commun, ] . however, corresponding protein analyses, which bridge from transcriptional control to cm function are still lacking. therefore, we aimed to map the landscape of nuclear protein expression in newborn and adult mice in order to complement and extend our epigenetic studies. methods and results: cardiac nuclei were isolated from homogenized adult and p mouse frozen hearts by sucrose gradient centrifugation. magnetic-assisted cell sorting (macs) with pcm- as a nucleus-specific marker was used to enrich cm nuclei to > %. proteins were extracted from nuclear lysate with % sds. quantitative protein data were obtained from silac-based liquid chromatographytandem mass spectrometry (lc-ms/ms) experiments with an ltq orbitrap xl mass spectrometer after in-gel digestion with trypsin. nuclear protein extracts from murine cell lines served as silac (lys /arg )-labeled internal standard. finally, protein data are correlated with corresponding mrna data obtained by rna-sequencing. we identified proteins, of which are annotated to the nucleus. %/ % (adult / p ) of nuclear proteins are annotated as dna-binding. . %/ . % belong to transcription factor complexes; . %/ . % are able to bind transcription factors. . %/ . % have chromatin modification functions; . %/ . % modify histones. nuclearenriched go terms include mrna processing and transport, transcription, nucleosome assembly and protein degradation. % of proteins are shared between p and adult nuclei. proteins are exclusive to p , are only found in adult. proteins are enriched ( . -fold increase in abundance) in p hearts, proteins in adult proteins related to heart development, gene silencing, and dna replication are more prominent in or exclusive to newborn mice. adult nuclei strongly express proteins related to regulation of actin fibers and cm function, proteins involved in protein degradation, and chaperones. although high mrna expression increases the chance of protein identification, a significant correlation between mrna and protein level could not be observed on a genome-wide scale. conclusions: we present a comprehensive and specific protein landscape of newborn and adult cm nuclei. young cm nuclei appear as a developing tissue, show the ability for proliferation, and indicate ongoing alterations in gene expression. adult cm nuclei prominently display a focus on regulation of contractile fibers and cm function, as well as chaperones and proteasomal proteins indicative of its arduous function. background: fibrosis is a hallmark of many myocardial pathologies and contributes to distorted organ architecture and function. recent studies have identified premature senescence as regulatory mechanism of tissue fibrosis. however, its relevance in the heart remains to be established. objective: to investigate the role of premature senescence in myocardial fibrosis. methods: murine models of cardiac disease and human heart biopsies were analyzed for characteristics of premature senescence and fibrosis. results: senescence markers p cip /waf , senescence-associated ß-galactosidase (sa-ß-gal) and p ink a were increased -, -and -fold (n= - ; p < . ), respectively, in perivascular fibrotic areas after transverse aortic constriction (tac) when compared to sham-treated controls. similar results were observed with cardiomyocytespecific β -adrenoceptor transgenic mice and human heart biopsies. senescent cells were positive for vimentin ( ± . %), platelet derived growth factor receptor α ( ± . %) and α-smooth muscle actin ( ± . %), specifying myofibroblasts as the predominant cell population undergoing premature senescence in the heart. conclusion: our data provide first evidence for an essential role of premature senescence of myofibroblasts in myocardial fibrosis. it is tempting to speculate that pharmacologic modulation of premature senescence might provide a novel therapeutic target for anti-fibrotic therapies in the heart. introduction: the endocannabinoid system is increasingly studied in cardiac research due to its role in fibrosis, inflammation and cell fate modulation. the deregulation of this system has been implicated in myocardial infarction (mi) and consequent heart failure development. a recent study suggests cannabinoid receptor (cb ) inhibition to improve cardiac function and to reduce adverse remodeling after cardiac stress, but the exact underlying molecular mechanisms of these beneficial effects are still unknown. micrornas (mirnas, mirs) provide a complex layer of post-transcriptional regulation modulating key biological processes such as tissue remodelling in heart failure. the aim of the present study was to explore microrna pathways in the chronic effect of cb receptor inhibition after angiotensin-fibrosis induction and left ventricular remodeling. methods and results: adverse cardiac remodelling was induced in mice by chronic administration of angiotensin ii (angii, , mg/kg/day) with osmotic minipumps for days. treatment with cb antagonist, or vehicle was performed every second day during the angii administration period. hemodynamic parameters were measured by echocardiography and cardiac pressure volume catheter and tissue samples were taken for molecular and histological analysis. after two weeks of angii infusion, left ventricular dysfunction was prevented by cb antagonist treatment. this was shown by significantly improvements of the myocardial performance index and end-diastolic pressure values. at the tissue level, anti-fibrotic effects of cb antagonist treatment was confirmed histologically and by expression analysis of pro-fibrotic genes. these beneficial effects were also observed in cb ko mice and in an aging mice model. the particular role of tissue fibroblast in aii-induced cardiac fibrosis was further explored. primary cardiac fibroblasts (cf) from each experimental group were isolated and analysed by next generation deep rna sequencing to identify differentially regulated micrornas. microrna- a/b family was downregulated by in vivo angii delivery and vice versa upregulated after cb antagonist treatment and foxb (a direct target of mir- a family) was differentially regulated, suggesting a possible mechanism of action for the benefits of cb receptor inhibition. conclusion: we found that in angii-induced cardiac remodelling, lv function is preserved by chronic cb antagonist treatment and that cardiac fibrosis is reduced with concomitant downregulation of fibrogenic genes. also, cf-enriched mir- a/b family seems to be sensitive to cb antagonist treatment, thereby affecting cardiac fibrosis. the current study employs a novel concept regarding chronic cb inhibitor treatment and may provide important details and novel targets for anti-fibrotic approaches in heart failure. background: low homoarginine (harg) was recently identified as an emerging biomarker for stroke, myocardial infarction, and heart failure in clinical and epidemiological studies. harg competes with arginine as a substrate for nitric oxide (no) synthase and weakly inhibits arginase. both mechanisms might lead to increased no formation in vivo. the aim of this study was to investigate whether harg effects the development of atherosclerosis as a potential underlying mechanism of cardiovascular diseases. methods: harg-deficient agat-knockout (agat -/-) and wildtype (wt) mice were crossed with apolipoprotein e (apoe) deficient mice and fed with high fat diet (hfd) for three months to induce atherosclerosis. harg plasma concentrations were determined using mass spectrometry. en face preparation of aortae followed by red oil staining of atherosclerotic plaques and quantitative evaluation of plaque areas was performed for female mice. endothelial function of male mice was tested with acetylcholine (ach) and nitroglycerin (ntg) after contraction with prostaglandin f α. background: the direct oral thrombin inhibitor dabigatran etexilate (dabigatran) is used for the prevention and treatment of venous thromboembolism. obese patients as well as patients with type diabetes mellitus (t dm) have an increased risk for thrombotic disease and show enhanced thrombin generation. besides its role in blood coagulation, thrombin is known to be involved in many pro-inflammatory processes. in obesity, adipose tissue (at) inflammation plays a crucial role in the development of insulin resistance and t dm and contributes to atherosclerosis development. the aim of the present study was to analyse the effects of dabigatran on at inflammation in a mouse model of diet-induced obesity in the context of accelerated atherosclerosis. methods: -week-old female low-density lipoprotein receptor-deficient (lldr -/-) mice were fed a high-fat diet containing mg/g dabigatran or respective placebo for weeks. results: analysis of visceral at revealed a significant increase in adipocyte size in dabigatran-treated mice, although body weight, fat mass, glucose tolerance, and insulin resistance were unchanged between groups. this effects seemed to be directly mediated by thrombin, as treatment with another thrombin inhibitor (argatroban) also resulted in the development of adipocyte hypertrophy. accordingly, in vitro studies in t -l cells revealed an inhibitory effect of thrombin on lipid accumulation in adipocytes. the amount of pro-inflammatory cd c-positive macrophages (atms) in visceral adipose tissue was significantly reduced, and the secretion of pro-inflammatory il- from visceral at was significantly lower in dabigatran-treated animals. in vitro studies using t l cells and primary bone marrow-derived macrophages revealed that the changes in macrophage polarization were not directly mediated by thrombin, but indirectly by a change in the secretion profile of adipocytes. a similar reduction in proinflammatory macrophages as detected in at could also be observed in the aortic wall of dabigatran-treated mice. conclusions: the direct thrombin inhibitor dabigatran inhibits at inflammation and the accumulation of pro-inflammatory macrophages in vat but also the aortic wall of ldlr -/mice. these anti-inflammatory effects of dabigatran might contribute to the known atheroprotective effects of dabigatran. background: sarco/endoplasmic reticulum ca + -atpase (serca a) and its inhibitor phospholamban (pln) are critical determinants of cardiomyocyte calcium cycling and hence, cardiac contractility. pln exists in an equilibrium between mono-and pentamers. while monomeric pln has been implicated in direct serca a inhibition, a functional role for the pentamers remains ambiguous. recently it has been shown that pln pentamers modulate pka-dependent phosphorylation of pln monomers in vitro. using transgenic mouse models we now investigated the effects of pln pentamers on pln phosphorylation, myocyte ca + cycling and contractility in cardiac myocytes. methods: phosphorylation patterns of pln were analyzed by western blot using phospho-specific antibodies as well as phosphate affinity sds-page. to assess the phosphorylation at baseline, pln knockout (pln-ko) mice expressing either wild type pln (tgpln) or the solely monomeric pln afa mutant (tgafa) transgene were deeply anesthetized, whereas pln phosphorylation by pka was induced using the betaadrenergic agonist isoproterenol. the consequences on myocyte ca + kinetics were measured in isolated, fura -loaded and electrically paced ( . hz) cardiomyocytes as the time to % decay of the ca + signal (t % ). the time to % baseline of sarcomere length (t % baseline) characterized the speed of myocyte relaxation. results: under basal conditions, we found stronger phosphorylation of pln pentamers than monomers, pointing at pentamers as the preferred pka target. pln afa monomers showed . -fold stronger phosphorylation signals if pentamers were absent (p< . ). consistent with a higher basal phosphorylation of pln afa monomers, measurements of calcium kinetics revealed a faster decay of calcium signals in tgafa compared with tgpln cardiomyocytes (t % [ms]: ± and ± , respectively, p< . ). notably, t % of pln-ko myocytes was ± ms (p= not significant versus tgafa), indicating that the strong basal phosphorylation of monomers leads to near complete inactivation of pln in tgafa. upon stimulation of pka, pln monomer phosphorylation and calcium kinetics of tgpln and tgafa mouse myocytes were indistinguishable, because monomer phosphorylation and the speed of cytosolic ca + clearance strongly increased only in tgpln. acceleration of sarcomere relaxation upon pka stimulation was also more pronounced in tgpln than in tgafa and pln-ko myocytes (increase of t % baseline [ms]: ± in tgpln versus ± in tgafa-pln and ± in pln-ko, p< . ). even high-dose isoproterenol induced phosphorylation of only about half of all protomers of pln pentamers suggesting a high capacity of pentamers to attenuate monomer phosphorylation by acting as a phosphate scavenger. conclusions: our data demonstrate that pln pentamers reduce basal phosphorylation of pln monomers in myocytes. nevertheless, pentamers allow strong phosphorylation of monomers during beta-adrenergic stimulation, thereby extending the range within which pln can modify diastolic ca + kinetics and myocyte relaxation. therapeutic inhibition of micrornas is a promising field in cardiovascular research. vector-based overexpression of an inhibitor construct (e.g. microrna sponge) is one approach to achieve sustained inhibition with potential applicability in humans. yet the strength of expression achieved by the currently available gene therapy vectors (e.g. aavs) in humans remains a limiting factor, therefore inhibitory constructs with increased potency would provide an improvement of this approach and bring it closer to therapeutic application. micrornas are believed to discriminate between potential binding sites, based on additional factors provided by the endogenous untranslated regions at the ' end of mrnas ( ' utrs) and the proteins that are bound to them. aim of this project was to investigate whether selected endogenous ' utrs can likewise increase the potency of microrna inhibitory constructs. to this end several known targets for a cardiac microrna were selected and their relative potencies of microrna inhibition was compared. to accurately assess changes in the activity of the respective micrornas we constructed dual-fluorescent reporter plasmids and established an automated fluorescent microscopy acquisition and analysis pipeline. among several tested utr constructs we found one which strongly increased the inhibitory potency of the microrna binding site in primary rat cardiac myocytes (nrcms). furthermore a similar effect was obtained when the binding site was exchanged for that of a different microrna and analyzed in the nih- t fibroblast cell line. we therefore conclude that endogenous utr contexts can indeed be successfully applied to increase the potency of vector-based microrna inhibitors. the g-protein-coupled protease-activated receptor- (par ) regulates inflammatory responses including monocyte migration and cytokine release. par is activated by the coagulation factor-xa or by the tissue-factor (tf)/factor viia complex. the immunomodulatory lipid sphingosine- -phosphate (s p) is released from activated platelets and interlinks blood coagulation and inflammation. this study investigates the impact of s p on the expression of par , tf and of the anticoagulant protein thrombomodulin (tm) in human monocytes and after pma-induced differentiation into macrophage-like cells. monocytic thp and u cell lines were used as human monocyte models. primary monocytes were isolated from healthy volunteers using a magnetic bead-based monocyte isolation kit. expression of par , tf and tm was measured by quantitative real-time pcr and western blotting. differentiation of monocytes into macrophage-like cells was induced by incubation with ng/ml pma (phorbol -myristate -acetate) over h. calibrated automated thrombin (cat) generation was determined in plateletrich plasma from healthy volunteers. in thp and u cells s p induced a time-( to h) and concentration-dependent ( . to µm) significant upregulation of par mrna and total protein expression. par total protein was upregulated maximally (about . -fold, n= ) with µm s p after h incubation. comparable effects were seen in human primary monocytes. in comparison, tf mrna and protein were only marginally elevated in non-differentiated thp monocytes and tm was not regulated by s p. after differentiation of cultured monocytic cells with pma into adhesive macrophage-like cells, incubation with s p resulted in a time-( to h) and concentration-dependent ( . to µm) significant upregulation of tf expression within to h of incubation. conversely, par total protein expression was reduced by about % after h s p incubation. the expression of tm was again not affected. the generation of thrombin in platelet-rich plasma was determined using pma-differentiated thp cells as tf source. timedependent incubation with s p ( µm) in differentiated monocytes shortened the time to the onset (lag time) of thrombin generation in plasma from . ± . to . ± . min and elevated total the thrombin generation capacity from ± to ± nm. peak thrombin formation was elevated from ± to ± nm/min (control versus s p for h, mean±sd, n= , respectively). these data suggest that s p induces an enhanced expression of par in undifferentiated human monocytes while tf and tm are not regulated. in differentiated monocytes/macrophages, s p does upregulate tf expression but attenuates par levels. since par is involved in regulation cell migration, s p may stimulate a phenotypic switching from a migratory to a procoagulant phenotype during differentiation of monocytes into macrophages. the pro-inflammatory cytokine interleukin- (il- ) plays an important role in vascular inflammation. coagulation factors such as the activated factor-x (fxa) may regulate local inflammatory responses of the vessel wall. in this study we investigated whether fxa regulates il- expression and secretion in human vascular smooth muscle cells (smc) as well as in failed thrombosed vein grafts. also, we analysed its possible prothrombotic impact on monocytes. il- mrna expression was determined in primary human saphenous vein smc by taqman® real-time pcr. secretion to the cell culture media was measured by elisa. tissue factor (tf) expression in monocytic thp- cells was determined by western blot. immunostainings for il- and the smc marker smoothelin were performed on paraffin embedded tissue sections from failed thrombosed vein grafts and control veins. incubation of cultured human venous smc with fxa ( nm) induced a time-dependent ( - h) increase in il- mrna expression. maximum expression was observed within h to a . ± . fold increase (mean±sd, n= , p< . ). incubation with an inhibitor of p map kinase (sb , µm) or pi k (ly , µm) significantly attenuated fxa-induced il- mrna expression (n= ). inhibition of p / mapk, rho kinase or nf-kb had no significant effect. stimulation with fxa for h resulted in a markedly increased il- secretion into the smc culture media from . ± . to . ± . ng/ml (p< . , n= ). stimulation of thp- cells with il- ( ng/ml) induced a time dependent ( - h) increase of up to . ± . fold (p< . , n= ) in tf protein expression. immunostainings of tissue sample of failed vein grafts revealed enhanced il- expression in smc-rich regions in vessel walls compared to non-thrombosed control veins suggesting an elevated il- regulation in thrombosed vein grafts in vivo. in conclusion, fxa induced il- expression and secretion in venous smc which may be regulated via p and pi k signaling. il- enhanced tf expression in thp- monocytes and was found in smc-rich regions in failed thrombosed vein grafts. fxa-stimulated il- release may be involved in regulating local pro-thrombotic processes during vascular inflammation and possibly vein graft failure. rationale: the transcription factors camp-response element binding protein (creb) and camp-responsive element modulator (crem) bind to camp response elements (cres) and mediate a camp dependent gene regulation. suppression of cre mediated transcription is linked to atrial remodeling in genetic mouse models. inhibition of creb target genes is associated with atrial fibrillation (af) susceptibility in patients. creb and crem affect histone acetylation recruiting the creb-binding protein (cbp/p ). the histone acetyltransferase (hat) activity of cbp facilitates gene transcription by loosening chromatin structure. histone deacetylases (hdacs) catalyze the inverse reaction: histone deacetylation with consecutive gene silencing. mice with heart directed expression of the human cardiac isoform crem-ib∆c-x (tg) show atrial dilatation, morphological and physiological alterations in atria preceding spontaneous-onset af. the hdac inhibitor (hdac i ) valproic acid (vpa) reduced atrial weight and af incidence in tg mice. here we tested the hypothesis, that vpa attenuates the structural remodeling in tg atria by reversing changes in atrial gene regulation due to the transgene. methods and results: tg and wt mice were treated from week - with vpa ( . % in drinking water, ad libitum) or vehicle (veh). atrial ultrastructure was studied by electron microscopy (em) (week and ). veh-treated tg atria showed a progressive dysorganisation of sarcomeres (sm) with less mitochondria and more collagen fibers between cardiomyocytes as compared to veh-treated wt atria. the fraction of sm structure in veh-treated tg atria was significantly reduced as compared to veh-treated wt atria (week : tg veh : ± %, wt veh : ± %; week : tg veh : ± %, wt veh : ± %, p< . ). vpa led to a more organized ultrastructure and restored, at least partially, the degradation of the sm in the tg atria (tg vpa at week : ± %, tg vpa at week : ± %, p< . vs. tg veh ). the structure of wt atria was not affected by vpa. we further analyzed the protein abundance profiles in the groups of all animals (wt veh , wt vpa, tg veh , tg vpa) by using lc-ms/ms. between veh-treated genotypes (tg veh vs. wt veh , p< . ) proteins were significantly changed while proteins were differentially abundant between vpa-treated groups (tg vpa vs. wt vpa, p< . ). proteins were regulated by vpa in wt atria (wt vpa vs. wt veh , p< . ), whereas vpa affected proteins in tg atria (tg vpa vs. tg veh , p< . ), out of which proteins were common. prominent changed proteins between veh-treated tg and wt atria were significantly regulated by vpa in tg atria in the opposite direction. a functional pathway analysis showed that pathways activated in tg atria such as cardiac fibrosis, mitochondrial dysfunction were inhibited by vpa treatment. conclusion: similar to human af, crem-tg mice present atrial dilatation, ultrastructural changes and impaired conduction and spontaneous af. while vpa had little to no effect in wt mice, valproate improved the tg phenotype by interfering with pathways involved in structural remodeling. this supports the idea that hdac inhibition by vpa antagonizes effects of crem expression in atria. in isolated mouse cardiac preparations, histamine is ineffective regarding inotropic or chronotropic effects, presumably because of lack of receptor protein expression. on the other hand, histamine can exert positive inotropic and chronotropic effects in humans via cardiac histamine h -receptors. hence, we have generated transgenic mice (tg) which overexpress the human h -receptor specifically in cardiomyocytes. in isolated left and right atrial preparations of these mice, we investigated the histamine metabolism on a functional level. preparations of wild type mice (wt) served as control. histamine induced positive inotropic effects (pie) and positive chronotropic effects (pce) in left and right atria of tg mice, respectively, but not in wt. interestingly, the inhibitor of histamine oxidation, aminoguanidine ( mm), shifted the concentration response curves for the pie of histamine from ec = nm to nm (p< . ). furthermore, the unspecific inhibitor of mono amine oxidase, tranylcypromine ( µm), shifted the pie of histamine from ec = nm to nm and increased the efficacy of histamine for the pie (p< . ). these data indicate that exogenously applied histamine is subject to degradation in the mouse heart by two different pathways namely via diaminoxidase and mono amine oxidase. drugs that inhibit theses enzymes could conceivably alter cardiac function also in the human heart. protein phosphorylation by kinases and dephosphorylation by protein phosphatases has a crucial function in cell signal cascades. it has been shown that cardiomyocyte specific overexpression of serine /threonine protein phosphatases pp , pp a, pp b (calcineurin) and pp in mice leads to cardiac hypertrophy and alters cardiac function. to examine the function of another important protein phosphatase in the heart we established a mouse model overexpressing protein phosphatase cβ (pp cβ) under control of the α-myosin heavy chain promoter. cardiac overexpression was demonstrated by western blotting. like other serine/threonine phosphatases, pp cβ can lead to cardiac hypertrophy. in transgenic mice (tg), relative ventricular weight was increased ( . ± . mg/g) compared to wild type (wt) littermates ( . ± . mg/g; p< . ) whereas weights of right and left atria were unchanged. therefore, relative heart weight was increased in tg ( . ± . mg/g) vs. wt ( . ± . mg/g; n= - ; - months of age; p< . ). left ventricular function, measured in vivo by echocardiography under isoflurane anesthesia was diminished in tg compared to wt (ejection fraction: . ± . % (tg) versus . ± . % (wt); n= - ; - months; p< . and fractional shortening: . ± . % (tg) versus . ± . % (wt); n= - ; - months; p< . ). the left ventricle was dilated (systolic diameter: . ± . mm (tg) versus . ± . mm (wt); n= - ; - months; p< . ; diastolic diameter: . ± . mm (tg) versus . ± . mm (wt); n= - ; - months; p< . ). in contrast, atrial function measured as response to β-adrenergic stimulation in isolated left and right atrial preparations was unchanged in tg vs. wt. in summary, our results indicate that pp cβ overexpression can lead to ventricular dysfunction and hypertrophy. the underlying signal transduction pathways need to be elucidated. the insulin-like growth factor binding protein (igfbp ) -a potential developmental gene is regulated upon cardiac stress m. wölfer background: cardiac remodeling is a complex biological adaptation process of the failing heart accompanied by a re-activation of embryonic gene expression, which so far has unclear pathophysiological relevance. we and others showed that insulin-like growth factor binding protein (igfbp ) is expressed in the early pre-cardiac region in mouse embryos and its up-regulation impairs cardiac progenitor differentiation. igfbp functions as an extracellular growth factor binding protein for igf and also has igfindependent activities. the role of this factor in the context of cardiac remodeling is still unknown. the aim of this study was to investigate the relevance of igfbp in cardiogenesis and cardiac remodeling and its role as a potential target for ameliorating stress-induced cardiac remodeling methods and results: we investigated the expression of igfbp in murine cardiac tissue at different developmental stages by qpcr normalized to tpt (tumor protein, translationally-controlled ). this analysis showed temporal changes of cardiac igfbp expression from developing to postnatal hearts, where a high expression was detected in early heart stages, which decreased during cardiac development and became low in the postnatal heart. the analysis of igfbp expression in different heart cells showed a very low igfbp in adult cardiomyocytes in contrast to a high expression in undifferentiated sca- positive cells. in a mouse model with cardiac specific wnt/βcatenin activation, which led to cardiac dysfunction, igfbp was found up-regulated (p< . ). further we found an increased igfbp expression after pressure induced cardiac hypertrophy using mice with transverse aortic constriction (tac) (p< . ). in line with this data, an in vitro model of human heart muscle hypertrophy using engineered cardiac heart muscle (ehm) showed an up-regulation of igfbp upon adrenergic activation via norepinephrine stimulation accompanied by a functional deterioration in comparison to untreated controls (p< . ). all these findings were further supported by rna-sequencing analysis from human aortic stenosis patient samples, where igfbp expression was found increased in patients with compensatory hypertrophy and in a higher extent in patients with heart failure in comparison to non-failing heart samples. interestingly, the expression of igfbp in angiotensin or norepinephrine stimulated neonatal murine cardiomyocytes, as well as in hearts of mice treated with angiotensin , showed the opposite results, namely a reduction in its expression (p< . ; p< . , respectively). summary and conclusion: our results show active igfbp transcription in the early developing heart but a low expression in the postnatal heart. a re-activation of expression was found in the process of pathological heart remodeling in mouse and human, in vivo as well as in vitro, indicate the participation of igfbp in a conserved manner. we hypothesize that igfbp may participate in the developmental gene program becoming activated in the diseased adult heart again. the functional role and regulation of igfbp is under investigation. we have recently shown that perivascular adipose tissue (pvat) plays a crucial role in obesity-induced vascular dysfunction. in pvat-free aortas isolated from male c bl/ j mice fed a high-fat diet (hfd) for weeks, the endothelium-dependent nitric oxide (no)-mediated vasodilator response to acetylcholine remained normal. in contrast, a clear reduction in the vasodilator response to acetylcholine was observed in aortas from obese mice when pvat was left in place. these results suggest that the reason for vascular dysfunction in diet-induced obese mice is a pvat dysfunction rather than an endothelial dysfunction. treatment of hfd mice during the last weeks with crataegus extract ws® ( mg/kg/day) completely normalized vascular function in pvatcontaining aorta. the expression of endothelial no synthase (enos) was not changed by ws® , neither in pvat nor in aorta. phosphorylation at serine is the most important positive modulation of enos activity. hfd-induced obesity was associated with a reduction in enos phosphorylation at serine in pvat, but not in aorta. ws® treatment significantly improved enos serine phosphorylation selectively in pvat but had no effect in aorta. a major upstream kinase for enos serine phosphorylation is akt. the activity of this kinase is inhibited in the pvat of hfd mice, which was largely reversed by ws® treatment. in addition, ws® treatment enhanced the mrna expression of the nad-dependent deacetylase sirtuin- (sirt ), known also as a longevity gene. the activity of sirt depends, among others, on the intracellular content of its cofactor nad. ws® treatment led to an upregulation of nicotinamide phosphoribosyltransferase (nampt), a rate-limiting enzyme in the salvage pathway of nad biosynthesis. one of the non-histone substrates of sirt is enos. deacetylation of enos at lysine residues and by sirt enhances the activity of the enos enzyme. currently, we are studying the effect of ws® on nad synthesis, sirt activity, and enos (de)acetylation. in conclusion, crataegus extract ws® reverses obesity-induced vascular dysfunction by improving pvat function. the molecular mechanisms may involve enos phosphorylation at serine and upregulation of sirt . the raf kinase inhibitor protein (rkip) inhibits g-protein-coupled receptor kinase (grk ) and the raf-erk / pathway. these two functions of rkip could counteract each other. while grk inhibition is cardio-protective, inhibition of the pro-survival erk / axis promotes signs of heart failure in patients and experimental models. in view of this ambivalent nature, the function of rkip in vivo is not clear. furthermore, rkip could have a pathophysiological role because heart specimens from patients with heart failure showed rkip up-regulation (ref. ). to investigate the impact of cardiac rkip upregulation in vivo, we generated transgenic mice with myocardium-specific expression of the human rkip gene (pebp ) under control of the alpha-mhc promoter. two different rkip-transgenic lines with . -fold and . -fold increased cardiac rkip protein level were generated (ref. , and jax id number ). we investigated the cardiac phenotype and found that tg-rkip mice developed cardiac hypertrophy with a significantly increased heart weight to body weight ratio and a decreased left ventricular ejection fraction relative to non-transgenic fvb controls, as early as weeks of age. histology analysis revealed progressive atrial and ventricular enlargement of tg-rkip hearts. ecg abnormalities, a lower maximum rate of left ventricular pressure rise, and a strongly decreased left ventricular ejection fraction of . ± . % (n= ; ±s.d.) were documented at an age of months. down-regulation of the transgenic rkip by lentiviral transduction of an rkip-targeting mirna retarded the cardiac phenotype of tg-rkip mice. thus, dual-specific inhibition of the grk and raf-erk / axis by the human rkip gene (pebp ) triggers signs of heart failure in vivo, and the documented upregulation of the cardiac rkip in heart failure patients could aggravate disease pathogenesis. these findings are in contrast to rodent rkip (pebp ), which does not seem to inhibit the raf-erk / axis in vivo but instead confers grk inhibition- heterodimerization between the at receptor (at r) for the vasopressor peptide, angiotensin ii, and the b receptor (b r) for the vasodepressor peptide, bradykinin, enhances angiotensin ii-stimulated signalling in cells. in addition, at r--b r heterodimerization has a major pathophysiological role and contributes to the angiotensin ii hypersensitivity in women with preeclampsia hypertension. to analyse the vascular function of the at r--b r heterodimer in vivo, we generated a transgenic model of at r--b r heterodimerization (tg-b r+) by transgenic expression of the b r gene (bdkrb ) in the b r-deficient tg-b r-/-strain. fluorescence resonance energy transfer (fret) imaging was applied to analyse the interaction between different gprotein-coupled receptors in the aorta of transgenic mice. we report here that fret imaging detected the close interaction between the aortic at r and b r at a distance of less than nm in tg-b r+ mice whereas the at r--b r heterodimer was absent in tg-b r-/-mice. in contrast, fret was not detectable between the endothelin eta receptor (etar) and the b r in the aorta of tg-b r+ mice, although immunofluorescence and immunohistology confirmed the aortic (co-)-localization of both, etar and b r. the efficient at r--b r heterodimerization in tg-b r+ mice was accompanied by an enhanced angiotensin ii at r-stimulated vasopressor response relative to that of tg-b -/-mice, which lack the at r--b r heterodimer. as a control, the endothelin- -stimulated vasopressor response mediated by the etar, which did not dimerize with b r, was not significantly different between tg-b r+ and tg-b r-/-mice. together these findings provide strong evidence that at r--b r heterodimerization occurs in vivo and enhances the angiotensin ii at r-stimulated vasopressor response. dysfunction of the cardiac energy substrate metabolism is a characteristic feature of late-stage heart failure. the dysfunctional cardiac substrate metabolism contributes to insufficient energy generation and has limited treatment options. in search for a treatment approach, we investigated whether inhibition of g-protein-coupled receptor kinase (grk ) could confer cardioprotection by targeting the dysfunctional cardiac substrate use. the impaired substrate metabolism of late-stage heart failure was reproduced in a transgenic model with myocardium-specific expression of fatty acid synthase (fasn), which is the major palmitate-synthesizing enzyme. experiments with a seahorse xf extracellular flux analyzer revealed that in an adult-like lipogenic milieu, fasn-transgenic cardiomyocytes reproduced the overall depressed substrate use of late-stage heart failure with a switch from fatty acid to predominant glucose utilization. the impaired substrate use was largely retarded by co-expression of a small peptide inhibitor of grk , grkinh. the grkinh-mediated protection against cardiometabolic remodelling required an intact raf-erk / axis and involved the erk / -dependent inactivation of the heart failure-promoting peroxisome proliferatoractivated receptor gamma (pparg) by phosphorylation of serine- . as a consequence of erk-dependent phosphorylation of pparg on serine- , the expression of heart failure-related pparg targets such as fatty acid synthase, resistin and adiponectin was decreased. the importance of pparg serine- phosphorylation was further shown in transgenic mice with myocardium-specific expression of the phosphorylation-deficient pparg serine- a mutant, which was resistant to the cardioprotective activity of grkinh. taken together our experiments show that grk inhibition could target cardiometabolic remodelling by inhibition of the heart failure-promoting transcription factor pparg. the effect of sodium valproate on the action potential of atrial myocytes of crem ib∆c-x transgenic mice introduction: in mouse, cardiomyocyte directed over-expression of transcription factor crem (camp response element modulator) causes an atrial phenotype characterized by hypertrophy, reduced contractility and increased duration of the monophasic action potential (map). moreover, this animal model (crem ib∆c-x) showed spontaneous atrial fibrillation (af) episodes as early as weeks of age in homozygous mice and - weeks of age in heterozygous mice (phenotype delayed towards adult stage). previous studies in heterozygous mice targeted hdac inhibition by sodium valproate (vpa, an anticonvulsant drug, acting also as inhibitor of hdac class i>ii). vpa treatment delayed significantly the development of atrial hypertrophy and the incidence of af episodes, without affecting cellular hypertrophy. our aim was to investigate the effect of chronic vpa treatment on the electrical activity of atrial myocytes isolated from crem ib∆c-x and wild type (wt) littermate mice. methods and results: atrial myocytes were isolated from weeks old wild type mice (wt) and heterozygous crem ib∆c-x transgenic mice with enlarged atria (tg), treated for weeks with vpa ( . mm in the drinking water) vs. water (vehicle control). action potentials (ap) were measured at room temperature using the patch-clamp technique. atrial myocytes of water treated tg mice had the ap amplitude significantly reduced by mv compared to water treated wt, and, in line with previous results for the map, the tg cells depolarized with a slower slope of . ± v/s (tg: n= cells) vs. . ± . v/s (wt: n= , p= . ). moreover, ap of atrial myocytes isolated from water treated tg mice had longer duration (apd) at % (tg: . ± . ms, n= vs. wt: . ± . ms, n= , p< . ), at % (in ms: . ± . vs. . ± , p< . ) and at % (in ms: . ± . vs. ± . , p< . ) repolarization. vpa treatment reduced ap amplitude in wt mice by mv (n= , p< . ) vs. water treated wt, without altering the slope of depolarization or the apd. in vpa treated tg mice the apd was reduced ( %: . ± . ms, %: . ± . ms, %: . ± . ms, n= , p< . vs. untreated tg), the amplitude was increased by mv (n.s.) and the slope of depolarization was increased by % (p= . , n.s.). membrane capacitance evaluation, as an estimation of atrial myocyte size, showed that in untreated tg mice the cells were larger than in wt (tg: ± . pf, n= vs. wt: ± . pf, n= , p< . ), in line with the occurrence of cellular hypertrophy in tg atria. chronic vpa treatment did not change the cell size in either genotype (wt-vpa: . ± pf, n= n.s. vs. wt; tg-vpa: ± pf, n= , p< . vs. wt-vpa; p< . vs. wt; n.s. vs. tg). conclusions: in hypertrophied atrial myocytes of crem-ib∆c-x, ap were characterized by smaller amplitude, slower onset of depolarization and increased duration compared to wt cells. despite having no effect on atrial myocytes size, vpa treatment reduced the duration and showed a tendency to increase the amplitude and the slope of depolarization of the action potential in tg mice to values similar to wt. these data suggest that chronic treatment with vpa restored partially the electrical activity of atrial myocytes and may reverse the electrical remodeling via hdac inhibition. (supported by the dfg) of the transcription factor crem (camp response modulator) icer, smicer and crem-ib∆c-x are inducible by β-adrenergic stimulation and code for similar or even identical proteins. thus, these isoforms are able to repress expression of respective target genes in response to camp and might play a role in an arrhythmogenic remodeling during the development of chronic heart diseases. here we test this hypothesis in a mouse model with transgenic expression of crem-ib∆c-x (tg). these mice develop not only spontaneous onset atrial fibrillation but likewise arrhythmogenic alterations in the ventricle. patch clamp experiments revealed an increased na + -ca + exchanger current (i ncx ) and decreased transient outward current (i to ) in tg ventricular cardiomyocytes (vcms) vs. wild-type controls (ctl). these alterations were associated with an increased arrhythmogenicity in tg vcms. action potentials were prolonged in tg vcms vs. ctls leading to an increased proportion of vcms displaying early afterdepolarizations. ca + imaging revealed that the transduction rate of spontaneous sub-threshold ca + -waves into supra-threshold transient-like ca + -events which is mediated by the ncx was increased in tg vcms. at the same time the serca mediated ca + transport rate (r serca ) was enhanced in tg vcms potentially limiting ca + extrusion by the ncx. underlining the in-vivo relevance of our findings ventricular extrasystoles (ves) were augmented in ecgs of tg mice (ves/mouse during - m isoproterenol challenge, tg: . *, ctl: . ; n= /condition). the increase in i ncx and r serca and the decrease in i to went along with an increase of ncx , serca a and decrease of kchip protein levels. however, the respective mrna levels (slc a , atp a and kcnip ) were unaltered between groups pointing to a post-transcriptional regulation of these genes. in a mrnasequencing approach we identified the downregulation of precursor mirnas inter alia for mir- (fold change in tg: . *) and mir- (fold change in tg: . *) (n= /condition). atp a is a predicted target of mir- and mir- has recently been shown to regulate ncx and i to related potassium channel subunits. (*p< . vs. ctl) our results demonstrate that transgenic expression of crem-ib∆c-x in mouse vcms leads to distinct arrhythmogenic alterations. they further indicate that the repression of micro rnas by short crem repressor isoforms may lead to the upregulation of genes in the context of an arrhythmogenic remodeling. since crem-repressors are inducible by chronic β-adrenergic stimulation our results suggest that the inhibition of credependent transcription contributes to the formation of an arrhythmogenic substrate in chronic heart disease. ( chronic overstimulation of cardiac β-adrenergic receptors (β-ar) is a major trigger for the development and maintenance of cardiac hypertrophy and heart failure. although the camp activated proteinkinase a (pka) is known as a prominent downstream effector of β-ar signaling, its functional contribution to pathological cardiac remodeling is neither well understood nor directly studied so far. to address this issue we used mice carrying a point mutation in the regulatory pka subunit riα (pkariαb), which prevents binding of camp and consequently diminished kinase activity. this dominant negative mutation was controlled by a tamoxifen (tam) inducible αmhc promotor driven cre transgene which allows a selective expression in the ventricular myocardium. the inducible and tissue specific gene expression was analyzed and confirmed by pcr, rt-pcr and immunohistochemistry. furthermore diminished phosphorylation of several pka targets verifies impaired pka activity in tam treated double transgenic animals. hypertrophic response in ventricular pka mutants was studied in genetic, pharmacological and surgical mouse models of heart disease as well. genetically induced heart failure was observed following tam treatment in mice expressing an inducible myocardial-specific cre transgene. this deleterious cardiac phenotype develops independently of the presence of the floxed transgene. days after tam treatment, controls displayed elevated heart weight to bodyweight ratio (hbr) and heart weight to tibia length (htr). hbr shifted from . (mg/g) in untreated control animals to . (mg/g) in tam injected mice. in contrast pka inhibited mutants displayed a minor increased hbr of . (mg/g). for the pharmacological induction of cardiac hypertrophy we implanted osmotic mini pumps, delivering a combination of isoproterenol and phenylephrine. control animals showed a significantly increased hbr ( . mg/g) compared to saline treated animals ( . mg/g) and pka mutants ( . mg/g). paradoxically, all pka inhibited animals displayed a consistent elevation in important hypertrophic markers like anp. surgical constriction of the aortic arch (transverse aortic constriction tac) led to a pressure induced hypertrophic response (hbr: . vs . mg/g) followed by a pronounced elevation in several hypertrophic factors such as anp, myh / ratio and myocytes size. in contrast pka mutants displayed an irregular progression of cardiac hypertrophy presented by two groups with either an unchanged ( . mg/g) or a strongly elevated hbr ( . mg/g). however, additional hypertrophic factors including anp, myh / ratio and myocyte size were significantly increased in both groups. to our knowledge this is the first report, which directly studies the role of ventricular pka activity in cardiac hypertrophy in a genetically altered mouse model. our results suggest that in an early stage of cardiac remodeling pka inhibition alleviates cardiac weight gain but provokes a detrimental shift during further progression, which implicates a protective role of ventricular pka activity in cardiac disease. acetyl-coa carboxylase catalyzes the first step in the biosynthesis of fatty acids in bacterial and eukaryotic cells, i.e. the conversion (carboxylation) of acetyl-coa into malonyl-coa. acc-generated malonyl-coa functions as a substrate for de novo lipogenesis and acts as an inhibitor of mitochondrial β-oxidation of fatty acids. because of its role in lipid metabolism this enzyme has become an interesting target in drug discovery in the field of metabolic diseases and cancer. despite this interest in acc, no attention has as yet been given to the role of acc in endothelial cells. we aimed to investigate the role of acc in two functional key aspects of angiogenesis: endothelial cell proliferation and migration. we used the acc inhibitor soraphen a, a polyketidic natural compound isolated from the myxobacterium sorangium cellulosum, as well as an rnai-based approach to inhibit the function of acc. primary human umbilical vein endothelial cells (huvecs) were used as in vitro model. first, we analyzed the action of soraphen a on cell viability. the compound did neither lower the metabolic activity of huvecs up to a concentration of µm after and h (ctb assay) nor increase in the apoptosis rate after , , or h up to µm. measuring adenosine triphosphate (atp) levels revealed that µm soraphen a does not alter the atp levels in huvecs after h treatment. in contrast, a h treatment significantly lowered the atp levels by %. also gene silencing of acc in huvecs attenuated the atp levels by %. mitochondrial membrane potential (mmp) assays showed decreased mmp levels ( %) in soraphen a-treated cells after h. interestingly, the compound inhibited the proliferation of endothelial cells with an ic value of µm. cell cycle analysis showed that soraphen a decreases the amount of cells in the g /g phase by % and increases the number of cells in the g /m phase by %. the compound also inhibited the activation of akt (western blot analysis). in a wound healing/scratch assay, µm soraphen a lowered the migration of endothelial cells by %. gene silencing of acc in huvecs strongly decreased endothelial migration, whereas a knockdown of acc had no influence. furthermore, boyden chamber assays revealed that soraphen a can also lower chemotactic migration by %. since actin rearrangement is necessary for migratory processes, we analyzed the factin cytoskeleton (microscopy) and found that soraphen a decreases the number of filopodia by % but did not influence stress fiber formation. surprisingly, soraphen atreated cells did not exhibit significant alterations in their capacity to form tube-like structures on matrigel. in summary, we could gather first hints that inhibiting acc has an immense impact on the proliferation and migration of primary endothelial cells. the mechanistic basis of this phenomenon will be investigated in future studies by analyzing the lipid profile and the transcriptome of endothelial cells. acknowledgement: this work was supported by the german research foundation (dfg, for , fu / - ) . introduction: statins are among the best examined drugs with excellent efficacy and safety profiles. lowered low-density lipoprotein (ldl) cholesterol goals, new indications for treatment and new knowledge about their pleiotropic effect have promoted a considerable increase in statin use. but as statin use becomes more widespread, awareness of their adverse effects as well as the recognition of statin intolerance problems increase. statin intolerance is a significant problem in the treatment of dyslipidemia, understood as the inability to tolerate a dose of statin required to reduce individual cardiovascular risk sufficiently and could result from different statin-related side effects. muscle-related adverse events, elevation of liver enzymes, cognitive problems and new onset diabetes mellitus have all been described, especially at higher doses. although muscle symptoms are the common side effects observed, excluding other adverse events might underestimate the number of patients with true statin intolerance. these patients represent a target population for the newest lipid lowering drug category i.e. the proprotein convertase subtilisin/kexin type (pcsk ) inhibitors. this work aimed to give an overview of published definitions derived from clinical studies, associations as well as major drug regulatory agencies. we discussed overlaps, differences and limitations in the current definitions. methods: literature based search included pubmed and uptodate publications in english and german language until october . we performed hand searches of the references retrieved and performed an overview. results: a definition of statin intolerance of the european medicines agency (ema) or the us food and drug administration (fda) is not available. in clinical studies, different definitions are chosen and the results are not comparable. also different associations, such as the american heart association (aha), the european atherosclerosis society (eas), the canadian working group or the national lipid association (nla) are not able to agree on one common definition. statin intolerance definitions included different types of muscle symptoms, integration of ck levels and minimal requirements of statin doses. there are currently no validated questionnaires or specific laboratory parameters available. in addition, the term 'myopathy' is often considered as a synonym to statin intolerance. overall, only a few major studies have been conducted with statin intolerant patients so far using inconsistent definitions. discussion and conclusion: there is an unmet need to find a robust and clear definition of statin intolerance as overemphasizing it might hinder appropriate clinical use of this important drug class. thus, further work is required to develop a consensus definition on statin intolerance or a more focused definition regarding statin-associated muscle symptoms only. subsequently, these definitions could be implemented in patient care and their relevance being analyzed and tested in future studies. background: development of cardiac hypertrophy is characterized by reactivation of genes involved in cardiac development. wnt/β-catenin signaling is essential for embryonic cardiac development and is known to be dysregulated in pathological heart remodeling. our previous work suggested a cardiac specific protein complex regulating wnt/b-catenin/tcf transcription in the adult heart. we aim to identify and to characterize this complex in order to find potentially interesting targets for pharmacological therapy preventing maladaptive cardiac remodeling and the onset of heart failure. results: we previously demonstrated that the krüppel-like-factor (klf ) is a βcatenin interaction partner and a cardiac specific nuclear inhibitor of the wnt/β-catenindependent transcription. because klf and β-catenin are ubiquitously expressed, we suggest the existence of cardiac specific co-factors responsible for cardiac specificity in this complex. we identified the basic leucine zipper and w domain containing protein (bzw ), a phylogenetically conserved protein, as a β-catenin and klf interaction partner using yeast-two-hybrid screen. in vitro overexpression experiments and coimmunoprecipitation validated these interactions, which were also confirmed by mass spectrometry. in the developing mouse embryo bzw mrna expression is detectable in the heart, neuronal tissue, somites, limbs and branchial arches as shown by whole mount in situ hybridization. in the adulthood expression of bzw is confined to the heart, predominantly in cardiomyocytes and in cardiac progenitor cells compared to cardiac fibroblasts (*p< . , cm n= , cfb n= , cpc n= ), and skeletal muscle. bzw was localized in both the cytosol and in the nucleus. mutation analysis showed the importance of the n-terminus of bzw , containing a putative bzip dna interaction domain, for the nuclear placement of the protein. bzw protein expression was significantly increased under cardiac wnt/β-catenin signaling activation in vivo in two mouse models (klf knockout (ko) mice **p< . n= , and in a cardiac specific β-catenin stabilized mouse model, **p< . n= ). a mouse model with constitutively bzw loss of function (bzw ko) showed cardiac specific upregulation of β-catenin on rna level (***p< . , p< . , ctrl n= , bzw ko n= ) and on protein level (**p< . , ctrl n= , bzw ko n= ). echocardiography analysis in eight-weeks-old bzw ko mice showed increased left ventricle wall thickness indicating a hypertrophic phenotype at baseline. we also observed increased levels of bzw expression in angiotensin ii treated mice as a model for cardiac hypertrophy (*p< . ctrl n= , angii n= ) as well as in human samples derived from patients with dilated cardiomyopathy and ischemic cardiomyopathy (*p< . ctrl n= , dcm n= , icm n= ). conclusion: these data demonstrated that bzw is associated with components of the canonical wnt cascade and suggest its relevance in the constitutive regulation of the wnt/β-catenin components specific in the heart. this study further contribute to the elucidation of the tuning of the wnt-off/-on states aiming to establish a proof-of-concept model for wnt-modulation as a therapeutic strategy in hypertrophic-induced heart failure. objective: sphingosine- -phosphate (s p) is involved in the regulation of cell growth, survival, migration and adhesion. it is formed by sphingosine kinases and degraded by phosphatases and s p lyase [ ] . mice that lack s p lyase are characterized by the accumulation of s p and sphingosine in their cells and tissues, and by lymphopenia, generalized inflammation, multiple organ damage, and a strongly reduced life span [ ] [ ] [ ] . on the other hand, embryonic fibroblasts from s p lyase-deficient mice (sgpl -/--mefs) are resistant to chemotherapy-induced apoptosis [ ] , in part due to an upregulation of multidrug transporters of the atp-binding cassette (abc) transporter family [ ] . interestingly, s p lyase-deficient mice have elevated plasma levels of cholesterol and triglycerides, while suffering from strongly reduced body fat [ ] . the aim of the present study was to analyze the link between s p lyase deficiency and altered cholesterol homeostasis using sgpl background: cardiac gene expression changes during cardiac development and under pathophysiological conditions. these alterations in gene expression are regulated by several processes and the exact regulation of gene expression is essential for the proper development and function of the heart. crucial steps in transcription regulation are rna polymerase ii (pol ii) recruitment and changes in pol ii activity. pol ii activity is tightly linked with phosphorylation at serine- (p-ser ) of the carboxyterminal domain of pol ii. thus, the aim of the present study was to identify cardiomyocyte-specific genomewide pol ii and p-ser -pol ii enrichments to get insight into pol ii activity and recruitment in development and disease. methods and results: to get insight into rna polymerase ii dynamics, genome-wide maps of rna polymerase ii occupancy were generated by chromatinimmunoprecipitation in cardiomyocyte nuclei purified from normal neonatal and adult mouse hearts. in addition, cardiomyocyte nuclei were obtained from adult hearts after weeks of pressure overload induced by transverse aortic constriction (tac). cardiomyocyte nuclei were isolated by magnetic beads with an anti-pcm antibody. nuclei were used for pol ii chromatin-immunoprecipitation followed by deep sequencing (chip-seq). to test if pol ii marks correlate with nuclear mrna expression in cardiomyocyte nuclei all coding genes were ranked according to their expression level. genes expressed in cardiomyocyte nuclei (> . fpkm, gene expression rank < , ) showed high pol ii enrichment at promoters as well as in genomic regions. many of the gene promoters showed high levels of pol ii accumulation at the transcription start site, as compared to genic regions, which have been associated with pol ii pausing. in contrast, p-ser -pol ii showed enrichment downstream of the transcription start site. the genomic region of troponin i type (tnni ) which is expressed in cardiac muscle only during development but not in adult cardiomyocytes, was enriched for pol ii in neonatal cardiomyocytes. at the tnni gene, pol ii was absent in adult or pressure-overloaded cardiomyocytes. in contrast, pol ii enrichment at the alpha actin (acta ) locus was only present in pressure-overloaded cardiomyocytes. these data are consistent with cellular rna-seq data showing an induction of acta after tac. furthermore no pol ii enrichment could be detected in the genomic region of biglycan (bgn), a matrix proteoglycan that is not expressed in cardiomyocytes. this confirms a high purity of cardiomyocyte chromatin. conclusions: this study provides, for the first time, cardiomyocyte-specific landscapes of rna polymerase ii occupancy in heart development and disease. cardiac myocyte maintenance dna methyltransferase is essential for embryonic heart development but is dispensable for cardiac function and remodeling postnatally t. nührenberg background: recent studies have identified dynamic changes in dna methylation in cardiac myocytes during development, postnatal maturation and in disease. however, the enzymes involved in shaping the cardiac myocyte dna methylome are only partially known. here, we explored the role of maintenance dna methyltransferase dnmt in cardiac development and in remodeling after chronic left ventricular pressure overload. methods: in mice, deletion of the dnmt gene was accomplished by use of two different cre recombinases. crosses of homozygous dnmt fl/fl mice with heterozygous dnmt fl/+ mice expressing a cre recombinase under control of the atrial myosin light chain gene promoter (myl -cre) resulted in embryonic deletion of dnmt (ko). embryos without myl -cre served as controls (ctl). embryos were dissected and genotyped at e . , e . , e . and e . . rna-seq and pyrosequencing of genomic dna was performed on e . hearts, histology on e . hearts and electron microscopic imaging on e . hearts. for deletion of dnmt in adult mice, homozygous dnmt fl/fl mice expressing an inducible cre recombinase (myh -mcm) were given tamoxifen i.p. over days. homozygous dnmt fl/fl mice not carrying myh -mcm as well as myh -mcm carrying mice without dnmt fl alleles were also injected with tamoxifen and served as controls. cardiac phenotyping including histology, echocardiography and qpcr was carried out without (sham) or with left ventricular pressure overload induced by transverse aortic constriction (tac). results: myl -cre mediated loss of dnmt resulted in progressive embryonic lethality with absence of living ko embryos after e . . ko embryos displayed loss of cardiomyocyte gene expression patterns, decreased promotor cpg methylation of aberrantly expressed genes and ultrastructural features of wide-spread cardiac myocyte cell death. in contrast, tamoxifen-induced ablation of dnmt in adult mice did not affect survival of ko mice. cardiac phenotyping of adult mice revealed no significant differences between ko and ctl mice under sham and tac conditions. conclusion: dna methyltransferase dnmt in embryonic cardiac myocytes is essential for proper heart development. in adult cardiomyocytes, dnmt is dispensable for normal cardiac function and for adaptation to chronic cardiac pressure overload. background: recent studies showed that mice with general deletion of the oxidoreductase tet involved in dna demethylation are embryonic lethal, the underlying cause remaining unknown. this prompted us to investigate wether embryonic lethality is caused by cardiomyocyte-specific loss of tet . methods: female mice homozygous for tet flox and male mice heterozygous for tet flox and heterozygous for myl -cre were mated and offspring were genotyped after weaning. mice homozygous for tet flox and heterozygous for myl -cre (ko) or homozygous for tet flox without myl -cre (controls) were sacrificed at weeks of age and ventricles were harvested. mrna of the ventricles was isolated and expression of cardiomyocyte-specific genes was evaluated by quantitative real-time pcr. cardiomyocyte-specific genomic dna from ko and control mice was obtained from facs-sorted cardiomyocyte nuclei and bisulfite-converted for analysis of dna methylation by pyrosequencing. results: ko mice showed embryonic lethality of nearly %. born ko mice developed without phenotypic abnormalities (normal heart weight/tibia length ratio) and displayed compensatory upregulation of tet and tet . cardiomyocyte genomic dna of ko mice showed significantly higher methylation levels in the body of the atp a gene and at the binding site of the transcription factor gata but not near the promotor and the binding site of the transcription factor tbx . higher methylation levels were not accompanied by changes in atp a expression. however, both myh and nppb were upregulated in ko mice compared to control mice. conclusions: our findings suggest that tet is involved in dna demethylation in cardiomyocytes. loss of tet expression resulted in embryonic lethality. compensatory upregulation of tet and tet isoenzymes may contribute to the incomplete penetrance of this phenotype. further studies are ongoing to investigate the functional relevance of tet in cardiomyocytes. to identify novel proteins secreted by the myocardium, we have previously conducted a genetic screen, which led to the identification of protease inhibitor (pi ). a recent gwas analysis showed an association of a genetic variant in the pi genomic locus (rs ) with chemerin plasma levels. here we tested the hypothesis that pi determines chemerin plasma levels through regulation of chemerin processing. we generated mice deficient for pi , which did not display an overt phenotype under basal conditions. plasma levels of chemerin were found significantly lower in pi -deficient animals compared to littermate controls. to investigate whether pi and chemerin interact, we performed co-immunoprecipitation experiments. indeed, we found pi to co-precipitate with chemerin from both murine plasma and cell culture supernatants. as chemerin is proteolytically processed and activated, we next asked whether the presence of pi would affect the processing of pro-chemerin to its processed forms in native tissue. western blot analysis on cardiac and adipose tissue lysates that detected both the unprocessed precursor and the processed forms of chemerin showed a significant shift towards the processed forms upon genetic deletion of pi . when we assayed for the activity of the chemerincleaving protease cathepsin k, we found recombinant pi to potently inhibit cathepsin k activity. taken together, we propose pi to act as a regulator of chemerin processing. the transient receptor potential canonical (trpc ) is a second messenger-gated cation channel, which mediates depolarization and ca + entry. it is known to be activated by diacylglycerol derivatives (dag, -oleoyl- -acetyl-sn-glycerol oag) [ ] in a pkc-independent manner and plays important roles in lung and kidney physiology. gain-of-function mutations in the trpc gene can cause focal segmental glomerulosclerosis (fsgs), a kidney dysfunction leading to end stage renal disease. [ ] thus, the discovery of potent inhibitors of trpc may help to develop new therapeutic strategies. urban et al. discovered that larixol, a natural product with a labdane skeleton found in the oleoresin of the european larch (larix decidua), blocks the oag-dependent activation of trpc . [ ] larixyl acetate, another component of the resin showed an even higher potency in trpc inhibition (ic = . µm) and a -fold selectivity compared to trpc . these findings led to the idea that further modifications of the larixol lead structure may reveal even more potent inhibitors. furthermore, changes in selectivity and efficacy of such compounds may also provide a deeper insight about relevant structural elements for channel binding. as larixyl carbamate was assumed to exhibit a higher metabolic stability as larixyl acetate, this compound was already investigated in previous studies. it showed a potent and subtype-selective inhibition of trpc . hence, the development of further carbamates was a priority objective. as an alternative to the use of different isocyanates for the introduction of a carbamate function at the c position of the molecule we found an elegant way via formation of an active ester with carbonyldiimidazole. this precursor allowed the design of several isosteric compounds like larixyl methylcarbamate, larixyl hydrazide and larixyl methylcarbonate, which were all able to block trpc with similarly low ic values. the introduction of more bulky side chains appeared to diminish the bioactivity of the compounds, the stereochemistry at the c position, however, seems to play no important role for the inhibition of trpc currents. larixyl methylcarbamate lead to trpc inhibition with an ic value of . ± . µm. compared to larixyl carbamate and larixyl methylcarbonate, which are also very potent blockers of trpc , this compound bears the benefit of high subtype selectivity towards trpc . even with concentrations up to µm of larixyl methylcarbamate, no complete inhibition of the ca + influx via trpc channels could be achieved. this fact distinguishes this larixol derivative as a very promising compound for further studies of trpc in health and disease. poisoning by organophosphorus compounds (opc) including pesticides and highly toxic nerve agents is based on irreversible inhibition of acetylcholinesterase (ache) resulting in an excess of acetylcholine causing accumulation. the subsequent overstimulation at nicotinic and muscarinic receptors finally leads to respiratory arrest due to paralysis of the respiratory muscles. therapy focuses on competitive antagonism at muscarinic acetylcholine receptors and reactivation of inhibited ache by bisquarternary pyridinium oximes. thereby, nicotinic malfunction is not directly approached. for that reason, an alternative strategy appears rational using nicotinic acetylcholine receptor (nachr) active substances to counteract the effects of accumulated acetylcholine and thus to restore the loss of function of nachrs. different bispyridinium-non-oxime-compounds (bps) have been demonstrated to be able to serve as target structures for the identification of new positive allosteric modulators of nicotinic receptors. unlike nicotinic agonists, positive allosteric modulators can reinforce the endogenous cholinergic neurotransmission despite of acetylcholine accumulation in the synaptic cleft. to this end, the following electrophysiological in vitro study investigated the effect of twelve diversely substituted bps on human α nachr using whole-cell patch clamping under voltage-clamping conditions (- mv) performed with planar electrodes in an automatic system (nanion technologies gmbh, munich). cholinergic currents of hα nachr that have been expressed in stably transfected cho cells were activated by the agonist nicotine. measurements of the effect of various bps concentrations in the presence of nicotine were performed to establish concentration-response relations. cholinergic inward currents were generated by human α nachrs in response to low nicotine concentrations. at high concentrations of the drug the currents were decayed reflecting both, desensitization of the receptors and presumably block of the open channel by high agonist concentrations. four out of twelve bps co-applicated with nicotine showed a concentration-dependent enhancement of peak agonist-evoked currents and, most pronounced, -tert-butyl-substitued-bp, also demonstrated a marked elongation of the evoked response. this suggests a positive allosteric effect of these compounds on the nicotinic receptor. however, at high bp concentrations in the presence of agonist, responses were decayed significantly, presumably resulting from an open channel block induced by bps. hence, further compounds have to be synthesized to identify promising candidate compounds for improvement of effective therapy against nerve agent poisoning. the transient receptor potential channels (trp) are a family of tetrameric nonselective cation channels, which are involved in a variety of physiological and pathological processes ( ). among the mammalian trp channels, the canonical channel (trpc ) is a ca + -permeable ion channel, which is predominantly expressed in the brain ( ) . many aspects of trpc function are still elusive although behavioral experiments with trpc -knock-out mice suggest a role in innate fear-response ( ) and some studies indicate a trpc -mediated down regulation of neurite outgrowth in nerve cells ( , ) . to elucidate trpc function on a cellular level, selective and potent compounds are required to acutely control channel activity. despite extensive research, trpc modulators often lack selectivity or exhibit toxicity, limiting their applicability in vivo ( , ) . thus, there is still a need for identifying novel and efficient trpc modulators. we therefore screened a compound library (chembionet) and identified a benzothiadiazine derivative (btd) as a novel, potent, and selective trpc activator. hek cells heterologously expressing trpc upon tetracycline induction (hek trpc ) show a btd-induced concentration-dependent activation in both ca + assays (ec = . µm) and in electrophysiological whole cell patch clamp recordings (ec = . µm). btd elicits currents with an n-shaped i/v curve, typical for trpc . the resulting activation is long lasting, reversible and sensitive to clemizole, a recently established trpc inhibitor ( ) . mtt assays revealed that incubating hek trpc cells for h with btd concentrations above µm results in a concentration-dependent decrease in viability and cell proliferation, indicating a ca + -mediated cytotoxic effect in consequence of sustained channel activity. non-induced control cells remain unaffected by btd at concentrations up to µm. ca + assays showed no influence of btd on closely related trpc channels, as well as trpc / / at concentrations up to µm. the same applied to more distantly related trpv and trpm channels. besides a homotetrameric organization, trpc subunits can also assemble to heteromeric channel complexes with their closest relatives trpc and trpc ( ) . trpc / and trpc / heteromers can also be activated by btd as evident from their typical i/v curves in patch clamp experiments, suggesting a high selectivity of btd for channel complexes bearing at least one trpc subunit. transient receptor potential canonical channels / and are controlled by membrane lipids and highly expressed in neuronal and cardiac tissues. the involvement of these channels in development and (patho)physiology of these tissues is well documented, while our understanding of structure-function relations, specifically in terms of the lipid sensing machinery, in these channel proteins is still incomplete. using a homology model of trpc , based on the recently available structural information on trpv , we performed structure-guided mutagenesis and identified a single residue in transmembrane domain (g ), which is conserved within the canonical family of trp channels. single point mutations at position in trpc largely eliminated lipid sensitivity. trpc g a expressed in hek cells was found resistant not only to activation via the phospholipase c pathway but also to direct administration of diacylglycerols. on the contrary, a synthetic agonist of trpc / / channels (gsk a) activated wild-type trpc and trpc channels as well as the respective lipid insensitive mutants (trpc g a, trpc g a ). interestingly, the synthetic activator was found to generate substantially enhanced trpc conductances in cells expressing the lipid-insensitive mutants as compared to wild-type proteins. closer inspection of sensitivity of the wt and mutant proteins to various gsk derivatives argue against a contribution of g to gsk recognition by trpc . our results demonstrate the existence of two different mechanisms of trpc / activation supposedly involving distinct gating movements in the channel complex. we suggest that lipid gating of trpc / involves a hinge-point and/or requires a certain level of flexibility within transmembrane segment s provided by g . lipids and synthetic activators of trpc / may be capable of initiating markedly different structural rearrangement in these channels. objective: organophosphorus compounds (opcs), i.e. nerve agents or pesticides, are highly toxic due to their strong inhibition potency against acetylcholinesterase (ache). inhibited ache results in accumulation of acetylcholine in the synaptic cleft and thus the desensitisation of the nicotinic acetylcholine receptor (nachr) in the postsynaptic membrane is provoked. as the therapeutic efficacy of oximes is limited, e.g. poisoning by soman or tabun, the direct targeting of nachr may be an alternative therapeutic approach. studies with the non-oxime bispyridinium compound (bp) mb ( , '-(propane- , -diyl) bis ( -tert-butylpyridinium) di(iodide)) demonstrated a therapeutic effect against soman in vitro and in vivo. consequently, studying the affinity of bps at muscle-type nachrs and functional effects are topics of interest. to identify potential candidates, homologous series of substituted and non-substituted analogues (linker c -c ) of mb were investigated by using binding and functional assays. experimental procedures: crude membranes from frozen electric organ of torpedo californica were purified by sucrose-gradient density centrifugation and used in both affinity and functional assays. in competition radio-ligand binding assays, the influence on [³h]epibatidine binding sites of torpedo muscle-type nachr was determined. functional assessments were carried out with a bilayer method to investigate the effect on the cholinergic signal induced by µm carbamoylcholine. results: bispyridinium compounds bearing unsubstituted pyridinium rings and long alkyl linkers (> c ) inhibited the binding of [³h]epibatidine and decreased the cholinergic signal of µm carbamoylcholine in the functional assay. mb and several bispyridinium structure analogues (mainly c -c linker) exhibited no regular displacement curves at [ h]epibatidine binding sites and enhanced the carbamoylcholine-induced signal. the results demonstrate that the described affinity and functional screening methods detected some structure-activity-relationships (sar). depending on linker length and substitution pattern, the investigated bispyridinium compounds seemed to interact as positive allosteric modulators. further research is necessary to verify this hypothesis. non oxime bispyridinium compounds with an effect on soman-blocked respiratory muscle function have no effect on normal muscle function the life threatening toxicity of organophosphorus compounds (op), like nerve agents or pesticides, lies in the inhibition of acetylcholinesterase (ache) which causes cholinergic crisis. the accumulated acetylcholine in neuromuscular synapses results in the desensitization of nicotinic acetylcholine receptors (nachr) and paralysis of respiratoric muscles. the -tert-butyl-substituted bispyridinium compound mb showed therapeutic efficacy in soman and tabun poisoned guinea pigs in vivo. partial restauration of neuromuscular transmission by bispyridinium compounds (bp), e.g. mb or mb , could also observed in soman paralysed respiratory muscles in vitro and was partly attributed to an interaction of bp with nachrs. however, it is unknown, whether these bp might affect normal respiratory muscle function in the absence of cholinergic crisis. therefore this study investigated the effect of bp on physiological rat diaphragm muscle function. force generation of rat diaphragm hemispheres was determined after incubation with increasing bp concentrations ( - µm) and compare to sham treatment. the diaphragm hemispheres were stimulated every ten minutes by an indirect electrical field ( , , hz). muscle force was analyzed as time-force integral and is expressed as percentage of the individual control values, measured at the outset of the experiment. the muscle force dropped during the experiment. the application of the bispyridinium compounds mb ( , ′-(propan- , -diyl) bis ( -tertbutylpyridinium) di(iodide)) and mb ( , ′-(propan- , -diyl) bis ( -ethylpyridinium) di(iodide)) in the tested concentration range ( - µm) did not change muscle force production compared to the sham treated muscle. this was equally true for low ( hz) and high ( and hz) stimulation frequencies. this study showed that bispyridinium compounds which can partially reverse somaninduced neuromuscular block in rat diaphragms show no effect on respiratory muscle function in absence of the op-induced neuromuscular block. these results suggest that the bp tested in this study interacted with desensitised nachrs only, but do not affect physiological neuromuscular transmission. this effect needs to be investigated with further, promising bp compounds. interaction of recombinant pain-relevant atp-and proton-gated ion channels in an expression system; potentiation of the p x receptor-induced current by the opening of asic channels g. stephan , p. illes universität leipzig, rudolf-boehm-institut für pharmakologie und toxikologie, leipzig, germany the p x receptor (r) is a ligand-gated cationic channel, which is activated by extracellular atp. the acid sensing ion channel (asic ) belongs to the enac/degenerin family and is gated by extracellular protons. despite their different amino acid sequences both ion channels share the same structure and pore architecture, by i.e. consisting of three identical subunits. besides, they are both located at partially overlapping subpopulations of dorsal root ganglia neurons and are implicated in acidic pain signaling. consequently, their physical interaction in the cell membrane or even the formation of heteromeric receptor channels from p x and asic subunits has to be taken into consideration. we transfected rat (r)p x r and rasic constructs individually or together in a ratio of : into cho cells. we further used the whole cell patch clamp technique to analyze the current responses either elicited by the application of α,β-methylene-atp (α,β-meatp) or by a decrease in the extracellular ph value. the functionality of the individually transfected p x r-and asic -constructs was verified by recording concentration-response curves for the agonists α,β-meatp and protons, respectively. after co-transfection of both ion channels, a ph-shift from . to . caused a rapidly desensitizing current response and a subsequent strong potentiation of the α,β-meatp-induced current. an even larger potentiation was achieved after a decrease of the ph value to . . the opening of asic channels failed to facilitate the p x r current when -guanidine- -methylquinazoline was used to stimulate a non-proton ligand-sensor of asic . then, we substituted ca + in the extracellular medium by ba + or decreased the intrapipette concentration of egta, to modify the free intracellular ca + concentration. in cells individually transfected with the receptor-channels, external ba + increased the effect of α,β-meatp but decreased the effect of protons. the lowering of intrapipette egta modified p x r-and asic -specific currents in a similar manner as external ba + . in cells co-transfected with p x r/asic , the ionic manipulations mentioned above abolished the potentiation of the α,β-meatp currents by asic activation. taken together, our results suggest that p x r and asic interact with each other, since the activation of asic had a marked impact on the p x r specific current response. further experiments are required to clarify the mechanism of this interaction, although it has been shown that extra-and intracellular ca + and the proton sensor of asic appear to critically participate in this process. university of duisburg-essen, institute for anatomy, essen, germany background: the sperm acrosome reaction is an all-or-none secretion process, mainly following the conserved principles of calcium-regulated exocytosis in neurons and neurosecretory cells. however, the relationship between the formation of hundreds of fusion pores and the required mobilization of calcium from the lysosome-related acrosomal vesicle has only been partially defined. hence, the second messenger, nicotinic acid adenine dinucleotide phosphate (naadp), known to promote efflux of calcium from lysosome-like acidic compartments, was analyzed for its ability to trigger acrosome reaction in mouse sperm. in addition, the expression of two-pore channel (tpc) proteins, which are primarily localized in lysosome-related acidic organelles and which present potential molecular targets of naadp were examined in mammalian spermatozoa. methodology/ principal findings: our results show that treatment of spermatozoa with naadp resulted in a loss of the acrosomal vesicle, which shows typical properties, described for tpcs: (i) registered responses were not detectable for its chemical analogue nadp, and (ii) where blocked by the naadp antagonist trans-ned- . in addition, (iii) two narrow bell-shaped dose-response-curves were found, with maxima either in the nanomolar or low micromolar naadp concentration range. performing immungold-electron microscopy with a tpc specific antibody, a co-localization with naadp-binding at the acrosomal region was detectable. moreover, quantifying loss of the acrosomal vesicle in tpc null sperm upon application of different naadp concentrations, responsiveness to low micromolar naadp concentrations was completely abolished. conclusions/significance: our finding that two convergent naadp-dependent pathways are operative in driving acrosomal exocytosis and that zona pellucida induced acrosomal exocytosis is prevented by trans-ned- support the concept that both naadp-gated cascades match local naadp concentrations with the efflux of acrosomal calcium, thereby ensuring reliable and complete fusion of the large acrosomal vesicle. since the acrosome reaction shares the same basic sequence of events typical for the conserved process of calcium regulated exocytosis, such as tethering, docking, priming and final vesicle fusion, the sperm model system may also be useful to comparatively examine whether the same convergence of naadp-dependent pathways is also operative in cellular systems with many secretory vesicles. walther-straub-institut, münchen, germany trpv channels are members of the vanilloid family of trp proteins. the channel is nearly ubiquitously expressed and can be found in brain, kidney, skin, heart, blood vessels as well as in the lung. pulmonary expression of trpv has been identified in endothelial cells ( ), epithelial cells ( ) and arterial smooth muscle cells ( ) . most interestingly, the channel is known to be involved in the development of several lung diseases such as cough, asthma and pulmonary edema formation, due to its activation by heat, changes in osmolarity and shear stress (reviewed in ). it is a matter of debate however if trpv activation in pulmonary endothelial as well as epithelial cells induces disruption of the barrier and an increased fluid leak into the alveolus as described for trpc ( ) which is also expressed in both cell types. to analyze the potential role of trpv on ischemia-reperfusion-injury(iri)-induced pulmonary edema formation we utilized the isolated perfused mouse lung model. much to our surprise, we detected a significantly enlarged edema formation after minutes of ischemia in trpv -deficient mice in comparison to wild-type (wt) mice. this effect was observed by constant weight measurements as well as wet-to-dry ratio gain and was not dependent on the initial perfusion rate. most interestingly, edema formation of trpv /trpc double deficient lungs was indistinguishable from wt lungs, indicating antagonizing effects of both channels, because trpc deficiency protected lungs from iri-induced edema ( ) . moreover, we identified reduced expression levels of aquaporin in trpv -deficient lung lysates compared to wt lungs. these findings raise the intriguing possibility that trpv might be involved in the regulation of aquaporin expression in lung endothelial cells. endosomes and lysosomes are cell organelles involved in transport, breakdown and secretion of proteins, lipids, and other macromolecules. endolysosomal dysfunction can cause storage disorders such as mucolipidoses, sphingolipidoses, or neuronal ceroid lipofuscinoses, but is also implicated in the development of metabolic and neurodegenerative diseases, retinal and pigmentation disorders, trace metal dishomeostasis, infectious diseases, and cancer. endolysosomal ion channels and transporters are highly critical for the tight regulation of the multiple endolysosomal fusion and fission processes including endo-and exocytotic events as well as the regulation of proton and other ionic concentrations in the lumen of endolysosomal vesicles. methods to patch-clamp endolysosomal organelles are continuously improving. yet until now it has not been possible to selectively enlarge endosomal or lysosomal organelles with pharmacological tools for patch-clamp experimentation. we show here by using a combination of two small molecules that we can selectively enlarge early endosomes to a degree sufficient for patch-clamp experimentation. the ability to more selectively patch-clamp intracellular organelles will substantially improve the functional investigation of endolysosomal ion channels under physiological and pathophysiological conditions. the transient receptor potential (trp) channels are a superfamily of non-selective ion channels involved in a variety of physiological processes and in the pathgenesis of many disorders. in the kidney, trp channels have been implicated to be involved in diabetic nephropathy, focal, segmental glomerulosclerosis, polycystic kidney disease, hypomagnesemia with secondary hypercalcemia and idiopathic hypercalcuria. the melastatin-like trp channel subfamily (trpm ) has been shown to be expressed in human kidney , . using newly developed anti-trpm antibodies, we are able to visualize trpm protein in epithelial cells of proximal tubule as well as collecting ducts in the mouse kidneys. therefore, we compared renal function of male, five month old mice lacking trpm (trpm the atp-gated p x receptor (p x r) is a non-selective cation channel widely expressed in epithelia, endothelia, and cells of hematopoietic origin. it plays a central role in cytokine release and studies in p x -/animals indicate its involvement in inflammatory and neurodegenerative diseases. in addition, accumulating data suggests a functional role in neurons and its involvement in neurotransmitter release in the brain. however, despite its importance as a drug target, its precise localization and its molecular and physiological functions remain poorly understood. in particular, the location and function of p x r in neurons remain a matter of ongoing debate. to clarify the cellular and subcellular distribution of the p x r and to investigate its physiological and pathophysiological role in the brain we generated bac transgenic mouse models in which murine polymorphic variants of egfp-tagged p x r are overexpressed under the control of the endogenous p x promoter. the egfp-tagged p x rs are efficiently overexpressed in the plasma membrane and can be directly visualized by green fluorescence or indirectly by anti-egfp antibodies. the obtained mouse lines show different expression levels but identical expression patterns with predominant expression in the cerebellum, hippocampus, and thalamus. using cell type-specific markers, p x -egfp was identified in almost all microglia and subpopulations of oligodendrocytes and astrocytes in the brain. in the spinal cord, numerous astrocytes in the white matter showed egfp immunoreactivity. so far, no egfp immunostaining was found on map -and neun-positive cells indicating that under non-pathological conditions, the p x receptor is not expressed in neurons of the cns. interestingly, a higher expression level of cd protein was observed in the p x r-overexpressing mice. these results suggests that overexpression of p x rs alone is sufficient to induce microglia activation, even under non-pathological conditions. since cd primarily localizes to lysosomes and endosomes, this further supports a role of the p x r in the regulation of phagocytosis. to validate these data, conditional knockout mice are generated. the current status of the project will be presented. the two-pore channels (tpcs) -tpc and tpc -are located in membranes of intracellular organelles of the endo-lysosomal system. the tpc-protein-monomer contains two homologous domains with six transmembrane α-helices each. a functional tpc probably consists of a dimer of two tpc-proteins resembling an ion channel architecture with the typical four domain organization like voltage gated na + or ca + channels or such as trp channels. due to their biophysical properties, tpc and tpc are assumed to be involved in the efflux of ca + from intracellular organelles and thereby contribute to fusion/fission processes of endosomes and lysosomes. thus, tpcs are supposed to be important regulators for vesicle trafficking, sorting and degradation/recycling processes. recently, it was shown that virus entry and replication of certain strains of filoviridae -such as ebola -depends on functional tpcs and that either block or genetic inactivation of tpcs reduces virus infectivity. a large family of bacterial protein toxins elicit their effects by modification of intracellular target proteins of host cells. these toxins are taken up by receptor mediated endocytosis and follow different endosomal routes to reach their final cytosolic destination. these toxins principally use two different intracellular routes: the first group uses an entry route via early or late endosomes (short-trip toxins), the second group takes a retrograde route via endosomes, golgi network and the endoplasmic reticulum (long-trip toxins) to get access to the cytosol. translocation of short-trip toxins -such as diphtheria toxin (dt), pasteurella multocida toxin (pmt) and bacillus anthracis lethal factor (pa/lf) -from early and late endosomes into the cytosol is driven by ongoing acidification. long-trip toxins -including cholera toxin (ct) -are retrogradely transported after endocytosis via the golgi apparatus to the endoplasmic reticulum (er). within the er a specific peptide-motif allows the translocation into the cytosol. due to the role of tpc for vesicle fusion & fission processes we investigated a potential impact of tpc on the uptake of bacterial toxins. first we determined the precise localization of tpc in intracellular compartments. to deduce its role for trafficking processes we performed co-localization and correlation studies with a whole set of established markers such as rab-gtpases and pips. second we intoxicated wild-type and tpc deficient cell lines with different bacterial protein toxins such as cholera (ct), diphtheria (dt) or pasteurella multocida (pmt) toxin. using cell viability and other intoxication assays we investigated the consequences of tpc -deletion on bacterial toxin uptake, translocation and cytotoxicity. universität des saarlandes, institut für experimentelle und klinische pharmakologie und toxikologie, homburg/saar, germany trpm ion channels are considered to be involved in hormone release from pancreatic islets and the pituitary gland , . the trpm gene encodes a number of different splice variants that differ in their permeation properties and their activity in response to agonists , . variations include the presence or absence of five stretches of to amino acid residues within the aminoterminus, long or short pore loops and long or truncated carboxytermini . furthermore, three different aminotermini of human and mouse proteins have been described , but presumably these differences are caused by the activity of different promoters. screening of a mouse pituitary gland cdna library identified variants that differ in exons , , , and . however, only variants bearing the short, ca + permeable pore loop were detected. ´ rapid amplification of cdna ends ( ´race) revealed that trpm proteins of the pituitary gland carry truncated c-termini exclusively. ´race identified five independent regions of transcription initiation within the trpm gene implying the presence of five independent promotors. the different transcripts encode four trpm amino termini α, β, γ and δ of - amino acid residues including those described in humans (β,γ). however, the activity of these variants after stimulation with pregnenolone sulfate varied largely just as their frequency in the pituitary with shortened γ-variants being most abundant (~ %). two-pore channels (tpcs) are a small family of ion-channels found throughout the endolysosomal system of eukaryotic cells. phylogenetically tpcs belong to the voltagegated ion channel superfamily sharing common traits with ca v / na v and trp channels. tpcs show a duplicated architecture with two homologous trans-membrane domains. each domain is build up by six membrane spanning alpha helices linked by short loops. it is very likely that tpcs form dimers maintaining the four-fold symmetry found in other members of the voltage-gated ion channel superfamily. due to their localization in the endolysosomal system tpcs are not accessible to conventional patch clamping. to investigate tpcs electrophysiological properties we use black lipid bilayer measurements. purified channels are integrated into an artificial phospholipid bilayer that separates two chambers enabling us to apply different buffers. upon activation by naadp ions flow through the channel and can thereby pass the diffusion barrier. the movement of charged molecules through tpcs results in currents which can be amplified and recorded. the controlled environment of the lipid bilayer setup allows testing of different ions as well as putative activating and inhibitory substances. one of the major drawbacks of conventional lipid bilayer setups are long preparation times between each measurement. stability of the phospholipid bilayer can pose another issue. often several membranes have to be established before a measurement can be performed making it very time consuming to achieve adequate experiment numbers. new multichannel systems resolve this issue supporting fast formation of bilayers while allowing measurement of up to different bilayers at a time. here we utilize the "orbit" multichannel system with a meca chip by ionera to measure tpc channel activity. we will present data generated by the "orbit" system and a conventional bilayer setup using different channel constructs and charge carriers. cardiac action potentials are generated and propagated through the coordinated activity of multiple ion channels, including voltage-gated sodium channels (nav ) and potassium channels (like kv , kv ). the voltage-gated na + channel nav . initiates the cardiac action potential (ap), is essential for rapid depolarization, and is also known to control the ap duration in cardiomyocytes. the voltage-gated k + channel kv . is responsible for the early repolarization of action potentials in human heart. similar to many membrane proteins, nav . and kv . have been found to be regulated by several interacting proteins. the transmembrane β subunit dipeptidyl aminopepidase-like protein (dpp) is known to interact with the kv . channel complex, modulating kinetics and voltage dependence. the overexpression of dpp in ventricular cardiomyocytes of rats revealed strong reduction of ap amplitude and significant slowing of ap upstroke velocity and ap duration, which could not be explained by the effects on cardiac kv channels. to study the potential influence of dpp on nav . channels, we performed whole-cell patch-clamp analysis of transiently transfected cho-k cells, expressing scn a alone or with dpp . surprisingly, we observed significant effects of dpp on nav . channel voltage dependence and kinetics. thus, the co-expression of dpp significantly shifted the half-maximal voltage of steady-state activation and steady-stateinactivation to more positive potentials compared to nav . channels alone. in addition we analysed the effects of dpp on the kinetics nav . currents. while time to current peak was not affected in cells co-expressing nav . and dpp compared to nav . alone, dpp slightly accelerated the inactivation. in addition, the time course of recovery from inactivation was clearly accelerated in cells expressing both nav . and dpp compared to nav . alone. in summary, we provide first evidence that dpp not only interacts with kv channels, but also influences nav . channels modulating the depolarization as well as the early repolarization phase of the cardiac ap. therefore, it becomes likely that these ion channels are part of large, multi-protein complexes, and that the pore-forming subunits kv . and nav . behave very differently depending on the expression of its associated proteins like dpp . cardiac fibroblasts (cf) comprise the most abundant cell type of the mammalian heart and it is known that they contribute to maladaptive cardiac remodeling processes. in response to pressure or volume overload, ischemia-reperfusion injury or myocardial infarction, cardiac fibroblasts proliferate and transdifferentiate into myofibroblasts which produce collagen and pro-hypertrophic cytokines influencing cardiomyocyte function and size. it was shown that β-adrenergic stimulation of cfs with isoproterenol leads to angiotensin ii (at-ii) production and autocrine stimulation of these cells (jaffre et al, ). activation of phoypholipase c triggered by at-ii leads to formation of inositol trisphosphate (ip ) and subsequent release of calcium from intracellular stores as well as calcium entry across the cell membrane. the focus of our research is the identification of the plasmalemmal channel proteins such as trpc channels mediating this calcium entry, and whether these calcium entry pathways in cfs contribute to pathological remodeling. to date the precise role of calcium entry for these pathological processes is largely unknown. trpc channels are candidates for the analysis of calcium homeostasis in cf. recently, we showed that trpc /c -deficient mice are protected from maladaptive cardiac remodeling after neurohumoral stimulation or pressure overload, respectively, which can be explained by a significant reduction of a background ca + -entry (bcge) pathway in cardiomyocytes; this bgce is enhanced by stimulation with agonists such as isoproterenol or angiotensin ii and it critically depends on trpc and trpc (camacho londoño et al., ) . nevertheless, the role of trpc /c for calcium homeostasis in cfs has not been analyzed so far. we established an in vitro model that allows the analysis of calcium release and entry triggered by several (patho)physiological agonists in cultured primary adult cfs from mice. cfs were isolated using langendorff-perfusion and were cultured for maximal days. our results show that there is no difference in nm at-ii induced ca + -release or ca + -entry in trpc /c -deficient cf compared to wt. to evaluate whether the lack of trpc /c can be compensated by other trpc channels we currently analyze cfs from trpc hepta ko mice lacking all seven trpc channel proteins concerning at-ii induced ca + -release and ca + -entry and we will also analyze the influence of other agonists on cfs which are known to evoke a longer lasting rise in the [ca + ] i like isoproterenol, -ht, thrombin and endothelin- . cardiovascular and metabolic diseases are currently the primary cause of morbidity and mortality in the western world and are spreading to the rest of the world following globalization. adipose tissue, in particular perivascular adipose tissue (pvat) is recognized as an important player in the development of these diseases. the release of relaxing factor(s) from the pvat has been a matter of interesting and highly spirited debates about its nature, the channels that govern its activities and its role in vascular dysfunction. data from our laboratory indicate that adipose-derived relaxing factor (adrf) is an important player, however the potential channels necessary for its downstream activities are still under study. our recent research primarily focuses on kv . channels, which are known to be expressed in vascular smooth muscle cells. k v . voltage-gated potassium channels are expressed in vascular smooth muscle cells (vsmc) of diverse arteries, including mesenteric arteries. based on pharmacological evidence using r-l (k v . opener), hmr , chromanol- b (k v . inhibitors), these channels have been suggested to be involved in the regulation of vascular tone. however, the specificity of these drugs in vivo is uncertain. we used kcnq -/-mice to determine whether k v . plays a role in the regulation of arterial tone. we found that r-l produces similar concentration-dependent relaxations (ec ~ , µm) of wild-type (kcnq +/+) and kcnq -/-arteries pre-contracted with either phenylephrine or mm kcl. this relaxation was not affected by µm chromanol- b, µm hmr or µm xe (pan-k v blocker). the anti-contractile effects of pvat were normal in kcnq -/-arteries. chromanol- b and hmr did not affect the anti-contractile effects of perivascular adipose tissue (pvat). isolated vsmcs from kcnq -/-mice exhibited normal peak k v currents. the k v . - opener retigabine caused similar relaxations in kcnq -/-and wild-type vessels. we conclude that k v . channels are apparently not involved in the control of arterial tone by alpha adrenergic vasoconstrictors and pvat. in addition, r-l is an inappropriate pharmacological tool for studying the function of native vascular k v . channels in mice. introduction: according to international guidelines [ ] [ ] [ ] [ ] [ ] [ ] [ ] , both human and animal skin in vitro models have been used and validated to predict percutaneous penetration in humans. excellent correlations have been found for domestic pig as surrogate for human skin [ ] [ ] . material and methods: tissue the skin samples are descended from female pigs of german landrace ( kg weight, approx. month). process approved under german welfare law. after narcotization and euthanization the animals were shaved with an electric shaver, washed and dried. microbiological investigation swabs from different skin area (fig. ) were taken before next preparation step. skin areas were cut, stretched and subcutaneous fatty tissue was carefully removed. the skin was harvested at thickness of . µm by dermatome. after dermatomization, samples were taken for histological examination. skin disks of mm were punched out from the frozen skin stripes and stored at - °c. hplc and skin absorption waters corporation hplc containing sample manager, binary gradient pump, pda detector (optional: electron spray mass spectrometer); column: nucleodur® - c ec mm x . mm id. hanson microette™ vision® diffusion test system (hanson vision® autoplus™ autosampler/autofill™ collector, -cell drive system with vertical diffusion cell "standard". the permeation experiment was performed over a period of h at °c. the dosage compartments of each cell were filled with approximately µl of the caffeine solution ( mg/ml). samples of ml are taken after , , , and hours from receptor medium of each cell. aliquots from each vdc are analyzed by hplc in duplicate. microbiology staphylococcus spp. were found explicitly on pig skin surface. bacteria are facultative anaerobe, gram-positive bacteria that are physiologically colonizing the skin, oropharynx and the gastrointestinal tract. histology and skin thickness he staining and mechanical skin thickness determinations confirmed intact dermatomizing process of skin (fig. ) . thickness was in the order of magnitude between to . µm and intra variations were less than %. caffeine skin absorption permeability coefficients and lag-phases recorded are in the same order of magnitude of previous work [ ], demonstrating intact barrier properties of the membranes after month storage process. until today, intra-assay variations are superior or equal to interregional and inter-animal variations. discussion: well characterized dps provides ready to use research tool for locally and systemic skin investigations. ongoing experiments will cover skin structure analysis by raman spectroscopy, biophotonics and storage impact on skin barrier function. respirable biopersistent granular dusts (gbs) should fulfill the criteria of i.) a negligible solubility in physiological lung fluid that ii.) do not exhibit a specific surface chemistryrelated toxicity at volumetric non-overload conditions in lungs. in , the mak commission derived a new threshold value of . mg/m for gbs with a density of , recognizing that at this concentration a chronic inflammation and increase of the lung cancer risk will not occur. -the objectives of the project were i.) to determine an experimental dissolution value for 'low soluble' gbs using six candidate dusts; ii.) in addition, to measure the inflammatory response in lung lavage fluid and to decide on the criterion 'inert dust'; iii.) to investigate whether nanoscaled dusts could possibly fulfill the criteria to be included in the gbs class. -six micro-and nanoscaled dusts (one of them a well-characterised inert tio dust (microscaled; rutile modification) were compared analysing the solubility in the lung fluid (day , and ) and the lung toxicity after intratracheal instillation in rats (day and ): tio (rutile, micro), tio (anatase, nano), eu o (micro-nano mixed), baso (micro), zro (micro) and amorphous sio (nano). two doses of . and . µl per rat were administered to wistar rats; these volume doses resulted in a non-overload and moderate overload of lungs, respectively. -the differential cell count showed only slight inflammatory cell levels after treatment with tio (rutile) and baso (pmn < % after days in the low dose group; < % in the high dose group; full recovery after days). in contrast, the tio (anatase) showed a stronger response (pmn > % after and days). the rare earth eu o (micro-nano) dust showed the strongest effect (approx. % pmn after and days) including a red-coloured lung lavage fluid. µ-zro and amorphous sio showed a strong acute response after days, however, mostly complete recovery after days. the low solubility criterion was met by the following dusts: tio (both) and zro . -similar volumetric lung burdens were deposited in a parallel validation experiment ( -day subacute inhalations). overall the physiological inhalation route confirmed the results obtained in the instillation study, thus suggesting the applicability of the latter as a tool for identification for gbs dusts. however, for nanoscaled dusts an individual toxicological characterization seems to be adequate. polycyclic aromatic hydrocarbons (pah) represent a complex mixture of compounds and occur in considerable amounts as contaminants in the environment and food. some pah have been demonstrated to be carcinogenic and mutagenic. benzo[a]pyrene (bp), the most known and studied member of the potent carcinogenic pah, is classified by iarc as group carcinogen and is present in a wide variety of food items. however, other non-carcinogenic pah such as pyrene (pyr) and fluoranthene (fa) are also found in substantial amounts in the diet and are strongly suspicious to cause interactive effects. reporter gene assays were used for analyzing interactive effects of a ternary mixture including bp, pyr and fa in relative proportions occurring in food on the nuclear receptors aryl hydrocarbon receptor (ahr) and constitutive androstane receptor (car). the observed activations were verified at the gene expression level in the human hepatoma cell line heparg. beside the well characterized ligand bp, µm of pyr and µm fa also activated the ahr, even though to a much lesser extent. no significant higher activation over the level of bp alone was reached when testing different pah mixtures. however, in heparg cells the analysis of cyp a gene expression as a model target gene of ahr showed synergistic effects after pah co-exposure. in addition, the activation of human car was analyzed. pyr and fa are each strong agonists, whereas bp was less potent. for the ternary pah mixture with bp a strong decrease of the induction was observed. this inhibiting effect was verified at the mrna level using the model car target gene cyp b in heparg cells. in conclusion, really occurring mixtures with non-carcinogenic pah can modulate the effects of carcinogenic pah. such effects warrant to be investigated in more detail to enhance our knowledge of interaction of pah mixtures at the molecular level. cytochrome p enzymes and transporters are important for the turnover of pharmaceutical compounds. their expression levels and activity influence bioavailability and convey drug-drug interactions. moreover, transporters mediate barrier maintenance of several organs such as blood-brain-barrier and placenta-barrier. overexpression of export transporters in tumors can lead to multiple drug resistance. however, membrane associated proteins are difficult to quantify by conventional bioanalytical methods such as sandwich immunoassays because of their hydrophobicity. antibody -based analysis of cytochrome p enzymes and transporters is challenging due to their sequence homology. therefore, we developed a test system for protein quantification which combines the sensitivity of immunoprecipitation and the specificity of mass spectrometry: this method is especially convenient for hydrophobic proteins because denatured samples are analyzed on peptide level. one peptide from each protein, which can be assigned unambiguously, is identified via tandem ms and quantified by means of an isotope labeled reference. prior to ms-read-out, the peptides are enriched by antibodies which recognize a very short c-terminal epitope. these epitopes are selected in such way that they are common in peptides derived from target proteins and therefore allow the analysis of protein groups with few antibodies. the major advantage of this method is that whole cell or tissue lysates -without preparation of microsomal fractions -can be used for quantification by lc-ms. also, samples from different model organisms can be analyzed with the same assay which enhances the comparability of experiments. physiologically based toxicokinetic modeling (pbtk) is an in silico tool to predict compound kinetics based on test substance related properties and physiological parameters of the organism. pbtk is a key element for inverse dosimetry to relate effect concentrations in vitro to external, e.g. oral doses. in our investigations, we use compartment models for the rat including adrenals and testes or ovaries. test substance specific properties taken for pbtk modeling are molecular weight and logp o/w as well as ivis based tissue specific partition coefficients, hepatic clearance, intestinal permeability and plasma protein binding. berkeley madonna software was applied to solve consequent differential equations. here we present the above described model for the test substances bisphenol a (bpa), fenarimol (fen) and ketoconazole (keto). using the lowest effect concentrations (loecs) of bpa, fen and keto from ) an in vitro yeast based assays with human estrogen and androgen receptor combined with a reporter gene and ) the interaction of steroidogenesis model calculations were made to relate in vitro concentrations to oral doses in the rat. model calculations, based on in vitro loecs of µm (bpa), µm (fen) and . µm (keto), for concentrations in target organs resulted in estimated oral loels of , and . mg/kg. when calculations were made for plasma levels oral loels were estimated to be , and mg/kg for bpa, fen and keto, respectively when compared to existing in vivo data with endocrine related loels of mg/kg bw day for bpa ( ), mg/kg day for fen ( ) and mg/kg day for keto ( ) , it can be concluded that for the exemplary test substances addressed, ivis related risk assessment approaches based on target tissues seems overpredictive, whereas plasma related loels were closer to the in vivo situation, to study the activation of the nuclear receptor pxr a gal /uas-based pxr transactivation assay was used. the pxr-mediated induction of cyp a promotor activity was investigated using a pxr-dependent cyp a reporter gene assay. cyp a induction was analyzed at the mrna and protein levels in hepg cells using qpcr and western blot. to cover the most frequently occurring pa structures (retronecine, heliotridine and otonecine type as well as monoester, open-chain diester and cyclic diester) the four pa senecionine, heliotrine, echimidine and senkirkine were selected as representative pa for initial analyses. out of the four investigated pa only echimidine activated pxr. accordingly, pxrmediated induction of cyp a promotor activity could only be detected for echimidine. cyp a induction by echimidine was verified at the mrna and protein level in hepg wildtype and hepg pxr-overexpressing cells. taking heinrich-heine universität düsseldorf, institut für toxikologie, düsseldorf, germany introduction: in higher concentrations, the blood pressure regulating hormone angiotensin ii (ang ii), leads to vasoconstriction, hypertension and oxidative stress by activation of the renin angiotensin system (ras). here we investigate if nadphoxidases are responsible for ras-mediated oxidative stress in kidney and heart. nadph-oxidases ( isoforms are known, nox - , duox & ) are membrane-bound enzymes that produce reactive oxygen species (ros) for example during the immune response and cell signaling. material and methods: to clarify the role of nadph-oxidases, wildtype mice and nox -, nox -and nox -deficient mice were equipped with osmotic minipumps, delivering ang ii in a concentration of ng/kg during days to stimulate high blood pressure. kidney and heart were investigated for steady-state ros levels and dna damage (dna single and double strand breaks). results: in wildtype mice, ang ii leads to hypertension, a declined renal function, formation of ros in kidney and heart and to dna single and double strand breaks in the kidney. all nox-knockout mice exhibited ang ii-mediated hypertension and albuminuria. the lack of nox and nox could neither protect from the formation of oxidative stress in the kindey nor from dna double strand breaks in the kidney. initial findings from the nox -knockout mouse do not show an increase in dna double strand breaks in the kidney. discussion: contrary to published results of nox -knockout mice, we observed a constant rise in blood pressure over the treatment period compared to the control. this can possibly be due to different ang ii doses. in nox -knockout mice we observed increased oxidative stress and increased renal dna damage already in untreated control animals, which is in line with reports suggesting a protective effect of nox . conclusion: separate eliminations of nadph-isoforms did not allow the identification of the enzyme which is responsible for ang ii-induced oxidative stress. a possible explanation is that oxidative stress is caused by more than one nox-isoform or other enzymes like xanthine oxidase or nitrogen synthase take major part in the formation of ros. of mice (rats, cats, dogs, monkey) and men -how to measure kidney biomarkers across species introduction and objectives: there is a need for reliable biomarker assays to detect organ toxicity induced by drug candidates. in the last years about drugs were withdrawn from the market due to liver and/or kidney damage. for the detection of druginduced organ injury, safety-tox studies in rodents, dogs, non-human primates and humans are mandatory in the drug development process. currently, several protein biomarkers for kidney, liver and cardiovascular organ toxicitity are being clinically validated by international consortia like the safer and faster evidence-based translation (safe-t) or the predictive safety consortium (pstc). we are developing mass-spectrometry-based immuoassays suitable for the detection of these markers in animal models to support these efforts method: urinary proteins are proteolytically digested to peptides using trypsin. subsequently synthetic isotope-labelled peptide standards are spiked in at known concentrations. we employ multi-specific antibodies (txp-antibodies) targeting cterminal amino acid motifs for the enrichment of peptides derived from the protein biomarkers. finally, the protein biomarkers are quantified using nanolc-parallel reaction monitoring-ms. the use of our group-specific txp-antibodies allows protein analysis of samples from different species using the same antibody. results and discussion: we established an ms-based immunoassay platform for the analysis of kidney (diki) injury protein biomarkers in urine across species; human, cynomolgus, mouse, rat and dog. we analyzed the potential diki biomarkers aquaporin , podocin, synaptopodin, retinol-binding protein , clusterin and osteopontin in urine samples from toxicity studies in cynomolgus monkeys, rodents and humans. conclusion: the application of group-specific txp-antibodies and mass spectrometry allows the quantification of biomarkers in urine of all relevant model organisms. the results strongly support the validation of translational drug-induced organ injury protein biomarkers. although effective anticancer-therapeutic regimen are available, they are accompanied by severe adverse effects on normal tissue, for instance chemotherapy induced peripheral neuropathy (cipn) caused by platinum compounds. the pathophysiology of this clinically highly relevant side-effect is still unknown and neither prophylaxis nor specific treatment is available. therefore, further research elucidating the underlying molecular mechanisms of platinating anti-tumor drugs leading to cipn is required as basis for future development of preventive or therapeutic strategies. in general, platinum compounds lead to cell death mainly via dna damage induction (mostly intrastrandcrosslinks) and through interference with the redox homeostasis of cells. here, we introduce and suggest the well-known nematode model organism c. elegans to elucidate mechanisms of neurotoxicity triggered by platinating agents. so far, we determined doses for cis-and oxaliplatin, which have only moderate effects on development, reproduction and body movement (muscular read-out). however, these doses are sufficient to trigger apoptosis in c. elegans and to induce a considerable amount of , -intrastrand crosslinks in dna (measured by south-western blotting). even more important they lead to strong neurotoxicity in a functional read-out (pharyngeal pumping). with regard to redox homeostasis, we determined the oxidative stress resistance showing that e.g. cisplatin sensitizes c. elegans to reactive oxygen species (ros), which could be prevented if worms were co-or pretreated with n-acetylcysteine. furthermore we determined the level of ros in living c. elegans after treatment with platinating agents and also in combination with protective compounds. using the advantages of c. elegans as a genetic model system, we will further clarify the relevance of different defense mechanisms, including dna repair (nucleotide excision repair, base excision repair), detoxification systems (antioxidative stress factors, metallothioneins) as well as drug transporters and signaling proteins. this will be achieved by using rna interference approaches that allow targeting either the whole animal or specific tissues (i.e. neurons) only. first results of this approach will be presented. finally we aim to use this setup to identify neuroprotective compounds that prevent chemotherapy induced peripheral neuropathy induced by platinating anti-tumor drugs. clostridium botulinum c exoenyzme (c ) exclusively adp-ribosylates rhoa, b and c leading to reorganization of the actin cytoskeleton and morphological changes. in addition to the enzyme-based inhibition of rho-gtpases, c promotes in an enzymeindependent manner axonal and dendritic growth in neurons. as c lacks the canonical binding and translocation domains of bacterial protein toxins, cell entry is currently not well understood. based on overlay assays and mass spec analyses the intermediate filament vimentin was identified as the putative membrane receptor for c . knock down of vimentin by sirna and application of the selective vimentin disruptor acrylamide led to a significantly delayed uptake of c . moreover, addition of extracellular vimentin to cells induced an enhanced uptake of c . proof of principle experiments in astrocytes and neurons from vimentin knock out mice showed c -induced morphological changes (astrocyte stellation and axon growth) to a reduced extent and a significantly delayed uptake of c compared to wild type cells. as vimentin knock out did not completely inhibit c uptake into cells, an additional uptake mechanism or additional receptor for c is likely. nevertheless, our data reveals that c employs a specific endocytosis mechanism with involvement of the intermediate filament vimentin to gain access to host cells. the primary target organ of organic hg species-mediated toxicity is the central nervous system (cns). humans are exposed to organic hg mainly in the form of methylmercury (mehg) via the consumption of contaminated fish and other seafood products. in terrestrial food sources hg is mostly found as inorganic hg. thiomersal is a further organic hg compound which is used as a preservative in medical preparations. exposure to organic hg promotes primarily neurological effects. the understanding of transfer mechanisms regarding the cns is an important precondition for an evaluation of hg species-induced neurotoxicity. thus, primary porcine in vitro models of the bloodbrain barrier and the blood-cerebrospinal fluid (csf) barrier were used to investigate effects of mehgcl, thiomersal and hgcl on the barriers as well as transfer properties into and out of the cns in vitro. the results show significant transfer differences of the various incubated species as well as in the different barrier systems. whereas the bloodbrain barrier seems to account for the transfer of organic hg species from the blood side to the brain side, these species are transferred in the contrary direction by the blood-csf barrier. inorganic hgcl was not transferred across both brain barriers towards the brain side but was able to leave the brain side across the blood-brain barrier. additionally, cytotoxic effects of the hg species by themselves as well as the combination of organic and inorganic hg species have been investigated in human astrocytes and human differentiated neurons. differentiated neurons were much more sensitive towards all hg species. organic species exerted stronger cytotoxic effects in both cell types as compared to hgcl . interestingly, a coincubation of organic and inorganic hg species led to an increased cytotoxicity in the astrocytes. this cocytotoxic effect is currently investigated in differentiated neurons. the species-specific differences with respect to both, effects on and transfer across the blood-brain and the blood-csf barrier in vitro as well as toxic effects in brain target cells, clearly emphasizes the necessity for comparative analyses. introduction: the neural crest is a multipotent stem cell population that arises at the neural plate border during early fetal development. neural crest cells (nccs) migrate to target sites in the periphery, where they differentiate into multiple cell types, including melanocytes, cranial bones and peripheral neurons. failure of ncc migration can lead to severe disorders, such as hirschsprung's disease. aim: to test whether toxicants interfere with human ncc migration, a high-throughput migration assay was established. this test system was used to screen an compound library of potential developmental toxicants. methods: nccs were derived from human embryonic stem cells. the cells were allowed to migrate for h before toxicants were added to the cells. migration and viability of the cells were then measured after another h by high-content image analysis and a custom-developed software package. results: the screening library was assembled by the us national toxicology program (ntp) and consisted of different substance classes, e.g. organophosphates, organochlorines, drug-like compounds, pesticides and polycyclic aromatic hydrocarbons (pahs). out of the tested potential developmental toxicants, compounds reduced ncc migration at non-cytotoxic concentrations. hit-confirmation testing confirmed of the compounds as concentration-dependent inhibitors of ncc migration. among the potential developmental toxicants identified here, there were several organophosphates (e.g. chlorpyriphos) and drug-like compounds as well as polybrominated diphenyl ethers (pbdes) and organochlorine pesticides (e.g. ddt and dieldrin), while none of the tested pahs inhibited ncc migration. the negative controls in the screening library, like acetylsalicylic acid, acetaminophen and saccharin, proved to be non-toxic. conclusion/outlook: the newly established test system allows screening of potential developmental toxicants in a high throughput manner for interference with human ncc migration. confirmation in other types of migration assays is ongoing, and selected compounds from amongst the screen hits are undergoing mechanistic evaluation. oxidative stress is regarded as a major trigger for neuronal dysfunction and death in the ageing brain and in multiple neurodegenerative disorders. how oxidative stress mediates neuronal death and whether the associated mechanisms are accessible for therapeutic intervention strategies is not clarified. increasing evidence suggests, however, that oxidative stress triggers molecular mechanisms of regulated necrosis that involve the activation of receptor interacting protein (rip ) independently of death receptor activation. here, we show that erastin-induced ferroptosis which involves inhibition of the glutamate-cystein transporter (xc -), glutathione depletion and lethal formation of reactive oxygen species (ros) , triggers mechanisms of regulated necrosis independent of tnfα-signaling. in hippocampal ht- cells erastin promotes activation of rip and subsequent rip -rip necrosome formation which has been investigated as a hallmark of regulated necrosis . in fact, silencing of rip by sirna or by the rip inhibitor necrostatin- prevents ferroptosis-induced cell death whereas the ferroptosis inhibitor ferrostatin- fails to protect cells against tnfα-induced classical necroptosis, a form of programmed cell death that is mediated by receptor interacting protein- (rip ) and rip kinases downstream of death receptor activation (e.g. tumor necrosis factor receptor tnfr) , . recently, a genome-wide sirna screen linked cylindromatosis (cyld) to rip /rip -dependent necroptosis and also in the present paradigm of ferroptosis, cyld depletion promotes neuronal survival and decreases rip -rip complex formation, suggesting a role of cyld in intrinsic pathways of regulated necrosis triggered by oxidative stress. the ns a inhibitor daclatasvir is used in combination with other antivirals such as the polymerase inhibitor sofosbuvir for treatment of chronic infection with the hepatitis c virus. daclatasvir is embryotoxic and teratogenic in rats and rabbits at exposures at or above the clinical exposure. in contrast, no teratogenic effects were observed in rat and rabbit developmental toxicity studies with ledipasvir, another ns a inhibitor. we studied these compounds in the embryonic stem cell test (est) alone and in combination with sofosbuvir. the ns a inhibitors were obtained from selleckchem, the main metabolite of sofosbuvir, psi- , was from medchem express. murine embryonic stem cells (es-d ) were obtained from atcc. they were kept in iscove's modified dulbecco's medium (imdm). substances were dissolved in dmso at a final dmso-concentration of . % in the culture medium. a cytotoxicity assay as well as a differentiation assay were performed. after days in culture the cells were evaluated. cytotoxicity was measured by an mtt test. differentiation into contracting myocardial cells was determined using direct phase contrast microscopy. the substances were tested at concentrations between . and mg/l, which is a broad coverage of the therapeutically relevant concentrations reached in patients. at a concentration of mg daclatasvir / l medium and higher the substance inhibited differentiation of cells. we observed contracting myocytes in , and wells out of wells in total at concentrations of , and mg/l. at mg/l no differentiation was observed. effects on cell viability were observed at mg/l. unexpectedly, we found a higher potency with ledipasvir. at the low, therapeutically relevant concentration of mg/l this nsa -inibitor showed a clear impact on differentiation with out of wells affected and no differentiation at higher concentrations. addition of sofosbuvir or its main metabolite psi- at concentrations up to mg/l had no influence on the concentration effect curves established for daclatasvir or ledipasvir. this is the first indication of an embryotoxic potential of ledipasvir. the difference to the results from the routinely performed animal experiments is unknown. possibly, metabolic activity in the maternal organism is responsible for this discrepancy. dimoxystrobin is a european-registered pesticidal active ingredient. biologically it is acting as an inhibitor of the fungal respiratory chain. for the purpose of european registration a full set of toxicological studies has been conducted with dimoxystrobin, including reproduction toxicity studies (according to the most recent oecd tg ) and developmental toxicity studies (oecd tg ) in rats and rabbits. dimoxystrobin interferes with the iron transport in rats and mice. this leads to lower serum iron levels and anemia in rats after repeated exposure. this holds true for treated dams and offspring in reproduction toxicity studies. furthermore, offspring effects seen at the high dose of the -generation toxicity study were a hypochromic microcytic anemia, impaired body weight development, which only developed postnatally, and reversible cardiomegalies in some -days old pups. for all effects clear noaels were determined. in the -generation toxicity study no dose adjustment during pregnancy and lactation was performed, which resulted in considerably higher food and compound intakes in dams and offspring during these lifestages. as a result, it seemed, that pups were more severely affected by body weight effects compared to the parental generation. by performing a life-stage specific comparison of body weight and substance intakes, as well as benchmark dose calculations (bmd) for these parameters, it could be demonstrated that the point of departures (pods) and the loaels for direct dimoxystrobin-related effects were comparable for offspring and parents. the heart effects (cardiomegaly), which were reversible, occurred only after direct dimoxystrobinexposure and are considered to be secondary to the detected offspring anemia. both effects (lower body weights and offspring cardiomegalies) only occur postnatally and are not the consequence of in-utero exposure, as no respective effects at higher doses in rat prenatal toxicity studies were seen. two new mechanistic studies ( -generation toxicity study and a -week study in young and adult rats, additionally investigating serum iron levels and anemia) confirmed, that pups and young rats were not more sensitive than adult animals to develop anemia or decreased serum iron levels. in , dimoxystrobin was classified with r (possible risk of harm to the unborn child) by the ecb, which was the european authority responsible for classification and labeling, before echa in helsinki was formed. the r (which has been translated into the ghs classification repr. , h d) was based on offspring body weight and heart effects seen in the -generation toxicity study. based on a comprehensive re-evaluation of existing and on new data of dimoxystrobin, the conclusion can be drawn, that a classification for reproduction toxicity is scientifically not justified and should be reconsidered. perfluorooctanesulfonic acid (pfos) and perfluorooctanoic acid (pfoa) are perfluorinated substances (pfas) which are used for the fabrication of surfaces with water-and dirt-repellent properties. due to their reprotoxic properties and their persistence in the environment, the use of pfos was restricted in and a restriction program for pfoa was initiated in . therefore, industry switches to pfoa and pfos substitutes, which are predominantly pfas with a shorter carbon chain length, or structure-related compounds. in contrast to pfoa and pfos only few toxicological data are available for their substitutes. aim of this study was to examine endocrine effects of the substitutes perfluorohexanesulfonic acid (pfhxs), perfluorobutanesulfonic acid (pfbs), perfluorohexanoic acid (pfhxa), perfluorobutanoic acid (pfba) and , , , -tetrafluoro- -(heptafluoropropoxy)propionic acid (genx) in comparison to pfoa and pfos. a hek- t cell-based dual-luciferase reporter gene assay was used to investigate the potential of these compounds to affect the activity of the human estrogen receptors herα and herβ. the reporter gene assay revealed no activation of herα or herβ by the pfas tested in this study. to investigate the potential inhibition of herα and herβ by pfas, a coincubation with the estrogen receptor agonist β-estradiol was performed. none of the tested pfas inhibited herα or herβ activity. however, in the case of herβ an enhancement of β-estradiol-stimulated activity was observed. thus, pfas do not directly activate or inhibit the human estrogen receptors but have an impact on herβ activity as they amplify the activation mediated by β-estradiol. further studies will be conducted to examine this synergistic effect in more detail. the xeer-reporter cell line: a novel dual-color luciferase reporter assay for simultaneous detection of estrogen and arylhydrocarbon receptor activation p. tarnow consumers are exposed to a multitude of anthropogenic and natural substances capable of activating or inhibiting ligand activated transcription factors, respectively. this in turn can lead to adverse health effects, particularly for substances acting on signalling pathways that are subject to regulatory crosstalk such as xenoestrogens and polycyclic aromatic hydrocarbons (pahs). xenoestrogens are known to activate human estrogen receptors (ers), whereas pahs or dioxins act on the arylhydrocarbon receptor (ahr). importantly, both receptor signalling pathways are interconnected by a complex crosstalk on multiple levels. this ranges from direct protein-protein interactions to competition for common co-factors. however, although this cross-talk has been long known we still lack a deeper understanding of its molecular mechanisms and physiological implications. one reason for this is a lack of tools to visualise and investigate receptor interaction in vivo. based on the breast cancer cell line t d we thus developed a dualcolour reporter assay which allows time-resolved simultaneous monitoring of the activation of er and ahr in living cells. the assay uses two beetle luciferases emitting luminescence in the red (slr) and the green (eluc) spectrum, respectively. while eluc is expressed under the control of a -fold repeated xenobiotic response element (xre) slr is subject to transcription regulation by a -fold repeated estrogen response element (ere). both constructs were stably transfected into t d human breast cancer cells, which endogenously express erα and ahr and are thus ideally suited for monitoring interactions with both receptors. the respective "xeer"-cell line has been successfully subjected to proof of principle studies, using prototypical er-and ahrligands as well as various phytochemicals and xenobiotics. besides e and tcdd ligands included various pahs, polychlorinated biphenyls, alpha-and betanaphthoflavone, cosmetic ingredients (butylparaben, benzophenone- and -mbc), bisphenol a, genistein, resveratrol, diindoylmethane as well as pharmacological antagonists of both receptors. asian women consuming soy rich food throughout life possess lower levels of betaestradiol (e ) in plasma (pl) than western women, whose diet is characterized by less soy consumption during early life and possible intake of soy based dietary supplements during adulthood. however, the impact of these soy exposure scenarios on estrogen (biotrans)formation and the consequence thereof in female mammary glands (mg) has not been investigated yet. thus, female august copenhagen irish rats were fed either isoflavone (if) depleted diet (idd, western exposure scenario without if supplement) or if rich diet (ird, asian exposure scenario) until the end of the study at postnatal day (pnd) . furthermore, rats fed idd until pnd were fed ird for days (idd+ird, western dietary exposure scenario with if supplement). estrous was determined histologically. levels of transcripts were determined by qpcr and e and estrone (e ) in pl and mg were quantified by gc-ms/ms. statistical analyses of estrogens were performed by kruskal wallis and unpaired wilcoxon tests and of transcript levels by linear regression models considering the explanatory variables tissue levels of e and diet (idd vs ird and idd vs idd+ird). e levels in pl and mg did not coincide with those predicted by estrous. furthermore, median levels of e and ratios e /e in mg and ratio of e levels in pl/mg were not affected by diet. in contrast, diet tended to affect e concentrations in pl (p= . ) due to an increase in the ird group (p= . ) whereas e levels in the idd+ird group only tended to be elevated (p= . ). in mg, ird and idd+ird increased e levels only weakly (p= . each). likewise, besides significant changes in transcript levels of cyp a and a , putatively decreasing oxidation of e to catechols, in the idd+ird group and (not significantly) also in the ird group, no changes in transcript levels putatively affecting e levels were observed. moreover, no decrease in levels of transcripts indicative for cellular (oxidative) stress (gclc, tp , mt a) was observed in the idd+ird group. e mg levels were significantly associated with an increase in transcript levels of areg and pgr, indicating activation of estrogen receptor (er). in contrast, ird was associated with a significant and idd+ird with a not significant decrease in pgr transcript levels. e levels but not diet were significantly associated with gata transcript levels, indicating tissue differentiation. furthermore, levels of transcripts involved in intercellular communication (egfr, wnt ) were significantly decreased by idd+ird and not significantly by ird and differed from that affected by e (increase in gdf , hgf, igf r, wnt a). bmf, a marker transcript for apoptosis was increased by ird, but not affected by e and even decreased not significantly by idd+ird. taken together, despite an increase in e levels in pl, less er activation was observed after dietary exposure to if. whereas e and transcript levels of enzymes involved in e (biotrans)formation as well as er activation and cellular communication were affected similarly but to a different extend in both asian and western if exposure scenarios, differences in apoptosis were observed between ird and idd+ird groups. supported by dfg le / - . august copenhagen irish (aci) rats with β-estradiol (e )-releasing implants are an accepted model to study the etiology of breast cancer, but neither e (biotrans)formation in mammary gland tissues (mg) during tumorigenesis, nor the impact of isoflavones (if) shown to affect tumorigenesis in aci rats, has been investigated, yet. therefore at postnatal day (pnd) and , placebo (-e ) or silastic implants containing mg e were implanted in female aci rats exposed to either if depleted diet (idd) or if rich diet (ird) since conception until the end of the study at pnd . palpable mg tumors (pt) and - mg per animal without pt were characterized histologically and categorized into normal (-e group, n= ), hyperplasia and non-pt and pt with and without solid tumors (+e group, n= ). e , estrone (e ), their hydroxylation products and methylation (meo-) products thereof, as well as conjugates of e and e in plasmas and mg were analyzed by gc-and uhplc-ms/ms, respectively. levels of transcripts involved in (biotrans)formation of e and estrogen receptor (er) activation were determined by taqman®-pcr. without exogenous e , plasma e as well as e and (borderline) e levels in mg were higher in ird. plasma e as well as e and e levels in mg were lower in the -e group than that in the +e group. e levels as well as e /e and e mg/plasma ratios were elevated in pt, accompanied by a significant increase in transcript levels indicative for estrogen receptor activation (areg, pgr) and proliferation (mki ). ird increased e /e ratio in pt and, although ird did not affect er activation (areg, pgr), ird increased differentiation (gata ) in normal and hyperplastic tissues and tended to decrease proliferation in hyperplastic (ccnd ) tissues. levels of e and -meo-e were highest in hyperplastic tissues, accompanied by an increase in transcript levels of hsd b (conversion e to e ) and cyp a . transcript levels of gstm and gstm were decreased in the whole +e group and of gstt and gstt in hyperplastic tissues, possibly decreasing inactivation of electrophilic metabolites. accordingly, maximum transcript levels of tp and mt a indicating cellular (oxidative) stress were observed in hyperplastic tissues. ird did neither affect levels of -meo-e nor cellular stress (gclc, mt a, tp ). of note, neither -meo-e , nor e catechols, nor e catechols nor methylation products of the latter were observed in any sample. furthermore, no conjugates of e or e were detected in plasmas and mammary gland tissues. thus, changes in transcript levels of conjugating enzymes induced by tumorigenesis and by ird were not related with detectable conjugate levels of e or e . taken together, whereas hyperplastic tissues were characterized by maximum oxidative metabolism of e and cellular (oxidative) stress, pt exhibited highest e levels and er activation. ird increased differentiation and decreased proliferation in normal and hyperplastic tissues but increased e /e ratio in pt. supported by dfg le- / - . level of beta-estradiol (e ) in human breast tissue is considered to affect breast cancer initiation, promotion and progression. although putatively beneficial and adverse effects of soy isoflavones (if) on the human mammary gland, in particular in western women, have been discussed extensively, the influence of if levels on estrogen formation in human mammary gland tissue has not been investigated yet. thus, glandular tissues were dissected from mammoplasty specimen obtained from women (age - years old) not taking estrogen active drugs. of these women had been exposed to if by their usual diet or by intake of a soy-based dietary supplement for days prior to mammoplasty. information on soy consumption and lifestyle were collected by questionnaire and tissues were characterized histologically. genistein, daidzein their conjugates (n= ) and bacterial metabolites (n= ) as well as the estrogens estrone (e )-sulfate, e , e and -methoxy-e were determined by uhplcand gc-ms/ms, respectively and transcript levels of enzymes involved in e (biotrans)formation were quantified by taqman®-pcr in glandular tissues. isoflavonoids were categorized into the if parameters aglycones (agl) and conjugates (con) of either genistein, daidzein or sum of both and were further statistically analyzed by spearman`s rank correlation analysis. a positive correlation of e /e ratio with agl(+con) was observed in glandular tissues (r= . , p= . ), accompanied by a significant negative correlation of e levels with agl (r=- . /p= . ), possibly due to reduction of beta-hydroxysteroid dehydrogenase (conversion of e to e ) expression as indicated by a weak negative correlation of transcript levels of beta-hydroxysteroid dehydrogenase with agl+con (r=- . , p= . ). further statistical analysis taking into account multiple variables using linear regression models will provide more insights into variables affecting e /e ratio. taken together, estrogen profile in human glandular breast tissue seems to be affected by if levels. supported by dfg le- / - . allergic contact dermatitis (acd) is a widespread disease often caused by substances in consumables. the eu prohibits the testing of cosmetic ingredients in vivo. this urges the development of reliable in vitro testing strategies. activation of dendritic cells (dcs) represents a key step during sensitization as they are essential for selection and priming of allergen specific effector t cells. in an integrated omics approach we aimed to further elucidate the molecular mechanisms of dc activation using quantitative metabolomics and proteomics. monocytic thp- cells were used as a model system and treated with the sensitizer , dinitrochlorobenzene (dncb; , and µm) and the irritant sodium dodecyl sulfate (sds; µm). samples were taken after , and hours. thp- activation was analyzed by measuring the established activation markers cd and cd after hours. a targeted lc-ms/ms approach was used to analyze metabolites including amino acids and lipids. protein levels were quantified by nano-lc-maldi-ms/ms after stable isotope labeling by amino acids in cell culture (silac). data sets were examined by multivariate analyses for identification of biomarker candidates. regulated metabolites and proteins were subjected to pathway analysis. the data presented might contribute to the further development of suitable in vitro testing methods for chemical-mediated sensitization. drug induced liver injury (dili) is one of the most frequent causes of acute liver injury and a main cause for drug withdrawals. currently there are no reliable models to test the dili potential of new compounds available. kupffer cells (kc) play an important role in hepatic cell stress mediated through chemokines and release of endogenous proteins. kc activation by damaged or stressed hepatocytes can lead to activation of the nf-κb signaling pathway transmitted by reactive oxygen intermediates (roi). we have recently established a liver model composed of primary human hepatocytes (phh) and kc which enables investigation of immune reactions after induction of hepatocyte stress (kegel et al., ) . aim of the present study was the kinetic investigation of hepatic cell stress induction and macrophage activation after treatment with subtoxic concentrations of hepatotoxic drugs. primary human hepatocytes (phh) and kc were isolated from human liver resectates using a two-step collagenase perfusion technique. initial kc activation was characterized by the dcf assay and immunofluorescence staining. phh were incubated with different concentrations of acetaminophen (apap) and diclofenac (dic) for different time intervals. cell stress was evaluated by measurement of oxidative stress (dcfassay) and viability (xtt-assay). in order to simulate macrophage activation following hepatocyte damage, kc and macrophages derived from the monocytic cell line thp- were incubated with supernatants of phh treated with hepatotoxic compounds. kc and thp- -macrophage activation were investigated by measuring intracellular formation of roi using the dcf-assay and cell activity using the xtt-assay. the characterization of kc activation revealed a donor and disease dependent kc activation resulting in kc differentiation to pro-and anti-inflammatory macrophages. therefore, kc were substituted by macrophages derived from thp- cells. evaluation of hepatic cell stress showed the strongest effect on thp- -macrophages when phh were incubated with apap or dic for h.treatment of kc and thp- -macrophages with supernatants of phh challenged for h with hepatotoxic compounds indicates that thp- -derived macrophages react similar to kc when treated with phh supernatants in terms of cell activity and roi-production. in conclusion, thp- derived macrophages might be a suitable alternative to kc concerning macrophage activation. the evaluated kinetic window of h covering hepatic stress induction and immune reaction allows to perform these measurements in a since the use of cerium dioxide nanoparticles is known to be beneficial e.g. in terms of reducing fuel consumption when added to diesel fuel it has become a frequently used nanomaterial. to compensate the concurrent lack of information on its toxicology a day nose-only inhalation study was initiated. by comparing the results to a combined chronic inhalation toxicity and carcinogenicity study using the same test items and experimental conditions (basf, ludwigshafen, germany) early indicators for genotoxic and carcinogenic effects should be determined. rats were exposed to , . , . , and mg/cm³ ceo as well as mg/m³ baso nanoparticles ( h/day, days/week, weeks). animal dissections were conducted at five time points (exposure day and ; recovery day , and ) aiming for endpoints mandatory according to oecd guideline . additionally, gene expression analyses in isolated pneumocytes type ii were performed using pathway arrays for inflammation, oxidative stress, genotoxicity, apoptosis and lung cancer. the given results intend the identification of marker genes displaying modulated expression in response to nanoparticle exposure. investigations on ceo and baso retention in the lung are also included in this project. in bronchoalveolar lavage fluid (balf) a time and dose-dependent increase of inflammatory cells has been detected up to the end of exposure. the amount of inflammatory cells decreased during post-exposure; however, in the high dose group a persistent inflammation up to days was detected by balf and histopathology examination. based on our current results effects of ceo nanoparticles on the respiratory system are suggested. its relevance in the context of long term effects such as tumor development needs to be estimated considering all investigations included in this study. the inhalt- project is funded by the german federal ministry of education and research (bmbf) - x a. core or coating material? what dictates the uptake and translocation of nanoparticles in vitro? nanoparticles are becoming increasingly important role in consumer-related products. understanding the interactions between nanoscaled objects and living cells is therefore of great importance for risk assessment. in this context, it is generally accepted that nanoparticle size and shape are crucial parameters regarding the potential of nanoparticles to penetrate cell membranes and epithelial barriers. current research in this field additionally focuses on the particle coating material. in order to distinguish between core-and coating-related effects in nanoparticle uptake and translocation behavior, this study investigated two nanoparticles equal in size, coating and charge but different in core material. silver and iron oxide were chosen as core materials to ensure similar nanoparticles characteristics after particle synthesis. nanoparticles were coated with poly (acrylic acid) (pas) and extensively characterized by tem (transmission electron microscopy), saxs (small-angle x-ray scattering), zetasizer tm and nanosight tm . for uptake and transport studies the widely used human intestinal caco- model in a transwell tm -system with subsequent elemental analysis (aas) was used. for evaluation and particle visualization transmission electron microscopy (tem) and ion beam microscopy (ibm) were conducted. although similar in size, charge and coating material, the behavior of particles in caco- cells was quite different. the internalized amount was comparable, but pas-coated iron oxide nanoparticles were additionally transported through the cells. by contrast, pascoated silver nanoparticles remained in the cells. our findings suggest that the coating material influenced only the uptake of the nanoparticles whereas the translocation was determined by the core material. in summary, a core-dependent effect on nanoparticle translocation was revealed. both the uptake and transport of nanoparticles in and through cells should be considered when discussing nanoparticle fate and safety. nanotechnology is having a great impact not only on basic research but also on many sectors of industry opening the market for numerous new applications ranging from electronics to the health care system. besides their great innovative potential, the large variety of existing synthetic nanomaterials used in the last decade represents a major challenge for scientists and regulators in terms of measuring and assessing the potential hazard caused by the materials or the products themselves. equally, consumers often miss reliable and easy-to-understand information on nanomaterials and nanotechnology and do not know where to get such information. therefore, the international dana . expert team brings together its expertise and knowledge from different research areas dealing with all aspects of nanosafety research in order to create and provide easy-to-understand, up-to-date and quality-approved nanomaterials' knowledge base on www.nanopartikel.info. this information platform covers the market-relevant nanomaterials focusing on their effects on the safety of humans and the environment.in order to manage and asses the rapidly increasing number of publications related to nanosafety issues, the dana . project developed a customised methodology «literature criteria checklist», which includes mandatory and desirable assessment criteria covering physico-chemical characterisation, sample preparation and (biological) testing parameters. this checklist facilitates the discrimination between high-and low quality publications and all positively evaluated literature is then fed into the dana knowledge base. accounting for the need to harmonise experimental practices, the dana team also developed a template for standard operation procedures (sop) to support careful scientific practice. validated protocols generated within the german bmbf-funded nanosafety research projects are presented together with results from the swiss ccmx project v.i.g.o. and available for download. another unique feature of the dana knowledge base is the integrated application-based database that provides a unique link between nanomaterials in real applications (e.g. environmental remediation or medical products) and their potential impacts/ toxicological effect(s) that can be easily accessed by the interested visitor. additionally, dana . provides a list of faqs, a link platform with contact data to other information portals and the opportunity to directly pose questions to our experts via e-mail. dana . is also present on twitter, follow us @nano_info. background: particulate matter of combustion processes enhances cardio-vascular diseases and increases associated mortality rates. around % of total pm emissions are emitted by wood burners (uba ) . how wood combustion aerosols (particles and gasses) can affect human lung cells and how such cellular responses depend on the usage of different wood types and burners is widely unknown. methods: in an exposure chamber imitating the human respiratory tract human alveolar cells (a ) were exposed at an air-liquid-interface (ali) to gasses and particles of wood combustion aerosols. log wood of beech, birch and spruce was burnt in a conventional oven and compared to the combustion of wood pellets in a modern pellet burner. the combustion aerosols were diluted : and directly delivered to the exposure chamber. after h exposure the lung cells were lysed and rna was isolated. in an array based transcription analysis of the whole genome the effects of the aerosol exposures on lung cells was assessed. in parallel, physical and chemical parameters of the combustion aerosols were analyzed. results: the combustion aerosol of wood pellets contained less organic substances than the log wood aerosols, but was higher in its zinc content. genome-wide we found a higher number of regulated genes with combustion of pellets compared to combustion of log wood. the gas phase alone (filtered aerosol) showed comparable gene regulatory activity as the particle-containing total aerosol. aerosol from log wood burning induced mainly genes of the xenobiotic metabolism and cellular signaling. pellet aerosols additionally regulated apoptosis and dna repair processes. conclusions: modern pellet burners reach better combustion efficiencies than conventional log wood ovens, but their emissions seem to stress human lung cells stronger. one reason might be the higher zinc content of wood pellet aerosols. multiwalled carbon nanotubes (mwcnts) may pose as a risk similar to asbestos in causing cancer, notably mesothelioma, which is a malignant tumor originating from mesothelial cells. to identify molecular cues leading to mesothelioma development, we performed genome-wide transcriptome analysis using microarrays in primary human peritoneal mesothelial lp cells treated with two different tumor-inducing tailor-made mwcnts (rat model; rittinghausen et al. part fibre toxicol : ), or amosite asbestos at µg/cm for h. specifically, we determined how the transcriptomic changes of the highly tumorigenic mwcnt a would differ from another tumor-inducing mwcnt with albeit lesser potency (mwcnt d), long amosite asbestos as positive control, and milled mwcnt a as material control. initial analysis using bioinformatic tools, revealed significantly differentially regulated genes for mwcnt a, for mwcnt d, for amosite, and for milled mwcnt. further analyses with ingenuity pathway analysis comparing the two different mwcnt types and amosite, found common as well as exclusive biomarkers. interestingly, we identified many differentially regulated genes implicated in cellular senescence, a growth arrest in response to different stressors including dna damage, disrupted chromatin, and strong mitogenic signals. paradoxically, cellular senescence can represent both tumor suppression and tumor promotion mechanisms. more important, we found differential expression of genes associated with senescence-associated secretory phenotype (sasp) such as inflammatory cytokines, chemokines, proteases, and growth factors, which were manyfolds up-and down-regulated in mwcnt a, compared to mwcnt d and amosite. the mechanisms leading to mesothelioma induction by mwcnts are from far clear, but the key information emerging from the present transcriptomic data, together with our previously identified senescence markers, indicate that cellular senescence has a likely role. nanotechnology offers great advantages for the food industry despite its partly unknown risks, whose enlightenment is the main target of nanotoxicology. due to variability in terms of size, material, shape, surface texture and several endogenous influences, the toxicity of most frequently used and ingested nanomaterials is difficult to estimate. therefore, the aim of this study was the in vitro investigation of toxicological endpoints such as cell viability, dna integrity and the induction of apoptotic processes in human colon carcinoma cells (ht ). for this purpose ht cells were exposed for hours with metal nanoparticles (gold, silver) and metal oxide nanoparticles (copper oxide, titanium dioxide, zinc oxide) in concentrations of - µg/ml. at first the cellular uptake of the nanoparticles by means of an icpms was determined. the influence of cell viability was demonstrated by the trypan blue staining and the mtt assay. the alkaline comet assay gave information about possible dna damages and the use of the repair enzyme formamidopyrimidine dna glycosylase (fpg) additionally allowed the detection of oxidized bases. the induction of programmed cell death was examined using by annexin v fitc assay. the icp-ms data showed a maximum particle content of . pg per cell for the used concentration range. the metal oxide nanoparticles resulted in a significant reduction of cell viability with a decrease up to % after copper oxide and zinc oxide treatment. for metal particles, only for silver a reduced cell metabolism of about % was detectable by the mtt assay. low genotoxic effects could be determined for silver nanoparticles (tail intensity about %; control about %), while for titanium dioxide the amount of oxidized bases was additionally increased (tail intensity about %; control about %) for concentrations above µg/ml. induction of apoptosis was determined for silver particles (up to % early apoptotic and % late apoptotic cells) as well as for titanium dioxide and zinc oxide ( % each early apoptotic cells), whereby the most significant increase in late apoptotic cells was detected for zinc oxide (up to %). the results obtained in our studies indicate a clear particle-dependent influence on cell viability and apoptosis-triggering processes, depending on the used material or the concentration deployed, while only minor changes of dna integrity were detected. the evolution in the field of nanotechnology led to a variety of novel materials at the nanoscale. among them are different carbon materials like buckyballs, graphene nanoplates and carbon nanotubes (cnts). cnts are hollow carbon fibres with either one (sw) or multiple sidewalls (mw). mwcnts usually show a diameter of up to nm and can be several micrometres long. because of their nanoscale diameter cnt-uptake can take place directly through the plasma membrane of cells by the so called nanoneedle effect [ ] . additionally cnts, like most nanomaterials, show a high surface to volume ratio and, because of their micro scale length, a potentially high loading capacity. these properties make cnts interesting for the potential use as drug delivery carriers (ddcs). mwcnts, produced via chemical vapour deposition with a diameter of nm and µm length, were used in three different forms, unmodified, acid oxidized (ox_cnt) and ground. cytotoxicity testing was performed in human umbilical vein endothelial cells (huvec). the cells were seeded in -well plates and exposed to doses of , , and µg/cm² growth area of the respective cnt type for h. the wst- assay was applied for testing cell viability and the ldh cytotoxicity assay to identify potential damage to the plasma membrane and to calculate overall cytotoxicity. the results show that an increased oxidation time for the ox_cnts, in a h so /hno mixture, leads to decreased cytotoxicity in huvec, compared to unmodified mwcnts. during the oxidation reactive oxygen groups are formed on the cnt surface [ ] . these groups lead to a reduced hydrophobicity of the cnt surface which could be responsible for the decline in cytotoxicity. future investigations will include the toxicological analysis of mwcnts functionalized with polyethylene glycol (cnt-peg). the hydrophilic polymer peg will be covalently bound to the cnt surface and is expected to further reduce the cytotoxic effect. for these investigations different analytical methods will be used. among others, cell cycle analysis, the brdu assay, pathway arrays and qrt-pcr for the investigation of gene expression and cytokines will be measured. these methods will involve a co-culture model of huvec and human umbilical vein smooth muscle cells (huvsmcs) for a better approximation to the cellular in vivo situation. additionally the peg modified mwcnts will be tested for their loading capacity and efficacy with the anticancer drug doxorubicin for a potential use as an intravenous drug delivery carrier in vivo. although aluminium is one of the most common elements in the biosphere, up to now little is known about its impact on human health. aluminium and its chemical derivatives are highly abundant in food, food contact materials and consumer products what leads to an exposition via the gastrointestinal tract (gi tract), the lung and via skin contact. recently, aluminium is hypothized to cohere with cancer and neurodegenerative disorders. lately, due to an increasing attentiveness on this topic, limiting values for food additives have been tightened by the eu commission. however, cellular effects of aluminium and especially aluminium-containing nanomaterials, are in the focus of our research activities, for example in the international solnanotox project. we established an in vitro simulation system of the gi tract, where nanomaterials undergo the different physiological, chemical and proteinbiochemical conditions of saliva, gastric juice and the intestine. the artificially digested nanomaterials, as well as soluble aluminiumchloride as ionic control substance, were subjected to several analytical and biochemical methods to characterize their change of appearance and their cytotoxic effects on intestinal cellular models. we observed the fate of the nanomaterials during typical ph-values of saliva, gastric and intestinal juice with dynamic light scattering measurements and icp-ms in the single particle mode. after observable disappearance at ph the particles recovered in the simulated intestinal fluid. the simulation of the gi tract, mainly the change of ph settings, may lead to a certain chemical activation of aluminium that can increase bioavailability in the intestine after oral uptake of aluminium-containing food products. in vitro assays like ctb, mtt and cellular impedance measurements showed that there were no acute cytotoxic effects measurable after a period up to h after incubation, comparable to undigested particles. in contrast, high amounts of aluminium ions showed additional effects on cell viability compared to non-digested aluminium ions. although toxicological potential of al ions to healthy tissue appears to be low, increased hazardous potential cannot be ruled out to pre-damaged tissue and can have a relevance for special consumer groups with for example chronical intestinal inflammation or dietary eating behavior combined with high exposure to al-containing food products. in the eu, there is a strong need for solutions to substitute halogenated flame retardant (hfr) additives, employed in the fabrication of fr thermoplastic and thermoset materials. these materials are used in diverse commercial products, applications and markets, such as electrical/electronic devices, low-voltage wires or household appliances. the phoenix project, funded by the european union th framework program (grant agreement no. ), therefore investigates e.g. several tailor-made, nano-layered hybrid particles as alternatives to hfr additives. considering "safer-bydesign" strategies, potential fr nanomaterials (nm) were physico-chemically characterized (e.g. particle size distribution, zeta potential) and screened early in development for their (geno)toxic and pro-inflammatory potential to timely reject nm with high health hazard. as inhalation is the most important exposure route for nm, lungrelevant cells were used as in vitro screening models. to better enable detection and differentiation of the biologic effects of the most promising nm, screening was started with primary rat lung alveolar macrophages (am; cells of first contact for inhaled nm), at a high concentration of µg/cm ( h of incubation), using membrane damage (lactate dehydrogenase release assay), direct dna-damage (alkaline comet assay), and il- liberation (elisa) as primary endpoints, and quartz dq and al o as particulate positive and negative controls, respectively. in this screening system, biologically inert nm could be differentiated from more active ones. thereby, mg(oh) nanoplatelets (mg ; mean lateral size: , - µm; mean thickness: - nm) represented the least, and pristine few layers graphene nanoplatelets (gr ; mean lateral size: µm; mean thickness: nm; graphene layers: ± . ) the most biologically active nm. clear concentration dependencies were detected for gr in follow-up experiments. mg and gr were further tested in other lung-relevant cell types, i.e. mrc- primary human lung fibroblasts and a lung adenocarcinoma epithelial cells. interestingly, mrc- cells were less sensitive towards biological effects of gr , compared to am, whereas a cells showed nearly no effect, keeping in mind that lung epithelial cells are the target cells of lung tumor development. to test the hypothesis that the observed cellspecific differences in sensitivity might in part be based on cellular uptake, cells were exposed for h to . or µg/cm of gr on chamber slides. slides were finally stained with dapi and analyzed by dark field microscopy. cells indeed demonstrated differences in uptake capacity and also showed unique pattern of cellular localization of gr , i.e. with tight perinuclear agglomeration in am, a more scattered cytoplasmic distribution in mrc- cells and limited uptake in a cells. additionally, biological activity of the diverse nm seemed also to correlate with cellular uptake, as determined by light and dark field microscopy in am and mrc- cells. in conclusion, an am-based screening system was able to differentiate biological activity of diverse nm, with morphology, physico-chemical characteristics, and related cellular uptake most likely to be key for nm-and cell type-specific hazard. lung carcinogenicity and putative systemic effects of low-dose life-time inhalation exposure to biopersistent nanoparticles were examined in a chronic inhalation study performed according to oecd test guideline no. with several protocol extensions. female rats ( /group) were exposed to cerium dioxide (nm- , . ; . ; ; mg/m³) for two years; a control group was exposed to clean air. after one year exposure, µg/lung was found in animals exposed to . mg/m³ and . mg/lung in animals exposed to mg/m³. histological examination of lungs revealed several adverse and non-adverse effects in the lung. the non-adverse effects comprised accumulation of particle-laden macrophages in alveolar/interstitial areas and in the balt, particle-laden syncytial giant cells in the balt and bronchiolo-alveolar hyperplasia (alveolar bronchiolization). the adverse effects included (mixed) alveolar/interstitial inflammatory cell infiltration, alveolar/interstitial granulomatous inflammation, interstitial fibrosis and alveolar lipoproteinosis. the incidence and severity of the effects were concentration-related. alveolar lipoproteinosis was not observed at low concentrations of . and . mg/m³ ceo . neither pre-neoplastic nor neoplastic changes were observed after -months exposure. a no observed adverse effect concentration could not be established in this study. the comprehensive histopathological examinations of lungs and other tissues will be finalized in . this project is part of the eu project nanoreg. moreover, german federal ministry for the environment, nature conservation, building and nuclear safety, german federal institute for occupational safety and health, german federal environment agency funded this project. lung carcinogenicity and putative systemic effects of low-dose life-time inhalation exposure to biopersistent nanoparticles were examined in a chronic inhalation study performed according to oecd test guideline no. with several protocol extensions. female rats ( /group) were exposed to cerium dioxide (nm- , . ; . ; ; mg/m³) and barium sulfate (nm- ; mg/m³) for two years; a control group was exposed to clean air. lung burdens and burdens in extrahepatic tissues were measured at various time-point. the two year exposure period was successfully terminated and animals per dose group were examined for organ burden and histopathology. the remaining animals currently are kept exposure-free for maximally additional months. up to two years exposure to both nanoparticles did not lead to body weight reduction compared to control animals. the mortality rates were in an acceptable range. macroscopically evident tumors were not detected after two years. the ceo lung burdens were maximally . mg/g lung tissue at the highest exposure concentration of mg/m³. in comparison, highest ceo burdens in organs remote to exposure were liver and spleen with maximally roughly x - g/g tissue. in brain, maximum ceo levels were x - mg/g lung tissue. baso lung burdens were comparatively low ( mg/g) within the first weeks of exposure and steeply increased to mg/g lung tissue after one year. the comprehensive histopathological examinations of lungs and other tissues will be finalized in . the european centre for ecotoxicology and toxicology of chemicals (ecetoc) 'nano task force' proposes decision-making framework for the grouping and testing of nanomaterials (df nano) that consists of tiers to assign nanomaterials to main groups, to perform sub-grouping within the main groups and to determine and refine specific information needs. the df nanogrouping covers all relevant aspects of a nanomaterial's life cycle and biological pathways, i.e. intrinsic material and systemdependent properties, biopersistence, uptake and biodistribution, cellular and apical toxic effects. use (including manufacture), release and route of exposure are applied as 'qualifiers' within the df nano to determine if, e.g. nanomaterials cannot be released from a product matrix, which may justify the waiving of testing. the four main groups encompass ( ) soluble nanomaterials, ( ) biopersistent high aspect ratio nanomaterials, ( ) passive nanomaterials, and ( ) active nanomaterials. the df nano aims to group nanomaterials by their specific mode-of-action that results in an apical toxic effect. this is eventually directed by a nanomaterial's intrinsic properties. however, since the exact correlation of intrinsic material properties and apical toxic effect is not yet established, the df nano uses the 'functionality' of nanomaterials for grouping rather than relying on intrinsic material properties alone. such functionalities include system-dependent material properties (such as dissolution rate in biologically relevant media), bio-physical interactions, in vitro effects and release and exposure. the df nano is a hazard and risk assessment tool that applies modern toxicology and contributes to the sustainable development of nano-technological products. it ensures that no studies are performed that do not provide crucial data and therefore saves animals and resources. the the grouping decisions of df nano for nanomaterials were validated against grouping by results of existing in vivo data and demonstrated concordant grouping decisions. serum concentrations in cell culture medium influence the effect of cerium oxide nanoparticles on human lung a cells: cytotoxicity, proinflammation, cellular uptake, and particle properties k. burchardt the wide use of cerium oxide (ceo ) nanoparticles (np), e.g. as fuel additive and for industrial and biomedical applications, evoked intense studies to understand the effects those particles might have on living organisms. contradictory results of ceo nanoparticle toxicity have been published as they depend on many variables like shape, size, diluent and others. in our work we used an in vitro model of ceo nanoparticles and lung carcinoma cells to investigate the role of serum content in the cell culture medium on cellular toxicity, particle uptake, proinflammation and particle characteristics. proinflammatory ceo np with an average diameter of - nm and different concentrations were diluted in cell culture medium with different fetal calf serum (fcs) concentrations ( . , . , and %) and were used to expose human lung adenocarcinoma cells (a ) for up to hours. at µg/ml ceo np showed little to no toxic effecton growth arrested a cells at fcs concentrations of % or below, but cell viability was decreased to about % in proliferating cells in cultures with % fcs. the proinflammatory effect of ceo np was investigated through measurement of il- mrna expression after hours and cellular il- secretion after hours. the qrt-pcr showed that the expression of il- mrna in cells treated with µg/ml ceo np in % fcs medium was three times higher than in cells treated with lower fcs concentrations. this finding correlated with the cellular il- secretion, which showed a stronger increase by cells treated at % fcs. differences in cellular uptake of ceo np was determined by fluorescence-activated cell sorting (facs) after and hours of exposition. after hours, the cells treated with ceo np in % fcs medium showed a lower mean granularity (∆gmean) as measure for cellular particle uptake than those with less fcs in medium. after hours all probes showed about the same granularity. to examine the effect the fcs concentrations on ceo np characteristics in cell cultures we used dynamic light scattering (dls) and phase contrast microscopy (pcm). dls measurements revealed an increasing hydrodynamic diameter of the particles with decreasing fcs concentrations (about nm ( % fcs) to nm ( . % fcs)), which was correlated by an increasing particle agglomeration shown by pcm. our results show that the fcs concentration in cell culture medium has a direct or indirect impact on the cytotoxicity, the proinflammatory effect, the facs parameter for cellular particle uptake as well as on particle properties, which should be taken into account when designing, performing and interpreting in vitro experiments to investigate the toxicity of nanoparticles. toxic and inflammatory effects of shape-engineered titanium dioxide nanoparticles in nr rat alveolar macrophages. knowledge about the contrasting toxicity of nanoparticles (np) of different chemical composition has steadily increased over the past decade. however, available literature often reveals considerable differences in effects within a specific type of nanomaterial. these contrasts have been contributed to different handling and testing protocols as well as to sample-specific differences in physico-chemical properties of np that could affect their mode of interaction with cells. within the nanometrology project setnanometro, the highly controlled generation and characterisation of a large set of shape-engineered tio np allows us to investigate the potential role of subtle shape-and surface structure changes on np toxicity. as inhalation represents the most relevant uptake route of np, the nr rat alveolar macrophage cell line was selected for in vitro toxicological testing. since oxidative stress and inflammation are considered as key biological pathways in nanotoxicity, we evaluated the expression of the oxidative stress marker genes heme oxygenase- (ho- ) and g-glutamylcysteine synthetase (g-gcs) as well as the pro-inflammatory genes interleukin (il)- β, il- , il- and inducible nitric oxide synthase (inos) by qrt-pcr. protein levels of il- β and tumour necrosis factor-α (tnf-α) were measured by elisa. cytotoxicity testing of the tio np by wst- assay overall revealed only minimal toxicity in comparison to sio np which were used as reference material. ho- and g-gcs mrna analyses indicated that specific tio np triggered a moderate induction of oxidative stress. il- was only induced after sio treatment, whereas il- was not affected by any of the tested np. in contrast, various tio np caused a significant induction of il- β mrna expression. however, no significant induction of il- β and tnf-α protein secretion was observed for any of the tio np. the results obtained from these and ongoing investigations will be linked to the physico-chemical database as being developed for all tio np within the setnanometro project, with the overall aim to build and model nano-structure activity relationships (nsar) for this widely applied type of nanomaterial. acknowledgements: the setnanometro project is supported by the eu-fp programme. specific types of tio particles were obtained by solaronix (switzerland), evonik and cristal. potential of silver and silver nanoparticles to reduce n-acetyltransferase (nat ) activity j. lichter , b. blömeke trier university, department of environmental toxicology, trier, germany humans are exposed to various kinds of engineered nanoparticles including silver, which is frequently used in consumer and biomedical product due to its bactericide properties. despite their widespread usage, knowledge about influences on cellular functions is still incomplete. n-acetyltransferase (nat ), an enzyme which is ubiquitously expressed in human tissues, catalyzes the transfer of an acetyl group to its substrates and although its endogenous function is not clear yet, it is well known to be involved in the n-acetylation of arylamines. in addition nat enzyme activity is known to modulated by non-substrates including metals and certain nanoparticles, however, the influence of silver on nat has not been analyzed yet. to address whether human nat is a target of silver nanoparticles and released irons on protein, purified nat was exposed to silver ions (agno ) and silver nanoparticles (ag -cooh, average size nm, carboxyl functionalized), and nat enzyme activity was analyzing the n-acetylation of the nat substrate para-aminobenzoic acid (paba). therefore, purified nat ( ng/µl) was co-exposed for min to paba ( mm) and agno or ag -cooh ( . , . , , and µg/ml each), resulting in a nat :silver relation of : . - (w/w). both, agno and ag -cooh inhibited the n-acetylation of paba in a concentration dependent manner. using equal amounts of silver and nat (w/w, µg/ml) enzyme activity was reduced about ± . % (agno ) and ± . % (ag -cooh). the lowest concentration analyzed ( . µg/ml) reduced nat activity about ± % (agno ) and ± % (ag -cooh). fifty percent activity reduction was caused by . ± . µg/ml of agno and . ± . µg/ml of ag -cooh, which is -fold lower compared to the published ic values for other metal oxide nanoparticles ( - µg/ml). these data indicate that both chemical silver species are able to modulate paba acetylation. further studies will be performed to clarify whether silver ions and/or silver nanoparticles could affect the specific n-acetylation of arylamines in human cells. colloidal silver has been used in medicine for centuries and nanosilver is present in many consumer-related products. however, despite of intense research in the past few years, the potential of nanosilver to induce effects different from ionic silver in vivo and in vitro, is still under debate. in this study, we compared proteomic effects of nanosilver (agpure™) and ionic silver (silver acetate) in the kidney of male rats after repeated oral delivery in a rat -days toxicity study. in order to avoid overt signs of toxicity, silver was dosed moderately in amounts of and mg/kg body weight for nanosilver and corresponding amounts of silver acetate ( . and . mg/kg). accordingly, no pathologic effects, including results from clinical chemistry and hematology, were reported. kidney tissue protein crude extract was separated by -d gel electrophoresis and differentially expressed spots were identified by maldi-ms. unique proteins, showing a log ratio of ≤ - . for downregulation and ≥ . for upregulation were identified in all treatment groups. protein lists were analyzed with ingenuity pathway analysis (ipa). when comparing effects of particulate and ionic treatments, similar alterations were indicated for canonical pathways associated with glycolysis, gluconeogenesis and tricarboxylic acid cycle. regarding inflammatory responses, stronger effects were derived for ionic treatments. for both types of silver exposures, changes of protein expressions were linked to changes of fatty acid metabolism and nrf -mediated stress. mitochondrial dysfunction was highlighted for both nanosilver treatments only, as well as activation of the insulin receptor. in the top-scored network of the higher dose nanosilver treatment, upregulated - - protein zeta (ywhaz) displays a central position. ywhaz, an important regulator of cell cycle and apoptosis, interacts with the insulin receptor and is well known to be envolved in many types of cancer. overall, both forms of silver treatment revealed similar patterns of affected cellular and molecular functions in rat kidney, supporting common and overlapping mechanisms of particulate compared to ionic silver. because of the widespread application of nanomaterials and the fact that for some nanomaterials effects on different organisms where shown, nanomaterials are still in focus of interest. moreover the fate of nps is only partiallyassessed over the lifecycle of products containing nanomaterials. while general toxicological properties of nps are well described in diverse in-vitro and in-vivo experiments, the distribution of these particles during the whole and complex process of waste incineration shows big knowledge gaps. in the "nanoemission"-project the entire route from the residual material via incineration, filtering of the exhaust gas up to a possible release into the environment are considered together with the toxicological evaluation of effects on humans and the environment. in these experiments the influence of thermal waste treatment on the toxicological profile of nanoparticles, contained in the waste, will be described. after a complete characterization of the two types of baso -nps from two different manufacturers by scanning electron microscopy/ energy dispersive x-ray analysis (sem/edx), measurement of the specific surface area (bet) and dynamic light scattering (dls) we investigated the impact of the pure baso -nps on primary cells (normal human bronchial epithelial cells (nhbec) and peripherial lung cells (plc)). both materials show statistically significant cytotoxic effects in the resazurin-assay (decreased viability below % for mg/ml after h). in general the effects of both nps were almost similar. additionally the effects of the baso -nps were compared more in detail. uptake of the baso was quantified by icp-ms after h and h as well as the release of ba-ions into the cell culture medium after centrifugal separation. the incubation with baso of plc and nhbec to . mg/ml over h and h leads to ~ µg baso / mio cells. the uptake is dose dependent but not time dependent. the impact on the secretion of inflammatory cytokines was determined by bead-based multiplex-elisa flow cytometry. tnf-α, il- and il- could be detected in nhbec and plc after np incubation. the possible induction of apoptosis was measured by flow cytometry as well. first investigations showed no induction of apoptosis for both materials. the impact of both nps on the intracellular glutathione level was measured by hplc and showed a decrease of gsh after h. summing up baso -nps showed toxic effects in primary human lung cell cultures after h for concentrations under mg/ml. c exoenzyme from c. botulinum is an adp-ribosyltransferase that inactivates selectively rhoa, b and c by coupling an adp-ribose moiety. rho-gtpases represent a molecular switch integrating different receptor signalling to downstream transcriptional cascades that regulate various cellular processes, such as regulation of actin cytoskeleton, cell proliferation and apoptosis. previous studies with the murine hippocampal cell line ht revealed a c -mediated inhibition of cell proliferation and a prevention of serum-starved cells from apoptosis (rohrbeck et al., ) . former results of studies on map kinase signalling indicated c -induced modulations of downstream signalling modules. therefore, ht cells treated with nm c for h were applied for screening of the activity of various transcription factors followed by luciferase reporter assays. five transcription factors namely sp , atf , e f- , cbf, stat were identified as significantly regulated in their activity. for validation of identified transcription factors, studies on the protein level of certain target genes were performed. western blot analyses exhibited an enhanced abundance of sp target genes p and cox- as well as a raise in phosphorylation of c-jun. in contrast, the level of apoptosisinducing gadd , a target gene of atf , was decreased. our results suggest that c is able to modulate the activity of transcription factors whose target genes are involved in the regulation of cell proliferation and apoptosis. via covalent binding of chemical compounds to dna, adducts can be formed. as a consequence, mutations may occur, which represent stages of chemical mutagenesis and carcinogenesis. the isolation of leucocytes represents an essential field of work within dna-adductomics of blood cells, in which adducts serve as markers of exposure for biological monitoring. peripheral mononuclear blood cells (pbmc) comprising monocytes and lymphocytes can be separated from other blood cells like granulocytes, erythrocytes (and dead cells) by isopycnic density gradient centrifugation of buffy coats (bc´s). bc´s are blood concentrates rich in leucocytes and thrombocytes, prepared from whole blood samples thorough removal of plasma and erythrocytes. for the experiments only bc´s from healthy donors were used. while erythrocytes contain no dna, lymphocytes, which constitute a subcategory of leucocytes, carry genetic information. a variety of commercially available fluids with a density of . g/cm , based on different polymers, exists. these materials form the gradient required for the centrifugation. furthermore, there are several methods available, which differ in the ratio blood vs. fluid, duration of the different work up steps, centrifugation parameters and others. the aim of this work was to compare the effectivity of the different fluids and optimize the workflow. for isolation of pbmc the fluid was put in a tube and then covered by a thin layer of blood. after centrifugation, five layers were obtained (figure ). the interphase was removed carefully, washed and the cells were counted. the yield is expressed as percentage of isolated vs. total leucocytes in the bc, which was based on the leucocyte count of the whole blood sample. as separation fluids, ficoll® paque plus (ge healthcare), histopaque® (sigma aldrich) and lsm® (ge healthcare) were tested. no significant differences between the different fluids were observed. although dilution is often recommended in literature, it was found that dilution had no effect on the yield. similarly, using different fluids (rpmi or pbs) for the washing steps did not reveal any differences. increasing centrifugation speed from to xg resulted in higher yields. in general, large variations in yield ( . % ± . %) among the various bc´s arose. this result is due to the different parameters such as age, storage, leucocyte and erythrocyte counts of the different bc´s used. therefore, the test conditions were optimized using the same batch of bc. the results show that the low priced separation fluids are comparable in performance tothe more expensive ones. by the direct lamination with bc (without dilution) the wastage could be minimized, the yield increased and thus the isolation made more efficient. the franz cell is a well-established model in lots of skin research fields. we adapted the diffusion cell system to additives and contaminants of consumer products which are designated for skin contacts. we were aimed to simulate real exposure as realistic as possible. to meet requirements like longevity, haptic properties and factory costs, different polymers are used as the raw material of choice and modified by a variable number of additives in the majority of commodities. besides additives with a defined function such as plasticizers, stabilizers, colorants and vulcanization accelerators, contaminants as well as decomposition products or so-called non-intentionally added substances (nias) can be part of the material, among them potentially harmful substances. in a first step, polymers of consumer products like flashlights or tools have been characterized concerning additive composition. possible breakdown products were identified by means of gc-ms/ms or pyrolysis-gc-ms. we focused on analytes of toxicological relevance including antioxidants such as n-phenyl- -naphthylamine (neozon d), which is suspected of causing cancer, and on degradation products like cresol and its derivatives (e.g., mesitol or -tert-butyl- -methylphenol). subsequently, analytes of interest were brought into direct skin contact using porcine, human and artificial skin models in the franz cell chamber assay. the analytes were either followed up layer by layer using tape stripping or examined utilizing cryo sections. for visualization purposes, analytical evaluation has been completed by use of imaging techniques like he staining or atr-ftir (attenuated total reflectance fourier transform infrared) microscopy. the latter was used with intrinsic markers for tissue specific distribution. our project provides evidence for the potential of polymer components to overcome the natural epidermal barrier and, in part, to enter the viable layers of the epidermis. during skin contact with consumer products several substances migrate out of the matrix and penetrate the skin, among them substances which are hazardous to health such as neozon d. depending on its dose, the blister agent sulfur mustard (sm) may lead to painful erythema, blistering with complicated wound healing, pulmonary edema, pulmonary bleeding and temporary blindness. sm is listed as a schedule chemical in the chemical weapons convention and thus its production, stockpiling and use is prohibited. sm still represents a serious threat for civilians and military forces, especially in asymmetric, terroristic and accidental scenarios. after exposure, the highly reactive molecule alkylates nucleophilic sites in endogenous biomacromolecules forming the characteristic and stable hydroxyethylthioehtyl (hete) residue. hence, bioanalytical methods targeting theses adducts in forensic post-exposure verification analysis are of high interest. herein, we present an optimized, accurate and comparably simple method to detect adducts of sm and human serum albumin (hsa) alkylated at its cysteine -residue. since albumin extraction from human plasma is a time-consuming and expensive step of an established procedure, an alternative method for direct proteolysis of human plasma was developed. plasma samples were cleaved directly with pronase resulting in the alkylated dipeptide hete-cysteine-proline (hete-cp) which is detected by micro-liquid chromatography-electrospray ionization high-resolution tandem-mass spectrometry (µlc-esi hr ms/ms). in order to optimize reproducibility and yield of proteolysis, kinetics were investigated for different kinds of plasma (edta-, citrate-and heparin-) as well as serum. two different mass spectrometers, a triple quadrupole system ( qtrap) and a hybrid quadrupole time-of-flight instrument (tt + ), were compared. the latter one proved to be the more selective and sensitive system. the method was successfully applied to in vitro and in vivo samples of real cases of sm poisoning. organophosphorus compounds (op), which were originally intended to be used as pesticides to increase agricultural yields at the onset of the th century, still represent a considerable threat to human health. by irreversible inhibition of acetylcholinesterase op lead to cholinergic crisis due to uncontrolled increase of acetylcholine in the synaptic cleft. finally, sudden death by respiratory failure may result if medical countermeasures are lacking. therefore exceptionally toxic compounds were designed und synthesized as chemical warfare agents (cwa), among which v-type nerve agents, i.e. vx, chinese vx and russian vx, belong to the most toxic artificial substances. recent events like the first gulf war, terrorist attacks in tokyo and the conflict in syria underline the need for ongoing and strict surveillance of cwa prohibition by the chemical weapons convention. unambiguous evidence of such substances (verification analysis) plays an important role with great political and legal impact. a variety of such bioanalytical methods have been established at the bundeswehr institute of pharmacology and toxicology in munich, which is accounted for medical chemical defence in germany. exhibiting quite short half-lives in vivo nerve agents can hardly be detected days or even weeks after exposure. accordingly, there is a great need for additional long-term biomarkers like specific protein adducts. consequently, the current work focuses on examination of adducts between nerve agents and human serum albumin (hsa) as its high abundance and stability in vivo provide relative ease of sampling. after incubation of hsa with v-type nerve agents in vitro the protein was subjected to proteolysis. subsequently resulting peptides were separated using microbore high-performance liquid chromatography (µlc) and detected on-line by modern high-resolution tandemmass spectrometry (hr ms/ms). this allowed unambiguous identification of already known phosphylated tyrosines as well as novel adducts between cysteine-proline dipeptides and the thiol-containing leaving group of v-type nerve agents. simultaneous detection of both biomarkers was realized by a new method, which was applicable even at the very low toxicologically relevant concentrations of v-type agents. therefore, this method represents a valuable and novel supplement of existing methods for verification. fatty acid esters of glycidol (glycidyl esters) are processing contaminants formed as byproducts of industrial deodorizing of plant fats or during other heating processes. following oral intake, glycidyl esters are mainly cleaved to release the reactive glycidol in the gastrointestinal tract. according to the national toxicology program (ntp), glycidol is carcinogenic, genotoxic and teratogenic in rodents. it is classified as probably carcinogenic to humans (iarc group a). the exposure assessment of the oral intake of glycidyl esters in humans is difficult, because the current data set for glycidyl ester contents in food is incomplete. we developed a method for the determination of the internal exposure to glycidol by mass spectrometric quantification of a hemoglobin adduct reflecting the total glycidol burden over approximately three months. a modified edman degradation was adapted for the cleavage of the valine residues from the n-termini of hemoglobin by fluoresceinisothiocyanat (fitc) (von stedingk et al. ( ) chem res toxicol , resulting in the formation of dihydroxypropyl-valine-fluorescein thiohydantoin (dhp-val-fth). the target analyte is purified with mixed-mode anion-exchange solid-phase extraction and analyzed by lc-ms/ms. a major advantage of the technique is the application to whole blood samples, which renders the time-consuming isolation of erythrocytes unnecessary. we synthesized dhp-d -val-fth as an internal standard for the quantification of the glycidol adduct by lc-ms/ms multiple reaction monitoring. a limit of detection of fmol per injection ( pmol adduct/g hemoglobin) was achieved. the application of this method will possibly allow future monitoring of the internal exposure of glycidol in human studies. glycidol and -monochloropropane- , -diol ( -mcpd) are carcinogenic food contaminants, which are present in heat-processed oils and fats mainly in form of fatty acid esters. the risk assessment concerning human consumption of these substances is complicated by various reasons. for example, the data on the occurrence in food stuffs is incomplete. also, the amounts of the proximate carcinogens released from ester hydrolysis in the gastrointestinal tract in humans are not known. monitoring of the internal exposure would be an alternative strategy to support the assessment of possible health risks related to the intake of glycidol and -mcpd and their fatty acid esters. for short-term monitoring of the internal exposure, urinary metabolites are suitable biomarkers. we study the potential use of two different substances as descriptors of the oral intake of glycidol and -mcpd. the metabolite , -dihydroxy mercapturic acid (dhpma) is generated following glutathione conjugation of both compounds. the second target analyte is -mcpd itself, which may also be formed from glycidol in the reaction with hydrochloric acid in the stomach. a method for the quantification of urinary -mcpd by gc-ms is currently developed. an lc-ms/ms multiple reaction monitoring technique was devised for the quantification of dhpma in urine samples with the isotope-labeled reference compound c -dhpma. the limit of quantification is µg dhpma/l. related to creatinine, the analyte was detected to be in a relatively small concentration range in urine samples from humans. the average concentration in urine samples (n = ) of one male volunteer collected over ten days was ± µg dhpma/g creatinine. a meal of a highly contaminated, commercially available frying fat (containing . mg of glycidol equivalents) did not lead to a visible increase of the urinary concentrations. the considerable background levels of dhpma in urine of humans and also in urine samples of other mammals support the hypothesis that dhpma may be also be formed from an endogenous c -metabolite, as already reported by eckert et al. furfuryl alcohol is a common food contaminant formed by acid-and heat-induced dehydration from pentoses. it induced renal tubule neoplasms in male b c f mice and nasal neoplasms in male f /n rats in a study of the national toxicology program (ntp). the neoplastic effects may originate from sulfotransferase (sult)-catalyzed conversion of furfuryl alcohol into the dna reactive and mutagenic -sulfoxymethylfuran. the incomplete data set of furfuryl alcohol contents in food does not allow estimating the human exposure. thus, we sought a method for the determination of the internal exposure. recently, the dna adduct of furfuryl alcohol n -[(furan- -yl)methyl]- ′deoxyguanosine was detected in specimen of human lung tissue. however, human biomonitoring of dna adducts has various disadvantages. for example, dna adducts are removed by various repair systems and human dna samples are usually not accessible in sufficient quantities. we decided to develop a biomarker for the internal exposure to furfuryl alcohol using blood proteins as dosimetric targets. bladder cancer (bc) is a smoking and occupation related disease showing a substantial genetic component. though the prognosis is generally good, a major problem is the frequent relapses affecting about half of the patients. n-acetyltransferase (nat ) is well-known to modulate bc risk in persons heavily exposed to carcinogenic aromatic amines. we aim to investigate the impact of nat genotypes, in particular, the ultraslow genotype, on relapse-free time after first diagnosis in bladder cancer cases. we used follow-ups of three case-control studies from lutherstadt wittenberg (n= ), dortmund (n= ) and neuss (n= ). nat was genotyped using seven characteristic polymorphisms (rs , rs , rs , rs , rs , rs , rs ). haplotypes were reconstructed using phase v. . . . we compared slow to rapid acetylators. additionally, we differentiated between the most frequent slow nat * b/* b and * b/other slow haplotypes as well as between the ultra-slow * a/* a and * a/other slow haplotypes compared to rapid acetylators. chi-square tests used to check the frequency of relapses in ultra-slow, slow and rapid acetylators. genotype differences in relapse-free time up to yr after first diagnosis of bc were analysed using cox proportional hazards models adjusted for age, gender, smoking habits, invasiveness and study group. a total of ( %) patients showed a relapse within the first yr after bc diagnosis. slow acetylators show a higher frequency of relapses than rapid acetylators ( % vs. %, p= . ). this frequency is even higher in ultra-slow acetylators ( %, or= . , p= . ) but not in slow * b/* b genotypes ( %, p= . ). ultra-slow acetylators had a significantly shorter relapse free time within yr after bc diagnosis than rapid acetylators (median . vs. . yr, hr= . , p= . ). this trend was not that pronounced in all slow acetylators combined ( . yr, hr= . , p= . ) nor in the subgroup of nat * b/* b genotypes ( . yr, hr= . , p= . ). the effect of ultraslow nat is even more pronounced in smokers (hr= . , p= . ) but absent nonsmokers (hr= . , p= . ). ultra-slow nat seems to be associated with a higher recurrence risk and a shorter relapse-free time, especially in smokers. slow nat in general seems to have less impact on recurrence. aalborg university, department of biotechnology, chemistry and environmental engineering, aalborg, denmark xenoestrogens with the potential for endocrine disruption like bisphenol a (bpa) may bind to the estrogen receptors (ers) and modulate expression of er target genes mimicking the natural ligand β-estradiol (e ). the potential for endocrine adversity is still predominantly assessed in vivo as existing in vitro tests have only limited value for an exposure-based risk assessment. thus, the development of reliable bioassays for the detection of endocrine disruptors is one of the paramount challenges faced by modern toxicology. a targeted metabolomics approach in mcf- cells treated with e or bpa revealed potential biomarkers for the estrogenic potency of the studied compounds. among them were several phosphatidylcholines and amino acids. we further addressed proline levels that were found to be strongly increased. investigations of proline levels over time showed a clear proliferation correlated concentration dependency after both e and bpa stimulation. furthermore, sirna knockdown experiments suggested an influence of the oncogenic transcription factor myc and the dependency of erα activation on the estrogen-mediated proline increase. our study demonstrates metabolomics as a powerful tool for biomarker identification and hypothesis generation. the results could be used further to develop bioassays for the detection of endocrine disruptive chemicals. children are considered to be more sensitive to most chemicals than the general population due to a variety of factors, including dynamic growth and developmental processes as well as physiological, metabolic and behavioral differences [ ]. however, only a few data are available on the magnitude of preschool children's exposure to most chemicals present in many consumer products. several of these chemicals are linked to endocrine disrupting effects in animal studies and are suspected to have also adverse effects e.g. on development and function of the reproductive organs as well as on neurological and behavioral development in humans. among of the chemicals that have been a major focus of discussion in the last years are phthalates, dinch, parabens, bisphenol a, and triclosan due to their suspected health effects. therefore we aimed to investigate exposure levels to metabolites of different phthalates and parabens as well as to bisphenol a and triclosan in urine samples collected from preschool children in german day-care centers from north rhine-westphalia (lupe iii; / ). urine specimens from children aged from to months from different day-care centers were analyzed. in total, preschool children were recruited with mean age of months. our study results show that nearly all children (> %) of the study population had urine concentrations equal to or above the limit of quantification for five most common phthalates metabolites (mnbp, mibp, oh-mehp, oxo-mehp, oxo-minp), for bisphenol a and methylparaben. triclosan was detected in % of the study population. in general, the median urinary concentrations of the above mentioned phthalate metabolites were about - µg/l in spot urine samples. the highest amount among the phthalate metabolites was observed for mibp with maximal values of about µg/l. median urinary concentration for methylparaben and bisphenol a were about µg/l and µg/l respectively. the maximum methylparaben, bisphenol a and triclosan level found were µg/l, . µg/l and , µg/l respectively. in conclusion, our study shows a widespread exposure of young children to various phthalates, parabens and bisphenol a in north rhine-westphalia, germany. a follow-up human biomonitoring study ( / ) has finished recruitment and is in the process of analyzing data. polycyclic aromatic hydrocarbons (pahs) represent a large group of organic compounds that are common environmental contaminants. they are formed by incomplete combustion of organic matter such as coal or crude oil and are often known to be carcinogenic, mutagenic and teratogenic. the acute toxicity of pahs is rather low, but because of their stability and lipophilic character those compounds can accumulate in the human body and cause severe chronic effects. additionally pahs may enter the food chain when preserving meat or fish by exposure to smoke. in the european union maximum levels of µg/kg benzo[a]pyrene and µg/kg as the sum of benzo[a]pyrene, benz[a]anthracene, benzo[b]fluoranthene and chrysene in the meat of smoked fish and smoked fishery products are set, respectively. smoked fish is often handmade in small fishery stores in schleswig-holstein, where self caught fish is prepared in smoke houses. this technique implies the danger of pahs to accumulate in smoked fishery products above allowed maximum levels. here, we report our findings of pahs in smoked fishery products bought in local convenience and fishery stores in schleswig-holstein and give a brief overview about actual contaminant levels. hplc with fluorescence detection was used to determine the quality and quantity of several toxic pahs in smoked fishery products made locally. pahs may constitute risks for human health when exposed to hazardous levels and therefore it is important to have knowledge about given contaminant levels. colorectal cancer (crc) is one of the most frequent cancers worldwide and is tightly linked to dietary habits. epidemiological studies provided evidence that the intake of red meat is associated with an increased risk to develop crc [ ]. red meat contains high amounts of heme iron, which is thought to play a causal role in tumor formation. the underlying molecular mechanism, however, remains elusive and may involve increased cell proliferation and dna damage induction by heme-iron. in this study, we set out to analyze the genotoxic and cytotoxic effects of heme-iron in human colonic epithelial cell lines. we used hemin (fe iii ) as commercially available heme source, which was compared to inorganic iron chloride (fecl ). first, the time-dependent internalization of hemin and fecl into hct cells was determined using icp-ms/ms analysis. treatment of cells with inorganic iron resulted in a maximum of intracellular iron content after h at all doses tested, while hemin particularly at high doses caused an iron accumulation up to h. hemin catalyzed the formation of reactive oxygen species (ros) in a dose-dependent manner in caco- and hct -cells as shown by flow cytometry. consistent with this finding, hemin dose-dependently induced the oxidative dna lesion -oxoguanine ( -oxog) as revealed by slot blot analysis and fpg-modified alkaline comet assay. using a pharmacological inhibitor of mutt homologue (mth ), which protects the nucleotide pool by hydrolysis of -oxogtp, -oxog dna adduct levels in hemin-treated cells were further enhanced. in contrast, inorganic iron hardly affected the cellular ros level and only slightly increased oxidative dna damage. subsequently, a time-and dose-dependent activation of the dna damage response (ddr) by hemin was shown in hct and caco- cells using western blot analysis, which was followed by a reduction in cell viability at high doses after h. finally, the cytotoxic effects of hemin and inorganic iron were tested using an ex vivo model of intestinal crypt organoids. preliminary results indicate that hemin is highly cytotoxic in organoids, whereas inorganic iron does not impair their viability. taken together, this study demonstrated that hemin induces oxidative stress and dna damage, resulting in the activation of the ddr and subsequent cytotoxicity. in contrast, inorganic iron displayed only modest effects. further in vivo studies using dna-repair deficient and proficient animals will shed light on the contribution of specific dna lesions to hemeassociated colorectal carcinogenesis. red and processed meat consumption is known to be a crucial risk factor in the development of colon cancer, which is one of the most common cancers worldwide. a diet rich in red and processed meat increases n-nitrosation within the colon leading to an increase in endogenously formed nitroso compounds. these compounds are able to alkylate dna of gastrointestinal tract cells, resulting in the formation of dna adducts such as -meg is repaired by mgmt, the potential of this enzyme to repair o -cmg in cells is not well characterized. additionally, adenomas containing a k-ras gc-at transition mutation have lower mgmt levels than adenomas without this mutation. therefore, the aim of this study is to determine the role of mgmt in protecting colorectal cells from genotoxicity by repairing o -cmg adducts. for this purpose, an mgmt-deficient non-transformed human colon epithelial cell line was established by stable transfection via rna interference to inhibit the expression and therefore the activity of mgmt. the transfected cell line was analyzed for complete mgmt gene silencing by activity and expression analyses and cell characterization. results confirmed that there was neither expression nor activity for mgmt in the transfected cells, and the cell characterization data showed that mgmt deficiency did not lead to differences in growth behavior or cell morphology or malignant cell transformation. cytotoxicity experiments performed in the transfected and nontransfected cell lines by treatment with dna alkylating agents suggest that the mgmtdeficient cell line is more sensitive to dna alkylating agents than the non-transfected cell line. these results were also supported by dna damage analysis via comet assay. asarones are secondary plant constituents mainly occurring in acorus calamus l. and guatteria gaumeri. the essential oil of the rhizome of acorus calamus l. is used for flavoring of food and alcoholic beverages. the concentration of β-asarone (ba) in these oils varies between . % and %. the bark of guatteria gaumeri, which is rich in αasarone (aa), is used as cholesterol-lowering drug and to treat gallstones in traditional mexican medicine. both, aa and ba are carcinogenic in rodents and mutagenic in the ames test after metabolic activation and thus classified as genotoxic carcinogens. [ , ] previously, the major metabolites in microsomal incubations with aa and ba were identified and synthesized in our laboratory. side-chain epoxides, the corresponding diols, side-chain alcohols and aldehydes were identified as the major metabolites of aa and ba. [ ] the investigation of the mutagenic potency in the ames fluctuation test showed positive results in the salmonella strain ta for aa and ba with metabolic activation and for aa-and ba-epoxide without metabolic activation. while it is known that epoxides are reactive against nucleophiles we set up the hypothesis of dna-adduct formation by epoxides to explain the mutagenic effect. this adducts are currently isolated, characterized by nmr-spectroscopy and used to quantify adducts in cells. at the same time primary rat hepatocytes were incubated with non-cytotoxic concentrations of aa and ba for h. after harvest and lysis of the cells, the dna was isolated by chloroform/phenol extraction and enzymatically hydrolyzed. [ ] the residual hydrolysates will be used to identify the dna-adducts formed in cells and to determine the adduct formation rate. preliminary experiments indicate that both, aa and ba also form dna-adducts in intact cells in a concentration-dependent manner. [ ] göggmann, schimmer ( ) . mutagenicity testing of α-asarone and commercial calamus drugs with salmonella typhimurium. mutation research. , [ ] [ ] [ ] [ ] wiseman et al. fatty acid esters of -chloro- , and of -chloro- , are food process contaminants that are formed, e.g., during refinement of vegetable oils. after ingestion, the esters are hydrolyzed in the gut, thereby releasing free -mcpd and -mcpd. -mcpd has been identified by the international agency for research on cancer (iarc) to be possibly carcinogenic to humans (class b) and is therefore in the focus of food safety authorities. to elucidate the toxicological properties of these compounds at the molecular level (mode of action) a proteomic study was conducted in which mg/kg b.w./day of either -mcpd or -mcpd were orally administered to rats over a period of days. total protein was extracted from different tissues of the animals and separated via twodimensional gel electrophoresis ( de). among others, the redox sensor protein dj- was identified to be deregulated in liver, kidney, testis, and heart of rats treated either with -mcpd or -mcpd. up to six different isoforms of dj- were identified by de-western blot analysis, all of them having the same molecular weight but different pi values, indicating protein modifications of low molecular weight but with a strong impact on the protein charge. treatment of the animals with either -mcpd or -mcpd predominantly led to a shift of the abundance between two dj- isoforms in the rat tissues. this effect was more pronounced with -mcpd compared to -mcpd. mass spectrometric analysis of these two isoforms identified an oxidation of a conserved cysteine residue (cys ) of dj- to a cysteine sulfonic acid to be the protein modification induced by treatment of the rats with either -mcpd or -mcpd. dj- is discussed to participate in a number of biological processes such as proteolysis, protein folding, or redox regulation. oxidation of cys was shown to be crucial for the activity of dj- , and the irreversible oxidation of cys to a cysteine sulfonic acid as observed in the present study was shown to result in a loss of protein function and was correlated with diseases related to oxidative stress such as parkinson's disease. thus, the potential impact of -mcpd and -mcpd on cellular oxidative stress and on associated neurodegenerative diseases has to be considered in the ongoing risk assessment of these food contaminants. pyrrolizidine alkaloids (pa) are secondary plant compounds and widely spread in plant kingdom. humans can therefore be regularly exposed via direct or indirect contamination of food, like herbal teas, honey, wheat or salad. , -unsaturated pa are known for their potentially harmful effects. an acute intoxication may cause venoocclusive disease leading to hepatomegaly, ascites as well as liver hardening resulting in high mortality. on the other hand, chronic pa intoxications due to regular consumption of small amounts of pa are characterized by hepatic necrosis, fibrosis and cirrhosis. an initial whole genome transcriptome study in hepatocytes revealed that molecular pathways related to cancer development, cell cycle regulation and cell death are regulated by the four structurally different pa echimidine, heliotrine, senecionine and senkirkine in short-term exposure ( h). additionally, lipid and bile acid metabolism was affected. however, the uptake of pa by food is more likely to be continuous than singular. therefore, the aim of this study was to investigate molecular effects of longterm exposure ( d) comparatively to short-term exposure ( h) in the hepatoma cell line heparg with the four structurally different pa. in this context we analyzed selected cellular parameters like cytotoxicity and morphology. in contrast to short-term exposure, structure-and concentration-dependent cytotoxicity was found for the long-term exposure (sn>he>em/ski). furthermore, obvious morphological changes such as destructuration and perforation of the heparg cell monolayer were seen after d of incubation. based on these findings, a possible induction of apoptosis or necrosis by pa was examined. effects of long-term exposure to pa on gene expression were investigated for a set of genes found to be regulated in the short-term whole genome transcriptome study. the identified regulation of gene expression was confirmed for both terms, with the strongest regulation for cyp a (down-regulation), a gene involved in cholesterol metabolism. therefore, the effects of pa on various parameters related to cholesterol metabolism were analyzed, showing pa effects on cholesterol levels and bile acid secretion. short-term exposure to pa did not affect cell viability. however, repeated doses of pa resulted in severe effects on hepatic cells, concerning viability and morphology. at the mrna level, short-and long-term incubation with pa seem to affect a wide range of signaling pathways. in conclusion, we show for the first time that heparg cells can serve as an in vitro model for hepatotoxicity following chronic intake of pa. shiga toxin-producing e. coli (stec) strains cause a diversity of enteric symptoms in humans, ranging from mild diarrhea to severe diseases such as the hemolytic uremic syndrome (hus). hus is a life threatening disease with microvascular endothelial damage in the gastrointestinal tract and the kidneys, which often leads to haemolytic anemia, thrombocytopenia and acute renal failure. shiga toxin plays a major role for the pathogenesis of hus but the subtilase cytotoxin, which was identified during an hus outbreak in in stec strains, might contribute as an additional potent enterotoxin. like shiga toxin, subab is an ab protein toxin. the pentameric binding/transport subunit (subb) delivers the enzymatic active subunit (suba), a protease, into the endoplasmic reticulum (er) of human target cells. in the er, suba cleaves the chaperone bip/grp , which results in cell stress and caspase / dependent cell death. recently, we discovered that higher concentrations of suba ( mg/ml) causes cytotoxic effects in human epithelial cells (hela, in the absence of subb. the cytotoxic effects were investigated in hela cells in more detail. here, suba binds in a concentration dependent manner to the cell surface and induces dramatic morphological changes as well as caspase-dependent cell death [ ] . in contrast to hela and caco- cells, cho fibroblasts did not respond to suba. currently, further cell types including macrophages are tested for suba effects and the molecular mechanisms underlying the cytotoxic effects caused by suba and the cell type selectivity are investigated. although there are strong cytotoxic effects caused by suba on some human epithelial cells in vitro, the situation in vivo and the pathogenic relevance of the newly observed suba effect are not known. thermal treatment of fat-containing foodstuff in the presence of salt leads to formation of -mcpd and its esters. high amounts of -mcpd esters detected in food raised toxicological concern. recent studies revealed that food may also contain significant amounts of structurally related -mcpd and its fatty acid esters. toxicological studies indicate genotoxicity in vitro and a carcinogenic potential of -mcpd, pointing to kidney and testes as main target organs. -mcpd esters were shown to cause similar, but milder effects. few unpublished data exist for -mcpd, showing similar organ toxicity compared to -mcpd and identifying heart as additional target organ. no such data exist for -mcpd diesters. in consequence, further toxicological data were required in order to complete risk assessment for -mcpd, -mcpd and their esters. hence, an oral -days study was performed in male rats in order to fill data gaps and provide essential information for risk assessment. a proteomic analysis was included in order to compare molecular effects induced by -mcpd and -mcpd and its dipalmitic ester in rat liver, kidney, testes and heart. in order to avoid overt toxicity, moderate doses of -mcpd + -mcpd ( mg/kg body weight), and -mcpd-dipalmitate ( + . mg/kg body weight) were applied. accordingly, no pathologic effects were reported. here, we present proteomic results obtained after analysis of heart tissue. after separation of heart tissue protein crude extract by -d gel electrophoresis , differentially expressed spots were identified by maldi-ms. a total of unique proteins deregulated at a log ratio of ≤ - . for downregulation and ≥ . for upregulation at p ≤ . were identified. comparing deregulated spots induced by different treatments revealed that a higher number of spots was deregulated by -mcpd versus -mcpd. dipalmitate treatment even caused more deregulation than -mcpd. upregulated cytochrome b-c complex subunit rieske (ucri) and downregulated acetyl-coa acetyltransferase (thil) were among the top deregulated proteins after -mcpd and -mcpd dipalmitate treatment. pronounced upregulation of respiratory chain protein ucri, not deregulated after -mcpd treatment, indicates an effect on energy metabolism. downregulation of thil, involved in ketone body metabolism, was only weakly affected after -mcpd treatment. protein dj- (park ), a multifunctional redoxsensitive chaperone and protease protecting the cell against oxidative stress, was significantly downregulated after treatment with -mcpd and the higher dose of the -mcpd diester. tropomyosin beta chain (tpm ) was commonly upregulated after -mcpd and -mcpd treatments, possibly indicating a tgf-beta induction of actin stress fibers. for rat heart, data show that similarities but also some significant differences of -mcpd-and -mcpd dipalmitate-induced proteomic changes exist compared to -mcpd, indicating different mechanisms of toxicity for this structural analogues. oxidation products (oxy) of cholesterol (chol) such as α-hydroxy-chol ( α-ho-chol), β-hydroxy-chol ( β-ho-chol), -keto-chol ( -o-chol), , α-epoxy-chol (α-epoxy-chol) and , β-epoxy-chol (β-epoxy-chol) are formed by autoxidation of chol and are discussed as biomarkers for inflammation and oxidative stress in human tissues to be used in the identification of risk factors for disease. however, oxy-chols are also present in processed foodstuff where β-ho-chol (milk) and -o-chol (fish, meat, and egg) tend to represent the main oxychols, whereas epoxy-chols, are generally minor constituents. thus, levels of oxychols in tissues may result from both endogenous formation as well as dietary exposure. since quantitative profiles of oxy-chols have not been determined in human adipose tissues yet, levels of oxychols and chol were quantified using gc-ms/ms (internal standards: deuterated oxychols) and gc-fid (internal standard: α-cholestan- β-ol), respectively in breast adipose tissues of healthy women undergoing mammoplasty. furthermore, tissue levels of fatty acids in adipose tissues were determined by gc-fid (internal standard: undecanoic acid) to assess relative levels of pentadecanoic acid, docosahexaenoic acid and elaidic acid, indicative for consumption of dairy products, fish, and processed fats, respectively. all oxychols were detected in all breast adipose tissues. the most abundant oxychol was β-epoxy-chol (median: . nmol/g tissue, range: . - . nmol/g), followed by α-epoxy-chol > -o-chol > α-ho-chol> β-ho-chol ( . nmol/g, range: . - . nmol/g). tissue levels of chol ( . micro mol/g, range: . - . micro mol/g) did not correlate (spearman's rank analysis) with that of epoxy-chols and correlated even negatively with that of α-ho-, β-ho-, and -o-chol (r = - . , p= . - . ) median oxychol/chol ratios ranged from . ( , to . ( β-ho-chol). -o-chol and -ho-chols correlated strongly with each other (r= . - . , p oxy-chol levels did not correlate with relative amounts of pentadecanoic acid and docosahexaenoic acid, whereas total oxychol, β-ho-chol, and β-epoxy-chol levels correlated with relative amounts of elaidic acid (r= . , . , and . , respectively, p= . , . , . , respectively) . no correlations of oxychol levels, individual or total oxychol/chol ratios with age or bmi were observed. taken together, oxychol profiles in breast adipose tissues were different from that usually present in food but could be affected by dietary habits. classification and labelling of hazardous substances and hazardous consumer products ( ) has proven to be a very efficient tool for risk communication. consumer products, such as glue, varnish, or washing and cleansing products need to be classified and labelled if they contain dangerous ingredients that render the mixture hazardous. personal care products, however, need not be classified and labelled if they contain dangerous substances above the thresholds for classification. they are excluded in the clp regulation. what would happen without this exception? when i applied the criteria for classification and labelling to a selection of cosmetic product formulas in a conservative approach, most products would have to be labelled and classified, mainly due to hazardous effects to the eye and to the skin ( ) . benefits of personal care products can go along with unwanted properties such as the hazards for the human health, and consumers should be informed about them ( ) . risk communication for every day products like personal care products should be clear, easily and quickly understandable. according to the cosmetic regulation ( ) the ingredients must be listed on the containers. it must be questioned whether the listing of the ingredients without hazard pictograms on the products could be considered as a clear, easily and quickly understandable risk communication instrument ( ). the results show that it is urgent to inform consumers better about the potential dangers of personal care products, because cosmetics need to be applied even with more care than any other consumer product. it is strongly recommended to delete the exception provision in the clp regulation for personal care products. the infochemical effect describes that anthropogenic substances can interfere with the chemical communication and influence organisms so that they perceive their chemical environments differently ( , ). infochemicals play a role in life history, habitat search, food related aspects and survival which shows that disturbed communication (infodisruption) could affect population vulnerability at various decisive points ( ). the classical ecotoxicological standard tests do not allow to observe the infochemical effect. systematic analyses are needed to find out more about the relevance of this new chapter in ecotoxicology for natural ecosystems. the first crucial step is to select suitable test substances. repellents (substances used to keep away target organisms, e.g. invertebrates such as midges or fleas via olfaction) enter surface waters mainly indirectly via wastewater discharges from sewage treatment plants or directly by being washed off from the skin and clothes of bathers. there are various indications that repellents which are not toxic for aquatic animals could induce effects like organismic downstream drift of non-target species (downstream dislocation of e.g. crustacean and insect larvae in streams). in a literature study, three repellents were identified to be suitable test compounds for proof of concept of the infochemical effect. deet (cas - - ), icaridine (cas - - ) and ebaap (cas - - ) ( ). these substances are investigated in new test designs in a subsequent experimental part of the project which are designed to detect and quantify the infochemical effect. persistent, bioaccumulative and toxic (pbt) substances or very persistent and very bioaccumulative (vpvb) substances are compounds with hazardous properties. the non-biodegradability (persistence) and high accumulation in organisms (bioaccumulation) may elicit long-term adverse effects in the environment. persistent and bioaccumulative substances concentrate in the environmental compartments (water, sediment, soil, air) and can be distributed in the food chains. ecotoxicological effects are strengthened by bioaccumulation and appear often in remote areas like marine and polar regions. in the framework of pbt assessment, contrary to the risk assessment, the substances are evaluated regardless of the emission into the environment. an evaluation of medicinal active ingredients under assessment in the german federal environment agency (uba) identified less than % as potential pbt candidates. due to data lacks in many cases a definite pbt classification is not possible. the poster presents results of an extensive literature research on the global occurrence of pharmaceuticals in the environment. data on measured environmental occurrences from more than , international publications have been transferred to a database, with more than , entries. according to the database, pharmaceuticals have been found in the environment of countries worldwide in all five un regions. most published data are for the compartments surface water and sewage effluent; less information is available for groundwater, manure, soil, and other environmental matrices. more than active pharmaceutical substances (or their metabolites and transformation products) have been detected in the environment. most findings have been published for industrialized countries. monitoring campaigns are increasingly being conducted in developing and emerging countries. these have revealed the global scale of the occurrence of pharmaceuticals in the environment. for example, diclofenac, a non-steroidal inflammatory drug, has been detected in the aquatic environment in countries worldwide. a number of globally marketed pharmaceuticals have been found in both developing and industrialized countries. the available ecotoxicological information indicates that certain pharmaceuticals pose a risk to the environment at measured concentrations. options for cooperative action to address the risk of are also presented. the aim of the research presented was to support the discussion of the proposed emerging policy issue pharmaceuticals in the environment under the strategic approach to international chemicals management (saicm), which is a global initiative of united nation environment programme (unep). the european chemicals' legislation reach aims to protect man and the environment from substances of very high concern (svhc). chemicals with (very) persistent, (very) bioaccumulative and toxic properties (pbt and vpvb compounds), substances that are carcinogenic, mutagenic and toxic to reproduction (cmr compounds), as well as chemicals of comparable concern like endocrine disruptors (eds) may be subject to authorization. the identification of eds is limited as yet, because specific experimental assessments are not required under reach. evidence is currently based on a combination of few experiments, expert judgement and structural analogy with known eds. structural alerts for the identification of potential eds: predictions of properties and effects from chemical structures are based on similarities with either active or inactive substances. structural alerts for the identification of potential estrogen/androgen-ed activities are relevant parts of the structures of compounds that are known to interact with estrogen and androgen receptors as either agonists or antagonists. in addition to the backbones of the chemical structures (pharmacophore) for steric fit to the receptors, modulating factors may be small substructures for local interactions with receptor binding sites and physicochemical properties related to the strength of binding to the receptor. comparison of evidence from in silico, in vitro and in vivo assays for potential eds: identification of potential eds based on findings from mammalian long-term reproduction studies, fish life-cycle tests, receptor assays, and chemical alerts were compared and differences analysed. agreement is limited because in vivo, in vitro and in silico methods address different aspects of potential effects on endocrine systems regarding toxicological targets, modes of action and functional similarity of chemical structures. a combination of toxicological and chemical assays can provide comprehensive and complementary information to support evidence-based identification of potential eds among the chemicals released into the environment. application of structural alerts for the identification of potential eds to the einecs inventory: more than discrete organic einecs compounds are within the applicability domain of the structural alerts for potential estrogen/androgen-ed activities. among them, chemicals (ca. %) are indicated as potential candidates for endocrine effects based on structural alerts. due to the possibility that these chemicals may interact with estrogen/androgen receptors they should be subject to further investigations regarding their potential for endocrine effects, eventually leading to regulatory actions. within the imi (innovative medicine initiative) project "intelligence-led assessment of pharmaceuticals of the environment " (ipie;http://i-pie.org/), a more intelligent environmental testing and tools for prioritisation of legacy compounds shall be developed. regarding the evaluation of fish toxicity, screening approaches in fish embryos are pursued. while the standard fish embryo toxicity (fet) test is restricted to lethal parameters more relevant as substitutes for acute effects, additional sublethal endpoints may provide expanded applicability of the fet assay for chronic effect assessment in fish. in this respect, the analysis of the metabolome could provide additional insights into biochemical processes elicited by pharmaceutical compounds and could potentially support the extrapolation from fish embryo to chronic fish toxicity as displayed in the standard early life stage (els) test. in the context of ipie a pilot study was performed with the aim to quantify and comparatively assess changes in the metabolic signatures of fish and fish embryos induced by the reference compound amikacin, an aminoglycoside antibiotic, in order to identify metabolic patterns applicable as biomarker profiles that can be linked to apical endpoints in terms of an integrated approach. therefore, toxic effects in fathead minnow embryos and els fish were investigated following a and days exposure, examining conventional endpoints and additionally using a combined direct injection and lc-ms/ms assay for metabolite identification and quantification. metabolic endpoints were found to be at least as sensitive as standard apical endpoints such as growth and mortality as detected in the longer-term fish study. furthermore, multivariate data analysis (pca-x, opls-da) revealed substance induced metabolic perturbations specific for fish and fish embryos, respectively. beyond that, the statistical approach of shared and unique structure (sus) identified some metabolites from the classes of amino acids, biogenic amines and lipids to be similarly changed in both developmental stages related to amikacin treatment, representing shared biomarker candidates. in this first pilot study, the integrated metabolomics approach yielded insights into the molecular consequences of amikacin exposure and provided indications for biomarkers for common effects in fish embryos and fish. due to the different exposure levels in the fet ( - mg/l) and els study ( . - . mg/l), more definitive conclusions regarding the predictivity of metabolic responses in fish embryos for apical endpoints in chronic fish tests are yet not possible. further studies are ongoing with a range of pharmaceutical compounds of different chemical classes which will reveal more substantial information on the applicability of this technology in the prediction of longterm effects in fish. the human cationic amino acid transporter hcat- (slc a ) represents the major uptake route for arginine and other cationic amino acids (such as the essential amino acid lysine) in most mammalian cells. it thus provides these amino acids for protein synthesis as well as for essential metabolic pathways. in endothelial cells, special attention has been given to the role of hcat- for supplying arginine to nitric oxide synthase. in spite of its wide distribution, hcat- expression is highly regulated both, on the transcriptional and post-transcriptional level. however, the genetic elements involved in transcriptional regulation a largely unknown. here we studied the expressional regulation of hcat- in human ea.hy endothelial cells. starvation of these cells from cationic amino acids led to a pronounced induction of both, hcat- mrna and protein. the mrna induction was almost completely abolished by the transcription elongation inhibitor drb ( , -dichloro- -β-dribofuranosylbenzimidazole), suggesting the involvement of transcriptional regulation. we thus aimed at identifying the promoter elements in the hcat- gene responsible for this regulation. to our surprise and in contrast to data published by others the chromosomal region up to kb upstream of the first hcat- exon exhibited no promoter activity in either endothelial or dld- colon carcinoma cells that exhibit a very strong endogenous hcat- expression. we could however identify a promoter element within the first intron of the hcat- gene. transcriptional activity of this element increased upon amino acid starvation in a similar way as endogenous hcat- expression. our results indicate starvation-induced transcriptional regulation of hcat- through newly identified promoter regions distinct from those published previously. the transport of a multitude of drug molecules into the cell is mediated by multispecific organic cation transporters (octs), belonging to the solute carrier group (slc). one of these families within this group of membrane transport proteins is the slc -family consisting of the two multidrug and toxin extrusion transporters mate- (slc a ) and mate -k (slc a ). while mate- is highly expressed in several different tissues like kidney, liver, skeletal muscle, adrenal glands, testes and heart, mate -k exclusively occurs in the apical membrane of proximal tubular epithelial cells within the kidney. both transport proteins translocate organic cations in exchange of protons into as well as out of the cell. to define the affinity of both transporters for the anti-diabetic drug metformin and to investigate their interactions with different anti-neoplastic agents comparative transport experiments with the model substrate -methyl- -phenylpyridinium (mpp) have been carried out. therefore stably transfected hek -cells expressing mate- or mate -k transport proteins generated by portacelltec biosciences gmbh and vector transfected hek -cells were used. the interaction analyses were carried out by determining the uptake of [ c]-metformin and the inhibition of [ university of basel, basel, switzerland drug transporters play a pivotal role in pharmacokinetics by modulating the cellular entry or efflux of compounds. one transporter facilitating the transport of bile acids, steroid hormones, and statins is the organic anion transporting polypeptide (oatp) b that is highly expressed in placenta, liver, and small intestine. especially its activity in small intestine and liver is assumed to be basis for specific drug-drug interactions. understanding mechanisms involved in pharmacokinetics is a prerequisite in drug development. to test whether there are species differences in transport activity we compared the expression and function of the human and rat orthologue. first, we determined the transport activity of the known oatp b substrate estrone sulfate (e s), and observed a significantly lower k m for mdck-hoatp b compared to . ± . µm, *p< . student´s t-test) whereas there was no difference in v max (mdck-hoatp b . ± . fmol/min/µg protein; mdck-roatp b . ± . fmol/min/µg protein). the human oatp b exhibits multiple binding sites for its substrates that may explain specific drug-drug interactions [ ] . to identify whether rat oatp b owns the same characteristics, the cellular accumulation of µm e s (low affinity site) or . µm e s (high affinity site) was determined in presence of specific inhibitors/substrates of oatp b . as observed for atorvastatin, a known inhibitor of both affinity sites, the rat transporter failed to exhibit the low affinity site. in detail, while atorvastatin reduced the accumulation of e s in mdck-hoatp b cells ( . ± . fmol/min/µg protein vs. . ± . fmol/min/µg protein, *p< . student´s t-test), there was no inhibition of e s accumulation in mdck-roatp b cells ( . ± . fmol/min/µg protein vs. . ± . fmol/min/µg protein). additionally, we observed a different membrane localization of both transporters as assessed by immunofluorescent staining showing an apical localization for rat oatp b while human oatp b is localized at the basolateral pole of the cellular model. absolute quantification of mrna (copy number per ng of total rna) in different tissues of rat revealed a high expression of oatp b in liver ( . ± . ), a moderate expression in small intestine ( . ± . ) and colon ( . ± . ), and a low expression level in kidney ( . ± . ) . the latter is in accordance with previous findings showing that oatp b is abundant in rat kidney as quantified by absolute proteomics technics [ ] . our data demonstrated species differences in localization and activity of the drug transporter. further studies are warranted to proof whether this knowledge will help in future drug development and which molecular cause is responsible for the observed data. background: intestinal drug absorption depends on various factors including aqueous volume, site-specific ph and intestinal motility. moreover, the expression of efflux-and uptake-transporters vary in dependence of the anatomical localization making the gut a complex barrier for drug transfer into the body. in a recent study, site-specific protein and mrna expression levels of drug transporters were determined along the entire length of the human gut. interestingly, discrepancies between mrna expression and protein levels were observed. moreover, there were quantitative differences between small intestine and colon. as a consequence the question arose if this observation could be explained by small non-coding rnas acting as highly tissue-specific posttranscriptional regulators of gene expression. hence, in our current study, we aimed to investigate the impact of mirnas on site-specific transporter expression along the human intestine. methods: total rna was isolated from biopsies obtained from six disease-free organ donors. tissue samples were acquired from the duodenum, the upper and lower jejunum, the upper and lower ileum, and the transversal or descending colon. the expression of mirnas was measured using rt-pcr based low density arrays. expression of all detected mirnas was correlated with transporter protein data recently determined by lc-ms/ms-based targeted proteomics. mirnas and transporter genes showing an inverse pearson's correlation between mirna and protein expression underwent an in-silico search (microcosm targets v. , targetscan . ) for putative mirna/mrna interaction. to investigate those interactions in more detail, reporter gene assays and site directed mutagenesis were conducted. results: out of mirnas, were detected in all tissue types. out of ten transporters five showed significant inverse correlations with putatively targeting mirnas (e.g. pept and hsa-mir- a, r= - . , p= . ). reporter gene assays indicated interactions of mir- a/b with pept '-utr (p = . and p = . ) as well as of mir- a with abcb '-utr (p = . ). the site-specific abundance of intestinal drug transporters is significantly affected by microrna-mediated post-transcriptional regulation under physiological conditions as exemplified for pept and abcb . background: the human uptake transporter nact [gene symbol slc a ; also known as mindy, the human orthologue of the drosophila indy (i´m not dead yet) transporter] is expressed in liver and brain. nact is important for energy metabolism and brain development and mediates the uptake of tricarboxylic acid (tca) intermediate such as citrate and succinate. reduced expression of this transporter, as studied in knock-out mice, mimics aspects of dietary restriction, promotes longevity and protects against insulin resistance and adiposity. furthermore, mutations in the human slc a gene are associated with epileptic encephalopathy with seizure onset in the first days of life, possibly due to the reduced uptake of tca intermediates into neurons. to gain more insights into the role of nact in drug transport and into structure-function relationships, we studied the inhibition of nact-mediated citrate transport by various drugs and analyzed the effect of known mutations in the slc a gene on nact-mediated transport. methods: drug inhibition studies were performed using hek cells stably expressing human nact with citrate as prototypic substrate. twenty-four drugs were used as potential inhibitors of nact-mediated uptake. the effects of eight mutations, three of them (nactp.g r, -p.t m and -p.l p) associated with epileptic encephalopathy, were analyzed using a transient transfection approach. furthermore, the first computational-based structural model of the nact transporter was established and the impact of all mutations on substrate transport was modeled. results: inhibitions studies demonstrated that only a few drugs (three out of tested) inhibited nact-mediated citrate transport at the tested drug concentration of µm. from these, benzbromarone shows the strongest inhibition with an ic value of . µm. furthermore, citrate transport was also slightly inhibited by pioglitazone and rosiglitazone. citrate transport of the mutated proteins nactp.g r, -p.g e,p.t m, -p.l p and -p.l p was totally abolished and the effect of these mutations could be explained on the basis of the structural model. conclusion: inhibition studies demonstrated that simultaneously administered drugs can inhibit nact-mediated uptake of the prototypic substrate citrate. nact-mediated uptake is abolished by mutations in the slc a gene associated with epileptic encephalopathy. the effect of these mutations can be explained on the basis of the first structural model of this uptake transporter. the atp-binding cassette subfamily b member (abcb ) is a drug efflux pump responsible for the classic multi-drug resistance phenotype in cancer cells. increased activity of abcb in cancer cells contributes to protection against a wide range of chemotherapeutic agents. this dramatically decreases therapeutic options and the chance of patient survival. knowledge of the underlying mechanisms for abcb deregulation is a critical step for the reversal of this unfavorable condition. of note, phosphatidylinositol- , -bisphosphate (pip ) is a known regulator of abcb . the protein "myristoylated alanine-rich c-kinase substrate" (marcks) is known for its ability to bind and sequester the phospholipid pip . in various forms of colorectal cancer the deregulation of marcks protein goes hand in hand with an increase in malignancy and therapeutic resistance. in this study, we characterized the enigmatic marcks as a modulator of abcb activity. we focused on a subgroup of colon cancers, where marcks resides in a hyperphosphorylated state for which the established cell line ht- served as a model. we employed various in-vitro methods for the measurement of abcb activity, in combination with imaging experiments, assays for cellular viability and classical methods of molecular biology. we combined these approaches with pharmacological inhibition of marcks phosphorylation state or rnai-mediated depletion of marcks. with these interventions we could modulate endogenous abcb activity and re-sensitize our cell model against chemotherapeutic agents like -fluorouracil which are known to be substrates of abcb . taken together, our findings suggest a new way how a cancer cell can gain a state of therapeutic resistance. the exploitation of marcks as modulator of abcb might be a new approach to target resistant tumors without interfering with the natural function of abcb in non-malignant tissue. background: in several large clinical studies low blood concentrations of homoarginine were identified as independent risk marker for stroke, cardiovascular events and mortality. experimental data suggest an active role of homoarginine deficiency in disease development. interference with l-arginine-dependent pathways and signaling has been implicated as a possible mechanism. it was the aim of the present study to identify transport mechanisms involved in the cellular uptake and tissue distribution of homoarginine, which is poorly understood, so far. the experiments focused on cationic amino acid transporters (cats) and possible interactions with known cat substrates. methods: uptake assays were performed using [ h]-labeled homoarginine as substrate and human embryonic kidney (hek ) cells stably overexpressing human cat [gene: slc a (solute carrier family )], cat a (slc a a) or cat b (slc a b). cells transfected with an empty vector were used as control. unlabeled known catsubstrates l-arginine and asymmetric dimethylarginine (adma) were investigated as inhibitors. results: compared to the uptake into control cells, uptake of homoarginine was significantly higher in hek cells overexpressing cat ( -fold), cat a ( . -fold) or cat b ( . -fold) after . min using µm substrate (each p< . ). apparent k m values for cellular net uptake of homoarginine were . µm for cat , µm for cat a and . µm for cat b. homoarginine uptake by the three cats could be inhibited by addition of l-arginine and adma. conclusion: the protective biomarker homoarginine is a substrate of the human cationic amino acid transporters cat , cat a and cat b. compared to other cat substrates, the cat -and cat b-mediated homoarginine transport is characterized by relatively high affinity. the uptake of homoarginine by all three cats can be inhibited by l-arginine and adma. taken together these findings make cat-mediated transport of homoarginine a possible target for interactions and pharmacological interventions aimed at homoarginine homeostasis. this project is supported by the doktor robert pfleger-stiftung. background and aim: organic cation transporter (oct , alternative name slcc a ) is a polyspecific membrane transporter, which has been suggested to play a role in absorption, distribution and elimination of drugs and toxins. besides endogenous compounds like thiamine (vitamin b ), known oct substrates are toxins like mpp + as well as drugs like metformin, o-desmethyltramadol, ranitidine, and sumatriptan. tissue distribution of oct has been shown to have strong inter-species differences. in rodents oct is strongly expressed in both the sinusoidal membrane of hepatocytes and the basolateral membrane of kidney epithelial cells. human oct is strongly expressed on the sinusoidal membrane of hepatocytes, but not in the kidney. furthermore, substrate specific differences have been observed between the human and rodent oct orthologs. the aim of this study is to characterize the mechanisms causing substrate specificity between rodent and human oct orthologs. methods: overexpression of oct orthologs in mouse, rat and human cells was performed by targeted chromosomal integration of t-rex™ . the cells were characterized in detail for their ability to transport the model substrates tea + , mpp + , and asp + , the drugs ranitidine, sumatriptan and fenoterol. results: mouse and rat oct orthologs showed similar transport activity for all the model substrates and drugs tested. however, significant differences were observed when rodent orthologs were compared to the human oct . human oct showed a fold higher v max for the uptake of asp + and -fold increase of v max for sumatriptan in comparison to the rodent oct orthologs. conversely, human oct showed a -fold lower v max for the uptake of fenoterol compared to rodent oct s. there was no difference between rodent and human oct in the uptake of ranitidine. these differences were further characterized in detail using chimeric mouse-human oct constructs and by comparing the effects of key point mutations in mouse and human oct orthologs. conclusions: these data demonstrate strong differences in the substrate specificity between rodent and human oct orthologs. therefore oct pharmacokinetic data obtained in mouse or rat models could not be directly extrapolated for use in human. furthermore, comparative functional analyses of orthologs may help reveal the mechanisms underlying polyspecificity of oct . ranitidine is a histamine h -receptor antagonist which is commonly used without prescription for the treatment of pyrosis and gastric ulcers. approximately % of the systemic clearance of ranitidine is via hepatic metabolism. ranitidine is known to be a substrate of the hepatic organic cation transporter oct [ ]. oct is expressed on the sinusoidal membrane of human hepatocytes and is highly genetically variable. sixteen major oct alleles are known [ ] . thereof alleles confer partial or complete loss of oct activity. the observed loss of activity was highly substrate specific and should be analyzed substrate by substrate. in this study we analyzed the effects of genetic polymorphisms in oct on the uptake of ranitidine. we used hek cells stably transfected to overexpress wild type or variant oct isoforms. the variant oct alleles oct * a (met val), oct * c (phe leu), oct * d (pro leu/met val), oct * (met del), oct * (arg cys), oct * (gly ser), oct * (gly arg/met del), oct * (cys arg/met del), oct * (ser phe), oct * (arg met), oct * (pro leu), oct * (ser leu), oct * (ile thr), oct * (ser leu) and oct * (thr met) were analyzed. we characterized in ranitidine uptake determined k m and v max for the different polymorphic oct isoforms. wild type oct showed a time and concentration dependent uptake of ranitidine with a k m of . ± . µm and a v max of . ± . pmol/min/mg protein. variants oct * , oct * , oct * and oct * completely lacked ranitidine transport activity. variants oct * , oct * , oct * and oct * showed v max values decreased by , , and %, respectively. oct * variant showed an increase of v max by %. there was no significant changes in ranitidine uptake by variants oct * a, oct * c, oct * d, oct * , oct * and oct * compared to the wild type. there were no significant differences in the k m values between the wild-type and variants. in conclusion, we confirmed ranitidine as an oct substrate and demonstrated that genetic polymorphisms in oct can strongly affect ranitidine uptake. the effects of oct polymorphisms on ranitidine pharmacokinetics in humans remain to be analyzed. otto-von-guericke university, institute for pharmacology and toxicology, magdeburg, germany serotonergic hallucinogens ( s hgs), such as lysergic acid diethylamide (lsd), induce profound alterations of human consciousness, which are thought to be mediated by activation of -ht a receptors. with repeated application, the mind-altering effects of most s hgs rapidly are undermined by tolerance. the only exception seems to be dimethyltryptamine (dmt), whose mind-altering effects for reasons unknown even with repeated application do not decrease. assuming that dmt differs from other s hgs in its capacity to regulate -ht a receptors, we here compare lsd and dmt with regard to processes of -ht a downregulation. in heat-exposed rats, lsd and dmt induce a marked increase in body core temperature (hyperthermia) accompanied by "defensive hypersalivation". both effects are mimicked by the -ht selective agonist dimethoxybromoamphetamine (dob) and can be blocked by the selective -ht a antagonists ketanserin and mdl . after repeated application, the temperatureregulatory effects of lsd are subject to tolerance, whereas the ones of dmt are not. tolerance to lsd (as measured by dob induced [ s]gtpуs binding) is paralleled by a desensitisation of frontocortical -ht receptors; for dmt, there is no such decrease. applying techniques of immunocytochemistry, transphosphatidylation, and quantitative real-time pcr to (ha- -ht a transfected) hek and (endogenously -ht a expressing) c glioma cells, respectively, we furthermore demonstrate that dmt in contrast to lsd fails to internalise -ht a receptors, fails to activate the phospholipase d (which is needed for -ht a internalisation), and fails to inhibit the synthesis of -ht a receptors. given that dmt unlike lsd turns out to be inactive as to all processes of -ht a downregulation investigated, our data suggest that the differential tolerance development noted for dmt and lsd indeed might be accounted for by differential regulation of -ht a receptors. lsd and dmt both have recently regained scientific attention as potential therapeutics in the treatment of depression and/or anxiety disorders. providing mechanistic insights into their action, thus, is of timely clinical relevance. increased gaba release in human neocortex at high intracellular sodium and low extracellular calcium -an anti-seizure mechanism? ] e , this reduction might induce an anti-seizure mechanism by augmenting gat-mediated gaba release, a mechanism absent in rats. aging is complex on the systems as well as on the molecular level. the process of aging is characterized by a progressive loss of physiological functions and accumulation of cellular damage. one hallmark of aging is an impaired protein homeostasis. the imbalance of the quality control of both de novo protein synthesis and protein degradation, therefore, is likely to contribute to the phenotype of aging. we investigated protein turnover rates with the state-of-the art techniques funcat (dieterich et al., ) and sunset (schmidt et al., ) in aging neuronal cell cultures . using these techniques we show a prominent decrease in protein synthesis and degradation that progressed gradually in aging neuronal cells cultured up to div . in order to rejuvenate the protein turnover in aged neuronal cells we applied the selective eukaryotic elongation factor- kinase inhibitor a and the polyamine spermidine and observed protein translation utilizing funcat and sunset. whereas both a and spermidine had no effect on de novo protein synthesis in juvenile neurons (div ) , both substances increased the de novo protein synthesis to a juvenile level in aged neuronal cultures (div ). this effect is seen in neuronal somata and dendritic spines. the molecular function of spermidine as an "anti-aging agent" is not defined yet. thus, additional pharmacological interventions are used for further examination of specific molecular spermidine targets. in conclusion, the described experimental setup is used to investigate impaired protein homeostasis as one hallmark of aging. agents with a presumed "anti-aging" effect can be tested for a potential rejuvenating effect on the level of protein homeostasis. a screening approach to test tolerability of multitargeted drug combinations for antiepileptogenesis in mice a large variety of brain insults can induce the development of symptomatic epilepsies, particularly temporal lobe epilepsy. in the latent period after the initial insult multiple molecular, structural, and functional changes proceed in the brain and finally lead to spontaneous recurrent seizures. prevention of these developments, called antiepileptogenesis, in patients at risk is a major unmet clinical need. several drugs underwent clinical trials for epilepsy prevention, but none of the drugs tested was effective. similarly, most previous preclinical attempts to develop antiepileptogenic strategies failed. in the majority of studies, drugs were given as monotherapy. however, epilepsy is a complex network phenomenon, so that it is unlikely that a single drug can halt epileptogenesis. recently, multitargeted approaches were proposed ("network pharmacology") to interfere with epileptogenesis. developing novel combinations of clinically used drugs with diverse mechanisms that are potentially relevant for antiepileptogenesis is a strategy, which would allow a relatively rapid translation into the clinic. we developed an algorithm for testing such drug combinations in a screening approach, modelled after the drug development phases in humans. tolerability of four repeatedly administered drug combinations was evaluated by a behavioral test battery: a, levetiracetam and phenobarbital; b, valproate, losartan, and memantine; c, levetiracetam and topiramate; and d, levetiracetam, parecoxib, and anakinra. as in clinical trials, tolerability was separately evaluated before starting efficacy experiments to identify any adverse effects of the combinations that may critically limit the successful use in preclinical studies on antiepileptogenesis and translation of these preclinical findings to the clinic. based on previous studies, we expected that tolerability would be lower in epileptic mice than in nonepileptic mice. therefore nonepileptic mice were used as a first step, followed by epileptic mice and mice during the latent period shortly after status epilepticus. except combination b, all drug cocktails were relatively well tolerated. in contrast to our expectations, except combination c, no significant differences were determined between nonepileptic and post-status epilepticus animals. as a next step, the rationally chosen drug combinations will be evaluated for antiepileptogenic activity in mouse and rat models of symptomatic epilepsy. major depression is one of the most common mental disorders worldwide, with serious social and economic consequences. there are many different hypotheses concerning the pathophysiology of this disease. the complex brain serotonin system and particularly the serotonin a receptors ( -ht a r) apparently play a pivotal role in the development of depression. the involvement of an altered, -ht a r-mediated signalling in adult neurogenesis is also discussed. however, in this context the effects of pre-and postsynaptically located -ht a rs have not been clarified yet. mice with a permanent overexpression of postsynaptic -ht a rs (oe mice) represent a unique tool to elucidate the effects of postsynaptic -ht a rs in adult neurogenesis and depressive-like behaviour. previous studies demonstrated an increased proliferation and survival of newborn cells in the adult dentate gyrus of female oe mice in comparison to controls. in the present study, we investigate the proliferation and survival of adult born cells after chronic treatment ( days) with the selective -ht a r agonist -oh-dpat ( , mg/kg/day) in young adult oe and wt mice. on the last three days of treatment, newly generated cells of oe and wt mice are labelled by injections with bromodeoxyuridine (brdu; mg/kg/day). mice are sacrificed either one day (proliferation) or days (survival) after the last injection. we hypothesise that the data we will present will confirm our previous results, with possibly more pronounced proneurogenic effects and differences in male mice. further immunohistochemical studies post-exercise and behavioural analyses are in progress to identify the relation between chronic postsynaptic -ht a r stimulation, depressive-like behaviour and hippocampusdependent learning. dystonia is a common movement disorder characterized by intermittent and prolonged muscle contractions resulting in involuntary movements and/or abnormal postures. the lack of knowledge of the pathophysiology of dystonia hampers the development of effective therapeutics. although benzodiazepines can improve dystonic symptoms, tolerance and side effects limit their use. there is evidence for striatal dysfunctions in human dystonia. gabaergic striatal interneurons (in) are important for the regulation of striatal signaling. in the dt sz mutant hamster, a model of paroxysmal dystonia, immunoreactive in were reduced at the age of maximum severity of dystonia ( days), but not after spontaneous remission (age days). as indicated by unaltered homeoboxprotein nkx . (cell density, mrna), the age-dependent deficit seems not to be related to a disturbed migration, but to a retarded maturation of in in mutant hamsters. here we further determined the maturation of striatal gabaergic neurons in the dt sz hamster compared to healthy controls. kcc and cavii mrna, used as markers for the gaba-switch, were unchanged in and day old mutant hamsters, indicating that there is no general delay in gabaergic maturation. as a retarded maturation seems to be specific for in, we used another marker for gabaergic maturation: the expression of specific gaba a receptor (gaba a r) subunits (mature striatal in express the alpha subunit). by stereological determination, we found a % decrease in alpha subunit expressing neurons. a lower immunoreactive intensity was restricted to the somata of dorsomedial striatal in ( %) of dt sz hamsters, indicating both a reduced density as well as a delayed maturation. these findings prompted us to examine the effects of the αlpha gaba a r preferring compound zolpidem in comparison with the benzodiazepine clonazepam. zolpidem ( . and . mg/kg i.p.) only exerted moderate antidystonic effects compared to the benzodiazepine clonazepam ( . and . mg/kg i.p.) in the dt sz hamster. examinations of αlpha gaba a r preferring compounds are ongoing. in summary our studies indicate that there is no general defect in striatal gabaergic maturation in the dt sz mutant but a specific alteration of striatal gabaergic interneurons which express αlpha gaba a r subunits. changes in αlpha gaba a r subunit expression and differences in the antidystonic efficacy of zolpidem and clonazepam indicate that further investigations on the role of gaba a r subunits could lead to new therapeutic approaches for the treatment of dystonia. ) . therefore, the hypothesis of the present study was that pregnenolone attenuates the inhibition of synaptic transmission elicited by cannabinoids. methods: µm-thick slices containing the cerebellum and the nucleus accumbens were prepared from the brains of mice and rats. spontaneous and electrically evoked gabaergic inhibitory postsynaptic currents (sipscs and eipscs) and evoked glutamatergic excitatory postsynaptic currents (eepscs) were analyzed in superfused brain slices with patch-clamp electrophysiological techniques. results: a) the synthetic cannabinoids jwh- ( x - m) and jwh- ( x - m) inhibited the spontaneous gabaergic synaptic input (sipscs) to purkinje cells in mouse cerebellar slices. the inhibition by jwh- was not affected by pregnenolone ( - m), the inhibition by jwh- was only marginally attenuated. b) the depolarization of the purkinje cells induced suppression of the gabaergic input to purkinje cells (dsi); pregnenolone ( - m) did not affect this endocannabinoid-mediated form of synaptic suppression. c) in rat nucleus accumbens slices, gabaergic and glutamatergic synaptic input to medium spiny neurons was activated by electrical stimulation of axons. ∆ -tetrahydrocannabinol ( x - m) suppressed the gabaergic and glutamatergic synaptic transmission in the nucleus accumbens. these suppressive effects of ∆ tetrahydrocannabinol were not changed by pregnenolone ( - m). d) finally, we tested whether we can observe neurosteroid-mediated effects in our brain slice preparations. tetrahydro-deoxycorticosterone (thdoc, - m) markedly prolonged the decay time constant (τ) of spontaneous gabaergic postsynaptic currents (sipscs), similarly as in previous experiments (ej cooper et al., j physiol : - , ) . the results show that inhibition of gabaergic and glutamatergic synaptic transmission by synthetic-, endogenous,-and phyto-cannabinoids is not changed by pregnenolone. therefore, it is unlikely that interference with cannabinoid-induced inhibition of synaptic transmission is the mechanism by which pregnenolone attenuates behavioural and somatic effects of ∆ -tetrahydrocannabinol in vivo. the hypothalamus is one of the key players in the regulation of the energy homeostasis. cold stress leads to an activation of neurons in the paraventricular hypothalamic nucleus (pvn) and increases thermogenesis. the thyrotropin-releasing-hormone (trh) neurons have an important function in this effect. however it is hardly understood which role the trh neurons exactly play and how they are connected to other regions of the brain. we have transduced neurons in the pvn of mice with a recombinant adeno associated virus which contains an activating "designer receptors exclusively activated by designer drugs" (dreadd) system under the control of a shortened trh promotor. two weeks after transduction the animals were injected with clozapine-n-oxide (cno). to analyse the physiological function of this neurons we performed indirect calorimetry, measured rectal temperature and thermogenesis in the brown adipose tissue (bat), analysed drinking feeding behaviour and the home cage activity. after stimulation we measured the expression of genes in bat as well plasma hormone levels of pituitary hormones. propranolol and the specific β -antagonist sr a were used to analyse the relevance of the sympathetic system. to further characterise the transduced neurons and their projections we used immunohistochemistry methods. after stimulation with cno the energy expenditure and body temperature were increased. these effects were mostly driven through an activation of the brown adipose tissue (bat). in dreadd transduced trh-receptor (trh-r ) knockout mice this effects were abolished. in parallel the plasma levels of tsh, the ucp mrna level in the bat, the home cage activity as well the food and water intake were increased. after the treatment with propranolol and sr a the effects on the thermogenesis were reduced, but the home cage activity was not affected. sr a treatment normalised the food intake and increased in parallel the plasma leptin concentrations after cno stimulation. transduced neurons project into the raphe nucleus, the medial part of the thalamus and the spinal cord. with our experiments we could provide strong evidence for a sympathetic connection of the transduced neurons in the pvn to the bat and for the involvement of thr neurons in these effects. therefore, this system is a suitable tool to investigate the metabolic relevance of trh neurons in detail. background & objective: during obesity development, tissue factor signalling contributes coagulation-independently to inflammatory and metabolic dysfunction of adipose tissue. adipogenesis involves proliferation and differentiation of preadipocytes, apoptosis and hypertrophic growth of differentiated adipocytes, angiogenesis and extracellular matrix reorganisation. the coagulant protease thrombin promotes similar processes in various cell types, through activation of protease-activated receptors par- , par- and par- . in human adipose tissue, par- is found in vascular stromal cells and par- in preadipocytes and differentiated adipocytes. thrombin stimulates mitogenic kinase signalling and induces inflammatory cytokine and angiogenic growth factor secretion in adipocytes. we have examined the contribution of thrombin receptor activation to adipogenesis processes in t l cells. results: differentiation of t l preadipocytes with insulin, dexamethasone and isobutylmethylxanthine increases leptin and pparg gene expression and accumulation of triglycerides and oil red o-stained lipids. par- is time-dependently upregulated in maturing cells while par- expression is detectable but not altered. in preadipocytes, thrombin ( u/ml) activates the mitogenic kinase erk / , promotes cell proliferation and induces gene expression of the maturation markers leptin and pparg and the inflammatory marker tumor necrosis factor alpha (tnfa). repeated stimulation of differentiating adipocytes with thrombin suppresses induction of leptin and pparg and attenuates lipid accumulation, while expression levels of the proliferation marker ki and the inflammatory cytokine interleukin (il)- are increased compared to differentiated control cells. similar proliferative and anti-adipogenic effects are seen with the selective par -activating peptide (gypgkf, µm) and cathepsin g, a proteolytic par- activator released from neutrophils and mast cells. repeated exposure of maturing t l cells to conditioned medium from degranulating mouse peritoneal mast cells (mccm) augments lipid accumulation and il- expression. pretreatment of mccm with a par- inhibitor further drives lipid accumulation, the induction of il- by contrast is suppressed. conclusion: par- activation by thrombin or inflammatory cell-derived cathepsin g appears to suppress adipogenesis, possibly by maintaining proliferative capacity and preventing the growth arrest essential for initiating matuation. increased par- expression in maturing adipocytes may instead support inflammatory changes, thereby promoting the onset of insulin resistance. the chemokine receptor cxcr antagonist amd exerts deleterious effects in endotoxemia in vivo s. seemann , a. lupp the chemokine receptor cxcr is a multifunctional receptor which is activated by its natural ligand c-x-c motif chemokine (cxcl ). although a blockade of the cxcr /cxcl axis revealed beneficial outcomes in chronic inflammatory diseases, its importance in acute inflammatory diseases remains contradictious and not well characterized. as cxcr seems to be part of the lipopolysaccharide sensing complex, cxcr agonists or antagonists may have a positive impact on tlr signaling. additionally, cxcr is involved in the production of pro-inflammatory cytokines, suggesting the receptor to be a promising target in terms of mitigating the cytokine storm. therefore, we aimed to investigate the impact of a cxcr blockade on endotoxemia by applying a sublethal lps dose ( mg/body weight) in mice. the selective cxcr inhibitor amd was administered intraperitoneally shortly after lps treatment to ensure an immediate effect after endotoxemia onset. hours after lps administration, the clinical severity score, the body temperature and the body weight of the animals were determined. afterwards, the mice were sacrificed and serum tnf alpha as well as ifn gamma levels were measured. furthermore, the oxidative stress in the brain, liver, lung and kidney tissue was assessed. in addition, the biotransformation capacity of the liver was evaluated and finally, the expression of gp phox as well as of heme oxygenase in the spleen and liver were determined by means of immunohistochemistry. the mice of the amd plus lps treatment group displayed a significantly impaired general condition, a reduced body temperature and a decreased body weight in comparison to the control and to the lps treated animals, respectively. tnf alpha levels were significantly increased by more than % or % when compared to the control or to the lps group, respectively, whereas ifn gamma levels were elevated by about % in comparison to mice which had received lps only. in all investigated organs, but especially in the liver and in the kidney, co-administration of amd and lps caused massive oxidative stress. furthermore, the protein contents and the activities of several cyp enzymes in the liver were significantly reduced. immunohistochemistry revealed gp phox to occur above average, whereas heme oxygenase expression was distinctly decreased. our results indicate that a blockade of the cxcr in endotoxemia is disadvantageous and even worsens the disease. co-administration of amd and lps impaired the health status of the animals, caused massive oxidative stress and diminished the biotransformation capacity. thus, handling acute systemic inflammation with a cxcr antagonist cannot be recommended, hence indicating the activation of cxcr to be an attractive treatment option. toll-like receptors (tlrs)recognizemicrobial pathogens and trigger inflammatory immune responses to control infections. in acne vulgaris, activation of tlr by propionibacterium acnes contributes to inflammation. although glucocorticoids have immunosuppressive and anti-inflammatory effects, acne can be provoked by systemic or topical treatment. enhanced tlr expression by glucocorticoids has been reported in undifferentiated keratinocytes, however, human skin cells of different epidermal and dermal layers have not been investigated. in this study, the modulation of tlr expression by dexamethasone was assessed in monolayer cultures of primary human keratinocytes and dermal fibroblasts, as well as the immortalized keratinocyte cell line hacat. constitutive tlr , tlr and tlr mrna and protein expression was confirmed in basal keratinocytes, calcium-induced differentiated keratinocytes, hacat cells and fibroblasts by qpcr and western blotting. dexamethasone induced tlr expression in a time-dependent and concentration-dependent manner and reduced tlr / expression in keratinocytes but not in hacat cells or fibroblasts. stimulation with dexamethasone in the presence of the pro-inflammatory cytokines tnfα or il- β further increased tlr mrna levels. gene expression of mapk phosphatase- (mkp- ) was also upregulated by dexamethasone. glucocorticoid-induced tlr expression was negatively regulated by p mapk signalling under inflammatory conditions through mkp- induction which functions to deactivate mapks. as expected, dexamethasone inhibited the immune responses linked to tlr signalling as demonstrated by reduced il- , il- β, mmp- and mcp- levels. however, the expression of traf , a critical cytosolic regulator of tlr-and tnf family-mediated signalling, was further upregulated by the tlr agonist hklm (heat-killed lysteria monocytogenes) in dexamethasonepretreated basal keratinocytes.conclusively, our results provide novel insights intothe molecular mechanismsof glucocorticoid-mediatedtlr expressionand function in human skin cells. psoriasis is a cutaneous chronic inflammatory disease characterized by increased amounts of il- cytokines and t helper (th ) related cytokines in lesional psoriatic skin. treatment with beta-adrenoceptor antagonists is associated with induction or aggravation of psoriasis, however, the underlying mechanism is poorly understood. previously, we could demonstrate a pivotal role for langerhans cells and dermal dendritic cells in antimalarial-provoked psoriasis by maintaining a potent th activity. in the present study, we investigated the effect of propranolol on human monocyte-derived langerhans-like cells (molc) and dendritic cells (modc) under inflammatory conditions. in the presence of il- β, propranolol induced the th priming cytokines il- and il- in a concentration-dependent manner. the increased cytokine release was not mediated by camp suggesting gpcr-independent pathways. in contrast, il- γ and lps failed to increase il- release in molc and modc in the presence of propranolol but further induced secretion of il- β. autophagy has been linked with the secretion of il- family cytokines that are upregulated in chronic inflammatory disorders such as psoriasis. propranolol upregulated the expression levels of the autophagy marker p and lc -i to lc -ii conversion, induced accumulation of lc positive vesicles, as well as expression of il- signalling downstream adapter molecule traf , indicating a late-stage block in autophagy. in summary, our results suggest a prominent role of cutaneous dendritic cell subtypes in psoriasis-like skin inflammation mediated by propranolol and possibly other beta blockers. langerhans cells (lcs) represent a highly specialized subset of epidermal dendritic cells (dcs), yet not fully understood in their function of balancing skin immunity. in the present study, we investigated in vitro generated langerhans-like cells obtained from the human acute myeloid leukaemia cell line mutz- (mutz-lcs) to study tlr-and cytokine-dependent activation of epidermal dcs. mutz-lcs revealed high tlr expression and responded robustly to tlr engagement, confirmed by increased cd and cd expression, upregulated il- , il- p and il- p mrna levels and il- release. tlr activation reduced ccr and elevated ccr mrna expression and induced migration of mutz-lcs towards ccl . similar results were obtained by stimulation with pro-inflammatory cytokines tnf-α and il β whereas ligands of tlr and tlr failed to induce a fully mature phenotype. despite limited cytokine gene expression and production for tlr -activated mutz-lcs, co culture with naive cd + t cells led to significantly increased ifn-γ and il- levels indicating th differentiation independent of il- . tlr -mediated effects were blocked by the putative tlr / antagonist cu-cpt , however, no selectivity for either tlr / or tlr / was observed. computer-aided docking studies confirmed non-selective binding of the tlr antagonist. taken together, our results indicate a critical role for tlr signalling in mutz-lcs considering the leukemic origin of the generated langerhans-like cells. the stagnation in the development of new antibiotics during the last decades and the concomitant high increase of resistant bacteria emphasize the urgent need for new therapeutic options. antimicrobial peptides are promising agents for the treatment of bacterial infections and recent studies indicate that pep - . , a synthetic antimicrobial and lps-neutralizing peptide (salp), efficiently neutralizes pathogenicity factors of gram-negative and gram-positive bacteria and protects against sepsis. in the present study, we investigated the potential of pep - . and the structurally related compound pep - lf for their therapeutic application in bacterial skin infections. primary human keratinocytes responded to tlr (fsl- ) but not tlr (lps) activation by increased il- production, as determined by elisa. western blot analysis showed that both salps inhibited fsl- -induced phosphorylation of nf-κb p and p mapk. furthermore, the peptides significantly reduced il- release and gene expression of il- β, ccl (mcp- ) and hbd- as assessed by qpcr. no cytotoxicity (mtt test) was observed at salp concentrations below µg/ml. in lps-stimulated monocyte-derived dendritic cells, the peptides blocked il- secretion, downregulated expression of the maturation markers cd and cd , as analysed by flow cytometry, and inhibited ccr -dependent migration capacity. similarly, monocyte-derived langerhans-like cells activated with lps and pro-inflammatory cytokines showed reduced il- levels and cd /cd expression in the presence of salps. in addition to acute inflammation, bacterial infections often result in impaired wound healing. since re-epithelialization is a critical step in wound repair, we tested whether pep - . affects keratinocyte migration using the scratch wound assay. the peptide markedly promoted cell migration and accelerated artificial wound closure at concentrations as low as ng/ml and was equipotent to tgf-β. conclusively, our data suggest a novel therapeutic target for the treatment of patients with acute and chronic skin infections. recently, we and others have shown that the transcription factor nuclear factor erythroid -related factor (nrf ), a major regulator of the cellular antioxidant defence system, is activated by mechanical ventilation. during ventilator-induced lung injury, nrf exerts a protective role by interaction with the stretch-induced growth factor amphiregulin. in the current study, we aimed to investigate the role of nrf in acid-induced lung injury, a model for aspiration-induced ards. methods: nrf -deficient (nrf -/-) mice and wild type (wt) littermates were tracheotomised and ventilated for min (v t = ml/kg, f= min - , peep= cmh o, fio = . ), before µl hydrochloric acid (hcl) with ph= . or ph= . were instilled intratracheally, controls received nacl. mice were then ventilated for further h under monitoring of lung mechanics and vital parameters. blood gases as well as proinflammatory mediators, neutrophil recruitment and microvascular permeability were examined to assess lung injury. results: instillation of hcl ph= . induced mild lung injury, indicated by hypoxemia (po /fio ~ mmhg) and continuously increasing lung tissue elastance (stiffness), from which nrf -/mice were protected. pulmonary inflammation, characterized by liberation of cytokines, chemokines and oedema formation, was attenuated in nrf -/mice. in contrast, hcl ph= . caused more severe lung injury (po /fio ~ mmhg) with a steeper incline in elastance and more severe inflammation in both wt and nrf -/mice. conclusion: we conclude that the presence of nrf augments mild acid-induced lung injury, but plays no role in more severe injury. these discrepant results will be elucidated in future investigations. uniklinik rwth aachen, institut für pharmakologie und toxikologie, aachen, germany fakultät für maschinenwesen der rwth aachen, werkzeugmaschinenlabor, aachen, germany rationale: reproducibility is key to science. in recent times, the reproducibility of biomedical research has been questioned increasingly. this reproducibility crisis also affects complex animal experiments, which -if not reproducible -might also be regarded as unethical and lose public acceptance. part of the problem is frequently that the provided documentation is not sufficient for reproduction. therefore, in this study we analyzed the potential of conventional quality management tools -used as standard in machine production -as an approach to improve the documentation and ascertain the quality of complex animal experiments. methods: quality management tools were transferred to an experimental animal set up -the mouse intensive care unit (micu) -which we use for lung injury studies. the tools included visualization of the experimental set-up, transfer of the experimental procedures to an event-driven process chain (epc) and statistical process control (spc) of all crucial pulmonary and cardiovascular parameters. data from ventilator-and acidinduced lung injury studies acquired in the micu were analyzed retrospectively. results: schematic visualization of the micu resulted in a chart comprising medical components, hardware, software and generated data types. the customized epc included all important activities and the resulting events for preparation of the mouse and the workplace, the actual animal ventilation experiment and sample-taking. in addition, checklists were provided for these activities and events, to ensure standardization of every work step. lung impedance and cardiac functions from ventilator-and acid-induced lung injury models were analyzed by spc and correlated with events in the epc. the spc proved to be suitable to identify outliers, predict processes and thereby validate the lung injury models. conclusions: conventional quality management tools were successfully adapted to analyze the quality of lung injury experiments in the micu. we suggest that this new approach is suitable to standardize animal testing procedures and increase the reproducibility of animal studies. background: a dysfunctional endothelial l-arginine-nitric oxide (no) pathway is a key pathomechanism of idiopathic pulmonary arterial hypertension (ipah) that can be provoked by hypoxia in cell culture models [ ] [ ] [ ] [ ] . the small peptide apelin is involved in the maintenance of pulmonary vascular homeostasis and angiogenesis although its precise mechanism of action is still unclear [ ] . asymmetric dimethylarginine (adma) is known to be an endogenous inhibitor of endothelial no synthase and is associated with several cardiovascular diseases [ ] . adma is degraded by dimethylarginine dimethylaminohydrolase and (ddah) enzymes [ ] . objective: to determine the effect of apelin on the l-arginine/no pathway in human pulmonary microvascular endothelial cells (hpmecs). methods: hpmecs were cultured under normoxic and ph-related hypoxic conditions and treated with apelin. the expression of regulators of the l-arginine/no pathway were analysed using real-time pcr. the effect of apelin on the phosphoinositide- kinase (pi k)/akt signalling pathway was determined using immunoassays and specific inhibitors[lh ] . apelin and adma concentrations were measured in cell culture supernatants using an enzyme-linked immunosorbent assay and a liquid chromatography-tandem mass spectrometry assay. results: treatment with apelin resulted in a reduced expression of the apelin receptor (aplnr) on hpmecs suggesting a negative feedback mechanism. apelin directly influenced the l-arginine/no pathway by increasing the expression of ddah and ddah enzymes. thus, the concentration of adma was decreased in hpmecs supernatant following treatment with apelin. the effect of apelin could be abrogated by modulation of the pi k/akt pathway. conclusion: apelin modulates the l-arginine/no pathway and mediates enhanced degradation of adma via an upregulated expression of ddah and enzymes. the pi k/akt pathway might play a decisive role in regulation of the effect of aplein. an apelin receptor agonist could be a novel and promising therapeutic option for ipah treatment. background and purpose: there is presently no proven pharmacological therapy for the acute respiratory distress syndrome (ards). recently, we and others discovered that the heptapeptide angiotensin (ang)-( - ) shows significant beneficial effects in preclinical models of acute lung injury (ali). here, we aimed to identify the best time window for ang-( - ) administration to protect rats from oleic acid (oa) induced ali. experimental approach: the effects of intravenously infused ang-( - ) were examined over four different time windows before or after induction of ali in male sprague-dawley rats. hemodynamic effects were continuously monitored, and loss of barrier function, inflammation, and lung peptidase activities were measured as experimental endpoints. key results: ang-( - ) infusion provided best protection from experimental ali when administered by continuous infusion starting min after oa infusion till the end of the experiment ( - min). both pretreatment (- - min before oa) and short-term therapy ( - min after oa) also had beneficial effects although less pronounced than the effects achieved with the optimal therapy window. starting infusion of ang-( - ) min after oa (late-term infusion) achieved no protective effects on barrier function or hemodynamic alterations, but still reduced myeloperoxidase and angiotensin converting enzyme activity, respectively. conclusions and implications. our findings indicate that early initiation of therapy after ali and continuous drug delivery are most beneficial for optimal therapeutic efficiency of ang-( - ) treatment in experimental ali, and presumably accordingly, in clinical ards. airway epithelium functions as a physicochemical barrier against dust, air pollutants and other pathogens and plays a critical role in physiological and pathological processes including modulation of the inflammatory response, innate immunity and airway remodeling such as in human asthma, copd and equine recurrent airway obstruction (rao). models of the airway epithelia are, indeed, missing for the horse; thus, we established long-term equine bronchial epithelial cell cultures using the rock inhibitor y- and cell growth and differentiation was characterized. bronchial epithelial cells (ebec) from adult horses were cultured in the presence and absence of µm y- under conventional and air-liquid-interface (ali) culture conditions. cell proliferation and differentiation were analyzed. formation of a functional epithelial barrier was investigated by transepithelial electric resistance (teer) measurement and immunocytochemical staining of the tight-junction-protein zonula occludens- (zo- ). under conventional culture, y- induced higher growth rate of primary ebec and increased the passage number up to passages with retained epithelial cell behavior. in the presence of y- , ebecs under ali showed higher teer values. expression of zo- correlated with the increase in teer, but in y- -treated ebec tight-junctionformation was more rapid, indicating accelerated differentiation, as well h/e-staining and scanning electron microscopic imaging showed a higher amount of cilia and microvilli and pas-positive cells. in conclusion, the data suggest that the rock inhibitor y- facilitates long-term culture of equine bronchial epithelial cells which can be used to study airway disease mechanisms and to identify pharmacological targets. leishmaniasis is a neglected disease of tropical and subtropical regions with millions of people at risk of infection with severe consequences including death. current antileishmanial drugs exhibit serious side effects and also development of resistances is rising. this disease is caused by protozoal organisms from the genus leishmania. in their insect vector they exist in the promastigote form, while in the mammalian host they survive as amastigotes inside the phagolysosomes of macrophages. this makes a specific pharmacotherapy complicated. due to the success of artemisinin in malaria therapy, it was of interest whether endoperoxides are also useful to treat leishmaniasis. in a previous study we demonstrated that ascaridole, an endoperoxide from chenopodium ambrosioides, can cure cutaneous leishmaniasis in a mouse model and exhibited ic values for the viability in the low micromolar range [ ] . even though in chemical model systems some basic ideas about the mechanism of activation of these endoperoxides exist, in biological systems including leishmania parasites this activation step has never been demonstrated. therefore, we set up experiments to identify primary drug intermediates formed from ascaridole by activation in leishmania tarentolae promastigotes using electron spin resonance spectroscopy in combination with spin trapping methods. ascaridole was activated in a cell-free system by fe + . the radicals were trapped by -methyl- -nitrosopropane (mnp). the resulting esr spectra consisted of the triplet of duplets. spectral simulations revealed coupling parameters of a n = . g, and a h = . g. these coupling constants are compatible with iso-propyl radicals as primary intermediates. in the cellular system, consisting of leishmania tarentolae promastigotes, instead of mnp the less cytotoxic , -dimethyl- pyrroline-n-oxide (dmpo) was used for spin trapping. without addition of fe + a six line esr signal was observed. spectral simulations of the dmpo spin adduct revealed coupling constants of a n = . and a h = . g. according to previously published data [ ] from other spin trapping experiments, this corresponds to the formation of carboncentered radicals from ascaridole by leishmania parasites. additional experiments using iron chelators and antioxidants as well as a comparison with the endoperoxide artemisinin were performed. in summary, this study for the first time demonstrated the activation of the endoperoxide ascaridole by a protozoal organism to its active intermediate as a prerequisite to understand its mechanism of action. [ ] l. monzote, j. pastor, r. scull, and l. gille. antileishmanial activity of essential oil from chenopodium ambrosioides and its main components against experimental cutaneous leishmaniasis in balb/c mice. phytomedicine : - , . nitric oxide (no), produced by the inducible nitric oxide synthase (inos) has many functions in physiological and pathophysiological pathways. after induction of inos expression by cytokines and other agents the enzyme produces high amounts of no in a ca + -independent way. this high no production can have beneficial microbicidal, antiparasital, antiviral and antitumoral effects. in contrast, aberrant inos induction may have detrimental consequences and seems to be part of many diseases such asasthma, arthritis, multiple sclerosis, colitis, psoriasis, neurodegenerative diseases, tumor development, transplant rejection or septic shock. analysis of the human inos-mrna structure revealed the existence of an upstream open reading frame (µorf) and a putative internal ribosome entry site (ires) in the ' untranslated region ( 'utr) in front of the start codon of the inos coding sequence (cds). to analyze the function of the µorf and the putative ires we cloned different egfp and luciferase reporter constructs and transfected them into the human colon carcinoma cell line dld . using a plasmid construct with the µorf fused with the egfp cds, we could show that the µorf can be translated. however, compared to the positive control plasmid less egfp was produced, which can be explained by a weak kozak sequence of the µorf. blocking the mrna cap-dependent translation by cloning a stem loop structure in front of the inos 'utr within a luciferase reporter plasmid led to a remarkable loss of luciferase production. thus, the expression of inos seems to be cap-dependent. furthermore, transfection experiments with dld cells using constructs coding for a bicistronic renilla-firefly luciferase mrna showed that there is no ires in front of the inos cds. taken together, the inos expression seems to be cap-dependent and without influence of an ires, while the µorf is translatable. therefore we speculate that inos expression is only possible due to a leaky scanning mechanism depending on the weak kozak sequence of the µorf. objectives: vascular oxidative stress is considered a pathophysiologic factor promoting cardiovascular diseases such as coronary artery disease, heart failure, diabetes and hypertension. there are several sources of superoxide in vascular smooth muscle and endothelial cells but whether an impairment of the catalytic function of enos and thus generation of oxidative stress is involved in blood pressure (bp) regulation and/or the development of hypertensive disease states is unknown. methods: we generated a mutant enos in which one of the two essential cysteines required for the coordination with the central zn-ion, correct dimer formation and normal activity is replaced by alanine (c a-enos). normal enos (enos-tg) or a novel dimer-destabilized c a-enos described previously (antioxid redox signal. sep ; ( ) : - ) were introduced in c bl/ in an endothelial-specific manner. mice were monitored for enos expression and localization, aortic relaxation, systolic blood pressure, levels of superoxide and several post-translational modifications indicating activity and/or increased vascular oxidative stress. some groups of mice underwent voluntary exercise training for weeks or treatment with sod mimetic tempol. results: c a-enos-tg showed significantly increased superoxide generation, protein-and enos-tyrosine-nitration, enos-s-glutathionylation, enos / phosphorylation and amp kinase (ampkα) phosphorylation at thr in aorta, skeletal muscle, left ventricular myocardium and lung as compared to enos-tg and wild type (wt) controls. the localization of enos-c a-tg was restricted to endothelium as evidenced by immunohistochemically staining for enos and an endothelial-specific marker cd . exercise training increased phosphorylation of enos at ser / and of ampkα at thr in wt but not in c a-enos-tg. aortic endothelium-dependent and endothelium-independent relaxations were similar in all strains. in striking contrast, c a-enos-tg displayed normal blood pressure despite higher levels of enos, while enos-tg showed significant hypotension. tempol completely reversed the occurring protein modifications and significantly reduced bp in c a-enos-tg but not in wt controls. conclusions: by means of a novel transgenic mouse model we demonstrated that vascular oxidative stress generated by endothelial-specific expression of a dimerdestabilized variant of enos selectively prevents bp reducing activity of vascular enos, while having no effect on aortic endothelial-dependent relaxation. these data suggest that oxidative stress in microvascular endothelium may play a role in the development of essential hypertension. the herbal medicinal product myrrhinil-intest ® consists of myrrh, chamomile flower dry extract and coffee charcoal. clinical data prove the effectiveness of this herbal preparation for inflammatory intestinal disorders. to further investigate the anti-inflammatory potential of the single components as part of a multi-target principle, an ethanolic (my) and aqueous (mya) myrrh extract, ethanolic chamomile flower extract (ka) and aqueous coffee charcoal extract (cc), were examined in an in vitro tnbs inflammation model using rat small intestinal preparations. the effect of the plant extracts on tnbs induced inflammatory damage was characterised based on tnfα-gene expression analysis, isometric contraction measurement and histological analysis. furthermore, tnfα-release from lpsstimulated thp- cells was determined. budesonide was used as positive control. additionally, microarray gene expression analysis was performed in lps/ifnγ stimulated native human macrophages to determine potential underlying mechanisms. the tnbs-induced overexpression of tnfα-mrna was reduced after ka ( . mg/ml) and mya ( mg/ml) treatment down to % and % resp.; tnbs-induced loss of contractility and reduction of mucosal layer thickness was inhibited after ka ( mg/ml) treatment by % and % resp.; after mya ( . - mg/ml) treatment by % and % resp. lps-induced tnfα release from thp- cells was inhibited concentrationdependently by my (ic = . μg/ml; % inhibition), ka (ic = μg/ml; % inhibition) and cc (ic = μg/ml; % inhibition). furthermore, ka ( µg/ml) and cc ( µg/ml) inhibited the lps/ifnγ-induced expression of genes associated with chemokine signalling up to -fold (for cxcl ). the presented study demonstrates further evidence for anti-inflammatory properties of the herbal components which contribute to the reported clinical effectiveness. introduction: the purine nucleoside adenosine, which is involved in a variety of physiological functions, regulates immune and inflammatory responses and acts as a modulator of gut functions. although it is present at low concentrations in the extracellular space, stressful conditions, such as inflammation, can markedly increase its extracellular level up to micromolar range. by activation of different receptor subtypes adenosine is able to induce anti-inflammatory or pro-inflammatory impacts. aim: the current study examined the impact of adenosine a a receptors (a ar) and adenosine a b receptors (a br) to regulate contractility in untreated and inflamed rat colon preparations using a specific a ar agonist (cgs ) and an a br antagonist (psb- ) on acute inflammation in rat colon preparations. further it focused on interactions of the multi-herbal drug stw with a ar as a possible mechanism of the protective effect of stw in gastrointestinal disorders. methods: inflammation was induced by intraluminal instillation of , , -trinitrobenzene sulfonic acid (tnbs). contractions were measured isometrically in an organ bath set up. gene expression was determined using rt-pcr. radio ligand binding assays (competition experiments) were carried out with rat brain homogenates. morphological changes were estimated after van gieson staining. results: all four adenosine receptor subtypes were expressed in untreated colon preparations. activation of a , a b, and a receptor with specific agonists reduced the acetylcholine (ach, µm)-induced contractions, while activation of a br enhanced it. after incubation with tnbs morphological damages in colonic mucosa and muscle walls were detectable followed by reduced ach-contractions. the tnbs-mediated decrease of ach-contractions as well as the morphological damages were partially normalized by co-incubation of tnbs with cgs ( µm) or with psb ( µm). the same effects with smaller intensity were found for stw ( µg/ml) in female but not in male colon preparations. these results are in accordance with ligand binding studies indicating that stw interact with the a ar. conclusion: anti-inflammatory mechanisms and cell protective actions of stw are partly due to the interaction with adenosine receptors. the results give a clear-cut correlation with symptom improvements in clinical trials and thereby highlight the relevance of stw as a therapeutic approach in ibs. (allescher ) . therefore, a multi-target approach is a promising therapeutic strategy, as is exemplified by stw (ottillinger et al. ) . stw (iberogast®) is a fixed combination of nine plant extracts with iberis amara (stw ) as one of its components. it is successfully used for treatment of functional dyspepsia and irritable bowel syndrome (ibs). to allow an overview of targets addressed by stw and the role of its components in relation to the different forms and causes of functional gi diseases, an evaluation of the data, which have been gained from more than pharmacological tests, is needed. all data from studies including stw alone, or stw and its components, were retrieved and sorted according to types of study models (human and animal systems, animal disease models, gi-preparations, cell cultures, in vitro-systems) and respective etiologic mechanisms related to fgds and then visualized in the form of d histograms (lorkowski et al. ) . results: more than pharmacological tests indicated anti-oxidative activity, electrophysiological effects, ulcer protection, anti-inflammatory actions, pro-kinetic and spasmolytic effects as well as reflux and acid reduction. moreover, the analysis indicated that the components of stw contribute differently to the overall effect of stw . altogether, the evaluation of the data shows that stw is active in response to multiple etiologic factors involved in fgds, especially functional dyspepsia and irritable bowel syndrome, and to which extent the herbal extract components of the combination are relevant for the different mechanisms of action and their translation to clinical efficacy. conclusion: multi step clustering allows the transformation of complex data sets. it makes the allocation of specific actions to the different components of stw manageable, so also giving support to its clinical use in patients with different symptoms. introduction: stw ii has been recently developed in an effort to reduce the number of active extracts in the mother multi-component herbal preparation, stw (iberogast ® , steigerwald arzneimittelwerk gmbh, darmstadt, germany) without affecting the overall therapeutic efficiency. stw consists of a mixture of standardized extracts: bitter candytuft (iberis amara), lemon balm (melissa officinalis), chamomile (matricaria recutita), caraway fruit (carum carvi), peppermint leaf (mentha piperita), liquorice root (glycyrrhiza glabra), angelica root (angelica archangelica), milk thistle (silybum marianum) and celandine herb (chelidonium majus), whereas stw ii lacks the last components. stw was shown to be effective clinically to treat functional dyspepsia ( ) and irritable bowel syndrome ( ) and was shown experimentally to be effective to guard against the development of radiation induced intestinal mucositis ( ) and in the management of ulcerative colitis ( ) . the present study was initiated to show whether stw ii with the reduced component extracts would also be as effective in the latter condition. this was induced in wistar rats by feeding them with % dextran sodium sulfate in drinking water for week when lesions were observed in the colon evidenced by histological examination as well as colon shortening and reduction of colon mass index. this was associated with a rise in myeloperoxidase and a fall in reduced glutathione, glutathione peroxidase, and superoxide dismutase in colon homogenates as well as a rise in tnfα in serum. oral administration of stw in doses of and ml/kg or stw ii in a dose of ml/kg for week before and continued during dss feeding tended to normalize all the changes in a fashion comparable to sulfasalazine, used as a reference drug in a dose of mg/kg. conclusions: the modified preparation, stw ii thus proved to be as effective as stw , thereby reflecting its potential usefulness in ulcerative colitis possibly by virtue of its anti-inflammatory and anti-oxidant properties. ( ) schmulson mj ( ) the emetic pathways include the action of neurotransmitters dopamine, serotonin and substance p in the emetic centers localized in the brainstem, area postrema and vagal nerve afferents. previous in vivo studies in beagle dogs revealed that the plant alkaloid lycorine potentially induce nausea and emesis. though antagonists of the tachykinin receptor (maropitant) and serotonin receptor (ondansetron) prevented lycorinemediated emesis, the molecular mechanism of nausea and vomiting remain still unknown. to study the mechanism of action of the emetic agents, we analyzed the effect of lycorine (direct activation of nk ) and channel opening (activation of ht ) on the intracellular calcium homeostasis (using fluorometric ca + analysis) and cell proliferation rates in endogenously nk and ht receptor expressing cell lines as well as in cho and hek cells stably expressing the receptors. neither endogenously receptor expressing nk or ht cells nor receptor overexpressing cells showed calciumflux or calcium mobilization after stimulation with lycorine. furthermore, we are measuring the receptor number and subtypes using radioligand binding studies. it is planned, moreover, to obtain fluorescent labeled constructs of the nk receptor to gain insights into the involvement of receptor internalization which might mediate emesis. by characterizing these molecular principles of the nk and ht receptors, we are attempting to obtain more information in predicting drug-induced side effects such as nausea and emesis. the intestinal epithelium is completely renewed every - days. this process is driven by stem cells, which reside within specialized niches in the intestinal crypts and give rise to several differentiated cell types, including enterocytes, paneth, enteroendocrine, goblet and tuft cells. however, the molecular mechanisms that establish and maintain differentiated cell numbers and proportions remain largely unknown. here, we systematically analyzed the intestinal expression of semaphorins and plexins, which constitute a ligand-receptor system that plays central roles in cell-cell communication in various biological contexts. we identified plexin-b and its semaphorin ligands to be highly expressed in intestinal epithelial cells. genetic inactivation of plexin-b in intestinal organoids strongly reduced the number of enteroendocrine cells. our data suggest that semaphorin-plexin-b signaling promotes differentiation of intestinal epithelial cells towards the enteroendocrine lineage. the gastric epithelium contains several types of differentiated cells, including foveolar cells that produce mucus, parietal cells that secrete gastric acid and intrinsic factor, chief cells that synthesize pepsinogen and gastric lipase, and enteroendocrine cells that release different hormones. these differentiated cell types all originate from multipotent stem cells, yet little is known about how this differentiation process is regulated on a molecular level. the gap protein rasal controls the activity of small gtpases of the ras family, and its expression levels have been shown to inversely correlate with progression of stomach cancers. however, functional studies on the physiological role of rasal in the gastric epithelium are lacking. here, we established and characterized a mouse line with inactivation of the rasal gene. we observed that these mice showed increased numbers of enteroendocrine cells in the gastric mucosa. conditional inactivation of rasal in enteroendocrine cells, using a mouse line in which cre expression is driven by the atoh promoter, further corroborated that rasal expression in enteroendocrine cells determines enteroendocrine cell numbers. these findings identify rasal as a regulator of gastric epithelial cell differentiation. ). the present study investigates the impact of two faah inhibitors (arachidonoyl serotonin [aa- ht], urb ) on a lung cancer cell metastasis and invasion. lc-ms analyses revealed increased levels of faah substrates (aea, -ag, oea, pea) in cells incubated with either faah inhibitor. in athymic nude mice faah inhibitors were shown to elicit a dosedependent antimetastatic action. in vitro, a concentration-dependent anti-invasive action of either faah inhibitor was demonstrated, accompanied with upregulation of tissue inhibitor of matrix metalloproteinases- (timp- ). using sirna approaches, a causal link between the timp- -upregulating and anti-invasive action of faah inhibitors was confirmed. moreover, knockdown of faah by sirna was shown to confer decreased cancer cell invasiveness and increased timp- expression. inhibitor experiments point toward a decisive role of cb and transient receptor potential vanilloid in conferring the anti-invasive effects of faah inhibitors and faah sirna. finally, antimetastatic and anti-invasive effects were confirmed for all faah substrates. collectively, the present study provides first-time proof for a pronounced antimetastatic action of the faah inhibitors aa- ht and urb . as underlying mechanism of its anti-invasive properties an upregulation of timp- was identified. regenerative activity in tissues of mesenchymal origin depends on the migratory potential of mesenchymal stem cells (mscs). the present study focused on inhibitors of the enzyme fatty acid amide hydrolase (faah), which catalyzes the degradation of endocannabinoids (anandamide, -arachidonoylglycerol) and endocannabinoid-like substances (n-oleoylethanolamine, n-palmitoylethanolamine). in boyden chamber assays, the faah inhibitors, urb and arachidonoyl serotonin (aa- ht), were found to increase the migration of human adipose-derived mscs. lc-ms analyses revealed increased levels of all four aforementioned faah substrates in mscs incubated with either faah inhibitor. following addition to mscs, all faah substrates mimicked the promigratory action of faah inhibitors. promigratory effects of faah inhibitors and substrates were causally linked to activation of p / mitogen-activated protein kinase (mapk), as well as to cytosol-to-nucleus translocation of the transcription factor, peroxisome proliferator-activated receptor α (pparα). whereas pparα activation by faah inhibitors and substrates became reversed upon inhibition of p / mapk activation, a blockade of pparα left p / mapk phosphorylation unaltered. collectively, these data demonstrate faah inhibitors and substrates to cause p / mapk phosphorylation, which subsequently activates pparα to confer increased migration of mscs. this novel pathway may be involved in regenerative effects of endocannabinoids whose degradation could be a target of pharmacological intervention by faah inhibitors. background: the hematopoietic disorder chronic myeloid leukemia (cml) is one of the most extensively studied neoplasms. it is caused by translocation between chromosomes and leading to the formation of the philadelphia chromosome and the bcr-abl fusiongene. first-line targeted therapy is still the tyrosine-kinase inhibitor imatinib (im), which led to tremendous success in treatment. however, the amount of therapeutic resistances is increasing, caused either by bcr-abl-dependent mechanisms (e.g. bcr-abl amplification/overexpression, point mutations) or bcr-ablindependent mechanisms. these might be linked to alterations in drug transporter expression or particularly, microrna-expression levels. in our previous study, we analyzed the changes of microrna expression profiles during the development of imresistances in the leukemic cell line k . an inverse correlation of mir- expression and protein levels of the efflux transporter atp-binding cassette transporter g (abcg ) was observed in cells resistant to different im-concentrations, pointing to a relation of mir- to im-resistance. hence, we investigate in current studies, how the influence of mir- on im-sensitivity could be explained. methods: we transfected k cells, sensitive treatment-naïve cells and cells resistant to various im-concentrations, either with mir-mimic pre-mir- or inhibitory anti-mir- , challenged them with im and analyzed effects on cell viability, activation of apoptosis and cell death using wst- -, caspase glo -assay and cell counting. in addition, we analyzed changes in abcg expression using flow cytometry and qrt-pcr and investigated alterations in im-efflux using hplc and hoechst efflux assay. results: under im-treatment, sensitive k showed an effect of mir- -inhibition using anti-mir- . this led to a significant promotion of cell survival apparent on the level of respiratory chain function (p< . ) and cell membrane integrity and reduced capase- activity (p< . ). furthermore, these mirna-effects are dose-dependent as confirmed in concentration row-experiments. regarding transport and abcg expression, we found that µm im-resistant k do not express higher amounts of abcg , but showed higher transport rates of im or the abcg -substrate hoechst . conclusions: overall, these experiments indicate that mir- does not only affect abcg -expression, but also influences cell sensitivity to im in a more direct manner. further analysis will now be performed to reveal the underlying mechanism, how cell sensitivity to im is altered and if these effects occur due to a direct regulation of abcg . in summary, these findings could be relevant in cml-therapy, overcoming imresistances with a better understanding of mirna-and drug transporter alteration in cml. acknowledgments: we would like to thank all the authors for their contribution to this project. this work was funded by the university hospital schleswig-holstein. oxidized silicon nanoparticles and iron oxide nanoparticles for radiation therapy s. klein radiation therapy often combined with surgery and/or chemotherapy is applied to more than % of patients at some point of their treatment. the cytotoxic effects of ionizing radiation occur from their ability to produce dna double-strand breaks through the formation of free radicals within cells. however, the curative potential of radiotherapy is often limited by intrinsic radio resistance of cancer cells and normal tissue toxicity. to overcome this resistance and enhance the effectiveness of ionizing radiation, radio sensitizers are used in combination with radiotherapy. in our studies we used amino functionalized, oxidized silicon nanoparticles (sinp), superparamagnetic iron oxide nanoparticles (spion) and iron doped silicon nanoparticles (fe( %)-sinp) to increase the formation of reactive oxygen species (ros) in cells. cancer and tissue cells loaded with the various nanoparticles were irradiated with a single dose of - gy using a kv x-ray tube. after irradiation, the formation of the different ros species including superoxide, hydroxyl radicals and singlet oxygen was investigated. sinps with sizes around nm can easily cross the cell and nuclear membrane. the positively charged amino functionalized sinps stick in all membranes as well in those of the mitochondria. irradiation of the mitochondria may cause the depolarization of the mitochondrial membrane, which enables the release of cytochrome c and simultaneously, an inhibition of the respiratory chain, which leads to an increased generation of superoxide. amino functionalized sinps, as being embedded in the outer mitochondrial membrane, evidently enhance the depolarizing effect of the x-ray radiation on the mitochondria and therefore increase the concentration of superoxide. [ ] oxidized sinps with larger sizes accumulate in the cytoplasm and generate mainly singlet oxygen after irradiation. spions enter the cells via endocytosis, whereas the uncoated spions remain in the vesicles and the citrate coated spions accumulate in the cytoplasm. cells loaded with citrate coated spions show no higher ros concentration than in media-cultured cells. but after irradiation, the ros formation increased drastically. this enhancing effect is explained with the impact of x-rays onto the surface of spions which is due to the destruction of surface structures. the freed spion surface contains easier accessible iron ions. this ions can participate in the fenton and haber-weiss chemistry and thus, catalyze the hydroxyl radical formation. [ ] to % iron doped sinp increase the formation of hydroxyl radicals as well as the generation of singlet oxygen after irradiation. chronic pain in response to tissue damage (inflammatory pain) or nerve injury (neuropathic pain) is a major clinical health problem, affecting up to % of adults worldwide. currently available treatments are only partially susceptible and are accompanied with therapy limiting side effects. thus it is important to elucidate molecular mechanisms of pain signaling in detail to obtain new insights in potential future therapies. recent data indicate that hydrogen sulfide (h s) contributes to the processing of chronic pain, however pro-as well as antinociceptive effects have been described so far. moreover the sources of h s production in the nociceptive system are only poorly understood. here we investigated the expression of the h s releasing enzyme cystathionine g-lyase (cse) in the nociceptive system and characterized its role in chronic pain signaling using cse deficient mice. paw inflammation and peripheral nerve injury led to upregulation of cse expression in dorsal root ganglia. however, conditional knockout mice lacking cse in sensory neurons as well as global cse knockout mice demonstrated normal pain behaviors in inflammatory and neuropathic pain models as compared to wt littermates. thus, our results suggest that cse is not critically involved in chronic pain signaling in mice and that sources different from cse mediate the pain relevant effects of h s. this work was supported by the deutsche forschungsgemeinschaft (sfb -a ) and in part by loewe-schwerpunkt "anwendungsorientierte arzneimittelforschung". heinrich-heine-universität, institut für toxikologie, düsseldorf, germany heinrich-heine-universität, urologie, düsseldorf, germany background: cisplatin (cispt) is frequently used in the therapy of advanced stage urothelial cell carcinoma (ucc). yet, inherent and acquired drug resistance limits the clinical use of cispt. here, we comparatively investigated the response of epithelial-like (rt- ) and mesenchymal-like (j- ) uc cells following cispt treatment. methods: upon selection with equitoxic doses of cispt for months, we obtained cispt resistant variants (rt- r , j- r ). cell viability was measured using the alamar blue assay. cell cycle distribution was analysed by flow cytometry. immunocytochemistry was used to quantify the number of nuclear γh ax and bp foci representing dna double strand breaks (dsbs), while western blot was used to unravel the role of dna damage response (ddr) to acquired cispt resistance. qrt-pcr was performed to analyse the mrna expression of genes associated with cispt resistance. j- and j- r cells were treated with different concentrations of lovastatin and selected ddr inhibitors to elucidate their influence on cell viability. results: untreated rt- cells showed an about - -fold higher resistance to cispt than j- cells. both cell lines differed in the expression pattern of genes that are associated with cispt resistance. rt- r and j- r revealed a - -fold increased cispt resistance as compared to the parental cells. during the selection procedure, we observed that acquired cispt resistance goes along with morphological alterations that resemble epithelial mesenchymal transition (emt). cell cycle analysis of rt- r cells disclosed a reduced apoptosis and enhanced g /m arrest following cispt exposure as compared to rt- wild-type cells. by contrast, induction of cell death was similar in j- and j- r cells. notably, j- r cells showed a reduced formation of cispt-induced dsbs. correspondingly, the related ddr was diminished in j- r as compared to their parental cells. this was not found when ddr was comparatively analysed between rt- r and rt- cells. data obtained from qrt-pcr analysis indicate that different mechanisms contribute to acquired drug resistance of j- r and rt- r . unexpectedly, j- r and rt- r shared the upregulation of xaf- . treatment of j- r cells with statins and protein kinase inhibitors revealed an enhanced sensitivity to pharmacological inhibition of chk- and, moreover, re-sensitization to cispt by chk- inhibitor. based on the data we suggest that mechanisms of acquired cispt resistance of epithelial and mesenchymal uc cell lines are different with apoptosisrelated mechanisms appear to be more relevant for epithelial-like rt- cells and ddr-related mechanisms dominating cispt susceptibility in mesenchymal-like j- cells. furthermore, our findings indicate that chk- might be an appropriate target to deal with acquired cispt resistance in ucc. in many patients, gastric cancer treatment with conventional cytostatic agents shows only limited clinical response. novel therapeutics, which inhibit rtk signaling by targeting c-met or her family receptors, have demonstrated some efficacy; however, primary resistance of gastric cancer cells against these inhibitors is still a major problem. in the present study we investigated the mechanism of heregulin (hrg)-promoted survival of gastric cancer cells after treatment with c-met inhbitors or sirna-mediated downregulation of c-met. we found that hrg treatment of gastric cancer cells with a c-met amplification partially rescued the cells from the antiproliferative effects of pharmacological c-met inhibition or sirna-mediated downregulation of c-met. moreover, c-met inhibition or downregulation led to an induction of her expression on mrna and protein level, whereas other her family receptors were unaffected. downregulation of her impaired the hrg-mediated rescue of cell survival upon c-met inhibition. in other tumor entities the chromatin organizer special at-rich sequence-binding protein (satb ) has been described as a regulator of her family receptor expression involved in adaptive responses of tumor cells. thus, we investigated the contribution of satb in the upregulation of her after c-met inhibition. of note, c-met inhibitors as well as c-met-specific sirnas markedly induced satb expression in gastric cancer cells, and the downregulation of satb by sirnas completely prevented the induction of her upon c-met inhibition. in contrast, her or her expression levels were not affected by satb -specific sirnas. the function of satb as transcriptional regulator is controlled by its phosphorylation status, which in turn is modulated by pkc activity. thus, we also tested the effect of pkc inhibitors on her expression after c-met inhibition. interestingly, the upregulation of her in gastric cancer cells was significantly reduced by pkc inhibitors. to summarize, satb and pkc are critically involved in the regulation of her expression in gastric cancer cells after treatment with c-met inhibitors and the oncogene her plays a crucial role for tumor cell survival in this context. thus, inhibition of pkc or satb may help to overcome resistance against c-met inhibition in this tumor entitiy. in the rising field of nanomedicine, development of new approaches in diagnosis and treatment of cancer is a challenging task. typically, a nanocarrier is synthesized and linked to functional compounds displaying either diagnostic or therapeutic effects in cancer models. recently, nanomaterials combining both diagnostic and therapeutic properties, so-called 'theranostics', became of primary interest. here we used a human albumin-polyethylene glycol (peg) copolymer (hsa) as a theranostic platform for molecular integration of the chemotherapeutic drug doxorubicin (dox) and the magnet resonance imaging (mri) contrast agent gadolinium (gd) yielding gd-hsa-dox nanoparticles. besides in vitro testing, which demonstrated cytotoxic efficacy of gd-hsa-dox, we used the chorioallantoic membrane (cam) of fertilized chick eggs as a preclinical xenotransplantation model. the cam assay, which in legal terms does not represent an animal experiment, allows testing of compounds in an in vivo setting. this model is particularly helpful to narrow the gap between in vitro and in vivo applications in rodents, because it can help to reduce number of elaborate experiments with typically nude mice, and it reduces or even avoids exposure of those animals to adverse effects and distress. treatment-resistant mda-mb- breast cancer cells stably transfected with luciferase were xenotransplanted onto the chorioallantoic membrane. after formation of solid breast cancer xenografts, gd-hsa-dox was injected intravenously and its antiproliferative effect was evaluated by ivis imaging of luciferase activity and by immunohistochemical analysis of the tumor xenografts for the ki- proliferation antigen. in comparison to conventional dox, gd-hsa-dox showed increased antiproliferative efficacy and reduced general toxicity in the cam assay. on the basis of these findings, a rodent model was established, where the mda-mb- breast cancer cells were orthotopically xenotransplanted into the mammary fat pads of female nmri nu/nu mice. in this model, we further investigated biocompatibility, as well as diagnostic and therapeutic properties of the engineered nanomaterial. after repeated administration of gd-hsa-dox into the tail vein of the animals, biocompatibility of gd-hsa-dox was confirmed by uncompromised liver, kidney and hematopoietic parameters. to warrant diagnostic properties, accumulation of the nanomaterial in tumor tissue is indispensable. by small animal mri of gd, kinetics of intravenously applied gd-hsa-dox in tumor tissue was monitored. an enhancement of the engineered nanomaterial in tumor tissue was detected for up to h after injection indicating successful enrichment of gd-hsa-dox within the tumor tissue, which can be ascribed to the enhanced permeability and retention (epr) effect observed in the microenvironment of many solid tumor tissues. we are currently investigating the antitumor efficacy of gd-has-dox in this mouse model and preliminary data seem to indicate a dose-dependent anticancer effect. supported by the volkswagenstiftung. tubulin-binding agents are the most important anti-tumoral drugs. due to the side effects and the development of resistances, the discovery of new agents is still of importance. recently, pretubulysin (pt), a naturally occurring precursor of the myxobacterial compound tubulysin, was identified as a novel tubulin-binding compound. in the dfg research group for , pt was characterized as anti-tumoral, antiangiogenic and vascular-disrupting compound. moreover, pt was also found to inhibit the formation of metastases in vivo. aim of the present study was to gain first insights into the mechanisms underlying this anti-metastatic effect by investigating the influence of pt on the interaction of endothelial and tumor cells in vitro. pt treatment of primary human endothelial cells (huvecs) strongly increased the adhesion of breast cancer cells (mda-mb- ) on huvecs, but limited their transmigration through the endothelium (transwell assay). based on this data, the gene expression of presumably involved adhesion molecules was determined by qrt-pcr: icam- , vcam- , e-selectin, n-cadherin, and galectin- . moreover, the chemokine system cxcl /cxcr was analyzed. it could be demonstrated that the mrna level of endothelial n-cadherin is upregulated by pt. while total protein expression of ncadherin was enhanced in pt treated huvec, its surface expression was not largely influenced by pt (western blot, flow cytometry). in line with this, blocking endothelial ncadherin by a neutralizing antibody revealed that this protein is not involved in ptevoked tumor cell adhesion. interestingly, pt strongly augmented the mrna and protein expression of cxcl in huvecs (qrt-pcr, western blot), whereas its endothelial secretion was not affected by pt (elisa). an autocrine action of cxcl could be excluded, since blocking the cxcl receptor cxcr on endothelial cells with plerixafor did not influence cancer cell adhesion. by microscopic analyses, we observed that pt treatment causes transient gaps in the huvec monolayer, where tumor cells prefer to adhere. since β -integrins on the tumor cells could mediate interactions between cancer cells and extracellular matrix proteins in the gaps (e.g. collagen), their influence in cell adhesion and transmigration assays was examined. both the pt-evoked increase in cell adhesion and decrease in transmigration was completely abolished when β -integrins were blocked on mdas by a neutralizing antibody. these results indicate that the anti-metastatic action of pretubulysin might be based on the trapping of tumor cells on the endothelium. whether this effect is also relevant in vivo, will be analyzed in future studies using intravital microscopy. this work was supported by the german research foundation (dfg, for , fu / - ). introduction: tyrosine kinase inhibitors (tkis) for the treatment of non-small cell lung cancer (nsclc) patients harboring activating mutations in the epidermal growth factor receptor have shown prominent success. nevertheless, patients treated with tkis eventually acquire resistance and relapse ( ). based on an evolutionary cancer model ( ) , weekly high dose-pulsed tki regimens were proposed to delay resistance. using data from nsclc bearing mice treated with erlotinib at different dosing regimens, we developed a semi-mechanistic pharmacokinetic/pharmacodynamic model for erlotinib effects on tumor killing and resistance development. methods: data was available from experiments in xenograft mice bearing nsclc tumors (pc and hcc cell lines; both erlotinib sensitive) ( ). plasma concentrations from two single-dose groups, mg/kg and mg/kg, were used for pharmacokinetic modeling. relative tumor volume changes in mice randomized to five dosing regimens ( mg/kg daily, mg/kg daily, mg/kg every days, mg/kg every days, or vehicle) was the pharmacodynamic endpoint. a tumor growth inhibition model was developed by testing linear, exponential and logistic models to account for the tumor growth kinetics, as well as fitting an emax model to explain the effect of exposure on killing the sensitive tumor cells, and resistance development. analysis was performed using nonmem . . results: absorption was dose dependent, and a precipitate compartment accounted for dissolution limited absorption for the mg/kg dose. a -compartment model with first order elimination kinetics described distribution and elimination. to describe tumor volume changes, a tumor was assumed to be a mixture of sensitive and resistant cells (represented by distinct compartments and ordinary differential equations). exponential kinetics best described natural growth (doubling times: and days, for sensitive and fully resistant cells, respectively). a tumor was found to transit through a less sensitive phase before acquiring full resistance. an e max model (less than linear) best described effect on the sensitive cells (ec = . μm for both cell lines), and on the partially sensitive transit phase (ec = . μm and . μm, for hcc and pc cell lines, respectively), urging to provide adequate trough erlotinib concentrations for optimal effects. conclusions & future perspectives: an exposure-driven tumor growth inhibition model accounting for the kinetics of resistance development was developed. the model emphasizes the need for establishing an adequate trough erlotinib concentrations to delay disease progression. extracts of the stem bark of ficus platyphylla (fp) have been used in traditional nigerian medicine to treat psychoses, depression, epilepsy, pain and inflammation. previous studies have revealed the analgesic and anti-inflammatory effects of fp in different assays including acetic acid-induced writhing, formalin-induced nociception, and albumin-induced oedema. in this study, we assessed the effects of the standardised extract of fp on hot plate nociceptive threshold and vocalisation threshold in response to electrical stimulation of the tail root in order to confirm its acclaimed analgesic properties. we also investigated the molecular mechanisms underlying these effects, with the focus on opiate receptor binding and the key enzymes of eicosanoid biosynthesis, namely cyclooxygenase (cox) and -lipoxygenase ( -lo). fp (i) increased the hot plate nociceptive threshold and vocalisation threshold. the increase in hot plate nociceptive threshold was detectable over a period of min whereas the increase in vocalisation threshold persisted over a period of min. (ii) fp showed an affinity for µ opiate receptors but not for δ or κ opiate receptors, and (iii) fp inhibited the activities of cox- and -lo but not of cox- . we provided evidence supporting the use of fp in nigerian folk medicine for the treatment of different types of pain, and identified opioid and non-opioid targets. it is interesting to note that the dual inhibition of cox- and -lo appears favourable in terms of both efficacy and side effect profile. despite the fact, that the enormous economic burden and individual suffering caused by gastrointestinal infections permanently persists in developing and newly industrialized countries, healthcare systems in first world countries underestimated its significance for a long time. the alarming prevalence of multidrug-resistant gram-negative bacteria, combined with a high epidemic potential of gastrointestinal pathogens, however, demonstrates the urgent need for new antibiotics and antiinfectives worldwide. , million deaths per year were actually caused by acute diarrheal infections. the most common causative agents of acute diarrheal infections, amongst others, are yersinia enterocolitica, campylobacter jejuni, salmonella spp., shigella spp., escherichia coli, vibrio cholerae, and clostridium difficile. the established treatment based on antibiotics is mostly ineffective or may even have adverse side effects and result in prolonged shedding. in either way, antibiotic treatment also eradicates at least parts of the intestinal microbiome, and thereby disrupts colonization resistance, fosters overgrowth of pathogens and prolongs shedding times. therefore, the development of future drugs should be focused on highly specific antiinfectives, which enable a direct pathogenspecific treatment. one very promising strategy is the inhibition of the biogenesis of outer membrane virulence factors. due to the fact that many decisive virulenceassociated outer membrane proteins (omps) of gram-negative enteropathogens are substrates of the periplasmic chaperone sura exclusively, we developed a new assay format to determine sura in vitro chaperone activity. previous publications by behrens et al., and buchner et al., documented an assay to determine sura in vitro chaperone activity with extremely limited sensitivity and minimal detectable concentration, which was not suitable for high throughput screening (hts). we now developed a luciferase-based screening assay. this highly sensitive and robust test system has been validated extensively and now gives reliable output with an appreciable z-factor of > , . in cooperation with the hzi braunschweig (germany) and the hzi saarbrücken (germany), we were able to screen over purified compounds and over extracts of myxobacteria. during the ongoing screening period, the assay generated four validated primary actives, which corresponds to a positive hit rate of , %. additionally, we developed an elaborate follow-up strategy to validate positive hits, which includes a well-established mouse infection model. we are looking forward to escalate our screening efforts and would like to use this abstract to invite all scientist who are interested in testing compound/natural extract libraries for an activity against the target structure sura. the potential atypical antipsychotic and dopamine d receptor partial agonist bromoterguride antagonizes phencyclidine-and apomorphine-induced prepulse inhibition and novel object recognition deficits in rats e. tarland objectives: schizophrenia is a disabling mental disorder affecting more than million people worldwide. available medical therapies are effective in the treatment of psychosis and other positive symptoms, however come with considerable side effects and often fail to ameliorate cognitive deficits and negative symptoms of the disorder. the dopamine d receptor partial agonist -bromoterguride ( -bt) has recently been shown to exhibit antipsychotic effects in rats without causing adverse side effects common to antipsychotic drugs [ ]. to determine its atypical character in vivo, the ability of -bt to antagonize the disruptive effects of phencyclidine (pcp) and apomorphine on sensory motor gating was determined in the prepulse inhibition paradigm. the effect of -bt on cognitive deficits was assessed in the novel object recognition (nor) test after object recognition memory deficits were induced by pcp treatment. method: week old male sprague-dawley rats were injected with -bt ( . or . mg/kg; i.p.) followed by pcp ( . mg/kg; s.c.) or apomorphine ( . mg/kg; s.c.). prepulse inhibition was measured in two sound-proof startle chambers. the attenuating effect of -bt ( . or . mg/kg; i.p.) on visual learning and memory deficits following subchronic administration of pcp ( . mg/kg; i.p. twice daily for days) was assessed in the nor task consisting of a min acquisition trial and a min retention trial separated by a h inter-trial interval. clozapine ( . mg/kg; i.p) or haloperidol ( . mg/kg; i.p) were used as positive controls. results: the dopamine d receptor partial agonist -bt ( . mg/kg) and the typical antipsychotic haloperidol successfully antagonized apomorphine-induced ppi-deficits. interestingly -bt also ameliorated the pcp-induced ppi-deficits to the same extent as the atypical antipsychotic clozapine. preliminary data from the nor test indicate that -btreduces subchronic pcp-induced cognitive deficits in novel object recognition analogous to clozapine. the disrupting effects of pcp on ppi are mediated by non-competitive antagonism at nmda sites indirectly influencing a series of neurotransmitter systems. our results indicate that -bt mediates actions at multiple neurotransmitter receptors as it successfully ameliorated both the pcp-and apomorphine-induced ppi disruptions in rats, showing an atypical antipsychotic character. furthermore, our preliminary results support the potential atypical antipsychotic effect of -bt as it restored performance in the nor test, a test with good predictive validity. due to the previously shown properties and antipsychotic-like effects of -bromoterguride [ ], this substance may be a promising candidate for treatment of schizophrenic patients. ongoing experiments investigate the potency of -bt to improve social deficits following a sub-chronic pcp regime in rats. background and objectives: cannabinoid- receptor signaling increases the rewarding effects of food intake and promotes the growth of adipocytes, whereas cb possibly opposes these pro-obesity effects by silencing the activated immune cells that are key drivers of the metabolic syndrome. pro-and anti-orexigenic cannabimimetic signaling may become unbalanced with age because of alterations of the immune and endocannabinoid system. methods: to specifically address the role of cb for age-associated obesity we analyzed metabolic, cardiovascular, immune and neuronal functions in . - . year old cb -/and control mice, fed with a standard diet and assessed effects of the cb agonist, hu during high fat diet in - week old mice. results: the cb -/mice were obese with hypertrophy of visceral fat, immune cell polarization towards pro-inflammatory sub-populations in fat and liver and hypertension, as well as increased mortality despite normal blood glucose. they also developed stronger paw inflammation and a premature loss of transient receptor potential responsiveness in primary sensory neurons, a phenomenon typical for small fiber disease. the cb agonist hu prevented hfd-evoked hypertension, reduced hfdevoked polarization of adipose tissue macrophages towards the m -like proinflammatory type and reduced hfd-evoked nociceptive hypersensitivity but had no effect on weight gain. conclusion: cb agonists may fortify cb -mediated anti-obesity signaling without the risk of anti-cb mediated depression that caused the failure of rimonabant. leishmaniasis is a neglected tropical disease caused by leishmania, eukaryotic protozoal organisms, which infect humans and other mammals. this disease is transmitted by sandflies of the genus phlebotomus. due to global warming the endemic region of these vectors expands further to northwards and threatens south european countries as well. the treatment of leishmaniasis is difficult due to toxicity and resistance development for current drugs. the so far unexplored inhibition of mitochondrial functions in leishmania by natural products or even food ingredients seems to be an interesting alternative. two food ingredients, resveratrol (res) and xanthohumol (xan), were widely studied in mammalian cells but little is known about their actions on protozoal parasites. therefore, we compared the influence of res and xan on the function of leishmanial and mammalian mitochondria. anti-leishmanial activities of xenobiotics were assessed in cell culture of leishmania tarentolae promastigotes (ltp), leishmania amazonensis amastigotes (laa) and compared to peritoneal macrophages from mouse (pmm) using viability assays. furthermore, mechanistic studies regarding mitochondrial functions were conducted in ltp, mitochondrial fractions isolated from ltp and bovine heart submitochondrial particles using oxygen consumption measurements, assays of individual mitochondrial complex activities, membrane potential and superoxide radical formation by photometry, fluorimetry and electron spin resonance spectroscopy. in ltp, xan inhibited the viability more effective than res (ic : xan µm, res µm). likewise, xan and res demonstrated anti-leishmanial activity in laa (ic : xan µm, res µm) while had less influence on the viability of pmm (ic : xan µm, res > µm). in contrast to res, xan strongly inhibited oxygen consumption in leishmania. further studies demonstrated that this is based on the inhibition of the mitochondrial electron transfer complex ii/iii by xan which was less pronounced with res. however, xan also demonstrated inhibitory activity on mammalian mitochondrial complex iii. in addition, xan caused no decrease of the membrane potential in leishmanial mitochondria, while res resulted in mitochondrial uncoupling. neither xan nor res increased mitochondrial superoxide release in ltp. these data show that res, a major polyphenol from red wine, and xan, an ingredient of hop-containing beer, may have selective anti-leishmanial activity. tryptophan hydroxylase (tph) is the rate-limiting enzyme in serotonin ( -ht) biosynthesis. its two existing isoforms are exclusively expressed in the periphery (tph ), or the raphe nuclei of the brainstem (tph ) and the respective -ht populations are distinctly separated by the blood-brain barrier, offering the possibility to pharmacologically modulate central and peripheral functions in an independent manner. peripheral -ht is mainly produced by tph -expressing enterochromaffin cells of the gut and taken up into platelets and transported in the blood stream. upon platelet activation, -ht is rapidly released and locally induces multiple effects, such as vasoconstriction, cell proliferation or fibrosis and is furthermore involved in the regulation of e.g. vascular tone, gut motility, primary hemostasis, insulin secretion and t-cell-mediated immune response. following the classical early drug development pathway, we developed a fluorescencebased tph activity assay and performed a high-throughput screening of about small chemical compounds. we discovered a novel class of tph inhibitors, which was thoroughly validated in a variety of in vitro assay setups. combining medicinal chemistry and x-ray crystallography, we further aimed to develop these inhibitors into preclinical drug candidates. to date we were able to generate and patent a series of novel tph inhibitors with optimized affinity and an in vitro ic in the low nanomolar range. this novel class of tph inhibitors could potentially be used to treat a variety of disorders with aberrant peripheral -ht signaling, such as gastrointestinal disorders (e.g. irritable bowel syndrome, crohn's disease, various forms of diarrhea), cardiac valve diseases, pulmonary hypertension, chronic respiratory diseases and some neuroendocrine (carcinoid) tumors. primary hepatocellular carcinoma (hcc) is the most frequent type of liver cancer. therapeutic options are rare. beside sorafenib, a tyrosinkinase inhibitor, which is only used in end stage liver cancer, the surgical intervention is the only successful clinical treatment option. hence there is an urgent need to develop new therapeutic strategies and to identify new drugs for therapy of hcc. hcc often arises in fibrotic or cirrhotic liver, which is accompanied by a change of the extracellular matrix (ecm) composition. in addition it was shown that hepatoma cells express different integrins, which interact with ecm and intracellular cell signaling, compared to hepatocytes. snake venoms have gained increased attention, as it was shown that some of their enzymes and peptides directly act on tumor cells and their multicellular arrangement or indirectly by influencing the stroma environment of the tumor. aim of the present study was to investigate the effect of snake venoms on liver cancer related cell lines as well as their specific action on the ecm-integrin axis. the effects of the snake venoms vipera palestinae (vp), calloselasma rhodostoma (cr) and echis sochureki (es) on a cellular level (mtt, ldh release), on cell-cellconnections (caco permeability assay) and on cell-matrix-interactions (adherence test) were investigated. cell-matrix interactions were tested with an adhesion assay using collagen i (c-i), collagen iv (c-iv), fibronectin (fn) and laminin (lm) as ecm compounds. in our in vitro models we used hepg as a hcc tumor cell line and the fibroblast cell line fi as stroma simulation. additionally caco cells were used, a colon carcinoma cell line representing colorectal liver metastasis. the toxicity of snake venom on liver cancer related cell lines was determined in the range of . - µg/ml and plotted into dose response curves. the noaels were calculated from these dose response curves: vp: . µg/ml -cr: µg/ml -es: µg/ml. performance of the caco -transwell permeability assay revealed no influence of the tested venom concentrations on the integrity of the cellular arrangement. investigations for integrin inhibition revealed that the venom from vp reduced adherence on lm coated plates and the venom of ec reduced adherence on lm and fn coated plates compared to untreated cells. there was no effect on the adherence on any matrix from the venom of cr observable. co-incubation of the snake venoms of vp and es (below or near noael concentrations) with -fluorouracil ( fu), which is used as a chemotherapeutic agent, caused a reduction of its ic values. the results indicate that components of vp and ec inhibit the formation of cell-matrixinteractions possibly acting as disintegrins. the co-incubation experiments demonstrated a synergistic effect of fu and snake venoms. further experiments should enable the isolation of therapeutic active venom compounds, identification of disintegrins and their role in synergistic mechanisms in liver cancer therapy. modulation of the blood-brain barrier with peptidomimetics to improve drug delivery s. dithmer after decades of research, the blood-brain barrier (bbb) still remains a major problem for successful delivery to the brain for the vast majority of drugs. the main component forming the bbb is the brain microvascular endothelium. the paracellular permeation is limited by tight junctions (tjs), a multiprotein complex composed of the members of the claudin family claudin- , - , - , - . claudin- is known to be the key tj protein tightening the bbb. therefore, claudin- has been selected as target to modulate the bbb. for this reason, drug enhancer peptides (peptidomimetics) were designed to modulate transiently claudin- and, thereby, permeabilize the bbb. by combining biochemical protein/peptide interaction and tissue culture methods, we identified, validated and optimized peptide sequences modulating claudin- containing barriers. the claudin- targeting peptides decreased the transcellular electrical resistance and increased the permeability through mdck-ii cell monolayers stably expressing yfpclaudin- and immortalized brain endothelial cells (bend. ). the peptides decreased the amount of claudin- and zo- at cell-cell contacts and changed the cell morphology from spindle-shaped to more round-shaped. all tested peptides showed no signs of toxicity on cell cultures and in vivo (intravenous injection). permeability measurements in mice proved enhanced permeation of na-fluorescein ( da) through the bbb, which was confirmed by magnet resonance imaging of contrast agents (gd-dtpa, da). in summary, we identified new peptides with the potential to enhance cerebral delivery of small molecules through the bbb. treatment of cerebral diseases is limited by the capability of pharmacologically active agents to penetrate the blood-brain barrier (bbb). this paracellularly tight diffusion barrier is formed by brain capillary endothelial cells. the paraendothelial cleft is sealed by tight junctions (tjs), a multiprotein complex. cerebral tjs predominantly consist of claudin- (cldn ) which tightens the bbb for molecules < da. consequently, cldn is a potential target for transient and size-specific modulation of the bbb to improve cns penetration for pharmaceutically active agents. in high throughput screening using a cldn assay, the barrier opener (bo ) was identified as a cldn modulator. initially, a significant removal of cldn from the plasma membrane was shown by confocal microscopy using epithelial and endothelial cell lines. measurement of transcellular electrical resistance and of paracellular permeability using lucifer yellow (mw da) demonstrated the effect of bo . concentration dependent treatment ( - µm) of cell monolayers with bo reduced tightness of the tjs between some hours and h. applying -hydroxypropyl-ß-cyclodextrin as a solubilizer, opening activityof bo became detectable in mice. due to short stability (< h) of bo in the bloodplasma repeated administration ( . mg/kg i.v.) was required to induce significantly increased permeability of the bbb for na-fluorescein(mw da). the small molecule bo is a promising new approach for transient opening of the bbb in vivo. further modification of the stability and solubility of bo is necessary to optimize its applicability. the complex of tight junction (tj) proteins is located between opposing epithelial or endothelial cells. tjs restrict the paracellular permeation of ions and other solutes. tricellulin (tric) tightens tricellular tjs (ttjs) and regulates bicellular tj (btj) proteins like claudins and occludin (occl). current data suggest an important role of ttjs at the blood-brain barrier (bbb). a main pharmacological problem is modulation of the bbb to improve drug delivery to the cns. therefore, tricsi has been developed as a peptide taken from tric to open tissue barriers specifically and transiently.initially, a recombinant protein was generated based on a sequence of an extracellular loop of tric, tagged with maltose binding protein. the fusion protein caused down-regulation of tric, internalization of both tric and occl (confocal laser scanning microscopy), and a significant decrease in transcellular electrical resistance (ter) of a human epithelial colorectal adenocarcinoma cell line. then, studies with the synthetic peptide tricsi indicated its capacity of cell barrier openingafter about h of incubation with concentrations varying from to µmaffecting the membrane localization of tricand occl. barrier opening was proven by decreasing ter, increasing permeability coefficient of lucifer yellow ( da) and fitc-dextran ( kda); the localization of tric elongated from ttjs towards btjs and cldn was weakened at btjs.physiochemical properties of tricsiexamined by circular dichroism spectroscopy suggested ß-strand structure and no helical propensity. taken together, a tric-derived peptide has been identified increasing the paracellular permeability of tissue barriers and redistributing the cellular localization of tj proteins. tricsi is a novel, promising tool to overcome cerebral barriers with the potential to improve drug delivery to the cns. further experiments are needed to better understand the role of tric in tissue barriers as well as to clarify the mode of action of tricsi. introduction: lung transplantation has become an established treatment option for a variety of end-stage lung diseases, but the long-term survival is often disappointing. the leading cause of death is generally chronic rejection which is characterized by inflammation and fibrous obliteration of the small airways, progressively leading to a reduction of the airflow. the mouse heterotopic tracheal transplantation model is widely used as an experimental model to study the development of obliterative airway disease. despite its widespread application, the heterotopic transplantation model does have a number of limitations, as for example the lack of airflow. the present study provides a description of the orthotopic tracheal transplantation mouse model, which shares more similarities with transplant situation in humans, and provides the analysis of airway obliteration via micro ct and histological evaluation. methods: a seven-ring donor trachea from balb/c mice was implanted into the recipient c bl/ mice. c bl/ mice without transplantation were used as normal controls. donor c bl/ mice to recipient c bl/ mice were served as the isograft group. days after transplantation, mice were scanned using an in vivo small animal µct (skyscan ). tracheal tissue was harvested and fixed in formalin, embedded in paraffin, cut and stained with hematoxylin and eosin (h&e) as well as sirius-red/fast-green. results and conclusions: histologic evaluation showed luminal narrowing with subepithelial inflammatory cell infiltrates and fibrosis, as well as partially damaged and flattened epithelium. the aerated volume of the allogeneic grafts, analyzed by micro ct was significantly reduced compared to the isogenic control grafts and normal controls. non-invasive imaging via micro ct may offer an option for longitudinal monitoring of the progression of obliterative airway disease as well as response to treatment. c. elegans is a well-established model organism to study the aging process as well as effects of various substances in vivo. its lifespan is regulated by multiple signaling pathways (e.g. insulin or mtor signaling), which are well conserved up to humans. the insulin/igf- pathway was the first pathway shown to effect ageing in animals. mutations that decrease the activity of daf- (igf r) lead to a significant increase of lifespan accompanied by a decrease of age pigment accumulation in c. elegans. the relevant effector of the insulin/igf- pathway is the transcription factor daf- (hfoxo a). inhibition of hmg-coa reductase (enzyme of mevalonate pathway) by statins, which are frequently used as cholesterol-lowering agents in the clinic, has been shown to attenuate protein prenylation and glycosylation. notably, prenylated-, membrane-bound small gtp-binding proteins are important for the regulation of the afore mentioned age-related signaling pathways like the insulin/igf- pathway. recently, a cohort study showed that a decreased mortality rate in humans between age - correlates with statin treatment, but is independent of total cholesterol levels. as c. elegans harbors the mevalonate pathway, but the branch leading to cholesterol synthesis is missing, it is a well-suited model to study cholesterol -independent effects of statins on aging-associated phenotypes and the underlying molecular mechanisms. here, we show that exposure of c. elegans to statins substantially decelerated the accumulation of age pigments. while the level of age pigments roughly doubled in control animals, there was only a slight increase in the lovastatin group. the use of atorvastatin gave comparable results indicating a more general effect of the inhibition of the hmg-coa reductase. the retarded accumulation of age pigments could be partly phenocopied using an inhibitor of the small gtpase rac or using rnai against the hmg-coa reductase. a reduced level of age pigments is prognostic for an elevated mean lifespan (about %) in c. elegans. a post reproductive treatment with lovastatin, mimicking the use of statins in patients of advanced age increased the mean lifespan in c. elegans even further. in addition, we could show a mild reduction of fertility and a developmental delay as well as a marked increase in acute thermal stress resistance mediated by lovastatin. besides the reduced accumulation of age pigments and the increased lifespan these are phenotypes which are usually observed under accumulation of daf- overactivity. consequently we found an increased nuclear localization of daf- in the presence of lovastatin and lovastatin completely failed to reduce age pigments in a daf- -ko mutant background. rt-qpcr brought jnk- , a known activator of daf- , into play as a possible effector induced by statins. this is currently under investigation. in summary, statin exposure induces a longevity phenotype in c. elegans, which might be daf- dependent. this findings indicates that a product of the mevalonate pathway might influence the insulin/igf- pathway and particularly the transcription factor daf- . the high-fat diet (hfd)-fed, streptozotocin (stz)-treated rat model is one of the experimentally-induced animal models of diabetes. this model is often used to evaluate the antidiabetic activity of several agents. according to srinivasan et al. ( ) , prolonged exposure of high-fat diet leads to insulin resistance, and the development of diabetes occurs only in insulin-resistant hfd-fed rats following low dose stz, because the hfd-fed rats are already mildly hyperglycemic due to insulin resistance ( ). in hfd/stz model, the rats are fed with high-fat diet for - weeks or for a relatively long time (≥ months) in order to simulate the insulin resistance and/or glucose intolerance. after induction of diabetes with multiple or single low-dose of stz ( - mg/kg), some of the diabetic rats receive treatment ( ) . in this way, the impact of treatment can be determined by comparing the differences between groups. despite the lack of methodological information concerning the feeding time in some studies, all rats should be allowed to continue to feed on their respective diets until the end of the study. but what would happen if the hfd was switched to normal pellet diet in these diabetic rats? in our experience, the feeding of npd for weeks significantly decreased fbg in diabetic rats compared to hfd-fed diabetic rats ( . ± . mg/dl vs. . ± . mg/dl, p < . ). although diet regulation could not restore normal blood glucose, such a decrease was unexpected. in addition, the body weights of the npd-fed diabetic rats were significantly lower than the body weights of the hfd-fed diabetic rats ( ± . gvs. . ± . g, p < . ). there was no significant difference in body weight between nondiabetic control rats and diabetic rats fed npd for weeks. further details can be found in table . diet regulation and weight loss may prevent, control and reverse diabetes. however, at later stages of the disease, it is difficult to improve blood glucose control without medication, because the disease progresses from insulin resistance to insulin deficiency ( ) . according to some diabetes researchers, the amount of residual functional betacells mass is an important issue, and another important question is whether hfd/stz rat mimics an early or late stage of type diabetes ( ). these preliminary findings suggest the possibility that hfd/stz rat model may simulate the characteristics of early stage more than the final stage of type diabetes, and hyperglicemia in the experimental model can partially reverse with diet regulation. references: . srinivasan, k., viswanad, b., asrat, l., kaul, c. l., ramarao, p. ( ) . combination of high-fat diet-fed and low-dose streptozotocin-treated rat: a model for type diabetes and pharmacological screening. pharmacol res ( ): - . . oztürk z, gurpinar t, vural k, boyacıoglu s, korkmaz m, var a. ( ) . effects of selenium on endothelial dysfunction and metabolic profile in low dose streptozotocin induced diabetic rats fed a high fat diet. biotech histochem ( ): - . . franz, m. j. ( ) . the dilemma of weight loss in diabetes. diabetes spectr ( ) animal models are pivotal for studies of pathogenesis and treatment of movement disorders. dystonia, characterized by sustained or intermittent muscle contractions causing twisting movements/postures, is regarded as a basal ganglia disorder. the pathophysiology is however poorly understood. in mouse models of dyt dystonia, which is caused by a gag deletion in tor a that encodes for the protein torsin a, ex vivo electrophysiological studies have shown an abnormal d receptor mediated release of acetylcholine from striatal interneurons. in these models, which do not exhibit a dystonic phenotype, the functional relevance of the increased d receptor mediated acetylcholine release has not been examined yet. the aim of present study was to ( ) generate more powerful tests to detect behavioural alterations in the dyt knock-in mouse and to ( ) examine the behavioral effects of the d receptor agonist quinpirole. for this purpose, a sequence of cognitive, motoric and sensorimotor tests were performed in this mouse model. only the adhesive removal test that explores sensorimotor connectivity revealed significant impairments in the dyt knock-in mice compared to controls. to induce a more characteristic and stronger phenotype, the "rotating beam test" was developed. this motoric test measures motor coordination and balance. interestingly, dyt knock-in mice showed significant motor deficits in the rotating beam test. based on these results, the acute effects of quinpirole ( . - mg/kg i.p.) were tested in dyt knock-in and wildtype mice. subsequent to the injections, mice were tested in the open field, the rotating beam test and the adhesive removal test, respectively. in the open field test, dyt knock-in mice showed increased thigmotaxis at a dose of . mg/kg quinpirole. in the rotating beam test, both groups showed a dose-dependently reduced performance. in the adhesive removal test, quinpirole improved the reaction time in dyt mice independently of dosage, while no effects were observed in the wildtype littermates. however, in vehicle follow-up (post-drug control), this effect remained consistent in the dyt model, suggesting a habituation effect. in conclusion, we generated a new test, i.e., the rotating beam test which improves the detection of mild motor impairments in dyt knock-in mice. furthermore, the adhesive removal test revealed sensorimotor dysfunctions in this animal model. these results represent an important step for our ongoing optogenetic examinations on the role of abnormal neuronal plasticity in dyt dystonia and for pharmacological studies. the first data on the effects of quinpirole do not indicate a critical role of d dysregulated acetylcholine release, but this has to be clarified by ongoing local striatal injections of quinpirole and by pharmacological manipulations of the cholinergic system. renal fibrosis is characterized by decreased nitric oxide (no) bioavailability and pronounced transforming growth factor β (tgfβ) signalling subsequently excessive extracellular matrix (ecm) deposition. here, the effects of the soluble guanylate cyclase (sgc) stimulator bay - after unilateral ureter obstruction (uuo) have been studied. kidney fibrosis was induced by unilateral ureter obstruction (uuo) in wild type (wt) and cgki knock-out (cgki ko) mice. starting one day after uuo, the sgc stimulator bay - was ( mg/kg/daily) i. p. injected for seven days. biomarkers indicating remodelling processes in the kidney were analysed via mrna expression and protein expression. bay - administration influenced the activity of the ecm degrading matrix metalloproteases (mmp and mmp ) and their inhibitor timp- , the expression pattern of extracellular matrix proteins (e.g. collagen and fibronectin) of profibrotic mediators (e.g. connective tissue growth factors (ctgf) and plasminogen-activator inhibitor- (pai- )) and the secretion of cytokines, e.g. il- . thereby, bay - increments the cgmp pool among others via modulation of endothelial no synthase (enos) expression. agents, which enhance no and cyclic guanosine monophosphate (cgmp) ameliorate the progression of fibrotic tissue. however, the molecular mechanism by which cgmp via cgki affects the development of kidney fibrosis has not fully been elucidated. accordingly, the present study investigates the functional role of sgc stimulation in regulating the fibrotic process, the signalling pathway and the underlying mechanisms involved. we hypothesize that the antifibrotic potential of bay - might be related to the increased cgmp pool and the inhibition of the mapk and smad signalling pathway. the elucidation of the signalling allows the development of new therapeutic options. infection of mice with listeria monocytogenes (lm) results in a strong t-cell response that is critical for an efficient defense. here, we demonstrate that the adapter protein sly (sh -domain protein expressed inlymphocytes ) is essential for the generation of a fully functional t-cell response. the lack of sly leads to reduced survival rates of infected mice. the increased susceptibility of sly ko mice was caused by reduced proliferation of differentiated t cells. ex vivo analyses of isolated sly ko t cells displayed a dysregulation of forkhead box protein o (foxo ) shuttling after tcr signaling, which resulted in an increased expression of cell cycle inhibiting genes, and therefore, reduced expansion of the t-cell population. foxo shuttles to the cytoplasm after phosphorylation in a protein complex including - - proteins. interestingly, we observed a similar regulation for the adapter protein sly , where tcr stimulation results in sly phosphorylation and sly export to the cytoplasm. moreover, immunoprecipitation analyses revealed a binding of sly to - - proteins. altogether, this study describes sly as an immunoregulatory protein, which is involved in the generation of adaptive immune responses during lm infection, and provides a model of how sly regulates t-cell proliferation (schäll et al., eur j immunol. ) . the catalytical isoforms p γ and p δ of phosphatidylinositol- , -bisphosphate kinase γ (pi kγ) and pi kδ play an important role in the pathogenesis of asthma. two key elements in allergic asthma are increased eosinophil and ige levels. whereas dual pharmacological inhibition of the catalytical subunits p γ and p δ reduces asthmaassociated eosinophilic lung infiltration and ameliorates disease symptoms, it has been shown that dual genetic deficiency in pi kγ and pi kδ in p γ ko δ d a mice increases serum ige and basal eosinophil counts in mucosal tissues and blood. this suggests that long-term inhibition of p γ and p δ might exacerbate asthma. here we analysed p γ/δ -/mice and determined ige and eosinophil counts in a basal state and the immune response to ovalbumin (ova)-induced allergic asthma. we found that serum concentrations of ige, il- and eosinophil numbers in blood, spleen and bone marrow were significantly increased in p γ/δ -/mice in comparison to single knock-out (ko) and wildtype (wt) mice. nevertheless, p γ/δ -/mice were protected against ovainduced infiltration of eosinophils, neutrophils, b cells and t cells into the lung tissue and the bronchoalveolar space. moreover, p γ/δ -/mice, but not single ko mice, showed a reduced bronchial hyperresponsiveness as measured with the isolated and perfused lung. we conclude that although the dual deficiency of p γ and p δ causes eosinophilia and ige hyperproduction, p γ/δ -/mice are not prone to develop ovainduced allergic asthma. an increase of plasma extravasation induced by activation of constitutively expressed endothelial bradykinin type receptors (b ) has been shown to contribute to the development of angioedema occurring as a sometimes life-threatening side effect of angiotensin-converting enzyme inhibitors such as enalapril (new engl j med : ; - ) . these drugs inhibit the degradation of bradykinin and increase its vascular steady-state concentration. hence, it is reasonable to assume that bradykinin may destabilise the endothelial barrier, i.e. may increase physiologic extravasation. while the commonly used miles assay provides a useful and relatively easy tool to study the effect of permeabilizing mediators in-vivo, it does not distinguish between intravascular and interstitial evan's blue dye. likewise, extravasation can only be quantified at one particular time point per animal, usually - min. furthermore, evaluation of physiologic extravasation is not possible. in contrast, non-invasive twophoton laser microscopy may allow separating the intravascular from the interstitial compartment and thereby investigations of changes of the physiologic endothelial barrier induced by drugs or transgenes. therefore, we have evaluated this methodology for its suitability to study endothelial permeability in mice in vivo. to establish this, we used two different fluorescent dyes of different molecular weight. a , kda dextran equipped with a green fluorescent chromophore which cannot leave vascular lumen was injected intravenously to visualize small dermal blood vessels of the mouse ear located approximately µm below the surface. after stabilization of the green fluorescent signal, a , kda dextran equipped with a red fluorescent chromophore which easily traverses the endothelial barrier was applied by intravenous injection. the red fluorescence permeates into the interstitium during physiologic extravasation and accumulates in the interstitial space. this process can be followed by measuring the decrease of intravascular red fluorescence over various time periods. using this methodology we have studied whether endothelial-specific overexpression of b changes physiologic endothelial permeability. this newly developed transgenic mouse line (b tg ) was established using a plasmid consisting of pbluescript ii sk+ -vector, the tie- -promotor, the human b cdna, the sv poly-a-signal and a tie- intron fragment. we observed that b tg showed a significantly stronger extravasation than their transgene negative littermates as evidenced by the more rapid extravasation of the , kda dextran at each time point (fig. ) .we conclude that two-photon laser microscopy is suitable to study endothelial permeability non-invasively in-vivo and that this methodology allows to study the effects of drugs and transgenes on the endothelial barrier under non-inflammatory conditions. furthermore, our results suggest that endothelial-specific overexpression of b increases physiologic extravasation. non-allergic angioedema such as angioedema induced by angiotensin converting enzyme inhibitors (acei) develops as a consequence of increased activation of bradykinin receptor type (b ). using a plasmid consisting of pbluescript ii sk+ -vector, the tie- -promotor, the human b cdna, the sv poly-a-signal and a tie- intron fragment a transgenic mouse line harbouring an endothelial-specific overexpression of b was generated and backcrossed to c bl/ for more than times (b tg ). lung mrna using primers specific for the human or the mouse b cdna revealed a . -fold stronger expression of human b in b tg (n= ), while the expression of murine b mrna was unchanged and similar to transgene negative littermates (b n ). we have evaluated the specificity of several antibodies directed against b and found that a rabbit monoclonal anti b antibody appears to be reliable, i.e. there was just a faint staining in lung tissue of b -/mice. however, this antibody primarily stains rodent b and has only little cross-reactivity to human b . hence, we were not able to detect a significant increase of b protein in tissues of b tg . previous experiments have shown that bradykinin induced concentration-dependent constrictions of aortic rings with a maximal effect at µm of bradykinin. the contraction due to bradykinin was completely inhibited by icatibant or diclofenac indicating that it is mediated by endothelial b activation and dependent on cyclooxygenase activity. in striking contrast to their transgene negative littermates b n , we found a significant icatibant sensitive aortic dilation in b tg following preincubation with diclofenac which indicates functional overexpression of b in conductance vessels of b tg . to evaluate whether this applies to dermal micro vessels we used the miles assay to quantify dermal extravasation of the albumin-bound dye evans blue following intradermal injection of µl of either vehicle, bradykinin, labradimil and histamine (control). increasing concentrations of bradykinin caused a significant increase of extravasation reaching . ± . fold at . nmol bradykinin in c bl/ (n= each, p< . vs. vehicle). a similar increase was found in b n ( . ± . fold, n= , p< . vs. vehicle), while there was a stronger response in b tg ( . ± . fold, n= , p< . vs. vehicle) which was significantly different to b n (p< . ) and c bl/ (p< . ). in another set of experiments the specific and ace-resistant b agonist labradimil ( . nmol) was used instead of bradykinin. labradimil increased extravasation by . ± . fold in c bl/ (n= each, p< . vs. vehicle), by . ± . fold in br n (n= each, p< . vs. vehicle) and . ± , fold in b tg (n= , p< . vs. vehicle) which was significantly different to c bl/ (p< . ) but not to b n (p> . ). the effects of bradykinin and labradimil were largely blocked by nmol icatibant (i.v.) in c bl/ , b n and b tg mice (p< . ) and hence mediated by activation of b . these data suggest that overexpression of b in b tg is functionally active in endothelial cells of large conductance and small dermal vessels. therefore, b tg represents a new animal model suitable for cardiovascular and non-allergic angioedema research. pharmacokinetic pharmacodynamic modeling of irreversible effects: the rituximab example f. keller universitätsklinikum, innere , nephrologie, ulm, germany background: for pharmacokinetic-pharmacodynamic modeling usually the sigmoid emax model is used as described by the hill equation. however, treatment regimens exist where the effect is only exerted as long as the drug concentration increases whereas decreasing concentrations produce no longer an effect. examples are the pulse anti-cancer therapy such as originally proposed by the devita protocols. methods: here, the new model for irreversible drug action is derived from the time dependent change of the concentration that must be larger than the time-dependent growth of the number of target cells (tumor or bacteria). the irreversible effect can be assumed if the is no further growth of the target cells occurs. de/dt = + dc/dt -dn/dt dn/dt = a solution for the above differential equation can be obtained by use of the integral exponential function iec based on the euler-mascheroni constant (gamma = . …). this model of an irreversible effect was applied.to the example of rituximab where the initial effect on cd + and cd + b-cells completely persists for month. to obtain a numerical solution, the following parameters are needed to be determined: the target concentration ctarget = mcg/ml and the infusion time t = hours. results: it can be shown that a plausible result for rituximab can be estimated only under the condition that a short distribution half-life is assumed of t / = hours (not shorter than the time t of infusion). with the terminal half-life of hours no plausible solution is obtained. under these conditions two observations are made: there is a negative effect both, initially for low concentrations and after cessation of the infusion when concentrations decrease (in reality this means no effect in both cases). the irreversible effect is proportional to the target concentration. the shorter the half-life comes out relative to the infusion time (t / < t) the stronger is the effect (figure) . occasionally there are specific questions occurring on the ruminant xenobiotic metabolism: ) are the observed metabolites ruminant specific and formed directly in the rumen? ) are ruminants able to cleave plant specific metabolites like glycosides to the respective aglycon? in the past new additional in vivo goat metabolism studies with at least one animal were performed. the aim of the project was to elucidate an alternative in-vitro method to replace the existing in vivo method in order to address robustly specific questions on xenobiotic metabolism in ruminants for registration of ppp. fresh sheep rumen fluid was incubated in-vitro > days by using rumen simulation technology (rusitec). the conservation of the physiological conditions were proven by measurement of ph (~ph . ) and redox potential (~- mv). the microflora composition and their viability (bacteria, protozoa and fungi) of the rumen were monitored by microscopy, incubation on agar plates and performing several viability tests (e.g. glycosidase-test, nh and short fatty acids). all the tests showed that the rusitec is a successful tool to maintain sheep rumen fluid for at least days in -vitro. the metabolic behavior and performance of the rumen fluid was tested by e.g. incubating c-triazol derivative metabolites (tdm) like triazol-alanin (ta); triazol-acetic-acid (taa) and triazol-lactic-acid (tla), which are usually formed in plants after application of triazol-containing fungicides. it was shown that ta was cleaved within h to . . .-triazol, while taa and tla were stable under these conditions. these data are in a good accordance with available in vivo data in cows. moreover glycosides ( c-polydatin, octyl- c-β-d-glucopyranosid) were cleaved within hour completely. all these data show, that the rumen fluid maintained its metabolic performance by using rusitec. basf identified the rusitec method, which is usually used in different areas (e.g. investigation of methane production in-vitro) as suitable and adapted this method for the purpose of investigation of ruminant xenobiotic metabolism. it was shown that rusitec is a robust method to analyze rumen xenobiotic metabolism and therefore can clearly substitute in vivo animal studies on ruminant metabolism studies beyond oecd and contribute significantly to animal welfare ( r: replacement). results: by using the training set, physicochemical (e.g. lipophilicity) and pharmacokinetic characteristics of mtx (e.g. v max for active tubular secretion) were slightly adjusted. using the gfr formula of morris et al. ( ) and including an empiric correction factor, mrd for the training set was . whereas bias was . µmol/l. by applying the developed pbpk model to the test set the respective values were mrd= . and bias= . µmol/l. for the covariates "at least one potentially interacting co-medication" and "trimethoprime/sulfamethoxazole" a significant impact on the prediction quality was found. conclusions: using the developed pbpk-model, a good prediction of the pharmacokinetics of hd mtx in severely ill children was found. by including additional factors influencing the prediction of mtx characteristics (e.g. co-medications) an improved prediction of mtx-sl might be reached. in prospective clinical trials, those more complex models should be evaluated and might be helpful to predict hd mtx pharmacokinetics and reduce unwanted side effects. hypericin is a natural polycyclic quinone found in hypericum perforatum. although hypericin reportedly has numerous pharmacological activities, only a limited number of studies have been performed on the absorption and transport characteristics of this compound, presumably, because hypericin is a highly lipophilic compound which is poorly soluble in physiological medium. recently we have shown that quercitrin and isoquercitrin, but not hyerosid, quercetin or rutin increased the uptake of hypericin in caco- cells. the major aim of this study was to get a detailed understanding of the exposure and fate of hypericin in the caco- cell system under different experimental conditions. the permeation characteristics of hypericin ( µm) in absence or presence of hypericum extract , . µg/ml) were studied in the absorptive direction. following application of hypericin to the apical side of the monolayer only negligible amounts of the compound were found in the basolateral compartment. the amount of hypericin in the basolateral compartment increased concentration-dependently in the presence of the extract (from to . %). the majority of hypericin was found after cell extraction ( % in absence and % in presence of the extract). the recovery was in the range of %, and significant amounts of hypericin found after cell extraction. fluorescence microscopy and imaging analysis revealed that hypericin is mainly accumulated in the cell membrane. the precise mechanism through which hypericin might overcome the hydrophobic barrier of cell membranes remains to be elucidated. however, our experiments demonstrated that the permeation characteristics of hypericin significantly improved in presence of the extract. background: the combination of gamithromycin (gam), a novel drug with the big advantage of a once weekly administration, and rifampicin (rif) is used in the treatment of lower airway disease in foals. both are effective in the therapy of infections with rhodococcus equi, a gram-positive coccobacillus bacterium, which is known to survive and reproduce within alveolar macrophages. macrolides are combined with rif to prevent resistance developing with single agent therapy. both drug classes reach high concentrations in the lung, penetrate into phagocytes and kill intracellular pathogens. methods: a controlled, single-and multiple dose study with four-periods was conducted in healthy foals ( ♂ and ♀, age: - days, body weight: - kg) which were treated once with rif alone ( mg/kg s.i.d., p.o., a) followed by the administration of gam ( mg/kg once weekly, i.v., b) for weeks. study period ("rif-gam acute", c) includes the administration of gam and rif for days with an administration interruption after the first rif dose for blood sampling. for the last study period ("rif-gam chronic", d) both gam and rif were coadministered for weeks. all periods were completed with blood sampling for pharmacokinetic analysis for ( . rif is also accumulated in the lung, but to a much lower degree than gam (elf/c h : . ± . ; balc/c h : . ± . ). conclusion: pharmacokinetic data of the present study provides surprising results. in previous studies coadministration of clarithromycin and rif show a dramatic decreased plasma exposure of the macrolide, whereas the balc/c h -ratio was unaffected (peters et al. ) . in contrast, systemic exposure of gam increase significantly in case of the combined therapy and the balc/c h -ratio was nearly halved. both macrolides have in common, that they are intensively accumulated in the lung (elf << balc). at the moment there is further research required (e.g. in vitro studies) for a better understanding of the very interesting in vivo data. institut für klinische pharmakologie, göttingen, germany background and aim: desvenlafaxine is a selective serotonin and norepinephrin reuptake inhibitor, which is approved in the usa (but not in europe) for treatment of major depressive disorder. desvenlafaxine is the major active metabolite of the antidepressant venlafaxine. desvenlafaxin is produced by o-desmethylation via cyp d . direct administration of desvenlafaxine should bypass the variability in venlafaxine pharmacokinetics caused by the highly polymorphic cyp d . however, desvenlafaxine is less lipophilic than venlafaxine and may require carrier-mediated transport to penetrate cell membranes. based on our in vitro data, desvenlafaxine is a substrate of the hepatic organic cation transporter oct and common genetic polymorphisms abolished desvenlafaxine cellular uptake. about % of caucasians are compound heterozygous carriers of loss-of-function oct polymorphisms. therefore, oct polymorphisms may cause substantial inter-individual variability in the hepatic uptake and plasma concentrations of desvenlafaxine. in this study we evaluated the influence of genetically-determined loss of oct function on the pharmacokinetics and pharmacodynamics of desvenlafaxine. primary aim was dependence of desvenlafaxine plasma concentrations (represented by auc as primary endpoint) on the number of active oct alleles. methods: mg desvenlafaxine (pristiq®) was orally administered to healthy subjects preselected according to their oct genotypes. oct * allele was regarded full active, oct * to * alleles were regarded loss of function. plasma concentrations of desvenlafaxine and its main metabolite didesmethylvenlafaxine were quantified in plasma sampled up to hours after administration using lc-ms/ms. pupillographic measurements were performed as possible surrogate markers for desvenlafaxine pharmacodynamics. results out of the subjects carried two active, one active, and zero active oct alleles. age, height and weight were . ± . years (mean ± standard deviation), . ± . m and . ± . kg with no significant differences among the oct genotypes. there were strong variations in the pharmacokinetics of desvenlafaxine and its metabolite didesmethylvenlafaxine. the auc -infinity of desvenlafaxine varied between . and . min*mg/l and auc -infinity of didesmethylvenlafaxine between . and . mg*min/l. however, neither desvenlafaxine nor didesmethylvenlafaxine pharmacokinetics significantly differed among the three oct genotypes. concerning pharmacodynamics of desvenlafaxine, pupil diameters at maximal constriction after a standardized light exposure were on average % greater around the time of t max than before administration. in line with the pharmacokinetic results there were no significant differences in maximal constriction or other pupillographic parameters among the oct genotype groups. conclusions: our results suggest that oct genotype does not affect the pharmacokinetics of desvenlafaxine and therefore no dose adjustment in respect to oct genotype should be considered. other factors like renal transporters or polymorphic glucuronidation may explain the great variability in desvenlafaxine pharmacokinetics. background: cardiovascular disorders and medication are highly prevalent in elderly ( ). due to age related changes in the body, the elderly are particularly vulnerable to side effects and adverse drug reactions. some psychotropic drugs are linked with reports of cardiac side effects. additionally, some cardiac drugs may also cause psychiatric symptoms. of these, angiotensin converting enzyme inhibitors, beta blockers, methyldopa and calcium channel antagonists can induce or exacerbate symptoms of depression ( ) . the aim of this study was to provide information on the concomitant use of cardiovascular drugs among elderly patients who took psychotropic medication. methods: we conducted a single-center, retrospective study between september and december using the medical records of elderly patients (≥ years of age) admitted to basin sitesi polyclinic, izmir ataturk research hospital, turkey. demographic characteristics of patients, diagnoses, prescription drugs were evaluated, and spss . statistical software was used for data analysis. number, percent, mean and standard deviation were used as descriptive statistics. results: a total of elderly patients with psychiatric disorders were identified. one in four patients receiving psychotropic medication took at least one cardiovascular agent concomitantly (n= ). median age was (min: , max: ), patients were female ( . %). according to medical records of patients, the most commonly used drugs were escitalopram, sertraline, mirtazapine, quetiapine, mianserin and risperidone. the proportion of the concomitantly use of cardiovascular drugs was higher among the patients who took more than one psychotropic drug ( . % vs. % . ) compared to patients taking psychotropic monotherapy. a higher percentage of women used diuretics ( . % vs. . %) and angiotensin receptor blockers ( . % vs. . %) concomitantly with psychotropic drugs when compared to men. the proportion of men using angiotensin-converting enzyme inhibitors and lipid-modifying agents was higher than women ( . % vs. %, . % vs. . %, respectively). conclusions: the world's population is ageing rapidly. according to world health organization, over % of elderly suffer from a psychiatric or neurological disorder. our data showed that use of cardiovascular drugs among elderly patients with psychiatric disorders was extensive. the effects and interactions of these drugs should be discussed and carefully evaluated before starting treatment in the elderly. further studies focusing on drug use in elderly will increase the success in geriatric pharmacotherapy. since the adoption of the ich e guideline [ ], the thorough qt (tqt) study has become a standard element of clinical drug development. however, with the iq/csrc study [ ] the ability to detect qtc-prolongations of about ms in a phase i setting has been demonstrated. as a consequence, regulatory agencies have begun to grant waivers for a tqt study based on negative qt findings obtained from first-in-man studies. a concentration-response model is the key tool that gives sufficient power to an analysis based on data from single or multiple ascending dose studies. this power has been investigated in subsampling studies that simulate situations comparable to those encountered in first-in-man studies [ , ] . other topics to be addressed are the assurance of sufficient quality of the ecgs obtained, in particular in doses that cause adverse reactions, and a replacement for the active control that is part of a tqt study. if a model based statistical analysis is used for confirmatory inference, it must be specified in advance. this pre-specification includes tests to ascertain that the model assumptions are met and alternative methods to be used in case they are violated. in particular linearity and the absence of a hysteresis, i.e. a delay between the drug concentration and the observed qt effect need to be tested. this is an area of active research. in this contribution, i will share current experience from a statistical perspective, both based on real data and on simulated studies. i will also discuss critical points in the design of first-in-man studies that are intended to be used to obtain a waiver for a tqt study. human platelets express the g-protein-coupled angiotensin receptor-like- (apj) receptor. apj is activated by apelin, which is produced as pre-apelin and cleaved into several bioactive peptides such as apelin- , - and - . apelin and apj are expressed in a variety of tissues such as the heart, the vessel wall, several tumor types, and in platelets. to date, there is no description or a suggested function of the apelin/apj system in platelets to date. here, we investigate apj expression and function in human platelets. apelin and apj expression were determined in platelet-rich plasma from healthy donors by immunofluorescence, western blotting and flow cytometry. in a pilot study apelin and platelet apj expression were analyzed in patients with nstemi, stemi patients and controls. here, platelet aggregation was analyzed by light transmission aggregometry (lta); platelet cd and apj expression by flow cytometry and circulating plasma apelin by elisa. in resting human platelets, apj receptor expression was observed predominantly in the outer cell membrane, as determined by immunofluorescence staining and flow cytometry. activation with a selective thrombin receptor-activating peptide (ap ) resulted in decreased apj protein levels determined by western blotting in platelet lysates compared to untreated controls. preincubation of platelets with different apelin isoforms for sec to min reduced platelet aggregation in lta studies by up to % for apelin- . this effect was inhibited by preincubation of the platelets with the enos inhibitor l-name ( µm), suggesting the involvement of a no-dependent mechanism. in patients with myocardial infarction the expression of platelet apj was significantly reduced compared to the control group ( , % ± , % in mi versus % ± , %in controls; p = . ). this reduction in apj expression on platelets was accompanied by decreased plasma levels of apelin- in patients with mi ( . ± . pg/ml versus . ± . pg/ml; p = . ). interestingly, the decreased apj expression on platelets in mi patients significantly correlated inversely with the troponin t plasma levels (r = - . ; p = . ). this may suggest an association of apj expression with lower plasma levels of troponin t and possibly tissue damage. in conclusion, our study shows for the first time the expression of apj and a possible function in human platelets. apj may act as an endogenous inhibitor of platelet aggregation in response to certain apelin isoforms, predominantly apelin- . upon platelet activation, apj is internalized and surface expression is reduced by about %. in mi patients, plasma levels of apelin- and platelet apj expression were reduced. this correlated inversely with troponin t levels. reduced circulating apelin- levels and platelet apj expression may be associated or partly account for platelet hyperactivity in mi patients. anticholinergic drug use, m receptor affinity and dementia risk-a pharmacoepidemiological analysis using claims data f. thome background: dementia is characterized by cumulative cognitive decline and progressive inability for independent living. the lack of suitable therapies for terminating the progression of this disease underlines the importance of the detection of risk factors. anticholinergic drugs have been shown to enhance cognitive decline in the elderly. the classification of anticholinergic drugs according to their anticholinergic burden, however, is inconsistent. since cholinergic transmission is mainly mediated by the m muscarinic acetylcholine receptor in the brain, we classified anticholinergic drugs from anticholinergic risk lists according to their affinity for the m receptor subtype and calculated the risk for the onset of incident dementia. methods: our analyses are based on claims data of the public health insurance fund aok. data include information of inpatient and outpatient diagnoses and treatment from to . inclusion criteria comprised the initial absence of dementia and age of years or older in . anticholinergic drugs were taken from three anticholinergic risk lists. the pdsp-database and literature search were used to define k i -values for the substances. hazard ratios were calculated using time-dependent cox regression including covariates like age, gender, and several comorbidities. results and conclusion: anticholinergic drug exposure increases the risk for dementia. we found that anticholinergics with small k i -values are at a higher risk than those with greater k i -values. furthermore, conventional risk factors for dementia (e.g. age, depression, stroke) could be confirmed. in conclusion, the intake of anticholinergic drugs increases the risk for incident dementia in the elderly. taking into account the m receptor affinity may be a contribution in determining the anticholinergic load in view of the risk for incident dementia. safety signal detection in a large german statutory health insurance databasefirst results of a feasibility assessment f. andersohn , , s. schmiedl , , k. janhsen , , p. thuermann , , j. walker background: during the last years, approaches to routinely screen health care databases based on electronic medical records or claims to identify drug safety signals were proposed. to evaluate the performance of such methods, reference sets of index drugs have been compiled consisting of ( ) drugs with a known association to a certain adverse event (=positive controls) and ( ) drugs without any evidence to cause this adverse event (=negative controls). the best possible signal detection method would identify a safety signal for % of the positive control drugs, and for none of the negative controls. ryan et al. developed drug reference sets for four adverse events of interest (acute myocardial infarction=ami, acute kidney injury =aki, acute liver injury=ali, and upper gastrointestinal bleeding=ugib) and have shown feasibility of using these reference sets in us health care databases. if the use of these us specific drug lists for evaluation of signal detection methods is also feasible within german health care databases, is unknown. aims: to evaluate if the drug reference sets developed by ryan et al. (drug saf. ; suppl :s - ) could be used for testing signal detection methods in a large german statutory health insurance database. methods: data source was the health risk institute (hri) database, an anonymized healthcare database with longitudinal health insurance data from approximately six million germans. new users (initiators) of index drugs in to were identified and followed-up for one year from their first prescription. exposed person-time to the respective index drug was assessed to estimate for which of these drugs an increase in risk of % (relative risk . ) compared to the background incidence of the respective adverse event (ami, aki, ali or ugib) could be identified with % statistical power. results: from a total of index drugs in the reference sets of ryan et al., ( . %) were also available on the german drug market and were used by at least one insurant in the hri database during the study period. a total of , , index drug initiators were included in the analysis. for a total of index drugs, a relative risk of . could be detected with % power. the numbers of index drugs for each of the outcomes of interest were: ami ( positive controls; negative controls); aki ( positive controls; negative control); ugib ( positive controls; negative control). as the background incidence of ali was low, no positive or negative control with sufficient power was identified for this outcome. conclusions: using the set of reference drugs proposed by ryan et al., the number of drug-event pairs with % power to detect a relative risk of . was low, despite the magnitude of the database used. this may be attributable to differences of drug exposure in germany and the us. hence, an adaptation of the drug list to the german drug market and consumption data might be relevant for future evaluations of signal detection methods using german databases. correlation of sativex™ doses to steady state concentrations of -nor- -carboxy- administration of the oromucosal spray sativex™ represents a therapy option for treatment of spasticity in patients with multiple sclerosis. sativex™ is an extract containing equal amounts of the cannabis-derived cannabinoids ∆ tetrahydrocannabinol (thc) and cannabidiol (cbd). in cases of cannabis abuse a long elimination half life of some thc metabolites is known. therefore, in patients receiving sativex™, the long elimination half life of these metabolites should allow a drug monitoring under conditions of steady state. due to the fact that immunologically based methods for thc determination are very common in medical chemistry, a monitoring might be simply performed even in patients under sativex™ therapy. in a preliminary observational study -nor- -carboxy-∆ -thc (thc-cooh) concentrations were measured with a commercial immunoassay in urine samples of patients with multiple sclerosis obtaining sativex™. in addition, thc-cooh, thc, cbd as well as the hydroxy metabolites of thc and cbd were measured by gc/ms in urine and blood samples. using this analytical technique, only an excessive dosing (as compared to the declaration by the patient) can be detected. as a result of this approach, thc-cooh concentrations determined by the immunoassay were found not to correlate to the daily applicated amount of sativex™ as indicated by the patients (spearman rang order test: p > . ). two patients mentioned not to have taken sativex™ on the day the samples had been taken, and one patient predicated an additional cannabis abuse. in three patients the immunological thc-cooh determination was negative or nearly negative. interpretation of the data is hampered by the fact that an incorrect declaration of sativex™ applications by the patients cannot be excluded. introduction: learning analytics seeks to enhance the learning process through systematic measurement and analysis of learning related data to provide informative feedback for students and lecturers. however, which parameters have the best predictive power for academic performance remains to be elucidated. objective: to analyze the potential of different learning analytics parameters to predict exam performance in undergraduate medical education of pharmacology. methods and results: hypertext preprocessor (php) as server-side scripting language was used to develop a learning analytics platform linked to a my structured query language (mysql) database for storage and analysis of data (www.tumanalytics.de). the database consisted of lecturer-authored multiple choice questions that were made available to a cohort of undergraduate medical students enrolled in a pharmacology course (winter term / ) at technische universität münchen (tum). the course consisted of a -day teaching period, followed by a -day self-study period and a final written exam. students' assessment data of tumanalytics was collected during the self-study period and correlated to the individual exam results in a pseudonymized manner. a total of out of ( %) students participated in the study. the coefficient of multiple correlation (r) was calculated for different parameters in relation to exam results as a measure of predictive power. of different parameters investigated, the total score and the score of the first attempt in tumanalytics had the highest positive correlation with exam performance (abb. ). no sex-specific differences were observed. summary and conclusion: in this study we systematically investigated the potential of different learning analytics parameters to predict learning outcome and exam performance. total score and score of the first attempt were identified as parameters with the highest predictive power. in conclusion, our study underscores the potential of learning analytics as valuable feedback source in undergraduate medical education of pharmacology. in educational settings, tests (e.g., written or oral exams) are usually considered devices of assessment. however, a recent and intriguing line of evidence from basic cognitive psychological research suggests that tests may not only help to assess what students know, but may also help to improve the learning and long-term retention of information. the goal of the present study was to apply such test-enhanced learning to pharmacological teaching. after the last lecture of a pharmacology class (n= rd -year medical students, n= th -year medical students: basic or clinical pharmacology, respectively), one week prior to the final exam, students were given the opportunity to voluntarily participate in online exam. because pilot work from previous semesters had revealed relatively low levels of participation in such formative exams, students were offered bonus points for (successful) participation as an incentive. the online exam consisted of items (i.e., selected pieces of information from the lectures and seminars) and was provided on the e-learning platform ilias. twenty of the items were presented as statements for restudy, items were tested using single-choice questions, and items were tested using short-answer questions. randomly a third of the students were assigned to different sets of questions. the summative final written exam for each group consisted of single-choice questions, questions of which had not been used before (as a standard of comparison). the remaining questions of the final exam were taken from the previous online exam, but were slightly reworded to avoid ceiling effects. each of of these reworded questions from the final exam corresponded to restudy items, single-choice items, and short-answer items from the online exam, respectively. the main points of interest were (i) whether the re-processing (rewording and asking for transfer of knowledge) of information in the online exam affected participants' performance on the final exam, and (ii) whether any effect depended on the specific type of re-processing (restudy vs. single-choice test vs. shortanswer test). if previous findings from basic cognitive psychological research on testenhanced learning can be generalized to more applied settings and educationally more relevant materials such as pharmacological information, students' performance in the final exam should be better for questions corresponding to previously tested items than for questions corresponding to previously restudied items. moreover, if more difficult tests lead to more test-enhanced learning than less difficult tests, as is suggested by recent findings from cognitive psychological research, performance for questions corresponding to (supposedly more difficult) short-answer items should even exceed that for questions corresponding to (supposedly less difficult) single-choice items. the present findings bear direct implications for educational practice. safe and rational prescribing is one of future physicians' key skills [ ] . in order to address the persisting prescribing deficiencies [ ] , we set out to develop a learning tool for pharmacotherapies of the most important diseases worldwide. the format, scope, information architecture, and functionalities of the app were identified through assessment of existing apps, literature analysis, app simulation-based student surveys, and expert advice. a fully functional offline app format for smartphones was selected based on the trends in using digital technologies for educational purposes [ ] and on the unreliable internet and power availability in many learning settings. a relational database based on semantic relationships was chosen to minimize information redundancy and to enable the retrieval of drug-related information in the context of mechanisms, contra-and indications, adverse drug reactions, interactions, and common prescribing situations. the usability was optimized using a simulation of the app evaluated by medical students from germany and tanzania, and by experts. a list of indications was assembled beginning with disease burden data for the seven who world bank regions. each disease accounts for at least . % of life years lost due to premature death or lived with ill-health or disability (daly) in at least one region. the list was further complemented according to expert recommendations. therapeutic recommendations are based on current guidelines, considering cheaper treatment alternatives provided in the who list of essential medicines. a novel dual-scale classification system lists drug mechanisms according to the affected physiological process and to the resulting therapeutic effect [ ] . contraindications, adverse drug reactions, and interactions were compiled using drug monographs of the european medical agency, the us food and drug administration, and health canada. unexpectedly, we found significant differences among these sources in respect of adverse drug reactions. this necessitated the ongoing verification through surveying general practitioners and specialists in internal medicine. during the dgpt meeting we will present the results of testing of the cardiovascular section comprising indications, drugs representing mechanisms, and up to adverse drug reactions. the european certified pharmacologists (eucp) programme was lauched in july by the federation of european pharmacological societies with the intention to identify experts in the field of pharmacology whose competency profile, in addition to their personal specialised scientific expertise, covers expert knowledge in all major fields of the discipline. seventeen ephar member societies have declared their active participation in the eucp programme so far (austria, croatia, czech republic, finland, france, germany, greece, hungary, italy, the netherlands, norway, poland, portugal, serbia, slovenia, spain, turkey) . eacpt, the european association of clinical pharmacology and therapeutics, has also recently decided to participate in the eucp programme. national programmes must meet all requirements of the eucp guidelines including a clear catalogue of criteria with respect to knowledge, practical awareness and skills, as well as general rules including rules for final assessment of candidates. such programmes may be based on existing diplomas or training schemes or may consist of a set of rules how applicants may submit credentials for their expertise with respect to the eucp criteria. so far, three ephar member societies have submitted a national eucp program: austria, italy and the netherlands. the programmes differ in structure and reflect the flexibility of the eucp programme with respect to the respective national conditions. while the italian programme is based on a catalogue of criteria where applicants have to certify and document their expertise on the basis of this catalogue and the dutch program is based on a structured phd training course, the eucp scheme submitted by the austrian pharmacological society aphar is based on the legally regulated diploma medical specialist (facharzt) in pharmacology and toxicology (aphar also plans to submit separate regulations for specialists in clinical pharmacology and for nonmedically qualified pharmacologists). the aphar eucp scheme has been approved by the eucp committee in november and its regulations are available on the eucp website (www.eucp-certification.org). the differently structured programs of the italian and dutch pharmacological societies will also be available at this website, once approved by the eucp committee and may thus serve as 'case studies' for other ephar and eacpt member societies wishing to take part in the eucp programme. introduction: the outstanding importance of pharmacovigilance (pv) for the safe use of medicines has increasingly been recognised during recent years. the multidisciplinary character of pv requires know-how in topics as different as pharmacology, clinical medicine, pharmacoepidemiology, information technology, pharmaceutical manufacturing, legal aspects, public health policies, and medical traditions in different regions of the world. in this complex situation there is a growing need for pv capacity building, in particular by professional training through high quality pv courses with different focuses and different levels of detailing. against this background, the world health organization (who) and the international society of pharmacovigilance (isop) have co-operated to create a curriculum for teaching pv which can be used for a wide range of audiences and in very different settings and situations. the purpose was to provide an inventory and systematically structured overview of pv including recent developments of topics like pharmacogenomics, consumer reporting of adrs, risk management and who-led international projects, and to propose a range of tasks for practical training. we made use of several relevant already existing packages of pv topics and concepts of pv teaching from national and international institutions. we also drew from extensive printed material as well as comprehensive reviews, textbooks and guidelines developed by international organisations which are often available online. results: the curriculum includes a main component consisting of a major part for theoretical lecture-based training and a minor component with suggestions for hands-on exercises. the theoretical part has a three-level hierarchical and modular structure with evenly divided tiers. there are chapters. each of them is divided into four sections and each section into four to six sub-sections. the practical part consists of twelve times three or four proposals for practical tasks which are related to the theoretical lectures. since its launch in it has successfully been used in several international courses. currently a pilot project is under way to explore its use for 'crowd sourcing': it is placed on the isop homepage with a programme allowing for institutions or persons experienced in pv teaching to upload any relevant presentations they may have with a link to related chapters, sections or subsections. these presentations will be offered for downloading by interested users. the curriculum provides a comprehensive coverage of almost all areas of pv. the structure and content allows almost every kind of focusing on specific issues and going into depth, while maintaining the overall context. it offers opportunities of tailoring courses specifically to the needs of different audiences and can be applied to various forms of training, such as broad, comprehensive and intensive courses, short overviews or focuses on specific narrow topics in perspective. according to the reach regulation (ec) no. / chemicals produced, marketed or used within the european union have to undergo a registration process, wherein the registrants have to provide information on hazard and potential risks presented by the substances. however, the standard information requirements defined in annexes vii to x of the regulation might be waived or adapted by the registrants if adequate documentation and justification according to criteria specified in annexes vii to xi are provided. to evaluate the data availability in registration dossiers of high tonnage substances (above tpa) and their compliance with the reach regulation, the federal institute for risk assessment (bfr) in cooperation with the federal environment agency (uba) developed a systematic web-based scheme. in total, dossiers were checked for selected human health and environmental endpoints such as repeated dose toxicity, genetic toxicity and ecotoxicity. a remarkable high rate, % to % depending on the endpoint, of the evaluated dossiers included waiving or adaptations from the standard information requirements. therefore, those dossiers were not concluded, but categorised as 'complex' (springer et al., ) . the use of waiving and adaptations in 'complex' endpoints were part of a follow-up project. herein, it was evaluated whether the given justifications were in accordance with the criteria set out in the respective reach annexes. the results will show the frequency and pattern of waiving/adaptation approaches for the human health as well as the environmental endpoints. besides this general overview, specific problems regarding the application of the reach regulation were identified and their significance with regard to remaining data gaps will be discussed. the german commission for the investigation of health hazards of chemical compounds in the work area has re-evaluated dimethylformamide, and classified it in the carcinogen category . this category is for chemicals with carcinogenic potential for which a non-genotoxic mode of action is of prime importance and genotoxic effects play no or at most a minor part provided the mak and bat values are observed. under these conditions no contribution to human cancer risk is expected. dmf was identified as a substance of very high concern by european commission. the amount of dmf manufactured and/or imported into the eu is, in the range of - t/y. n,n-dimethylformamide is a hepatotoxin in humans and rats. the carcinogenicity studies in both mouse and rat were conducted with test material of an acceptable purity and physical form. the critical study involved administration of dmf via inhalation, which is relevant to human exposure. there is conclusive evidence that dmf induces significant increases of hepatocellular carcinomas in rats after exposure to ml/m³ and in mice in response to ml/m³ and higher. several in vitro and in vivo studies have indicated that dmf is not genotoxic. the results of the long-term studies reveal that the tumors develop in the liver only after chronic toxic inflammatory and degenerative changes have developed in this organ. the commission concluded that the tumors are a result of chronic liver damage, occurring at high exposure concentrations. the available evidence therefore suggests that there is a threshold dose for the carcinogenic effects of dmf. accordingly, dmf was classified in carcinogen category with a mak-value of ml/m³, an exposure concentration which does not induces liver toxicity and as a consequence is not associated with an increased cancer risk. today, a large majority of people is constantly exposed to electromagnetic radiation. many studies have been performed to investigate whether this type of radiation has a potential to affect biological systems at low intensity levels. even though no complete consensus has been reached so far in this issue, most of the investigations do not indicate a harmful potential of this radiation. two questions remain open until today, i. e. long-term effects and specific effects on children. it has been demonstrated that in comparison to adults, children absorb far higher doses of mobile phone radiation in the skull, particularly in the bone marrow, where hematopoiesis takes place. these absorptions occasionally exceed the recommended safety limits. the aim of this study was to elucidate, whether cells of the hematopoietic system can be affected by different forms of mobile phone radiation. as biological systems, two cell types were investigated, hl- cells as an established cell line, and human hematopoietic stem cells. the radiation was modulated according to the two major technologies, gsm ( mhz) and umts ( mhz). additionally, lte ( . mhz) modulation was applied because this technology is used worldwide already but has not been studied sufficiently. cells were exposed for a short and a long period and with different intensities ranging from to w/kg. studied endpoints included oxidative stress, differentiation, dna repair, cell cycle, dna damage, histone acetylation, and apoptosis. appropriate negative and positive controls were included and three independent replicate experiments were performed. exposure to radiofrequency radiation did not induce any alterations of cell functions, measured as oxidative stress and cell cycle. cell death in the form of apoptosis was not observed. primary dna damage was not induced and dna repair capacity for nucleotide excision repair was not changed. epigenetic effects (measured as histone acetylation) were not observed. finally, differentiation was not affected. the effect of treatment with various chemicals as positive controls was different in the two cell types. all in all, mobile phone radiation did not induce effects on human hematopoietic cells. in who published guidance on evaluating and expressing uncertainties in human health hazard characterisation (hc). in this approach, the outcome of hc is expressed as an interval or distribution rather than a "traditional" deterministic point estimate, such as a reference dose (rfd), thereby communicating potential uncertainties more clearly. risk management protection goals, such as the acceptable magnitude of effect (m) and incidence (i) in the population, are made explicit quantitatively along with the confidence with which they are achieved, e.g. by an rfd. specifically, the goal of this approach to hc is to estimate the "hd m i" , i.e. the "true" human dose associated with m and i (e.g. body weight decreased by ≥ % (m) in % (i) of the population). if uncertainties are expressed by providing estimates of the hd m i as confidence intervals or uncertainty distributions, both the "degree of uncertainty" (ratio of upper and lower limit of the interval/relevant distribution segment) and the "coverage" (statistical confidence) associated with a given rfd value can be characterised. alternatively, one may start from a chosen coverage and calculate the associated "probabilistic rfd". uncertainty in each hc aspect, e.g. inter-/intraspecies or time extrapolation, can be characterised by a "generic default uncertainty distribution" which has been derived from historical data, but may be replaced by a substance-or effect-specific distribution (analogous to a chemical-specific adjustment factor in the "traditional" deterministic approach), where available. the uncertainty distributions for the individual hc aspects can then be combined into an overall uncertainty interval/distribution in ( ) a simple non-probabilistic way, ( ) a more refined "approximate probabilistic analysis" (aproba, a free spreadsheet tool for easy implementation also by non-statisticians is available from the who website), or ( ) a fully probabilistic monte carlo analysis. the hc aspects contributing most to overall uncertainty are also identified and may be prioritised for refinement in a next assessment tier. the who approach uses a tiered strategy which may start with evaluating the uncertainties in the outcome of a "traditional" deterministic hc. in this way it represents a unified framework integrating deterministic and probabilistic hc methodologies. moreover, the concept can be easily combined with exposure uncertainty assessment. risk managers may use the additional information in better weighing potential health effects against other interests. when they consider the overall uncertainty larger than desirable in view of the problem formulation, they may decide to ask for a more refined (higher tier) assessment. if all possibilities for refinement are exhausted, the new approach can also aid in the selection of new data which might need to be generated. due to a constant improvement in analytical methods an increasing number of substances are found in drinking water. the joint project toxbox aimed for the development of a reliable test battery, allowing for a rapid evaluation of single substances in water. eleven partners either from the research (nine) or the business sector ( ) formed the project. the attention was focused on genotoxic, neurotoxic and endocrine effects, which are considered to be of most concern to the consumer. by the end of the project a set of guidelines is published that describes the analytical methods in detail. the project was based on a theoretical concept, called "health-related indicator value" (hriv), which was developed by the german federal environment agency (uba) for the assessment of substances with incomplete toxicological data. depending on the type of effect a hriv between . and . µg/laws derived for the substance which had to be evaluated. during the years an increasing amount of substances as well as an increase in finds was observed in drinking water. this called for the creation of an evaluation scheme that offers rapid and at the same time reliable evaluations of chemicals for which there are no data available. the concept is in accordance with tox , which envisages the trustworthy evaluation of relevant endpoints by two or three in vitro assays. in the context of toxbox this was provided for the endpoints genotoxicity, neurotoxicity and endocrine effects. in all cases a hierarchic strategy is applied that enables a first assessment via relatively simple test assays and only when these test give a hint towards an effective more elaborate techniques are applied for a final assessment. the ames test and micronucleus assay in combination with the umu test will form the panel for genotoxicity testing. neurotoxicity will be assessed by comparing necrotic and apoptotic effects as well as the development of reactive oxygen species in human nervous cell with human liver cells. additionally neuron specific assay like the neurite outgrowth test are performed. this is complemented by measuring the activity of acetyl choline esterase activity and the development of the side line organ in zebra fish (danio rerio). the test battery for endocrine effects consists of hormone specific reporter gene s assays in addition to the h r assay. when necessary a reproduction assay in the mud snail potamopyrgus antipodarum is carried out. during the project some substances were evaluated. this allowed for the development of a reliable test strategy. currently the guidelines for performing the required tests are in the making. metabolomics has gained increasing interest over the last years with numerous possible applications ranging from strain optimization for industrial production over drug discovery to improved toxicity testing. however, regulatory acceptance of this promising approach is still not reached, mostly because standardization and evaluation of reproducibility are still mostly lacking. the metamap®tox database has been developed by basf over the last ten years containing toxicity and metabolome profiles of more than different compounds. to ensure maximum reliability, data was gained from plasma samples of highly standardized week rat studies. animal maintenance and treatment, sampling and work-up of plasma, measurement of the metabolome as well as data interpretation and storage were standardized including thorough documentation, the compliance with sops and safe data storage. data from more than control groups with each males and females were analyzed to assess variability. an in depth analysis of this showed a high stability and robustness of the metabolome over a period of ten years. after artificially splitting the groups of control groups into groups of five animals and comparing the number of statistically significantly regulated (false positive) metabolites, the peak of the distribution curve was located to the left of the exact (gaussian) center, but tailed off to the right more than expected under the normal distribution. from this analysis we were able to calculate density distributions (relative ratio and standard deviation) for the control values of each metabolite, which can serve as a historical control displaying the range of changes which can be expected as normal. during the course of our project we have used more than ten exact repeats to show reproducibility and reliability of the metabolome analysis (kamp et al., ) . comparing these exact repeats at different levels of statistical significance, we noted that at a level of statistical significance of approximately p = . , the best balance between matches (metabolites regulated in the same direction) and mismatches (metabolites regulated in opposite directions) was obtained. the high quality standards applied as well as the examination of control data increase the robustness of this approach, going also hand in hand with improved data quality. this significantly facilitates decision making based on the gained data. due to these improvements a new level of transparency is reached, which might allow inclusion of metabolome data in a regulatory environment. hydroxycitric acid (hca) is a fruit acid naturally occurring in fruits of the tropical plant garcinia cambogia. a number of dietary supplements intended for weight loss contain hca, which is added in form of g. cambogia extracts. the composition of these extracts is often not clearly specified. health concerns about safety of the hca-containing supplements have been raised, based on results from animal studies, which observed toxic effects on the testis and on spermatogenesis after administration of preparations containing high hca doses. in the current risk assessment, the possible health risks associated with consumption of hca-containing dietary supplements (hca doses of approximately to mg per day) were evaluated based on relevant animal and human studies with the focus on testicular toxicity as a critical endpoint. in several published animal studies, repeated (short-term or subchronic) ingestion of certain hca-preparations (g. cambogia-extract or ca + -hca salt) induced testicular atrophy (i. e. atrophy of seminiferous tubules, degenerative changes of sertoli cells at histological examination) and impaired spermatogenesis (i. e. decreased sperm counts) in male rats at high doses (noael and loael of and mg hca/kg body weight & day, respectively). animal studies with other hca-preparations (ca +/ k + -hca salt) found no such effects at the highest hca-doses tested (noael: , mg hca/kg bwt & day). human intervention studies which addressed the safety of hca in healthy test persons reported no substancespecific adverse effects after ingestion of hca doses up to mg per day over the period of up to weeks. however, the question of possible adverse effects of hca on the human testes was not adequately addressed in studies with human volunteers. in a single clinical study with male test subjects, no significant changes in endocrinologically relevant parameters such as serum inhibin b or fsh were observed after consumption of mg of hca for weeks. however, no investigations of direct parameters that might inform on potential effects on spermatogenesis, such as sperm quality and sperm count, were conducted in this study. considering the serious adverse effects on the testes observed in several animal studies as well as in view of lack of the adequate human data on the safety of the long-term use of hca-preparations, it is concluded that knowledge gaps and substantial uncertainties exist regarding the safety with respect to human health of high amounts of hca found in commercially available food supplements, particularly with regard to the human male reproductive system. a critical look on the passing-bablok-regression b. mayer universität ulm, institut für epidemiologie und medizinische biometrie, ulm, germany background: the passing-bablok (pb)-regression is a commonly used approach to prove the equality of different analytical methods when studying quantitative laboratory data. it is based on the assumption that the measurements of two methods are linearly related. if then one method is regressed onto the other and the respective confidence intervals of the intercept and the slope include and , respectively, it is assumed to have a proof of methods equality. however, this conclusion is problematic in respect of an essential principle of statistical hypothesis testing. methods: in this talk the general idea behind the pb-regression is discussed critically. although the method makes use of confidence intervals a decision is made, which is why it is important to discuss how the results of a statistical hypothesis test have to be interpreted. moreover, alternative statistical approaches to investigate agreement in biometrical practice are pointed out by means of a practical example and their advantages and limitations are addressed. results: all approaches applied to a sample data set led to the same conclusions. demonstrating methods equality though necessitates an a-priori definition of an appropriate equivalence margin. conclusion: the pb-regression may give useful advice when comparing two measurement methods towards equality. however, its results are statistically inconclusive, since the pb-method does not follow the principle of equivalence testing. alternative measures of agreement should be applied instead to ensure results which are not attackable and serve as a statistical proof. insulin is an important parameter both in toxicology (toxicity to the endocrine pancreas) and pharmacology (models of diabetes and metabolic syndrome). currently available elisa and ria methodologies for insulin often require up to µl plasma or serum for a single measurement. in order to meet the general trend to include more relevant parameters in animal studies and restrictions through animal welfare requirements to limit the volume of interim blood draws we explored the rat/mouse insulin singleplex assay of meso scale discovery (msd) as an alternate assay consuming only µl serum or plasma or less for a single measurement. the assay is a sandwich immunoassay, whereby insulin in the sample binds to the capture antibody immobilized on the working electrode surface at the bottom of each well and recruitment of the labeled detection antibody (anti-insulin labeled with electrochemiluminescent compound, msd sulfo-tag™ label) by bound analyte completes the sandwich. voltage applied to the plate electrodes then causes the label bound to the electrode surface to emit light the intensity of which is a quantitative measure of insulin. the msd insulin assay was characterized by a robust calibration and only small variations within repeated measurements. the assay presented a broad dynamic range and differences in insulin levels of normal and rats suffering from metabolic syndrome could readily be demonstrated. furthermore, the high sensitivity may even allow the use of smaller sample volumes. these features render this assay an attractive alternative for the measurement of insulin. the lack of corresponding quality control samples for internal quality control may be considered as a relative drawback. however, the cross reactivity of the assay with human insulin provides the opportunity to use qcs designed for human assays and to possibly participate in ring trials for human insulin for external quality control if needed. surfactants are main constituents of different consumer products, e.g. detergents or cosmetic cleansing products. since surfactants show an intrinsic skin irritation potential, dilutions are used in the final products to avoid adverse effects like irritant contact dermatitis from product use. in addition, mixtures of different surfactants are typically formulated, as it is a long-standing experience that those mixtures exhibit much lower acute irritation potential than expected from the mere summation of their individual irritation potential, an effect coined surfactant antagonism. only few studies were performed to gain a more fundamental understanding of the effect, and it's mechanistic basis remains unclear. however, a thorough understanding of the surfactant antagonism is not only of value for the formulation of products that are considered 'mild to the skin'. it is also important for the classification of products according to the clp regulation in cases when data of the mixtures is missing, because summation of the ingredients' irritating effects usually results in over-classification as skin irritant. due to the progress in the development of alternatives to animal testing, different in vitro methods have become available to determine skin irritating properties of substances. methods like the oecd tg and especially aim at deriving a classification for skin irritation/corrosion effects according to the clp regulation. however, even though these methods became the preferred test methods for skin irritation testing, to our knowledge hitherto isolated investigations on the surfactant antagonism were only performed either by human patch test studies or by non-standard in vitro assays. in this study, the irritation potential of binary mixtures of sodium dodecylsulfate (sds), linear alkylbezene sulfate (las), cocamidopropyl betaine (cabp) and alkylpolyglucosid (apg) compared to the single compounds was investigated using open source reconstructed epidermis (os-rep) models. combinations of sds or las with cabp and apg, respectively, resulted in a clear decrease of the irritation potential compared to the irritation exerted by the single surfactants, even though the total surfactant concentration was higher in the mixtures. in addition, the effect of surfactant antagonism was also observed in a mixture of cabp and apg. the reduced irritation potential of mixed surfactants came along with both reduced skin penetration of fluorescein and reduced release of ldh. since no surfactant antagonism is observed in monolayer cultures of keratinocytes that were exposed to mixtures of surfactants, it is assumed that keratinocytes in the viable parts of the reconstructed epidermis are promptly damaged by the surfactants once the model's barrier is destroyed. hence, surfactant antagonism appears to be primarily driven by the mixture's lower ability to damage the skin model's barrier. the micronucleus (mn) test is a reliable method for the detection of cytogenetic damage in proliferating cells. in recent years, substantial progress has been made on automated, thus faster and more objective scoring of mn test samples, i.e., methods based on flow cytometry. the aim of the present study was to use the adherently growing human bladder cancer cell line rt to carry out a comparison between traditional (fluorescence microscopy) and automated (flow cytometry) mn scoring. for this purpose, different substances which are known to be positive controls were used. rt cells were either continuously incubated for h (approximately . cell cycles duration) with methyl methanesulfonate (mms; - µm), benzo[a]pyrene (b[a]p; . - µm), vincristine ( - nm) , and colcemid ( - nm) or cells were irradiated with xrays ( . - sv) and then cultured for h. for standard mn scoring, cells were harvested, subjected to hypotonic treatment, fixed with methanol/acetic acid, placed on glass slides, stained with acridine orange and observed by fluorescence microscopy. for the flow cytometric method, harvested cells were stained in two sequential steps. intact cells were subjected to ethidium monazide bromide followed by photoactivation ( w, min) to label dead or dying cells. then, cells were lysed and stained with sytox green for a pan-dna labelling and analyzed on a flow cytometer. both, chemically-and radiation-induced treatment led to a dose-dependent induction of mn when evaluated by fluorescence microscopy. when the flow cytometry-based method was applied, clearly positive results including a dose-dependent induction of mn, however, were obtained only for out of the treatments (vincristine, colcemid and x-rays); whereas, treatment with mms and b[a]p led to only minor increases in relative mn frequencies (≤ -fold), even at the maximum concentrations. in summary, flow cytometry-based mn scoring has been successfully applied in rt cells. however, our initial results suggest that flow cytometry-based mn scoring is less sensitive than microscopic scoring when rt cells are used. so far, only few adherently growing cell lines have been applied to flow cytometry-based mn scoring. further substances (positive and negative controls) and possibly other adherent cell lines need to be tested to expand our knowledge on the effectiveness of automated mn scoring in vitro and compared to traditional approaches. background: the platinating agent cisplatin is commonly used in the therapy of various types of solid tumors, especially urogenital cancers. its anticancer efficacy largely depends on the formation of bivalent dna intrastrand crosslinks, which impair dna replication and transcription. these crosslinks stimulate mechanisms of the dna damage response (ddr), thereby triggering checkpoint activation, gene expression and cell death. the clinically most relevant adverse effect associated with cisplatin treatment is nephrotoxicity, which mainly results from damaged tubular cells. here, we analyzed the influence of the hmg-coa-reductase inhibitor lovastatin on the cisplatin-induced geno-and cytotoxicity in the rat renal proximal tubular epithelial cell line nrk- e. methods: cell viability was determined by using the alamar blue assay, as well as by electrical impedance measurements via the icelligence system. alterations in cell cycle progression were assayed by flow cytometric analysis. the formation of pt-(gpg) intrastrand crosslinks was determined via southwestern blot. the amount of dna double-strand breaks (dsbs) was quantified by measuring the level of s phosphorylated h ax (γh ax) via immunocytochemistry as well as by western blot. additionally, neutral and alkaline comet assays were performed to determine the amount of dna single-and dna double-strand breaks. mechanisms of the ddr were analyzed by western blot as well as by quantitative real-time pcr. results: the data show that pretreatment of nrk- e cells with a subtoxic dose of lovastatin reduced the cytotoxicity evoked by high doses of cisplatin by protection from cisplatin-stimulated apoptotic cell death. moreover, lovastatin had extensive inhibitory effects on cisplatin-induced ddr, as reflected on the level of p-atm, p-p , p-chk , p-chk and p-kap . furthermore, activation of mitogen-activated kinases (mapks) was also reduced. the lovastatin-mediated mitigation of cisplatin-induced ddr was independent of the initial formation of dsbs as well as of pt-(gpg) intrastrand crosslinks. lovastatin protects nrk- e cells from cisplatin-induced cytotoxicity by interfering with proapoptotic mechanisms of the ddr independently from initial dna damage formation. with respect to the clinic, the data indicate that lovastatin might be useful to mitigate cisplatin-induced nephrotoxicity. the influence of oxidant tert-butylhydrochinone (tbhq) on endothelial cell migration in wrn-deficient cells k. laarmann , g. fritz institut für toxikologie, düsseldorf, germany introduction: wrn is a dna helicase and possesses a ´- ´exonuclease and atpase activity as well as a single strand annealing activity. it is involved in dna repair, by interacting with proteins of base excision repair (ber) and nucleotide excision repair (ner). defects of wrn are marked by genome instability which, in turn, is caused by defects in dna damage repair. patients with a mutation in the wrn gene show premature aging and early mortality. the latter is mainly caused by arteriosclerosis. furthermore, wrn participates in the regulation of genotoxic stress responses stimulated by reactive oxygen species (ros) and alkylating agents. the aim of this study was (i) to investigate whether endothelial cell migration and adhesion were effected by sub-toxic (ic ) and moderate toxic (ic ) concentrations of the oxidant tertbutylhydrochinone (tbhq) and (ii) whether wrn influences migration and adhesion in the presence or absence of tbhq. methods: endothelial-like ea.hy cells were treated with different concentrations of the redox cycling and thus ros producing oxidant tbhq. viability was measured by the alamar blue assay. ic and ic were determined after h permanent treatment. to investigate the influence of wrn on endothelial cell migration and adhesion, a wrn knock-down was performed in ea.hy cells using rna interference. to measure migration, a confluent cell monolayer was scratched using a pipet tip, h after permanent tbhq treatment. pictures were taken at the time points h, h and h after performing the scratch. the non-closed area was measured. in a second part, adhesion of the calcein-labeled colon adenocarcinoma ht- cell line on the ea.hy monolayer was investigated. wrn-deficient or non-deficient cells were treated with µm and µm tbhq or with tnfα. results: for ea.hy cells, µm and µm tbhq were determined as ic and ic , respectively. performing the migration assay, ea.hy cells showed % gap closure, whereas wrn-deficient cells showed a closure of only % after h. the gap was closed of % and % after µm and µm tbhq treatment. in wrndeficient cells no remarkable effect on migration was observed after µm tbhq treatment, whereas the treatment with µm tbhq showed a slight decrease in migration of about % compared to wrn-deficient cells. no effect on adhesion was observed after tnfα treatment. after µm tbhq treatment a slight increase of adhesion was detected in ea.hy cells. the influence of moderate tbhq concentration on adhesion was reduced in the absence of wrn. conclusion: wrn influences endothelial cell migration. in contrast to wild-type ea.hy cells, no significant effect of tbhq was observed on migration of wrndeficient cells. furthermore, the moderate toxic concentration of tbhq showed slightly increased ht- adhesion to ea.hy , which was not found in wrn-deficient cells. outlook: in forthcoming studies we analyse the effect of alkylating agents on migration and adhesion. data will be presented and discussed. the aim of the present work was to compare the sensitivity of leukemia cell lines (hl , jurkat and tk ) and hematopoietic stem cells with regard to the response to genotoxic agents. chromosomal damage was analyzed by evaluation of the micronucleus frequency. furthermore, changes in the proliferation index and the frequencies of apoptotic and mitotic cells were assessed. several cytostatic drugs with different mechanisms of action were used as genotoxic agents. doxorubicin was used as an intercalator, radical producer and topoisomerase ii inhibitor. also, the effects of vinblastine, a mitosis-inhibiting drug and of methyl methanesulfonate, which forms dna-adducts and stalls replication forks, were analyzed. in general, a difference in sensitivity between the different substances was observed. with regard to the formation of micronuclei after treatment with doxorubicin, jurkat and tk cells showed similar increasing trends, whereas hl cells showed a much higher increase in micronucleus frequency. a clear decrease in proliferation and the frequency of mitotic cells was observed at the highest concentration ( nm doxorubicin) investigated, and only a slight increase in the number of apoptotic cells could be shown. the biggest differences in formation of micronuclei could be detected after treatment with vinblastine. hl cells showed only a slight increase of micronuclei, but the effect on jurkat cells was stronger. the highest micronucleus frequency after vinblastine treatment was detectable for the tk cells. the results for the highest investigated concentrations ( . nm and nm vinblastine) showed a significant reduction of the proliferation index. this effect is reflected by the increasing numbers of apoptotic cells in all cell lines. the results for methyl methanesulfonate demonstrated only a small increase in micronucleus formation for the jurkat cells, but higher values for the tk cells. in contrast the hl cells did not lead to a concentration-dependent effect with methyl methanesulfonate. these results are complemented by preliminary findings in hematopoietic stem cells at selected compound concentrations. the different results between the leukemia cell lines and the stem cells might possibly originate from the different p status of hl (null), jurkat (multiple mutations), tk (wild type) and hematopoietic stem cells (wild type). this difference might also cause differences in cell cycle control or repair mechanisms, and needs further investigations. hyperinsulinemia is thought to enhance cancer risk. a possible mechanism is induction of oxidative stress and dna damage by insulin, here, the effect of a combination of metformin with insulin was investigated in vitro and in vivo. the rational for this were reported antioxidative properties of metformin and the aim to gain further insights into mechanisms responsible for protecting the genome from insulin mediated oxidative stress and damage. comet assay, micronucleus frequency test and a mammalian gene mutation assay were used to evaluate the dna damage produced by insulin alone or in combination with metformin. for analysis of antioxidant activity, oxidative stress and mitochondrial disturbances, the cell-free frap assay, the superoxide-sensitive dye dihydroethidium and the mitochondrial membrane potential-sensitive dye jc- were applied. accumulation of p and pakt were analysed. as an in vivo model, hyperinsulinemic zucker diabetic fatty rats, additionally exposed to insulin during a hyperinsulinemic euglycemic clamp, were treated with metformin. in the rat kidney samples, dhe staining, p and pakt analysis, and quantification of the oxidized dna base -oxodg was performed. metformin did not show intrinsic antioxidant activity in the cell free assay, but protected cultured cells from insulin mediated oxidative stress, dna damage and mutation. treatment of the rats with metformin protected their kidneys from oxidative stress and genomic damage induced by hyperinsulinemia. metformin may protect patients from genomic damage induced by elevated insulin levels. this may support efforts to reduce the elevated cancer risk that is associated with hyperinsulinemia. the human skin is the primary barrier against environmental and chemical impacts. as such it shields us against a plethora of xenobiotics such as potentially carcinogenic polycyclic aromatic hydrocarbons (pahs). at the same time it is the second most densely populated organ, harbouring more than bacterial species and population densities of up to cfu per cm . yet little is known about this microbiome's potential to metabolise and toxify pahs such as benzo[a]pyrene (b[a]p). previous work at the bfr showed that degradation of b[a]p and other pahs is a universal feature of the skin's microbiome (sowada et al., ) . the corresponding metabolites only partly overlap with those known from eukaryotic metabolism and possess cytotoxic as well as genotoxic properties. excretion of these metabolites will lead to exposure times of - hours or longer for full and partial metabolisers, respectively. while in vitro studies show the corresponding substances to exert their effects synergistically, an assessment of their potential impact on human carcinogenesis is pending. one obvious mode of action would be direct genotoxicity. however, another option is interference with uv-damage repair. ultraviolet radiation (uvr) from sunlight is regarded the main causative factor for the induction of skin cancer. it induces two of the most abundant mutagenic and cytotoxic dna lesions, that is cyclobutane-pyrimidine dimers (cpds) and - photoproducts ( - pps). these lesions are repaired primarily by nucleotide excision repair (ner), a system that is also responsible for the removal of pah-derived dna adducts. we therefore wanted to know whether and to what extent bacterial b[a]p metabolites have the capacity to interfere with ner, potentially contributing to uv-induced dna-damage. to investigate this selected genotoxic metabolites were examined for their potential to affect the dna repair capacity of skin cells (hacat). following treatment with uva/b and bacterial b[a]p-metabolites the skin's repair capacity was assessed using a modified comet-assay. ionizing radiation (ir) is a well-established model to induce dna double-strand breaks (dna-dsbs), but it also generates a broad range of other dna lesions including dna single-strand breaks as well as oxidative dna base modifications. furthermore, ir is able to modify membrane components and triggers the activation of epidermal growth factor receptor. a more specific dsb-inducer is cytolethal distending toxin (cdt), which is produced by a variety of gram-negative bacteria and harbours an intrinsic dnase-like endonuclease activity [ ] . dsbs are potent cytotoxic lesions and promote genomic instability, e.g. by formation of chromosomal aberrations. a cellular mechanism to prevent genomic instability and maintain cell homeostasis could be autophagy. this process is highly regulated involving the lysosomal degradation of damaged organelles and proteins. here, we study autophagy induction following dsb generation in human colorectal cancer cells as well as in primary human colonic epithelial cells (hcec) and analyzed regulatory mechanisms. first, the autophagy-specific marker lc b was shown to increase in a dose-and time-dependent manner after treatment with both cdt and ir as assessed by confocal immunofluorescence microscopy and western blot analysis in hct . similar results were obtained in sw and hcec cells via western blot. these findings are in agreement with the enhanced formation of autophagosomes and the dose-dependent decrease of the autophagy substrate p as observed by flow cytometry and western blot analysis in hct , sw and hcec. cdt-and irinduced autophagy rates in hct increased over time correlating well with the dsb induction. importantly, a dnasei-defective mutant of cdt did neither cause dsbs nor induce autophagy. additionally, the time-dependent accumulation of the lysosomal associated membrane protein (lamp- ) was observed by confocal immunofluorescence microscopy. dsb-induced autophagy was blocked by chemical inhibitors. next, we showed that both ir and, to a lesser degree, cdt induce the phosphorylation of akt at ser . pharmacological inhibition of akt in hct cells enhanced the cdt-and ir-induced autophagy shown by accumulation of lc b and lamp after h and increased autophagosome formation. upregulation of dsbinduced autophagy by akt inhibition resulted in a decreased cytotoxicity after h and significantly lower apoptosis/necrosis rates after h, which were determined by mts cell viability assay and annexin-v/pi staining. ongoing studies will evaluate the impact of other dna damage response pathways and the potential protective role of autophagy against genomic instability. mustard agents are potent dna alkylating agents. among them, the bi-functional agent sulfur mustard (sm) was used as a chemical warfare agent due to its vesicant properties. although the use of sm in warfare has been banned in most countries of the world, its use in terroristic attacks or asymmetrical conflicts, such as the syrian civil war, still represents a realistic and significant threat. on the other hand, especially nitrogen mustards, such as cyclophosphamide or melphalan, have been used as chemotherapeutic agents due to their cytostatic properties. thus, mustard-induced dna damage, in particular dna crosslinks, can trigger complex pathological states, as it is observed in sulfur mustard exposed victims, but on the other hand also lead to the chemotherapeutic effects of clinically-used nitrogen mustards. mass spectrometric monitoring and quantitation of mustard-induced dna adducts can help to unambiguously identify and verify sm-exposed victims and to monitor the efficiency, as well as potential side-effects of mustard-based chemotherapy. up to now, the verification of mustard-induced nucleic acid damage is mainly based on immunohistochemical methods, which have several drawbacks such as limited specificity, sensitivity, and low dynamic range of quantitation. with this project, we aim to develop a (hplc/uplc)-ms/ms-based platform for the quantitation of the most common mustard-induced dna adducts including bis(n -guanine-ethyl) sulfide dna crosslinks. up to date, we established methods for the quantitation of the several common dna adducts induced by the mono-functional sulfur-mustard derivative chloroethyl ethyl sulfide ("half mustard", cees). for that reason purification protocols, chromatographic conditions and mass spectrometric settings were developed to detect n -ethylthioethyl- ´desoxyguanosine (n -ete-dg) and n -ethylthioethyl- ´desoxyadenosine (n -ete-da) and their thermal hydrolysis products n ethylthioethyl-guanine (n -ete-gua) and n -ethylthioethyl-adenine (n -ete-ade), respectively, and the sensitivity was compared to immunohistochemical methods. additional non-radioactive isotope-labelled standards are being synthesized, which will be spiked into samples to account for technical variability during sample work-up and to improve ms-based quantitation. this procedure requires minimal cellular material and therefore should be transferred to quantitation of dna adducts in human blood samples. this will allow to monitor dna adducts as biomarkers of exposure in potential smexposed victims as well as in mustard-based chemotherapy. this method also sets a basis to investigate specific mustard-induced dna repair mechanisms and their cellular consequences. the γh ax assay vs. comet assay for genotoxicity testing universitätsmedizin mainz, institut für toxikologie, mainz, germany dna damage leads to activation of the cellular dna damage response (ddr). this signalling network results in activation of various dna repair proteins and chromatin structure modulators. a frequent manifestation of ddr is the phosphorylation of histone ax (gh ax), which can be visualised as gh ax foci by immunocytochemistry. in the present study, we tried to assess if gh ax is a reliable biomarker for detecting the cellular response to dna damage. we selected well-characterised genotoxic compounds and compared them with non-genotoxic chemicals in the wellcharacterised cho cell system. we measured quantitatively γh ax by manual and automatic scoring of γh ax foci, and by flow cytometry counting of γh ax positive cells. the cytotoxicity dose-response was determined by the mtt cell proliferation/viability assay. we show that a) all genotoxic agents were able to induce dose-dependently γh ax in the cytotoxic range whereas no induction was observed after treatment with non-genotoxicants; b) manual scoring of γh ax foci and automated scoring gave similar results, with the automated scoring being faster and more reproducible; c) data obtained by foci counting and facs analysis of γh ax positive cells showed a significant correlation. further we compared dna damage induced by selected genotoxins at the time-points using the alkaline and neutral comet assay. significant correlation with the alkaline and neutral comet assay was observed for some but not all genotoxins and, predominantly, at earlier time points. we suggest that comet assays detect mainly primary dna damage, whereas γh ax assay detects a specific response to dna damage which can persist longer. the γh ax foci and flow-cytometry assays allow for a rapid and reliable determination of genetic damage in mammalian cells and can be used as additional genotoxicity assays. available in vitro methods to investigate the genotoxic potential of drugs fall short of throughput, specificity and mode of action information. a set of mechanistic biomarkers for clastogenic, aneugenic or apoptotic effects may help to overcome these limitations. thus, a staining assay amenable to flow cytometric analysis is being developed by litron laboratories, rochester, ny, supported by international collaborators. the experimental design of this assay consists of stages. the objective of this work is the evaluation of this assay in the laboratories of bayer pharma ag. the biomarkers covered by the assay are associated with dna damage response pathways that have potential for class discrimination (clastogen/aneugen/cytotoxicant) of in vitro genotoxicants: dna double strand breaks (γh ax), nuclear division (phospho-h , dna content), apoptosis (cleaved parp). based on the pilot work at litron laboratories, tk cells were introduced to the genetic toxicology of bayer pharma ag. cells were exposed for and hrs in triplicates on a microwell plate to one reference clastogen (etoposide, eto), aneugen (vinblastine, vb) and cytotoxicant (carbonyl cyanide -chlorophenylhydrazone, cccp). after staining, the samples were analyzed with the flow cytometer bd accuri c (bd biosciences, heidelberg, germany). the reference substances yielded the responses expected from the pilot study at litron laboratories: vb showed distinct increases of phospho-h events at and hrs and polyploidy at hrs time point. eto induced a clear increase of yh ax with a simultaneous reduction of phospho-h at and hrs. finally, cccp caused a reduction of phospho-h events, increased cleaved parp events and did not influence γh ax. moreover, benchmarking experiments under pilot work conditions were performed with high content imaging analysis. we compared yh ax and phsopho-h pilot study results as well as cleaved parp with caspase / . in addition, the tunel assay (click-it ® tunel alexa fluor, thermofisher) was executed to benchmark cleaved parp. the benchmarking results support the selected biomarkers of the multiplexed assay. in stage , additional reference compounds (three aneugens/clastogens/cytotoxicants) were investigated. so far, the chosen biomarkers of dna damage response appear useful for class discrimination and provide additional information to existing genotoxicity tests. cell-cell contacts are involved in keeping a physiological balance between proliferation, differentiation and apoptosis. far less is known about the role of cell-cell-contacts in regulating necrosis, for instance in response to oxidative stress. previous findings of our group demonstrated that, in contrast to semi-confluent proliferating cultures, confluent murine fibroblasts (nih t , mef) and human keratinocytes (hacat) are protected against necrosis induced by tert-butyl hydroperoxide (t-booh). comparison of confluent cells (g /g = ~ %) and semi-confluent cultures, similarly arrested in the g /g phase by serum-starvation or the mek inhibitor u , ascertained that the resistance against t-booh is mediated by cell-cell contacts and not by cell cycle arrest. we further revealed that confluent cultures are protected against t-booh-induced dna double strand breaks as assessed by the neutral comet assay and against mitochondrial damage detected by flow cytometric analysis of dioc staining. to better understand the protective role of cell-cell-contacts in ros-mediated necrosis, we started characterizing the signaling cascade induced by t-booh in semi-confluent proliferating cultures. in accordance with the observed formation of dna double strand breaks in response to t-booh, we detected phosphorylation of the checkpoint kinase chk . however, inhibition of atm, the kinase responsible for chk activation, did not influence t-booh-induced cell death. interestingly, first experiments gave a hint for the participation of rip , since the chemical rip kinase inhibitor necrostatin- (nec- ) blocked cell death up to averagely %, what is described as a specific marker for regulated necrosis. in line with this observation, t-booh-induced cell death could not be blocked by the pan-caspase inhibitor z-vad-fmk strongly indicating that caspase activity is not required. moreover, parp- and p are probably not involved. deeper analyses could give evidence that nec- did not block formation of dna double strand breaks nor mitochondrial damage indicating that the kinase blocked by nec- , possibly rip , acts downstream of dna double strand breaks and / or mitochondrial damage. in the end, we could identify a crucial role of ca + signaling for t-booh-mediated toxicity. as the calcium chelator bapta-am was able to completely block not only cell death, but also mitochondrial damage and dna double-strand break formation, there is a strong need for further investigations of the possible interplay between regulated necrosis and calcium, regulated by cell-cell contacts among oxidative stress. the work was supported by the hoffmann-klose-stiftung, the promotionsförderung rheinland-pfalz, the johannes gutenberg-university and the university medical center of the johannes gutenberg-university. the mammalian target of rapamycin (mtor) forms two multiprotein complexes (mtorc and mtorc ) and influences cell growth, proliferation, survival and metabolism. constitutively activated mtor was found to be deregulated in several cancer types, which makes it an interesting target for therapeutic cancer strategies. rapamycin is able to inhibit mtor and its downstream targets and is currently studied for its anticancer properties in clinical trials. despite previous evidence, there are studies that show an adverse effect in cancer treatment causing tumour growth, evolving the question of the effectiveness of the drug in cancer treatment. therefore, we examined the transformational potential of rapamycin in a balb/c cell transformation assay (cta) as well as markers of proliferation and protein synthesis. the balb/c t cell transformation assay mimics different stages of in vivo carcinogenicity (initiation, promotion, post-promotion phase) and is a promising alternative to rodent bioassays. balb/c fibroblasts are treated for days with the tumour initiator mca ( -methylcholanthrene) followed by days with the promotor tpa ( -o-tetradecanoylphorbol- -acetate). upon treatment with these chemicals cells are transformed into morphologically aberrant foci and can be visualized after six weeks by giemsa staining. it is possible to apply additional substances during the whole assay or in several phases of transformation and evaluate the colony formation. furthermore, our improved protocol allows additional westernblot or immunofluorescence analysis. the influence on cell proliferation of different concentrations of rapamycin was investigated by cell counting (living and dead) to choose a suitable concentration for the cta. performances of balb/c ctas with nm rapamycin showed, contrary to expectations, an increase in cell transformation. by administration of rapamycin only in the promotion phase we could detect an increase in colony formation, whereas a treatment with rapamycin in the post-promotion phase with already established foci, seemed to reveal its therapeutic properties. to better understand the role of mtor in our cell transformation system we used another mtor inhibitor called osi- . surprisingly, an incubation with µm osi- led to a decrease in colony formation. we are now able to investigate the underlying mechanism with westernblot and immunofluorescence analysis and can compare regulations of downstream targets like the marker of protein synthesis p-s . our investigations revealed different cell transformation outcomes by comparing the two known mtor inhibitors rapamycin and osi- , which need to be further evaluated. in the ongoing project we want to detect differences between rapamycin and osi- by protein analysis and identify key proteins, which are involved in this opposed colony formation of the balb/c cells. these results can be helpful to better understand mtor inhibition in matters of tumour therapy. introduction: over the past years, the biguanide compound metformin has been widely prescribed as an insulin sensitizer in type diabetes mellitus. interestingly, recent meta-analyses of epidemiological studies have shown that metformin might be involved in risk reduction of carcinogenesis. in vitro studies have described amp-activated protein kinase (ampk)-dependent, by inhibition of the respiratory chain complex i, as well as ampk-independent actions of metformin. however, the detailed molecular mechanisms by which metformin affects cell proliferation and carcinogenesis have not been well identified up until now. method: to evaluate the protective potential of metformin, balb/c t cell transformation assays were performed. this valid toxicological method is an alternative to in vivo carcinogenic testing and mimics the different stages of cell transformation during carcinogenesis. in detail, mouse fibroblasts are treated with metformin and/or the tumour initiator -methylcholanthrene (mca) and the tumour promotor -otetradecanoylphorbol- -acetate (tpa). in the first experiment several metformin concentrations ( . - mm metformin) were applied answering the question of an effective metformin concentration. next, metformin treatment during the different phases of carcinogenesis (initiation, promotion, post-promotion phase) was done determining the most effective phase for an intervention, i.e. chemopreventive or chemotherapeutical properties of metformin. additionally, the effect of metformin on the energy metabolism of the cells was analysed using various methods like immunoblot and oxygen measurement by clark electrode. results/discussion: analysis of different metformin concentrations revealed a concentration-dependent effect of metformin. in detail, decreased colony forming potential of balb/c cells was most prominent using mm metformin. this effect was not caused by growth inhibition of metformin itself since mm metformin showed no growth inhibitory properties in a cellular growth pretrial. interestingly, the phase cell transformation assay showed that the metformin effect is more pronounce in the postpromotion phase than in the initiation and promotion phase pointing to a chemotherapeutical potential. investigating several energy metabolism parameters, the results indicate that metformin may affect cell respiration as well as energy-dependent mechanistic markers like ampk. the presented results support rather the idea of the chemotherapeutic potential of metformin than a chemopreventive, using mm metformin. the initial analysis of energy metabolism markers discovered interesting starting points for further investigations. johannes gutenberg university, institute of toxicology, mainz, germany nvp, widely used e. g. as a monomer for polyvinylpyrrolidones (pvp) with applications in food technology or cosmetics is a known hepatocarcinogen in rats after inhalative exposure to , , and ppm for years. nvp is tested in a battery of genotoxicity assays (e.g. ames, hprt, mouse lymphoma, uds, chromosome aberration, cell transformation, micronucleus test (mnt) in mice bone marrow) [ ]) that all yielded negative results. however, nvp induces cell proliferation in liver (loaec: . ppm) after whole body exposure to vapor [ ] . to confirm the absence of genotoxicity in the context of a potentially non-genotoxic mode of action, a five day whole body inhalation study to nvp vapor with concentrations of , , , ppm was conducted in wistar rats (six animals per gender and group, ethyl methanesulfonate mg/kg bw p.o. as positive control). genotoxicity was investigated by the mnt in bone marrow and the comet assay (± fpg) in liver and lung. further investigated endpoints related to possible non-hepatogenotoxic moa were: enzyme induction (erod, prod, brod), oxidative stress (gsh-, gssg-, non-protein sulfhydryl group level), and peroxisome proliferation (cyp a, cyanide-insensitive palmitoyl-coa-oxidase). at carcinogenic inhalative doses, the results of this study proved the absence of genotoxicity in lung, liver and bone marrow as neither the tail intensity in the comet assay nor the number of micronuclei in the mnt was increased compared to the controls. however, also the non-genotoxic parameters (cyp-enzyme activity, glutathione levels, cyanide-insensitive-palmitoyl-coa-oxidase) were not affected by nvp-treatment. as potential metabolic activation cannot be excluded and may essentially contribute to the understanding of the carcinogenic mechanism, in vitro investigations in rat liver systems (subcellular fractions, hepatocytes, precision cut liver slices (pcls)) were performed additionally. up to now, -pyrrolidone is the only identified in vitro metabolite. as these results cannot mimic the in vivo situation of two described, ring-and vinylmajority containing unidentified metabolites [ ] detailed investigations on metabolism may be a future perspective to approach the overall understanding of the carcinogenic mechanism of nvp. introduction: in ischemic conditions such as wound healing and myocardial infarction, new vessels are generated by vasculogenesis and angiogenesis. these processes are stimulated by the signalling peptide vascular endothelial growth factor which therefore has been proposed as a promising compound for the treatment of ischemic conditions. however, results of respective clinical studies have not been fully convincing yet. here, we investigated principles underlying the selforganization of newly formed vessels to functionally adequate microvascular networks indispensable for proper tissue substrate supply. intravital microscopy of the chick chorioallantoic membrane (cam), a non animal model as defined by the american national institutes of health's office for protection from research risks, was used to study peripheral expansion of existing arteriolar and venular trees by recruiting segments of the dense polygonal capillary mesh. this process we call "emerging angiogenesis". methods: white leghorn chicken eggs were put into incubators on embryonic day (e ) at . °c and % humidity. on e , the eggs were cracked open and transferred into petri dishes. on e , cam microcirculation was recorded using time-lapse intravital videomicroscopy at discrete time points for up to hours. to improve the visibility of the capillary mesh, videorecordings were processed offline by generating coefficient of variation images of pixel grey values over time. changes of network topology during the observation time were investigated. results: in the cam, a sequence of specific events leading to extension of existing vessel trees was observed: in a capillary mesh region near terminal branches of existing vessel trees, homogeneous flow distribution is transferred to inhomogeneous flow distribution: preferred flow pathways through the mesh evolve carrying most of the blood. over time, these flow pathways exhibit diameter increase, straighten and connect the mesh to arteriolar and venular trees. in contrast, less perfused parallel mesh flow pathways and transversal mesh segments exhibit progressive decrease of flow and diameter resulting in vessel regression. as a result, hierarchical vessel tree structures are extended into the mesh region. while newly generated tree extensions are located above the mesh at the beginning, they sink to a lower level at later stages until they are finally covered by a reconstituted mesh network. the cam ex ovo model is well suited for studying emerging angiogenesis. vessel tree extension occurs via parallel processes of vessel maturation and capillary mesh segment regression. at later stages, newly formed vessel tree branches sink and the capillary mesh is reconstituted above. in the next step of our project, we will implement these phenomena in a computer simulation and use theoretical modeling to further investigate and better understand principles underlying microvascular network maturation. this will allow us to derive effective therapeutic strategies which could be tested in the cam model. chemicals are able to induce cancer in a wide range of organs. therefore, it is very important to investigate the toxic properties of chemical substances, especially their carcinogenic potential. in this context the number of animal experiments will drastically increase in the future. in order to avoid the use of expensive and time consuming animal experiments for long-term carcinogenic studies, the development of an in vitro system to test the carcinogenic potential of a high number of chemicals in a highly reproducible manner within a short period of time is imperative. by combination of the well-established balb/c cell transformation method with the soft agar colony formation assay, we developed a high-throughput in vitro system to identify effects of chemicals on cell transformation for the first time. balb/c mouse fibroblasts are treated with -methylcholanthrene as a tumour initiator and -o-tetradecanoylphorbol- -acetate as promotor for several days, whereby foci of transformed cells are developed. after the promotion phase of the common balb/c cell transformation assay, cells are transferred into soft agar to further monitor the anchorage independent growth of transformed cells only. the established soft agar transformation assay reproduces the foci growth of previous experiments and is performed in -well plate format. hence, we can analyse the carcinogenic potential of several chemical substances in parallel and are also searching for alternative endpoint analysis, e.g. the usage of fluorescing cells stably expressing irfp, instead of the former time-consuming microscopic assessment. the here presented new technique is a high-throughput and low priced alternative for the evaluation of the carcinogenic potential of chemical substances in a short period of time without animal testing. the effort to develop new or refine established in vitro test systems rises due to animal welfare, scientific and/or regulatory reasons (e.g. the animal testing ban concerning the risk assessment of cosmetic product ingredients in march ). this progress, among others, leads to an increased performance of cell-based assays. the majority of model cell lines are routinely cultured using medium supplemented with fetal bovine serum (fbs) in amounts between - %. the application of serum-substitutes will provide a reduction of the animal number needed, which corresponds to the guiding principles of the three r's ( r), described by russel and burch in . in addition, chemically defined serum-substitutes have the potential to reduce the inter-experimental variability of test conditions caused by the inherent differences in chemical composition across fbs batches , resulting in a refinement of in vitro testing. in this study, human tk cells were gradually adapted to serum-free conditions, where they show comparable growth gradients at the exponential phase. for cells under serum-free conditions a mean doubling time of . (± . ) h was observed while fbs supplemented cells showed a doubling time of . (± . ) several non-animal test methods addressing key events in the sensitization process have passed formal validation and oecd (draft) test guidelines are available. one of these methods is the direct peptide reactivity assay (dpra) assessing the ability of a chemical to bind to proteins to form a complete antigen (oecd tg c). the test is used to obtain a yes/no answer on whether the substance has a protein-binding potential. for a complete risk assessment, however, an estimation of a chemical's potency is also needed. in this study we examined if an assessment of potency could be achieved by ) determining reactivity class cut offs based on published data on substances for the dpra performed according to oecd c to predict un ghs sensitizer classes, ) a variant of the dpra assessing reaction kinetics (time and concentration) for substances or ) an extended protocol testing several test substance concentrations for reference substances and estimating the concentration of a test substance that is needed to cause a peptide depletion of . % (ec . %). results of the first approach indicated that cut offs to differentiate the un ghs sensitizer classes a and b could indeed be defined. secondly, evaluating the reaction time based assay in which several time points between min and hours were assessed, it was found that not all reactions followed ideal kinetics. hence further investigations are needed to eventually derive a reaction time based prediction model. the results of the rd approach (the standard protocol of the dpra was amended by testing three concentrations i.e. , , and mm) indicated that potency classes could be assigned using the ec . % value to assess potency. in summary, using quantitative information derived from the dpra in particular using ec . % value may support the assessment of the skin sensitizing potency. identification of pre-and pro-haptens with non-animal test methods for skin sensitization since pro-haptens may be metabolically activated in the skin, information on xenobiotic metabolizing enzyme (xme) activities in cell lines used for testing of sensitization in vitro is of special interest. metabolic activity of e.g. n-acetyltransferase (nat ) and esterase in the keratinocyte (keratinosens tm and lusens) and dendritic cell-like cells (u and thp- ) was previously demonstrated. aldehyde dehydrogenase (aldh) activities were found in keratinosens tm and lusens cells. activities of the investigated cytochrome p -dependent alkylresorufin o-dealkylases, flavin-containing monooxygenase, alcohol dehydrogenase as well as udp glucuronosyl transferase activities were below detection in all investigated cell lines. a set of putative pre-and pro-haptens (no obvious structural alert for peptide reactivity but positive in vivo) was routinely tested using the above mentioned cell lines as well as in the direct peptide reactivity assay (dpra). of the compounds were unexpectedly positive in the dpra und further analyzed by lc/ms techniques to clarify the reaction mechanism leading to true positive results in this assay. oxidation products like dipeptide formations or the oxidation of the peptide-based sulfhydryl group led to positive results for benzo[a]pyren or -amino- -methylphenol, respectively. in contrast, covalent peptide adducts were identified for putative pre-haptens, indicating the dpra to be suitable for compounds requiring abiotic oxidation to get activated. for some dpra negatives, the keratinocyte and dendritic cell based assays provided true positive results. a combination of dpra, keratinosens tm and h-clat within a ' out of ' prediction model provided a high sensitivity of % for the set of the pre-/pro-haptens. the sensitivity of this combination of non-animal test methods in the ' out of ' prediction model in a set of direct haptens was comparable (sensitivity = % when compared to llna skin sensitization testing is mandatory for all substances produced or marketed in volumes larger than tonne per year under the european reach legislation. with reach supporting in vivo testing only "as a last resort" and the marketing ban for finished cosmetic products with ingredients tested in animals, attention has been given to developing integrated testing strategies combining in vitro, in silico and in chemico methods. key challenges are which tests to select and how to combine non-animal methods into testing strategies. this study suggests a bayesian value of information (voi) approach for developing non-animal testing strategies, which consider information gains from testing, but also expected payoffs from adopting regulatory decisions on the use of a substance, and testing costs. the 'value' of testing is defined as the expected social net benefit from decision-making on the use of chemicals with additional, but uncertain information from testing. the voi is calculated for a set of individual nonanimal methods including dpra, oecd qsar toolbox, are-nrf luciferase method covered by keratinosens and lusens, and hclat, seven battery combinations of these methods, and two-test and three-tests sequential strategies consisting of nonanimal methods. their voi is compared to the voi of the local lymph node assay (llna) as the animal test. we find that battery and sequential combinations of nonanimal methods reveal a higher voi than the llna. in particular, for small prior beliefs (i.e. a chemicals is, prior to testing, assumed to be a non-sensitiser), a battery of dpra + lusens reveals the highest voi. if there are strong beliefs that a chemical is a sensitizer, a sequential combination of the battery dpra + lusens, followed by keratinosens + hclat at the second stage and by the oecd qsar toolbox at the third stage performs best. for given specifications of expected payoffs the voi of the nonanimal strategy significantly outperformed the voi of the llna, for the entire range of prior beliefs. this underlines strong economic potential of non-animal methods for skin sensitization assessment. a chemical series to predict the proarrhythmic potential of drugs with low solubility for which no reliable purkinje fiber results could be obtained. these validation results showed that this cardiosafety in silico model can successfully be applied in r&d to predict the proarrhythmic potential of drug candidates within the model ad. introduction: the use of p-phenylenediamine (ppd) and derivatives (tab. ) in oxidative consumer hair dye products is considered as key in hair dye allergic contact dermatitis [ ] [ ] [ ] [ ] . in recent supplement, -methoxymethyl-ppd (me+) shows significantly reduced sensitizing properties [ , ] . since overcoming the skin barrier is a prerequisite for sensitization, numerous in vitro an in vivo studies on skin penetration of ppd and derivatives have been performed. the aim of the present study is the in silico prediction of the penetration of ppds, because such computations may help in understanding the processes involved in sensitization. for the first time, software dskin [ ] is challenged to simulate this class of compounds. in silico results are retrospectively compared to previously published experimental data and may assist in future tailoring of in vitro experiments. material and methods: the permeabilities, lag-times and the time-dependent accumulated amounts of ppds were computed using dskin. input parameters for the latter were a concentration of mg/ml ( %), finite dosing and min in use incubation periods. molecular structures were optimized ab initio and the condensed fukui functions (ff) were estimated from mulliken population analyses [ ] and electrostatic potentials using gamess [ ] . results: initial results agree with experimental results using ppd in white petrolatum, demonstrating the applicability of dskin to ppds. the four ppds exhibit only small differences in permeabilities in silico (tab. ). toluene- , -diamine shows a higher accumulated mass due to increased lipophilicity (fig. ) . in general, the ff were very similar for all ppds and indicated that the n atoms would be the preferred targets for radical and electrophilic attack. discussion and outlook: in silico methods may be used to model the permeation of ppds despite their low molecular weight and low lipophilicity. the low amounts of ppds under in use conditions result from oxidative conditions. computed me+ permeation was not different to other ppds, therefore other properties account for the reduced sensitization potential. the very similar ff values hint at similar reaction pathways. furthermore, ppd and its derivatives are prone to n-acetylation in living skin resulting in metabolites exhibiting higher molecular weight and greater lipophilicity than the parent compounds. the effects of n-acetylation and reactions of ppd and its derivatives with histidine and cysteine residues are subject of upcoming computations. dermal absorption is an important factor in regulatory science regarding the registration of chemicals, agrochemicals and cosmetics. the issue has gained importance since it has been realized that the skin is not completely impenetrable for chemical substances. [ ] the different ways to assess dermal absorption range from qsar models to complex in vivo studies including a complete toxicokinetic examination. the choice of method depends on the question that has to be answered as different systems give different results: absorption as % of applied dose in in vivo studies or permeability coefficient and lag time in infinite dose in vitro studies [ ] . ideally both data would be available. since the oecd has adopted a guideline for assessing dermal penetration in vitro in the number of in vitro studies is rising continuously. depending on the chosen method results may vary in reliability and in acceptance by regulatory authorities. the skinab database [ba ] contains data for about substances on dermal absorption which has been found through the echemportal [ ] and extended with data from the edetox database. for selected substances with a broad spectrum of data available further analysis has now been started. chemicals have been investigated in a comparable test system; from these were shown to have a low dermal absorption of less than % and compounds showed a high absorption rate of more than %. for the assessment of dermal exposure either the absorbed dose in percent or the flux can be measured. data analysis showed that only for substances both is available: flux data from in vitro studies and absorption data from in vivo studies. this data could be used to clarify which parameter would be most useful for exposure assessment regarding dermal exposure. seven substances in the dataset were conspicuous for their range of absorption rates in different studies: less than % to more than %. an in depth analysis revealed the complex influence that different exposure parameters have on the results of dermal absorption studies. for some chemicals the influence of exposure time on increasing absorption values could be clearly demonstrated. beside other factors such as the chosen vehicle, and the (non-)occlusion of the site of exposure especially the choice species introduced a high variability; this holds even for the most common laboratory animals t. a review published by jung in [ ] which comes to the conclusion that i.e. hairless species are usually not a good model to predict dermal absorption in humans. [ ]who ( ) ehc dermal absorption [ ] scholz et al ( ) naunyn-schmiedeberg´s arch pharmacol (suppl ):s [ ] www.echemportal.org [ ] http://edetox.ncl.ac.uk [ ] jung et al ( ) in-silico methods have evolved to indispensable tools in various areas of life sciences. several stages in drug development including hit identification and lead optimization, for instance, highly benefit from an accurate estimation of binding free energies associated with biological host-guest systems. as a consequence, the need for laboratory experiments including in-vivo experiments and animal testing is considerably reduced. another field profiting from free energy calculations is human as well as ecotoxicology. upon the development and risk assessment of new chemicals, transformation products arising from biotic or abiotic degradation of the parent substance have usually been neglected. however, since few years, the risk assessment of new chemicals often includes transformation products probably causing more harm than the parent substance itself. such studies as well are mostly carried out on the basis of in-vitro and in-vivo tests. moreover, many metabolites can be detected but neither enriched nor synthesized in amount sufficient for toxicological evaluations. at this stage, computational methods come into play. using classical molecular dynamics simulations in combination with an empirical linear prediction model, we have investigated several metabolites of the drugs sulfamethoxazole and carbamazepine and prioritized them according to their estimated binding affinities to potential biological target proteins. consequently, a couple of metabolites were identified that bind to one or more human cytochrome p variants and the bacterial enzyme dihydropteroate synthase, respectively, which are known to be sensitive to the two drugs. the investigations were carried out in the framework of the bb r poject funded by the german government through bmbf. instituto superiore di sanità, environment and primary prevention dept., rome, italien introduction: in vitro methods have been increasingly used to characterize pharmacological and toxicological properties of substances. to address the problem of nominal versus actual concentrations, in vitro biokinetic studies were recently undertaken (truisi et al., toxicol lett : - , ) . we use those data as input into a physiologically based human kinetic model (pbhkm) to model the in vivo doses leading to the in vitro measured concentrations. methods: a pbhkm was used to simulate the concentration time profile of ibuprofen in the hepatic vein after oral administration. the details of the model and the physiological parameters used have been described elsewhere (abraham et al., arch. toxicology : - , ) . we modelled the concentration time profile exploring the dose which would lead to a concentration at hour and at hour as similar as possible to the concentration measured in the supernatant of human freshly prepared cell cultures after dosing the culture with ibuprofen. we parametrized the pbhkm with the parameters which have been estimated from the in vitro kinetic studies (clearance between and µm /sec (truisi et al., ) and an absorption of % and an absorption rate of /h (cristofoletti and dressman, j pharm sci : - , ). results: the data of the in vitro study with µm ibuprofen could well be modelled. when assuming a clearance of µm /sec the dose of mg resulted in an hour concentration of . µm in the hepatic vein of pbhkm equal to . nmol/well (volume of the well = ml) in the in vitro study in which the measured concentration was . nmol/well. the concentration at hours of . µm (equal to . nmol/well) corresponded with the in vitro concentration ( . nmol/well). the modelling approach was less successful with in vitro dosing of µm. the fold higher dose of mg lead to nearly double the concentration at hour than measured in vitro. with a dose of mg/kg an approximation was feasible resulting in . µm in the hepatic vein at hour which is equal to . nmol/well whereas the measured concentration in vitro was . nmol/well. even with a clearance value as low as the . percentile ( µm /sec), the concentration at hours was modelled to be lower than the in vitro measured value (in vivo model: . µm which corresponds to . nmol/well; measured in vitro concentration: . nmol/well). discussion: this is the first attempt to use kinetic data obtained in vitro to feed in in a pbhkm for reverse dosimetry finding the dose which corresponds in vivo to the in vitro situation. in the case presented here, the in vitro dose assumed to be low in vitro ( µm) corresponds to a dose of mg (note: the highest approved daily dose is , mg). for the high in vitro dose modelling was successful only for the concentration hour after dosing and a dose of , mg. conclusions: in vitro kinetic parameters, such as clearance, can successfully be used for parametrizing a pbhkm. it is of utmost importance for the relevance of in vitro finding to assure that the concentrations used in vitro can be obtained with relevant in vivo doses. in this case, the in vitro concentrations were within (low dose) and . fold above (high dose) the in vivo relevant therapeutic concentration range. introduction: a variety of drug residues have been detected in sewage plant run-offs, rivers and lakes, but also in groundwater and tap water samples. studies have yet to identify a risk for human health from these contaminants, but adverse health effects have been reported for various species, including fish and birds. it has recently been suggested that for a comprehensive risk assessment toxicologists should also consider transformation products (tps) of such water contaminants that may arise from abiotic and biotic (metabolic) reactions. with aciclovir (acv), a well-known antiviral drug, as the parent drug we tried an in-silico approach to identify tps that might be of interest due to some mutagenic or carcinogenic toxicophores. methods: from a literature and database search we picked up acv-tps. predicted acute toxicities and mutagenic / carcinogenic properties for these tps were derived from an expert system analysis using the lazar portal (http://lazar.in-silico.ch/) as front end. results: two of the identified acv-tps could not be handled by lazar because of insufficient training data in one out of eight queried categories. the highest score ( positive out of possible genotoxicity categories) was assigned to of the tps, including acv itself. this is a rather low score when compared to other water-borne drug residues, e.g. carbamazepine. cofa, an imidazole derivative of acv seen in advanced oxidation processes, had shown antiproliferative effects in several ecotoxicologic screening assays, e.g. [ ], but was unremarkable in our tests. additionally, a computer-based simulation of the respective tps interacting with human cyp isozymes did not support concerns that these tps may pose a risk for human health. conclusions: our in-silico analyses of acv-tps did not provide evidence for any adverse health effects in the micromolar concentration range. further studies are needed to clarify if the biological activity of some acv-tps in ecotoxicological assays may eventually affect yet unidentified biological targets in the human body. sulfur mustard (sm) is a chemical warfare agent which was first used in world war i, but has found use in several conflicts afterwards. although sm is prohibited by geneva protocol, terroristic attacks cannot be ruled out. latest news give rise to concern that is may be in the possession of sm and is willing to deploy it. even years after the initial synthesis of sm its mode of action is not fully unraveled. thus, no antidote does exist. however, chemosensing ion channels have been shown to be activated by highly toxic chemicals and might represent a specific therapeutic target. previous studies have shown that the sm-surrogate cees (mono-functional alkylating agent) is able to activate transient receptor potential ankyrin (trpa ) channels that are known to affect mapk cell signaling. mapk-pathways, especially perk / , are known to increase protein biosynthesis through activation of transcription factors binding to the serum response element (sre). it is unknown whether alkylating agents have also impact on mapk signaling mediated through trpa activation. our results demonstrate that aitc resulted in phosphorylation of the mapk perk / and increased protein biosynthesis of sre-regulated genes in hek cells overexpressing htrpa . cees increased perk / levels already after . min which could be prevented by the trpa blocker ap . activation of target genes through perk / signaling was also evident, but less pronounced compared to aitc. our results demonstrate that alkylating agents have impact on cell signaling through trpa channel activation. thus, trpa might represent a promising target for counteracting sm toxicity. sulfur mustard (sm) is a chemical warfare agent that provokes severe inflammation and blistering upon exposure to the skin accompanied by disturbed wound healing. the potential use of sm in terroristic assaults amplified the interest in understanding the underlying cellular and molecular pathomechanisms in order to improve therapeutical intervention. autophagy is a highly conserved catabolic pathway in eukaryotes that ensures the degradation and recycling of cellular components through the lysosomal machinery. autophagy is important for cell survival in physiological and pathological stress situations. emerging knowledge indicates that imbalanced regulation of autophagy disturbs basal cell functions including proliferation, differentiation and migration, thus contributing to the pathophysiology of various diseases. after penetration into skin cells, sm alkylates and thereby modifies nucleic acids and proteins thus forming aggregates of dysfunctional proteins destined for autophagic disposal. in our studies, we analyzed the influence of sm on protein expression (western blotting) of autophagy-related (atg) genes as well as proliferation (wst- ) of primary normal human keratinocytes (nhek) and primary normal human dermal fibroblasts (nhdf). preliminary results demonstrate that sm strongly dysregulates the biosynthesis of atg proteins that may contribute to the diminished cell migration and proliferation under these conditions. our findings suggest that sm affects autophagy in correlation with an impairment of physiological functions in keratinocytes and fibroblasts that are essentially required for normal tissue regeneration. thus, application of pharmacological modulators of autophagy might be useful in the treatment of the delayed wound healing in skin upon exposure to sm. exposure of the respiratory tract to airborne particles is a major risk to human health. due to the ubiquitous application of these particles in the field of pharmacy, industry and in daily life, there is a strong necessity to investigate the toxic properties and the underlying pathomechanisms of these inhalable substances. in addition, the eu chemicals regulation requires not only that all substances placed on the market have to undergo a toxicological characterization, including the identification of potential toxic inhalation hazards (reach), but also that animal testing shall be undertaken only as a last resort (" rs" principle) and the promotion of the development of alternative methods. thus, the development, establishment and validation of alternative in vitrobased test systems for the assessment of pulmonary toxicity are in the focus of current research. until now, most of the available in vitro cell culture models are limited to some extent as in those studies the exposure is either done under submerged conditions, not resembling the exposure conditions in vivo, or a homogeneous particle distribution is not guaranteed. the cultex ® radial flow system (rfs) is a specially designed in vitro modular exposure system that overcomes these limitations. it enables the homogenous exposure of human lung epithelial cells at the air-liquid interface (ali), thereby mimicking the physiological conditions of the alveolae. however, further optimizations are needed for the enhancement of the cultex® methodology. aim of this study was first the optimization of the test methodology in general (i.e. focus on clean air controls of the human lung epithelial cell line a ), and second the improvement of cultivation conditions. parameters such as handling of the cultex® device (proper closing and opening operation of the cultex® rfs module; improved washing conditions and media supply), treatment of the incubator controls, adjustment of clean air pressure and flow rates, and integration of two additional filters were sequentially adjusted in order to enhance the methodical setup. our results show that the test parameters for clean air exposure of the a cells were successfully optimized resulting in more accurate and robust data. cultivation conditions were improved by changing from closed-wall cell culture inserts to open-wall cell culture inserts. the openwall inserts turned out to be more suitable for exposure experiments as they provided a better medium supply and preserved humidity. deductive, the change of the cell culture inserts was identified as the deciding factor for the improvement of cell morphology. hence, we have successfully optimized the cultex ® rfs methodology for clean air exposure of a cells. human primary hepatocytes represent the gold standard in in vitro liver research. due to their low availability and high costs, alternative liver cell models with comparable morphological and biochemical characteristics have come into focus. the human hepatocarcinoma cell line hepg is often used as a model for liver toxicity studies. however, under two-dimensional ( d) cultivation conditions the expression of xenobiotic-metabolizing enzymes and typical liver markers is very low. cultivation for days in a three-dimensional ( d) matrigel culture system has been reported to strongly increase the metabolic competency of hepg cells. in our present study we extended previous studies and compared hepg cell cultivation in three different d culture systems: collagen, matrigel and alvetex culture system. cell morphology, albumin secretion, cytochrome p monooxygenase (cyp) enzyme activities, as well as expression of xenobiotic-metabolizing and liver-specific enzymes were analyzed after , , , and days of cultivation. our results show that the previously reported increase of metabolic competency of hepg cells is not primarily the result of d culture but a consequence of the duration of cultivation. hepg cells grown for days in d monolayer exhibit comparable biochemical characteristics, cyp activities and gene expression patterns as all d culture systems used in our study. however, cyp activities did not reach the level of heparg cells. in conclusion, the increase of metabolic competence of the hepatocarcinoma cell line hepg is not due to d cultivation but rather a result of prolonged cultivation time. in vitro assessment of the neurotoxic potential of arsenolipids arsenolipids are organic, lipid-soluble arsenic compounds, which occur mainly in marine organisms. major human exposure routes are fatty fish including herring or fish oil-based food supplements. about different arsenolipids have been identified so far. thereby, arsenic-containing hydrocarbons (ashc) and arsenic-containing fatty acids (asfa) represent two subgroups of the arsenolipids [ ]. our in vitro studies have demonstrated high cellular bioavailability and a high cytotoxic potential of ashcs in human liver and bladder cells [ ] , whereas asfas were less toxic [ ] . a substantial transfer across an intestinal barrier model (caco- ) indicated that ashcs are highly intestinal available. in comparison, asfas showed lower intestinal bioavailability and underwent a presystemic metabolism [ ] . moreover, in drosophila melanogaster ashcs exerted late developmental toxicity and accumulated in the fruit fly's brain. these results suggest that ashcs might pass the blood-brain-barrier due to their amphiphilic structure [ ] . in order to assess the neurotoxic potential we currently investigate the toxicity of several arsenolipids in differentiated, human neurons (luhmes). after h incubation with ashcs or asfas, cell number (hoechst) as well as cellular dehydrogenase activity (resazurin) were measured, with the latter endpoint turning out to be more sensitive. ashcs showed substantial cytotoxic effects (ic ~ - . µm) in a concentration range comparable to that of arsenite (ic ~ . µm), whereas asfas were less cytotoxic (ic > µm). after incubation with ashcs the cellular arsenic concentrations increased - fold as compared to incubation with arsenite. further studies indicated that one possible toxic mode of action of arsenolipids could be a disruption of the cellular energy level. therefore, the mitochondrial membrane potential was investigated after incubation with the arsenic compounds in differentiated neurons. whereas arsenite did not exert an impact, ashcs reduced the mitochondrial membrane potential significantly. this might be due to interactions of the amphiphilic ashcs with mitochondrial membranes. currently we investigate the impact of the arsenolipids on neurite outgrowth as a developmental toxicity endpoint. standard treatment of poisoning by organophosphorus compounds (op; e.g. nerve agents and pesticides) consists of co-administration of atropine and an oxime-based reactivator of inhibited cholinesterases. due to lack of efficacy of clinically used oximes against various op-inhibited human acetylcholinesterase (ache) (e.g. soman) research started focusing on new therapeutic approaches. several research groups conducted in silico screenings [ , ] in order to identify new non-oxime reactivators, presenting amodiaquine as a promising candidate for paraoxon-inhibited hache. for decades, antimalarial drugs like amodiaquine and chloroquine have been closely investigated regarding their side effects, thereby discovering interaction with cholinesterases, which could pose a new potential therapeutic benefit for inhibited cholinesterases. therefore, in this study interactions between antimalarial agents in presence or absence of ops were examined spectrophotometrically by a modified ellman assay. reversible inhibition of cholinesterases was observed with both antimalarial agents. amodiaquine had higher inhibitory potency for hache than human butyrylcholinesterase (bche), being confirmed by ic values of . ± . µm for hache and . ± . µm for hbche. ic values with chloroquine were . ± . µm for hache and . ± . µm for hbche, thus representing a weaker inhibition of hache than amodiaquine. furthermore, reactivation of paraoxon-(pxe), sarin-(gb), cyclosarin-(gf), and vxinhibited hache and hbche by amodiaquine and chloroquine was determined. after minutes, only paraoxon-inhibited hache ( %) and cyclosarin-inhibited hbche ( %) were reactivated by µm chloroquine. on the contrary, µm amodiaquine reactivated all tested ops after minutes in the following order: pxe > vx > gf > gb. in contrast, with hbche the highest reactivation was generated with µm amodiaquine in the following order: vx > gb > gf > pxe. due to the high reversible inhibitory potency of amodiaquine, an increased concentration does not result in a higher reactivation of op-inhibited hache. in summary, our results show that amodiaquine is a reactivator of op-inhibited cholinesterases. in the future, non-oxime reactivators that are structurally-related to amodiaquine should be further investigated. [ ] bhattacharjee, a.k., marek, e., le, h.t., gordon, r.k., eur. j. med. chem., , , - . [ ] katz, f.s., pecic, s., tran, t.h., trakht, i., schneider, l., zhu, z., ton-that, l., luzac, m., zlatanic, v., damera, s., macdonald, j., landry, d.w., tong, l., stojanovic, m.n., chembiochem., , , - bundesinstitut für risikobewertung, lebensmittelsicherheit, berlin, germany development of mammary gland tumors is connected to a deregulation of breast epithelial cell differentiation, a complex process which cannot be reproduced in vitro under standard cell culture conditions. however, cultivation of cells in a tissue-like environment in an in vitro three dimensional ( d) model can mimic general architecture, function and differentiation of mammary bulks. in this project, a d model was used consisting of the permanent breast epithelial cell lines mcf a (er -, estrogen receptor negative) and mcf a (er + , estrogen receptor negative) grown in matrigel tm , which mimics the complex extracellular matrix in vivo. the d culture of mcf a and mcf a cells in matrigel tm results in the formation of growth-arrested, polarized spheroids with a lumen (acini-like organoids). in order to perform a semi-quantitative estimation on the influence of substances on the differentiation of the breast cells for the identification of non-genotoxic carcinogens a scoring method was developed. this scoring method provides information about substance-induced morphological changes of the spheroids during differentiation based on the following parameters: size of the spheroids, the formation of the lumen, and the degree of polarization. furthermore, the model allows distinguishing between erdependent (mcf a) and er-independent (mcf a and mcf a) effects. the d in vitro model is a useful tool for toxicologists to study substance effects on differentiation processes. the system will be used to examine the potential of e.g. food contaminants such as phthalates or perfluorinated substances (pfas) to disrupt the differentiation process of breast epithelial cells and will therefore serve as a valuable in vitro tool to assess their carcinogenic potential. inflammatory episodes occur erratically throughout life and are likely to play a critical role in the alteration of the individual susceptibility of a person to idiosyncratic druginduced liver injury (idili), a particular severe form of drug-induced liver injury (dili). in concordance with the inflammatory stress hypothesis, modest inflammatory stress can lower the threshold for hepatotoxicity and make an individual susceptible to develop liver injury during exposure to therapeutic doses of a drug. in order to evaluate the role of immune cells and its secreted factors during drug therapy, we established an in vitro test battery consisting of two cell culture systems in presence or absence of proinflammatory factors (lps, tnfα): (a) the monoculture of human hepatoma (hepg ) cells and (b) co-culture systems of human monocytic or macrophage-like (thp- ) and hepg cells. with these different test settings we aimed to identify whether the introduction of inflammatory immune cells and/or pro-inflammatory factors could increase the sensitivity of liver cells towards idili compounds. three reference substance pairs were tested, namely troglitazone -rosiglitazone, trovafloxacin -levofloxacin, and diclofenac -acetylsalicylic acid, each of them being composed of a compound that is known to induce idili and a partner compound of the same substance class that does not induce idili. first, all compounds were tested for cytotoxicity towards the single cell systems using the wst-assay. co-culture experiments with hepg and thp- monocytes or macrophages as well as co-exposure experiments with lps or tnfα were then done at about % cytotoxicity of the respective substance in the most sensitive cell type. subsequently the results were compared to the experiments in the monoculture of hepg . we observed that every idili compound showed a significant increase in cytotoxicity in a minimum of one exposure combination while this effect was not observed with the corresponding non-dili partner compound. in conclusion, a combination of different culture systems and co-exposures with proinflammatory factors is needed for a valid differentiation between non-dili and idili compounds. this test battery could provide a useful tool for the prediction of inflammation-associated idiosyncratic drug-induced hepatotoxicity. furthermore, our results support the inflammatory stress hypothesis and points to an involvement of proinflammatory factors in the development of idili. extensive animal models of carcinogenicity ensure a safe usage of chemicals. to elucidate fundamental molecular mechanisms of carcinogenicity these methods are expensive, time consuming and above all too complex. in contrast, most in vitro methods are rather simple and detect only selected endpoints, like dna damage, mutations or changes in proliferation. the balb/c cell transformation assay is a validated toxicological method to identify potential tumour initiators and promotors. first, balb/c mouse fibroblasts form a monolayer culture and get contact-inhibited after reaching confluence. upon treatment with a tumour initiator ( -methylcholanthrene) and promotor ( -o-tetradecanoylphorbol- -acetate) transformed cells do not stop proliferation and grow as morphologically aberrant foci over the monolayer of normal cells. after fixation with methanol at day , morphological aberrant foci can be visualized with giemsa staining. because the balb/c assay mimics different stages of the malignant cell transformation process (initiation, promotion and post-promotion phase) and detects with the colony formation a late endpoint of carcinogenicity we improved this method for mechanistic cancer research. using the example of insulin-signalling pathway we can show that ( ) several substances have a different impact on the transformation process, ( ) it is possible to identify for each substance the phase with the greatest effectiveness and ( ) we can detect additional endpoints to elucidate the mechanistic mode of action. therefore we used several compounds (linsitinib, metformin, rapamycin, …) to manipulate the insulin-signalling pathway on different levels (insr, ampk, mtor, …) and analysed a number of characteristic endpoints of carcinogenesis. changes on protein level and signalling (westernblot, immunofluorescence, flow cytometry) or parameters of energy metabolism (oxygen consumption, glucose or atp measurement) are measurable and enable new insights into the process of cancer origin. summing up, the balb/c t assay proves to be a cheap and short-time alternative to rodent bioassays. although this method does not mimic the whole in vivo neoplastic process, it can be used to provide essential information regarding key proteins and their signalling, during the different stages of transformation. is there hope to correctly classify severe ocular irritant agrochemical formulations using in vitro methods: a proof of concept using the isolated chicken eye test, two modified bcop protocols and an epiocular™ et protocol while some in vitro methods addressing ocular irritancy have gained regulatory acceptance, to date the draize rabbit eye test (oecd tg ) is the only world-wide regulatory accepted test for the determination of the full range of eye irritation potential. further although several in vitro methods for the severe eye irritation have gained regulatory acceptance, agrochemical formulations are nor explicitly included nor excluded from the applicability domain to predict severe ocular irritant formulations. systematic analyses are only available for e.g. the hen's egg test-chorioallantoic membrane (het-cam), and bovine corneal opacity and permeability (bcop, oecd tg ) assays both showing that the used protocols do not provide sufficient sensitivity to reliably predict severe ocular irritating formulations. the purpose of this study was to evaluate whether the regulatory accepted isolated chicken eye (ice, oecd tg ) test including corneal histopathology (as suggested for evaluation of the depth of injury), as well two modified protocols of the bcop and/or an et (exposure time reducing viability of treated tissue to %) protocol using the reconstructed cornea model epiocular™ are useful to predict severe ocular irritant agrochemical formulations. a proof of concept comprising the testing of ten to twelve agrochemical formulations with available in vivo data in each assay was conducted. in summary, based on the ice evaluation described in oecd tg , one of the five severe ocular irritant formulations (un ghs cat ) was predicted correctly. using both modified protocol versions of the bcop the result for one of the four tested un ghs cat formulations was just above the un ghs cat classification border for using one of the modified protocols. lastly and most promising, the epiocular™ et predicted four of five tested un ghs formulations correctly with the fifths being close to the classification border. additional agrochemical formulations will be tested to further evaluated the epiocular™ et protocol to identify severe ocular irritant agrochemical formulations. drug-induced pancreatic toxicity comprises effects on the exocrine and/or the endocrine pancreas, which both can have serious clinical implications, e.g. acute pancreatitis or diabetes mellitus. adverse effects on the pancreas are occasionally observed during drug discovery and development and often prohibit further development. hence, there is a need for reliable in vitro models to early on identify the pancreas-toxic potential of drug candidates. permanent cell lines and primary cells have many shortcomings, e.g. loss of cell-to-cell and cell-to-matrix relationships or changes in cell physiology due to the isolation procedure. pancreas tissue slices are a potential alternative, circumventing most of these limitations. their preparation is rather elaborate which may explain its rare use. so far, pancreas tissue slices have predominantly been used to address physiological or pharmacological questions, although they might also serve as valuable in vitro model for toxicological applications. therefore, this work aimed to establish and characterize rat pancreas tissue slices as in vitro model for studying drug-induced pancreatic toxicity. results will be compared to the responses of the permanent endocrine (ins- e) and exocrine (ar j) pancreatic cell lines to evaluate a potential added value. rat pancreas tissue slices were prepared by a protocol adapted from marciniak et al. (nat protoc, . ( ): p. . briefly, pancreas was infused and embedded with agarose. tissue sections of app. µm were prepared using a vibratome and maintained in cell culture medium for up to days. cell viability was determined by daily measurement of lactate dehydrogenase (ldh) in medium supernatants and by microscopic evaluation following fixation in % formalin and h&e staining. functional integrity of acinar and beta cells were assessed by cell-type specific secretory responses (i.e. insulin, amylase, lipase) to physiological stimuli. moreover, the effects of the pancreas toxins streptozotocin (stz), alloxan (all), and the cholecystokinin (cck) analogue cerulein on the viability and functional integrity of tissue slices were compared to the respective responses of the cell lines. we were able to establish an optimized isolation and cultivation procedure for rat pancreas tissue slices applying minor modifications to the original protocol. cell viability declined over the cultivation period. stimulation of the cell lines with glucose or cerulein increased secretion of insulin (ins- e cells) or amylase/lipase (ar j cells), respectively. the pancreas slices responded to both stimuli, demonstrating functional integrity of endocrine and exocrine cells. treatment of ins- e islet cells with the betacell toxicants all or stz only slightly affected islet cell viability, whereas treatment of ar j acinar cells with cerulein at supraphysiological concentrations had no effect. this set of experiments is currently completed by investigating the effects of all, stz and cerulein on the viability of acinar and islet cells in pancreas slices. our preliminary data demonstrate feasibility to prepare and cultivate rat pancreas tissue slices over a period of days thereby maintaining functional integrity to some extent. coculture of human monocytes with the keratinocyte cell line hacat in serumcontaining medium leads to higher sensitivity to weak contact allergens: an improvement for the loose-fit coculture-based sensitization assay (lcsa) a. sonnenburg , j. the loose-fit coculture-based sensitization assay (lcsa) has proved reliable for the in vitro detection of contact sensitizers in the past. however, the use of primary human keratinocytes has some disadvantages. to facilitate high throughput screening of chemicals, we replaced primary keratinocytes from the original assay setup (setup a) by the human keratinocyte cell line hacat. these cells were cocultured with monocytederived dendritic cells in serum-free medium (setup b) or fetal calf serum (fcs)containing medium (setup c). upregulation of the dendritic cell maturation marker cd assessed by flow cytometry served as endpoint. we have tested four substances known as sensitizers and four non-sensitizers in both new setups as well as in the original setup with primary cells. three out of four sensitizers ( , -dinitrochlorobenzene, -mercaptobenzothiazole, and coumarin) , and three out of four non-sensitizers (glycerol, monochlorobenzene, and salicylic acid) were correctly assessed under all culture conditions. the weak sensitizing potency of resorcinol was only detected by setup b with fcs supplemented medium. a false positive reaction to caprylic (octanoic) acid in all three setups confirms earlier results from our laboratory that some fatty acids are able to induce cd on dendritic cells in vitro. culture in fcs supplemented medium led to generation of dendritic cells showing a more pronounced upregulation of cd after application of substances with rather high sensitization potency compared to dendritic cells which are formed under serum-free conditions. therefore, we characterized dendritic cells from setups b and c by flow cytometric measurement of additional dendritic cell surface markers. dendritic cells from the original setup a had been characterized extensively before (schreiner et al., toxicology ; : - ) . dendritic cells generated in fcs supplemented medium were cd a+/cd c+, whereas dendritic cells from serum free culture conditions were cd a−/cd c− regardless whether cocultured with primary human keratinocytes or hacat. populations with cd a+/cd c+ dendritic cells in coculture seem to show a higher sensitivity to weak sensitizers, which proved beneficial for the identification of resorcinol. in conclusion, modification of the lcsa protocol led to an increased sensitivity of the assay. due to ethical and social reasons, in vitro assays are being developed to replace animal tests for addressing e.g. toxicological questions. for the induction of skin sensitization by chemicals, resulting in tolerance or allergic contact dermatitis after repeated exposure, prerequisites are the induction of inflammatory responses in keratinocytes supporting maturation of dendritic cells (dc), which is needed for the t cell response. although related in vitro assays consisting of one single cell type have good hazard prediction capacities, they have limitations in predicting sensitization potency. one drawback could be the lack of communication between keratinocytes and dc. with respect to the activation of keratinocytes and maturation of dc, intercellular communication between these two cells may include the release of danger molecules such as cytokines, damage-associated molecules such as atp, and metabolized chemicals. beside this, microrna (mirna), among them those that can regulate dc activation or maturation, can be differentially expressed upon stimulation but can also be transferred between cells. for skin sensitizers, we reported already that cross talk between hacat keratinocytes and thp- cells, as model for dc, enhanced cyp enzyme activity in hacat cells exposed to benzo[a]pyrene (b[a]p) and eugenol, belonging to a subgroup of chemicals (prohaptens) whose sensitizing potential depend on prior metabolic activation e.g. via cytochrome p (cyp) enzymes. furthermore, coculture clearly increased the upregulation of the cell surface molecule cd on thp- cells after incubation with these prohaptens and also several other skin sensitizers. in this study we further elucidate the cross talk between thp- cells and hacat cells by analyzing the impact of hacat cells on the expression of mirnas in thp- cells by microarray technology. we identified differentially expressed mirnas in cocultured thp- cells compared to monocultured thp- cells irrespective of the treatment (medium, . % dmso as solvent control, b[a]p). in the presence of dmso and b[a]p (after h) additional mirnas are differentially expressed. up to now it is not clear whether the cross talk between hacat and thp- cells comprises the exchange of mirna between the cocultured cells or whether it influences the expression of these mirna in thp- cells, or both. given that one mirna has several gene targets these results illustrate that the cross talk between thp- and hacat cells also impacts on the mirnome. walther-straub-institut der lmu-münchen, münchen, germany transient receptor potential (trp) proteins represent a large superfamily of nonselective cation channels sensing toxic stimuli in the human body. trpa expresses a high number of aminoterminal ankyrin repeats and is the only member of the trpa family. channel monomers form homotetramers in the plasma membrane with six transmembrane segments (tm) and a pore forming loop between tm and . trpa has been extensively described in sensory nerve endings as an important cellular detector for toxic stimuli and as an oxygen sensor (reviewed in ). although recently two reports identified trpa in pulmonary epithelial and endothelial cells ( , ) , its expression in non-neuronal tissues is still a matter of debate. after isolation and identification of different murine lung cells we were able to identify murine trpa protein in primary endothelial cells, pneumocytes type ii (atii) and fibroblasts by using specific antibodies in a western blot analysis, but not in cells from trpa -deficient mice. atii cells were identified by specific cell markers such as surfactant protein c and were further differentiated to ati cells characterized by their specific expression of podoplanin. quantitative trp expression patterns will now be evaluated by quantitative reverse transcription (rt)-pcr as well as utilizing nanostring ® technology in different lung cells. to characterize trpa on a cellular level we cultured a hek cell line stably expressing trpa ( ) . allylisothiocyanate (aitc) a specific activator as well as hypoxia and hyperoxia was able to induce ca + -influx in this cell line, which was blocked by the specific inhibitor a . in the future, we will utilize the isolated perfused lung model ( ) to quantify toxin-induced edema formation in ex vivo lungs from wt and trpa -deficient mice after exposure to potential toxic inhalation hazards (tih see ) to challenge the hypothesis of trpa as an important toxin sensor in the lung. by this strategy we hope to understand trpa function in lung cells and to evaluate trpa proteins as potential pharmacological targets for a specific therapeutic intervention during toxin-induced edema formation. metabolism by the intestinal microbiota is likely to contribute essentially to the plasma metabolite profile of the mammalian host organism and it requires adequate identification of effects of the microbiome on the endogenous plasma metabolite patterns. the current investigations present insights in the mammalian-microbiome cometabolism of endogenous metabolites. antibiotics have a profound effect on the micro-organism composition of the microbiome and hence on the mammalian-microbiome co-metabolism. the consequences, however, on the functionality of the microbiome (defined as the production of metabolites absorbed by the host) and which of these changes are related to the microbiome are not well understood. to identify plasma metabolites related to microbiome changes due to antibiotic treatment, we have employed a metabolomics approach. to this purpose broadspectrum antibiotics belonging to the class of aminoglycosides (streptomycin, neomycin, gentamicin), fluoroquinolones (moxifloxacin, levofloxacin) and tetracyclines (doxycycline, tetracycline) were administered orally for days to male rats including blood sampling for metabolic profiling after , and days. fluoroquinolones and tetracyclines can be absorbed from the gut whereas aminoglycosides cannot. to distinguish between metabolite changes caused by systemic toxicity of the antibiotics and microbiome related changes, the metabolites identified in the metabolome pattern were compared to a list of metabolites known to be produced by the gastro-intestinal micro-organisms. beside changes mainly concerning amino acids and carbohydrates, hippuric acid and indole- -acetic acid were identified as key metabolites being affected by antibiotic treatment. for each class the following gut metabolites were found to be unique: indole- -propionic acid for aminoglycosides, taurine for fluoroquinolones, indoxylsulfate, uracil and allantoin for tetracyclines. for each class of antibiotics specific and selective metabolome patterns could be established. the results suggest that plasma based metabolic profiling (metabolomics) could be a suitable tool to investigate the effect of antibiotics on the functionality of the microbiome and to obtain insight in the mammalian-microbiome co-metabolism of endogenous metabolites. drug-induced liver injury (dili) is still a major reason for termination of clinical trials and thus is an important concern in drug development. identification and prediction of dili in the clinic and in preclinical safety testing still relies on the classical clinical chemistry panel and histopathology with known limitations in sensitivity and specificity. in the last years bile acids (bas) have been studied as potential biomarkers to better characterize drug-induced liver injury with promising results (ellinger-ziegelbauer et al., ; luo, schomaker, houle, aubrecht, & colangelo, ; yamazaki et al., ) . to evaluate whether a targeted bile acid profiling via lc-ms/ms in plasma and liver tissue can improve assessment of liver injury, methapyrilene (mpy) a known hepatotoxin, or corresponding vehicle, was administered daily to male wistar rats at a low ( mg/kg) and a high ( mg/kg) dose. rats were sacrificed following , , or consecutive daily doses, or after recovery days following consecutive administrations of mpy or vehicle. in addition to bile acids which were determined both in plasma and tissue, conventional preclinical safety endpoints (histopathology and clinical chemistry) assessment and gene expression profiling was performed in liver to obtain mechanistic information about potential changes in regulation of bile acid levels. conventional findings included periportal necrosis, inflammation and biliary hyperplasia, and increased liver enzyme activity and bilirubin levels during the treatment phase. the bile acid pattern showed increased levels of conjugated and unconjugated bile acids in low dose and high dose groups compared to the controls after administration of methapyrilene. furthermore, although liver enzyme activity and bilirubin levels in serum were decreased again in the recovery groups, suggesting recovering liver injury, bile acid concentrations remained elevated with no signs of recovery. analysis of transcriptomics data revealed decreased levels of mrna encoding α-methylacyl-coa racemase (amacr) and days after dosing, a gene responsible for bile acid synthesis. membrane transport systems for bile acids like sodium/taurocholate co-transporting polypeptide (ntcp) and organic anion transporting polypeptide (oatp ) expression were down regulated as well, indicating that the increased bile acid concentrations in plasma and tissue could be attributable to reduced uptake by the hepatocyte. in summary the data suggest that targeted bile acid profiling could be used as potential biomarkers to enhance assessment of drug-induced liver injury. photorhabdus asymbiotica is an entomopathogen and emerging human pathogen causing soft tissue infections in humans. photorhabdus asymbiotica produces the bacterial protein toxin patox, which is cytotoxic for various cell lines and kills insect larvae. previous studies have established that patox harbors two enzymatic active domains, a glycosyltransferase and a deamidase domain. the glycosyltransferase domain inactivates host gtpases of the rho family by glcnacylation of a tyrosine residue in the effector binding loop, which results in the disassembly of the actin cytoskeleton. the deamidase domain deamidates a crucial glutamine residue in heterotrimeric gα i and gα q/ proteins, which renders the g proteins constitutive active. sequence and structural homology analyses of patox revealed a third domain (patox p ) resembling peptidases of the c protease family. patox p contains the conserved catalytic triade (c/h/d) of papain-like cysteine proteases and shares sequence similarity with effectors from yersinia pestis (yersinia outer protein yopt) and pseudomonas syringae (avirulence protein avrpphb). transient expression of patox p in hela cells induces cell rounding and indicates a cytotoxic potential of patox p . incubation of patox p with linearized bovine serum albumin (bsa) results in cleavage products of bsa assuming proteolytic activity of patox p . mutation of the catalytic cysteine in patox p prevents cleavage of bsa and blocks cytotoxicity. we were not able to observe autocatalytic cleavage of patox constructs under various conditions. the intracellular activity of the protease domain is most likely involved in the pathogenicity of patox. vitamin d metabolism -involved in triazole fungicide toxicity? a. lehmann background: in a -day rat feeding study with the azole fungicides cyproconazole (c), epoxiconazole (e), propiconazole (p), tebuconazole (t), prochloraz (pz) as well as combinations c+e and c+e+pz, a reduction of vitamin d (vitd) receptor mrna levels was reproducibly observed in adrenals for c, e and p. transcription of various enzymes related to vitd homeostasis (including cyp r , gc, cyp a, ugt a) in liver was also affected, while initial indications for modulation of renal cyp a and renal and hepatic cyp b could not be confirmed. a possible induction of parathormone (pth) was noted for the high dose of c, but statistical significance could not be shown. we have now performed supporting analyses for serum vitd levels, measured additional transcript levels and will provide a framework for the interpretation of the findings. methods: male wistar rats (n= for single substances, n= for combinations) were treated for days at dose levels tested based on noaels from -day subchronic feeding studies and ranged from noael/ to noaelx . quantitative rt-pcr analyses were performed on organ samples obtained at sacrifice. serum vitd levels were determined using the total ( -oh) vitamin d elisa (drg instruments gmbh, marburg, germany). results: the elisa established for diagnostic analysis of human serum and plasma samples could be applied to rat serum. vitd levels in control animals (n= ) were . ± . ng/ml (min/max: / ng/ml), i.e. in the range of values reported previously for rats. for the high dose of c ( ppm in food, n= ), there was a statistically nonsignificant reduction of vitd levels to . ± . % of the concurrent control (n= ). however, for of animals of this group, measured vitd level were below the range observed in pooled controls (n= ). an according follow-up is ongoing. qrt-pcr analysis of adrenal tissue showed deregulation of apoptosis related genes (p for c, e and pz; cdk and gadd a for e; cdkn c for c), which is in agreement with an involvement of vitd in the autocrine/paracrine regulation of cell proliferation. conclusion: reduction of circulating vitd levels would be plausible as a result of induction of hepatic cyp a / and ugt a. however, this could not be confirmed by elisa as a general mechanism for all azole fungicides under investigation. only for rats fed with ppm cyproconazole, there were indications for a moderate reduction of -oh vitamin d, which would correlate with the previously reported moderate increase in serum pth for this group. hansen's disease during pregnancy and lactation: two babies born to a mother using antileprosy drugs z. ozturk hansen's disease, also known as leprosy, during pregnancy has been rarely reported in europe and united states. early diagnosis is important, and medication can decrease the risk of those living with leprosy patients from acquiring the disease. this report presents a case of multidrug antileprosy therapy during pregnancy and lactation. a -year-old multiparous woman with a known case of multibacillary leprosy presented with unplanned pregnancy. her pregnancy was discovered in the th week, and she has been taking a multidrug therapy (dapsone mg/day, rifampicin mg/month, clofazimine mg /day and clofazimine mg/month) for the past months. diagnosis of leprosy was established in her previous pregnancy. the patient was informed about the risks of drugs used in pregnancy. the treatment was continued unchanged during pregnancy. a detailed fetal ultrasonography was offered to scan the development of the fetus at about weeks. in the th, nd, th weeks of pregnancy, prenatal sonographic examinations revealed normal fetal growth and amniotic fluid volume. at weeks pregnant, she was diagnosed with gestational diabetes. diabetes did not cause any symptoms during pregnancy, and it was controlled with a reduced-calorie diet in a week. the patient delivered a healthy baby girl by vaginal birth in the th week of gestation without perinatal complications. the baby was also healthy (apgar - , g, cm), and its growth and development were normal during a -month follow-up period. the patient decided to breastfeed while taking medication. she had a previous experience with use of anti-leprosy drugs while breastfeeding, her other child was months old and healthy. as well as in the first child, skin discoloration was observed in newborn due to clofazimine during lactation. after months, she stopped breastfeeding, and the infant's skin changes were reversed. for pregnant women and practitioners, treatment of leprosy in pregnancy can be complicated. physical and neurological damage may be irreversible even if cured. multidrug therapy consisting dapsone, rifampicin and clofazimine is highly effective for people with leprosy and considered safe, both for the mother and the child. antileprosy drugs are excreted into human milk but there is no report of adverse effects except for skin discolouration of the infant due to clofazimine. therefore, multidrug therapy for leprosy patients should be continued unchanged during pregnancy and lactation. methods: individuals included in the analysis were participants of the berlin initiative study (bis). bis is a population-based prospective cohort study initiated in in berlin, germany, to evaluate kidney function in people ≥ years. medication was assessed through personal interviews and coded using the anatomical therapeutic chemical classification system. for estimation of glomerular filtration rate (egfr) we used the ckd-epicr equation. predictor analysis was conducted via logistic regression. results: figure illustrates the percentage of drug use for the three noacs and phenprocoumon, the most common vitamin k antagonist in germany, over the course of four years. table shows the characteristics of patients for each oral anticoagulant group during the four-year follow-up visit (from january until april ). the probability of dabigatran use rose with increasing age (+ %), and the probability of phenprocoumon use rose in case of egfr < ml/min/ . m (+ %) or male sex (+ %). discussion: our data show that also in the elderly noac use increased over the past years. characteristics such as age, sex or kidney function had an impact on the choice of oral anticoagulation. objective: orthostatic hypotension (oh) is an important factor in determining cardiovascular mortality especially in older age. different factors were discussed to influence oh. arterial stiffness, medication and frailty were demonstrated as modifying factors of oh. the aim of this study was to assess prevalence of and influencing factors on oh in nursing home residents (nhr) in germany. methods: systolic (sbp) and diastolic (dbp) blood pressure as well as pulse pressure (pp) and pulse wave velocity (pwv) as markers of arterial stiffness were measured in nhr aged ≥ years in nursing homes in berlin, germany. measurements were first performed in the sitting position and then repeated after standing up. oh was defined as a sbp decrease of > mmhg and/or dbp decrease of > mmhg within min after standing up. hypertension was defined as the presence of diagnosis arterial hypertension, the prescription of at least one antihypertensive drug, or mean sbp values > mmhg and/or mean dbp > mmhg. information about antihypertensive medication was received from interviews and medical records. frailty was determined by geriatric assessments, e.g. "timed up and go test" (tug) or barthel scale. results: oh testing could be performed with nhr (mean age = . ± . years). in total, subjects ( . %) had oh. the mean change in sbp from sitting to standing was . ± mmhg (range + . to - . mmhg) in patients with oh and . ± . mmhg (range + . to - mmhg) in patients without oh. mean sbp was significantly higher ( . ± . mmhg) in people with oh than in those without ( . ± . mmhg). all of the nhr with oh were hypertensive compared to % of the nhr without oh. sex, mean age, pwv and pp was not significantly different between individuals with or without oh (p> . ). medication data was available for patients. all individuals with oh and nhr without oh ( %) had antihypertensive medication. more than different antihypertensive drugs were present in patients with oh ( . %) and in patients without oh ( . %). the intake of beta-blockers had no impact on oh development. geriatric assessments did not differ significantly between the oh group and the non-oh group. more than % of patients in both groups reached points as maximum in barthel scale defining a need for assistance and tug analyses demonstrated that around % of patients with oh as well as patients without oh needed more than sec showing a motor slowing. conclusion: we found a relatively low prevalence of oh in our very old patient cohort and the overall bp control was good. similar to earlier publications mean sbp was significantly higher in nhr with oh. all of the other investigated factors were not associated with the occurrence of oh. the small cohort size might have limited the detection of cardiovascular, epidemiological or geriatric associations. in addition, important confounding factors such as the inability to stand of some nhr and the lack of standardized fraility assessments must be addressed. impact of reticulated platelets on the initial antiplatelet response to thienopyridine loading in patients undergoing elective coronary intervention c. stratz , t. nuehrenberg are known to be involved in cell metabolism pathways and therefore ccrcc is supposed to be a metabolic disease. in order to facilitate a better understanding of cancer metabolism and to support tumor classification on the metabolite level we have developed a novel analytical approach for comprehensive metabolomic profiling of small molecules and lipids in kidney tissue. the method was established and validated based on porcine tissue and, as proof of concept, applied to a small cohort of human normal and ccrcc tissue samples for molecular tissue differentiation. methods: five fresh frozen ccrcc samples and corresponding normal tissue were used for cancer-specific metabolomic profiling and were derived from patients who underwent partial or radical nephrectomy. metabolites and lipids were recovered from tissue samples by a two-step extraction protocol. tissue homogenization and extraction of polar metabolites was performed in methanol/water (aqueous extract) by a beadbeating approach. lipids were recovered by consecutive extraction of the pellet with methanol/methyl tert-butyl ether (organic extract). metabolites in aqueous extracts were separated by hydrophilic liquid interaction chromatography whereas compounds in organic extracts were separated by reversed phase chromatography prior high resolution mass spectrometry. results: reproducibility of tissue extraction and metabolite analysis was assessed by the analysis of multiple individually prepared porcine kidney samples. more than metabolic features including amino acids, nucleotides, small organic acids, phospholipids, sphingolipids, glycerolipids and fatty acids could be reproducible (cv ≤ %) analyzed with the novel non-targeted metabolomics approach. the validated protocol was applied for metabolomic profiling of kidney tissue derived from ccrcc patients. based on unsupervised multivariate statistics, a clear differentiation between cancerous and normal tissue for the small metabolites profile as well as for the lipid profile could be observed. a first subset of differentially regulated metabolites responsible for tissue differentiation could be tentatively identified. conclusion: metabolomic profiling of kidney tissue extracts enables differentiation between ccrcc and normal kidney tissue samples based on the lipid and small molecule metabolomic profiles. further studies on larger and independent sample groups are necessary to confirm and validate our preliminary findings. in summary, the presented approach provides a first basis for comprehensive metabolomics studies in human kidney tissue and thus offers great potential for the metabolic characterization of ccrcc with important prognostic and therapeutic implications in the future. introduction: clomiphene (clom) citrate as mixture of trans-and cis-isomer ( : ) is the first line therapy for the treatment of infertility caused by the polycystic ovary syndrome. treatment schedule includes dose escalation from mg/d clom citrate to up to mg/d in case of non-ovulation. however, therapy outcome is variable and approximately - % of patients do not benefit from clom treatment. the pro-drug clom is bioactivated via -hydroxylation of trans-clom by the highly polymorphic cytochrome p (cyp) d leading to the major active metabolite trans- hydroxyclomiphene (trans- -oh-clom) [ ] . recently, we identified a less active trans- -oh-clom which is also formed by cyp d . besides the formation of the active metabolites, their plasma concentrations are influenced by their clearance e.g. via glucuronidation and sulfation. here we investigated the glucuronidation and sulfation of both hydroxyl-metabolites. methods: isoforms of udp-glucuronosyl-transferase (ugt) and sulfotransferase (sult) responsible for conjugation of oh-clom were identified using commercially available supersomes. glucuronidation and sulfation kinetics were determined in pooled human liver microsomes. conjugated clom metabolites were quantified in plasma and urine samples obtained from healthy female volunteers who received a single dose of mg clom citrate. results: incubations with human liver microsomes revealed an almost -fold higher glucuronidation rate for trans- -oh-clom, which is exclusively catalyzed by ugt b , compared to the more potent trans- -oh-clom. for the latter a pattern of multiple ugts was identified. in contrast, the intrinsic clearance of trans- -oh-clom to its sulfate is -fold higher compared to -oh-clom. for both metabolites a participation of sult a and sult e was identified. these results were in line with previous studies, which identified the same sults [ ] and ugts [ ] responsible for the conjugation of the structurally related trans- -hydroxytamoxifen. in addition, in vivo data from plasma and urine samples confirmed the reverse regioselective glucuronidation and sulfation of trans- -oh-clom and trans- -oh-clom. overall, concentrations of clomglucuronides were significantly higher than those of sulfates. highest concentrations in plasma and urine samples were measured for trans-clom- -o-glucuronide. conclusion: our results suggest a new metabolic route via trans- -oh-clom which appears to be a potential inactivation pathway of clom. institut für pharmakologie und toxikologie der bundeswehr, münchen, germany for decades the biological effect of sm has been investigated. it is well known how sm interacts and destroys cells. unfortunately, it is still unknown if and how a cell can become resistant against sm. within the here described experiments we investigated a new approach adapting cells to the presence of sm. over a time period of nearly three years the cells were cultivated in presence of sm with increasing concentrations. before starting the initial sm sensitivity was investigated. at the beginning cells were cultivated with a concentration of . µm sm (ic ). today the cells are able to tolerate a concentration of . µm sm (ic ), which reflects to a concentration of which % of the original cells would have died. to determine cellular characteristics, the resistant cells were compared with wildtype cells. the following cell characteristics were investigated: proliferation, apoptosis, clonogenicity, size of nuclei and cytoplasm, cell-cell contacts, dna adducts formation, secretome, screening of mirna expression, next generation sequencing, vital observation and scratch assay, nad(p) + /nad(p)h, h o , glutathione, ca + -influx, mdrchannels, resistance to other alkylating agents and the reversibility of the resistance. the resistant cells demonstrate smaller nuclei and cytoplasm, less dna adducts, a higher clonogenicity as well as proliferation and less apoptosis. the secretome analysis showed an up-regulation of anti-apoptotic acting cytokines timp and ang and the proproliferative acting cytokines timp and pdgf-aa. in contrast, immunologically active cytokines were down-regulated. concerning cell-cell contacts no differences were seen. in the mirna screening significant up-regulated and significant down-regulated mirnas have been observed. noteworthy was the regulation of various members of different families. during vital observation and in a scratch-assay the resistant cells were show to have disadvantages. the observed resistance was not unique for sm but also towards other alkylating agents and cytostatic drugs. by analyzing the reversibility cells stayed resistant over more than weeks. in conclusion, many aspects investigated in this study have an influence on the sm resistance, pointing out that it is a combination of various effects that are involved to switch on resistance. more likely, there are many aspects working together. the present results are an important step in the characterization of the sm-resistant cell line and further studies may be able to directly use these as a start for target identification in antidote or prophylactic agent discovery. the arylhydrocarbon receptor (ahr) is localized in a cytosolic complex that contains several co-chaperones and associated factors. the protein is shifted into the nucleus in response to endogenous and xenobiotic ligands. however, a transient nuclear transport does also occur in the absence of any ligands, while the predominant cytoplasmic compartmentalization is maintained by parallel export. we have analyzed the interplay between this basal nucleo-cytoplasmic shuttling and ligand induced transport in hepg cells, using a yfp-tagged fusion protein that is capable to respond to ligands and to trigger the induction of cyp a expression. basal import was assessed in cells that had been treated with leptomycin b (lmb), an inhibitor of crm -mediated nuclear export. interestingly, the apparent ahr import rate in lmb-treated cells was comparable with nuclear import as trigged by xenobiotic (b-naphthoflavone) or endogenous (kynurenine) ligands. this observation was confirmed for endogenous ahr in hepg cells, since both ligands and lmb showed comparable effects on nuclear compartmentalization. however, the basal nuclear import rate in lmb-treated cells was strongly increased by ahr ligands. ligand-induced nuclear transport was therefore confirmed as an import step in receptor activation. interestingly, lmb did also accelerate nuclear import of ahr after pretreatment of cells with ahr ligands. these data suggest that nuclear export of the ahr is maintained in the presence of ligands. receptor activation might therefore comprise several rounds of shuttling, thereby involving both accelerated import and continued export of the ahr protein fraction that has not already undergone interactions with arnt or dna. we suggest that nuclear export provides an additional kinetic control of ahr activation and function. mitochondrial toxicology: rescuing mitochondria in wilson disease avoids acute liver failure h. zischka institut für molekulare toxikologie und pharmakologie, ag zischka, neuherberg, germany in wilson disease (wd) functional loss mutations in the hepatocyte atp b gene cause dramatic copper overload leading to acute liver failure, posing an unmet therapeutic issue. we find that the pathology of severe wd cases is mirrored in lpp (-/-) rats carrying a functional loss atp b mutation. this is especially apparent in the hepatocyte mitochondrial compartment. a progressive copper deposition increasingly harms the lifesustaining mitochondrial membrane integrity. thus, depleting this devastating mitochondrial copper burden is a core requirement for a treatment strategy against acute liver failure in this wd animal model. preparation for the master degree program in toxicology started in as a cooperation of charité universitätsmedizin berlin with the university of potsdam and other institutions of the region. first enrollment of students was done in . the program was accredited in by the central evaluation and accreditation agency. it offers a modern curriculum encompassing a wide variety of scientific aspects with an interdisciplinary character. this training program in toxicology is organized in modules and ends with the degree "master of science" (m.sc.). the goal of this program in toxicology is to teach the basis of the interactions between substances at toxic concentrations and living organisms, as well as the molecular mechanism of the adverse effects of chemicals. the understanding of the mechanism of a toxic action is an important prerequisite for the scientifically based evaluation of a hazard associated with a substance. furthermore, only with the knowledge of the mechanism of action and a deduction of structure activity relationships it is possible to predict toxic effects of new substances. this knowledge should enable students to perform a risk evaluation of chemicals or to predict the adverse effects of chemicals with the aim that human beings and the environment can be protected from harmful consequences of chemical exposure. the program allocates places per year to an average of applicants. most applicants have a basic training in the fields biology, chemistry, pharmacy, veterinary medicine and nutritional sciences. about % of the students are female. the majority of them have a bachelor's degree before starting the master program, other degrees are diploma and state examination as pharmacists or physicians. ninety percent of the students pass the final examination consisting of the master's thesis and disputation at the end of the four semesters. afterwards, most of the graduates aim to obtain a phd degree. the program is well established in the education of toxicologists in germany. respiratory injury due to chlorine developed from consumer products. still an issue in germany u. stedtler , m. hermanns-clausen uniklinikum freiburg, vergiftungs-informations-zentrale, freiburg, germany objective: in the last decades strong effords have been took to improve product safety, especially in products intended for domestic use. hypochlorite-containing cleaners may develop chlorine gas when acidified e.g. by adding an acid sanitary cleaner. usually these cleaners contain sodium hydroxide or other strong alkalines to avoid this reaction. we analysed reports to our poisons center concerning inhalation exposure to chlorine developed from hypochlorite-containing mixtures. method: retrospective search in the case database of the poisons center. human inhalative exposures to chlorine released from mixing hypochlorite as well as human inhalative hypochlorite exposure alone were analysed. frequency and symptoms were compared. results: from to in total cases of human exposures to chlorine developed from mixtures of hypochlorite and acids ( . of cases) were registered. in cases the exposure was due to mixtures of products intended for domestic use. % of the exposed patients reported symptoms. only in two cases the symptoms were not considered to be caused by the inhalation accident. most frequent symptoms reported were (percent of symptomatic patients): cough ( %), dyspnea ( %), irritated upper airway ( %), abdominal discomfort (pain, nausea, vomiting) ( %), thoracic pain ( %), irritated eyes ( %), dizziness ( %), and bronchospasm ( %). further symptoms were malaise, headache, irritated nose, sweating, muscle pain, and others. in patients ( %) the symptoms were graded as moderate severe. main symptoms in this group were dyspnoea ( % ), cough, and irritated airway. one third of the patients experienced bronchial obstruction. all symptomatic patients developed symptoms while exposed or shortly after exposure. there were no severe or fatal cases (especially no lung edema) and all symptoms were expected to resolve completely. because hypochlorite containing procucts sontanously release "chlorine-like" smelling gases, we additionally analysed inhalation exposures to hypochlorite solutions alone in the same period. there were patients in the same period exposed to hypochlorite evaporation alone. of them ( %) had symptoms of which in cases these were considered to be caused or possibly be caused by the hypochlorite. most frequent symptoms were irritated upper airway ( %), nausea or vomiting ( %), cough ( %), irritated eyes ( %). dyspneoa was less fequent than in the mixture group ( %). all symptoms were considered mild. there was no bronchospasm or thoracic discomfort. conclusion: respiratory injuries by chlorine from hypochlorite-containing solutions still occur despite clear warning on the label. the majority of cases was due to products for domestic use. symptoms develop shortly after exposure. the γh ax assay for genotoxic and nongenotoxic agents: comparison of h ax phosphorylation with cell death response perturbation of mitosis through inhibition of histone acetyltransferases: the key to ochratoxin a toxicity and carcinogenicity? regulation of chromatin by histone modifications transcriptomic alterations induced by ochratoxin a in rat and human renal proximal tubular in vitro models and comparison to a rat in vivo model in vitro gene expression data supporting a dna non-reactive genotoxic mechanism for ochratoxin a fragment ion patchwork quantification for measuring site-specific acetylation degrees combinatorial patterns of histone acetylations and methylations in the human genome inroads to predict in vivo toxicology -an introduction to the etox project value of shared preclinical safety studies -the etox database acknowledgements: support of the bfr through grant - is gratefully acknowledged. acknowledgement: supported by the robert bosch foundation, stuttgart, germany. [ ] mürdter t, et al. hum mol genet, , : - [ ] nishiyama t. et al., biochemical pharmacology, , : - [ ] sun d. et al., drug metabolism and disposition, , : background: infections are a major problem in patients with burn diseases (bd). due to severe injuries of their total body surface area (tbsa), burn patients have altered pharmacokinetic characteristics. therefore, insufficient plasma concentrations may be achieved, when standard dosing schedules are applied for antibiotics such as piperacillin. for time-dependent antibiotics, the duration how long drug concentration exceeds the minimal inhibition concentration (mic) is crucial for their antibacterial effects. since pseudomonas spp. is the main problematic pathophysiological bacterium for bd patients. the aim of the present study was to monitor the plasma concentrations of piperacillin during piperacillin/tazobactam treatment in bd patients. patients from intensive care units (icu) served as controls. methods: bd patients ( / m/f, . ± . y, tbsa . ± . %) and patients ( . ± . y) from the icu were included in this observational study. blood samples were taken within the rd interval of the h-lasting dosing period of piperacillin/tazobactam ( / . g within . h) at , and . h after the end of infusion. total and free piperacillin concentrations were determined in plasma using hplc-uv after deproteinisation with acetonitrile and by ultrafiltration, respectively. pharmacokinetic parameters and dosing simulations were calculated by tdmx (www.tdmx.eu). free plasma concentrations of piperacillin exceeding at least xmic but preferably xmic over the whole dosing interval were considered to be sufficient for antibiotic efficacy (mic mg/l for pseudomonas spp.,www.eucast.org). results: the pharmacokinetic parameters of total piperacillin, calculated for each bd or icu patient using the concentrations at , , and . h, were as follows: c max . ± . vs. . ± . mg/l, p< . , half-life . ± . vs. . ± . h, p> . , clearance . ± . vs. . ± . l/h, p< . , volume of distribution . ± . vs. . ± . l, p< . . free concentrations (which were included in tdmx calculations) were ± vs. ± % (p< . ) of total concentrations. duration per day while concentrations exceeded xmic ( . ± . vs. . ± . h, p< . ) or xmic ( . ± . vs. . ± . h, p< . ) were lower in bd than in icu patients. moreover, tdmx simulations predicted that the duration per day for xmic could be enhanced to . ± . h if the piperacillin amount will be increased to x g/d and the infusion duration to h. pharmacokinetic parameters have, however, to be determined in a pilot study with bd patients to ensure predicted values. conclusions: standard dosage regimens for piperacillin/tazobactam could result in suboptimal plasma concentrations of piperacillin in bd patients as well as in icu patients. drug monitoring and tdmx simulation of kinetic parameters may easily help to improve piperacillin treatment in bd patients. background: high dose methotrexate (hd mtx), defined as > mg mtx/m bodysurface-area (bsa) is used in children to treat a variety of malignant diseases since the s. clinicians observe relevant rates of severe unwanted side effects. identifying patients having an increased risk for toxicity due to altered mtx pharmacokinetics is urgently needed. we aim to develop and evaluate a physiology-based pharmacokinetic (pbpk) model for hd mtx in children using pk-sim® (bayer technology services gmbh, leverkusen, germany) with a special emphasize on relevant covariates. methods: in this non-interventional observational study, children receiving hd mtx intravenously at two major german pediatric oncology departments during the years - were included if at least one mtx serum level (mtx-sl) was determined during clinical routine. patients aged - years (male = , female = ) with following diagnoses were included: acute lymphoblastic leukemia, non-hodgkin lymphoma, burkitt lymphoma, brain stem glioma and glioblastoma multiforme. in total, mtx treatment cycles corresponding to mtx-sl were used in this study. patients were randomized into two patient sets (training set and test set). based on literature data, mtx pbpk-models were developed and slightly adapted taking into account mean relative deviation (mrd) and bias of predicted versus observed mtx-sl of the training set. the pbpk model with the lowest mrd and bias was chosen and finally evaluated using the test set. the impact of the covariates urine ph < . , trimethoprime/sulfamethoxazole, proton-pump-inhibitors, non-steroidal anti-inflammatory drugs and ß-lactam antibiotics on the prediction quality was assessed using the mann-whitney u test. ochratoxin a (ota) is a wide-spread food contaminant and one of the most potent renal carcinogens [ ] . recent data by our group demonstrate that ota inhibits histone acetyltransferases (hats), thereby causing a global reduction of lysine acetylation of histones and non-histone proteins [ ] . based on these findings and the importance of specific histone acetylation marks in regulating gene transcription [ ] , we speculated that repression of gene expression as the predominant transcriptional response to ota [ , ] may be linked to loss of histone acetylation. in this study we therefore used a novel mass spectrometry approach, which is based on chemical acetylation of unmodified lysine residues of histones using c-labeled acetic anhydride and subsequent calculation of the degree of acetylation based on the measured intensities of heavy and light acetylated isotopologues [ ] , to identify and quantify site-specific alterations in histone acetylation in human kidney epithelial (hk- ) cells treated with ota. our results demonstrate ota-mediated loss of acetylation at almost all important lysine residues at histones h a, h b, h and h . we further selected acetylation at histone h lysine (h k ), a well-known euchromatic hallmark that is elevated at promoter regions of transcriptionally active genes [ ] and which was reduced from ~ % in controls to < . % in response to ota, to establish a link between loss of h k acetylation and expression of genes consistently shown to be down-regulated in response to ota [ , ] . using chromatin immunoprecipitation followed by quantitative real-time pcr (chip-qpcr), we observed ota-mediated loss of h k acetylation at promoter regions of the selected genes (% of controls: amigo : %, clasp : %, ctnnd : %). overall, these data provide first evidence for a mechanistic link between h k hypoacetylation as a consequence of ota-mediated inhibition of hats and repression of gene expression by ota. a new paradigm to assess the proarrhythmic potential of drugs is proposed by the cipa (comprehensive in vitro pro-arrhythmia assay) initiative combining a suite of a priori in vitro assays ( most important ion channels for cardiac activity) coupled to in silico reconstructions of cellular cardiac action potential (ap). the etox consortium has developed a multiscale simulation in silico model based on o'hara/rudy incorporating the principles of this new paradigm. the core model simulates the effects of drugs on a virtual cardiac tissue composed by different types of cardiomyocytes. the input of this model, the blockade of a set of ion channels (ikr/herg, iks, ical), can be obtained experimentally or predicted using advanced d-qsar models. the system predicts the % change of the qt interval at different drug concentrations in order to facilitate risk assessment. this in silico model was validated using purkinje fiber assay results (input: ap prolongation and arrhythmogenic risk assessed by early after-depolarisation occurrence) from in-house drug candidates. the validation showed that predictivity is highly dependent on the model's applicability domain (ad): for some chemical series the proarrhythmic potential could not be identified, for others, however, most of the positive drugs were correctly predicted with sensitivities up to - % (average prediction accuracy was %). retraining of this model with additional internal data should help to improve the model ad and predictivity. it is important to note that ap prolongation was correctly predicted for many proarrhythmic drugs with only low (> µm) in vitro herg inhibition. furthermore, the model showed high additional benefit for read-across within bayer pharma ag, investigational toxicology, berlin, germany etox [ ] [ ] started in and is a public-private partnership project within the european innovative medicines initiative (imi) [ ] . the etox project is building a toxicology database relevant to pharmaceutical development and to elaborate innovative strategies and software tools. the overall goal is to better predict the toxicological profiles of new chemical entities in early stages of the drug development pipeline based on existing in vivo study results contributed by the participating efpia * companies in the consortium. the etox database is a relational database with a specifically designed schema to store complex and comprehensive preclinical safety data like the study design, toxicokinetics, adme data, clinical chemistry, hematology, gross necropsy, histopathological findings and general toxic effects. in addition relevant data from public sources has been included into the database. the primary focus for data collection are systemic toxicity (up to week) repeated dose studies, mostly in rodent. overall more than study reports for approximately investigated compounds. in order to optimize the usage and mapping of data from different sources the development of common ontologies was a key task within the project. this timeconsuming step was necessary to make a high quality read-across analysis possible and valuable. therefore the ontobrowser [ ] tool was developed to curate and harmonize the verbatim terms to standardize terms which are used within the etox database. until now more than million verbatim terms were curated. additionally to the toxicology database, a web-based user interface called etoxsys was developed to allow the retrieving of toxicity information, as well as the prediction of toxic endpoints for chemical compounds. due to the complex search capabilities, the database can be queried for structural similarity, similar target classes and specific toxicological endpoints. approximately prediction models based on public data are available and first models based on in vivo data are in development. the etox database therefore represents a valuable tool for early animal-free assessment of drug candidates [ ] . * european federation of pharmaceutical industries and associations cell lines background: consumers are constantly exposed to chemical mixtures e. g. to multiple residues of different pesticides via the diet. this raises questions concerning potential cumulative effects, especially for substances causing toxicity by a common mode of action. since substances are tested for regulatory purposes on an individual basis at generally high dose levels, there is only limited data available on potential mixture effects especially in the low dose range. with more than active substances approved for being used in pesticides and over chemicals registered under reach there are more possible combinations than one could test with classical animal experiments. the development of in vitro tools for assessment of mixture effects consequently is of tremendous importance. methods: as a first step in the development of such in vitro tools we used a group of fungicides, (tri-)azoles, as model substances in a set of different cell lines from known target tissues, basically liver (human: hepg , heparg, rat: h iie) and adrenal gland (human: h r). concentrations were taken from measured tissue concentrations in vivo to ensure that used concentrations of the (tri-) azoles reflect realistic effect levels. the cell lines were exposed with the triazoles cyproconazole and epoxiconazole as well as with the azole prochloraz as individual substances and in binary or ternary combinations of these substances at three dose levels and three different time periods. the effects of the substances were subsequently analysed by transcriptomics and metabolomics. a support vector machine will be utilized to integrate the data from the different sources to gain a complete picture of affected adverse outcome pathways and mechanistic information about the applied fungicides. first results indicate combination effects of the substances also at the omics level depending on the specific endpoint and the concentration used. some of these are comparable to effects found with similar methods in a standard toxicity test, a -day feeding study in the rat, thus raising hope for the development of in vitro methods suitable to detect combination effects. background: plant protection and biocide products are chemical mixtures, which contain one or more active substances as well as several co-formulants (e.g. solvents, wetting agents, thickener or preservatives). nevertheless, to this day extensive toxicological testing is performed only with the individual active substances, while the plant protection products are only evaluated for acute toxicity, ie, a single dose group experiment with rats is performed as well as testing for skin-and eye-irritation. current pesticides regulation foresees testing of potential harmful mixture effects but only when adequate methods are available making the development of such methods a high priority. several published studies both in vitro and in vivo have shown fortified toxic effects of plant protection products compared to individual active substances. methods: here we present effects of plant protection products as a whole as compared to the individual active substances or co-formulants in a set of human cell lines of hepatic and renal origin (hepg , heparg, hek ). cytoxicity has been analysed by wst- and nru assay as well as gene expression of several marker genes involved in xenobiotic metabolism. additionally reporter gene assays have been conducted for nuclear receptors such as ahr and car. results: while some active substances showed lower toxicity as compared to the respective products, this cannot be confirmed as a general rule for all endpoints for all of the analysed fungicides or herbicides containing active substances such as epoxiconazole, cyproconazole, azoxystrobin or glyphosate. chemical compounds may induce skin sensitization in humans, resulting in tolerance or allergic contact dermatitis after repeated exposure. mechanistically, the activation of dendritic cells is one of the prerequisites for the induction of skin sensitization. a subgroup of sensitizing chemicals, prohaptens, need metabolic activation, e.g. via cytochrome p (cyp) enzymes. thus, xenobiotic metabolism may crucially impact on a chemical's potential for the induction of skin sensitization by activation, but also deactivation of reactive molecules via conjugation, which determines the concentration and the chemical species available for protein haptenation and cell activation. we established a coculture model consisting of hacat keratinocytes and thp- as surrogate dendritic cells for the detection of sensitizing chemicals and found enhanced cyp enzyme activity in hacat cells exposed to benzo[a]pyrene (b[a]p) and eugenol as well as clearly increased expression of cell surface molecule cd on thp- cells after incubation with these prohaptens (hennen et al., ) . here, we studied the impact of intercellular cross talk on activation and conjugation capacities in more detail. treatment of thp- with b[a]p and eugenol in coculture with hacat cells augmented cyp a and/or cyp b mrna levels, while this was not found for thp- monoculture. augmentation of cyp a mrna needed continuous presence of hacat cells. in coculture, levels of -oh-b[a]p as exemplary cyp-dependent metabolite were increased compared to single cultures. in contrast to this, total glutathione contents as well as n-acetyltransferase enzyme activities in both cell types were not modulated in coculture, furthermore the capacity for sulfation/glucuronidation of -oh-b[a]p was maintained in coculture. additionally, the decrease of the total glutathione content in thp- cells by , -dinitrochlorobenzene (dncb) was much less pronounced when exposed in coculture with hacat cells, showing that hacat cells provide additional targets for cysteine-reactive chemicals such as dncb, diminishing the total amount of chemicals available for thp- cells.overall, results indicate that the cross talk between keratinocytes and antigenpresenting cells enhances their capacities for metabolic activation of chemicals, while hacat cells also provide supplementary capacities for phase ii reactions. references: hennen j et al. cross talk between keratinocytes and dendritic cells: impact on the prediction of sen-sitization. toxicol sci : - .toxicology -toxic pathway analysis/aop background: reticulated platelets are associated with impaired antiplatelet response to thienopyridine treatment. this interaction might be caused by intrinsic properties of reticulated platelets or a decreased drug exposure due to high platelet turnover reflected by reticulated platelets as surrogate. we investigated the impact of reticulated platelets on antiplatelet response to thienopyridines and if this effect is linked to platelet turnover. methods: this study randomized elective patients to loading with clopidogrel mg or prasugrel mg (n= ). adp-induced platelet reactivity was assessed by impedance aggregometry to minutes and day after loading but before intake of the next dose of thienopyridines. immature platelet count (ipc) was assessed as marker of reticulated platelets by whole blood flow cytometry. results: platelet reactivity increased with rising tertiles of ipc (figure) . this effect was more pronounced in patients on clopidogrel as compared to patients on prasugrel. overall, ipc correlated well with on-treatment platelet reactivity at min (r= . ; p< . ). this correlation did not change over time indicating an effect independent of platelet turnover (comparison of correlations min/day : p= . for clopidogrel, p= . for prasugrel). conclusion: a high immature platelet count is associated with impaired response to thienopyridine loading. this effect is independent of platelet turnover indicating a relation to intrinsic properties of reticulated platelets. introduction: one of the biggest drawbacks of protein-based therapeutics with intracellular targets is their inability to enter the cytosol. targeted toxins are known to be used in drug delivery. aim of the study was to target epidermal growth factor (egf) receptor overexpressed on pancreatic carcinoma using a novel well-defined targeted toxin consisting of egf fused to the toxic plant ribosome-inactivating protein dianthin and a glycosidic triterpenoid (so ) as efficacy enhancer. methods: the enzymatic activity of dianthin-egf was verified by an adenine release assay. the kinetics of cytotoxicity were evaluated in pancreatic adenocarcinoma bxpc- and miapaca- cells in comparison to the non-target cell line nih t with an impedance-based real time cell analyzer (xcelligence) and final cytotoxicity analyses with conventional end-point mtt assays. acute toxic of dianthin-egf was studied in male balb/c mice. a xenograft solid tumor model was developed in male nude mice by injecting bxpc- cells into the dorsal part subcutaneously. dianthin-egf was administered at the vicinity of the tumor and so by subcutaneous injection at the neck. after the tumor reached a diameter of to mm in size treatments were given in total. tumor volumes and body weight shifts were observed twice weekly to determine the potency of dianthin-egf when given alone and in combination with so in comparison to placebo. immunohistochemical detection of egf receptor was performed according to the manufacturers's advice (dako, glostrup, denmark, k ). complete blood count analysis was done by labor gmbh, berlin. results: the adenine release mediated by dianthin-egf was . pmol adenine/pmol toxin/h. the in vitro efficacy of the targeted toxin was proven by an ic value of approximately nm for egf receptor expressing miapaca- and bxpc- cells as compared to nm for non-target nih t cells. real time measurement of the cytotoxicity showed a dose-dependent decrease in cell viability from pm to µm. toxicity studies in balb/c mice revealed . µg/mouse to be non-toxic and maximum tolerated dose (mtd) whereas µg caused moribundity accompanied with white ocular discharge. efficacy studies were performed for a period of days. the combination therapy showed that the average tumor volume measured by a digital vernier caliper was found to be % less than for placebo whereas single therapy using dianthin-egf alone caused a further increase in tumor volume which was although yet % less when compared to placebo. immunohistochemistry slides showed egf receptor expression in each of all untreated xenograft tumors, which further confirms the presence of egf receptor overexpression in the target bxpc- cell line. enlarged spleen was only observed in untreated xenografts. no significant change in various blood parameters (rbc counts, wbc counts, hgb, hct, mcv, mch and mchc) were observed on hematological analysis except for the platelet (plt) counts in comparison to healthy male nude mice. conclusion: combination therapy with so proves to be a promising approach for the targeted delivery of toxins instead of single therapy administering targeted toxin alone. the strategy is specific for egf receptor overexpressing tumors such as pancreatic cancer. introduction: moringa oleifera (mo) is a popular herbal supplement used for treatment and management of diverse diseases in sub-saharan africa. its intake among individuals infected with hiv/aids has increased recently due to the purported immune boosting property. limited information, however, is available regarding its potential to cause interactions with commonly prescribed medications that are substrates of cyp a and p-glycoprotein. methods: the methanol extract and four fractions of mo were tested on recombinant cyp a at different concentrations with and without nadph to determine the ic shift reduction. the crude methanol extract of mo was incubated with testosterone (tst) and cryopreserved hepatocytes to evaluate its influence on clearance of tst. effect of mo on the efflux transporter, p-glycoprotein was investigated by incubating the methanol extract with mdr -mdckii cells. virtual screening was conducted to predict physicochemical properties, bioavailability and interaction potential of phytochemical compounds unique to mo using combination of molinspiration version . and admetsar. results: fractions (f -f ) indicated ic shift reduction ≥ post-incubation with and without nadph. mo showed moderate interaction (auc i /auc = . ) with tst in cryopreserved hepatocytes. also, mo mildly inhibited the transport of digoxin (ic = . µg/ml) across mdr -mdckii cells. niaziminin indicated . % bioavailablity via the human intestinal membrane with % chance of inhibiting cyp a . βsitostenone showed strong p-gp inhibition ( . %) with % absorption via the intestine. conclusions: mo has the potential to inhibit the metabolism or excretion of other medications that are eliminated by cyp a or p-glycoprotein, respectively, if adequate amounts of the active constituents such as niaziminin and β-sitostenone enter the circulation. background: herb-induced liver injury (hili) has attracted attention in the past years due to an increasing number of publications reporting cases of hepatotoxicity associated with use of phytotherapeutics. here, we present data on hili from the berlin case-control surveillance study fakos. methods: fakos was initiated in to study serious toxicity of drugs including hepatotoxicity. potential cases of liver injury were ascertained in more than departments of all berlin hospitals from october until december . through a standardised face-to-face interview and review of medical charts information on all previous intakes of drugs or herbals, on co-morbidities, and demographic data was ascertained. inclusion criteria were an elevation of alanine aminotransferase or aspartate aminotransferase threefold above the upper limit of normal or an elevation of total bilirubin higher than mg/dl. excluded were patients with underlying liver disease (e.g., alcoholic fatty liver disease). drug or herbal aetiology was assessed based on the updated council for international organizations of medical sciences (cioms) scale. results: of all cases of hepatotoxicity included into the fakos study, herbs were involved in ten cases ( . %). demographic, clinical, and laboratory characteristics of these ten cases are illustrated in table . among the six patients with available liver biopsy results, five patients showed signs of necrosis, either disseminated or predominantly near the central vein. portal inflammation was more common than lobular inflammation, and the infiltrates contained mostly lymphocytes, neutrophil or eosinophil granulocytes. herbal aetiology was judged two times as probable (ayurvedic herb in patient , pelargonium sidoides in patient ), and eight times as possible (valeriana in patients , , , , , mentha piperita in patient , hypericum perforatum in patient , eucalyptus globulus in patient ). in nine cases other non-herbal drugs were also suspected as potentially hepatotoxic (exception: patient ). seven cases occurred in the ambulatory setting requiring hospitalisation, three cases occurred during hospital stay. discussion: this case series provides further information on laboratory and clinical aspects of hili. it corroborates known risks for valeriana and ayurveda treatment, and suggests that further herbals rarely or never associated with liver injury before such as pelargonium sidoides, hypericum perforatum or mentha piperita could also exhibit a hepatotoxic potential. clinical routine often requires to evaluate the cause of a newly occurring adverse event. if this event is regarded to be iatrogen, further information of the association between the drugs in the current medication list and the adverse event is needed. this information should ideally reflect the true risk and allow ranking of the drugs according to this risk to identify which drug to discontinue first. we discuss the summary of product characteristics (spc), the sider side effect resource and openvigil as possible sources of information. spcs are becoming more and more a vindicative charter for pharmaceutical companies that contain misleading information which is not based on evidence (ref. ). since it relies on the spcs, sider inherits these shortcomings and flags warnings that result from confounding factors (ref. , fig. ). furthermore, if any rates are given, they are not easily comparable since they stem from different studies. pharmacovigilance data are biased by the very nature of the data and the collection method. however, once confounders are eliminated, pharmacovigilance offers better information om how to rank the drugs than spcs/sider. we present decision-guiding information obtained by sider and by openvigil for one of our patients ( fig. & ) and discuss how this information was used to modify the therapy. institut für naturheilkunde und klinische pharmakologie, universität ulm, ulm, germany background: differences (polymorphisms) in target genes or genes encoding drug transport proteins or drug metabolizing enzymes may be responsible, among other factors, for observed variation in patients' response to medications. pharmacogenetics aims at identification of patients at higher, genetically determined, risk of adverse drug effects or ineffective medication, to modify dosage or switch to alternative therapy. there is, however, a lack of awareness of pharmacogenetic-based clinical practise guidelines. methods: a systematic literature review was conducted which focused on published guidelines on genotype-based (germ-line genetic variants) dosage modification or selection of drugs. we serched the medline and the pharmacogenomics knowledgebase (pharmgkb) databases. prescribing information was also screened for pharmacogenetic guidance. results: the systematic review revealed recommendations for drugs (table) that enable the translation of genetic test results into actionable prescribing decisions. for % of these drugs the respective german drug labels recommend or even require pharmacogenetic testing (table, rd column). although pharmacogenetic testing is recommended, the prescribing information not always provides guidance on how to adjust the drug dosage based on the pharmacogenetic test result. compared with the german or european drug labels, the fda drug labels povide more detailed information on pharmacogenetic dose modifications. conclusions: academic working groups have a front-runner role in the development of prescribing recommendations based on genetic markers. to date, drug labels rarely contain detailed guidelines how available genetic test results should be used to adjust drug dosage. because pharmacogenetics has a growing role during drug development and pre-prescription genotyping will become more widespread, it is expected that specific pharmacogenetic guidance for the treating physicians will become increasingly important. bisphenol a (bpa) is a high production volume compound mainly used as a monomer to make polymers for various applications, including food-contact applications. people are exposed to low levels of bpa because very small amounts of bpa may migrate from the food packaging into foods or beverages. however, other potential sources of exposure, such as dermal contact have also been identified (efsa, ) . a substance evaluation process (corap) was initiated for bpa by the european chemicals agency (echa). as part of the safety evaluation of bpa, a study was required by echa to assess absorption and metabolism of bpa following dermal exposure to human skin. an in vitro study with human skin was requested according to oecd tg under consideration of the scientific committee on consumer safety (sccs) criteria for the in vitro assessment of dermal absorption. to investigate potential dermal bpa metabolism fresh human skin was used. abdominal skin was obtained fresh from surgery from different donors. split-thickness human skin membranes were mounted into flow-through diffusion cells (n= per dose and donor) and the receptor fluid was pumped underneath the skin at a constant flow rate. the skin surface temperature was maintained at °c throughout the experiment and electrical resistance barrier integrity testing was performed at the start ( h) and end of the experiment ( h). four test preparations at final bpa concentrations of . , , , and mg/l were investigated. the highest concentration was chosen based on the maximum solubility of bpa in water and the lowest concenration was chosen based upon the specific activity of the radiolabelled [ c]-bpa that could be used for mass balance. percutaneous absorption was assessed by collecting receptor fluid (tissue culture medium (dmem), containing ethanol (ca %, v/v), uridine '-diphosphoglucuronic acid (udpga, mm) and '-phosphoadenosine- '-phosphosulfate (paps, µm)), at multiple time points througout the experiment. at termination the skin was removed from the cells and the stratum corneum was removed with successive tape strips. the exposed epidermis was separated from the dermis using a scalpel. metabolism was investigated for the highest concentration ( mg bpa/l) only, using a hplc with in-line radiodetection and confirmed bpa-glucuronide (bpa-g) and bpa-sulfate (bpa-s) standards for comparison. no metabolism was observed in any of the epidermis samples, however some metabolism is observed in dermis and receptor fluid samples. metabolites were identified with retention consistent with bpa-g and bpa-s, and also some more polar components. the mean total absorbed dose (receptor fluid + receptor chamber wash + receptor rinse) was between . and . % of the applied dose and the mean dermal delivery (epidermis + dermis + total absorbed dose) was between and % of the applied dose, with the majority of the radioactivity associated with epidermis samples compared to dermis and receptor fluid samples. a linear dose-response relationship is observed over the whole concentration range. anastrozole is a well-known non-steroidal aromatase-inhibiting drug approved for the second-line treatment of breast cancer after surgery and for treating postmenopausal women. treatment with the only available dosage form, anastrozole film-coated tablets for oral administration, is frequently associated with concentration-dependent unwanted side effects like hot flashes, fatigue, joint pain, joint stiffness, vaginal dryness, hair loss, skin rash, nausea, diarrhea and headache. in order to minimize the local gastrointestinal as well as systemic side effects, a system for transdermal anastrozole delivery has recently been developed. in this study, we describe the first experimental in vivo application of a transdermal therapeutic system (tts) to beagle dogs and, as a necessary prerequisite for the analysis of the time course of anastrozole release and uptake, a simple, sensitive and accurate lc-ms method for quantifying anastrozole in plasma. the detection of fragment ions at m/z and instead of the molecule ions (m/z and ) generated from the elevated collision energy, and the use of a deuterated internal standard resulted in increased relative abundances and improved signal-to-noise ratios.the lower limit of quantification and the limit of detection were . ng/ml and . ng/ml, respectively. the developed method was successfully applied in a pharmacokinetic study of anastrozole plasma levels in beagle dogs, measuring percutaneous drug absorption from an experimental, newly designed glycerol-based patch / tts. a distinct time course was observed, with an initial linear increase over hours and a plateau thereafter. this offers promising strategies for the transdermal application of anastrozole with improved pharmacokinetics. background: the monocarboxylate transporter (mct ), encoded by the slc a gene, mediates h + -coupled transport of lactate across the plasma membrane. for cells with high glycolytic activity lactate export is of major importance for the maintenance of the glycolytic metabolism and for the prevention of intracellular acidification. in glycolytic tumor cells, the acidic extracellular environment resulting from export of lactate and h + , furthermore promotes anti-apoptotic effects and metastasis. clear cell renal cell carcinoma (ccrcc) is the most common subtype of renal cell carcinoma (rcc) and is characterized by a metabolic shift towards enhanced aerobic glycolysis and hence, increased lactate production. mct and its epigenetic regulation by slc a promoter methylation has previously been identified as prognostic marker for ccrcc outcome and as target for ccrcc treatment. since metastatic ccrcc is associated with poor overall survival and represents a major challenge for treatment, mct /slc a might represent a promising prognostic marker and a target for therapeutic intervention also for metastatic disease. methods: mct protein expression was analysed in paraffin embedded tissue samples of distant metastases derived from different organs by immunohistochemical staining of tissue microarrays. protein expression was evaluated semi-quantitatively using tissue studio v. . (definiens ag). dna methylation in the slc a promoter, specifically at the previously identified cpg site with prognostic potential in primary ccrcc, was analysed in paraffin embedded metastasis samples by maldi tof-ms. mct protein expression data and dna methylation at the specific cpg site in the slc a promoter were correlated with clinicopathological parameters and outcome data. results: distant metastases of primary ccrcc showed high mct protein expression irrespective of the affected organ. the most frequently affected organs like lung or bone, with approximately % and % in our cohort respectively, showed similar expression levels as less frequent metastatic sites such as thyroid gland or spleen. accordingly, dna methylation at the identified cpg site in the slc a promoter was low in metastatic tissue in all investigated organ sites. an association of low promoter dna methylation level at the previously identified prognostic cpg site in metastases with poor tumor-specific survival of the patients was observed. conclusion: from these results we hypothesize that dna methylation at specific cpg sites in the '-regulatory region of mct may not only serve as a predictor for patient outcome and as potential novel target for therapeutic intervention in primary, but also for metastatic disease. tamoxifen is used to treat pre-and postmenopausal women with estrogen-receptor (er) positive breast cancer. as a prodrug, tamoxifen undergoes extensive hepatic metabolism resulting in a complex mixture of metabolites with estrogenic and antiestrogenic effects. while endoxifen and (z) -hydroxytamoxifen are the most potent antiestrogenic metabolites, bisphenol and both isomers (e) and (z) of metabolite e are the most potent compounds with estrogenic properties at the er. the mixed antagonist/agonist pharmacodynamic effects of the selective estrogen receptor modulator tamoxifen at the er have been mainly attributed to tissue specific action of er coregulators, yet little is known about agonistic metabolites contributing to its estrogenic actions. the aim of the present study was to clarify whether there is a genetic component for interindividual differences in the formation and clinical effect of agonistic tamoxifen metabolites. a genome-wide association study (gwas) was conducted on steady-state agonist plasma levels in postmenopausal breast cancer patients of european origin who were treated with mg/day of tamoxifen for at least months. plasma concentrations of estrogenic metabolites bisphenol, (e), and (z) metabolite e were quantified using a recently established lc-ms/ms method . promising snps for an association between genotype and either plasma metabolite concentration or clinical outcome were confirmed for their relevance in an independent patient cohort of premenopausal breast cancer patients mainly of european descent , . twelve snps close to or above genome-wide significance (p < e- ) were found to be associated with allele-dependent variable (e) or (z) metabolite e plasma levels, while no genomic hit was found for the tamoxifen metabolite bisphenol. here, positive intergenic or genic regions mapped to chromosomes , and for (e) metabolite e and to chromosomes and for (z) metabolite e. upon genotyping of the validation cohort, two genetic loci with minor allele frequencies < % were confirmed as putative candidates: rs was associated with a - % variant allele-dependent increase of (e) and (z) metabolite e isomers (p< . ), and rs , mapping to a gene encoding zinc finger protein znf , was associated with increased risk of reccurrence or death (hr carriers . , % ci: . - . ; p < . ). these findings suggest the existence of genetic loci that may contribute to the formation and clinical effect of estrogenic tamoxifen metabolites and therefore could explain therapeutic failure of tamoxifen and/or the occurrence of adverse events during treatment. introduction: metabolomic monitoring of endogenous biomarkers is of increasing importance for the assessment of drug safety and efficacy during clinical drug development. myrcludex b, a novel lipopetide-based entry inhibitor for the therapy of hepatitis b and d, exerts its function through inhibition of the hepatic bile acid transporter na + -taurocholate cotransporting polypeptide (ntcp). in order to assess a myrcludex binduced metabolomic response in humans, lc/ms-based monitoring of endogenous metabolites was performed in blood and urine samples from healthy individuals before and during treatment with myrcludex b. methods: plasma and urine samples were collected from healthy volunteers participating in clinical phase i trials to evaluate safety, tolerability, and pharmacokinetics of single doses of the ntcp inhibitor myrcludex b. using quadrupole time-of-flight mass spectrometry coupled to reversed-phase chromatography (lc-qtof-ms) a set of known ntcp substrates (bile acids) was quantified by targeted metabolomics. protein precipitation was performed in the presence of deuterium-labeled internal standards (istds) which allowed absolute bile acid (ba) quantification in low amounts of plasma. ba profiling in urine was performed after dilution with methanol/water ( : ) in the presence of istds. both methods were validated according to fda guidance and applied to monitor the effect of myrcludex b treatment on human bile acid homeostasis. results: dynamic quantification in plasma and urine was achieved in the range from . nm to nm depending on the ba species analyzed. intraday-and interday accuracy and precision were in the % tolerance range for all analytes in all matrices. matrix effects were between - % (plasma) and - % (urine), apparent recoveries in plasma were above %. basal plasma ba level (mean ± sd) in fasting healthy subjects were ± nm (unconjugated bas), ± nm (glycine-conjugated bas) and ± nm (taurine-conjugated bas). urinary ba level (nmol/g creatinine) were ± nm (unconjugated bas), ± nm (glycine-conjugated bas) and ± nm (taurine-conjugated bas). myrcludex-induced ntcp inhibition resulted in significantly elevated amounts of conjugated ba species demonstrating a spillover of ntcp substrates into the systemic circulation. furthermore, higher urinary ba level were observed during treatment indicating accelerated elimination of excessive bas from the body. conclusion: lc/ms-based monitoring of endogenous biomarkers has been successfully established and applied to study the effect of myrcludex b treatment on human ba metabolism. the results obtained by our assay demonstrate that a myrcludex-induced ntcp inhibition drastically affects human ba homeostasis. this observation provides valuable insights into the drug´s mode of action and will be indispensable for the assessment of side effects and dose-finding processes during future clinical trials. further studies are required to assess a possible role of ba modification (e.g. sulfation) in the process of ba detoxification during myrcludex treatment. key: cord- -xta e j authors: nan title: deutsche gesellschaft für experimentelle und klinische pharmakologie und toxikologie e.v. date: - - journal: naunyn schmiedebergs arch pharmacol doi: . /s - - - sha: doc_id: cord_uid: xta e j nan nucleoside diphosphate kinases (ndpks) are multifunctional enzymes involved in a variety of cellular processes including cancer metastasis and heart diseases. the plasma membrane content of the three major ndpk isoforms ndpk a, b and c is increased in human heart failure. we have previously shown that the ndpk b isoform regulates camp levels and cardiac contractility through a receptor-independent gprotein activation involving direct g protein β subunit phosphorylation. the precise role of ndpk c in the heart is unknown and was the object of this study. ndpk c function was assessed in neonatal (nrcm) and adult (arcm) rat cardiomyocytes with real-time pcr, immunoblotting, and quantification of camp content. heart failure was induced by chronic treatment with isoproterenol (iso, . mg/kg/d days) via minipumps. chronic iso increased mrna levels of ndpk c by . ± . -fold and its protein levels by . ± . -fold. immunoprecipitation of the g protein β subunit resulted in coimmunoprecipitation of ndpk c and the stimulatory gαs subunit: iso enhanced this interaction. upon iso stimulation, ndpk c translocated from the cytosol to the plasma membrane within hours in both nrcms and arcms. adenoviral overexpression of ndpk c in nrcms caused a . -fold increase in basal and iso induced camp synthesis, whereas sirna mediated knockdown of endogenous ndpk c decreased camp levels by ~ %. our results establish ndpk c as a novel and critical regulator of camp synthesis and gs signaling in the heart. the up-regulation of ndpk c and the increased responsiveness to iso in failing hearts point to ndpk c as a potential counterregulatory factor in the onset of heart failure. uptake and metabolism of methylated myricetin derivatives: studies in cell culture and c. elegans ackermann d. , , büchter c. secondary plant compounds like flavonoids that are ubiquitary present in fruits and vegetables are believed to exert health protective effects in terms of lowering the incidence of widespread diseases such as cardiovascular diseases and cancer. besides their antioxidative effects, flavonoids may also modulate cell signaling pathways and thereby performing their disease-protective actions. though this class of dietary polyphenols has become increasingly popular as dietary supplements, only little is known about their metabolic fate in vivo. therefore, we investigated the absorption and metabolism of several flavonoids such as myricetin and its methylated derivatives laricitrin, syringetin, and myricetin- ', ', '-trimethylether in the human colon carcinoma cell line hct and the human hepatoma cell line hepg as well as the model organism caenorhabditis elegans. all flavonoids were rapidly taken up by both cell lines as shown by hplc analyses. the intracellular amount of myricetin and laricitrin did not increase with time and was only half of that of syringetin and myricetin- ', ', '-trimethylether. interestingly, no metabolites of these flavonoids could be detected which might at least in part be due to their low intracellular amounts. absorption as well as intracellular distribution was also evidenced by using the fluorescent dye "naturstoff reagent a" nsra) . fluorescence microscopy indicated a predominant cytosolic distribution of the employed flavonoids. in the model organism caenorhabditis elegans, the flavonoids were exclusively distributed in the intestine as visualized by nsra. the antioxidative capacity of the four flavonoids (measured by using the cell free teac assay and the h dcf-da assay in hct cells) decreased with increasing methyl groups in the b-ring. myricetin- ', ', '-trimethylether (three methyl groups) was the least effective radical scavenging flavonoid in both the cell free system and in hct cells compared to myricetin (no methyl groups). in conclusion, for exerting their biological effects, uptake and distribution as well as metabolism of flavonoids in certain organs such as liver and gut are important and more research in that field is warranted. munich heart alliance, münchen, germany signaling through g protein-coupled receptors is affected by receptor polymorphisms, yet the molecular basis for the functional differences of individual receptor variants is unclear. to investigate the impact of the frequent gly arg variant of the β -adrenergic receptor (β ar) on receptor conformation we used β ar-sensors capable of fluorescence resonance energy transfer (fret). these sensors retained the pharmacological and functional characteristics of the native receptors. upon stimulation of the sensors we determined the activation characteristics of the polymorphic receptors in real time and in living cells. we found the β ar variants to behave similar upon a single stimulation with an agonist, but to differentially respond with a change of their activation kinetics during subsequent stimulations. while the arg -β ar did not show altered activation kinetics after prestimulation, the gly -β ar became slower compared to the initial stimulation suggesting that β ars possess a memory of previous activation. we then permeabilized β ar-sensor-expressing cells with saponin to remove soluble cytosolic factors. upon permeabilization the β ar variants did not display receptor memory, suggesting that the β ar memory depended on the interaction of the receptors with soluble cytosolic factors upon their initial activation including the phosphorylation of agonist-bound receptors by protein kinase a or g protein-coupled receptor kinases. our findings suggest an intrinsic, polymorphism-specific property of βars that alters activation kinetics upon continued stimulation and that might account for individual drug responses. micro-rna replacement therapy: nanoparticle-mediated in vivo delivery of mirna- or mirna- a exerts antitumor effects in colon carcinoma xenograft mouse models weirauch u. micro-rnas (mirnas) control the expression of various genes, and under pathological conditions several mirnas are up-or downregulated. previous in vitro studies have established a pro-apoptotic and anti-proliferative role of mir- , which shows decreased levels in colon carcinoma. in contrast, while mir- a is only weakly expressed in several tumors as well, its role in cancer has not been analysed so far. in this study, we demonstrate the tumor-relevance of mir- a and identify the protooncogenic kinase pim- as a target of mir- a. pim- harbours a highly conserved mir- a binding site within its '-utr, and seed mutagenesis of this target sequence abolishes the mir- a-mediated downregulation of pim- . the knockdown of pim- by rnai or mirna transfection inhibits proliferation in leukemia and in colon carcinoma cells by decelerating cell cycle progression, thus establishing a tumor inhibitory function of mir- a. we furthermore introduce polyethylenimines (peis) for the therapeutic application of mirnas in vivo, which is critically dependent on the development of appropriate delivery tools. peis are able to form non-covalent complexes with mirnas, leading to mirna protection after systemic application in combination with an attractive biodistribution profile and the efficient uptake in target organs/cells. therapeutic effects of pei-mediated mirna delivery were demonstrated in subcutaneous colon carcinoma xenograft mouse models. the in vivo application of mirna- through systemic or local injection of pei/mirna complexes resulted in efficient mirna delivery and in antitumor effects, based on the concomitant repression of erk . likewise, tumor growth inhibition was observed upon treatment of tumor-bearing mice with pei-complexed mir- a. this is due to the mir- a-mediated downregulation of pim- expression and resembles the pim- knockdown through rnai / pim- sirnas. taken together, in tumor xenograft mouse models we establish mirna replacement therapy through the pei-complexation of mirnas as a novel therapeutic strategy and demonstrate that mir- and mir- a may be promising mirnas in colon carcinoma therapy. md , a hybrid of chloroquine and primaquine, is a potential drug against infectious diseases such as malaria. since one moiety of the hybrid, the known antimalarial drug chloroquine, is a known intercalator, the potential of md to intercalate into dna was determined. due to the ability of intercalators to cause frame shift mutations, the mutagenic potential of md was also investigated. the potential of md to intercalate into dna was investigated by means of fluorescence based micro plate assay using ethidium bromide (eb) and isolated calf thymus double stranded dna. as a positive control chloroquine was used. the potential of md to cause gene mutations was determined using the hypoxanthine-guanine phosphoribosyltransferase (hprt) test in chinese hamster v lung fibroblasts (v cells). v cells were treated with . µm, . µm and . µm md or the positive control, the direct mutagen -nitroquinoline-n-oxide (nqo, µm) for h. on day , mutants exhibiting loss of hprt function were selected with -thioguanine . whereas at µm chloroquine, a % decrease in fluorescence intensity of eb (indicating dna intercalation) was observed, µm md were needed to observe a similar decrease ( %) in fluorescence intensity of eb. therefore, md is fold less potent to intercalate into dna than its moiety chloroquine. the frequency of spontaneous -tg resistant mutants per colony-forming cells was ± . as expected, µm nqo caused a significant increase in the mutant frequency (mf, ± ) . in contrast, mf was not significantly affected by treatment with md at both noncytotoxic ( . µm: ± ) and cytotoxic ( . µm: ± ) concentrations. in conclusion, md is a less potent intercalator than the known antimalarial drug chloroquine. furthermore, md does not cause gene mutations in the hprt test. since current studies show that various metabolites of md are formed in vitro, their mutagenic potential is currently under investigation as well. -conducting channels but depends critically on the membrane potential. trp channels form cation entry channels thereby either contributing to ca + entry or depolarisation. recently, we showed that trpm acts as a ca + -activated non-selective cation channel and critically determines the driving force for ca + influx in mast cells following fcεri-stimulation ( ) . in addition to trpm we also identified the expression of other trp transcripts in bone marrow derived mast cells (bmmc) including those encoding trpc , trpc , trpc , trpc and trpm . in peritoneal mast cells (pmc), rt-pcr indicated expression of trpc , trpc , trpc , trpc , trpm , trpm and trpm . to identify the functional role of those trp channel proteins for mast cell activation we analysed ca + signaling using microfluorimetry in bmmcs and pmcs after stimulation with substances known to activate trpc channels in other cell systems such as the diacylglycerol analogue oag, the hyperforin analogue hyp- and flufenamic acid (ffa), but could not evoke a rise in the [ca + ]i in both pmc and bmmc. sphingosine phosphate and lysophosphatidylcholine, which were reported to activate trpc channels, induced only minor rise in [ca + ]i in bmmcs, respectively. here, we will present our analysis of ca + signaling following stimulation of the fcεri receptor and application of secretagogues that are supposed to affect ca + -dependent mast cell activation such as adenosine, endothelin- , substance p and compound / in bmmcs and pmcs derived from mouse lines with inactivation of trpc , trpc , trpc , trpc or trpc since specific antagonists are still lacking for these trp channels. the α a-ar is the main ar in the central nervous system and it plays a crucial role in regulating norepinephrine (ne) release from nerve terminals via presynaptic feedback inhibition. it is also associated with a number of physiological effects, including hypotension, pain perception, sedation and modulation of mood. ne, once released in the synaptic space, binds to α a-ars and induces a rearrangement of the receptors from the inactive state into an active conformation. this allows the binding and activation of the cognate gi-protein and, hence, the transduction of the transmembrane signal to the downstream effectors. generally, α a-ar activation has been deduced from the stimulation of a receptor-mediated biological response that could be easily followed experimentally. however, most of these approaches do not employ living cells and are normally applied under equilibrium conditions that need prolonged incubation periods incompatible with the physiological temporal dynamics of ne. here, we monitored the ne-mediated α a-ar and gi-protein activation by using a fluorescence resonance energy transfer (fret)-based approach in living cells. to examine the effects of increasing concentrations of ne on the speed and extent of α a-ar activation with very high temporal resolution, we took advantage of the previously described α a-ar flash/cfp sensor [ ] . the results indicate that in our system the efficacy of ne in eliciting α a-ar flash/cfp activation increases in a time-dependent way and reaches the maximum with a half-life of ~ ms. the ec values decrease in an exponential manner and arrive at ~ µm with a half-life of ~ ms. next, we analyzed the ability of increasing concentrations of ne to trigger a downstream intracellular response after α a-ar stimulation by monitoring the kinetics and amplitude of gi activation in living cells. we applied the previously well characterized gi cfp/yfp sensor [ ] . the results show that both the efficacy and the potency of ne in inducing gi activation reach the steady state slower compared to receptor activation (half-life ~ ms and ~ , ms respectively). in conclusion, we were able to monitor ne-mediated events occurring in the millisecond time scale and reaching the equilibrium in a time interval compatible with physiological conditions. sphingosine- -phosphate (s p) is an immune modulator produced by sphingosine kinase (sphk ) and sphingosine kinase (sphk ) and de-phosphorylated or degraded irreversibly by s p phosphatases and a lyase, respectively. we recently showed that tlr -induced il- p is selectively counter regulated by sphk , s pr and its extracellular ligand s p. on the other hand, spiegel et al. have demonstrated that specific, sphk -dependent, binding of s p to traf enhances the tnf-alpha signaling. therefore we were interested whether the tlr/tir and tnf-alpha-signaling pathways are interfered with each other and are modulated by s p. in a first approach we focused our investigations on sphk effects on both traf and traf stimulatory signals and cytokines produced downstream. experimentally, with gm-csf expanded, bone marrow-derived dcs we first desensitized the lps-tlr or cpg-tlr signal by a defined time period of costimulation with tnf-alpha. the initial results showed a partial decrease of il- p secretion in tnf-alpha-co-stimulated dcs in contrast to lps stimulation alone. this might indicate that traf activated via tnf-alpha interacted with the traf pathway to reduce il- p . further series with dcs derived from sphk -deficient mice confirmed our former results that il- p in contrast to other cytokines is specifically sensitive to sphk -s p feedback, but did not change the effects of tnf-alpha on wt dcs il- p release. in comparison, cpg-tlr -induced il- p release reached only % of lps-induced il- p levels and was less sensitive to tnf-alpha costimulation. however, sphk -deficiency strongly augmented cpg-dependent il- p production. in ongoing experiments we started to analyze the details of traf /rip and traf /tak activation by ubiquitination blots in wt, sphk -and s plyase-deficient dcs. in conclusion, we hope to unravel possible mechanisms of the observed differential effects of s p and its enzymes on inflammation and cancer-relevant cytokines. identification of the kh type splicing regulatory protein (ksrp) as a new important mediator of the anti-inflammatory effects of resveratrol art j. , besche v. , bros m. , li h. , handler n. , bauer f. , erker t. , behnke f. , mönch b. , förstermann u. , dirsch v. m. , werz o. , kleinert h. , pautz a. university of vienna department of pharmacognosy, althanstr. , wien, austria resveratrol, a polyphenol derived from different plants, possesses multiple pharmacological functions such as anti-oxidative, anti-diabetic, cardioprotective, anticancer, neuroprotective and anti-inflammatory properties. many of these effects have been attributed to its anti-oxidative activity but resveratrol also modulates signal transduction pathways like the p mapk pathway or the activity of different transcription factors like nf-κb redox-independently. moreover, the histone deacetylase sirtuin (sirt ) is an important mediator of resveratrol effects. nevertheless the direct molecular target of resveratrol remains unclear. in target fishing experiments we identified the rna-binding protein ksrp as direct resveratrol binding partner. ksrp is an rna-binding protein that controls proinflammatory gene expression on the post-transcriptional level by modulation of mrna stability. moreover, it is involved in the biogenesis of mirnas. resveratrol treatment of human dld- cells resulted in a decreased mrna expression of a number of well known ksrp target mrnas and enhanced mirna- function. downregulation of ksrp expression by sirna prevented the mrna destabilizing effect of resveratrol. as the activity of ksrp is mainly regulated on the post-translational level by phosphorylation of different serine and threonine residues we analyzed whether resveratrol changes ksrp activity by altering the phosphorylation of the protein. indeed, our immunoprecipitation experiments demonstrated that resveratrol reduces the p mapk-mediated phosphorylation of threonine residues in the ksrp protein and thus leads to an increase of ksrp activity. interestingly, resveratrol does not block p mapk activation or activity. in addition we have evidence that sirt is not involved in the resveratrol mediated activation of ksrp. so we believe that activation of ksrp by resveratrol is the major mechanism mediating the anti-inflammatory effects of resveratrol. in vitro testing of oecd reference nanomaterials (nm-series) in rat precision cut lung slices aumann a. the oecd has defined reference nanomaterials (nm) to be tested in different endpoints concerning human health and environmental safety ( ) in order to evaluate if the toxicity of nanomaterials can be linked to their physico-chemical properties. for nanomaterials, inhalation presents the major exposure route of concern and can be assessed using acute inhalation toxicity studies in rodents. however, these in vivo studies are resource intensive and animal consuming. the oecd working party on nanomaterials has named several alternative methods as being of particular interest for testing of nanomaterials; among them is the precision-cut lung slices model (pcls) to estimate respiratory toxicity. we have tested all nm in pcls measuring cytotoxicity, apoptosis, oxidative stress and inflammatory response of the tissues as well as observing them histological. for in vitro exposure of pcls the test material was dispersed in medium. since it is the nature of these materials to change their surface characteristics and agglomeration state in different environments, a standardized dispersion method (nanocare) using bovine serum albumin as a stabilizing agent, was used. particle size-distributions of the nanomaterial dispersions were characterized via analytical ultracentrifugation and found the nanomaterials well dispersed. silver and zinc oxide but none of the other nm showed cytotoxicity to the lung tissue in the tested concentrations. however, differences in cytokine profiles among the nm were observed and showed several correlations to the results obtained in in vivo inhalation or instillation studies. universität des saarlandes institut für molekulare zellbiologie, gebäude , homburg, germany tmem proteins show similarities in their primary sequence to motifs that are conserved amongst various members of the trp protein family. based on hydropathy analysis these proteins exhibit to membrane spanning domains. in contrast to trp channels there is no evidence that these proteins form ion channels in the plasma membrane following overexpression of their cdna in hek cells. tmem -/mice are viable and show no obvious signs of disease, but exhibit increased pancreatic amylase and lipase plasma levels. microfluorimetric measurements using fura- revealed that the elevation of the cytosolic ca + concentration after stimulation with carbachol and the cholecystokinin analogue caerulein is unchanged in tmem -deficient acinar cells. tmem is expressed in several cell types including pancreatic acinar cells, cardiac myocytes, cardiac fibroblasts, but their subcellular localization is still unkown. we generated several constructs encoding tmem fusion proteins with fluorescence protein tags by fusing eyfp, mcherry and tagrfp-t to the n-and c-terminus of the protein, respectively. based on western blot experiments and expression in hek cells the tmem -eyfp construct was most suitable for further colocalisation analysis and generation of viral vectors including adenovirus and semliki forrest virus. in contrast to the prediction by the psort ii algorithm tmem -eyfp could not yet be identified in the plasma membrane of fibroblasts, cardiac myocytes or acinar cells but showed a vesicular subcellular localization pattern. we localized tmem -eyfp in acidic compartments and predominantly in lysosomes (pearson coefficient (pcc) , ± , , n= using lysotracker ® dye). analysis of subcellular localization with independent tmem fusion constructs and additional vesicular markers will be presented as a framework to get insights towards the cellular function of tmem and to reveal the mechanisms underlying increased amylase release from acinar cells of tmem -/mice. the small molecule bcl- /mcl- inhibitor tw- shows single-agent cytotoxicity in neuroblastoma cell lines bachmann h. s. , akdeli n. high-risk neuroblastoma (nb) remains a therapeutic challenge in paediatric oncology. pro-survival bcl- family proteins critically regulate apoptosis, and may represent important therapeutic targets in nb. primary nb tumours heterogeneously express mcl- or bcl- , with high expression correlating to high risk phenotype. co-expression can be detected in approximately % and is correlated to reduced survival. recent studies with two inhibitors that predominantly target bcl- and other proteins, but not or to a lesser extend mcl- , elucidated the importance of mcl- inhibition for cytotoxicity in nb. tw- is a small molecule inhibitor that showed almost equal affinity to bcl- and mcl- . to explore the effect of combined bcl- /mcl- inhibition on neuroblastoma cells, four cell lines (sk-n-as, imr- , sy y and kelly) were treated with tw- and changes in growth properties were determined. furthermore, nude mice with kelly (human neuroblastoma cell line) xenografts were treated with tw- . using sirna, we investigated the functional relevance of mcl- and bcl- in kelly cells. for in vitro cell viability we observed ic values of . ± . µmol/l. on treatment with µmol/l dose of tw- , all neuroblastoma cell lines analyzed showed significantly reduced proliferation and increased apoptosis rates. bcl- as well as mcl- knockdown induced apoptosis in kelly cells. interestingly, tw- was able to reduce, but not to abrogate growth of kelly neuroblastoma xenografts in nude mice. in conclusion, combined inhibition of bcl- and mcl- using tw- exhibits strong single-agent antitumor activity on human neuroblastoma cells in vitro, but limited single-agent activity in vivo. therefore, inhibition of bcl- /mcl- may represent an interesting therapeutic strategy, most likely in combination with conventional chemotherapy and other specific inhibitors. localization and functional characterization of membrane transporters for sulfated steroid hormones in the human testis bakhaus k. , wapelhorst b. circulating sulfated steroid hormones like estrone sulfate (e s) or dehydroepiandrosterone sulfate (dheas) are delivered to the testis via membrane uptake carriers such as the sodium-dependent organic anion transporter (soat). inside the cell these sulfated steroids can be metabolized to active steroid hormones by the catalytic activity of the steroid sulfatase (sts), which shows high enzymatic activity in the testis ("sulfatase pathway"). in addition to soat, other candidate carriers like the organic solute carrier protein (oscp ) and the organic anion transporting polypeptides oatp a and oatp c are predominantly expressed in the human testis and demonstrate transport activity for sulfated steroids. we aimed to evaluate the cellular expression of soat and the other steroid sulfate carriers and their co-localization with the steroid sulfatase (sts) in human testis. furthermore we want to perform functional transport studies with the steroid sulfate carriers in stably transfected hek cells. we detected soat by rt-pcr and western blot analysis in the human testis. single cell analysis and in situ hybridization revealed pachytene primary spermatocytes to express the soat mrna. soat expression in specimens showing maturation arrest at the level of early round spermatids seems to be severely reduced or absent. sts mrna was detected by rt-pcr in testis homogenates. preliminary immunohistochemical data showed that sts may be expressed in germ cells and interstitial leydig cells. hek cells stably expressing the soat carrier protein showed significant transport activity for dheas. this was demonstrated by using a radiolabeled [ the hepato-intestinal induction of the detoxifying enzymes cyp a and cyp a by the xenosensing pregnane x receptor (pxr) constitutes a key adaptive response to oral drugs and dietary xenobiotics. in contrast to cyp a , cyp a is additionally expressed in several, mostly steroidogenic organs, which creates potential for induction-driven disturbances of the steroid homeostasis. using cell lines and mice transgenic for a cyp a promoter we demonstrate that the cyp a expression in these organs is noninducible and independent from pxr. instead, it is enabled by the loss of a suppressing yin yang (yy )-binding site from the cyp a promoter which occurred in haplorrhine primates. this yy site is conserved in cyp a , but its inhibitory effect can be offset by pxr acting on response elements such as xrem. taken together, the loss of yy binding site from promoters of the cyp a gene lineage during primate evolution may have enabled the utilization of cyp a both in the adaptive hepato-intestinal response to xenobiotics and as a constitutively expressed gene in other organs. our results thus constitute a first description of uncoupling induction from constitutive expression for a major detoxifying enzyme. they also suggest an explanation for the considerable tissue expression differences between cyp a and cyp a . serum albumin adducts as biomarkers for systemic bioavailability of active metabolites of various glucosinolates in animal models and humans barknowitz g. , engst w. glucosinolates (gls) are natural pesticides of brassicales, which comprise many important food and feed plants. upon physical damage to the plant, the enzyme myrosinase can convert gls to reactive metabolites (e.g. isothiocyanates). the same reaction can also be catalyzed by enzymes of the intestinal microbiota. modification of sensor proteins (e.g. keap- ) by some gls metabolites leads to adaptive responses, resulting in enhanced detoxification of reactive metabolites and other protective reactions. at least in experimental models, this mechanism can be exploited for chemoprevention of carcinogenesis induced by various chemical carcinogens. however, own research revealed that certain reactive gls metabolites can covalently bind to dna in vitro and in vivo, involving possible genotoxic and carcinogenic risks. animal models are useful for studying beneficial and adverse effects of gls. however, it has to be taken into account that the toxicokinetics of gls may differ between rodent and humans and that the active metabolites are short lived and thus difficult to detect and quantify. likewise, exposure of humans to gls and their breakdown products may enormously vary depending on (i) food preferences, (ii) cultivars, growth conditions and preparation of plants consumed and (iii) variations in human xenobiotic metabolizing system and composition of intestinal microbiota. in order to estimate individual levels of systemic exposure to reactive gls metabolites, blood protein adducts may be useful. we have developed lc-ms/ms methods for quantifying serum albumin adducts formed by glucoraphanin, glucotropaeolin and neoglucobrassicin. the method involves digestion of the protein to amino acids and the usage of isotope-labelled amino acid adducts as internal standards. serum albumin adducts were detected in mouse models after feeding broccoli and pak choi respectively, as well as after administration of purified gls and breakdown products. likewise, gls adducts were detected in human blood plasma after consumption of broccoli or cress. this work was financially supported by the bundesministerium für bildung und forschung (grant d) . barlow s. harrington house, harrington road, brighton, bn re, great britain values for cancer endpoints for the application of the ttc approach have been derived by linear extrapolation of results from animal carcinogenicity studies to calculate "virtually safe doses" (vsds). a vsd is defined as an exposure that represents an estimated upper bound increase in risk of in a million of developing cancer during a lifetime. in , from consideration of a range of vsds for carcinogens, the us food and drug administration (fda) proposed and adopted a value of . micrograms/kg of diet ( . ppb) as a threshold of regulation to be used for substances present in food contact materials. the fda considered that if exposure to a substance in the diet was below this value, consumers would be protected "with reasonable certainty of no harm" and no toxicological data on the substance need be submitted. the value of . ppb is equivalent to . micrograms/person per day, assuming that g of food and g of fluids diet might be consumed daily. this value was subsequently incorporated into the ttc approach to be used for assessment of substances without a structural alert for genotoxicity. in , kroes and colleagues further explored cancer as an endpoint and recommended a lower ttc value of . micrograms/person per day for substances with a structural alert for genotoxicity and exclusion from the ttc approach of certain groups of high potency carcinogens (with vsds below this value). in this presentation, the data underpinning these ttc values will be discussed from the perspective of their reliability for risk assessment of substances with low exposures, for which there are no toxicity data, but which may in fact be genotoxic or non-genotoxic carcinogens. stable conjugates which are recognized by the immune system. in the current investigations, the ability of chemical pre-treatment to interfere with antibody-protein binding has been investigated using ovalbumin (ova) as the model protein and naturally occurring anti-ova igg from healthy human donors. preparation of conjugates: ova ( mg/ml) was dissolved in sodium borate buffer ( . m, ph . ) . for chemical treatment various amounts ( - mg) of -fluoro- , dinitrobenzene (dnfb; sensitizer) or , -dichloro- -nitrobenzene(dcnb; non-sensitizer) was added and stirred for hrs at room temperature. unbound compound was removed by consecutive dialysis against phosphate buffered saline (pbs) and distilled water. inhibition elisa: plates were coated with µg/ml ova and blocked with % fcs in pbs. ova samples (native or conjugated; . - µg/ml) were pre-incubated with polyclonal antibodies (ab) from pooled normal human serum for min and then added to the plates. human anti-ova igg abs were detected by colorimetric analysis using ortho-phenylendiamine as a substrate. the concentration of soluble native ova or conjugate required to displace % of ab to plate-bound ova (ic ) was calculated (minimum of n= independent experiments). treatment of ova with the chemical sensitizer and protein reactive dnfb resulted in increased ic value, whereas mock treatment resulted in comparable ic values to native ova. treatment with dcnb showed that the presence of a chemical per se (and possible denaturation) was not sufficient to alter the ic value; conjugation of the compound to the protein was required. the analysis is not test chemical specific (specific ab against compound-protein conjugates are not required) and the colorimetric analysis is unaffected by absorbance of the compound itself. thus, this method may have utility for the identification of chemical sensitizers which are directly protein reactive. ´-deoxy-camp in human cell lines: another second messenger? beckert c. , hinz c. we have shown that ´-deoxy-camp (dcamp) is synthesized by recombinant human soluble adenylyl cyclase (sac). here, we report that dcamp can be detected and quantified by liquid chromatography coupled to mass spectrometry (lc-ms) in various human cell lines. in most cells a ratio of camp : dcamp of ~ was observed. as a remarkable exception, in hl- promyelocytic leukemia cells, the dcamp concentration exceeded the camp concentration more than -fold. a differential regulation of camp versus dcamp was determined upon replacement of the incubation medium (proliferating condition with serum / serum-free resting condition). for example, camp was dramatically reduced in hek cells after hours under resting conditions whereas dcamp was significantly increased. in cellular subfractions of hek cells ac assays (mn + /forskolin-or mn + /bicarbonate-stimulated) with either atp or datp as substrate revealed that comparable amounts of camp and dcamp accumulated. in addition to sac, membranous acs such as ac v were capable of forming dcamp with vmax and km values for datp comparable to those for atp. we also analyzed the substrate-specificity for several human phosphodiesterases. pde and pde hydrolyzed dcamp more effectively than camp. taken together, these data point to a putative second messenger role of dcamp in human cells. we are currently investigating the regulatory role(s) of camp and dcamp in apoptosis of hek cells. as a novel tool for these studies, we will use the cellpermeant dcamp-acetoxymethylester which penetrates the plasma membrane and releases dcamp intracellularly. the biogenic amine histamine is recognized by target cells via four different histamine receptors subtypes (h r -h r), which all belong to the family of g-protein-coupled seven-transmembrane receptors. histamine plays a crucial role in allergic reactions such as rhinitis or conjunctivitis and also in allergic asthma. previously, we showed an interaction of the effects of antagonists at the h r and h r in a mouse model of allergic asthma. however, not much is known about the signaling pathway activated by murine h r and h r. in order to analyze these signaling pathways, we established a cellular model using transfected hek cells which stably express recombinant mh r or mh r. proper expression of the receptors was verified by western blot analysis and flow cytometry. in functional assays we demonstrated that histamine stimulation results in the increase of intracellular ca + concentration ([ca + ]i) in cells expressing either of both, mh r (pec = . ) or mh r (pec = . ). as a second readout, we analyzed the modulation of forskolin-induced camp-accumulation. in mh r-expressing cells the intracellular camp concentration was increased by stimulation with histamine, while in mh r-expressing cells forskolin-induced camp accumulation was reduced. the histamine-induced effects in h r-expressing cells were blocked by the h r antagonist mepyramine ([ca + ]i: pkb = . ) and those in the h r-expressing cells by the h r antagonist jnj ([ca + ]i: pkb = . ) or by pertussis toxin, which selectively blocks receptor gi-protein coupling. jnj , which behaves as a partial mh r agonist in the steady-state gtpase assay using membranes of infected sf cells, was without effect on [ca + ]i and forskolin-induced camp-accumulation in the mh r-expressing cells. currently, we are investigating mitogen-activated protein (map)-kinase pathways activated by the h r and the h r. using a phospho-map-kinase array, histamine dependent phosphorylation of erk / , p , jnk, creb, pkb (akt), and mkk / were detected in cells expressing either of both, mh r and mh r. in summary, the hek cell lines stably expressing selective histamine receptors are very useful tools to investigate hxr signaling pathways in-vitro. enhanced fibroblast motility in the absence of the β regulatory subunit of voltage-activated calcium channels belkacemi a. cavβ subunits of voltage-activated ca + channels are required for trafficking the poreforming cavα subunit to the plasma membrane and modulate the kinetics of its current. mouse embryonic fibroblasts (mefs), acutely isolated cardiac fibroblasts (cfs) and nih t fibroblasts do express cavβ and cavβ subunits, but we could not detect any voltage-activated ca + influx. whereas in mouse cardiomyocytes or hek cells coexpressing cavβ and cavα subunits a dihydropyridin-sensitive voltage-activated ca + influx was readily detectable. apparently, cavβ subunits serve functions in fibroblasts unrelated to voltage-activated ca + influx. among the proteins potentially interacting with cavβ are the inositol , , -trisphosphate receptors (ip rs) [ , ] . we therefore coexpressed mouse cavβ and mouse ip r type , or in cos- cells and found coimmunoprecipitation of ip rs using an antibody for cavβ and vice versa. to study the release of ca + from ip -sensitive stores we performed fura- measurements on fibroblasts isolated from wild type and cavβ -deficient mice either in the presence of thapsigargin or after stimulation of gq-coupled receptors by par- , lpa or bradykinin. receptor-activated ca + release was more pronounced in β -deficient mefs and cfs, whereas thapsigargin-induced ca + release was the same in cells from both genotypes. in addition, ip production measured by a radioreceptor assay was already increased in β -deficient cells under basal conditions. fibroblasts are migrating cells and involved in various physiological and pathophysiological processes. we therefore started in vitro assays for proliferation, migration and angiogenesis as well as in vivo assays for skin wound healing. angiogenesis and proliferation were apparently not different in both genotypes but migration (measured as transwell migration and in scratch assays) and wound healing were affected in different ways. fluorescent staining of cytoskeleton and quantification of the f-actin/g-actin ratio show similar results in both genotypes, suggesting that the increased migration rates and wound repair in β knockout may result, in part, from the increased amount of ip -releasable ca + . [ ] berggren, yang, murakami, et al., removal of ca channel β subunit enhances caoscillation frequency and insulin exocytosis. cell , ( ) - . [ ] müller, haupt, bildl, et al., quantitative proteomics of the cav channel nanoenvironments in the mammalian brain. pnas , ( ) - . bender-sigel j., closs e. i. universitätsmedizin mainz institut für pharmakologie, obere zahlbacher straße , mainz, germany human cationic amino acid transporters (hcat) are a family of multimembrane spanning proteins that mediate the transport of cationic amino acids through the plasma membrane. our earlier results have demonstrated that activation of either protein kinase c (pkc) by pma or cdc by egf leads to an internalization of these transporters. in addition, in a recent collaboration with the group of alexander sorkin (university of colorado denver) we found that ubiquitination and clathrin-dependent endocytosis are necessary for the down regulation of hcat- -mediated arginine transport by pma (vina-vilaseca et al, j biol chem : ) . this mechanism requires nedd e ligases, but hcats do not contain a ppxy motif to bind the ligases, suggesting that an adaptor protein takes part in this process. however, an involvement of the adaptor protein beta-arrestin in this mechanism could be excluded. using sirna against pkc alpha we now show that pkc alpha is the major isoform that induces the reduction of arginine transport in human u glioblastoma cells overexpressing hcat- a-egfp. in addition, sirna-mediated knock down of cdc prevented the decrease of hcat- amediated transport induced by pma. taken together pkc seems to negatively influence the constitutive cycling of cats by activation the ubiquitination machinery and clathrinmediated endocytosis. cdc is part of this pathway. converging of the classical mitochondria-related pathway in parkinson and nuclear dna-repair signaling? scherr a. -l. parkinson disease is the second most neurological disorder worldwide. despite the fact that most cases are idiopathic and only few can be traced back to specific genes, general progression between both tracks of the disease is comparable. the variety of clinical symptoms in motor control like tremor, rigor and postural problems all originate from loss of dopaminergic neurons in the substantia nigra pars compacta of the brain. several proteins mutated in pd are involved in surveillance pathways, monitoring functionality and integrity of proteins and organelles either by the proteasome degradation machinery or by clearance of mitochondria via autophagy (mitophagy). disturbed calcium-and redox-homeostasis seems to play a major role in susceptibility to cell death signals in dopaminergic neurons, but if this is a preceding or successive event in cell death related to pd progression is not known. on the other hand, experimentally elicited parkinsonism by oxidative stress inducers like paraquat or rotenone and mptp (inhibitors of mitochondrial complex i) lead to damage in nuclear dna and activation of the dna repair protein poly(adp-ribose) polymerase . by consuming substantial amounts of its substrate nad + , this enzyme can drastically decrease energy levels and disturb the redox balance within a cell, also sensitizing it to stress induced cell death. inhibition of poly(adp-ribose) polymerase has been proven to be beneficial to some extent in cell culture models as well as in experimental pd in mice. our research focuses on a putative crosstalk mechanism we recently discovered between the two pathways of experimentally induced cell death in culture models, i.e. mitochondrial signaling and parp -dependent poly(adp-ribosyl)ation. both converge on two mitochondrial chaperones, mortalin and trap . whereas mutations in mortalin have been reported recently to be responsible for some parkinson disease cases in humans, trap is a specific target of the kinase pink (pten induced putative kinase), which is often mutated in autosomal-recessive forms of the disorder. pink is a central regulator of the mitophagy process, tagging mitochondria with dissipated membrane potential for destruction. we could show now that both chaperones bind to short-chain poly(adp-ribose) specifically synthesized by poly(adp-ribose) polymerase . we will present our most recent findings about regulation of these two chaperones after application of parkinson-inducing toxins. aldrich). the effect was reversible within ten minutes when cells were re-incubated in regular cell culture medium. stimulatory effects were not due to osmolarity or cell stress due to medium exchange. analysis of different components of both media (table ) revealed that bicarbonate stimulates accumulation of ccmp and cump besides cgmp and camp in a time-and concentration-dependent manner. bicarbonate is known to activate soluble adenylyl cyclase (sac) and particulate guanylyl cyclase g (pgc-g), regulating sugar metabolism, sperm motility and olfaction by synthesis of camp and cgmp, respectively. in order to identify a responsible cyclase for ccmp and cump generation after bicarbonate treatment, we are currently analyzing transiently and stably transfected hek cells overexpressing various known adenylyl and guanylyl cyclases (sac, mac , mac , mac , soluble guanylyl cyclase, pgc-g, pgc-a, and pgc-d) for their pyrimidinyl and purinyl cyclase activity in vivo and their regulation by bicarbonate. in addition, cell fractions will be analyzed for the detection of specific cyclase compartments. question: long term ventricular pacing, especially at the right ventricle (rv), results in left ventricular (lv) failure. there are several lines of evidence that disturbed ca + homeostasis is involved in the pathophysiology of human heart failure. in this study we examined if ventricular pacing affects the na + -and ca + -channels and the expression of ca + -handling proteins and investigated if there is a differential effect between right ventricular free wall (rvfw) pacing and left ventricular apex (lva) pacing. methods: after av-node ablation minipigs underwent ventricular pacing at beats/min (ddd mode) for one year. minipigs were paced from the rvfw and minipigs from the lva, respectively. minipigs with normal sinus-rhythm served as control group. patch-clamp-experiments were studied to measure na + -and ca + currents. western-blots were carried out to investigate the expression of the ca +handling proteins l-type ca + -channel, serca and phospholamban. results: both rvfw-and lva-pacing led to significant decreased ca + -currentdensities in cardiomyocytes of the lv compared to the control group. the plateau phase of the action potential was significantly shortened after ventricular pacing in relation to control minipigs. furthermore cardiomyocytes of rvfw-and lva-paced minipigs had significant lower na + -current-densities than control minipigs. the action potential amplitude was significantly decreased after rvfw-and lva-pacing whereas the diastolic potential remained unchanged. the expression of the l-type ca + -channel was significantly reduced after ventricular pacing, regardless of the pacing site. in contrast rvfw-and lva-paced minipigs showed significant increased serca -expression. the expression of phospholamban remained unchanged after rvfw-and lva-pacing compared to control minipigs. conclusion: in a chronic animal model ventricular pacing leads to remodeling of ionchannels and ca + -handling-protein-expression, regardless of the pacing site. investigation on metabolic competence of dermal systems: native human skin, in vitro skin models and keratinocytes blatz v. the implementation of reconstructed human skin equivalents (rhes) as an alternative method for dermal toxicity testing became very prominent in the last decades. their advantages are e. g. the human cell origin and an organ-like d structure. already regulatory accepted methodologies are widely in use for testing the skin corrosion (oecd ) and irritation (oecd ) within rhes. but there are still some questions open, one of them the metabolic competence of such dermal systems. in this context, enzyme activities of oxidizing (cyp; fmo; adh; aldh) and conjugating enzymes (nat; ugt) were investigated in subcellular fractions of in vitro systems such as keratinocytes and rhes (epidermis model epiderm tm (mattek), full-thickness skin models epiderm tm ft (mattek) and phenion ® ft (henkel ag)) and compared to those of native human skin. activities of cyp a, b and a isoenzymes were measured fluorometrically by oxidative desalkylation of alkoxyresorufines. fmo / activities were evaluated by hplc/fld detection of n-oxygenated product of benzydamine [ ] . adh and aldh activities were investigated by photometrical detection of nadh generation during ethanol (adh) [ ] or propanal (aldh) oxidation [ ] . nat activity was followed by hplc/uv detection of acetylated p-aminobenzoic acid. ugt activity was quantified fluorimetrically by glucuronidation of methylumbelliferone [ ] . during the course of this study the following results were observed: (loq = limit of quantification) since the metabolic competence of rhes is confirmed, these in vitro systems are estimated as suitable for further toxicity tests (e. g. genotoxicity by comet assay), where metabolic activation of substances may play a crucial role. however, for the data assessment, the determined metabolic profiles should be taken into account. we acknowledge bmbf funding this project ( d). signalling pathway indicates that in b cells the adenine receptor couples to a gqprotein followed by activation of phospholipase c pathway. these findings represent a new signalling pathway of the adenine receptor and allow the assumption that different adenine receptor subtypes exist in the rat brain. in the scope of the project lexukon ("foodborne exposure to environmental contaminants -data analysis to support and standardise exposure assessments based on nvs ii") exposure to the heavy metals cadmium (cd), lead (pb) and mercury (hg) via food consumption has been assessed for the german adult population based on the national nutrition study ii ( the updated intake assessments show that especially foods regularly consumed such as vegetables and grain contribute mainly to exposure of cd that is about . µg/kg body weight (bw) per week for average consumers over all food groups. this corresponds to % of the tolerable weekly intake (twi) of . µg/kg bw for cd defined by the european food safety authority (efsa) in . beverages and vegetables are the food groups most relevant for exposure to pb. about . µg pb/kg bw is taken up by average consumers that is below the benchmark dose for renal toxic effects ( . µg/kg bw) defined by efsa for the weekly pb intake. for hg the intake amounts for all population groups examined were significantly below the toxicological reference values. for average consumers the weekly intake of hg is . µg/kg bw that is primarily taken up by eating fish and fishery products. however, individual population groups and high consumers reach and/or exceed the toxicological reference values for the daily intake amounts for cd and pb. high consumers almost reach the twi for the cd with %. for pb a weekly intake of µg/kg bw was estimated for high consumers that exceeds the benchmark dose for renal toxic effects for the weekly pb intake. the results show that data collection should also focus on highly consumed and not only on highly contaminated foods. further, uncertainties in concentration levels should be reduced e.g. by lowering and standardizing the analytical limits. it's recommended to consider further measures in view of the reduction of contents of environmental contaminants in foods. however, other sources can also contribute to the intake of the mentioned heavy metals (e.g. smoking). major cell biological processes are regulated by rho-gtpases, actin-mediated processes in particular. amongst others, rho-gtpases are stimulated by the receptormediated activation of gα / and gαq via specific rhogefs. the p rhogef is activated by gαq and plays a major role in the acute response of vascular smooth muscle cells to angiotensin ii treatment. the aim of the present study was to establish a fret-assay between gαq-cfp and venus-p rhogef and characterize the dynamics of p rhogef-gαq-interaction in single living cells. the fusion of p rhogef with venus resulted in a functional gαq-regulated p rhogef-protein as determined by means of rho-luziferase-assays. whereas no specific fret signal was observed between the two interaction partners in the absence of receptor stimulation, a robust and rapid fret signal developed in response to stimulation of histaminergic h and cholinergic m -recptors. the onset of this signal after rapid application of agonist paralleled gαq activation kinetics. similar to the kinetics of gαq-protein deactivation the dissociation of p rhogef and gαq after withdrawal of agonist was slow (tens of seconds). the specificity of the fret signal between gαq-cfp and venus-p rhogef was verified by introducing point mutations rendering p rhogef unable to bind to active gαq. furtehrmore we observed a robust acceleration of the dissociation of p rhogef and gαq upon cotransfection of rgs , suggesting a very short lifetime of the p rhogef-gαq-complex or the ability of rgs to bind to p rhogef-associated gαq. taken together, fret-based imaging of the interactions between p rhogef and gαq revealed fast interaction kinetics closely resembling g-protein activation kinetics, both of which can be regulated by rgs . toxicity of silver nanoparticles in intestinal cells boehmert l. the rapid development of nanotechnology has been accompanied by an increased concern for the safety of nanomaterials. especially silver nanoparticles are used in many manufacturer identified consumer products including silver coated food contact materials or hydrosol silver supplements. these products lead to an intentional or unintentional oral uptake of silver nanoparticles and hence to a contact with the intestinal barrier. the human cell line caco- is a well established model system in studying effects on human enterocytes. although these cells are colon carcinoma cells and exhibit typical features of cancer cells when they are kept sub confluent, these cells have the capability to differentiate into polarized cells with morphological and biochemical properties of small enterocyte cells. we investigated the effects of silver nanoparticles on these colon carcinoma (proliferating) and small intestinal epithelium like (differentiated) caco- cells. the silver nanoparticles agpure were commercially available from rent-a-scientist gmbh. the behaviour of these silver nanoparticles in cell culture medium were characterised using asymmetric flow-fild flow fractionation (a f), small ankle x-ray scattering (saxs) and dynamic light scattering (dls). we investigated the particle toxicity on both cell states using cell titer blue assay, xcelligence impedance measurements, annexin-v and caspase measurements, diclorofluorescein assay and antioxidant pre incubated cells. the agpure stock solution is an aqueous suspension of silver particles with a metal core radius of . nm stabilised with tween- und polyoxyethylenglycerol trioleate. agpure silver nanoparticles were toxic for proliferating as well as differentiated caco- cells in a time and concentration depending manner. the presence of foetal calf serum in the incubation medium has a minor influence on the toxicity. prior to cell death, morphological abnormal adherence characteristics and morphological changes in the cells were observed using microscopy and quantified by xcelligence impedance measurement. it is concluded that cell death is caused by an oxidative stress related mechanism rather than apoptosis. the release of sphingosine- -phosphate from human platelets during acute coronary syndrome is attenuated by aspirin böhm a. , polzin a. in addition to atherosclerosis ttp knock out mice develop more cardiovascular dysfunctions. in tail vein bleeding assays we monitored a significant difference in the bleeding times of ttp deficient mice in comparison to wildtype mice, triggered by a stronger granulopoeisis. our results leed us to the assumption that the chonic inflammation seems to be more improtant for the development of cardiovascular diseases in ra patients than the traditional risk factors. differentially expressed cardiac genes in a mouse model with heart-specific overexpression of pp a bollmann p., makarova e. a., gergs u., neumann j. institute for pharmacology and toxicology medical faculty, magdeburger str. , halle (saale) , germany in transgenic (tg) mice with cardiac myocyte-specific overexpression of the catalytic subunit of protein phosphatase a (pp a) reduced cardiac protein phosphorylation, cardiac hypertrophy and impaired cardiac contractility were noted compared to wild type (wt) littermates. the hearts of tg mice also suffered from ventricular dilatation and a diminished response to β-adrenergic stimulation. analyses of mrnas expressed in tg and wt hearts (n= ) using affimetrix mouse genome microarray chips resulted in several candidate genes possibly differentially regulated. in this study, we focussed on verifying the mrna data of selected genes important for stress response and signal transduction on protein level in cardiac homogenates by western blotting. hearts from wt littermates were used as control. compared to wt heat shock protein (hsp ) and calcium calmodulin dependent protein kinase type ii (camkii) mrnas were upregulated in tg but only hsp protein was increased (p< . , n= - ) but not camkii (p> . ). protein phosphatase type (pp ) and superoxide dismutase (sod) were downregulated on mrna level in tg but on protein level this could be found only for sod (p< . , . in contrast, pp protein was upregulated (p< . , in tg compared to wt. for comparison the regulatory a-subunit of pp a and hsp were studied. both genes were unchanged on mrna level in tg: western blotting revealed the same results for the corresponding proteins. in summary, mrna expression data could only partially be confirmed on protein level elucidating the importance of western blotting studies. these data indicate that increased pp a activity is associated with modified gene expression in tg hearts possibly affecting stress response and regulation of cell signalling. (supported by the deutsche forschungsgemeinschaft) center for regenerative therapies, technische universtität, dresden, germany cell therapy in the form of beta cell replacement to cure diabetes has been practiced for decades without become a routine clinical therapy. more widespread clinical application is hindered by the scarcity of suitable organ donors, a dramatic loss of transplanted cells within the first days post-transplant, the requirement of long term immunosuppression to maintain graft survival, and despite this, a loss of graft function from a recurrence of autoimmunity in some patients. research is currently dedicated to overcome each of these limitations. additional beta cell sources investigated include embryonic stem cell derived insulin producing cells, human insulin producing cells lines, and xenogeneic beta cells. parallel to these efforts are the development of encapsulation devices to protect these sources from immune and inflammation mediated destruction, and transplantation into new sites such as the muscle and bone marrow to infuse beta cells. additional therapy to reduce immune suppression includes the infusion of t regulatory cells to control autoimmune and alloimmune response, and cytokine and chemokine receptor directed compounds aimed at blocking early inflammation or autoimmunity. these efforts are likely to lead to an expansion of clinical activity to replace beta cells in diabetes, and to novel pharmaceutical therapies that may be more generally applicable in patients with diabetes. pdgf-bb induces the h s producing enzyme cystathionine-γ-lyase via a rosdependent mechanism in rat renal mesangial cells boosen m. , hassan m. there is increasing evidence that hydrogen sulfide (h s) that is endogenously produced in several cell types serves as a potent gasotransmitter in a wide variety of physiological processes involving vascular homeostasis and inflammation. in the present study we investigate the expression and the regulation of the hydrogen sulfide synthesizing enzyme cystathionine γ-lyase (cse) in cultured rat renal mesangial cells. as demonstrated by qpcr and western blot experiments, mesangial cells showed a marked time-and dose-dependent upregulation of cse mrna and protein levels after treatment with platelet-derived growth factor (pdgf-bb). the cse upregulation by pdgf-bb is accompanied by a marked increase in reactive oxygen species (ros) formation. interestingly, co-administration of the ros scavenger n-acetylcysteine, the glutathione peroxidase mimetic ebselen and the nadph oxidase inhibitor diphenylen iodonium chloride (dpi) drastically reduced pdgf-induced cse expression, indicating a role for endogenously produced ros in mediating regulation of cse. as demonstrated by electrophoretic mobility shift (emsa) experiments pdgf-bb induces binding of the redox-sensitive transcription factor nf-e -related factor (nrf ) to a consensus antioxidant response element and this effect was also diminished by co-administration of antioxidants (dpi, nac, ebselen) . furthermore, lps/ifnγ-as well as pdgf-bb-induced cse upregulation was nearly completely abolished in nrf -/spleen macrophages and mesangial cells, respectively. as a consequence of the elevated cse levels we could demonstrate increased h s levels and a higher cse enzyme activity in mesangial cells after stimulation with pdgf-bb by using the colorimetric methylenblue method and a cse activity assay. importantly, in a rat model of anti-thy- -induced proliferative glomerulonephritis we observed a marked upregulation of cse protein during the course of the disease paralleled by a stabilization of nrf protein. from our data, we hypothesize that pdgf-bb-mediated regulation of cse via a redox-mediated activation of nrf may constitute a protective mechanism during glomerular inflammatory disease. rac knockout protects from acute hepatic damage following doxorubicin treatment bopp a., wartlick f., fritz g. heinrich-heine-universität institut für toxikologie, universitätsstr. , düsseldorf, germany rac belongs to the best characterized members of the ras-homologous (rho) family of small gtpases, which are key regulators of the actin cytoskeleton. furthermore, rac is part of the activation of the nadph oxidase, which produces reactive oxygen species and regulates the activity of stress kinases (e.g. sapk/jnk) and transcription factors such as nf-κb and ap . anticancer drugs cause dna damage, which in turn stimulates the dna damage response (ddr) regulating dna repair, cell cycle progression and, in case of non-repairable dna damage, triggers apoptosis. so far, a role of rac in the ddr has not been reported. based on its exceptional function as a regulator of transcription and because of its recently found ability to translocate to the nucleus, we hypothesize that rac may be involved in the ddr. to study the in vivo function of rac we used an inducible cre-based knockout mouse model (rac flox/flox/mxcre ). mice were treated with different doses of doxorubicin for different periods of time. we monitored gh ax foci formation as a marker of dna strand breaks, used the masson-goldner staining for the detection of collagen accumulation, analyzed phosphorylated histone as a marker of mitotic events and performed a tunel assay to detect apoptotic cells. in the absence of rac the basal mrna expression of pro-fibrotic ctgf was decreased. collagen levels were increased and mmp mrna expression was reduced in the liver of rac -/animals as compared with rac proficient animals. in addition we found more apoptotic cells in rac -/mice. hours after treatment with the anthracycline derivative doxorubicin the number of gh ax foci in rac -/animals was reduced in comparison to rac +/+ animals. we also found lower level of ctgf mrna expression and reduced amount of collagen in rac -/mice. none of these protective effects resulting from rac deficiency could be detected after administration of three consecutive doxorubicin injections over a time period of days. there were no significant differences in the number of gh ax foci or collagen accumulation. the mrna expression of ctgf was even higher in rac -/animals. furthermore the number of mitotic events was almost two times higher in the rac -/mice compared to the rac +/+ mice. summarizing, our findings show that impaired hepatic expression of rac protein is hepatoprotective against acute damage following doxorubicin exposure, but does not protect against doxorubicin-induced subacute toxicity. in vitro cytotoxicity of tbhq (tert-butyl-hydroquinone) braeuning a., vetter s., schwarz m. institut für experimentelle und klinische pharmakologie und toxikologie toxikologie, wilhelmstrasse , tübingen, germany at high concentrations, tert.-butyl-hydroquinone (tbhq), a phenolic antioxidant frequently used as a food preservative, exerts cytotoxic effects, which are closely linked to its ability to form reactive oxygen species as a consequence of redox cycling processes. here we describe that treatment of murine t cells with tbhq in -well culture plates induces the death of untreated cells in neighboring wells on the same plate. the mechanisms underlying that effect were investigated. death of the seemingly untreated neighboring cells was caused by a more toxic and volatile tbhq oxidation product which was formed in a non-enzymatic process involving metal ions and oxygen. the unexpected perturbation of cytotoxicity testing by the volatile tbhq metabolite shows that not only metabolic processes, but also non-enzymatic mechanisms have to be considered as important parameters for in vitro assays. furthermore, our data show that even cells several wells distant from the site of treatment do not necessarily constitute proper "untreated" controls when cells are treated with tbhq, e.g. in assays aimed to analyze the activity of the tbhq-inducible nrf pathway. s angiotensin ii causes oxidative stress and dna damage in mouse kidneys via the angiotensin ii type receptor brand s. , amann k. , schupp n. universität würzburg institut für pharmakologie und toxikologie, versbacherstr. , würzburg, germany universität erlangen-nürnberg institut für pathologie, krankenhausstraße , erlangen, germany angiotensin ii (ang ii), the reactive peptide of the renin-angiotensin-system, causes vasoconstriction and, in higher levels hypertension, which is connected with an increased cancer risk in the kidney. treatment of male c bl/ mice with ang ii results in the formation of superoxide radicals and dna damage in the kidney as well as in the heart. to answer the question if the dna damage is caused by hypertension or by elevated ang ii concentrations, mice were treated with different compounds: the angiotensin-converting-enzyme blocker ramipril, the ang ii receptor blocker ramipril, the ang ii receptor candesartan, the antioxidant tempol and the vasodilator hydralazine. the effect on blood pressure and renal function of ang ii-treated c bl/ mice was examined. treatment with ang ii led to a significant increase in blood pressure. candesartan and hydralazine led to a decrease, whereas intervention with ramipril and tempol had no effect. equal conditions could be found by examining renal function regarding the excretion of urinary albumin, which was ameliorated by candesartan and hydralazine. in addition, histopathological changes were investigated. there was significant glomerular damage and tubulointerstitial damage in ang ii-treated animals compared to control animals, which was significantly improved by candesartan and tempol. hydralazine and ramipril mitigated the observed renal damage but were less effective than candesartan. furthermore, the ang ii-induced formation of superoxide radicals in the kidney and the heart was slightly affected by all interventions. genomic damage, in the form of double strand breaks was prevented by the ang ii receptor antagonist candesartan and the antioxidant tempol. to sum up, the results from this study show that ang ii induces the elevation of markers of kidney failure and dna damage, which is prevented by substances lowering blood pressure like candesartan, showing the receptor responsibility for the induction of dna damage. actually by substances not lowering blood pressure like tempol, the oxidative stress and dna damage was ameliorated, showing the involvement of reactive oxygen species. optimization of the balb/c- t cell transformation assay by coupling a drug metabolizing system brauneis m. d., steinberg p. stiftung tierärztliche hochschule hannover institut für lebensmitteltoxikologie und chemische analytik, bischofsholer damm , hannover, germany the analysis of the carcinogenic potential of chemicals plays an important role in toxicology. up to now the acquisition of such data requires a large amount of animal experiments. the aim of this study is to reduce the number of experimental animals being used by further optimizing the balb/c- t cell transformation assay, an already well-established in vitro method. this method, which is also well suited for high throughput screening applications, allows a quantitative analysis of the aforementioned carcinogenic potential. the incubation of balb/c- t cells (murine embryonic fibroblasts) with mutagenic compounds leads to a loss of contact inhibition between these cells, which results in the development of so-called foci. these foci can be distinguished by characteristic changes in cell growth behaviour, a result of the treatment with carcinogenic compounds, and their number is therefore directly related to the genotoxic potential of the latter. a major disadvantage of the "classic" balb/c- t cell transformation assay is that a number of compounds initially require a metabolic transformation to gain their full genotoxic potential. hence, without prior metabolic transformation many chemicals are not detected as carcinogenic in the abovementioned test system. to overcome this drawback the balb/c- t cell transformation assay has been coupled to a drug metabolizing system, in this case the so-called liver s . in a first step the well-known genotoxic agents benzo[a]pyrene, aflatoxin b and nnitrosodimethylamine were tested in this assay. all three compounds led to a concentration-dependent increase in the number of foci formed, whereby this concentration-dependent increase was observed in a non-cytotoxic concentration range. in a next step the balb/c- t cell transformation assay will be coupled to further drug metabolizing systems as well as to the soft agar assay. this study is being financially supported by the stiftung set and the doerenkamp-zbinden foundation. adenylyl cyclases (acs) synthesize the second messenger camp. the family of acs consists of nine membranous and one soluble isoforms with ac and ac being the predominantly expressed isoforms in the heart. in the heart, acs integrate β-adrenergic (β-ar) signaling as the main physiological mechanism to improve cardiac performance. although ac and ac share high sequence homology, opposing effects on cardioprotection have been reported, where disruption of ac , as well as overexpression of ac both exerting beneficial effects in heart failure. prospective pharmacological treatment of heart failure on the level of ac is under investigation. our study explored the impact of ac ko on ac-activities in the heart at a functional level. complementary, mrna expression studies of the β-ar-g-protein-ac signaling cascade were performed to detect possible compensatory alterations. hearts from - week old homozygote ac knockout and wild-type male littermates were examined in this study. ac activities where measured in cardiac membrane preparations from left ventricles. ac activities were assessed under β-ar and g-protein (g s) stimulation by isoproterenol, guanosine '-triphosphate (gtp) and '-o-( thiotriphosphate) (gtpγs) as well as for direct activation by forskolin. relative mrna expressions for ac - , gs-, gi-a and β -, β -ar where measured by quantitative realtime pcr. surprisingly, assessment of basal, β-ar and g-protein-mediated ac-stimulation as well as direct activation by forskolin revealed no changes in ac activities. besides from detection of the ac knockout, mrna expressions analysis of ac - , gs-, gi-a and β -, β -ar did not detect any compensatory alteration. these findings suggest that proximal adrenergic signaling in the heart does not necessarily require ac . whether physiological integration of beta adrenergic signaling in the heart is mediated by both isoforms ac and ac , or can be attributed to one main isoform remains to be elucidated. melanocortin-promoted pka activation decreases ampk activity via erk- / and lkb- in hypothalamic gt - cells breit a., ellen d., gudermann t. goethestrasse , münchen, germany α-melanocyte stimulating hormone (α-msh)-induced activation of the melanocortin- receptor (mc r) in hypothalamic neurons increases energy expenditure and inhibits food intake. intrahypothalamic injection of melanocortins decreased food intake due to the inhibition of amp-activated protein kinase (ampk) that has recently been reported to enhance food intake in rodents. until now, it is not clear if α-msh affects ampk via direct intracellular signaling cascades or if the release of paracrine factors is involved. herein, we used a murine, hypothalamic cell line (gt - cells) and monitored ampk phosphorylation at thr which has been suggested to increase ampk activity. we found that α-msh dephosphorylated ampk at thr and consequently decreased phosphorylation of the established ampk substrate acetyl-coa-carboxylase at ser . inhibitory effects of α-msh on ampk were blocked by specific inhibitors of protein kinase a (pka) or extracellular-regulated kinases- / (erk- / ), pointing to an important role of both kinases in this process. since α-msh-induced activation of erk- / was blunted by pka inhibitors, we propose that erk- / serves as a link between pka and ampk in gt - cells. furthermore, down-regulation of liver kinase b- (lkb- ), but not inhibition of calcium-calmodulin-dependent kinase kinase-β or transforming growth-factor-beta-activated kinase- decreased basal phosphorylation of ampk and its dephosphorylation induced by α-msh. thus, we propose that α-msh inhibits ampk activity via a linear pathway including pka, erk- / and lkb- in gt - cells. given the importance of the melanocortin system in the formation of adipositas detailed knowledge about this pathway might help to develop drugs targeting obesity. autism spectrum disorder (asd) is a complex neurodevelopmental disorder with dysfunction of social interaction and communication. a hitherto unknown complexgenetic principle of origin probably underlies asd. so far, more than candidate genes were identified in literature. the patients affected with the monogenic timothy syndrome show multiorgan dysfunction including lethal arrhythmias, immune deficiency, skeleton-dysplasia, syndactylia and autism. this single gene disorder serves as a model disease for asd, giving insights in a possible pathophysiology. here, a point mutation in a highly-conserved region of the pore-forming subunit of the voltage-dependent calcium channel (ca v) cav . gene (cacna c) results in incomplete inactivation of the l-type calcium currents (splawski et al., cell ; : - ) . functionally similar biophysical effects can be induced by structural variation β -and β -subunits of the voltagedependent calcium channels (herzig et al., faseb j. ; : - ; jangsangthong et al., pflugers arch. ; : - ) . supported by findings in a meta-analysis of linkage data of asd patients (trikalinos et al., mol psychiatry. ; : - ) , we are investigating a function-based candidate gene hypothesis linking the β subunit gene (cacnb ) with asd. we performed a case control study sequencing all exons and flanking intronic regions of cacnb in patients with asd. we found three rare missense mutations in asd patients, but not in unaffected controls. all three mutations occur at highly conserved positions and might alter protein function; additionally results one amino acid substitution highly probable in a post-translational modification by phosphorylation. so far, we characterized two of these mutations and also a phosphorylation-mimicking mutant in electrophysiological studies. all variants show a decelerated and incomplete time-dependent inactivation of the co-transfected cav . subunit. furthermore, two variants exhibit a significant increased slope factor of voltage-dependent steady-state inactivation. we here present mutations in the β subunit gene of asd patients that result in a retardation of inactivation behavior, thus phenocopying the monogenic timothy syndrome mutations of cav . . β subunit mutations may influence neuronal function or development in some asd patients. jangsangthong, w., kuzmenkina, e., khan, i.f., matthes, j., hullin, r., and herzig, s. ( ) . inactivation of l-type calcium channels is determined by the length of the n terminus of mutant beta ( ) subunits. pflugers arch , - . herzig, s., khan, i.f., grundemann, d., matthes, j., ludwig, a., michels, g., hoppe, u.c., chaudhuri, d., schwartz, a., yue, d.t., et al. ( ) . mechanism of ca(v) . channel modulation by the amino terminus of cardiac beta -subunits. faseb j , - . splawski, i., timothy, k.w., sharpe, l.m., decher, n., kumar, p., bloise, r., napolitano, c., schwartz, p.j., joseph, r.m., condouris, k., et al. ( ) . ca(v) . calcium channel dysfunction causes a multisystem disorder including arrhythmia and autism. cell , - . trikalinos, t.a., karvouni, a., zintzaras, e., ylisaukko-oja, t., peltonen, l., jarvela, i., and ioannidis, j.p. ( ) . a heterogeneity-based genome search meta-analysis for autism-spectrum disorders. mol psychiatry , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] background: brain serotonin ( -ht) has been implicated in the regulation of food-intake. the ingestive effects of -ht are mediated by a number of different receptor subtypes under which the -ht a-receptor plays a central role. former in vivo studies have shown an increased intake of food, elicted by -ht-receptor agonists. the aim of this behavioural pharmacologic project was to determine if the hyperphagic effect is mediated by presynaptic -ht a autoreceptors in the raphe nuclei or by postsynaptic -ht a heteroreceptors in serotonergic terminal structures. methods: the effect of the -ht a receptor agonist -oh-dpat ( . , . or . mg/kg) was investigated on feeding behaviour in non-food-deprived young-adult and adult nmri and transgenic l mice. l mice are characterized by an overexpression of postsynaptic -ht a receptors. results: the administration of the -ht a receptor agonist induced hyperphagia in all groups of mice, except for the adult transgenic mice which showed no drug effect. conclusion: the results confirm a key role of the -ht a receptor in food intake. further, we make the assumption that the hyperphagic effect of -oh-dpat is mediated by presynaptic -ht a autoreceptors in the raphe nuclei which decreases -ht function in the central nervous system. it can be speculated that the aberrant feeding behaviour of the adult transgenic mice refers to a possible opposite role of the postsynaptic -ht a receptors. these receptors might affect the release of neuropeptides in the hypothalamus. the efflux transporter abcc (mrp ) expressed at different compartment barriers is important for the elimination of various endogenous and exogenous compounds. with some evidence inflammatory processes regulate abcc expression and cause changes of absorption, distribution and clearance of a number of xenobiotics. the investigation of the influence of interleukin (il) β on abcc mrna and protein expression in various cell lines representing specific tissues is the aim of our study. a further aim is to characterize the signaling pathways regulating abcc expression while inflammation. three different cell lines a) hepg cells (liver tissue) and b) caco (colon tissue) both without naïve il β expression and c) skhep cells representing physiological liver tissue with naive il β expression, were stimulated with different concentrations of il β (range pg/ml to ng/ml). over a period of h samples were taken at defined time points. abcc mrna and protein expression were quantified by qrt-pcr and western blot analysis, respectively. by using small molecule kinase inhibitors for signal transduction proteins (p mapk, akt, erk and jnk) we analysed the signal transduction pathways associated with il β-mediated transcriptional abcc regulation. on abcc mrna level an up-regulation in caco cells ( , fold) and hepg cells ( , fold) within the first hour after stimulation with ng/ml il β was shown. in contrast skhep cells demonstrated a decreased abcc mrna expression ( , - , fold) in comparison to unstimulated controls. the abcc protein expression exhibited a time and il β dependent regulation as well. the analysis for the signal transduction showed for p mapk a moderat time dependet down regulated phosphorylation ( %) in hepg cells whereas it showed no effect in caco cells. concluding, the expression of abcc is regulated moderately by il b in a concentration and time-dependent manner. interestingly, the effects are strongly tissue-dependent concerning abcc expression and signal transduction pathways and show partly contradictory results. the regulation of the different signaling pathways is currently subject of ongoing investigations. introduction: despite the remarkable success of imatinib treatment of chronic myeloid leukemia (cml), therapy resistance emerged as a major clinical problem. the aim of this study was to identify micrornas, which may serve as biomarkers for therapy response or predict pathways involved in pharmacoresistance of imatinib treatment. methods: blood was collected from cml-patients, ten of whom responded to imatinib therapy. after rna extraction from leukocytes, we performed a taqman low-density array screen to determine the expression of micrornas. statistical analysis using the -∆ct method was performed. micrornas showing a p-value< . and a fold change> were considered to be significantly differently expressed. in addition, by using microrna target prediction databases (targetscan , mirdb , pictar , microcosm , diana microt ), selected putative target genes were further functionally investigated by the david bioinformatics database . results: comparing treatment-naïve responders and non-responders four micrornas were identified to be deregulated that were predicted to target genes, especially transcription regulators ( %). pathway analysis showed that six of the predicted genes are relevant in cancer pathways, four of which play a role in cml (smad , nras, rb , raf ). when comparing patients' expression profiles before and under treatment, seven micrornas were identified to be deregulated in responders and five micrornas in nonresponders. ninety-nine targets of the latter include transcription regulators ( %), but also cellular transporters ( %, especially uptake transporters of the slc-family). most target genes are involved in mapk signalling or endocytosis pathways. conclusion: analysis of microrna expression profiles revealed four micrornas involved in imatinib-response and micrornas deregulated during imatinib treatment. predicted target genes code mainly for transcription factors as well as oncogenes relevant for cml and are involved in transporter expression and endocytotic processes. dissociations in the effects of beta -adrenergic receptor agonists on camp formation and superoxide production in human neutrophils brunskole i. , buschauer a. activation of the β -adrenergic receptor (β ar), a classically gs-coupled receptor, in neutrophil granulocytes results in an inhibition of inflammatory responses [ ] , which could be further therapeutically exploited. the aim of the present study was to evaluate the effects of various β ar ligands on cyclic adenosine ', '-monophosphate accumulation (camp assay) and n-formyl-l-methionyl-l-leucyl-l-phenylalanine (fmlp)induced superoxide anion production (o •assay) in isolated human neutrophils, which are a physiologically relevant native test system. camp concentration in neutrophils was determined by hplc/tandem mass spectrometry, and o •formation was assessed by monitoring the superoxide dismutase-inhibitable reduction of ferricytochrome c. (-)-isoproterenol, (-)-adrenaline, salbutamol and dobutamine were more potent in inhibiting fmlp-induced o •production than in stimulating camp accumulation. (-)-ephedrine and dichloroisoproterenol were devoid of any agonistic activity in the camp assay, but could partially inhibit fmlp-induced o •production at higher concentrations. moreover, (-)-adrenaline and dobutamine were equi-efficacious in both assays whereas the efficacy of salbutamol was more than two fold higher in the o •assay. this suggests that salbutamol is able to stabilize a different receptor conformation than the other two ligands. thus, ligand-directed signaling via β ar can also occur in human neutrophils. in addition, differences between the data from neutrophils and recombinant test systems [ , ] were noticed, pointing to the problem of insufficient comparability of effects in recombinant and native test systems. the investigation of β ar antagonists on neutrophil granulocytes is subject of ongoing work, in order to find out whether pkb values of β ar antagonists in the camp assay and the o •assay are different. such differences were previously reported for β ar antagonists in other test systems [ ] . moreover, studies with protein kinase a inhibitors should give deeper insight into the signaling events in neutrophils that result in inhibition of fmlp-stimulated o •production and clarify how camp increase interferes with this events. agonist-selective internalization of the human -ht a receptor buchborn t., kahl e., höllt v., koch t. otto-von-guericke-universität magdeburg institut für pharmakologie und toxikologie, leipziger straße , magdeburg, germany the serotonin a ( -ht a) receptor is a g protein coupled receptor and the molecular target of lsd-like hallucinogens. downregulation of -ht a receptors is an adaptive process considered relevant for the therapeutic action of diverse serotonergic antidepressants, such as ssris. since the antidepressant targeting of -ht a receptors, however, is largely restricted to indirect agonists and/or antagonists, little is known about the mechanisms and implications of their regulation by direct agonists. in the present study we, therefore, investigated the capacity of various agonists to regulate the human ha-tagged -ht a receptor by internalization. using immunocytochemical techniques in stably transfected hek cells, we show that agonists differ in their capacity to internalize the receptor. serotonin, quipazine and doi are the agonists most efficaciously internalizing the receptor, dmt and methysergide, on the other hand, hardly internalize; other agonists like psilocin, ergotamine and lsd induce low to intermediate internalization. the specificity of the agonistic effect was demonstrated by the -ht a selective antagonist ketanserin, which blocked the agonist-induced internalization. in additional experiments, we show that the internalized -ht a receptors colocalize with a -labelled transferrin receptors, and that the internalization can be blocked by high molar sucrose; these results are indicative of a clathrin associated sequestration of -ht a receptors in recycling endosomes. also, we demonstrate that the proteinkinase c activator pma efficaciously induces -ht a internalization in the absence of an agonist, and that the doi-induced internalization can be blocked by the proteinkinase inhibitor staurosporine. we, thus, confirm previous findings that the activation of proteinkinases seems to be necessitated for the -ht a internalization to occur. overall, we conclude that the internalization of the human -ht a receptor is agonist-selective, and employs a proteinkinase (possibly pkc) dependent, clathrincoated endosome associated pathway. as there is recent evidence that the regulation of -ht (a) receptors by agonists might have antidepressant (-like) properties, knowledge about the agonist-selective processing of -ht a receptors could help to identify agonists most promising for future (pre-)clinical research. non-clinical safety assessment of homeopathic medicinal products: criteria for establishing a first safe dilution buchholzer m. -l., werner c., knoess w. bfarm bundesinstitut für arzneimittel und medizinprodukte zulassung , kurt-georg-kiesinger-allee , bonn, germany like all human medicinal products the homeopathic medicinal products for human use must demonstrate adequate safety. in general, they are regulated according to the analogue non-clinical safety principles (points to consider on non-clinical safety of homeopathic medicinal products of botanical, mineral and chemical origin, adoption by hma ). one particular approach is the recently introduced concept of a first safe dilution (fsd; introduction to the list of first safe dilutions, adoption by hma ) . this contribution summarizes the first experiences in establishing fsds of a selection of given homeopathic preparations by bfarm. for a given preparation the major toxicological concern and available data set is identified. this determines the safety assessment route: food regulation, permitted daily exposure (pde), threshold of toxicological concern (ttc) or lowest human recommended dose (lhrd/ ). finally the acceptable amount/tolerable daily intake is derived and the respective fsd is calculated. for example the draft evaluation for reserpinum (ph. eur. method . . ) and for atropine (ph. eur. method . . or . . ) based on lhrd leads in each case to a suggested fsd of d related to g of preparation. furthermore, the draft evaluation for potassium iodide (ph. eur. method . . or . . ) based on food legislation emerged a proposed fsd of d related to g of preparation. the concept of fsd combines a scientific and at the same time pragmatic approach in differentiated risk assessment of homeopathic medicinal products. impact of myricetin and its methylated derivatives laricitrin, syringetin and myricetin- `, `, `-trimethylether in c. elegans büchter c. , ackermann d. polyphenolic compounds ubiquitously present in herbal food are discussed to contribute to the health beneficial effects of a diet rich in vegetables and fruits. additional to a strong antioxidative activity of various flavonoids, most of these substances display a variety of other pharmacological properties. we investigated the flavonoid myricetin found in several species of berries, as well as the methylated derivatives laricitrin, syringetin and myricetin- `, `, `-trimethylether. in this study caenorhabditis elegans was used as a model to explore the impact of myricetin and its methylated derivatives in vivo and to investigate molecular modes of action. myricetin ( µm) caused an increase in mean and median adult lifespan of c. elegans. this longevity effect was associated with a decrease of the aging marker lipofuscin as well as a decrease in ros induction, measured by using the h dcf-da assay. however, myrictin failed to improve heat stress resistance, an attribute often associated with longevity in c. elegans. the methylated myricetin derivatives ( µm) showed a decrease in lipofuscin accumulation and ros induction and they further improved the heat stress resistance. in order to elucidate the basis of the life prolonging action of myricetin, we investigated its influence on factors known to have important functions in stress response and the regulation of aging, namely the foxo homologue daf- , the nad + -dependent protein deacetylase sir- . and the heat-shock transcription factor hsf- , respectively. lifespan extension by myricetin disappeared in daf- and sir- . loss of function mutant strains, showing the effect is at least partially dependent on these signaling molecules. by using a hsf- loss of function mutant strain of c. elegans, it was further shown that the life prolonging effect of myricetin is independent of hsf- . in conclusion, our results indicate that the life prolonging effect of myricetin is at least in part dependent on daf- and sir- . , probably due to a modified expression of target genes. stimulatory and inhibitory control of phospholipase c-gamma bühler a., walliser c., becker l., gierschik p. universität ulm institut für pharmakologie und toxikologie, albert-einstein-allee , ulm, germany activation of phospholipase c-γ (plcγ ) upon b cell antigen receptor (bcr) stimulation has been implicated to be a critical step in the bcr-mediated calcium signaling. therefore it is important to understand the mechanisms of how the activity of plcγ is stimulated and inhibited. the mammalian plcs are divided into six subfamilies, designated β, γ, δ, ε, ζ, and η. within the plcγ subfamily, the two plcγ isoforms share a number of features that are distinct from those of the other plc subfamilies. the most striking difference is the insertion of additional domains between the catalytic subdomains x and y. this specific array (sa) contains a second, split pleckstrin homology (spph) domain, consisting of two halves separated by two src homology (sh ) domains and one src homology (sh ) domain. there is abundant evidence in the literature that plcs are autoinhibited in their basal state by structural elements within their x/y linker, pointing to a conserved role of the x/y linker in autoinhibitory regulation of plc isozymes. data from our group show that plcγ is also regulated by autoinhibitory elements within its specific array (walliser et al., ; everett et al., ) . our recent data demonstrated that plcγ is negatively regulated by its sa. specifically, within the sh domain tandem, the c-terminal sh (sh c) and the sh domain in combination, but not either one alone, cause the strongest autoinhibitory control of plcγ . plcγ has been shown to be phosphorylated at tyrosine residues and upon bcr stimulation (kim et al., ) . both tyrosines are located in the linker between the sh c and the sh domain, which we have shown to be the major elements involved in autoinhibitory regulation of plcγ . interestingly, a novel phosphorylation site in plcγ was found in non-small cell lung cancer (nsclc) tissue which is located at tyrosine residue (rikova et al., ) . in this work, we demonstrate, for the first time, the activation of plcγ by phosphomimetic mutations in these three positions and the functional interplay of the three tyrosine phosphorylation targets. most interestingly, mimicking phosphorylation of tyr is critical to fully activate the enzyme. the results not only point to a crucial role of plcγ in pulmonary tumorigenesis, but also prompt and stimulate the search for the protein kinase involved in phosphorylating plcγ at tyr . molecular characterization of hepatotoxic effects of perfluorooctanoic acid (pfoa) buhrke t., scharmach e., lampen a. bundesinstitut für risikobewertung (bfr) lebensmittelsicherheit, max-dohrn-str. - , berlin, germany perfluorooctanoic acid (pfoa) is an industrial chemical that is used for the fabrication of numerous products with oil-, dirt-and water-repellent properties. pfoa is resistant to chemical, thermal and biological degradation and has become a global contaminant of soil, water, air and food in the meantime. the toxicological data of pfoa give cause for concern as the substance was shown to damage the liver of rodents and to impair embryo development. currently, the hazard potential of pfoa for humans is controversially discussed. in this study the human liver cell line hepg was employed to analyse the hepatotoxic effects of pfoa on the cellular and on the molecular level. pfoa was shown to stimulate cellular proliferation at concentrations in a range between µm and µm. at concentrations higher than µm the substance was cytotoxic to the cells (ic µm). cytotoxicity was not due to apoptotic mechanisms as no increase of caspase activity was detected up to a level of µm pfoa. on the molecular level pfoa is known to act as an agonist of the peroxisome proliferator-activated receptor alpha (pparα), and the observed hepatotoxic effects in rodents are associated with pfoa-mediated pparα activation. here we show that pfoa has the capacity also to activate the human isoform of pparα. additional human nuclear receptors were tested for activation by pfoa, and pparγ as well as the pregnane x receptor (pxr) were shown to be activated at high concentrations of pfoa whereas pparδ and the liver x receptor alpha (lxrα) were insensitive to activation by pfoa. notably, we observed a significant inhibition of the activity of the hepatocyte nuclear factor α (hnf α) by incubating the cells already with moderate concentrations of pfoa at a level of about µm. these findings indicate that additional, pparα-independent mechanisms may contribute to the observed hepatotoxicity of pfoa. the elucidation of novel modes of action of pfoa is relevant for the ongoing risk assessment of the substance. s human breast stem cells as a toxicological model for endocrine disruptors, such as soy isoflavones stempin s. , bumke scheer m. (kao et al., ) . two daughter cell lines were developed from m sv after x-ray irradiation (m sv r ) and an additional transfection with a mutated erbb oncogene (m sv r -n ), resulting in high and low tumorigenicity respectively and showing a change in estrogen response after growth in minimal media (wang et al., ) . isolated isoflavones are currently widely used in the treatment of postmenopausal symptoms of women. according to the stem cell theory of carcinogenesis, breast stem cells are the ideal target for the proposed research. however, the epidemiological data to the effect of isoflavone intake on breast cancer is contradictory. therefore, we want to develop a toxicological model using these human breast stem cell lines to test the effect of endocrine disruptors, such as soy isoflavones in a human relevant model. in the present study we analyzed the effect of the phytoestrogen genistein, the most intensively studied soy protein, on the differentiation of the hbec lines. the expression of different luminal epithelial cell markers, estrogen receptors and stem cell markers was measured on the mrna level by quantitative real time pcr. the analysis of several of these markers was also performed on the protein level using western blot. additionally, a broad number of genes related to breast cancer and estrogen receptordependent signal transduction were studied using a commercial pcr-array. in parallel we are also analyzing the changes on the protein level using d gel electrophoresis. we want to use this panel of different markers to establish a toxicological model that can be used in the future to analyze a wide range of different endocrine disruptors. kao cy, nomata k, oakley cs, welsch cw, chang cc. ( ) bronchial asthma is a common inflammatory disease of the airways whose occurrence has increased dramatically over the past decades. histamine plays an important role in mediating the inflammatory response leading to characteristic symptoms like wheezing, coughing, chest tightness, and shortness of breath. since antagonizing the histamine h -receptor (h r) shows no ameliorating effects on asthmatic symptoms, h r antagonists may be new drugs for asthma therapy. in addition to the h r-and h rselective antagonist thioperamide, the selective h r antagonist -[( -chloro- h-indol- yl)carbonyl]- -methylperazine (jnj ) is used in pharmacodynamic studies. a correct interpretation of the collected data requires the detailed knowledge of the pharmacokinetics of the applied substances. for this reason, we developed a fast and robust method based on high performance liquid chromatography coupled to tandem mass spectrometry (hplc-ms/ms) which allows the simultaneous quantitation of thioperamide and jnj as well as the selective h r antagonist -({ - [ -(piperidin- -yl) propoxy]phenyl}methyl)piperidine (jnj ) in murine plasma and lung tissue. the treatment of plasma samples based on protein precipitation performed with a mixture of methanol and . m znso using µl of plasma. analyte extraction from lung tissue was achieved by treating - mg of tissue with a mixture of ethanol and water followed by rigorous mixing using a fastprep-system. ten µl of the extracted samples were transferred to a synergy polar-rp a mercury column ( x mm; µm) connected to a polar-rp security guard. chromatographic separation was performed via a gradient using an acetate buffer (ph ) and methanol at a flow rate of . ml/min. the analytical run-time was minutes. for plasma samples the assay was linear over a concentration range of . - µg/ml for jnj and jnj , and . - µg/ml for thioperamide, respectively. in tissues, thioperamide could be quantified in a concentration range of . - µg/sample, jnj and jnj in a range of . - µg/sample. our results show that the developed hplc-ms/ms method is suitable for the quantitation of all tested histamine-receptor ligands in murine plasma and lung tissue. the functionality of the heart greatly depends on strict homeostasis and interplay of a range of signalling cascades. deregulation of either one is always harmful and eventually detrimental for life. some of the most relevant signals in the adult heart are triggered by the stimulation of g protein-coupled receptors such as adrenergic or angiotensin receptors. those in turn are modulated by a small subset of kinases, the g protein-coupled receptor kinases (grks). interestingly, grks, which for the longest time were believed to regulate only g protein-coupled receptors were shown to modulate also non-receptor-mediated signalling pathways. by now it is well documented that grk plays important roles in both the physiological as well pathological setting of the adult heart. in spite of the important functions of grks in the adult heart, it must be assumed that grk , one of the two main cardiac grks, may also be involved in signal modulation in the course of heart development. deregulation of grk -dependent pathways may very well be causal for impaired cardiac development up to congenital heart disease. in fact, grk is already expressed during embryogenesis and we can detect it in the developing heart. however, grk has not been studied yet for a potential function during embryonic development in general or heart formation in particular. we have established zebrafish as a very time and cost efficient vertebrate model to investigate the role of grk on cardiac signalling and development. tools for grk specific loss-as well as gain-of-function analyses have been developed in our lab. we revealed an unexpected role of grk in the development of left-right asymmetry in zebrafish. clinically, this has been associated with disorders such as heterotaxy and other syndromes linked to ciliary dysfunction. many of those disorders are known to affect proper heart development resulting for example in septum defects or in detrimental translocation of the outflow tract. precisely, depletion of the close homolog of human grk in zebrafish mirrors the human syndrome called heterotaxy by displaying randomized placement of inner organs, aberrant heart looping and disrupted valve formation in the heart. in addition, loss of zebrafish grk results in a lower heart rate as well as dilatation of the embryonic heart at later stages of development. therefore, we believe that grk may potentially serve as a candidate gene for congenital heart disease. identification of a hcn interacting protein in mouse brain cao-ehlker x., hammelmann v., zong x., fenske s., biel m. department of pharmacy, center for drug research ludwig-maximilians-universität, munich center for integrated protein science cipsm, butenandtstr. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] münchen, germany hyperpolarization activated cyclic nucleotide-gated cation channels (hcn) pass a depolarizing current (ih) that is involved in cardiac pacemaking and the control of numerous basic functions in neuronal circuits. the four hcn channel types (hcn - ) display specific expression pattern in brain suggesting that each channel fulfills a distinct physiological function. while hcn , hcn and hcn channels have been studied in quite some detail there is only little information on the particular role of the hcn channel. as an important step towards achieving a better understanding of hcn function we set out in this study to identify proteins that are assembled with hcn in brain tissue. to this end we performed a yeast two hybrid screen with a mouse cdna library using the hcn c-terminal domain as bait. several proteins were obtained and confirmed for interaction with hcn using heterologous coexpression in hek cells. here, we provide an in-depth analysis of the functional interaction between hcn and one of the identified interacting proteins (hip . ). we show that hip . physically binds to hcn channels in vitro and in vivo. still several open issues remain to be clarified i.e. the precise function of tpc and its tissue-specific and subcellular distribution. therefore we established a mouse model with a general deletion of tpcn and generated a series of tpc antibodies. using these tools we investigate the closer molecular and vesicular environment by different biochemical approaches i.e. affinity purification from native tissue derived from wild-type and as a control from knock-out mice, density gradient based vesicle separation, fluorescence activated organelle sorting (faos), total internal reflection fluorescence (tirf) and confocal microscopy. so far, we confirmed the tpc knock-out model by our self-generated tpc antibodies. tpc knockout mice are viable and do not show any obvious deficits. to isolate tpc containing vesicles or protein complexes, tissue or cell culture derived material was prepurified by sucrose density gradient centrifugation. for a subsequent mass spectrometric analysis this preparation was taken as a source material for coimmunoprecipitation or faos respectively. in another approach the migration pattern of tpc containing endosomes on linear density gradients was compared with a series of endolysosomal markers i.e. different rab-, er-, golgi-and lysosomal antibodies. potentially interesting markers were then in turn analyzed for their co-localization with tpc by confocal microscopy/tirf. by combining these results with that from mass spectrometric analysis of faos samples we collect data to get detailed information on the precise endolysosomal distribution pattern of tpc . foxos are involved in a wide spectrum of cellular functions, including cell proliferation, apoptosis and regulation of oxidative stress. in order to identify novel target genes of foxo transcription factors and to achieve further insight into their role in cancer cells, dna microarray analysis was performed using wild type mcf- breast cancer cells and mcf- cells overexpressing foxo. we found that several genes involved in the tnf receptor/nf-κb pathway were differentially regulated. one of the genes that was identified to be up-regulated in foxo overexpressing cells was a a negative regulator of nf-κb signaling pathway. at both mrna and protein level foxo -dependent up-regulation of this ubiquitin modifying enzyme was confirmed. to determine whether a is a direct target of foxo , a luciferase reporter containing a . kb of the a promoter was co-transfected with different amounts of foxo wild-type expression construct. foxo induced a dosedependent increase in a promoter activity, supporting the assumption that a is a direct transcriptional target of foxo . overexpression of foxo led to decreased activity of nf-kb signaling pathway as confirmed by reporter gene and nf-kb specific elisa assays. in addition, mcf- cells can be sentisized to tnf-α mediated cytotoxicity which is assocciated with a dimineshed activation of nf-κb. altogether, we identified a , an ubiquitin modifying enzyme, as a novel foxo target gene. our data implicate that sustained foxo expression may be involved in regulation of tnf receptor/nf-κb pathway and leading to reduced cell survival. trpm is a bi-functional protein consisting of a transient receptor potential ion channel segment linked to an α-type protein kinase domain. trpm is essential for motility, proliferation and cell growth. up-regulation of trpm function is involved in anoxic neuronal death, cardiac fibrosis and tumor cell proliferation. recently, we have demonstrated that the recombinant trpm channel is inhibited by the known modulators of sk - channels such as antimalarial plant alkaloid quinine, cyppa, dequalinium, ns , ska , ucl . the most potent of these compounds, ns (ic . µm), interferes with the regulation of trpm by cytosolic mg + . here we show that ns ( µm) fully and reversibly inhibits native trpm -like currents in hek cells, freshly isolated smooth muscle cells, primary podocytes and ventricular myocytes. furthermore, we examined whether targeting of the native trpm currents by ns would impact cellular processes known to be affected by a genetic inactivation of trpm . we found that ns ( - µm) suppressed motility of hek cells without a detectable effect on cell viability. taken together, our findings indicate that ns is a potent and reversible inhibitor of endogenous trpm currents and may be a good candidate drug for pharmacological targeting of trpm . sulfur mustard ( , '-dichlorodiethylsulfide; sm) is a highly toxic and mutagenic warfare agent classified as a weapon of mass destruction. as soon as sm was first used as a warfare agent, research aimed at the development of an effective antidote was launched. early studies with first-generation inhibitors of poly(adp-ribose) polymerases (parp) have revealed promising therapeutic potential in sm-induced skin injury, but the underlying mechanism remains elusive. the current renaissance of parp inhibitors in cancer chemotherapy has revived the discussion on their use for treatment of sm injury. thus we established a comprehensive study aiming the elucidation of the role of parp in sm pathology based on model substance -chloroethyl ethyl sulfide (cees), which is not classified as warfare agent. we have recently demonstrated that parp becomes rapidly activated in living human keratinocytes (hacat) after treatment with cees. the maximal parp activity was observed minutes after treatment with mm cees. the activation was transient and dose dependent. to our knowledge this is the first demonstration of parp activation after treatment with mustards in the context of live cells. an important question is how parp- becomes activated upon treatment with mustards. parp- is a first-line protein involved in the cellular response to dna strand breaks. however, mustards do not directly induce large numbers of such lesions. one possibility is that parp- is activated by dna breaks incorporated as base excision repair (ber) or nucleotide excision repair (ner) intermediates. thus, we performed knockdown experiments of ape and ercc , i.e. endonucleases involved in ber and ner, respectively. the reduction of ape expression had no effect on parp activity. surprisingly, the knockdown of ercc almost completely abolished the cellular par production after cees treatment. the functional consequence of the errc -parp cross-talk with regards to adduct removal is under investigation. however, our present data indicate that parp activity is not obligatory for the survival of cells upon cees treatment, as revealed by the lack of effect of the potent parp inhibitor abt- . expression and activity of g protein coupled receptor kinase (grk ) are elevated in several conditions of compromised heart function. although grk inhibition has been characterized as a promising therapeutic strategy in heart failure, a specific grk inhibitor is not available. raf kinase inhibitor protein (rkip) inhibits raf but it also acts as a physiological inhibitor of grk upon phosphorylation by pkc at serine . a detailed understanding of the rkip/grk interaction may help to identify inhibitory compounds for grk . since phosphorylation often induces homo-oligomerization of proteins, we investigated whether this could be implicated in switching rkip from a raf -into a grk -inhibitor. co-immunoprecipitation assays showed that rkip self-association was substantially increased after pkc-mediated phosphorylation of rkip. rkip mutants either lacking or mimicking s phosphorylation confirmed that this phosphorylation is indeed a prerequisite for rkip/rkip association. cross-linking experiments with myc-tagged rkip in living cells or with purified rkip revealed that rkip phosphorylation by pkc promotes rkip dimers -not oligomers. to test whether dimerization is a critical step for the association of rkip with grk , we generated a peptide to inhibit rkip dimerization. intriguingly, the peptide did not only prevent rkip dimerization but also attenuated rkip/grk association. this implicates, that dimerization of rkip is essential to bind grk . to determine whether rkip dimers consequently inhibit grk activity, we established rkip mutants with high tendency to form dimers. subsequent functional analyses demonstrated that enhanced dimerization of rkip indeed translates into increased grk inhibition. we conclude that pkc-mediated phosphorylation of rkip is important for dimerization and that these dimers are essential for grk binding and inhibition. our results reveal new insights in the molecular mechanism of rkip/grk interaction and will help to develop specific grk inhibitors. expression and function of trpm ion channels in epithelial mdck cells dembla s., meiser j., philipp s. university of saarland institute for experimental and clinical pharmacology and toxicology, kirrberger str. , homburg, germany trpm proteins build ca + permeable cation channels [ ] activated by steroids [ ] and sensitive to increased temperatures [ ] . trpm channels are expressed in pancreatic ßcells as well as neurons of the dorsal root ganglion, where they act as mediators of insulin release [ ] or as nociceptors of noxious heat, respectively [ ] . however, northern blots and in situ hybridization experiments revealed that trpm is also expressed in epithelial cells of the choroid plexus and the ciliary body [ ] as well as in the kidney. pcr analysis of different epithelial cell lines indicated that trpm is also expressed in madin-darby canine kidney (mdck) cells. quantitative analysis of trpm expression by qrt-pcr revealed a ~ fold upregulation in mdck cells grown in confluency compared to well separated, proliferating cells. in contrast the level of expression of trpm , a related ion channel described as regulator of proliferation in other cell types, remained constant. hek cells overexpressing trpm channels did not proliferate in the presence of the trpm agonist pregnenolone sulphate. however, as indicated by impedance analysis, the proliferation of mdck cells in the presence pregs was only slightly affected. when we analysed the transepithelial resistance (ter) of mdck epithelial cells in transwells as a measure for the formation of tight junctions, we found that the ter of cells grown in the presence of pregs was reduced. interestingly, ca + imaging experiments using fura revealed that pregnenolone sulphate induces ca + entry in well separated mdck cells but not in cells growing in confluency. we hypothesize that trpm might act as a regulator of cell proliferation and/or the formation of tight junctions in mdck cells. inhibition of grk by rkip improves cardiac contractility and structure in a transgenic mouse model of heart failure denzinger s., schmitt j. p., lohse m. j., lorenz k. institut für pharmakologie und toxikologie pharmakologie, versbacher str. , würzburg, germany the raf kinase inhibitor protein (rkip) has been identified as a physiological inhibitor of g-protein coupled receptor kinase (grk ). grk initiates g protein coupled receptor (gpcr) desensitization. since expression and activity of grk are upregulated in human heart failure, it has been proposed that grk inhibition may resensitize badrenergic receptor activity in heart failure patients. in this study, we evaluated chronic grk inhibition by rkip as a potential strategy to improve cardiac function in heart failure. to analyse the effect of rkip on heart failure, rkip transgenic mice were crossed with mice carrying a mutation in phospholamban (pln r c ). pln r c causes severe heart failure and premature death in humans and transgenic mice. cardiac function was significantly improved in the presence of rkip as shown by left ventricular catheterization and echocardiography. expression of heart failure marker genes anf and bnp was indistinguishable between wild-type mice and mice co-expressing rkip and pln r c . in line with these findings, the life span of double transgenic mice was significantly prolonged compared to pln r c transgenic mice. slow calcium transport into the sarcoplasmatic reticulum was characterised as cause for dilatated cardiomyopathy of pln r c transgenic mice. since western blot analyses of rkip transgenic heart lysates showed increased phosphorylation of important regulators of cardiomyocyte relaxation, we analysed calcium transients and contractility of isolated cardiomyocytes as possible mechanism of the rkip mediated rescue. in the presence of rkip, calcium reuptake into the sarcoplasmatic reticulum was accelerated and cardiomyocyte relaxation improved. furthermore, coexpression of rkip significantly attenuated pathological cardiac remodelling. interstitual fibrosis and apoptotic cells were quantified in histological sections after sirius red-and tunel-staining. this study revealed a protective function of rkip in a genetic mouse model of human dilated cardiomyopathy by improving cardiac contractility and attenuating interstitial fibrosis and apoptosis. a detailed understanding of this rescue may help to find a new therapeutic strategy to improve cardiac contractility in heart failure. gαi-proteins comprise a group of three highly related members characterized by specific expression patterns. based on previous work of gi-mediated signaling pathways in cardiomyocytes and platelets, we checked gαi expression in mouse heart and platelets. the analysis revealed the presence of gαi and gαi with gαi as the predominant isoform. gene-targeted mice lacking either gαi or gαi were analyzed to unravel the physiological role of gαi-proteins in the cardiovascular system. extraordinarily prolonged bleeding times in gαi -deficient animals were an obvious phenomenon. detailed analysis using isolated platelets gαi -deficient mice exhibited reduced platelet activation and attenuated aggregation in response to stimulation by various agonists accompanied by reduced thrombus formation and diminished stability on a collagen-coated surface. employing in vivo injury/thrombosis models revealed abrogated thrombus formation selectively in gαi -deficient mice. comparable results were obtained in experiments using mice with megakaryocyte/platelet-specific gαi deficiency. to assess the pathophysiological consequences of platelet gαi function, we challenged these mice in experimental models of myocardial and cerebral ischemia. the results clearly show that platelet-gαi -deficient mice were protected from both, myocardial and cerebral ischemia. in contrast, conventional gαi -deficient mice subjected to the heart ischemia/reperfusion model exhibited a significantly increased susceptibility to ischemic injury as compared to wild type controls. in contrast, gαi deficient mice were strongly protected from injury. thus we suggest that gαi and gαi play distinct roles in major cardiovascular disorders pointing to specific, non-redundant functions of these two highly related gαi isoforms. the cgmp signaling pathway is activated by nitric oxide (no), natriuretic peptides (anp, bnp & cnp), and cgmp-elevating drugs. it regulates several physiological functions such as smooth muscle relaxation, platelet inhibition, and cell growth and differentiation. recent studies indicate that cgmp signaling might also play a role in tumorigenesis, but the cellular and molecular mechanisms of cgmp's potential pro-and/or anti-tumor activities are not well understood. this study has examined the expression and function of components of the cgmp pathway in melanoma cells of murine and human origin. we have found that mouse b melanoma cells specifically express the alpha isoform of the cgmp-dependent protein kinase type i (cgkialpha) but not the beta isoform. treatment of intact cells with the membrane-permeable cgmp analog -br-cgmp induced the phosphorylation of cgki substrates, vasodilator stimulated phosphoprotein and phosphodiesterase . anp and cnp, ligands of the membrane-bound guanylyl cyclase gc-a and gc-b, respectively, did also activate the endogenous cgmp/cgki pathway. however, b melanoma cells did not respond to dea-no, which stimulates no-sensitive soluble guanylyl cyclases. interestingly, activation of cgmp/cgkialpha signal transduction was associated with an increase in erk / and p phosphorylation, growth and migration of b melanoma cells. similar results were obtained with wm human melanoma cells. in conclusion, we have identified a gc-a/gc-b/cgmp/cgkialpha pathway in melanoma cells, which stimulates tumor cell growth and migration in vitro. pharmacologic inhibition of cgmp signaling may offer a promising strategy for the treatment of melanoma. an increasing body of evidence supports important roles for voltage-gated calcium channels in idiopathic generalized epilepsies (iges), however which calcium channels participate in ige pathogenesis and how has yet to be fully understood. recently, it has been proposed that cav . (r-type) and t-type calcium channels jointly contribute to oscillatory bursting in the reticular thalamus (rt) , which is associated with absence epilepsy. cav . is one of the two t-type calcium channels known to be expressed in the rt . it has been demonstrated that ablation of either cav . or cav . reduces susceptibility to experimentally induced epilepsy ; and in addition that both channels share several pharmacological properties [ ] [ ] [ ] . to gain further insight into interacting mechanisms of these two channels in epilepsy, we tested cav . (-|-), cav . (-|-) and cav . (-|-)xcav . (+|-) mice side-by-side in the kainic acid model of epilepsy. we provide first in vivo data supporting a synergistic mode of action for cav . and cav . calcium channels in epileptogenesis. the deubiquitinase cyld regulates mechanisms of rip /rip -dependent necroptosis in neuronal cells diemert s. , krieg s. vivo model of cerebral ischemia, we found, that cyld -/-mice exhibit significantly reduced infarction volume compared to control littermates. overall, these data reveal a role for cyld in rip / -dependent mechanisms of necroptosis in a model of glutamate toxicity in neuronal cells and further suggests cyld-mediated mechanisms of neuronal cell death as a potential therapeutic target after acute brain injury in vivo. cyanamide-mediated inhibition of n-acetyltransferase dierolf d., bonifas j., blömeke b. university trier department of environmental toxicology, universitätsring , bldg. n, trier, germany cyanamide has been used for decades for medical purposes in the treatment of alcoholism and for agricultural purposes as a plant growth regulator and bud-breaking agent. its therapeutic effect is mediated by reversible inhibition of aldehyde dehydrogenase and it was reported to be metabolised in vivo mainly via coenzyme a dependent n-acetylation by n-acetyltransferases. reported to be a substrate for n-acetyltransferases, cyanamide has a different molecular structure to arylamines and hydrazines, the preferred substrates for nacetyltransferases. therefore a more detailed investigation of its interrelations with nacetyltransferases was performed. we analysed nat enzyme activities after incubation of thp- cells with cyanamide for h, and found that the metabolic conversion of the classic substrate para-aminobenzoic acid was significantly reduced at physiologically relevant concentrations. in detail a significant dose-and time-dependent nat protein inhibition was observed for and µm cyanamide using over-expressed human recombinant nat (insect cell cytosol containing recombinant human nat * ). however, no inhibition was found in the presence of recombinant nat * . as we also provide evidence that cyanamide is not metabolised via coenzyme a dependent nacetylation in vitro by human nat or nat cytosol, by thp- cells or by human liver cytosol, we can conclude that this inhibition is not based on substrate-dependent downregulation of nat . further mechanistic and kinetic studies indicated that cyanamide reacts with the active site cysteine residue of nat , leading to its rapid inhibition. since the presence of the reduction agent dithiothreitol did not reverse the results it could be that it is possibly not caused by oxidative processes. in sum these data indicate that cyanamide is able to interact with cysteine residues of human nat , which causes its inhibition but not by a substrate-dependent mode of action. taken together our results show, that cyanamide is not n-acetylated by human nats, but might modulate nat dependent detoxification and activation of arylamines. dissecting the signal transduction pathway of acute hypoxic vasoconstriction (hpv) in precapillary pulmonary arterial smooth muscle cells ( low levels of oxygen in the pulmonary airways induce acute hypoxic pulmonary arterial vasoconstriction (acute hpv) redirecting blood flow to normoxic areas of the lung to assure optimal uptake of oxygen during ventilation. acute hpv lasting several minutes occurs predominantly in the precapillary region of the pulmonary vascular tree [ ] . therefore, precapillary pulmonary arterial smooth muscle cells (pasmc) have been suggested as sensor as well as effector cells and trpc a member of the classical transient receptor potential (trpc) ion channel family was identified to be essential for the initiation of ca + influx and the subsequent contraction of pasmc [ ] . however, the underlying oxygen sensor and the exact signal transduction pathway(s) in pasmc have not been fully elucidated yet. by using gene-deficient mouse models as well as downregulation of potential candidate proteins by specific small interfering rnas (sirnas), we aim to dissect signaling cascades of trpc channel activation in acute hpv. for pasmc isolation and culture from mice we use a technique based on magnetic separation of intrapulmonary arteries originally developed in rats [ ] . trpc-expression in freshly isolated and passaged pasmc cultured in low ( %) and high fetal bovine serum ( %) was analyzed. interestingly higher passage numbers resulted in a significant down-regulation of trpc and trpc the most predominantly expressed channel monomers in pasmc, while different serum concentrations resulted in no significant changes in their expression rates. sirnas were designed, transfected and successfully tested in hek cells and pasmc. initial results of the dissection of the signal transduction pathway activating trpc and inducing acute hpv in pasmc will be presented. references [ ] staub, n. c. ( ) . site of hypoxic pulmonary vasoconstriction, chest , s- s. [ ] weissmann, n. et al. ( ) . classical transient receptor potential channel (trpc ) is essential for hypoxic pulmonary vasoconstriction and alveolar gas exchange, proc. natl. acad. sci. u.s.a. , - trpv is a highly selective calcium channel expressed in various tissues amongst others in placenta. the channel may be involved in transcellular calcium transport in epithelial tissues thereby playing some role in calcium homeostasis of the body. in the placenta trpv is assumed to contribute to the maternal-fetal calcium transport. most probably trpv is part of a multiprotein channel complex but most of the components of this complex are unknown so far. our aim is to find interaction partners of the trpv protein in the placenta that might contribute to the regulation of the trpv protein function. therefore we expressed the intracellularly located n-and c-terminal parts of trpv (aa - and - ) as trpv -gst (glutathione-s-transferase)-fusion proteins and used the purified recombinant proteins for pulling down proteins from human placenta cell extracts. the proteins pulled down by this approach were analysed by mass spectrometry. we identified several potential trpv interacting proteins which were not associated with the gst protein only. one of the proteins which was highly enriched with the n-terminal part of the trpv protein is calpain . in contrast to the classical calpains (calcium activated cystein proteases), calpain is unique in that it lacks the cysteine residue in the active site. calpain is mainly expressed during embryogenesis and is reported to be involved in cytoskeleton stabilisation but with unknown function in placenta. the interaction between trpv and calpain was confirmed in reciprocal pulldown experiments and the trpv binding region for calpain was narrowed down by using overlapping n-terminal trpv -gst fusion proteins. after injection of trpv crna into xenopus laevis oocytes calcium uptake into the oocytes was measured; this uptake was largely reduced after co-injection of the calpain crna. in further experiments we want to study potential regulatory effects of the trpv protein on the calpain function in cell culture models. comparative studies on the effects of the human carcinogen inorganic arsenite and its recently identified thiolated metabolite thio-dma v on human urothelial cells ebert f., leffers l., unterberg m., schwerdtle t. universität münster institut für lebensmittelchemie, corrensstr. , münster, germany it has been demonstrated that chronic ingestion of - µg/day inorganic arsenic is associated with an increased risk for cancers of the skin, the lung and the bladder, but until now the underlying toxic modes of action are still under debate. in this context, in the last five years one main focus has been given to the role of human inorganic arsenic metabolism and nowadays it is generally accepted that human biomethylation contributes to inorganic arsenic induced carcinogenicity. due to further improvements in arsenic speciation techniques recently a new thiolated arsenite metabolite, the thio analogue of the well known metabolite dimethylarsinic acid (dma v ), the so called thiodimethylarsinic acid (thio-dma v ), has been discovered in human biological samples. after synthesizing and analytically characterizing this metabolite (bartel et al. , j toxicol. we investigated its toxic effects in direct comparison with ias iii in human urothelial cells. thereby cell cycle studies revealed a g /m-and s-phase arrest as well as subg peak formation in case of thio-dma v . moreover, thio-dma v induced apoptosis (subg , caspase activity) at lower concentrations and earlier time points as compared to ias iii . most likely this is partly due to the higher cellular bioavailability of thio-dma v (aas/icp-ms). regarding genotoxicity, a generation of dna single strand breaks (alkaline unwinding technique) as well as an increased formation of reactive oxygen species (ros, dcfh-da-fluorescence) occurred only at high cytotoxic concentrations. however, thio-dma v strongly increased h o induced ros formation at very low nanomolar concentrations, which might result in cogenotoxic effects. since our earlier studies have shown a strong inhibition of h o induced poly(adp-ribosyl)ation especially by trivalent methylated arsenic metabolites, actual studies investigate the impact of thio-dma v on cellular poly(adp-ribosyl)ation, parp- gene expression, protein level and cellular cleavage, which might hopefully give further hints regarding the mode of action behind thio-dma v induced apoptosis. mitochondrial dysfunction in models of alzheimer´s disease eckert a. universitäre psychiatrische kliniken basel neurobiology laboratory, wilhelm klein strasse , basel, switzerland the histopathological characteristics of alzheimer's disease (ad) are amyloid-ß (aß) containing plaques and neurofibrillary tangles (nfts) as well as neuronal and synaptic loss. until today, the underlying mechanisms of the interplay of plaques and tangles remained unresolved. there is increasing evidence that mitochondrial dysfunction might be a possible link, as revealed by studies in several app and tau transgenic mouse models. recently, we examined mitochondrial function in a novel triple transgenic mouse model (pr /app/ps ) -triplead mice -that combines both pathologic features of the disease in brain. using comparative, quantitative proteomics (itraq) and mass spectroscopy we found a massive deregulation of proteins, of which one-third were mitochondrial proteins mainly related to complexes i and iv of the oxidative phosphorylation system (oxphos). remarkably, deregulation of complex i was related to tau, whereas deregulation of complex iv was aß dependent, both at the protein and activity levels. the triplead mice showed synergistic effects of aß and tau already at the age of months, resulting in a depolarized mitochondrial membrane potential. at months, the strongest defects on oxphos, synthesis of atp and reactive oxygen species were exhibited in the triplead mice, again emphasizing synergistic, ageassociated effects of aß and tau in impairing mitochondria. evidences from ad post-mortem brain as well as cellular and animal ad models indicate that aß and tau protein trigger mitochondrial dysfunction through a number of pathways, such as impairment of oxidative phosphorylation, elevation of reactive oxygen species production, alteration of mitochondrial dynamics, and interaction with mitochondrial proteins. moreover, recent reports indicate that aß may also interact directly with intracellular proteins such as the mitochondrial enzyme abad (aß binding alcohol dehydrogenase) in executing its toxic effects. mitochondrial dysfunction occurs early in ad, and aß's toxicity seems to be in part mediated by inhibition of abad. in total, a vicious cycle as well as several vicious circles within the cycle, each accelerating the other, can be drawn emphasizing the synergistic deterioration of mitochondria by tau and aß. olesoxime is a novel mitochondrial-targeted compound that is orally active and crosses the blood brain barrier. the cholesterol-oxime targets proteins of the outer mitochondrial membrane and represents a promising drug candidate for neurodegenerative diseases . we evaluated olexoxime's neuroprotective effects against mitochondrial dysfunction in an animal model for alzheimer`s disease (ad). dissociated brain cells (dbc) and mitochondria were isolated from brains of c /bj -thy -appsl (ad-mice) mice that were fed with mg olesoxime/kg feed for months. drug plasma levels reached approx. ng/ml. respiration of isolated mitochondria were significantly diminished in ad-mice due to reduced complex i, i+ii and cox activities. consequently, mitochondrial membrane potential (mmp) was significantly reduced in dbc from ad-mice. olesoxime normalized respiration chain complex activities and the mpp. to further evaluate the beneficial effects of olesoxime on complex i activity, we challenged dbc with rotenone ex vivo and observed that olesoxime treatment was protective. to further clarify the mode of action, we analyzed the ability of olesoxime to prevent opening of the mitochondrial permeability transition pore (mptp) in vitro using energized brain mitochondria by measuring ca + -and atractyloside (atr) induced swelling. the opening of mptp precedes apoptosis and can be induced by mitochondrial dysfunction due to calcium overload, oxidative stress, elevated phosphate concentrations or adenine nucleotide depletion. olesoxime prevented ca + -as well as atr induced mitochondrial swelling. atr inhibits the adenine nucleotide translocase (ant) that requires appropriate membrane properties to mediate mitochondrial permeability transition (mpt). since cholesterol (cho) and its derivates represent potent modulators of membrane viscosity, we related the effects of cho and olesoxime on mptp opening to membrane properties. both, cho and olesoxime reduced the flexibility of membrane acyl-chains in energized mitochondria and prevented atr induced mptp opening. however, cho didn`t prevent ca + -induced mptp opening, indicating a different mode of action for olesoxime. our data confirm olesoxime as drug candidate against mitochondrial dysfunction, which is considered to play a pivotal role in neurodegenerative diseases. the work was supported by the european union under the th framework program for rtd -project mitotarget -grant agreement health-f - - . several inflammatory glomerular kidney diseases are accompanied with a massive production of reactive oxygen species (ros) that may attack the glomerular filtration barrier by affecting podocyte function and may contribute to apoptotic or necrotic cell death of mesangial cells. otherwise, ros also trigger fine-tuned signaling processes that may result in cell proliferation or cell migration. to define such redox-driven signaling devices, we performed a non hypothesis-driven proteomic approach, to identify homo-or heteromeric protein complexes induced by ros. to this end, protein lysates of human podocytes were treated with or without hydrogen peroxide ( µm) for min. thereafter, the cell lysates were subjected to diagonal d gel electrophoresis and putative redox-affected proteins were analyzed by ms/ms-analysis. by this approach, we could identify a series of proteins that form interprotein-disulfide bonds in a redoxdependent manner. one of those proteins could be characterized as the regulatory subunit of protein kinase a (r-subunit of pka), which belongs to the family of serine/threonine kinases. to evaluate whether ros is capable to activate pka also in a more physiological setting, we treated rat mesangial cells with pdgf-bb to induce ros formation and we could demonstrate that pdgf-bb induces dimerization of r-subunits in a redox-dependent manner. to demonstrate whether pdgf-bb induces pkadependent pathways, we analyzed the effects of pdgf-bb on phosphorylation of serine of vasodilater stimulated protein (vasp) a classical target of pka. in fact, pdgf-bb induced vasp phosphorylation independently of intracellular camp levels. moreover, elevating camp levels via activation of adenylate cyclase with forskolin did not change the dimerization state of r-subunits. pdgf-bb-induced dimerization of the r-subunits and subsequent phosphorylation of vasp was blocked by diphenyljodonium (dpi), indicating activation of a nadph oxidase is essential for pka activity. taken together, we demonstrate a redox-dependent activation of pka by pdgf-bb and this may hint also for a probably protective role of ros in rat mesangial cells. testing the potential sensitizing capacity of chemicals is currently done by using the murine local lymph node assay (llna). animal welfare and eu cosmetics directive demands alternative methods to animal tests. the purpose of this study was to establish an in vitro assay for the prediction of skin sensitizers. based on the finding that the majority of skin sensitizers are electrophilic or have the potential to be metabolized to electrophilic substances, it is assumed that they can activate the nrf -keap -antioxidant response element (are) regulatory pathway. here, we report the results obtained from the lusens assay that detects electrophilic chemicals using the nrf pathway. the cell line lusens was derived from immortalized keratinocyte hacat cells and carries a luciferase reporter gene under the control of an are-element from the rat nadph quinone reductase nq . the lusens assay was in house validated with a panel of chemicals and cosmetic ingredients including the performance standard substances of the local lymph node assay. the predictivity of this assay was compared to the predictivity of the murine llna and to human patch test data and can be considered as reliable screening approach (accuracy of % compared to human data). however, in order to cope with the complex multi-step mechanism of skin sensitization, an integrated approach of in vitro assays mimicking several steps was designed; thereof, the are-dependent gene activation represents one module. time-resolved fluorescence ligand-binding assays for parathyroid hormone receptors emami-nemini a. ligand-binding studies represent essential tools for pharmacological research on g protein-coupled receptors. in recent years, time-resolved fluorescence gained significant relevance as readout for ligand binding studies. however, ligand-binding assays for parathyroid hormone receptors (pthrs) utilizing fluorescent parathyroid hormone (pth) were missing. therefore, we generated various fluorescent pth analogues which exhibit properties of native pth in terms of affinity, potency and internalization. for the purposes of academic and commercial research, we utilized labeled pth to set up three time-resolved fluorescence assay formats: (i) classical separation binding assay, based on time-resolved fluorescence and suitable for native receptors; (ii) homogeneous timeresolved fluorescence resonance energy transfer (htrf) based on tag-lite technology for high through-put screening; (iii) htrf based on antibodies, a synergistic approach using htrf with minimized receptor modification. this work will facilitate the development of new drugs directed to the pthr as well as fundamental research on the pthr. anandamide production in eosinophilic granulocytes is independent of il- and eotaxin stimulation engeli s., reinke j., zörner a., tsikas d., jordan j. medizinische hochschule hannover institut für klinische pharmakologie, carl-neuberg-straße , hannover, germany introduction: some animal and in vitro studies suggest that endocannabinoids exert anti-inflammatory effects. specifically, inhaled anandamide reduced the obstructive effect of leukotrien d in airways, and a specific cannabinoid receptor agonist significantly reduced pulmonary inflammation in guinea pigs. we have recently shown that segmental bronchial allergen challenge is associated with significant increases of anandamide concentrations in bronchoalveolar fluid of patients with asthma. the concomitant increase in eosinophilic counts, eotaxin and il- concentrations in bronchoalveolar fluid led us to hypothesize that anandamide is produced by eosinophilic granulocytes in response to chemotactic stimuli. peripheral eosinophilic granulocytes were isolated from whole blood by means of percoll gradient centrifugation and magnetic separation employing cd antibodies conjugated to magnetic beads. isolated cells were counted and anandamide measurements were typically performed in whole cell lysates of . x eosinophils. we stimulated eosinophils with varying concentrations of il- , eotaxin- (ccl ), eotaxin- (ccl ), and eotaxin- (ccl ). to prevent anandamide degradation, a specific fatty acid amide hydrolase (faah) inhibitor (oloxa) was employed. anandamide concentrations and faah activity were determined by stable isotope dilution using lc-ms/ms protocols. results: first, we confirmed the ability of eosinophilc granulocytes to synthesize anandamide. however, cellular anandamide content could only be measured when faah was effectively blocked with oloxa, and strong faah activity was demonstrated in eosinophils. with oloxa, typical anandamide concentrations were in the range of - pm/ . x eosinophils. neither il ( - pg/ml), nor any of the eotaxins ( ng/ml either alone or in varying combinations) did stimulate anandamide production after min of incubation. our results suggest that chemotactic molecules like eotaxin and il- are not responsible for increased anandamide formation in eosinophils during allergen challenge. in a next step, the effects of anandamide on eosinophils and bronchial epithelial cells need to be determined. the suprisingly high faah activity in eosinophils may point to an alternative pathway facilitating prostaglandin and leukotriene synthesis by production of arachidonic acid. screening methodology for estimatation of dermal absorption in vitro fabian e., goth c., guth k., mehling a., van ravenzwaay b., landsiedel r. basf se experimentelle toxikologie, carl-bosch-strasse , ludwigshafen, germany dermal absorption is used in the evaluation of the effectiveness of pharmaceutical or cosmetic formulations, but often dermal absorption is a critical parameter in risk assessment of pesticides or chemicals. therefore, knowledge of dermal absorption is e.g. helpful in formulation development. skin absorption is routinely measured in vivo or in vitro following oecd tg or . however, these tests are complex, time consuming and expensive. therefore, a method was developed to allow simple and rapid screening. the experiment uses dermatomized skin in modified franz type diffusion cells. µl of test substance preparation are applied to the skin preparation. after h, the skin is washed and the amount of penetrated substance is quantified. the receptor fluid and the washing solutions are optimized for subsequent analyses by lc-ms. we performed dermal absorption screenings in parallel to our routine guideline studies and demonstrated a good correlation of the results of both study types: the total recovery found in the screening studies is somewhat lower than in the corresponding guideline studies but is always in an acceptable range above %. the efficacy of the skin washing procedure is lower than under routine conditions, most probably due to the change to an lc-ms-compatible washing solution. overall, the dermal absorption screening is an easy, fast and cost-effective screening method for the estimation of dermal absorption of a wide variety of test substances and formulations. the p tumor suppressor protein is frequently inactivated in human cancers by diverse mechanisms. owing to its fundamental role in the maintenance of genomic stability and cancer prevention, p is an attractive target in cancer therapy and several approaches were pursued to restore p function in tumor cells. polyamidoamine (pamam) dendrons are positively charged molecules with a systematically branched structure that interact with the negatively charged cell membrane, inducing cellular uptake via endocytosis. in the present study, biotin-labeled p protein was attached to a dendronized streptavidin nanocarrier to facilitate its internalization into different tumor cell lines. first, biotin-substituted pamam dendrons were conjugated with streptavidin to allow formation of the dendronized streptavidin nanocarrier. the nanocarrier displayed uptake into hela cervix carcinoma and a lung adenocarcinoma cells without detectable cytotoxicity. biotin-labeled p was then conjugated to the dendronized streptavidin, preserving its specific dna-binding in vitro. immunoblot analysis revealed efficient internalization of biotin-p into hela cells in the presence of dendronized streptavidin. in line with this finding, specific cellular uptake of biotin-p was observed by confocal microscopy, which showed a cytoplasmic and peri-nuclear localization in hela, a and saos osteosarcoma cells. the internalized biotin-p also partially co-localized with early endosomes. importantly, the delivery of biotin-p into p -deficient saos cells resulted in impaired cell viability and upregulation of caspase / activity, demonstrating its biological functionality. this study intriguingly demonstrate the efficient delivery of functional biotin-p into different tumor cell lines using the novel streptavidin nanocarrier, which can be further modified to allow cell-type specific targeting and combined with cytotoxic drugs such as doxorubicin. identification of novel ahr target genes in rat liver oval cells faust d. , vondracek j. the aryl hydrocarbon receptor (ahr) is a transcription factor involved in physiological processes, but also mediates most, if not all, toxic responses to , , , tetrachlorodibenzo-p-dioxin (tcdd), some polycyclic aromatic hydrocarbons (pahs) and dioxin-like polychlorinated biphenyls (pcbs), such as pcb . activation of the ahr by these ligands leads to its dimerization with arnt and transcriptional activation of several phase i and ii metabolising enzymes. while it is generally accepted that many pahs are thereby transformed to genotoxic metabolites, this classical signalling pathway so far failed to explain the tumour promoting effects of the nongenotoxic compounds tcdd and pcb . thus, there is an urgent need to define genetic programmes orchestrated by ahr to unravel its role in physiology and toxicology. we have recently shown that treatment of rat liver oval cells with tcdd or pcb leads to a release from contact-inhibition involving activation of the ahr, elevation of jund protein levels and transcriptional activation of cyclin a ( , ) . loss of contact-inhibition is one hallmark of tumour promotion. to better understand ahr-driven pathways we identified the transcriptional programme using high density microarrays in response to pcb . already h after treatment, genes were found to be upregulated and genes downregulated indicating that these are direct ahr-dependent target genes. david analysis revealed that these genes are involved, for instance, in drug and lipid metabolism, cancer pathways, tgf-b signalling and cell-cell communication. ten of the genes were selected for confirmation by semi-quantitative rt-pcr. using the ahr inhibitor ch- and sirna directed against ahr and arnt, we further demonstrated that ahr-and arnt-function is required for transcriptional activation of the selected genes. finally, we identified the transcription factor foxq as a novel ahr target protein in rat liver oval cells. although the function of foxq is poorly understood, it has been shown very recently that foxq is overexpressed in colorectal cancer and is involved in epithelial-mesenchymal transition in breast cancer cells. its function in ahrmediated tumour promotion, however, remains to be determined. the practical relevance of histamine h and h receptors in the brain can be easily deduced since h receptor antagonists of the first generation have a sedative effect and an inverse h agonist, pitolisant (close to its introduction to the market), is active against excessive daytime sleepiness associated with narcolepsy. in this context the question arises whether also h and h receptors possess a practical relevance in the brain. to this end, we examined whether the electrically evoked h-noradrenaline release in superfused human cerebral cortex slices is affected by agonists at the above receptors. the h agonist impromidine µm failed to affect noradrenaline release in human cortex slices although it facilitated noradrenaline release in guinea-pig cortex slices; the maximum extent of facilitation was %, the pec was . and the pa of the h antagonist ranitidine against impromidine was . . with respect to h receptors there is some controversy in the literature whether they occur in the brain at all. however, we were able to detect h receptor mrna in the human and mouse cortex by the reverse transcriptase polymerase chain reaction. in cortex slices of either species, noradrenaline release was not affected by the h agonist -methylhistamine - µm but inhibited by histamine µm via h receptors by and %, respectively. in mouse cortex membranes, -methylhistamine µm also failed to affect s-gtpγs binding although r-α-methylhistamine µm, acting via h receptors, increased it by %. in conclusion, h receptors facilitating noradrenaline release are detectable in the isolated guinea-pig but not human cortex. despite the presence of h mrna in the brain, functional readouts of this receptor, i.e. modulation of noradrenaline release (humans, mice) and modulation of s-gtpγs binding (mice), could not be shown. murine cx promoter activity is dependent on the transcription factor creb fels b., nunes f., schmitz w., müller f. u. westfälische wilhelms-universität münster institut für pharmakologie und toxikologie, domagkstraße , münster, germany connexin (cx ) is a gap junction protein expressed in atrial myocytes and the ventricular conduction system, mediating the electrical intercellular communication in the myocardium. alterations in cx function were linked to the pathophysiology of atrial fibrillation and heart failure. in the heart, camp dependent gene transcription is regulated by members of the creb/crem/atf family which bind to camp responsive elements (cres). similar to the human cx gene promoter, the murine promoter contains one cre. cardiomyocyte-specific overexpression of a crem-isoform (crem-ib∆c-x) led to cx down-regulation, suggesting that creb related transcription factors are involved in cx gene regulation. in order to study the functional role of creb in the regulation of the cx promoter we have generated a luciferase based murine cx promoter reporter gene construct and monitored its activity in a permanent cell line upon overexpression of constitutive-active creb (cacreb) and a non-phosphorylatable dominant-negative creb (dncreb) isoform. surprisingly, overexpression of cacreb and dncreb both lead to a reduction of cx promoter activity (cacreb % ± % vs control % ± %; p< . vs control , n= ), dncreb % ± % vs control % ± %; p< . vs control, n= ). the activity of the murine connexin promoter is modulated by creb. both cacreb and dncreb led to cx down-regulation, which could be explained by induction of inhibitory transcription factors creb/crem/atf transcription factor family, which in turn could suppress cx promoter activity. (supported by the dfg) results: fxa increased par- mrna, protein and cell-surface expression and augmented par- -mediated mitogenesis. par- expression was not influenced. the regulatory action of fxa on par- was concentration-dependent and mimicked by a par- selective activating peptide. the thrombin inhibitor argatroban or par- gene silencing did not influence fxa-stimulated par- expression. fxa increased oxidative stress and expression of the nadph oxidase subunit nox- in smc. nox- gene silencing prevented fxa-stimulated par- regulation, as did ebselen and catalase. exogenous hydrogen peroxide increased par- expression and mitogenic activity. fxa induced nuclear translocation and par- dna binding of nuclear factor kb (nfkb). inhibition of nfkb prevented fxa-stimulated par- expression. in separate studies, fxa promoted par- mrna stabilisation through increased human antigen r (hur)/par- mrna binding and cytoplasmic shuttling. hur gene silencing abolished fxa-stimulated par- expression. conclusion: expression and mitogenic activity of vascular par- , but not par- , is upregulated by fxa. this action involves transcriptional and post-transcriptional mechanisms mediated through nox- -containing nadph oxidase and its downstream effectors hydrogen peroxide, nfkb and the mrna stabilising protein hur. continued generation of fxa by the mural thrombus, and the autoregulatory feedback control of par- may maintain the inflammatory and proliferative state of the injured vessel, thereby promoting vascular remodeling. the mrna stabilising factor hur is a critical regulator of human proteaseactivated receptor aim: we recently reported that functional expression of par- thrombin receptors is induced in human saphenous vein smc exposed to high glucose. this effect could be attributed in part to transcriptional mechanisms mediated through nfkb but the contribution of post-transcriptional effects such as mrna stabilisation is not known. this study explored the potential role of the mrna stabilising factor human antigen r (hur) in the regulation of par- . methods: human saphenous vein smc were serum-deprived prior to study. gene expression was determined by realtime pcr, protein expression by western blotting. gene silencing utilized commercially available sirna. hur binding to par- mrna was determined by immunoprecipitation ("pull-down") pcr. results: high glucose ( mm vs . mm) slowed par- mrna degradation in the presence of actinomycin d. par- mrna decay was not affected. hur binding to par- mrna and nucleo-cytosolic shuttling was enhanced by high glucose, total hur protein expression was not affected. hur sirna abolished the high glucose-stimulated induction of par- mrna. hydrogen peroxide (h o ) also induced cytosolic hur shuttling and increased par- mrna and total protein expression. the role of endogenously generated h o in the regulatory effect of high glucose on par- expression was investigated with the nadph oxidase inhibitors apocynin/diphenyliodonium (to prevent h o generation) and cell-permeant catalase (to degrade cellular h o ). both approaches prevented the stimulatory effect of high glucose on par- expression. cyclic amp has been reported to suppress hur activation and in the present study, the cyclic amp stimuli forskolin and cicaprost (prostacyclin analog) suppressed basal hur shuttling and par- transcript stability. cicaprost also attenuated high glucose-induced hur binding to par- mrna and as a consequence normalised par- expression and inflammatory signalling in high glucosetreated cell. conclusion: the regulation of par- thrombin receptors in human vascular smc is critically dependent on the mrna stabilising actions of hur. through activation of hur, high glucose and other hur stimuli such as ang ii and exogenous h o , increase par- expression, while cyclic amp agonists such as prostacyclin oppose this effect. such interactions could potentially represent a fine-tuning mechanism to control par- expression and ultimately also the mitogenic and inflammatory actions of thrombin in the vessel wall. nucleoside diphosphate kinase b (ndpk b) is a member of a family of ubiquitously expressed enzymes required for nucleoside triphosphate synthesis. thus, they are involved in the regulation of a variety of cellular processes, e. g. g protein mediated signal transduction. however, whether ndpk b has a specific role in the regulation of angiogenic processes in endothelial cells is unknown. therefore, we studied the function of ndpk b in the vasculature in a developmental, an ischemia-induced and an in vitro model of angiogenesis. firstly, depletion of ndpk b expression was achieved by morpholino-mediated knockdown in zebrafish embryos in which the developing vasculature can be visualized by egfp expression in the endothelium. h post fertilization, ndpk b knockdown larvae showed a dramatic inhibition of intersegmental and dorsal longitudinal anastomatic vessel formation compared to control injected fish. this phenotype could be rescued by early re-expression of ndpk b. secondly, ischemia driven angiogenesis was studied in ndpk b-depleted mice and wildtype littermates after excision of the left femoral artery. hind limb blood flow was assessed by laser doppler perfusion imaging immediately before and after ligation (day ) and on postoperative days , , , , , and . a significant reduction of recovery was observed in the ndpk b depleted mice at days and . thirdly, in vitro-sprouting angiogenesis was analyzed in human umbilical vein endothelial cell (huvec) spheroids with and without sirna-mediated ndpk b knockdown. vascular endothelial growth factor (vegf)induced sprouting was significantly attenuated by ndpk b knock down by more than % in comparison with control transfected huvec. we conclude from these results that ndpk b is an essential regulator of angiogenesis. the loss of ndpk b may specifically interfere with the vegf-induced migration and proliferation during endothelial sprouting. ethylene oxide in blood of ethylene-exposed volunteers ethylene (et) is a commercially important high volume industrial chemical. inhaled and endogenous et is metabolized in mammals to ethylene oxide (eo), which is carcinogenic in rats and mice. until now, no data on the oxidation of et in et-exposed humans has been published. in the present study, we investigated the formation of eo in four male adult volunteers exposed for hours to constant atmospheric et concentrations of , , or ppm by means of a breathing mask. during exposure, et concentrations were measured in inhaled and exhaled air by gc/fid and eo concentrations in venous blood by gc/ms. rates of et metabolism were obtained from the product of the differences in the et concentrations with the pulmonary ventilation. in each subject, linear correlations were found between the et exposure concentration and the rate of et metabolism or the eo concentration in blood. mean rate of et metabolism was . ± . nmol/h/ppm/kg body weight. steady-state concentrations of eo in blood differed by a factor of between the volunteers. these inter-individual differences likely reflect the polymorphism of glutathione s-transferase theta, the main eo metabolizing enzyme in human liver. mean eo concentration in blood at steady state was . ± . nmol/l blood per ppm of et. the data will be used for validating a physiological toxicokinetic model which will describe the et related eo tissue burdens in rodents and humans. the model predictions will support risk evaluations of et. financially supported by the lower olefins sector group of cefic. in vitro effect of stw on human dendritic cells fink c. , bonaterra g. a. extracts of echinacea (purple coneflower) are used in the prevention and therapy of infectious diseases. the medicinal product stw contains the extract from purple coneflower, and in addition, extracts of monkshood, venom of honey bee and bushmaster snake in homeopathic dilutions. previous studies showed a stimulation of the cellular and humoral immune response. dendritic cells (dcs) are antigen presenting cells that act at the interface of the innate and adaptive branches of the immune system. during stages of dc differentiation, the ability to internalize antigens varies and decreases during maturation. in this study, we determined the influence of stw on the expression of maturation related genes (cd a, cd ), cytokines (tnfα, il- , il- ), chemokines (ccr ), major histocompatibility complex ii(mhc-ii) and toll-like receptors (tlr , tlr ). in mature (mdc) and immature dc (idc) using real-time rt-pcr. peripheral blood mononuclear cells (pbmcs) were isolated from buffy coats of human volunteers by densitygradient (ficoll ® ) and seeded in well plates. non-adherent cells were eliminated. to induce idc development, ng/ml il- and ng/ml granulocyte macrophagecolony stimulating factor (gm-csf) were added. at day , maturation was induced by addition of lipopolysaccharide (lps) at a final concentration of µg/ml and cultured for additional days. after incubation with different concentrations ( . in idc, compared to control, we found a significantly increased expression of cd ( . - . fold) and tnfα ( . - . -fold) genes after treatment with . - % stw , respectively, but no effect was found on the expression of cd a, il- , il , adam , cd c, cd ,tlr , tlr , mhc-ii and ccr . in summary, these data demonstrate a stimulatory effect of stw in idc and especially in mdc, concerning an increase of various genes related to maturation (cd ), immunomodulation (tnfα, cd ), adhesion (ccr ) and antigen presentation (mhc-ii) and are in accordance with the therapeutic use in infectious diseases. waterproofing sprays are widely used consumer products containing for example fluorinated polymers or silicon based compounds dissolved in alcohols or volatile petroleum distillates. there have been repeated reports on cases of severe acute respiratory disorders especially when using products that newly entered the market. it is hypothesized that impairment of the pulmonary surfactant by deposition of inhaled respirable particles of the active compound is one of the main causes of the acute lung injury. since the inhalation toxicity cannot be predicted a priori based on the physical and chemical properties of the formulation, proper test strategies are required to ensure consumer safety. we propose to combine screening tests addressing both, exposure and acute lung toxicity. the exposure potential of the spray product is characterized by determining the release fraction of the active compound in the respirable particle size range under conditions relevant for the product application. this is carried out by spraying defined quantities of the product into a control volume and measuring the concentration of health related size fractions. this procedure takes into account spray ageing, especially size reduction of the droplets due to solvent evaporation. the isolated perfused lung is used as a model for testing acute toxicity. ventilated rat lungs are exposed to aged aerosols with proper particle size of approximately µm mmad generated from the liquid spray formulation. lung compliance and lung resistance are continuously monitored during exposure. dose dependent deviations from the normal values (without exposure) are used as read-out parameters. using the combined procedure, different sprays could be ranked according to their realistic exposure risk and, most importantly, sprays with known lung toxicity could be uniquely distinguished from those that have been shown to be safe. in its current stage of development the simple test method is recommended for screening of substances only. induction of oxidative damage in calf thymus dna by the fusarium mycotoxin zearalenone after metabolic activation with liver microsomes fleck s. c., pfeiffer e., metzler m. kit -institute of applied biosciences chair of food chemistry, adenauerring a, karlsruhe, germany zearalenone (zen) is an estrogenic mycotoxin produced by fusarium species. the adverse effects of zen and its reductive metabolite zearalenol (zel) are often compared to those of -beta-estradiol (e ) and estrone (e ). these endogenous estrogens are associated with an increased risk for cancer, which may be mediated by two mechanisms, i.e. (i) hormonal activity and (ii) genotoxic effects by p -catalyzed metabolic activation to catechols (wang et al., chem res toxicol , . like e and e , zen and zel exhibit marked estrogenicity and also undergo aromatic hydroxylation to catechol metabolites (pfeiffer et al., mol nutr food res , . the aim of the present study was to examine the formation of catechol metabolites of zen by liver microsomes of various species and their potential for redox cycling. catechol metabolites are frequently associated with the generation of reactive oxygen species and subsequent oxidative damage of dna, for which -oxo- , -dihydro- 'deoxyguanosine ( -oxo-dg) is a common biomarker. the propensity of the catechol metabolites of zen and zel to cause the formation of -oxo-dg in isolated calf thymus dna was determined using a lc-esi-ms/ms method. to this end, zen was incubated with microsomes from human, rat, mouse, bovine and porcine liver as well as with human cyp a , and the incubations were extracted with ethyl acetate. the extract was analyzed with lc-ms and then added to a solution of calf thymus dna in the presence of copper(ii) ions and nadph. the formation of -oxo-dg could be demonstrated with each extract. the levels of -oxo-dg correlated directly with the extent of catechol formation, which increased from steer to swine to human to mouse to rat microsomes. -hydroxylated zen/zel, which is the major catechol, was more reactive than -hydroxylated zen/zel to form -oxo-dg. in conclusion, our study has shown that the catechol metabolites of zen are highly reactive and give rise to oxidative dna damage in vitro. in addition, recent research from our laboratory revealed that the catechols of zen are less efficiently inactivated by catechol-o-methyl transferase than the catechols of e and e . the genotoxic potential of zen may constitute another biological activity in addition to the well-known estrogenicity. supported by deutsche forschungsgemeinschaft (grant me / - ) and "food and health" of kit. thrombin regulates expression of sphingosine kinase- (sphk- ) in human vascular smooth muscle cells -inhibition by dabigatran reduces vascular sphk- expression and atherosclerotic burden in vivo flößer a. results: thrombin induced a time-and concentration-dependent ( - nmol/l) increase in sphk- mrna and protein expression in human saphenous vein smc, n= - . this was mimicked by a synthetic par- ligand. inhibition of sphk- attenuated thrombin-induced smc proliferation but not smc migration (n= ). the regulatory action of thrombin on sphk- expression was suppressed by sirna against the mrna stabilisiserhur. in thrombin-stimulated smc, hur binding to sphk- mrna and subsequent nucleo-cytosolic shuttling was enhanced. accordingly, thrombin induced sphk- mrna stabilisation in smc in the presence of actinomycin d. in apoe-deficient mice, long-term treatment with the direct thrombin inhibitor dabigatran significantly reduced aortic sphk- expression by % (n= ) and plaque size by % compared to control animals (n= ). conclusions: thrombin induces sphk- expression and s p synthesis in vascular smc via the mrna stabilising protein hur. this leads to increased smc proliferation. inhibition of thrombin by dabigatran treatment in vivo attenuates progression of plaques possibly by reducing sphk- expression. mycotoxin contamination and cytotoxicity of grain mill products typical grain mill products from north-rhine westphalia, i.e. grains, flour, wholemeal flour and bran (from wheat, rye and spelt) as well as typical by-products (outsourced fractions) from the milling process were analysed for their mycotoxin content by lc-ms/ms. the cytotoxicity of sample extracts with known mycotoxin composition was then assessed in v cell cultures by means of the neutral red uptake assay, in parallel with pure reference mycotoxin mixtures. extracts from flour and wholemeal flour samples with low levels of deoxynivalenol and enniatin b (from not detectable to . µg/g don and . µg/g ennb) induced no measurable cytotoxicity. on the other hand, although mycotoxin contamination levels were also rather low in bran, these samples induced strong cytotoxicity: extracts of bran derived from rye and spelt were more cytotoxic than those of wheat bran. the cytotoxic effects of the bran samples cannot be related to their mycotoxin content as comparable concentrations of pure mycotoxins and mycotoxin combinations tested in parallel were not cytotoxic. by-products from certain stages of the milling process (sorting and waste fractions) were found to contain mycotoxins at rather high levels: enniatin b was detected in nearly all samples, and also t- toxin, ht- toxin, ergotamine, ergocornin and deoxynivalenol were present. waste sample extracts with notable mycotoxin levels (up to µg/g don, µg/g ennb, µg/g ergotamine, ng/g ht- toxin) exert pronounced cytotoxicity in v cells. the cytotoxicity of these samples was somewhat stronger than expected when compared with mixtures of reference mycotoxins tested in parallel. in summary, the tested flour and wholemeal flour extracts contained only low levels of mycotoxins and were not cytotoxic. in contrast, bran samples showed cytotoxicity which cannot be explained solely by their mycotoxin content. this unexpected observation in real samples and combination effects of mycotoxin mixtures require further studies. the ahr is a ligand-activated transcription factor that mediates the toxicity of dioxins and related compounds. upon ligand binding the ahr translocates into the nucleus and dimerizes with arnt to modulate gene expression, e.g. of cyp a . recently, we have shown that uvb irradiation of human keratinocytes results in activation of the ahr and associated egfr signaling leading to an enhanced expression of cyp a and proinflammatory cox- , respectively. the initial step is the uvb induced intracellular formation of the tryptophan photoproduct -formylindolo [ , b] carbazole (ficz), a high affinity ahr ligand. thus, the ficz activated ahr is an important mediator of the dna damage independent part of the uvb response. our current study aims to identify further aspects of ahr mediated uvb responses. therefore, we analysed changes in protein expression, proliferation and apoptosis in ahr+/+ and ahr-/-keratinocytes (nctc ) by western blot, flow cytometry and brdu incorporation. uvb exposure of nctc cells led to a dose-dependent increase in apoptosis. compared to ahr+/+ cells, ahr-/-cultures exhibited an increased amount of apoptotic cells. this finding was confirmed in irradiated ahr+/+ cells, pretreated with the ahr antagonist 'methoxy- '-nitroflavone. moreover, the proliferation of sham as well as uvb irradiated ahr-/-cells was significantly decreased. in ahr-/-cells we found a reduced expression of checkpoint kinase (chk ), an important cell cycle regulator that arrests the cell in g /m upon dna damage. interestingly, uvb exposure led to a higher net phosphorylation of chk in ahr-/-cells, indicating that this pathway is responsible for the observed ahr-dependent differences in proliferation and apoptosis. further expression analyses of chk client proteins emphasize our hypothesis. in conclusion our study identifies the ahr as an anti-apoptotic player in uvb irradiated human nctc cells. therefore, we propose that the ahr is a suitable target to prevent uvb induced skin diseases. synthetic progestins exert divergent effects on thrombosis in a murine model of atherothrombosis background: medroxyprogesterone acetate (mpa), a synthetic progestin often used in postmenopausal hormone replacement therapy, has previously been described to be pro-thrombotic in a murine model of accelerated atherosclerosis. however, nothing is so far known about effects of progestins with receptor profiles different from mpa (i.e. agonism or antagonism of mineralocorticoid-or androgen-receptors), such as drospirenone, levonorgestrel and norethisterone acetate. methods: apo -/mice were bilaterally ovariectomized (ovx) and substituted subcutaneously with mpa, drospirenone, levonorgestrel and norethisterone acetate as well as the respective placebo pellets for days on a western-type diet. subsequently, thrombosis was induced by photochemical injury to the right carotid artery using rose bengal and a green light laser. results: compared to placebo, animals substituted with mpa showed significantly shortened times to occlusion of the right carotid artery (placebo mpa: . ± . min. vs. mpa: . ± . min., n = - , p < . ). in contrast, drospirenone, levonorgestrel or norethisterone acetate did not alter thrombotic responses. however, at least drospirenone (placebo drospirenone: . ± . min. vs. drospirenone: . ± . min., n = - ) and levonorgestrel (placebo levonorgestrel: . ± . min. vs. levonorgestrel: . ± . min., n = ) showed a trend towards shorter times to stable occlusion. furthermore, analysis of aortic gene expression revealed that in aortas of mpa-treated mice expression of matrix-metalloproteinase (mmp- ) was induced as compared to placebo-treated mice. conclusion: mpa, a progestin with glucocorticoid effects, exerts a pro-thrombotic effect that is either progesterone-or glucocorticoidreceptor-dependent while progestins with receptor profiles different from mpa do not show a significant pro-thrombotic effect. furthermore, the pro-thrombotic effect exerted by mpa may be associated with increased expression of mmp- , a metalloproteinase being known to destabilize atherosclerotic plaques and make them more prone to rupture. rapid screening for mitochondrial toxicity in vitro using an oxygen-sensitive phosphorescent probe freyberger a. bayer healthcare bph gdd ged toxikologie -p & cp, aprather weg , wuppertal, germany impaired mitochondrial function has been implicated with disease, aging, and druginduced toxicities. analyzing mitochondrial respiration (mr) rates is one of the most informative ways to assess mitochondrial function as it provides information on the the bioenergetic capacity of a tissue, however, previously measurements using polarography (clark electrode) were cumbersome with only low throughput. in this work we explored luxcel's water-soluble phosphorescent oxygen-sensitive probe mitoxpress tm for the assessment of mitochondrial toxicity in freshly isolated male rat liver mitochondria (rlm) in a -well plate format using glutamate/malate ( mm/ . mm) and succinate ( mm) as respiratory substrates. inhibition of mitochondrial complexes i to iv, adenosine triphosphate synthetase and the adenosine diphosphate (adp) / adenosine triphosphate (atp) antiporter by rotenone, thenoyltrifluoracetone (ttfa), antimycin a, potassium cyanide, oligomycin, and atractyloside was readily detected in adp-stimulated rlm. use of the two different substrates in parallel allowed to discriminate complex i inhibition by rotenone from complex ii inhibition by ttfa, whereas downstream of these complexes inhibition by the other model inhibitors occurred independent of the substrate used. decoupling of mr from oxidative phosphorylation by carbonylcyanid-p-trifluormethoxyphenylhydrazone (fccp) was detected best in the absence of adp. compared to polarographic measurement, the use of an oxygen-sensitive probe is superior with regard to assay cycle time and sample throughput and offers new opportunities to characterize and screen for mitochondrial toxicity, but also to support studies on mitochondria-mediated modes of action of new chemical entities. the murine local lymph node assay (llna) and the guinea pig maximization test (gpmt) have been used to study the sensitization potential of a series of unsaturated compounds by kreiling et al. ( ) . we have examined the same substances in the loose-fit coculture-based sensitization assay (lcsa), developed by our working group (schreiner et al., ) . eight unsaturated compounds [oleic acid (oa), linoleic acid (la), linolenic acid (lna), undecylenic acid (ua), fumaric acid (fa), maleic acid (ma), squalene (sq), -octyn- -ol (oc)] and succinic acid (sua) were investigated using a coculture of keratinocytes and dendritic cell-related cells (dcrc). sensitization potential was quantified by flow cytometry measuring the increase of cd expressed on dcrc (ec = half maximal effective concentration). a pronounced induction of cd at low concentrations was seen with la, lna and oa (ec : , and µmol/l, respectively). ua exhibited an intermediate response (ec : µmol/l). with oc and ma, we observed effects at higher concentrations only (ec : and µmol/l). no significant increase of cd was observed with fa, sua and sq. because of poor solubility, sq could not be studied adequately. induction of cd was generally observed at concentrations which did not cause a major impairment of cell viability. our results show a high degree of concordance with those obtained by the gpmt, except for oa. in comparison with the results of the llna, those compounds which showed a strong effect in the llna (oa, la, lna) also induced an increase of cd at low concentrations, whereas those with low stimulation indices in the llna induced no significant increase of cd (fa, sua) or only at higher concentrations (ua). we observed a discrepancy between the tests with ma and oc, causing a strong stimulation of the murine lymph nodes, while the expression of cd was increased at high concentrations only. we assume that ma and oc might be false-positives in the llna, because they were also negative in the gpmt. background: the first step in elimination of many cationic drugs is their uptake from the blood into hepatocytes and renal proximal tubular cells by the organic cation transporter (oct ) and oct , respectively. the pivotal role of octs in the excretion of cationic drugs raises the possibility of drug-drug interactions in which one drug reduces octmediated elimination of a second drug. although many psychoactive drugs are cationic at ph . and some of these have already been recognized as oct inhibitors, a systematic screen of this class of compounds is missing. methods: we screened a drug library of most frequently prescribed psychoactive drugs (inpatient prescriptions in germany, at least million ddd each) for their inhibitory interaction with oct and oct . human embryonic kidney (hek) cells stably overexpressing oct or oct and the prototypical oct substrate -methyl- phenylpyridinium (mpp+) were used as a test system. cells transfected with the empty vector were used as controls. results: at µm, % and %, respectively, of the tested compounds significantly decreased oct -and oct -mediated uptake of mpp+. the most potent inhibitors (inhibition > %) of oct were chlorprothixen and clomipramine, whereas olanzapine, clomipramine and doxepin were the most potent inhibitors of oct . in contrast, neither at µm nor at µm carbamazepin, haloperidol, lithium, moclobemide and valproic acid did significantly inhibit mpp+ uptake into hek-oct or hek-oct cells. there was a significant correlation between the degree of oct and oct inhibition (p conclusions: our results demonstrate that inhibition of oct function by psychoactive drugs has to be considered as a potential mechanism underlying drug-drug interactions. considering estimated peak sinusoidal concentrations e.g., of chlorprothixen and clomipramine between and µm in humans, inhibitory interactions of these compounds with hepatic oct have to be taken in account. our data will help to create a chemoinformatic model to predict potential oct-dependent interactions of psychoactive drugs with the hepatic or renal elimination of coadministered drugs. this project is supported by the german federal ministry of education and research (bmbf), project grant no. ex b. cardiac gene expression is altered during the development of hypertrophy and heart failure compared to the healthy heart. the molecular mechanisms controlling gene expression in cardiac failure are only partially known. dna methylation is one epigenetic mechanism that regulates long-term changes in gene-expression. to elucidate whether dna methylation is altered during the development and progression of chronic heart failure, genome-wide dna methylation profiles were determined in myocardial biopsies from control patients and patients with cardiac hypertrophy or failure. cardiac biopsies were obtained from patients with aortic aneurysm who served as control and did not show clinical signs of chronic heart disease (ef: ± %, n= ) and from patients with aortic stenosis. the latter group was subdivided according to the ejection fraction into hypertrophic (ef: ± %, n= ) and failing patients (ef: ± %, n= ). after bisulfite conversion of extracted dna, the methylation status of genomic dna was quantified using the infinium® humanmethylation beadchip (illumina). this microarray allows analysis of more than , methylation-sites throughout the whole genome at single-base-pair resolution. these experiments identified cpg sites in hypertrophic samples and cpg sites in failing samples which were differentially methylated compared to control specimens (delta > %; p< . ). cpg sites were significantly altered in both aortic stenosis groups compared with control hearts. from these cpgs, sites were altered concordantly in hypertrophic and failing samples. analysis of regions harbouring distinct cpg densities revealed that most changes occured in shelf regions of cpg islands whereas the methylation status in the cpg islands was more stable. further analysis showed that differences in methylation were most frequent in gene body, enhancer and `utr regions. specifically probes spanning a cpg-island at the promotor region of the muscle-specific serine kinase (srpk ) showed diminished cpg-methylation in hypertrophic (- . ± . %) and failing (- ± . %) as compared to control biopsies. remarkably, no alterations of dna-methylation were observed in loci of classic marker genes of chronic heart failure like nppa, serca, ctgf, myh or myh . these results indicate that dna methylation is specifically altered in chronic heart disease but does not affect classic marker genes of chronic heart failure. gliomas are the most abundant type of primary brain tumor in the central nervous system in adults. the current standard of glioblastoma multiforme (gbm) therapy is surgery followed by radiotherapy and chemotherapy. however the morbidity and mortality of gbm remain very high and the median survival period is only months even with treatment. therefore it is important to identify novel drugs to reduce gbm cell proliferation. purine-analogues (pa) are well known for their anti-proliferative effects on eukaryotic cells. in this study novel pa were synthesized and the library of substance-derivatives was tested using different gbm cell lines namely ln , u -mg and gl . the effect on proliferation and viability was assessed by using brdu and resazurin assays. using these in vitro methods we were able to identify several compounds with cytotoxic and anti-proliferative effects in vitro showing ic values in the deeper µm range. cytotoxicity of selected compounds was further analyzed by assessment of caspase and propidium iodide based cell cycle facs analysis to discriminate between apoptosis and cell cycle arrest. based on these data purine-derivatives might inhibit proliferation and induce apoptosis in glioma cells. as a result we hypothesize that these compounds could be potentially interesting for the drug-development of gbm therapy and therefore a clue for chemical modifications. further studies are required to identify the exact underlying mechanism of action of the tested purine-analogues. the biological role of adenosine receptors in brown adipose tissue gnad t. brown adipose tissue (bat) is responsible for basal and inducible energy expenditure in mammals. bat contains large amounts of mitochondria and is highly vascularized. bat lipolysis and thermogenesis are stimulated by sympathetic neurons. importantly, recent findings indicate that adult humans possess metabolically active bat . here, we analyzed the expression and function of adenosine receptors in bat. adenosine receptors (ador) are members of the superfamily of g protein-coupled receptors. there are four subtypes of adors in humans referred to as adora , a a, a b and a . they are widely expressed in tissues and mediate a variety of cellular functions, mostly due to their regulation of camp levels within cells. interestingly, it has been shown that adenosine can either inhibit or stimulate lipolysis in white adipocytes through adora or a a, respectively . however, the role of adenosine in the differentiation of brown preadipocytes to adipocytes and in bat function is not clear. to analyze the role of adors in bat, we use preadipocytes isolated from bat of newborn mice and subjected them to a differentiation protocol. abundance of adora , a a, a b and a mrna was measured using qpcr. all four receptor subtypes are present in preadipocytes with adora b being the most abundant. adora , adora a and adora are significantly transcriptionally upregulated -albeit at varying degree -during differentiation. adora is upregulated . fold (+/- . fold) and fold (+/- . fold) at day and at day , respectively, as compared to preadipocytes (n= ). adora a is fold (+/- . fold) upregulated at day and fold upregulated (+/- . fold) at day , respectively (n= ). adora was found upregulated . fold (+/- . fold) at day (n= ). in contrast to this, ador b was downregulated to . fold (+/- . fold) at day and to . fold (+/- . ) day compared to preadipocytes (n= ). to investigate the functional role of ador in bati cells, we analyzed lipolysis in mature cells after acute treatment with specific agonists and antagonists. we observed that adora a activation by cgs significantly increased lipolysis by % (+/- . %) compared to untreated control. moreover, adora antagonist psb increased lipolysis by % (+/- . %) (n= ). in conclusion, ador are highly regulated during brown fat cell differentiation. lipolysis of mature brown fat cells is significantly increased by adora a agonist or adora antagonist, respectively. munich heart alliance, münchen, germany activation of the sympathetic nervous system and the subsequent activation of βadrenergic receptors (βars) through catecholamines represents the strongest mechanism to increase cardiac function. however, long-term activation of cardiac βars is clearly detrimental and β-blockers have been introduced as an effective treatment modality in cardiac failure. despite their central role in cardiac physiology and disease, our knowledge about the intracellular mechanism of βar stimulation is confined to a few targets and is likely incomplete. here, we report a functional proteomics approach to directly assess the entire phosphoproteome of βar-stimulated mouse hearts in vivo. to identify proteins that are phosphorylated in response to β-adrenergic stimulation in vivo, we treated mice with isoproterenol or, as a control, with propranolol. after lysis of hearts and tryptic digest, phosphopeptides were enriched by tio or immobilized metal ion affinity chromatography (imac). subsequent analysis of eluated peptides by tandem mass spectrometry (ms/ms) mapped several phosphopeptides to cardiac proteins, among which known mediators of βar signaling such as phospholamban, troponin i and myosin binding protein c. we then employed multiple reaction monitoring (mrm) as a quantitative approach to assess changes of phosphorylation after βar stimulation. using this combination of ms approaches, we identified peptides with pka consensus phosphosites that were more abundantly detected under βar stimulation. among those, we found myozenin- (myoz ) and g protein signaling modulator (gpsm , also termed ags ) as proteins previously not related to βar signaling. we validated the βar-dependence of phosphorylation at these sites in isolated cardiomyocytes by in vivo labelling or phosphoepitope-specific antibodies. current efforts aim at the functional characterization of these novel candidate mediators of βar signaling in the heart. taken together, we report the β-adrenergic phosphoproteome of the mammalian heart in vivo. we have identified several new targets of βar signaling that may represent essential factors in cardiac physiology and disease. background: drug measurement in autopsy material is normally used to investigate the cause of death. in our study it was possible to measure concentrations of drugs that were part of a regular treatment without connection to the cause of death. metamizole is used as an analgetic and spasmolytic agent. the active metabolite maa ( -methyl-aminoantipyrin) is metabolized by the liver and eliminated by the kidney. hepatic and renal dysfunction can therefore influence maa clearance. methods: maa concentrations were measured in different samples of the autopsy material (heart blood, venous blood, urine, liver, kidney and brain) using an hplc-ms/ms method. information about the dosage and time of drug application as well as information about existing renal or hepatic disorders were taken from the corresponding patient records. because of the low number of cases an explorative single-case study was necessary. results: cases with oral intake of metamizole in a customary continuous dosage could be indentified. the maa distribution into body liquids and organs depended on the time between last oral intake and death. in two cases without renal or hepatic diseases maa blood levels were below µg/ml. five cases with combined renal and hepatic disorders showed either increased blood levels of - µg/ml or prolonged maa elimination half-life of up to hours. in one case with manifest hepatic insufficiency an maa concentration of more than µg/ml was measured in venous blood. two cases with renal insufficiency alone had maa venous blood levels of less than µg/ml. (pet) . pet detects the positron emission of neutron-deficient radioactive nuclides and allows their external localization in vivo. fet, a modified amino acid, is not incorporated in proteins but accumulates in glioblastomas. one pathway responsible for its accumulation is the preferential transport into the tumor cells, probably via amino acid transporters. we investigated in more detail (a) which individual, cloned amino acid transporters accept fet as substrate and (b) which transporter is responsible for the major fet transport into glioblastoma cells. studies with xenopus laevis oocytes, expressing individual human amino acid transporters, revealed that system l, y + l and b + amino acid transporters recognize fet as substrate (lat and , y + lat , and b + at, respectively). in contrast, y + lat and atb ,+ did not transport fet. rna expression studies using qrt/pcr revealed that lat is the dominant amino acid transporter in all glioblastoma cells investigated (ln /u /u mg/u /a /t g). a strong lat expression was also shown on the protein level. to find out whether lat is the main transporter responsible for fet accumulation, we first studied transport of the parent amino acid l-tyrosine in ln glioblastoma cells. [ h] tyr uptake was completely na + -independent and inhibited by leu, phe and trp, but not by arg, pro or ser. sirna-mediated down-regulation of lat in ln cells led to a concomitant decrease of lat mrna and tyr transport (down to % and %, respectively). these results indicate that tyr is exclusively transported by lat in ln cells. we are currently performing transport studies using [ f]fet to investigate whether fet transport is also exclusively mediated by lat in glioblastoma cells. a further question is if lat , a sodium-independent transporter, can be responsible for the accumulation of fet observed in glioblastoma cells. if true, other amino acid derivatives that are lat substrates might also proof useful in cancer diagnosis. telmisartan reduces adipose tissue inflammation and biglycan accumulation in diabetogenic ldl-receptor knockout mice grandoch m., nagy n., fischer j. w. institut für pharmakologie und klinische pharmakologie, universitätsklinikum der heinrich-heine-universität düsseldorf, moorenstraße , düsseldorf, germany in addition to lowering blood pressure some of the angiotensin ii at receptor antagonists (arb) such as telmisartan have additional beneficial effects on the onset of type diabetes mellitus and obesity. this was contributed mainly to peroxisome proliferator activated receptor (ppar)γ modulating activity. hyaluronan (ha), a high molecular weight polysaccharide and the small leucine rich proteoglycans, decorin and biglycan, are known to be involved in atheroprogression. mechanistically these matrix components contribute to inflammatory processes via toll-like receptor-signalling and are supposed to modulate lipid retention. the aim of this study was to elucidate the effects of telmisartan in comparison to valsartan, an arb without pparγ activity, on extracellular matrix remodelling and inflammation in atherosclerosis and the interrelationship with adipose tissue inflammation using the ldlr-/-model of accelerated atherosclerosis. male ldlr-/-mice were fed either a diabetogenic diet alone or in combination with telmisartan ( mg/kg), valsartan ( mg/kg) or valsartan ( mg/kg) from weeks of age for weeks. all treatment groups except of the lower valsartan dose showed significant effects on reducing the aortic plaque score. the content of ha, collagen and decorin in the aortic root were not changed. however, telmisartan reduced the content of biglycan in the aortic root significantly in contrast to valsartan. in addition, a trend towards decreased mac -positive macrophages in abdominal adipose tissue was detectable after telmisartan treatment as well as a strong reduction in the adipose tissue mrna expression of biglycan. finally, telmisartan reduced the expression of hyaluronan catabolizing enzymes potentially leading to an increase of high molecular weight ha in the adipose tissue, which is thought to be homeostatic and antiinflammatory. in summary, the results of this study underline the pronounced anti-inflammatory capacity of telmisartan on atherosclerosis and adipose tissue inflammation in comparison to valsartan and strongly suggest that biglycan might be an additional target of telmisartan not only concerning matrix composition of atherosclerotic lesions but also concerning the structure of adipose tissue and metabolic effects of the compound. human primary malignant cancer cells derived from peritoneal effusions of a patient with colorectal carcinoma, as assessed by comet assay. the primary cancer cells were more efficient in dsb repair than ht- cells, and their doxorubicin ic was four times higher. comparative protein expression levels showed that the primary cells had less rad and as well as less topoiiα, while ku and levels were similar. another very interesting protein is the mrn (mre -rad -nbs ) complex that initializes the phosphorylation of atm and thereby starts the signalling cascade. the newly described mrn-atm pathway inhibitor mirin interrupts mrn activity by inhibiting the exonuclease activity of mre . the toxicity of mirin in ht- cells was measured using a luminescence-based assay detecting the amount of atp, which is correlated with cellular viability. mirin did not show any toxic effects up to a concentration of µm and incubation times of hours, indicating that mirin can be used under these conditions without detrimental effects. we are currently investigating the effect of mirin on the toxicity of topoiiα inhibitors. the inhibition of dna repair may be a valuable strategy to enhance the effect of dnadamaging anticancer drugs. since tumours (even of the same entity) are not only heterogeneous, but also polyclonal, a broad selection of response modifiers of anticancer drugs would be helpful to individually enhance chemotherapeutic effectiveness. evidence has been provided that diet and environmental factors directly influence epigenetic mechanisms associated with cancer development in humans. epigenetics play an important role in the control of gene expression. epigenetic mechanisms comprise modulation in dna methylation, histone modification and non-coding rna. several polyphenols have been reported to possess histondeacetylase (hdac) inhibitory properties [ ] . histone deacetylation is generally linked to transcription repression. furthermore, hdac belongs to the group of small ubiquitin-related modifier (sumo) substrate proteins. sumoylation of hdac is associated with a modulation of its biological activity [ ] . little is known so far about the mechanism by which hdac sumoylation mediates inhibition of gene transcription. we addressed the question whether sumo e and hdac expression and whether potential hdac-sumoylation will be affected by polyphenols such as chlorogenic acid, genistein and (-)epigallocatechin- -gallate (egcg). chlorogenic acid, genistein and egcg decreased sumo e protein level in the human colon carcinoma cell line ht after h of incubation measured with western blot analysis. egcg exhibited the most pronounced effect at concentrations ≥ µm. hdac expression was also affected by these polyphenols. the direct impact of polyphenols on the hdac sumoylation is detected by co-immunoprecipitation experiments with the respective antibodies against hdac- and sumo e . these experiments are still under investigation. in conclusion, chlorogenic acid, genistein and (-)-epigallocatechin- -gallate influenced the sumo and hdac expression in vitro. in further studies the direct impact on subtract-sumoylation will be investigated. these studies contribute to a better understanding of potential chemopreventive effects of dietary polyphenols on specific epigenetic alterations may provide chemopreventive strategies for reducing cancer risk. the no/cgmp cascade is thought to be essential for penile erection. within the smooth muscle of corpus cavernosum, nitric oxide activates the no-sensitive guanylyl cyclase (no-gc) which raises the intracellular concentration of cgmp. this second messenger activates the cgmp-dependent protein kinase i (pkgi) and subsequent phosphorylation of target proteins leads to relaxation of cavernosal smooth muscle. knock out of key enzymes of the no/cgmp cascade has led to discrepant results: the deletion of pkgi in the mouse has been shown to lead to erectile dysfunction whereas mice lacking neuronal no synthase are fertile. to investigate the role of the no receptor in fertility we have generated mice lacking no-gc (gcko), a bottleneck enzyme of the no/cgmp cascade. we have shown that lack of no-gc resulted in arterial hypertension concomitant with a totally abolished no responsiveness of vascular and gastrointestinal smooth muscle. in addition, we generated a mouse line in which no-gc was specifically deleted in smooth muscle cells (sm-gcko). using these ko strains we here examined the role of no/cgmp signaling with regards to the smooth muscle relaxation of corpus cavernosum. no failed to affect corpus cavernosum from gcko in organ bath experiments: neither exogenously produced no by no donors nor endogenous no release from neurons induced by electrical field stimulation led to relaxation. similar results were observed in the corpus cavernosum of sm-gcko mice. to our surprise, the gcko animals were fertile and produced offspring albeit at a reduced rate compared to wt animals. our data show that interruption of no/cgmp signaling results in complete absence of no-induced relaxation of penile corpus cavernosum in mice and reduces the ability to produce offspring but does not abolish fertility. novel modes of invasive cell motility regulated by the formin class of actin nucleators khan j., grosse r. philipps-universität marburg, pharmakologisches institut, karl-von-frisch-str. , marburg, germany pathological invasive cell migration essentially reqires actin polymerization. formins are the largest group of rho-gtpase effectors involved in actin nucleation and assembly as well as microtubule dynamics. here we studied the role of formins in cytoskeletal regulation during homotypic cancer cell invasion. we identified the actin-dependent steps and structures involved for this process. using live cell analysis we characterize the distinct actin dynamics controlled by formin-like and rho function. the specific involvement of this signaling module will be discussed. formin-driven nuclear actin assembly controls mal/srf activity baarlink c., wang h. polymerization of actin in the cytoplasm is tightly linked to transcriptional activation of the srf cofactor mal (also known as mrtf-a) through release of actin/mal interactions and subsequent nuclear accumulation of mal. formins directly promote assembly of actin filaments thereby efficiently regulating mal-dependent transcription for cell shape, adhesion and motility. here we show that formins assemble f-actin and promote mal activation inside the mammalian nucleus. the rho-gtpase effector mdia rapidly enters the nucleus in a signal-dependent fashion and an active mdia confined to the nucleus potently promotes release of g-actin from mal to specifically activate srf. live cell imaging reveals formin-mediated nuclear actin dynamics. moreover, using actin assembly assays we find that inhibition of endogenous mdia formins controls f-actin turnover in isolated nuclear extracts. thus, formin activity is dynamically compartmentalized to the mammalian nucleus to potently regulate actindependent mrtf function. in women the placenta becomes the main source of maternal estrogens during pregnancy. placental estrogen biosynthesis is located in the syncytiotrophoblast, a syncytium that builds the main part of the placental barrier and limits the transfer of substances between the fetal and maternal compartment. since the human placenta is unable to convert cholesterol into -oh-pregnenolone, the placenta tissue highly depends on the supply of c- steroids for their conversion into c- estrogens. in contrast to lipophilic unconjugated steroids that penetrate the cell membrane passively via diffusion, circulating sulfated steroid hormones are delivered to the placenta via carrier-mediated transport, followed by their reactivation via the catalytic activity of the steroid sulfatase (sts). dheas of maternal and fetal origin contributes about equally to the placental formation of estrone (e ) and estradiol (e ), while αoh-dheas supplied by the fetus contributes to over % of placental estriol (e ) synthesis. soat, a member of the slc family with highest expression in hormone-responsive tissues such as testis, placenta, and mammary gland has been shown to transport the sulfoconjugated steroid hormones dehydroepiandrosterone sulfate (dheas), estrone sulfate (e s), and pregnenolone sulfate (pregs) [ ] . aim of this project is to investigate the role of soat for placental estrogen synthesis by means of the choriocarcinoma cell line jeg- as in vitro model for the human syncytiotrophoblast. therefore, we characterized a jeg- cell line that transformed dhea into e and αoh-dhea into e . by qrt-pcr we found expression of sts and aromatase, both essential for estrogen synthesis in these cells. upon transient transfection of soat the carrier was located in the cell membrane of transfected jeg- cells. currently we investigate the transformation of dheas of these soat-jeg- cells by lc-ms-ms. we could demonstrate transport of αoh-dheas for stably transfected soat-hek cells. in situ hybridization and immunohistochemistry showed coloured syncytiotrophoblasts and vascular endothelial cells in late term placenta. in conclusion, soat-mediated transport of sulfated steroids could play a pivotal role for placental estrogen synthesis from sulfated steroid hormones. developing non-animal test systems for evaluation of toxicity was important in the past and will remain essential in the future. here we present a toxicity test using the chicken yolk sac area vasculosa (cav) of fertilized white leghorn chicken eggs [ , ] and compare it to hen's egg test on chorioallantoic membrane (het-cam) [ ] for polymer toxicity testing. fertilized chicken eggs were incubated and after h explanted shell less into sterile petri dishes. test substances were applied on the cav and the appearance of different effects (vascular lysis, haemorrhage, aggregation of blood components, lethality) was determined by light microscopy after - h (fig. ). these effects were combined to a cav test score based on the irritation score calculation used for het-cam evaluation. different polymers like poly(ethylene glycol) (peg; neutral), poly(ethylene imine) (pei; cationic) and dextran sulphate (ds; anionic), as well as guideline-conform (recommended het-cam protocol from the interagency coordination committee on the validation of alternative methods) negative ( . % nacl) and positive controls ( % sodium dodecyl sulphate (sds) and . n naoh) were investigated. additionally ld values for different cationic polymers have been determined. within the selected incubation times ( - h) , effects such as vessel lysis and blood component aggregation could be detected. additionally to het-cam, lethality as well as recovery of the cav could be observed. differences between neutral, positively and negatively charged polymers were obtained. pei showed strong vessel lysis and aggregation of blood components whereas ds and peg showed none of these effects. lethality was found to increase from peg < ds < pei and is concentration and time dependent. the results demonstrate that differences, regarding the toxicity of the used polymers, can be shown with this test. these findings in the cav test can be well correlated with already existing data. in summary, the cav test provides same data (testing control substances) and more information (recovery and lethality) compared to het-cam and could be a suitable model for toxicity testing of polymers. risk characterisation of chemicals consists of three steps ( ) hazard identification and characterisation, based on substance-specific toxicological hazard data, ( ) estimates of the level of exposure toward the substance and ( ) the comparison between the toxicologically safe level and the exposure level. in contrast to the classical risk assessment approach, the threshold of toxicological concern (ttc) approach is developed as a tool to assess the risk of substances without toxicity data. its application requires ( ) information on human exposure, for which it is essential that exposure is fully captured and ( ) knowledge of the chemical structure to assess whether the chemical is not excluded from the application of the ttc concept. instead of chemical specific no observed (adverse) effect levels (noels/noaels), the ttc approach utilises knowledge on the empirical distribution of several hundreds of noels/noaels, originally , based on toxicological testing in animals (munroe et al., ) . with the basis on noaels, the ttc concept builds on the fundamental principle of toxicology, that toxicity is a function of dose and that a dose exists, below which no adverse effects of the substance can be detected. it is assumed that exposures below this level will not result in health risks. three separate ttcs were derived (munroe et al., ) by classifying the chemicals into three toxicity classes using a decision tree based on a series of questions related to chemical structure, and on natural occurrence in food and in the body (cramer et al., ) . the ttc values are derived from empirical distribution of the noels/noaels in the class taking the th percentiles and dividing them by the default uncertainty factor of . it is assumed that the probability is very low that the unknown noael of a not tested chemical will be lower than the value of the th percentile in the distribution of the known noels/noaels. hence, at exposures below the ttc values, the probability of adverse effects on human health is considered to be very low. introduction: kibra, mainly expressed in kidney and brain tissue, is involved in brain development and memory formation as a postsynaptic scaffold protein. in podocytes, kibra is proposed to regulate cell motility as a linker between components of the cytoskeleton and polarity protein complexes (duning et al, jasn ) . furthermore, kibra has been identified as key regulator of the hippo pathway, which is involved in organ size control and tumorigenesis. in the current study, we focused on the identification of kibra gene expression regulation and functional promoter characterization. methods: serial promoter deletion constructs were generated by cloning bp of the 'flanking region of kibra into the pgl -vector system. deletion constructs were transiently transfected into human neuroblastoma cells (sh-sy y) and immortalized human kidney epithelial (ihke) cells. potential transcription factors (tfs) were investigated in cotransfection experiments. transcriptional start sites (tss) were determined by rapid amplification of 'cdna ends ( 'race) . tss utilization between cell lines was assessed by semiquantitative pcr. transcriptional activity (ta) of the kibra promoter p was separated by ~ bp into two distinct regions, promoter p a and p b. deletion constructs harbouring promoter p b were transcriptionally active only in ihke cells. 'race revealed two alternative tss in both cell lines upstream of the annotated tss (nm_ ). exclusively in ihke cells, two additional tss were detected in intron , resulting in two alternative exons. deletion constructs harbouring the putative regulatory regions (p and p ) of both exons were transcriptionally active only in ihke cells. overexpression of full length tcf l (transcription factor -like [t-cell specific, hmgbox]) resulted in a ~ -fold increase of promoter p a and intron promoter p ta. kibra gene expression is driven by a complex alternative promoter system comprising the constitutional promoter p and three alternative promoters p b, p and p . the tss utilization is cell type-specific. subsequent usage of an alternative translation start site within exon could result in truncated kibra protein isoforms. tcf l is involved in the differential kibra gene expression regulation. resulting kibra protein isoform and their cellular function will be assessed in further studies. skin absorption in vitro based on the study of human/animal skin ex vivo or reconstructed human epidermis, respectively, is an alternative method which is accepted by the oecd. guideline tg and a corresponding technical guidance document (gd ) give technical guidance how to perform valid experiments , . the requirements include integrity evaluation tests for the skin samples. different tests are proposed to ensure an exclusively use of undamaged skin. to decide which test suites best to our routine test strategy, we investigated the correlation between integrity test results and absorption profiles of various penetrants (logp range: - . - . ). finite dose experiments using rat and human skin were performed with c-labeled testosterone, caffeine, mcpa ( -chloro- -methylphenoxyacetic acid) and its -ethylhexyl-ester mcpa- ehe. for each experiment at least three of the five following integrity tests were conducted: transepidermal electrical resistance (teer), transepidermal water loss (tewl), transepidermal tritiated water flux (³h o), transepidermal absorption of methylene blue (blue) , transepidermal absorption and flux of a ³h-labeled internal standard (istd); ³h-testosterone or ³h-mannitol was used as istd. the applied radioactivity of the ³h-istd was selected to show no analytical interference with the c-penetrants. teer, tewl and ³h o represent pre-study, istd concurrent and blue post-study tests. calculated maximal permeability constants (kp) and absorbed doses (ad) of the penetrants were compared to the results of the integrity tests. individual linear regression analysis was used to evaluate the correlation the correlations varied over a wide range for all five methods and four penetrants. the best correlations in average were achieved with the istd. no inverse correlations were obtained for the istd, but partly for tewl, teer, ³h o and blue. in conclusion, the istd represents best the achieved absorption profiles of the test compounds and is based on that the most suitable integrity test for our dermal absorption studies. we will further confirm its effectiveness and generate a sufficient historical database in order to include the istd in our routine test protocol. investigation of mirna expression and dna methylation in focal and non-focal brain tissue of therapy-resistant epilepsy patients haenisch s. background: resistance to anticonvulsants affects one third of all epilepsy patients. limited bioavailability of the drug at the target site caused by increased expression of efflux transporters on the blood brain barrier or alterations of target genes are potential mechanisms for therapy resistance. however, these mechanisms alone cannot completely explain the observed resistance and it is likely that multifactorial alterations lead to pharmacoresistance. there is increasing evidence that expression of micrornas probably caused by dna modifications is deregulated in many neuronal diseases. we hypothesize that mirna regulation of target genes is involved in drug resistance in epilepsy. methods: hippocampal focal and cortical non-focal brain tissue samples from patients diagnosed with mts (mesial temporal sclerosis) who underwent neurosurgery have been screened for mirna expression using taqman low density arrays. in silico approaches for both a hypothesis-based (efflux-transporter and target gene) as well as a hypothesis-free approach were used to identify potential phenotype-relevant target genes. using the program r (bioconductor) a mann-whitney-u test was performed to compare mirna expression between brain regions. pyrosequencing was performed to investigate methylation status '-upstream of dna regions encoding for selected candidate mirnas. results: out of mirnas, were detected in both tissue types. the expression of one mirna was . fold higher (q= . ) and another was . fold lower (q= . ) in the hippocampus relative to the cortex. evidence could be found that down-regulation of the latter is possibly caused by hypermethylation of '-flanking region of its encoding dna locus. bioinformatic analysis has identified eight genes important for neuronal regulation and signal transmission (e.g. sox , mecp , bsn), as well as one abc effluxtransporter, as potential targets for these differentially regulated mirnas. conclusion: differential regulation of two mirnas could contribute to an altered function of several genes resulting in an imbalance between neuronal excitation and inhibition that is independent from mechanisms presently targeted by anticonvulsants. this work was supported by a fellowship from dfg and nih grant gm . recently, it has been reported that human b cells express and secrete the cytotoxic protease granzyme b (grb) after the combined stimulation of the il- -and the b cell receptors. grb produced by b cells is enzymatically active and b cells deliver grb to sensitive cancer cell lines, thereby inducing apoptosis. to date, there is little experimental evidence on the mechanisms involved in grb expression, or its function in b cell biology. as experimental transgenic murine systems should enable us insights into these issues, we assayed for grb in c bl/ b cells using an extensive array of physiologically relevant stimuli, but were unable to detect either grb expression or its proteolytic activity, even when antigen specific transgenic b cell receptors were cross-linked. similar results were also obtained with b cells from dba/ , cba or balb/c mice. in vivo, infection with either influenza virus or murine γ-herpesvirus induced the expected expression of grb in cytotoxic t lymphocytes, but not in b cell populations. we also investigated a possible role of grb on the humoral immune response to np-klh, but grb-deficient mice produced normal amounts of antibody with typical affinity maturation and heightened secondary response, demonstrating conclusively the redundancy of grb for antibody responses. our results highlight the complex evolutionary differences that have shaped the immune systems of mice and humans and demonstrate the need to develop novel in vivo systems to study human humoral immune responses. investigations of the cholinergic neurotransmitter system in dyt mice hamann m. early-onset torsion dystonia is an autosomal dominant inherited movement disorder associated with the dyt gene defect with deletion of a glutamic acid residue in the protein torsina. despite the gene defect, the pathophysiology is poorly understood. animal models can help to understand the underlying mechanisms and thereby to develop new therapeutic strategies. sharma et al. ( , j. neurosci. [ ] , - ) initially described a transgenic mouse model (dyt mice) with overexpression of mutant torsina. previous studies in these mice pointed to alterations in the cholinergic system. to investigate the functional relevance of these in-vitro findings, we carried out pharmacological in-vivo experiments and determined the density of striatal cholinergic interneurons as well as the expression of choline acetyltransferase in different brain regions. the acute intraperitoneal administration of the cholinomimetic drug pilocarpine ( , and mg/kg) as well as a long-term treatment over days ( mg/kg/d) did not induce pronounced effects in dyt mice compared to wildtype controls. the higher incidence of epileptic seizures in dyt mice compared to controls after repeated local striatal applications of pilocarpine ( and µg/ . µl/hemisphere) let presume an altered synaptic plasticity in dyt mice. the immunohistochemical investigations revealed a moderately reduced density of striatal cholinergic interneurons in the dorsomedial subregion of dyt mice compared to wildtype controls, while significant differences in other striatal subregions were not detected. western blot analysis did not show clear differences in the expression of choline acetyltransferase between dyt and wildtype control mice. these results indicate that the cholinergic system seems not to play a key role in this line of dyt mice. ongoing receptor autoradiographic analysis of binding to different muscarinic receptors subtypes have to further clarify the existence of possible alterations within the cholinergic system of these dyt mice. inhibitors direct against cell cycle-regulatory kinases are being tested in clinical trials as anti-proliferative agents. thus, the atp-competitive kinase inhibitor pd which inhibits cdk and cdk is currently tested in patients with solid tumors such as glioma. we found that pd suppressed il- -induced expression of il- suggesting that cdk or cdk may have unknown anti-inflammatory properties. to study the effects of cdk on the il- -signaling network, we established a bidirectional doxycyline-inducible system to express a constitutively active mutant of cdk , cdk s p, in asynchronized hela cells. cdk -expressing cells were identified by gfp which was expressed from the same promoter, isolated by laser-microdissection and analysed for mrna expression using a down-scaled rt-qpcr assay. compared to the uninduced state, cdk s p enhanced il- -induced il- and il- mrna expression. moreover, shrna-mediated suppression of endogenous cdk confirmed a role of this kinase in regulation of maximal il- -induced gene expression of il- . these data also revealed that the contribution of cdk to inflammatory gene expression is highest in g , when activity of endogenous cdk is activated by d-type cyclins. these findings were corroborated in hela cells expressing fluorescent ubiquitin-dependent cell cycle indicator (fucci) proteins. hela-fucci cells from g , g /s, g or mitotic states were isolated by laser-microdissection and analyzed by rt-qpcr for tnf-inducible gene expression. stable knockdown of cdk in hela fucci or inhibition by pd suppressed inducible il- expression. microarray experiments identified many additional genes that required active cdk for maximal il- -or tnf-inducible gene expression. we also found that cdk co-immunoprecipitated with p nf-κb, colocalized with p in the nucleus and was recruited together with the p subunit to the proximal il- promoter as assessed by chip and re-chip experiments. collectively, these results suggest an unexpected control of inflammatory gene expression through a classical cell cycle regulatory pathway. these results also imply that pharmacological targeting of cdks may have effects and side-effects on the immune system in addition to inhibition of cell cycle progression. trp channels form a heterogeneous family of calcium-permeable channels, which play major roles in physiological functions ranging from sensory reception to cellular signal transduction. members of the trpc subfamily (classic transient receptor potential channels) are downstream targets of hormone receptors. of particular interest is the biological role of trpc channels. they are directly activated by diacylglycerol due to phospholipase c-driven signalling pathways which are involved in smooth muscle contractility, neuronal plasticity, keratinocyte differentiation and renal function. their impact in renal function became evident from analyzing patients suffering from familial forms of focal segmental glomerolusclerosis (fsgs) which could be linked to trpc mutations. since the first descriptions at least different pathogenic mutations have been identified in humans to cause fsgs. in order to study the underlying pathophysiological mechanisms of trpc mutations, we have analysed all mutated trpc channels known to date heterologously expressed cells. one set of mutations showed a gain-of-function phenotype which has been previously suggested to cause an increased intracellular calcium load and subsequent cell death. hence, gain of function mutations fit to the current paradigm of fsgs pathophysiology. however, another set of mutations found in the patients showed a loss-of-function phenotype. our results enable a change in the current paradigm for the role of trpc in renal pathophysiology and may provide a basis for our understanding of the pathophysiology of loss-of-function mutations in familial focal segmental glomerolusclerosis. karlsruher institut für technologie (kit) institut für angewandte biowissenschaften, abteilung lebensmittelchemie und toxikologie, adenauerring a, karlsruhe, germany risk assessment for genotoxic carcinogens is an important challenge in toxicology. even though manifold attempts have been made to substitute carcinogens and to reduce exposures, their complete elimination appears to be not possible. thus, low concentrations of known or suspected genotoxic carcinogens are present at workplaces, in the environment and in food. in order to deal with this situation and to set priorities for risk management, different concepts have been established such as the alara principle (as low as reasonably achievable) and the margin of exposure (moe), based on the ratio between concentrations being carcinogenic in experimental animals and the actual exposure of humans for example via foodstuff. while usually linear doseresponse-relationships have been used as default assumption, analytical methods are now available to assess the induction and repair of dna lesions on low exposure conditions, including environmental background exposure, and to relate the extent of exposure-induced dna lesions to endogenous dna damage. this may be an important prerequisite to establish health-based limit values for selected genotoxic carcinogens. within this workshop, different examples will be discussed and research need will be identified. dendritic cells from h r-deficient mice lose their ability to properly stimulate t lymphocytes hartwig c., seifert r., neumann d. mhh pharmakologie, carl-neuberg str. , hannover, germany the incidence of allergic airway diseases is increasing throughout the world, especially in western countries. although histamine (ha) is found at high concentrations in asthmatic lungs, a role for ha in bronchial asthma is still a neglected topic in clinical research. in particular, the capacity of ha to modulate the underlying immune reaction is far from being understood. the histamine h -receptor (h r) is involved in acute inflammation and th cytokine production. consequently, we intended to analyze the role of h r in a murine th lymphocyte transfer-based model of asthma. specifically the ability of h r expressed on dendritic cells (dcs) to modulate t cell function was analyzed. ova-specific cd + t cells were polarized in vitro under th -favoring conditions with ova peptide-pulsed dcs, obtained either from wild-type or h r -/mice. analysis of the polarized t cells after in vitro restimulation revealed a marked decrease of il- production in t cells polarized in the presence of h r -/-dcs compared to those polarized in the presence of wild-type dcs. thus, on dcs, the h r is essential for proper stimulation of spleen t cells and for directing their polarization towards a th phenotype. the transfer of in vitro polarized t cells into recipient mice and subsequent provocation elicited an asthma-like disease. the h r on dcs not only affects in vitro polarization of t cells, but also the in vivo function of the obtained polarized t cells. a parameter indicating allergic inflammation is the enhanced influx of inflammatory immune cells into the lung tissue, mainly driven by eosinophils, which are virtually absent in non-asthmatics. when analyzing the number of eosinophils, a dramatic difference due to the polarizing conditions of t cells occurs. in bal fluids of mice that received t cells polarized in the presence of wild-type dcs, about % eosinophils were detected. in contrast, the transfer of t cells polarized in the presence of h r -/-dcs yielded only about - % eosinophils in bal fluids. in summery, the h r on dcs plays an important role for t cell polarization and consequently affects the allergic reaction during sensitization. since the lack of the h r on dcs reduced their ability to stimulate proper th polarization of cd + t cells, we conclude that ha via the h r significantly affects the manifestation of asthmatic inflammation. antioxidant polyphenols and their effects on nrf (skn- ) signalling in a cell culture system and the model organism c. elegans havermann s., wätjen w. heinrich-heine-universität düsseldorf institut für toxikologie, p.o. box , düsseldorf, germany oxidative stress has been connected with a variety of diseases, (e.g. alzheimer`s and parkinson´s disease), cancer and ageing over the last years. certain polyphenols were shown to have an antioxidant capacity as well as being able to activate the protective nrf signalling pathway. compared to direct radical scavengers modulators have the advantage of building up a permanent defense against oxidative insults whereas scavengers do not protect any more after consumption or may even cause stress due to redox cycling. we have employed cell culture based assays (dcf, western blot, gfp reporters) to analyse the effects of polyphenols. further we tested the coumpounds in vivo in the nematode c. elegans where skn- is the nrf homologue. baicalein and caffeic acid phenethylester (cape) protected cells and the nematode from ros accumulation after application of stress (shown by dcf assay). activation of nrf signalling is correlated with translocation of the transcription factor into the nucleus. in both systems nrf ::gfp accumulation in the nuclei could be observed after incubation with baicalein (fluorescence microscopy). but while cape is a potent activator of nrf in cells, it has no effect on skn- localisation. further the effect on the nrf protein amount was investigated by western blot analysis. the expression of target genes can be investigated by differing means: while pcr methods and western blotting are standard for in vitro studies, the vast number of available gfp reporter strains offers opportunities for research using c. elegans. we have performed congruent assays in a cell culture system and the model organism c. elegans to compare antioxidative capacity and effects of polyphenols on nrf signalling. therefore, depending on the substance tested, c. elegans is a suitable model system to investigate effects of natural compounds in an organism. being associated with adverse health effects, the human exposure to dehp is subject to concern. quantifying the population's exposure and determining the contributions of different exposure routes is a key task of environmental health risk assessment. the study presented comprises a review of the available data on dehp levels in foods, consumer products, and house dust. extensive survey data, e.g. from the current national nutrition survey ii and the eu rapex system were processed for modeling the exposure by the oral, inhalative and dermal path of the population in germany. the study also included analytical analyses of dehp levels in selected foods and consumer goods (incl. migration rates for mouthing). probabilistic techniques allowed elucidating the exposure's variation and the relevance of different routes. mean exposure estimates for german children and adults to dehp are and µg/(kg d), resp. for children, food accounts for % of the total exposure, followed by mouthing ( %) and house dust ( %). the adult exposure is almost entirely ( %) due to food. as dietary exposure is a result from concentration and consumption, foods exhibiting high contamination e.g. butter ( %) and dressings (mayonnaise) ( %) as well as highly consumed foods e.g. bread and bakery ( %) and vegetables ( , %) contributed significantly. the mean estimate of children's dehp exposure via mouthing revealed , µg/(kg d). high exposures were estimated ( th percentile) up to , µg/(kg d). on average people in germany are exposed to dehp below the current tdi of µg/(kg d). however, individual exposures exceeding the tdi still cannot be excluded. current data on dehp and other plasticizers in foods are scarce, which warrants broader monitoring. our findings highly facilitate further exposure modeling focusing on dehp substitutes and risks of combined exposure. this study was funded by the federal ministry for the environment, nature conservation and nuclear safety in the frame of the environmental research plan (umweltforschungsplan, förderkennzeichen (ufoplan) ). on the basis of the available measurements of dehp, the exposure assessment has been focused on food categories characterising a selection of the most important food groups covering all major food classes of the german population. based on an extensive literature survey, the analysis considered the available data of dehp measurements in food, as well as the official german food control data taken from the national food consumption survey. the high amount of considered data allowed the consideration of several exposure assessment tiers (deterministic and probabilistic by using monte carlo simulation). a quantitative evaluation of the uncertainties of the estimate of the food categories groups has been performed by means of a sensitivity analysis by using the methodology proposed within the who ipcs guidance document of characterising and communication uncertainty in exposure analysis. qualitative uncertainty analysis (tier ) was applied to determine the most important sources of uncertainty, i.e. concentration of dehp in all food categories. the probabilistic monte carlo simulation (tier ) was then used to rank the cumulative probability distributions of the exposure assessments of food categories on the basis of the food categories that appear to dominate. sensitivity analyses were applied to prove the impact correlation of food groups for uncertainties. by a scenario based concept, the aggregation of the food groups to groups has been evaluated, as well as the sensitivities by characterising particular scenarios. for this purpose, particular "meals" have been described as fixed combined scenarios and. the aggregation leads to a considerable higher exposure estimate which can be explained by the combination of high contaminated foods with others of high consumption. the evaluation confirms the considerable role of possibly high contaminated foods e.g. fats, or mayonnaise. the evaluation shows that quantitative probabilistic sensitivity analysis is a suitable and pragmatic tool for uncertainty analysis in exposure assessment. the transcription factor camp response element (cre)-binding protein (creb) plays a critical role in regulating gene expression in response to activation of the campdependent signaling pathway, which is implicated in the pathophysiology of heart failure. we observed creb knock-out cardiomyocytes to be larger than wildtype cardiomyocytes (cell area in µm ; mean±sem; creb-ko ± vs. wt ± ; n= / cells, n= mice, p< . vs. ctr.). the nuclear factor of activated t-cells c , nfatc , is another transcription factor involved in the development of heart failure and also a known positive regulator of hypertrophy. hence, we investigated whether inhibition of the cre-dependent transcriptional activation has an impact on the nfatc signaling pathway. we first studied the effects of an overexpression of a dominant negative creb mutant (dncreb) or of nfatc on the activity of a nfat-dependent model promoter in a permanent cell line. overexpression of dncreb evoked an . ± . fold increase of the nfat model promoter activity (n= ; n= transfections), but had no impact on kv . promoter activity which is known to be regulated by nfatc ( . ± . fold; n= ; n= ; p< . vs. ctr.). nfatc overexpression led to a . ± . fold increase of the nfat-dependent model promoter activity (n= ; n= ) and to an inhibition of kv . promoter activity ( . ± . fold; n= ; n= ; p< . vs. ctr.). we conclude that creb is a negative regulator of nfat-mediated gene transcription and that activation of nfatc might contribute to the observed hypertrophy of creb-ko cardiomyocytes. neuroleptika der perazin-klasse sind potente modulatoren des p x -rezeptors -perazine-type neuroleptic drugs are potent modulators of p x receptors hempel c., nörenberg w., urban n., sobottka h., schaefer m. universität leipzig rudolf-boehm-institut für pharmakologie und toxikologie, härtelstraße - , leipzig, germany p x receptors belong to a family of atp-gated, non-selective cation channels, which play an important role in immune cell activation, inflammatory hyperalgesia and neuropathic pain. they differ from other p x family members by the low atp affinity, and by the ability to form or recruit dilated pores in the sustained presence of atp. owing to its involvement in many diseased states, p x is a promising target for pharmacological intervention. accordingly, p x blockers are currently tested in phase ii clinical trials. in an attempt to identify p x -modulating properties of approved drugs or natural compounds, we performed a medium-throughput screen, using an appropriate compound library (spectrum collection) and a stably transfected hek hp x cell line. with ic values of - µm, the tricyclic antipsychotics prochlorperazine and trifluoperazine showed a high potency and efficacy to block the atp ( mm)-triggered increases in the intracellular ca + concentration ([ca + ]i) that was mediated by human p x (hp x ). the closely related phenothiazine-class neuroleptic drugs, such as chlorpromazine or triflupromazine did not have an appreciable effect on hp x mediated ca + influx. whole-cell inward currents, measured at - mv, were blocked by more than % by - µm prochlorperazine. the inhibitory effects of perazines developed within about ms, hinting to a direct mode of action by binding to the p x protein. prochlorperazine added intracellularly via the patch pipette did not substitute for the extracellularly applied drug, indicating that its binding site is accessible from the extracellular side. in addition, both compounds blocked yo-pro- uptake when preincubated before p x stimulation with mm atp or when applied subsequent to the agonist. interestingly, when added to a hek cell line expressing the rat p x , perazines potentiated the atp-induced increase in [ca + ]i. measurements in human monocyte-derived macrophages confirmed the ability of prochlorperazine and trifluoperazine to inhibit atp-evoked increases in [ca + ]i, changes in yo-pro- permeability and whole cell currents. taken together, we conclude that perazine-type neuroleptics impede on p x activity in a species-specific manner, presumably by binding to an extracellularly accessible binding site of recombinant or natively expressed p x . similarly, pre-treatment with lov also lowered dox-induced stabilisation of p and phosphorylation of chek and sapk/jnk. while lov had no influence on ir-induced initial dna damage formation in huvec and rat cardiomyoblasts (h c ), it decreased dox-and eto-induced phosphorylation of histone h ax, which is a surrogate marker of dna-double strand breaks. this indicates that lov specifically protects against the genotoxicity of topoisomerase type ii poisons. in an acute and subacute balb/c mouse model lov protected from ir-induced toxicity. this effect rested on inhibition of pro-inflammatory and pro-fibrotic processes as measured via quantification of mrna levels of il , ctgf and tnfα. the same was true for dox-induced toxicity, i.e. heart and liver damage. similar to the in vitro experiments, dox-induced hepatic dna-damage was attenuated by lov treatment. overall, liver and heart toxicity were reduced by lov as mirrored by the serum levels of gldh/gpt and ctn-i, respectively. both in liver and in heart we observed collagen rich perivascular areas following dox treatment. under situation of lov-co-treatment these areas occurred more rarely and were less pronounced, pointing to a lowered level of fibrosis. pcr-array-based mrna analyses showed inhibitory effects of lov on dox-triggered expression of genes involved in oxidative stress response, drug transport, dna repair, cell cycle progression and cell death. for instance, up-regulation of p , wee , cjun/fos and hmox- following dox administration was attenuated by lov. altogether, we suggest that including lov in current cancer therapeutic regimen might widen the therapeutic window of anticancer therapeutics by lowering normal tissue damage. the p values. in addition, array data underwent cluster analysis for identification of substantial differences of gene regulation among the three different types of biopsies. results: of particular interest in our study was the expression of genes coding for metabolism and transport proteins. therefore genes from the significant differentially regulated genes, were selected for the qrt-pcr analysis. genes coding for abcb and abcg transport proteins showed higher expression in the jejunal tissue one year after surgery compared to the duodenal tissue (fold change . and . ). moreover, cyp a mrna involved in metabolic processes is higher expressed in postoperative jejunum than in the jejunum tissue taken during the surgery (fold change . ). in conclusion roux-en-y gastric bypass operation leeds a change of mucosal gene expression profile in the jejunum during one year. there was also a significant differential gene expression between the original duodenum and jejunum one year after surgery. these results give strong evidence that jejunum not exposed to pancreatic but only to gastric fluids may change its gene regulation. background. numerous genome-wide association studies (gwas) identified polymorphisms located in transporter genes such as slc a , abcg , npt , and urat as predicitive for the serum levels of urate . these genes encode membrane proteins expressed in the apical membrane of human kidney proximal tubule cells and are assumed to facilitate tubular exchange of urate , , , . importantly several single nucleotide polymorphisms (snp) located in vicinity of slc a have been identified as highly associated with serum urate levels. little is known about the transcriptional regulation of slc a . therefore, the aim of our study was to investigate which sequences in the slc a gene harbour ciselements and regulate its gene expression. we also asked whether intronic snps influence gene expression at the transcriptional level. methods and results. performing dual luciferase reporter gene assays we found gene regulating modules in the slc a gene. dna from human kidney samples was then genotyped for rs being part of this region. next total slc a mrna-expression levels of the samples were determined using real-time quantitative rt-pcr assay. male samples with two minor alleles of snp rs showed lower slc a mrna levels than samples with the wild type alleles. the effect was not seen in females. reporter gene constructs with either minor or major allele of rs were then used in luciferase assays, however showing no significant difference in activity. furthermore mrna-expression levels of other urate transporter genes were determined in kidney samples. after linear regression a positive correlation of mrna-expression of slc a , urat , npt , and oat , respectively was observed. conclusion. our data suggest that the slc a snps rs and rs might influence slc a mrna-level without controlling the transcriptional activity. it needs to be elucidated whether those snps alter mrna stability. however, the mrna coexpression of slc a and other urate transporter might be attributed to a common gene regulating pathway of an "transportosome" controlling urate homeostasis. sulfotransferases mediate the bioactivation of methyleugenol to a reactive sulfate ester binding to dna in vitro and in vivo herrmann k. methyleugenol (me) is a secondary metabolite occurring in many herbs and spices. although me is hepatocarcinogenic in rodents, standard genotoxicity tests were negative. this may be due to the lack of critical activating enzymes responsible for the terminal bioactivation of me to a genotoxicant. me is initially hydroxylated by cytochrome p enzymes yielding ´-hydroxymethyleugenol ( ´-ohme). this alcohol can be further activated by sulfotransferases (sults) to an electrophilic sulfate ester that can be easily attacked by dna. the dna adducts formed could lead to mutation and further carcinogenicity observed in animals. the aim of the present study was to clarify whether individual human (h) and murine sult forms are involved in the activation of me to a genotoxicant. in order to identify critical sults, mutagenicity tests including bacteria expressing different sult forms were conducted. (±)- ´-ohme (separated into its enantiomers) served as test compound. we could show that hsult a , standing out due to its high expression level in many tissues, can efficiently activate both enantiomers even at low concentrations. furthermore, dna adduct formation in hsult a -proficient and sult-deficient bacteria was examined after incubation with µm of (+)-or (-)- ´-ohme. for selective detection and quantification of me-derived ´-deoxyadenosine (da) and ´-deoxyguanosine (dg) adducts we developed a sensitive tandem mass spectrometry method including stable isotope dilution analysis. adduct formation was only observed in bacteria expressing hsult a . the concentration dependence of adduct formation in hsult a -proficient bacteria was examined for (+)- ´-ohme. both adducts turned out to be concentrationdependent. to check the extent and organ specificity of adduct formation in vivo we administered mg/kg bw (±)- ´-ohme (i.p.) to mice carrying the hsult a / a gene cluster. mice getting only the vehicle served as controls. animals were sacrificed and dna from eight organs was extracted. by means of tandem mass spectrometry adducts were measured and quantified. da and dg adduct formation was observed in all tissues studied, but not in untreated animals. furthermore, adduct levels were higher than in experiments using wild-type mice. altogether, we herein could show that sulfo conjugation leads to bioactivation of me to a dna-binding intermediate in vitro and in vivo. this work was financially supported by bundesinstitut für risikobewertung. objective: the soluble adenylyl cyclase (sac) activates the na + /k + -atpase in renal epithelial collecting duct cells. nuclear sac constitutes a functional complex with camp response element binding protein (creb), suggesting a more general role of sac in overall gene regulation. we determined the chromatin binding capacities of sac at cre sequences and its influence on genes, which play a role in aldosterone signalling. furthermore, we functionally characterised expression relevant promoter portions of sac and the influence of aldosterone and camp mediated signalling pathways on sac gene regulation. design and methods: in vascular endothelial cells (ea.hy ) and in human kidney cell lines (hek t; ihke), we performed chromation immunoprecipitation (chip) assay with antibodies against sac and creb. we conducted transfection with a cre luciferase reporter vector and sac promoter constructs, following treatment with sac inhibitors and aldosterone. total rna of ea.hy cells, which were treated with sac inhibitors and aldosterone, was isolated and subsequently analysed by real-time pcr for expression of genes involved in aldosterone signalling. in vivo binding of sac at cre motifs was shown using cre consensus sequences in chip experiments. specific pharmacological inhibition of sac led to a significant decrease of transcriptional activity of the cre control vector in endothelial and kidney cell lines. furthermore, we were able to show the different effects of sac on the expression of downstream targets of aldosterone signalling, e.g. mineralocorticoid receptor and na + /k + -atpase alpha and beta and sac itself. regulation of sac itself is mediated by two different promoter portions, which are influenced by aldosterone and inhibition of sac and differentially accessed in kidney and endothelial cells. sac has transcriptional trans-acting properties as it interacts with cre sites and potentially influences the expression of genes, which play a role in aldosterone signalling. transcription of sac is regulated via aldosterone and camp. the location of promoter ta is cell type-and stimulation-specific. the role of the sodium-calcium exchanger (ncx ) in cardiac pacemaking herrmann s., stieber j., ludwig a. friedrich-alexander-universität erlangen-nürnberg institut für experimentelle und klinische pharmakologie und toxikologie, fahrstrasse , erlangen, germany the mammalian heart is driven by the sinoatrial node, the primary cardiac pacemaker. the unique feature of sinoatrial node (sn) cells is the ability to generate a spontaneous diastolic depolarization that periodically initiates action potentials which set the heart rhythm. the molecular origin of this cardiac pacemaker activity is still a matter of debate. recent findings point to a coordinated interplay between intracellular ca + -cycling processes and plasma membrane-localized ion channels which determines the origin, periodicity and rate modulation of pacemaker potentials. in this study, we investigated the contribution of the cardiac sodium-calcium exchanger (ncx ) to pacemaking. ncx is a key sarcolemmal protein for the maintenance of calcium homeostasis in the heart. it was speculated that the membrane depolarizing current incx, whose activity is dependent on intracellular ca + -fluctuations, represents a main determinant of the spontaneous diastolic depolarization. we used an inducible and sinoatrial node-specific cre-transgene to delete ncx in the murine pacemaker system. the successful creation of a cardiac pacemaking and conduction system specific ncx knockout (cpncx ko) was demonstrated by transcript quantification as well as immunofluorescence experiments. telemetric ecg recordings of cpncx ko displayed a distinct cardiac phenotype. mutant animals were deeply bradycardic and lost their capability of maintaining a stable heart beat as demonstrated by various ecg abnormalities like sn arrhythmia, sn pauses, av block and ventricular tachycardia. analysis of the spontaneous activity of isolated sn preparations showed a slower and arrhythmic contraction rate of the mutant tissues strips confirming that the bradycardia and arrhythmia induced by deletion of ncx results from a slower and arrhythmic intrinsic pacemaker activity. a battery of experiments using different heart rate lowering as well as increasing drugs revealed an altered heart rate modulation in cpncx ko animals as compared to controls. in conclusion, these initial results establish ncx as a major contributor to cardiac pacemaking. a wide variety of contaminants are ingested through food, among them the procarcinogenic polycyclic aromatic hydrocarbon benzo[a]pyrene (bp) which is resorbed and partially metabolized in the enterocytes of the small intestine. previous in vitro studies revealed that bp phenols are excreted as phase ii metabolites including bp glucuronides and bp sulfates. this export is mediated by the breast cancer resistance protein (bcrp/abcg ). the ultimate carcinogenic phase i bp metabolite anti-bp- , dihydrodiol- , -epoxide (bpde) can be detoxified by glutathione conjugate formation catalyzed by various glutathione s-transferases. in the present study, differentiated human intestinal caco- cells were used as a model for the human small intestine to investigate the detoxification of bpde and the subsequent transport of the stereoisomeric glutathione conjugates in the presence of an inhibitor (acivicin) of the glutathione-cleaving enzyme gamma-glutamyl transpeptidase (ggt) at the surface of the cells. the results indicate that the glutathione conjugates of bpde are formed and excreted mainly to the apical and to a minor extent to the basolateral side of the polarized caco- monolayer. to stimulate the transport rate several inducers known to enhance gene expression of xenobiotic-metabolizing enzymes as well as transport proteins were used (quercetin, oltipraz, butyrate). however, solely oltipraz substantially increased the efflux of bpde glutathione conjugates after inhibition of ggt. inhibition studies revealed that the multidrug resistance-associated proteins (mrps/abccs) are involved in the transport of the bpde glutathione conjugates. stable abcc , abcc and abcc knockdown cell lines were generated allowing to demonstrate that abcc mediates the basolateral, abcc the apical excretion of the bpde glutathione conjugates. in conclusion, the ultimate carcinogen bpde is detoxified via glutathione conjugation and subsequently excreted by caco- cells in both apical and basolateral directions. .this finding is equivalent to a transport into the feces as well as blood system in the in vivo situation. signaling via irag regulates store operated calcium entry (soce) in aortic vsmc hieke b., hüttner j., schlossmann j. university of regensburg department of pharmacology and toxicology, universitätsstr. , regensburg, germany the mechanisms involved in the activation of store operated calcium entry (soce) through depletion of intracellular stores and their regulation are not yet fully understood. we examined the effect of inositoltriphosphate-receptor associated cgmp-kinase substrate (irag) on soce. aortic vascular smooth muscle cells (vsmc) from wild type (wt) and irag-knock-out (ko) mice were loaded with the calcium indicator fura -am and soce was measured as a change in the intracellular calcium concentration. in experiments with vsmc from wt mice soce was attenuated by the application of -br-cgmp. this effect was not observed in vsmc isolated from irag-ko mice. these differences in the strength of the soce-signal were abolished by the replacement of extracellular sodium with n-methyl-d-glucamine. the observed sodium dependence of the soce regulation via irag suggests, that an alternated sodium conductance might be responsible to some extent for the differences detected in wt and irag-ko vsmc. as a change in sodium conductance might result in a changed membrane potential we tried to track these changes with the flipr membrane potential assay kit while executing the soce protocol with and without -br-cgmp. no significant differences in membrane potential could be detected in the various stages of soce. in conclusion, our results indicate that irag exhibits a dual action on calcium regulation as it inhibits not only the intracellular calcium release but also the extracellular calcium influx through soce. induction of the icer promoter in vascular smooth muscle cells hildebrandt i. tokyo metropolitan institute of gerontology, tokyo japan several transcription factor isoforms are encoded by the crem (camp response element modulator) gene. one prominent isoform is the inducible camp early repressor (icer), which acts as a transcriptional repressor on so called camp responsive elements (cres) in its target gene promoters. the icer mrna expression is regulated by an intronic promoter of the crem gene. in vascular smooth muscle cells (vsmcs) crem/icer is involved in the regulation of cell proliferation and apoptosis with physiological consequences in vivo. for instance crem-knockout mice, in which none of the known isoforms can be expressed, exhibit an increased neointima formation after carotid ligation as well as an increased atherosclerotic plaque formation after high fat diet on an apoe background. these observations were associated with an increased proliferation rate in isolated crem deficient vsmcs. on this background we wanted to clarify the specific role of icer isoforms in the vasculature. in first experiments we examined the inducibility of icer in primary vsmcs and smooth muscle cell lines. reporter luciferase assays showed that the activity of the icer promoter is induced at the maximum of fourteen fold after hours of stimulation with forskolin (fsk) in immortalized rat vsmcs ( . ± . ; n= from isolations). in a r rat smooth muscle cells the icer promoter showed a maximum stimulation of . ± . fold after two hours of fsk stimulation (n= from isolations). these pilot experiments showed that the icer promoter is inducible in vsmcs by camp dependent pathways. further experiments have to be carried out to elucidate the role of icer in the vascular system for example by stimulation of primary vsmcs and analysis of icer knockout mice. (supported by the dfg) overexpression of transmembrane channel-like proteins (tmcs) uncouples receptor-mediated calcium mobilisation hill k., urban n., straub i., schaefer m. universität leipzig -universitätsmedizin rudolf boehm-institut für pharmakologie und toxikologie, härtelstr. - , leipzig, germany the family of transmembrane channel-like proteins (tmcs) consist of members (tmc -tmc ) all tmc genes are predicted to encode transmembrane proteins with at least six membrane-spanning helices. mutations of tmc cause deafness in human and mice whereas tmc and tmc (also referred to as ever and ) are linked to epidermodysplasia verruciformis (ev), a skin disorder, which is characterised by an enhanced susceptibility towards cutanous infections by human papillomaviruses. the cell biological and physiological functions of tmc proteins still remain elusive. we have overexpressed tmc and tmc in hek cells to get insights into their physiological function. all tmcs were located within the endoplasmic reticulum (er) after overexpression of yfp or cfp-tagged constructs. ratiometric calcium imaging revealed that after overexpression of tmc or tmc , stimulation of gq-coupled receptors with carbachol and atp resulted in a greatly reduced amount of calcium release from the er. moreover, challenging tmc -or tmc -expressing cells with the serca pump inhibitor thapsigargin was also not followed by a release of er-based calcium within the cell. to test whether the lack of calcium release was caused by a reduced calcium content within the er, we investigated calcium dynamics within the er using an er-targeted fret-based calcium indicator (d er cameleon). the experiments revealed that the amount of calcium within the er was reduced upon overexpression of tmc or tmc . recently, it has been reported that tmc and tmc might influence intracellular zinc distribution in human keratinocytes. we could confirm the presence of tmc and tmc mrna in a human keratinocytes cell line (hacat). upon overexpression of tmc , hacat cells revealed the same phenotype as described above for the hek cells with an uncoupling of the receptor-mediated calcium mobilisation due to a depletion of the er calcium store. the mechanism by which overexpression of tmc proteins causes a reduced calcium concentration within the er remains unclear. considering that the presumed topology of the tmc proteins distantly resembles those of other ion channel superfamilies such as anoctamins, one might speculate that a conductance through the tmc protein itself leads to a calcium leak from the er. terahertz radiation is defined as radiation between . thz and thz. a number of applications are currently being developed using radiation in this frequency range. these applications will lead to exposure of the general public, making it very important to study potential effects on biological systems. historically, only a few studies on effects caused by terahertz radiation have been conducted because of the lack of suitable generators and detectors. during the last decade, a number of studies on effects caused by radiation with frequencies around ghz have been published. the present study investigated the genotoxic potential of terahertz radiation at three different frequencies, . thz, . thz and . thz. two skin cell types were used, primary human dermal fibroblasts (hdf) and a keratinocyte cell line (hacat). the cells were irradiated applying different exposure times and different power intensities. two genotoxicity tests were applied: the comet assay quantifies dna strand breaks as well as alkali-labile sites whereas the micronucleus test quantifies chromosomal damage. all experiments were performed and evaluated under blinded conditions as three independent replicate experiments. positive (mms-treated) and negative (untreated, sham-exposed) controls were included. in the comet assay no dna damage was observed as a consequence of the exposure under all experimental conditions. the same was true for the chromosomal damage investigated with the micronucleus test. the latter finding was particularly interesting for the experiments at . thz, because this type of radiation had been reported to cause mitotic disturbances. therefore these experiments were extended, applying higher power intensities and longer exposure periods. also with these modifications, no genomic damage was observed in the form of micronucleus formation. all in all, terahertz radiation did not induce genomic damage under the applied experimental conditions. this result is in line with published findings on genotoxicity of low-frequency terahertz radiation around . thz. the question, why the reported mitotic disturbances do not lead to manifest genomic damage remains open and requires further research. introduction: tea flavonoids derived from camomile and green tea such as apigenin and epigallocatechin gallate (egcg) can inhibit intestinal neoplasia. recurrences of adenomas and cancers were reduced in patients with resected colorectal cancer by treatment with tea bioflavonoids after tumor operation [ ] . to clarify the biomolecular pathway for suppression of neoplasia we investigated the anti-inflammatory effect of a nutritional supplement flavo natin® (fn) which had been used in the clinical study on tertiary tumor prevention and of egcg in a colon tumor cell line. the aim of our study was to investigate if tea flavonoids are capable to suppress the inflammatory markers produced by tumor cells after cytokine stimulation. method: we studied the cytotoxicity of fn in the colon cancer cell line t- by resazurin fluorescence and compared it with the placebo supplement. additionally, the t- cells were incubated with fn, egcg or placebo and stimulated with tnf-alpha, if-gamma and il- -beta. after the cytokine stimulation the mrna expression of ip- , il- and tnf-alpha was measured by quantitative real-time pcr (qrt-pcr). results: stimulation of t- cells increased the expression of ip- (gamma-interferon inducible protein ), tnf-alpha and il- . by preincubation with fn at µm the mrna expression of ip- was strongly reduced (log -ratio - ). the tnf-alpha mrna was also but less decreased by fn. egcg displayed an inhibition pattern similar to fn. placebo did not influence the mrna expression of the chemokines and tnf-alpha. discussion & conclusion: clinically useful dietary tea bioflavonoids inhibit the expression of inflammatory genes in a colon cancer cell line. down-regulation of inflammatory gene products could be achieved in vivo by botanicals without clinically relevant side effects. [ ] h. hoensch, b. groh, l. edler, w. kirch ( ) . prospective cohort comparison of flavonoid treatment in patients with resected colorectal cancer to prevent recurrence. world j gastroenterol, , - . the cxcr c-terminal domain mediates efficient cxcl uptake and degradation hoffmann f., müller w., schütz d., schulz s., stumm r. universitätsklinikum jena institut für pharmakologie und toxikologie, drackendorfer str. , jena, germany cxcl -signaling mediated by the g protein-coupled cxcr receptor plays a key role during embryonic development and disease states including cancer and inflammation. the second cxcl -receptor cxcr modulates cxcl /cxcr -signaling by acting as a cxcl -scavenger. given the distinct functions of cxcr and cxcr , we hypothesized that trafficking and receptor stability are differently regulated by the distinct c-terminal domains. here, we examined epitope-tagged wild type and c-terminal mutant receptors expressed in human embryonic kidney cells (hek ) with respect to trafficking, stability, i-cxcl radioligand degradation, and g protein-coupling. we found that the c-terminal residues of cxcr were sufficient for cxcr to undergo rapid spontaneous internalization. replacement of the cxcr c-terminal domain with that of cxcr (cxcr - tail mutant) abolished spontaneous internalization but permitted ligand-induced internalization in conjunction with c-terminal phosphorylation. conversely, replacement of the cxcr c-terminal domain by that of cxcr caused ligand-independent internalization of cxcr . receptor-mediated i-cxcl -uptake, release of i-cxcl -degradation products, and degradation of the receptor protein itself were accelerated with receptors bearing the cxcr c-terminus. while the cxcr c-terminus was sufficient to abolish g protein coupling in the cxcr - tail mutant, replacement of the cxcr c-terminus, cxcr second intracellular loop or both domains with the corresponding cxcr domain did not generate a g protein-coupled cxcr chimera. taken together, we provide evidence that the cxcr c-terminal domain influences the ligand-uptake/degradation rate, g protein-coupling, and stability of the receptor. this suggests that heterologous regulatory pathways targeting the cxcr -c-terminal domain may effectively control cxcr functions. soluble guanylyl cyclase is a key mediator of brown adipocyte differentiation hoffmann l. s. brown adipose tissue (bat) uses energy to produce heat by inducible thermogenesis. recent studies show that active bat is present in adults and involved in human energy balance, suggesting that the energy consuming property of bat might be exploited to fight obesity and related diseases. the no/cgmp signaling pathway is a key player in diverse physiological processes. recently, we have shown in bat that cgmp signaling is connected with insulin signaling and abrogation of cgmp signaling leads to impaired bat differentiation and function (haas, b. et al., sci signal, ). here we investigated the role of the cgmp generating enzyme soluble guanylate cyclase (sgc) in bat differentiation in vitro. mesenchymal stem cells isolated from bat of newborn sgcβ -/mice and wt littermates were differentiated in vitro into brown adipocytes in the presence or absence of cgmp. abundance of sgc isoforms was determined by qrt-pcr and western blotting. bat differentiation was assessed by redo staining of accumulated intracellular lipids, measurement of triglyceride (tg) content, determination of expression of bat marker proteins pparγ, c/ebpα, ap and bat marker genes ucp , pgc α, cidea. the α and β isoforms of sgc were highly expressed in bat whereas α sgc could not be detected. redo staining of wt brown adipocytes showed basal differentiation which was increased upon addition of -pcpt-cgmp. in contrast, staining was lower in sgcβ -/cells compared to wt under control conditions and increased in the presence of cgmp. tg measurement showed that sgcβ -/brown adipocytes contain approximately % less lipids than wt cells under basal conditions. addition of cgmp doubled tg content in both genotypes. similar results were observed for marker protein expression. deletion of sgc resulted in - % decrease in c/ebpα, pparγ and ap expression compared to wt. again, cgmp roughly doubled protein expression in sgcβ -/and wt cells compared to control. under basal conditions, bat marker gene expression was decreased by approximately % in sgcβ -/cells compared to wt cells. this decrease was prevented by addition of cgmp. these results show that sgc deletion leads to dysfunctional bat differentiation and emphasize the central role of cgmp signaling in bat differentiation. further investigation of sgc/cgmp signaling in bat might reveal new drugable targets bringing bat-dependent pharmacological therapy to treat obesity and related disease into closer reach. comparative inhalation toxicity of carbon-nanomaterials (multi-wall carbon nanotubes, graphene and carbon black) hofmann t. carbon black is a spherical carbon anomaterial whereas multi-wall carbon nanotubes (mwcnt) are cylindrical and graphene is a laminar allotrope of carbon. processing and handling as well as abrasion processes can set free inhalable cnt particles. results of rodent studies collectively show that regardless of the process by which cnts were synthesized and the types and amounts of metals they contained, cnts were capable of producing inflammation, epithelioid granulomas, fibrosis, biochemical and or toxicological changes in the lungs (lam et al. , muller et al. , ma-hock , pauluhn . graphene possess similar physical properties as cnt but may different toxicological property. we performed short-term inhalation studies in rats to compare the toxic potency of four different cnt, two graphenes and one carbon black. the materials are characterized thoroughly according to the oecd list. the four mwcnt caused morphological changes as descriped above. several biochemical and cytological parameters in the broncho-alveolar lavage fluid were strongly increased consistent with the histological findings. two mwcnt exhibited a higher toxic potency than two other mwcnts and findings caused by one graphene typ were even less severe. the graphene with lower surface area as well as low surface area carbon black did not cause any adverse effects up to mg/m . the short-term inhalation studies were able to descriminate different toxic potencies of carbon-based nanomaterials and is hence used for the selection of less toxic materials for further product development as well as to define and prioritize higher-tier toxicological testing of nanomaterials. synthesis and analytical assessment of possible dna adducts formed after activation of the tobacco alkaloid myosmine högg c., zwickenpflug w., gudermann t. walther-straub-institut abt.: toxikologie, nußbaumstraße , münchen, germany myosmine represents one of the minor tobacco alkaloids and its effective uptake from smokeless tobacco or tobacco smoke, as well as by consumption of food is not understood in detail. myosmine can be activated by peroxidation and n-nitrosation yielding -hydroxy- -( -pyridyl)- -butanone (hpb) which is well known as reactive intermediate during activation of a variety of tobacco specific n-nitrosamines (tsna). therefore, myosmine may be a potential candidate for possible mutagenic or carcinogenic risk to human health. furthermore, myosmine n-nitrosation yields the tobacco specific nitrosamine n-nitrosonornicotine (nnn), which is classified as carcinogenic to humans. considerable efforts have been undertaken, especially in organic synthesis, to verify and elucidate the significance of the hpb precursor the pyridyloxobutyl (pob) intermediate and its dna-adducts, which were analysed only in animal experiments till now. the formation of -pyridylmethanol was observed under myosmine peroxidation and identified as a metabolite in rat urine after application of myosmine to rats. the formation of the -pyridylmethanol intermediate, might provide for the reactive electrophilic picolinium ion which could interact with dna. these possible adducts might be of special interest to elucidate the role of myosmine concerning its uptake by smokers and passive-smokers in contrast to non smokers ingesting the substance by consumption of food. dna adducts may help to obtain more information about possible risk assessment of myosmine and to differentiate between the activation from the other nicotinoids and tsna. the specific dna adducts, -methyl- , -bispyridin- -ylmethyl- h-pyrimidin- , -dion and -( ''-picolyl)thymidine have been synthesised as reference substances. the former was prepared by addition of diisopropylazodicarboxylate (diad) to a mixture of -pyridylmethanol, thymine and triphenylphosphine. the reaction product was identified using nmr ( h, c, cosy, hmqc, hmbc). for synthesis of -( ''-picolyl)thymidine the hydroxyl groups of thymidine were initially acetylated using acetic acid anhydride and dimethylaminopyridine (dmap). the existence of this thymidine adduct was confirmed using nmr. this adduct was labelled with -([ , ,-dichlorotriazin- -yl]amino)fluorescin (dtaf) to enhance the sensitivity using hplc-fluorescence chromatography and used as reference substances for analysis of dna samples from biological tissue. supported by dfg grant (ty / - ). cancer and cardiovascular diseases such as atherosclerosis are the most important causes of death in western societies. common to both diseases is a deregulation of cell death, with significant contribution of inflammatory processes. enhanced oxidative stress plays a dominant role in such events as it forms a vicious cycle with inflammation and controls multiple forms of cell demise. therefore, anti-oxidative enzyme systems gained considerable interest since control of reactive oxygen species (ros) has the capacity to regulate cell death in either direction. the human enzyme family of paraoxonases consists of three members, known as pon , pon and pon . while pon is found predominantly in the circulation, pon and pon are intracellular enzymes with established anti-oxidative functions. it has been shown that both pon and pon are protective against atherosclerosis. underlying mechanisms of their protective and antioxidative functions however remained elusive. here we demonstrate that both enzymes locate to the endoplasmic reticulum (er) and mitochondria where they fulfill vital functions in the control of ros generation. in particular, pon and pon were shown to interact with coenzyme q which diminishes mitochondrial ros formation. as a consequence, these enzymes reduce execution of mitochondrial apoptosis, such as cardiolipin peroxidation, cytochrome c release and caspase activation. moreover, pon and pon reduced er stress-triggered cell death, i.e. by diminishing jnk signaling and chop expression. while these results elucidate their protective role in cardiovascular diseases, it also establishes a relevant function in survival of tumor cells. in accordance, we demonstrate that both enzymes are frequently found overexpressed in various tumors. in cancer cell culture studies, overexpression of both enzymes granted considerable resistance against chemotherapeutics. in turn, knock-down of pon caused spontaneous apoptosis of several cancer cell lines. finally, our analyses also revealed that pon -knockout mice show severe alterations of the hematopoetic stem cell compartment, which implies a significant role in leukemias where these enzymes are frequently found overexpressed. together, our results propose pon and pon as new putative anti-tumor candidates and demonstrate the efficacy of interventions targeting cellular redox-balance. steigerwald arzneimittelwerk gmbh wissenschaftliche abteilung, havelstr. , darmstadt, germany stw (iberogast ® ), a multi-component herbal drug, is successfully used in the therapy of functional dyspepsia and irritable bowel syndrome (ibs). previous studies revealed effects of stw on disturbed motility and inflammatory processes. although the antiinflammatory properties of stw are well examined, the contribution each of the individual herbal constituents to the anti-inflammatory effect remains unclear. therefore, we studied the effects of stw and its components on inflammation-induced cell death and on the release of the pro-inflammatory cytokine tnf-α. the aim of these investigations was to analyse additive or synergistic effects of the components. the experiments were carried out on caco- cells after stimulation with lps ( ng/ml) for hours. cytotoxicity was evaluated using a commercially available ldh (lactate dehydrogenase)-assay. furthermore, the release of tnf-α after lps ( ng/ml) stimulation of differentiated thp- cells was measured using a commercially available elisa. stw ( . - . µg/ml) reduced lps ( ng/ml)-induced cell death in a concentration-dependent manner with a maximum inhibition of . %. the herbal components in equivalent concentrations contributed to the inhibitory effect of stw to different extents. the maximum inhibition differed in a wide range between the components. stw ( . µg/ml) reduced significantly the release of tnf-α by % in lps ( ng/ml)-stimulated differentiated thp- cells while having no effect in untreated cells. in concentrations equivalent to stw caraway, milk thistle, lemon balm and greater celandine had no effect on the lps-induced increase in tnf-α release. bitter candytuft, peppermint, chamomile, liquorice and angelica reduced the tnf-α release, though less pronounced as compared to stw , indicating a possible synergistic effect. our results indicate a multi-target effect of stw . the anti-inflammatory effect may be due to a reduction of the cytotoxic effect on intestinal mucosa cells and to an inhibition of the release of the pro-inflammatory cytokine tnf-α from immune cells. the individual herbal components seem to contribute by different mechanisms of action to the overall effect of stw . immune cell-induced local steroidogenesis in the lung: implications for asthmatic disease and therapeutic intervention hostettler n. , brunner t. glucocorticoids are steroid hormones with potent anti-inflammatory properties. synthetic glucocorticoids are frequently used for the therapeutic treatment of inflammatory disorders, such as asthma. endogenous glucocorticoids are predominantly produced in the adrenal glands in response to emotional, physical and immunological stress. recent years, however, revealed several alternative sources of these immunoregulatory steroid hormones. thus, we have found that the intestinal epithelium is a rich source of glucocorticoids and intestinal glucocorticoids contribute to the maintenance of local immune homeostasis ( ) . as the intestinal and the pulmonary epithelium have much in common, i.e. barrier functions and transport of nutrients, resp. gases, we wondered whether the lung mucosa is also capable of synthesizing immunoregulatory glucocorticoids in response to immune cell activation. the murine lung was found to expresses all enzymes required for the synthesis of corticosterone from cholesterol. while most enzymes where expressed in a constitutive manner, cyp a , encoding p ssc, was strongly induced in response to immunological tress. treatment of mice with t cell-activating anti-cd antibody of macrophage-activating lipopolysaccharides induced strong local glucocorticoid synthesis, which was effectively blocked by the corticosterone synthesis inhibitor metyrapone, indicating that glucocorticoids measured were produced bona fide in the lung tissue. surprisingly, allergen-induced allergic airway inflammation failed to trigger local glucocorticoid synthesis despite the massive infiltration of neutrophils, eosinophils and t cells. in contrast to that in the intestinal epithelium local glucocorticoid synthesis in the lung was found to be dependent on the presence of adrenal glands as adrenalectomy abolished pulmonary steroidogenesis. in line with the notion that the lung metabolizes steroid precursors we found that ex vivo cultured lung tissue metabolized h- during the last decade small rna molecules has been identified as important regulators for gene expression. these micrornas (mirna) are single-stranded transcripts, which are expressed in many cell types, where they modulate rna stability and translation and, therefore, controlling various cellular mechanism and tissue development. against this background, in the present study the mirna expression and potential target genes were studied in the human placenta as a tissue demonstrating various developmental changes in a limited period of time. taqman®array microrna cards for profiling of mirnas in placentas of different gestation times revealed a significant expression of mirnas by comparing placentas of early gestation (< .week), preterm (< .week) and term (> .week). when comparing the analyzed groups, of these mirnas expose a continuous up-(including mir- a, mir- ) or downregulation (including mir- , mir- a). while comparing placentas of early gestation to term placentas, % (e.g. mir- , mir- ) were up-and % (e.g. mir- , mir- ) were downregulated. by focusing on the latter group with early preterm placentas the ratio is opposed. here, % of mirnas showed higher (e.g. mir- ) and % lower expression (e.g. mir- , mir- ). with emphasize on these mirnas, a computational prediction algorithm using the mirò database predicted a potential interaction between corticotropin-releasing hormone (crh) and the mirnas mir- and mir- . specific expression analyses validate an inverse expression between mirnas and target with a reduced expression of mir- ( %) and mir- ( %) in term placentas, while crh is upregulated fold. this interaction was verified on functional level by reporter gene assay. a significantly suppressed luciferase activity of the reporter plasmid containing the ´-utr sequence complementary to crh was exhibit for the predicted mir- binding site. here, the mirna-mrna-interaction reduces the luciferase activity by ~ %, whereas the decreased luciferase activity for the predicted mir- binding site is not significant. the results demonstrate a gestation dependent expression of placental mirnas, which may help to explain gestational changes in gene expression and highlight the potential of mirnas as biomarker for pregnancy-related pathologies. since homo sapiens era wound treatment by early civilizations was based on the use of local flora. scilla indica knuth liliaceae (s.i) is a perennial herb with a pear shaped, tunicated bulb, bearing fibrous roots, white flowers and leaves on the stem resembling u. maritima. it is used as cardiac tonic, against inflammation, ulcers and sinus diseases. traditionally the powdered bulb is used topically for warts treatment, while roasted and crushed bulbs are applied to corns of the feet soles. the plant contains steroid glycosides (bufadienolides - %), glucoscillarene a, proscillaridine a, scillarene a, scillcyanoside ,scilliglaucoside ,mucilage and alkyresorcinol derivatives exerting skin healing properties similar to wheat bran products. the study is focused on the investigation of the restoration quality of a skin dorsal incision wound in wistar rats in three groups, control (a ) and experimental (a ,a ) of rats weighing - g local application of dichloromethane extract of s.i in vehicle olive oil preparations of and mg were used. animals were anaesthetized ( ether pro narcosi ), trauma cm long and mm deep was performed by lancet on depilated dorsal area skin until the muscular aponeurosis and were treated for days with λ of each preparation. group a showed increased remodelling of trauma area (limited width). granulomatous tissue was more pronounced in length, width and surface in group a . the macroscopic ulcus dimensions were better in group a while the microscopic view were similar in a and a and better in comparison to control a . a tendency to trauma remodelling was observed, without statistical significance (t = . ) in recent years, it has been suggested that nanoparticles generated from combustion processes (e.g. diesel engine exhaust particles), may contribute to the pathogenesis of neurodegenerative diseases such as alzheimer's disease (ad). the aim of our current study was to investigate the effects of subchronic exposure to diesel engine exhaust (dee) in the xfad mouse model, which is characterised by progressive behavioural deficits as well as amyloid plaque formation and neuron loss. ten weeks old female xfad mice and their nontransgenic littermates were exposed by whole body inhalation to diluted dee (~ mg particles/m ) or clean air (controls) for or weeks ( days/week and hour/day). subsequently, all animals were subjected to a series of behavioural tests. at ten days post-exposure, mice were sacrificed to investigate lungs and brain tissues for pathological and biochemical and molecular-biological changes. in line with the expectations, the xfad mice displayed typically age-dependent behavioural deficits and amyloid plaque formation in cortex and hippocampus. a significant dee exposure-related effect was observed for the string suspension test, representing a measure of motor coordination/grip strength. dee exposure was also associated with mrna expression changes of markers of inflammation and oxidative stress in specific brain regions, including the olfactory bulb. histopathology of plaqueload in cortex and hippocampus (from a limited number of animals investigated so far) did not reveal clear evidence for increased plaque formation due to the dee exposures. further research is needed to evaluate the effects of long term exposure to nanoparticles in the central nervous system. this work is supported by funds from the research committee of the medical faculty of the university of düsseldorf ( - ), the dfg graduate school grk and the rivm -centre for environmental health, bilthoven, netherlands. mono-glucosylation of (h/k/n)ras by clostridium sordellii lethal toxin (tcsl) blocks critical survival pathways, including the pi k/akt, the ralgef/ral, or the raf/erk, and results in apoptosis. in this study, ras glucosylation is presented to result in expression of the cell death-regulating small gtpase rhob based on transcriptional activitation. rhob expression depends on k-ras inhibition, as sirna-mediated knock-down of specifically k-ras (neither h-ras nor n-ras) provokes rhob expression. rhob expression further depends on inhibition of pi k/akt, as activation of pi k/akt using a pi k activator prevents rhob expression downstream of inactivated ras. newly synthesized rhob is gtp-loaded and rapidly degraded in a proteasome-and a caspase-dependent manner, providing first evidence for caspase-dependent degradation of a rho family protein. although often characterised as a pro-apoptotic protein, rhob suppresses caspase- activation in tcsl-treated fibroblasts. conclusions: . rapid and efficacious ras inactivation by tcsl turns out to be particularly useful in the characterisation of ras inactivation-induced rhob expression as an immediate-early gene response. . the finding on the cytoprotective activity of rhob in tcsl-treated cells re-enforces the concept that rhob exhibits cytoprotective rather than pro-apoptotic activity on cellular background of inactive ras. the activity of the rhob promoter is suppressed by rhoa (through a not yet identified pathway or ras (through the pi k/akt pathway. ras glucosylation by tcsl results in de-suppression of the rhob promoter and rhob expression. the calcium-binding protein annexin a (anxa ) is involved in diverse cellular processes including e.g. vesicular transport, ion channel regulation and transcriptional regulation. upon ca + binding anxa undergoes conformational changes, which lead to oligomerization of anxa to homotrimers and cause an increased affinity for membrane phospholipids, which in turn provokes the translocation from the cytosol and the nucleoplasm to plasma and nuclear membranes. in order to examine the effect of anxa on cre-and creb-(camp response element-binding protein) dependent transcription, a series of transient transfections using a luciferase reporter gene driven by the creb target promoter of the inducible camp early repressor (icer) was carried out. when the luciferase reporter construct was co-transfected with anxa , there was significant reduction of basal (dmso) luciferase activity ( the implantation of drug eluting stents (des) after coronary artery intervention was an important step treating coronary artery disease. indeed, cytotoxic compounds like sirolimus used on des are responsible for reduced in-stent restenosis in comparison with bare metal stents. pimecrolimus, a potent anti-inflammatory drug has also been investigated for its efficacy on des. preclinical studies in pigs revealed promising antiproliferative effects of pimecrolimus on neointima formation. however, in humans, pimecrolimus coated stents exerted adverse effects. we hypothesize that compared to the highly active sirolimus, pimecrolimus may influence additional cellular processes leading to the worse outcome. in order to identify those processes we conducted in vitro studies in human coronary artery endothelial cells (hcaec) and smooth muscle cells (hcasmc). brdu in vitro assays of hcaec treated with pimecrolimus examined an ic value of . µm [ . to . ] which is much higher than ic values of sirolimus described in literature [ . nm to nm]. genechip array analysis comparing gene expression in pimecrolimus and sirolimus treated hcaec and hcasmc showed significant induction of several genes involved in interferon signaling. in detail, the expression of ifnβ activated genes like irf and ifitm was up regulated in cells treated with pimecrolimus while no or oppositional effects were observed with sirolimus. gene regulatory effects were validated by real time pcr. incubation with ifnβ itself showed similar effects in up regulation of genes involved in interferon signaling. furthermore, we were able to demonstrate a significant increase of ifnβ secretion in hcasmc and hcaec treated with pimecrolimus. however, comparison of ifnβ and pimecrolimus on proliferation of hcaec and hcasmc revealed different cellular responses. while ifnβ significantly decreased hcasmc and increased hcaec proliferation, treatment with pimecrolimus lead to anti-proliferative effects on both cell types. in conclusion, pimecrolimus activates pathways involved in interferon signaling but exerts different pharmacological effects, compared to the endogenous compound suggesting that infβ secretion is not the major factor contributing to the difference in pimecrolimus function. identification of novel phospho acceptor sites of the mu opioid receptor regulating receptor internalization illing s., just s., doll c., schulz s. uniklinikum der fsu jena pharmakologie und toxikologie, drackendorfer straße , jena, germany the opioid alkaloid morphine is among the most potent clinically used analgesics. however, the clinical utility of morphine to treat chronic pain is limited by its rapid development of tolerance and dependence. the stimulation of the mu opioid receptor (mor) with damgo or morphine results in different patterns of receptor phosphorylation and trafficking. so far, the three major phosphorylation sites of the mu opioid receptor namely serine (s ) threonine (t ) and serine (s ) have been identified using phosphosite-specific antibodies. however, mutations of these three residues to alanine (s a/t a/s a) did not prevent agonist-dependent internalization. in the present study, we have constructed a series of phosphorylationdeficient mutants showing that mutation of at least six residues (s a/t a/s a/t a/t a/t a) was required to completely block agonistdriven endocytosis. consequently, we generated phosphosite-specific antibodies to t and t which enabled us to provide direct evidence for agonist-dependent phosphorylation of these sites. our analysis of time-and dose-dependent phosphorylation of mor revealed that s was the primary phosphorylation site, whereas t , t and t were secondary sites. moreover, the partial agonist morphine induced only the phosphorylation of s but not of t , t of t . our results show, that mor phosphorylation occurs in a hierarchical and agonist-selective manner directly regulating receptor sequestration. assessment of mda effects from toxicity studies with regard to endocrine modulation jäger r. methylene dianiline (mda; cas no. - - ) is on the usepa list for endocrine disruption screen testing. in a yeast androgen screening (yas) assay (in vitro assay on receptor binding or blocking) mda revealed a significant anti-androgenic activity at a concentration of . mm. to assess the biological relevance of this observation under in vivo conditions the existing data from animal toxicity studies with mda were reviewed for indications of possible endocrine modulating (em) effects (weight-of-evidence approach). in addition, literature was searched for relevant studies on mda and structural analogues using the american chemical society scifinder client-server software. structural analogues indicated several drivers for em activity, notably the diphenolic ring structure. however, in vitro receptor binding assays showed mda had no androgenic or anti-estrogenic activitiy. one report described a weak estrogenic binding at . mm mda, while another described no effect at similar concentrations and a rat estrogen receptor binding study found no effect at . mm. overall it is concluded that mda does not bind to the estrogen receptor. endocrine related effects of mda were investigated in several species and dose routes in unvalidated research-type protocols. guideline subchronic and chronic toxicity studies with rodents revealed neither adverse organ weight changes nor histopathological alterations of sex organs. the main systemic effects from the oral -week studies were body weight reduction and histopathological changes of the thyroid and bile duct (lowest loael: . mg/kg bw). mda was carcinogenic to rats and mice (thyroid and liver) after oral administration over years. non-neoplastic effects in the thyroid (rats and mice) were observed (lowest loael for systemic toxicity: mg/kg bw). mda inhibits iodide oxidation which with concomitant decreased thyroid hormone formation is known to induce thyroid tumors. in summary, in vitro screening tests revealed no consistent endocrine related effects of mda. in vivo, there was an effect on the thyroid gland, possibly by enzyme inhibition. there were no histopathological changes of gonads and accessory sex organs and no evidence of sex hormone related em. the evidence from the full dataset on mda does not indicate androgenic or estrogenic effects. overall, based on a weight of evidence assessment there are insufficient alerts for em activity to suggest further testing should be done. the gtpase arfrp controls the assembly of apoa-i to and the lipidation of chylomicrons in the golgi of intestinal epithelium jaschke a. background: the uptake and processing of dietary lipid by the small intestine is a multi-step process that involves luminal digestion, cellular uptake of fatty acids by the mucosa, and subsequent synthesis and export of chylomicrons. the gtpase adpribosylation factor related protein (arfrp ) is a member of the arf-family and controls the arf-like (arl )-mediated golgi recruitment of grip domain proteins which in turn bind several rab-gtpases. the aim of the study was to define the role of arfrp in intestinal nutrient absorption. methods: for the generation of intestine-specific null mutants arfrp flox/flox mice were crossed with mice expressing the cre recombinase under the control of the intestinespecific villin promoter (vil-cre) and arfrp expression was suppressed by sirna in caco -cells. the phenotype in respect to lipid absorption and chylomicron production in the intestinal epithelium and in caco -cells, respectively, was analyzed. results: arfrp vil-/mice were viable but showed an early postnatal growth retardation (mean body weight of arfrp vil-/was . ± % lower than that of control mice at the age of days) arfrp vil-/mice displayed reduced triglycerides, free fatty acids and glucose plasma levels indicating that the growth retardation is the result of a malabsorption. uptake of glucose and amino acids were unaffected by the deletion of arfrp . in contrast, lipid uptake as elucidated by oral fat tolerance tests was impaired in arfrp vil-/mice. arfrp vil-/enterocytes as well as arfrp mrna depleted caco- cells absorbed fatty acids normally but secreted chylomicrons with a markedly reduced triglyceride content. in addition, while the release of apolipoprotein a-i (apoa-i) was dramatically decreased apoa-i accumulated in the arfrp vil-/epithelium and was predominantly colocalized with rab . our results demonstrate that the growth retardation of arfrp vil-/mice is a consequence of impaired intestinal fatty acid absorption. we suggest that arfrp is required for the assembly of aopa-i to the chylomicrons and for the further lipidation of chylomicrons in the golgi of intestinal epithelial cells. this finally leads to an secretion of chylomicrons with a markedly reduced triglyceride content. rhoa influences adhesion and spreading of cardiac fibroblasts via complex regulation of cytoskeletal proteins jatho a. the monomeric gtpase rhoa is thought to be involved in the pathology of heart diseases, however, its role in cardiac cells is not well defined. therefore we intended to analyze the effect of rhoa in cardiac fibroblasts by using a lentivirus-based knockdown approach. by doing so, we could show that the knockdown of this gtpase by about % resulted in a massive change in cell morphology, but displayed no effect on cell viability. the appearance of the cells in the d-culture changed from a fibroblast-typical stretched morphology with intracellular stress fibers to a more epithelial-like cell morphology. by morphometrical analyses we demonstrate that fibroblasts with reduced rhoa expression display an increase in cell surface by . -fold and in perimeter by . fold. moreover, the depletion of rhoa significantly influences the adhesion velocity, as within the first hour after cell detachment about % of the rhoa knockdown cells reattach, but only % of the respective control cells. based on these findings, we investigated the distribution and composition of different cytoskeletal proteins by immunofluorescence stainings and immunoblot analysis. we found, that the amount of b-actin is not reduced in rhoa knockdown cells, however, the distribution is markedly changed. in these cells internal star-shape bundles of actin could be found instead of the commonly appearing stress fibers in control cells. in contrast, the cortical actin fibers, mainly consisting of g-actin, were not affected. in addition, smooth muscle-actin, which is characteristic for myofibroblasts, was clearly reduced in rhoa knockdown cells compared to control cells by %. this reduction might be responsible for the more relaxed cell shape. in summary, the knockdown of rhoa influences cell adhesion and the morphological characteristics of cardiac fibroblasts, without obviously affecting cell proliferation and viability. using site-specific fluorescence labeling to study uptake of pasteurella multocida toxin into eucaryotic cells jehle d., aktories k., orth j. institut für experimentelle und klinische pharmakologie und toxikologie abteilung , albertstraße , freiburg, germany pasteurella multocida is an opportunistic pathogenic bacterium living in the nasal pharyngeal space of animals. p. multocida is of particular importance in the livestock management of pigs. under special conditions infection of pigs with p. multocida leads to an atrophic rhinitis, which is characterized by the atrophy of nasal turbinate bones accompanied by a shortening and twisting of the snout. the causative agent of the atrophic rhinitis was found to be the bacterial protein toxin pmt (pasteurella multocida toxin). after entering the cell the -kda toxin activates various signal transduction pathways by stimulating heterotrimeric g proteins of the gαq, gα and gαi family. the underlying mechanism of the activation of heterotrimeric g proteins is a deamidation of an essential glutamine residue leading to a constitutive activation of the g protein. the uptake of pmt into cells is not comprehensively understood. therefore, we utilized a recently described technique, called "sortagging" (popp mw et al. nat chem biol. ; : ) , to specifically couple fluorescence tags to the n-or c-terminus of pmt. the enzyme sortase a (srta) from staphylococcus aureus attaches proteins to the bacterial cell wall. the substrates are recognized by an lpxtg motif. srta cleaves the peptide bond after the threonine and adds a glycine-containing nucleophile. we introduced these motifs into pmt to express srta-recognized toxin and coupled the toxin with fluorescence tags, respectively. fluorescently labeled pmt was used to study the uptake of the toxin into eucaryotic cells by laser scanning microscopy. munich heart alliance, münchen, germany cells within the myocardium communicate by secreted factors and this has been suggested to contribute to cardiac remodeling. to identify novel factors secreted by the myocardium, we have previously reported a genetic yeast screen which led to the identification of protease inhibitor (pi ). here we report the generation of a mouse line where pi can be deleted globally or conditionally using the cre/loxp system. after electroporation of the pi floxneo targeting vector in embryonic stem cells and injection into murine blastocysts we gained a mouse line that carried the targeted modification of the pi allele. global pi deficiency (pi lox/lox ) per se did not lead to a cardiac phenoytpe (hw/bw (mg/g): pi +/+ = . ± . (n = ), pi lox/lox = . ± . (n = ), p > . ; fibrosis (%): pi +/+ = . ± . (n = ), pi lox/lox = . ± . (n = ), p > . ; fractional shortening (%): pi +/+ = . ± . (n = ), pi lox/lox = . ± . (n= ), p > . ). in addition we carried out an immunohistochemical analysis of pi expression using pi -deficient mice as negative controls. pi localized to cardiac fibroblasts, to the epididymis and the trachea. in the failing heart we detected accumulation of pi preferentially in fibrotic areas. we are currently applying cardiac stress models to gain a broader understanding of the function of pi and its potential as a therapeutic target molecule. enantioselective determination of r-and s-hyoscyamine in mammalian plasma and urine samples john atropine (atr) is the racemic mixture of the tropane alkaloids s-and r-hyoscyamine (hyo). s-hyo acts as a competitive acetylcholine antagonist at the muscarinic receptors (eutomer) inducing mydriasis, excitations, hallucinations, coma and ultimately death, whereas r-hyo does not (distomer). atr is used for clinical intervention of poisoning with organophosphorus (op) pesticides or nerve agents. despite well known differences in pharmacological behavior, individual pharmacokinetics of both atr enantiomers have rarely been addressed in the literature [ , , ] . therefore, we initially developed a nonchiral liquid-chromatography-electrospray tandem mass spectrometric method (lc-esi ms/ms) that allows quantification of atr and additional natural and synthetic tropane alkaloids from plasma after simple deproteinization [ ] . to discriminate both atr enantiomers the sample preparation step was expanded by an enzymatic pretreatment. samples were incubated either with diluted human serum (not containing atropinesterase, atre, procedure a) or with diluted rabbit serum (procedure b). rabbit serum contains atre (ec . . . ) which is suitable for stereospecific hydrolysis of shyo into tropine and tropic acid while r-hyo remains unaffected. after sample precipitation, hyoscyamines were quantified by the lc-esi ms/ms method. following procedure a the concentration of total hyo and following procedure b remaining r-hyo were determined. s-hyo was calculated by the difference between these concentrations [ ] . the impact of potential matrix ingredients that may appear in samples from oppoisoned patients under atr therapy were evaluated (oximes, op agents, carbamates) [ ] . the assay was applied to diverse toxicological and pharmacological samples. i) measurement of natural s-hyo in an extract of atropa belladonna leaves as well as in plasma and urine of a female patient who was poisoned after ingestion of such leaves revealed that no biotransformation to r-hyo occurred. ii) analysis of plasma obtained from an op-poisoned female patient under atr therapy revealed faster elimination of shyo when compared to r-hyo [ ] . iii) in contrast, no enantioselective differences were obvious in healthy anaesthetized swine after intravenous injection of atr [ ] . data indicate that the enzymatic enantioselective procedure represents a useful tool to characterize in vivo behavior of r-and s-hyo allowing to reveal individual kinetics. failing hearts are unable to adequately supply the body with blood and oxygen. common therapeutic strategies interfere with cardiac remodelling, reduce cardiac preand afterload or aim at direct improvement of cardiac contractility. cardiac contractility is mainly controlled by β -adrenergic receptors. resensitization of b-adrenergic receptors by inhibition of g-protein coupled receptor kinase (grk ) is discussed as a potential strategy to treat heart failure. we characterized raf kinase inhibitor protein (rkip) as a physiological inhibitor of grk and found rkip to increase contractility of neonatal cardiomyocytes. the present study evaluated the role of rkip in heart failure. we assessed its effects on cardiac function in pressure overload induced heart failure and determined the expression patterns of rkip in failing hearts of humans and mice. transverse aortic contriction (tac) was performed on -week-old c /bl mice to induce heart failure by pressure overload of left ventricles. after three weeks of tac, echocardiography showed distinct signs of decreased cardiac function in wild-type mice. fractional shortening was reduced and left ventricular diameters were increased. histological analyses revealed increased interstitial fibrosis, caspase-and tunelassays indicated myocyte apoptosis. western blot analysis showed significant upregulation of rkip expression in failing hearts compared to non-banded control hearts. interestingly, this upregulation of rkip expression was also detected in failing human hearts and in samples of patients with aortic valve stenosis but not in healthy control samples. to assess the effects of rkip overexpression on heart failure, we analysed heart function and structure of rkip transgenic mice and wild-type mice weeks after tac. while left ventricular hypertrophy was increased to similar extents in wild-type and rkip trangenic mice, rkip mice did neither develop dilatation of the left ventricle nor a decrease in fractional shortening. in contrast to wild-type mice, the expression of the heart failure markers bnp and anp was not upregulated in banded rkip transgenic mice after weeks of tac. taken together, cardiac overexpression of rkip prevented left ventricular dilatation and loss of contractile function in a mouse model of pressure overload induced heart failure. therefore, increased rkip expression may be an interesting target to prevent detrimental effects from increased left ventricular pressure. the main focus of this study was the chromatographic separation. the most frequently prescribed antibiotic drugs clarithromycin, erythromycin, clindamycin, cefuroxim, doxycyclin, amoxicillin, levofloxacin, and ciprofloxacin were selected for the screening method. method: a relatively short operation time and a sufficient separation were reached by column, eluent, and gradient optimization with poplc (phase optimized liquid chromatography). in the first step columns with the five stationary phases c eps , c sh , phenyl , cn , and c were used to determine the retention times of the drugs in an isocratic mode. the stationary phases, the column length and the retention times were fed in the poplc software and the optimal column was calculated. this column contained different stationary phases and was compared with customary columns. results: using the optimal column a sufficient chromatographic separation of the eight antibiotic drugs was reached. that was not possible with the customary columns. with the optimal column the time of measurement was too long. using a mobile phase gradient the measuring time could be reduced. discussion: with lc-ms/ms a complete chromatographic separation of all analytes is not necessary. but when measuring many transitions in a biological matrix two problems should be excluded: ion suppression and a too small number of measurement points per peak. especially when using positive and negative ionization in the ms a good separation is mostly necessary. to determine only the eight antibiotic drugs an optimized column is not necessary, but for a screening method with more than twenty drugs the poplc system is very helpful. we have investigated the uptake mechanism of cdt and in particular the intracellular membrane translocation of cdta. our data indicate that cdt requires acidification of the endosomal lumen for translocation of cdta across endosomal membranes into the cytosol. bafilomycin a , an inhibitor of endosomal acidification protects vero (african green monkey kidney) cells from intoxication with cdt. consistently, translocation of cdta was observed when the acidic conditions of the endosomal lumen were mimicked at the cytoplasmic membrane of intact cells. next, we tested whether host cell factors are involved in membrane translocation of the toxin. radicicol, a specific pharmacological inhibitor of the chaperone heat shock protein hsp as well as cyclosporine a, an inhibitor of cyclophilins delayed the intoxication of cells with cdt but not with toxins a and b [ ] . this result was confirmed by analyzing the adp-ribosylation status of actin from such cells in the presence or absence of the inhibitors. in addition, we excluded that the inhibitors of hsp and cyclophilins have any effect on receptor binding, endocytosis or enzyme activity of cdt. the data strongly suggest that the participation of hsp and cyclophilin is crucial for translocation of cdta into the cytosol. comparable results were obtained for the related binary iota toxin of c. perfringens. in vitro purified immobilized hsp and cyclophilin a specifically bound to the enzyme components of cdt and iota toxins. in conclusion, the results imply a common hsp /cyclophilin a-dependent translocation mechanism for the family of binary actin-adp-ribosylating toxins. our current investigations focus on the participation of fk -binding proteins (fkbps), another group of peptidyl-prolyl cis/trans isomerases in the membrane translocation step of these toxins. [ ] kaiser, e., kroll, c., ernst, k., schwan, c., popoff, m. r., fischer, g., buchner, j., aktories, k. and barth, h. ( ) . complex multicellular in vitro coculture models represent a promising tool regarding e. g. cellular interactions with nanoparticles, since they more closely mimic the cellular composition of the body. therefore, we used our developed coculture model of the alveolar-capillary barrier composed of lung epithelial cells (nci h ) on top and microvascular endothelial cells (iso-has- ) on the bottom side of a filter-membrane to study nanoparticle cytotoxicity and cellular uptake. with a coculture period of about days the cells achieve a more differentiated and polarized state and develop a tight barrier, which can be measured via ter (transepithelial electrical resistance). regarding cellular uptake of fluorescently labeled amorphous silica nanoparticles (asnps, nm) the coculture took up much less asnps than conventional monocultures. besides, we could not verify a specific uptake mechanism (e. g. clathrin-, caveolae-mediated endocytosis) via immunofluorescence staining of the cells. however, we detected asnps incorporated in flotillin- and - labelled vesicles. former studies concerning cytotoxicity (lactate dehydrogenase assay) of amorphous silica nanoparticles (asnps, nm) revealed that our coculture behaved much more robustly compared to conventional monocultures. however, regarding inflammatory responses (e. g. sicam, il- increase) the coculture responded more sensitively than conventional monocultures. in a further development we added a third cell type, the alveolar macrophage (am), to our coculture. since ams embody the front-line of alveolar defense against inhaled pathogens and particles, they play a central role in regulating lung immunity. as model we applied the human acute monocytic leukemia cell line, thp- (prestimulated with nm pma for d) apically to the epithelial monolayer of the coculture. our preliminary studies concerning inflammatory responses of the tripleculture (h /iso-has- with thp- ) revealed a higher sensitivity of the triple-culture compared to the double coculture. the triple-culture responded with an increased il- release upon lps or tnf-a stimulation. in conclusion, this triple-culture model offers a promising prospect to mimic more closely realistic cell interactions with nanoparticles in the distal lung. ethanol is a component of many herbal fluid preparations [ ] , since it is an excellent extraction solvent for the phytochemical components of herbal drugs and contributes to the stability of these medicines. toxicological and pharmacokinetic evaluations [ ] have shown that the small amounts of ethanol applied with therapeutic doses are safe even in children. despite that these medicines have been used safely since many decades, they have occasionally been subject of discussion in the public, triggered by the increasing problem of recreational abuse of alcoholic beverages by children and young persons [ , ] . therefore, there is a growing need of a systematic evaluation of pharmacovigilance data on these medicines. for evaluating the experience gained from the therapeutic use of these medicines, pro-and retrospective studies with herbal medicinal products containing ethanol at doses of to mg per single dose, depending on the age group, have been analyzed, covering patients of - years of age. in these studies, altogether adverse drug effects have been described, none of which was attributable to the ethanol content of the medicines. in a survey of the worldwide use of these and other herbal medicinal products it was shown that during the past few years, more than mio daily doses have been sold, corresponding to more than mio of patients (data obtained from manufacturers; figures available partly from onwards, partly from / onwards). from the packages sold in germany in the years between and , . mio were attributable to self-medication, . mio to prescriptions reimbursed by health insurance (ims, frankfurt). as non prescription medicines are reimbursed in germany only in children, at least the latter part of the prescriptions can be attributed mainly to children. all of these medicines are registered or licensed by regulatory authorities. adverse effects are covered by the pharmacovigilance system, and no adverse effects attributable to the ethanol content have been reported. this set of data supports the conclusion drawn from the experience of a safe use over decades, i.e. that the ethanol content of herbal medicinal products does not give any causes for concern regarding their safety even in children. dedication: this contribution is dedicated to prof. dr. hilke winterhoff, institute for pharmacology and toxicology, university of münster, who died on may . she has initiated this work. prucalopride was introduced as a new selective -ht receptor agonist that is approved for treating obstipation. whereas one could expect -due to the fact that -ht receptors are functionally expressed in the human heart -in clinical trials prucalopride did not show cardiac effects. this is quite surprising because other -ht receptor agonists have been withdrawn from the market just for that reason. in this study we used prucalopride for in vitro studies with atrial preparations from transgenic (tg) mice with cardiac myocyte-specific overexpression of the human -ht a receptor. isolated electrically driven ( hz) left and spontaneous beating right atria of tg mice were compared with those of wild type (wt) littermates. moreover, we used isolated electrically driven ( hz) human right atrial preparations from patients undergoing cardiac surgery. finally gr , a -ht receptor antagonist, was added. prucalopride exhibited a dose dependent positive inotropic effect in left atria and a positive chronotropic effect in right atria of tg mice with a logec of - . and - . , respectively (p< . vs. wt, n= ). in human atrial tissue prucalopride also acts as an agonist, leading to an inotropic effect. all effects could be antagonized by µm gr . we could demonstrate that prucalopride acts as an agonist at the -ht receptor in our transgenic mice model and also in human right atrial preparations. these findings suggest that tachycardia and arrhythmias are possible side effects, which should be carefully looked for. the involvement of psychological factors, especially stress, are known to play an important role in functional gastrointestinal diseases ( , ) probably by affecting the brain-gut axis. based on the good correlation between stress & functional dyspepsia (fd), many animal models for fd have been developed where animals are subjected to various kinds of psychological stress either during the neonatal period or in adulthood. this stress was found to induce gastric motor dysfunction resembling symptoms of fd. two models for stressinduced fd were performed in order to choose the more adequate one for studying sensitivity changes of the fundus to various mediators as well as changes in some relevant hormone levels in the blood for subsequently testing the efficacy of treatment with stw (a component herbal preparation of proven clinical efficacy in fd and in irritable bowel syndrome ( , ) ). in one model, maternal separation ( ) was performed on weanling rats starting from postnatal day for h each day for weeks. rats were then allowed to mature to an adult age. the other model was that of restraint stress (rs) ( , ) . adult animals were restrained for min/ day for week. the animals of both models were eventually sacrificed, the stomach fundus was isolated and its sensitivity in vitro to carbachol, potassium chloride, serotonin and adrenaline was tested. blood samples were taken to assess levels of ghrelin, corticosterone releasing factor (crf) and corticosterone. the sensitivity of fundus strips from restrained rats towards the agents tested, partly representing autonomic responsiveness, was more depressed than those from maternally separated ones. levels of ghrelin, crf and corticosterone were also more elevated in the rs model. that model was therefore chosen to test the efficacy of stw in restoring the deranged parameters. a group of animals received stw orally once daily starting treatment week before exposing them to rs and continuing treatment for a further week during subjection to rs. stw was effective in normalizing the depressed stomach fundus responses exhibited by animals subjected to rs and to normalize to a large extent the deranged blood levels of ghrelin, crf and corticosterone. the findings lend further evidence to the role of the brain-gut axis in fd and gives supportive evidence for the first time for the clinical usefulness of stw in this condition. there is good evidence that oxidative damage to dna leads to down-regulation of transcription of affected genes and epigenetic gene silencing in a mechanism dependent on the -oxoguanine dna glycosylase (ogg ), which generates harmful repair intermediates [ , ] . we have recently shown that the magnitude of inhibition of transcription of an egfp reporter gene by single -oxo- , -dihydro-deoxyguanosine ( -oxodg) varies between the opposing dna strands of the gene [ ] . we now have addressed the question, to which extent the transcription inhibitory potential of -oxodg depends on its position in the gene and on the dna microsequence surrounding the modified nucleobase. to investigate the effect of position, we produced plasmid vectors containing single -oxodg or dg (underlined) in the second position of an agc trinucleotide. measurements of egfp expression in transfected mammalian host cells showed that a single -oxodg caused a strong inhibition of gene transcription. the magnitude of this effect for -oxodg situated in the transcribed dna strand of the gene was the same as in the non-transcribed dna strand. similarly, there was no quantitative difference between the effects of -oxodg present in the ′-versus ′-utr of the gene. the results thus indicate that inhibition of transcription by this base modification does not depend on the position in the gene. further comparisons were done between the effects of -oxodg nucleobases localised in the same dna strand but within different sequence contexts. gene expression analyses in the repair proficient host cells showed that the degree of inhibition of transcription caused by single -oxodg was dependent on the neighbouring nucleotides. among three tested sequence motifs, the minimal effect on the gene transcription was found to correlate with a significantly less efficient base excision by the purified human ogg protein. the results thus support the initiatory role of ogg in the mechanism of transcriptional repression. in addition, the finding of the effect of dna sequence on the base excision activity of ogg suggests that repair rates of single base modifications in genome could also be heterogeneous. allgayer j., kitsera n., epe b., khobta a. johannes gutenberg university of mainz institute of pharmacy and biochemistry, staudingerweg , mainz, germany interference of dna base modifications with gene transcription is an important biological consequence of genotoxic damage [ ] . an efficient method for incorporation of a single -oxo- , -dihydroguanine ( -oxog) at a defined position in the egfp gene in a plasmid vector was recently developed in our lab. the method relies on the availability of adjacent sites for a sequence-specific nicking endonuclease, which allow the insertion of a synthetic oligonucleotide containing -oxog in a chosen position. we further showed that this single lesion inhibits transcription after excision by ogg [ ] . in order to determine to which extend the observed effect depends on the position of the modified base in the gene, we constructed several new plasmid vectors which allow incorporation of the same dna oligonucleotide containing -oxog or g in different positions in different dna-strands. dna sequence cassettes were designed to contain a ′-cattgcttcgctagcacg nucleotide sequence in different orientations, which was flanked by two unidirectional bsrdi recognition sites ( ′-gcaatgnn). adapters containing the restriction sites for directional cloning into the ′-or the ′-utr of the plasmid-borne egfp gene were added. the produced plasmid vectors thus allow the insertion of a single -oxog in the same sequence context into opposing dna strands in the 'utr and in the 'utr. keratinocytes are the major cell type of epidermis and are responsible for the formation of an outer barrier, the statum corneum, to protect an organism against harmful environmental influences. for generation of this barrier, keratinocytes pass through a complex differentiation program that is accompanied by synthesis of lipids, like cholesterol and ceramides. finally, the differentiation of keratinocytes leads to apoptosis. another function of keratinocytes is to sense environmental factors, some of which are decoded by members of the transient receptor potential (trp) ion channel family. trpv , for example, is predominantly expressed in keratinocytes, and decodes different chemical and physical stimuli like the terpenoid-derived ligands camphor and thymol or temperatures above °c. less is known about the influence of cholesterol on trpv signalling. we modified the cholesterol content of hek cells stably transfected with trpv and performed flipr-based calcium measurements. these experiments revealed that cholesterol enrichment robustly potentiates trpv by sensitizing it to lower agonist concentrations. we verified these results with whole-cell patch-clamp measurements. in contrast, trpv , another heat-sensing channel, was not affected by cholesterol modification. since former studies showed a defective formation of epidermal barrier in trpv -/mice, our results imply that a cholesterol-regulated trpv signalling may contribute to the progression of differentiation or initiation of apoptosis of keratinocytes. ischemia-reperfusion injury causes severe problems in the early period after lung transplantation. since transient receptor potential (trp) channels are important regulators of vascular permeability and tone, we investigated the influence of a trpc blocker on pulmonary function after simulated transplantation, using an ex vivo model of isolated perfused and ventilated rabbit lungs. to this end, heart-lung blocks were excised and mounted in an artificial thorax chamber. negative pleural pressure ventilation was initiated, and lungs were perfused with albumin-containing tyrode-solution ( ml min - ). after equilibration in a stable perfusion and ventilation mode for min, lungs were flush-perfused with perfadex ® solution, followed by an ischemic storage for h on ice. subsequently, ventilation and perfusion were re-initiated to simulate a transplantation situation. in the oxygenated group, pulmonary artery po was adjusted to mmhg, in the deoxygenated group, the perfusate inflow was gassed with nitrogen to achieve a pulmonary artery po of mmhg. both transplantation conditions were conducted in the absence or in the presence of the trpc blocker larixol acetate ( µm). hemodynamic and ventilatory parameters were continuously monitored. the weight of deoxygenated lungs steadily increased during h of reperfusion from . ± . g to . ± . g. this weight gain was inhibited by supplementation of the trpc blocker (from . ± . g to . ± . g h after reperfusion). in contrast, oxygenated lungs showed no marked weight gain after reestablishment of perfusion (from . ± . g to . ± . g h after reperfusion), and the trpc blocker had no additional effect (initial . ± . g, h reperfusion . ± . g). we conclude that a trpc -blocking compound to the lung perfusate during simulated transplantation counteracts the endothelial permeability increase and the resulting post transplant weight gain. the results indicate a role for trpc in the regulation of pulmonary vessel permeability and support the concept that perfusion of donor lungs with trpc blockers may prevent edema formation caused by ischemiareperfusion injury shortly after lung transplantation. regulation of human inducible nitric oxide synthase (inos) expression by noncoding rnas (ncrnas) kleinert h., schmitz k., koch k., hahn s., pautz a. universitätsmedizin der johannes gutenberg-universität mainz institut für pharmakologie, obere zahlbacher str. , mainz, germany the transcriptome analyses of human cells showed that additionally to the . protein coding sequences the human dna codes for much more ( . ?) non-coding rnas . beside the ribosomal, and transfer rnas (rrna and trna) involved in the mechanism of translation there are also short (snrna, sorna, mirna) and long noncoding rnas (ncrnas) implicated in regulation of gene expression (splicing, translation, chromatin packaging etc.). matsui et al. described regulation of il- β-induced inos expression by an antisense rna (as- -utr-rna) in rat hepatocytes . a promoter located on the antisense strand ' to the last exon (exon ) of the rat inos gene drives the expression of an as-rna complementary to the '-utr of the rat inos mrna. using different sense primers with homology to the '-utr sequences of the human inos gene for specific rt-pcr reactions (detecting only an as-rna) we were not able to detect such an as- -utr-rna in human cells. in addition, transient transfection analyses using constructs containing a . kb fragment of the '-flanking genomic sequences (as used by matsui et al. in the rat system) of the human inos gene in front of a luciferase reportergene into dld- cells revealed no promoter activity of these sequences. korneev et al. described the expression of a kb anti-inos-ncrna in different brain tumors and showed reciprocal expression to the inos mrna in human embryonic stem cells . analyzing the expression of this anti-inos-ncrna in cytokine-treated dld- cells also showed a basal expression of this as-rna and an enhancement of the expression by cytokine-treatment. downregulation of the anti-inos-ncrna by sirnas reduced whereas overexpression enhanced cm-induced inos expression in human dld- cells. this indicates that this anti-inos-ncrna regulates cytokine-induced inos expression in a positive manner. rna , rna , - rna , ( . using directed evolution to improve functional expression of class b g-protein coupled receptors klenk c., scott d. j., plückthun a. universität zürich institut für biochemie, winterthurerstrasse , zürich, switzerland the class b of g-protein coupled receptors (gpcrs) comprises peptide hormonebinding receptors which regulate important endocrine and neuroendocrine functions of the human body. several of these receptors are implicated in the pathogenesis of severe diseases such as diabetes, osteoporosis, growth disorders and depression, which makes them attractive targets for drug therapy. to develop new compounds targeting these receptors a detailed understanding of the molecular structure is required which has not been succeeded to date. structural studies of proteins by x-ray crystallography or nmr spectroscopy generally require large and homogenous quantities of protein. for gpcrs this prerequisite is difficult to achieve as the vast majority of gpcrs exhibits low endogenous expression and is very unstable in solution. therefore, improved expression conditions are necessary for the efficient characterization of new gpcr structures. here we present a method to optimize class b gpcrs for improved heterologous expression and increased thermostability by means of directed evolution. libraries of class b gpcrs were obtained by random mutagenesis and were expressed in a heterologous expression system in which functional gpcr is targeted to the inner membrane of e. coli. mutants that display increased receptor expression levels and ligand binding were selected by flow cytometry using fluorescently labeled ligands. repetitive cycles of randomization and selection allow to gradually increasing the level of protein expression and stability. with this evolutionary approach key residues within the receptor sequence can be identified rapidly that are responsible for improved biophysical properties without affecting the pharmacological features of the receptor. such gpcr mutants will become a valuable tool on the way to express high quantities of stable receptor protein for subsequent structural studies. pasireotide (som ) is currently under clinical evaluation as a successor compound to octreotide for the treatment of acromegaly, cushing's disease and carcinoid tumors. whereas octreotide acts primarily via the sst somatostatin receptor, pasireotide was designed to exhibit octreotide-like sst activity combined with enhanced binding to other somatostatin receptor subtypes. somatostatin and octreotide stimulate the complete phosphorylation of at least six carboxyl-terminal serine and threonine residues namely s , s , t , t , t and t , which in turn leads to a robust endocytosis of the sst receptor. surprisingly, pasireotide failed to phosphorylate the four threonine residues and induced only a detectable phosphorylation of s and s . somatoprim and ke are recently developed somatostatin analogs capable of binding to four of five somatostatin receptor subtypes. here, we performed an in vitro study comparing the effects of pasireotide, somatoprim and ke on sst somatostatin receptor binding, phosphorylation, internalization and signaling. further somatostatin, octreotide, pasireotide, somatoprim and ke were tested for functional selectivity at sst receptor mutants, which possess a carboxyl-terminal sst tail. this approach allows detection of receptor activation by phospho-specific sst antibodies. compared to octreotide, somatoprim activates sst but has a higher activity on sst . ke and pasireotide are partial agonists at the sst receptor. pasireotide, ke and somatoprim show comparable effects on sst receptor. however, none of these new pan-somatostatin analogs behaves like natural somatostatin on the sst receptor. cadmium modulates ahr-associated gene expression in the rat intestine after oral exposure kluxen f. m. , , höfer n. cadmium has been shown to mimic steroid estrogen effects in vivo and in vitro. we have recently identified cross-talk of estrogen receptor (er) and aryl hydrocarbon receptor (ahr) in the rat uterus where beta-estradiol (e ) and cdcl modulate ahrassociated genes via er after i.p. injection in a similar fashion (kluxen et al., arch toxicol doi . /s - - -x). however, the predominant route of exposure to cadmium in non-smokers is via diet. moreover, uterus expresses mainly the receptor subtype eralpha, whilst small intestine and colon express mainly erbeta. thus, we now investigated by real-time rt-pcr the effects of cadmium ( mg/kg b.wt cdcl (cd )) or steroid estrogen ( . mg/kg b.wt. alpha-ethinylestradiol (ee )) on ahrassociated gene expression (i.e., ahr, cyp a , gsta , nqo ) in the small intestine of rats after oral exposure ( days gavage). the animals were also co-treated with cd and pure anti-estrogen ( . mg/kg b.wt zk (zk)) or ee , to asseess whether ermediated processes are involved. we also measured cyp a mrna expression in two estrogen receptor negative colon cancer cell lines (ht- and caco ) treated for days with cdcl ( µm) and e ( . µm). the dose-dependency of cadmium induced ahr target gene modulation was studied in a second animal experiment, with administration of cadmium in drinking water for days ( . - mg/kg b.wt cdcl equivalent to , , and ppm) and ee ( . mg/kg b.wt) as steroid reference. in summary we present two major results: the metalloestrogen cadmium modulates dose-dependently the ahr-associated gene expression in the intestine after oral exposure. yet, since the cadmium induced modulation of ahr target genes was not antagonized by anti-estrogen in the small intestine in vivo and was also found to occur in er-negative colon cells in vitro, we propose that er-independent mechanisms might play a role in this effect. meg) is the most cytotoxic lesion. if not repaired by o -methylguanine-dna methyltransferase (mgmt), o meg/t mismatch is recognized by the mismatch repair system (mmr) that performs futile repair cycles. during this process secondary lesions (i.e. dna single-strand brakes) are formed, which block dna replication in the next replication cycle, leading to dna double-strand breaks (dsbs). these dsbs eventually signal to apoptosis and other genotoxic endpoints. here, we wished to address the question whether autophagy is part of the cellular response triggered by o meg. we also assessed whether autophagy influences apoptosis induced by tmz in glioma cells. we show that tmz induces autophagy in u- mg and ln- glioma cell lines. the maximum amount of autophagy was observed several days ( h) after tmz treatment and mgmt proficient cells did not display significant autophagy. thus, the data show that mgmt protects against tmz induced autophagy, pointing to o meg as the critical lesion responsible for induction of autophagy. using colon cancer cell lines proficient and deficient in mmr, we show that mmr is required for tmz-induced autophagy. because dsbs, which emerge during the processing of o meg, are repaired preferably by homologous recombination (hr) ln- cells stably down-regulated for hr were tested for autophagy induction. the data indicate that dsbs are involved in tmz-induced autophagy. because autophagy following tmz treatment occurs earlier than apoptosis we hypothesize that autophagy protects glioma cells against apoptosis. using an early stage autophagy inhibitor -methyladenin we have shown, that autophagy inhibition sensitized glioma cells to tmz-induced apoptosis. taken together our data point out that tmz induces autophagy in glioma cells and that autophagy protects glioma cells against tmz-induced apoptosis. o meg is the lesion responsible for autophagy induction. furthermore, the data also shows that mmr and dsbs are involved in the induction of autophagy after tmz treatment. work was supported by bmbf ( nuk ). retinitis pigmentosa (rp) is a severe human retinal disease characterized by a progressive degeneration of rod photoreceptors and a secondary loss of cone function. in most cases, rp finally leads to legal blindness. mutations in the regulatory subunit of the rod cyclic nucleotide-gated (cng) channel (cngb a) have been found in patients suffering from rp. we used cngb -deficient (cngb -/-) mice to establish a gene replacement therapy as a potential treatment for rp by means of recombinant adenoassociated viral (raav) vectors. the packaging limitations of raav vectors required a capacity-optimized vector of the large cngb a cdna (approx. kb). therefore, we replaced regulatory elements within the expression cassette and used a short mouse rhodopsin promoter element for rod-specific expression. after injection of therapeutic raavs (serotype ) into the subretinal space of -week-old cngb -/mice, we assessed the restoration of vision by analyzing i) protein expression and localization, ii) retinal function and morphology and iii) vision guided behavior. we found that treated cngb -/mice expressed full-length cngb a and cnga , which were previously downregulated. both proteins co-localized in rod outer segments and formed regular cng channel complexes in the treated area of the cngb -/retina. using electroretinography (erg) we observed a distinct rescue of rod-driven responses. moreover, cngb replacement significantly preserved outer segment morphology and delayed retinal degeneration. finally, treated cngb -/mice performed significantly better than untreated mice in a modified water maze task designed to test for rod-mediated, vision-guided behavior. in summary, this work provides a proof-of-concept for the treatment of rod channelopathyassociated retinitis pigmentosa by raav-mediated gene replacement. most endocrine disruptors interact with hormone receptors or steroid biosynthesis and metabolism, thereby modifying the physiological function of endogenous hormones. here, we present an alternative testing paradigm for detection of endocrine modes of actions that replace and reduce animal testing through refinement. receptor mediated endocrine effects were assessed using the yeast based receptor mediated transcriptional activation yes/yas assays and effects on steroid hormone biosynthesis were assessed using the human cell line h r in the steroidogenesis assay. in our testing paradigm we propose to complement the in vitro assays with a single in vivo repeated dose study in which plasma samples are analyzed for their metabolome profile. the combination of these methods does not only contribute to refinement and reduction of animal testing, but also has significantly increased the efficient allocation of resources and allows for a sound assessment of the endocrine disruption potential of compounds. thus, this proposal constitutes a potentially attractive alternative to epa's endocrine disruptor screening program. data on reference substances for which the in vitro yes/yas and steroidogenesis assays and the in vivo metabolome analysis were performed to assess their putative endocrine mode of action is presented here. the bovine corneal opacity and permeability (bcop) test has been adopted by oecd for the identification of corrosive and severe ocular irritants (ghs category ) for single component substances and multi-component formulations. eye irritation tests (eit) using human reconstructed tissue models (such as epiocular) have been described to predict ocular non-irritants (ghs no category). thus the ultimate repaltement of the draize rabbit eye irritation test (oecd tg ) by a combined or tiered testing strategy could be possible. the purpose of this study was to evaluate whether the bcop with additional corneal histology together with the eit could be used to predict eye irritancy of agrochemical formulations according to different classification schemes including un ghs and epa systems. we have performed the bcop (plus histology) and the eit of agrochemical formulations for which in vivo eye irritation data were already available (for registration purposes). using the oecd tg guideline evaluation scheme for opacity and permeability in the bcop was not predictive for the agrochemical formulations assessed here, while corneal histology grades and the epiocular tissue viabilities were useful predictors of eye irritancy potencies and could be applied for the different classification schemes. the nanomaterials offers extraordinary opportunities. the nano-structure can change the physical and chemical properties, and often also alters the biological effects. hence the toxicity of a nanomaterial can differ from its larger-scale material; but as of today, no new quality of a general nano-specific toxic effect has been observed. therefore the established testing methods are generally suitable. it is, however, difficult to apply the nanomaterials to in vitro test systems, since it is the nature of these materials to change their surface properties and agglomeration stateand the uptake and distribution in the body may differ from their larger-scale materials. while the methods for topical effects may be used for nanomaterials without further modification, the in vitro methods for genotoxicity testing require the dispersion in culture media. the use of reproducible and well-documented dispersion protocols andthe characterization of its particle size distribution is de rigueur . [ ] . for many nanomaterials published genotoxicity studies did not give a consistent picture [ ] and therefore there are rather effects of individual nanomaterials than nano-genotoxicity per se. modern toxicology is based on the insight into toxic pathways. for nanomaterials a testing strategy will include testing for their primary effects (which might be only a handful: particle effects, catalyzing the formation of reactive molecules and ion release) and their uptake, distribution and clearance. the use of dermal penetration studies in vitro for the risk assessment of sunscreen nanomaterials has been demonstrated [ ] . in vitro methods for specific effects (such as inflammation, pulmonary toxicity, sensitization) are currently awaiting validation (for both chemicals/molecules as well as nanomaterials). in the meantime alternative short-term in vivo studies with optimized biological readouts can deliver information on the toxic pathways as well as the biokinetics and dose-response relation of nanomaterials in the body. a short-term inhalation test for nanomaterials has already been used successfully [ ] . testing strategies based on those methods engage less animals and provides more significant data than classical testing. moreover data from these methods will serve as a benchmark and a validation for the in vitro models under evaluation. nanoparticles (np) are increasingly used in various field of industry which necessitates evaluation of their safety. also in the food industry, nps have gained strong interest, for example as food additives or to improve food packing. however, the potential risks of ingested np have been rarely investigated. inhalation studies have revealed that inflammation and oxidative stress may represent unifying mechanism for the induction of adverse health effects of toxic np. in the present study, a co-incubation model of human polymorphonuclear neutrohils (pmns) and caco- human intestinal epithelial cells was used as a model to address potential genotoxic effect of np during intestinal inflammation. oxidative dna damage induction (measured by the fpg-modified comet assay) was induced in the caco- cells by activated pmn and this effect increased with increasing pmn to caco- cell ratio. the crucial involvement of the phagocyte nadph oxidase complex could be demonstrated using treatment of caco- cells with bone marrow-derived pmn from nadph oxidase deficient mice. dna damage by pmn as well as h o was increased in buthionine sulphoximine (bso) pre-treated caco- cells, illustrating the importance of the cellular glutathione (gsh) status in these target cells. gsh depletion in caco- cells could also be shown upon treatment with various types of np. our data suggests that ingested np may increase the susceptibility of the colon mucosa to genetic damage during the occurrence of intestinal inflammation. the ingestion of seafood contaminated by acute toxic doses of the marine toxin okadaic acid (oa) is responsible for diarrhetic shellfish poisoning. it is recently known that both the rat and the human hepatic cytochrome p monooxygenases (cyp) are able to metabolize this toxin. currently, there is a lack of data about the toxicity and mode of action of oa after xenobiotic metabolism. the aim of our study was the measurement of the toxicity and oxidative stress status in hepg cells incubated with oa in the absence and presence of s mix. pure oa, as well as oa pre-activated with liver homogenisates (s mix) were used to treat human hepg cells that have nearly undetectable levels of functional cyp but express phase ii enzymes. the experiments were performed with both human and rat s fraction plus cofactors of phase i enzymes. the cell viability was measured after h using mtt-test and xcelligence real time cell monitoring system. furthermore, levels of intracellular reactive oxygen species (ros) were detected by ´, ´-dichlorofluorescein diacetate and additionally by measuring the intracellular glutathione content. in the presence of both human and rat s mix oa showed a higher toxicity than the parental substance. oa pre-incubated in rat s mix was toxic at nm oa. strong effects could be observed when oa was pre-activated with human s -mix at a concentration of nm oa. pure oa was non-toxic in that concentration range. we could also detect an increase of oxidative stress in hepg cells treated with oa in the presence of all investigated s -mix. these results suggest that oa is activated after oxidative xenobiotic metabolism into metabolites which possess a higher cytotoxic activity and increase the amount of intracellular ros in hepg cells. ballast water treatment -emerging health risks werschkun b., banerji s., krätke r. bundesinstitut für risikobewertung, max-dohrn-strasse , berlin, germany the introduction of invasive marine species into new environments by ships' ballast water, via ships' hulls and other vectors has severe impacts on the oceans. in the international maritime organisation (imo) launched the international convention for the control and management of ships ballast water and sediments which requires ballast water to be treated in order to eliminate alien aquatic species. ballast water treatment may include mechanical, physical or chemical measures. any ballast water management system using active substances needs imo approval. therefore identification of active substances, relevant chemicals and submission of specified datasets on their physical, chemical and toxicological properties is required in order to assess the safety for the aquatic environment and for human health. the bfr is the german federal agency responsible for health risk assessment and has been involved in more than twenty assessment and approval processes so far. the majority of imo approved systems are based on oxidative principles such as chlorination and ozonation. these methods can generate disinfection by-products (dbps), which are a mixed group mostly of halogenated organic substances like trihalomethanes, haloacetic acids and haloacetonitriles. the formation of dbps is well known from the disinfection of drinking water. some dbps are regulated under drinking water directives because of their long-term toxicity but many are unregulated and have unknown toxicological properties. the formation of dbps may vary significantly depending on the treatment system as well as on environmental parameters like temperature, ph and composition of the organic matter within the aquatic environment. in sea water, sources for dbp formation besides ballast water treatment are aquaculture and the cooling systems of coastal power plants. in order to address possible health and environmental risks from dbps formed during ballast water treatment a conference on emerging risks from ballast water treatment was held at bfr in october . here we summarise the main conference findings and identify areas for future research. two presentations corroborated that significant amounts of dbps can be formed in sea water and a presentation on the toxicological properties of dbps pointed out that many have genotoxic properties. accordingly, the determination of dbp species and generated concentrations under different ballast water treatment conditions was seen as a mayor task. different approaches for health and environmental risk assessments were also discussed. appropriate human exposure scenarios and methods for exposure assessment, taking into account common approaches used in risk assessment were presented during the conference. a suitable approach based on derived pec-values for exposure quantification was proposed in order to improve the procedure available for risk assessment of chemical agents used for ballast water treatment. agonist-selective signaling of µ-opioid receptors in t lymphocytes kraus j., börner c., lanciotti s., koch t., höllt v. inst. für pharmakologie und toxikologie, leipzigerstr. , magdeburg, germany opioids are the most potent analgesics and irreplaceable for the treatment of severe pain. in addition to their central effects, opioids modulate a great variety of immune effector cell functions, which may result in unwanted side effects during opioid treatment. the effects of most of the commonly used opioids are mediated by µ-opioid receptors, which belong to the superfamily of g protein coupled receptors. recent data support the concept that g protein coupled receptors function as dynamic entities, which may occupy multiple conformations and activate multiple signaling pathways in a ligand-dependent manner. consequently, different ligands activating the same receptor may have different cellular effects, which has been termed "agonistselective signaling". little is known about agonist-selective signaling of µ-opioid receptors in immune effector cells. in a first attempt to understand if and why such different profiles among different opioids occur we investigated effects of different opioids in human jurkat t cells. we report that the µ-opioid receptor ligands fentanyl, methadone, loperamide and betaendorphin induce internalization of a µ-opioid receptor-green fluorescent reporter construct, whereas morphine and buprenorphine did not induce internalization. the internalization was dependent on p mapk and phospholipase d . in line with this, we observed marked phosphorylation of p mapk and activation of phospholipase d induced by the internalizing opioids, but no or little such activity by morphine and buprenorphine. as a physiological result, fentanyl, methadone, loperamide and betaendorphin treatment of primary human t cells and jurkat t cells resulted in a strong, up to fold induction of il- , which was dependent on p . in contrast, morphine and buprenorphine only showed a weak, approximately one order of magnitude lower induction of il- . by inducing il- opioids significantly modulate the t helper cell balance into the type direction, which influences various immune responses, e. g. the antiviral, t helper cell type -mediated response. considering the vital necessity of opioid use in humans, it is an intriguing goal to identify analgetically feasible opioids that have little or no immunosuppressive or -modulatory effects. modulation of cgmp signals by phosphodiesterases in smooth muscle cells krawutschke c., koesling d., russwurm m. ruhr-universität bochum pharmakologie und toxikologie, universitätsstrasse , bochum, germany within the cardiovascular system, cgmp mediates smooth muscle relaxation and inhibition of platelet aggregation. cgmp is formed by particulate guanylyl cyclases and nitric oxide-sensitive guanylyl cyclases, that are activated by natriuretic peptides or nitric oxide (no), respectively. besides the cgmp-forming enzymes, the cgmp-degrading phosphodiesterases strongly determine amplitude and shape of cgmp signals. in vascular smooth muscle cells, three phosphodiesterases are considered to be responsible for cgmp degradation: pde , the cgmp-specific phosphodiesterase is activated directly by cgmp binding to its gaf domains; this activation if further stabilized by cgmp-dependent protein kinase-mediated phosphorylation. pde , the ca +/calmodulin-stimulated pde constitutes the majority of cgmp-hydrolyzing activity in smooth muscle cells at least in the presence of high intracellular ca + concentrations. and lastly pde , the cgmp-inhibited pde displays some cgmp-degrading activity, although cgmp binding to its catalytic domain is primarily thought to inhibit the campdegrading activity of pde . cgmp signals measurable in radioimmunoassays require stimulation with cgmpincreasing vasodilator concentrations that are orders of magnitudes higher than those required for relaxation. thus, we developed fluorescent sensors for real-time measurement of cgmp signals in single cells. by using these indicators, we analyzed the contribution of different cyclic nucleotide-degrading phosphodiesterases to the modulation of cgmp signals elicited by physiologically relevant vasodilator concentrations. hyaluronan (ha) is a major component of extracellular matrices and is thought to control cellular phenotypes such as proliferation and migration. therefore, ha synthesis may play an important role in the pathophysiology of atherosclerosis. there are three major ha-synthase isoenzymes (has - ). the has gene is alternatively spliced. has transcript variant (has v ) encodes the smallest has isoenzyme which has a different c-terminus and contains only two transmembrane domains compared to has transcript variant . the aim of the present study was to investigate whether has v is expressed by vascular cells, how it is regulated and where it is localized in cells and whether it indeed synthesizes ha. has v mrna expression was monitored by quantitative real-time rt-pcr. protein expression was determined by western blotting using a polyclonal antibody that was raised in rabbit. an n-terminal eyfp-has v fusion protein and a ddk-tagged has v construct were expressed for subcellular localization studies and co-immunoprecipitation (co-ip). endogenous has v mrna was expressed in both vascular smooth muscle cells (vsmc) and endothelial cells. furthermore, western blotting revealed has v protein expression in vsmc and platelets. in vsmc has v mrna expression was strongly up-regulated in response to interleukin- β (il- β, ng/ml) whereas stimulation with interleukin- ( ng/ml), platelet-derived growth factor-bb ( ng/ml), transforming growth factor β ( ng/ml), tumor necrosis factor α ( ng/ml) and interferon-γ ( ng/ml) had no effect. transfection of hek cells with eyfp-has v fusion protein revealed localization to the endoplasmic reticulum but not to the plasma membrane. furthermore, co-ip experiments showed that tagged has v proteins were precipitated together suggesting formation of multimeric has v complexes. transfection of has v did not cause increased secretion of ha into the cell culture supernatant in hek cells. in conclusion, has v is present in vascular cells and responds to inflammatory cytokines such as il- ß. because of the intracellular localization and the lack of ha secretion in has v transfected cells, has v may serve intracellular functions apart from ha synthesis. the tubulin antagonist pretubulysin shows strong vascular-disrupting properties in vitro several epidemiological studies indicate a correlation of human exposure to ultrafine particulate air pollution caused by incomplete combustion processes and an increase in the incidence of pulmonary immune diseases like asthma. as a possible mechanism behind this pathological phenomenon, the adjuvant effect of lung inflammation induced by poorly soluble environmental particles has been hypothesised. the aim of our study was to investigate the causal link between carbon nanoparticle-induced lung inflammation and modulations of immune cell populations during processes leading to sensitization, and allergic immune responses of the airways. therefore mice were treated with ovalbumin (ova) alone or in combination with carbon nanoparticles (cnp) by pharyngeal aspiration. the induction of inflammation and the immune adjuvant activity were studied in the lungs and lung draining peribronchial lymph nodes (pbln) at the level of sensitization, and at the level of the immune response. ova-specific ige antibodies were measured in blood serum, and the development of allergic airway inflammation was studied after ova challenge. results at the level of sensitization showed that cnp-induced immediate airway inflammation had immune adjuvant activity resulting in an increase of specific cell populations in pbln and in a stimulation of asthma-specific th cytokines. a specific reduction of the neutrophilic lung inflammation by application of the compatible solute ectoine significantly reduced the adjuvant effects of cnp. in ova-sensitized mice, application of cnp hours prior to allergen challenge, led to a significant increase in inflammatory cell infiltrate and respective cytokines in broncho-alveolar lavages. coapplication of mm ectoine together with cnp reduced the particle-induced effects. our data show a link between neutrophilic lung inflammation and adjuvant effects of cnp. a specific reduction of neutrophils by the application of ectoine attenuated this np induced adjuvant effect, indicating that particle-induced lung inflammation rather than the direct interaction of nanoparticles with immune cells is the critical step in environmentally modulated pulmonary immune diseases like asthma. introduction: drug-eluting stents (des) are commonly used in the treatment of acute artery occlusion. however, even if released cytotoxic drugs reduced neointimal proliferation significantly there is still the risk of in-stent thrombosis. it is presumed that this is due to reduced reendothelialization. it has been suggested that coating the stent with biomolecules may provide a new approach to circumvent the lack of healing of the endothelial layer. one approach would be the use of biomolecular signals, such as (poly)peptides and growth factors. rgd and redv are peptide motifs, known to enhance cell attachment and spreading. aim: the aim of our study was to evaluate the efficacy of proteins, derived from elastin-like proteins (elp) and artificial modified by incorporating with the amino acid motifs rgd,redv and p , in terms of endothelial healing on stents and other cardiovascular devices. results: in this work, we generated vectors encoding for different biopolymers consisting of various bioactive signal molecule sequences. the peptides, e.g. based on the elastinlike matrix (vpgig) -vpgkg-(vpgig) , were synthesized using heterologous expression. after optimizing culture conditions and extraction procedures their biological activity was assessed using human umbilical vein endothelial cells (huvec). elastin like proteins with differently incorporated bioactive signals (redv, rgd and a small p peptide) were linked covalently via carbodiimide coupling to poly(l-lactide) (plla) films. huvec growth was determined on these modified surfaces using the brdu assay (cell proliferation) and resazurin assay (cell viability). the chemically modified plla surfaces conferred higher cell viability after h adhesion ( %) and an enhanced proliferation ( %, h adhesion, h cultivation) in comparison to the unmodified plla. these results indicate that the synthesized elp incorporated with amino acid motifs promote an accelerated endothelialization of the biodegradable stent material plla. discussion: in summary, we were able to generate elastin-like proteins modified by bioactive sequences. those sequences enhanced endothelial cell proliferation and adhesion. further studies are warranted to determine the activity on smooth muscle cells of these peptides. ( ) the failing heart is characterized by excessive extracellular matrix production by myofibroblasts (myocfs) causing fibrosis and myocardial stiffening. myocfs represent phenotypically transformed cardiac fibroblasts (cfs) and are characterized by the expression of contractile proteins like a-smooth muscle actin (α-sma) and enhanced secretion of growth factors (e. g. ctgf). identification of intracellular enzymes that modulate this transformation process is desired to therapeutically modulate pro-fibrotic progression in heart failure. we show that pde a, a phosphodiesterase isoform, that is able to hydrolyse cgmp and camp, is markedly upregulated in failing hearts from patients with end-stage heart failure ( - -fold, p< . , n= ). notably, pde a protein abundance is -fold higher in myocfs compared to cardiomyocytes from neonatal rat hearts (p< . , n= ). to this end we tested whether pde a modulates the transformation of cfs isolated from neonatal rat hearts to myocfs. indeed, as assessed by immunoblotting and fluorescent microscopy (α-sma, phalloidin, dapi), adenoviral pde a overexpression induced α-sma expression ( . -fold p< . , n≥ ) and to a lower extent ctgf synthesis ( . -fold, p< . , n≥ ). mechanistically, pde a showed preferential subsarcolemmal localisation with diminished total cgmp levels (- %, p< . , n≥ ). consistently, parallel stimulation with atrial natriuretic peptide (anp), a selective activator of membrane-bound guanylyl cyclase, normalized ctgf synthesis indicating that pde a controls cgmp in a discrete subdomain near the plasma membrane. moreover pde a overexpression diminished the protein levels of vasodilator-stimulated phosphoprotein, a membrane cytoskeletal component (- %, p< . , n≥ ). these data implicate pde a-dependent subsarcolemmal cgmp regulation in myofibroblast formation and potentially cardiac fibrosis. therefore, targeting pde a may lead to regression of the fibrotic remodeling associated with heart failure. several anorganic nanoparticles (np) causedhigher inhalation toxicity than the corresponding coarse particles (oberdoerster et al. ) . we examined an organic pigment and a polymer dispersion each as nanomaterial and as the chemical identical coarse material in short-term inhalation studies in malerats. the polymer was an anionic acrylic ester copolymer containing free carboxylic groups. three different particle sizes were synthesized by varying polymerization conditions: , or nm. although polymeric acrylic ester was reported to be irritating to the respiratory tract at mg/m , all three tested polymers -including the np ( and nm) -did not cause any changes in lavage fluid and in histopathology at mg/m . the organic pigment was a poorly soluble pyrrol with an intense orange color. the np ( to nm width and to nm length) and the coarse pigments ( to nm width and . to µm length) are both needle-like. they were tested at , , and mg/m for the np and , and mg/m for the coarse materials. mild and partly reversible morphological changes were observed in lung and lymph nodes at the highest concentrations, but the more pronounced effect were found in rats exposed to the coarse material. likewise there was an increase of lavage parameters in rats exposed to thecoarse material but not to the np. these data demonstrate that inhalation of finer np is not necessarily associated with higher toxicity compared to the coarse material. the results were obtained with two organic particles of rather different size and composition but are in contrast to the more severe effects seen with several anorganic np when compared to the corresponding coarse particles. within the nanocare project a standard short-term inhalation test to examine the toxicity of inhaled aerosols from nanomaterials has been developed. the inhalation toxicity of nano-andpigmentary materials was studied: baso , zno, ceo , al-doped ceo , zro , amorphous silica, surface-coated amorphous silica, titania, carbon black and three multi-wall carbon nano tubes, all with complete phys-chem-characterization as planned for the oecd sponsorship program. quartz dust tio and zno were tested as pigmentary materials. rats were exposed nose-only to three concentrations of one of these materials, h a day for five consecutive days. positive controls were exposed to quartz dust or pigmentary zno, negative controls to clean air. a wide range of endpoints for pulmonary toxicity were evaluated immediately after the last exposure and after days and weeks after the last exposure. among these parameters, polymorphnuclear granulocyte count in bronchoalveolar lavage fluid is the most sensitive early parameter indicating inflammation process in lung, while histological examination reveals the type and localization of inflammation. among these substances, we identified baso as having the lowest toxicity. all mwcnts were most potent in producing progressive inflammation in the lung; granulomas in lung and lung associated lymph nodes were observed without indication for fibrosis. the noaecs of the substances ranged between < . and > mg/m . generally the material was only found in the lung (surface and macrophages) and in the draining lymph nodes. surface modified amorphous silica was also found in the spleen. the data demonstrate that the method is able to differentiate the toxic potential of different nanomaterials and to indicate regression or progression of the effects effects. moreover the lung burden and potential translocation to other tissues was detecable. comparing the material properties and effects of the materials, no general relationship between the toxicity and either particle size, specific surface area or aerosol particle number concentration was found. hence we must not expect to find a gerneral "nanotoxicology" or a unifying dosimetry for all nanomaterials. we must rather be prepared to test individual nanomaterials for their effects. and to develop grouping concepts not only based on material properties but also on biopersistence, biokinetics and biological effects. part of this studes has been sponsored by bmbf (nanocare). endpoint-centric search for toxicological information and data to support the information retrieval for regulatory programs landsiedel r. , wächter t. the eu reach regulation no / requires industry to ensure the safety of chemical use and manufacturing. all substances manufactured or imported in quantities above one tonne per year must be registered. information requirements for the dossiers increase with increasing tonnage or once hazards are suspected. searching for substance specific literature and the compilation of hazard data for safety assessments are highly challenging procedures. the novel web-based search engine go r, accessible free of charge at www.go r.org, has been created to allow quickly finding relevant hazard information and data. go r provides an endpoint-centered literature search to all scientists and regulatory authorities seeking for toxicological information. furthermore, go r specifically highlights information on animal testing alternatives. search results are presented automatically linked to an "intelligent table of contents" which enables the user to sort the literature listed in pubmed or the toxicology data network (toxnet) in a fast and comprehensive manner. retrieved documents are automatically organized in categories relating to the iuclid chapters. hereby, the user can browse directly through the entire million documents without even having to start the search with an initial query. the semantically enriched platform supports the user during query formulation, allows for bibliographic analysis, and specifically highlights information related to the replacement, reduction, and refinement of animal experiments. search results in go r are shown in an dynamic table of contents (left) making them browsable for the contained information on animal testing alternatives and toxicologogical endpoints. towards a differentiation therapy of acute myelogenous leukemia with histamine h -receptor agonists laue s., burhenne h., seifert r. hannover medical school institute of pharmacology, carl-neuberg-str. , hannover, germany acute myelogenous leukemia (aml) is a devastating malignancy characterized by a differentiation block of myeloid progenitor cells. recently, histamine dihydrochloride in combination with interleukin has been approved as orphan drug for the consolidation treatment of aml ( ) . it is assumed that histamine exerts its effects by activating the histamine h -receptor (h r) in human neutrophils, resulting in improved anti-tumor function of t killer cells by inhibiting nadph oxidase-catalyzed superoxide formation. previous studies had shown that histamine also induces myeloid differentiation ( ) . considering the fact that all-trans-retinoic acids constitutes a powerful differentiation therapy of acute promyelocytic leukaemia, a specific subtype of aml ( ), we initiated a study to explore the possibility that h r-mediated myeloid differentiation provides an alternative or complementary strategy to treat leukemias associated with differentiation blocks. as model system, we used hl- cells. in hl- cells, histamine and various h -receptor agonists induced concentrationdependent increases in camp levels. interestingly, ligands differentially increased cytosolic calcium concentration and extracellular receptor kinase (erk) pathways, indicative for ligand-specific h r conformations. h r activation resulted in myeloid differentiation as assessed by enhanced formyl peptide receptor-mediated increases in cytosolic calcium concentration. h r agonists showed no signs of cytotoxicity. intriguingly, following h r activation, the majority of the formed camp was exported into the extracellular space via multi-drug resistance protein (mrp) , indicating that export is a more important pathway for signal termination than cleavage of camp by phosphodiesterases. despite effective camp export, even a short-term exposure ( minutes) of cells was sufficient to induce expression of functionally active formyl peptide receptors. these data indicate that in contrast to previously held dogma, induction of myeloid differentiation does not require continuous presence of a camp signal. from a therapeutic point of view this is very important since "spike" therapy with campincreasing substances may be sufficient to induce a therapeutic effect in aml, thereby also reducing toxic side effects. currently, we are systematically exploring the effects of h r agonists on signal transduction pathways and differentiation in various myeloid cell types to identify highly efficacious compounds. introduction. activation of gαq/ protein-coupled receptors of postsynaptic neurons can elicit the production of endogenous cannabinoids (endocannabinoids), which in turn inhibit transmitter release from axon terminals by activating presynaptic cb receptors. the aim of the present experiments was to study the mechanism of the endocannabinoid production. specifically, we wanted to clarify the role of ca + release from intracellular stores in triggering endocannabinoid production. methods. patch-clamp-and ca + imaging experiments were performed on purkinje cells in mouse cerebellar brain slices. glutamatergic excitatory postsynaptic currents (eepscs) were elicited by electrical stimulation of parallel fibers. the gαq/ proteincoupled metabotropic glutamate receptor (mglur ) was activated by superfusion of (rs)- , -dihydroxyphenylglycine (dhpg) or -more physiologically -by burst stimulation of the parallel fibers. results. both dhpg superfusion and burst stimulation of parallel fibers elicited an increase in intracellular ca + concentration in the postsynaptic purkinje cells. dhpg superfusion and burst stimulation suppressed eepscs, and this suppression was abolished in the presence of the mglur antagonist cpccoet. the suppression of the eepscs was also sensitive to the cb receptor antagonist rimonabant, pointing to involvement of endocannabinoids and cb receptors. the suppression of the eepscs was attenuated after depletion of the endoplasmic reticulum ca + stores by thapsigargin, cyclopiazonic acid and ip . the results indicate that after activation of the gαq/ protein-coupled metabotropic glutamate receptor (mglur ) of the postsynaptic neuron ca + is released from the endoplasmic reticulum. this ca + release significantly contributes to the production of endocannabinoids. the endocannabinoids diffuse in the synaptic cleft retrogradely to the terminals of afferent axons and inhibit transmitter release there through presynaptic cb receptors. the guanine nucleotide exchange factor dock controls reelin dependent cdc effects on radial migration pichler m. the regulation of blood glucose levels is under tight control of a complex system including hormone and neurotransmitter signalling. many of these cellular signalling pathways are initiated by binding of the ligand to a g-protein coupled receptor (gpcr), e.g. noradrenaline inhibits insulin secretion upon binding to a gi-coupled receptor. upon gpcr activation the heterotrimeric g-protein is activated and both the α-subunit and βγdimers are released and interact with their specific target proteins. by the usage of bordetella pertussis toxin (ptx) as a common gαi inhibitor gαi-dependent signalling pathways are interrupted which leads to increased insulin secretion, and significantly improves glucose tolerance. since the gαi-isoform specific roles in the regulation of glucose homeostasis are still debated we studied the glycemic control in gαi -deficient mice. surprisingly and in contrast to the ptx data, glucose tolerance was unchanged in the gαi -deficient mice compared to wild type controls. however, the plasma insulin levels were significantly reduced upon glucose challenge. these findings point to disturbed islets function and improved peripheral insulin sensitivity. analysing gαi deficient islets we show that islet size and number of nuclei are reduced. nevertheless, in vitro insulin secretion is improved at low ( mm) and high ( mm) glucose concentrations and can be further stimulated upon ptx-treatment. these data indicate that gαi proteins influence islet development and inhibit insulin secretion. in addition, these findings support our hypothesis that gαi -deletion influences peripheral insulin sensitivity. therefore, we investigated glucose homeostasis and pakt-levels after two hours feeding ad libitum in gαi -deficient mice. under feeding conditions no differences in plasma insulin levels were visible although blood glucose levels were significantly reduced in gαi -targeted mice. pakt-levels of liver and skeletal muscle were unaltered, whereas akt phosphorylation in white adipose tissue was significantly increased, indicating improved glucose uptake of adipocytes. in conclusion, gαi is a negative regulator of both insulin secretion and peripheral insulin sensitivity and important for the maintenance of glucose homeostasis. , ) in a radioligand binding test and to determine their functional effects with a membrane potential test using the dye r (molecular probes, . mg/ml, excitation nm, emission nm). the affinity of compounds in radioligand binding was slightly higher in sur b than in sur a-type channels, but the enantiomeric ratio in sur a channels matched that one determined for the sur b-type indicating some conformity of the binding pockets of sur a and sur b-proteins. surprisingly, however, the membrane potential tests revealed that the ( r, s)-enantiomer acted as agonist (a) whereas the ( s, r)-enantiomer acted as antagonist (b): ( r, s)-bms- induced membrane hyperpolarisation whereas ( s, r)-bms- repolarised cells prestimulated with submaximally effective concentrations of diazoxide. concluding, bms- is not selective for sur a as compared to sur b-type k atp channels. its enantiomers activate and block sur -type katp channels in a stereospecific manner. thieno-thiadiazine derivatives with full agonistic activity at sur b-type katp channels act as partial agonists at cardiac sur a-subtypes oldenhage c., grittner d., schmidt c., lemoine h. heinrich-heine universität, inst. für lasermedizin, mol. wirkstoff-forschung, universitätsstr. , düsseldorf, germany new potassium channel openers (kco) of the thieno-thiadiazine(ttd)-type initially developed as agonists for the sur -type katp channels (nielsen et al., j med chem : , ) were characterized in sur b-type katp channels as agonists and antagonists, if r contains a quaternary (methyl-cycloalkyl) and a tertiary (r = cycloalkyl) carbon, respectively (lemoine et al., this journal , r , ) . to investigate the selectivity of ttd-derivatives for myocardial katp channels the membrane potential actions of compounds were tested in hek (kir . /sur a)-cells and compared to hek (kir . /sur b)-cells as a model for smooth muscle-type katp channels. membrane potential was measured by fluorescence (excitation nm, emission nm) using . mg/ml of the dye r (molecular probes). standard-kco induced hyperpolarisation with ~ -fold smaller potency (pec ) in sur a. ttd-compounds with ch -cycloalkyl residues not only lost potency but also intrinsic activity for channel activation (emax) in sur a. possibly, this loss of emax would be much greater in native heart cells with a normal channel density. in contrast, ttd-compounds with cycloalkyl residues acted as antagonists of cells pre-hyperpolarized with diazoxide with similar affinity in sur a and sur b-type katp channels. concluding, selectivity of kco for katp channel-subtypes cannot only be achieved by a different affinity but also by a selective stimulation of the channel of interest. small-conductance calcium activated potassium (kcnn/sk/kca ) channels maintain neuronal calcium homeostasis, shape synaptic functions and prevent excitotoxic neuronal death. so far, little is known about the function of kca channels in nonneuronal cells. the aim of this study was to investigate the expression of kca channels in microglial cells and their potential function in microglial activation and maintenance. expression of kca channel subtypes in microglial cells was assessed by mrna analysis, western blots and immunocytochemistry. lipopolysaccharide (lps)-induced microglial proliferation was evaluated by the xcelligence impedance-based system and mtt assays, and immunogenic activation of microglia was determined by measuring cytokines and nitric oxide (no) release into the cell culture medium. the kca . and kca . channel activator cyppa ( µm) and specific inhibitory peptides ( µm) were applied to distinguish effects mediated by the kca channel subtypes. all kca channel subtypes were detected on mrna and protein levels in resting and in lps-activated microglial cells. xcelligence real-time measurements and mtt assays demonstrated that lps ( ng/ml) induced microglial proliferation. the kca . /kca . channel activator cyppa reduced lps-induced microglial proliferation in a concentration-dependent manner. specific peptide inhibitors of kca . channels, but not of kca . channels, reversed the cyppa-effects on lps-induced microglial proliferation. cyppa alone did not alter the production of tnf-alpha or il- , but strongly reduced the lps-dependent cytokine production. interestingly, chelation of extracellular calcium by edta induced differential cytokine kinetics by decreasing lps-dependent il- production while tnf-a production was not affected. moreover, using inhibitory sk /kca . channel peptides, we demonstrated that sk /kca . channels modulate lps-induced cytokine il- production in a calcium-dependent manner, while the tnf-a release was independent of extracellular calcium. in summary, the present study revealed that kca . channel stimulation reversed microglial activation. thus, kca . channels may serve as a therapeutic target for reducing microglial activity and related inflammatory responses in cns diseases. ( r, s)-( s, r)- intracellular amyloid beta (aß) oligomers and extracellular aß plaques are key players in the progression of sporadic alzheimer disease (ad). still, the molecular signals triggering aß production are largely unclear. we asked whether mitochondria-derived reactive oxygen species (ros) are sufficient to increase aß generation and thereby initiate a vicious cycle further impairing mitochondrial function. complex i and iii dysfunction were induced in a cell model using the respiratory inhibitors rotenone and antimycin resulting in mitochondrial dysfunction and enhanced ros levels. both treatments lead to elevated levels of aß. presence of an antioxidant rescued mitochondrial function and reduced formation of aß demonstrating that the observed effects depended on ros. conversely, cells overproducing aß showed impairment of mitochondrial function such as comprised mitochondrial respiration, strongly altered morphology, and reduced intracellular mobility of mitochondria. again, the capability of these cells to generate aß was partly reduced by an antioxidant indicating that aß formation was also ros-dependent. moreover, mice with a genetic defect in complex i, or ad mice treated with a complex i inhibitor, showed enhanced aß levels in vivo. several lines of evidence show that mitochondria-derived ros result in enhanced amyloidogenic amyloid precursor protein processing, and that aß itself leads to mitochondrial dysfunction and increased ros levels. we propose that starting from mitochondrial dysfunction a vicious cycle is triggered that contributes to the pathogenesis of sporadic ad. comparison of methods to derive health-based guidance or limit values for chemicals licht o., voss j. -u., mangelsdorf i. fraunhofer item chemikalienbewertung, nikolai-fuchs-str. , hannover, germany health-based guidance or limit values are derived for chemicals to compare measured or estimated exposure concentrations with these values. if the exposure is below the limit value, adverse effect for human health can be regarded as negligible, e.g. the exposure is expected to be tolerable. in germany such values have been derived since years for chemicals that can be found in soil, water and air as well as human blood and urine (biomonitoring). in a research project sponsored by the german umweltbundesamt several methods used by the agency are compared to the method laid in reach guidance document r. to derive a derived no effect level (dnel). the aim was to identify possibilities for standardization as well as to figure out specific elements in individual methods. in addition to extrapolation factors the public availability of guidance and specific derivations as well as procedures for consensus on the limit value were evaluated. the comparison of extrapolation factors revealed that, although they are named differently such as extrapolation, safety or assessment factors, they are used in a comparable manner. factors for interspecies and intraspecies extrapolation are presented in more detail. the standard factor for such extrapolation is in most cases. in the reach guidance this factor consists of a part for allometric scaling and remaining differences. other factors are only defined in some methods, like a factor for extrapolation from loael to noael, data quality or data gaps. a factor for data quality is not laid down in the basic scheme for setting of indoor air guidance values, but is used in some of the recent derivations of limit values. also the who guidelines for drinkingwater quality use comparable factors to account for adequacy of studies or database and nature and severity of effect. a transparent and documented derivation is necessary for acceptance of the value. the derivation methods as well as the evaluation document on a specific substance are available through publications or the internet in nearly all cases. for the dnel only the numeric value is available at the echa website, but not any information on starting point and extrapolation factors. although all guide or limit values are derived in a comparable way, differences, however, exist in some details. in most cases detailed explanation is lacking when deviating from standard or default assumptions. often such deviation is based on expert judgement. hepatocellular carcinoma (hcc) is the fifth most common cancer in the world and has a poor prognosis with limited therapeutic options. up to now, no curative systemic therapy exists emphasizing the high clinical importance of new therapies for hcc. therefore, the identification and characterization of novel drugable targets is a relevant goal. cyclin-dependent kinase (cdk ) is well characterized for its function in cns development and disease. recently, few reports indicate functions of cdk in cancer. cdk was shown to regulate tumor growth, and our group discovered that cdk regulates angiogenesis. since hcc is a highly vascularized tumor and anti-angiogenic treatment (sorafenib) has shown some therapeutic benefit, we hypothesize that cdk is an interesting target for hcc therapy. the aim of this study was to characterize the function of cdk in hcc. histology of tissue micro arrays indicates an increased expression of cdk in human hcc tissue in comparison to healthy liver tissue of the same patient. to investigate the function of cdk in hcc, we analyzed the impact of both pharmacological inhibition of cdk and specific downregulation of cdk with rna interference on hcc cells. pharmacological inhibition of cdk with the small molecule roscovitine (r-roscovitine, seliciclib) decreased proliferation and clonogenic survival, induced g /m cell cycle arrest and cell death, and reduced motility of huh and hepg cells. transient downregulation (sirna) and stable knockdown (shrna) of cdk also reduced proliferation, clonogenic survival, migration and invasion of huh cells. in a subcutaneous hcc xenograft model, treatment with roscovitine reduced tumor growth and angiogenesis, indicated by decreased tumor weight and volume, and reduced vessel density. moreover, cotreatment of hcc cells with roscovitine and tumor necrosis factor related apoptosis inducing ligand (trail) resulted in an over-additive additive effect on the induction of apoptosis. this coincided with reduced phosphorylation and activity of the anti-apoptotic transcription factor stat at ser that is directly phosphorylated by cdk , and tyr . in line with this, the expression of the antiapoptotic protein mcl- is reduced by inhibition of cdk . our results point to an important function of cdk in hcc and suggest cdk as an interesting pharmacologically druggable target for hcc therapy. delivery of mono-biotinylated rnasea into macrophages with streptavidinconjugated clostridium botulinum c toxin lillich m. , chen x. clostridium botulinum produces the adp-ribosyltransferase c , which modifies and thereby inactivates exclusively the small gtp binding proteins rho-a,-b and -c. recently, we discovered a specific endocytotic internalization of c toxin in macrophages and myeloid leukaemia cells, but not in epithelial cells [ ] . thus, c toxin provides a tool to target cells of the monocyte/macrophage lineage, which are involved in various diseases and are of great clinical interest. we used a biochemical crosslinking approach to design a delivery system based on an enzymatic inactive c bot mutant (c mut) and streptavidin. the c portion mediates uptake of the transporter into monocytes/macrophages and streptavidin allows for binding of biotinylated cargo molecules to the transporter. in vitro, the generated c mut-streptavidin bioconjugate showed specific and concentration dependent binding to biotinylated oligonucleotides as demonstrated by electrophoretic mobility shift assay. cell fractionation experiments indicated an uptake of the bioconjugate into the cytosol of j a. macrophages. in the next step, mono-biotinylated bovine pancreatic ribonuclease a (rnasea) was used as a model cargo for the delivery of macromolecules by the bioconjugate. rnasea is a highly stable, well studied protein which catalyzes the degradation of rna. mono-biotinylated rnasea interacts in a specific and concentration dependent manner with the c mut-streptavidin bioconjugate in vitro as analysed with dot blot technique. the c mut-streptavidin bioconjugate efficiently mediates the internalization of biotinylated rnasea into j a. macrophages as analyzed with laser scanning microscopy in fixed cells. this finding was also confirmed by live cell imaging. furthermore, cell fractionation showed a cytosolic delivery of biotinylated rnasea in the presence of c mut-streptavidin. as expected we could not observe a cytotoxic effect of biotinylated wild-type rnasea on j a. macrophages, which is attributable to the presence of ribonuclease inhibitor protein in mammalian cells. in summary, the c mut-streptavidin bioconjugate mediates the efficient internalization of biotinylated (macro)molecules into macrophage like cells, and therefore represents a useful tool for the transduction of exogenous molecules into macrophages. in addition, cytotoxic rnasea mutants are available and will be used in further studies. organometal compounds such as cisplatin or the second generation complexes carboplatin and oxaliplatin have become more and more important as antitumor agents. nevertheless there is still an increasing demand for novel metal-based compounds. this is necessary due to severe side effects and the occurence of resistent tumour cells. in this context we investigated the cytotoxic effects of imidazole-based phosphane gold(i) complexes as potential agents for cancer treatment. initially we have used the mtt-assay to examine the toxic potential of the gold complexes in h iie rat hepatoma cells. in this context cw (a diphosphane ligand with azoyl substituents r p(ch ) pr , r= thiazol- -yl) turned out to be the compound with the highest cytotoxic potential with an ic value of , mm ( h incubation). further investigations revealed that cw induced an apoptotic cell death in h iie demonstrated by the activation of caspase / ( h incubation with mm cw ). in addition the induction of apoptosis was confirmed by the dna ladder formation ( h incubation with mm cw ). in connection with the molecular mechanisms of apoptosis induction we used the comet assay to analyse the generation of dna strand breaks as well as the dcf-assay to detect the formation of reactive oxygen species. however neither dna strand breaks nor increased levels of reactive oxygen species were detected after h of incubation. furthermore we analysed if the compound influences intracellular signalling pathways such as the jnk pathway and the pi k/akt but after h of incubation neither pakt nor jnk were influenced. the imidazole based phosphane gold (i) complex cw shows strong toxic effects in h iie cells and turned out to be a promising compound as a potential agent for cancer treatment. the high and inappropriate intake of loop diuretics in hypertensive elderly reported in former studies has again been confirmed. remembering that inappropriate intake of loop diuretics can lead to exsiccosis and electrolyte loss especially in elderly, better medical education has to follow these alarming results to improve the pattern of diuretic prescription. furthermore, our results lead us to assume a high estimated number of unreported cases of torasemide use in uncomplicated arterial hypertension in elderly. this loop diuretic agent shows a longer duration of action compared with furosemide (elimination half-life: - hrs vs. hr) and is effective in decreasing blood pressure in subdiuretic doses. it must be pointed out that loop diuretics are still frequently inadequately prescribed because current guidelines recommend loop diuretics only in complicated arterial hypertension. the role of cgmp/cgki signaling and trpc channels in regulation of vascular tone loga f., domes k., hofmann f., wegener j. pharmakologie und toxikologie for , biedersteiner str , münchen, germany signaling by intracellular cgmp and cgmp-dependent protein kinase i (cgki) is the major pathway in vascular smooth muscle, by which endothelial no regulates vascular tone. recent evidence suggests that trpc channels are targets of cgki in smooth muscle and mediate, at least partially, the relaxant effects of cgmp. we tested this concept by investigating the role of cgmp/cgki signaling on vascular tone and peripheral resistance using cgki-, trpc -, and trpc -, and trpc /c -double knock-out mice. we found larger contractile responses to α-adrenergic stimulation in intact aorta from cgki-, trpc -, and trpc /c -double knock-out mice as compared to aorta from ctr and trpc -knock-out mice indicating a functional link between cgki and trpc channels. no differences were found if the vasodilator tone, provided by the no generation in the vascular endothelium, was inhibited by l-name. likewise, no differences were observed in the increase in peripheral resistance by α-adrenergic stimulation using the hind limb perfusion system. activation of cgki by -br-cgmp diminished aortic tone and peripheral resistance to a similar extent in control, trpc -, trpc -, and trpc /c -double knock-out mice. no effect of -br-cgmp was observed in preparations from cgki -/mice. to test the co-localization of cgki and trpc channels, we performed immunocytochemistry on isolated smooth muscle and endothelial cells from aorta of ctr, trpc -, and trpc -knockout mice. trpc could be detected in both smooth muscle and endothelial cells whereas trpc was only detected in endothelial cells. the results suggest that absence of cgki or trpc impairs the vasodilator tone induced by endothelial no production but that cgki and trpc channels are not functionally coupled in vascular smooth muscle. we thank profs birnbaumer (nih) and freichel (homburg) for providing us with trpc -/-, and trpc -/mice and prof. flockerzi (homburg) for the antibodies against the trpc channels. whole genome microarray analysis of the effects of tcdd and pcb in human hepatic cell models lohr c. , neser s. after the treatment with tcdd, however, a total of genes were more than -fold up regulated in hepg e.g. cytochrome p a (cyp a ) ( -fold) a sensitive marker for ahr activation. additional up regulated genes in hepg were; arylhydrocarbon receptor repressor (ahrr) -fold, aldehyde dehydrogenase a (aldh a ) -fold, and cytochrome p b (cyp b ) -fold. genes were more than -fold down regulated in hepg cells e.g. -proprotein convertase subtilisin/kexin type . markedly different findings were obtained in hheps, i.e., genes were up regulated, the highest up regulated gene was cyp b with a -fold increase in gene expression, followed by cyp a ( -fold) and aldh a ( -fold). only a small group of genes were significantly down regulated ( in total), e.g., solute carrier family (facilitated glucose transporter). comparing both human cell types, there was an unexpected small overlap of genes being up or down regulated. interestingly, in both cell types, only in common genes were up regulated, including cyp a , cyp a , cyp b and aldh a . only platelet-derived growth factor receptor, beta polypeptide, was down-regulated in both hepg and hheps. in conclusion, our data indicate pronounced differences in the patterns of tcdd-regulated genes between hepg cells and hheps. detection of redox modified proteins in nociceptive processing lorenz j. e. , kallenborn-gerhardt w. recent data indicate that redox modifications of proteins induced by reactive oxygen species (ros) contribute to sensitization of pain pathways during persistent pain. however, little is known about the targets of ros in pain processing, because the relatively unstable nature of many reversible protein oxidation states hampers the reliable detection and identification of modified proteins. here, we used the quantitative thiol trapping technique termed oxicat to identify proteins which are redox modified during nociceptive processing. we investigated spinal cords of untreated mice, after zymosan injection into a hindpaw (inflammatory pain model) and after spared nerve injury (neuropathic pain model). we identified several proteins with marked changes in their redox states after nociceptive stimulation. our results show that the oxicat method is an efficient method to detect redox modifications in proteins and that redox modifications seem to play a role in pain processing. supported by the deutsche forschungsgemeinschaft (sfb /a ). additive antinociceptive effects of a combination of vitamin c and vitamin e after peripheral nerve injury lu r., kallenborn-gerhardt w., geisslinger g., schmidtko a. pharmazentrum frankfurt/zafes institute of clinical pharmacology, goethe university, frankfurt am main, germany accumulating evidence indicates that increased generation of reactive oxygen species (ros) contributes to the development of exaggerated pain hypersensitivity during persistent pain. in the present study, we investigated the antinociceptive efficacy of the antioxidants vitamin c and vitamin e in mouse models of inflammatory and neuropathic pain. we show that systemic administration of a combination of vitamins c and e inhibited the early behavioral responses to formalin injection and the neuropathic pain behavior after peripheral nerve injury, but not the inflammatory pain behavior induced by complete freund's adjuvant. in contrast, vitamin c or vitamin e given alone failed to affect the nociceptive behavior in all tested models. the attenuated neuropathic pain behavior induced by the vitamin c and e combination was paralleled by a reduced p phosphorylation in the spinal cord and in dorsal root ganglia, and was also observed after intrathecal injection of the vitamins. moreover, the vitamin c and e combination ameliorated the allodynia induced by an intrathecally delivered ros donor. our results suggest that administration of vitamins c and e in combination may exert synergistic antinociceptive effects, and further indicate that ros essentially contribute to nociceptive processing in special pain states. - , - and - ) replaced by leucine residues. both amino acids are comparable in terms of hydrophobicity, volume and the preference for forming α-helices, but only methionine is oxidizable to a sulfoxide, in contrast to leucine. in the present study we examined the protein-protein interaction (ppi) of recombinant ac , expressed in sf insect cell membranes, with cam, cam- , - , - and - by measuring the catalytic activity of ac . cam-mutants show a - -fold lower potency than cam, but they are more efficacious than cam. most prominently, cam- was % more efficacious than cam. such striking differences between cam and cam-mutants have not yet been observed for other mammalian effector proteins. as a result of the exchange of all methionine against leucine residues in cam- , it is more hydrophobic than cam and this leads to a better ppi with ac . in future studies we will examine the effects of cam inhibitors, antidepressants and antipsychotics on cam/ac interaction. furthermore we will analyze the effects of oxidized cam and cam-mutants on the catalytic activity of ac . because oxidative stress is of great importance in aging, it is important to know more about the abovenamed interaction in view to the demographic change. taken together, our data point to a unique cam/ac interaction that may be selectively targeted by small molecules. in particular, enhancers of these interaction could be useful to improve memory and learning. gender differences in fat distribution and diabetes prevalence in nzo mouse lubura m., scherneck s., zucker a., schürmann a. deutsches institut für ernährungsforschung experimentelle diabetologie, arthur-scheunert-allee - , potsdam, germany background: excessive fat accumulation in visceral but not subcutaneous fat depots as well as ectopic fat storage in liver, skeletal muscle and pancreas are associated with an increased risk for the development of type diabetes in humans. in this study we aimed to examine the influence of early fat distribution on onset of type diabetes in mice. methods: nzo mice are regarded as insulin resistant model in which only males become diabetic. we used male and female mice fed with high-fat and standard diet. we determined fat distribution by computed tomography for three times and conducted oral glucose tolerance tests on two different time points. besides we assessed body weight and blood glucose levels on weekly basis. results: contrary to previous findings, we observed that not only male nzo mice on high-fat diet develop diabetes. blood glucose levels at the th week of age and total pancreatic insulin content indicated diabetes prevalence of % in males and % in females these results lead to the conclusion that high-fat diet counteracts protective action of estrogens against diabetes. inversely to the findings in humans, female mice tend to store more fat in abdominal region than males. there was no relationship between early accumulation of fat in abdominal region and onset of type diabetes. however, visceral fat was associated with liver fat in males as well as in females. furthermore, at the age of ten weeks hepatic fat content correlated with blood glucose levels (r² = . ) indicating that the early hepatosteatosis is a predictor for hyperglycemia. however, there was no correlation between hepatic insulin sensitivity (indicated by quantitative insulin sensitivity index-quicki) and amounts of hepatic fat we conclude that early hepatosteatosis does not predict for glucose intolerance in nzo mice. in the nzo mouse, the amount of liver fat but not the early fat distribution predicts for the later onset of type diabetes. further experiments are needed to examine the gender dependent differences in the diabetes prevalence of this mouse strain. with a prevalence of about - % non-alcoholic fatty liver disease (nafld) represents the most common liver disorder in europe. nafld manifestation ranges from steatosis through steatohepatitis (nash) to fibrosis and cirrhosis, followed in some cases by liver failure and hepatocellular carcinoma. fatty degeneration of liver cells, increased oxidative stress with concomitant lipid peroxidation and an induction of pro-inflammatory cytokines are proposed as possible causes for developing inflammation and fibrosis, but the exact pathogenesis of the progression of nafld into nash is still unknown. thus, besides life style modifications and weight reduction interventions, no established pharmacological therapy exists so far. to gain further insights into the pathogenesis of nafld and nash and to develop new therapeutic strategies, appropriate animal models are essential. thus, in the present study three different dietary animal models for nafld were evaluated and compared to the biochemical and metabolic alterations seen with nafld and nash in man. male adult lewis rats were given standard food or one of three different diets: fatty liver diet [fld] , methionine/choline deficient diet [mcd] or methionine/choline deficient plus high fat diet [mcd+hf] . after , , , or weeks of treatment, animals were sacrificed and body and liver weights, laboratory parameters (asat, alat) as well as histopathological changes in the livers and different parameters indicating oxidative stress or representing the biotransformation capacity of the livers were analyzed. with fld and mcd+hf a normal body weight gain was observed, whereas with mcd body weight gain was strongly impaired. liver weights were mainly increased after mcd+hf. elevation of asat and alat values and hepatic steatosis were more pronounced after mcd and mcd+hf than after fld. all three diets caused an increase in the oxidative stress in liver tissue, but especially with mcd a tremendous elevation in the hepatic levels of lipid peroxidation products was seen. with regard to liver biotransformation capacity, with all three diets mainly an induction of the cytochrome p e and a isoforms expression and activity was observed, which was most pronounced after mcd and mcd+hf. in summary, the changes induced by mcd or mcd+hf most closely resemble the alterations described in literature for nafld in man and thus should be preferred over fld in future investigations on nafld and nash. ep receptors for prostaglandin e convey stimulatory and inhibitory effects. e.g., their stimulatory effect leads to vasoconstriction in the human pulmonary artery and their inhibitory activity to reduction of neurotransmitter release from neuron endings. the aim of our study was ( ) the pharmacological characterization of ep receptors in human pulmonary arteries and ( ) the examination of the involvement of these receptors in the regulation of the neurogenic tachycardia in pithed rats. l- served as the ep antagonist. experiments were performed in human pulmonary arterial rings isolated from patients undergoing lobectomy during resection of lung carcinoma and in pithed and vagotomised rats. the ep /ep agonist sulprostone ( nm - mm) concentrationdependently contracted human pulmonary artery rings (pec and emax; . ± . and . ± . %, relative to the contraction induced by kcl mm). the concentrationresponse curve of sulprostone was not affected by the ep antagonist sc ( µm) but shifted to the right by l- ( µm) (apparent pa . ). extending the exposure time to l- from . to h increased its antagonistic potency to . (schild plot-based pa ; concentrations . , and µm). in pithed rats electrical stimulation ( . hz, ms, v for s) of the preganglionic sympathetic nerve fibers or intravenous isoprenaline ( . nmol/kg) increased heart rate (hr) by beats/min. sulprostone ( - nmol/kg) did not affect the isoprenaline-induced increase in hr but inhibited the neurogenic tachycardia dose-dependently, maximally by %. l- ( µmol/kg) diminished the inhibitory effect of sulprostone nmol/kg on the neurogenic tachycardia by %. in conclusion, ep receptors ( ) located postsynaptically strongly contract human pulmonary arteries and ( ) located presynaptically on sympathetic nerve fibres supplying the heart of rats strongly inhibit the neurogenic tachycardia. - -bromo-n- [ -( - voltage-gated ca + channels of the central nervous system control a multitude of ca + dependent processes such as neurotransmitter release, neuronal excitability, neurite outgrowth, synaptogenesis, plasticity and neuronal survival. the cav . ca + channelalso known as p/q-type channel -belongs to the subfamily of high voltage activated ca + -channels. ca + influx via cav . ca + channels located at presynaptic nerve terminals triggers vesicle fusion and transmitter release at brain synapses and at the neuromuscular junction. thus, cav . ca + channels play a crucial role in synaptic transmission. the global cav . knock-out phenotype is characterized by severe ataxia, dystonia and lethality during the first postnatal weeks and is therefore an unsuitable model to analyze the importance of cav . ca + channels for learning and memory. therefore, we crossed a floxed cav . mouse line with nex-cre transgenic mice to establish a viable, forebrain specific knock-out mouse line (fbko-mice). results from western blot analysis confirmed an efficient knock out of cav . in hippocampal and cortical preparations, whereas the expression level in the cerebellum was not altered. to investigate the specific role of cav . channels in hippocampus and neocortex dependent behavior, we performed tests for motor functions and sensory abilities and in particular learning and memory tasks. mice with a forebrain specific cav . knock-out show significant deficits in spatial learning & reference memory and a significant reduced recognition memory as revealed by the morris water maze and an object recognition task. the fbko-mice exhibit no obvious locomotor deficits during behavioral tasks in the open field test and elevated plus maze. some fbko-mice demonstrate episodes of seizures in the morris water maze and during different rotarod tasks. to assess motor-function of fbko-mice in a stress reduced environment, we performed home cage based running-wheel motor-learning tasks. in summary, the diverse phenotypes of the forebrain specific knock-out mouse line emphasize the critical importance of cav . for learning and memory. helicobacter hepaticus-infected rag -/mice emulate many aspects of human inflammatory bowel disease (ibd), including the development of colitis and colon cancer [erdman et al., , pnas : - . toward the goal of elucidating mechanisms of inflammation-induced carcinogenesis and developing biomarkers of inflammation, we undertook a comprehensive analysis of macromolecular damage products during disease progression in h. hepaticus-infected rag -/mice. infected mice developed severe colitis and hepatitis, accompanied by infiltration of myeloperoxidase-positive neutrophils and f / -positive macrophages, by wks postinfection (pi), progressing into colon carcinoma by wks pi. qpcr array-based gene expression profiling revealed that pathophysiological changes were associated with characteristic alterations in the expression of genes related to inflammation, dna repair, and oxidative stress response. to study inflammation-related macromolecular damage, colon and liver tissues were analyzed by isotope-dilution chromatography-coupled mass spectrometry to quantify a battery of different dna, rna and protein damage products thought to represent the full spectrum of inflammation-related chemistries. our data revealed a significant predominance of chlorinated dna-, rna-, and protein damage products by weeks pi. in contrast, levels of damage products arising from oxidation, nitration and nitrosation changed only modestly or remained unchanged. our analyses also revealed higher levels of damage products in rna than in dna and demonstrated organ-specific differences of oxidative damage products, such as -oxo-dg and its oxidation products spiroiminodihydantoin and guanidinohydantoin. collectively, these results suggest that neutrophil and myeloperoxidase-induced chlorination chemistry may serve as a biomarker of ibd and may play important roles in the pathophysiology of ibd and colitis-associated cancer. characterization of a membrane protein expressed in mouse heart and brain mannebach s. recently, a novel membrane protein in drosophila was shown to be localized in presynaptic vesicles. it appears to mediate a ca influx after vesicle fusion with the plasma membrane. disruption of the corresponding gene leads to endocytic defects in drosophila [ ] . apparently, this protein plays a role in exo-and endocytosis and could serve as a ca channel supplying ca required for endocytosis. we have identified a protein in mouse, c rf , which shares , % amino acid sequence identity with the drosophila protein. it covers amino acid residues. using rt-pcr the full length transcripts could be identified in brain, kidney, pancreas, heart, spleen, thymus and mast cells. coexpression of c rf and the ca v . channel in xenopus oocytes reduced the amount of the α b and cavβ subunits of the ca + channel in the plasma membrane but did not affect the gating properties of the cav . channel. expression of c rf alone did not yield any channel activity. we therefore started to produce recombinant protein using the his-sumo-prokaryotic expression vector. the protein was efficiently expressed as his-sumo-c orf -fusion in e.coli (yield mg at mg/ml). we are currently preparing the c orf part of the his-sumo-c orf fusion protein by ulp -protease digestion followed by various chromatographic steps. the purified recombinant protein will be used to immunize rabbits to get antibodies. in parallel we generated antisera by immunizing rabbits with peptide fragments derived from the c orf sequence. we could not identify any homologues of c orf in the mouse genome and to analyze its function we are currently generating c orf deficient mouse lines by gene targeting. we have chosen a strategy for conditionally inactivation of the gene with the option to study the cellular localization of c orf by expression of the bgalactosidase gene under the control of the endogenous c orf promoter. by southern blot analysis we´ve already identified homologous recombinant embryonic stem cell clones out of analyzed ones and we will proceed with blastocyst injection to get chimeric mice and finally mice carrying the introduced mutations in the c rf gene. parps are involved in various biological processes such as regulation of dna repair, cell cycle progression, and cell death. consequently, several parp inhibitors are currently in clinical development as chemo-and radiosensitizers as well as monotherapeutic agents following the concept of synthetic lethality. pharmacological and toxicological studies call for an accurate analysis of parp activity in terms of a detailed knowledge of the structure of par and a reliable method for its quantification. we have developed a sensitive, precise, and accurate bioanalytical method based on liquid chromatography coupled to electrospray tandem mass spectrometry (lc/ms-ms) to characterize and quantify par with femtmol sensitivity: par is extracted from cells and hydrolysed to specific monomeric units, i.e., ribosyladenosine, which is characteristic for linear par, diribosyladenosine, which is characteristic for branching points, and adenosine, which represents the terminal part of the polymer. using this method, we are currently analyzing par levels in different cell lines and in primary human peripheral blood mononuclear cells (pbmcs) both under physiological conditions as well as upon genotoxic stress and in the presence of potent parp inhibitors. we expect that after completing method validation this assay will be useful for a wide range of applications in pharmacology and toxicology. gene mutagenic potential and metabolite profile of β-estradiol in cultured v cells expressing human cytochrome p a martínez jaramillo d., lehmann l. university of wuerzburg, institute of pharmacy and food chemistry section of food chemistry, am hubland, wuerzburg, germany oxidative metabolism of the female sex hormone β-estradiol (e ) is considered to play a major role in the initiation of hormone-induced carcinogenesis. in extrahepatic tissues, e undergoes metabolic activation by cytochrome p -dependent monooxygenase (cyp) isozyme a to -hydroxy-( -ho) and to a lesser extent to -ho-e . if not conjugated, these catecholestrogens (ce) can further oxidize to electrophilic quinones (q), which may react with dna and induce thereby mutations. conjugation of these ce in extrahepatic tissues is mainly catalyzed by catechol-omethyltransferase. in order to identify possible mutagenic metabolites (i) the induction of gene mutations by e was determined in male chinese hamster lung fibroblasts (v cells) expressing human (h) cyp a and (ii) the metabolite profile of e in these cells was analyzed via gas chromatography/mass spectrometry after solid phase extraction of the cell suspension in the culture medium. (i) gene mutations were assessed using the hypoxanthine-guanine phosphoribosyltransferase assay. the promutagen benzo[a]pyrene (bap) served as positive control requiring metabolic activation by hcyp a and dimethylsulfoxide as solvent control. v hcyp a were treated with nm e for weeks and the resulting -thioguanine ( -tg) resistant mutants selected at weeks (w) and . the frequency of spontaneous -tg resistant mutants per colony-forming cells ranged from ± (w ) to ± (w ). as expected, . µm bap induced a significant increase in mutant frequency (mf, ± , w and ± , w ) . treatment with nm e resulted in a -fold ( ± , w ) and a -fold ( ± , w ) increase in mf, suggesting slight mutagenic activity. in culture medium of v hcyp a treated with nm e , -ho-e , -methoxy-(meo)-e , -o-methyl- ho-e and -meo-e (suggesting intracellular formation of -ho-e ) were detected. while -meo-e concentration remained constant over the exposure period, the concentration of the other metabolites increased in a timedependent manner. the maximum concentration increase was reached at w for methylcatechols and at w for -ho-e , correlating with the maximum increase in mf, observed after weeks as well. in conclusion, e possessed a slight mutagenic potential after hcyp a -mediated activation to -, -ho-e and their corresponding methylcatechols. cumulative effects of three triazole fungicides in a broad dose range in vitro rieke s., kneuer c., bumke scheer m., lampen a., hirsch-ernst k., marx-stoelting p. bundesinstitut für risikobewertung chemikaliensicherheit, max-dohrn-str., berlin, germany consumers are exposed to multiple residues of different pesticides via the diet. this raises questions concerning potential cumulative effects, especially for substances causing toxicity by a common mode of action. the aim of this work was to investigate potential combination effects of the three triazole fungicides epoxiconazol, tebuconazol and flusilazol for selected parameters in a broad dose range in vitro. parameters investigated were cytotoxicity, hormone synthesis ( -β estradiol, progesterone and β-hcg), expression of a panel of androgen-or estrogen-responsive genes in the human placental choriocarcinoma cell-line jeg- and transactivation via estrogen receptors α and β in stably-transfected hek cells. the ability to inhibit steroidogenesis was analysed by measuring the concentrations of β-estradiol and progesterone in cell culture supernatants of jeg- cells. additionally, the placental peptide hormone β-hcg was measured. while no change in β-hcg and β-estradiol concentrations were observed, all triazoles induced a dose-dependent decrease in progesterone concentration and a cumulative effect was observed implying dose additivity at individual doses of > . µg triazole/ml. significant activation of erβ by the three triazoles, especially by flusilazol, was observed at µg triazole/ml and combined exposure showed additive effects, while no significant activation of erα was observed. based on the data, our findings suggest dose-additivity of triazole pesticides with the same mode of action for selected parameters in vitro. no significant effects were observed at lower doses [ ng - µg triazole/ml] neither for substances applied individually nor in combination. transient receptor potential channels as mediators of catecholamine release mathar i. trp proteins form cation channels that are regulated through strikingly diverse mechanisms. recently, genetic association studies identified many trp genes including trpm as risk factors for disease states such as arrhythmias, hypertension and cardiomyopathy. the melastatin trp channels trpm and trpm have distinct properties within the trp channel family; they form non-selective cation channels activated by intracellular calcium ions and are expressed in heart, aortic endothelial cells, kidney and adrenal gland. disruption of the trpm gene in mice leads to increased basal blood pressure without evidence for impairment of endothelium-or smooth muscle-dependent regulation of contractility of peripheral resistance vessels, the renin angiotensin aldosterone system, basal cardiac output or body fluid homeostasis. instead, trpm -deficient chromaffin cells exhibit increased acetylcholine-induced exocytosis of catecholamines which is associated with elevated level of epinephrine in the plasma and its metabolites in the urine. this indicates that trpm serves as an inhibitory regulator of exocytotic catecholamine release, at least in chromaffin cells. whether catecholamine release is also regulated by trpm in other cells of the sympathetic nervous system such as perivascular neurons still needs to be clarified as well as the molecular mechanism underlying how trpm regulates catecholamine release. besides trpm we recently identified transcripts encoding additional trp channels including trpc and trpc in chromaffin cells isolated by laser capture microdissection but their functional role in these cells is still unknown. measurements of the time course of the intracellular calcium concentration before and during acetylcholine stimulation ( µm) of catecholamine release as well as the analysis of the number of released vesicles in chromaffin cells relvealed no changes in trpc /c /c triple knock out mice compared to wildtype controls. although it seems that these trpc proteins are not directly involved in catecholamine release from chromaffin cells induced by acetylcholine application in our hitherto existing experiments, their contribution to the modulation of catecholamine release by agonists of gq-coupled receptors still needs to be analysed. aims: sulfonylureas (sus) are among the most widely used oral hypoglycaemic drugs that stimulate insulin secretion. in addition, sus have pleiotropic effects on other tissues. regarding the effects of sus on adipocytes conflicting findings were reported. we have now investigated the actions of glimepiride and glibenclamide (=glyburide) in primary human adipocytes. methods: primary cultured human white pre-adipocytes were differentiated in vitro according to a standard protocol. lipid accumulation was assessed by oil red o staining and determination of triglyceride content; gene expression was measured by real-time pcr and western blotting. results: we initially characterized the genes regulated during human preadipocyte differentiation by a global microarray analysis. treatment with glimepiride and glibenclamide caused a strong accumulation of lipid droplets and an increase in triglyceride content. genes involved in lipid metabolism were induced, chemokine expression was decreased. interestingly, the effects of sus were over all qualitatively and quantitatively similar to pioglitazone. in direct comparison glibenclamide was more potent than glimepiride in respect to the induction of fabp (ec . vs. . µm), an important adipocyte marker gene. su-induced differentiation was virtually completely blocked by the pparγ-antagonist t but not affected by diazoxide, indicating pparγ activation by sus. repaglinide, causing insulin liberation like sus but being structurally different, had no effect on adipocytes. conclusions: in primary human pre-adipocytes, glibenclamide and glimepiride strongly induced differentiation, apparently by activating pparγ . thus, sus but not repaglinide may be used to influence insulin resistance beyond their effect on insulin liberation. the role of at a and at b receptors as mechanosensors in myogenic vasoconstriction blodow s. , schneider h. arterial myogenic tone denotes the intrinsic property of vascular smooth muscle cells to constrict in response to an elevated intraluminal blood pressure. this physiological reaction is more distinct in small resistance arteries than in large conduit arteries. understanding the underlying mechanisms should provide useful information for the treatment of diseases like anaphylactic shock and systemic hypertension in which this reaction is altered. whereas the underlying signaling cascade has been extensively studied, the molecular identity of the mechanosensory elements still remains elusive. recent studies at the cellular level suggest a sensory function for a subgroup of gprotein coupled receptors (gpcrs) coupling to gq/ -proteins. by determining mrnaexpression levels of selected gpcrs in consecutive pairs of resistance and conduit vessels, we could identify a subset of gq/ -coupled receptors such as angiotensin ii at b, vasopressin v a, endothelin eta and etb and α a adrenoceptor significantly enriched in resistance vessels. by pharmacological blocking of those highly expressed gpcrs by different antagonists and inverse agonists, we evaluated their influence on the formation or the intensity of myogenic tone, as measured in isolated murine mesenteric arteries ex vivo. while blocking of v a receptor and α a and α ab adrenoceptors showed no differences of myogenic tone, blocking of at a and at b receptors by losartan and candesartan, eta receptor by bq and α a adrenoceptor by prazosin caused significant reductions of the vascular response. analyzing the myogenic response of at a -/mice with and without additional blocking of at b receptors by candesartan suggested that especially at b receptors play a dominant role for mechanosensitivity in mice. this was further supported by investigating the myogenic response of at b -/mice. these findings suggest that mechanosensitive gq/ -protein coupled receptors, especially at b receptors, play a dominant role for the development of myogenic vasoconstriction. trpm ion channels are activated by steroidal compounds and noxious heat and are considered to be involved in insulin secretion and pain perception. the expression of the trpm gene generates a variety of different transcripts which arise by alternative splicing and the use of different promoters [ ] . they encode a substantial variety of isoformes and so far we have identified more than distinct trpm proteins in mouse and rat each varying in exons , , , , , and . these variants differ enormously in their biophysical properties. for example splicing within exon affects the channel pore and causes significant changes of the ionic selectivity of trpm channels [ ] , whereas splicing of nucleotides encoded by exon leads to dormant trpm proteins. however, the frequency of these different isoformes in trpm expressing tissues is completely unknown. to get insight into the significance of the different trpm isoformes we investigated the abundance of alternative trpm transcripts in different tissues and cell types by reverse transcription quantitative pcr (rt-qpcr). we found that the frequency of splicing within exon ranges from up to % in different cell types and tissues. furthermore we analyzed the trpm transcriptome in the choroid plexus of the brain and the pituitary gland, tissues in which trpm transcripts are most abundant. for that purpose we sequenced more than clones, each. corresponding to our rt-qpcr result, we found a significant number of transcripts lacking exon . in cells of the choroid plexus nearly all ( / clones) carried the short ca + permeable pore. furthermore, we identified seven variants spliced in exon encoding truncated trpm proteins. however, the composition of the trpm transcriptome in the choroid plexus and pituitary gland differed enormously, indicating the importance of alternative splicing for trpm function in different tissues. the concept of "thresholds of toxicological concern" (ttc) defines tolerable dietary intakes for chemicals without toxicity data and is widely applied to chemicals present in food in low concentrations such as flavorings. based on a statistical evaluation of the results of many toxicity studies and considerations of chemical structures, the ttc concept derives a maximum daily oral intake without concern of , or µg/person/day for non-genotoxic chemicals depending on the allocation to so-called cramer classes i, ii or iii. for substances with a structural alert for genotoxicity a ttc value of . µg/person/day might be used. recently, it has been investigated, whether the ttc values, which were derived based on mostly chronic oral dietary rodent studies would cover all relevant toxicities (neurotoxic, repeated dose, reproductive and developmental, immune effects and endocrine-related effects). several authors using different specific databases have confirmed that the ttc values derived using cramer classes are also covering immunotoxic, neurotoxic, reproductive and developmental effects. a respective decision tree is going to be presented, also considering substances or substance classes which shall be excluded from the ttc approach. there are several areas in which the ttc concept is already used, or a ttc approach is considered useful, to assess low-level human exposures, or help in prioritizing toxicological testing; as for example the assessment of plant metabolites and degradates of pesticide active substances, feed and food additives, chemicals with a low exposure profile under reach, residues, metabolites and impurities in plants, chemicals, plant protection products or pharmaceuticals. if no structural alert for genotoxicity is given or standard genotoxicity tests are negative the cramer class iii value of µg/person/day, which corresponds to a dose of . µg/kg bw is considered to represent a chronic tolerable daily intake of the test substance. examples for current and future uses of the ttc concept in regulatory toxicology are presented. objective: hyaluronan (ha), synthesized by three ha-synthases (has , - , - ), is a prominent matrix component of atherosclerotic lesions. the aim of the present study was to identify the has isoenzyme that is associated with ha-matrix remodeling in inflammatory regions of atherosclerotic plaques. furthermore the underlying regulatory pathways were determined and functional aspects of this regulation in vascular smooth muscle cell (vsmc) were addressed. methods and results: during atherosclerosis in apoe deficient mice the peak of macrophage invasion at weeks coincided with ha deposition and induction of has in aortic root plaques. in human symptomatic carotid artery plaques has was by far the most prominent has isoenzyme as determined by quantitative real time rtpcr. in vitro, in human vascular smooth muscle cell (vsmc) has was specifically induced via activation of nfkb by interleukin- β (il- β) and tumor necrosis factor alpha (tnfa) as shown by chip assay and utilization of nfkb inhibitor bay - . has was also upregulated in a co-culture system by activated macrophages via paracrine release of tnfa and il- β as verified by neutralizing antibodies. in human atherosclerotic lesions nfkb positive vsmc were frequently detected in close proximity with ha and f / positive macrophages as shown by immunohistochemistry. to study the effects of has mediated ha synthesis in human coronary vsmc, lentiviral overexpression and knockdown of human has were employed. overexpression of has resulted in increased migration and proliferation whereas knock down had the opposite effect. the effects of has were mediated by both pi k signaling and mapk signaling via hyaluronan receptors cd and rhamm. conclusion: the present results suggest that has -dependent ha synthesis is induced in human vsmc by inflammatory cytokines released from activated macrophages. moreover, has -mediated ha production induced phenotypic activation of vsmc. pulmonary inflammation and airway remodeling are major features of chronic obstructive lung disease (copd). in addition, pulmonary hypertension is a common comorbidity, which is associated with a poor prognosis of the disease. recent studies in a guinea pig model of allergic asthma have shown that increased arginase activity, which converts larginine into l-ornithine and urea and competes with nitric oxide synthases for the common substrate, contributes to allergen-induced airway inflammation, hyperresponsiveness and remodeling. there is evidence that cigarette smoke and lipopolysaccharide (lps), both involved in the pathogenesis of copd, increase the expression of arginase, however, its role in the pathogenesis of copd is currently unknown. this study aimed to investigate the role of arginase in pulmonary inflammation and remodeling, using a guinea pig model of lps-induced copd. to this aim, guinea pigs were instilled intranasally with lps or saline twice weekly for weeks and were pretreated by inhalation of the arginase inhibitor ( )s-amino-boronohexanoic acid (abh) or pbs. repeated lps exposure increased lung arginase activity, resulting in increased lornithine/l-arginine and l-ornithine/l-citrulline ratio's. both ratio's were reversed by abh treatment. repeated lps exposure also induced increased il- levels, neutrophils, goblet cells, hydroxyproline and airway collagen content in the lung, which were all abrogated by abh. moreover, repeated lps exposure increased right ventricular mass, indicative of pulmonary hypertension, which was similarly prevented by abh. in conclusion, increased arginase activity contributes to pulmonary inflammation, airway remodeling and right ventricular hypertrophy in a guinea pig model of copd, indicating that arginase inhibitors may have therapeutic potential in the treatment of this disease. (supported by msd). behavioral abnormalities in hcn -deficient mice michalakis s., schöll-weidinger m., mader r., cao-ehlker x., fenske s., wahl-schott c., biel m. center for integrated protein science munich (cipsm) department of pharmacy -center for drug research, ludwig-maximilians-universität münchen, butenandtstr. - , münchen, germany hcn encodes a hyperpolarization-activated and cyclic nucleotide-gated channel, which is expressed in various brain regions including thalamic, hypothalamic and habenular nuclei as well as brain stem and olfactory bulb. in this study we performed a comparative analysis of hcn -/and hcn +/+ mice using a battery of behavioral tests and telemetric biopotential measurements to evaluate a potential role of hcn in central nervous system function. in general, the knockout mice showed normal motor function as assessed by the rotarod and open field tests. telemetric home cage activity and core body temperature measurements confirmed a normal circadian behavior, but revealed a lower basal activity that concurred with decreased body temperature during the light phase and the light-dark transition phase. hippocampus-dependent spatial learning was normal. by contrast, hcn knockout mice showed more immobility than control mice on day two of the porsolt forced swimming test, which could reflect increased depressionlike behavior. however, center exploration in the open field test as well as performance in the light-dark transition and the elevated-plus maze tests was normal in hcn -/mice. this suggests that general anxiety was not changed in the knockout mice. in addition, hcn knockout mice were less active on the second day of the open field test, which supports a habituation phenotype. finally, hcn -/mice had higher burying scores in the marble-burying test, which is a test for certain aspects of obsessive compulsive disorder in rodents. taken together, genetic deletion of hcn in mice results in distinct behavioral abnormalities related to behavioral despair and expression of repetitive behaviors in response to mild stressors. mielke h. , gundert-remy u. alcohol consumption when breast feeding is discussed controversially. some groups recommend breast pumping before alcohol consumption and feeding the stored milk instead of breast feeding after drinking alcohol. this study was performed to simulate the blood concentration in the breastfed baby and to assess the health impact. method: we established a physiologically based kinetic model. its parameters were calculated (partition coefficients tissue/blood ; schmitt, ) silva et al., ) . we simulated . the alcohol concentration in a breastfed neonate and a -month-old suckling infant after the nursing mother had consumed alcohol, . the alcohol concentration in utero/fetal compartment during pregnancy assuming the identical alcohol consumption of the pregnant woman . the alcohol concentration during infant´s treatment of bloating by an approved herbal drug containing alcohol. results: peak maternal alcohol concentration was . ‰ after consuming . l of wine, peak concentration was . ‰ in the newborn, . ‰ in the -month-old infant and . ‰ in the utero/fetal compartment. the peak concentration after herbal drug treatment was . ‰ in the neonate and . ‰ in the -month-old infant, respectively. we discuss the results of the simulations and compare it with doses and published concentrations measured in experimental animals or in vitro studies. conclusions: we conclude that the recommendation " to glasses of wine on occasion" (agence nationale d'accréditation et d'Évaluation en santé, assante ) is in accordance with the simulation results presented here whereas stricter rules are not scientifically sound. ( ) http://www.has-sante.fr/portail/upload/docs/application/pdf/ breastfeeding_guidelines.pdf da silva et al. ( ) adenylyl cyclases (ac) mediate physiological responses in virtually all cells, where their regulation through receptors and g proteins results in the modulation of camp. in the present study we focused on the kinetics of interactions between the alpha-subunit from inhibitory g protein type (gαi ) and adenylyl cyclase type v (ac ). these proteins were labeled with cfp and yfp, respectively. the dynamics of their interactions was monitored by means of high temporal resolution fret imaging in hek cells expressing unlabeled a a-receptor and gβg subunits. to activate the signaling pathway, we applied agonist using a rapid superfusion device. application of norepinephrine resulted in the development of a fret signal, indicating interaction between gai -cfp and yfp-ac . after withdrawal of agonist the fret signal recovered with a remarkably slow time course compared to the deactivation kinetics of gi proteins reported previously (bünemann et al. ) . to further analyze the properties of the dissociation between gai and ac we measured in parallel the offset kinetics of the interaction between gai -yfp and gβg-cfp (gi -fret) after agonist withdrawal under comparable conditions. in addition we tested to what degree the coexpression of rgs accelerated the deactivation of gi proteins and the dissociation of gai -cfp from yfp-ac . these experiments revealed that in the absence of rgs the dissociation of gai from ac after agonist withdrawal takes about times longer than the deactivation of gi proteins. in the presence of rgs this difference is even larger due to the pronounced acceleration of g protein deactivation. the dissociation of gai from ac was only marginally accelerated by rgs . these observations lead us to hypothesize, that ac might trap activated g protein-subunits and thereby affect the g protein cycle by shifting the equilibrium towards activated g proteins. if this hypothesis is true, it should result in a left-shifted dose response curve compared to g protein activation dose response. in support of this hypothesis we found that the concentration response curve for gai -ac interaction was several-fold leftward-shifted compared to the concentration-response curve of gi-protein activation under very similar conditions. influencing the dynamics of the g protein cycle by effectors may represent a novel and powerful mechanism for finetuning the sensitivity of receptor evoked responses in an effector-specific manner. obesity, the excessive accumulation of white adipose tissue (wat), has reached pandemic dimensions. the factors that determine fat mass are not fully understood, but adipocyte hypertrophy and adipokine secretion are thought to be important. in present study, we investigated the role of the cyclic gmp (cgmp) signaling pathway focusing on cgmp-dependent protein kinase i (pkgi) in white adipocytes. pkgi is expressed in wat, preadipocytes and differentiated adipocytes as demonstrated by real-time pcr, western blot and immunochemistry. differentiation of pkgifl/fl preadipocytes, using an optimized protocol, resulted in an enhanced lipid accumulation as evidenced by oil red o staining. deletion of pkgi in pkgifl/fl adipocytes infected with a cre lentivirus (lv-cre, pkgi / ) exhibited reduced differentiation. analysis of the triglyceride (tg) content revealed a significant decrease of tg levels by % ± % in pkgi / as compared to pkgifl/fl adipocytes. western blot analysis of white adipocytes showed a significant decrease of c/ebpalpha ( % ± . %), ppargamma ( % ± . %) and ap ( % ± . %) expression in pkgi / cells as compared to pkgifl/fl. treatment of t -l cells with cgmp resulted in increased lipid accumulation and enhanced expression of fat marker genes. lentiviral overexpression of pkgi further increased differentiation. importantly, pkgi significantly induced mitochondrial biogenesis in t -l cells. concomitant activation of pkgi in t -l preadipocytes and treatment with the demethylating agent -aza-deoxycytidine significantly increased expression of uncoupling protein- (ucp- ) -a unique protein of brown fat cells. we found rhoa as major target of pkgi signaling with increased phosphorylation of rhoa at ser- in pkgi overexpressing cells. moreover, pkgi-dependent phosphorylation counteracts the effects of rhoa on insulin signaling as well as adipokine expression. taken together, pkgi is a key player in white adipocyte differentiation that regulates cell size and has an anti-inflammatory effect. pkgi decreases the secretion of proinflammatory adipokines via inhibition of rhoa signaling. in addition, activation of pkgi can establish a brown fat cell like phenotype during white adipocyte differentiation if the ucp- promoter is accessible. the rag gtpases, raga, ragb, ragc, and ragd form a subfamily gtpases of the ras-related superfamiliy. rag proteins are characterized by a modified ras-like gtpbinding domain and a unique c-terminal region lacking a lipid modification motif. interestingly, rag proteins have been proposed to function as heterodimeric complexes consisting of raga or ragb associated with ragc or ragd. rag gtpases have been implicated in the control of mammalian target of rapamycin (mtor) function, in particular in regulation of the nutrient-stimulated and/or hormone-regulated mtor activity. the protein kinase mtor is found as the catalytic subunit of two larger protein complexes referred to as mtor complex and , mtorc and . under amino-acidrich conditions, activated mtorc promotes protein synthesis and inhibits autophagy, while under starvation autophagy inhibition is released. increasing evidence suggests that activation of rag gtpases contributes to mtorc function. thus, rag proteins were found to be associated with a protein complex termed ragulator, a major regulatory protein of mtorc function and guanine nucleotide exchange of rag gtpases within the rag-ragulator-complex were described to promote mtorc translocation to its functional lysosomal compartment. however, the guanine nucleotide exchange properties of rag proteins are poorly characterized, and it is currently unknown, how amino acids promote rag proteins to facilitate the formation of the active, raptor-binding state of the rag heterodimers. to characterize the guanine nucleotide exchange properties of the rag gtpases as momomers or heterodimers in more detail, recombinant rag proteins were expressed in bacteria and purified from this source to near homogeneity. first, the parameters of gdp/gtp exchange of each of these proteins were compared using the non-hydrolysable gtp analogon gtpgs. the results showed that the rag isoforms are distinct in their guanine nucleotide exchange activities. in particular, nucleotide exchange on raga and ragc, but not on ragb and ragd, was only observed at low concentrations of gdp and mgcl in extraction and assay buffers, i.e. conditions favoring the gdp/gtp exchange. these findings may indicate that guanine nucleotide exchange on raga and ragc is controlled by guanine nucleotide exchange factors and suggest specific functions of the individual rag gtpases within individual rag heterodimers. in in-vitro studies on rat and canine mast cells and human mast cell leukemia cells hmc . bz at micromolar concentrations inhibited mediator release which appeared to be related to an inhibition of the intracellular camp pathway. in order to identify potential targets on/in mast cells at which bz may cause an inhibitory effect on mast cell activation, the , -bz flunitrazepam (flu), clonazepam (clo) and chlorodiazepam ( -cd) were selected because of their different affinity and selectivity to/for the gaba-a-receptor and the translocator protein (tspo): flu and clo bind with nanomolar affinity to gaba-a receptors, whereas -cd is a selective ligand at tspo with nanomolar affinity to tspo but only micromolar affinity to gaba-a receptors. flu also possesses nanomolar affinity to tspo, whereas clo has no or only micromolar affinity to tspo. after incubation of hmc . cells with -cd, flu and clo for , and hours up to genes were significantly differently expressed in a substance-specific and timedependent manner. comparison of the genes differently expressed at hours revealed that the expression of genes was regulated by both flu and clo but only genes were regulated by both -cd and flu suggesting that flu and clo induce gene expression by acting at a target site different from that of -cd. the difference between the gene regulation by flu and clo on the one hand and that of -cd on the other hand is also reflected in pathway analysis. since it was conceivable that the beneficial effects of the , -bz could be mediated by the recognition sites targeted by the , -bz, i.e. the gria -encoded ionotropic glutamate receptor ampa , we investigated by quantitative pcr whether hmc . cells express gria , tspo, the genes encoding the subunits of the gaba-a receptor and the gaba-forming enzyme glutamic acid decarboxylase. tspo, gabra , gabrb , gabre and gabrd were moderately expressed. in addition, there was a week or very week expression of gabra , gabra , gabrb , gabrg and gabrr . expression of gria was not detectable. taken together, it cannot be decided yet from our data whether the inhibitory effect of benzodiazepines on mast cell activation is due to an action at tspo or at gaba-a receptors of a novel subunit composition. monien b. h., glatt h. german institute of human nutrition (dife) department of nutritional toxicology, arthur-scheunert-allee - , nuthetal, germany -hydroxymethylfurfural (hmf) and furfuryl alcohol (ffa) are common constituents of foodstuffs in which they are formed by heat-and acid catalyzed reactions from carbohydrates. hmf and ffa have been reported to induce the formation of hepatocellular adenomas in female mice and renal tubule neoplasms in male mice, respectively. we studied whether the carcinogenic effect of these hydroxymethylsubstituted furans may originate from sulfotransferase (sult)-catalyzed formation of electrophilic esters. hmf was inactive in in vitro mutagenicity tests using standard activating systems. in contrast, it was mutagenic in v cells genetically engineered for expression of human sult a suggesting that hmf is converted into the reactive sulfooxymethylfurfural (smf). following incubation of mutagenic smf with porcine liver dna in vitro, specific methylfurfural adducts were detected using liquid chromatography tandem mass spectrometry (lc-ms/ms), i.e., n -(( -formylfuran- -yl)methyl)- 'deoxyadenosine (n -ffmda) and n -(( -formylfuran- -yl)methyl)- '-deoxyguanosie (n -ffmdg). these adducts were also detected in dna from v -sult a cells incubated with hmf. in order to determine sulfo conjugation of hmf in mice in vivo, we conducted pharmacokinetic measurements showing that about ppm of the hmf dose was converted to smf and reached the circulation. like hmf, ffa was negative in the standard ames test and various other in vitro genotoxicity tests. we showed that ffa is mutagenic in salmonella typhimurium ta engineered for expression of human sult a . the putative mutagen -sulfooxymethylfuran was synthesized and incubated with porcine liver dna, in which various nucleoside adducts were found. the main adducts, -mfda were detected in dna of ffa-exposed salmonella strain ta -sult a and in dna of liver, lung and kidney of fvb/n mice that had received about mg ffa/kg body weight per day via the drinking water for days. in summary, both furan derivatives form mutagenic sulfate esters in vitro and in vivo. in the future, we will use genetically engineered mice to characterize the role of single murine and human sult forms in the bioactivation of the furan derivatives and the contribution to tumor induction. background: micrornas are small non-coding rnas that can negatively regulate gene expression on a post-transcriptional level and have been shown to interact with epigenetic mechanisms like dna methylation. mecp (methyl cpg binding protein ) is a protein that binds methylated dna cpgs in the promoter region of genes and can thus regulate their expression. otherwise, mecp is known to be a target gene for several micrornas including the cluster mir- / in the brain. recently, our group could show that mecp expression is downregulated in human heart failure suggesting that mecp might be involved in cardiac pathogenesis. the aim of this project is to study the upstream regulation of mecp by the cluster mir- / in the heart during cardiac hypertrophy in-vitro and in-vivo. methods and results: to test whether hypertrophic stimuli can induce mir- / expression, we treated cultured nrcms with µm norepinephrine for hours. this induced cardiomyocyte hypertrophy and expression of the hypertrophy marker nppa, but also of mir- ( . ± . -fold of untreated cells, p< . ) and of mir- - p ( . ± . -fold of untreated cells, p< . ) and downregulated mecp mrna and protein levels ( . ± . -fold of untreated cells, p< . ). to check whether mecp downregulation also occurs by direct mir- / activation we increased levels of mir- and mir- in cardiac myocytes by transfecting precursor mir- and mir- - p molecules. again, we observed nrcm hypertrophy, nppa mrna upregulation and mecp mrna and protein downregulation ( . ± . -fold of control, p< . ) after mir- overexpression. similar results were obtained by overexpression of mir- - p. to test the effects of adrenoceptor activation on the mir- / -mecp axis in-vivo, wild-type mice received isoprenaline and phenylephrine via osmotic minipumps ( mg/kg/day each). after days, cardiac ventricles were analyzed. nppa gene expression ( . ± . -fold of control animals), mir- and mir- - p levels ( . ± . and . ± . -fold of control animals, p< . and p< . , respectively) were increased while mecp protein levels decreased to % (p< . ) conclusion: these results suggest that in-vitro and in-vivo adrenoceptor stimulation leads to the activation of mir- / expression and to downregulation of mecp in cardiac myocytes in-vitro and in-vivo. leopold-franzens-universität, innsbruck, austria at- receptor antagonists block the angiotensin ii-enhancing effect on noradrenaline release from sympathetic neurons. in a cell-free assay the binding affinity of the at- receptor antagonists telmisartan and valsartan to the gamma peroxisome proliferatoractivated receptor (pparγ) is close to that of the pparγ selective agonists thiazolidinediones (tzds). we tested whether the tzds rosiglitazone and pioglitazone would also modify the prejunctional facilitatory effect of angiotensin ii. left ventricular slices of rats were incubated with tritiated noradrenaline, perifused and electrically stimulated. the negative logarithm of the drug concentration that caused a % increase of control (pec %) was calculated. angiotensin ii caused a concentration-dependent increase of tritium overflow induced by electrical stimulation [pec %= . ± . (mean±sem, n= ); maximum increase= ± %]. neither rosiglitazone nor pioglitazone ( . - µm) had a direct effect. the concentrationresponse to angiotensin ii in the presence of fixed concentrations of rosiglitazone was shifted to the left with increase of the maximum (pec %= . ± . , . ± . and . ± . ; maximum increase= ± %, ± % and ± %, in the presence of . , and µm of rosiglitazone, respectively, n= - , each). in contrast, pioglitazone in concentrations up to µm had no effect on the release-enhancing effects of angiotensin ii. results show that rosiglitazone but not pioglitazone potentiates the noradrenalinerelease enhancing effect of angiotensin ii. this action might contribute to the risk for myocardial infarction from rosiglitazone use but not from pioglitazone use. deleted in liver cancer (dlc ) is a tumor suppressor whose allele is lost in % of liver, breast, lung and % of colon cancers. despite its significance, the molecular mechanisms that drive cancerous transformation upon dlc loss remain unclear. we found that the transcriptional coactivators megakaryoblastic leukemia and (mkl / ) are constitutively localized to the nucleus in hepatocellular and mammary carcinoma cells that lack dlc . moreover, dlc loss and mkl nuclear localization correlated in primary human hepatocellular carcinoma. nuclear accumulation of mkl in dlc -deficient cancer cells was accomplished by activation of the rhoa/actin signaling pathway and concomitant impairment of erk-mediated mkl phosphorylation. dlc loss led to constitutive activation of the mkl-dependent, tumor-relevant target genes ctgf, cyr , myl and myh . furthermore, we identified a novel target gene, integrin a , with a key role in cell migration and metastasis, that exhibited a dlc -and mkldependent regulation. depletion of mkl / suppressed not only cell migration, but also cell proliferation and anchorage-independent cell growth induced by dlc loss. our data provide insight into the mechanism by which dlc loss initiates tumorigenesis. as mkl and have a key role in this process, this pathway may provide promising pharmacological targets for cancer therapy. universität bonn, pharma-zentrum bonn pharmazeutisches institut, pharm. chemie i, an der immenburg, bonn, germany membrane receptors activated by purine and/or pyrimidine nucleotides ("p receptors") are widely distributed in the body and constitute novel (potential) drug targets. they are subdivided into g protein-coupled p y receptors (p y , , , , , , , ) , and homo-or heterotrimeric ligand-gated ion channel or p x receptors (subunits: p x - ). we have been interested in the identification and development of potent and subtype-selective ligands -as tool compounds and potential drugs -for the various p y and p x receptor subtypes. our strategy involves (i) establishment of a proprietary compound library consisting of synthetic small molecules and natural products; (ii) development of screening assays suitable for medium throughput screening; (iii) careful analysis of structure-activity relationships at each target and systematic optimization of the lead structures; (iv) pharmacological evaluation of selected compounds. this approach has led to new biological tools for several targets, including p y and p x receptors [ ] [ ] [ ] [ ] [ ] [ ] . fine particles in particulate matter (pm) are effective vehicles to transport toxicants into the lung; depending on their size, smaller particles may reach the bronchiolar or alveolar space. in recent years the pm fraction pm . has especially been correlated with both pulmonary and cardiovascular diseases. in order to better characterize pm emission and distribution of environmental tobacco smoke (ets) from cigarettes (reference cigarette (rc), brand cigarette (bc)) we have developed an ets emitter to simulate human smoking emission and measured pm . concentration in a telephone booth ( , m volume) as an example for small indoor spaces like cars. fine particulate matter was measured using an aerosol spectrometer with sec time resolution; laser scatter allowed a size resolution from , µm to µm. for the pm . concentration the following values were calculated: cumulative pm . concentration as auc-pm . (µg/m /sec), peak pm . concentration as cmax-pm . (µg/m ) and average pm . concentration cmean-pm (µg/m ). in closed door condition both cigarettes produced particulate auc-pm . values of ± µg/m /sec (rc) to ± µg/m /sec (bc after myocardial infarction (mi) inflammatory cells and cardiac fibroblasts (cf) determine the remodeling response. interleukin- (il- ) is induced in the ischemic myocardium and is known to stimulate the differentiation of fibroblasts to myofibroblasts. hyaluronan (ha) is an extracellular matrix component synthesized by ha-synthase isoenzymes (has - ) and is also known to control fibroblast phenotypes. however, it is presently unknown whether il- participates in the remodeling of the ha-matrix or whether the ha-matrix modulates the responses to il- . therefore, the aim of the present study was to elucidate whether il- regulates the expression and function of ha-matrix in cfs. cells were isolated from c bl/ j mice and used during passage - for experiments. cfs were stimulated with il- or hyper-il- which is a fusion protein of il- and soluble il- receptor (sil- r). after and min signal transducer and activator of transcription (stat ) was phosphorylated in response to hyper-il- but not in response to il- . rt-pcr revealed rapid upregulation of has ( . ± . fold of unstimulated control, h) in response to hyper-il- . has was induced to a lesser degree ( . ± . fold of unstimulated control, h) whereas has was not responsive ( . ± . fold of unstimulated control, h). in contrast, il- had no effect on transcript levels of has isoenzymes. in turn, expression of has and has in response to hyper-il- was inhibited by ag , which indicates the involvement of stat signaling. interestingly, despite induction of has and has the amount of secreted ha as determined by an elisa-like assay was not affected by hyper-il- . this may indicate that il- regulates the cell surface associated ha-matrix of cfs. in conclusion, the present data demonstrate that cardiac fibroblasts respond to il- trans-signaling (hyper-il- ) via the soluble il- r and subsequent stat signaling with increased ha-synthesis. the fact that il- had no significant effect suggests that the expression of the non-signaling membrane-bound il- α-receptor (il- r) in cultured murine cardiac fibroblasts is not sufficient to induce has and - gene expression. therefore, il- trans-signaling mediated by il- and the circulating sil- r might be necessary to mediate the il- -induced has expression in vivo. mrgprd receptor endogenously expressed in dorsal root ganglia: evidence for an activation by -aminoisobutyric acid müller s., hoffmann k., von kügelgen i. universität bonn institut für pharmakologie und toxikologie, sigmund-freud-straße , bonn, germany the gpcr mrgprd (mrgd) is highly expressed in small diameter dorsal root ganglion (drg) neurons and has been implicated to play a role in nociception. the receptor was previously shown to respond to β-alanine. in the present study we searched for agonistic activity of structural analogues of β-alanine. for further characterization of the receptor we used fura- fluorimetry, a nfat luciferase reportergene assay and the determination of the inhibition of forskolin-induced camp production ([ h]-camp affinity assay). first, we confirmed the activation of the receptor by β-alanine and gaba. in reportergene experiments we then identified -dlaminoisobutyric acid as an agonist, with similar potency but weaker affinity when compared to β-alanine (ec µm). fura- fluorimetry showed an increase in intracellular ca + levels by -dl-aminoisobutyric acid ( µm). moreover, -dlaminoisobutyric acid reduced the forskolin-induced camp production by up to % (ec µm). in addition to -dl-aminoisobutyric acid, we identified -dl-aminobutyric acid as a weak agonist acting at the mrgprd. other closely related substances failed to show significant responses. next to the agonists we further characterized antagonists inhibiting the response to βalanine mediated by mrgprd. chlorpromazine shifted the concentration-response curve of β-alanine to the right with an apparent pkb of . (nfat assay), thioridazine with an apparent pkb of . (nfat assay) and . (camp assay) and rimcazole with an apparent pkb of . (nfat assay) and . (camp assay). in conclusion we show for the first time that -dl-aminoisobutyric acid is an agonist at the mrgprd and that the structure-activity relationship of agonists at mrgprd is very close. the sdf- -chemokine receptor cxcr plays a key role during embryogenesis and regulates functions of immune and stem cells in adult life. furthermore, cxcr is involved in disease states including inflammation and cancer. it is well established that sdf- -stimulated cxcr receptors activate gi protein-dependent signal transduction pathways and undergo c-terminal phosphorylation and internalization. because the cxcr c-terminal domain contains serine and threonine residues, it is incompletely understood which of the potential phosphorylation sites contribute to homologous and heterologous regulation of cxcr . here, we analyzed the phosphorylation pattern of cxcr at c-terminal sites after stimulation of the receptor with sdf- and after pma-induced activation of the pkc pathway as a model for heterologous receptor phosphorylation. using phospho-specific antibodies against s / , s / and s / in immunoblot analyses, we showed that the sites were phosphorylated after stimulation with sdf- or pma. stimulation with egf or forskolin did not induce phosphorylation at these sites. sdf- -induced phosphorylation at s / , s / and s / was reversible after wash out of the ligand. time course analyses revealed that phosphorylation occurred first at s / and then at s / and s / . taken together, these results indicate that the c-terminus of cxcr is phosphorylated at multiple sites by homologous and heterologous pathways and that phosphorylation at the different sites may be hierarchically organized. human milk represents the best form of nutrition for infants early in life. however, it can also contain toxic contaminants that may adversely affect infant's development. the nephrotoxin ochratoxin a (ota) is present in human milk (tab. in [ ] ), but information on transfer from maternal blood to milk is scarce: published data [ ] indicate that levels of ota in milk are roughly one tenth ( . ) of those in blood. but, the efficiency of the ota-transfer at various stages of breastfeeding may vary since studies in animals revealed that transfer of ota is apparently time-and dose-dependent. thus, the aim of this study was to assess the ota transfer from blood to milk at different stages of breastfeeding in humans. in a small chilean cohort, lactating women were asked to provide blood and milk on the same day. these samples were collected on four different occasions within the first months after delivery and analyzed using hplc with fluorescence and/or tandem mass spectrometric detection [ ] . the transfer of ota from blood to milk was quantitatively assessed by measuring the milk to plasma ratio (m/p). the average ota level in blood plasma was ± ng/l, and no major variations were observed over time (p = . ). on the other hand, ota levels found in colostrum ( ± ng/l) were higher than in mature milk (p < . ). in line with these data, higher m/p ratios (table) were obtained with samples collected in the first six days after delivery. this study showed that the transfer of ota from blood to milk was more efficient with colostrum (m/p . ± . ) than with mature milk. thus, a higher exposure to ota can be expected for neonates than for infants at later stages of breastfeeding. moreover, the lactating women have lower average ota levels in plasma than non lactating women from chile [ ] , indicative of milk as additional excretion route. acknowledgement: this work has been supported by a stipend from conicyt/daad to km. exposure of infants to ochratoxin a (ota) deserves particular attention since ota is nephrotoxic, and one of the most potent rodent renal carcinogen studied to date [ ] . moreover, infants may be more vulnerable to the toxic effects of contaminants than adults. ota-levels in plasma of infants are indicative of an early exposure in life [ ] . but blood sampling is an invasive method not readily applicable for breastfed infants. thus, the aim of this study was to implement a non invasive biomonitoring method to assess ota-exposure in this group. to assess the exposure to ota, breast milk and infants' urine specimens were collected, from two different cohorts: chile (n= ) and turkey (n= , only urine). analysis of the samples was performed using enzymatic hydrolysis prior to extraction and hplc-ms/ms [ ] . the magnitude of infants' exposure was assessed by calculating the ota-daily intake with human milk and relating it also to urinary ota levels. calculations of the daily intake with human milk [ ] showed that infants may be exposed to ota at high levels, exceeding the tolerable daily intake (tdi) of ng/kg-bw/day set for adults [ ] . in both cohorts, most of the urine samples tested positive for ota (chile %, turkey %). ota levels observed in urine samples from the turkish infants (range: - , ng/l) were fold higher than levels found in chilean samples (range positive samples: - ng/l). further analysis of phase ii metabolites in urine confirm the excretion of ota as conjugate (glucuronide) in highly exposed infants. in conclusion, ota exposure of infants early in life was documented. given that otaintake by several infants exceeded the tdi for adults, further biomonitoring in this vulnerable group is advised including also suitable biomarkers of effect. a mixture of (e)-and (z)-clomiphene citrate is the first line therapy of female infertility. however, up to % of patients do not respond. (e)-clomiphene is structurally closely related to another selective estrogen receptor modulator, tamoxifen which is frequently used for the treatment of hormone receptor-positive breast cancer. like tamoxifen, clomiphene is extensively metabolised by the cytochrome p system. using the estrogen receptor response assay (e)- -hydroxyclomiphene and (e)- -hydroxy-n-desethylclomiphene (ec : . and . nm, respectively) turned out to be times more active at the er compared to the parent drug isomers and de-ethylated metabolites. using recombinant expressed human cyp isoforms and inhibitory antibodies, cyp d revealed to be the major isoenzyme involved in the formation of -hydroxlated metabolites. n-deethylation was catalysed by cyps a / , d , c and c . rates of -hydroxylation in microsomes from human liver donors correlated with the number of functional cyp d genes. these in vitro results were confirmed in a pharmacokinetic study with female healthy volunteers receiving a single dose of clomiphene. in carriers of two non-functional cyp d genes (poor metabolizers) cmax of (e)- -hydroxyclomiphene and -hydroxy-ndesethylclomiphene was and times lower, respectively, when compared with subjects with at least one fully functional cyp d allele. in contrast, half-life of (e)-clomiphene and (e)-n-desethylclomiphene was and -fold higher, respectively, in poor metabolizers. our data provide first evidence of a pharmacogenetic rational for the variability in the response to clomiphene treatment. among the tested compounds, compound proved to be the most active derivative, showing a significant toxicity at a concentration of , µm. compounds and showed significant toxic effects at a concentration of µm. the compound showed no toxicity up to a concentration of µm. all derivatives , and have a ec between and µm. we further proved the induction of apoptosis by apo-one assay (caspase / activity) and life/dead-assay (fluorescence microscopy). in conclusion, these gold complexes exhibit an example of interesting potential candidates for future anticancer pharmaceuticals due to relatively high cytotoxicity. gene regulating effects in mouse liver subsequent to treatment with selected dioxin-like compounds and pcb using whole genome microarray analysis neser s. , lohr c. , van ede k. i. , andresen k. interaction with the aryl hydrocarbon receptor (ahr), with , , , -tetrachlorodibenzo-pdioxin (tcdd) being the most potent congener amongst the ahr agonists. recent risk assessments have employed the toxic equivalency factor (tef) concept. the current eu-project systeq aims at developing, validating, and implementing human systemic tefs as indicators of toxicity for dl-compounds. at present, the best known parameter of ahr mediated effects is the induction of cytochrome p isoenzymes (cyps), i.e., cyp a , a , and b . one of the major objectives of the systeq project is the identification of novel quantifiable biomarkers. in a three day study, female c bl/ mice were treated with single doses of six dl-congeners (tcdd, pcb , pcb , and pcb ) , and the 'non-dioxin-like' (ndl) pcb . quality tested (agilent ® bioanalyzer) mrna isolated from livers was analyzed using the agilent ® mouse whole genome array ( x k) system. the quantity of genes affected (≥ fold) was highly heterogeneous amongst the dl-compounds. whereas tcdd-treatment upregulated genes, and down-regulated , -pncdf-treatment had impact on (up), and (down). treatment with pcb led to marginal numbers of up and down-regulated genes. with (up), and (down) genes shared, the most extensive overlap occurred between tcdd-and -pncdd-treatment. no overlap was found due to treatments with the ndl pcb ( up, down) and tcdd. when comparing the effects of all dl-congeners, minor numbers of genes of up, and down-regulated remain, most of them being related to drug metabolism. while pcb regulated only genes involved in drug metabolism, omission of pcb -regulated genes resulted in consistently (up), and (down) regulated among dl-compounds. in conclusion, our findings suggest that the pattern of gene regulation in mouse liver elicited by pcb was strictly different from tcdd, while a very limited coincidence of genes was found even among dl-compounds. comparison of these 'core' genes with data from human models is required with respect to determination of novel biomarkers. introduction: proper use of antibiotics is essential with regard to effective treatment of bacterial infections. providing adequate information for patients can contribute to achieve this aim. materials and methods: data was collected from the relational database of the drug information service at dresden university of technology. the patients, who used the service, were interviewed concerning socio-demographic characteristics, reason for enquiry, number and kind of drugs taken, and diseases. possible contact paths were phone, e-mail or letter. in the present evaluation, all enquiries from the years and were analyzed descriptively focussing primarily on systemic antibiotics as reason for the enquiry. results: in the evaluated period, enquiries were registered in total. in . % of those enquiries systemic antibiotics were named with a total number of drugs. . % of those antibiotics were found to be the direct reason for the enquiry. most common information requested by patients corresponded to adverse drug reactions ( . %), diagnosis/treatment ( . %), drug application ( . %) and drug-druginteractions ( . %). the majority of the requesting patients ( . %) was born before . a correlation between incidence of enquiries especially concerning antibiotics and quarterly statistics could not be detected. conclusion: mainly patients aged years or more seem to need or search for further information about antibiotic medication. advice is required especially regarding adverse drug reactions and diagnosis or treatment. in order to this, the advisory service can help patients to lose their insecurity and to gain more confidence in handling antibiotic drugs. colon cancer is one of the most frequent cancers in the industrialized nations. epidemiological studies show a correlation between highly processed meat and the development of colorectal tumours. it is assumed that the risk of developing colorectal cancer, among various different factors, is related to the uptake of toxic substances contained in food such as heterocyclic aromatic amines that arise during the processing of fish and meat. phip is the most abundantly formed heterocyclic compound, and therefore has the biggest impact. in a previous study, we measured the absorption of phip in different intestinal segments of the rat. in the present study we focussed on the potential mechanisms by which phip is reabsorbed. the unidirectional phip transport from the mucosal to the serosal compartment (j ms) and in the opposite direction (jsm) was examined using the ussing chamber technique and c-phip as a radiotracer. the proximal jejunum and distal colon of male fischer rats in short-circuit current chambers was clamped, so that mucosal and serosal compartments were built. the phip flux rates were determined at defined intervals over min. the experimental conditions were selected in such a way that negative net flux rates (jnet = jms-jsm) were indicative of an active secretion. both intestinal segments showed large differences. while in the jejunum jms and jsm of phip were not significantly different, there was an active secretion in the colon. in a next step the transport proteins involved in this process should be examined. introduction: human organic anion transporter , oat (slc a ), is abundantly expressed in kidney and liver and mediates the sodium-independent uptake of clinically relevant drugs like -fluorouracil, paclitaxel, bumetanide, tetracycline, and zidovudine. while immunohistochemical studies have localized human oat to the basolateral membrane of kidney proximal tubules, its hepatic localization is currently unknown. we, therefore, firstly determined oat localization in human liver. because interindividual variability of oat expression may affect hepatobiliary drug uptake and elimination, we next systematically investigated the influence of genetic and non-genetic factors on hepatic oat expression. methods: an expression profile of oat for human tissues was determined by realtime quantitative polymerase chain reaction (taqman). oat mrna expression was analyzed in well-characterized human liver samples from caucasians that were accompanied by detailed demographic and clinical data. oat was localized in human liver cryosections using a commercial rabbit polyclonal antibody and hepatic oat protein levels were determined. resequencing, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and genome-wide single nucleotide polymorphism microarray technology served to genotype variants in the slc a gene region. results: oat mrna was expressed in several human tissues, including liver. moreover, a new alternatively spliced variant of oat was identified in human liver. hepatic expression of full-length oat mrna and oat protein varied -fold and fold, respectively. oat mrna and protein levels did not correlate with each other. oat was localized to the sinusoidal membrane of human hepatocytes. no novel variants in the exons, the '-flanking region, or the '-untranslated region of the slc a gene were identified. univariate analysis showed that oat mrna is reduced in patients diagnosed for cholestasis (p= . ) and is affected by genetic variants. whereas the influence of genetic variants on hepatic oat expression appears to be limited, cholestasis significantly contributes to the variable interindividual oat expression. this indicates consequences for hepatic drug elimination of and response to oat drug substrates such as paclitaxel or tetracycline. the life threatening toxicity of organophosphorus (op) nerve agents is caused by the inhibition of the acetylcholine esterase (ache). oximes were shown to be potent reactivators of inhibited ache, but in poisoning by some compounds, e.g. soman, they have only a small therapeutic effect. for such cases, an alternative new strategy may be the intervention at nicotinic acetylcholine receptors (nachr). previous studies with the bispyridinium non-oxime mb demonstrated therapeutic effects against soman in vitro and in vivo which was partly attributed to its direct interaction with nachrs [ ] . we investigated the interaction of mb and several structure analogous at the orthosteric binding site of human α nachr (hα nachrs), a subtype which appears to be widespread in the human body, and compared the results with data obtained from torpedo-nachrs, which show a high degree of homology with human muscle-type nachrs. interaction of compounds with the orthosteric binding site of hα nachrs were investigated with radioligand binding experiments performed as high-throughput method [ ] . membrane preparations of gh c cells stably expressed hα nachrs were incubated with the nachr agonist [³h] epibatidine and appropriate concentrations of the unlabelled competitors e.g. bispyridinium compounds. after incubation, bound and free [³h] epibatidine were separated by rapid vacuum filtration. ki values of the competing compounds were calculated with nonlinear regression. three bispyridinium compounds, mb , mb and mb exhibited ki values at micromolar concentrations while three other compounds, mb , mb and the pharmacological inactive mb (negative control) did not show any interaction with the orthosteric binding site of hα nachrs. with torpedo-nachrs, ki values were in similar orders of magnitude -except mb which indicated significant subtype selectivity. interestingly, the affinity of monomeric pyridinium derivates did not correlate with their bispyridinium structure analogues. obviously, no correlation between the affinity to the orthosteric binding site and the functional improvement of neuromuscular transmission exists, although species-related differences cannot be excluded. in this study, we analysed the cytotoxic and clastogenic effects of the anticancer drug nimustine (acnu) in cells deficient in repair proteins involved either in homologous recombination (hr) or non-homologous end-joining (nhej). we show that hr mutants are extremely sensitive to acnu as measured by the induction of apoptosis and colony formation as well as the induction of chromosomal aberrations. the nhej mutants were slightly sensitive to acnu and differed in their sensitivity, with the ku mutants being moderately sensitive and the dna-pkcs mutant resistant, comparable to the wild-type (wt). cell death was mostly executed via the caspase-dependent apoptotic pathway with involvement of caspase- and - , and necrosis was also induced. further, we investigated the kinetics of dna double-strand break (dsb) formation that resulted from the repair of acnu-induced interstrand cross-links by means of γh ax and bp foci analysis in wt and mutant cells. cells mutant in hr did not repair dsb and went into the apoptotic or necrotic pathway, whereas wt cells were able to repair most of the dsb. cells deficient in ku formed at early times after acnu treatment less γh ax and bp foci compared to the corresponding wt, which might be due to a reduced capacity of recognising dsb. at later times after treatment, ku mutant cells show foci levels similar to the wt indicating restitution of h ax phosphorylation. we also analysed whether dsb formation after acnu treatment was replication-dependent using synchronised cells. we determined the formation of γh ax and other dsb marker in wt cells that passed through the first cell cycle after demecolcine synchronization. the level of γh ax foci increased significantly in the s-phase and remained at a high level during g where a fraction of cells remained arrested. rad , atm, mdc- and rpa- foci were also formed and shown to co-localize with γh ax. these foci were ameliorated significantly in s-and g -phase, which was similar to the time course of γh ax foci formation. in western blots, we confirmed a higher phosphorylation level of atm and chk and less phosphorylation of chk in hr mutants. the data indicate that acnuinduced dna cross-links give rise to cyto-and genotoxicity via the formation of dsbs that activate the cellular dna damage response. the endocannabinoid system has been established as a mediator of numerous central and peripheral biological functions. cannabinoids have emerged as attractive alternatives or supplements to therapy with opioids for chronic pain states. however, in human the activation of cannabinoid receptors is associated with side effects. for clinical exploitation of the analgesics properties of cannabinoids, a major challenge is to devise strategies that reduce or abolish their adverse effects on cognitive, affective and motor functions without attenuating their analgesics effect. in animal studies, the anti-nociceptive efficacy of cannabinoids has been unequivocally demonstrated in several models of inflammatory and neuropathic pain. however, there are marked inconsistencies between different reports with respect to the locus of these pain-protective effects. we are working towards establishing the contribution of cb receptors expressed on the peripheral terminals of nociceptors to cannabinoid-induced analgesia. using cb globally knock-out animal as background, we induce the expression of cb specifically in nociceptive neurons localized in the peripheral nervous system and test the analgesic effects of cannabinoid systemical delivery in these mice. our results support the development of peripherally acting cb analgesic agonist with reduced central side effects. furthermore, we are utilizing proteomics approach to identify protein complexes that interact with cb receptor which hold promise in understanding cannabinoid signaling in health and disease. most chemoattractants, including chemokines, complement c a, fmlp, and leukotriene b are signaling through heterotrimeric g proteins of the pertussis toxin (ptx)-sensitive gi family. the functional inactivation of all gαi proteins with ptx leads to a fulminant decompensation of the immune system, whereas the constitutive inactivation of a single gαi coding gene results in mild phenotypes in mice. we are mostly interested in the nonneuronally found gαi and gαi isoforms and their redundant and specific roles in immune function and infection. for this purpose cellular in vivo and ex vivo models and in vivo infection model with listeria monocytogenes are being used. macrophages were isolated from the peritoneal cavity of wild type (wt) and gαi-deficient mice days after i.p. injection of % thioglycolate that induces peritonitis in vivo. we confirmed previous observations that in gαi -deficient mice the migration of macrophages into the peritoneal cavity was reduced after induction of peritonitis. regarding the expression levels of gαi and gβ isoforms in the lavage samples, the predominant gαi isoform gαi was upregulated in gαi -deficient macrophages. vice versa gαi was upregulated in gαi -deficient macrophages. concerning gβ isoforms, both gβ and gβ were strongly reduced in the gαi -deficient macrophages which resulted in a reduced total amount of gβ. surprisingly, the gαi -deficient macrophages showed reduced gβ protein levels only which caused a change in the gβ / gβ quotient in favour of gβ . we are currently establishing an in vivo infection model with l. monocytogenes in gαi and gαi -deficient mice. our previous in vitro infection studies in mice embryonic fibroblasts provided us with information about possible distinct roles of these two isoforms as far as the uptake of l. monocytogenes in the cells is concerned. challenging the immune system of gαi-deficient mice with this pathogenic organism will give us new insights into the systemic immune response in these mice upon bacterial infection. our data indicate that we may surmount the redundancy between these two isoforms and focus on their distinct and specific roles in pathogen defense. fret-based β-arrestin biosensors reveal conformational changes upon binding to the β -adrenergic receptor in real time and living cells nuber s., zabel u., ziegler n., hoffmann c., lohse m. j. institut für pharmakologie und toxikologie pharmakologie, versbacherstr. , würzburg, germany β-arrestins are multifunctional adapter proteins that regulate seven transmembranespanning receptor ( tmr) signaling and initiate also alternative signaling pathways. studies have shown that β-arrestins undergo conformational changes upon receptor stimulation, which are thought to be necessary for its downstream actions. to investigate these conformational changes in living cells we constructed fret based biosensors of β-arrestin , in which cfp was fused to the c-terminus and the flashbinding motif (ccpgcc) was inserted to different positions within the n-or c-domain of β-arrestin . upon β -adrenergic receptor (β ar) stimulation we observed a decrease of the intramolecular fret signal between cfp and flash at the n-domain (β-arrestin flash ), indicating a conformational change moving the c-terminus and the ndomain of β-arrestin relative to each other. kinetic analysis revealed that this conformational change immediately follows β-arrestin /β ar interaction on a timescale of seconds. a β ar mutant that was previously shown not to interact with β-arrestin was utilized as control and did not induce a conformational change in the β-arrestin molecule. our data provide evidence that β-arrestin changes it`s conformation upon binding to the activated β ar in living cells. the β-arrestin flash sensor could serve as universal biosensor for gpcr activation. studies on the physiological role of annexin a in the heart nunes f. the calcium binding protein annexin a has been examined in the context of heart failure in the past. annexin a expression level was found to be elevated in ventricles of human failing heart in comparison to expression levels in non-failing ventricles. furthermore the intracellular localization pattern in atrial cardiomyocytes was found to be altered in the failing human heart (moravec and matteo, cardiovasc res ). in order to gain insight into the possible physiological significance of these findings we utilized an annexin a gene trap model (gt) in which the annexin a protein content was not detectable in ventricles and atria. measurements of sarcomere shortening and calcium transient kinetics in isolated ventricular cardiomyocytes revealed a prolonged calcium transient decay at stimulation frequencies of . hz, hz and hz as well as an increased sarcomere shortening at hz and hz in anxa gene trap animals in comparison to wild type (wt) ( the effects of the β-adrenoreceptor agonist isoprenaline (iso) on the shortening of ventricular cardiomyocytes was increased in gt as compared to wt ( , ± . vs. . ± . , *=p< . vs. wt; n= - / ). western blot analyses indicated that the expression of the sarcoplasmic reticulum (sr) ca + -atpase (serca a) and the phosphorylation status of its regulator protein phospholamban (plb) did not differ between groups (n= ). however, co-immunoprecipitation experiments suggest, that anxa is able to interact with hax , which acts as a repressor of serca a (n= ). we performed force measurements in isolated and electrically stimulated left atria in response to rising isoprenaline concentrations ( - m- - m). the positiv inotropic effect of isoprenaline was significantly increased in gt atria (rel. force at - m iso [%]: wt: ± ; gt: ± *= p< . vs wt; n= - ). in conclusion, annexin a contributes to the regulation of cardiomyocyte contractility. the anxa up-regulation might therefore contribute to diminished cardiac performance in heart failure. matteo rg, moravec cs. immunolocalization of annexins iv, v and vi in thefailing and non-failing human heart. cardiovasc res. mar; ( ) background: pregnane x receptor (pxr) is considered the most important sensor of natural and anthropogenic xenobiotics in vertebrates. in contrast, the amphibian ortholog is involved in neural development and irresponsive to xenobiotics. instead, the xenopus laevis constitutive androstane receptor (car) was recently found to possess pxr-like properties, featuring low basal activity and a pronounced ligand spectrum. thus a structural and functional characterisation of x. laevis car may provide further insights into human car basal and ligand-induced activity. methods: the time-point of origin of car genes was determined by macrosynteny analyses of car, pxr, and vdr (vitamin d receptor) gene loci, which form the nr i subfamily of nuclear receptors. based on a -dimensional protein model of xenopus laevis car, docking studies with structurally diverse agonists were conducted. proteinligand-interactions as well as sequence comparisons were performed in order to select amino acids to be mutated towards human car. the organ response to car activators was determined in xenopus laevis using rna microarrays. results: car emerged together with pxr and vdr from an ancestral nr i gene in early vertebrates via two whole-genome duplications. this was followed by losses of car from the fish lineage and of pxr from sauropsida (reptiles and birds). amino acids important for ligand binding were identified. structural features responsible for the pronounced basal activity in human constitutive androstane receptor are not present in x. laevis car. in human pxr the inter-helical loop in front of helix is part of the ligandbinding pocket and supposed to be responsible for the wide substrate spectrum. in amphibian car this inter-helical loop plays no role in ligand binding. car agonists resulted in a pronounced induction of antimicrobial peptides in the ovary. conclusions: car emerged already in early vertebrates and it is conserved in land vertebrates, whereas xenosensing pxr is found only in the fishes and mammals. we provide a comprehensive modeling and mutational analysis of this first reported amphibian xenosensor. the induction of antimicrobial peptides by car activators suggests a link between xenosensing and innate immunity. the latter one may play a previously unrecognized role in the amphibian reproduction. background: retigabine belongs to a novel class of potent anticonvulsant drugs and is currently being investigated in clinical routine. the therapeutic range of retigabine serum concentration is unknown. a therapeutic drug monitoring (tdm) is used for most other anticonvulsant drugs. the aim of this study was to develop a method for the determination of retigabine in serum of patients and to compare the effect and the side effects of retigabine with the blood levels of the drug. method: a hplc method with tandem mass spectrometric detection for the sensitive determination of retigabine was developed. solid-phase extraction (spe) of µl serum with oasis hlb cartridges allowed a reliable quantification down to ng/ml. in order to develop an assay with high sample throughput and to obtain maximum response for the analytes we required the shortest possible retention time. to implement the determination of retigabine in a second step in the routine tdm of anticonvulsant drugs the corresponding hplc method was selected: a purospher rp column ( mm x mm; µm, merck) and a mobile phase with a steep acetonitrile gradient. results: the great advantage of having analytes with different molecular masses and similar retention times in combination with ms/ms detection enabled us to aim at a minimum separation that might remove some salts or matrix components that can suppress or interfere with the analyses from the target components, while maintaining good sample throughput. the method was validated. the assay is precise, accurate, fast, sensitive, and selective. discussion: the developed method is suitable for therapeutic drug monitoring of retigabine. the correlation of the serum concentration and the effect of the drug and thus the necessity of tdm have to be tested. targeting inflammatory t lymphocytes with conditional chemokine receptor antagonist expression for a tissue-specific therapy of chronic inflammatory disorders ogrissek n., giegold o., pfeilschifter j., radeke h. h. uniklinikum der goethe-universität pharmazentrum / zafes, theodor-stern-kai , frankfurt am main, germany chemokines and their receptors are known to be involved in the pathogenesis of chronic inflammation and autoimmune diseases. several approaches tried to use chemokine receptor antagonists as therapeutics to reduce exagerrated immune response, however, due to compensation and systemic side effects clinical trials often failed. in previous experiments our group identified three promising antagonists. cxcl ( - ) has antagonistic function for cxcr , cxcl (p g ) is able to inhibit cxcr and the herpesvirus encoded protein vmip-ii interferes with ccr , - and - as well as with cxcr , - and cx cr . their expression and secretion was confirmed in pichia pastoris and antagonistic function has been proven by a reduction of t cell migration. the aim of this project is to develop a cell-based therapy for chronic inflammation with a treatment that is based on the collective effect of cxcl ( - ), cxcl (p g ) and vmip-ii. with targeting of stable transduced memory t cells these antagonists should be conditional expressed and secreted directly in the centre of inflammation, resulting in inhibition of further inflammatory t cell accumulation. to realize this project we first cloned constitutive lentiviral constructs containing these antagonists and optimized transduction of t cells, such as the ova-specific memory th- cell clone if with the potential to initiate antigen specific nephritis in scid mice. next we investigated expression and secretion of cxcl ( - ), cxcl (p g ) and vmip-ii with pcr, western blot and elisa. at the moment we want to measure the inhibition efficiency of t cell migration in vitro with chemotaxis and flow chamber assays. construction of an inducible lentiviral vector plasmid to ensure expression of the antagonists only upon t cell activation, is also part of our current work. finally we would like to test the chemokine receptor antagonists in vivo in two relevant mouse models of type- -diabetes and contact dermatitis. small heterodimer partner (shp- ) is a member of the superfamily of nuclear receptors (nrs). in contrast to other nrs this orphan receptor lacks the dna binding domain. however, shp- is known to inhibit activity of several nrs by direct proteinprotein interaction. importantly several of the interacting nrs have been shown to directly regulate shp- expression, suggesting that shp- is involved in negative feedback loops of various metabolic pathways, such as cholesterol-, bile acid-and drug metabolism and glucose homeostasis. recently binding sites for nrs were identified in the promoter region of shp- , including hnf α, lrh , lxr, fxr, srebp c and pparγ. the aim of our study was to identify single nucleotide polymorphisms (snps) in the promoter region of shp- and to determine their impact on the transactivation of shp- . dna samples from subjects of the population based cohort study of health in pomerania were analyzed by sanger sequencing, thereby we identified four snps namely - t>c (rs ), - g>c, - c>t (rs ) and del- ctga (rs ). subsequently those polymorphisms were tested for their functional consequence performing cell based reporter gene assays testing all above mentioned modulators (lrh , lxr, fxr, srebp c and pparγ) of shp- expression. only the transactivation by hnf α was decreased in the presence of the - c>t polymorphism to % and the - g>c polymorphism to %. in conclusion we described snps with impact on transactivation. it will be aim of future studies to determine the potential impact on physiological processes or disease development. autosomal recessive polycystic kidney disease (arpkd) is a rare genetic disease, afflicting about in . individuals. arpkd is characterized by cystic fusiform dilatations of the renal collecting ducts leading to massive enlargement of the kidneys and ultimately loss of renal function. in addition, the patients suffer from congenital hepatic fibrosis (chf), possibly leading to portal hypertension and liver enlargement. so far, there is no cure for arpkd. therapy is focussing on controlling the disease symptoms [ ] . mutations in the pkhd gene cause arpkd. more than different mutations in this gene have been reported, all leading to the same phenotype, though there are differences regarding the severity of the disease [ ] . in animal models of autosomal dominant polycystic kidney disease (adpkd) as well as arpkd elevated levels of camp were shown [ ] [ ] [ ] . in isolated kidney cells camp stimulates cl-secretion and activates the b-raf /mek/erk pathway. these both are important factors for cyst development and disease progression [ , ] . intracellular camp regulation is based on conversion of atp to camp by adenylyl cyclases (acs) and degradation by phosphodiesterases . referring to this, we asked the question if there are differences in the activation and expression pattern of acs in pck rats, an animal model of arpkd [ ] and in sprague dawley rats. therefore, we examined membranes in a radioactive ac activity assay using various stimulatory compounds, e.g. forskolin, a direct ac activator, or hormones like glucagon and vasopressin to characterize acs. furthermore, we examined ac isoform expression on the mrna level via rt-pcr. we observed that in pck rats ac activity was decreased in general in comparison to sprague dawley rats. in future experiments we are aiming to obtain further knowledge about the influence various hormones exhibit on pck rat acs and to biochemically characterize acs. the major pathogenicity factors tcda and tcdb from clostridium difficile monoglucosylate and thereby inactivate small gtp-binding proteins of the rho subfamily after entering host cells via receptor-mediated endocytosis. although the intracellular mode of action of the toxins is well understood, far less is known about binding structure and internalization pathway of tcda and tcdb. since antibodies directed against the c-terminal located clostridial repetitive oligopeptides (crops) are able to neutralize toxin cytotoxicity the crop domain is acknowledged to mediate receptor binding. however, we recently demonstrated that crop deletion mutants of tcda (tcda - ) and tcdb (tcdb - ) enter host cells and exhibit full cytopathic potency though lacking the proposed receptor binding domain. we therefore refute the accepted opinion of a solely crop-mediated toxin uptake and re-evaluate the role of the crops in toxin endocytosis. tcda - and tcdb - induced time and concentration dependent cell rounding and rac -glucosylation. however, depending on the cell line, truncated toxins exhibit up to -fold reduced potency towards host cells compared to the respective full length toxin. the observed difference in toxin potency might reflect the recognition of different receptor structures or the use of various endocytotic routes. interestingly, pre-incubation of cells with the isolated crop domain enhances binding as well as cytotoxicity of subsequent applied truncated tcda indicating that the crops primarily determine toxin uptake. in fact, competition experiments revealed that tcda and tcda - predominantly use different receptor structures corroborating the notion of alternative internalization processes utilized by tcda. different routes for cellular uptake might enable the toxins to enter a broader repertoire of cell types leading to the observed multifarious pathogenesis of c. difficile. thus, characterization of alternative endocytotic pathways used by the c. difficile toxins might therefore be the basis to investigate the opportunity of toxin uptake inhibition as therapeutic option. in neurodegenerative diseases, such as alzheimer´s disease and parkinson´s disease, mitochondrial pathways of apoptosis are considered as major features of the underlying neuronal cell death. such mitochondrial mechanisms of apoptosis are mediated by the bh -only protein bid, a member of the bcl- family that triggers mitochondrial permeabilization and the subsequent release of death-promoting proteins into the cytosol. the pivotal role of bid in apoptotic cascades of neuronal cells has been shown in our previous studies showing a neuroprotective effect of bid sirna and small molecule bid inhibitors such as bi c in vitro. in vivo, however, the available bidinhibitors failed to protect brain tissue likely because the compounds were not bioavailable or did not cross the blood brain barrier. therefore, chemical modifications of bi- c were generated resulting in new structures and molecules with different pharmacophors. the aim of the present study is to identify novel potent bid inhibitors available for applications in model systems of brain damage in vivo. for the first screening of compounds we used a model of glutamate toxicity in immortalized mouse hippocampal neurons (ht- cells). in this model system, glutamate induces a decrease of intracellular glutathione levels resulting in lipoxygenase activity and enhanced formation of toxic reactive oxygen species (ros). to investigate the compounds' ability to prevent glutamate induced cell death, we first analyzed the cell viability by the mtt assay. in addition, we examined the cell survival by using real time monitoring of cell impedance (xcelligence system) to determine the neuroprotective potency of the new structures. using these assays, we identified novel molecules that significantly prevented glutamate-induced toxicity in ht- cells. further we were able to express and to purify recombinant bid in a high amount. in the ongoing study the purified bid protein will be used for co-crystallization with the identified neuroprotective structures for further optimization of novel bid inhibitors for therapeutic applications in experimental models of neurodegenerative diseases in vivo. polymorphic enzymes, urinary bladder cancer risk and structural change in the local industry ovsiannikov d. , selinski s. in the s, an uncommonly high percentage of glutathione s-transferase m (gstm ) negative bladder cancer cases ( %) was reported in the greater dortmund area (golka et al., ) . the question arose whether this uncommonly high percentage of gstm negative bladder cancer cases was due to environmental and occupational exposure decades ago. thus, years later, another study on bladder cancer was performed in the same area after the coal, iron and steel industries had finally closed in the s. in total bladder cancer patients from the st.-josefs-hospital dortmund-hörde and controls with benign urological diseases were investigated by a questionnaire and genotyped for gstm , gstt and the n-acetyltransferase (nat ) tag snp rs . the frequency of the gstm negative genotype was % in bladder cancer cases and thus much lower, compared to a previous study performed from - in the same area ( %). nat genotypes were distributed equally among cases and controls ( % slow acetylators). less gstt negative genotypes were present in cases ( %; controls %). apoptosis inducing factor (aif) has been identified as a key factor in intrinsic pathways of caspase-independent neuronal death in model systems of acute brain injury and neurodegenerative diseases, such as alzheimer's disease and parkinson's disease. aif is a mitochondrial intermembrane flavoprotein with the capacity to translocate to the nucleus where it induces chromatin condensation and large-scale dna fragmentation. previous studies revealed that aif deficiency leads to protective effects in different models of neuronal death in vitro and in vivo. however, aif also plays an important physiological role for the integrity and function of the mitochondrial respiratory chain. thus, aif deficiency may significantly alter mitochondrial functions and metabolic homeostasis thereby preconditioning the cells to tolerate subsequent stress stimuli. the present study addresses this hypothesis and investigates whether neuroprotection by aif depletion was attributed to a preconditioning effect, i.e. protecting mitochondrial function and integrity. as model system we use glutamate induced oxytosis in immortalized mouse hippocampal ht- neurons. silencing of aif expression by sirna ( nm) protected mitochondrial morphology and integrity against glutamate induced damage. microscopy analysis of the mitochondrial morphology revealed that aif sirna prevented mitochondrial fission. furthermore, facs analysis confirmed that mitochondrial membrane potential was stable in cells with aif silencing. this protection of mitochondrial morphology and integrity by aif depletion was associated with preserved atp levels and inhibition of cell death as detected by an mtt assay. pronounced formation of lipidperoxides as another indicator of mitochondrial damage was also attenuated in cells preconditioned by aif sirna. these protective effects of aif sirna were highly similar to effects obtained with low doses of rotenone ( nm), which was applied as an inhibitor of complex i and mediated comparable preconditioning effects in the ht- cells. overall, these findings support the conclusion that aif depletion mediates a preconditioning effect protecting neuronal cells from a subsequent glutamate toxicity. in order to link these preconditioning effects to complex i functions, protein expression and functional analysis of complex i are being analysed to identify the molecular mechanisms of aif dependent control of neuronal life and death. -dependent inactivation and display very slow voltage-dependent inactivation. both properties are of crucial importance in ribbon synapses of retinal photoreceptors and bipolar cells, where sustained ca + influx through cav . channels is required to couple slowly graded changes of the membrane potential with tonic glutamate release. mutations in the gene coding for cav . cause severe impairment of retinal circuitry function and have been linked to congenital stationary night blindness type a (csnb ), aland island eye disease (aied) and cone-rod dystrophy type (cordx ). the clinical phenotypes of these eye diseases vary substantially regarding the ratio of rod to cone functional impairment. the reasons for this variability are not known. to gain more insights into the pathophysiology caused by loss of cav . function we analyzed the visual phenotype of cav . -deficient mice. to this end, we combined immunohistochemistry, electroretinography (erg) and vision-dependent behavioral testing. immunohistochemical analysis using synaptic and postsynaptic markers revealed severe synaptic defects in cav . -deficient mice. heterozygous cav . mice showed mosaic synaptic defects most probably caused by random x-chromosomal inactivation of the healthy allele. electroretinography revealed a loss of scotopic and photopic photoreceptor function. this loss of retinal network function resulted in impaired performance of cav . knockout mice in a water maze-based behavioral test of rod and cone function. in conclusion, loss of cav . channels strongly impairs rod and cone retinal function and vision in mice. lsr is the host cell receptor for clostridial iota-like toxins papatheodorou p., aktories k. albert-ludwigs-universität freiburg institut für experimentelle und klinische pharmakologie und toxikologie, albertstr. , freiburg, germany the human enteric pathogen clostridium difficile is the most serious cause of antibioticassociated diarrhea and pseudomembranous colitis. hypervirulent strains of the pathogen, associated with more severe disease and increased death rates, produce the binary actin-adp-ribosylating toxin cdt (c. difficile transferase) in addition to the rho glucosylating toxins a and b. cdt is member of the family of clostridial iota-like toxins, including c. spiroforme toxin (cst) and the eponym c. perfringens iota toxin. the toxins induce depolymerization of the actin cytoskeleton and the formation of microtubulebased cell protrusions that increase adherence and colonization of clostridia. using a haploid genetic screen, we identify the lipolysis-stimulated lipoprotein receptor (lsr) as the target molecule for entry of cdt into host cells. in addition, we present evidence that lsr is shared as a cell entry point by all members of the iota-like toxin family. identification of the toxin receptor provides a most valuable basis for antitoxin strategies. bisphenol a (bpa) is a chemical of high interest due to its endocrine activity. controversy exists concerning the blood concentration due to normal exposures. some authors claimed to have measured concentrations in the ng/ml range which is in contrast to kinetic properties of bpa. bpa is excreted in the urine as glucuronide and sulfate metabolites. recently, data on the in vitro metabolism of bpa by recombinant udpglucuronyltransferase b enzymes (ugt b ) revealed that ugt b . and ugt b . had markedly lower intrinsic clearance as compared to ugt b . (hanioka et al., ) . using the in vitro metabolism data, we scaled the kmand vmaxvalues in an established human physiologically based toxicokinetic (pbtk) model (mielke and gundert-remy, , mielke et al., ) to the values of the variants. for oral doses at relevant exposure levels, the maximum blood concentration (cmax) for the ugt b . variant (v ) was fold and those of the ugt b . variant (v ) was fold higher than that of the ugt . variant. with dermal exposure at a relevant exposure level, the cmax values were . (v ) and . fold (v ) of ugt . variant. a combined exposure of oral and dermal exposure, an exposure scenario, which occurs in daily life, resulted in . fold (v ) and . fold (v ) higher cmax values as compared to ugt . variant. the values for the area under the blood concentration time curve (auc) were for a relevant oral dose . fold (v ) and . fold (v ), for relevant dermal exposure . fold (v ) and . fold (v ), and for combined exposure . fold (v ) and . fold (v ) of ugt . variant. from the results we conclude: ( ) polymorphism of udpglucuronyltransferase ( b . and b . ) has an impact on the blood concentrations which, however, is less than fold for cmax and for auc. the effect is more pronounced for oral as compared to dermal or combined exposure. ( ) polymorphism of metabolism does not explain the blood/plasma concentrations in the ng/ml range measured by some authors. hanioka n, oka h, nagaoka k, ikushiro s, narimatsu s. effect of udpglucuronosyltransferase b polymorphism on bisphenol a glucuronidation. arch toxicol. , ( ) : - . mielke h, gundert-remy u. bisphenol a levels in blood depend on age and exposure. toxicol lett. ; ( ) : - . mielke h, partosch f, gundert- the heart responds to maladaptive pro-hypertrophic stimuli by stimulating intrinsic signals that contrast and dampen the onset and development of hypertrophy. cyclic guanosine monophosphate (cgmp) and its downstream effector cgmp kinase i (cgki) have been suggested to be an important anti-hypertrophic signaling pathway ( ) . intracellular levels of cgmp can be raised by the action of nitric oxide (no) and natriuretic peptides (anf, bnf), or by inhibiting cgmp-degrading phosphodiesterases (pde). a growing body of evidence suggests that the pde specific inhibitor sildenafil (sil) prevents and reverses hypertrophy and chamber remodelling in the heart of mice subjected to thoracic aorta constriction (tac) by elevating cgmp levels and cgki activation ( ) . in contrast, using a mouse model that lacks cgki expression in every cell type except smooth muscle cells (βres mice; see ref. ), we recently showed that the absence of this kinase does not alter the onset of hypertrophy induced by tac or isoproterenol infusion ( ) . sil is believed to increase cardiac cgmp levels, although it is unclear, if its target (pde ) is expressed in cm ( ). sil may act on other pdes, such as pde c which is abundant in cms. it is also unclear if sil effects are mediated by other cardiac cell types, in particular by cardiofibroblast. to answer these questions, we are currently investigating whether sil is able to prevent hormone induced cardiac hypertrophy in the absence of cgki in cm. preliminary results on βres mice show that even in the case of chronic angii infusion, lack of cgki in cm does not alter the induction of hypertrophic response, at least in the initial phase ( days of angii infusion at mg/kg/day). interestingly, βres mice showed impaired cardiac function, as indicated by decreased fractional shortening. sil was able to partially block the onset of cardiac hypertrophy in wt animals, but not in βres mice, indicating a requirement of cgki in this process. in particular, sil was able to block the transcription of pro-fibrotic genes such as tgfβ, ctgf, collageni and fibronectin. the overexpression of the somatostatin receptors sst and sst in neuroendocrine tumors provides the molecular basis for therapeutic application of the stable somatostatin analogs octreotide and pasireotide. whereas the phosphorylation of the carboxyl-terminal region of the sst receptor has been studied in detail, little is known about the agonist-induced regulation of the human sst receptor. here, we have generated phosphosite-specific antibodies for the carboxyl-terminal threonines (t ) and (t ), which enabled us to selectively detect either the t -or the t -phosphorylated form of sst . we show that agonist-mediated phosphorylation occurs at t , whereas t is constitutively phosphorylated in the absence of agonist. we further demonstrate that the pan-somatostatin analog pasireotide and the sst -selective ligand l- , but not octreotide or ke were able to promote a clearly detectable t phosphorylation. however, none of these compounds was able to stimulate t phosphorylation and sst internalization to the same extent as the natural somatostatin. agonist-induced t phosphorylation was dose-dependent and selectively mediated by g protein-coupled receptor kinase (grk ). like that observed for the sst receptor, phosphorylation of sst occurred within seconds. however, unlike that seen for the sst receptor, dephosphorylation and recycling of sst were complete within minutes. we also identify protein phosphatase g (pp g) as sst receptor phosphatase. together, we provide direct evidence for agonist-selective phosphorylation of carboxyl-terminal t . in addition, we identify grk -mediated phosphorylation and pp g-mediated dephosphorylation of t as key regulators of rapid internalization and recycling of the human sst receptor. termination of signaling of activated g protein-coupled receptors (gpcrs) is essential for maintenance of cellular homeostasis. although the regulation of agonist-induced phosphorylation has been studied in detail for many gpcrs, the molecular mechanisms and functional consequences of receptor dephosphorylation are far from understood. recent studies have shown that phosphatase inhibitors, such as okadaic acid and calyculin a, can block the dephosphorylation of a number of gpcrs including the ß adrenergic receptor, d dopamine receptor, parathyroid hormone receptor , thromboxane a receptor and the vasopressin receptor . however, a specific phosphatase has not been identified so far. in present studies, we have examined the mechanism and function of receptor dephosphorylation using the sst a somatostatin receptor and the µ-opioid receptor (mor) as models. within those analyses, we have identified protein phosphatase beta (pp ß) as the phosphatase for the cluster of phosphorylated threonines ( ttetqrt ) within the sst a somatostatin receptor carboxylterminus using sirna knock down screeening. those phosphorylation sites mediate ß-arrestin binding. we have also identified protein phosphatase gamma (pp γ) as mor phosphatase that catalyzed t and s dephosphorylation at or near the plasma membrane within minutes after agonist removal. here, we show the different activated phosphatases with functional selective mutants. we examined tailswap mutants which specify the different phosphatase activities. therefore we produced a mor-rsst a chimera with the rsst a c-terminal tail and a rsst a-mor chimera with a mor c-terminal tail. detoxification by conjugation of glutathione? formations of dna adducts of patulin activated by glutathione pfenning c., lehmann l. university wuerzburg, institute of pharmacy and food chemistry section of food chemistry, am hubland, wuerzburg, germany as a frequent contaminant in apple juice, the mycotoxin patulin (pat) has shown mutagenic potential in cultured mammalian cells at concentrations which are equivalent to those found in marketable foods. this fact is in contrast with the assumption that conjugation to the major intracellular nucleophile glutathione (gsh) leads to detoxification of the electrophile pat. although pat reacts readily with gsh, previous studies showed that co-incubation of pat with model thiols and amine compounds increased the reactivity of pat towards amines forming mixed-type adducts. thus, we hypothesise that the potential to react with dna bases after being activated by gsh might contribute to the mutagenicity of pat. adduct formation of dna bases (adenine, guanine or cytosine) with pat in the presence and absence of gsh was studied under neutral conditions. liquid chromatography coupled with electrospray ionization tandem mass spectrometry was applied for identification and structure elucidation of putative adducts. besides published as well as hitherto unknown pat-gsh adducts, several pat-dna base adducts were formed both in presence and absence of gsh. in addition, with each of the three dna bases one product exhibiting a mass to charge ratio and fragmentation pattern suggesting a mixed thiol-amine adduct was detected. based on the fragment ions of adducts formed with gsh and chemically modified derivatives, we postulate a cyclic structure of the pat-gsh-dna base adducts, resulting from the reaction of the α-amino group of the glutamic acid residue with the c -carbonyl function of pat. the exocyclic amino group of the dna base is linked to c of the pat backbone by an amid bond. thus, the present study demonstrates the reactivity of pat towards dna bases and the participation of gsh in adduct formation. the postulated structures of dna adducts could be used as biomarkers for the determination of the internal exposure to pat in humans. prescribing information detailed in the summary of product characteristics (spc) forms the officially approved basis for safe prescribing of drugs. in a project funded by the german federal ministry of education and research (bmbf) we aimed to derive an internationally valid data set for safe prescribing of psychiatric drugs and therefore analyzed and compared the content of internationally available prescribing information. a team of pharmacists and clinical pharmacologists performed an in-depth comparison of the german, swiss, british and us-american spcs of top prescribed psychiatric drugs. for drugs (of identical pharmaceutical form) the spcs from the same manufacturer were available for all countries, whereas for three drugs spcs were only available from different companies. in these cases the most recent prescribing information from each country was included in the comparison. in spcs individual data points ( . ± . per individual spc) were compared. between countries the timeliness of prescribing information for an individual drug varied by a median of . (range: - ) months. the respective spcs covered on average . ± . % (range: . - %) of all mentioned indications and . ± . % (range . - %) of all mentioned contraindications. the warnings and precautions section of an individual spc covered on average . ± . % (range: . - . %) of all mentioned warnings and precautions for that drug. the variation observed was only marginally improved when restricting the analysis to the spcs of the drugs available in all four countries from the same manufacturer. across countries, the summary of product characteristics of individual psychiatric drugs show substantial variation in crucial prescribing information. as different manufacturers are unlikely to explain much of the observed variation, these data argue for a better international cooperation and standardization of the content of summary of product characteristics. this project is supported by the german federal ministry of education and research (bmbf), project grant no. ex b. protein kinase ck (former name 'casein kinase ') is a highly conserved serine/threonine kinase, which acts as a component of regulatory networks implicated in many cellular processes but is also linked to various types of human cancer [ , ] . elevated ck activity has been associated with aggressive tumor behavior and results in growth advantage, enhanced survival and dynamic adaption to stress of cancer cells [ ] . ck is a heterotetramer consisting of two catalytic subunits (ck α) attached to a dimer of regulatory subunits (ck β) [ ] . ck β stabilizes ck α against denaturation and modulates the substrate specificity of the catalytic subunit [ ] . due to the relatively small and hydrophobic ck α-ck β interface ( Ų) [ ] , low molecular weight inhibitors are able to interfere with the ck subunit interaction and thus affect the kinase activity [ ] . such inhibitors might exhibit an increased specificity in comparison to those compounds interacting with the conserved atp binding site [ ] . we have developed an elisa-based ck α-ck β binding assay using recombinant human ck subunits. different blocking reagents were analyzed to minimize nonspecific binding. the optimized binding assay was then applied to screen for inhibitors of the ck subunit interaction. primary hits were further characterized by determination of the parameters ic and ki as well as by comparing the results from the binding assay with literature-known or recently obtained crystal structures. numerous epidemiological studies have shown associations between exposure to ultra fine particles and an increase in cardiovascular diseases such as atherosclerosis, coronary heart disease and myocardial infarction. ultra-fine particles have an aerodynamic diameter of < . µm and are highly diverse with impurities of transition metals and organic compounds (e.g. polycyclic aromatic hydrocarbons). posttranslational modification (ptm) of proteins, particularly phosphorylation, is a key element in the regulation of cell function and any disturbance can lead to multiple diseases. the present study focused on the proteomic-based identification of phosphorylated proteins to understand the mechanism behind ultrafine particle exposure and cardiovascular disease development. as one of the major sources for ufp emissions are diesel exhaust, therefore to mimic the diesel particles, carbon black (cb) and benzo[a]pyrene loaded carbon black (cb+) were used in the present study. cells of the endothelial cell line ea.hy were exposed for days to ng/ml cb and cb+. phosphoprotein extraction of whole cell lysates was carried out by the method developed in our lab . the obtained proteins were then separated by two-dimensional gel-electrophoresis followed by maldi-tof-ms (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry) analysis of differently expressed proteins. to further validate the results invasive potential of cells were monitored by plating exposed cells for hrs on top of matrigel-coated inserts. differential expressions of phosphoproteins were found in cb-treated cells while an altered expression of phosphoproteins was observed in cb+-treated cells. the maldi-tof analysis revealed proteins involved in the regulation of the endothelial permeability and the cellular plasticity such as vimentin, actin and transitional endoplasmic reticulum atpase. further, the invasion assay supported these results as the cb-exposed cells showed a high invasive potential as compared to control. [ ] pink m. et. al. , precipitation by lanthanum ions: a straightforward approach to isolate phosphoproteins, j.protomics, , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] application of the ttc approach in cosmetics platzek t. bundesinstitut für risikobewertung, max-dohrn-straße - , berlin, germany regulatory toxicologists in europe have been discussing the ttc approach since more than a decade, e.g. the previous scientific committee on food in . since then, the concept was further developed and is now applied in the eu for the assessment of flavouring substances by efsa. two committees are discussing possible applications: the efsa scientific committee prepared an opinion exploring options for the application in food and feed, e.g. for impurities of food additives, thermal reaction products, food contact materials, contaminants etc. in addition, an eu non-food expert committee consisting of members of sccs, scher and schenir discussed the ttc concept in general as well as additional possible fields of application with the focus on cosmetics and an opinion was already published for public consultation. the proposal to apply the ttc approach also for cosmetic ingredients was introduced by a paper published in by a colipa (the european cosmetics association) supported working group. major aspects to be considered are the following: . applicability domain. the chemical space of the ttc dataset (> compounds) has to be compared with that of cosmetic ingredients (> compounds). route to route extrapolation. since no adequate dermal toxicity database is available both data on oral intake used in the ttc approach and on dermal exposure to cosmetic ingredients have to be transfomed to internal exposure figures. gastrointestinal and dermal bioavailability as well as route specific differences in metabolism have to be integrated. exposure. the reliability of exposure estimation is the second pillar of the ttc approach. compared to food, data on exposure to substances in cosmetics and consumer products is scarce. a pragmatic step forward is the comparison of ttcs and noael-derived safe exposure levels for cosmetic ingredients. this work was already done with substances in food contact materials and chemicals from the elincs list. for cosmetic ingredients a similar european project is ongoing. further refinement of the ttc approach is needed taking into account the up-to-date toxicological knowledge. with cosmetics specific problems may arise in praxi: according to the new eu cosmetic legislation the safety of cosmetic products available on the market has to be assessed by the manufacturer or importer and also assessors with limited toxicological experience may apply the ttc approach, e.g. by running the toxtree software. plöttner s., marczynski b., käfferlein h. u., welge p., groth h., engelhardt b., schmitz k., erkes a., brüning t. institut für prävention und arbeitsmedizin der deutschen gesetzlichen unfallversicherung -institut der ruhr-universität bochum (ipa) toxikologie, bürkle-dela-camp-platz , bochum, germany polycyclic aromatic hydrocarbons (pahs) comprise several hundred compounds with different carcinogenic potentials, typically occurring in complex mixtures. due to a lack of data risk estimations for pah mixtures are usually based on those for benzo[a]pyrene (b[a]p). the aim of our present study was to explore the suitability of a permanent human lung cell line as tool for future studies on genotoxicity of pah mixtures. in this pilot study we investigated the time-and concentration-dependent generation of specific anti-benzo[a]pyrene - , -diol- , -epoxide (anti-bpde)-dna adducts as well as cytochrome p (cyp) a / b enzyme activities after b[a]p-incubation in vitro. we used metabolically competent a lung carcinoma cells which display several characteristics of alveolar epithelial type ii cells. after h and h incubations with different b[a]p-concentrations cytotoxic effects were assessed with the neutral red assay and cyp a / b activities using luminescent tests. the formation of specific anti-bpde-dna adducts was determined by hplc with fluorescence detection. a time-and concentration-dependent formation of anti-bpde-dna adducts was observed with maximum rates of . ± . and . ± . anti-bpde/ nucleotides after incubation with µm ( h) and µm b[a]p ( h), respectively. however, the mean adduct rates decreased at higher b[a]p-concentrations. the reduction was more pronounced after h than after h. increased cyp a / b activities were observed at > . - µm ( h) and > . - µm ( h). a clear decrease of enzyme activities was observed at higher concentrations for both incubation times. in the neutral red assay no more than % cytotoxicity in relation to the negative control were found after h incubation with ≥ µm b[a]p and after h with ≥ µm b[a]p. overall, incubation of a cells with b[a]p resulted in a time-and concentration-dependent increase of cyp-activities and anti-bpde-dna adducts. this clearly shows that a cells are able to generate mutagenic dna-adducts. thus, the in vitro model used in the present work appears suitable for genotoxicity studies of individual pahs and pah mixtures, and therefore may be a useful tool for research on syncarcinogenesis. the β subunit of the cav . channel complex has been shown to play a key role in cav . channel trafficking and channel characteristics like opening probability. furthermore, the last exon of the cacnb gene coding for cavβ , exon , contains several potential phosphorylation sites, e.g. for protein kinase a or ca + /calmodulin dependent camkii. pka-dependent phosphorylation mediates β-adrenergic stimulation of cav . . potential phosphorylation sites are ser and ser (in the β subunit in rat, perez-reyes et al. a ) and ser in the c-terminus of the poreforming α c subunit (lemke et al. b ) . in cardiomyocytes camkii regulates ca + release and reuptake from and into the sr and is involved in the facilitation of the calcium channel. potential interaction sites between the cav . channel complex and camkii are thr in the β subunit (in rat, grueter et al. c ) and ser and ser in the cterminus of the α c subunit (blaich et al. d ) . however, the exact pathways remained widely unclear up to now. to clarify these mechanisms and to identify the relevant phosphorylation sites for pka and camkii we established a mouse line carrying a stop codon in exon after aa pro . this mutation prevented translation of the cavβ c-terminus containing the corresponding potential phosphorylation sites mentioned above (cavβ stop mouse). these mice were viable, showed unaltered expression of the truncated of cavβ protein and unchanged ecg and echocardiography. electophysiological analysis of isolated cardiomyocytes showed no differences in current density, the effect of isoproterenol, the time course of inactivation and the facilitation property when compared to cells isolated from littermate controls. for further investigations we bred the cavβ stop mice with s a mice (lemke et al. b ) lacking the pka phosphorylation site s in the α c subunit (s aβ stop mouse) or with sf mice (blaich et al. d ) lacking the camkii phosphorylation sites s and s in the α c c-terminus (sfβ stop). both mouse lines were viable and showed unchanged echocardiography recordings compared to their control littermates and unaltered ecg for s aβ stop mice. electrophysiological investigations on cardiomyocytes of s aβ stop mice showed unchanged β-adrenergic stimulation with isoproterenol compared to littermate controls. these results suggest that β adrenergic regulation of the cardiac cav . channel is not mediated by these phosphorylation sites. introduction. chronic atrial fibrillation (caf) is marked by increased fibrosis which contributes to the perpetuation of the disease. in addition to the role of fibrosis in structural remodeling of cardiac tissue, fibroblasts can couple with cardiomyocytes via gap junction thereby altering the electrophysiological properties of the later and potentially participating in atrial electrical dysfunction. in order to understand the importance of fibroblasts in the pathophysiology of caf, we compared the electrical properties of atrial fibroblasts isolated from patients in sinus rhythm (sr) and caf. methods. fibroblasts were isolated by outgrowth culture from right atrial biopsies and cultivated in medium containing % fetal calf serum. we used whole-cell patch clamp techniques to investigate ion currents and membrane potential. results. sr and caf fibroblasts showed similar capacitance (sr: . ± . pf, n= ; caf: . ± . pf, n = ) and membrane potential (sr: - . ± . mv, n = ; caf: - . ± . mv, n = ) . in both groups, we observed fast activating outward currents with a mean threshold at - mv. interestingly, current amplitude was significantly larger in sr than caf cells (sr: . ± . pa/pf, n = ; caf: . ± . , n = ; p < . ). when maintained in culture for - weeks, cells from both groups developed na + currents. surprisingly, the fraction of caf cells displaying such currents was larger than the sr counterpart (caf: %; sr: %). furthermore, na + current amplitude was significantly larger in caf fibroblasts (sr: . ± . pa/pf, n = ; caf: . ± . pa/pf, n = ; p < . ). na + currents were not altered by nm tetrodotoxin (ttx), but µm ttx reduced current amplitude to % of control, suggesting that the channel involved is the cardiac ttx-resistant isoform nav . . conclusion. in the context of caf, fibroblasts undergo electrophysiological changes which need to be thoroughly described. understanding whether those changes contribute to the af substrate might provide new therapeutic targets for the treatment of caf. the initial recruitment of gi to the alpha a-adrenergic receptor is affected by gprotein-coupled receptor kinases and arrestins prokopets o. s., krasel c., marburg, germany most g-protein-coupled receptors undergo homologous desensitization after agonist stimulation. in this process, agonist-activated receptors are phosphorylated by gprotein-coupled receptor kinases (grks), followed by binding of arrestins to the still agonist-occupied, phosphorylated receptors. it is assumed that arrestin competes with heterotrimeric g-proteins for the receptor molecule and thereby causes desensitization of g-protein-mediated responses. we tested this idea by investigating the effect of grks and arrestins on the recruitment of g-proteins by the alpha a-adrenergic receptor. hek t cells were transfected with yfp-tagged alpha a-adrenergic receptor and the three subunits of gi . the g(beta) subunit was cfp-tagged. upon stimulation with noradrenaline, a very rapid, robust increase in fret between the receptor and the g(beta) subunit was observed, confirming previous observations that the alpha aadrenergic receptor recruits gi with a half-life of around milliseconds. when arrestin and grk were also co-transfected, the half-life of gi recruitment was substantially delayed, increasing to around seconds. there seemed to be no effect of grk +arrestin cotransfection on a second stimulation; neither the kinetics nor the extent were altered compared to the first stimulation. interestingly, grk alone seemed to cause a similar but slightly less pronounced delay of g(beta) recruitment to the alpha a-adrenergic receptor. in corresponding experiments, we measured the recruitment of arrestin -cfp to yfp-tagged alpha a-adrenergic receptors in the presence of grk . upon stimulation with noradrenaline, we observed a robust increase in fret between the receptor and arrestin , confirming previous observations that the alpha a-adrenergic receptor recruits arrestin . co-transfection of the three subunits of gi had no effect on the kinetics of arrestin binding to the alpha a-adrenergic receptors. based on previous results with other receptors, an attenuation of gi recruiting to the alpha a-adrenergic receptor is quite unexpected. our data suggest that the relation between receptors, g-proteins, grks and arrestins may be more complex than previously postulated. introduction: human urinary bladder expresses mrna of the three known badrenoceptor (b-ar) subtypes (b , b , b ) in detrusor and urothelium. we have shown previously that only b -ar is involved in human detrusor relaxation. to investigate the urothelium-induced modulation of b-ar-mediated relaxation, we have examined systematically whether other b-ar subtypes are involved. human detrusor tissue samples were obtained from patients undergoing radical cystectomy for the treatment of bladder cancer. detrusor strips were studied with and without an intact mucosa layer. muscle strips were precontracted with µm carbachol and relaxation was studied in response to the b-ar agonist ne. a-ar mediated processes were blocked with the a-ar antagonists phentolamine and prazosin. selective b-ars antagonists were used to investigate b-ar mediated relaxation. at the end of each experiment µm forskolin was used to determine maximum camp-mediated relaxation. the presence of intact urothelium reduces potency but not effectivity of ne indicating involvement of urothelium-mediated processes not only during detrusor contraction but also during relaxation. ne-mediated detrusor relaxation is mediated through b -ar in the absence of urothelium. but in intact detrusor strips b -ars seem to have an additional inhibitory effect. affinity of the selective b -ar antagonist l , is unchanged, therefore the intact urothelium does not interact with the function of b -ars. aldosterone causes oxidative stress and dna damage independent of blood pressure in vivo queisser n., schupp n. universität würzburg institut für toxikologie, versbacherstr. , würzburg, germany background: an inappropriate increase of the mineralocorticoid aldosterone (ald) can be induced by a stimulated renin-angiotensin-aldosterone system. epidemiological studies exploring the connection between hypertension and cancer found higher cancer mortality and an increased risk to develop kidney cancer in hypertensive individuals. we recently showed that ald produces oxidative stress, activates transcription factor nf-kb and is genotoxic in kidney tubule cells. objectives: this study investigated the capacity of ald to induce oxidative/nitrosative stress, dna damage, dna repair, apoptosis, cell proliferation and the activation of nf-κb in rat kidneys. methods: mineralocorticoid-dependent hypertension was induced by ald/salt in sprague dawley rats. dna damage and oxidative/nitrosative stress markers were detected immunohistochemically. results: ald/salt treatment caused increased blood pressure compared to untreated rats. tempol, an antioxidant, and hydralazine, a vasodilator acting independent of the renin-angiotensin-aldosterone system, could lower the blood pressure, while the mineralocorticoid receptor (mr) antagonist spironolactone was administered in a subtherapeutical dose not lowering the blood pressure. ald/salt treatment caused oxidative and nitrosative stress, structural dna damage, double strand breaks, dna repair and nf-κb activation. spironolactone decreased these markers significantly. tempol was also able to reduce these markers, while hydralazine had no effect. ald/salttreated kidneys showed a tendency to lower apoptosis and to increased cell proliferation compared to control rat kidneys. discussion: this study provides a first hint of blood pressure-independent effects of ald. the mr and the production of ros seem to be crucial for the damaging effects of ald. an aberrant or long-term activation of nf-κb by persistently high ald levels could support resistance to apoptosis and the survival of cells with damaged dna, and increase cell proliferation. these actions could contribute to the increased cancer incidence in hypertension by initiating carcinogenesis. grant support by the dfg is gratefully acknowledged. creb regulating transcriptional coactivator (crtc ) is a transcriptional coactivator of the transcription factor creb. we have recently shown its expression in cardiomyocytes and its activation by beta-adrenergic signaling. beta-adrenergic signaling contributes to the pathogenesis of cardiac hypertrophy, leading to heart failure, as evidenced by the therapeutic success of the beta-adrenoceptor antagonists. in order to investigate if crtc is involved in this process, we investigated the expression of crtc in hypertrophied myocardium from mice and humans. methods: protein lysates from mouse and human samples were investigated for crtc protein expression. we distinguished between an acquired and an inherited form of cardiac hypertrophy. acquired cardiac hypertrophy is an adaptation of the heart to an increased cardiac workload and can be found in patients with an aortic valve stenosis. in mice this kind of hypertrophy can be evoked by transverse aortic constriction (tac). the inherited form of cardiac hypertrophy is caused by mutations in genes coding for proteins of the sarcomeric apparatus and is referred to hypertrophic cardiomyopathy (hcm). as a model for hcm, transgenic mice with a mutation in the mybpc gene, coding for the sarcomeric protein cardiac myosin-binding protein c (cmybp-c), were used. these transgenic mice were characterized by a hypertrophied heart. in case of human samples we distinguished between patients with an acquired form of hypertrophy due to an aortic valve stenosis, and patients with a hypertrophic obstructive cardiomyopathy (hocm), a special form of hcm. results: in the tac mice and in the myppc mutant mice the expression of crtc was significantly higher than in the controls ( . -and . -fold, respectively; n= ). in both forms of human hypertrophy, we found a significantly -fold upregulation of crtc protein level in comparison to human samples from non-failing myocardium or samples from patients with a dilatative or ischemic cardiomyopathy (n= - for each group). conclusions: crtc is upregulated in cardiac hypertrophy with acquired or geneticbased origin. the evaluation of the role of crtc in the heart may help to elucidate the role of beta-adrenergic signaling in the development of cardiac hypertrophy. microarray gene expression profiling reveals up-regulation of the cardiac lipid metabolic process at the onset of heart failure fu x., abd alla j., quitterer u. eth zürich molekulare pharmakologie, winterthurerstrasse , zürich, switzerland atherosclerosis and chronic pressure overload are major cardiovascular risk factors for the development of heart failure in patients. to mimic those risk factors in experimental models we used atherosclerosis-prone apolipoprotein e (apoe)-deficient mice, and chronic pressure overload was imposed by abdominal aortic constriction (aac). cardiac function was monitored by echocardiography. severe atherosclerosis in aged apoedeficient mice or chronic pressure overload induced signs of heart failure as evidenced by a significantly reduced cardiac ejection fraction (< %). to investigate pathomechanisms underlying the development of heart failure, microarray gene expression analysis and qt-rt-pcr were performed of failing heart tissue relative to age-matched controls. gene ontology analysis of the microarray data revealed that the onset of heart failure, in two different experimental models, was characterized by a strong up-regulation (≥ -fold) of the cardiac lipid metabolic process and lipid overload. lipid metabolism genes were involved in lipid synthesis, storage and oxidation. the major palmitate-synthesizing enzyme, fatty acid synthase, was causally related to the development of cardiac dysfunction by enhancing cardiomyocyte apoptosis. taken together the data support that the onset of experimental heart failure is characterized by a dysfunction of the cardiac lipid metabolism promoting cardiomyocyte death. at -receptor blockers (arbs) are established for the treatment of high blood pressure and new onset of diabetes is reduced by arbs. in the past years evidence increased that body weight may also be lessened particularly in rats and mice. however, less data are available whether arbs still reduce weight, when treatment was initiated not until animals became obese by diet. prior to drug treatment, spontaneously hypertensive rats were fed for months with chow but also with a cafeteria diet (cd) to develop obesity. controls received only chow (conchow). cd-fed shr were treated for months with telmisartan (tel mg/kg/d) or amlodipine (aml, mg/kg/d), whereas controls received vehicle (concd). systolic blood pressure (sbp), feeding behaviour, body weight, abdominal fat mass (by mrt) and energy expenditure (by indirect calorimetry) was monitored. leptin sensitivity was assessed by measuring energy intake and expenditure after repetitive injections (s.c.) of leptin. insulin sensitivity was functionally determined by glucose and insulin tolerance tests. due to cd feeding body weight was increased after months by more than g. tel normalized sbp whereas it remained > mmhg in concd and conchow. tel additionally reduced cd-induced increase of body weight and abdominal fat mass. food intake was diminished during the first weeks, but raised beyond control levels during the last weeks of treatment. the shift of the respiratory index to lower levels indicated improved energy expenditure. in response to exogenous leptin, the food intake of concd was higher compared to conchow, indicating a leptin resistance. this assumption is further supported by high triglyceride concentrations of concd. after tel, leptininduced food intake was reduced and energy expenditure was increased compared to concd, indicating that leptin sensitivity was at least partially restored. accordingly, triglycerides were reduced. compared to concd, the insulin sensitivity was improved by tel since maximal increases in plasma concentrations of glucose and insulin in response to glucose challenge were reduced, but glucose response to insulin challenge was diminished. even though reduction in blood pressure was almost similar between tel and aml, metabolic and antiobese efficacies of aml were markedly attenuated. we conclude that telmisartan reveals wide efficacies in improving all symptoms of the metabolic syndrome. the pleiotropic effects are not related to the hypotensive action of tel. a β -adrenoceptor -camp mediated, immediate stimulation of β -adrenoceptor gene expression in human lung fibroblasts is opposed by a delayed up-regulation of inhibitory factors racké k., lamyel f., kämpfer n., schütz i., warnken m. univ. bonn dept. pharmacol. &toxicol., bonn, germany based on their bronchodilatory effects, β -adrenoceptor agonists constitute essential elements in the treatment of bronchial asthma and copd. however, treatment with long-acting β -adrenoceptor agonists has been associated with possible worsening of airway hyper-reactivity, possibly because of loss of β-adrenoceptor function. therefore, the molecular regulation of β -adrenoceptor expression was addressed here. mrc- human lung fibroblasts were cultured for up to h in absence or presence of test substances, followed by β -adrenoceptor mrna determination by qpcr. β -adrenoceptor mrna decreased with a half-life of min after inhibition of mrna synthesis with actinomycin d ( µm), but increased by ± %, ± % and ± % (means±sem) within . , and . h, resp. after inhibition of protein synthesis by cycloheximide ( µm). the β -adrenoceptor agonists formoterol and olodaterol ( - nm) induced a rapid increase in β -adrenoceptor mrna (maximally within h by ± % and ± % at nm, resp.). however, after h exposure to nm formoterol or olodaterol a reduction in β -adrenoceptor mrna by ± % and ± %, resp., was observed. both, the stimulatory and inhibitory effects of β adrenoceptor agonists were mimicked by forskolin ( µm, increase by ± % and inhibition by ± %) and cholera toxin ( ng/ml, increase by ± % and inhibition by ± %). the formoterol-induced up-regulation of β -adrenoceptor mrna was blocked by actinomycin d, but not by cycloheximide. moreover, in presence of cycloheximide, β adrenoceptor agonist induced inhibition was converted into a marked stimulation. in conclusion, expression of β -adrenoceptors in human lung fibroblasts is highly regulated at transcriptional level. the observations with cycloheximide indicate that the β -adrenoceptor gene is under strong inhibitory control of short-living, not yet identified suppressors. although both, the time-dependent up-and down-regulation of the β adrenoceptor gene expression by β -adrenoceptor activation appears to be mediated via adenylyl cyclase -camp signalling, only the stimulatory effect appears to be a direct action on the β -adrenoceptor gene. et- appears to be involved in the pathogenesis not only of pulmonary hypertension, but also in fibrotic remodeling associated with chronic obstructive airway diseases. since human lung fibroblasts (hlf) are a source of et- and have been shown to be controlled by muscarinic receptors and β-adrenoceptors, a possible muscarinic and βadrenergic modulation of et- expression in hlf was explored. mrc- hlf were cultured for up to h in absence or presence of test substances, followed by prepro-et- (ppet- ) mrna determination by qpcr. the muscarinic agonist oxotremorine ( µm) induced an increase in ppet- mrna by %, an effect prevented by nm tiotropium. the β -adrenoceptor agonist olodaterol (up to nm) caused a reduction of ppet- mrna expression by %. the effect of nm olodaterol was prevented by ici , ( µm), but not affect by cgp ( µm) . the pka agonist -bnz-camp ( µm) caused a reduction in ppet- mrna expression by %, whereas the epac agonist -cpt- '-o-me-camp ( µm) caused only a marginal inhibition by %. olodaterol ( nm) strongly opposed the stimulatory effect of µm oxotremorine. an increase in ppet- mrna expression by % caused by . ng/ml tgf-β was effectively opposed by and nm olodaterol, resulting in an inhibition comparable to that in absence of tgf-β. however, the increase in ppet- mrna caused by a maximally effective concentration of tgf-β ( ng/ml, increase by %) was not significantly affected by or nm olodaterol. likewise, the pkaagonist -bnz-camp ( µm) opposed the increase in ppet- mrna expression caused by . ng/ml tgf-β, but not that caused by ng/ml tgf-β. tgf-β caused, with an ic of . ng/ml, a marked down-regulation of β -adrenoceptor mrna expression, maximally by % within h. et- expression in hlf is stimulated by muscarinic receptors and inhibited by β adrenoceptors. the effect of β -adrenoceptors may be mediated via pka. et- expression in hlf is markedly up-regulated by tgf-β, but only effects of sub-maximally effective concentrations of tgf-β are opposed by the β -adrenoceptor -pka pathway, in part because of tgf-β-induced down-regulation of β -adrenoceptors. since et- can promote pro-fibrotic features in hlf, inhibition of et- expression could contribute to long-term beneficial effects of long-acting β -adrenoceptor agonists such as olodaterol and long-acting muscarinic antagonists such as tiotropium. cardiac hypertrophy leads to up-regulation of dipeptidyl aminopeptidase-like proteins in human and rat radicke s., hutschenreuther a., schaefer m. universität leipzig rudolf-boehm-institut für pharmakologie und toxikologie, härtelstr. cardiac hypertrophy is a major risk factor for heart failure and associated morbidity and mortality. functional down-regulation of k + currents is a prominent feature of cells isolated from failing ventricles. a marked decrease in the transient outward potassium current ito has been shown in various models. changes in the k + channel expression differ depending on the species, and the mechanism of induction of heart failure. to study the regulation of ito channel subunits we compared the hypertrophic responses in human ventricular tissues from failing hearts with doxorubicin-induced hypertrophy in rats and in h c embryonic rat cardiac cells. specifically, we quantified mrna expression of the pore-forming subunits kv . and kv . , the cytosolic β-subunit kchip and the transmembrane subunits dpp and dpp using rt-pcr. treatment with doxorubicin ( µm) induced hypertrophy and increased the mrna expression of the hypertrophy marker genes anf, bnp and beta-mhc in h c cardiac myoblasts. while kv . was detected in h c cells and hearts from human and rat, kv . mrna was only expressed in adult rats. during hypertrophy kv . was downregulated in human tissue as well as in doxorubicin-treated h c cells compared to the controls. in rat hearts kv . expression was increased after doxorubicin treatment. interestingly, kv . was also found to be up-regulated in rat heart tissues and h c cells after treatment with doxorubicin. as previously shown, kchip mrna expression was significantly reduced in tissues of failing hearts. in contrast, kchip mrna was upregulated in hypertrophic rat hearts and h c cells. the expression of dpp and dpp was observed only in human hearts. but mrna levels of both were significantly increased in failing tissues. dpp and dpp were not expressed in the adult rat heart or h c cells, whereas in rats with doxorubicin-induced cardiac hypertrophy and in doxorubicin-treated h c cells, the mrna of dpp and dpp was up-regulated. h c cells showed almost identical hypertrophic responses to those observed in human ventricles and rat hearts. this finding validates h c cells as a model for in vitro studies of cardiac hypertrophy. in further studies we will investigate the consequences of a knock-down of dpp and dpp in doxorubicin-induced hypertrophic h c cells. in preliminary experiments specific short-hairpin rna, targeting dpp and dpp , has been designed and tested in heterologous expression systems. the nonsynonymous c. t>c germline genetic variation in the liver-specific organic anion transporter slco b is associated with methotrexate pharmacokinetics in pediatric acute lymphoblastic leukemia radtke s. background: methotrexate (mtx) plasma concentration is related to its clinical effect. transport proteins, such as abcc , slco a , and slco b , have been implicated in the disposition of mtx. here we investigated whether common reduced-function variants in abcc , slco a , and slco b contribute to the interindividual variability in methotrexate pharmacokinetics in children with acute lymphoblastic leukemia (all). we analyzed mtx pharmacokinetics (mtx plasma concentration at the end of infusion c h, mtx auc - h, and mtx clearance cl) in an unselected population of children with all from the all-bfm trial (clinicaltrials.gov: nct ) who received courses of mtx at g/m as h infusions. the contribution of genes (genetic component, rgc) to the interindividual variability in mtx pharmacokinetics was estimated according to the method of kalow et al. ( ) . abcc c.- c>t (rs ), slco a c. g>a (p.his arg, rs ), slco b c. t>c (p.val ala, rs ) and slco b a>g (p.asn asp, rs ) genotypes were analyzed by taqman polymerase chain reaction. there was substantial interpatient variability in average (± sd) mtx c h ( . ± . µmol/l), auc - h ( . ± . h*mg/l), and cl ( . ± . ml/min/m²). the rgc values of c h, auc - h, and cl ranged from . - . suggesting that variation in mtx pharmacokinetics has a substantial genetic component. after adjustment for age and sex by multiple regression, the slco b c. t>c snp was significantly associated with c h (p< . ), auc - h (p< . ), and cl (p= . ) of mtx. compared with the wildtype genotype, in patients with the tc genotype c h and auc - h increased by % (p= . ) and % (p= . ), respectively, whereas cl significantly decreased by % (p= . ). pharmacokinetic variables significantly changed with increasing number of variant c. t>c alleles (p< . , jonckheere-terpstra), suggesting a per allele effect consistent with a co-dominant model of association. in contrast, the abcc c.- c>t, slco a c. g>a, and slco b a>g polymorphisms did not show an association with mtx pharmacokinetics. conclusions: the nonsynonymous c. t>c polymorphism in slco b contributes to the variability of mtx pharmacokinetics in this study of high-dose mtx in pediatric all. this project is supported by the johannes und frieda marohn-stiftung. the antitumorigenic mechanism of the selective cyclooxygenase- (cox- ) inhibitor celecoxib is still a matter of debate. using human lung cancer cell lines (a , h , h ), the present study investigates the contribution of cox- and peroxisome proliferator activated receptor γ (pparγ) to apoptosis elicited by celecoxib. celecoxib was found to cause apoptotic cell death in a concentration-dependent manner ( - µm), whereas structurally-related cox- inhibitors (etoricoxib, rofecoxib, valdecoxib) were inactive in this respect. apoptotic cell death by celecoxib was suppressed by preincubation of tumor cells with the selective cox- inhibitor ns- , the pparγ antagonist gw- and by sirna targeting cox- or pparγ. celecoxib-induced apoptosis was paralleled by a time-and concentration-dependent upregulation of cox- and pparγ at both mrna and protein level. using an established cox- activity assay monitoring immediate conversion of exogenously added arachidonic acid to the respective prostaglandins (pgs), ns- was shown to suppress celecoxib-induced cox- activity when added prior to arachidonic acid. among the cox- -dependent pgs analyzed, pgd and its dehydration product -deoxy-∆ , -pgj were found to induce cytosol-to-nucleus translocation of pparγ as well as pparγ-dependent apoptosis. celecoxib-elicited translocation of pparγ was inhibited by preincubation of cells with ns- which itself did not alter celecoxib-induced total pparγ protein expression. finally, a cox- -and pparγ-dependent proapoptotic mechanism of celecoxib was confirmed in primary tumor cells obtained from brain metastases of two lung cancer patients. together, our data demonstrate a proapoptotic mechanism of celecoxib involving initial upregulation of cox- and pparγ and a subsequent nuclear translocation of pparγ by cox- -dependent pgs. uncoagulated ppp contained - nm thrombin throughout. fxa was initially measured in clots at nm. this level declined over time while clot-conditioned pbs accumulated fxa. exposure of human aortic smc to clots or native unclotted ppp for h only marginally influenced smc apoptosis but increased mitogenesis over -fold. this was reduced by all inhibitors. clot-stimulated induction of tnfα and interleukin- mrna was also attenuated by the inhibitors. denatured ppp (no protease activity) increased smc mitogenesis to a level seen in smc exposed to clot and combined hirudin + dx a, reflecting the well-known mitogenic actions of serum alone. conclusion: coagulation of human plasma generates nanomolar amounts of thrombin and fxa, sufficient to stimulate the proliferative and inflammatory properties of adjacent smc. our observations validate the use of purified thrombin and fxa at nanomolar concentrations for in vitro studies, and support the individual and coagulationindependent roles of these proteases in cell proliferation and inflammation. antithrombotic therapy with argatroban or rivaroxaban may limit the cellular effects of clot-derived thrombin and fxa, while normal anti-platelet therapy would not. this aspect should be considered in the clinical use of these agents, specifically in healing processes after vessel injury. rhogef mediates cgmp/cgk induced adherence and relaxation of vascular smooth muscle cells rauch j. , stephan-schnatz k. the guanine nucleotide exchange factor rhogef is the only gef known so far to be directly activated by cgmp-dependent kinase. it is expressed in various types of smooth muscle cells and has been shown to play a role in the regulation of cell integrity. in a previous investigation we showed that the knockdown of rhogef by a shrna approach caused a loss of actin stress fibers and a subsequent change of smooth muscle cell morphology that finally resulted in cell rounding. we now provide evidence that the expression level of rhogef influences the re-attachment of cultured rat aortic smooth muscle cells (rasmc) to a surface after detachment. although rhogef depleted rasmc were still able to adhere and spread, their cell surface area remained considerably smaller than that of control cells in the first hours after seeding. cell counting revealed that to hours after seeding the percentage of adherent cells was significantly lower in the rhogef knockdown group compared to the control group. these data indicate a delay in attachment. interestingly, the knockdown of rhogef was paralleled by a loss in rhoa and cadherine expression. as rhogef mediated a cgmp-induced activation of the small gtpase rhoa in rasmc, we studied the effect of a stable cgmp analogon ( -pcpt-cgmp) on the adhesion process. in accordance with previously published data, cgmp treatment accelerated the attachment of rasmc to the surface within the first hours. in contrast, the adhesion of rasmc after rhogef knockdown was no longer stimulated by cgmp. as these data indicate that rhogef mediates cgmp-dependent signalling in a physiological process we wondered whether this protein might also play a role in the regulation of cgmpdependent relaxation of vascular smooth muscle cells. thus, we performed a collagenbased contraction assay with rhogef depleted rasmc. while the contraction in response to serum was not affected by the depletion of rhogef , we observed a slight decrease in basal contractility. interestingly, cgmp was not able to counteract the serum-driven contraction of rhogef knockdown cells. there was no cgmp-induced relaxation in these cells. we conclude that rhogef is involved in the cell adhesion of vascular smooth muscle cells and likely promotes the expression of specific proteins. its activation is required to mediate cgmp-induced signalling in terms of vascular smooth muscle cell adherence and relaxation. human eosinophil and neutrophil granulocytes are cells of the innate immune system. they both express formyl peptide receptors (fpr) und histamine h receptors (h r). activation of fpr leads to a release of reactive oxygen species (ros). h r activation results in an increase of intracellular '- '-cyclic adenosine monophosphate (camp) concentration and inhibition of fpr-mediated ros release via adenylyl cyclase activation. in this study we compared the effects of various h r ligands on camp accumulation and formyl peptide n-formyl-l-methionyl-l-leucyl-l-phenylalanine (fmlp)induced ros release in isolated eosinophils and neutrophils. camp concentration was determined by hplc/tandem mass spectrometry, and ros release was assessed by monitoring superoxide dismutase-inhibitable reduction of ferricytochrome c. in eosinophils, histamine, amthamine and -methylhistamine exhibited similar potencies and efficacies with regard to camp accumulation and inhibtion of ros release. in marked contrast, in human neutrophils, we observed dissociations in potencies and efficacies of ligands at increasing camp accumulation and inhibition of ros production. our data suggest that in human eosinophils, but not neutrophils, camp mediates inhibtion of ros production. in a broader context our data provide a compelling example of the context-dependency of the pharmacological properties of g-proteincoupled-receptors. specifically, one has to be cautious when extrapolating experimental observations from one cell type to another, even when they are very closely related to each other. comonomers and monomers are used as dental restorative materials (e.g. in dental composites). unconverted compounds can be released from dental composites and can enter the body in humans. comonomers can induce various side effects in humans. this study was evaluated to qualify and to quantify eluted compounds from various dental composites. following composites were tested (producer in parentheses): els extra low shrinkage (saremco), synergy duo shade (coltène), grandio (voco), tetric evo ceram (vivadent), venus (kulzer), gradia (g.c.), and premise (kerr).polymerized composites ( mg) were incubated in gc vials with ml dest. water or ml methanol, each at °c for hours. aliquots were taken, and eluted compounds were analyzed with the method of gas chromatography/mass spectrometry (gc-ms) and liquid chromatography/mass spectrometry (lc-ms).from all composites different compounds were found. following comonomers were quantified (µg/ml; mean ± s.d.; n= )( fig. ).following range of the eluted and detected comonomers from dental composites was found (dest. water; decreasing elution): venus > gradia > synergy duo shade > tetric evo ceram premise > grandio > els extra low shrinkage. * n.d. = not detectable (below limit of detection). triphenylstibane was detected in tetric evo ceram ( ± µg/ml). reimann c., lupp a., schulz s. institut für pharmakologie und toxikologie universitätsklinikum jena, drackendorfer str. objective: cxcr is a plasma membrane chemokine receptor which is involved e.g. in organogenesis, hematopoesis and inflammation. in the adult organism cxcr is physiologically expressed on various cell types, in particular on lymphocytes. with respect to neoplastic tissues, in the current literature an over-expression of cxcr is described in different types of tumors, especially in breast and prostate cancer. additionally, an involvement of cxcr in tumor metastasis is discussed. subsequently, detection of cxcr expression in a given human tumor sample would provide a valuable predictive information on disease prognosis and possible therapeutic intervention. however, previous attempts to localize cxcr using poorly characterized mouse monoclonal or rabbit polyclonal antibodies have yielded predominant nuclear and occasional cytoplasmic staining, but did not result in the identification of cell surface receptors. thus, the aim of the present study was to reassess the cxcr expression in a panel of formalin-fixed and paraffin-embedded human normal and neoplastic tissue samples by means of immunohistochemistry using the well characterized novel rabbit monoclonal anti-cxcr antibody umb- . methods: in comparison to negative and positive control samples (cxcr -knockout and wild-type mouse embryos) the extent of staining in the different normal and neoplastic tissue specimens was scored from zero (no expression) to three (high expression). results: cxcr was found to be expressed in all neoplastic tissue entities analyzed. in many cases, the receptor was predominantly localized at the plasma membrane of the tumor cells. however, in all cxcr -expressing tumor entities a huge interindividual variability both in the percentage of positive cells and in the intensity of staining was noted which strongly differed also between the various types of cancer. the most intense (score three) staining was found in the samples of (small cell) lung cancer, ovarian cancer and of pheochromocytoma. additionally, lymphatic organs such as lymph nodes, spleens and tonsils were cxcr positive, with mainly b cells displaying a distinct staining of the plasma membrane. the rabbit monoclonal antibody umb- may prove to be of great value in the assessment of cxcr expression in different human tumor entities and of the mechanisms underlying the formation of metastases, thus helping to find new targets and strategies in cancer therapy. link between β -adrenoceptor-mediated inhibition of formyl-peptide-induced o β -adrenoceptor (adrb )-agonists are in daily use for asthma therapy. although most cases of asthma are controlled by standard medication, a subpopulation of asthmatics remains difficult to treat. the adrb gene contains a total of polymorphisms. this variability could cause part of the ~ % genetically-determined differences in therapy response. genetic and corresponding functional data on adrb can help to understand the complex disease and, in cases of severe asthma, optimize therapy with adrb agonists for each individual. (ortega et al. ; chung et al. ) our present study connects sequence data with pharmacological data of prototypical adrb -ligands, namely, (r)-isoproterenol, (r,r)-and (s,s)-fenoterol, (r)-and (s)salbutamol and (r,r)-formoterol. as a pathophysiologically relevant cell type, we analysed human neutrophils from peripheral blood of healthy volunteers. formyl-peptide-induced o .production and its inhibition by the agonists are examined in a -well cytochrome-c assay. characteristic pharmacological values (pic , emax) are obtained for each individual. the data-set for each individual is supplemented by a differential blood cell analysis and an asthma-related questionnaire. most importantly, each volunteer's adrb -sequence variant is determined by sanger-sequencing. complete determination of a , bp sequence, including the entire adrb exon and part of the flanking '-and '-untranslated regions, allows mapping of the most common, but also of new or rare polymorphisms. first data demonstrate the inter-day and intra-individual robustness of the functional data. in the next step, we will link sequence variants and functional differences within a population of sixty volunteers with sufficient statistical power. collectively, this study represents a straightforward approach to link functional and genetic data of a clinically relevant receptor. cyclic nucleotide phosphodiesterases (pdes) are classified into eleven families and are essential for second messenger metabolism in human cells. recently, we have shown that several human pdes possess a much broader substrate-specificity than previously assumed, being capable of hydrolyzing not only the purine nucleotides cyclic adenosine ', '-monophosphate (camp) and cyclic guanosine ', '-monophosphate (cgmp), but also pyrimidine nucleotides such as cyclic uridine ', '-monophosphate (cump). these data were obtained using a highly sensitive hplc mass spectrometric assay which is quite expensive and whose technical requirements are available only in few laboratories. in our present study we developed a fluorimetric pde activity assay using '-o-(n'methylanthraniloyl) (mant)-substituted cyclic purine and pyrimidine nucleotides that can be used more broadly in the scientific community. human pde a is important for the regulation of platelet aggregation, oozyte maturation, vascular smooth muscle relaxation and contractility of cardiac myocytes. moreover, this pde shows a broad substrate-specificity, hydrolyzing camp, cgmp, cump and cyclic inosine ', '-monophosphate (cimp). using this enzyme, here, we demonstrate that various mant-substituted cyclic nucleotides are substrates of pde a and undergo a significant change in fluorescence whilst being hydrolyzed, thus allowing a quantitative analysis of catalysis via fluorescence detection. in fact, not only native cump but also mant-cump is a substrate of pde a. this finding is consistent with data published by hardman and sutherland who described a cump-degrading pde activity in homogenates from beef and dog heart, leading to the assumption that the pde activity described there could be attributed to pde a. as cump has furthermore been proven to be present in mammalian cells , to differentially activate camp-and cgmp-dependent protein kinases and to be synthesized from utp by mammalian soluble guanylyl cyclase , our present study supports the hypothesis that this cyclic nucleotide could play an important role in cell metabolism. the newly established fluorescence assay with mant-cump facilitates future studies on pde a and the assumed second messenger function of cump. rhoa is reportedly involved in stat-dependent transcription. however, the pathway connecting the gtpase and stat signaling has not been characterized. we made use of bacterial toxins, which directly activate rho gtpases to analyse this pathway. cytotoxic necrotizing factors (cnfs) are produced by pathogenic escherichia coli strains and by yersinia pseudotuberculosis. they activate small gtpases of the rho family by deamidation of a glutamine, which is crucial for gtp hydrolysis. we show that rhoa activation leads to phosphorylation and activation of stat and identify signal proteins involved in this pathway. rhoa-dependent stat stimulation requires rock and junkinase activation as well as ap -induced protein synthesis. the secretion of one or more factor/s activate/s the jak-stat pathway in an auto/paracrine manner. we identify ccl /i- as an essential cytokine, which is produced and secreted upon rhoa activation and which is able to activate stat -dependent signaling pathways. the knowledge about the connection between rhoa and stat signaling is crucial for understanding several deseases, especially cancer. acid sphingomyelinase-deficient mice are protected from the lethal cardiovascular effects in tnf-induced septic shock reiss l. k. christian-albrechts universität institut für immunologie, michealisstrasse , kiel, germany introduction: the cytokine tumor necrosis factor (tnf) is a mediator of septic shock. sepsis is a major cause of acute respiratory distress syndrome (ards), a heterogeneous lung disease with a mortality of about %. the present study was designed to investigate the effects of high systemic tnf-levels on the lung and on the systemic circulation in wildtype and acid sphingomyelinase-deficient (asm -/-) mice. the enzyme acid sphingomyelinase generates the signaling molecule ceramide that plays a critical role in edema formation and vasodilatation. material and methods: asm -/and wildtype mice were ventilated mechanically at vt= ml/kg and f= min - with fio = . and peep= cmh o while lung mechanics were followed. half of the mice received µg of murine tnf intravenously. blood pressure was stabilized by intra-arterial fluid support and body temperature was kept at °c to prevent lethal shock and to allow investigation of blood gases, lung histopathology, pro-inflammatory mediators and microvascular permeability hours after tnf application. results: tnf induced septic shock in wildtype mice, as indicated by metabolic acidosis, high serum levels of the sepsis marker procalcitonin, decreasing blood pressure and reflex tachycardia. interestingly, asm -/mice were protected from the tnf-induced cardiovascular effects and mortality. in the present study, circulating tnf failed to cause lung injury. lung mechanics stayed stable during ventilation in all groups and also pulmonary histopathology, cytokine levels and microvascular permeability were unaffected. conclusion: circulating tnf alone is not sufficient to cause acute lung injury. we conclude that the cardiovascular effects in tnf-induced septic shock are partly mediated by acid sphingomyelinase. cyclophilin a sirna provides mitoprotection and prevents aif-dependent neuronal cell death reuther c. cyclophilin a (cypa) is a peptidyl-prolyl-cis-trans isomerase which is localized in the cytosol. recent data suggested that neuronal cell death involved cytosolic cypa translocation to the nucleus, where it formed a pro-apoptotic complex with apoptosis inducing factor (aif). this cypa-aif complex induced caspase-independent chromatin condensation, dna degradation and cell death in various paradigms of apoptosis. on the basis of these data, the selective inhibition of the aif-cypa complex was proposed as a potential strategy to prevent aif-dependent cell death in neurons. therefore, the aim of this study was to determine effects of cypa silencing in a model of glutamate toxicity in immortalized hippocampal ht neurons. first, we addressed the interaction of aif and cypa by immunoprecipitation and their translocation to the nucleus by immunohistochemistry and confocal fluorescence microscopy. after exposure of ht- cells to glutamate the translocation of aif and cyp a occurred prior to cell death. cypa sirna attenuated glutamate-induced cell death as detected by the mtt-assay, impedance measurements (xcelligence system), and by facs analysis after annexin v/ propidium iodide staining. most intriguingly, cypa sirna also preserved the mitochondrial membrane potential as shown by facs analysis after tmre staining. further, confocal microscopy showed that cypa silencing prevented mitomorphology alterations and blocked the release of mitochondrial aif to the nucleus. the inhibition of the aif translocation to the nucleus was also shown by western blot analysis. in summary this study demonstrates that silencing of cypa prevents mitochondrial disruption and attenuates glutamate toxicity in vitro. thus, cypa is a promising target for mitoprotection as a basis for novel strategies of neuroprotection. up to now the question is unresolved how the ingested dose influences the absorption and metabolism of chlorogenic acids (cga) from food. so far no studies have been performed on the impact of the dose on cga absorption, circulation and excretion. recently we performed a dose-response study in a randomized, double-blinded, crossover design with five ileostomy subjects. in three trials the volunteers consumed after a two day polyphenol free diet coffee with varying cga content (high µmol; medium µmol; low µmol). the cga concentrations in plasma, ileal effluent and urine were subsequently identified and quantified by hplc-esi-ms, hplc-esi-ms/ms and hplc-dad. the results showed that the consumption of higher cga concentrations lead to a faster ileal excretion measured in the ileal effluents. this corresponded to the renal excretion of . ± . % (high), . ± . % (medium) and . ± . % (low) of total cga and metabolites. we found that cgas with a caffeic acid moiety are predominantly sulphated and those with a ferulic acid moiety are predominantly conjugated via glucuronidation prior renal excretion. furthermore, in the ileal effluents, sulphation of both structural units dominated. in plasma samples (after enzymatic deconjugation) the auc values were determined by the major cga classes in coffee, the caffeoylquinic acids: . ± . nm*h - (high); . ± . nm*h - (medium) and . ± . nm*h - (low). no major differences in the metabolic pattern were observed. additionally, we were able to identify new metabolites of cga in urine and ileal fluids. we conclude that the consumption of high concentrations of cga via coffee might influence the gastrointestinal transit time and consequently affect cga bioavailability. this study was supported by the nestlé research centre (lausanne, switzerland). interaction of antagonists with the atp binding pocket at the human p x ion channel helms n., riedel t., illes p. rudolf-boehm-institut für pharmakologie und toxikologie, härtelstraße - , leipzig, germany the homomeric p x receptor (p x r) is a rapidly activating and desensitizing cation channel, gated by extracellular atp. it consists of three homomeric subunits. this representative of the p x receptor family is highly expressed on sensory afferent neurons and plays a significant role in chronic pain, bladder reflexes and taste sensation. therefore, the development of selective antagonists for p x receptors and knowledge about the binding of these antagonists are of great significance for future pain therapy and therapy of urge incontinence. to simulate the shape of the rapidly desensitizing agonist-induced current responses via p x receptors, we created a specific markov model to describe the binding of agonists and competitive antagonists. this model can be used to prove the competitive character of inhibition and to calculate the association and dissociation constants of the antagonists. furthermore we use this model to fit current responses at p x wild type receptors and their mutants to α,βmethylene atp in the presence of different antagonists. whole-cell patch-clamp recordings were performed on hek cells, heterologously expressing the human p x receptor, to determine the concentration-response relationship of different antagonists. by applying increasing concentrations, differences of antagonist potency could be observed at the wild type receptor. afterwards, we chose amino acid residues for replacement by alanine, which seem to be important for agonist binding and should be so for competitive antagonist binding as well, based on our homology model, developed from the zebrafish p x r crystal structure and previous mutagenesis studies. we intend to identify those amino acids which are important for competitive antagonist binding by monitoring the altered antagonist potency on the mutated receptor when compared with the wild-type receptor. analysis of the p x agonist binding site by double mutant cycles riedel t., wiese s., illes p. rudolf-boehm-institut für pharmakologie und toxikologie, härtelstraße - , leipzig, germany purinergic p x receptors belong to the family of ligand-gated ion channels. they are non-selective cation channels, activated by extracellular atp. one of the seven members of the p x receptor family, the p x receptor, is localized at the plasma membrane of sensory neurons and is involved in pain perception. therefore, this receptor is a possible target for new drugs in pain treatment. the development of such drugs can be supported by an exact knowledge of the receptor structure and function. there are many hints to the atp binding site, but the interaction of the p x receptor with its agonists and antagonists remains still unknown. in this study, we investigated the effects of single alanine substitutions of amino acid residues in the supposed atp binding site of the homomeric human p x receptor on the effect of nucleotide analogues. the mutant receptors were expressed in hek cells and the nucleotide effects were measured by means of the whole-cell patch-clamp method. modifications in the receptor binding site changed the concentration-response dependency as well as the current kinetics during fast pulsed agonist applications. based on this fact, we were able to distinguish binding from gating, conductance, and desensitisation, using a markov model that describes the complete channel behaviour by a matrix of rate constants. the results were also checked for consistency with a structural hp x model that we developed from the known zebra fish p x crystal structure in the closed state. voltage-dependent modulation of alpha a adrenergic receptor signaling rinne a., birk a., bünemann m. philipps-universität marburg institut für pharmakologie und klinische pharmazie, karlvon-frisch str. , marburg, germany g protein-coupled receptors (gpcrs) are proteins that regulate numerous signaling pathways by activation of intracellular g proteins. gpcrs are activated by extracellular stimuli, such as light, hormones and neurotransmitters. recent evidence suggests that some gpcrs exhibit voltage-sensitivity leading to a modulation of their activity by the membrane potential (vm). we used a fret-based biosensor of the α a adrenergic receptor to analyze receptor activation at defined membrane potentials in hek cells by means of voltage-clamp recording. the biosensor was stimulated either with the partial agonist clonidine or with the full agonist norepinephrine (ne) and receptor activation was measured as decrease in the ratio of acceptor-/donor-fluorescence. receptor stimulation by ne was inhibited at depolarizing membrane potentials but enhanced by hyperpolarization. inhibition of ne activated receptors was strong at low concentrations ( nm: % inhibition) but almost absent at saturating agonist concentrations ( µm: % inhibition). both agonist-induced and hyperpolarizationinduced receptor activation exhibited a similar monoexponential time course and speed of activation was primarily dependent on agonist concentration for both activation modes. the latter indicates that depolarization lowers the apparent affinity of the ne receptor interaction and thus causes receptor deactivation by means of ne release. application of clonidine ( µm, vm=- mv) resulted in a fret response that was inhibited by % at + mv. in contrast to ne, strong receptor inhibition at + mv was present even at super-saturating concentrations of clonidine ( µm), suggesting that voltage alters the equilibrium between active and inactive conformations of the receptor. voltage-dependence of the a a adrenergic receptor also modulated downstream receptor signaling: g protein activation or the recruitment of arrestins, which we determined in fret assays that directly detect gαi protein activation or receptor-arrestin interactions, were both substantially inhibited at vm = + mv. therefore we conclude that negative membrane potentials promote active conformations of the a a adrenergic receptor, increase affinity of full agonists and enhance receptor signaling. in conclusion the present data show that scc cells extrude more ha, possibly related to increased levels of has , in comparison to keratinocytes. increased amounts of ha appear to be essential for the uvb induced tumourgenesis of sccs in mice. this effect might be related to the pro-proliferative property of high molecular weight ha. furthermore biological active ha fragments derived from ha degradation by hyaluronidases (hyal , ) are thought to be pro-angiogenetic, anti-apoptotic and proinflammatory, thus possibly also promoting tumour growth and malignancy. estradiol induced paracrine release of egf from keratinocytes protects the dermal hyaluronan/versican matrix during photoaging röck k. , meusch m. hyaluronan (ha) and versican are key components of the dermis and are responsive to uvb induced remodeling. the aim of the present study was to investigate the molecular mechanisms of estrogen (e ) mediated effects on ha-rich ecm during actinic aging. weeks of uvb irradiation ( x med ( mj/cm ), weekly) of hairless skh- mice caused a marked decline of dermal ha, which was aggravated by ovariectomy (ovx). subcutaneous substitution of estrogen (e ) by means of controlled release pellets abolished these effects confirming the stimulatory role of e . the increase of dermal ha correlated with induction of ha synthase has by e . in addition the ha-binding proteoglycan versican was induced by uvb and further increased by e . however in cultured skin fibroblasts e reduced the expression of versican and had no effect on has . therefore, direct upregulation of has and versican in fiborblasts by e was excluded. however, e increased the expression of egf in uvb irradiated skin in vivo and in keratinocytes in vitro. egf in turn upregulated the expression of has and versican in dermal fibroblasts. furthermore the supernatants of estradiol treated keratinocytes led to the same effects in dermal fibroblasts, which could be abolished by previous treatment of the supernatant with neutralizing egf antibody or treatment of the fibroblasts with egf receptor blocker erlotinib. functionally, dermal ha and versican induction by e correlated positively with proliferation and negatively with accumulation of inflammatory macrophages in the dermis. collectively these data suggest that e treatment increases the amount of dermal ha and versican via paracrine release of egf which may be implicated in the pro-proliferative and anti-inflammatory effects of e during photoaging. differential pro-and eukaryotic toxicity of silver released from nanocomposite surfaces increases the therapeutic window of silver in antibacterial treatments röhl c. , hrkac t. silver has been used since ancient times as antimicrobial agent. recently, silver gained new attention due to its higher effectiveness in its nanoform. this led to new developments of silver nanomaterials, e.g., for medical devices and consumer products. though, it is generally assumed that silver is less toxic for eukaryotes than for prokaryotes, concern is raised, if nanosilver at the same time might also increase mammalian cytotoxicity. in our study we examined the toxicity of silver released from nanocomposite surfaces with that of silver from agno solutions for adherent bacteria and mammalian cells. therefore, we established an in vitro reference system which enabled us to compare the therapeutic window between prokaryotic toxicity and eukaryotic integrity in both exposure settings. we focussed especially on the comparability of the bacterial and mammalian cell systems and the development of characterized ag/tio nanocomposite coatings with well-defined silver filling factors and silver surface release, which could be varied over a wide concentration range. as reference cells the e. coli sar strain and human dermal fibroblasts, which are of special relevance in the context of medical devices like implants or wound dressings, were chosen. bactericidal effects were determined by direct growth visualization of the gfp-producing e. coli strain by epifluorescence microscopy. mammalian cell growth and toxicity was determined by the mtt assay, protein measurements and phase contrast microscopy. the ag/tio samples were prepared by sputter co-deposition from two separate magnetron sources. the silver surface concentration release was determined by xps and the silver release by icp-ms. in solution a concentration-dependent constant silver concentration could be determined between and at least hours at the surface. while lowest bactericidal and cytotoxic concentrations of ag + from agno solutions with . and . mg/cm , respectively, differed only slightly, the therapeutic window increased significantly if ag + was released from the nanocomposite surface. while the toxicity on the fibroblasts was unchanged the bactericidal potency increased at least one order of magnitude. taken together, it can be concluded that local exposure factors i) can be modulated by silver nanocomposites and ii) play an important role for the differential toxicity of surface silver on bacteria and mammalian cells. charite -universitätsmedizin institut für integrative neuroanatomie, funktionelle zellbiologie, philippstr. , berlin, germany c exoenzyme (c bot) a clostridial adp-ribosyltransferase does not possess a cellbinding/-translocation domain. nevertheless, c is able to efficiently enter intact cells, including neuronal cells but the mechanism of uptake is not yet understood. in the present work, binding of c bot to the hippocampus-derived ht cell line was characterized by means of binding and blot overlay assays as well as mass spectrometry analysis to identify binding partners of c bot. the binding assays established that c bot bound in a concentration-dependent manner to ht cells. in the overlay assay we detected one clear band of kda. to elucidate whether glycosylation is important for the c bot-protein interaction, ht cells were incubated with glycosidase f resulting in a decreased binding of c to the kda band. to explore the involvement of phosphorylation in the binding of c to the putative binding protein, blot was pre-treated with cip (calf intestinal phosphatase) before overlay with c bot. pretreatment greatly reduced the c bot-protein interaction. moreover, inhibition of dephosphorylation by vanadate before in intact cells showed an increased level of c botprotein interaction in the following overlay. thus, interactions between c bot and ht cell proteins may require phosphorylation. to further characterize the kda band as binding target of c bot, the kda band was digested with trypsin and then subjected to lc-orbitrap mass spectrometry analysis. from this kda single gel band proteins were identified. further analysis of the identified proteins will provide a possible interaction partner of c bot. in sum, protein overlay assays revealed that phosphorylation and glycosylation are critical for efficient c bot-protein interaction. s solvent effects on enzyme kinetics in vitro rokitta d., pfeiffer k., gerwin h., streich c., fuhr u. uniklinik köln institut für pharmakologie, gleueler str. , köln, germany kinetic parameters provide essential quantitative information for characterisation of drug metabolising enzymes. such enzymes are located in an a partially queous environment, but to solve potential lipophilic substrates for in vitro measurements organic solvents are regularly needed. to preserve the enzymes from denaturation and other solvent related effects, the concentration of these solvents must be kept low. data on nature and extent of such solvent effects is sparse. in this study, we investigated the effects of methanol, ethanol, acetonitrile and dimethylsulfoxide ( % to %) on the assessment of k m, vmax and clint with regard to the -hydroxylation of midazolam via cyp a and the cyp a catalyzed metabolism of caffeine to paraxanthine in vitro. the presence of acetonitrile showed the highest vmax value for paraxanthine formation but the lowest values for -hydroxymidazolam formation. the km value for midazolam showed no systematic effects of organic solvents, while for caffeine km was up to eightfold lower for solvent free samples compared to solvent containing samples. the present example suggests that the presence of organic solvents may considerably influence enzyme kinetic parameters beyond a mere change in apparent activity. these effects are differing between enzyme-substrate systems and solvents. it remains to be determined to which extent such effects compromise in vitro -in vivo extrapolations, and which solvents are most appropriate. atrial remodeling and arrhythmia induced by the transcription factor er rommel c. , rösner s. introduction: the transcription factor er (ets related ) belongs to the large family of ets-transcription factors that are involved in developmental processes and in the pathogenesis of cancer. er is activated by gq-and gs-coupled receptors leading to a phosphorylation of the transcription factor by map-kinases and protein kinase a, respectively. cardiac er mrna expression is increased in failing human hearts. however mechanical unloading by a left ventricular assist device leads to normalization of er expression. thus, the aim of the present study was to investigate the cardiac function of er in genetically modified mouse models. we previously generated transgenic mice overexpressing er under control of the cardiomyocyte-specific α-myosin heavy chain gene (αmhc) promoter by pronuclear injection and established independent transgenic lines. electrocardiography (ecg) was assessed in mice at day after birth (p ) and in adult mice ( months) during isoflurane anesthesia and by ecg telemetry in awake mice, respectively. ecg analysis revealed no differences between the genotypes at day after birth. however, we found a decreased heart rate, a replacement of regular p-waves by an undulating baseline and frequent supraventricular extrasystoles in adult er αmhc transgenic mice. next, isometric contractile force measurements on isolated left atria were carried out in organ baths. while wt left atria responded to increasing concentrations of isoprenaline, nkh and calcium with an increase in contractility, the maximal positive inotropic responses to these substances were severely blunted in er αmhc atria. we performed western blots to identify potential aberrations of calcium handling and regulatory proteins. phosphorylation of serine of phospholamban (pln) was reduced in er αmhc mice. in addition, protein phosphatase (pp ) expression was significantly increased in er αmhc mice, which is consistent with the increased dephosphorylation of phospholamban. furthermore, we found a decreased expression of calsequestrin and serca a protein in er αmhc atria. electron microscopy revealed the significant structural remodeling of er αmhc atria at months of age. conclusion: increased cardiac expression of the ets-transcription factor er leads to structural and electrical remodeling of the atria. thus, er may play an important role in the pathogenesis of cardiac arrhythmias in chronic heart failure. signal transduction pathway of atp and utp in neonatal rat cardiac myocytes rothkirch d., gergs u., neumann j. institute for pharmacology and toxicology medical faculty, magdeburger str. , halle (saale), germany extracellular atp and utp can be released from the heart during pathological conditions such as ischemia or hypoxia. in humans, atp and utp levels are increased during myocardial infarction. atp and utp can act via p -purinoceptors which are further divided in p x - and p y - -receptors. as previously shown atp and utp can induce inotropic effects in cardiac preparations of mice and man. for rat articular chondrocytes and human intestinal cells it has been demonstrated that the mapk cascade can be activated by atp and utp. therefore, the cardiac effects of atp and utp on force of contraction probably occur via the mapk pathway. to investigate the signal transduction pathway involved, we studied the effects of atp and utp on mapk phosphorylation in isolated neonatal rat cardiac myocytes using phosphorylation-specific antibodies. µm atp as well as utp transiently increased phosphorylation of erk / and p mapk with a maximum effect at to minutes after application of atp and utp in neonatal cardiac myocytes (n= preparations each). the maximum phosphorylation of p increased with atp to % ± % (p< . ) at minutes and with utp up to % ± % (p< . ) at minutes. the phosphorylation with erk / mapk increased with atp to % ± . % (p< . ) at minutes and to % ± % (p< . ) with utp at minutes of basal values, respectively. after minutes, predrug values of mapk phosphorylation were reached again. in summary, we noted an atp-and utp-induced phosphorylation of erk / and p mapk in isolated neonatal rat cardiac myocytes. the involved receptor subtype(s) and the link between mapk phosphorylation and inotropic effect of atp and utp need to be elucidated. hameel: use of elearning in teaching pharmacology and toxicology -the halle experience rulf k. , gergs u. introduction: during the past three years, our faculty has started to integrate items of elearning into the standard curriculum of a classical medical school: the "hallesches medizinisches elearning -hameel". our hypothesis was that these new elearning tools would improve the willingness of students to spend more time into learning and this would lead to an improved outcome (in multiple choice tests). methods: hence, we offered medical students ( th or th semester) additional learning environments. the courses for students (experimental pharmacology and toxicology or clinical pharmacology) were existed of a weekly lecture and in addition tutorials (problem-based-learning style, paper cases) or classical seminars. furthermore, we offered the possibility to use an online multiple choice quiz (involving - previously used tests) and/or an online module on heart failure each week. we used the learning management system ilias software in combination with the content management system stud.ip. all students were subjected to an introductory test (to assess knowledge prior to our teaching section and allowing us to exclude a conceivable bias due to previous knowledge, involving basic items from prior teaching opportunities), a mid-term test and a final test to assess gain of knowledge. a maximum of points could be obtained as a sum of both tests. results: in the means % of students used the new elearning tools (quizzes, heart failure module). however, there was no association between the use of self-assessment quizzes and examination results. the usage of the online quizzes increased in the periods before the exams. however, usage of the heart failure module was accompanied by significantly increased scores in exams. moreover, in a formalized evaluation system, students positively commented on our elearning efforts. conclusions: while usage of our quizzes did not improve test marks, another more sophisticated clinically oriented elearning module seemed to be improving test outcomes marginally. targeting of erk thr phosphorylation attenuates cardiac hypertrophy but preserves the anti-apoptotic effects of erk / ruppert c., vidal m., lohse m. j., lorenz k. institut für pharmakologie und toxikologie pharmakologie, versbacher str. , würzburg, germany background and aims: the extracellular regulated kinases and (erk / ) play an important role in cardiac hypertrophy and cell survival. erk / are phosphorylated at the so-called tey motif, which in turn activates erk / . hypertrophic stimuli lead to an additional autophosphorylation threonine (thr ). this autophosphorylation of erk / stimulates activation of nuclear erk targets, which are known to induce hypertrophy. the aim of this study is to investigate whether specific targeting of erk thr phosphorylation affects both erk functions -erk mediated hypertrophy and cardioprotective cell survival. methods and results: for the analysis of cardiomyocyte hypertrophy in vitro, we stimulated cardiomyocytes with phenylephrine and measured the incorporation of tritiated isoleucine. cardiac hypertrophy was assessed by echocardiography before and after transverse aortic constriction (tac). for analysis of cell survival, caspase activity and dna fragmentation was determined upon hydrogen peroxide stimulation in vitro and in response to tac in vivo. to differentiate between inhibition of erk / activity and prevention of erk thr phosphorylation, we either inhibited erk activity with pd or overexpressed a mutant of erk , which cannot be phosphorylated at thr phosphorylation. while inhibition of overall erk activity with pd attenuated cell survival and hypertrophy in vitro, specific targeting of erk thr phosphorylation by overexpression of the phosphorylation deficient mutant (erk t a ) attentuated phenylephrine induced hypertrophy, but preserved the anti-apoptotic effects of erk. cardiac overexpression of erk t a significantly reduced tac-induced hypertrophy compared to wild-type erk overexpressing mice. in line with the in vitro experiments, erk thr inhibition only prevented hypertrophy in the tac model without promoting apoptosis. conclusions: these results show that blockade of erk thr phosphorylation attenuates cardiomyocyte hypertrophy but preserves anti-apoptotic effects of erk / . therefore, specific targeting of erk thr phosphorylation might be a promising strategy for the treatment of pathological hypertrophy. intracellular camp levels are determined by interplay of camp formation by adenylyl cyclases and camp degradation by phosphodiesterases (pde). eleven families of pdes are known. one of the most recently identified pdes is pde , a pde in principle capable of hydrolysing camp as well as cgmp. pde contains a tandem of so called gaf domains in its n-terminal regulatory domain that mediate activation by camp. because current knowledge about the tissue distribution of pde was mostly based on the analysis of mrna distribution, we generated antisera against pde to analyze tissue distribution of the protein level. using these antibodies, we found a prominent occurrence of the enzyme in testis and in brain, where it was confined to the striatum. thus, pde displays a comparably restricted tissue distribution which is in contrast to that of many other pdes. low camp levels in so called medium spiny neurons of the striatum have been implicated in schizophrenia. furthermore, studies using the nonspecific pde inhibitor papaverine as well as specific pde inhibitors suggest pde as a target for the treatment of schizophrenia. here we set out to analyze the contribution of pde to camp degradation in striatum, to identify the physiological pathways pde is involved in and to clarify the functional impact of the proposed phosphorylation of the enzyme. identification of cgmp-dependent kinase i substrate complexes salb k., schlossmann j. universität regensburg lehrstuhl pharmakologie, universitätsstrasse , regensburg, germany the cgmp-dependent kinases (cgks) are components of the no/cgmp/cgk-signalling pathway and have a great physiological importance in a multitude of tissues and organs such as smooth muscles and platelets. two isoforms of the cgki and the cgkii are known. cgkiα and cgkiβ differ only in their first ~ amino acids which constitute the leucine zipper and the autoinhibitory domains. the n-terminal leucine zipper domains mediate homodimerization of the kinase and the interaction with diverse substrate proteins. since cgkiα and cgkiβ express different n-termini they interact with different substrates. the cgkiβ isoform is assembled in a macrocomplex at the endoplasmic reticulum (er) with the intracellular calcium release channel inositoltrisphosphate receptor i (insp r-i) and the inositol-trisphosphate receptor associated cgmp kinase substrate (irag). we investigated, whether irag also interacts with the insp r-ii and the insp r-iii in murine platelets and tissues. additionally, we analyzed the interaction between the amino acid peptide phospholamban (plb), which is also located at the er and regulates the er calcium reuptake by the sarco/endoplasmic reticulum ca + -atpase (serca), and the two cgki isoforms. we performed cgmp-agarose experiments with murine wt and irag-ko platelets to examine the irag-insp r interactions. the insp r-ii isoform was neither bound to cgmp-agarose nor detected in the anti-irag immunoprecipitate. on the other hand, insp r-iii from wt but not from irag-ko platelets was bound to cgmp-agarose. hence, insp r-iii interacts directly with irag but not with cgkiβ in murine platelets. however, in colon smooth muscle lysate, insp r-iii not only interacted with the irag protein but was also detected in the anti-cgkiα-immunoprecipitate. phospholamban from wt and irag-ko platelets was also bound to cgmp-agarose. subsequent immunoprecipitation experiments with the respective antibodies against the two cgki isoforms revealed that plb interacted both with cgkiβ and cgkiα. these results were supported by analysis of colon smooth muscle tissue from wt and irag-ko mice. in conclusion, irag interacts with insp r-i and insp r-iii but not with insp r-ii in murine platelets and colon smooth muscle tissue. moreover, phospholamban is an interacting partner of both the cgkiα and the cgkiβ isoform. the human immunodeficiency virus type enhancer binding protein (hivep ) is regulated by proinflammatory stimuli and statins salomon a. , , schmitz b. objective: hivep binds nf-ĸb and other proinflammatory consensus sequences, and is suggested to be involved in inflammatory processes. we recently identified two tagging snps, one positioned kb upstream (rs ) and another in exon (rs ) of the hivep gene, to be replicatively associated with venous thrombosis in gwas and follow-up studies (ajhg, ; plos one, ) . methods: total rna isolation was performed after treatment of vascular endothelial cells (ea.hy ) with proinflammatory cytokines or statins ( h). serial hivep promoter deletion constructs were cloned into the pgl -basic vector, a potential enhancer fragment, harbouring rs c/t, into the pgl -promoter vector. in ea.hy cells and thp monocytes, reporter gene assays were performed by transient transfection and overexpression of transcription factors. chip and bandshift assays were performed to identify candidate transcription factors. results: in ea.hy cells, endogenous hivep expression was increased by proinflammatory cytokines tnfα and il- β. simvastatin ( . and . µm) and atorvastatin ( µm) -but not pravastatin or aspirin -both dose-dependently decreased basal and tnfα-stimulated hivep expression. the construct harbouring rs t exerted significantly higher transcriptional activity (ta) compared to rs c (p< . ). for an intronic modulator, reporter gene assays demonstrated a regulatory effect on hivep expression in ea.hy and thp cells. cotransfection of sp and egr led to an increase in ta, while wt exclusively upregulated ta of constructs comprising the intronic modulator. chip and bandshift assays combined with specific antibody detection revealed binding of sp to the '-flanking region and the intronic modulator of hivep . conclusion: increased hivep expression during inflammatory conditions can be repressed by simvastatin and atorvastatin, and not by pravastatin or aspirin. basal hivep expression is regulated by sp combined in a transcription factor module with egr and wt under basal and/or inflammatory conditions. the rs site harbours potential activational capacity for hivep gene transcription and may communicate with the sp /egr /wt module. to date, the treatment of various movement disorders of the central nervous system is still insufficient. in most cases this is due to the sparse knowledge of the pathophysiology. l-dopa-induced dyskinesias (lid) represent a severe complication of long-time pharmacotherapy in parkinson's disease that deserves novel therapeutics. an increased activity of striatal projection neurons, which express kv . / channels, seems to be involved in the pathophysiology of these spontaneous involuntary dystonic and choreatic movements. previous studies demonstrated an antidyskinetic effect of the kv . - . channel opener retigabine after acute and chronic treatment in a rat model of lid. in order to clarify if this effect was based on the modulation of kv . / channels, we examined the acute effects of the preferred kv . / channel opener ica on lid in this animal model. four weeks post -ohda lesioning of the left forebrain bundle, dyskinesia was induced by chronic treatment with mg/kg l-dopa and mg/kg benserazide for days. three subtypes of dyskinesia (limb, axial and orolingual) were rated according to a score system from to over min. for drug testing, ica ( , and mg/kg) was administered intraperitoneal additionally to l-dopa (or vehicle). effects of drug action in comparison to vehicle controls were detected by adding up the severity scores of each observation time. additionally, effects on parkinsonian symptoms were examined min after drug administration using the block and the stepping test. ica reduced the severity of dyskinesia significantly at all doses while no negative impact on the antiparkinsonian effect of l-dopa was observed. whereas the antidyskinetic effect was restricted to the first min after the application of mg, it lasted up to min in rats treated with mg ica . a higher dose of mg did not further enhance the antidyskinetic effect. the results of our study suggest that the antidyskinetic effect of the k v channel opener retigabine was based on its action on striatal kv . / channels. in line with the results of previous studies with retigabine, this action does not seem to interfere with the antiparkinsonian effect of l-dopa. this study was supported by the micheal j. fox foundation. background: skeletal muscle toxicity is the major side effect of hmg-coa-reductase inhibitors (statins) and can be simulated in engineered skeletal muscle. statins are known to exert "pleiotropic" effects, e.g. reducing endothelial dysfunction by inducing no synthases and no production. the role of no synthases in skeletal muscle under statin treatment is largely unknown. interestingly, some skeletal muscle pathologies (e.g. duchenne muscular dystrophy) may be exacerbated by increased inos activity. here we tested whether or not statin-induced skeletal muscle toxicity would be associated with enhanced no synthesis. we generated engineered skeletal muscle (esm) from rat skeletal muscle cells, matrigel and collagen. esms displayed typical skeletal muscle properties (differentiated muscle fibres, tetanic contractions). under baseline conditions esm expressed enos most abundantly, followed by inos and nnos (n= - ). myotoxic cerivastatin ( . , . , µm for days) caused a concentration-dependent decrease of contractile force (p< , , n= - ) paralleled by an increase in inos transcript (mean±sem: . µm ± . -fold, n= p< . ; . µm . ± . -fold, n= p< . ) and protein ( . µm . ± -fold, n= p< . ; . µm . ± . -fold, n= p< . ). mevalonic acid fully prevented the inos increase suggesting that the induction is hmg-coa reductase-dependent. to test whether inos may contribute to the decrease in contractile force we co-treated esm with w, a specific inos inhibitor. we applied µm of w, a concentration found to potently reduce lipopolysaccharide (lps)induced no-production in cultured myotubes. however, we did not observe a rescue effect (n= - ). also, l-name ( mm), an unspecific nos inhibitor, did not improve contractile function, instead we observed increased myotoxictiy (n= - , p< . ). to further investigate the role of no for muscle function we treated the esms with increasing concentrations of the no-donor snp. only high concentrations of snp ( µm) caused a reduction of contractile force. combined treatment with cerivastatin and . µm snp showed a tendency towards improved force development in esm. conclusions: statins increase inos activity in our skeletal muscle model (esm). however, this does not seem to functionally contribute to myopathy in esm. increased production of no may in fact be a protective measure. esm may help to dissect clinically relevant functional changes in statin myotoxicity. characterization of primary skin fibroblasts of patients with m syndrome and mutations in the cul gene meyer k., hieber m., engelhardt s., sarikas a. technische universität münchen institut für pharmakologie und toxikologie, biedersteiner str. , münchen, germany m syndrome is an autosomal-recessive disorder characterized by pre-and postnatal growth retardation (< - sd), facial dysmorphism and skeletal anomalies. the majority of patients harbor missense mutations of the cul ( %) or obsl ( %) gene, respectively. cul constitutes an e ubiquitin ligase that is involved in the regulation of the insulin-like growth factor (igf- ) signaling pathway via ubiquitin mediated degradation of insulin receptor substrate (irs- ). to investigate the role of cul mediated irs- degradation in the pathogenesis of m syndrome. primary skin fibroblasts of seven m syndrome patients (six with cul mutations, one with a obsl mutation) and control fibroblasts were analyzed for proliferation rate (cell counter), cell cycle profile (facs), cell morphology and cellular senescence (histochemistry), irs- protein concentrations and activation of the igf- signaling pathway (western blot). the proliferation rate of m patient fibroblasts was significantly increased when compared to control cells. in contrast, irs- protein levels and activation of the pi k/akt and erk mapk pathway were only increased in a subset of m cells that carried cul mutations, but not in cells from a patient with the obsl mutation. no significant differences in cell cycle profile, cell morphology or cellular senescence were observed in m patient fibroblasts when compared to control cells. to determine the pathogenetic contribution of increased irs- levels to the observed phenotype, human imr fibroblasts were stably transfected with retroviral vectors encoding irs- . despite -fold overexpression of irs- compared to empty vector controls, no significant effect of igf- stimulation on proliferation rate or pi k/akt and erk mapk signaling was observed. skin fibroblasts of m patients with cul mutations displayed an increased proliferation rate and enhanced activation of the igf- signaling pathways. despite accumulation of irs- in fibroblasts from a subset of m patients with cul mutations, no pathomechanistic role for irs- could be demonstrated. collectively, our data indicate that a dysregulated igf- signaling may contribute to the pathogenesis of m syndrome, yet in an irs- independent manner. pharmacases.de -a student-centered elearning project of clinical pharmacology zollner b., berg c., gros n., muß n., oestreicher d., engelhardt s., sarikas a. technische universität münchen institut für pharmakologie und toxikologie, biedersteiner str. , münchen, germany pharmacases.de is a novel e-learning website of clinical pharmacology that presents clinically relevant aspects of pharmacology and toxicology in an interactive and multimedial manner. the aim of the project pharmacases.de was to develop an innovative concept for creating high quality elearning content that i) integrates and promotes the theoretical and cooperative skills of final year medical students and ii) is easily adoptable by cooperating institutes and hospitals. a peer-teaching concept was developed in which final year medical students with the elective pharmacology (pj wahlfach pharmakologie) independently researched and wrote elearning lessions ("pharmacases"). subject-specific expertise was acquired by consulting elective students of other disciplines. at present ( / ) , this "peer network" consists of elective students of nine cooperating institutions (pathology, microbiology, radiology, cardiology, psychiatry, dermatology, neurology, ophthalmology, pediatrics) at the technische universität münchen. the average time for the generation of one elearning lession by the peer network was days. to date, the website consists of pharmacases that are available to all students online (http://www.pharmacases.de). the website also contains a discussion forum and evaluation form for direct feedback. on average, pharmacases.de has visitors per month with the following evaluation results: "excellent": %, "good": % and "satisfactory": % (n= ). the didactic concept of pharmacases.de enabled the efficient generation of high quality elearning content in a student-centered and interdisciplinary manner. the peer-teaching approach supports the collaborative skills of final year medical students and facilitates the transfer of theoretical pharmacological knowledge into clinical practice. improved glucose tolerance, less chronic adipose tissue inflammation and reduced adipose tissue mass in mice with adipocyte-specific loss of tak sassmann a., offermanns s., wettschureck n. max-planck-institut für herz-und lungenforschung pharmakologie, ludwigstr. , bad nauheim, germany tgf-β activated kinase (tak ) is known to be involved in numerous inflammatory processes by linking receptors for inflammatory stimuli like lps, interleukin- or tnfa to ikk, p and jnk activation. chronic inflammation of white adipose tissue is one of the major causes for the development of insulin resistance and impaired glucose tolerance in states of obesity. to investigate the role of tak in white adipose tissue, we crossed the tamoxifen-inducible white adipocyte-specific cre mouse line adipoqcreer t with animals carrying floxed alleles of the tak gene. adipoqcreer ; tak fl/fl animals and cre negative control littermates are viable and fertile and do not show any developmental defects. after tamoxifen induction and high fat diet feeding adipocytespecific tak knockout mice show improved glucose tolerance and lower fasting insulin levels compared to control animals. in line with this, serum levels of the adipose tissuespecific hormone resistin are reduced in adipocyte-specific tak knockout mice. these findings are accompanied by a lower state of chronic inflammation of adipose tissue as indicated by a dramatic reduction of adipose tissue macrophage number and lower serum levels of tnfα and interleukin- . stimuli like tnfα, interleukins and tgf-β released from macrophages and adipocytes are known to promote obesity-related adipose tissue inflammation. when stimulated with these substances tak deficient adipocytes show reduced activation of jnk and p which both play an important role in the development of insulin resistance. interestingly, we observe a lean phenotype in adipocyte-specific tak knockout mice when fed a high fat diet which reflects a reduction of white adipose tissue mass. currently we are investigating the molecular mechanisms underlying the reduced adiposity and lower state of chronic inflammation in adipose tissue. growth of small cell lung cancer (sclc) cells is regulated via the autocrine stimulation of g protein coupled receptors (gpcrs), i. e., neuropeptide and muscarinic acetyl choline (ach) receptors. the activation of gq/ and calcium-dependent gpcr pathways results in the stimulation of erk signaling which is necessary for the mitogenic effects of neuropeptides or ach on sclc cells. in contrast, the role of calcium-independent gpcr signaling and its interplay with gq/ -regulated pathways in sclc cells are less well defined. the aim of our studies was to characterize the molecular make-up and the interaction of these pathways, and to delineate the phenotypic effects of calciumdependent and -independent signaling cascades in sclc cells. using a panel of sclc cell lines, we found that the stimulation of neuropeptide receptors led to an increase of calcium which was independent of extracellular calcium and could be prevented by depleting internal calcium stores. this calcium increase was sufficient to activate the tyrosine kinase pyk and subsequently the erk / cascade. the role of pyk for the growth of sclc cells was further supported by the fact that inhibition of pyk using a sirna approach or a novel specific inhibitor, pf , exerted pronounced cytotoxic effects on sclc cells, whereas non-sclc cells were less sensitive. interestingly, the inhibition of g / signaling by sirna-mediated g(alpha) or g(alpha) knockdown also markedly reduced the growth of sclc in vitro or in subcutaneous tumor xenografts, and increased the sensitivity of sclc cells towards certain cytostatics. to further define the role of calcium-dependent signaling via pyk versus the role of calcium-independent signaling via g / , we tested the effect of pyk inhibition in cells with impaired g / signaling. notably, pyk and g / double inhibition led to an even increased proliferation. thus, we propose that dysbalanced g protein signaling favoring either pyk activation or g / -dependent cascades inhibits the growth of sclc cells, whereas the parallel inhibition of both pathways restores again the balance and the growth capacity in this tumor entity. dendritic cells (dcs) are essential for the initial immune response and for the defence against inhalated pulmonary toxins and carcinogens in lung. to differentiate dcs, the cell line thp- were used for days and stimulated with various cytokines (il- , gm-csf, tnf-a, ionomycin). the dcs were characterized by flow cytometry with different typical dendritic cell markers (for example cd c, cd , cd ) and by immunfluorescence compared to monocytes. the bronchial tract contains up to dcs per mm² and therefore we established a triple culture model to mimic the situation in vivo. the triple culture consists out of primary human epithelial cells from small bronchi (hbec) and lung fibroblasts which are cultured under air-liquid conditions on filter membranes for weeks and dcs which were added after the differentiation phase of the bronchial cells. during the cultivation time the hbec formed an epithelial layer expressing both tight and adherens junctions. they also produced mucus, formed functional cilia with a beat frequency of between to hz and the transepithelial resistance values were stable between to Ω·cm². pathomechanisms of pulmonary toxicity in vivo are difficult to investigate, so the tripleculture model is the basis for investigations of the toxic effects at cellular level. lungtoxic substances such as organophosphates are usually absorbed through inhalation. organophosphates are dangerous nerve agents for the human organism. at high concentrations organophosphates damage in the coculture without dcs the cell-cell contacts of the epithelial layer. in the triple culture dcs firstly respond to inhaled organophosphates and seem to compensate effects on the other cells. in summary, it is very important to understand the pathogenic mechanisms of lung injury in relation to the role of dendritic cells in lung. they could play an essential role in therapy against damage of organophosphates in the lung. co-purification of arf gtpase-activating protein git and cavb schalkowsky p., wissenbach u., fecher-trost c., flockerzi v. universität des saarlandes institut für experimentelle und klinische pharmakologie und toxikologie, kirrbergerstraße, homburg, germany high-voltage activated ca channels are assembled from pore-forming α subunits and two distinct types of auxiliary subunits, cavβ -β and, maybe, α δ -δ . by a cavβ -specific antibody based affinity chromatography the cavβ protein was highly enriched from rat brain microsomal membranes. proteins associated with cavβ were identified by mass spectrometry (lc-esi-ms/ms) and include α -subunits, α δ-subunits and β-subunits. in addition to these expected interacting proteins additional proteins were co-purified with the cavβ protein, including the g protein-coupled receptor kinase-interactor (git ). the aa git is a ubiquitously expressed multidomain protein which may serve as a scaffold to bring together molecules to form signaling modules controlling, for example, vesicle trafficking, cytoskeletal organization and cell migration. in rat brain lysates the git and cavβ proteins were co-immunoprecipitated by the antibodies for cavβ and git, respectively. we cloned the git cdna from mouse brain and co-expressed it with the cavβ subunit in hek cells. like in brain lysates the git protein was retained by cavβ precipitated by the antibody for cavβ and cavβ was retained by the git protein precipitated by the antibody for git . both proteins, cavβ and git are endogenously co-expressed in mouse embryonic fibroblasts (mef). we could not observe potassium-induced voltage-activated ca influx in these acutely prepared cells. accordingly, mefs can be used as a model system to study the impact of cavβ -git interaction in the absence of functional cav channels. in addition, using mefs from cavβ -deficient mice enables us to control the impact of cavβ on git function. vice versa down-regulation of git by specific sirnas might allow to control the impact of git on cavβ function. as read-outs we use cell migration assays and monitor receptor-dependent and receptor-independent calcium signaling in these cells. effects of sphingosine- -phosphate and fty on epidermal hyperproliferation and inflammation in an imiquimod induced mouse model of psoriasis the sphingolipid sphingosine -phosphate (s p) is a mediator that modulates various physiological functions of skin cells. s p has distinct direct effects on keratinocytes as it diminishes proliferation and induces differentiation which is a classical goal of psoriasis therapy. furthermore, s p modulates the function of various immune cells, mainly to an anti-inflammatory direction. thus, the strategy of targeting immune cells with locally acting s p was explored in an experimental animal model of psoriasis vulgaris, the recently established imiquimod induced psoriasis mouse model and in the mouse tail test. topical administration of imiquimod onto back and ear skin led to a distinct inflammatory response characterized by epidermal hyperproliferation, scaling and redness which was scored with a modified pasi (psoriasis area and severity index). the positive control diflorasone diacetate and s p, but not fty reduced the epidermal hyperproliferation by topical administration onto ear skin, indicating a mode of action for s p via the s p receptor, which is not activated by fty . there was also a moderate reduction of inflammatory cell influx and edema formation in ear skin by s p treatment, which was even more pronounced by treatment with diflorasone diacetate. the pasi determined on back skin was, however, only significantly reduced by diflorasone diacetate. the discrepancy between outcome on ear and back skin remains elusive. in the mouse tail assay, the influence of s p in stratum granulosum formation (orthokeratosis) was tested compared to the positive control calcipotriol. whereas topical administration of calcipotriol led to the expected significant increase of stratum granulosum in mouse tail epidermis, s p lacked such an effect, indicating a different mode of action in epidermal differentiation. taken together, these results imply that topical administration of s p might be a new option for the treatment of mild to moderate psoriasis lesions. inhalation of toxicants such as sulphur mustard (sm), an alkylating chemical warfare agent, cause pulmonary complications like respiratory failure, pulmonary edema and secondary pneumonia. in order to investigate pathomechanisms of pulmonary toxicity, an in vitro alveolar-capillary co-culture model has been established recently by our group. in this model the human lung adenocarcinoma epithelial cell line (h ) is mimicking the epithelial site of the alveoli while the human hemangiosarcoma cell line (iso-has) represents the endothelial site. acute respiratory injuries are accompanied by disruption of the alveolar-capillary barrier that can be detected by the use of biochemical markers (e.g. ldh) and electrochemical indicators (e.g. transepithelial resistance). sm-mediated pulmonary injury is characterized by the increased secretion of proinflammatory mediators (e.g. il- ). a shortcoming of this model is the missing inflammatory component in the lung. aim of the present project is the addition of macrophages to the established co-culture model to improve the model and to investigate the relevance of inflammatory processes in toxic lung injury. the effect of sm on this triple-culture model is characterized with special regard to the interaction of epithelial cells and macrophages. the human acute monocytic leukemia cell line (thp- ) was stimulated to allow differentiation into macrophages. validation of the cellular differentiation was checked by specific clusters of differentiation (e.g. cd ) using flow cytometric analysis. after successful differentiation into macrophages, these inflammatory cells were added to the co-culture model before and after exposure with sm, respectively. the cytotoxicity of sm on the triple-culture model was evaluated by xtt assays and ter measurements. furthermore, immunohistochemical staining of tight junction proteins (e.g. zo- ) and of adherens junction proteins (e.g. e-cadherin) was conducted to enhance the knowledge of the function of the intercellular junction in injured and rejuvenated regions as well as the interaction of epithelial cells and macrophages. for the contact allergen para-phenylenediamine (ppd) we showed that concentrations above µm are accompanied with inhibition of nat activity in human keratinocytes [ ] . in the following we investigated the impact of ppd on nat activity in antigenpresenting cells using dendritic cell-like cells, namely the monocytic thp- cells. measured nat activity of thp- was comparable to those found in primary keratinocytes. a h treatment of thp- cells with physiologically relevant concentrations of ppd ( - µm) led to a % reduction of nat activity. comparable results were found for mono-acetylated ppd (mappd) whereas di-acetylated ppd demonstrated no inhibition. time-dependent studies found a significant decrease in enzyme activity already h after application of ppd or mappd while nat mrna levels were not modified. these results are indicative for a substrate-dependent inhibition. further investigations concentrated on the restoration of nat activity after treatment with ppd or mappd. here we found that n-acetylation capacities were restored after h cultivation of the treated cells in fresh medium. independent of the enzymatic activity, certain compounds are known to oxidise the catalytic cysteine or form adducts with the nat protein. therefore we studied whether ppd and/or oxidised ppd including the trimer bandrowski´s base interact additionally with recombinant nat protein itself in the absence of acetyl-coenzyme a. we found that all compounds but mappd bind to nat protein after h. the greatest inhibition was found for oxidised ppd (up to %). due to the greater inhibition by oxidized ppd we propose that oxidation products interact with the protein whereas ppd itself modulates nat enzyme activity in a substrate-dependent mode of action. overall we demonstrated that ppd can inhibit nat in two different ways. the work was partially financed by federal office of public health (foph), switzerland and stiftung zur förderung begabter studierender und des wissenschaftlichen nachwuchses objective: fabry's disease is a rare progressive multisystem disorder resulting from deficiency of the lysosomal enzyme alpha-galactosidase a (gla, ec . . . ). we hypothesize that genetic gla variants, especially those in its promoter region are of pathophysiological relevance for the development and progression of fabry's disease phenotypes. this study focuses on the characterization of the gla promoter, identification of functional genetic variants and impact of transcription factor eb (tfeb), a regulator of lysosomal genes. we screened bp of the '-flanking region of gla in patients with fabry's disease and controls for genetic variants. serial promoter deletion constructs for reporter gene assays were designed and identified genetic variants were introduced by site-directed mutagenesis. constructs were transiently transfected into immortalized human kidney epithelial (ihke) cells and human vascular endothelial cells (ea.hy ) to determine transcriptional promoter activity (ta). sequencing of patients' dna revealed five genetic variants in the 'flanking region of gla, significantly more frequent in fabry's patients compared to control group (rs ; rs ; rs ; rs ; rs ; all minor alleles p< . ). we identified two regions, a proximal one between - and - and a distal region between and - with significant ta, in both cell lines. cotransfection with tfeb activated ta of both regions significantly up to . -fold (p< . ). in ihke cells, insertion of the minor t allele (rs ) significantly enhanced basal ta of the proximal promoter region (p= . ), while insertion decreased basal ta (p< . ) of the distal promoter portion. the combined insertion of the minor c alleles (rs ; rs ), which were in complete linkage disequilibrium, significantly increased basal ta of the distal promoter region (p= . ). our results indicate that three genetic variants, overrepresented in fabry's patients, are located within transcriptionally active regions, possibly altering tf binding sites and therefore, affecting gla expression. future analysis will assess the impact of gla promoter variants and gla regulation by tfeb with respect to fabry's phenotypes. multiple sclerosis (ms) and its animal counterpart experimental autoimmune encephalomyelitis (eae) have a major inflammatory component that drives and orchestrates both diseases. ceramides (cer) are known as mediators of inflammatory processes, but until now their role in ms was not elucidated. we measured the ceramide levels in the cerebrospinal fluid of ms patients and control patients using lc-ms/ms. interestingly, the c : -cer levels were . fold increased in ms patients. this translates into the finding that c : -cer levels were also significantly elevated in the lumbar spinal cord of eae mice. the raised c : -cer levels in the lumbar spinal cord were caused by a transiently increased expression of ceramide synthase (cers) in macrophages. nitric oxide (no) and tumor necrosis factor alpha (tnf-α) secreted by interferon gamma (infγ ) induced macrophages play an essential role in the development of ms. astonishingly, rnai experiments reveal that cers and its product c : -cer are mediators of inf-γ induced no/tnf-α release in raw macrophages. moreover, treatment of eae mice with l-cycloserine prevented the increase of c : -cer and of inos/tnf-α expression and caused a remission of the disease. in summary, cers plays a critical role in the initial phase of ms, most likely by regulating the no and tnf-α synthesis. this let us speculate, that a substance designed to inhibit cers and therefore to limit the inflammatory effects of c : -cer may represent a new drug in ms therapy. role of cgmp-dependent protein kinase i for kidney fibrosis schinner e. cgmp is synthesized via nitric oxide-or natriuretic peptide-stimulated guanylyl cyclases and exhibits pleiotropic regulatory functions also in the kidney. hence, the integration of cgmp signaling via cgmp-dependent protein kinases (cgk) might play a critical role for renal physiology. both isozymes were detected in arterioles, mesangium and within the cortical interstitium. in contrast to cgkiα, the β isoform was not detected in the juxtaglomerular apparatus and medullary fibroblasts. here, we focused on the function of cgki in the renal interstitium emphasizing a functional differentiation of both isoforms. the interstitium exists mainly of fibroblasts playing a prominent role in the interstitial fibrosis. accordingly, cgki could also be involved in this pathophysiological process. therefore, we studied whether cgki influences renal fibrosis which was induced by unilateral ureter obstruction (uuo). at first we analysed the role of the no/cgmp signaling by application of cgmp increasing yc or isdn. thereby we detected antifibrotic effects of these substances. subsequently we tested whether these effects are mediated by cgki by using mutant mice. on the one hand we examined αsm-rescue mice (expressing cgkiα only in smooth muscle under the control of the sm promotor with a cgki-ko background) and cgki-ko mice (expressing no cgki). on the other hand we used tgtg mice expressing more cgkiα in smooth muscle than wt mice (transgenic cgkiα under the control of the sm promotor). due to the steeply increased use of nanomaterials for commercial and industrial applications, toxicological assessment of their potential harmful effects is urgently needed. moreover, the continuous development of novel materials requires the implementation of hazard-predicting models to prevent potential health effects resulting from human exposure. in the present study, we studied the toxic potential of a set of nanoparticles (np) with varying physicochemical properties in human a lung epithelial cells, hepg liver epithelial cells and hk- proximal tubule epithelial cells. the used nanomaterials incorporated five tio samples, two zno samples (i.e. uncoated and coated), two multi-walled carbon nanotube (mw-cnt) samples and a nanoparticulate ag sample. cells were treated with np at doses ranging from . to µg/cm for cytotoxicity and from to µg/cm for genotoxicity. dna damage was evaluated using the alkaline comet assay while concurrent cytotoxicity was determined by the wst- assay. marked contrasts in cytotoxic and dna damaging properties were observed among the different materials. the overall strongest responses were observed with the uncoated zno-np sample and with ag-np, although effects were found to depend on the cell type. notably, the dna damaging effect of ag-np could at least partly be attributed to its dispersant. present results form part of a growing data set which are generated in the framework of the eu fp project enpra (fp -nmp) to establish dose-response relationships and a mathematical model to predict the hazard of nanoparticles. increased spontaneous hprt mutant frequency in v cells expressing human cytochrome p b schlechtweg a., esch h. , martínez jaramillo d., lehmann l. university of wuerzburg/institute of pharmacy and food chemistry section of food chemistry, am hubland, wuerzburg, germany the hypoxanthine-guanine phosphoribosyltransferase (hprt) assay in chinese hamster v lung fibroblasts (v cells) represents a widely-used mammalian test system to detect gene mutations. since v cells do not express any cytochrome-p dependent monooxygenase (cyp) isozymes, usually an activating system has to be added. therefore, v cells expressing human (h) cyp isozymes have been commercialized. to test these v cells for their use in the hprt test, v h a and h b cells were characterized regarding (i) spontaneous frequency of -thioguanineresistant clones per clonable cells (smf), (ii) the stability of which over weeks (w), and (iii) the mutational spectrum (ms) of cdna from mutant clones. ms of cdna was determined by isolation of total rna, reverse transcription/amplification of the coding region by polymerase chain reaction and sanger sequencing of the amplification product. activity of cyp isozymes was verified by ethoxyresorufin-o-deethylase (erod) assay. (i)/(ii) whereas the smf of v cells (w : ± ; w : ± ) and v h a (w : ± ; w : ± ) only varied within the range of historical controls, smf of v h b increased continuously over time (w : ± ; w : ± ). (iii) although the smf of v and v h a were similar, the mutational spectrum of v cells was characterized by as many transversions as transitions and deletions of exon or exon + , whereas the mutational spectrum of v h a was characterized exclusively by transversions and deletion of exon + . surprisingly, with out of cdnas derived from v h b mutant clones, no amplification product was detected. first results indicate that there is at least one gene mutation in the untranslated region before and behind the coding region precluding amplification with the original primers. (ii)to reduce the smf of v h b , cells with wildtype hprt activity were cloned and one clone with an erod activity which did not differ significantly from the original cell population was further characterized. initially, smf of the clone varied between . ± . and . ± . . yet its smf was unstable reaching up to ± . in conclusion, the mutational spectrum differed between the v cell lines. furthermore, h b expression seemed to enhance smf in v cells. even though a temporary reduction of the smf by cloning was possible, smf of v h b cells was unstable. we wanted to investigate the possible antithrombotic effects and elucidate the chemical identity of the active principles involved in inhibitory effects against adp-induced aggregation of human platelets by wild garlic, allium ursinum l. method: bioassay-guided isolation procedure was used followed by spectrometric identification of pure active compounds. for the bioassay, blood was taken from healthy human volunteers and platelet rich plasma (prp) was prepared for turbidimetric platelet aggregation tests. prp, stimulated with µm adp, was treated with extracts of different polarities, fractions and isolated single compounds from allium ursinum. the extracts were investigated by thin layer chromatography, hplc, mass spectroscopy, esi-ms and d/ d h/ c-nmr spectroscopic techniques. for references the adt-antagonist mes-amp was used. result: fresh allium ursinum leaves were extracted with ethanol, which was the potent form that effectively inhibited adp-induced aggregation of human platelets. this ethanolic extract was subjected to liquid-liquid partition. whilst the aqueous phase containing the moiety of cysteine sulphoxide and thiosulphinate derivatives showed only weak activity on platelet aggregation, the ethyl acetate and especially the chloroform partitions showed highest aggregation inhibiting potency. thus, in our bioassay effects of alliins/allicins could be neglected. the chloroform phase, possessing the strongest activity, was separated into fractions by gradient elution open cc on silica gel. the most active fractions - were separated again yielding subfractions. this afforded , -di-o-α-linolenoyl- -o-β-d-galactopyranosyl-sn-glycerol and β-sitosterol- -o-β-dglucopyranoside, the structures of which were determined by esi-ms and d/ d h/ c-nmr spectroscopic techniques. furthermore, the diminutive amounts of volatile oil of a.ursinum leaves obtained by steam distillation according to ph.eur. could be evaluated as a third aggregation inhibiting principle. conclusions: at the first time two active, non-sulphur-containing constituents of wild garlic, namely a galactolipid and a phytosterol, could be identified exhibiting inhibitory action on adp-induced aggregation in human blood platelets. as a major constituent, the galactolipid , -di-o-α-linolenoyl- -o-β-d-galactopyranosyl-sn-glycerol, not yet found in allium spec., appears as a new, highly useful marker substance for a.ursinum drugs, or their pharmaceutical preparations. in recent years, public attention focused more and more on risk factors which may impair sperm quality and thereby human reproduction. in this context, for example pesticides, alcohol, cigarettes, and even mobile phones are discussed. a variety of parameters exists including sperm counts as well as sperm motility, which are considered to be two of the most important parameters to evaluate sperm quality in animal models with the final aim to assess human risk. in recent years computer assisted sperm analysis (casa) devices mostly replaced the formerly used manual counting and manual motility assessment. however, although casa offers multiple opportunities and can allow for an objective and more detailed evaluation, several pitfalls exist which can alter the results profoundly and consequently compromise the quality of the data and ultimately the validity of a study. the aim of the present study was to establish and validate the casa device tox ivos sperm analyzer from hamilton thorne and thereby to gain detailed knowledge about the practical advantages but also intricacies which may alter the obtained results. in this regard healthy adult male rats ( - weeks old) were used. ultrasonic sound resistant sperm heads were isolated from the testis and in addition, sperms were isolated from the cauda epididymis. testicular sperm head counts and sperm motility were assessed using different isolation procedures and/or instrument settings. results different instrument settings modulate both -sperm motility and testicular sperm counts. in this regard, a wide range of results including slight changes as well as false positive/negative results were obtained. in addition, the modification of the isolation procedure can lead to variable results especially for sperm motility. conclusion isolation procedures as well as instrument settings can alter the results. consequently, in an experimental setting, potential adverse effects can be confounded with methodologically mediated apparent findings exerted via inappropriate use of the device -depending on the respective conditions in the test laboratory. this study demonstrates the relevance of standardization of testing conditions adopted for computer assisted sperm analysis and the need for a robust validation prior to use in experimental settings. orai and stim proteins have been identified as central components of the highly ca + selective, store-operated current in immune cells (icrac). the molecular basis of selective orai-mediated activation of the calcineurin/nfat pathway and the crosstalk with other channel and scaffold molecules of the trpc family are still incompletely understood. using patch clamp recordings complemented by fluorescence and tirf microscopy we investigated interactions between orai and trpc in plasma membrane microdomains of rbl- h mast cells. orai -mediated crac currents, activated by passive store depletion, were found significantly reduced by over-expression of trpc . this negative impact of trpc on icrac was independent of channel function as the trpc pore dead mutant (e k) inhibited icrac to a similar extent as wild type trpc . importantly, despite a reduction in icrac, nfat translocation in trpc overexpressing rbl cells remained unchanged, or was even slightly promoted. store depletion-induced nfat translocation in rbl cells was as well unaffected by trpc e k but substantially reduced by trpc mutants with either i) eliminated fkbp /calcineurin binding (p q) or ii) deficiency in pkc phosphorylation (s a). moreover, inhibition of pkc phosphorylation by (gfx x; µm) strongly suppressed nfat signaling. we suggest trpc as a scaffold that links orai-mediated ca + -entry to nfat/calcineurin signaling within plasma membrane microdomains. neurally-induced bronchoconstriction in human and guinea pig precision-cut lung slices schlepütz m. , rieg a. d. introduction: precision-cut lung slices (pcls) are well suited to study peripheral airway responses in different species. airway tone is under close control of the autonomic nervous system and dysregulation may contribute to airway hyperresponsiveness as observed in human lung diseases such as asthma. hence, the aim of the present study was to characterize neurally induced bronchoconstriction (bc) in guinea pigs (gp) and to compare the results with those in human pcls. methods: pcls were prepared from gp or human lung tissue. nerve endings in pcls were activated by electric field stimulation (efs) or capsaicin addition. cholinergic nerve responses were proven by atropine. capsaicin was used to show excitatory nonadrenergic non-cholinergic (enanc) responses. ruthenium red or skf were used to confirm transient receptor potential (trp) channel contributions upon enanc activation. results: gp and human pcls were both sensitive to efs and airways contracted to ± % of the initial airway area (%-iaa) and ± %-iaa, respectively. in frequency response curves half maximal response was found at . ± . hz for guinea pig pcls and . ± . hz for human pcls. efs-induced bc was inhibited by atropine in both species. capsaicin contracted gp to ± %-iaa. % of human pcls were responsive to capsaicin and airways contracted to ± %-iaa, respectively. in gp ruthenium red and skf blocked capsaicin-as well as efs-induced bc. conclusion: gp and human pcls contain atropine sensitive cholinergic and capsaicin sensitive enanc nerve endings. since gp pcls were sensitive to trp channel inhibitors, the involvement of those channels can be characterized with respect to lung diseases. in conclusion, gp pcls resemble the human distal lung innervation and represent a useful model to study neural airway pharmacology. the erk / -pathway is involved in pkc-induced nox up-regulation schlufter f., xia n., förstermann u., li h. universitätsmedizin mainz institut für pharmakologie, obere zahlbacher straße , mainz, germany nadph oxidases (nox) are major producers of reactive oxygen species in the vascular wall and nox is the most abundant nox isoform in human endothelial cells. we have previously shown that treatment of human ea.hy endothelial cells with phorbol myristate -acetate (pma) for h leads to an up-regulation of nox expression. this effect of pma is mediated by protein kinase cα, because it is preventable by the pkc inhibitor gö and by pkcα-sirna. the present study is aimed to investigate the signal transduction cascade downstream of pkcα. pma-induced nox up-regulation can be attenuated by pd , (an erk / inhibitor), but not by sp (a jnk inhibitor), indicating in the involvement of erk / . consistently, pma treatment leads to a sustained activation of erk / , and sirnamediated knockdown of erk / markedly reduces the pma-induced nox up-regulation. h , an inhibitor of the mitogen-and stress-activated protein kinases (msks) has no effect on the pma-stimulated nox expression, indicating that msks are not the target molecules of erk / in this scenario. on the contrary, knockdown of the transcription factor elk- by sirna significantly reduces the pma-induced nox up-regulation. in conclusion, erk / and elk- are involved in the pkcα-induced nox up-regulation. determination of spontaneous mutation frequencies in normal human mammary gland tissue using the random mutation capture technique schmalbach k., lehmann l. university of wuerzburg section of food chemistry, am hubland, wuerzburg, germany annually, over , women develop breast cancer in germany. the accumulation of genetic mutations in mammary gland tissue during lifetime may be reasonable for developing breast cancer. in particular mutations in tumor suppressor genes, e.g. p , seem to play an important role in developing cancer. up to now, lack of a method sensitive enough to determine the expected very low spontaneous mutation frequency (smf) in normal mammary gland tissue precluded the investigation of the role of spontaneous mutations acquired in the p gene in epidemiological studies. the only test with the potential to determine low smfs was the random mutation capture (rmc) assay, a genotype selective method which detects mutants that render the mutational sequence non-cleavable by the taqi restriction enzyme after accumulation of the target sequence. therefore, the suitability of the rmc assay to determine smf in p gene in normal human mammary gland tissue was evaluated. thus, the rmc assay was optimized concerning (i) dna isolation, (ii) pcr conditions, and (iii) amount of mammary gland tissue. (i) genomic dna from normal human mammary gland tissue, obtained from healthy women who underwent mamma reduction surgery for cosmetic reasons, was isolated using an extended proteinase k digestion prior to chloroform extraction. (ii) the target sequence in intron of p gene was captured by hybridization with a complementary uracil-containing dna-probe synthesized via polymerase chain reaction (pcr), followed by magnetic separation from the remaining genomic dna. the copy number of the target sequence was quantified by competitive pcr. the number of mutants was detected after cleavage of the target dna with taqi by means of pcr with a primer set flanking the restriction site. (iii) with g of normal mammary gland tissue a smf of . ± . x - per base pair was determined indicating the rmc assay suitable for smf determination. in conclusion, the smf in the p gene in normal human mammary gland tissue was determined for the first time, enabling the future investigation of factors influencing the smf during breast cancer development. cigarette smoke-induced release of pro-inflammatory cytokines including interleukin- (il- ) from inflammatory as well as structural cells in the airways, including airway smooth muscle (asm) cells, may contribute to the development of chronic obstructive pulmonary disease (copd). despite the wide use of pharmacological treatment aimed at increasing intracellular levels of the endogenous suppressor cyclic amp (camp), little is known on its exact mechanism of action. we report here that next to the β -agonist fenoterol, direct and specific activation of either exchange protein directly activated by camp (epac) or protein kinase a (pka) reduced cigarette smoke extract (cse)-induced il- mrna expression and protein release by human asm cells. cse-induced iκbαdegradation and p nuclear translocation, processes that were primarily reversed by epac activation. further, cse increased extracellular signal-regulated kinase (erk) phosphorylation, which was selectively reduced by pka activation. cse decreased epac expression, but did not affect epac and pka expression. importantly, epac expression was also reduced in lung tissue from copd patients. in conclusion, epac and pka decrease cse-induced il- release by human asm cells via inhibition of nf-κb and erk, respectively, pointing at these camp effectors as potential targets for antiinflammatory therapy in copd. however, cigarette smoke exposure may reduce antiinflammatory effects of camp elevating agents via down-regulation of epac . polycyclic aromatic hydrocarbons (key marker substance benzo[a]pyrene (bap)) have been assumed to play a role in the development of bladder cancer. the objective of the present study was to unravel cellular and in particular cytoskeletal response to bap. to follow the sequential steps of chemical carcinogenesis the differential proteomic profile was analyzed at early and late time points. the study was carried out in a superficial human bladder cancer cell line (rt ) exposed to . µm bap, a subacute concentration based on results of proliferation (brdu) and dna damage (tunel) tests. cells of a human bladder cancer cell line (rt ) were exposed to . µm bap for h (n= ), wk (n= ) and wk (n= ). proteins of whole cell lysate were separated by twodimensional electrophoresis (fig. ) . differentially expressed proteins were identified by matrix-assisted laser desorption/ionization-time of flight analysis. cortactin, actin and tubulin were immunohistochemical stained. changes in migration and colony forming ability were assessed by scratch wound-healing assay and soft-agar colony formation. results: by using several databases (uniprot, reactome, panther) the identified proteins were categorized into different functional classes such as mrna processing, translation, protein metabolic process, or several areas associated with the organization of the cytoskeleton. % of the differentially expressed proteins after h of treatment are involved in the processing of pre-mrna ( %) and protein metabolism ( %). this pattern changed after wk of treatment. then, % of the proteins affected the cytoskeleton whereas still % were categorized to protein metabolism and only % to pre-mrna processing. in the immunhistochemical staining, the treated cells appeared after wk of exposure larger and rounder predominantly due to the modifications of the actin cytoskeleton. merged images of actin and cortactin revealed that both proteins colocalized in invadopodiae. after wk, a higher number of treated cells moved toward the centre of the wound and they formed more soft-agar colonies compared to vehicle conditions, suggesting a transformation of the cells. in conclusion, bap exposure causes in an early phase an activation of the spliceosome which can led to an epithelial-tomesenchymal transition. two coordinators of a cell-type-specific splicing program, epithelial splicing regulatory proteins and , are currently being validate by pcr. fused master gel : representative -de gel of rt cells exposed to . µm bap for wk. protein spots which were differentially expressed compared to control and identified were marked. cannabinoids stimulate mesenchymal stem cell migration via a mitogen-activated protein kinase pathway schmuhl e. , ramer r. mesenchymal stem cells (mscs) are known to be involved in various regenerative processes such as cardiac, ocular, skin and bone tissue healing. however, little is known about the pharmacotherapeutical options aiming at tissue healing steps such as the mobilization and homing of mscs. here, we show that cannabidiol (cbd), a nonpsychoactive cannabinoid, stimulates the migration of human adipose-derived mscs in both boyden chamber and in vitro scratch wound assays. in boyden chambers cbd ( . - µm) was shown to promote cell migration in a time-and concentration dependent manner. this promigratory action was inhibited by am- (cb receptor antagonist) and by o- (g protein-coupled receptor [gpr] agonist). moreover, cbd activated the mitogen-activated protein kinase (mapk) pathway as evidenced by increased phosphorylation of extracellular signal-regulated kinase (erk) / . blockade of erk activation by pd prevented cbd-stimulated msc migration, whereas inhibition of p mapk by sb was inactive in this respect. furthermore, am- and o- were found to attenuate cbd-induced erk activation. an erk-dependent promigratory action was likewise demonstrated for the phytocannabinoid ∆ tetrahydrocannabinol and for the hydrolysis-stable anandamide analogue r(+)methanandamide. we conclude that cbd promotes msc migration via receptordependent erk activation, possibly contributing to tissue healing. the duffy antigen receptor for chemokines (darc) binds promiscuously many inflammatory chemokines without showing intracellular signal transduction. it is mainly expressed on endothelial cells of postcapillary venules and on red blood cells, where it acts as a transendothelial transporter of chemokines and as a chemokine sink, respectively. surprisingly, as shown for human and mouse brain, darc is also expressed at high density in the cerebellum. however, nothing is known about the function of darc in this location. we addressed this question by subjecting c bl/ wildtype and darc-deficient mice to a series of behavior experiments including morris water maze-, elevated plus maze-, rotarod-and actometer tests. while the results from the water maze experiments are ambiguous, elevated plus maze trials show a strong aversion of darc -/mice to walk to the end of the open arm, which is consistent with anxiety-like behavior. moreover, darc -/mice show greatly reduced locomotor activity, which is at least partly caused by episodes of reduced mobility occurring more frequently than in the corresponding wildtype controls (elevated plus maze, actometer). finally, darc -/mice spend a significantly reduced time on the rotating rod compared to c bl/ wildtype controls, which may indicate an impaired cerebellar function. we conclude that darc in fact modulates brain function. surprisingly, this appears to be happening under homeostatic conditions, although darc binds for the most part to inflammatory chemokines. it remains to be elucidated, how this effect can be caused by a non-signaling chemokine receptor. it may be an indirect consequence of altered brain chemokine concentrations or of as yet unknown signaling pathways activated by darc. transporter gene expression in human head-neck squamous cell carcinoma and epigenetic regulation mechanisms schnepf r. hals-nasen-ohren-klinik, kopf-und halschirurgie, friedrich-alexander-universität erlangen-nürnberg, waldstraße , erlangen, germany background: membrane transporters may affect the disposition and thereby treatment efficiency of anticancer drugs in human head-neck squamous cell carcinoma (hnscc). the gene expression profile of transporters in hnscc, however, is unknown and was evaluated in this study. moreover, we evaluated mechanisms by which transporters are regulated in hnscc. we focused on the role of the nuclear pregnane x receptors (pxr, nr i ) and epigenetic mechanisms. methods and results: real-time rt-pcr revealed a significantly increased mrna expression of slco a and slco b and a significantly decreased expression of transporters such as slco b , slco a and abcc in human hnscc tissue samples compared to adjacent normal mucosa. moreover, an association between slco b mrna levels in tumor tissues and five-year survival of hnscc patients was observed (χ = . ; p= . ; n= ). bisulfite sequencing revealed that promoter cpg islands of abcc and slco a were not methylated and thus these genes were not epigenetically silenced in hnscc tissues. in the hnscc-derived umscc- and scc- cell lines, transcript expression of transporters (e.g., abcc , slco a ; p< . ) and pxr (nr i ; p< . ) was markedly induced by the dna methyltransferase inhibitor decitabine. cotreatment with the prototypical pxr activator rifampicin significantly reversed decitabine-induced abcc and slco a expression. conclusions: transporter expression profiles significantly differed between hnscc and normal mucosa and expression levels of slco b may serve as a marker for prognosis. modulation of the epigenome with the anticancer drug decitabine substantially affects transporter expression in umscc- and scc- cells, suggesting epigenetic regulation mechanisms. moreover, interactions between epigenetic and nuclear receptor-mediated mechanisms in transporter regulation occur. this work was in part supported by the johannes und frieda marohn foundation. the role of hcn in neuropathic and inflammatory pain schnorr s. , eberhardt m. the pacemaker current ih is carried by hyperpolarization-activated cyclic nucleotidegated cation channels (hcn - ) and contributes to cellular excitability in the heart and the nervous system. hcn and hcn are the two most abundant hcn subunits in peripheral sensory neurons with hcn being the prevalent isoform in nociceptive small sized c-fibre dorsal root ganglion (drg) neurons. we examined the role of hcn for peripheral sensitization and spontaneous neuronal activity in neuropathic and inflammatory pain. we generated a conditional deletion of hcn by using a nociceptor specific cre-transgene driven by the nav . promoter. the nociceptor-specific knockout of hcn in drg neurons (nosphcn ko) was confirmed by quantitative rt-pcr and western blot. immunohistochemical staining revealed that the deletion of hcn was mainly restricted to small sized drg neurons. the conditional loss of hcn resulted in a significant reduction of ih positive small diameter drg neurons pointing to a central role of this isoform to the hcn current in nociceptive neurons. behavioral studies showed that the lack of hcn did not influence basal pain responses but led to a significant reduction in mechanosensation in both neuropathic and inflammatory pain models. however, thermosensation of the mutants was only decreased in neuropathic pain conditions. in wild-type animals, intraperitoneal, intraplantar and even intrathecal injection of the hcn channel blocker zd nearly eliminated tactile allodynia caused by inflammation in contrast to thermal hyperalgesia which remained unaffected. in contrast, pain thresholds in nosphcn ko mice did not significantly increase after pharmacological block of ih. additionally, experiments revealed that the inflammatory condition induced an upregulation of hcn protein in the spinal dorsal horn compared to saline injected mice. our results suggest that hcn might be a new target in the treatment of neuropathic and inflammatory pain. the proper functioning of the central, as well as the peripheral nervous systems is of outstanding importance to the survival and well-being of humans. yet, the integrity of neuronal systems is constantly challenged by a plethora of environmental and occupational toxins. some of these toxins preferentially target neural cells. these neurotoxins can exert their devastating effects by very different modes of action. neurotoxins may induce apoptosis or necrosis of neurons, or interfere with axon growth and elongation. these processes can be identified by specialized in vitro tests. furthermore, neurotoxins have been described to alter glial function which may compromise the viability of surrounding neurons. as another important mode of action, several neurotoxins act on neurotransmitter receptors, thereby altering signal propagation within neuronal networks. countless natural and synthetic substances have been characterized for their effects on neurotransmitter receptors and today can be used for detailed studies of receptor function. however, environmental toxins of anthropogenic origin and occupational toxins that both represent constant sources for human exposure are still poorly studied with respect to their effects on neurotransmitter receptors. thus, the need for a better understanding of the susceptibility of neurotransmitter systems for toxic effects exerted by these substances is of outstanding importance for the protection of human health. here, we introduce an imaging-based approach for the screening of the effects of potential and known neurotoxins on neurotransmitter receptors of intact cells in vitro. different neuronal cells were tested for their sensitivities for classical neurotransmitters using life-cell imaging experiments. in more detail, we examined the proportion of responding cells and determined the ec values for the most prominent neurotransmitters in cell lines widely used for in vitro neurotoxicity studies on the one hand, namely sh-sy y and lumes cells, and primary mouse neurons on the other hand. with these data at hand, we are now able to identify and characterize the effects of neurotoxins on receptor function in chronic, as well as acute exposition paradigms. the use of an in vitro imaging-based physiological test system is at the interface between non-functional in vitro approaches and in vivo toxicity tests, thus, giving mechanistic insight into neurotoxic processes without requiring animal experiments. apomorphine acts on trpa channels scholze a., schaefer m., hill k. universität leipzig -universitätsmedizin rudolf boehm-institut für pharmakologie und toxikologie, härtelstr. - , leipzig, germany apomorphine is a non-narcotic derivative of morphine which acts as a dopamine agonist and is clinically used to treat "off-states" in patients suffering from parkinson´s disease. adverse effects of apomorphine treatment include dopaminergic effects such as nausea, but also ulceration and pain at the injection site. we wanted to test whether an activation of trp (transient receptor potential) channels in sensory neurones contributes to the perception of pain after apomorphine injection. while the warm/heat receptors trpv , trpv , trpv , and trpv and the cold receptor trpm were insensitive towards apomorphine treatment, trpa could concentration-dependently be modulated by apomorphine. low micromolar apomorphine concentrations potently activated heterologously expressed trpa channels in a stably transfected cell line (hek -trpa ), as well as natively expressed trpa in cultured dorsal root ganglion neurones. on the other hand, when using higher concentrations of apomorphine, we observed inhibition of trpa activity. previous studies have shown that subcutaneously administered apomorphine produces a biphasic dose response relationship in rats, inducing hyperalgesia at low doses whereas high doses of the substance cause antinociception. from our studies we conclude that such in vivo effects of apomorphine are presumably mediated by activation/inhibition of trpa expressed in sensory neurones agonist binding to a g protein-coupled receptor (gpcr) induces a conformational change of the receptor protein, which results in the activation of receptor-associated heterotrimeric g proteins [ ] . in radioligand binding studies, conducted to investigate ligand binding to specific gpcrs, receptors are usually probed with radioantagonists. as in other gpcrs [ ] , agonists of the muscarinic m receptor exhibit biphasic kinetics and biphasic competition curves with radioantagonists, indicating a more complex situation probably caused by g protein interactions. here, we present a detailed study of the binding of agonists to muscarinic m receptors including the novel super-high affinity agonist iperoxo and a differential chemical knockout of g proteins. in addition to membrane homogenates living cells were employed. we demonstrate that the high affinity fraction in biphasic curves does not differ between selected full agonist and is sensitive to pertussis toxin, thus indicating that this receptor population is associated with gi proteins. however, despite promiscuous signalling properties of m receptors, the low affinity fraction is not associated with any other g protein, since low affinity binding is insensitive to high concentrations of guanylnucleotides and cholera toxin. moreover, high affinity agonist binding appears solely in membrane homogenates but not in experiments conducted with living cells, probably due to their high intracellular concentration of guanylnucleotides. taken together the chemical knock-out of g proteins revealed that the high affinity binding of agonists in membrane homogenates is associated with the interaction of the muscarinic m receptor with gi proteins. the low affinity binding cannot be related to another g protein, although the muscarinic m receptor exhibits promiscuous g protein signalling properties. interestingly data obtained with living cells do not reveal any high affinity binding of agonists. prolonged stress leads to a dysregulation of the hypothalamus-pituitary-adrenal (hpa)axis and may affect the sensitivity of pain perception. however, it is not yet known whether the alterations of hpa-axis and increased pain sensitivity are related. to create a long lasting stressful situation, male wistar rats were exposed to a restraint-stress for h daily over a period of two weeks. the effect of stress on the hpa-axis was determined by adrenal morphology and stress hormone levels, the influence on mechanical pain sensitivity was evaluated by the randall-selitto paw pressure test. on day the animals exhibited a significant mechanical hyperalgesia. they also showed increased acth and corticosterone plasma levels and an enlarged zona fasciculata of the adrenal gland, indicating a dysregulation of the hpa-axis. for testing the correlation of hpa-axis dysregulation and hyperalgesia a persistent increase in plasma corticosterone in wistar rats was generated by the administration of corticosterone via the drinking water for two weeks. these animals also showed an increased mechanical nociceptive sensitivity with an accompanied decrease of the adrenal glands and reduced acth levels. the results show that chronic stress leads to a dysfunction of the hpa-axis with an accompanied mechanical hyperalgesia which can be mimicked by oral administration of corticosterone. thus, this in-vivo test system may provide a new animal-friendly pharmacological model for stress-related pain disorders. the alternaria mycotoxins aoh and ame induce cyp a and apoptosis in murine hepatoma cells dependent on the aryl hydrocarbon receptor mycotoxins are secondary metabolites of fungi including the genus alternaria (black mold). alternaria fungi are known to infest different types of foodstuffs and produce diverse toxins amongst them the mycotoxins alternariol (aoh) and alternariol methyl ether (ame) which are potential carcinogens. as planar compounds, aoh and ame are preferentially metabolized by cytochrome p (cyp) a and a . the most prominent regulator of cyp a is the dimeric transcription factor complex ahr/arnt, which is activated by planar ligands. therefore we studied the activation of ahr/arnt by aoh and ame and monitored cyp a induction in murine hepatoma cells (hepa- c c ). indeed, aoh and ame enhanced the levels of cyp a in hepa- c c cells but not in cells with inactivated ahr (hepa- c c ) or arnt (hepa- c c ). furthermore, we studied the cytotoxicity of aoh and ame. by using a fluorescence-based microscopic readout we measured effects on cell counts, apoptosis, senescence and micronuclei formation. both aoh and ame reduce the cell number and the cell nuclei show drastic morphological changes e.g. enlargement after aoh treatment or micronuclei formation. the observed effects where, except for the induction of apoptosis, independent of ahr/arnt. in summary, aoh and ame activate the ahr/arnt pathway to induce cyp a expression and apoptosis. however, the predominant cytotoxic effect of aoh and ame in hepatoma cells is a profound reduction in cell numbers, which is independent of the ahr/arnt pathway. special purpose databases are the first place for researchers in the life sciences to obtain expert curated data. naturally, such resources are limited in terms of timeliness and comprehensiveness. the literature database pubmed alone lists more than , , scientific abstracts, and , are newly added every year. the protein sequence database uniprotkb stores over , , sequences, a hundred times more than ten years ago. turning these data into meaningful information and making it accessible to both humans and computers have become an essential part of biological discovery and biomedical research. text mining techniques have proven useful to extract the missing links in areas such as drug-target and drug-disease prediction, the extraction of mutation-phenotype associations, or the prediction of protein-protein and protein-ligand interactions. by systematically extracting information from available literature, reports or patents, text mining techniques can help to refine existing categorical knowledge stored in databases, and hence will support drug repositioning, the discovery of novel cancer biomarkers, or help to understand causes of hereditary diseases. in the area of regulatory toxicology we developed go r, the first knowledge-based search engine for alternative methods to animal experiments. the system not only helps retrieving information on the availability of alternative methods that allows for replacing, reducing or refining animal experiments, but also provides an endpoint-centered literature search to all scientists and regulatory authorities seeking for toxicological information and data. the up-to-date taxonomicstructured "table of contents" provided by go r allows for search in the literature listed in pubmed or the toxicology data network (toxnet) in a fast and comprehensive manner. the semantically enriched platform supports the user during the query formulation, allows for bibliographic analysis, and reveals existing relations to replacement, reduction, and refinement of animal experiments. impaired cardiac excitation-contraction-coupling in mice with complete inactivation of the crem gene schulte j. s., tekook m., schmitz w., müller f. u. westfälische wilhelms-universität institut für pharmakologie und toxikologie, domagkstraße , münster, germany the structurally related transcription factors camp response element binding protein (creb) and camp response element modulator (crem) mediate a regulation of gene transcription in response to camp and are expressed in the heart. mice with complete inactivation of crem display impaired cardiac contraction and relaxation, decreased expression of serca and down-regulation of β -adrenoceptors. to elucidate the underlying functional mechanisms on the cellular level we here investigated cellular electrophysiology and ca + -cycling in ventricular cardiomyocytes from crem ko mice (ko). adult ventricular cardiomyocytes were isolated from ko and wildtype (wt) mice (age - weeks) and subsequently used for experiments within hours after isolation. action potentials (aps) were recorded from ventricular cardiomyocytes (perforated patch, whole cell current clamp inactivation of crem seems to have no consequences for ap duration and possibly associated ion channels but leads to impairment of ca + cycling and sarcomere shortening under basal conditions well explaining the previous findings in vivo. our results show that crem is essential for a regular excitation-contraction coupling in the mouse heart. (supported by the izkf münster) new mechanistic insights in no/cgmp actions in the vasculature schulte k., koesling d., universitätsstr. , bochum, germany hypertension, a major risk factor for cardiovascular diseases, is associated with vascular changes resulting in increased vascular contractility und vascular peripheral resistance. a prominent factor in the development and maintenance of hypertension is the reninangiotensin-aldostrerone system. angiotensin ii (ang ii) is the main mediator of this system and a powerful vasoconstrictor. ang ii increases the intracellular ca + concentration thereby activating myosin light chain (mlc) kinase, which enhances mlc phosphorylation and subsequent vascular contraction. opposite to angii-induced vascular contraction, no/cgmp pathway promotes vascular relaxation by decreasing ca + concentration and lowering mlc phosphorylation. responsible for mlc dephosphorylation is the mlc phosphatase (mlcp), whose activity is regulated by different phosphorylations. phosphorylation of mlcp via rhoa-activated rho-kinase enhances phosphatase activity while phosphorylation via the cgmp-dependent protein kinase has been proposed to decrease enzymatic activity. to investigate the interplay of angii with the no/cgmp pathway, we treated wild-type and ko mice lacking the cgmp forming no receptor, no-gc , with angiotensinii ( . mg / kg bw / d) for two weeks. in addition to various cardiovascular parameters, physiological changes in vascular reactivity of aortic rings of angii-treated wt and no-gc ko mice were assessed in organ bath experiments and correlated with biochemical parameters as the phosphorylation state of mlc, mlcp and rho-kinase activities examined by immunoblot analysis. analysis of cgmp levels revealed that angii treatment decreased cgmp in wt mice to levels comparable to those of the ko mice which were unaltered by the treatment. our study will provide further mechanistic insights in the molecular interactions between constrictor and dilator stimuli in the vasculature. nanomaterials are already used today and offer even greater use and benefits in the future. the progress of nanotechnology must be accompanied by investigations of their potential harmful effects. for airborne nanomaterials, lung toxicity is a major concern and obviously the particle size is discussed as a critical property directing adverse effects. while standard toxicological test methods are generally capable of detecting the toxic effects, the choice of relevant methods for nanomaterials is still discussed. we have investigated two genotoxic endpoints -alkaline comet assay in lung tissue and micronucleation in polychromatic erythrocytes of the bone marrow -in a combined study hours after a single instillation of µg gold nanoparticles (np) into the trachea of male adult wistar rats. the administration of three test materials differing only in their primary particle size ( -, -and -nm) did not lead to relevant dna damage in the mentioned tests. the measurement of clinical pathology parameters in bronchoalveolar lavage fluid (balf) and blood indicated neither relevant local reactions in the animals' lungs nor adverse systemic effects. minor histopathology findings occurred in the lung of the animals exposed to -nm and -nm sized nanomaterials. in conclusion, under the conditions of this study the different sized gold np tested were non-genotoxic and showed no systemic and local adverse effects at the given dose. platelet dense granule secretion mediates platelet-dependent enhancement of tumor cell transmigration and formation of metastases schumacher d., strilic b., wettschureck n., offermanns s. mpi für herz-und lungenforschung offermanns, ludwigstr. , bad nauheim, germany tumor cell metastasis to distant organs is the primary cause of mortality in cancer patients. tumor cells leave the primary tumor, intravasate, survive in the circulation and extravasate through the endothelial cell layer to grow in the target organ. it has long been known that blood platelets play an important role in tumor cell survival and dissemination, but the mechanism by which platelets promote metastasis remained unclear. given that platelets are found closely associated with tumor cells shortly after vascular arrest, we explored whether platelets can facilitate the transmigration of tumor cells through the endothelium and thereby promote extravasation of tumor cells into the organ parenchyma. the ability of various mouse and human tumor cells like lewis-lung carcinoma cells (llc ), b f melanoma cells or human neuroblastoma cells (sh-sy y) to transmigrate through an endothelial cell layer was strongly enhanced by seeding tumor cells together with mouse or human platelets onto the endothelial cell layer. this indicates that platelets facilitate tumor cell transmigration in vitro. we found that platelet granule secretion is involved in this process as supernatant from platelets incubated with tumor cells but not from resting platelets was sufficient to enhance tumor cell transmigration. additionally, no platelet-mediated increase of tumor cell transmigration was observed in dense granule secretion-defective platelets of munc - deficient mice. thus, dense granule secretion is required for platelet-dependent tumor cell extravasation in vitro. while the growth and weight of primary tumors after subcutaneous injection of llc and b cells was indistinguishable between wild-type mice and animals lacking munc - , the number of metastases in the lung was strongly reduced in munc - -deficient animals. the strong decrease in formation of metastases in munc - deficient mice was also observed after i.v. injection of llc and b f cells. thus, platelet dense granule secretion plays a critical role in tumor cell metastasis by enhancing tumor cell transmigration through the endothelial cell layer. formation of dna adducts in mouse tissues by the brassica ingredient methoxy- -indolylmethyl glucosinolate and its break-down product -methoxy- indolylmethyl alcohol schumacher f. , engst w. glucosinolates are secondary metabolites present at substantial levels in cruciferous vegetables, such as broccoli and cabbage. after injury of plant tissue they are activated by the enzyme myrosinase to form various electrophilic degradation products like isothiocyanates. we previously showed that -methoxy- -indolylmethyl ( -mim) glucosinolate (or neoglucobrassicin) is a potent genotoxicant in bacterial and mammalian cells after activation by myrosinase. the induction of mutations could be correlated with the formation of dna adducts [ ] . we have identified and synthesized the major dna adducts n -( -mim)-dg and n -( -mim)-da. moreover, we developed a highly sensitive uplc-esi-ms/ms method for selective mrm quantification of these adducts using stable-isotopic labeled adducts as internal standards. while the plant enzyme myrosinase is probably almost completely inactivated after cooking the vegetables, the glucosinolates reach the gut mostly intact due to their good heat and ph stability. enzymes of individual intestinal bacteria are able to cleave the glycosidic bond of the glucosinolates, which leads to the formation of reactive metabolites within the gut lumen. we were able to detect significant levels of n -( -mim)-dg and n -( -mim)-da in a dose-dependent manner in the large intestine of mice treated orally with isolated -mim glucosinolate. the peak levels of n -( -mim)-dg and n -( -mim)-da in the murine large intestine were reached h after a single administration of µmol -mim glucosinolate/ kg body weight. the oral application of the relatively stable metabolite -mim alcohol to mice led to the formation of identical dna adducts. this benzylic alcohol can be activated by sulfotransferases to an electrophilic sulfo conjugate. in contrast to the intact glucosinolate the orally administered -mim alcohol generated significant levels of n -( -mim)-dg and n -( -mim)-da not only in the large intestine but also in other tissues, such as the liver, of mice. [ ] h. glatt, c. baasanjav-gerber, f. schumacher, b. h. monien, m. schreiner, h. frank, a. seidel, w. engst, chem.-biol. interact., ( ) human pregnane x receptor genotype of the donor but not of the recipient is a risk factor for delayed graft function after renal transplantation schwab m. , , schaeffeler e. delayed graft function (dgf) is an important immediate complication in renal transplantation significantly contributing to decreased long-term allograft survival. in addition to donor-and recipient-related risk factors altered renal excretion of xenobiotics by membrane transporters may influence dgf as well. using recipients' and donors' dna, we assessed the impact of genetic variants on dgf for the transporter proteins, pglycoprotein (abcb ) and multidrug resistance protein (abcc ), and the nuclear pregnane x receptor (pxr/nr i ), regulating the transcription of drug metabolizing enzymes and membrane transporters. in our local cohort of transplant patients (n= ) dgf occurred in . %. logistic regression analysis using four different genetic models (i.e. co-dominant, dominant, recessive and log additive) indicates that only the donor's pxr rs tt genotype was significantly associated with dgf (recessive model: or, . ; %ci, p= . unadjusted) , even after correction for multiple testing (p= . holm-adjusted). when we performed multivariate analysis including genetic and clinical co-variates (i.e. age, gender, hla mismatches, panelreactive antibodies, immunosuppression using cni, t cell-depleting agents, anti-il- receptor antibody and steroids, cold or warm ischemia time, living vs deceased donors or graft loss) again dgf was significantly associated only with the pxr rs tt genotype of the donor (recessive model: or, . ; % ci, . - . ; p= . unadjusted) which held true after correction for multiple testing (p= . ). for abcc variants only the donor rs t>a genotype correlated with dgf by univariate (or, . ; %ci, p= . unadjusted) as well as by multivariate analysis (or, . ; %ci, p= . ; table ) but not after correction for multiple testing (p= . ). for variants in the abcb gene no significant associations with dgf were detected for both the donor's and the recipient's genotype. in summary, our findings suggest for the first time that pxr may be a risk gene for the development of dgf independently from previously identified risk factors. supported by the robert-bosch foundation, stuttgart, germany, the bmbf grant is c (berlin, germany) and by the ferdinand eisenberger grant of the german society of urology (id krs /fe- ). formation, morphology and structural requirements for formation of microtubule protrusions by clostridium difficile toxin cdt schwan c., kruppke a. s., nölke t., aktories k. institut für experimentelle und klinische pharmakologie und toxikologie i, albertstr. , freiburg, germany clostridium difficile is an anaerobe, gram-positive pathogen. it causes antibioticassociated diarrhoea and pseudomembranous colitis by production of the rho gtpaseglucosylating toxins a and b. recently emerging hypervirulent clostridium difficile strains additionally produce the binary adp-ribosyltransferase toxin cdt (clostridium difficile transferase). cdt is taken up via receptor-mediated endocytosis after binding to the lipolysis stimulated lipoprotein receptor (papatheodorou et al., pnas ) . in the cytosol, cdt adp-ribosylates actin at arg , thereby actin polymerization is blocked, resulting in disruption of the f-actin network. cdt and other binary actin-adp-ribosylating toxins induce redistribution of microtubules in the cell interior and formation of long (> µm) microtubule-based protrusions at the surface of intestinal epithelial cells which increase bacterial adherence (schwan et al., plos pathog ). the clostridial actin-adp-ribosyltransferases influence the dynamicity of microtubules and their capture at the cell cortex by indirectly affecting different microtubule regulating proteins like clasp and acf . besides the influence of cdt on microtubule regulatory proteins, the formation of protrusions depends on plasma membrane composition. depletion of cholesterol, the breakdown of sphingomyelin or inhibition of sphingolipid-synthesis reduce the formation of microtubule-based protrusions. surprisingly, most of the cdt-induced processes contain membrane-tubules derived from the endoplasmatic reticulum (er). the remodeling of the er is microtubule dependent and is mainly mediated by stim that usually functions as a calcium sensor in the er and activates the store operated orai calcium ion channels in the plasma membrane. the data suggest that toxin-induced changes of the microtubule system including alterations of the er, may affect trafficking and er-dependent signalling. bilobalide is a neuroprotective constituent of ginkgo biloba with an unknown mechanism of action. in the present study, we first used microdialysis in mice to evaluate changes in the extracellular fluid of the brain during and after stroke. microdialysis probes were implanted into the striatum of cd- mice, and dialysates were obtained while a monofilament was inserted for min via the common carotid artery (cca) to block perfusion through the middle cerebral artery (mca). while glucose levels dropped immediately upon middle cerebral artery occlusion (mcao), glutamate concentrations in the microdialysates -as measured with a cma analyzer -rose extensively during ischemia to more than % of baseline level. both glucose and glutamate levels recovered rapidly when mcao was terminated after min. when bilobalide ( µm) was perfused into the striatum through the microdialysis probe during mcao, glucose levels dropped but the neurotoxic rise of glutamate was significantly attenuated and reached only % of baseline level (p< . ). in the following experiments, we investigated the activity of mitochondria in ischemic brain. ischemia was induced by mcao, and ischemic as well as "healthy" tissue from the opposite hemisphere was obtained. mitochondria were isolated and mitochondrial respiration was monitored using the oroboros ® oxygraph. significant deficits of respiration were observed after ischemia. in the healthy hemisphere, the respiratory states (leak i+ii, complex i+ii+iv, oxidative phosphorylation (oxphos) and electron transport system (ets) capacity) showed a decrease of oxygen consumption to - % of sham-operated mice. in the ischemic hemisphere, several values were lower at % of sham-operated mice (leak i+ii, complex ii+iv,oxphos and ets) whereas complex i showed a remarkably low respiratory capacity of % of baseline. direct addition of bilobalide ( µm) to post-ischemic mitochondria caused a -fold increase of complex i activity in vitro. pretreatment of mice with bilobalide ( mg/kg i.p.) one hour before mcao caused a significant, -fold improvement of complex i respiration when measured ex vivo. these data clearly indicate that bilobalide targets mitochondrial processes within the respiratory chain, preserving complex i function during ischemia. this action likely explains its neuroprotextive activity in vivo. unreacted resin monomers such as -hydroxyethyl methacrylate (hema) are environmental stressors released from dental composites after incomplete polymerization. the production of reactive oxygen species (ros) is a major response of cells to monomer exposure. moreover, adverse effects of monomers including delayed cell differentiation or mineralization processes, dna damage or apoptosis are associated with increased ros production. the intracellular redox homeostasis is controlled by the major non-enzymatic antioxidant glutathione (gsh), and antioxidant enzymes. here, we hypothesized that cells exposed to hema responded by a differential expression of antioxidant enzymes such as superoxide dismutase (sod- ), catalase (cat) or glutathione peroxidase (gpx / ). raw . mouse macrophages were exposed to hema ( - mm) for h, and protein expression was analyzed by western blotting. to study the influence of intracellular gsh on enzyme expression, gsh synthesis was reduced by the inhibitor buthionine sulfoximine ( µm bso), or enhanced by -oxothiazolidine- -carboxylate ( mm otc) and n-acetyl cysteine ( mm nac). expression of sod- found in untreated cultures was decreased in the presence of hema and even further reduced by bso. in contrast, nac counteracted hemainduced inhibition of sod- expression. cat expression was not detected in untreated cells, however, the enhanced expression of cat in cells exposed to hema indicated the decomposition of abundant levels of hydrogen peroxide. the minor influence of bso or otc showed that expression of cat was independent of gsh levels while a decrease of hema-induced cat expression in the presence of nac indicated reduced oxidative stress. gpx / was expressed in untreated cultures, and its down-regulation by bso indicated that this enzyme was primarily responsible for h o decomposition. the inhibitory effect of hema on gpx / expression was enhanced by bso but counteracted by otc or nac. these findings indicate that h o is the predominant reactive oxygen species generated in the presence of dental resin monomers like hema. abundant h o production leads to the activation of cat expression and a feed-back inhibition of sod- expression. the hema-induced reduction of gpx / expression is most likely a consequence of reduced gsh levels because of the formation of glutathione disulfide (gssg) or by gsh-hema adducts. the life-threatening effects of certain organophosphorus compounds such as soman or fenamiphos cannot be antagonized adequately by the treatment with atropine and oximes. alternative approaches are necessary. since the adequate restoration of disturbed muscle function is considered to be life-saving, a model is needed for screening of potentially therapeutic substances. an established model for the development of such new therapies is the diaphragm preparation. however, this model requires a large number of animals and experimental available time frame is limited to some hours. here, the organotypic nerve-muscle co-culture may be an appropriate alternative, because a large number of specimens with low numbers of animals and a long period of investigation over several days is possible. in the present study, the restoration of vx paralysed muscle function with obidoxime was investigated by using both models. slices of spinal cord and muscle tissue were dissected from mice embryos (e - ) , fixed to coverslips and incubated in roller tubes for about - weeks. spontaneous muscle activity was recorded by video microscopic techniques and was quantified offline. muscle force production in mice diaphragm preparations was elicited by indirect field stimulation technique in a chamber organ bath and quantified as time-force diagrams (auc) that were expressed as relative changes of the muscle force compared to the control data. application of the nerve agent vx ( . µm) resulted in a strong reduction of muscle activity in the co-culture and of muscle force production in the diaphragm muscle. after obidoxime ( µm) was added spontaneous muscle activity in the co culture recovered from . ± . hz to . ± . hz (control . + . hz) . muscle force remained stable over the next days. the vx-blocked muscle force of diaphragm was restored to . ± . % by obidoxime compared to control. muscle force production after indirect stimulation was stable for hours only. our results suggest that the organotypic nerve-muscle co-cultures may be an appropriate tool for the screening of new therapeutic approaches in restoration of blocked neuromuscular transmission. moreover, the model offers an additional advantage as long-term experiments may be performed and pre-and postsynaptic effects may be assessed directly. additionally, the number of experimental animals could be reduced. the modulation of gene expression by the transcription factor crem (camp responsiveelement modulator) represents a fundamental mechanism of gene control in response to elevation of intracellular camp levels. in vascular smooth muscle cells (vsmcs) crem is involved in the regulation of cell proliferation and apoptosis supporting its relevance for vascular proliferative diseases. mice with a global inactivation of crem (cko) showed a significant increase in neointima formation after ligation of the carotid artery and an increase of atherosclerotic plaque formation after high fat diet on an apoe knockout background compared to wildtype controls (wt). on the cellular level a crem deficiency was associated with a . fold increased proliferation rate of primary vsmcs after stimulation with the platelet-derived growth factor (pdgf; n= from isolations). microarray analysis and subsequent realtime-rt-pcr validation revealed that the alpha-type platelet-derived growth factor receptor (pdgfra) the regulator of g-protein signaling (rgs ) and peptidylprolyl isomerase a (ppia) were . - . fold upregulated in pdgf treated cko vsmcs compared to wt vsmcs (n= ). transcripts of rgs ( . fold, ) and ppia ( . fold, were also upregulated in the carotid artery of cko mice in comparison to wt mice (n= - ). we conclude that crem deficiency is associated with transcriptional changes of rgs , pdgfra, ppia, which might explain the increased proliferation rate in cko vsmcs and the increased responsiveness to pathophysiological conditions. (supported by the izkf münster). the role of non-catalytic p and p subunits in regulating phosphoinositide kinase γ by gβγ and h-ras shymanets a. phosphoinositide -kinase γ (pi kγ) controls a plethora of cellular responses. pi kγ, a heterodimer formed by non-catalytic p or p and catalytic p γ subunits, is regulated by gβγ and ras. earlier we speculated that p binds to gβγ to translocate cytosolic pi kγ to the plasma membrane, enabling direct activation of p γ (brock et al., j. cell biol. ) . however, the p subunit does not function as gβγ adapter (kurig et al., pnas ) . since the impact of each non-catalytic subunit in regulating p γ by gβγ and ras still remains elusive, we studied their role in detail. gβ γ variants harbouring mutations in positions involved in interaction with gα subunit were purified from sf cells and tested for their ability to activate pi kγ. we observed that p , but not p , was able to rescue the stimulatory activity of gβ γ mutants incapable to activate p γ (shymanets et al., biochem. j. doi: . /bj ). to further study the functional impact of the non-catalytic subunits on pi kγ regulation, we have designed phospholipid vesicles containing similar amounts of recombinant pi kγ variants. although p /p γ exhibited stronger sensitivity to gβ γ than p γ, the activity of vesicles-associated p /p γ was significantly higher as compared to vesicles-associated p /p γ or p γ in the absence and in the presence of gβ γ . to study an effect of ras proteins on pi kγ variants, recombinantly expressed h-ras was purified from sf cells. the posttranslational processing and lipidation of the protein was verified by mass spectrometry analysis. the impact of h-ras on regulation of p /p γ and p /p γ differed, which may explain integration of pi kγ variants in different signalling pathways. taken together, p and p subunits implement discrete functions in respect to (i) membrane recruitment of pi kγ and (ii) regulation of enzymatic activity by gβγ and h-ras. preparation of consolidated exposure scenarios for mixtures under reach sica m., dorn s., mostert v. dr. knoell consult gmbh, marie-curie-str. , leverkusen, germany under reach, formulators of mixtures need to include substance-related information into extended safety data sheets (esds), if mixtures are classified as dangerous according to the dangerous preparation directive (directive / /ec). one way to add information on substances into esds of mixtures is to generate exposure scenarios (ess) for mixtures. in order to fulfil this task, two approaches have been developed for the identification of the risk-determining substances (lead substances) in the mixtures: the critical component approach (cca) relies on dnels and pnecs for all substances, their concentrations in the mixtures as well as substance and use-specific availability parameters (echa, ) . in contrast, the dpd+ method is based on the legislation for classification of preparations (directive / /ec). the dpd+ method defines a lead substance for each route of human exposure and for the aquatic environment (cefic, ) . however, each of these methods has certain limitations. the aim of the present work is to improve the preparation of consolidated ess for a number of mixtures and provide information about their safe use. to this end, we first adopted information on risk management measures (rmms) and operational conditions (ocs) of the lead substances using the dpd+ methodology. at the same time, we considered the specific conditions of use of the mixtures (e.g. spraying, brush painting). we then conducted risk assessments by deriving dnels for the mixtures and using exposure modelling tools recommended under reach (e.g., ecetoc tra, riskofderm, art). we compared the outcome of these assessments with results obtained from the application of the dpd+ methodology. the work presents the results of application of dpd+ approach and the cca and indicates possible improvements for the risk assessment of mixtures. to check for seasonal and weather dependent influences in the prescription rate of drugs used to treat cardiovascular and respiratory diseases (atc codes c and r) a survey covering all prescriptions of a specimen german general regional health insurance (aok plus, data for saxony, largest health insurance service, approx. % of all saxonian citizens are inscribed to this service) for the years to was analysed on a monthly basis. the number of prescriptions for cardiovascular drugs changed approximately +/- % around the mean for the different month without a clear seasonal pattern. for respiratory drugs only the systemic anticholinergics and drugs used to treat obstructive lung disease displayed a distinct seasonal pattern with a - % above average prescription figure during spring time (february to may) and a to % trough in late summer/autumn (july to october). the data have to be analysed for further cofounders (e.g. influenza prevalence, environmental conditions etc.) to fully understand the fluctuations observed. the prescription rate for cardiovascular drugs and respiratory drugs seems to be influenced by multiple factors aside seasonal influences. pasteurella multocida toxin prevents osteoblast differentiation by activation of heterotrimeric g proteins siegert p., aktories k., orth j. institut für experimentelle und klinische pharmakologie und toxikologie abt. i, albertstr. , freiburg i. br., germany pasteurella multocida toxin (pmt) is a major virulence factor of pasteurella multocida causing pasteurellosis in man and animals and is responsible for atrophic rhinitis in pigs. the toxin modulates various signaling pathways by acting on the heterotrimeric g proteins gαq, gα / and gαi. pmt activates gq to increase inositol phosphate production via phospholipase cβ and alteration of gene expression via the jak/stat pathway. the toxin also activates rhoa via gαq and gα / family proteins. we showed that the underlying mechanism of the activation of heterotrimeric g proteins is a deamidation of an essential glutamine residue leading to a constitutive activation of the g protein. because pmt is the causative agent to induce progressive atrophic rhinitis in pigs, which is characterized by loss of nasal turbinate bones leading to a twisting and/or shortening of the snout, we studied the effect of pmt on bone cells. here we studied the effect of the toxin on osteoblast differentiation in st- cells and in primary osteoblasts from rat calvaria. st- cells are stromal derived cells, which can be differentiated into osteoblasts or adipocytes. the toxin inhibits the differentiation of st- cells into osteoblasts studied by determination of specific osteoblast markers. additionally, pmt represses the induction of transcription factors essential for osteoblast differentiation. moreover, the principal pathways activated by pmt to induce these effects were investigated. ventilator-induced lung injury (vili) is a serious problem in intensive care medicine. its mechanisms are only incompletely understood, although it is widely accepted that ventilation-induced inflammation (biotrauma) makes an important contribution. the isolated perfused mouse lung (ipl) is a valuable tool to investigate the mechanisms of vili. several studies have shown considerable differences between various mouse strains with respect to lung mechanics and inflammatory responses. therefore, we hypothesized that the pulmonary responses to mechanical ventilation differ between c bl/ and balb/c mice. in addition, this study introduces the novel half lung technique that allows to obtain lung tissue from the same mouse at two different time points. isolated perfused mouse lungs from c bl/ or balb/c were subjected to high ( cmh o) or low pressure ( cmh o) ventilation for minutes. after minutes the left lung was removed and used for western blot analysis. the right lung was ventilated for another minutes. by the end of experiment the right lung was removed and qrt-pcr performed. during the whole experiment perfusate sample were taken from the venous catheter and used for protein quantification by elisa. it was possible to remove half of the lung and to further ventilate the other half without acute changes in lung mechanics. in both strains high pressure ventilation elicited a significantly higher cytokine release than low pressure ventilation. c bl/ mice showed higher tnf, il- β and amphiregulin levels after high pressure ventilation, whereas balb/c exhibited increased production of cxc chemokines (cxcl- , cxcl- ) and il- . kinase activities (jnk, akt, erk / , p map kinase) were increased in high pressure ventilated animals, but were strain independent. the novel half lung technique builds on the well established ipl method. it permits to separately analyze the left and the right lung at different time points during continual ventilation. this method reduces animal numbers by % and allows statistical within subject analysis. using this method, the present study showed that inflammatory response to mechanical ventilation differ between c bl/ and balb/c. these findings show that the biotrauma response in mice depends on the strain that is studied. macrophages play an important role as an integral part of the first line of immune defense. two different macrophage populations have been described. m macrophages produce proinflammatory cytokines and are involved in inflammatory processes. by contrast, m macrophages release anti-inflammatory cytokines and extracellular matrix components. they can enhance wound repair and angiogenesis, but they can also promote tumor progression. recently, industrial nanoparticles have raised concerns because of their putative toxic effects. on the other hand, specifically designed nanoparticles can be used as clinical diagnostics and as drug carriers for pharmacotherapy. thus, investigations on the interactions of engineered nanoparticles with living cells and organisms are of great importance. macrophages as phagocytosing cells scavenge nanoparticles circulating in the bloodstream. therefore, we analyzed how nanoparticles with different surface functionalization might affect functions of human macrophages. monocytes were isolated from buffy coats and differentiated to macrophages with macrophage colony-stimulating factor. carboxy-(ps-cooh) and amino-(ps-nh ) functionalized polystyrene nanoparticles were produced by the miniemulsion polymerization process and the average particle size, the polydispersity index and their zeta potential were determined by dynamic light scattering. the macrophages were cultured in the presence or absence of different concentrations of ps-cooh and ps-nh nanoparticles for up to days. analysis of cell viability revealed that ps-nh but not ps-cooh concentration-and time-dependently reduced the macrophage viability. by annexin v/propidium iodide double staining we could show that ps-nh trigger apoptosis in macrophages. we further polarized macrophages to either m or m using ifn-γ and lps or il- , respectively. these macrophage populations were characterized by their expression of extracellular markers by flow cytometry and their production of cytokines by elisa. the effects of functionalized polysterene nanoparticles on the cytokine production and surface marker expression of m and m macrophages were analyzed. our data indicate that surface functionalization is a critical parameter in the nanoparticle-induced toxicity in human macrophages. this work was supported by the dfg spp . loos c., lunov o., syrovets t., simmet t. ulm university institute of pharmacology of natural products & clinical pharmacology, helmholtzstr. , ulm, germany nanoparticles are currently used for various medical applications including imaging, diagnosis and drug delivery. due to particle size and surface area, their fundamental properties differ significantly from those of corresponding bulk materials. nanoparticles circulating in the blood are mainly sequestrated by the reticuloendothelial system that consists predominantly of phagocytic macrophages. macrophages express a variety of cellular receptors for sensing and internalizing particular material like viruses, microorganisms, and foreign particulate matter including nanoparticles. therefore, a detailed understanding of the intracellular fate and processing of the nanoparticles by macrophages is indispensable for controlled biomedical applications of nanoparticles. introducing distinct surface modifications, one might control nanoparticle uptake by different cell types and thereby target specific tissues and cellular compartments. tumor cell lines are frequently used as models for primary cells to analyze the effect of nanoparticles on cells. here we show that carboxy-(ps-cooh) and aminofunctionalized (ps-nh ) polystyrene nanoparticles of ~ nm in diameter are internalized by human macrophages, thp- monocytic leukemia cells, and by pmadifferentiated thp- cells via different mechanisms. in buffer, macrophages and thp- rapidly internalize both types of nanoparticles, yet, the carboxy-functionalized particles were taken up to a higher extent. the uptake of both nanoparticles was drastically reduced in media containing serum. using pharmacological and antisense in vitro knockdown approaches, we showed that the specific interaction between cd receptors and the particles determines the macrophage uptake of particles by phagocytosis, whereas particle internalization by thp- cells occurred via dynamin iidependent endocytosis. by contrast, pma-differentiated thp- cells took up the particles via macropinocytosis. in line with the in vitro data, more intravenously applied ps-cooh particles accumulated in liver tissue, whereas ps-nh were preferentially targeted to tumor tissue. these data show that the amount of particle internalization, the uptake mechanisms, and kinetics differ significantly among primary cells and model tumor cells, whether differentiated or not, and that they are further critically dependent on the particle opsonisation by serum proteins. this work was supported by the dfg spp . specifically designed and functionalized nanoparticles hold great promise for a variety of biomedical applications. to ensure their safe application, such particles require a rigorous analysis of their effects on cell functions. here we demonstrate that aminofunctionalized polystyrene nanoparticles (ps-nh ) of ~ nm in diameter in contrast to carboxy-(ps-cooh) and nonfunctionalized (ps) particles induce an nlrp inflammasome activation and the subsequent release of il- β in human macrophages. amino-functionalized ps nanoparticles induced time-dependent lysosomal destabilization followed by release of lysosomal enzymes. this resulted in mitochondrial damage and formation of reactive oxygen species. accumulation of mitochondrial reactive oxygen species was accompanied by oxidation of thioredoxin, a protein playing a central role in maintaining the cellular redox balance. upon oxidation, thioredoxin dissociated from the thioredoxin-interacting protein (txnip). liberated txnip, in turn, interacted with the nlrp protein resulting in a conformational change of the pyrin domain of the nlrp protein as predicted by molecular modeling. txnip interaction with nlrp led to assembly of the nlrp inflammasome complex, to caspase- activation, and release of il- β. using an in vitro knockdown approach, we showed that ps-nh induced activation exclusively of nlrp , whereas other inflammasomes remained unaffected. treatment of macrophages with n-acetyl-l-cysteine, a scavenger of reactive oxygen species, abolished both, the caspase- activation and the subsequent release of il- β caused by ps-nh nanoparticles. these data reveal a novel mechanism of the nlrp activation induced by amino-functionalized nanoparticles and provide a strategy as to how such an effect can be functionally antagonized by supplementation with a radical scavenger. this work was supported by the dfg spp . the semi-permeable barrier of the endothelial cell lining of the blood vessels has important synthetic and metabolic functions including transport of cells and biomolecules, regulation of vascular smooth muscle tone, and control of hemostasis. plasmin is a serine protease, which is generated from its zymogen plasminogen under physiological and pathological conditions. small amounts of plasmin are produced in the context of contact activation during inflammation. consistently, increased generation of plasmin has been reported during atherosclerosis. we have shown previously that plasmin, in addition to its role in fibrinolysis, could induce proinflammatory activation of various cells including monocytes, macrophages, and dendritic cells. therefore, we analyzed how plasmin might affect the functions of endothelial cells, which could be relevant during inflammation and atherosclerosis. using flow cytometry, western immunoblotting and fluorescent microscopy, we show that endothelial cells of different origin express the plasmin receptor complex composed of annexin a and s a . addition of plasmin to human umbilical vein endothelial cells (huvec) induced timeand concentration-dependent cytotoxic effects in the cells. in addition, within min plasmin triggered a rapid and prolonged expression of free radical oxygen species (ros) in endothelial cells as analyzed by microscopy and fluorometry using the rossensitive dye carboxy-h dcfda. the ros production in endothelial cells was accompanied by cell detachment. fluorometric and western blot analysis of caspase activation in the cells treated with plasmin showed that plasmin induced apoptotic cell death in endothelial cells, which was evident already several hours after exposure to plasmin. thus, plasmin might induce production of ros in endothelial cells, their detachment and apoptosis, events which might be relevant for the development of atherosclerosis. this work was supported by the dfg. sesquiterpene lactones (stl) comprise a large group of secondary plant metabolites that constitute the active principle of a number of traditional anti-inflammatory phytomedicines. specifically helenalin and parthenolide have recently gained considerable attention as lead compounds or putative therapeutics for the treatment of inflammation and possibly cancer. both compounds have been shown to interfere with the signal transduction through inhibition of the nuclear factor κb (nf-κb). whereas the inhibitory effects of the stl on nf-κb are undisputed, their molecular mechanism of action remains a matter of debate. surface plasmon resonance (spr) analysis allows label-free measurement of molecular interactions. yet, analysis of the interaction of immobilized recombinant proteins with small molecular ligands remains a technically challenging task. in the present study we used spr technology to investigate the molecular interaction of the stl helenalin with putative intracellular target proteins such as the nf-κb protein p /rela, the catalytic subunits of the ikk complex, namely ikkα and ikkβ, and the intracellular antioxidant glutathione (gsh). at physiological ph . , helenalin interacts with rela (k d = . µm), yet it failed to bind either ikkα or ikkβ. hence, when dna with nf-κb binding consensus sequence was immobilized on sensor chips, the binding of rela was inhibited by helenalin with an ic of . µm. moreover, we provided several lines of evidence that stl may modify rela on cysteine by a michael-type addition. this interaction was confirmed by molecular docking that identified the best matching interaction between rela and helenalin with predicted hydrogen bonding interactions between helenalin and residues arg , lys , gly and ile of rela. consistent with our hypothesis that helenalin interacts with sulfhydryl groups at ph . , helenalin was also able to interact with reduced, but not oxidized, glutathione with a kd of µm, though no significant interaction was observed at ph . . thus, we showed that the sesquiterpene lactone helenalin interacts with the nf-κb protein rela but not with ikkα or ikkβ. moreover, at physiological ph, helenalin does not interact with glutathione to any significant extent. direct interaction of helenalin with rela leading to inhibition of rela-dna binding and transactivation might present the molecular mechanisms underlying the anti-inflammatory effects of stls. although nanosized materials are quickly taken up by macrophages, our understanding of the involved processes is still rather limited. therefore, we analyzed the uptake of diagnostically used carboxydextran-coated iron oxide nanoparticles of two different sizes, superparamagnetic iron oxide nanoparticles of nm (spio) and ultrasmall superparamagnetic iron oxide nanoparticles of nm (uspio), by human macrophages. by pharmacological and in vitro knockdown approaches, the principal uptake mechanism of macrophages for both particles was identified as clathrin-mediated, scavenger receptor a-dependent endocytosis. further, we created a mathematical model of the nanoparticle uptake by macrophages that permitted determination of key parameters of endocytotic process, such as the uptake rate, the mean uptake time, the number of particles taken up by a cell, and the correlation between the number of internalized particles and their extracellular concentration. the model also provided information on the individual and collective wrapping time of the nanoparticles and described the relation between biophysical parameters such as cytoskeletal forces, membrane elasticity, and the uptake time. finally, we gained information on the minimal linear spacing between simultaneously acting neighboring endocytotic pits that contain single nanoparticles and govern the collective uptake process. the calculated parameters were further confirmed experimentally using spinning disc confocal microscopy. thus, the new model provides important insights into the biophysical processes involved in endocytosis of nanoparticles by human macrophages. this work was supported by the dfg spp . prostaglandins (pg) are hormones which are formed during inflammatory processes from arachidonic acid by cyclooxygenases and prostaglandin synthases [ ] . in the subsequent metabolism, in which the five-membered ring is dehydrated, α,β-unsaturated carbonyl compounds are generated [ , ] . these come along with mercapto groups of amino acids in a michael addition reaction associated with activation of cellular enzyme cascades [ ] that potentially contribute to their possessed antiinflammatory, antineoplastic and antiviral effects [ ] . however little is known so far about possible adverse health effects.we addressed the question whether selected cyclopentenone prostaglandins (cypg) exhibit potential mutagenic and genotoxic properties in the hamster lung fibroblast cell line v . induction of dna damage was investigated by single cell gel electrophoresis assay (scge). the impact of cypg on cellular redox status was detected by total glutathione (tgsh) assay. the induction of micronuclei and apoptosis was determined by staining with ', -diamidino- -phenylindole (dapi). furthermore the hypo-xanthine-guanine phosphoribosyltransferase (hprt) assay was used for mutagenicity testing. , followed by prostaglandin a (pga ), showed the most distinctive genotoxicity, i.e., induction of micronuclei, and apoptotic effects. furthermore, the dpgj and pga -induced significant decrease in the tgsh level in v may contribute to the observed increase in oxidative dna-damage. however, none of the tested cypg exhibited mutagenic properties in the hprt assay. in conclusion, a potential in vitro genotoxicity of cypg has been observed which may be involved in carcinogenesis associated with chronic inflammation. parabens and methylisothiazolinone are used as preservatives in personal care products. sensitization to parabens and methylisothiazolinone is relatively rare considering their wide use in cosmetics, but only few quantitative or clinical data exist. therefore, we have tested methyl-, ethyl-, propyl-, isopropyl-, butyl-, isobutyl-, pentyl-, phenyl-and benzylparaben , and methylisothiazolinone in the loose-fit coculture-based sensitization assay (lcsa) developed by our working group. the coculture of primary human keratinocytes and allogenic dendritic cell-related cells (dc-rc) in this assay emulates the in vivo situation of the human skin. sensitization potential of the test substances was determined by flow cytometric analysis of the dc-rc maturation marker cd . determination of the concentration required to cause a half-maximal increase in cd -expression (ec ) allowed a quantitative evaluation. the irritative potential of the substances was assessed by -aad ( -amino-actinomycin d)-staining. the concentration required to devitalize % of the examined cells compared to a zero control was termed ec %. parabens exhibited weak (methyl-, ethyl-, propyl-and isopropylparaben) or strong (butyl-, isobutyl-, pentyl-and benzylparaben) sensitizing potential, phenylparaben was found to be a moderate sensitizer, with ec -values ranging from . µmol/l (pentylparaben) to . µmol/l (methylparaben). due to a pronounced cytotoxicity (ec % = . µmol/l), we could not estimate an ec -value for methylisothiazolinone. sensitization potential of parabens correlated with side chain length. parabens showed no (methyl-and ethylparaben) or weak irritative potential (propyl-, isopropyl-, butyl-, isobutyl-, phenyl-and benzylparaben) , only pentylparaben was rated to be irritative. apart from phenyl-and benzylparaben, irritative potential also correlated with side chain length but did not correlate strictly with the sensitization potential. overall, we were able to demonstrate and compare the sensitizing potential of parabens in this in vitro test. it was weak for methyl-and propylparaben, the most commonly used parabens. furthermore, we showed an irritative potential for most of the perservatives. thus the lcsa is a useful in vitro test to compare the sensitizing potential of xenobiotics. phosphorylation of the neurodegeneration-related septin by protein kinase dyrk a at serine affects protein stability soppa u. septins are gtp-binding proteins forming heterooligomeric complexes and filaments by interactions of the family members. these complexes have important functions by building scaffolds for proteins involved in cell cycle or cell polarity but their subcellular distribution as well as their regulation remain largely unclear. septin (sept ) was found in neurodegeneration related protein aggregates and is associated with migration of cortical neurons. here we first describe a potential mechanism for regulation of sept by phosphorylation via dual specificity tyrosine-phosphorylation-regulated kinase a (dyrk a). dyrk a is overexpressed in down syndrome and supposed to be involved in neurodevelopment and neurodegeneration. by site directed mutagenesis of flag tagged mouse sept and overexpression in hela cells we identified serine as the major phosphorylation site of dyrk a and generated a phosphospecific antibody. transient coexpression of sept and dyrk a in hela cells increased phosphorylation of serine by % in relation to basal phosphorylation. in contrast, cotransfection of the kinase deficient dyrk a mutants k r and d n did not increase serine phosphorylation. moreover we could show, that inhibition of kinase activity by the dyrk a inhibitor harmine reduced phosphorylation of exogenous sept at serine about % in hela cells. furthermore, down regulation of dyrk a by rna interference lead to decreased phosphorylated serine . these results indicate that endogenous dyrk a contributes to sept phosphorylation in hela cells. finally we analyzed protein stability of wild type sept compared to the phosphorylation resistant s a mutant in hela cells by inhibition of translation with cycloheximide. we found that in living cells the sept s a mutant is more stable than wild type sept . in summary, our results suggest phosphorylation at serine by dyrk a as a novel mechanism to regulate sept stability and indicate a possible link of these proteins in cellular processes. is formaldehyde a good example for a "genotoxic carcinogen" with a threshold mode of action? speit g. institut für humangenetik, universität ulm, ulm, germany formaldehyde (fa) induces toxic and genotoxic effects in directly exposed cells (site of contact). several studies in which fa was administered to rats by inhalation showed evidence of tumor induction in nasal epithelium. there is also some epidemiological evidence that fa causes nasopharyngeal cancer in humans. although fa is a known mutagen, it is still a matter of discussion whether carcinogenesis is primarily mediated via a mutagenic mode of action. there is evidence that cytotoxicity and induced proliferation are the main causes for tumor formation. however, a decisive role of mutagenesis cannot be excluded and a mutagenic mode of action has to be considered for risk estimation. the basic assumption is that mutagens have a non-threshold mode of action. a threshold mode of action for a chemical is likely when a substance with a known mutagenic potential does not induce mutations at low concentrations due to a specific type of reaction with the genetic material and / or physiological protective mechanisms. because fa is a directly acting dna-reactive substance, a threshold mode of action may only be considered because of physiological protective mechanisms. after inhalation, pre-lesion protection occurs by unspecific binding to mucus, cellular proteins and glutathione. furthermore, fa is efficiently inactivated by enzymatic pathways. if fa reaches and damages the nuclear dna, dna repair mechanisms act as efficient postlesion protection mechanisms. in vivo inhalation studies with rats indicated that fa induces primary dna damage in the nasal epithelium but increased mutation frequencies were not measured. data are now available to show the relative distribution of endogenous versus exogenous dna adducts in different locations of the nose and other organs. considering the fa concentrations present in every living cell and the background levels of endogenous dna adducts, appropriate risk assessment and the identification of practical thresholds for fa-induced genotoxicity become feasible. fraud and misconduct in clinical trials steffen c. formerly federal institute for drugs and medical devices clinical trials unit, kurt-georg-kiesinger-allee , bonn, germany plagiarism in scientific publications has been the subject of public ("guttenplag") and scientific debate. plagiarism violates, however, "only" the intellectual property of its authors. in clinical trials, misconduct may also endager the health of patients. the suppression or falsification of data in clinical trials may mislead patients and doctors to use worthless treatments. clinicians may be provoked to repeat these trials, thereby wasting time and money. in clinical trials, misconduct includes everything from suppression or their repeated publication, the "correction" of unwanted results to the complete invention of the data, their intentional or negligent misinterpretation leading to the publication of biased conclusions from otherwise correctly performed clinical studies. the peer review system cannot protect against fraud, as few reviewers will have the means and the time to reevaluate original data. they will have to rely on these data, the calculations and statistics that are presented to them. some kinds of fraudulent behavior, such as double publications, can be found in the review process, but this is also time-consuming and depends on a helpful librarian. other kind of fraud, as the suppression of patient data in clinical trials (the deletion of "non-responders", according to cinderella: the good ones go into the pot, the bad ones go into your crop) can only be detected by the national competent authorities performing an inspection according to good clinical practice. even if the results of such an inspection become public as in the case of ukrain (gansauge et al. ) , there is no institution that will further analyze and publish the misconduct or initiate the retraction of the incriminated paper. a german agency similar to the us office of scientific integrity could foster good clinical practice in germany. although it is sometimes very difficult to decide whether improper results arise from fraud or error, a bias for the source of funding is obvious. trials funded by for-profit organizations were significantly more likely to recommend the experimental drug as treatment of choice (als-nielsen et al. ) . medicinal products with disputed efficacy such as orally applied enzymes for systemic action, bacterial preparations for irritable bowel syndrome and food supplements are to be reviewed with special attention. further examples from the author's experience will be presented. ]i favors further kca opening, kca may establish a feed-forward regulation of ca + influx. in the present study we analyzed whether kca channels of sk and bk type play a role in sustaining ca + oscillations at g -to s-phase transition of primary mouse tumor cells and in human breast cancer cells, aiming towards a better understanding how kca modulate tumor cell proliferation. methods: kca expression was quantified by qpcr in human breast cancer biopsies, transgenic mmtv/c-neu + mouse mammary tumors and primary tumor cells derived thereof. the identity of the tumor cells was verified by gliolan staining. proliferation of the primary mammary tumor cells in the presence or absence of bk and sk modulators was tested using a real time cell monitoring system. the cellular dna content as a measure for cell ploidity was determined by propidium iodide staining and flow cytometry. changes in [ca + ]i oscillations and peak amplitude were determined using ca + indicator fura- am. results: sk , but not bk, expression is detectable in human and mouse breast cancer biopsies and in primary tumor cells derived from the mmtv/c-neu + mouse model. sk inhibition by tram- dose-dependently ( , to µm) inhibits the growth of primary mammary tumor cells probably by a g cell cycle arrest. ca + oscillations in proliferating mmtv/c-neu + tumor cells were ablated upon pharmacologic inhibition of sk channels. -dependent cell cycle progression is dependent on sk activity. blocking sk disrupts a feed-forward loop that coordinates ca + influx via trp or crac channels in tumor cells. the consequences of sk inhibition in mammary tumors in vivo will be discussed. synthesis of a triphenylphosphonium substituted derivative of -hydroxymethyl- -methylpyrroline n-oxide stolze k. esr combined with spin trapping is a well-known analytical approach to detect free radicals formed in various biological systems, e.g. superoxide, hydroxyl and a series of carbon-centered free radicals, which are involved in oxidative stress. our aim was to modify the established spin trap , -dimethyl-pyrroline n-oxide (dmpo) with a functional side chain, which can be used further as anchor for moieties enabling the spin trap to penetrate mitochondrial membranes, such as the positively charged triphenylphosphonium substituent. several synthetic routes were tested to introduce a -carboxybutyltriphenylphosphonium-substituent to the spin trap -hydroxymethyl- -methylpyrroline noxide (hmmpo). while the activation of the carboxy group via the corresponding chloride was not successful, the use of a mixed anhydride with acetic acid appeared to be a promising way, although the reaction is considerably slower. preliminary spin trapping experiments have been performed with model systems generating superoxide, hydroxyl-, and carbon-centered radicals. othman e. m., stopper h. universität würzburg toxikologie, versbacher str. , würzburg, germany type diabetes mellitus (dm ) is a growing health problem affecting more than million people worldwide. it is associated with severe acute and chronic complications that negatively influence both the quality of life and survival of affected individuals. epidemiological studies clearly indicate that the risk of several types of cancer (including pancreas, liver, breast, colorectal, urinary tract and female reproductive organs) is increased in diabetic patients. diabetic patients are exposed to oxidative stress which plays a pivotal role in the pathogenesis of both micro-and macro-vascular complications. this is due to a decreased antioxidant capacity and chronic exposure to increased levels of reactive oxygen species (ros). since the insulin resistance in dm leads to hyperinsulinemia we studied the cellular consequences of the elevated insulin level and showed that it generates superoxide anions (o -) and dna damage by a nadph oxidase dependent mechanism in cultured cells. in addition, we found elevated genomic damage in the lymphocytes of diabetic patients as well as oxidative stress and genomic damage in kidneys of diabetic rats. this effect of insulin may contribute to the pathogenesis and progression of dm complications including the elevated cancer risk. the classical transient receptor potential (trpc) channel subfamily is regarded as nonselective, calcium permeable cation channels involved in a wide range of physiological events that require calcium signaling. until now, the specific roles of trpc channels in neuronal function are still elusive. given that trpc is able to form receptor-operated heterotetrameric channel complexes with other trpc channel subunits, we investigated the role of trpc for receptor-operated calcium influx in the heterologous expression system as well as in neurons. for this electrophysiological whole-cell measurements, fluorimetric calcium measurements, mn + quenching and qpcr analysis were applied. furthermore, the effect of trpc knock-down on neuronal migration was monitored performing scratch assays, videomicroscopy and g-actin/f-actin assays. employing these techniques, we found that recombinant trpc was not able to function as a homomeric channel. instead, trpc subunits formed functional receptor-operated heteromeric channel complexes with trpc , , , , and . heteromers containing trpc subunits showed significantly decreased calcium permeation in heterologous cell systems. mutation of amino acids in the putative pore forming region of trpc further reduced calcium permeability. in gnrh neurons endogenously expressing trpc , , , and , downregulation of trpc by shrna resulted in increased basal cytosolic calcium concentrations and elevated calcium permeability. trpc was not involved in store-operated cation influx in gnrh neurons. moreover, trpc suppressed the migration of gnrh neurons without affecting cell proliferation. these findings suggest a novel regulatory mechanism relying on the expression of trpc and the subsequent formation of heteromeric trpc channel complexes with reduced calcium permeability, thereby fine-tuning neuronal migration. the transient receptor potential melastatin- (trpm ) is a calcium permeable nonselective cation channel that can be activated by the neurosteroid pregnenolonesulfate (pregs) or heat. trpm is expressed in various tissues, including insulin-secreting βcells and a subset of sensory neurons from dorsal root (drg) and trigeminal ganglia. the ability of pregs to evoke trpm -like currents in pancreatic β-cells and to induce insulin secretion indicated its involvement in blood glucose regulation. however, trpm -/mice show so far no metabolic deficits but further investigations are recommended to evaluate its function in insulin secretion. further studies showed that trpm is a nociceptor channel involved in sensing heat and inflammatory thermal hyperalgesia. we performed a calcium-based screening of a compound library (spectrum collection) that identified several natural compounds as trpm blockers. the most potent blockers were the citrus fruit flavonoids hesperetin and naringenin as well as ononetin, a chalcon from ononis spinosa. the ic values of the substances are in the low micromoles ranges. electrophysiological whole cell measurements as well as calcium measurements confirmed the potency of the trpm blockers. furthermore, we could show that these blockers are effective on endogenous trpm in drg neurons from mice and isolated β-cells. by drinking grapefruit juice naringenin could be consumed in concentrations that are sufficiently high enough to block trpm activity in vivo. in sensory neurons, trpm may exert similar functions as trpv . thus, trpm blocker could bear a therapeutic potential for analgesic treatment. xtt-based cell viability assay was used to determine the half-maximum effect concentration (ec ) for the investigated composite components in hgf. following concentrations of substances were used to determine the induced double strand dna breaks (dsbs): / × ec , / × ec , / × ec , and × ec . each experiment was performed at least four times. hgf were incubated with various concentrations of substances for a period of hours. induced dna double-strand breaks (dsbs) were tested by the γh ax focus assay, which is a direct marker for dsbs using anti γh ax antibodies. for quantitative γh ax analysis foci in cell nucleus were counted by eye down using a fluorescence microscope. each experiment was performed at least four times. in the xtt test following ec values of substances were found (mmol/l;mean +/-sem): tmp(eo) ta a, b . ± . ; , -hddma b, c . ± . ; etma a, c ; . ± . ; a significantly (p < . ) differently to , -hddma, b significantly (p < . ) differently to etma, c significantly (p < . ) differently to tmp(eo) ta. after six hours of exposure with tmp(eo) ta at . mm there were induced . γ-h ax foci-formations in hgfs, at . mm . foci, at . mm . foci and at . mm . foci. after exposure with , -hddma at . mm there were induced . γ-h ax foci, at . mm . foci, at . mm . foci and at . mm . foci. after exposure with etma at . mm there were induced . γ-h ax foci, at . mm . foci, at . mm . foci and at mm . foci. the negative controls dmso and medium cultures displayed . - . γ-h ax foci/cell. it was found that the induction of foci/cell were concentration-dependet for all xenobiotics in the order of: , -hddma > tmp(eo) ta > etma. these results show that dental composite components can induce dsbs in primary oral cells and therefore these substances demonstrate a genotoxic potential. effects of antioxidants on the dna-toxicity of dental (co)monomers in human gingival fibroblasts styllou p. , scherthan h. unreacted (co)monomers can be released from restorative dental materials and may show biologic activity after ingestion in the human organism. in previous studies the mutagenic/carcinogenic effect of dental monomers/co-monomers (e.g. methacrylates) on the human dna was demonstrated. in this study the effects of the antioxidants vitamin c and n-acetylcysteine on the dna toxicity of the (co)monomers triethylenglycol-dimethacrylate (tegdma) and -hydroxyethyl methacrylate (hema) was investigated. the induction of dna double-strand breaks with (co)monomers alone and in combination with antioxidants was investigated in human gingival fibroblasts (hgf). hgf were incubated with substances without or with antioxidants for a period of hours. induced dna double-strand breaks (dsbs) were tested by the γh ax focus assay, which is a direct marker for dsbs using anti γh ax antibodies. for quantitative analysis of the γ-h ax test, foci were counted by the same investigator by eye down the fluorescence microscope. each experiment was performed at least four times. the halfmaximum effect concentration ec (mmol/l) of triethylenglykol dimethacrylat (tegdma) and -hydroxyethyl methacrylat (hema) was taken from of a previous study after using xtt-based cell viability assay. tegdma induced significantly (p < . ) higher dsbs compared to hema ( . ± . vs . ± . ). the mean number of cells scored and the standard deviation (sd) were calculated. when cells were exposed to tegdma in combination with the antioxidant vitamin c an increase of dsbs was observed ( . ± . ), compared to tegdma alone. when cells were exposed to hema in combination with vitamin c an increase of dsbs was observed ( . ± . ), compared to hema alone. when cells were exposed to tegdma in combination with the antioxidant nacetylcysteine a decrease of dsbs was observed ( . ± . ), compared to tegdma alone. when cells were exposed to hema in combination with n-acetylcysteine a decrease of dsbs was observed ( . ± . ), compared to hema alone. these results show that dental (co)monomers can induce dsbs in primary oral cells. it also shows for the first time that the genotoxic potential may be reduced by the addition of the antioxidant n-acetylcysteine. purpose: we aimed to investigate the role of superoxide and peroxynitrite generated by genetically destabilized enos for the development of endothelial dysfunction and vascular remodelling. methods: a mutant of bovine enos in which cys was replaced by ala (c a) resulting in destabilization of enos has been generated (enos-c a). transgenic mice carrying c a were generated on a c bl/ background using the endotheliumspecific tie- promoter. by breeding these mice with enos knockouts (enos-ko), mice that express enos-c a (enos-ko/enos-c a-tg) exclusively in the endothelium were obtained. unilateral common carotid artery ligation experiments were performed in c bl/ , enos-ko, and enos-ko/ enos-c a-tg to study a role of destabilized enos for vascular lesion formation. results: western blot analysis confirmed the expression of enos in enos-ko/enos-c a-tg in aorta ( . ± . %, n= ), skeletal muscle ( . ± . %, n= ) and myocardium ( . ± . %, n= ) and revealed an increased phosphorylation of enos on ser / ( ± %) as compared to c bl/ (p< . , n= ). endothelium-specific overexpression of destabilized enos induced a large increase in superoxide and peroxynitrite formation in the aorta and the heart of enos-ko/enos-c a-tg (p< . , n= - ), which was abolished by nos-inhibitor l-nitroarginine (l-na) suggesting enos-c a as a source of elevated radical generation. endothelium-specific introduction of enos-c a at ~ % of c bl/ level almost completely restored aortic endotheliumdependent relaxation. experiments with l-na, soluble guanylyl cyclase inhibitor odq, peg-catalase and no-scavenger fe(detc) indicated that endothelium-dependent relaxation in enos-ko/enos-c a-tg is nos-and cgmp-dependent and nomediated. four weeks after the carotid artery ligation, neointima formation, media thickening and luminal narrowing were observed in the ligated arteries of all studied genotypes (p< . , n= - ). consistent with vasoprotective roles of enos, neointima formation was accelerated in enos-ko (n= - , p< . ). despite significantly higher vascular levels of nitrotyrosine and peroxynitrite, neointima formation in enos-ko/enos-c a-tg was substantially lower then in enos-ko and tended to be similar to c bl/ . conclusions: increased vascular superoxide and peroxynitrite formation caused by destabilization of enos does not induce endothelial dysfunction in healthy mice and has negligible effect on neointima formation. fenton reactivity as a determining parameter for the interaction of manganese oxide nanoparticles with lung epithelial cells sydlik u. , bieschke c. nanoparticles consisting of manganese oxide have been suggested for several innovative technological approaches, including the use in nanomedicine and diagnostics. therefore, the interaction of such nanoparticles with human target cells is of particular interest for the success of nanomedical approaches but also with regard to unintended side effects. to address this problem, we tested different kinds of manganese nanoparticles (mnnp) in an in vitro system which we earlier evaluated for proliferative, apoptotic, and pro-inflammatory endpoints induced by carbon nanoparticles (cnp). mnnp were synthesized by hydrothermal treatment of manganese salt solutions. the particles were subsequently characterized by scanning electron microscopy and dynamic light scattering. biological and toxic effects of the generated particles were studied in comparison to carbon nanoparticles (cnp) in experiments with rat and human lung epithelial cells (rle- tn and hbe o-). cytotoxicity was determined as measures of membrane damage (lactate dehydrogenase release) and metabolic activity (water soluble tetrazolium conversion). the oxidative capacity of the particles as well as the generation of intracellular oxidative stress was monitored using dichlorofluorescein diacetate in cell free experiments and flow cytometry assays (facs), respectively. the particle-specific phosphorylation of src family kinases (sfk) and mitogen activated protein kinases erk / were investigated using western blot techniques. after physico-chemical characterization, a set of three mnnp consisting of mn o or mno with significant differences in size and shape were selected. according to the different oxidation stages of manganese, the particles showed significant differences in fenton reactivity in the cell free system. these data did not reflect the capacity of the particles to induce intracellular oxidative stress. the characteristic to trigger membranedependent signaling processes, however, was correlated to the intrinsic oxidative capacity of mnnp than to the ability to induce intracellular ros. furthermore, the metabolic activity (wst) was negatively correlated with intracellular ros, indicating a link between mitochondrial activity and ros generation. none of the particles had effects on the membrane integrity of the cells. the data demonstrate that mnnp, unlike other poorly soluble nanoparticles (e.g. cnp), mainly trigger adverse health effects through ros production via the fenton reaction. acute ozone induced airway inflammation does not effect resting human sympathetic nerve traffic tank j. numerous mediators released in inflammatory and neuropathic pain states activate gprotein-coupled receptors (gpcrs) and modulate nociception via activation of gs, gi/o, g / , or gq/ g proteins. each of the g protein-coupled receptor pathways is involved in nociceptive modulation and pain processing, but the relative contribution of the individual signaling pathways in vivo has not yet been worked out. the gq/ signaling branch is of particular interest in pain research because it leads to the activation of phospholipase c, protein kinase c, and the release of calcium from intracellular stores. using a conditional gene-targeting approach we generated double-deficient mice lacking gaq and ga selectively in nociceptors to investigate the contribution of the entire gq/ signaling pathway in nociceptors towards the regulation of pain. we observed that mice lacking gq/ in nociceptive neurons show normal development of the nociceptive circuitry. the nociceptor-specific loss of gq/ results in reduced pain hypersensitivity following paw inflammation or spared nerve injury. surprisingly, our behavioral and electrophysiological experiments also indicated defects in basal mechanical sensitivity in gq/ deficient mice, suggesting a novel function for gq/ in tonic modulation of acute nociception. patch-clamp recordings revealed changes in voltagedependent tetrodotoxin-resistant and tetrodotoxin-sensitive sodium channels in nociceptors upon a loss of gq/ , whereas potassium currents remained unchanged. our results indicate that the functional role of the gq/ branch of g-protein signaling in nociceptors in vivo not only spans sensitization mechanisms in pathological pain states, but is also operational in tonic modulation of basal nociception and acute pain. provocation of arrhythmic events in single primary isolated adult mouse ventricular cardiomyocytes tekook m., fehrmann e., schulte j. s., schmitz w., müller f. u. westfälische wilhelms-universität institut für pharmakologie und toxikologie, domagkstraße , münster, germany ap duration and ca + cycling are altered in cardiomyocytes of different genetic mouse models. here, we systematically tested various protocols to study the inducibility of arrhythmic events in mouse cardiomyocytes. adult ventricular cardiomyocytes were isolated from wildtype (wt) mice by enzymatic digestion and subsequently tested to trigger arrhythmic events within hours after isolation. in patch clamp experiments (perforated patch, whole cell current clamp) aps were stimulated for second ( hz; hz; hz + s -stimulus after - ms) followed by a s rest period. the resting-membrane-potential (rmp) was observed over cycles. we observed rmp-fluctuations of different length (amplitude < mv; % of observed cardiomyocytes, mean events/cell; < s: %, . ; < s: %, . ; > s: %, . ), spontaneous depolarizations (> mv; %, . ) and spontaneous aps ( %, . ). intracellular ca + transients and sarcomere shortening were measured after loading cardiomyocytes with indo- /am. after preconditioning ( min/ hz) cells were measured under basal and continuous isoprenaline ( - m) stimulation (iso). a min pacing period was followed by a min interval of no pacing. pacing frequency was reduced after each cycle ( hz, . hz, . arrhythmic events were provocable with stimulation/rest protocols both by field stimulation and direct stimulation via patch pipette. however, low stimulation frequencies seem to lead to distinct destabilization of cardiomyocytes probably due to ca + overload. we conclude that the tested stimulation protocols are able to provoke arrhythmic events even in wt single adult mouse ventricular cardiomyocytes and may serve as a tool to test for the relevance of potential proarrhythmic substrates in mouse models. ruhr-universität bochum pharmakologie ma nord , bochum, germany cgmp is a second messenger involved in many (patho-)physiological processes such as smooth muscle relaxation, platelet inhibition, and the development and plasticity of the nervous system. however, it is not fully understood how cgmp regulates these and other processes on a mechanistic level. in particular, the existence and functional relevance of global and local cgmp signaling domains is not clear. recently, highly specific genetically-encoded optical biosensors for cgmp have been developed. these cgmp indicators are either based on fluorescence resonance energy transfer (fret), with cgmp-binding domains sandwiched between fluorescent proteins with overlapping spectra, or they consist of a single fluorescent protein fused to cgmp-binding domains. with these cgmp indicators, the spatiotemporal dynamics of cgmp signals, which result from the interplay between cgmp-producing guanylyl cyclases, cgmp-binding effectors, and cgmp-degrading phosphodiesterases (pdes), can be monitored in living cells. here, we report the generation of transgenic mice expressing the fret-based cgmp indicators cgi and cgi with apparent cgmp affinities of nm and nm, respectively. one mouse line expresses cgi driven by a cmv promoter in neural cells. fret experiments were performed with isolated cerebellar granule neurons, hippocampal neurons, and astrocytes. we observed nitric oxide (no)-induced cgmp transients and analyzed the capability of other agents (natriuretic peptides, glutamate) to induce cgmp responses. in another mouse line, the sm alpha promoter directs cgi expression specifically to smooth muscle cells (smcs). fret experiments have been performed with smcs isolated from aorta, bladder and colon, as well as with intact vessels in the retina and cremaster muscle of transgenic animals. in primary smcs we studied responses to no, atrial and c-type natriuretic peptide (anp,cnp). in different smc types we observed differences in the overall ability to react to these stimuli and in the kinetics of the induced cgmp transients. we also studied the effects of pde inhibitors on the no-, anp-, and cnp-induced cgmp signals. importantly, we were able to detect cgmp transients upon no stimulation in intact vessels of the retina and cremaster. we conclude that the cgi transgenic mouse lines are valuable tools to visualize cgmp signals in living cells in vitro and, possibly, also in vivo in the intact animal under physiological and pathophysiological conditions. current research data dealing with pharmacotherapy of α-ama intoxication shows a particularly high variability regarding the protective effect of silibinin. the aim of this study was therefore to evaluate the influence of the frequently used clinical antidotes benzylpenicillin, silibinin and their combination in human hepatocyte culture intoxicated with α-ama. cytotoxicity and apoptosis testing were performed after two and five days of simultaneously exposure to α-ama and/ or tested antidotes. to quantify apoptosis, necrosis and cell viability, we used cell death detection elisa plus®, toxilight® bioluminescence assay and cell proliferation kit ii (xtt). furthermore, we analysed the ways of apoptosis by using immunohistochemistry (differential detection of caspase , and , activated caspase , and aif). exposure of hepatocytes to α-ama at concentrations of , µm, , µm and µm resulted in disorder of cell cultures, apoptosis and reduction in cell viability compared with unexposed hepatocytes. in hepatocyte cultures treated with benzylpenicillin at concentrations of µm and mm, silibinin at µm and µm and a combination of both ( µm benzylpenicillin and µm silibinin, mm benzylpenicillin and µm silibinin), toxilight® values in the supernatant and xtt values were not significantly different from untreated cultures. simultaneous exposure to α-ama (at all tested concentrations) and benzylpenicillin, silibinin or combination of both showed higher cell viability and lower values of necrosis compared to the cultures exposed to α-ama alone (exept µm silibinin at , µm α-ama); however, in both groups dosed with benzylpenicillin the highest hepatocyte viabilitiy was observed. this protective effect was particularly revealed at high α-ama concentrations ( , µm and µm). in conclusion, our data suggest that benzylpenicillin in monotherapy is more effective than in combination with silibinin or silibinin alone. glucocorticoids (gcs) are important hormones in the regulation of metabolic homeostasis. synthetic gcs, such as dexamethasone (dex), play a fundamental role in the treatment of inflammatory diseases. there are numerous side effects of a dex therapy, e.g. the development of hypertension. in the pathogenesis of hypertension oxidative stress is a crucial factor. glucocorticoid-induced hypertension has been shown to be associated with an imbalance between nitric oxide (no) and superoxide. however, the source of this elevated superoxide production is unknown. we hypothesize that an uncoupling of the no synthase (enos), a key mediator of vascular homeostasis, may contribute to dex-induced oxidative stress. incubation of human endothelial cells (ea.hy ) with dexamethasone led to a decrease in enos expression at mrna and protein levels. this effect of dex was timeand concentration-dependent. since the major cause of enos uncoupling is a deficiency of its co-factor tetrahydrobiopterin (bh ), we analyzed the amount of bh in ea.hy by hplc. a concentration-dependent reduction of bh and also bh (dihydrobiopterin) could be demonstrated in response to treatment with dexamethasone. bh can be synthesized endogenously by two different pathways -the de novo pathway (from gtp with gtp cyclohydrolase i, gch , acting as the rate-limiting enzyme) and the salvage pathway (conversion of sepiapterin to bh involving dihydrofolate reductase, dhfr). treatment of ea.hy cells with dex decreased mrna and protein expression of both gch and dhfr. because bh is the major "coupling switch", an enos uncoupling is likely to occur in dex-treated cells. in summary, we showed that dex treatment led to a reduced availability of the important co-factor bh which could lead to enos uncoupling. the uncoupled enos may possibly contribute to glucocorticoid-induced vascular oxidative stress. the cellular oncoprotein c-fos is a major component of the heterodimeric transcription factor ap- and has been commonly found over-expressed in tumors and cancer cells of different origin. previous work showed that mouse cells lacking the immediate-early gene c-fos are hypersensitive to ultraviolet (uvc) light. here we demonstrate that in human telomerase-immortalized vh tert foreskin fibroblasts (behaving like primary cells) and sv -immortalized gm fibroblasts, uvc-triggered induction of c-fos protein is a delayed and long-lasting event. sustained up-regulation of c-fos went along with transcriptional stimulation of the nucleotide excision repair (ner) gene xpf, carrying an ap- binding site in the promoter. c-fos mrna was induced in a biphasic manner. an immediate c-fos mrna expression ( - min after exposure) was not translated into the protein, the second wave of transcription ( - h after uvc exposure) resulted in c-fos protein expression, - h post-uv. the stress-activated/mitogen-activated protein kinases (jnk, p k and erks) were immediately induced upon uvc exposure and stayed active for at least h. inhibitor experiments revealed that c-fos was phosphorylated by erks and jnk. the activation of c-fos preceded re-synthesis and the induction of xpf mrna, which was observed - h post-uvc, resulting in the increased expression of the xpf protein. cells over-expressing c-fos showed an accelerated induction of xpf mrna, and consequently a faster repair of cyclobutane pyrimidine dimers (cpds). sirna-mediated silencing of c-fos (transient c-fos knockdown) resulted in abrogated uvc-triggered induction of xpf, attenuated repair of cpds and increased apoptosis. finally, we observed that the removal of cpds but not of photoproducts was significantly faster when cells were pre-exposed to a low uvc dose, indicative of an adaptive response to dna damage. the work was financed by deutsche forschungsgemeinschaft (dfg ch / - ). the addition of clopidogrel to aspirin reduces ischemic events in patients with acute coronary syndrome and in those undergoing percutaneous coronary intervention (pci). however, recurrent ischemic event occurrence during dual antiplatelet therapy remains a major concern. variability in the pharmacodynamic response to clopidogrel is well recognized, and patients with higher platelet reactivity while receiving clopidogrel are at increased risk of ischemic cardiovascular events. clopidogrel is an inactive prodrug requiring biotransformation to form the platelet inhibiting metabolite. interindividual differences in clopidogrel metabolism are the major source of variability in antiplatelet response. polymorphically expressed cytochrome p (cyp) enzymes play a critical role in the metabolism of clopidogrel. these findings gave rise to the concept of personalized antiplatelet therapy -i.e. individual platelet function testing and correction of insufficient platelet inhibition to reduce ischemic events in patients with high on-clopidogrel platelet reactivity (hcpr). gravitas was the first study to test this concept by comparing double-dose clopidogrel to standard-dose clopidogrel in patients with hcpr. gravitas failed to correct hcpr consistently in the study arm, which coupled with a low overall event rate precluded demonstrating a substantial benefit from improved platelet inhibition. the trigger-pci trial tested the effectiveness of the more potent thienopyridine prasugrel versus clopidogrel in patients with hcpr after elective pci with implantation of drug-eluting stents (des). switching from clopidogrel to prasugrel in patients with hcpr afforded effective platelet inhibition. however, given the low rate of adverse ischemic effects using contemporary des after pci in stable ischemic heart disease, the clinical utility of this strategy could not be demonstrated and the study was terminated prematurely for futility. multiple studies have shown that both heterozygotes and homozygotes for loss-offunction cyp c alleles have higher rates of adverse cardiovascular events as compared with noncarriers on approved maintenance dosing of clopidogrel ( mg qd), albeit carriage of cyp c loss-of-function alleles accounted for only a minor proportion of the variability in on-clopidogrel platelet reactivity. results of ongoing studies with antiplatelet treatment stratified by cyp c genotyping are awaited to assess the clinical benefit of this approach. the organic cation transporter novel type (octn /slc a ) represents a high affinity uptake system for carnitine. besides metabolic disease like severe system carnitine deficiency, genetic variants within the slc a gene have been associated with inflammatory diseases like colitis ulcerosa. against this background, we characterized octn expression in peripheral blood cells thereby identifying its expression in all cell types. in the present work we studied octn expression in monocytes and thp- cells as an in vitro model for this cell type. in addition we examined transcriptional regulation of the carnitine transporter in lps activated thp- and investigated the effect of carnitine and its analog mildronate on the respective cytokine response. octn expression was characterized on monocytes and thp- cells on mrna and protein level. transporter mrna expression could be shown in both cell types by realtime pcr. however, the protein expression was analyzed by western blot, flow cytometry and immunofluorescence microscopy demonstrating octn specific signals as well as a localization in the plasma membrane. following thp- cells were activated using lps ( ng/ml) for up to h, thereby indicating the expected cytokine response as demonstrated by increased tnfα ( fold induction) and il- β ( fold induction) mrna levels. in addition, octn expression was analyzed identifying an initial reduction of around % compared to untreated cells. in parallel activated thp- cells were coincubated with increasing concentrations of the octn substrate carnitine or its analog resulting in reduced cytokine release as shown by elisa for tnfα. here, the tnfα effect was diminished by % in the presence of mm carnitine. this effect does not rely on a direct neutralization of lps by carnitine since it was also present in cells only preincubated with carnitine. in the present work we could show that thp- cells represent a useful model to study octn expression and function. in addition, we demonstrate immunosuppressive effects of octn substrates like carnitine. further experiments will be necessary to identify the underlying mechanism of this observation. castor oil has been used for more than years for its laxative effects and also to induce labor in pregnant women. despite its wide-spread use, the mechanism of action remained unknown. the active metabolite of castor oil is ricinoleic acid which is released from castor oil by intestinal lipases. we have found that exposure of meg- cells to ricinoleic acid caused an increase in [ca + ]i, an effect which was dose-dependent and abolished by pretreatment of cells with pertussis toxin, suggesting the involvement of a g-protein coupled receptor. to search for a putative receptor, we determined ricinoleic acid-induced [ca + ]i increases in cells transfected with a sirna library directed against human gpcrs. in this way, we identified prostaglandin e receptors ep and ep as mediators of ricinoleic acid-induced effects. to test if ep and ep receptors mediate pharmacological effects of castor oil in vivo, we analyzed laxative effects induced by castor oil in wild-type (wt) mice, ep -deficient (ep -/-) or ep -deficient mice (ep -/-). while ep -/mice responded similarly to the wt mice, ep -/animals were totally insensitive to castor oil-induced laxation. moreover, mice lacking the ep receptor only in the smooth muscle cells did not respond to castor oil, in contrast to mice which lack ep receptor only in epithelial cells of the intestinal mucosa. similarly, ricinoleic acidinduced contractions of isolated ileal segments were absent in segments lacking ep , consistent with a preferential expression of the ep receptor in the longitudinal muscle layer of the intestine. also, ricinoleic acid-induced contractions of isolated uteri were dependent on the expression of ep receptor in the myometrium. these findings identify the cellular and molecular mechanism underlying the effects of castor oil and indicate a role of the ep receptor as a pharmacological target to induce laxative effects. introduction: patients seek health information from various sources. they are facing the challenge to differentiate between reliable and untrustworthy sources and at the same time identify the best drug therapy for them. furthermore generalised health information confuses more than they benefit or rather unsettle. patients are not necessarily qualified to assess the evidence of statements properly. there is thus a need for providing competent drug information, which is offered by the independent drug information service at the institute of clinical pharmacology in dresden, germany. for the present descriptive evaluation we selected drugs (arimidex ® , cipralex ® pentalong ® , onbrez ® and pradaxa ® ), that were affected by new referenceprice formation, generic registration, warnings or directions in . in specified time frames we assessed the increase in and the cause of enquiries. deductively we draw conclusions for a perspicuous presentation of patient information. since generic registrations of the aromatase inhibitor arimidex ® enquiries on side effects of this drug were stable, but additional consultations were held on generic changeovers. the antidepressant cipralex ® as well as the long-acting β-agonist onbrez ® were assigned to reference-price groups, which resulted in an -times (cipralex ® : → ) and -times (onbrez ® : → ), respectively, increase in enquiries. main aspect was to give background information on reference prices and point out therapeutic alternatives (cipralex ® : of ; onbrez ® : of ). an additional amount of conversations were carried on the fictive registered drug pentalong ® after health insurance companies advised practitioners to avoid recourse by not prescribing this organic nitrate. notable insecurity was aroused by media reporting on lethal bleeding after taking pradaxa ® for anticoagulation. every tenth enquiry in the evaluation period was focussing on these instigative reports ( of ). patients are confronted by current changes, but often do not get enough background information from their health care providers to become acquainted with the tidings. health seekers may find eligible data from media coverage. however the individual assessment as well as the risk-benefit-relation may not be feasible for them. the drug information service for patients is a convenient helpline to reduce lack of knowledge and uncertainties and therefore support shared decision making. insulin effects on hyaluronan production -a possible link between diabetes and cancer? twarock s., fischer j. w. institut für pharmakologie und klinische pharmakologie, universitätsklinikum der heinrich-heine-universität düsseldorf, moorenstraße , düsseldorf, germany background: epidemiological studies have shown an elevated incidence of certain tumor entities in diabetes type and type patients. to reveal the underlying mechanisms we focused on the effects of increased glucose uptake in cancer cells with respect to matrix production. abundant production of hyaluronic acid (ha) in the vicinity of gastrointestinal cancer cells is a hallmark in tumor development. esophageal cancer is a rare but severe kind of gastrointestinal cancer which is differentiated in adenocarcinoma and squamous cell carcinoma (scc) of the esophagus. we studied the effects of increased glucose levels on ha production in an scc cell line (osc ). in starving and full media, elevated glucose concentrations increased the production of ha secreted to the medium in h as measured by an ha-binding protein linked assay (starved, g/l glucose: ± . %; g/l: . ± . %; g/l: . ± . %; full medium ± . %; . ± . %; . ± . %). surprisingly, total ha concentrations were about . - . fold higher under starved conditions. to investigate whether this effect might be due to insulin actions, starved cells were treated with mg/l insulin for h. we observed a dosedependent decrease in ha production following insulin treatment (control vs insulin, g/l glucose: ± . % vs . ± . %; g/l: ± . % vs . ± . %). this finding might suggest that insulin directs glucose usage to the glycolytic pathway thereby diminishing ha synthesis. a premise to this assumption is the ability of osc cells for insulin independent glucose uptake. to verify this thesis, mrna expression levels of insulinindependent and insulin-dependent glucose transporters (glut , glut ) were analyzed by qrt-pcr. the relative abundance was . ± . in favor of glut indicating the presence of insulin-independent glucose transport. conclusion: in osc the absence of insulin actions caused increased ha production which might be due to diminished insulin driven glycolysis, thus leading to the use of early glucose metabolites for ha production instead of energy gain. this finding could be important in the context of diabetes type , where insulin actions are also diminished because of insulin resistance. since increased ha production is of critical importance for cancer growth and spread, the cellular shift in glucose usage from glucose catabolism to ha anabolism could therefore indicate a possible link between diabetes type and cancer progression. schwarz m., unterberger e. universtität tübingen, institut für klinische und experimentelle pharmakologie und toxikologie abteilung toxikologie, wilhelmstraße , tübingen, germany chemical hepatocarcinogenesis is a multi-stage process triggered by an intitiating mutation in a gene encoding an important cell-regulatory protein. tumour initiation may be caused by genotoxic substances which directly interact with the dna, causing mutations. cells carrying permanent mutations experience clonal expansion which may be accelerated by exposure of the experimental animals to tumour promoters during the following step of tumour promotion. it has been shown that substances which constantly activate certain nuclear receptors act as tumour promoters in rodent liver, such as the model tumour promoter phenobarbital which, amongst others, activates constitutive androstane receptor (car). since these tumour promoters do not seem to directly interact with dna causing mutations they can be regarded as non-genotoxic carcinogens. however, the molecular mechanisms of non-genotoxic carcinogenesis are still widely unknown which also poses a major problem in preclinical drug-development. the aim of the marcar (biomarkers and molecular tumour classification for nongenotoxic carcinogenesis) project is to establish early biomarkers for non-genotoxic carcinogenesis by creating a comprehensive molecular profile of tumours generated by a regimen including model tumour promoters such as phenobarbital. the ultimate aim is to differentiate spontaneous liver tumours from tumours generated by non-genotoxic carcinogens. this molecular profile includes mutational analyses, immunostaining for known tumour-specific markers, phospho-proteome analyses, genome wide and promoter-specific dna methylation analyses, as well as mirna analyses. mutation analyses were carried out with mouse and rat tissue from phenobarbital promoted liver tumours to identify mutations which phenobarbital provides a growth advantage for. furthermore, real time pcr measurements show that the expression of a particular non-coding rna and mirna precursor is up-regulated in tissue isolated from phenobarbital promoted mouse liver tumours. additional in-situ-hybridisation experiments demonstrated the localisation of this transcript in ctnnb -mutated tumours. larch-derived diterpenes are potent and selective trpc blockers urban n. , kübler w. the transient receptor potential channel trpc is a poorly ca + -selective cation channel that is activated by the membrane-resident second messenger diacylglycerol (dag). consistent with the major sites of trpc expression, its activation has been implicated in pulmonary and renal diseases, such as pulmonary hypertension, lung edema, chronic obstructive lung disease, allergic airway disease, and focal segmental glomerulosclerosis. amongst various plant extracts, conifer oils and resins are traditionally used to treat pulmonary ailments. therefore, we reasoned that they may contain constituents with a biological activity to modulate trpc activity. the true turpentines, oils and resins of various coniferous genera were tested with respect to a possible inhibition of dag-or receptor-induced activation of trpc and trpc . indeed, turpentines and resins, but not coniphere oils blocked trpc and trpc in a concentration-dependent manner. interestingly, the larch-derived turpentine exerted a trpc -prevalent inhibition. we identified larixol and its mono-and diacetates as the specific compounds that are contained in larch resin and give rise to a trpc -selective block. larixol acetates displayed an ic towards the dag-or receptor-stimulated trpc activity of about . - µm, but did not strongly inhibit a number of other trp channels, including trpv , trpm , trpm , trpm , or trpa . selectivity for trpc compared to its closest relative, trpc , was about -fold. unlike conipherous oils, which contain toxic pinenes, the resin constituent larixol ant its acetates exerted no significant cellular toxicity at concentrations that are required to block trpc . electrophysiological analysis confirmed the highly potent block, which was voltageindependent and reversible. in a murine hypoxia-induced pulmonary vasoconstriction (hpv) model, larixol acetate abrogated the euler-liljestrand mechanism and, thus, mimicked the phenotype of trpc -/mice. we conclude that trpc blockers and, more specifically, larixol-related derivatives may provide novel therapeutic strategies to treat or prevent pulmonary diseases. dioxin is an environmental contaminant, believed to affect basic biological equilibria such as calcium and iron homeostasis. however, the molecular mechanisms underlying these effects are still largely unknown. this strongly hampers the estimation of the hazard to humans associated with dioxin exposure and necessitates further studies aimed at the clarification of these mechanisms. it has been suggested that nearly all biological and biochemical processes are mediated by protein complexes. the most commonly used technology for monitoring changes in the expression of complex protein mixtures is still d gel electrophoresis, but this method suffers from poor expression of low or moderately abundant proteins. blue native page and subcellular fractionation form an ideal partnership when it comes to enrichment and analysis of intracellular organelles and low abundant multiprotein complexes. the aim of the study is to identify and characterize multiprotein complexes by blue native page to elucidate the network of protein-protein interactions that regulate protein function after dioxin exposure. sample preparation and subcellular fractionation rt cells were cultured in mccoy's a medium. cells at confluence were harvested and fractionated into cytosolic, membrane/organelle and nuclear fraction by using the proteoextract subcellular proteome extraction kit. first dimension (bn-page) mg of protein sample was mixed with % of coomassie blue g- (cbb g- ) and loaded in each lane of - % polyacrylamide native gradient gels. the lanes from the first dimension were cut into individual strips and were placed into a % sds gel. the gels were stained with coomassie and the spots were picked up for mass spectrometry. bn/sds-page combined with ms led to the identification of proteins involved in the regulation of both calcium and iron homeostasis in dioxin-exposed cells. these results demonstrate for the first time that dioxin exposure simultaneously affects calcium and iron metabolism. since important iron and calcium requirement changes occur during the regulation of cell growth, the protein expression changes observed in our study may be associated with dioxin-dependent cell-fate decisions. the murine protease inhibitor serpina n inhibits mechanical allodynia in a model of neuropathic pain vicuna l. , , simonetti m. several chronic diseases are accompanied by strong, long-lasting pain. a majority of chronic pain diseases are not well understood yet and cannot be controlled by conventional analgesics or non-pharmacological approaches. therefore, there is a major need to develop novel therapeutic principles. using a genetic screen, we identified serpina n, a serine protease inhibitor, which is homologous to human a -antichymotrypsin, to be a determinant of low neuropathic pain. we found that serpina n is expressed in the dorsal root ganglia (drg) and spinal cord and that it is upregulated in these tissues in mice developing neuropathic pain. importantly, we observed that spinal delivery of recombinant serpina n inhibits mechanical allodynia in a mouse model of neuropathic pain. we identified a novel serine protease substrate for serpina n, which is upregulated in the spinal cord in mice undergoing neuropathic pain ('enzyme e'). recombinant enzyme e delivered intrathecally to the spinal cord of mice elicited rapid and long-lasting allodynia, which was fully blocked by concomitant administration of serpina n. our results suggest that serine protease-serpin signaling modulates spinal neuronal and glial cell networks involved in processing pain and that activity-induced spinal release of serpina n constitutes an endogenous defence mechanism against establishing chronic pain hypersensitivity. these data have important implications for the pathophysiology of pathological pain and potentially hold therapeutic relevance. gβγ subunits are involved in β-adrenergic receptor induced cardiac hypertrophy vidal m., lohse m. j., lorenz k. institut für pharmakologie und toxikologie pharmakologie, versbacher str. , würzburg, germany introduction. activated β -adrenergic receptors and their g protein gαs induce the development of cardiac hypertrophy. however, the hypertropic effects of direct activation of downstream effectors, such as adenylyl cyclase, camp or pka, are controversely discussed. recently, a hypertrophic pathway involving a g protein βγ subunit induced phosphorylation of the mitogenic kinases erk / at threonine (erk thr phosphorylation) has been described to mediate erk-induced hypertrophy. this study aims to investigate whether erk thr -phosphorylation is involved in cardiac hypertrophy triggered by β-adrenergic receptors. -phosphorylation was detected in hek cells overexpressing β -receptors, murine hearts and neonatal rat cardiomyocytes after isoprenaline treatment. we performed [ h]-isoleucine incorporation assays to assess cardiomyocyte hypertrophy in vitro. neonatal rat cardiomyocytes (nrcms) overexpressing wild-type erk showed a significant increase in [ h]-isoleucine incorporation after isoprenaline treatment. in contrast, nrcms transfected with erk thr -phosphorylation deficient mutants (erk t a and erk t s ) or pretreatment with the erk inhibitor, pd , significantly attenuated cardiomyocyte hypertrophy. for in vivo studies, isoprenaline was given subcutaneously for days to wild-type mice and transgenic mice overexpressing either wild-type erk t t or erk t s . echocardiography and histological analyses revealed that erk t s mice developed less left ventricular hypertrophy than control mice. hypertrophic target proteins of erk (e.g. elk ) are located in the nucleus. western blot and confocal microscopy analyses showed that overexpressed erk t a or erk t s are retained in the cytosol and prevented elk -phosphorylation after isoprenaline stimulation. co-immunopreciptation assays in hek cells and nrcms underlined the direct involvement of g protein βγ/erk interaction upon isoprenaline stimulation. in line with this finding, direct activation of adenylyl cyclase by forskolin did not lead to gβγ induced erk thr -phosphorylation. conclusion. taken together, gβγ-subunits participate in β -adrenergic receptor mediated hypertrophy by enhancing erk thr -phosphorylation. these findings add important insight to the molecular signaling of g proteins in cardiac hypertrophy. the protein tyrosine kinase src and its role upon alpha-toxin stimulation of human platelets vogel k. , burke m. introduction: alpha-toxin, a kda calcium pore forming exotoxin, is a major virulence factor in the pathogenesis of staphylococcus aureus infections. alpha-toxin affects human blood cells such as platelets and induces aggregation that is accompanied by multiple changes in platelet protein tyrosine phosphorylation and dephosphorylation ( ). in the present paper, we focused our interest on the protein tyrosine kinase src, the most abundant member of the src-family kinases present in platelets ( ) . by the use of various inhibitors, we studied src and its role in α-toxin-induced platelet aggregation. methods: isolated human platelets from healthy volunteers were stimulated with α-toxin in the presence or absence of the src-family member inhibitors pp , pp or su (referred as src inhibitors). src and autophosphorylation of src were analyzed by sds-page and western blotting using specific antibodies against src and tyr- -phospho-src from calbiochem and cell signaling, respectively ( ). furthermore, calpeptin, an inhibitor of the calcium-dependent protease calpain, was used. platelet aggregation was measured by the method of born. staphylococcal α-toxin induced platelet aggregation in a concentration-dependent manner ( . - . µg/ml of toxin). pre-incubation with src inhibitors (pp , pp or su ) reduced α-toxin-induced platelet aggregation by about %. similar inhibitory effects have been observed by the use of calpeptin that acts as an inhibitor of the src degrading protease calpain. with respect to src itself, a-toxin induced autophosphorylation at tyr- followed by a fast and complete dephosphorylation within min. while calpeptin modified the time course of dephosphorylation, only little effect of the src inhibitors has been seen on tyr- phosphorylation/dephosphorylation. the typical calpain-dependent degradation of src can be blocked by calpeptin ( µm), but also by depletion of extracellular calcium indicating that it is a calcium-dependent process. conclusion: taken together, our data demonstrate that α-toxin of staphylococcus aureus induces platelet aggregation accompanied by src degradation and autophosphorylation at tyr- typically observed in activated platelets. inhibition of the cellular tyrosine kinase src as well as the protease calpeptin reduces aggregation indicating an important role of src and/or other src-family members in α-toxin-induced platelet stimulation. the five subtypes of muscarinic acetylcholine receptors belong to the superfamily of gprotein coupled receptors. the even-numbered subtypes m and m prefer coupling to gi proteins, whereas the odd-numbered receptors m , m and m prefer coupling to gq proteins. with respect to ligand binding and m receptor activation, the conserved epitope trp . at the beginning of tm displays remarkable functional features. it is located at the junction between the orthosteric and the allosteric binding site of the m receptor [ ] . in the inactive m receptor, it provides subtype-independent baseline affinity for allosteric antagonists [ ] . in the active receptor, m trp . affords binding affinity for the full agonist acetylcholine and intrinsic efficacy for the partial agonist pilocarpine [ ] . to study the role of trp . for m receptor activation, agonist-induced formation of dmyo-inositol-monophosphate was measured in cho-cells transfected with the respective human receptor-cdna. surface receptor expression measured by radioligand binding was similar in hm wild-type-cells and hm trp . →ala-cells, amounting to . and . x receptors per cell, respectively. the intrinsic efficacy of acetylcholine was not influenced at m trp . →ala relative to m wild-type, whereas potency was reduced about tenfold. these findings resemble those made previously in m and the corresponding mutant. in the case of pilocarpine, replacement of trp . by alanine in m did not reduce intrinsic efficacy. this finding is in contrast to m , where the corresponding mutation induced a loss of pilocarpine's intrinsic efficacy. the potency of pilocarpine was diminished about tenfold at the m trp . →ala mutant relative to m wild-type. this finding is also in contrast to m , at which pilocarpine's potency was not sensitive to the trp . →ala mutation. taken together, the diverging sensitivity of pilocarpine to the trp . →ala mutation between the m and the m receptor suggests that the role of this epitope for receptor function may differ between even-and odd-numbered muscarinic acetylcholine receptors. in vivo experiments for inhalation toxicity are time and animal consuming. thus several in vitro methods aim to replace or reduce and refine the in vivo experiments. human dtissue models are commercially available reconstructed from different donors (normal, smokers, chronic obstructive pulmonary diseases), which show a normal human bronchiole tissue that reveals a pseudostratified epithelial structure, numerous microvilli and cilia on the apical surface of the cultures. the presence of tight junctions and mucus secretion has also been confirmed comparable to the in vivo situation. these d-models are cultured on a porous membrane as air-liquid interface. test substances can be applied apically, either as solution or with an aerosol-inducer. in our in house validation to test the strengths, handling and reproducibility of such dmodel systems as well as determining the correlation between in vivo inhalation data, we have assessed the epiairway tm model from mattek, usa. a set of substances were selected with known in vivo toxicity data and mode of action. the substances were tested in the epiairway model an in parallel, in t and a cell lines to assess putative unspecific cytotoxic effects of the test substances. a comparison of toxicity data from the d-model and the in vivo data revealed, that the model is only predictive of respiratory toxicity in vivo for a subset of substances with specific modes of action. the epiairway tm model has proven to be robust, showing high reproducibility between pre-and main-tests as well as in the concurrent controls but it will need a strict definition of its applicability domain or further development of the test protocol to achieve a wider applicability. remodeling of intracellular ca + handling and cyclic amp-dependent signaling in atrial myocytes from patients with chronic atrial fibrillation. voigt n. background: in atrial myocytes ca + entry through l-type ca + channels (ica,l) triggers a larger ca + release (ca + transient,cat) from the sarcoplasmic reticulum activating contractile myofilaments. reduced ica,l is a hallmark of atrial remodeling in chronic atrial fibrillation (caf) and is supposed to contribute to action potential shortening and contractile dysfunction. however, the coupling efficiency between ica,l and cat and its regulation by camp-dependent signaling in caf patients are unexplored. methods: ica,l (voltage-clamp) and cat (fluo- ) were measured simultaneously in rightatrial myocytes from sinus-rhythm (ctl) or caf patients. a saturating concentration of the non-selective β-adrenoceptor (ar) agonist isoprenaline (iso, µm) and the nonselective phosphodiasterase (pde) inhibitor -isobutyl- -methylxanthine (ibmx, µm) were used to increase cellular camp content. camp content was assessed with immunoassay. results: in caf amplitudes of ica,l ( . ± . pa/pf, n= / [myocytes/patients] vs . ± . pa/pf, n= / , p< . ) and cat ( . ± . nm vs . ± . nm, p< . ) were lower than in ctl myocytes, whereas diastolic [ca + ]i levels were unchanged (caf, . ± . nm; ctl, . ± . nm). the coupling efficiency between ica,l and cat was similar in ctl and caf. application of iso increased ica,l amplitude to . ± . pa/pf (n= / ) in caf and to . ± . pa/pf (n= / ) in ctl. the corresponding cats increased to . ± . nm in caf and to . ± . nm in ctl. although the amplitudes of ica,l and cat also increased after pde inhibition with ibmx, the magnitude of these increases was smaller than the iso-induced enhancements. both iso and ibmx had no effect on diastolic [ca + ]i and coupling efficiency. however, the relative iso-induced increases in ica,l (caf, + . ± . % vs ctl, + . ± . %, p< . ) and cat (caf, + . ± . % vs ctl, + . ± . %, p= . ) were significantly higher in caf compared to ctl myocytes and a similar tendency was found for ibmx. basal camp levels were higher in caf compared to ctl (caf, . ± . pmol/mg, n= vs ctl, . ± . pmol/mg, n= , p< . ), pointing to an increased camp-dependent signaling in caf patients. conclusions: these data point to remodeling of camp-dependent signaling in caf patients which likely contributes to the stronger relative increases of ica,l and cat amplitudes after β-ar stimulation and pde inhibition. remodeling of camp-dependent signaling might be a novel contributor to af pathophysiology. direct visualisation of g-protein-coupled receptors and heterotrimeric g-proteins using single-molecule microscopy wagner j. g-protein-coupled receptors (gpcrs) form the largest family of membrane-bound receptors and mediate the effects of several extracellular stimuli. although the basic mechanisms of gpcr signalling have been extensively studied, a full characterization of the involved protein-protein interaction is still missing, largely due to technical limitations. in this study, we developed new methods for labelling gpcrs and g-protein subunits based on snap-and clip-tags and visualise them with single-molecule sensitivity. the snap-tag is a mutant of the dna repair protein o -alkylguanine-dna alkyltransferase that reacts with fluorescent benzylguanine derivatives, whereas the clip-tag is reacting specifically with o -benzylcytosine derivatives. these tags allow labelling proteins directly in living cells with very high specificity and low background. snap/clip-tagged receptors and g-proteins were covalently labelled with small organic fluorophores and visualised by total internal reflection fluorescence microscopy, which allows to selectively illuminate only fluorescent molecules located on or immediately underneath the cell surface. particles were automatically analysed with previously published as well as newly developed algorithms. the results indicated that both receptors and gproteins, although diffusing with high speed on the cell surface (diffusion coefficients: receptors ~ . mm /s, g-proteins ~ . mm /s), can be visualised and correctly tracked. a variable fraction of receptors and g-proteins are immobile or show hop movements, possibly suggesting their interaction with cytoskeletal or other membranebound proteins. our data also suggest the feasibility of performing two-colour analyses with snap-and clip-tagged proteins aimed at directly visualizing transient interactions between receptors and g-proteins or among g-protein subunits. in-vitro screening systems are particularly well suited to preclinical toxicology testing at an early stage of drug development as they have the advantage of being fast and requiring only a small amount of test substance. the demands for in-vitro screening assays for systemic toxicity are multiple and include the need of organ specific cell systems, the use of optimal cell numbers, cell passages and incubation times. even minimal changes in the conditions of the test system may lead to significant changes of the biological system. therefore a reliable normalization compensating biological variability is crucial prior to any interpretation of results generated from a biological system. basf has developed an in-vitro metabolite profiling assay and a subsequently tuned normalization strategy allowing the prediction of specific organ toxicity. the in-vitro assay consist of exposing cells lines to test substances and to determine the metabolite profile using chromatography coupled to mass spectrometry systems. herein, we compare five different normalization strategies referring to their suitability in the application to in-vitro metabolite profiling data. the strategies comprise statistical approaches, approaches referring to reference values from each individual sample or samples generated in dependent batches. best results were achieved by an individual strategy using a new reference value correlating well over a large range of cell counts previously used for generating corresponding cell extracts. statistical analysis revealed the normalization based on the new reference value greatly improved the quality of the results compared to non-normalized samples as well as to all remaining strategies. generation and application of this new reference value and the corresponding normalization strategy will be presented the first time. validation will be featured on the basis of extracts of the human hepatocellular carcinoma cell line hep g . molecular mechanisms of the inhibitory function of rhoh in phospholipase cmediated signalling walliser c., löschmann y., ziegler v., kühne e., schilling p., rasonabe z., bühler a., vatter p., gierschik p. universitätsklinikum ulm institut für pharmakologie und toxikologie, albert-einstein-allee , ulm, germany rho gtpases are a subfamily of ras gtpases regulating diverse signalling pathways, for example those regulating the reorganisation of the actin cytoskeleton. among them, rac and rhoh show an expression restricted to the hematopoietic lineage. rhoh is constitutively active, because it carries mutations in two positions (s and n ) known to be important for gtp hydrolysis. hence, rhoh is controlled on the level of protein expression and, possibly, by tyrosine phosphorylation. rhoh has been implicated in human malignancies, since the gene is subject to somatic hypermutation in its noncoding regions and to translocation to the gene encoding laz /bcl or to other genes in human b-cell lymphomas. furthermore, rhoh is overexpressed in primary human chronic lymphocytic leukemia (cll) cells. these findings suggested that rhoh is involved in the initiation and/or progression of cll. we previously showed that rhoh acts as a potent inhibitor of both rac -mediated phospholipase c-β (plcβ ) and plcγ activation in intact cells. the aim of this study was to elucidate the molecular mechanisms of the inhibitory effect of rhoh on plc activity. the results showed that rhoh directly inhibited the activity of constitutively active variants of plcγ , plcβ , and plcδ , but that it had little or no effect on the activity of plcγ and plcε. the amino acid residues s and n , likely to be the cause for the gtpase-deficiency of rhoh, are not required for the inhibitory function of rhoh. furthermore, the switch-i and switch-ii regions of rhoh are not necessary for the inhibitory effect of rhoh, since rhoh mutants carrying switch-i or switch-ii regions of rac caused inhibitory effects on rac -mediated plcβ and plcγ stimulation indistinguishable from wild-type. interestingly, rhoh seems to interact with regions of plcγ distinct from those which are necessary for rac interaction, as the split pleckstrin homology domain of plcγ , which is essential for its interaction with activated rac , is dispensable for the inhibitory effect of rhoh. in summary, our results indicate, that rhoh acts as a plc-isozyme-specific negative regulator of the activity of plcβ and plcγ , both of which are specifically expressed in hematopoietic cells. these findings suggest a novel mechanism of plc isozyme regulation by rhoh. the results also suggest that rhoh plays an important role in b cell maturation, function, and leukemogenesis by modulating b-cell-receptor-mediated plcγ activation. effect of rac inhibition on doxorubicin mediated cell response wartlick f., fritz g. heinrich-heine-universität düsseldorf institut für toxikologie, universitätsstrasse , düsseldorf, germany background: the small gtpase rac is a well characterized member of the rashomologous (rho) family. rac is not only a key regulator of the actin cytoskeleton but also regulates the activity of nadph oxidase, stress kinases and transcription factors (e.g. nf-κb, ap ). furthermore, rac can translocate into the nucleus and interacts with topoisomerase type ii (topo ii). yet the general nuclear function of rac is still unclear. here, we address the question how rac influences the genotoxicity of the topo ii poisons doxorubicin and etoposide. methods: to study the function of rac , human hepatoma cells were pretreated with the rac -inhibitor eht before they were exposed to doxorubicin, etoposide or, for control, ionizing radiation (ir). to check the influence of rac inhibition on the outcome of genotoxin treatments, cell viability and cellular stress response were analyzed by the wst-assay, western blot (wb), co-immunoprecipitation experiments, facs-analysis and the alkaline comet-assay. results: as compared to the control, cells that have been pretreated with the rac inhibitor showed a higher viability, less phosphorylation of h ax (s ) and a reduced dna damage formation (measured by alkaline comet-assay) after treatment with doxorubicin and etoposide but not after treatment with ir. furthermore inhibition of rac resulted in a reduced phosphorylation of topo iiα (s ) and an increased interaction of topo iiα with hsp in doxorubicin treated cells. the data indicate that inhibition of rac protects human hepatoma cells against topo ii poisons due to interference with topo iiα function. the presence of drugs or other potential toxic substances in milk has enormous toxicological and nutritional consequences for consumers of dairy products. the atpbinding cassette (abc) transport protein breast cancer resistance protein (bcrp; abcg ) is expressed in alveolar epithelial cells of the mammary gland in cows, sheeps and goats. bcrp is known to play a major role in the active secretion of a variety of xenobiotics into human milk. so far there is little information about the transport activity and substrate specificity of dairy bcrp. therefore we aimed to establish a mdck cell in vitro model expressing bcrp of dairy animals. bcrp mrna was isolated from bovine, caprine and ovine mammary gland. full-length clones were generated using race (rapid amplification of cdna ends) pcr. the final full-length bovine, ovine and caprine abcg cdna-clone sequences were submitted to the ncbi genebank (eu , gq and gq ). stable transfection of bcrp in mdck cells was performed and the subcellular localization of bcrp at the apical plasma membrane was identified by confocal laser scanning microscopy. bcrp-mediated transport of the substrate hoechst was measured and the selectivity was determined by the bcrp inhibitor ko . inhibition studies using hoechst identified various drugs including the antibiotic enrofloxacin or anthelmintic agents like oxfendazole as substrates of bovine, caprine and ovine bcrp. to further characterize bcrp carrier activity, bidirectional transport studies were performed with transwell® filter inserts that allow studying drug transport between an apical and basolateral compartment. cell monolayer integrity was checked by measuring teer values as well as by measuring the paracellular flux marker atenolol by lc/ms. bidirectional transport studies with enrofloxacin were performed to characterize the bcrp transporter activity. our results may contribute to increase the understanding of carrier associated drug transport into the milk of dairy cattle and therefore enlarge consumer protection. acrolein and acrylamide: excretion of mercapturic acids after consumption of potato chips watzek n., scherbl d., berger f., feld j., eisenbrand g., richling e. technische universität kaiserslautern fachbereich chemie; fachrichtung lebensmittelchemie & toxikologie, erwin-schrödinger-str. , kaiserslautern, germany acrolein (ac) and acrylamide (aa) may be formed from food constituents during heating of food. ac is supposed to be generated via heat induced formation from glycerides/glycerol, aa is known to arise during the maillard reaction from asparagine and reducing carbohydrates. ac also has also been suggested to be formed by endogenous metabolism as a side product of carbohydrate and/or amino acid turnover or by oxidative desamination of polyamines [ ] . as an α,b-unsaturated aldehyde, ac forms , -michael-adducts with biomolecular nucleophiles, such as sulfhydryl and amino groups. in the organism, ac and aa are preferentially conjugated to glutathione and are excreted as mercapturic acids (ma), n-acetyl-s-( -hydroxypropyl)-cysteine , n-acetyl-s-(carboxyethyl)-cysteine (cema), (n-acetyl-s-( -carbamoylethyl)-cysteine (aama), and (n-acetyl-s-( -hydroxy- -carbamoylethyl)-cysteine (gama). data on human exposure to ac and its occurrence in the diet are scarce. in general, contents in heat treated foods are considered to be in the low ppb range (µg/kg) [ ] . nevertheless, in a pilot study in humans urinary -hpma excretion of non-smokers was reported to be about three fold higher, as compared to aama [ ] . in the present human intervention study we monitored the excretion of mas in five healthy volunteers (male) after ingestion of commercially available potato chips ( g), equivalent to an uptake of µg aa (absolute amount), together with an as yet unknown amount of acrolein [ ] . urinary ma contents were monitored by hplc-ms/ms following solid phase clean-up of urine for up to h after test meal uptake. the results demonstrated kinetics of -hpma and cema excretion in human urine to be clearly related to ingestion of the potato chip meal. on the basis of auc values, total excretion of -hpma plus cema exceeded that of aama plus gama by a factor of about four. the results confirm earlier findings on urinary mas, suggesting markedly higher human exposure to dietary ac / potential ac precursors than to aa. it is an as yet unresolved question, whether and to what extent concomitant substantial ac exposure may influence toxicology of such dietary heat-induced toxicants. [ ] stevens, j.f. and maier, c.s. ( ) molecular nutrition & food research ; [ ] osorio, v. m. and de lourdes cardeal, z., ( ) journal of chromatography a ; [ ] schettgen, t., musiol, a., and kraus, t., ( ) rapid communications in mass spectrometry ; [ ] ewert, a., granvogl, m., and schieberle, p., ( ) lebensmittelchemikertag protective effects of increased nad + levels in human peripheral blood mononuclear cells exposed to dna damaging agents weidele k., beneke s., bürkle a. university of konstanz molecular toxicology group, department of biology, jacob-burckhardt-str. , konstanz, germany the dna damage-activated enzyme poly(adp-ribose) polymerase (parp- ) acts as a nick sensor and modifies target proteins by covalent attachment of poly(adp-ribose) [par] using nad + as substrate. the intracellular levels of par and nad + are important parameters for biological responses to genotoxic stress and influence diverse cellular functions including dna repair or maintenance of genomic stability. notably, loss of genomic stability is a hallmark of both carcinogenesis and the ageing process. here we analysed the impact of elevated nad + levels in human blood peripheral mononuclear cells (pbmc) with regard to (i) poly(adp-ribose) formation, (ii) cell death, (iii) initial dna damage and subsequent repair, as well as the influence on (iv) genomic stability under genotoxic stress. after ex vivo supplementation of pbmc with low concentration of nad + precursor nicotinic acid (na) intracellular nad + level significantly increased up to fold in unstimulated [ ] and . fold in mitogen-stimulated cells. after dna damage infliction, parp activity was dramatically increased in supplemented cells, necrotic cell death was reduced and dna strand break repair was significantly affected. furthermore the frequency of micronuclei decreased significantly after irradiation damage, emphasizing the fundamental role of adequate nad + levels in maintaining genomic integrity. the cyclic purine nucleotides adenosine ': ' monophosphate (camp) and guanosine ': ' monophosphate (cgmp) are well-examined second messengers with many proven biological functions. in a recent study, using a highly sensitive and specific mass spectrometry method, we have shown that cyclic ': ' cytidine monophosphate (ccmp), a pyrimidine nucleotide, is naturally occurring in several mammalian cells [ ] . ccmp activates both camp-and cgmp-dependent protein kinases with low potency [ ] but the physiological function of ccmp is still very poorly understood. in an effort to delineate the function of ccmp, we analyzed expression of the early response gene egr . we chose this gene because it is regulated by numerous stimuli including camp [ ] . in our first study, we showed that dibutyryl-ccmp and ccmp failed to increase egr gene expression levels after stimulation of kb cells under various experimental conditions using real-time pcr (taqman®). we have now changed the experimental set-up using hela cells and the new ccmp analogue, ccmp-acetoxymethyl ester (ccmp-am), still focusing on gene expression of egr . esterification of the negatively charged cyclic phosphate of ccmp allows better transport of the nucleotide across the cell membrane, thus augmenting possible intracellular effects. hela cells were stimulated in cell culture medium with extracellularly applied ccmp-am ( , , , µm) over to min h after seeding. analysis of real time pcr (taqman®) experiments, using β-actin as a housekeeping gene, showed a significant increase of egr expression in a time and concentration dependent manner. these effects were specific for stimulation with ccmp-am but not the control phosphate trisacetoxymethyl ester. hela cells were also cultured in serum free resting medium (mcdb , sigma) that induces growth arrest, one to eight hours prior to stimulation. here, even higher egr expression levels through ccmp-am stimulation could be seen. these results suggest that ccmp could function as a second messenger just as camp and cgmp do. studies are in progress to further examine the mechanisms of the ccmp-am effects on egr expression in hela cells. methyl-cpg-binding protein (mecp ) recognizes methylated dna, it is involved in chromatin remodeling and it acts as a transcriptional repressor or activator. we have previously shown that expression of mecp is diminished in murine and human heart failure. prevention of mecp downregulation in transgenic mouse models aggravated cardiac hallmarks of heart failure. in patients with rett syndrome, which is caused by mutations in the mecp gene, mitochondrial function was found to be altered in the central nervous system. as the impact of mecp on mitochondrial function in the heart is unknown, the aim of the present study was to characterize the significance of mecp of cardiac mitochondria in mouse models with cardiac myocyte-specific expression or ablation of mecp . in order to investigate the cardiac function of mecp , two genetically modified mouse models were previously generated, including mice with inducible transgenic expression of mecp in cardiac myocytes under control of the tetracycline-system (mecp -tg) and mice with targeted ablation of mecp in myocytes (mecp mlccre ). these mice were analyzed under basal conditions and after chronic transverse aortic constriction (tac). at baseline, cardiac-specific overexpression of mecp did not cause any difference in cardiac function as compared to control mice using millar catheterization. isolated interfibrillar mitochondria showed a decrease in citrate synthase activity. after chronic pressure overload, the decrease in cardiac mecp expression could be completely prevented by the mecp transgene. cardiac contractility and relaxation were significantly decreased in mecp -tg animals. upon electron microscopical investigation, transgenic mecp expression was associated with a significant reduction of interfibrillar mitochondria, clustering of mitochondria in the perinuclear region and smaller mitochondrial cross sections as compared with control specimens. in contrast, cardiac myocyte-specific ablation of mecp caused a rightward shift in the size distribution of mitochondria as compared with mecp -tg hearts. epigenetic processes, including the recognition of dna methylation by mecp , may play an important role in the control of mitochondrial gene expression, structure, subcellular localization and function in the heart. thus, precise control of mecp expression and function is essential to prevent deterioration of metabolic function during chronic heart failure. normalisation of blood pressure does not prevent angiotensin ii-induced dna damage in kidney and heart of ren rats weissenberger s., hey v., lau d., schupp n. universität würzburg institut für pharmakologie und toxikologie, versbacher strass , würzburg, germany increased activity of the renin angiotensin system (ras) with enhanced levels of angiotensin ii (angii) leads to oxidative stress with endothelial dysfunction, hypertension and atherosclerosis. epidemiological studies revealed a higher cancer mortality and an increased kidney cancer incidence in hypertensive patients. we could show in vitro and in vivo that angii causes structural dna damage dose-dependently in kidney cells and in the kidney. elevated angii levels therefore might contribute to carcinogenesis of the kidney. in a model of high angii organ levels, the transgenic ren rat, carrying an additional renin gene, dna damage in the kidney was analysed in animals of and weeks. untreated ren rats exhibit increased blood pressure from the age of weeks on. therefore, the line is kept on angiotensin i converting enzyme inhibitor therapy, which normalizes blood pressure and kidney function to values of control sprague dawley rats. despite this normalized blood pressure of the ren animals, a significant higher superoxide production could be observed in kidneys already in week old animals. also a higher frequency of structural dna damage and double strand breaks could be detected in the comet assay and with an antibody against the double strand break marker γ-h ax in kidneys. further, fittingly, an increased dna repair activity exists in kidneys of ren rats compared to control rats. as another organ affected by hypertension the heart of the ren animals was analysed for oxidative dna damage. although only a marginal increase of superoxide production could be found, also in the heart a significant higher frequency of dna double strand breaks and cells positive for dna repair activity could be observed. our data let us conclude that normalization of blood pressure in a state of activated ras is not sufficient to prevent angii-induced genotoxicity. this further implies that also patients with treated hypertension still might suffer from endorgan-damaging effects of elevated angii levels. the d a pore mutation leads to complete inactivation of trpv channels in epididymis weißgerber p. , kriebs u. replacement of aspartate residue by alanine (d a) in the pore of trpv channels in mice disrupts ca + absorption by the epididymal epithelium resulting in abnormally high ca + concentrations in epididymal luminal fluid and in a dramatic but incomplete loss of sperm motility and fertilisation capacity raising the possibility of residual activity of channels formed by trpv d a proteins (sci signal , ra , ) . it is known from other cation channels that introducing pore mutations even if they largely affect their conductivity and permeability can evoke considerably different phenotypes compared to the deletion of the corresponding protein. to gain insights whether the trpv d a pore mutant still contributes to residual channel activity and/or channelindependent functions in vivo, we compared important fertility-parameters between trpv -/and trpv d a/d a mice: the fertilization rate observed in permanent matings, the in vivo fertilization rate as judged by the rate of embryos isolated from plug positive females of matings with males homozygous for either trpv mutation as well as the motility, in vitro fertilization capacity and viability of sperm were reduced to the same extent in both genotypes. also, no differences were observed in copulatory behavior between trpv -/and trpv d a/d a males. the profound reduction in ca + uptake by the epididymal epithelium was identical in trpv -/and trpv d a/d a males. this direct comparison of these parameters indicate that deleting trpv does not further aggravate the phenotype observed in trpv d a/d a mice, and -in our opinion -allows the conclusion that the d a pore mutant of the trpv protein leads to complete inactivation of the trpv channel activity or channel-independent scaffolding functions in epididymal epithelium. characterization of a naturally occurring c-terminal mutation (n i) on herg channel function in hek cells sellmaier v. , moretti a. long-qt-syndromes (lqts) are acquired or inherited disorders which predispose patients to cardiac arrhythmias and sudden death. in affected individuals, the electrocardiogram shows a prolongation in the qt interval, due to an unstable repolarization of the action potential. acquired forms of lqts are often the result of treatment with medications that block cardiac potassium channels, such as class iii antiarrhythmic drugs or antihistamines. inherited lqts are caused by mutations in cardiac ion channels. congenital long qt syndrome (lqt ) is caused by loss-offunction mutations in the human ether-á-go-go-related gene herg (also known as kcnh or kv . ). herg encodes the pore-forming α-subunit of the rapid delayed rectifier potassium current i kr, whose physiological role is to repolarize the late phase of cardiac action potentials. herg channel α-subunits exist as isoforms ( a and b) that are identical except for structurally divergent n termini. native cardiac ikr channels are tetraheteromers containing of each α-subunit types. a loss-of-function can be due to either defects in a) channel opening (gating), b) ion permeation or c) protein maturation and trafficking. we have identified a so far uncharacterized dominant missense mutation in the herg gene (n i) in a patient with lqt. both herg a and herg b subunits were cloned from a human heart cdna library and the specific n i mutation introduced by site directed mutagenesis. hek cells were transiently transfected with equal amounts of mutated herg a and wild type (wt) herg b cdnas and the resulting potassium current compared to herg a/b wt. whole-cell patch-clamp analysis showed similar current densities for wt versus mutated channels. also the voltage-dependence of activation was unchanged with a halfmaximal activation at - mv for wt and - mv for the mutated channel assembly. differences were found for the deactivation and inactivation kinetics. the deactivation was faster in the mutated channels with t fast = ms and tslow = ms versus tfast = ms and tfast = ms in wt channels, determined from the tail current at - mv after a -second pulse to + mv. the half-maximal steady-state inactivation of the tail current was shifted by mv to the depolarizing direction in the mutated channel compared to the wt. a defect in channel gating appears to be the most likely explanation. background: abcc is adjacent to p-glycoprotein the most important efflux transporter for various endogenous and exogenous compounds and is expressed at several compartment barriers. by increasing evidence it is shown that the abcc polymorphisms are of clinical significance. the aim of our study is to analyze the epigenetic regulation of distinct abcc haplotypes by the influence of micrornas. methods: abcc cdna clones containing five distinct haplotypes were generated by site-directed mutagenesis, cloned into pires-zsgreen expression vectors and transfected into different cell lines for further functional analysis. one modified vector set contained a short 'utr sequence of abcc whereas the other contained the full length (fl) sequence. mirnas potentially interacting with abcc were identified form an mirna array after rifampicin stimulation of hepg cells. results: we could demonstrate that there is no difference in the basal protein expression level comparing the two vector types (fl 'utr vs. mod. 'utr) concerning the - c/ g/ c (cgc) abcc wt in hepg cells. using the fl vector construct, the expression level of cac haplotype protein was increased ( , % ± %). transfection assays with the mir- , which was identified as a candidate mirna targeting abcc mrna, and the two different vector constructs harboring the cac or the cgc (wt) haplotype, confirmed that mir- was able to down regulate the abcc protein expression. there was no significance in downregulation for one abcc haplotype, respectively (modified 'utr: cgc - %; cac - %, fl 'utr: cgc - %; cac - % down regulation compared to mir-negative control). discussion: differences of abcc protein expression level are due to the epigenetic regulation of abcc haplotypes. to further characterize the effect of mir- on tcg, tgt and cgt abcc haplotypes, transfection assays are currently performed using cell cultures as well human primary leukocyte cultures. sex differences affect the pathophysiology and pharmacology of leukotriene biosynthesis werz o. friedrich-schiller-university jena, institute of pharmacy, philosophenweg , jena, germany inflammatory diseases affect more females than males. thus, women suffer more often from asthma, rheumatoide arthritis, alzheimer´s disease, and many autoimmune diseases than men. of interest, sex differences also exist in drug responses, with respect to both pharmacodynamics and pharmacokinetics. we have recently discovered a sex bias in the biosynthesis of pro-inflammatory leukotrienes (lts) due to testosterone, which may represent a molecular basis for gender differences in inflammation and asthma. interestingly, testosterone downregulates lt biosynthesis and also causes a sex bias in the efficiency of lt synthesis inhibitors, which demands for the clinical evaluation of a gender-tailored therapy with anti-lts. we found that certain inhibitors of lt biosynthesis were more efficiently in females than in males, and that androgens are responsible for these gender differences. in fact, the flap inhibitor mk effectively reduced ltb pleural levels in female but not in male rats treated with carrageenan, and mk increased survival only of female mice in the lt-related disease model of paf-induced lethal shock. administration of testosterone to female mice abolished the protective effects of mk . in view of the current active development of lt synthesis inhibitors as therapeutics in respiratory and cardiovascular diseases, our data prompt for consideration of gender issues in the development and use of such drugs, in order to optimize medical therapy both for men and women. considering the complexity of deposition and kinetics of air-borne nanomaterials in the lung, potential pulmonary toxicity of biopersistent nanomaterials should be evaluated by inhalation studies. those studies demand special equipment and large quantities of test material. intratracheal instillation appears as a simple and low substance-consuming alternative, although bolus dosing and the more central distribution of the particles in the lung are a well known trade-off. we compared the inflammatory response of the lung to amorphous silica (as) after instillation and inhalation. for inhalation the established short-term protocol for nanomaterials was employed (ma-hock et al. inhalation toxicology, : , ): male wistar rats were exposed to the test items for h/day on five consecutive days. the lungs were evaluated by analysis of bronchoalveolar lavage fluid (balf) and by histopathology three days and three weeks after the end of the exposure. in a parallel study, rats were intratracheally instilled and equally evaluated three days after instillation. assuming a deposition rate of %, the instilled dose corresponded to the aerosol concentration of mg/m used for inhalation. results show that inhalation and instillation of nominally equal mounts of amourphous silica elicit different results in the lung with inhalative treatment being less harmfull. this difference may be due to the bolus effect inevitable linked to instillation. instillation stuldies with amorphous silica may, therefore, be of limited value with respect to doseresponse assessment. sunscreen products containing uv filters protect consumers from the harmful effects of uv exposure. pigmentary grades of metal oxides like zno result in an opaque whiteness as a result of scattering visible light, whereasnanoparticles result in transparent products for better consumer acceptance and thus improved protection of human skin against uv-induced damage. in addition scatter uv light is most efficiently reflected at a nanosize of - nm. in the last years the toxicological properties of nanozno in comparison with pigmentary zno were examined, the results of these comprehensive studies are presented. all tests were performed according to oecd guidelines, which were modified, especially in regard of substance preparation where appropriate. nanosized zno showed no acute toxicity after dermal application, in the bcop assay as well as in the epiderm assay it showed no corrosion / irritation potential. nanosized zno does not penetrate the intact as well as the sunburned skin. a dermal absorption test in rats (oecd ) with zn-labelled test item as well as penetration tests in weanling pigs after uv radiation did not show a penetration of the zno nanoparticles through the skin. genotoxicity was tested in vitro in the ames test, in the chromosomal aberration test in v cells, both showing negative results whereas the mouse lymphoma mutation test / l y/tk+/-cells was positive. in vivo no mutagenic effect was detected in two mouse micronucleus tests, on with intraperitoneally application and another after repeated inhalation. nanosized zno was tested in -days, -days and -days inhalation studies, in all studies the predominant effects were reversible local inflammatory changes in the nasal cavity and lungs, with a noaec of . mg/m in the -day study. in a prenatal developmental toxicity study according to oecd tg , with repeated inhalation exposure to female wistar rats from gestation day through , maternal toxicity was observed by increase lung weights and inflammations in the lungs. but no substance-related effects on reproductive parameters (conception rate, corpora lutea, implantation sites, preimplantation loss, postimplantation loss, resorptions, dead fetuses) and no increase in external and soft tissue malformations and variations could be detected. the overall result of all the toxicological studies with nanosized and pigmentary zno is that the toxicological profile of both is very similar. studies were sponsored partly by cefci lri and partly by basf se. use of reach registration data for improving thresholds of toxicological concern (ttc) wieneke n., dorn s., jakupoglu c., schäfer c., sica m., wiegand c., mostert v. dr. knoell consult gmbh, marie-curie-str. , leverkusen, germany the threshold of toxicological concern (ttc) concept is utilised to identify human exposures that are so low that in-depth toxicological investigations are expendable. this is called "exposure-based waiving". exposure-based waiving serves to focus available resources on substances with relevant human exposure potential. important work into establishing ttc values has been published by munro et al. ( ) . the initial report used a database of organic substances compiled from publicly available sources. in total, noels were collected in this fashion. the munro concept used the cramer classification to categorise substances according to their hazard potential. we broadened the ttc database by including noaels published on the echa website as per november , containing data for more than registrations. only nongaseous mono-constituent substances with oral noaels were included in the ttc database. organophosphates and genotoxic substances were excluded from the database as well as noaels obtained for surrogate substances. noaels for all systemic endpoints (general toxicity, developmental toxicity, fertility, neoplasia) were taken into account. where appropriate, default assessment factors of up to were used to establish chronic noaels for each substance. for every eligible substance, we collected the published clp category for acute oral toxicity as a potential predictor of overall hazard potential. this gives rise to five categories of acute oral toxicity. a ttc is calculated from the th percentile of noaels in each of these categories using the reach rules for establishing dnels for workers and the general population. this poster presents the preliminary results for more than substances. the results indicate that the ttc concept becomes more robust when using the very broad echa database. it also suggests that acute oral toxicity categories can be used as a predictor for the overall hazard potential of a substance. comparison of different in-vitro models for inhalation toxicology with respect to the effects of cigarette smoke total particulate matter wiese j. , schumann b. b-and l-moc can be cultivated up to days without loss of viability, as determined by resazurin-assay. viability of cell cultures was determined by mtt-assay after incubation with increasing doses of tpm. exposure of h to tpm ( mg/l) reduced viability to % or % after or h, respectively. in a viability was % after h with tpm ( mg/l). the same dose of tpm lead to a decrease in viability to % ( h) or % ( h) in nhbec and to % ( h) or % ( h) in plc. as a marker of oxidative stress the level of intra-cellular glutathione (gsh) was determined by hplc. in both the tumor cell lines analysed gsh-level was increased by tpm ( mg/l). in h the induction was . and . fold after or h, respectively. while in a it was . ( h) and . fold ( h). in nhec and plc tpm ( mg/l) did not have a significant effect on gsh-levels after h. in n-moc tpm ( mg/l) also did not modulate gsh after h, but it diminished gsh-level by . fold upon prolonged contact ( h). in b-moc gsh concentrations also decreased to . or . fold the level of controls after or h incubation. the results presented show that in-vitro models of varying complexity and origin within the respiratory tract clearly differ in their response to tpm, which was used a model inhalative toxicant. the tumor cell lines used seem to be better adapted to chemical stress, while the models closer to the in-vivo situation are more vulnerable. the nongenomic effects of the mineralocorticoid receptor in transgenic mouse heart winter s. , schreier b. within the renin-angiotensin-aldosterone system (raas), the mineralocorticoid receptor (mr) is important for the regulation of fluid and electrolyte balance in the kidney, salivary glands, sweat glands and colon. however, survival of patients with severe heart failure is increased when mr blockage is combined with standard therapy suggesting aldosterone, the mr ligand, as a key factor in the development of cardiovascular diseases, but the mechanism is not yet fully understood. in recent years, evidence accumulated that besides its function as a hormone-activated transcription factor the mr also functions via nongenomic pathways. to investigate the function of the nongenomic effects of the mr in cardiovascular dysfunction, we generated a transgenic (tg) mouse model expressing a truncated human mr lacking the dna-binding site (hmr def ) under control of the cardiac specific α myosin heavy chain promoter (αmhc), a model for nongenomic effects of the mr in the heart. in this mouse model no enhanced mortality could be observed. body weight (bw), heart weight and relative heart weight were not different compared to wild type (wt) while left atrial weight/bw was increased by % (wt , ± , mg/g vs. tg , ± , mg/g, p< . , n= ). compared to wt mice neither surface electrocardiographic experiments nor echocardiographic experiments revealed modified parameters for tg mice under basal (i.e. unstimulated) conditons as well as under β-adrenergic stimulation by isoproterenol (iso, µl mm iso intraperitoneally applied). to uncover the role of aldosterone in the development of cardiovascular diseases treatment with aldosterone and high-salt diet ( %) was performed. after days cardiac function and heart dimensions were analyzed, surface electrocardiographie uncovered increased p duration ( ± . ms vs. ± . ms, p< . ) and qtc interval ( ± ms vs. ± ms, p< . ) in tg (n= ) compared to wt (n= ) animals. these findings probably indicate more sensitive conduction pathways to aldosterone in tg mice. oligomerization is important for regulation of phospholamban activity wittmann t., lohse m. j., schmitt j. p. institut für pharmakologie und toxikologie, versbacher straße , würzburg, germany phospholamban (pln) is a heart specific protein located in the membrane of the sarcoplasmic reticulum. it inhibits the ca + -atpase serca a, thereby decelerating cytosolic ca + clearance during diastole of the cardiac cycle. upon phosphorylation the inhibitory activity of pln on myocyte ca + transport is attenuated. further, it is believed that phosphorylation favors the formation of (rather inactive) pentamers and that pln pentamers itselves were an inferior substrate for phosphorylation compared to monomers. this would suggest an important role of pln oligomerization in the regulation of pln activity. to prove this hypothesis, we are investigating the patterns and kinetics of pln phosphorylation in the context of alterations in pln structure. the introduction of specific point mutations into the transmembrane region of pln yielded mutants that are purely monomeric (l a, i a and c f) or favor pentamer formation (i a, v a). transfected hek cells expressing these mutants or wildtype pln were stimulated with forskolin to induce pln phosphorylation before lysis of cells and western blot analysis using antibodies directed against phosphorylated pln. surprisingly, phosphorylation was increased for both monomeric and pentameric pln after stimulation with . µm forskolin for less than one minute. at increasing forskolin concentrations phosphorylation signals increased in parallel for monomers and pentamers. for measurement of phosphorylation kinetics stimulation of cells with . µm forskolin was stopped at different time points. we found phosphorylation of both pln monomers and pentamers within seconds of stimulation. differences in phosphorylation patterns became more pronounced when assays were performed at low temperature ( °c). intriguingly, preliminary analyses suggest that pka dependent phosphorylation occurs first in pentamers and that phosphorylation of monomers may catch up only after pentamer phosphorylation is almost complete. our data suggest that both pln pentamers and monomers are suitable substrates for pka dependent pln phosphorylation. unlike the prevalent assumption, kinetics of pentamer phosphorylation seem to be at least as fast as that of pln monomers in transfected hek cells suggesting an important role of pln pentamers in the regulation of pln activity. regulation of cardiac contractility by nucleoside diphosphate kinases in zebrafish wolf n. m. , , abu-taha i. in the heart, nucleoside diphosphate kinases (ndpks) can interact with heterotrimeric g proteins, thus regulating camp synthesis in a receptor independent manner and thereby influencing contractility in cardiomyocytes. we further investigated the interaction of ndpk isoforms with heterotrimeric g proteins in the heart in vivo and in vitro using zebrafish embryos and embryonic fibroblast from ndpk a/b double knockout mice (ndpk a/b ko mefs). in zebrafish the morpholino-mediated knockdown of ndpk a did not lead to an obvious phenotype, although the total ndpk activity was reduced. depletion of ndpk b caused a cardiac phenotype characterized by severely impaired atrial and ventricular contractility and insufficient blood flow. the depletion of ndpk b was associated with a significant decrease of protein levels of the heterotrimeric g protein subunits gβγ, gα s and gαi. the knockdown of ndpk c led to a more restricted cardiac phenotype with markedly reduced pumping function of the ventricle, while the atrium was unaffected. in accordance to the reduced cardiac pumping function, camp levels were significantly diminished in the ndpk b and ndpk c morphants. similar findings were obtained in ndpk a/b ko mefs. the absence of ndpk a and b resulted in a decrease of the plasma membrane content of gβ and gαs and a significant reduction in camp synthesis. the protein expression of the isoform ndpk c was also significantly reduced in the ndpk a/b ko mefs. the re-expression of ndpk b but not ndpk a rescued the basal camp production and the membrane content of g proteins. interestingly, the overexpression of ndpk c led to a -fold enhancement of the camp level and a significant increase of the membrane content of gβ and gα, and thus rescued the knockout phenotype. our data indicate, that the ndpk isoforms b and c are essential for cardiac contractility, most likely by forming a signaling complex at the plasma membrane including ndpk b, ndpk c and heterotrimeric g proteins. the isoform ndpk c, with its n-terminal hydrophobic region, might serve as a membrane anchor for the ndpk/g protein complex. induction of apoptosis via pka-dependent and pka-independent pathways by cyclic purine and pyrimidine nucleotides in mouse lymphoma cell lines wolter s. camp is a second messenger that plays an important role in intracellular signal transduction of various hormones and neurotransmitters. a major function of camp in eukaryotes is the activation of camp-dependent protein kinase a (pka). pka is involved in the control of a variety of cellular processes. pka exists as an inactive tetramer of a dimeric regulatory (r ) and two catalytic (c) subunits that releases the active c-subunits upon binding of camp. stimulation of the mouse t-lymphoma cell line s wild-type (wt) with dibutyryl (db)-camp induces apoptosis by an intrinsic, mitochondria-dependent mechanism. apoptosis induced by db-camp occurs via a pka-dependent mechanism, since s kincells lacking the catalytic subunit of pka are resistant to db-camp-mediated cell death. db-camp is cleaved by esterases into the biologically active compound n -mb-camp and into '-o-mb-camp. other cyclic nucleotides (cnmps) in addition to camp, like ccmp and cump can also function as second messengers and activate pka and cgmp-dependent protein kinase (pkg) . therefore, we investigated the effects of a series of membrane-permeable analogues of camp, cgmp, ccmp and cump in s wt und s kincells on apoptosis. stimulation with db-ccmp or db-cgmp induced neither apoptosis in s wt nor in s kincells. interestingly, we observed apoptosis in s wt and s kin cells after incubation with membrane-permeable nucleotide acetoxymethyl ester (am)-analogues of cgmp, ccmp, cump and also camp. induction of apoptosis occurs via pkadependent and also pka-independent pathways. a potential role of pkg and of the exchange protein activated by camp (epac) in the induction of apoptosis is unsolved and will be explored by specific pkg-and epac-activators in this system. investigations are on the way to identify the targets, the involved signal transduction pathways and the mechanisms of pro-apoptotic actions mediated by cnmp-ams. ( ) wolter s, golombek m, seifert r ( ) differential activation of camp-and cgmpdependent protein kinases by cyclic purine and pyrimidine nucleotides. biochem biophys res commun. in press apoptosis in s cells : induction of apoptosis in s wt and in s cells lacking the catalytic subunit of pka (s kin-) with a) db-cnmps and b) cnmp-ams for h. nebivolol reduces vascular inflammation in spontaneously hypertensive rats wu z., xia n., förstermann u., li h. institut für pharmakologie, obere zahlbacher str. , mainz, germany nebivolol is a third generation β receptor blocker with additional effects on endothelial nitric oxide production. the aim of the present study is to investigate the antiinflammatory effects of nebivolol in vivo. spontaneously hypertensive rats (shr) were divided into two groups: control or nebivolol treatment group. nebivolol treatment ( mg/kg/day for days) significantly reduced the blood pressure and the heart rate in shr. the drug had no effect on coagulation. aorta from nebivolol-treated rats showed significantly improved endothelial function. nebivolol did not change the expression levels of aortic nf-kb, but significantly reduced its dna binding activity. furthermore, nebivolol decreased the expression of adhesion molecules (e.g. icam- , vcam- ) and pro-inflammatory cytokines (e.g. il- ). in conclusion, nebivolol reduces vascular inflammation in experimental hypertension. it inhibits nf-kb activity, decreases the expression of adhesion molecules and pro-inflammatory cytokine, and improves endothelial function. characterization of the cellular activity of pde inhibitors using two novel pde reporter cell lines wunder f., quednau r., barg m., tersteegen a. bayer pharma ag lead discovery wuppertal, aprather weg a, wuppertal, germany cyclic nucleotide-specific phosphodiesterases (pdes) play an essential role in cellular signal transduction by regulating the intracellular levels of camp and cgmp and, therefore, are important pharmacological targets. we report here the generation and pharmacological characterization of two novel pde reporter cell lines. plasmid constructs encoding human pde b or pde d were stably co-transfected with the beta -adrenoceptor in a parental camp reporter cell line expressing a cyclic nucleotide-gated (cng) cation channel, acting as the biosensor for intracellular camp. in this reporter cell line, camp levels can be monitored in real-time via aequorin luminescence stimulated by calcium influx through the cng channel. by using different pde and non-pde inhibitors, we could show that our novel pde b and pde d reporter cell lines specifically monitor pde inhibition with high sensitivity. pde -selective inhibitors alone did not increase basal luminescence levels in this experimental setting. however, these inhibitors induced concentration-dependent luminescence signals in combination with the adrenoceptor agonist isoproterenol. in contrast, in a stable beta -adrenoceptor reporter cell line with no recombinant pde expression, pde inhibitors had no effect on isoproterenol-stimulated luminescence signals. we compared the cellular activity of different pde inhibitors with the in vitro inhibition of full-length and truncated (catalytic domain) pde d from cell lysates. two different groups of pde inhibitors could be identified. the first group, including the allosteric inhibitors pmnpq and d , showed high cellular activity and much better inhibition of full-length versus truncated pde d . the second inhibitor group, including classical competitive inhibitors like roflumilast, cilomilast and piclamilast, showed comparably lower cellular activity and similar inhibitory activity on full-length and truncated pde d . the results imply that these novel pde reporter cell lines are well-suited for the characterization of the cellular activity of pde inhibitors and may also support a better understanding of the complex pde pharmacology. plexin-b is required for kidney regeneration after acute renal failure xia j. , gröne h acute renal failure is a common clinical problem with unsatisfactory therapeutic options and high mortality in humans. therefore, unraveling the mechanisms that promote kidney regeneration and repair may provide new therapeutic strategies for acute renal injury. plexin-b belongs to a family of transmembrane receptors which mediate the cellular effects of semaphorins. while plexins have first been described in the context of axon guidance, several recent studies have established them as key regulators of organogenesis, the immune system and cancer. we have recently found that plexin-b is highly expressed in the adult kidney, particularly in tubular epithelial cells which are most sensitive to acute ischemic injury. to study the role of plexin-b during kidney regeneration we generated mice lacking plexin-b specifically in tubular epithelial cells. under physiological conditions, these mice displayed normal kidney morphology and function. in contrast, following ischemia/reperfusion injury, plexin-b conditional knockout mice exhibited severely impaired kidney regeneration. while the renal function of control mice fully recovered within weeks after injury, plexin-b knockout mice had strongly elevated serum creatinine and urea levels associated with increased morbidity and mortality. this was accompanied by hyperproliferation of tubular epithelial cells and obstruction of tubular lumina. we conclude that plexin-b is required for regeneration after acute ischemic renal injury and that pharmacological interventions activating plexin-b might represent a new therapeutic strategy in acute renal failure. the nadph oxidase enzyme complex consists of two membrane-bound catalytic subunits (a nox protein and p phox) and several cytosolic regulatory components including p phox, p phox, p phox and the small gtpase rac . we have previously shown that treatment of apolipoprotein e knockout mice with resveratrol led to a downregulation of nox and nox in the heart. our recent data demonstrated that resveratrol also reduced the enzymatic activity of cardiac nadph oxidase. because activation of nadph oxidase enzyme complex is induced by translocation of the regulatory subunits, we studied whether the reduced enzymatic activity is due to an inhibition of such a translocation. indeed, resveratrol treatment prevented rac membrane translocation from cytosol. resveratrol is known as an activator and expression enhancer of the longevity gene sirtuin (sirt ). we then wanted to find out whether the effect of resveratrol on rac was mediated by sirt . sirt is a histone/protein deacetylase. in vitro incubation of rac with sirt led to a reduction of lysine acetylation. deacetylation of rac on lysine could be identified by mass spectrometry analyses. the lysine lies within the p phox-binding region of rac . consistently, in vitro incubation of rac with sirt markedly reduced its binding activity to p phox. in conclusion, we provide evidence that rac is a direct target molecule of sirt . sirt deacetylates rac on lysine and thereby inhibits its interaction with p phox. this is a novel mechanism of nadph oxidase inhibition by sirt /resveratrol. mutational analysis of the effects of the cardioprotective drug dexrazoxane on topoisomerase ii beta in vitro yan t., deng s., frensch i., gödtel-armbrust u., wojnowski l. universitÄtsmedizin der johannes gutenberg-universität mainz institut für pharmakologie, obere zahlbacher straße , mainz, germany dexrazoxane (drz, icrf- ) is the only approved drug shown to protect against anthracycline-induced heart failure. the protection is usually ascribed to the ironchelation by drz, which is thought to reduce anthracycline-induced oxidative stress. however, similarly to anthracyclines, drz is also an inhibitor of the anthracycline target topoisomerase ii (top ). we hypothesized that the cardioprotective effects of drz are mediated by the prevention of the anthracycline-induced dna damage mediated by top b, the dominant cardiac top isoform. this was investigated using top b mutants resistant to drz, which were expressed in cells depleted of wild-type top isoforms. top b-mediated double-strand dna breaks were assessed as γ-h ax. the levesl of dsb generated by the top b mutants in response to the anthracycline doxorubicine (dox) was indistinguishable from that mediated by a wild-type top b. preincubation with drz depleted wild-type top b and this was accompanied by a decrease in the dna damage following a subsequent exposure to dox. in contrast, neither top b depletion nor the reduction of dsb by drz was seen in drz-resistant top b mutants. furthermore, the cardially ineffective drz analog icrf- , capable of iron chelation but not of top binding, affected neither the stability of top b, nor the dox-induced dna damage mediated by this enzyme. these results indicate that drz may exert its cardioprotective effects by reducing the dna damage mediated by doxpoisoned top b rather than by iron chelation. they also suggest a cardioprotective function of top b, which is currently under investigation using cardiomyocyte-specific top b mouse knockouts. aminoglycosides are important antibiotics in the treatment of life-threatening infections, especially those caused by gram-negative bacteria. their nephrotoxic and ototoxic potential is well-known, but little is known about the effects of aminoglycosides on the male reproductive system. we studied the effects of four aminoglycosides on sertolicells in vitro. rat sertoli-cells from the cell line serw were cultivated for , , and days in dmem supplemented with three different concentrations of amikacin, streptomycin ( mg/l, mg/l, mg/l), gentamicin or tobramycin ( mg/l, mg/l, mg/l). we determined the expression of two junctional proteins (connexin , ncadherin) and one protein of the cytoskeleton (vimentin) by western blot. cells were solubilized in lysis buffer. lysates were separated by sds-page and electroblotted on a pvdf-membrane. after incubation with primary antibodies overnight and horseradish peroxidase-conjugated secondary antibody the visualization was achieved by a chemiluminescence-detection system. in addition, proteins were detected by immunohistochemistry. after three days in culture amikacin caused the most pronounced effect. at the lowest concentration tested ( mg/l) connexin and n-cadherin were reduced to ± % and ± % of the controls (n= ). no change was recognized for vimentin ( ± %). effects obtained with streptomycin were less pronounced for these these proteins ( ± %, ± %, and ± %, respectively). similar, but less pronounced effects were observed with gentamicin and tobramycin at a concentration of mg/l (connexin : ± % and ± %; n-cadherin: ± % and ± %; vimentin: ± % and ± %) and mg/l (connexin- : ± % and ± %; n-cadherin: ± % and ± %; vimentin: ± % and ± %). after incubation for and days the effects occurred in the same range. the substances showed no influence on the viability of serw sertoli-cells up to mg/l in the mtt assay. by immunohistochemistry we showed that the localisation of the proteins -connexin and n-cadherin at the cell membrane and vimentin in the cytoplasm -was not influenced by the aminoglycosides. large conductance calcium-and voltage-gated potassium (bk) channels play an important role in controlling membrane potential and calcium influx, and are strongly modulated by protein kinases at multiple sites. the stress-regulated exon (strex) adds to the bk channel c terminus a cysteine-rich insert of amino acids that inverts the channel regulation by protein kinase a (pka) from excitatory to inhibitory. here we investigated the mechanisms by which the strex insert influences bk channel regulation by protein kinase c (pkc). activity of bk channels without strex insert (bk-zero), transiently expressed in hek cells, was inhibited by pkc in inside-out membrane patches (~ % inhibition). bk channels with strex insert (bk-strex), however, were insensitive to pkc. phosphomimetic mutation of a pkc phosphorylation site (s e) in bk-strex, resulted in a ~ % reduction of basal channel activity, whereas the s a mutant retained normal activity. to examine whether palmitoylation, and thus association of the strex domain with the plasma membrane, prevents pkc inhibition of bk channel gating, palmitoylation was abolished by either site-directed mutagenesis (c : a) or by pharmacological inhibition of palmitoyl transferases with -bromopalmitate ( -bp). both, mutation and pretreatment with -bp resulted in the expression of bk-strex channels which were sensitive to pkc (~ % inhibition of channel activity). no inhibitory pkc effect was observed in patches of the bk-strex s a channel mutant pretreated with -bp. in a clonal rat somatomammotroph pituitary cell line (gh b ), in which pcr products without (zero) and with the bp strex exon could be identified, the pkc activator pma blocked channel activity by ~ %. this inhibition was increased to over % when gh b cells were pretreated with -bp, indicating that both channel isoforms were functionally active. in summary, the present study demonstrates that palmitoylation of strex prevents bk channel regulation by pkc, which is mediated by phosphorylation of ser , probably by steric hindrance. our results provide further evidence for a cross-talk between palmitoylation and phosphorylation as a crucial mechanism underlying the dynamic regulation of ion channels. human pleural mesothelial met- a cells are a limited in vitro model system in determining potential asbestos-like genotoxic effects of multiwall carbon nanotubes ziemann c. , reamon-büttner s. multiwall carbon nanotubes (mwcnt) are nanomaterials with important technological impact. depending on their diameter, length, and biopersistence, however, some mwcnt seem to exhibit a fiber-like cytotoxic and genotoxic potential, similar to asbestos. thus, a project funded by the german federal ministry of education and research (bmbf contract no. x a) focuses on potential adverse biological effects of different types of mwcnt to enlarge the knowledge base about toxicity determining parameters. this project comprises both in vitro (rat) and in vivo endpoints with long amosite asbestos as a positive control. as mesothelial cells are target cells for adverse effects of asbestos, in particular mesothelioma development, the human sv transformed, non-malignant pleural mesothelial cell line met- a was chosen as the main in vitro model in this project. in the present study part, met- a cells were characterized concerning their usefulness as an in vitro model to study potential asbestos-like cytotoxic and genotoxic effects of different mwcnt varieties. using an mwcnt-optimized lactate dehydrogenase liberation assay and proliferation parameters derived from cell counts, concentration-dependent cytotoxicity of long amosite asbestos ( , , and µg/cm ) was demonstrated in met- a cells. cells also showed asbestosinduced increase in dna-strand breaks and oxidative dna-damage in the hogg modified comet assay. thus, met- a cells were responsive to asbestos treatment. owing to asbestos potential to induce aneugenic effects and spindle fiber damage, micronucleus induction, determination of numerical chromosome aberration, and altered meta-, ana-, and telophase morphology were planned as in vitro endpoints. met- a cells were thus initially characterized in this regard and were found to exhibit highly variable chromosome numbers with lower than % cells exhibiting a normal diploid chromosome set, an up to twentyfold higher spontaneous micronucleus frequency, as compared to polychromatic bone marrow erythrocytes in rodents, and a profound number of aberrant meta-, ana-and telophases with bridges, lagging chromosomes and multipolar divisions. in conclusion, met- a cells are of only limited value as an in vitro model system to study potential asbestos-like effects of mwcnt and also biopersistent fibers. the cells are indeed responsive to asbestos, but unfortunately demonstrate marked genomic instability and thus limited significance concerning genotoxic effects. waixenicin a inhibits cell proliferation through magnesium-dependent block of trpm channels zierler s. transient receptor potential melastatin (trpm ) channels represent the major magnesium-uptake mechanism in mammalian cells and are key regulators of cell growth and proliferation. they are abundantly expressed in a variety of human carcinoma cells controlling survival, growth and migration. these characteristics are the basis for recent interest in the channel as a target for cancer therapeutics. we screened a chemical library of marine organism-derived extracts and identified waixenicin a from the soft coral sarcothelia edmondsoni as a strong inhibitor of overexpressed and native trpm . waixenicin a activity was cytosolic and potentiated by intracellular free magnesium (mg + ) concentration. mutating a mg + -sensitive site on the trpm kinase domain reduced the potency of the compound, whereas kinase deletion enhanced its efficacy independent of mg + . waixenicin a failed to inhibit the closely homologous trpm channel and did not significantly affect trpm , trpm , and crac (calcium release activated calcium) channels. therefore, waixenicin a represents the first potent and relatively specific inhibitor of trpm ion channels. consistent with trpm inhibition, the compound blocked cell proliferation in human jurkat t-cells and rat basophilic leukemia cells. based on the compound's ability to inhibit cell proliferation through mg + -dependent block of trpm , waixenicin a or structural analogs may have cancer-specific therapeutic potential, particularly since certain cancers accumulate cytosolic mg + . release of metals from different sections of domestic drinking water installations zietz b. p., richter k., laß j., suchenwirth r., huppmann r. governmental institute of public health of lower saxony division of environmental medicine and environmental epidemiology, roesebeckstraße - , hanover, germany different metals were used as important piping materials in the drinking water supply for a long time. due to corrosion metals can leach into the tap water. of special importance is the toxic element lead. however other heavy metals in drinking water such as copper, nickel and cadmium can also give reason for health concerns. in this study it was investigated in which amount relevant metals were released from different parts of domestic installations into the cold water. for the spatial allocation of the emission sources a sequential water sampling protocol was used after three hours of stagnation time representing the first litre of the water column. after stagnation ten sample volumes were collected in series. existing facilities of domestic installations constructed with different plumbing materials were examined predominantly from residential buildings. the elements al, as, cd, cr, cu, fe, mg, mn, ni, pb, sb, se, u and zn were detected by means of icp-ms. in total water pipe strands of domestic installation systems were examined. they comprised single water samples and single parameters. depending upon the type of plumbing different courses and concentration ranges of the elements could be measured in the tap water samples. terminal taps or installation parts were frequently responsible for a release of nickel and in several cases of cadmium. the concentration courses of the element zinc proved as a good indicator for the allocation of the emission source to a brass containing section of the installation (zinc as an alloy component of brass). one can conclude that an investigation by means of a sequential water sampling protocol and multi-element detection can be a valuable non-destructive method for drinking water-hygienic investigations of domestic installations. novel interaction partners of the murine trpc protein zimmermann j., beck a., flockerzi v. universität des saarlandes experimentelle und klinische pharmakologie und toxikologie, kirrberger str. , homburg, germany in this work novel interaction partners of the murine protein transient receptor potential canonical (mtrpc ) were identified. the trpc protein is the major subunit of a cation channel, residing in the plasma membrane. it comprises six trans-membrane domains and cytosolic amino and carboxyl termini. two major splice variants of the trpc gene exist, trpc a ( aa) and trpc b ( aa), trpc b lacks aa to of the trpc a variant. both trpc variants are co-expressed in endothelial cells, intestinal smooth muscle and brain. to identify trpc -interacting proteins a yeast two-hybrid system, cytotrap®, which allows identification of protein-protein interactions within the cytosol was used. a premade mouse brain cdna library was screened by the cytosolic amino and carboxyl terminal parts of mouse trpc a (aa to ; aa to ). for the carboxyl terminal part fourteen proteins were identified. to independently prove the interaction, the fulllength cdnas of all fourteen proteins were cloned, fused to a flag-tag and coexpressed with trpc in hek cells. co-immunoprecipitations were performed for all candidates using both the anti-flag-antibody and the antibody for trpc . in addition, changes of cytosolic calcium were monitored and trpc currents were recorded in hek cells expressing the candidate cdnas and stably expressing the trpc a or trpc b and the muscarinic receptor type cdnas. the tarbp protein, one of the candidates shown to interact with trpc , changed calcium influx when coexpressed with trpc . in order to identify the domains of trpc responsible for its interaction with the tarbp protein, six trpc -gst-fusion proteins covering the carboxyl terminal aa of trpc a were expressed in e. coli and used for pull-down experiments. by this approach two domains of trpc could be identified to interact with tarbp . one of these domains is well conserved within the trpc protein, corresponding to the result, that trpc and tarbp effectively co-immunoprecipitate, too. the tarbp protein has been shown to be a component of the risc loading complex, also known as the micro-rna loading complex which is composed of dicer , eif c /ago and tarbp (chendrimada et al. ) . by its interaction it may link trpc to pre-microrna processing. increased levels of angiotensin ii provoke dna damage and have influence on dna repair in mouse kidneys zimnol a., brand s., schupp n. universität würzburg institut für toxikologie, versbacherstr. , würzburg, germany the renin-angiotensin system (ras) plays a crucial role concerning the blood pressure, electrolyte balance and cardiovascular homeostasis. angiotensin ii (ang ii), the active hormone of the ras, in higher concentrations leads to vasoconstriction, oxidative stress and hypertension. hypertensive patients have an increased risk to develop cancer, especially kidney cancer. we have shown in vitro and in vivo, that ang ii is capable to cause an elevation of blood pressure as well as dna damage dose-dependently. to investigate whether the high blood pressure or the enhanced levels of ang ii are responsible for dna damage, male c bl/ -mice were equipped with osmotic minipumps, delivering ang ii in a concentration of ng/kg · min during days. additionally they were treated with ramipril, an angiotensin-converting-enzyme blocker, with the ang ii receptor antagonist candesartan, the vasodilator hydralazine, and the antioxidant tempol. dna damage was analysed with the comet assay. we measured the base excision repair (ber)-related dna repair in the kidney with a comet-based in vitro repair assay. furthermore, the distribution and expression of the ang ii-type (at ) receptor in the kidney was analyzed by immunohistochemistry. treatment with ang ii led to a significant increase of blood pressure, whereas the medication with candesartan decreased the systolic blood pressure. the intervention with hydralazine lowered the blood pressure only for a short time. the other substances had no effect at all on the blood pressure. genomic damage, quantified with the comet assay, was augmented by ang ii and improved by all interventions, particularly by candesartan and tempol. beyond that, ang ii showed a tendency to reduce dna repair. treatment with candesartan, hydralazine and tempol increased the repair capacity. furthermore, ang ii tended to result in a downregulation of the at receptor in kidney tubule cells. candesartan and ramipril, especially were able to augment the expression of the at receptor, whereas hydralazine achieved the opposite. these results demonstrate that ang ii leads to dna damage in the kidney independent of blood pressure. apparently elevated levels of ang ii affect dna repair and expression of at receptor. to confirm these findings we are going to examine more precisely the manifestation of other enzymes, which are implicated in dna repair. regulation of hcn channel activity by cyclic cytidine ´, ´-monophosphate zong x., krause s., chen c. -c., gruner c., cao-ehlker x., fenske s., wahl-schott c., biel m. lmu münchen, department pharmazie, pharmakologie für naturwissenschaften center for integrated protein science munich (cipsm), butenandtstr. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] münchen, germany hyperpolarization-activated cyclic nucleotide-gated (hcn) channels play a key role in controlling cardiac pacemaker activity and are essential for normal function of neuronal circuits. hcn channels are principally gated by voltage but are coactivated by the cyclic nucleotides camp and cgmp which directly bind to a c-terminal binding domain. recently, cyclic cmp (ccmp) was shown to be present in various cell lines and tissues at concentrations that are comparable to cellular cgmp levels. moreover, there is recent evidence that ccmp can activate camp-and cgmp-dependent protein kinases in vivo. here, we examined whether ccmp exerts effects on hcn channels. to this end, we recorded hcn channel-mediated currents (i h) in hek cells that stably express hcn , hcn , hcn or hcn , respectively. currents were measured either with a standard patch-clamp setup or by employing the planar patch-clamp technology. in hcn and hcn channels, ccmp shifted the membrane potential for half maximal activation (v . ) to more positive values. in addition, ccmp accelerated activation while it slowed down deactivation kinetics. the ec for ccmp was µm which is about times higher than the ec of cgmp. cyclic cmp is a partial agonist of hcn channels since it activates only % of the maximal current obtained with camp or cgmp. to identify in vivo effects of ccmp we recorded ih of murine sinoatrial node (san) cells in the presence and absence of mm ccmp. like for heterologously expressed hcn channels, ccmp shifted v . of ih by about + mv. importantly, the steepness of the diastolic depolarization of san pacemaker potentials which is mainly determined by the amplitude of ih was profoundly increased by ccmp, compared to control conditions. as a consequence of the upregulation of ih the frequency of san pacemaker potentials was increased by about % in the presence of ccmp. our results suggest that ccmp is a physiological regulator of hcn channel activity. a d actinic keratosis like construct for assessment of innovative tumour therapeutics zoschke c. the incidence of actinic keratosis (ak) has increased dramatically in the last decades and it is considered the most frequent carcinoma in situ today. especially immunosuppressed patients are at high risk to develop invasive squamous cell carcinoma (scc) [ ] which asks for most efficient and well-tolerated ak therapy. yet, current measures do not fit with these demands. nucleotide analogues, recently identified by molecular modelling [ , ] , outperformed the current standard for the therapy of actinic keratosis, -fluoruracil, when tested in the tumour cell line scc , in normal human keratinocytes and fibroblasts [ ] . as next step in pre-clinical drug assessment, we aimed to characterise the effect of the most selective nucleotide analogue oxbu in reconstructed human tumour skin. based on the d construct of scc developed by höller and co-workers [ ] for start we introduced several adaptations with respect to keratinocyte, fibroblast and scc seeding to grow an aklike construct with scc cells forming nests in particular in the epidermis. in addition, first experiments with the oxbu on the ak like constructs showed promising results for an efficient treatment of actinic keratosis. efficacy was derived from immunohistology (marker for proliferation: ki- , marker for scc: cytokeratin- , axl, marker for invasion: mmp , marker for apoptosis: caspase- , nuclei were stained with dapi) as well as the effects on the secretion of cytokeratin- and its caspase-induced cleavage product into the culture medium following drug exposure for up to days. efficacy of a . % oxbu solution proved close to or even better when compared to both a . % fluorouracil and . % aphidicolin solution. the former being the gold standard of current ak therapy, the latter is a frequently used inhibitor of human polymerase alpha and delta, however, failed to be introduced into clinical use. -in fact, in monolayer cultures aphidicolin proved most toxic for normal human keratinocytes which was not true with oxbu [ ] . -therefore, these d tumour constructs offer a new approach to pre-clinical drug assessment and may be added to other d models of skin diseases currently gaining increased interest as test platforms. ima , a multi-peptide cancer vaccine for advanced colorectal cancer, induces multiple cd + and cd + t-cell responses associated with improved survival walter s. , kuttruff s. ima is a novel peptide-based vaccine consisting of hla-a* and hla-dr binding synthetic peptides that were identified based on natural presentation on human colorectal cancer (crc) samples. ima was characterized in a phase i/ii trial in advanced/metastatic crc patients being at least clinically stable after weeks of first-line oxaliplatin-based therapy. all patients received a single application of low-dose cyclophosphamide for immunomodulation. this was followed by repeated ima vaccinations in combination with low-dose gm-csf (cohort ; n= ) or ima / gm-csf plus topically applied imiquimod (cohort ; n= ). before and post vaccination, patients were analyzed by hla-multimer assay and intracellular cytokine (ics) assay for cd + t-cell responses and by ics assay for cd + tcell responses. as immune status biomarkers, phenotypically defined myeloid derived suppressor cell populations (mdsc - ) were analyzed prior to immunotherapy. tumor status of patients was monitored repeatedly by ct/mri according to recist and corresponding tumor scans were reviewed centrally. clinical assessment included disease control rate (dcr), time to progression (ttp), progression-free survival (pfs) and overall survival (os). ima overall was immunogenic in / ( %) evaluable patients, with % and % of patients mounting multiple cd + and cd + t-cell responses, respectively. patients that received the immunomodulator imiquimod presented with significantly more multiple cd + cell responses as detected by ics (p= . ). multiple cd + and multiple cd + responses were individually associated with significantly better clinical outcome. the association was most pronounced for patients with both multiple cd + and multiple cd + responses. these patients had significantly higher dcr at months (p= . ), improved ttp (p= . ) and improved pfs (p= . ) than other patients. most importantly, a trend for prolonged os was also observed in these patients (p= . , hazard ratio . ). in the study population, levels of different mdsc phenotypes were significantly increased as compared to age/gender matched controls. high mdsc levels were associated with fewer immune responses and for mdsc and mdsc high frequencies were associated with shorter os (p= . and p= . , respectively). to summarize, both hla-a* and hla-dr restricted peptides in ima were immunogenic. a significantly better clinical outcome of multi-tumap responders in comparison to patients with one/no tumap response strongly indicates clinical activity of ima . acrylamide (aa), a genotoxic carcinogen (iarc class a) is formed in food by thermal treatment from different precursors. after oral ingestion, aa is metabolically epoxidized in the liver by cyp e into glycidamide (ga). ga binds to dna, forming covalent adducts, primarily at n of guanine (n -ga-gua). both, aa and ga undergo conjugation to glutathione (gsh) to be excreted via urine as mercapturic acids (ma), namely nacetyl-s-( -carbamoylethyl)-cysteine (aama), and n-acetyl-s-( -hydroxy- carbamoylethyl)-cysteine (gama). in a dose response study, encompassing the dosage range from human dietary exposure levels up to mg/kg bw, female sprague dawley (sd) rats on a diet devoid of detectable aa content were gavaged with single doses of aa. formation of urinary mas and of n -ga-gua dna adducts in liver, kidney and lung was measured h after application, a time point where cmax of n -ga-gua was reached. the untreated control group was found to excrete about . nmol (aama plus gama) in the urine ( h), indicating a background of endogenous aa formation. compared to untreated control, the lowest dosage of . µg aa/kg bw neither resulted in significantly enhanced ma excretion, nor in a detectable n -ga-gua adduct levels in any organ tested (limit of detection, lod, . adducts/ nucleotides). at the tenfold higher dose ( µg/kg bw), adducts were found in kidney (about adduct/ nucleotides) and lung (< adduct/ nucleotides), but not in liver. at and µg/kg bw, adducts were found in all three organs, at levels not significantly different to those found at µg aa/kg bw (about - adducts/ nucleotides). the results of this in vivo study and of further recent research on aa toxicology will be discussed with respect to risk assessment. exposure of rats to single doses of aa in the range of human dietary exposure ( . - µg/kg bw ) leads to n -ga-gua adduct levels in the tissues monitored obviously not exceeding the range of steady state background dna lesions associated with endogenous/exogenous exposure to various genotoxic electrophiles. thus, the question of significant impact on human background dna damage resulting from exposure to a given genotoxic carcinogen, and on potentially ensuing biological consequences may become a highly relevant issue in risk assessment. pharmaco-economic impact of price, volume and demographic development böcking w., kirch w. institut für klinische pharmakologie, medizinische fakultät der tu dresden, fiedlerstr. , dresden health insurance costs in germany have grown by % p.a. over the last ten years and amount to approx. bn eur in . while costs for stationary treatment as the largest cost category have been intensely analyzed over the past years, pharmaceutical expenses have been analyzed in less detail, mostly focusing on the statutory health insurance side, even though pharmaceutical expenses have grown almost twice as much as costs for ambulant treatments. therefore, the question was asked how pharmaceutical expenses in a large german private health insurance company are allocated with respect to age and indication groups, and how those have developed from to . the data of a private health insurance company with more than . customers was split into price and volume effects per age group to understand if price or volume drives the cost development. additionally, the two largest indication groups are analyzed in detail. as a result, both price and volume effects drive an overall cost increase. these effects are even stronger in older age groups. this cost increase is not sustainable for the german health insurance system over a longer period of time and will even further increase due to the ageing of the german population. a novel animal replacement system for the detection of endocrine disruptive capabilities in sexual development scheider j. , , winter p. alternatives to animal testing for prediction of local toxicity and genotoxicity have been recently established. however, currently these methods are not suitable for measuring endocrine effects in developing organs such as e.g. embryonic gonads. here we present a phenotypic anchoring of a comprehensive study on sex-specific gene expression analysis accompanied by histological analysis of endocrine disruption in chicken embryo gonads, having the potential for an animal replacing system for endocrine disruptive toxicologic and ecotoxicologic examinations of chemicals. chicken embryos were inoculated with different amounts of tributyltin (tbt) and bisphenol-a (bpa). embryos were incubated and their gonads analyzed histologically d prior to hatching. from identically treated embryos right and left testes and ovaries were separated and genome-wide transcription profiles generated using supertag digital gene expression (st-dge, supersage) profiling. male and female gonadal tissues both revealed histological aberrations in response to tbt and bpa. female gonads became masculinized in response to tbt and, viceversa, bpa-treated male gonads underwent feminization whereas in female gonads clearly visible structural aberrations occurred. in both chemicals mortality increased especially in the most affected sex (tbt: females, bpa: males). the expression profiles of more than million mrnas revealed massive effects of both chemicals, tbt and bpa, on important cellular signaling pathways. gene expression differences were most pronounced in the phenotypically most affected sex. our results demonstrate that endocrine disruptive chemicals exert their effects on several levels including but not restricted to known hormone-based pathways. together with an ongoing study of gene expression differences in very young life stages and different chemicals these data will form the base for a blow-by-blow analysis of sexspecific gene expression of embryonic development. the project builds on already existing and further to generate data with the aim of the development of an in vitro method for testing chemicals at chicken eggs for ) replacement of tests on juveniles and (sub-) adult rodents, ) stages with impossibility of sensation of pain in the individuals, ) highly sensitive prospects of modes of action of chemicals, which ) might show consequences in the next generations. channels are critical for oscillatory burst discharges in the reticular thalamus and absence epilepsy differential distribution of three members of a gene family encoding low voltage-activated (t-type) calcium channels hippocampal seizure resistance and reduced neuronal excitotoxicity in mice lacking the cav . e/r-type voltage-gated calcium channel transcriptional upregulation of cav . mediates epileptogenesis in the pilocarpine model of epilepsy structure and functional expression of a member of the low voltage-activated calcium channel family a molecular determinant of nickel inhibition in cav . t-type calcium channels histidine residues in the is -is loop are critical for nickel-sensitive inhibition of the cav . calcium channel substrate recognition and translocation by polyspecific organic cation transporters proton pump inhibitors inhibit metformin uptake by organic cation transporters (octs) structural determinants of inhibitor interaction with the human organic cation transporter oct (slc a ) functional characterization of the human organic cation transporter variant p. ala>ser extra-adrenal glucocorticoid synthesis in the intestinal epithelium: more than a drop in the ocean? local glucocorticoid production in the mouse lung is induced by immune cell stimulation biomimetic materials in tissue engineering biomaterials offer cancer research the third dimension synthetic biomaterials as instructive extracellular microenvironments for morphogenesis in tissue engineering injectable self-assembling peptide nanofibers create intramyocardial microenvironments for endothelial cells directed growth of fibroblasts into three dimensional micropatterned geometries via selfassembling scaffolds novel pcl-based honeycomb scaffolds as drug delivery systems for rhbmp- tissue engineering spatio-temporal vegf and pdgf delivery patterns blood vessel formation and maturation presentation of rgds epitopes on self-assembled nanofibers of branched peptide amphiphiles controlling mammalian cell interactions on patterned polyelectrolyte multilayer surfaces langmuir avintegrins as receptors for tumor targeting by circulating ligands heparin binding nanostructures to promote growth of blood vessels tirrell endothelial cell adhesion to the fibronectin cs domain in artificial extracellular matrix proteins design and bioproduction of a recombinant multi(bio)functional elastin-like protein polymer containing cell adhesion sequences for tissue engineering purposes stimuli-responsive thin coatings using elastin-like polymers for epac as a novel effector of airway smooth muscle relaxation ) camp inhibits airway smooth muscle phenotype modulation functional roles of epac and pka in human airway smooth muscle phenotype plasticity assessment of the sensitizing and irritative potential of preservatives by loose-fit coculture-based sensitization assay (lcsa) sonnenburg a nsc- (ukrain) in the palliative treatment of pancreatic cancer. results of a phase ii trial association of funding and conclusions in randomized drug trials: a reflection of treatment effect or adverse events a general method for the covalent labeling of fusion proteins with small molecules in vivo robust single-particle tracking in live-cell time-lapse sequences correlation of structural class with no-observed-effect levels: a proposal for establishing a threshold of concern trbp recruits the dicer complex to ago for microrna processing and gene silencing index a , , , , objective: hypertension and arterial stiffness is influenced by environmental and genetic factors. high plasma sodium concentration leads to mechanical stiffening of endothelial cells resulting in endothelial dysfunction and elevated blood pressure. here we investigated whether endothelial cell stiffness of ex vivo preparations of human arteries is linked to plasma sodium concentrations and functional genetic variants of the mineralocorticoid receptor (nr c ), rs modulating blood pressure, renin, and aldosterone levels, and rs , which alters a mirna binding site. design and methods: twenty patients were enrolled after a vein stripping procedure and collateral arterial blood vessels were prepared for atomic force microscopy (afm). plasma sodium concentration was routinely determined and dna for genotyping was extracted from edta blood samples. sodium levels > mmol/l were defined as 'high'. after application of µm amiloride, a specific blocker of the endothelial sodium channel (enac) changes in endothelial cell stiffness, were defined as 'weak' (≤ %), or 'strong' (> %). statistical analyzes were performed by anova. results: in ex vivo artery preparations of patients with high sodium levels (n= ), mechanical stiffness of endothelial cells was tend to increase (∆ amiloride) (p= . ). both nr c variants were associated with a change > % in endothelial stiffness after amiloride treatment. the rs c allele was significantly associated with a strong amiloride response (p= . ), while the rs a allele only showed a trend towards stronger amiloride effects (p= . ). conclusion: our findings indicate that high plasma sodium concentration results in an increased endothelial amiloride response and thus influencing mechanical stiffness, modulated by functional nr c variants. our novel approach linking patients' sodium levels and genetic status to endothelial stiffness by afm will be further evaluated in larger clinical settings. protein expression changes in bap-exposed human bladder cancer cells from spliceosome activation towards redistribution of the cytoskeleton after long-term exposure to subacute concentration schmitz-spanke s., pink m., jeske e., stempelmann k., rehn s., verma n., rettenmeier a. w. universitätsklinikum essen institut für hygiene und arbeitsmedizin, hufelandstr. , essen, germany deregulation of the β-catenin signaling pathway plays an important role in the development of hepatocellular tumors. activating mutations in ctnnb (encoding β-catenin) are frequently observed in murine and human liver tumors (e.g. human hepatoblastomas). activation of β-catenin signaling induces an overexpression of several cytochrome p (cyp) enzymes, including cyp e . cytotoxicity of acetaminophen (aap) is based on its cyp e -catalyzed metabolism to the electrophilic compound n-acetyl-p-benzo-quinone imine, which forms covalent adducts with cellular macromolecules if depletion of glutathione occurs. treatment with aap should therefore lead to a selective damage of cyp e -overexpressing ctnnb mutated hepatoma cells. mice were injected with a single dose of the liver carcinogen n-nitrosodiethylamine (den) and subsequently treated with the tumor promoter phenobarbital to select for ctnnb -mutated tumors. administration of a single dose of aap ( mg/kg of body weight) followed the tumor promotion protocol. two days after treatment immunohistological analysis of the livers showed about % necrotic tissue in the larger tumors which were positive for glutamine synthetase (gs), a marker for ctnnb -mutated tumor cells. by contrast, gs-negative tumors remained unaffected. at later time points we observed regeneration processes with infiltration of the necrotic tissue by inflammatory cells followed by fibrotic cells. proliferation of normal hepatocytes surrounding the damaged areas could also be observed. however, repopulation of parts of the former tumor areas by remaining gs-positive tumor cells was also detected. these results suggest that treatment with aap might serve as a future therapeutic possibility to selectively poison cyp e -overexpressing hepatoma. release of , -epoxyeicosatrienoic acid ( , -eet) upon neuronal activity induces trpa -dependent mechanical pain hypersensitivity sisignano m. , epoxyeicosatrienoic acids (eets) are cyp-epoxygenase (cyp ) derived metabolites of arachidonic acid (aa) which act as endogenous signaling molecules in multiple biological systems. we investigated the specific contribution of , -eet to transient-receptor potential-(trp)-channel activation in nociceptor neurons, and its consequence for nociceptive processing. we found that during capsaicin-induced nociception , -eet-levels increased in the drg and it is released from activated sensory neurons in vitro. , -eet potently induced a calcium flux [ nm] in cultured drg-neurons which was completely abolished when trpa was deleted or inhibited. in spinal cord slices , -eet dose-dependently enhanced the frequency, but not the amplitude of spontaneous excitatory postsynaptic currents (sepsc) in lamina ii neurons that also respond to mustard oil (aitc), indicating a presynaptic mechanism. furthermore, , -eet-induced enhancement of sepsc frequency was abolished in trpa null mice, suggesting that , -eet pre-synaptically facilitates spinal cord synaptic transmission via trpa . finally, intrathecal injection of , -eet caused mechanical hyperagesia in wild type but not trpa null mice. we conclude that , -eet is synthesized upon acute activation of nociceptors and leads to mechanical hypersensitivity via trpa at central afferent terminals in the spinal cord. sisnaiske j. , hardelauf h. introduction: neurite outgrowth and plasticity of neuronal networks are essential processes e.g. during brain development and learning. thus, morphological readouts of neuronal connectivity are thought to be important endpoints to assess neurotoxic effects of environmental chemicals as well as when discovering new drugs. to analyze neurite outgrowth and connectivity level rapidly and easily in vitro we developed the network formation assay (nfa) (pct/ep / ). this platform requires a spatially standardized hexagonal array for culturing neuronal networks with no need to fix or stain the cells to visualize neuritic processes. methods: to demonstrate the feasibility of the nfa we performed experiments in which we disrupted mature neurite networks or inhibited generating networks of human sh-sy y cells with different concentrations of acrylamide (acr). we also observed the counteracting effects of brain-derived neurotrophic factor (bdnf) and calpeptin in these systems. to create the hexagonal array we used a poly(dimethylsulfoxide) bilayer stencil comprising through holes for adhesion spots and interconnecting tracks. plasma stencilling a peg-coated glass substrate produces adhesive nodes for the neurons and micron-scale-tracks for guiding neurite outgrowth and connectivity. results: in both systems, the developing and mature network, we found not only a concentration dependant effect of acr and bdnf but also a time dependant effect with a limited capability of the developing system to regenerate, even in the presence of acr. the co-treatment of the cells showed that inhibition of calpains by calpeptin might reduce the effect of intracellular elevated ca +, a known neurotoxic mechanism of acr. moreover, the neurothrophin bdnf acts via trkb receptors on pathways stimulating neurite outgrowth and thereby counteracting the adverse effect of acr. conclusion: with the nfa we provide a rapid and simple way to analyze neurite outgrowth and connection formation in real time. by spatially standardizing the array we provide assay coordinates to streamline the analysis process and bring it towards high throughput testing. furthermore preliminary data showed that modification of the surface with biomolecules allows cell adhesion of other neuronal celltypes (e.g. primary mouse neurons) and extracellular matrix proteins (e.g. laminin) stimulate neurite outgrowth via integrins. transcriptional regulation of nox by histone deacetylases siuda d. , , zechner u. , prawitt d. nox is a member of the nadph oxidase family, which represents a major source of reactive oxygen species (ros) in the vascular wall. nox -mediated ros production mainly depends on the expression levels of the enzyme. the present study is aimed to investigate the regulation mechanisms of nox transcription by histone deacetylase (hdac). in cultured human ea.hy endothelial cells, treatment with the pan-hdac inhibitors (scriptaid, trichostatin a, tsa, and suberoylanilide hydroxamic acid, saha) leads to a drastic decrease in nox mrna expression. a similar down-regulation of nox mrna expression can be achieved with sirna-mediated knockdown of hdac . hdac inhibition in endothelial cells is associated with enhanced histone acetylation in the human nox promoter region, with no significant changes in dna methylation. consistently, scriptaid-treated cells show increased chromatin accessibility in nox promoter. in addition, we provide evidence that c-jun plays an important role in controlling nox transcription. knockdown of c-jun with sirna leads to a marked downregulation of nox mrna expression. in response to scriptaid treatment, the binding to c-jun to the nox promoter region is reduced despite the open chromatin structure. in parallel, the binding of polymerase iia to the nox promoter is significantly inhibited as well, which may explain the reduction in nox transcription. in conclusion, hdac inhibition decreases nox transcription in human endothelial cells by preventing the binding of transcription factor(s) and polymerase(s) to the nox promoter. this is very likely because of a hyperacetylation-mediated steric inhibition. cyclopentenone prostaglandins induce oxidative dna-damage in hamster lung fibroblast v cells solecki g. m. key: cord- - r ndzv authors: nan title: posters date: - - journal: glia doi: . /glia. sha: doc_id: cord_uid: r ndzv nan university of colorado school of medicine, aurora, united states oligodendrocyte progenitor cells (opcs) migrate long distances before differentiating and myelinating axons. it is well established that both short and long-range soluble guidance factors, as well as contact with the surrounding extracellular matrix (ecm) and neighboring cells, can modulate opc migration and differentiation. however, the influence of cell-matrix interactions on opc responses to guidance molecules is less clear. previous in vitro studies of opc migration in response to semaphorins and netrin- yielded inconsistent results, but these studies were performed on a variety of ecm substrates, i.e., explants, collagen, poly-d-lysine. in the current study, we systematically studied opc migration in response to guidance factors on several different ecm substrates, to better understand the influence of ecm interactions on opc responses to chemotropic molecules. understanding the regulation of opc migration and differentiation has important implications for the treatment of demyelinating diseases such as multiple sclerosis (ms). for example, semaphorins a and f are expressed in active, but not chronic, ms lesions, and in demyelination/remyelination studies in animal models, semaphorins influence opc migration into lesions, affecting the rate at which remyelination occurs. in addition, aberrant expression of ecm ligands occurs in and around ms lesions. we performed live imaging experiments of opc migration on different ecm substrates in response to gradients of chemotropic molecules. these studies are combined with ihc, co-ip, western blot and rt-pcr analyses to determine the effects of ecm substrates on opc migration and signaling pathway responses to chemotropic molecules. our preliminarily results suggest that ecm substrate can regulate both the direction and rate of opc migration. semaphorin a is repulsive to opcs on laminin , while on fibronectin opc migration remains uniform in the presence of semaphorin a. since laminin is the ligand for a b integrin heterodimers, while opc migration on fibronectin is mediated by avb integrins, the repulsive effect of semaphorin a appears likely to function through b integrin. thus, signaling through the neuropilin-plexin receptor complex that underlies the repulsive effects of semaphorin a on opc migration may be modulated by interactions with b integrins. x. bai university of saarland, homburg, germany secondary injury processes after acute brain trauma involve activation of different cell types like astrocytes, oligodendrocytes and microglia cells. a complex and yet not completely understood sequence of cellular responses initiate functional recovery after the neurodegeneration process. by using double-transgenic mice gcpb (gfap-egfp plp-dsred ) mice, we were able to identify a particular type of activated glia expressing astro-and oligodendroglial properties simultaneously (ao cells) that transiently appeared after three types of acute cortical injuries (stab wound injury, pial vessel disruption and middle cerebral artery occlusion). ao cells could be labeled by oligodendrocyte precursor cell (opc) markers olig and sox , but not for markers of mature astrocytes or neurons. two-photon live imaging of gcpb mice revealed that ao cells originated from plp-dsred-positive oligodendrocyte lineage cells. electrophysiological inspection of ao cells in acutely isolated brain slices revealed the expression of delayed rectifying, voltage gated k currents, a typical property of ng glia. therefore, we classified ao cells as opcs. to follow the fate of ao cells which disappeared about days after the injury, we generated gfap-n-cre plp-c-cre rosa eyfp triple transgenic (crec) mice. under non-injury conditions, few recombined cells were observed. however, numerous recombined cells appeared after week of stab wound injury adjacent to the lesion site, and lasted over weeks. we found that - % of recombined cells were gfap-positive, - % of them were pdgfra-positive and - % were positive for the oligodendrocyte marker gstp. in line with that, about % of newly generated gfap-positive astrocytes were observed from ng -creert x rosa tdtomato mice after week of stab wound injury. these results provide a strong evidence for opcs giving rise to astrocytes after acute cortical injuries. to further understand the molecular mechanism, we performed intra-cerebral injection of bone morphogenetic protein (bmp , known to promote astrocyte, but blocking oligodendrocyte differentiation) into gcpb and crec mice. bmp , but not leukemia inhibitory factor (lif), significantly increased the number of ao cells as well as astrocytes in the respective mice. in conclusion, ng /opcs display strong potential to differentiate into astrocytes after acute cortical injury. for instance, sox and sox have a crucial role in opc specification and differentiation, respectively. using microarray analysis of oligodendrocyte lineage cells, we previously identified sox as a major regulator of oligodendrocyte development (sohn et al., ) . in opc cultures, sox expression was maximal at developmental corresponding to cell cycle exit and onset of differentiation. these findings, suggest that sox has critical functions in the controlling of opc cell cycle exit and differentiation. in order to decipher the functional role of sox in oligodendrocyte development, we generated a tet-sox :sox rtta double transgenic mouse line. this mouse strain allows a sox overexpression specifically in sox cells in a doxycycline dependant manner (tet-on system). in the cns of dox-treated transgenic animals, we showed that sox overexpression was specifically targeted in sox oligodendroglial cells. in the developing cns, sox overexpression was targeted in pdgfra /nkx . opcs and in olig /cc differentiated oligodendrocytes. to assess the effect of sox gain-of-function on oligodendrocyte development and myelination, we analysed opc proliferation, apoptosis and differentiation, after doxycycline induction from e . to p . in the embryonic spinal cord, we showed that overexpression of sox did not modify cell proliferation or the number of pdgfra opcs. these data strongly suggest that sox is not involved in early steps of oligodendrocyte development. interestingly, we found that sox gain-of-function drastically reduces the density of cc mature oligodendrocytes at postnatal stages, correlating with a delay in myelination in transgenic mouse spinal cords. altogether, our data reveal critical functions of sox in oligodendrocyte lineage progression and myelination. supports: arsep and nmss (usa). m.f is a phd fellow of the french mesr (ed c). m. rocha, p. fernandes, a.c. manhães, p.c. barradas, f. tenorio universidade do estado rio de janeiro, instituto de biologia, rio de janeiro, brazil nutrient restriction during the perinatal period exerts a profound influence on brain development. previous studies using an undernutrition protocol ( % protein diet) during the first half of the lactation period in rats showed that the offspring presented an altered feeding pattern, which reflected a metabolic imprinting effect on feeding behavior. there is a delay in the pattern of npy expression in the arcuateparaventricular (arc-pvn) pathway, reflecting an effect on the development of the hypothalamic circuitry, leading to metabolic imprinting. here we studied the effects of undernutrition on glial differentiation using the same model. we used rats from to postnatal (p) days of age whose dams were either fed a % protein diet (dg) or a normoprotein diet (cg) from p to p . we assessed ki , vimentin, gfap, ed- and cnpase distribution in hypothalamic nuclei using immunohistochemistry. our results showed impairment in cell proliferation, more evident in the pvn and in the lateral hypothalamus (lh), where dg animals showed a lower number of ki- positive (ki ) cells at p when compared to cg. however, at p and p , the number of ki cells was significantly increased in dg. from p onwards, staining intensity remained relatively stable and similar in all groups. dg animals showed a delay in astroglial differentiation, presenting a decrease in gfap expression and a peak of vimentin expression at p and p accompanied by an increase in vimentin/ki cells. an increase in the number of ed positive cells next to the third ventricle was also shown at the same ages in dg. cnpase staining was very faint in most nuclei and did not show any obvious difference in dg animals. our results suggest that glial differentiation is delayed in dg, which may contribute to the changes in hypothalamic circuitry that causes metabolic imprinting. recent studies have demonstrated that neural precursor cells (npcs) residing in the adult subventricular zone also exhibit the capacity to generate new oligodendrocytes following experimental demyelination. the relative capacities of these two cell types to contribute to oligodendrogenesis in this context remain largely unexplored. we have adopted transgenic approaches that enable either the specific labeling of npcs or oligodendrocytes and the interrogation of their subsequent fate within the corpus callosi of mice subjected to central demyelination induced by cuprizone. adult nestin-creer t : r r-eyfp mice were administered tamoxifen ( . g/kg/day) for days by oral gavage, inducing the permanent expression of yellow fluorescent protein (yfp) in npcs and their progeny. mice were subsequently fed . % cuprizone for weeks followed by weeks recovery on normal food to enable the analysis of remyelination. expression of yfp and other cellular markers was examined immunohistochemically to determine the fate and migratory potential of npcs in the remyelinating corpus callosum (cc). rostrocaudal analyses of cc in the cuprizonechallenged mice demonstrated that approximately % of yfp npcs commit to an oligodendroglial fate. there was robust recruitment of npc-derived oligodendroglial cells, with a -fold increase in yfp sox cells compared to unchallenged mice. most of these cells were mature oligodendrocytes (yfp cc ) and their density in the remyelinating cc was highest adjacent to the dorsolateral corner of svz and decreased proportionally with distance in the mediolateral axis. on the other hand, fate mapping of opcs using pdgfracreer t : r r-eyfp mice revealed that oligodendrocytes derived from parenchymal opcs were distributed in a converse manner to svz-derived oligodendrocytes. in addition, we found that, independent of the cellular source, many oligodendrocytes repopulated the corpus callosum as linear arrays of clonally derived cells, in a manner that recapitulated the postnatal development of these structures. these results demonstrate that lineage determination and maturation of oligodendroglia from npcs occurs efficiently in situ. we also reveal that remyelination occurs in a coordinated way, most likely secondary to interactions between a restricted pool of axons and a sentinel opc induced to proliferate in situ. play an important role in developmental oligodendrogenesis and myelination and mounting evidence from rodent models of demyelination suggest that they also promote remyelination. the therapeutic application of thyroid hormone to demyelinating disorders is limited by the potential for cardiac side effects mediated by thyroid hormone receptor (thr) a signaling. here we report that gc- , a thyromimetic with selective thrb action promotes in vitro oligodendrogenesis from both rodent and human oligodendrocyte progenitor cells. in addition, we used pdgfar-creer;rosa -eyfp double-transgenic mice to examine the effect of gc- on the fate of oligodendrocyte progenitor cells and find that treatment with gc- during developmental myelination promotes oligodendrogenesis in vivo. these results indicate that a b receptor selective thyromimetic can enhance ol differentiation in vitro and during developmental myelination and warrants further study in demyelinating models. the olfactory epithelial layer contains multipotent horizontal basal cells (hbcs) that differentiate into olfactory sensory neurons. we generated olfactory sphere (os) cells in cultures that were derived from adult rat olfactory mucosa. fluorescence-activated cell sorting and immunofluorescence analyses showed that os cells were derived from hbcs. os cells underwent neuronal and glial differentiation in vitro. to examine multipotent differentiation in vivo, os cells were transplanted into injured rat spinal cords. the transplanted cells integrated into host tissue and underwent glial differentiation. our data showed the stem cell properties of hbcs. oligodendrocyte progenitor cells (opcs) comprise the main cycling cell population of the cns parenchyma during the early postnatal period and at adult stages. however, the molecular mechanisms implicated in opc divisions are still by large obscure. with the aims to unveil i) whether opc divisions exploit the same molecular machinery of neuronal precursors, and ii) whether distinct opc subsets exist with different cell division machineries, we focused on a mutant mouse where citron-kinase, a crucial regulator of cytokinesis in neuronal precursors, is germinally ablated (cit-k ko). these mice have severe cns developmental abnormalities, reduction of selected neuronal populations due to apoptosis triggered by defective divisions, display ataxia, epileptic seizures and die by the third postnatal week. interestingly, they were reported to have myelin defects, suggesting that the cit-ko affects oligodendroglial cells. notably, already early after birth we found a -fold decrease in opc density in both the cortical grey (gm) and white matter (wm), compared to wild-type mice (wt). here, most opcs were hypertrophic and multinucleated, indicating a cytokinesis failure after nuclear division. at later ages, opcs progressively disappeared from the cortical gm, while in both wm and ventral areas of the telencephalon (i.e. striatum, hypothalamus), uni-and multi-nucleated opcs could be detected, although at lower densities compared to wt. these data suggests opc heterogeneity in both cit-k requirement for cytokinesis and susceptibility to death. however, even where normal uninucleated opcs persisted, we could not find pre-myelinating or myelinating cells, in line with additional differentiation defects. to discriminate the contribution of cell-autonomous vs. environmental factors in differentiation impairment, we performed homochronic grafts of wt fluorescently tagged (gfp ) cells into perinatal cit-ko mice. strikingly, gfp opcs invaded the whole cit-k ko brain and actively divided. however, they hardly ever differentiated into more mature cells at difference with cells grafted in the wt, indicating that altered environmental signals contribute to myelination defects in cit-k ko. in conclusion, our results show that cit-k is involved also in glial cell divisions and suggest distinct cit-k requirement and proneness to death of dorsal and ventral opcs. additionally, cit-k ablation results in pervasive nervous tissue alterations affecting opc maturation. further studies will clarify the molecular mechanisms underlying these alterations. knowledge of how these cells act in repair is lacking, partly due to a limited understanding of their behaviour in normal developmental processes. radial glia cells are a unique population of neural progenitors that have recently been suggested to function in neurogenesis in the cerebral cortex. however, many questions still remain regarding their specific role in the developing spinal cord. using an organotypic spinal cord slice culture model and two-photon microscopy we directly visualized radial glial cell behaviour in the developing spinal cord. mm slices of rat embryonic spinal cord (e -e ) were cultured in order to preserve the d cytoarchitecture of the cellular environment. using electroporation, slices were transfected with a brain-lipid binding protein (blbp) driven gfp expression plasmid, which specifically labels endogenous radial glial cells. slice cultures were then imaged using two-photon time-lapse microscopy, allowing for high spatially and temporally resolved d imaging. we were able to follow individual gfp expressing radial glial cells over time ( hours to days) and characterized precise morphological changes. at early developmental ages, gfp expressing cells initially exhibited a radial morphology with the soma located in the ventricular zone and processes extending out to the pial surface. over time, many cells developed a multipolar morphology and migrated away from the lumen, primarily using somal translocation. using immunohistochemistry we identified populations of transfected cells which also expressed gfap, representing the differentiation of blbp-expressing radial glia cells into astrocytes. we also found that radial glial cells differentiate into oligodendrocytes (olig and cnpase positive) but found little evidence for gfp co-localization with neuronal markers (neun, dcx, biiitubulin). using this model we can directly observe complex developmental behaviours of specific cell populations and elucidate the relevant genetic regulation and molecular mechanisms that govern spinal cord development. by understanding these intricate events, we will gain insight into how a simple tube of undifferentiated neuroepithelial cells generates different cell populations that will eventually develop into the adult spinal cord. methods: we used the new cre-expressing mouse strain cx cr creer to express a diphtheria toxin receptor (dtr) specifically on microglia/macrophages. in these mice, administration of tamoxifen leads to cre-mediated excision of a loxp flanked transcriptional stop element in both macrophages and microglia, thus allowing for the expression of the dtr as well as an yfp reporter. due to their fast turnover macrophages are replaced by unaffected precursors whereas microglia persist in the modified stage. these mice were used for in vivo and ex vivo analysis of microglia by facs and histology. results: our optimized protocol with two tamoxifen injections at the age of weeks resulted in - % of reporter-positive microglia in adulthood, whereas all peripheral macrophages were reporter-negative. with additional three successive dt injections we could achieve a microglia ablation efficiency of %. interestingly, histological as well as facs analysis revealed % of microglia repopulation already days after depletion. two weeks after depletion microglia numbers were even higher compared to unaffected controls. on day , we found a cd . high ly c population, indicating replenishment by peripheral monocytes. in bm chimera experiments, where peripheral cells were labeled with a congenic marker, we obtained striking evidence showing that almost all of the repopulating cells are of peripheral origin. conclusions: after depletion microglia were rapidly repopulated, which emphasizes their critical role in brain homeostasis and function. even though ms has an onset in young patients, it persists throughout life and progresses along adulthood and aging. therefore, mscs from aged individuals should be tested for their pro-oligodendrogenic activity before moving into the clinical trials. this is insofar of clinical relevance, since the brain's capacity for self-repair and remyelination strongly declines with aging. thus, the aim of this study was to determine whether mscs from aged individuals maintain their known prooligodendrogenic activity. mscs were obtained from bone marrow of young ( months) as well as old ( - months) rats and analyzed their stem cell properties as well as their pro-oligodendrogenic activity. as expected, mscs derived from aged bone marrow presented diminished stem cell properties and differentiation capacity. to test the msc prooligodendrogenic activity, npcs were incubated with msc conditioned medium. surprisingly, soluble factors derived from old rats mscs maintain their ability to induce an increase of mbp-expressing oligodendrocytes on npcs. moreover, aged mscs conserve their oligodendrogenic activity also on npcs derived from old donors ( months). these results indicate that mscs keep their oligodendrogenic activity regardless of age and, therefore, this study provides a rationale for clinical trials of autologous msc transplantation in ms therapy. munich cluster for systems neurology (synergy), munich, germany astrocytes fulfill essential functions under physiological conditions and are involved in various beneficial and adverse functions after brain injury (reviewed by sofroniew ). in order to improve functional recovery after injury, it is essential to delineate the beneficial from adverse effects. however, little is yet to know which extent distinct sets of astrocytes perform distinct functions or whether all astrocytes behave in a uniform manner. live imaging of astrocytes after stab wound injury in the adult cerebral cortex in vivo revealed a striking heterogeneity of astrocyte behaviour with distinct subsets extending long processes towards the site of injury, others proliferating and yet other subtypes undergoing only hypertrophy. most strikingly, the vast majority of astrocytes proliferating were located with their somata directly at blood vessels (capillaries or post-capillary vessels). this close proximity was confirmed at ultrastructural level and identified as juxtavascular positions, sometimes adjacent to pericytes located in the perivascular space. this novel population of reactive astrocytes at juxtavascular positions is of particular interest, as the capacity of astrocytes to reactivate proliferation ( cell stem cell, in press). therefore we examined whether this population of juxtavascular astrocytes would also preferentially proliferate in even more severe injury conditions, such as hour of middle cerebral artery occlusion (mcao). the proliferation of juxtavasular astrocyte was analyzed immunohistochemically in fixed brain sections co-stained for s b/gfap/ki /cd at different time points after injury. at days post injury, % of all astrocytes were actively dividing (ki ). strikingly the majority of these was again located at juxtavascular positions ( %), suggesting a general bias of astrocytes at this position to proliferate after injury, while astrocytes further distant from blood vessels virtually fail to divide. we shall further present data from conditional knock-out mice with reduced proliferative activity of juxtavascular astrocytes in order to elucidate the function of this highly specific astrocyte population and their proliferative reaction after injury. a.d. rivera, a. butt university of portsmouth, portsmouth, united kingdom the generation of oligodendrocytes (ols) from their precursors (opcs) occurs throughout adult life and is particularly important following demyelinating insults, such as occurs in multiple sclerosis (ms). glycogen synthase kinase -b (gsk b) acts as a constitutively active negative regulator of multiple pathways that regulate proliferation, survival and differentiation of opcs. lithium is a potent inhibitor of gsk- and is used for the treatment of bipolar disorder, but in addition is neuroprotective in a wide range of diseases, including stroke, amyotrophic lateral sclerosis, and alzheimer's disease. lithium inhibits gsk- by binding directly to the enzyme's magnesium-sensitive site and indirectly by enhancing its phosphorylation. notably, by inhibiting gsk b, lithium acts as a wnt mimetic to promote b-catenin-dependent transcriptional events, including enhancement of genes involved in apoptotic inhibition. here, we have examined the effects of lithium on oligodendrocytes in the adult mouse optic nerve, a typical cns white matter tract; all procedures were in accordance with the animals scientific procedures act ( ) . transgenic mice aged postnatal day (p) in the developing and adult cns multipotent neural stem cells reside in distinct niches. specific carbohydrates and glycoproteins are expressed in these niche microenvironments that are important regulators of cell fate determination and stem cell maintenance. in previous studies we described lewisx (lex) glycan as a specific marker of neural stem precursor cells (nspcs) in developing brain and showed it to be involved in nspcs migration and proliferation. using lex glycosylation as a biomarker we aimed to identify the lex carrier glycoproteins which could have a functional relevance for cns development. in addition to already known lex carriers we have found lrp as new major lex carrier and for the first time showed it's expression in nspcs and developing brain. lrp is essential for the normal neuronal function in adult cns, whereas the role of lrp in development is still unclear, mainly due to the lethality of the lrp knock-out in mice. in the current study we investigated the basic properties of lrp knock-out nspcs created by means of cre-loxp mediated recombination. the elimination of lrp in vitro was induced by the addition of cell permeable cre-recombinase to nspcs derived from embryonic brain of lrp flox/flox mice. the functional status of lrp -deficient cells was subsequently studied using proliferation, migration and differentiation assays. lrp knock-out cells maintained as neurospheres, retained the ability to migrate and differentiate. interestingly, lrp knock-out nspcs generated -times less opcs in comparison to lrp wt/wt cells. this suggests that lrp is involved in the control of the oligodendroglial differentiation. astrocytes are multifaceted cells in the adult central nervous system and react to pathology of the adult brain with a finely graded continuum of morphological and behavioral changes. given that astrocytes in healthy adult brain rarely divide outside the stem cell niches, but activate both proliferation as well as a stem cell potential after injury (for review see robel et al., ) , it is important to understand their exact mode of cell division. indeed, a defining hallmark of a stem cell is the capacity to self-renew -often by asymmetric cell division. therefore we set out to image reactive astrocyte divisions in vitro by developing a primary culture system for reactive astrocytes obtained from the somatosensory cortex of adult mouse at days after stab wound injury. continuous live imaging and single cell tracking (costa et al., (costa et al., , allowed us observing the mode of cell division mode as well the cell fate of their immediate progeny. results of our study reveal (i) that reactive astrocytes can undergo multiple rounds of cell division, (ii) the shh signaling has a profound influence on this behavior, (iii) reactive astrocytes divide with distinct modes of cell division, which can be influenced by the local microenvironment. taken together, this new culture system allows close examination of signals on the mode and multitude of reactive astrocyte cell divisions and provides a great tool to identify signals derived from the extracellular matrix, other cell types or as diffusible signals. interestingly, even without injury temporary depletion of proliferating opcs leads to behavioral deficits which will be described. to identify the molecular mechanisms by which ng cells may react and influence other cells after brain injury, we isolated sox -gfp cells by facs either from the intact or the lesioned cerebral cortex at three days after stab wound injury. comparative genome-wide expression profiling revealed exciting candidates for opc function in the physiological and pathological brain. oligodendrocytes are the myelin forming cells of the central nervous system (cns) that enable fast salutatory signalling transduction and mediate trophic support and protection of axons. oligodendrocytes originate from a population of precursors cells (oligodendrocyte precursor cells, opcs) that are formed at distinct developmental stages. the biological process leading to opc differentiation involves a cascade of molecular events that eventually constitute the complex and multifaceted cellular program that determines the generation of mature oligodendrocytes. to identify the earliest detectable transcriptional correlates of differentiation we used a microarray approach investigating changes in gene expression occurring in primary rat opcs within the first hours after purification. amongst the most regulated factors that were detected was hairless (hr), a nuclear receptor co-repressor that has been associated with thyroid hormone signalling in the neonatal cns. validation of the microarray results by rt-qpcr, western blot and immunochemistry confirmed a rapid increase of hr expression during opc differentiation in media containing thyroid hormone. in the absence of thyroid hormone, hr expression was suppressed and associated with impaired opc differentiation. similarly, rnai mediated gene silencing of hr induced an impairment of opc differentiation demonstrating an important functional role of hr in the differentiation program. thyroid hormones regulate hr expression via the thyroid hormone receptors thra and thrb as rnai-mediated gene silencing of each of the receptors resulted in a decrease in hr expression. hr is known to negatively regulate rar-related orphan receptor alpha (rora) and vitamin d receptor (vdr). however, rnai-mediated gene silencing of rora and vdr did not change the course of opc differentiation. interestingly, an interaction between hr and histone de-acetylases (hdacs), of which hdac and are known regulators of opc differentiation, has been noted in the past. using an in situ protein-protein interaction assay we were able to demonstrate a direct interaction of hr with hdac and hdac in differentiating opcs. moreover, chip qpcr analysis revealed hr-hdac recruitment to the opc differentiation inhibitor hes . taken together these data show that thyroid hormone promotes opc differentiation via hr by modulating hdac and function. as iodine deficiency results in hypothyriodism our results give an example of a developmental disease that is triggered by environmental factors (the absence of iodine) causing epigenetic dysregulation. c. stagni, a.d. rivera, a. butt university of portsmouth, portsmouth, united kingdom astrocytes respond to all forms of cns insults by reactive astrogliosis, which involves changes in their molecular expression and morphology, and in most severe cases glial scar formation. reactive astrogliosis is potentially neuroprotective, but can also act as chemical and physical barrier to axon growth and repair. however, mechanisms underlying this process are still poorly understood. the enzyme glycogen synthase kinase -b (gsk- b) is a key regulator of many signalling pathways involved in cell proliferation, survival and differentiation, including wnt signalling. lithium is a potent inhibitor of gsk- and acts as a wnt mimetic to promote b-catenin-dependent transcriptional events. wnt signalling is altered following cns injury and in a range of neurodegenerative diseases, such as stroke, amyotrophic lateral sclerosis, and alzheimer's disease. notably, lithium is neuroprotective in these same diseases. here, we have examined the effects of wnt a and lithium on astrocytes of the adult mouse optic nerve, a typical cns white matter tract; all procedures were in accordance with the animals scientific procedures act ( ). we used transgenic mice aged postnatal day (p) inwhich gfap drives the expression of egfp. optic nerves (ons) were isolated with the retina intact and maintained in organotypic culture for days in vitro (div), in control medium, or medium containing lithium chloride, or wnt a. both agents resulted in a significant increase in the number of astrocytes, compared to controls (p < . , unrelated t-tests), but lithium and wnts a had markedly divergent effects on astrocyte morphology. in the presence of lithium, astrocytes became highly polarised, extending long thin processes that traversed the nerve perpendicularly to the long axis. in contrast, wnt a treatment resulted in the generation of morphologically simple astrocytes, with short, fine branching processes that extended radially from a centrally located cell body. genome wide microarray analysis indicated wnt a and lithium differentially altered chondroitin sulphate proteoglycans (cspgs), a family of extracellular matrix molecules highly expressed at cns injury sites. further experiments are required to define the molecular phenotypes of the novel astrocytes generated by these treatments, but the results provide evidence that wnt signalling regulates astrogenesis in situ and is therefore a potential molecular target to modulate astrogliosis. the contrasting effects of lithium suggest it is acting via other pathways, which require further study. studies of mouse schwann cell culture. the proliferation of scs is regulated by axonal signals and several growth factors. growth factors that are essential for rodent sc survival in culture include heregulin-b and forskolin. in the case of rat scs, these factors are also sufficient to allow a robust proliferation. in contrast, the proliferation of mousederived scs in culture requires the presence of other growth factors. we studied effect of several factors known to be expressed in scs after nerve injury on mouse sc proliferation. these included fibroblast growth factor type (fgf), pituitary extract (pe), epidermal growth factor, platelet-derived growth factor bb, and transforming growth factor b . we found that heregulin-b and forskolin are essential for mouse sc to survive in culture, fgf and pe were the best combination of growth factors promoting the mouse sc proliferation, and that neuronal axons provide effective mitogenic environment for mouse scs. the expression of the large myelin-associated glycoprotein in pns was studied in scs cultured alone and in cocultures of scs and drgns. when schwann cells are alone without axons, expression of the cytoplasmic domain of l-mag is seen in the nucleus of schwann cells. when schwann cells make contact with neuronal axons, the location of the expression shifts out of the nucleus to the perinuclear and cytoplasmic regions; the extracellular domain is not visible. until the schwann cells initiate the myelination programme. these results support the hypothesis that l-mag might have role in the regulation of the myelination programme in the pns. it has been postulated that trophic support provided by either stem cells or precursors is actuallyone of the most beneficial effects in the cell-based therapies. to address this issue, a comparison between the neuroprotective properties of opcs and the stem cells derived from warton jelly was carried out in the co-culture system with ischemically injured organotypic hippocampal slices. those two cell types were selected in respect of the advancement in their differentiation process. while the mesenchymal stem cells residing in warthon jelly are still pluripotent, the opcs are already the glia-committed precursors. to evaluate their neuroprotective properties, we set -up an ex vivo co-culture system of either stem cells or opcs isolated from neonatal rat brain with organotypic hippocampal slices subjected to a short oxygenglucose deprivation (ogd). the cell death, as well as the number of the newborn cells has been quantified in slices co-cultured for one week. a microarray analysis of a broad spectrum of trophic factors and cytokines expressed by opcs was performed for the purpose of finding the factor(s) contributing to the observed effect. three of them were selected for the subsequent blocking experiments with specificl antibodies to verify their influence on the injured tissue. the results obtained in the study prove that both cell types secrete soluble factors and are potent in exerting the neuroprotective effect in a paracrine-like manner, promoting cell survival and proliferation in damaged hippocampal slices. in conclusion, the observed advantageous qualities of stem cells and oligodendroglia-biased progenitors significantly contribute to anticipating a clinical improvement in cell replacement therapies. ng is a type i transmembrane glycoprotein and also known as chondroitin sulphate proteoglycan (cspg ). in the central nervous system ng -expressing cells have been identified as a novel type of glia with a strong potential to generate oligodendrocytes in the developing white matter. for temporally-controlled gene targeting of ng glia in vivo, we generated a mouse line in which the open reading frame of the tamoxifen-inducible form of cre recombinase (creert ) was inserted into the ng locus by homologous recombination. here, we investigated the differentiation potential of ng glia at different developmental stages of the forebrain. cre recombinase activity was induced at embryonic day . , postnatal day (p ), p , p , p and p by intraperitoneal tamoxifen injections into novel tgh(ng -creert ) mice crossbred to rosa -tdtomato and rosa -eyfp reporter lines that helped to identify ng glia and its progeny. recombination and cell identification were determined to days after the first tamoxifen injection. induction of recombination at embryonic stages revealed that embryonic ng cells mainly generated more ng glia and oligodendrocytes, however, a significant number of astrocytes could be detected as well. recombined cells were predominantly restricted to the ventral brain. in contrast, after tamoxifen injections at p and p recombined cells were widely found in all brain regions, thereby suggesting the presence of a distinct subpopulation of ng cells after birth. interestingly, only very few recombined astrocytes could be found, which are probably derived from few remaining embryonic ng glia. starting from p , ng glia stopped generating astrocytes, with the progeny largely restricted to the oligodendrocyte lineage. however, consistently, we found recombined neun cells with the morphology of neurons in the ventral cortex after administration of tamoxifen at p . even more of such cells were present in the whole cortex when cre activity was induced in adult mice. the morphology, the presence of neun immunoreactivity and our electrophysiological characterization that demonstrated bona fide action potentials clearly identify these cells as functional neurons. our results suggest that ng cells display a broad differentiation potential that appears to be highly age-dependent during development. brdu-assay) and the number of surviving cells (by counting dapi-stained nuclei). after we had observed, that the choice of the basal medium (neurobasal, rpmi or dmem) exerts a pronounced effect on opc proliferation we here investigated the effect of cell density on opc proliferation. starting from a small seeding density ( . cells/cm ) a tenfold increase in seeding density only resulted in a fourfold augmentation of the density of surviving cells after days in culture. using plating densities of . ; . and . cells/cm we observed, that an increase in seeding density led to a decrease in the proliferation rate, as tested with a brduassay and monitored after days in culture. similarly, the percentage of a b -positive, immature, cells was lower, the higher the cell density. we then tested whether this finding is due to proliferation inhibiting factors secreted in cultures with high seeding densities and whether the remaining microglia population could be involved. to this aim we seeded opcs at low density ( . cells/cm ) and cultivated them with the medium supernatant of cultures with high density ( . cells/ cm ). furthermore we seeded opcs in low density ( . cells/cm ) and co-cultivated them with an added density of microglia as counted in cultures of high density ( . cells/cm ). the resulting cell counts after days showed, that the percentage of brdu positive cells after cultivation with the supernatant was significantly reduced compared with control cultures, although the number of a b -positive cells was not influenced by this treatment. the addition of microglia to the cultures lead to a strong reduction of surviving cells, even though the percentage of brdu and a b positive cells were not altered. so apoptosis, necrosis or phagocytosis might be responsible for the reduction of surviving cells. our findings suggest that the reduction of surviving opcs in high density cultures is due to secreted factors inhibiting opc proliferation and that microglia could be involved in this effect. in neuropathological studies, it is important to detect microglial cells; however, a specific microglia marker has yet to be determined. [methods] in this study, we examined and compared the usefulness of various microglia markers, including human glucose transporter- (glut- ), mhc class ii, cd , and iba- . [results] all of the microglia markers consistently stained for microglia in paraffin sections fixed with formalin. cd staining of microglia in the gray matter was not as well defined as the other microglia markers. moreover, cd did not clearly stain the microglial processes. with regard to mhc class ii, it was difficult to observe the microglial processes in the white matter. in contrast, both glut- and iba- clearly stained for microglia in the gray and white matter, and provided detailed staining of microglial processes. although it has previously been shown that perivascular cells are negative for glut- , we found perivascular macrophages to be glut- positive. [conclusions] nevertheless, glut- and iba- may be considered as markers suitable for routine histopathological staining procedures. neurons and glia of the enteric nervous system (ens) originate from a pool of sox progenitors. in addition to being a scaffold for enteric neurons, enteric glial cells (egcs) have been suggested to function in mucosal integrity, neuroprotection, adult neurogenesis, neuro-immune interactions and synaptic transmission. currently, diverse glial populations are known to reside within the ens. however, whether, specific functions are carried out by specialized glial subtypes, with characteristic morphologies and molecular markers and within specific locations in the gut remains elusive. to address this question we used a repertoire of genetic tools, immunostaining and imaging techniques. for high single-cell resolution study of egcs, we mosaic analysis with double markers (madm) in conjunction with sox -cre. we analyzed adult gut tissue to characterize the different glial populations based on morphology, position in the ens and their relationship with the intestinal epithelial and vascular system. moreover, we used reporter mice (sox -icreer t ; r r-yfp and gfap-icreer t ; r r-yfp) to quantify the expression of three glial markers -gfap, s b and sox in myenteric plexus (mp) preparations of adult mice. our investigation on madm-mp preparations, revealed three subtypes of enteric glia in the mp of adult mice. type i and type ii glia, present in the primary plexus of mouse ens have been previously defined. in addition, we characterized a third subtype in the extraganglionic space of the mp of adult mice, at present termed as type iii-extraganglionic glia. furthermore, our analyses using d-imaging, on madm-gut sections allowed us to characterize the relationship of enteric glia located within the mucosa, with the intestinal epithelium and vascular system of the villi. our marker expression analysis showed that while the majority of glial cells co-expressed gfap and s b, a large fraction of glia within the myenteric ganglia was positive for either s b ($ %) or gfap ($ %). we also found that s b /gfapglia were abundant outside the ganglia. most glial cells co-expressed sox with gfap and s b yet a significant proportion ($ %) of mainly extra-ganglionic glia (type iii) expressed only sox . surprisingly, $ % of the cells that were s band sox displayed high gfap expression. use of gfap-icreer t ; r r-yfp mice showed that about $ % of yfp-labelled cells did not express gfap, implying dynamic regulation of gfap expression. overall, our high resolution studies reveal extensive heterogeneity of enteric glial cells in terms of morphology, position and expression of molecular markers suggesting differential functional roles. our future experiments will address whether different physiological roles of egcs can be assigned to specific subtypes. university of washington, seattle, united states m€ uller glia are the major type of glia in the retina, spanning its entire thickness. it is known that bmp signaling promotes astroglial differentiation in cortex, but its role in m€ uller glial development in the retina has not been studied. the purpose of this study was to investigate the role of bmp-smad / / signaling in m€ uller glial development. c bl/ mouse retinas were collected at various ages between postnatal day (p ) and p in order to analyze the expression and activation of smad / / . smad / / was transiently but robustly activated in the inner nuclear layer, including developing m€ uller glia between p -p . the timing of this transient activation of smad / / corresponds with the timing of m€ uller glial differentiation and maturation. to test the hypothesis that activation of bmp-smad / / signaling is essential for m€ uller glial development, retinas were explanted at p or p and treated with bmp or a bmp inhibitor dorsomorphin (dm) for days in order to activate or inhibit smad / / , respectively. when p and p retinal explants were treated with dm for days in vitro, there was a significant reduction in m€ uller glial markers (cralbp, gs, sox , and sox ). smad / / activation and cralbp expression were also significantly attenuated in vivo day after intraocular injection of a bmp inhibitor dm at p . our data suggest that activation of bmp-smad / / signaling is necessary for the development of m€ uller glia. this work was supported by r ey to tar. huazhong university of science and technology, wuhan, china rho-associated kinase (rock) has been identified as an important regulator of proliferation and cell cycle progression in a number of cell types. although its effects on astrocyte proliferation have not been well characterized, rock has been reported to play important roles in gap junction formation, morphology, and migration of astrocytes. in the present study, our aim was to investigate the effect of rock inhibition by [( )-(r)trans- -( -aminoethyl)-n-( -pyridyl) cyclohexanecarboxamide dihydrochloride] (y ) on proliferation and dna synthesis in cultured astrocytes from rat spinal cord and the possible mechanism involved. western blots showed that treatment of astrocytes with y increased their expression of cyclin d , cdk , and cyclin e, thereby causing cell cycle progression. furthermore, y -induced astrocyte proliferation was mediated through the extracellular-signalregulated kinase signaling cascade. these results indicate the importance of rock in astrocyte proliferation. here we used cell transplantation experiments to determine to which extent this differential behavior is regulated by environmental signals differing between the wm and gm, or intrinsic fate determinants restricted to one or the other cell population. these experiments indicate that the wm promotes differentiation of opcs to mature oligodendrocytes, as the rate of differentiation of gm cells increased when exposed to this environment. interestingly, cells derived from the wm retained their higher differentiation rate also in a less supportive environment like the gm. these results reveal not only important environmental differences for opc maturation, but also support the concept of intrinsic heterogeneity among adult opcs, persisting even when exposed to more inhibitory environments. thus, this work provides the basis to identify molecular pathways regulated by distinct niche/environmental signals and involved in the heterogeneity of adult opcs. multiple sclerosis (ms) is a chronic inflammatory and neurodegenerative demyelinating disease of the central nervous system (cns) characterized by inflammation, which leads to formation of demyelinating areas due to loss of oligodendrocytes, astrogliosis and, finally, axonal degeneration. different studies have suggested that - -cyclic adenosine monophosphate (camp) levels might play an important role in neuroprotection and neuroinflammatory response so the modulation of this nucleotide intracellular levels might control the neuroinflammatory pathological process and, consequently, to delay the progression of ms. intracellular camp levels depend on its synthesis by adenylyl cyclases, and on its degradation by cyclic nucleotide , -phosphodiesterases (pdes). specific inhibitors for the different isoforms of pdes family, particularly camp-specific pde, emerge as putative treatments of this kind of disease. pde has emerged as a new therapeutic target, not only for a variety of immunological and immunodeficiency conditions to alleviate chronic inflammation, but also for several neurodegenerative disorders, including ms, in which both immune system and cns are implicated. in the present work, we have detected the expression of pde in oligodendrocyte precursor cells (opcs) isolated from cerebral cortex of p and p mice and from adult human samples derived from neurosurgery of epilepsy. opcs are abundant in the mice and human cns during development but they also represent - % of the total number of cells in the adult cns, where they serve as a source of new oligodendrocytes to replace those which die. however, in ms these endogenous opcs are not able to effectively replace dead oligodendrocytes. in vitro, we have demonstrated that two newly-developed pde selective inhibitors (tc . and vp . ) favour opc survival and differentiation towards mature oligodendrocytes, being both more effective than the commercial pde inhibitor brl- . erk intracellular pathway has been identified as a key in these cellular processes related with the camp/pka/creb pathway. moreover, we have observed that both specific inhibitors (vp . and tc . ) increase the differentiation of human opcs. these findings, combined with the already known anti-inflammatory effect carried out by these pde inhibitors, point them as potential therapeutic agents to treat ms. the knowledge of factors that affect the biology of murine opcs and even more, human opcs, are critical for possible remyelination in this kind of pathologies. their role in cns remyelination has not been fully investigated. previous studies using genetic fate mapping techniques revealed their recruitment into injured spinal cord and differentiation into astrocytes and oligodendrocytes. in this study, we used aninducible cre-floxed-stop-rosa -yfp mediated lineage tracing strategy to characterize the fate of foxj expressing ependymal cells during remyelination of toxin-induced demyelination. we found that in unlesioned spinal cord, foxj labelled ependymal cells. however, it also labeled a population of cells in the spinal roots with immnochemical features of fibroblasts. following lysolecithin-induced demyelination in the dorsal funiculus, there were few yfp expressing cells in the lesion area at and days post lesion (dpl) indicating that the ependymal cells were not recruited in demyelinated area. however, in ventral white matter lesions, which were adjacent to damaged nerve roots, many cells were detected expressing yfp. the distribution of yfp positive cells overlapped that of remyelinating schwann cells and a number of yfp expressing cells colocalised with mature schwann cell marker periaxin. these data revealed a foxj expressing population in peripheral nerve which migrates into demyelinated lesions andcontributes to cns remyelinaiton. the generation of myelinating cells from multipotential neural stem cells in the cns requires the initiation of specific gene expression programs in oligodendrocytes (ols). we reasoned that micrornas (mirnas) could play an important role in this process by regulating genes crucial for ol development. here we identified mir- a as one of the highly enriched mirnas in oligodendrocyte precursor cells (opcs), overexpression of which in either neural progenitor cells (npcs) or embryonic mouse cortex promoted the generation of ol lineage cells. blocking the function of mir- a in differentiating npcs led to a reduction in ol number and an expansion of neuronal populations simultaneously. we also found that overexpression of this mirna in purified opc cultures promoted cell proliferation and inhibited further maturation. in addition, mir- a might exert the effects just mentioned partially by directly repressing proneuronal differentiation factors including pax and neurod , or prool genes involved in oligodendrocyte maturation. these results suggest that mirna pathway is essential in determining cell fate commitment for ols and thus providing a new strategy for modulating this process in ol loss diseases. a. guzman de la fuente , j. huang new sheaths can be regenerated following the recruitment of adult neural progenitor cells (opc) into areas of injury and their differentiation into myelinating oligodendrocytes. although this regenerative process (called remyelination) occurs efficiently in the early stages of multiple sclerosis, its efficiency gradually declines leaving axons demyelinated and vulnerable to degeneration. several signalling pathways are involved in regulating opc differentiation and subsequent myelination. understanding their mechanisms is necessary to design regenerative therapies to overcome remyelination failure. we have identified rxrc as a key positive regulator of opc differentiation and remyelination (huang et al., ). rxrc is a nuclear receptor forms a heterodimer with other nuclear receptors to activate its downstream signalling cascades. here we describe rxrc binding partners expressed in both opc and oligodendrocytes, and the binding of vdr, rarb and pparc to rxrc within oligodendrocyte lineage cells by coimmunoprecipitation. vdr-rxrc heterodimer is necessary for the oligodendrocyte differentiation. treatment of oligodendrocytes with vdr antagonist inhibits opc differentiation and abrogates the effect of -cis retinoic acid, an rxrc agonist, in opc differentiation. our data suggests a model in which rxrc is an essential anchor for different nuclear receptors, including vdr, with a shifting and complex rxrc signaling in oligodendrocyte lineage cells. cleavage of type i membrane proteins by a-and c-secretases to yield biologically active fragments is best exemplified for the evolutionarily conserved protein notch. here we show that the protein ng , a type i membrane protein which has homologues in mouse (ng ) and drosophila (kontiki) and is expressed by multiple immature cell types including glia, is processed in a similar fashion to notch. in both mouse and drosophila this generates a major kd shedded ectodomain, a c-terminal fragment (ctf) and a small intracellular domain (icd). the ng icd is present after overexpression in cells of biochemical isolates of nuclear fractions, and can be visualized in the nucleus by immunofluorescence of cultured murine glial cells. in glial nuclei of developing drosophila larva, a striking intranuclear staining of the kontiki icd is observed. levels of expression of ng mrna and protein in mouse glial cells are regulated by the neurotransmitter glutamate and overexpression of cleaved fragments modulates protein expression in murine glial cells. these results demonstrate that cleavage of ng takes place in mouse and drosophila glia and that this generates fragments with signaling properties. modulation of ng expression by glutamate provides a way for neuronal activity to regulate ng signalling. our goal is to understand the glial signaling that controls inflammatory responses in the central nervous system (cns). this glial response can play both a detrimental and beneficial role. toll like receptor (tlr) signaling is implicated in responses to pathogens or endogenous signals. tlr signaling mediates immune response by inducing cytokines, including type i interferons (ifn). ifn-beta, a member of the type i ifn family, is a first-line therapeutic for multiple sclerosis. type i ifns signal through a common receptor, ifnar. the aim of the present study was to investigate the in vivo response of microglia and astrocytes to cns administration of tlr ligand/agonist, and to examine whether this response involves type i ifn signaling. mice were administered tlr ligands/agonists by injection to the cisterna magna. we analyzed leukocyte entry to the cns by flow cytometry, and their localization by immunostaining. the induction of ifn-beta was examined by in vivo imaging of an ifn-beta reporter mouse. astrocytes and microglia were sorted by facs and gene expression was measured using quantitative real time-pcr. injection of ligands/agonists for tlr , and resulted in infiltration of cd leukocytes after hrs, most strongly by tlr . immunostaining showed parenchymal localization of infiltrating cells in cerebellum. facs sorted astrocytes expressed equivalent levels of tlr mrna to microglia but lower levels of tlr or . astrocytes were induced by tlr signaling to express interferon regulatory factor , which regulates the induction of type i ifn. the induction of ifnbeta in cns in response to tlr signaling was verified in ifn-beta reporter mice. tlr , and signaling led to increased levels of mrna for glial cxcl . together these results suggest the involvement of type i ifn signaling. however, unlike cxcl gene expression, that was dependent on ifnar signaling, tlr -induced leukocyte infiltration was not affected in ifnar deficient mice. these studies point to a role for tlr signaling in the innate glial response that regulates cns inflammation. microglial phagocytosis plays a key role in neuroprotective and neurodegenerative responses of the innate immune system in the brain. here we investigated the regulatory function of the signaling protein phosphoinositide -kinasec (pi kc) in phagocytosis of bacteria and zymosan particles by mouse brain microglia in vitro and in vivo. using genetic and pharmacological approaches our data revealed pi kc as an essential mediator of microglial phagocytosis. unexpectedly, microglia expressing lipid kinase deficient mutant pi kc exhibited similar phagocytosis as wild-type cells. detailed analysis disclosed pi kc dependent stimulation of camp phosphodiesterase as a crucial mediator of phagocytosis. both stimulation of protein kinase a and epac were shown to convey inhibitory effects of camp on phagocytosis. together our findings indicate pi kc-dependent suppression of camp signaling as an indispensible regulatory element of microglial phagocytosis. v. kovaleva, e. berezhnaya, m. rudkovskii, a. uzdensky southern federal university, rostov-on-don, russian federation photodynamic therapy (pdt) is currently used in oncology, particularly, in treatment of brain tumors. the possible role of no-mediated signaling in photodynamic injury and protection of neurons and surrounding glial cells (gc) was studied. crayfish stretch receptor consisting of a single neuron enveloped by gc was photosensitized with alumophthalocyanine photosens ( nm, min incubation) and irradiated with laser diode ( nm, . w/cm ). application of no generators notably mm sodium nitroprusside and mm nonoate reduced pdtinduced necrosis of gc and showed the same tendency in neuronal necrosis. nonoate significantly increased pdt-induced apoptosis of gc. inhibitor of neuronal no-synthase l-name ( mm) significantly increased the percentage of necrotic glial cells after pdt but did not influence neuronal necrosis. this confirmed the anti-necrotic effect of no in glial cells and involvement of neuronal no synthase in protection of glial cells. mm l-name and mm l-nna (another inhibitor of neuronal no-synthase) as well as lm s-methhylisothioharnstoff sulfat (inhibitor of inducible no synthase) protected gc from pdtinduced apoptosis. therefore no may be involved in pdt-induced apoptosis of glial cells. inhibition of no-activated protein kinase g with lm kt decreased the percentage of necrotic glial cells but not neurons. therefore protein kinase g appeared to be involved in pdtinduced necrosis of gc, possibly independently on no. kt also increased the level of apoptosis of gc indicating the anti-apoptotic role of protein kinase g. thus no is involved in regulation of pdt-induced necrosis of neurons and glial cells as well as in apoptosis of glial cells. g. tyzack, t. cymes, n. lau, a. lakatos university of cambridge, cambridge, united kingdom background and question. emerging evidence suggests that signalling by damaged neurones leads to reactive astrocyte response that may have a fundamental impact on synaptic reorganisation. soluble factors, such as cytokines, have been described to induce such astrocyte activation. however, rapid contact dependent signalling between damaged neurones and astrocytes is also a possibility. in light of recent findings of ephb upregulation on injured neurons closely surrounded by reactive astrocytes, we hypothesized that ephb can signal to ephrin-b expressing astrocytes. methods. to test this in simplified in vitro assays, purified astrocyte cultures were treated with clustered-ephb -fc, and the characteristic features of glial activation, such as cytoskeletal protein expression, stat phosphorylation, and p-stat nuclear transfer were monitored. results. we have demonstrated that ) astrocytes express ephrin-b , ) ephb triggers a two-fold increase in gfap and ezrin expression, ) ephb induces both stat phosphorylation and nuclear transfer by a three-fold value, and ) all effects were significantly diminished by knocking down ephrin-b in astrocytes. conclusions. our data show that ephb induces astrocyte activation by triggering ephrinb -mediated reverse signalling via stat activation. this work therefore suggests a potential novel route in neuron-astrocyte communication after injury. a further characterisation of this signalling pathway may provide a better understanding of mechanisms underlying glial activation and its role in plasticity. n. kramann , r. pf€ ortner , u.-k. hanisch , k. hagemeier , t. kuhlmann , w. br€ uck , c. wegner university medical center g€ ottingen, department of neuropathology, g€ ottingen, germany university hospital m€ unster, institute of neuropathology, m€ unster, germany introduction: laquinimod (laq) is an oral well-tolerated molecule that has been shown to reduce brain atrophy, disability progression and relapse rate in patients with relapsing-remitting multiple sclerosis. recent experimental data indicate that laq minimizes demyelination, inflammation and axonal damage in mice with cuprizone challenge. aim: since astrocytic nfjb activation plays a crucial role in cuprizone-induced demyelination, we investigated the effects of laq on cns cells in vitro and in vivo. methods: in vitro experiments used primary cells to test effects of laq on oligodendroglial survival as well as on cytokine secretion and nfjb activation in astrocytes and microglia. primary mouse astrocytes and microglia were pre-treated with , . and . mm laq and stimulated by pro-inflammatory cytokines. a dual-luciferase reporter assay was used to measure nfjb activity. for in vivo experiments, -week-old male c bl/ j mice were challenged with . % cuprizone and treated daily with laq ( mg/ kg) or water. to assess astrocytic and microglial nfkb activation in vivo, nuclear translocation of nfjb p was assessed in astrocytes and microglia by double immunofluorescence with antibodies against p and iba or gfap. results: in vitro, astrocytic but not microglial nfjb activation was markedly reduced by laq as evidenced by nfjb reporter assay. in astrocytes, pre-treatment with . and . mm laq significantly reduced the induced nfjb activity after tnfa stimulation compared to stimulated controls. pre-treatment with . mm laq also significantly reduced nfjb activation after stimulation with the combination of il- b and ifnc compared to untreated stimulated controls. oligodendroglial viability and survival were not affected by laq treatment. to confirm the in vivo relevance of these findings, we also examined astrocytic and microglial p translocation in mice with and without laq treatment after cuprizone challenge. the proportion of astrocytes with nuclear p immunoreactivity was significantly reduced in laqtreated mice ( . % . %) compared to untreated controls ( . % . %). microglia did not display marked translocation of nfjb p after cuprizone challenge. conclusion: our data indicate that laq prevents cuprizoneinduced demyelination by attenuating astrocytic nfjb activation. in vitro, laq reduced the astrocytic nfjb activation by up to % as evidenced by nfjb reporter assay. similar quantitative findings were obtained in vivo when laq treatment also led to a % reduction of astrocytes with nfjb activation, as evidenced by nuclear translocation of p . these findings suggest that targeting the astrocytic nfjb pathway might have therapeutic effects in demyelinating cns disorders. . we asked whether brain-derived neurotrophic factor (bdnf), known to be highly expressed in a circadian manner in scn, might be involved in the mechanisms governing these astroglial rearrangements. using an analog-sensitive kinase allele murine model (trkb f a ) and semi-quantitative electron microscopy, we found that the pharmacological blockade of the tropomyosin-related kinase receptor type b (trkb), the high affinity receptor of bdnf, abolished the day/night changes in glial coverage of vip dendrites. the bdnf/trkb signaling pathway therefore exerts a permissive role on the ultrastructural rearrangements that occur in scn across the day/night cycle. in contrast, the extent of glial coverage of non-vip dendrites was not different at daytime and nighttime in trkb f a mice submitted to trkb inactivation or not receiving any pharmacological treatment. considering the well-established role of bdnf in the clock's photic synchronization process, these dataprovide strong support to the view that the daily astroglial rearrangements in scn could be involved in the photic entrainment of circadian rhythms. above all, they highly suggest that they are necessary for the modulatory action of bdnf on the scn responses to light. features of this form of neurodegenerative ataxia, including a striking reduction in lifespan when polyq atrophins are expressed in the glia. a sensible hypothesis is that affected glia will act non-autonomously on neurons as in other neurodegenerative pathologies. this is an emerging field that yields new and important therapeutic possibilities. we now wish to elucidate the mechanisms through which degenerating glia impairs organism functions, and identify genes involved cell-autonomously and non-autonomously in generating this aspect of the pathology. first, we investigated the impact of glial polyq atrophin on lifespan. using different glia marker, we have demonstrated that the expression of polyq atrophin in glia, or specifically in astrocyte-like cortex glia, has a dramatic effect on drosophila lifespan while brood brain barrier glia do not have any phenotype. we will then investigate the effect of other subtypes of glia looking at lifespan and motility to identify the relevance of each subtype in drpla. second, to identify new regulators of gliopathology we will conduct unbiased genetic screens for mutations in neurons that modify the lifespan of drpla flies, expressing polyq atrophin within the glia. our studies will be paramount in deciphering the complex glia-neurons interactions in ataxias and may be beneficial for several ataxias. they will open up new avenues for therapies aimed at targeting glianeuron interactions during disease progression. in alzheimer's disease, microglial clearance of beta-amyloid (ab) is considered neuroprotective. whether ab uptake regulates microglial cell responsiveness, and whether macrophages participate, remains unclear. here we investigated whether in vivo phagocytosis of endogenously-produced ab disturbs the turnover of microglia and macrophages by changing rates of cellular proliferation and apoptosis. using app swe /ps de tg mice, we show that plaque-associated microglia and cd b cd high macrophages bind and ingest endogenously-produced ab in vivo. plaqueassociated microglia and cd leukocyte-like cells were also observed in brains from patients with alzheimer's disease. phagocytic microglia and macrophages had a reduced rate of proliferation compared to abcells. instead, high proportions of apoptotic annexin v microglia and macrophages were found in aged app/ps tg mice. phagocytic microglia and macrophages had greater caspase activity and increased p expression after in vivo uptake of ab, than cells that did not phagocytose ab. in vitro, we found that ab uptake induced caspase activation in microglia, whereas blocking ab uptake triggered apoptosis-induced cell death in macrophages. our data demonstrate that ab uptake impairs microglial/ macrophage viability and responsiveness. amyloid clearance may fail as phagocytic microglia and macrophages undergo ab-induced apoptosis. f. michetti, s. ceccariglia, a. d'altocolle, f. pizzolante, m. barba, a. delf a, c. gangitano universit a cattolica del s. cuore, rome, italy trimethyltin (tmt) is a neurotoxicant producing neuronal degeneration in the mammalian central nervous system , especially in the hippocampus (for reviews, , ). magnetic resonance imaging (mri) investigation in tmt-treated rats has evidenced dilation of lateral ventricles, possibly correlated to alterations in blood brain barrier permeability. we have investigated in the hippocampus and cortex of rats the expression of aquaporin (aqp ), a glial water channel protein which is regarded to play a role in brain oedematous conditions, to explore the molecular mechanisms involved in the phenomenon. tmt-treated aqp expression was tested both by real-time pcr and western blotting analysis in hippocampus and cortex homogenates. to confirm molecular results and visualize the aqp cell distribution, double-label immunofluorescence for aqp and gfap was performed. real-time pcr and western blotting data show a significant upregulation of aqp starting from days of tmt treatment in the hippocampus and, at a lower degree, in the cortex. accordingly, the immunofluorescence shows an intense astrogliosis and aqp immunoreactivity diffusely pronounced in the hippocampal and cortex areas starting from days after intoxication. aqp immunolabelling was localized in astrocytic end-feet encircling the blood vessels, as expected. the study of the rhodamine b fluorescent tracer, intraperitoneally administered, also revealed an intense vascular reaction, characterized by hypertrophic vessels with abnormal course and dimensions in the brain of tmt-treated rats, indicating a vascular involvement in the tmt-induced neurodegenerative processes. aqp over-expression and the concurrent astrogliosis occurring in the brain of tmt-treated rats might be candidated to play a role in alterations of vascular permeability and brain oedema formation evidenced by mri studies. amyotrophic lateral sclerosis (als) is a neurodegenerative disease affecting predominantly motoneurons but with a particular pathological role of non-neuronal cells. previous research suggested that increased concentration of extracellular potassium could lead to motoneuron dysfunction. the na /k -atpase should regulate the homeostasis of potassium ions and thus help maintain regular neuronal activity. this homeostasis may be regulated by na /k -atpase in both neurons and astrocytes. in order to reveal the possible role of the na /k -atpase in als pathology, we explored the expression of the alpha catalytic subunit of na /k -atpase in neurons and astrocytes from different brain regions of the sod g a rat model of als. labeling immunofluorescently the alpha isoform of the na /k -atpase revealed a decreased signal in the trigeminal nucleus and cerebellum of the als rat. double immunofluorecence with neun for neurons showed that the reduced alpha was on the neuronal surface in the trigeminal nucleus. in addition, the alpha ring-like staining of cerebellar granule neurons was lower in als as compared to the wild type rat. in comparison to neurons, astrocytes from the wild type rat were characterized with a lower expression of alpha . in the examined brain regions we did not observe a prominent difference in alpha expression between astrocytes of wild type and als rats. based on the findings of this study the prominent reduction of na /k -atpase level observed in neurons and not in astrocytes could contribute to further understaning of impaired ion homeostasis affecting proper neuronal physiology in als. the role of glial na /k -atpase needs to be further studied with markers of other alpha subunit isoforms. although commonly used as nutritional supplements, excessive intake of bcaas might favour the establishment of neurotoxic conditions as indicated by the severe neurological symptoms characterising inherited disorders of bcaa catabolism such as maple syrup urine disease (msud). recent evidence indicates that bcaas induce excitotoxicity through mechanisms that require the presence of astrocytes. in the present study, we evaluated the effects of bcaas on microglia, the main immune cells of the brain. as an experimental model we used primary microglial cells harvested from mixed glial cultures that had been kept in normal or high bcaa medium (h-bcaa). we show that h-bcaa microglial cells exhibit a peculiar phenotype characterized by a partial skewing toward the m state, with enhanced il- expression and phagocytic activity but also increased free radical generation and decreased neuroprotective functions. we suggest that such an intermediate m /m phenotype might result in a less efficient microglial response, which would promote the establishment of a low grade chronic inflammation and increase the likelihood of neurodegeneration. although based on in vitro evidence, our study adds on to an increasing literature indicating that the increasing use of dietary integrators might deserve consideration for the possible drawbacks. in addition to excitotoxicity, the altered immune profile of microglia might represent a further mechanism by which bcaas might turn into toxicants and facilitate neurodegeneration. the proteolipid protein gene (plp ), located on the x-chromosome, undergoes alternative splicing to produce the most abundant proteins of central nervous system myelin: plp and dm . related to the extent of myelin defect, mutations in the plp gene in humans are associated with a spectrum of x-linked disorders, from the severe pelizaeus-merzbacher disease (pmd), to the mild form spastic paraplegia type (spg ). phenotype-genotype correlation exists, large duplications of plp gene are predominantly found in pmd and null mutations in spg . plp is almost % conserved across mammalian species, and the large spectrum of phenotype severity is also found plp transgenic mice. experiments on these models have contributed to our understanding in the pathophysiology of plp related disorders. in one hand, mice with extra copies of the plp gene ( plptg mice) have neurological symptoms and cns pathology similar to those found in pmd patients while mice with plp gene inactivation (plp null mice) share similarities with spg patients. then, these two mouse models represent very useful tools to evaluate the efficacy of a therapeutic strategy for the severe and the mild form of the plp related disorders on the neuropathological and behavioral aspects. olesoxime (tro ), a cholesterol-like small molecule, has been shown to rescue motor neurons from cell death and to promote axonal regeneration both in vitro and in vivo. more recently, olesoxime has also been shown to promote central remyelination by accelerating oligodendrocyte maturation both in vitro and in vivo. altogether, these results strongly suggest that olesoxime may be a potential drug candidate for the treatment of white matter neurodegenerative disorders and notably to plp related disorders. here we treated plptg mice with olesoxime for months starting at birth, as well as plp null mice from to months of age (presymptomatic treatment) or from to months of age (symptomatic treatment). behavioral and neuropathological analysis revealed that olesoxime failed to correct the development of symptoms in mice modeling the severe form of plp related disorders. on the contrary, treatment of plp null mice with olesoxime resulted in a -month delay in the onset of abnormal behaviors related to anxiety and in the slowing of central nerve conduction. these beneficial effects are observed only when the drug is administered before disease onset. treatment with olesoxime also reduces the effect of aging on rotarod performance decline in both wt and plp null old mice. altogether these results suggest that olesoxime could be of interest as a treatment to delay the onset of symptoms in plp related disorders provided that patients suffer from a mild form of the disease and are treated before the establishment of symptoms. objectives: glial cells play an important role in amyloid-beta (aß) clearance, however little is known about the cellular routes involved in internalization of different aß aggregation forms by adult human microglia and astrocytes. therefore, we evaluated the uptake mechanism of aß - by primary human adult microglia and astrocytes isolated from brain tissue of non-demented control and ad cases. methods: cells were preincubated for hour with different inhibitors, before exposure to fluorescence labelled (fam) oligomeric and fibrillar forms of synthetic aß. the number of amyloid positive cells was quantified by flow cytometry. inhibitors of various pathways tested, include cytochalasin b (inhibits actin polymerization; general endocytosis inhibitor), nocadozole (inhibits tubulin depolymerization; general endocytosis inhibitor) and fucoidan (scavenger receptor inhibitor). results: cytochalasin b inhibited aß fibril uptake ( % reduction, p < . ) and to a lesser extent oligomer ( % reduction, p < . ) uptake in microglia, whereas no effect was seen on aß uptake by astrocytes. in contrast, nocodazole was found to inhibit aß fibril uptake ( % reduction, p < . ) and oligomer ( % reduction, p < . ) uptake in astrocytes, whereas no effect was seen on aß uptake by the microglia. interestingly, fucoidan prevented aß uptake in both astrocytes (oligomers: % (p < . ); fibrils: % (p . ) and microglia (oligomers: % (p < . ); fibrils: % (p < . )). conclusions: these data indicate that both human astrocytes and microglia use scavenger receptors to internalize aß oligomers and fibrils. whether different types of scavenger receptors are involved in aß uptake by astrocytes and microglia will be further investigated. tel-aviv university, tel aviv, israel alzheimer's disease (ad) accounts for the majority of cases of dementia worldwide, affecting over % of the population over . the disease is characterized by a progressive memory loss, cognitive deterioration, behavioral disorders, deposit of beta-amyloid (ab) peptides, neurofibrillary tangles, reactive astrocytosis and activation of microglia cells. mutations in the genes that encode app (amyloid precursor protein) and components of the proteases that generate amyloid beta cause familial forms of ad. microglia, the resident immune cells of the central nervous system (cns) were suggested to play both beneficial and harmful roles in the course of the disease. therefore, modulating the microglial response is being considered as a promising approach for the treatment of this disease. we have recently shown using in vitro and in vivo systems that the ectoenzyme cd , regulates microglial activation. in view of the significant role of activated microglia in ad and the important role played by cd in microglial activation, we hypothesized that cd deficiency would affect the course of the disease. in order to test this hypothesis, we implicated the app swe ps de transgenic mouse model for ad, and compared the cognitive performance (as evident by behavioral studies) of aged app swe ps de mice, to app swe ps de cd -/mice. in addition, abload, accumulation of microglia and astrocytes was assessed in the brains of these mice. our preliminary results show that cd deficiency has a neuroprotective role in this ad mouse model as indicated by improvement in cognitive performance and dramatic reduction in ab load. oxidative stress induced by reactive oxygen species (ros) is associated with various pathological conditions, including aging, neurodegenerative disorders, traumatic and ischemic insults. the mechanism by which the gap junction protein connexin (cx ) contributes to oxidative stress-induced cell death or the "rescue" of the dying cells is still unclear. cx is highly expressed in astrocytes. we have previously shown that astrocytic cx is critical for neuroprotection during ischemic insults in vivo. to investigate the protective effect of cx in oxidative stress pathways, we induced oxidative stress in wild-type and cx knockout astrocytes using hydrogen peroxide (h o ). cx knockout astrocytes exhibited increased h o -induced cell death as measured by the loss of membrane integrity using the dye rhodamine b dextran, supporting a cell protective effect of cx ; this effect was not observed in wild-type astrocytes. to examine the involvement of cx in h o -induced cell death, we studied the expression and distribution of cx in response to h o in wild-type astrocytes. h o treatment altered the expression level and phosphorylation of cx . this was accompanied with changes in cx distribution and gap junction plaque formation. hemichannel activity was measured using dye uptake. h o ( . mm) increased dye uptake, an effect that was blocked by gap (syntethic mimetic peptide, mm) or carbenoxolone ( mm), two connexin hemichannels blockers. however, h o treatment did not result in any measureable gap junction activity in cx knockout cells as measured by a scrape-loading dye transfer assay. these findings suggests that gap junction activity contributes to cx -mediated protection in response to ros. one of the receptors found on microglia is the triggering receptor expressed on myeloid cells (trem ), which signals intracellularly via an adapter molecule referred to as tyro protein tyrosine kinase-binding protein (tyrobp, known also as dap ). in humans, loss of function of either trem or tyrobp leads to nasu-hakola disease, which is characterized by neuroinflammation and bone cysts. by using a trem knock-out (ko) mouse line, we provided further insights on the trem function in neurodegenerative diseases. first, we observed mild signs of dopaminergic neurodegeneration and a slight increase in microglial iba -immunoreactivity in different brain regions of months trem ko mice compared with the wild type (wt) control animals. however, the transcription levels of inflammatory cytokines (tnfa, il b) or inducible nitric oxide synthase (inos) were unaffected in mice of different ages ( , or months). to shorten the time required for neurodegeneration to occur, we have injected the mice intraperitoneally with lipopolysaccharides (lps) on four consecutive days and analyzed the dopaminergic neurodegeneration in the substantia nigra after a three weeks period. while the quantification of dopaminergic neurons in the substantia nigra showed a slight decrease in number for wt animals, a higher loss was recorded in the trem ko mice after systemic lps challenge. concomitantly, significant increase of microglia activation was detected in the lpstreated trem ko mice compared with lps-treated wt animals. our results are pointing towards a neuroprotective effect of the microglial trem receptor under chronic and systemic inflammatory conditions. increasing evidence suggest that brain energy metabolism is severely altered in multiple sclerosis patients, which may contribute to the process of neurodegeneration. imaging studies reveal a disturbed cerebral perfusion and glucose uptake and metabolism in ms patients. moreover mitochondrial dysfunction is recognized to play a crucial role in ms pathology. to date little is known about the distribution of glucose transporters (gluts), key molecules for the cellular uptake of glucose, in human brain and their role in the pathogenesis of ms. therefore, the aim of this study was to provide a complete overview of the distribution and expression levels of glucose transporters in control and ms brain. our data so far show that mrna levels of glut , and as well as their regulators hypoxia inducible factor (hif)- a, hif- a and peroxisome proliferator-activated receptor gamma coactivator (pgc)- a, are increased in normal appearing white matter (nawm) of ms patients compared to controls. immunohistochemical analysis revealed that brain endothelial cells not only express glut but also glut and glut . glut , and are present in resting microglia and highly expressed in activated microglia and macrophages in active ms lesions. interestingly reactive astrocytes expressed increased levels of glut and glut compared to control, in addition glut positive astrocyes were observed in ms brain. axons expressed high levels of glut in active ms lesions, which was evidently reduced in chronic lesions. strikingly, all glucose transporters studied were down regulated in chronic ms lesions. together, these data emphasize the extent to which glucose metabolism appears to be affected in all stages of ms. a better understanding of these metabolic changes, especially in the nawm and chronic stage of the disease, is essential to develop strategies to improve neuronal survival and to gain more insight in the mechanisms underlying lesion formation. pericytes are vascular mural cells which are enriched within the capillary basement membranes of the brain. recent studies have provided evidence that pericytes regulate the blood-brain barrier (bbb) and modulate cerebral blood flow. neurovascular dysfunction and bbb damage are hallmarks of alzheimer's disease (ad), but the role of pericytes in ad pathogenesis is still unclear. we have examined post-mortem samples of middle frontal gyrus in collaboration with the university of cambridge after ethical approval by the local irb. ten cases with neither history of neurological disorder nor evidence of neuropathology were compared with ad cases provided by the neurological foundation of the new zealand human brain bank. we performed immunostainings for laminin and plateletderived growth factor receptor (pdgfr)-b which mark vessels and pericytes, respectively. we also stained for ab - for evidence of amyloid pathology. quantification of pericyte and vessel density was performed using stereological methods, which are considered as gold standard in morphometric quantification. the spaceballs method was used to measure the density of capillaries. pericyte density was assessed using the optical fractionator. the age of the subjects did not differ between the control and ad groups ( , , vs. , , years). the cortex of ad cases contained more capillary vessels than controls (mean sd: vs. mm/mm ; p < , ; fig. a ). the capillary density increased in correlation to the total plaque load (r , ; p < , ). in contrast, we could not observe a significant difference in the number of pericytes per mm capillary length between control and ad cases ( , , vs. , , cells/mm; p , ; fig. b ). relative to tissue volume, the cortex of ad individuals showed a tendency to contain more pericytes than controls ( vs. cells/mm ; p , ; fig. c ). this is the first study using state-of-the-art techniques to investigate the relationship between capillaries and pericytes in ad brains. impaired clearance of b amyloid across the pericyte-regulated bbb and deficient microvascular regulation of blood flow may contribute causally to ad pathogenesis. in consequence, loss of pericytes could contribute to disease progression. however, our results show that ad cortical pathology is characterized by an increase in the density of capillary vessels without deficit in pericyte coverage. thus, they do not substantiate the notion that pericyte loss is a significative feature of ad. m. noack, j. leyk, c. richter-landsberg carl von ossietzky universit€ at oldenburg, oldenburg, germany histone deacetylase (hdac ), a member of the class ii hdacs, is a unique cytoplasmic a-tubulin deacetylase that interacts with the microtubule (mt)-associated protein tau. in healthy human brain six tau isoforms are expressed generated by alternative mrna splicing. these contain either three ( r-tau) or four ( r-tau) microtubule binding repeats regulating mt assembly and stability. tau hyperphosphorylation impairs its mt-binding activity. tau deposits in nerve cells and glia are the characteristic hallmark of a number of neurodegenerative diseases termed tauopathies. in progressive supranuclear palsy and corticobasal degeneration, pathogenic glial cell inclusions are observable in oligodendrocytes, the myelin forming cells of the cns. hdac plays an important role in the degradation and accumulation of misfolded proteins by promoting transport of aggregated proteins to the aggresome, localized at the microtubule-organizing center where protein aggregates are deposited and processed by autophagy. the present study was undertaken to elucidate the functional significance of hdac in oligodendrocytes and its role in pathological protein aggregate formation. towards this cultured rat brain oligodendrocytes and oln-t cells, an oligodendroglial cell line expressing the longest human tau isoform, were used. treatment of primary oligodendrocytes with tubastatin a (tst), a selective inhibitor of the tubulin deacetylation activity of hdac , caused a significant increase in the level of acetylated tubulin, as determined by indirect immunofluorescence and immunoblot procedure. mts appeared more bundled and stabilized. inhibition of hdac attenuated the phosphorylation of tau at the phf- -epitope (serine / ) and its enhancement at the e -epitope (serine , located within the microtubule binding repeat region ). furthermore, altered protein levels of tau isoforms containing either three or four mt binding repeats were observed. while r-isoforms were reduced, r-isoforms were increased in primary oligodendrocytes suggesting an impact on microtubule binding ability. hdac knockdown in oln-t cells revealed no alteration in tau phosphorylation at phf- -epitope, but also augmented phosphorylation at the e -epitope. cells were morphologically damaged and mt organization was disturbed. the data indicate that hdca modulates tau expression and phosphorylation, its inhibition leads to the accumulation of r-tau which is also prominent in tauopathies with oligodendroglial pathology. to test the hypothesis, that these cells are part of a possible compensatory mechanism to cope with the -ohda-induced loss of dopaminergic neurons, we studied the expression of tyrosine hydroxylase (th), the rate-limiting enzyme in the catecholamine synthesis pathway, in the cortex of -ohda-lesioned animals. performing immuncytochemistry at d after the lesion we demonstrated a -fold increase in the number of th-positive (th somata in rat cortex following -ohda injection) compared to sham-lesioned animals. th somata in the parietal cortex were restricted to the layers ii-v with most of the cells being bipolar. combining th immuncytochemistry with classical nissl stain yielded complete congruency. a small fraction of th cells co-expressed calretinin thus pointing to an interneuron affiliation. virtually none of the th cells expressed other markers of interneurons (calcium binding proteins, neuropeptides) or the glial markers gfap and nestin. in contrast, we found a co-localization of th with markers of glial progenitor cells (sox and s b) and with psa-ncam, which has been shown to be expressed in immature, but not recently generated cortical neurons in cortical layer ii of the cat (varea et al.; front. neurosci. : ). taken together, these findings indicate that -ohda lesions might induce a glial de-differentiation followed by a fate re-direction towards neuronal committed cells. future studies have to be performed to confirm this and to find out if the th cells are capable of dopamine synthesis. objective: using the mouse optic nerve (mon) wm model, we tested whether hydrogen in drinking water reduced functional wm ischemic injury, and if it reduced cellular lipid peroxidation and mitochondrial dysfunction including mitochondrial and nuclear dna oxidation. methods: functional integrity of mon was determined by quantitatively monitoring the area of mon compound action potential (cap) before, during and after a standardized min period of oxygen and glucose deprivation (ogd), the ischemia equivalent ex vivo. nuclear -oxoguanine (nu -oxog) was used as a marker of oxidative dna damage. results: a min period of ogd caused prompt loss of the cap, followed by an average % recovery. in mice that had received hydrogen-containing drinking water for - days, the cap area did not disappear during ischemia and recovered to a significantly greater extent during reperfusion (normal oxygen and glucose levels). immunostaining of axonal neurofilament by smi- showed significant protection in mons from mice drinking hydrogen-water. accumulation of nu -oxog was observed mainly in oligodendrocytes after ogd. the levels of -oxog after ogd were significantly reduced in optic nerves from hydrogen-water drinking mice. the 'protection' induced by - days of hydrogen-water drinking lasted several days. conclusions: our results show that several days of hydrogen exposure reduced the extent of cns wm irreversible injury associated with ogd. the importance of these observations is that oligodendrocytes may play an important role in the protective effect against ischemic injury. these observations raise intriguing therapeutic options. amyloid-b (ab) deposits in the brain extracellular space (ecs) and neurofibrillary tangles are typical features of alzheimer's disease (ad). both affections are expressed in triple transgenic ( xtg-ad) mice, making them a useful model for studying the pathophysiology of ad. in these mice, significant changes in astroglial morphology appear in the limbic brain structures [ , ] , which may affect the diffusion of neuroactive substances through the ecs. using the real-time iontophoretic method, we determined the ecs volume fraction a (a ecs volume / total tissue volume) and the geometrical factor tortuosity k (k free / apparent diffusion coefficient) in the ca region of the hippocampus, dentate gyrus (dg) and prelimbic cortex (plc) in brain slices obtained from -month-old ( m) and month-old ( m) xtg-ad mice and age-matched wild-type (wt) controls. to study potential diffusion anisotropy, the measurements were performed along orthogonal axes: medio-lateral, rostro-caudal and ventro-dorsal. astroglial morphology and the presence of ab were assessed using immunohistochemical staining for gfap and ab. we found no anisotropy in the ca region, dg, or plc, thus the data for each structure along the three orthogonal axes were pooled. the ecs diffusion parameters measured in the ca are shown in table . in the ca of m mice, there was no significant difference in the ecs diffusion parameters between wt and xtg-ad mice. in m mice, a decreased by % in wt but increased by % in xtg-ad mice in comparison with the values found in the younger animals. during aging, there was also a significant decrease in k in wt but not in the xtg-ad mice. a similar pattern of changes in the ecs diffusion parameters during aging was also observed in the dg and plc. unlike in the ca , we found significant differences in young animals: a was lower in the dg and k was higher in the dg and plc of xtg-ad mice than in wt mice. in m xtg-ad mice, ab deposits accompanied with astroglial atrophy or hypertrophy were observed. we suggest that the amyloid load in xtg-ad mice and the subsequent prevailing astroglial atrophy affect the normal aging process by inducing an increase in the ecs volume during aging instead of the normal decrease. this leads to a larger ecs space in aged xtg-ad mice than in age-matched wt mice, which may alter the efficacy of extrasynaptic as well as synaptic transmission and contribute to the impaired cognitive functions in ad. the study was supported by the grants: gacr p / / , gacr p / /g . significant differences between m and m mice of the same group are marked with *, differences between wt and xtg-ad mice of the same age are marked with . sequence or any treatment. however growing evidences are in favor of the involvement, besides neurons, of several partners such as glia and muscles. to better characterize the time course of pathological events in an animal model that recapitulates human als symptoms, we investigated functional and cellular characteristics of hsod g a mice. we have evaluated locomotor function of hsod g a mice through dynamic walking patterns and spontaneous motor activity analysis. we detected early functional deficits that redefine symptoms onset at days of age, i.e. days earlier than previously described. moreover, sequential combination of these approaches allows monitoring of motor activity up to disease end stage. to tentatively correlate early functional deficit with cellular alterations we have used flow cytometry and immunohistochemistry approaches to characterize neuromuscular junctions, astrocytes and microglia. we show that ( ) decrease in neuromuscular junction's number correlates with motor impairment, ( ) astrocytes number is not altered at pre-and early-symptomatic ages but intraspinal repartition is modified at symptoms onset, and ( ) microglia modifications precede disease onset. at pre-symptomatic age, we show a decrease in microglia number whereas at onset of the disease two distinct microglia sub-populations emerge. we are now establishing the transcriptomic profile of microglia over the course of the disease. in conclusion, precise motor analysis updates the onset of the disease in hsod g a mice and allows locomotor monitoring until the end stage of the disease. early functional deficits coincide with alterations of neuromuscular junctions. importantly, we identify different sets of changes in microglia before disease onset as well as at early-symptomatic stage. this finding not only brings a new sequence of cellular events in the natural history of the disease, but it may also provide clues in the search for biomarkers of the disease, and potential therapeutic targets. in order to gain more knowledge about the disease pathophysiology, two mouse models for vwm are developed in our lab. co-cultures between astrocytes (wt and vwm) and wt oligodendrocyte progenitor cells (opcs) are done to investigate the effect of astrocytes on opc maturation. furthermore, induced pluripotent stem cells (ipscs) from both vwm and wt mice are compared in their glial differentiation efficiency in vitro and in vivo. the astrocyte-opc co-cultures show that vwm astrocytes inhibit opc maturation. this suggest that astrocytes might have a crucial role in the development of the oligodendrocyte and myelin pathology in vwm. currently our research focuses on rescuing the maturation inhibition in vitro, which can aid the development of stem cell therapy for vwm. particularly in response to b-amyloid. using the appps alzheimer's disease mouse model, we observed the production of the common interleukin (il) and il- subunit p by microglia. genetic ablation of various il- /il- signaling molecules, of which deficiency in p had the strongest effect, resulted in a drastic decrease in cerebral amyloid burden. although deletion of il- /il- signaling from the radiation-resistant glial compartment of the brain was most efficient in mitigating cerebral amyloidosis, peripheral administration of a neutralizing p -specific antibody likewise resulted in reduction of cerebral b-amyloid in appps mice. furthermore, intracerebroventricular delivery of antibodies to p significantly reduced soluble ab species and reversed cognitive deficits in aged appps mice. our results suggest that inhibition of il- /il- signaling reduces cerebral amyloidosis and cognitive dysfunction, and may pose a novel potential pharmacological target to combat alzheimer's disease. charit e -universit€ atsmedizin berlin, berlin, germany question: microglia are attracted to and surround b-amyloid deposits, the main hallmark of alzheimer's disease (ad), suggesting a role for these cells in disease pathogenesis. however, recent in vivo data using the cd b-hsvtk system for microglial depletion suggests that resident microglia are not sufficiently capable of restricting and clearing bamyloid plaques. to underpin the cd b-hsvtk system and shed more light into the biological mechanistics of microglial depletion mediated by ganciclovir (gcv), we aim to intravitally monitor the depletion process. methods: we are using cd b-hsvtk transgenic mice crossed to fractalkine-gfp /mice harboring green fluorescent microglia. a chronic cranial window is implanted onto the head of the offsprings followed by depletion of microglia and subsequent in vivo two-photon ( p) imaging. as p imaging is able to visualize the upper mm of the cortex, a new topical depletion approach of cd b-hsvtk microglia was established. here, gcv is applied directly onto the brain surface in the area of the cranial window through a catheter. results: manipulation of the brain skull by installation of the cranial window and placing a catheter for topic gcv application resulted in strong depletion of microglia up to % in fractalkine-gfp /mice crossed to cd b-hsvtk mice. conclusions: taken together, we established a minimal invasive technique for visualization of the microglia depletion process in cd b-hsvtk mice using intravital p microscopy. these tools will allow the underpinning of the cd b-hsvtk system as well as the study of relevant biological questions involving resident and peripheral derived microglia in ad. (icrea) , barcelona, spain x-linked adrenoleukodystrophy (x-ald) is a metabolic genetic disorder of the central nervous system characterized by demyelination in brain and/or axonopathy in the spinal cords, adrenal insufficiency and accumulation of very long-chain fatty acids (vlcfa) in plasma and tissues. the disease is caused by inactivation of the abcd transporter with the consequence production of oxidative stress mediators and damage. here we aimed to determine: i) the existence of endoplasmic reticulum (er) stress and the ensuing unfolded-protein response (upr) in brains and fibroblasts from x-ald patients, and in the x-ald mouse model (the abcd null mouse); and ii) whether the early and rampant oxidative stress formerly identified accounts for the er stress. indeed, here we report signs of er stress and upr response in human and mouse tissues. a common feature is the activation of atf and lower amounts of ire , two er transducers constituting the core signature of upr in x-ald. chaperone grp and grp expression, by contrast, differ between variants and stages of the disease. we highlight how chaperones are down-regulated in x-ald mice at months of age, pointing to selective depletion of upr-related factors overtime. treatment of the mouse model with a combination of antioxidants reversed the initial activation of transducers and chaperones, indicating that oxidative stress caused er stress. altogether, we postulate that oxidative-stress elicited er stress occurs in x-ald, and that a defective upr may contribute to axonopathy progression. thus, potentiation of the upr may be a therapeutic avenue in x-ald. representing opc-like cells with the potential to differentiate upon growth factor withdrawal and addition of triiodothyronine (t ) into premyelinating oligodendrocytes. upon differentiation, significant fewer cg _wt-a-syn cells express the myelin basic protein (mbp; see figure a) as a marker for terminal differentiation and additionally, mrna levels were remarkably reduced compared to control cells (see figure b ). as mrna levels of the myelin protein proteolipid protein (plp ) as well as the major myelin-gene regulatory factor (mrf) are also reduced in differentiated cg _wt-a-syn cells (see figure b) we hypothesized that the process of differentiation is delayed upon a-syn expression. using various differentiation-promoting agents targeting distinct maturation-associated pathways we aimed to dissect the a-synmediated effect on differentiation in more detail and evaluate potential disease-modifying approaches for this devastating neurodegenerative disorder. methods: two nutritionally distinct groups of male newborn ratswell-nourished (w; n ) and malnourished (m; n ) were treated from the th to the th day of postnatal life with daily intraperitoneal injections of saline (sal) or cona (sigma-aldrich) at doses of mg/kg (group w-l and m-l ) or mg/kg (groups w-l and m-l ). two groups of "naive" (non-injected) pups were used as additional controls. when the pups reached adult age ( - days), under ip anesthesia ( g/kg urethane mg/kg chloralose) they were submitted to the csd recording (ecog and slow potential change) in two points of the parietal cortical surface. csd was triggered every minutes by applying % kcl for min at a point in the frontal cortex. this study was approved by the ethics committee of our university, case no.: . / - . results: compared to w-sal controls (mean sd velocity in mm/ min . . ), m-sal rats presented significantly higher velocities ( . . ; p < . ). in both nutritional conditions l and l treated groups presented significantly lower csd velocities ( . . and . . for the w-l and w-l , respectively, and . . and . . for the m-l and m-l ). this effect was dose-dependent, and was greater in the m-condition. the na€ ıve-and salgroups had similar csd velocities. conclusions: the lectin cona administered early in life to rats decelerated csd in a dose-dependent manner at adulthood, suggesting a long-lasting effect. the largest relative reduction was observed in the m group, suggesting modulation of the effect by the nutritional status. the involvement of glial cells in this effect is discussed. the endocannabinoid system is of growing interest as a therapeutic target in neurological diseases. the two major endocannabinoids arachidonoylglycerol ( -ag) and n-arachidonoyl ethanolamine (anandamide, aea), are the endogenous ligands for the cannabinoid receptors (cb ) and (cb ). cb is mainly expressed in the brain and associated with neuroprotective effects, whereas cb is primarily found in hematopoietic cells and associated with anti-inflammatory effects. in neurodegenerative diseases, e. g. amyotrophic lateral sclerosis (als) and parkinson's disease (pd), an increased cb expression on microglia has been reported and is therefore an interesting therapeutic target. furthermore, an increase of endocannabinoid levels occurs in the affected neuronal tissues. stimulating this disease-related increase in endocannabinoids might thus be of therapeutic interest. in our project, we evaluated the novel, highly selective inhibitor kml of an enzyme central to the degradation of the endocannabinoid -ag, the monoacylglycerol lipase [ ] . an in vitro monoacylglycerol lipase inhibitor screening assay for the comparison of the utilized inhibitor with others was performed. additionally, behavioral experiments including body temperature measurement, pain threshold measurement and motor activity analysis in healthy control mice were performed. furthermore, endocannabinoid system baseline levels of healthy control mice vs. sod (superoxide dismutase ) mice as an als mouse model were compared at the gene expression and protein level. in vitro inhibition of the monoacylglycerol lipase by the recently published compound kml resulted in lower ic values and therefore indicated a higher potency of kml when compared to the other monoacylglycerol lipase inhibitors jzl and cpc applied in the assay. in vivo administration of the reported monoacylglycerol lipase inhibitor kml resulted in significant decrease in body temperature, increase in pain threshold in b sjlf /j mice as well as in impairments in the nocturnal motor activity of c bl/ j mice when compared to vehicle-treated mice. in summary, our data show a comparatively high potency in vitro and significant dose-dependent effects on physiological parameters in vivo of the monoacylglycerol lipase inhibitor kml and therefore lay the foundation for a future therapeutic study targeting neuroinflammation in mouse models of als and pd. glaz/apod protects against oxidative stress and promotes axon regeneration after injury, but its mechanism of action is unknown. we hypothesize that glaz/apod modulates membrane dynamics and oxidation. to contrast this hypothesis we study glaz protective role on the polyq-based spinocerebellar ataxia type i (sca ) model in drosophila. human polyq-ataxin expression in fly photoreceptors triggers glaz up-regulation. overexpressing glaz in photoreceptors or in retinal support cells rescues neurodegeneration. when expressed in retinal support cells, the glaz rescue is dependent on lipocalin-receptor expression by photoreceptor neurons, and the glaz-gfp fusion protein co-localizes with photoreceptor markers, suggesting the existence of receptor-mediated endocytosis of glaz. upon neurodegeneration, oxidative stress and induction of autophagy coexist. to test whether the glaz beneficial effects are mediated by the modulation of autophagy we quantified atg a expression and monitored the accumulation of ubiquitinated proteins and p . overexpression of glaz reduces atg a induction, but concurrently reduces the accumulation of ubiquitinated proteins and p . upon autophagy stimulation by rapamycin treatment, the levels of sca -dependent accumulation of ubiquitinated proteins and p are further reduced by glaz. in addition, glaz decreases the sca -dependent induction of gsts , a sca genetic modifier contributing to the clearance of lipid peroxides. our data support that glaz enters the degenerating neurons by a receptor-mediated mechanism and promotes the resolution of autophagy, increasing its flow and helping to clear polyq-induced protein aggregates. moreover, the beneficial effects of glaz are linked to lipid peroxide clearance, either directly or through receptor-mediated modulation of protective gene networks. micinn(bfu - ); jcyl(va a - ); fund. rodr ıguez-pascual. we investigated the effect of three different intensities of running exercises on the survival of sod g a mice. at the early-symptomatic stage (p ), males were isolated and randomly assigned to conditions: sedentary groups ("sedentary" and "sedentary treadmill" placed on the inert treadmill), and different training intensity groups ( cm/s, cm/s and cm/s; min/day, days/week). we first demonstrated that an appropriate "control" of the environment is of the utmost importance since comparison of the two sedentary groups evidenced an . % increase in survival in the "sedentary treadmill" group. moreover, we showed by immunohistochemistry that this increased lifespan is accompanied with motoneurons survival and increased glial reactivity in the spinal cord. in a second step, we showed that when compared with the proper control, all three runningbased training did not modify lifespan of the animals, but resulted in glial and motoneuronal changes. conclusions/significance: we demonstrate that increase in survival induced by a slight daily modification of the environment is associated with motoneurons preservation and strong glial modifications in the lumbar spinal cord of sod g a . using the appropriate control, we then demonstrate that all running intensities have no effect on the survival of als mice but induce cellular modifications. our results highlight the critical importance of the control of the environment in als studies and may explain discrepancy in the literature regarding the effect of exercise in als. alzheimer's disease is the most prevalent neurodegenerative disorder, characterized by occurrence of senile plaques, neurofibrillary tangles and aberrant function of classical neurotransmitters and peptide messengers, such as neuropeptides and growth factors. in the last years a role of astrocytes in mediating and amplifying the progression of neurodegenerative disorders has been proposed. moreover, a growing body of evidence has shown that astrocytes can release non-peptide and peptide transmitters to influence neuronal development, function and plasticity. here we analyzed the impact of the main component of senile plaques, the amyloid-b (ab), on two key components of peptide vesicles, carboxypeptidase e (cpe) and secretogranin iii (sgiii), in astrocytes in vitro and in vivo. we consistently located cpe and sgiii proteins in cultured and in situ astrocytes. traffic and secretion of astroglial cpe and sgiii were analyzed under basal and stimulated conditions in primary cultures. we found that exposure of astrocytes to ab - markedly impairs expression, traffic and secretion of glial cpe and sgiii. protein levels were decreased in the media, but increased into the cells, by ab - incubations. the effect of ab on cpe and sgiii in glial cells was also investigated in vivo using the amyloid-forming transgenic mice appswe/ps de . an aberrant accumulation of cpe and sgiii was found in numerous activated-astrocytes surrounding senile plaques through all the cns of aged transgenic mice. taken together, the present study shows that ab dramatically alters expression, traffic and secretion of cpe and sgiii. because cpe and sgiii are essential in the process and targeting of neuropeptides and growth factors, an involvement of the astroglial secretory pathway in the pathology of alzheimer's disease is suggested. glia cytokine that is produced subsequent to h-i, that collaborates with other cytokines to stimulate the production of astrocytes from subventricular zone glial progenitors. the specific goal of this study was to evaluate gliogenesis after h-i when the tgfb receptor alk is antagonized in the vanucci p h-i rat model. by q-pcr, the relative amount of tgfb mrna peaked days after h-i. therefore, sb- , and alk antagonist, was given intraperitoneally (i.p.) days after the injury to a group of rats while another group received pbs. animals were sacrificed at different time points and brain samples processed for western blot analysis. validating the importance of alk- signaling, sb reduced the levels of phosphorylated smad / that increased after h-i. in another study, sb- was administered i.p. from p to p and animals sacrificed at p for immunohistochemistry for gfap, iba- , gstpi and mbp. gfap and iba- positive cells were dramatically increased in the injured striatum and corpus callosum after h-i and mbp staining was decreased. by contrast, both the extent of injury and the degree of reactive gliosis was decreased by sb- treatment. altogether, our results indicate that sb- inhibits alk signaling in the damaged brain. furthermore, sb- administration decreases the extent of microgliosis and astrogliosis while preserving myelination in the damaged brain after neonatal h-i. supported by nih r hd and a grant from the leducq foundation awarded to swl. [ ] . despite efforts to improve air quality, dep persists as a problem and the share of diesel engined passenger cars in western europe is increasing steadily. honey bees are an important pollinator of food crops and wild flowering plants. they not only contribute to food security by providing pollination services of an enormous economic value but also play an important ecological role. over the last five years, bee keepers worldwide have reported a decline in honey bee populations. the reasons for this decline remain unknown, but it is thought to be multi-factorial [ ] . honey bee colonies are confronted with a number of stressors, for example infection with the mite varroa destructor or exposure to pesticides. to forage successfully honey bees rely on their learning and memory abilities. our research aims to establish whether dep might be a factor contributing to declines in honey bee populations through impairment of healthy cns function. methods: we have investigated the impact of dep exposure on the learning abilities of the honey bee using the proboscis extension reflex and classical conditioning. western blotting and histology were used to determine regional expression levels of stress proteins in the brain in response to an acute diesel exhaust exposure. we are examining the morphological appearance of glial cells to investigate the impact of dep in the honey bee cns of dep exposed, and control, forager bees. results and conclusions: we hypothesize that dep acts as a stressor in the brain of the honey bee causing changes in glial cells that are detectable using morphological and molecular techniques -similar to microglial activation or immunological responses of astroglia in the mammalian brain. this work will further our understanding of how dep acts on the brain and how it might impact on the health of the animal. current results indicate that diesel exhaust pollution impacts on the neurobiology of the honey bee and this may contribute to decline by impairing cns functions. hepatic encephalopathy (he) is a serious neurological disorder caused by liver failure. the prime candidate responsible for he pathology is an increased ammonium concentration; and following acute liver failure, ammonium can reach levels of up to mm in the brain. astrocytes are a major target of ammonium toxicity and earlier studies suggested that this toxicity includes a disturbance in intracellular calcium signaling. in the present study, we analyzed the effect of acute hyperammonia on the intracellular calcium concentration of astrocytes in tissue slices of mouse brain, using quantitative intracellular calcium imaging with the indicator dye fura- . astrocytes were identified by staining with the vital dye sr ; cerebellar bergmann glial cells were identified based on their morphology. in all brain regions studied, the hippocampal ca area, somatosensoric cortex, and cerebellar cortex, bath perfusion with mm ammonium for min induced a small, but persistent elevation in intracellular calcium, averaging about nm. in addition, a fast and transient increase by on average nm that lasted about minutes was seen in a small percentage of astrocytes in hippocampal and cortical slices at the onset of ammonium perfusion. this transient increase was never observed in cerebellar bergmann glial cells. both the transient, as well as the plateau phase of ammonium-induced calcium changes were unaltered in the presence of ttx, indicating that they are independent from action potential generation by neurons. furthermore, ammonium-induced calcium increases largely persisted upon removal of extracellular calcium, indicating that they are caused by release from intracellular stores. taken together, our experiments demonstrate that ammonium evokes complex changes in the intracellular calcium concentration of astrocytes in the intact tissue, which differ both between cells in one preparation as well as between different brain regions. because of the central role of astrocyte calcium in gliotransmission, the observed ammonium-induced calcium dysbalance might result in disturbed neuronglia interaction and contribute to the pathology of he. it has been shown in a number of animal models that microglia in the degenerating brain are primed to show exaggerated cytokine responses to subsequent stimulation with toll-like receptors agonists such as lps and poly i:c. it is not clear whether the degenerating brain shows similarly exaggerated responses to pro-inflammatory cytokines. in the current study we hypothesised that glial cells in the hippocampus of animals with chronic neurodegenerative disease (me prion disease) would display abnormal responses to central cytokine challenges. in normal animals it has previously been established that intracerebral cytokine challenges elicit specific pathways, i.e. il- b a cxcl- a neutrophil recruitment or tnfa a ccl- a monocyte recruitment. unilateral ll doses of tnfa ( ng/ll), il- b ( ng/ll) or saline were administered intrahippocampally via pulled glass microcapillary, in normal (nbh) and me mice. these animals were terminally perfused for formalin fixation and paraffin embedding at , and hours post challenge. at hours post challenge me microglia produced il- b following either il- b or tnfa challenge while nbh microglia did not. furthermore, there was very robust nuclear localisation of the nfkb subunit p in the astrocyte population and this was associated with very marked astrocytic synthesis of the chemokines cxcl- and ccl- in response to both cytokine challenges in me animals. conversely, very limited expression of these chemokines was apparent in nbh animals similarly challenged. thus astrocytes are the primary chemokine synthesizing brain cell and in the prion-diseased brain are they primed to produce exaggerated chemokine responses to acute stimulation with pro-inflammatory cytokines. this abnormal pattern of chemokine expression is predicted to have consequences for leukocyte infiltration and the ramifications of an altered cellular infiltration pathway for the degenerating brain will be discussed. these findings suggested that the presence of mutated htt in astrocytes alters glial glutamate transport capacity early in the disease process and may contribute to excitotoxicity. to decipher whether astrocytic mutated htt expression was directly associated with msns dysfunction, we investigated the functional properties of individual msns in proximity of mutated htt expressing astrocytes in acute slices. results from whole-cell patch clamp technique showed that msns surrounded ( - microns distance) by astrocytes expressing mutated htt vs. astrocytes expressing normal htt did not differ with regard to passive membrane properties (input resistance, resting membrane potential), action potential firing rates nor spontaneous excitatory postsynaptic current properties (averaged amplitude and frequency). thus, astrocytic expression of mutated htt does not lead to readily apparent functional consequences in msns in these conditions. our data show that month old tg mice displayed a significant increase of ab-plaques in the brain, compared to wt mice. furthermore, we show that the a c subunit colocalizes with % of the abpositive plaques. the cellular expression of a c was predominantly found in activated gfap astroglia around plaques. qpcr profiles of the hippocampus revealed expression of the majority of calcium channel subunits, which was stable during aging. the auxiliary beta subunit was expressed in these mice but did not co-localize with a c astroglia around plaques. in cultures of primary astrocytes, ab( ) slightly enhanced the a c mrna expression after days. we are currently underway to characterize this expression in more detail. in summary, our data provide evidence for a largely stable expression of most ltcc subunits in alzheimer tg mice during aging. finally, the expression of a c but not beta is upregulated in activated astrocytes located around ab-plaques and ab( ) may directly induce astroglial ca v . calcium channels. this study was supported by the sonderforschungsbereich sfb f -b and f -b of the austrian science funds. increasing evidence indicate that both physical and cognitive stimulation induce plastic changes in the brain, such as increase in synaptic and spine densities or neurogenesis, and counteract b amyloid (ab) pathology recovering cognitive function. in the present study we, for the first time, demonstrate the effects of psychostimulation on glial fibrillary acid protein (gfap) architecture in the dentate gyrus (dg) of xtg-ad, a well-established mouse model of ad. starting from months of age, xtg-ad and their corresponding controls (non-tg) were housed either in the presence of a running wheel (run) or in the enriched environment (enr) for months. as reported previously (olabarria et al., ), in standard housing, xtg-ad animals showed marked atrophy of gfap-positive profiles, evidenced by significant reduction in gfap surface area, by %, and volume, by %, compared with non-tg mice. however, physical and cognitive stimulation affected astrocyte morphology by increasing their cytoskeleton surface area and volume, both in non-tg and in xtg-ad mice. the surface area of gfap-ir astrocytes increased by % and % in xtg-ad mice housed in run and enr, respectively, compared with transgenic mice kept in standard housing. in addition, the cell volume of gfap-ir astrocytes was higher by % and % in xtg-ad mice housed in run and enr, respectively. the hypertrophy was also evidenced by an increase in the surface area and the volume of astroglial somata and processes in both non-tg and xtg-ad mice. further correlation analysis between xtg-ad and their corresponding control mice housed under the same conditions, revealed that run and enr not only reversed the genotype-induced astrocytic atrophy, but even induced a hypertrophy in xtg-ad mice, with gfap positive profiles rising to the levels of non-tg mice. thus our study indicates that long-term exercise and enriched environment restore and improve astroglial plasticity in the context of adlike pathology, which may recover cognitive functions by compensating disease-associated degeneration of neuronal-glial network. methods: intraperitoneal administration of wa at a dosage of mg/ kg of body weight was initiated from postnatal day (p ) till end stage in sod g a mice, from months till end stage in sod g r mice and from months of age in tdp a t mice. results: wa was able to improve the survival by more than a week in sod g a and by two weeks in sod g r familial mouse model. in addition wa administration also conferred significant neuroprotection in tdp a t transgenic mice model. beneficial effects of wa in sod g a mice model was accompanied by reduction in loss of motor neurons and also by reduction in level of misfolded sod protein in the spinal cord as detected by immunoprecipitation with antibody specific to misfolded sod . wa was found to be an inducer of heat shock protein (hsp- ), which could possibly explain reduced level of misfolded sod and increased neuroprotection in wa treated mice. moreover real-time imaging with the use of biophotonic sod g a transgenic mice carrying luc (luciferase) and gfp (green fluorescent protein) reporter genes under the control of the murine gap- promoter revealed that wa was able to reduce neuronal stress at post symptomatic stage. conclusion: taken together, our results suggest that wa may represent a promising therapeutic drug for treatment of als. the temporal lobe epilepsy (tle) is the most common form of epilepsy that originates from the hippocampus and then propagates to other limbic areas such as the amygdala and entorhinal cortex. the pathological feature associated with tle is hippocampal sclerosis which is characterized by atrophy, induration, gliosis and loss of neurons in ca , ca and the dentate hilar regions. some animal models, albeit do not exactly match the complex etiologies identified in humans, are found to recapitulate most of the pathological features observed in tle. there is evidence that administration of kainic acid can cause seizures in the ca region of the hippocampus that can lead to loss of neurons and astrogliosis characteristic of tle, but the underlying mechanisms associated with the degeneration of neurons remain unclear. since lysosomal enzymes, cathepsins b and d, can have important roles in the loss of neurons in a variety of experimental conditions, we evaluated their potential roles along with the insulin-like growth factor-ii (igf-ii) receptor, which is involved in the intracellular transport of these enzymes, in the kainic acid treated rats. our results clearly showed that systemic administration of kainic acid evoked severe loss of neurons along with hypertrophy of astrocytes and microglia in the hippocampal region of the adult rat brain. the levels and expression of cathepsins b and d as well as igf-ii receptor increased progressively with time in the hippocampus of kainic acid treated rats compared to control rats. the activity of both cathepsins b and d was also found to be enhanced in the hippocampus of treated rats. our double labelling studies revealed that expression of both cathepsins were initially increased in the pyramidal neurons and then decreased with time. this was accompanied by the expression of immunoreactive igf-ii receptors as well as cathepsins b and d in a subset of gfap-labelled activated astrocytes and iba -labelled microglia in kainic acid treated rats. these results, taken together, suggest that enhanced levels/expression and activity of lysosomal enzymes may have a role in the loss of neurons observed in kainic acid treated rats. in addition, increased ng glia immunoreactivity and myelin loss were found specifically in the gray matter of patients' motor cortex and spinal cord ventral horn. furthermore, oligodendroglial specific monocarboxylate transporter expression is decreased not only in als mouse models but also in patients. given that oligodendrocytes not only facilitate saltatory conduction of action potentials by myelinating axons, but also support neuronal functions metabolically through monocarboxylate transporter (mct ), the injury to oligodendroglia in als may have pathogenic consequences. methods. to further investigate whether oligodendrocytes play a role in als development, we selectively removed mutant human sod (g r) from postnatal ng cells in pdgfar-creer;loxsod (g r) mice. results. the administration of -hydorxytamoxifen ( ht) caused significant decrease in the expression of mutant human sod transgene in ng glia as determined by quantitative pcr analysis. we found that excision of mutant hsod from ng glia dramatically delayed disease onset and early disease, and significantly prolonged animal survival. in addition, at disease onset stage, activated astroglial and microglial responses were delayed in the animals received ht treatment. moreover, the removal of mutant sod helped to preserve mct expression at disease onset. conclusions. these data indicate that expression of mutant sod in ng cells and their oligodendrocyte progeny has a deleterious effect on motor neuron survival, and suggest that a key negative consequence of mutant sod expression in oligodendrocytes is to diminish their capacity to provide metabolic support to neurons. recent studies suggest that innate immunity might be also involved in the pathogenesis of neurodegenerative diseases, cerebral ischemia and brain injury. although the recent works demonstrated that adaptive immune system is involved in the motor neuron disease process, the role of innate immune system in motor neuron disease was not fully investigated. to assess the contribution of innate immunity in the pathogenesis of amyotrophic lateral sclerosis (als), the gene expression profile of lumbar spinal cord from symptomatic mutant sod mice was obtained by microarray approach and subsequent pathway analysis indicated the involvement of innate immune pathway. next, to test the role of innate immunity in the pathogenesis of als, sod g a als model mice were mated with myd and trif (tir domain-containing adaptor inducing ifnb) deficient mice. myd and trif are the essential adaptor proteins for toll-like receptor mediated signaling pathway. as compared with sod g a mice, myd /trif double-deficient and trif-deficient sod g a mice exhibited the substantially shorter survival times with accelerated disease progression. the disease duration was shortened by % in trif-deficient sod g a mice. in contrast, elimination of myd in sod g a mice showed marginal effect in survival time. in addition, the expression levels of pro-inflammatory chemokines, ccl and cxcl- were significantly suppressed in the spinal cord of trifdeficient sod g a mice, as compared with sod g a mice. to determine the cell type in which trif-dependent pathway contributes to the production of these chemokines, we examined the expression of poster abstracts s glia chemokines in lps-stimulated primary microglia or astrocyte derived from trif-deficient or myd -deficient mice. trif-dependent induction of these chemokines was observed only in lps-stimulated microglia. moreover, we found that infiltration of t-lymphocytes and other immune cells were significantly decreased in the spinal cord of symptomatic trif-deficient sod g a mice. these results suggest that the basal level of trif-dependent innate immune activation of microglia is beneficial to slow disease progression of als models through the maintenance of pro-inflammatory chemokines and the infiltration of the immune cells to the spinal cords. the detailed analyses to clarify the role of these infiltrating immune cells are underway. alzheimer's disease (ad), the most common age-dependent neurodegenerative disorder, causes a chronically progressive decline in cognitive functions. there is growing evidence that glial changes are early involved in this pathology. pdapp mouse, a well-defined model of ad, accumulates toxic soluble and deposited ab, derived from proteolytic processing of the amyloid precursor protein (app) and develops adlike synaptic deficits and cognitive impairment. on the other hand, autophagy has been associated to the neurodegenerative process, in particular with the clearance of aggregation-prone proteins like ab. the amyloid plaques, mainly located in cortex and hippocampus, are closely surrounded by reactive and hypertrophic gfap astrocytes. the aim of this work was to evaluate the potential astrocyte autophagic activity during the progression of ad in pdapp mice from to months (m) of age. lc ii/i ratio was studied by western blot. amyloid deposit load, stained with congo red, exhibited a continuing rise according to age in transgenic mice. the markers gfap and lc were analyzed by immunofluorescence and confocal microscopy on hippocampal sections showing an increasing colocalization that reached the top at m, where . . % of plaque associated gfap cells were lc . at m, this proportion was lower. conversely, the subpopulation of astrocytes located far from ab deposits were gfap /lc -, besides a decreased cell volume compared with control mice astrocytes. additionally, the density of astroglial cells in the stratum radiatum diminished with aging ( compared to m, total gfap cells, p < . ) along with higher plaque load, in a more prominently manner than in control mice. our results clearly show that ) astroglia is strongly affected during ad progression: two different morphologic subpopulations -close to or far from deposits-are distinguished in the hippocampus. aging correlates with a glial density reduction; ) a gradual increase of autophagic activity in plaque associated-astrocytes is verified by the first time, suggesting a contribution to ab clearance until m in pdapp mice, with a significant decay after this age. it also shows widespread neuron loss and gliosis in the brain. interestingly, in cstb-/-mice pronounced microglial activation has been detected in selected brain areas already in presymptomatic mice, preceding astrocytosis and neuronal death. in this study, we examine the microglial contribution to the neuronal dysfunction and death in epm . we aim to characterize the functional properties of cstb-/-microglia and their effects on the survival of cstb-/-neurons. our results show that the cstb mrna level in cultured wild type mouse microglia analyzed by real time quantitative pcr is high in relation to primary astrocytes or neurons. a gene-expression profiling of microglia from cstb-/-mice and from wild type mice has been obtained by using the affymetrix mouse exon . st array and reveals a down-regulation of interferon-regulated pathways in cstb-/-microglia. the stimulation of primary wild type mouse microglia with the endotoxin lipopolysaccharide (lps) up-regulates cstb mrna expression measured by real time quantitative pcr. cstb-/-microglia show an altered inflammatory response to the stimulation with lps by increased secretion of chemokines demonstrated by cytokine array analyses. moreover, the release of nitric oxide from cstb-/-microglia measured by griess assay is elevated. our data indicate an altered response of primary cstb-/-microglia to inflammatory stimuli, which may contribute to neurodegeneration in epm . the data provide a basis for further detailed studies on the pathophysiology and therapy of this devastating disease. activation of astrocytes and microglia surrounding amyloid extracellular plaques in the brain is a hallmark of alzheimer's disease (ad). increasing evidence suggests that chronic neuroinflammation and elevated levels of several cytokines play important roles in ad development. beside astro-and microgliosis, demyelination and oligodendrogenesis can be observed in human patients as well as in murine models of ad. oligodendrocyte progenitor cells, also termed ng cells because they express the neuron/glial antigen (ng ) proteoglycan, comprise about % of total cell number of the adult brain and are the main proliferating cells present. ng cells receive gabaergic and glutamatergic synaptic input and can secrete pro-and antiinflammatory substances upon activation by microglia-derived cytokines. overall, ng cells are highly reactive cells and one of the first cells which respond to changes in the cns. however, their role during ad pathology is still uncompletely characterized. using immunohistochemistry, we have analyzed their expression pattern in a transgenic murine model of ad (coexpressing a mutated form of the amyloid precursor protein (app) and the presenilin (ps ) gene). we focused on the spatial arrangement of ng cells in the hippocampus of transgenic mice at , and months of age and age-matched controls (n / time point). our goal is to provide an anatomical and structural basis for elucidating the role of ng cells in ad. the first results indicate an uniform distribution of ng cells in the hippocampus of app/ps mice, with an accumulation at the sites of plaque deposits in close association with microglia. ng cells are normaly absent from the pyramidal and granular cell layers. however, when plaques deposits were found at these principal cell layers, ng cells could be seen surrounding them, suggesting an active migration. changes in ng cell phenotype were noted from months of age on. like microglia, ng cells assume an activated morphology, as in a more "bushy" appearance. in numerous plaques, ng immunoreactivity was strongly increased, mostly in the intermingled processes that encircle the core of the plaques. whether these activated ng cells proliferate and differentiate into oligodendrocytes or communicate with activated microglia in the plaque microenvironment remains to be investigated. further, levels of the glutamatergic ampa receptor expression will be evaluated. ultimately we aim at understanding how ng cells and microglia interact in ad, which could be usefull in the development of novel therapies. accumulating evidence supports an increasing role of astrocytes in the initiation or progression of a variety of neuropathological conditions. in alzheimer's disease (ad), numerous data indicate that astrocyte properties are modified with potential deleterious effects on neurons. accordingly, we have described changes in the expression of astroglial connexins, the gap junction channel and hemichannel forming proteins, in the vicinity of amyloid-b (ab) plaques in brains from ad patients and murine models of ad. also ab peptide was shown to trigger astroglial cx hemichannel activation in culture and in acute slices leading to neuronal degeneration. hence, we have investigated hemichannel function of astroglial connexins in - months old app swe /ps de mice that exhibit ab plaques in the cortex and hippocampus. ethidium bromide (etbr) uptake performed in acute brain hemisphere slices was used as an index of hemichannel activation and was quantified in astrocytes identified by gfap immunostaining. in app swe /ps de mice, compared to wildtype mice of the same age, an increased uptake of etbr was detected in the overall population of astrocytes. this uptake was blocked by carbenoxolone and lanthanum ions, indicating connexin hemichannel involvement. such activation may be linked to the increase in resting astroglial [ca ] i described in this mouse model. interestingly, etbr uptake was higher in reactive astrocytes contacting ab plaques than in non-reactive astrocytes located far from (! mm) these deposits. we are currently investigating their respective pharmacological profiles. moreover, in app swe /ps de mice knock-out for cx generated in our facility, etbr uptake was comparable to that observed in app swe /ps de mice, suggesting that cx is the major contributor to the hemichannel activity observed. altogether, these results indicate that neuroglial interactions could be affected by astroglial hemichannel activation and could account for neuronal alterations or death observed in neurodegenerative diseases. since it is well established that amyloid plaques are associated with activated astrocytes we asked whether concentrations of the astrocyte-derived proteins glial fibrillary acidic protein (gfap) and s b in human csf might serve as additional biomarkers. to functionally link the role of astrocytes to mechanisms involved in memory formation, we asked whether the amount of gfap-positive astrocytes correlates to the level of ltp in the hippocampus of aged ad transgenic mice. we used elisa kits for the quantitative analysis of gfap (ibl-international, hamburg, germany) and s b (ibl). in a mixed disease cohort (n diseased patients, n controls), we correlated standard biomarkers (abeta and tau-protein) and gliaderived proteins. furthermore we compared mean levels between groups of ad patients (n ) and healthy control patients (n ) and correlated levels of astroglial proteins in the csf and cognitive performance (mmst values). in transgenic (app/ps transgenic mice), aged ( - months) mice (n ), we performed ltp experiments at the schaffer collateral synapses in the ca region of the hippocampus. the number of gfap-positive astrocytes was quantified contralaterally. results: in the mixed cohort, we found a significant correlation of t-tau and gfap levels in csf (r . , pbetween the s b levels and the patients cognitive performance as measured by the mmst values (r - , ) as well as between the gfap levels and the mmst values (r - , ). when correlating the number of astrocytes in the ca region to the level of ltp, min after induction, we found a significant correlation of the number of astrocytes per mm to the level of ltp (r , , p conclusions: the astrocyte-derived proteins gfap and s b can be reliably detected in human csf. measurement of astroglial biomarkers might allow an improved pathobiological staging of ad, also since astrocytes appear to play a functional role in memory formation in the context of an ad-like pathology in mice. retinitis pigmentosa is a group of inherited disorders affecting photoreceptors or retinal pigment epithelium (rpe) that leads to progressive loss of vision. the pde b rd mouse model is a very well-known animal model for retinal degeneration, in which rods carry a mutation in b subunit of rod cgmp-phosphodiesterease. glial cell line-derived neurotrophic factor (gdnf) has already been shown to rescue morphology as well as function of rod cells in pde b rd mouse. this effect was indirect, through stimulation of m€ uller glial cells (rmg). to better understand the neuroprotective effect of gdnf, primary retinal rmg were stimulated in vitro with gdnf and the secreted proteome was analyzed by proteome profiler arrays. among others, cyr /ccn , a member of ccn family, was found strongly induced upon rmg stimulation with gdnf. when applied directly to medium, cyr significantly reduced photoreceptor death in organotypic ex vivo cultures of pde b rd retinas. in order to identify the target cells of cyr we treated, arpe and mio-m cell lines with cyr , and observed an increase in phosphorylation of akt and erk / signaling molecules. these results suggest that stimulation of rpe and rmg cells may have a protective influence on photoreceptor survival in organotypic ex vivo conditions. we postulate cyr as a novel potential candidate for future therapeutic approaches in neurodegenerative retinal disorders. since astrocytes react to multiple physiological and pathological stimuli in the microenvironment of the brain, they could be mediators for gene x environment interactions in the pathogenesis of psychiatric disorders. altered gene/protein expression and regressive changes have been reported for astrocytes in post-mortem studies of psychiatric disorders, i.e. schizophrenia (scz), bipolar disorder (bp) and autism spectrum disorders (asd). by contrast, no genetic link to astrocytes has emerged from the extensive genomic analysis of psychiatric disorders. genetic findings mostly relate to neurons for disorders rooted in neurodevelopment (asd, scz). it is timely to perform a formal analysis of genomic information emerging for psychiatric disorders for a contribution of genes expressed by astrocytes. methods: we generated a meta-list of genes highly expressed in rodent astrocytes using published gene lists and literature mining. datasets were prepared for candidate genes and genes from gwas in attention deficit/hyperactivity disorder (adhd), asd, bp and scz, by using literature and databases (szgene, sfari base, dbgap). datasets for copy number variations (cnvs) associated with neurodevelopmental delay were also assembled. then the overlap between the meta-list of genes highly expressed in astrocytes and gene datasets for psychiatric disease was determined. overlapping genes were pooled and subjected to david bioinformatics analysis. results: the meta-list of genes highly expressed in astrocytes contained n , genes ( . % of the genome). datasets for risk genes in psychiatric disorders showed random overlap with the meta-list (adhd %; asd %; bp %; scz %). cnvs related to neurodevelopment were not enriched for astrocytic genes ( %). when the overlapping genes were pooled from all disorders, a small set of shared astrocytic genes emerged (n ). cntnap , nrxn and sdc were linked by david analysis (p . ). conclusions: this correlative analysis makes it unlikely that genes highly expressed in astrocytes make a major contribution to the genetic risk in four major psychiatric disorders. no genetic link was found between astrocytes and neurodevelopmental delay. changes in gene/ protein expression and pathology in astrocytes in the adult brain in scz and bp may be explained by chronic neuronal dysfunction, chronic medication or acute changes. the neuronal ceroid lipofuscinoses (ncls, batten disease) are a group of autosomal recessively inherited lysosomal storage disorders affecting children and young adults, each of which is caused by a mutation in a different gene. a common feature across all ncls is the early, localised glial activation that occurs long before the onset of neuron loss. this glial activation appears to be an accurate predictor of which neuron populations are vulnerable and will subsequently die. both astrocytes and microglia express the ncl gene products and it is likely that normal glial cell function may be compromised. given the close functional relationship between neurons and glial cells it is possible that glial activation and/or dysfunction may affect neuronal health. we have begun to explore the biology of astrocytes and microglia in the juvenile form of ncl using primary cultures from cln -/mice. defects in both astrocytes and microglia, including altered protein secretion profiles and impaired morphological and proliferative responses were apparent. co-culturing with mutant microglia and astrocytes influenced the survival and morphology of wild type neurons, with more profound effects upon cln -/neurons. intriguingly, these effects were largely reversed by substituting wild type glia, which rescue mutant neurons. a pilot study has provided similar evidence for glial dysfunction in infantile ncl, including and altered protein secretion and response to stimulation. these changes appear to be different from those observed in juvenile ncl and we are in the process of exploring whether the glial cell phenotypes discovered so far, and their impact upon neurons are specific to the different forms of the disease. the accumulation of aggregated proteins in nerve cells and glia underlie the pathogenesis of many neurodegenerative diseases. glial pathology is characteristically observed in tauopathies, such as corticobasal degeneration and progressive supranuclear palsy, and alexander disease, a primary disease of astrocytes caused by mutations in the gfap (glial fibrillary acidic protein) gene. irreversibly damaged proteins can be degraded by the proteasomal system. for proteasomal degradation they are covalently linked to ubiquitin in a three-step enzymatic pathway (e , ubiquitin-activating enzyme; e , ubiquitin-conjugating enzyme; and e ubiquitin ligases). the polyubiquitin chain needs to be removed before the translocation of the substrates to the lumen of the proteasome. this is achieved by deubiquitinating enzymes (dubs), which oppose the functions of e -ligases and assist in targeting substrates to specific pathways. to assess whether impairment of dubs may contribute to age-related neurodegenerative diseases and the regulation of cell death and survival, we have subjected primary cultures of rat brain astrocytes to pr- , a dub inhibitor with broad specificity. immunoblot analysis demonstrates that after a h treatment, pr- caused the induction of heat shock proteins, hsp and hsp , and an increase in p , which is considered a cargo receptor for ubiquitinated proteins and a common constituent of ubiquitinated protein inclusions. as analyzed by indirect immunofluorescence, protein aggregates positively stained by antibodies against ubiquitin and p assembled in the perinuclear region at the microtubule organizing center (mtoc). these aggregates were surrounded by a cage-like network of gfap intermediate filaments, thus resembling aggresomes. using mitotracker and lysotracker staining procedures, the fluorescent images demonstrate that after dub inhibition lysosomes and mitochondria are recruited to the mtoc. this process was dependent on an intact microtubule network, since it was interrupted after treatment with nocodazole, a microtubule destabilization drug. furthermore, pr- caused mitochondrial fragmentation which may be a stress response to enable the removal of damaged mitochondria by mitophagy. in conclusion, dub inhibition impairs the cellular architecture, leads to protein aggregate formation and mitochondrial alterations in astrocytes. our data sustain the hypothesis that an imbalance in dub activities may contribute to the pathogenesis of neurodegenerative diseases. shown that the collapsin response mediator protein (crmp- ), which play a significant physiological role in neuronal cell bodies and axons within the cns, is phosphorylated during the neurodegenerative phase of the inflammatory disease. methods: we investigated the limitation of axonal degeneration by transducing retinal ganglion neurons with a phosphorylation mutant of crmp- utilising an intraocular adeno-associated virus (aav ) delivery system in eae-induced mice. the other group of mice injected with aav consisting of the green flourescent protein reporter only (aav -gfp) with eae was used as a control (n per each group). results: we showed substantial preservation of axons of the optic nerve in the eae-induced mice injected with the aav carrying the crmp- phospho-mutant compared ($ -fold difference) with those mice injected with aav -gfp. the aav -gfp transduced axons showed significant degeneration during the peak stage of eae. conclusion: our data suggest that phosphorylation of crmp- may be a central mechanism that governs axonal degeneration during inflammatory demyelination of the cns (as occurs in ms) and inhibition of phosphorylation of crmp- may be of therapeutic potential for the progressive phase of the disease in ms patients. y. shinozaki, s. koizumi univ. of yamanashi, yamanashi, japan atp, a major gliotransmitter integrating neuron-glia networks, is known to be released or leaked from injured cells. in the cns, atp dramatically changes glial phenotypesand could affect pathogenesis of brain disorders. however, whether such glial responses facilitate or rather inhibit the diseases is still under debate. to address this issue,we studied the neuroprotective role of atp/purinergic signaling using in vivo stab injury model on mouse cerebral cortex. without injury or the contralateral side of the injured brain exhibited no fluoro-jade(fj)-positive neuronal death, cd infiltrating leukocytes or gfap reactive astrocytes. three days after injury, fj signals, cd cells and reactive astrocytes were increased in the ipsilateral side of the brain. the cd cells were enclosed by reactive astrocytes-formed glial scar. administration of an atp-degrading enzyme apyrase, a broad p receptor antagonist ppads, and a p y receptor antagonist mrs significantly reduced the stabincreased fj signals. immunohistochemical analysis revealed p y receptor was expressed in gfap astrocytes. p y receptor knockout (p y ko) mice also exhibited reduced fj signals and infiltration of cd cells. they exhibited higher level of gfap expression, more remarkable hypertrophy of astrocytes and tighter glial scar formation than wild type mice did. the accelerated glial scar formation reduced cd cell number and inhibition of astrocytes by fluorocitrate increased cd cell number. although glial scar formation was accelerated, no enhanced proliferation was observed. we then analyzed the mechanism of enhanced glial scar formation and neuroprotection using in vitro scratch-wound model. in cultured astrocytes, the scratch induced a transient increase in extracellular atp, which was followed by decrease in extracellular atp mainly due to an increase in ectoatpase activity. suppression of purinergic signaling by either apyrase, ppads or mrs enhanced scratchevoked astrocytic migration rather than proliferation. neither activation nor inhibition of p y receptors in cultured cortical neurons affected the scratch-induced damages. our data suggest that the decrease in atp/ p y receptor-mediated signal should be a trigger that transforms astrocytes into migratory phenotype, which forms tighter glial scar structure thereby suppressing neuronal death. background: oligodendrocyte damage and loss are key features of multiple sclerosis (ms) pathology and oligodendrocytes appear to be particularly vulnerable to ros. in vitro studies showed that ros induce cell death and prevent the differentiation of oligodendrocyte precursor cells (opcs) into mature myelin-producing oligodendrocytes. hence, a potential therapeutic strategy to protect these cells from ros-mediated damage is urgently needed. here we investigated the efficacy of several compounds that are able to boost antioxidant enzyme production, including monomethyl fumarate (mmf), tert-butylhydroquinone (tbhq), sulforaphane (sfn) and protandim. these compounds are thought to exert their protective function via activation of the nuclear-factor-e -related factor- (nrf ) transcriptional pathway, which is involved in the production of antioxidant enzymes necessary for oxidative stress defense. methods: primary rat oligodendrocytes were treated with different concentrations of mmf, tbhq, sfn and protandim. expression of antioxidant enzymes were analyzed by pcr and western blot analyses. to study the beneficial effects of the different nrf activators, oligodendrocytes were first incubated with nrf activators and subsequently exposed to various concentrations of hydrogen peroxide. oligodendrocyte cell survival was measured by a live/dead cell viability assay. results: sfn, mmf and protandim are well-tolerated and induce nrf driven antioxidant enzyme production in oligodendrocytes. protandim was the most potent compound with regard to antioxidant enzyme induction and protected oligodendrocytes against ros-induced cytotoxicity. conclusions: our findings indicate that several nrf activators are able to induce antioxidant enzyme production in oligodendrocytes. interestingly, protandim, a dietary supplement consisting of herbal ingredients, was the most potent protector of primary rat oligodendrocytes. in future experiments we will determine whether protandim can also promote the differentiation of opcs under oxidative stress and test the clinical efficacy of protandim in an experimental animal model for ms. the arising of the "tripartite synapse" concept and the discovery of the astrocytic excitability have been highlighting astrocytes as active elements concerning information flow in the brain. however, the impact of such remarkable features in complex brain function is still under-explored mostly due to the difficulty of studying neuron-astrocyte interactions in vivo. the aim of this work was to use an in vivo model of astrocytic dysfunction and investigate the impact of this treatment in complex cognitive functions. the rationale consisted in studying behavioral performance that relies on the prefrontal cortex, such as behavioral flexibility and working memory in an animal model of astrocytic dysfunction. for that purpose, we used a pharmacological model in which wistar-han rats were subjected to bilateral intracranial injections of aminoadipate in the prelimbic portion of the medial prefrontal cortex to cause astrocyte depletion specifically in this region, mimicking pathological states such as depression, in which marked decreases of gfappositive cells are observed. this animal model was tested for its cognitive abilities by the attentional set-shifting task (asst) and water maze-based tests. a clear impairment of cognitive function was observed in the animals treated with aminoadipate. a detailed morphological and histological analysis showed that along with the astrocytic lesion, neurons were also affected at the site of lesion. this seems to contribute to the cognitive decline observed in this model and may explain similar observations under pathological states. a. sternotte, e. hermans universit e catholique de louvain, brussels, belgium amyotrophic lateral sclerosis (als) is an adult disease characterized by a selective loss of motor neurons, resulting in progressive paralysis and death within - years. in several inherited cases, the disease is caused by point mutations in the gene encoding for superoxide dismutase (sod ) which promotes its aggregation, leading to biochemical damages, including mitochondrial dysfunction. alteration of mitochondrial membrane potential and/or respiratory chain leads to atp depletion and these metabolic dysfunctions could contribute to motor neuron death in patients and animal models of als. in motor-neurons, the combination of altered axonal transport with mitochondrial dysfunction likely leads to considerable decrease in atp levels at distant neuromuscular junctions. commonly know as the fuel gauge of mammalian cells, amp-activated protein kinase (ampk) is a key enzyme in the control of cellular atp and energy homeostasis. in this study, the implication of ampk in the progression of als was specifically investigated by measuring the expression and activity of this enzyme in the spinal cord of mice overexpressing the mutated human sod (hsod g a ), a commonly used experimental model of als. no consistent modification of the enzyme was detected in spinal cord samples throughout the progression of als. indeed, such experiment did not discriminate between cell types present in the tissue. hence, a more detailed characterization of ampk in cultured astrocytes derived from als animals revealed a robust induction of the enzyme activity, which correlates with decreased atp levels in these cells. in a second part of our study, we have investigated the consequences of ablating ampk in the mouse model of als by breeding ampk knock out mice with hsod g a mice. while we did not detect any difference in the lifespan of ampk(-/-)/hsod g a mice as compared to hsod g a mice, we observed substantial alterations in the progression of the disease in selected behavioural tests. in particular, analysis of the gait (catwalk) which enables early detection of muscle weakness revealed that the onset was delayed by up to days while the progression of the disease after onset was accelerated. further studies will be conducted to establish the importance of atp-in the disease, and to validate this enzyme as a putative pharmacological target in als and in other neurodegenerative diseases involving mitochondrial dysfunction. institute of neuroscience, sibs, cas, shanghai, china ng glia is the fourth type of neuroglia in vertebrate nervous system, which also known as oligodendrocyte progenitor cell (opc) in a developmental point of view. in adult brain, ng glia has a small cell body with highly branched extensions and represents a new type of glial cell differing from other glial cell types such as astrocyte, microglia and mature oligodendrocytes. published studies by others have shown that ng glia is able to promptly respond to brain injury and activated in several neurodegenerative disease animal models including -ohdainduced rat parkinson's disease model. however, the pattern and function of these activated ng glia remain largely unknown. here, we performed a time-course study of ng glia activation in mptp mouse model of parkinson's disease using immunohistochemistry. it was found that ng glia was activated as early as hours after the last mptp injection in the substantia nigra (sn) of mptp-treated mice. this temporal character is very similar to those of microglia, indicating ng glia respond to mptp challenges very quickly. the activation of ng glia became stronger over time and reached the peak at about days after the final mptp injection. then, the extent of ng glia activation declined gradually and diminished days after mptp injection. interestingly, we found that prominent ng glia activation could only be observed in the substantia nigra. the dorsolateral striatum which is the projection target of nigral dopaminergic neurons, however, was devoid of ng glia activation in all the time-points examined. in summary, we characterized the ng glia activation in mptp mouse model of parkinson's disease. these data suggest that activated ng glia may play a role in the degenerative process of dopaminergic neurons in pd. hallmarks of cns inflammation, including microglial and astrocyte activation, are a feature of post-mortem tissue from amyotrophic lateral sclerosis (als) patients and in transgenic mice overexpressing mutant superoxide dismutase- (sod g a ). administration of glucocorticoids does not significantly alter disease progression, but this may reflect poor cns delivery. here, we sought to discover whether cns-targeted liposomally-packaged glucocorticoid would inhibit the cns inflammatory response and reduce motor neuron loss. sod g a mice were treated with saline, free methylprednisolone (mp, mg/kg/week) or glutathione pegylated liposomal mp ( b - , mg/kg/week) and compared to saline treated wild-type animals. animals were treated weekly with intravenous injections for weeks from days of age. animal weights and motor behaviour were monitored during this period. at the end of the experimental paradigm ( days) mice were imaged using t -weighted mri for brainstem pathology, and brain and spinal cord tissue was collected for histological analysis. all sod g a animals showed a significant decrease in motor performance compared to baseline from $ days. sod g a animals showed a significant increase in signal intensity on t weighted mr images, which may reflect the combination of neuronal vacuolation and glial activation in these motor nuclei. treatment with b - , but not free mp, significantly reduced t hyperintensity, which correlated with significantly reduced histopathological manifestations in the brainstem motor nuclei, but not the spinal cord. interestingly, there was a significant reduction in astrogliosis but not microglial activation following treatment with b - . the results of this work indicated that the cns-targeted anti-inflammatory agent b - has therapeutic potential in als. the toxic effects of new antiretroviral drugs on the central nervous system (cns) are unclear. because these drugs penetrate the brain even at low concentrations, it becomes crucial to determine the doses which can be toxic for the cns resident cells. moreover, after the recent introduction into clinical practice, it is unclear whether the efficacy of the antiretroviral drugs of new generation may also derive from their ability to exert extravirological effects on factors responsible for the development of hiv brain injury, e.g. matrix metalloproteinases (mmps). objective: to investigate on the toxicity of four different antiretroviral drugs and their ability to modulate the expression of gelatinase b (mmp- ) in astrocyte cultures. methods: primary cultures of rat astrocytes were activated by exposure to mg/ml lipopolysaccaride (lps) (positive control) and simultaneously treated for h with increasing doses ( - - - . mm) of: efavirenz (efv); darunavir (drv); maraviroc (mvc) or raltegravir (ral). mmp- mrna expression was assessed by rt-pcr. quantitative determination of mmp- expression was done by computerized scanning densitometry. single drug toxicity was assessed by the mtt test. each drug was considered toxic at the concentration able to induce a percentage of cell survival above %. results: the treatment with antiretroviral drugs inhibited mmp- mrna expression in lps-activated astrocytes in a dose-dependent manner. in particular, a statistically significant inhibition of mmp- expression was observed when astrocytes were treated with mm efv ( % of inhibition) or with mm ral ( % of inhibition). as assessed by the mtt test, the toxicity of the antiretrovirals ranges from and mm. in particular, efv was toxic for astrocytes at the concentration of mm, mvc at mm, while drv and ral were toxic at the concentration of mm. conclusions: the present results indicate that efv and ral directly inhibit mmp- expression in lps-activated astrocytes with mechanisms that are independent from their antiviral activity. the toxic doses of antiretrovirals are much higher than those found in the csf of hiv-positive patients. our results highlight some beneficial/deleterious extra-viral effects of the antiretroviral drugs that may be useful to improve the development of new therapeutic strategies for the management of hiv infection. we used a parahippocampal kainate injection mouse model to induce mtle-hs and to study early expression patterns of kir . and apq after se. mice were sacrificed h, h and d after kainate injection. immunhistochemistry of the hippocampus was performed to assess kir . and aqp expression, and gfap staining was used for identification of astrocytes. gfap expression was increased compared to saline injected controls, suggesting that kainate injection induces astrogliosis. comparison of kir . and aqp staining showed a similar change in the expression pattern. our data indicate a drop in kir . and aqp expression within hrs after se followed by an upregulation within the first week. altered expression of kir . and aqp could contribute to dysregulation of potassium and water homeostasis and play a role in the early phase of epileptogenesis. c. l€ o€ ov, a. erlandsson uppsala university, neuroscience/neurosurgery, uppsala, sweden we have previously shown that astrocytes effectively engulf dead cells after trauma both in vitro and in vivo, but that they store the ingested material rather than degrade it. compared to macrophages, which degrade engulfed, dead cells within hours, our data show that astrocytes store the ingested material for weeks before the degradation is completed. to further study the routes of degradation and the possibilities to speed up this process we have used a cell culture model where uvtreated, dead cells are added to stem cell-derived astrocytes. by labeling the dead cells with the ph-sensitive dye phrodo prior to the engulfment, we demonstrate that the ph in the astrocytic phago-lysosomes is higher than in professional phagocytes. interestingly, lamp and lamp , which are involved in the phago-lysosome fusion, are both highly expressed in the astrocytes, particularly around the ingested material. dendritic cells are known to express the inhibitory protein rab a that slow down the degradation in order to preserve antigen for presentation. rab a prolongs the actin coating around the phagosomes, which physically inhibit the phago-lysosome fusion, and interacts with nox- , which leads to an increased consumption of protons. western blot analysis shows a high expression of rab a in our cell cultures which may explain the slow degradation. moreover, dead cells in astrocytes are surrounded by actin rings for a long period of time after the ingestion. to counteract this, the astrocytes were treated with . or mm of the actin inhibitor latrunculin b. the number of actin rings were significantly lower in the cultures treated with mm of latrunculin b compared to controls, but the intensity of phrodo was unaltered indicating that the prolonged degradation may be due to a higher ph in the lysosomes rather than a delayed actin coating. next we will investigate the effect of rab a expression on the degradation process by performing sirna experiments. one of the promising strategies for the treatment of spinal cord injury is the transplantation of glial cells obtained from the olfactory system; olfactory ensheathing cells (oecs). effective proliferation and migration of oecs are essential for optimizing clinical applications and there is a need for identification of small molecules that regulate glial cell biology. curcumin is a natural polyphenol compound found in the spice turmeric, which is known for its neuro-protective properties and has been reported to have an effect on neurogenesis and nerve regeneration. however, the effect of curcumin on oecs has not been determined. we have examined the effect of curcumin on oecs at the cellular level using fluorescence and timelapse microscopy. oecs were purified from s b-dsred transgenic mice in which oecs express the fluorescent protein dsred. different treatments: (i) control medium, (ii) curcumin ( . to mm), (iii) g commercial growth factor, or (iv) a combination of g and curcumin were used to determine the effect of curcumin on oecs proliferation and migration. cell proliferation was quantified at two different times by cell counting and mts proliferation assay. timelapse microscopy was used to visualize changes in cell morphology and cell migration. cell branching, number and area of lamellipodia and speed of migration per cell were measured for each treatment. we found that lower concentrations of curcumin ( . and mm) increase oec proliferation. as well, combination of g and curcumin showed the highest proliferative effect on oecs suggesting a possible synergistic relation between g and curcumin. live cell imaging analysis showed that curcumin increased the number and area of lamellipodia resulting in faster migration of oecs. these results suggest that curcumin can regulate the proliferation, morphology and migration of oecs which could improve the therapeutic use of oecs for spinal cord injury repair. methods: the dams were divided in four groups and had received ml of nonalcoholic or alcoholic beer solution -control, vehicle (nonalcoholic solution), etoh % (nonalcoholic solution %v.v ethanol) or etoh % (nonalcoholic solution %v.v ethanol) during gestational day up to weaning (postnatal day ). male offspring ( - days old) were sacrificed and hippocampal acute slices were prepared. we analyzed gfap content, s b and glutamate uptake as astrocyte parameters. we tested the animals in the plus-maze discriminative avoidance task to check anxiety and memory. results: gfap content decreased after pnee for etoh % and etoh % treatment (f , . , p < . ). interestingly, we found that only the etoh % treatment showed an increase of hippocampal s b content (t . , p . ) and in the csf (f , . , p . ), but no difference in secretion. glutamate uptake was significantly decreased for etoh % group (f , . , p . ). to perform the plus-maze discriminative avoidance task we selected pups from control, vehicle and etoh % treatment on pnd . two-way anova revealed significant effects of treatment (f , . , p . ), arm type (f , . , p < . ) and treatment x arm type interaction (f , . , p < . ). in the test session, only a significant prenatal treatment group effect (f , . p < . ). bonferroni's post-hoc test revealed that only control group presented significantly less time in the enclosed aversive arm than nonaversive one. conclusions: in this study we showed that pnee with moderate doses could alter the structure and functional astrocytes balance. thus, high levels of s b are indicated in some neurodegenerative disorders. taken this data together we could demonstrate that moderate ethanol exposure can be harmful to fetal brain. aging has been associated to neuroinflammation in the central nervous system, however it is not known whether microglial changes induced by aging are affected by early effects, such as litter size and sedentary life style. in addition, the lateral septum has been recognized by its complementary role in the memory processing for tasks of object recognition for identity and spatial location. in the present report, we investigated whether aging cognitive decline and microglial morphological changes in the lateral septal region are influenced by changing litter size early in life and by a sedentary life style. to assess these questions, wistar rats suckled in litters of either six or pups per dam were raised sedentarily in groups of up to , from the st post-natal day onwards. at (young adult) or (aged) months-old, half of the sedentary rats underwent progressive daily treadmill exercise for five weeks, while the others remained sedentary. after performing the tests of recognition for spatial localization and object identity all of the animals were sacrificed and their brains were processed for selective microglia/macrophages immunolabeling with anti-iba- antibodies. a representative sample of the immunolabeled cells in the lateral septum was analyzed after three-dimensional reconstruction with neurolucida software (microbright field inc.) and morphological features of each cell were quantified by neuroexplorer (microbright field inc.). it was found that sedentary life style of wistar rats maintained in standard laboratory cages is associated with spatial memory deficits in both mature and aged subjects no matter the litter size, and that exercise decreased these effects in aged subjects raised in small but not in large litters. on the other hand, all sedentary aged rats, despite of the litter size, presented impairment of object recognition memory for identity, and exercise decreased this effect in animals from both large and small litter size groups. microglial morphological analysis revealed that cell soma area, perimeter and branches volume seem to be more intensely affected by aging and that these changes are mainly associated with animals raised in large litters. furthermore, it was observed important shrinkage and thickening of the microglial branches in aged individuals on a higher proportion in the sedentary group suckled in large litters. taken together the results suggest that litter size and sedentary life style may affect object recognition and spatial memories in both adult and aged rats and that exercise minimizes these effects on animals suckled in small but not larger litters. in addition, we suggest that these changes are related to distinct effects on the soma and branching patterns of septal microglia from young and aged subjects. huntington disease (hd) is a dominant inherited neurodegenerative disorder caused by an unstable expansion of a cag repeat within the gene encoding for huntingtin protein (htt). this mutation induces formation of a poly-glutamine tract in the mutant htt (mhtt) protein that prones its aggregation into the cells. hd is characterized by an initial and massive degeneration of the medium spiny neurons in the striatum with later neuronal loss in cortex, globus pallidus, and other structures leading to motor and cognitive symptoms. alterations of energy metabolism contribute to hd pathogenesis but the mechanisms responsible for these alterations are not well understood. a recent pet study provided evidence for a selective impairment of striatal glycolytic and not oxidative metabolism of pre-symptomatic hd patients. in the brain, glycolysis is predominantly an astrocytic metabolic process, whereas oxidative metabolism is primarily neuronal raising the question that metabolic defects in hd could originates from astrocytes. the purpose of our study was to characterize ( ) the metabolic alterations in vivo in a transgenic mouse model of hd (bachd mice) and ( ) the respective roles of astrocytes and neurons in metabolic defects occurring in hd using an in vitro strategy. bachd mice express the human full length htt protein with glutamine repetitions under the human htt promoter into a bacterial artificial chromosome. bachd mice present a slow disease progression with motor deficits occurring at months and progressive neuronal aggregates from months, reflecting human pathology. first, we performed [ c]- -deoxyglucose autoradiography experiments in vivo to assess energy metabolism in months bachd (n ) and wt (n ) mice. we performed a d analysis of the whole brain glucose uptake without any regional a priori. using statistical parametric mapping (spm), a voxel-wise statistical analysis approach, we showed that compared to age-matched wt mice, months bachd mice present hypometabolism in the striatum, like pre symptomatic hd patients, and also in the hippocampus. hypermetabolism was also detected in the hypothalamus. second, we performed an in vitro study to dissect out the cellular mechanisms responsible for these metabolic changes. by using cell insert, we realized neurons/astrocytes co-culture without any physical contact. this culture system allowed us to grow neurons in presence of wt or bachd astrocytes. we showed that both bachd and wt neurons have a decreased [ c]- -deoxyglucose uptake when cultured in presence of bachd astrocytes but not wt astrocytes. these results collectively suggest that energy defects in bachd mice are due to noncell autonomous mechanisms by which astrocytes expressing mhtt induce neuronal metabolic dysfunction. naag and naa are abundant metabolites in the brain which can be utilised by oligodendrocytes to make myelin. but elevated levels of naag and naa are associated with myelin loss in the leukodystophies canavan and pelizaeus-merzbacher-like disease, whereas lower levels of naa are observed in brain tissue of ms patients. currently, the physiological function of these two metabolites as well as their role in white matter pathology is unknown. naag and naa can also act as signalling molecules as they can affect glutamate receptors. oligodendrocyte precursor cells (opcs) express nmda receptors and activation of these receptors may be important for their differentiation and efficient myelination. in disease opcs are recruited to demyelinating lesions where they proliferate and differentiate into new myelinating oligodendrocytes. we therefore investigated the effects of both physiological and pathological concentrations of naa and naag on opc biology in vitro. we used calcium imaging to see whether naag or naa can induce intracellular calcium changes in opcs as they are known to act on neuronal nmda receptors. mm naa or mm naag evoked intracellular calcium changes in opcs (df[ / ] . . and . respectively), but only one third of the cells responded which corresponds to the proportion of nmda responsive cells in the cultures. however, not all opcs that responded to nmda did also respond to naag or naa. therefore, it seems that the naa and naag evoked calcium raise is via an nmda receptor independent pathway. naag and naa evoked a small but significant increase in intracellular calcium in opcs, which even after prolonged application of pathological levels of naa and naag did not affect the cells' viability as propidium iodide staining of opcs showed no significant difference in viability. over % of the opcs survived hrs application of mm naa and mm naag. naag and naa are produced and released by neurons but it is not known how opcs take them up from the environment and how it affects them. we therefore addressed which possible transporters opcs express and the effect naa and naag have on opcs' proliferation and differentiation. it is unlikely that naa and naag even at high concentrations have a detrimental effect on opcs. perhaps these high concentrations of naa and naag found in canavan and pelizaeus-merzbacher-like disease indicate rather opcs' deficiency to transport and utilise naa and naag for energy and myelin formation. vanishing white matter (vwm) disease is a severe leukoencephalopathy, classically starting in childhood. the disease is characterized by neurological symptoms, including uncoordinated movements and balance disturbances which are slowly progressive. episodes of rapid deterioration are triggered by febrile infections and minor head trauma, which can lead to coma. a treatment is unavailable and eventually all patients die within a few years upon diagnosis. the disease is caused by mutations in any of the five genes encoding the subunits of the eukaryotic translation initiation factor b (eif b). although this protein has an important function in all cells, mainly the cells in the brain white matter (astrocytes and oligodendrocytes) are affected by these mutations. pathologically the brain shows cystic degeneration with meager gliosis, dysmorphic astrocytes, lack of myelin and an increased number of oligodendrocyte and astrocyte progenitor cells. we have developed two mouse models for vwm to further unravel the disease mechanisms at molecular level and to develop and test possible treatments. the b mouse line is homozygous for a mutation in the eif b gene, encoding the eif bd subunit, and the b mouse line is homozygous for a mutation in the eif b gene, encoding the eif be subunit. both mutations have been described in human patients and are associated with a severe form of vwm. preliminary data show that these mice models recapitulate the clinical and neuropathological phenotype observed in vwm patients. pathologically, the central nervous system white matter is vacuolated and shows lack of myelin while astrocytes are dysmorphic and immature. these transgenic mice appear to be a representative model for vwm. microglial cell lines were differentiated from ips cells and represent in vitro models for human microglia. rt-pcr and flow cytometry analysis of ipsdm showed gene transcription and protein expression of cd respectively. the protein expression was increased by breaking the cis-interacting sialic acids on the microglial glycocalyx using sialidases. cd -fc fusion protein was designed and purified via the fc tag to study binding patterns of cd to various cell types expressing different glycocalyx structures. additionally, ipsdm exhibiting either an over expression or knockdown of cd will be used to determine protein functions. furthermore, preliminary immunohistochemical analysis of control brain tissue compared to ad patients showed increased cd protein expression in cd positive cells, in the latter. in summary, data show that cd is expressed by human microglial cell line enabling further characteristic and functional analysis of the protein. furthermore, preliminary analysis on primary brain tissue corroborates the association of cd -expressed on microglia -to lateonset ad. the neurotrophins are essential for peripheral nervous system development and myelination. we have previously demonstrated that the neurotrophin bdnf exerts contrasting influences upon myelinationacting through neuronal p ntr to enhance myelination, but inhibiting it via neuronal trkb. recently we have generated a small peptide called cyclo-dpakkr that structurally mimics the region of bdnf that binds p ntr . here we aim to investigate whether utilising cyclo-dpakkr to selectively target p ntr is an approach that could exert a unified promyelinating response. like bdnf, cyclo-dpakkr promoted myelination of ngf-dependent neurons in vitro, an effect dependent on the neuronal expression of p ntr . importantly, cyclo-dpakkr significantly enhanced the myelination of bdnf-dependent neurons in vitro, whereas bdnf inhibited it. local injection of cyclo-dpakkr adjacent to the neonatal sciatic nerve in vivo significantly enhanced myelin protein expression and increased the number of myelinated axons. we found that injection of cyclo-dpakkr also significantly upregulated the expression of neuregulin type-iii, a key factor required to induce peripheral myelination. thus, our data demonstrate that cyclo-dpakkr promotes peripheral myelination in vitro and in vivo. to investigate whether cyclo-dpakkr promoted the remyelination of peripheral neurons, we utilized the ean, a rodent model of peripheral demyelinating neuropathy. our data show that administration of cyclo-dpakkr significantly delayed the disease onset and reduced clinical severity in ean. this improved disease phenotype is supported by reduced demyelination, as cyclo-dpakkr substantially reduced the extent of myelin damage in myelinated peripheral nerves subjected to ean. collectively, our data demonstrate that using cyclo-dpakkr to selectively target p ntr promotes peripheral myelin development, and is effective at ameliorating ean disease and protecting against myelin damage. our findings suggest that selective targeting of p ntr is a strategy worthy of further investigation for the treatment of peripheral demyelinating diseases introduction huntington's disease (hd) is a neurodegenerative disease characterised by huntingtin protein misfolding and the intra-cellular accumulation of mutant huntingtin (mhtt). accumulation of mhtt is most pronounced in neurons and is thought to underpin neuronal dysfunction. oligodendrocytes have been shown to accumulate mhtt aggregates, which may contribute to cellular dysfunction in these cells. evidence from diffusion tensor mri (dt-mri) studies shows that white-matter changes are amongst the earliest pre-symptomatic changes observed in hd. these findings have been taken to implicate axonal and or glial aberrations prior to disease onset. methods we have used a variety of techniques, which include, emulsion in situ hybridisation, whole brain sub-fractionation, immunohistochemistry, meso scale cytokine assay, and western blots. results our investigations using the r / mouse model show that mhtt aggregates result in a specific down-regulation of the small heat shock protein (shsp) hspb (alpha-b-crystallin). localisation of hspb by whole brain sub-fractionation shows that hspb is enriched in the myelin fraction. emulsion in situ hybridisation further shows localises hspb in oligodendrocytes. detergent extraction of myelin fractions show differential hspb partitioning between soluble and insoluble fractions. cumulatively, these findings suggest that hspb is constitutively expressed in oligodendrocytes and is present in two distinct pools. recent studies implicate hspb as being a negative downregulator of inflammation. as such down-regulation of the shsp in r / mice may promote increased oligodendrocyte vulnerability to mhtt cytotoxicity. to investigate the role of hspb as a negative regulator of inflammation, we obtained transgenic hspb knockout mice. the knockout mice are viable and show very little phenotypic difference against littermate controls. we have analysed the inflammatory profiles of these mice using the meso scale. to validate our findings in the r / mice, we are currently investigating human tissue from distinct vonsattel stages of disease, to see if hspb downregulation is also observed in the human disease. conclusion as hspb has several critical cellular roles, its downregulation may compromise oligodendrocyte structure and function, which ultimately has negative consequences for neurons. it is therefore worthwhile to investigate it's specific role in the cns. the myelin sheath is attached to the axon through multiprotein complexes that form the axon-glial interactions. they consist of cell adhesion molecules and voltage gated ion channels and divide the axon into distinct domains: the node of ranvier, the paranode, the juxtaparanode and the internode. this molecular organization is crucial since recent studies identified distinct perinodal proteins as autoantigens in ms patients: nodal and paranodal neurofascin isoforms and juxtaparanodal tag- /contactin- . tag- is a cell adhesion molecule expressed both by axons and glial cells. in the adult nervous system, tag- organizes the juxtaparanodal domain of the fiber, where it interacts with caspr and the potassium channels (vgkcs). question: in this study we have analyzed the alterations of the juxtaparanodal proteins caspr , tag- and vgkcs in relation to paranodal caspr and nodal sodium channels upon the onset and progression of experimental autoimmune encephalomyelitis (eae) and in the cuprizone model of toxic demyelination. methods: we immunohistochemically and biochemically analyzed both models of cns demyelination. results-conclusions: our results show a higher paranodal susceptibility to disruption compared to juxtaparanodes in eae. as eae progresses there is subsequent compensatory efforts for myelinated fiber restoration that include paranodal protein re-clustering and increased heminodal formation, without subsequent juxtaparanodal protein aggregation. in contrast, juxtaparanodal organization was observed only when remyelination occured in the cuprizone model of toxic demyelination. supported by: imbb-forth scholarship, manassaki scholarship (university of crete), the project "myelintag" ( ) is implemented under the "aristeia" action of the "operational programme edu-cation and lifelong learning" and is co-funded by the european social fund (esf) and national resources. multiple sclerosis (ms) is a chronic disease of the human central nervous system that is characterized by focal lesions with inflammation, infiltration of immune cells, demyelination and axonal damage. activation of cannabinoid cb /cb receptors is considered a potential therapeutic strategy for the treatment of ms, based on the evidence that exogenous cannabinoid agonists exert neuroprotective and immunosuppressive effects in experimental models of the disease. nevertheless, the therapeutic use of synthetic and/or plant-derived agonists acting on brain cannabinoid receptors is limited by the possible adverse responses related to memory and learning impairment. an alternative approach that could avoid this limitation consists of enhancing the concentration of the main endocannabinoids (aea, -ag) by increasing their synthesis or decreasing their degradation. the main objective of this study was to analyze the effects of jzl , a selective inhibitor of -ag hydrolyzing enzyme monoacylglycerol lipase (magl), in the chronic eae model of ms. mice were treated daily with jzl ( mg/ kg and mg/kg) or vehicle from onset of the motor symptoms to the end of the experiment. comparison of the motor score curves indicated that both doses of jzl ameliorated the deficits observed in vehicletreated mice during the disease course. nevertheless, the beneficial effect of the mg/kg dose was no longer evident in mice scored at dpi. chronic treatment with mg/kg jzl reduced the conduction latency of the corticospinal tract measured at the end of the experiment, as well as the number and size of inflammatory lesions in spinal cord, whereas chronic administration of the high dose of the magl inhibitor had no effect on these parameters. importantly, treatment with the mg/kg dose of jzl reduced the coupling ability of cannabinoid receptors to g i/o proteins, measured by [ s]gtpcs autoradiography, in all the brain areas analyzed. finally, incubation with jzl prevented cytotoxicity by activation of ampa-type glutamate receptors in oligodendrocyte precursors in vitro. this protective effect of the jzl was blocked by the cb receptor antagonist am . our findings point to the involvement of cb receptors in the in vivo therapeutic effects of jzl , and suggest that chronic administration of magl inhibitors may be a promising strategy for the treatment demyelinating disorders in which oligodendrocyte excitotoxicity plays an important role as mechanism of white matter damage. under disease conditions, connexins can also release these signaling molecules into the extracellular milieu through hemichannels. since astrocytes are involved in the disease progression of als, we are exploring if abnormal gj activity can serve as a potential mechanism for the reported astrocyte-mediated toxicity. we used the sod g a mutation as a als mouse model to address the role of connexins in als. we observed that at the end stage of the disease, spinal cords of sod g a mice exhibited a significant increase in expression of connexin protein compared to their spinal cords of control mice. this increase in cx co-localizes primarily to astrocytes in the gray matter of the spinal cord. importantly, human post-mortem tissue from als patients compared to control patients exhibited a significant increase in cx rna and protein levels in the motor cortex and a trend for increase in cx in the cervical spinal cord. this is an important finding in pursuing cx as a potential candidate contributing to astrocyte-mediated toxicity in als. in addition, when astrocytes were isolated from wt and sod g a mice, endogenous levels of cx rna and protein were elevated in sod g a mice compared to the wt derived astrocytes. we are currently investigating if functional properties of astrocytes from sod g a mice are altered in addition to biochemical changes. in vitro studies using cx specific blockers to assess neuroprotection are also being conducted in co-cultures of neurons and astrocytes. these findings will be novel and an important step towards harnessing therapeutic strategies for als. the pdz protein omi/htra is localized in the intermembrane space of mitochondria. it is involved in mitochondrial homeostasis and may act as a chaperone. mutations in the serine protease domain of omi/ htra (mnd mice), leads to massive loss of striatal neurons and neuromuscular disorders in mice confirming the protective function within mitochondria. upon cellular stress, omi/htra is translocated from mitochondria into the cytosol when the mitochondrial outer membrane is permeabilised. in the cytosol, omi/htra blocks the action of the inhibitors of apoptosis proteins (iaps) by competitive binding or by degradation via the protease activity. this leads to caspase activation and apoptosis induction. omi/htra also regulates apoptosis in a caspaseindependent manner via its protease activity; recent studies have defined multiple substrates of the serine protease. in a yeast twohybrid screen we identified the ng proteoglycan as a binding partner of omi/htra . ng is expressed by oligodendrocyte progenitor cells (opc). in the presence of hydrogen peroxide, which is a model of cellular oxidative stress, omi/htra is released from mitochondria and can bind to ng in opcs. binding of ng to omi/htra via the pdz domain may thus be a way to sequester or regulate the serine protease. oligodendrocyte-lineage cells are known to be particularly sensitive to cellular stress and white matter diseases of new borns is a major pathology in situations where premature infants are exposed to unphysiological oxygen concentrations. it has been recently shown that glioma cells often express the ng protein which may play a protective role. our results indicate that the interaction between omi and ng may help protect opcs from cellular stress. microglia are the immunocompetent cells of the cns that continuously survey the extracellular milieu for foreign antigens and play an instrumental role in brain inflammation. in contrast, astrocytes extend highly ramified processes between neurons and synapses and play a vital role in regulating brain homeostasis, synaptic function and plasticity. interestingly, both microglia and astrocytes adopt a specialized 'activated' or 'reactive' state when exposed to adverse brain conditions. the changes in their molecular profiles include the release a diversity of proteins such as pro-and anti-inflammatory cytokines and extracellular matrix molecules that could impact each other's behavior and regulate the properties of nearby neurons. in alzheimer's disease (ad), activated microglia and reactive astrocytes are detected around plaques in mouse models of ad and human ad tissues. however, exactly how these glial types contribute to the onset or progression of ad still remains an open question. to gain a better understanding of the bidirectional communication and response of microglia and astrocytes in the development of ad-like symptomatology, we investigated the properties of these cells types in the crnd transgenic ad mouse model by applying confocal imaging and western blot analysis. crnd is an early-onset model of ad-like symptomatology showing ab deposits present at months of age and neuritic pathology by months. our results show complex spatio-temporal changes in the interaction of microglia and astrocytes around ab plaques during the progression of the disease in both cortex and hippocampus. we found that subtle rearrangements of microglial morphology and astrocyte reactivity were detected as early as month when ab plaque deposition is absent. by - months, low numbers of activated microglia and reactive astrocytes begin to surround small ab deposits. the establishment of complex 'reactive glial domains (rgds)' can be observed at months, when amoeboid microglia fully encompass large ab plaques and become surrounded by reactive astrocytes forming an elaborate outer shell-like structure. intriguingly, after months, the domains become progressively disrupted due to the reorganization and recruitment of additional microglia and astrocytes. we also observed gradated inflammatory phenotypes of glial cells. these are first restricted to rgds in the early and middle stages of ad but spread locally in late stages when disorganized rgds are observed. interestingly, neuronal dystrophy was correlated with the severity of the inflammation. we conclude that communication between microglia and astrocytes may be a key process in the ability of the brain to surmount a reparative response to early neurodegenerative conditions but may be disrupted or become overwhelmed in later stages of ad. supported by alzheimer society of canada (db) and by cihr (kkm and rq). poster topic extracellular matrix and cell adhesion molecules opcs exist in mature rodent and human central nervous system (cns; around - % of the total cells) and constitute an interesting source regenerative therapies in demyelinating diseases, like multiple sclerosis (ms). different molecules are involved in opc morphofunctional development during embryogenesis and postnatal stages, and some of them are upregulated in ms lesions, suggesting their involvement in the pathogenesis of the disease. this is de case of anosmin- , an extracellular matrix glycoprotein coded by the kal gene and responsible for the x-linked form of kallmann syndrome. the best known mechanism of action of anosmin- seems to be mediated through the interaction of this protein with fibroblast growth factor receptor (fgfr ) and the modulation of the activation of this receptor by fgf . this protein participates in the adhesion, migration and differentiation of various cell types in the cns; among others, anosmin- promotes the adhesion of neurons, neurite outgrowth, axonal guidance and branching of different cns projection neurons, as well as having a role in the migration of different types of neuronal precursors, immortalized gnrh-producing neurons and embryonic opcs. in addition, previous results of our group also suggest a role of anosmin- in demyelinated lesions from ms patients and point to the feasible pharmacological and genetic manipulation of the fgf /fgfr- /anosmin- system on endogenous and/or exogenous opcs in demyelinating lesions. in the present work we studied the functional implications of anosmin- and fgf- on neurobiology (cell death, proliferation, motility, migration) of postnatal/adult murine opcs isolated from cerebral cortex (p , p , p ) and of opcs isolated from adult human biopsies, using both in vivo (immunohistochemistry) and in vitro (chemotaxis chambers, migration, brdu uptake, tunel, immunocytochemistry) techniques. these results would be useful for the design of effective neuroreparative therapies in ms and other demyelinating diseases. funded we generated ipsc lines from fibroblasts isolated from pitx -gfp knock-in mice which allowed facs-purification of mature midbrain da neurons upon in vitro differentiation based on the expression of the mature da marker pitx . subsequently, mouse pitx -gfp knock-in ips cells were transduced with lentiviral vectors containing genes encoding for hl and for stx, the enzyme that add psa onto ncam. next they were differentiated in vitro into da neurons. the effects of hl and psa-ncam on differentiation, survival and outgrowth of ipsc-derived da neuron were analyzed in vitro and in vivo. indeed, we were able to obtain a pure suspension of mature ipscderived da neurons upon in vitro differentiation and fac sorting. transfection of pitx -gfp knock-in ips cells with hl or stx did not affect the pluripotent potential of these cells and did not interfere with the process of dopaminergic differentiation itself. we continued to establish the beneficial effect of l and psa-ncam expression on survival and outgrowth of ipsc-derived da neurons in vitro and after grafting in the striatum of parkinsonian rats. vascular endothelial growth factor (vegf) is a major regulator of neurovascular remodeling following brain injury. while much have been learnt about vegf functions on target endothelial cells, little is known about its intracellular trafficking, mode of secretion and matrix interactions in "source" cells, i.e. mainly reactive astrocytes. here, we generated a vegf::gfp fusion protein to follow the distribution of vegf during its trafficking in primary astrocytes and cos cells. we found that vegf::gfp forms dimers and gets glycosylated similarly to wild type, and as expected, the molecule follows the endoplasmic reticulum-golgi pathway. however, its post-golgi trafficking suggests a unique route with features that do not conform the classical constitutive secretory pathway: the secretion of vegf occurs even at c and shows a ca -and pkc-induced increase. we investigated the distribution of vegf in polarized primary astrocytes in an in vitro scratch wound assay. in these cells, vegf::gfp appeared to follow a vectorial distribution and accumulated behind the leading edge. the accumulation appeared to be on the extracellular surface, moreover, ultrastructural immunogold labeling revealed that extracellular vegf::gfp remains associated with discrete areas of the cell membrane and is accumulated in caveolae as well as in microvesicular shedding elements. this particular localization corresponds to fibrillar adhesions (fb), where vegf::gfp is co-localized with fibronectin. we also observed that vegf::gfp is targeted to adherens junctions between astrocytes, however, at these sites vegf::gfp was not co-localized with fibronectin. finally, we show in frap experiments that integrin turnover is decreased in vegf associated fbs, which raises the possibility of an autocrine/paracrine regulation of astrocytic functions. together, these findings have strong implications for understanding focal coordination of angiogenesis and vascular remodeling by astroglia derived vegf. after invading the embryonic cns, microglia migrate extensively to occupy their final positions. however, the cellular and molecular mechanisms of microglial migration are unknown. therefore, this study aims to elucidate the microglial migration behavior, cellular interaction partners and ecm-integrin interactions essential for migration in the developing neocortex. methods: immunohistochemical stainings and immunogold labeling for transmission electron microscopy were carried out on fixed brain tissue of c bl/ cx cr /egfp mice embryos (embryonic day (e) . - . ). multicolor facs analyses were performed on homogenized age-pooled cx cr /egfp embryonic cortici. microglial migration was recorded in cx cr /egfp -hgfap-cfp acute brain slices using multiphoton time-lapse excitation and analyzed using mtrackj in imagej. results: laminin (lm) is expressed as punctuate dots near the pia and in the ventricular zone at e . and disappears at e . to remain only in blood vessels and the pia. fibronectin (fn) is present as aggregates from e . until e . . both ecm proteins follow the course of radial cells. preliminary data indicate a transient upregulation of the lm receptor (lmr) on e . and e . . in addition, the percentage of microglia expressing the fn receptor (fnr) tends to decrease during development. ex vivo time-lapse recordings show that embryonic microglia are dynamic cells that migrate in random patterns. these cells seem to make brief contacts with the processes of radial glia. conclusions: embryonic microglia express laminin and fibronectin receptors and possibly change the expression level during development. they are dynamic cells that migrate in random patterns, and possibly make occasional contact with radial glia. knowledge about ecm-integrin interactions during microglial migration could reveal targets for intervention in pathological settings, such as multiple sclerosis and alzheimer's disease. multiple sclerosis (ms) is a chronic demyelinating disease of the central nervous system in which astroglial cells become hypertrophic, produce various extracellular matrix (ecm) proteins which become aggregated when secreted. these aggregated ecm proteins contribute to a non-permissive environment for repair. tissue transglutaminase (tg ) expression is enhanced in astrocytes in ms lesions. this enzyme is well-known for protein cross-linking capacities. moreover, tg can act as a co-receptor for b-integrins to bind to ecm proteins, thereby mediating cell adhesion processes. in this study we questioned whether astrocyte-derived tg affects ecm protein production and/or aggregation, and if astrocyte-derived tg mediates cell adhesion onto various ecm proteins. primary rat astrocytes were transduced with an empty lentiviral vector (mock) or with a lentiviral vector expressing human wildtype tg . alternatively, primary rat astrocytes were transduced with a lentiviral vector expressing scrambled shrna or tg shrna to knockdown endogenous rat tg . the rat primary astrocytes overexpressing human tg showed an increased production of fibronectin and collagen v. conversely, knocking-down tg showed a decrease in fibronectin and collagen production. interestingly, overexpression of tg resulted in enhanced aggregation of extracellular laminin. also, human astrocytoma cells (u ) were transduced with an empty lentiviral vector (mock) or with a lentiviral vector expressing human wildtype tg and allowed to adhere onto ecmcoated wells. astrocytoma cells overexpressing human tg showed increased adhesion onto laminin-, and fibronectin-coated wells. we conclude that astrocyte-derived tg affects production/aggregation of fibronectin and laminin. moreover, the astrocyte-derived tg mediated elevated production/aggregation of fibronectin and laminin coincides with more adherence of astrocytes onto these ecm proteins. we hypothesize that tg -mediated enhanced production/aggregation of fibronectin and laminin is related to increased astrocyte adhesion which together could contribute to the non-permissive environment for repair in the central nervous system of ms patients. a. tripathi, z. parikh, p. pillai the m.s. university of baroda, vadodara, india background: oligodendrocytes originate as progenitor cells (opcs) in discrete areas of the developing brain which undergoes a complex and precisely timed program of proliferation, migration, differentiation, and myelination in cns. during migration it interacts with its surrounding environment through integrins. purpose: to evaluate the interactions between integrins and growth factors (gfrs) that promote the molecular events such as proliferation, cytoskeletal reorganization and migration in oligodendrocytes leading to oligodendrocyte mediated myelination. methods: cortical cells were prepared from p rat cerebral cortex following standard protocols. immunocytochemistry (icc), western blot (wb), immunoprecipitation (ip), migration assay and real-time rt-pcr analysis were performed. for dissecting out the roles of different intracellular signalling proteins in the regulation of events downstream of receptor activation, use of pharmacological inhibitors was carried out. results: tnfa, pdgfa and estradiol significantly increased cell proliferation and cell processes. data also suggest differential effects of extracellular matrix (ecm) on signalling component activation. significant increase in erk expression was found following gfrs-integrin interactions. ecm proteins bind to integrin family members and mediate bidirectional signalling between the cellular interior and the external environment through the activation of focal adhesion complexes. conclusion: integrin switching, oligodendrocyte proliferation, cytoskeletal reorganization and differentiation profile is highly influenced by ecm and gfrs. integrin governs the signalling cascades underlying oligodendrocyte differentiation and myelination. this study was performed in strict accordance with the recommendations for the care and use of laboratory animals. all the protocols were duly approved by the institutional ethical committee, the m.s. university of baroda. topography, roughness and mechanical properties of micro environment are crucial parameters influencing cell adhesion/motility, morphology and mechanics as well as the development of cells e.g. stem/progenitor cells, and neuronal cells [ ] [ ] [ ] [ ] [ ] . atomic force microscopy is a powerful tool not only to study the morphology in terms of high resolution imaging and roughness measurements, but also to map mechanical and adhesive properties of the sample/cells and tissues. combining these remarkable abilities with advanced optical microscopy allows for extensive characterization of biomaterials and tissue slices [ ] [ ] [ ] . however, commercially existing technical solutions are either time consuming, or only limited practicable for soft, sticky, or fragile samples. therefore, we developed a new force curve based afm mode -quantitative imaging (qi tm ). the novel qi tip movement algorithm prevents lateral forces and controls the vertical forces for nondestructive imaging. to demonstrate the performance and flexibility qi tm mode we investigated topography, adhesion properties, and young's modulus local distribution of living dorsal root ganglion cells. a specialized platform -cellhesion v r -has been developed to run single cell force spectroscopy (scfs) with a need for long-range cellsurface and cell-cell binding experiments with up to microns pulling length. using single cell force spectroscopy (scfc), cell-cell adhesion can be quantified [e.g. ], and the contribution of different components e.g. from the extra cellular matrix, can be assessed [ ] . a new technical solution will be demonstrated if a cantilever attached cell can be transferred through the liquid-air interface. this approach allows to measure the adhesion of the same individual single cell on different materials within different dishes as it will presented for cho cell adhesion on plastic as well as albumin coated surfaces. we want to present the progress in automatic data processing and display to investigate topography, young's modulus and local adhesion phenomena on transparent and non-transparent biomaterials like titanium, peptide functionalized surface or cell layers, and tissue slices. l. vargova , m. cicanic , , e. sykova , charles university, nd faculty of medicine, prague, czech republic institute of experimental medicine, v.v.i., department of neuroscience, prague, czech republic bral- is a link protein that is localized in distinct brain regions, where it stabilizes the perineuronal nets of the extracellular matrix (ecm). our previous studies showed that qualitative and quantitative ecm changes, might evoke changes in the diffusion properties of the extracellular space (ecs), thus affecting the movement of neuroactive substances in the cns. in the current study, we determined by the real-time iontophoretic method the extracellular space volume fraction a (a ecs volume / total tissue volume) and the geometrical factor tortuosity k (k free / apparent diffusion coefficient) in coronal brain slices obtained from the sensorimotor cortex and the ventral posteromedial thalamic nucleus of sixteen-month-old bral- deficient mice (bral- -/-), age-matched bral- / controls and young adult ( -month-old) bral- / controls. the diffusion parameters in the cortex of young adult bral- / mice were: a . . (mean s. e. m.) and k . . , n , n (n -number of slices, n -number of animals). in the thalamus, there was a smaller a and a larger k ( . . and . . , respectively, n , n ). in young adult mice, we found no significant differences in the ecs diffusion parameters between bral positive and negative mice in either the cortex or the thalamus. in the cortex of aged bral- / mice we found a smaller a ( . . , n , n ) than in the cortex of young adult mice. during aging, there was no significant difference in the thalamic diffusion properties in wild-type animals, but in the thalamus of aged bral- -/-mice, a significantly decreased ( . . , n , n ) in comparison to the younger bral -/-animals as well as to age-matched controls. immunohistochemical analysis of the ecm in the ventral posteromedial thalamic nucleus revealed no positivity for bral protein in either group of bral -/-mice. in the youger mice, bral deficiency was associated with a small attenuation in the positivity of brevican and another link protein, ctrl ; however, in aged bral -/-animals, brevican and ctrl positivity was increased in comparison with the age-matched controls. in contrast to the cortex, we found no decrease in a in the thalamus of aged wt animals, suggesting that the process of aging may differ between the cortex and the thalamus. a decrease in the ecs volume fraction in the thalamus of bral- -/-aged mice may result from a disruption of the perineuronal nets and rearrangements of the molecular assembly, which seem to differ in young adult and aged animals. here we analyzed the global transcriptome of cultured astroglial cells incubated with activators of camp pathways. a bulk of astroglial transcripts were differentially regulated by camp signaling. camp analogs strongly upregulated genes involved in typical functions of mature astrocytes, such as homeostatic control, metabolic and structural support to neurons, antioxidant defense and communication, whereas they downregulated a considerable number of proliferating and immaturity-related transcripts. moreover, genes typically activated in reactive cells, such as immunological mediators and scar components, were repressed by camp. gene set enrichment analysis and evaluation in situ of gene expression in astrocytes in different states showed that camp signaling conferred a mature and in vivo-like transcriptional profile to cultured astrocytes. these results indicate that camp signaling is a key pathway restricting developmental and activation features of astrocytes and promoting their maturation. a positive modulation of camp signaling is suggested to suppress the mechanisms of activation driven by pathological situations and to promote the physiologically normal state of differentiated astrocytes. question: microglia, the innate immune system of the central nervous system, are crucial to maintain homeostasis by cleaning debris and attacking stressors. in the ageing brain, increased numbers of microglia with ameboid morphology and increased inflammatory cytokine levels are reported. the altered microglial phenotype in the ageing brain is characterized by hyperresponsive to immune stimuli, which is called immune priming. immune priming could be a potential cause of age-associated neurodegeneration. the aim of this study is to characterize the immune pathways and biological processes that are altered in ageing-induced immune primed microglia and to find genes potential drivers of this process. design/methods: the effect of ageing on microglia has been addressed in naturally aged mice ( months old) and in ercc transgenic mouse model in which accelerated ageing is induced by dna damage accumulation. from each of these models a pure population of microglia was isolated. microarray technology was used to obtain information about the genome wide gene expression profile. weighted gene co-expression network analysis (wgcna), uses correlational structures to find clusters of genes to make optimal use of a dataset in biological interpretation. results: using weighted gene co-expression network analysis, a very robust microglial immune priming gene module was discovered. in this module, there is a significant enrichment of the complement system, antigen presentation, interferon type- signaling and phagocytosis-machinery. the average expression of this module is stronger up-regulated in ercc transgenic than in normally physiologically aged mice, supporting the notion that ercc is characterized by excessive immune priming. conclusion: microglial priming which occurs in normal ageing or in accelerated ageing models is marked by increased expression of several immune signaling pathways. methods: we investigated three single nucleotide polymorphisms (snps) (rs (arg gly), rs (gln gly) and rs ) and five haplotypes in adrb to explore whether these polymorphisms were associated with ms progression in well phenotyped ms patients. genotyping was done using illumina bead microarray and missing genotypes were imputed using beagle. results: a trend towards a decreased frequency of rs c allele ( gln) was in people with progressive ms than in people with relapsing remitting ms. this was seen in both sp-ms (p (uncorrected) . ) and in pp-ms (v . , p (uncorrected) . ). however, those p values did not reach statistical significance after correction for multiple testing. neither haplotypes nor snps were significantly associated with altered msss, age of onset, time between fist and second relapse, processing speed (sdt) or brain atrophy. conclusion: we found no evidence that polymorphisms in adrb influence severity in multiple sclerosis. here we examine the expression of these brain-specific pgc- a isoforms in neurons and glia cells. we characterize the abundance of the different isoforms of pgc- a in primary murine cell cultures of neurons, glia cells, including oligodendrocytes, astrocytes and microglia under control conditions and metabolic stress. we show that pgc- a is differentially expressed in the different cell types, e.g. neurons and oligodendrocytes express much higher levels of the novel brain-specific isoforms of pgc- a. the different expression levels might reflect the different metabolic needs of the different cell types. s. marques, g. castelo-branco karolinska institutet, stockholm, sweden oligodendrocytes (ol) are neuroepithelium-derived cells that insulate neuronal axons through myelin-containing membranes, essential for axonal integrity and impulse transmission. defects in this process are present in several diseases, including the highly debilitating multiple sclerosis (ms). the process of remyelination can be promoted by ol precursor cells (opc) endogenously and occurs during a short window of time at earlier stages of ms, ultimately failing as the disease evolve. the understanding of development and differentiation regulation of ol is essential to clarify the reasons behind the defective myelination during pathologies and to open the pave to new remyelinating cell therapies. opcs start to be specified early during embryogenesis but regardless of the origin, their terminal differentiation and functional maturation occurs only at post-natal stages. in this project, we are characterizing the transcriptome of opcs during development, which will allow us to better characterize the heterogeneity of these cells and point out what might be the key factors involved in transition between states. recent studies suggest that non-coding rnas can interact with proteins complexes containing chromatin modifying enzymes and transcription factors, regulating their activity, acting as scaffolds and/or recruiting them to specific loci in the genome. therefore, we are investigating their role in opcs by rna interference and how they might modulate the function of key transcription factors. neural stem cells (nscs) represent a potentially valuable resource when considering repair strategies for cns damage. unlike their embryonic counterparts, adult neural stem cells (anscs) in the two neurogenic niches have an astrocyte-like identity, raising the question of what regulates this ansc state. some early postnatal astrocytes in the parenchyma also retain some nsc-like characteristics and in vitro display self-renewal and multipotency, though these properties are lost following in vivo maturation. interestingly, following injury, adult astrocytes can become reactive and reacquire nsc-like properties, whilst others can generate new neurons through forced expression of specific transcription factors. we are aiming to elucidate the gene regulatory networks underlying specific nsc and astrocyte states and transitions between them using transcriptomic and epigenomic tools alongside computational methods in different nsc and astrocyte populations. we will describe our latest findings from an in vitro system that aims to model immature versus mature astrocytes derived from a common progenitor. from microarray data, we have identified candidate genes and pathways involved in regulating astrocyte potential versus differentiation, including a role for specific pro-inflammatory signalling. we will also describe the associated epigenetic signatures that accompany cell state and discuss the implications of our latest findings. microglia are the cns immune-related cells and associated with the pathogenesis of several types of neurological diseases. following peripheral nerve injury (pni), spinal microglia become activated and have crucial roles in generating neuropathic pain. we have previously shown that interferon regulatory factor- (irf ), upregulated in spinal microglia after pni, regulates gene expressions that switch to a reactive phenotype. here we identified irf as a crucial factor of reactive microglia that works cooperatively with irf . we found that pni increased expression of irf in the spinal cord, the expression of which was restricted to microglia. forced expression of irf in cultured microglia markedly increased expression of irf in a manner that depended on its ability to bind dna. furthermore, irf -deficient mice failed to increase the expression of irf in spinal microglia after pni, indicating irf -dependent expression of irf in microglia in vivo. interestingly, upregulation of p x receptor (p x r; an atp-gated channel crucial for producing neuropathic pain) by forced expression of irf in cultured microglia was markedly suppressed by knockdown of irf expression. in a model of neuropathic pain, suppressing upregulated irf protein by spinal administration of sirna targeting irf alleviated pain hypersensitivity. altogether, the irf -irf axis drives a program of gene expression that promotes p x r hi microglia gating neuropathic pain. astrocytes are the most abundant glial cell type. they express a range of neurotransmitter receptors localized along their processes that cover synapses and constitute "microdomains" for signalling pathways. to investigate astroglial purinergic p y and glutamatergic nmda receptors we generated knockout mice in which gene recombination of "floxed" receptors was controlled by an astrocyte-specific and tamoxifen-inducible cre recombinase (glast-creert ). here, we present data on the dna recombination efficiencies of glast-creert x floxed p y mice and glast-creert x floxed glun mice in different brain regions (cerebellum, hippocampus, brainstem, optic nerve and cortex). recombination is determined by quantitative real-time pcr of genomic dna using primers across the loxp sequences flanking exon of the p ry gene and exon - of the grin gene, respectively. dna recombination was induced in young adult mice by intraperitoneal tamoxifen injection for three consecutive days and analyzed three weeks later. primers were designed such that reduction of the pcr signal reveals increasing recombination (i.e. gene excision and knockout) compared to control. the degree of reduction in the investigated brain regions is expected to be similar in both mouse lines; allowing conclusions ( ) about the reliability of the glast-creert /loxp system in general (i.e. recombination efficiency at different chromosomal locations), and ( ) gives a percentage of glastpositive cells (predominantly astrocytes) in the given brain area. first data demonstrate % recombination in the cortex, which is well in line with the percentage of astrocytes in this brain region. these results could be confirmed by using a reporter mouse line expressing the red fluorescent protein tdtomato (r -tdtomato) and using immunohistochemistry with astrocyte markers. in conclusion, the combination of quantitative real-time pcr analysis of gene recombination and cell-specific cre mouse lines represents a reliable approach to reveal the percentage of a given cell type in a given tissue region. the transcription factor creb is protective in injury and neurodegeneration, but the contribution of neuronal vs astrocytes is unknown. we have generated a mouse with targeted over-expression of a constitutively active creb (vp -creb) in astrocytes by crossing teto-vp and tta-gfap mice. vp -creb/gfap mice show expression of vp in astrocytes -as detected by immunohistochemistry -restricted to cerebellum, hippocampus and outer layers of cortex. we determined the role of astrocytic creb in the outcome of brain injury by subjecting wild type (wt) and vp -creb/gfap (tg) mice to a focal cryolesion (c) in the frontal cortex. at days post-lesion, wt mice presented a necrotic region filled with macrophages (labeled with lectin) surrounded by a rim of tissue with robust gliosis (labeled with gfap). tg mice showed reduced macrophage infiltration as compared with wt mice. a dna array analysis of the damaged zone (agilent) has revealed differentially expressed genes in paired comparisons. wtc/wt: , ; wtc/tgc: , ; tgc-t: ; tg/wt: (statistical linear model in limma., p values computed by empirical bayes moderated t-statistics at . ). there were significantly enriched kegg pathways regulated by the cryolesion, according to gsea and hypergeometric tests set at p < . . vp -creb had an effect on of the pathways. upon increasing the analysis stringency by setting the cut-off at p < . , the number of significantly enriched kegg pathways regulated by vp -creb was reduced to . we selected of these pathways related to functions: energy metabolism, inflammation, structural reorganization, genomic stability and apoptosis, which we define as the core signature of the vp -creb-elicited change. the differential expression of representative genes related to this signature was validated by real-time pcr. thus, vp -creb rescued the decreased expression of enzymes regulating energy metabolism, while decreasing the expression of pathways related to apoptosis, extracellular matrix and inflammation. the latter is consistent with the decreased macrophage infiltration detected by immunohistochemistry in tg mice. we posit that vp -creb protects the brain against bionergetic failure and secondary damage due to macrophage infiltration, while rendering the tissue more conducive to neurite regeneration by reducing extracellular matrix components. the study supports the notion of astrocytes as therapeutic targets in brain injury. furthermore, ca /cn/nfat dependent mmp expression was confirmed in pure astrocyte cultures derived from neural stem cells (ast-nsc), demonstrating that the induced mmp expression occurs in astrocytes, and not microglial cells. in an in vivo stab-wound model of brain injury, mmp expression was detected in nfatc -positive scarforming astrocytes. since [ca ] i increase is an early event in most brain injuries, these data support an important role for ca /cn/ nfat-induced astrocyte mmp expression in the early neuroinflammatory response. understanding the molecular pathways involved in this regulation could provide novel therapeutic targets and approaches to promoting recovery of the injured brain. the posterior hypothalamus (ph) is crucial for maintaining wakefulness whereas the anterior hypothalamus (ah) constitutes a sleep-promoting region. consequently, the energy needs of ah and ph should probably change throughout the sleep-wake cycle. the glycogen mobilization altogether with the astrocyte-neuron lactate shuttle (anls) likely constitutes two major mechanisms by which astrocytes ensure the neurometabolic coupling. therefore, we hypothesized that astrocytes of ah and ph may display differences in genes expression involved in these mechanisms during the sleep-wake cycle. to investigate this, we measured the gene expression levels in astrocytes obtained from transgenic mice expressing the fluorescent protein egfp under the control of the astrocyte-specific gene gfap promoter. mice were sacrificed at four time, zt , zt , zt and zt (zt corresponding to light-on). after brain dissection, "punches" of ah and ph were micro-dissected from slices. cell suspensions were obtained from a pool of samples by enzymatic digestion and cell trituration. then, gfp-positive cells, which were highly enriched in astrocytes, were sorted by facs. total rna was extracted from these cells and gene expression levels were assayed by qrt-pcr and normalized by cyclophylin mrna levels. seven genes related to anls were tested: the alpha sub-unit of the na-k atpase (atp a ), the two sub-unit of the lactate dehydrogenase (ldha and ldhb), the glutamate transporters (glast and glt ) and two monocarboxylate transporters (mct and mct ). levels of mrna encoding genes related to glycogen metabolism such as ptg, the glycogen synthase (gs) and the glycogen phosphorylase (gphos) were also measured results showed that the levels of mrna encoding ldha, mct and mct expressed significant circadian variations which can reach % for mct at zt , compared to zt levels in ah. the glt mrna levels as well as mct and mct mrna levels also displayed significant circadian variations in ph. levels of expression of genes encoding the gphos, gs and ptg did not display major regional circadian variation. this study shows that astrocytes displayed some transcriptional regulation in hypothalamic areas involved in the sleep-wake cycle. the difference in the pattern of expression of anls related genes in ah and ph, such as glt , suggests that neuro-metabolic coupling capacity of each structure might be different and might play a functional role in the sleep-wake regulation. , are a heterogeneous cell population that is despite its functional importance still not well defined. while astrocytes are no longer just considered the supporting cells in the cns and are recognized to play a significant functional role in e.g. synapse formation, regulation of the blood brain barrier and synaptic transmission, little is known whether specific subtypes of astrocytes fulfill these functional tasks differently. studies in our laboratory described brain region specific differences in the expression of astrocytic glutamate transporter (glt- ) with significantly increased levels of glt- in the spinal cord compared to brain. the mechanisms of glutamate transporter regulation are not well defined, and to date little is known about the factors that are responsible for regulating protein expression and transporter activity and whether this regulation is specific for certain subtypes of astrocytes. to study glt- regulation in more detail, we generated a family of transgenic mice using increasing lengths of the non-coding region of the glt- gene controlling expression of tdtomato. these mice were crossed with full length bac-glt- -egfp mice to compare astrocytic expression of the reporter genes. interestingly, only a subpopulation of gfp-positive astrocytes was found to have strong tdtomato fluorescence signal, when we crossed an . kb eaat promoter fragment mouse with the bac-glt- mouse, suggesting the existence of astroglial subtypes that are differentially regulated for eaat expression. such differences might account for variance in local glutamate-dependent excitotoxicity to motor neurons. to this end, we purified both tdtomato /gfp and tdtomato-/gfp astrocytes from multiple cns regions by fluorescence-assisted cell sorting (facs) and analyzed their transcriptomic profiles using microarray analysis. a list of candidate genes, mainly transcription factors and cell membrane molecules, was complied from this analysis and validated by both quantitative rt-pcr and immunofluorescence. pax and kir . were found to be highly expressed in astrocytes, consistent with previous findings. more importantly, kir . mrna was noted to be selectively enriched in cortical tdtomato /gfp cells, compared to gfp only cells. it suggests kir . may serve as an astroglial subtype marker. . we have hypothesized that microglial c/ebpb is a potential target to attenuate the neurotoxic effects of neuroinflammation. in order to test this hypothesis in vivo, c/ebpb knockout mice are not suitable because they show multiple phenotypic alterations as a result of the widespread expression of c/ ebpb. mice with specific c/ebpb deletion in microglia would be a better model. unlike gfap promoter for astrocytes, there is not a gold standard promoter for cre expression in microglia. cd b, cx cr or lysozime m (lysm), among other promoters, have been used to this end. we have generated lysm-cre/c/ebpb fl/fl mice with two main aims: ) to validate the use of lysm as a suitable promoter for microglial gene deletion by cre-lox recombination ) to analyze the effects of the absence of microglial c/ebpb in vitro and in vivo lysm-cre/c/ebpb fl/fl mice which were fertile and viable and did not show the female infertility and high perinatal mortality described in c/ ebpb knockout mice. western blot experiments revealed the presence of c/ebpb in protein extracts from wild-type but not from lysm-cre/c/ ebpb fl/fl microglial cultures. immunocytochemistry experiments confirmed this observation. thus, c/ebpb immunoreactivity was present in all cells in wild-type microglial cultures and it was absent in all microglial cells in lysm-cre/c/ebpb fl/fl microglial cultures. the microglial specificity of lysm-cre recombination was demonstrated in primary mixed glial cultures. whereas in wild-type primary mixed glial cultures c/ebpb immunoreactivity was observed both in astrocytes and microglia, in lysm-cre/c/ebpb fl/fl mixed glial cultures it was detected in astrocytes, but never in microglia,. to assess the functional outcome of microglial c/ebpb deficiency, we analyzed lps ifncinduced no production and we observed a marked decrease in lysm-cre/c/ebpb fl/fl microglial cultures. this decrease was also observed in mixed glial cultures in fitting with the microglial origin of no production in this model. in summary, these findings show that lysm-cre promoter causes recombination in % of microglial cells in primary culture and it is therefore suitable for microglial-specific gene deletion in vitro. experiments are in progress to determine the degree and specifity of microglial c/ebpb deletion in vivo in these mice. supported by grants pi / and pi / , instituto de salud carlos iii, spain. to study the functional role of cpeb , transgenic mice overexpressing cpeb in astrocytes were generated. cpeb overexpression without the endogenous untranslated region (utr) led to the downregulation of astrocytic connexins (cx & cx ). consistently, in the hippocampus of cpeb overexpressing mice, intercellular gap junction coupling was strongly impaired. cpeb overexpression also led to downregulation of glutamate transporter (glt- ) and glutamine synthetase (gs), key players involved in extracellular glutamate clearance. we have now raised mice overexpressing cpeb with the endogenous utr. in these mice, we observed the downregulation of cx , cx , glt- and gs. since interastrocytic coupling and astrocytic glt- and gs activities are downregulated in epilepsy patients with hippocampal sclerosis, we hypothesize that upregulation of cpebs in astrocytes may contribute to the pathogenesis of epilepsy by causing astrocyte dysfunction. to study the influence of the p gene in the structural and quantitative characteristics of astrocytes in the mice retina methods: adult mice of the c bl/ strain ( months old) were distributed into two groups: ) mice with two extra copies of p ("super p "; n ) and ) wild-type p age-matched control, as the control group (wt; n ). retinas were immunohistochemically processed with gfap. gfap astrocytes were quantified. results: in comparison con wt: i) retinal-astrocyte distribution followed established patterns; however, morphological changes were seen through the retinas in relation to p availability: astrocytes were more robust and secondary processes were more evident; ii) astroglial density was significantly higher in the sp retinas, both in the whole-retina (p < , student's t-test) and in the intermediate and peripheral concentric areas of the retina (p < . student's t-test). conclusion: the astrocyte changes in the sp retinas might improve the resistance of the retinal cells against oxidative stress and its downstream signalling pathways. however, neuronal damage caused due to excessive microglial activation is a well known phenomenon in neurodegenerative diseases. mirnas are novel regulators of gene function, controlling several biological processes. recent reports show altered mirna expression in immune-mediated pathologies, suggesting that mirnas have roles in modulating immune responses. thus, the present study was initiated to identify micrornas and their target mrnas which can modulate microglial inflammatory responses. a pilot study was designed to understand the role of mirna- b in modulating microglia-mediated immune response as this was found to be localized in the microglia. recently, mirna b has been shown to target several proteins including c-jun, the substrate of jnk mapk (mitogen-activated protein kinase) which mediates the release of proinflammatory cytokines in activated microglia. qrt-pcr revealed the decrease of mir- b expression level in activated bv microglia. loss-of-function and gain-of-function studies confirmed c-jun to be the target of mir- b in microglia. overexpression of mir- b in activated microglia resulted in a decrease in c-jun and jnk expression and activity, thereby dysregulating the mapk-jnk pathway and proinflammatory cytokines. further, treatment of neuronal cells, mn d with conditioned medium obtained from activated microglial cells, resulted in increased inflammatorymediated cell death upon knockdown of mir- b. overexpression of mirna- b also reduced the phagocytic ability in activated microglia. taken together, these results demonstrate the important role of mir- b in modulating the mapk pathway via c-jun which in turn affects different aspects of the inflammatory process accompanying microglia activation including cytokine response, no production, phagocytosis and neuronal cell death. thus, mir- b may prove to be a useful target for developing therapeutic strategies to control microglial mediated inflammation in neurodegenerative diseases. further in order to understand the global mirna changes contributing to microglial activation, a global mirna microarray was carried out using control, lps-activated and amyloid b-activated primary microglia. this screen identified several families of mirnas differentially expressed between the different treatment groups enabling us to analyze the mirna signature in activated microglia. national university of singapore, singapore, singapore an inflammatory process in the brain as a result of an injury or infection is mediated by the microglia, the prime immune effector cells of the central nervous system (cns). microglia have also been shown to display chronic inflammatory responses leading to neurodegenerative disorders. small ubiquitin-like modifier- (sumo- ) is a post-translational protein modifier, which has been shown to be associated with nuclear factor kappa b (nfjb)-mediated inflammatory pathway. in this study, we showed the expression of sumo- in microglia and characterized the roles of sumo- in activated microglia. we found that sumo- is specifically expressed in the amoeboid microglial cells of the early postnatal rat brain. secondly, lipopolysaccharide-mediated activation of microglia led to a significant increase in the expression as well the nuclear translocation of sumo- in microglia in vitro. in order to establish a function for sumo- in activated microglia, we sought to silence its expression using sirna-mediated gene knockdown. sumo- knockdown in lps-treated bv- microglia in vitro subsequently showed the decreased nuclear translocation of nfjb and expression of tumour necrosis factor-alpha (tnf-a) level. further, we performed an in silico screening to identify the targets of sumo- in the nfjb pathway. our study thus, suggests that sumo- regulates nfjb translocation into the nucleus for the transcription of pro-inflammatory genes including tnf-a in activated microglia. microglia are the main resident immunological cells the cns. in the healthy brain, microglial cells are in a surveillance state whereas upon rupture of cns homeostasis they enter activated states. in ischemic stroke, activated microglia is one of the most important cellular components of post-stroke neuroinflammation, which mainly occurs in and around the area of the infarct. microglia activation is characterized by morphological alterations, functional and transcriptional remodeling, which account for the acquisition of immune phenotypes. purinergic receptors are known to play important roles in microglia activation, and p x r have been suggested as potential therapeutic target to limit microglia-mediated inflammatory responses associated with brain diseases. in the present study, we have developed an experimental approach based on laser micro-capture to isolate microglia from the penumbra area at different post-lesion times (from h to days post-lesion). the repertoire of genes expressed by microglial cells was determined through cdna microarray analysis. with this approach we have been able to determine clusters of genes that are co-regulated thus revealing the time course of microglia activation in this model. in addition, we have compared the transcriptional remodeling of microglia in wild-type and p x -deficient mice. our results suggest that wild-type and p x r ko mice present specific transcriptional profiles, which only partially overlapped. thus our data suggest that p x r play significant roles in the regulation of microglial cells functions. z. chen , m. ghosh , r. sattler , m. robinson , j. rothstein johns hopkins university, baltimore, united states university of pennsylvania, philadelphia, united states question: astrocytes, the most abundant cell type in the central nervous system (cns), are a heterogeneous cell population that is despite its functional importance still not well defined. while astrocytes are no longer just considered the supporting cells in the cns and are recognized to play a significant functional role in e.g. synapse formation, regulation of the blood brain barrier and synaptic transmission, little is known whether specific subtypes of astrocytes fulfill these functional tasks differently. studies in our laboratory described brain region specific differences in the expression of astrocytic glutamate transporter (glt- ) with significantly increased levels of glt- in the spinal cord compared to brain. the mechanisms of glutamate transporter regulation are not well defined, and to date little is known about the factors that are responsible for regulating protein expression and transporter activity and whether this regulation is specific for certain subtypes of astrocytes. methods: to study glt- regulation in more detail, we generated a family of transgenic mice using increasing lengths of the non-coding region of the glt- gene controlling expression of tdtomato. these mice were crossed with full length bac-glt- -egfp mice to compare astrocytic expression of the reporter genes. results: interestingly, only a subpopulation of gfp-positive astrocytes was found to have strong tdtomato fluorescence signal, when we crossed an . kb eaat promoter fragment mouse with the bac-glt- mouse, suggesting the existence of astroglial subtypes that are differentially regulated for eaat expression. such differences might account for variance in local glutamate-dependent excitotoxicity to motor neurons. to this end, we purified both tdtomato /gfp and tdtomato-/gfp astrocytes from multiple cns regions by fluorescence-assisted cell sorting (facs) and analyzed their transcriptomic profiles using microarray analysis. a list of candidate genes, mainly transcription factors and cell membrane molecules, was complied from this analysis and validated by both quantitative rt-pcr and immunofluorescence. pax and kir . were found to be highly expressed in astrocytes, consistent with previous findings. more importantly, kir . mrna was noted to be selectively enriched in cortical tdtomato / gfp cells, compared to gfp only cells. it suggests kir . may serve as an astroglial subtype marker. conclusions: our data clearly suggest the existence of molecular subtypes of astrocytes. factor expressed by activated astrocytes and microglia. studies with non-glial cells have shown that c/ebpd is able to regulate the expression of proinflammatory genes. however, the role of c/ebpd in neuroinflammation has not been established. we have here analyzed the effects of c/ebpd absence on glial activation in vitro and in vivo. in primary mixed glial and microglial-enriched cultures the expression of the pro-inflammatory genes nos- , cox- and il- induced by lps ifnc, but not by lps alone, was attenuated in the absence of c/ebpd. chip experiments revealed binding of c/ebpd to the promoters of these genes in activated, but not in control, mixed glial cultures. in neuronal-microglial co-cultures the neurotoxicity elicited by lps ifnc-activated microglia was completely abolished when microglial cells were devoid of c/ebpd suggesting that this transcription factor plays a key regulatory role of the expression of potentially detrimental proinflammatory genes by activated microglia. to analyze the role of c/ebpd in neuroinflammation in vivo mice were treated systemically with lps ( ug/mouse; i.p.). this treatment induced a marked increase in c/ebpd mrna and protein brain levels. double immunofluorescence experiments showed the presence of c/ebpd in astrocytes and microglia in lps-treated mouse brain. in c/ ebpd -/-mice, systemic lps-induced brain expression of nos- , tnfa, il- b and il- was attenuated. finally, in mouse experimental autoimmune encephalitis (eae) c/ebpd was upregulated in the spinal cord and the severity of eae symptoms was reduced in c/ebpd -/-mice. altogether these findings demonstrate that c/ebpd plays a major role in the regulation of proinflammatory gene expression in neuroinflammation and point to microglial c/ebpd inhibition as a novel strategy to reduce the detrimental effects of neuroinflammation. supported by pi / and pi / from instituto de salud carlos iii, spain gene targeting strategies have become a powerful technology for elucidating mammalian gene function. the recently generated knockout (ko)-first strategy produces a knockout at the rna processing level and also allows for the generation of conditional ko alleles by combining flp/frt and cre/loxp systems, thereby providing high flexibility in gene manipulation. however, this multipurpose ko-first cassette might produce hypomorphic rather than complete knockouts if the rna processing module is bypassed. moreover, the generation of a conditional phenotype is also dependent on specific activity of cre recombinase. here, we report the use of an efficient molecular biological approach to test pannexin (panx ) mrna expression in global and conditional panx ko mice derived from the ko-first mouse line, panx tm a(komp)wtsi . using qrt-pcr, we demonstrate that tissues from wild-type mice show a range of panx mrna expression levels, with highest expression in trigeminal ganglia, bladder and spleen. unexpectedly, we found that in mice homozygous for the ko-first allele, panx mrna expression is not abolished but reduced by % compared to that of wild-type tissues. thus, panx ko-first mice present a hypomorphic phenotype. crosses of panx ko-first with flp deleter mice generated panx f/f mice. further crosses of the later mice with mgfap-cre or nfh-cre mice were used to generate astrocyte-and neuronal-specific panx deletions, respectively. a high incidence of ectopic cre expression was found in offspring of both types of conditional panx ko mice. our study demonstrates that panx expression levels in the global and conditional panx ko mice derived from ko-first mouse lines must be carefully characterized to ensure modulation of panx gene expression. the precise quantitation of panx expression and its relation to function is expected to provide a foundation for future efforts aimed at deciphering the role of panx under physiological and pathological conditions. agarwal, e. hughes, d. bergles johns hopkins university, baltimore, united states astrocytes elevate intracellular ca in response to stimulation of metabotropic receptors. these ca transients are linked to processes as diverse as synaptic plasticity and functional hyperemia, but the relevance of this signaling remains uncertain. although most studies of ca signaling in astrocytes have focused on somatic events, anatomical and functional studies indicate that ca signaling in astrocytes may be compartmentalized into functionally isolated "microdomains." to facilitate analysis of ca signaling, we generated transgenic mice in which the cytosolic and membrane anchored variant of genetically encoded calcium indicator gcamp , referred as cgcamp and mgcamp respectively, can be expressed conditionally in a cell specific manner. a ubiquitous cag promoter is used to control cgcamp and mgcamp expression, and a "stopper" sequence flanked by loxp sites was placed upstream of the coding sequence, preventing expression until cre excises this dna. these constructs were targeted to rosa locus to ensure widespread expression. to evaluate whether cgcamp and mgcamp were expressed at levels sufficient to resolve ca transients, we bred r -lsl-cgcamp and r -lsl-mgcamp mice to glast-creer mice. double transgenic offspring were injected with tamoxifen at weeks of age and analyzed - weeks later. histology revealed that cgcamp and mgcamp were expressed in astrocytes throughout the brain. astrocytes in acute cortical slices exhibited large amplitude, spontaneous increases in both cgcamp and mgcamp fluorescence. the signal to noise ratio of the fluorescence intensity was greatly improved in astyrocytes expressing mgcamp , partially due to the ability of mgcamp to better resolve near membrane ca flux. ca transients were typically restricted to small regions of an individual astrocyte, consistent with ca elevations within microdomains. spontaneous ca transients were occasionally observed in the soma of astrocytes expressing cgcamp but not mgcamp , and were not coincident with activity in the processes, suggesting that these regions are functionally uncoupled. to determine if gcamp expression is sufficient to visualize ca signaling in vivo, we made cranial windows over the somatosensory cortex and performed -photon imaging. microdomain ca transients were prominent in cortical astrocytes, indicating that these mice provide new opportunities for understanding the significance of this form of signaling. in the trigeminal ganglion (tg), satellite glial cells (sgcs) surround neurons and are able to release substances that may affect sensory transmission through the tg and contribute to mechanisms underlying craniofacial pain. the aims of this study were ) to investigate whether sgcs could be provoked to release glu in vitro and ) to study the in vivo response properties of trigeminal afferent fibres upon artificial elevation of glu concentration in the tg. methods: confocal microscopy was used to assess glu content in and the expression of excitatory amino acid transporter (eaat ) and eaat by sgcs of the tg. glu release was investigated in vitro, where sgcs were isolated from the tg of adult male sprague-dawley (sd) rats and treated with control medium or medium containing mm kcl together with , . , , mm of the eaat / inhibitor tfb-tboa. in vivo electrophysiology experiments were conducted in additional anaesthetized adult male sd rats to determine the effect of repeated intraganglionic injections of glu ( mm, ml , min apart) on the response properties of tg neurons that innervated either the temporalis or masseter muscle. cumulative afferent discharge was calculated as the sum of action potentials (ap) min post injection subtracted from the sum of ap min prior to injection. an electronic von-frey hair was used to measure afferent mechanical threshold (mt) at min intervals for min prior to the first and again min after the second microinjection of glu. results: most sgcs were found to be glu positive and express eaat and eaat . treatment with mm kcl resulted in a $ -fold increase in glu concentration, compared to control ( . . vs. . . mm, respectively; p . ). treatment with mm tfb-tboa significantly reduced the glu concentration to . . mm (p . ), which was further lowered to . . mm when mm was applied (p . ). cumulative afferent discharge evoked in tg neurons by the second injection of glu ( ap) was not significantly different from that evoked by the initial injection ( ap; p . ). glu injections significantly reduced muscle afferent mt (baseline: g) by % (p . ). these results indicate that glu can be released from sgcs through eaats, and that increased glu concentrations in the tg excite neurons and induce a state of peripheral sensitization. this may hypothetically be a mechanism, whereby activated tg sgcs could contribute to e.g. migraine headache. in an accompanying poster, we present data demonstrating that in cultured neurons l-lactate induces plasticity-related genes expression through a nmda-dependent mechanism (i.e. arc, zif and c-fos). here, we describe the effect of l-lactate on neuronal excitability by performing electrophysiological recordings of cultured cortical neurons. we observed that application of l-lactate ( mm) triggers an inward current with an amplitude of . /- . na and slow kinetics, with a peak reached at /- s (called ilac). the membrane current decreases gradually to reach a plateau, with a mean inward current of . /- . na persisting up to s ( minutes) after addition of l-lactate (called ipyr). in order to assess l-lactate specificity on the generation of these currents l-pyruvate, another monocarboxylate, as well as d-lactate, the non-metabolized enantiomer of l-lactate) were tested. in contrast to l-lactate, l-pyruvate ( mm) induced a low-amplitude sustained current ( . /- . na) similar to the late-phase slow current evoked by l-lactate (i.e ipyr), but no current similar to ilac. dlactate on its side did not elicit any specific current. pharmacological characterization of ilac and ipyr currents demonstrate that ilac is generated by nmda receptors (as it is blocked by mk ) and relies on active nmda receptors (as it is blocked by either glutamate or glycine binding sites inhibitors). in contrast, generation of ipyr by both l-lactate and lpyruvate is prevented by diazoxide, a katp opener, whereas ilac was unaffected by the treatment. in order to determine the importance of katp for plasticity-related gene expression, the katp closer glibenclamide ( um) was applied to neurons and mrna gene expression of arc and zif quantified. results obtained demonstrate that arc and zif mrna expression are unaffected by glibenclamide. as a whole this set of data demonstrates that l-lactate generates two types of inward currents in neurons i.e. nmda-dependent (ilac) or katp-dependent (ipyr). moreover, they highlight the nmda-dependent ilac current as the key mediator, in contrast to katp, of l-lactate-induced plasticity gene expression. this is further sustained by the observation that l-pyruvate does not reproduce the effect of l-lactate on gene expression (see accompanying poster). this set of results provides novel insights into the mechanisms of action of l-lactate as an inducer of plasticity gene expression, and an important signaling molecule for neuronal plasticity. the reliance on drg neuron co-culture for sclcs fate commitment stands as a major hurdle for the therapeutic application of this finding. thus, we aim to unravel the mechanisms underlying sclcs fate commitment in order to develop a drg neuron-free condition for generating fate committed schwann cells. we hypothesised that the switch is brought about by the membrane bound neuregulin (nrg), nrg type iii, but not the other soluble isoforms of neuregulin. as our results show that sclcs treated with soluble neuregulin did not show significant changes in morphology nor marker expression when compared with untreated sclcs. nrg type iii expression was observed on purified drg neurons by immunocytochemistry, western blot analysis as well as reverse transcription pcr for the mrna. mammalian expression constructs for nrg type iii were made by transfection into human embryonic kidney cells, hek t, and mouse embryonic fibroblasts (mef) with the aim of generating surrogate cell types for co-culture with sclcs to pursue cell-specific effects of nrg type iii on differentiation of sclcs. the findings promise a way for generating fate committed schwann cells for autologous transplantation for recovery after nerve injury. ( ) the co /hco buffer system. the latter being an open buffer system, because biomembranes are usually permeable to co , and, in addition, most cells express hco transporting membrane proteins. the enzyme family of carbonic anhydrases (cas) catalyses the reversible reaction from co and h o to hco and h , and thereby modulates the intracellular h buffer dynamics.we have performed calibrated in situ live-cell imaging with the proton-sensitive fluophore bcecf in glial cells and neurons of acute cerebellar slices of wild-type and knockout mice. the studies were used to quantify the intracellular buffer capacity and to dissect the contribution of the intracellular caii and the extracelluar caiv by comparing intracellular h shifts in glial cells and neurons from wild-type mice and mice deficient in their caii or caiv gene. we found that only one proton in is unbound and thereby chemically active, and that about % of this buffer capacity is mediated by the co /hco buffer system and the other % by intrinsic buffers. the rate of co -induced change in intracellular h concentration was increased by intracellular caii in both glial cells and neurons. the extracellular caiv on the other hand affected primarily neurons, but also showed unexpected intracellular activity (schneider et al. , pnas ). the rates of acidification and alkalinisation in wild-type and knockout mice were significantly reduced in the presence of the ca blocker -ethoxy- -benzothiazolsulfonamid ( mm). we confirmed ca protein expression by western blot and could also show that the expression of the remaining ca is not upregulated. these results provide new insights into cellular proton-coupled processes in acute neural tissue, which shows some new complex roles of carbonic anhydrases in shaping proton dynamics in cells and tissues. objective: it is becoming increasingly evident that inflammatory mechanisms promote neuronal hyper-synchronization and epileptogenesis following brain insults. our recent studies identified serum albumin as a key player in the inflammatory cascade leading to epilepsy, at least partially through the activation of tgf-b signaling pathway in astrocytes. in this study we set out to explore the mechanisms by which tgf-b induces glial activation and epileptogenesis. methods: primary cultures of microglia and astrocytes were treated with tgf-b and gene expression analysis along with protein quantification were performed by quantitative pcr and elisa. electrophysiological response to il- was obtained in acute brain slices (field potential recordings) and in-vivo (sub-dural corticoencephalography) following to a continuous administration of il- into the lateral ventricle for days. results: in glia cultures tgf-b induced early and rapid up-regulation of il- at both mrna and protein levels whereas the upregulation of other pro-inflammatory cytokines such as il- b and tnf-a was milder and at a later time point. notably, smad / -dependent tgf-b signaling induced the expression of il- primarily in astrocytes and to a significantly lesser extent in microglia. il- was sufficient to induce epileptiform activity in acute brain slices and recurrent seizures within - days a following continuous intracerebroventricular injection of il- . conclusions: we thus suggest astrocytic release of il- as a potential key mechanism in epileptogenesis. okayama univ., dept. of brain science, okayama, japan dopamine transporter (dat) is expressed not only in catecholaminergic neurons but also in astrocytes. we previously showed that repeated l-dopa treatment markedly induced expression of dat and apparent dopamine (da)-immunoreactivity in the reactive astrocytes in the striatum of animal models of parkinson's disease. it is thought that astrocytes can uptake both l-dopa and da via neutral amino acid transporter lat and dat, respectively. therefore, uptake and metabolism of l-dopa and da in the striatal astrocytes may influence their availability in dopaminergic system of parkinsonian patients. to clarify uptake and metabolism of l-dopa and da in striatal astrocytes, the contents of l-dopa, da and their metabolites were measured after the l-dopa/da treatment using primary cultured astrocytes. first, we revealed the expression of neutral amino acid transporter lat and aromatic amino acid decarboxylase (aadc) in the striatal astrocytes. the level of l-dopa in astrocytes was markedly increased after -hr l-dopa exposure, but da was not detected in the astrocytes or hr after the treatment. on the other hand, the da treatment for hr increased levels of da and its metabolites, especially dopac. these results indicate that uptaken da into astrocytes is rapidly metabolized and that uptaken l-dopa never be converted to da in astrocytes. furthermore, level of uptaken l-dopa in cultured striatal astrocytes was rapidly decreased after removing extracellular l-dopa. taken together, the present results suggest that striatal astrocytes act as a reservoir of l-dopa to uptake or release l-dopa depending on the extracellular l-dopa concentration, but have less ability to convert l-dopa to dopamine. s. limmer, c. kl€ ambt universit€ at m€ unster, m€ unster, germany neuronal function consumes a large amount of energy and thus the brain requires a constant but regulated supply of metabolites. in invertebrates, such as drosophila, the nervous system is floating in the hemolymph and all metabolites that enter the brain have to be transported across the blood brain barrier (bbb). as in primitive vertebrates, the drosophila bbb is generated by glia. a layer of so-called subperineurial glial cells completely encapsulates the nervous system and thus, all metabolites reach the neurons en route through glia. the main energy supply of the inner organs of drosophila is provided by trehalose that is found in high concentrations in the hemolymph. trehalose is a nonreducing disaccharide, in which two dglucose units are linked via a a,a- , -glycosidic bond. the uptake of trehalose is mediated by two trehalose transporters, which are members of the slc a gene family. the subperineurial glial cells then either secrete c sugars to the neurons -or alternatively supply energy to neurons by secreting c metabolites as described for the bee retina. to discriminate between these possibilities we have followed a number of different approaches. the relevance of trehalose transporters in the different cell types has been determined by mutant analysis and rnai studies. furthermore, we have studied the role of all other putative sugar and monocarboxylate transporters in glial cells by rnai knockdown. to determine whether c or c carbohydrates are secreted by glial cells we suppressed the expression of core enzymes regulating glycolysis or components of the respiratory chain in either glial cells or neurons. moreover, we have ablated mitochondria specifically in glia or in neurons through expression of a restriction enzyme targeted to mitochondria. we will summarize our results and discuss approaches towards identifying neuronal signals that control glial carbohydrate secretion during energy homeostasis of the brain. microglia have long been known to respond to virtually any insult to the central nervous system. they quickly migrate to the site of the insult, and play many important roles, such as barricading the injury site, phagocytosing debris, and releasing cytokines. the role of microglia in the normal brain, however, has only recently begun to be appreciated. with the advent of in vivo imaging, microglia have been shown to be constantly surveying their environment with rapid extensions and retractions of their processes. subsequent studies have shown that these 'resting' microglia (now known as 'surveying microglia') are indeed active throughout development and adulthood. during development, microglia have been shown to be involved in synaptic monitoring and pruning as well as synaptic stripping after injury. thus far, many studies have focused on the role of microglia at the synapse. however, we report here, for the first time, that in the cortex of normal adult rodents, a small percentage of microglia are specifically associated with the axon initial segment (ais). the ais is characterized by a high density of voltage-gated ion channels and plays an important role in initiation of the action potential and maintenance of neuronal polarity. this interaction seems to be limited to the 'surveying' phenotype of microglia and much less frequent in 'activated' microglia. this overlap of processes appears early in development and continues throughout adulthood although the function of microglia at the ais is still currently unknown. x. liu , j.-m. petit , , p.j. magistretti , , c. giaume cirb, collège de france, paris, france lndc, brain mind institute, epfl, lausanne, switzerland centre de neurosciences psychiatriques, chuv, prilly, switzerland astrocytes act as modulators of neuronal activity through mechanisms such as neurotransmitter uptake, 'gliotransmitters' release, and metabolic supply. recently, astrocytes were shown to modulate sleep by vesicular release of atp. besides the cellular mechanism, we are interested in the network behavior of astrocytes in sleep-wake cycle. indeed, astrocytes form networks of communicating cells via gap junction channels constituted by connexins (cxs). recently, we have demonstrated that astroglial networking is regulated by neuronal activity and that neuronal activity is affected by deletion of the two main astroglial cxs (cx , cx ). to investigate how astroglial networks behave in sleep-wake cycle, we used pharmacological treatments and sleep deprivation to manipulate sleep-wake status of mice and determined whether astroglial networks would change in response. astroglial networks in acute cortical slices were revealed by "dye coupling", i.e. by loading of a patch-clamped astrocyte with sulforhodamine b that diffuses into neighboring astrocytes through gap junction channels. also, cxs expression at mrna and protein levels was measured as an index of connections among astrocytes. the results are as follows. first, i.p. injection of modafinil, a potent wakefulness-promoting drug, increased both mrna and protein expression of cx in the mouse cortex. superfusion of modafinil also enhanced dye coupling among astrocytes in acute coronal slices of the mouse somatosensory cortex. this effect was abolished by ttx treatment, which demonstrates neuronal activity is necessary for the effect of modafinil. in contrast, c-hydroxybutyric acid (ghb), a sleep-promoting agent, and propofol, a general anesthetic, decreased astroglial coupling in cortical slices. interestingly, the effect of ghb was not affected by ttx, suggesting a different pathway of action from modafinil. next, we used a "gentle" sleep deprivation model to sleep deprive mice for hours from the onset of the light period. as a result, mrna of cx , but not cx , was increased in both the cortex and hippocampus. finally, dye coupling in cortical astrocytes was enhanced in mice after sleep deprivation compared to mice after normal sleep. this increase in dye coupling, however, was not observed in slices from cx knock out mice after the same sleep deprivation treatment. altogether these results indicate that astroglial networks are bidirectionally regulated by perturbations in sleep-wake cycle and that cx is the major cx sensitive to such perturbations. in neurons, synaptic and extrasynaptic gaba a receptors (gaba a rs) differ in their subunit composition, conferring them distinct functional and pharmacological properties. oligodendrocyte precursor cells, also called ng cells, are contacted by bona fide neuronal gabaergic synapses. however, we recently showed a synaptic to extrasynaptic switch in the mode of transmission between gabaergic interneurons and ng cells during postnatal development of the somatosensory cortex. we therefore hypothesized that the postnatal switch of gabaergic transmission in ng cells is accompanied by changes in the expression of gaba a r subunits. to test for this hypothesis, we stimulated neuronal fibers to evoke gaba a r-mediated responses in ng cells recorded in acute slices of the somatosensory cortex of ng -dsred transgenic mice. the effect of zolpidem and a ia on evoked gabaergic responses reveals the predominance of functional a -and a -containing gaba a receptors, respectively, at interneuron-ng cell synapses early in development. however, the expression level of a decreases when responses rely exclusively in extrasynaptic transmission at more mature developmental stages. more importantly, specific pharmacology for the c subunit, a crucial molecular component for the clustering of gaba a receptors at postsynaptic sites, demonstrated a down-regulation of this subunit in ng cells prior to the complete loss of gabaergic synaptic activity. in keeping with the synaptic nature of the c subunit in neurons, this molecular change in ng cells correlates with the switch from synaptic to extrasynaptic transmission. to corroborate these functional data, we performed single cell multiplex rt-pcr of a - , b - , c - and d subunit mrnas in ng cells of the second and fourth postnatal weeks. despite the large heterogeneity of subunit mrna expression at both developmental stages, we found that the number of cells expressing c mrnas dramatically decreases in the fourth postnatal week. in conclusion, the expression loss of the c subunit is an important molecular determinant impacting the change of transmission modes between interneurons and ng cells during cortical development. financial support: frc, anr blanche, arsep, dfg (sfb/tr ) and eu (fp - neuroglia). university of rochester, medical center, rochester, united states synaptic plasticity is a critical process throughout the lifespan for achieving and maintaining normal and efficient operation of the nervous system. while much is known about the functional and structural changes that occur at the synapse during synaptic plasticity, the mechanisms implementing these changes are poorly understood. despite being classically characterized as immune cells, microglia have recently been shown to play a role in normal brain function by restructuring and removing synapses. given the novelty of this role, few details are known about how microglia behave to implement such a role during periods of plasticity. to characterize the changes in microglial behavior during ocular dominance plasticity in the developing visual cortex, we examined microglial morphology in fixed brain sections as well as in vivo using two-photon microscopy. for analysis of microglia in fixed sections, c /bl mice were monocularly deprived for either hours, , , , or days during the visual critical period (p -p ). fixed coronal sections were stained with iba , a specific marker for microglia, imaged on a confocal microscope and analyzed using imagej. microglial density decreased rapidly (within hours) and remained low for the day deprivation period. characterization of microglial morphology revealed an increase in the complexity of the microglial process arbor after hours, which steadily decreased back to baseline by days. this suggests that microglia do undergo a rapid change in morphology during plastic events that is reminiscent but distinct from the traditional activation pattern observed during pathological events. for analysis of microglia in vivo, heterozygous cx cr /gfp (jung ) mice were monocularly deprived directly preceding the first imaging session, the binocular segment of primary visual cortex was imaged, and the same cortical area was re-imaged and days later. while no change in microglial density was observed, analysis of microglial process motility showed a decrease in motility after periods of monocular deprivation. these results, combined with the observed morphological changes, show that microglia undergo behavioral changes after a period of monocular deprivation that are inconsistent with pathological activation, suggesting that microglia take on different roles and activities during normal brain function. g. baltazar, j. oliveira, t. roxo, c. fonseca faculty of health sciences, cics-ubi, covilhã, portugal neuroinflammation is a pathological hallmark in patients and experimental models of parkinson's disease. both present the classical features of inflammation, with evidence of an uncontrolled process. moreover, microglia may become activated early in the disease process and remain primed, responding strongly to subsequent stimuli, and thereby enhancing inflammation-induced oxidative stress and cytokinedependent toxicity in vulnerable neuronal populations. we have previously demonstrated that glial cell line-derived neurotrophic factor (gdnf), released by ventral midbrain astrocyte cultures, potently prevents microglial activation induced by a pro-inflammatory agent. therefore, gdnf is an important mediator in the astrocytemicroglia crosstalk keeping microglia in a resting state. however, in the brain, microglia and astrocytes are not alone and other cell types, e.g. neurons, may interfere and influence this astrocytic control of inflammation. therefore, it is important to investigate if the presence of neurons alters gdnf-mediated astrocytic control of microglial activity. in this work we aimed at investigating whether the presence of neurons, injured or not, changes the ability of astrocyte-derived gdnf to prevent microglial activation. for that purpose, we used primary cultures of ventral midbrain astrocytes and microglia, as well as neuronastrocyte mixed cultures from the same brain region. neuron-astrocyte cocultures were exposed to vehicle (control) or to the da neurotoxin mpp . the media conditioned by control and mpp -challenged neuron-astrocyte mixed cultures, or by astrocyte cultures, was collected and transferred to ventral midbrain microglia cultures, which were then exposed to the pro-inflammatory agent lipopolysaccharide. the effect of conditioned media obtained from the different cell culture systems on microglial activity was determined by analyzing the production of nitric oxide by the griess reaction and of the phagocytic activity by determining the engulfment of fluorescent microspheres by fluorescence microscopy. university of lausanne, lausanne, switzerland lactate is produced by astrocytes and used by neurons as metabolic substrate during activity. recent evidence indicates that lactate may not only serve as energy substrate but also as modulator of glutamatergic or gabaergic neurotransmission. we tested this hypothesis using primary cultures of cortical neurons obtained from wild type and gad -gfp knock-in mice (c bl/ ). neuronal excitability was monitored by electrophysiological recordings and calcium imaging using the fluorescent probe fluo- am. spontaneous action potentials and rhythmic calcium transients were present in more than % of cultured neurons, which were either gabaergic or glutamatergic cells. in the presence of mm glucose, l-lactate application reversibly diminished calcium transient frequency in a concentration-dependent manner in both cell types (glutamatergic neurons ic . . mm and gabaergic neurons ic . . mm). to test whether lactate effects were dependent on metabolism, we applied the closely related substrate pyruvate ( mm) or switched to different glucose concentrations ( . or mm). none of these conditions altered the calcium transient frequency. in contrast, the application of d-lactate, thought not to be metabolized by neurons, decreased the spiking activity in the same concentration range as l-lactate (ic . . mm). we determined that d-lactate was taken up by neurons, however more than two-fold less efficiently than l-lactate. these results suggest that the mechanisms of lactate sensitivity are not purely metabolic. since d-lactate has the same potency as l-lactate but it is less internalized, it is likely that lactate acts as an extracellular ligand that triggers the reduction of neuronal activity. this hypothesis is currently under investigation. this modulating effect of lactate represents a novel role for the compound that has to be taken into account in the context of the interactions between astrocytes and neurons. ben-gurion university, ber-sheva, israel microglia integrate within the neural tissue with a distinct ramified morphology through which they scan the surrounding neuronal network, a process which appears to contribute to the integrity, maintenance and functioning of the brain. since microglia are long-lived, they are subjected to senescence processes which may severely compromise their function with age. here we used a digital tool for the quantitative morphometric characterization of fine cortical microglia structures in mice. we thus followed the morphological changes microglia underwent with aging and with the progression of alzheimer's-like disease. compared with microglia in young mice, microglia in old mice are less ramified and possess less branches and fine processes, a phenomenon which was associated with increased expression of pro-inflammatory genes. notably, a similar microglial pathology appeared - months earlier in mouse models of alzheimer's disease (ad). we thus demonstrate that in addition to promoting neurotoxic inflammation, amyloid plaques attract the microglia and modify their structure. this, in turn, causes a severe microglial process deficiency, which possibly results in compromised neuronal function and repair. blood-borne glucose is the main energy substrate for the brain under physiological conditions. however, the question remains unresolved if the two main cell types in the brain -neurons and astrocytes -consume glucose upon their individual needs or if glucose is mainly taken up by one cell group and glycolytically degraded to energy-rich substrates (e.g. lactate), which are then shuttled to the other cell group. a novel genetically encoded fret glucose sensor (bittner et al., , ) together with two-photon microscopy now provides for the first time the opportunity to determine intracellular glucose concentrations in single cells in the intact organism. cortical microinjections of an adenoviral vector (aav and aav ) coding for the sensor flii pglu md with the promoter sgfap for astrocytes and synapsin for neurons were performed in mice and chronic windows were implanted above the somatosensory cortex. after two weeks, specific expression in astrocytes and neurons was observed. the ratiometric fret signal in both cell types increased after intraperitoneal glucose injection and decreased after insulin application demonstrating the functionality of the construct in vivo. the astrocytic sensor displayed a large linear range at blood glucose levels ranging from to mmol/l. in a second set of experiments, we examined the dynamic behavior of cellular glucose concentrations upon increased brain activity. during electrical hindpaw stimulation % of examined astrocytes showed a . % decrease of the fret signal, whereas neuronal glucose levels did not exhibit activity-dependent fluctuations. to be able to draw conclusions about metabolic rates of substrate oxidation we recently started with experiments where cellular glucose uptake was transiently inhibited using different blockers of glucose transport. drugs were applied systemically and intracortically. we could demonstrate this principle on first measurements on astrocytes. glucose fret sensors allow measurements of the dynamics of glucose concentrations of single neurons and astrocytes in the intact organism. measurements of cellular glucose transients during transport blockade hold the potential to compare glycolytic rates of neurons and astrocytes. this tool may substantially contribute to uncover the complex organization of brain energy metabolism. neuron-glial communication in the cns is fundamentally important for many brain processes including synaptic transmission and plasticity. perisynaptic astrocytic processes (pap) contact excitatory synapses, forming tripartite structures with neurons. in the hippocampus, the morphology of pap has been shown to remodel rapidly and continuously but the mechanisms and roles of this form of structural plasticity remain unknown. this study investigated the physiological mechanisms driving pap movements and their role during long-term potentiation (ltp). pap and spines contacts were labeled by viral gene delivery of farnesylated fluorescent proteins in hippocampal slice cultures and imaged by confocal microscopy. electron-microscopy of infected slices confirmed that pap-synapse morphology is preserved in organotypic cultures. pap movements adjacent to dendritic spines were evaluated with an index of motility (mi). increasing neuronal activity by schaffer collaterals (sc) stimulation elevated pap mi and this was prevented by both ttx and mglur inhibition, suggesting that neuronal activity modulates pap movements. we next investigated whether intracellular calcium (ca i ) in astrocytes influenced pap motility. bapta-am bulk-loading specifically chelated ca i in astrocytes and reduced pap movements. when exogenous gq-coupled receptors mrga or mrgc were specifically targeted to astrocytes, their agonist fmrfa induced ca i increases and accelerated pap motility. moreover, fmrfa delivery at the synaptic level by two-photons flash photolysis was sufficient to elevate pap mi. we then investigated pap dynamics in relation to ltp. application of theta-burst sc stimulation initially increased pap motility, but then resulted min later in a decrease of pap motility. interestingly, the reduction of pap movements correlated with both spine enlargement and increased pap's spine coverage. to assess the consequences of these changes, we then specifically triggered elevation of pap movements at the synaptic level by flash photolysis. spine stability analysis hour after uncaging revealed that spines in contact with activated paps were more stable than others. this study suggests that excitatory synapses control the motility of surrounding paps through the triggering of neurotransmitter-evoked astrocytic ca i elevations. increased pap motility seems to be necessary to elevate their coverage of the synapse during ltp, leading to higher synapse stability. astrocyte reactivity occurs in response to many pathological brain situations. reactive astrocytes display morphological and functional changes that could result in concentration variations of some brain metabolites localized both in astrocytes and in neurons. such changes could be detected by magnetic resonance spectroscopy (mrs), allowing non-invasive monitoring of astrocyte reactivity and thereby neuronal dysfunction in vivo. one of the major peak detected by mrs in the brain corresponds to neuronal metabolite n-acetyl-aspartate (naa). although its function is unclear, it is considered as a biomarker for neurodegeneration and a decrease in its concentration is interpreted as neuronal death or dysfunction. however, there is not clear evidence that other cell types, specifically astroglia, could not contribute to change in naa levels in the brain. in this study, we used a rat model of astrocyte activation by stereotaxic injections of lentiviral vectors encoding for either the cytokine ciliary neurotrophic factor (lenti-cntf) into the right striatum or betagalactosidase (lenti-lacz) into the left striatum, as a control. mrs data were acquired on a t magnet from two voxels placed over each injected striatum. a diffusion-weighted laser sequence with echo time te ms was used. concentrations were measured using lcmodel software for total n-acetyl-aspartate (naa naag), myo-inositol (ins), total choline (cho), glutamate and taurine relative to total creatine. lenti-cntf injection promotes a sustained, extensive and selective activation of astrocytes, as evidenced by overexpression of gfap and vimentin and cellular hypertrophy (fig. ) . importantly, neurons displayed unaltered morphological, molecular and electrophysiological features, as described previously (escartin et al., ; beurrier et al., ). mrs evidences changes in metabolite concentration: in the lenti-cntf injected striatum, levels of the astrocyte metabolites ins and cho were increased, whereas levels of the neuronal metabolite naa and glutamate were decreased. increased levels of ins and cho are generally found associated with astrocyte reactivity, however, the decrease in naa is unexpected given that neurons are not dysfunctional with cntf. to understand how astrocyte reactivity can influence naa levels, we are currently characterizing the molecular changes occurring in the naa metabolism using quantitative pcr, biochemistry, histology and hplc. our data suggest that reactive astrocytes alone result in alterations of metabolite concentrations detectable by nmr. although the mechanisms by which activated astrocytes regulate neuronal metabolism remain to be elucidated, our work challenges the mrs dogma that decreased neuronal metabolites can be used as a biomarker for neurodegeneration. microglial phagocytosis is a vital phenomenon for the clearance of damaged and death cells or infectious agents in a context of brain injury or infection, respectively. in addition, microglia can boost synaptic plasticity by the phagocytosis of axon terminals and dendritic spines. therefore, it is crucial to better understand the mechanisms involved in microglia clearance in order to devise new strategies to promote an efficient brain repair. recently, we showed that histamine modulates microglia motility and cytokines release. in this work, we aimed to investigate the role of this molecule and its receptors in microgliainduced inflammation by evaluating microglial phagocytic activity and reactive oxygen species (ros) production. for that purpose, an iggopsonized latex bead assay was performed in n murine microglial cell lines exposed to lipopolysaccharide (lps) or histamine , and mm. we showed that histamine significantly stimulated phagocytosis of opsonized latex beads via h receptor activation. this effect was accompanied by the rearrangement of cytoskeleton analyzed by phaloidin and acetylated tubulin expression and cellular distribution. the phagocytic activity was significantly reduced after pretreatment with apocynin, a putative napdh oxidase (nox) inhibitor. all nox isoforms were expressed by microglial cells, but only the expression of nox was increased by treatment of histamine. rac , an important subunit for the activation of nox , was also activated by histamine. in addition, histamine induced ros production via h and h receptor activation. in conclusion, histamine plays an important role in the regulation of microglial phagocytosis, which is mediated by nox and rac activation. multiple sclerosis (ms) is a chronic inflammatory disease of the central nervous system characterized by demyelination and axonal damage, involving both white (wm) and gray matter (gm) areas. furthermore, cortical pathology correlates with ms progression and cognitive dysfunction. we previously found altered expression of gap junction (gj) proteins connexin (cx ) and cx in oligodendrocytes and cx in astrocytes in wm lesions and normal appearing wm (nawm) in brain samples from ms patients, and in a mouse model of experimental autoimmune encephalomyelitis (eae), implicating uncoupling of myelinating cells as an important aspect of ms pathology. here we studied cx and cx and their astrocytic partners cx and cx in and around cortical lesions and the normal appearing cortex (nacx) in ms samples and non-ms controls. we examined their expression by real-time pcr, quantitative immunoblot and immunohistochemichal analysis. compared to non-ms controls, expression levels of cx and cx mrna were reduced within lesions whereas cx and cx were increased. in nacx, all four connexins were upregulated with cx being significantly increased. immunoblot analysis of ms cortex revealed reduced cx and increased cx levels, while cx and cx showed no significant change. immunohistochemical analysis showed severely reduced cx gj plaques within lesions with gradual increase toward nacx. in contrast to nawm, cx gj plaques were not increased in nacx. both astrocytic gj proteins were increased in ms, but in distinct patterns: cx gj plaque counts were higher within lesions and perilesional areas. cx was increased in ms cortex compared to non-ms controls, but in contrast to nawm, cx gj numbers were higher in nacx than in lesions. similar alterations were found in the eae mice, in which cx and cx gj counts were reduced whereas cx and cx gjs were increased. our results indicate that oligodentrocytic as well as astrocytic gjs are affected not only in ms wm but also in the gm. loss of oligodendrocyte gjs and in parallel increase of astrocytic gjs reflecting astrogliosis occurs throughout the ms brain and may negatively affect the prospects of repair and remyelination, worsening disease progression. acknowledgements: this project was funded by the cyprus research promotion foundation (grants access/ / and health/bios/ / to kak) and by cyprus telethon ( - grant to kak). post-mortem human brain tissues were kindly provided by the uk multiple sclerosis society tissue bank, imperial college london, funded by the uk multiple sclerosis society. the mainly astroglialocated enzyme glutamine synthetase is required to synthesize glutamine from the re-uptake of either glutamate or gaba. thereby, this enzyme significantly contributes to the control of brain glutamate and gaba levels. there is good evidence that both neurotransmitter systems are dysregulated in depression. hence, we decided to study the cellular expression of this enzyme in subjects with major depression and bipolar disorder. material and methods: we investigated the numerical density of glutamine synthetase immunoreactive astroglial cells in different brain areas (prefrontal cortex, anterior insula) of subjects with major depression, subjects with bipolar disorder and matched control cases. results and discussion: we found that compared with controls in cases with major depression there was a significant reduction of the density of immunostained glial cells in all brain regions studied, whereas bipolar cases showed a normal expression of the enzyme. our findings point to a profound disturbance of the glutamateglutamine-gaba cycle in major depression, which is in good agreement with neuroimaging observations and molecular biologic data of others. m.-c. marx, d. billups, b. billups university of cambridge, cambridge, united kingdom perisynaptic astrocytes are thought to play a key role in the recycling pathway of glutamate by supplying the precursor glutamine to the presynaptic terminal. using fluorescent ph i measurements of astrocytes and direct patch-clamp recordings from the calyx of held synapse in rat brain slices, we have investigated the transport mechanisms that may mediate this release of glutamine from astrocytes in situ and assessed the role of glutamine in maintaining glutamatergic neurotransmission. glutamine transporter currents were elicited by puff application of mm glutamine from a nearby pipette. for imaging astrocytic ph i , hpts was included in the patch pipette. system a (sa) transporter function was probed using the substrate inhibitors meaib, alanine and serine, while system n (sn) function was isolated using its unique ability to transport li in place of na . histidine was used as a substrate inhibitor of both sa and sn. glutamine-induced membrane currents i gln ( . . pa, n ) were blocked by bath applied alanine ( mm) and meaib ( mm), as well as substitution of external na with li . fluorescent ph i measurement revealed an alkalinisation during puff application of glutamine. while bath application of histidine ( mm) abolished the ph change, meaib and serine ( mm) only attenuated it. most of the ph change ( %; n ) was insensitive to the substitution of na with li . in presynaptic terminals, puff application of glutamate did not induce a membrane current indicating a lack of direct glutamate uptake; however, puff application of glutamine resulted in a i gln which was eliminated by the removal of external na and by meaib. miniature epscs recorded from postsynaptic neurons ( . . pa) were inhibited by meaib ( . . pa; n ) following turnover of the presynaptic glutamate pool. single epscs were unaffected by meaib indicating no direct effect on postsynaptic ampa receptors. together, these data identify two glutamine transport systems within astrocytes. properties of the glial i gln indicate expression of sa while ph imaging reveals a non-electrogenic glutamine transporter with properties identifying it as sn. sn is known to be readily reversible under physiological conditions and we propose that this mediates the astrocytic glutamine release mechanism within the glutamate-glutamine cycle. furthermore, system a mediates glutamine transport in glutamatergic nerve terminals and is important in maintaining the supply of glutamate for excitatory neurotransmission during periods of high frequency stimulation. a. briens, m. schwalm, f. docagne, d. vivien inserm u , caen, france astrocytes are key players in the brain, cooperating with neurons to modulate crucial processes. in particular, they are able to uptake and release neurotransmitters and neuromodulators to control their synaptic level. in our study, we hypothesize that astrocytes can regulate some effects of the serine protease plasmin in the cns through a mechanism of uptake. conversion of the zymogen plasminogen to the serine protease plasmin by the activators (tpa or upa) is the basis of fibrinolysis in the vasculature. but cerebral functions for the plasminogen activator/plasmin system have recently arisen. it has been shown that plasmin was essential to activate the brain derived neurotrophic factor (bdnf) in its mature form, promoting long-term hippocampal plasticity. in alzheimer's disease, plasmin plays a protective role by participating in beta-amyloid clearance. plasmin can also lead to neuronal injury by degrading extra-cellular matrix proteins. in animal models of multiple sclerosis, plasmin can also help removing intraparenchymal deposits of fibrin, thus limiting axonal damage. these data suggest that a system of regulation of plasmin effects in the brain is necessary. in this study, we showed that astrocytes can actively and specifically uptake plasminogen and plasmin in a time-dependent and a dose-dependent manner. this mechanism involves clathrin pits formation in astrocytes and the lysine-binding site of plasminogen/plasmin. plasminogen and plasmin, following endocytosis are found in early endosomes, late endosomes, lysosomes and recycling endosomes. finally, we noticed that astrocytes uptake plasmin more efficiently than plasminogen. this study brings out a mechanism of glial uptake of plasminogen and plasmin. this could control their extra-cellular levels and regulate their effects within the brain. further investigations should determine whether this mechanism can modulate plasminogen activation and synaptic plasmin activity. besides, plasminogen activators (tpa) and cerebral substrates of plasmin (pro-bdnf) are also taken up by astrocytes. a link could thus exist between these processes to regulate the amount of synaptic mature bdnf. this would reveal a cross-talk between neurons, releasing precursors in the synapse (pro-bdnf and plasminogen) and astrocytes, regulating their activation and their clearance. understanding and modulating plasmin uptake could be very important in pathological conditions where the plasminogen/plasmin system is involved, such as alzheimer's disease, multiple sclerosis or stroke. healthy brain aging is characterized by neuronal loss and cognitive decline being inflammation a major causative factor. microglial activation is considered to be a major driver for the neuropathological findings in alzheimer's disease (ad). we evaluated whether young vs. aged microglia responded differently to ab soluble oligomers (abo) and to conditioned media from abo-treated hippocampal neurons. microglia from day cd pups were incubated for h at (young) and (old) days in vitro (div) with abo (ab - at and nm). in parallel, e mice hippocampal neurons were treated with abo at (young) and (old) div for h, and the incubation media used as conditioned media to treat microglia, at respective div, for further h. cell migration (boyden chamber), extracellular content in glutamate (commercial kit), and activity of matrix metalloproteinases (mmp) and (gelatin zymography) were assessed. an increased migration towards abo and atp was observed in young microglia but almost undetectable in aged cells (p < . ). interestingly, microglia exposure to conditioned media from nm abotreated neurons markedly decreased both young and old microglia migration, while treatment with media from nm abo-treated neurons enhanced migration, mainly in the young microglia (p < . ). concerning glutamate, aged cells released less than young ones upon abo treatment (p < . ). in addition, when exposed to neuronal conditioned media, only the young microglia were able to exert a neuroprotective role by reducing the media glutamate content. curiously, while abo promoted a microglia release of mmp- , independently of cell age, it only induced mmp- secretion in old cells in an abo concentrationdependent manner (p < . for nm abo). in contrast, conditioned media from abo-treated neurons elicited mmp- release only from young microglia. together, our data point to a loss of microglia function towards abo with ageing and are unique in suggesting that senescent microglia may release high levels of mmp- in ad. in addition, our results indicate that abo-treated neurons enhance the release of mmp- even by young microglia. whether this finding may contribute to enhance inflammatory stress and the onset of ad will be the aim of further studies. human immunodefeciency virus- (hiv- ) productively infects microglia within the central nervous system (cns). viral replication within microglia and the resulting immune response and neurotoxicity leads to significant cognitive impairments not limited to dementia, neurocognitive abnormalities, and memory deficits. combination antiretroviral therapy reduces the cognitive impairments associated with hiv- , but the regulatory hiv- tat protein is still produced in the cns during treatment, even in the absence of productive infection. our laboratory analyzes the effect of tat on the murine cns by stereotactically injected into cortex, which mimics in part the neuroinflammation and neurodegeneration characteristic of hiv- associated neurocognitive disorders (hands). additionally, in vitro tat-treated bv- microglial cells exhibit activation associated with increased cytokine expression and phagocytosis. mixed lineage kinase (mlk ) has recently been implicated in the tat-mediated activation of microglia. mlk activity is increased by the presence of tat protein, which results in microglial activation and increased production of inflammatory cytokines. conversely, the brain penetrable, small molecule, new chemical entity urmc- inhibits the mlk pathway. we hypothesize that urmc- attenuates tat-induced microglial activation as measured by microglia process length, phagocytosis assay and cytokine expression. in vitro application of urmc- decreased tatinduced production of ccl and il- , microglia process length, and phagocytosis of charged and uncharged beads in bv- microglia cells. these data, in aggregate, strongly support a role for the mlk pathway in neuroinflammation and suggest that inhibition of this pathway with urmc- may have significant anti-inflammatory effects in an inflammatory cns milieu. in conclusion, this work will advance our understanding of mlk activity and may provide a viable drug therapy for hands. the contributions of glial cells to the modulation of adult synaptic plasticity are becoming increasingly more appreciated. astrocytes in the supraoptic nucleus (son) of the hypothalamus demonstrate prominent temporary structural changes during physiological events such as dehydration and lactation. such structural remodeling is characterized by a reduction in the astrocytic coverage of oxytocin neurons and their synapses. these astrocyte morphological changes also contribute to the observed functional changes in synaptic activity during dehydration and lactation. in order to elucidate the underlying mechanisms regulating the astrocytic contributions to synaptic plasticity we have established an astrocyte protein expression profile for structural changes in the son during lactation and hyperosmotic challenge. for this, son from virgin, lactating, and hyperosmotic rats were collected from acute hypothalamic slices and analyzed by mass spectrometry to identify proteins differentially regulated by the changes in synaptic structure induced by lactation and hyper-osmolarity. our mass spectrometry data analysis has placed particular focus on regulation of astrocyte-specific proteins and the roles of these proteins in molecular pathways known to be involved in son plasticity, such as cytoskeleton remodeling, cell adhesion, and cell signaling. gene ontology analysis revealed over representation of pathways known to be highly active in astrocytes including metabolism and antioxidant activity. promising target proteins for future functional studies will be discussed. here we elucidate a novel merlin isoform (merlin-iso )-specific function in maintaining axonal integrity and propose that reduced axonal nf gene dosage leads to nf -associated polyneuropathy. we identify a merlin-iso -dependent complex corresponding to activation of the gtpase rhoa, enabling downstream rho-associated kinase to promote neurofilament heavy chain phosphorylation. in vivo, specifically merlin-iso knockout mice exhibit impaired locomotor capacities, delayed sensory reactions, and electrophysiological signs of axonal neuropathy. sciatic nerves from these mice and sural nerve biopsies from nf patients show reduced nf-h phosphorylation, decreased interfilament spacings and irregular-shaped axons. a. catenaccio, p. silva, f. court pontificia universidad cat olica de chile, santiago, chile axonal degeneration is an active process involved in a variety of neurodegenerative conditions triggered by diverse stimuli. this degenerative process can cause permanent loss of function, so it represents a focus for neuroprotective strategies. we have recently shown that axonal degeneration triggered by distinct mechanical and toxic damage is dependent on the activation of the mitochondrial permeability transition pore (mptp), which probably represents a central execution program of axonal degeneration, upon which several pathways converge (barrientos et. al., journal of neuroscience ). nevertheless, the participation of other cellular types in this degenerative process has been largely overlooked. after axonal damage in the peripheral nervous system (pns), schwann cells enveloping axons have been regarded as responsible for clearing degenerating axonal debris and to mount an inflammatory response for tissue clearance by invading macrophages. nevertheless, a possible function of glial cells in modulating axonal degeneration has not been addressed so far. here we explored the possibility that glial cells actively participates not only in the clearance of degenerating axons, but directly in the axonal degeneration program during wallerian degeneration. we found that axonal injury activates an early response of schwann cells that results in the fragmentation of their associated axons by actin-rich cytoplasmic domains of schwann cells known as schmidt-lanterman incisures (sli). this first step of schwann cell fragmentation of axons is dependent on the erk signaling pathway, wich control the dedifferentiation program initiated in schwann cells after nerve injury (napoli et. al., neuron ). in the axonal compartment, injury triggers a parallel cell autonomous degenerating program dependent on mitochondrial mptp activation, which is cell non-autonomously assisted by schwann cells through the activation of a cell extrinsic pathway of axonal destruction by tnf-alpha secretion. we showed for first that time that axonal degeneration in the pns proceeds by axonal autonomous mechanisms, as well as cell non-autonomous ones dependent on scs. therefore, effective targets for neuroprotection should consider not only the axonal compartment, but also the associated glial cell. glutamate is the major excitatory neurotransmitter in the brain. its concentration is the synaptic area is highly regulated in order to maintain point-to-point transmission and to prevent excitotoxicity associated with chronic activation of glutamate receptors. as there is no endogenous extracellular enzyme to degrade glutamate, the clearance of glutamate is ensured by transporters located on the surface of astrocytes. among these surface transporters glt- ensures almost % of glutamate uptake at synapses. it is widely accepted that many receptors and transporters on the surface of neurons and glia display surface trafficking properties and are not simply static proteins. the basic surface trafficking properties of glutamate receptors at the synapse have been studied in depth and have allowed us to understand many important trafficking events that occur during synaptic plasticity. the action of glt- is fundamentally important in synaptic communication. surface trafficking of this transporter may play a pivotal role in the spatial and temporal properties of glutamate diffusion at the synapse. however, it is still unknown whether glt- is dynamically regulated on the surface of astrocytes. thus we set out to uncover the surface trafficking of glt- in astrocytes by using a combination of high-resolution imaging, such as single particle (quantum dot [qd]) tracking, molecular biology and electrophysiological approaches. here we have demonstrated that glt- is indeed subject to surface trafficking on astrocytes. using pharmacological approaches we have shown that this diffusion is subject to regulation by the activity of the transporter itself as well as neuronal activity. furthermore, we have demonstrated that the speed of glt- diffusion is greatly reduced at excitatory synapses. this leads us to the conclusion that glt- surface diffusion is highly regulated at the synapse where it can effectively carry out its role as the principal glutamate transporter at the synapse. finally, to elucidate the physiological role of glt- surface diffusion, we have carried out electrophysiological experiments in which glt- trafficking is effectively reduced using a cross-link (x-link) protocol. we observed that a reduction in the surface trafficking of glt- resulted in an increase of neuronal excitability. thus glt- surface trafficking on astrocytes has an implicit role in regulating basal neuronal activity through glutamate uptake at the synapse. o. chever, e. dossi, n. rouach collège de france, paris, france astrocytes play crucial roles in brain physiology by dynamic interactions with neurons. they form plastic and extensive networks mediated by gap junction channels. it has recently been shown that astroglial networks limit neuronal network activity. however, it is currently unclear how astroglial networks influence neuronal excitability and population activity. to investigate how astroglial assembly regulates neuronal network activity, we performed electrophysiological recordings in hippocampal slices (ca and ca areas) from mice with disconnected astrocytes, in which both astroglial gap junction forming proteins, connexin (cx ) and connexin (cx ), are knocked out (gfap-cre cx -/-cx fl/fl , dko). synchronized excitatory discharges were recorded in an acute pharmacological model of epileptic-like activity. in this model, spontaneous bursting discharges are characterized by a sharp and large amplitude shift in field potential, generated by a major depolarization and firing of all putative neurons. pyramidal cells depolarized up to mv during seconds and such depolarization is followed by a long-lasting undershoot of the membrane potential. we found that the frequency of bursting activity in dko hippocampal slices is drastically increased. however, the duration of neuronal depolarizing bursts is severely reduced. furthermore, pyramidal neurons are more depolarized due to an increase of synaptic bombardment. this suggests that increased synaptic background promoting resting membrane potential depolarization facilitates triggering of neuronal bursts, but compromises the strength of synchronized events. to investigate local dynamics of bursts generation, we performed multielectrodes arrays recordings in the ca area of the hippocampus. in slices from dko mice, bursting activity in the ca region is generated within a more restricted area, indicating that the number of neurons to be recruited is highly decreased. altogether, these results indicate that gap junction-mediated astroglial networks strengthen the coordination of neurons during synchronized events. acutely loaded hypoxia increases ventilation, and increased ventilation does not immediately return to the pre-hypoxic level, but persists for a while after resuming room air inhalation. this phenomenon is referred to as short term potentiation or plasticity of breathing, and is thought to contribute to the stabilization of ventilation. although extensive studies have been conducted to clarify the cellular mechanism of respiratory plasticity focusing on neurons and synapses, its precise mechanism remains unknown. on the basis of the recent discovery that astrocytes are actively involved in neural plasticity in various brain regions, we hypothesized that the post-hypoxic potentiation of breathing is mediated by astrocytes. we used unanaesthetized unrestrained adult mice. ventilatory parameters (respiratory frequency, tidal volume and minute ventilation) were measured by whole body plethysmography. to test the hypoxic response, mice breathed room air, hypoxic gas ( % o , % n ), and again room air. we also analyzed the hypercapnic response that was conducted under a hyperoxic condition to eliminate the influence of the carotid body. to test the hypercapnic response, mice breathed pure o , hyperoxic hypercapnic gas ( % o , % co ), and again pure o . after recording of the hypoxic and hypercapnic responses with their recovery time courses, arundic acid (ono- ) was injected intraperitoneally. arundic acid is a compound that suppresses the function of astrocytes through the inhibition of s b production in astrocytes. arundic acid did not affect ventilatory augmentation induced by either hypoxia or hypercapnia. however, arundic acid suppressed the short term potentiation induced by hypoxia (but not by hypercapnia) in a dose-dependent fashion. high dose arundic acid completely eliminated hypoxia-induced short term potentiation. these results suggest that the post-hypoxic potentiation of breathing is mediated by astrocytes with the involvement of s b. the mechanism of hypercapnia-induced potentiation is different from that of hypoxia. we propose a model to explain the cellular mechanism of post-hypoxic potentiation: hypoxia stimulates the respiratory neuronal network in the brainstem through the excitation of the carotid body. neurotransmitters spilled from excited neuronal synapses activate nearby astrocytes. the activation of astrocytes is sustained, and these astrocytes persistently excite respiratory neuronal network by releasing gliotransmitters. monosynaptic c-fibre evoked excitatory postsynaptic currents (epscs) were recorded in lamina i neurons using whole-cell patchclamp in rat transverse spinal cord dorsal-root slice preparations. spontaneous epscs were recorded simultaneously. in all experiments bicuculline and strychnine were added to the bath solution to block gaba a -and glycine-mediated synaptic currents. the superfusion of spinal cord slices in vitro with fkn results in a rapid facilitation of evoked epscs in spinal lamina i neurons receiving monosynaptic input from primary afferent c-fibres. both the microglial cell inhibitor minocycline and a fkn neutralising antibody, completely prevented fkn-induced changes in synaptic transmission. in addition, the inclusion of the ca chelator bapta or the nmda receptor antagonist mk in the pipette solution completely prevented fkn-induced enhancement of synaptic strength, suggesting a post-synaptic ca -dependent mechanism of ltp induction. analysis of paired-pulse ratio (ppr) indicated that fkn-induced facilitation of synaptic strength is accompanied by a decrease in ppr suggesting a pre-synaptic mechanism of ltp expression. in support of this finding, fkn increases the number of spontaneous epscs recorded from lamina i neurons. these data indicate that stimulation of microglial cells in order to induce a pro-nociceptive activity state is sufficient for facilitation of synaptic strength within the dorsal horn. both pre-and post-synaptic mechanisms contribute to fkn-induced facilitation of synaptic strength. this work was funded by a wellcome trust flexible travel fellowship to akc. the organization and connectivity of the developing neocortex remains largely unresolved. the establishment of neuronal microcircuits is a complex process that partly depends on the diversity of neuronal cell types and their ultimate role in the mature brain. this complexity is increased by the existence of bona fide synaptic connections between neurons and oligodendrocyte precursor cells, also called ng cells, which properties and function remains largely unknown. to unravel the physiological characteristics of gabaergic synapses between interneurons and ng cells, we performed paired recordings in the developing somatosensory cortex. experiments were carried out during the second postnatal week in vgat-venus;ng -dsred double transgenic mice that allowed us to identify interneurons (venus ) and opcs (dsred ) in acute slices. our results demonstrated that ng cells are synaptically contacted by fast-spiking (fsi) and non-fast-spiking (nfsi) interneurons. however, a predominant input was received by fsi, a class of interneuron that constitutes only a minor population in the second postnatal week. on the contrary, only a small proportion of the abundant nfsi were connected to ng cells. all the connections showed paired-pulse depression, low probability of response, and preliminary analyses suggest that each interneuron form a single synaptic contact onto ng cells. paired recordings, extensively used to examine the synaptic properties of neuronal microcircuits, may open a new perspective to understand in detail the mechanisms of gaba release and signaling between interneurons and ng cells in the healthy and pathological developing brain. first, we characterized a microglial cell line obtained from cd mouse cortex (n ), in order to clarify the mechanisms involved in the activation pattern of these cells. for that, we incubated n microglia with ng/ml of lipopolysaccharide (lps) for h. morphology (anti-iba staining), phagocytic ability (fluorescent latex beads) and chemotaxis to atp (boyden chamber) were evaluated. we observed that n microglia is ramified in control conditions and after lps treatment they change to an amoeboid morphology, which is accompanied by increased phagocytosis and decreased migratory ability, indicating that lps induces activation of n cell line. next, we investigated the role of the released factors from a mn-like cell line expressing human sod with g a mutation (nsc- / hsod g a) on microglia functions. nsc- expressing human sod wt were used as control. thus, n microglia were incubated for and h with nsc- conditioned media that had been collected at and days of differentiation (div) and reactivity tests were performed as above. although after h of incubation no significant changes were observed, data evidenced that incubation for h with nsc- / hsod g a conditioned media collected at div changed the bipolar morphology of the n microglia into an active amoeboid state, decreased phagocytic ability ( %, p < . ) and induced apoptosis ( %) (fig. ) . moreover, microglia have shown to not be attracted by the released factors from degenerating mn, as determined by the chemotaxis assay. together, our results evidence that released factors from nsc- / hsod g a degenerating cells although inducing microglia morphological activation trigger a reduction in cell phagocytic ability and loss of viability, suggesting a role for microglia along als progression. organotypic explants culture has a pivotal role in studying the complex structure of neuronal tissue. although embryonic tissue explants and slices are used to obtain long term culture, most applications such as drug testing require adult tissue for reliable results. in this study, we show that nanostructured metal-oxide arrays can be employed to yield long-term organotypic culture of adult neuronal tissue explants as demonstrated for the adult guinea pig retina and adult murine brain slices. even after days of culture, the thickness and the layered structure of the retina were well maintained. quantitatively, the number of nuclei within the inner and outer nuclear layers was comparable to freshly isolated retinae and no significant change in the densities of the nuclei within the layers was observed. the outer plexiform layer, as the site of synaptic connection between photoreceptors and neurons, was still well preserved. moreover, the thickness of the photoreceptor layer was conserved, indicating that the inner and the outer segments of the photoreceptors survived well during two weeks culture. this is hardly observed in long-term cultures since the outer segments were cultured without retinal pigment epithelium. concerning the adult murine brain slices from neo-cortex, after days of culture, the neurofilaments and cell nuclei were still well preserved and the neuronal network displayed the typical arrangement of long axons. however, preservation of the tissue depends strongly on the geometry of the nanostructured array surface such as roughness and spacing, whereby organotypic brain slice culture requires different geometries than retina culture. since organotypic culture of adult tissue could only be obtained for about days with conventional techniques, our new substrate material could be the breakthrough for in vitro studies on tissue regeneration, neuroplasticity, as well as drug testing. the intrinsic optical signal (ios) is widely used for mapping neuronal activity in afferent activated brain areas. ios evoked by afferent stimulation in vitro is generally attributed to glial cell swelling via activation of na /k /clcotransporter that follows postsynaptic activation. despite the phenomenon is known for decades, the relative contribution of different cell types and the underlying molecular mechanisms have not been disclosed in detail. our goal was to explore the glial and neuronal correlates underlying in vitro ios genesis. we characterized ios initiated by schaffer collateral stimulation in the rat hippocampal slice using simultaneous high-frequency imaging and field potential recordings. we used a -element photodiode-array device (pda) that enables ios detection with . ms time resolution, making it achievable to align optical and electrophysiological signals. ios was primarily observed in the proximal region of the stratum radiatum and stratum pyramidale of the hippocampus. ios was decreased by blockade of neuronal activity by voltage-gated na channel inhibitor tetrodotoxin and was significantly enhanced by suppressing inhibitory signaling with the gaba a antagonist picrotoxin. we found that ios was predominantly initiated by postsynaptic glutamate receptor activation and progressed by the activation of astroglial glutamate transporters and mg -independent astroglial nmda receptors. in agreement with previous studies, furosemide, the blocker of both the neuronal k / clcotransporter kcc and the glial na /k /clcotransporter nkcc decreased the ios amplitude. to decide which cotransporter is responsible for this effect of furosemide, we applied selective blockers of nkcc and kcc , bumetanide and ( )[r]-dioa, respectively. we evidenced, that nkcc did not contribute to the ios generation, in contrast to previous suggestions. enhancement and slight inhibition of ios through anion and volume-regulated anion channels, respectively, were also depicted. major players of ios mechanisms disclosed by high-frequency ios imaging imply that spatiotemporal ios reflects glutamatergic neuronal activation and astroglial response, as observed within the hippocampus. our model may help to better interpret in vivo ios and support diagnosis in the future. question: a poor x polyunsaturated fatty acids (x pufa) status, favored by the low x /x ratio in western diets, seems to contribute to cognitive decline in the elderly, but mechanistic evidence is lacking. we therefore explored the impact of x status on the evolution of glutamatergic transmission, astroglial regulation and neurogenesis in the hippocampus during ageing in rats. these processes are involved in memory formation and their dysregulation participates to the agerelated brain damage leading to cognitive decline. methods: we have compared groups of rats aged to months fed x -deficient, x /x -balanced, or x (fish oil) supplemented diets: young x balanced (yb), deficient (yd) or supplemented (ys), and old x balanced (ob), deficient (od) or supplemented (os) rats. we have evaluated synaptic efficacy and plasticity (electrophysiological recording), astroglial regulations (glutamate uptake and gfap expression), neuronal markers (glutamate transporters and receptors), neurogenesis (proliferation of neuronal precursors in the sub granular zone), and analyzed brain fatty acids composition. results: dietary modulation of x intakes efficiently modified the incorporation of docosahexaenoic acid (dha, the main x in cell membranes) in brain ( % deficient vs balanced, % supplemented vs balanced). ageing induced a % reduction of synaptic efficacy due to decreased pre-synaptic glutamate release, and a % decrease in the astroglial glutamate uptake associated to a marked astrogliosis ( % gfap). x deficiency further decreased these hallmarks of ageing (od vs ob rats: % synaptic efficacy, % glutamate uptake, % gfap). on the opposite, x supplementation increased synaptic efficacy ( % os vs od) and seems to abolish astrogliosis (os vs ys : no change in gfap). neurogenesis was altered by x deficiency but not by supplementation. conclusion: our results characterize some specific age-related alterations of the glutamatergic synapse in the hippocampus that are aggravated by a dietary deficit in x and attenuated by x supplementation. methods: sgcs were isolated from the trigeminal ganglion (tg) of adult male wistar rats (n ). animals were deeply anaesthetized and both tg were extracted and digested first in mg/ml collagenase for min and then in . % trypsin containing dnase for min. in initial experiments, isolated sgcs were treated with control medium or medium containing . , , or mm cis for h. in the subsequent experiments, sgcs were pretreated for h with control medium or medium containing mm ibu or mm skf. afterwards, sgcs were stimulated for hours with medium containing mm cis. pge concentration was determined by elisa. results: cis caused a concentration-dependent increase in pge release from sgcs. treatment with mm cis significantly increased pge concentration to . . pg/ml, compared with control . . pg/ml (p . ). pge concentration was further increased to . . pg/ml (p . ) following mm cis. pretreatment with ibu significantly lowered the pge concentration compared with cis-stimulated sgcs ( . . pg/ml vs. . . pg/ml, respectively; p . ). a more pronounced inhibitory effect was observed following skf pretreatment, where it reduced the pge concentration to . . pg/ml (p . , compared with cis-stimulated sgcs). conclusion: these findings suggest that cis contributes to an inflammatory response in the tg by provoking release of pge from sgcs, which may lead to development or maintenance of peripheral sensitization involved in cipn. the data also suggest that treatment with agents that target pge release cascade may prove beneficial for preventing development or maintenance of cipn. functional alterations in synaptic contacts have often been described as a hallmark of major depressive disorder (mdd). antidepressants (ads) have been shown to restore neuronal circuits through an enhancement of synaptogenesis in some regions of the brain. nevertheless, the underlying mechanisms are still unclear. glia cells have been since long acknowledged as active partners of neurons in orchestrating molecular signals crucial for the proper arrangement of neuronal circuits in the developing and adult brain. therefore, understanding how both neurons and astrocytes respond to antidepressants (ad) is of high interest. using rat c glioma cells and primary cultures of astrocytes and neurons, we showed a time-dependent cell-autonomous modulation of the erk/mapk signalling pathway after acute treatment with various classes of ads in both cell types. specifically, glia cells responded to ads with the simultaneous and fast activation (after min treatment) of both erk / , in contrast to treatment with antipsychotics or mood stabilizers. this activation induced hrs later an increased release of gdnf, a factor involved in synapse formation and axonal wiring, which was mapk-dependent. on the contrary, hippocampal neurons showed a reduction in erk activity that correlated with neuronal activity inhibition after short term ( min) ads administration, as demonstrated by quantification of c-fos expression after kcl stimulation. interestingly, this inhibitory effect was reversible after long term ( hrs) ads treatment. to further identify whether gdnf released from astrocytes treated with ads might be responsible for long term changes in neuronal synapses, we examined how ads influenced synaptic densities in neurons alone or co-cultured with astrocytes. strikingly, we found that the number of synapses was reduced at hrs after ad treatments, but only in the presence of intact astrocytes and not in cultures of neurons alone or neurons treated with astrocyte-conditioned media. this effect was reversed after hrs treatment and rescued by the soluble form of the specific gdnf receptor gfralpha , but not by gdnf alone. moreover, live imaging of astrocytes showed dramatic morphologic changes of their processes in response to ads that might underlie the observed synaptic remodelling. our analysis of changes occurring at the neuronglia interface upon ad treatments might elucidate novel mechanisms of ads action that may open the avenue for unravelling the role of astrocytes in psychiatric diseases and implement pharmacologic treatment regimens for mdd patients. here we extend these findings showing that the neurotrophin nt- , which also belongs to the putative factors secreted from glial cells after t -stimulation, also increases the navd in neuron enriched cultures. the effects of nt- and fgf- are, however, not additive. antibodies against nt- potentiated the effects of fgf- , suggesting that by converging signal cascades nt- could reduce the effect of fgf- . in neuron-glia mixed cultures antibodies against nt- enhanced the t -induced up-regulation of navd, suggesting, that a co-secretion of nt- from glial cells could limit the effects of fgf- . in a second series of experiments we investigated, whether the well-known effect of t on the expression of na /k -atpases, thought to contribute to the effect of thyroid hormone on metabolism, is also influenced by glial cells. we found, that the effect of t on [h]ouabain-binding and on the expression of na / k -atpase alpha subunits detected by western-blots was highest in neuron-mixed cultures, suggesting that a neuron-glia interaction is also involved in the t effects on na /k -atpase expression. in the healthy brain microglia engage in bi-directional interactions with neurons and other brain cells, which is crucial for maintaining normal physiology and cognitive function of the brain. disturbances in homeostasis and cell-cell communication may predispose to age-related dysfunction and neurodegenerative disease. for decades it has been speculated that microglia exhibit phenotypic diversity allowing specialisation to a variety of brain microenvironments. previous observations of regional variations in microglial densities and morphologies support the existence of microglial heterogeneity. however it is clear that a more comprehensive understanding, in particular of the molecular basis of microglial phenotypic and functional diversity on a global scale, is required. the limitation of recent gene expression studies is the use of mixed whole brain or dissected mixed cell extracts which preclude the detection of cell-specific gene expression profiles. thus, our aim was to validate an efficient extraction process for microglia, ideally minimising the impact on the resting state. the method developed is based on existing procedures and was refined to ensure consistency and to maximise cell yield and purity. the extraction of highly pure microglia as a single cell type was achieved by a two-step isolation technique using density-gradient and magnetic bead separation. purity between - % was verified by flow cytometry and immunohistochemistry. quantitative pcr also demonstrated expression of specific microglial genes indicating that isolated cells are microglia. cytokine assays suggest that no or only minimal activation of cells was induced by the isolation. hence, the microglia retained their ability to respond to immunogenic stimuli and produce il- b as a key inflammatory cytokine. overall, the purification procedure was shown to achieve consistent high purities and retain crucial functional properties. thus, the extracted microglia are likely to adequately represent the real in vivo state and enable the study of microglial phenotypes and functionality in the healthy and ageing whole brain and accordingly brain regions. early diversity experiments showed region-specific microglia phenotypes in the brain of healthy and immune challenged animals as well as differences between unchallenged and challenged animals. question: activation of nmda receptors increase the respiratory frequency in mammals. astrocytes are in intimate contact with neurons, specially in glutamatergic synapses, and are able to sense neuronal activity, react to, and even influence nearby neurons. furthermore, they can sense changes in h or pco , and in response to these stimuli, they can release molecules like atp and d-serine, which will activate neuronal circuits. then d-serine, an agonist of the glycine site of the nmda receptor, can affect the respiratory rhythm during hypercapnia. our aim was to study the probable role of d-serine in the modulation of the respiratory rhythm in mice neonates. methods: fictive respiration was recorded with suction electrodes from c -c ventral roots in "en bloc" (brainstem-spinal cord) preparations from p -p cf mice. superfusion was done with artificial cerebrospinal fluid equilibrated with o :co %: %, (ph . , c) containing different d-serine concentrations ( . - mm) in presence or absence of d-amino acid oxidase (daao), which degrades d-serine. in addition, hypercarbic acidosis (switching equilibration from to % co , changing ph from . to . ) was done in absence or presence of daao. results: application of d-serine increased the frequency of the respiratory rhythm in a concentration-dependent way. application of dao attenuated the effects of d-serine application. likewise, daao reduced slightly the effect of hypercarbia on the respiratory rhythm. conclusions: our preliminary experiments indicate that d-serine is a potent agent increasing the respiratory rhythm frequency in in vitro neonatal preparations, and likely, in association with atp, is mediating the respiratory response to hypercarbia. there, we showed that mac- positive microglia accumulate within the motoneuron area and phagocyted apoptotic bodies at the onset of motoneuron developmental cell death (from embryonic day . -e . ). motoneuron developmental cell death and microglia accumulation in the motoneuron area increased at e . , decreased at e . and disappeared at e . . since it was supposed that microglia promote developmental cell death in the developing central nervous system, at least in vitro, we determined to what extent it was also the case in the embryonic spinal cord in vivo. to address this issue, we analysed in vivo the progression of developmental neuronal death in the spinal cord of transgenic mouse embryos lacking microglia (pu. -ko mice). as opposed to what is observed in the sc of wild type mouse embryo, we found that activated caspase- staining was detectable before e . in pu. -ko mouse embryos. in pu. -ko mouse embryos, activated caspase- staining was not restricted to the motoneuron area, as several positive cells are also present in the dorsal region of the spinal cord and in the progenitor zone. at e . , activated caspase- staining was times greater in the spinal cord ventral area of pu. -ko mouse embryos when compared to wild type littermates. moreover, and opposed to what occurs in wild type animals, caspase- staining in the motoneuron area of pu. -ko mouse embryos increased at e . and persisted at e . , being likely partly due to the accumulation of apoptotic bodies in the absence of microglia. our results suggest that, in addition to their phagocytic role, embryonic microglia are likely to protect neuron from death at the onset of developmental cell death and to promote survival of progenitor-like cells in the embryonic sc in vivo. accordingly, microglia could have an opposite effect on cell survival in the embryonic sc when compared to postnatal snc developmental stages and pathological conditions in the adult. we recently demonstrated a key role of the a-secretase tace, also known as adam , in peripheral nervous system (pns) myelination by tace-mediated cleavage and subsequent inhibition of neuregulin (nrg ) type iii activity. unlike the pns in which nrg type iii is an essential instructive signal for myelination, oligodendrocyte (ol) development and myelination in the central nervous system (cns) are likely controlled by several growth factors some of which undergo cleavage by secretases. to study the role of tace on opc development, immunopanned a b opcs were cultured in proliferating or differentiating medium and treated with a soluble form of tace (rhtace). when ols were scored according to their morphology, we observed enhanced opc differentiation upon addition of rhtace to proliferating opcs. addition of rhtace to differentiating opcs also increased the number of ols with a complex morphology and already presenting membrane sheets, suggesting that tace might promote opc differentiation. to further investigate the role of neuronal tace in cns, we used an in vitro ol myelinating coculture system. when cocultured with tace null drg neurons, rat wild type opcs never myelinate, suggesting that neuronal tace might control opc differentiation. in agreement, preliminary ultrastructural analyses of transgenic mice lacking tace in cns neurons showed hypomyelination and signs of myelin degeneration. accordingly, conditional transgenic mice lacking tace in ol are normally myelinated. these studies suggest that in the cns the a-secretase tace promotes opc differentiation and might regulate cns myelination. a better understanding of the role of tace will provide novel insights into the mechanism regulating cns myelination. in the excitatory network glutamate is released in the synaptic cleft during synaptic activity. glutamate clearance by astrocytes is mainly performed by na -dependent glutamate transporters. the na load during glutamate uptake evokes an activation of the na /k atpase, which dramatically increases energy demands in astrocytes. astrocytes are also involved in the regulation of extracellular potassium (k e ) which significantly increases during the repolarization of excitatory neurons. in this study we evaluated how these fundamental functions of astrocytes coexist and if they interact. we investigated the impact of altering k e concentrations on glutamate transporter activity measuring intracellular sodium (na i ) using the na sensitive fluorescent dye asante sodium green loaded in cultured astrocytes. we observed that glutamate uptake caused a reversible rise in na i , which was tightly modulated in amplitude, slope and recovery rate by k e levels. in order to evaluate the impact of physiologically relevant k e fluctuations on the kinetics of the glutamate transporter the na /k atpase was inhibited. therefore we found that low k e evoked a significantly faster influx of na , whereas high k e markedly slowed down glutamate capture, indicating that k e directly modulates the kinetics of glutamate transporters. we then investigated the energy demands of astrocytes caused by k e alterations. atp hydrolysis was indirectly measured through the changes in intracellular free mg using the mg sensitive fluorescent probe magnesium green. we found that low k e led to higher energy demands of astrocytes during glutamate uptake. overall these results indicate that k e acts as a negative feedback on glutamate uptake in astrocytes. the physiological consequences of these findings have to be considered in the context of k buffering and of maintenance of neurotransmitter and energy homeostasis. recovery was significantly improved by nf fractions, as well as tubulin (tub), added after lpc removal: compared to cultures treated with lpc alone the number of olp (a b cells), and their proliferation increased significantly, as well as the number of differentiated (cnp ) and mature (mbp ) ol. moreover, nf and tub protected ol from lpc toxicity when added at the time of lpc treatment: they increased olp proliferation, as well as the number of cnp and mbp ol significantly, compared to cultures treated only with lpc. on the contrary irrelevant proteins (actin, and skin proteins) were ineffective, demonstrating the specificity of the cytoskeleton proteins effects. importantly, nf and tub increase olp survival and proliferation when challenged with lpc, without delaying differentiation and maturation. we hypothesize that release of nf and tub following axonal damage in ms could participate in the regulation of remyelination through this process. this putative phenomenon might be complexified by alterations of axon protein expression, or of proteolysis, known to occur in ms models. purpose: microglial proliferation is commonly accepted as a hallmark of glial activation. an automatic method that allows assessing the number of microglial cells to analyze the proliferative microglial behavior in a laser-induced ocular hypertension (oht) model has been developed. methods: adult albino swiss mice were organized in two groups: naive (age-matched control; n ) and lasered (n ). retinal wholemounts were immunostained with anti-iba and images of iba- microglias were recorded with a fluorescence microscope. new algorithms of segmentation and control of distances were developed in matlab to obtain the number of iba- microglial cells. automatic results were compared with those obtain by direct human observation of the samples. results: the automatic method detected the number of iba- cells in the inner and outer plexiform layers of the retina in both, na€ ıve and oht-retinas. the number of cells present in the samples was not an obstacle for the program to run properly. the time required for counting iba- cells decreased from the human guide to the program based counting method from days to one hour. results show a strong correlation between both automatic and manual method (pearson correlation test, r . ; p . and r . ; p . for outer and inner plexiform layer respectively) indicating the consistency of the automatic counting. conclusions: a new, reliable and quick algorithm was developed with matlab to obtain the number of iba- microglial cells and also cellular density maps through retinal wholemounts in both na€ ıve and other proliferative conditions (i.e. glaucoma). due to this new automatic method, a bigger set of images or samples could be included in future studies to analyze the behavior of microglial cells. we are interested to understand whether scs in the peripheral nerves of mammals express functional ionotropic glutamate receptors, and whether scs use these receptors for communication with axons. to start addressing this question, we established a preparation of mouse live sciatic nerve slices employing "know-how" of live brain slices technique. using this preparation, we performed whole-cell patch-clamp recordings of scs at different stages of development (embryonic and early postnatal). we included a fluorescent dye into the pipette solution in order to perform post-recording morphological analysis of single scs. first, we recorded current responses of scs to a series of depolarizing voltage steps, aiming to test whether all scs in the nerve are electrophysiologically akin. we found that mouse scs differ in their passive membrane properties and expression of voltage-gated k channels: depending on the age, to cell types could be distinguished. post-recording morphological characterization of the recorded cells showed that scs in the mouse sciatic nerve differ in their size, as well as in the number and length of their processes. next, we used fast pressureinduced application of glutamate to investigate whether scs possess ionotropic glutamate receptors, and whether electrophysiologically distinct sc types differ in their expression of glutamate receptors. application of mm glutamate caused an inward current in at least two types of scs, and preliminary analysis revealed a peak current amplitude in the range of - pa (n ). glutamate-induced currents in scs were blocked by gyki , indicating that mouse scs carry ionotropic glutamate receptors of ampa/kainate type. currently we are studying which subunits of ampa receptors are present in scs, and how these receptors get activated in situ. although cx is expressed by myelinating schwann cells in the peripheral nerves, patients with cmt x develop relatively early axonal degeneration, which correlates best with chronic disability. we found in previous studies of gjb -null mice, a model of cmt x, that the diameter of myelinated axons was progressively reduced at the age of months compared to wild type (wt) littermates, resulting from progressive dephosphorylation of axonal neurofilaments and increased packing density. fast axonal transport was slower in distal axons of mutant compared to wt animals, with impaired mobility of synaptic vesicleassociated proteins and accumulation of beta-amyloid precursor protein and tau. how cx -formed gjs are involved in the regulation of axonal cytoskeleton dynamics remains unclear. methods-here we investigated the signaling mechanisms regulating axonal cytoskeleton focusing on major kinases and phosphatases involved in neurofilament phosphorylation by immunohistochemistry and immunoblot. in addition we examined the localization of axonal mitochondrial, which also depend on axonal transport. we focused on nerve pathology in month-old gjb -null mice, when demyelination and inflammation is minimal, in order to identify the direct effects of gj loss on the axon. results-our results show that erk / kinase and pp a phosphatase were localized in non-compact myelin areas, including the schmidt-lantermann incisures and paranodes, where gjs are normally formed by cx . another kinase, cdk , was more diffusely localized along the axon. biochemical analysis showed altered amounts of erk / , pp a and cdk as well as their upstream activators in gjb -null nerves. the pp a inhibitor phapi was also localized in non-compact myelin areas and significantly increased in gjb -null nerves. assessment of axonal mitochondria by porin immunostaining showed significantly increased density in gjb -null nerves, consistent with slower axonal transport. conclusion-our findings clarify some mechanisms of early axonal pathology that are independent of demyelination in this mouse model of cmt x. several components of the signaling pathway regulating axonal cytoskeleton and axonal transport appear to be functionally related to gjs at the non-compact myelin sheath. loss of myelin gjs results in impaired regulation of axonal cytoskeleton, energy supply, and axonal transport. we have recently discovered that oligodendrocytes secrete exosomes containing a distinct set of proteins as well as mrna and microrna. intriguingly, oligodendroglial exosome release is stimulated by the neurotransmitter glutamate indicating that neuronal electrical activity controls glial exosome release. in this study we examined the role of exosomes in neuron-glia communication and its implications in glial support. results: to analyze the transfer of oligodendroglial exosomes to neurons, we exposed cultured cortical neurons to fluorescently labeled oligodendroglial exosomes. indeed, cultured cortical neurons internalized and accumulated oligodendroglial exosomes in the neuronal cell soma in a time-dependent manner. addition of endocytosis inhibitors or expression of dominant negative dynamin interfered with neuronal exosome internalization indicating that exosome uptake is mediated by clathrin-dependent endocytosis. furthermore, neuronal internalization of exosomes resulted in functional retrieval of exosomal cargo in vitro and in vivo upon stereotactic injection of exosomes. to investigate the influence of oligodendroglial exosomes on neuronal gene expression we performed a microarray screen of neuronal mrnas after exosome exposure. interesting candidates were further validated by qrt-pcr. conclusion: taken together, our results represent a proof of principle of exosome transmission from oligodendrocytes to neurons suggesting a new route of horizontal transfer in the cns. astrocytes are essential for the regulation of neuronal excitability, synaptic plasticity, and the vascular coupling of brain metabolism. unlike neurons, they are electrically silent, but produce a complex repertoire of ca events that coordinate their major functions. however, the canonical cellular mechanism for ca integration in astrocytes is unknown. using cultured astocytes and astrocytes in brain slices expressing ca sensor, gcamp , we report a candidate principle for ca integration in single astrocytes. analysis of the spatiotemporal dynamics of ca signaling in cultured astrocytes demonstrated that ca event distribution can be described by a power law. we show that metabotropic glutamate receptor activation or changes in extracellular ca regulate the power law exponent. these findings demonstrate scale-free ca dynamics in astrocytes that can provide a potential computational theory for brain operation and metabolism. the biogenesis of the axon-myelin unit is controlled by reciprocal interactions between oligodendrocytes and neurons, which continue throughout life. oligodendrocytes secrete endosome-derived microvesicles termed exosomes, implicated in intercellular communication. these exosomes carry a specific set of proteins as well as rna species. here, we show that the neurotransmitter glutamate stimulates exosome secretion from oligodendrocytes by provoking ca entry through oligodendroglial nmda and ampa receptors. furthermore, active neurons evoke exosome release from oligodendrocytes, indicating that neuronal activity controls oligodendroglial exosome release. in turn, neurons internalize oligodendroglial exosomes and functionally retrieve the exosomal cargo. thus, oligodendrocytes may influence the neuronal metabolism by an exosome-dependent transfer of bioactive substances to neurons. axonal degeneration resulting from lack of glial support occurs in plp and cnp deficient mice. intriguingly, both proteins are components of oligodendroglial exosomes. a proteomic approach revealed that amount and composition of exosomes derived from plp -/and cnp -/oligodendrocytes are altered, supporting the hypothesis that disturbed intercellular transfer of substances by exosomes may contribute to axonal degeneration in these mouse models. a. shahraz, j. kopatz, h. neumann university of bonn, bonn, germany microglial cells are the resident macrophages of the brain, which are derived from the embryonic haematopoiesis of the yolk sac. recently, we established a protocol for in vitro differentiation of pluripotent stem cells into microglial precursors by mimicking the embryonic haematopoiesis in a neural microenvironment. we now succeeded to produce microglia from human induced pluripotent stem (ips) cells. these human microglial cells were responsive to amyloid-b by increasing reactive oxygen species (ros) production. in addition, we produced primitive neural stem cells (pnsc) from ips cells that differentiated with suitable growth factors towards neurons. in human microglia-neuron co-culture, amyloid-b treatment resulted in shorter neural branches length compare to the control group. furthermore, results showed that the inhibitory human lineage specific receptor sialic acid binding immunoglobulin-like lectin- (siglec- ) was expressed on human microglial lines. we are now studying the neuroprotective role of siglec- in the amyloid-b-mediated microglia-neuron co-culture model. by expressing the human-lineage specific receptors, these microglial cells will be a suitable model to investigate these receptors in a human microglianeurons co-culture system.x purpose: organotypic retinal culture is a useful tool to perform preclinical drug testing, in which cellular interactions as well as tissue threedimensionality are preserved. the present study aimed to provide a detailed characterization of the morphologic changes of macro and microglial cells in this culture system. methods: retinas were isolated from days old crl: cd(sd) rats with the retinal pigment epithelium attached as described previously (arango-gonzalez et al. ). explants were cultured for , and days (div , div and div ). glial cell populations were characterized by immunostaining cryosections and wholemounts of age-matched control and cultured retinas using specific antibodies against gfap, vimentin and cd -b. results: in div and div cultures, gfap-positive astrocytes were more robust and not homogeneously distributed as observed in vivo: in some retinal areas the astrocytic network was thicker and in other regions astrocytes were sparsely distributed. at div only few thinned gfap-positive astrocytes remained. gfap immunoreactivity of m€ uller cells was up-regulated in culture. however, no major difference was observed in vimentin staining between both, in vivo and in vitro retinas. m€ uller cells extended processes from the inner to the outer limiting membrane were observed for all examined retinas. microglial cells stained with cd -b at div and div had somas more robust than in vivo and cell processes were thicker and retracted. same cells at div exhibited mainly a rounded morphology. conclusions: progressive morphological changes were observed in glial cell populations in retina explants. these changes include reactive macrogliosis, rearranged astrocytic distribution and microglial activation. since glial response is a hallmark of several retinal diseases, our organotypic retinal culture is a valuable resource for future investigations in retinal degenerative processes and therapy. it is widely recognized that grouped housing in enriched environment (enr) impacts the animals' brain structure and function. for instance, rats reared in enr exhibit enhanced neurogenesis in the dentate gyrus, improved hippocampus-dependent spatial memory task performance, and spine density increases of ca and ca pyramidal neurons. we found that that enr housing for four weeks after weaning induces an enhancement in gamma band local field potential oscillation power and an increase in spine density in the right ca stratum radiatum of long-evans rats. the mean spine size, as assessed by serial measurements of postsynaptic density areas, did not change substantially by laterality or rearing condition. one standing question is how glial processes are organized in enr treated rats. unlike cerebellar parallel fiber synapses, the astrocytic coverage of a hippocampal synapse varies from synapse to synapse. our preliminary electron microscopic observation suggests that right ca str. radiatum excitatory synapses tend to have larger degrees of astrocytic coverage in enr treated rats than singly caged rats.we are currently making morphometric measurements to quantitatively assess the peri-synaptic glial changes. it is becoming more and more evident that proper functioning of the neuron-astrocyte-microglia triad is fundamental for the functional organization of the brain, and thus it is of the utmost importance to better characterize their interactions in physiological and pathological processes. we recently demonstrated, in rat models of normal brain aging and lps-induced acute inflammation, that astrocytes and microglia actively collaborate in the clearance of apoptotic neurons and neuronal debris associated with programmed cell death. here we studied the interactions between neurons, microglia and astrocytes within the ca region of the hippocampus after bilateral common carotid artery occlusion (bccao) in the rat, a valid model of chronic cerebral hypoperfusion which leads to persistent ischemic conditions and ultimately to neuronal death. male wistar rats were subjected to permanent bccao. a group of rats was infused into the jugular vein with dipyridamole ( days, mg/kg/day by an osmotic minipump). sham-operated animals were used as controls. three months after bccao, immunohistochemical studies were performed on brain coronal slices, focussing on the hippocampal ca region. using an antibody against glial fibrillary acidic protein (gfap) no astrogliosis was detected. we found a significant increase in the number of total microglia, visualized using the iba antibody, in bccao-treated rats in comparison to the sham group ( %, p < . , one way anova and newman-keuls) and this effect was completely reverted by dipyridamole (p < . , one way anova and newman-keuls). as exemplified in figure , in the ca and str. radiatum of bccao-treated rats, many neurons (anti-neun antibody, red) showing signs of degeneration were closely apposed to and infiltrated by astrocyte branches (anti-gfap antibody, green) which appeared to be bisecting the cell body into cellular debris, and microglia cells (anti-iba , antibody, blue) were actively phagocytosing the damaged neurons. this finding is consistent with the scavenging activity of microglia upon dying neurons or debris, a possible mechanism that prevents further injury to neighboring neurons. it will be interesting to investigate which intercellular communication mechanisms allow the recruitment and activation of different glial cells in a well-organized reciprocal interaction to scavenge the damaged neurons and to verify the mechanisms of the protective effects of dipyridamole found in this chronic cerebral ischemic model. astrocytes are glial cells which provide important metabolic support to neurons, actively tune synaptic activity and influence brain microcirculation [ ] . one of the key processes which sustain astrocyte communication with neighbouring cells is regulated exocytosis mediating the release of gliotransmitters (peptides, amino acids, atp), delivery of membrane transporters, channels and other molecules to the plasma membrane. the exocytic gliotransmitter release is thought to be associated with snare complexes. these consist of four a-helices where one of these is contributed by syntaxin- , one by synaptobrevin- (sb ) and two by snap- in astrocytes. out of these, sb is a trans-membrane protein present on the secretory vesicle [ ] . however, the subvesicular nano-architecture and the number of sb molecules per vesicle are unclear. moreover, it is also unknown how many sb molecules are involved in the fusion between the vesicle and the plasma membrane in astrocytes. to study single vesicles and their association with sb in live astrocytes, we generated a ph-sensitive indicator yellow synapto-phluorin (ysph) as a marker for sb and as a functional readout for monitoring the properties of fusion pores, which are formed following the merger between the vesicle and plasma membranes. in an acidic environment ysph is non-fluorescent and becomes fluorescent upon alkalinisation, permitting the study of fusion of vesicles with the membrane during gliotransmitter release [ ] . our preliminary results, obtained by confocal fluorescence microscopy (with the resolution limit of about nm) and by a super-resolution microscopic technique structured illumination microscopy (sim; with the resolution limit of about nm) show that the new probe (i.e. ysph) efficiently reports the fusion pore establishment in astrocytes and the configuration of sb molecules in a vesicle. the number of schwann cells is fitted to the axonal length in the peripheral nerves. this setting is lost when tumorigenic stimuli induce uncontrolled schwann cell proliferation, generating tumours such us neurofibromas and schwannomas. schwann cells proliferate as well during wallerian degeneration. in both cases proliferation is finally arrested. we show that in neurofibroma, the induction of jmjd removes trimethyl groups on lysine- of histone-h and epigenetically activates the ink a/arf-locus, forcing schwann cells towards replicative senescence. remarkably, loss of function of this mechanism allows unrestricted proliferation, inducing malignant transformation of neurofibromas. interestingly, our data suggest that in injured nerves, schwann cells epigenetically activate the same locus and go as well into the senescence program. indeed, when this pathway is genetically blocked, cell proliferation results increased after nerve injury. we postulate that the ink a/arf-locus is expressed as part of a physiological response that prevents uncontrolled proliferation of the de-differentiated schwann cells generated during nerve regeneration, a response that is also activated to avoid overproliferation after tumorigenic stimuli in the peripheral nervous system. university of rochester medical center, rochester, united states synaptic plasticity is critical for normal neurodevelopment and proper circuit function in the adult nervous system. recent evidence suggests that microglia, classically studied in neuroinflammation, play critical roles in neurodevelopment and synaptic plasticity. however, the mechanisms driving these roles are not well understood. to start elucidating the molecular players involved in microglial communication with neurons during plasticity, we decided to first explore the role of purinergic signaling in this process. while purinergic signaling has been implicated in microglial behaviors, studies have primarily focused on neuroinflammatory roles. however, noninflamed microglia highly express the purinergic receptor, p y , which has known functions in microglial chemotaxis in early inflammation and can stimulate the release of cytokines implicated in synaptic plasticity. thus, we posited that purinergic signaling could contribute to the microglial motility that underlies synapse surveillance in the non-inflamed brain. this in turn may be critical for microglial roles in synaptic refinement during development. to explore this possibility, we took advantage of the p y knock-out (ko) mouse and examined the morphology and motility of microglia in the visual cortex as compared to microglia in wildtype mice. first, we used immunohistochemistry for the microglia specific ionized calcium-binding adapter (iba- ) protein to stain microglia in fixed sections. we then used confocal imaging and scholl analysis to investigate the complexity of the microglial processes. we also used -photon microscopy to monitor microglial motility in p y ko mice by crossing them with cx cr -gfp knock-in mice that allow visualization of microglia including fine processes in vivo. our preliminary evidence suggests that p y disruption alters basal microglial morphology and behavior making microglia less complex and altering their motility patterns. we are now investigating whether microglia-synapse interactions, as well as functional plasticity elicited by visual experience are altered in p y ko mice. our studies will expand our understanding of emerging roles for microglia in neurodevelopment and will pioneer new non-pathological roles for microglial purinergic signaling. a. dvorzhak, a. wojtowicz, r. grantyn charit e -university medicine, berlin, germany in neurodegenerative diseases, the afflicted brain is both an important object of study and an opportunity to characterize a given cellular interaction from a pathophysiological perspective. this dual approach is particularly advantageous when human disease is based on a monogenetic defect and an appropriate animal model becomes available for detailed investigation, as in case of z_q _ki (q ), a new knock-in mouse expressing a mutant form of murine huntingtin. electrophysiological recordings of gabaergic unitary ipscs from striatal output neurons (sons) in sagittal slices from wild-type and homozygous q challenged the current viewpoint that gabaergic transmission is enhanced in the hd striatum. quantal analysis in combination with high frequency stimulation and paired pulse tests revealed that synaptic gaba release is in fact tonically suppressed resulting in disinhibition of striatal output activity (dvorzhak et al j physiol ). the underlying mechanism involves a retrograde endocannabinoid signalling pathway linking postsynaptic mglur with presynaptic cb and gaba release. in addition to this deficit, z-q _ki homozygotes exhibited a significant reduction of tonic inhibition via extrasynaptic gaba(a) receptors. using pharmacological tools to alter the ratio of ambient glutamate and gaba concentrations made clear that the hd-related depression of synaptic and extrasynaptic gabaergic actions depends on the state of both the astrocytic glutamate transporter glt- and the gaba transporter gat- . in support of h eja and colleagues, our imaging experiments suggest that in normal striatal astrocytes gat- is in a releasing mode and driven by the activity of glt- . however, the latter activity is much lower in the hd striatum. sbfi recordings of glt- -related na transients and patch clamp recordings of glt- transporter currents revealed a marked reduction (less than % of wt level) in sr-labelled astrocytes from symptomatic hd mice. together, our results suggest that the deficiency of astrocytic glutamate uptake might represent a pathophysiological key mechanism underlying the observed disinhibition in the striatum of mice affected by huntington's disease. nodes of ranvier are highly enriched in voltage-gated sodium channels (nav), allowing rapid action potential propagation on myelinated axons. cell adhesion molecules (neurofascin- (nfasc ) and contactin) and scaffold proteins (ankyring and bivspectrin), which are also enriched at the nodes, have a critical role in assembly and/or stabilization of na v clusters. oligodendrocytes have been described to induce nav channel clustering at nodes in the central nervous system (cns); however, the nature of the signal(s) involved in nodal formation remains mostly unknown in the cns. to gain insight into cns node assembly, we developed hippocampal neuronal cultures from e rat embryos. during the first week, as expected, na v channel accumulation was detected at the axon initial segment (ais), co-localized with ankyring and nfasc , whereas expression along the axon was diffuse and hardly visible. in contrast, at days in vitro (div), regularly spaced clusters of na v channels, ankyring and nfasc were detected along the axon. the percentage of hippocampal axons with clusters increased from . . % at div, to . . % and . . % at and div, respectively (mean sd). importantly, these clusters were formed in the absence of myelin and no accumulation of the paranodal protein caspr was detected at this stage. to study the role of extrinsic versus intrinsic pro-aggregating factors, we analyzed nearly pure hippocampal neuronal cultures. these purified neurons still formed ais, while only few axons formed na v clusters. interestingly, when these purified neurons were co-cultured with oligodendrocytes or with conditioned medium from oligodendrocytes, the percentage of neurons with nascent nodes was greatly enhanced. taken together, these results confirm that some glial soluble factor(s) might be involved in na v channels clustering, as previously shown for retinal neurons (kaplan et al., ). furthermore, nascent nodes were detected on gabaergic neurons, but not on glutamatergic hippocampal neurons, thus arguing also for the role of intrinsic neuronal factors in their establishment. using hippocampal sections from gad -gfp mice, we showed that some gabaergic interneurons were myelinated in vivo. moreover, we observed that nascent node formation prior to myelination also takes place in vivo, strengthening physiological relevance. the electrophysiological properties of neurons with nascent nodes will be studied. microglia in the degenerating or aging brain are primed by aspects of pathology to produce exaggerated pro-inflammatory responses to subsequent central and systemic inflammatory insults. however, the mechanisms by which microglia become primed, rather than adopting an m phenotype, are not clear. triggers for priming may include altered expression of complement, recognition of amyloid or neuronal/synaptic debris for phagocytosis, decreased engagement of microglial regulatory molecules such as cd r, trem or fractalkine receptor. there is also evidence that loss of basal neurotransmitter tone may contribute to this state. microglia are known to express the nicotinic cholinergic a receptor, which has been shown to influence macrophage and microglial reactivity. in the current study we performed limited lesions of the basal forebrain cholinergic system using the murine-p -saporin immunotoxin (mu-p -sap), to investigate the degree to which loss of cholinergic tone predisposes the microglia to subsequent inflammatory stimulation and to assess the consequences of this for cognitive function in the vulnerable brain. intracerebroventricular injection of mu-p -sap ( . lg) depleted cholinergic neurons in the basal forebrain and decreased cholinergic innervation of the hippocampus, but left performance on hippocampal-dependent reference and working memory tasks relatively intact. however, decreased cholinergic innervation of the hippocampus conferred increased susceptibility to cognitive deficits induced by systemic lps ( lg/kg) days after lesioning, but microglia were not primed days after % lesions. to investigate the regional, temporal and neurochemical basis of this we induced more severe lesions of the basal forebrain (using . lg mu-p- sap) and assessed microglial priming at or days post-lesion. these data demonstrate that microglia are primed by low dose challenge at but not days and, when more robust lesions are induced, priming remains even at days. these responses are observed at the site of injury (medial septum) but more profoundly at the site of denervation, the hippocampus. indicating that neuronal injury and, particularly, loss of cholinergic tone have a profound effect on the reactivity of the microglia to subsequent inflammatory challenge. these data expand our knowledge of microglial priming and have clear implications for the interaction of cholinergic tone and inflammation in cognitive dysfunction such as dementia and delirium in which cholinergic dysfunction is implicated. ). however, glucose may also be metabolized to glycogen or oxidized to co , so it is not obvious that the stimulation of glut and hexokinase leads to lactate release. the objective of this work was to investigate whether lactate may be released by cultured mouse astrocytes within the time frame of the interstitial lactate rise observed in vivo. a first approach with an enzymatic assay showed a significant increase in extracellular lactate after minute of exposure to mm k or lm glutamate. to obtain better time resolution we developed a "lactate sniffer", a hek cell that expresses the fret lactate nanosensor laconic (san mart ın et al., plos one ). seeded on top of an astrocytic monolayer, the sniffer cell responded within seconds of exposure to elevated k mm and a similar response was elicited by mm ba . the response to k and ba supports a role for the na /bicarbonate co-transporter nbce , previously shown to mediate the stimulation of astrocytic glycolysis (ruminot et al., j. neurosci., ). the sniffer cell detected an equally fast increase in extracellular lactate when the culture was exposed to lm glutamate. previous work had shown that glutamate does not stimulate glucose consumption in the short-term (bittner et al., j. neurosci. ), and therefore the rapid release of lactate came as a surprise. this phenomenon may relate to glycogen mobilization or perhaps to inhibition of astrocytic oxygen consumption (azarias et al., j. neurosci. ). k , ba and glutamate did not produce detectable effects on the sniffer cell in the absence of astrocytes. the rapid release of lactate by astrocytes exposed to k and glutamate supports a role for these cells in the fast increase in interstitial lactate concentration that accompanies synaptic activity. ] i ) are regularly occurring as a result of uptake of glutamate from the synaptic space. glutamate uptake occurs via the glutamate/na co-transporters glast and glt- , where one glutamate is accompanied by na and h in exchange for k . the salt pump, na,k-atpase (nka), is responsible for the maintenance of the trans-membrane na gradient by exporting na and importing k for each atp. nka is dose-dependently inhibited by ouabain. astrocytes express two isoforms of the catalytic nka a subunit, the ubiquitous a and in the cns the glia-specific a . the isoforms differ with regard to na affinity, which is lower for a and with regard to ouabain affinity, which is higher for a than for a . although a mutations give rise to neurological symptoms -familial hemiplegic migraine type , the relative roles of the a and a isoforms in astrocytes are still incompletely understood. we hypothesized that the low na affinity of a makes it more suitable to cope with transient large increases in na i . to test this, we compared the effect of mm glutamate for min on real time changes of [na ] i in primary astrocytes, which, following transient transfection, predominantly expressed either the a or the a isoform. in a cells glutamate caused a significantly larger increase in [na ] i and longer recovery time to base line [na ] i than in cells expressing a . glutamate uptake dependence on either a isoform was determined by aspartate uptake in astrocytes expressing endogenous a and a . in the presence of ouabain concentrations that selectively inhibit a , we found a modest ( . mm) increase in [na ] i , accompanied by a disproportionately large decrease ( %) in glutamate uptake. it has been suggested that astrocyte glutamate transporters are clustered in microdomains where they may interact with nka. in gst pull down assays on rat brain we found that both glt- and glast interacted with the st intracellular loop of both a and a , but the interaction was significantly stronger for the a isoform. no interaction was found with other segments of the a molecule. the data indicate that the nka a isoform, which has a more restricted expression than a , plays a specific role for the interaction with the glutamate transporters and for sodium homeostasis in astrocytes. this specificity may be attributed to low sodium affinity of a and to the relatively high capacity of a to interact with the astrocyte glutamate transporters. ozone, a major component of air pollution, has considerable impact on public health. besides its well described inflammatory and dysfunction effects on the respiratory tract, there is accumulating evidence indicating that ozone exposure also affects brain functions. however, the mechanisms through which ozone exerts toxic effects on the cns remain poorly understood. we previously showed that in addition to lung inflammation,ozone exposure caused a neuronal activation in the dorsolateral regions of the rat nucleus tractus solitarius (nts, a sensory nucleus involved in visceral information processing) overlapping terminal fields of lung primary afferent running in the vagus nerves to investigate this hypothesis, we used electron microscopy and immunoblot techniques. in ozone-exposed animals, the astrocytic coverage of nts glutamatergic synapses is increased while the astrocyte volume fraction and the astrocyte membrane densities are not significantly increased. moreover, the expression of specific astrocyte markers gfap, s beta and ezrin did not change in ozone-exposed animals. altogether, our results indicate that ozone inhalation induces glial plasticity, which is restricted to the peri-synaptic coverage. . we recorded pp-gc synaptic activity after eae induction via adoptive transfert (at-eae) and found that mepsc frequency was increased compared to control animals. moreover synaptic alteration in at-eae mice depended on astrocytic tnfr because the effect was absent in tnfr -/-mice but reappeared upon tnfr re-expression in astrocytes. nr b receptors are also necessary because in vivo ifenprodil injection prevented synaptic alteration. overall, our study reveals a specific mechanism responsible for hippocampal synaptic dysfunction possibly relevant for cognitive impairment in ms. research supported by snsf grant a- and nccr "synapsy" to av. we patched ng cells in the hippocampal ca region of ng -dsred mice ( - days old) and investigated in current clamp mode how sodium and potassium channels affected synaptic depolarizations. epsps and ipsps were evoked from a resting membrane potential of mv by injecting mock epsc or ipsc waveforms derived from miniature currents. epsps which depolarized ng cells to more than mv were increasingly shortened by vgcs. at mv, corresponding to a quantal content of , the half width of epsps was shortened to % while the amplitude was hardly altered. application of ttx, -ap and tea showed that most of the observed shaping of epsps was primarily due to activation of a-type k current rectifying the depolarizing input. na currents only slightly ($ %) amplified epsp input, but this effect was outcompeted by the dampening effect contributed by a-type current ($ %). tea was almost without effect on epsps. due to their slower time course ipsps were very differently shaped by vgcs: while recruitment of vgcs by mock ipsps started at a similar threshold voltage, the maximal suppression of ipsp amplitude was much stronger. when depolarizing ng cells to mv, corresponding to a quantal content of , ipsp amplitudes were suppressed to % and the duration of ipsps was not shortened but increased to %. a-type k current was the primary contributor for rectifying as well. interestingly, if a-type k current was blocked, ipsps with a quantal content of unmasked a small ttx-sensitive spikelike depolarization of mv (duration: ms). for comparison, while synaptic responses to simultaneous release of glutamatergic vesicles were hardly altered by vgcs, the synaptic response of gabaergic vesicles was substantially low-pass filtered by a-type potassium currents. altogether, our results suggest that vgcs in ng cells normalize the synaptic strength of excitatory and inhibitory inputs and support their differentiation by ng cells through further emphasizing their pre-existing kinetic differences. to explore the involvement of this pathway in pain and analyze its impact separately in sensory neurons and sgcs, we tested pharmacological inhibitors and transgenic mice in an orofacial pain model. transient inflammation in the submandibular region of mice was induced using complete freund's adjuvant (cfa) and tactile sensitivity was quantified using von frey filaments. although inflammation was resolved by days after injection, tactile hypersensitivity persisted in wildtype mice for at least days, consistent with chronic post-inflammatory pain. tactile hypersensitivity in mice was reversed by systemic injection of the panx /gap junction blockers mefloquine or carbenoxolone at days (peak inflammation) and at days after cfa-injection, suggesting the contribution of these channels to tactile hypersensitivity. in dissociated trigeminal ganglia (tg), which innervate the submandibular skin, bzatp-induced yopro uptake into neurons and glia was prevented by mefloquine, demonstrating the functional presence of the p x r-panx complex in tg. moreover, tg of cfa-injected mice showed more atp release and immunostaining of tg revealed higher expression of panx at week after cfa injection compared to controls. development of hypersensitivity was prevented in p x r-null and in panx -null mice, and was attenuated in mice with glia-specific deletion of panx (gfapcre-panx f/f) but not with neuron-specific deletion (mnfhcre-panx f/f), emphasizing the importance of glial panx signaling in pain. because p x r and panx both have a key role in the immune response by activating the inflammasome, we are currently dissecting the relative importance of neuron-glial communication compared to inflammatory responses in the development and maintenance of orofacial pain. overall, our results show that the p x r-panx complex likely plays a major role in signaling events contributing to tactile hypersensitivity. synaptic plasticity is central to the process of learning and memory and is accompanied by strengthening of existing synapses and/or formation of new synapses. numerous studies have investigated the molecular mechanisms of learning and memory with neurons as the primary interest. given the tight coupling between synaptic activity and brain metabolism, it seems reasonable to assume that synaptic plasticity changes occurring at the synaptic level may also occur at the metabolic level in order to keep up with the augmented demand at the synapse. in this study we investigated the importance of genes involved in brain energy metabolism, and in particular those involved in neuronglia metabolic coupling during fear-motivated inhibitory avoidance learning. using ( c) -deoxyglucose ( -dg) technique we first defined the brain areas that are involved in this learning process. context-dependent avoidance behavior was tested in c bl/ mice using the step-through inhibitory avoidance paradigm (ia). regional brain metabolic activity, as measured by the dg uptake, was quantified in several brain regions. brain metabolic mapping revealed increased glucose utilization in hippocampus, amygdala [(basolateral complex (bla) and the central nucleus (cea)], and anterior cingulate cortex and mammillary bodies. quantitative mrna levels were assessed in dorsal hippocampal tissue at different time points following ia learning. results obtained demonstrate that learning modulates the expression pattern of genes involved in brain energy metabolism, i.e. glycogen synthesis and degradation, pyruvate metabolism, the pentose phosphate shunt and the astrocyte-neuron lactate shuttle (anls) in a time dependent manner. we found a late-phase of enhanced dorsal hippocampal gene expression, particularly for anls related genes, including monocarboxylate transporter (mct ). furthermore, we found that mice deficient in mct transporter display impaired long term memory in the inhibitory avoidance and spatial learning in morris water maze. these observations indicate that metabolic adaptation shown by neuron-glia metabolic coupling might be a relevant phenomenon following learning in conjunction with synaptic plasticity. in order to systematically as well as functionally analyse rmg reactivity to retinal degeneration and thus explore the rmg-derived signalling molecules in depth, we aim at comprehensively profiling proteomewide cellular responses to stimuli. we thus developed a proteomics approach screening specifically for cell surface and membrane proteins as well as secreted protein expression profiles and in addition monitor quantitative changes induced by prototype inflammatory inducer lipopolysaccharide lps. this workflow utilises sugar residues of cell surface proteins on intact rmg which are mildly oxidized and then covalently coupled to biotin. biotinylated proteins are affinity purified and glycosylated peptides are specifically released by pngasef followed by protein identification and quantification by label-free lc-msms. this workflow resulted in the identification of more than proteins on cell surfaces complemented by more than proteins identified in the rmg secretome. bioinformatic analysis allocates % of the cell surface proteins to be truly membrane or extracellular matrix proteins. this comprehensive cell surfaceome includes transmembrane receptors, transporters, adhesion molecules, signalling molecules and proteases, including cd markers, integrins, solute carriers, two ephrins, five ephrin receptors and six plexins. treatment of cells with lps results in a highly reproducible significant shift of cell surface proteome with upregulation of proteins and downregulation of proteins. among those lps-induced cell surface expression changes are proteins that suggest an active role of these glial cells in inflammatory processes. the specific changes will be discussed in detail. cell surface biotinylation on glycosyl-residues in combination with label-free lc-msms is a sensitive and reproducible method to profile cell surface proteomics and sheds light on the biological properties of cells. background: neuronal activity alters calcium ion (ca ) dynamics in astrocytes, but the physiologic relevance of these changes is controversial. to examine this issue further, we generated an inducible transgenic mouse model in which the expression of an inositol , , -trisphosphate absorbent, "ip sponge", attenuates astrocytic ca signaling. results: attenuated ca activity correlated with reduced astrocytic coverage of asymmetric synapses in the hippocampal ca region in these animals. the decreased astrocytic 'protection' of the synapses facilitated glutamate 'spillover', which was reflected by prolonged glutamate transporter currents in stratum radiatum astrocytes and enhanced n-methyl-d-aspartate receptor currents in ca pyramidal neurons in response to burst stimulation. these mice also exhibited behavioral impairments in spatial reference memory and remote contextual fear memory, in which hippocampal circuits are involved. conclusions: our findings suggest that ip -mediated astrocytic ca signaling correlates with the formation of functional tripartite synapses in the hippocampus. they are the only non-neuronal cell type in the brain that receives synaptic input from neurons. ng -cells exhibit a variety of ca -signalling pathways, which might be triggered by pre-synaptic neuronal activity. however, no global increase in the intracellular ca -concentration ([ca ] i ) was detected in ng -cells employing the minimal stimulation technique at neuron-glial synapses. therefore we assume that post-synaptic ca -microdomains might be activated under physiological conditions. these ca -signals are locally restricted to the plasma membrane. membrane bound ca -sensors with a high signal-to-noise ratio, such as lck-gcamp , are best suited for optimal visualisation of ca -microdomains. here, we set out to express lck-gcamp in ng cells together with the cytosolic reporter dye dsred. we generated transgenic mice which express a bidirectional promoter encoding both proteins under the control of a tet-off system. this strategy allows an exclusive expression of the transgenes only in the presence of a tet-responsive transcriptional activator (tta). the construct was successfully cloned and expressed in hek cells. as expected, lck-gcamp was located only at the inner plasma membrane while dsred was expressed all-over the cytosol. functionality of the ca -sensor was tested in vitro. elevation of [ca ] i reliably increased the fluorescence intensity of lck-gcamp . after zygote injection seven positive founder animals could be identified and will be crossbred with a ng -tta mouse line. taken together, the presented construct appears suitable for functional imaging of ca -microdomains in post-synaptic ng -cells. according to the "lactate shuttle" hypothesis, glycogen stored in astrocytes can be converted to l-lactate and exported to neurones as preferred energy substrate. we use optogenetics to investigate signalling between astrocytes and noradrenergic (naergic) neurones of the locus coeruleus (lc). to selectively activate g s -protein-mediated signalling in astrocytes, we generated a viral vector to express a chimera of rhodopsin and b -adrenoceptor (optob ar). we confirmed that this construct activates camp-mediated signalling in astrocytes. in cultured astrocytes, excitation of optob ar resulted in acidification as detected using snarf- indicator, and this acidification could be blocked by pre-incubation with an inhibitor of glycogen breakdown , -dideoxy- , -imino-d-arabinitol (dab), suggesting that the acidification was due to build-up of l-lactate. fast scan cyclic voltammetry (fcv) was used to measure noradrenaline (na) release in organotypic slices cut at the level of the lc in which astrocytes were expressing optob ar. light stimulation of astrocytes powerfully induced release of na which could be prevented by pre-incubation with dab or application of d-lactate. bath application of l-lactate (! mm) in absence of optogenetic stimulation triggered the release of na. in patch clamp experiments, l-lactate depolarised lc neurones and evoked vigorous firing of action potentials. these experiments suggest that activation of g s -protein-coupled receptors in astrocytes could potentiate the release of na from lc neurones via l-lactate. the cellular and molecular mechanisms of this signalling mechanism are currently under investigation. supported by the british heart foundation. rgcs. in addition to this, the organization of astrocytes in the retina of a rat model of glaucoma was found to be significantly different to those of a healthy retina. these alterations in glial cell morphology may reflect changes in their relationship with rgcs and could signify profound changes in their secretome, which may influence rgc survival. in this study we investigated labeling of astrocytes by sr in acute slices from the ventro-lateral medulla and the hippocampus of transgenic mice expressing egfp under the control of an astrocyte-specific promoter. while sr efficiently labeled egfp-expressing astrocytes in hippocampus, we found that sr -staining was very weak in the ventro-lateral medulla and rather unspecific. thus, sr is not a reliable marker for brainstem astrocytes. although carbenoxolone decreased the labeling of astrocytes in the hippocampus significantly, mefloquine which blocks pannexin and connexin hemichannels, was unable to prevent sr uptake in hippocampal astrocytes. time-lapse -photon imaging revealed that in hippocampus both astrocytes and neurons showed temporary sr -loading. in astrocytes the rise of sr fluorescence was slower as compared to egfp-negative cells and sr was quickly removed from non-astrocytic cells during washout, while it was retained in astrocytes. in brainstem astrocytes, however, only a very weak and transient sr -labeling was observed. to test if sr is actively removed from astrocytes in the brainstem, we applied mk- to block the multi-drug resistance transporters mrp- . we did not observe any increase of sr -labeling in brainstem astrocytes. in contrast, astrocytic sr labeling was significantly reduced by substrates of organic anion transport, probenecid, estron- sulfate and dehydroepiandrosterone sulfate suggesting that sr is actively transported into hippocampal astrocytes by an organic anion transporting polypeptide (oatp). additionally, the data suggest that astrocytes modulate extracellular concentration of neurosteroids in the hippocampus. as an alternative approach we have used scanning electron microscopy (sem) to obtain high-resolution back scattered images (bsi) of serial ultrathin sections over a wide-area. in tem the size of the specimen is limited to less than mm , whereas in sem it can be expanded to - mm , allowing us to mount and examine more than serial ultrathin sections on the same stage, collecting d information about many nodal structures simultaneously. here we demonstrate a variety of nodal architectures assembled from bsi-sem (su ,hitachi). in our preliminary study using this technique, the pattern of cellular processes approaching the perinodal axolemma is quite different among axons even in only four nodes examined in optic nerve. in the present study, we analyzed perinodal elements at whole nodes in rat optic nerves and reconfirmed this variety of perinodal glial elements include the followings: ) perinodal space with extracellular matrix ( - %); ) closely abutting astrocytic processes ( - %) ; ) unidentified glial cell, presumable ng glial processes ( - %), but could not find ) pre-synapse-like pseudo-neuronal processes, which was observed at the perinode in the cns grey matter (cerebral cortex) in the previous study. additionally, we found unexpected structures, small teardrop-like protrusion(s) of axolemma at out of nodes examined ( %). in nodes out of these nodes ( . %), glial elements encircled or densely contacted with the teardrop protrusions. a typical case of d reconstructed image is shown in the figure. nodal axon (ax, red), unidentified glial process (yellow; ug, presumable ng cell) and teardrop-like protrusion (asterisks) are demonstrated. outer part of the protrusion from the position indicated two arrows is encircled by the process of ug cell. based on these observations, we propose that some glial cells, including astrocytes and/or ng cells, might contribute to remove wasted membranous elements from the axon at the node of ranvier, which is a new type of neuron-glial interaction. to understand what makes this ssc network more synchronous and excitable, we performed a morphological study of neurons and astrocytes by estimating densities of neun and s -positive cells respectively. although we found similar densities of neurons and astrocytes between gaers and non-epileptic controls, the thickness of the ssc was % smaller in gaers. western blotting quantification of gfap, another astrocytic marker, was significantly higher at p -p , as compared with non-epileptic control. moreover, gfap-immunolabeling suggested that these astrocytes were reactive. we hypothesized that these modifications are linked with an alteration of astrocytic and/or neuronal physiological calcium excitability that could lead to the occurrence of epileptic seizures. to better evaluate the role of astrocytes and neurons networks during the development of ssc between p and p , we used two-photon microscopy imaging coupled with eeg recording in animal under anesthesia and neuroleptanalgesia. we first analyzed neuropile, representing ascendant projection of deeper cortical layers, astrocytes and neurons calcium activities. preliminary results obtained from animals under isoflurane anesthesia showed similar neuropile activities. astrocytic and neuronal activities were too weak to highlight differences between gaers and non-epileptic control. this is likely due to the mode of anesthesia known to strongly decrease astrocyte calcium signaling and neuronal synchronization. current experiments are therefore performed using neuroleptanalgesia know to allow the occurrence of swd. the expected data should lead to a better understanding of neuron-astrocyte interactions during brain development and the mechanism underlying epileptogenesis in a genetic model of epilepsy. purpose: to study the effects of laser-induced ocular hypertension (oht) in the astrocytes of contralateral eyes two weeks after lasering. methods: adult swiss mice were divided into two groups: na€ ıve (n ) and lasered (n ). retinal whole-mounts were immunostained with antibodies against gfap. the gfap-labelled retinal area (gfap-ra) and the number of astrocytes were quantified. results: in comparison with na€ ıve: i) astrocytes were more robust in contralateral eyes. in oht-eyes, the astrocyte population was not homogeneous, given that astrocytes displaying only primary processes coexisted with astrocytes in which primary and secondary processes could be recognized, the former having less intense gfap-ir (p< . ). the mean percentage of astrocytes in which only primary processes could be detected was . %; ii) gfap-ra was increased in contralateral (p< . ) and decreased in oht-eyes (p < . ); iii) the mean intensity of gfap-ir was higher in oht-eyes (p< . ), and the percentage of the retinal area occupied by gfap cells with higher intensity levels was increased in contralateral (p . ) and in oht-eyes (p< . ); iv) the astrocyte number did not differ significantly among the eyes analyzed. however, in oht-eyes the number of astrocytes in which primary and secondary processes could be observed were decreased (p< . ). conclusions: two weeks of laser-induced oht caused changes in the gfap-labelled retinal area but not in astrocyte number in both, contralateral and oht-eyes. on the basis of the astroglial changes detected in the present work, the use of the contralateral eye as an internal control in experimental model unilateral oht should be reconsidered. astrocytes show a high level of functional and morphological heterogeneity and are involved in many aspects of neural function, e.g., formation of the blood-brain barrier, regulation of ion homeostasis, synaptogenesis, and synaptic plasticity. despite the diversity of this cell type, the various astrocyte subpopulations are not well characterized, which is mainly due to the lack of markers that would allow for classification of astrocyte subsets. our study aimed at phenotyping of astrocyte subpopulations based on the expression of cell surface markers, which are associated with certain functions. we used two novel monoclonal antibodies, acsa- and acsa- (acsa: astrocyte cell surface antigen), directed against extracellular epitopes of astrocyte-specific cell surface markers to identify astrocyte subtypes. the acsa- antibody was generated by immunization of glast knockout mice and specifically detects the astrocyte-specific l-glutamate/l-aspartate transporter glast (eaat , slc a ), whereas the acsa- antibody results from an immunization of rats with astrocytes isolated from gfap-egfp transgenic mice. a mass spectrometric approach for the ligand-based identification of the acsa- antigen pointed to a number of candidates, which have to be validated by further experiments. the antibodies acsa- and acsa- were carefully analyzed by costaining experiments with commonly used intracellular astrocyte markers. we found that both antibodies specifically detect astrocytes in the developing and adult central nervous system. in contrast to antibodies against intracellular markers, such as gfap, s ß, or glutamine synthetase, acsa- and acsa- can be used to detect and isolate living astrocytes, which enables further analysis and culture of the separated cells. flow cytometric analysis of acsa- and acsa- antigen expression on cells from different brain regions, as well as immunohistochemical analysis, revealed differences in the expression patterns. this allowed us to define different astrocyte subtypes especially in the cerebellum and olfactory bulb of the neonatal mouse brain. future work will focus on the identification of additional astrocytespecific cell surface proteins for a comprehensive classification of astrocyte subtypes based on cell surface marker expression or expression patterns. the reciprocal communication between neuronal and glial cells represents the key component of the immunosurveillance system of the brain. it has been proposed that the neuro-glial interaction is impaired in human alzheimer's disease. in this study we focus on neuronal "on" and "off" signalling molecules in the transgenic rat model for alzheimer's disease. using enzyme-linked immunosorbent assay we determined the level of cd , cx cl ("off" signalling molecules) and mmp , trem ("on" signalling molecules) in brain homogenates. our results demonstrated significantly up-regulated level of cd (p < , ) in our ad rat transgenic animal model. on the other hand, quantification of mmp level revealed significant decrease of mmp expression (p < , ) in tested transgenic animals. previous studies showed that cd and mmp are predominantly expressed on neuronal cells in cns where cd functions normally as a marker of "self" to protect intact body component or "don't eat me" molecule which protect cd -expresing cells from phagocytosis. our results indicate that overexpression of cd on neuronal cells expressing pathologically modified truncated tau protein can be used as a neuroprotective mechanism potentiating spread and accumulation of pathological modified tau protein in neurons affected by ad pathology which in final stage support progression of the developing disease. in conclusion, we showed, that pathologically modified tau protein can modulate specific "on and off" signalling molecules and thus affects neuron-glia interaction in ad. field-excitatory post-synaptic potentials (fepsp) were recorded from the ca area of hippocampal slices prepared from wistar rats ( - weeks old) as before (fontinha et al., ) . after obtaining a stable value of the fepsp slope for at least min, ltp was induced by delivery of trains of pulses at hz, each train being separated by ms. ltp magnitude (% increase of fepsp) was evaluated - min after induction. ltp magnitude in control conditions was . %, whereas in a second independent pathway of the same slices but in the presence of bdnf ( ng/ml) it was . % (n ; p < . ). ltp was completely abolished when hippocampal slices were superfused with the gliotoxin fluorocitrate (fc, mm), which selectively reduces the astrocytic metabolism decreasing intracellular ca signalling and the release of gliotransmitters. interestingly, in presence of fc, the facilitatory action bdnf ( ng/ml) upon ltp was completely prevented. noteworthy, when fc ( mm) treated slices were superfused with the selective adenosine a a receptor agonist, cgs ( nm), the facilitatory action of bdnf ( ng/ml) upon ltp was rescued (n ; p < . ), so that dltp caused by bdnf (ltp in the presence of bdnf -ltp in the absence of bdnf in two independent pathways of the same slices) in fc cgs treated slices ( %) was similar to that observed in control slices ( % in the absence of fc and cgs ). the results suggest that the facilitatory action of bdnf upon ltp is controlled by astrocytes, and that this role of astrocytes results from their contribution to the extracellular accumulation of adenosine allowing a a receptor activation to gate plasticity actions of bdnf. several studies demonstrated the ability of astrocytes to sense, respond to and regulate neuronal function. among the many functions of glial proteins, glutamate (glu) and gaba transporters play important roles in balancing excitatory and inhibitory signals in the brain. here we show that astrocytes regulate the tonic inhibition of neurons by the concerted action of glu and gaba transporters, thereby protecting both neurons and glial cells from overactivation. we demonstrate that the uptake of glutamate triggers the reverse function of glial gat- / transporters by elevating the intracellular na concentration in astrocytes. the released gaba significantly contributes to the tonic inhibition of neurons in a network activity-dependent manner. we also describe the source of the releasing gaba that is synthesized by an alternative pathway from polyamines. moreover, in the low-[mg ] model of epilepsy, we show that blockade of the glial glu/gaba exchange mechanism increases the duration of seizure-like events and also results in increased activity of astrocytes, demonstrating the neuroprotective impact of the mechanism. finally, we show that the released glial gaba modulates the power of gamma range oscillation in vivo, suggesting that the glu/gaba exchange mechanism is also functioning in the intact hippocampus under physiological conditions. revealing this novel molecular mechanism by which astrocytes provide an adjustable, in situ negative feedback on the excitability of neurons is expected to broaden our understanding about the regulation of neuronal activity by astrocytes and may open up new targets for the treatments of pathological conditions, such as epilepsy or ischemia. chronic stress is well recognised to decrease the number of gfap astrocytes within the prefrontal cortex (pfc). recent research, however, has suggested that our understanding of how stress alters astrocytes may be incomplete. specifically, chronic stress has been shown to induce a unique form of microglial remodelling, but it is not yet clear whether astrocytes also undergo similar structural modifications. such alterations may be significant given the role of astrocytes in modulating synaptic function. accordingly, in the current study we have examined changes in astrocyte morphology following exposure to chronic stress in adult rats, using three-dimensional digital reconstructions of astrocytes. our analysis indicated that chronic stress produced profound atrophy of astrocyte process length, branching and volume. we additionally examined changes in astrocyte-specific s b, which is both a putative astrocyte marker, and a protein whose expression is associated with astrocyte distress. while we found that s b levels were increased by stress, this increase was not correlated with atrophy. we further established that while chronic stress was associated with a decrease in astrocyte numbers when gfap labelling was used as a marker, we could find no evidence of a decrease in the total number of cells, based on nissl staining, or in the number of s b cells. this finding suggests that chronic stress may not actually reduce astrocyte numbers and may instead selectively decrease gfap expression. together, these results provide a significantly more elaborate picture of how chronic stress alters the pfc. the drosophila nervous system is ensheathed by several types of glial cells, one of which, namely the subperineurial glia, forms pleated septate junctions (psj) to build a tight blood-brain barrier (bbb). hence, nutrients from the surrounding hemolymph must cross these glial cells to reach the neurons. the amount of nutrients entering the nervous system is likely to be tightly controlled through neuron-glia communication to ensure optimal metabolic supply while avoiding disturbance of the extracellular homeostasis. we aim to elucidate signals involved in this regulatory interaction. to this end, an rnai-based screen in glia of adult flies is performed using the gal /gal ts system. we analyze the intake of blue-dyed food by photometric measurement. since impairment of the metabolic supply of the brain should severely affect the organism, compensatory changes in feeding behavior are expected. for instance, knockdown of nutrient transporters specifically in glia would then induce excessive feeding in normally nurtured individuals. the first set of rnai lines screened includes genes that have previously been found to be essential in glia for survival or locomotion of the animal. furthermore it comprises metabolic enzymes, all carbohydrate transporters, neuropeptides and their corresponding receptors. candidate genes will be further characterized regarding their role in controlling the energy homeostasis of the nervous system. the discovery that activation of non-neuronal cns microglia plays a causal role in spinal processing of nociceptive signaling has shed new light on the processes underlying neuropathic pain facilitation. however, there remains much uncertainty as to the necessary contribution of microglia to enhanced pain states. we aim to define the particular role of microglia for the initiation of neuropathic pain and by answering this question also learn if the function of peripheral myeloid cells is distinct or redundant in this process. methods: to specifically investigate spinal microglia and peripheral macrophages in the pathogenesis of neuropathic pain, we model chronic pain by performing partial ligature of the sciatic nerve in cd b-hsvtk mice engrafted with gfp bone marrow (gfp>cd b-hsvtk). cd b-hsvtk /mice allow the exchange with peripherally-derived, gfp macrophages upon central depletion of endogenous cd b microglia. following this depletion/ repopulation paradigm, behavioral analyses of mechanical and thermal allodynia are conducted. results: we established a selective tool to exchange cns parenchymal microglia with peripheral gfp myeloid cells. in chronic pain tests for mechanical and thermal hyperalgesia, gfp>cd b-hsvtk /mice show considerable decreases in paw withdrawal thresholds in response to mechanical, but not thermal stimuli ipsilateral to the injury, indicating distinct roles of microglia and macrophages in the facilitation of thermal hyperalgesia. conclusions: we identified a differential contribution of resident spinal microglia vs. peripheral myeloid cells in the development of neuropathic pain in gfp>cd b-hsvtk chimeras. future studies aim to examine the exact mechanisms underlying the distinction between these two populations. a deeper understanding of the processes involved in the compartmentation of brain energy metabolism is of major interest. not only to enwrap pathomechanisms of brain diseases associated with metabolic deficits but also for a more accurate interpretation of functional neuroimaging signals which often use metabolic signals as surrogate marker for brain activity (e.g. fdg pet). large amount of knowledge in the field has been obtained from small animal neuroimaging studies. but due to their invasiveness they often need anesthesia. anesthetics heavily alter physiological read-outs. the aim of this study was .) to test if metabolic imaging using two-photon microscopy ( pm) and genetically encoded glucose sensors is feasible in the awake, head-fixed mouse and .) to examine the effects of isoflurane anesthesia on the signals. in two mice, a recombinant adeno-associated virus as a carrier for the genetically encoded glucose sensor flii pglu md was injected into the whisker barrel cortex. by using an astrocyte-specific promoter (short gfap) astrocytic expression was ensured. in addition, a chronic window and a head post were implanted. behavioral training started a few days after surgery. animals had to learn to tolerate head fixation without spontaneous motor activity. within a single trial animals were trained not to move for seconds (imaging period) to receive a water reward. animals were subjected to water restriction during the behavioral training. animals were trained for three weeks before pm imaging started. animals learned to tolerate head fixation up to minutes allowing acquisition of up to trials per session. upon oral glucose administration (per single water reward $ mg glucose was administered) fret signal started to increase within minutes and went up to a maximum of %. surprisingly, isoflurane anesthesia led to an even higher increase of the signal (mean increase of %) compared to the awake state. in some experiments the barrel cortex was activated by vibrotactile single whisker deflections generated by a piezo bending actuator. fret signal did not show a difference between baseline and stimulated condition ( . . vs. . . ). in conclusion, the presented experiments demonstrate for the first time the feasibility of awake pm imaging for metabolic measurements on a single cell level. the signal increase upon peroral glucose administration unambiguously supports the functionality of the sensor. isoflurane exhibits a large effect on intracellular astrocytic glucose concentration distinctly revealing the need for experiments in awake animals for unbiased results. future experiments will now be expanded to neuronal glucose sensors to enable comparisons between astrocytes and neurons. our previous studies indicate that neuronal electrical activity controls glial exosome release resulting in subsequent neuronal uptake and functional retrieval of the exosomal content. proteomic analysis of oligodendroglial exosomes revealed a list of candidates with potential neurotrophic action in neurons. to elucidate the impact of oligodendroglial exosomes on the neuronal metabolism, we analyzed the metabolic activity of neurons after co-culture with oligodendrocytes or direct treatment with exosomes. methods: neurons were subjected to different stress paradigms such as oxidative stress, hypoxia, and nutrient deprivation. neuronal vitality was assessed by mtt assay and the mitochondrial membrane potential was visualized by staining with mitocapture. results: neurons grown under optimal conditions were not affected by the presence of exosomes. intriguingly, when neurons were subjected to stress (oxidative stress, nutrient deprivation, oxygen-glucose deprivation) their metabolic activity was significantly increased in the presence of exosomes. when challenged with oxidative stress prior to exosome treatment, neurons were not able to recover. mitocapture staining demonstrated that oligodendroglial exosomes prevent the breakdown of the mitochondrial membrane in nutrient-deprived neurons. conclusions: our results indicate that exosome supply is protective for neurons but is unlikely to support their recovery. we suggest that oligodendroglial exosomes carry neuroprotective substances, which protect neurons from stress. interestingly, this effect of lactate on synaptic plasticity mechanisms was not fully mimicked by glucose, suggesting that the effect was not only related to supporting the potential increase in energy demands related to plasticity, but that l-lactate could act as a signaling molecule for the regulation of expression of plasticity-related genes. we have followed up on these observations and show that l-lactate significantly stimulates mrna expression of key immediate early genes (iegs), in a time-and concentration-dependent manner, in cultured neurons. following one hour of treatment with mm l-lactate, arc, zif and c-fos mrna levels were increased by . , . and . folds, respectively. in addition, the increased iegs mrna expression levels induced by l-lactate are correlated at the protein level with respectively . , . and . fold increase compared to control values at the same time point. these effects are specific for llactate, since d-lactate (non-metabolized enantiomer of l-lactate), l-pyruvate and d-glucose (at equicaloric concentrations) have no effect on gene expression. interestingly, we observed that, in similar culture conditions, l-lactate-induced iegs expression is only observed in neurons but not in astrocytes, implying a cell specific effect. characterization of the underlying molecular mechanisms of l-lactate action on iegs expression demonstrate the involvement of nmda receptors. finally, we obtained evidence that such regulatory mechanisms of ieg expression also operate in vivo as direct intracortical injections of l-lactate ( mm) into the somatosensory-motor cortex areas resulted, within hour of application, in a significant stimulation of arc, zif and c-fos expression (by %, % and %, respectively) as compared to d-lactate ( mm)-injected contralaterally areas. taken together these findings demonstrate that l-lactate acts as a direct signaling molecule which regulates neuronal plasticity-related iegs expression both in vitro and in vivo. interestingly, the underlying mechanism of action of l-lactate involves nmda receptors.this set of observations therefore reveals a novel signaling role of lactate which may represent a likely mechanism to account for the role of astrocyticderived l-lactate on ltm formation. neuronal activity in the brain is associated with a transient increase in the extracellular k concentration. the excess k is removed from the extracellular space, primarily by the surrounding glial cells, leading to an intracellular accumulation of k via mechanisms not fully identified and/or quantified. post-stimulus recovery of [k ] o has been proposed to be dependent on kir . -mediated spatial buffering and/or to na /k -atpase activity. to resolve the molecular mechanisms involved in k clearance, we initially determined the contribution from the different k -transporting mechanisms present in primary culture of rat astrocytes and their k . for k . the na /k / clcotransporter, nkcc , increased its activity within a physiological concentration range of k , while the na / k -atpase saturated at much lower concentrations. consequently, a concentration-dependent switch occurs between the two mechanisms of k uptake in cultured astrocytes. in addition, nkcc was capable of mediating robust k -induced astrocytic cell swelling. thus, nkcc could potentially act as a molecular mechanism responsible for clearance of the stimulus-evoked rise in [k ] o and the associated shrinkage of the extracellular space. to determine the contribution of each of the three molecular mechanisms to k -clearance in native brain tissue, we used rat hippocampal brain slices and employed ion-sensitive microelectrodes in association with high-frequency electrical stimulation as well as focal appliances of k by ionophoresis. inhibition of kir . ( um bacl ) or nkcc ( um bumetanide) failed to show a significant effect on the rate of k removal from the extracellular space. in contrast, inhibition of the a and a isoforms of the na /k -atpase significantly delayed post-stimulus recovery of [k ] o and this delay was further potentiated by additional inhibition of the a isoform. the na /k atpase emerged as the primary factor responsible for stimulus-evoked k -clearance with no evidence in favor of kir . and nkcc involvement. further characterization of the different na /k -atpase isoforms, by heterologous expression in xenopus laevis oocytes, revealed that the a isoform reached its maximal activity already at resting k concentrations, hinting at a "housekeeping" function. the a subunit displayed voltage-sensitivity and increased turnover rate along physiologically relevant increments in extracellular k concentration. these a -related features may render this astrocyte-specific subunit variant specifically geared for post-stimulus recovery of [ as a model of ischemic injury, and sham-operated mice were used as controls. we isolated gfp cells from the dorsal part of the lv of sham-operated-and post-ischemic brains and employed a neurosphereforming assay in order to estimate their ability to proliferate and selfrenew. furthermore, we followed their immunocytochemical and electrophysiological properties during in vitro differentiation and compared the differentiation potential of gfp cells isolated from the controls to those isolated from post-ischemic brains. the gfp cells isolated from the dorsal part of the lv of controls formed neurospheres and differentiated only into a glial phenotype. the gfp cells isolated from the dorsal part of the lv of post-ischemic brains were able to form neurospheres as well; however, besides their differentiation into a glial phenotype, they also gave rise to cells with the properties of neuronal precursors. we also performed in situ immunohistochemical/electrophysiological analyses of gfp cells in the adult brain of controls and those after mcao. in situ analyses revealed that gfp cells expressed the phenotype of adult nscs or neuroblasts in controls or following ischemia and that their number was increased in both hemispheres following mcao. compared to controls, we found that the number of gfp/doublecortin-positive cells significantly increased in the dorsal part of the lv as well as the number of gfp cells in the olfactory bulb, where they probably differentiated into calretinin interneurons. our results indicate that gfp cells with an active mdach gene in the dorsal part of the lv play an important role in the increased production of neuroblasts after injury and that this process is also enhanced in the contralateral hemisphere. collectively, our results reveal the involvement of the mdach gene in adult neurogenesis. cells expressing this gene exhibit the properties of adult nscs or neuroblasts and respond to mcao by enhanced neurogenesis. experimental cerebral ischemia leads to activation of microglia and astrocytes, which subsequently release pro-and anti-inflammatory factors. furthermore, during the earliest periods of neuronal damage, large quantities of dopamine are released, which may exacerbate the vulnerability of the lesioned tissue. on the other hand, delayed treatment with levodopa has been proven beneficial in stroke patients, suggesting a more complex effect of dopamine. recent studies on astrocytic involvement in neuroinflammation have shown a remarkable modulation of the inflammatory response over the dopamine d receptor (d r). after stroke, reactive astrocytes in the astroglial scar were found to be immunopositive for d r. in this study, we report the expression of d r in activated microglia, both after experimental stroke in vivo and after oxygen-glucose deprivation (ogd), an in vitro model of ischemia. using immunohistological stains, we analyzed the expression of d r in iba positive cells , , , , and days after middle cerebral artery occlusion (mcao; n - mice/time point). for each animal, > pictures were taken from the ischemic core as well as from the contralateral, non-lesioned cortex from sections. a total of > (control) and > (ischemic core) iba cells were analyzed for each time point. in the ischemic core, $ % of cells immunopositive for iba expressed d r in contrast to < % in control areas. a strong depletion of d r immunoreactivity was observed in the ischemic striatum, accompanied by an increase in d r expression in the adjoining cortex and an elevated presence and activation of microglia. for in vitro experiments, we used fluorescence activated cell sorting to isolate cd low /cd microglial cells. no significant d r expression at mrna level could be detected by rt-pcr in this population, while we were able to detect d r in non-microglial cells. interestingly, cultured primary mouse microglia expressed low quantities of d r, as revealed by western blotting and rt-pcr, suggesting that culturing is sufficient to induce d r in microglial cells. upon in vitro activation of primary microglia through minutes of ogd, we found a . fold increase in d r expression after hours (n ). furthermore, treatment of primary microglia with the selective d r agonist pramipexole showed a dose-dependent tendency to increase lipopolysaccharideinduced tnfa release (n ). our studies indicate that activated microglia express a functional d r, which may contribute to the pro-inflammatory reaction of microglia after ischemia. white matter (wm) is injured in most strokes and axonal injury and dysfunction contribute to disability associated with clinical deficits. in young wm, the damage from ischemic injury involves the sequence of energy depletion (ionic pathway), excessive glutamate release (excitotoxicity), generation of reactive oxygen species and oxidative stress (oxidative pathway). in older wm the injury is mediated by ca -independent excitotoxicity due to an earlier and more robust glutamate release. because excitotoxicity leads to oxidative stress in wm we investigated whether blocking nitric oxide synthase (nos) activity before or after a period of oxygen glucose deprivation (ogd) promoted axon function in an age-dependent manner. acutely isolated optic nerves from young and old ( and month) swiss webster (sw) or c bl/ (bl ) mice were used to ascertain quantitative measurements of wm function and structure. to support a biological basis for nos inhibitor nitro-l-arginine methyl ester (l-name) action in themonpreparation, we evaluated the expression and localization of brain nos (bnos) using immunohistochemistry in conjunction with confocal imaging. the expression of bnos co-localized with gfap ( ) astrocyte nuclei, cytoplasm, end-feet as well as nf- ( ) axons. the pattern of bnos expression paralleled astrocyte morphology with age and became more punctate in appearance. evoked compound action potentials (caps) recovered to . . % (n ) after min of oxygen glucose deprivation (ogd) in young bl mons. pretreatment of mons with l-name ( mm) promotedcaprecovery to . . % (n ) compared to ogd while caps recovered to . . % (n ) when l-name was applied after the end of ogd. pretreatment of mons from or month old sw with l-name improvedcaprecovery to . . % (n , vs ogd . . %, n ) or to . . % (n , vs ogd . . %, n ) respectively. l-name application after the end of ogd failed to promote aging axon function ( . . %, n ). changes in nos activity help unveil agedependent oxidative injury mechanisms in ischemic white matter. question: cerebral endothelial cells have been reported to exert a protective effect against brain damage. does transplantation of healthy endothelial cells have a beneficial effect on the outcome of ischemic brain damage? methods: we injected endothelin- (et- ) into the rat internal capsule to induce lacunar infarction. seven days after et- injection, microvascular endothelial cells (mvecs) prepared from adult rat cerebral cortices were transplanted into the internal capsule. meningeal cells prepared from adult rat cerebra or . % bovine serum albumin-hank's balanced salt solution were injected as controls. two weeks later, the footprint test and histochemical analysis were performed. results: we found that mvec transplantation improved the behavioral outcome based on recovery of hind-limb rotation angle (p < . ) and induced remyelination (p< . ) compared with the control groups. also the inflammatory response was repressed by mvec transplantation, judging from fewer ed- -positive activated microglial cells in the mvec-transplanted group than in the other groups. conclusions: elucidation of the mechanisms by which mvecs ameliorate ischemic damage of the white matter may provide important information for the development of effective therapies for white matter ischemia. objective: though lactate is a known neuroprotective agent, its mechanism of action is not known. we hypothesized that lactate can provide neuroprotection by activating trek channels. the effect of lactate on the electrophysiology of trek channels was studied both in an in vitro brain slice model, after blocking the activity of other ion channels and in a heterologous expression system. results: whole cell patch clamp experiments, on ca stratum radiatum astrocytes, in acute hippocampal slices, significantly increased trek channel activity by . . % and hyperpolarized their resting membrane potential by . mv, on bath application of mm lactate. comparison of rectification index at negative potentials between control and mm lactate treatment showed no significant difference, suggesting that the conductance activated by lactate is outward rectifying. lactate-evoked increase in trek channel activity was reversibly inhibited by mm quinine, a potent inhibitor of trek channels. further, lactate was unable to increase trek channel activity after disruption of intracellular lactate uptake with mm a-cyano- -hydroxy cinnamate, a blocker of monocarboxylate transporter. this was confirmed by inside-out patch clamp experiments on human trek channel expressed in hek cells. conclusion: the experimental observations suggest uptake of lactate by astrocytes to activate trek channels intracellularly. regenerative responses occurring after different types of postnatal brain injury often involve expansion of an endogenous neural progenitor cell pool, but the molecular mechanisms underlying this process are still poorly understood. we studied expansion of the glial progenitor cell pool in a mouse model of neonatal hypoxia (hx) that reproduces the hallmarks of perinatal brain injury in infants, including diffused white matter injury (dwmi). we have previously shown that hx causes proliferation of wm oligodendrocyte progenitor cells (opcs), and that this process is regulated by the cyclin-dependent kinase (cdk ) pathway. our analysis in wm in vivo and in cultured cells demonstrated: i) higher expression of cdk , cycline, prb / and e f in wm opcs after hx; ii) activation of the cdk pathway after hx, and iii) reduced hx-induced opc proliferation in cdk -null mutant mice. here, we studied the upstream molecular mechanisms that regulate activity of the cdk signaling pathway resulting in opc proliferation after hx. we show that sirtuin (sirt ) histone deacetylase activity is a major regulator of cdk signaling and of the regenerative response of opcs after hx. under hypoxic conditions, sirt is upregulated in wm, and interacts with both rb and cdk . patterns of posttranslational modifications of cdk or sirt proteins after silencing either of these genes in cultured cells from normoxic and hypoxic wm indicate that cdk is a substrate for deacetylation by sirt , and that sirt is phosphorylated by cdk . also, sirt causes rb deacetylation resulting in dissociation of e f from the rb/e f complex, which ultimately leads to higher opc proliferation. knockdown of sirt in hypoxic wm cell cultures suppresses opc proliferation. finally, inhibition of sirt activity by sirtinol accelerates opc differentiation to mature oligodendrocytes, and reduces the number of opcs. we are currently analyzing the effects of hx on opc proliferation and differentiation in the wm of sirt -null mice. our results provide evidence that sirt is an essential regulator of the cdk -dependent regenerative response observed in wm opcs after hx, and that inhibition of sirt activity facilitates oligodendrocyte regeneration from opcs, which might ultimately promote wm recovery after hx. supported by nih r ns , p ns and iddrc p hd . gonadal steroid hormones reveal a potential neuroprotective role in acute brain ischemia. in rat in vivo studies using the transient occlusion of the middle cerebral artery as ischemic stroke model, we have shown that ß-estradiol and progesterone reduce the infarct area by more than % given h after the onset of stroke, prevent neuronal death and behavioral deficits. an intriguing aspect of this study was that the application of steroid hormones reduced the number of microglia and microglia-related markers in the penumbra during the first h after the onset of stroke. besides microglia, penumbral astroglia coevally appeared as cellular target for steroid hormones by reducing the expression of proinflammatory-and molecules necessary for the attraction and activation of microglia and lymphocytes. this suggests that protective steroid hormones influence glia cell cross-talk and activity during early damaging conditions in the lesioned brain site. by using an in vitro hypoxic approach, we have now demonstrated that microglia express steroid hormone receptors and directly respond to ischemic conditions and steroid hormone treatment by changing expression and secretion of inflammatory compounds, trans-signaling factors and by modulating phagocytotic activity. this clearly shows that microglia is an important direct target for steroid hormones but also indirectly influenced via cross-talk by adjacent astrocytes at the damaged brain site. this generally points at an extensive bidirectional crosstalk between both glial cell types to convey steroid-mediated neuroprotection in ischemic brain tissue. in summary, the balance of inflammatory astrogliamicroglia interactions within the injured brain tissue during an early phase of ischemic destruction is a pivotal step for tissue repair. cannabinoids are considered as key regulators in the pathology of various diseases, including ischemic stroke. we previously demonstrated that post-ischemic treatment of two naturally occurring phenylpropanoids, trans-and cis-hinokiresinols (hrs) significantly reduced ischemic injury in rats subjected to middle cerebral artery occlusion (mcao) . in studies to screen their cellular targets, we found that hrs selectively bind to both type and type cannabinoid receptors (cb r and cb r). in the cb r reporter gene assay, both hrs demonstrated antagonistic activity. in cb r-overexpressing u os cell lines, both hrs increased forskolininduced camp accumulation, suggesting their inverse agonism. since camp induces expression of heme oxyagenase- (ho- ), a well-known antioxidant stress protein, we further investigated the effect of hinokiresinols on ho- expression in mixed cortical neuron/glia cultures. trans-hr increased astroglial expression of ho- greater than cis-hr did. a selective ho- inhibitor, tin protoporphyrin ix significantly reduces the neuroprotective effect of trans-hr in cortical cultures exposed to oxygenglucose deprivation. in addition, both hrs reduced the number of ed- immunopositive cells (i.e., active microglia and macrophages) in ischemic lesions. also, both hrs significantly reduced microglial migration induced by an endocannabinoid, -arachidonoylglycerol. the present study suggests that hrs reduce ischemic injury via regulation of glial cbrs. , the main anaplerotic enzyme in the brain, is predominantly located in astrocytes. in the adult brain, ischemia is known to reduce pyruvate carboxylation. moreover, reperfusion after ischemia leads to production of reactive oxygen species (ros). the pentose phosphate pathway (ppp) is, by making nadph necessary for the regeneration of reduced glutathione, a major pathway in the protection against ros. however, astrocytic and neuronal metabolic function in the neonatal brain after hypoxic-ischemic brain injury (hi) remains to be explored. methods: hi was induced in -day-old rats by unilateral severing of the carotid artery followed by hours recovery and subsequent exposure to hypoxia ( %o ) for min. min after end of hypoxia (the early reperfusion phase), rats were injected with [ , - c]glucose and decapitated min later. one group was sham-operated, and not exposed to hypoxia, thus reflecting normal metabolism in the neonate. extracts of ipsilateral hemispheres from hi and sham animals were analysed with h-and c-nmr spectroscopy. results: the sham animals had similar glutamine content, but lower amounts of c labelled glutamine compared to corresponding values from adult rats, reflecting a generally lower rate of glucose metabolism in the neonatal astrocytes. however, approximately equal amounts of c labelled isotopomers of glutamine were derived from pc and pyruvate dehydrogenase (pdh), resulting in a relatively high pc/pdh-ratio compared the same ratio in adults. following hi and reperfusion, a reduction in glucose metabolism and mitochondrial metabolism was seen by increased glucose and reduced incorporation of c labelling in glutamate, glutamine and aspartate via pc and pdh. however, the pc/pdh-ratio in glutamine was similar in hi and sham. total mean values of glutamate, glutamine and aspartate were decreased, but only the latter to a significant degree. labelling in lactate via the ppp was reduced following hi. conclusion: the low labelling of lactate via the ppp indicates that the flux through the ppp was in fact reduced in the early reperfusion phase after hi. moreover, mitochondrial metabolism of pyruvate from glucose was reduced in both astrocytes and neurons. impaired anaplerosis in astrocytes was confirmed by lower amounts of aspartate. however, the proportionally similar decrease in c labelling in glutamate and glutamine via pc, and the maintained pc/pdh-ratio implies that astrocytes continue to provide metabolic support for the neurons during the early reperfusion phase. ret receptor tyrosine kinase is the signaling component of the receptor complex for the family ligands of the glial cell line-derived neurotrophic factor (gdnf). ret is involved in the development of enteric nervous system, of sympathetic, parasympathetic, motor and sensory neurons and it is necessary for the postnatal maintenance of dopaminergic neurons. ret expression has been as well demonstrated on microglia and several evidence indicate that gdnf regulates not only neuronal survival and maturation but also certain functions of microglia in the brain. here we demonstrated that isolectin ib , commonly used as a microglial marker in the brain, binds to the glycosylated extracellular domain of ret both on the surface of living nih t fibroblasts cells stably transfected with ret than in adult rat brain sections as revealed by immunoblotting. further, confocal immunofluorescence analysis demonstrated a clear overlap staining between pret and ib labeling in primary microglia cultures as well as in adult rat sections obtained from control or postischemic brain after permanent middle artery occlusion (pmcao). interestingly, ib staining identified activated or amoeboid retexpressing microglia under ischemic conditions. collectively, our data indicate ret receptor as one of the ib -reactive glycoconjugate accounting for the ib stain in microglia under physiological and ischemic conditions. astrocytes can adapt to lower oxygen tensions by enhancing their glycolytic rate of energy production. in this case, adequate expression of monocarboxylate transporters (mcts) may be essential to sustain prominent lactate efflux and prevent glycolysis inhibition. here we show that oxygen level is an important factor controlling mct expression in primary cultures of mouse cortical astrocytes. indeed, while mct is clearly expressed by astrocytes in vivo, its basal in vitro expression in primary cultures of mouse cortical astrocytes is undetectable under classical culture conditions ( % air, %co ). exposing astrocytes to more physiological brain oxygen tensions (hypoxic chamber flushed with %o / %co / %n , leading to a concentration of dissolved oxygen in the culture medium of - mmhg) restored mct expression. mct mrna expression became detectable after hours under lower oxygen tension and was still present after days with a maximum at hours of incubation. this effect was paralleled by the appearance of the mct protein which reached a plateau between hours and hours, with a further enhancement at and days of incubation. this induction was specific for mct since mct expression was not altered. lactate release was significantly enhanced after hours of incubation and further increased up to days compared to astrocytes maintained under atmospheric conditions. treatment with dimethyloxalylglycine (dmog), a compound that mimics hypoxia by stabilizing the hypoxia-inducible factor alpha (hif- a) , produced a similar effect. both mct mrna and protein induction reached their maxima when astrocytes were treated with a concentration of dmog ranging between to . mm, which is also the range for greatest hif- a expression. again, no effect was detectable on mct expression. transfecting astrocyte cultures with a sirna against hif- a significantly reduced the effect of lower oxygen tension on mct expression. our results suggest that ) under physiological conditions, changes in local oxygenation might regulate the capacity of astrocytes to supply lactate to neighboring cells via notably alteration in mct expression ) under pathological conditions, mct may be upregulated as a consequence of hypoxia and contribute to neuroprotection by favouring the release and accumulation of lactate in the extracellular space. indeed, this process might give rise to the beneficial effect of lactate for neurons observed during the recovery from hypoxic/ischemic conditions in vitro and in vivo. early onset dementia and. the loss-of-function of trem or its coreceptor dap is responsible for the recessively inherited nasu-hakola disease (also known as plosl). the brains of plosl affected patients show strong microglial activation in the cerebral white matter. reports demonstrate that trem -mediated phagocytic function of microglia is required for debris clearance and cns tissue homeostasis. perinatal brain injury is the underlying etiology for a host of developmental disabilities that includes spastic motor deficits and cognitive, behavioral and learning difficulties. hence, our aim is to characterize the expression of trem in the control of neuroinflammation following hypoxia/ ischemia (hi) in the newborn brain. we performed hi brain damage in postnatal day (p ) c /bl mice by permanent left carotid occlusion and litters were exposed to % of oxygen balanced with nitrogen for minutes in a hypoxic chamber with controlled humidity and temperature maintained at c. pups were then returned to their dam until sacrifice. the age-matched controls and samples from hours to days after hypoxia were collected and processed for immunohistochemistry. in control animals, trem expression was observed in the corpus callosum and in the subventricular zone at p that almost disappeared at p . following hi, an increase in trem staining was observed in the corpus callosum, hippocampus, caudate-putamen, fimbria, cortex and thalamus in the ipsilateral (il) hemisphere, following a similar regional pattern of microglia activation. the increased expression of trem was observed from hours to days post-hypoxia. trem colocalized mainly with microglia markers, such as iba- and cd . oligodendrocyte expression of trem was also analyzed. in conclusion, hi produced an increase in trem expression on the il damaged regions. these results suggest that the modulation of trem might be a possible target for damage control in neonatal brain. increasing evidence shows that astrocytes play a critical role in neuronal protection during an ischemic insult. in the vertebrate retina, most of astrocytic functions are executed by m€ uller glial cells, the major macroglial cell type in this tissue. the aim of this study was to evaluate the role of m€ uller glia in the onset and progression of retinal ganglion cell (rgc) degeneration after acute ischemic injury in the mouse eye in vivo. here, we used the selective gliotoxin fluorocitrate (fc) in order to transiently impair m€ uller glial metabolism at different time points during and after induction of an acute retinal ischemia/reperfusion by means of elevated intraocular pressure (iop). the effect of fc ( mm) on metabolism was determined by measuring retinal glutamine levels, aconitase activity and mitochondrial membrane depolarization after intravitreal injection of the gliotoxin. retinal ischemia was unilaterally induced by elevating iop for min. fc was intravitreally injected h before onset of ischemia and after , or h of reperfusion. saline was injected in the contralateral eye and served as control. surviving rgcs were quantified by means of confocal scanning microscopy and automated cell counting-based analysis on retinal wholemounts days after lesion. fc treatment reduced glutamine levels to - % as compared to control - h after injection. inhibition was completely reversed h after injection. aconitase activity was reduced to $ % from control levels h after fc injection. mitochondrial membrane potential was reduced by - % as indicated by reduced intensity of the specific stain mitotracker red cmxros. one week after ischemia, only % of rgcs survived as compared to unlesioned controls. impairment of m€ uller glial metabolism during ischemia further reduced rgcs by % as compared to ischemia alone. fc treatment did not significantly modify rgcs survival and h after lesion. results support the notion for a critical neuroprotective role of m€ uller glial cells during ischemia. in the acute phase following ischemia, however, m€ uller glia inhibition does not further affect rgcs survival. further research is required to establish the time point for this switch in m€ uller glial function and the underlying mechanisms. extract has been widely investigated in animal models and clinical studies for various diseases including ischemic stroke. its major metabolite, cordycepin, has been reported to act as a selective a adenosine receptor agonist and to protect neurons against ischemic injury. however, their underlying mechanisms remain unclear. in the present study, we report that the standardized c. militaris extract, wib- c, which contains cordycepin % of total dry weight of the extract, markedly reduced ischemic injury by inhibiting post-ischemic inflammatory responses. postischemic treatment with wib- c (orally administered twice at and h after onset of mcao) significantly reduced infarct size and edema in rats subjected to transient middle cerebral artery occlusion (mcao, . h) and subsequent reperfusion ( h). wib- c also significantly improved integrity of glial cells in ischemic lesions, as evidenced by reduced white matter degeneration and loss of blood-brain barrier. importantly, wib- c significantly ameliorated neurological deficits in not only transient but also in permanent stroke models. moreover, wib- csignificantly improved long-term survival of mcao rats over days. as we previously reported with selective a receptor agonists introduction: cd & cd r are immune inhibitory molecules that have been shown to be involved in inducing immune tolerance and in contributing to immune privileged status of the cns. the developing brain exhibits distinct morphological as well as physiological characteristics determining a peculiar response to injury showing an aggravated susceptibility to excitotoxicity and pro-inflammatory cytokines, along with an exacerbated inflammatory response. previous studies from our group have described the expression of cd -cd r in brain during development showing a distinct pattern of expression in the cortex and the hippocampus. hence, the aim of this study is phenotypic characterization of cd r microglia/macrophages and cd neuronal cells following hypoxia/ischemia (h/i) in neonatal mice brain. wild-type c /bl mice postnatal (p) day , , , , , , and adult were used for developmental studies. p mice was used for h/i injury that was administered using vannucci model modified for neonatal mice ( % o , min) and samples were collected h, h, h, h, h & days after hypoxia for immunofluorescence staining. results: phenotypic characterization of cd r microglia/macrophages showed them to express markers of pro-or anti-inflammatory phenotype in a temporal fashion post lesion. they expressed m phenotype as observed by colocalization with cd cells throughout the time points studied but a subpopulation of cd r cells exhibited m phenotype as exhibited by mhcii , cd cells from - hrs post lesion. calretinin (cr) used for the characterization of cd neurons showed that most cr neurons were cd especially the interneurons in the innermolecular layer of hippocampus, layer i of the neocortex and the hilus at most age groups studied. after h/i, cd immunolabeling was increased in the hippocampal fissure until days post-lesion. this followed a change in cd r microglial cells described above. to conclude, a balance between m /m phenotype of cd r microglia/macrophages and expression of cd by cr cells are involved in evolution of h/i induced brain injury in neonatal mice. objective: the cytokine interleukin- (il- ) and its naturally occurring receptor antagonist (il- ra) play a key role in determining neuronal cell death and survival in focal cerebral ischemia. the objective of this study was to determine the cellular production of il- /il- ra, and to test the neuroprotective potential of post-surgically injected il- ra overexpressing bone marrow (bm) cells in a mouse model of focal cerebral ischemia. materials and methods: c bl/ mice were injected i.v. with bm cells isolated from sil- ra overexpressing mice, min after permanent middle cerebral artery occlusion (pmcao). physiological parameters and behaviour were recorded post-surgically, infarct sizes were estimated, and microglial-leukocyte expression of il- /il- ra was analyzed by flowcytometry, in situ hybridization and immunohistochemistry. results: we identify microglia, and not recruited leukocytes as the major producers of il- ra after pmcao in mice, and we show by using il- ra knock out mice that microglial-produced il- ra is neuroprotective. we report that the sil- ra produced by the post-surgically injected bm cells, potentiates the neuroprotective effect of microglialderived il- ra, at both h and days, which is consistent with behavioural improvement at days, and detection of recruited bm cells in the ischemic area . h after pmcao. interestingly, we also find that the sil- ra overexpressing bm cells stimulate microglial production of il- ra h after pmcao, at which time both il- a and il- b is upregulated. conclusion: our results provide proof of principle that increasing the production of il- ra by recruited bm cells can counteract the effect of il- a/b, increase neuronal survival and improve motor function alone or through induction of microglial-produced il- ra after pmcao in mice. bogomoletz institute of physiology, kyiv, ukraine short-term oxygen-glucose deprivation (ogd) is known to activate excitatory glutamatergic synapses, induce long-term potentiation and result in neuronal and synaptical ultrastructural remodelling in organitypic cultured hippocampal slices (ochs). in this work we studied changes in glial synaptic coverage following min ogd episode using d reconstruction of ochs confocal and electron microscopic images. propidium iodide (pi) and mitotracker orange cmtmros (mto) were used to estimate cell viability and their mitochondrial potential. astroglial and microglial cells were identified using immunohistochemical staining with anti-gfap and anti-iba- antibodies. the study demonstrated that astroglial and microglial cells retain viability (there was no significant increase in the amount of pi-labbelled cells). d reconstruction revealed significant increase in excitatory synapses glial coverage as early as hour following ogd accompanied by a significant increase in number of astroglial and microglial processes. the deprivation also resulted in gradual increase of mitochondrial potential from hr to hr in both astroglial and microglial cells. it was previously shown that pyramidal neurons under the same conditions lose mitochondrial potential and became pi-positive after and reoxygenetion, indicating damage of cytoplasmic membrane, the effect prevented by application of nmda receptor antagonist d-ap . thus, unlike neurons astroglial and microglial cells are ogd-resistant and demonstrate marked mitochondrial activation -a process that might be involved in maintaining neuronal function during oxygen-glucose deficiency. using a microfluidic high throughput qpcr platform, we measured the expression of selected genes encoding astrocytic and polydendrocytic markers, ion channels, transporters and receptors that participate in maintaining k and glutamate homeostasis in each of individual gfap/egfp-positive glial cells. in this study we show how the expression of these selected genes changes during development (postnatal days , , and ) as well as , and days after middle cerebral artery occlusion (mcao). self-organizing maps and principal component analyses divided the cells according to their similarity in gene expression into three subpopulations of astrocytes present within the first - days of postnatal development (p -p ). the first subpopulation comprises immature glia mainly from p , characterized by the high transcriptional activity of all of the studied genes, including polydendrocytic markers. the second subpopulation is dominated by cells from p and displayed low transcript levels of all of the studied genes. the third subpopulation represents mature astrocytes mainly from p and p . three, seven and fourteen days after ischemia (d , d , d ), additional astrocytic subpopulations appear: resting astroglia (mainly from p and d ), transcriptionally active early reactive astroglia (mainly from d ) expressing the mrna of polydendrocytic markers, and, presumably, permanent reactive astroglia (solely from d ). following focal cerebral ischemia, reactive astrocytes undergo pronounced changes in their expression of aquaporins, nonspecific cationic and potassium channels, glutamate receptors and markers of reactive astrocytes and polydendrocytes. from the data obtained from single cell pcr analysis, we calculated the spearman correlation coefficients between pairs of genes, which revealed interesting positive gene expression correlations with polydendrocytic markers (gria - , grik - , grin a, kcnj , kcnk and kcnk genes). this study was further supplemented by an immunohistochemical analysis of nmda receptor subunits and pdgfar, which confirmed that changes in gene expressions correlate with immunodetected proteins. ga cr - s, astf - , gauk . reactive astrogliosis is often regarded as universal type of astroglial response to various forms of injuries. among its most characteristic features one may include: glial fibrillary acidic protein (gfap) up-regulation, cellular hypertrophy and proliferation and gliotic scar formation. an important element of glial cells response is their ability of de-differentiation, which is related with their re-gaining of immunological and molecular properties characteristic for earlier developmental forms. the origin of cells proliferating in response to various types of brain injury is still a subject of debate. among others it is related to the pathomechanism of lesion, affected brain structure and developmental stage of the nervous system. aim: in our study we aimed to assess the differences of de-differentiation and proliferation potential of astroglia localized in the cerebral cortex and striatum to the transient focal brain ischemia. material and methods: transient focal brain ischemia was evoked in adult male wistar rats by placement of the monofilament surgical thread into the internal carotid artery and occlusion of the middle cerebral artery for h. the postoperative survival period was up to weeks. immunocytochemical double-staining for astrocytic marker (gfap), developmental (pax ) and proliferative (ki- ) markers was performed and subsequent qualitative and quantitative study was done by means of confocal microscopy. results: double-labeled gfap/pax and gfap/ki reactive astroglial cells were revealed in the cerebral cortex and striatum of the ischemic region, starting from h after initiating the ischemia. the apparent difference in the intensity of morphological changes, quantity of de-differentiating and proliferating astroglial cells was observed between ischemic cerebral cortex and striatum. intensity of astroglial response and proliferative potential were much higher in the striatum than in the cerebral cortex. summary and conclusion: apparent difference in proliferative response between the cerebral cortex and striatum may be consequence of metabolic and molecular differences between astroglial cells populating two mentioned above structures. it may be also consequence of uneven decrease of cerebral blood flow and resulting different metabolic conditions influencing astroglial function in various fragments of ischemic area. reassuming, the differentiated potential of astroglial proliferative response to ischemic changes in different brain regions must be taken into account while analyzing clinical consequences of cerebral infarcts of different localization. triggering receptor expressed on myeloid cells- (trem ) is a microglial surface receptor involved in phagocytosis. clearance of apoptotic debris after stroke represents an important mechanism to re-attain tissue homeostasis and thereby ensure functional recovery. the role of trem following stroke is currently unclear. as an experimental stroke model, the middle cerebral artery of mice was occluded for minutes with a range of reperfusion times (duration of reperfusion: h/ h/ h/ d/ d/ d). quantitative pcr (qpcr) revealed a greatly increased transcription of trem after stroke ($ fold). we subsequently analyzed the expression of proinflammatory cytokines, chemokines and their receptors in trem knockout (trem -ko) mice via qpcr. microglial activation (cd , iba ) and cd -positive t-cell invasion were analyzed via qpcr and immunohistochemistry. functional consequences of trem knockout were assessed by infarct volumetry. the acute inflammatory response ( h reperfusion) was very similar between trem -ko mice and their littermate controls. however, in the sub-acute phase ( d reperfusion) following stroke, trem -ko mice showed a decreased transcription of pro-inflammatory cytokines tnfa, il- a and il- b, associated with a reduced microglial activity (cd , iba ). furthermore, trem -ko mice showed a reduced transcription of chemokines ccl (mcp ), ccl (mip a) and the chemokine receptor cx cr , followed by a diminished invasion of cd -positive t-cells. no effect on the lesion size was observed. conclusions although we initially expected an exaggerated pro-inflammatory response following ablation of trem , our data support a contradictory scenario that the sub-acute inflammatory reaction after stroke is attenuated in trem -ko mice. we therefore conclude that trem appears to sustain a distinct inflammatory response after stroke. metabotropic glutamate receptors (mglurs) are seven transmembrane domain g-protein-coupled receptors for l-glutamate, the main excitatory neurotransmitter in the mammalian brain. in addition, glutamatemediated excitotoxicity plays a central role in mediating neuron and oligodendrocyte death during ischemic episodes. in vitro, developing oligodendrocytes (ols) in particular have been shown to be highly vulnerable to excitotoxicity and oxidative stress. notably, activation of group mglurs has been shown to attenuate ol excitotoxicity in vitro and in brain slices has been shown to reduce ischemia-mediated impairment of astrocytes. here, we have examined the functional expression of mglurs and their role in acute ischemic injury in developing cns white matter of the mouse optic nerve. calcium imaging experiments were performed in optic nerves from p - wild-type mice. optic nerves were isolated intact, loaded with fluo- am and visualized using a zeiss lsm pascal confocal microscope; images were collected in optical z-sections and changes in fluorescence intensity were measured. the application of mglur agonists acpd and dhpg showed that group i mglur mediate a rise in glial [ca ] i . the action of the group i mglur antagonist aida versus acpd indicated that the rise in [ca ] i also involves group ii (and possibly group iii) mglur. immunohistochemistry confirmed glial expression of group i subunit mglur and group ii subunits mglur and mglur in the optic nerve, although further analysis of cellular localization is required. to examine the role of mglurs in white matter ischemia, optic nerves from p - mice in which fluorescent reporters are driven by astrocyte (gfap-egfp) or oligodendrocyte (sox -egfp) genes were isolated intact into artificial cerebrospinal fluid (acsf) and subjected to oxygen glucose deprivation (ogd) for hour. activation of group i or group ii mglur with the agonists acpd, dhpg or ly protected astrocytes and oligodendrocytes from ischemic cell death. further experiments are required to determine the mechanisms involved, but these findings demonstrate functional expression of mglur in optic nerve glia and indicate that activation of mglur is a possible therapeutic strategy to protect against glial damage in response to ischemia. the cerebral cortex is one of the most often affected areas after stroke, but there are no studies comparing the outcome in different cortical regions after focal ischemia. the aim of this investigation was to evaluate the patterns of glial activation, tissue degeneration and neuronal loss in different survival times following cortical ischemia. focal ischemia was induced by stereotaxic microinjections of endothelin- (et- ) into the somatosensory, motor and association cortices of adult rats (n ). the control animals were injected with the same volume of sterile saline (n ). the animals were perfused at , and days after ischemia. gross histopathology was evaluated using cresyl violet staining. lm sections were submitted to immunohistochemistry for astrocytes (anti-gfap), activated microglia/macrophages (anti-ed ) and microglia (anti-iba and ed ). tissue loss and glial activation were more intense in the somatosensory cortex than in other cortical areas. the motor cortex was the second more affected area. the association cortex was the less damaged area, which was confirmed by quantitative analysis (anova-tukey). the results suggest that an ischemic lesion of the same intensity induces a differential pattern of tissue loss and neuroinflammation, depending on the cortical area, and that the primary sensory and motor areas are more susceptible to ischemia than association areas. tlr is required for ipc-induced neuroprotection. in order to elucidate the mechanisms by which microglial tlr contributes to ipc, we carried out cell-targeted genomic analyses specifically on microglia exposed to either ischemic conditions in vitro or ipc in vivo. for our in vitro model, we exposed wt and tlr -/mouse primary microglia to hypoxic/hypoglycemic conditions for hours. for our in vivo model, we carried out transient middle cerebral artery occlusion (mcao) or sham surgery as our ipc pulse on wt and tlr -/adult male mice. three days later mice were sacrificed and microglia acutely isolated from ipsilateral cortex using magnetic affinity chromatography cell separation and ex vivo flow cytometry. microarray analysis was carried out on rna from both in vitro and in vivo microglia. results from both datasets identified robust expression of type and/or type interferon (ifn)-stimulated genes (isgs) as the predominant transcriptomal feature of ischemia-exposed microglia. in vitro, we found that out of the ( %) individual genes that were significantly up-regulated > fold by hypoxia/hypoglycemia in wt, but not tlr -/-, microglia were isgs including mx , oas , irf and ccl . promoter analysis demonstrated that the top four most active transcription factors were isgf , irf , irf and irf ; all ifn regulatory factors (irfs). a similar pattern emerged from the in vivo dataset with multiple irfs again among the most activated transcription factors in sorted microglia from ipcexposed mice. however, unlike the isg response in vitro, the isg response from the in vivo dataset was not tlr -dependent. the ifn family of cytokines is recognized as a key component of the innate immune response to infection and other types of injury. the role of microglial ifn-signaling in ipc is unknown. these results suggest that broad activation of isgs may be a key feature of the microglial response to ischemic conditions. astrocytes respond to central nervous system (cns) injury by the formation of a glial scar that develops within few days after insult and is characterized by astrocyte proliferation and cellular hypertrophy. reactive astrocytes change their immunohistochemical profile, such as increasing expression of gfap, nestin and vimentin; however, they also markedly alter the expression of ion channels, receptors and transporters, which participate in the maintenance of ionic-, water-and neurotransmitter homeostasis, such as k channels, aquaporins or glutamate transporters. here, we aimed to characterize the gene expression profile and functional properties of reactive astrocytes weeks after global cerebral ischemia (gci) or focal cerebral ischemia (fci) with a specific focus on k channels or non-specific cationic channels. gci was induced in adult rats by bilateral, -minute common carotid artery occlusion combined with low oxygen, while fci was induced in adult egfp/gfap mice by permanent middle cerebral artery occlusion. using the patch-clamp, we investigated the membrane properties of astrocytes in situ weeks after gci in the rat hippocampal ca region and weeks after fci in the mouse cortex. regardless of the type of ischemic injury, astrocytes from both cns regions depolarized their membrane potential by $ mv weeks after ischemia. when compared to astrocytes from the non-injured ca region or the cortex, the post-ischemic astrocytes displayed large hyperpolarizationactivated time-and voltage-dependent, non-inactivating inward currents, the current density of which increased -fold in response to gci or fci. in addition, these non-specific cationic channels were sensitive to zd and to a low extracellular na concentration, suggesting that they may belong to the family of hyperpolarization-activated cyclic nucleotide-gated (hcn) channels. we have taken advantage of fluorescently labeled astrocytes in egfp/gfap mice and isolated egfp cells by facs from non-injured as well as ischemic regions and performed gene expression profiling using single-cell rt-qpcr. our data revealed that cortical astrocytes after fci express, besides the increased expression of gfap, gfapd, aqp and , extremely high levels of hcn - transcripts that encode hcn - channels. moreover, our immunohistochemical analyses confirmed the presence of hcn - in reactive astrocytes, especially hcn , and channels. in summary, our results show that reactive astrocytes from post-ischemic tissue express hcn channels and that their activity might significantly contribute to na influx, possibly maintaining atpase function and/or contributing to intracellular na -dependent events, such as glutamate transporter or na /ca exchanger reversal. ga cr - s, p / /g and gauk . the optimal operation of electrogenic astrocytic transporters and exchangers for some well-defined astrocyte brain homeostatic functions depends on the presence of k channels in the cell membranes and the hyperpolarized membrane potential. our previous study showed that astrocytes functionally express two-pore domain k channel trek- , which helps to set the negative resting membrane potential. however, the roles of trek- on astrocytic function under normal and ischemic conditions remain unclear. in this study, we investigated the effects of trek- activity on astrocytic function and neuronal death under ischemic conditions. in an in vitro hypoxia model with astrocytes culture, trek- immunoreactivity was up-regulated after hypoxia. suppression of trek- activity inhibited the glutamate clearance capability, enhanced the inflammatory secretion of astrocytes derived s b and led to increased neuronal apoptosis after ischemic insult. after middle cerebral artery occlusion induced focal ischemia in rats, the trek- expression was significantly increased which correlated with reactive astrogliosis. application of lineonic acid, a trek- agonist which could potentiate the astrocytic conductance and hyperpolarization membrane potential, up-regulated the expression of astrocytic glutamate transporter glt- , inhibited the micro-glial inflammatory response and attenuated neuronal damage. our results suggest that trek- activity is involved in astrocytic function and neuronal survival. this would provide evidence showing astrocytic trek- involvement in ischemia pathology which may serve as a potential therapeutic target in stroke. oligodendrocytes are the myelinating cells of the central nervous system (cns). they are responsible for ensheathing myelin layers around axons, which contributes to improve conduction of neuronal impulses and additionally provides axonal support. oligodendrocytes are generated by oligodendrocyte precursor cells (opcs), a self-renewing and proliferating adult stem/precursor cell population in the cns. this process occurs not only during developmental myelination but also mediates cns remyelination, a regenerative process that restores myelin sheaths to demyelinated axons. although energy metabolism in the oligodendrocyte lineage is thought to be very active to support the lipid production specific to this cell type, it is still poorly characterized. as a first step towards a detailed characterization of the energy metabolism associated with specific lineage stages we investigated how glucose -the main cerebral energy substrate -is metabolized at an immature opc stage and in differentiated oligodendrocytes. primary cultures of opcs and oligodendrocytes were incubated with medium containing [ , - c]glucose for h and metabolites in both cell extracts and culture medium were collected and analysed for excess % c enrichment using gas-chromatography-mass spectrometry (gc-ms) as well as for total amounts using hplc. the significant c-enrichment in citrate, malate, and aspartate in cell extracts indicate that oligodendrocytes oxidize glucose-derived metabolites extensively in the tricarboxylic acid (tca) cycle. moreover, they release c-labelled aspartate and glutamate to the extracellular medium, although a significant consumption of aspartate, glutamate and glutamine from the medium was also observed. enrichment of extracellular lactate and increased alanine synthesis in oligodendrocytes as compared to opcs suggests that the rate of aerobic glycolysis is increased in immature as compared to differentiated cells. in conclusion, these data confirm that both opcs and oligodendrocytes are highly metabolically active and, in addition to extensively oxidizing glucose, synthesize and release distinct metabolites. we anticipate that further studies on this subject will contribute to a better understanding of the physiological role of oligodendrocytes in the cns and the role of glycolysis and mitochondrial metabolism during their maturation. schwann cells in the peripheral and oligodendrocytes in the central nervous system require a tremendous amount of lipids to be capable of producing all cell membranes forming the myelin structure. furthermore, when compared to other cells, myelin membrane presents an extremely high lipid content ($ % of dry weight) coupled to an unique lipid composition. the importance of myelin lipid composition towards myelin formation and maintenance is highlighted by the frequent appearance of myelin defects in lipid-metabolism human disorders. one of the main represented class of lipids in myelin are phospholipids, of which fatty acids (fas) represents the building blocks. most cells preferentially uptake circulating free fas. however, cells under increased membrane production, e.g. highly proliferating cells, can endogenously produce them through the enzyme fatty acid synthase (fasn), which synthesize palmitate. in these cells, fasn is transcriptionally augmented via the mtorc pathway. thus, we hypothesized that due to their increased lipid demand myelinating cells would require fasn activity towards myelin production. the functional role of fasn in the metabolic control of myelin membrane lipid composition and myelination is unidentified. thus, we addressed it experimentally by conditionally deleting fasn in myelinating cells in vivo. we show that endogenous fas biosynthesis in myelinating cells is essential to maintain myelin membrane lipid homeostasis. furthermore, maintaining a correct lipid homeostasis is critical for myelination. thus, we analyzed whether fasn is required to allow palmitoylation of myelin proteins and their targeting to the myelin membrane. in addition, we show that interfering with myelin fas homeostasis results in the activation of systemic compensating metabolic loops promoting fas uptake, although not sufficient to preserve efficient myelination. taken together our data demonstrate that in myelinating cells fas homeostasis is under the synergistic control of endogenous metabolic fas production via fasn and systemic functional regulation of free fas uptake. most importantly, maintaining a correct homeostasis of fa-derived lipids in myelin is critical for myelination. homeostasis and signaling, respectively ( ). in mammals, two cytosolic and two mainly mitochondrial located glutaredoxins have been identified ( ). we already described an essential role for the formation of a functional neuronal network during embryonic development of one of these proteins, the vertebrate specific glutaredoxin ( ). here, we will focus on the contribution of glutaredoxin on basic cellular functions of oligodendrocytes, the myelinating cells of the central nervous system, under physiological and pathophysiological situations, e.g. multiple sclerosis, which is characterized by inflammatory axonal demyelination ( ). oligodendrocyte progenitors and mature oligodendrocytes are very sensitive against oxidative stress induced by release of nitric oxide by activated microglia ( ) . using a b positive glia restricted progenitor cells as well as organotypic cerebellar slice cultures, we found that increased levels of glutaredoxin protect against cell death induced by either activated microglia or s-nitrosoglutathione, a physiological nitric oxide donor. under physiological conditions elevated levels of glutaredoxin increased migration of a b positive cells nearly three fold. western blot analyses, quantitative real time pcr, as well as immunocytochemistry revealed that glutaredoxin inhibited further differentiation of ng positive oligodendrocyte progenitor cells. in summary, our data indicate the importance of specific thiol redox signaling during cellular protection and function in this type of glia cells. current multiple sclerosis therapeutics act as immunomodulators and/ or immunosuppressors. this strategy is largely ineffective at preventing progression of the disease. significant benefits to multiple sclerosis patients might be seen with a therapeutic agent which induces remyelination, however, to date no such agents have been developed. using in vitro and in vivo models we have identified nefiracetam as a potential remyelinating therapeutic. to study remyelination in vitro, hippocampal organotypic cultures were exposed to lysophospatidylcholine (lpc) for hrs to induce demyelination before being allowed to recover. cultures were treated with nefiracetam for hrs of this recovery period. in all cases immunofluorescent labelling of myelin basic protein (mbp) was used as a measure of myelin. nefiracetam was shown to increase mbp expression, relative to untreated controls, accelerating recovery from lpc. two different models were used to investigate the efficacy of nefiracetam in vivo; an inflammatory model, experimental autoimmune encephalomyelitis (eae), and a toxin induced model of demyelination, achieved through the use of cuprizone chow. nefiracetam treatment did not alleviate the symptoms of eae. however, nefiracetam did restore the amount of myelin in cuprizone exposed animals, compared to saline-treated controls. in combination, these observations suggest that nefiracetam is capable of accelerating remyelination through a mechanism of action which is independent of immunomodulation. we conclude that nefiracetam encourages remyelination both in vitro and in vivo. these pro-myelin properties identify nefiracetam as a potential therapeutic for demyelinating disorders. university of naples "federico", neuroscience, naples, italy changes in intracellular [ca ] i levels have been shown to influence the developmental processes that accompany the transition of human oligodendrocyte precursor cells (opcs) into mature myelinating oligodendrocytes and are required for the initiation of myelination and remyelination processes. in the present study, we explored whether calcium signals mediated by the selective sodium calcium exchanger (ncx) family members ncx , ncx , and ncx , play a role in oligodendrocyte maturation. functional studies, as well as mrna and protein expression analyses, revealed that ncx and ncx , but not ncx , were divergently modulated during opc differentiation into oligodendrocyte phenotype. in fact, while ncx was down-regulated, ncx was strongly up-regulated during the oligodendrocyte development. the importance of calcium signaling mediated by ncx during oligodendrocyte maturation was supported by several findings. indeed, whereas the knocking down of the ncx isoform in opcs prevented the up-regulation of the myelin protein markers cnpase and mbp, its overexpression induced an up-regulation of cnpase and mbp. furthermore, ncx knock-out mice exhibited not only a reduced size of spinal cord but also a marked hypomyelination, as revealed by the decrease in mbp expression and by the accompanying increase in opcs number. collectively, our findings indicate that calcium signaling mediated by ncx plays a crucial role in oligodendrocyte maturation and myelin formation. to explore the role of kif b in myelination in vivo, we generated conditional knock-out mice with kif b specific ablation in either schwann cells or oligodendrocytes. we found that kif bfl/fl p cre mouse nerves are hypomyelinated with reduced myelin thickness, thus confirming in vitro data. in the pns, axonal neuregulin (nrg) type iii is one of the main signaling promoting myelination and remyelination after injury. nrg type iii binds to erbb /erbb receptors in schwann cells, which signal through a plethora of downstream signaling pathways also including the pi k/akt complex. the pi k/akt signaling pathway is attenuated by the pten phosphatase, which dephosphorylates ptdins( , , )p (s. goebbels et al., ). dlg is believed to interact with pten and potentiate its ability to dephosphorylate ptdins( , , )p , thus negatively modulating the akt-mtor pathway (l. cotter et al, ). as kif b interacts with dlg in schwann cells, we investigated the pi k/akt pathway in kif b-null nerves and surprisingly, we found that dlg expression levels are increased whereas akt phosphorylation is decreased in mutant nerves. our findings suggest that kif b negatively regulates dlg activity and acts as a promoter of myelination in the pns. on the contrary, when kif b is lost specifically in oligodendrocytes, myelin sheaths are thicker. at the molecular level, the hypermyelination is consistent with increased akt phosphorylation. we are currently investigating the molecular mechanisms at the basis of the kif b/dlg interaction in both schwann cells and oligodendrocytes. we have identified the nootropic nefiracetam as a potential novel remyelinating therapeutic using in vivo and in vitro models of demyelination. in this study we investigate the mechanism of action of nefiracetam in the context of remyelination as regulated by wnt/b-catenin signalling and spontaneous activity. to investigate whether nefiracetam acts by modulating wnt signalling, organotypic hippocampal cultures were exposed to nefiracetam or the wnt/b-catenin inhibitor cardamonin. both increased immunofluorescent staining for the opc marker ng . myelin formation, as assessed by mbp staining, was enhanced by nefiracetam but was not affected by cardamonin. thus, modifying wnt signalling appears to impact opc development without effecting differentiation, suggesting the pro-myelinating effects of nefiracetam involve an additional mechanism. we next investigated the effect of nefircatam on opc differentiation using purified opc cultures. nefiracetam increased the ol:opc ratio, as assessed by immunofluorescent staining for mbp and ng respectively, suggesting nefiracetam is capable of promoting ol maturation through a direct action on opcs. finally, we investigated the effect of nefiracetam on spontaneous activity. organotypic hippocampal cultures were exposed to lysophospatidylcholine (lpc) for hrs to induce demyelination before being allowed to recover. cultures were treated with nefiracetam for hrs of the recovery period. spontaneous calcium activity was then measured using fluo -am. cultures not exposed to lpc displayed low levels of activity which were unaffected by nefiracetam. lpc exposure caused an increase in activity which was enhanced by nefiracetam. additionally, in these cultures, lpc decreased mbp staining compared to control while nefiracetam post-lpc caused a return to control levels. these data suggest increased activity may be an adaptive response related to myelin repair which is enhanced by nefiracetam. together, these findings suggest a complex mechanism of action for nefiracetam as a remyelinating agent involving modulation of wnt signalling, spontaneous activity and opc differentiation. in an assay in which cortical oligodendrocytes ensheath dorsal root ganglion cells we found that nrg and nmda receptors interact to regulate myelination. without nrg, blocking nmda receptors had no effect on myelination. adding nrg increased myelination at all timepoints analysed ( - weeks), suggesting that nrg did not merely accelerate myelination. strikingly, nrg led to the majority of myelination becoming dependent on activation of nmda receptors and action potential activity. nrg's effect was associated with a -fold increase in nmda receptor current in oligodendrocyte lineage cells. thus, nrg switches myelination from a default programme, which is independent of neuronal activity, to a mechanism that is regulated by glutamate released from active axons. the effects of nrg were associated with an increase in the phosphorylation of akt and the transcription factor creb, but not associated with changes in the phosphorylation of erk. these data reveal a function for oligodendrocyte nmda receptors. the absence of nrg in multiple sclerosis lesions, and enhanced remyelination by added nrg, suggest a role for neuregulin/nmda receptor dependent remyelination after pathology. low-density lipoprotein receptor-related proteins (lrps) are endocytic receptors that internalize several ligands and are involved in lipoprotein homeostasis in adult brain. lrp- , also known as megalin, is involved in the development of the central nervous system (cns) and in the transport of molecules across the blood brain barrier (bbb). in a previous work, our group revealed that megalin is selectively involved in sonic hedgehog-mediated effects on oligodendrocyte precursor cell migration and proliferation during the development of the cns. although there are some reports describing the presence of lrps in acute lesions of multiple sclerosis (ms) patients and pointing to an important role of shh in the pathogenesis of this disease, there are not data about the expression of megalin in ms tissue. in the present study, we analyse the distribution and the cellular characterisation of megalin in samples of human brain from ms patients to elucidate the role of this receptor in the pathogenesis of demyelination. changes in megalin distribution parallel the different ms histopathological lesions: we detected megalin in active demyelinating lesions and in the periplaque of chronic-active lesions, areas where remyelination spontaneously occurs. on the contrary, megalin was absent in the demyelinated region of chronic lesions. in addition, we observed the up-regulation of megalin in a subpopulation of perivascular astrocytes within the normal appearing grey matter (nagm) of ms patients. moreover, megalin was up-regulated in blood vessels within active lesions, in the periplaque of chronic-active lesions and in the nagm. in parallel, we also analysed the bbb profile to determine structural and functional alterations. altogether, we suggest that megalin is an important receptor expressed in diverse areas of the cns of ms patients representing different aspects of ms development: i) a potential target to improve remyelination; ii) an indicator of a functional rather than a structural disruption of the bbb. therapeutic strategies to modulate specific lrps would be useful to effectively treat ms. bdnf is known to promote central nervous system myelination both in vitro and in vivo. adopting in vitro myelination assays, we identified that bdnf acts through oligodendroglial-expressed trkb receptors to promote myelination. this was verified in vivo, as deletion of trkb in mature oligodendrocytes (in trkb fl/fl mbp-cre mice) resulted in a hypomyelinating phenotype during development. question: here we investigate the consequence of trkb deletion in oligodendrocyte progenitor cells (in trkb fl/fl cnpase-cre mice) and the signalling pathways downstream of trkb that promote myelination. methods and results: trkb fl/fl cnpase-cre mice were born in mendelian ratios and were phenotypically indistinguishable from littermate control mice. surprisingly, analysis of myelinated axonal tracts in the spinal cord and optic nerve revealed normal myelin development. in addition, the number of cells in the oligodendroglial lineage was the same as control mice during postnatal development. these data indicate that the timing of trkb deletion in the oligodendroglial lineage is critical, as deletion in opcs results in no significant phenotype, whereas deletion in mature oligodendrocytes results in a hypomyelinating phenotype. these data suggest that opcs are able to compensate for the loss of trkb and myelinate normally in the absence of bdnf signalling. our in vitro data have shown that the promyelinating influence of bdnf strongly correlates with erk / activation. here we show that overexpression of erk in opcs exerts a more significant promyelinating effect than erk . as erk / are known to exert some of their effects through direct transcription factor phosphorylation, we have undertaken an in silico analysis of myelin-specific transcription factors and identified several that contain potential erk / binding domains and phosphorylation sites. co-immunoprecipitation experiments show an interaction between erk / and these transcription factors. investigation into the nature of these interactions and their functional consequences are ongoing. conclusions: this work suggests a novel role for erk / signalling within oligodendrocytes that regulates cns myelination, possibly via activation of myelin-specific transcription factors. as erk / is activated by multiple receptors, other receptors could potentially compensate for the loss of trkb in opcs, resulting in a normally myelinated cns in vivo. myelin is vulnerable to impairment by genetic, toxic, and immunological triggers, but the molecular basis for many aspects of myelin pathology has remained unknown. to identify molecules required for the structural integrity of central nervous system myelin we have systematically analyzed its protein composition by quantitative proteome analysis. based on the high abundance of filament-forming septins in myelin, we hypothesized that septin filaments may constitute a cytoskeletal membrane cortex in myelin. we find that septin filaments localize to the non-compact adaxonal myelin compartment, which is considered relevant for the metabolic maintenance and the turnover of myelin. for functional analysis, we generated mice lacking the most abundant septin of myelin, sept , from myelinating glia. our results indicate that sept , sept , sept and sept multimerize with sept and that this interaction is essential for their incorporation into myelin. myelin lacking septin filaments display pathogenic outfoldings emerging from adaxonal myelin. we propose that this phenotype reflects that myelin septins provide long-term structural integrity to the adaxonal compartment of cns myelin. here, we used primary opc cultures to investigate the mechanisms underlying gpr endogenous regulation. this is quite important since gpr is upregulated at injury sites in both a demyelinating in vivo model (boda et al., glia, ) and in multiple sclerosis (ms) patients (chen et al., nat neurosci ), which, could, in turn, cause defective myelination. in line with these findings, we have recently shown that gpr forced over-expression at late differentiation stages, obtained by transfecting cultured opcs with a gfp-gpr fusion vector, indeed impairs terminal cell maturation. this suggests that the receptor needs to be down-regulated/ desensitized to allow opc terminal maturation. physiologically, gpr downregulation may occur through agonist-induced receptor phosphorylation via g-protein coupled receptor kinases (grks) (daniele et al., j pharm exp ther, ; frantangeli et al., j biol chem, ), which are, in turn, controlled by mtor kinases, and are altered in ms. our in vitro data show that rapamycin, an inhibitor of mtor kinase, reduces grk levels, with parallel increases in gpr expression and strong impairment of opc maturation. globally, these data suggest that dysregulation of these interconnected pathways leading to aberrant gpr overexpression may prematurely block opc maturation. analysis of gpr in vivo in ms models will confirm if the receptor is dysregulated during disease development and will help finding ways to re-establish myelin repair in demyelinating diseases. loss of central nervous system myelin, and the failure of remyelination by oligodendrocytes, contributes to the functional impairment that characterizes neurodegenerative disorders and demyelinating diseases such as multiple sclerosis (ms). since incomplete remyelination will irreversibly damage axonal connections, treatments effectively promoting (re)myelination are pivotal in precluding progression of disease. although the reasons for remyelination failure are likely multifactorial, one of the major impediments to successful remyelination is the altered lesion microenvironment. our previous findings suggest that fibronectin aggregates, as an environmental factor, contribute to remyelination failure. here, we aim at elucidating whether exogenous gangliosides, i.e., cell surface lipids with a potential to modulate fibronectin-cell surface interactions, could overcome fibronectin-mediated inhibition of myelination. of the gangliosides tested, only addition of gd a, perturbed adherence of oligodendrocyte progenitor cells to fibronectin, but not to poly-l-lysine, without affecting cell viability. gd a slightly increased growth factor-mediated proliferation on fibronectin, but in the presence of the lipid migration of oligodendrocyte progenitor cells on fibronectin was reduced. furthermore, addition of gd a increased myelin-like membrane formation on (aggregated) fibronectin, whereas gm reduced oligodendrocyte differentiation, as reflected by a decrease in the number of mbp positive cells. most interestingly, gd a counteracted the myelination inhibiting effect of aggregated fibronectin in co-cultures of dorsal root ganglion neurons and oligodendrocytes. also, gd a enhanced remyelination in demyelinated cerebellar slice cultures that contained fibronectin aggregates. taken together, gd a is able to overcome the inhibiting effect of aggregated fibronectin in remyelination. given the persistent presence of these aggregates in ms lesions, gd a might therefore act as a potentially novel molecular tool to selectively modulate the impeding signaling environment, apparently detrimental to remyelination. its mechanism of action and exploration of gd a's potential in the treatment of ms, are currently investigated. in the peripheral nervous system (pns) it is secreted by schwann cells and fibroblasts and its expression is strongly induced upon injury. we are interested in the function of apod in the molecular events that take place after a pns injury. we have studied in vivo the process of wallerian degeneration following sciatic nerve crush and we have assayed ex vivo the phagocytosis of labelled myelin from wt and apod-ko mice by flow cytometry. the analysis of cellular processes and protein or mrna expression of a group of signalling molecules induced by injury shows that the lack of apod results in an exacerbated mcp- and tnfa-dependent macrophage recruitment. at the injury site, free aa decreases in the wt. lack of apod results in higher basal levels and a stronger injurytriggered depletion of aa. control by apod of the availability of aa to produce the lipid mediators involved in macrophage recruitment is therefore a key mechanism that conditions both wallerian degeneration and injury resolution later on. on the other hand, the phagocytosis of apod-ko cns myelin is less efficient than the phagocytosis of wt myelin. these results indicate that there are also genotype-dependent differences in myelin composition and/or in the interaction between myelin and macrophages. an electron microscopy analysis of crude myelin preparations reveals that the cns myelin from apod-ko mice has abnormal periodicity and shows defective myelin compaction. lipid analysis of apod-ko myelin shows altered phospholipid composition, particularly in phosphoinositid species. a study of the function of apod in the myelination process is currently underway, our results demonstrate that apod function is relevant for both, myelin membrane properties that influence myelin-macrophage interactions, and the control of the lipid-mediated signalling events controlling the extent of macrophage recruitment. adhesion g protein-coupled receptors (ad-gpcrs) are a subgroup of gpcrs that facilitate cell-cell and cell-matrix interaction. structurally, they differ from other gpcrs by the presence of an extremely larger extracellular n-terminal region that is separated from the -transmembrane region by a gpcr autoproteolysis-inducing (gain) domain that in most ad-gpcrs facilitates an auto-catalytic process to produce an nand c-terminal fragments during the maturation process. gpr is a member of the ad-gpcr family and its mutations are responsible for a human brain malformation known as bilateral frontoparietal polymicrogyria (bfpp). individuals with bfpp present with changes in the white matter tracts in the form of hypomyelination, in addition to cortical malformation. here, we show that gpr knockout mice and zebrafish exhibit a reduction of myelin-specific protein expression, fewer myelinated axons and decreased number of mature oligodendrocytes in the central nervous system (cns). during cortical development, gpr functions together with its ligand collagen iii to regulate neuronal migration and cortical lamination. however, it is not clear whether collagen iii is also the ligand of gpr during cns myelination. gpr has a very long and poorly characterized n-terminal fragment, allowing for the possibility of multiple binding partners that mediate cell-extracellular matrix interactions. indeed, gpr also binds to tissue transglutaminase (tg ) in melanoma cells. we have begun to study the possible role of collagen iii and tg in oligodendrocyte development and cns myelination. the likelihood of these two proteins as being the ligand(s) of gpr during oligodendroglial development will be discussed as will our ongoing work to define the role of gpr in cns myelination. proper control of cell cycle, proliferation and differentiation is fundamental to regulate oligodendrocyte (ol) development and recruitment of oligodendrocyte precursors (opc) after brain damage. failure in this process results in severe dysmyelinating disorders and defective brain repair. the molecular machinery that couples differentiation to proliferation withdrawal plays therefore a critical role in brain development and repair and is regulated by multiple extracellular cues including extracellular matrix (ecm) and growth factor derived signals. however, how these signals are integrated into ol to control cell cycle progression and differentiation is only partially understood. we are investigating the role of jun activating binding protein (jab ), an intracellular signalling molecule, shuttling between nucleus and cytoplasm, involved in the control of cell proliferation, survival and gene transcription. recent evidence showed that jab function is regulated by ecm and growth factors. we demonstrated that jab is expressed in glial cells in peripheral and central nervous system (cns), and conditional inactivation of jab in schwann cells results in dysmyelinating neuropathy. here we present preliminary data showing that jab plays a role in cns development. mice with conditional inactivation of jab in ol, by cnp-cre tansgene, show cns hypomyelination, including corpus callosum, optic nerve and spinal cord. concomitant fiber degeneration was observed. our preliminary data suggest that jab expression in ol plays a role in cns myelination and possibility axonal survival. c. gonsior, j. trotter university of mainz, mainz, germany myelin basic protein (mbp), a major component of central nervous system (cns) myelin, is essential for oligodendroglial function and myelination in the cns and its expression is tightly regulated in time and space. the mrna of mbp is sorted to cytoplasmic rna granules and transported up to the distal processes of oligodendrocytes in a translationally silenced state. we and others could show that, dependent on axonal signals, activation of the non-receptor tyrosine kinase fyn leads to a release of translational inhibition allowing synthesis of mbp protein at the axon-glial contact site. a key factor in this pathway is the fyn target and rna-binding protein heterogeneous nuclear ribonucleoprotein (hnrnp) a , that recognizes the a response element (a re) in the mbp utr and orchestrates the dynamics of the mbp mrna granule. moreover, we identified hnrnp f as an additional target of fyn kinase present in these ribonucleoprotein complexes and contributing to posttranscriptional regulation of mbp. cell stress conditions, as present in various disease states such as multiple sclerosis, may result in the formation of cytoplasmic rna-containing stress granules (sgs), potentially influencing the fate of myelin transcripts including their translation.mbp mrna was shown to be sorted to these cytoplasmic foci. we here identified the hnrnps a and f to be associated with oligodendroglial sgs. de-regulation of such factors involved in mbp mrna metabolism could affect stress dependent synthesis of mbp and thus (re-)myelination and myelin maintenance. we performed the experiments in biozzi abh mice known to develop a chronic relapsing-remitting form of eae following immunization with myelin oligodendrocyte glycoprotein. our findings suggest that in demyelinating lesions, myelin layers are pulled off from the myelin sheath and form bulb-like structures that we call "myelinosomes". we can detect the formation of such structures in acute and in chronic stages in the animal model, as well in biopsies from multiple sclerosis patients. we are currently investigating the molecular interactions that underlie the formation of myelinosomes to gather further insight into the mechanisms that underlie immue-mediated demyelination. adenosine is a potent neuromodulator which can be released from most cells and its extracellular concentration increases dramatically after brain injury. it acts on four g protein-coupled receptors, a , a a , a b and a , and their activation is involved in myelination as well as in apoptosis linked to neurodegenerative diseases. in this study, we initially found that rat oligodendrocytes in vitro express all four adenosine receptor subtypes, adenosine transporters ent and ent and the adenosine degrading enzymes adenosine deaminase and adenosine kinase. activation of adenosine receptors caused mitochondrial dysfunction and oligodendroglial apoptosis. notably, selective activation of adenosine receptors revealed that a receptors are the main subtype mediating oligodendrocyte death. thus, a receptor agonist -ci-ib-meca caused a robust increase in ros levels, mitochondrial membrane depolarization and caspase-dependent apoptosis. in turn, selective a receptor activation induced mapk modifications compatible with cellular damage, which was prevented by the selective antagonist mrs . the modulation of g protein and adenylate cyclase suggested that -ci-ib-meca acts via camp-pka. moreover, selective activation of a receptor triggered intracellular calcium mobilization via plc pathway. finally, we used cerebellar organotypic slices and optic nerve ex vivo to investigate adenosinemediated oligodendroglial death in more integral preparations. consistent with data in dissociated cultures, a receptor activation induced a significant damage in the optic nerve as well as in the cerebellum white matter, an effect that was prevented by caffeine, an adenosine receptor blocker. together, these results indicate that adenosine can trigger oligodendrocyte death via activation of a receptors, and suggest that this mechanism may contribute to the etiology of demyelinating diseases. question-one of the commonest forms of inherited neuropathy, xlinked charcot marie-tooth disease (cmt x) leads to progressive distal muscle weakness and sensory loss. the disease is caused by a large number of mutations in the gjb gene encoding the gap junction protein connexin (cx ). cx is expressed by schwann cells in peripheral nerves and forms important channels of communication between the layers of the non-compact myelin sheath and is necessary for the homeostasis and survival of the myelinating cells and the axon. transgenic disease models generated in our lab as well as clinical-genetic analysis of large number of patients have demonstrated that most cmt x mutations result in loss of cx function and failure to form gap junctions. cx ko mice offer a well characterized and relevant model of the disease to study future therapies for cmt x, for which gene replacement may be an effective treatment strategy. methods-for this purpose, we have generated a novel rd generation lentiviral vector to allow gene delivery to peripheral nerves. in this vector, the cx open reading frame is placed under the control of cell-specific promoter for schwann cells, the rat mpz/p promoter. the expression cassette also includes a green fluorescent protein (egfp) as a marker. we have successfully produced lentiviral vector particles and delivered them to the sciatic nerves of -month old wild type (wt) and cx ko mice, immediately distal to the sciatic notch. the expression of egfp on wt background and of cx on ko background was determined by western blot analysis and immunohistochemistry of teased nerve fibers at different time points after injection. results-our initial studies in wt mice (n ) show that more than % of schwann cells from different nerve fascicles expressed egfp as far as mm distal to the injection site. expression started a week after the intraneural injection and remained stable for up to months. when the lentiviral construct was injected into the sciatic nerves of cx ko mice (n ), virally delivered cx was expressed and was properly localized at the non-compact myelin areas including the paranodal regions and incisures of myelinated fibers, where gap junctions are normally formed. poster abstracts s glia conclusions-thus, we have generated lentiviral vector that leads to efficient gene expression specifically in schwann cells. these results indicate that gene replacement therapy is feasible and may lead to correction of the gap junction loss in myelinating cells of the pns. perinatal inflammation is intensively discussed to have a long-term impact on various cell populations and on the development of autoimmune diseases such as multiple sclerosis (ms) in adulthood. in order to determine long-term effects of perinatal inflammation on the course of demyelination in adulthood we mimic a bacterial inflammation by exposure to lipopolysaccharide (lps) of either pregnant or newborn mice. demyelination as a "second hit" was induced by feeding adult mice with the oligodendrocyte toxin cuprizone (bis-cyclohexanone oxaldihydrazone). a single prenatal lps injection at e . did not affect the course of demyelination in adulthood. in contrast, serial postnatal lps injections lead to a delayed demyelination and a reduced number of activated microglia in the corpus callosum, suggesting a long-lasting effect of perinatal inflammation on the function of microglia. surprisingly, mice exposed to lps after birth showed an enhancement of early remyelination accompanied by an increased number of newly differentiated oligodendrocytes. furthermore, independent of cuprizone administration the perinatal lps injections seem to induce a long-term impact on the blood-brain barrier (bbb) by decreasing the number of claudin- positive vessels. in summary, perinatal inflammation mimicked by the administration of lps has long-lasting effects on the bbb, microglia activation, and remyelination. question: epigenetic control is crucial for the differentiation of a variety of cells including oligodendrocytes, the myelinating glial cells of the central nervous system. however, studies about the implication of epigenetic factors in peripheral nervous system maturation are just emerging and we were wondering whether methylation of histones is involved in this process. methods: we describe the function of ezh in primary schwann cells and drg cocultures using immunohistochemistry, transfection and lentiviral transduction, shrna, qrt-pcr analysis, morphological analysis, western blotting and chromatin immunoprecipitation. results: here, we demonstrate for the first time the impact of a histone methyltransferase, encoded by the enhancer of zeste homolog (ezh ) gene, on schwann cell differentiation. in sciatic nerves, ezh expression was found in schwann cells and to peak perinatally. suppression of ezh expression in cultured primary rat schwann cells reduced the length of cell processes. these morphological changes were accompanied by widespread alterations in the gene expression pattern, including downregulation of myelin genes and induction of p kip , which we have recently identified as an intrinsic inhibitory regulator of schwann cell maturation. in addition, we show that ezh suppression in dorsal root ganglion cocultures interferes with in vitro myelination. chromatin immunoprecipitation analysis revealed binding of ezh at the p kip promoter and reduction of histone h k trimethylation upon gene suppression. ezh suppression-dependent effects on morphology and myelin genes could be reversed by concomitant suppression of p kip , indicating that p kip is a downstream effector of ezh . furthermore, we describe hes as transcriptional repressor of myelin genes in schwann cells, which was induced upon ezh suppression and downregulated in p kip -suppressed schwann cells. conclusions: we have identified a molecular link between histone methylation and control of schwann cell differentiation and demonstrate that this epigenetic mechanism is crucial for glial differentiation to proceed. previous studies, performed primarily in cell culture, suggest that electrical activity may regulate multiple stages of oligodendrocyte development including proliferation, migration, initial wrapping and myelination. in this study, we have examined the requirement for electrical activity in oligodendrocyte development in vivo using zebrafish as a model system. results: we find that electrical activity is not required for proliferation, migration, or the initiation of axon wrapping. rather, our data indicate a specific role for electrical activity in oligodendrocyte differentiation by regulating a subset of myelin genes, including myelin basic protein (mbp). silencing electrical activity with tetrodotoxin reduced mrna expression of mbp, as assessed by rna in situ hybridization and quantitative rt-pcr. in contrast, myelin protein zero, claudin k, claudin a, ugt , and k levels were not regulated by electrical activity. ongoing experiments are aimed to uncover the mechanism of activity-dependent mbp mrna expression during oligodendrocyte differentiation. conclusions: taken together, these data indicate that electrical activity can modulate myelin gene expression in vivo but do not support the notion that electrical activity drives key axon-glia messengers that are required for the initial stages of myelination. (fancy et al., ) . eight-week-old lgals -/-and wild type (wt) mice were fed a diet containing . % cpz w/w during weeks, after which cpz was withdrawn in order to evaluate remyelination weeks after. according to our results, cpz-induced demyelination in lgals -/mice showed an exacerbated astrocytic and microglial response as compared to wt littermates. electron microscopy showed a significant lack of myelinated axons in lgals -/-mice as compared to controls. furthermore, the few myelinated axons present were nearly % less myelinated than those of controls and were found to be collapsed. remarkably, the remyelination process seemed to be faster in lgals -/-mice than in wt. remyelinated lgals -/-mice showed a higher myelin basic protein (mbp) recovery rate as compared to their controls. flow cytometry assays showed a sharper microglial response in lgals -/-mice, which was supported by an exacerbated number of cd b and cd cells. however, electron microscopy images from remyelinated lgals -/-animals showed, again, collapsed axons with a defective myelin wrap, as compared to wt mice showing normal axons without any relevant myelin wrap disruption. behavioral performance observed during cpz treatment recovery correlates with alterations in the morphological studies, which show that neither lgals -/-nor wt mice reach basal myelination levels. we have shown that new oligodendrocytes (ols) continue to be generated in healthy adult mice after months of age, even in the optic nerves, which are considered to be completely myelinated already [ ] . this raises the question of whether the adult-born ols and myelin are engaged in remodeling existing myelin (e.g. intercalating among the existing myelin sheaths), or replacing ols that die in use. to ask whether ols die and are replaced during healthy adulthood we generated transgenic mice that express icreer t under the transcriptional control of the myelin basic protein promoter (mbp -icreer t ). the $ kb mbp upstream fragment that we used is reported to direct transcription to ols but not to either ol precursors (ops) or schwann cells [ ] . we confirmed that our mbp transgene targets cre recombination specifically to differentiated ols by crossing to ros -yfp conditional reporters; when tamoxifen was administered to double-transgenic offspring, essentially all yfp cells in the cns were also cc . when mbp -icreer t is crossed to tau-mgfp conditional reporter mice (mgfp is membrane-tethered) we can visualize differentiated ols and their myelin internodes in white and grey matter, allowing us to follow the fates of myelinating ols for extended periods of time after tamoxifen labeling. following tamoxifen administration to mbp -icre t : tau-mgfp mice on postnatal day (p ) or p , a large proportion of ols became gfp-labelled in the corpus callosum, optic nerve and spinal cord. examining these mice at increasing times post-tamoxifen will reveal if and how the number and fraction of mgfp-labeled ols/ internodes changes with age -whether there is net loss or gain of ols or whether new ol synthesis is balanced by the rate of ol death (i.e. there is ol turnover). our preliminary results indicate that new ol synthesis exceeds ol death, so the function of adult ol genesis is not simply to replace dying ols. this study aims to identify protein networks that participate in peripheral myelin formation and maintenance. we use a cell culture system, where c bl/ j mouse embryos (e . ) are used for the isolation of dorsal root ganglia neuron explants. explants are cultured in matrigel, and endogenous schwann cells appear in the culture within a few days. myelin formation is induced by adding myelination-promoting substances to the culture medium. immunocytochemistry is used to evaluate the myelination efficiency and the developmental timetable of myelination in culture. electron microscopy is used to study structural features of myelin in culture and to compare its ultrastructural features with those of myelin in situ. rna microarray analysis is performed to study gene expression changes during myelin development in culture. the rna expression data indicates the changes in the rna expression levels, but it does not give information about the amount of final protein product, their posttranslational modifications or stability. we combine the rna expression analysis with a proteomics approach. proteins are extracted for -dimensional electrophoresis analysis at the same time points, and protein spots exhibiting time-dependent changes in their expression levels are analyzed by mass spectrometry. we show that mouse dorsal root ganglion explant cultures produce substantial amounts of mature myelin in weeks after the induction of myelination. ultrastructural analysis shows that myelin in our cell culture system has the same structural characteristics as those observed in peripheral nerves in situ. rna microarray analysis reveals changes in myelin-, nervous system-and schwann cell-related genes. the most significant changes in gene expression occur at the induction of myelination. at the protein level, peaks of specific proteins can be observed at the first and the last time points, which complement the results obtained using microarray data. we have developed a reproducible and efficient method to study molecular changes in myelination at the proteomic and genomic levels. this approach will be exploited further for the characterization of the molecular networks involved in the myelination process. results: our findings show that primary rat schwann cells respond to ivig stimulation with altered cell morphologies accompanied by an accelerated growth of cellular protrusions in early stages of the differentiation process. stimulation with ivig was also found to reduce cellular proliferation rates without affecting cell survival. furthermore, we observed that ivig treatment transiently boosts myelin gene expression of immature cells, whereas myelin gene expression of differentiating schwann cells is promoted on a long-term scale. importantly, myelin gene expression responses cannot be detected upon application of igg control antibodies (herceptin, synagis, avastin). in addition, we could demonstrate that primary rat schwann cells express the cd fc receptor and that in differentiating schwann cells cd levels were significantly increased. on the other hand, we could also reveal a specific binding of the human ivig on the schwann cell surface. conclusions: we therefore conclude that schwann cells are not only able to respond to but also specifically bind immunoglobulins and that ivig stimulation can promote their differentiation. here, we demonstrate that cnp deficient mice exhibit alterations in mechanical and thermal sensation. histological analyses of cnp deficient mutants reveal loss of sensory axons when quantified in the dorsal root and the predominantly sensory saphenous nerve. in contrast ventral roots remain unaltered. moreover, electron microscopical assessments of saphenous nerves display a hypermyelination of small to mid-sized caliber axons and an overrepresentation of the smallest fibers. the myelin pathology of sensory axons is reflected by alterations of sensory nerve conduction velocities. thus, our data demonstrate that, contrary to findings in the central nervous system, cnp has an impact on myelination and is especially important for the integrity of small to mid-sized caliber axons within the sensory peripheral nervous system. to unravel the pathogenetic mechanism of the s del mutation, we generated transgenic mice that express p s del protein and manifest a demyelinating neuropathy similar to cmt b. p s del is a misfolded protein retained in the er, where it activates a chronic unfolded protein response (upr). the upr is a cellular response to er stress, aimed to reduce the load of abnormal proteins by attenuating protein translation, increasing the er folding capacity and stimulating protein degradation. among the degradation strategies upregulated by the upr is the er-associated-degradation (erad) pathway. in erad, proteins destined for degradation are retrotranslocated from the er to the cytosol, ubiquitylated and degraded by the proteasome. microarray analysis of s del nerves revealed that several erad genes, such as derlins, are strongly upregulated and derlin- expression returns towards normal in association with the amelioration of demyelination produced by ablation of chop, indicating a possible involvement of this pathway in cmt b neuropathy. derlins elicit particular interest for exerting a protective role in several er stress-mediated diseases. in s del nerves, derlins co-immunoprecipitate with p s del protein and, finally, derlin- overexpression reduces the level of er stress in cos cells expressing p s del protein, suggesting a positive role in the modulation of p s del protein retrotranslocation. these observations indicate that derlins may be part of the schwann cell adaptive response that attempts to cope with chronic er stress by modulating p s del clearance. we are now in the process of investigating the role of derlins, in the pathophysiology of pns myelination using in vitro, ex vivo and in vivo approaches. during the myelination process, the oligodendrocyte extends processes to and wraps around multiple axons of different diameter, keeping the number of wraps proportional to the axon diameter. this is one unique example of how cells in the central nervous system establish asymmetry. transport of mrna and local regulation of protein synthesis represent one mechanism by which such cellular asymmetry can be generated and the different requirements for myelin sheath at each axo-glia interaction can be controlled. the mrna of a key myelin sheath protein, myelin basic protein (mbp), is known to be transported into the oligodendrocyte processes, and a tight temporal and spatial control of mbp mrna translation is thought to be required for normal myelination. the molecular basis for the tight control of mrna transport and translation is the assembly of large mrna-protein complexes. prior work has identified a number of proteins that interact with the mbp mrna, including hnrnp-a , hnrnp-k and hnrnp-e . however, it is currently unknown how these protein functions together to prevent translation during transport. it is also unknown how targeting of the mbp mrna to the myelin sheets occurs. to delineate the precise role of the individual binding protein in regulating mbp mrna translation, we have identified regulatory elements within the utr of the mbp mrna involved in translational inhibition, and we have identified binding sites in the mrna for hnrnp-k and hnrnp-e . furthermore, we have analyzed the effect of sirna-mediated knockdown and overexpression of these proteins in primary oligodendrocytes. together, our results allow us to propose a model, in which the individual mrna binding proteins are assigned distinct roles for correct spatial and temporal expression of mbp. remarkably, this manipulation was sufficient to achieve both the expansion of oligodendrocyte precursor cells (opc) and the de novo myelination of a large fraction of enlarged parallel fiber axons in the cerebellar molecular layer, independent of axonal neuregulin- expression. by laser-guided microdissection and gene expression profiling of cerebellar gc, we identified neuronal factors that promote opc proliferation and myelin synthesis in vitro. these findings suggest that oligodendrocytes and their precursors respond to multiple 'instructive' signals expressed by cns neurons in vivo, but only on axons with diameters > . lm that became 'permissive' for myelination. l. marziali, p. franco, j. pasquini university of buenos aires, caba, argentina promyelinating effects of both apo-transferrin (atf) and thyroid hormone (th) on rat brain are well documented. th effects are mediated by nuclear receptors which act as transcription factors and regulate different cell processes. previous results from our laboratory showed that th promotes the commitment of oligodendrocyte progenitors (opcs) to mature myelinating oligodendrocytes (olg). on the other hand, atf is able to promote the commitment of neural stem cells (nsc) to cells of the oligodendroglial linage and favors olg maturation after an intracranial injection. our previous demonstration that th administration is able to up-regulate tf mrna expression leads us to hypothesize that both factors converge in the control of oligodendrogenesis. to test if the combined effects of both atf and th are required for proper myelination in the rat brain, studies were done at p and p for each experimental condition. a hyperthyroid state was rendered by daily subcutaneous th administration from the day of birth (hyper). hypothyroidism was achieved by giving propylthiouracil to the mother from gestational day to the end of the experiment (hypo). half of the hypo pups received an intracranial injection of atf at postnatal day (hypo atf). a set of euthyroid animals received an intracranial injection of atf at postnatal day (atf). control groups received an intracranial injection or subcutaneous saline solution (c). at p , hyper animals showed an up-regulation of % in tf mrna expression and % in protein levels as compared to controls. hypo showed a decrease of % in tf mrna levels. this effect was not affected by exogenous administration of atf. at p , hyper showed no differences in the expression of tf mrna or protein levels as compared to controls. hypo showed a decrease of % in tf mrna and a % decrease in protein levels. this effect was not affected by exogenous administration of atf. no differences were observed in the expression of insulin growth factor i (igf-i) or igf-i receptor between groups at p or p . immunohistochemical analyses were performed at p in all groups usingspecific markers anti caii, anti mbp, anti rip and pdgfra. our conclusion is that, in a hypothyroid state, tf is not able to produce olg and myelin maturation at the corpus callosum, which indicates that th is necessary for atf action. however, the finding that hyperthyroid animals showed a significant increase in tf mrna strongly suggests that tf could be involved in th effects. new experiments are carrying out in order to knock down the tf gen to probe this last piece of evidence. h. yigit, l. meyer, c. gonsior, p. hoch-kraft, j. trotter johannes gutenberg university, mainz, germany myelination is the prerequisite for fast saltatory conductance of action potentials. in the central nervous system, the myelin sheath is produced by oligodendrocytes which extend processes and wrap the axons. this structure is subsequently compacted by the help of certain proteins. of major importance here is the myelin basic protein (mbp) which interacts with the cytoplasmic leaflet of the plasma membrane. mbp is a highly basic protein and binds to the negatively charged plasma membrane with high affinity. it has isoforms in mice produced by alternative splicing. the mostly studied isoforms are kd, kd, , kd and , kd. interestingly, exon-ii containing kd and , kd isoforms are actively transported into the nucleus while kd and , kd isoforms are rather localized to the distal part of the plasma membrane. transport to the nucleus depends on temperature and energy. recently, it was reported that exon-ii contains a non-canonical py nuclear localization signal. as a cytoplasmic protein, mbp is essential for proper myelination. furthemore mbp is supposed to have multiple additional functions, e.g. in calcium homeostasis and cytoskeleton dynamics. it was also shown that the expression level of mbp exon-ii containing is changed in schizophrenia patients. selectively transport of exon-ii containing isoforms to the nucleus raises the question of its nuclear functions which have not been elucidated yet. this study aims to unravel the role of exon-ii containing mbps in the nucleus. gene expression profiling of the microdissected svz regions revealed that wnt-responsive genes were enriched in the postnatal dorsal svz compared to the lateral svz. we performed inhibition of gsk b by intraventricular infusion of ara to initiate wnt-signalling. several approaches confirmed activation of the wnt-signalling pathway in the dorsal svz as well as within ops. thus, gene expression profiles of wnt-target genes of the dorsal svz indicated that axin , lef , fzdl and tcf , but not those other pathways, were dramatically up-regulated. furthermore, nuclear b-catenin was transiently up-regulated in the dorsal svz including in a large population of sox -egfp expressing cells following pharmacological wnt-activation. gene expression profiling and immunostainings revealed that the densities of nscs, cycling progenitors and subsequent op differentiation in the dorsal svz were augmented. in parallel, we genetically manipulated wnt-signalling in stem and progenitor cells of the dorsal wall of the lateral ventricle. targeted dorsal svz electroporation of cre-gfp plasmids in floxed stabilised b-catenin (gain-of-function) and floxed b-catenin (loss-of-function) transgenic mouse lines were performed and resulted in a phenocopy of the results observed following ara-intraventricular infusion. in summary, our findings illustrate that wnt-signalling endogenously persists in the dorsal svz after birth. pharmacological as well as genetic interventions reveal an important role of this signalling pathway in the regulation of op genesis during the period of myelination. in the cerebellum the generation of neurons and glia and the relationships between these two lineages are poorly understood. all the cerebellar neuronal phenotypes derive from two distinct embryonic germinal neuroepithelia that are restricted regarding the neuronal cell fate: the rombic lip (rl) gives rise to the glutamatergic neurons, whereas the ventricular zone (vz) generates all gabaergic neurons. in particular, different types of inhibitory interneurons are generated during the postnatal life from a common pool of progenitors expressing the transcription factor pax . on the other hand, the glial development is relatively unexplored. part of the oligodendrocytes derives from extracerebellar sources, whereas some of them derive from endogenous precursors. astrocytes, instead, may derive from progenitors that delaminate from the vz and continue to divide in the prospective white matter (pwm) up to postnatal life. given the vn origin of both astrocytes and interneurons, we wondered whether a lineage relationship exists between these two cell populations and whether they share the same bipotent progenitor. in order to address these issues, we performed a genetic fate mapping study of glastcreert mice. we showed that both cerebellar gabaergic interneurons and astrocytes derive from proliferating glast precursors. in particular, we found that these progenitors are able to produce interneurons at earlier developmental phases, whereas after p they appear restricted to the glial lineage. proliferating glast cells that may produce interneurons comprise bergmann glia and pwm progenitors. however, when recombination was specifically induced in bergmann glia, we found that these cells were not neurogenic. therefore, we concluded that glast progenitors reside in the pwm. to get further insight in the behaviour of these precursors, we injected in the pwm a gfp-containing lentiviral vector with a specific tropism for astroglial cells. some days after, infected cells generated pax -interneurons that were able to mature into interneurons of different cortical layers. moreover, in vitro and in vivo clonal analysis of pwm derivatives indicates that the pwm is neurogenic and both astrocytes and interneurons may derive from a common progenitor. evidence is accumulating that neurogenesis in the subventricular zone (svz) is boosted after trauma or ischemia, also through the interaction with surrounding parenchyma or niche cells. nevertheless, the number of newborn neurons that survive and integrate in the damaged areas is negligible, suggesting a non-permissive environment. thus, understanding the complex signaling network guiding neuroblast generation/survival could help identifying strategies to limit negative inputs and promote regeneration. extracellular nucleotides (ents) are among the hypothetical modulators of svz cell functions, especially under pathological conditions where their concentrations raise several folds and they contribute to reactive astrogliosis. few literature data have recently pointed for a role of the p y receptor subtype (one of the known g protein-coupled nucleotide receptors) in controlling the proliferation and differentiative potential of svz cells. thus, we tested the ability of adpbs, a stable p y agonist, to modulate stem cell properties in the adult brain, with a focus on the possible modulatory effects exerted by reactive astrocytes. the administration of adpbs in the lateral ventricle of adult mice led to reactive astrogliosis in the surrounding brain parenchima, and to a massive reaction of gfapexpressing precursors and astrocytes in the svz. also proliferation was increased, paralleled by a significant expansion of the population of mash transit-amplifying cells and of doublecortin neuroblasts. thanks to the conditional glast::creert yfp mouse model, we also demonstrated that adpbs promoted the proliferation of glast-expressing progenitors in the neurogenic niche, and sustained their progression towards the generation of rapidly dividing transit-amplifying cells. in vitro the nucleotide analog increased the proliferation of svz cells grown as neurospheres, and their differentiation towards neurons, fully confirming in vivo data. interestingly, a significant enhancement in neurosphere generation was detected when svz cells were initially grown in the supernatant of astrocytes exposed to adpbs, and then shifted to normal medium. this suggests that adpbs stimulates the release of yet-to-be identified astrocytic mediator(s) whose removal from the culture medium boosted proliferation of svz cells. our results further strengthen the notion that the purinergic system is a key regulator of the neurogenic potential of svz cells, both directly and through the involvement of reactive astrocytes. cambridge stem cell institute, cambridge, united kingdom oligodendrocytes, one of the principal glial cell-types of the central nervous system, are critical for neuronal function and represent the primary target of many neurological diseases, including multiple sclerosis and leukodystrophies. despite the recent progress in stem cell research and the possibility to generate various neural cell types in vitro, the derivation of oligodendrocytes from human pluripotent stem cells remains inefficient and unreliable. we employed a new experimental approach termed "direct cellular reprogrammng" which was already succesfully applied for the generation of different types of neurons and neural stem cells. aided by bioinformatic filtering we have generated a pool of seventeen candidate reprogramming factors that could potentially convert fibroblasts directly into oligodendrocyte lineage cells. combinatorial testing of the candidate factors in overexpression experiments has revealed a combination of just two transcription factors that was sufficient to convert mouse embryonic fibroblasts directly into o -expressing oligodendrocyte precursors. supplementation of additional factors and multicistronic gene delivery strategies render the reprogramming process more efficient and widen the applicability of this protocol. cellular reprogramming offers a new tool for the generation of oligodendrocyte precursors. this approach, that circumvents the protracted specification and differentiation process inherent to the oligodendrocyte lineage will foster the quest for a culture system of human oligodendrocyte lineage cells. transplanted are derived from progenitors within the intestine. to gain further insight in possible stem cell niches the compelling evidence that mouse enteric glia can also be neuronal precursors was related to human tissue. human colon from infants with hirschsprung's disease was investigated with a panel of glial and stem cell markers. tissue samples from infants with hirschsprung's disease were investigated with glial and stem cell markers along the gut axis. the tissue samples from ganglionic, aganglionic and transient segments were immuonstained either for s /nestin, s /p , gfap/nestin and gfap/p . in all samples investigated, nestin positive ganglia could be found, even in the distal parts with a severe hypo-or aganglionosis. beside nestin positive cells in all segments, there were also different expression pattern glial markers within the ganglia, indicating that distinct phenotypes of glia cells could be found. neural and glial precursor cells are present in the ganglionic as well as in the hypoganglionic segments of hirschsprung's colon, suggesting that these cells might be suitable for ncsc generation and further transplantation. the subventricular zone (svz) of the lateral ventricle, the largest adult stem cell niche, has a striking pinwheel organization specific to regions of adult neurogenesis. the pinwheel's core contains the apical endings of b cells and in its periphery two types of ependymal cells: multiciliated (e ) and biciliated (e ) cells. previous studies showed that svz cells are reactivated in response to demyelination. however, the mechanisms inducing svz reactivation in response to inflammatory demyelination such as in multiple sclerosis (ms) are poorly understood. we hypothesize that b and e, which are in direct contact with csf through cilia, may play a crucial role in initiation of svz reactivation. in the present study, we examined the svz reactivation in mice in which we targeted an ms-like pathology to the forebrain white matter (t-eae). animals were immunized with subclinical doses of myelin oligodendrocyte protein and after days, received a stereotaxic injection of a cytokine cocktail consisting of tumour necrosis factor (tnfa) and interferon (ifn c) into the corpus callosum. to obtain an en face view of the ventricular surface, we dissected whole mounts of lateral ventricle of control and eae animals at , , and days post-eae induction (dpi). we investigated by immunohistochemistry whether and how the pinwheel architecture of the svz is modified and ependymal cells are reactivated during disease course of eae. in svz niche of t-eae animals we found that the number of b cells increased and peaked at dpi compared to control. the number of e cells and pinwheels decreased and then reached control level at dpi. while the number of e cells remained steady, their surface area increased at dpi and then returned to normal size over time. in addition, the number of their ciliary basal bodies increased during eae time courses, and e cells highly expressed gfap in eae animals compared to controls. thus, the findings of our ongoing research suggest that neuroinflammation induces some changes in cell number and morphology of svz niche as well as reactivation of ependymal cells, which altogether may contribute to svz reactivation mechanisms. further study will provide detailed information on the homotypic and heterotypic interactions between pinwheel cells in response to t-eae. to test whether hypothalamic tanycytes act as bona fide neural stem/ progenitor cells in vivo, we analysed their immumoprofile and proliferative potential, and lineage-traced them in vivo. for this, we exploited our earlier findings that in the adult hypothalamus, fibroblast growth factor (fgf ) is expressed exclusively by tanycytes (hajihosseini et al. mcn : - ) , a radial glial-cell like population that lines the floor and ventral walls of the third ventricle. using a line of fgf -lacz reporter mice, we found that fgf beta tanycytes express a panel of neural/stem progenitor markers such as nestin, musashi , blbp and sox . fgf tanycytes also incorporated brdu under cumulative brdu-labelling paradigms, and formed neurospheres in vitro. to directly test the potential of fgf ve tanycytes, we lineage-traced them at p and p by activating the constitutive expression of the marker gene, tomato-dsred, in these cells and their descendants. this was achieved by applying tamoxifen to fgf -creer-t ::rosa r-tomato double transgenic mice. after short survival time-points, tomato was detected exclusively in tanycytes, whilst with longer survival time-point, tomato cells emerged in the neighbouring parenhcyma. the majority of these were neun neurons with fine arborizations. we noted that the newly-generated neurons become associated mainly with hypothalamic nuclei that regulate appetite and energy-balance. moreover, they respond to appetite/energy balance regulating signals such as acute food deprivation and leptin administration. our findings establish fgf beta-tanycytes as a putative population of neural stem/ progenitor cells that generate appetite/energy balance regulating neurons in the postnatal and adult hypothalamus. we used an inducible transgenic mouse line (hgfap-creert ) to conditionally label and fate map putative gfap( ) nscs populations in the adult svz and spinal cord (sc), and compare their self-renewal and multipotential properties. we evidence that a population of gfap cells that share the morphology and antigenic properties of svz-nscs reside in the dorsal and ventral aspects of the central canal (cc) throughout the spinal cord. these cells are non-proliferative in the intact spinal cord, but incorporate edu, an s-phase marker following spinal cord injury. multipotent yfp-expressing neurospheres (i.e. which derive from recombined gfap-expressing cells) were successfully obtained from both the intact and injured spinal cord, confirming their early gfap origin. notably, only spheres isolated from the injured spinal cord revealed long-term self-renewal properties resembling those of svz neurospheres. altogether, this work highlights discrepancies in the identity of nscs that reside in distinct regions of the adult cns. our observations however reconcile divergent views on the location and identity of spinal cord-nscs by showing a population of multipotent gfap progenitors with limited self-renewal properties co-existing alongside with multipotent, self-renewing ependymal cells within the spinal cord central canal. during development neural precursors (nps) both divide, to expand the cell population, and produce many different kinds of neurons and glia. this balance appears to be regulated by par complex proteins, which polarize neural precursors and can thereby direct daughter cells for different fates. how par complex proteins are regulated to appropriately polarize nps remains unknown. in recent years, regulation of gene function by micrornas has emerged as an important mechanism during development. using bioinformatics we identified the polarity gene pard as a candidate target for microrna (mir- ). mir- is specifically expressed in the developing central nervous system (cns) of vertebrates and mir- -deficient zebrafish embryos have a deficit of oligodendrocytes, the myelinating glial cells of the cns. because a disruption in polarity could affect the types of cell divisions that nps undergo, thus altering the balance of cell types that arise, we hypothesize that neural precursor maintenance is regulated by modulation of polarity cues through mir- . by using an in vitro reporter assay we found that mir- can downregulate expression of a luciferase gene fused to the pard utr. reduction of pard function in zebrafish embryos suppressed the oligodendrocyte phenotype resulting from loss of mir- function and injection of a morpholino oligonucleotide designed to block binding of mir- to its pard target sequence phenocopied mir- knock down. together, these data provide strong evidence that pard is a functionally relevant in vivo target of mir- . to further investigate the role of mir- function we tested expression of np, radial glial and neuronal markers in mir- -deficient embryos. the number of cells expressing sox , a marker of nps and neural stem cells, was significantly increased upon mir- knockdown. concomitantly, mir- -deficient embryos had a dramatic deficit of radial glia and late born neurons. these data provide evidence for a new mechanism of np regulation, in which mir- regulates pard levels, thereby regulating the transition of dividing neural precursors to differentiated neurons and glia. in demyelinating diseases such as multiple sclerosis myelin repair activities based on recruitment, activation and differentiation of resident progenitor and stem cells can be observed. however, the overall degree of successful remyelination is limited. it is therefore of considerable interest to understand oligodendroglial precursor cell (opc) homeostasis and maturation processes in order to develop remyelination therapies. mesenchymal stem cells (msc) were shown to exert positive immunomodulatory effects, to reduce demyelination, to increase neuroprotection and to promote adult neural stem cell differentiation towards the oligodendroglial lineage. we here addressed whether msc secreted factors can influence primary opcs in a myelin non-permissive environment. methods: to this end we analyzed cellular morphologies, expression and regulation of key genes/proteins involved in oligodendroglial cell fate and maturation upon incubation with mesenchymal stem cell conditioned medium. results: this demonstrated that msc derived soluble factors promote and accelerate oligodendroglial differentiation, even under astrocytic endorsing conditions. accelerated maturation featured elevated levels of , -cyclic nucleotide -phosphodiesterase and myelin basic protein expression, reduced glial fibrillary acidic protein expression and was accompanied by downregulation of prominent inhibitory differentiation factors such as id and id . conclusions: we thus conclude that besides the previously established immunomodulatory and neuroprotective roles of mscs these cells can also positively influence oligodendrogenesis in the adult central nervous system. in this study, we evaluated the effects of epo and the epo splicing variant (vepo) on murine neurogenesis ex vivo. for this purpose, we analyzed the survival of cultured neural stem cells (nscs) either exposed transiently to recombinant protein, or transduced with lentiviral vectors to achieve stable protein expression. in comparison to non-exposed controls, recombinant epo and vepo enhanced survival of nscs ex vivo (epo: %; vepo: % survival). nscs transduced with pcl -mscv-epo-and pcl -mscv-vepo showed also higher viability ex vivo compared to nontransduced nscs (epo: % vepo: % survival). furthermore, pcl -mscv-epo, but not pcl -mscv-vepo, increased the proportion of differentiated neurons when compared to non-transduced controls. thus, stable expressed vepo may have rather an effect on nsc survival than on nsc differentiation. our results demonstrate that vepos are neuroprotective in vitro. vepo may serve as therapeutic protein for treatment of neurodegenerative disease associated with impaired neurogenesis such as huntington disease. in the subependymal zone (sez) cytogenic niche of the adult mouse brain neural stem cells drive the continuous generation of new cells, mostly of olfactory bulb interneurons, but also of cells of the oligodendroglial lineage. previous reports have shown that newly-born cells exit the sez and migrate to the adjacent corpus callosum (cc) where they differentiate into oligodendroglial cells. however, the real contribution of these niche-derived oligodendrocyte progenitor cells and oligodendrocytes (nopcs and noligos) to the oligodendroglial population of the cc has not been assessed so far. in this study we used the hgfap-cre ert rosa -eyfp double transgenic mice in order to label specifically the progeny of sez-located adult neural stem cells. we found that cells expressing markers of opcs, such as pdgfra and olig , are constantly generated in the sez and migrate to the proximal fraction of the cc. interestingly though, these cells do not remain in the cc for more than days with their contribution to the total population of oligodedroglial cells remaining stably below % even in the ageing brain. moreover, immunostaining for markers of cell cycle revealed that a higher fraction of nopcs undergoes mitosis, when compared with local opcs; hence, they constitute almost % of proliferating opcs of the cc. notably, during their transient presence in the cc, nopcs express markers of maturing oligodendrocytes and are incorporated in established local cell structures. when the cc is challenged with a focal demyelinating insult the local and the niche-derived oligodendroglial machineries exhibit differential cell kinetics, nopcs increase their contribution to the total pool of oligodendroglial cells; however, their presence remains transient. in the human and mouse retina m€ uller glia (mg) are well known to undergo gliosis in all major types of retinal diseases -which sometimes may even lead to scar formation due to proliferative gliosis. some studies suggest that in the mouse retina mg derived neuronal regeneration can be stimulated, but only to a very limited extent. here, we started to find out, if conditional immortalization might stimulate mg derived proliferative gliosis and /or neuronal regeneration. others and we reported that in the juvenile mouse retina, after retinogenesis is finished, some m€ uller glia shift from a quiescent differentiated state into a proliferative state upon damage of retinal explants ex vivo. we now observed that this process is differentially inducible by mitogens like egf (epidermal growth factor). edu (s-phase marker) pulse chase experiments revealed a tremendous increase in mg proliferation within the first two days, a peak of edu positive cells at day ( sem, n ) and a massive decrease until day ( . sem, n ) per mm of a central retina section. next, we used transgenic mice with tightly and temporally controlled expression of the proto-oncogene sv large t-antigen (csv lt). it is well reported that csv lt binds several proteins including the tumor suppressor p and retinoblastoma and bypasses cell cycle checkpoints. induction of the csv lt for days ex vivo led to an overall increase in proliferation compared to control. the number of edu cells was . fold increased (csv lt: sem, n ; control: . sem, n ; p our results so far suggest that induction of csv lt not only overcomes the proliferative restriction of m€ uller glia but also maintains its progeny in the cell cycle over extended period of time. surprisingly, major parts of the generated cell progeny formed gliotic cell clusters, which were all located within the boundaries of the retina. further, we present data of successful m€ uller glia isolation at high purity by fac-sorting. in our current and future work we study the m€ uller glia and its derived progeny to find out the underlying mechanisms that enable neuronal regeneration and prevent gliotic scar formation. in mammals, regeneration capacity of the central nervous system (cns) is considered too low to recover the neurological functions after traumatic injury. in fishes and amphibians, however, the spontaneous regeneration is vigorous enough to complete the anatomical reconstruction and functional recovery even after severe damage of cns, in which the ependymal cells play the central role to exhibit proliferation, epithelial-mesodermal transition, cell migration, support of regenerating axons and neurogenesis to contribute to recovery from injury. these positive roles of the ependymal cells observed in fish and amphibian damaged cns may be good candidates for treating mammalian cns injury. in this study, we attempted to control the activity of the ependymal cells in the adult rodent spinal cord by virus-mediated gene transfer for imitating the reactions that are observed in the ependymal cells of fish and amphibian damaged cns. we introduced the gene known to be expressed in both the ependymal cells and neural stem cells (nscs) and found the proliferation of ependymal cells. in this case, the ependymal cells expressed glial fibrillary acific protein suggesting that this gene regulates proliferation and differentiation into astrocytes. in addition, introduction of another gene, also known to be expressed in the ependymal cells and nscs, caused cell proliferation without differentiation tendency. these findings indicate that the cell fate of the ependymal cells in the adult rodent spinal cord can be modulated by artificial intervention. future studies will clarify whether cell fate modification in the ependymal cells can contribute to anatomical reconstruction and functional recovery in the adult mammalian damaged spinal cord. methods neural stem cells are isolated from different parts of the human gastrointestinal tract, and also from different compartments (muscle, submucous and mucous layer) using enzymatical digestion approaches. generated neurospheres were screened for neuronal, glial, neural stem cell and pluripotency markers, prior and after differentiation. additional experiments were performed where neurospheres were either co-cultured with tissue blocks from different parts of the brain, or transplanted into brain slices from the rat. results neurospheres could be generated from all areas investigated and differentiated on glas coverslips. nestin and musashi- could be found in all neurospheres. moreover sox- , nanog and oct- could be found in the differentiating cultures. after differentiation tubulin and pgp . positive neurons could be found. transplantation experiments showed that cells from the transplanted spheres migrated into the brain slices and differentiated into neurons or glial cells. neurospheres co-cultured with tissue blocks from cortex, cerebellum or hippocampus showed a significant higher outgrowth than neurospheres cultured without any brain tissue. conclusions in general, the ens is a suitable tissue for the isolation of neural stem cells in the individual patient and might so be an appropriate source for autologous neural stem cells. neurogenesis. at the end of embryogenesis, oligodendrocytes and astrocytes appear during the process of gliogenesis. although in the last decades diverse extrinsic and intrinsic factors involved in these developmental processes were identified, our understanding of detailed mechanisms is still comparatively low. in order to increase the knowledge about the cellular and molecular mechanisms that underlie central nervous system development, transfection of nscs showed to be a promising method to understand the function of specific genes and proteins. to overcome the well-known difficulties to transfect cells of the central nervous system, we improved the electroporation conditions to achieve high efficiency transfection and survival rates for embryonic and adult nscs isolated from mouse forebrain structures. one day after electroporation transfection and viability rates of around % were achieved in murine nscs (bertram et al. j neurosci meth ). due to our interest in differentiation and maturation of oligodendrocytes we are currently improving the transfection conditions of oligodendrocyte precursor cells (opcs), since a sufficient protocol for the transfection of opcs derived from neonatal rat cortices is currently not established. the opcs are isolated from rat cortical tissue by a shaking protocol and transfected with the d-nucleofector (lonza). when we used the same pulse protocol that successfully works on nscs, oligodendrocytes were sensitive to the transfection process, and many cells died or did not undergo normal differentiation. this demonstrates that other pulses are needed to electroporate opcs. in cooperation with lonza we tested several recommended pulses to increase the transfection and survival rates of opcs. a dramatic increase of the viability rate has already been achieved. in further experiments the pulse protocol will be improved to obtain transfection rates above %. by establishing this transfection protocol we want to accomplish transfection and viability rates comparable to murine nscs. this improved protocol for the transfection of sensitive opcs will allow for reliable studies of gene function by over-expression or knockdown experiments, and will enhance our understanding of cell biological mechanisms important for oligodendrocyte development. recent studies demonstrate that astroglial cells isolated from non-neurogenic brain regions have the potential to be reprogrammed into functional neurons through forced expression of transcription factors known to instruct neurogenesis. based on our previous studies on the potential of the neurogenic gene cend in directing neural stem/precursor cells (nsc) to exit the cell cycle and acquire a neuronal phenotype, in parallel with evidence demonstrating activation of cend expression by genes of the neurogenin family, we explored the combined effect of cend and neurogenin- on their reprogramming potential on postnatal cortical astrocytes. to this end, forced expression of either cend , neurogenin- or both, resulted in an important increase of two morphologically distinct subpopulations of gfapcells with elongated morphology, that strongly expressed the radial glial marker glutamate transporter glt- . further characterization revealed that a subpopulation of these cells differentiates towards the neuronal lineage, as they were exhibiting a differentiated neuronal morphology and expressed b-iii tubulin, as well as neuronal subtype-specific markers, including gaba and th. further analysis using long time live cell imaging and brdu cumulative labeling revealed that the majority of cend -transduced astrocytes undergo - cell divisions before differentiating to gabaergic neurons, whereas most ngn astrocytes directly transdifferentiate giving rise to th neurons. additionally, only in the doubletransduced cultures, cend /ngn astrocytes seemed to take a step back forming colonies of small round glast /nestin cells, detected h following transduction. a day later, these colonies grew as three-dimensional spheres of high proliferative potential attached to the culture dish. when 'astrospheres' were isolated and cultured under nsc conditions, they grew as neurospheres and were propagated for over ten passages. importantly, as soon as 'astrospheres' were cultured in the absence of growth factors, they differentiated into neurons, astrocytes and oligodendrocytes, implying that they possess neural stem cell properties. at the moment we are performing electrophysiology recordings to assess the functionality of cend derived neurons in vitro, while studies are in progress to explore the potential role of cend and ngn on astrocytic reprogramming in vivo following cortical brain injury. average membrane potential (v m ) was mv and input resistance (ir) was mx, while dcx or map cells expressing fast activating outwardly rectifying k currents (ka), and delayed outwardly rectifying k currents (kdr) were defined by a v m of mv and high values of ir ( mx). ng cells with a complex current pattern expressed inwardly rectifying k (kir) currents, in addition to kdr and ka currents, and their v m was mv and ir was mx. the electrophysiological analysis revealed that the inhibition of the wnt signaling pathway significantly increased the incidence of cells with a complex current pattern, while the number of cells with the marked expression of outwardly rectifying k currents declined. wnt signaling inhibition also resulted in increased kir and decreased ka current amplitudes. oht-treated cells were also hyperpolarized when compared to controls. immunocytochemistry using antibodies against map and dcx showed that neuronal progenitors in oht-treated cultures were less developed, having fewer and less branched processes. in summary, our data imply that the canonical wnt pathway in the neonatal svz promotes nsc differentiation into cells with neuronal characteristics. gacr p / / ; p / /g . adult neurogenesis plays a critical role in the overall health of, and repair processes occurring within the nervous system. while a great deal of information has been gained about the elements of the neurogenic niche and the factors that mediate neurogenesis, many unanswered questions remain. specifically, studies suggest that microglia have a significant impact on adult neurogenesis, but no unifying theory exists to explain their exact role in this process. in order to determine if microglia play a pivotal role in adult hippocampal neurogenesis and to examine the mechanisms through which microglia exert their effects on the hippocampal neurogenic niche, we employed a transgenic mouse model that allows for the selective ablation of microglia in the central nervous system (cns), the cd b-herpes simplex virus thymidine kinase (cd b-hsvtk) mouse. using this system, we removed microglia from the adult hippocampal neurogenic niche and evaluated the effects of this manipulation on neurogenesis and hippocampal homeostasis, including immunohistochemical analyses of stem cell survival and proliferation, as well as analyses of neuroblast development, migration and electrophysiological activity. taken together, these studies help to provide a better understanding of the factors governing adult neurogenesis and further our knowledge about the increasingly diverse role of microglia in the cns. the current assumption is that ependymal damage, caused by viral infections may result in reactive gliosis in the subventricular zone (svz), leading to sgns. however, as the svz is currently recognized as one of the neurogenic niches in the adult human brain, and svz astrocytes have been identified as neural stem cells (nscs), the assumption of sgns being reactive astrocytes has to be reconsidered. therefore, we have immunophenotyped the cell types present in these structures in the brains of individuals of various ages. in addition, we assessed nodule incidence in the svz in cases infected with hiv, where cells in the svz are likely to be to be affected. we found sgns in almost % of all cases, independent of age or hiv infection. we determined that cells in the sgns express adult nsc markers, rather than markers for reactive astrocytes. we hypothesized that direct contact of the nscs with cerebrospinal fluid (csf) may induce nodule formation. indeed, we could show that human ventricular csf stimulated the proliferation of human nscs in culture. from our data, we conclude that sgns most likely are formed upon ependymal damage in response to exposure to csf and represent local expansions of the svz. . in experimental murine model of demyelination, the repair capacity is robust and sufficient, even if it might decrease with aging. one major therapeutic goal in ms is to favour endogenous myelin repair, to prevent neurodegeneration and disability progression. using a transcriptomic approach on pure populations of opcs, we try to identify mechanisms specifically involved in opcs activation. we set up a method to isolate a purified population of opcs, by fluorescent-activated cell sorting from pdgfar::gfp mouse brains. we created the gene expression profile of neonatal opcs, adult opcs and adult oligodendrocytes (ols) in control conditions and of adult opcs in cuprizone-induced demyelinating conditions. we analyzed the transcriptomic profile of adult opcs, neo-natal opcs and ols, and identified more than differentially expressed genes. after demyelination, we identified more than adult-opc-specific genes that are either up-or down-regulated compared to control conditions. preliminary analysis suggests that adult opcs are more "mature" than neonatal opcs and partially revert to a more immature profile after demyelination. bio-informatic analysis is ongoing, with the perspective of identifying key candidates for myelin repair capacity, then develop functional experiments to validate their involvement in remyelination. in parallel, we plan to compare the transcriptomic profile of adult opcs from aged versus young mice, to get insight into age-related decrease of repair efficacy. question: after damage, astrocytes are activated and exert specific roles for central nervous system (cns) repair. among these, astrocytes within the injury site may represent a novel source of cells with stem cell potential. in this study, we used sedimentation field-flow fractionation (sdfff) method to rapidly sort well preserved astrocyte subpopulations and we identified stem cell properties within one of these subpopulations. methods: cells were collected from newborn rat cortex, separated mechanically and subjected to sdfff. cell fractions obtained were analyzed by immunocytofluorescence using antibodies against specific epitopes: glial fibrillary acidic protein (gfap), o , b iii tubulin, and cd , labelling respectively astrocytes, oligodendrocytes, neurons, and microglial cells. then, proteomic analysis was performed on astrocyte subpopulations and their behaviour in culture was analyzed, particularly under culture conditions usually allowing neurosphere development. results: three main fractions (f , f , and f ) were isolated and compared with the total eluted population (tp). after one week in vitro, tp and f derived cell cultures contained respectively . % and . % of gfap expressing astrocytes while this percentage was extremely high in f and f derived cell cultures ( . % and . % respectively). f derived cells displayed higher migratory capacities than those of f . proteomic analysis of f and f derived cells indicated a high expression of vimentin in f derived cells. in the presence of epidermal growth factor (egf), f derived cells formed neurospheres containing cells expressing gfap and nestin (figure) that, when placed in appropriate conditions, were able to generate the three major cell types of the cns (neurons, astrocytes and oligendrocytes). moreover, colony-forming cell assay in a collagen-containing semi-solid matrix allowed in the presence of egf the formation of large colonies. in addition, days after cortical lesion in adult rats, cells presenting similar stem properties were obtained after sdfff cell sorting. conclusions. our study demonstrates that sdfff enables the isolation of almost pure astrocyte subpopulations after one week in vitro. in addition, after sdfff cell sorting, we isolated an astrocyte subpopulation expressing vimentin and displaying the self-renewal and multipotency properties of neural stem cells. our data support that sdfff is an efficient tool to explore the different astrocyte subpopulations of the cns, particularly those involved in cns repair. experimental studies have shown that loss of myelin results in axonal loss and disability. so, finding of an expandable and autologous source of myelin-forming cells to enhance remyelination is required. induced pluripotent stem cell-derived neural progenitor cells (ipsc-npcs) have been developed recently. the remyelination potential and safety of these cells still remained to be well-addressed. the main goal of this study is to characterize mouse ips-npcs in vitro and in vivo after transplantation. we used embryonic mouse neural progenitor cells (mnpcs) as control and characterized mouse ips-npcs for their expression of major markers of immature or mature cells by immunostaining. rt-pcr was performed to analyze expression of nestin, olig , b -tubulin, olig , sox , and nkx . mrnas. furthermore, to investigate the fate of these cells in vivo, we injected the lysolecithin to induce focal demyelination in the spinal cord of adult nude or shiverer mice. we transplanted cells in the lesion site days after demyelination. animals were sacrificed days, , , and weeks post transplantation to assess survival and differentiation of the grafted cells. our preliminary results showed that mips-npcs similar to mnpcs were nestin , ki , olig , b -tubulin but o -and map -. mips-npcs, were more proliferative than mnpc. moreover, mips-npc expressed all mrna transcripts except sox . a first line of mips-npcs, was transplanted in the newborn shiverer:rag forebrain or the demyelinated spinal cord of immunosuppressed shiverer mice with a constant failure in terms of cell survival beyond days of transplantation, highlighting the fragility of some of the reprogrammed cell lines. cells of a second line of mips-npcs were transplanted in nude mice to be sacrificed at , and weeks. an additional serie of transplantation was performed in the demyelinated shi:rag spinal cord. data at and weeks indicate excellent survival and integration of the mips-npc at both time points. some of the grafted cells expressed gfap, olig and ki (few only) and so far, no tumors were observed at these timepoints. immunohistochemistry with other markers and analysis of weeks post transplantation animals, are in progress. our preliminary results show that although mips-npcs have very similar immature phenotype of mnpcs in vitro, they may have different survival capacities in vivo. future studies will reveal whether transplanted cells which showed short-term survival after grafting are capable of differentiation into myelin-forming cells. . however these putative stem cells have only been partly characterized in vitro and their therapeutic potential for cns repair has not been addressed. we cultured adult mouse drg cells in sphere-forming conditions. in parallel, we derived spheres from adult spinal cord of the same animals and compared their properties. this last tissue contains well-described stem/progenitor cells whose repair capacities have already been proven for spinal cord injury but not for primary demyelination. in vitro, regardless of their origins, all tissues generated self-renewable spheres up to passages. rt-pcr and immunocytochemistry revealed that sphere-forming cells actively proliferate and express neural stem cells markers. drg spheres were multipotent: as they differentiated into schwann cells, neurons and myofibroblasts, whereas spinal cord spheres differentiated into astrocytes, neurons and oligodendrocytes. we then compared the integration, differentiation potential and remyelination capacity of primary sphereforming cells isolated from actin-gfp transgenic mice after their engraftment in two distinct environments: the adult nude lpc-demyelinated spinal cord and the developing newborn shiverer forebrain. both cell types survived but with distinct integration and differentiation patterns. in the mbp deficient mutant, spinal cord cells exhibit a substantial tropism toward white matter tracts and differentiated mainly into myelinating oligodendrocytes while drg cells were largely distributed in the parenchyma in close association with blood vessels and differentiated in vascular smooth muscle cells. surprisingly, after focal demyelination, in the adult spinal cord, drg cells migrated extensively towards the lesion and differentiated into remyelinating schwann cells whereas adult spinal cord cells migrated less efficiently, and gave rise to oligodendrocytes and a large population of astrocytes. our study reveals that both cell types present different but promising potential for cns remyelination. funding by ela foudation, dim nerf, arsep foundation. ischemia-induced progenitor cell proliferation is a prominent example of the adult mammalian brain's ability to regenerate injured tissue resulting from pathophysiological processes. a key locus of regeneration is the neurogenic niche located in the subgranular zone of the dentate gyrus (sgz). within the sgz, radial glia-like progenitor cells generate neural precursor cells which migrate into the granule cell layer (gcl) of the dentate gyrus, mature, and integrate into the hippocampal synaptic network. in order to better understand and ultimately exploit the cell signaling mechanisms that regulate ischemia-induced proliferation in the sgz, we tested the role of the p / mitogen-activated protein kinase (mapk) cascade effector ribosomal s kinase (rsk) in this process. using the endothelin- ischemia model in c bl/ mice, we report that intrahippocampal cerebral ischemia triggered rsk activation in sgz progenitor cells. further, microinjection of a rsk inhibitor significantly reduced ischemia-induced sgz progenitor cell proliferation. using the neurosphere assay, we also show that sgz-and subventricular zone (svz)-derived adult neural stem cells (nsc) exhibit a significant reduction in proliferation in the presence of rsk and mapk inhibitors. taken together, these data indicate that rsk functions as a cell-autonomous regulator of ischemia-induced progenitor cell proliferation. here we demonstrated that the inpcs not only possess npc-specific marker genes, but also have qualitites of primary brain-derived npcs (wt-npcs), including tripotent differentiation potential, mature neuron differentiation capability and synapse formation. moreover, the mature neurons derived from inpcs exhibit significant physiological properties, such as potassium channel activity and generation of action potential-like spikes. importantly, besides survival, the transplanted inpcs exhibit the migratory property responding to inflammatory stimulation in vivo. these results suggest that directly reprogrammed inpcs closely resemble wt-npcs, which may suggest an alternative strategy to overcome the restricted proliferative and lineage potential of induced neurons (incs) and broaden applications of cell therapy in the treatment of neurodegenerative disorders. the pathogenesis of neurodegenerative disorders, including human immunodeficiency virus (hiv) associated neurocognitive disorders (hand), is exacerbated by an imbalance between metalloproteinases (mmps) and their inhibitors, tissue inhibitors of metalloproteinases (timps). as the timps exhibit diverse non-classical functions including anti-apoptotic effects, the induction of timp- in neuroinflammatory conditions likely serves multiple roles in addition to modulating mmp activity. our work demonstrates differential timp- expression in acute versus chronic activation of astrocytes and hiv- associated dementia (had) brain tissues. the timp- promoter harbors five consensus ccaat boxes. ccaat enhancer binding protein (c/ebp)b levels were elevated in brain specimens from hiv- patients and this transcription factor regulated astrocyte timp- expression. hiv-relevant stimuli increased c/ebpb expression in human astrocytes and localized the factor to the nucleus. overexpressing c/ebpb in astrocytes increased timp- promoter activity, mrna and protein expression, while knockdown of c/ebpb decreased timp- mrna and protein expression. erk / activation is critical for il- b-mediated astrocyte timp- expression and p k activation contributes to il- b-induced astrocyte timp- and c/ebpb expression. these data suggest that erk / signals downstream of c/ ebpb to facilitate il- b-induced astrocyte timp- expression. the role of astrocyte-timp- as a neurotrophic factor was examined. interestingly, cotreatment with timp- protected neurons from apoptosis and reversed neuronal morphological changes induced by staurosporine (sts) and hiv- ada virus. further, the anti-apoptotic effect was specific to timp- and partially independent of mmp-inhibition. additionally, timp- modulated the bcl- family of proteins and inhibited opening of mitochondrial permeability transition pores induced by hiv- or sts. together, these findings describe a novel function, regulatory mechanism and direct role of astrocyte-timp- in neuroprotection suggesting its therapeutic potential in had and possibly in other neurodegenerative diseases. mounting evidence and our pervious study has also confirmed that the axon outgrowth inhibition receptor ngr was also expressed on microglia, and regulated cell adhesion and migration behavior in vitro. however, microglia has been proposed to play a pivotal role in the neuroinflammation through expressing a range of proinflammatory enzymes and proinflammatory cytokines under pathological stimulus. these findings initiate the possibility of nogo/ngr signal on mediating neural inflammation processes during cns injury beside neurite outgrowth inhibition. in the present study, we found that nogo-p , a -aa core inhibitory peptide sequence of nogo- , was able to induce microglia expressing cox- , inos, as well as releasing proinflammatory cytokines including il- b, no and pge , which could been reversed by nep - or pi-plc treatment. microglia stimulated with nogo-p increased the phosphorylation lever of signal transducer and activator of transcription (stat ). the activation of stat further mediated the promotion of nogo-p on microglia expression proinflammatory factors. furthermore, administration nep - was capable to attenuate the inflammation response in a mouse spinal cord compression injury model by reducing the expression of several proinflammatory mediators, as well as attenuating the activation of stat . taken together, our results demonstrated, for the first time, that nogo- may directly take part in cns inflammation process by influencing microglia expressing proinflammatory factors, which was related to ngr /stat signal pathway. these results imply that the interaction of nogo- with ngr expressed on microglia may participate in diverse cns diseases related to neural inflammation, including trauma, stroke, and neurodegeneration diseases, etc. our results indicate that ccl- is one of the key molecules in pathogenesis and ccl- /ccr- signaling system can be a potential target for drug development in the treatment for neuropathic pain. demyelination plays a central role in the pathophysiology of multiple sclerosis (ms) not only because it disrupts nerve conduction, but also because of effects that compromise axonal survival. there is a growing consensus that any effective treatment for ms must include a component that restores or enhances remyelination by endogenous oligodendrocyte progenitor cells (opc). however achieving this goal requires a far better understanding of why remyelination fails in the first place. we now report that fgf is selectively up regulated in demyelinating ms lesions and acts to inhibit (re)myelination in vitro. to explore the mechanism involved we compared the activity of fgf to that of fgf , another member of the fgf family with well characterised effects on opc differentiation and myelination. fgf acts directly on opc to stimulate proliferation, while at the same restricting their differentiation into mature oligodendrocytes. in contrast, fgf is neither an opc mitogen, nor is it able to inhibit opc differentiation directly. however, opc differentiation and myelination in myelinating cultures were inhibited by supernatants harvested from fgf treated astrocytes, even in the presence of an excess of fgf neutralising antibody. microarray studies were performed to identify astrocyte derived factors responsible for this inhibitory effect. this approach identified several potential candidates that were significantly up regulated by fgf in astrocytes. these included lif, a member of the gp /il cytokine family that is generally described as having pro-myelinating effects, although our own studies indicate when present in excess relative to other gp /il family members it will inhibit myelination in vitro. our experiments demonstrate that fgf -mediated signal transduction in astrocytes disrupts their ability to maintain a growth factor milieu that can support myelination. been shown that inflammation is primarily mediated by macrophages that are recruited to the tissue. similarly, a recent study demonstrated reactive microgliosis in the hypothalamus of mice fed a high-fat diet. in the cns, however, the relevance of microglial activation in response to a high fat diet remains unclear. we aim to determine if microglia activation occurring in response to overnutrition is a cause or consequence of dysregulation of energy homeostasis and obesity. to determine if the absence of microglia (and therefore microglia-mediated inflammation) may influence the metabolic response to a high fat diet, we used a transgenic mouse model, the cd b-hsvtk mouse (tk), which allows for an inducible, specific ablation of microglia in response to treatment with the nucleoside analog, gancilovir (gcv). mice were treated with gcv for weeks during which time they were maintained on a diet high in fat ( %) or a respective control diet. body weight, food intake, locomotor activity and energy expenditure were measured and compared to wildtype mice. analyses revealed alterations in the metabolic phenotype of mice fed both hfd and control diet in the absence of microglia cells. our findings point towards a physiological role of microglia in energy homeostasis, the study of which will be the focus of further experiments. interleukin- (il- ), a cytokine classically related with anti-inflammatory and protective functions in the central nervous system (cns), has been reported to be produced by astrocytes and microglia in different neurodegenerative and neuroinflammatory conditions. in order to study the specific role of il- in the cns, we have created a new transgenic mouse with astrocyte-targeted production of il- (gfap-il tg). the aim of this study was to evaluate if production of this cytokine in the cns has any effect on the phenotype of glial cells. for this purpose, coronal cryostat free-floating sections from the brain of both adult transgenic mice and their corresponding wild-type (wt) littermates, were processed for the study of astrocytes using gfap immunohistochemistry and microglia using antibodies against iba and several markers commonly related to the activated phenotype of these microglial cells, such as cd / (fc receptor), f / , cd b, cd , cd and mhc-ii. our results showed that astrocyte-targeted production of il- induces an increase in the area covered by gfap immunolabelling. microglial cells in these gfap-il tg animals displayed also morphological changes in specific areas of the brain, including the hippocampus, cortex and cerebellum, characterized by coarser processes and a notable enlargement of the cell body. distinctively, in the hippocampus, microglial cells adopted an elongated morphology parallel to the dendrites of pyramidal neurons. in addition, in the aforementioned areas, microglia exhibited a statistically significant increase in iba , cd / , f / and cd b markers and "de novo" expression of cd , whereas no detectable levels of either cd or mhc-ii was found. in conclusion, these results indicate that in specific areas, glial cells are influenced by the presence of astrocyte-targeted il- . further studies are necessary to evaluate whether after injury these transgenic animals will present changes in the microglial and astrocyte activation that can influence the evolution of the lesion. networks. brain-resident macrophages, microglial cells were considered ''resting,'' but it has recently become evident that they actively sense neuronal microenvironment with their motile and ramified processes. they develop specific activities (cytotoxic, neuroprotective or phagocytic) depending on brain molecular context. for example, they contribute directly to the development of neuronal networks by eliminating or maintaining synapses in an activity-dependent manner. morphofunctional plasticity of microglial cells is finely tuned in order to keep brain homeostasis and to ensure such developmental activity. like macrophages at the periphery, microglial cells indeed adopt m or m phenotypes, distinguishable through their pattern of cytokine expression and of specific cell surface markers, a m phenotype being more pro-inflammatory while a m phenotype is more related to phagocytosis. linoleic acid (la, : n- ) and alpha-linolenic acid (ala, : n- ) are essential fatty acids that cannot be synthesized de novo by mammals and have to be provided through the diet. they are precursors of arachidonic acid (aa; : n- ) and docosahexaenoic acid (dha; : n- ) respectively which are key structural components of brain cell membrane phospholipids and precursors of bioactive lipid messengers involved in the regulation of inflammation and microglial activity. most aa and dha accumulate in the brain during the perinatal period via placenta and milk. we previously demonstrated that depleting the diet in n- pufas from the first day of gestation alters some neuronal functions of the offspring at adulthood. we here hypothesized that this is due to an alteration of microglial mrophofunctional activity in the developing brain. to test this hypothesis, mice were submitted to a diet deficient or not in ala throughout gestation and lactation. microglial morphology, phenotypes and functions were determined in the brain of pups at post-natal day (pnd ). we show that dha levels is decreased in membrane phospholipids of microglia. in addition, microglial cells from mice fed with an ala deficient diet present a drastic increase of the cd marker expression combined to a dysregulated cytokine/chemokine expression profile and an altered phagocytic activity. all together, our results show that dietary n- pufas deficiency is likely to impair brain innate immune system activity at pnd . the outcome of such microglial impairment on brain function is discussed. in recent years, one of the most promising therapeutic means for ad was thought to be immunization with specific antibodies against ab. several therapeutic antibodies were developed, however, most of the clinical trials were canceled due to unexpected deaths and neuroinflammatory responses in some patients. the causes and mechanisms of such responses are currently only partially understood. we have recently raised monoclonal antibodies against oligomeric forms of ab and were investigating whether these antibodies would prevent the neurotoxicity of ab oligomers in primary neuronal-glial cerebellar granule cell (cgc) cultures. antibodies were not directly toxic to cgcs when applied at concentrations relevant to concentrations reported to be in blood serum. however, surprisingly, the antibodies when in complexes with ab oligomers or firbrils dramatically increased the neurotoxicity of ab. similar effects were also observed with antibodies to other oligomeric proteins: hamster polyomavirus major capsid protein vp , human metapneumovirus nucleocapsid protein and measles virus nucleocapsid protein strongly potentiated the neurotoxicity of their antigens. the neurotoxicity of antibody-oligomeric antigen complexes was abolished by removal of the fc region from the monoclonal antibodies or by the removal of microglia from cultures, and was accompanied by inflammatory activation and proliferation of the microglia in culture. in conclusion, we find that immune complexes formed by ab oligomers or other oligomeric antigens and their specific monoclonal antibodies can cause neuronal death in primary neuronal-glial cultures via fc-dependent microglial activation. the results suggest that therapies resulting in antibodies to oligomeric ab or oligomeric brain virus proteins should be used with caution or with suppression of microglial activation. additionally, if endogenous antibodies contribute to neuronal loss in ad or viral encephalitis, suppression of microglial activation may be therapeutic. antidepressants have often been recommended for potential treatment of chronic pain, especially for neuropathic pain caused by nerve damage. it is known that nerve injury-induced activation of microglia and astroglia can be a cause for the development of neuropathic pain. recent data indicate that abnormal neuronal activity mediated by glia activation may influence the level of neuroimmune signaling molecules in chronic pain. however, it remains unclear whether the mechanism underlying antidepressant effects is related to glia activation. this study investigated the potential pain-relieving properties of milnacipran and venlafaxine, selective serotonin/noradrenaline reuptake inhibitors (snri), in neuropathic pain and attempted to resolve how these antidepressants affect the factors synthesized by the activated glia in neuropathy. chronic constriction injury (cci) was performed in wistar rats by loose ligation of the sciatic nerve. after a single intraperitoneal (i.p.) injection of antidepressants, the responses to mechanical and thermal stimuli in neuropathic rats were evaluated days after cci by the von frey test and the cold plate test, respectively. to confirm the influence of antidepressants on microglia and astroglia, western blot analysis of iba- (microglia marker) and gfap (astroglia marker) in the spinal cord and dorsal root ganglions (drg) was performed. the experiments were carried out according to the iasp and local bioethics committee rules. our findings confirmed that the administration of milnacipran ( mg/kg) and venlafaxine ( mg/kg) elicited antiallodynic and antihyperalgesic effects in cci-exposed rats. western blot analysis showed that milnacipran elevated the level of iba- protein in the spinal cord (but not in drg) in cci-exposed rats. venlafaxine decreased the cci-induced iba- protein level in the spinal cord and in drg. we observed no changes in gfap in the spinal cord and drg after milnacipran and venlafaxine administration. these results provide a substantial evidence that both used antidepressants belonging to snri attenuated allodynia and hyperalgesia in an animal model of neuropathic pain, although their effect on microglial cells and astrocytes was different, namely, milnacipran increased the microglia activation, in contrast to venlafaxine. this effect can be explained by comparing the effects of these drugs during their repeated application, and relevant experiments are currently in progress. myeloid cells can respond to intra-and extracellular danger/stress signals by inflammasome-mediated activation. this process consists of two steps: toll-like receptor-induced production of pro-il- b followed by inflammasome-mediated cleavage and secretion of bioactive il- b. inflammasome-mediated activation is strictly regulated by expression of regulatory proteins and by inhibitory processes like autophagy. recent studies describe that microglia are markedly different from other myeloid cells. they originate from a separate progenitor, are chronically exposed to the neural microenvironment and their activation may be regulated differently. the objective of this study was to determine the expression profile of inflammasome components in primary microglia and to compare inflammasome-mediated microglial activation and its regulation with other myeloid cells. primary microglia, bone marrow-and blood-derived macrophages (bmdms) of adult rhesus macaques of the same donor were profiled for all nod-like receptors (nlrs), inflammasome-associated adaptor proteins, caspases, and regulatory proteins by qpcr. inflammasome function and kinetics were assessed by exposing lps-primed microglia to atp, silica or msu (danger-associated molecular patterns: damps) followed by measuring the transcription of pro-il- b-encoding mrna and the secretion of il- b. primary microglia expressed nlrs (nalp - , nod / / , aim , ipaf, naip), adaptor proteins (asc), caspases ( / - / / ), and regulatory proteins (a ), and the expression profile closely resembled that of bmdms. priming of microglia with lps induced high levels of pro-il- b-encoding mrna, but il- b protein was only secreted in response to subsequent stimulation with various damps. interestingly, in lpsprimed bmdms the window for inflammasome-mediated activation was - hours, while lps-primed microglia remained sensitive for inflammasome-mediated activation for at least hours. in conclusion, primary microglia express multiple inflammasome components closely resembling the expression profile of bmdms and they can be induced to form functional inflammasomes. importantly, primed microglia remain sensitive to inflammasome-mediated activation for much longer than other macrophages. we will present data on whether this reflects a difference in negative regulation of the inflammasome, or differences in autophagy or apoptosis sensitivity. objective: the susceptibility of the aging brain to neurodegenerative diseases may in part be attributed to the intrinsic senescence of the microglia. cellular aging or senescence is linked to telomere shortening/dysfunction. evidence is accumulating that aging microglia show morphological changes, referred to as microglial dystrophy, which may be accompanied by accumulation of the iron-binding protein, ferritin. here, we investigated the effect of telomere shortening on microglial morphology, numbers, and activation state in telomerase deficient (terc -/ -) mice, a model of premature aging due to shortened telomeres. methods: male terc / mice and rd generation terc -/mice, which show a clear aging phenotype, were perfusion-fixed with % paraformaldehyde. brains were processed into lm thick horizontal sections. every fourth section was used for stereological estimation of cd b microglia in the molecular layer of the dentate gyrus by the optical fractionator method. sections were also stained for the microglial markers iba- to monitor microglial morphology, cd and cd to study microglial activation, and ferritin to study microglial aging. further sections were stained for the apoptosis marker cleaved poly (adp-ribose) polymerase (cparp). results: unlike resting microglia in littermate terc / mice, which display thin, finely branched processes, microglia in terc -/mice exhibited partial retraction and hypertrophy of processes. microglial cd and cd immunoreactivity were similar in terc / and terc -/mice. absolute numbers of mac- microglia were significantly reduced in young and old terc -/versus terc / mice. an increased microglial density in old terc -/versus terc / mice could be attributed to a volume reduction of the dentate gyrus. we also observed an increased number of cparp cells in old terc -/versus terc / mice. this increase was most prominent in the sub-granular zone, an active site of neurogenesis, but also occurred in the molecular layer of the dentate gyrus and might reflect microglial apoptosis. we observed no difference in ferritin staining in terc -/versus terc / mice. conclusion: our findings suggest that telomere shortening impairs microglial capacity for self-renewal. surprisingly, changes in microglial morphology in terc -/mice occurred without differences in cd and cd expression. ongoing studies will show if increased numbers of microglia in terc -/mice co-express cparp or ferritin as evidence of increased microglial aging. tetrahydrocannabivarin (d thcv) and cannabidivarin (cbdv). we have developed a series of new cannabinoid quinones, among them the cbg quinone (vce- ) that shows pparg and cb receptors agonism. in addition we have found that vce- activates the nrf /are pathway in neuronal cell lines. in the present study, we investigated the therapeutic potential of vce- in the experimental autoimmune encephalomyelitis (eae) model of multiple sclerosis (ms) by immunization con mog - . vce- ( mg/kg ip, daily) was administered to susceptible c /bl mice at the onset of symptomatology. clinical score and weights of mice were daily recorded until the day of sacrifice ( days post-immunization). vce- treatment delayed the onset of disease and ameliorated the symptomatology. histological analysis of spinal cord of eae mice treated with vce- showed decreased microglia reactivity and reduced cellular infiltrates, in particular cd t lymphocytes. double labeling with neurofilament and the myelin protein, rip indicated that vce- diminished the axonal damage. demyelination was evaluated by luxol fast blue labeling. changes in the expression of several cytokines, adhesion molecules and nrf -dependent genes were determined by qrt-pcr. the implication of pparg and cb receptors in the beneficial effects of vce- in the eae model of ms is being investigated by using specific receptors antagonists. taken together our results support the potential of vce- for the treatment of ms and other chronic inflammatory diseases. we found that the lipid molecule lactosylceramide (laccer), is upregulated in ms patients. to better understand the role of laccer in ms, we used an animal model of spms, and found that laccer synthase (b galt ) is upregulated during disease progression by astrocytes, and that its inhibition halts the progressive phase of the disease, attenuating cytokine and chemokine production by the astrocytes, and reducing the recruitment of inflammatory ly c high monocytes to the cns. moreover, it also skewed the microglia and monocytes towards a m phenotype, and shifted the astrocyte phenotype to support re-myelination and axonal growth. in vitro experiments, using primary cells demonstrated that the inhibition of laccer synthesis directly inhibits cytokines and chemokines production in astrocytes. importantly, similar results were also obtained with human astrocytes, reinforcing the biological importance of our findings. in summary, we identified a novel lipid-signaling pathway that promotes the activation of local cns immunity and neurodegeneration in experimental spms and is a potential therapeutic target for spms. converging clinical studies report an increased prevalence of comorbid neuropsychiatric symptoms, particularly major depressive disorders, in a number of conditions (e.g., aging, obesity) and diseases (e.g., atherosclerosis, congestive heart failure, rheumatoid arthritis) sharing inflammation as a common denominator. by using an experimental approach in mice exposed to innate immune system (iss) stimulation, we demonstrated that induction of depressive-like behavior is mediated by cytokine-induced brain activation of a tryptophan-catabolizing enzyme, the indoleamine , -dioxygenase (ido). in the metabolic syndrome (mets), a widely accepted concept that identifies a cluster of individual risk factors for type diabetes and cardiovascular disease (including obesity, metabolic dysregulations and basal low-grade inflammation), neuropsychiatric symptoms emerge as significant factors for aggravation of the disease and related outcomes. recently, we demonstrated in a model of mets, the diabetic and obese db/db mice that cognitive alterations and increased anxiety-like behavior are related to hippocampal inflammation. on the other hand, depressive-like behavior is not affected by basal inflammation displayed by db/db mice. given the data linking increased depressive-like behavior and ido activation by cytokines in conditions of iss stimulation, the question arises as to whether depressive-like behavior is increased in those conditions in db/db mice. to answer this question, we measured in db/db mice and in their healthy db/ littermates the effect of a lipopolysaccharide (lps) challenge ( mg/mouse, ip) on behavioral reactivity in a depression test (the forced swim test, fst), plasma levels and hippocampus expression of inflammatory cytokines and related neuronal targets, and brain ido activity. plasma levels and hippocampus expression of il- b, il- , tnfa and il- are similarly increased h after lps in both db/ and db/db mice. as expected, brain ido activity and duration of immobility in the fst are increased in lps-treated db/ mice h after lps. on the contrary, induction of brain ido activity is significantly blunted in db/ db mice compared to their db/ counterparts and no increase of depressive-like behavior is observed. moreover, some data suggest an impairment of the neuroimmune interactions in db/db mice. we need now to understand how obesity and related neurobiological alterations impair ido activation by cytokines and their consequences in terms of vulnerability to infections in mets. in this study, we tested it in a collagenase-induced mouse intracerebral hemorrhage (ich) model using tlr knock-out (ko) mice. to induce ich, collagenase or blood was injected into the right caudate putamen in - week old male mice. tlr expression was upregulated in the ipsilateral hemorrhagic tissues of the collagenase-injected mice. brain injury volume and neurological deficits following ich were reduced in the tlr ko mice as compared to the wild type (wt) mice. heterologous blood-transfer experiments show that tlr signaling in the brain-resident cells, but not leukocytes, contributes to the injury. in our study to elucidate underlying mechanisms, we found that damage in the blood-brain barrier (bbb) integrity following the ich was attenuated in the tlr ko mice compared to the wt mice, which may be due to reduced mmp- activation in brain astrocyte. the reduced bbb damages accompanies with reduced neutrophil infiltration and proinflammatory gene expression the injured brain parenchyma, which may account for the attenuated brain damage in the tlr ko mice after ich. conclusively, these data demonstrate that tlr contributes to brain injury following ich by compromising bbb through activating mmp- in brain. the pathological relevance of this autoantibody response is unknown, but it is generally assumed that it exacerbates demyelination via activation of complement and/or cell meditated effector mechanisms. however in a majority of cases the mog-specific autoantibody titre is far lower than that required to induce widespread demyelination and exacerbate disease severity in animal models of ms. to explore the possibility mog-specific antibodies may play other more subtle roles in disease pathogenesis we investigated possible effects in myelinating cultures derived from embryonic rat spinal cord; a model system that allows us to explore antibody-dependent effects in the absence of exogenous complement and effector cells. myelinating cultures were treated continuously with monoclonal antibodies specific for either mog (clone z , igg a), sulphatide (clone o , igm), proteolipid protein (plp) (clone o , igm), or an appropriate isotype control from days in vitro onwards. in the absence of an exogenous source of complement none of these antibodies induced demyelination, but by div had all had a significant inhibitory effect on myelination compared to cultures treated with appropriate isotype control antibodies. to investigate possible mechanisms contributing to this inhibitory effect qpcr arrays were used to determine if this complement-independent effect on myelination was associated changes in expression of immune mediators. unexpectedly we found recognition of antigens exposed at the surface of the myelin sheath induced a rapid increase in expression of three chemokines known to be involved in recruitment of effector t cells into the cns. within hours of adding antibody to the cultures expression of ccl , ccl and cxcl increased by at least three orders of magnitude and then declined to baseline over the following five days despite continuous presence of antibody. this transient increase in mrna transcripts for these cytokines resulted in sustained protein synthesis and secretion of biological active products as demonstrated by analysis of culture supernatants. these results challenge the traditional view that myelin-specific autoantibodies contribute to the pathogenesis of diseases such as ms and adem by virtue of their ability to initiate immune-mediated demyelination. we now demonstrate that even in the absence of recruited immune effector mechanisms myelin-specific autoantibodies not only inhibit myelination but trigger secretion of chemokines predicted to trigger or exacerbate inflammation within the cns. the blood-brain barrier (bbb), comprising specialized brain endothelial cells, is crucial in maintaining a controlled environment within the central nervous system (cns) to safeguard normal neuronal function. in multiple sclerosis (ms) the function of the bbb is disturbed, leading to uncontrolled influx of metabolites and immune cells and neuroinflammation. therefore, restoring the function of the bbb may be a novel therapeutic tool to halt disease progression or new lesion formation. we previously showed the importance of the vitamin a metabolite retinoic acid (ra) in bbb function during cns development , where fetal astrocyte-derived ra is needed to induce bbb function during cns embryogenesis. considering the possible involvement of developmental pathways that could regenerate the disrupted bbb, we investigated the possible role for ra in the protection or reinstatement of disrupted bbb function. we show that ra counteracts the deleterious effects of inflammatory cytokines such as tumour necrosis factor (tnf)-a and interferon (ifn)c on bbb function in vitro. moreover, ra diminishes the induction of inflammation-related genes in human brain endothelial cells, and induces general immune quiescence in resting brain endothelium. our preliminary data as well as a recently described study in the cuprizone animal model for demyelination suggest that ra synthesis in the cns is increased under inflammatory conditions. the impact of cns-derived ra on the inflamed bbb, as well as the cellular source is currently under investigation. insight into the ra-synthetic pathway and its protective effect on the bbb may lead to the development of novel therapies which are aimed to restore bbb function and reduce the inflammatory cascade in ms lesions. tissue transglutaminase (tg ) is a multifunctional enzyme whose expression and activity is enhanced during inflammatory processes. we previously observed that the expression of tg is increased in monocytes in lesions in post-mortem material of ms patients. moreover, tg activity and expression is enhanced in chronic-relapsing experimental autoimmune encephalomyelitis (cr-eae) in rats. in this same ms model, pharmacological inhibition of tg activity dramatically reduced clinical symptoms and attenuated the influx of monocytes. in the present study we question whether tg plays a role in mouse eae, as transgenic mice have to be used for subsequent in vivo imaging of leukocytes/monocytes during eae. methods and results: eae was induced in tg -/mice and littermate wildtype mice (c bl/ ) using mog - . during the early phase of disease (day - ), the clinical symptoms observed were significantly less severe in tg -/compared to the wildtype mice. moreover, the maximal clinical score was significantly lower in the knockout mice. there was no difference in the day of onset of disease symptoms between the two groups. secondly, we aimed at visualizing leukocytes/monocytes in the spinal cord of lysm-gfp and cd c-yfp mice suffering from mog - induced eae using intravital video microscopy. a permanent window was fixed on top of the spinal cord of mice to allow reimaging of the same field of view in the same mouse over the disease course. in a pilot experiment we observed numerous gfp positive cells in the white matter around the imaged blood vessel in the spinal cord of a lysm-gfp eae mouse. although equally present, this was less prominent in a cd c-yfp mouse suffering from eae. subsequent immunohistochemical analysis revealed that various cell types had infiltrated the spinal of cord of both mice. outlook: to further address the role of tg in the infiltration of leukocytes/monocytes into the spinal cord of eae mice in vivo, specific inhibitors for tg activity will be administered to the transgenic mice and leukocytes/monocytes will be visualized using intravital video microscopy. z. muneer , c. wiesinger , g. regelsberger , j. berger , s. forss-petter medical universty of vienna, center for brain research, vienna, austria medical university of vienna, institute of neurology, vienna, austria x-linked adrenoleukodystrophy (x-ald) is the most frequently occurring peroxisomal neurodegenerative disorder and is often associated with cerebral demyelination and inflammation. x-ald is caused by mutations in the atp-binding cassette sub-family d member (abcd ) gene encoding adrenoleukodystrophy protein (aldp), which is a transporter for coa esters of very long-chain fatty acids (vlcfa) across the peroxisomal membrane. adrenoleukodystrophy related protein (aldrp), another member of the abcd sub-family, encoded by abcd , is the closest homologue of abcd . upon over-expression, abcd has been shown to compensate for abcd deficiency in vitro and in vivo. several lines of evidence imply an important role of microglia/macrophages in the disease progression. here we used mouse peritoneal macrophages (mpmu) to correlate the gene expression levels of candidate modifiers with x-ald associated defects, like accumulation of vlcfa and decreased peroxisomal b-oxidation, in abcd and abcd single ko mutants as well as in abcd /abcd double deficient (doko) mice. by quantitative rt-pcr analysis, in mpmu from wild type mice the abcd mrna was present at about half the level of the abcd mrna. abcd ko mpmu showed elevated accumulation of vlcfa (c : ) compared to the mpmu from wild type mice as measured by gc-ms. the vlcfa levels of abcd ko mpmu were similar to wild type amounts. interestingly, there was much higher accumulation of vlcfa in the mpmu from doko mice. there were no differences in the mrna expression level of the elovl gene, encoding the enzyme catalyzing the elongation step of vlcfa biosynthesis. peroxisomal boxidation activity was decreased in abcd ko mpmu compared to wild type level. this defect was strongly enhanced in mpmu from abcd / abcd doko mice. these results show that the increased accumulation of vlcfa in mpmu from doko mice as compared to abcd ko mice was due to a more severe defect in peroxisomal b-oxidation in doko mpmu. we conclude that a substantial expression of abcd mrna prevents a more severe metabolic phenotype in abcd deficient mouse peritoneal macrophages. this study also supports the validity of abcd up-regulation as a potential therapeutic target in x-ald. j. claude, b. linnartz-gerlach, h. neumann reconstructive neurobiology, bonn, germany microglia have innate immune receptors recognizing pathogens and disease-associated molecular patterns but also molecules that could sense the intact tissue. a subfamily of these receptors is the inhibitory signaling sialic acid-binding immunoglobulin-like lectin (siglec) group including siglec-e that has an immunoreceptor tyrosine-based inhibitory motif (itim) in the cytoplasmic tail to suppress activatory microglial signals. in this study, we used primary and stem cell-derived microglia that were modified by lentiviral vectors. here we show that siglec-e is expressed on microglia and is up-regulated following interferon-c (ifnc) treatment. we performed lentiviral knock-down and overexpression of siglec-e. lentiviral overexpression of siglec-e decreased, while knock-down increased the phagocytosis of neural debris and its associated reactive oxygen burst. the extracellular domain of siglec-e linked to a fc-part of immunoglobulin bound to the sialic acid residues of the neuronal glycocalyx. therefore, we co-cultured these modified microglia with primary hippocampal neurons. overexpression and knock-down of siglec-e showed an increase and decrease in relative neurite-length, respectively. the neuroprotective effect of siglec-e was abrogated after removal of the sialic acid residues on the neuronal glycocalyx. treatment with the anti-oxidant trolox abolished the neurotoxic effect of the siglec-e knock-down on neurite length. in summary, our data suggests an immunomodulatory function of siglec-e on microglia which leads to a neuroprotective phenotype by decreasing the production of reactive oxygen species and a reduced phagocytosis rate of neural debris. a. nadjar, c. madore, a. sere, a. aubert, s. lay e university of bordeaux , bordeaux, france many epidemiological studies have linked maternal exposure to infections during pregnancy to later development of cognitive disorders in the descendants. these alterations are likely to originate from a generalized neuroinflammation in the fetal brain following activation of the maternal immune system. this neuroinflammatory response relies on microglial cells activation. these latter normally contribute to the development of neuronal networks especially by eliminating/maintaining synapses, a phenomenon also known as synaptic stripping, in optimal conditions of brain development. neonatal inflammation may alter the synaptic stripping capacity of microglial cells. thus, targeting this persistent inflammatory microglial activation could represent an original and promising strategy to improve cognitive performances in the descendants. omega- are known as immunomodulators and target microglial cells. we thus hypothesized that enriching the diet with omega- from the first day of gestation may impair the development of neurological deficits at adulthood, through limitation of microglial inflammatory response in favor of its synaptic stripping activity. to characterize the beneficial effects of omega- on microglial activity, pregnant mice were fed with an omega- -deficient diet, or a balanced omega- / omega- diet or supplemented with omega- and received a peripheral injection of either lps or saline at e of gestation. using the golgi method, we first evaluated the morphology of dendrites and quantified the dendritic spines density as a measure of neuronal networks maturation in the hippocampus. we found that a prenatal exposure to lps increased the number of immature spines and this was reversed by an omega- supplemented diet. to correlate these data with alterations of microglia-neurons interactions, we took advantage of the cx cr -gfp mice and injected them with a neuronspecific lentivirus containing dsred protein, in the hippocampus, days prior to two-photon experiments. we focused on microglia general dynamic and microglia-neuron physical interactions and found an alteration in microglial behavior and microglia-neurons interactions in pups from lps-treated females. these alterations were reversed by an omega- supplemented diet. overall, our data show that alterations of microglia-neurons interactions in the developing brain may explain the neurological disorders observed in the descendants of females with prenatal inflammation. these deleterious effects may be prevented by nutritional strategies. , which is characterized by a relapsing phase with inflammatory cell infiltrates and a remitting period, where patients partially recover. among the different cell types involved in the necessary immunomodulation to allow the relapsing-to-remitting transition, the role of myeloid-derived suppressor cells (mdscs) gains importance. mdscs form a heterogenic population of immature myeloid cells that is able to suppress the inflammatory response. this cells act, among other mechanisms, through arginase-i (arg-i) activity on tcells. a previous study of our group showed that arg-i -mdscs transiently enter the spinal cord of eae mice and takes part in the immune response control by inducing t cell apoptosis around the peak of the clinical score. therefore, changes in the mdsc population during eae should modify the evolution of the disease. the retinoid acid family molecules are used for the treatment of different leukaemia due to their role as mdsc differentiation factors into diverse cell populations, abolishing t cell immunosuppression. am , a synthetic analogue of the retinoic acid with a higher bioavailability and less side effects than the natural ones, has controversial actions on eae depending on its administration period. in this work, we administered am specifically in the critical moment for immune modulation (around the maximum clinical score). drug administration affected mdsc population and clearly worsened the eae course. our results point to endogenous strengthening of mdsc population as a new and promising therapeutic strategy to treat ms by speeding up the transition from the relapsing to the remitting period. sildenafil induces cgmp accumulation by pde inhibition. we have demonstrated that sildenafil decreases microgliosis and astrogliosis and proinflammatory cytokines expression in a demyelination model. however, little is known about mechanisms of sildenafil neuroprotection. since nfjb plays an important role in the regulation of glial activation, we examine the hypothesis that nfkb is part of the mechanism underlying the sildenafil neuroprotective effects. five male mice (c bl/ ), weeks-old, were used per group. the groups received for four weeks: . % cuprizone (cpz) mixed into the chow; cpz into the chow plus sildenafil (viagra v r , pfizer, mg/kg) in the drinking water, starting concomitantly (sild-t ) or fifteen days (sild-t ) after initiation of cpz; controls received pure chow/water. cerebella were processed for western blotting and immunofluorescence (if). results showed that cpz increased gfap expression compared to control (astrocytes activation). sild-t (but not sild-t ) significantly decreased gfap compared to cpz group. in controls, microglia showed resting phenotype, with thin and branched processes positive to iba /nfjbp (inactive fraction) double labeling. cpz treatment decreased inactive-nfjb expression, indicating that this transcription factor was activated. in agreement, ikba (nfjb inactivating protein) was also decreased. if showed increase of iba- expression, indicating microglia activation. sild-t strongly increased the nfjbp and its inhibitory protein, ikba, suggesting inactivation of nfjb. if showed decreasing in iba- labeling, suggesting inactive microglia. the delayed treatment (sild-t ) did not decrease iba- or increase inactive-nfjb and ikba expression. in conclusion, treatment with sildenafil concomitant with cpz exposure prevents micro-and astrogliosis in mice, possibly through ikba-nfjb signaling pathway. sildenafil may be suitable to improve neuroinflammatory/neurodegenerative diseases treatment. financial support: cnpq, capes, facepe, fapesp. mt- & levels have been found to be increased in several human neurodegenerative diseases including alzheimer's disease (ad). moreover, mt are also upregulated in different ad mouse models, for example tg mice, which show a significant up-regulation of these proteins in the vicinity of the amyloid plaques. in the present study we generated a double transgenic mouse line that develops ad-like pathology in addition to having an overexpresion of mt , in order to determine the role of mts in different aspects of ad pathology. the results show that the overexpression of mt does affect the mortality rate of the tg and control mice in a gender-dependent manner and partially reverses the behavioural phenotype of young ( - months) tg mice, reducing the exploratory activity and improving the learning process; in contrast, mt does not cause relevant changes in deambulations and anxiety. on the other hand, the amyloid cascade and neuroinflammation is increased in the hippocampus of old ( - months) apptgmt mice, but the lower level of gliosis in the hippocampus of young male apptgmt mice suggests that the overexpression of mt is capable of reducing inflamatory response well before amyloid plaques are formed but not afterwards. further molecular and immunohistochemistry analyses are underway to give further insight into the role of mts in amyloidosis and neuroinflammation in this ad mouse line. exposure to prenatal inflammation is a risk factor for neurodevelopmental and neurobehavioural abnormalities that manifest in later life in disorders such as autism, schizophrenia and seizure development. the amygdaloid complex is composed of more than nuclei with subdivisions that have different cytoarchitectonic, chemoarchitectonic, and projection characteristics and that are responsible for the processing of higher functions such as memory consolidation and emotional conditioning. it is also known that the basolateral nucleus of the amygdala is implicated in seizure propagation and initiation. seizure onset later in life may be linked to abnormal development of the amygdalaoid complex. neuropeptide y (npy) and its receptors are known to be involved in a number of important functions such as feeding behaviours, anxiety and seizure modulation. npy y and y receptors are the most abundant in the brain and are expressed at different stages of development. during foetal development different brain regions are populated by neurones and glia at different times and the interface between mother and foetus plays a crucial role in the development process. lipopolysaccharide (lps) induced maternal inflammation is a known animal model for studying the inflammatory effects on the developing foetus. in this study we are investigating the effects of an acute prenatal systemic insult using the lps model on amygdala morphology and structure. embryonic day (e ) c bl j pregnant mice receive an intraperionteal injection of lps ( mg/kg) or . % sterile saline. embryos at e , e , e and pups at p , p p and p are taken and used to study the developing hippocampus and amygdala. for each age and treatment group foetal brains, maternal brains, maternal serum and placentas are collected. incidence of preterm birth, resorption rate, litter size and weights are also assessed. immunohistochemistry, western blot and rt-pcr are then used to assess the expression of neuronal and glial markers in the amygdala and surrounding limbic structures. specifically alterations in expression, activation and distribution of npy and its receptors, microglia and astrocytes in the developing amygdala and hippocampus at different stages following lps treatment are being investigated. it is hoped that this study will further enhance our understanding of how the maternal environment influences brain development and, if disturbed at a critical time in the development of the amygdala, may predispose individuals to amygdala related disorders in later life. in vivo imaging of transgenic fluorescent mice in the cns by twophoton laser-scanning microscopy ( p-lsm) has become a powerful tool in neuroscience. for in vivo imaging of the cortex a craniotomy of the skull has to be made. in addition to anaesthesia and analgesia treatment, we occasionally apply anti-inflammatory drugs to prevent immunological activity that can obscure the images. as anti-inflammatory drug we used carprofen ((rs) -( -chloro- h-carbazol- -yl)propanoic acid), a known cyclooxygenase- (cox- ) inhibitor. adult cx cr egfp mice in which microglia are labeled by expression of the green fluorescent protein egfp were treated with a single dose of carprofen or with vehicle hours before the craniotomy. in all mice we exposed the right somato-sensory cortex, and quantified the microglial response to a laser-induced micro-lesion. this micro-lesion was caused by increasing the power of the laser for second in the middle of the region of interest. results -conclusions unexpectedly, we observed that carprofen reduced the microglial process motility significantly. the speed with which the processes approached the lesion dropped from . mm per minute in the untreated mice to . mm per minute in the treated mice. a molecular understanding of microglia response in inflammatory processes and how anti-inflammatory drugs modify normal microglia response will provide a strong impact in developing treatment strategies for diseases with strong inflammatory components. resting and activated (lipopolysaccharide, lps) cultured cstb-/-and control microglia were analyzed for cytokine production, chemotaxis and phagocytosis. microglia extracted directly from the mouse brains were studied for expression of mhc ii and m -m polarization using flow cytometry. mouse brain cortex (p ) was analysed for m -m microglial phenotypes and the presence of other immunological cells from the periphery. moreover, we have checked myeloid cell population in bone marrow and spleen of p animals as well as cytokines' level in blood serum. our results from cultured microglia show that secretion of proinflammatory chemokines is elevated by cstb-/-microglia. also, cstb-/microglia are chemotactically more active compared to the controls whereas their phagocytic activity is decreased. cstb-deficient microglia show decreased expression of mhc ii on the cell surface indicating reduced antigen presentation. activated cstb-/-microglia directly extracted from the brain has predominantly proinflammatory m phenotype. cstb-/-mice display inflammatory changes in the peripheral tissues at their early postnatal period. we have registered high concentrations of proinflammatory chemokines and cytokines in blood serum of p cstb-/-pups, but no difference in amount of neutrophils and lymphocytes between brains of wild-type and knockout mice. also, in bone marrow and spleen amount of granulocytes is enhanced in cstb-/mice during early postnatal period as well as level of granulocyte macrophage colony-stimulating factor in their blood serum. our results suggest a role for cystatin b in regulation of immune response and that cstb-deficiency is associated with early inflammatory processes both in the brain and the peripheral tissues. therefore, we consider that epm should be treated as a disorder combining neuropathological and immunological features. the contribution of glial cells in the pathophysiology of epilepsy is increasingly valued. furthermore, clinical and experimental evidence suggests a direct relationship between epileptic activity and cns inflammation, which is characterized by accumulation, activation and proliferation of microglia and astrocytes. concomitant glia-mediated mechanisms of action of several aeds have been proposed. however, their direct effects on glial cells, especially microglia, have jet been hardly investigated. we aimed to investigate the influence of commonly used aeds on the glial viability and microglial in-/ activation state in a physiological and inflammatory modified in-vitro astroglia/ microglia coculture model. methods:primary astrocytic cultures were prepared from brains of postnatal (p -p ) wistar rats and cocultured with a physiological amount of % (m ), as well as % (m ) microglia in order to mimic inflammatory conditions. cocultures were treated for hours with valproic acid (vpa), carbamazepine (cbz), phenytoin (phe) and gabapentine (gbt) with a concentration of , , and mg/ml. viability and proliferation was measured using the tetrazolium (mtt) assay. the microglial activation state was determined by immunocytochemical labeling using a monoclonal antibody to the ed marker. results:m and m cocultures showed a dose-dependent, significant reduction in glial viability after incubation with phe and cbz. furthermore, low doses of vpa led to highly significant microglial activation in m cocultures. however, cbz significantly reduced the amount of activated microglial cells and increased the total number of inactivated microglia in the inflammatory modified m cocultures. conclusion: cns inflammation is characterized by a disturbance of glial cell functions. strong microglial activation, a typical hallmark of inflammation, was induced by vpa in m and continued in m cocultures. with regard to the direct relation between cns inflammation and seizures, vpa seems to be unsuitable to reduce inflammatory condition. the reverse effect was achieved after cbz. we noticed significant microglial inactivation, after incubation of the m cocultures. as it has been demonstrated for levetiracetam before, we assume a beneficial therapeutic effect of anti-epileptic drugs (aed) with an antiinflammatory glial potential in epileptic patients with persistent inflammation. microglial cell activation due to homeostatic imbalances of external and/or internal etiology implies, among others, secretion of proinflammatory cytokines. the fact that microglial activation, inflammation and neurodegeneration often coexist, suggests that proinflammatory cytokines might induce and/or enhance neuronal damage. we investigated the neurotoxic impact of microglial activation in organotypic hippocampal slice cultures by exposing them to the toll-like receptor ligand, lipopolysaccharide (lps), for hours. microglial activation was characterized by unbiased estimation of their number (stereology) and quantification of soma and branching morphology (neurolucida v r -based cell reconstructions). moreover, proinflammatory cytokine and nitrite levels in the culture supernatant were estimated by elisa and griess. the neurotoxic impact was assessed by morphology (nissl staining, fluoro-jade v r b) and extracellular electrophysiological recordings of spontaneous and evoked neuronal activity in the hippocampal ca subregion. lps exposure induced microglial population expansion, morphological changes, such as process thickening and somatic enlargement, as well as substantial secretion of proinflammatory cytokines (tnfa, il ) and nitrite accumulation in the culture medium. however, these changes did not coincide with neurodegeneration and were associated with only minor effects on neuronal function, as demonstrated by the amplitude of evoked neuronal responses and short-term plasticity properties. we conclude that, in contrast to what has been observed in vivo, chronic microglial activation by lps is not sufficient to drive neuronal death and dysfunction in organotypic hippocampal slice cultures. the neonatal brain is particularly susceptible to oxidative stress. our group has previously shown that following hypoxic-ischemic injury, hydrogen peroxide (h o ) levels rise significantly particularly in the neonatal brain and are sustained for up to hours. this rapidly accumulated h o is detrimental in the iron-rich immature brain as it can lead to the generation of dangerous free radicals that can cause extensive injury. to date, there is limited literature on the effects of increased h o levels, particularly on microglial cells, which have been extensively indicated in the mediation of ensuing injury. here we describe the effects of a continuous exposure of microglia to h o , as generated using the glucose oxidase-catalase (gox-cat) system. this system allows us to generate and continuously maintain for up to h pathophysiological levels of h o > um. microglial cultures were derived from the p mouse brain and exposed to either bolus concentrations of h o [ - mm] or varying concentrations of gox-cat for varying lengths of time. conditioned medium was collected from cells at , and h of treatment and analyzed for secreted cytokine levels using the -plex cytokine microbead array kit. treated cell extracts were processed for protein and fixed cells were labeled with m and m a phenotype markers. continuous exposure to very low levels of h o can produce - -fold higher (over control) pro-inflammatory cytokine protein levels of il , il , il p , il p , il , il and ifng and at least a -fold higher level of il a, il b, il and tnfa by h. anti-inflammatory cytokine (il , il and il ) and chemokine (rantes, g-csf and gm-csf) protein levels were also increased by h. interestingly, no prominent cytokine responses were seen with bolus treatment at any of the time points studied. low, continuous h o promoted a predominantly m a microglial phenotype by h. we conclude that studying continuous exposure effects will help delineate the various specific effects h o can have on microglial cells. these specific effects can then be used to clarify when and how microglial cell responses can be beneficial or detrimental following injury in the immature brain. we have previously shown that natural ( -deoxy-d , prostaglandin j , d) and synthetic (pioglitazone) agonists of peroxisome proliferator activated receptor-c (ppar) potentiate intrinsic cellular mechanisms protecting oligodendrocyte (ol) progenitors (ops) from oxidative insults and promote their differentiation to ols. in addition, ppar-c agonists potentiate mitochondrial activities, as the mitochondrial respiratory chain activity and the regulation of cytoplasmic ca waves, which are known to be crucial for ol differentiation. in the present study we sought to investigate whether ppar-c agonists can protect ol cultures from conditions causing mitochondrial stress. first, to specifically induce a mitochondrial impairment, we used the complex i inhibitor rotenone. as expected rotenone, at concentration not affecting cell viability, significantly inhibited ol differentiation, as indicated by the reduced number of cells expressing specific markers of differentiation (o and o ). in ppar-c agonist-treated ols the inhibitory effects of rotenone were significantly attenuated, suggesting a protective effect of the agonists against the mitochondrial toxin. we next examined a condition mimicking inflammatory stress by challenging op cultures with tnf-a, an inflammatory cytokine known to retard the differentiating program of ops. in parallel with the expected reduction of the percentage of o and o positive cells, the cytokine induced a significant reduction of mitochondrial membrane potential (mmp), suggesting an impairment of the mitochondrial functions. the simultaneous treatment with tnf-a and ppar-c agonists ( d or pioglitazone) significantly reverted both tnf-a dependent reduction of ol differentiation and mmp. at the molecular level, we found that in op cultures, ppar-c agonists increased the expression of the uncoupling protein- (ucp- ), a mitochondrial protein known to contribute to the protection of mitochondria against oxidative stress. these findings suggest that ppar-c agonists protect ols and promote myelination through several mechanisms, including those involving mitochondrial functions. our studies support the therapeutic potential of ppar-c agonists in brain diseases in which mitochondrial alteration, oxidative stress and demyelination occur and point to the need to better understand the role of ppar-c and its agonists in ol biology. it is characterized by lesions of demyelination and inflammation, which can be classified according to the activation state of the microglia/macrophages. well before any myelin and blood-brain barrier damage clusters of activated microglia are noticeable. these clusters are considered to be the first stage of lesion formation and therefore called 'pre-active lesions. however, they do not always develop into demyelinating lesions. here we postulate that stressed oligodendrocytes play an crucial role in pre-active lesion formation, by producing factors that will attract and activate microglia to either a more pro-inflammatory (m ) or antiinflammatory (m ) activation status. this can contribute to the amount of damage to the environment and further lesion formation. this study sets out to identify the best method to mimic this early lesion formation in vitro. first, human primary isolated microglia are stimulated to either a m or m phenotype and characterized by marker expression and cytokine profile. oligodendrocytes are isolated out of the human brain. medium of (stressed) oligodendrocytes will be used as attractant as well as activator for microglia. by immunohistochemical staining the activation status of microglia in pre-active lesions will be determined. preliminary data reveal that human microglia are able to be skewed towards a pro-and anti-inflammatory phenotype. first experiments of primary human oligodendrocyte cultures, showed pure oligodendrocytes. thus far our data support our hypothesis, however further research is required to make our conclusions more robust. question: although cell transplantation is increasingly suggested to be beneficial for the treatment of various neurodegenerative diseases, the therapeutic application of such intervention is currently hindered by the limited knowledge regarding central nervous system (cns) transplantation immunology. in this study, we aimed to investigate the early post transplantation innate immune events following grafting of autologous mesenchymal stromal cells (msc) in the cns of immune competent mice. methods and results: first, the survival of grafted luciferase/egfpexpressing msc (msc-luc/egfp) was demonstrated to be stable from on day post implantation using in vivo bioluminescence imaging (bli), which was further confirmed by quantitative histological analysis of msc-luc/egfp graft survival. additional histological analyses at week and week post grafting revealed the appearance of (i) graftsurrounding/-invading iba microglia and (ii) graft-surrounding gfap astrocytes, as compared to day post grafting. while the density of graft-surrounding astrocytes and microglia did not change between week and week post grafting, the density of graft-invading microglia significantly decreased between week and week post implantation. however, despite the observed decrease in microglial density within the graft site, additional phenotypic analysis of graftinvading microglia, based on cd b-and mhcii-expression, revealed > % of graft-invading microglia at week post implantation to display an activated status. although microglial expression of cd b and mhcii is already suggestive for a pro-inflammatory m -oriented phenotype, the latter was further confirmed by: (i) the expression of nos by microglia within the graft site, and (ii) the absence of arginase -expression, an enzyme known to suppress no activity in m oriented microglia, on graft-surrounding and -invading microglia. conclusions: in summary, we here provide a detailed phenotypic analysis of post transplantation innate immune events in the cns of mice, and warrant that such intervention is associated with an m -oriented microglia response and severe astrogliosis. it is well known that cell surface immune receptors play a critical role in regulating immune and inflammatory processes in the central nervous system (cns). cd f is a novel immunoreceptor that dampens inflammatory reactions in experimental autoimmune encephalitis (eae) and diverse allergy models. we have analyzed the function of cd f immunoreceptor in an nmda excitotoxicity model and in a traumatic brain injury model (tbi). for this purpose, we determined the pattern of expression of both cd f and its putative ligand(s) in the cns, as well as the neuroprotective role of cd f after both acute brain injuries. first, we used a human and rat cd f-ig soluble protein to show by confocal microscopy the presence of endogenous ligand(s) for this receptor mainly in cns white matter oligodendrocytes and on the surface of oligodendrocytes, fibrous astrocytes and neurons in vitro. interestingly, when we analyzed the in vitro expression pattern of rcd f in brain cells by q-pcr and immunohistochemistry, in addition to the expected expression in microglial cells, we detected expression of cd f in oligodendrocytes and neurons. in vivo, after a tbi, cd f is expressed at early timepoints ( d) in small round macrophages, and at later time-points ( d) in neurons of the penumbra. q-pcr studies showed significant upregulation of the mrna for rcd f at and d after tbi. interestingly, a delayed ( hours after the lesion) intraparenchymal injections of a non-viral modular recombinant gene therapy vector termed nlsct overexpressing hcd f induced a decrease in the lesion volume d after nmda injection or tbi when compared to control gfp over-expression. in addition, the over-expression of hcd f decreased the long-term sensitive functional deficits observed by the "sticky tape" test. in order to validate these results with the endogenous molecule, we cloned the rat ortholog of cd f protein. on-going positron emission tomography (pet) studies using -[ f]-fluoro- -desoxiglucosa ( f-fdg) are being used to evaluate the long-term functional recovery after tbi. the overexpression of rcd f receptor had a comparable neuroprotective effect as the human molecule after nmda injection. these data suggest that cd f may be important in neuron-glia inflammatory and trophic interactions. in addition, our results suggest that delayed cd f overexpression by means of a modular recombinant gene therapy may constitute an interesting therapeutic strategy for acute brain injuries. depending on the type of signals produced after an inflammatory event, microglia develops specific activities, including cytotoxic, neuroprotective or phagocytic activities in order to come back to brain homeostasis. as described for macrophages, microglia can adopt m (proinflammatory) or m (phagocytic) phenotypes distinguishable through their pattern of factors expression and specific cell surface markers. interestingly, polyunsaturated fatty acids (pufas) are precursors of bioactive lipid messengers involved in the regulation of inflammation and in particular, docosahexanoic acid (dha, : n- ) an n- pufa, presents anti-inflammatory properties. the aim of our study was thus to investigate the effect of an increase of n- pufas on the inflammatory response and cognitive abilities after a peripheral immune challenge. to increase n- pufas, we took advantage of transgenic mice carrying the fat- gene from the roundworm caenorhabditis elegans. this fat- transgenic mouse is capable of producing n- fatty acids from the n- type endogenously, eliminating confounding factors of the diet. this conversion leads to abundant n- fatty acids with reduced levels of n- fatty acids in tissues, including the brain. to induce a neuroinflammation, we injected mice intraperitoneally with lipopolysaccharide (lps), components of gram negative bacteria walls. we first studied the microglial phenotype h after lps injection and found that microglia in fat- mice presented a significant increase of m phenotype markers. we then measured cytokine expression in the hippocampus after a lps challenge and found a significant decrease in il- b mrna in fat- mice compared to wildtype littermates. in terms of hippocampal memories abilities, only control mice presented a deficit in the y-maze task whereas fat- mice were able to perform the test correctly. our results indicate that h after a lps injection, fat- mice presented a decreased of the inflammatory response and normal hippocampal memory abilities compared to wild-type littermates. glia integrity and homeostasis as they have prominent role in blood brain barrier formation and maintenance, as well as in active regulation of an immune response. however, astrocytes are affected in neuroinflammation, when their protective roles might be suppressed and when they might be shifted towards pro-inflammatory phenotype. experimental autoimmune encephalomyelitis (eae) is a widely used model of autoimmune neuroinflammation. chemokine cxcl produced by astrocytes and endothelial cells has profound anti-inflammatory and anti-encephalitogenic role in eae. nitric oxide (no) produced by inducible no synthase (inos) within the cns is considered as disease promoting, while interleukin (il)- as protective in eae. the aim of this work was to investigate effects of no and il- on cxcl gene expression in astrocytes. methods: astrocytes were stimulated with supernatant collected from concanavalin a-stimulated splenocytes (conasn) and simultaneously exposed to no donor, sodium-nitroprusside (snp) or anti-il- antibody. also, astrocytes were stimulated with ifn-c il- in the absence or presence of il- . peritoneal macrophages isolated from healthy rats were co-cultivated with astrocytes. after h incubation period cxcl gene expression in astrocytes was measured by rt-qpcr. phosphorylation of p mapk and nf-jb was assessed by immunoblot in astrocytes exposed to snp. results: we found that no released from snp or macrophages significantly reduced cxcl gene expression in astrocytes. this reduction was mediated by inhibition of p mapk activation, but not of nf-jb signaling. on the contrary, il- stimulated and anti-il- antibody suppressed cxcl gene expression in astrocytes. conclusions: these results imply that expression of cxcl in astrocytes is inversely regulated by no and il- in the central nervous system affected by inflammation. having in mind, profound regulatory role of cxcl in neuroinflammation, these data contribute to our understanding of pro-and anti-inflammatory nature of no and il- , respectively. in the presence of pathological insults, they acquire reactive phenotypes aimed at re-establishing brain homeostasis and minimizing neuronal damage. however, reactive microglia produce several factors, typical of an inflammatory response, with a potential neurotoxic effect. consequently, the progression and resolution of microglial activation have to be tightly controlled to avoid detrimental secondary effects. signals arising from neuronal cells play an important role in the activation state of microglial cells. among them, inhibitory mechanisms, such as neuronal cd and microglial cd r interaction, keep the proinflammatory phenotype of microglia under control. alterations in the expression of cd and cd r have been described in pathological conditions, and the modulation of cd -cd r signalling could be an interesting target to be considered in therapeutic approaches against neuroinflammation occurring in neurodegenerative diseases. little is known on the molecular mechanisms involved in the regulation of cd and cd r expression. the aim of the present work is to study the possible modulation of cd and cd r by ppar-c agonists, and the involvement of cd -cd r in the anti-inflammatory and neuroprotective effects of ppar-c activation. we have used mouse microglial, mixed glial and neuronal cell cultures, as well as neuron-microglia co-cultures. cd r expression was detected in microglial cells, and it was decreased in response to pro-inflammatory stimuli such as lps/ifn-c. the ppar-c agonist -deoxy-d , -prostaglandin j ( d-pgj ) abrogated the inflammatory response in lps/ ifn-c-treated microglial cells and prevented cd r expression inhibition. cd expression was detected in neuronal cultures and, at a lesser extent, in astrocytes in mixed glial cultures. lps/ifn-c-treatment did not modify cd expression in neuronal cultures, but it induced an increase in mixed glial cultures. this increase was inhibited by d-pgj pre-treatment. d-pgj also protected against lps/ifnc-induced neurotoxicity in neuron-microglia co-cultures, but this effect was abolished when cd -cd r interaction was interrupted using an anti-cd r blocking antibody. these results show that ppar-c agonists modulate cd and cd r expression in reactive glial cells, and that cd -cd r interaction is necessary for the neuroprotective effect of ppar-c agonists. to gain insight into the contribution of the cwbc pathway to remyelination in ms, we analyzed the expression pattern of tcf l , a downstream target of the cwbc pathway and coactivatior of b-catenin in ms lesions. tcf l was expressed in ol and astrocytes in a subset of early ms lesions, but was also observed in tissue samples from patients with inflammatory, non-demyelinating diseases. in contrast, in chronic lesions, no expression of tcf l was detected. by wb we found slightly increased b-catenin levels in ms lesions compared to normal appearing white matter. aspirin (ass), a non-steroidal anti-inflammatory drug, attenuates bcatenin signaling activity by inhibition of protein phosphatase a (pp a) via increased phosphorylation. therefore, we determined the effect of ass on ol differentiation and myelin gene expression (mge). as a control for the cwbc pathway activation or inhibition the selective cwbc pathway inhibitor (icg- ) was chosen. icg- binds specifically to cbp and causes a disruption of the b-catenin/cbp interaction. exposure of ol to . mm icg- for hours increased mge and had a positive effect on ol differentiation. in contrast, ass had no positive effect on process formation and did not promote mge in primary murine ol. our results suggest that icg- , but not ass increases the expression of myelin genes. further experiments are required to determine the functional role of the cwbc pathway for ol differentiation and remyelination in ol and ms. (braak et al., ) . furthermore, neuroinflammation, i.e. activation of microglial cells in the substantia nigra (sn), has been widely implicated in pd progression (mcgeer et al., ) . in the present study, we question whether microglial activation occurs in brain regions beside the sn which are affected in pd patients. methods: to this end, we studied microglial activation and protein pathology in the ob and hc of clinically and neuropathologically verified pd patients (braak - ) and control subjects (braak ). human post-mortem formaldehyde-fixed material of pd patients included sn (n ), ob (n ) and hc (n ), and material from age-matched control subjects without neurological deficits included sn (n ),ob(n ), and hc (n ). results: the presence of a-syn pathology concentrated in the anterior olfactory nucleus of the ob and in the ca - region of the hc. furthermore, using cd as a microglial marker, we observed a significant increase in the number of immunopositive microglial cells, with an activated, amoeboid morphology, in the sn as well as in the ob and hc of pd patients compared to control subjects. co-localization studies indicated that these activated microglia were found in the proximity of a-syn inclusion bodies and neurites, but did not co-localize suggesting that the microglial cells do not actively phagocytose a-syn. conclusion: we conclude that microglial activation occurs in other brain regions beside the substantia nigra of pd patients. the functional role of the microglial cells in those brain regions and their contribution to pd pathology remains to be established. in the present study, we question whether ccl and cx cl and its receptors are present in hippocampal lesions of ms patients. to this end, semi-quantitative rt-pcr was performed on cdna transcribed from rna isolated from hippocampi of ms patients and control subjects. moreover, immunohistochemical analysis of ccl , cx cl and its receptors ccr and cx cr was performed on post-mortem, formalin-fixed hippocampal lesions (plp -) from ms patients and on hippocampal material (plp ) from control subjects. ccl and ccr mrna was significantly enhanced in demyelinated ms hippocampal homogenates compared to non-demyelinated and control hippocampal homogenates. cx cr mrna was significantly increased in demyelinated ms hippocampal homogenates compared tot non-demyelinated hippocampal homogenates, but cx cl mrna levels showed no difference between groups. ccl , ccr , cx cl and cx cr immunoreactivity was present mainly in white matter of control and ms hippocampi and was clearly enhanced in hippocampal grey (cornu amonis) and white (stratum lacunosum, stratum radiatum, alveus) matter lesions. the intensity of the ccl and cx cl immunoreactivity was most pronounced when active lesions were present. co-localization studies indicated that ccl and cx cl are present in astrocytes, whereas cx cr and ccr are present in or on microglial cells. we conclude that the chemokines ccl and cx cl and their respective receptors are enhanced in hippocampal ms lesions. as no clear influx of leukocytes is observed in the grey matter lesions, we propose that astrocyte-derived ccl and cx cl , via interaction with their receptors, affect microglial activation and/or function thereby contributing to neuronal dysfunction in the hippocampus as seen in ms patients. , is a kda cytokine-inducible protein, produced by activated macrophages during chronic transplant rejection and in inflammatory reactions. in central nervous system (cns), iba is a sensitive marker associated to activated microglia and is upregulated following neuronal death or brain lesions. iba -like factors have been described in several metazoan and share a well conserved amino acid primary structure throughout evolution suggesting a common, functional role. the medicinal leech hirudo medicinalis is able to regenerate its cns after injury, leading to a complete functional repair. similarly to vertebrates, leech neuroinflammatory processes are linked to microglia activation and recruitment at the lesion site. we investigated the expression of hirudo iba to track the activation state of leech microglial cells involved in nerve repair events. results: we recently identified a gene, named hmiba , coding a . kda protein showing high similarity with vertebrate iba factor. quantitative rt-pcr analyses showed that hmiba is constitutively expressed in cultured nerve chains. a weak down regulation was observed in the days following experimental injury. gene transcripts rise back to basal level one week later. cultured nerve chains stimulated with atp shows a significant increase of hmiba transcript hours after treatment. immunoblot analysis, performed with anti-hmiba polyclonal antibodies, revealed an immunopositive band at the predicted size. the presence of hmiba protein in na€ ıve and experimentally challenged tissues was evaluated by immunohistochemistry. the protein is constitutively present in spread, stellar shaped microglial cells, distributed in connective fibers and in segmental ganglia. a few hours after experimental injury of cns, the amount of immunopositive microglial cells increases at the lesion site and at the cut end of nerve fibers until. the amount of hmiba cells in connectives rapidly increases in atp treated nerve chains. this augmentation is visible in ganglia microglia and in connective fibers, where cells located between axon fibers display an elongated and stretched shape. conclusion: hmiba is a good marker of activated microglia. like in vertebrates, the atp induces its expression in leech cns. also if the functional role of hmiba has to be further elucidated, this molecule appears as a good activation marker of microglia and an interesting tool to study and follow the activity of such cells during nerve repair in leech. hypertension is the single most important risk factor for cardiovascular disease. despite significant advancements, - % of all hypertensives remain resistant to all current available pharmacotherapy. these patients exhibit elevated sympathetic drive, increased norepinephrine spillover, and dampened parasympathetic drive. the dysfunctional autonomic nervous system of these resistant patients indicates a neurogenic origin for their pharmacotherapy resistance. previous studies have proposed that neuroinflammation in the autonomic brain regions plays an important role in neurogenic hypertension. this coupled with the emerging interest in microglia led us to hypothesize that activation of microglial cells in the brain could be a critical event in the initiation and establishment of hypertension pathophysiology. we have previously established that chronic low dose angiotensin ii (ang ii) induced hypertension involves activation of microglia, and increase in proinflammatory cytokines and reactive oxygen species (ros) in cardioregulatory brain areas, such as the paraventricular nucleus of the hypothalamus (pvn). intracerebroventricular (icv) delivery of minocycline, an anti-inflammatory antibiotic that inhibits microglia activation, decreased activated microglia, inhibited increase in cytokines and ros, and attenuated hypertension. in addition, inhibition of brain mitochondrial ros attenuated hypertension, microglial activation, and the overexpression of brain pro-inflammatory cytokines. a time-course experiment demonstrated that there was a significant increase in activated microglia before the increase in blood pressure was detectable by radiotelemetry. furthermore, the origin of microglia in established hypertension appears to be both from resident and bone marrow derivation. these observations indicate that the activation of microglia is a critical player in the development of neurogenic hypertension. they suggest that activation of microglia in cardioregulatory regions of the brain is an early occurrence that may initiates a cascade of signaling events leading to increase sympathetic nerve activity and initiation of hypertension. this research is supported by nih grant (r hl to mkr). our results demonstrate significant inflammatory activation of the neurons and their sgc not only in drg associated but also non-associated with injured nerve. a distinct expression of cytokines and their receptors was identified in sgc surrounding largesized drg neurons. significant inflammatory reactions of sgc were found in all drg of pac-treated rats. moreover, inflammatory activation sgc was also observed in drg of sham-operated rats indicating other kinds of trigger than traumatic nerve injury. the nerve injury and pac-treatment also triggered socs expression in sgc to control stat activation. inflammatory activation of sgc significantly contributes to ectopic activation of the drg neurons not only associated with injured nerve, and is involved in npp induction. aging is associated with reduced function and degenerative changes of the central nervous system (cns). increasing evidence suggests that changes in microglia cells, i.e. the resident macrophages of the cns, contribute to the age-related deterioration of the cns. the most prominent age-related change of microglia concerns enhanced sensitivity to proinflammatory stimuli of microglia in mice, rats and primates, called priming. we have addressed this issue in ercc mutant mice, ercc d/mice, a progeroid, dna repair deficient mouse model that displays features of accelerated aging in multiple tissues including the cns. in aged ercc d/mice, microglia showed increased proliferation and enhanced immune function including expression of cytokines and antigen presentation molecules, increased phagocytosis and production of reactive oxygen species (ros). this microglial functionality was found to be surprisingly hyper-responsive to immune stimuli indicative of microglia priming. transcriptome analysis revealed an expression pattern that characterizes microglia priming, featuring genes associated with increased antigen pattern recognition and antigen presentation and phagocytic-, adhesion-and chemotactic ability. in mice where the ercc related dna repair deficit was targeted to forebrain neurons, microglia priming was restricted to forebrain areas, suggesting that the dna damage-induced changes in neurons provided the signals leading to microglial immune priming. acidosis is a clinical consequence of many major diseases. non-infectious diseases are increasing in prevalence and many have been shown to have an inflammatory component, which in the absence of infection, will be sterile. the nlrp inflammasome, a multimeric protein complex which induces processing of il- b into its active form, is a well established mediator of sterile inflammation and is known to drive the worsening of brain injury in numerous experimental disease paradigms. we sought to investigate whether acidic conditions, typical of a disease environment, affected il- b processing in primary mouse glial cells. mixed glial cultures were grown from wild type and nlrp knockout mice. culture media was reduced to ph . and il- b release was measured following addition of activators of the nlrp inflammasome (calcium pyrophosphate dihydrate crystals, monosodium urate crystals, atp) with or without pre-treatment with caspase- or cathepsin d inhibitors (yvad-cho or pepstatin a respectively). subsequently, il- b release was measured following addition of lactic acid with or without pre-treatment with the above inhibitors. at ph . , activators of the nlrp inflammasome (calcium pyrophosphate dihydrate crystals, monosodium urate crystals, atp) induced the release of il- b from mixed glial cultures. the il- b released at this low ph was kda in size in addition to the mature kda il- b. this kda il- b release was maintained in nlrp deficient cells and was not significantly altered by pre-treatment with the caspase- inhibitor yvad-cho. lactic acid, itself released during disease and a common cause of acidosis, also induced the release of kda il- b from mouse glial cultures. as with the nlrp activators under acidic conditions, the lactic acid-induced kda il- b release was maintained in nlrp knock-out cultures and with pre-treatment with yvad-cho. pre-treatment with the cathepsin d inhibitor pepstatin a, however, significantly reduced the kda il- b released with the nlrp activators and lactic acid. here we show that under disease relevant conditions (low ph), kda il- b is released from mouse glial cultures and this kda il- b is independent of the classical nlrp inflammasome/caspase- pathway and likely mediated by cathepsin d. further investigation of this caspase- -independent il- b pathway may in future provide novel targets for the treatment of inflammatory disease. the phoneutria nigriventer (ctenidae-araneaeomorpha) spider venom (pnv) causes reactive gliosis, neuroinflammation and blood-brain barrier (bbb) impairment. nitric oxide(no)-soluble guanylate cyclase(sgc)-cgmp has been implicated in pnv-induced cavernosal relaxation. we investigate whether no-sgc-cgmp signaling acts on astrocytes/microglia activation and neuroinflammation induced by pnv. male wistar rats ( - -week-old) were pre-treated (i.p.) with sgc inhibitor (odq, mg/kg), nnos inhibitor ( -nitroindazole- ni, mg/kg), no donor (nitroprussiate-ntp, mg/kg) or pde inhibitor (sildenafil, mg/kg). after minutes, the venom ( . mg/kg) was injected in the tail vein (i.v.). saline (i.v.), pnv alone or dmso (i.p) followed by pnv were used as controls. one hour after injection, cerebella were processed for immunofluorescence or western blotting. pnv increased gfap, iba- , ifn-c and sgc, compared to saline control, indicating astrocytes and microglia activation, neuroinflammation and sgc-cgmp involvement. the sgc inhibition by odq intensified the pnv effects, increasing even more gfap, iba- and ifn-c levels. this indicates that sgc-cgmp production can be inhibited by venom. in agreement, the cgmp accumulation by sildenafil inhibited the pnv effects, decreasing gfap, iba- , ifn-c and sgc. the increase of sgc by venom could result from a feedback mechanism, when sgc activity is inhibited and its expression is increased. ni and ntp pre-treatment did not show difference compared to venom alone, suggesting that no per se is not involved in the inflammatory effects of venom. however, it is not discarded no and sgs-cgmp coupling in the mediation of pnv effects. we suggest that pnv induces glial reaction and neuroinflammation by sgc-cgmp inhibition. the study gives evidence that sgc-cgmp, coupled or not to no signaling, mediates pnv cerebellar effects including the bbb impairment. pnv can be a useful tool for studies on the mechanisms involved in glial regulation. cnpq/fapesp/faepex support. j. engele, m. puchert, v. € odemis university of leipzig, leipzig, germany it is currently believed that cxcl predominantly signals through cxcr . in addition, it is assumed that the alternate cxcl receptor, cxcr , represents a non-classical g protein-coupled receptor which primarily acts as a modulator of the function of cxcr . discrepant from this view, we demonstrated recently that in primary rodent astrocytes and human glioma cell lines, sdf- exclusively signals through cxcr by a g protein-dependent mechanism. we now provide evidence that cxcr is essential for cxcl /i-tac signalling in primary rodent astrocytes and some human glioma cells. treatment of cultured rat astrocytes with cxcl for min resulted in the dose-dependent activation (phosphorylation) of erk / and akt with maximum activation in the presence of ng/ml of the chemokine. cxcl- -dependent activation of both signalling molecules persisted in astrocytes in which expression of the established receptors for cxcl , cxcr and cxcr , were inhibited by rnai. however, cxcl -dependent activation of erk and akt was abrogated following sirna-mediated inhibition of cxcr . likewise, cxcl failed to activate erk and akt in cortical astrocytes cultured from a cxcr -/-transgenic mouse line. moreover, similar to primary astrocytes cxcl activated erk and akt in the human glioma cell line, a . again activation of both signalling molecules was abrogated following rnai-mediated inhibition of cxcr . cxcl -dependent activation of erk and akt further remained undetectable in a non cxcr -expressing subpopulation of a cells, previously isolated by flow cytometry. together, these findings unravel a unique processing of cxcl and cxcl signalling in primary astrocytes which is preserved at least in some malignant astroglial cells. long-lasting activation of gfap-positive astrocytes occurs in brain tissue exposed to injuries that promote epilepsy development. studies in experimental models of epilepsy showed that activated astrocytes release various molecules, such as proinflammatory cytokines and danger signals, that play a role in seizures generation and recurrence. these activated cells also loose their ability to buffer extracellular k and glutamate. this set of evidence suggests, therefore, that astrocytes may contribute to epileptogenesis (i.e. the post-injury phase prodromal to generation of spontaneous seizures), thus representing a putative biomarker of epilepsy. to test this hypothesis, we set up an in vivo longitudinal study using h-magnetic resonance spectroscopy (mrs) to measure the hippocampal levels of metabolites that could reflect the extent and the duration of astrocytes activation after an epileptogenic brain injury. status epilepticus (se), which provokes epilepsy, was induced by pilocarpine in adult male rats. h-mrs measurements were done in the hippocampus every h for d post-se, thus encompassing the epileptogenesis phase, and in chronic epileptic rats using a tesla bruker biospec. spectra were processed and analysed using jmrui and tarquin freeware softwares. we studied changes in myo-inositol (mins) and glutathione (gsh), which reflect astrocytes activation. we found a progressive ( -fold, pwhich was maintained in epileptic rats. immunohistochemical analysis (ihc) of s ß and gfap confirmed the concomitant activation of astrocytes in separate timematched groups of rats. gsh levels during epileptogenesis showed a negative correlation with the frequency of spontaneous seizures which developed after se. a negative correlation was also found between gsh and mins levels during epileptogenesis and the extent of neurodegeneration in hippocampus of epileptic rats. ihc done in epileptic rats at the end of the mrs experiments, showed that hippocampal s ß levels positively correlated with spontaneous seizure frequency. since this is a soluble protein, further investigations of its csf and blood levels are warrented. our mrs findings show that gsh levels during epileptogenesis could serve as a predictive biomarker of the ensuing seizure frequency (thus of epilepsy severity) and, together with mins levels, also predict the extent of neuronal cell loss which develops following se in epileptic tissue. notably, ihc-detected s ß levels in the epileptic tissue also reflect seizure frequency. these findings highlight the potential use of serial h-mrs analysis of astrocyte activation for predicting the severity of epilepsy and the extent of neuropathology in the clinical setting. interleukin- (il- ) is a highly plurifunctional cytokine, with many pleitropic actions, considered one of the main cytokines controlling the immune system and coordinating it with the nervous and endocrine systems. il- is produced in multiple cell types in the cns, and in turn many cells do respond to it. it is therefore important to ascertain which the contribution of each cell type is in the overall role of il- during both physiological and pathological conditions. astrocytes are major responders to il- as well as one of the main cns producers of il- . for this work we used astrocytary il- ko (ast-il ko) mice, which we already proved to have an important role in physiological conditions (like body weight control and exploratory/ locomotion behavior), in order to test astrocytary il- role during a neuroinflammation situation. for this purpose, we induced either an extensively used animal model of multiple sclerosis, experimental autoimmune encephalomyelitis (eae), or a traumatic brain injury (cryolesion) in our mice. regarding eae, results indicate that lack of astrocytary il- delays clinical course of eae and ameliorate eae symptomatology in ast-il ko respect littermate controls in a gender-dependent way. further immunohistochemistry analyses confirm a decreased number of cellular and lymphocytes infiltrates and lesser demyelination, angiogenesis and gliosis in spinal cord of ast-il ko animals. regarding traumatic injury, astrocytary lack of il- facilitates microgliosis, lymphocytes infiltration and a faster decrease of the injured area. all these results are likely to bring some answers to astrocytesecreted il- involvement in neuroinflammation pathways and eae pathogenesis. interleukin- (il- ) is a counterregulatory cytokine that plays an important role in controlling inflammatory and immune reactions. in the central nervous system (cns), production of il- has been demonstrated in activated astrocytes and microglia after different types of injuries. the specific role played by il- inmodulating glial responses is however still unclear. hence, the objective of this study was to evaluate the effects of local astrocyte-targeted production of il- on glial reactivity using the axonal anterograde degeneration paradigm. for this purpose, unilateral perforant pathway transection (ppt) was performed on adult gfap-il transgenic (tg) animals and their corresponding wild types (wt) littermates. at and days post-lesion (dpl), animals were intracardially perfused with % of paraformaldehyde, brains frozen and parallel free-floating sections processed for glial analysis using immunohistochemistry for iba- (microglia) and gfap (astrocytes). our results showed that in comparison with wt animals, microglial cells in the dennervated dentate gyrus molecular layer of gfap-il tg showed differential reactivity changes. after lesion, astrocytic reactivity presented a higher hypertrophy in gfap-il tg animals when compared with their corresponding wt littermates. in conclusion, this study demonstrated that local production of il- inthe cns can modify the glial response associated with ppt. further studies are warranted to evaluate if these alterations in microglial and astrocytic reactivity have any repercussions for the evolution of the lesion and the associated axonal sprouting. astrocyte and microglia become reactive under many brain pathological conditions, making this process of neuroinflammation a surrogate marker of neuronal dysfunction. several studies have reported that reactive microglia overexpress the translocator protein kda (tspo, formerly known as peripheral benzodiazepine receptor). positron emission tomography (pet) using radioligands of tspo is thus considered as a potent technique to detect reactive microglia in situ. however, it is still controversial whether tspo pet imaging is also able to monitor reactive astrocytes in situ. this is an important question as reactive astrocytes and reactive microglia play very different roles in brain physiology and could impact disease progression in opposite ways. to address this question, we used a model of selective astrocyte activation through unilateral lentiviral gene transfer of the cytokine ciliary neurotrophic factor (lenti-cntf) in the rat striatum. cntf induced an extensive activation of astrocytes, which overexpressed gfap and became hypertrophic. microglia, on the contrary, displayed a minimal increase in the expression of markers of reactivity. cntf-activated astrocytes overexpressed tspo at the mrna and protein levels. pet imaging experiments demonstrated a significant and specific binding of two tspo radioligands [ f]dpa- and [ c]ssr in the lenti-cntf-injected striatum. we show that reactive astrocytes can be monitored by tspo-pet imaging in the rat brain. this technique is thus well suited to monitor reactive microglia as well as reactive astrocytes, the two cell types involved in neuroinflammation, but would not allow their discrimination in situ. chronicity might establish a neuroinflammatory ganglion profile with inflammatory cells contributing to the hypersensitive phenotype. we first investigated whether, in trigeminal sensory ganglia, cytokines such as tnfa might contribute to a local inflammatory phenotype of a transgenic mouse model of familial hemiplegic migraine type- (fhm- , cacna a r q knock-in mice). with respect to wild-type, r q ki trigeminal ganglia were enriched in activated macrophages and expressed higher mrna levels of il b, il , il and tnfa cytokines and the mcp- . functional consequences of crosstalk between macrophages and sensory neurons were studied in primary ganglia cultures, where larger release of soluble factors and larger currents mediated by pain-transducing atp-gated p x receptors were found. consistently, we observed that, following lps injection, tnfa expression and macrophage occurrence were significantly higher in r q knock-in ganglia with respect to wild-type ganglia. our data suggest that, complex cellular and molecular environment of sensory ganglia could support a new tissue phenotype compatible with a neuroinflammatory profile. we propose that, in selected patients, this condition might contribute to pain pathophysiology through release of soluble mediators, including tnfa and atp that may modulate the crosstalk between sensory neurons and resident glia, underlying the sensitisation process. cultures of astrocyte/microglia are stimulated with ifn-gamma and then infected with tachyzoites of neospora caninum they release nitric oxide (no) that controls parasite proliferation. in order of elucidating the immune response of cns against this parasite, this study investigated the participation of inducible nitric oxide synthase (inos) in the control of its proliferation in co-cultures of neuron-glia obtained from rat brain. the cells were stimulated with ifn-gamma ( iu/ml- h) then supplemented with l-ng-nitroarginine methyl ester/l-name, an inhibitor of inos ( . mm/ml - min) before infection with tachyzoites of n. caninum ( : parasite:cell). after h, in the cultures supplemented with l-name, it was verified, a reduction of tachyzoites number in . % (control cells) and . % (ifn-y stimulated cells). however, in infected co-cultures, it was not observed any release of no, even when cells where modulated by ifn-g. additionally, in cultures infected and treated with l-name, the levels of il- were increased in six fold (non stimulated) and nine fold (ifn-g stimulated). these data suggest that no should not participate as a mediator of n.caninum control in neuron/glia co-culture but, the regulatory pattern of immune response must play a role in its down modulation regulating the inflammatory mediators released during the cns infection, perhaps to protect neurons. methods: adult albino swiss mice were organized in two groups: naive (age-matched control; n ) and lasered (n ). anti-iba was used in retinal whole-mounts immunolabelled to analyze the distribution, morphology, density and arbor area of iba microglial cells. results: in na€ ıve, contralateral and lasered eyes, iba microglias were distributed throughout the retina in the: subretinal space (ss), outer plexiform layer (opl), inner plexiform layer (ipl), nerve-fibre layer (nfl) and ganglion-cell layer (gcl). in na€ ıve eyes, iba microglias: i) had rounded bodies with fewer and shorter processes in the ss; ii) exhibited a branched morphology emanating from small cell bodies in the opl and ipl; iii) were less ramified and were related with the retinal vessels in the nfl and in the gcl. by contrast, the morphological features exhibited by iba cells in contralateral and oht-eyes differed from na€ ıve: i) somas were more robust; ii) processes were thicker and more branched in contralateral eyes and thicker and retracted in oht-eyes. in oht-eyes and contralateral untreated eyes the iba microglial number was increased in comparison with na€ ıve (p < . and p < . respectively, t-test). in comparison to na€ ıve retinas the iba- cell arbor in the opl and ipl decreased in both oht-eyes (p < . and p < . respectively, t-test) and in the contralateral untreated eyes (p < . and p < . respectively, t-test). conclusions: two weeks of laser induced-oht triggered microglial retinal changes suggestive of activation in the contralateral untreated and oht-eyes. the microglial activation in contralateral eyes could be related to the immune response. this behaviour in contralateral untreated eyes, lead us to suggest that the use of contralateral eyes as internal controls in experimental unilateral oht, should be reconsidered. interleukin- (il- ) is a pleiotropic cytokine involved in inflammatory and non-inflammatory responses. among the latter, it participates in the regulation of body weight and metabolism. il- -deficient mice develop mature onset obesity and have higher blood glucose, as well as impaired glucose tolerance and elevated blood leptin levels, suggesting that il- participates in suppressing adiposity in mice. moreover, transgenic mice with astrocyte-targeted production of il- challenged with a high-fat diet are resistant to high-fat diet-induced obesity, highlighting the role of centrally produced il- in the regulation of body weight. in this context, we hypothesize that mice lacking il- produced by astrocytes (ast-il ko) will be more prone to develop obesity. we therefore challenged ast-il ko mice (obtained with the cre-lox technology) with a high fat diet ( % kcal from fat) for weeks and compared their body weight and food intake with those of mice fed a standard diet ( % kcal from fat). on weeks and , the metabolic status was evaluated by an insulin tolerance test (itt) and an oral glucose tolerance test (ogtt), respectively. regarding body weight gain, we see a clear increase in ast-il ko females on a high-fat diet in comparison to their controls with no apparent difference in food intake, which is also already apparent in the control diet. in males a similar tendency is observed. part of this difference can be attributed to the heavier subcutaneous white adipose tissue depots (relative to body weight) in ast-il ko mice (fig ) . insulin and oral glucose tolerance are compromised in mice fed a high-fat diet, but no differences between genotypes are observed. taken together, these results indicate that centrally produced il- has indeed a major role in the regulation of body weight, affecting subcutaneous white adipose tissue, but without significantly altering peripheral glucose metabolism. we are currently working on assessing hypothalamic neuropeptides with in situ hybridization to study the effects of astrocytic il- deficiency at the central level. minocycline is an agent with pleiotropic properties that targets multiple proteins and cellular processes associated with development of neuropathic pain, including inhibition of the injury-induced glia activation. the aim of our study was to examine the effects of minocycline on the injury-induced changes in immune factors and on morphine effectiveness in a rat model of neuropathic pain. chronic constriction injury (cci) to the sciatic nerve in rats was performed according to bennett and xie ( ) and behavioral tests were conducted to measure allodynia (von frey test) and hyperalgesia (cold plate test). minocycline was administered intraperitoneally h and h before cci and then twice daily for days. the studies were performed using competitive rt-pcr in the spinal cord and drg from control, cci-exposed and minocycline-treated cciexposed rats. morphine was administrated i.t. (chronic catheterization according to yaksh and rudy, ) and ip h after the last minocycline injection. the experiments were carried out according to iasp rules (zimmermann, ) . repeated administration of minocycline attenuated allodynia and hyperalgesia when measured days after cci. minocycline downregulated the spinal and drg level of several immune factors: c q, mmp- , mmp- , il- beta, il- , il- , nos and nos which were increased in consequence of the injury. moreover, chronic administration of minocycline improved the response to i.t. and i.p. morphine injections. our results demonstrate that minocycline reduces the level of pro-inflammatory factors and increases the effectiveness of morphine, which may have clinical significance for enhancing analgesic effects of opioids in neuropathic pain. to date, there is no standardized simple model available to investigate the biology of human microglia. the aim of this study was to establish a new in vitro microglia model using blood-derived precursor cells. for that purpose, human peripheral blood monocytes were cultured in serum free medium in the presence of a mixture of cytokines and chemokines (m-csf, gm-csf, ngf and ccl ) to generate monocyte-derived microglia (m-mg). monocyte-derived dendritic cells (m-dc) were also generated as a control population using gm-csf and il- . the human microglia cell line hmc was used as control. m-mg were clearly different in morphology, phenotype and function from m-dc, but shared many properties with hmc cells. m-mg acquired a ramified morphology with primary and secondary processes, comparable to hmc . they expressed very low levels of cd , cd and hla-dr, cd b and cd c; but a distinct pattern of chemokine receptors, including ccr , ccr , ccr , ccr , ccr , cxcr , cxcr , cx cr . similar to hmc , under non-activated condition, the m-mg secreted of il- and il- . in comparison with m-dc, m-mg displayed lower t-lymphocyte stimulatory capacity, as well as lower phagocytosis activity. in summary, we have established a new protocol for the generation of human monocyte-derived microglia, which is is feasible, well standardized and reliable, as it uses well defined culture medium and recombinant cytokines, but no serum or conditioned medium. this model will certainly be very helpful for future studies investigating the biology and pathology of human microglia. w. schaafsma university of groningen, neuroscience, section medical physiology, groningen, netherlands background: microglia are the innate immune cells of the cns. like other tissue macrophages, they can activate and commit to distinct reactive phenotypes in response to tissue damage and infections. however, the microglial response to tissue damage may change over age and by past experience. these changes in activation patterns may be caused by epigenetic modifications which in turn can result in changes in gene expression of, for example inflammatory cytokines. a well studied condition with an altered responsiveness of innate immune cells is 'endotoxin tolerance' (et). innate immune cells that are pre-exposed to endotoxins are dampened in their inflammatory response upon re-exposure to these endotoxins. while et is well described for peripheral macrophages/monocytes, very little is known about et in relation to microglia. objective: in our experiments we set out to investigate the presence of et in microglia and if an 'epigenetic' memory is involved. design/methods: in our in vitro experiments, microglia cells were stimulated with lps ( ng/ml). naive microglia only received one lps stimulation at the end of culture, pre-stimulated microglia received an h pre-stimulation of lps ( ng/ml) and a second stimulation days later. in vivo, male c bl/ mice received an i.p. injection with either pbs or lps ( mg/kg) and weeks later again an i.p. injection with pbs or lps. transcription levels and secretion of il -b and tnf-a were assessed by use of quantitative pcr and elisa. in order to answer if there is a long lasting epigenetic memory involved in this tolerance we used chromatin immune-precipitation (chip), to investigate changes in covalent histon tail modifications associated with either permissive or repressed chromatin. results : in vitro, we show a 'tolerant' phenotype in microglia which receive a second lps stimulation after days. a reduced expression level of pro-inflammatory genes il -b and tnf-a and reduced secretion of tnf-a were observed in response to a second lps challenge. furthermore, with in vivo experiments we showed that mice which received a second lps injection after weeks, showed a dampened response in their microglia inflammatory reaction at the level of il -b and tnf-a gene transcription. in both in vitro and in vivo experiments we showed a corresponding pattern for histon tail modifications in the enrichment of activation marks h k me and ach at the il -b and tnf-a promotors. we are the first to show a long lasting 'tolerant' phenotype in mouse microglia in vitro and in vivo and the involvement of epigenetic changes at the level of histon modifications. morphological criteria and dual immunofluorescent labelling confirmed expression of er stress protein in neurons, astrocytes, microglia or oligodendrocytes. an intriguing finding was localisation of crt to the rim of oro-positive myelin fragments and the 'patchy' nature of crt staining seen when tissue was dual-labelled with crt and gfap or iba . these results are the first demonstration of significantly higher levels of crt in rodent eae. chop and p-eif a data has also not been reported in rat eae. this study highlights the potential importance of er stress in inflammatory demyelination. the authors acknowledge support from the irish research council and from the foundation office of nui, galway. ( ). considering the responsible signaling pathways regulating adult neurogenesis ( ), we observed differential regulation of wnt members in the hippocampus on transcriptional level, e.g. wnt dependent transcription factors (axin , tcf ) and ligands (wnt , a, b and ), as well as on translational level represented by the wnt signaling cascade (active-bcatenin, phospo-glycogen synthase kinase b) during acute and chronic states of disease. by using in vitro studies in primary hippocampal cultures, we further show the role of transforming growth factorbb (tgfb ), a key cytokine early involved during eae ( ), in regulation of wnt ligand expression and wnt signaling activity. furthermore we are able to visualize the connection between tgfb and wnt signaling by using the axin -lacz mouse, a wnt reporter mouse, for functional and histological investigation of the hippocampus. taken together our results suggest a cross talk of inflammation and wnt signaling for dysregulated hippocampal neurogenesis. interleukin- (il- ) is a cytokine with major regulating effects of the inflammatory response. moreover, il- is a neuropoietin that has neurotrophic effects related to neuronal survival and protection. to establish the importance of il- produced only in the central nervous system, we have generated mice producing il- essentially only in the brain by crossing gfap-il mice (transgenic mice with astrocyte-targeted production of il- ) with il- ko mice (il- -deficient). we studied the inflammatory response in a traumatic brain injury model, cryolesion, after and days post-lesion gfap-il -il ko mice, comparing them with appropriate controls. in basal conditions wt and il- ko mice showed a similar phenotype. this was also the case for gfap-il and gfap-il -il- ko mice, which showed prominent astrogliosis, microgliosis, increased recruitment of t lymphocytes and vascularisation compared with the other two groups. in response to cryolesion of the cortex an increased astrogliosis, microgliosis, recruitment of t lymphocytes and vascularisation was observed. il- deficiency produced an altered inflammatory response, in a time-and gender-dependent manner. this study with il- ko and gfap-il transgenic mice indicates that during an acute neuropathological insult such as traumatic brain injury il- , from either the brain or the periphery, has an important role on the inflammatory response. increasing evidence suggests that iron accumulation in the brain might contribute to neurodegeneration. iron is a potential source of free radicals as it can catalyze the production of hydroxyl radicals under oxidative conditions. this can lead to amplification of tissue injury caused by oxidative damage. we thus characterized iron storage within the central nervous system (cns) of animal models of different neurodegenerative diseases. we examined animals with acute inflammation mediated by cd or cd positive t cells and animals suffering from t cell and antibody mediated chronic inflammation due to active immunization. further, we studied lps induced lesions which represent cns disease caused by the innate immune system. similarly, we characterized oxidative damage in the different models for inflammation. we did not find evidence for the presence of oxidized phospholipids or oxidized dna in the experimental lesions, which is in contrast to our results of ms lesions. none of these models showed iron accumulation in glial cells comparable to what is seen in ms tissue. however, some iron positive microglia and perivascular macrophages were observed in acute and chronic models. in preliminary experiments we found evidence that the presence of iron exacerbates h o induced cell death in purified glial cells in vitro. as a next step, we wanted to study a possible amplification of oxidative damage and neurodegeneration by iron in a more elaborate system. for this purpose we treated myelinating spinal cord cultures with iron chloride (fecl ) or ferritin. we observed iron loading in microglia in both experimental setups. moreover we found a selective decrease of microglia in iron chloride treated cultures but not after ferritin application. we did not find iron loaded oligodendrocytes in this in vitro model. iron accumulation is only minimal compared to the human brain in all the tested animal models. these observations necessitate the search for additional animal models mimicking the human situation more closely. objectives: microglia are a brain resident population of immune cells, involved in homeostatic surveillance of the brain parenchyma, with high importance in the regulation of inflammatory processes after injury. there are unresolved questions regarding microglia origin, and their similarities to peripheral macrophage populations. our objective was to compare the capacity of microglia and bone marrow derived macrophages (bmdms) to adopt different phenotypes, and how this influenced cell death after brain injury. methods: we compared the phenotype of bmdms and microglia (both bv microglial cell line and primary microglia) after treatment with well known polarising agents. lipopolysaccharide (lps) was used as an inducer of the classical m phenotype and il- was used to induce an alternative m phenotype. microglia or bmdms, of different phenotypes, were added to hippocampal organotypic slices (hosc) subjected to oxygen glucose deprivation (ogd) as an in vitro model of brain injury. results: bmdms and microglia (bv and primary microglia) both adopt m or m phenotypes after treatment with lps or il- respectively. however, addition of polarised microglia or bmdms onto hosc resulted in different outcomes. addition of activated bmdms, of either m or m phenotypes, led to cell death in control hosc and increased death when combined with ogd. on the other hand, addition of bv -microglia did not induce any cell death in control hosc, and were protective after ogd, except for m microglia which were toxic. endogenous microglia within the hosc were also able to adopt different phenotypes and exert neuroprotection after il- treatment. these ex vivo data correlated with the in vivo situation where we observed increases in microglial activation early after experimental stroke, although the vast majority of these activated cells did not express markers of the m phenotype. conclusions: this study highlights functional differences between macrophages and microglia, especially in response to brain injury. although microglia, like peripheral macrophages, can adopt different demyelination, thereby creating an unfavorable environment for remyelination. exploring efficient ways to prevent or overcome tlr-induced fibronectin aggregation by astrocytes might pave the way for new approaches in remyelination-targeted therapeutic strategies in ms. to study the tlr response in-vivo, sod g a mice were crossed with the tlr -luc-gfp reporter mice, a transgenic mouse bearing the dual reporter system luciferase and green fluorescent protein under the transcriptional control of the murine tlr promoter. double transgenic mice and littermates controls were monitored longitudinally using in-vivo biophotonic/bioluminescent imaging to measure microglial activation over the course of the disease. surprisingly, this analysis did not reveal an increase of activation in sod g a mice as the disease progressed, with only weak signal coming from olfactory bulb and brain area. however, quantification of this signal suggested a lower level of tlr activation in the sod g a mice compared to wild-type littermates. to further study the microglial impairment observed in the sod g a mice, these transgenic mice were given lps with i.p. injections at mg/kg and were followed for h by bioluminescence imaging, both at a pre-symptomatic age ( days) and closer to paralysis ( days). the level of tlr activation was lower in the sod g a mice than the wild-type littermates, both at days and days. immunostaining of olfactory bulbs reveal the same phenomenon, which is less iba and less tlr -positive cells in the sod g a tissue. mrna analysis by in-situ hybridization confirms a similar pattern of microglial response. in a parallel study, primary microglial cell culture, derived from adult mice ( days) were similarly stimulated with lps to further understand the altered sod mutant microglia response. here again, sod cells appears less responsive to lps stimulation as observed in our mice studies. these combined results suggest that sod g a microglial cells might have a pre-symptomatic suboptimal immune response. the role of reactive glia in the etiology and progress of neurological diseases is still unknown. reactive glia produce several factors, typical of an inflammatory response, with a potential neurotoxic effect, highlighting the relevance of a strict control of the progression and resolution of glial activation. under some circumstances glial activation does not resolve, but it exacerbates or becomes chronic resulting in detrimental secondary effects. it is necessary to develop strategies to halt the negative outcome of glial activation in these situations, by controlling the pro-inflammatory phenotype of reactive glial cells and potentiating their beneficial effects. we studied the temporal pattern of inflammatory reaction in spinal cord and brain regions in the experimental autoimmune encephalomyelitis (eae) model of multiple sclerosis. special attention was paid to the involvement of members of the c/ebp family of transcription factors, which regulate the expression of pro-inflammatory genes in reactive glia, and cd , cd r and trem- , membrane-associated inhibitors of the pro-inflammatory response in resting/surveillant microglia. mice were scored for signs of eae for up to days postimmunization (dpi). eae symptoms were present from dpi, reaching score peak at dpi. in the spinal cord, we observed a sustained increase in c/ebpa and a transient increase in c/ebpb and c/ebpd expression peaking at dpi. cd expression decreased at dpi, remaining below control values at dpi. cd r and trem- expression increased at dpi, thereafter this effect progressively attenuated. no alterations were observed in the brain. an acute increase in the expression of pro-inflammatory genes (il- b, il- , tnf-a, inos, cox ) occurred in the spinal cord at dpi. our results suggest that cd expression decreases before onset of eae symptoms resulting in downregulation of the microglia inhibitory signal mediated by cd -cd r , which would facilitate the proinflammatory response observed after onset of the eae symptoms. the increase in cd r expression observed after the onset of eae symptoms suggests a concomitant intend of microglia/macrophages to resolve the inflammatory response. the transient increase in c/ebpb and c/ebpd expression could be related to the acute increase in proinflammatory gene expression, while the sustained increases of c/ebpa expression and trem- could be involved in the resolution of the inflammatory response. recently, we showed a beneficial effect of myelin reactive t cells on oligodendrocyte precursor cell differentiation in zones of axonal degeneration in the hippocampal dentate gyrus, which mirrors grey matter injury in multiple sclerosis. this effect was associated with a marked expression of t-cell cytokines, such as interferon-c and interleukin- , enhanced microglial clearance of myelin debris, and an enhanced sprouting of calretinergic axons. to gain better insight in which cytokines and signaling pathways are elevated in response to the t cell enhanced regenerative responses, we performed a mrna microarray study, in which the transcript profile in hippocampi from mice with adoptively transferred proteolipid protein specific t cells combined with an axonal lesion were compared to the transcript profile from mice with axonal lesion. thereby we identified transcripts, which were differently expressed. we identified the interleukin- (il- ) signaling pathway as a potential target by performing pathway analysis using david and amigo databases. we discovered that il- a, il- b and il- receptor antagonist (ra) mrna as well as the il- signaling pathway were highly upregulated in response to myelin reactive t cells. in order to determine if il- b mrna was translated into protein, we performed immunohistochemical stainings on tissues from mice with days post lesion survival. expression of il- b was observed in the molecular layer of t cell infiltrated, but not in t cell na€ ıve mice with axonal lesion, suggesting that myelin reactive t cells stimulate il- b protein expression. ongoing studies will determine the expression of il- a and il- ra protein and determine the cellular expression of the il- signaling pathway in t-cell infiltrated versus non-t cell infiltrated mice with axonal lesion. understanding the regulation of the expression of il- a, il- b and il- ra in t cell compared to non-t cell infiltrated cns may lead to a better understanding of the functional consequences of inflammatory responses in the cns. when io were challenged with kainate in the presence of conditioned medium (cm) from astrocytes, susceptibility to kainate-induced cell death was reduced, but cm from l-ap -treated astrocytes reverted kainate toxicity. in contrast, treatment with cm from control or lpsactivated microglia, either untreated or pre-exposed to l-ap , was not able to affect kainate-induced io cell death. to establish whether mglur activation could modulate the antigen presenting activity of microglia, the bv microglia cell line was used. treatment with l-ap ( lm/ h) did not affect changes in morphology induced by activation with lps ( . lg/ml/ h), but significantly reduced the percentage of cells expressing mhc class ii, as detected by flow cytometry. these data suggest that during neuroinflammation, activation of mglur directly on io or through astrocytes and microglia may contribute to a protective effect resulting in survival of the oligodendrocyte lineage. mirnas are small non-coding rna molecules that modulate gene expression at a post-transcriptional level and their role in the regulation of biological processes makes them an emerging class of therapeutic targets. dysregulation of mirna networks has been linked to neurodegeneration and immune dysfunctions and has become a research focus in the context of alzheimer's disease (ad). in this work, we demonstrate that mir- expression is increased in the brain of trg ad transgenic mice, prior to senile plaque formation, and co-localized with intraneuronal app accumulation in the cortex and hippocampus. in view of the pro-inflammatory functions of mir- and aiming at clarifying its role in ad, we evaluated the levels of mir- in ab-stressed astrocytes and microglia cultures, two brain-related cell types involved in neuroinflammation. we show that mir- expression levels are increased in astrocytes and microglia following activation with ab fibrils but not with ab oligomers. these findings correlate with the observed decrease in socs- expression, a mir- validated target, and with the increase in the production of tnf-a, il- b and il- by these cells. importantly, we show that mir- expression is regulated by c-jun, since silencing of this transcription factor led to the reduction of mir- levels following astrocyte and microglia exposure to ab fibrils or lps. c-jun was also found to be upregulated in the trg ad transgenic model since early ages, which can help to explain the observed early increase in mir- expression. overall, our results demonstrate the important role of mir- in ad and show that silencing of c-jun in glial cells may constitute an interesting and promising anti-inflammatory therapeutic strategy towards this disease, by targeting mir- -mediated inflammatory responses. here we demonstrate that tgfb is an important endogenous factor promoting quiescence of microglia in vitro. inhibition of microglial tgfb signalling resulted in a microglia polarisation towards the classical activation state. moreover, tgfb is able to significantly decrease ifnc-induced classical activation of primary microglia and to enhance il -induced alternative microglia activation. we further provide evidence that il -mediated alternative microglia activation is dependent on active tgfb signalling. these results demonstrate the essential functions of tgfb as an important regulator of microglia activation states and further suggest that tgfb might be an interesting therapeutical molecule to modulate microglial functions during development and progression of neurodegenerative diseases. we found that young mice lost weight only in the first h after infection, whereas aged mice exhibited a biphasic pattern of weight loss, with the second phase of weight loss beginning week after infection and continuing for approximately = weeks. aged mice also demonstrated a co-ordination and balance deficit in the static rod test week after infection compared to uninfected or week post infection aged mice, whereas young mice were unimpaired at any timepoint. s. typhimurium infection caused a significant, progressive reduction in open field rearing activity at week and weeks after infection in both young and aged mice. a similar but not significant trend was apparent in novel object performance. forced swim test data showed a trend towards depressive behaviour at week after infection, but not at weeks. analysis of pro-inflammatory mediators in the hippocampus did not indicate significantly upregulated il- b, tnf-a, cox- or cox- transcript at any timepoint, suggesting that microglia might not play a significant role in mediating these behavioural effects, or that the changes in these molecules were too subtle to reliably detect using qpcr in the hippocampus. the effects of ageing on s. typhimurium induced weight and co-ordination changes may be a result of regional differences in the sensitivity of microglia to peripheral infection within the cns or increased sensitivity to activation of neuronal circuits involved in weight loss or co-ordination with age. we have studied microglial cells with anti-iba and anti-cd immunohistochemistry in the brains of subjects with pretangle neuropathology ranging from to years of age. regions examined included the locus coeruleus and surrounding brainstem areas, entorhinal cortex, hippocampal formation, and other parts of the mesial temporal lobe. ten of the subjects (median age ) showed severe neuroinflammation secondary to infectious disease (hiv, sepsis, toxoplasmosis) and cns trauma, while subjects (median age . ) died from non-infectious conditions and lacked neuroinflammatory changes (controls). we also examined two -year-old subjects with down syndrome both of which showed braak stage vi neurofibrillary degeneration, where one had comorbid sepsis and the other one did not. pretangle neuropathology was staged a and b in all subjects, i.e. they exhibited primarily subcortical tau pathology in the locus coeruleus and only sporadic lesions in the transentorhinal cortex. our findings show that rampant microglial activation in the ten subjects with infections and trauma was coincident with the same level of tau pathology as seen in the control subjects, demonstrating that neuroinflammation does not initiate or exacerbate neurofibrillary degeneration. in addition, our findings show that the extent of neurofibrillary pathology seen in down syndrome is unrelated to neuroinflammation since the down subject without comorbid sepsis revealed a complete absence of microglial activation. overall we conclude that neuroinflammation is not causally involved in the development of neurofibrillary degeneration which is most characteristically associated with alzheimer's disease dementia. it has been suggested that peripheral infection/inflammation may play a role in the onset or development of not only neurodegenerative diseases but also depression, autism and chronic fatigue syndrome through inflammatory responses in the brain. to investigate the role of microglia in this hypothesis we used a model of neuroinflammation induced by an intraperitoneal (i.p.) injection of the toll-like receptor (tlr ) agonist, polyinosinic:polycytidylic acid (poly i:c) in rats. as we reported previously an injection of poly i:c (i.p) decreased the daily amounts of spontaneous running wheel activity to $ % of the preinjection levels until day . microglia were morphologically activated in the prefrontal cortex (pfc) until hrs after the injection of poly i:c. pretreatment with minocycline for the consecutive days ( mg/kg/ day) blocked the poly i:c-induced decrease in the running wheel activity as well as microglial activation. quantitative analysis of mrna levels demonstrated that interferon-a (ifn-a) increased in the pfc on both days and . we found in the present study that poly i:c injection induced increases in mrnas relevant to tlr-signaling such as tlr , interferon regulatory factor (irf ) and irf in the pfc. furthermore, direct application of poly i:c to the primary cultured microglia also induced an enhanced expression of ifn-a, tlr , irf and irf . it has been reported that permeability of blood-brain barrier is increased after poly i:c injection (wang et al., ) . therefore, it is possible that the peripheral poly i:c enters into the brain to induce neuroinflammation by activating microglia. interestingly, intracerebroventricular (i.c.v.) injection of primary cultured microglia activated by poly i:c, but not by lipopolysaccharide, effectively produced a significant decrease in spontaneous activity in rats. taken together, these findings suggest that microglial activation is important for poly i:cinduced neuroinflammation through inducing tlr -related genes. roles of each gene in the behavioral changes will be further studied. results: microglia produced nanogram levels of pgrn ( fold higher in microglia than in astrocytes), and pgrn release from microglia was suppressed by the tlr ligands or il- /ifnc, but increased by il- or il- . unexpectedly, while astrocytes stimulated with proinflammatory factors released large amounts of slpi, none were detected in microglial cultures. we also identified mmp- as a pgrn proteolytic enzyme, and slpi as an inhibitor of mmp- -induced pgrn proteolysis in human microglia. conclusions: our results establish microglia as a significant source of pgrn. mmp- and slpi are modulators of microglial pgrn proteolysis. negative and positive regulation of microglial pgrn release by the proinflammatory/th and the th stimuli, respectively, suggests a fundamentally different aspect of pgrn regulation compared to other known microglial activation products. microglial pgrn appears to function as an endogenous modulator of innate immune responses. at pathological condition such as injury or ischemia to the central nervous system (cns), microglial cells and astrocytes become activate and inflammation occurs like proliferate, migrate and secrete some proinflammatory cytokines, il- ß, tnf-a or il- . however, it is unclear that the reason why inflammation is induced in the cns, and whether or not it is necessary for the brain during recovery from pathological situation. to know the molecular mechanisms for inflammation occurs in activated glial cells, we made a stab wound mouse model to the brain. we performed microarray analysis for rna extracted from the brain tissue around stab wound site compared among day after , , and . it revealed that most of the genes from top genes at high expression level around lesion site were concerned in immunological or inflammatory functions. we successfully identified and focused on to osteopontin (opn), which is an inducer of pro-inflammatory cytokine production expressed not only in reactive microglial cells but also in reactive astrocytes around the injured cns. furthermore, we also found receptors against opn functioning in the injured brain. however, functional role of opn in reactive astrocytes is not yet clarified. to analyze the central role of opn in reactive astrocytes, we examined primary culture of astrocytes from opn-deficient (opn/ko) mice and found that the morphology of these cells was unusual. by the stimulation with lipopolysaccharide to the primary culture of astrocytes from opn/ko mice, some of the pro-inflammatory cytokine expression was altered. moreover, reactivity of astrocytes caused by stab wound to the brain was examined using analogue of nucleic acid, bromo-deoxy-uridine (brdu), and clarified that it was decreased in opn/ko mice. from these results, we conclude that opn might play a major role in the activation of astrocytes under inflammation occurred to the cns. here we determined the influence of a hfd on eae. we show that feeding mice with a hfd exacerbates eae severity through the induction of the brain renin angiotensin system (ras), resulting in an increased immune cell infiltration into the cns, oxidative stress and th cytokines. moreover, peripheral inflammation was boosted upon a hfd due to a ras independent mechanism, indicating that a hfd exacerbates neuroinflammation in various manners. these data suggest that nutritional modulation may have an important influence on the course of ms and other neuroinflammatory conditions. the brain's immune privilege has been attributed to a lack of dendritic cells (dc) within its parenchyma and the adjacent meninges implying maintenance of antigens rather than their presentation in lymphoid organs (lo). using cd c-gfp mice, we have recently reported the existence of a cd c/iba- /cd b -population in the juxtavascular parenchyma which extent their processes into the glia limitans, an ideal position for antigen-presentation. therefore, we phenotypically compared them to the cd c /cd population (all isolated with the same protocol used for brain cd c cells) from lung, liver and spleen in healthy mice using -color flow cytometry. we found unique, sitespecific expression patterns of f / , cd , cd , cx cr , ccr , flt and mhc-ii. as described before, two different cd -populations (cd high and cd int ) in the brain can be separated, whereas liver, lung and spleen exhibit a more homogeneous cd high population. a higher percentage of the brain's cd /cd c -cells were f / -positive compared to spleen, liver and lung, but expressed it at lower levels. within the cd int /cd c -population most cells expressed the microglia marker cx cr and ccr low (marker for inflammatory, motile cells). most importantly, compared to spleen and liver, cd int /cd c -cells from the brain almost completely lacked mhc-ii expression and cd high /cd c -cells from the brain have a lower percentage of mhc-ii -cells. since the cd int cells are widely regarded as the resident, intraparenchymal microglial population and cd high as dcs and perivascular macrophages, our data confirm the view that a small subpopulation of microglia (cx cr high , f / ,ccr low ) share established immune phenotypical characteristics of dcs (cd c ). additionally we showed that intraparenchymal cd c microglia are unique in their low expression of mhc-ii. their weak expression of cd is in line with a tolerogenic phenotype. methods: we performed a series of experiments, where we examined the expression of pro-inflammatory factors in resident microglia and infiltrating macrophages after fluorescence-activated cell sorting (facs) at different eae stages. in order to properly discriminate the two cell types we used a set of markers where resident microglia were defined as cd b cd int ly c -, and infiltrating macrophages cd b cd hi ly c . using this protocol we have analyzed the rna expression levels of mhcii (cd , h -aa), co-stimulatory molecules (cd , cd , cd , cd ), pro-inflammatory genes (il- b, tnf-a) and inflammasome-regulated cytokines in these immune cells with quantitative pcr. followed by analysis of mhcii, cd and cd at the protein level with flow cytometry. results: our results indicate that infiltrating macrophages have a pronounced activated (cd and h -aa) and pro-inflammatory phenotype (il- b and tnf-a) at rna level. this is supported with an increase of the expression of mhcii and the key co-stimulatory molecules cd and cd at rna as well as protein level. in contrast, microglia display a decrease in the expression of the co-stimulatory molecules cd and cd at the rna level and do not up-regulate expression of il- b and tnf-a during the progression of eae. conclusion: our data suggest that upon eae, infiltrating macrophages display an inflammatory profile whereas microglia are only mildly immune activated. background: age-related hearing loss (presbycusis) affects half of people by the age of . it has a large impact on quality of life and is associated with accelerated cognitive decline. however, presbycusis is not an inevitable process of aging, suggesting that its progression could be minimised. hearing relies on sound vibrations from the middle ear to elicit movement of the basilar membrane in the cochlea, resulting in excitation of hair cells. spiral ganglion neurons relay the inner hair cell signals to the central auditory system via the vestibulocochlear nerve. sounds are integrated in the central auditory pathway and then processed by cognitive areas of the brain. degeneration of cochlear structures, including hair cells and spiral ganglion cells occurs in presbycusis. in the central auditory system there are alterations in neuronal plasticity and neurodegeneration may occur. chronic inflammation has previously been correlated with agerelated hearing loss in humans, implicating immune cells in the progression of presbycusis. microglia within the central nervous system are susceptible to priming by neurodegeneration or aging, both factors in presbycusis. it is feasible that microglia in the cochlea, an organ with similar immune privilege as the brain, could also become primed. priming increases the chance of microglia becoming activated by subsequent inflammatory events, such as 'flu, which may cause them to contribute to neurodegeneration in the cochlea and central auditory system. hypothesis: our working hypothesis is that as age-related hearing loss progresses, microglia in the cochlea and central auditory pathway will become primed. we predict that systemic inflammation will cause primed microglia to be activated to a pro-inflammatory damaging phenotype, which will increase the rate of cochlea and auditory pathway degeneration and hence hearing loss progression. methods: c bl/ j mice are genetically predisposed to develop age-related hearing loss. young and middle-aged mice will be challenged with lps or saline (control) and microglial phenotype assessed in the cochlea and central auditory system. neurodegeneration will be evaluated at corresponding points in the cochlea and auditory pathway using molecular techniques. conclusion: if our hypothesis is proved this would suggest that rigorous control of infection in older individuals would reduce progression of hearing loss by minimising degeneration in the auditory system. this receptor triggers the response via both myd -and trif-dependent signalling pathways, leading to production of cyto-and chemokines and thereby alarming and attracting the immune cells of the periphery to invade the brain. however, tlr is only fully functional in complex with several co/receptors, such as cd . even though cd has been traditionally considered simply as a lps affinity provider, recent data indicate that cd is also necessary for the endocytosis of tlr , which links tlr to intracellular signalling via the adapter protein trif. our latest data reveal the critical role of this receptor for the profile as well as the magnitude of microglial cyto/chemokine production in response to diverse lps variants. cd increases the sensitivity towards lps in a cell type-specific manner, making microglia far more sensitive to lps than bone marrow and peritoneal macrophages. while striatal applications of low lps doses reveal less incoming neutrophils into a brain of cd ko mice, as compared to wildtypes, high doses lps lead to an excessive neutrophil infiltration. this phenomenon is well correlating with the observations in vitro, where cd absence in microglia stimulated with high doses lps causes an excessive production of selected chemokines, with the highest impact on the neutrophil chemoatractant cxcl . these regulatory activities of cd require its membrane insertion and prolonged functionality, pointing to an involvement of signalling. in order to reveal candidates for such signalling mechanisms, we tested the role of syk and plc. even though these enzymes were described to play a role in cd -dependent signalling in dendritic cells, they have no contribution in microglia, further pointing to a cell typespecific organization of the cd /tlr complex in microglia. importantly, we identified receptor systems that impose influences on cd expression itself, which would thereby determine the extent and impact of a cd -mediated regulation of tlr functions. supported by the dfg (for ). in neurodegenerative disease misfolded proteins and cues released by dying cells can attract immune cells by chemotaxis and alter their phenotype. to better understand and differentiate between the effect of these cues, we aim to elucidate the spatiotemporal immunological events in the brain in vivo. zebrafish are an excellent model system to study leukocytes in vivo, and we previously showed by live imaging how engulfment occurs in the developing zebrafish brain (van ham et al., curr biol ). here we use genetically targeted cell ablation to specifically induce cell death of target brain cells. we subsequently track dying cells and immune cells by single and multiphoton d imaging using fluorescent reporter genes. to analyse immunological infiltrate and resident cells in an unbiased fashion, we use large-scale electron microscopy (em) of complete zebrafish cross-sections in parallel as a complementary approach. we find that within a day after onset of cell death multiple types of phagocytic cells, including microglia, accumulate in areas where cell death occurs, engulfing dying cells. granulocytes, do not migrate towards these dying cells and are not found inside the brain at these stages. we also find total numbers of immune cells, microglia in particular, are increased. in addition to microglia, we find phagocytic peripheral cells to be involved in clearance of dying cells. our findings provide an initial in vivo characterization of the immune response to programmed cell death in the brain, indicating involvement of resident and peripheral immune cells. we show spatiotemporal recruitment of these different immune cell types, and proliferation of microglia. the latter together with the recruitment of peripheral leukocytes are likely triggered to increase capacity to dispose of dying cells efficiently. our results indicate zebrafish provides a model to tease apart basic dynamic properties of immune maintenance of the diseased brain in vivo. our current studies focus on identifying the sequence of immunological events in resolving brain injury in vivo and how this is controlled, to ultimately help identify which of these aspects are harmful during brain damage or promote tissue recovery. in humans, lif and osm both signal through the lif receptor (lifr), however osm can also activate its specific receptor, osmr. lifr signaling promotes the survival of glial cells and neurons, and is thought to limit the development of pathogenic t helper cells while promoting differentiation of protective regulatory t cells. osmr signaling is also neuroprotective, however the effects on immune cells are not yet elucidated. in this study, the immunomodulatory effects of lifr and osmr signaling in humans are investigated. we determined which immune cells express the receptors for lif and osm in healthy donors and compared this to the expression levels in ms patients. we found that in blood of healthy donors approximately half of the monocytes express the lifr and osmr, while - % of the t cells and b cells express the receptors. more importantly, in untreated ms patients higher numbers of t cells and b cells express both the lifr and osmr as compared to healthy controls and treated ms patients. available treatments suppress the immune system, resulting in less activated t and b cells, which could explain the lower receptor expression. indeed, we measured the receptor expression on t cells and b cells after activation and demonstrated this strongly induces expression of both receptors. currently, we are investigating the effect of lif and osm on proliferation, cytokine production and antigen presentation of the different immune cell subsets. as blood circulating immune subsets of ms patients have a higher expression of lif and osm receptors, patients show increased susceptibility for modulation by these cytokines. thus, their immunoregulating properties together with the protection of glial cells and neurons indicates that these cytokines are promising candidates for the treatment of ms and other neuroinflammatory diseases. interleukin- (il- ) and interleukin- (il- ) are key cytokines with an important role in the regulation of the inflammatory and immune responses. in the central nervous system (cns), increased expression of both il- and il- occurs in a wide range of pathological conditions. meanwhile il- is recognized as a cytokine with a dual role acting as a pro-inflammatory or anti-inflammatory signal inducing glial activation, il- is well known by its ability to counteract the inflammatory and immune reactions. the objective of the present study was to evaluate the effects of local production of either il- or il- on the microglial response and the neuronal degeneration induced by facial nerve axotomy, a sterile neuronal injury model. to accomplish that, we used two transgenic mice, gfap-il tg and gfap-il tg, which express either il- or il- under the gfap promoter on astrocytes, i.e. a local production within the cns. unilateral facial nerve mm resection was performed, one set of transgenic and wild-type (wt) axotomized animals were sacrificed at , , , and days post injury (dpi) and cryostat free-floating sections were processed for immunohistochemical analysis. another set of animals were sacrificed at dpi to study neuronal survival. our results showed that, at days after facial nerve axotomy, in comparison with the usual neuronal death observed in wt, selective cns il- production had a detrimental effect on neuronal survival, whereas, il- production was able to reduce neuronal death. these effects on neuronal survival correlated with changes in the expression of different molecules associated with microglial activation such as iba , cd b, cd / , mhc class ii, and some integrins like osteopontin and its receptors (cd and a ), along the different time points after injury. remarkably, when compared with their wt littermates, cd b expression was higher on gfap-il tg mice, whereas remains lower on gfap-il tg animals along all the time-points analyzed. similarly, cd expression, which increases after axotomy in wt mice, was higher on gfap-il tg mice at dpi and dpi, whereas no induction of this molecule was detected on the gfap-il tg axotomized animals. in conclusion, our results indicate that astrocyte-targeted il- and il- production has a direct impact on neuronal survival, glial activation and integrin mediated signaling, producing a specific outcome of facial nerve axotomy. methods: we used bv- cells, a murine microglial cell line. in a first experiment aimed at determining the time course of the induction of expression of the lipoxygenases and receptors, they were incubated with lps. in a second experiment aimed at determining the effects of the resolvins and lipoxin on the production of il- , il- b, il- and tnfalpha, they were firstly incubated with lxa , rvd and rve and then incubated with lps. results: our results indicated that lps only enhanced the expression of alx, the receptor for rve and lxa ( i ) (p < . ). the expression of the -lipoxygenase was also affected by lps (p < . ) and varied during the time course, reaching a maximum after h of incubation (p < . ). the production of the proinflammatory cytokines decreased after h of incubation: il- b with rvd ( nm, p < . ) and rve ( nm, p < . ); il- with rve ( and nm, p < . and p < . ); tnfalpha with rve ( and nm, p < . and p < . ). the production of the anti-inflammatory cytokine il- increased only when cells were incubated with lxa after h ( and nm, p < . ). conclusions: these results suggested a potential role of pufa-derivates in the resolution of inflammation. methods: the expression of a panel of m and m markers on human monocyte derived m and m macrophages was analyzed using flow cytometry. this revealed that cd and mannose receptor (mr) were the most distinctive markers for m and m macrophages, respectively. we next examined the activation status of macrophages/microglia in ms and control tissue using a panel of m and m markers. results: our data show that m markers, were abundantly expressed by microglia in normal appearing white matter and by activated microglia/macrophages throughout active demyelinating ms lesions. m markers, mr and cd , were expressed by myelin-laden macrophages in active lesions and perivascular macrophages. double stainings using anti-cd and anti-mr revealed that % of the results and conclusions: the aim of the present study was to evaluate the specific binding of [ h]pk , a compound targeting tspo, to human tissue sections from patients with ms, and to correlate these findings with presence of tspo microglia in different types of lesions. specific binding of [ h]pk was evaluated using autoradiography, and histological analysis was performed on tissue sections to evaluate lesion type, tspo expression and presence of microglia. there was a clear correlation between level of specific binding of [ h]pk and lesion type, such that there was an increased binding to tissue with active or chronic-active lesions compared to control tissue or tissue with chronic lesions. this was concomitant with an increased tspo expression and increased presence of microglia in tissue sections with active or chronic-active lesions. in conclusion, specific binding of [ h]pk is correlated with lesion type, tspo expression and presence of microglia in tissue sections from ms patients. being astrocytes key regulators of microglial cell activation. during inflammatory response, glial cells secrete cytokines such as tnfa, il b and tgfb, as well as nitric oxide (no), and activate phagocytosis, chemotaxis, increased expression of receptors that bind cytokines and inflammatory ligands. among ligand receptors are induced pattern recognition receptors such as scavenger receptor (srs), including class a receptor (sra), expressed by astrocytes and microglia. we will evaluate if sra has active roles in ab clearance and inflammatory activation of glia. methods: we evaluated the role played by sra in glial cell activation, and the regulatory capacity of astrocytes upon microglia in vitro, assessing production of no griess assay and cytokines by elisa; phagocytosis and activation of activity identity markers of signaling pathways by western blot. results: in primary glial cultures from wild type (wt) and knockout mice (ko) for sra, it was found that lps-stimulated sra ko astrocytes release % more no than sra wt astrocytes. these differences were associated with an extended activation of erk signaling pathway. in contrast, evaluation of lps-induced cytokines production, only wt astrocytes released il b, whereas production of tnfa and tgfb reached similar levels in wt and sra ko animals. the abolition of il b release by sra ko astrocytes appeared to be associated to the inhibition of activation of jnk and p signaling, and to the delayed activation of ijb/nfjb pathway in response to lps. microglia from sra ko mice also showed several differences in response to lps stimulation compared with their wt counterparts. they failed to show activation-associated morphological changes and were unable to secrete il b, although they showed an increased release of no. in terms of activation pattern, sra ko microglia showed increased expression of mhc-ii, indicating that sra could be involved in a mechanism inhibiting mhc-ii expression that has not been previously documented. conclusions: our results suggest that sra participates in the activation of specific inflammatory responses by astrocytes and microglia, and the activation of signaling pathways involved in the production of soluble molecules, being also needed for astrocytes in order to be able to modulate microglia activation. microglia are the resident macrophages of the brain and the first responders to disturbances of brain homeostasis. they exist in one of two broadly defined states, a rather inaptly named "resting state" in which they exhibit a ramified morphology and, upon tissue disturbance, an "activated state" in which they exhibit an amoeboid morphology. the characterization as "resting" is in stark contrast with the frantic morphological changes that ramified microglia undergo in brain slices and in vivo, constantly extending and retracting processes. while the purinergic receptor p y has been identified as a key receptor via which microglial processes are guided to a source of atp or to the site of an injury, the signals and pathways guiding microglial processes in the healthy tissue remain elusive. here, by imaging microglia in acute hippocampal brain slices, we assessed the baseline and targeted motility of microglial processes in the presence of different pharmacological agents targeting purinergic signaling. we found that while bath application of a p y specific blocker alone (psb- at nm) or of a p y specific blocker alone (mrs at mm) did not affect the baseline motility of microglial processes, bath application of a p y / / blocker (mrs at mm) strongly reduced the baseline motility of microglia, leading them to retract most of their processes. moreover, a strong rebound of motility was observed after washing out mrs that did not lead to microglial activation within the time window of the experiment (up to minutes). in experiments measuring the chemotactic response of microglia to a pipette containing mm of atp inserted into the slice, we found that chemotaxis was completely abolished by psb- (bath applied at mm) but not by mrs ( mm). finally, no chemotaxis was observed in response to a pipette containing udp-glucose ( mm), an agonist of the p y receptor. these results confirm that p y alone controls the targeted chemotactic response of microglial processes to atp, with no discernable involvement of p y , or , but suggest that p y , and might collectively play a role in controlling the baseline motility of microglial processes in the healthy tissue. how the different p y receptors interact with each other to control microglial movements remains to be determined. supported by an eu marie curie fellowship, the wellcome trust and the erc. the ubiquitin-proteasome-system (ups) plays a central role in degradation and clearance of short lived regulatory as well as misfolded or damaged proteins. upon stimulation by interferons (ifn) the catalytic subunits b , b and b of the s proteasomes (s-proteasomes) can be replaced with b i/lmp , b i/mecl- and b i/lmp subunits, giving rise to the catalytically active immuno-proteasomes (i-proteasomes), respectively. since i-proteasomes play an important role in clearance of oxidant-damaged proteins that preferentially accumulate upon inflammatory stimulation, and many neurodegenerative diseases, including alzheimer's disease (ad) and parkinson's disease (pd) are associated with a chronic inflammatory reaction, we tested the impact of i-proteasome deficiency on the pathogenesis and progression of ad and pd. while appps mice, a model for ad associated extracellular plaque pathology, exhibited increased levels of i-proteasome subunits in the brain, genetic ablation of the b i/lmp subunit, which significantly impairs i-proteasome function, resulted in a reduction of cerebral plaque burden, indicating a disease exacerbating role of iproteasomes in ad. in contrast, transgenic alpha-synuclein (tgasn) mice, a mouse model for intracellular alpha-synuclein pathology associated with pd, crossed to b i/lmp subunit deficient mice, highlight an increase of aggregated alpha-synuclein accompanied by worsening of pathology in tgasn mice lacking functional i-proteasomes, suggesting a protective role of i-proteasomes for intracellular amyloid pathologies. future studies will aim at resolving the mechanistic underpinnings of the seemingly opposing effects of i-proteasome deficiency on the pathogenesis and progression of extracellular and intracellular protein aggregation to aid better understanding and guide novel therapeutic opportunities for neurodegenerative diseases. in the most severe form x-linked adrenoleukodystrophy (x-ald) is a fatal neurodegenerative disorder with inflammatory demyelination of the brain caused by a deficiency of the adrenoleukodystrophy protein (aldp), encoded by the abcd gene. aldp, a member of the abc transporter subfamily d, is located in the peroxisomal membrane and transports very long-chain fatty acids (vlcfa) as coa-esters for degradation into the peroxisome. currently, the only curative therapies are allogeneic hematopoietic cell transplantation (hct) or genetically corrected autologous hematopoietic cd stem cell therapy (hsct). the success probably relies on the settlement of microglia and perivascular macrophages derived from the myelo-monocytic donor cells in the brain of the patient. therefore, we explored the phenotypes of the major immune cell types derived from the cd stem cell. immune cells from peripheral blood of controls and x-ald patients were isolated by magnetic activated cell sorting (macs). purity of cell isolations was verified by flow cytometry. in purified cells qrt-pcr analysis was used to determine mrna levels of the abcd transporters and gc-ms was used for the quantification of vlcfa levels, a diagnostic marker. the three peroxisomal abc transporters were differentially expressed in cd derived cell types of healthy controls: abcd and abcd mrna were inversely expressed in all cell types, whereas abcd was equally distributed. abcd , the closest homolog of abcd could be expected to compensate for the loss of abcd function. however, the expression pattern of abcd was unchanged in x-ald patients; accordingly, cell types lacking abcd mrna (e.g. monocytes) displayed the most severe biochemical phenotype concerning vlcfa catabolism, whereas cells with substantial abcd expression (e.g. t-lymphocytes) were barely affected. thus, not all investigated immune cells present an intrinsic metabolic defect. therefore, we propose that the beneficial effect of hct and hsct may rely on the replacement of those cells lacking sufficient abcd expression in abcd deficiency. in addition, these findings support the concept that abcd is a target gene for pharmacological induction, as an alternative treatment strategy, to rescue vlcfa metabolism and possibly halt the inflammation in x-ald patients. interleukin- (il- ) is a cytokine that has important functions in inflammatory and autoimmune diseases. little is known, however, about il- in the brain. we analyzed expression of il- in the mouse brain during embryonic and postnatal development. being undetected until late embryogenesis, il- was highly expressed during the first two weeks of postnatal life, after which expression gradually decreased and was then absent from the normal adult brain. astrocytes and oligodendrocyte precursors expressed il- , but not neural progenitors or neurons. the vast majority of il- positive cells displayed nuclear staining. apart from its function as a pro-inflammatory cytokine, a role for nuclear il- , potentially in transcriptional regulation, is also emerging. the important role of neuro-inflammation in traumatic brain injury is being increasingly recognized, and local cytokine production can mediate the inflammatory response. because of its potential for attracting inflammatory cells we analyzed il- expression in a cortical contusion infarction model (cci). we found that il- expression was induced by injury, with a peak of expression three days after cci, and at six days the levels declined. il- positive cells showed an overlap with astrocytic and oligodendrocytic lineage markers, but not with neurons, neural progenitors, endothelial cells or microglia/macrophages. a similar time course for il- expression in human brain trauma was found, using cerebral microdialysis, which allows continuous sampling of the parenchymal concentrations of molecules over several days in vivo. the peak of released il- for the patient analyzed was days post trauma. il- binds to a heterodimeric receptor composed of st and il- racp. following injury of st knockout mice we found a general reduction of inflammatory cells at the site of injury, compared to wild type animals, and there were fewer microglia at the trauma site of mice lacking il- receptors. our data show that il- expression is under tight regulation in the normal brain, but is induced by traumatic injury where is important for attracting inflammatory cells. the small heat shock protein alpha b-crystallin (cryab) is expressed by oligodendrocytes (ol) and astrocytes at several stages of multiple sclerosis (ms) lesions. cryab has been shown to be protective and therapeutic in the eae model of ms, possibly due to its anti-apoptotic functions and regulation of inflammatory pathways. in this study, we further investigated the role of cryab in de-and remyelination in vivo, applying the cuprizone model of demyelination to cryab -/mice. surprisingly, we found that cuprizone-induced lesions are significantly smaller, less severe and less inflammatory in cryab -/mice, compared to wild type (wt) controls. staining for microglia and astrocytes revealed that astrocytes are the main inflammatory cell type that is affected by cryab expression: lesions in cryab -/mice display less reactive astrogliosis than wt controls. conversely, although less severe, lesions in cryab -/mice also contain fewer oligodendrocyte progenitor cells (opc) than in wt mice. in addition, our data suggest that remyelination is less efficient in cryab -/mice than in wt controls and that remyelinated areas in cryab -/mice contain fewer ol than remyelinated areas in wt mice. together, we hypothesize that cryab, due to its anti-apoptotic actions, mediates protection in both astrocytes and ol, enabling on one hand reactive astrogliosis, which contributes to demyelination, and on the other hand ol survival, facilitating remyelination. additional in vitro experiments support this hypothesis, revealing that cryab -/astrocytes are less reactive than wt astrocytes and that sirna-mediated knockdown of cryab affects ol survival. interestingly, analyzing phosphorylation patterns of cryab in cuprizone lesions revealed that cryab is differentially phosphorylated in astrocytes and ol. furthermore, we found that cryab is differentially phosphorylated in astrocytes in active demyelinating ms lesions, indicating that phosphorylation of the protein might underlie its (pathogenic) role in active demyelination. these paradoxical findings not only suggest a role for cryab as a potential target in a regenerative approach for ms treatment, but also point to a possible pivotal role for reactive astrogliosis in cuprizoneinduced demyelination and, more importantly, in early ms pathology. in a comprehensive immunohistochemical survey, we compared cortical ms lesions to those of inflammatory (tuberculous meningitis, rasmussen's encephalitis, b-cell lymphoma, and meningitis) and neurodegenerative (alzheimer's disease) diseases. although complex and fulminant immune responses were seen in many disease cases, primary demyelination was only detected in ms. in search of ms-specific molecular mechanisms, we performed whole-genome microarrays on micro-dissected archival formalin-fixed paraffin-embedded material. apart from cortical ms lesions, we also included brain tissue from patients suffering from tuberculous meningitis (inflammatory control) and alzheimer's disease (neurodegenerative control). additionally, control cases without brain pathology were included. using a restrictive cut-off, we identified genes differentially expressed in ms lesions. more than % of these candidate genes could be assigned to t-cell mediated inflammation, microglia activation, oxidative stress, tissue injury, dna damage/repair as well as remyelination and regenerative processes. subsequent neuropathological analysis confirmed that significantly more oxidatively damaged neurons (stained positive for oxidized phospholipids) were present in cortical ms lesions than in any other examined disease. tunel stainings for the detection of dna strand breaks showed that neurons and oligodendrocytes with tunel-positive nuclei were most abundant in ms lesions containing zones of active demyelination and tissue injury. interestingly, immunohistochemical stainings for radical producing enzymes revealed that oxidative stress-mediated tissue damage in cortical ms lesions seems to be driven by nadph oxidases rather than by nitric oxide synthases. taken together, we were able to show that ms-specific primary demyelination is driven by mechanisms of tissue injury that differ from any other investigated inflammatory and neurodegenerative disease. among these mechanisms, oxidative tissue injury seems to play a major role. f. labombarda , s. gonzalez , i. jure , a. de nicola ibyme/conicet/uba, buenos aires, argentina ibyme/coni-cet, buenos aires, argentina reactive gliosis and inflammatory mediators are implicated in demyelination and secondary damage after spinal cord injury (sci). we have previously reported that after sci, short-term progesterone treatment ( days) stimulates oligodendrocyte precursor cells proliferation and decreases reactive gliosis, whereas chronic treatment ( days) differenciates oligodendrocytes precursor cells into mature oligodendrocytes and enhances remyelination. presently, we further studied whether progesterone was able to modulate the inflammatory reaction and cytokine production by astrocytes and microglial cells. thus, the timecourse mrna expression of pro-inflammatory cytokines (il b, tnfa and il- ) and pro-inflammatory enzymes (cox- and inos) was studied by pcr in real time. results showed that the highest increase in cytokine and pro-inflammatory enzymes mrna production occurred in rat spinal cord h after sci. progesterone treatment significantly decreased the early rise of proinflammatory mediators mrnas at h. as progesterone action in spinal cord involves multiple mechanisms, the role played by the classical progesterone receptor (pr) on the progesterone inhibitory effects on cytokines, was assessed in prko mice. in agreement with data obtained in the rat model, sci strongly stimulated tnfa, il b and il- mrna levels in spinal cord of both wild type and prko mice. however, whereas progesterone treatment inhibited the mrnas of cytokines in wild type mice h after sci, it was ineffective in prko mice, involving pr in the inhibition of cytokines production. finally, we measured astrocyte and microglial cells density by immunohistochemsitry after h of progesterone treatment. the steroid administration significantly decreases gfap and ox- cells, which correlated with cytokine and pro-inflammatory enzymes inhibition. we conclude that progesterone attenuates reactive gliosis and inflammatory reaction, probably reducing the secondary spinal cord damage and favouring remyelination. in the steady state they are rarely observed in the cns parenchyma. however, during cns inflammation they cross the blood-brain barrier where together with microglia (cns-resident apc) they can present antigen to infiltrating t cells. the goal of this study was to compare two subpopulations of microglia: cd c-and cd c with brain dc in terms of promoting different t cell responses. two subpopulations of microglia: (cd c-cd dim cd b ) and (cd c cd dim cd b ) as well as (cd c cd hi) bdc have been sorted from the cns of c bl/ mice subjected to eae. sorted cells were used either for ex vivo proliferation and cytokine assays or for qrt-pcr. our results show that bdc and cd c microglia are similar with regard to ability to induce proliferative t cell response from both primed and na€ ıve t cells. nevertheless, they differ in their cytokine profile and ability to promote cytokine release by t cells. our findings suggest that different subtypes of apc in the cns promote different immune responses. this work has been supported by lundbeckfonden. melanocortin receptor (mc r) is predominantly expressed in the brain and it is the only mcr expressed in astrocytes. our previous results showed that a-melanocyte-stimulating hormone (a-msh) the anti-inflammatory action is mediated by mc r in astrocytes and in the hypothalamus of male rats and that these effects may lead to neuroprotection. we have already demonstrated that ndp-msh (an a-msh analogue) increased brain-derived neurotrophic factor (bdnf) expression through the camp-pka-creb pathway in astrocytes. in the present study we examined the participation of mitogen activated protein kinases (mapk) and phosphatidylinosotol- kinase (pi k)-akt pathways in mc r signaling in astrocytes. we also investigated the effect of a-msh on bdnf expression in vivo in brains of male rats.we preincubated cultured rat primary astrocytes with mapk (p , jnk and erk) and pi k-pdk -akt inhibitors min before addition of mm ndp-msh for h. we found that ndp-msh-stimulating effect on bdnf expression assayed by qrt-pcr was abolished only in the presence of erk and pi k inhibitors. accordingly, levels of phospho-erk / determined by western blot were increased by ndp-msh whereas phospho-akt levels were not modified at min. ndp-mshinduced erk / activation was decreased by adenylate cyclase and pi k inhibitors but not by a pka inhibitor, suggesting a camp and pi k involvement in this effect. we also investigated if a-msh was able to induced bdnf expression in vivo. for that purpose we injected male rats with a-msh ( . mg/kg, ip) or vehicle (saline) once and sacrificed them after h. rats were also injected twice daily and for two days and killed h after the first injection. we observed that bdnf mrna levels increased after a-msh treatment at h in the hypothalamus whereas in the cortex bdnf expression was not modified. at h bdnf expression was not modified by a-msh. we showed by immunohistochemistry that mc r and bdnf co-localizes with neurons and astrocytes in the brain. since melanocortins have anti-inflammatory and neuroprotective effects in the brain, the mechanisms described in astroglia help understand mc r action. our results indicate that melanocortins induce bdnf expression in astrocytes through erk and pi k, suggesting that this effect could be involved in the neuroprotective actions of melanocortins. the fact that bdnf expression also increases in vivo in the hypothalamus reinforce this idea. methods: we analyzed microglia activation and the role of nmda receptors in this process using the microglia cell line bv and cortical slice cultures. the in vivo analysis was performed in two models: first, in odtr mice that express the diphtheria toxin receptor specifically in oligodendrocytes (odcs) which allows induced killing of odcs (locatelli et al., ) and second in mice that were immunized with mog/ cfa to induce eae. results: the proinflammatory cytokine il- a plays a pivotal role in the pathogenesis of ms and eae. we showed il- a activates microglia in vitro. therefore, we activated the microglia cell line bv and cortical slice cultures with il- a to and subsequently treated the cultures with the nmda receptor antagonists mk and ap to block nmda receptor signaling. this treatment inhibited il- a-mediated activation of microglia and in particular ros production, proliferation and migration. additionally, we detected increased secretion of il- and g-csf. furthermore, il- a enhanced activation of the nmda receptor through phosphorylation leading to higher influx of calcium upon ligand binding. to further analyze nmda receptor-depended microglia activation we used two different mouse models that provoke different mechanisms of microglia activation. whereas in eae a strong inflammatory process activates microglia, in odtr mice, activation occurs due to induced odc death. interestingly, in the context of eae microglia activation was clearly diminished when mice were treated with mk whereas inhibition of the nmda receptor had no effect on microglia activation in the odtr model. in line with the in vivo data we show that inhibition of microglia activation takes place only when the cells were stimulated with il- a and not upon stimulation with lps. conclusions: our findings show that nmda receptors are not involved in general microglia activation but rather play a role in microglia activation in an inflammatory context with specific cytokines such as il- a. aims of the study: to investigate whether cx cl -cx cr signaling contributes to microglia migratio ctivation and subsequent astrogliosis following msc transplantation in the cns. methods: first, in order to allow localization of grafted msc in vivo, wt c bl/ msc were transduced with a lentiviral vector encoding the blue fluorescent protein (bfp). next, bfp msc were injected into the cns (striatum) of cx cr /-(n ) and cx cr -/-(n ) transgenic mice. these mice have respectively one or both copies of the cx cr gene replaced by the egfp reporter gene. at days posttransplantation, histological analyses were performed to determine iba microglia and s b astrocytes within and surrounding the bfp msc graft site. astrogliosis was determined based on staining for gfap. cx cr expression was evaluated based on egfp expression. all histological evaluations were quantified using tissuequest and/or imagej cell analysis software. results: (i) bfp msc graft survival is observed in both cx cr -/and cx cr /mice; (ii) microglia migration towards grafted msc occurs independent of functional cx cr -cx cl signaling; (iii) down-regulation of egfp gene-expression (and thus also cx cr gene expression) is observed in both cx cr -/and cx cr /mice on msc graft-invading microglia, but not on msc graft-surrounding microglia; (iv) the absence of functional cx cr -cx cl signaling in cx cr -/mice results in significantly less astrogliosis around the msc graft site, as compared to the msc graft site in cx cr /mice. conclusions: here we identified cx cr -cx cl signaling as a potential target to modulate and/or control astroglial scarring following (stem) cell grafting in the cns. the latter is of significant importance as grafted cells can only functionally interact with (injured) brain tissue in the absence of a graft-surrounding astroglial scar. this accumulation is known to be essential for the usual sprouting of injured axons and leads to a functional nerve repair. because of the insignificant infiltration of macrophages in the injured leech cns, we investigate the chemotactic mechanisms of the recruitment of only resident microglial cells in order to explore in fine the crosstalk between damaged neurons and activated microglia leading to the leech cns repair. methods: in vitro and ex vivo chemotactic assays were performed by using recombinant form of hmc q on microglial cells which were pre-incubated or not with anti-cc qr or anti-gc qr antibodies. then, affinity purification analyses using biotinylated c q were performed from leech microglial cell protein extracts. finally, immunohistochemistry analyses were located the production of newly characterized c q receptors in microglial cells. results: we demonstrated that rhmc q contributes to the recruitment of some leech microglial cells. chemotaxis assays showed that the blocking of respective receptors, homologous to human gc qr and cc qr, inhibits the hmc q-dependent recruitment. importantly, affinity purification analyses demonstrated the interaction between both receptors and hmc q. finally, those receptors were located on distinct microglial subsets. conclusion: this work is used to specify the activation processes of only resident microglial cells. the results show the importance of hmc q in the microglial accumulation leading to the nerve repair. in h. medicinalis, hmc q activity is driven through two different receptors which are not present on the same cells suggesting that hmc q activates two distinct microglial cell subpopulations. the question whether blood-borne immune cells are infiltrating brain areas afflicted with neurodegeneration in ad and other tauopathies yielded contradictory results. some authors showed that the chronic neuroinflammation in ad was provided almost exclusively by resident cns cells without any apparent influx of leukocytes from the blood while others reported that hematopoietic cells can enter the brain in alzheimer's disease and may contribute to an increased inflammatory burden. in order to address this issue we have used two independent transgenic lines expressing human misfolded truncated tau in two different genetic background (wistar and shr). we have shown that tau neurodegeneration can induce inflammatory responses mediated by reactive microglia in both transgenic lines, however the microglial responses showed a striking difference between the lines. we have identified two significantly different inflammatory responses to the same inducer. moreover, we found that the genetic background significantly modified the molecular cascade influencing leukocyte influx into the brain. in both transgenic lines, the majority of the blood cells entering the brain belonged to antigen presenting cell family -monocytes and dendritic cells. at the proteomic level we found striking differences between the lines. while in the wistar transgenic line we observed significant increase of cytokine-induced neutrophil chemoattractant- , in shr transgenic line, tissue metalloproteinase was significantly reduced. these findings suggest that blood cell infiltration of the brain affected by tau neurodegeneration is modified by genetic background. this inter-individual variability should be taken into the consideration in the development of novel drugs with anti-inflammatory properties. acknowledgement: this work was supported by axon neuroscience and research grants vega / / , / / , / / , apvv - , apvv - and structural fund . charit e -universit€ atsmedizin, berlin, germany toll-like receptors (tlrs) are key molecules of the innate and adaptive immune response in vertebrates. the original protein toll in drosophila melanogaster regulates both host defense and morphogenesis during development. tlrs recognize host-and pathogen-derived stimuli. single-stranded rna is sensed by tlr localized to the endolysosomal compartment of immune cells. we found that both extracellular viral rna and host-derived micro-rnas induce cell-autonomous and microglia-mediated neuronal cell death through the endosomal receptor tlr in vitro and in vivo. however, the exact intracellular signalling cascade and the cellular mechanisms through which tlr leads to tissue injury in this context remained unclear. unc b is a molecule specifically involved in trafficking of nucleotide-sensing tlrs, such as tlr , in immune cells of both humans and mice. unc b physically interacts with this receptor in the endoplasmic reticulum (er), and the function of the membrane protein unc b is to deliver the nucleotidesensing receptors from the er to endolysosomes. making use of real-time pcr, immunocytochemistry, and histochemistry we systematically examined the expression of unc b in the murine cns. whereas a distinct expression for this molecule was observed in microglia and astrocytes, expression of unc b in cultured neurons was negligible. however, expression of this molecule was detected in cortical neurons of brain sections. moreover, unc b was strongly regulated during different embryonic, postnatal, and adult stages of the developing mouse brain. neurons of various brain regions were identified as the main cell type expressing unc b in the developing brain. taken together, our data reveal a specific expression pattern of unc b in the cns, in particular in a developmental context, and lay foundation for further investigation of the pathophysiological significance of this molecule for both injurious and developmental processes in the central nervous system of vertebrates. has benefited from a number of studies that precisely depicted different types of plaques in white or grey matter areas. also, both inflammation and diffuse axonal loss in the normal appearing white matter (nawm) were extensively described. however, little attention was given to the so-called periplaque, which is usually defined as a partially demyelinated ribbon of tissue surrounding the plaque. whether periplaques correspond to expanding lesions, sites of ongoing remyelination or areas of tract degeneration remains uncertain. methods: in this context, our study aimed to bring quantitative insights to the neuropathology of ms periplaques in the spinal cord of primary or secondary progressive ms patients. a neuropathological quantitative assessment of inflammation, axonal loss and myelin loss was performed concurrently in the periplaques, plaques and normal-appearing white matter (nawm) of cervical spinal cord samples from patients with progressive ms. results: periplaques formed large areas of incomplete myelin loss that extended distant away from the border of plaques. axonal loss was quantitatively similar in plaques and periplaques but signs of axonal dystrophy were predominantly observed in plaques. surprisingly, axons that remained myelinated in periplaques presented a thicker myelin sheath than myelinated axons of the nawm. inflammation in the periplaque was mainly characterized by an accumulation of macrophages/microglia that were closely apposed to myelin sheaths but exerted poor phagocytic activity. finally, we found that neuropathological features of periplaques were overall disconnected from that of plaques with regard to size, shape, inflammation and axonal integrity. conclusions: our work indicates that in ms spinal cords, periplaques correspond to demyelinating rather than remyelinating lesions. it further suggests that in progressive forms of ms, periplaques are likely to impact significantly on neurological disability. finally, we propose that tract degeneration might not be the only cause of periplaque extension and that slowly-expanding demyelination, temporally remote from plaque formation, might occur in periplaques. molecular results will be presented that support these hypotheses. cell-adhesion between endothelial cells at the blood-brain barrier (bbb) makes the endothelium impermeable to blood-derivatives and immune cells. to establish and maintain this barrier during development, adulthood, and during disease, brain endo-thelial cells must develop and sustain these strong adhesive contacts, through expression of tight junction molecules. however, we do not know whether netrin supports inter-endothelial cell adhesion at the blood-brain barrier. given this, we hypothesize that netrin tightens the bbb during development, adulthood, and protects it during disease. methods: to test this, we used both human adult primary brainderived endothelial cells and newborn netrin- knockout mice and evaluated netrin's effect on inter-endothelial cell adhesion and barrier permeability. we also assessed netrins' therapeutic potential to maintain the barrier and limit immune cell infiltration into the central nervous system (cns) during experimental autoimmune encephalomyelitis (eae). results: our results demonstrate that brain endothelial cells express netrins. they help to form a tighter bbb during development. they also maintain and protect the adult barrier by increasing the expression of endothelial junction molecules, thus promoting inter-endothelial adhesion and reducing protein leakage across the barrier. netrins also reduce bbb breakdown and diminish initial myeloid cell infiltration into the brain and spinal cord during eae, which delays disease onset and ameliorates disease severity. discussion: we conclude that netrins enhance bbb stability, and protects the bbb during neuroinflammatory disease. microglial cells were shown to play an important role in many diseases of the central nervous system, not least due to their capability to become activated upon signs of brain injury. they migrate to lesion sites, proliferate, release cytokines and chemokines, and phagocytose cells or cellular debris. therefore, the microglial cytoskeleton has to be highly rearrangeable. one major element of the contractile system in nonmuscle cells is nonmuscle myosin ii (nm ii). as the role of nm ii in glial cells is poorly characterized, it is an aim of this project to study nm ii-dependent functions in microglia. we found that inhibition of nm ii by blebbistatin, which is a highly selective inhibitor of nm ii adenosine phosphatase, prevents any morphological shaping in freshly plated microglia and leads to functional deficits during migration and phagocytosis of fluorescence-labeled beads. using confocal microscopy, we could find diffuse expression of nm ii in resting microglia in vitro, whereas activation with bacterial lipopolysaccharide (lps) led to perinuclear accumulation of nm ii protein. in an activated state, microglial cells release pro-inflammatory cytokines and reactive oxygen species; interestingly, in the presence of blebbistatin the release of nitric oxide (no) as well as rearrangement of nm ii was prevented. taken together, these results illustrate a key role for nm ii in microglial shaping and functioning. ongoing studies will improve our knowledge on spatial and functional aspects of the actomyosin complex during demyelinating processes and open targets for development of therapeutic strategies towards remyelination in the cns. prolonged activation of the nucleus basalis of meynert (nbm), the primary source of cholinergic projection to the cerebral cortex has been reported to cause significant increases in cerebral cortical blood flow (cbf) in rodents. while the nbm also gives rise to gabaergic and glutamatergic projections to the cerebral cortex, the nbm-driven increase of cbf has been described to be dependent in part on muscarinic acetylcholine receptors. lately, several groups reported that astrocytes modulate local cbf via intracellular ca signaling. considering that cortical astrocytes express machrs and in vivo activation of the nbm leads to machr-dependent ca surges in astrocytes, cholinergic modulation of cbf via astrocytic ca surges is conceivable. we report here that a single train stimulation of the nbm (stnbm; hz, . s, ma) induces a biphasic (a rapid increase, followed by an overshooting slower decrease that goes back to baseline within a minute) laser doppler flowmetry (ldf) response in the somatosensory cortex. moreover, the stnbm-induced ldf response was sensitive to the machr antagonist atropine. stnbm with a weaker stimulation intensity ( ma) induces a transient decrease. surprisingly, we find that ip r knockout mice, which lack cytosolic ca surges in astrocytes, show similar ldf responses to stnbm. a.-c. boulay, c. giaume, m. cohen-salmon cirb, paris, france astrocytic endfeet are specialized processes, which enwrap blood vessels and express a large molecular repertoire dedicated to the physiology of the vascular system. one of the most striking properties of astrocyte endfeet is their enrichment in the gap junction proteins connexin (cx) and cx allowing for direct intercellular trafficking of ions and small signaling molecules through perivascular astroglial networks. however, the role of these proteins at the gliovascular interface is not fully understood. we have recently demonstrated that astroglial perivascular cxs expression starts after birth during bloodbrain barrier maturation and controls its integrity (ezan et al. cx d/d purified vessels showed essentially a strong upregulation (fold change > ) of sgcg. this gene encodes gamma-sarcoglycan, a membrane glycoprotein associated with the dystrophin-dystroglycan complex which is essential for muscle integrity and is impaired in duchenne muscular dystrophy (petrof et al., ) . at the gliovascular interface, the dystrophin-dystroglycan complex anchors astrocyte endfeet to the basal lamina (nico et al., ) , and is crucial to the bbb integrity. however, expression of sgcg and its role in the brain has until now only been poorly documented. moreover, we found an upregulation of s a and s a (fold change , and , , respectively) encoding the calgranulin a and b, two proteins present in neutrophils and monocytes, and involved in their migration to inflammatory sites, attachment to endothelial cells and extravasation (ryckman et al., ) . altogether, our results suggest that cx might modify the dystrophin-dystroglycan complex thereby influencing blood-brain barrier properties, and control the attachement as well as the transmigration of leukocytes through the endothelium, thus contributing to the regulation of inflammatory processes in the brain. the blood-brain barrier is supported by the perivascular glia. a crucial event during the gliogenesis is the replacement of radial glia by astrocytes. immunohistochemical staining of nestin visualized the first gliovascular connections after e , and by e the radial glial endfeet cover the vessels completely. by e numerous nestin immunoreactive astrocyte-like cells participated in it. in parallel, nestin immunoreactivity also appeared in the wall of the vessels. by about p both radial glia and nestin immunopositive astrocytes disappeared and gave place to astrocytes immunoreactive to s protein and glutamine synthetase, markers of mature astrocytes. in the radial glia these markers were not found, and they colocalized with nestin only scarcely. the s and glutamine synthetase immunoreactive cells appeared at first in the middle zone of the pallium about p , and their population gradually extended to both the pial surface and the deep cortical zones, colonizing the whole thickness by p , in parallel with the disappearance of nestin immunoreactive glial elements, either radial or astrocytic ones. at p gfap immunoreactive cells were still confined to the most superficial and deepest cortical zones, so showed no colocalization with s and glutamine synthetase, unlike that found after p . whether this glial 'takeover' results in a transitory increase of leakage of the blood-brain barrier, it remains to be answered. it seems however, to underly that cerebrovascular fibronectin and laminin immunoreactivities disappear at first from the middle zone of the developing cortex and only later from the superficial and deep ones, between p and p . according to krum and rosenstein ( ) disappearance of these immunoreactivities refers to the fusion of the glial and vascular basal laminae, the maturation of the definite gliovascular connections. surrounded by astroglial scars that inhibit such growth. little is known about how astrocytes assemble to form this scar, and a deeper understanding of the cellular and molecular mechanisms that control this process is needed if this goal is to be achieved. in vitro data suggest that certain bioactive molecules can alter astrocyte morphology into a more growth supporting state. other molecules have been shown to promote axon growth. clinical application of many of these molecules would require neurosurgical delivery directly into the spinal cord. injectable biomaterials have the potential to serve as depots and scaffolds for in vivo delivery of bioactive molecules. we are developing di-block co-polypeptide hydrogels (dch) as fully synthetic biomaterials that could safely and easily be injected into specific sites after sci to deliver molecules in order (i) to understand better, and (ii) to manipulate, the mechanisms that control glial scar formation. we have previously shown that dch are biocompatible after injection into brain or spinal cord and selfassemble into structures with finely controllable properties. our current work shows that dch depots can deliver diffusible bioactive growth factors that influence local neurons or astrocytes in predictable ways and form gradients that are effective up to several mm away from depots. we are now testing the ability of dch depots to deliver specific growth factors that will manipulate scar-forming cells when dch are injected at clinically realistic sub-acute times after sci. to investigate the molecular phenotype of esdm, we performed a whole transcriptome analysis of esdm in comparison to primary cultured microglia, flow cytometry-sorted microglia, different myeloid cells, t cells, astrocyte-and neuron-enriched cultures. while being distinct from t cells, neurons and astrocytes, esdm were closest related to primary cultured microglia followed by flow cytometry-sorted microglia and other myeloid cells. esdm and primary cultured microglia showed a strong overlap in their transcriptome by only having gene transcripts differentially expressed. most of these differentially expressed genes were immune-related genes that were up-regulated in primary cultured microglia. this indicates that esdm were in a less activated state rather reflecting the in vivo biology of these cells. flow cytometry analysis of cell surface markers selected from the microarray confirmed the close relationship between esdm and primary cultured microglia. esdm resemble primary microglia not only in respect to surface marker expression and functional properties, but also display a molecular phenotype similar to primary microglia. our data suggest that esdm are a reliable tool for the replacement of primary microglia. spinal cord injury (sci) is a devastating condition which usually leads to paralysis. this outcome of injury in most cases is permanent due to the cascade of immunological factors that create an anti-repair environment around the site of injury and due to the limited capacity of central nervous system (cns) axons to regenerate, creates a daunting challenge to establish an effective therapy. from many years of research it is apparent that a combination of therapies would be the best course of action for treatment of sci; this however greatly increase the animals cohorts and time to establish the most effective course of treatment. for this reason we have developed an in vitro model of spinal cord injury as a moderate throughput screen, which is in accordance with the r's. this culture consist of dissociated mixed rat embryonic spinal cord cells plated onto a monolayer of astrocytes this is essential for the spinal cord cells, obtained from rats of embryonic day , which then develop into a carpet of neurites that over time become myelinated forming internodes of myelin interspaced with nodes of ranvier. this mixed cns culture was utilized using a scalpel blade to axotomise the axons and a persistently neurite-free area is created which is accompanied by many features of sci, including demyelination and reduced neurite density adjacent to the lesion and the presence of and reactive astrocytes. these finding are congruent with changes seen in vivo after spinal injury. data will be presented regarding the effects from proven and novel pharmacological agents on neurite outgrowth and myelination looking to understand the mechanism in which they act. as well as modifications of the culture system to incorporate methods to align axons and isolate cell bodies from their processes for a more detailed analysis of regeneration. the role of increased activity of astrocyte networks in structural synaptic plasticity following remote axonal injury is unexplored. we set out to investigate the efficiency of synaptic rearrangements in models where astrocyte reactivity is selectively diminished by the inhibition of the main cytokine pathway in transgenic mice (tg) in comparison to that observed in wild type mice (wt). first, synapse densities were compared in the facial nuclei two weeks after unilateral facial nerve transection by synaptotagmin- /psd- immunolabelling and ultrastructural analysis. we found that the recovery of synapse density was decreased by % in tg mice compared to wt mice, and that the reduction in synaptic density correlates with a relative loss of neuronal coverage by reactive astrocyte end-feet in the same conditions. specifically, we observed a % reduction in excitatory synapse density in the tg mice. second, we developed an organotypic entorinho-hippocampal slice culture system in which astrocyte activation and synaptic plasticity could be more closely monitored in real time. this was assisted by two-photon microscopy, using glial ca -imaging and dendritic spine motility analysis after perforant pathway transection. in summary, we demonstrated that full astrocyte activation is necessary for partial structural recovery of synapses after axonal injury. our work, using simplified models, may help experimental approaches aimed at optimizing adaptive plasticity in cns injuries. pathological loss of myelin in diseases like multiple sclerosis (ms) is usually followed by a phenomenon of remyelination, in which oligodendrocytes synthesize new myelin sheaths to envelope exposed around axons in the adult central nervous system (cns). the importance of remyelination arises from the fact that this process not only restores saltatory conduction, but also protects axons from different insults and thus limits clinical disability in demyelinating diseases. in this scenario, the mammalian subventricular zone (svz) has garnered attention as a potential source of replacement cells after injury. this zone harbours stem cells and supports long-distance migration, and is activated in ms patients to promote gliogenesis. although ng precursor cells are the first to react to demyelination and show the highest proliferation rate in comparison with other cells, the relative contribution of the svz with respect to the inflammatory demyelination induced by the theiler's virus infection and oligodendrogenesis has never been addressed. in the present study, we have investigated the behavior of the svz in theiler's murine encephalomyelitis virus -induced demyelinating disease (tmev-idd). we report that in this viral model for ms, there is a preclinical phase of the disease with demyelination in the corpus callosum that is followed later by an attempt of remyelination. this phase is accompanied by an activation of the svz with no apparent activation of ng precursors, and a strong and clear stain- galectins (gals) are a family of soluble lectins that, when binding to b-galactosides, form multivalent complexes with glycoconjugates of the cellular surface and induce a modulation of intracellular signaling pathways for differentiation and survival. recently, galectin- has been described as a microglial deactivator which prevents neurodegeneration and promotes neuroprotection in an eae model. however, its action at neuronal level is poorly understood. spinal cord injury (sci) also remains a major challenge to neurological research, as several factors such as extracellular matrix molecules inhibit axonal regeneration. among them, semaphorine a (sema a) contributes to the inhibition of axonal regeneration by acting on microtubules and actin cytoskeleton. neuropilin- (nrp- ) is a neuronal receptor whose binding of sema a generates repulsion of axonal growth. in addition, nrp- has been described as a target of gal- in non-neuronal systems. the aim of this work was to study the role of gal- intraumatic sci, considering that sema a expression is "turned on" in sci and prevents axonal regeneration via nrp- . gal- knockout mice (lgals -/-) were submitted to a full traumatic sci at thoracic level (t -t ) and treated with different concentrations of recombinant gal- . we demonstrated that lgals -/mice treated with gal- have a dose-dependent functional recovery of motility as early as days post-treatment, which is structurally associated to neuronal repopulation and high axonal regeneration. these effects were only observed in animals treated with gal- with dimerizing capacity, but not in mice treated with a mutant gal- , only present in a monomeric form. in addition, we observed that microglial response was "turned off" in gal- -treated mice. moreover, exogenous dimeric gal- bound to the surface of injured motoneurons and promoted the spread of nrp- . d confocal microscopy reconstruction showed gal- and nrp- spatial proximity. to confirm this result, we carried out immunoprecipitation assays, which allowed us to determine that only the dimeric form of gal- interacted with nrp- and induced a decrease in the levels of sema- a bound to nrp- . our results show that gal- -based treatments combine its neuronal effect on nrp- with its deactivating effect on microglia, which leads to a better restorative process after medullar lesion. the findings presented here support the potential of dimeric gal- as a therapeutic agent for human sci patients. theophylline and caffeine. it depresses activation of microglial cells and astrocytes which is associated with neuronal damage during inflammation and hipoxia. it is largely known that ethidium bromide (eb) injection into the cns induces local oligodendroglial and astrocytic death, resulting in primary demyelination, blood-brain barrier disruption and schwann cell invasion, and thus serving as an experimental demyelinating model in animals. the aim of this study was to evaluate if prop had the capacity of affecting glial cell behaviour during the process of demyelination and remyelination following gliotoxic injury with eb. wistar rats were divided into groups: i-rats injected with microlitres of . % eb into the cisterna pontina and treated with prop; ii-rats injected with eb and not treated with the xanthine. prop (agener) treatment was done using . mg/kg/ day by intraperitonial route during all experimental period. the rats were euthanized from to days after eb injection and brainstem sections were collected and processed for light and transmission electron microscopy studies. results from both groups were compared by using a semi-quantitative method developed for documenting in semithin sections the extent and nature of remyelination in gliotoxic lesions. in general terms, by - days following gliotoxic lesion, the center of the lesion was expanded and filled with huge amounts of myelin debris among foamy macrophages and demyelinated axons. no astrocytic processes were found in this site and some lymphocytes were seen in the neuropil and around blood vessels. the most prominent feature of the th day was the initial association at peripheral locations between naked axons and remyelinating cells. schwann cells were associated with one or multiple demyelinated axons or already forming thin myelin lamellae around single axons in astrocytic-free areas. oligodendrocytes began to form thin myelin sheaths in areas completely or partially filled with astrocytic prolongations. by - days, it became evident that rats treated with prop presented a small improve in the repair process when compared to untreated animals, the latter presenting greater amounts of myelin-derived membranes in the central area and a lesser extension of oligodendrocyte remyelination, but without any apparent difference regarding to schwann cells. by days results showed that prop administration following eb injection seemed to stimulate oligodendroglial remyelination (mean remyelination scores of . peripheral axotomy results in a disconnection of the neuronal cell body from the target organ. a complex rearrangement of the nerve microenvironment, the so called wallerian degeneration, takes place distally to the lesion and involves schwann cell proliferation as well as macrophage infiltration. the process results in the formation of the bands of b€ ungner which guide the axonal sprouts throughout the nerve distal stump. although this process has been extensively studied, a thorough understanding of the three-dimensional nerve tissue reorganization is not completely known. such knowledge may in turn lead to more effective strategies aiming at the repair of peripheral nerves following transection or crushing. in this sense, the tetrahydrofuran (thf)-based tissue-clearing technique has been adapted for sciatic nerves of mice and used for subsequent observation to p-lsm. transgenic mice that express fluorescent proteins in neurons, astrocytes, schwann cells and microglia/macrophages as well as double and triple hybrids were used. additionally, by immunohistochemical analysis, it was possible to observe the structure of the nerve after injury by the expression of neurofilament and s b, in non fluorescent mice. also, increased non neural cell responsiveness after injury was studied with anti-p ntr immunostaining. transmission electron microscopy allowed the evaluation of the ultrastructure of the nerve microenvironment, as well as the regenerative process by , and days after injury. microscopic examination of cleared tissues of transgenic mice through p-lsm consisted in comparing the different cell type interaction before and after injury, with particular interest on the schwann cells, macrophages and growing axons. question: mass spectrometry (ms) has become nowadays an essential tool for the detection and identification of biomolecules in various contexts of biological problematic. ms can be performed from all types of biological materials ranging from biological fluids to tissues. end of 's it was shown that with certain instrumentation such as those equipped with a maldi ion source it was possible to record ms spectra directly in situ from tissue sections. more, point to point automated acquisition of maldi ms spectra from a whole tissue section was shown to allow for reconstruction of the d images of biomolecules contained in the section distribution through ion density maps (caprioli rm et al., anal. chem. ). methods: maldi ms imaging (maldi msi) is a molecular imaging modality that presents several advantages including possible imaging of hundreds of biomolecules in one step acquisition and the untargeted character of the method allowing to study molecules without any prerequisite knowledge or use of probes. results: over the past decade maldi msi has demonstrated its potentiality in terms of applications for biology and clinics (franck j et al., mol. cell. proteomics. ). maldi msi can be use to study both endogenous molecules namely metabolites, lipids, peptides and proteins or exogenous ones such as drugs or xeniobiotics and was applied to various problematic. understanding of physiological or physiopathological processes requires knowledge on the identity and localization of molecules within a tissue. maldi msi reseals a clear potential to answer this objective and give a unique contribution. indeed, more recently maldi msi has gained new strategies for identification of molecules in situ on tissues. in particular, many efforts were given to develop confident identification of proteins from tissue sections. conclusion: here we will review this novel technology and discuss some of its current and potential contributions for neurosciences application (wisztorski m et al., dev. neurobiol. ). this will be illustrated by presentation of the investigation of invertebrate neuroimmune response as well as vertebrate neuropathological disorders using either fresh frozen or formalin fixed paraffin embedded tissues. the sphingosine -phosphate (s p) signaling pathway is known to influence astroglial reactivity in experimental autoimmune encephalomyelitis and the synthetic s p analog fty has been shown to provide neuroprotection in experimental models of acute stroke. however, the effects of a manipulation of s p-s p signalling at later time points after experimental stroke have not yet been investigated. we examined whether a relatively late initiation of a fty treatment has a positive effect on long-term neurological outcome with a focus on reactive astrogliosis, synapses and neurotrophic factors. we induced photothrombotic stroke (pt) in adult c bl/ j mice and allowed them to recover for three days. starting on post-stroke day , mice were treated with fty ( mg/kg b.i.d.) for days. behavioral outcome was observed until day after photothrombosis and periinfarct cortical tissue was analyzed using tandem mass-spectrometry, taqman v r analysis and immunofluorescence with especial emphasis on markers of astroglial reactivity. fty treatment results in a significantly better functional outcome persisting up to day after pt. this is accompanied by a significant decrease in gfap-immunoreactivity (-ir) and an increase in psdsize. however no change in gfap-mrna, gs-ir or cs -ir was observed. while fty -treatment leads to a decrease in s p concentration, it increases vegf-mrna in the periinfarct cortex. the initiation of fty treatment in the convalescence period has a positive impact on long-term functional outcome, probably mediated through reduced astrogliosis, a modulation in synaptic morphology and an increased expression of neurotrophic factors. due to the restrictions in regenerative capacity of the brain the tissue most severely damaged after traumatic brain injury (tbi) will end up as a fluid filled cavity. presently, it is an impossibility to effectively restore lost brain tissue, but ongoing research on the stimulation of endogenous neural stem cells has increased the hope to viably and functionally repopulate the injured parenchyma. however, it is crucial that possible therapies can show a long-term effect on both regeneration and functional recovery to be of clinical interest. in this study the enhanced induction of neurogenesis in rats after experimental tbi was evaluated three or six weeks after injury. severe focal tbi was performed using the controlled cortical impact model after which a combination therapy of intracerebroventricular administration of epidermal growth factor (egf) for seven days followed by deposition of extracellular matrix scaffold, containing vascular endothelial growth factor, into the cortical cavity. the treatment was devised to accomplish an optimal effect on the stem cell regeneration. the animals with six weeks survival time were functionally evaluated using the morris water maze (mwm). before sacrifice the animals were injected with bromodeoxyuridine (brdu) to identify newly generate cells. sections were made and immunohistochemically double stained for brdu and cell type markers for neurons, astrocytes and blood vessels. the sections were micrographed and analyzed for hemispheric tissue loss as well as number of new astrocytes, neurons and blood vessels. three weeks after injury there was a significant treatment effect as shown by an increase in neuronal and astrocytic regeneration. however, after six weeks there was no difference in the number of newly generated neurons and astrocytes. evaluation of tissue loss and spatial learning in the mwm corroborated that the treatment had no effect at the later time point. the results clearly show the importance of longterm studies in rodents to ensure that a promising effect on tissue regeneration and functional outcome is not merely temporary. astrocyte activation to extracellular matrix (ecm) -producing cells is inhibitory to regeneration in cns injury or disease. here we show that the cleaved neurotrophin receptor p ntr controls transforming growth factor (tgf)-beta-mediated astrocyte activation via regulation of nucleocytoplasmic shuttling of smad . loss of p ntr completely rescues tgf-beta-induced hydrocephaly, astrocyte activation, and ecm production in gfap-tgf-beta transgenic mice. nuclear fractionation of tgfbeta-treated p ntr knock out astrocytes revealed reduced nuclear phosphorylated smad than their wt counterparts. in accordance to p ntr depletion, inhibition of tgf-beta-induced gamma-secretasemediated p ntr cleavage also reduces nuclear accumulation of smad and the secretion of proteoglycans that inhibit neurite outgrowth. taken together, our results identify regulated intramembrane cleavage of p ntr as a mechanism for astrocyte activation resulting in the regulation of tgf-beta signaling and scar formation in the diseased brain. the reaction of glial cells toward brain injury comprises a proliferative response with some reactive astrocytes even reacquiring stem cell potential as indicated by forming long-term self-renewing and mutlipotent neurospheres (for review: robel et al., ). here we addressed first the question to which extent the type of injury selectively affects the proliferative response of ng glia and astrocytes and to which extent the proliferative reaction of astrocytes may be linked to the stem cell response. interestingly, while both astrocytes and ng glia mounted a profound proliferative response after acute invasive injury, such as stab wound and mcao, non-invasive injury models, such as amyloid plaque deposition or widespread neuronal death could not activate the proliferation of these macroglial cells, while microglia proliferated actively in all these lesion models. intriguingly, the proliferative response of astrocytes correlated to the activation of their potential to form self-renewing, multipotent neurospheres with a steep decline with age. we then set-out to determine the signals regulation reactive astrocyte proliferation and neurosphere formation after invasive injury and demonstrate that invasive lesions result in entrance of sonic hedgehog (shh) into the brain from extraneural sources, such as the cerebro-spinal fluid, and activate astrocyte proliferation and neurosphere formation. this response can be blocked by systemic injection of the shh-signaling antagonist cyclopamine as well as inducible astrocyte-specific deletion of the receptor smoothened by glast creert . interestingly, shh-agonists can boost the proliferative response of astrocytes in vivo and their subsequent neurosphere-forming capacity, thereby providing a new approach how to activate this response in conditions where it is limited, as e.g. in age or noninvasive injury conditions. taken together, our work highlighted for the first time differences in the proliferative and stem cell response of reactive astrocytes in different injury paradigms and identified the shh signaling pathway as a key signal in this response. tumor necrosis factor (tnf) is a pleotrophic cytokine involved in normal brain function and inflammation, apoptosis, and other responses that occur following injury and disease. there is a considerable amount of confusion as to whether or not tnf activation is beneficial or detrimental following injury to the cns. genetic studies in mice have suggested that inflammation in disease models involves soluble tnf (soltnf) and that maintenance of innate immune function involves transmembrane tnf (tmtnf). these findings suggest that the outcome of selective pharmacologic inhibition of tnf may depend on whether the pharmacologic intervention is targeted towards soltnf or tmtnf. therefore, we took advantage of a dominant-negative inhibitor of soltnf, xpro , that selectively inhibits soltnf and compared the effect of this inhibitor to a more widely used igg fc-tnf receptor (tnfr) fusion protein etanercept, an inhibitor of both soltnf and tmtnf, that has been a successful treatment strategy for several autoimmune diseases. however, etanercept has been shown to display severe side effects by increasing the risk of infections. female c bl/ mice were subjected to a spinal cord injury at t level. mice were divided into three groups, receiving etanercept, xpro , or saline. the drug was delivered directly onto the lesion site using alzet mini osmotic pumps for days starting immediately after introduction of the lesion. mice were allowed to survive days. functional evaluation was performed using the basso mouse scale, rung walk, open field test and hargreaves test. in contrast to etanercept, xpro significantly ameliorated recovery of hind limb function (evaluated by motor recovery score) compared to saline when administered continuously to the lesioned cord, whereas s.c. injections every days for weeks following sci had no effect. our study suggests that selective inhibition of soltnf is beneficial following sci when administered directly to the contused spinal cord and raises the possibility that selective inhibition of soltnf may represent a new therapeutic strategy for the treatment of sci without compromising the innate immune response. here, we show that expression of oncostatin m (osm) is upregulated after spinal cord injury (sci). to reveal the relevance of increased osm signaling in the pathophysiology of sci, osm was applied locally after hemisection. acute osm-treatment significantly improved locomotor recovery after mild and severe sci. delayed osm-treatment in the chronic phase was ineffective. improved recovery in osm-treated mice was associated with a reduced lesion size, less astrogliosis and diminished neuroinflammation. the underlying mechanism involves neuroprotective effects of osm. furthermore, osm promoted nerve fiber regeneration both in vitro and in vivo. thus, local production of osm after sci is an endogenous response that limits cns lesion propagation and promotes recovery. the university of western australia, crawley, australia following neurotrauma, cells beyond the initial trauma site undergo secondary degeneration, with excess ca a likely trigger for loss of neurons, compact myelin and function. treatment of secondary degeneration by limiting excess ca entry into cells using inhibitors of specific ca channels showed promise in preclinical studies, but clinical trials were disappointing and combinatorial approaches are acknowledged as necessary. we assessed efficacy of every possible combination of three ca channel inhibitors at reducing secondary degeneration months after partial optic nerve (on) transection in rat. we used lomerizine to inhibit voltage gated ca channels (for months); oxidised adenosine-triphosphate (oxatp) to inhibit purinergic p x receptors and/ or -[ -( h-imidazol- -yl) -nitro- , -dioxo- , , , -tetrahydro quinoxalin- -yl]acetic acid (inq) to inhibit ca permeable a-amino- hydroxy- -methyl- -isoxazolepropionic acid (ampa) receptors (both for the first weeks after injury). only the three ca channel inhibitors delivered in combination completely preserved visual function to levels not different from normal animals. in order to determine the mechanism/s by which the three inhibitors delivered in combination preserved function, we assessed neuroprotection, nerve swelling, myelin compaction and node/paranode complexes. preservation of retinal ganglion cell (rgc) somata and axons is unlikely to have accounted for the full recovery of function as only partial neuroprotection of rgcs vulnerable to secondary degeneration was achieved. a range of the ca channel inhibitor combinations prevented swelling of optic nerve vulnerable to secondary degeneration, with no one agent selectively effective. each of the treatments involving lomerizine significantly increased the proportion of axons with normal compact myelin and decreased the proportion of axons with partially decompacted myelin, indicating the importance of sustained control of ca flux via voltage gated ca channels for prevention of chronic myelin decompaction. nevertheless, limiting decompaction of myelin or formation of atypical node/ paranode complexes was not sufficient for preservation of function in our model. it is likely that the additional prevention of the lengthening of the paranodal gap that was only achieved by treatment with the three ca channel inhibitors in combination was an important effect that contributed to the associated preservation of visual function by this combinatorial treatment strategy. to generate myelinating oligodendrocytes opcs undergo a distinct series of morphological and molecular changes. these are regulated by a combination of extrinsic and intrinsic factors, which have only been partially been revealed so far. in the present study we identify a novel signaling mechanism as potent negative regulator of opc maturation. ephrin b , a member of the b-class family of ephrins, inhibits opc process formation and blocks the formation of mature oligodendrocytes in vitro. the presence of ephrinb results in activation rhoa and pkca signalling and deactivation of fak kinase in opcs in vitro. moreover, epha rtk, a receptor responsive to ephrinb in opcs, is also expressed by opcs in demyelinating lesions. infusion of recombinant ephrin b into demyelinating lesions results in a failure of remyelination caused by an inhibition of opc maturation. in contrast, antibody-mediated masking of ephrin b epitopes in vitro as well as in demyelinating lesions of aged rats promotes opc differentiation and accelerates myelin regeneration. our results demonstrate an important role of ephrin b for the process of remyelination. after peripheral nerve injury, axons rapidly degenerate and start regrowing only after a few days. full functional recovery, however, is rare. a progressive loss of intact axons also determines the clinical phenotype of chronic demyelinating peripheral neuropathies, such as charcot marie tooth disease type a (cmt a). cmt a is the commonest inherited neuropathy, caused by a duplication of the peripheral myelin protein kda (pmp ) gene. we demonstrate that the transgenic neuronal overexpression of the growth factor neuregulin- type i enhances axonal regeneration and functional recovery after experimental acute peripheral nerve injury. similarly, neuregulin- type i overexpression preserves axonal loss and improves nerve function in a mouse model for cmt a. importantly, neuregulin- type i induced axonal preservation in cmt a mice was independent of myelination, as myelin sheath thickness was not altered. we conclude that axonal recovery and preservation relies on common supportive factors and that paracrine neuregulin- type i constitutes a beneficial signal in peripheral nerve disorders. g. szab o university of tuebingen, tuebingen, germany question: the enormous capacity for repair of the cns after lesion is one of the most amazing property of lower vertebrates. in the mammalian central nervous system (cns) the astrocytes reveal the so-called orthogonal arrays of particles (oaps); the clusterization of the aqp molecules at the astrocytic endfeet. in lower vertebrates these structures are lacking. in the case of the amphibia -anolis carolinensis-the caudal spinal cord has the capability for regeneration after loss of the tail, an ability which might be in relation with the presence of tight junctions (tj) between the glial cells like the olfactory ensheating cells act in mammalian olfactory system through the lifespan. the growing axons of the fish optic nerve are embraced by astrocytes which are also interconnected by tjs. tj connected glial cells could contribute to a growth-supportive microenvironment which might reinforce our hypothesis namely the mutual exclusion of aqp occurrence in astrocytes and regenerative growth of nerve fiber. methods: we performed immunocyto-and immunohystochemical staining, western blot, freeze-fracture electron microscopy and pcr on cultured aqp knockout and wildtype astrocytes and also on formalin fixed paraffin embedded tissue sections regarding junctional proteins, such as occludin, claudin , , . result: in the light of our experiments, there is no mutual exclusiveness between the presence of aqp and tight junctions in astrocytes, at least in mammals. further investigations need to be completed. glutamate and electrical activity influence opc differentiation and myelination in normal development. opcs receive synaptic input from unmyelinated axons, possibly to initiate myelination. both opcs and mature oligodendrocytes respond to glutamate via ampa and nmda receptors. activation of glutamate receptors in vitro increase opc migration but decreases proliferation. here we examine the role of glutamate signalling in remyelination following experimental demyelination in the adult rodent cns. we voltage-clamped opcs in brain-slices of adult rat cerebellar peduncle containing focal areas of primary demyelination and post-identified these by ng -immunolabelling. recruited opcs mainly expressed ampa receptors at the peak of the opcs proliferation ( - days postlesion), as over % of the glutamate evoked current was blocked with nbqx (ampa/kainate receptor antagonist) and unaffected by ap (nmda receptor antagonist). the demyelinated axons continued to propagate action potentials with latencies similar to those in unmyelinated axons during development. critically, the demyelinated axons established synapses with opcs expressing voltage-gated sodium currents. these synaptic inputs had identical decay times to those recorded during developmental myelination. pharmacological inhibition of neuronal activity or glutamate signalling decreased remyelination efficiency by impairing differentiation. these results indicate that ) glutamate currents are mainly mediated via ampa and kainate receptors during the opc recruitment ) opcs establish synaptic contact with demyelinated axons and ) the neuronal activity is essential for remyelination. together, these data suggest that demyelinated axons remain active and communicate with opcs through glutamate release during the regenerative process to guide their remyelination. there has been much recent interest in the use of intraocular stem cell transplantation to slow or reverse visual loss in glaucoma. however, migration and integration of stem cells into the retina is limited by reactive gliosis, a major barrier to retinal engraftment. our aim was to identify glial modulators of low toxicity and measure their potential to facilitate stem cell entry into the host retina. an explant organotypic culture system was used as a stem cell transplantation model and screening tool for candidate drugs. gfp mouse mesenchymal stem cells (mmscs) were co-cultured on the surface of the explants for days.the microglial contribution to mscinduced reactive gliosis was investigated by assessing microglial activation and proliferation using immunohistochemistry for f / and edu. distribution of chondroitin sulphate proteoglycans (cspgs) and their role in the barrier was also explored by immunostaining for chondroitin sulphate (cs- ) and by chondroitinase abc(chabc) treatment. stat inhibitor vi and jak inhibitor were used to suppress glial fibrillary acidic protein (gfap) expression. the efficacy of each drug to overcome the barrier was evaluated by immunohistochemistry for the glial markers gfap. mmsc integration was assessed by simultaneous immunostaining for gfp. cell viability after drug treatment was investigated by measuring the preservation of retinal thickness and by immunostaining for the neuronal marker neun and for caspase . co-culture of retinal explants with mmscs caused a . . fold increase in gfap expression. microglial activation and proliferation was not markedly affected by the presence of grafted mmscs on retinal surface. cspgs, highly expressed in the outer retina but only moderately in the inner retina, do not contribute substantially to the barrierto inner retinal engraftment. in contrast, interfering with the jak/stat pathway successfully reduced gfap expression by % % compared to control and facilitated mmscs integration into the host tissue. no toxic effect on cell viability was detected. on the contrary, over the days ex-vivo culture, stat inhibitor vi treatment resulted in a neuroprotective effect on the outer nuclear layer, whose thickness ( . . mm) is better preserved compared to control ( . . mm) the results show that microglia and cspgs do not play a dominant role in the barrier to retinal engraftment, but the jak/stat pathway represents a promising pharmacological target.targeting modulators of glia reactivity to promote stem cell integration in the host tissue may facilitate stem cell therapy in glaucoma and other neurodegenerative diseases. oecs possess many characteristics favorable for the repair of sci including the secretion of growth and survival factors, the expression of cell adhesion molecules and the ability to integrate with astrocytes. oecs have also been reported to promote exceptional outgrowth of severed axons across the inhibitory environment of the lesion scar. the unique combination of regenerative properties suggests oecs would be beneficial for the repair of ventral spinal root avulsion injuries. after ventral root avulsion anterior horn neurons often fail to regenerate axons back into the surgically re-implanted nerve root resulting in poor functional recovery. to evaluate the efficacy of oec transplants for the repair of ventral root avulsions, the ability of oecs to promote neurite outgrowth from spinal cord neurons was investigated in a spinal cord organotypic co-culture. the integration of oecs with the spinal cord tissue was also examined by confocal fluorescence microscopy. oecs were harvested from adult sprauge dawley (sd) rats, cultured for days and transfected with gfp. slices of cervical spinal cord were prepared from p sd pups and cultured on collagen coated membrane inserts. each slice was surrounded by a collagen gel with or without the addition of gfp oecs and glial derived neurotrophic factor (gdnf). our results indicate that oecs alone and in combination with gdnf have a positive effect on neurite outgrowth and morphology. the aim of this study was to obtain an hydrogel for the controlled delivery of vascular endothelial growth factor (vegf-a ). hydrogels are a class of liquid-gel biomaterials with high water content, typically composed of cross-linked, three-dimensional hydrophilic water-soluble polymer networks. the advantages of injectable hydrogel systems are: biocompatibility, biodegradability, adjustable shape, low cost and similarity to the extracellular matrix; moreover, they can be used as growth factor and cell delivery systems for tissue engineering applications. agarose/gelatin (a/gl) hydrogel was cross-linked with genepin. rheological measurements show that a/gl hydrogel can be injected through a syringe into the inner cavity of a nerve guide. in vitro assays performed to evaluate adhesion, proliferation, viability and migrationusing a fibroblast cell line (nih t ), neonatal olfactory bulb ensheathing cells (nobec) and a schwann cell line (rt -d p t)show that hydrogel allows cell adhesion, proliferation, viability and migration. vegf-a is a potent angiogenic factor and angiogenesis has long been recognized as an important and necessary step during tissue repair. vegf is known to have a positive effect on schwann cell proliferation and migration, neuron survival and outgrowth of regenerating nerve fibers. the hydrogel preparation protocol was optimized to be functionally efficient at physiological conditions, allowing the loading of vegf without the risk of its denaturation. different amounts of vegf ( - - ng/ml) were incorporated in the a/gl hydrogel and the releasing rate in vitro up to days was quantified by elisa . released vegf bioactivity was validated in human umbilical vein endothelial cells (huvec) and in nobec by evaluating phosphorylation of vegf-receptor (vegfr ), akt and erk - . moreover, dorsal root ganglia explants were cultured on hydrogel containing different amounts of vegf, and an increased neurite outgrowth was quantitatively assessed. these results demonstrate that vegf can be successfully incorporated and bioactive released from a/gl hydrogel inducing vegfr activation and neurite outgrowth. in vivo test are ongoing on adult rats: one centimetre lesions on medial nerve were performed, followed by implantation of porous polye-caprolactone tubes filled with a/gl hydrogel containing vegf-a . functional tests and histological analysis will be carried out to evaluate peripheral nerve regeneration. .spinal cord injury (sci) often leads to death of oligodendrocytes and considerable demyelination. demyelination of axons compromises signal conduction, and contributes to the functional deficits following sci. enhancing remyelination may serve to restore conduction and likely prevents axonal degeneration. here we focussed on a potential therapeutic intervention to promote remyelination, the transplantation of human oligodendrocytes precursor cells (opc)s capable of differentiating into mature oligodendrocytes and remyelinating denuded axons. human platelet derived growth factor (pdgf)-responsive neural precursor (prp) cells were isolated from the fetal forebrain and had been previously shown to differentiate into mature oligodendrocytes with a higher efficiency in vitro than traditional egf/fgf responsive neural stem cells (chojnacki and weiss ; deleyrolle et al. ). therefore, we tested the potential of human prps to differentiate into new oligodendrocytes and remyelinate axons in a rodent thoracic contusion spinal cord injury model. one week following injury, human prps were transplanted rostral and caudal to the lesion site. subsequent histological examination at two, seventeen and forty-two days post-transplantation demonstrated that human prps survived and integrated well into host tissue, particularly when nk cells were depleted. transplanted human prp's labelled with the mature oligodendrocyte marker apc, and % of the cells colabelled with the oligodendroglial marker olig . astrocyte differentiation was also observed, suggesting human prps function as a multipotent glial progenitor following sci. human-specific immunoreactive processes were seen in close association with axons expressing the myelin protein mbp, providing evidence of remyelination by the transplanted cells. overall, these findings indicate that human prps are a source of new oligodendrocytes capable of producing myelin in response to spinal cord injury. remyelination is a regenerative process during which demyelinated axons are enwrapped by newly formed myelin sheaths. in the central nervous system (cns), this process takes place in two stages: first, during the recruitment stage, oligodendrocyte precursor cells (opcs) divide, migrate and engage the demyelinated axons. then, during the differentiation stage, opcs differentiate to myelin-forming oligodendrocytes. under pathological conditions such as multiple sclerosis (ms), remyelination often fails and as a consequence chronic demyelinated areas accumulate in ms. post mortem evidence suggests that opc differentiation is often impaired in ms patients. however, the reason why opc differentiation fails in ms is not completely understood. it has been proposed that ms lesions contain factors that act as specific inhibitors of opc differentiation. amongst them, myelin associated inhibitors play an important role. we have demonstrated that the inhibitory effect of myelin protein extracts (mpe) is mediated by activation of signalling cascades including pkca-marcks in vitro. furthermore, modulation of pkca has been shown to rescue opc differentiation in the presence of inhibitory mpe. here we demonstrate that tamoxifen, a drug that is used in various clinical settings because of its pkc-inhibitory activity, is able to promote opc differentiation in vitro. in a series of in vivo experiments, we tested the hypothesis that inhibition of pkca by tamoxifen promotes cns remyelination. demyelination was induced in the caudal cerebellar peduncle (ccp) of young-adult sprague-dawley rats by the administration of ethidium bromide. animals received daily doses of tamoxifen. although tamoxifen treatment did not induce changes in the number of opcs expressing immature markers (ng and pdgfr-a), a significant increase in the number of cells expressing mature marker of oligodendrocytes (cc /olig and plp) was detected. furthermore, analysis of resin-embedded tissue demonstrated that tamoxifen significantly promotes remyelination in the cns when compared to controls (mann-whitney's test, p < . ). although further investigations are required to elucidate the mechanisms by which tamoxifen exerts cns remyelination, our results demonstrate that administration of tamoxifen represents a potent and clinically accessible strategy to promote endogenous myelin regeneration. induced pluripotent stem (ips) cell technology now allows development of autologous oligodendrocyte precursor cells for putative remyelination cell therapy for multiple sclerosis (ms). we have successfully developed mouse and human ips cells and converted these into oligodendrocyte precursor cells (opcs) that can differentiate into fully mature oligodendrocytes. furthermore, we have shown that (intracerebral) implantation of ips-derived opcs in mouse ms models enhances remyelination.before implantation-induced remyelination therapy with autologous ips-derived opc can proceed to a clinical trial for remyelination cell therapy in ms patients, we have to provide solid evidence for long term efficacy and to establish the safety thus ultimately excluding contamination of teratogenic (pluripotent) stem cells. these issues are best examined in a nonhuman primate model. the biomedical primate research center (bprc), rijswijk has developed a welldocumented experimental autoimmune encephalomyelitis (eae) model in marmoset monkeys that most closely approximates the pathology of ms in humans. in this animal model we will verify that intrathecally administered ips-derived opcs migrate to and remyelinate eae lesions and determine their long-term fate.we have developed an efficient protocol to induce marmoset ips cells from skin fibroblasts. for that we have used a lentiviral polycistronic construct, containing the four (human) reprogramming genes oct , klf , sox and cmyc, that is excisable using cre-recombinase. the pluripotent nature of the marmoset ips cells was established based on the endogenous expression of pluripotency transcription factors and the ability to differentiate in-vitro (via embryoid bodies) and in-vivo (subcutaneous injection for teratoma formation) into all the different tissues of the three germ layers. presently we are developing protocols for the differentiation of these marmoset ips cells into an oligodendrocyte cell lineage. published protocols have been modified for production of opcs for research purposes, and produced cells are used for characterization of opcs. ultimately the aim is to define an opc population with optimal myelination capacity. results and discussion: currently, human oligodendrocytes and opcs can be produced from pluripotent stem cells. these cells are routinely characterized in protein expression level, and methods for more comprehensive characterization are under development. produced opcs can be purified with fluorescence-activated cell sorting based on ng protein expression in order to study if this cell population is heterogeneous in terms of myelination capacity. conclusions: oligodendrocytes and opcs can be produced from human pluripotent stem cells. studies are ongoing to determine the molecular characteristics and myelination capacity of the differentiated and purified opc-population. remyelination, the regenerative process in which myelin is restored to demyelinated axons, is carried by oligodendrocyte precursor cells (opcs). in a previous study we have shown that oligodendrocyte precursor cells are multipotent and they can differentiate into oligodendrocytes as well as to schwann cells and occasionally astrocytes in the remyelinating lesions. we observed that schwann cells derived from opcs in the central nervous system occupied almost exclusively tissue around blood vessels in astrocyte deficient areas. therefore, we postulated the occurrence of specific niches or microenvironments that modulate opc fate. the aim of present study was to identify the factors and their downstream effectors that significantly discriminate vascular and nonvascular niche and determine the fate of oligodendrocyte precursor cells. we microdissected tissue from vascular and non-vascular regions of lesion areas at , and days after bilateral stereotaxic injection of ethidium bromide into the brain white matter of adult rats and performed microarrays to profile the whole transcriptome of both niches separately. this approach allowed us to distinguish between mrnas that are enriched within two separate environmental niches within the same lesion area. the study identified differentially expressed transcripts. comparative bioinformatic analysis of global gene expression combined with signalling transduction pathway structure and genes function analysis revealed specific candidate signaling pathways differentially expressed in local peri-vascular niche compared to nonvascular one. results of rt-pcr-based validation of microarray data have proven that expression gradient of bmp , bmp , their antagonist sostdc , as well as wnt b, wnt , dhh and scube act as the major regulators of opcs fate in the vascular niche. moreover, we observed that blood vessels undergo substantial regeneration in the course of remyelination and postulated the important role of blood vessels reconstruction in creating the specific microenvironment of neurovascular niche during tissue regeneration. our analysis shown that unique properties of tissue around blood vessels differ from the rest of the lesion which can be one of the factors favoring opcs alternative differentiation in this area of lesion. . however, key aspects of dynamic behavior, such as cell migration or process orientation and motility, can only be monitored by live imaging. to elucidate these key aspects of opc behavior after injury, we used repetitive in vivo two-photon laser scanning microscopy ( plsm) to follow ng -cells after smaller and larger stab wound injuries in the mouse somatosensory cortex. we therefore used the bac transgenic mouse line sox icreert (simon et al., ) crossed to the inducible reporter line cag-gfp (nakamura et al., ) selectively labeling cells of the oligodendrocyte lineage. gfp opcs were imaged through a cranial window at the day of injury and at different time points thereafter. identification of the same cells was possible based on blood vessels labeled by rhodamine dextran as stable landmarks. live imaging revealed that the majority of opcs reacts within days after injury with hypertrophy (enlarged soma and processes), polarization (elongation and concentration of processes towards the injury site), directed migration towards the injury site (defined as movement of the cell body for at least mm) and proliferation (defined by a single cell being replaced by or even more daughter cells). only a small proportion of cells within $ mm of the injury did not react in any detectable manner. we noted that polarization and hypertrophy occur rather fast after inflicting the injury, while proliferation peaks days after injury. taken together, the live observation of opcs reacting to stab wound injury supports the concept of opc heterogeneity and reveals new insights into the functional role of these cells after injury: the fast process orientation towards the injury site implies a contribution to wound closure and their substantial proliferation sometimes for several rounds amplifies the number of ng -cells surrounding the injury site with implications for scar formation. the aim of this study was to create a non-invasive tool for cell visualization and cell population tracking that can be used in long term studies. methods: this study was performed using human pluripotent stem cell lines derived in institute of biomedical technology (former regea), university of tampere, finland. cells were differentiated to neural cells using neural differentiation protocol described earlier by lappalainen and colleagues. fluorescent probes used in this study were ) chloromethylfluorescein diacetate (celltracker green cmfda, ct) and ) , -dioctadecyl- , , , -tetramethylindodicarbocyanine perchlorate (did). cell viability and proliferation of the labeled populations was briefly studied. also, the labeled cells were characterized using immunocytochemistry and neuronal functionality was studied using micro electrode array (mea). results: during labeling optimization a wide range of different labeling parameters was tested for both probes to obtain long term labeling. most suitable parameters for human neural cells were mm ct with hours incubation time and mm did with hours incubation time. with these concentrations the labeling was visible up to weeks and had no statistical effect on cell viability. ct labeling was not affecting to cell proliferation but with did slight decrease in proliferation was seen in long term culture. labeled populations contained both map- positive neurons and gfap-positive astrocytes. also, the ct or did labeled population and their mixed-cultures expressed normal spontaneous network activity when cultured on mea-platform. conclusions: this study indicated that fluorescent probes ct and did are suitable for long-term labeling of human derived neural cultures in vitro. the labeling had no negative effects on cell behavior with the exception of did labeled cells having slight decrease on cell proliferation. importantly, the labeled neural networks were electrically functional. axonal regeneration after spinal cord injury (sci) is limited mainly due to the presence of an inhibitory microenvironment, especially reactive astrogliosis and glial scar formation. overcoming this inhibitory barrier of reactive astrocytes and inhibitory substances making the microenvironment of the damaged neurons permissive for axonal regrowth might be crucial for cns repair. single-dose irradiation has been proved to be neuroprotective in cns trauma through elimination of reactive astrocytes, however, the irradiation conditions with singledose protocols are not in clinical use. in the present study, we aimed to investigate whether low-dose-fractionated irradiation, which is currently being used widely for clinical treatment of several tumor types, could regulate cell cycle elements of reactive astrocytes just like its use in cancers, inhibit glial scar formation, attenuate astrogliosis-mediated inhibition of axonal regeneration and facilitate recovery of motor function after spinal cord hemi-section in beagle dogs. as expected, our results demonstrated that low-dose-fractionated irradiation reduced reactive astrogliosis, attenuated astrogliosis-associated neural injuries, ameliorated microenvironment of axonal regeneration, and benefited recovery of the animals subjected to spinal cord trauma. accordingly, irradiation might be a promising therapeutic strategy targeting at excessive astrogliosis for sci in the near future. mesenchymal stem cell transplantation has been proven to have beneficial effects in various degenerative diseases, including demyelinating models. both in our lab as well as others have shown that they express a number of trophic factors that are capable of inducing remyelination, mainly by activating nearby oligodendrocyte progenitors towards mature oligodendrocytes that in turn remyelinate the damaged area. however, this effect was only observed locally, in the area surrounding the graft, thus in order to achieve general remyelination in various brain structures simultaneously, we decided to perform bone marrow-derived mesenchymal stem cell injections into the lateral ventricles. in this manner, the cells may attach to various areas such as the hippocampus, corpus callosum and fornix, among others, all of which are demyelinated. previous to the graft, the cells were incubated with iron nanoparticles. this way, it is possible to track in vivo the grafted cells by magnetic resonance imaging (mri). as a result, the cells were observed at different time points ( - - - - days) by mri. also, the demyelinated areas can also be visualized and myelin quantified using image analysis software. this allows the quantification myelin density in the same individual mice at different moments before and after transplantation. j. muenzel , , c. becker , t. becker , a. williams university of edinburgh, centre for regenerative medicine, edinburgh, united kingdom university of edinburgh, centre for neuroregeneration, edinburgh, united kingdom central nervous system (cns) remyelination is important for the restoration and protection of proper nervous system function in demyelinating diseases such as multiple sclerosis but is poorly understood, and inefficient in humans. in contrast, zebrafish are able to regenerate many tissues very efficiently. as zebrafish are being increasingly used to study myelination, we aimed to develop and characterise an adult zebrafish model of cns de-and remyelination, to answer the question if remyelination is also very efficient, to describe the biology and to identify similarities and differences with mammalian remyelination. this then may allow us to better understand why zebrafish have a high regenerative capacity compared to rodents and humans. previous studies in teleost fishes have used optic nerve crush as a paradigm of myelin injury to investigate myelin repair. this, however, involves the de novo myelination of regenerated axons, rather than deand remyelination of the same axons. lysophosphatidylcholine (lpc) is a detergent-like toxin, which is widely used in rodent models to demyelinate axons. we administer lpc to the optic nerve of adult zebrafish and observe less myelin by both immunoreactivity and electron microscopy at the lesion site at days post lesion (dpl) and microglia activation along the optic pathway. immunoreactivity and electron microscopy suggest complete regeneration of myelin at dpl. we cannot identify any axonal injury in this model. in young zebrafish (aged - months), the myelin thickness of remyelinated fibres shows no difference to the pre-lesion state, which is different to mammals, where the myelin thickness is reduced. however, in old fish (aged months), although the axon diameter is the same as pre-lesion and in young animals, , remyelinated fibres have thinner myelin, suggesting that the regenerative capacity of zebrafish declines with age. we believe that this new zebrafish model of cns remyelination can be added to the suite of current models to better understand the remyelination process, why it fails, and to test for compounds to improve it, with the added benefit that zebrafish are rapid breeders, transgenesis is easy and there is a high potential for live imaging. although often described as a hard-wired component of the vertebrate body, the nervous system is a plastic and considerably fluid organ system that reacts to external stimuli in a consistent, stereotyped manner, while maintaining incredible flexibility and plasticity of its core components. unlike the cns, the pns is capable of significant repair, but we have only just begun to understand the cellular and molecular mechanisms that underlie this phenomenon. peripheral nerves are composed of axons surrounded by layers of glia and connective tissue. they are ensheathed by myelinating or non-myelintaing schwann cell glia, which are in turn wrapped into a fascicle by a cellular sheath called the perineurium. this structure forms from centrally-derived glial cells and serves as a protective barrier that is essential for nerve function. following an injury, adult peripheral nerves have the remarkable capacity to remove damaged axonal debris, regenerate and re-innervate targets. schwann cells have been shown to play an important role in this process by trans-differentiating, proliferating, clearing debris, and guiding re-growing axons, but less is known about the potential role of perineurial glia. to investigate the role of perineurial cells in pns regeneration, we have developed an injury response assay that uses a micropoint laser to create injuries along the motor nerves in live transgenic zebrafish. time-lapse imaging of injured nerves reveals that perineurial glia rapidly respond to nerve injury and extend processes toward the injury zone. this is in contrast to schwann cells, which we observe orienting towards the distal stump where they engulf and clear axonal debris. these data demonstrate that perineurial glia respond immediately to motor nerve injuries in a manner distinct from schwann cells, and future work is aimed at defining the molecular mechanisms that mediate the cellular responses of perineurial glia and schwann cells, as well as determining if developmental paradigms are recapitulated in these glial populations during the regenerative process. we have previously shown that in schwann cells the transcription factor c-jun acts as a master regulator of wallerian degeneration and is required for successful repair following nerve injury (arthur-farraj et al., methods. we here investigate on the release of the gliotransmitter glutamate from superfused isolated purified glial processes (gliosomes) obtained from adult rat cerebellum. intracellular ca levels were measured by a microfluorimetric technique. immunocytochemical confocal and western blot analysis were also carried on cerebellar gliosomes. results. confocal and western blot analysis confirmed that cerebellar gfap-positive gliosomes are a purified preparation of glia processes. more then % of gliosomes were positive for brain-specific lipid binding protein (blbp), indicating that they derived from bergmann cells. by measuring the release of glutamate we obtained evidence for the presence of glutamate release-facilitatory ampa receptors on the bergmann glial processes. in fact, ampa evoked [ h]d-aspartate or endogenous glutamate release which was abolished in ca -free medium; effectiveness of the selective inhibitor naphthylacetyl spermine indicated the involvement of ca -permeable, glua -lacking ampa receptors. ca imaging confirmed the presence of ca -permeable, glua lacking ampa receptors on bergmann glia processes. activation of the glua -lacking ampa receptors triggered vesicular exocytotic glutamate release: inhibition of vesicular loading by the vesicular glutamate transporter (vglut) inhibitors rose bengal or trypan blue, or by the h -atpase inhibitor bafilomycin a prevented the ampa-evoked glutamate release (see figure) . confocal analysis confirmed that blbpand gfap-positive processes expressed vglut and vglut . conclusions. we here report evidence that: . functional processes of bergmann cells are recovered in purified preparation of adult rat cerebellar gliosomes; . ampa receptors located on bergmann glia processes show features of ca permeable, glua -lacking ampa receptors; . activation of the ampa receptors evokes ca entry in isolated bergmann glia processes and ca -dependent vesicular glutamate release from the processes. activation of ca -permeable, glua lacking ampa receptors coupled to vesicular glutamate release might represent a mechanism by which bergmann glia processes modulate synaptic transmission at parallel fibers/purkinje cell synapses. the formation of brain edema, which accompanies ischemic or traumatic brain injuries, originates from a disruption of ionic/neurotransmitter homeostasis that leads to extracellular k elevation and neurotransmitter accumulation in the extracellular space. an increased uptake of these osmotically active substances, predominantly provided by astrocytes, is accompanied by intracellular water accumulation via aquaporin- (aqp ). previously, it has been shown that the removal of perivascular aqp via the deletion of alpha-syntrophin, a protein responsible for anchoring aqp on the astrocytic membrane, delays edema formation and k clearance (amiry-moghaddam et al., , pnas ; ( ): - ). therefore, we aimed to elucidate the impact of alpha-syntrophin deletion on astrocyte volume changes in the cortex during pathological states, such as hypoosmotic stress or oxygen-glucose deprivation (ogd), using three-dimensional ( d) confocal morphometry in situ. in addition, single cell rt-qpcr profiling was carried out to reveal possible differences in the expression profiles of ion channels/transporters that participate in maintaining ionic/neurotransmitter homeostasis. in order to visualize individual astrocytes that lack alpha-syntrophin, double transgenic mice were generated by crossbreeding gfap/egfp mice ( . d-confocal morphometry revealed that alpha-syntrophin deletion results in significantly smaller/slower astrocyte swelling when induced by min hypoosmotic stress ( mosm), min ogd or by high extracellular k ( mm), while alphasyntrophin deletion had no effect on the astrocytic shrinkage evoked by hyperosmotic stress ( mosm). the volume recovery of cortical astrocytes from gfap/egfp/alpha-syntrophin knockout mice was significantly slower following their exposure to hypo-or hyperosmotic stress, whereas no differences were found in astrocyte volume recovery following ogd or after their exposure to high extracellular k . compared to the cortical astrocytes of gfap/egfp mice, single cell rt-qpcr analyses revealed that astrocytes from gfap/egfp/alpha-syntrophin knockout mice express higher mrna levels for two-pore domain k channels (twik- , task- , task- ), inwardly or outwardly rectifying k channels (kir . , kir . , kv . , kv . ) and chloride channels (clc ,clc ), while mrna expression for the glutamate transporter glt- is lower. in summary, the deletion of alpha-syntrophin slowed down astrocyte swelling during hypoosmotic stress, ogd or high k ; however, it also resulted in alterations in astrocytic gene expression profiles. supported by grants ga cr - s and p / /g from the gacr. microglia undergo a process of activation in any type of pathology which is controlled by many factors such as cytokines, chemokines or growth factors, but also by neurotransmitters. we found that a subpopulation ( %) of freshly isolated microglial cells from the adult brain respond to the muscarinic acetylcholine receptor agonist carbachol with a ca response, indicating the expression of functional receptors. the carbachol-sensitive population increased in microglia/ brain macrophages isolated from the tissue of mouse models for stroke ( %) and alzheimer's disease ( %), but not for glioma and multiple sclerosis. microglia cultured from adult and neonatal brain contained a similar carbachol-sensitive sub-population ( % and %) which was increased by treatment with interferon-c to % within hours, and was sensitive to blockers of protein synthesis. carbachol was a chemoattractant for microglia and decreased phagocytosis activity, indicating that microglia are a functionally heterogeneous population. t - a astrocytes and s p receptors the family of sphingosine -phosphate receptors (s prs) are g protein-coupled comprising five subtypes (s p r-s p r). these receptors are expressed in cells of the immune, cardiovascular, and central nervous systems (cns), in addition to others. s prs play important roles in celular proliferation, differentiation, survival and migration. recently, the immunomodulatory drug, gilenya v r has been approved as the first oral therapy for multiple sclerosis (ms), after proving efficacious in clinical trials. the active ingredient of gilenya v r is the phosphorylated compound fty (pfty ), which is a potent agonist on all s prs, except s p rs. pfty has been suggested to work as a 'functional antagonist' causing s p r internalisation in lymphocytes, thus limiting t cell auto-immunity. in addition to regulating the immune system, the lipophilic nature of the pro-drug fty allows it to readily cross the blood-brain-barrier (bbb) where it may also activate s prs expressed on both neurons and glia. here, the role of s prs in the cns was investigated, by specifically investigating their role in astrocyte function. a range of methods were used to examine the effects of s pr activation and their roles in astrocytes, including: (i) s p r subtype trafficking, (ii) transient and continued intracelular signalling, (iii) astrocyte cell migration, (iv) release of pro-inflammatory cytokines, (v) oligodendrocyte cell differentiation and survival and (vi) myelination state in brain slice cultures. the data showed that activation of the s p r subtype leads to (i) its internalisation and (ii) continued signalling in astrocytes, and that s p rs were found to (iii) promote astrocyte migration, (iv) limit release of pro-inflammatory cytokines, (v) promote oligodendrocyte survival and (vi) limit demyelination. these studies demonstrate a role for s p rs in regulating astrocyte function and suggest their use a drug targets for neuroinflammtory and neurodegenerative disorders. in particular, both astrocytes and oligodendrocytes express kir . , which are essential for setting their strongly negative rmp, as well as the maintenance of [k ] o following neuronal activity. notably, strong expression of the kir . subtype was determined during a whole genome microarray analysis of the mouse optic nerve, a typical cns white matter that contains mainly astrocytes and oligodendrocytes. the kir . subunit is known to be involved in potassium transport in epithelial cells and has been identified in cerebellar purkinje neurons, but has not been reported in glia. here, we examined functional expression of kir . in mouse brain and optic nerve. rt-pcr and western blot confirmed expression of kir . mrna and protein expression in the brain and optic nerve. positive kir . immunostaining was observed in astrocytes and oligodendrocytes in brain sections and in optic nerve explant cultures and the results indicated a developmental increase in kir . expression. in astrocytes, kir . was localised to perivascular end-feet, supporting a potential role in k regulation. in oligodendrocytes, kir . were localised to cell somata, suggesting a developmental role in setting their rmp. in addition, oxygen and glucose deprivation (ogd) experiments were performed on isolated intact optic nerves from mice aged p and examined for the effects of the kir . channel blocker vu . after min ogd, blockade of kir . resulted in increased cell death of optic nerve glia, measured using propidium iodide (pi) labelling, from in controls to in vu (n nerves per group, fov per nerve; p < . , anova). our results demonstrate expression of kir . in glial cells, and indicate they are important in protecting glial cells from ischemic damage. is an eight transmembrane domain protein highly expressed in brain. human mutations in mlc lead to the rare genetic disorder "megalencephalic leukoencephalopathy with subcortical cyst" (mlc). characteristic for the disease are the onset of macrocephaly within the first year of life, and a slowly progressive loss of motor skills with epilepsy and cognitive decline. at the cellular level, countless fluid-filled vacuoles occur within myelin sheaths surrounding axons and in astrocytic endfeet. in cell culture models, mlc mutations are associated with defects in chloride currents and cell volume regulation. thus far, the expression and function of mlc in intact murine and human brain are still controversial. to understand the role and developmental expression of mcl in the brain, we have developed a mlc mutant null mouse model carrying an egfp reporter gene under the mlc promotor. we now show the exclusive expression of mlc in astrocytes throughout the brain with specifically higher expression around blood vessels, at the sub-ventricular zone and at the glia limitans. the functional loss of mlc in mutant mice recapitulates in part the human mlc disease. ko mice show macrocephaly with high brain wet weight. water filled vacuoles develop in the white matter of the cerebrum and in large fiber tracts of the brainstem. by contrast, the heterozygous loss of mlc has no consequences on myelin structural integrity. both heterozygous and homozygous mlc ko mice have dysmorphic peri-vascular and peri-ventricular astrocytes. in contrast to humans, ko mice have no motor deficits, but are hyperactive and show an anxiety-like behavior. in the mlc ko mouse, a dysfunction of an astrocytic protein causes loss of myelin structural integrity leading to vacuolating myelinopathy. therefore, the mlc mutant mouse could be a key model to study the astrocytic involvement in brain water and ion homeostasis. gaba activated slowly desensitizing responses in ng cells which were mimicked by muscimol and inhibited by bicuculline. to elucidate the subunit composition of the receptors we tested its pharmacological properties. co-application of pentobarbital, benzodiazipines and zolpidem all significantly increased the gaba responses. the presence of small tonic currents indicated the presence of extrasynaptic gaba a receptors. to further analyze the subunit expression, single cell transcript analysis was performed subsequent to functional characterization of ng cells. the subunits a , a , b , c and c were most abundantly expressed, matching properties resulting from pharmacological characterization. importantly, lack of the c subunit conferred a high zn sensitivity to the gaba a receptors of ng cells. to determine the effect of gaba a receptor activation on membrane potential, perforated patch recordings were performed. in the current-clamp mode, gaba depolarized the cells to about mv, indicating a higher intracellular clconcentration (about mm) than previously reported. gaba-induced depolarization in ng cells might trigger ca influx through voltage-activated ca channels. schwann cells (sc) play important roles in the development and regeneration of the peripheral nervous system (pns) following injury. several molecules such as neurosteroids and neurotransmitters have been suggested as potential pharmacological targets in regulating sc physiology and regenerative potential. nevertheless, the slow growth rate and difficulties in harvesting limit sc applications in regenerative medicine. adipose-derived stem cells (asc) can be differentiated into a sc-like phenotype (dasc) sharing morphological and functional properties in the present study, we analysed changes in glutamate transporter expression and function following a mechanical lesion in organotypic slice cultures of the mouse hippocampus using immunohistochemistry, western blots and dynamic imaging. the lesion was positioned perpendicular to the stratum pyramidale in the ca area and comprised the entire hippocampus proper. after three-six days, a glial scar had formed along the lesion site. activated astrocytes in close proximity ( - mm) to the lesion ("scar cells") directed long, palisading gfap-positive processes towards the lesion, had significantly swollen somata and lost their ability to take up sr . furthermore, some exhibited distinct clustering of glast and glt- immunoreactivity. scar cells showed greatly diminished increases in intracellular sodium in response to application of d-aspartate, an agonist for glutamate transporters. astrocytes in the periphery to the lesion, in contrast, maintained their ability to take up sr and showed only slight upregulation of gfap, as well as less swollen cell bodies. cells in the periphery displayed only marginal changes in glutamate transporter immunoreactivity and unaltered amplitudes of sodium changes in response to d-aspartate. taken together, our data show that mild astrogliosis in the periphery of a mechanical lesion is not accompanied by a significant change in glial glutamate uptake capacity. at the scar itself, a strong clustering of glutamate transporters is observed that apparently goes along with a severe functional reduction in glutamate uptake. transport of base equivalent (hco -) across the membrane is a crucial process to maintain the intracellular and extracellular proton concentrations within a narrow physiological range. the electrogenic sodium-bicarbonate cotransporter (nbce , slc a ) is a major base transporter in eukaryotes and expressed in many tissues, especially in epithelial cells. nbce may transport significant amounts of bicarbonate into and out of the cell. in the brain, nbce is predominantly expressed in glial cells and operates with a transport stoichiometry of na: hco -. we have studied the bicarbonate sensitivity of nbce , in primary cultured mouse cortical astrocytes (c bl /n) using live cell fluorescence imaging with confocal microscope and bcecf-am as a proton sensitive fluorescence probe. we have also used the primary cortical astrocyte culture from nbce -ko mouse to compare the nbce -mediated processes in wt astrocyte culture. bicarbonate dose response protocols suggest that nbce has a very high affinity for bicarbonate (k m < mm). due to this high affinity for bicarbonate, nbce can sensitize even residual bicarbonate concentrations of - lm present in nominally bicarbonate-free extracellular solutions (buffered with hepes). the removal of residual bicarbonate by saturating extracellular buffer (hepes) with % o reduced these nbce -mediated intracellular proton changes significantly. in astrocytes of nbce -ko mice, cytosolic h changes could not be detected at hco concentrations lower than mm. it has been reported that astrocytic nbce activity mediates the activation of astrocytic glycolysis by a mechanism dependent of intracellular bicarbonate and ph (ruminot et al j neurosci : - ). using a genetically encoded fret-based glucose nanosensor, we show that glucose metabolism can be activated at low millimolar bicarbonate concentration, which was largely absent in astrocytes from nbce -ko. our results demonstrate the highest bicarbonate sensitivity yet described in animal cells, mediated by nbce in cortical astrocytes. nbce may function as a bicarbonate sensor in these cells, e.g. for modulating energy metabolism. supported by the deutsche forschungsgemeinschaft de / - . inward currents which were significantly blocked by the l-type calcium channel antagonist nimodipine. to better investigate the role of l-type calcium channels in ng glia at different developmental stages of the cns, we take advantage of the inducible cre-lox system by breeding homozygous cav . floxed mice and cav . flexed mice with ng -creert knock-in mice. we are currently investigating how the removal of cav . / . alters proliferation, migration and survival of ng glia. a particular focus lies on the analysis of behavioral abnormalities generated by cav . / . -deficient ng glia. chronic neuropathic pain is a frequent consequence of spinal cord injury (sci). yet despite recent advances, upstream releasing mechanisms and effective therapeutic options remain elusive. previous studies have demonstrated that sci results in excessive atp release to the peritraumatic regions and that purinergic signaling, among glial cells, likely plays an essential role in facilitating inflammatory responses and nociceptive sensitization. we sought to assess the role of connexin (cx ) as a mediator of cns inflammation and chronic pain. to determine the extent of cx involvement in chronic pain, a weight-drop sci was performed on transgenic mice with cx /cx deletions. sci induced robust and persistent neuropathic pain including heat hyperalgesia and mechanical allodynia in wild-type control mice, which developed after weeks and was maintained after weeks. notably, sciinduced heat hyperalgesia and mechanical allodynia were prevented in transgenic mice with cx /cx deletions, but fully developed in transgenic mice with only cx deletion. sci-induced gliosis, detected as upregulation of glial fibrillary acidic protein in the spinal cord astrocytes at different stages of the injury, was also reduced in the knockout mice with cx /cx deletions, when compared with littermate controls. in comparison, a standard regimen of post-sci treatment of minocycline attenuated neuropathic pain to a significantly lesser degree than cx deletion. these findings suggest cx is critically linked to the development of central neuropathic pain following acute sci. since cx /cx is expressed by astrocytes, these findings also support an important role of astrocytes in the development of chronic pain. mast cells (mcs) are immune cells that reside in normal brain tissue. they store in granules an array of pro-inflammatory mediators, which are rapidly released to the extracellular milieu upon activation through an extracellular ca -dependent mechanism. neurotoxic amyloid peptides are known to induce mcs degranulation but the membrane transduction mechanism remains unknown. here, the possible role of pannexin hemichannel (panx hc) known to be permeable to ca was studied. objective: since ca influx is essential for mcs degranulation, we evaluated if the neurotoxic amyloid fragment ab - peptide promotes mcs degranulation via activation of panx hcs. methods: primary na€ ıve mcs were differentiated from bone marrow precursors of wild type (wt) and panx knock out (panx -/-) c bl/ mice using il- present in wehi conditioned medium. the presence of panxs , and mrnas presence was evaluated by rt-pcr analyses. the hc activity was assessed using the ', -diamidino- -phenylindole (dapi) uptake assays and measurements of membrane current using whole cell patch clamp while intracellular ca signal was evaluated using fura- am. degranulation was assessed by quantification of extracellular histamine as well as release of toluidine blue (tb) from tb preloaded mcs from wt and panx -/mice. results: only panx mrna was detected in wt mcs. stimulation with ab - but not ab - peptide induced histamine and tb release. it also enhanced dapi uptake and total membrane current in wt mcs. these responses were drastically reduced in wt mcs pretreated with mm carbenoxolone and mm panx , blockers of panx hcs and were absent in panx -/-mcs. in wt mcs treated with ab - increase the intracellular ca signal and was drastically prevented by panx , absence of extracellular divalent cations and in panx -/-mcs. conclusion: these findings indicate that mcs express functional panx hcs, which are essential for ca influx required for the degranulation response induced by ab - . thus, inhibition of panx hcs might avoid spreading of mc-dependent inflammatory mediators released during alzheimer's disease progression. gliomas are the most frequent primitive cns tumors and are thought to derive from astrocytes or from neural progenitors/stem cells. however, the precise identity of the cells at the origin of gliomas remains a matter of debate because no pre-neoplastic state has been yet identified. tgf alpha is frequently over-expressed in the early stages of glioma progression. sharif & al (oncogene. ( ): - ) previously demonstrated that prolonged exposure of normal astrocytes to tgf alpha is sufficient to trigger their reversion to a neural progenitor-like state. when astrocytes de-differentiated with tgf alpha were submitted to oncogenic stress using gamma irradiation, they acquired cancerous properties: they were immortalized, showed cytogenomic abnormalities, and formed high-grade glioma-like tumors after brain grafting. our study aims to identify and characterize the protein signature of those in vitro transformed cells in an attempt to understand their neoplastic behavior and the effect of transformation on metabolic processes. this involves a global proteomic analysis using the d-dige methodology and a study of the metabolism of these cells (carbon source, lactate, glucose and glutamine use, ros metabolism…) by hand c-nmr nmr quantification of metabolites. such approaches are expected to provide information allowing understanding the metabolic reprogrammation that occurred during transformation. the comparative d-dige proteomic analysis of normal and transformed astrocytes shows that during transformation, the cells increase their expression of glycolytic enzymes, thus acquiring the ability to use aerobic glycolysis (warburg effect). moreover, the transformed cells reduce their capacity for tricarboxylic acid oxidation and for neurotransmitters (glutamate and gaba) metabolism. ingenuity pathway analysis indicates major effects on carbohydrates, amino acids and nucleotides metabolic components. using enzymatic activity measurements and the detection of protein isoforms by d-western blot and zymography, we document a change in expression and activity of various isoenzymes that may be responsible for those metabolic reprogrammations. r.e. k€ alin, a. jarczewski, f. apel, r. monk, s. kraft, j. radke, f.l. heppner charit e -universit€ atsmedizin berlin, neuropathology, berlin, germany question: glioma are the most frequent malignant primary tumors of the brain. glioma survival and growth is determined by the interaction with brain parenchyma, which includes intense tumor angiogenesis. this process is supported by glioma-invading myeloid (gim) cells, namely brain resident microglia or brain-invading macrophages. depletion of gim cells leads to a significant decrease in glioma volume. in a previous study, we established the novel neuroendocrine hormone apelin as an angiogenic factor and described its upregulation in human glioblastoma multiforme. our aim is thus to study the role of apelin signaling in tumor angiogenesis but also its possible effect on brain monocytes for the regulation of glioma growth. methods: to investigate apelin signaling during gliomagenesis we took advantage of an orthotopic mouse model for tumor formation. intracerebral xenotransplantation of the human glioma cell line u mg, expressing lentiviral control or apelin shrna, allows us to study the specific contribution of glioma-derived apelin to gliomagenesis. to further dissect a putative immune function of apelin, we used the isogenic mouse glioma cell line gl for intracerebral implantation into immunocompetent mice lacking (apelin-ko) or overexpressing apelin (apelin-tg). results: loss-of-apelin expression in u mg cells resulted in a reduced glioma volume and attenuated the formation of the xenograft vasculature. gl implants in apelin-ko mice produced a similar phenotype. in addition, invasion of glm cells was significantly reduced. interestingly, expression analysis showed an upregulation of the apelin receptor apj in brain myeloid cells making them competent to respond to glioma-derived apelin. conclusions: our findings demonstrate that apelin plays an important role in tumor angiogenesis and glioma growth. we show here that both, glioma cell-derived apelin and apelin expressed by the tumor neovasculature are contributing to tumor angiogenesis, gim invasion and glioma growth. we anticipate that our study will provide insights whether apelin signaling may serve as a future novel target for antiangiogenic tumor therapy. tumor infiltrating microglia/macrophages (tims) constitute the largest population of infiltrating cells in glioblastoma (gbm), the most aggressive brain tumor. data from the clinic and from experimental work performed in murine and human models indicate that tims play a significant role in gbm biology as they support proliferation, migration and invasion of tumor cells. evidence is amounting that tumor cells actually polarize tims towards this m -like, tumor-supportive phenotype. we showed that toll-like receptor ligand reverses this m -into a m -like phenotype. pre-activated, m -like polarized tims incubated with spheroids of gbm cells reduced migration, killed and phagocytosed tumor cells over a day-period, indicating a sustained m activation of tims in absence of exogenously added stimuli. in order to analyse in more detail tims-gbm cells interactions, we have undertaken two approaches. we use spheroids of cells (tumor with/without tims) embedded in collagen matrix, in which tims can be implanted, as an experimental model. this three-dimensional in-vitro system is well suited for a qualitative and quantitative monitoring of cell proliferation, death, migration and invasion. data experimentally generated are then implemented in a mathematical model that is, to our knowledge, the first one proposed to take into account tumor cells and tims in order to simulate gbm progressive behaviour. in a first step, the capacity of spheroids made of murine glioma cells to invade collagen matrices was evaluated in absence and presence of microglia. invasion was monitored by photography of the spheroids and images were processed with photoshop cs software. in order to achieve a precise determination of invasion, we chose to measure the diameter of the core plus the invasive rim as a read out of expansion rate. untreated microglia promoted growth and invasion of tumor cells. data generated through the proposed in-vitro system were comparable to and reproduced the insilico simulations obtained with the mathematical model, hence validating our mathematical and experimental approaches. we currently evaluate the rate of proliferation and death of tumor cells in various settings that modulate tims polarization, using flow cytometry and confocal (live) imaging. data contributed by these two approaches should facilitate the delineation of a predictive model for tumor progression in a tims-enriched microenvironment with possible therapeutic fallouts. sialic-acid-binding immunoglobulin-like lectins (siglecs) are type membrane proteins displaying an amino-terminal immunoglobulin-like variable (v-set ig-like) domain that binds sialic acid and variable numbers of immunoglobulin-like constant region type (c -set ig-like) domains. siglec-h is a recently identified mouse-specific cd -related siglec that signals via the itam linked adaptor protein dap . so far the function of siglec-h and its ligand are unknown. in this project, we demonstrate gene transcription and protein expression of siglec-h by mouse microglia after interferon-c (ifn-c) treatment or polarization into a m -subtype. targeting of beads to siglec-h by specific antibodies triggered the phagocytic uptake of the beads by the microglia. a siglec-h fc fusion protein generated by our laboratory selectively bound to cells with an altered glycocalyx, particularly glioma cells, but not to normal control cells. m -polarized microglia phagocytosed glioma cells and also limited their proliferation in a co-culture system. interestingly, no signs of apoptosis were observed before phagocytosis of the glioma cells by siglec-h expressing microglia. phagocytosis of glioma cells was reduced after shrna mediated siglec-h knock down in microglia. furthermore, microglial clearance of glioma in vitro was dependent on the interaction of siglec-h with its corresponding adaptor protein dap . our data show that m -polarized microglial cells can engulf glioma cells via a dap -mediated and siglec-h dependent uptake. glioblastomas (gbm) are the most common brain tumors in humans. although advances in the chemotherapeutic management of glioblastoma have been made, almost % of the patients die within the first years after diagnosis. on the basis of these observations, the identification of new molecules that can counteract the gbm growth and invasiveness appears relevant. previously, we have demostrated that m muscarinic receptor agonist arecaidine inhibited cell proliferation in a time and dose-dependent manner and induced a severe apoptosis both in two glioma stable cell lines (u and u ) and in primary cultures derived from human biopsies. in order to clarify the mechanisms causing the decreased cell proliferation, we have evaluated the ability of m receptor activation to counteract the notch- and egfr pathways. the analysis by real time pcr and western blot have demonstrated that, in both cell lines, m receptor caused a decreased expression of egfr and notch- and its ligands (e.g. delta- , jagged- and ), suggesting that the decreased cell proliferation may dependent on altered expression and activity of these pathways. although facs analysis have showed that arecaidine induced an arrest of cell cycle progression, we observed, in both cell lines, a significant increase of dividing cells already after hours of treatment. in particular, the number of metaphases increased significantly, while the percentage of anaphases diminished. furthermore we observed in both cell lines, a dis-regulation of mitotic spindle assembly, misalignment of chromosomes and the presence of multipolar spindles. moreover, due to prolonged activation of the promethaphase/methaphase mitotic checkpoint, chromosomes appeared more condensed in cells treated with arecaidine. facs analysis and immunocitochemistry using anti-histone c-h ax, a marker of double strand breaks, have also demonstrated that arecaidine treatment induced dna damage. on the other hand, m agonist induced also oxidative stress, followed by an increased expression of the enzyme superoxide dismutase and sirtuins (sirt and sirt ). in conclusion, the data obtained suggest that the activation of m receptor induces cytostatic and cytotoxic effects in glioblastoma cell lines, opening new therapeutic perspectives for this receptor in glioblastoma therapy. univdersidade federal do rio de janeiro, rio de janeiro, brazil glioblastoma (gbm) is one of the most aggressive human cancers. despite current advances in multimodality therapies, such as surgery, radiotherapy and chemotherapy, the outcome for patients with high grade glioma remains fatal. there is now a growing awareness that the main limitations in understanding and successfully treating gbm might be bypassed by the identification of a distinct cell type that has defining properties of somatic stem cells, as well as cancer-initiating capacitybrain tumor stem cells, which could represent a therapeutic target. in addition, experimental studies have demonstrated that the combination of antiangiogenic therapy, based on the disruption of tumor blood vessels, with conventional chemotherapy generates encouraging results. emerging reports have also shown that microglial cells can be used as therapeutic vectors to transport genes and/or substances to the tumor site, which opens up new perspectives for the development of gbm therapies targeting microglial cells. finally, recent studies have shown that natural toxins can be conjugated to drugs that bind to overexpressed receptors in cancer cells, generating targeted-toxins to selectively kill cancer cells. further, extensive effort is being dedicated to characterize the molecular basis of gbm resistance to chemotherapy and to explore novel therapeutic procedures that may improve overall survival. we show that a non-cytotoxic concentration of equinatoxin ii (eqtx-ii), a pore-forming toxin from the sea anemone actinia equina, potentiates the cytotoxicity induced by temozolomide (tmz), a first-line gbm treatment, and to etoposide (vp- ), a second-or third-line gbm treatment. we also suggest that this effect is selective to gbm cells and occurs via pi k/akt pathway inhibition. finally, magnetic resonance imaging (mri) revealed that a non-cytotoxic concentration of eqtx-ii potentiates the vp- -induced inhibition of gbm growth in vivo. these combination therapies constitute a new and potentially valuable tool for gbm treatment, leading to the requirement of lower concentrations of chemotherapeutic drugs and possibly reducing, therefore, the adverse effects of chemotherapy. a growing body of evidence indicates that glioblastoma stem-like cells (gscs) play a central role in glioblastoma development and resistance to current therapies. we recently described a cluster of micro-rnas, the mir- - , which induces the exit of gsc from their stem state and suppresses their tumorigenic properties in an irreversible manner (fareh et al, ). we postulated that such a drastic change in the cell phenotype should be accompanied with metabolic alterations that could be instrumental in the loss of functional properties of gscs induced by the micro-rna cluster. metabolome profiling by mass-spectrometry of gscs and gsc-mir- - pointed to changes in the gaba synthesis pathway (see abstract el-habr et al). remarkably, exposure of na€ ıve-gsc to metabolites of the gaba synthesis pathway found to be overproduced in gsc-mir- - reproduced at least in part the effects of mir- - , including inhibition of clonality, down-regulated expression of selfrenewal markers, and loss of self-renewal properties. the metabolites studied acted by interfering with progression of the cell cycle and the nuclear localization of transcription factors crucial for maintenance of gsc stem-like properties. studies assaying the effects of the metabolites on gsc tumor-initiating properties are under way. these results demonstrate that metabolic regulations can participate in the control of gsc properties, and opens novel paths in therapeutic targeting of glioblastoma. *lgd and eae contributed equally. glioblastomas are the most frequent and aggressive form of adult primary brain tumors. glioblastoma stem cells (gscs) are thought to play key roles in the development and resistance of the tumor to existing radio-and chemotherapies. our previous results showed that the cluster of micrornas, mirna- - , induces loss of gsc stem-like and tumor-inducing properties (fareh et al. ). to further understand the molecular pathways controlling the peculiar properties of gscs, we sought for metabolic changes likely to accompany the drastic change in cell phenotype induced by mir- - expression. we measured the intracellular and secreted levels of metabolites by mass spectrometry. reconstitution of the metabolomes revealed significant and coordinated changes in components of the krebs cycle, and the glutamine/glutamate neuropeptide metabolic pathway that suggested altered turnover of the gaba synthesis pathway. exon array hybridization and western blot analysis, revealed changes in expression of several enzymes of the gaba synthesis pathway in mir- - -gscs. of note, none of the analyzed enzyme transcripts appeared as a direct target of mir- - . our results could be integrated in a complex multistep schema compatible with enhanced turnover of the gaba synthesis pathways, resulting in decreased cell gaba levels and increased levels of gaba by-products, as observed in mir- - gscs. further studies showed that these metabolic changes participate in the induced loss of gsc properties (see abstract by dubois et al). * eae and lgd contributed equally colonization of the brain by carcinoma cells is barely understood, in particular when considering interactions with the host tissue. we recently demonstrated that microglia assist carcinoma cell invasion. current findings indicate that this is part of a danger response of the entire brain tissue. in the brain slice coculture model, contact with both benign and malignant epithelial cells induced a response by microglia as well as astrocytes comparable to that seen at the interface of human cerebral carcinoma metastases. this response tries to protect the brain from intrusion of benign epithelial cells by inducing apoptosis. however, this is ineffective against malignant cells which did not undergo apoptosis and actually exploited this reaction to invade instead. gene expression and functional analyses revealed that c-x-c chemokine receptor type (cxcr ) and wnt signaling are involved in this process. furthermore, cxcr regulates the recruitment of microglia towards brain injury in a zebrafish model and was expressed in human stroke patients, suggesting a conserved role of cxcr in various danger reactions. we propose that glia-assisted malignant invasion takes advantage of the physiological two-stage glial defense reaction. carcinoma cells escape the second step of cell killing and misuse the damage response to colonize the brain. university of tuebingen, tuebingen, germany aquaporin- (aqp ), the main water channel of the brain, is highly expressed in animal glioma and human glioblastoma in situ. in contrast, most cultivated glioma cell lines do not express aqp , and primary cell cultures of human glioblastoma lose it during the first passages. accordingly, in two glioma cell lines of the rat (c cells and rg cells) and in sma mouse glioma cell lines we found no aqp expression. this let us consider the possibility that aqp expression depends on brain microenvironment. aqp negative rat glioma cells were implanted into rat brain. within two weeks, a tumor developed. aqp staining of the tumor cells was positive. however, if the identical cells were implanted into the rat's flank, they did not express aqp . in contrast to the normal brain, where aqp staining is polarized in the astrocytic endfoot membranes, the aqp staining in c and rg tumors was distributed over the whole glioma cell as in human glioblastoma. we conclude that the micro-environment is crucial for aqp expression in brain and brain-tumor. glioblastomas (gbm) are infiltrative and highly vascularised brain tumors containing a subpopulation of multipotent stem-like cells. here we questioned whether notch pathway activation in these cells influenced the extent of tumoral vascularisation and dissemination. we used two cd sox gbm stem-like cultures generating highly infiltrative but poorly vascularised tumors upon intracranial grafts. the use of a hes promoter reporter construct indicated that the notch pathway was only active in a small fraction of cells. sustained notch activation in these cultures, using an activated form of the notch- receptor (nicd), induced a profound alteration of their morphology, phenotype and properties. a severe decline of their migration and proliferation rates was observed in vitro and in vivo. intracranial and subcutaneous transplantation revealed that nicd triggered an extensive vascularisation of tumoral cells indicated by the presence of wellformed vessels with large lumens. close interactions between gbm cells and host endothelial cells were readily observed but no evidence for endothelial transdifferentiation was found either in vitro or in vivo. microarray and proteomic analyses showed that nicd induced the expression of vascular adhesion proteins (icam- , vcam- , itga ), proangiogenic cytokines (plgf, il- , hb-egf), metalloproteinase (mmp- ), a-smooth muscle actin and dll , a notch ligand essential for vascularisation. this was associated with a remarkable transcription factor switch whereby cells became klf snai while ascl , olig , and sox were reduced or lost. thus in addition to its well-described role in vascular development and remodelling, the notch pathway is also central in controlling proliferation, migration and vascularisation of gbm-stem like cells. t - b extracellular space diffusion parameters in the mouse thalamus in bral knock-out mice retinal wholemounts were immunostained with antibodies against gfap, iba- and mhc-ii. results: in the na€ ıve retinas, weak constitutive mhc-ii expression was scarcely found in some iba- microglial cells and rarely in gfap astrocytes. only a small dendritiform subpopulation of iba- cells, located in the juxtapapillary area and in the marginal region of the retina, had a strong mhc-ii immunoreaction. in comparison with na€ ıve both, in contralateral and in oht-eyes: i) gfap was upregulated in m€ uller cells and microglia was activated; ii) mhc-ii was upregulated on macroglia and microglia. in microglia, it was similarly expressed in contralateral and ohteyes. by contrast, in macroglia, mhc-ii upregulation was observed mainly in astrocytes in contralateral eyes and in m€ uller cells in oht-eyes conclusions: both, contralateral and oht-eyes had macro-and microglial retinal changes in mhc-ii expression after two weeks of laser-induced oht approximately % of the nmo patients harbor antibodies against the water-channel aquaporin- , aqp -igg (seropositive nmo), expressed mainly by perivascular astrocytes. however, some patients diagnosed with nmo don't have aqp -igg (seronegative nmo). the aim of this work is to compare in vitro the neuroinflammatory profile of igg from seropositive (igg-nmo ) and seronegative (igg-nmo-) nmo patients. pool of purified igg from igg-nmo and igg-nmo-patients fulfilling diagnostic criteria of , and from multiple sclerosis (igg-ms) patients and healthy controls (igg-hc) were include in the study. mixed glial cell cultures of astrocytes ( %), oligodendrocytes ( %) and microglia ( %) were obtained from spinal cord of postnatal c bl /j mice. after days in vitro, cells were treated with the igg pools for hours for rt-pcr, and hours for western blot and immunocytofluorescence (if) igg-nmo caused a stronger upregulation of c/ebpb mrna and nos protein than igg-ms and igg-nmo-. all these data show that igg-nmo and igg-nmo-induce a different proinflammatory glial activation. the proinflammatory pattern of igg-nmo-is quite similar to that induced by igg-ms patients. these findings raise the question if igg-nmo-could be a different disease. such as gdnf, artemin, bdnf and sonic hedgehog, adhesion molecules including p ntr, n-cadherin and n-cam and the transcription factor olig . c-jun also controls the structure of the regeneration tracks. in schwann cell c-jun null mice, the initial rate of axonal regeneration after injury is impeded and long term recovery of function measured by footprint analysis, toe pinching , von frey hairs, and the hargreaves test is not seen weeks after injury. there is widespread death of both large and small drg neurons and although spinal cord motor neurons survive there is widespread failure of both sensory and motor neurons to reinnervate target muscles united states mutations in the neurofibromatosis (nf ) gene cause neurofibromatosis type (nf ) characterized by formation of multiple schwannomas and meningiomas. nf encodes a tumor suppressor protein called merlin. loss of function of merlin is associated with an increased level of active rac and p -activated kinases (pak). the lim domain kinases (limk and ) are rac-pak substrates and modulate actin dynamics and cytoskeletal organization by phosphorylating cofilin, an actin severing and depolymerizing agent acknowledgements: this work was supported by grant ncn / /b/ nz / and statutory funds. phenotypes, their functions are fundamentally different in a model of brain injury. these data provide new insights into the protective role of microglia after brain injury. . although the pathogenesis is still not clear, activation of the immune system at some point of the disease process plays a crucial role. it is known that myelin-specific autoreactive t-cells and monocytes cross the blood-brain barrier (bbb) and migrate into the cns. within the cns, these cells secrete proinflammatory cytokines which stimulate local glial cells to produce inflammatory factors, including reactive oxygen species that contribute to demyelination and ultimately axonal loss. the destruction of the bbb and the influx of monocytes and t-cells from the circulation are key events in the progression of ms pathology. tissue transglutaminase (tg ) is a multifunctional enzyme that has been shown to play a role in monocyte/macrophage adhesion and migration onto extra cellular matrix proteins in vitro. this binding occurs via interaction with bintegrins. previously, we observed the appearance of tg in monocytes in active human post-mortem ms lesions in the cns.in the present study we question whether tg expression is regulated in primary human monocytes by inflammatory mediators and whether it is modulated in ms patients. methods: to this end, tg in monocytes was detected by semiquantitative rt-pcr.results and conclusions: our preliminary results show that activation of primary human monocytes with lps or lps ifnc results in a time-dependent increase in tg mrna whereas the expression of factor xiiia (another member of the transglutaminase family) remains stable over time. moreover, the tg mrna level is higher in unstimulated monocytes derived from ms patients compared to control subjects while factor xiiia mrna level remains unaffected.these initial data are promising with respect to a possible role for monocyte-derived tg being involved in adhesion to and migration across the bbb under inflammatory conditions (e.g. multiple sclerosis). this hypothesis has not been proven yet. in addition, by increasing the number of patients, a consistent difference in tg expression in monocytes between ms patients and control subjects may point to tg as a novel biomarker for disease. an important aspect of chronic neurodegenerative diseases, such as alzheimer's, parkinson's, huntington's and prion disease, is the generation of an innate inflammatory response within the central nervous system (cns). microglial and astroglial cells play a key role in the development and maintenance of this inflammatory response, showing enhanced proliferation and morphological activation. using a laboratory model of chronic neurodegeneration (me murine model of prion disease), we studied the time-course and regulation of microglial proliferation. our results show that resident microglial cells have an increased proliferation rate during the development of the disease, leading to a significant increase in the population, without a contribution from circulating cells. microglial proliferation is differentially regulated in diverse regions of the cns, pointing to a heterogeneous development of the pathology. we have identified novel molecular regulators of the proliferative response, and addressed the significance of the contribution of microglial cells to the pathological course of the disease by modifying their proliferation. we also found a correlation of our results with the scenario present in chronic human neurodegenerative conditions variant creutzfeldt-jakob disease (vcjd) and alzheimer's disease. our results demonstrate that microglial proliferation is an important feature of the evolution of chronic neurodegenerative disease, with direct implications for understanding the contribution of the cns innate immune response to disease progression. here, we examined the underlying mechanism of fibronectin aggregation and address whether inflammatory mediators, such as toll like receptor (tlr) agonists or pro-inflammatory cytokines induce fibronectin aggregation by astrocytes. our findings reveal that only tlr and agonists, including the endogenous tlr agonist stathmin, increased stable fibronectin aggregation. interestingly, only white matter-derived astrocytes displayed fibronectin aggregation whereas in cortical astrocyte cultures fibronectin remained unaffected, suggesting regional differences in the functional response of astrocytes. furthermore, in rat cerebellar slice cultures, the tlr agonists only induce aggregation of fibronectin after lysolecitin-induced demyelination. in this manner, efficient remyelination is impeded and, consequentially, progressive neuronal decline occurs.taken together, tlr and agonists promote fibronectin aggregation by primary rat astrocytes. since tlr on astrocytes and stathmin are both upregulated in ms lesions, stathmin, as an endogenous tlr agonist, might play a role in inducing fibronectin aggregation after c-jun reprograms schwann cells of injured nerves to generate a repair cell essential for regeneration. neuron. ( ): - ). in this study we identify a novel role for c-jun in the activation of notch signalling in the denervated schwann cell. we find that c-jun is required to activate notch signalling, leading to upregulation of the bhlh protein hes . hes then plays two functions in the denervated cell, promoting myelin breakdown and acting as part of a negative feedback loop to reduce c-jun levels. as a result of this, ablating notch signalling specifically in schwann cells acts to increase c-jun levels. we show that this upregulation of schwann cell c-jun accelerates axon outgrowth, target re-innervation and remyelination by generating a cell, which promotes faster than normal functional recovery. these results identify novel functional links between the c-jun and notch signalling pathways. they also show that not only is schwann cell c-jun necessary for successful nerve regeneration, but that nerve repair can be improved by enhancing normal c-jun signalling. it is well known that cell surface immune receptors play a critical role in regulating immune and inflammatory processes in the central nervous system (cns). after injury of the peripheral nervous system (pns), schwann cells and macrophages phagocyte myelin debris in wallerian degeneration in order to regenerate axons to distal targets. the immunoreceptor cd f is normally expressed in the myeloid line and in nervous system in microglia, oligodendrocytes and neurons under certain conditions. however, little is known about the cd f ligands. by using a cd f-fc fusion protein we have analyzed the expression of the cd f ligands in the pns. moreover, we have also analyzed the possible role of the cd f immunoreceptor in peripheral nerve regeneration by blocking the interaction between cd f and its ligands with the same fusion protein. thy -yfp-h mice sciatic nerves were injected with cd f-fc, control migg a or pbs immediately before a crush injury. the results show that cd f ligand is expressed in schwann cells. moreover, after a sciatic nerve injury, animals injected with the cd f-fc protein show a lower number of yfp-positive fibers growing into the tibial nerve after days post-lesion (dpl) than control groups. moreover, the cd f-fc group shows a higher number of macrophages and cd -positive cells at dpl when compared to control groups. we do not see these differences in axon regeneration and macrophage infiltration after dpl. we have also evaluated the mrna expression of the pro-inflammatory cytokine il- b at hours after crush and injection of the fusion protein. q-pcr shows an up-regulation of the mrna in control groups and a lower mrna expression level in the cd f-fc protein group. together these results show that the pair cd f receptor and ligand is implicated in some aspects of wallerian degeneration and nerve regeneration such as the modulation of both the influx and phenotype of macrophages. in response to the central nervous system (cns) injury, hematopoetic cells migrate to the lesion where they can potentially contribute to tissue regeneration. following cns injury, these cells can secrete a plethora of immunomodulating cytokines and/or pro-regenerative growth factors as well as phagocyte proinflammatory tissue debris. nevertheless, the role of primitive hematopoetic stem/progenitor cells (hspc) -derived from bone marrow -to support cns regeneration has not been studied in detail. therefore, the aim of the present study was to examine characteristics of hspc in vitro as well as to test proregenerative potential of these cells to support cns repair in vivo.highly pure population of primitive hematopoetic stem/progenitor cells was isolated from the bone marrow and the cns with fluorescence activated cell sorting (facs). the number of hspc in the cns lesion was significantly increased compared to intact cns tissue. sorted-bone marrow hspc were co-cultured with primary astrocytes to examine their phenotype according to the presence of specific antigens and gene expression as well as to test their phagocytosis potential. in the presence of astrocytes, bone marrow-derived hspc differentiated in vitro into microglia-like cells expressing specific myeloid/microglia markers and showing high ability to ingest fluorescent microbeads. the in vivo potential of hpsc for supporting regeneration was examined by their transplantation into the cns lesion.results of our study demonstrated that primitive hematopoetic stem/progenitor cells are the source of microglia-like cells which can support regeneration in the central nervous system. when the brain or spinal cord is injured, glial cells in the damaged area undergo complex changes resulting in the formation of the glial scar. whether the scar is beneficial and detrimental to recovery remains controversial. the signals that initiate the formation of the glial scar are unknown. because both canonical and non-canonical wnts are increased after spinal cord injury (sci), we examined the role of canonical wnt signaling in the glial reactions to cns injury. to disrupt b-catenin-dependent wnt signaling specifically in opcs, we created transgenic mice carrying an opc-specific conditionally deleted bcatenin gene. after moderate contusion injuries to the thoracic spinal cord of control mice, opcs proliferate and accumulate in the penumbra region surrounding the injury epicenter. in the b-catenin-depleted mice, there is reduced proliferation and accumulation of opcs after sci, reduced accumulation of activated microglia/macrophages and reduced astrocyte hypertrophy. using a crushed optic nerve model, we show that these reduced glial reactions create an environment that is permissive for axonal regeneration. these results suggest that canonical wnt signaling in glia after cns injury is necessary for the formation of the glial scar and they identify wnt signaling as a new therapeutic target for promoting axon regeneration. the crucial role of central nervous system (cns) glial cells in the integrity and physiology of neuronal networks, is well known. however, little is known about glial cells in the peripheral nervous system (pns). one of the main glial cell types in the pns is the perineuronal satellite glial cells (sgcs), that surround drg neurons in an envelope-like fashion. despite their abundance in peripheral nerves, having stem cells properties and playing a role in neuropathic pain, there is limited information on their physiology. although sgcs have similarities to astrocytes in terms of purinergic-and gap junction-mediated signaling, their membrane properties and ionotrophic glutamate receptor expression are more or less unknown. our aim was therefore to characterize the membrane properties and glutamatergic ion channel expression of sgcs. we performed patchclamp experiments on sgcs wrapped around sensory neurons in the rat dorsal root ganglion (drg) in vitro. whole-cell recordings revealed that sgcs are slightly hyperpolarized in comparison to the neurons they ensheath, with a resting membrane potential of approximately $ mv and a high input resistance (&gt; gx). moreover, upon membrane depolarization no detectable voltage-gated na , ca or k currents were detected. extracellular application of glutamate agonist, kainic acid, n-methyl-d-aspartic acid (nmda) and -amino- -( hydroxy- -methyl-isoxazol- -yl) propanoic acid (ampa) during the recording, did not evoke any response from the cells, indicating their lack of ionotropic glutamatergic receptor (iglur) expression. double immunocytochemistry on lucifer yellow (ly) filled scgs revealed that they express the scip/oct transcription factor, which is also expressed by promyelinating schwann cells.drg ganglion sgcs differ from cns perineuronal cells as they lack glutamatergic receptors, but are similar to astrocytes as they have no voltage-gated ion channels, are gap-junctionally coupled and express glutamine synthetase. thus, we are currently addressing if sgcs respond to neuronal activity in a similar manner to astrocytes in the cns with the use of calcium imaging. these studies will provide further insights into the physiological role of sgcs in the pns. multiple sclerosis (ms), a chronic neuroinflammatory disease with presumed autoimmune etiology, is the most common demyelinating disorder of the central nervous system among young adults. progressive neurological impairment of the patients are associated with accumulation of characteristic brain lesions characterized by inflammation, demyelination, gliosis and axonal damage. demyelination and loss of oligodendrocytes contributes to axonal loss and permanent neurological deficits. remyelination occurs but is limited; one contributing factor is the incapability of oligodendrocyte precursor cells (opc) to differentiate and form new myelin sheaths. k channels are not only known to predominantly set and maintain the membrane potential but also to be important regulators of various cell functions like proliferation and maturation. specific regulation of these cellular functions is based on high channel diversity and their ability to form heteromers as well as alternate mrna splicing and posttranslational modifications. so far the expression and function of potassium channels in cells of the oligodendroglial lineage has not been clearly identified. previous studies indicate that an unspecific blockade of k channels in opc suppresses maturation as well as proliferation. moreover, for one distinct channel (k ir . ) a functional relevance has been shown, as deficiency of kir . leads to altered opc maturation and hypomyelination in mice. here, we elucidate the expression and functional relevance of k channels in oligodendrocytes. our first electrophysiological recordings from primary murine opcs revealed the presence of different k channels with both inward and outward rectification. in future experiments, we aim at identifying k channel subtypes that specifically regulate opc cell functions and might influence cell maturation under neuroinflammatory conditions. thereby, we want to offer new insights in the functional role of k channels in opcs/oligodendrocytes and contribute to novel therapeutic strategies for the treatment of ms. aqp ) is the predominant water channel in the mammalian brain and is mainly expressed in the perivascular glial endfeet at the brain-blood interface. aqp has been described as an important entry and exit site for water during formation of brain edema and regulation of aqp is therefore of broad interest. phosphorylation of some aquaporins has been proposed to regulate their water permeability via gating of the channel itself. protein kinase (pk)-dependent phosphorylation of ser has been reported to increase the water permeability of aqp expressed in an astrocytic cell line. this possibility was, however, questioned based on the crystal structure of the human aqp . our study aimed to resolve if ser was indeed a site involved in phosphorylation-mediated gating of aqp . the water permeability of aqp -expressing xenopus oocytes was not altered by a range of activators and inhibitors of pkg and pka. mutation of ser to alanine or aspartate (to prevent or mimic phosphorylation) did not change the water permeability of aqp . pkg activation had no effect on the water permeability of aqp in primary cultures of rat astrocytes. molecular dynamics simulations of a phosphorylation of aqp .ser recorded no phosphorylation-induced change in water permeability. a phospho-specific antibody, exclusively recognizing aqp when phosphorylated on ser , failed to detect phosphorylation in cell lysate of rat brain stimulated by conditions proposed to induce phosphorylation of this residue. thus, our data indicate a lack of phosphorylation of ser and of phosphorylation-dependent gating of aqp . undocked connexons may form open hemichannels in the plasma membrane when exposed to specific stimuli, e.g. reduced extracellular concentration of divalent cations, and allow passage of fluorescent molecules with masses lower than kda. a range of physiologically relevant molecules, smaller than the assumed molecular cut-off of kda, have therefore been proposed to permeate connexin (cx ) in its hemichannel configuration. however, the permeability profile of cx hemichannels remains unresolved as does the molecular substrate for hemichannel activity in astrocytes. exposure of cx -expressing xenopus laevis oocytes to divalent cation free solution induced a gadolinium-sensitive uptake of the fluorescent dye ethidium. in spite thereof, a range of smaller biological molecules, such as water, glutamate, lactate, glucose, and atp, did not gain detectable access through the pore. in contrast, permeability of glutamate, glucose and atp was observed in oocytes expressing the other major astrocytic connexin, cx . exposure to low divalent cation solutions also induced a robust membrane conductance in cx -expressing oocytes but none in cx -expressing oocytes. expression of cx in c glioma cells failed to induce hemichannel activity. our results thus call for caution when assigning molecular identity to astrocytic channel activity and when interpreting hemichannel-mediated dye-uptake as equal to permeability of physiologically relevant molecules. atp-gated p x receptor channels expressed in spinal microglia actively participate in central sensitization, making their functional regulation a key process in chronic pain pathologies. p y metabotropic g q -coupled receptors, also expressed in microglia, are involved in the initial response to nerve injury, triggering phagocytosis upon activation by udp. it has been reported recently that expression of both p x and p y is upregulated in activated microglia following nerve injury. we show here, in resting as well as lps-activated primary microglia, that p y decreases p x -mediated calcium entry and inhibits the dilation of p x channels into a large-conductance pore measured with a yo-pro- uptake assay. furthermore, p y activation modulates the atp-dependent migration of microglia, a process likely involved in their shift from migratory to phagocytic phenotype.reconstituting the p x -p y interaction in recombinant systems shows that p y activation decreases p x current amplitude, activation and desensitization rates, and reduces p x channel permeability to the large cation nmdg . phospholipase c-mediated hydrolysis of the phosphoinositide pi( , )p , a necessary cofactor for p x channel function, underlies this inhibitory crosstalk. as extracellular levels of both atp and udp are increased in the spinal cord following nerve injury, the control of p x activity by p y might play a critical role in regulating neuropathic pain-inducing microglial responses. with sc, thus representing a valid sc alternative. we have previously shown that dasc express c-aminobutyric-acid (gaba) receptors (i.e. gaba-a and gaba-b receptors), which modulate their proliferation and neurotrophic potential, although little is known about the role of other neurotransmitter systems in asc.in this study we investigated the expression of purinergic receptors in dasc. using rt-pcr, western blot analyses and immunohistochemistry we have demonstrated that asc express p x , p x and p x purinoceptors. interestingly, differentiation of asc towards glial phenotype was accompanied by up-regulation of p x and p x receptors. using ca imaging techniques, we have shown that stimulation of purinoceptors with adenosine- -triphosphate (atp) results in intracellular ca signals, indication functional activity of these receptors.. moreover, we have shown that the increase of intracellular ca leads to sc death, an effect that can be prevented using specific p x or p x antagonists.altogether, these results show, for the first time, the presence of functional purinergic receptors in sc-like derived from asc and their link with critical physiological processes such as cellular death and survival. the presence of these novel pharmacological targets in dasc might open new opportunities for the management of cell survival and neurotrophic potential in tissue engineering approaches using dasc for peripheral nerve repair. university of freiburg, freiburg, germany intracellular ph homeostasis is a vital function shared by all cells and is mainly regulated by the coordinated action of several acid-base transporters. in the brain, ph homeostasis is of particular importance since changes of extracellular ph (pho) are events associated with both physiological conditions and pathological states in brain function. transient pho changes usually accompany physiological processes, including neuronal activity, and astrocytes respond to a rise in extracellular k with plasma membrane depolarization and intracellular alkalinization. on the other hand, loss of brain ph homeostasis can lead to severe pathological conditions and significant changes of pho have been observed during seizures and spreading depression.in the present study, we sought to investigate regulation mechanisms of the electrogenic sodium/bicarbonate cotranspoter , nbce , and of the vacuolar h -atpase (v-atpase) in primary hippocampal astrocytes following extracellular acid-base changes, following neuronal activity, and using the -aminopyridine ( -ap) model of epilepsy in vitro.we show that extracellular acidosis, but not extracellular alkalosis, increased the number of activated astrocytes. our data also show differential regulation of acid-base transporters in the hippocampal glial cells during extracellular acid-base changes. intracellular ph measurements revealed a crucial role for v-atpase in regulation of intracellular ph, a process accompanied by increased membrane expression of the transporter, as assessed by immunofluorescence and surface biotinylation. nbce -b is the nh -terminal variant of nbce predominantly expressed in glial cells and is transcriptionally regulated followed neuronal activity or treatment of the cells with -ap.these data propose transcriptional and post-translational modification of acid-base transporters as putative regulatory mechanisms in astrocytes to cope with changes of extracellular ph serving the maintenance of intracellular ph homeostasis. these astroglial networks fulfil a variety of functions in the brain, e.g. potassium buffering and metabolite transport. in this study we compared glial networks in different brain regions to investigate site specific effects on network formation. we combined electrophysiology and immunohistochemstry with semi-quantitative rt-pcr and western blot analysis to investigate connexin expression, gap junction coupling and antigen profiles. experiments were performed in wild type and transgenic mice with glia specific fluorescence labelling as well as in cx ko mice. astrocytes were investigated between postnatal days - . gap junction networks in the ca region of the hippocampus and the ventrobasal thalamus show abundant coupling in hgfap-egfp mice. intriguingly, we found significant coupling between oligodendrocytes and astrocytes in the thalamus, while in the hippocampus panglial coupling was less abundant. other glial cells did not participate in the networks. we also found that a fluorescent glucose analog, -nbdg, propagates through the thalamic panglial network. the function of these panglial networks remains largely unclear.in heterozygous cx -ecfpki mice, deletion of one allele of the major hippocampal cx significantly reduced the number of coupled astrocytes only in the hippocampus, while the thalamic networks remained unchanged. sr labelling of astrocytes and subsequent p microscopy identified a significant subset of thalamic sr cells lacking cx -ecfp expression. sr did not label oligodendrocytes as analysed in plp-gfp mice. semi-quantitative rt-pcr and western blot analysis revealed stronger expression of cx in thalamic nuclei while cx levels were higher in the hippocampus. this indicates a minor role for cx in gap junction coupling of astrocytes in the thalamus. consistent with these findings, the thalamus of cx ko mice displayed a strong decrease in astrocytic cell coupling compared to wild type littermates.together, these results indicate that thalamic astrocytes differ in various aspects from their counterpart in other brain regions and support the emerging concept of astrocyte heterogeneity. the mechanism of secondary damage spread after brain trauma remains unresolved. in this work, we redirected the attention to astrocytic communication pathways. using an in vitro trauma model that consists of a scratch injury applied to a rat astrocyte monolayer, we found a significant induction of connexin hemichannel activity, demonstrated by ethidium uptake, in regions distal from the injury ($ mm away and maximal $ h after injury). this response was abolished by two connexin hemichannel blockers, la and the peptide gap . in addition, the trauma-induced increase in hemichannel activity was prevented by inhibition of purinergic p receptors. the scratch-induced increase in hemichannel activity was absent in astrocytes from cx knockout mice. this activity took place with a singular spatial distribution, since cells located at $ mm away from the scratch gained connexin hemichannel activity. however, the functional state of gap junction channels (dye coupling) was not significantly affected in the same locations. the connexin hemichannel activity was also enhanced by the acute extracellular application of mm k . the increase in hemichannel activity was correlated with an increment in apoptotic cells, measured by immunofluorescence to annexin v at h post-trauma, which was totally prevented by gap peptide. these findings could open a new approach to prevent or reduce the secondary cell damage due to brain trauma. l. schlosser, a. scheller, f. kirchhoff institute of physiology, saarland university, homburg, germany current research suggests that astrocytes represent heterogeneous neuroglial populations in different regions of the brain. this is particularly evident when the expression pattern of various transmitter receptors is analyzed. hippocampal astrocytes, for example, do not express ampa receptors, while cerebellar bergmann glia is loaded. functional analysis of glial receptors requires dedicated efforts for each distinct brain region and developmental age. unfortunately, the molecular identification using antibody approaches is often hampered by either poor antibody quality or abundant receptor expression on adjacent neuronal membranes. therefore, we are using cre/lox-mediated gene ablation for proper functional analysis.here, we are focusing on the characterization of astrocyte-specific gene deletion of the gaba b receptor. metabotropic gaba b receptors consist of two subunits gaba b and gaba b forming a heteromeric receptor complex of which gaba b is essential. in contrast to neurons, activation of astroglial gaba b receptors leads to transient rises of intracellular ca . the functional impact of these glial gaba b -receptors is largely unknown.to address the gaba b receptor function in astrocytes we took advantage of the cre/lox system and crossbred glast-creert knockin mice with floxed gaba b receptor mice. first immunohistochemical analysis reveals a selective deletion of gaba b on a majority of astroglial processes. functional studies addressing the role of these receptors in astroglial ca signaling are in progress. purinergic signaling is the most diverse system in astrocytes to communicate with other glial cells and neurons. astrocytes express a variety of p x (ionotropic) and p y (metabotropic) receptors. especially in case of brain injury, atp levels and receptor expression on astrocytes are increased. atp is immediately degraded to adp, amp and adenosine. these nucleotides/nucleosides act back on the preferred purinoreceptor expressed on glial and neuronal cells. in the last decade researchers tried to evaluate the functional impact of astrocytic purinoreceptors on the complex information flux at the tripartite synapse. a prominent role has been suggested for the p y receptor subtype. we generated conditional mouse mutants to investigate the function of p y receptors in astrocytes in vivo and crossbred mice carrying the floxed p y gene with mice that express the inducible dna recombinase (creert ) under the control of the glast (l-glutamate/ l-aspartate transporter) locus. ablation of the p y receptor is induced in development and adulthood by intraperitoneal tamoxifen injections. successful recombination of the targeted p y receptor is evaluated by qrt-pcr on genomic dna and mrna usually days after tamoxifen application. at the genomic level we find $ % and $ % recombination in astrocytes of the cerebellum and hippocampus, respectively. similar recombination frequencies are observed in young (p ) or adult mice ( - weeks). when looking at the transcript level, the mrna is reduced to % in the cerebellum of adult mice and to % in the hippocampus. in young mice we determined a reduction to % and % mrna expression in the cerebellum and hippocampus, respectively. given the high level of p y expression on other cells of the brain these findings indicate a successful astrocyte-specific knockout. further fluorescence-activated cell sorting (facs) of recombined cells expressing the red fluorescent protein td-tomato will be used to verify the knockout. the potential function of these astroglial receptors in neuronal networks will be as well investigated using histological (em and light microscopy), twophoton ca imaging in situ and in vivo and behavioral approaches. we recently showed that they invade the cortex at . days of embryonic age (e . ). they first accumulate at the pial surface and within the lateral ventricles, after which they spread throughout the cortical wall, avoiding the cortical plate region in later embryonic ages. the absence of the expression of mac- and mhc ii suggest these cells have a na€ ıve/quiscent phenotype during embryonic development of the cortex. besides immunohistochemical markers is the presence of different k channels on microglia also an indication of their activation stage. however most studies have been conducted on postnatal and adult microglial cells. therefore we aimed at determining the electrophysiological activation phenotype of these embryonic microglia.at the age of e . and e . , microglial cells display a small inward rectifying k current and this independent of their location in the embryonic cerebral cortex and their cell morphology. these cells also express functional p x receptors, which based on the profile of the response are most probably p x receptors. time-lapse analysis showed that embryonic microglia are highly dynamic cells. suggesting that these cells are already very active during fetal development. rapid extracellular removal of glutamate, the major excitatory neurotransmitter in the cns, is essential for normal brain function. this task is primarily accomplished by the action of the sodium-dependent, high-affinity transporters glast and glt- (rodent analogous of eaat and eaat ), which are mainly expressed by astrocytes. impairment or failure of glast and glt- plays an important role in many pathological conditions. question: pelizaeus-merzbacher-like disease (pmld) is a hypomyelinating leukodystrophy caused by mutations in the gjc gene encoding the gap junction (gj) protein connexin (cx ). cx is expressed in oligodendrocytes and forms most gj channels to astrocytes and to other oligodendrocytes. since pmld-associated gjc mutations cause loss of cx function, gene replacement strategies may be promising for developing future treatments. a mouse model for plmd, the cx / cx double knockout (ko), is characterized by early onset of severe leukodystrophy at weeks of age leading to death by weeks, offering the possibility to test therapies. the aim of the present study was to generate a lentiviral vector to allow gene delivery specifically to oligodendrocytes, and to examine the transduction efficacy, distribution, duration and levels of egfp reporter gene and cx expression throughout the cns, in order to establish a method for oligodendrocyte-targeted gene therapy.methods: the expression cassette with the cx gene along with the ires-egfp as a reporter gene under the control of oligodendrocyte-specific cnp promoter was cloned into the lentiviral vector pcclsin.ppt.hpgk.gfp.pre. mock vectors lacking the cx gene were also generated as controls. viral particles were produced to high titers and purified, before injection. lentiviral vectors were delivered into the brain of wild type (wt) and cx ko mice at postnatal day (p ), intraventricularly and in the stratum radiatum of the dorsal hippocampus. egfp and cx expression was assessed using immunochemistry and immunoblot analysis at different time points post injection.results: we found widespread expression of virally delivered egfp in different brain regions colocalizing with oligodendrocyte markers (cc and olig ). the expression of egfp was detected from p until months post-injection. on average . . % of oligodendrocytes were egfp positive, with highest rates in the subventricular zone ( . . %, n ) and olfactory bulb ( . . %, n ) and lower rates in the cortex ( . . %, n ) and corpus callosum ( . . %, n ). in cx ko brain expression of virally delivered cx was also found after p with formation of gj plaques in a subpopulation of oligodendrocytes.conclusions: our results show that neonatal lentiviral gene delivery may result in stable and widespread cns expression targeted to oligodendrocytes by using cell specific promoters. thus, gene therapy approaches using lentiviral vectors may be feasible for future treatment of leukodystrophy and should be further studied. recent reports identified mrna coding for different cav subtypes like . and . in hippocampal ng glia. using acute brain slices prepared from developing and mature ng -eyfp mice, we also found that all ng glia upon depolarization in different brain regions elicited a. grimaldi, g. d'alessandro, c. lauro, m. catalano, c. limatola sapienza university of rome, rome, italy glioblastoma (gbm) is one of the most aggressive tumor of the central nervous system, being characterized by a great invasiveness and a very low survival rate of patients. this work wants to better define the role of tumor microenvironment in the modulation of tumor invasiveness. it is known that tumor migration and invasiveness can be modulated by the chemokine cxcl and its receptor cxcr . the expression of both these proteins has been demonstrated in various human gbm cell lines and it has been shown that, blocking this pathway, tumor cells greatly reduce their invasive ability. we have recently demonstrated that an important molecule directly implicated in tumor migration is the intermediate conductance calcium-activated potassium channel (kca . ). given the expression of these channels also in other cell types infiltrating the tumor mass, like microglia, we investigated the effect of kca . inhibition on microglia-glioblastoma interaction. preliminary data indicate that kca . activity on microglia is involved in the movement of these cells toward the tumor mass. a growing body of evidence indicates that glioblastoma stem-like cells (gscs) play a central role in human glioblastoma development and resistance to current therapies. we recently described a cluster of micro-rnas, the mir- - , which induces the exit of gscs from their stem state and suppresses their tumorigenic properties in an irreversible manner (fareh et al, ) . to elucidate the gene networks that govern gsc exit from their stem state, we used chip-seq (chromatin immunoprecipitation followed by next generation dna sequencing), to identify gene loci showing enrichment of the active (h k me ) and repressive (h k me ) epigenetic marks in gsc-mir- - as compared to na€ ıve gscs. functional significance of the chip-seq results was evaluated with confrontation of the chip data with the transcriptomes of the corresponding cells derived from exon array hybridization. western blot analysis showed that global h k me and h k me expression levels were similar in gsc and gsc-mir- - . chip-seq analysis revealed similar gross number of gene loci associated with h k me or h k me ( and genes, respectively) in either cell type. interestingly, % ( genes) of the analyzed genes exhibited a change in either h k me or h k me , or both in mir- - -gscs as compared to na€ ıve gscs. these changes resulted into a transcript variation in % of the cases ( / ) considering as significant an increase or a decrease of at least -fold and the % of these genes translated into congruent alterations in the corresponding transcript levels.analysis of the functional significance of the changes observed using functional annotation databases identified novel gene networks, likely to participate in the regulation of gsc properties. studies under way focus on members of these networks specifically activated in mir- - gscs that might alter gsc dialog with their microenvironment. malignant gliomas are the most frequent primary tumors of the brain with poor clinical prognosis. infiltrating peripheral macrophages and resident microglia contribute significantly to the tumor mass. we have previously shown that microglia as the intrinsic immune competent brain cells promote glioma expansion by up-regulating metalloprotease mt -mmp through toll-like receptor (tlr) and its adaptor protein myd . in this study we identified tlr , as the main tlr controlling mt -mmp expression and pro-tumorigenic signaling in microglia. glioma-derived soluble factors and synthetic tlr specific ligands induced mt -mmp expression in microglia from wt mice but not tlr -/-mice. by using the organotypic brain slice model, we found that tumor expansion depended on both parenchymal tlr expression and the presence of microglia. tlr is also highly expressed in human gliomas and inversely correlates with patient survival. in search for an endogenous tlr ligand released from glioma cells, we screened glioma conditioned medium (gcm) by mass spectrometry for endogenous tlr agonists produced by glioma cells and found versican, an extracellular matrix proteoglycan and a reported ligand of tlr . to examine if versican is the factor mediating the glioma-microglia interaction, we silenced versican expression in gl cells. primary microglia were then stimulated with gcm from versican sirna and non-target sirna transfected gl cells and microglial mt -mmp expression was analyzed by real-time pcr. an almost -fold up-regulation in microglial mt -mmp expression was observed using gcm from non-target transfectants while the expression was reduced with gcm from versican knock-down gl transfectants. our results show that tlr activation is an essential part of the signaling cascade employed by glioma cells to convert microglia into a pro-tumorigenic phenotype and tlr thus may be a novel target for glioma therapies. two ipsdms were used to determine their responses in the presence of glioblastoma cells. using lc-ms/ms method, proteomic profiles were generated and compared between ipsdms exposed to human glioblastoma cells and ipsdms exposed to normal human astrocytes as control.results: immunolabeling of the two ipsdm cell lines showed specific expression of microglial markers such as iba- , cd b, and cd . the comparative proteomic analyses indicated that microglia exposed to glioblastoma cells showed differential protein expressions relevant for cytoskeletal activity, energy production, and cell survival compared to control.conclusion: this is the first study showing the proteomes of human induced pluripotent stem cell-derived microglia exposed to glioblastoma cells, and the findings could provide further insight of the interaction between microglia and glioblastoma. key: cord- -pol qm authors: nan title: third international congress on the immune consequences of trauma, shock and sepsis —mechanisms and therapeutic approaches date: journal: intensive care med doi: . /bf sha: doc_id: cord_uid: pol qm nan this issue of the journal contains the abstracts for the third international congress on the immune consequences of trauma, shock and sepsis -mechanisms and therapeutic approaches. we hope that the information contained in this special issue will stimulate you to participate in the congress, to contribute to the knowledge being developed in this field and to use this information to help you in providing better care for your patients. we thank the editors and the editorial board and publishers of the journal for their interest and support in preparation of this special issue. we also, on behalf of the scientific committee, welcome you to the third international congress in munich on - march . when, in the mid- s, we thought of having a worldwide congress, we hoped to bring together investigators to discuss this theme. the explosion of knowledge occurring around that time provided an excellent background against which the first conference in provided stateof-the-art information and consensus on factors involved in injury and sepsis. in , the second congress was held at the time of another resurgence of research, study and information on injured and operated patients. it seemed then that there would be a lull in the development of new information and therapy, and that another state-of-the art conference might not be necessary until or . however, the explosion in molecular biology has continued. the wonderful world of cytokines has gone from ill to il- to il- , il- and il- and beyond. the vast amount of information about mediators and their importance in disease is impressive. this has all suggested a magic bullet that might be used to alter or block inflammatory responses. this has not happened, however, and the question is "why not"? our science is powerful, but our therapy is still weak. what are the issues, then, in , to be dealt with at this symposium and congress? ( ) proposals for new terminology. there have been a number of proposals for new terminology and new classifications of injury, sepsis, inflammation and various other problems related to human illness. the question is whether this is the way to go. will this contribute to better clinical trials, information basis and better research? the pros and cons of this development will be reviewed by those making the proposals and those questioning the need for and wisdom of this effort. ( ) magic bullets: the prospect of a magic bullet to deal with inflammation in injury and infection seemed highly promising earlier. many preclinical trials and a lot of animal research suggested the possibility of a great breakthrough in clinical care. what has become, then, of all the expensive and extensive multi-institution randomized, placebo-controlled, double-blind clinical trials of agents that block mediators and endotoxin. many such studies have yielded equivocal, marginal or negative results. the reasons for this and the future of clinical research will be the subject of presentations and discussions to set the stage for further work. ( ) should future clinical trials be based on new classifications of illness such as mods, sirs, apache iii, sap ii, mrm, etc., or should trials be dedicated to specific diseases -urinary tract infections, pneumonia, trauma patients, cardiac surgery and other specific problems, rather than generalized problems of sepsis, the sepsis syndrome and other classifications? in other words, should we now begin to have clinical trials on specific diseases with causes that are known and can be attacked? the causality of disease becomes an important consideration in this regard. ( ) a multitude of potential therapeutic agents has been proposed on the basis of animal studies. how should we decide which of them should be brought to clinical trial? the possibilities are endless as we develop new clinical information about the mechanisms and pathogenesis of human disease. ( ) information on the pathogenic mechanism of disease states and of injury continued to emerge in an explosive fashion, and in light of our gathering knowledge we can look forward to working out a cohesive system of response to injury. ( ) additional information will be provided in plenary sections, many symposia and free communication sessions and posters, which will update the participants on a variety of relevant topics presented by many of the leading in-iv vestigators in these fields. topics will range from molecular mechanisms, such as signal transduction, through the explosive growth of information on the role of cytokines and pathophysiology, to practical considerations in the design of immunomodulatory therapeutic regimens. these merely touch on a few areas, from the basic to the clinical, which will be the subjects of those symposia. all this information will fit into the jigsaw of this exciting area and its stimulus to further research study. this promises to provide an exciting, educational programme with experts and participants from all over the world. we hope it will set the stage for many years to come and will increase our understanding of trauma, shock and sepsis and help us to provide better therapy for those of our patients who are affected by such problems. a. the clinical syndrome of mods versus mof will be reviewed in detail by those who have made these proposals. b. an extensive review of the design and interpretation of clinical trials in patients with shock and injury will be provided. the reasons why so many clinical studies in the recent past have been negative will be reviewed. the therapeutic strategies that are being developed for the treatment or prevention of mods or mof will be the subject of another panel discussion by experts who have been involved in and contributed to this area. a consensus conference or controversy conference will be presented about various aspects of mods or mof, including the benefits of supernormal oxygen delivery, bacterial translocation, parenteral nutrition, the immune response and other aspects. the successes and failures of completed clinical trials will be presented by those who are involved in these clinical trials, with a refreshing review of the problems related to that injury. there will be late news about studies just being completed at present or after the beginning of and where they stand. c. the mechanisms and biochemical profiles of specific organ dysfunction or failure will be reviewed. what are the definitions? what are the mechanisms? how can organ dysfunction and/or failure be defined? an extensive review of the biological mechanisms involved in production of injury by mediators will be presented. a session will be devoted to how future ongoing trials might be better designed and what can be done about the studies recently completed, many of which are negative. d. the immunological or inflammatory pathways resulting in organ injury will be reviewed in detail in presentations and a panel discussion. we look forward to welcoming you to an exciting and rewarding conference, which undoubtedly possesses the potential to become a landmark event and major reference point for any scientific discussion about the complex of host defense dysfunctions following trauma, shock and sepsis. studies over the past years have established that the contact system, which forms bradykin/~, is gax important mediator in hypotensive septicemia. in addition to hradyk{nln, another product of the contact system, kailikrein, can mediate inflammation by virtue of its chemotaetic mad neutrophj/activating properties. using functional and immunochemical tech~ ques, we have demonstrated activation of the contact system in the adult respiratory distress syndrome in typhoid fever and clin/cal sepsis. we have also been able to inhibit the hypotension but not the disseminated intravaseular coagulation in a model of primate sepsis by the use of a monoclonal antibody directed agsi~st factor xii, the initiating protein of the contact system, in volunteers given e. coil endotoxin, who did not develop hypotension, we were also able to demonstrate activation of the contact system with a rise of alpha- macrogiebulin-kalllkrein complex. we have also examined, j~ an i~tensive care situation, patients with sirs. we found that serial measuremezzts of the contact system were useful in eva~u~ting prognosis+ these studies suggest that inhibition of kalllkrein a~d l e r bradykinin actions might be useful i~ obviating many .of the features seen in sepsis and septic shock. dextran sulfate (dxs) activates the contact system and, in vivo, produces transient hypotension. in order to better define the mechanisms underlying the dxs-induced hypotension, we investigated the effects of either the plasma kallikrein inhibitor, des-pro -iarg] ]aprotinin (bay ) or the b kinin antagonist, hoe on the hypotensive response to dxs. in the first study, anesthetized miniature pigs ( pigs/group, randomly assigned) were given one of the following treatment protocols: ) dxs ( mg/kg), - ) dxs plus bay ( , , , or rag), or ) saline. dxs alone produced a profound but transient systemic arterial hypotension with a corresponding reduction in plasma kinin-containing kininogen. circulating kinin levels, complement fragment c adesarg and fibrin mom)mer were all increased. bay produced a dose-dependent delay or attenuation in these effects with the highest dose completely blocking dxs-induced hypotension and elevations of kinin, c adesarg and fibrin monomer levels. thus, the effects of dxs are solely dependent on contact system activation and this activation is sensitive to bay . llowev~:r, contact system activation is known to produce changes in a variety of vasoactive mediators, all of which can affect blood pressure. in a second study, two groups of pigs ( /group) were given either dxs alone ( mg/kg) or dxs minutes after a bolus injection of hoe ( #g/kg). dxs alone produced transient hypotenmon. this response was completely blocked by hoe pretreatment. both groups had identical reductions in kinin-containing kininogen. we conclude that dxs-induced hypotension is produced by activation of the contact system which results in the production of bradykinin. liberation of bradykinin is both necessary and sufficient to produce all of the hemodynamic changes observed. dr. matthias siebeck, department of surgery, university of munich, klinikum lnnenstadt, nussbaumstrasse , d- munich, germany in experimental animals exposed to i.v. injection of endotoxin accumulation of leukocytes in various organs as lungs and the liver is a prominent feature. as a part of these morphological changes damages of endothelial ceils are regularly seen. this process, which is a part of endothelial-cellular interaction, leeds to exposure of the sub-endothelial basement membran. the basement membran is known f r its capacity to activate the contact system of plasma. during this cascade activation, coagulation factor xii is converted to the active factor xii. this activation might produce increased plasma kallikrein activities and thereby give release of the vasoactive substance bradykinin. using a porcine model we have noticed that endotoxin infusion ( , mg/kg) induces elevated plasma kailikrein activities within two hours after the start of the infusion. this enzyme activity remained increased during the next hours and reached value of up to u/ . in patients with sepsis we also have observed elevated plasma kallikrein activities with enzyme activities up to u/ . in order to further elucidate the significance of these elevated enzyme activities, we prepared human plasma kallikrein and injected it intravenously in anaesthetized pigs ( ). when very small plasma kailikrein activities ( , u/kg bodyweight) were given intravenously a % decrease in arterial blood pressure was seen in the animals. in the patients with sepsis also decreases in prekallikrein values and functional plasma kallikrein inhibition are frequently seen. furthermore, degradation of high molecular weight kioinogen is found in these patients indicating formation of bradykinin. these experimental and clinical studies underline that contact activation in sepsis might results in the release of very powerful mediator substances which can be of pathophysiological importance in this disease. a number of pathological disorders as reperfusion injury, bone marrow transplantation, polytrauma and septic shock are associated with capillary leakage. as the activation of the complement system and the contact phase play a major role in these diseases we investigated whether cl-lnhibitor (c -inh), which inactivates cl-esterase, kallikrein and clotting factors xii and xl, could abolish vascular leakage. a capillary leakage was induced in rats by the administration of interleukin- ( x iu/kg). the increased vascular permeability was monitored for one hour as the extravasation of fitc marked rat serum albumin from a mesenterial vessel by a video-image processing system. ci-inh (berinert®, behringwerke) given as a single i.v. bolus in concentrations of , or u/kg dose-dependently prevented the capillary leakage. carrageenaninduced inflammation in the rat leads to vascutar leakage and to edematous swelling of the paw. ci-inh in this model leads to a dose-dependent decrease in paw edema formation. finally, we investigated the effect of ci-inh (infusion ( - u/kg x h) on a lps-induced shock in the rat by combination therapy with the antithrombotlc agents antithrombin ill (kybernin®) or rec. hirudin (both substances from behringwerke). in this animal model mortality was % in the untreated control. both antithrombotic agents decreased mortality rates by inhibiting formation of dic; a further significant improvement of survival was achieved by the treatment with ci-inh. thus+ it could be concluded that c -inh has a beneficial effect in diseases associated with a vascular leakage. iclb and laboratory for experimental and clinical immunology, university of amsterdam, the netherlands; thrombosis research center, temple university, penn., usa; oklahoma medical research foundation,. ok. city, usa. to evaluate the contribution of the contact system to activation of other mediator systems in an experimental model of sepsis, we investigated the effect of mab c b which inhibits activation of factor xli, on activation of complement and fibrinolytic cascades and activation of neutrophils in baboons suffering from a lethal sepsis. activation of the complement system was assessed by measuring circulating levels of c b/c and c b/c, and a significant reduction was observed in animals that had received a lethal dose of e. coli together with mab c (treatment group), compared to animals that had received a lethal dose of e. coil only (control group). activation of the fibrinolytic system as reflected by circulating plasmin-= antiplasmin complexes and tissue plasminogen activator, and activation of neutrophils, assessed by measuring circulating elastase-=l-antitrypsin complexes, was also significantly less in the treatment group. we conclude that activation of the contact system protein factor xll during the inflammatory response to a lethal dose of e. coil in this baboon model, modulates directly or indirectly activation of the complement and fibrinolytic systems and that of neutrophils. in a prospective study, plasma levels of c a, c , and c a were measured in patients from an internal intensive care unit. patients were clinically septic defined by the criteria of bone et al.(l) . the remaining patients were critically ill but didn't fulfill the clinical criteria of sepsis. from both groups of patients blood samples were taken over a l days period. during the first days blood samples were drawn every h, on day - every h and the last days once daily. mean plasma concentrations of c a within the first h after clinical onset of sepsis were + pg/ml, whereas non-septic-patients exhibited mean values of only +_ p_g/m/. c levels were lower for septic-patients ( + lag/ml) than for non-septic-patients ( _+ lag/ml). the most profound difference between both groups was found, when the c a/c ratio was compared ( . + . for septic-patients and . _+_ . for the control group). no significant differences between both patient groups were observed in c a plasma levels ( . + . ng/ml in septic-patients vs. . _+ . ng/ml in control patients). in of cases of clinically defined sepsis causative organisms like bacteria, protozoa or fungi could be cultured from blood, bronchoalveolar lavages and/or section materials. application of the complement parameters to survivors (n= ) and non-survivors (n=l ) within the septic-group revealed, that the c a/c ratio could also be used as a prognostic parameter for clinical outcome. the possibility of rapid and easy measurement of c a and c in only - minutes ( ) and the significant difference of the c ajc ratio between the septic and non-septic group renders this parameter a good candidate for early diagnosis of sepsis in the intensive care unit. hirudin, a single polypeptide chain composed of amino acids with cysteine residues (mr daitons), is the most potent and specific thrombin inhibitor, which is now available as a genetically engineered product (rec. hirudin -hbw , behringwerke; marburg). the aim of our study was to establish a rabbit model of tissue factor (tf) induced activation of the extrinsic pathway of coagulation and to evaluate the therapeutic efficacy of rec. hirudin. coagulation was induced in female nzw rabbits by infusion of . p.g/kgxh thromboplastin for hours. development of disseminated clotting was manifested by a decrease of fibrinogen and platelets to . % and , % respectively, and by an increase of fibrin monomers from . to > . ~tg/ml. we administered rec. hirudin to rabbits in different concentrations ( . , . and . mg/kg); treatment started simultaneously with the infusion as an i.v. bolus. rec. hirudin significantly prevented the decrease of fibrinogen, platelets and the increase of fibrin monomers. this effect was dose dependent and long lasting, even hours after the administration of rec. hirudin, clotting was still significantly reduced. as could be drawn from the plasma levels, rec. hirudin had been cleared from plasma at this time. in a post-treatment study we administered rec. hirudin ( . , . and . mg/kg i.v. bolus) as late as hours after the start of tf infusion. at this time there was already a prominent activation of coagulation. even in this post-treatment regimen rec. hirudin significantly prevented disseminated clotting. hence, it was concluded, that rec. hirudin by inkihiting thrombin could be effective in the prevention of coagulation disorders including disseminated intravascular clotting (dic) induced by a septic disease. research laboratories of behringwerke ag, marburg, germany $ novel protease inhibitory activities of the second domain of urinary trypsin inhibitor (r- ) and its effect on sepns-lnduced organ injury in rat atsuo murata , hitoshi toda , ken'ichi uda , hidewaki nakagawa , takesada mori , hideaki morishita , tom yamakawa , jiro hirese , atsushi ni~ , nariaki matsuura osaka university medical school, osaka, mochida pharmaceutical co. ltd. tokyo, wakayama medical schoof, wakayama, japan inhibitory-activities of the second kuntz-type inhibitor domain of human urinary trypsin inhibitor (uti) and its effect on sepsis-induced organ injury in rat were investigated by using the recombinant protein. uti is a glycoprotein with a structure in which kunitz-type inhibitor domains are linked in a row. we isolated the gene encoding the second kunitz-type inhibitor domain of uti, and then constructed expression plasmids by ligating it to the e. coli phoa signal peptide gene. these plasmids expressed the second domain in e. coil strain je which lacks the membrane lipoprotein. the recombinant second domain (r- ) innb[ted trypsin, plasmin, neutrophil elastase and chymotrypsin. in addition it inhibited blood coagulation factor xa and plasma kallikrein in a concentration dependent and competitive manner. the in vivo effect of the recombinant r- was investigated in a rat model of septic shock induced by cecal ligation and puncture. the administration of r- significantly improved the survival rate of the rats and attenuated the pathological changes of lung and iiver. we found out the novel protease inhibitory activities of the second domain of uti and its protective effects on sepsis-induced organ injury. macrophages are known to secrete lysosomal proteinases,mainly cathepsin b and cathepsin l, and also ~-proteinase inhibitor (pi),related to acute phase proteins.disturbances of proteinases/ proteinase inhibitors correlates with inflammatory process,leading sometimes to noncontrol "pathglogical" proteolysis (jochum et ai., ) . the cathepsin l-like and cathepsin b-like activity were measured in serum of patients with chronic bronchitis ( -with obstructive, -with nonobstructive bronchitis),acute bronchitis ( ) and healthy persons.simultaneously the level of~pi was determined in the same groups.cysteine proteinases were measured with help of fluorogenic substrates,as was presented earlier (korolenko et ai., ) , ~pi with help of immune enzyme method. it was shown increase of cathepsin l-like and cathepsin b-like activities during aggravation of chronic bronchitis comparatively to the controls ( - fold) .after treatment there was a tendency to normalization of indices,but the increase was about - % more than the control values.~pi level in this group was also increased (two-fold),in patients with acute bronchitis - - -times more comparatively to the control.it is possible to conclude that chronic bronchitis induced increased secretion both cysteine proteinases and d{pi into blood. some peculiarities of ratio were noted in patients with emphysema. endotoxins are microbial products derived from the outer cell membrane of gram negative bacteria. the active component of endotoxin is lipopolysaccharide (lps), a complex macromolecule consisting of polysaccharide covalently bound to a unique lipid, termed lipid a. now recognized to embody the endotoxic principle of lps, lipid a consists of a/ - diglucosamine backbone, both ester and amide linked fatty acids, some of which are acyloxyacylated, and charged constituents such as phosphate, phosphorylethanolamine and amino arbinose lps, exerts its biological effects in vivo by noncytotoxic interactions with a variety of host inflammatory mediator cells, primarily the mononuclear phagocyte and the endothelial cell, although other host cells also participate. these interactions are modulated by lps-specific binding proteins found in plasma, including lps-binding protein (lbp) scd and perhaps other proteins as well. specific receptors for lps have been identified on mammalian cells which mediate signal transduction via multiple pathways. lps-activated host cells are stimulated to secrete or express multiple proinflammatory mediators, including tnf-a, illa, il- / , ifn-a, il- , il- , il- , paf, pge, ltb and procoagulant activity. the overproduction of these proinfiammatory mediators results in the manifestations of endotoxemia, observed experimentally as fever, hypotension, disseminated intravascular coagulation and death. modulation of activity of these mediators protects animals against lethality. similar pathways are thought to be operative in gram negative sepsis, and control studies with human volunteers support such conclusions. immunotherapeutic approaches in clinical gram negative sepsis have, to date, been less successful. in vitro experiments and studies in animal models have recently shown that several proteinaceous bacterial exotoxins can evoke cytotoxic effects that ultimately lead to cardiovascular collapse and shock. since the possible relevance of bacterial exotoxins in the pathogenesis of septic shock has received very little attention in the past, an attempt will be made here to provide a brief overview of this generaily neglected topic. protein toxins act intracellularly or they dz~nage the integrity and function of the plasma membrane. major representatives of the former group are the adenosine diphosphate (adp)-ribosylating toxins, e.g. cholera and cholera-like toxins, diphtheria toxin), and the neurotoxins. most medically relevant toxins of this category have been studied in great detail. although often responsible for severe and sometimes fatal disease, their association with septic shock is rare. in contrast, experimental evidence is accumulating for a role of membrane<lamaging toxins in the pathogenesis of inflammatory lesions. formation of transmembrane pores is probably the most widespread mechanism via which such physical membrane perturbation can occur ( , ), the present discussion will be restricted to this category of toxins. the author shall first discuss basic properties of pore-forming proteins, and consider the factors that direct their attack to specific targets. thereafter, attention will be drawn to diverse functional consequences that may follow membrane damage so that a general concept of how pore-forming toxins may contribute towards the development of shock will evolve. i. bhakdi, s. and tranum-jensen. j. gram positive bacteria produce exotoxins that, at least in part, exhibit the unusual properties of superantigens. superantigens (sag) activate v/ selectively t cells to produce lymphokines including i - and tnf/lymphotoxin. systemic application of the sag staphylococcal enterotoxin b (seb) to d-galactosamine sensitized mice causes lethal shock within hours. seb reactive v/ + t cells accumulate within - min mrna for the ii- , i - , tnfai~ and ifn- ,. parallel in time high concentrations of bloodborne i - and tnf are noted. anti tnf t~// neutralizing mab protect mice against seb induced t cell dependent lethal shock thus indicating a key role of tnf in effecting lethal toxicity. within - h distinct levels of tolerance to seb become discernable on ligand reactive v/ t ceils, dependent of the dose of seb used. these levels include anergy, t cell receptor downregulation, loss of cd , cd , cd accessory molecules and programmed cell death. m.freudenberg, y.yaegashi, p.nielsen, c.galanos. the sensitivity towards endotoxin (lps) is genetically determined, however it may be increased under different experimental conditions. these include treatment with hepatotoxic agents such as d-galactosamine, and with live (infection) or killed bacteria. it is well established now that d-galactosamine sensitizes to lps by increasing the susceptibility of the host to toxic activity of lps-induced tnf~. sensitization by bacteria, especially by gram-negative once is of especial interest because it implies that infecting microorganisms not only produce lps, but als sensitize the host to its lethal activity. sensitization to lps by bacteria proceeds in all lps-sensitive (lps n) mouse strains that have been investigated so far. it also proceeds in lps-resistant (lps d) c h/hej mice. the absence of sensitization to lps in lps d c bl/ sccr mice was identified as being due to their inability to produce interferon- (ifn?). administration of exogenous ifn? to these mice conferred sensitivity to lps. conversely, administration of anti-ifn? to lpsn; mice inhibited the development of sensitization'by bacteria. it may be concluded therefore that ifn is the mediator of sensitization to lps by bacterial infection. the ifn? defect of c bl/ sccr mice concerns only the ifn response to bacteria, since the response to the t-cell mitogen con a and to cd monoclonal antibodies was not impaired. the ifn?producing cells themselves were shown to be intact. on closer investigation the impaired ifn? response was identified as the result of a defective accessory function of macrophages. macrophages of c bl/ sccr mice are incapable of producing interferon-~ (ifn~) which we could show to be obligatory for the production of ifn? in response to bacteria. although antibiotics are required to clear bacteremia, they do not reverse evolving shock, because endotoxin free in the bloodstream or exposed on the surface of circulating bacteria continues to activate the production of inflammatory mediators, furthermore, antibiotics have been shown to induce release of endotoxin from gram-negative bacteria during the treatment of severe infection. in an in-vitro study, using live escherichia coil, we have observed the release of endotoxin upon treatment with several antibiotics like cefuroxim, imipenem, ceftazidime and aztreonam. incubation with imipenem or ceftazidime induced an early lyeis and a mild rise in endotoxin levels, whereas incubation with cefuroxime or aztreonam resulted in the formation of filaments with a late but dramatic rise in endotoxin levels. in a second study we studied the influence of different antibiotics on the tnf-= production of e.coli stimulated human monocytes. we found again great differences in the production of this important cytokine among the different antibiotics according to their capacity to induce liberation of endotoxin. these differences in the amount of endotoxin release are probably caused by penicillin-binding-proteins (pbp) specificities of the antibiotics with resultant differential morphological changes, in a rat sepsis model treatment with imipenem or ceftazidime resulted in a transient increase in il- levels, whereas treatment with aztreonam resulted in progressively increasing levels of il- and a significant increase in mortality. the increased mortality in pbp- specific aztreonam treated rats might have been the result of filament formation in the abdominal cavity of the septic rats. the endotoxin sequestered at local sites may account for the precipitation of gram-negative shock. finally, we've also demonstrated that in patients under treatment for septic shock endotoxin release can be related to the administration of antibiotics. carbapenem-and cephalosporin-lnduced release of lps from smooth and rough pseudomonas aeruginosa: in-vivo relevance j. jackson and h. kropp, merck research laboratories, rahway, nj b-lactam (cell-wall active) antibiotics are generally deemed the class of antibiotics most responsible for the release of free endotoxin (lps) from gram-negative bacteria due to their cell lytic actions although all antibiotic classes studied thus far induce release of endotoxin. we have recently shown in-vitro that antibiotics within the b-lactam class (i.e. imipenem , meropenem and ceftazidime , with equivalent mics) differ in their propensities to release excessive amounts of lps from both smooth-(s-) and rough-(r-) lps laboratory and clinical and antibiotic-sensitive and -resistant p. aeruginosa isolates and that lps release is independent of cell lysis. additionally, variations in the induction of endotoxin release is antibiotic concentration dependant and correlate with antibiotic penicillin-binding protein (pbp) affinities which result in morphological changes. these studies also show that lps differences measured via chromogenic lps (c-lal) assay correlate with lps lethality in lps-sensitive mice. release of lps was compared to changes in cfus, cell mass, morphology and antibiotic pbp-specificity. corresponding in-vivo studies in cd mice against s-lps p. aeruginosa isolates show that, despite equivalent in-vitro protection, antibiotic efficacy in mice differ as much as -to lo -fold. to establish a possible in-vivo role for antibiotic-induced release of free lps we compared antibiotic efficacy results in mice challenged with virulent parent s-lps and its relatively avirulent r-lps mutant (lacking only its side chain) p. aeruginosa isolates. results show the relevance of quantity and physiochemical composition (bioreactivity) of free lps to virulence and in-vivo antibiotic efficacy. in other in-vivo studies chemical agents that destroy cells (m~) which mediate lps lethality in-vivo were used to pretreat mice prior to antibiotic therapy and bacterial challenge with wild type pseudomonas. we conclude that circumstances in which antibiotic-induced filamentation occurs are also conditions that yield excessive lps release, the in-vivo effects of which are shown through the requirement of larger amounts of antibiotic to afford equivalent protection. bacterial endotoxins have been reported to play a significant role in the pathogenesis of gram negative sepsis by their interaction with, and stimulation of, host inflammatory mediator cells to produce cytokines, biologically active lipid intermediates, and procoagulant activity (tfa). although both microbe-associated and free endotoxin are capable of stimulating mediator production, studies from our laboratory suggest that free endotoxin may be the more potent stimulus for tnf and tfa. further, our studies suggest that the majority of the proinflammatory activity released from antibiotictreated e. coli coisolates with lps by two different fractionation techniques. to assess whether actions of endotoxin directly contribute to lethality in gram negative sepsis, an experimental model was developed in which mice were made hypersusceptihle to the lethal effects of endotoxin by treatment with d-galactosamine (dgaln). challenge of cf- dgaln-treated mice with e. coli olll:b resulted in an lds of approximately . x cfu. resolution of the peritonitis and bacteremia by treatment with cell wall-active antibiotics resulted in an increase in the ldso. ceftazidime and imipenem, which differ in their mechanism of action and in their capacity to effect endotoxin release in vitro, also differed in their in vivo capacity to provide chemotherapeutic protection ( fold vs fold respectively, p< . ). parallel studies with endotoxin hyporesponsive c h/hej mice indicate significantly greater levels of protection with imipenem (> fold vs saline controls). collectively these data suggest that endotoxin may contribute directly to the pathogenesis of experimental gram negative sepsis. bacterial lipopolysaccharides (lps) are the endotoxins of gram-negative bacteria and represent their major surface antigens. lps is made up of three chemically, biologically and genetically disctinct regions, i.e, the o-chain, the core region and the lipid a moiety whereby the latter represents the endotoxic center. it is our current understanding that lps is responsible for many of the pathophysiological events observed during gramnegative infections and that one of the major mechanisms leading to shock and death is the lps-induced activation of macrophages resulting in the production and release of lipid and peptide mediators, among which tumor necrosis factor seems to be the most important. therefore, in the fight against the lethal outcome of gram-negative infections, modern strategies, in addition to antibiotic treatment, aim at i) the neutralization of tumor necrosis factor, ii) the inhibition of the production of tumor necrosis factor or iii) the neutralization of the activation potential of lps for macrophages by monoclonal, preferably human antibodies. the latter approach, to be effective against a broad spectrum of gram-negatives, must be directed against common structures of lps (lipid a and core region). the molecular basis of this approach and the controversy in this field will be discussed. passive immunotherapy has been used since , when von behring described the administration of immune horse serum to treat a patient with diphteria infection. even if this therapy was sometimes successful in bacterial infections, it has been largely replaced by antibiotics. however, antibiotics have their limitations, especially in critically-ill patients. to improve outcome, adjunctive therapies such as immunotherapy with polyclonal and monoclonal antibodies particularly against endotoxin are again considered. the role of humoral immunity in host defenses against bacterial infections is weu known. for instance, tile importance of antibodies in the defense against gramnegative infections has been established clinically by studies relating the outcome of patients with gram-negative bacteremia to tilers of antibodies directed at the offending pathogens at the onset ofbacteremia (mccabe ; pollack ) . ever since we know the role of endotoxins in the pathophysiology of sepsis, antibodies against the s-and r-lps have also been detected in sepsis patients. the aim of the administration of iv/g to the sepsis patient is as follows: ) enhancing of opsonization and phagocytosis(antibactericidai activity) ) synergistic effects with [ - actam antibiotics ) neutralization of endotoxin, the main pathogenic mediator of gram-negative sepsis ) modulation and/or inhibition of cytokine release the enhancement of opsonic-and phagocytic-activity especially with igg via fc and c receptors has been well documented. monoclonal antiendotoxin antibodies, proven in clinical studies, do not appear to neutralize endotoxin in vitro and are not reproducibly protective in animal models of sepsis. also they can not suppress endotoxin-induced tnf-~, il- release in mice (baumgartner , corriveau and danner ) . in conlrast, recent studies of a polyclonal immunoglobulin preparation, containing high levels of antibodies against gram-negative bacteria and their o-antigen of lps in igg, igm and iga classes (pentaglobin®) provide evidence to neutralize endotoxin. this effect is demonstrated in vitro (berger (berger , , in animal models (stephan , berger and also in prospective, randomized, controlled clinical trials (schedel , poynton , behre . furthermore mortali b' was reduced statistically in patients with septic shock and endotoxemia by using this preparation, as has been demonstrated by sehedel. anti-core lps monoolonal antibodies: binding specificity and biological properties f.e. di padova, r. barclay, e.th. rietschel. bacterial lps and cytokines are responsible for the pathological processes of gram-sepsis and are suitable targets for therapeutic interventions. chemical characterization and structural analysis of different lps have revealed common features. the inner core region of lps shows a high degree of similarity among e. coli, salmonella and shigella. among a large number of broadly cross-reactive murine anti-core lps mab one of these igg ak) has been selected and chimerized into a human igglk (sdz - ). in elisa and in immunoblots on purified lps both sdz - and wni - show a strong reactivity with all smooth lps from e. coli and salmonella. reactivity with all the known complete core structures from e. coli and salmonella (ra) is evident. reactivity with re structures or free lipid a is not observed. this mab cressreacts with all clinical e. coli isolates from blood, urine and feces and with other enterobacteriaceae. sdz - and wni - have biological activity as they inhibit the lal assay and the secretion of monokines (il- and tnf) by mouse and human macrophages. moreover, sdz - and wni - inhibit the release of il- and tnf in vivo. in vivo sdz - as well as wni - neutralize the pyrogenic activity of e. coli lps and protect mice from lethality in d-gain-sensitized mice. the possibility to use wni - as a capture antibodies in the immunolimulus assay opens the possibility to differentiate the origin of the lps in patients with endotoxemia. franco di padova, sandoz pharma ag, ch basel, $chweiz $ presentation of lps to cd by lps binding protein peter s. tobias, julie gegner, katrin soldau, lois kline, loren hatlen, douglas mintz, and richard j. ulevitch. the activation of myeloid cells by lipopolysaccharides (lps) has been shown to require the serum glycoprotein lps binding protein (lbp) and binding of lps to membrane bound cd (mcd ). other cells such as human umbilical vein endothelial cells (huvec), smooth muscle cells, and some epithelial cells, which do not express mcd but nevertheless respond to lps in the presence of serum, have receptors for complexes of lps with the soluble form of cd (scd ). these complexes of lps with scd are only formed efficiently in the presence of lbp. we have begun to characterise the mechanisms by which lbp enables lps to bind to cd , either soluble or membrane bound. with the use of fluorophore and radiolabelled reagents we have developed procedures for quantitative measurement of the association of lps with lbp and of lps-lbp complexes with cd . these results show that the delivery of lps to scd is catalysed by lbp, i.e., lbp is not included with the lps-scd complex. in contrast, on the surface of cells, lbp does not dissociate from the cells after lps binds to mcd . the kinetics, equilibria and stoichiometry of these reactions will be discussed in the context of models for cellular activation by lps and cellular uptake of lps. supported by nltt grants gm , ai , ai , gm , and assistance from the pharmaceutical research institute of johnson and johnson. the scripps research institute, imm- , n. torrey pines rd. la jolla, ca usa . modulation of endotoxin-induced cytokine production by lps partial structures h.-d. flad, h. loppnow, t. mattern, and a.j. ulmer department of immunology and cell biology, forschungsinstitut borstel, d- borstel lipid a constitutes the active moiety of endotoxin (lps) of gramnegative bacteria. it activates mononuclear phagocytes to produce cytokines, such as tnf, i _- , and il- , which are the major mediators of the endotoxic effect of lps in vivo. lipid a precursor la (synthetic compound ) does not induce cytokines, but is able to specifically antagonize lps-or lipid a-induced mediator production in human mononuclear cells, vascular endothelial cells, and smooth muscle cells. furthermore, we present evidence for the first time that t-lymphocytes proliferate in response to lps and express mrna for interleukin- and interferon-~ and that these responses are also antagonized by synthetic lipid a precursor la. when comparing the agonistic and antagonistic activity of lipid a and different partial structures at the functional and binding level, the number and length of the fatty acids and the number of phosphoryl groups were pound to be of crucial importance. unexpectedly, lipid a precursor la, although biologically inactive, turned out to be both the most potent antagonist and competitor in inhibiting the binding of lps. taken together, our results provide evidence for a model in which lipid a partial structures compete with lps for specific cell surface receptor(s). in this sense, biologically inactive lipid a analogues may be good candidates as therapeutic agents for the prevention of gram-negative septic shock. two mammalian lipid a-binding proteins have been identified that are believed to have important roles in mediating the host response to endotoxin: lipopolysaccharide-binding protein (lbp) and bactericidal/ permeability-increasing protein (bpi). human lbp shares a % amino acid sequence identity with human bpi. despite the sequence homology, the two lipid a-binding proteins have very different functional activities. lbp is an acute phase serum protein that markedly potentiates the proinfiammatory host response to gram-negative infection by a mechanism which involves binding of the lbp-lps complex to cd receptors on monocytes, neutrophils and endothelial cells. in contrast, bpi is a neutrophil granule protein with potent bactericidal and lps-neutralizing activities. the divergent functional properties of these two lps-bindlng proteins can be explained by the inability of bpi-lps complexes to bind to cell-surface cd receptors. a recombinant protein (rbpi ), corresponding to the amino terminal kd fragment of human bpi, has been shown to retain the potent biological activities of the hdlo protein and may represent a novel therapeutic agent for the treatment of gram-negative infections, sepsis and endotoxemia. for therapeutic effectiveness in many clinical situations, rbpi will have to successfully compete with relatively high serum levels of lbp ( - ~g/mi) for binding to endotoxin and gram-negative bacteria. to evaluate this issue, experiments were conducted to compare the relative binding affinities of rbpi and human recombinant lbp (rlbp) for lipid a. the binding of both proteins to iipid a was specific and saturable with apparent kd's of . nm for rbpi and nm for rlbp. in a competition assay format rbpi was approximately -fold more potent than rlbp in inhibiting the binding of nsi-rlbp to lipid a. these results demonstrate that rbpi has a significantly higher affinity for endotoxin than does rlbp and may explain the potent inhibitory activity of low concentrations of rbpi in a variety of in vitro functional assays for lps activation of cells despite the presence of high lbp levels. for example, rbpi at . ~tg/mi was able to totally inhibit lps-induced tnf release from monocytes despite a -fold weight excess of rlbp over rbpi . and for heparin binding. three separate domains which inhibit the lal reaction to lps and bind to heparin were identified in amino acid regions - , - and - . a single synthetic peptide ( - ) was bactericidal. these results suggest that rbpi contains three separate functional domains which may contribute to its high affmity interaction with gram-negative bacteria and heparin. the individual activity of each domain and the cooperative interaction among domains provide the basis for developing rbpi analogues with increased biologic efficacy. a considerable body of experimental data has accumulated implicating tumour necrosis factor (tnf) as a principal mediator of the pathophysiological features of septic shock. these data prompted the development of clinical strategies designed to limit excess (inappropriate) tnf production. monoclonoal antibodies (mobs) were developed and a phase ii dose escalation trial in patients confirmed that the mab was safe, and suggested that it was having a beneficial effect on certain parameters. preliminary results of a large phase iii study indicated that (a) the mob was safe; (b) that it was of no discernible benefit in non-shocked patients; (c) that it reduced mortality in shocked patients, especially during the first days. an alternative strategy was to take advantage of the high binding affinity of soluble receptors for tnf (stnfr). stnfr-iggfc constructs were made for both the p and p receptors. both were effective in animal models of lps challenge, but when a clinical trial was done with the p stnfr-fc there was unexpected mortality in the treated arm. using an animal model of live e.coli sepsis, we have shown that this may have been due to the release of bound tnf from the construct. plasma enhances while bpi inhibits lps-induced cytokine production from peripheral blood mononuclear cells (pbmc). pseudomonas species produce cytokine-inducing substances which are different from lps as indicated by the fact that polymyxin b blocks only % of the cytokine-inducing activity of these pyrogens. we now tested the effect of plasma and bpi on the il- [ -inducing activity of pseudomonas maltophilia -derived pyrogens (pmp). bacteria were cultured to the log phase and filtered ( kd) to obtain prop. dilutions of pmp or lps were added to pbmc alone or to pbmc in % plasma +/-bpi ( ng/ml). pbmc were incubated for hours at °c and total il-i~ was measured by ria. results: il-i[~ in ng/ml (n= , mear~+sem, *p< . vs control). control . _+ + bpi . + % plas. . _+ + bpi . _+ pmp (ng/ml) lps (ng/ml) . _+ . _+ . _+ . _+. . +. . _+. . _+. " _+ " . _+ " . _+ " . _+. . + _+ _+. * _+ " . +. " . + -+ . -+ " . _+. " cba, c bl/ , balb/c, akr, dba, swiss mice, guinea pigs, rabbits have been used in research work. the toxicity, immunogenicity, mitogenic and immunomodulating activity of lps have been studied. the possibility of reduction of the toxic activity of lps on macroorganism by bioglycansimmunomodulators obtained from sea invertebrates anymals (crenomytilus grayanus, stromhus gigas) have been investigated too. lps has been shown to induce specific antibody response of laboratory animals. cba mice are high responsive to lps. lps stimulates humoral immune response of mice to tdependent and t-independent antigens and suppresses intensity of the delayed hypersensitivity. the small doses of lps stimulate functional activity of macrophages, the large doses of lps -decrease one and show the cytotoxic effect. the bioglycans enhance the resistance of mice to the lethal effect of lads and provide protection - % of mice. one opens possibility to use of bioglicans for reduction of toxinemia in generalizated forms of pseudotuberculosis. thus, lps from y.pseudotuberculosis is immunogen and immunomodulator wich has influence on humoral and cellular factors of immunity and plays the important role in immunopathogenesis of infection. endotoxaemia is implicated in the pathophysiology of obstructive jaundice. the lirnulus lysate (lal) assay is the gold standard method for measuring endotoxin concentrations, but inherent biochemical and technical problems limit the usefulness of this assay. the endocab elisa is a novel assay which measures endogenous antibody (igg) to the inner core region of circulating endotoxins (acga). objectives we evaluated the significance of endotoxaemia in biliary obstruction using the endocab assay and subsequently the specificity of the humoral response to endotoxin compared with an exogenous antigenic challenge [tetamls toxoid (tt) ]. materials and methods in experiment i three groups of male wistar rats ( - g) were studied [no operation (n= ) , sham operation (n= ), and bile duct ligation for days (bdl)(n= )]. plasma was collected and assayed for bilirubin, endntoxin(lal) and acga(endocab). in experiment ii rats were actively immunised with tetanus toxoid ('it) and then randomised to have no op(n= ), sham op(n= ) or bdl(n=i ). blood was taken at this time (to) and days later(t at sacrifice for acga concentrationslendocab] and igg produced to tt(ttab) [elisa] . antibody concentrations are expressed as % increase from control values.results in bdl rats, acga concentrations were significantly increased compared with controlslp< . , mann-whitney]. endotoxin concentrations were sporadically elevated in the jaundiced rats but the rise was not significant. in experiment [i there was no difference between the acga or ttab concentrations in the fllree groups at to, bdl rats had a significant rise in acga concentrations by t [p< , ,paired t-test] and humoral response to tt was significantly impaired in bdl rats compared with control groupslp< . , paired ttest data plasma endotoxin was measured by means of an endotoxinspecific endospecy test after pretreatment of the plasma with a new perchloric acid method that we developed. the normal value of plasma endotoxin is less than . pglml. polymyxin b was administered at a dose of , u every hours. plasma endotoxin rapidly decreased to the normal range in of the patients. body temperature fall significantly. apache ii scores were also significantly improved. tumor necrosis factor-o~ and interleukin decreased in survivors, while in high values tended to persist in patients died. no side effects were observed in any of the patients. in conclusion, intramuscular injection of minute of polymyxin b was useful in the treatment of endotoxemia. - uchimaru, morioka , japan. l e v a n t g r a m n e g a t i v organisms. m e t h o d s : u n d e r general anesthesia, n o r w e g i a n b r e d landrace pigs ( - kg) of either sex, pr group, u n d e r w e n t t r a c h e o s t o m y a n d w e r e v e n t i l a t e d on a / air a n d o x y g e n m i x t u r e a i m e d at m a i n t a i n i n g a n o r m a l p h a n d a isocapnic level. ventilation w a s not readjusted d u r i n g the observation period. the anesthesia w a s k e t a m i n e . m g / k g h a n d d i a z e p a m . m g / k g h i n t r a v e n o u s l y . h e m o d y n a m i c m o n i t o r i n g of m e a n aorta, p u l m o n a r y artery, central v e n o u s a n d p u l m o n a r y capillary w e d g e pressures w a s p e r f o r m e d w i t h a f s w a n -g a n z catheter a n d an aorta catheter. a continous infusion of r i n g e r ' s acetate ( m l / k g h ) w a s g i v e n intravenously. w h e n stabilised, the a n i m a l s w e r e g i v e n . x l cfu of e colt intraperitoneally as a bolus in ml saline, the a n t i b o d y g r o u p received in a d d i t i o n m g / k g e a n t i e n d o t o x i n i n t r a v e n o u s l y over h o u r via a n infusion p u m p at the start of the observation period. the a n i m a l s w e r e observed for hours. results : a t a n d hours, the o x y g e n c o n s u m p t i o n increased by % in the a n t i b o d y treated g r o u p w h e r e a s there w a s a significant fall of % in the sepsis group. in the a n t i b o d y group, the arterial p h a n d the cardiac index were also significantly h i g h e r at the s a m e p o i n t s in time. there w a s no significant difference in arterial po . in severe bacterial infections it would be beneficial to neutralize the plasma endotoxin content with complex forming compounds. the phenothiazines are able to form complexes with endoto×in and the existence of these complexes were already shown in differential speetrophotometry and animal experiments, however, the mechanism of partial neutralization was not clarified. therefore some representative phenothiazines and structurally related compounds were tested for anti-endotoxin activity. the endotoxin neutralizinb effects of several benzophenothiazines were investigated in differential speotrophotemetry, tnf induction and in the conventional limulus test. in animal experiments some beneficial effect of complex forming compounds was found. the benzophenothiazines were not able to inactivate the biological effect of endotoxin in the limulus test. the recent findings indicates that a multifocal effect can be responsible for "anti-endotoxin action in vivo". effects of tnf inducing effect of endotoxin in leukocytes and bypotensiv action in experimental animals were reduced by some phenothiazine derivatives. monophosphoril lipid a was without effect. of microbiology, albert szemt-gydrbyi medical university, odm t~r lo, h- szeged~ hunbary involvement of streptococcus pyogenes erythrogenic toxins in the induction oflstreptococcal toxic shock syndrome heide mgller-alou~* , joseph e. alouf , die [er gerlach , ~atherine fitting., and jean-marc ca~aillon . unit des toxines microbiennes and "unit d'immuno-allergie, institut pasteur, , rue du docteur roux - paris (france) ; institut f~r experimentelle mikrobiologie, jena (germany). superantigen erythrogenic toxin a (eta) is thought to be involved in toxic shock syndrome in humans by inducing massive release of cytokines by patient immune cells. the cytokineinducing capacity of eta w~:s £:ompa~ed to that of lps, a gram-negative bacterial cell wall component. eta elicited weak production of il- d and ~, tnf ~ and il- in purified human monocytes whereas lps stimulated the production of high amounts of these cytokines. in the presence of t cells, eta elicited the production of significant amounts of il-i~, il-i~, il- and il- . however, the most preponderant cytokine was tnf~, which peaked at i ng/ml after stimulation with i ~g eta. comparable amounts of tnfd (ca ng) were induced by .i ~g eta and .i ~g lp$. in contrast to lps, eta was a strong inducer of tnf~ which was produced only in marginal amounts by lps. these results suggest that the septic shock induced by gramnegative bacteria (lps) and by gram-positive bacteria {extracellular superantigens) follows different pathogenic pathways. lps-induced shock is mainly mediated by monocytes and monocyte-produced cytokines (il-i and tnf). the eta-induced shock is mediated by t-cells or depends on t cell help for the production of monocyte-liberated cytokines. production of t cell cytokines such as tnf~ and interferon in addition to the other cytokines contribute very likely to the severity of the toxic-shock resulting from s. auzeus and s. pyogenes infections in humans. the present study was utidertakc~l to cvalu~tlc the effect of soluble chemically modified giucan during septic shock. carboxylnethyl-b-i, -glucan (ram ) was injected twice and h before the shock i.v. in a dose of ing/kg. shock was induced in u~?esthetizcd (sodikm~. l)mntobarbital) rats by i.v. injection of endotoxin of escherichia colli bs, mg/kg. aiiofcmg pretreated ruts survived during first haher ¢ndotoxine, while in controi shock group the lethality was %. the concentration of ~col)terin in serum was significantly elevated hafterthc second cmginjection (appare~tly % if compare with the control rats), but didu't chartged rain and s rain after endotoxin injectjom cardiac output in cmogroup was higher a* the i and min after endotoxine onset ( i % trod ~, respectively of initial level) than in the control shock group ( % and % at the same time). pretreatment of rals with soh~ble giucan w~ts associated with beneficial effects o~ the hepatic c~ergy $ia[tls after h after challenge of endotoxiae: the tissue level of lactale was ahnost twice lower than in the control ruts, me~mthne the tissue atf in cmg pretreated group was higher at %. twice injected macrophage stimuhttor soluble glucan can prevent the endotoxic shock, and extremely ir~creased survival rate after endotoxine injection. the national committee of surgical infections of the spanish association of surgeons have produced a computer program for the collection and analysis of information on surgical infections. the program is suitable for ibm compatible hard disk personal computers and works through the ms-dos system. the main menu is called up on the screen when the operating disk has been installed; it reads as follows: i. new record; . modify records; . erase records; . searches; . reports; . configure; o. ouit. if you ask fdr a new record the screen will prompt you to enter the number of case, record number, hospital, age and sex. the next screen will come up and the words "topographic diagnosis" will flash. a menu of areas or organs will be displayed. then, the words "type of pathology" (inflammatory, neoplastic, traumatic and other). days of postoperative period. type of surgery (programmed and emergency). type of operation (clean, clean contaminated, contaminated and dirty). duration of surgery. this is followed by "order of operation" and the "type of anaesthesia (general, regional or local). you are then required to supply the "diagnostic code of who" (icd ) and the "procedure code of who. analytic and concurrent illnesses (total proteins, albumin, haemoglobin, haematocrit, leucocytes, red corpuscles, glucose and bilirubin). the next screen asks for "risk factors" (obesity, uraemia, neoplasia, malnutrition, urinary catheter, distant infection, artificial valve, immunosuppressive drugs, over years and anergy. this is followed by a screen headed "postoperative complications". "evolution" (the questions asked are drainage, systemic antibiotics, and on each ocasion a choice of antibiotics is displayed), local antiseptics, reoperation, etc. under "microhiology" is a choice of organisms and the chance of identifyin organisms. finally, "sepsis score". our recent work had shown that renshen-fuzi-chaihu mixture could increase the survival rate in experimented study. the purpose of this study was to determine the effect of combined administration of renshen-fuzi-chaihu mixtuer and antibitics (sa) in patients with septic shock. the result showed that, in sa group ( cases), the total effective rate was , %, in the contral group (combined administration of gentamycin and dexamethasone, cases) the total effective rate was %. however the obviously effective rate in sa group % was significantly higher than in contral group % (p<o.os). sa group appeared to be more obvious than the contral group in raising arterial pressure, recovering consciousness and pulse, improving cold lilmbs and reducing fever (p<o.o ). it was found that sa could inhibit the pathogeneses changes of septic shock such as the decreases of superotide dismutase (sod) and glutathione peroxidase (gsh-px) in erythrocytes and the increases of plasma free hemoglobin (phb), plasma malondialdehyde (mda) and -glucuronidase ( -gc) occured in septic shock. sa had stronger effect than gd in inhibiting the changes mentioned above. hence, these results suggest that sa has beneficial effect in the treatment of septic shock. sa could diminish the decrease of the antioxdase activities and inhibit lipid peroxidization and stabilize cellmembrance. this may be one of the principal mechanisms of the beneficial effect of sa in the treatment of septic shock. donna l. lehman, heather a. childers, irshad h. chaudry and alfred ayala. the thymus is thought to be a privileged sight for tcell generation. here the t-lymphocytes are either selected for their potential to recognize and react competently to foreign antigens or de-selected, due to their potential to react to auto-antigens, de-selected. tcells with this latter capacity are thought to be deselected through a process referred to as programmed cell death or apoptosis. while it is known that thymic apoptosis is elevated by a variety of stressers associated with infection and inflammation, little or no information is available concerning its presence in polymicrobial sepsis. it was, therefore, the aim of this study to determine whether thymocytes exhibit increased apoptosis during sepsis. to assess this, thymocytes were harvested from c h/hen mice at i and h following cecal ligation and puncture (clp; to induce sepsis) or sham-clp (sham). thymic yields were assessed and the extent of apetosis determined by staining the cells with propidium iodide (a dna intercalating dye) for facs analysis or by examining the extracted dna electrophoretically for the endogenous endonuclease activity the results (n= /grp) indicate that at i h post-clp there was no marked changes in any of these parameters. however, at h the thymic cell yield from clp mice was significantly decreased (sham: . ! . x l vs clp: . i . x * cells; *,p< . ), the % of cells apoptotic by facs analysis increased from . i . % in sham to . ± . %* in clp, and the septic mouse genomic dna exhibited dna degradation these results indicate that clp, but not simple laparotomy, induces marked thymic apetosis, which may contribute to the lymphocytic immune deficiency seen in these mice the purpose of this retrospective study was to evaluate effectiveness of irrigation to treat septic knee joints from longterm results. from to patients with septic knee joints have been treated. treatment in acute cases started with asp#at[on of the effusion to evaluate for cultures. without awaiting the result treatment was continued with immediate joint irrigation under arthroscopic control. three redon tubes ( ch ) were introduced for continuous irrigation and for intermittent distention of the joint cavity. in case of chronic joint infection additionally all foreign and synthetic material was removed. systemic antibiotics were given. patients could be re -examined with a mean follow-up of years. re-examination showed that immediate arthroscopic lavage started at the onset of septic sings had best results (less signs of osteoarthr[tis, less synovitis, less pain while loading and less range of motion loss ). patients ( %) out of patients with s tre_=.tsd acute se.~tic knee joint had excellent functional recovery, whereas all patients with chronic septic knee joints showed poor results. additionally, in of these patients with chronic septic knee joint after irrigation a further procedure was necessary due to recurrent septic knee joint. the concept that bacteria can translocate across the intestinal mucosa under a variety of circumstances is well established. however, due to the inherent limitations of in vivo studies, the cellular mechanisms underlying the process of bacterial translocation (bt) across the epithelial barrier are poorly understood. this is especially true in those circumstances in which there is no merphologic evidence of mucosal injury. consequently, we have utilized an in vitro cell culture model system to investigate the cellular events involved in the translocation process. in this model system, a polarized monolayer of intestinal cells (caco- cell line) is grown on a micron filter in a two compartment system. after tight junction integrity is established, bacteria are placed in the apical surface chamber and bt across the caco- monolayer is measured by culturing the basal chamber. the results of our studies utilizing the caco- system indicates that; l) several strains of non-pathogenic bacteria will translocate across the caco- monolayer in a time-dependent and dose-dependent fashion. however, the incidence and magnitude of bt are highest for those strains of bacteria (gram-negative enterics) that are most commonly cultured in vivo. ) caco- cells are actively involved in the transcellular passage of bacteria across the caco- monolayer, since inhibition of caco- microtubule or microfilament function decreases the magnitude of st. ) lectin but not integrin receptors are involved in bacterial translocation of e. col~ across the caco- cell monolayer. ) caco- cells have the ability to ingest and kill certain strains of bacteria. the mechanisms involved in caco- bactericidal activity appear similar to that of pmns to the extent that both are oxidant but not nitric oxide mediated. these in vitro studies suggest that caco- cells can function as "nonprofessional" phagocytes and that both bacteria and enterocytes are involved in the translecation phenomenon. bacterial translocation due to gut barrier dysfunction is considered to be a major cause of septic complications and multiple organ failure following trauma, burn injury, hypoxia, shock and shock-like states. the invasion of bacteria into the circulation from the gastrointestinal tract or even from other sources, however, should not necessarily result in systemic infection or organ cohinisation, as long as the humoral and cellular defense mechanisms are not impaired. thus, a reduced res clearance capacity and hepatic clearance function of bacteria and toxins can be observed under the same conditions which enable bacterial translocation from the gut. in order to evaluate the influence of circulatory, metabolic and humorul disorders on the elimination of bacteria out of the blood circulation, a series of experiments were performed in anesthetized and ventilated rabbits which received defined numbers ( . x colony-furming units) of escherichia coli as a bolus injection. in unaffected control animals the intravenously injected bacteria are eliminated about . % within the first minutes and are totaiy cleared within minutes. % offue total cfu counted in the cultures of the organ homogenates were found in liver and spleen, whereas only very low numbers could be detected in the lungs and kidneys. systemic injury of the animals by hemorrhagic shock, endotoxinemia, hypoxia, coagulation disorder, systemic vasoconstriction by norepinephrine and other types of injury reduced the elimination rate of bacteria and changed the pattern and degree of their dissemination and tissue distribution. the bacterial clearance was delayed mostly in rabbits subjected to hypoxemic hypoxia in which about cfu of viable e.eoli per ml blood could still be counted hours following their injection. the number of viable bacteria was some ten powers higher in organs of hypoxic rabbits in comparison to control ardmais, and an excessive colonisation of the lungs and kidneys with % respectively % of the total cfu of the cultared organ homoganates was observed. unilateral iscbemia of the kidney prior to the intravenous injection of e.coli only moderately delayed the bacterial clearance, bowever, caused an intense eohinisation of the affected reperfused organ. in contrast to this, inhalation injury did not promote the accumulation of bacteria in the lungs. conclusion: shock, systemic and local affections reduce the clearance of bacteria from the blood circulation and enable their dissemination and accumulation in vital organs. the degree of this reduction and the organ pattern of the bacterial distribution varies with the type of injury. thus, these factors seem to be essentially involved whether or not bacterial translocation and multiple organ failure will follow the invasion of bacteria into the circulation as in the ease of gut barrier dysfunction. movement of enteric baaterla from gut to extralumeaal sites (bacterial tra~sloeation) m~y play a role ~. nraltipl~ orga~ failure. traumatic stress (shock, hffectiort, and im~lammation) and severe illness are common alateeedents. tnt~st~aal lleus is e common proximme causal factor. we have exanlined the hypothesis that bacterial ttanslecation from the gut in expe~me~tal paaerestitis is due to bacterial overgrowth as a cuas~ucnee of a deere~ae in intestinal transit. ne~rotit_.~g pancreatitls fbliowlng bile duct iigation in the opossum is associated with colonization of the pa,se ~re, aa by gut derived organisms or salmonella of biljary origin in infected animals (previously reported). to ti~rther define the mechanism of transloemdon in panereati* inflammation, pancteatitis was induced hy iigatlon of t~e lower bile duet distal to the pancreatic duets m the rat. intestinal transit, enteric bacteriology, and tromsloeatien of bacteria to mesanterie lymph nodes, blood stream and splanelmie organs wen deletmhaed at (n~ ), g (n ~ ), and (n~ ) hours with the fallowing results: ly~testinai,..transfi". geometric mean of fluorescent marker - % at hour~ with return to % of gut leugth (similar to ~m) at hour~. bacterial overgrowt_hff -increase in total bsctetia ~ad gram negative rod.~ at ,; hours with return to sham control at hours ( log cfu/g veraus log cfu/g ia ileum). translocation to mesenteric nodes - , , and percent at , , and fi hours compared to b in sham controls (n=l of ). conclusion -bacterial translocation following panetoatieobiliary duct ljgation induced panereatitls ha the rat is tighlly linked to intestinal trausit and bacterial overgrowth within the gut lumen. speclrjlation -the ilens in in/~arm~ation may be related to activation of eytokiaes and mediated by nitric oxide. preliminary evidence for this notion will be discussed. in a series of studies we investigated: a) the time course of bacterial translocation (bt), b) the kinetics of lps and cytokine appearance (tnf, il- , soluble tnf receptor [stnf-r] ) in the circulation, and c) the role of lps and/or tnf with regard to haemorrhagic shock (hs) related pathophysiologie alterations and mortality in baboons and/or rats. method: baboons were subjected to femur fracture, soft tissue trauma (acute experiment), and hs ( mmhg mean arterial pressure: map for - h terminated at an accumulated oxygen debt of - ml/kg followed by resuscitation). a chronic model of hs was set up by administration of zymosan activated plasma (zap) before and after hs but without trauma. hs was induced in rats by bleeding the animals to map of - mmhg - rain followed by resuscitation. results and conclusion: in rats, lps increased in the systemic circulation, plasma tnf levels were found to be significantly elevated at min and were still markedly increased at rain postshock. plasma tnf, il- , and stnf-r levels increased already during the shock period in baboons, while tnf was not detectable. our data suggest that haemorrhagic shock may lead to early bt in the intestinal wall (at min following resuscitation in rats subjected to hs for min, similarly at h after the end of oxygen debt period in baboons ) and transient access of gut-derived lps into the circulation (levels became significant at min, while in baboons higher levels up to pg/ml were found at the end of shock and h after reinfusion, partially accompanied by bacteremia). in addition, since the treatment of rats with anti lps measures or anti tnf therapy significantly improved the -hour survival rate it can be assumed that endogenous lps and lps-induced mediator(s), in particular tnf, may lead to organ injury and mortality following haemorrhagic shock. alteration of the mesenteric blood flow and the consequent ischemia / reperfusion injury to the intestine appear to be one of the earliest events in the systemic inflammatory response following severe thermal injuries. the causative relationship between the subsequent disruption of the intestinal integrity and the potentiation of the translocation of indigenous bacteria and/or endotoxins to remote body organs and the systemic circulation has been deeumented in several studies. among other effects, endotoxemia solely or acting synergistically with thermal injuries has been shown to promote bacterial translocation. as these sequences continue without adequate intervention, multiple organ failure and eventually death may become inevitable. hence, the crucial need of treatment modalities with the capacity of modulating the intestinal blood flow and preventing the manifestations of these pathological incidents. although the underlying mechanisms of this process have not yet been fully elucidated, there is accumulating evidence that various interacting vasomediators are involved. the actions of these mediators can probably be modulated by either nonspecific or selective agents. among the nonspecific agents, the composition, volume and timing of the resuscitation fluids appear to play an important role in this process. nitropmsside, a nonspecific vasodilator, has been shown to prevent the decrease of the postbum mesentefic blood flow when selectively infused in the mesenteric artery. the nonspecificity of various vasodialators with their possible undesirable systemic effects emphasizes the importance of the identification and modulation of selective vasomniiators. thromboxane a (txa ) and angiotensin ii (a-ii) appear to be the primary mediators of the postburn vasoconstriction. both mediators were found to be elevated following thermal injuries. in a previous study, pretreatment with oky- , a thromboxane synthetese inhibitor was found to attenuate the postbarn mesenteric vasoconstriction. in a bum/sepsis porcine model the efficacy of dup , an a- receptor antagonist as a posthum treatment modality was evaluated. treatment with dup prevented the early posthum and the late posteodotoxin elevation in the mesenteric vascular resistance and subsequently abrogated the fall in the mesenteric blood flow. the incidence of burn & endotoxin induced bacterial translocation was significantly reduced by dup from % to % (p< . , fisher's exact test). the indigenous flora of the gastrointestinal tract exerts a potent influence on the developmental maturation of normal systemic immunologic responsiveness. moreover, both oral and intraportal administration of experimental antigen is associated with a systemic state of immunologic hyporesponsiveness, known as oral tolerance. since trauma and critical illness are associated with changes in the flora of the gut and with altered systemic immune reactivity, we have hypothesized that the interactions of an altered gut flora with immune tissues in the gut and liver play a role in the pathogenesis of the immunologic derangements of critical illness. prolonged feeding of two clinically relevant killed organisms-pseudomonas and candida-to a rat results in significant impairment of cutaneous delayed hypersensitivity (dh) reactivity. conversely, measures directed against gram negative bacterial overgrowth in experimental peritonitis result in partial restoration of impaired dh reactivity. to ascertain the role of the liver in the pathogenesis of these alterations, we studied the differential immunologic sequelae of portal versus systemic (ivc) infusion of killed bacteria. experimental infusion of live or killed gram negative organisms into the portal vein produces dh suppression in rive, and profound suppression of mitogen-driven lymphocyte proliferation in vitro; systemic administration of the same organism has no effect on these phenomena. suppression is tgf -dependent, and mediated by a soluble factor released by fixed tissue macrophages of the lung and spleen. the suppressive influence appears to be mediated at the level of interactions between il- and its high affinity receptor. release of this factor can be induced by a second circulating factor present in the plasma of portally-, but not systemically-infused animals two hours after bacterial administration. these studies suggest that the release of inducible immunoregulatory factors from the liver, and possibly from the gut, may play a role in the pathogenesis of the profound derangements of systemic immune homeostasis that accompany trauma and critical illness. acad.hospital st. radboud,postbus , lib nijmegen introduction. the central question being tested in this study was whether liposome encapsulated dichloromethylene-diphosphonate (ci mdp) mediated elimination of liver and splenic maorophages would influence zymosan induced systemic toxicity (st) and bacterial lranslocation (bt). materia|s and methods. male swiss (icr) mice were used weighing - g elimination of liver and spleen macrophages was accomplished by intravenous injection of / suspension of ci mdp-liposome suspension. control mice received an i.v. injection with pt phosphate-buffered saline (pbs) . two days later all mice were challenged with an i.p. injection of zymosan suspended in paraffin (z) at a dosage of either mg/g, . mg/g or . mg/g body weight (n= in each group), signs of st and bacterial translocation bt were measured hours later the st was assessed by blindly grading the degree of lethargy, conjunctivitis, diarrhea, and ruffled fur of each mouse on a four point scale. bt to the mesenteric lymph nodes (mln) and systemic organs (liver, spleen, lung and blood) was tested by culturing on blood and macconkey's agar at the time of sacrifice sections of the distal ileum were taken for histology. an additional two control groups (n= in each group) were tested to verify that neither paraffin nor pbs or ci mdpqiposome suspension alone induced bt or st. furthermore survival over a -day period was assessed in a control and macrophage-depleted group with a dose of d mg/g z. results. neither the paraffin nor ci mdp-tiposome suspension induced bt or st the incidence of bacterial translocation to the mln was similar between macrophage depleted and control groups. however, macrophage depletion was associated with a significantly higher incidence of systemic spread of bacteria. data expressed as positive tissues/total tissues tested: / vs. / at . mg/g zymosan (p~o. ); vs. at mg/g zymosan (p~q ); / vs. / st . mg/g zymosan (p~o.o ).the magnitude of bt however did not show any differences. although systemic bt was greater in the macrophage depleted groups, the systemic toxic response was significantly less at doses of . mg/g zymosan ( . -+ . vs. . -+ , p~o. ) and . mg/g zymosan ( . -+ . vs. . -+ . , p<_.o. ) . the day mortality after . mg/g zymosan was % in the control group and % in the macrophage depleted group (p=o. ). histology did not show any differences between the control and macrophage depleted groups. conclusion. the lethal and toxic effects of zymosan in this model appear to be more related to the excessive activation of macrophages than to the systemic spread of bacteria. bacterial translocation (bt) in hemorrhagic shock was studied using a porcine model. in this study three groups were randomized: one control (n= ) and two experimental (n= each). a portal vein catheter was placed and remained in situ hrs. a femoral artery and vein catheter were in place hrs. the arterial eaanula was used for monitoring mean arterial pressure (map) in all animals and to bleed the experimental animals and the venous cannula to repelfuse with ringer's lactate (rl) or autologous blood (ab). baseline samples and values were taken in this period. control animals were observed for a sham shock period of . hrs., given more hours of anesthesia, and studied for further hrs. % of the estimated blood volume was withdrawn from each experimental animal over min. these animals were kept in shock for . hrs. at the end of the shock one group was perfused with rl and the other with ab. two hours later they were extubated and further studied for hrs. samples from portal blood for cultures and measurement of leukotriene b (ltb ), prostaglandin e (pge ), and nitric oxide (no) metabolites were taken from all animals at intervals beyond baseline up to hrs. after hrs. under general anesthesia a second laparotomy was performed and samples for culture from mesenteric lymph nodes (min), liver and spleen were taken; as well as samples for histologic study with light and scanning electron microscopy were taken from jejunum, ileum, and colon. animals were then euthanized. results: significant shock was induced as indicated by map and arterial blood gases. significant bt to min was observed in all groups. no significant bt was observed in cultures from liver and spleen in any of the groups. increased values for ltb , pge and no metabolites were found during the period of shock. these results suggest that the isehemic phenomenon triggers a significant inflammatory response, and that bt to min may be an epiphenomenon without apparent significant influence on the inflammatory response. objective: to study the potential effects and mechanisms of action of systemic administration of monoclonal antibodies anti-endotoxin (ha- a) in a animal model of gut-origin sepsis. materials and methods: exoeriment a. balb/c mice were transfused with allogeneic blood (from c hb-iej mice). five days post-transfusion the animals were gavaged with lxl viable escherichia coil and randomized into groups (n= /group) to receive a sham burn (sb group) or a % thermal injury immediately followed by the systemic administration, via a tail vein, of monoclonal antibodies ( mg/kg) (ha- a group) or a % burn injury and administration of equal volume of sterile saline (c group). all groups were resuscitated by intraperitoneal injection of . ml of saline. the animal survival rate was observed for days post-burn. experiment b, transfusion and burn procedures were identical to experiment a but the mice (n= /group) were gavaged with viable e.coli labeled with mmndium oxine. four hours after burn the mesenteric lymph nodes (mln), liver, lungs and blood were harvested to determine plasma endotoxin levels and the magnitude of transtocation of labeled bacteria as measured by the residual radioactivity (dpm) in the organs. circulating endotoxin levels were determined by limulus colorimetric assay. diamine xidae(dao) is an enzyme existing in the mucosa of small intestines, but its activity is low in the plasma. it is evoked in the blood with the intravenous injection of much heparin. it is well known that da declines under the chronic mueosal injury when the small intestines are cut off or the mucosae become atrophied. aim of this paper is to confirm that da goes up to the measureable level by the intravenous injection of a small amounts of low molecular heparin(lmh objectives: the aims of this study is to investigate the inhibitor" effect of the prolease inhibitor, urinastatin, on endotoxin induced bacterial transleeation in mice. materials and methods:lcr mice were divided in six groups as the fotlows, group i: control mice receiving intraperitoneal injections (ip) of saline . and hr prior to sacrifice, group ih treated mice receiving ip of utinastatin . hr and endotoxin ( mg/kg) hr prior to sacrifice,group ilk sham reated mice receiving ip of saline . hr and endotoxin ( mg/kg) hr prior to sacrifice,group iv: saline control mice receiving ip twice during rain intervals,group v: mice received ip of utinasmtin, and min later they were received ip of endotoxin ( mg/kg), group vi: mice received ip of saline,and min later they received ip of endotoxin ( mg/kg). in group l,ii and ill ,the mice were killed by cervical dislocation.using stetile techniques,a middle-line incision was made and the mesenteric lymph nodes were harvested in order to culture on selective agars for quantitation of bacterial trauslocation. in group iv, v and vi, survival was recorded for days. unnastatin pretreatment could rednce mortality significantly. first surgey, shiga university of medical science, seta ,ohtsu - ,japan increased gut permeability correlates with inflammatory response in multiple trauma aptients. pape, h.-c. dwenger, a. grotz, m. regel, g. purpose: disturbances of intestinal permeability have been advocated as a pathogenetic factor of posttranmatic multisystem organ failure (mof). a close relationship with impairment of the stationary reticulocndothelial system (res) and a systemic inflammatory response (sir) was discussed. these tactors were investigated prospectively in patients with multiple trauma showing posttraumatie complications (multiple organ failure, mof). methods: polytranmatized patients were studied prospectively. according to a mof-score (goris) those with posttrattmatic complications were selected (> points at days), others were excluded. every second day gut permeability according to the ratio of urine concentrations of lactulose and mannitol (l/m) was evaluated (enteral application). at parallel time points res clearance capacity (k-value, invasion constant, normal range . - . mind) was studied after i.v. injection of mbq rotehuman albumin. liver perfusion was calculated from these data, total serum bilirubin (/zmol/l) was documented. serum elastase (#g/l) levels were determined enzymatieally. results . + + liver perfusion did not ehangu, bilirubin showed progressive worsening indicating mof. a positive correlation was present between l/m and k (r= . ) and between l/m and ela (r= . ). conclusions: there is a positive correlation between the time pattern of intestinal permeability dysfunction and res hyperactivity as well as between intestinal permeability and the systemic intlammatory response (elastase levels). the results speak in favor of an interaction between intestinal and extraintestinal inflammatory systems, which in eombiuation are likely to be responsible for post~anmafic complications. endotoxemia, il- release and consecutive acute phase reaction are observed as a host response to surgical trauma. as well vasodilative prostaglandins (pg) and thromboxane (tx) are released after abdominal meaenteric traction (mt). the following hypotension and acute hypoxeraja are duo to prostacyelin (pgiz) arm can be avoided by perioperative cyclooxygenase inhibition. we therefore focused on the effect of pg and tx liberated following mt on the induction of endotoxemia. methods: in a prospective, randomized double-blinded protocol patients, who were scheduled for major abdominal surgery (pancreatic or infrarenal abdominal surgery), were studied. ibuprofen ( mg i.v.) or a placebo equivalent was administered minutes before skin incision. mt was applied in a uniform fashion. baseline values were obtained before induction of anesthesia. further measurements followed before the incision of the peri[onenm (tl) and , , , min, . the plasma concentrations (,pc) of -keto-pgft,, txb: and-ki- -pgf ~ (stable metabolites of pgi , txa and pge~) were determined by ria. we measured endotoxin pc by limulus-amoebocyte-lysate test and il- levels by elisa. data are given as mean+sem (* p< . placebo vs. [ibuprofen] ). results: endotoxin plasma levels increased before incision of the peritoneum tl both in the ibuprofen pretreated and in the placebo group. peak pc were observed minutes after mt. endotoxin pc were significantly higher in the ibuprufen treated group (t . + . e[ . + . ] eu/ml). il- pc demonstrated an increase continuously from t to t (t + [ + ] ng/l) in both groups. after intentional abdominal mesenteric traction we observed a marked increase of -keto-pgf~,, pc up to h after mt in untreated patients with a peak of *[ ] ng/ at tl. also txb: and kh pge pc showed a considerabe increase up to h after mt in the placebo group. in ibuprofen pretreated patients the pg and tx pc remained within the normal range. discussion: our data clearly indicate a significant endotoxemia and il- release following major surgical trauma which is not initiated either by prostaglandin or thromboxane release. moreover endotoxemia is accentuated by ibuprofen pretreatment. therefore we hypothesize that in major abdominal surgery prostacyclin release-after mt may play a crucial physiological role in maintaining splanclmic microcirculation and thus preserving gut mucosal barrier function. objectives of the study it has been shown recently that parenteral and certain euteral diets promote the translocation of gut flora to the mesenteric lymph nodes (mln) and systemic organs, a process termed bacterial translocation (bt). in chow fed rats bt usually does not occur without further promoting factors. the goals of the present study were to determine whether the provision of defined amounts of standard lab chow during iv-tpn administration wotfld redane the incidence of bt, materials und methods male spf spragnle-dawley rats were divided into groups. group received standard laboratory chow feeding ad lib. in group a central venous catheter was placed, ligated and secured by a spring coil tether attached to a swivel allowing free movement in the housing cage and chow was fed ad lib. in group % of the calculated daily required calory intake (drci) ( /kcal/kg) was given by iv-tpn ( % glucose, , % amino acids) and % by limited chow administration. groups and received % and % of the drci by i.v. tpn and % and % respectively by chow feeding. group received iv-tpn only. after days the rats were sacrificed and the mln, liver, spleen and cecum removed aseptically, homogenized and cultured for bt samples of distal ileum were taken for light microscopy. the group with the least amount of chow shown to be protective against bt received the amount of non-fermentable fiber of that chow regimen during iv-tpn feeding and bt was studied. , + , , - , , / + ~ " , -+ , , -+ ~ - , / +~ + _+ , + , , - , -+ + , ~ , , -+ ~ conclusions: the administration of % of drci by chow feeding during iv-tpn significantly reduced the incidence of bt and maintained gut barrier function. the addition of the respective amount of dietary fiber of this group did not prevent iv-tpn-indueed bt. dr. med. m naruhn., dep. of general surgery, eberhard-karls-university, hoppe-seyler-str. previous experimental studies have suggested that a disturbed ecology of the enteric bacterial population might contribute to the development of bacterial translocation from the gut in acute liver failure (alf). in the present study, the effect of oral administration of lactobacillus reuteri r lc and oat fiber on bacterial overgrowth and translocation was investigated in rats with acute liver failure induced by subtotal ( %) liver resection. the oatmeal soup base was anaerobically inoculated with lactobacillae and fermented for hours, after which the animals were fed with either fermented or unfermented oatmeal or saline daily for days prior to the operation. bacterial translocation to mesenteric lymph nodes (mln) and the systemic circulation was determined, as well as the intestinal bacterial flora and enterocyte protein content. the incidence of bacterial translocstion to the systemic circulation was nit in rats subjected to sham operation and saline treatment and % in animals subjected to % bepatectomy and lreatment with fermented oatmeal, while - % and - %, respectively, in rats subjected to hepatectomy and treatment with either saline or unfermented oatmeal. only one rat with fermented oatmeal demonstrated bacterial growth in mln (p < . vs hepatectomy and treatment with saline or unfermented oatmeal). the enterocyte protein content significantly decreased (p < . ) in salinetreated animals following % hepatectomy, while there was no significant difference between bepatectomized animals with oral administration of fermented or unfermented oatmeal. the number of anaerobic bacteria, gram-negative anaerobes and lactobacillus significantly decreased and the number of e.cnli increased in the distal small intestine and colon in hepatectomized animals with enteral saline or unfermented oatmeal as compared with animals subjected to sham operation or bepatectomy with fermented oatmeal. our results thus show that the occurrence of bacterial translocatiou from the gut in % hepatectomy-induced alf could be prevented by enteral administration of fermented oatmeal, maybe partly due to a positive effect on the enteric bacterial ecology. _+ " +_ " . " data=mean_+sd, * stats anova p< . vs control. l+air and lap groups, both exposed to exogenous i.ps shnwm:t m significant increase (p<. ) in lps gut translocation compared to control and l+co . this correlated with a significant increase in peritoneal inflammatory responses (o -,tnf) above that of the control and l+co groups, while mac- and cr opsonized phagocytosis were significantly impaired. the absence of significant differences between l+air and lap groups indicates that lps rather than wound factors is the principle mediator. thus, lps plays a significant role in regulating peritoneal responses in the early post-operative period dept of surgery, rcsi, beaumont hospital, dublin , ireland brlke e, berger d, staneseu a, buttenschsn k, vasilescu c, seidelmann m, beger hg in patients undergoing a colonoscopy, endotoxin, endotoxin neutralizing capacity (enc), thromboxane b o (stabile metabolite of tbmomboxane ~), -keto-prostaglansin, leueotriene c , interleukin and the incidence of bacteremia were determined before and then every five minutes during the procedure. twenty-one of patients showed a significant increase of endotoxin plasma levels during colonoscopy (p= . ), whereas only one patient had a positive blood culture with bacteria obviously derived from the gastro-intestinal tract. the enc decreased significantly five minutes after the beginning of eolonoscopy and was diminished further thereafter. the baseline values were reached after hours. ~hromboxane b o levels also increased after five min. from to pgyml peaking at min. with pg/ml. -keto-prostaglandin,leucotriene c , ii- and crp remained unchanged. a control group of i volunteers who were not subjected to endoscopy, were prepared for eolonoscopy by orthograde lavage. the blood sampling procedure remained identical. no differences were seen in all described parameters for the controls. these data show that the gut barrier can be compromised by mininml invasive procedures, at least, concerning bacterial products. living bacteria, on the contrary, do not pass the gastro-intestinal wall. endotoxin, when determined by enc, is more sensitive than the conventional limulus-amebocyte-lysate test. no acute-phase reaction was induceri by the observed endotoxin translocation. it can be speculated from the dramatically enhanced thromboxane b levels, together with its hemodynamie effects, that the thromboxane release may support translocation of bacterial products. sepsis is common after hemorrhagic shock. this study aims to demonstrate that hemorrhagic shock alone can promote translocation of gut bacteria from intestinal tract to its regional nodes and subsequently to blood. one hundred twenty mice, divided into groups were subjected to , and minutes of %, % and % of hemorrhagic shock. on the specified time, blood cultures were taken and mice were sacrificed. the intestinal tract were histologically examined for any changes which allows translocation and its regional nodes were quantitatively cultured for translocated bacteria. there was a direct relationship between duration and degree of hemorrhagic shock and incidence of translocation (p . ). there was a high incidence of gut bacterial translocation to the mesenteric and mesocolic nodes in all degrees of shock (p . ). bacterial growth in the regional intestinal nodes increased and blood cultures were positive in direct proportion to degree and duration of shock. histologic evaluation of segments of git showed submucosal congestion to allow bacteria normally contained within the gut to cause systemic infections. translocation of gut bacteria in untreated hemorrhagic shock is clearly shown in this study on animal models. in this study, guotobiotic rats with known species of bacteria were subjected to total parenteral nutrition(tpn) and subsequent hemorrhagic shock. the purpose of the study was to observe the impairment of gut barrier function following tpn and hemorrhagic shock and to study the mechanism of enterogenic infection induced by tpn and shock.the results were as follows: .long term( - days) tpn induced impairment of gut barrier function, evidenced by atrophy of intestinal mucosa, significant decrease in diamine oxidase activity of intestinal mucosa and blood, and marked microecologic imbalance of the intestinal mucosa flora with dorminant growth of aerobes and relative decrease in anaerobes. the degree of mucosal damage were proportional to the duration of tpn. .in tpn+shock groups, failure of gut barrier function was found. ri,~ere were further damage in the mucosa, with a large number of gramnegative organisms invading mucosa and submucosa and a significant decrease in dao activity as compared with each relative tpn groups. these changes were significantly correlated with enhanced bacterial translocation, elevation of lps and mda levels in the plasma. these findings suggested that long term standard tpn impaired the gut barrier function, precipitating posttraumatic gut barrier failure. thus infec. fion following shock might be oi'iginated from the gut and it was obviously related to the impaired gut defence resulted from antecedent tpn. the determination of plasma dao activity might provide a valuable tool for the ear. ly diagnosis of gut injut;y during tpn and after trauma. in our earlier studies we have investigated the dynamics of granuloayte infiltration of the ischemic/reperfused s~all intestine (g. illy~s, j. hamar int. j. exp. athol. . . .) . there was a increasing infiltration of the mucosa c m~nating at the d to th hours of reperfusion. in the present series we have studied sc~e of the conseqn/ences and the possible role of this cellular reaction. ~in isehemia was followed by a hour reperfusion in the anesthetized rat. arterial ~/ad mesenteric venous blood samples were collected at m_in, i, ~ , and hours of reperfusion. elastase and lactate concentrations were determined and hamoculture was carried out from the blood samples, and tissue pieces from the heart, lung, liver and kidney were collected for histological analyses at the above mentioned times of reperfusion. all blood samples were free of cell bacteria. staphylococci appeared only occasionally at the th hour in the arterial blood .and at the d and th hours in the venous blood, respectively. arterial and venous elastase activities were high throughout the reperfusion, venous concentrations being higher at all times. lactate concentrations of the arterial and mesenteric venous blood samples increased during shock. ~ranuloeyte infiltration of all organs studied appeared during the d hour and it increased at later times of reperfusion. it is concluded that heavy infiltration of the intestinal mucosa can block bacterial translocation in most of the cases during reperfusion. granulocytes activated either by the reperfused area or by the released cytokines infiltrate other organs contributing by this way to the mesenteric shock s!rndrc~e. intestinal motility plays an important role for maintaining nutrient transport and absorption and for balancing the enteric bacterial population. disturbances of intestinal motility may be one of the earliest notable changes in intestinal function. in the present study, we aimed at determining early alterations in intestinal transit time following ischemia-reperfusion injury induced by occlusion of the superior mesenteric artery in the rat. intestinal ischemia was induced for and minutes by applying a microvascular clip on the superior mesenteric artery followed by reperfusion , and hours after clip removal. intestinal transit time was measured by the propulsion of a radiolabelled solution (cr ). light microscopy was performed on intestinal samples. macroscopical pathological changes were not observed. however, microscopically, mucosal epithelial oedema, degeneration or slight ulceration occurred in rats hours after reperfusion in ischemia- rain group and and hours after reperfusion in the ischemia- rain group. delayed small intestinal transit time was seen from hours and on after intestinal ischemia for both and rain ischemia followed by reperfusion. the distribution of radioactivity demonstrated that most radioactivity was accumulated in the first two segments following intestinal ischemia and reperfusion, significantly differing from what was seen in animals subjected to sham operation (p < . ). the distribution of radioactivity in segments and in the group with repeffusion hours after intestinal iscbemia for rain was significantly higher than that noted in the group with repeffusion hours after intestinal ischemia for min (p < . ). q'he results indicate that a delayed intestinal transit time may be one of the earliest pathophysiological alterations noted, associated with duration of gut ischemia, and a potential factor for the development of bacterial overgrowth, gut barrier failure and bacterial translocation, in hypovolemic conditions. bacterial infections still constitute a major cause of morbidity and mortality in patients with acute liver failure. the present study aimed at evaluating the effect of ethylhydroxyethyl cellulose (ehec) on bacterial translocation following surgically induced acute liver failure. acute liver failure was induced by subtotal hepatectomy ( %) in the rat. water-soluble ehec was administered orally and hours prior to hepatectomy. the incidence of bacterial translocation from the gut to mesenteric lymph nodes (mlns) and systemic and portal circulation was evaluated and the number of isolated bacteria from these samples and from intestinal content were determined. intestinal transit time, bacterial adherence onto the intestinal surface, intestinal mucosal mass, bacterial growth and dna synthesis, bacterial surface characteristics (hydrobiology: hydrophobicity, hydrophilicity and neutrality; surface charges: positive, negative and neutral) were also determined. hepatectomized animals showed a - % translocation rate to mlns or blood and hours after operation, while only - % of rats subjected to sham operation or animals with % hepatectomy and pre-treatment with ehec (p < . ). bacterial overgrowth, increased bacterial adherence onto the intestinal surface as well as decreased intestinal mucosal masses were observed in animals with subtotal liver resection alone, alterations that were prevented by enteral ehec treatment. a delay in intestinal -hour transit time occurred in both groups with subtotal liver resection, with or without enteral ehec. ehec inhibited bacterial growth and dna synthesis, and altered bacterial surface properties following hour incubation with bacteria. in conclusion, the findings in the present study imply that ehec alters enterobacterial capacities for metabolism, proliferation and invasion by effects on e.g. bacterial surface characteristics. furthermore, ehec seems to possess a trophic action on the intestine, rather than exerting its effect by enhancing intestinal motility. department of surgery, lund university hospital, s- lund, sweden disturbances in intracellular calcium signalling can potentially result in impairments of cellular responses vital to the functional integrity of both immune and non-immune cells, and thus contribute to a decrease in host resistance against infection and to multiple organ system failure during sepsis. studies in our laboratory have focused on assessments of intracellular ca ÷ regulation and ca~+-depended cellular responses in the liver, skeletal muscle and splenic tlymphocytes harvested from rats subjected to gram-negative intraabdominal sepsis. cytosolic ca + concentration, [ca *]i, and ca + fluxes were measured by the use of fluorescent ca + chelating dyes (fura- or indo- ) and ca respectively. to assess sepsis-related changes in ca + dependent cellular responses, we measured the acute phase protein response in the liver, the regulation of protein and sugar metabolism in the skeletal muscle, and the proliferation response in the splenic tlymphocytes. altered ca + i signalling with sepsis was correlated with an exaggerated inappropriate acute phase protein response ( % ¢) in the liver, and a blunted insulin mediated sugar utilization ( % ) and increased proteolysis ( % ~) in the skeletal muscle. in t-lymphocytes, a decrease in mitogen induced elevation of [ca +]i by - % was correlated with a significant depression in their proliferative capacity. these studies clearly suggest that altered calcium signalling is correlated with disturbances in cellular responses in both immune and non-immune cells during sepsis. the altered cellular responses adversely effect the outcome of the septic injury. (supported by nih grant gm ). alfred ayala, ping wang and irshad h. chaudry. changes in macrophage capacity to respond to foreign pathogens are thought to be central to the developing immunosuppression associated with traumatic injury. in this respect, the suppression seen in m~ functions following hen (a common component of traumatic injury) may be mediated by the direct or indirect inhibition of their capacity to perceive external stimuli (e.g., opsonized & non-opsonized bacteria, and their cellular components, etc.} due to the breakdown of the receptormediated signal transduction system. results of a number of studies by our laboratory and others indicate that this inability to respond to external stimuli is in part due to the loss of cell surface receptors. decreases have been documented for not only la antigen, but also c b, fc, and tnf receptors following hem in mice. furthermore, studies which have examined second messenger generation in these cells indicate that m~ derived from the peritoneum and spleen exhibit a decreased capacity to mobilize ca + from intracellular stores. this protein kinase dependent process of [ca+ ] i mobilization appears to be linked to the inability to synthesize inositol triphosphate. of interest, the depression in ca + signal generation appears to be inversely related to presence of elevated levels of camp in m~ from hen mice. we have reported that m~ priming agents, such as ifn- (which exhibits salutary effects on m~ function following hem), appear to restore cell signal transductive capacity while reducing the levels of camp. nonetheless, the extent to which depressed receptor signal transduction in hem, is due to receptor loss~dysfunction or elevated antagonistic second messenger levels remains to be determined. conclusions: significant impairment of calcium signaling occurs at all time-points prior to and following pha stimulation in trauma patients. tcell activation failure can, in part, be explained by the inadequacy of this essential intracellular second messenger system. restoration of immunocompetence following trauma will have to address strategies to better assess and restore this vital step in the activation sequence leading to proliferation during the antigen recognition process. patrick a. bseuerle institute biochemistry, albert-ludwigs-university, hermann-herder-str. , d- freiburg, germany the active form of the transcriptional activator nf-~b is a heteredimer composed of a and kda polypeptide. in this form, nf-'<b can bind with high affinity to decameric sequence motifs (consensus: "-gggpunnf'ypycc- ") found in enhancers and promoters of many genes activated upon a wide variety of pathogenic conditions, including viral and bactedai infections, acute phase response, oxidative stress and uv and gamma irradation. clinically important target genes for nf-k:b encode inflammatory cytokines (il- b, tnf, [l- , il- , gm-csf etc), cell adhesion molecules (icam- , elam- , vcam- ), acute phase proteins and immunoregulatory ceil surface receptors. in most cell types, nf-~:b is sequestered in the cytoplasm and, therefore, unable to initiate transcription of these genes. the cytoplasmic form of nf-~:b contains one additionai subunit, referred to as h,;:b. i~b can reversibly suppress dna binding and nuclear uptake of nf-~b by virtue of its association with p . upon stimulation of cells, ikb is proteolytically destroyed, allowing nf-kb to enter the nucleus and activate genes upon binding to transcriptional enhancers. we have demonstrated that various antioxidative compounds inhibit the activation of nf-~b in response to many inducers. moreover, nf-~:b was shown to be activated upon treatment of cell cultures with p.m-amounts of hydrogen peroxide. these data suggested that nf-~b prinicipaliy is an oxidative stress-responsive transcnption factor. the notion is supported by numerous reports on the induction of oxidative stress by many nf-nb-inducing agents. nf-~b activition can likewise be suppressed by protease inhibitors. these drugs apparently inhibit a yet unidentified protease responsible for the inducible decay of inb. we propose to use nf-nb inhibifors as novel drugs with a very broad application under acute phases el inflammation, injury and infection. northern blot experiments revealed that expression of the recently discovered lipopolysaccharide binding protein (lbp), a / kd glycoprotein, in the liver rises substantially during the acute phase response. experiments with an in-vivo acute phase model using new-zealand-white rabbits and also in-vitro experiments employing stimulated hep-g cells showed that lbp transcripts could be induced to ford within hours after induction with cytokines. by using a newly established nonradioactive nuclear run-on technique we show that induction of lbp during the acute phase is transcriptionally regulated. furthermore we screened a human genomic library and were able to clone the lbp gone, containing the promoter . kb upstream. several liver-specific and "acute-phase-typical" potential binding sites were found fn the promoter region and reporter gone assays were set up. several caat-boxes, the il- responsive elements hstf-hsp . and h-apf- rs as well as the transcription factor hnf- and the glucocrticoid-responsive elements oct- -d and gcre were found, which correlates with the induction pattern of lbp mrna in hep-g cells by il- , il- and dexamethasone. pcr-based analysis of the exon-intron pattern of the lbp gene revealed similarities with the genes of two other lps binding proteins, namely bpi and cetp. further analysis of this novel acute phase protein, that also has an important role in endotoxin recognition and immunomodulation will help in understanding the complex acute phase mechanism and potentially lead the way to new therapeutic strategies. lps activation of nucle?/r fa-ctor:~b " (nf:~bj"in cd .transfected fi'broblasts does not appear to require tyrosine kinase activity d. t. golenbock, r. delude, r. savedra, s. vogel and m. j. fenton lipopolysaccharide (lps) is the major constituent of the outer leaflet of gram-negative bacteria. during the course of serious infection, shock may occur as a result of the interaction of lps with macrophage lps receptors. cd , a kda glycosyl phosphatidylinositol (gpi)-linked protein, functions as an lps receptor. a critical issue in lps activation concerns the mechanism by which cd , which has no transmernbrane domain, transduces a sig,nal after lps binding. evidence exists which suggests that cd may signal cells after lps binding by interacting with an lps signal transducer with tyrosine kinase (tk) activity. wild-type chinese hamster ovary fibroblasts (cho) normally do not respond to lps, but can be activated following transfection with cd (cho/cd ). stimulation of cho/cdi with as little as ng of lps/ml resulted in the release of h-arachidonic acid ( h- : ) into the culture supernatant. in addition to h- : release, we examined lps-imiuced activation (transl cation) of the transcription factor nf-~b. similar to lps-induced h- : release, these responses were observed only in cho cells lransfected with cd closely resembling the same response in the marine macrophage cell line raw . . maximum nf-~cb expression occurred at minutes. in contrast to lps-induced h- : release, dexamethasone, cycloheximide, and actinomycin d had no effect on activation of nf-r,.b in cho/cd , suggesting that nf-r,b activation begins early after lps binding to cd and does not require transcription or translation. we then examined cells for lps-induced tk activity by western blotting cellular ly~ates with anti-phcsphot-'rosin:~ anl',bodie~" although tk activity was seen in lps-stimulated raw cells and phorbol ester stimulated cho/cd , lps-induced tk activity in cho/cd was not observed. although the tk inhibitor herbimycin inhibited the release of h- : in cho/cd and raw cells, neither herbimycin nor genestein effectively blocked nf-r,b activation in either cell type. these results suggest that tk activity is involved in a portion of the signaling pathway that is distal to initial signal transduction. the absence of observable lps-induced phosphotyrosine residues in cho/cd cells as well as the failure of tk antagonists to inhibit lps-induced nf-~cb activation suggest that the proposed cd -related lps signal transducer in cho/cd is not a tymsine ldnase. dimished cardiac inotropic function and response to -adrenergic receptor ( -ar) stimulation are frequently seen in septic patients, these cardiac defects are also seen in animal models of entoxemia. to determine the characteristics of the decreased transmembrane signal associated with the decreased beta adrenergic response we measured the force and rate of contraction of atria harvested from sprague-dawley rats exposed to endotoxin ( : gm/kg). to stimulate various components of the transmembrane signal cascade of -ar was isoproterenol (i -ar), acetylcholine (m ach receptor), naf(gs and gi proteins), forskolin (adenylate cyclase), and calcium (intracellular contracttile signal) were used. to eliminate the transmembrane control of calcium and test the intrinsic contractile response, hypothermia ( °c) was used the results showed alterations in the inotropic function with no significant difference in chronotropic response. the inotropic reponse for the endotoxin exposed atria and controls is shown below these results show that there is a transmembrane signal defect both for the i~-ar signal and for ca +. these data indicate that the level of the defect appears to be at the g proteins. ischemia-reperfusion of the rat intestine produces an injmry both to the intestine and to distal organs, notably the lungs and liver. prior studies have shown that h of intestinal ischemia leads to a nemtrophil independent reperfusion injury of the gut. thus, rats rendered neutropenic by pl~l antiserum have been shown to express a severe local gut injury when subjected to ischemia and reperfusion. this gut injury is postulated to he mediated at least in part by the complement system. the soluble (s) cri receptor which binds to c b and cdb was given to rats at a dose of mg/kg by intravenous bolus, min before reperfusion of the ischemic gut. a control group was given pbs buffer. at h of rmperfmsion the animals were sacrificed. pbs treatment led to a significant activation of both classical and alternate complement pathways while scri inhibited both pathways (fall to zero of chbo ). intestinal mucosal injury score, assessed by histological grading was reduced by scri ( . ± . to . _+ . ,p< . ) . intestinal neutrophil sequestration estimated by assay of myeleperoxidase was also reduced ( . ± . to . ± . u/g). c was detected in the gut hy i~unohistochemistry. remote organ injury was modified that is lung permeability (ration of concentration of radiolabelled albumin in broncho-alveolar lavage fluid to that in blood) was reduced ( . ± . x i to . ± . x , p<o. ). however, neutrophil sequestration by the lungs was unaffected. this remote protection is thougth to be related to prevention of ic b deposition on imng andotheli~ which activates adherent neutrophils. these findings support the hypothesis of complement mediated local and remote injury. we postulate that complement fragments may be deposited either as a result of the ischemia induced loss of membrane bound complement regulatory proteins, such as decay accelerating factor or membrane cofactor protein, which allow complement fragments to accumulate on the endothelial cell surface. alternatively, increased expression of p-selectin on the endothelial cell surface may, by means of its complement binding domains, act as a lattice for complement deposition. indeed, p-selectin has been detected on the surface of endothelial cells ans platelet-neutrophil aggregates recovered from the intestine of these animals. ischemia ( hr) followed by reperfusion (dhr) of rat hind limbs resuits in local (muscle) injury as well as in remote (lung) injury, which is characterized by increased vascular permeability (leakage of j sl labeled albumin) and neutrophil accumulation (tissue content of myeloperoxidase, mpo). within minutes of reperfusion following ischemia, tumor necrosis factor-o~ (tnfc , interleukin-i ( l-i) and il- plasma levels increased significantly, reaching maximum levels after hours ofreperfusion. polyclonal antibodies to tnfet and l-i provided significant protection against muscle and lung injury. muscle and lung vascular permeability decreased in anti-tnfct treated rats by % and % respectively and mpo was reduced by % and %. antml- yielded a protection in muscle and lung vascular permeability of . °, and . % respectively whereas muscle mpo was reduced by . % and lung mpo by . °, . these results were confirmed by the use of soluble tnf~ receptor and il-i receptor antagonist (il-ira). when icam-i expression in vivo was examined using mabeled antmcam-i, constititive expression of icam- was evident only in lung but not in muscle. during reperfusion icam- expression increased by . %. treatment with anti-tnfcx or il-ira reduced icam-i upregulation by . °, and . % respectively. frozen sections of normal rat lung revealed no evidence of e-selectin expression, whereas lung sections obtained after ischemia and reperfusion immunhistochemically demonstrated expression of e-sdectin. lungs from rats pretreated with anti-tnfct were negative for e-selectin staining. our data indicate a role for cytokines in the upregulation of adhesion molecules in ischemia/reperfusion injury. adherence of neutrophils to the coronary endothelium is associated with significant myocardial injury following min of ischemia (mi) and . h of reperfusion (r). p-selectin is expressed by activated endothelial ceils and platelets within min and tethers polymorphonuclear leukocytes (pmns) to the endothelium. we examined the cardioprotective effects of a monoclonal antibody (mab) designated pb . to p-selectin in a feline model of mi+r. pb . ( mg/kg) administered rain post-occlusion ( min prior to reperfusion) significantly attenuated myocardial necrosis compared to a non-blocking mab (nbp . ) for p-selectin ( + vs + % of area-at-risk, p< . ). endothelial dependent relaxation to acetylcholine was also significantly preserved in ischemic-reperfused coronary arteries isolated from cats treated with pb . compared to nbp . ( + vs + , p< . ). furthermore, pmn adherence to ischemic-reperfused coronary endothelium was significantly attenuated in pb . treated cats ( + vs + pmns/mm z, p< . ). also, normal coronary endothelium activated with thrombin ( u/mt) had increased pmn adherence under conditions of shear stress, an effect which was significantly (p< . ) blocked by pb . , but not by nbp . . furthermore, p-selectin is markedly upregulated - min post-reperfusion in coronary arterial and venous endothelinm. similar results were obtained in a rat model of mesenteric ischemia/reperfusion, including upregulation of p-selectin on mesenteric vascular endothelium. these results demonstrate that tethering of neutrophils to endethelium by p-selectin is an important early consequence of reperfusien injury, and a specific monoclonal antibody to p-selectin exerts significant endothelial preservation and cardioprotection in ischemia and reperfusion states. as recent interest has increased in the role of extracellular matrix proteins in inflammation, we examined the role and relationship of laminin and fibronectin to the processes of cell infiltration in a rat model of heart isografts. changes occurring in this system are on a nonimmunological basis and may be an effect of the initial surgical manipulation and the warm and cold ischemic times of transplantation. lewis (lew) donor hearts were transplanted into lew recipients, naive lew acted as naive controls (nc). grafts were harvested , , , , , , , and h after transplantation (n= /time point) . snap frozen sections of organs were stained for cd- (w / ), cd- (ox ), t-lymphecytes (tl) (ox- ), macrophages (ed , ed ), neutrophils (pmn) (mom), icam-i (iap ), laminin and fibrinogen. results were evaluated visually by counting cells/field of view (cf) for w / , ox , ox , ed , ed and mom, staining for icam , lain and fib was assessed visually using a scale (ve= - ). as early as h after transplantation, fibrinogen and laminin expression increased in i, peaking h after transplantation (ve= . ; nc: ve=i. ) . at and h in i, on both vessels and interstitial cells laminin and fibrinogen expression had declined to baseline (ve=i. ) but rose again at h, peaking at h (ve= ) and remaining elevated thereafter (ve= ). the high expression at h correlated well with the maximal pmn infiltration at that time (cf= ) in i returning to baseline at h (cf= . ) and thereafter. the second peak at h correlated with the onset of macrophage and t-lymphocyte infiltration. thus, laminin and fibrinogen seem to guide leucocyte infiltration following graft ischemia during the time of reperfusion. background. reperfusion of ischemic kidneys with xenogeneic blood leads to activation of endothelial cells and circulating leucocytes with the final consequence of microvascular thrombosis. in concordant systems, the mechanisms of rejection can be studied due to cross-reactivity of mediators with anti-human monoclona~ antibodies. aim of the study. to obtain information about the kinetics of proinflammatory cytokines and production of soluble adhesion molecules in the acute phase of reperfusion, eight kidneys from rhesus monkeys were perfused ex-vivo with human blood (group b/o) for one hour in a closed system. blood levels of il-lb, il- , tnf-a, soluble icam and e-selectin were measured in elisa-technique under steady-state conditions. results. cytokine levels rose significantly within the minute interval (il-lb: , __+ , to , __+ , pg/ml; il- : . - , to , __+ , pg/ml; tnf-a: , __+ , to , + , pg/ml; p< . ). immediately after the beginning of reperfusion, soluble icam- and selectin levels were abnormally high and constantly rising throughout the observation period, reaching significance at minutes. discussion. high levels of proinflammatory cytokines may lead to an induction of adhesion molecules, thus upregulating the leukocyte-endothelial interaction in an complement-independent mechanism. specific pretreatment with monoclonal antibodies against icam- , lfa- or other soluble mediators may be useful in downregulating hyperacute rejection in transspecies transplantation. prolonged ischemia has been associated with later dysfunction of solid organs as it may cause upregulation of adhesion molecules, production of cytokines on vascular endothelium and migration of host cells into the graft. these early events may lead to irreversible organ injury. this study evaluates the effects of global ischemia on early immunohistological changes in cardiac grafts transplanted in rats. isografted hearts (lewis->lewis) were were divided into ischemic and non-ischemic groups (n= /group). all donor hearts were flushed immediately with cold saline. non-ischemic hearts were then transplanted within rain, ischemic hearts were stored in cold ringer's solution for hours before revascularization. representative grafts were removed after . , hrs, and days, and evaluated immunohistologically (cells/field of view=c/f). restitution of ventricular activity was significantly delayed in ischemic grafts ( vs rain). after hrs, all ischemic grafts exhibited an extensive interstitial edema, declining slowly thereafter. at the same time, numbers of pmn peaked ( vs c/f in non-ischemic grafts), whereas edl+macrophages ( vs c/f) and tnfe expression peaked by hrs. by hrs t-lymphocytes began to enter ischemic myocardium and icam- was moderately increased. after days cellular infiltration had returned to baseline, and no differences were seen among both groups after days. global myocardial ischemia inhibits initial graft function, and engenders a brisk inflammatory reponse, primarily pmn and macrophages, with increased mhc class ii and cytokine expression. leukocyte -endothelial interactions are the result of endothelial activation, leukocyte activation or combination of both, which are accompanied by nee-expression, upregulation or shedding of adhesion molecules (selectins, inlegrins). such interactions differ with regard to the stimulus (e.g. thrombin or histamine for p-selectin, endotoxin or tnf/il- for e-selectin), the time course of response (minutes versus hours) and the localisation in different organs. recently assays are available for circulating soluble fragments of the cell bound adhesion molecules e.g. se-seleetin was found to be increased in plasma concurrent with high circulating endoloxin and cytokine levels. the importance of adhesion molecules for the sepsis event is evident, while effectiveness of anti-adhesion inolecu]e therapy is controversial e.g. beneficial anti-e-selectin therapy in baboon bacleremia but deleterious effects of amti-cd treatment in the same model. in other species similar controversial results with anti-cd therapy in sepsis were reported. steven l. kunkel,theodore standiford* and robert m. stricter. the migration of leukocytes to the lung during endotoxemia is dependent upon the coordinated expression of lung vascular adhesion molecules and the subsequent production of appropriate leukocyte chemotactic proteins. in experimental animals, neutrophils accumulate within the lung soon after the administration of endotoxin, while mononuclear cell infiltration occurs in a more distal manner. a kinetic analysis of lung leukocyte levels revealed a -fold increase in neutrophil numbers associated with dispersed lullg tissues hours after lps treatment, while macrophage levels increased by -fold at the hour time point. thus, the recruitment of different leukocyte populations to the lungs during endotoxemia is likely directed by different mechanisms. recent studies have identified a supergene family of small inducible chemotactic cytokines (chemukines) which possesses chemotactic and activating properties for neutrophils. the prototype of this family is interleukin- (il- ). interestingly, a related supergene family has been identified which possesses activity for recruiting mononuclear cells. examples of this group of inflammatory chemukines are monocyte chemotactic protein-i (mcp-i) and macrophage inflammatory protein-i alpha (mip-i). in initial in viva studies we examined whether mip-i was expressed systemically or in a compartmentalized fashion post lps challenge. assessment of plasma cytokine levels revealed maximal tnf levels occurred i hour post lps administration, returning to baseline by hours, while mip-i levels were maximal at hours ( , ng/ml), with a second peak at hours after lps challenge. interestingly, aqueous extracts of liver homogenates from lps treated animals demonstrated no mip-i levels, while aqueous extracts of lung revealed a -fold increase in mip-i levels over control lungs. immunohistochemical analysis of the lungs from hour lps treated animals demonstrated the alveolar macrophage was a rich source of mip-i protein. cell-associated mip-i was also expressed by blood monocytes adherent to the pulmonary vascular endotheliun, however the expression of monocyte-mip-i was observed by hours post lps administration. immunohistochemical analysis also demonstrated that mip-i antigen is associated with the extracellular matrix on the interstitial side of the endothelium. this suggests that the extracellular matrix, which is produced during inflammation, can bind mip-i and this may serve as a depot for the prolonged presence of nip- . in additional studies we have demonstrated that the intratracheal instillation of rmui [ip-l(loong) activation of polymorphonuclear leukocytes by inflammatory stimuli may contribute to the development of multiple organ failure in septic patients. thereby pmnl are proposed to avidly adhere to vascular endothelium causing damage by the subsequent release of toxic agents. as cellular adhesion is primarily mediated by -integrins and lselectins, the present study compares the expression of these adhesionmolecules on pmnl in septic patients and healthy volunteers. methods: expression of -integrins and l-selectins on pmnl was measured in whole blood by flow cytometry using the monoclonal antibodies ib and dreg , baseline values were determined immediatley after drawing blood. in addition cells were incubated min at °c to allow for spontaneous regulation of adhesion molecules. blood specimens from septic patients were obtained during the course of their illness. control values were determined in healthy volunteers. results: baseline expression of -integrins and l-selectins was not signifcantly different in septic and in healthy subjects. in contrast, there was a significant upregulation of g -integrins and shedding of l-selectins of pmnl in septic patients (sp) compared to healthy volunteers (hv). the local or systemic production of inflammatory cytokines, such as tumor necrosis factor alpha (tnfc~), can serve to modulate multiple aspects of neutrophil function. the ability of neutrophils to leave the circulation and migrate to areas of infection is one essential component of host defense. l-selectin, a leucocyte-associated adhesion molecule, is responsible for the initial reversible contact between neutrophils and endothelium and the subsequent roiling action of neutrophils along the vessel wall. in contrast to other adhesion molecules, l-selectin expression is rapidly down-regulated after neutrophil activation. the loss of l-seleclin may thus be a critical determinant of how neutrophils become unbound from their endothelial attachments and enabled to proceed towards an underlying extravascular area of infection. we hypothesize that the shedding of l-selectin is a strictly controlled process, occurring primarily at localized sites of inflammation, which may be modulated by tnf~, a flow cytometric method of staining neutrophhs by monoclonal antibodies in whole blood is described whereby the kinetics of l-selectin shedding may be followed in real time. the dose response and time course of in-vitro l-selectin shedding by neutrophils from normal human subjects was assayed after exposure to n-formyl-methionylleucyl-phenylalanine (fmlp) and tnfc~. either singly or in combination, our results show that l-selectin shedding can be reliably followed over time. a significant percentage of cells shed l-selectin after exposure to pg/ml tnfc~ or nm fmlp (but not at pg/ml tnfc~ or nm fmlp). greater numbers of cells were able to shed their l-selectin when fmlp and tnf~x were presented in combination rather than alone. high levels of tnfc~ did not appear to alter the threshold concentration of fmlp required to induce shedding, we conclude that the extent and rapidity of l-selectin shedding may be modified by different combinations of ligands and that shedding, by vidue of the high concentrations of cytokines or chemotactic factors required, is a process localized to sites of infection or inflammation. we prospectively studied patients with severe sepsis syndrome; group a : septic shock with or without adult respiratory distress syndrome lards) (n = , bacteremia = ); group b : sepsis syndrome without septic shock (n = , bacteremia = ). serial plasma samples obtained on day , , , , and , were assayed using elisas method (british biotechnology), normal control levels of soluble icam- and e-selectin, obtained from healthy volunteers, were respectively ± . ng/ml and ± . ng/ml (mean _+ se), acute lung injury was quantified dally on a tour-point score system (murray, am rev respir dis, ) . compared to control mean values, initial levels of groups a and b were significantly higher for icam- (p < - ) and e-selectin (p < - ). comparisons of group a and [] (* = p< . ; ** = p< . t) soluble icam- levels of group a enhanced significantly (p< . ) during the first hours, and a sustained high levels was of bad prognosis ( % of survivors at day ). the evolution of soluble icam- and e-selectin levels were significantly correlated with murray's score (spearman test : p < . ). conclusion: these results suggest that endothelial adhesion molecules are released into the plasma of patients with severe sepsis syndrome. soluble icam- and e-selectin are correlated with endothelial lung damage, and loam- seems to be a better indicator of the severity of endothelial injury. introductory remarks to anti-adhesion molecule strategies as a therapeutic modality ch wortel, repligen corporation, one kendall square, building , cambridge, ma , usa. the development of antimicrobial therapy represented a major breakthrough in the struggle against disease. it strengthened the notion that disease could be overcome by eliminating foreign invaders threatening the host. this paradigm has proven to be very successful, the threat of many infectious diseases has significantly changed, some have even been eradicated. nevertheless, sepsis has remained a severe condition, increasing in incidence while mortality remained very high. more recently, it has become increasingly clear that besides the nature and treatment of an exogenous agent, the reaction of the host defense itself plays a pivotal role in the outcome of the event. endogenous mediators, such as tnf, il-i, il- and il- , govem many of the actions of the host defense system. while the expression of these cytokines more often than not benefit the host, (over)-expression can cause severe damage. based on this hypothesis,anticytokine strategies, such as those targeted against tnf or il- , have been evaluated for the treatment of sepsis. results of these early studies have not yet indicated success in improving the outcome of the disease. it has been difficult to define a patient population where a benefit could be reproducibly shown. furthermore, it has been documented that synergy between cytokines occurs, but detailed knowledge of the cytokine network is not yet available. it is conceivable, that neutralization of one cytokine prompts the induction of another which will evoke the intended response in the host. recent data obtained in human endotoxemic volunteer models seem to confirm this. if this turns out to be the case, neutralizing a single cytokine may not be a successful approach. cytokines in tum, induce various adhesion molecules, such as icam- . such molecules regulate for instance the neutrophil-endothelial cell interactions, which are thought to play an important role in the pathogenesis of systemic organ injury. the potential for monoclonal antibodies to adhesion proteins to reduce vascular and tissue damage has been studied in a large number of experimental models. protective effects have been observed in a wide variety of inflammatory, immune, and ischemia-reperfusion injuries. thus, altering the host response by modulating the function of adhesion molecules may attenuate the inadvertent injury caused by inappropriate behavior of host defense cells. targeting cellular surface interactions has been added to the efforts to change the outcome of disease. modulation oftheseprocesses seems very promising, but may temporarily leave the host without effective defense mechanism. great care therefore, must be exerted when studying this powerful two-edged sword in a clinical setting. our knowledge of the role of adhesion molecules in the intlammatory response has increased rapidly due to the availability of new reagents and mice geneticly deficient in adhesion molecules. these molecules are important in interactions of leukocytes with endothelial cells, other leukocytes, platelets, and epithelial cells. when these molecules are engaged, they can also play a role in activating leukocytes and their effector functions. in the venules of the systemic circulation, adhesion often occurs through a series of sequential interactions. initial interactions are mediated by members of the selectin family to loosely associate the leukocytes with the endothelium and are followed by firm adhesion requiring members of the integrin and immunoglobulin family. later interactions with endothelium may require pecam. adhesion molecules are usually required for leukocyte emigration in response to extravascular stimuli and for neutrophil-mediated endothelial cell injury. they are critical for host response in many diseases including infections. however, when the inflammatory response results in damage to host tissues, patients may benefit from blocking the leukocyte response. anti-adhesion molecule agents are an important potential antiinflammatory therapy. the focus of anti-adhesion therapy may be at any step of the sequence. diseases where anti-adhesion molecule therapy may benefit patients include ischemia/reperfusion injury in many organs, ards and mof, and transplantation, both to protect the donor organ from ischemia/reperfusion injury and to inhibit graft vs host disease. many strategies have been considered and include: ) blocking the ability of adhesion molecules to recognize their ligand using antibodies that have been humanized or soluble receptors linked to igg to prolong their circulating halflife, ) blocking the ligands for adhesion molecules using soluble adhesion molecules, peptide analogues, or oligosaccharides, and ) blocking the production of the adhesion molecule using anti-sense oligonucleotides. because the synthesis of adhesion molecules is usually regulated by cytokines, inhibiting the action of cytokines is another potential site for interrupting the adhesion process. although important issues of safety must be evaluated, the potential for modulating the inflammatory response make this an exciting area of improvement in health care delivery. claire m. doerschuk, m.d.; riley hospital for children, room ; barnhill drive; indianapolis, in usa. modulation of neutrophil-endothelial cell adhesion with anti-cdl i/cd monoclonal antibodies as a therapeutic modality. ch wortel, repligen corporation, one kendall square, building , cambridge, ma , usa. the central role of inflammatory cells in the pathogenesis of lung and systemic organ injury is well recognized. binding of neutrophils to endothelial cells and migration into the parenchyma are largely regulated by complementary adhesion molecules. the leukocyte integrins are glycoproteins expressed on the neutrophil surface and in the cytoplasmic granules. integrins consist of a common beta or cluster differentiation (cd) chain covalently linked to one of three different alpha chains (cdlla, cdllb, cdilc) and exist on the cell surface as three distinct heterodimers. cdlla/cd is expressed on all leukocytes, whereas cd b/cd and cd c/cd . are restricted to cells of myeloid origin. cd i / cd interacts with intracellular adhesion molecule- (icam-i), its ligand on endothelial cells. the potential for monoclonal antibodies to adhesion proteins to reduce vascular and tissue damage has been studied in a large number of experimental models. protective effects with anti-cd antibodies have been observed in a wide variety of inflammatory, immune, and isehemia-reperfusion injuries, such as arthritis, burns, endotoxic shock, bacterial meningitis, autoimmune diabetes, nerve degenemrion, allograft rejection, allergic asthma, acute lung inflammation, skin lesions, and ischemia-reperfusion models of the intestine, myocardium, lung, skeletal muscle, and central nervous system. protective effects have also been observed in animals resuscitated following hemorrhagic shock. blockage of cd , however, would affect all leukocytes, as would antibodies to cdlla/cdi . targeting cdllb/cd would affect cells of the myeloid lineage only, which could prove to be beneficial. cd b/cd is not only involved in transendothelial migration, but is also implicated in adherencedependent formation of reactive oxygen species. blocking cd lb/cd may therefore not only reduce the numbe r of leukocytes accumulating in the tissue, but also attenuate the oxidant stress of infiltrated neutrophils. anti-cd b treatment has been used effectively to reduce tissue injury initiated by ischemia-reperfusion, complement activation and endotoxemia. altering the host response by modulating the function of adhesion molecules may attenuate the inadvertent injury caused by leukocytes, but may also temporarily leave the host without effective defense machinery. overall, animal studies suggest that it may be safe to inhibit neutrophil adhesion for a limited period of rime. these observations will have to be confirmed in carefully designed clinical trials. c, arbobydrams are ubiquizom constir~uts of cell sv.rfaees, and possess many c~xssfies ttm~ m~,e ~em ide~. canaidates for r~ognifioa mole~ule& in m~y systems whe,~ cer udhesioa ~lays a critical ro~ car~hydram l:~dtag ~otegas have been shown to b~ad tocell surfa~ earbohydzaxes ~nd pzrl~pate in cell-ceil lumtaefion& such sys,.ems include ~rti~za~io=, deveaopmeat, l~thoge~-hcet reeog--ition ~d i~zmmadon_ in particular, tb.z recent di%~ve~ of lhe selec~ and th~ impo.~a~c~ in teukccy~udo~lelium adh~ion has -~f~m av.c~on ok l~in m~ted cell adhe~on. s~vere/poten~s/cs.rbohydr~ l~ga~s hrve ~e~l ~u~ilied for ~he s~lcc~ins. the,~ c~u be broadly di,,sded la~o ~wo m'oups -sibyl l~wis x m~ mh~.~l oligo~chadd~s, ~d sf/~ ca~ohydmma, all ~:~ ~l~dns bind m siflyl l~wis x (sie$ o!igos~ccb.e.rkms, zlthou~ w~ differing avi~re~. 'we have i~¢n~ed the functional g~oups a s~ex ~n~ med/a~ ~he b~u ~di~g of ~h~ c~b hydmm = e-se/sedm we have used ~hat iv.formation to sya~esize sle ~ '~mt gs r.he, t focus on replacing slslic ~sd ~nd fuc s¢ wi~ simpler, more stable strunt~es. a[~ou~a ~ proeer~ is ongoing, we hve been ~ucee,.~ful a~ rep~aein t.ke si~ic a~id. residue wi~ std.fzte. ~ce~ or la~c amd groupa we t'we ex aninad &e ten, bunion of ezed~ hydroxyl group of the fizeose residue ~ billding of e-, l-~nd p-selees..u. we have also found m~fi~fio~ of the reducing end ~¢.cha'i~ ~z increase mtagovsst activity. the, m¢ond. group of figs,rids a.r eontzin su~a~ u a ea.rbohydr~t¢ support,, und seem to bi~.d to t~e sele~ti~s wi~ dlf:ferem characteristics c .an does sle:, s=h compounds are m ogniz~d by l-selects. md p-selectia, bur., in genera/, not e, selecti~ these dam may mdicam r.hat l-and p-s~ ¢at~ h~d via o, second ~te thaz operates lu~.ead of, or in conjunction with ~tc sle" b~ding ~iite. dam rela~&~g to ±e, se two types of ,ml~ liga~ds have beam t~ed to desig~ potential the ~peutics for i~fi~anmat ry disease. lr:rng maimai models of acute lung lu ury we can demo~trate that eompmmds that inhibit seleetiu birding ~ ~i~o hzve ber~ficial effects when uc~d in rive. progressive microvascular damage in the tissue adjacent to a cutaneous burn injury results in extension of burn size. the role of leukocytes in the pathogenesis of microvascular injury was investigated by inhibition of their adherence to the microvascular endothelium using monoclonal antibodies directed to leukocyte cdi or its endothelial ligaud, intercellular adhesion molecule- (icam- , cd ). a model of thermal injury was developed using new zealand white rabbits. two sets of three full-thickness burns separated by two x -mm zones were produced by applying brass probes heated to °c to the animals' backs for sec. cutaneous blood flow determinations carried out with a laser doppler blood flowmeter were obtained for hours. there were five experimental groups: controls given saline alone; animals given monoelonal antibody to the cd r . prior to burn injury (pre-r . ); animals given r . min after burn injury (post-r . ); animals given a monoclonal antibody to icam-i, r . prior to burn (pre-r . ); and animals given the r . min postburn injury (post-r . ). blood flow in the marginal "zone of stasis" between burn contact sites was significantly higher in the antibody-treated animals. administration of the antibodies min after injury was as effective as preburn administration in preserving blood flow. at hr post-burn all antibody -treated animals had blood flow in the areas at risk for progression (i.e., the zone of stasis) at or above baseline levels while the control animals had levels equal to . _+ % of baseline (p < . by analysis of variance and mann-whitney u test). these results indicate that leukocytes play an important role in the pathogenesis of burn wound progression, and that this progression can be attenuated by moduiating adherence to endothelial cells. a wealth of information now supports the hypothesis that inhibition of cell adhesive mechanisms will nter the course of immunologicand inflammatory processes. what remains unclear is whether inhibition of specific mechanisms wfl[ be of therapeutic benefit in any specific human disease. current data derived from animal models are not inconsistent with the hope of therapeutic benefit, but techniques for inhibition (e.g., antibodies, antisense oligonucleotides, inhibitory peptides, inhibitory carbohydrates, smaii synthetic inhibitors, etc), tissue and species differences in the relative contributions of adhesion molecules to the inflammatory process, and the cascade model of adhesive interactions are all confounding issues, making predictions of therapeutic benefit in any specific human disease process very difficult. additional concerns involve the potential roles of adhesive mechanisms in host resistance to infection. as human therapeutictdals are initiated, more exact information on the roles qf specific adhesion molecules in human disease should emerge. inhibition of leukocyte adherence to endothelial cells can represent a novel therapeutic approach to septic shock. we performed a pilot study to evaluate the safety and tolerability to cy- , a monoclonal antibody against human e-selectin, in patients with septic shock. septic shock was defined by clinical signs of sepsis, a documented source of infection, and fluid-resistant hypotension requiring the use of vasopressors. eleven patients entered the study, but patients who died during the first hours were excluded, as this was part of the protocol. cy- was administered as a single intravenous bolus of . mg/kg (n= ), . mg/kg (n= ) or i mg/kg (n= ) mg/kg. the antibody was well tolerated. none of the patients died during the day follow-up period. organ failure was assessed for organs (cns, lungs, liver, kidneys and coagulation). the mean number of organs failing, which was initially . ± . , decreased to . ± . at the end of the study (p<o.o ). these results are therefore encouraging. administration of an antibody to e-selectin in patients with septic shock requires further study. the importance of cytotoxic t lymphocytes (ctl) in the host defense mechanism against viral as well as parasitic and bacterial diseases is becoming increasingly appreciated. the design of vaccines to generate cytotoxic t ceils from nonviable or subunit constructs, however, poses challenges due to the unique characteristics of antigen processing and presentation by class i major histocompatibility complex (mhc) molecules. since the final antigen recognized by ctl are short peptides capable of high affinity binding to class i mhc molecules, we have designed strategies: a) to define the structural characteristics of peptides that are required for their high affinity binding to mhc; and b) to formulate these peptides into immunogenic vaccines that are capable of generating cytotoxic t lymphocytes. the strategy and results to accomplish these two goals will be discussed. amnon altman and erich gulbins ras proteins represent essential intermediates in receptor-mediated growth and differentiation pathways. they couple signals initiated by receptor or nonreceptor protein tyrosine kinases (ptks) at the cell surface to cytoplasmic targets which coordinate signal transduciion to the nucleus. ras also participates in t lymphocyte activation initiated by the antigen-specific t cell receptor (tcr)/cd complex, which leads to lymphokine production and proliferation. receptor ligation causes activation of associated ptks of the src and syk families as the earliest identifiable event in this pathway. the importance of ras in t cell activation is indicated by the findings that an oncogenic ras protein activates the interleukin- (il- ) gene promoter; conversely, a transdominant inhibitory ras mutant inhibits this event and the activation of map kinases. the rate-limiting step in ras activation is the exchange of bound gdp for gtp, which is catalysed by guanine nucleotide exchange factors (gefs). we identified the sh /sh domain-containing vav protein, which is selectively expressed in hematopoietic cells, as a ras-specific gef coupled to several hematopoietic receptors. vav can be activated by two pathways, i.e., by ptk-mediated phosphorylation or by binding of diglyceride second messengers to a cysteine-rich, protein kinase c (pkc)homologous vav domain. these pathways are independent as demonstrated by structure/function analysis and the use of pharmacological inhibitors. the two activation pathways may enable vav to integrate signals from distinct hematopoietic cell receptors, and couple them to ras. t cells express another, ubiquitous ras gef, i.e., sos, which is known to be coupled to activated tpk receptors. t cell activation leads to membrane association of a signaling complex consisting of sos and two other signaling proteins, grb- and shc, via direct binding of the shc sh domain to the tyrosine-phosphorylated chain of the tcr/cd complex. thus, multiple receptors, ptks and ras activators participate in signaling pathways that lead to hematopoieitc cell growth and differentiation. the interactions and function of these various signal transducers will be reviewed. phagocytes. immunity is mediated by specific t lymphocytes, cytokines produced by these specific t cells activate antimicrobial capacities in mononuclear phagocytes, thus contributing to protection. other immune mechanisms also participate in the acquisition of resistance. on the other hand, t cells are also crucial for many pathologic sequelae of intracellular bacterial infections. although ifn-y is central to macrophage activation, synergistic and antagonistic effects with other cytokines occur. the cd/ t cells, representing the major t cell population in experimental mice, are the main mediators of antibacterial immunity; an auxiliary role for y/~ t cells appears likely. the c~// t cell population further segregates into cd t cells which recognize microbial peptides in the context of gene products of the major histocompatibility complex (mhc) and cd t cells which see their peptide in the context of mhc class i. the -y/~ t cells are generally double-negative. knock-out mice in which the genes for various t cell receptor chains, for mhc class i or mhc class ii molecules, for cytokines or cytokine receptors had been deleted, have provided extremely helpful tools for analyzing the role of t cell subsets and cytokines in antimicrobial immunity. using these mutant mice, the role of distinct t cell subsets and cytokines in bacterial elimination and granuloma formation was analyzed in detail. these studies underline the central role of a// t cells and ifn-y in antibacterial immunity and, furthermore, show a distinct function of /$ t cells. moreover, these studies show that the relative contribution of cd or cd t cells to antimicrobial immunity varies in response to different pathogens. apoptosis is an active form of cell death in which the cell participates in its own demise, providing the biochemical pathways that trigger and mediate the complex events of the process. although this phenomenon has been studied for many years, it is only recently that significant progress has been made towards the elucidation of the molecular mechanisms that control apoptosis. once such mechanism came as something of a surprise: in a number of eases, apoptosis can be promoted by the action of the c-myc protein, a protooncogene product that had previously been associated only with cell proliferation (and presumably, survival) . expression of c.myc in cells can cause them to enter an apoptotic pathway or proliferate, mad this "decision" depends upon additional signals, such as growth factors that inhibit apoptosis and allow the ceils to proliferate. thus, c-myc directs cells out of rest and the choice of proliferation or death depends upon the presence or absence of survival signals. other survival signals are generated by oncogene products that can cooperate with my¢, including bcl- and abl there are implications of this interplay between apoptotie and anti-apoptorlc signals in cell iransformation and the resistance of some tumor ceils to therapeutic agents capable of inducing apoptosis. oncogene interactions therefore control the life or death of transformed ceils but also have important roles in normal, development processes. in the immune system, developing t ceils are "screened" for potential self-reactivity by a process of negative selection, in which activation via the t cell receptor (tcr) induces apoptosis. this process can be mimicked in t cell hybridomas, and appears to depend upon the expression of the c-mye protein. evidence that this represents a requirement for the myc/max heterodimer will be presented. as above, these cells are presented with the "choice" to proliferate or die and this depends not only upon myc but also other signals. thus, expression of functional v-an kinase in these ceils can render them resistant to the activation of apoptosis via tcr ligation, surprisingly, bcl- does not appear to be capable of blocking this form of apoptosis, and the reasons for this will provide new insights into the development of the immune system and the process of apoptosis. although the oncogene products discussed here probably do not directly participate in the biochemical process of apoptosis, they represent molecular controls on this complex mechanism. as such, they gave us new ways to look at the life and death of a cell recent attention has focused on macrophage release of cytokines, such as il- , il- and tnf as important mediators of septic shock. however, serum cytokine levels have correlated poorly with outcome of septic shock. the purpose of this study was to compare the functional status of macrophages to serum cytokine levels in early sepsis by an analysis of splenic macrophage protein synthesis during abdominal sepsis. macrophage total protein distribution and biosynthetic activity was assessed by large format -d page, autoradiography of s-labeled proteins and computer assisted densitometric analysis. sepsis was induced in sprague dawley mts by cecal ligation and puncture (clp). untreated and sham operated rats served as controls. splenic macrophages were harvested at and hrs. following clp. serum il- levels were determined at , , and hours by the tdi bioassay. by hrs. % of the clp mls exhibited multi-microbiaj bacteremia. control or sham operated rats did not show bacteremia. clp resulted in a significant % elevation (p < . ) in serum il- levels at hrs. there was no difference in il- at and hrs. when compared to the control groups. d page studies examined splenic macrophage total protein distribution and biosynthetic activity at and hrs. post-clp. at hrs. macrophages from clp rats showed a substantial decrease in total protein pattern ( protein spots vs protein spots) when compared to non-septic control macrophages. a decrease in the number of lower molecular weight proteins (< kd) was observed. at hrs. post-clp, splenic macrophages from clp rats showed a substantial increase in the synthesis of low molecular weight (< kd) proteins, when compared to non-septic controls. we conclude that: l) fulminate abdominal sepsis induces an early ( hrs.) staining with cd and cd mabs will identify two monocyte subpopulations in human blood: a major population of regular monocytes, which strongly expresses the cd antigen (cd ++) and a minor population with weak expression of cd and expression of the cd antigen lcd +/ cd + cells). in healthy control donors(n= ) the latter cells account for + cells/mm and %+ % of the monocytes. in sepsis patients, the cd] +/cd + cells can become a major population with more than % of all monocytes in of patients and with more than cells/mm in of cases. there was no correlation of cd +/ cd + cells to any clinical parameter except for cd +/cd + percentage and body temperature (p= . ). the cd ++ monocytes showed a substanttial decrease in cd antigen density in of patients. three-color-if shows that the cd +/ cd + cells in sepsis patients when compared with the cd ++ monocytes exhibit a higher level of class ii antigen and a lower level of cdiib and cd antigens, consistent with a more mature nature of the cd +/cd + cells. levels of il- were increased in sepsis patients: of patients with high numbers of cdi +/cd + cells ( /mm ) had high levels of il- ( u/ml). these data suggest that septicemia may lead to substantial changes in blood monocyte composition and this may be related to elevated levels of cytokines such as il- . human peritoneal macrophages were exposed to increasing doses of lipopolysaccharide (lps) or a synthetic lipid a analogue (mrl ) and production of the cytokines il-lb, il- , tnf-a and g-csf was assessed at the protein-and mrnalevel. cells were also stimulated with low doses of lps and mrl to study their "adaptation" to a secondary challenge with high doses of lps. tnf-a production after stimulation with lps could be almost completely relieved by preincubation of cells with low doses of lps and similarly, il- production was suppressed. surprisingly, however, "adapted" cells were able to synthesize even larger quantities of of g-csf and il-lb when exposed to a secondary lps challenge. the changes in tnf-a, il- and g-csf protein synthesis could a/so be detected on the mrna level, whereas transcript levels for il-lb remained unaltered as assessed by northern blot analysis. high doses of the synthetic lipid a analogue mrl were also able to "adapt" macrophages for a secondary lps challenge by downregulating tnfa-and il- -production, while priming secretion of g-csfand il-lb as well. taken together, here we describe for the first time the discordant adaptation of human peritoneal macrophages to a secondary lps stimulus in vitro. these findings appear to bear ramifications for the in-vivo endotoxin response during inflammation and gramnegative septicemia. shock states with inadequate cellular oxygen delivery may contribute to macrophage (mo) activation or dysregulation. in this study we compared the effect of transient hypoxia and endotoxin pretreatment (lps ) on mo mediator release with a second endotoxin stimulus (lps ). methods: in vitro cultures of marine peritoneal exudate mo were exposed to hr. of hypoxic or normoxic conditions, then incubated hr under identical normoxic conditions _+ ng/ml of lps pretreatment. during the final hours all m~l were exposed to a range of lps concentrations. m~i supernatants were assayed for tnf, il- , il- , pge , nitric oxide (no), and superoxide release. results: lps markedly inhibited mo tnf release by lps but hypoxia had no effect on lps -triggered tnf release. hypoxia increased mo il- production in the absence of lps , but inhibited lps augmentation seen under normoxic conditions. pretreatment with lps increased no production from lps under normoxic conditions, but bypoxiainhibited this effect. pretreatment with lps signifcantly augmented mo release of il-i and pge , but hypoxia had no significant effect (data not shown) superoxide production was increased by lps under normoxic conditions, but hypoxia significantly inhibited superoxide release (not shown a. lepape, c. docile, f. arpin, m. c. gutowski, v. banssillon and j. bienvenu. i aim of the study. increased levels of tnf are inconsistently correlated with the severity of sepsis. one possible explanation is a decrease ability of ceils to produce eytokines. the objectives of this study were m compare at different levels of of clinical severity the responsiveness of cytokine producing cells. this was evaluated by the response to a stimulation by lps as well as to the inhibitory effect of ptx. the technic used is a whole blood ex-vivo model, as described by desch et al. ( ) . h materials and methods. different groups of icu patients, postoperative (n= ), sepsis (n= ), septick shock (n= ), and healthy control (n= ), were studied. blood was drawn in endotoxio free heparinized robes. stimulation was achieved by addition nf pl oflps ( ng/ml) to id of whole blood in presence of various concentrations of ptx ( , " , - , " , i " m). after hrs incubation at °c, the tubes were centrifuged and supematants frozen at - °c. tnfa and/l were measured by elisa (medgenix r and immunotech r respectively). monocytes were quantified on a technicon h . the results are expressed as median values. non parametric tests are used for testing differences between groups. hi results, tnftx and , , corrected for monocytes count, are given as % of their initial value, the baseline before stimulation or inhibition being %, ) stimulation by lps. the control group is much more stimulated (> % for il , > % for tnfa). blood samples taken postoperatively and in patients with simple sepsis are significantly less stimulated (> % for il , > % for tnfa ). the lowest stimulation was observed in patients with septic shock (median = %), some patients being not stimulated at all. )effects of ptx.the inhibitory effect of ptx on tnftx production is effective in all groups at - m (reduction to less than '¼ of the median values), and is almost complete at " m. the septic shock group has a decreased sensitivity to ptx. il production exhibits a lesser reduction at - m (~ 'a to ½ of the median values), further increased at - m. the septic shock group is again less sensitive to ptx. iv conclusion: the reduced ability of circulating monocytes to produce cytokines during severe infections is confirmed here. ptx is able to reduce significantly tnfc~ at - m and the inhibition is nearly complete at - m. surprisingly, there is a lesser, but significant suppressive action of ptx on il , not found in experiments using purified monocytes. one possible explination could be the interplay between cytokines production. ( ) lymphokine research ( ) cdna sequencing constitutes a powerful method of measuring steady-state mrna levels for all genes transcribed in a given cell or tissue at a particular stage of differentiation. by comparing transcript abundance both prior to and following differentiation, individual genes can be identified whose transcription is regulated both positively and negatively. in order to examine monocyte activation, the human monocyte line thp- was induced with phorbol ester ( h) and activated for h with lipopolysaccharide (lps) after which polya + rna was purified. the rna from control and lps-treated cells were each used to construct a cdna library under identical conditions, and all resulting clones were selected for cdna sequence analysis. each clone sequence was evaluated by matching with both genbank and our own gene databases. very different patterns of gene expression were seen in the two libraries, the latter reflecting very high levels of known inflammatory mediators such as il- and tnf. a second set of libraries were made from umbilical vein endothelial cells (huvec), both with and without lps stimulation, and were analyzed in a similar fashion. the effects of lps induction on specific gene transcription in both cell types will be discussed. t. tadros, md, th wobbes, me) phd, rja goris, md phd to investigate whether the preactivation of regional macrophuges by liposomes containing muramyl tripeptide (mtp-pe) can counteract the detrimental effect of blood transfusions on both anastomotic repair and host susceptibility to infections. methods eighty lewis rats received lmg/kg of either empty or mtp-pe encapsulated liposomes, intraperitoneally (ip). twenty-four hours thereafter, the animals underwent resection and anastomosis of both ileum and colon, and received ml of either saline or blood from brown norway donors,iv. the animals were killed or days after surgery and examined for septic complications and anastomotic repair. the average anastomotic strength, as assessed by bursting pressure (+sd), was significantly diminished in the transfused animals, as compared to the non-transfused animals (ileum;day ; -+ vs + , p< . ). transfused animals pretreated with mtp-pe encapsulated liposomes showed a significant improvement of their anastomotic bursting pressure ( + , p< . vs transfusion). pretreatment with mtp-pe encapsulated liposomes decreased significantly the incidence of anastomotic abscesses in transfused animals ( from % in ileum on day to %, p< . ). conclusions preactivation of regional macrophges by intraperitoneal administration of mtp-pe encapsulated liposomes prevents the detrimental effects of transfusions on anastomotic repair and reduces the incidence of intraabdominal sepsis. academic hospital nijmegen, dept of general surgery, pb i, hb nijmegen, the netherlands. leukemia cell line, teip- . robin s. wa, gner*, perry v. halushka "~, and james a. cook*, departments of physiology , pharmacology "l" and medicine "t, medical university of south carolina, charleston, s.c. . adherence of monoeytes to endothelium and extracella/ar matrix proteins is essential for accumulation at sites of inflammation. txa , an arachidonic acid metabolite, inhibits human monocyte chemotactic responses suggesting that txa may alter monocyte adhesiveness. we selected the thp- cell line, a human monocytic leukemia cell line to further investigate the effect of txa on adhesion. we tested the hypothesis that txa alters lpsinduced adhesion of thp- cells and that txa exerts its effect on adhesion via a camp dependent mechanism. thp-i cells were exposed to s. enteritidis endotoxin (lp.g/ml) _+ the cyelooxygenase inhibitor lndomethacin (in), the txa mimetic i-bop ( . .tm,) or txa receptor antagonists bms and l ( ~m). cells were allowed to adhere for hours and adherent protein/well was determined. lps-induced a significant (p< . ;n= ) increase in adherence of thp- cells (basal, . + . gg protein/well; lps, . +_ . p.g protein/well). the amino acid glutamine is an essential compound for synthesis of purine and pyrimidine basis and therefore necessary for rna-and dna synthesis. in human plasma the concentration of glutamine is between . - . mm, and is reduced in septic patients up to % ( . - . mm). monocytes play a central part in the inunune system and it was of interest, whether glutamine is involved in the modulation of cell surface markers and phagocytosis of these cells. human peripheral blood mononuclear ceils were obtained from ml heparinized blood of apparently healthy donors by ficoll-paque density gradient and isolated by counterflow elutriation. the puritiy was more than %. subsequently cells were cultured in phenolred-free rpmi medium with various concentrations of glutamine ( . , . , . , . , . , , mm) in teflon-fluorinated ethylene propylene bottles to exclude cell adhesion and possible cell activation. aider seven days culture, cell viabilty was determined by trepan blue exclusion and varied between and %, independent of glutamine concentrations. cell surface markers were detected by flow cytometry, noaspecifie phagoeytosis was measured with latex beads and specific phagocytosis with opsonizied e.eoli using a facscan. lower concentrations of glutamine decreased the expression of hla-dr and icam- /cd on monocytes in a dose-dependent manner. the receptor for fc'/rucd as well as the receptors for complement cr /cdllb and cr /cdllc were down-regulated. cr /cd which is only slightly expressed on monocytes was not influenced. furthermore, no effects on the expression of cdi , the receptor for transferrin cd and fc'friii/cd were seen. our data indicate, that lower concentrations of glutamme influence the phenotype of monocytes. we are now interested to study whether glutmnine influences non-specific phagocytosis, or whether specific phagocytosis correlates with the decreased expression of fc'/r and complement receptors. we investigated immunologically more than patients who were admitted to icu because septic syndrom during the last four years. patients were immunologically followed up - times per week until release from icu. the expression of hla-dr antigen on monocytes turned out to be the best prognostic parameter. the persistence (> days) of low hla-dr expression (< %) predicts fatal outcome (mortality > %). the altered phenotype was associated with a functional deactivation of monocytes (diminished apc, ros formation, cytokine secretion). we called this phenomenon "immunoparalysis". ifn-gamma and gm-csf were able to restore the altered phenotype and function in vitro. however, addition of autologous plasma from septic patients with "immunoparalysis" to these cultures prevented the cytokine-induced restitution. the inhibitory activity could not be removed by dialysis. therefore, we started a study to prove the therapeutic efficacy of plasmapheresis. indeed, [ of patients recovered from "immunoparalysis" following repeated plasmapheres; of them survived ( %). patients recovered temporarely and patients did not respond (all died). the survival rate in the control group of septic patients with persistent "immunoparalysis" was of ( %; p< , ). in summary, plasmapheresis in association with immune monitoring may be an alternative strategy to improve survival rate in severe sepsis. taurolidine, a synthetic taurine-formaldehyde derivative has antiadherent, bactericidal and anti-lps properties functioning primarily through binding of the lipid a region of the lps molecule. the active derivative of taurolidine, taurine, modulates calcium channel activity, critical to the initiation of a number of immunostimulatory pathways. we hypothesised that taurolidine may have direct immunostimulatory activity. the aim of this study was to investigate the immune effects of taurolidine on peritoneal macrophage (pmo) function and then determine the role of taurine in this response. study : in vivo stimulation:cd- mice (n= ) were randomized to receive taurolidine ( mg/kg bw/i.p.) or saline cor~trol. peritoneai cells were harvested after hours and were assessed for pm function [superoxide anion generation (o -), nitric oxide (no), tumor necrosis factor (tnf), fc/cr -mediated phagocytic function (phago) study : control pm were harvested and cultured in vitro with taurine ( . mg/ml for hrs), after which time they were assayed for -and tnf release. in vivo stimulation with taurolidine taurolidine has specific immunological effects on m . release of the inflammatory mediators -and tnf, and fc/cr -mediated phagocytosis were significantly increased, while release of the endothelial relaxing factor no was significantly reduced. in addition, the amino acid taurine, which is released as a byproduct of taurolidines breakdown has an immunostimulatory effect on pmo and may be the active moeity of the compound tanrolidine. in sepsis, a number of mediators which affect vasomotor tone and cardiovascular function are produced. inasmuch as sepsis causes decrease in systemic vascular resistance (svr), attention is usually focussed on vasodilators such as lactate, tumor necrosis factor, interleukin-i & , and nitric oxide. but injury and inflammation als cause production of several vasoconstrictors whose effect may not be evident in changed svr, but may significantly affect organ blood flow or function in the paracrine environment. endothelin (et) is a amino acid peptide vasoconstrictor produced by ischemic or injured endothelial cells (ec's). et is also a potent constrictor for renal mesangial and coronary vessels, an endocrine regulator, and a negative cardiac inotrope. systemic et levels increase significantly in hypoperfusion and ischemia. while et is principally produced by ec's, we asked if human monocytes might also produce et and thereby regulate vasomotor tone in areas of inflammation. monocytes from healthy donors were separated on ficoll, resuspended in rpmi + % fetal calf serum and stimulated with i ug/ml endotoxin (lps). et was measured by radioimmunoassay. lps-stimulated monocytes produced ! fm of et/ cells (vs. unstimulated controls of < ). this calculates to - % of the amount of et observed in patients with low cardiac output, sepsis or ischemia. we conclude that et is a cytokine produced by both ec's and monocytes with potent effects on numerous cells and organs in the critically ill. wuppertal , germany we and other authors showed that fatal outcome in septic disease is associated with a decreased capacity of peripheral blood monocytes for the in vitro production of proinflammatory cytokines, especially tnf-alpha. we found that this monocytic deactivation is completed by a persistent and marked decrease of hla-dr expression on monocytes (< % hla-dr+ monocytes) and a diminished antigen presenting activity whereas the capacity to form the antiinflammatory il- receptor antagonist remains high. in order to evaluate the in vivo situation and to determine at which level tnfproduction/secretion is altered we assessed the tnf-alpha mrna expression in freshly isolated peripheral blood mononuclear cells (pbmnc) from septic patients. tnf-mrna was onty rarely detected by semiqaantitative polymerase chain reaction in pbmnc's from septic patients with monocyte deactivation. meanwhile, it was found in almost all pbmncs from septic patients without monocytic deactivation. we wondered, whether il-i , which ,is known to depress monocytic proinflammatoly response and mhc class ii expression, could be one of the mediators in fatal sepsis. in fact, we found that il- message in pbmncs of septic patients peaked in the beginning phase of monocytic deactivation. in further investigations we found that tnf-administration can induce monocytic deactivation in a murine model/n vivo and provoke il- message in human pbmncs in vitro. these results support our hypothesis that an excessive delivery of proinflammatory cytokines in a first phase can induce an overwheiming inhibitory feedback, mediated by immuninhibitory mediators like il-l , which leads to often fatal monocytic deactivation in a second phase. interferon-gamma which is known to counteract il- production and the effects of il- on monocytes restores the function and phenotype of monocytes from septic patients with monoq, te deactivation in vitro and could be a possible therapeutic agent in otherwise fatal sepsis. our laboratory previously reported that lps dependent macrophagederived tnf-a production can be enhanced by pretreatment with lps at substimulatory lps priming doses coincident with a suppression of lps dependent nitric oxide (no) production (zhang and morrison, j. exp. med : , ) . in order to extend the characterization of these lps priming effects in mouse macrophages, we examined the capacity of substimulatory lps to modify lps dependent il- production. macrophages were obtained from peritoneal exudate of thioglycollate treated c heb/fej mice and cultured in rpmi medium containing % fetal bovine serum. macrophages were pretreated with various subthreshold stimulatory concentrations of lps (olll:b ) for hours, washed three times, and then stimulated with the effective stimulatory concentration of lps for hours. the amount of il- in the supernatant was measured by il- dependent cell line (b and td ) proliferation assay. il- was produced by macrophages at lower threshold doses of lps than those required for tnf-o~ or no production. subthreshold doses of lps modulated il- production in a biphasic manner characterized by an initial suppression and then potentiation. higher doses resulted in secretion of il- during the initial incubation with lps and subsequent desensitization. il- , like tnf-~ and no, is, therefore, also affected by lps pretreatment. moreover, tnf-a and il- shared the similar potentiational pathway, but differed by the fact that only il- was inhibited. (supported by r ai and po a .) department of microbiology, molecular genetics and immunology and the cancer center, wahl east, university of kansas medical center, kansas city, ks - . korolenko t.,urazgaliev k.,and arkhipov s. the role of macrophage (mph) stimulation in mechanism of protective effect of new immunomodulators yeast polysaccharides -heteropolysaccharide cryelan and homopolysaccharide mannan rhodexman (both produced by petersburg chem.-pharm. inst.) was studied. in vitro according to nst test incubation of murine peritoneal mphs with cryelan or rhodexman, ~g/ml, min was followed by increase of potencial microbicidic activity of mphs. in vivo mph stimulation by immunomodulators studied included increase rate of carbon particles phagocytosis during single i.v. or i.p. mode of administration to mice - days after (peak at nd day for i.v. and th day for i.p. mode of administration of the same dose of mg/ g b.w.).the preliminary injection of cryelan ( mg/ g, or h before) to mice with acute cold stress (- ° c, h) revealed protective effect restorating the value of depressed phagocytosis up to the normal level;the positive effect on ultrastructure of hepatocytes was noted also.there was no changes of plasma corticosterone level between group with acute cold stress and mice with cryelan + acute cold stress (several fold increase comparatively to the control mice).as was suggested, the mechanism of protection can include mph stimulation and secretion of some acute phase proteins responsible for positive effect of immunomodulators. new yeast polysaccharides cryelan and rhodexman can be used for macrophage stimulation,especially in pathological states. immunomodulators were shown to increase production and secretion of lysosomal enzymes (like zymosan). secreted enzymes,especially cysteine proteinasescathepsins b and l -involve in the process of inflammation;however, excessive release of these enzymes may lead to noncontrolled proteolysis followed by tissue degradation (assfalg-machleidt et al., ) .the effect of zymosan,bcg and new immunomodulator carboxymethylglucan (cmg), second fraction on secretion of lysosomal enzymes by murine peritoneal macrophages was studied. zymosan increased the secretion of n-acetyl-~-d-glucosaminidase and ~-galactosidase into the culture medium ( - fold); bcg possessed similar effect.cmg in the same concentrations ( /~g/ml) increased release of these enzymes only saightly ( . times).it's known that zymosan-induced secretion reflects the enzyme release from formed lysosomes (warren, ) .it was suggested that cmg activated macrophages via interaction with scavenger-receptors,followed by weak secretion of lysosomal enzymes and as a result decrease of tissue damage. in vivo zymosan induced stimulation of mononuclear system of phagocytes followed by increase of cysteine proteinases activity in liver at the th day. in the same time in blood n-acetyl-~-d-glucosaminidase and n-acetyl-~-d-galactosidase activity increased - fold. it was concluded that in drug design it's possible to select such immunomodulators,e.g. cmg,which can activate mononuclear system of phagocytes and do not damage tissue. endothelin-i (et-i) is produced by injured/ ischemic endothelium, mobilizes intracellular ca ++ and is a potent vasoconstrictor. it is also a ca ++ agonist for anterior pituitary or renal mesangial cells and monocytes. et-i causes monocytes to produce interleukin-l, , , prostaglandin e , and substances which trigger neutrophil superoxide production. et-i levels increase in shock and et may play a role in activating leukocytes post shock causing reperfusion injury. but blood flow experiments suggest splanchnic circulation changes more profoundly in shock than peripheral circulation. we therefore asked if et- (or vic), the et which predominates in splanchnic vessels, had any effect on monocyte cytokine production. human monocytes from health~ blood donors were separated on ficoll. . x ucells/ ml in rpmi + % fcs were incubated i min., & hrs. with - m et-i, - m vic or i ug/ml of lps. supernatants were assayed by elisa. we have shown that low dose endotoxin pretreatment (lps ) for hrs markedly inhibits the macrophage (mo) release of tumor necrosis factor (tnf) and increases interleukin- (il-i) in response to a subsequent endotoxin stimulus (lps ). in this study we examined the kinetics of lps inhibition of tnf and augmentation ofil- . methods: murine peritoneal exudate mo from balbc mice were exposed in vitro to medium or ng/ml of lps for intervals of to hours. culture medium was then replaced with , or ng/ml of lps for hrs. tnf and il- in mo supernatants were measured by specific bioassays. during sepsis endotoxin (lps) activates macrophages (mo) to release mediators such as tumor necrosis factor (tnf), interleukin- (il- ), interleukin-i (il-i) and prostaglandin e (pge ). we showed that preexposure to lps (lps ) alters the response of murine m~i to subsequent lps stimulation (lps ). we hypothesized that in vitro cytokine release by lps in human monocytes (mo) is also be altered by preexposure to lpsi. methods: human peripheral blood mo were obtained from healthy volunteers (n= ), cultured in vitro hrs, then pretreated hr _+ lps -cultures were then stimulated with lps and mediators in mo supernatant measured: tnf, il-i, and il- by specific bioassays, pge by immunoassay kit. serum cytokine levels (specific elisa kits) were compared to in vitro supernatant levels. data is expressed as % control_+sem, lps = ng/mh the table shows that all mediators were increased, in the absence of lps . pretreatment with lps resulted in complete inhibition of lps -triggered tnf release. in contrast, lps significantly increased mo secretion of il- , il- and pge (data not shown). serum cytokine levels were as follows: tnf _+ , il-i + , and il- . -+ . ng/ml. these serum levels were low, showed an extremely wide variation, and did not correlate with in vitro lps -triggered mediator production. conclusion: human monoeyte mediator production is differentially regulated by preexposure to lps . provocative in vitro testing of monocytes may ultimately be clinically useful to identify prior in vivo lps exposure or mo macrophages release numerous secretory products involved in host defense and inflammation. activated macrophages with cytokines produced have been implicated in tissue damage in sepsis and multiple organ dysfunction. aimed to elucidate the organ-association phenomena,this study is to compare peritoneal macrophage(pm),alveolar macrophage(am), and kupffer cells(kc) during sepsis in terms of cellular protein contents as symbol of activation by flow cytometry analysis. sepsis were produced by cecal ligatien and perforation (clp) in wistar rats weighing - g.pm were obtained by peritoneal lavage,am by bronchial lavage and kc by incubating the collegenase digested liver with pronase-e. leukocytes have been implicated as a mediator of the microvascular dysfunction associated with reperfasion of ischemic tissues. a role for ieukocytes is largely based on observations that rendering animals anutropenic with anti-neutrophil serum or preventing leukocyte adhesion with monoclonal antibodies attenuates the increased fluid and protein leakage from the vaseulature that is normally observed in postischemic tissues. we have recently undertaken studies designed to determine the relationship between leukocyte-endothelial cell adhesion and albumin leakage ia rat mesenterlc venules exposed ~o ischemia-reperfusion (i/r). leukocyte adherence and emigration as well as albumin extravasafion were monitored in single postcapillary venules using iatravital fluorescence microscopy, lschemia was induced by complete occ!usion of the superior mesenteric artery and ~dl parameters were monitored at various intervals following reperfusion. the magnitude of the leukocyte adherence and emigration, and albumin leakage elicited by i/r was positively con-elated with the duration of ischemia. the albumin leakage response was also highly correlated with the number of adherent and emigrated leukocytes. monoclonal antibodies against the adhesion glycoproteins cd , cdllb, icam- and l-selectin, but not p-or e-selecdn, reduced i/r-induced leukocyte adherence and emigration as well as albumin leakage. phauoidln, an f-aetin stabilizer, largely prevented the emigration (but not adherence) of leukocytes and greatly reduced, the raicrovascular protein leakage. plateletleukocyte aggregates were formed in postischemic vemdes; the number of aggregates was reduced by antibodies against p-selecdh, cdilb, cd , and icam- , but not e-selectin or lselectin. a significant fraction of the mast ceils surrounding the posteapillary venules degranulated in response to ischemia/repeffusion, but mast cell stabilizers did not afford protection against the albumin leakage elicited by i/r. these results indicate that reperfusloninduced albumin leakage is tightly coupled to the adherence and emigration of leukocytes in posteapillary venules. this adhesiomdependent injury response is primarily mediated by cdllb/cdi on activated neutrophils and icam- on venular endothellum, and appears to require l-selecda dependent leukocyte rolling. mast cell degranulation does not appear to conwibate to the vascular pathology associated with i/r. m.d. rod=iek, boston, ma, usa the polymorphonuclear neutrophil (pmn) has long been known to pa~tlcipats in the inflammatory rebpons~ as a phagocyte and killer of invading organisms, but little attention has been given to its potential as a participant in the in~une interaction of lymphocytes and macrophages. we and others have shown that the pmn may have i~m~/nomcdulatory effects both in vitro and in vlvo. more recently it has been proven that the pmn can make mrna for and secrete the proinflammatory oytokines illa, il-ib, tnfs, il- and il- as does the other major circulating phagocyte, the monocyte/macrophags. furthermore it has been shown to make the potentially autoregulatory oytokines gcsf and gmcsf. these functional capabilities suggest that the pmn is not an wend cell ~, but one which has a potential role in regulation cf ~he immune response and that this potential ~cle should no longer be ignored when considering the immune abnormalities existing in patients following majo~ injury or surgery. we have investigated the proinflaznmatory oytokine secretion patter~ by pmn in patients following major ~hermal or tra~matic injury and in volunteers fellowinq endotoxemia. ?ollowing major injury there is variable pmn secretion of these cytokines when stimulated in vlero. following endotoxemia in a group of human volunteers pmn showed a hypo=esponsivenesa to lps hrs following endotoxin infusion followed at hre by an overshoot. pretreatment with steroids modulated this overshoot phenomenon, suggesting that receptors for steroids are involved in the regulation of cytokin® secretlon by fmn. these results sugges~ that the pmn, the most numerous cell in the circulation and the first to respond to an ins~l~ may be a so~rce of the prolnflammatory cytokine cascade following injury that has been recognized as significant in the process which often leads to multiple o;gan failure, the immunosuppresslon which occurs following major thermal injury may predispose these individuals to infection and sepsis, which remain a significant cause of morbidity and mortality. included among the many immune aheratlons are the p integrln (cdlla, b,c/cd ) dependent activities of adhesion, chemotaxls, diapodesls, and phagocytosls. our investigations indicate that, following major thermal injuries, the expression of the [~ integrlns, but not cd , is significantly decreased on neutrophlls (pmns). it remains unclear if pmns from thermally injured patients respond normally to lps, the effects of treatment in vitro with lps and f-met-leu-phe (fmlp) on the expression of cdtlb was examlned on pmns from the peripheral blood of healthy volunteers and non-septic burn patients (> ~; total body surface area, >ls~ full thickness), the pmns were incubated with lps (]ng- p.g/ml) or f'mlp ( " to " m) et oc for mln, in ~; human ab serum, the expression of the ]ntegrins was detected using monoclonat antibodies and flow cytometry. lps and f'mlp resulted in a slight increase ( fold) in the expression of cd b on pmns from burned patients compared to an and fold increase, respectively, on pmns from healthy individuals. this inability of lps or fmlp to increase cd b expression was not due to the amount of lps bound to the two cell populations. because the same defect is seen after either lps or fmlp stimulation, it is speculated that the defect must be in the amount of preformed cd ] b or its transport to the plasma membrane. platelet-activating factor (paf) and neutrophils have been implicated in the patbophysiology of ischemia-repeffusion injury, in addition, paf stimulates neutrophi[ (pmn) oxidative metabolism in vitro. the present study examined the potential role of paf in repeffusion injury in an in viva rabbit model. eight anesthetized rabbi~s underwent retroperitoneal exposure of the infrarenal abdominal aorta after percutaneous insertion of a catheter through the jugular vein into the infrahepatic inferior vena cava. doppler flow probes were placed around the abdominal aorta and the right common femoral artery to assess flow through these vessels. an occlusive ligature was placed around the abdominal aorta (superior to the flow probe) at t = and total occlusion of blood flow to the lower extremities was maintained for g mins., after which the ligature was released allowing for reperfusion of the ischemic lower limbs. effluent blood from the ischemic hind-limbs was collected through the ivc catheter at the times indicated below and assayed for paf by a direct radioimmunoassay. in addition, neutrophil h production was determined by a previously described ' '-dichlorofluorescein flowcytametric assay. _+ amean _+ s.e.m, pg/ml blood; brelative fluoresenee (% of baseline); caortic and femoral artery flow (% of baseline); *p < . vs. baseline; "p < . vs. baseline. a significant elevation of paf was observed in ischemic hind-limb effluent blood at min. after release of the aortic ligature during the repeffusion phase, as compared to baseline levels. in addition, pmn h production was increased by . -fold above baseline values by hour after ligature release during the reperfusion phase. both of these elevations were transient and returned toward baseline by hours post-isehemia. tatar occlusion of hind-limb flow was achieved as evidenced by the absence of aortic or femorat flow at rain. post-ischemia, however after release the ligature a significant reactive hyperemia was observed by mln. into the rapeffusion phase. histolog[c examination of reper[used gastrocnemius muscle revealed moderate pmn infiltration into the interstitium. in conclusion, these data indicate that paf is released into the circulation during repeffusion, and is likely involved as a mediator in the observed pmn oxidative burst activity, thereby contributing to reperfusien injury. following thermal injury and infection granulocyte function ts abnormal. to elucidate the mechanism by which thermal injury and infection affect the granulocyte's ability to polymerize and depolymedze actin, we serially measured f-actin levels in granulocytes from burned patients (mean age , +_ . years, mean burn size . % _+ . %) during the first s weeks post injury. six of the patients had infections during the course of the study, (septicemia, wound invasion and pneumonia). actin levels in granulocytes from eleven healthy volunteers (mean age years) were measured repeatedly and served as controls. lysecl white blood cell preparations were brought to c and incubated with n-formyl-met-leu-phe (stim) or with dulbecco's phosphate unbuffered sellne (unstim). the cells were concomitantly stained and fixed with formaldehyde, lysoleclthln and fiuoresceln phafioidin. actin depolymedzation (depol) was measured by incubating stimulated cells at °c before the stain-fixative was added. baseline (base) f-actln levels were assessed by adding stsln-fixatlve to icecold unstimulated cells. fluorescence was estimated in a facscan and expressed as ilnesr mean channel fluorescence_+ sem (mcf). figure displays granulecyle fectln levels in infected and uninfected patients as compared to controls. f-actln levels were consistently lower in control cells than in those from burned or burn-infected patients under all measured conditions. granulocytes from infected burned patients demonstrated a significant decrement in their ability to depofymerlze f.actin compared to both uninfected burned patients and controls, while there were no significant differences between infected and ,~ uninfected patients in the baseline, unstlmuleted and stimulated conditions. those results indicate la that grsnulocytas from burned and bum-infected patients contain higher levels of polymerized actln than ~ , s control cells. in order to study tumor necrosis factor (tnf) receptor sensitivity in septic critically ill patients we investigated blood samples of such people in reaction of leucocyte migration inhibition. migration of their polymorphonuclear leucocytes (pmns) was studied with stimulation with human recombinant tnf in concentration of . u/ml (recommended by manufacturer is the range of - o/ml) and without such. ten healthy blood donors formed control group. the results obtained showed diminished pmn reactivity to tnf in patients (migration inhibition was absent) oscaring with significantly increased migration ability of their pmns ( . % of that in control group). at the same time normal pmns in control group did show migration changes upon tnf stimulation. considering all the above we come to a conclusion that externally added tnf fails to activate pmns in critically ill patients more than they are by their endogenous tnf. moreover, this tnf no longer serves a positive chemotactic factor for such pmns. these findings may suggest that in critically ill septic patients reactivity of pmns to tnf is deeply altered. tnf receptors of pmns are either exhausted as such by excessive stimulation with endogenous tnf or further transmission of their message is impossible due to "fatigue" of the cell's activation mechanisms. we express our gratitude to reanal factory of laboratory chemicals for generously providing us with a tnf com~rcial sample. ~-sanguis medical, ekaterineburg russia; s-urals med.lnst. activated neutrophils infiltrating the local site of inflammation following trauma release high amounts of destructive lysosomal enzymes into the extracellular space. cytokines were discussed to be involved in regulation of this early process. the task of this investigation was to evaluate the possible regulatory role of interleukin- (il- ) and its potential immunosupressive opponent, the transforming growth factor-&, in regulation of neutrophil degranulation. we analysed the concentration of the al-proteinase-inhibitor complex of the lysosomal elastase as marker for the degranulation of neutrophils as well as the levels of il- and tgf- in the plasma probes of patients undergoing multiple trauma and severe surgeries. the time courses of il- and elastase were found to be highly correlated, wheras the concentrations of the cytokine tgf-e~ were found to be not significantly altered in comparison to the control group. this close temporal correlationship was confirmed by investigation of fluids derived from sites of inflammation. interstingly, the inhibitory potential (~zcproteinase inhibitor, antithrombin iii) was dramatically reduced in the early inflammatory phase. to prove this in vivo findings, the effects of il- and tgf-i~ on the degranulation of isolated human neutrophils of healthy donors was investigated in vitro. pathological high concentrations of rhll- up to u/ml (as detected in fluids derived from local inflammatory site) were found to be capable to induce a significant release of lysosomal elastase in a concentration-dependent manner, whereas the degranulation of neutrophils was uneffected by tgf- . in conclusion, these data suggest a contribution of il- in regulation of neutrophil activation at sides of inflammation. the immunosuppressive cytokine tgf-i&~ seems to have no direct regulatory effect beside its described chemotactic function on neutrephils. postirradiation chan~es of adhesive properties arid supercoiled nucleoid dna structure of blood leukocytes were studied in macaca nemestrina andrats. the dynamics of membrane chan~es after nonlethal irradiation of rats demonstrated the temporary increase of the leukocyte adherence at h followed by return of this parameter to normal levels at h. after lethal irradiation of both animal species the increase in adhesive leukooytes fraction was detected as early as at h. this hi~her index persisted until the end of experiments ( days). the early ( - h) temporary loosin~ of supercoiled dna structure was demonstrated in the leukocytes of nonlethally irradiated animals. this phenomenon seems to be connected with the lymphocyte fraction chan~es. this process was not dependent on altered adhesive properties of leukocyte membranes. the membrane chan~es of leukocytes preceded decondensation of supercoiled dna after lethal irradiation of animals, in this case loosin~ of supercoiled dna pro-~ressively increased at h and at the later terms of postirradiation period. the systemic inflammatory response syndrome (sirs) involves many inanunological reactions of the host including acfivatinn of inflammatory mediator cascades and depression of cellular reactivity in t-lymphecytes ( ). there are reports of nentrophil dysfunction in inflammatory disorders of the skin ( ), are there dysfunctions concerning the unspecific host defense in sirs, as well? in this study, we examined the reactivity of neutrophil granolocytes from patients suffering from sirs. twenty-one patients (apache ii-score ± ) with diagnosis of sirs entered the study. granulocytes were prepared as reported previously ( ) . in parallel, granulocytes from healthy individuals were tested. two granulocyte functians were studied in vitro: . migration of the ceils in a boyden chamber through a filter matrix following stimulation with different receptor dependent stimuli (c a, intefleukin- , platelet-activating-factor, leukotrien b , fmlp). . release of glucuronidase following stimulation with the aforementioned activators. the results demonstrate, that the release of -glucuronidase in patients suffering from sirs was comparable to the enzyme release of granulocytes prepared from healthy individuals. each stimulant induced release of p-glucuronidase in a characteristic dose dependent fashion. all granulocyte preparations from the healthy donors showed a positive chemotaxis response in the migration-assay. in contrast, only ten out of twenty-one patients had granulocytes migrating after stimulation. the two groups of patients displaying reactive or non-reactive granulocytes differed clinically: the nonreactive group consisted of patients with multiple organ failure ( / ) and nonsurvivors ( / ), whereas / patients in the reactive group survived. thus, the in vitro chemotaxis of granulocytes is impaired in a subgroup of patients with sirs. this defect of the non-specific host defense may contribute to poor prognosis and outcome of these patients. dermatol. : - , klinik ffir an~isthesiologie und operative intensivmedizin der cau kiel, schwanenweg , kiel, germany. objectives of the study: major emphasis has been given to the analysis of interactions of antibiotics with microorganisms. effects of antibiotics on cells of primary host defense mechanisms, such as the neutrophils, are less well known. therefore, attention has been focused on clindamycin, a member of the lincoseamide family. materials and methods: the effect of clindamycin (i -i ~g/ml) on granulocyte functions (healthy volunteers) such as random migration, chemotaxis (agarose method), ingestion (radiometric assay), superoxide (cytochrom c reduction) and hydrogen peroxide production (phenol red oxidation), lucigenin-and luminol-amplified chemiluminescence (luminometry) and degranulation (turbidometry with micrococcus lysodeicticus) were investigated in vitro. results: motility and degranulation were inhibited, ingestion of saccharomyces cerevisiae, zymosan-induced lucigenin-and luminol-amplified chemiluminescence, superoxide and hydrogen peroxide production were stimulated in a dose dependent fashion. conclusion: clindamycin has granulocyte function modulating properties. recognition of immunomodulating effects of antibiotics may have therapeutic significance, especially in patients with long-term antibiotic therapy or immune deficiencies. the intense muscle activity (ea) of rats resulted in increase of neutrophil influx in muscles during the recovery. we investigated neutrophil proteinases involvement in neutral proteinases balance of skeletal muscles by na. the rats were submited to swim with the load ( % of body mass) till exhaustion. immediately after na the neutrophil antiserum was injected i.p. to rats of experimental group. saline was injected to control animals° injections were repeated in h of the recovery and cytosol proteolytic activity (ph . ; fitc-casein) was determined. isolated soleus muscles were incubated also in vitro and proteolytic activity of incubation media was measured. it was found that there was - -fold proteinases activity increase in cytosols of all investigated muscles (soleus, white and red portions of quadriceps) of control animals by h of the recovery (the comparison was done with the sedentary rats). in h cytosol proteolytic activity decreased and then increased again by h of the fast. antiserum injections resulted in relible decrease of the proteolytic activities at every investigated time. when incubating m. soleus in vitro the activities of proteinases in incubation media turned out reliably less if soleus muscles were isolated from the animals to which antiserum was injected. the conclusion is that neutrophil proteinases can be involved in the balance of rat skeletal muscle neutral proteinases after ~a. a lot and new clinical problems complicating the outcome of polytrauma, burn and septic patients in surgical intensive care units, have arisen as the care improvement prolonged the patient's survival: a progressive degradation of organ and system functions often develops, usually making its first clinical appearance by ards, followed by the other organ failure (mof) and sepsis symptoms. the clinical picture is polymorphic, the end result of a complex systemic pathophysiological reaction trigg~ed off by trauma consequences (tissues disruption, hypo~xygenatiun and necrosis). nowadays there is not a preventi~ or specific therapy to lower the mortality rate ( - %) and-'mdy-a~ early, aggressive surgical approach .-evacuating haematomas, stopping bleeding, toileting all septic, necrotic foci and restoring anatomic continuity-, seems to be of some help this complex clinical entity has not an univocal denomination yet. the proper labelling of an illness should come from the full understanding of its pathopysiology and suggest the proper treatment choice. clinical and experimental studies demonstrated that pathophysiologic mechanisms involved in the past-traumatic illness, share the same anatomo-pathological elemem: the interstitial edema, due to a generalised endothelial micro circulatory injury. this alteration, as constantly seen in polytrauma patients, develops in a few hours after trauma as a consequence of the deregulation of the homoeostatic and immune mechanisms. in fact the overproduced oxygen free radicals and r~ombinam cytokines (il ,tnf), together with the complement degradation fragments, the proteolytic enzymes and many other mediators are all strongly h~l ~ ,_he e,,j,yheha! ceils. our~osect, atim~,-bnsed on examination of autopsical specimens from polytraanm patients, showed that such endothelial damage, supporting the interstitial edema, is widely and simultaneensly distributed, ensues shortly arer trauma and shows its effects in different organs at different times, only because each apparatus has different fimctienal reserves: the lung is the first organ to fail just because its ah, celocapillary membrane is one of the most delicate bodily structure, and its function is irroplace~le. we think it will be of a great help, in planning a preventive therapy, to chose a denomination focusing the physician's attention on the earl)" generalized endothelial injury and its effects, as in trauma patients it is present -even if latenflysince the first few hours. we would like to see the generalised endothelial microcircolatory injury properly highlighted when considering the best definition and the optimal nomenclature for the post-traumatic s mdrome. the presence of interleukin (il)- in bronchoalveolar lavage fluid of critically ill patients correlates clinically with the development of the adult respiratory distress syndrome lards), and inhibition of il- in animal models can attenuate lung injury. collectively, evidence to date suggests that il- attracts and activates neutrophiis (pmn), which are then responsible for the capillary leak of ards. however, an alternative explanation is that il- is directly toxic to the endothelial cell (ec). in this study, we have hypothesized that il- can disrupt endothelial integrity independent of pmn. meth ods: human umbilical vein (huv) ec monolayers were cultured to confluency on collagen-coated micropore filters. to assess ec integrity, .albumi n leak was quantitated by measuring the counts which crossed the monolayer, using a gamma counter. il- (lpg/ml) was incubated in the culture medium with .albumi n for hrs. the il- dose was not cytotoxic. to determine the involvement of protein synthesis in this process, selected monolayers were pretreated with cycloheximide (ch) prior to .- addition. statistical analysis was performed using anovmfisher plsd. we have previously shown that platelet activating factor (paf) enhances cdt expression and primes pmn's for subsequent generation. both are important steps in pmn mediated injury and are assumed to occur in concert. following major trauma non-specific pmn inflammation is activated, however, unbridled systemic pmn activity needs to be minimized. since circulating catecholamines are high early post-injury, we hypothesised that they downregu/ate cd expression and pmn priming via the [ adrenergic signal transduction pathway. methods: normal human pmns were primed with paf ( ng/ml for min) or pre-treated with - m of isoproterenol (i) or forskoklin (f) for rain and then primed with paf. cd expression was measured by flow cytometry (fig.l) and -generation in response to -rm fmlp was determined as sod inhibitable reduction of cytochrome c ( fig. holler** and georg w. bornkamm* lymphocyte-endothelial interactions are crucial for various immune responses, including cytokine driven inflammatory processes. protein kinase c (pkc)-inhibitors on the other hand are discussed as potential cytokine antagonists. in the present study we investigated the influence of the pkc-inhibitor gf x on cytokine-and endotoxin induced expression of intercellular adhesion molecule (icam- ) and on adhesion of lymphocytes to cytokine activated endothelial cells. we found that tumor necrosis factor alpha (tnfo -and lipopolysaccharide (lps)-induced icam- expression on human endothelioma celts (eahy ) were unaffected by the pkc-inhibitor and thus appeared to be independent of pkc activation. in contrast, gf x significantly reduced icam- expression induced by interferon-y (ifn-?) and interleukin- (il- ). the functional relevance of these findings was evaluated in an adhesion assay using human umbilical vene endothelial cells (huvec) and peripheral blood mononuclear cells (pbmc). in fact, the ifn-? and il- induced adhesion of pbmc to cytokine treated huvec could be downregulated by the pkc-inhibitor, whereas tnfc~-and lps-mediated adhesion was not influenced. additionally, the il- driven icam- expression on eahy cells as well as the il- induced adhesion of pbmc to huvec was found to be tnf-dependent, since both effects could be inhibited by an anti-tnf monoclonal antibody ( f) . these in vitro data further support the idea of examining pkc-inhibitors, such as gf x, for their biological relevance in cytokine related dysregulations. seiffge, d., bissinger, t., laux, v., during inflammation there are some key processes, which occur in the microcirculation: the release of mediators from various cell types, the migration of inflammatory cells towards a chemotactic stimulus in the tissue, the expression of adhesion molecules on different cells, and the extravasation of plasma proteins. the aim of the present study was to elucidate the mediator induced interaction of leukocyte adhesion and plasma leakage in postcapillary venules. using an analogous video-image analysing system we have studied the effect of different mediators on leukocyte adhesion and macromolecular permeability in the mesentery of the rat. the increase in permeability was measured as changes in optical density. we found that topical administration of leneotriene b (ltb , x " tool/l) or intravenous injection of interleuldn- (il- , - iu/kg b.w.) and lipopolysaccharide (lps, mg/kg b.w.) resulted in a significant extravasation of fitc-labelled rat serum albumin (fitc-rsa) in venules but not in arterioles. we could correlate the changes in vascular permeability with a locally increased number of rolling and sticking leukocytes in venules. both effects were dose dependently inhibited by different drugs. pentoxlfylline inhibits lps-indueed fitc-rsa extravasation and leukocyte adhesion at a dose of mg/kg b.w., superoxid-dismutase (sod, . iu/kg b.w.) was able to decrease the ltb effect, and the immuumodulating drug leflunomide (hwa ) exerted inhibitory effects on il- -induced permeability at a dose of mg/kg b.w.i.v. the obtained results demonstrate that lps, ltb or il- induced extravasation of fitc-rsa is mediated by activated leukocytes and can be deminished following administration of different drugs. platelet-endothelial cell adhesion molecule-i (pecam-i), a member of the immunoglobulin superfamily, is constitutively expressed at high levels on the endothelial cell surface. in vitro data have suggested that pecam-i functions as a vascular adhesion molecule, specifically in neutrophil transmigration across the endothelium. this current work is the first demonstrating the in vivo role of pecam- in neutrophil migration. blocking antibodies to human pecam- , in which the antibodies are crossreactive with rat pecam- , were able to block the movement of neutrophils into the rat lungs after igg immune complex deposition. furthermore, when human foreskin was transplanted into mice with severe combined immunodeficiency and the site injected with tnf-alpha, anti-pecam-i blocked neutrophil emigration into the dermal interstitium. it has already been established that neutrophil recruitment is dependent upon selectin mediated rolling, followed by firm adherence that is icam- / integrin mediated. these data suggest, for the first time, that a third endothelial adhesion molecule (pecam-i) is involved in the coordinated recruitment of neutrophils in vivo. to test whether trauma causes generalized activation or priming of pmns, cdi adherence receptors were measured with iinmunomonitoring in whole blood after lps stimulation ex vivo. anesthetized (fentanyl) mongrel pigs ( - kg) were subjected to % arterial hemorrhage + soft tissue injury and after liar, resuscitated with all the shed blood + supplemental fluid. blood was collected at hr intervals from unanesthetized animals with indwelling catheters, pmns were counted, and lps was added ( , , , i.tg/ml) ex vivo. after hr incubation at - °c, %cd (+) pmns were determined with fitc-ib and flow cytometry from mean channel fluorescence histograms. ± # p< . vs baseline * p< . vs sham $p< . vs no anesthesia these observations provide direct evidence for time-dependent changes in pmn priming following major injury because cd expression was depressed for at ]east hr after trauma relative to sham but by hr, was enhanced, relative to sham, and because fentanyl anesthesia at hr had a greater effect on cd expression in trauma vs sham. neutrophil (pmn) adhesion to vascular endothelial cells (•c) is a key element in the inflammatory response and tissue injury. inflammatory mediators such as lps (exogenous) and tnf (endogenous) can promote pmn-ec interaction which is believed to be responsible for capillary leakage and subsequent organ injury. however, the mechanism of this injury remains unclear.we hypothesised that the mechanism of tissue injury is due to ec necrosis with release of toxic products and that activated pmn are responsible. human pmn were obtained from healthy donors, separated by density gradient, and activated with lps ( ng/ml), tnf( ng/ml), and lps/tnf( ng/ ng/ml). cultures of the human ec tine(ecv- ) were used as surrogates of the microvasculature, were exposed to either lps, tnf, lps/tnf and pmn activated with lps, tnf, lps/tnf and incubated for , , , and hrs. ec necrosis was assessed by a cr release cytotoxicity assay. pmn activation was assessed by cd lb receptor expression and respiratory burst activity hr _+ . -+ -+ . _+ _+ . _+ _+ . _+ . hr + . _ _+ . _+ _+ _+ " +_ +-- . " lghr - . _+ +_ - " o:fo , " ~ +- . * hr _+ . - -+ +_ * _+ _+ * _+ _+ " data = ec % necrosis mean_+sd stats: student's t-test with significance (*) set at p< . vs control. ( our previous studies have indicated that despite the increased cardiac output and maintenance of tissue perfusion, hepatoceliular dysfunction occurs during early sepsis. nonetheless, it remains unknown whether vascular endothelial cell function (i.e., the release of endothelium-derived relaxing factor/nitric oxide) is depressed under such conditions and, if so, whether endothelial cell dysfunction also occurs at the microcirculatory level. to determine this, rats were subjected to sepsis by cecal ligation and puncture (clp), following which these and corresponding shams received ml/ g bw normal saline. at hr after clp (hyperdynamic sepsis) or sham operation, the thoracic aorta was isolated, cut into rings, and placed in organ chambers. norepinephrine (ne, xi - m) was used to achieve near-maximal contraction. responses for an endothelium-dependeut vasodilator, acetylcholine (ach, via nitric oxide), were determined. in additional studies, the small gut was isolated at hr post-clp. after pre-contraction of blood vessels in the isolated gut with xl m ne, vascular responses to ach ( x m) and an endotheliumindependent vasodiiator, nitroglycerine (ntg, xl - m), were determined. total vascular resistance (tvr, mmhg/mi/min/ g) was then calculated as pressure/ perfusinn rate. ach-induced relaxation (%, n= /group) in the aortic rings were: ach lxl i~s, st-in ~ ~ significantly at hr post-clp (i.e., increased *p(o vs. sham; n- per group. tvr) in the absence of any changes in ntginduced relaxation (fig. a) . thus, the vascular endothelial cell dysfunction observed in the aorta in early sepsis also occurs at the microcirculatory level. introduction: the cytokine-mediated adherence of leulcooytes to vascular endothelium is considered as an early step in the cascade of pathologic reactions culminating in the "systemic inflammatory response syndrome" (sirs); the purpose of this study was to evaluate the influence of interleakin- on leukooyteendothelial cell-interactions and microoirculation in the liver after hemorrhagic shock by means of intravital microscopy. methods: in anesthetized female sprdrats co.w. - g) shook was induced by fractionated withdrawl of arterial blood within rain and maintained for h (map at mm hg, cardiac output % of baseline). rats were adequately resuscitated with % of shed blood and twice the volume in ringer's solution additionally. following h of reperfusinn (map > mm hg, co > % of baseline) the microcirculation in liver lobules was examined by intravital fluorescence microscopy after labelling of leukocytes. continuous administration of il-lra (synergen, boulder, colorado, mg/kg/h) was started at different time points in a randomized and blinded manner. the animals in group p (n= ) received the il-lra as pretreatment beginning min prior to shock induction. in the group t (n= ) the application of il-lm started at the beginning of the reperfusion period with a bolus injection of mg/kg and was followed by continuons administration of mg/kg/h. the control group c (n= ) received equal volumes in nac , %, the sham-operated group s (n= ) was not exposed to shock. results: macrohemodynamics were comparable in all shook groups. the increased percentage of permanendy adherent leukocytes after hemorrhagic shook (s: , % + , %; c: , % _+ , %) was significantly reduced by pretreatment or treatment with il-lra (p: , % -+ , %; p< . , t: , % -+ , %, p< . , anova). temporary adhesion of leukocytes was unaffected by application of il-lra. liver microcirculation measured by volumetric blood flow in liver sinusoids and sinusoidal diameters was impaired after hemorrhagic shock in all groups and was not affected (c: iam /s + um /s, p: llm /s + }am /s, t: ams/s -+ lam /s, s: am /s -+ am /s). di.seu~sinn: the results demonstrate that permanent adherence of leukocytes to endothelium is in part regulated by il- . pathological adherence could be reduced by application of illra, even given at die time of resuscitation. the effect of ll-lm on permanent adhesion is a specific event and might be caused by reduced expression of specific receptors on sinusoidal endothelial cens and leukocytes. objectives of the study. the adhesion of activated neutrophils (pmn) to endothelial ceils (ec) and the concomitant production of reactive oxygen metabolites (rom) initiates organ damage after trauma, sepsis, shock and organ reperfusion. aien of this study was to investigate the effect on adhesion and rom production of the highly water-soluble, membrane-permeable and physiological ascorbic acid (asc). materials and methods. adhesion of pmn to nylon fiber (cell count) and simultaneous rom production (chemiluminescence-cl-response) were measured up to retool/ asc as well as adhesion, rom production and ec damage (lllln-release from labeled ec) of endotoxin-activated pmn to cultered ec moanlayers. in an in vivo animal model (sheep with lung lymph fistulas) the effect of asc ( g/kg bw bolus, followed by . g/ kg-h infusion) on the endotoxin-induced ( . ixg/kg bw) neutropenia (cell count), lung capillary permeability damage (lung lymph protein clearance) and rom production of neutrophils (zymosan-induced cl response) was measured. results. asc scavenged rom dose-dependently during adhesion of pmn to nylon fiber (p< . at mmol/l asc), adhesion itself was unchanged. during the activated pmn/ec interaction asc scavenged rom (p< . at mmol/l asc) and reduced the adhesion dose-dependently (p< . at mmol/l asc); ec damage was also reduced (p< . at retool/ asc). in the in rive model asc increased the endotoxin-induced blood pmn decrease (p< . ), decreased the protein clearance (p< . ) as well as the zymosan-induced rom production (p< . ), indicating the asc-mediated reduction of adhesion, rom production and lung tissue damage processes. conclusions. by in vitro and in rive experiments ascorbic acid reduced the adhesion-and rom production-initiated tissue damage. therefore, i.v. administration of ascorbic acid is recommended for oxidative stress-associated states after trauma, sepsis, shock and organ reperfusion. for neut rophi l-accumulat ion and activation. we investigated the influence of or to the activation and the expression of lecam-i and cdiib,cdi on neutrophils and lymphocytes. methods: from blood samples (n= ) all white blood cells (wbc) and neutrophils (nc) were isolated and cultured. or were produced via the xanthine oxidase/hypoxanthine system. after , , , , and minutes a giemsa-staining to determine the granulation of neutrophils (n: normal, r : reduced ) and a facs-analysis with monoclonal antibodies detecting cdiib,cdi and lecam-i was performed. results: under the influence of or a degranulation of neutrophils starting at min was observed in wbc-cultures (n/r: min / , min / , min / , min / , min / ). these data were confirmed in the dot-plots of facs-analysis. only in wbc-cultures or induced a significant increase of lecam-i expression on neutrophils up to min followed by a decrease to normal values at min. lecam-i on lymphocytes disappeared totally during the observed period. cdllb,cdl -expression was not altered. conclusion:increased lecam-i expression on neutrophils due to or could enhance the 'rolling' of neutrophils along the endothelium which is a prerequisite for neutrophil sticking and migration. further or are able to activate neutrophils without endothelium. these changes seem to be mediated by other wbc. introduction. multiple organ failure (mof) has been hypothesized to be the result of an excessive uncontrolled autedestructive inflammatory response. since the complement system is an important mediator and initiator of the inflammatory response, interruption of this cascade could theoretically lead to an attenuation of mof. in order to test this hypothesis we evaluated the response of c -delicient mice in a model of zymesan indt~ed mof. materials and methods. c -deficient b d /oid and c -sufficient b d /new mice were used in this study. on day all mice received an intraperitoneal injection with zymosan suspended in paraffin in a dose of mg/g body weight. between day and , biological parameters (temperature, body weight and clinical condition) were measured daily and mortality was monitored. clinical condition was assessed by blindly grading the degree of lethargy, conjunctivitis, diarrhea, and ruffled fur of each mouse on a two point scale (maximum score= ). on day all surviving mice were sacrificed and relative organ weights of lungs, liver, spleen and kidneys (relative organ weight= (organ weight/body weight)x ) wore calculated. earlier experiments with our model have shown a good correlation between histological organ damage and relative organ weights. statistical analysis of biological parameter was performed using the koziol curve analysis. analysis was divided in an acute phase (day - ) and a late phase (day - ). relative organ weights were analyzed using wilcoxon's test and mortality rate using fischor's exact test. results. all zymosan injected mice showed a typical triphesic illness. deterioration of the clinical condition as indicated by the symptom score and the decrease in temperature and body weight in the acute phase were all significantly lass severe in c deficient mice (all p< . ). in the late phase no differences could be noticed in the courses of biological parameters. overall mortality was / ( %) in c deficient mice and / ( %) jn c sufficient mice (p= . ), a difference mainly due to a difference in the acute phase. organ damage assessed as the relative organ weights did not show any statistical differences for any organ between both strains. conclusion. complement factor c appears to play an important role in the acute hyperdynamic septic response in this model but deficiency of c could not prevent organ damage in the late mof phase. this suggests that other factors could be more important in the development of the inflammatory response leading to mof. proinflammatory cytokines are thought to play a critical role in the pathophysiology of multiple organ failure (mof). in mice, zymosan-lnduced generalized inflammation (ztgi) leads to mof. therefore we performed a sequential study into plasma levels of, and macrophage production capacity for, four cytokines during the development of mof in the zigi model. male young-adult c bl/ mice received zymosan ( mg/g body weight) intraperitoneally. groups of animals were killed after , , , and h and subsequently at each day until day . plasma was collected and peritoneal macrophages were isolated and cultured overnight with or without lipopolysaccharide (lps). interleukin -ct, and - (il-lc~,~,), and tumour necrosis factor-o~ (tnf-c were measured in plasma and culture fluid by means of a ria (detection limit . ng/ml). interleukin- (il~) levels were assayed using the b hybddoma cell proliferation assay. zymosan induces a three-phase disease in mice. after an acute phase the animals recover. around day , they start to develop clinical signs which resemble mof. plasma tnf-~ peaked within h after zymosan injection and disappeared within h. from day onwards, tnf levels started to rise again. plasma il- behaved almost similarly in the acute phase, but in the mof phase plasma il- remained low. no circulating il- could be detected at any time point. macrophage lps-stimulated production of il-lcq il- ~ and tnf--c~ was suppressed immediately after zymosan injection. production of il- and tnf-~ was normalized within h, while production of il-lc~ remained lower than that in macrophages from untreated control mice. only at day did production of il-i~ reach control values. il- production was higher than control values from day onwards. il production was similar to that of ili-il the production of tnf-ct was strongly elevated between days and and again during days to . the development of mof-like symptoms during zlgi in mice is accompanied by increased plasma levels of tnf-ct without enhanced il- or il- . also, the ability of macrophages to produce excessive amounts of il- and tnf--~, as well as the suppressed capacity to produce il-lcq could be important mechanisms in the pathophysiology of mof. when conjugated to an asialoglycoprotein, dna and oligonucleotides are specifically taken up by the hepatocytes via the asialoglyccprotein receptor which is unique to the liver. human asialoglycoprotein (~ -acid, asgp) was derivatized with low molecular weight poly(l)lysine(pll) and complexed with antisense dna's (as) complementary to the ' region of the il- gpl receptor. the antisense were '-agtttagggatgagg- ' (asl), '-atcttcatcttctgaat- ' (as ), '-aagtgaatgattaaaacact- ' (as ), '-aaacctttataggcg- ' (as ), and '-cgttctacaactgcaacgt- ' (as ). using hepg , the biological effects of these antisense complexes on the high affinity il- receptor were evaluated by scatchard analysis, cellular proliferation, and acute phase protein expression by radioimmunoprecipitation and two dimensional gel electrophoresis. scatchard analysis demonstrated that high affinity receptor expression was inhibited by incubation of cells with asgp-pll-asi for h. underivatized asl was less effective and the complex, asgp-pll-as , had minimal effects on high affinity binding. when the cells were treated with the conjugates and stimulated with il- (i units) asgp-pll-asi alone showed a dose dependent ( .i- . ~m) inhibition of ss fibrinogen synthesis. two dimensional gel electrophoresis showed that expression of other acute phase proteins was also blocked. these results indicate that the targeted delivery of antisense molecules via conjugates recognized by the asialoglycoprotein receptor can block the cytokine stimulated acute phase protein response in hepatocytes, this approach may be relevant to the therapeutic management of patients with severe injury and sepsis. it has been established that immune cells are able to express neuropeptide genes and to release products that were considered to be of neuroendocrine origin. we have shown that proenkephalin (penk), a neuropeptide encoding gene, is expressed in lymphoid cells in culture. to study the physiological significance of these observations we have used the model of experimental endotoxemia. in this model, a disease state is induced by bacterial lipopolysaccharide (lps), that activates the immune system, the adrenocortical axis and the nervous system. we found that the expression of penkmrna is markedly enhanced in vivo immediately after lps injection both in the adrenal glands and in the lymph nodes. in situ hybridization analysis combined with immunohisto-chemistry indicated that the induced penk expression is confined to macrephages within the lymph nodes and chromaffin cells in the adrenal medulla. furthermore, this expression in lymph nodes is modulated by ligands of the adrenergic system. our results strongly support the notion that immune derived opioids participate in the bidirectional communication between the nervous and immune systems. of neurology hadassah university hospital, jerusalem , israel. objectives of the study: multiple-organ-failure is recognized as the most severe, and often lethal, complication after multiple trauma. however there is no adeqate animal model available. our goal was to develop an animal model, in which reproducable irreversible failure of parenchymal organs is achieved in the late phase after insults in the early phase (trauma). materials and methods: l female merino-sheep were included (mean weight: kg). day : hemorrhagic shock (mean arterial pressure (map) mmhg for hrs.), closed femoral nailing (ao-technique), day - : bolusinjection of endotoxin (et) ( , ~tg/kgbw) und zymosan-activated plasma (zap) ( ml) every hrs., day - : observation. bronchoalveolar lavage (bal): day , , . the course of representative parameters of organ function was documented: cocardiac output (i/min), svr -systemic vascular resistence (dyn ~ s cm- ), pap -putm.art.pressure (mmhg), pap -arterial oxygen pressure (mmhg), bill -bilirubin (;xmov ), crci -creatinin clearence (ml/min) statistics: data as means+sem, *significant from baseline (wileoxon test; p< ) results: baseline day day day day heart: co , _+ , , _+ , , _+ , , _+ , * , _+ , * svr _+ + _+ +_ " +- " lung: pap , _- , , _+ , " , +- , " , + , " , +- , ' pap , + , , +- , , _+ , , +- , , +_ , * liver: bill , _+ , , _+ , ' , _+ , ' , _+ , " , _+ , " kidney:crcl , +_ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , _+ , , + , , + , histologic specimens showed all signs of fulminant mof. combination of hemorrhagic shock, femoral nailing, et und zap (insults in the early phase) lead to an irreversible organ failure in the late phase. prostaglandin e (pge) levels are elevated by trauma, shock or sepsis and can profoundly affect the immune response. pge is produced by many cell types including fibroblasts, macrophages, monoeytes, follicular dendritic cells, and epithelial cells and is induced by il-i, bacterial lps, components of the complement cascade, tnf, il- and crosslinking of surface fc receptors for igg, iga and ige. our research has shown that pge inhibits b cell activation (specifically enlargement, class ii ~c and fc~ rii expression), proliferation, igm and igg responses, t cell proliferation, and il- synthesis in the mouse model. in contrast, pge greatly promotes class switching to ige,the isotype responsible for type i allergic hypersensitivity. thus, our model mirrors th~ general immunosuppression and elevated ige titers of the trauma or sepsis patient. pge increases the number of cells secreting ige and iggl, acts on surface igm positive b cells, synergizes with il- and lp$ to induce preswitch germline transcripts, and induces more rapid expression of mature vdj~ mp~a than in eontro~ pge intracellular signalling occurs through cyclic adenosine monophosphate (camp) levels and can be mimicked by camp-inducing agents and blocl~ed by an inhibitor of campdependent protein kinase a. pge action requires de novo protein synthesis and candidate pge-inducible regulatory proteins have been identified by d gel eleetrophoresis. thus, pge inhibits a number of immune mechanisms while promoting ige production. a deeper understanding of pge immune regulation may lead to more effective treatment of immune perturbations as sequelae of trauma, shock or sepsis. during infrarenai aortic surgery mesetueric traction (re.t.) results in prostacyclin (pgi:) release and consecutively in hemodynamic disturbances (decreased systemic vascular resisteace, mean arterial pressure; increased cardiac output, heartrate). these symptomes are bypassed by cyclooxygenase inhibition. hemodynamic symptoms vanish after - rain even without cyclooxygenase inhibition although pgi levels remain elevated. to study the endocrine vasopressor system in a prospective double blinded protocol, we investigated patients undergoing major abdominal surgery as compared to ibuprofen ( rag, i.v.) pretreated (ibu) patients. the surgeon applied m.t. in a uniform fashion. we chose a general anesthesia combined with a supplemental thoracic epidural anesthesia. at the points in time , , , , , , , rain after and before (to) mesentzrie traction we determined the plasma concentrations (pc) of -keto-pgf~o~pr~, epinephrine, norepinephrine, dopamine, renin, aldosterone, adh and cortisol. pc of -k-pgf~,tp~, peaked minutes after m.t. ( _+ , ibu: _+ , to: +i ng/l) and declined monotonously over h ( +_ , ibu: _+ ng/ ). catecholamine pc "s did not exceed the reference range during the observation period. reninpc peaked after rain ( _+ , ibu: + , to: -+ /~u/ml); aldosteronc also presented a maximum after rain ( + , ibu: -+ , to: +- pg/ml), whereas cortisol demonstrated irrespectively of circadian rhythms a maximum h after m.t. ( +_ , ibu: -+ , to: +_ ~g/ ). adh pc peaked min after m.t. ( + , ibu: -+ , to: +_ pg/ml) and showed analogously to -k-pgft~j~ pc a monotone decline over the observation period. our data demonstrate a counteractive reaction to pgiz mediated vasodilation via adh secretion. the second regulative is the renin-angiotensin-aldosterone system (raas), which is activated min after m.t., the aldosterone pc does not paratlel the cortisol pc, which peaked post operafionem in both groups, probably due to the end of anaesthesia. a regulative release of catecholamines could not be documented. the activation of adh and raas after mt is not a hormonal response primaryly related to surgical trauma and/or stress but a counterregulation to systemic vasoditafion induced by prostacyclin. although adh and raas support systemic circulation, angiotensin and vasopressin may compromise local organ blood flow (e.g. splancimic vascular bed). insfitut f. klin. chemic, anaesthesiologie ~, chirurgie l*, univ. ulm, elm, expression of c-fos protein in rat brain following occlusion of superior mesenterie artery. takanobu there is general agreement that neurologic abnormalities are seen in sepsis. the aim of this study is to examine what effect does the brain receive in case of sma occlusion by immunohistochemistry using antibody to c-fos, an immediate early gene, which is recently recognized as a genetic marker of activated neurons. moreover, we investigated the correlation between c-fos induction in the brain and plasma endotoxiu level. rats of them received sma clipping and others wee used as control. control and treated rats at , , , hours were perfused and fixed. the brain were sectioned at pm and stained by abc method using c-fos antibody. plasma endotoxin level of rats were measured at , , , , hours after the treatment by chromogenic limulus method. immunohistochemical study showed scarcely no immunoreactivity in control rat brain. in treated rat brain, the significant expression of c-los was detected in specific nuclei including the habenula, some hypothalamie nuclei, amygdala, locus ceruleus and nucleus tractus solitarii. such immunoreactivities were increased in time curse, which well corresponded plasma endotoxin levels. the mean plasma endotoxin level of , , , , hours after the treatment were . ± . , . _- - . , . _+ . , . ± . and . ± . pg/ml, respectively. the results indicate that limbic and hypothalamic-brainstem systems are involved in sma occlusion, and suggest that such neuronal actival.jon may precede the elevation of plasma endotoxin icy.el. systemic vascular resistance and increased cardiac output accompanied presumingly by a increased pulmonary shunt (qs/qt). this response is induced by prostacyclin (pgi ). we examined oxygen transport after traction on the mesentery root and the transpulmonary prostacyclin levels in a prospective placebo controlled study with intravenous ibuprofen. methods: with approval of the human [nvestigadon review board we studied patients in a prospective, randomized double-blinded protocol who were scheduled for major abdominal surgery. ibuprofen ( mg i.v.) or a placebo equivalent was administered minutes before skin incision. pulmonary artery thermodilution and radial artery catheters were placed after induction of anesthesia. mt was applied in a uniform fashion. baseline values preceded the incision of the peritoneum (to). fulther assessments followed , , , , . tile plasma concentrations (pc) of -keto-pgft, (stable metabolite of pgi ) were determined in arterial and mixed venous blood by radioimmunoassay. at all points in time we measured arterial and mixed venous blood gases. qs/qt was calculated by standard formula. data are given as median (p < . placebo vs. [ibuprofen] [ ] mmhg (*p< . i). these changes were accompanied by a marked increase of -keto-pgf~ pc up to rain after mt in arterial and mixed venous blood of untreated patients with a peak of *[ ] ng/l tl (*p< . ol). there was no difference between arterial and mixed venous pc. ibuprofen pretreated patients (n=zr) demonstrated stabile qs/qt and pao while -keto-pgf~ pc remained within the normal range. discussion: our data clearly indicate that mesenteric traction response includes a critical rise in qs/qt followed by significant decrease of paov stable oxygen transport determinants following cyclooxygenase inhibition signify an action mediated by prostacyclin. an indicative transpulmonary gradient for -keto-pgft~ was not detectable. a splanchnic vascular source for pgi release seems to be likely, but could not be proved by our current data. department of anesthesiology, cliu. chemistry * and surgery*; university clinics uim, prittwitzstral]e , ulm, germany it is unclear whether injuries like bums, in general, directly result in alterations of cell-mediated immunity that, in turn, promote endotoxic and bacterial translocation or, alternatively, whether these conditions allow increased bacterial invasion that, in turn, inhibits cmi. aim: to determine whether infectious challenge, as clp alone or combined with ti causes further immune abnormalities in the days following clp. study plan: on day , two groups of n= week old aj mice were subjected to either a % scold burn (ti), or were untreated (c) n= . on day , mice (ti+clp) and mice (clp) were subjected to clp. the two other groups (ti and c) were untreated. at days , and after thermal injury splenocytes (sp) were harvested and cultured with cona for an assay of il- and adherent splenocytes (as) were cultured with lps for il- , tnf, il- and pge . results: either ti + clp or clp alone result in significantly decreased secretion of all cytokines tested. in the ti group almost every cytokine production determined was elevated in comparison to ti + clp and prosmcyclin (pgi ) has been implicated in the pathophysiology of septic shock. however, pgi~'s role in the inflammatory response to sepsis is not well-defined. the purpose of this study was to identify which acute septic events are mediated by pgi during graded bacteremia. methods: eleven ~nesrhetized, hemodynamically monitored adult swine were infused iv with aeromonas h. ( /ml) at rates increased incrementally from . to . mi/kg/hr over hours. animals were studied in two groups: septic control (sc), graded bacteremia only (n= ); pga (n= ), graded bacteremta plus anti-pgiz antibody, ml/hr iv, beginning at hours. mean systemic (map) and pulmonary arterial (pap) pressures and arterial po , mmhg, cardiac index (ci), l/min/m , oxygen delivery index (do i) and consumption index (vozi), ml/min/m , and oxygen extraction (er), %, )latelet aggregometry (plt), %max., plasma pg -keto f alpha ; in the first instance~ peak values of lt ~ after i~ hrs post infarction were times higher than in the controls and excess leucocyte infiltration was noted at the infarction zone. in second instance two levels of lt b led to weak infiltration of the infarction zone by leucocytes. a. mo~e~o, in~.~p~siolo~,d~t.e~.cardiolo~,bogotsolets , ~ev , ukrmne systemic lesion$of erythron in traumatic disease and possibilities of their regulation by opioid peptides. redkin y. v., fominih s. g. using clinical ( patients) and experimental material( rats and dogs) we revealed general regularities of erythron lesions after hard mechanical trauma of various genesis as well as some mechanisms of development of posttraumatic anemia and possibilities of its correction with preparations of opioid peptides. the condition of central and peripheral compartments of erythron was studied with unified morphologic, immunogematological, biochemical and radiological methods. it was revealed that irrespective of the experimental animal species (dogs, rats) or in clinical experiments (patients) and irrespective of the injuring factor type (skeletal trauma, craniocerebral trauma, loss of blood) in erythron can be observed one-directed unspecific reaction realized by the considerable lowering of hemoglobin concentration, erythrocytes number and hematocrit. in the initial period ( - days) in the system of erythron prevail processes of distraction and elimination of er~zthrocytes relatively to the general production of stimulated erythropoiesis. the primary alterating factor is the prolonged intensification of peroxydation of membrane iipids of erythrocytes with simultaneous lowering of reserves of reduced glutathione. the distraction of erythrocytes is supported by the developing phenomena of autoallergization of organism that becomes apparent by the appearance of sensitized t cells and antierythrocyte antibodies. the intensified production of erythropoietin rules to the realization of he program of fetal and terminal (reserved) erythropoiesis. failure of erythropoiesis function is supported by disturbances of the processes of the injuring of cell metabolic apparatus. using of dalargin ( microgram per kilogram of body mass intrap'eritoneally within days after the trauma) showed the precise pharmacotherapeutic effect revealed by the diminishing of anemia of experimental rats, more . fiberbronohoscopic procedures are known to produce "peep-like" effects and to increase pulmonary artery (pa) resistance [ ] . peep can affect rv function by reducing preload and ejection fraction (ef) [ ] . since changes of rv function during bronchoscopy in septic patients are not reported, we measured rv parameters before, during and after fiberoptic bronchoalveolar lavage (bal). method: this -year-old patient (apache-ii: ) developed a hyperdynanlic septic state due to staphylococcus aureus (blood culture). we inserted a "fast response" thermistor pa-catheter (baxter-edwards) to evaluate rv performance [ ] . the therapeutic procedure included volume replacement, vasopressors (dopamine , dobutamine gg/kg/min. iv) and analgosedatior/. before bronchoscopy (olympus bf- , od= mm) the patient received pancmonium for muscle relaxation. ventilation was not changed during the procedure (endotracheal tube: id= ram, bennett a, pressure controlled mode, pm~x= mbar, peep= mbar, i:e=i:i, fio = . ). we measured rv enddiastolic volume (edv), stroke volume (sv), ef, heart rate (hr), cardiac index (ci) and mean pa pressure (mpap gerlach h, gerlach m, clauss m, falke kj renal hypoxia and/or ischemia initiates the development of a deteriorated medullary perfusion based on fibrin deposition in the peritubular capillaries, vasoconstriction, and perivascular edema, which is followed by a swelling of the tubular epithelial ceils, intraluminal tubular obstruction, and a backleak of fluid through the injured tubules into the renal interstitium, finally leading to an acute tubular necrosis (atn) [ ], clinically diagnosed as acute renal failure (arf). one important pathway for induction of enhanced vascular procoagulant activity and permeability is based on the synthesis and expression of macrophage-derived cytokines, which bind to specific endothelial cell surface receptors. we recently described the identification and purification .of a new , dalton polypeptide, which is synthesized and expressed by murine macrophages after stimulation with lipopolysaccharide, and exerts procoagulant activity on cultured endothelial cells [ ] . in the presented study, we demonstrate that the new polypeptid is also synthesized by macrophages under hypoxic conditions. the protein binds to specific receptors, which are expressed by endothelial cells dependent on the environmental oxygen tension. animal studies were performed after approval by the local committee for animal safety; the animals were anesthetized, treated and supervised in accordance with the guidelines of this committee. in contrast to other authors, who performed long-term hypoxia experiments in awake animals, we preferred to implement the studies under anesthesia for ethical reasons, although regulatory functions for ventilation might be influenced. animal studies demonstrated that the intravenous injection of the polypeptide initiates fibrin formation in the peritubular vessels. keeping the animals under hypoxic conditions induces similar effects, which are reduced by a rabbit-antiserum against the new protein. in conclusion, the new polypeptide obviously contributes to the pathogenesis of acute renal failure by tubular necrosis during and after hypoxic events. the use of verapamil as cardioprotective agents for management of patients with acute ischemic/reperfused heart is based on the assumption that the increased intracellular ca+ level is a key factor in causing cell death. our in vitro study was designed to focus on effects of verapamil on the metabolic potential of cardiac slices after reversible ischemia in rats. the material consisted of two main groups : group a (non ischemia/reperfusion group) and group b (ischemia/reperfusion group), each is subdivided into two subgroups (a and b). each subgroup included rat hearts. group aa is the control group, group ab is verapami] added group. group ba is ischemia group without verapamil. group bb is verapamil added group. ischemic cardiac slices were obtained from rats subjected to min. haemorrhage to induce reversible global ischemia. both nonischemic and ischemic cardiac slices were placed in well oxygenated krebs ringer phosphate buffer containing mg% glucose & gm% bovine albumin and incubated in dubnoff shaking water bath for min at °c the results revealed that there was an enhancement in release of free fatty acids (ffa) ( %) and lactate ( %) and in glucose uptake ( %) in group ba as compared with group aa. these metabolic alternations produced by ischemic cardiac slices were reversed by verapamil addition ( ml%) but in group ab verpamil did not alter the release of ffa & lactate from non-ischemic cardiac slices, whereas it inhibited glucose uptake from these slices by %. the improvement of the metabolic intervention of ischemic myocardium indicates that verapamil may be of importance in reducing the extent and severity of acute myocardial ischemic injury in acute haemorrhage. severe endothelial dysfunction occurs following injury to carotid arteries which is characterized by a decreased ability of these arteries to dilate when challenged with ach or a , but not with a direct vasodilator (nano ). this failure to relax to ach and a reflects an inability of endothelium to generate edrf, but relaxation recovers gradually to control values by weeks. exogenous no donors (e.g., c - or spm- ), accelerate the recovery of the injured endothelium in rat carotid arteries. intravenous infusion of an no donor ( p.g/day) with an implanted osmotic pump significantly accelerated the recovery of regenerated endothelium to produce edrf at days. rat carotid artery rings relaxed only + % and + % to gm ach in vehicle treated rats and in inactive no donor treated rats respectively days following injury compared with + % in no donor rats (p< . ). relaxation to gm nan was normal in all groups indicating that the differences in relaxation were not the result of damage to vascular smooth muscle. contraction to l-name ( mm) was markedly reduced by injury, but was protected by no donors (p< . ). thus, exogenous no donors enhance the ability of the endothelium to regenerate and to release edrf in response to endothelium-dependent vasodilators. this may be due to an anti-proliferative and anti-mitogenic effect of no on vascular smooth muscle cells, allowing the endothelium to regenerate without intimal thickening. no also has been shown to inhibit platelet aggregation, and to attenuate neutrophil adherence and activation. the superoxide scavenging effect of no is not the basis for these effects since hsod is inactive in preserving endothelial function in injured arteries. thus, no exerts a variety of cytoprotective effects which may be of importance in protecting against vascular injury. much evidence has now accumulated to show that the excess production of the vasodilator nitric oxide (no) in sepsis is an important contributor to the hypotension and multiorgan failure characteristic of this condition. various cytokines play an important role in this process through their ability to induce the production of one of the enzymes responsible for no synthesis, the inducible no synthase (inos). we have studied the effects of cytokines on the induction of this enzyme both in vitro using vascular smooth muscle cells, and in a murine model of gram-negative sepsis. tn smooth muscle ceils, the cytokines il- , ifnq', and tnf-oc show strong synergy with one another in the production of inos. in order to define the molecular basis for this synergic effect, we have linked the promoter of the inos gene to a "reporter" gene, chloramphenicol acetyl transferase (cat), and transfected these constructs into vascular smooth muscle cells. assays of cat activity reflect the activity of the promoter in this system, and by generating sets of deletion mutants of the promoter sequence we have been able to define the area within the promoter which mediates the synergic effect of these cytokines. in addition to stimufatory effects on inos production, certain cytokines are able to down-regulate the production of inos in vascular smooth muscle cells, and the effects of these counterregulatory cytokines will be discussed. the interaction of these cytokine effects in the whole organism has been studied in a murine model of gramnegative sepsis. widespread induction of inos occurs in this model as assayed by enzyme activity and through use of specific antisera to inos. neutralizing antibodies to tnf-~ and tfn-y are both able to prevent death in this model, but it is only the anti-ifn-y which attenuates the induction of inos assayed in the liver. clearly there is some redundancy in the effects of cytokines on the production of inos in sepsis, and greater understanding of the most important factors in inos production is required in order to target anti-cytokine therapy most appropriately. effects of nitric oxide on hepatocyte metabolism in inflammation. j. stadler, department of surgery, tu mqnchen, frg hepatocellular nitric oxide (no) synthesis is induced by proinflammatory mediators such as tumor necrosis factor, interleukin- and interferon gamma or by bacterial toxins such as lipopolysaccharide. stimulation of the hepatocytes (hc) with a combination of these agents leads to an output of no in quantities which are not seen in any other celltype. it has been demonstrated by various investigators that important effects of these cytokines and bacterial toxins on hc metabolism can be attributed to the action of no. in contrast to other celltypes hc seem to be relatively resistant to suppression of basic metabolic functions such as energy metabolism by no. therefore, cell damage has not been described to a significant extent following exposure to no. however, no does inhibit total protein synthesis. the exact biochemical mechanism of this phenomenon has not been uncovered yet, but it has been demonstrated for some specific proteins that their production is inhibited at a posttransscriptional level. as in many other celltypes cgmp generation is elevated in hc by no through activation of the soluble guanylate cyclase. cyclic gmp may possibly exert a plethora of metabolic functions, but it is interesting to note that most of the cgmp seems to be transported out of the cell. some very specific effects of no on hc metabolism include the inhibition of the glyceraldehyde- -phosphate dehydrogenase (gapdh) and the cytochrome p (cyp) enzymes. inhibition of gapdh activity is mediated through nitrosylation of critical domains of the enzymes by no which enhances auto-adpribosylation. this effect on gapdh activity might be responsible for the inhibition of gluconeogenesis by no, which has been described recently. finally, no-mediated inhibition of cyps may help to explain the suppression of hiotransformation processes which is a characteristic featur,'~ r ~ "~flamed liver. nitric oxide (no) is an endogenous inhibitor of polymorphonuclear leukocyte (pmn) adhesion which limits pmn-endothelial cell interactions under normal conditions. we have previously demonstrated that following ischemia, no production by the vascular endothelinm is dramatically reduced. accordingly, we investigated the effects of no-donors on pmn accumulation and tissue injury following hemorrhagic shock and ischemia. hemorrhagic shock was induced in anesthetized rats by bleeding to mmhg for hours followed by reperfusion. segments of superior mesenteric artery (sma) were isolated and suspended in organ baths. in rats receiving saline sma relaxation to acetylcholine (ach, nm) was reduced by % compared to control sma segments (p< . ) while relaxation to sodium nitrite ( gm) was unaffected. in addition, mesenteric tissue pmn accumulation as determined by myeloperoxidase (mpo) activity was significantly elevated compared to controls (p< . l). interestingly, treatment with the no-donating agent, s-nitroso-n-acetylpenicillamine (snap) significantly preserved sma relaxation (p< . ), attenuated mesenteric mpo (p< . ) activity, and significantly improved survival compared to saline vehicle. in anesthetized, open-chest dogs we investigated the cardioprotective actions of a novel no-donor, spm- (schwarz pharma), following regional myocardial ischemia ( hour) and reperfusion ( . hours) . treatment with spm- ( rim) significantly reduced myocardial necrosis by % (p< . ) compared to an no-deficient analog of spm- , spm- . furthermore, mpo activity within the ischemic-reperfused zone was also significantly (p< . ) reduced following treatment with spm- compared to spm- ( . + . vs. . + . u/ mg tissue). these data strongly suggest that no is a potent inhibitor of pmn-mediated tissue injury following hemorrhagic shock as well as in acute myocardial ischemia-reperfusion injury. overproduction of nitdc oxide (no') may contribute to sepsis-induced hypotension. during septic shock, excess no" is produced by an isoform of nitric oxide synthase (nos) which is induced by inflammatory mediators. nonselective nos inhibitors have been proposed as a new therapeutic approach to treating hypotension in septic shock. we studied the differential hemodynamic effects of n~-methyi-l-arginine (l-nma), a nos inhibitor, in normal canines versus those challenged with endotexin (lps) and compared the activity of this drug across the venous, pulmonary and systemic vascular beds. awake canines were challenged with lps ( mg/kg, n= : mg/kg, n= ; or mg/kg, n= ) and treated with l-nma ( , , , , mg/kg/hr) for hours following a , , or mg/kg loading dose. animals were resuscitated with iv ringers solution ( ml/kg/hr). hemodynamic data were collected at , , , , , and hours using intravascular catheters and radionuclide heart scans and analyzed by anova. in both normal and endotoxemic animals, l-nma at all doses studied similarly increased mean arterial pressure (p= . ), and systemic vascular resistance index (p= .ol) and decreased cardiac index (p= . ) and oxygen delivery index (p= . ). in contrast, the effect of l-nma on mean pulmonary artery pressure, central venous pressure, pulmonary capillary wedge pressure, and pulmonary vascular resistance index was greater in lps-challenged canines compared to normal animals (p< . ), but this differential effect on the venous and pulmonary circulation occurred, > hours after lps challenge. l-nma did not significantly increase survival rates or times at any of the doses studied ( , , , or mg/kg/h) in either the low ( mg/kg) or high dose ( mg/kg) lps-challenge groups. a nonsignificant (p> . ) trend toward a beneficial effect on survival ol low dose l-nma ( mg/kg/h) in animals given the mg/kg lps-cha[lenge was not enhanced by increasing the lethality of the model or by administering higher l-nma doses. at the highest l-nma dose used in this study ( mg/kg/h), survival time decreased significantly for both the low and high dose lps-challenge animals (p< . ). this increased mortality was not explained by changes in plasma concentrations of either lps or tnfc~. thus, l-nma did not have a greater effect on the systemic arterial circulation in endotoxemic compared to normal canines. however, in the venous and pulmonary vascular beds, the effect of l-nma increased with time after endotoxin-challenge these data suggest the induction of nos activity by endotoxin in canines may be relatively greater in venous and pulmonary vessels compared to systernic arteries. l-nma, a nonselective nos inhibitor, did not decrease mortality in endoloxemic canines and the highest dose studied was harmful. pulmonary hypertension (ph) and arterial hypoxemia are characteristic features of the adult respiratory distress syndrome (ards). reducing pulmonary vascular pressures may promote the resolution of pulmonary edema. intravenously infused vasodilators lower ph in ards, but, as a result of their general vasodilatatory effects, systemic mean arterial pressure may also decrease. furthermore, blood flow may be increased to non-ventilated or poorly ventilated lung areas resulting in a rise of intrapulmonary shunt, thus causing a further fall in pad . recently, short term inhalation of low concentrations of the gas nitric oxide (no), an endogenous endothelium derived relaxing factor, which is rapidly inactivated in blood by hemoglobin, was reported to decrease ph without causing systemic vasodilation in sheep [ ]. similar changes have been observed in patients with severe ards during repeated short term inhalation of no ( and ppm), which rapidly and selectively decreased the mean pulmonary artery pressure (pap) and, in contrast to intravenously infused prostacyclin, induced a remarkable increase of pad [ ] . this improvement in oxygenation was caused by a redistribution in blood flow away from intrapulmonary shunt areas to normal ventilated lung regions. continuous no inhalation ( - ppm) consistently lowered the pap and augmented the pao /f.o for up to days. no negative side effects were observed during the whole time span examined. in particular methemoglobin levels always remained below . %. following these investigations, it could be shown that these effects may also occur using concentrations in the parts per billion range [ ] , which may reduce possible toxic side effects. however, in the same study it was demonstrated that the dose-response curves for pa and pap have different patterns. whereas pap presented a continuous dose-dependent downward tendency with an eds o of approximately - ppm, the improvement of oxygenation had a maximum at ppm and, at higher doses, drifted back towards the baseline data. the ed~o was estimated at approximately ppb, i.e. more than ten times lower than for the reduction of pap. in conclusion, inhalation of no by patients with severe ards may result in persistent and reproducible decreases in pap associated with an evident improvement in pad , thus allowing reduction of the f.o . no inhalation should be performed using low concentrations which are less toxic, although any possible risks still have to be considered carefully. dose-response studies for the individual patients are recommended urgently. finally, controlled randomized studies are required to demonstrate that additional no inhalation is able to reduce mortality of ards. inhibition of the activity of glyceraldehyd- -phosphate dehydrogenase (gapdh), an enzyme of the glycolysis/gluconeogenetic pathway, through adp-ribosylation is promoted by nitric oxide (no). since no is produced in the septic liver and hypoglycemia is a major problem of late sepsis, it was investigated whether no interferes with gluconeogenesis of hepatocytes. hepatocytes (hc) were isolated from sprague-dawley rats using a collagenase perfusion technique and differential centrifugation. exogenous no was applied by incubation with the no-donors s-nitrosyl-acetylpenicillamine and sodium-nitroprusside. endogenous no synthesis was induced by incubation with cytokines (tnfcq il- , ifnj and lipopolysacchafide (lps). hrs later the incubation medium was changed to a solution containing lactate, ornithine, lysine, ammoniumchloride and glucagon for optimal conditions of gluconeogenesis. after more hrs glucose and nitrite levels were determined spectrophotometrically. gapdh activity was measured by the nadh-dependent conversion of , -diphosphoglycerate to glyceraldehyde- -phosphate. incubation of hc with no-donors led to a concentrationdependent inhibition of gluconeogenesis and gapdh activity. however, gapdh activity was about times more sensitive to the inhibitory effect of exogenous no. incubation of hc with cytokines and lps induced nq synthesis as measured by an increase in nitrite concentrations. endogenously produced no suppressed gluconeogenesis by _+ %. in contrast to exogenously applied no, the effect of endogenous no synthesis was less on gapdh activity resulting in an inhibition of only _+ %. in conclusion, exogenous and endogenous no inhibited gluconeogenesis as well as gapdh activity. however, there was no correlation between the extent of inhibition of these two parameters of hepatocellular glucose metabolism. we have shown that inhibition of hepatocyte (hep) synthesis of nitric oxide (no) potentiates cell injury in a model of acetaminopheninduced oxidative stress and the extent of damage was paralleled by depletion of reduced glutathione (gsh) stores. to clarify the role of no in modulating the redox state of hep, we studied the effect of inhibition of cytokine-mediated no production on hep gsh stores, in a system of isolated rat hep in primary culture, no synthesis was induced (stim) by exposure to il- , tnf, ifn, and lps for hours. , , and ~m of n-monomethyi-l-arginine (nmma), a specific inhibitor of no synthesis, was added. cells incubated in media alone served as controls (cont). the no metabolite (no ); aspartate aminotransferase (ast), an indicator of cell injury; and gsh were assayed. (data presented as mean + sem; n= .) gsh (nmovma orotein) ..~ (nmol/ma orotein) cont . + . + . # stim . + . + stim+ o tzm nmma . + . + . # stim+ ~m nmma . _..+ . * + . # stim+ pm nmma . + . * + . # stim+ )lm nmma . + . * + . # anova , . (* p < . versus stim, # p < . versus stim; anova with neuman-keuls) gsh in cont+ i~m l-nmma was equivalent to that of cont ( . vs. . ). ast release was equivalent in all treatment groups. these data show that inhibition of hep synthesis of no depletes intracellular stores of reduced gsh. we conclude that hepatocyte no production modulates cellular gsh homeostasis and as a result, may be hepatoprotective in oxidative injury. nitric oxide (no) is a modulator of immune response and may be involved in the changes in immune reactivity after major trauma and operations. we investigated no-generation in rat and mice spleen cells (sc) after partial hepatectomy (ph). c bl/ mice and lew rats underwent a % and % ph, respectively. sc were prepared - days after ph and plated at to x ecells per well. after h incubation at °c, no-production was measured as nitrite levels (griess reagent). normal mouse sc did not produce no, neither basal nor in response to lps or con a starting at the second day after ph, we found a substantial production of no. in rats, also sc from control animals were able to generate no; both basal and stimulated no-generation were further enhanced after ph (table, values expressed as mean --se). after shame operation, there was only a modest elevation of noproduction in rat and mouse sc. in first experiments we could demonstrate no-production also in phagocytes from a patient days aider liver partial resection ( . nmol nitrite/ cells) enhanced no-production in macrophages may contribute to the changes of immune reactivity after partial hepatectomy. nitric oxide (no) is recognized as an important mediator in endotoxemia and sepsis. increased synthesis of no has been demonstrated in septic humans and animals, and no inhibitors have been used in the treatment of septic shock. recent reports have, however, suggested that this form of therapy may cause serious organ damage. in the present investigation circulatory and metabolic changes in the liver were studied during treatment with the no-synthase inhibitor n-nitro-l-arginine-methyl ester (l-name) in endotoxemia. methods: juvenile pigs were randomized to one of the following treatment groups: ) encletoxin and l-name, ) endotoxin, ) naci and l-name, ) nach preliminary results from groups (n= ) and (n= ) are presented. catheters for pressure measurement were introduced into the aorta, hepatic and portal veins and ultrasonic transit time flow probes were placed on the hepatic artery and portal vein. a catheter was introduced into the pulmonary artery. endotoxin ( . gg/kg/h) was given as a continous portal infusion over the entire observation period of hrs. l-name ( mg/kg) was given as a bolus after hrs. of endotoxemia. results: endotoxin transiently reduced portal vein flow (pvf) by %* and hepatic artery flow (hal e) by %*, while l-name caused a further and lasting reduction in flow (pvf %, haf %)*. transhepatic (portal-hepatic vein) vascular resistance increased to times baseline value during endotoxemia while l-name caused a further marked increase in resistance to times initial value. portal oxygen saturation (so ) decreased by %* during endotoxemia. l-name caused a reduction in portal so by %*. arterial so was unchanged in both groups. hepatic oxygen uptake was not changed by endotoxin, but was markedly reduced after addition of l-name. endotoxin caused a % reduction in cardiac output (co). the addition of l-name reduced co by a total of %*. *: p < . . conclusion: is the present model of endotoxemia treatment with the nitric oxide synthase inhibitor l-name markedly reduced liver perfusion and portal oxygen supply. this might explain the increased liver damage reported in previous studies using no-inhibitors. the increase in transhepatic resistance found after l-name treatment will tend to cause pooling of blood in the splanchnic veins, resulting in reduced filling of the heart and thus contribute to the observed reduction in cardiac output. institute for surgical research, rikshospitalet, the national hospital, university of oslo, oslo, norway. we have investigated the role of tumour necrosis factor (tnf) and interleukin-i (il-i) in the induction of nitric oxide synthase (nos) by bacterial endotoxin (lipopolysaccharide; lps; mg kg -i i.v.) in vivo. in anaesthetized rats, pretreatment with a monoclonal antibody for tnf (tnfab; mg kg -i s.c., at h prior to lps) or with an il-i receptor antagonist (il-ira; mg/kg bolus and . mg/kg/h infusion) ameliorated the fall in mean arterial blood pressure (map) at - min after lps. for instance, endotoxaemia for min resulted in a fall in map from -+ (control) to -+ mmhg (p< . ; n= ). in contrast, animals pretreated with tnfab or il-ira prior to lps injection maintained significantly higher map at min when compared to lps-control: -+ mmeg (n= ) and -+ mmhg (n= ), respectively (p< . ). three hours of endotoxaemia significantly reduced the contractile effects of noradrenaline (na) in the thoracic aorta ex vivo. the hyporeactivity to na was partially restored by in vitro treatment of the vessels with ng-nitro-l-arginine methyl ester (l-name, min, x - m). pretreatment of rats with tnfab or il-ira significantly (p< . ) prevented the lps-induced hyporeactivity of rat aortic rings ex vivo. l-name did not alter or only slightly enhanced the contractions of aortic rings obtained from tnfab or il-ira treated lps-rats, respectively. at min after lps there was an induction of calcium-independent nos activity in the lung ( . -+ . pmol citrulline/mg/min, n= ), which was attenuated by tnfab and !l-ira by -+ % and -+ %, respectively (n= ; p< . ). thus, the production of both tnf and il-i contributes to the induction of nos by lps in vivo. the protective effect of agents which inhibit the release or action of tnf or il-i in shock may be, in part, due to inhibition of nos induction. neal garrison, md objective: sepsis is often accompanied by organ dysfunction, in part due to impaired microvascular perfusion. recently, nitric oxide (no) has been described as an important mediator of the hemodynamic changes of sepsis, and no synthase (no-s) inhibitors have been advocated for treatment of septic shock, but their visceral microcirculatory effects are inadequately characterized. we postulated that no-s inhibition would exacerbate the impaired organ perfusion of sepsis. methods: six groups ofdecerebrate rats were studied. bacteremia was induced with live e. coli, which consistently increased cardiac output - % above baseline (bl). the no-s inhibitor nm-nitro-larginine methyl ester (l-name, mg/kg iv), prevented this increase and elevated map by - %. in the first groups, total hepatic blood flow (thbf, ml/min by time transit flowmetry) and microvascular perfusion (mi-ibf, ¼ bl by laser doppler flux) were measured. in the other groups, in vivo videomicroscopy was used to observe renal microvascular responses (ila=interlobular artery, aff=afferent arteriole, eff=efferent arteriole; % bl for all). results: data are rains after e. cob. n= - /group. * p< . vs bl by remanova and § p< . vs e. coli alone by anova. ec+l-name -+ - _+ " § - _+ * § - _+ * § - + * - + * § conclusions: l-name administration in controls decreased renal blood flow, indicating no contributes to basal renal tone. bacteremia decreased mtlbf but not thbf, and mi-ibf was further impaired by no-s inhibition. e. coli caused renal preglomemlar, but not postglomerular constriction and reduced flow. l-name exacerbated these e. coli-induced alterations and caused eff constriction. these data indicate that no-s inhibition exacerbates bacteremia-induced impairment of renal and hepatic blood flow, suggesting that no is an importam compensatory dilator mechanism in these organs during sepsis. irf (iron responsive factor) is the central regulatory protein of intracellular iron metabolism able to bind to responsive rna elements (ires) present atthe 'untranslated region (utr) of ferritin mrna and 'utr of transferrin receptor mrna. binding of irf to ires results in repression of ferritin mrna translation and increased stability of transferrin receptor mrna leading to enhancement of transferrin receptor translation. we describe here that either tetrahydrobiopterin dependent stimulation as well as cytokine (ifn-~)/lipopolysaccharidemediated induction of nitric oxide synthase activates irf, which is due to direct interaction of nitric oxide with the iron-sulphur-cluster of irf. this was shown by gene expression studies using a plasmid containing a ferritin ire and a cat indicator box which was transfected into k myelomonocytic cells, which were shown to have a constitutive form of nitric oxide synthase (nos). furthermore, the increased binding of re to irf due to irf activation of irf by nitric oxide was demonstrated by gel shift assays. irf activity was much more increased in cellular extracts from murine macrophages (j ) where a cytokine inducible form of nos has been characterized earlier as compared with irf activity in k cells, where nos was stimulated by increasing the availability of the essential nos cofactor , , , -tetrahydrobiopterin. we then demonstrated that activation of irf by nitric oxide is accompanied by alterations in ferritin translation as checked by metabolic labeling and immunoprecipitation. these results suggest a reasonable mechanism for the regulation of iron disturbances under chronic inflammatory disorders, characterized by increased concentration of immune activation parameters like ifn- or neopterin and low serum iron and hemoglobin concentrations. taken nitric oxide, no, the putative endothelial derived relaxant factor, edrf, has been shown to be a potent inhibitor ofplatelet aggregation in vitro. in vivo evidence however, is scarce. accumulation of platelets in the lungs has been shown to occur during extracorporeal circulation. the aim of the present study was to investigate the effect of inhaled no on this reaction. materials and methods: the animals were divided into two groups, each consisting of pigs. platelets were selectively labelled with luln-oxine. dialysis was instituted via catheters in the femoral vessels. in group , no, ppm, was added to the inhaled gas from the start of dialysis. in group no was not given. the activity over the lungs was followed dynamically with a gamma camera. central hemodynamics was monitored via a swan -ganz catheter. results: the activity was significantly lower in group , from minutes after start of dialysis and onwards, indicating diminished accumulation of platelets in the lungs. parallel to this the hemodynamic response in terms of increased pulmonary artery pressure and pulmonary vascular resistance was blunted in this group conclusion: inhaled no in this model seems to affect pulmonary platelet sequestration. an associated attenuation of the changes in central hemodynamics was also seen. previous studies from our laboratory have demonstrated that vascular contractility decreased in endothelium-intact blood vessel rings in early and late stages of sepsis. although endothelium removal in early sepsis restored vascular contraction, the depressed smooth muscle contractility observed in late sepsis was not restored by endothelium removal. this indicates that impairment of smooth muscleper se may be responsible for such dysfunction in late sepsis. the aim of this study, therefore, was to determine whether or not smooth muscle-derived nitric oxide (no) plays a role in producing vascular smooth muscle dysfunction during late stages of sepsis. to study this, rats ( - g, n= - /group) were subjected to sepsis by cecal ligation and puncture (clp). septic and shamoperated rats then received rrd/ g bw normal saline. the animals were killed at , , or h post-clp ( h post-clp=early sepsis; - h post-clp=late sepsis), and thoracic aortic rings were prepared for contraction studies using organ chambers. the complete removal of endothelial cells was tested by the absence of any significant acetylcholine-induced vascular relaxation. contractile responses to norepinephrine (ne, to - m) were determined in the aortic rings without intact endothelium. ng-monomethyl-l-arginine (l-nmma, /~m, an inhibitor of no synthase) was then added to the organ chamber and ne-induced peak contraction was determined before and after the addition of l-nmma. the peak contraction (rag/rag tissue, mean_+sem) is shown below: the results indicate that the addition of l-nmma did not significantly affect ne-lnduced peak contraction in endothelium-denuded vessel rings at and h after clp. in contrast, l-nmma administration produces an % increase (p< . ) in peak contraction during late sepsis. therefore, the vascular smooth muscle contractile dysfunction observed at h post-clp is partially due to smooth muscle-derived no over-production. thus, unlike macrophages in which inducible nitric oxide synthase (inos) is observed in early sepsis, the inos in vascular smooth muscle appears prominent only in the late stages of sepsis. in three cases of human septic shock in which ng-monomethyi-l-arginine, (l-nmma) a nitric-oxide-synthase-inhibitor was applied, we isolated three completely different types of pathogens: candida, pseudomonas aeruginose and multiresistant coagulase-negative staphylococci. this observation suggests that endotoxin alone is not the main factor triggering hypotension in septic shock by the nitric oxide pathway. in a -years-old woman in severe septic shock due to a candida and pseudomonas aeruginosa infection complicated by adult-respiratorydistress-syndrome conditions deteriorated despite adequate conventional therapy. in this trial, effects of l-nmma on cytokin-levels were investigated. the study-protocol was approved by the ethical committee of the department of surgery. after two boll of mg of l-nmma, a continuous infusion was installed ( . mg/minute and kg body weight l-nmma). as expected mean arterial blood pressure rose ( to mmhg}, heart rate stayed stable ( + b/rain), systemic vascular resistance increased ( to dyne.sec/cm ), cardiac output decreased ( to . l/rain), and cardiac index declined ( . to . l/min/m }. before and after minutes while the infusion of l-nmma, blood samples for immunological measurements were taken and processed together. pulmonary-shunt-volume was observed before the application of l-nmma, after one hour and after matutes. neopterine increased from . to . ng/ml, tumour-necrosis-factor-a increased from . to . pg/ml and intedeukin- increased from . to . pg/ml. immunoglobulines a, g, and m ( . to . , . to . , . to . g/i), complement factor c- c and c- ( . to . , . to . g/i), alpha-l-antitrypsine ( . to . g/i), c-reactive-protein ( . to . rag/i), interleukin- ( pg/ml) and soluble interleukin- ( to units/ml) did not change significantly. pulmonary-shuntvolume decreased from . % to . % within one hour and to . % after minutes. in septic shock blocking nitric oxide as an intervention at the end of a not ~,et ful!y understood cascade might have important influences on pulmonary-shunt-volume and inter-cell-communication. department of surgery, pharmacy* and immunology**, university hospital of zurich, r~imistrasse , zurich, switzerland we previously reported that hypoferremic cba mice had an increased resistance to salmonella infection, and that injection of ammonium ferric citrate (afc) to these mice led to enhanced infection (ganthier et at. . microbiol.immuno : ) . because nitric oxide (no) is involved in the antimicrobial activity of routine macmphages towards various inttacellular pathogens, we investigated the influence of iron on the bactericidal activity of cba mouse macrophages towards s.typhimurium and on the production and activity of reactive nitrogen intermediates (rni). peritoneal macrophages hum cba mice were cultured in the presence (or not) of afc ,um, ifn-,/ u/ml, lps fig/m/, ngmonomethyl-l--arginine (mmla) ram. nitrite (no -) content of the supematants was determined by a standard griess reaction, and h release was measured by the peroxidese dependant oxidation of phenol red. for intracellular killing, macrophages monolayers were infected, and, at various intervals, lysed by triton x- , and surviving bacteria enumerated by colony counting on agar. for in vivo experiments, mice were infected ip with . ml of a suspension of . ~" s.typhimurium, strain c , and injected with aminoguanidine (ag) mg/ml in saline. our results show that the rn[ inhibitor ag strongly accelerates the mortality of infected mice, the survival rate decreasing from % in the control group to % in the treated group, days after challenge. correlatively the rni inhibitor mmla induces in vitro a decrease in the rate of bacterial killing, fxom % to %, in macrophages triggered with ifn-? + lps. the cultivation of macrophages in the presence of afc leads to a decreased no -accumulation, . nmole/well v.s. nmole/well. conversely h production is enhanced from nmole/well up to , nmole/well. nevertheless, macrophages cultivated in the presence of afc exhibit an increased tale of intracellular killing, % in iron exposed macrophages v.s, % in control macrophages. when triggered with ifn-~, alone, macrophages have a reduced antibacterial activity ( % v.s. %) whereas the addition of afc to these macrophagas restores an elevated ( %) rate of killing. in conclusion, the results show that bactericidal activity of cba macrophages towards s.typhimurium depends on the production of no by these macrophages ; but they also demonstrate that no is not the only reactive species involved in the intracellular kil/ing of s.thyphimurium ; indeed afc which strongly inhibits rni production, stimulates h release by these macrophages and increase their bactericidal activity in vitro. nevertheless afc may promote bacterial growth in vivo. crssa. unit de microbiologie. bp . la tronche cedex france. henning jahr, ulrike noack, karin braun the large amounts of no produced by the inducible no synthase in rat macrophages have direct antimicrobial effects, but inhibit the activation of the lymphocyte-dependent host defense system. the aim of this study was to investigate if complement activation influences no-generation. spleen cells from lew rats were incubated at °in tcm- / % fcs, with or without additional rat serum. after h, nitrite (end product from no metabolism) was measured by oriess reagent. in rat spleen cell preparations, most of the no is produced by macrophages. complement activation in vivo was carried out by i.v. injections of u cobra venom factor/kg b.w. at days and . significantly higher (p<o.o ) lps-stimulated no-generation was found in spleen cells prepared at day ( . - . nmol nitrite/ cells, n= ) compared to spleen cells from non-treated animals ( . ± . nmol, n= ) complement activation was effective also in vitro. when incubated in % zymosan-activated rat serum, both basal and lps-stimulated noproduction were enhanced in spleen cells (table, values increased production of nitric oxide (no) is associated with sepsis, however, the effect of no inhibition on survival during sepsis is unclear. we examined the effect of no inhibition on host's ability to eliminate bacteria and survival in mice sepsis model. sixty female balb/c mice were injected with e.coli ( qbody) into peritoneal cavity. the treated group received n-ultro-l-arginine-methyl-ester (l-name), an inhibitor of nos, one hour before bacterial challenge. thirty mice were observed for survival after e.coli challenge and the other mice were sacrificed at hours. blood was obtained by cardiac puncture and peritoneal lavage fluid (plf), liver and lung were harvested. the number of peritoneal exudative cell (pec) was counted and colony forming units (cfld of bacteria in the tissues, blood, and plf were also determined. in addition, pec was cultured in vitro with or without lipopolysaecharide (lps). tnf levels in the blood, plf, and supematants of cultured pec were measured by l bioassay. survival rate (%) qrql/,p n hr hr dav control (normal saline . ml/body i.p.) n (l-name mg/kg i. administration of l-name before e.coli injection increased the number of bacteria in plf and tnf level in plasma, the result suggests that nos inhibitor may depress the host's ability to kill the injected bacteria and that it may induce greater systemic tnf production. we conclude that nos inhibitor is detrimental to animals in sepsis. the hypothesis that sod prevents reperfusion injury to the liver by protecting the endogenous endothelium derived vascular relaxing factor (no) from being inactivated by oxygen free radicals should be investigated. male wistar rats were subjected to min of partial liver ischemia ( %) and min of consecutive reperfusion in situ. some rats were pretreated with the no synthase inhibitor nitro-l-arginin (nla: i mg/kg). sod, when used, was given as mn-containing preparation at a dose of u. i.v. i min prior to ischemia. results (sod/ nla+sod/ ni~,, respectively): bile flow (~i/g/min) : . ± . "#/ . ± . / . ± . . alt (u/ ) • ± ~#/ ± / ± . gldh (u/l): . ± . e#/ . ± . #/ . ± . . in absence of nla, sod enhanced postischemic bile flow and reduced leakage of alt and gldh into the plasma, while the effects of sod disappeared, when endogenous ~synthesis was inhibited by nla. nla had no ~ffect on untreated animals submitted to the same protocol. these results may suggest, that oxygen free radicals interfe~:e with the endogenous vasodilator no and that the benefit of sod can be attribuated in large part to a protection of no during reperfusion. nitric oxide (no) reduces pulmonary vascular resistance and increases oxygenation by inhalation in ards patients [ ] . animal studies on the endogeneous no production proved that no is exhaled after synthesis in the respiratory tract [ ] . with approval by the hospital ethics committee, we studied healthy volunteers and ventilated patients by measurement of inand exhaled no by no/no -chemiluminescence in segments of the upper respiratory tract. we found that no is synthesized predominantly in the nose and in the upper nasopharynx, leading to inspiratory tracheal no concentrations of . - . parts per million in non-smoking volunteers; data during mask ventilation per nose were as follows: non . . . . , parts per million (ppm) no, mean, n = ; ~ p < . (t-test) between smokers and non-smokers; p < . (t-test) between in-and expiration. about % of no is resorbe.d by the lung, and the exhaled no -in contrast to former presumptions -is the remaining part of the autoinhaled no. intubated and ventilated patients are deprived of no autoinhalation, and the exhaled no in the trachea decreases more than % {o. ppm before intubation vs. . ppm after intubation, ~, < . ), whereas the nasal no concentration increases by cumulation. hence, low-dose no inhalation in ards patients might be considered as a no replacement, especially since the tracheal no concentrations we measured in volunteers are similar to those which could be demonstrated to be effective for ards in former studies [ ] . the data demonstrate that no is synthesized in the nose, and inhaled and resorbed by the lung. this phenomenon of no autoinhalation, which is reduced in smokers and in ventilated patients, does possibly contribute to the regulation of the ventilation-perfusion ratio in the lung. kikuchi , yoshihiro inoue , masao yoshida critical care and emergency center, and department of bacteriology, iwate medical university. nitric oxide (no) is produced by macrophages and vascular endothelial cells. it is thought to be the true form of endotheliumderived relaxing factor, and to be involved in the fall of blood pressure during septic shock. we have reported previously that endotoxin (lps), tnf-c~ and il- contribute to the occurrence of septic shock (circ shock : ; . the present study investigated the in vitro effects of lps, tnf-o~, il- and ifn-'i on the production of no by macrophages. peritoneal macrophages were cultured with lps, il- , tnf-~ and ifn-¥. in culture with lps, no was produced in a dose-dependent manner, and the production was suppressed by a no production inhibitor, l-nmma. ifn-y, il- and tnf-c~ used alone did not promote the production of no, but helped lps to facilitate no production. a combination of il- and tnf-c( enhanced lps-facilitated no production to a greater extent than il- or tnf-c~ alone. the above results suggest that these cytokines are involved in endotoxin shock through the mechanisms that facilitate no production. - uchimaru, morioka . japan. t. yolk , , i. ioannidis , w.j. kox and h. de groot objectives: nitric oxide (no.) is known to play an important role in neurotransmission, vasoregulation, and bactericidal as well as tumoricidal cellular defense systems. by means of no-donating agents we studied the mechanism of no-mediated cytotoxicity against rat liver microvascular endothelial cells. materials and methods: -morphoiinosydnonimine-n-ethylcarbamide (sin-l) and sodium nitroprusside (snp) were used as no. donators, ldh release and trypan blue exclusion were applied to indicate cell viability. results: sin- ( mm), which releases both no. and o -., caused a time-dependent cytoreduction of % and % after and hrs, respectively. this effect was not inhibited by superoxide dismutase (sod), which removes --to form h and . in contrast, the addition of catalase (cat), which removes h to form h and , diminished the cytotoxic effect ot sin- to %, equivalent to high concentrations of snp ( mm), which solely releases no.. in addition, the amount of h formed by mm sin- equaled the amount ot enzymaticany produced h by mu/l glucose oxidase (god). whereas god in this concentration and snp in low concentration ( mm) alone were not toxic to endothelial cells, the combination caused an % reduction of viability, comparable to the effect observed with sin- ( mm) alone. moreover, toxicity mediated by high concentrations of snp ( mm and ram) was reduced by % under hypoxic conditions indicating synergism with no-and . conclusions: we conclude, that no.-induced toxicity against endothelial cells is enhanced slightly in the presence of oxygen and is not due to an interaction with -. however, toxicity increases dramatically in the presence of h . this may possibly occur either by a chemical formation of more potent reactive hydroxyl radicals or by independent actions on different cellular targets. although it is becoming increasingly clear that nitric oxide (no) is associated with otxan dysfunctions in septic patients, little is known as to die kinetics of no production in these patients to clarify these kinetics, we have investegated serum arid monocyte associated no in septic patients. five patients who had undergone gastrointestinal surgeries mid were complicated postoperatively with severe sepsis served as the subjects in this study (sgroup), using twelve healthy volunteers as a control group (c group). serum no and no levels were measul*d sliectrophotometricallymonocyte cultures were done for itrs with or without lps+ifn-t stimulation mid no and no levels in the supernat,'utts were also measured spectrophotometrically. furthermore,using an no selective electrode,time course of the lps+ifn-t induced no production by monocytes was studied. l) serum no levels in s group were slightly lfigher than dmse in c group,bnt there were no significoatt differences between the two. )both no levels in the supematmlts of monocytes cultured with lps+ifn-t mid no , no levels ill lhe supernatants of monocytes cultured without lps+ifn-y were significantly higher in s group. further,the time required for the no production by enltnred monoeytes was siglfificantly shorter also in s groap. conclusions l)in severe septic patients, monocyte no productions wei~ not only primed, but also activated concomitantly, suggesting that these monocyte activations m'e strongly associated with organ dysfunctions in sepsis. )based on the restdts of serum no nteasuremeuts, it can be deduced that no p~'oduced by monocytes in septic patients affects tissues and org~s ioeoregiomflly. objectives of the study: nitric oxide (no) is an important messenger which accounts for a wide spectrum of physiological and pathophysiological processes, e.g. mediating vasodilation in endoloxin shock investigating the signal lransducfion pathways involved in the regulation of the inducible nitric oxide synthase (inos), we report the involvement of phosphatidylcholinespecific phospholipase c (pc-plc) and proteinkinase c (pkc). materials and methods: no-production was induced in the murine macrophage cell line j with lipopolysaccharide (lps) [lljg/ml] and interferon ;f (ifny) [ u/ml]. after hours of incubation no-release was determined as nitrite (no ) by using a colorimetric assay based on the griess-reaetion. results: preincubation of cells with the pkc-inhibitors staurosporine (stp), chelerythrine, bisindolylmaleimide (bim) and d , that recently has been identified as a selective inhibitor of pc-plc, diminished significantly lpsand ]fnt-induced no -production (p<o, ). although we observed no toxic effect on cell viability, no -production was related to protein concentrations to exclude any inhibitory effect on celt growth. celts preincubated with stp [lo nm] released % less no in response to lps and ifn,[ compared to control cells cultured without inhibitor. chelerythrine [ }jm] and bim [ioijm] reduced the no -formation by % and %, respectively. interestingly, the most potent inhibitor of no-production turned out to be the xanthate d cells pretreated with d [ ~g/mt] released % less no than stimulated controls the inhibitory effect on no production decreased the later the inhibitore were added after stimulation; e.g. bim added and hours after stimulation reduced no -production only by % and %, respectively. accordingly, inqs activity present in stimulated cell lysates supplemented with l-arginine and co-factors was not suppressed by the inhibitors tested. conclusions: our findings suggest, that the induction of inos but not its activity is a pkc and particularly pc-plc dependent process. dr. k tschaikowsky, institut for anaesthesiologie, universital erlangen--nqrnberg, krankenhausstr , erlangen interleukin- (il- ) affects nearly every cell type by increasing the expression of a variety of genes associated with the promotion of inflammatory processes. these include upregulation of cyclooxygenase and nitric acid synthases. the il- ri transmits a signal(s) through the cytoplasmic domain which is structurally related to the fruit fly toll protein. we have recently cloned the promotor most proximal to the ' utr of the human il- ri gene. the genomic sequences reveal a lack of tata and caat boxes but rather a considerable ( %) gc rich region. the translation of the type i il- r appears under a significant suppression and may limit the biological activities of il- by low level of expression. on the other hand, the type ii il- r, lacking a significant cytoplasmic domain, does not transmit a signal and acts as a decoy receptor preventing the binding to the type i receptor. there are several approaches to reducing il- activity; inhibition of il- converting enzyme which cleaves pro-il-l[ , anti-il- ri, the il-i receptor antagonist (il- ra) and soluble (extracellular) il- ri and il- rii. il- ra is structurally related to il- and binds at °c to the il- ri with near equal affinity as that of bona fide il-i; however, il- ra does not transduce a signal. il- ra has been given to humans in pharmacologic levels (mean plasma levels of gg/ml) without evidence of agonist activity. in addition, there has been no affect on normal homeostatic parameters, suggesting that il-i does not play a role in health. in healthy humans given intravenous endotoxin, il-ira reduced endotoxin-associated neutrophilia by % and completely reversed endoloxin-iuduced immunosuppression, il-tra is produced naturally and is elevated in the circulation in several diseases. in a variety of diseases, plasma levels of il- ra are in -fold molar excess to those of il-i [ . it is unclear whether these endogenous levels of il- ra are sufficient to block il- activity in disease. a single injection of ]l- or ifna in humans does not induce il- but rather high levels of il- ra ( ng/ml). prof.dr.l.a. aarden interleukin (il ) is a multifunctionai cytokine with a central role in host defense mechanisms and consequently in inflammation. il is thought to be involved in the pathogenesis of various diseases. therefore, specific inhibitors could have therapeutic value. a human-mouse chimeric monoclonal antibody to il has been applied in a phasei clinical trial in patients with multiple myeloma and in primate models of sepsis. no toxicity was observed and the most remarkable outcome of this study was the build up in the circulation of il -anti-il complexes, suggesting that plasma il measurements represent a gross underestimation of il production in the patient. until now no natural antagonists of il have been described. the biological activities of il results from binding to a lowaffinity il -receptor (il r). the il /il r complex induces disulfide-linked homodimerization of the signal transducing receptor component, gp . studies on the structure-function relation of il revealed that disruption of the gpl -interaction site on il gives rise to a mutant il that, by virtue of its intact binding to the il r, acts as a potent antagonist on human hepatocytes and on human b cells. in vivo studies using this molecule will be initiated in the near future. the evolution of infla~ation involves a dynamic series of cellular events controlled by specific signals which are important to the initiation, maintenance, and final resolution of the inflammatory response. the various phases of inflammation are influenced via the expression and subsequent regulation of a number of key mediators which play a predominant role during each particular stage of the developing lesion. for example, the initial elicitation of blood born leukocytes to on inflamed area is a complex system that is dependent upon the expression of early response cytokineso these proximal mediators, including both interleukin-i (il-i) and tumor necroisis factor (tnf), are important in the initiation phase by activating both immune and non-immune cells and establishing a series of cytokine networks, thus, the above proximal mediators can stimulate endothelial cells, epithelial cells, smooth muscle cells, and fibroblasts to generate more distal cytokines. these latter cytokines include interleukin- (il- ), macrophage inflammatory protein-i (mip-i), and monocyte cbemoattractant protein-i (mcp-i). the importance of the above chemukines can be found in the role they play in leukocyte elicitation, as well as wound repair. numerous investigations have shown the diverse cellular sources of il- and the ability of il- to elicit neutrophils in vitro at pm to nm concentrations, however, recent studies have sho%~ an additional role for il- during the resolution phase of the inflammatory process. using corneal pocket animal models of angiogenesis/neovascularization, il- was found to be a potent angiogenic factor at concentrations ranging from - ng/ml. sequential fluorescein angiograms demonstrated that il- induced neovascularization by days, which regressed by weeks. in further analyses, both conditioned media recovered from either inflamed human heumatoid synovial tissue macrophages or lps-stimulated peripheral blood monocytes were found to possess potent angiogenic activity, which was effectively blocked by neutralizing antibodies directed against human il- . in addition, an il- antisense nligomunclentide blocked the expression of a functionally active angiogenic factor from lps treated monocytes. the above data demonstrate that il- has multifumctional activities during the initiation, maintenance, and resolution of a local inflammatory response. inhibits specific t-cell responses to soluble protein antigens and alloantigens. the proliferative responses of th , thl and th cd ÷ t-cell clones and cytokine production by these t-cell subsets are equally well inhibited. the inhibitory effects of il- are indirect and related to inhibition of antigen-presenting capacity and accessory function of the antigen-presenting cells (apc). however, il- also directly inhibits t-cell proli/eration induced by cross]inked anti-cd or anti-cd mabs (e.g. in the absence of professional apc), by specifically inhibiting il- gene transcription and il- protein production. il- also inhibits the production of the pro-inflammatory cytokines il-l-a, ~, il- , and tnf-a and the chemokines il- and mip-i-u, which are strong chemo attractants for neutrophils and macrophage, respectively. in contrast, il- enhances the production of the il- receptor antagonist, a cytokine with anti-inflammatory activity. il-l produced by monocytes downregulates class ii mhc expression and il- production by these cells in an autoregulatory fashion. collectively, these in vitro data indicate that il- is a powerful suppressor factor for immune-and inflammatory responses and therefore may have potential clinical utility as an anti-inflammatory agent. this notion is supported by recent studies which indicated that il- also prevents lethal lps-induced toxic shock in mice. five of the chamekines (hip-i~,era~tes. ip-io and il ) also chemoattraet t lymphocytes, while others act on mast cells. we have studied the effaces of these chemokines on t cell subsets. il preferentially chemoattracts uns~imulated t cells. raises ~ttracte resting as well as activated cd and cd memory t cells. ip- chemoattracts only preactivated (not resting) cd or cd t cells. hip-l~ preferentially chemoattracts gd t calla, while hip- more readily attracts the cd t call subset. the chemoklnes and ip-io were also sho~rn to promote the adherence of ~he same subsets of anti-cd activated t cells to ili activated human umbilical vein endothelial cells (hitvec). this was not associated with a~y i~crease in the number of lymphocyte adhesion molecules, bu~ could be inhibited by neutralizing monoclonal antibodies re lfa-i and vla- . these chemokines also rapidly increased ~ha adhesion of resting t l~phoeytes to plates coated wi~h integrlns (i-cam or v-cam) or matrix proteins such as f~bronectln. this induction of adhesion began wiehln ', peaked by hr and was completed in hr. this suggests that chemoklnee rapldly increase the binding affinity of adhesion prote~ns on t cells. we also recently observed ~hat mcaf/mcpi and rantes chemoattract unatimulated mast calls. furthermore, igs actlvared mast calls were chemoattraeted by mcaf, ra~tes, pf and mip.i~. however. ~he chemoklnee did not induce mast cells ~o release histamines. presumably, chemokines as regulators of the adhesion, migration and homing of all kinds of leukocytes participate in many types of inflammatory diseases. natural killer cell stimulatory factor or interleukin- (il- ) is an heterodimerie cytokine formed by two covalently linked glycosylated chains of kd and kd. il- was originally purified from the supermatant fluids of ebv-transformed human b ly~phobiastoid cell lines. however, in addition to b cells, phagocytic cells, i.e. monocytes, macrophages, and neutrophils, have been identified as the major physiological producers of il- . phagocytic cells produce il- in response to bacteria, bacterial products such as lps, and intracellular parasites. the cell types on which il- exert biological activity are t and nk cells. the major direct biological function of il- on these cell types are the induction of cytokine production, primarily ifn-y, the enhancement of a generation of cytotoxic cells, and a mitogenic effect on antlgen-activated lymphocytes. in part thromgh its ability to induce ifn-y production, il- , produced in response to infections, represents an important functional link between effector cells of innate resistance, phagocytic cells and nk cells, and effector cells of adaptiye resistance, t and b lymphocytes. the ability of il- to induce ifn-y production from circulating. nk cells and at least a proportion of t cells determines a rapid, antigen-independet production of ifn-y during infections. ifn-y acts as a potent enhancer of phagocytic and bacteriocidal activity of phagocytic cells, participating early in infection to activate some of the inflammatory mechanisms that represent the first innate resistance against infection. this antigem-independet activation of phagocytic cells that involves, in addition to il- , other monocyte derived cytokines such as tnf and il-l, has been shown to be important in experimental animals for the resistance against infection by microorganisms such as limteria monocytogens and toxoplasma gondii. in addition to this role, early in infection in the antigen-independet phagocytic cell activation, il- has a major effect in the ensuing antigenspecific admptive immune response by facilitating the generation of t helper type (th-i) response and suppressing a th- response. the presence of il- during antigen stimulation in vitro using human peripheral blood lymphocytes, or both in vivo and in vitro in experimental animals, induces generation of th-i cells producing ifn-y and il- , and inhibits the generation of th- cells producing il- . il- induces a direct differentiative effect on th-cells, priming them for high ifn-y production. ~is priming effect is stable and maintained even when t cell clones are cultured for extended periods in the absence of il- , however, il- needs to be present during the first few days after stimulation with antigen or mitogen and established th clones are resistant to the priming effect of ifn-y. unlike the generation of high ifn-y producing clones, the il- -mediated inhibition of il- producing cells in probably due to selective mechanisms, which may include a selective mitegenic effect of il- for thl cells and an inhibitory effect of ifn-y on th cell proliferation. in several experimental systems, it has been shown that the enhancing effect of il- on th-i responses is dependent on production of if~-y and on the presence of nk cells, possibly needed for the early production of ifn-y. the importance of phagocytic cells and nk cells in determining the characteristics of the th response during antigenic stimulation indicated that the mechanisms of innate resistance have a determining influence on the subsequent antigen-specific immune response: il- appears to have a key role in mediating the interaction between phagocytic cells. nk cells, and t lympocytes in these proccessem. trinchieri, g. , interleukin- and its role in the generation of t e cells, imm~ol. on peripheral blood monocyte/macrophages, il- behaves like il- in a variety of assays, and shows both similar and contrasting, activties with either il- or ifn-y. il- reduces inflammatory monokine and il- production. it reduces the pyrogen-induced expression of tissue factor (herbert et el, , febs lett. , ) . it mpregulates manmose receptor and ~c class ii expression, while reducing cdi expression. il- produces morphological changes (extensive cell processes, aggregation, giant cell formation) which have previously been observed with il- . it inhibits niv-i production (montaner et el, , i. exp. mad. , ) , and interferes with hiv-induced cell fusion. the activities of il- and il- differ on t-ly•ocyte and large granular lymphocyte population. in particular il- does not exhibit the suppressive effects of il- on thl-type-helper functions (ifny production) and cytotoxic functions (perforin production). data will be presented showing that il- and il-l share a common receptor on a nu~er of cell types, but not on t-lympocytes (see also zmrawski st el. , embo j. . - ), explaining both thai common and divergent activities. the levels of il- ~a in activated peripheral blood lymphocytes are greater than or equivalent to those of il- mp~ja and the two ml{nas are expressed by different t lympocyte population: in particular, t cytotoxic lymphocytes express markedly higher levels of il- ~rna. in vivo, il- may thus be contributing, significantly to some of the activites previously ascribed to il- il- . recently, we cloned the human cdna encoding interleukin- (il- ). human il- is a non-glycosylated protein of aa with a molecular mass (mr) of kd and has biological activities on monocytes and b cells, il- , like il- , strongly enhanced the expression of class ii mhc antigens on monocytes and induced expression of cd (fc~rii). furthermore, il- , like il- and il~ , inhibited the production of pro-inflammatory cytokines il- , ]l- , tnfo~ and chemokines miplc~ and tl- by lps activated monocytes. both il- and il- enhanced the production of il-lra by monocytes. in contrast, il- lacked the t cell stimulating activities of il- . the responses of monocytes to il- and il- were not additive or synergistic, supporting the hypothesis that il- and il- receptors are complex and share a common component which is important for signal transduction, taken together, these data indicate that il- , il~ and il- have important anti-inflammatory activities on human monocytes. in addition, these results indicate that il- is unique in that it can modify inflammatory reactions without stimulating (as does il- ) or inhibiting (as does il- ) t cell responses. transforming growth factor beta i (tgf-fll) belongs to a family of proteins that have potent immunoregulatory properties ranging ftom effects on the growth and dfflerentiafion of primitive stem cells to the differentiated functions of immune system effector cells. tgf-i~i is synthesized as a large secretory precursor polypeptide of amino acids that is inactive until processed to a disulfide linked homodimeric molecule of t amino acids each. recently it has been found that tgf-[~i can have autocrine as well as paracrine effects on the immune system indicating that immune cells can activate the inactive secreted form of tgi~-~i. in addition, tgf-i~i has differential intraceuular effects on cell surface receptor modulation, tyrosine phosphorylation and cytokine gene transcription as well as cell mediated cytotoxicity. the systemic administration of tgf- has proven beneficial in a variety of animal models such as septic shock, allograft rejection and experimental forms of autoimmunity including collagen induced arthritis and experimental autoimmune encephalomyelitis. moreover the increased levels of tgf-ill and other tgf- isoforms found in several disease states associated with immunosuppression such as different forms of malignancy, chronic degenerative diseases and aids implicate the involvement of tgt~-i~ in the pathogenesis of some diseases. hopefully, well designed clinical trials will define the potential roles of the tgf-[ family of proteins in the treatment of diseases of man. patient development of systemic inflammatory response syndrome (sis) after severe trauma is accompanied by a wide range of immune aberrations and altered cytokine production. in particular, t lymphocyte proliferation and ifn, t production is suppressed while monocyte (mo) tnfc~, il- , pge and tgf~ production is massively increased concomitant to decreased antigen presenting capacity. recently, two new cytokines, il- and il- , have been characterized as critical in regulation of mo tnfa and il- , as well as in induction of t lymphocyte proliferation and ifn production. in this study, peripheral blood leukocytes (pbl) from severe trauma patients (injury severity score > ) were analyzed for their il- levels, their in vitro proliferation to mitogen (pha) and their response after il- addition. since il- produced either by mo or by t lymphocytes can depress m~ antigen presenting capacity, inhibit t cell ifn,/production and directly diminish t cell proliferation, it might be suggested that immunosuppressed patients' mo and/or t lymphocytes would have increased il- levels. increased patient il- production might also be resulting from the high levels of tnfa a known stimulator of il- . conversely, since il- augments mo antigenpresenting capacity, thl induction and proliferation, post-trauma leukocytes might be il- deficient. pbl of trauma patients were compared to normals' pbl, either unstimulated or ptta induced, and their levels of il- found to be dramatically and significantly reduced. patients' isolated m~, either stimulated with the bacterial cell wall analogue, mdp, or unstimulated, also had depressed il- production concomitant to elevated tnfa production when compared to normals' mo. mechanisms for the depressed patients' mo il- were explored. increases in tgf[ may have partially contributed to the patients' depressed il- level, but elevated pge had no effect. addition of il- to patients' pbl significantly increased their mitogen responses. these data imply that sis is characterized by disruption in the interactions between mci and t lymphocytes so that patients' m~i produce excesses of some mediators (tnfa, il- , pge ) and a dearth of other monokines (il- , il-io). t lymphocytes are not activated and, therefore, unable to function in both immune defense and monocyte regulation. it is known that lge receptor-mediated or ca-ionophore-induced activation of mouse bone marrow-derived mast cells ( mmc) may result in the production of different cytokines including the interleukins (il) , , , and as well as gm-csf and tnf-a. in the present study we analyzed the effects of exogeneously applied pro-inflammatory cytokines (il- , l- , tnf-c as well as various mast cell growth factors (il- , il- , il- , il- , ngf, kl (kit ligand)) on cytokine production in primary mouse bmmc using a standard activation protocol (lxl bmmc/ml; ll.um ionomycin; - h). the actixdties of bmmc supernatants were assessed in specific biological (il- , il- il- , l- ) and/or elisa assays (il- , il- ). here we show that homogeneous populations of bmmc (> %alcian blue+/safranln-; in vitro age: weeks) generated in the presence of recombinant (r) rail- from normal balb/c mice produced modest amounts of l- and low or undetectable levels of il- , - , and - after induction with lp.m ionomycin only. however, a dramatic increase ( -to -fold) of these cytokine activities was noted, when in addition to ionomycin also human ( ) rll-la was provided during the induction period. this il- effect was dose dependent with a maximgm at - u/ml hrll-la and specific, as pre-incubation (lh) of bmmc with ng/ml hrll- receptor antagonist abolished the action of u/ml hrll-lcc similar effects were noted with hrll-lg or rurll-lb (lng/ml, respectively), but not with rhll- or rmtnf-~. both mrll- and hrll- substantially enhanced ionomycin-induced l- production of bmmc in the absence or presence of il- . il- significantly enhanced il- and il- production while decreasing il- activities to abont - % of control levels, when il-i was provided in the presence of il-l/ionomycin. a monoclonal anti-nfil-t antibody (ascites : ) abrogated the effects of mrll- . other mast cell-active cy~okines (] ,- , il- , l- , ngf, or kl) added to ionomycia-or l- /ionomycin-treated bmmc had no major effects on cytokine production. il- and il-i did not induce significant cytokine release in the absence of ionomycin suggesting tlmt cadependent signalling was required. at doses of " m, dexamethasone, corticosterone, or hydrocortisone almost completely abolished ionomycin/il- /ll- induced cytokine production. the inducer cocktails per se did not interfere with the cytokine bio-assays. in case of il- inducibility of this cytokine in bmmc was confirmed at the mrna level by northern blot analysis. hence our data show that activated mast cells are a source of il- previously recognized as a product of th type lymphocytes only. moreover, our study reveals novel functional roles for i-l-i, il- , and ghicecorticoids in the regulation of cytoldne production in mast ceils. accumulating data suggests that cytokines, peptides involved in regulation of both physiological and pathological immunological responses, predominantly are produced at the local site of antigen stimulation. a new method was used to detect cytokine-producing cells in haman tissue at the protein level. single-cell production of different httman cytokines, ilia, ill [ , illra, il , il , il , il , il , ils, ill , gm-csf, tnfa, ifn and tgf[ . , was identified by indirect immunohistochemical staining procedures and use of carefully selected cytokine-specific mab's. frozen sections were fixed with % paraformaldehyde and permeabilized by . % saponin treatment, eluting cholesterol from the membranes. the intracellular presence of all cytokines except ill, illra (late) and tfg[ _ , could be demonstrated by a characteristic perinuclear configuration in producer cells. in addition, the immunoreactivity extended over a large extracellular area encompassing the producer cell. a localization of the cytokine to the golgi-organelle was established by use of two culour staining including a haman golgi complex specific mab. this staining pattern was only evident in producer cells because injection of recombinant human cytgkines into the tissue caused a membraneous and extracellular staining pattern. both the extra-and the intracellular types of staining reaction could, however, be blocked by preincubating the cytokine specific mab with pure human interleukins. oxygen radicals (or) directly induce lipid peroxidation, indirectly they trigger adhesion and activation of pmn leukocytes. we investigated whether or also lead to a release of acute-phase response cytokins such as tnf-alpha, il-i beta or il- in whole blood cultures to maintain the induced inflammatory reaction. methods: blood samples from healthy volunteers (n= ) were incubated at °c. or were produced by the xanthine oxidase (xo)/ hypoxanthine (hx) system. after , , , , and minutes plasma levels of tnf-alpha, il-i beta and il- were determined with elisa kits. results: under the influence of or tnf-alpha plasma levels increased from , pg/ml at min to pg/ml, pg/ml, pg/ml after , and min. il-ibeta ( , pg/ml, , pg/ml, , pg/ml, pg/ml and pg/ml after , , , and min) and il- ( , pg/ml, l,lpg/ml, , pg/ml, pg/ml and , pg/ml after , , , and min) plasma levels were increased min later than tnf-alpha. summary: these data suggest that or do not only play an important role in initial accumulation and activation of pmn leukocytes but also lead to a stimulation of monocytes to produce the acute phase reaction cytokins tnf-alpha, il-i beta and il- to maintain and strengthen the inflammatory reaction. department of general surgery, steinhsvelstr. , ulm, germany jan k. horn md, greg a. hamon md, robert h. mulloy md, greg chen bs, rebecca chow bs, and christof birkenmaier md. transforming growth factor-i~l (tgf- ) is released from inflammatory ceils following injury and in sepsis. in vitro experiments have confirmed that low concentrations of tgf- ( . - . ng/ml) are chemoattractive for monocytes, whereas higher levels of tgf- (> . ng/ml) potentiate production of the immunedepressive prostaglandin e . other investigators have shown that tgf-] can cause the appearance of cd (fc immunoglobulin receptor) on monocytes exposed to ng/ml of tgf-[~i for hours. monocytes also express on their surface a glycoprotein that binds complexes of lipopolysaceharide (lps) and lpsbinding protein (lbp). such binding is associated with generation of proinflammatory cytokines such as tumor necrosis factor alpha. we have shown that cd is depressed in septic patients and therefore we hypothesized that tgf- could account for the down-regulation of cd observed in these individuals. we incubated normal human monocytes with platelet-derived tgf-[ for and hours at °c and examined ceils for cd and cd expression using flow cytometry after immunnfluoreseent staining with appropriate monoclonal antibodies. monocytes were selected on the by usual criteria for size and granularity. non-viable ceils were excluded with the use of propidium iodide. two populations of monocytes could be found afcer incubation at °c alone. one displaying high density of cd had increased fluorescence over the homogeneous expression of cd in cells maintained at °c (baseline). the other population displayed decreased cd expression relative to the baseline cells. tgf-i~i ( - ng/ml) caused a shift of ceils from the high density into the low density cd population. this trend was observed within hours of incubation and was complete by hours. we observed a net decrease in cd expression f % for all subjects studied (p< . vs controls). phorbol myristate acetate ( ng/ml) also caused down-regulation of cd to a similar degree as tfg-i~i. we also confirmed that monocytes could be induced to express cd after incubation with tgf- ( ng/ml) for hours. these studies demonstrate that monocytes incubated with immunodepressive levels of regulation of cd by tgf- deplete their surface expression of cd while generating cd . this down-regulation of cd by tgf- correlates with our clinical observations of lower cd expression on monocytes obtained from septic patients. for over years, activated t lymphocytes have been considered to be the cellular source of mif. we recently isolated and cloned the murine homolog of mif after identifying the specific secretion of this protein by lpsstimulated pituitary cells in vitro and in vivo. however, further experiments showed that mif protein is detectable both in t-cell deficient (nude) and hypophyseetomized mice, suggesting that yet additional cell types may produce mif in vivo. since monocytes/macrophages are a major source of the cytokines that appear in response to lps administration, we examined the possibility that mif also is expressed in cells of the monocyte/macrophage lineage. we found that mif is expressed constitutively in the murine macrophage-line raw . and in thioglycollate-elicited peritoneal macrophages. significant amounts of mif mrna (rt-pcr) and protein (western blotting) were observed in cell lysates. in raw . cells, mif secretion was induced by as little as pg/ml of lps (e.coli l:b ), peaked at ng/ml, but was not detectable at lps concentrations > txg/ml. similar data were obtained with elicited macrophages, but higher lps concentrations were required, unless the cells had been preincubated with ifn . production of mif by lps-stimulated (l ng/ml) macrophages peaked at hr. expression ofmif mrna and tnf mrna by lps-stimulated raw . macrophages was investigated by rt-pcr. as expected tnf mrna expression increased over the range of lps concentrations ( pg/ml to p_g/ml). in contrast, levels of mif mrna correlated inversely with lps concentration. by competitive pcr, mif mrna was observed to increase approximately -fold after lps induction ( pg/ml). mif secretion also was induced by tnfoc ( ng/ml) and ifn? ( iu/ml), but not by il- and il- (up to ng/ml). lps and ifn had additive effects in inducing mif secretion. in separate experiments, macrophages stimulated with recombinant mouse mif ( gg/ml) were found to secrete bioactive tnf~ (> pg/ml by l cytotoxicity). we conclude that the macrophage is an important albeit overlooked cellular source of mif in vivo. mif secretion is induced by lps, tnfc~ and ifn?. mif also stimulates macrophages to secrete tnf. taken together with previous observations that anti-mif antibody protects against lethal endotoxemia, these data implicate mif as a critical mediator of inflammation and septic shock. inflammation is characterized by an exacerbation of proinflammatory cytokine production. cytokines such as il- , il- , and tgf , have been identified as anti-inflammatory mediators thanks to their ability to down regulate the production of il- , il- , il- , tnfc~ by activated monocytes / macrophages. however, other cells, including polymorphonuclear cells (pmn) do contribute to the release of pro-inflammatory cytokines. we investigated the capacity of the so-called anti-inflammatory cytokines to control the release of il- by activated neutrophils. human pmn were purified following glucose-dextran sedimentation and ficoli-hypaque centrifugation. the cells were cultured at °c for h in the absence or presence of lipopolysaccharide (lps) or tnfa. il- release was measured in the supernatants using a specific elisa. among tested cytokines, il- was the most efficient inhibitor of il- production by lps-activated pmn. il- was also active, whereas no down regulation was noticed with tgfp~i. when tnfa was used as a triggering agent, none of the cytokine could prevent il- production. northern analysis are under investigation to precise the level of the il- -and il- -induced inhibition of il- production by pmn. our data illustrate that il- and il- possess the capacity to down regulate the production of il- by both monocytes and pmn, whereas tgfb has a more limited inhibitory activity. ciliary neurotrophic factor (cntf), a member of the il- superfamily, has recently been shown to promote axonal growth and neuronal healing. cntf production is also increased during neuronal and muscle damage, associated with soft tissue injury or trauma. we postulated that production of cntf may explain the loss of skeletal muscm protein that occurs in inflammation. female, wistar ( - gm) rats received either or pg/kg bw s.c. injections of recombinant rat cntf for seven days, or received sham injections and were freely-fed. additional animals were pretreated with mg/kg ibuprofen lp prior to pg/kg bw cntf. rats treated with ,ug/kg bw cntf lost . _+ . gms bw as compared to freely-fed controls which gained . _+ . gms (p<o. by anova and lsd), and ibuprofen treatment had no effect on cntf-induced weight loss (- . _+ . ). animals pair fed to the high dose cntf gained body weight ( . -+ . ), although weight gain was less than seen in freely-fed controls. rats treated with pg/kg bw had reduced body weight gain (+ . +_ . gin). food intake was also reduced by % in pg/kg cntf (from . +_ . gm/day to . _+ . gm/day; p<o. ) and was also unaffected by prior ibuprofen treatment ( . +- . gm/day). despite the loss in body weight noted in the high dose cntf animals, there was no appreciable hepatic acute phase response, since liver weight ( . + , gms vs. . _+ . gins), total protein ( . +- . gms/l vs . +- . gins/l) and albumin ( . + . gms/l vs. . +_ . gms/l) were not different between cntf and freely fed animals. we conclude that cntf causes anorexia, and increases body weight loss in excess of the associated anorexia. unlike the cachexia associated with il- and tnf infusions, which is associated with an acute phase response, cntf acts predominantly on food intake and carcass weight through a prostaglandin-independent mechanism and has only minimal acute phase inducing properties, n. joseph espat, md. university of florida, department of surgery, j-box , gainesville, fl. . of cytokines by neurotrophic factors, the neuroendocrine system and phenotiazines. cytokines, including tnf and il- , have a pathogenetic role in various diseases related to infection and inflammation. the action and/or production of cytokines can be regulated by drugs or endogenous mechanisms that involve the cetral nervous system (cns) we found that the antipsychotic chlorpromazine (cpz) potently inhibits tnf production and protects mice against endotoxic shock. cpz also inhibits brain tnf production in mice intracerebrally injected with endotoxin, whereas cpz analogs that do not cross the blood-brain barrier inhibit only perypheral, not brain, tnf. tnf production and susceptibility to the toxicity of endotoxin, tnf and il- are increased in adrenalectomized mice, indicating that endogenous corticosterone (cs) controls cytokine production and toxicity, in a feedback fashion since endotoxin and cytokines increase serum cs. increased tnf production was also observed in mice where the hypothalamus-pituitary-adrenal axis was blocked by chronic administration of dexamethasone, following a two-day washout. administration of cytokines acting through the gp signal transd,uction protein, including il- and ciliary neurotrophic factor, cntf potentiated il-l-induced elevation of cs. il- and cntf also induced hepatic acute-phase proteins. thus il- and cntf might have antiinflammatory activity, by potentiating the feedback responses of the neuroendocrine axis. these data indicate how complex can be the interregulatory relationships between the brain and the immune system, how they can be used to pharmacomodulate cytokine action and how their disregulation might exacerbate il- -and tnf-mediated pathologies. lipopolysaccharide (lps) constitutes a well established activator of monocytes/ macrophages (mizi) and murine b lymphocytes. the hydrophobic part of lps, termed lipid a, represents the endotoxic principle of lps. we now demonstrate that lps is also capable of inducing proliferation of human t cells at a dose of to pg/ml. the specificity of this stimulation was analysed by the following experiments: i) the synthetic hexaacyl escherichia coiltype lipid a (compound ) also stimulated human t cells; ii) the synthetic tetraacyl lipid a precursor (compound ) or the phosphono-oxyethyl analogue pe- , structures known to antagonize lps-induced monokine production in human mz, also inhibited lpsinduced t-cell proliferation. the t-cell proliferation expressed the following features: i) cd ÷-as welt as cd ÷ t cells proliferate in response to lps, ii) limiting dilution analyses reveal that the frequency of responding cells was between / to / cells; iii) purified t cells alone cannot be stimulated by lps, but need the help of monocytes. this help cannot be replaced by b cells or fixed monocytes. furthermore, for activation a direct cell-to-cell contact between t cells and monocyte is neccessary; iv) t cells stimulated with lps express mrna for il- and ifnf, but not for il- or il- (determined by pcr). in supernatants, ifnf was detectable (elisa); v) lps-activated cd ÷ as well as cd + t cells express cd , the high affinity il- receptor. our results indicate that in contrast to previous reports human t cells respond to lps with il- receptor expression, lymphokine production, and proliferation. the reactive cells appear to belong to the t, cell type, the finding that human t cells can be activated by lps is of important relevance for the understanding of the pathomechanisms of infections by gramnegative bacteria. this may lead to new strategies for the therapy of sepsis. we have recently shown that human eosinophils produce mrna for interleukin (il)- when stimulated with calcium ionophore (eur. j. immunol. ( ), ). we now present evidence that human eosinophils contain preformed il- protein although they do not express mrna for il- as measured by rt-pcr. il- protein expression was determined using specific anti-human il- monoclonal antibodies by western blotting, facs analysis and immunohistochemistry. moreover, preformed il- was re/eased into supernatant by % pure eosinophils after priming with gm-csf or il- and subsequent -min stimulation with paf, rantes, ionomycin or pma, however not with il- or il- , as measured by elisa. this observation implies that activation of protein kinase c and/or intracellular calcium mobilization are necessary events for il- release in human eosinophils. the determined amounts of preformed il- protein was higher in patients with asthma compared to normal individuals suggesting a role for eosinonhi! derived il- in asthma. since the eosinopnit is the ,,r~:=z~mmant cell in the asthmatic airways, we determined il- concentrations in bronchoaiveolar lavage (bal. ~ids from normal !i~.~ijvtduals and asthmatic patients. indeed, il- concentrations in bals from both groups were similar to those seen in the supernatants released by activated eosinophils. the role for il- in asthma remains to be determined. based on a well established rat model named activity-induced ulceration (asu) or activity based anorexia (aba), we have developed a rat model for chronic stress. male rats were exposed to a feeding schedule in order to reduce body weight by - % within days. while one group of rats was kept sedentary, the other had access to a running wheel. food restricted rats allowed to exercise developed highly increased activity as compared to ad libitum fed rats in wheels (ca. versus kin/day). they developed elevated plasma corticosterone levels ( _+ gg/dl) at their circadian peak, increased adrenal weight and decreased thymus and spleen weight as compared to control rats and weight matched sedentary rats, establishing this paradigm as a model of chronic stress. no differences in plasma acth were seen among the various groups, suggesting increased sensitivity of the adrenal glands to acth. the effect of chronic stress accompanied by hypercorticism on baseline and endotoxin-induced cytokine levels was studied in this model. under baseline conditions,splenic il- a was suppressed in both food-restricted groups. this was more pronounced in rats with access to a running wheel. il- and tnf activity in plasma was not affected. after administration of .tg/kg lipopolysaccharide (lps), tnf activity in plasma was suppressed by food restriction but there were no differences between sedentary or exercising rats. in addition, no differences were found among groups for corticosterone, spleen il-lc~ and plasma il- activity. we conclude, that the food restriction-induced hyperactive rat model is a useful model for studies on the effects of chronic stress. in this model, under basal conditions il-le~ in tissues may be subject to downregulation by glucocorticoids, whereas after lps-stimulation of cytokine production only tnf activity is regulated by fast acting feedback loop between cytokines and the hypothalamo-pituitary-adrenal axis (hpa-axis). this is supported by strong correlations between plasma tnf activity and corticosterone in stressed rats. cytokine antagonists modulate cytokine actions and it appears that a balance between production of cytokines and cytokine antagonists is important to control of host responses. recent observations show that during myocardial infarction there is an increase in circulating cytokines such as tumor necrosis factor (tnf) and interleukin (il- ). we tested the idea that compensatory increases in cytokine antagonists likewise occur and investigated changes in plasma concentrations of tnf and interleukin i (il- ) and those of their specific antagonists interleukin- receptor antagonist (il- ra) and soluble tumor necrosis factor receptor type i (s tnf r-i). we also measured plasma concentrations of ~-melanocyte-stimulating hormone (~-msh), an endogenous neuropeptide that occurs in pituitary, brain, skin, gut and other tissues. when administered to laboratory animals, this peptide has potent antiinflammatory and antipyretic activity, perhaps by mimicking an endogenous modulatory influence on cytokine actions. samples were obtained from patients with acute myocardial infarction at presentation in the coronary unit, every three hours for hours, and daily thereafter until discharge. we found elevations in the plasma cytokines il- and tnf only in a proportion of subjects whereas cytokine antagonists c~-msh, il-lra, and stnfr were elevated in all patients, o~-msh was elevated at presentation but it decreased progressively in successive tests, reaching normal values within hr whereas stnfr and l-lra peaked at - hr or later. there were significant correlations between plasma concentrations of cytokine antagonists and enzymes released from injured myocardium, including creatine kinase (ck), aspartate serum transferase (ast), and lactate dehydrogenase (ldh). the data show that cytokine antagonists increase in the circulation of patients with acute myocardial infarction likely to modulate prninflammatory effects of cytokines. objects of the study: interleukin- (il- ) is a multifunctionai cytokine known to be involved in important homeostatic processes. il- levels are increased in many pathological situations, and correlates with disease activity and patient outcome. to exert its effect il- binds to its receptor on the membrane of its target cell. this receptor is also present in a soluble form in plasma and in urine. in this study we quantitatively measured the availability of soluble il- receptor (sll- r) in biological fluids in healthy volunteers. materials and methods: blood, urine, cerebrospinal fluid (csf), and synovial fluid (sf) are obtained from healthy volunteers, sil- r and il- are measured using elisa's. both elisa's are tested for interference of sil- r mid il- . the effect of sll- r in the il- bio-assay (b ) is tested. results: baseline levels are (mean + sd): . -+ . ng/ml in serum, . -+ . ng/ml in urine, . _+ . ng/ml in csf and . + . ng/ml in sf. there is no interference of sll- r in the il- elisa or bio-assay and no interference of l- in the sil- r elisa. conclusions: in all investigated biological fluids sll- r can be found. these data provide baseline values for investigations concerning pathological situations. both elisa's described are useful tools to do these investigations. trauma-associated immune aberrations coincide with augmented release of immunoregulatory and proinflammatory cytokines. the present study examines the effect of thermal trauma on the capacity for specific (receptormediated) response of lymphoid cells to interleukin (il ) and to turnout necrosis factor ~x (tnfc . methods. bum patients (n= , -> % total body surface area) were studied weekly up to days post-injury. the limulus amoebocyte lysate (lal) test was used to measure plasma endotoxin levels. the percentage of il ~-and tnfcz-binding t(cd ) lymphocytes was assessed by flow cytometry analysis. levels of il receptor antagonist (il lra) in patients' plasma and cultures of peripheral blood ceils (pbc) were determined by immunoassay. results. plasma endotoxin concentrations were significantly (p< . ) increased up to weeks post-bum (means . + in non-surviving and . + . u/ml in surviving patients vs < u/ml in the control). within weeks of bum, the percentage oft ceils expressing receptors for tnfa and il [~ constitutively was elevated (by - fold). in contrast, the capacity for de novo receptor expression by activated pbc was reduced. serum levels of il ira were significantly increased (range . - x j pg/ml vs < . x j pg/ml in the control). in all patients, high concentrations of il lm were released spontaneously in unstimulated cultures of adherent ceils (range - x - pg/ml vs - x j pg/ml in the control). however, its secretion was decreased in lps-stimulated parallel preparations. conclusions. in the bum patient, susceptibility to the immunoregulatory effect of tnfcz and tl ~ may be modulated by infection-related products. alterations in the capacity for receptor expression and secretion of l lra may affect il -regulated biological responses including specific immune reactions. while studies suggest that il- is an important lymphokine involved in cell-mediated immunity, little is known about this mediator's role in hem-induced immunesuppression. our aims, therefore, were to determine: i) if il- contributes to depressed t-cell responses seen following hem; and ) how other agents, known to play a role in hem, effect il- release. to study this, c h/hen mice were bled to and maintained at a map of mmhg for h and then adequately resuscitated. mice were killed h post-hem to obtain splenic t-cells (nylon-wool purified). il- 's immunosuppressant role was demonstrated by the ability of monoclenal antibody (mab) to il- to markedly improve the t-cell proliferative response [ . #g the marked increase in capacity of t-cells from hem mice to produce il- was significantly reduced by treatment with either ibu or mabs. since ibu, tgf-~, as well as il- are all reported to directly/indirectly influence prostanoid synthesis, this implies that eicosanoids play a major role in inducing il- release by t-cells following hem which depresses t-cell function. the mechanisms underlying immunosuppression induced by thermal injury and alcohol ingestion are in part due to cytokine dysregulatinn. il- down-regulates production of eytokines by maerophages and may be an important regulator of the initiation of the immune response. il- has also been demonstrated to inhibit the production of no by macrophages. this study examined the alterations in eytokine production and effect of inhibition of no production on immunologic function in a routine thermal injury model. methods: balb/c mice (n= ) were randomized to groups: saline-sham(ns-sham), alcohol-sham(etoh-sham), ns-bum, etoh-bum. animals received % etoh or ns daily for days by gavage. a % full thickness bum was induced hrs after the last dose of etoh or ns. animals were resuscitated, then sacrificed days post bum. splenic lymphocytes were cultured for days with lps, and lps with two concentrations of n-monomethyl-l-arginine, a nitric oxide inhibitor (l-nmma . ug/ml, ug/ml). splenocyte production of il- , interferon-gamma, il- , pge were measured, and lymphocyte proliferative response examined. results: il- production was significantly suppressed in thermal injury. exogenous l-nmma normalized the suppression of .- in a dose-dependent manner, indicating nitric oxide may modulate il- and interferon-gamma production in thermal injury. il- production is normal in etoh-burn animals. conclusion: il- and interferon-gamma production is altered in this murine thermal injury model, and may contribute to this injury-induced immunosuppression. inhibition of no synthesis normalizes il- production and should be investigated further as an immanomodalator in thermal injury. surgery, infection and inflammation results in the production of pro-inflammatory cytokines which mediate metabolic and immunologic host responses. the aim of this study was to characterise the elaboration of cytokine release following a variety of surgical procedures. twenty one patients undergoing elective intermediate, hip, knee and major gastrointestinal surgery were studied. levels of interleukin- (i - ), interleukin- (i - ), the interleukin- receptor antagonist (i - ra) and the acute phase c-reactive protein (crp) were measured in bloods drawn , , , , , , and hours following operation. a portion of the results are shown (mean -+ sem). + -+ _+ one and two factor anova; *p< . , #p< . , §p< . , ¶p< . , for differences between groups i - was not detected at any time point. both ii-ira and i - increased after surgery. maximum responses occurred following major git and hip surgery, minimal responses were seen after intermediate and knee surgery. ii-ira levels increased within two hours and remained elevated for hours; the b-ira increase was a thousand fold greater than the rise in i - levels. i - levels increased up to hours after surgery. crp levels reflected maximum ii-ira and i - levels (r =. , p< . and r =. , p< . respectively). high ii- ra and i - levels reflect major surgery, however the ii-ira response is more rapid and of greater magnitude. the strong i - ra correlation with crp may indicate that this regulatory cytokine is itself a mediator of host responses to surgery. dept. of surgery, meath/adelaide hospitals, heytesbury st., dublin , ireland. change of il- and soluble il- receptor levels after surgery s. hisano, k. sakamoto, s. mita, t. ishiko, m. ogawa [objectives] under surgical stress, il- plays a main role in producing acute phase proteins and contributes to host defense mechanism. soluble il- receptor (sll- r) is considered to be agonistic to il- , unlike other soluble type receptors of cytokines. here we measured il- and sll- r levels in the serum and drain fluid from surgical field in order to investigate the changes of il- and sll- r after surgery and their origins. [materials and methods] serum and drain fluid samples from cases ( of esophagectomy and of gastrectomy ) were serially collected before and after surgery. il- and sll- r levels were measured by elisa. [results] ( ) serum il- : all cases reached the maximum level on pod-l, more precisely - hours after operation. ( ) il- in the drain : maximal il- levels in the drain were recognized - hours after operation, at almost the same time as serum il- . furthermore the il- values in the drain were much higher, about times, than those in serum. ( ) sll- r in the serum : all cases reached minimum levels - hours after operation and recovered to the preoperative levels a few days later (decrease ratio : . + . ~,, range : - ~'). ( ) sll- r in the drain : sll- r levels in the drain showed almost the same value and change as serum sll- r. [conclusions] ( ) il- is produced from the cells gathering around operative fields whereas sll- r is considered to be produced in the cells which do not gather around the operative fields. ( ) there may be a mechanism that down-regulates sll- r in the early stage of surgery. [objectives] il- plays an important role in host defense in the early stage after surgery. in the present study, we examined changes in il- concentration after major thoracoabdominal surgery and elucidated the effect of surgical trauma and factors influencing postoperative elevation of serum il- . [materials and methods] thirty-eight patients undergoing elective surgery of the thoracoabdomen were classified into groups according to the location of the operation. bloods and drain fluids were serially obtained and samples were frozen until measured, keukocytes were simultaneously collected for northern blot analysis. concentration of il- was measured by elisa and il- mrna was detected by northern blotting after total rna was extracted by the acid guanidium phenol chloroform method. [results] ( ) serum il- levels reached the maximum concentration on the st postoperative day in all patients. ( ) the il- peak was significantly correlated with surgical trauma as defined by the operation length and the volume of blood loss during operation (r= . , p< . , r= . , p< . , respectively). ( ) the peak concentration of serum il- in patients undergoing esophagectomy was significantly higher than in those undergoing pancreaticoduodenectomy (p< . ), despite a similar degree of surgical trauma. ( ) peak l- concentration observed in a patient who underwent esophagectomy was about fold greater in the drain fluid of thorax than in the peripheral blood. ( ) il- mrna was demonstrated in leukocytes from thoracic and abdominal exudate at , and hours after surgery. in contrast, il- mrna could not be detected in leukocytes from the peripheral blood. [conclusion] il- is mainly produced in the operative field and subsequently enter the peripheral blood to induce cytokinemia. the operation length, volume of blood loss and thoracotomy are factors influencing the concentration of cytokine in the blood. zaragoza spain age may be an important factor influencing the function of immunocompeteut cells releasing cytokines after both accidental and surgical trauma the aim of the present paper is to ascertain if patients (pts) over years old show a different serum level cytokine pattern than pts under after a standard surgical procedure considered as a "medium strength trauma". patients and methods: pts( females males)with gallstone disease were perspectively studied, pts were allotted in two groups: gr.a: pts under years(mean age: . +- )gr.b: pts over years(mean age: . _+ ). all pts underwent cholecystectomy and cholangiography. pts in gr.a and pts in gr. b underwent common duct exploration. spbintercctomy was performed in each group. on the day of surgery (pre) and on the st and th postoperative day(leo, po) : percentages of cd , cd , cd , cd and cd cells we measured by means of flow cytometry using moab. and levels of il- , il- , il- and tnf "in vivo" by elisa using moab. results: ere: cd % was . _+ in gr.a and . objectives of the study. after surgery for esophageal cancer multiple organ damage has been reported to be caused by polymorphonuclear leukocyte (pmn)-mediated injury. we measured serum granulocyte colony-stimulating factor (g-csf) and interleukin (il- ) levels to determine a role of g-csf and il- in pmn function after surgery for esophageal cancer. materials and methods. peripheral pmn counts, peripheral pmn chemiluminescence, serum g-csf levels, and serum il- levels were measured before and after surgery in patients with esophageal cancer (ec), and patients of gastric cancer (gc). esophagectomy with thoracotomy and laparotomy were performed for patients with ec, while subtotal gastrectomy with laparotomy were performed for patients with gc. results. peripheral pmn counts (p< . ) and peripheral pmn chemiluminescence (p< . ) of patients with ec were significantly decreased compared to those of patients with gc at and hours after surgery. serum g-csf levels of patients with ec were significantly (p< . ) increased compared to those of patients with gc at and hours after surgery. serum il- levels of patients with ec were significantly (p< . ) increased compared to those of patients with gc at , and hours after surgery. significant inverse correlations (p< . l) between peripheral pmn count and serum g-csf and il- levels were seen at hours after surgery. conclusion. these results suggest that many circulating pmns, which are excessively activated by g-csf and il- , may adhere to the endotherial cells and then migrate into the tissues, and cause multiple organ damage after surgery for esophageal cancer. immunnogical changes in patients with severe brain trauma receive increasing attention since morbidity and mortality ere still high. interleukin- (il- ) was previously detected in the cerebrospinal fluid (csf) during different pathologies of the nervous system ( , , ). in our study we monitored il- and nerve growth factor (ngf) production in the csf after human brain trauma. since astrocytes within the brain constitute one of the major cell type contributing to the inflammatory response through the release of cytokines and other factors after injury, we investigated the functional relationship of il- and ngf on a single cell niveau using cultured astrocytes. methods csf was obtained from patients with severe brain injury (glasgow coma score (gcs) < and ct abnormatities or gcs < over hours) after implantation of intraventricular icp monitoring device for therapeutic purpose and collected over hours csf and serum. il- and ngf were assayed by elisa. astrocytes were isolated from neonatal mouse brain as described ( ) . ngf production by cultured astrocytes was measured by elisa in the presence of csf, il- and il- antibody. astrocyte migration was tested in a chemstaxis chamber. results head trauma patients were included in this study (approved by the university hospital medical ethics board) and the csf was obtained through intraventricular catheters. high levels of il- were detected in the csf of these patients when compared to serum during the first days after brain trauma. furthermore ngf could be found inside the intracerebral compartment. csf containing high levels of il- could stimulate ngf production in cultured astrocytes. this effect could be [nhibited partially by il- antibodies, purified il- exposed to cultured astrocytes in vitro, stimulated the migratory activity of these cells in a dose response fashion. il- was found in the csf of brain injured patients, suggesting a role for this cytokine in the pathophysiology of brain injury. since astrocytes are involved in maintaining the homeostasis of the brain, we further investigated the possible role o il- on astrocyte functions, il- promoted ngf production in vivo and in vitro, thus contributing to neuronal cell survival and regeneration. furthermore il- stimulated astrocyte migration in a dose response fashion, potentially contributing to astrocytosis following brain injury and inflammation, these results show that il- represents a key cytokine in traumatic human brain injury with possible systemic effects, which are at preserlt under investigation. we studied a) the role of tnf and b) the therapeutic effect of a mab to tnf with regard to haemorrhagic shock (hs) related ,pathophysiologic alterations and mortality in rats. method: a prolonged hs was induced by bleeding to a blood pressure of - mmhg for pin followed by reinfusion of shed blood (sb) and resuscitation with two times of sb volume of ringer's lactate over rain. animals received a bolus dose ( mg/kg) of tnf mab (celltech, berkshire, uk) at min after resuscitation (tn ). the control group (n = ) was treated similar to the tn group but received ringer's lactate (con). results: at min the prolonged hs resulted in a metabolic acidosis indicated by a significant decrease of blood ph ( . + . ), hco -( . ___ . mm), and base excess (- . + . ram) values with pco ( . + . mmhg) and po ( . + . mmhg) in the tn with no difference to the con group. immediately after resuscitation ( min) plasma endotoxin levels were found to be increased in both groups ( . + . in tn vs . _ . pg/ml in con group) . prior to the treatment with tnf mab ( min) there was also no difference between plasma tnf levels of the two groups ( . + . in tn vs + . pg/ml in con group). treatment with the tnf mab at rain post-hs improved the hour survival rate to . % as compared to . % in the control group. macropathologic evaluations revealed frequency of intestinal bleeding in oniy animals in the tn vs in the con group. no bleeding in the kidneys was found in the tn but in rats in the con group. the significant increase in lung wet weight observed in non-survivors in the con (n = ) was prevented in animals which died in the tn (n = ) group (( . +_ . vs . +_ . g/kg). conclusion: our data suggest that tnf formation induced by hs in rats is an important mediator for pathophysiologic alterations leading to multi organ failure and lethality. antibodies to tnf might be a useful agent in the treatment of haemorrhagic shock related disorders. -+ n=ll*$ -+ n= _+ n= * * p< . vs baseline :~p< . no anesthesia vs anesthesia thus ) tnf production increased - fold by - hrs following trauma in unstimulated blood, but was reduced or not changed after lps stimulation, so circulating leukocytes are probably not an important source of tnf post trauma; ) anticd had no obvious effect on tnf production in unstimulated or lps stimulated blood, relative to vehicle, which suggests that the protective mechanism of anticd does not involve tnf suppression; ) fentanyl anesthesia at hrs following trauma unexpectedly decreased lps-evoked tnf production, which suggests that anesthesia alone can influence an inflammatory response. proinflamrnato~ cytokines have been shown to play a signific~t role in the pathogenesis of sepsis, which is a very common occurrence in born injury. tnfa is infrequently detected in the blood of burned patients, the ability to detect the shed receptors of stnfg has not been determined. serial serum mmples from burn patients were collected from the time of admission until death from septic shock. these samples were analyzed using an enzyme-linked immunosorbent assay (elisa) for stnfr, l-ira, tnf-a, and il-ib. the patients ranged in age from to yeas of age. the percentages of bum ranged from % - %. cytokine concenlrntions vmled from patient to padent irrespective of bum size. tnfa levels were consistentiy in the range of pgjml - pg/ml. peaks in the tnfa values were above pg/ml and were also associated with a peak in the stnfr levels. these levels began at < , pghnl within the in,st ins of injury and gradually increased with time. clinically. ti~ appearance of eytoklnes was independent of positive wound, blood, or respiratory cultures however peak values in tnfa and stnfr were ~ialed with a fluid requirnmenl levels of il-i ra were also elevated independent of clinical findings as well as extent of injury. in pl there is a significant corresponding peak in il-trn (> ~ /ml) at the same time as t/~:a and stnfr levels. we aimed to characterise the pattern of secretion of interleukin- beta l-ii ), intefleukin- (il- ) and tumour necrosis factor alpha (tnfa) in multiply injured patients and to relate these results to their clinical condition and outcome. two hourly blood samples were taken from ten patients from the time of injury until hours. cytokine levels were measured using sandwich enzyme-linked immunosorbent assays (elisas). injury severity scores (iss) were calculated and haemorrhage was assessed from the blood transfusion requirement over the hours. patients' ages ranged from to years. iss varied from to and transfusion requirement from to units. five patients died after the study period. ] ,- was raised in / patients (max level , pg/ml) but was unrelated to condition or outcome. / showed a rise in il- b (max level pg/ml) which was negatively correlated to iss (i=- . , p< . ). tnfa was raised in / (max level pg/ml). peak tnfc~ was positively correlated with iss ( = . , p< . ) and haemorrhage (i= . but p< . ). il-ib and tnfa production was mutually exclusive. there was no common cytokine profile for these patients. unlike elective surgery there was no correlation between peak ,- and severity of injury: tissue damage may not be the stimulus for the cytokine response to multiple injury. periods of ischemia or hypoxia produce endothelial damage in peripheral organs. tumor necrosis factor-alpha (tnf) plays a central role for regulation of endothelial physiology during septic events, taking influence on vascular permeability and coagulant activity [ ] . animal experiments demonstrated a synergism between hypoxia and septic shock on letality, leading to the hypothesis that low oxygen tension leads to enhanced sensitivity of target cells for tnf [ ] . radioligand binding studies with ~ odid-tnf on cultured human endothelial cells were performed after incubation in several environmental oxygen tensions (pc ) for hours. data were achieved by nonlinear regression of an idealized saturation curve according to the equation: b = n " k./( + k,); b = totally bound tnf; k,: association constant (concentration for half-maximal binding); n: number of binding sites per cell. p_o o (mm h¢i): _k, (nm}: n (molecules/cell): - . ± . _+ - . ± . + - , ± . -+ - . + . -+ presented are calculated values on the idealized curve + % percentiles. hypoxia induces enhanced binding of tnf to specific receptors on the endothelial cell surface in a time-and dose-dependent manner by a mechanism, which is not dependent on oxygen radicals, as shown by additional protocols with radical-scavenging drugs. with respect to former findings about a correlation between growth and tnf receptor affinity [ ] , these data lead to the hypothesis that enhanced tnf binding during hypoxia is due to a biochemical conversion of the receptor protein from the low affinity to the high affinity state, possibly by posttranslational phosphorylation of the binding protein by intracel)ular kinases. the proposed involvement of tnf-dependent pathways in pathogenesis of organ dysfunction and multiple organ failure after hypoxia/ischemia may provide a basis for understanding the initiation of hypoxic vascular injury, as manifested by increased permeability and prothrombotic tendency, and, thus, merits further attention. the levels of activity of circulating cytokines (ill, il- and tnf-alpha) which are believed to play important regulatory role in response to trauma are determined (by hioassays and respective anti-cytokine antibodies) in mice and rats subjected to scald injury ion c, see, ° v bsa, ld ) and ( c, see, ~ b ~^)~ , respectively. biphasic increase of cytokine activity was noted in mice: initial increase of il-i and il- , - hr following injury and of try activity hr after scald, followed by elevated levels of il-i and il- at hr, with tendency of decrease of activity at later time points. increased activity of tnf was noted hr following injury, in rats, initial, short-lived increase of il-i and tnf activity was detected lhr following injury, folowed by increase on days i and postburn. il- increase peaked - hr after scalding and levels remained elevated - days following injury. similar kinetics of appearance of proinflammatory cytokines (il-i and tnf-alpha) both in lethal and ncnlethal injury concomitant with differential profile of circulating il- activity (early,short-lived increase and later slow decrease of activity in lethal burn injury) with late persistent high levels of activity in nonlethai injury demonstrated in the present study highlight the need for investigation the relationship of these cytokines in burn-injury induced inflammation. zikica jovicic,lnstitute for medical research, mma,crnotravska , belgrade~yu. asadullah k ( ), woiciechowsky c ( ), liebenthai c ( ), doecke wd ( ), volk hd ( ), vogel s ( ), v. baehr r ( ); depts. of med. immunology ( ) and neurosurgery ( ) , medical school (char#d), humboldt university berlin, frg in patients after polytrauma or major abdominal surgery a hyperinflammatory phase seems to be followed by the development of a phase of monocyte inactivation. the latter is charaeterised by a decrease of monocytic hla-dr expression and a shift to anti-inflammatory cytokine production. as shown, by us and others, this phenomenon indicates severe immunodepression with a high risk of infection. however, the mechanisms leading to monocyte inactivation in the above mentioned syndromes may be multiple. to elucidate the influence of a selective, sterile trauma to the central nervous system (cns) on immune reactivity the neurosurgieal patient is an interesting model. initially, patients who developed a systemic inflammatory response syndrome following neurosurgery were analysed. in all of them a marked decrease of monocytic hla-dr expression was observed soon after the operation. these results suggest that neurosurgery alone can induce immunodepression and lead us to conduct a prospective study, in which we closely monitored l patients undergoing neurosurgery from the first preoperative day until at least day after the operation. hla-dr expression was decreased hi all patients to various extent only hours after surgery. in one patient only we found a persistently reduced hla-dr expression and this was the only patient to develop sepsis syndrome. this suggests that a prolonged, postoperatively decreased hla-dr expression is predictive of infection following cns trauma. in order to assess, whether a decrease of hla-dr expression was associated with a preceding inflammatory response, local cytokine release in the cns was compared with systemic cytokine release. for this purpose, paired samples of earebrospinal fluid (csf) from a vantricle drainage and peripheral blood plasma were obtained. in the csf extremely elevated futerleakin (il)- levels, peaking already a few hours after the operation were found. in plasma, by eontrast, il- ( and tnf-alpha) was detectable not until days later and only if infection was present. the antiinflammatory ili-ra, on the other hand, was also present in csf but peaked after il- and was detectable in peripheral plasma too. we believe there is an association between the inflammatory response in the cns and the following depression of hla-dr expression on peripheral blood monocytes. our results suggest that even a sterile cns-trauma by itself may contribute to general immunodepressinn leading to septic complications. the aim of this study was to evaluate the effect of haemorrhagic shock (hs) a) on total capacity of the host, and b) the circulating blood cells to produce tnf immediately after bleeding. in vivo studies: baboons were subjected to a limited oxygen deficit ( - ml/kg) hypotension phase (mean arterial pressure = map of - mmhg for - hours followed by adequate resuscitation). rats subjected to hs (map of - mmhg for rain followed by reinfusion of shed blood and fluid resuscitation) were challenged with endotoxin ( ~g/kg i.v.) at the end of shock (rhs group). the control group (rco) received the same dose of endotoxin as rhs group but without prior bleeding. in vitro studies: whole blood (wb) obtained from both baboons and rats before and at the end of hs were incubated with endotoxin ( ng/ml) for hrs at °c. results: at min post-lps challenge we found significantly higher plasma tnf levels in rats that were subjected to hs prior to the endotoxin challenge as compared to the control group ( _+ vs + pg/ml) . after hs the tpc was significantly decreased in in vitro stimulated cbc of both rats ( + post-hs vs + ng tnf/ml pre-hs) and baboons ( ± post-hs vs ± pg tnf/ml pre-hs). in contrast, the il- productive capacity was increased in baboons cbc (not yet analysed in rats) stimulated at the end of hs ( ± pre-vs ±_ pg il- /ml post-hs). conclusion: from our data we suggest that despite of down regulation of the cbc to produce tnf the overall tpc is enhanced at the early stage of i-is. with regard to the related literature (chaudry's group) it can be assumed that among the macrophage/monocyte populations, as the main source only the kupffer cells (kc) exhibit enhanced tnf production capacity following haemorrhage. the mechanisms of down/up regulation of cytokine response of cbc and/or kc following hs remain to be examined. d. eg~er, s. geuenich °, c. dertzlin~er °, e. schmitt*, r. mailhammer, h ehrenreich #, p. drrmer, and l. h mer gsf-instimt fox experimentelle h~znatologie, °medizinische kliulk iii, klinikum groghadern, munich, *institut for immunologic, johannes gutenberg universit/it, malnz, and #psychiatrische k/in& der georg-aagust-universi~t, grttingen, germany. it has been shown previously (ehranreich et al., , new biol. : ) that mouse bone marrow-derived mast cells (bmmc) synthesize and secrete endothelin- (et-i) and express eta-type endothelin receptors (eta). so far, however, no functions of et- /et a in bmmc have been described. in the present study we investigated the effect of exogeneously administered et- on the release of histamine, serotonin, and leukotriene c (ltc ) by primary mouse bmmc (in vitro age: weeks) caltured with different recombinant mttrine cytokines (interleukin (il- ) and/or kit ligand (kl) in the presence or absence of il ) for two weeks prior to activation. et- ( x - to lxl - m) induced an extremely rapid (_<lmin) and dose-dependent release of histamine and serotonin (up to % and % of total mediator content, respectively, comparable to ige/ag-indueed mediator release) from bmmc cultured with il- and il- . only when cultured in the additional presence of il- rather than in medium containing kl or kl plus il- alone, bmmc released histamine and serotoaln upon challenge with et- .furthermore, et- stimulated ltc biosynthesis up to -fold in bmmc cultured in the presence of il- . in contrast, the peptide was without major effect on ltc biosynthesis in bmmc culturd without il- . the release of histamine and sereotonin was not altered if bmmc were preincubated for i h with il- prior to activation with et-i, suggesting that a differentiation process rather than a functional priming effect had been initiated by ]l- . et- ( " m) -induced histamine and serotouln release from bmmc was inhibited by an eta-specific antagonist (cyclic id-asp-pro-d-val-leu-d-tel ) in a dose-dependent manner, with a complete inhibition observed at an antagonist concentration of - m. crude peritoneal cells (containing - % serosal mast cells) obtained from normal balb/c-mice released % histamine upon challenge with et- ( - m; rain). taken together, these resu/ts demonstrate that et- can directly function as a histamine and serotohin secretagogue and as a stimulator of ltc production in mast cells. il- appears to be involved in the differentiation of immature mast cell prec~trsors towards an et-l-reactive functional phenotype. when inflammatory reactions are advanced, type ii phospholipase a (type ii pla ) is produced in high levels by various cells at the site of inflammation. cytokines such as tnf-a and il-i activate type ii pla and promote the production of eicosanoid, which is one of the mechanisms by which they enhance the inflammatory reaction. in the present study, the blood levels of type ii pea , endotoxin, tnf-c~, il- and il- were determined in patients with sepsis to examine the relationship between these levels and the patients' pathological conditions. the levels of type ii pla and tnf-a in these patients were . ± . ng/ml and . ± . pg/ml, respectively, the two parameters being significantly correlated (r = . , p = . ). there were also a significant correlation between the level of type ii pla an il- (r = . , p = . ). there was no significant correlation between the level of type ii pla and il- . these in vivo findings suggest that tnf-a may be involved in the production of type ii pla . a. filzmaver, g. reisbach, e. schmitt °, r. mailhammer, and l. hiittner. gsf-iustitut ff~r experimentelle h~imatologie, munich, and °iustitut fttr immunelone , johannes gutenberg universit~t, mainz, germany the product of c-kit, a transmembrane tyrosin ldnase receptor, and a corresponding kit ligand (kl) are critically involved in the control of different hemopoietic cell lineages including the proliferation, maturation, migration, and function of mast cells. it has been reported previously that interleukin (il)- down-regulates kit receptors in human primary myeloid leukemic cells and in a human mast celt line (sinaber et al. , j. immunol. : ) . transforming growth factor (tgf) gl, was also found to down-modulate c-kit mrna and kit receptor proteins in human aml blasts, a mechanism probabely contributing to the anti-proliferative effect of tgfih on kl-stimulated aml ceils in vitro (de vos et at. , cancer res. : ) . in porcine endothelial cells transfected with a retroviral construct carrying the human (hu) e-kit gena, exogenous hu kl transiently down-regulated kit receptors (blume-jeusen et al. , embo j : ) . in the light of these previous findings we analyzed the effects of these exogenously applied cytokines (il- , tgfgl, kl) on kit receptor expression and kl-mediated proliferation and chemotactic migration of primary mouse bone marrow-derived mast cells (bmmc) (in vitro age: weeks). our results show that in bmmc cultures initiated with recombinant (r) marine (mu) il- the additional presence of rmull- for or days did not change the expression of kit protein (assessed by facs analysis with the anti-mouse c-kit antibody ack- ) nor kl-mediated proliferation or chemotaxis. in contrast, il- syeergistically increased kl-stimulated growth of bmmc in a short-term proliferation assay (mtt-tes . pre-incubation of bmmc with rhutgfl~ ( . , . , or . ng/ml) for h had no effect on kit receptor expression, although tgffil at concentrations of . - . ng/ml completely suppressed the proliferative action of kl on these ceils. similar anti-proliferative effects of tgfg were observed in various factor-dependent mast cell lines stimulated with different mast cell growth factors (il- , i,- , il- , and/or il- ). moreover, pre-incuhation ( h) of bmmc with tgf-gl (> pg/ml) significantly enhanced spontaneous undirected cell movement (chemokinesis) and synergistically increased il- -or kl-induced chemetaxis. when bmmc were preancuhated with rmukl ( ng/ml) for , . or days, a transient down-modulation of kit receptors with a maximum effect on day was demonstrated by facs analysis and correlated well with a decreased chemotactic response of these cells. in conclusion our results show that neither il- nor tgfi affect expression of kit receptors in primary murine bmmc. it is reasonable to suggest that c-kit expression is controlled in a cell type-specific manner.interestingly, tgfgl is obviously able to dissect the proliferative from the migrational signal transducted by kl in these cells. objectives of the study: antisense strategies using dna-otigonucleofides (odn) to modulate the cytokine response are presently under investigation. odn are thought to act very specifically with little or no relevant negative side effects. we now report that odn unspeeifically protect wehi cells from tnf-mediated cytolysis. material and methods: wehi subclone ceils ( x ), that are highly sensitive to the cytolytic activity of tnf, were grown on -well culture plates in rpm medium. after hours, phosphorothioate(ps)and partially ps-modified-odn as well as phesphodiester-odn ( - bp) were added ( . , and pm). four hours after incubation with odn, ce(i lysis was induced by recombinant murina tnf. after hours the plates were washed and stained with crystal violet cell lysis was determined by reading the absorbance (abs) at nm. results: wehi ceils incubated with tnf ( - ng/ml) were completely lysed after hours ( % abs). interestingly, wehi cells incubated with tnf and odn resisted complete lysis, eg cells incubated with . ng/ml tnf and jm odn showed still % of the absorbance observed in control ceils without tnf ( % abs). the protective effect of odn started at . pm, reached a maximum at ,um, and diminished at jm. with increasing amounts of tnf the protective effect of qdn decreased and no protection was detectable at ng tnf per ml conclusions: dna-oligonucleotides were found to unspecifically inhibit tnf-induced cytolysis. we hypothesize, that this protective effect of qdn results from an inhibition of the binding of tnf to its receptor, or from interference of odn with the subsequent signal transduction mechanisms. as a consequence, to discriminate the specific effect of odn in biologic systems, several control odn should be used. secondly, whether dna released by degradation of tumor cells or leukocytes can significantly impair tumor-and immune-defense mechanisms merits further investigation dr. med. michael meisner, institut for anaesthesiologie der universitat erlangen-nqmberg, krankenhausstral~e , d- erlangen. in this study we investigated the involvement of serine protease and free radical generation in the systemic release of tumor necrosis factor-alpha (tnf) and interieukin i(il- ), in the sepsis model of lipopolysaccharide (lps, mg/kg i.p.) induced hepatitis in galactosamine (gain, rag/mouse, i.p.) sensitized mice. treatment of gain-sensitized mice with lps (gain/lps) led to dramatic increase in serum cytokine (tnf and il-i) ievels and transaminase activity at hr and hr respectively. pretreatment of serine protease inhibitor, c~jantitrypsin (a j-at, mg/kg i.p.), rains prior to gain/lps treatment, fully protected the animals against the hepatotoxic challenge with significantly reduced serum tnf and il- levels. in order to block and scavenge superoxide generation, the mice were pretreated with xanthine oxidase inhibitor, allopurinol (al, x mg/kg i.p.) and pyran polymer-conjugated superoxide dismutase (sod, x unit/mouse i.v) r spectively. pretreatment with al and sod ( and hr prior to gain/lps) prevented gain/lps hepatitis and blocked lps induced released of tnf and il- into serum of the mice. the protective agents like cq-at or al/sod did not protect the mice against th~ hpp~totoxi£ ch~llpn-e indllee b'~ th~ recombinant mmlse tnf-o' ( . ~/rno~e j.p.) ~d oi~lps ~ caln-.~dlfa%aed mlce. it-l cett~aged la tnf (x/gain treated mjde was not detectable in animals pretreated with oq-at or al/sod. our study suggests that a serine protease sensitive to cq-antitrypsin is responsible in regulating tnf release, possibly by proteolytic cleavage of a tnf-precursor or membrane bound tnf. in addition our evidence suggest that the balance of extracellular protease/antiprotease activity may be regulated by free radical generation, possible superoxide anion, resulting in inactivation of the antiprotease. il- release may be subsequent to tnf release. objective: during sepsis one can observe a dramatically impaired production of proinflammatory cytokines like the tumor necrosis factor alpha (tnf-a), interleukin i-alpha (il-la), intedeukin i-beta (il-i&) and interferon gamma (if~) upon in vitro stimulation of circulating cells. however there is also evidence of a decreased ability to produce cytokines in other immuno-deficient states. in this study we compared the capacity to secrete proinflammatory cytokines upon in vitro stimulation of patients in severe sepsis and patients with malignant tumors. methods: heparinized blood samples of ten patients ( + years) in severe sepsis (sepsis score > according to e}ebute and stoner) were drawn at onset of disease, from fifteen patients with solid growing carcinoma ( + years) blood was drawn at diagnosis prior to any therapy. controls were obtained from fifteen healthy volunteers. pl of whole blood were incubated either with / of a standard medium or with pl of a standard medium and pl of phytohemagglutinin (pha) a potent mitogen. after an incubation period of hours plasma concentrations of tnf-a, il-la, il- and if-~ were determined by elisa. comments: our results suggest that down-regulation of cytokine secretion or of cell responsiveness to non-specific mitogens during sepsis has occurred. we observe a similar phenomenon for the group of carcinoma patients vs control significant for stimulated tnf-a and stimulated if-t. sustained immunological interactions between tumorcells and cytokine producing cells could effect responsiveness of the latter, a general increased immuno-tolerant state in patients with carcinoma has to be discussed. however we found significant differences between sepsis and cancer concerning the in vitro capacity of responsable cells to produce il-la and il-i#. the dramatically decrease of the ability to produce il-i upon in vitro stimulation could be more sensitive for a septic state than stimulated tnf-a or if- ,. objective: tumor necrosis factor alpha (tnf-a) has been implicated as a central mediator of sepsis and its sequelae. increased systemic levels of this cytoklne seem to be correlated with severity of sepsis and outcome. however mechanism of action and metabolism of tnf-g are not fully understood. in most studies blood samples for tnf-a determinations are obtained either by peripheral venipuncture, a central venous catheter or by an indwelling arterial catheter. very often blood samples are taken in different manners within the same study. in this study we measured circulating tnf-a and the amount of tnf-a released upon in vitro stimulation in arterial and central venous blood. methods: heparlnized arterial and central venous blood samples of ten patients ( males, females, mean age +_ ) with severe sepsis (sepsis score > , elebute and stoner} were drawn on day , , , , and of disease. blood was immediately placed on ice and processed within hour. pl of whole blood were incubated with pl rpmi-medium supplemented with antibiotics and l-glutamlne or with pl of rpmi-medium and pl phytohemagglutinin (pha) a potent mitogen. after an incubation period of hours samples were centrifuged and plasma was harvested and stored at - ° celsius before assessment of tnf-a concentration by elisa. statistical analysis was performed with the paired student-t-test. results: we found a significant difference (p < , ) for circulating mean arterial tnf-a concentration ( pg/ml _+ sem} and central venous tnf-a ( pg/ml +_ sem). upon in vitro stimulation there was also a significant difference (p < , ) between released arterial tnf-~' { pg/ml _+ sem) and venous tnf-a ( pg/ml +_ semi. conclusions: these results are difficult to interprete but could reflect the influence of pao and sao on tnf a release. it could also be the result of different concentrations of tnf-o release influencing factors like for example endotoxin, interferon-f or prostaglandin. a possible pulmonary and/or a hepatic metabolism of tnf-n and tnf-a producing cells cannot be ruled out. however for better interpretations of tnf-a release in septic states it is necessary to use either arterial or venous blood samples. early inflammatory processes following trauma and/or infections were found to be associated with the secretion of high amounts of proinflammatory cytokines. besides intedeukin-t (il- ), tumor necrosis factor-a (tnf-c and interleukin- (il- ) the multifunctional cytokine intedeukin- (il- ) was described to be a central regulatory element of the primary cellular and humeral defence reaction. the previously described close temporal correlation of pathologically elevated il- -concentrations and the extracellulary release of lysosomal enzymes from activated pelymorphnuclear neutrophils suggests, that il- may be a potential substrate of these preteases. the serine preteases elastase (ec . . . ) and cathepsin g (ec . . . ) derived from the azurophilic granules were assumed to be mainly involved in unspecific proteolysis at sites of inflammation by cleavage of structural as well as soluble proteins at random sites, if the inhibitory potential is decreased. the possible proteolytic activity of elastase and cathepsin g toward the proinflammatory cytokine interleukin- (il- ) was investigated. the addition of purified neutrephil elastase and cathepsin g to recombinant human il- leads to a rapid sequential degradation in vitro. at least two intermediate products could be detected by silver staining and western blotting following protein separation under reducing conditions. the serine protease inhibitor g-anitrypsin was shown to prevent the proteolytical degradation of intedeukin- . furthermore the loss of the biological activity of both, recombinant and natural human il- , was demonstrated by determination of the capacity of protease-treated il- to stimulate hybddoma growth ( td bioassay). these data suggest a possible downregulation of pathologically elevated il- levels by proteolytic activity of extracellulary released enzymes at sites of inflammation. the aim of the study was to compare circulating levels of three cytokines -il- , il- , _- -between critically ill subjects who developed gram-negative sepsis and who did not. materials and methods: the patient population consisted of patients admitted to an intensive cars unit, with different underlying diseases. sepsis diagnosis was given according to pre-estabilished cdteda. nineteen cases were enrolled in sepsis group, twenty in control group. serum sampling was collected in sterile tubes at study entry and every three days until study dismissal. serum concentrations of il- , _- and il- were measured using commercially available test kits, based on the dual immunometric sandwich principle. results: the causative patogens of sepsis were: pseudomonas aeruginosa, acinetobacter, eseherichia co~i, serratia marceseens, proteus mirobilis and citrobacter freundl the time of observation was equal to days, for a total of four tests performed (to, tl, t , t ). i .- was not detected in any samples. the serological profiles of the two cytokines .- and _- were similar; augmented levels were found at study entry and throughout the observation period, peaking at t and decreasing at t . however, in patients with sepsis, il- and _- concentrations were significantly higher in respect to control group. conclusion: our observations shown that in icu patients increased il- and il- release may be induced by cdtical illness; however, in subjects in which sepsis occurred, il- and il- production appears more significantly elevated, suggesting a role of il- and _- in the pathophysiology of sepsis. the fact that ii. objective: to check whether continuous veno-venous haemofiltration (cvvh) could remove the cytokines, namely tumour necrosis factor alpha (tnfc and interleukin (il- ) from the circulation of critically ill patients with sepsis ad multiple organ failure (mof). setting: the intensive therapy unit of the medical school teaching hospital. patients: nine critically ill patients with sepsis and mof treated with cvvh. methods: blood samples were collected before the cvvh had been started. then, blood and ultrafiltrate samples were collected simultaneously after hours and every hour. tnfct and il- levels were measured using the bioassays with cell lines wehi- ci and td , respectively. other data were recorded from the patient notes and intensive therapy unit charts. results: no measurable concentrations of tnfct were detected in either blood or ultrafiltrate samples. il- was found in all the patients' plasma samples and five patients' ( . %) ultrafiltrate samples. the il- blood level ranged from . to . u/ml (mean . , sd . ). the il- level in positive ultrafiltrate samples ranged from . to . u/ml (mean . , sd . ). conclusions: our preliminary results suggest that il- is present in bloodstream of septic patients. we assume we could not detect tnfa in any sample because we usually started observations when septic state had developed. cvvh could extract cytokines from the circulating blood. it remains under discussion, whether that extraction may be beneficial to patients with mof. the pattern of some significant cytokines tnf, il- and il- and their pharmacomodulation were evaluated in an experimental model of polimicrobial sepsis induced in cd- mice by cecal ligation and puncture (clp) in order to understand their roles. this model of sepsis, which resembles the clinical situation of bowel perforation, was also compared with that induced by administration of pure endotoxin (lps). tnf was detectable in serum and tissues during the first h with a peak h after clp at a significantly lower level than after lps. il- was measurable in serum only after h, significantly increased in spleen and liver after and h and in mesenteric lymphonodes from to h after clp compared with shammice. il- was significantly increased in serum throughout the first h after clp. pretreatment with dexamethasone (dex), ibuprofen (ibu) and nitro-l-arginine (n-arg) significantly reduced the survival time while chlorpromazine (cpz) and tnf did not affect it. only the antibiotics and pentoxifylline (ptx) significantly increased the survival in clp. however cpz and dex protected from lps-mor~ality. in conclusion, by inhibiting tnf with dex, cpz, ptx a reduced, unchanged and increased survival time was observed and by increasing tnf with ibu and tnf administration the survival was decreased or unchanged respectively suggesting that the modulation of this cytokine does not seem to play a significant role in clp unlike lps_ moreover the negative effects of ibu and n-arg suggest an important and protective role by prostaglandins and no in clp. to gain more insigths on the contribution of tnf~, il-i~ and if to lps toxicity, we explored the time-course of the cytokine production in ealb/c mice given different doses, from the lethal (= ld ) to the sublethal (= / ld ) of three different lps (e.coli oiii:b and :b ; p.aeruginosa r ) endowed with different degree of toxicity cytokines were measured in serum and organs with specific elisas up to i h after lps administration. results demonstrate that i) circulating and organ levels of tnf~ do not reflect lps toxicity. in fact, the lethal dose of lps :b induced as much tnf~ as the sublethal dose of lps :b ; furthermore, lps r , whose cytokine inducing capability is far lower than that of lps from e.coli, induced higher tnf~ levels at the sublethal than at the lethal dose. in addition, policlonal anti tnf ab, that were able to protect mice from e.coli lps induced mortality, failed in mice treated with lps r ) circulating il-i~ levels are generally low and increase significantly only in muribond animals. on the contrary, in spleen and lung very high levels of il-i~ are persistent from i to h post lps administration moreover, the treatment with mgr of neutralizing policlonal anti il-i~ ab, did not modify survival in lps challenged mice. ) circulating and organ levels of if are proportional to the dose and degree of toxicity of all the administered lps even if lps r was again a less efficient cytokine inducer than lps from e.coli. csa is an immunos~ppressive drug, able to inhibit gene expression for many cytokines, including if . to study the effect of cytokines modulation on lps toxicity, csa was administered to mice twice at the oral dose of i mg/kg before the challenge with lps. mice were monitored in terms of mortality and tnf~, il-i~ and if production. together with the total ablation of if , the strong reduction of tnfu and unmodified il-i~ levels, a significant increase of lps toxicity was also observed. these results suggest the hypothesis that the numerous factors that jointly mediate lps toxic effects, can also be protective, the final outcome depending on their relative ratio rather than on the absolute amount interleukin- (il- ) mediates the septic shock syndrome and affects intestinal secretion in vitro. we studied the intestinal production of il-t and its effects on diarrhea during endotoxic shock. cd- mice were randomized to mg/kg e.coli :b lps or saline infusion (i.p. or i.v.). diarrhea invariably occurred following lps infusion. mice were sacrificed at , ', lh, . h, h, h, h, and h ( mice/group/time-point). the small bowel was compressed and the intestinal contents were weighed and expressed per g sb weight. the small (sb) and large bowels (lb) were eventually frozen, weighed, and homogenized for either cytosolic protein or total rna. il-i~ (cell-associated agonist) was measured with a radioimmunoassay specific for mouse il-l~ (detection limit pg/ml) and expressed as ng/g weight + sem (lowest detectable amount ng/gwt). northern analysis of total rna and in sfu hybridization of paraformaldehyde-fixed frozen tissue were done with [ ~- p]-iabeled mouse il-lc~ cdna probes. only sb had il-i~ constitutively present ( . + . ng/gwt). lps i.p. or i.v. induced elevation of il-lc¢ in both organs in a biphasic pattern; lps i.v. induced -fold more il-i~ than lps i.p. following lps i.p., il-i~ in sb was . + . ng/gwt at lh, reached maximal levels at . h ( . -+ . ng/gw-i) and returned to baseline at h. saline controls maintained their constitutive il-i~ levels. sb had fold more il- ¢ than lb and identical kinetics, but lb showed a clearer doseresponse. northern analysis of sb-total rna showed induction of il-i~ mrna by lps in correlation with il-lc¢ kinetics. il-i~ mrna producing cells were mononuclear cells in the lamina propda and epithelial cells at the bottom of the crypts of ueberkuhn. mucus and fluid were increased in the small bowel post-lps in correlation with intestinal il-lc~ kinetics (r = . ). separate mice were pretreated with saline i.p. orthe il- receptor antagonist (irap, mg/kg bolus i.p.) and were challenged rain later with . mg/kg lps i.p. or saline i.p. specific blockade of il- by irap decreased intestinal secretion at h and h post-lps challenge (p<_. . , student's-t-test). these data indicate that local (intrinsic) intestinal il-i~ mediates sepsis-induced intestinal changes. inflammatory cytokines initiate the host response to endotoxemia, causing severe physiological and hemodynamic changes which may lead to septic shock. among the regulatory systems that play an important rote in controlling host inflammatory responses is the pituitary. it has been known for many years for example, that hypophysectomized animals are extremely sensitive to lps lethality. while investigating the possibility that protective, pituitary mediators might explain this phenomenon, we identified the cytoldne mif to be a specific secretory product produced by pituitary cells in vitro and in vivo after lps challenge. analysis of serum mif levels in control, t-cell deficient (nude), and hypophysectomized mice revealed that pituitary-derived mif contributes significantly to the rise in serum mif that occurs after lps administration. of note, pituitary mif content ( . % of total pituitary protein) and peak serum mif levels ( - ng/ml) were determined to be within the range observed for other pituitary hormones that are released after pituitary stimulation. to investigate a possible beneficial role for mif in septic shock, we co-injected mice with purified, recombinant murine mif (rmif) together with lps ( mg/kg). surprisingly, rmif markedly potentiated lps lethality compared to control mice that were injected with lps alone ( % vs. %, p = . ). to confirm these results, mice were treated with anti-rmif antibody prior to injection of a high dose of lps ( . mg/kg). anti-rmif antibody fully protected mice against lps lethality, increasing survival from % to % (p = . ). serum levels of tnf,~, the first cytokinc that appears in the circulation after lps challenge, were reduced by . _+ . % in anti-rmif-treated mice. we conclude that pituitary derived mif contributes significantly to circulating mif in the post-acute response in endotoxemia and may act in concert with other pituitary mediators to regulate both pro-and antiinflammatory effects. moreover, mif may play a critical regulatory role in the systemic host response in septic shock. our results suggest that anti-rmif antibody might be of potential therapeutic use in the treatment of septic shock. although anti-interleukin- (il- ) antibodies and il- receptor antagonist have been shown to improve survival in animal models of endotoxemia and abrogate the lethal effects of tnf, the presence of il- in the serum does not correlate well with outcome. we hypothesized that this may be because il- acts mainly in a paracrine fashion and is metabolized before it diffuses into the circulation. methods: we measured the il-i~ mrna expression with the differential reverse transcription polymerase chain reaction (rt-pcr) using g-actin as internal standard in the peritoneal macrophages and lung tissue in normal controls and mice after cecal ligation and puncture (clp). clp resembles human intra-abdominal sepsis in that it is characterized by very slight elevations of serum il- levels. results: il-lg mrna levels after clp are expressed as % of normal (mean+sem, n= in several experimental models of infection exacerbation of disease was observed, when infected animals were depleted of tuajor necrosis factor (tnf). after sublethal cecal ligation and puncture (clp) leading to peritonitis and sepsis the survival of mice also critically depends on tnf as demonstrated in earlier studies, when clp-treated mice injected with anti-tnf antibody died, whereas mice injected with a control antibody survived after clp (echtenacher et al. , j. inununol. : ) . from a panel of different cell types (macrophages, neutrophils, t lymphocytes, natural killer cells, mast cells) able to produce tnf upon activation~ the mast cell is apparantly the only one capable of storing in cytoplasmic granules preformed tnf-ct which is rapidly released following challenge. in the present study-we analyzed serum tnf after lps injections as well as the outcome of clp in severely mast cell deficient mutant mice (wav v) as compared to syngeaeic wild-type littermates (+/+). we proposed that concentrations and/or kinetics of serum tnf should be different between wavv mutants and wild-type mice, if mast cell-derived tnf significantly contributes to the rise in serum tnf levels following systemic stimulation with endotoxin. although similar levels of increased tnf were detected in the sera of both genotypes after and hours of lps injection ( btg/ . ml / mouse i. p.), mast ceil-deficient mice indeed showed decreased serum tnf levels iron after injection amounting to only to % of the concentrations observed in the corresponding sera of normal wildtype mice. in the clp model of septic peritonitis we found that mast celldeficient mutant mice were dramatically more sensitive to clp than syngeneic normal mice resulting in % mortality in w/w v versus % mortality in +/+ mice . days after initiation of clp. further experiments with w/w v mutants selectively reconstituted with cultured bone marrow-derived mast cells from normal syngeneic wild-type mice and the use of an antibody specifically blocking the action of tnf tn vivo should clarify a potential protective function of mast cells in this model of septic peritonitis. interleukin- (il- ) inhibits cytokine production, including tumor necrosis factor (tnf), by lipopolysaccharide (lps)-aetivated maerophages. we recently observed that lps injection (e.coli :b , gg ip) into balb/c mice induces the rapid release of circulating il- ( ± u/ml at min). blocking endogenous il- using monocional antibody (jes - a , mg, h before lps) resulted in a massive increase in tnf production ( ± in lps+anti-il- treated mice vs ± ng/ml in lps alone, p< . , n= to mice per group) and an enhanced lps-induccd lethality ( % vs % in anti-il- +lps or lps alone respectively, p= . , n= mice per group). irrelevant igg rat monoclonal antibody (lo-dnp) did not influence neither tnf production nor lethality associated with endotoxin shock. this led us to study the production of il- during human septicemia. plasma samples were obtained from patients with gramnegative (gns, n= ) or gram-positive septicemia (gps, n= ) and from healthy volunteers. among these patients, suffered from septic shock at the time of sampling. il- levels were measured by elisa (detection limit: i pghrd). we found that patients ( %) had increased il- plasma levels (range to pg/nd). patients with gps had il- levels similar to the ones observed in gns (median: vs . pg/m, respectively). patients with septic shock had higher il- values (median: pg/ml) than septicemic patients without shock ( pg/ml, p= . ). no il- was detected in plasma from healthy volunteers. we conclude that il- is produced daring human septicemia. our experimental data suggest that il- might be involved in the control of the inflammatory response induced by bacterial products. dr arnand marchant, immunology department, hopital erasme, route de lennik, brussels, belgium. to provide information about the role of tnf in sepsis and mods we measured tnf and stnfr-i levels in septic patients and investigated if there is a relation between plasma concentration of these molecules and the severity of sepsis evaluated by two scores (apache i and sss). patients and melhods: septic patients fullfilling sepsis criteria of american college of chest physician and society of critical care medicine were studied. tnf-cc and stnfr-i ( kda) were measured by enzyme immuneassays (norms values = + pg/ml and . _+ a ng/ml respectively). results: the mean tnf and stnfr-i values for each patient (mean+sd) were + pg/ml and . + . ng/ml respectively. these values are approximately seven and ten times greater than those observed in normal healthy volunteers (p< . ). mean tnf concentrations for each patient were significantly greater in non survivors ( + vs _+ pg/ml p< . ); stnfr-i levels also were greater in this group, but the difference was not statistically significant ( . + . vs . _+ . ng/ml). plasma tnf and stnfr-i concentrations were significantly correlated (r = . p< . ). mean tnf levels were significantly correlated with apache ii (r = . p< . ) and sss (r = . p<o. ); stnfr-i levels were likewise correlated with both scores (r = . p< . and r = . p< . respectively). tnf and stnfqgi levels increase with the number of dysfuctional organs; in particular tnf and stnfr-i increase when hemodynamics deteriorate and kidney failure ensues. conclusions: the correlations between tnf and stnfr-i levels and sepsis severity scores suggests that tnf is involved in sepsis and may influence the occurrence of mods and finally clinical outcome. tnf and stnfr-i are in fact especially increased when mods occurs. in particular their concentrations are elevated when cardiovascular system and kidney failure ensue, suggesting that tnf can have a direct role in hemodynamic derangements caused by sepsis and that kidneys are involved in tnf and stnfr-i excretion. lif is a pleiotropic cytokine that has been implicated in the pathogenesis of sepsis in animal models. we conducted a prospective study in septic patients to correlate lif plasma levels with patient's clinical and biological data (among them il- ). plasma was drawn daily from day one to .ten and lif presence was determined with a specific elisa and with the dai,a bioassay (sensitivity of pg/ml and ngml respectively). risk factor associated wftt~ with elevated lif plasma level was determined by an univariate analysis. a multivariate stepwise logistic regression analysis was subsequently performed with a bmdp statistical software. lif was detected with the elisa and with the bioassay in and out of the patients, respectively. lif peak plasma levels were statistically associated with high creatmin levels, temperature and il- . multivariate regression analysis showed that high serum creatinin level was the major independent risk factor associated with elevated lifplasma levels. this correlation was also found for il- . peak plasma levels of both lif and il- correlated inversely with patients survival duration. cox's analysis for plasma levels of lif > pg/ml yelded a hazard ratio of [exp ( . )= . ]. our study indicates that lif levels were associated with clinical and biological parameters of illness severity and significantly increased (cut-off value pg/mi) in patients with fatal outcome. current consensus exists about the central role of tumor necrosis factor (tnf) alpha in initiating the systemic inflammatory response syndrome (sirs). a correlation with sirs has inconsistently been found. tnf effects its pleiotropic reactions upon two distinct cellular receptors. soluble extracel]ular fragments of the human kda tnf receptor (stnfri) and the kda receptor (stnfrii) are detectable in the circulation. the kinetics of these endogenously produced tnf-inhibitors were measured to evaluate their role in patients with sirs. fourteen patients of an operative icu were included with the diagnossis of sirs (mean apache ii score: points). serial blood samples were obtained within h after diagnosis of sirs, every hrs for the first hrs and every hrs thereafter until patients died or recovered. soluble tnfri and stnfrii were assayed by an enzymed-linked immunological binding assay. soluble tnfri and ii could be detected in all samples with a significantly higher level (p<o,o ) compared to healthy controls (normal value: tnfrh , ng/ml; tnfrii: , ng/ml). the serum concentration of tnfri ( , ng/ml) as well as tnfrii ( , ng/ml) were significantly higher in nonsurvivors (n= ) compared to survivors ( , and , ng/ml; n= ) at the onset of sirs and during the course of the inflammation response (p< , ). a positive correlation exists between both receptors (r- , ; p < , ). increasing levels and maximal values of stnfri ( ng/ml) and i ( ng/mi) were detected only in patients who died. the serum concentrations of patients who recovered did not change and are remaining at the initial level. tnf alpha bioactivity was detected in out of patients. no significant correlation exists between the concentration of both receptors and tnf alpha as well as the apache ii score at the onset of sirs. elevated serum concentration of stnfri and are found in patients with sirs. these naturally occuring antagonists reflect the inflammatory process. the measurement of soluble tnf receptor levels may be useful in monitorina sirs and in the prediction of outcome. - is given to patients with advanced melanoma or renal carcinoma. however, this therapy is accompanied by severe side effects, including the vascular leak syndrome (vls) that closely resembles septic shock. to investigate the involvement of cytokines and phospholipase a z in the pathogenesis of the vls we collected serial plasma samples from patients during the first hours after administration of il- . in these patients we monitored temperature, blood pressure and heart rate and assessed levels of the cytokines tnf, il- and il- using immuno assays. in these plasma samples we also measured phospholipase a activity using an enzyme activity assay, since this enzyme is beleived to play a major role in the generation of lipid mediators. we demonstrate that during il- therapy the cytokines tnf, il- , and il- are produced and that the release of these cytokines is followed by an induction of phospholipase az activity in the plasma of these patients, we further discuss the role of these mediators in the development of the vascular leak syndrome observed in patients receiving il- . objectives of the study: sepsis caused by candida albicans (ca) is no longer a clinical rarity but a common consequence of immunosuppression and surgery, and is associated with high mortality. tumor necrosis faetor-c¢ (tnf-<~), interlcukin-lg (il-lg) and interleukin- (il- ) are thought to play an important role in the pathogenesis of the sepsis syndrome. we therefore studied the in vitro production of tnf-<x, il-ib and il- by human peripheral blood mononuclear cells (pbmc) stimulated by ca compared to stimulation by bacterial lipopolysaccharide (lps). materials and methods: pbmc were isolated from venous blood of healthy volunteers and cultured at a concentration of . • ~ eells/ml in culture medium with a dose-range of either heat-killed ca or lps. supernatants were harvested after hours. using elisa, tnf-c~, il-ib and il- concentrations were measured. ca was isolated from a patient with ca sepsis and cultured under sterile conditions. results: ca and lps stimulate the production of tnf-~t, il- ] and il- by human pbmc. we observed a dose-dependent synthesis of these three cytokines in human pbmc. tnf-c¢, il-ib and il- in ng/ml as mean _+ standard error. ca/ml . . ( ) we constructed a single-chain fv-fragment (sc-fv) from the variable domains of a neutralizing antibody to human tumornecrosisfactor-alpha (tnf) because sc-fv should have some advantages in vivo. the sc-fv has a similar affinity ( , x - m) as the fab-fragment ( , x - m) but bind the antigen with a eightfold lower avidity compared to the parental mab ( , x - ). the parental mab as well as its fragments can compete the binding of rhutnf to both tnf-receptors. this was shown by competitive binding to the cell line hl- and to immobilized rtnf-receptors. in the nutralization assay on the cell line, l , the sc-fv can neutralize tnf but in comparison to the mab or its fabfragment, the capacity was three-fold lower as expected from the molar ratio of the dissociation constants. the in rive-neutralizing capacity was tested in a mice receiving toxic dose of rhutnf. the sc-fv showed a -fold lower neutralizing capacity in comparison with the fab-fragment. this could be explained in different manners: i) partial instability of scfv at c, ii) shorter half-life because sc-fv but not fab-fragments are eliminated via kidney, iii) elimination of tnf-immunocomplexes may be improved by opsonization mechanisms which requires some structures of the constant region (as in fab or mab). in summary, despite the encouraging in vitro experiments, the in vivo data suggest that sc-fv are not the right strategy for neutralizing tnf in vivo. tnf production in endotoxin non-responder mice, effect of a glucocorticoid antagonist g. iazgtr jr, e. duda, j. ol~th, e. husztik, g. iazar glucocorticoids inhibit lipopolysaccharide (lps)-induced tnf production and tnf can stimulate pituitary adrenocorticotropin secretion, resulting in the release of corticosterone. earlier studies ( ) reveales that the glucocorticoid antagonist ru , nufepristone, sensitizes both endotoxin responder and nonresponder, c h/hej, mice to endotoxin lethality. we have also shown that the glucocorticoid antagonist ru sensitizes endotoxin-tolerant (endotoxin-pretreated) and normal mice to the lethal effect of septic and endotoxin shock. on this basis, we set out to study the influence of ru on tnf production induced by endotoxin in endotoxin responder and non-responder mice. one and hrs following lps injection, ru significantly increased the tnf concentration in the serum, liver and spleen of treated animals as compared with the control and methylprednisolonetreated groups. in tissue cultures, ru induced the tnf synthesis of myeloid cells and increased the tnf production of genetically modified hela cells, which synthesize tnf constitutively. normal and tumor cell cultures exhibited an increased sensitivity toward tnf in the presence of ru . however, the same dose of lps did not induce tnf production in the serum, liver and spleen of endotoxin non-responder mice and this was not influenced by concomitant ru treatment. these studies suggest that ru aggravates lps lethality via factors other than tnf in endotoxin non-responder, c h/hej mice. the cytokine response to a novel exnacellular mitogenic factor, mf, produced by the group a streptococci (gas), and to the streptococcal pyrogenic exotoxins (spe) a and b, was determined by in vitro stimulation of peripheral blood mononuclear cells (pbmc) obtained from healthy blood donors. the induction and kinetics of different cytokines was studied at single-cell level using cytokine specific monoclonal antibodies and intracellular immunofluorescent staining. all three mitogens induced a massive ifny and tnfi response in - % of the pbmc after - hours of cell stimulation. in contrast, il and tnfc~ containing cells was only detected in - % of the pbmc. the induction ofil , il , il , and ill , was weak or completely absent. thus, the response indicates activation of th cells. however, illc~, illl , illra and il , were consistently found and gradually produced, peaking at hems in approximately - % of the pbmc. mf induced an equally extensive cytokine as well as proliferative inducing capacity, as did spea and speb, which suggests that also mf is a superantigen. a marked inter-individual variation could be noted between lymphocytes isolated from different individuals, both in the toxin induced proliferation and cytokine induction. this may be one explanation for the varying clinical severity noted in patients after infection with clonal gas strains. introduction -tumour necrosis factor (tnf) is released by mononuclear cells in response to inflammation and sepsis. it has been implicated as a possible mediator in inflammatory bowel disease (ibd) but its pathogenic role remains uncertain. this study assessed the relationship between tnfct and disease activity by measuring plasma and faecal tnf concelnmtions in an experimcntai model of ibd. methodologo' -colitis was induced in male wistar rats by intmcolonic instillation of rag , , -trinitrobenzenesulphunic acid in . ml % ethanol (n= ). control animals received an isovohlmetric instillation of normal saline (n= ). faecal pellets were collected before induction of colitis (day ) and throughout the experiment. after days the colon was excised, ueighed and the colon macroscopic score (cms) assessed plasma tnf (ptnf) samples were assayed by bioassay and elisa. faecal samples were vortcxed in water and the supernatant removed for estimation of protein and faecal tnf (itnf) content (elisa). there is a significant increase in itnf on day -#p= alld the total ftnf measured during the study correlatcs with the cms oll day -p= . there is no correlation between ftnf and ptnf. conclusions -therc is evidence of coloific tnf excretion by macroscopically normal animals and following induction of colitis there is increased faecal tnf which correlates with disease actmty. there is no significant elevation in bioactive or imnmnoreactive plasma tnf in colitie animals this data would suggest that faecal tnf is a marker of colonic inflamrnatiort and serial tnf assessment inay be useful as an indicator &severity in ibd. to study the effect of in-vivo hypoxia/reoxygenation on cytokine elaboration by routine peritoneal macrophages. materials and methods: adult male cba mice ( - g) were primed intraperitoneauy with either lipopolysaccharide (lps) ug]anlmal or saline. five days later, the animals were subjected to hypoxia ( % ) for , followed by of normoxia. harvested peritoneal macrophages were plated in-vitro, culture supernatants were collected at , , and and assayed for tumor necrosis factor (tnf), prostaglandin e (pge ) and nitric oxide (no). control animals underwent the same procedures, but were maintained in normoxia ( %o disease , berlin, germany the precise mechanisms of reactivation of latent human cytomegalovirus (cmv) infection are still unknown. apart from the permissive effect of diminished cellular immunity there is evidence of a cytokine mediated cmv reactivation as it is known for other systemic virus infections (e.g. hiv). we found that tumor necrosis factor-alpha (tnf) -in contrast to all other cytokines tested -can stimulate the activity of the cmv-ie-enhancer/promoter region in the human monocytic cell line, hl . we wondered whether our in vitro results were actually reflected by the incidence of cmv reactivation in diseases which go together with high tnfalpha levels. cellular immunodeficiency as well as at least temporarily increased tnf levels are known to occur in septic disease. we tested for the presence of cmv infection in peripheral blood mononuclear cells (pbmnc) by means of h centrifugation culture, immunocytological detection of different cmv antigens (apaap-technique), and pcr technique (cmv-ie dna). in striking contrast to healthy controls almost all examined septic patients were positive, irrespective of the method for cmv-ddtection applied, including the less sensitive immunocytology. follow-up studies showed that cmv-antigen positive pbmnc ceased to be detectable in patients with good outcome once septicaemia had been overcome. to further back up our hypothesis, we looked for a coincidence of enhanced tnf-alpha serum levels and cmv antigenaemia (immunocytology) in some other diseases which are known to have either enhanced tnf serum levels or increased incidence of active cmv infection and found the majority of patients with b-cell chronic lymphocytic leukemia, liver cirrhosis, and common variable immunodeficiency to be positive for these two parameters. we now wondered whether, apart from cmv reactivation, tnf-alpha could also cause cellular immunodeficiency this way promoting cmv replication and spreading and eventually causing cmv disease. in summary, we were able to show that a diminished cellular immune response results from/n vitro or in vivo methylprednisolone (mps) is used to suppress both inflammatory and immune responses. il- receptor antagonist (il-lra) and tnf binding protein (tnfbp) are naturally occuring anti-cytokine proteins, possibly modulating inflammatory and immunological responses. to define mps modulation of cytokine and anticytokine responses, we examined effects of mps on il-lra production and tnfbp generation as well as il-i~ and tnfa production by human peripheral blood mononuclear cells (pbmc) stimulated with lipopolysaccharide (lps). pbmc were purified from the blood of healthy volunteers, then incubated with lps ( ng/ml) supplemented with different concentrations of mps ( , pg/ml, mg/ml). after hrs incubation, the supernatants were collected for rias of secreted cytokines. the cell lysates obtained by cycles of freeze-thaw, were also collected for r ias of cell-associated cytokines. both the supernatant for secreted cytokines and the cell lysates for the cell-associated cytokines were subjected to rias for il-i~, tnfa, il- ra, and tnfbp-i (tnfsrp ). il-la, il- ra, and tnfbp were produced mainly as cell-associated forms in response to lps, whereas most tnfa was secreted into the supernatant of lps-stimulated pbmc. mps supplement resulted in reduction of il-la, il-lra, and tnfa production. il-la production was reduced up to . + . %( mg/ml) by mps in a dose dependent fashion, whereas about % decrease in tnfc, and il-lra production was observed at both high and low dose mps supplement. however, total tnfbp generation was not reduced significantly by mps supplement. furthermore, tnfbp secretion was increased times more in the supernatant of lps-stimulated pbmc with gg/ml of mps. these data suggest that mps could effectively block tnf activity by both reducing its production and up-regulating tnfbp generation and that mps works as an anti-inflammatory drug as well as an immunosuppressant by modulating cytokine and anticytokine responses. in severe gram-negative sepsis, excess of proinflammatory cytokines is associated with increased morbidity and the mortality. we have found that immunocytes possess functional -adrenergic receptors (~-ar) which, when activated, down regulate the lipopolysaccharide (lps) induced gene expression and subsequent secretion of tnf-~ in vitro. in the present study, we used a lethal endotoxic model in mice to test the hypothesis that -ar stimulation will down regulate the gene expression thus decreasing the circulatory levels of proinftammatory cytokines and improve the survivals in severe endotoxicosis. the animals (iso group) were injected times with ! -ar agohist isoproterenol ( . mg/kg i.m. and . mg/kg s.c.) , and hours prior to a lethal dose of lps injection ( mg/kg i.p.). control group (ctl) received same volume of saline at the same times before the lps insult. at the appropriate time points after lps injection animals were sacrificed and venous blood was collected from the inferior vena cava. the plasma levels of tnf-cc and il-loc were determined by elisa. in the mortality study, each grou the data showed that isoproterenol significantly decreased the mortality and the circulatory levels of tnf-c~ and il-lcc (fig. ) . these data suggest that -ar activation of immunocytes down regulates cytokine gene expression, decreasing circulatory eytokine levels thus reducing endotoxicosis mortality. these results provide a new pharmacological approach to reduce the excess of proinflammatory cytokines and mortality in sepsis. introduction: cytokines released from monocytic cells are thought to play an important role in the pathophysiology of sepsis. during the last few years several investigations have been performed aimed at modulating this mediator response. in the present investigation using a full blood model we have studied the effect of a standard immune globulin and an antitipopolysaccharide (lps) immune globulin preparation and a monoclonal anti-lps antibody in modulating the endotoxin induced cytokine response. methods: citrated blood from healthy human donors was incubated at °c in rotating polystyrene tubes. samples were taken before addition of x ng/l e. col± endotoxin and after hours. plasma was assayed for the cytokines minor necrosis factor alpha (tnf-~) and interleukin- (il- ) using elisa methods. the eodotoxin concentration was assayed by lal and end-point chromogenic substrate technique. the blood was preincubated with standard immune globuline (human igg from pooled plasma, gammagardr, baxter) or with an anti-lps immune glubuline (human anti-lps igg obtained by screening of blood donors, novo nordisk) in the concentrations of mg/ml or mg/ml or with the monoclonal lipid a igm antibody ha- a for ten minutes before the endotoxin was added. results: two hours after endotoxin addition markedly elevated tnf-~ and il- values were found.in the plasma. cytokine values at hours: tnf-ec pg/ml (mean) and l- pg/ml (mean). preincubalion with mg/ml standard immune globulins reduced il- values by % (mean) while there were no effects on tnf-c~ values at hours. a slight reduction in il- values was also found with the lower concentration, mg/ml, but this was not statistically significant. preincubation with anti-lps immune globulins had no effect on il- nor on tnf-cc values. preincubation with the monoclottal lipid a igm antibody ha- a in variable concentrations had no effect on cytokine release in this model. in this full blood in vitro model standard immune globuline reduced endotoxin induced l- release while tnf-cc values were unchanged.the anti-lps immune glubuline preparation and the monoclonal lipid a igm antibody ha- a had no effect on the il- or tnf-ct release. the effect of anti-tnf treatment in severe haemorrhagic/traumatic shock was investigated in a conscious rabbit model. the jugular vein (for administration of substances and blood withdrawal) and right femoral artery (for measurement of bp) were cannulated and an aortic thermistor probe inserted into the left; femoral artery for the measurement of cardiac output (co). the following day, upto % of the estimated blood volume was withdrawn over - rain, to reduce bp to - mmttg and co by / mad withheld for rain. shed blood ( %) was then reinfused followed by lactate solution to give a total potential dreulat%ag fluid volume of %. zymoeen activated plasma ( , gg/ml) was given rain prior to haemorrhage and during blood r~n%eion, all ~,~ir, ak were killed at h and o~gane removed for histology. rabbita received either monoclonal antirabbit tnf (r , rat igg , mgkg i.e. n= ) or emine (nffi ), rain prior to haemorrhage. the cardiovascular and biochemical changes during the haemorrhage and hypotensive phase (is. haemorrhagic stimulus) were similar in both groups. upon reinfusion, haematccrit and white cell count remained sigutfia~utly (p< . ) higher in saline compared to r -treated animals, indicating a relative haemoconcentration (i,e. reduced circulating volume) in saline animals. the lettkocytosis persisted in the saline animals upto h. plasma glucose, lactate and asparteto .m~-o transferase all rose similarly in both gorups but plasma ¢rea~!~+-e levels were markedly (p< . ) higher in ealine ve r animals at and h indicative of kidney damage, using a macro and micro. pathological scoring system, major organ damage was seen in the lung, liver and kidney which was significantly (p< . ) attenuated in r treated animals. in the hmg and liver, this was primarily due to a reduced bleeding and pmn infiltrate and to an additional maintenance of tubular integri~:y in the kidney. hence, in tlais model anti-tnf treatment markedly reduced the biochemical and histological indices of severe ,, ~gan damage during liaemorrhage and ranerfn~ion. thermal injuries and systemic bacterial sepsis produce a wasting diathesis with anorexia and marked loss of lean tissue and body fat. endogenously produced cytokines, including interleukin- (il- ), are thought to mediate many beneficial as well as pathologic host changes that occur during burn injury and infection. to evaluate whether bluckade of il- in vivo would affect survival and attenuate the anorexia and cachex[a associated with a thermal injury and chronic fulminant gram negative infection, sprague-dawley rats were studied. anesthetized rats were divided in groups, implanted with alzet r minipumps and randomized to receive the following treatment for days:i: gg/kgbw/min of il-lcc; ii: ~tg/kgbw/min of il- receptor antagonist (il-ira); iii: buffered vehicle. the animals were subjected to a % bsa, full thickness scald burn and the wounds then infected with + cfu/ml pseudomonas aeruginosa. eight additional rats were anesthetized, but were not burned, and served as healthy controls. . il-hx reduced food intake transiently but was otherwise without effect however, il-lra significantly attenuated weight loss over the first days and improved food intake between days and . we conclude that blockade of il- after a thermal injury with superimposed infection does not adversly affect the progression of the disease, but does attenuate the anorexia and weight loss associated with this model. these data indicate that il-i receptor blockade may offer a means to minimize the morbidity associated with catabolic illness. tumor necrosis factor (tnf) and its downstream induced mediators have pivotal roles in the pathogenesis of sepsis and septic shock. the suppressive effect of pentoxyfillin (ptx) on tnf production may be of clinical importance. when peripheral white blood ceils were stimulated with either lps or staphylococcus aureus, pentoxyfillin inhibited tnf production. in contrast, no inhibitory effect was observed on the induction of il- . ptx inhibited not only the tnf production of monocytes but also the tnf secretion of both granulocytes, and unseparated whole blood. the facs analysis revealed that the expression of cd antigen on monocytes was not influenced by pentoxyfillin. the in vitro tnf and il- producing capacity in septic patients were higher than in the control group, but decreased on subsequent days especially in fatal cases. administration of ptx ( mg/day) in septic patients resulted in tnf and il- productions similar to those found in control donors. it also subsequently led to an improvement of the clinical status. a further improvement in apache score could be achieved by administration of pentaglobin ° (biotest)( ml/kg/day ). the prevention of lpsinduced in vitro tnf and il- production by pentaglobin ° could be demonstrated in in vitro experiments involving the use of whole blood rather than purified lymphocytes, very probably because of the presence of lps binding protein in the plasma. the level of soluble icam- in sera of septic patients was significantly higher ( - ng/ml) than in normal individuals ( - ng/ml), but it decreased following the ptx and pentaglobin ° therapy. it is presumed that ptx and pentaglobin ° can antagonize eytokine production at different levels, resulting synergistic action being beneficial in the treatment of sepsis. albert szent-gy rgyi medical university, institute of microbiology, szeged, hungary h- ddm t. . e~i~im~/tii, septic sh ~ '. tnf alfa/pge, and somatostatln lands ji., alvarez j., gram m., llanos k., alcalde j., sanchez ja., balibr~d jl. taurine, a semi-essential sulphur-containing t:~-amian acid, promotes neutrophil phagocytic activit ' against yeasts and enhances intracellular killing of e.coli. paradoxically it protects against hypochlorous acidmediated biomembmne oxidatio~ and abrogates the pyrogenic effects of intracerebral endotoxin. it is therefore possible that taurine mediates some of these effects by modulating the cytokine cascade. aim to assess the cytekine responses in a rat model of intraperitoneal sepsis, pretreated with supplenmntary taurine, by measuring tnf and il- concentrations. methodology female wistar rats (wt - g) (n- per group) were prefed with % tanrine, . % glyciuc ( in tap water) or tap water (tw) for days prior to caecal ligation aud puncture (clp). normothermia was maintained intraoperatively and the animals received . % saline subcutaneously ( mls/ g) post-procedure. animals were venosected by direct cardiac puncture at or hours post-operation and the blood placed in endotoxin-free tubes. centrifilgation and storage of plasma at - °c was completed within hours. tnf and il- were measured using wehi and b bioassays respectively. results (means ± sem) bioactivc tnf concentrations were not significantly different between clp or sham groups. however plasma il- estimation revealed a statistically significant reduction at hours in tanrine-treated animals compared to glycino and tw controls ( objective: to evaluate the effects of allogeneic blood transfusion, thermal injury and bacterial garage on interteukin (il- ), tumor necrosis factor alpha (tnf) production and host mortality and to study if the administration of thymopentth (thy) could affect these events. materials and methods: balb/c mice were transfused with allogeneic blood (from c h/hej mice) and treated daily with thy (i mg/kg)(t+thy) . control animals were transfused but not treated (t). after days systemic blood was harvested to determine the circulating levels of il and tnf. a second group was treated with thy for days and then received a gavage wide lxl escherichia coli and a % burn injury (bg+thy). one hour post-bum, blood was collected to measure cytokins. control animals received the same treatment but thy (bg). a third group was transfused, treated with thy for days and then received gavage and burn (tbg+thy). one hour post-burn, blood was collected to measure cytokins. control animals received the same treatment but thy (tbg). all treated and control groups had mice/group. in separate groups (n= /group), receiving the same treatments, mortality was observed for days post-burn or transfusion. the production and release of oxygen radicals by phagocytic leukomytee is one of the most relevant and important functions of these substances in biology. when neutrophilic granulocytes or certain macrophages phagocytize they produce super oxide and hydrogen peroxide and form secondary products of these activated species. all of these substances are released in to the phagosome. it has been appreciated since that the amount of oxygen consumed by phagocytes in producing free radicals is prodigious. what has not been totally appreciated is the significant contribution this consumption contributes to total body oxygen consumption. our understanding of increased oxygen consumption following sepsis is compounded by a paradox of increased cardiac index and a narrow av difference. it was mclean in a study reported in that showed an increase in cardiac index and a narrowing of the avo z difference in humans following sepsis. other studies documented similar changes in humans and it has recently been speculated that increasing o~ delivery might have a favorable impact on outcome. at least three different explanations have been put forward as to why there are possible alterations in energy metabolism during sepsis. these include decreases in oxygen supply, peripheral shunts and direct action o~ cellular mediators on o~ delivery, a provocative review by hodgkiss and nark in shewed that in experimental animals sepsis did not cause any evidence of bioenergatic failure in muscle i liver, heart or brain tissue. they further showed that the increase in serum lactate is mot necessarily due to cellular hypoxia. they conclude that cellular hypoxia and defects in energy producing metabolites are not the cause of metabolic abnormalities in sepsis. we set out on a series of experiments to try to explain the paradox in cellular metabolism and whether or not white cells might contribute to total body oxygen consumption during sepsis. the first set of experiments involved growing rat cardiac myocytes on tissue culture. pyruvate was added to the medium as substrata and physiologic levels of peroxide were also added simultaneously, carbon labeled co~ production was measured as was nucleotides and degradation products from the cell. the peroxide induced a significant inhibition of pyruvate dehydrogenase. in addition, there was a loss of cellular atp and a corresponding rise in the release of inosine and adenine from the cell. we concluded from this first sst of experiments that physiologic levels of peroxide are a potent inhibitor of pyruvate dehydrogenase in isolated cardiac mitochendria as well as the cultured myocyte. we further showed that pyruvate dehydrogenase inhibition blocks the aerobic oxidation of glucose and leads te adenine nucleotide metabolism with adenosine and inosine release from the cell. other enzymes within the tca cycle did not appear to be specifically blocked by peroxide. this would explain the increase in lactate and the ability of the cell to continue to make high energy phosphate by entry of metabolites in to other entry points of the tca cycle, the second set of experimbnts was designed to estimate the magnitude of whole body reactive oxygen generation via nadph oxidase following granuloeyte activation invivo. using a guinea pig model baseline oxygen consumption was determined. ~iaphenyl iodinium (dpi) was used as a specific inhibitor of granulocyte nadph oxidase. animals were divided into two groups; those that received dpi and those that served as control, all animals were then injected with phorbcl myristate acetate (pma) intravenously. pma is a potent stimulus for granulocyte activation and subsequent reactive oxygen generation. whole body oxygen consumption measurements were then repeated after the p~la injection. in addition, arterial blood gas amalysis was performed, circulating neutrophils were collected and assayed for their ability to generate hydrogen peroxide and the r~ght lung was removed for wet and dry weight measurement. total body oxygen consumption increased by percent after pma injection. arterial oxygen tension fell significantly in the control animal. neutrophil hydrogen peroxide generation rate doubled and lung water in the untreated animals increased by percent. in this animal model we could show that granulocyte activation substantially increases whole body oxygen consumption to approximately percent of control values. the nadph oxidase inhibitor, dpi, decreases granulocyte hydrogen peroxide generation invivo and prevents the pulmonary injury induced by pma. using this animal modal it is possible to transpose this to the human septic state. if we assume there are , neutrophils per cubic millimeter of blood, a kg man would have ~ . xi u circulating neutrophils. when activated ixl ~ neutrophils generate one nanemole of hydrogen peroxide per minute. t~erefore, the circulating neutrophil pool could generate - . xi nanomoles of hydrogen peroxide per minute. this corresponds to an oxygen consumption of - . ml/ojmin by the circulating neutrophil pool alone. this does not take into account the production of oxygen by macrophages or the amount of oxygen consumed to produce nitric oxide. manipulation of oxygen delivery in human sepsis may or may not be beneficial. irshad h. chaudry, ping wang, and alfred ayala life threatening blood loss, due to soft tissue and bony injuries, is a frequent complication in trauma victims. although numerous organs and cells are adversely affected by hemorrhage, the focus here will be to discuss whether or not there are any differential alterations in splenic and hepatic immune functions. in particular, m~ antigen presentation (ap) and associated processes will be described since one of the important functions of the m is to activate resting t-cell lymphocytes by processing and presenting foreign antigens in a mhc class ii-restricted fashion. studies have shown that splenic m and kupffer cell (kc) ap was significantly depressed following hemorrhagic shock and remained so for at least h alter resuscitation. the presentation of antigenic fragments associated with mhc class ii antigen itself was not decreased following hemorrhage, but the catabolism of antigens in antigen-presenting cells was decreased. this is likely due to a significant loss of cellular atp. although m ap was depressed in splenic, as well as kc, only kc showed an enhanced capacity to produce inflammatory cytokines, il- , il- , and tnf. this difference in response between kc and splenic md may be due to differences in the microenvironment in which these cell populations are found, as well as potential differences in the effects that hemorrhage has on the environment from which these cells are obtained. splenic m from hemorrhage-resuscitated mice exhibited a significantly reduced cytotoxic response, whereas kc showed an enhanced cytotuxic capacity. the increased kc cytotoxicity is probably mediated through the increased expression of cell-associated tnf and the release of reactive nitrogen. the enhanced production of reactive nitrogen may play an important role in the clearance of microbes translocated from the gut following hemorrhage. however, excessive release of reactive nitrogen by kc may have deleterious effects on the adjacent hepatncytes. such an effect could, in part, explain the reduction in active hepatocellular function, which is seen following hemorrhage-resuscitation. thus, hemorrhage induces differential effects on splenic and kc m populations; it decreases splenic m but increases kc capacity to mount a cytotoxic response. the depressed splenic md and kc ap was, however, significantly improved by posttreatment of animals with atp-mgcia, chloroquine, diltiazem or interferon-'/. these agents also decreased the susceptibility to sepsis following hemorrhage. thus, the use of atp-mgci~, dfltiazem, chloroquine or interferon- offers new therapeutic modalities in the treatment of immunosuppression and for decreasing the susceptibility to sepsis, which is observed following severe blood loss. the production of macrophages from bone marrow precursor cells is a dynamic and highly regulated process. the differentiation of myeloid lineage-restricted progenitor cells is initially controlled by interleukin- , which drives the progenitors along a maturation pathway that leads to their response to specific lineagerestricted colony-stimulating factors(csfs) we have previously hypothesized that thermal injury can initiate a self perpetuating cycle of production of defective immune cells: thermal injury can initially affect circulating immune cells. then these cells, by unbalanced production of colony stimulating factors and other mediators, can cause a derangement of hematopoiesis resulting in an abnormal production profile of tnf, il-i, and il- and cytotoxic activity of bone marrow derived cells. the results of our studies so far have supported parts of this hypothesis. il- and il- +m-csf treatments increased the production of tnf in burn day macrophages and progenitor cells compared to normal cells. il- +m-csf+il- increased the production of il- by burn progenitor cells compared to normal macrophagee. preliminary results for the ratios of tnf/~ actin mrna indicate that il- +m-csf increased the mrna for tnf in the -day macrophages derived from burned animals compared to macrophages derived from normal animals. ratios of il- /~ actin mrna indicated that il- , il- +m-csf, and m-csf increased the il- ~rna in progenitor cells from burn animals compared to progenitor cells from normal animals. these preliminary results support our hypothesis that bone marrow derived macrophages and progenitor cells from burned animals act differently compared to cells from unburned animals regarding the production of inflanunatory cytokines. more specifically, the results suggest that the different cell types are regulated in different ways by cytokines, and cells from burned animals are regulated differently than cells from unburned animals. it i,~ to ~ssume that the translneatinn is due to a ixx~h,v functioning ~astroiutestlnal surface pro|ection system, which leads to an unacceptably high load ¢ln the imrsunc dcfcncc system, partlcul~ly the local system in the gastrointestinal wall, leading tu exh~ustiou of ihi~ system. tlae g&~lrointesliual i]'ac( can be regal'deal a.s a t~ servojf of appr m ~ separating i~ eucayotic ceils of the hosl ttom l ~ bacterial culls. in the human body the the myriad of cndngan¢nts micrix)rganistns, exogenous itl,~ttdittg micro~ganisms altd taxies m-'e sepia"areal li'otll lhe rest of the body by a simple epithclhd layer. the barrier function is heavily tlcpcnding tm =~ proleclive layer cmtsisting ie. surl~:emts, nluclts, probiotic bacteria, and ,~ub~tances with strong antioxid~lt, antipfotease al~d other i~rotective eft'ecru, the surfaet~ml htycr consists at tile besl if i(.) bimolecular layers, fl is thus very iltil~, the epilhelial layer is renewed every hours , and tile tanuwer of the mucus is ~so fa,~t, all attdphy of die epithelial layei induced by g&sttointestinal st~'vation does also include the mu~usprndocing cells, gnhlet cells, which leaduhg lu slmnage aad luwer quality of gi mucus, altered viscoelas,jc properties and reduced protection, ]~'obioiic bactcrla keeps the ppm's low, produce nutrients for the intestinal mllcosa, eliminates tuxia~ and stimulates the local immune system, the gl surface is conslanlly under hc wv wearing by ingested bacteria and their enzymes, ingested substaucas, chefftie,'ds at~d toxins. 'll~e n~tective flora is reduced in disease, by stress and by cm'clcss antibiotic therapy. wc have made attempts tn recondition the i,,.astroiniesli!l~d. tnucosa by rcstlpply of surfactants or sttrfactanfliko glyc~lipids, gels with pseudomuein [until.oil, and probiotl¢ laetobaeilli. by supply of glycolipids we have bt:cn able m prevent and heal intlammatory and tlleerallve injuries (and io p)'cveut microbe transhteatkln) in uppe~' and lower (. u'acl, this c~al be fu~'ther augmentented by shnultaueos supply el' a p.~uedomucin,likc gel like pectin. ()at contain~ one hundred times more ot membrane lipid$ th~a =nay other food, much of fiber, ~spceially watcrsnlnhlc betaglucaal~, at~d has an close to ideal amino acid cornpo~ition, <')at, termented by species-specific lactobacilli, has a strol~ ability to prevent local inflammatory and ulcerative chunges,transh)cadnn and sepsis in experimeut~d attimals mid to pl~ven~ revert mof in ba,nan.~.lt seems us, that it the mucos~lnucus/probiotic bacterial filnclinns cat'= %' restated by mucosa reconditioning, even in severely sick iudividuals) the hx:~d iuuuuue ~y,~teme will be capable tu pr~)tec! the ix~y ag~lls~ sepsis ~nd mof. the impaired immune response associated with multiple system organ failure (msof) has been most widely studied following surgery for inflammatory disease and trauma. the majority of late deaths following trauma are due to infection and msof. not all patients with multiple organ failure have concomitant infection present, yet most have been septic at some time in their hospital course. their generalized inflammatory condition often persists whether or not organisms have been recovered. immune failure probably precedes msof with or without overt infection and indeed may perpetuate such widespread sepsis. macrophage tumor necrosis factor-alpha (tnf) production is thought to represent an important pathogenic mechanism by which this is mediated. cecal ligation and puncture (clp) is a clinically relevant murine model of intra-abdominal infection and sepsis. serum tnf and endotoxin levels, and peritoneal macrophage tnf production are only slightly elevated in this model even in endotoxin sensitive animals. mortality in this model, therefore, does not appear to be attributed to overwhelming endotoxemia and/or tnf production. it has therefore been postulated that tnf and other cytokines may act in a paracrine fashion to cause local injury. tnf messenger rna (mrna) expression was therefore measured and found to be increased in the lung and liver following clp. a different pattern of expression was seen after endotoxin administration, characterized by a more rapid rise and fall, and enhanced tnf mrna expression in the spleen as well. route of spread of infection as well as the type of stimulus may determine the organ specific cytokine response. these data support the concept of locally produced tnf as a contributing factor in tissue damage and multiple organ failure during sepsis. the estimation of histamine's role in sirs changed over the years based on increased knowledge in shock pathogenesis, evidence later found to be faulty, and the discovery of other, more fashionable mediators. over the decades, the prevailing view has been that histamine is a noxious mediator contributing to the fatal outcome of shock. the analysis of experimental data on exogenously administered, endogenously released and/or newly formed histamine in septic/endotoxic shock suggests that histamine contributes to the early cardiovascular changes via hi-receptor vasoconstriction thus excerbating (directly and indirectly) the effects of catecholamines. in the later phase of septic shock (low out-put, high resistance states), however, it can be assumed that histamine exerts strong vasodilator and positive inotropic effects via h -receptors. these effects are considered beneficial in counteracting or attenuating the effects of sympathetic responses of catecholamines and other mediators. thus, a blockade of the early reaction with hi-antagonists and a stimulation of the h -receptors by h -agonists are worthwhile therapeutic approaches. shock produc~s ~ij alterations, an encrgy crisis and eventual c~ll damage, however, shock in itse|f do~ not lead freqoenfly to r~mote organ damage. in animal ~xpcrhnents and clinical expariez:ces in korea and vietnam, shock i~r s¢ was not associated with lung damage, renal failure or ards. the same is true with g.l bleeding. however, when shock is due to trauma or associated with tlssus injmy, then organ malfunction m~d damage is frequent. shock is bclleved to increase the frequency of infection, but clinical evidcnc~ for this is scant. hemonhsgio shock akme is an unusual o~orrenee dinica[ly. immune dysfolletion dons occur witl~ experhnental hypovolemic shock and in pailcnts with hypotcnsion or after receiving blood ~ansfosions. tbc most significant factor affecting reoorrcnce of resented ca,lorectal liver mclastasis was hypotensivc episodes during operation. blood transfosions depress immune function, improve transplant ~raft survival and increase ttll'rmr rec~r'tcnos ill co[eli and head and nc~k cancers. in experimental hemorrhagic shock, we found decreased response of [ymphocytes to mitogens, el:clad mixed lymphocyte reactilln and i[,- decreased. othe~.x hsvc found decreased macrophage and t and b ¢¢lt function, deci~ased re,ease of/l~i, increased poe v depressed macrophagc antigen presanlatiota, increased ien y, decreased il- , and , and shared mscrophags funclion with loss nr f and c b r~ccpto~s. this is inhibited by chlornqoine, ibuprofen, dihiazem, and atp. shock may activate ncutrophils contributing to ilappitlg in cspillarics, no refluw, increased adhesion, cytotoxicity and organ damage. this may be related to decreased clearance of bacteria after shock. t~lls shock, hypotcnsion and decreased microcirculatory blued flow initiate or set the stage for immune dysfmtction. they, along with tissue injury trigger inflammatory cascades. this also contributes to immunologic dysfonctitm, which is associated with increased lufoction. thus altered bh,od flow and mediator cascades are bolh involved, the question now is can wc rapidly improve microcirculatory blood flow, and dg'masa inflsrmnatory fosponss, which is also necessary for survival the effe~ of ~nterferon ~amma @n wour~ healing was ewaluated ~n a routine mo~el. ~mma ~n~erferon we g~ven in doses of ~ . to ~ , u~%$ synchronous with ereatio~ of a pa~splnal woumd then daily for is ~lays. ~mteet~on ~amma impaired tensile strength a% all ~oses relative to control. m zphology suggested ~reas~ ¢oll~gen deposition and r~du~-~ neovag~ulaeit¥ ~n t~eat~ animals, interferon ~amma was a so ~valuated in a murine mo~el of cecal llgatlon and punneure. one hundred to ,s uni~ vere ~iv.n following ~nju~ an~ than daily. interferon qamma was associated with inco:ea~ed mortalit~ and earlier death a~/a~n ~n a dose dependant manner (p< , ). afmlnistra~ion the proinfiammatory cytokines tumor necrosis factor-c~ (tnf-c , interleukin- (il-ii ), interleukin- (il- ), and interleukin- (il- ) have been implicated in the pathogenesis of the multiple organ dysfunction syndrome (mods) following shock and trauma. these mediators which are released in high concentrations by activated macrophages, endothelial cells, and connective tissue cells cause a systemic inflammatory response syndrome (sirs), thus leading to a shock-like state and tissue destruction. recently, a number of proteins have been characterized in in vitro and in vivo studies demonstrating potent anti-inflammatory properties. these latter cytokines include interleukin- (il- ), interleukin- (il= ), transforming growth factor- (tgf-j ), and the newly sequenced interleukin- (il-i ). they are considered antiinflammatory, since in vitro studies using purified macrophage cultures or whole blood assays revealed an inhibitory effect of these antmnfiammatory cytokines on ~he synthesis and release of tnf-cl and il-u . in addition, they reduced the severity of disease and reduce plasma levels of tnf-c~ and il-ii when administered to animals with infection or inflammation. furthermore, naturally occurring substances have been described that specifically neutralize the bioactivity of il- b and tnf-a. the il- receptor antagonist (il-i ra) is related structurally to il-i and binds to the same cell-surface receptors, but does not stimulate the cell. in animal models ore. cob-induced shock, inflammatory bowel disease, and peritonitis, injection of il-lra significantly reduced the severity of disease. the cytotoxicity of tnf-c~ can be inhibited through two types of soluble tnf receptors (stnfr) type i (p ) and type ii (p ). these stnfrs are proteolytic cleavage products of the cell-bound tnfreceptors. when given to baboons to combat e. coil-induced shock, soluble receptm's to the tnf type [ receptor decreased the severi~ of the shock-like state. it has been well accepted that shock and severe injury result in an increased release of proinflammatory cytokines with a high incidence of sirs and mods. our studies investigating plasma levels of anti-inflammatory mediators (il- , il-lra, stnfrs) in patients after severe injury further suggest that shock and injury lead to an activation of anti-inflammatory processes with altered plasma levels of these mediators. however, while plasma levels of il- and l-lra were significantly increased in trauma patients compared to healthy volunteers, stnfrs did not differ from control samples. thus, shock and severe injury result in different activation patterns of anti-inflammatory processes. m,l. rodrick, boston, b~usa following severe thermal injury significant suppression of the immune response has been demonstrsfed. these changes are associated with increased susceptibility not only to co,on human pathogens, but to organisms not normally pathogenic in the uncompromlsed host. early observations included prolonged graft rejection and skin test anergy in patients following burn injury. work from our laboratory and that of numerous others has shown that the~e is a profound £mmunoeuppresslon following severe thermal injury. i~mune deficiency has been identified in these patients hyz ii lack cf t nell responses to mitogens, ) early decreases in total t and helper t cells in the peripheral bloo~ } lack of response to tetanus toxoid i~uniza~io~ in spite of increased non-speoifi~ i~k~unoglobulin praduction and ) severe and prolongei deficiency of interleukin- (il- ) prcductlon by peripheral blood mononuclear cells (pbmo). this il- deficiency could be an underlyin~ cause of all the afo~ementloned immune defects by failing to activate both helper and suppressor t cell functions. mo~a recent work has focused on the molecular mechanisms of this il- deficiency end shown that the defect lies post-transcrlptionally since mrna for il- is also depressed following thermal injury. we have also investigated the potential of a defect in ii- receptor status on peripheral blood mononuclear uolls from patients fqllowing burn injury and in a murine model. in both oases no decrease in il- r was found either by phenotypic markers or by funational assays. another hallmark of thermal injury is hyperao~ivatlon of proinflammatory ~ytok~ne secretion, which may play a significant role in multiple organ failure following burn injury. therapy o~ major burn injury may reasonably be aimed, therefore, at up-regulating t cell ~unction and down-regulating hyperactive secretion of proieflaam~ntory cytokines. the majority of patients dying of bums succumb to sepsis which coincides with dysfunction in the specific and non-specific host defences. generally, concepts relating to the mechanism(s) of specific immune failure in thermal trauma imply that inhibition of t (cd +) cell responses is imposed by the injury or infection-related factors. however, the same factors elicit strong biological reactions within the lymphoid compartment. recent evidence indicates that systemic activation is inherent in the burn patient. to establish the relation of molecular and cellular events to the development of post-bum anergy we examined the state of and the capacity for t cell activation with regard to: ) occurrence of activation-induced cell death (aicd), ) activation-related phenotypic and functional_ diversity in the pool of peripheral cd + t cells. initially, (up to days post-bum), peripheral blood lympliocytes from severely injured (> % total body surface area) patients exhibited high levels of constitutive expression of surface receptor for ]l (cd ) and spontaneous blastogenesis. the presence of activation-related t cellproducts in bum plasma was also apparent. subsequent impairment of the t cell receptor (tcr)-regulated t cell responses in vitro was accompanied by significantly increased dna fragmentation that is associated with cell death by the mode of apoptosis. using molecular markers we established that flesh peripheral blood ceils from immunosuppressed patients also contain large numbers of apoptotic cells. fluctuations in the number of viable (pi-) peripheral blood lymphocytes involved primarily cd +/cd ro+ (memory) subset of t ceils. the above observations suggest that thermal trauma-associated t cell anergy develops through aicd, a phenomenon commonly associated with the tolerogenic activity of bacterial superantigens. persistence of staphylococcal infections in the burn patient may support this assumption. response following trauma jane shelby, ph.d. the immune system is integrated with other physiologic systems, and is exquisitely sensitive to changes in nervous and endocrine systems changes following traumatic stress challenge. the immune, nervous and endocrine systems interact via both direct and indirect pathways which utilize neuro and endocrine hormones, neurotransmitters, neurepeptides and immune cell products. it is now known that the immune system may be affected by all of the neuroendocrine products produced during a stress response, with evidence for innervation of iymphoid organs, lymphoid cell receptors for neuroendocdne products, and leukocyte production of chemicals which are virtually identical to certain neuroendocdne peptides (acth, endorphins). trauma induced alterations in the equilibrium of various neuropeptides and neuroendocdne hormones have a significant impact on immune response potential, affecting control of proliferation, differentiation and function of immune cells. for example, the neurohormone melatonin is thought to be a natural antagonist to counteract glucocorticeid associated immunosuppression resulting from stressful challenges, such as surgery and trauma, plasma melatonin levels are known to be significantly reduced in burn patients. the administration of exogenous me[atonin improved cellular immune response following burn injury in an animal model. melatonin was also shown to have in vivo cytokine regulatory activity, increasing the potential for il- secretion and downregulating excessive il- and ifn~ in burn injured, stress susceptible mice. the regulatory interactions between the immune, nervous and endocrine systems provide mechanistic pathways for trauma associated immune dysfunction. increased knowledge of these interactions will enhance the potential for the design of novei clinical interventions to improve immune response and decrease the risk for infection in trauma and surgical patients. . animals receiving e were given a single dose daily of either . g/kg of e in a % solution by garage (ge), or . g/kg of sterile ive in saline. four hours following the last dose, bum animals were subjected to a % body surface area bum injury to their dorsum. twentyfour hours following injury, the animals were sacrificed and spleen cells were harvested for assessment of lymphocyte function. splenocytes were prepared by mincing the spleen, followed by incubation on glass petri dishes to remove adherent macrophages. non-adherent cells were then tested for proliferative response to t-cell mitogen concanavalin a (con a) and b-cell mitogen lipopolysaccharide (lps). data were analyzed by anova. results: chronic alcohol exposure and burn injury independently inhibit lymphocyte response to con a but not to lps. the combination of e plus bum injury, however, pmfouedly decreases this response to both con a and lps as outlined in the this data clearly identifies the synergistic impairment of immune function produced by ethanol and bum injury. it is furthermore apparent that ibis effect is gut mediated and that gastrointestinal exposure to alcohol is necessary to produce this effect. further studies will work to identify cellular and subcellular mechanisms to explain this effect. in experimental animal studies and investigations on human volunteers endotoxin infusion is mgulary accompanied by the release of the cytokine tumor necrosis factor a (tnf-~) determined by elisa technique. in patients with menigococcal sepsis also elevated tnf-a values have been found using a functional assay. we have studied the role of tnf-et in surgical icu patients with sepsis. using functional technique, we were not able to detect tnf-~ activities in the patient plasmas. when this cytokine, however, was determined by immunochemicai technique (el sa) elevated tnf-e~ values where frequently oberserved. in order to further elucidate these observations, we studied shedding of tnf receptors in the patients. in these studies, we noticed that shedding of tnf receptors oecured regulary in the patients. at the time of diagnosis, soluble tnf receptor p and p were both - fold higher than values found in plasma samples obtained prior to die diagnosis of sepsis. we also observed that the sepsis patients revealed higher maximum values of p and p during the icu stay compared to values found in surgical icu patients without sepsis. these observations indicate that soluble tnf receptors are available in sufficient amounts to bind tnf-ot which is released in surgical patients developing sepsis. this mechanism may explain why functional tnf-c~ was not detected in the patients. institute for surgical research, rikshospitalet, the national hospital, university of oslo, oslo, norway. decker, d., sch ndorf, m., bidlingrnaier, f., hirner, a., yon rfcker, a. the advantage oflaparoscopic cholecystectomy over conventional open surgical approaches in the treatment of symptomatic cholelithiasis has been shown convincingly by clinical studies. in order to facilitate comparisons of different surgical approaches, we evaluated the cell biological characteristics of tissue trauma by measuring changes in various cell surface markers on leukocytes and eytokines in plasma as a possible means to assess tissue trauma in choleeystectomy. patients recruited into our study had experienced at least one typical bifiary colic, had ultrasound-proven cholelithiasis (stages -ii according to me sherry), were - years old, and presented for elective choleeysteetomy. patients could choose between laparoscopic and conventional eholeeystectomy after being informed about the advantages and disadvantages of each procedure. cell surface markers on leukoeytes were determined using whole blood techniques with the help of commercially available fluorescent monocloml antibodies and flow cytometry. shed cell surface markers in plasma and cytoldnes were measured with the help of sandwich-elisa kits. blood samples were drawn h before surgery, immediately before incision (after anaesthesia), h and h after incision. seventeen cell surface markers were examined on different cell populations and cellular subsets in laparoscopic and open-surgery patients. three soluble cell surface markers and six cytokines were monitored. by statistical analyses (multivariate regression analysis, student's t test, wilcoxommann-whituey's rank sum test) the six markers/cytekines that best distinguished open surgical from laparoscopic procedurea were determined. these were . the interleuldn- receptor and im soluble form (cd /scd ); . the activation antigen fd- and its soluble form (cd /scd ), a member of the nerve-growth-factor receptor family; . the cd ro epitope which characterizes t memory ceils; . the trausferrin receptor cd ; . the soluble adhesion molecule icam- ; and . the cytokines interieukin- and interleuldn- . on the basis of these results, a tissue trauma activation (tta) index was calculated by combining the marker/cytoldne measurements by simple multiplication. anaesthesia and pre-ineision maneuvers did not significantly change cell marker or cytokine levels in either surgical approach as compared to h before surgery. h after incision the tra index in open cholecystectomy showed a distinct - fold increase, whereas in laparoseopic surgery a mere - fold increase was noted. h after incision, the tra-index returned to near pre-surgery levels. in conclusion, our results demonstrate that changes in cell surface markers and cytokines can help evaluate the magnitude of tissue trauma in diffei'ent surgical approaches. the relationship between lymphocyte subpopulation changes after thermal injury and the increased susceptibility of burned patients to infection is unclear. in this study, we have attempted to correlate such subpopulation changes with the presence of infection in burned patients. peripberal blood from patients was monitored for lymphocyte subpopulation changes three times weekly for three weeks postburn and weekly thereafter for three additional weeks. mean bum size was . % (range %- %) of total body surface and mean age was years. infection was diagnosed by carefully defined clinical and laboratory criteria and its presence or absence noted each time blood was drawn. samples taken when patients had wound infection, bacteremia, or pneumonia were compared with samples taken in the absence of systemic infection. whole blood samples were stained with four monoclonal antibodies, the red blood cells lysed and the leukocytes fixed and analyzed by flow cytometry. for each patient sample, the proportion of lymphocytes falling within the light scatter gates was determined as the percentage of cells negative for cd and most strongly positive for cd . this percentage was used to correct each sample for the presence of debris or nonlymphocytic cells. the proportion of cd and cd positive cells was slightly greatc~ in the samples from infected patients, while the proportion of b cells (cd +) was unchanged and nk (cd +) cells were decreased by ahnos[ % compared to sampie~ li'om uuiuleclcd patients. the percentage of cells positive for cdilb (c~ integrin) decreased sharply and cd ro (memory cells) decreased slightly in samples from infected patients while the expression of the lymphocyte homing receptor and cd were unchanged. cd (il receptor) and cd (early activation marker) were significantly increased in the samples from the infected patients while hladr was unchanged. these changes in lymphocyte phenotype correlate with the presence of infection. if they closely precede or occur during the early development of infection they may be valuable clues to the mechanism of susceptibility following thermal injury. trauma patients are subjected to an immediate massive impact on their host defense integrity due to the combined effect of tissue trauma, shock and endotoxemia. cytoldnes are playing a crucial role within the course of an impaired cell mediated immune response (cmi) resulting from a disruption of intact m%/tcell interaction. the current study was undertaken to further elucidate the mechanisms of dysfimctional cmi following major burn and mechanical trauma -via comparative analysis of mrna expression and protein release. the major regulatory levels for different cytokines were determined in mitogen, respectively lps stimulated peripheral blood mononuclear cell (pbmc) cultures of trauma patients on consecutive days ( ) t, , , and post injury. we analyzed the cumulative data for interleukin- beta (il-i[ ), il- , il- as well as tumor necrosis factor alpha (tnf-~) and saw a considerable impairment of the protein release in the stimulated pbmc cultures until d post-trauma and recovery thereafter. *p < . , ** p < . vs control comparing the autoradiographies of the specific cytokine mrna expression with the protein release in the supernatants, we saw a good correlation between mrna signal intensity and protein synthesis for il- and ,- , suggesting that for these cytokines the main regulatory mechanisms are located at the pre-/transcriptional level. for the other cytokines investigated one has to suppose posttranseriptional mechanisms. the analysis of our data clearly indicates a severe impairment of forward regulatory immune mechanisms following trauma. most likely the regulatory mechanisms, that are involved are greatly different among the cytokines investigated. it may be concluded, that depressed cmi responses post-trauma are partly due to an impaired pro-inflammatory cytokine production. the severity of the injury (iss) correlated with the development at multiple organ failure (mof-score; r= . ). the levels of mediators and markers of the inflammatory response were generally higher in the more severely injured group (iss> , n= ). i - , - , g-csf, fpa, and c a -levels differed significantly (p< . ) between the iss-groups (>-< iss ) at the time of admission, whereas on day tnfa, c a, - , and ealpi showed significant differences. beyond the first week, major differences were restricted to pge and c a. the formation of two groups with respect to later multiple organ failure (mof < ; mof > n= ) yielded similar results. leukocyte-facs analysis revealed significant differences mainly in the cd (monocytes), cd /cd (i - r + t-cells), and cd /cd (th calls) populations. summarizing our findings we were able to detect some alterations in the surface antigens of immunocompetent cells. the inflammato d response, however, seemed to be more pronounced and correlates wi~ the further clinical course. using an experimental bum model in rodents, we have demonstrated that administration of a full thickness, scald burn involving % or more of the total body surface area (tbsa) elicits systemic responses which are characterized by numerous alterations in t-ceu function (i.e., lymphokine production and contact hypersensitivity (ch) responses) plus an enhanced susceptibility to bacterial infection. in the present study we questioned whether the apparent systemic effects mediated by large burns would be elicited as site-specific alterations in immune function following administration of small area burn trauma ( % tbsa). following a % tbsa burn, ch responses to contact sensitizing antigens were found to be altered. the depression in ch responses could be induced independent of the site used for topical skin sensitization. following a % tbsa thermal injury, development of ch responses were affected in a site-specific manner. immunization of % tbsa thermally injured mice in a site near the position of the burn resulted in depressed responsiveness, whereas immunization through a contralateral site resulted in responses that displayed both the intensity and kinetics of a ch response equivalent to sham-bumed mice. similar systemic and site-limited changes in lymphokine production were observed with % and % tbsa thermal injuries, respectively. a % tbsa injury affected the lymphokine producing potential of all cells regardless of which lymphoid tissue the cells were isolated from. the effect of a % tbsa burn was significant but site-specific. thus, ceils from lymph nodes receiving drainage from thermally injured tissue were specifically affected, whereas lymphokine production by cells from lymphoid organs receiving drainage from unaffected skin was normal. it was concluded that modulation of lymphokine production and cellular immune responses may be a normal consequence of burntrauma regardless of the size of the burn. changes in immune competence can be mediated either regionally or systemically in direct proportion to the area of skin exposed to the burn injury. this work is supported by phs grant gm and the office of navy research n - -j- . division of cell biology and immunology, department of pathology, university of utah school of medicine, salt lake city, ut . post spleneetomy septic sequelae may be fatal, but the mechanisms remain unclear. the objectives ef this study were to assess the mortality from concomitant splen-'etomy and ]~eritoneal bacterial challenge and to elucidate the local cetkdar responses. cd- mice were randomised to receive laparotomy and sham splenectomy (l) or splenectomy (s) with simultaneous ca'-cal ligation and "):mcture and the survival patterns assessed. subsequently, cd- mice were randomised into control (c), l or s groups and peritoneal cells studied at hours for bacterial phagocytosis and killi:~g, superoxide ( -) and tumour necrosis factor (tnf) production and macrophage activation vsing mac-i(cd- b) receptor in~.ensity expressed es mean channel of fluorescence (mcf). these resides indicate that sf!enectomy predisposes to nrortal~ty from bacterial sepsis ia the early pos~ operative period compared to sham operated animals. failure ~f p'.acrophages to kill bacteria in the splenectomv group '~:cured in t?~e absence of impairment of oxygen freeradical or tnf pred:~ctien. the macrovh~ge ac!ivotion marker mac- was significantly reduced in both l and s groups and impaired phagocytosis of bacteria oceured in both operative groups compared to controls. laparotomy a!one reduces macrophage activity in terms of surface re:eptor mac- expression and !ingestive capacity. splenectomy however s~gnificantiy ~mpairs r-acrophage-wediated l~,acterial killing and this qefect rttav co~tribut~ sig~ifjcav'ly to th-~ dissemination of local infection and to n':ortalit). depts of haem~ tology & surgery, beaumont hosoital, dub!in ,eire. introduction: loss of cell membrane integrity appears to be a common pathway of injury to tissues subjected to high-voltage electrical shock. the cell membrane is the most heat labile structure in the cell, and is also the most vulnerable to externally-imposed electrical forces. skeletal muscle and nerve cells are particularly susceptible to electroporation by clinically relevant electric fields. restoration of membrane integrity is essential for cell survival in victims of electrical shock. we have studied the effect of non-ionic triblock copolymers ( poloxamer class) on the transport properties of isolated rat skeletal muscle cells following electroporation-induced membrane disruption. - mm long adult skeletal muscle fibers were isolated by enzymatic digestion from the rat flexor digitorium brevus and maintained under standard culture conditions. they were loaded with the calcein-am dye and placed in a ,c chamber for recording by real-time video confocal microscopy. the cells were subjected to msec, v/era, a field pulses with a low duty cycle to allow thermal relaxation. peak temperature rise was , .c. the uye content of the cell was monitored in real time. experiments were carried out in calcium-free phosphate buffered saline, with mm mg%. experiments were repeated with mm neutral dextran ( the aim of the present paper is to ascertain if thuracotomy induces a different pattern of variations of cytokines, immunocompetent cells and antibodies from laparotomy in the early postoperative period. patients ( males females,mean age: . _+ ) with gallstone disease and with non neoplastic pulmonary disease were studied. none of these patients received blood transfusion, biological response modifiers, radiotherapy or surgery for at least months before being included in our study. anaesthetic procedures were similar in all patients and none were matnourished. on the day of surgery and on the st and th postoperative days (pre, lpo, po) percentages of cd , cd , cd , cds, cdi were measured by means of flow cytometry using moab., and levels of ig a, lgg, igm, ige. by nephelometry cytokine levels in peripheral blood(il- , il- , il- , il- , tnf) were measured in pts. of each group by means of elisa using moab. _r. esults:variations of il- and il- were not s.s.. il- increased but differences between groups were not statistically significant (s.s). il-i decreased on po and increased on po in both groups but were only s.s. in the th.g., and therefore, the differences between groups were s.s (p< . ).tnf decreased in the l.g. and increased in the th.g. on the po, the difference was s.s(p< . ); on po, tnf decreased in the l.g. and decreased in the th.g. but these variations were not s.s. cell percentages decreased an lpo and increased on po, except for %cd cell that increased on lpo and decreased on po ,in both groups of pts. differences were not s.s. ig a, igm decreased and ige increased in both groups (p< . i), but differences between them were not s.s. in contrast, igg decreased on po (p< . ) and increased on po in both groups, but the decrease iu the th.g. was greater than in the l.g. twenty male children,aged from six months to years,admitted for elective inguinal operation were studied. the operations were performed under balanced combined anaesthesia (fentanyl,thiopemtone,vecuronium, % nitrous oxide in oxygen) and blood samples were collected before flunitrazepam premedication,after anaesthesia, and hours after anaesthesia. cells from the wound were collected with cellstick sponge which was removed from the wound or hours after anaesthesia. the study was approved by the local ethical committee. the percentage of neutrophils was increased and that of lymphocytes was decreased in perpheral blood after the operation.the values in the wound were close to the values found in peripheral blood. the percentage of t-lymphocytes (cd ) and helper-t-cells (cd ) decreased in peripheral blood being lower in the wound than in peripheral blood after the operation. the percentage of t-eytotoxic cells (cd ) also decreased in peripheral blood and was similar to that in the wound. b-lymphocyte (cd ) percentage was increased in pe~pheral blood after the operation and was higher than in the wound. the percentage of activated t-cells (cd +hla-dr-positive cells) in peripheral blood increased while that of natural killer cells (cd +cd +leu -pos) was increased just after anaesthesia being decreased at g and hours after the operation. spontaneous lymphocyte proliferative responses didn't change while phytohemagglutinin a and concavalin a induced responses were decreased in peripheral blood samples hours after the operation with recovery at hours.pokeweed mitogen induced lymphocyte proliferative responses were decreased at hours (p<o. ) and hours after the operation we previously reported that plasma ige increased after traumatic injury. in this study, we measured plasma ige ievels in trauma patients on hospital admission and x weekly in the intensive care unit to evaluate ige kinetics. patient characteristics were: mean age -+ years ( m, f), mean injury severity score (iss) + , sepsis ( patients), sepsis syndrome ( patients), ards ( patients), renal failure ( patients). ige increased in patients ( %). the mean increase was ng/ml (range to ng/ml; % ci was to ng/ml). increase in plasma ige was significantly greater in patients with sepsis syndrome (p= . ) and renal failure (p< . ). although mean plasma ige was higher with ards, pneumonia, hepatic dysfunction, and shock, these differences were not significant (p> . ). plasma ige increase was not related to severity of injury by iss score (p = . ). the mean day to highest ige was . -+ . . the day sepsis was first observed preceded the day of highest ige by . + . days. there was a significant association between the day of sepsis onset and the day of highest ige (p= . ). eight of nine patients with sepsis syndrome had > % increase in plasma ige from admission. one patient's ige levels were normal ( - ng/ml) for days and then increased to ng/ml over the next days, after onset of sepsis syndrome. changes in ige plasma levels may reflect the action of cytokines, such as il- , which concurrently regulate production of ige and il- receptor antagonist in a response to sepsis. sepsis remains a leading cause of late mortality in trauma and hs. although hs-induced bacterial translocation is supposed to be the major cause of sepsis and mof, depression of the res increases susceptibility to infection after injury. the purposes of this study were: a) to evaluate the res in the lung, spleen and liver after hs and subsequent hypertonic saline (hsl) treatment, and b) to document the patterns of phagocytic activity in these organs during hrs. adult male wistar rats ( +_ gin) were submitted to hs (sbp tort) and after t hr (shock i hr) and hrs (shock hrs) hsl (nac . %, . ml/kg) treatment, e. coli (i ) was injected into the portal vein ~tci (n_> ). twenty minutes later, the lungs, spleen and liver were harvested and scintilographic counts obtained. data is depicted as mean_%+sem * p< . , ~" p< . and statistical analysis was performed by analysis of variance and wilcoxon tests. one hr after treatment, lung uptake was increased and liver and spleen uptake were reduced compared to sham. twenty four hrs after treatment, all organs, except lung uptake, returned to normal values. radioautographic histological analysis revealed radiolabeled particles inside phagocytic cells of all organs. we conclude that pulmonary phagocytic activity increases after hr of hs hsl reatment, diminishing by hrs although still above normal values. in contrast, res suppression occurs in liver and spleen after hr hs hsl treatment, returning to normal values by hrs. these results may explain lung complications and immunosuppression after trauma. infusion of endotoxin as well as major surgery is followed by lymphopenia in peripheral blood. the purpose of this study was to investigate to which tissues the lymphocytes are redistributed in response to endotoxaemia and major surgery. in addition changes in lymphocyte subpopulations and expression of mecii was measured. lymphocytes were isolated from peripheral blood of rabbits, labelled with indium-tropolene and reinjected intravenously into the rabbits, i rabbits received an infusion of escherichia coli endotoxin ~g/kg, while i rabbits were subjected to a major sham operation and i rabbits served as a control group. the redistribution of lymphocytes were imaged with af gamma camera, and calculated with an interfaces computer before, and , and hours after major surgery or infusion of endotoxin or saline. interleukin-l~ and serum cortisol were measured. in addition we followed cd , cd , cdlla/b, cdis, cd , cd , mhcii and cd /cd ratio. following endotoxaemia interleukin-lf~ increased significantly, following endotoxaemia as well as major surgery serum cortisol increased significantly. following major surgery as well as endotoxaemia there was significant lomphocytepenia in peripheral blood with a decreased cd /cd ratio while the cd positive subpopulation increased. in addition there was a decrease in the expression of mhcii on the lymphocytes peripheral blood. the radioactivity of the lymphatic tissue in and around the intestine increased to % of initial values following endotoxaemia and to % following major surgery. the results indicate that endotoxaemia as well as major surgery induces redistribution of lymphocytes from peripheral blood to lymphatic tissue. among the lymphocytes staying in peripheral blood there was a decreased expression of mhcii and a relative decrease in cd cells compared to cd positive lymphocytes. in order to analyze the effects of immune suppressive substances on expression of mrna of interleukin- (il- ) and interleukin- reeeptor(il- r), this study was carried out. twenty male rabbits with comminuted fracture were used in the study. ten ml blood were taken at , i, , , days after injury. the sera were tested for the effects on lymphocyte blastogenesis and induction of il- stimulated by concanavalin a(con a): the sera from the rabbits days after injury were analyzed with sds-page gel eleetrophoresis, and divided into three groups by ultrafiltration (ufpi ttk, kd,milipore; centricon- , kd,amicon), that are less than kd, between i and kd, and more than kd. each group of the substances also was tested for the expression of il- and il- r by the dot blot hybridization. the results showed that: i) all sera from the rabbits after injury had significant suppression on lymphocyte proliferation and secretion of il- by the con a-stimulated splenocyte in mice; ) the sera from the rabbits days after injury had more profound suppression than other injured sera; ) there was a marked band at about kd in sera from the rabbits days after injury, but nothing at the same position in normal sera analyzed with electrophoresis; ) the substance with molecular weight of about iokd had more obvious suppressive action on expression of mrna of il- and il- r than other groups substances, of which molecular weights are more than kd. it is concluded that: i) the sera from the injured rabbits can reduce immune response; ) there is kind of substance, of which molecular weight is about kd, it is probable the main factor involved in the pathogenesie of postinjury suppression immune; } the substance can depress the expression of mrna of both il- and il- r. research institute of surgery daping, chongqing, p. r. china acute ethanol uptake prior to injury modulates monocyte tnfo~, production and mononuclear cell apoptosis. g. szabo, b. verma, p. mandrekar, d. catalano monocytes (mo) have been shown to contribute to immunosuppression after both major injury and alcohol consumption. we reported that acute ethanol exposure of m( results in decreased antigen presentation, induces tgf- and pge while inhibiting inflammatory monokine production. we also showed that post-trauma immunosuppression is mediated by hyper-elevated mo tnfc~ and il- . consequently, here we investigated rnonokine production in trauma patients (n= ) who had elevated (>o.lmg/dl) or had no blood alcohol level (n=t ) at the time of emergency room admission. none of the patients had chronic alcohol use history. met tnfc~ production from trauma patients with prior alcohol uptake was undetectable during days - post-injury in contrast to patients without alcohol exposure. furthermore, decreased tnf~x levels were found in alcoholic patients' mci after mdp or ifny + mdp induction. however, mcl tnfc~ levels during the - days post injury period became higher in alcoholic trauma patients. furthermore, over days post-injury, alcoholic trauma patients showed significantly elevated mci tnfo~ production after adherence isolation, mdp, or ifn+mdp stimulation compared to patients without alcohol. these results suggest that acute ethanol uptake prior to injury decreases tnf(x inducibility in the early post-trauma period, but these patients' mo produce hyper-elevated tnfa levels later post-injury, thereby prolonging their cytokine shock risk. tnf ng/ml - days post-injury days post injury stimulus ale. pt. pt . . . . immunosuppression might also be increased by the elevated apoptotic activity found in trauma patients' mononuclear ceils, which was even greater in alcoholic trauma patients' cells. in non-alcoholic trauma patients' preactivated mo, in vitro acute ethanol ( - mm) exposure resulted in a significant down-regulation of tnfc~ (p< . ) and il- (p< . ) production. in contrast, in vitro ethanol exposure increased the production of inhibitory monokine, tgfi]. these results provide both in vivo and in vitro evidence for the effect of acute ethanol exposure increasing immunosuppression and cytokine shock. the 'systemic inflammatory response syndrome' (sirs) with consecutive septic multi-organ dysfunction represents the major cause of late death following major mechanical and burn trauma. systemic hyperinflammation and concurrent depression of cell mediated immune response (cmi) render the traumatized host anergic, resulting in profound susceptibility to opportunistic infection. monooytes/macrophages (mo) play a central role within the host defense system in developing and manifesting states of injury, shock and sepsis. the mechanistic scrutiny of the synthesis patterns of crucial cccytokines appears to be a helpful tool to further analyse mo behaviour in the compromised individual. the objective of this study was to further dissect the characteristics of cytokine regulation in pbmc under stressful conditions, via analysis of the expression of cd + receptor, the proinflammatory mediator il- , the macrophage activating factor ifn- ,, and neopterin (npt) a metabolite of activated mo. we investigated pbmc's on consecutive days , , , and after mechanical trauma of and after bum trauma of patients (mean age ~ years; mean iss ± pts). in trauma patients we saw a massive increase of pha induced neopterin synthesis compared to controls. however, when discriminating the npt levels in the supernatants for the amount of mo stimulated, the npt output of the individual cell was lower compared to mo of nontraumatized individuals. interestingly there was a contrary coarse in the cumulative protein release patterns of il- and ifn- in mechanical versus burn trauma patients. wheras in burn patients ifn-y was decreased significantly ( + u/ml) compared to controls ( + u/ml) as well as mechanical trauma ( + u/ml). il- showed a significant suppression following mechanical trauma ( + u/ml) vs control ( + u/ml) and bum patients. the rt~,na signal intensity for beth eytokines was in concurrence with the protein release in more than % of the individual patients investigated. from these data we can conclude that the inadequate low npt synthesis predominantly in bum patients appears to be a sign of cellular immaturity and is probably partly due to low t-cell ifno t signals. in addition we could state that the quality of trauma is apparently responsible for the different synthesis patterns of ]l- and ifn-q,. it has been postulated that bacterial invasion or endotoxemia are necessary for cytokine production following burn injury. we studied the organ distribution and kinetics pattern of il-fc~ (cell-associated il- agonist) in eutrophic rats subjected to either % tbsa cutaneous scald injury (bi), muscle scald injury of equivalent % tbsa (mbi), sham muscle bum (resection of skin only, up to % tbsa) (smbi), and sham cutaneous burn (sbi), followed by saline resuscitation ( mukg i.p.). separate rats were infused with mg/kg e.coli :b lps or saline lv. unmanipulated rats were baseline normal controls. liver, lung, spleen, ileum, thymus, kidney, skin, and plasma were harvested at various time-points within the first h. tissues were frozen, weighed, homogenized, the homogenates centrifuged and the supernates assayed with a radioimmunoassay specific for rat il-l(z (detection limit pg/rnl). il-lc~ was expressed as ng/g weight + sem (lowest detectable amount . ng/gwt). il-lo~ was constitutively present only in the skin ( + . ng/gwt). cutaneous burn and sham cutaneous bum induced biphasic elevations of il-lcc in the liver and lung only, with maximal levels at . h (in the liver, bi = . _+ . ng/gwt, sbi = . + . ng/gwt, p _< . ; in the lung, bi = . + . ng/gwt, sbi = . + . ng/gwt, p -< . ). of note, both bi and sbi rats had detectable il-i~ in the liver at timepoint already ( min real-time). these levels increased in parallel until min and became eventually different by log at - . h. all other organs as well as plasma were below detection limits. muscle burn injury and sham muscle burn (skin resection) induced similar elevations of il- ~ in the liver at lh, indistinguishable from each other and from cutaneous burn. in contrast, lps challenge induced dramatic elevation of il-t~ in all organs tested except for the kidney; the spleen was the most responsive organ to lps-induced il-lo~ production. these data indicate that thermal or mechanical injuries induce very early and organ specific production of il- c~ in vivo by mechanisms other than endotoxemia. injury-induced complement and platelet activation may be involved as well as the neuro-endocrine axis, which may explain the low levels of il-lo~ induction observed in all rats at the very early time-points. trauma services, massachusetts general hospital, and department of surgery, harvard medical school. fruit, st, boston, ma . j. f. schmand *#, a. ayala* and i. h. chaudry* studies indicate that i.v. infusion of the colloid hes in normal animals does not adversely affect non-specific immunity. it remains unknown, however, if lies affects cell mediated, specific immune functions after trauma and hemorrhage (hem). to study this, non-heparinized c h/hen mice underwent midline laparotomy to induce trauma and were then bled to and maintained at a bp of mmi-ig for rain. the animals were then resuscitated with either times (x) the shed blood vohune as lactated ringer's solution (lrs) or x lrs + lx % lies. sham mice were neither hemorrhaged nor resuscitated. at or hours post hem serum, peritoneal (pm~) and splenic macrophages (sm~) were obtained. bioassayes were employed to assess the levels of ii-l, il- ( alternatively pmqb showed no differences in il- release between all groups at and h, while sm~ from the lrs + hen group showed a depression at h. tnf production by pm~ was depressed in all groups at h and remained so in the lrs + hes group at h. sm~b showed decreased tnf release values in both hem groups at and h. in summary, the levels of inflammatory cytokines (particularly the values of circulating il- ) after trauma/hem are positively influenced by the administration of hes. this might be due to a protective effect on pmqb and sm~, but also on other cytokine producing cells, e.g. kupffer ceils. we conclude that hes is not only a safe, but also beneficial agent in the resuscitation of patients atler trauma/bemorrhagic shock. this study investigated endotoxemia and consecutlve immune response in patients with multiple trauma (median injury severity score = , ). blood samples.were collected shortly after injury and after , , , , s and l days. endotoxin was measured with limulus-amebocyte lysate test and the specific antibody content (sac) against endotoxins of the classes igg, igm and lga by elisa-technique. five antigens were used: lipopolysaccaride (lps) of e.coli (ec), lipid a of e.coli (la), lps of pseudomonas aerog. (pa), lps of vibrin cholerae (vc) and cx-hemolysin of staphylococcus anreus (oth). a nephelometer indicated the total concentrations of igg, igm and iga. differences were checked with wilcoxon-test and p< , s was considered significant. cross-reactivity was calculated with rank correlation coefficients. results: endotoxemia peaked shortly after injury ( - h) at , eki/ml (median), decreased thereafter to , eh/ml at day s and remained on this level. sac oflgmclass increased to all endotoxins and peaked at day revealing the lfighest level to la followed by pa (= % of la-sac), ec (= % of la-sac) and vc (= % of la-sac). lga antibodies increased as well but only slightly and not significant (exception: sac to la was elevated significantly at day ). igg antibodies increased similar to iga class only slightly and again only sac to la was significantly higher at day and . however sac to (xh of all ig-classes remained continuously on the same level troughout the observation time. correlation analysis revealed strong cross-reactivity (r> , ; p< , ) most often between antibodies of igm-elass ( %) followed by igaclass ( %) and lgg class ( %]. conclusions: multiple trauma is associated with temporary endotoxemia. endotoxins probably translocated from the gut cause specific increase of anti endotoxin antibodies in blood of the igm-class. endotoxins cause no increase of antibodies to gramposilave bacteria. igm antibodies are most unspecific. during cardio-pulmonary bypass, as well as postoperatively, high levels of endotoxin, interleukin- (ii- ) and c-reactive protein (crp) were measured in patients. i female and male, ageing from to with a median age of . blood sampling was done preoperatively, immediately after induction of anaesthesia, after thoracotomy, after cannulation of the aorta and right atrium after the first half of the reperfusion phase, after closure of the thorax, and hours after the operation and then every morning until the th postoperative day. blood was drawn into heparinized tubes (i iu/ml) which were free of endotoxin. crp levels were determined through the use of the behring nephelometer. - levels were measured by using commercially-available elisa test. the endotoxin level was determined by a chromogenic modification of the limulus amebocyte test. the statistical analysis was done using the wilcoxon ranks test and correlation analysis. a significant increase {p . ) in endotoxin plasma occurred during surgery, culminating in a peak (median value of . eu/m!) during reperfusicn. plasma levels of endotoxin continued to be slightly raised till the th day after surgery, whereas those of interleukin- rose at the end of the operation and were at their highest hours later (median value of . pg/ml). crp levels were also high postoperatively with a median value of mg/l, and were markedly raised on day ( mg/l). a definite, statistically significant correlation between the plasma levels of endotoxin and - during the operation was establisthed (p . ), leading us to conclude that the endotoxin liberated during cardiac surgery acts as the main trigger in the releasing of - , and thus induces the postoperative acute phase reaction. there was no evidence of a correlation between crp and endotoxin or - plasma levels. impaired immune function is well described following trauma and hemorrhagic shock (hs). prior studies have utilized peripheral blood or spleen cells to index immune function following hs. however, changes in mucosal immunity are not weii characterized in this setting. gut origin sepsis is thought to be an important cause of organ failure and death following trauma. a rodent model was utilized to allow comparison of mucosal-associated immune function vs, systemic compartments after hs. fischer rates underwent hs (map ± mm hg) for minutes followed by resuscitation with shed blood and lr. sham animals were instrumented only. rat tears were collected at and hours following hs for quantitation of slga by ria. animals were sacrificed at hours and spleen (spl), peripheral lymph nodes (pln), and mesenteric lymph nodes (mln) harvested for cell population analysis using flow cytometry and mitogen stimulation analysis. cell marker expression analysis revealed no changes in t or b ceil populations following hs. mitogen mucosal immune function appears relatively spared following hs. the mechanism(s) for this variability in immune function requires further investigation. we have found that transplantation of bone marrow in a hind-limb graft to syngeneic lethally irradiated recipient is followed not only by rapid repopulafion but also overpopulation of bone marrow cavities. the question arises whether this unexpected phenomenon could be the result of stimulation of stem cells by factors (cytokines) released from surgical wound at the site of anastomosis of graft with recipient. aim of the study was to investigate which tissues damaged during the procedure of limb transplantation may be a potential source of humoral factors accelerating in vivo bone marrow proliferation. methods. experiments were carried out on lew rats in groups. in group i, the hind limb was transplanted orthotopically to a syngeneic recipient; in group ii, sham operation was performed; in group iii, a four-cm long cutaneous wound was made on the dorsum; in group iv, limb skin was harvested, fragmented and implanted into peritoneal cavity; in group v, bm from femur and tibia was implanted intraperitoneally. bm, lymphoid tissues and blood were sampled and days later for cell concentration and phenotype evaluation. results. the yield of nucleated cells from tibia was on day in the control . + . , in group . + . , in group ii . + . , in group iii . + . , in group iv . _+ . , in group v . _+ . x ( ). the evident increase in bmc yield in all groups continued until day . increase in weight and total cell count of spleen and mesenteric lymph nodes in all but group iii was also found. no differences in percentage of maturing erythroid cells, but higher of mature myeloid cells and lower of lymphocytes were observed. conclusions. trauma of skin, muscles, and bone brought about an increase in bone marrow cellularity and acceleration of maturation of myeloid lineage. transplantation of bm ceils alone did not produce this effect. transplantation of bm in limb graft is a good model for studies of natural factors reaulatin~ bm hemormesis. this study sought to determine a relationship, if any, between the degree of hypochclesterolemia upon trauma patients' admission and their subsequent outcome. all blunt and penetrating trauma patients admitted to a level i facility from through , and who had serum cholesterol assayed during the first hrs were retrospectively studied for development of death or significant organ dysfunction. the mantel-kaenzel chisquared test was used to determine significance of data at the p< . level. results: trauma patients were admitted during the four-year period who had serum cholesterol assays performed in the first hrs. patients had cholesterol levels less than mg/dl; of these ( . %) died, ( . %) developed ards, ( . %) developed acute renal failure, and ( . %) developed multisystem organ dysfunction; hypocholesterolemia in these patients was not due to liver injury or massive fluid administration. the risk of death was times greater and risk of multi-organ failure times greater in this group than in those with a normal serum cholesterol (>if mg/dl; patients; p< . ). conclusions: admission serum cholesterol level in the trauma patient serves as a powerful marker for those at risk of subsequent organ failure or death. hypocholesterolemia in this setting may result from organ hypoperfusion and humeral mediator release. lung tissue contains many immunocompetent cells. resection, therefore, is expected to activate extensively inflammatory mediators such as pmn-elastase, pmstanoids and pteridines. in a prospective clinical study we compared patients (pts) undergoing either thomcotomy with or without lung tissue msectioh and tboracoscopic lung resection concerning activation of inflammatory response. material & methods: group a pts (n= ) had thoraantomy but no lung tissue injury; group b pts (n=ls) had thoracotomy and lung tissue resection due to benign diseases; group c (n= ) represents group b tissue resection but using a thomcoscopic procedure. the following parameters were determined pre-, peri-, and postoperatively: elastase and crp as indicators of activation of pmn-leukocytes and injury severity; prostacyclin (pgi ) and thromboxane (txa~) as parameters of lung endothelial response; prostaglandin f ~ (pgf~) and pgm representing pulmonaly metabolic activity; pge a and neopterin as proof of macmphage activation. statistics were performed using analysis of variance for repeated measures. results: group b pts revealed postoperatively an increase in crp (p< . ) indicating a higher injury severity in comparison to the thoracoscopic procedure (c). both, controls (a) and group c pts did not show pmn-activation, whereas group b demonstrated a reversible increase in elastase. surgical trauma caused in all groups a release of pgi z and txa which was more pronounced in c (p< . ) and most in b (p< . ). similar results were found for pge~ and pgf =. there was no activation of maerophages since neopterin did not increase. apparently, metabolic lung function was not impaired because there was no marked rise in pgm except in b (p< . vs. c). discussion: our results demonstrate that lung tissue injury aggravates the mediator release induced by thoracic traum. these mediators among others are able to increase capillary pressure and hence lung edema formation. impairment of lung function, however, seems dependent on the extent of the liberation. therefore, the maximal release reactions occured in group b and c after lung tissue resection, whereas the controls showed the highest levels immediately after the incision. we conclude that thoracoscopic procedures are superior in reducing the resection trauma per se and hence might prevent severe mediamr-induced (pulmonary/systemic) sequelae. in a prospective study we investigated patients using radiochemical method according to sch~dlich (s) and photometric method according to hoffmann (h). serum of severly traumatized patients was withdrawn directly after admission at our emergency room and in narrow time intervals during first hours after trauma. follow up control samples were taken daily until day ten. whereas no elevated pla-ca was found during first hours, a peak was regularly observed around day four. there was high correlation between pla-ca and iss (r= . , p<o.o ) except patients suffering from thoracic trauma, thoracic trauma {tt) provoked similar gons-score-and pla-ca-values compared to multiple trauma (mt) despite significantly lower iss: pla-ca (h) mt . + . tt , +_ , n.s. pla-ca(s) mt . +_ . tt . _+ . n.s. goris mt . - . tt . _+ . n.s. iss mt - tt - p< . we found delayed elevation of pla-ca after severe trauma, however detection of delayed pla-ca in the serum of traumatized patients cannot give reliable information about the exact role of pla in the inflammatory reaction after trauma as latest results show that pla is secreted early but bound at lipophilic sites of vascular membranes. hern~mdez m j, amiguet ja, monreal ji, vid~n jr, jim nez f j, l pez-vivanco g acute phase reactant proteins increment in serum of patients with traumatisms and inflammation has been previously reported. alpha- antitrypsin (aat), alpha- macroglobulin (amg) and ceruloplasmin (cp) are evaluated in the following of patients, males and females (mean age: years), with compound fractures. % of them were located in tibia and fibula. intercurrent infection developed in patients and internal fixation rejection in one. measurements were made by radial immunodiffusion on days , , , , , and . a control group of healthy volunteers was also evaluated. aat and cp showed increased values from day which kept elevated untill the end of the study. amg elevated from day . when infection developed, aat and cp values were even higher whereas amg values decreased. fixation rejection caused aat, amg and cp increment. aat and cp increment in non complicated fractures might be due to accompanying inflamation, by means of cytokines release and aprps hepatic synthesis induction. therefore, increment is higher when infection or rejection develop. amg behaviour at early stages of evolution is secondary to hiperconsumption, considering mean life shortening after proteases ligation. amg modifications in fixation rejection remain obscure. aprps increment modulates inflammation and bony callus formation by means their antyprotease and antioxidant properties. clinically, aprps might be useful parameters in determining complications onset in fractures evolution. hemorrhagic shock (hs) is a common complication of trauma, as well as some major surgical procedures. the alteration of the immune system by hs is thought to contribute to the increased septic morbidity and mortality seen in these patients. multiple mediators are released following hs. il- , il- , tnf, pge and tgf-b have all been implicated in these immune system changes. in vitro, pge causes many of the immune system changes following hs, yet the time course of pge release has not been well deliniated. the aim of this study was to determine the time course of pge release by peritoneal and splenic macrophages following hs. c h/hen mice were bled to a mean bp of + mm hg for minutes. the animals were resuscitated with the shed blood and normal saline. control animals were cannalated, but blood was not withdrawn. the animals were sacrificed at , , , , and hours following hs. peritoneal and splenic macrophages were harvested from each animal and cultured with lps for hours. the supernatants were collected and frozen until assayed. pge was assayed using an elisa (cayman chemical). results are shown below for the peritoneal macrophages: pge release by peritoneal macrophages was significantly elevated at , and hours over control. it was maximal at hours, but by hours it had started to decline. splenic macrophage pge release was much more variable. (data not shown.) there was less consistency between time points and no clear trend was seen. in conclusion, pge is significantly elevated at hours following hs, and reaches a peak at hours. pge release may be responsible for the immune system changes seen in the first few days following hs. splenic and peritoneal macrophages respond differently in their release of pge following hs; which may be due to differences in their milieu. there is an obvious association between the altered immunocompetence of patients following traumatic injury of various types and the increased riskto develop complications. the present investigation was undertaken in order to analyze the value of both neopterin (n) and c-reactive protein (crp) in monitoring disease course in patients with multiple trauma. we were interested to compare nconcentrations as marker of t-cell/macrophage activation with crp levels as indicator of the inflammatory acute-phase response daily serum concentrations(n and crp) were measured in patients ( male, female, mean age . ), admitted to our icu following serious trauma (mean iss= ) and in control subjects. the clinical course of each patient was evaluated with apacheii score according to knaus. mean stay in icu was . (range - ). patients didn't develop organ failure(nof-group), patients survived, developing mof (s,mof-group), patients died with mof (ns, mofgroup). three patients(one s and two ns) showed signs of septicemia and septic shock. the serum samples were measured for neopterin (immu-test-ria, henning-berlin) and for crp (immunoturbidimetric method) -at the university hospital for internal medicine, innsbruck, austria. the highest crp levels were measured h after trauma, but they were not accompanied by significant increase in n-values. we found significantly elevated n-concentrations from the days - . n and crp levels showed a parallel release peaks on the days - and the values discriminated significantly among the three outcome groups(the second crp peak is lower in comparison to the first posttrauma peak). by the ns-group we found constant and parallel increasing crp and n levels, reaching extremly high values - h and preceding clinical signs of mof and sepsis. the values increased dramatically before death. the serum levels by all control subjects were upper the normal limit for n and crp.. the parallel occured peak changes in n and crp concentrations showed a significant correlation with clinical status, using a disease activity score (apac h eli). our findings suggest that both-cellular and humoral mediated immune mechanisms are activated after multiple trauma and that simultaneous determination of n-crp may be of advantage as diagnostic and prognostic parameter in polytrauma-patient monitoring panel. it is apparent, that measurement of very high levels may be of value as a srong indicator in developing of mof or septic complications and thus offer the possibility for earlier therapeutic intervention. the modification of t-cell/macrophage and acute-phase responses may be potentially useful in the treatment of icu patients, resuscitated after severe trauma, and suggest that n-crp may be relevant indicator for testing experimental immunomodulating therapies. although several in vitro studies suggested the catalytic action of iron in oxygen free radical generation, few data are available concerning iron-metabolism following ischemia-reperfusion injury and hemorrhage in vivo. aim of the present studies was to evaluate iron metabolism following mild and severe hemorrhage in the presence and absence of intraperituneal hematoma. methods. male lewis rats ( g) divided in groups (observation time hrs): a: mild hemorrhage by sequential blood sampling ( . ml each)( , , , , , , , , hrs). b: a with hemoperitoneum (hp)( ml/ g), c: hemorrhagic shock: blood withdrawal of ml/ g over min and resuscitation with ringer's lactate ( x shed blood) and isogenic donor blood; d: c with hp. parameters: serum iron ([g/dl], ferrozin-method); fe labeling of erythrocytas (application of ci fe i.a. into a donor rat, days later bleeding of the rat and injection intraperitoneally in experimental rat; plasma transferrin ([g/ml], ria). t-tests and mann whitney. data are expressed as mean + sem. results. compared to baseline, mild and severe hemorrhage caused a significant increase in serum iron at and hrs, respectively ( + vs. + ; +_ vs. +_ ). hp reduced this increase ( +_ vs. + ; + vs. +- , p<. ). i-ip significantly increased hemoglobin between to hrs in mild ( . +_. vs. . +. ) and severe hemorrhage ( . +. vs. . +. ). in mild and severe hemorrhage comparable maximal resorption ( fe labeled erythrocytes in circulating blood) of intraperitoneal hematoma occured at hrs (mild: +_ vs. severe: +- counts/rain). hp increased survival-rate following hemorrhagic shock (no hp: / ; hp: / ). in mild hemorrhage no alterations of plasma transferrin concentrations. in shock group without hp, there was at hrs a significant higher transferrin level than with hp alone ( . +_. vs. . + . ). in conclusion, hemoperitoneum with hemorrhage prevents increase in plasma iron levels by increasing hemoglobin through resorption of erythrocytes and thereby suppressing iron mobilization from endogenous iron resources, e.g. liver. this suggests that intraperitoneal hematoma not necessarily promotes iron-mediated oxygen free radical production, especially since survival in these groups was improved. increased transfarrin levels in group c at the end of the experiment suggest increased iron turnover which was not observed in shock groups associated with hemoperitoneum. primary immune response to thymus-dependent (srbc) and thymus-independent (vi-polysaccharide) antigens was determined during days after cct. antigenic challenge was made on day i, , or . number of spleen plaque forming cells (pfc) and serum haemagglutinin (ha) titre were studied on day after immunization. cct stimulated immune response to t-dependent, but not to t-independent antigen. enhanced response was noted when srbs were given in posttraumatic day i, it was higher than preceding one after injection on day and reached a maximum when antigen was presented on day after trauma (pfc number . times exceeded the level of immunized, but nontraumatized rats). the rise of pfc number in spleen correlated with increased ha level. thymectomy had been performed days before cct completely abolished posttraumatio stimulation of immune response to t-dependent antigen. standard thoracotomy also enhanced humoral response when srbc were administered on day after the operation. thymus trauma modeled by pincett squeezing of its right lobe and performed at the same time with thoracotomy fully abrogated as well as thymectomy stimulation of immune reaction. thus, different experimental chest traumas are able to increase antibody production via thymus-dependent mechanisms. endothelin is a amino acid peptide produced by ischemic or injured endothelial cells. in vitro and in animal studies, et is also a potent constrictor for renal mesangial and coronary vessels, a negative cardiac inotrope and an endocrine regulator. our lab has shown that et is an agonist for monocyte production of interleukins i, & , prostaglandin e (pge ) , and substances which trigger superoxide production. systemic et levels increase in hypoperfused and septic patients, and et may be yet another important cytokine in critical illness. but while et has been shown to be a potent vasoconstrictor in healthy dogs, systemic vascular resistance does not increase in critically ill patients with high et levels. (we hypothesize that this might be due to pgez-mediated vasodilation predominating over et-mediated vasoconstriction.) we next asked what happened to systemic et levels in patients with major burns (> %.) ten hemodynamically stable patients resuscitated by a modified parkland formula to a urine output > cc's per hour had et levels drawn on admission, at i, , , and hrs. et levels were measured by radioimmunoassay. mean levels were elevated at ± pg/ml at all time points versus levels in healthy controls of ± . in summary, systemic et levels increase significantly in patients with major burns. et may be yet another cytokine playing a significant role in the immune, inflammatory and multiorgan dysfunction observed with major burns. restoration processes in an organism after ischemic damage are realized through ~n~lammatory mechanisms~ the intensity of which is significantly defined by blood levels of neuropeptides. myocardial infarction (mi) was chosen for studyin these processes since it eradicates the influence of infectious factc~rs. dogs~ in whom mi underwent different forms o¢ healer, g; bhn~ed ~h~t during the acute phase of the disease there was a characteristic rise of ne!~ropeptides in the blood. these neuropeptides had nociceptive and antinociceptive effects. particularly substance p and -endorphins triggered off the development of compensatory and adaptive mechanisms and defined the intensity of inflammatory reaction at the zone of ischem~t: damage-notable fall in substance p levels after an ~nitial increase, while the ~-endorphins stayed high was an important condition for non complicated healing of mi. on the other hand high levels of substance p with low ~-endorphin concentrations lead to increased infiltration o~ neutrophils into the infarction zone and weakened the activity of synthetic processes~ thereby leading to left ventricular aneurysm. at the same time low intitial levels of substance p slowed down the development of necrotic processes which lead to delay in refunctioning of the heart and complicated the healing process. thus, regulation of the levels of neuropeptides in the blood in trauma forms a perspective method of its treatment. of laparascopic versus open choleocystectomy c. schinkel, s. zimmer, v. lange, d. fuchs, e. faist the impairment of immune function due to surgical trauma may be followed by deleterious septic sequelae. compared to open abdominal surgical procedures (lap), laparaseopic surgery (lsc) is associated with a decrease in hospital stay and in accelerated patient recover. the aim of the study was to evaluate the sensitivity of the immune sermn parameters of il- , saa and neopterin, the percentage of cd + cells, the in-vitro il- synthesis after mitogen stimulation and lymphocyte proliferation, in order to purposefully discriminate differences in the severity of trauma. we investigated the blood of patients with cholecystolithiasis undergoing either laparascopic ( ) or open (i ) cholecystectomy on consecutive perioperative days - , , and . there was no significant difference between the two groups concerning age and sex. patients with clinical signs of acute cholecystitis were excluded from the study. operation time and hospital stay were obviously longer in lap patients ( versus minutes, versus days) compared to the lsc group. concerning the unspecific acute phase reaction we could show no difference in the increment of senun amyoid a (saa) synthesis in the lsc group (d-i + lng/ml, d + ng/ml) versus lap group (d- + ng/ml, d + ng/ml), while in serum il- levels we saw a less steep increment in the lsc group ( -fold from d- to d ) compared to the lap group ( -fold from d- to d ). the analysis of cd + receptor expression and serum neopterin did not reveal any difference between the groups. lymphocyte function showed an impairment of proliferation to antigen stimulation in lap (d - : . + . cpm, d : . + . cpm) compared to the lsc group (d -h . + . cpm, d h . + . cpm). in both groups il- synthesis was decreased post-operatively. our data indicate that laparascopic cholecystectomy reusults in a less distinct unspecific acute phase reaction post-trauma compared to that following lap. neopterin serum levels and cd receptor expression show that these parameters apparently are less useful markers to detect differences of surgical trauma severity while it appears that the impact of lap is reflected most impressively on the lymphocyte compartment. trauma alters the host resistance of organism and is accompained by appearence of excgenic and endogenic proteins in the body. to understand the molecular mechanisms of host resistans disorders in trauma, as a first step, the genetic regulatory mechanisms of immune response after antigen injection has been studed. the appearence of specific protein factors ( - and kda), in the nucleus of rat splenic and brain cells, accordingly, was shown after immunization with sheep erythrocytes. the stimulatory effect of these factors on the il- mrna and il- production was detected. the nucleotide sequences of the human il- gene regulatory region bounding by the splenic nuclear proteins were determined between + - b.p. the il- trans-factors shows the affinity to splenic and thymic lymphocytes in vitro. thus, the antigen causes the appearence of specific protein factors in the cells,which act on the gene level,stimulate il- production and the host resistance. these results cause the next step of experiments using the same model, but after trauma. these investigations will let us verify the hypothesis that the protein il- gene trans-factors may play a definite role in the decrease of the cell immune responce after trauma. confronted with the routine procedure of prophylactic treatment of candidates for surgery in a rural african hospital, we initiated studies on the fre'quency of post-surgical malaria. in tanzania non-pregnant patients from rural areas were followed. of preoperative patients % had a parasitaemia and those maintaining it showed no increase or complaints. nine percent of patients without detectable parasitaemia before surgery came down afterwards and one-third had malaria-like complaints. spinal and general anaesthesia were equally applied in these last patients. in burkina faso we studied patients of which % had a parasitaemia on admission and % had postoperative malaria. half of the surgical patients came from rural areas, whilst only % of those with malaria lived in the city (with much less exposure and immunity). % underwent major surgery and % minor. bloodtransfusions ( % with parasites) never evoked a parasitaemia in recipients. post-surgical malaria is thus a reality in about % of the adult cases, both in east and west africa. surgery evokes a cascade of factors, varying from cortison to interleukines and acute phase proteins; immune responses may temporarily be suppressed. clinical attacks of malaria in otherwise immunes could be evoked by one of these factors. though malaria can easily be cured, the differential diagnosis is difficult because of post-surgery fevers; we found that % was treated without justified indication. the involvement of "student-doctors" a. this study examines glucose uptake and hexose monophosphate (i~ip) shunt activity in normal human peripheral lymphocytes and polymorphonuclear leukocytes (pmn). glucose uptake was determined by measurir,g the uptake of tritiated deoxyglucose, a non-metabolized glucose analogue. adsorption of co derived from [i- c] glucose was used to determine knp shunt activity. in vitro assays were carried out in hormone concentrations approximating normal and elevated trauma blood levels. (normal -cortisol . ~g/ml, glucagon #g/m , epinephrine ~g/ml, insulin t~u/ml; traumaeortisol . ~g/ml, glucagon /*g/ml, epinephrine ~g/ml, insulin ~ij/ml. analysis of twenty subjects showed a reduction of ° ~mp shunt activity by lymphoeytes and a ] % reduction in glucose uptake by p~n in normal vs. trauma hontc,nes p < . . lymphocyte glucose uptake was also reduced by trauma hormones p~ . . it ha~ be.ea~ suggested thgt idiopatno pulmonary fibrous (y.pf) [s a consequence of severe alveolar epithelial injury and is associated with an nveolar irnammamry reactio~ and the presence f.neutr phils. there~bre, neutr pk~ chemoattra~ant~ are probably important in the genegs oft.he infial lesions of ipf. the obse,"wson that stimulated macrophages are or~n histologically promin~t in fibmfio [-~gs ~.nd am capable of p~oducmg a v~dery f flbrogenic pep'ides also a~gues for their role ~n the pathogenic prc~e~ oflpf. the observation that stimume~ maerophages ere often histologica[iy prominent in fibrotio lungs and ~re ~pable of producing a varie~, offibroge.~e peptide~ also argues for tkek role in the pathogenic process, therefore, we ha-~e tested the potentn for iater!eukln- (i ..- ) and mo~tocyte chemotacde pop, de (x¢cp- ) to induce neutro~hil ~d mononuclear phagocyte accumuhdon in lungs of pafient~ with pulmonary .~r~idosis and i~f. brenet~o.alveolar lavabo (bal) fluids from ipf and sar~qidosis patient were conexntratea by reversed-phase chromatography, ~d ii. arid mcp-i asso.~ed by ells& ehemotaxis mad enzyme-reieasing ~ssas's on msnocyte~ and neatrophiis. elisa revealed significenfly elevated b al-eoneentrations o£mcp-i ( . ng]mg aibumm) in purisms with p~monary sarcoidodis artd in ipf ( . ng!mg) in comparises to . normal individuals ( . ng/mg) and to patients w~th obreic bronentis (cb) (~, rig/rag). similarly, chemota*dc ac~a~' for monocles (mcp- e.qu/va]ent) was strongly increased in sareoidosis ( . ngjmg) as well as ~n f pag,nts ( . ng/mg). norra.al indlvidu~s and cb patiants hzd a . or -fold lower ~cn%i~y, re~peefively. patients with ipf and sarcoidosi~ also h~l eievated il- ievei~ ( . and . rig/rag, respe~veiy; nomzls: . rig/rag; cb: . ng/mg) mad nvatropmi ohemotax~ ( . ~'~d . nnmg, res!z~ztiveiy; aormals: . ng,'mg; cb: l ngmg). these data suggest that increased ievels of born mcp. ~d il- may be oharacted~tie for ~arcoidosis or ipf_ it appears iikely that both ehernoattraetants ~ontribute to the influx ofmonocytes and neutrophils into the pulmonary alveoius and interstit~um in these dlsea~es. we have recently shown that the combined administration of noninjurious doses of lps and paf in the rat produce ards-like lung injury characterized by neutrophil adhesion to lung capillary venules, neutrophil accumulation in lung parenchyma, pulmonary edema, and increased protein and neutrophil count in bal fluid. this new paradigm of lung injury was associated with elevated serum tnfc~ and pretreatment with anti tnfa mab dose-dependently prevented these responses. also, the combined administration of lps and paf induced lung mrna levels of tnfe~ ( fold vs. lps or paf alone), ll-lg ( fold), kc ( fold) and il- . taken together, these data suggest that this new paradigm of lung injury is cytokinemediated and that lps/paf in vivo can functionally couple to the activation of gone expression of a multi-cytokine network system, all of which may be involved in the pathogenesis of ards. materials and methods. the sheep model included hemorrhagic shock and closed femoral nailing at day , hourly injections of e. coli endotoxin and zymosan-activated autologous plasma at clays - and further observation and measurements at days - . from venous blood and bronchoalveolar lavage(bal)fluid of ten merino sheep (mean weight kg) neutrophil counts ( e pmn/ml blood or epithelial lining fluid-elf-), the elf/ plasma ratio of albumin (r), and the zymosan-induced (stim) and non-induced (spont) chemiluminescence response (cl) of blood ( e cpm/ , pmn), and of blood-and bal-isolated pmn ( e cpm/ , pmn) were measured. for statistical calculations the wilcoxon test was used. data of the changes in polymorphonucleur leukocyte (pivinl) metabolism have been suggested to play a pivotal part in the post-traumatic systemic inflammatory response syndrome. the underlying cellular mechanisms which control this response are not yet completely understood. since the 'ca + second messenger'-system has been shown to be involved in regulation of pmnl-'respiratory burst', we investigated changes in pmnl-ca z÷ regulation in relation to oxygen free radical mediated injury. methods. in polytranmatized patients (mean injury severity score = ) arterial and venous blood samples during days. daily evaluation of horowitz-quotiant (po /fio ), plasma lactate (mg/dl) and body temperature ( results. body temperature peaked at day and (day : +. ; day : . +. ). plasma lactate was significantly increased at day l ( + ) and day ( . + ). hurowitz-quotient (day : + ) was low at day ( + ) and day to ( + )(p<. ). at day a substantial rise in venous pmnl-superoxide production (day : . +_. , day : . +. , day : . +_. ), oecured with significant increase in plasma lipid peroxidation (day : . + . ; day : . + . ). pivin~-myeloperoxidase activity was high at day ( . +--. ) and then continuously declined (day : . +. ). plasma antiexidant activity (glutathione pemxidase) was reduced by % at day (day : . +. ; day : . +_. ; day : . +. ). whereas basal ca + concentration remained unchanged (day : +_ , day : +_ ), fmlp-stimulated cytosolic ca + mobilization increased at day (day : + , day : , day : + ). conclusion. the present study in polytraumatized patients shows, that seven days after injury the agonist-induced pmnl ca + mobilization is significantly enhanced. at the same time, pmnl-oxygen free radical release and phagocytotic activity, systemic fever response and lactate concentrations were maximal. these observations were accompanied by post-tranmatic respiratory failure and in some patients by clinical signs of multiple organ failure. preliminary data from an ongoing study using hes-and dextran-infusions in these patients show attenuation of this inflammatory response. stefan rose, m.d., trauma surgery, univ. of saarland, homburg/saar donnelly sc, haslett c, dransfield i, robertson ce, grant is, carter c, ross ja, tedder tf. dept's of respiratory medicine, accident & emergency, intensive care, surgery, university of edinburgh, scotland and dept. tumor immunology, dana farber cancer institute, boston. the selectins are a family of adhesion molecules (l-selectin, e-selectin, pselectin), all of whom are implicated in inflammatory cell transendothelial migration. they, as a family can be proteolytieally cleaved from their parent cell and exist in a soluble form within the circulation. ards is a disease state in whic neutrophils and neutrophil transendotheliat migration have been implicated. in this study we wished to investigate whether the levels of these circulating soluble receptors from patients at-risk of ards at initial hospital presentation, correlated with subsequent ards progression. eighty-two patients were enrolled (pancreatitis (n= ), perforated bowel (n= ), and multiple trauma (n= )), of whom progressed to ards. assays for soluble l,p & e-selectin were performed on collected plasma samples via a sandwich elisa. (ns = not significant, **** = p<o. ) we have found a highly significant inverse correlation between a low circulating soluble l-selectin (and not soluble e & p-selectin) and subsequent ards. these results further our understanding of neutrophilendothelial interactions in the early stages of ards pathogenesis and offer the promise of sl-selectin in combination with other markers of inflammatory events being used to identify individuals at particularly high risk of ards progression on initial hospital presentation. it has been suggested that polymorphonuclear leukocytes aggregate in the lung capillaries of patients developing the adult respiratory distress syndrome (ards) and account for the endothelial damage while releasing toxic metabo-iites such as o.a. free oxygen radicals. among many antioxidants which have been investigated in in vitro systems and in animal models, n-acetylcysteine (nac) has been established to be useful as a free radical scavenger in vivo. to assess the influence of n-acetylcysteine (nac) on the activation of neutrophils in patients undergoing cardiopulmonary bypass (cpb) surgery with extracorporeal circulation (ecc), we evaluated the plasma levels of elastase and myeloperoxidase (mpo) throughout the operation and up to hours after surgery. four hours after surgery also a bronchoalveolar lavage was performed. a spectrophotometric method was used for the detection of plasma elastase activity and mpo levels were measured using a radioimmunoassay. we found that pretreatment with intravenous nac prevented the increase in elastase activity ( _+ pmol/i vs _+ ; p - . ), but not the release of neutrophil related mediators ( . _+ . ng/i vs . _+ . ; p= . ). besides, nac had the capability to prevent the enhancement of neutrophil numbers in lavage fluid as is normally seen during cpb ( . + . % vs . _+ . ; p = . ). although not significant, nac prevented also the increase in elastase levels in lavage fluid ( . + . pmol/i vs . + . ; p = . ). it is concluded that nac is able to protect against the inactivation of a -proteinase inhibitor, the main inhibitor of elastase activity, and that nac interferes with adhesion and migration of neutrophils. therefore nac may be useful in the prevention of tissue damage. this study is supported by inpharzam belgium. sodium nitroprnsside (snp) has previously been shown to attenuate the neutrophil induced lung injury associated with limb ischaemia and repeffusion. glyceryl trinitrate (gtn), already widely used in aortic surgery, also increases nitric oxide but has a less marked splanchnic "steal" effect. we examined the effect of gtn on lung injury, neutrophil infiltration and activation in a model of aortic occlusion ( rain) and repeffusion ( min). sprague dawley rats were randomized into: control (n=i ); ischaemia repeffusion (ir) (n= ); and ir treated with gtn ( ug/kg/min) during repeffusion (n= ). myeloperoxidase activity (mpo) measured pulmonary neutrophil influx. pulmonary endothelial permeability was measured by wet to dry weight ratio (w/d), broncboalveolar lavage protein (bal) and neutrophil counts (bal pmn). neutrophil superoxide release (mean channel fluorescence mcf) was measured by flow cytometry in a further ir v gtn experiment (n= per group). control _+t "* _+ _+ ** bal pmn/mm l+- " + # _~ *p< . , #p< . v control/gtn;**p< . v ir. students t test the significant increase in mpo activity produced by ir was attenuated by gtn. the increase in pulmonary microvascular leakage after reperfusion was also prevented by gtn. neutrophil superoxide release increased significantly from . + . to . _+ . mcf after minutes repeffusion (p<. ). this increase in superoxide was prevented in the gtn treated group. these results indicate that gtn inhibits neutropbil superoxide release during limb reperfusion, thus preventing pulmonary vascular leakage and neutrophil infiltration. these data suggest that the beneficial effects of gtn in aortic surgery may be due in part to a protective effect onpulmonary microvasculature. department of surgery, beaumont hospital, dublin , ireland. lipton adult respiratory distress syndrome (ards) is marked by increased vascular permeability of the lung to white blood ceils (wbc). indeed, influx of wbc into the lung is believed to be the central mechanism in acute lung injury of ards. evidence is developing that the neuropeptide c~-melanocyte stimulating hormone (c~-msh), a proopiomelanocortin derivative, inhibits inflammatory reactions in animai models of inflammatory responses in humans. to test the influence of a-msh on experimental ards, the peptide was administered to rats after intratraeheal infusion of endotoxin (s. typhosa, #g in . ml saline). three groups (n= each) received: intratracheai infusion of endotoxin plus ip injections of o~-msh ( ~tg in . ml saline) at , , and hr post-endotoxin treatment; intratracheal endotoxin infusion plus saline injections; control intratracheal saline infusion plus saline injections. the bal data showed overall differences among groups (f , = . , p<. ), o~-msh treatment inhibited endotoxin-induced wbc migration into the pulmonary tree (p<. ). circulating wbc counts were markedly lower in both groups given endoroxin than in the control group (p<. ), but there was no difference in wbc count between these two endotoxin-treated groups. the results indicate that c~-msh can reduce wbc migration in experimental lung injury. in further experiments we tested the hypothesis that c~-msh, administered alone or in combination with an inhibitor of gram-negative bacterial growth, increases survival in a model of peritonitis/endotoxemia/septie shock. cecal ligation and puncture were performed under sodium pemobarbital anesthesia ( mg/kg, i.p.) in female balb/c mice ( - g) that were then randomly assigned ( - mice per group) to receive at time and hr later: control saline thjectioas (i.p.), injections of ~-msh ( ~tg), gentamicin sulfate (i mg/kg), or both ~-msh and gentamicin. one ml of normal saline was given s.c. to each animal for fluid replacement. both ix-msh and gentamicin treatments administered singly improved survival; when given in combination survival was even greater these findings suggest that the anticytokine ot -msh molecule might be useful for treatment of systemic inflammation, perhaps particularly when used in combination with agents that inhibit bacterial growth. previous studies showed a close relationship between xanthine oxidase activation, membrane damage and postischemic cardio-respiratory failure following aortic clamping and reperfusion. aim of the present study was to evaluate alterations of polymorphonuclear leukocyte (pmnl)-metabolism and signal lransduction systems during ischemia/reperfusion induced by aortic clamping in man. methods, in patients ( female, + years) with elective reconstruction of infrarenal aortic anearysms, arterial (a) and venous (v) blood samples were obtained before clamping and declamping, and min after declamping. arterial pla concentrations were higher than venous ( + vs. + ). while cytosolic basal pmnl ca + concenlxation was not altered at the end of ischemia (v: . + ; a: _+ ) and during reperfusion (v: + ; a: + ), fmlp-stimulated cytosolic ca + mobilization showed a significant arterial increase as compared to venous ( _+ vs. + , p < . ). these presented data indicate that the reperfusion syndrome after infrarenal aortic clamping is characterized by ) pulmonary membrane damage possibly due to pmnl-degranulation, ) activation of pmnl-superoxide radical production with release of activated pmnl in arterial circulation, and ) a significant arterial increase of pmnl cytosolic ca + mobilization after fmlp-stimulation parallel to pmnl-activation. in conclusion, ischemia/reperfusion in man induces pulmonary damage and activation of pmnl superoxide production possibly due to an altered pmnl-ca + messenger system by inflammatory mediators (e,g. ii- , tnf, paf). the septic lung diseases (i.e. ards) generally show distinct alveolar damage and surfactant disturbances. it is thought that alveolar macrophages are involved in specific release products like h and cytokines like tnf. in this pathway the type ii alveolar epithelial cell (p cell) is not only an important target, but as recently shown it is also capable of producing h . the aim of this study was to investigate the influence of endotoxin on h release of p cells. we obtained p cells from lungs of female wistar rats and investigated the effect of lipopolysaccharid (lps) on h release of fresh isolated cells and on cells cultured for days in a fibronectin matrix. furthermore we conditioned isolated alveolar macrophages for hours with p.g/ml lps and tested the conditioned medium (mcm) on cultured p cells. p cells h ex vivo were _> % pure, _> % vital and had an basal h release of . _+ . nmol h per hour and million cells. adding p.g/ml lps to the incubation medium the h release decreases slightly but significantly to . _+ . nmol. adding . p.g/ml phorbol myristate acetate (pma) to the basal incubation medium the h release increased -fold to . _+ nmol. pma induced h release decreased to . + . nmol after addition of p.g/ml lps. after culture days the p cells were _> % pure and showed a pma inducible h release of . _+ . nmol addition of p.g/ml lps had the inverse effect as on freshly isolated cells as it increased the h release up to . _+ . nmol. addition of mcm to cultured p cells increases pma-stimulated h release to . +_ . nmol. the release decreased to . _+ . nmol when an murine anti-tnf-alpha antibody was added. vitality of cultured cells was > % in all experiments. the results show that lps has an direct effect on p cells cultured on fibronectin. we conclude that the observed additional stimulatory effects of mcm seems to depend on tnf-alpha. the induction of h release of p cells could be important for generating internal oxidative stress in p cells before external oxygen radicals exceed. the produced h did not necessarily damage p ceils, but it can effect surfactant metabolism, especially when extracellular h release of alveolar macrophages following an immune response is increasing. introduction: primary stabilization of femoral shaft fractures in patients with multiple trauma is beneficial. however, in patients with associated lung contusion we have found an increased incidence of ards, apparently associated with primary reamed femnral nailing (rfn). previous animal studies revealed, that perioperative disturbances of lung ftmetion appear to be related to the reaming procedure, ix~ssibly due to pulmonary embolizafion of bone marrow fat. in a prospective clinical analysis we compared effects of intrameduuary nailing with and withont reaming on parameters known to be related to ards-pathoganesis. in order to gain further insight into the role of endotoxin and cytokines in the pathogenesis of the adult respiratory distress syndrome (ards), we enrolled patients with severe lung injury after sepsis ( ) or polytrauma ( ) and obtained multiple blood samples ( days) for endotoxin, tumor necrosis factor e (tnfa), interleukin (il- ) and interleukin (il- ) determination. to evaluate the cytokine releasing capacity of the blood, plasma concentrations of tnfe, il-l and il- were also determined after the "in vitro" stimulation of the whole blood samples with lipopolysaccharide (lps, . ng/ml) for hours at c (stimulated values). the difference among stimulated cytokines levels and the basal plasma concentrations were defined as "delta values", an expression of the cytokine releasing capacity of the blood. the pao /fiao quotient was used as an index of the severity of lung injury (sli). the endotoxin plasma level was significantly higher in patients with sli < ( . ± . eu/ml, mean values ± sem) versus the patients with a sli > ( . ± . eu/ml, p<o. ). the basal plasma concentration of ll- also correlates with the sli index (p<o.o ). the delta tnfe and delta il- values were s~gnificantly decreased in patients with a severe lung injury. compared with the survivors (n: ), the deceased patients (n= ) showed higher endotoxin level, a reduced tnfa and il- releasing capacity of the blood and higher basal il- levels. we can conclude that endotoxemia, high il- plasma level and a decreased capacity of the blood to release tnfa an il- under endotoxin stimulation associates with the severity of lung injury. [objectives] experimental and clinical findings implicate the involvement of inflammatory cytokines in the pathogenesis of the decreased oxygenation in the lung after major surgery, though the mechanism remains unclear. in the present study, we elucidated the involvement of il- in the pathogenesis of lung injury defined by deterioration of pulmonary oxygenation after esophagectomy. [materials and methods] seventeen patients underwent subtotal esophagectomy through a right thoracotomy. in all patients, tbe alveolar-arterial oxygen tension difference (aado ) was measured everyday and bronchoalveolar lavage fluid (balf) samples were obtained from a single segment ($ or $ ) of the right and left lung through a bronchoscope on days l, , and days after surgery. they were then examined for cell analysis, measurement of cytokines by elisa, and measurement of neutrnphil elastase (ne) by elisa. [results] aado unexceptionally deteriorated postoperatively and the mean aado in patients reached the maximum (mean ± sem; _+ mmhg) on the rd postoperative day. the mean concentrations of il- , ne and neutrophil count in balf also increased postoperatively and reached their maximum level ( + pg/ml, -+ gg/l, and ± x cells/ml, respectively) on the rd postoperative day. these increases in balf were recognized in both the right and left lung. a highly significant correlation was observed between il- and ne levels in balf (r= . , p= . ). also, significant correlations between aado and the neutropbil count, and the concentration of ne or l- were observed. in contrast to il- , neither il- , il- nor tnfa was detected in balf. [conclusion] major surgical trauma such as esophagectomy may induce recruitment of neutrophils to the lung and cause lung injury. il- increased in the lung is an important mediator of neutrophil recruitment and activation in the lung. phorbol myristate acetate (pma) is commonly used to cause edema and other tissue damages in the lung. lung injuries induced by the administration of pma has been shown to be mediated mainly by neutrophils. neutrophils recruited to the lower respiratory tract may damage lung tissues by releasing reactive oxygen species, neutral proteases and lysosomal enzymes. the present study was conducted to investigate whether c~tocopherol, entrapped in dipalmitoylphosphatidylcholine liposomes and delivered directly to the lung, could counteract some of the pma-induced lung injuries. plain liposomes or c~tocopherol-containing liposomes ( mg c~-tocopherol/kgbody wt.) were instilled into the lungs of rats h prior to pma exposure ( pg/kg, intratracheally) and treated rats were killed h later. lungs of control animals exposed to pma developed increases in lung weight and lipid peroxidation as well as decreases in lung angiotensin converting enzyme (ace) and alkaline phosphatase (akp) activities. pma treatment also caused an increase in myeloperoxidase (mpo) activity in the lung, suggestive of neutrophi] infiltration. pretreatment of pma-treated rats with plain liposomes had no effect on pma-induced injuries. in contrast, pretreatment of rats with liposomal c~-tocopherol, h prior to pma administration, resulted in a significant elevation of pulmonary a-tocopherol concentration, accompanied by a concomitant reduction in mpo activity and reversal of pmainduced changes in lung edema, lipid peroxidation, ace and akp activities. these results appear to demonstrate that the intratracheal administration of a liposome-associated lipophilic antioxidant, such as a-tocopherol, can significantly ameliorate the toxic effects of reactive oxygen species, released from pmastimulated pulmonary target cells and infiltrating neutrophils. jk wojcik, g palasiewicz, w roszkowski immunology department,institute of tuberculosis and lung diseases~ warszawa, poland, m~larhospital, eskilstuna, sweden. introduction:the critical point in neutrophil migration to the alveolar space in the course of ards is the attachment between giyeoproteins of neotrophil membranes(sialyl lewis x carbohydrate epitope containing u-l-fucose) and lectin domains of endothelial p and e selectins (gmp- ,elam-i). a potential beneficial therapeutic strategy may be designep to suppress neutrophil recruitment to the lungs by preventing their rolling and attachment to the pulmonary endothelial cells. objeclive:to investigate if tz-l-fueose prevent experiments pulmonary hypertension in ards as effective as metylpredniselone. methods:only healthy rabbits (n- ) weighing .d- . kg oaselinepao> kpa and mean pulmonary arterial pressure (mpap)<i. kpa were included in this study. anaesthesia was induced with thiopental - mg/kg bw and "blind" jntubation was performed. a f swan ganz catheter was introduced via the left jugular vein into the pulmonary artery. pma, as was used in our experiments activates neutrophils and produces acute respiratory failure with pulmonary hypertension and pulmonary edema. eight rabbits serving as control animals received pma ug/kg bw iv only. sixteen other animals were divided into two groups receiving either ~-l-fucose mg iv or metylprednisolone mg iv min before pma admininstration. results:after pma administration notable physiological changes including marked increase in mpap( . -+o.~ kpe ..lere found. the increase of npap was observed to be completely blocked in both groups of animals which received ~-l-fucose or metylprednisolone. conclusion:in aur rabbit model ards cz-l-fucose pretreatment prevents pulmonary hypertension after pma administration. the effect is comparable with high dose metylprednisolone. the possible explanation is that g-l-fueose molecules block iectin domains of gnp- or elam-i preventing the adhesion of the neutrophils to the pulmonary endotheliom. bartens c, elmadfa i, steltzer h, fischer m, druml w introduction: various micronutrients and vitamins are particulary effective in modulating immune functions and host defense. the nutritive ant±oxidants vitamin c, e and b-carotene, as well as selenium lower the burden of reactive free radicals and protect the structural integrity of cells and tissues of the immune system, as well as other systems in the body. certain cells of the immune system like polymorphonuclear leucocytes (pmn's) use free radicals as a weapon to destroy invading pathogens. these reactive molecules, when released into the surrounding area, mediate lipid peroxidation and tissue injury. there is growing evidence that pmn's are activated in vivo by complement or immune complexes in disorders such as ards. the respiratory host defense mechanisms of patients with adult respiratory distress syndrome (ards) may be defective. the aim of this study was ta evaluate the status of the free radical scavengers and their activity in ards and to investigate the respiratory burst of ards patients. methods: seven ards patients ( sepsis, trauma) with an fie of . + .( , a peep of . ± . , and a murray score of . ± . were included in this study. healthy adults served as controls. superoxide anion production in granulocytes was measured photometrically, and the hydrogen peroxides by fluorimetric assay. vitamin a, e, and g-carotene concentrations were assessed by hplc, and plasma vitamin c photometrically. plasma selenium was determined by dectrothermal aas. plasma lipid peroxidation pruducts, expressed as malondialdehyde (mda), were determined by thiobarbituric acid assay and hplc. results: mda-plasma (/xmol/l) . ~: . * . ± . *= < . , **=p< . condusion: ards patients showed significantly decreased plasma concentrations of vitamin c, e and g-carotene, as well as selenium. these nutritive ant±oxidants are consumed during increased oxidative stress. mda, a final product of lipid peroxidation, is increased. however, a difference in rates of release of hydrogen peroxides and superoxide-anion of pmn could not be detected. therefore, oxygen radical accumulation is more likely a result of excessive inspiratory oxygen concentrations (f.io ), pro-oxidant drugs, and a high settlement of pmn in the lungs, and not an increased release of free radicals of the single pmn. kd glycoprotein secreted by the alveolar type ii cells. recent study showed that sp-a exists in the sera from normal healthy adults and that the significantly elevated serum sp-a levels were observed in the patients with interstitial pneumonia and pulmonary alveolar proteinosis. these findings means there may be a mechanism that the sp-a produced in the alveoli escapes into the bloodstream. here we examined the serum sp-a levels in the patients with critical illness including sepsis and ards. ]clinical subjects & methods] a total of serum samples were collected from patients hospitalized in the division of critical care medicine(icu), sappom medical college hospital. serum sp-a levels were measured by an enzyme-linked immunosorbent assay using monoclonal antibodies to human sp-a. ]results] there was no significant difference between the septic (n= ) and non-septic (n= ) patients. patients with ards (n= , . + . ng/ml) and with respiratory disease other than ards (n= , . +- . ng/ml) showed significantly higher levels of serum sp-a than those with non-respiratory disease (n= , . _+ . ng/ml). as to the ards patients, it was likely that the serum sp-a levels were getting higher in their clinical courses. however, there was no significant correlation between the serum sp-a levels and the pa%/fio ratio. ]discussion] at the present time, the exact mechanism of the escape of sp-a into the bloodstream remain to be unclear. our results may suggested that there was some relation between this leakage of sp-a and the alveolar-capillary permeability because of the higher sp-a levels observed in the ards patients. however it is also likely that higher serum sp-a level represents the proliferation of alveolar type ii cells as the regeneration of damaged lung. further studies are now being done. neutrophils contain a number of enzymes within intracellular granules,potentially damagingto lung tissue.release of these enzymes into the lung tissue from the sequestred activated neutrophils is felt to play significant role in lung injury in the course of aros. using -hours acute animal model of aros the activity of cathepsins,beta-glucuronidase and acid phosphatase was determined in the lung tissue. results of this research were compared with neutrophil proteases activity in serum and broncho-alveolar lavage.also hemodynamic,respiratory and biochemical investigations were performed before beginning of experiment,and in a two-hours time intervals of its duration.after hours was determined total and free activity of cathepsins,beta-glucuronidase and acid phosphatase in full homogenate of the lung tissue,mitochondriallysosomal fraction and the final supernatant. in the course of our experiment we observed an increase ( .i- . %) both total and free activity of neutrophil proteases in all fractions of the lung tissue.generally accepted current pharmacological treatment of aros only attenuated this increase but never caused reversion to the level before beginning of our investigations. the presence of aros was histologically proved.an activity of acid phosphatase in serum and the lung tissue fractions was compared with histochemical pictures of the lung. results of our research indicate that neutrophil proteases are a very important mediators in aros and they are a cause of the lung injury. in addition neutrophil-derived proteases can amplify the inflamatory process by enzymatitally activating of many other mediators and they also may to increase an oxidant induced injury by imparing of antioxidant defenses. oepartment of general surgery, sk~odowskiej a - ~ia~ystok, poland, tel/fax + . in vitro studies demonstrated that paf activates polymorplionuclear leukocyte (pmn) - ipoxygenase, but the generation of leukotrienes (lts) is critically dependent on exogenous arachidunic acid (aa) disposal (f. grimminger et at., mol. pharmacol. : , ) . we investigated the features of paf-evoked lt synthesis in a model of pulmonary microvascular leukostasis, in the presence and absence of intravascular n- -and n- -prescursor fatty acid supply. human neutrophils were infused into isolated, ventilated and perfused rabbit lungs to achieve microvascular sequestration of ligand-responsive leukecytes. leukotrienes (lt) and hydroxyeicosatetra(penta)enoic acids (het(p)e) were quantified by itplc techniques. intravascular application of paf ( um) or arachidonic acid (aa;ioum) provoked the generation of limited quantities of -series lts and -hete (total sum of - ipoxygenade products rd. and rd. pmol/ml.; both in pmn-preloaded and non-preloaded lungs). combined administration of pat r and aa caused amplification of - ipoxygenase product formation with predominance of cysteinyl-lt synthesis both in lungs without (total sum rd. pmol/ml) and, much more strikingly, with pro-application of neutrophils (total sum rd. pmofml). eicosapentaeooic acid ( um) elicited exclusive generation of -series lts and -hepe (total sum rd. pmol/mi). dual stimulation with paf and epa provoked amplification of epa-derived - ipoxygenase product formation, again with predominance of cysteinyl-lts, in lungs without (total sum rd. pmul/ml) and in particular in those with preceding microvascular pmn entrapment (total sum rd. pmol/ml). we conclude that the paf-evoked - ipoxygenase product formation in the neutrophil-harbouring lung capillary bed is critically dependent on intravascular precursor fatty acid supply, with epa representing the preferred substrate as compared to aa. pmn-related transcellular eicosanoid synthesis is suggested to underly the predominant generation of cysteinyl-lts. these findings may be relevant for lipid mediator profiles provoked by infusion of n- -versus n- enriched lipid emulsions in diseases with pmn-related inflammators events. adult respiratory distress syndrome (ards) involves damage to the alveolar epithelium and microvascular endothelium. products released from neutrophils (pmn) are thought to be among the chief causes of this damage, while the role of mononuclear cells (mnc) is uncertain. we studied patients at risk of or with acute ards. we measured the concentration of myeloperoxidase (mpo) and elastase (el) within circulating pmn as an index of pmn activation, elastin degradation products (edp) in the plasma as an index of tissue damage, as well as the cytotoxici~y of pmn and mnc to endothelial cells (ec) in vitro. cells and plasma were prepared from venous blood of healthy controls; or systemic arterial blood of patients on ventilators in intensive care but not at risk of ards; at risk of ards because of sepsis; at risk because of multiple transfusion/major trauma; or with acute ards. lung injury, multi-organ failure and apache h scores were recorded. to measure cytotoxicity, human umbilical vein ec were labelled with cr- , incubated with either pmn or mnc for hours, and the supernatant counted. the adherence of the pmn and the mnc to the ec was also measured. standard assays were used to measure mpo and el in the pmn, mad edp in the plasma. the mnc cytotoxicity did not differ between the groups. the pmn cytotoxicity was higher in the transfusion/trauma patients compared to each other group, which did not differ from each other. there was no correlation between cytotoxicity and adherence for any group. the mpo and the el concentrations were significantly decreased in the pmn from each of the patient groups, including the patient controls, compared to healthy controls. the edp were only increased in patients at risk of or with ards. thus pmn degrannlation occurred in patients not at risk of ards, but tissue damage as measured by increased edp only occurred in patients with or at risk of ards, suggesting that factors additional to pmn activation and degranulation are required for the progression to ards. there was no correlation between the parameters measured and the indices of disease activity, or outcome. these measures of pmn and mnc activation and function are not of value as prognostic indicators in patients with or at risk of ards. the liver is made up of several different cell types which subserve distinct functions in vivo. in addition to the different functions of the distinct cell types, it is now evident that even within the population of hepatocytes substantial functional heterogeneity exists. this heterogeneity occurs in a very organized fashion in the normal liver based upon the position of the hepatocyte in the acinus. although it has been proposed that the acinar heterogeneity of hepatocytes arises from migration of immature hepatocytes from the periportal region to the perivenous region where they become senescent and die, many of the heterogeneous responses can be accutely reversed by reversal of the oxygen gradient via retrograde perfusion. under normal conditions in which the acinus is exposed to unidirectional flow, the periportal hepatocytes are exposed to relatively high levels of oxygen, substrates and hormones. as these substances are extracted along the aeinus their concentration decreases leaving relatively low concentrations for the perivenous hepatocytes. in the normal liver, the heterogeneity of response is attenuated by extensive communication via gap junctions. the major liver gap junctions are made up of hexamers of connexin (cx ) which forms channels between cells capable of pas.~ing any molecule smaller than d. coupling is sufficient to allow diffusion of molecules such as atp or camp from one hepatocyte into > adjacent hepatocytes within seconds. during stress conditions such as endotoxemia or zymozan inflammation, expression of cx is markedly decreased while the secondary gap junction protein cx is either unchanged or even increased. while cx readily effects electrical coupling, molecules > d pass only very slowly. this would result in restriciton of transmission of moecules the size of atp or camp. since inhibition of gap junctions also attentuates metabolic response to hormone or nerve stimulation, it is evident that modulation of hepatocyte hetereogeneity by gap junctions must be considered in determining the mechanisms of metabolic alterations during stress. already minor haemorrhage decreases portal venous blood supply to the fiver and the reduction in portal blood flow becomes more pronounced with more profound btood loss. severe hacmorrhagic hypovolemia also reduces hepatic arterial blood supply which, however, is maintained over a vide range of haemorthage. the net effect of blood loss is a reduction in liver oxygee supply and this reduction is in proportion to the vulume iossed. however, oxygen supply to the liver exceeds the demands of the normal liver and this is the ca~ stilt following reduction of % of blood volume. the situation in sepsis is more complicated. po~l venous supply to the liver is redur.~i fairly early following normovolemic sepsis while hepatic arterial blood supply is maintained at le,~t initialiy, oxygen saturation might be maintained in arterial blood but may also be slightly reduced during sepsis, oxygen saturation of portal venous blood is significantly reduced during sepsis due to increased extraction of the intestines. therefore oxygea delivery to the liver during sepsis becomes sigalfkzntly reduced. at the s,~ne time and for mai.v.ly unknown reasons the need for oxygen becomes significantly increased in the ~-~ptic liver. as a consequence liver oxygen consumption becomes flow dependent and the liver is likely to suffer from ischemia during septic conditions. $ although liver failure is well recognized in sepsis, it is generally thought to be a late complication following pulmonary and renal failure. jaundice, hypoglycemia, encephalopathy and bleeding secondary to low levels of liver-synthesizing clotting factors are, however, signs of rather severe end-stage hepatic failure. furthermore, elevated liver enzymes (sgot and sgpt) represent hepatucyte damage and not hepatocellular dysfunction. in view of this, a more sensitive indicator of hepatic function is desirable in order to detect early hepatic abnormality. in this respect, indocyanine green (icg) is a tricarbocyanine dye that possesses several properties which makes it particularly valuable inthe assessment ofhepatic function. this dye is bound m albumin and is cleared exclusively by the liver through an energydependent membrane transport process and is nontoxic at lower doses. we propose that maximal velocity (vm~,) of icg clearance is a valuable measure of active hepatocellular function, since the total concentration of functioning receptors is directly proportional to vm~. we have utilized a fiber optic catheter and an in vivo hemoreflectometar to continuously measure the administered icg in vivo and consequently determine its clearance without the need of blood sampling. using this technique, we have found that in the early stages of sepsis (i.e., and h following cecal ligation and puncture), the vm~ and kinetic constant (k=) of icg clearance was significantly depressed. it should be noted that at this stage of sepsis, there was no elevation in serum enzyme levels. furthermore, hepatic blood flow and cardiac output increased at the above mentioned time points. thus, the extremely early depression in active hepatocellular function in sepsis, despite the increased hepatic blood flow and cardiac output, may form the basis for cellular dysfunctions leading to multiple organ failure during sepsis. additional studies indicated that following hemorrhage, active hepatocellular function was markedly depressed. this returned to prehemorrhage levels after ringers lactate resuscitation, however, this function was not maintained and decreased significantly after fluid resuscitation. nevertheless, the depressed active hepatocelinlar function following hemorrhage was markedly improved by post-treatment of animals with either atp-mgci , peutoxifylline or diltiazem. thus, the use of icg clearance provides an early sensitive indicator of hepatic abnormality during sepsis and following hemorrhage and this method should be used, not only experimentally, but also in the clinical arena for the early detection of hepatocellular abnormality. although multiple organ dysfunction syndrome (mods) remains a major cause of mortality and morbidity in intensive care units, very little is known about the mechanisms that precipitate its development. since an episode of inadequate tissue oxygenation is considered to be the trigger for mods, we have proposed that a primary localized injury such as ischemia/reperfusion may be sufficient to cause a change of gene expression of remote and apparently unaffected organs. such modulation of remote organ gene expression may decrease the organ's tolerance to a subsequent stress contributing to the development of mofs. to test this hypothesis, rats were subjected to hepatic regional ischemia by clamping the blood flow (hepatic artery and portal venous inflow) of the left and median liver lobes. intestinal congestion was prevented by allowing flow through the smaller right and caudate lobes. after minutes of ischemia, the clamp was removed and the blood flow restored. the animals were allowed to recover for , and hours. kidneys were removed, total rna was isolated and poly(a) ÷ selected by affinity chromatography on oligo(dt) columns. message was in vitro translated using rabbit reticulocyte iysates in the presence of radioactive amino acids. the gene products (radiolabeled polypeptides) were fractionated by two dimensional gel electrophoresis, and visualized by fluorography. analyses of the two dimensional fluorograms indicate that there is a dramatic change in the electrophoretic pattern of in vitro translated products in samples corresponding to kidneys obtained after minutes of hepatic ischemia and hours of reperfusion with respect to kidney samples obtained after sham operation or from control rats. the latter were not subjected to any surgical manipulation. these studies suggest that the gene expression of the kidneys is specifically modified after a remote organ injury (hepatic ischemia/reperfusion). we speculate that this change of gene expression in kidneys after an indirect injury may be part of the early events leading to the development of mods. a priming event, e.g. local ischemia, in combination with a second insult, e.g. sepsis, may amplify a host's response and lead to multiple organ failure. to better understand the mechanisms involved in the pathophysiology, male fischer rats were subjected to min of hepatic ischemia followed by reperfusion (rp) and injection of . mg/kg salmonella enteritidis endotoxin (et) at min of rp. et injection potentiated the postischemic liver injury as indicated by histopathology and an increase of plasma alt activities from + u/l (i/rp only) to + u/l at h rp. inhibition of kupffer cells (kc) with gadolinium chloride ( mg/kg) attenuated liver injury in this model by %, however, monoclonal antibodies (cl , wt ) directed against adhesion molecules ( integrins, cd ) on neutrophils had no effect on the injury despite the substantial accumulation of neutrophils in the liver at that time ( + pmns/ hpf; baseline: + ). isolation of kc and neutrophils from the postischemic liver indicated a -fold increase of the spontaneous superoxide formation only in the kc fractions [ . + . nmol o -/h/ %elts (kc ); . _+ . (kca) ] at h rp compared to control cells. in addition, stimulation with phorbol ester or opsonized zymosan revealed a substantial priming of kc for reactive oxygen formation. in contrast to the short-term experiments ( h), the antibody wt ( mg/kg) attenuated liver injury by % at h of rp and improved survival. conclusion: liver injury during the early rp phase is mediated mainly by kc generating excessive amounts of reactive oxygen while neutrophils are primarily responsible for organ damage during the later rp period. (es- and gm- ) tumor necrosis factors (tnf) are cytokines which are cytotoxic towards some tumors in vivo and certain tumor lines in vitro. moreover, these polypeptides are powerful immunomodulators and have been found to be distal mediators in several models of septic shock and septic organ failure. one of the best-characterized experimental systems is the hepatitis caused by lps or tnf in galactosamine (galn)-sensitized mice. here we describe a cell culture system, in which the direct toxicity of tnf towards mouse hepatocytes was examined. the toxicity of tnf, as determined by ldh-release or formazan-formation, was dose-and time-dependent. the threshold of toxicity was ng/ml, which corresponds to serum concentrations found in mice after lpsinjection. toxicity was only observed in hepatocytes sensitized with transcriptional inhibiters such as galn, actinomycin d (actd) or cxamanitin. sensitization was neither observed with different translational inhibitors nor with various other metabolic inlaibitors or toxins. inhibitors of protein synthesis or protein processing such as cycloheximide, puromycin, tunicamycin and ricin protected actdsensitized hepatocytes from tnf-induced cytotoxicity. tnf induced apoptotic changes and dna-fragmentation in sensitized hepatocytes which is in line with the above findings that cell death is dependent on protein synthesis. thus tnf may be a trigger of programmed cell death during inflammatory organ damage. with the purpose of studying the role of complement activation in tissue injury after ischaemia and reperfusion we blocked the complement cascade in a model of rat liver isehaemia and reperfusion, either by administration of soluble human complement receptor type (scri), mg/kg iv after vascular occlusion (n= ) or by depleting the complement system using cobra venom factor (cvf), . mg im, and hours before ischaemia (n= ). non-ischaemic rats (n= ) and ischaemic non-treated rats (n= ) were used as controls. the experimental procedure consists of the temporary interruption of arterial and portal blood flow to the left lateral and medial lobes of the liver during minutes, followed by reperfusion, recording the liver blood flow and haemoglobin saturation with a laser doppler flowmeter and photometer during one hour after declamping; alt levels were assayed and immunoperoxidase stainings for c and c were performed. there were statistically significant differences between the experimental ~roups and the untreated ischaemic control group in terms of post-isehaemic blood flow (p< . ) and haemoglobin saturation (p< . ). c and c were present in the endothelium of the ischaemic control group. no deposits of c or c were found in the cvf group. few c and no c were found in scri treated rats. these results show that the effect of reperfusion injury in the rat liver is ameliorated either by depleting complement with cvf or by regulating complement activation with scri. hepatic dysfunction, a major cause of mortality following hemorrhagic shock, has not yet been well characterized. the present study was designed to assess the effects of liver blood flow and cytokine levels on hepatic function following resuscitation from severe hemorrhagic shock in normal and cin-hotic rats. methods: aftor pentobarbltal anesthesia, control and cirrhotic sprague-dawley rats were subjected to severe hemorrhage to reduce their systolic blood pressure to + mm hg. this level of hypotension was maintained until the skeletal muscle transmembrane potential (era) depolarized by %.; the animals were then resuscitated with ringer's lactate solution in three times the volume of the shed blood. serial blood samples for tumor necrosis factor (tnf) determination (a modified flow-cytomeuic wehi cell bioassay) were obtained at baseline, during hemorrhage and following resuscitation. liver blood flow measurements by low dose galactose clearance (glc) and functional bepatocyte mass (fhm; defared as galactose elimination capacity [gec] from the zero order portion of the plasma disappearance curve following an intravenous galactose bolus [ mg/kg], divided by liver weight) were measured before shock and after resuscitation. results: higher survival rates (p < . ) were observed in control as compared with cirrhotic rats. shock produced a significant reduction in gec (to < . ); fhm ( < . ); and liver blood flow (p < . ) in normal and cirrhotic rats. decreases in gec and fi-im were greater (p < . ) in cirrhotic rots. tnf levels were higher (p < . ) in cirrhotic rats at baseline and during induction of shock. pre gap junctions provide pathways for metabolic signals between cells. in the liver, the majority of gap junctions are composed of connexin (cx ) polypeptide subunits, and are regulated by gluconeogenic hormones. since sepsis and other inflammatory states alter hepatic glucoregulatory control, we have evaluated the contribution of gap junctional conductance to the metabolic dysregulation in the liver. an acute inflammation was induced in rats by injection with e. coli endotoxin (lps lmg/kg). northern blot/hybridization analysis of total rna isolated from livers after endotoxin injection show a decrease in the steady state transcript levels of cx to % of sham controls. immunostaining of liver sections using anti-cx revealed punctate fluorescent staining on the plasma membrane at regions of call-cell contact in saline injected animals, whereas, staining was only observed in cytoplasmic vesicles hrs after animals were treated with lps, suggesting the internalization of cx without replacement on the cell surface. the staining was quantitated and expressed as % of pixels above threshold. at hr post injection . % ofpixels exceeded threshold, compared to . % in sham controls. functional gap junctional communication was assessed by dye coupling using lucifer yellow in an isolated perfused liver under intravital fluorescence microscopy. dye diffusion was markedly decreased hr after endotoxin injection. this suggests that decreased metabolic coupling after lps injection results from decreased gap junction abundance. the present data suggest that metabolic dysregulation during sepsis may arise in part from changes in intercellular communication caused by a decrease in gap junctional expression and communication. given the marked metabolic heterogeneity of hepatocytes with respect to acinar location, metabolic signaling via gap junctions most likely serves to moderate this heterogeneity, contributing to a coordinated metabolic response. altered cellular ca ÷ regulation might be a critical step in organ dysfunction during sepsis and ischemia/reperfusion events. the aim of the present study was to evaluate hepato-ceuular ca ÷ regulation in isehemiah'eperfusion after hemorrhage and to assess effectiveness of tnfc~-monoclonal antibody (tnfo~-moab). methods. male sprague-dawley rats ( g, n>_ /group; pentobarbital mg/kg) with hemorrhage for rain at mm hg. reperfusion by ringer's lactate ( x maximal bleed out/ min) and % of citrated shed blood. tnfcz-moab (tn , ceutech, mg/kg in . % nac ) infused during flrst min of reperfusion. at baseline, end of ischemia and min of reperfusion, hepatecyte isolation by liver collagenase perfusion. " hepatocyte incubation ( mg w.w./ml) with caci ( . + + + mbq/ml) for rain (ca influx [slope, /mini; ca uptake [nmol ca /mg protein]) w/ and w/o epinephrine (epi, nm). hepatecyte resuspension in radioisotope-free medium and farther incubation (exchangeable ca + (ca +ex) [nmol ca +/mg protein]; ca + membrane flux [nmol ca +/mg protein'min]). during incubation, aliquots ( ~tl) were centrifuged through oil/lanthanum gradient and acivity measured by scintillation counting. statistics: anova. mean + sem. results. hepatocyte ca +ex and membrane ca + flux were significantly increased at both, the end of ischemia ( . +. ; . +. ) and reperfusion ( . +. ; . +. ), as compared to sham-operated animals ( . +_. ; . +. )( <. ). tnfc~-moab treatment significantly prevented reperfusion-induced increase of ca +ex ( +. ) and membrane ca + flux ( . +. )(p<. ). fast ca + influx was significantly increased by epinephrine in hepatecytes from sham-operated rats ( . +. vs. epi: . +. , p< . ). this hormone effect was not observed in isehemia ( . +. , epi: . !-_. ) or reperfusion (untreated: . +. , epi: . +. ; tnft~-moab: . _+. , epi: . +. ). conclusion. the present study clearly demonstrated hepato-cellular ca + overload in ischemia and reperfusion as a result of hemorrhagic shock. analysis of membrane ca + fluxes and hormone ca + mobilization suggests disturbances of membrane ca + transport mechanisms, e.g. through ca +-atpases. reperfusion-induced oxygen free radical generation which affect exchange kinetics of cellular ca + buffering compartments might also be operative. prevention by tnfct-moab indicates the pivotal role of tnf as an early inflammatory mediator of hepatocellular alterations in signal transduetion mechanisms and cellular homeostasis. although the precise mechanism has not yet been elucidated, bacterial translocation and endotoxin absorption have been frequently shown after burn, and have been postulated to be one of the underlying processes of sepsis. the purpose of the current study is to define the hemodynamic response of the liver to endotoxin release in burns, in correlation to bacterial translocation. twelve female minipigs, weighing - kg, underwent a laparotomy & transition time ultrasonic flow probes were positioned on the portal vein, the common hepatic artery, and the superior mesenteric artery. . fr catheters were inserted in the superior mesenteric vein and the left hepatic vein. a jejunostomy was also performed. after five days all animals were anaesthetized and randomized to receive % of tbs a third degree burn. eighteen hours after burn. gg/kg e. coli lps was intravenously administered over rain. ali animals were studied for additional hours and then sacrificed. several recent data suggest that in severe injuries, such as shock state, the gradual activation of kupffer cells and the excessive release of destructive and immunosuppresive products from macrophages may contribute to the development of "multiple organ failure". in in vivo experiments in mice, the effect of kupffer cell phagocytosis blockade on the correlation between the tissue distribution of lps, endotoxin sensitivity and lps-induced tnf production was investigated. to depress the activity of the kupffer cells, gadolinium chloride (gdc ) or carrageenan was used. th~e studies indicate the dissociation of tissue localisation of cr jllabelled endotoxin and endotoxin lethalithy. both gdc and carrageenan depressed kupffer cell activity, but endotoxin sensitivity was enhanced only by carragenan treatment. however, there was a close correlation between the sensitivity to lps and lps-induced tnf production as measured in the serum, since lpsinduced tnf production was enhanced only by carrageenan treatment. on the other hand, gdc pretreatment significantly increased tnf production in the spleen. these results support our earlier findings that gdc -indueed kupffer cell phagocytosis blockade leads to activation of the spleen, and may explain some of the immunological effects of gdc . inositol(l, , ) triphosphate (ip ) has been proposed as a second messenger for calcium mobilization. the addition of ip at low concentration has been shown to cause calcium release from intracellular microsomal store in rat hepatocytes. the effects of sepsis on the ip binding from microsomal fraction of rat hepatocytes during sepsis were investigated. sepsis was induced by cecal ligation & puncture (clp). control rats were sham-operated. three microsomal fractions (rough, intermediate and smooth) were isolated from rat liver. study of ip receptor binding was performed with tridium label ip . the results shewed that the ip binding was significantly depressed by - % (p< . ) during late sepsis ( hrs after clp), but not in early sepsis ( hrs after clp). the ip binding depression during late sepsis was most significant on rough and intermediate endoplasmic reticulum (p< . ), but not on smooth subfraction. since ip binding plays an important role in the regulation of intracellular calcium homeostasis in hepatocytes, an impairment in the calcium release due to depressed ip binding on smooth and intermediate endoplasmic reticulum during late sepsis may have a pathophysiological significance in contributing to the development of altered hepatic metabolism during septic shock. septic organ failure is currently recognized as an overactivation of the nonspecific immune system by bacterial stimuli giving rise to proinflammatory mediators. little is known about the mechanisms of the resulting cellular injury. here, a synergism is described between tnf as a major mediator of septic organ injury released by macrophages and hydrogen peroxide (h ) as a representative of reactive oxygen species as formed by e.g. neutrophils. rat hepatocytes are only slightly sensitive to either agent alone. when treated with a conbination of tnf and h# a stronq synergistic toxicity was found, especially w~e~ tnf-treatment preceeded challenge with h~o~. we have recently described a coculture model bfzrat liver macrophaqes and hepatocytes where lps induces hepatocyte cell death partially mediated by macrophage tnf release. when h was also employed in fhis more complex cellular system a similar synergism was found: the ecc~ of lps was consecutive patients with liver cirrhosis admitted to the department of surgery over a year period from january to december were studied for their complement profiles in relation to other parameters of liver function, the aim of the study was to determine if a direct correlation existed between low complement levels and end stage liver cirrhosis. cirrhotic patients were divided into child's a, b and c categories using child's classification. complement levels (c , c ) were measured and functional assay for complement (ch ) were performed in each of these groupings in addition to normal blood donor controls. these results show that the qualitative c , c and the functional chs complement assays have good predictive values in assessing deteriorating liver function• in particular, the functional assay for complement (ch ) showed marked impairment in child's c patients (p< . ) confirming the impaired immunological status of these patients. sera from this group of patients (child's c) were titrated with pig red blood cells (rbcs) in a haemolytic assay. the results showed that there were significantly less haemolysis of pig rbcs in these patients (p= . ) as compared to the controls. this findings strongly support an impaired immunological status in child's c liver cirrhosis and may explain the high incidence of sepsis as a terminal event in these patients. aim:kupffer cells(kc) have an importamt play to cause hepatocellular injury in sepsis, because these cells release many kinds of substances. we reported that oxygem radicals released by kcs stimulated by lipopolysaccharide (lps) caused hepatocellular injury. aim of this study is to investigate the relationship between imtracellmlar calcium(ca) concentration of cultured rat kcs stimulated by lps and release of oxygen radicals, and effect of prostaglandin e~ (pge~) on imtracellular ca concentration. production of acute phase proteins (c-reactive protein, crp, transferrin, tf) and £erritin (f) in rat hepatocytes (hps) and its dependence on extracellular matrix components were studied. hps isolated from the liver by collagenase perfusion were cultured at ~o per . ml medium fi +dmem ( : ) with % fetal calf serum for days on uncoated or type i collagen coated plastic surface or in the presence of dextrane sulphate in the medium. hps were stimulated by conditioned medium (gm) from i~ia-p or e. coli lps preineubated human blood mononuclear cells. production of crp, tf and f by hps was detected by elisa. it was found that both cms decreased tf synthesis in hps by - % (p<o.os). the level of f synthesis didn't change or only slightly decreased. the addition of cms led to the enhancement of crp synthesis more than . times. the most reproducible results were obtained when plastics had been coated with collagen. dextran sulphate markedly increased crp synthesis. thus using this model of an acute phase reaction in vitro we found the enhancement of crp synthesis and the decrease of tf synthesis. our data correspond with nublished materials on the acute phase in vivo and point to relevance of the proposed model. metabolism of hepatocytes under traumatic illness has not been sufficiently investigated. therefore we have applied absorption and fluorescent cytophotometry techniques to study the content of rna, glycogen and its fractions in hepatocytes of patients with severe mechanical trauma, both with and without endointoxication at various stages. for these purposes slides were prepared from the repeated aspiration biopsy material according to special techniques (kudryavtseva et al., ) . slides were stained by gallocyanin-chromalum to measure rna or underwent fluorescent pas-reaction to measure glycogen and its fractions (kudryavtseva et al., (kudryavtseva et al., , the ability of metabolites derived from the xanthine-xanthine oxidase system to inhibit mitochondrial function was examined using freshly isolated rat liver mitochondria. under uncoupled conditions, mitochondria exposed to free radicals exhibited a significant decrease in consumption supported by nad+-iinked substrates, but showed almost no change in consumption in the presence of succinate and ascorbate. the activity of electron transfer complex i (nadh oxidase and nadh-cytochrome c oxidoreductase) and the energy-dependent reduction of nad+ by succinate were unaltered by oxidative stress. exposure to free radicals also had an uncoupling effect at all three coupling sites. the degree of mitochondrial swelling was closely correlated with the inhibition of state oxidation of site i substrates and with the increase in state oxidation of succinate. the immunosuppressive agent cyclosporin a (cya) completely prevented the mitochondrial damage induced by oxygen free radicals (swelling, ca + release, sucrose trapping, uncoupling, and selective inhibition of the mitochondrial respiration of site i substrates). the same protective effect was found when ca + cycling was prevented, either by chelating ca + with egta or by inhibiting ca + re-uptake with ruthenium red. these findings suggest that the deleterious effect of free radicals on mitochondria in the present experimental system was triggered by the cya-sensitive and ca ÷-dependent membrane transition, and not by direct impairment of the mitochondrial inner membrane enzymes. correspondence should be addressed to: naoshi takeyama the aim of this study was to clarify the critical role of reduced glutathione (gsh) on the progression of lipopolysaccharide (lps)induced liver damage of mouse. the conditions of gsh-depletion and -richness were produced by intraperitoneal administration of buthionine sulfoximine (bso), a specific inhibitor of gsh synthetase, and of gsh-monoethyl ester (gsh-me), respectively. gsh-me, which was esterified the caboxyl group of the glycine residue, is readily transported into cells and is hydrolyzed intracellularly; this leads to greatly increased the cytosolic and mitochondrial levels of gsh. bso and gsh-me were administered intraperitoneally mmol/kg and mmollkg, respectively, and lps ( mg/kg) given h later. hepatocytes were isolated by in situ perfusion of the liver with collagenase h after lps injection. the degree of hydrogen peroxide (h ) synthesis and mitochondrial membrane potential were measured by flow cytometry using fluorescence probe dichrolofluorescein diacetate and tetrametyl rhodamine ethyl ester, respectively. the degree of mitochondria membrane permeability transition (mmp) was assessed by [ ]sucrose entrapment. we found that lps treatment results in a decrease in hepatic gsh level and mitochondrial membrane potential, and an increase in h synthesis and mmp. lps administration to bsq-pretreated mouse worsened at[ these parameters. in contrast, gsh-me pretreatment ameliorates. all together, gsh may acts an important role in cellular defence against lps-mediated liver damage, and mmp may result in the decrease in mitochondrial membrane potential. it has been shown, up to now, that disturbances of enzymatic processes in the cell organella are the basic factor of the changes taking place during endotoxin shock it has been observed that lysosomes are biochemically changed as result of the effect of lipopotysacharides of gram negative endotoxic bacteria. the purpose of the experiment was: .evaluation nfthe course of the ees due to the biochemic changes .determination of the influence of the ees on the activity of lysosomal enzymes in liver cell .deterrmnatian of the influence of h and d on the activity of some lysosomal enzymes in liver cell during ees material and method: examinations ware carried out on dogs. the shock was evoked by i.v. administration of endotoxin e.coli. the dogs were divided into groups: icontrols, i[ -dogs with untreated shock, iii-dogs in shock treated with h, ivdogs in shock treated with d, v -dogs in shock treated with h and d. the following parameters were determined: .activity of acid phosphatase in the venous blood serum. - .total and free activity of acid phosphatase and catepsins in the full liver cell homogenate, in mitochondrial-and-lysosomal fraction, and in the superuatunt (all after hours of the experiment). conclusions . essential increase of activity of lysosomal enzymes was observed in the ees. . increased activity of lysosomal hydrolases in the liver cell homogenate corresponds, as a result of the endotoxin effect, with increased enzymatic activity in the peripherial blood. extensive investigations from our laboratory of the pathophysiology of hepatic no-flow ischemia ( min) -reperfusion injury demonstrated substantial kupffer cell activation with reactive oxygen formation during the first few hours of reperfusion as indicated by increases in plasma glutathione disulfide (gssg) levels in vivo (am. j. physiol. : g , ) and enhanced superoxide formation by kupffer cells (kc) isolated from the postischemic livers (free radic. res. commun. : , ) . the early kc-induced injury initiated the later, primarily neutrophilmediated reperfusion injury (faseb j. : , ). to test whether or not similar mechanisms are operative during hepatic ischemia induced by hemorrhagic shock, male fischer rats ( - g) were subjected to rain of hemorrhage (mean arterial pressure mm hg) followed by resuscitation (rs) (reinfusion of shed blood). hepatic atp levels declined from . + . txmol/g to . _+ . ( min shock) indicating severe ischemia, and recovered to . -- . at min rs. to test for potential oxidant stress during rs, plasma gssg conc. were measured. although gssg levels increased by % compared to baseline levels ( . !-_ . i.tm), there was no significant difference to shamoperated controls. spontaneous superoxide formation of isolated kc was . + . nmol o -/min/ ecells (kc ) and . + . (kc ) in controls and did not change significantly after shock and rain rs. addition of phorbol ester or opsonized zymosan to isolated kc did not indicate a priming of these cells. conclusion: in striking contrast to our previous findings with hepatic no-flow ischemia, there is no evidence for a significant kc activation after min of hemorrhagic shock and up to rain of resuscitation. objectives-this study investigates morphometric changes in kc nltrastmcture which might account for this diminution in phagacytosis. materials and methods -three groups of wistar rats were studied: control, sham-operated [sham] and bile duct ligated [edl] . after weeks animals were sacrificed and livers were "perfusion fixed" with % glntaraldehyde and processed for transmission electron microscopy and image analysis. results -in the the bdl group kupffer cell numbers were greatly increased. biliary ductular proliferation was evident and sinusoidal spaces were compromised. eleven nuclear and cytoplasmic parameters were measured and statistically significant results are summarised in the results: ascorbic acid levels were slightly elevated after the hs and returned to basal values after rain. glutathione concentrations were increased significantly after the hs and the gsh levels kept rising continuously to as much as -fold of basal levels at the end of min. mda showed no significant variation before and after the hemorrhagic shock. plasma concentrations of ratine, teucine, isoleucine, phenylalanine, proline, threonine and serine showed significant increase over basal levels during the whole ischemic period. glutamic acid was slightly elevated at the onset of hs and remained the same for the rest of ischemic period. hydroxyproline was significantly below the basal value at the mid-point of ischemic period. alanine, glycine, histidine and aspartate did not change significantly throughout experimental time course. conclusions: ( ) oxidative stress might not be severe in the systemic hemorrhagic shock for pigs since no significant variations were observed for ascorbic acid and mda ( ) the increase of gsh might be that it was released from hepatocytes when the animal was under systemic ischemia. ( ) celzain amino acids might be acting as transmitters in the event of ischemia. however, fm"lher investigations are needed to clarify the detailed mechanism in regard with antioxidants and amino acids. dr. fang-ku p'eng taichung veterans general hospital talchung, taiwan , r.o.c. jia shi yong, huang zhi oiang and men xian jun. in patients underi~ent major hepatic surgery,obstructive jaundice has been implicated with fnoreased susceptibility to sepsis and liver failure. jaundice was said to he associated with derangement of iamune function and result in expression of pr,:.inflar...aatory cytoki~es that cause hepatooellular damage. this study is ~.o investigate the effects ,.,t lip,~polysaccharide(lps)rats. jaundice were produced in healthy ~istar rats of either sex weighing - m~ by common hi l~ duet l igation(bl)l).seven days post l igation, rats were given lps( .gg. ) . rag/kg intraperitoneally. at the end ,:,f l,gand hours after lp$ ehanllenge, liver were removed and prepared for forzen sections immediately. i)emonstration of inf in liver parenchyna were performed by method of abu i~munohistochemistry using r~onoolonal antibody against tnf, leve of mrna for tnf ~ere measured by in-situ hybridization using biotin-labeled edna probe. results sho'~ed that tnf stained granules appeared in kup~'fer eell~ and polymorphonuclear leukocytes(pnnl,but not in hepatoeytes nor endothelial eel is. they ~'ere located within cytoplasms and peaked at - hours after lps ohal lenge. there was vocomitant tnfmrna expression but peaked earlier hour post lps challenge. tnfmrna vcere detected notably in necrotic area and barely in bdl rats ~ithout lps challenge. it is therefore postulated that production of tnf by inflammatory effeetor cells-kupffer cell and phn in site, in the obstructive jaundice rats during sepsis may atribute to the hepatocellular damage. it also provide evidence for the beneficial effects of early release of biliary obstruction. in the present study, an animal model of a c in wlstar rats was developed by tigatlng the common bite duct and injecting d. mt solution of e. goli % b, (g× ' efm/mt) into the bite duct. the mortality rate was ~ at h after aoc. at , , , h after aoc, kcs were isotated and cultured atone or cocuttured with hepatocytes to evaluate the modulation effect of kgs on hepatoeyte protein synthesis. the results showed that tactodehydrogenase leakage was increased progressively as the injured k~ function and structure developed. tnf and il-i tn the supernatant were detected with the peak values at h after aoc. at h after aoc, ~-teueine incorporation was obviousty decreased in the normal hepatocytes eocuttured with stimulated kcs compared to the uormai hepatocyte cultured group (p< . ), but it was slgnificantiy increased in the group cocultured with normal kcs. in the a c group, 'h-teuclne ineorporatien was decreased progressively in the injured hepatoeytes cocuttured with stimulatedkgs and it was markedly elevated when cocuttured wlthnormat kcs at h after a c (p< . og). it is concluded that the impaired hepatocyte proterin synthesis was directly mediated hy the stimulated kc function during aoc. such a mechanism possibly may be involved in the patho ensis of hepatic dysfunction and m f following h . " the project supported by nsfc. in the past few years it has become evident that cytokines are implicated in various pathophysiological mechanisms. therefore measurement of cytokines and their potential inhibitors in biological fluids may be helpful in diagnosing and monitoring of diseases. however, dependent on the type of assay used investigations performed in different laboratories have often yielded conflicting results. in order to assess reliability and reproducibility of cytokine measurements two comparative studies for the determination of cytokines in biological fluids were launched by several investigators interested in this field: one national austrian ~tudy and one european study in the context of the european workshop for rheumatnlogy research. serum and synovial fluid samples were distributed by the organisers, and participants had to measure one or several of the following cytokines in all samples: il- , il- , l- , i , tnf-alpha, ifn-gamma, gm-csf, and the soluble receptors for il- and tnf; both commercially available immunoassays and home-made assays were allowed. so far, the main conclusions emerging from these preliminary studies are: ( ) the same immurzoassay (elisa) yielded comparable results in different laboratories; ( ) immunoassays from different manufactureres usually measured the highest concentrations in the same samples, yielded similar relative values but disparate absolute values; ( ) assays for soluble receptors yielded less discrepant results; ( ) some assays seem to be more suitable for measuring cytokines in biological fluids than others. the mean retrieval of exogenous recombinant human cytokines was approximately % for most cytokines tested. however, it must be borne in mind that natural and recombinant cytokines may behave differently in immunoassays. this raises the question as to the necessity of setting up international standards for all cytokines. possible interferences by serum components such as (lipo)proteins, complement, rheumatoid factors, etc., which may either decrease assay sensitivity or yield false positive results, must also be considered. in addition, natural cytokine binding proteins (e.g. soluble receptors) might interfere with cytokine determination in immunoassays. the problem of immunoassays versus bioassays has not yet been approached, but is without any doubt worth indepth investigation. thus, these studies are a promising first approach to deal with the difficulties of cytokine analysis in biological samples, and it is hoped that they will help to overcome some of the major methodological problems in the near future. tumor necrosis factor (tnf) is a pleiotropic cytokine which plays a key role in a number of immunologically mediated disorders like septicemia or cachexia. tnf receptors are found on the surface of a variety of cells, where they provide molecular signals for the cytokine effect. there exist two types of soluble tnf receptors (stnf-rs), tnf-r p and tnf-r p . these stnf-rs can compete with the cell surface receptors and block their activity. the expression on and shedding from the cell surface of stnf-rs is enhanced by interferon gamma. increased concentrations of stnf-rs can be found in patients with infectious as well as malignant disorders. in patients undergoing immune stimulatory therapy with interleukin- and interferon alpha a simultaneous increase of tnf and its soluble receptors can be seen. in patients with hiv infection a strong correlation exists between the levels of stnf-rs and markers of immune activation like neopterin or beta- -microglobulin. likewise patients with hematological disorders show elevated stnf-r levels. patients with weight loss have higher concentrations than patients with stable weight. the final value of stnf-rs within the complex network of cytokines is not yet clear. however, there exist striking parallels between infectious and malignant disorders pointing to a key role of endogenous immune activation. biochemicatly, d-erythro-neopterin derives from guanosine triphosphate. in vitro studies show that large amounts of neopterin are produced by human monocytes/macrophages stimulated with interferon-j (ifn-j). neopterin is biologically stable, and its halflife in the blood seems to solely depend on renal excretion. specimen for neopterin determination can be stored without special precautions. increased concentrations of neopterin have been described in body fluids of patients suffering from diseases which are apparently associated with an activated cellular immune response and with enhanced endogenous formation of ifn-~', e.g,, ) increasing neopterin levels predict rejection episodes in allograft recipients ) virus infections induce increased neopterin concentrations, and the degree of neopterin elevation predicts prognosis in these patients which is particularly true in hiv- infection ) in autoimmune disorders such as systemic lupus erythematosus or rheumatoid arthritis neopterin levels reflect activity of the disease ) increased neopterin concentrations in patients suffering from certain types of cancer indicate poor prognosis. significant associations between changes of neopterin and decrease of hemoglobin as well as the development of weight loss and cachexia, e.g., in cancer patients and in patients with hiv- infection, support the concept that endogenously formed cytokines such as ifn-~" and tumor necrosis factor-~( are involved in the pathogenesis of such symptoms. in conclusion, neopterin monitoring is a sensitive tool to detect the activation status of the cell-mediated immune system in vivo. repair and healing or perpetuation of acute inflammation is characterized by a complex network of interacting cellular and humoral defence mechanisms. of the numerous inflammatory mediators/effectors investigated hitherto, the proteolydc lysosomal enzyme pmn elastase has turned out to be highly destructive when released extracellularly thus contributing considm:ably to organ dysfunctions in severe posttraumatic and postoperative courses. besides various cytokines, elastase in complex with its main antagonist c¢ -proteinase inhibitor (a pi) is also supposed to induce the shedding of intercellular adhesion molecules from inflammatory cells (pmns, monocytes/macrophages, endothelial cells). these soluble adhesion molecules may modulate the inflammatory process in many different ways, e.g. via acting as chemotaxins, blocking neutrophil activation or competing with the membrane-bound forms in cell-cell adhesion. thus, measuring circulating or local levels of elastase-a pi and soluble adhesion molecules (icam, e-selectin,-pselectin, l-selectin) may be a helpful tool in judging the severity of an acute inflammatory process. in several clinical studies on surgical patients suffering from multiple trauma and/or sepsis we could demonstrate that the measurement of these parameters in consecutive plasma samples and local body fluids was highly valuable for early diagnosis and prognosis of multiple organ failure. prof. dr. m./ochum, institut fdr klinischechemie und biochemie, lmu miinchen, nufibaumstrage , m nchen, germany procalaitonin (proct) a aa peptide is dramatically increased in the blood of patients with various sepsis and infections. the increase begins as soon as hours after the andoto:edn release and readied maximal level with a to b-fold increase h after li~ edrmrdstration. prcc, alcitorfin response was temporally different from these of tnf~ and ii- , that reached peak levels at h and h respectively. at the opposite of eytokine (tnfcz, ild, j, is), proct is a very stable molecule. the half iife of proct in ~he blood is surprising if we consider the half llfe (about nine mirtute) of mature ¢alcitonin (co. we have found that it is due to the binding of proct with a protein on the n-termktal part. thin complex of about daltons is unable to cross the kidney and the c_n barriers and the half life is at least elghteen hours. this long half llfe is very useful in the evaluation of a sepsis. a very sensitive and specific sandwich in'ununoassay has been developped. the results cart be delivered in two hours. at time, we know that in the healthy adults, the values are less thau . ng/ml. it is also clear that proct is not increased ~n patients wlth inflammatory dleeasea without lnfectlon. for instance, proct is not signlffcat vely increased in purpura rhumatom, arthritis, crohn's disease, rectocolitis, also in various cancer patients with inflammatory components. am other interesting finding, is the absence of increase or a very low increase in the viral infections. at the opp site, in all the patients with bacterial sepsis or in the malaria pat/ants, progt was dramatically increased, from i to fold. p~oct can be useful to the follow up of patients with sepsis. actually, we don't know the origin of this proct production, in the united states, will also be discussed. the septest portion of this study will eventually enroll over patients tentatively diagnosed as septic. the results of septest will be compared with commonly accepted symptoms and criteria associated with sepsis in order to determine the clinical utility of this endotoxin assay. preclinical results of patients and normals be presented as well as data on the time course of endotoxemia and clinical outcome of selected septic patients. preparation of dna libraries clifford w. schweinfest, ph.d. a fundamental tool of the molecular biologist today is the ability to work with purified dnas representing genes of interest with respect to the investigator's field of study. originally applied almost years ago to the genetic dissection of simple organisms such as viruses and bacteria, molecfllar cloning was rapidly applied to study the more complex genetics of the mammalian cell. today, virtually any study of cell physiology which depends upon specific gene expression is approachable using the tools of the molecular biologist. therefore, stress induced gene expression (such as the heat shock gene, hsp ) or the regulation of the nitric oxide synthetase gene or the induction of cytokines and growth factors as a result of trauma or injury lend themselves to investigation at the level of the gene. during these workshops, we will discuss the means by which cdnas and genes are isolated, amplified and identified. we will discuss the strategies and methods for cloning cdnas and genomic dnas, for organizing such cloned libraries, for finding specific genes and for characterizing those genes. this speaker will focus on the techniques and considerations involved in the synthesis of cdna libraries. topics include rna isolation, mrna quality, reverse transcription, choice of cloning vectors and library quality. genomic dna library synthesis will also be discussed with respect to the vector/host systems available and applications which are specific to genomic dna. finally, polymerase chain reaction (pcr) will be discussed briefly with regard to its impact on dna cloning. the identification of the genes or gene products that have a role in cellular processes is fundamental to our understanding of genetic control and methodologies to achieve this aim are currently available. all levels of the various signal transduction pathways can b~ molecularly analyzed to define the mechanism by which critical steps in each pathway are achieved. the questions that are unresolved in each area of investigation serve to direct the scientist towards analyses of gene products or gene regulation of the gene itself. thus, selection of the type of library (genomic or cdna) to be used is dependent upon the specific goals of each investigator. to facilitate resolution of this question, an overview of the uses for the different types of libraries will be presented. at least four methodologies are currently available for screening a library, which are dependent upon specific interaction between nucleic acids, nucleic acids and proteins, antibodies and antigens or between different proteins. the use and advantages of each of these methodologies will be discussed along with a description of probe preparation. sequence methodologies required to begin characterization of gene clones will be presented. hopefully, the participants in the workshop will help define areas of emphasis and allow time for directed interaction to discuss specific applications based upon their own experimental systems. alterations of physiological conditions, such as those occurring after shock and sepsis, induce changes in gene expression of cells composing the major organ systems. the challenge is to detect and characterize these changes in geae expression and relate them to the injury. the development of cloning techniques has emerged as the methodology of choice. changes in gene expression are usually characterized by variations in the concentration of cellular messages because synthesis of the final product "the polypoptide" is usually proportional to the concentration of mrna. due to the rapid regulation of gene expression the cellular concentration of messages changes very quickly. this is commonly referred to as steady-state levels, a balance between transcription and degradation. steady-state levels of mrna can be detected in a single cell (in situ hybridization) or in total rna isolated from cells or organs and separated in agarose gels (northern blots). although analysis of mrna steady-state levels are very useful, they do not provide information about the mechanisms that regulate gene expression. changes in gene expression primarily occur at the level of transcription; characterization of the rna species being synthesized at a given time is performed by the nuclear run-off technique. when there is no a priori information about the gene that may be regulated after a particular situation such as sepsis, the total pool of messages present in cells at given time can be characterized by a combination of in vitro translation of the cellular messages and fractionation of the in vitro translated products by two dimensional gel electrophoresis. such approach permits the visualization of the general pattern of gene expression, a "finger print", of the physiologic state. in summary, researchers now have access to a great variety of techniques to identify and characterize genes expressed in response to a physiological condition that may be utilized as "molecular tools" to study the organism's responses to shock and sepsis. the acute phase proteins are a set of plasma proteins with greatly increased plasma concentrations during acute inflammations and after tissue trauma, e.g. after major surgery. the proteins counteract the source of injury, facilitate clearance of infectious particles by phagocytes, assist immune responses and initiate wound healing. the corresponding genes are expressed in liver hepatocytes and regulated by the inflammatory mediators interleukin and interleukin (ill, il ). class acute phase genes are mainly controlled by ill and by combinations of ill plus il ; class genes mainly by ii, . the human c-reactive protein(crp), serum amyloid a(saa) and complement c genes will be discussed as examples of class genes, and rat c~ macroglobulin as a prototypical class gene. both classes of genes use different types of ¢ytokine response elements in their promoter-proximal transcription control regions; class genes use the transcription factor nf-il or c/ebp as the key factor to mediate cytokine responsiveness; class genes use a different, so far unidentified factor to achieve responsiveness to il . ongoing efforts to purify this factor, called the il -response factor (il -rf), from rat liver nuclear protein extracts will be described. the il -rf apparently mediates the effects of il in a broad range of cell types, including b lymphocytes and other hematopoietic cells. it is therefore likely to be of equal general importance for gene regulation by il as nf-il . the importance of transcription factors as potential targets for novel bioactive substances and possibly therapeutic agents will be discussed with the help of several recent examples. new methods for altering the germ lines of mice provide powerful tools for understanding the roles of individual genes in normal and pathological processes, and for reproducing specific genetic mutations that result in congenital disease. three approaches will be discussed. conventional transgeulc mice are frequently constructed using artificial transgenes that express genes from a promoter other than the normal gene promoter. this paradigm is used to produce very high levels of a gene product, for example, with a viral promoter, or to attempt to regulate gene expression using an inducible promoter. alternatively, normal promoter sequences can be hooked to a gene whose expression is lethal to the cell (e.g., diptheria toxin a chain) to ablate all cells expressing that gene. another approach uses an intact gene with its normal regulatory sequences. here, an elevated level of the gene product is delivered from the correct cells in the correct tissues at the correct developmental stages or in response to natural signals. this approach has been refered to as "genetic pharmacology," and provides a precise delivery of the gene product to the target. however, to be most effective, the transgene should contain arl regulatory sequences as welt as the entire genomic segment containing gene coding regions. the introduction of conventional transgenes is limited to roughly kilobases, while genes containing all regulatory sequences frequently stretch over dozens or even hundreds of kilobases. to overcome this problem, procedures have been developed recently to introduce yeast artificial chromosomes (yacs) into mouse embryonic stem (es) cells. yacs can include over mb of human dna, large enough to encompass the largest known human genes. es cells are also useful for targeted gene deletions and mutations. homologous recombination can be targeted to any known gene in es cells. when these modified cells are injected into a mouse blastocyst, they can be incorporated into all tissues of the resulting mouse, including germ cells, so subsequent generations will pass the genetic modification as an inherited trait. assessment of the effects of a null allele on development can provide valuable insights into its normal role. ultimately, these technologies may be adapted to human cells -not for embryonic intervention, but to provide modified stem cells for various tissues (e.g., gut, eye, hematopoietic system) which could then be applied to somatic therapies. with major illness/injury that respirswy faihne fi'equently followed sepsis, tilney el al nse, d the term "soqueutial syste~ failure" for patients with renal failure after solmir of ruptured abdominal aortic ~aeurysms, i then reviewed our experiences after inju~ and oporstion and suggested that multiple, progsesslve or sequential systems or organ failure was a syndrome to be reckoned with in the, 's. eiseman et al then wrote about "multiple organ failure", especially with liver failure. polk el m found that renmte orgau failure often signalled occult anita-abdominal infection. fry et al ampbasized the role of uncontrolled infection in organ failure, border et ai described metabolic alterations with mof and used the term multiplesystems organ feilmo (msof). other early conltibutots to our knowledge of mof include faist, trunkey and miller, marshall and dimic&, cassone, catlleo, meakins and marshall, (}otis et at., mater and many others. mof or msof were used eommoifly. cerra coined the terms "post-trsumatic septic sfndromc" and "hyper metabolism otgau failure complex", and border et al, used lhe ex ~ression "gut origin seplic states" to illdioate important eurrem aspects of abe gt.'nerlc problem of organ failure and death, other terms include acute organ-system failure, multiple organ injury syndrome, post-traumatic multi-system organ failure nod post-~aumatic organ system infection s~dmmc (fi~sis), i bdieve the latin "maltiple organ failure" is clean, neat and says it all, now there is a proposal for a new set of torminolugios -mods and sirs. will they be helpful in the study and esro of patients? our speakers in this session will review thcsa proposals and the evidence as we strlvo for eoasensns. it has been well demonstrated that the administration of pro-inflammatory cytokines, and especially tumor necrosis factor (tnf) can result in a picture similar to septic shock with secondary multiple organ failure. it is also clear that tissue hypoxia can lead to organ damage. we believe that it is the interaction between the two phenomenons that can lead to mof. on the one hand, the inflammatory response is amplified in the presence of hypoxia. on the other hand, the release of the mediators of sepsis can increase tbe oxygen demand ef the tissues, alter their oxygen extraction and decrease myocardial contractility, and the combination of these elements accounts for the development of septic shock. this hypothesis is supported by two lines of evidence. first, mof is usually preceded by an episode of acute circulatory failure (even though frank circulatory shock may not be evident). second, recent experimental and clinical studies indicated that immunotherapy (especially antibodies to tnf) is particularly effective in the most severe forms of sepsis, associated with circulatory shock. hence, an effective way to prevent mof may be a combination of aggressive cardiovascular management and immunotherapy. development of the multiple organ dysfunction syndrome (mods) in the critically ill follows an heterogeneous group of stimuli including infection, sterile inflammmion, extensive tissue injury, ischemia, intoxication with a variety of substances, and immune system activation. whether the resulting physiologic abnormalities reflect a common pathologic process, or are simply a non-specific manifestation of tissue injury is unknown. moreover, the lack of clearly-defined functional criteria for the characterization of the syndrome on the one hand, and of reliable biochemical markers of its evolution on the other, has made attempts to define its epidemiology and natural history difficult. using a numeric scoring system to quanfimte the clinical severity of mods, we demonstrated that, although mods is more common in patients who have significant infection at the time of icu admission, a much stronger correlation is evident between the severity of mods and the subsequent development of nosocomial icu-acquired infection. furthermore the severity of mods correlates strongly with the severity of the clinical septic response, and with the degree of immunologic compromise evident on delayed type hypersensitivity skin testing. to determine the relative importance of the initial stimulus, and the host response to that stimulus, to the subsequent emergence of mods, we undertook a matched cohort study of critically ill patients admitted to the icu with infection, matched by age, sex, and apache ii score to uninfected controls. organ dysfunction scores were higher in patients admitted with infection, but were also significantly associated with the expression of a septic response at the time of icu admission, independent of the presence of infection. by stepwise regression analysis, the magnitude of the septic response was the most powerful predictor of the severity of mods in both infected and uninfected patients. these data suggest a model of mods in which a stimulus (eg infection) and the host response to that stimulus (eg the septic response) result in a state of altered systemic homeostasis (manifested in this example by immunologic dysfunction). moreover they suggest that the principle determinant of the emergence of mods is the response of the host, rather than the nature of the stimulus, and are consistent with the hypothesis that a stereotypie systemic response to an heterogeneous group of injurious stimuli underlies the pathogenesis of mods. arthur e. baue, m.d, ever since the attorapt build the tower ef babel, as desclibcd in the old testament of the biblc, it has been difficult to got agreement o;x common definitions or language, although mof has served well, there will always he now classifications proposed with good reasons for them, thus mods and sirs are not the final oxpressinns in the lexico~aphic sweepstakes. attcmpts to develop standard animal shock and scpsis models (hemorrhage, endotoxin, foes[ pellets, cecal ligation and puncture, e. colt hlf sioi to evaluate therapy have net been suoca.ssf~. clinical syndromes of mof, sepsis, sepsis syndrome and athers have not been aeacpte.d by all. our problem is that we arc tryittg tu d¢ffino a non-emily. mof or mods or sirs is net a disease or oven a syndrome. it is a series of complications of injury, disease and intensiva care which loads to death for many patients in intensive care units, it is not a fixed entity, but an ewilving picture. we have lumped (ugethet for therapy and definition a number of diseases and problems according to clinical manifestatieos rather than the eause of ihe problem, the search for unified mechanisms has not bean successful. will we benefit oleo from a more precise classification and definition of a non-outjty, a hodgu-podge of human disorders which have charsmeristic.s and manifestations of tissue injury, inflammation and infactian? re.hi clinical trials of potential "magle ballets" suggest that we should go in a different direction--instead of lumpors, we should be splitters, to seek effective therapy, we must fucus on specific disorders such as trauma, specific infections, inflammatory disease and chronic or acute organ damage (copd -renal failure, etc.), i will say more aboet this later ha the me.ethag. thcre is cue potentially important pert of the mods proposal, and that would be do~mcntation of organ dysfunction before fsiiure occurs and before tile need for external supporl dovelope. this could lead to hatter methods of preventing organ failure. preveution is ultimately the only answer to organ failure, m mof, mods and sirs. have wo reached a consensus?--only that we arc dealing wilh a diffl~x:lt probtolr looking for solutlons. acute lung injury (ali) and its most severe form, acute respiratory distress syndrome (ards) characterized by severe hypoxemia, diffuse infiltration of both lungs, a reduction of compliance and a wedgepresure lower than mm hare related to direct or indirect injury. when aliresults from direct injury (toxic agents, lung contusion, pneumonia), the lesions of epithelial barrier and the activation of lung macrophages are the first events leading to the release of inflammatory mediators wh:ch are responsible for alteration of the membranar permeability and for invasion of the alveoli by fluids, proteins and cells. an inflammatory reaction develops, with injury to endothelial cells, adhesion of polymorpbonuclear leucocytes (pmn) and the release in blood stream of intlammatory mediators dispersing the inflammatory reaction to other organs and tissues. the failure of lung function also results into hypoxia in other organs, leading to tissular ischemia, triggering an inflammatory reaction leading to organ dysfunction. frequent complications of direct lung injury are septic pneumonia with endotoxin release, phagocytes activation and cytokine production disseminating inflammation. by this way, direct lung injury can be at the origin of the multiple organ dysfunction syndrome (mods), the frequence of which having been estimated to % and is increased when all has occured in patients alreadypresenting an organ dysfunction (immunodepresslon, cancer...). when ali results from redirect or extrathoraclc injury (trauma, infections, hypovolemic shock, acute pancreatitis...), inflammatory mediators and micro-aggregates released in particular organs or tissues reach the lung via the blood and lymph streams, are filtered in the organ or trapped in the lung capillaries, and are responsible for a local inflammatory reactaon (endothelial cell activation, pmn trapping and adhesion, platelet aggregation, release of mediators). sepsis acts by the way of endotoxiu and sequential eytokines release (tnf, ill,...) while trauma with important blood loss acts especially by the way of hypoperfusionreperfusion phenomena leading to a disseminatedinflammatory reaction and to a possible bacterial transloeation when intestinal hypopeffnsion is present. in these conditions of indirect injury, all (or ards) is a iocal early (often the first) manifestation of a general phenomenon of altered membrane permeability and inflammatory reaction, easily and rapidly recognized by its clin~cai signs. the iung is one of the numerous organs participating to the mods, in these conditions, the mortality rate is high, reaching more than % when sepsis is present or when at least organs are implicated in mods. by direct or redirect injury, lung is so a target organ for mods and injured lung can be the cause of mods or simply a participant to this syndrome. jorge cervts-navarro main determinants for brain dysfunction are breakdown of the blood-brainbarrier and raise of intracranial pressure (icp), in second place decrease of systemic blood pressure, disorders of blood coagulation and respiratory function. the blood-brain-barrier of cerebral blood vessels can be opened by stroke, cerebral trauma, inflammation, convulsions, acute hypertension and changes in the blood osmolarity. the consequence is brain oedema, increase of lop and decrease of the perfusion pressure. a close correlation between intraventricular pressure and the fate of patients with severe brain injuries has been established. regional cbf values of to ml/ g/min are reflected in isoelectric eeg, and values about t ml/ g/min, result in loss of somatosensory evoked potentials. for ionic pump failure the threshold has been determined by values of about ml/ g/min and is reflected in massive release of intracellular potassium. in stroke and in brain trauma when the oedematogenous condition is initially localized the tissue pressure variance within and between the hemispheres lead to tentorial and falciform herniations are the common cause of death even before the critical threshold of intracranial pressure is reached. the increase of the icp over the level of the perfusion pressure leads to the arrest of the cbf. if this persists {or periods exceeding seconds of global brain ischemia loss of conciousness and developing seizures appear. if it exceeds min irreversible injuries of the brain appear. following cerebral ischemia and after severe head injury a tendency to increased coagulation can be detected. vascular occlusion may develop either as a result of local thrombus formation or from emboli caused by circulating platelet aggregates. the formation of microthrombi is triggered by the plateletactivating factor a mediator in central nervous injury also responsible for aggravation of posttraumatic brain edema. acute hemorrhagic leucoencephalitis and brain purpura are known to arise as complications of acute viral infections and following gram-negative septicaemia. institute of neuropathology, free university of berlin, hindenburgdamm , berlin, germany undoubtedly, attempts to quantify as objectively as possible the degree of dysfunction of the various organs is very valuable. however, cardiovascular failure has a peculiar place in the context of sepsis. the development of acute circulatory failure (shock) is of paramount importance as a cause rather than as a consequence of organ failure. it is therefore essential to clearly separate patients with and without circulatory failure. once this has been done, attempts to list the cardiovascular system among the other systems have been unconvincing. in a number of studies, cardiovascular complications are assimilated to a low systemic vascular resistance (svr), but since svr is basically the ratio of blood pressure over cardiac output, and since hypotension is already included in the definition of shock, then a low svr is basically equivalent to a high cardiac output, which is a characteristic rather than an ominous complication of severe sepsis. development of a low cardiac output unresponsive to fluids is usually a terminal event. it is not advisable to list other problems such as severe arrhythmias, myocardial infarction or tamponade as complications of severe sepsis. host defense dysfunction following trauma, shock and sepsis e. faist, g. f. babcock * major accidental, burn and operative injury often precipitates a profound multicentric immunologic depression that is believed to predispose patients to become anergic and thus to develop towards a higb probability of infection. anergy or a lack of immunologic reactivity stands for a total elimination of skin test reactivity and recall antigen responses in the inununocompromised patient. anergy following overwhelming trauma or major septic conditions results from a massive impairment of cell mediated immunity (cmi) together with a whole body malignant inflammationiike state. we and others have demonstrated clearly that the skeration of cmi following trauma is mainly due to the disruption of intact monocyte (moo) / t-cell interaction. within this phenomenon we see a shift of the cell ratio in the compartment of peripheral blood mononuclear cells (pbmc) with a considerable increase of prostaglandin e synthesizing m~ and a simultaneous decrease of functionally competent cd + and cd + lymphocytes. t-cell dysfunction in states of profound stress is characterized by impaired synthesis of two crucial lymphokines -interleukin- (il- ) and gamma-interferon (y-ifn). the inability to produce adequate amounts of il- , most likely attributable to alterations in the transmission of signals from the cell membrane to the nucleus, results in incomplete proliferative t-cell responses to antigenic stimuli, while a lack of ?-ifn results in inadequate m~ antigen processing and presentation. the role of the two functionally distinct t-helper subsets, t-helper- (thl) secreting principally il- and ?-ifn and t-helper- (th ) acting principally in inducing antibody formation with a secretion of il- , il- , il- and tnf-[ has still to be established within the context of major trauma. antibody formation to common protein antigens is impaired, the predominant finding is a shift from igm to igg synthesis. although excessive secretion of prninflammatory cytokines (il-u , tnf-c~, il- , il- ) has been considered to be deleterious for the host in states of major inflammation and infection, in-vitro and whole blood investigation of me function has clearly revealed that there is initially a marked suppression of moo from septic patients to synthesize and secrete proinflammatory cytokines to endotoxin. this probably will result in immunodeficiency of traumatized as well as septic patients, since these cytokines in low concentrations are involved in the upregulation of the essential humoral and cellular immune functions. abnormalities of pmn function noted after serious injury have been interpreted by some investigators as signs of unresponsiveness of this cell population, others have demonstrated signs of pmn byperactivation. latest findings revealed that there is a clear pmn dysfunction in circulating blood: % of all pmn's in this compartment show downregulated or non existent ~-integrins within hours posttrauma -these cells do not phagocytose, have a reduced respiratory burst and appear to be completely degranulated. restoration of these cell functions within days post-trauma is significantly correlated to survival of traumatized patients. other reports however stress increased expression of cr + receptors (cdll/cd complex) on circulating pmn's, increasing the ability of these cells to adhere to intercellular adhesion molecules, thus contributing to play a major role in inducing the pulmonary capillary leakage. our knowledge about the perturbation of host defenses associated with serious injury and sepsis is continuously increasing and represents the precondition for the design of effective therapeutic measures to prevent and counteract the resistance to invading microorganisms. dept. of surgery, klinikum grosshadern, ludwig-maximilians-university, munich, germany. * dept. of surgery, university of cincinnati, cincinnati, ohio, usa lg thijs~ ce n~ck sepsis is the most common cause of acute disseminated intravascular coagulation (dic) and coagulation disorders as well as abnormalities of fibrinolysis are common in septic patients. some clinical and experimental studies indicate that dic is a pathogenetic factor for the development of multiple organ failure. endotoxin and various mediators (tnf, il-i) enhance tissue factor and decrease expression of thrombomodulin on endothelial cells in vitro. also, tpa activity is suppressed and release of pai-i is stimulated. in such a way the endothelial surface becomes procoagulant whereas fibrinolysis is inhibited. following administration of endotoxin to normal volunteers levels of prothrombin fragments (fi+ ) and thrombin-antithrombin (tat) complexes increase, indicating thrombin generation. also the fibrinolytic system is activated as evidenced by a rise of levels of tpa and plasmin-antiplasmin (pap) complexes but this is counteracted by a subsequent release of pai-i. in severely ill septic patients levels of tat complexes are increased and concentrations of the natural inhibitors of coagulation (at iii, protein c) are usually decreased. also, tpa levels snd pap complexes as well as concentrations of pai-i are elevated. the severity of many of these alterations is related to mortality. thus, both coagulation and fibrinolysis are activated in sepsis, but not in a balanced way, thereby promoting coagulation. one of the hallmsxk pathological alterations associated with sepsis is the development of microvascular thrombosis, an entity ~lmre~erizod by microvas~tlar plugbing with leukoeyies (predominately nentrophils) and fibrin deposits. preranably, ischemia remlt~g fibm ve~-'ttlac ow,.luwion contributes sisni.ficantly to end orgen darnaje and consequent dysfatlctlon. p, er~t srldies haw focused pmdominately on the role of adhesion molecules in the pathogenesis of neu~ophil sequestration during infection and s~sis. this preparation will review the mechanisms underlying intravak-'v.ler i~brin deposition and its contribution to organ injury. the normal endothelial cell sure is generally conddered o be anticos~am. this state is maintained by two major anticoag~lam molecules on the cell so,ace: hepax'an su,lfale and thmmbomodulin. studies performed over the past decade have do~mentod a general shii~ towards a procoa~qtlant staw during gram negative hffeodon, aft e~| mediated mainly by changes in the endothalial cell surface. antt-tf antibodies have been shown to be pro~ective during l~'ml endotexemia. in summary, ~erova~'~ler fibrin deposition, in ~rt mediated by ~rcgulation of cellular tf, is a consistemt patholo~cal flndin~ during sepsis and contributes to organ injury. strategies designed to abrogate this event may n~nimjze the magnitude of erg~ dysftm~on. n, v, christou md phd, department of surgery, megill university, montreal, quebec, canada interactions between immune calls and the endothelium vie adhesion molecules serve to modulate immune cell traffic between the blood strum and the extrmvascular space, these families of molecules acting in a cascade and closely integrated with the oytoklna network, blood coagulation and the complement system, are responsible for the homing of leukocytes to areas of inflammation and the realrculatlon of tymphocytes. there are three types of immune ceils that must exit the blood stream into the extravascular space: lymphocytes ; polymorphonuclear leukocytes; and non-lymphocyta mononuclaar cells (monooytes, basophlls, aoslncphils). b lymphocytes exit the blood stream and recircu}ate mostly via the high endothelial venule (hev) in lymphatic tissue, rarely, in chronic }nfectlon=, they may recirqulate vie hev in non-lymphoi:t tissue. t-lymphocyte recircutation on the other hand can occur via hev in lymphoid tissue, as well as, the endothalium of the skin, returning to peripheral lymph nodes via the lymphatic channels. lymphocyte recirculation is the mechanism which allows ¢ontaot between the immune repertoire and pathogens, and homing delivers these cells to the appropriate environment for activation and for effeotor function, pmns exit the blood stream through endotbelium in close proximity to the site of inflammation after appropriate endothelial membrane receptor expression. they must reach the site of pathogen tnveslon quickly in order to be effective. their exudation to the inflammatory site can be modulated by their intravascular pdmlng, which results from appropriate receptor expression. because endotoxin leak from the gut has been postulated as an important trigger mechanism for the systemic inflammatory response syndrome seen after serious injury, the immunologic and metabolic effects of bacterial endotoxin infusion into normal volunteers can be expected to shed considerable light on the validity of this hypothesis. we and ethers have used tbis model to show that endotoxin causes a temporary paralysis in the ability of circulating monocytes to prudce the proinflammatory cytokines interleukin-i (il-i) and tnf-a. endotoxin, however, causes increased production of pge by the same cell population. endotoxin infusion causes significant suppression of circulating t-cell numbers and a profound suppression in the ability of the circulating t-cell population to proliferate and to produce interle~in- (il- ) upon in vitro stimulation. after endotoxin circulating t-cells are also more sensitive to the inhibitory action of exogenous pge . administration of cyclooxygenase inhibitors at the time of endotoxin infusion largely abrogates the effect of endetexin on t-cell proliferation and il- production. endotoxin infusion also causes the release of increased levels of circulating catabolic hormones including cortisol. administration of cortisol alone or cortisol along with endetowin to volunteers has allowed dissection of cortisol effects from those of endotoxin itself. cortisol infusion alone causes a diminution in the circulating t-cell population but the remaining ceils continue to produce il- after appropriate stimulation. cortisol infused along with endotoxin blocks the overshoot in monocyte proinflammatory cytokine production seen - hours after endetoxin infusion. at present, one may conclude that administration of sublethal doses of bacterial endetoxin to human volusteers produces alterations in cytokime production and tlymphocyte activation which are both similar and dissimilar to those seen following serious injury. hemodynamic support is based first on intravenous fluids and second on vasoactive agents. colloids are usually preferred to crystalloids, for their more rapid hemodynamic effects and their lesser passage in the interstitial space. in the presence of hypotension, the pharmacological profile of dopamine makes it the first agent of choice. it is only when high doses of dopamine are insufficient to restore a sufficient tissue perfusion pressure that norepinephrine should be added. even when cardiac output is not decreased, a dobutamine infusion is often indicated to counteract the myocardial depression, prevent vasoconstriction, and increase oxygen delivery to the tissues. the benefit derived from the administration of "renal" doses of dopamine is doubtful, but stimulation of dopaminergic receptors may increase blood flow also to the gut. in any case, none of these catecholamines has been shown to significantly improve the oxygen extraction capabilities of the tissues. agents with antiinflammatory properties but also with some vasodilating properties, such as n-acetylcysteine, prostaglandin el or pentoxifylline represent more attractive options to increase oxygen extraction. cardiovascular support should aim not only at the restoration of a sufficient arterial pressure but also at the maintenance of an optimal oxygen delivery to the cells. it should thus be guided also by measurements of cardiac output, mixed venous oxygen saturation and oxygen extraction, and indexes of cellular hypoxia, such as blood lactate levels, gastric intramucosal ph or vanearterial pc gradients. supranorma[ delivery contributes to the prevention of tissue hypoxia tissue hypoxia is considered to be the common final pathway in the pathogenesis of multipte systems organ failure. it may directly contribute to cellular injury and organ dysfunction as well as enhance further mediator activation and release by a positive feed back mechanism. prevention of tissue hypoxia, is therefore, one of the most important aims in the supportive therapy of critically ill patients, especially in those with sepsis and septic shock. mismatch of cellular o= supply and demand in these patients may resuit from the impairment of the cardiocircu[atory system on all levels. . myocardial depression with a drop in delivery (do~) . redistribution of blood flow between the different organ systems . inadequate nutritive blood flow due to tissue edema and endothelial damage (gic, leucocyte swelling etc.) these pathological changes may go along with increased metabolic needs. cellular and organ supply may therefore be inadequately matched with cellular needs in patients with normal and even supral normal doz. hyperdynamic circulation is often present in adequately volume resusucitated patients. it is most likely that one of the bodys main compensatory mechanisms would maintain cellular o= supply in the face of increased metabolic needs and impaired oz extraction capabillities. this pathophysiology may explain the findings in several clinical investigations that patients with normal or even supranormal doz can have signs of inadequate regional tissue oxygenation. it is also consistent with the results of different studies which demonstrated that the achievement of supranormal doz levels, either spontanuously or due to adequate supportive therapy, resuits in better patient outcome. any attempt to prevent or treat tissue hypoxia in these patients, however, has to take into account the effects of therapy not only on global dgz but especially on regional and microcirculatory blood flow. r. rossaint, d. pappert, k. lewandowski, h. gedach, k. slama, k.j. falke klinik for anaesthesiologie und operative intensivmedizin, ukrv, freie universit §t berlin, germany important contributory factors to the high mortality of severe acute respiratory distress syndrome lards) are the aggressive therapies required, such as mechanical ventilation with high inspiratory oxygen concentrations and airway pressures. as specific therapies for reducing or preventing the general inflammatory reaction of the lung resulting in severe hypoxemia is unknown, today's therapy is limited to procedures which predominantly support or maintain pulmonary function, i.e. pressure limited ventilation with peep and permissive hypercapnea, avoiding or treatment of fluid overloading, and positional maneuvers. extracorporeal lung assist combined with low frequency positive pressure ventilation has been recommended, when conventional therapy fails. today, extracorporeal gas exchange may be carried out using a system internally coated with covalently bound heparin. this allows for a lower level of systemic heparinzation reducing the risk of severe hemorrhagic complications of extracorporeal respiratory support. from april to august patients, aged from months to years, were transferred to our intensive care unit (icu) for treatment of severe ards. all fulfilled at least the slow entry criteria of the u.s. national ecmo study. due to hypoxemia patients required veno-venous extracorporeal membrane oxygenation (ecmo) immediately after transfer to our icu. the other patients were first treated with pressure controlled mechanical ventilation, permissive hypercapnea, prone position, and dehydration, if necessary. in out of these patients, in which the gas exchange did not improve in the following days, veno-venous ecmo with heparin-coated systems was additionally performed. heparin was given to achieve a partial thrombin time between - s and an activated clotting time between - s. if surgery was performed during ecmo, heparin infusion was interrupted. no severe bleeding complications related to anticoagulation for the extracorporeal bypass could be observed, however, in some patients, after three weeks of ecmo thrombi in the drainage cannula were observed. a problem of of membrane leakage shortened the life span of the membrane lungs to a mean of about four days. in the conventionally treated group % survived and in the additionally with ecmo treated group % survived, representing a % survival rate in patients whose risk of death is considered more than %. on the basis of these experiences, we conclude that the described strategy, including veno-venous ecmo with heparin-coeted systems, may improve the survival in patients suffering from severe ards. abnormally high skeletal muscle po in septic patients and its normalization during recovery: evidence against tissue hypoxia but for impaired oxygen metabolism of skeletal muscle? p. boekstegers, k. werdan department of medicine i, gro hadern university hospital, munich, germany a pathological oxygen uptake supply dependence was suggested to indicate tissue hypoxia in sepsis-a hypothesis which is discussed controversially. because the detection of reduced oxygen transport to tissue and &tissue hypoxia would have important implications for therapy, skeletal muscle oxygenation was studied in patients with sepsis. intermittent and continuous determination of skeletal muscle po by polarographic needle or catheter probes provided no evidence for skeletal muscle hypoxia even in severe stage of sepsis (boekstegers et al., crit care med ) . in contrast, mean skeletal muscle po was found to be abnormally high in patients with sepsis ( _+ , mmhg, n= ) compared to patients with limited infection ( , + , mmhg, p< . , n= ) , to patients with cardiogenic shock ( , + , mmhg, p< . , n=ls) and to healthy subjects ( , _+ , mmhg, p< . , n= ). furthermore, serial measurements in patients with sepsis showed that skeletal muscle po was higher ( , _+ , mmhg) on days with severe sepsis than on days with intermediate state ( , _+ mmhg) and than on days without sepsis ( , _+ , i mmhg) . thus, a decrease of abnormally high skeletal muscle po was related to improvement of sepsis. this was also demonstrated by comparing the decrease of abnormally high skeletal muscle po with the decrease of apache ii score or elebute score as well as interleukin- serum levels in a separate study (boekstegers et el., shock, in press). in summary, our data provide no evidence for skeletal muscle hypoxia in patients with sepsis but support the assumption &impaired oxygen metabolism in severe sepsis. neutropnlic emigration from the vascular space into the extracellular matrix is important for the response to tissue injury or contamination by providing a large recruitable pool of tissue-based phagocytes. the consequences of neutrophil exposure to intravaseular chemoattractants, likely relevant for il- and c a in sepsis, trauma, and in burn injury, are suppression of neutrophil attachment to endothelial cells and matrix proteins. it is probable that proteoglycan-bound attractants are of considerable biologic relevance, and may result in enhancement of responses to subsequently encountered cytokines. in the presence of connective tissue proteins expressing integrin-specific binding ligands, neutrophils respond to tnf-cq gm-csf and other cytokines by producing large amounts of hydrogen peroxide. this response is likely important in the digestion of contaminated or injured tissue by facilitating activity of neutrnphil granular proteases and inactivating plasma anti-proteases. this matrix-dependent oxidative response is important in defining potential novel targets for therapeutic intervention. the response appears to involve signalling by integrin-linked tyrosine kinases. the net effect of matrix protein injury through this mechanism is enhancement of the fibrotic response which underlies much of the prolonged ventilator dependence of patients with the adult respiratory distress syndrome. this adherence dependent response has also been implicated in direct hepatocyte injury, and may represent an early component of the syndrome of multisystem organ failure. study purpose: in this patient (pat.) population with a considerable sepsis morbidity and mortality, parameters proposed for sepsis assessment (elebute sepsis score [ele], sirs) were investigated with regard to their use in the early classification of pop. pat. at risk for developing sepsis. patients and methods: in a previous observational study on consecutive pat. after elective open-heart surgery, we had determined ele daily for at least pop. days (in pat. with a longer stay: until icu discharge). after publication of the newly proposed sirs definition, these criteria were retrospectively calculated and compared to ele. results: on the total of n = patient-days, both ele and sirs showed significant (p< . ) correlations with parameters reflecting the general and sepsis-related severity of the pop. clinical course. compared to sirs, the ele correlations were better (icu mortality: . vs. . ; icu treatment days: , vs. . ; days on mechanical ventilation: , vs. . ; days with vasopressor requirement: . vs. . ; number of microbiological findings: . vs. . ), the optimal classification criteria for the mainly sepsis-related outcome gave maximal youden indices of . for ele (ele >_ on >_ days, accuracy: %) compared to . for sirs (sirs present on > days, accuracy: %). accordingly, ele roc curve areas for both overall ( . ) as well as sepsis-related prognostic evaluation ( . ) were significantly (p< , ) larger compared to sirs ( . and . , resp.), this higher overall accuracy of the ele criterion was primary due to a more valid assessment already on the first and second pop. day, where sirs still had a high false positive classification rate ( % and %, compared to % and %, resp.). conclusion: in the early postoperative course after cardiac surgery, the sirs definition displayed a high false-positive classification rate (low specificity) for subsequent sepsis-related mortality compared to better classification results obtained by the elebute sepsis score. from the departments of medicine i and of "cardiac surgery, grosshadern university hospital, marchioninistr. , d- munich, frg. correlation between physiological and immunological parameters in critically ill septic patients. ma rogy, h oldenburg, r trousdale, s coyle, l moldawer, sf lowry a relationship between physiological parameters of severe sepsis and immunological function has not been established. in an effort to assess such a relationship we prospectively evaluated nine severely ill septic patients. physiological risk was assessed by the apache iii score , while one component of immunologic function was evaluated by peripheral blood mononuclear cells (pbmc) eytokine production after in vitro lps stimulation . four of the nine patients died. apache iii scores at h were lower in survivors (s) than in non-survivors (ns), ( -+ vs -+ p< . ), while apache iii scores at admission were not significant different between s and ns ( -+ vs -+ ). down regulation of cytokine production by pbmc upon lps stimulation was a transient event in s. while s demonstrated an fold increase of tnf-a bioactivity with[r~ hours, ns did not demonstrate any increase at all. a similar pattern was demonstrated for il- [ and il- immunoactivity. tnf was measured by wehi bioactivity, il- [~ and il- immunoactivity were determined by elisa. the sensitivity was pg/ml for tnf, pg/ml for il-ll and pg/ml for il- , respectively. in conclusion, both physiological as well as immunological functions of severe critically ill septic patients demonstrate predictive value for ultimate survival. while patients biological status seems to be more predictable by apache iii at day , p< . , the pattern of cytokine production by pbmc upon lps stimulation over the first h might be a reliable predictor as well. introduction: therapy of sepsis and its sequelae depends largely on its early recognition. many studies have investigated the change of certain mediators during sepsis and their potential to predict multiple organ failure and outcome. it was the objective of this study to investigate whether the onset of sepsis can be predicted by alterations of levels of interleukin- (il- ), tumour-necrosis-factor (tnf), pmn-elastase and c-reactive protein (crp). materials and methods: over a one year period, polytraumatized patients were prospectively studied (mean age y, % male, iss ). serum and edta-plasma samples were taken in h intervalls until the patient left the icu. il- , tnf, elastase, and crp were determined immunologically. sepsis was defined according to the criteria of 'systemic sepsis' (veterans" administration study, ) with at least of clinical signs: ( ) tachycar-dia> /min, ( ) temperature > , °c, ( ) blood pressure < mmhg, ( ) mechanical ventilation, ( ) leukocytosis > . /ml, ( ) thrombocytopenia < . /ml and ( ) presence of an obvious septic focus. clinical parameters, sepsis severity and serum levels were documented on a daily basis, beginning on day after trauma. results: of patients developed a systemic sepsis ( . %), and died. all mediator levels were elevated under septic conditions. the clinical severity of sepsis correlated well with the respective levels of mediators. in patients, who developed a sepsis the following day, il- ( vs. ng/l; p= . ), crp ( vs. mg/l; p= . ) and tnf ( vs. ng/l; p= . ) were significantly increased as compared to those patients who remained non-septic. elastase levels were considerably elevated but did not reach the level of significance. we conclude that il- , tnf and crp appear to be sensitive markers for prediction of septic complications in polytraumatized patients. objectives of the study: the assessment of liver function in polytraumatized patients who are at risk of developing mof is too inaccurate and late by using conventional biochemical parameters. methats: the injury severity of the patients (n= ) was determined by the injury severity score (iss). lidocaine is given at a dose of mg/kgbw over rain. i.v. and is metabolized in the liver by a cytochrome p- mechanism to monoethylglycinexylidide (megx). the metabolite is measured by a fluorescence polarization immunoassay. serial determinations of the test were performed between the ~t and the ~ day after trauma and were compared with other liver function tests (bilimbin, gldh, alt, ast). the systemic inflammatory response syndrome (sirs) is still a challenge concerning early diagnosis, therapy and prognosis. therefore, evaluation of inflammatory and disease activity becomes more important. c-reactive protein (crp) is a well established acute phase protein in chronic inflammatory diseases. recent reports suggest an induction of crp by interteukin- (il- ), a cytokine involved in the mediator cascade of sirs. on the other hand, tumornecmsisfactor alpha (tnfcx) is a very early released mediator in sirs removed very rapidly from circulation. in addition, soluble tnf receptors (stnfr~ , stnfr ) are released into circulation in the acute phase response. this study examines the kinetics of five acute phase proteins (crp, il- , tnfot, stnfr , stnfr ) in patients suffering from sirs. eighteen patients entered the study after diagnosis of sirs. blood samples were drawn every six hours during the first two days and every twelve hours thereafter. crp was measured in an routine turbimetric assay. il- was detected in an biological assay using the/l- dependent -cell line / . detection of tnfc~ was performed in an elisa system using a monoclonal antibody" for tnfo~. soluble tnf receptors were also measured by elisa. crp levels were elevated (> mg/l) in all patients and at all time points. crp values did neither differ significantly in patients with ( ± mg/l) or without ( a: ) multiple organ failure (mof) nor in survivors ( ± ) or non-survivors ( :t: ). in contrast, l- was elevated in patients wilh mof (mean pg/ml, range - pg/ml). il- levels correlated especially with lung dysfunction. tnf(x levels were consistently elevated in patients with mof. crp, il- and tnfoc did not correlate with each other. in contrast, levels for both stnfr showed a positive correlation (r= . ). patients could be divided into two groups by values for stnfr~ and stnfr : the group with higher soluble tnf receptor levels showed increasing values combined with a poor prognosis. the group with lower levels of soluble tnf receptor consisted of patients surviving mof or without mof. in conclusion, crp does not monitor the course of sirs adequately. in contrast, il- correlates with mof and episodes of high disease activity. high stnfr levels may indicate poor prognosis. klinik f r an/isthesiologie and operative intensivmedizin der cau kiel, schwanenweg , kiei, germany. ch. waydhas, md; d. nast-kolb, ivid; m. jochum, phi); l. schweiberer, mi) objective: to evaluate the irfflarranatory response after different types of orthopedic operations and compare them with the systemic effects of accidental trauma of varying severity. patients: in consecutive patients with multiple injuries (iss . ) the inflammatory response to trauma was prospectively studied. the patients were divided into groups according to their iss points. additionally, the alterations after secondary operations (> hr) were determined (msteosynthesis of the femur (n= ), pelvic girdle (n=ll) and spine (n= ), facial reconstruction (n= ), smaller osteosynthesis (n= ) and others (n= )). methods: specific and unspecific parameters of the inflammatory response were determined in the trauma patients every h, beginning on admission of the patient to the emergency room for a period of hr, and in the operative patients on the morning of the operation, at the end of the procedure and every hr during the first two days. results: lactate, neutrophil elastase, heart rate, po /fio -ratio, and other parameters discriminated significantly between the injury severity groups during the first hr (kruskal-wallis-test, p<. ). the degree of postoperative changes differed significantly (kmskal-wallis-test, p<. ) between the types of operations for lactate, heart rate, po /fio -ratio, nitrogen excretion and showed a strong discriminating tendency for neutrophil elastase and c-reactive protein. the extent of changes were highest after operations of the pelvic girdle, followed by procedures on the femur, spine, smaller bones, and the facial region. the postoperative changes after osteosynthesis of the femur or pelvis were comparable to the alterations noticed after smaller (iss to ) or moderate (iss to ) accidental trauma for neutrophil elastuse, heart rate, po /fio -ratio and parameters of the coagulation system. conclusions: there is a considerable inflammatory response to operative procedures that varies with the type of surgery. large operations cause changes in the body homeostasis that resemble those after multiple injuries. it remains to be established whether the inflammatory sequelae of surgical trauma are additive to the changes caused by accidental trauma. objective of the study: we retrospectively compared characteristics of elderly patients (~ years) and yeunger patients admitted to a surgical {sicu) and a medical intensive care unit (micu). we further studied the relations between advancing age, chronic disease, sepsis, organ system failure (osf) and mortality in the elderly group. material and methods: during a -year period, patients were consecutively admitted into the icu; and during a -year period, patients were consecutively admitted to t~mich. criteria for chronic disease, sepsis, osfsi.e. cardiovascular (cf), pulmonary (pf), renal (rf), neurological (nf), haematological (hf), hepatic (lf), and gastrointestinal failure (gf)-were derived from the literature. results: patients from the sicu and~cu were similar in age, number of osf, and length of stay. however, when compared to sicu patients, micu patients had more cf (p<o.o ), sepsis (p<o.o ), and mortality rate ( % vs. ii~, p<o.ool). in contrast, sicu patients had more hp and nf, and prior chronic disease (all, p<o.o ). of the total group of , patients, there were ( %) patients years of age or older. elderly and younger patients had comparable length of stay in the icj, but elderly patients had significantly more chronic disease, sepsis, and number of osf (all, p<o.o ). all osfs, except hf and nf, were more ~o~mon i~ mdeljl, eompard to you-ger patients. ~he mortality is also higher in elderly patients ( % vs. % in younger patients). after multiple logistic regression, only number of osf increased mortality by a factor of . ( % confidence limits, . - . ), age did not increased [odds ratio . (i. i.i )], while admission to the sicu reduced risk of death by a factor of . ( . - . ). conclusions: based on these results, we conclude that age does not have any impact on icu outcome in the elderly. the only prognostic factor is the extent of acute disease, as measured by the number of osf. hence, prevention of oaf may influence outcome in elderly patients with critical illness. the lower risk of death in sicupatients may be explained by the large proportion of postoperative elective patients with relative mild chronic disease, and the lower incidence of sepsis. department of surgery, free university hospital, de boelelaan , ~v amsterdam, the netherlands. septic shock and low output syndrome h.miyakawa, t.noguchi, s.hattori, a. mizutani, m. mori and n.honda objectives: cytckines are thought to cause the acute phase reactions under several critical conditions. we had investigated the relationships between cytokines network which included il- , l- ,acth and cortisol,and the outcome of the patients with septic shock or low output syndrome (los). materials and methods: in the septic group and los group,nine and four patients were enrolled respectively. furthermore, the patients in the septic group were divided into two groups,survival (n= ) and non-survival group (n= ).tn these patients,ll- , l- ,acth and cortisol had been measured every day after diagnosis of septic shock or los.the total number of measurements were points in survival septic group, l l points in non-survival septic group and points in los group.statistical analysis was used student's t-test to compare the mean of each group,and the changes were reported as clinicat significant if p< . . results: il- levels in non-survival septic group were higher than in both survival septic and los group.ll- levels in non-survival septic group were more significant than in both survival and los group.otherwise,there were no differences with respect to acth and cortisol among three groups. conclusions: we assumed that los would make several organs dysfunction as well as septic shock. but our results showed septic shock, especially non-survival,had different cytokins network mechanism for organs failure from los. lasson ,~, g/ ransson j, jijnsson k. eady diagnosis of complications after trauma is imperative to decrease morbidity and mortality. for this purpose an easy, cheap and fast diagnostic method is needed. the aim of this study was to analyse if complications after surgical trauma of various extent could be diagnosed earlier using preoperative or daily postoperative measurements of various plasma proteins than by using climcal signs of complications. material and methods: two acute phase proteins (c-reactive protein and antichymotrypsin), lbur main plasma protease inhibitors (alpha- -maeroglobulin, c estemse inhibitor, antithrombin ill and alpha- -antiplasmin) and one collagen precursor (procollagen iii peptide) were measured preoperatively and then once dally for days postoperatively. for the plasma protease inhibitors immunorcactive as well as functional levels were measured. haemoglbin, albumin and the white blood cell count were also measured. measurements were done preoperatively in patients and continued postoperatively in patients, % of the patients were operated on for malignancy. resulls: preoperative plasma c-reactive protein levels were higher in patients developing postoperative complications. this discrimination increased when plasma was sequentially analysed postoperatively, when a remaining high level ora second increase in plasma levels depicted a complication - days earlier than clinical signs. high levels were seen both in patients developing local as well as in patients developing general complications. there was no clear correlation between plasma protease inhibitory levels and complications, neither pre-nor postoperatively. procollagen iii paptide levels were slightly (but not significantly) higher postoperatively in patients developing complications. contusions: the acute phase response, especially concerning c-reactive protein, predicts postoperative complications better than both plasma protease inhibitors or collagen precursors. preoperative plasma c-reactive protein levels indicate the risk of postoperative complications, while daily postoperative measurements more readily depicts a complication, usually preceeding the clinical signs of complication by - days. dept. of surgery m'alm general hospital, s- i malmr, sweden. mof is a major threat to the extensive burned patients and associated with a high mortality rate. the role of endotoxemia in the pathogenesis of mof in burned patients remains controversial. in this study, we examined the relationship of plasma endotoxin levels to development of mof, and outcome in patients with thermal injury. methods: a prospective cohort study of patients admitted with burns covering more than per cent of body surface area was undertaken. circulating endotoxin concentrations were measured by modified limulus amebocyte lysate assay in serial samples of plasma. results: out of burned patients developed mof according to multiple criteria. the plasma endotoxin concentrations of patients with mof were . -- . eu/ml, significantly higher than that of patients without mof ( . - . eu/ml) on , , and days postburn (p< . -- . ). significantly more incidence of positive endotoxin test (>_ . eu/ml) was found in patients who developed mof as compared to that of non-mof during the observation period (p< . ). as the mean endotoxin levels increased, the prevalence of mof and death also increased (see table below), persistent endotoxemia carried a poor prognosis. conclusions: the present investigation provide further evidence that endotoxemia in severely burned patients commonly occur. cimulating endotoxin has also been found to be strongly associated with development of mof and mortality following major burn injury. multiple hemostatic changes occur in sepsis mad multiple organ failure (mof). to evaluate the role of platelcts in patients with sepsis and mof, we examined changes in surface glyeoproteins on circulating platelets of t patients with suspected sepsis and mof. the severity of sepsis and mof was assessed by eiebute and apache i scoring system, respectively.using flow cytometric techniques and platelets specific monoclonal antibodies, platelet surface expression of fibrinogen receptor on gpiib-iiia, ofvon willebrand receptor gpib, and of granula glycoproteins (thrombospondin, gmp- , and gp ) was measured. receptor density of gpiib-illa mad gpib on circulating platelets was not affected by sepsis or mof. in septic patients surface expression of activated fibrinogen receptor (libs expression) was significantly elevated (p< . ) and correlated well with severity of disease (f . ). no significant change in surface expression ofthrombospondin, gmp- or gp was noted in septic patients. in contrast, degranulation ofgraanle glycoproteins was significantly elevated in mof (! < . ) that correlated well with severity of mof (gmp- , r= . ; thrombospondin, r= . ).we speculate, that platelets in sepsis circulate in a hyperaggregable (fibrinogen receptor activation ) but still reversible state that results in increased risk of microthrombotic events. in the course of the disease, irreversible platelet degranulation might occur and may play an important role in development of mof. abdominal sepsis is still associated with high morbidity and mortality. the present study aimed at evaluating patients with abdominal sepsis treated at our surgical intensive care unit during a -year period with the aim of identifying potential prognostic factors, bacteriological cultures, diagnostic procedures, treatment and outcome. during the period - i patients with abdominal sepsis were treated at the icu at our university hospital. patients were women and men with a mean age of ( - ) years. in cases, the abdominal sepsis occurred as a postoperative complication. the patients were scored according to apache ii and bacteriological cultures and the occurrence of organ failure were noted. the patients were hospitalized in median for (- ) days out of which (- ) in the intensive care unit. out of patients ( %) died in median after ( - ) days. the primary cause of mortality was multiple organ failure ( / ; %). apache ii scoring could not predict a fatal outcome. abdominal bacterial cultures were dominated by bacteria of enteric origin ( %) and in % cultures grew multiple bacteria. patients bad organ failure and multiple organ failure. / patients ( %) had abdominal sepsis due to diffuse peritonitis despite a morphologically intact gastrointestinal tract and the absence of localized abscess formation. mortality in this group was significantly higher as was the percentage of positive blood cultures and the occurrence of multiple organ failure. abdominal sepsis is still associated with a high mortality, predominantly caused by multiple organ failure. abdominal culture findings are dominated by bacteria of enteric origin. in about / of patients with severe abdominal sepsis a diffuse peritonitis with intact gastrointestinal tract without localized abscess formation was found. in this group the mortality was increased as well as the risk of developing multiple organ failure. during the period from january to september patients, mean age + years were referred to our department of resuscitologywith the diagnosis of eclampsia. all the patients were delivered by cesarian section and were mechanically ventilated for . _+ . days. diagnosis of sepsis was confirmed in cases by clinical and microbiological methods. patients were divided in two groups: lnon septic patients, -patients with sepsis, the control group consisted of patients after cesarian section without symptoms of eclampsia or infection. we determined plasma concentrations of immunoglobulins a,g,m(a,g,m), complement factors (c ,c ), alphal-antitrypsin (aat), trausferrin (trf) and albumin (alb) using beckman (usa) analyzer,protein concentration, using kone (finland) analyzer. a(mg/dl) g(mg/dl) m(mg/dl) c (mg/dl) c (mg/dl) k +- + _+ + +- -+ " -+ * _+ " -+ ' _+ " +_ '* -+ ** -+ "* -+ "* _+ " in a prospective study we investigated serum of severly traumatized patients withdrawn directly after admission at our hospital (tr i). follow up controls were taken daily until day ten after trauma (tr ii). two control groups were performed: serum of healthy volunteers (co, n = ) was investigated as. well as serum of patients undergoing elective herniotomy (n= ) hours before (op i) and hours after operation (op ii). serum bactericidal index (sbi) was determined using a hemolytic e.coli strain :k :h . / suspension with a final concentration of - cfu were incubated with l oopl serum. after overnight incubation sbi was calculated according a special formula. results: co . _+ . opi . _+ . opii . _+ . * tri . _+ . "* trii . + . ** (*:p< . ; **:p<o. ) sbi represents a simple and reproducible method to detect immunobiological alterations after trauma and elective surgery. patients undergoing elective surgery showed slight but significant diminuation of sbi h after operation. deterioration of sbi was observed in serum of traumatized patients directly withdrawn after admission at our hospital. ten days after trauma significant improvement of sbi was found. further investigations have to be done to proof sensitivity and prospectivity of this simple but reliable test. a sepsis like syndrome (sls) occurs in i % of our patients following cardiac surgery. sls is characterized by a severe decrease in systemic vascular resistance requiring the use of vasopressors to maintain adequate hemodynamics in the presence of negative blood cultures. in order to determine whether preoperative factors predict the occurrence sls we retrospectively studied patients with sls (group i: age . ~ . years); they were compared to control patients (group : age ± . years). treatment of sls consisted of aggressive fluid replacement and norepinephrine infusion. rydrocortisone ( mg/ hrs) was given on the first day. antibiotics were switched prophylactic to cover gramnegative bacteria in pts. preoperatively pts in group revealed a significantly lower cardiac index ( . ± i/min/m ) compared to group ( . ± . i/min/m ; p < . ) higher left atrial pressure (group i: . ~ . mmhg; group : . ~ . mmhg; p < . ) and lower ejection fraction (group i: . z . %; group . ± . %; p < . ). bypass time, minimum and mean blood pressure on bypass and aortic clamp time were not different between the groups. immediately postoperatively (pop), the white blood count was elevated (group i: , ± , /ui; group : , ~ , /ui; p < . ) and core temperature hours pop was higher (group i: . . °c; group : . ~ . °c; p < . ). serum lactate was already elevated on arrival on the icu pop (group i: , z . mmol/l, group : . ~ . mmol/l) but dropped within hours (p < . ). icu stay (group i: . ! . ; group : . ± . days; p < . ) and mortality over months (group i: . %; group : . %; p < . ) were significant higher in group i. sls is observed more frequently in patients with preoperative cardiac congestion and results in longer icu stay and higher mortality. although intraoperative hemodynamics are similar in both groups, the white blood count and serum lactate levels suggest an intraoperative cause for sls. further investigations will focus on possible intraoperative causes of sls. the objectives of the study were to estimate the problem of stress ulcers in critically ill patients and to compare ranitidine and omeprazole to determine their efficacy in the prophylaxis of stress ulcers. patients who were admitted to the intensive care units between january and july ' with polytrauma, sepsis or those requiring ventilatory support for more than hours were selected for the study. the patients were randomized into groups to receive ranitidine, omeprazole or a placebo. the changes in gastric ph were monitored hourly and the patients underwent an upper gi endoscopy hours after the start of the study to look for stress ulcers and hemorrhage. the drugs were withdrawn one week after the start of the study. of the patients selected for the study, had undergone cardiac surgery, had polytrauma and had sepsis. the change in average pre and post drug gastric ph was + . in the omeprazole group, + . in the ranitidine group and -). in the placebo group (p < . ). endoscopic evidence of stress ulcers was seen in % of patients not on prophylaxis, . % of those on omeprazole and . % of those on ranitidine. while all the patients in the placebo group who had stress ulcers showed endoscopic evidence of hemorrhage, % of the patients with ulcers in the ranitidine group and none of the patients with ulcers in the omeprazole group showed hemorrhage (p < . ). in critically ill patients the incidence of stress ulceration is high. an alkaline gastric ph is protective against stress ulcers. omeprazole when used for prophylaxis against stress ulcers in critically ill patients or those on short term ventilation will reduce the incidence of formation and hemorrhage from stress ulcers. the early and accurate diagnosis of sepsis is of key importance for critically ill patients survival. many studies have shown that tumor necrosis factor ~x (tnf ~) and interleukin- (il- ) are mediators of the inflammatory response in sepsis. bacterial antigens interact also with antigen specific t cell receptors and the activation of t ceils takes place. activated t cells release soluble interteuldn- receptors (sil- r). the aim of this study was to evaluate: the significance oftnf c~, il- and sll- r in the diagnosis of bacterial sepsis and to evaluate the correlation of clinical and laboratory parameters with serum levels of tnf ~x, il- and sll- r. we examined patients with clinical signs of sepsis treated in the intensive care units of university medical centre ljubljana. venous blood was drawn from each patient within hours after the first clinical signs of sepsis. clinical data (body temperature, blood pressure), laboratory dam (peripheral white blood cell count with differential, platelet count, c-reactive protein), and quantitative blood cultures were recorded. serum levels of tnf c~, ,- and sil- r were measured in septic patients and in adult healthy controls. tnf ct, il- and sil- r serum levels were measured by commercially available ria and elisa (amersharn and eurogenetics) kits. the differences in serttm levels of tnf ~, il- and sil- r between healthy control and patients and correlation between clinical and laboratory parameters were calculated. we found that only sll- r serum levels were significantly (p< . , student t test) increased in patients in comparison with healthy controls. of all indicators of sepsis that we compared, only hypotension was significantly correlated with tnf ~x levels and leukocytosis with ] ,- levels. we conclude that only the increased serum sil- r levels were predictive of bacterial sepsis within hours after the first septic signs. tnf c~ and il- levels were not significantly increased at that time in lifts small group of patients. in order to create high precision prognostic system for patients with sepsis and septic shock we have made prospective and retrospective analis of cases of sepsis. patients were included in this analis according bone's criterions of sepsis. comparing with apache-h, we have excluded such phisiologic variables as:temperature,na+ ,k+ ,}it, ph,which had a little influence on prognos of illness. we have added the other, more important variables: biilirubin, plateles,pti. we took into account: the seat arrangement, methods of respiratory support, comorbid conditions as: caneer, diabets,obersity. in general new score has headings: -physiologic variables, -age, -seat arrangement, -chronic comorbid conditions, -methods of respiratory support. roc -analis have showed a greater precision of our score, compared with apache-ii score. there is significant difference between the roc-curve of apache-ii scare. the k.p.tit~v.,i.e.gurmanchuk. objectives of the study. sepsis is a severe complication of the local infection, th e fever and the systemic inflammatory response syndrome may take place in such a case as manifistations of the inflammation induced by bacteria. the systemic fever observed in infection is known to develop due to the action of certain cytoldnes. other systemic manffistation of inflammation are the production of acute phase reactants, acute phase proteins, the activation of the complement system. ivratertals and methods. we observed children with light and children with severe course(l- class of serionuess) of the sepsis. the ftmetional activity of the complement system (ch , level of c -c , factors b told d, c a), level of c-reactive protein (crp} and tumor necrosis factor (tnf) were investigated, results. the level of crp was increased in the children with more severe class of the septic process in both group ( %- st and ioo%- nd}( with light and hard eours of disese) of patients. parameters of the complement system -the activity of cl, c , c components, activity of b and d factors-were increased in the seram of children with light course of sepsis. in children with severe course of sepsis these parameters were signffieantty decreased, especially in the group of chiidrellwith higer level of crp. we found the negative correlation of the tnp-alfa concentration with the functional activity of classical pathway components (cl~ ) and the positive onewith the funfional activity of the alternative pathway factors b-and d of the complement system. conclusions. thus, in children with different course of sepsis certain changes in levels and functional activity of an acute phase o[ inflammation proteins -crp and the complement system ones -&ire characteristic. the relationship between the tnf-alfa level and the activity of classical and alternative pathways proteins indicates on its role in the complement proteins genes expression. schr der j, braun j, rennekampff ha, schr der dw various alterations of the immune response are known in trauma, but the course will not be complicated by multiple organ dysfunction syndrome (mods) in all patients. phenotyping of cells offers a rapid system of evaluation certain aspects of the immune response. different subsets of lymphocytes and neutrophils were determined to evaluate their prognostic role in developement of mods. in trauma patients with an injury severity score (iss) > (mean iss = ; mean age years) lymphocyte and neutrophil phenotypes cd (t-cells), cd (t-helper cells), cd (t-suppressor cells), ratio cd /cd , cd b (receptor for cr ) and cd (fcriii) were measured on day , , , , and post trauma. the expression of class ii histocompatibility antigen (hladr) on monocytes (hladr+ cd ) and il -receptors on t-helper cells (cd /cd were determined as well. the percentage of cells was monitored by immunofluorescence using monoclonal antibodies and three color cytometry. the percentage of hladr+ cd were significantly lower an day , , and in patients who developed mods (p< , ) compared to patients without mods and a healthy control (p<o,o ). the expression of il receptors on cd was significantly different only on day . no differences could be measured in lymphocyte phenotypes cd , , and cd /cd -ratio as well as in expression of cd b and cd on neutrophils. class ii histocompatibility antigen (hladr) on monocytes offers the best ability to reflect cellular immunosuppression in trauma. this parameter allows the best differentiation of patients with and without mods. we retrospectively studied the relative importance of age, prior chronic disease, sepsis and organ system failure (osf) to determine outcome in critically ill patients with acute renal failure (arf). arf was defined as serum creatinine > /zmol/i, a twofold creatinine rise in prior renal insufficiency or the need of acute renal replacement therapy. definitions for prior chronic disease and other osfs -i.e. cardiovascular (cf), pulmonary (pf), neurological (nf), haematological (hf), hepatic (lf), and gastrointestinal failure (gf)-were derived from the literature and described previously. of the consecutively admitted patients to a surgical and a medical intensive care unit during -ye r period, ( %) had arf. arf mortality was %. ninety-eight percent had other osf. overall, cf, pf, gf, and nf was significantly more common in nonsurvivors than in survivors (all, p<o, ). although both the number of osf before and after arf was higher in nonsurvivors than in survivors (p< . ), the number of osf before was higher compared to after arf in both groups of patients (p< . ). furthermore, age, cf and pf before the ontset of arf, nf thereafter, and renal replacement therapy were related to mortality (all, p< . ). however, prior chronic disease was not related to mortality and chronic renal insufficiency was inversely related to outcome (p< . ). after multiple logistic regression, advancing age, cardiopulrnonary failure and sepsis before arf, and nf after arf remained significant. these results show the high prevalence of arf in critically ill patients and that arf rarely occurs alone. the prognosis of arf in critically ill patients is poor, especially if associated with advancing age, additional osf, particularly cardiopulmonary failure and sepsis before arf, and nf after arf. hence, prevention of cardiopulmonary and sepsis before the outset of arf may influence outcome in arf in patients with critical illness. the search cominnes for a magic bullet ta help critically ill petienta, bat recent elini ',.ml rials of agents that showed ixomise in animal studies raise questions about such trials. expcrlmemai uvidan~ :in anlmats and human volanteet~ for concepts, mechanisms and treatment of inju~ ur illness can be substentlal, l~rauasive and exciting but the positive effects of potential therapy suggested by such animal and ctinieal research may be difficult to prove in sick patients. efficacy of a new txeatment can be difficult to prove became elinie.al situations, hi spi~e of important biologic phenomena, are v~iable and conrplex. there is redundancy and overlap of mediators ~d problems of timing of therapy. a majt~r cause of this dilemtns is not with the trials or hypotheses, but rather the promem of the cause or the causability of the human dise~.se that is trebled. this is/hu "causa nets" or '*specific cause" as described by stehbens, when prevention or tre.atmcnt is based em the sauna nero, extinetiem of the disease is ix~ssible as with saul/ pox. only elimination of the cause wl cure the disease. careful animal studit~s evaluate single pcrturbmiuns for survival or other changes. an ldso preparation may be infiuancad aignificantly by a ~mall clangs which may be insignific~lt ht pstiants, most human experiments ate carried out in normal volunleers with a single porlurbation, such as a low-level, non-lethal dose of a substance. such studies are huportant in defining mechanisms attd mediators of disease, in our world of injm'ed, operated, infected and inflslned patients, there are many diseases, mof is not one of lhem. it isn't even a syndrume, it is the final pathway for a number of diseaaes~ each of which has a cause. mecormock believes thal a "multi-causal" etiology is a euphemism for ignoranc# or a synonym fo~ unknown etiology. admission iv sn icu, a high apache seer% the sepsis syndrome, mof, muds, or sirs and having predictors indicating potential mortality, are not diseases. the inmpors are getting negative results in trials of agents on eiek or septic icu patients and aru now becoming splitters, dafining subgaoups at higher risk for death. such an approach may produce positive results if the diseases are identified and the treatmeul is specific for them, a major challenge in deterraintng the efficacy of new therapies for sepsis nnd shock is identifying patients whose e#temtc inflammatory response is appropriate from those in whom the mediator| are contributing to end organ system damage end death, traditional and recently revised categorical patient descriptions such as sepsis syndrome, sepsis syndrome with shock, multiple organ system failure, or the recently proposed systemic inflammatory response syndrome (sirs) may not be sufilcient since they identify individuals at varying levels of risk for short term mortality, the efficacy of new lmmunotherapy may be directly related to the patient's severity end risk of mortality at entry to the study. we recently reported (jama ; : z - ~ ) a comprehensive risk assessment approach for patients with sepsis syndrome, a common eategarlcal description used as an entry eriterlon for many trials. by screening s database of , recent admissions to hospitals in the united states and western europe, we identified , patients who met sepsis syndrome criteria but were not enrolled tn clinical trinh, we then developed a survival model end severlty assessment method to estimate their g.day mortality risk. k..ente physiologic abnormalities were the most important prognostic factors ( % of total chl square) influencing outcome. the specific disease resulting in ice admission and the days the patient spent in the hospital and icu before qualification were independent risks, as were age and a clinical history of cirrhosis. the predictive model was cross-validated within the original , patients with a receiver d_perator characteristics (roc) area of . and in two independent databases, the first independent database contained hospitalized patients with gram-negative infection meeting criteria for sepsis syndrome (roc= . ); in the second, the placebo patients withtn the recently completed phase ill e~aluatlan of il- ra (rocffi . ). in all databases the risk model was able to accurately risk stratify patients throughout the range of risks that varled from a very low % to a very hlglt %. by drawing on the ready avatlabillty of large clinically aeuurate, current databases and using the power of modern statistical techniques to model the bodfs response to sepsis, we are able to produce very aeettrate pre-treatment risk estimates, these prospectively defined and continuous risk estimates reduce reliance on multiple subgroups and data dredging that can compromise a stady's integrity. they cart provide a nnlform method for patient description across clinical trials of different attempts at immmlotherapy. risk calculations can also be useful in developing entry criteria for phase ii evaluations in order to initially test efficacy on a limited number of high risk patients, fonowing sueeessinl completion of a definitive trial, the risk estimates can be used to develop specific |ndlcattons for s drug's use and can monitor the impact of both new and multiple compounds on patient outrome, when dealing with therapeutic trials for the treatment of sepsis, there are ways of checking the comparability of the groups (placebo and treated groups) -to use several description items such as age, sex, preexisting disease, number and nature of organ failures, physiology score, such as saps il ( ) -to use a model for risk of death either from a score (apache ii, lii, ( ) saps ii) or directly (mpm ii system ( )) before using the model for risk of death in septic patients, is it mandatory to validate it (by calibration, i.e. goodness of fit, and discrin'dnation, i.e., roc curves) first on a data base using the same definition for sepsis as in the trial and secondly on the placebo group of the trial ? or is it better to use a specific model for each trial ? the general models usually do uot fit for septic patients. there are methods to make them fit : either to add variables to the original score (model for sepsis using the apache iii system ( )) or to customize the model. for saps ii the customization is applied to the equation regression, using the same saps ii score. for mpm i each component of the model can be customized. several remarks : only one model is published to be applied at the icu entry ( mpm ii ) before any therapy. -most of the published scores are calculated from the lsl icu day. applying these scores to the let day of sepsis could have a very different significance. -is the accp classification of sepsis useful at the bedside to select oatients ? ( the pathophysiology of sepsis involves a dynamic interaction between the host and the infecting microorganism(s). a multitude of biochemical and hemodynamic interactions occur which function in concert to determine the ultimate outcome of the septic episode. the complexities of the septic process preclude outcome analysis by in vitro systems or computer models, animal studies have become indispensable in evaluating new therapeutic agents in the treatment of sepsis. these studies provide valuable information on safety, pharmacokinetics, relative efficacy and dosing strategies for potential therapeutic agents in the treatment of sepsis. there are three basic types of animal models: ( ) toxicity models with injection of bacterial endotoxin or exotoxins; ( ) live or killed bacterial challenge models; ( ) actual infection models. all models employ some form of external manipulation under controlled conditions to induce sepsis and to analyze potential therapeutic efficacy. these experimental requirements may limit the ability of animal models to accurately predict the clinical value of new agents in human sepsis. four major end points are analyzed in the various animal models: ( ) hemodynamic parameters; ( ) modulation of host-derived inflammatory mediators; ( ) resolution of end organ injury; or ( ) lethality. each animal model has certain advantages and limitations. species-specific differences in physiologic and immunologic features make it difficult to directly extrapolate the results of animal experiments into clinical medicine. while no ideal animal model exists, consistent benefit of a new immunotherapeutic agent in multiple animal systems provides the most compelling preclinical evidence of its therapeutic potential in septic patients. consensus-assisted development of a study protocol on sepsis: an important difference from previous randomized trials there have been several, and perhaps too many metaanalyses of cliuical trials on surgical infections, but their results have only rarely been incorporated into the design of new randomized trials in sepsis. metaanalysis is retrospective. it seems more important to analyse and to criticize the design of tria/s before they are ongoing. incorporation of that into development of a study protocol is the aim of the following procedure: the time-schedule of about two years should include further cell culture and animal experiments after the first successful studies showing effectiveness. exhaustive evidence for a dose-dependent cost-banefit and near complete explanations of the pathomechanism should not be awaited for development of the protocol of the clinical study. however, a discussion forum of experts should be performed in a recently published, almost perfect trial in order to see the frontiers of the present research in cell and molecular biology, clinical pharmacology, statistics and decision making. this should be followed by a metaanalysis of at least study designs on sepsis with methods of formalized medical decision making (decision tree). clinical algorithms -developed by objective methods including nominal group processes -should be developed to standardize any concomitant treatment in the centers considered for a multinentre trial. the latter is usually necessary in the field of sepsis. finally, a pilot study should be performed to demonstrate not only feasability, but to learn about all possible types of interventions which modify endpoints as response variables. especially mortality is not free from such effects. the time for the individual phases of this consensus-assisted process of protocol development have to be shortened by their temporal hiterloeldug. due to the fast discovery of new chemical factors in sirs it wilt otherwise be impossible to come up with results of randomized trials which are fascinating enough to be incorporated in daily routine treatment. severity scoring systems are intended to provide a population-based estimate for the risk of an unwanted outcome resulting from some disease process. in the area of infections and the "septic" response, this has routinely been defined as death. for anti-infective trials, there has been a reasonable correlation of severity (apache ll) with outcome measured as persistent or recurrent infection, but this is not as robust as the association with mortality. it is expected that non-lethal failure would not immediately correlate with disturbed physiology: the basis for antiinfective failure depends on a variety of factors not measured in severity scoring, such as type of infecting organisms, density and susceptibility to administered antimicrobia[s, and anatomic features of infection (e.g., pancreatic). severity scoring is important in differentiating these "categorical" variables from continuous (physiologic) variables. there are, additionally, a series of problems related to the cause of death in relation to severity scoring. in those tdals in which cause of death is analyzed, high severity scores did not routinely predict death as an immediate sequelae of sepsis/septic shock. many of the predicted deaths occurred remotely of progressive background diseases. other important problems with severity scoring have to do with lead-time bias: hew long patients were ill before entry into the study. statistical techniques for condensing various categorical and continuous variables, as well as factoring in information requiring the pharmacodynamics of agents which affect outcome, are being developed to further refine deviation from predictive equations as an outcome measure. stepwise logistic regression analysis has identified the presence of shock, intm-abdominal or unknown source of infection, hospital acquired infection, age greater than years, neutropenia and inappropriate antimicmbial therapy as independent variables associated with increased mortality in sepsis. maldistribution or" these variables is a concern in targe randomized clinical studies. it should be noted that only the selection of antimicrobiai therapy could be addre.~'sed and altered at the time of enrollment in a sepsis clinics] trial. inappropriate antimicrobial therapy has been noted in %- % of septic patients. well designed, randomized sepsis trials do not assure the absence of imbalance between treatment and control groups with regard to inappropriate antibiotic therapy, especially with subgroup analysis. statistical manipulation to correct for any imbalance is appropriate but avoiding imbalance is ideal. selection of antibiotics in septic patients requires such considerations as clinical setting (history, physical examination and underlying disease), appropriate laboratory tests (such as gram stain), identification of source and associated bacterial flora, antibiotic susceptibility patterns for that hospital and likelihood of resistant organisms. determination of inappropriate antibiotic therapy is made retrospectively after culture results are available. in some circunmtances the organism and antibiotic sensitivities cannot be predicted, while in others it can. leibovici et al identified hospital acquired infection, antibiotic treatment before a bacteremic episode, endotracheal intubetion and thermal trauma as independent variables which predicted isolation of a multiresistant organism. the same authors developed a predictive index for resistant organisms, which when compared to prescriptions of attending physicians, improved antibiotic selection in critically ill patients. including a standard antibiotic regimen for all septic patients in a clinical trial is impractical. there is too much interhopsital variability in flora and antibiotic susceptibility. in addition, the aggressive broad spectrum coverage necessary for some patients is inappropriate for others. a protocol which guides a prescribing physician through a logical chain of reasoning might improve antibiotic selection in critically ill patients and could be included in the design of a clinical trial in sepsis. although this approach might minimize potential for maldistribution of inappropriate antimierobial therapy, it would likely create multiple problem areas. will the patient's physicians agree to the protocol choice? what is the availability of speciftc antibiotics at a participating hospital? what is the aplpiieability of study results in typical clinical situations? if there is agreement that thin is an appropriate antibiotic protocol, should it not be used in all seriously ill septic patients in the hospital? in other conditions such as ards, the success of protocol therapy is dependent upon measurable goals such as pao and highest plateau pressure, while measurable goals are not as clear in choosing antibiotics. based on the above confounding issues, a complex and extensive algorithm covering many potential possibilities of error and specific antibiotics seems impractical; however, an algorithm focusing on three to four major common errors in antibiotic selection and dealing with classes of antibiotics is plausible for inclusion in sepsis clinical trials. in this session we will attempt to outline the methodology of such a future study, emphasizing the immense difficulties involved in its proper conduction including: design, selection of patients, surgical considerations, supportive treatment, the "human factor" and ~nd points. only a close cooperation between a few dedicated surgeons, obsessively studying a large number of well selected and stratified patients over years, could make such a study possible. algorithmis have frequently been mistaken for the graphic format in which they are presented. however, an algorithm is neither a flow chart nor a list of instructions; it is a step-by-step approach to solving a problem. likewise, a clinical algorithm is a step-by-step approach to solving a clinical problem. cas are deterministic, which does not mean that they are rigid. they are practical rather than mathematical algorithms, that tan be used only with clinical judgment, and must be tailored to each patient. there are a number of types of, or uses for, cas that can improve sepsis management, including: diagnostic or severity scorm cas, a management ca for managing sepsis constructed according to recently published standards for use in teaching research protocol algorithms that must be used to collect data or guide implementation of a clinical trial aimed at validating one or more dicisions in the management ca; finally, protocol-style cas drawn from the management ca and should be validated using matching outcome studies. organisational problems peculiar to multicentre trials the organisation of any randomised clinical trial is fraught with difficulties, and that of a multicentre trial particularly so. there are problems associated not only with the principles on which the investigation is based but also with the detailed organisation and analysis. are all the participants truly unbiased about the relative merits of the regimens being studied? is like being compared with like -are all the end points and the standards for measuring them clearly defined so that they cannot be misunderstood? are all eligible patients being studied, and their results reported? is truly informed consent being sought that will satisfy not only the patient's right to choose what happens to him, but also the law of the land? and, finally, how can the participants be sure that the trial will be stopped at the right time, to ensure that no treatment is given to any patient that might be harmful? these questions may seem insoluble, but until we tackle them we shall never be certain. dr.m. ev~s, scar~mu~ hospital, scarborough, nonhyo~s~ y ql, u.k. increasing knowledge of the pathogenesis of sepsis and septic shock has led to the development of many new therapies and technologies capable of preventing, blocking or even reversing the deleterious cycle of events. recent results of subsequent multicenter trials testing some novel therapeutic drugs, however, did not confirm the promising sometimes overwhelming effects obtained experimentally. it is postulated that this is largely due to inappropriate design and conduct of multicenter trials as currently performed. one of the shortcomings emphasized in this paper is the "treatment mix" problem. multicenter trials in sepsis are performed because no single center is able to recruit the necessary number of eligible subjects within a reasonable time. each single center (icu) has its own individual policy which cannot be standardized for the trial (treatment mix). even if we ascribe that each icu did the "best" for their patients, it is highly probable that the outcome differs between the icus. this translates to the effect of the new therapy under investigation. ]t is therefore worthwhile to consider that icus must first demonstrate a certain standard based on the patients' outcome (audit) before becoming accepted as a participating center. this prerequisite ensures comparable quality between participating centers, led to the reduction of "noise level" and increases the probability of positive study results. the riyadh-icu program based on standard mortality ratios is recommended as an audit instrument. during the last decade most clinical trials of potential therapeutic agents in systemic sepsis have failed to demonstrate benefit from the drug under study. although in many instances it is entirely possible that the agent in question does not have clinical efficacy, an equally plausible explanation for the multitude of negative results is that the studies were improperly designed and thus could not show therapeutic benefit, if it existed. problems which have been identified in these various trials include inappropriate or unrealistic definitions of response to the drug; inadequate sample size, with attendant insufficient statistical power to demonstrate a difference, if it existed; insufficient standardization of customary therapeutic maneuvers in septic patients; imprecise criteria for patient entry into the study, which creates unacceptable inhomogeneity between control and treatment groups and invites unjustified post-study subgroup analysis; the lack of a formal sequential monitoring procedure for interim data analysis, which could potentially affect patient safety; and a primary analysis of study results that excludes protocol deviants and is not according to intent-to-treat. these and other problems have impacted upon the validity of the various trials conducted to date and may well have prevented the resolution of controversies surrounding the use of certain agents in the treatment of septic patients. the complexity of bacterially-induced septic shock involves a cascade of events that evolve over time, beginning with the proliferation of offending organisms and followed by the release of toxic mediators from the bacteria. endotoxins [e.g. lipopolysaccharides) from gram-negative bacteria initiate septic shock by precipitating a systemic inflammatory response syndrome (sirs) through release of a broad spectrum of hostderived mediators. over the last century, hundreds of these endogenous mediators have been proposed to serve as pathophysiological substances in this "alphabet soup" of cytokines, eieosanoids, biogenic amines, kinins, peptides, ions and hormones. an evaluation of the literature on septic shock leaves the investigator with a sense that, although many pieces of the septic shock puzzle have been presented, no clue was provided to indicate how these pieces fit together, in an attempt to assess objectively the causal role of individual mediators, we recently completed a collective meta-analysis of mediators that were individually reviewed and subjected to koch-dale analysis of causality by distinguished authorities in the field ( ). in summary, our attempt analyze available evidence to support a cause-and-effect relationship for putative mediators of septic shock revealed that only eight of the mediators analyzed could be strongly inferred as playing a causal role in septic shock. evidence supported the involvement of endotoxln, endorphins, complement, interleukins, tumor necrosis factor, eieosanoids, platelet activating factor and calcium. other mediators either lacked adequate supportive evidence or were not conclusively involved. posttraumatic outcome may be reduced by an amplified stress response mediated by neural and humoral stimuli. the classical hormonal catabolic response is predominantly mediated directly or indirectly by neural stimuli, while most changes in inflammatory responses such as leukocytosis, acute phase proteins and changes immunofunction are mediated by humoral mediators. clinical experience from controlled studies suggest afferent neural blockade and modification of stress response to improve organ function and postoperative outcome, and limited experimental studies also suggest neural blockade to be of beneficial value in shock states. of special value may be the improved gastro-intestinal motility after neural (visceral) blockade. no outcome studies are available from patients with major truma, multiple organ failure or sepsis, conditions where humorai mediators may be more important to modify than neurally mediated responses. future studies should investigate the clinical outcome effect of combined neural and humoral mediated blockade, as well as the potential role of an initial neural blockade in elective trauma (first hit) to improve the metabolic scene and outcome if a postoperative complication (second hit) should occur. neutrophil elastase is the predominant protease of the human neutrophil. this enzyme, and a variety of other enzymes, including neutrophil collagenase, are released by activated neutrophils in the setting of high concentrations of reactive oxygen metabolites. in addition, in this extracellular environment, the normal antiprotease defense mechanisms of the organism may have been severely inactivated oxidatively especially along capillary endothelial cells. accordingly, release of neutrophil elastase which mediates inflammation and degrades most extracellular matrix proteins, including elastin, fibronectin and many forms of collagen, may contribute to tissue injury and organ failure in a variety of diseases ranging in severity and acuity from pulmonary emphysema to the adult respiratory distress syndrome (ards). as a result, significant efforts by the pharmaceutical industry have been directed not only toward the development of human neutrophil elastase inhibitors, but also toward their characterization in a variety of animal models of neutrophil mediated diseases, and their evaluation as potential therapeutics for the treatment of a variety of human clinical conditions. data from animal models of organ injury and failure along with data collected from a series of patients at risk for ards and with ards will be presented and discussed as a rationale for inhibition of neutrophil elastase. parameters that need to be monitored to obtain success in future clinical studies will also be presented in this context. have turned out to initiate and maintain organ dysfunctions in severe posttraumatic and postoperative courses. such proteolysis-induced disorders are especially rendered possible by the concurrently arising consumption of inhibitory regulators (e.g. a vproteinase inhibitor=a pi, antithrombin iii=at iii) via cofnplex formation and/of proteolytic/oxidative inactivation. besides the well-known destructive potency against protein substrates, proteinases such as elastase or thrombin (active or in complex with the serpin inhibitors ~ pi or at iii) are also supposed to increase the inflammatory reaction by inducing cytokine release or shedding of soiuble adhesion molecules from various inflammatory cells (pmns, monocytes/macrophages, endothelial cells). those cell-derived mediators may provoke further cell activation through an autocrine/paracrine loop thus increasing significantly the proteinase burden at the inflammatory focus. therefore, this noxious circle might be interrupted by a convenient proteinase inhibitor therapy to ameliorate the inflammatory process. to verify this hypothesis, high dosis of at iii (mean plasma levels up to %) were applied in first controlled randomized studies on surgical patients suffering from multiple trauma or severe sepsis. in control patients we could clearly demonstrate the sequential appearance of proteinase/serpin-complexes (fa pi, tat), cytokines (il- , il- ), and soluble intercellular adh sion molecules (icam, elan, p-selectin) in the circulation. these factors turned out to be a helpful tool for early diagnosis and prognosis of forthcoming organ dysfunctions. interestingly, m at iii-treated patients a significantly reduced release/production of inflammatory mediators became obvious concomitant with the improved clinical course in comparision to an increasing deterioration in control patients. soluble mediators of inflammation are cytokines (e.g. il- , il- and tnfe) as well as breakdown products of complement proteins (e.g. c a and terminal complement complex). therefore, attenuation of both, release and generation of these synergistically functioning mediators together with stimulation of release of antagonists including soluble forms of receptors are potentially beneficial in all kind of inflammatory diseases. intravenous immunoglobulins (ivigs) are known to have an anti-inflammatory effect in humans (nih consensus conference on wig, ) . the mechanism of action becomes more and more clear. in single cells stimulated by anti-cd mab wig was able to down-regulate production of il- , il- , ifn% and tnfj~ significantly. igg coated solid-phase stimulates release from monocytes of interleukin- receptor antagonist (il-lra) but not of il- ; this observation is very much in contrast to the effect of endotoxin in the same in vitro system. furthermore, naturally occurring anti-cytokine antibodies can be demonstrated in apparently healthy individuals and in wig prepared from plasma pools. some of these antibodies can be detected by antigen/antibody reactions in fluid phase (anti-ll-le, anti-il- ), some only by solid-phase detection systems (anti-ll- , anti-ifn% anti-tnfc . infusion of wig to patients suffering from inflammatory disorders have resulted in a decrease in il- in circulation. during infusion wig might induce an acute but transient rise of tnfo~, il- , and il- . self limitiation of this process might be due to demonstrable instant release of il- ra and soluble tnfo~ receptors. in vitro ivig has also been shown to attenuate complement activation via the classical pathway. furthermore, acid haemolysis of erythrocytes in paroxysmal nocturnal haemoglobinuria could partially be prevented by ivig. ivig was able to attenuate forssman shock in guinea pigs. we have seen a patient with congenitally elevated turnover of alternative pathway of complement who repeatedly showed an increase of haemolytic titres after administration of ivig. thus, wigs apparently have multiple potencies including attenuation of soluble mediators of inflammation and might therefore be of benefit in trauma, shock, and sepsis. significance of sinusoidal liuing cells and of humoral factors on hepatic function following sepsis and major surgery hirata k, katsuramaki t, yamaguchi h, mukaiya m, yamashiro k. regeneration of the liver after hepatic necrosis or partial removal has been extensively studied, but uncontrolled mechanisms to irreversible hepatic failure following sepsis or major surgery of liver or with hepatic dysfunction have so far not been well documented. we suggested the importance of the intrasinusoidal aggregation between sinusoidal lining cells and intrasinusoidal running ceils in the mechanisms of hepatocyte dysfunction. thus, the purposes of this study was to investigate the changes in each sinusoidal lining cell during this process and the effect of cytokine on hepatocyte under various oxygenation. [ materials and methods ] ( ) clinical study : thirty two patients with histologically proved malignant tumor underwent major hepatectomy were devided in two groups, i_e. the cirrhotic liver group and the non-cirrhotic [aver group. most of the former were the patients with hepatocellular carcinoma and most of the latter were the patients with metastatic carcinoma of colorectal cancer. five patients were complicated septic conditions with hepatic failure. following operation in each patient, a verous blood sample was taken for measurement of serum il- , il- , tnf and c-reactive protein concentration. and hepatic biopsies were sometimes undergone in patients with anomalous hepatic function. ( ) experimental study : kupffer ceils, sinsoidal endothelial cells and hepatocytes were separated from mouse ( c hhen ) liver. and also polymorphonuclear cells and platelets were purified from blood and peritoneal macrophages were from peritoneal cavity of mouse. co-cultures between or among these cells with iipopolysaccharide were performed. cytokine concentrations in media and hepatocytic function were compared. [ results and conclusion ] our findings in above experiments demonstrated the cleanly different concept for the mechanism of hepatocyte dysfunction following sepsis or major surgery. namely, the effect of hepatoeytie function by cytokines or superioxide were significantly affected under low oxygenation, but not significant under good oxygenation. hirata m.d.,nishi ,hokkaido,japan $ immune complexes as .mediators of sepsis and immune dyafumcffon laa k horn, chxistof birkenmaier, and greg h mon departmen~ of surgery, san frandsco general hospital, urdversr , of calif(~rrda, san frandsco. immune complexes (ic) a;:e commonly found circulating in parians during infection and sepsis; and following traumm, bums, and massive l~ensfusion. clearance of c occurs by phago~'tmsis of esll-bou_nd or opsonized p~tlcles. monocyte activation du~jng phmgocytos/s could lead ~o release of cytokilies ieletexiot~ to the hosh to examine tb/s phenomenon, we formed insoluble ic by precipitating iow-endotoxin bovine serun~ albumin (bsa) with rabbit ant/-bsa igg. we have ~town that these ic are ingested by monocytes and concomitantly stimv/ate re~pizatory bu~t activity measuxed by assay of in%racellul~ peroxldaffon. we have shown that ic ingesffon produces signlqcant el,era,lore in the monoey~e phenotype. spedacally, cdi is down-regu/ated, along wl~ cd and cd . thus responsiveness to lipopolysacchmdde ( ) a~xd i=m~unoglobulin fe st~mu/mtlon is reduced. in cert,+rest, the complement rote.p tot cdc is u~-regulamd, indicaling mcremsed re~ponalx=~ness to opsonized pat,rotes, we examined monocyte superna~n~s for production of hzmor necrosis factor alpha (tnfg) following ic stimulation and showed that ic are capable of provoking ~elaase of hrmaunl)reac~ve tni~. j[c also increase mono=yte produchon of prnstaglandin e . in summary, ~/~ese results demonstrate lhmt ic are capable of indudng production of ¢ytokines and the imrau-nosuppressive prostaglandin pge . ic also allez ~he surface expression of important receptors involved in actlvaffon by lp~ and phagocyiosis. thus, ic aze competent for indud/on of the mepl/e s! otlse. by examining m n cyte phenotypms, it may be possible to d~mrmkne extent ohn'u~u~e complex activation and predict immune depression iu criffcally ill pa~ents. a great deal of experimental work has focused on preventing and/or treating sepsis and organ failure following injury. many of these strategies have tried to attack mediator substances released during the systemic inflammatory response following injury. sugermann has demonstrated the improvement of pulmonary function, but not eappillary leak using [buprofen in the porcine model. in a clinical trial, faist demonstrated the effectiveness of indomethacine in amelierating the post trauma of cellular amenity using in vitro parameters. morbidity and mortality, however, were not different between control and treatment groups. baue und chaudry have suggested that atp magnesium chloride may have protective effect in renal failure um kupfer cell dysfunction seen after shock. pentoxiphyllin has been found to be effective in improving tissue oxygenation and oxygen consumption following hemorrhage, and there may be promise for this drug in the ischemia reperfusion injury. monoclonal antibodies against endothxin and inflammatory mediators seemed promising at first glance; however, preliminiary clinical data of the treatment of sepsis with either ha-ia or e monoelonoal antibodies against bacterial cell wall have been disappointing. initial studies with il-i receptor antagonist also appeared encouraging, but no difference was seen in the most recent trial. currently studies are ongoing with monoclonal antibodies to tumor necrosis factor which may have more efficacy given the fact that it is a macrophage mediator released by endotoxin. although we seem closer to understanding and preventing the problems of sepsis and organg failure after trauma, we must continue to appreciate the complex interrelationships and delicate balances inherent in the host defense system. overzealous attempts at restoring immunity in the absence of understanding pathophysiology may be just as dangerous at post-traumatic immlmospremsion. severe acute pancreatitis was caused by different etiologic factors, but once pancreatic tissues were infected, patients show a similar pattern of aggravation and develop organ failure. remarkable elevation of serum il- was unexceptionally observed in patients with severe acute pancreatitis. the serum levels were more than pg/ml (normal: less than pg/ml) during the first one or two days after onset. there was a significant correlation between the peak serum il- level and the number of organs showing failure (r= . ). tnf-a production by isolated peritoneal macrophages following lipopolysaccharide (lps) stimulation was significantly increased in cerulein-induced pancreatitis rats. serum tnf-a activity was elevated following intraperitoneal administration of lps as the septic challenge both in pancreatitis rats and in control rats, being significantly higher in the former (p< . ). histological findings and liver function tests revealed that lps induced more severe liver damage in pancreatitis rats than in control rats. these results indicate that increased tnf-a production by peritoneal macrophages in acute pancreatitis augmented lps-induced liver injury. organ failure in severe acute pancreatitis could be caused, at least in part, by increased cytokine production. it is clear now, that in mods, organ injury is not directly due to exogenous factors, such as bacteria or toxins, but instead is largely a consequence of the host's own endogenously produced mediators. there is an extensive body of literature on the inhibition of mediator production, release and action in different cascade systems by glucocorticoids. they can serve as a host protection against overactive defense reactions if used adequately. a new understanding of the regulation of cytokine synthesis, especially tnf, may lead to an insight of the conflicting findings between the benefits of glucocorticoid therapy in animals and its current failure in recent clinical trials. glucocorticoid shares its role with other novel therapeutic approaches also shown to be successful only in experimental settings. a mete-analysis of steroids in sepsis, however, revealed a beneficial effect in the subgroup of patients with gram-negative infections. there is now a series of arguments to reevaluate the indication for steroid thera-py and/or prophylaxis in mods and sepsis. honjo i- - , kumamoto , japan in septic shock j. briegel, m. halter, h. forst, w. kellermaun, m. bittl, k. peter imrodnetlea and methods: hydrocortisone (hc) at an infusion rate similar to the maximal secretory rate of cortisol in man improves hemodynamics in human septic shock ( ). we hypothesized that the hc-induced hypercortisolemia prevents a harmful overshoot of immune reactions. to test this hypothesis we prospectively investigated patients admitted to the icu in refractory septic shock. after resuscitation (mechanical ventilation, fluid resuscitation, titration of vasopressors, and initiation of antimicrobial therapy) hc was started with a loading dose of mg/ rain, followed by a continuous infusion ( mggh). with hemodynamic improvement (dopamine < gg/kg/min) the infusion rate nfhc was tapered over a period of days. phospbolipase a (pla ), c-reactive protein (crp), and elastase-proteinase inhibitor complex (e-pi) were deierminated daily. results: according to our previous results hemodynamics improved during hc infusien. after days dopamine requirements decreased to less than gg&g/min in all patients. this corresponded to an attenuation of the systemic inflammatory response syndrome (sirs) indicated by the abrogation of fever and the decrease in heart rate and inflammatory mediators. pla activity and e-pi concentrations decreased to near normal values and crp concentrations decreased as well. after cessation of hc infusion (between day and ) a reinforcement nfthe sirs was observed despite cortisol levels within the normal range. this was first indicated by e-pi, followed by fever, increases in heart rate, crp, pla , and finally by the relapse of septic shock in four patients on days , , and o. a second infusion of hc again resulted in shock reversal and in a decrease in pla , crp, and e-pi in the patients under study. discussion: there is growing evidence that the endogenous steroid hc and proinflammatory cytokines integrate a complex immunoregulatory feec~ack circuit. the elaboration of tnf, il- , il- , and il- is attenuated by- rag hc prior to endotoxin challenge in humans ( , ) . cytokines like tnf, il- , il- , and il- are potent proinflammatory signals for acute reactants (--, clip), neutrophil degranulation (--, e-pi), and pla synthesis and secretion. our data provide evidence that hc at an infusion rate similar to the maximal secretory rate of cortisol in man attenuates the systemic inflammatory response in human septic shock. to evaluate the feasibility of a nigh-dose regimen of antithrombin iii (at iii) treatment in patients with multiple injuries regarding blood levels of antithrombin iii and undesired side effcts and to analyse effects of the initibitortherapy on the systemic inflammatory response. patients and methods: patients with an iss nf more than points who could be treated with at iii administration within rain after trauma were randomized to receive either at iii or placebo. at iii was given to reach an antithrombin iii inhibitory capacity in plasma of % by using the following calculation: at iii to administer = ( % -at iii measured) x body weight. at iii was given every six hours for a period nf hours and on the th day. patients were followed up throughout their stay in the icu but at least days. in this abstract the data of the first patients are included. results: the patients' median injury severity score was points with a median age of years. the patients were equally distributed between verum and placebo groups with respact to age , iss, prehospital treatment, blood pressure, hemoglobin and at nmevel on admission, and time from the accident until the test solution was given. the patients of the at iii group received , units of the inhibitor on average during the first days. with our regimen, the at iii levels could be kept between % and % of normal, whereas control patients an at hi concentration of % to % was observed. patients with at ni treatment required less red blood cells bur received the same amount of ffp and fluids. there were no differences with respect to platelet count, partial thromboplastin time, l~rothrombin time and prothrombim. we did not observe bleeding disorders or any other side effect with peak concetrations of up to % of normal. white blood count, pmn--elastase, interleukin and and c-reactive protein tended to be lower in the at iii group. there were no differences with respect to renal and hepatic function parameters but the duration od mechanical ventilation was shorter in the at ii group ( . vs. . days) conclusions: we conclude that our treatment protocol (a) was able to restore at iil levels to the desired range of %, (b) did not lead to coagulation disorders, (c) did not increase transfusion requirements, (d) did not reveal other undesired side effects, and (e) showed a tendency towards reduced posttranmatic inflammation and mechanical ventilation requirements department of trauma surgery, essen, germany antithrombin-lll (at-ill) acts as an inhibitor of thrombin, further proteases and the alternative pathway of the complement system. inhibiting thrombin, at-ill works as a functional antagonist of paf~ therefore, in state of hypevolemic-traumatic shock, continuous supranormal serum -and tissue -concentrations of at-ill may be efficient to reduce the local posttraumatic reactions resulting from complement and pmn-activation. patients with severe multiple trauma ( treatment [at-ill] -- controls [c]) were investigated. study cdteda were age > and < years, injury severity (iss) > points and glasgow-coma-scale > points; randomization and treatment has to be started within hours after trauma. permission for the clinical study was given by the local ethic committee. bradykinin (bk) and related kinins are potent inflammatory peptides which possess the ability to induce, vasodilation, increased vascular permeability and hyperalgesia. cp- , a novel homodimer bk antagonist has previously been shown to increase survival in rat and rabbit models of lethal endotoxin shock and is now in clinical trials for sepsis. we have now evaluated the effect of cp- in other models of inflammation. male rats were precannulated with a catheter in the carotid artery. h later bk was injected ia and the pain score ranked from (no responses) to (vocalization). cp- at . umoles/kg completely inhibited the pain responses for a period of . - h. cp- at . umoles/kg s.c. was also found to inhibit the increase in paw volume and hyperalgesia induced in rats over a - h period by an intraplantar injection of . % carrageenan. the abdominal constriction response o an intraperitoneal injection of kaolin was inhibited in a dose-dependent manner by cp- . when ul of . % formalin was injected into the paw of a mouse a characteristic licking response was observed which was biphasic in nature. cp- significantly inhibited both the first ( - min) and second ( - min) phase responses. ]n a rat burn model, where the hind paw is immersed in water at °c for sec the increase in paw volume was significantly reduced by pretreatment with cp- , . umoles/kg s.c. finally cerebrai edema was induced in rats by applying cold (- °c for sec) to the dural surface following a craniectomy. cp- at . umoles/kg s.c. produced a significant reduction in the amount of edema compared with sham controls h later. these data suggest that bk is an important mediator of inflammation and hyperalgesia and that the bradykinin antagonist, cp- , may be useful in the treatment of such inflammatory, hyperalgesic disorders. partial hepatectomy in humans is associated with a considerable morbidity due to hemodynamic and metabolic derangements, which increase the risk for organ failure and mortality. we hypothesized that endotoxemia may play a pivotal role in these complications. we therefore, investigated whether peri-operative infusion of rbpi , a recombinant protein of the human neutrophil bpi with bactericidal and endotoxin-binding capacity, could prevent postoperative derangements following partial hepatectomy. male wistar rats ( - g.) received a % liver resection (phx) or a sham operation (sh), and a continuous intravenous infusion of either . mg/kg/hr rbpi (phx-bpi, n= ; sh-bpi, n= ) or the (iso-electric, iso-kd) control protein thaumatin (phx-con, n= ; sh-con, n- ). various parameters were measured h after the resection or sham operation. mean arterial pressure, cardiac output and heart rate were significantly decreased in phx-con rats compared with sh rats, which effects were not observed in phx rats treated with rbpi . blood ph was significantly decreased in the phx-eon group, whereas the leucocyte count, hematocrite and il- levels were significantly increased compared to sham levels. in the phx-bpi group, these parameters were restored to near sham levels. in vitro experiments with rat plasma and human mononuclear cells (mncs) revealed that plasma of phx-con rats is highly capable of activating mncs, accompanied by the release of cytokines. this activation is attenuated with phx-bpi plasma. in vitro added acd or polymyxin b was able to reduce the activation by phx-con rat plasma to the levels of phx-bpi rats thus, these data suggest that systemic endctoxemia, possibly of gut origin, is a major cause of postoperative hemodynamic and metabolic derangements following phx and that rbpizz can prevent these changes. more recently we reported a transient appearance of both endotoxin and tnf in the circulation of rats subjected to the haemorrhagic shock (hs) already at - rain. similar to bpi, recombinant bpi was found to bind lps and inhibit tnf formation in vitro. the aim of this study was to investigate the effects of rbpi (kindly provided by xoma corporation, berkeley, ca) against haemorrhage related endotoxemia and mortality in rats. method: a prolonged hs was induced by blood withdrawal to a mean arterial pressure of - mmhg for rain followed by reinfusion of shed blood (sb) and resuscitation with two times of sb volume of ringer's lactate over rain. rbplg. was administered at a total dose of mg/kg i.v. ( . mg/kg at the -eginning followed by two doses of . mg/kg each at end of shock and the end of resuscitation). the control group was treated similar to the bpi group but received thaumatin as a protein control preparation at the same dose as rbpi . results: imrffe?diately after resuscitation ( min) the detected plasma endotoxin levels in the control group (mean = , range = - pg/ml) were almost neutralized by rbpi treatment (mean = , range = - pg/ml) . plasma tnf levyis were not significantly influenced by rbpi treatment at the two time points and min of experiment (means: and in bpi vs , pg/ml in the control group). the -hour survival rate was improved from / ( . %) in the control to / ( %). conclusion: these data suggest that haemorrhagic shock may lead to bacterial translocation and/or transient endotoxemia with concomitant cytokine formation that may play an important role in the pathogenesis after shock and trauma, rbpi might be a useful therapeutic agent against endogenous bacterfal/endotoxin related disorders in hemorrhagic shock. morbidity and mortality after hypoxia of the vital organs had been correlated to the production of oxygen radicle which is mediated by xanthine oxidase activity, in this study we have evaluated the survival rate after allopurinol. rabbits weighed + grams divided into two groups. group i included tabbits were treated with allopurinol mg/kg for seven days before induction of haemorrhage. group ii as a control included rabbits. all rabbits were subjected to % arterial blood loss through the central ear artery for one hour then resusciatation was done by the heparinized withdrawn blood through a marginal ear vein. during the experiment blood pressure and heart rate were monitored through the central ear artery. also uric acid, lactic acid, glutathione activity were estimated. animal survival was followed for days. postmortem vital organ histochemistry and histopathology examinations were done. in group i the survival after three days was out of while in group ii it was two out of . our conc|usion, allopurinol had increased the survival in aiiopurinol pretreated rabbits which may indicate the value of allopurinol premedication for patient prepared for elective bloody surgical intervention . h receptor antagonists are commonly used for stress ulcer prophylaxis, but their actions on the septic response are largely unknown, in an experimental model, pigs were first anesthetized, then injured with joules of energy to the posterior thigh, then hemorrhaged - % of their blood volume. after i hr of shock, all the shed blood plus x the hemorrhage volume as lactated ringers was infused. following resuscitation, ranitidine ( . mg/kg iv twice daily) or saline placebo was begun. the treatment group was randomly assigned in a blinded fashion. after hrs, a septic challenge was administered ( bg/kg of e. coil endotoxin (lps)). serial gastroscopy, gastric ph, hemodynamics, abg's, physiologic dead space ventilation, leukocyte counts, and tumor necrosis factor (tnf) levels were recorded for min. baseline values and units were cardiac index _+ ml/min/kg (ci), arterial po + mmhg(pao ), base excess . -+ meq (be), physiologic dead space fraction +_ % (pds), and tnf . + . units/ml. baseline gastric ph was . -+ . and . _+ . in the placebo and ranitidine groups, respectively. the gastritis following hemorrhage was marginally attenuated in the ranitidine group. following lps infusion the following were obtained: ci pao * be* gastric* pds* peak* rain rain rain ph min tnf ranitidine _+ _+ - . ± . bum injury results in hypermetabolism, fever and nitrogen wasting. endotoxin (lps) has been proposed to mediate these effects, either directly or via activation of macrophages to produce cytokines such as interleukin- (ii- ). this study was designed to clarify the role of lps and - in the metabolic response to bum injury. twenty-five burn patients ( -+ %; + % ft bsa burn; _+ years old) were studied serially for three weeks post bum. patients underwent partitional calorimetry to assess metabolic rate and compartmented heat loss. nitrogen was assayed using chemiluminescence. lps and i - were measured with limulus amebocyte lysate assay and elisa. patients were excluded if they suffered smoke inhalation, showed any sign of sepsis or failed to rapidly meet their nutritional needs via the enteral route. ten patients received intravenous polymixin b ( , u/kg/day to bind lps). these patients did not differ for the remainder. all patients were hypermetabolic and febrile in proportion to the size of their bum wound but were not endotoxemic ( . +_ . pg/ml; normal < pg/ml). i - did demonstrate a significant correlation with cole temperature (tr~ = . + . ogi - , p= . ) and with nitrogen excretion (nou t = - . - . ogi - + . tr, p= . ). administration of polymixin b had no effect on metabolic rate, temperature or i - levels but did reduce nitrogen excretion resulting in more positive nitrogen balance ( .t grn/day vs. - . gm/day, p= . ). although bum injury does not produce an obligatory endotoxemia, i - does appear to play a role in the fever and nitrogen wasting seen with such injuries. the effect ofpolymixin b on nitrogen excretion suggests that lps may play a role either locally or in the portal system. introduction: there is substantial evidence that release of inflammatory mediators by activated kupffer cells contribute to the course of a systemic inflammatory process, e.g. after shock or lrauma. besides the systemic effects of mediators such as tnf, paf or interleukines, local actions on hepatic microvasculature and hepatic inflammatory response have to be considered. our aim was to assess the role of tnf and paf by blocking their effects using anti-tnf monoclonal antibody, pentoxifylline and a paf antagonist. methnds: in anesthetized sprd-rats, hemorrhagic shock was induced by withdrawl of arterial blood within rain and shock state was hold for h at a map of mm hg (cardiac output of %). following adequate resuscitation with % of shed blood and twice of this volume as ringer's solntion, animals recovered to map > mm hg and co > %. hepatic microcirculation and sinusoidal leukocyte-endothelium interactions were examined by intravital epi-fluorescence microscopy at , , or hours after resuscitation. in a blinded fashion, a rat-specific monoclonal anti-tnf antibody [ mg/kg, celltech, uk) , pentoxffylline (ptx, mg/kg, hoechst, d), and a paf antagonist (web , boehringer, ingh., d) were given either as pretreatment or at the time of resuscitation (n= - group bolla. k*., duchateau, j., hajos, gy., mbzes, t., hern~di, f. prevention of temporary/secondary immune deficiencies or reduction of their severity and/or duration as well as the reduction of the perifocal inflammatory processes belong to the rational targets of posttraumatic/pedsurgical medication. such a targeted medication can result in less frequently occurring nosocomial infections, and in reducing the duration of the intensive care and convalescence period. the results of in vitro studies performed with the amino acid sequence - of thymopoietin, i.e., with thymocartin in whole blood and peripheral mono-nuclear celi(pbnc) cultures clearly show some characteristic effects of this immunomodulator. preincubation with the tetrapeptide significantly (p<o.ol) and concentration-dependent reduced the endotoxin(lps)-induced tnfalfa production from , to about pglml, but did not abolish it. the inhibition of the lps( . ug/ml)-induced tnf production occured already in presence of , pg peptide/ml and peaked at the concentration of , ng/ml. higher concentrations did not increase this effect. in contrast to the other two small thymic peptides (tp- and tp- ), thymocartin did not enhanced the pwm-induced pge production in pbmc cultures up to a concentration of ng/ml, inclusive. the selection of thymocartin (tp- ) for perisurgical medication and/or for treatment of trauma-induced temporary immune depression has been strongly supported also by targeted animal experiments. thus, mortality of about % registered in mice infected with pseudomonas aeruginosa after bum injury could be reduced with the tetrapeptide treatment to a level which did not differ significantly from that of the infected non-burned controls. moreover, observations gathered first of all with the inflammatory model of air poach/croton oil grenuloma (selye) clearly showed, that a very definite, antiexudative effect can be achieved with this thymic peptide. as to this effect, thymocartin has been confirmed, e.g., to be equipotent with locally administered fluocinolone acetonide and/or s.c. injected prednisolone. the interim results of two explorative double-blind clinical studies (involving more than patients after polytrauma and hip-fracture untill now, respectively) correspond to the expectations. the treatment significantly reduced the occurence of nosocomial infections, the days spend in intensive care and on antibiotics as well as it shortened the bed-out time and reduced the additional medication. objective of this prospective randomized study was to further delineate the mechanisms leading to these alterations and to investigate the effects of immunomodulatory intervention. patients and methods: patients (pts) formed control group a. group b pts received x mg indomethacin (indo) from the first (dl) to the fourth postoperative day (d ) intravenously to block prostaglandin e induced suppression of cmi response as well as mg thymopentin (tp- ) subcutaneously preop., on d and d to augment cmi reactivity. in vitro investigations preoperatively, on dl and d included measurements of the percentage of cd + t-helper (th) cells, pbytohemagglutinin (pha)induced synthesis of interleukin (il)- as one cytokine primarily produced by thl-cells, and pha-induced synthesis of il- as one cytokine primarily produced by th -cells. delayed type hypersensitivity (dth) skin response to an antigen skin test battery and specific antibody (ab) production following tetanus vaccination preoperatively and on d were used as in vivo tests for thl-and th -induced immune response respectively. results: postoperatively, group a pts showed a persistent significant reduction of cd + th cells, il- /il- ratio, and dth skin response as compared to baseline values (bl), while ab production increased (p< . vs.bl). in group b pts no change in cd + th cells, il- /il- ratio, or dth skin response was observed (p< . vs.group a), and postoperative ab production increased significantly (n.s. vs. group a). conclusions: .these results clearly indicate a significant suppression of th induced cmi response, while th induced response remains normal following ohs. .these alterations may gain clinical significance whenever a postoperative infection requires a th response and may explain the susceptibility to opportunistic microorganisms observed in pts after ohs. the aim of this study was to find out whether the establishment of administration of thymostimulin (tp-i) in jaundiced and anergic rats would return the rats to the state of immunocompetence. material and methods. male wistar rats weighing - g were injected with haemocyanin (klh). all rats with a positive response were included in the study and we evalu ated the t cell subpopulations and il- . they underwent laparotomy and the common duct was ligated and divided. those rats whic become jaundiced and anergic one week after the operation, were divided in two groups: control group ( objective of study. the goal of this study was to prove the necessity of thymus derived immunomodulators treatment in acute period of infantine sepsis, materials and methods. immune status was investigated in o infants. clinical conditions of children were defined by syndrome of multiple polyorgan]c insufficiency. the heaviness nf condition was estimated as the sum of points according to efforts needed to restore of basic living functions -respiration, hemodynamjcs, hemocoagulatlon, metabolism, functions of liver and kidneys. it was expressed in form of clinic condition classes from to . results.it was found that in % of infants with - classes of heaviness immunode[iciency took place simultaneously with ii and iii stages of hemocoagulattve syndrome. amounts of t cells and t helpers were decreased more then times, ratio th/ts was reduced to . or lower. concentration of lgg and igawas reduced almost a hull in comparison to control. phagocytic and metabolic activities nf neutrophils were depressed. complement system state was changed: the functional activity of the alternative pathway factors b, d and the level or c a subcomponent were increased but the level of c , c ,, c and c components of the classic pathway were decreased. during the development of coagnlopathy the direct correlation between chso, levels of plasminogen and antithrombine iii was found. in greater extent the function of immune system was disodered in accordance with point metabolic violations and hemocoagulative disturbances considered as disseminated inmtravaseular clotting syndrome on ii or ili stages. conclusions. the rehabilitation of the immune system functions was promoted by taetivine and thymaline in doses of i- mcg/kg per day and mg/kg per day accordingly during a week in children with - classes el heaviness. aiter - days tactivine had advantage. when eoagulopathy took place thynaaljne promoted restoration of ( - )-~-d-glucan (bgc) is a major cell wall compornent of pathogenic fungi and shows many immunopharmacological activities on experimental animals. lps is also derived from gram-negative bacteria and have in~ttnomodulating activities on immune systems positively or negatively. because of difficulty to cure contaminating lps from bgc preparations, it is very hard to evaluate the exact activities of bgc. in this study, we compared the immunological activities of lps ans highly purified bgc on murine system. materials and methods: c h/hen or c h/hej mice, to wkolds of both sexes were used through experiments. highly purified ( - )-{ -d-glucan (gurdran; bgc)was courteous gift from seikagaku kogyo co. tokyo, japan and resolved in endotoxin-free ultra-pure water. escherichia cell lps was purchased from sigma chemicals, usa. mitogenic activities of bgc or lps on splenic cells were measured by mit dye assay. in vitro activation of peritoneal exudate macrophage (pen) have moniotored by phagocytosis and intraphagocytic killing of pseudomonas aeruginosa (z~b- . induction of cytokine productions from pem or spleen cells induced by bgc or lps were detected by cytotoxic assay. results and (bnclnsions: i ) in vitro cultures of pem with bgc have induced enhanced phagocytic and bactecidal activities in c h/hen and c h/hej mice, whereas lps only induced such activities in c h/hen mice. most effective dose of bgc was ug/ml. ) bgc-dependent proliferative response of c h/hen splenic cells was not detected under the culture condition used. ) induction of tnf~ prodction was apparently observed in bgc-stimulated c h/hej pd cultures, but not in lps stimulated one. these findings indicate that activation mechanisms of pr and splenic cells by bgc are different from that by lps. to investigate the bsc-induced p~ activation, intracellular signaling pathways of p~ by bgc-stimulation are under consideration. yamagami k, uedono y, kawakami k, kitazawa y and tanaka t according to the latest reports of use of il- in a burned-sepsis model, the dose was too high to adjust for human therapy. adoptive transfer of l~ymphokine-activated killer (lak) cells has been demonstrated as a means of enhancing therapy in malignant tumors. we therefore attempt to use lak therapy on burned-infected mice instead of il- . under anesthesia, -weekold male c h-hej mice were subjected to a % scald or sham burn and then resuscitated. sham burned mice served as controls. scald burn mice were subjected to peritonitis by intraperitoneal innoculation of organisms of p. aeruginosa hr after burn. burned-infected-mice were divided into two groups. one group had no further treatment, and the other group received lak cells ( x ) intraperitonelly after septic challenge. the mice were scored for survival daily. on day after burn, using moabs dual-color flow cytometry, we studied lymphocyte expression in mice with use of blood and spleen. simultaneousely we studied the alteration of il- and il- in their serum using immunoassy kits. sham burned animals had no mortality. the mortality of the lak-treated mice was significantly reduced when compared with that of the burned-infected mice. the most consistent changes observed were depressions in percentages of thyl, positive cells and a remarkable depression of nk cells in spleen. after lak cell treatment, those two percentages of cells significantly increased. all the data of assay of ill and [l were not detected on sham burn mice serum. due to burn and septic challenge the levels of illand il (n= ) were raised to . _+ . and _+ pg/ml, while the levels in lak treated mice were reduced to . _+ . and _+ pg/ml, respectively. the results reported here demonstrate that lak therapy is able to improve cell-mediated immunity in burned-infected mice. this is associated with a significantly improved survival rate, since ill has been demonstrated to mediate immune and inflammatory responses. also a cause effect relationship between elevated endetoxin levels and increases of [l has been recently established. the aim of our present study was to investigate the effect of parenteral indomethacin on the immune system of patients under major operations. our study included operated patients, of which received mg indomethacin before and , and days after surgery (group a) and patients received no antiinflammatory therapy (group b). we measured total t-cells helper (cd ), suppressor (cd ), nk-cells and interleukin- and tnf production by pha or endodoxin (lps) stimulated peripheral blood mononuclear cells at day and , and days after operation. in group a we detected a significant (p< . ) increase in cd /cd ratio on day while no differences were ~oted in nk-cells or cytokine production in both groups (table ) . it seems, therefore, that parenterai indomethacin may have a benefitial effect on the immune system of patients under major operations by increasing the t-helper /t-suppressor cell ratio while having no effect on the production of cytokines by peripheral blood mononuclear cells. this study was undertaken td dete~nnine the role of prostaglandin synthesis inhibitor on survival post traulna sepsis. analysis of the effects of prostaglandin synthesis inhibitor on survival of four groups of mice each were made after laparatomy and intravenous administration of live e.coli. post trauma sepsis, group a received no treatment; group b received antibiotics im; group c received prostaglandin synthesis inhibitor im and group d received both antibiotics and prostaglandin synthesis inhibitor im. survival time was longest in group treated with prostaglandin synthesis inhibitor and antibiotics post trauma sepsis. the singular use of either antibiotic or prostaglandin synthesis inhibitor did not alter the outcome on survival. mortality rate was lowest in the group treated with combined prostaglandin synthesis inhibitor and antibiotics. use of this combination showed a reduction in bacterial growth in peritoneal and blood samples, mild morphologic changes secondary to sepsis in the heart, lungs, liver, kidney and stomach and marked enhancement of mean level of activity. the study indicates that addition of prostaglandin synthesis inhibitor in treatment of trauma-sepsis has clear beneficial effects. interferon-? (ifn-y) is a potent immunostimulant which has been shown to be detrimental when administered during septic shock. blockade of this cytokine however is beneficial during the acute septic response. the aim of this study was to evaluate the cellular effects of this cytokine and its blocking monoclonal antibody (moab) in lethal sepsis following injury. female cd- mice were randomized into two groups: septic=lps vs injury=hind limb amputation (hla vs igg. nstats= anova=p< . vs lgg ifn-y was detrimental when given to control septic animals. however its blocking moab was protective (p< . ). conversely ifn-y was proteetive in injured septic animals compared with anti-ifn-? (p< . ). these findings demonstrate that the immune stimulant ifn-? has therapeutic potential in the immunosuppressed injured host predisposed to sepsis, while blockade of this cytokine is required for protection in the septic host with a normal immune response. dept of surgery, rcsi, beaumont hospital, dublin , ireland. oxidants play a role in physiology. the potential noxious oxidant biochemistry is restrained by chemically distinct antioxidants. these antioxidants, which may reside in different cellular compartments, can act synergistically. in normal physiology an oxidant/antioxidant bai&nce exists. unbalance in favour of the oxidants is called oxidative stress. oxidative stress has been implicated in many pathologies including lung diseases(e.g, doxorubicin induced cardiotoxicity), ischemia/reperfnsion damage and central nervous system trauma. transition metals such as iron are involved in the early events leading to oxidative damage in these pathologies. this has been underlined by the finding that postischemic cns pathology closely resembles that caused by a chemical oxidative insult (e.g. iron microinjection). moreover, a protective effect of oxygen-radical scavening agents or compounds that inhibit lipid peroxidation (antioxidants) in brain ischemia/trauma is apparent. some known drugs (e.g. anti-arrhythmics or histamine h -antagonists may act as non-specific antioxidants. howe~er, recently, interesting new membrancespecific antioxidants (e.g. -aminosterdids) have been designed. subt&e effects on iron redox chemistry contributes to the potent antioxidant activity of these aminosteroids. since a few years, one area that greeted enthusimastlcally in clinical medicine is that of reoxygenatlon injury or ischemia-repeffusion, a phenomenon accepted to make a majo:" contribution to organ dysfunction in trauma, shock and sepsis through the formation o( oxygenated free radical species. however, thei detection in vivo always remains a difficult challenge. efforts have been made recently to directly establish the formation of free radicals in biological samples as complex as blood, plasma or tissue with the use of electron spin resonance (esr) spectroscopy. using spin trapping agents, the presence of reactive carbon-and oxygen-centered radicals has been demonstrated in animals blood submitted to heart, renal and intestinal ischemia-repeffusion or to liver transplantation. recently, the in vivo formation of free radicals in the cerebra; extracellnlar fluid during cerebral isehemia-repeffusion has been assessed by the spin trapping technique coupled to brain microdialysis. esr investigations have also been conducted to detect endotoxin-induced free radical generation in rat or baboon liver and oxygen free radicals (ofr), produced both at intra-and extra-cellular levels, seem to play a major role in the pathophysiology of tissue injury and organ dysfunction in sepsis, through induction of lipid pemxidation in cell membranes. oxidative damage can result both to an ofr-overpmduction or to a depletion of endogenous antioxidant defenses, which are represented by scavenger enzymes (e.g. sod), hydrosoluble and liposoluble antioxidants (e.g. ascorbate, vitamin e), and other mechanisms such as metal-ion sequestration. in animal models, oxidative damage has been proved by detection of increased levels of lipoperoxidative products, and a depletion of antioxidant defenses was found both in plasma and tissues. in the few clinical studies a similar results have been reported, and oxidative damage appears to correlate with dic, with organ dysfunction and with red blood cell deformability. in a group of critically ill septic patients we found increased levels of conjugated dienes (cd) (bioproducts of lipoperoxidation) and decreased plasma levels of alpha-tocopherol and coenzyme qlo (coqi )(endogenous liposoluble antioxidants). a relationship between cd and uric acid (p< . ) was found, may be related to xantine-dehydmgenase to xantine-oxidase conversion. antioxidant depletion is due both to overconsumption and to a nutritional deficiency, even if a reduced synthesis can also be present. in fact one more relationship between coqi and plasma cholesterol (p< . ) demonstrates the commun hepatic biosynthetic defect. is there any role for antioxidative therapy in sepsis? can it achives a significant improvement in the septic course, organ dysfunction, and final outcome? several antioxidant drugs, have been evaluated in experimental and clinical sepsis: scavenger enzymes, natural antioxidants, alloporinol, -aminosteroids or lazaroids, have been proved to reduce lipopemxidative damage, to protect organ dysfunction, and in some cases to improve survival. several questions must be addressed in evaluating safety and utility of antioxidative therapy. more specific and standardized methods must be applied to detect and quantify the lipoperoxidative damage. antioxidant drags must act selectively on ofr-production, without interference with other than that are beneficial for the organism. antioxidants must be able to achive in adequate amount the tissue or cellular compartment where their action is required. many antioxidants have also a pro-oxidative effect which can he detrimental in some conditions. the timing of administration, the dosage used and the association wih others antioxidant drugs must be defined. nutrition plays an important role as antioxidative therapy, since it supplies all compounds needed to maintain adequately levels of endogenous antioxidant or to support their synthesis. thiobarbituric acid reactive substances (tbars) concentration in serum has been determined in a large population of healthy subjects and in patients suffering acute hepatitis and chronic cases of hepatitis c, as well as several other processes. the correlation with different physiological and clinical parameters in these populations is also presented. treatment with interferon of the chronic active hepatitis c patients, x u three times a week during two months, led in those patients whose sgpt activity normalized in serum, to a concomitant decrease in serum tbars content. the possible theoretical involvement of peroxidation and antioxidants in this beneficial effect of i~terferon in hepatitis c patients is discussed. the results presented confirm the value of tbars as laboratory test in the management of disease and as a useful tool for the study of pathogenic and/or therapeutic mechanisms. -hydroxyalkenals are among the different substances measured by the tbars assay. these compounds show several biological effects that have been demonstrated mainly in different experimental models. we have studied the capacity of different tissues to conjugate these compounds patients surviving the initial subarachnoid hemorrhage (sah) from a ruptured in~raeranial aneurysm are prone to develop cerebra] ischemia from vasospasm which is associated with significant morbidity and mortality. among the many treatments that have been prol~osed to treat this condition, none has been shown to be satisfactorily effective. recently, a new drug, namely the -aminosteroid tirilazadmesylats, has been studied in a large, prospective, multicentor trial for its effectiveness to improve the outcome of patients with aneurysma! said. the study was conducted in europe and australia in neurosurgical centers and included patients. all patients received presently accepted standard treatment, including nimodipine. patients were randomized to four different groups: placebo and groups of tirilazad in escalating dosage ( . - mg/kg/day). the clinical outcome was compared for these groups and cerebral a~giography was performed in % of patients on day - follovang sah, to study the effect f'the dflf~ off cerebral vasospasm. the study reached its planned endpoint months ago. preliminary results ~how a significant improvement with regardto mortality in those patients receiving the highest tirilazad dose as compared to placebo and the two lower dose groups. additional beneficial effects were seen in the rag/day groups with regard to the occurence of symptomatic vasospasm and good clinical outcome. although the final analysis of all data is still pending, these results suggest that tirilazad-mesylate could represent a major improvement for the treatment of patients with sail. experimentally lipidperoxidation products (lpo) are increased in acute pancreatitis (ap) due to oxygen radicals (or). the purpose of this clinical study was to determine the antioxidant status and lpo in patients suffering from ap and to evaluate the effect of antioxidant treatment with sodium selenite (se) thus improving the function of the se dependant glutathione peroxidase which is the major detoxifying system for or. materials and methods: in z patients sufferin s from severe ap (c-reactlve protein > me/l) we determined on day and day after admission the lpo ma!ondialdehyd (mda), conjugated dishes (cd), reduced (gsr) and oxidized (gssg) glutathione, the vitamins a,c,e and se. moreover the patients were evaluated clinically using the ranson and the apache ii score. i patients were randomly treated with ug/day of se for days. results: all patients suffered from a severe depletion of antioxidants,especially a low concentration of se (only / of normal). thereby the increase in lpo correlated with the clinical course. during se treatment lpo decreased and the levels of antioxidant vitamins improved. se had no influence on leth-slity the lenl or the chan in rs or ap ii. background: since reperfusion injury occurs when oxygen is reintroduced into ischemic tissue, the ideal timing for administration of therapeutic compounds aimed at ameliorating oxygen radical mediated injury is at the time of initial fluid resuscitation. currently used colloid or crystalloid preparations do not provide optimal, or even significant, anti-oxidant protection. systemic iron chelation affords protection against the iron catalyzed components of oxygen and lipid radical mediated tissue injury. the conjugate resulting from chemical attachment of the clinically approved iron chelator, deferoxamine (dfo, desferal ®, ciba), to hydroxyethyl starch (hes) represents a novel approach to colloid based fluid resuscitation. hes-dfo contains % hes and % chemically bound dfo. the polymer-drug conjugate has a lower molecular weight than that of hes in order to allow more rapid excretion. results: preclinical and initial clinical trials indicate that hes-dfo is well tolerated, even at high doses. in animal studies, fluid resuscitation with hes-dfo does not significantly improve central hemodynamic recovery beyond that observed with hes, but hes-dfo seems to afford better protection of microcirculation in organs at risk (lung, liver and gut), possibly by decreasing neutrophil sequestration. in a burn model, total fluid requirements are lower and oxygen utilization higher in hes-dfo treated animals compared to hes controls, suggesting decreased vascular leak and improved tissue perfusion. conclusion: hes-dfo represents a means by which potent antioxidant protection can be administered at resuscitation. iron has been suggested to play a pivotal role in oxygen flee radical mediated tissue injury. in vitro experiments indicated its critical role as a katalyst in hydroxyl free radical generation fenton-reaction). since iron chelator deferoxamine administered in shock alone demonstrated severe side effects, a hydroxyethylstarch (hes)daferoxamine (dfo)-conjugute was used to modulate oxygen free radical injury during the ischemia/reperfi~ion syndrome induced by hemorrhagic shock. methods. female lewis rats ( - g, n> ; pentobarbital anesthesia mgjkg), in hemorrhagic shock ( the aim of the study was to elucidate ( ) whether the generation of or would affect lung and kidneys as primary shock organs in the very early phase of sepsis and ( ) whether dfo-hes could prevent this tissue damage. methods: in rats sepsis was induced by cecal ligation puncture (clp) peritonitis. the animals were randomly assessed to groups: one group was treated with ml dfo-hes ( mg/kg iv), the other rats received solely ml of the carrier starch solution. , , , and min after induction of sepsis respectively, the animals were sacrificed, the organs collected, and tissue contents of glutathione (gsh), malondialdehyde (mda), myeloperoxidase (mpo) and conjugated dienes (cd) determined. plasma samples were obtained for analyses of endotoxin (chromogenic lal test). blood pressure (map) was measured via a carotid artery catheter. results: clp caused sepsis with high (> . eu/ml) endotoxin levels. map in both groups decreased slightly but significantly during sepsis regardless any treatment. in the lungs mpo concentration was increased (p< . ) in the lies group already min after sepsis induction. concomitantly, tissue gsh level decreased and lipid peroxidation was pronounced as shown by elevated mda and cd levels. dfo-hes diminished tissue pmn accumulation and mpo concentration. moreover, at each time point lung mda and cd levels were lower (p< . ). histomorphological examination showed marked micro-atelectases, destruction of the alveolar septa, and splicing of the basal membranes in the lies group. in contrast, in dfo-hes treated rats the alveoli remained well-ventiiated and only some enlarged reticular fibers without splicing were observed. almost similar results were found for the kidneys. mpo levels differed neither within nor between both groups. the slight decrease in gsh levels seen after min in the dfo-hes group seems to demonstrate an oxidative stress to a lesser degree. the most impressive effect of iron chelation, however, was revealed by the lipid peroxidation products. at each time point, mda and cd levels were lower (p< . ) compared to the hes group. light and electron microscopic examination disclosed tubulotoxic and mitochondriat damages while dfo-hes lxeatment prevented that alterations. conclusion: both the biochemical and histological results of this study reveal an early and remarkable generation of or in peritonitis-induced sepsis. thereby, these or obviously cause pulmonary and renal tissue damages, intravenous application of dfo-hes may, however, benefit by preventing early lipid peroxidation of the tissue. the proteolytic irreversible conversion of xanthine dehydrogenase (xd) to xanthine oxidase (xo) is triggered by calcium flux. the aim of our study is to clarify ~he link between intracellular ca + levels and xo activity determined by uric acid release, and to evaluate the efficacy of verapamil, on the generation of hydrogen peroxide associated with reperfusion by assaying lactate & pyruva~e release and the levels of cytosolic free nad /nadh ratio. experimental protocol consisted of :(a) non ischemic/reperfused experiment in which normal cardiac slices of rats were perfusated with oxygenated kreb's ringer phosphate buffer containing glucose ( mg%) and bovine albumine ( gm%) for min at °c.it composed of groups, group aa (control group), and groups ab & ac (perfusate supplemented with verapamil in the dose of loo& mi% respectively). (b) ischemic reperfused experiment in which ischemic cardiac slices were obtained from rats subjected to min ~aemorrhage.lt was also divided into two groups; group ba and bb (verapam~/ mi% added to perfusate}. verapamil stimulated uric acid release from normal rat cardiac slices were % in group ab and % in group ac(dose related). rates of uric acid release is enhanced by verapamil in group bb. moreover, rates of uric acid release in groups ac & bb are insignificant. in verapmil added groups (group ab, ac & bb), increase uric acid release is associated with an enhancement in pyrurate release and with increase levels of cytosolic free nad+/nadh ratio, although it is not evident ~ ischemic group (group ba).it is concluded that the conversion of xd to xo is calcium independent. eicosanoids like thromboxane a , leukotriene b and leukotriene c are known as promoters of initial inflammatory reactions. we investigated whether oxygen radicals (or) are able to induce a release of these eicosanoids in whole blood. blood from healthy volunteers was incubated with xanthine oxidase/hypoxanthine to generate oxygen radicals. after , , , and minutes plasma levels of thromboxane b (txb ), leukotriene b (ltb ) and leukotriene c (ltc ) were determined via elisa technique. another volunteer had taken mg aspirin one day before taking the blood sample (no ). results: txb plasma levels increased from pg/ml at min to pg/ml, pg/ml, pg/ml and pg/ml at , , and min (p< , ) . ltb and ltc plasma levels showed an increase during the first few minutes (ltb : min: llpg/ml, min: pg/ml; ltc : min: pg/ml, min: pg/ml (p< , )) followed by a decrease to normal values at min. in the sample no the cyclooxigenase-pathway was completely inhibited, the txb plasma-levels did not alter at all, whereas ltb and ltc -plasma levels weren't affected. opallogeneic blood transfusion jane shelby, ph.d., and edward w, nelson, m.d, there have been numerous investigations dudng the last two decades examining the effect of surgery, anesthesia, blood loss and transfusion on vadous immune parameters in humans and animal models. there appears to be concurrence among several well controlled studies that transfusion of whole blood (containing leukocytes), has regulatory effects on immune ceil function which include decreased cell mediated immune response, and inhibition of il- secretion. these effects occur following transfusion alone and in con.cart with the distinct immune effects of surgery, trauma and anesthesla, the clinical consequences of this immune modulation by transfusion include decreased allogeneic response to transplanted organs, which has been exploited clinicelly in renal transplant patients. additionally, there is evidence for a strong association with increased risk for infection in transfused patients following surgical procedures. aiiogeneio blood transfusions have been shown to inhibit cellular anti.bacterial mechanisms, causing increased susceptibility to bacterial pathogens, in humans and in animal models. there is also concern that allog~neic transfusion may adversely affect cancer patients, resulting in decreased disease-free survival. several stategies have been proposed to minimize the adverse effects of blood transfusion. there is evidence that the risk of immune mediated infectious complications associated with transfusion may be greatly minimized wlth the use of autologous blood and leukocyte free allogeneic blood.products in surgical and trauma patients, it also appears that the inhibition of cellular immune response and il- productiorl following atlogeneic blood transfusion may be mediated by increased prostaglandin e secretion, and that immune response may be preserved in allogeneio whole blood transfused subjects receiving c lc~oxygenase inhibitors such as ibuprofen. among these are various alterations in immune function. efforts have therefore been made to utilize alternatives to homologous transfusions. these include the use of autologous predonation, supplemental iron therapy, and recombinant human erythropoietin. although initially considered innocuous, these therapies are now recognized to have potential deliterious immune sequelae. erythropoietin, by its ability to lower serum iron levels, can impair both lymphocyte and nk cell activity. autologous donation impairs nk cell function. finally, supplemental iron therapy can stimulate bacterial growth and increase the rate of infectious complications. this talk will present a discussion of these factors as well as a weighting of their importance. r.l rutan, rn;bsn, shriners burns institute and the university of texas medical branch, galveston tx, usa the serious sequelae of homologous blood transfusions have resulted in vigorous efforts at identifying alternate therapies for correcting red blood cell (rbc) deficits. erythropoietin (epo) was hypothesized to exist in the early th century, however the protein was not isolaled until . the human gene was identified and cloned in , which permitted the production of epo through recombinant techniques. the earliest clinical trials were performed in anemic end-stage renal failure palients on hemodialysis. treated patients experienced increases in erythropoiesis with normalization of hematocrit and hemoglobin levels, cessation of lrans-fusion requirements and improvement in general wellbeing. these studies, however, identified side effects of epo treatment such as hypertension, seizures and ee deficiency. volunteer trials have established that the hypertension is not a direct pressor effect but rather the result of abnormally rapid increases in red cell mass in the face of the incompetent volume-controlling mechanisms of the end stage renal failure patient. lower doses of epo and the subsequent gradual increases in red cell mass are associated with significantly lower incidences of hypertensive complications of epo therapy. likewise, seizure activity is not the result of a direct epileptogenie effect but parallels the incidence of hyper-tensive-related sequelae during high.dose epo treatment. in cross-over designed studies, pre-existing iron deficiency has been demonstrated to decrease or negate stimulated erythropoiesis but effective-hess can be restored with appropriate fe supplementation. exogenous epo is effective whether given by iv or sq routes and dose response curves do not vary with route of administration. increases in rbc mass are directly related to the dose of epo, both in amount and frequency of administration although there is a - day time lag between the first epo dose and laboratory indications of its action (i.e. increase in the number of reticulceytes in peripheral wood). epo is currently labelled for use in the treatment of anemias associated with end-stage renal disease and aids. however, its use in the surgical population has been explored because of its unique direct dose-response, epo has been used to effectively increase the blood harvest amounls in autologous pre-donation, significantly increase hematocrils in children following thermal trauma and successfully increase red blood cell mass following essential surgical procedures in patients with religious aversion to transfusion. by blood transfusion in colorectal cancer surgery mm heiss md, ch delanoff md, r stets md, j hofinann, e faist md, kw jauch md, fw schildberg md allogeneic blood transfusions are associated with an increased risk for postoperative infections in colorectal surgery when compared with autologous blood transfusions. attribution of this effect to immunomodulation was suspected in our previous study (lancet ; : - ) . task of the recent investigations was to analyze which specific effector systems were affected in-vivo by this transfusion-associated modulation. for global in-viva assessment of cell-mediated immunity (cmi) multiple recall skin-reactions were applied prior and post-operative. the specific humoral immune mechanisms were investigated by applying tetanus-toxoid one day preoperatively and deterimnating the quantitative igg-response. for indication of macrophage stimulation in-vivo tnf-levels were determinated by bioassay. dth-responses were significantly suppressed (p< . ) in patients receiving allogeneic blood (n= ) or operated without blood transfusions (n= ). dthresponses were not suppressed and tendentiously increased in patients with autologous blood transfusions (n= ). in contrast, specific igg-levels increased sigmficantly (p< . ) in patients receiving allogeneie blood (from . + . to . _+ . ie/ml) whereas in patients receiving autologous blood a smaller increase (from . + . to . + . ; p= . ) was observed. tnflevels demonstrated a similar pattern with a higher increase in patients receiving allogeneic transfusions (l . + . to . + . u/ml) compared to those patients with autologous blood ( . + . to . + . ). in conclusion these data indicate that allogeneic blood transfusions lead to a remarkable macrophage/rhs stimulation. this is corroborated by the boostered humoral igg-response which was initiated before onset of surgical trauma and blood transfusion. concerning cmi this caused a substancial suppression probably due to a stimulated secretion of immunosuppressive monokines. objective: firstly, to analyse the concentrations of the cytokines tumor necrosis factor (tnc), interleukin- (il-i), interleukin- (il- ) and coagulatioo/fibrinolysis parameters in postoperatively retrieved blood from a surgical area, secondly to characterize the correspanding cytokine patters in the patients and thirdly to study cytokine concentrations in the initial portion of drainage blood from a surgical area. materials and methods: blood retrieval was performed in a closed-loop system without anticoagulant during - hours after surgery in patients undergoing arthroplasty ( hips and knee). kf, il- , it- , thrembin-antithrombin complexes (tac) and antithrombin (at) ~ere determined in shed blood. patient plasma tn v, il-i and il- concentrations ~ere analysed at the beginnlqg and end of the - hour blood retrieval period. in a separate study ( hip arthroplasties) f~f, il-i and il- ~ere determined in the initial portion of drainage blood. cytekine analyses ~re performed usiog ipmuooassays. an omidolytic method was used for at determinaf.ion and tac was analysed by elisa. n~n-poram~tric tests was used for the statistical comparison. results: the patient plasma il- coocemtratiems rose from a median value of to pg/ml, p<o. , during the blood ret.rieval period. in c was detectable in patients, range: - pg/ml. il-i was not detectable in the patients. in retrieved blood tag was > mg/ml in all samples (ref:< . mg/ml) and at was . - . units/ml (ref:o. - . ) . the il- concentrations in retrieved blood was > pg/ml in all samples. tn v or il-i was not detectable. in the separate study, (n= ), characterlzing eytokine content in the initial portiere of drainage blood, in= (range: - pg/ml) and il-i (range: - pg/ml) ~re present in all samples but ii- (range:o- pg/ml) was detectable in o.qly one semple. conclusion: theses findings indicate that hypereoagulability and hic~ ccrcentratioos are present in retrieved blood. the cytokine pattern in the initial portion of blood from a surgical area differed from these observed in retrieved blood and in the systemic circulation. to identify the role of both autologous and homologous blood on postoperative infections in elective cancer surgery. materials and methods: patients with colo-rectal cancer submitted to curative elective surgery were prospectively studied. on hospital admission the following nutritional measurements were assessed: serum level of albumin, cholinesterase, delayed hypersensivity response , total lymphocyte count and weight loss, as were age and sex, duration of operation , operative blood loss, amount and type of blood given, pathological dukes' stage of the disease and the attending surgeon were also recorded. results : eighty-four patients ( . %) were perioperatively transfused. thirty-six ( . %) patients were given autologous blood , while ( . %) received homologous blood. no patients received both autologous and homologous blood. twenty eight ( . %) patients developed postoperative infections. non transfused patients had a . % infection rate , those receiving autologous blood had a . % infection rate, whi]e in the homologous blood group the infection rate was . % (p < . ). univariate analysis showed that infections were significantly related to operative blood loss (p< . ), length of operation (p< . ) blood transfusion (p< . ) and attending surgeon (p< . ) . multivariate analysis identified homologous blood transfusion as the only variable related to the occurrence of postoperative infections , while the other variables failed to reach statistical significance. blood transfusion (bt) remains an essential life-saving treatment for surgical patients. however, besides the beneficial short-term impacts, negative longer-term effects are observed, which include various alterations in the immune responsiveness. in surgical patients these alterations may contribute to the increased risk for infections and cancer recurrence. since relatively few data demonstrate immunologic changes occurring in other lymphoid compartments than blood after bt, we studied the effect of et on the frequency and responsiveness of immune cells in bone marrow (bm), spleen (spl) and blood (b) in a rat model. normovalemic, month old rats were transfused intravenously with syngeneic heparinized venous blood ( x ml, every other day), and , and days after the last transfusion bm cells ( leh is an experimental oxygen-carrying resuscitation fluid. since leh is cleared from the circulation primarily by the mps, its effect on the development of sepsis and the nature of its relationship with the mps remain a major concern. preliminary in vivo data from our laboratory failed to show any leh effect on the hemodynamic and hematologic responses to endotoxin lipopolysaccharide (lps) in the rat. in contrast, leh exacerbated the lps-induced tnfa production and early mortality. the exacerbation of early mortality by leh was attenuated by pretreatment with the tnfu synthesis inhibitor rolipram. ex vivo, peritoneal macrophages from rats treated with leh and lps have shown increased il-lg mrna signal as compared to lps alone. also, leh increased tnftx production by peritoneal macrophages in response to lps stimulation in vitro. additionally, recent pilot studies indicate that leh attenuates pma-induced superoxide production from rat peritoneal macrophages and that leh augments fmlp-induced migration of human monocytes. taken together, these data strongly support possible interactions of leh with the mps and therefore the nature of such interactions should be further explored. over the last decade, we have developed liposome encapsulated hemoglobin (leh) as an artificial oxygen carrying fluid, or blood substitute. our efforts have focused on studies to define the safety and efficacy of this resuscitative solutions. leh consists of distearoyl phosphatidylcholine, cholesterol, dimyristoyl phosphatidylglyeerol, and alpha tocopherol in a : : . : . mole ratio and can encapsulate hemoglobins of different origin (bovine, human, recombinant human). leh is fabricated using hydrodynamic shear to create an average particle size of . microns. leh can be lyophilized using disaccharides and stabilized in the dry state and easily reconstituted before administration. histopathology and clinical chemistries indicate that leh rapidly accumulates in tissue resident macrophages in small animals injected in the tail vein, principai y in the liver and spleen. the consequences of accumulation in the reticuloendothelial system are manifest by transient increases in liver transaminases (ast, alt), bilirubin, and bun over - hours with no change in biliary function (ggt, ap) . clearance through the liver and spleen is observed over the course of - -weeks. more recent attention has been focused on secondary consequences of leh administration especially with regard to inflammatory eytokines. leh does not elicit expression of tumor necrosis factor in vivo and in isolated macrophage cultures, but does result in a transient increase in serum il- . we have also examined the interaction of leh with lps in vitro macrophage culture to further understand how this blood substitute may effect the immune system. we have labeled leh with technetium- m ( mtc) to study the biodistribution of leh non-invasively in anesthetized rabbits. rabbits were infused with a % topload of leh ( mg of phospholipid, . g of hemoglobin per kg of body weight) and imaged continuously with a gamma camera. at hours, images were again acquired. animals were then sacrificed and tissue counts obtained, images revealed an initial rapid uptake bythe liver, % at minutes and % by hours. the spleen accumulated activity at a slower rate, % at minutes and % at hours. at hours, autopsy biodistribution studies revealed that approximately . % of the dose is in the blood pool, . % in liver, . % in spleen, . % in lungs, . % in muscle and . % in urine, with trace levels in kidney, brain and heart (< °/o). in a hypovolemic model, rats were % or % exchange transfused with mtc-leh. in the % exchange model, mtc-leh was rapidly taken up by the liver and spleen with minimal activity in the circulation at hours. with the % exchange, % of the leh was in circulation at hours. the interaction of leh with platelets labeled with indium- was also studied. after infusion of leh, the labeled platelets rapidly moved from the circulation to the lungs and liver. over the next minutes, the platelets gradually returned to circulation. this effect was not seen with iiposomes of the same lipid composition but containing no hemoglobin. non-invasive imaging is proving to be a very useful tool for the investigation of leh. the need for a safe, efficacious and commercially viable blood substitute is unequivocal. of the several strategies pursued to invent an adequate blood substitute, liposome entrapped hemoglobin (leh) has been already established as a leading possibility. major advances in liposome technology have already resulted in liposome preparations compatible with clinical use for drug delivery. recent technological advances made by the u.s. naval research laboratories resulted in the capacity to entrap hemoglobin into liposomes in a way which secludes hemoglobin from interacting freely with biological systems. the leh produced has already been tested in in vivo systems and was foun.d to be well tolerated. moreover, the leh originally produced as a solution can be transformed into a lyophilized form which can be reconstituted and delivered as a fresh solution. while important milestones in leh development for a practical blood substitute have been achieved, several issues remain to be explored. most notably, the long term consequences of leh on host defense mechanisms and, in particular, immune cell function. in addition, it is important to understand more fully the metabolic fate and repercussions of leh delivered at clinically relevant dose/schedule regimens. finally, while leh is a highly promising strategy for a blood substitute, the present formulations consist of human hemoglobin derived from human blood, to improve the safety profile, a recombinant preparation for liposome entrapment will be much desired, aa-ginine, a semi-essendai dietary amino acid, possesses several unique and potentially pharmacologic properties. argirdun is a potent secretagogue for pituitary growth hormone and prolacfin and for pancreatic insulin and glueagon; it modulates host protein metabolism by increasing nkmgen retention and enhancing wound collagen synthesis. it also is a potent t call function regulator. ait of these effects coupled with its relative lack of toxicity and safety make it an a~antive nulritionai pharmacologic agem (t). rodents fed supplemeutal arginine exhibit increased thymsc weight which is due to increased numbers of thymic lymphocytes present in the gland. thymic lymphocytes from animals fed supplemental ar~e demonstrate increased blastogenesis in response to coma. and pha ( ) . peripheral blood lymphocytes from humans given supplemental arginine also have heightened mitogunic responses to mitogen or antigens ( ) . in postsurgery padents supplemental arginine abrogates or diminishes the deleterious effects of trauma on lymphocyte responsiveness and restores peripheral blood lymphocyte responses much faster than observed in controls. overall host immunity is also enhanced by arginine. allograft rejection is enhanced and septic animals survive longer when given supplemental arginine ( ) . tumor bearing urginine-supplemented animals have decreased tumor growth and enhanced survival (i). lastly, asgmine can induce t cell maturation and t cell mediated responses in athyrnic nude mice. arginine also has remarkable effects on host nitrogen metabolism post-injury. in increases nitrogen retention in healthy human volunteers and in surgical patients. this beneficial effect on overall nitrogen metabolism is accompanied by a unique effect on the healing wound. supp]emental arginine increases wound collagen synthesis which also translates into increased wound breaking strength ( ) . arginine has no effect ou epithelialization. douglas w. wilmom, m.d. boston, ma gintamine is the most abundant amino acid in the body, but it has long been considered a nonessential amino aeid because it is synthesized in many tissues. fohov~g st,~'vation~ injury or infection, skeletal muscle pmteln inoresses its net tale of degradation and releases amino acids into the blunds~mm at an aocelerared rate. app~o)~mately one-third of the amino nitmgea is ghitamine, which is metabolized by the kidney where it parth:~pates in acid-base homeostasis, is the primly ~ for lymphocytes, mac~optmgcs and untexocyms, and contm'butcs to the synthesis of giumth~une. olmamine degrades slowly while in ~olu~ou, especially at usual room teml~mtums. because giulamine was considered nonessential, it has beer absent r'om nil intravenous and most gluts.mine should be considered a cendittona]ly essential nutrient for individuals with serious ilinesses, uspccially those confoanded by infcctinn and inflammation. over the uc~:t - years, glutamine will be incorgorated into most feeding formulas designed for patients with critical illness. o]~ga- pufa there continues to much interest in the application of the mega- pufa in clinical nutrition. the basic principle has been that the mega- pufa will displace arachidunic acid and result in a decrease in eic san id production. in addition these changes in pufa will after the physical characteristics of the membrane including flujdity, receptor function and transmembrane signals. animal studies have shown that there is omega- incorporation with continuou~ enteral feeding both in control and endotoxic animals within days. this includes the liver, spleen, circulating and alveolar marc phages and the lung. this incorporation resuls in significant changes in the eicosan id production including pgf and ket -pgflalpha. there is improvement in the cardio-vascular reep nse of these animals with ~ecreamed lactic acidosis and improved cardiac contractility. as well there is improved immune function with improved t cell response to mit gens. the ~ of a mumber of pharmacological agents blocking cicosanoid production can enhance the cell effects of mega- pufa. clinical studies using short term entsral nutrition with mega- either alone or with other enteral supplements in a number of clinical settings have shown significant mesa- incorporation and decreased eicosan id production. these positive results must be discussed with the additional evidence that long term omega- supplementation decrease eic san id production but als induce a state of immune suppression that is capable of increasing transplant sunvival. these ng te~ inune effects may benefit clinical conditions including rheumatoid arthritis and cr hn' disease early enteral nutrition instituted i~mediately afte~ injury will decrease the entry of bacteria into the intestinal wall and decrease the number of bacteria that translocate into the portal blood. these reductions are associated with & decreased catabolic response, decreased plasma cortisnl levels, end decreased vma excretion in the urine and prevention of mueosal atrophy. sdecific nutrients also affect the transloeation process. addition of arginlne to the diet significantly improves the ability to kill translocated organisms. however. translooetion across the gastrointestinal barrier is not affected. in contrast, glutamine diminishes the rate of translooation across the imtestinal barrier and also improves killing of the beetarla that do translooate. the omega fatty acids in the form of fish oil slightly decrease the rate of translocation but more significantly increase the ability of the animal to kill translo~ated organisms, all three dietary additives, i.e. argini~e, glu=amine and fish nil. significantly improve survival, hut adding glyoine or medium chain triglyeeridem do not, combinations of srginine and glutamlns, glutamine and fish oil, and fish ell end arginine each improve survival, and to a greater degree than a combination of all three. these studies add further evidence that translocation is an important determinant of survival after injury, early feeding with immunonutrlent enriched dices will improve survival and dsarease transloeation to varying degrees, depending upon the nutrients provided. objectives: we studied effects of supplementing a commercial enteral diet, impact r (imp, sander nutr lnc), with fiber (imp/fib) or alanyl-glutamine (imp/ag, exogenous glutamine (gln) gms/l) on influencing the incidence of bt to mesenteric lymph nodes (mln) in burned mice. fiber has been shown to improve gi integrity under certain stress/treatment conditions. the dipeptide ag is a water-stable source of gln, which is a specific fuel for many cells including enterocytes. traumacal (trcal), a high-protein, high-fat enteral diet (mead johnson iuc), was also studied, as well as rodent chow (harlan teklad inc), which contains very high protein & fiber. methods: anesthetized cf- mice aged - wks received % tbsa fullthickness dorsal burns & were resuscitated with cc ip saline. diets were allowed ad lib; caloric intakes were comparable in all gps except fasted gp (fast hrs, chow hrs). at hrs postburn mln were sterily removed, homogenized and plated on heart brain infusion agar; cfu/g mln tissue were determined. bt was analyzed by fishers exact test, cfu/g by anova-bonferroni. * p< . , ** p< . compared to imp and burn-fast gps. background. infectious complications following trauma, major operation, or critical illness adversely affect hospital cost and length of stay (los). some key nutrients have been shown to possess immune enhancing properties. this multicenter trial was conducted to determine if early administration of an enteral formula supplemented with arginine, dietary nucleotides and fish oil can decrease los and infectious complications in icu patients. methods. this was a prospective, randomized, double-blind study of adult icu patients who required enteral feeding for > days. patients entered the study within hr of the event, were stratified by age and disease, and were randomized to receive either the supplemented formula (impact®) or the conventional formula (osmolite ® hn). feedings were initiated at full strength and advanced to at least ml/hr by hr after event. results. both groups tolerated administration of formula well. for patients fed > days, the median los was % shorter (p=o.ol) for the--supplemented group ( days) compared to the conventional group ( days). the incidence of most infectious complications was lower in the supplemented group, but this difference reached significance only for urinary tract infections (p=o.o ). the supplemented group had a significantly shorter los from onset of infectious complication until discharge for patients with pneumonia ( vs. days) and skin/soft tissue infection ( vs. days). conclusions. administration of the supplemented formula was safe and well tolerated. when fed > days, it reduced the incidence of most infectious complications, and significantly reduced los. materials and methods: twenty-seven patients were randomised into groups ( n= each) to receive either a standard enteral formula, the same formula enriched with arginine, rna and omega fatty acids (enriched group) or isonitrogen, isocaloric parenteral nutrition. early enteral nutrition was started within hours following surgery ( ml/hour). it was progressively increased reaching a full regimen on day . on hospital admission and on post-operative day and , the following parameters were assessed: serum level of transferrin , albumin , prealbumin, retiool binding protein (rbp), cholinesterase. delayed hypersensitivity response, igg, igm, iga, lymphocyte subsets and monocyte phagocytosis ability were evaluated on admission and on post-operative day , , . the three groups were comparable for sex, age, cancer stage, type and duration of surgery, intra-operative blood loss and amount of blood transfused . in all groups a significant drop in all the nutritional and immunological parameters was observed on postoperative day . comparing post-operative day versus day a significant increase of prealbumin (p< . ) and rbp (p< . ) was found only in the enriched group. with respect to immunological variables an increased phagocytosis ability (p< . ) and a significant recovery in delayed hypersensitivity response (p< . ) was observed only in the enriched group. conclusions : these data are suggestive for a more effective post-operative recovery of both. nutritional and immunological status in cancer patients fed with enriched enteral formula. gastrointestinal intolerance was equivalent ( % in each group) and laboratory screening confirmed that both diets were safe. when analyzing clinical outcome for all patients, there were no significant differences in septic complications (immun-aid = % vs vivonex ten = %), mean mof score (immun-aid = l.b vs vivonex ten = . ), or mortality (immun-aid % vs vivonex ten = %) . kowever, when analyzing the subgroup of patients with severe injury (iss or ati _> ), patients receiving immun-aid appeared to have fewer septic complications ( % vs %) and their mean mof was significantly lower ( . _+ . vs . + . , p = . , student's t-test) . these preliminary data indicate that immun-aid is tolerated well when aggressively delivered immediately postinjury. the ultimate affect on clinical outcome appears ~avorable for immun-aid, but needs to be confirmed in larger patient groups. kemp?n, m., neumann, h.a., he i[michh b: as both increased, normal and reduced phagocytic capabilities of polymorphonuclear leukocytes (pmn) and monocytes in acute batterial infections have been reported, the role of phagocytes in patients with severe sepsis is less clear.we examined pmn and monocytes from patients in septic shock and heailhy votunteers for phagocytic function. phagocytosis was determined by flow cytometry (facscan) and was measured by the ability of pmn and monocytes to phagocytose e.coli marked with fluorescent antibodies. a septic shock was defined by the presence of a ~ource of i, nfoctiqn with a known bacteriology, distinct signs of a systemic response and defined minimum scores in icu scoring systems indicating the presence of a multiple organ failure. additionally we examined how phagocytosis is influenced when a new enteral diet formulation containing substrates suggested to improve immune function or arginine, one of its major compononts, is added in vitro in defined concentrations and incubated for minutes. pmn (p{o, ) and monocytes (p<o, ) of the septic patients showed a significantly enhanced phagocytosis compared tolhe phagocytes of the healthy group. the incubation with the enteral diet in vitro was followed by a higher dose-related rate of phagocytosis, especially in the healthy group, that was not statistically significant. after addition of l-argintne we atso observed an iqcrease in phagocytosis, that was lower than in the group with the complete diet. using a modern immunefluorescence-cytometric essay we founfl an improved phagocytic function in septic shosk. there are signs for an influence of nutrient substrates on phagocytosis which seems to be complex and should be further investigated. arg and gln were lower in the %-bcaa vs %-bcaa group; arg was related directly to arg dose and inversely to bcaa dose (p<o.o for all). clearances of bcaa increased with increasing bcaa dose (p<o.o ); arg and gln clearances were directly related to the bcaa clearances (rz=o. , p<o.o i). relationships between arg, gln and other aa seem to be ruled by patterns involving specific intracellular aa transport systems. regulation of arg and gln clearances through bcaa administration may be related to an increased flux of aa into synthetic processes. intestinal segments from rats immunized by infection with the nematode trichinella spiruus undergo intestinal anaphylaxis or type i hypersensitivity when challenged in vitro with parasite antigen. immunized rats exposed to t-radiation ( gy) and sustained on rat chow,fail to fully express type i hypersensitivity up to days post irradiation (dpi). we hypothesized that enteral nutritional support immediately following irradiation may be effective in preserving mucosal immune responsiveness or in reducing the recovery time after radiation-induced injury. rats were infected with x t.spiralis larvae. thirty to fifty days later rats received gy t-radiation from a co source as total abdominal irradiation (tai). immediately following tai, rats were fed an elemental amino acid diet (eaad) and at various dpi sacrificed and tested in ussing chambers for local anaphylaxis, expressed as antigen-induced ci secretion. the normal ci secretory response to antigenic challenge which is suppressed after irradiation,was restored by dpi when rats were placed on the eaad. antigen-induced c[ secretion is dependent on the interaction o~ antigen with specific ige antibodies on the surface o mucosal mast cells and their subsequent degranulation. mast cell-derived -ht,histamine and pg, together effect ci secretion.in irradiated rats on rat chow,the failure to respond to antigenic challenge appears to be associated with the destruction of mast cells. they are undetectable with standard staining procedures and mucosal histamine levels are drastically reduced. also,el secretion can be induced by direct stimulation of epithelium with cr secrctagogues and circulating ige levels are normal. despite a more rapid recovery of immune responsiveness in irradiated rats receiving eaad,mast cells were not restored. we conclude that the eaad contributes to an adaptation that supports the expression of local anaphylaxis in the absence of mast cells. total parenteral nutrition (pn) and bowel rest may influence the host response to infection. this study examined the different host response to infection in parenterally and enterally fed animals. forty-three rats were divided into pn group and total enteral nutrition (en) group. they received identically constituted isocaloric isonitxogenous diets for seven days. animals were then challenged intraperitoneauy with xl(y escherichia coli and survival were observed. in other experiments, they were sacrificed before and two hours after bacterial challenge. peritoneal cavity was lavaged with ml saline and peritoneal exudative cells (pec) were harvested and cultured in vitro for hours with and without lipopolysaccharide (lps). colony f(m, ning units of bacteria (cfu-b) in the peritoncal lavaged fluid (plf) were determined and tnf in the plf, serum and pec culture supernal,ants were measured by l bioassay. twenty-fonr hour survival rate in en group was significantly greater than that in pn group ( % vs %). the data suggest that local immune responses of pn-treated animals may be depressed with consequent disturbance in the clearance of bacteria. this may lead to a greater systemic cytokine response and resulting in a poor survival after bacterial challenge in pn group. the effects of intragastfic tube feeding of diets enriched with m- polyunsaturated fatty acids (pufa) from safflower oil (so) or (o- pufa from menhaden oil (mo) on eicosanoid production, and onthe membrane phospholipid fatty acid composition were investigated in a severe acute septic shock model. the percent calories from fat maintained at % in the diets was adjusted to provide % each of saturated, monounsaturated, and pufa using olive oil and palm oil as filler fats. after feeding for days, lipopolysaccharide (lps, mg/ g) was injected through the tail vein. the percent of animals surviving in the mo group was % ( / ), and did not differ significantly compared to % ( / ) in the so group. the plasma concentrations of tnf ct for the groups of animals fed so ( . + . ng/ml) and those fed mo ( . + . ) did not differ significantly. the levels of prostaglandin(pg)e , -keto-pgflc~ and tumor necrosis factor-ct (tnf-c were determined min after lps injection. the fatty composition (relative mole%) of the liver was determined before (lps-) and h after the lps administration (lps+). the data (mean_+sd; n= ) are as follows. group these data indicate that a marked reduction in the plasma levels of poe and -keto-pgflct for rats fed mo diet was associated with a reduction in aa which was displaced by epa in the membrane phospholipids. further, in these rats, we observed that there was a . % reduction in the percent of epa following lps challenge compared to the basal levels. however, for the group of rats maintained on so diet, the liver membrane phospholipid fatty acid composition before and after lps administration was unaltered. these finding suggest that although a reduction in aa was associated with a decrease in the production of pro inflammatory eicosanoids after days of continuous mo feeding, there was no marked effect on the survival following lps challenge. animal models have been used to examine the effects of various nutrients and nutrient combinations upon the immune system. we studied the effects of standard and specialized enteral formulations on the response of mice to infectious challenge with listeria monocytogenes, influenza a or candida albicans in order to test the efficacy of specialized ingredients. cf- outhred female mice ( - g) were fed purina # chow, commercially available osmolite hn ®, perative ® or impact*. mortality and tissue burden of pathogen were examined during the d period following induced infection. the results indicate that there were no differences between the groups fed the liquid formulas with regards to mean survival time or % survivors in these models of infection. examination of spleens in the groups challenged with l. monocytogenes and kidneys in the groups challenged with c. albicans revealed no differences in tissue burden of pathogenic organisms. alterations in the length of pre-feeding from to days prior to infection did not affect these results. these data demonstrate that, contrary to previous reports, the use of specialized nutrients did not improve resistance to disease in mice challenged with l. monocytogenese, influenza a or c. albicans as compared with mice fed osmolite hn. animals fed standard chow tended to be more resistant to infectious disease than those fed the liquid formulas perhaps due to the form of diet or complexity of ingredients. the results indicate that these animal models of infectious challenge may be of limited use to distinguish effects of modified nutrient composition of enteral formulas. laboratories the beneficial effects of sesame oil (ses) have been attributed to the presence ofsesamin, a non-fat constituent which is claimed to iultibit a- desaturase activity. we investigated the effects of ad libidum feeding of ses enriched diet on fatty acid composition of phospholipids from plasma, liver, on eicosanoid production, and on the protective effects in mice after cecal ligation and puncture (clp) and compared with safflower oil (so) diets. olive and palm otis were added as filler fats to the so diet to maintain the ratios of saturated, and unsaturated fatty acids similar to ses diet. thus, while the total fat remained at wt%, the only difference between the two diets is the presence ofsesamin in ses diet. the animals were fed for weeks. the incorporation of dihomo-y-linolealc acid (dgla: : o)- ) in the phospholipids from plasma and from the liver for the group of mice fed so diet ranged between - % compared to the undetectable levels for those fed so diet. there were no significant differences in the incorporation of other fatty acids either in plasma or in the cell membranes between the two groups. following an intraperitoneal administration oflps ( p.g/kg body wt), the plasma levels of both pge , and txb , were significantly decreased (p< . ) by nearly % for the group of animals maintained on ses diet compared to those maintained on so diet. these findings suggest that sesamin inhibited the release of arachidonic acid (aa) which upon accumulation is retroconverted to dgla which could reflect a modest increase in the content of dgla. failure to decrease the formation of aa could mean that the effects of sesamin on chain elongation process may be after aa is formed, and that sesamin may not inhibit a- desaturase activity. on the other hand, our data suggest that sesamin present in sesame oil inhibited the activity of cyclooxygenase which converts aa to its metabolites), or alternatively could block the activity of phosphalipase a (pla ) (which releases aa from membrane phospholipids) as is evident from a marked decrease in eicosanoid production. the percentage of animals surviving after cecal ligation and puncture in rite group of mice fed ses diet was % ( / ) which was markedly higher (p< . ) compared to only % ( / ) for the so group. increase in survival in the group of mice fed ses diet was associated with nearly % reduction in the production of tnf in response to lps i.p. challenge, for ses group compared to so fed animals. our data suggest that sesamin, present in sesame oil, decreased the production tnf~, inhibited the activity ofpla and consequently offered a significant protection against clp. thus sesamin or sesame oil appear to be novel antiinflammatory agents which are present naturally in many edible products. [dept. surgery, ~e. deasoness hosp.,harvard medical school, boston, usa] stress causes alterations in the homeostasis of an organism with major changes in various organs, lifespans of cells, protein turnovers, metabolic pathways of activation or stimulation, energy mobilization, etc. punctual changes have been described in several organs and various cell types of the nervous, endocrine and immune systems. many stored or newly synthesized proteins are released to the bloodstream to be transported to target organs or to be disposed of. as stress alters protein synthesis and requirements in the targets, the bloodstream is a useful system for monitoring the relevant changes. we present here the pattern of newly synthesized or released serum proteins in c bl/ mice that have been stressed by immobilization. the proteins have been labelled with ssmethionine in vivo and the study was performed by two dimensional gel electrophoresis based on the method originally described by o'farrel. the electrophoretic run was carried out in an isodalt apparatus. a molecular dynamics laser scanner and the kepler image analysis system (lsb, rockville, usa) were used for modelling the radiofluorographic images. the radiofluorographic images of the gels from serum samples obtained from mice injected with i mci of ss-methionine reveal about protein spots, from which a small fraction -about are altered in both qualitative and quantitative manner. the major changes are in the range of to . d. the analysis displays a highly reproducible pattern, where clear differences between stressed and non-stressed conditions are shown. differences between patterns attributed to chronic vs. acute stress will be presented. patients with severe trauma or major surgery are known to acquire alterations in neutrophil functions which contribute to their enhanced susceptibility towards microbial infections. we analyzed neutrophil functions from fourty patients admitted for major surgery days and days preoperatively and on the ist and th postoperative days (study-days i, , , and ) in a randomized placebo-controlled double-blind study. patients were divided into two groups, one ~eceived days preoperatively an enteral nutrition enriched with omega- fatty acids, arginiee, and rna (impact), the other an isocaloric, not enriched control nutrition (placebo). meutrophils were isolated from the peripheral blood and stimulated with the ca-ionophor a ( . pm). the capacity of the respective neutrophils to generate leukotrienes was analyzed by rp-hplc. neutrophils from impact group generated significantly higher amounts of ltb (mean values . ng/l x i~ cells) compared to the placebo group ( . ng) at study-day [p, . ). in contrast, the capacity of neutrophils to produce ltb was not significantly different in both patients populations at study-days and . the omega-oxidation of ltb to its biologically less active metabolites oh-ltb and cooh-ltb was not significantly different in both groups. however, neutrophils from group f patients generate more ltc at study-day (p( / ). the capacity of the patients'neutrophils to generate reactive oxygen radicals upon stimulation with fmlp, pma or the caionopbore a exhibited patient dependent differences but no correlations to the repective nutritional supplementation as was measured by luminol dependent ohemiluminescense= these data indicate that an enteral nutrition enriched in omega- fatty acids lead to alterations in distinct parameters of neutrophil functions. clinical patient characteristics and mean caloric intake were similar between the two groups. both formula were tolerated well and the incidence of diarrhea was low. the number of infectious and wound complications as well as the apache ii score was evaluated for the two study groups. although the number of complications in the group supplemented with arginine, rna and omega- fatty acids was lower no significant difference in the occurance of infectious and wound complications has been observed between the two groups. however, the apache ii score indicated a significantly improved clinical outcome in patients with supplemented diet. in conclusion, early enteral nutrition with an arginine, omega- fatty acids and rna supplemented diet helps to recover more rapidly after surgical total parenteral nutrition (tpn} is associated with intestinal atrophy and dysfunction attributed to the absence of the non-essential amino acid glutamine in current parenteral nutrition solutions. in this animal study with male wistar-rats we tested the hypothesis that glutamine-dipeptide supplementation during tpn for days would restore small bowel atrophy and tpn induced dysfunction. a conventional tpn solution ( kcai/kg bw) was compared to an isocaloric and isonitrogenous tpn ( , g n/animal/d) supplemented with alanin-glutamin (ala-gin) dipeptide ( , g n derived from ala-gin = % gin-n). an enteral fed cqntrol group was included. mucosal thickness, villous height and architecture, absorption of water and glucose as well as dissacharidase activity of maltase and alkaline phosphatase were evaluated. total parenteral nutrition in rats causes villous atrophy, a significantly reduced absorption rate and a decreased activity of villous enzymes compared to an enteral fed control (p<o, ). addition of ala-gin to parenteral nutrition solutions prevents intestinal atrophy and restores functional alterations with a significantly increased rate of water and glucose absorption as well as a higher dissacharidase activity compared to the standard tpn group (p< , ). there was no significant difference found between the enteral fed control and the dipeptide supplemented animals concerning intestinal structure and absorption, the enzyme activity was significantly decreased in the ala-gin supplemented group compared to the enteral fed control (p< , ). in this rat model supplementation of parentera] nutrition solutions with ala-gin dipeptide restores tpn induced intestinal atrophy and dysfunction. boston. ~ usa injury, infection and major o ~'ations s~nulate a variety of factors whi~ initiate the body's response to th~ st~..sses. 'i"hese reactions are m~liated by a cbmage ia the neta'al hormonal, eac.ranmemt, by the elaboration of eytokines and tl~ougb the stimu/ation of other inflammatory mediators. this eatabolio setting zesulis in body protein loss, which g-taduai y erodes both fimetianal and stm~ tissue. wiu _b the normally nom'ished individual can withslzod "d~ response for a brief period of time, ircolonged eatabollsm l~ associated with ~,nmunosuppmssion, poor wound healing, and proioagcd convalescence. surgeons have rej ed on intraveaous or eater~ feedings to ~everse this process. a va~ety of data now indicates that it is extremely di~edt to replete protein-containing tissue d~g the eataholie state of illness; the umutt response to hypercaloise feedings in this setting is the incxeased accretion of body fat md water. administration of gxow~ ho~moae ~cesults i.a n shift of the body's oxidative machinery to fat rather than eazbohydmte utiq~afic~; ¢oncomitaatiy, protein synthesis is stimulated. this metabolic state may have clinical utility in p~e.n.ts who need to heal large wotmds, in those who need to gain strength in order to be w~ed ore veatilatory support, or those patients who ~ve mulfiorgan failure and nee~ to ealaaace protein syatsesis to in.suze recovery. als are now in progress to dcterm~e the benefit of growth hormone in these and other disease state.s, rn the fature, the increased use of growth hormone, will occur;, this and other growth factors will serve to su~ po~ the host and shorten convalesceace following catabolic diseases. our objective was to study effects of pre-trauma growth hormone (gh) treatment on liver and skeletal muscle metabolism in a trauma model in piglets. twenty-four piglets were randomized to three treatment groups; one group was pretreated with gh iu daily three days before the experiment (gh- ), another group treated with gh ie at the start of the surgical procedure (gh- ) and one group served as untreated controls (con). they underwent a standardized surgical procedure to place sampling cathethers in the portal, hepatic and femoral veins and doppler flow probes around the portal vein, the hepatic and the femoral arteries. all pigs recieved a primed constant infusion of u- c-glutamine. substrate fluxes were measured one (lh) and five ( b) hours after termination of the surgery. nitrogen excretion was significantly decreased in the gh treated animals (gh- : . + . mg/kg, gh-i: . +_. . mg/kg, con: . +- . mg/kg, p= . ). in the hindieg, there was reduced net glutamine efflux due to decreased absolute glutamine release (gh- : . + . gmol/min at lh and - . + . in.tool/rain at h, gh-i: - . + . i.tmol/min at lh and - . + . pmol/min at h, con: - . +_ . g mol/min at lh and - . + . /amol/min at h, p= . , p= . ), whereas the absolute uptake of glutamine in hindleg was unchanged (p= . ). glucose uptake in the hindleg was decreased (p= . ). net glutamine uptake in liver was attenuated (p= . at h), whereas net glutamate release from liver was increased (p= . ). thus, pre-trauma treatment with gh improved nitrogen economy, attenuated net glutamine loss from hindieg due to decreased absolute release, and attenuated hindleg glucose uptake. liver net glutanaine uptake was decerased and net glutamate release increased. patients with major abdominal surgery exhibit a considerable catabolic response with negative nitrogen balance and protein breakdown, which may have detrimental effects on outcome. we investigated the effects recombinant human growth hormone on substrate metabolism in parenterally fed patients. metabolic healthy patients were randomized to receive either placebo or hgh in a dosage of , ; , or , i.u. per kg bw. substrate oxidation rate, energy expenditure as well as short life proteins were measured daily. six patients were excluded from analysis as drop outs, due to surgical complications. we could find a dose-dependend effect of growth hormone on resting energy expenditure, carbohydrate oxidation, fat and proteine oxidation. with increasing growth hormone resting energy expenditure increased from . calories per kg body weight to . calories. this was mainly due to an increased carbohydrate and fat oxidation, while proteine oxidation decreased. this was in accordance with a decreased urea.production rate and an improvement in cumulative nitrogene balance, which increased from minus . to plus . g n / days. in consequence short life proteins were also increased. the only negative side effect was an increased blood glucose concentration in some of the patients, making insuline administration mandatory in patients. our results are in accordance with other studies showing improvement of nitrogen balance, maintainance of lean body mass and muscular strength. if growth hormone administration may have any impact on morbidity, mortality and length of hospital stay in these patients it should be evaluated in a larger number of patients. in patient after liver transplantation (oltx) the effect of recombinant human growth hormone (rhgh) on protein metabolism and hormonal changes was studied. patients and methods: the underlying disease in all patients was end stage liver cirrhosis, non of the patients included in the study was suffering from malignancies. the patients were randomly allocated to receive either u of rhgh bid (sc) or no alternative medication. the study period lasted days. from daily hrs udne collection urinary nitrogen loss and daily loss of urea were determined. body compostion analysis (bca, bioimpedance, akern-ltaly) was performed preoperativly and at the postoperative days and to evaluate changes in body water and body cell mass (bcm). the effect on resting energy expenditure (ree) was analysed using indirekt calorimetry wrhin the same intervalls (metabolic monitor, datex). blood levels of gh, igf and igf , igfbp were measured daily during the study period, on day a hour profile of gh was.performed. samples were collected each minutes. results: cumulative udnary nitrogen and urea loss were not signifcantly different for the whole study period but for the first study days. bia indicated for the rhgh-group a loss of bcm to a lesser extent than for the controls (n.s.), changes in bodywater where similar for both groups. ree changes (kcal/kg bcm) were not significantly different for the two groups. blood levels of gh and igf differed significantly between the groups for the whole study period, but not igf and igfbp- . the circadian rhythm of endogenous gh-excretion had not been altered by gh, but absolute levels of gh were increased. conclusion: during the first days after oltx we could proof the protein sparing effect of gh treatment. although the differences between the two groups were not significantly different for the whole study period the results showed on all days a less catabolic situation for the rhgh treated patients. to possibly improve protein balance after major abdominal surgery we studied the effects of gh on protein metabolism after ltx. excluding malignant diseases, candidates for ltx were randomized to either postoperatively receive the usual treatment (co=control group, n= , age range - yr, bmi . + . kg/m ) or for days additional gh ( x i.u.s.c.) (gh group, n= , - yr, . + . kg/m ). metabolic studies (calorimetry and lsc-leucine infusion) were performed (test a) and days (test b) after surgery. tracer analysis was done by gas chromatography-mass spectrometry. during the -week study clinical parameters did not differ significantly between groups; however, there was a tendency for increased glucose levels, insulin need, and energy expenditure at the time of test b in the gh group. leucine oxidation (x+se) during test a;b was + ; + (co) and + ; ± (gh) pmol/kg ffm/h (n.s.). protein breakdown was ± ; + (co) and + ; + (gh) pmol/kg ffm/h (n.s.). nonoxidative-leuine disposal was ± ; + (co) and ± ; + (gh) pmol/kg ffm/h (p< . for the rise in the gh group). tracer data were supported by cumulative urea nitrogen excretion (days - : gh + vs co ± g; p< . ). we conclude that after major abdominal surgery gh lowers nitrogen excretion by increasing total body protein synthesis. it is not known which organs contribute to this effect. in several investigations it has been shown that the protein saving effect of recombinant human growth hormone (rhgh) in the postoperative state is accompanied by raised igf- levels in plasma. it was concluded that the anabolic effects of rhgh were probably, at least in part, mediated by igf- . this study was aimed at detecting the efficacy of igf-i on modulation of the protein metabolism in the catabolic state. patients and methodes: patients (both sexes, age: - years, bmi - kg/m ) with major upper gastrointestinal surgery (gastrectomy or partial gastrectomy) received ug rhlgf- /kg body weight/twice daily or placebo during treatment days beginning on the first postoperative day in a double-blind, randomized study. all patients received hypocaloric and hyponitrogenous total parenteral nutrition ( g chng, o. g aa/kg) troughout the whole study period. cumulated nitrogen balance, -methyl-histidin exretion in urine as well as serum-igf- levels have been determined. the two tailed wilcoxon test was used for statistical analysis results: first results will be presented previous studies have suggested that growth hormone (gh) potentiates various activities of leukecytes. however, it is not clear whether gh can improve survival of animals with gram-negative sepsis. we investigated the effects of gh on host response to infection in septic mice model. methods balb]c mice were randomized to groups (n= /gronp): gh-high ( + mg/kg/day), gh-iow ( a mg/kg/day) or control (saline). following subcutaneous treatment (every hours for days) with gh or saline, mice were challenged i.p. with e.coli (lxl /body). survival rate was recorded every hours. in the next experiment, gh-high and control animals were sacrificed hours after bacterial challenge. peritoneal cavity was lavaged with ml saline and the peritoneal exudative cells (pec) were harvested and counted. colony forming units (cfu) of bacteria in the peritoneal lavaged fluid (plf), liver, lung and blood were determined. pec were cultured in vitro for hours with lipopolymccharide ( p.g/ml in normotensive adults growth hormone (gh) rises when clonidine challenge is performed. recently evidence was shown for gh to accelerate wound healing. while gh secretion patterns are inconstant the gh-dependant insulin like growth factor (igf- ) and the insulin like growth factor binding protein (igfbp- ) reflect the integrated gh secretion over days. since we administer clonidine to patients with acute alcohol abuse we determined plasma levels of igf- and igfbp- . materials and methods: patients sheduled for esophagus resection were investigated once they had given written informed consent. patients showed acute alcohol abuse and were treated with clonidine postoperatively• patients with no history of acute alcohol abuse served as controls. besides liver enzymes igf- and igfbp- were determined. results: both groups had a comparable hepatic capacity of synthesis with decreased cholinesterase levels as to be expected after a major operation. regression analysis of igf- and igfbp- levels showed no differences between the groups. discussion: igf- and igfbp- plasma levels are not increased in normotensive patients who are treated with clonidine for prevention of postoperative alcohol withdrawl syndrom. further studies are required to discriminate whether our results are due to impaired postoperative liver function or if clonidine has not any impact on the gh-igf-axis in patients with alcohol abuse. recent studies have revealed that igf-i has several immanoregulatory roles. however, little is known about its effects on septic animals. we tested whether igf-i could increase the survival o f mice challenged intraperitoneally (i.p.) with e. coli. m~thqd$ balb/c mice received either high dose igf-i (n= , mg/kg/day), low dose igf-i (n= , . mg/kg/day) or saline ( = ) every eight hours subcutaneously for six days. animals were then challenged i.p. with lx e. coli. survival was observed every two hours. in the other experiment, mice that received high dose igf-i or saline were sacrificed four hours after bacterial challenge. peritoneal cavity was lavaged with ml saline and the peritoneal exudative ceils (pec) were harvested and counted. colony forming units (cfu) of bacteria in the peritoneal lavaged fluid (plf), liver, lung and blood were determined. in addition, pec were cultured/n vitro for hours with lipopolysaccharide ( ).tg/ml). tumor necrosis factor (tnf) in the supernatants of pec culture, plf and serum were measured by l bioassay. results igf-i improved the survival rate at a dose of mg/kg/day. severe chronic neutropenia (scn) is a disorder characterized by severe neutropenia secondary to either a maturational arrest of myelopoiesis at the level of promyelocytes (kostmann-syndrome; scn) or regular cyclic fluctuations in the number of blood neutrophils with a median absolute neutrophil count (anc) of less than /~tl (cyclic neutropenia). patients suffering from scn have an impaired acute inflammatory response to injury and an increased susceptibility to infections, i.e. bacterial or fungal infections, resulting in chronic oral inflammation, severe pneumonia, abscess formation and bacteraemia. we have treated patients ( scn, cyclic neutropenia) with r-methug-csf (filgrastim). thirty of patients with scn and all cyclic neutropenia patients responded to this treatment with an increase of the median anc to greater than /~tl. the dose of r-methug-csf needed to achieve and maintain the response varied between . and ~tg/kg/d. long-term treatment did not exhaust myelopoiesis: the anc remained stable after up to years of treatment and antibodies to r-methug-csf were not detected. the increase of anc was associated with dramatic clinical responses: significant reduction of severe bacterial infections, reduction of intravenous antibiotic treatment, and reduction of hospitalizations. no severe bacterial infections occurred in any of the r-rnethug-csf responders during longterm treatment. severe adverse events, most likely due to the underlying disease, included the development of mds/leukemia in two patients, and osteopenia/osteoporosis in patients. these results demonstrate the beneficial effects of r-methug-csf treatment in severe chronic neutropenia patients. the newborn is predisposed to mortality during infections due in large part to developmentally immature host defenses. recently cytokines have been identified that regulate the development of these defenses. one such cytokine is specific for neutrophils and is termed granulocyte colony stimulating factor (g-csf). in the studies to be discussed, we have attempted to impact hematopoiesis in the fetus using exogenous rhg-csf delivered through the pregnant dam. our rationale was that an enhanced myeloid system may result from such in utero treatment leading to, perhaps, enhanced protection to microbial insult. rhg-csf crossed the placenta and reached peak fetal serum concentrations hours after administration. these levels were -fold lower than the levels detected in the adult dams serum. white blood cell counts were elevated approximately - -fold over control newborn blood. this increase was due to circulating numbers of neutrophils. the marrows from these pups were hyperplastic consisting of predominately immature and mature neutrophilic cells. no changes were seen in the thymus or livers. pups born to mothers treated with rhg-csf since day of gestation (parturition in rodents occurs on approximately day ) survived a lethal challenge of group b- hemolytic streptococcus. in contrast their untreated yet infected counterparts did not survive. recent clinical developments have led to trials of rhg-csf in certain neutropenic states including cyclic and chronic neutropenia. we have identified several of these patients who were pregnant during treatment with rhg-csf. biologically and anfigenically identifiable rhg-csf was detected at increased levels in the cord serum of these neonates. we will discuss our findings with these patients in light of our animal model of neonatal myeloid development. we have previously demonstrated decreased g-csf production and g-csf mrna expression from activated mononuclear cells from newboms compared to adults (cairo, pediatr res : , ) . we have also recently observed that administration of a single dose of rhg-cse to one-day-old newborn rats induced significant neatrophilia, while administration for days induced neulrophilia and increased the bone marrow (bm) neutrophil storage and progenitor pools (cairo, blood : , cairo, pediatr res : , ) . we embarked on a phase i trial exploring the safety, pharmacokinetics, and biological efficacy of g-csf in newborns with presumed sepsis. infants < hrs old with a diagnosis of presumed sepsis were stratified into three groups: very preterm ( - wk), preterm ( - wk), and term (> wk) and randomized to receive either a placebo or , , and gg/kg/qd or and p.g/kg/bid of rhg-csf infused by pump over hour for consecutive days. cbcs were obtained at , , , , and hrs. tibial bone marrow aspirations were performed hrs after study entry and differential counts and cfu-gm pools were determined. c bi expression was determined at and hrs after rhg-csf, and g-csf pharmacokinetics were performed after the first dose of rhg-csf utilizing a sandwich elisa. a significant increase in the anc was observed at , and hrs following administration of both and ~tg/kg/d of rhg-csf. the maximum increase in the anc occurred hrs after and ~tg/kg/d ( - %) (p< . ) and ( % -+ %) (p< . ), respectively. there was a significant dose-dapendeat increase in the bm neutrophil storage pool ( _+ % vs. + %) (p< . ) (placebo vs. ~tg/kg/d). there was no significant difference in the nantrophil proliferative pool. an increase in cfu-gm and cfu-gemm was seen at all doses tested, compared to placebo ( . _+ . vs. -+ ) (colonies/l(p cells/plate). c bi expression was significantly increased hrs after bg/kg/d of rhg-csf ( + % vs. +- %) (p< . ). peak serum g-csf levels occurred at hrs and were dosedependent. the half-life of rhg-cse was . + . hrs. most importantly, there was no observed toxicity from g-csf in all patients studied. of patients were on ventilators prior to administration of rhg-csf and there was no increase in pulmonary toxicity. these preliminary data suggest that rhg-csf is well tolerated at all gestational ages in newborns with presumed sepsis. a multi-center phase ii/iii randomized double-blindad placebo controlled trial is required to determine the efficacy of rhg-csf in this clinical setting. we investigated the effects of recombinant canine granulocyte-colony stimulating factor (g-csf) on survival, cardiopulmonary function, serum endotoxin levels and tumor necrosis factor (tnf) levels in a canine model of lethal bacterial septic shock (clinical research. : , ) . methods: awake ylo beagles had serial cardiopulmonary and laboratory studies before and for up to days after intraperitoneal placement of an e. celi infected clot. nine days before and daily until days after clot placement, animals received high (n= ) or low dose (n= ) g-csf or protein control (n= ) subcutaneously. results: survival in high dose g-csf animals ( / ) was significantly improved compared to low dose ( ) and controls ( ) (p< . wilcoxon). high dose g-csf also improved cardiovascular function evidenced by a higher mean left ventricular ejection fraction (day after clot, p< . ) and mean arterial pressure (day , p< , ) compared to low dose and controls. high dose rcg-csf increased (p< . ) peripheral neutrophil numbers both before and after clot implantation ( hours to days) compared to low dose and controls. in addition, high dose rcg-csf produced a more rapid (p< . ) rise (day ) and fall (day ) in alveolar neutrophils determined by bronchoalveolar lavage compared to low dose and controls. lastly, high dose rcg-csf decreased serum endotoxin ( to h, p< . ) and tumor necrosis factor (tnf, h, p< . ) levels compared to low dose and controls. discussion: these data suggest that therapy with g-csf sufficient to increase peripheral neutrophil numbers during peritonitis and septic shock may augment host defense and endotoxin clearance, reduce cytokine levels (tnf) and improve cardiovascular function and survival. the use of g-csf in sepsis prophylaxis in neutropenic patients is well established and has been ascribed to accelerated recovery in granulccyte counts. here, an additional sepsis-prophylactic property could be demonstrated in healthy volunteers: eleven volunteers were employed in a sinqle-btind, controlled study and were given uq g-csf or saline placebo via subcutaneous injection. blood was withdrawn immediately before and or hours later. lps-inducible tnf, il- , stnf-r p and il-lra were assessed in the supernatant of whole blood incubations stimulated with ug/ml lps from salmonella abortus equi. similarly to previous animal studies, lps-inducible tnf was attenuated by about % hrs. after treatment. the same was true of il-lb. in contrast, lps-inducible stnf-r p which was indetectable in blood incubations from untreated donors increased dramatically hrs. after g-csf treatment. il-lra found after lps challenge was increased tenfold by g-csf treatment. it is concluded that g-csf treatment switches peripheral leukocytes to an antiinflammatery state characterized by an attenuation of il-i and tnf releasing capacity and an augmentation of the release of cytokine antagonists. this findinq minht offer a novel concept in septic shock prophylaxis. objective.the aim of the study was to investigate the effect of recombinant human g-csf (rhg-csf) on survival, bone marrow neutrophil myelopoiesis, neutrophil counts, levels of bacteria and some important sepsis mediators in a model of rat abdominal sepsis. lethal peritonitis was induced with a mm coecal perforation (cp) in male wistar rats. rhg-csf was administered as /.tg/kg iv every h, first dose at sepsis induction. bone marrow neutrophi] progenitors were determined as blast colonies, cfu-gm and cfu-g. neutrophils and bacteria were determined in peripheral blood and peritoneal fluid. lps, tnf, endothelin and lactate were measured in blood from femoral vein. mortality rates were registered with g-csf treatment starting either or days before or hours after cp. results. mortality was reduced from % to about % with rhg-csf intervention and there was no difference between the pretreatment and treatment groups. bone marrow blast colonies were not influenced while neutrophil myelopoiesis was augmented at the stages of cfu-gm and cfu-g. neutrophils in blood and peritoneal cavity were enhanced and numbers of bacteria in the same compartments were substantially reduced. circulating lps, tnf, endothelin and lactate were attenuated the first hours after cp. neutrophil myelopoiesis is augmented with increased number of neutrophils in blood and peritoneal cavity, resulting in enhanced clearance of pathogens. lps, tnf, endothelin and lactate are suppressed the first hours during sepsis course. a. wendel, j. barsig, g. tiegs gm-csf stimulates the proliferation and differentiation of granulocytic and monocytic progenitor cells. in addition the hemopoietic cytokine activates the inflammatory response in mature leukocytes. the priming effect of gm-csf towards lipopolysaccharide (lps)-induced cytokine production in vitro has been described, but little is known about proinflammatory gm-csf effects in vivo. we detected gm-csf in plasma of lps-challenged mice with kinetics similar to tnf, reaching peak levels h after lps administration. gm-csf pretreatment ( ~tg/kg i.v.) enhanced mortality in mice challenged by a sublethal dose of lps. plasma levels of tumor necrosis factor (tnf) and interleukin- (il- ) were significantly enhanced. a monoclonal antibody, which neutralizes gm-csf bioactivity, rendered mice less sensitive towards lethal lps-challenge. tnf-and il- -tevels were reduced in these mice compared to control animals without antibody treatment. in addition, severalfold potentiation of lps-induced cytokine release by gm-csf was observed in vitro in murine bone marrow cell cultures. these data demonstrate the proinflammatory capacity of gm-csf and suggest that the hemopoietic cytokine plays also a role as an endogenous modulator of lps toxicity. immune dysfunction, developing in the wake of multiple trauma, overwhelming infection and other forms of critical surgical illnes% is associated with increased infections, morbidity and mortality. the mechanisms responsible for alterations in immune regulation are incompletely understood but monocyte appear to play a central role. polymorphonuclear leukocytes (pmn) are known to play a central role in the inflammatory response of the host toward invading microrganisms. reports of defects in all the aspeots of pmn function have been accumulated in recent years. the possible role of gm-csf in modifing the state of immuno suppression detected in severe intraabdominal infected pt~. inspite of surgical appropriate procedures and in reducing the expected mortality is investigated. the safety of rh-gm-csf administration in sepsis is also evaluated. a double blind randomized study is proposed. this study include icu patients who do not exhibit signs of shock and/or ards, with clinical signs and symptoms of abdominal infection. immunodepressed patients-aids, chronic chemotherapy or chronic steroid administration do not partecipate to the study. patients will receive rgm-csf (l~g/kg/day) or placebo in hs. continuous infusion for days. safetyandefyieacy will be assessed till to day . the apache ii score is adopted for risk stratification of patients because it is reliable and validated, objective and composed of information that is indipendent of diagnostic criteria. patient's entry criteria is apache ii > (score corresponds to expected mortality rate of %).in this protocol the surgeons report the judgement of the efficacy of surgical procedure to remove or not the focus of infection. objectives: infections and subsequent septic responses remain the leading cause of death among surgical intensive care (sicu) patients despite tmprovetaunts in supportive care and brond-epectrum antibiotics. usually invading bacteria are efficiently cleared by neutrophil granulocytes. however, during sepsis various neatrophil dysfunctions have been demonstrated, leading to impaired host defense. granulocyte colony-stimulating factor (g-csf) induces a sustained increase in circulating neutrophils and enhances various noutrophil functions. it was the purpose of the present study, to evaluate the safety and efficacy of g-csf (filgrastim) in sicu patients at risk of sepsis. materiel a.d methods: the study was designed as an open-label phase-ll study of filgrastim. ten consecutive slcu patients, with a therapeutic interveotion score greater than , were included in the study. filgrastim was given by daily continuous intravenous infusion for days or discharge from the sicu. apache ll-score, multiple-organ-failure (mof) score, definitions of infections, sepsis, systemic inflammatory response syndrome (sirs), and acute respiratory failure were applied daily. a response to filgrastinl th_erapy was defined as an improvement in disease severity quantified by a decrease of > apache i score points on day after onset of treatment. results: none of the patients developed a sepsis or mof later on and no patient died during hospitalization. specific postoperative complications occured in one patient ~jth a leekage of the oesophagou-gastric anastomosis after oesophageus resection. at study entry the leucocytes amounted to . + . /~tl (mean + sem) and reached a level of . +_ . /tal at day after onset offilgrastim therapy. the apache ii score initally was + . (mean + sem) and as an indicator of filgrastim response a decrease of points ~dthin days oceured in out ot patients. filgrastim was well tolerated, side effects were not noted. growth of solid tumors might be modulated by the activity of inflammatory and/or immune effector cells of undefined specificity. in this study patients undergoing surgical treatment for gastric (n= ) or colorectal (n= ) cancers were evaluated for endogenous serum levels of granulocyte colony-stimulatingfactor (g-csf) during a pre-and postoperative time period. from the same blood specimens mononuelcar cells (mnc) were prepared. the release of ifn-%, and il- , which are secreted by thl cells, were stimulated in vitro by pha during a cell culture period up to hours. the patients were further classified for their immunreactivity by responses in dth skin testing to seven different antigens (e.g. tetanus toxoid, ppd, diphtheria toxin, trichophyton, streptococcus, candida and proteus antigens). dth testing has been repeated in each patient two remarkable results were obtained. the serum levels of endogenous g-cse showed a biphasic increase with maximum values of pg/ml (preoperative < pg/ml) on day and day to after surgical treatment. similar patterns of g-csf production were found in both groups of patients with gastric or colorectal cancers. high serum levels of g-csf were significantly (p < , ) correlated with infectious complications in patients whh gastric cancer (n= / ). secondly patients could be arranged into two groups according to an anergic (n= ) or normergi¢ (n = ) responsiveness in dth testing. the frequency of anergi¢ responsiveness was similar in both patients with gastric (n= / ) or colorectal (n= / ) cancers. interestingly we found a significant correlation (p < , ) between low serum levels of g-csf and anergy during the postoperative period in both groups. stimulation of mncs from anergic patients (n= ) within the pre-and postoperative period resulted in reduced mean values (about %) for ifn-ff release (preoperative means llo pg/nfl), if compared to patients with normergic dth (n= , preoperative means pg/ml). similar, but less significant results were obtained for il- secretion. our results confirm a correlation between infectious complications and g-csf in the postoperative period, however elevated levels were also found in some patients without any signs of infections. more interestingly there might be an association between cytokine (c~csf, ifn-% and il- ) release and dth, which is known to be mediated by activated thl calls. to recognize anergic dth as a possible higher risk in the postoperative outcome of cancer patients extended periods of observation are needed. objectives of the study effects of recombinant huraan granulocyte colony-stimulating factor(rhc-csf)a galnst severe septic infections were investigated by its single use or by its corn b{nation with cephera antibiotlcs.we examined its effects on the mortality,and circulating blood neutrophyis counts and functlons,such as phagocytic activity and h production using the rat severe septic model. rats were subcutaneously administsrd rhc~csf(s orl o ~ g/k~ body wt)after on set of peritonitis brought about by cecal ]igation and one puncture withe -gaug e needle once a day for three days.in addjtlon,cefmetazol na(cmz)( m$/k bo dy wt)was injected intrarnustularly to the rats tv~ce a day for three days. cirehlatlng blood neutrophyls counts were determoned electronically with a hem ocytometer,and blood smears stained with may~runwaldm.qlemsa~taln. neutrophyls functions in vltro,such as phagocytic activity and h producti on using the rat severe septic model was analyzvd by automated flow cytometri c single cell-analysis methods. the reortallty rate after weeks was significantly decreased by administratlon of rh~-csf(p< , ).ln addjtion,a combination therapy of rhg-csf wlte cephern ant~biotics(cmz)showed a significantly survive] advantage and the rate had b een reached . %. nextly,treatn%ent wlth rhg-csf(s ~ $/k body wt)increased the nuzaber of the peripheral blood neutrophjls slgn[fieantly(p< . ). iv~oreover,functions of neutrophlis which were phagocytic activity and h p roduction were remarkably enhanced by admlnlstratlon of rhg-cs~( ~ /ks b ody wt) (p< .( ). these findings suggest that combination therapy of rhcrcsf with cephern antib iotlcs(cmz)is an efficient regime against severe infectlons.and the increased ne utrophils counts and enhanced neutrophiis functions were played a important ro le about the survival advantage. granulocyte macrophage colony-stimulating factor (gm-csf) is a haematopoietic growth factor active on neutrophils and macrophages. leukopenia often occurs following renal transplantation and can be associated with infection and/or the myelosuppressive effect of azathioprine. aim: we report the use of gm-csf in renal allograft recipients with leukopenia. nonglycosylated recombinant gm-csf was obtained from e. coli transvected by human gm-csf gene. m~terial ~,nd methods : written informed consent was obtained from all patients. patients were suffering from toxic neutropenia (neutrophils < /mm ) with medullar hypocellularity on bone marrow aspiration, or leukopenia (neutrophils < /ram ) with cytomegalovirus infection requiring ganciclovir administtation. gm-csf was given subcutaneously at a dally dose of to mcg/kg/day, according to renal function. results : in all cases, neutrophil counts returned to normal levels within to days. in most of them, spectacular correction was observed within hours, with a single injection. adverse events due to gm-csf at this dose were mild and easily managed ( cases of bone pain treated with paracetamol). one acute rejection episode was observed after correction of leukopenia. conclusion : on the basis of this study, it appears that gm-csf at a dose below mcg/kg/day is an effective treatment for renal transplant recipients with leukopenia associated with cmv infection or toxic neutropenia. department of nephrology, , rue de s~vres, hopital necker, paris, france. changes in serum g-csf and il- after surgical intervention hitoshi toda , atsuo murata , hidewaki nakagawa , takesada mori , nariaki matsuura osaka university medical school, osaka, wakayama medical school, wakayama, japan we measured serum immunoreactive interleukin (il- ) and granulocyte colony-stimulating factor (g-csf) levels of the patients undergoing major thoraco-abdominal surgery for esophageal cancer. serum samples were collected from eight patients on the day before surgery, at the time of operation, and thereafter at suitable intervals for one week. il- and g-csf were measured by means of enzyme linked immunoassay. the normal range of serum ]l- was less than pg/ml and g-csf less than pg/ml. values between groups were compared with linear regression analysis. both serum g-csf and il- levels reached their maximal levels at the first postoperative day and decreased thereafter. the correlation between g-csf (y) and il- (x) was y= . x+ . (r= . , n= , p< . ), showing a significant correlation. in the case who suffered from aspiration pneumonia and ards at the second postoperative day, the peak level of il- was pg/ml and g-csf pg/ml respectively. the estimated value of g-csf was pg/mi by the regression equation. this means the real g-cse level was less than half of the estimated value. it suggests that low responsiveness of g-csf is one of the reason of immunodeficient state after the major surgery, neutrophils from injured patients ingest and kill bacteria less efficiently as compared to those of healthy individuals, probably reflecting the suppression in respiratoly burst which occurs after severe trauma. one of the main mechanisms of killing bacteria by neutrophil granulocytes is production of oxygen radicals (respiratory burst). granulocyte colony-stimulating factor (g-csf), a kilodalton cytokine, leads to a sustained, dose-dependent increase in circulating neutrophils. thus, it was investigated whether filgrastim (recombinant human granulocyte colony-stimulating factor, rhg-csf) therapy fits for prophylaxis of sepsis in postoperative/posttraumatic patients, and whether, besides an expected increase in neutrophil count, filgrastim would also augment neutrophil function. material and methods: this study was designed as an open label, prospective phase ii study of filgrastim and performed in a surgical intensive care unit (sicu) (university hospital). postoperative/post-traumatic patients with a therapeutic intervention scoring system (tiss) score greater than were treated with filgrastim ( . - l.tg/kg/day) for prophylaxis of sepsis on days or until discharge from the sicu. production of oxygen radicals can be quantified by analysis of fmlp-and zymosan-induced chemiluminescence. neutrophil oxygen radical production was tested by fmlp-and zymosan-induced chemiluminescence by the polymorphonuclear cells (pmn) of these patients in multiple blood samples over a period of up to days. results: none of the patients treated with filgrastim for prophylaxis of sepsis developed sepsis. in vitro fmlp-induced ( - reel/l) neutrophil oxygen radical production was significantly increased under therapy with filgrastim by a maximum of % +- % ( % - %) compared to pretreatment values of %. tapering of filgrastim resulted in a reduction of fmlp-induced neutrophil oxygen radical production within hours. in contrast, zymosan-induced neutrophil oxygen radical production was not affected by filgrastim treatment. conclusions: besides its quantitative effect on neutrophil counts enhanced neutrophil function, documented here as increased fmlp-induced oxygen radical production, may account for the beneficial effect of filgrastim for prophylaxis of sepsis in posttraumatic/post-operative patients. granulocyte colony stimulating factor (g-csf) and granulocytemacrophage colony stimulating factor (gm-csf) have been recently introduced in the treatment of chemotherapy-induced neutropenia. effects of these csfs on cellular immune system were evaluated in neutropenic gynecological cancer patients during chemotherapy. g-csf and gm-csf were equally able to induce a rapid recovery of white cell count within one or two days. g-csf treatment resulted in a significantly higher concentration of leukocytes measured in the peripheral blood although by gm-csf a sufficient effect was achieved (p< . ). before initiation of csf treatment urinary neopterin was similar in both groups of patients ( +/- and +/- lamol/mol creatinine for gm-csf and g-csf respectively expressed as mean +/-one sd). in g-csf treated patient only a marginal induction of neopterin was observed. on day the mean value was about % above the basal level (p< . ). on the other hand gm-csf treated patients were characterized by a pronounced increase in urinary neopterin levels. in comparison with the basal level a more than fold induction was noted and the difference between g-csf and gm-csf was highly significant (p< . ). this effect was confirmed in vitro by investigating the effects of these csfs on interferon-gamma mediated pathways in thp- human myelomonocytic cells. results suggest activation of immune effector cells by gm-csf which may help the organism to overcome infections. however, activated macrophages produce several growth factors which may increase malignant proliferation, and augmented neopterin production as sign of macrophage activation has also been associated with poor prognosis m several malignancies. more data are therefore necessary to clarify whether csf mediated immune activation is beneficial or deleterious for cancer patients but considering our results caution in applying csfs in oncology seems advised. from a historical perspective, the development of humoral immunity to bacterial endotoxin has assumed a prominent position in the spectrum of therapeutic approaches which have been explored for the treatment of gram negative septic shock. predicated upon the fact that rough strains of bacteria manifest lps containing exclusively conserved structural features common to lps from all gram negatives, specific antibodies were elicited which conveyed cross protective immunity in experimental models of bacteremia and endotoxemia. such studies culminated in a well-conducted, randomized, double-blind placebo-controlled clinical trial using passively administered human polyclonal antiserum to treat patients with suspected gram negative sepsis. the efficacy of treatment established in that trial spurred efforts to develop monoclonai reagents which, to date, have not been uniformly successful in reproducing those earlier studies with polyclonai antibodies. nevertheless, the numerous successes which have been documented in experimental models of endotoxemia continue to foster promise for this immunotherapeutie approach. several recent studies with human polyclonalimrnunoglobulin preparations containing antibodies reactive with lps and lipid a have yielded promising results in treatment of patients with sepsis. in addition, the recent development of an antiidiotypic monoclonal antibody which reflects an internal image of a kdo specific monoclonal antibody has provided an alternative experimental approach to generate anti-lps antibody. immunization of mice with the antiidiotype provides significant protection against subsequent lps lethality consistent with the development of circulating immunoglobulin specific for lps. thus, the use of polyclonal immunoglobulins contrives to provide an alternative and potentially cost effective method for the treatment of endotoxin shock. supported by r a and pot ca . john holaday, anne fortier, shawn green, glenn swartz, john madsen, carol naey, and jan dijkstra entremed, inc.. rockville, md, . at the time of diagnosis, the signs and symptoms of septic shock are an indication that the systemic inflammatory response is well underway; thus, it has been argued that the endotoxin "cat is out of the bag", and that subsequent passive immunization may be too late to achieve therapeutic benefit. our approach has been to evaluate active immunization as a prophylax~s against sepsis. mice were inoculated twice (two weeks apart) with liposomes containing dmpc[i. ], dmpg[ . ], cholesterol [ . ] , and monophosphoryl lipid a [ - gg/txmole phospholipid] by several routes (i.p., i.m.), and serum was collected - days after each inoculation. after a single injection, highest tilers of ab were produced in mice inoculated i.p., but mice inoculated by all routes produced anti-lipid a ab. following the second injection. ab levels were roughly equivalent in mice inoculated by all routes, regardless of lipid a concentration. mice vaccinated i.p. with liposomes containing , or gg lipid a were treated with cyclophosphamide to produce neutroperda and then challenged with e. cole in an infection model of gram negative sepsis. the lds for control (liposomes with no lipid a) mice was x bacteria; ld for mice vaccinated with p.g was x ( -fold increase in resistance) and with ~tg was x bacteria ( -laid increase in resistance). mice vaccinated as before were also treated with actinomyein d to increase sensitivity to lps (salmonella minnesota) challenge in an endotoxemia model of grain negative sepsis. the ld for control (liposomes with no lipid a) mice was ng lps; the ld for gg lipid a was rig lps ( -fold increase in resistance) and for xg was ng lps ( -fold increase in resistance). mice were similarly vaccinated and challenged with an aggressive gram negative pathogen, francfsella tularensis. the ld of franciseua in normal mice or mice inoculated with liposomes without lipid a was - bacteria. in contrast, mice vaccinated with liposomal lipid a ( ggl survived challenges as high as , bacteria, ( logs of protection). the impressive protective capacity of this vaccine did not correlate with ab liter in any of the sepsis models, nor did it correlate with classic nonspeeific events, such as macrophage activation. maerophages harvested from the peritoneum of mice vaccinated and protected against sequelae of gram negative infections did not spontaneously kill the bacteria in vitro, but could be activated by ifn-y for antimicrobial activity equivalent to that of macrophages from unt#eated mice. research is underway to defme the protective mechanism(s) activated by this liposomal-lipid a vaccine. intervention by monophosphoryl lipid a in septic shock jon a. rudbach, ribi immunochem research, inc., hamilton, montana, usa monophosphoryl lipid a (mla), the clinical form of which is called mpl®-immunostimulant, has been tested extensively as an intervenient material in septic shock. mla is protective when given to experimental animals prior to a live microbial challenge or challenge with lethal doses of microbial products or certain cytokines. this is shown with gram negative and gram positive bacteria, gram negative bacterial endotoxins, and gram positive bacterial exotoxins. furthermore, animals treated with a regimen of mla which results in a refractory state to a lethal dose of gram negative bacterial endotoxin concomitantly display increased resistance to a live bacterial challenge. thus, both endotoxin tolerance and nonspeciflc resistance to infection can be manifested simultaneously. also, prophylactic doses of mla do not interfere with other therapies given subsequently; an additive or a synergistic protective effect can be demonstrated with certain combinatorial treatment regimens, such as mla followed by antiendotoxin monoclonal antibodies. the preclinical studies were extended to human trials wherein the safety of agonistic doses of mla was verified. furthermore, when mla was administered to human volunteers hr before challenge with a pharmacologically active dose of reference endotoxin, febrile, cardiac, tnf, il- , and il- responses were all decreased significantly as compared with the responses of subjects pretreated with a control solution and challenged with endotoxin. human trials with mla are being extended into patient cohorts which have high probabilities of developing septic shock; this will expand the safety base and establish clinical efficacy for mpl®-immunostimulant. a considerable body of in vitro evidence supports the concept that the effects of lps on cells of the immune/inflammatory systems are controlled by interactions of lps with cd . to evaluate if blocking lps-cd interactions has potential as a therapeutic in septic shock we have evaluated the effect of anti-cdi monoclonal antibody (mab) on lps-induced cytokine production and physiologic changes in an experimental model of endotoxin shock performed in cynomolgus monkeys. a novel model has been established where animals were treated with interferongamma for three days prior to infusion of highly purified lps over an eight hour period. in this model lps challenge resulted in marked release of eytokines in the blood, substantial hemodynamic changes, release of liver enzymes and alteration in lung permeability observed over a hour period. to evaluate the effect of treatment with anti-cd mab, animals were given either nothing, an isotype control or anti-cd mab ( mg/kg) rains, prior to the beginning of the lps infusion. evaluation of physiologic changes including mean arterial blood pressure and cardiac output, quantitative analysis of eytoldne levels including tnfct, il- , i,- , il- and il- , and liver enzymes during a hour period revealed that treatment with anti-cd mab markedly attenuated all parameters of injury including decreased mean arterial blood pressure, increased cytnkine levels and the release of liver enzymes observed in animals given the isotype control mab or those not treated. administration of anti-cd mab to interferon-gamma treated animals not challenged with lps did not induce any detectable physiologic changes or increases in cytoldnes. these studies suggest that strategies to block lps-cd interactions will have utility in diseases such as septic shock or ards where lps plays a central role in initiating injury. preclinical studies with recombinant bactericidal/permeability increasing proteins (rbpi and rbpi ). p.w. "frown, dept. of preclinical science, xoma corporation, berkeley, california, usa. bactericidal/permeability increasing protein (bpi), from neutrophils, binds to and neutralizes lipopolysaccharide (lps); it also specifically kills gram-negative bacteria (gnb). these properties, which reside in the n-terminal half of the molecule, indicate potential therapeutic application in the treatment of gram-negative sepsis. the gene for human bpi has been cloned and recombinant holoprotein (rbpi) and a kd n-terminal fragment (rbpi; ) have been produced in sufficient quantities for preclinical studies. both rbpi and rbpi bind to lipid a and neutralize the biological activities of lps derived from a variety of organisms, rbpi has equivalent antibacterial activity to bpi against rough gnb but is up to x more potent than bpi vs. serum-resistant and smooth gnb. rbpi and rbpi compete with lps-binding protein (lbp) for binding to lps under physiological conditions. consequently, both rbpi and rbpi block the cd -dependent lpsinduced synthesis of the cytokines tnf, il- , el- and il- in vitro. rbpi has also been shown to inhibit the lps-induced synthesis of reactive metabolites, endothelial adhesion molecules and the procoagulant molecule tissue factor. in animals, rbpi has been reported to increase survival of endotoxin-challenged rats and mice, to inhibit the dermal schwartzman reaction in rabbits and to increase survival of neutropenic rats with pseudomonas bacteremia, rbpi increases survival and decreases cytokine production in endotoxin challenged mice and rats. it normalizes lps-induced changes in hemodynamic, pulmonary and/or metabolic parameters in lps-induced rats, rabbits and pigs. treatment with rbpi also increases survival and decreases cytokine production in bacterial challenge models in rats and mice. rbpi was not toxic to rats after daily consecutive i.v. doses of mg/kg. this combination of properties indicate that recombinant bpi may be useful in the treatment of sepsis. phase i/ii clinical trials of rbpi have begun. the discovery of lps binding protein (lbp) and subsequent identification of cd as a receptor for lps or lps-lbp complexes has resulted in a new understanding o£ how lps responsive ceils are stimulated. cd is found either as a glycosylphosphatidyl-inositol (gpi)-anehored membrane glycoprotein (mcd ) of myeloid cells or as a soluble serum protein (scd ) lacking the gpi-anchor. binding of lps to mcd triggers cell activation while binding of lps-scd complexes to cells such as endothelial or epithelial cells that normally do not express mcd activates these cells. these pathways are shown in schematic form below. ~di mcd plays a crucial role in presentation of lps to additional membrane components that make up a functional lps receptor. an immediate consequence of engagement of this functional receptor is protein tyrosine phosphorylation. the molecular mechanisms leading to these events will be discussed. understanding of these pathways will lead to the development of new therapeutic approaches to controlling host responses to lps. pretreatmen t posttreatment (before or after tnf peak) d) with different antibody dosages: mg/kg --- . mg/kg pretreatment with anti-tnfab prevented death in most model situations (except peritonitis), but also posttreatment up to h after sepsis induction was successful in the few studies performed. there is additional evidence that low-dose tnfab is partially effective. especially baboon anti-tnfab studies provided many insights into the pathophysiological sequences of sepsis induction, due to crossreactivity with human reagents. those events include the cytokine sequence with tnf-dependent il-i, il- , or il- , but also il-lra or stnf receptor release. granulocyte as well as endothelial cell activation were shown to be partly tnf related, and the procoagulatory response was influenced by anti-tnf treatment. from many animal studies the concept that tnf plays a pivotal role in sepsis is clearly evident and therefore anti-tnf therapy is a major candidate tbr clinical studies. the beneficial or harmful effects of tnf-mediated inflammatory responses depend on the clinical context. decreasing exaggerated tnf-mediated inflammatory responses may be useful in some patients with organ failure. tnfr:fc (immunex, seattle, wa) is a recombinant human protein composed of two identical extracellular p tnf receptors linked by the fc region of iggl. it neutralizes tnf with an affinity for tnf<z of - m and has a t / of • h in normal humans. methods: normal volunteers were randomized to receive either tnfr:fc vehicle (n= ), or low dose (ld) tnfr:fc ( mg/m iv, n= ), or high dose (hd) tnfr:fc ( mg/m iv, n= ) infused over rain prior to endotoxin (e) (escherichia coil : ; ng/kg iv) administration. plasma cytokines were measured using elisa and tnf bioactiv'dy. systemic hemodynamics were evaluated with indwelling arterial and pulmonary artery catheters and radionuclide heart scans and compared before and after fluid loading in subjects given vehicle with hd tnfr:fc. results: endotoxin-related symptoms were unchanged after ld or hd. peak temperature occurred at a later time point ( - h) in subjects given tnfr:fc compared to vehicle ( h) (p=. ). at h, h (post fluid load ), and h, subjects given vehicle developed increased cardiac index and heart rate and decreased systemic vascular resistance index (all p=. ). hd tnfr:fc did not alter this hyperdynamic cardiovascular response. ld and hd inhibited the e-induced teukopenta at i h post endotoxin (p<. ) and later leukocytosis (p< . ). peak tnf bioactivity in subjects given vehicle was + pg/ml and < pg/ml in the ld or hd tnfr:fc groups (p<. ). two immunoassays for tnf were performed because the detection of receptor bound tnf varies with elisa. tnf (biosource) was decreased in ld vs vehicle or hd (p=. ) whereas tnf (r&d systems) was increased after hd or ld vs vehicle (p<. ). decreased levels of il- (p=. t), granulocyte colony stimulating factor (p=. ), and il- receptor antagonist (p=. ) were found after ld or hd vs vehicle. il- levels were lower after ld vs vehicle or hd (p=. ). il- levels were similar among subjects given vehicle, ld, and hd tnfr:fc. conclusions: ld and hd tnfr:fc delayed the febrile response to e but did not alter the early hyperdynamic cardiac response. this suggests that mediators other than tnf play an important role in the initial cardiovascular response to e in humans. tnfr:fc attenuated the release of some e and tnf-induced cytokines. ti~e antiinflammatory responses of tnfr:fc may be useful in some patients with disease processes resulting from excessive tnf-mediated inflammatory responses. the biosynthesis of tnf within macrophages is regulated both at the transcriptional and the translational levels. the ' flanking region of the tnf gene contains several transcriptional control elements, including two kb enhancers similar to those of immunoglobulin genes and a "y box" similar to that of the mhc class ii promoter. these elements are critical for the enhancement of transcription noted after stimulation of macrophages with lps. despite these and other elements, transcriptional regulation alone accounts neither for the absence of tnf protein during normal homeostasis nor the profound increasein tnf production following stimulation with lps or other secretagogues. the translation of tnf mrna into protein is also tightly regulated via mechanisms involving the octameric "ttatttat" motif present in the 'untranslated region of tnf, human inducible no synthase, and several other cytokine genes. this region not only destabilizes mrna, but also confers a translational blockade so that tnf protein is not normally synthesized despite leaky basal transcription. lps stimulation releases translational blockade and, together with enhanced trancriptional rate, accounts for significantly increased tnf secretion. opportunities for inhibition of tnf production are available at each of these two levels. agents which increase intracellular camp (pentoxif¥ ine, amrinone) inhibit accumulation of tnf mrna; in contrast, corticosteroids act primarily at the level of translation, inhibiting lps induced translational activation. understanding the regulation of tnf biosynthesis may assist in clinical strategies designed to regulate tnf secretion. the serine protease thrombin is formed by enzymatic conversion from its proenzyme form after coagulation activation and may enhance the inflammatory response in various ways. in the last step of the coagulation cascade, thrombin cleaves its substrate fibrinogen, thus forming fibrin. the fibrin clot may prevent phagocytosis of bacteria and promote local bacterial growth. thrombin can also stimulate a specific th.rombin receptor that exists on a variety of cells. for example, minute amounts of thrombin ma), stimulate platelets to develop tissue factor and boost the activation of more thrombin, thus initiating a positive feedback loop. other cellular effects of thrombin receptor stimulation include increase in intracellular calcium of platelets and monocytes, contraction of smooth muscle cells, increase in endothelial permeability, thromboxane generation by neutrophils and lymphocytes, pulmonary vasoconstriction, and enhanced cytokine production. animal experiments have been conducted with a variety of thrombin inhibitors including heparin, antithrombin ili, hiradin, and hirudin-like peptides. an alternative approach is the inhibition of the coagulation cascade at an earlier point in the cascades, or administration of the anticoagulant enzyme protein c work from our own group in a porcine model of lipopolysaccharide-(lps)-induced shock with disseminated intravascular coagulation has shown that prelreatment with recombinant desulfatohirudin (r-h) or with a preformed complex of human donor antithrombin iii with heparin or post treatment with r-h are able to block the degradation of fibrinogen and the formation of fibrin monomer. in addition, after pretreatment with r-h it was found that lps-induced lung injury was reduced. since both natural hirudin as well as r-h were well tolerated by healthy volunteers, adminislration of r-h may be a promising therapeutic approach in patients with sepsis and sirs. corticosteroids have been used in the management of various shock syndroms including sepsis. the data concerning the effects of this therapy is, however, conflicting and in large prospective studies, the use of early administration of high doses of corticosteroids have been found to give no beneficial effects in patients with sepsis. we have particularly been engaged in studies on possible effects of corticosteroids on the plasma cascade systems and on the mediator release from leukocytes. in in vitro studies, high doses of methylprednisolone were found to facilitate endotoxin induced generation of plasma kallikrein. furthermore, plasma prekallikrein and kallikrein inhibitor values decreased together with formation of kallikrein-c inhibitor complexes ( ). in this in vitro plasma model we also found that methylpredoisolone alone in doses of mg/ml induced contact activation with the formation of kallikrein-c inhibitor complexes. methylprednisolone was also found to have an additive effect on endotoxine induced decreases in kallikrein inhibitor functions. in the same experimental model on endotoxemia methylprednisoione was found to give an additive effect on activation of the initial part of the complement cascade ( ) . activation of the terminal part of complement, however, was inhibited by the same dose of methylprednisolone. addition of methylprednisolone alone to plasma was also found to activate the initial part of complement. using a full blood model, we studied the effect of methylpredoisolone in modulating the endotoxin induced cytokine response. when ng/l e. coli endotoxin was added to citrated blood from healthy human donors preinkubation with gg/ml methylprednisolone significantly reduced the release of tumor necrosis factor et ( %) and interleukin- ( % ) at two hours after exposure to endotoxin. in conclusion, these studies show that corticosteroids might facilitate contact activation of plasma and reduce the function of major protease inhibitors. furthermore, we also observed that this drag in in vitro conditions facilitated activation of complement. on the other hand, corticosteroids were found to reduce endotoxin induced cytokine release from leukocytes in whole blood. these studies also showed a dose dependence of the effects of corticosteroids on endotoxin induced cellular and cascade system activation. different predisposing conditions like extensive trauma, infection or pancreatitis result in a remarkably similar inflammatory response. although thls inflammatory response primarily aims at the removal of devitalized tissues, destruction of bacteria and reestablishment of the integrity of host tissues, it may potentially become unregulated and generalized and thereby producing deleterious effects to vital organs or the body" as a whole. cytokines play an important role in the development of the systemic inflammatory response. most of their biological effects, however, are not directly mediated but exerted through other mediator systems. when the inflammatory response results in the formation of capillary leak syndrome and refractory hypotension, the unregulated activation of complement and contact systems appears to be the cause. both systems are almost exclusively regulated by c - nhibitor. am unbalanced degradation of the inhibitor protein was found to be associated with the onset of capillary leakage in various conditions such as bone marrow transplantation, severe burns, and septic shock. these observations suggest a relative deficiency of biologically active c - nhibitor in these septic shock like syndromes. therapeutic intervention studies were initiated using high doses of c - nhibitor concentrate. first results are promising, as in most patients the administration of the drug was followed by a quick and marked improvement of the patient's hemodynamics. whether the administration of c - nhibitor alone or in combination with other drugs is also sufficient to improve long-time survival has to be investigated in double-blind, placebocontrolled studies. salutary effect of human soluble complement receptor type- in models of adult respiratory distress syndrome g. feuerstein, m. dimartino, and *r. rabinovici, smithkline beecham pharmaceuticals, king of prussia, pa, usa, and *jefferson medical college, philadelphia, pa, usa adult respiratory distress syndrome (ards) is a severe pulmonary insufficiency syndrome associated with diverse diseases and injuries. ards is believed to be the consequence of a massive acute inflammatory reaction consisting of activated neutrophils infiltration into the lung. neutrophil activation is likely the result of multiple factors of which complement is believed to play a major role. a potent complement regulatory system in the form of the complement receptor type- (cr- ) exists on many cells where protection against complement injury is provided by inhibition of both the alternative and classical pathways for c /c convertase formation. we have recently purified the human recombinant cr- in a truncated form (brl , tp-io, scr- ) and tested this soluble protein for protective effects in several models of ards. specifically, we have used the following rat models: ) endotoxin-induced ards in paf primed animals; ) .- induced ards; ) thermal injury ( % body surface)-induced ards; ) acid aspiration-induced ards. ards-like features included edema (increase in lung wet weight and water content), neutrophil accumulation (histological inspection and myeloperoxidase assay) and increase in cells and protein in the bronchoalveolar lavage (bad. scr- , - mg/kg, i,v., given as prophylactic (and in some cases, therapeutic) treatment significantly inhibited edema formation and leukocyte infiltration and, in some cases (e.g., endotoxin/paf or il- ), virtually completely. these comprehensive studies strongly support the therapeutic potential of scr- in ards due to diverse injuries. the xanthine derivative pentoxifylline (ptx) and several analogs of ptx have been evaluated for their protective effect in various animal models of septic shock. data from these in vivo studies demonstrate that ( ) ptx suppresses lps-induced tnf release from activated macrophages; ( ) ptx prevents tnf-induced pmn activation; ( ) ptx attenuates lps-or tnfinduced acute lung injury as evidenced by prevention of increased pulmonary vascular permeability and detoriation of gas exchange; ( ) ptx exerts its .protective effects even when administered following initiation of sepsis. the sum of these actions suggest that administration of ptx in sepsis may have therapeutic potential. hans hoffmann md, dept. of surgery, klinikum grosshadern, ludwig-maximilians-universit~.t, marchioninjstrasse , d- m nchen, germany. pentoxifylline [pof] and related xanthins are drugs of known haemorrheologieal activity and widely used clinically. recently, new pharmacological aspects of these established drugs could be demonstrated. it was found that pof selectively inhibits the formation of tnf but not il- most likely by causing accumulation of intraceliular camp via inhibiting phosphodiestersse activity, and, secondly, by inducing prostacyclin in different cell types. furthermore, pof is able to counteract tnf-mediated adherence of neutrophils to vascular endothelium and to inhibit fmlp-indueed respiratory burst in neutrophils. we and others have, therefore, studied its effect in different animal models. it was found that pof protected from increased pulmonary vascular permeability and sequestration of neutrophils into the lung in different animal models of acute lung injury. moreover, in pigs with induced faecal peritonitis the drug improved haemodyrmmle variables as well as pulmonary compliance and reduced the number of pulmonary neutrophila and lymphocytes. consequently, as a general outcome, pof could be found to improve survival in different models of haemorrhagie and endotoxie shock. the protective effect of pof was dose-dependent and observed even when pof was given up to hours after endotoxin. in order to evaluate these pharmacological effects of pof in humans, we studied pof under controlled conditions of endotoxlnemia in volunteers. we found that pof selectively inhibits the lps-indueed endogenous formation of tnf without affecting il- and il- . moreover, pof counteracts the lps-indueed initial leukocytopenia by interfering with the adherence of neutrophils in the microvasculature. meanwhile the inhibition of endogenous formation of tnf by pof could be demonstrated under different clinical conditions of acute and chronic cytokine release syndromes, {okt first-dose reaction, cancer-or tuberculosis-induced cachexla}. it appears worthwile and promising to investigate wether pof exhibits beneficial effects also in septic patients. objectives: to determine the specific role of paf in both lethal primate bacteremia and nonlethal human endotoxemia. materials and methods: two double-blinded placebo controlled studies were performed. first, ten baboons ( . +. kg), were randomized to a control group receiving ° cfu/kg of intravenously administered e. co/i (n= ) and a treatment group (n = ), pretreated with . mg/kg of paf receptor antagonist ro - two hours prior to e. co/i infusion. in a second study, ten healthy male volunteers were randomized to a control group receiving ng/kg of intravenously administered endotoxin (lps) (n= ) and a treatment group (n= ) pretreated with mg of ro - eighteen hours prior to lps administration. physiologic parameters and cytokine levels were measured continuously in both studies for hours. results: baboons pretreated with the paf antagonist had improved oxygenation ( + mm hg v -+ in controls, p < . ) and creatinine clearance hours post e.coli ( . + . ml/min v . _+ . in controls, p < . ). similarly, volunteers pretreated with the paf antagonist experienced fewer symptoms, including rigors and myalgias at hour post lps. this was in concert with a diminished peak cortisol ( +_ mmot/l v +- mmol/l in controls, p < . ), epinephrine secretion ( + nmol/l v + nmol/l in controls, p < . ). hemodynamics and circulating tnfa levels (data not shown) were unaffected in either study by prior paf antagonism. concluslons: paf influences some components of the multi-organ failure syndrome in lethal gram negative sepsis and the counter-regulatory hormonal system in human endotoxemia through tnf-independent mechanisms. paf antagonists may serve as adjuncts to cytokine blockade in the treatment of gram negative sepsis. the mediator role of platelet-activating factor in gramnegative septic shock pierre g. braquet, david hosford and matyas koltai institut henri beaufour, le plessis robinson, france considerable evidence has been accumulated to support the concept that a paf/cytokine autogenerated feedback network is responsible for priming and amplification of inflammatory mediator release in septic shock. cytokines, such as tnf~x, il- and other interleukins interact with paf which then amplifies cytokine production, therefore, paf may be the principle mediator in endotoxin shock. a single factor identified as tnf is suggested to play a pivotal role in the fatal phase of septic shock. presumably excess tnf release is the result of amplification process primarily triggered by paf. one may assume that the high tnf concentration in the blood of 'non survivors' is the consequence rather than the cause of cell death. the dubious results of clinical trials with anti-tnf antibodies and il- ra, a naturally occurring il- antagonist protein, have risen debate around the exceptional role of cytokines in the pathomechanism of septic shock or systemic inflammatory response syndrome (sirs) induced by gramnegative microorganisms. there are similar problems with the interpretation of the results obtained in clinical trials of monoclonal igm antibodies against e. coli endotoxin, ha- a and e . future studies will answer the question as to the effectivity of inefficiency of this treatment. perhaps when the plasma concentrations of lps and cytokines exceed a critical level, treatment fails to save the patients life. with regard the causal role of lps in septic shock, the putative role of bacterial porins may have to be taken into consideration. the marked release of paf induced by endotoxin leading to excess txa production may be responsible for the microvascular failure and death in septic shock. antagonists of paf markedly protect against the release of txa , and may interrupt the vicious circle of amplification process. no produced by the inducible no synthase plays a pivotal role in causing vascular paralysis in lps-induced sirs. to explore the relationship of paf to no synthesis will provide better understanding of the pathomechanism of septic shock. several paf antagonists, including bn , have undergone phase ii and phase iii clinical trials. the strategy in the treatment of sirs, however, remains multifactorial, since little is known about the sirs caused by microorganisms other than gram-negative bacteria. evidence has begun to accumulate which suggests that in sepsis excess production of lps and cytokines can lead to uncontrolled production of inos and that this might in part explain the severe unresponsive hypotension seen in some septic patients. a number of strategies have been explored in an attempt to limit excess no production. a favourite target has been competitive inhibition of nos by arginine analogues such as l-nmma. in animals, some investigators have shown that the haemodynamic effects of lps challenge can be attenuated by l-nmma, but there is a narrow toxic-therapeutic ratio. in a model of live e. cell sepsis, we have been unable to reproduce these findings. localisation of no production by immunohistochemistry suggests that no production is compartmentalised, and more subtle methods of regulation may be required if this approach is to have clinical valuc. anti-adhesion molecule strategies ch wortel, repligen corporation, one kendall square, building , cambridge, ma , usa. the neutrophil has been implicated as a pivotal player in the pathogenesis of multiple organ dysfunction. an important first step in the process of neutrophilmediated organ injury involves the binding of neutrophils to endothelial ceils. this process is largely regulated by complementary adhesion molecules, some of which are present constitutively on the cell surface and others that can be upregulated in response to chemotactic and pro-inflammatory stimuli. several different adhesion molecules have been described. l-selec tin is expressed on the plasma membrane of neutrophils and is shed from the surface early in the inflammatory process. it is therefore thought to mediate the initial interactions with endothelial cells. e-selectin and p-selectin are endothelial adhesion molecules. e-selectin is not constitutively expressed but is upregulated by inflammatory mediators, particularly il- and tnf. p-selectin is present in the weibel-palade bodies within the endothelial cytoplasm and can be upregulated rapidly in response to thrombin, histamine, and oxidants. all selectins recognize oligosaccharides, particularly sialylated lewis x antigen. this carbohydrate moiety is expressed on many proteins, including the selectins themselves. the leukocyte integrins are glycoproteins expressed on the neutrophil surface and in the cytoplasmic granules. integrins consist of a common beta or cluster differentiation (cd)i chain covalently linked to one of three different alpha chains (cd a, cd lb, cd lc) and exist on the cell surface as three distinct heterodimers. cd l a/cdi is expressed on all leukocytes, whereas cd l b/ cd and cd lc/cd are restricted to cells of myeloid origin. cd i/cd interacts with intracellular adhesion molecule- (icam- ), its ligand on endothelial cells. icam- is a member of the immunoglobulin family of adhesion molecules. it is constitutively expressed on endothelial cells and can be upregulated by cytokines. the potential for monoclonal antibodies to adhesion proteins to reduce vascular and tissue damage has been studied in alarge number of experimental models. protective effects have been observed in a wide variety of inflammatory, immune, and ischemia-reperfusion injuries such as arthritis, bums, endotoxic shock, bacterial meningitis, autoimmune diabetes, nerve degeneration, allograft rejection, allergic asthma, acute lung inflammation, skin lesions, and ischemia-reperfusion models of the intestine, myocardium, lung, skeletal muscle, and central nervous system. protective effects have also been observed in animals resuscitated following hemorrhagic shock. since modulating the function of adhesion molecules may temporarily leave the host without effective defense machinery, safety evaluations are of utmost importance in evaluating this therapeutic approach. treatment of gram-negative septic shock with an immunoglobulin preparation: a prospective, randomized clinical trial ingolf schedel, ursula dreikhausen; birgit nentwig; marion hockenschnteder, dirk rauthmann, salim balikcioglu, rolf coldewey, helmuth deicher, md endotoxin may play a major role in the pathogenesls of muitiorgan failure in the context of the toxic process in septicemia induced by gram-negative pathogens. the hypothesis which has led to a prospective clinical study consisted in the idea of inhibiting the biological effects ofendotoxin during the early phase of septic process, and thereby attampting to attain a positive effect on the clinical course of the disease. -objecave: to evaluate the effectiveness ofa polyclonal immunoglobulln (ig) preparation containing igg, lgm, and iga as an adjunctive therapy for septic shock. -desigru prospective, randomized clinical trial. patients: fifty-five patients w~th septic shock were randomly allocated to two groups according to criteria of septic shock. -intervention: one group of patients (n = ) received a commercially available immunoglobulin preparation (containing high titers of antibodies specific for determinants to bacteria endotoxin) during the first days after inciosion in the study. the other randomized group (n = ) did not receive any immunogiobulin preparation. -measuremems and main resmts: during the period of < wks after the beginning of clinically apparent septic shock, death related to the septic process occurred in one ( %) of patients who received immunoglobulln. by comparison, nine ( %) of control group patients died during this period (p <. ). within the first hrs after onset of the clinically apparent septie process, significaptly increased activity of circulating endotoxin and simultaneously decreased specifie igg serum titers to lipid a were detected in the group of nonsurvivors. the rationale for the use of polyvalent intravenous immunogiobulins (ivig) to treat severe infections stems from in vitro and animal studies documenting that high-dose igg enhance opsonization and phagocytosis of pathogenic bacteria. moreover, recent clinical studies have shown that in septic patients the phagocytic function of circulating polymorphonuclear neutrophils (pmn) is defective; the degree of such impairment correlates to the severity of the septic state, the beneficial effect of administering high dose igo in septic patients can be two-fold: i) ivig provides large quantities of antibodies against invading pathogens; the igg bound to the pathogens can opsonize their phagocytosis; ) the opsonization of phagocytosis by exogenously administred polyvalent igo would be of special importance in those patients who present a significant defect of their phagocytic function, when they are challanged by a large bacterial invasion. a prospective, randomized, double-blind study was carried out comparing the administration of ivig (sandoglobulin@) vs, paeebo (human albumin) in severely septic surgical patients, only patients with gepis score >_ (meaning a mortality risk > %) were accepted into this protocol. patients were randomized to receive . g/kg of ivig or placebo on day (when they reached sepsis score> ), repeated on day + and + . at the beginning of icu treatment, the two groups of patients were similar for severity of sepsis, age, concomitant disease, type of surgical procedures, antra and perioperative procedures, antibiotic administration. the results of the study indicated a significantly reduced mortality in patients with severe surgical sepsis treated with ivig as compared to placebo control patients (mortality: % vs, % respectively; p< , ). in conclusion, the results of our study in patients with severe surgical sepsis were the following: ) ivig plus multimodal treatment of sepsis, including antibiotics, reduce mortality significantly', ) the reduction of mortality seems to be due to a decreased incidence of lethal septic shock. despite substantial clinical research, the avallable data regarding the effectiveness of supplemental immunoglobulin (ig) treatment in sepsis in adult patients do not yet allow definitive conclusions. in view of the persistently high sepsis mortality there is a need to continue clinical investxqations regarding supplemental sepsis treatmen~ in general, as well as concerning ig administration in particular. we present and discuss the protocol of the ongoing ,,score-based-immuneglobulin therapy of sepsis (sbits)" study. the protocol (theoret surg ( ) - ) of this multicenter, randomized, prospective and double-blind trfal relies on the results of an observational trial on i.v. igg treatment in patients with sepsis and septic shock (infection ~ ) - ), carried out as a prerequisite for the present trial. using microcomputer-based bedside routine score monitoring, we regard quantitative measures of severity of disease and sepsis: only patients with a certain degree of both severity of disease (apache ii score - ) and severity of sepsis (elebute sepsis score - ) will be included. by observing these previously validated inclusion criteria, this trial snould iqentify a priori and include patients with potentially optimal response to therapy, consisting o~ either placebo ( .i % albumin) or polyglobin n" - ml ( . g)/kg on day and ml ( . g)/kg on day i. with an anticipatedpopulation size of patients the study should comply with the statlstical requirements (estimated mortality: %, with a % reduction in -day mortality in the treatment groupl to prove or disprove the question of igg effectiveness in sepsis in terms of improved prognosis. up to november , more than patients had been included; patient enrollment will be finished in . previous studies have demonstrated rhll-i ra, a naturally occurring antagonist of il- , increases survival in animal models of andotoxemia and eschehchia coli bacteremia and attenuates the decrease in mean arterial pressure resulting from challenge with both gram-negative and gram-positive bacteria. previously, in patients, rhll-lra was demonstrated to increase survival in patients with sepsis syndrome and septic shock in a dose-dependent manner. methods: a randomized, double-blind, placebo-controlled, malticenter, clinical trial enrolled patients at academic medical centers in europe aad north america. eligible patients received either placebo (vehicle) or rhil-lra (anakinra) . or . mg/kg/hr by continuous intravenous infusion for hours. the presence of organ dysfunction (i.e., ards, dic, renal, and hepatic) at study entry was determined prospectively by a clinical evaluation committee using definitions which were developed a-priori. survival time was evaluated over days utilizing a linear dose-response model, assuming a log-normal distribution. results: patients had one or more sepsis-induced organ dysfunction(s) at study entry. a dose-related increase in survival time was observed with rhll-lra compared to placebo in patients with ards, dic, and renal dysfunction (p --< . endotoxin infusion releases platelet-activating factor (paf), a potent phospholipid mediator which leads to an autocatalytic amplification of cytokine release. bn (ginkgolide b), a natural paf receptor antagonist, has provided significant protection against sepsis in different animal models• a randomized, placebo-controlled, double blind, multicenter trial on efficacy (mortality at d ) and tolerance of bn ( iv infusion of mg x /day over days) in severe sepsis has enrolled pts. the day mortality rate was % for the placebo group and % for the bn group (p = . ). the efficacy of bn was greater in pts with gram-negative sepsis: the -day mortality rate was % for the placebo group and % for the bn group (p = . ). bn also reduced mortality among pts with gram-negative septic shock (mortality was % for placebo vs % for bn ; p = . ). using statistical adjusments for pronostic factors, the relative risk of death of the bn group was . ( . - . , % confidence interval; p = . ). this risk corresponds to an adjusted reduction in mortality of % for pts receiving bn . no differences in mortality rates were found between the placebo and the bn groups in the absence of gram-negative sepsis• there were no differences in adverse events between the placebo and the bn groups. bn is a safe and promising treatment for patients with severe gram-negative sepsis. a confirming study, focused on gram negative sepsis, is in progress. v~ lliam a. kanus m.d. and the rhll-lra it has been traditional within the field of infection and sepsis to think in terms of specific indications for drugs based on the type of infecting organisms, advances in antibiotic therapy now control or ltnflt the growth of bacteria. the majority of deaths are now caused by either an initial overwhelming response to infection or subsequent multiple organ system failure attributed, in part, to the effects of intrinsic biologic responses of the host. type of organism, therefore, may not be as critical as determining the exact severity of the host's severity or risk of death from infection. we also know that both the relative benefit of a new treatment across groups and its absolute benefit for an individual patient will vary with their risk in a predictable fashion. we recently iuve~iguted the relationship between one measure of host response, the acute risk of death as prospectively estimated by u comprehensive risk mode[ for -day mortality (jamb. ; : , - ) , by its retrospective application to the results from the phase in evaluation of recombinant human intcrlenkin- receptor antagonist (rhll. ira). we found that there was a significant interaction between the patient's predicted risk of mortality at the time of entry to the study and the ability of rhil-lra to prolong survival time (x = . , p [] . , log.normal) for all patients in the trial• survival benefit began st approximately % baseline risk of -day mortality. for the $ patients with a predicted risk > %, there was a % reduction (p= , $ log normal). when we examined the variation in patients above and below the % risk level with hazard functions, i.e., their daily risk of death during the study period, we found that placebo patients with < % risk had lltile acute daffy risk during the hlltial two days follawh~g study entry and this risk was little affected by rhil-lra, in contrast, patients with > % risk had high daily mortality risks during the tuttlal two days that high dose rhtl-lro substantially reduced. these results are compatible with our current understanding of outcome from sepsis and the proposed mechanism of action o£ immunotherapy, the earliest deaths from sop sis are secondary to an immediate inflammatory response followed closely by deaths secondary to multiple organ system failure, later deaths (after days) are not as closely related to the acute effeete of the inflammatory cascade. because of the timing and action of most proposed tmmunotherapy, they may be capable of preventing mortality primarily in these initial two phases. in this study, an independent predicted risk of mortality reflected this mortality pattern ned illustrated the potential benefit of immtmotherapy. use of a predicted risk of mortality in the design and analysis of clinical trials could improve our understanding of the clinical benefit of these new therapeutic approaches. the systemic inflammatory response syndrome (sirs) is a term recently proposed to describe patients with systemic inflammatory responses to insults such as infections (sepsis), trauma, burns, pancreatitis, and other initiating events. patients with sirs may have similar activation of inflammatory mediators and similar outcomes independent of the initiating event. these outcomes include organ dysfunction and failure, shock, and death. challenges to the successful conduct of clinical trials in sirs include the complexity of illness in these patients and the important--but limited--clinical benefits of novel compounds that may be limited to selected patient subsets. addressing these challenges will require new tools and approaches. these will include more sensitive and appropriate endpoints, and the use of methods such as baseline risk adjustment, to allow detection of drug risk interactions not captured adequately by categorical definitions, such as sepsis syndrome. on the basis of supportive preclinical and phase i safety studies, we have initiated phase ii clinical trials of a novel bradykinin antagonist, cp- , in four sirs subcategofies: sepsis, multiple trauma, burns, and pancreatitis. each of these studies is designed to measure the effect of cp- on mortality, organ dysfunction and failure, and activation of mediators. in addition to investigating rates of organ failure using standard definitions--a new endpoint--a continuous summary measure of organ dysfunction (the acute physiology score of apache tm iii) is being used to quantify the degree of organ dysfunction and the speed and pattern of recovery of physiologic stability. in the sepsis study, another new approach--a study specific risk model based on the apache ill database--has been developed which will be used to assign a pre-treatment baseline risk to each patient enrolled. the primary outcome variable will be risk adjusted survival time to days. this type of risk-adjusted analysis may allow for more efficient and powerful trials and more accurate and useful indications for use. study purpose: in post-cardiac surgical patients (pat.) at risk for sepsis, the efficacy of early i.v. immunoglobulin (ig) treatment was compared to a matching historical control (con.) population. postoperative risk assessment: using apache ii scores lap) (first postoperative [pop.] day) in a pilot study phase, we were able to differentiate between the large population ( . %) of pop. low-risk pat. (ap< ; mortality: %) and the small groups of pop. pat. at risk lap= - ) and high risk lap_ ) with a significantly higher mortality ( % and %, mainly due to sepsis). subsequently, among consecutive pop. pat. we prospectively identified and treated these pat. iq treatment reqimens: first study period (n = ): (gg (psomaglobin n a, tropon biologische pr~parate, cologne, frg, day : ml/kg, day : ml/kg). second study period (n= ): iggma (pentaglobin r, biotest, dreieich, frg, ml/kg on days to ). results: ig pat. and con. were comparable in demographic data, operation characteristics and baseline disease severity lap and elebute sepsis scores). in contrast to con. (risk: n= , high-risk: n- ), the ig pat. showed a marked improvement in disease severity (fall in ap), especially in the high-risk group (igg, n= : p<o. ; iggma, n= : p: . ). in this group, ig led to higher (p< . ) response rates, defined as an ap score decrease -> within four days (igg: %, iggma: %; con.: %), and reduction in mortality (igg: %, iggma: %; con.: %), statistically significant (p< . ) for ig treatment as a whole (igg and iggma). conclusion: given the good comparability of the study groups, our results indicate, despite the non-randomized design, that early supplemental ig treatment can improve disease severity and may improve prognosis in prospectively apache ii score-identified high-risk patients after cardiac surgery. objective. elevated plasma levels of endothelin (et) have been demonstrated in both experimental and human sepsis. et has been proposed as a sepsis mediator leading to vasoconstriction with tissue hypoperfusion and organ failure. the aim of the study was to determine the effects of sepsis treatment with volume resuscitation, antibiotics and the anti-lps monoclonal antibody es® on big et and active, aminoacids et (et ) in rat abdominal sepsis. methods. lethal peritonitis was induced with a mm coecal perforation (cp) in male wistar rats. plasma levels of big et and et were determined with amersham tm endothelin rias , and h after sepsis induction. experimental groups: . cp control, . volume replacement (vr); , % saline ml/kg/h continous iv infusion started after h, . antibiotic; imipenem mg/kg iv after h, . e ®; mg/kg iv after h, . vr + imipenem + es® after h. results. high concentrations of both big et and et could be demonstrated after h and lasting for h after cp. neither volume replacement nor imipenem did influence the elevated plasma et. e ® significantly reduced et both , and h after sepsis induction, but did not reduce big et. when es® was combined with vr and imipenem, reduction of et was the same as for e ® alone. these results strongly suggest that bacteria and hypovolemia per se are not decisive stimuli for et production during sepsis. e ® reduces circulating lps and tnf which is the probable mechanism of the suppressed et synthesis. the unaltered big et fraction after e ® treatment indicates conversion of big et to et as the site of action responsible for reduced et . conclusion. lethal peritonitis in the rat is followed by elevated plasma levels of big et and et . e ® anti-lps antibody significantly reduces plasma et while volume resuscitation and antibiotics failed to do the same. es® did not reduce plasma big et. pmx treatment on severe endotoxemia with multiple organ failure was safety and effect in prognosis, and sepsis related parameters. it was certified that reduction of plasma endotoxin was effective in severe endotoxemia. a. lechleuthner,s. aymaz, g. grass, c. stosch, s. dimmeler, m. nagelschmidt, e. neugebauer. ii. dept. surgery, university of cologne, germany. introduction: the cardiovascular therapy of hypodynarnic shock states is a challenging problem. in clinical as well as experimental studies beneficial functions of a new hg-agonist bu-e- in congestive heart failure has been demonstrated aumann, ). therefore, we investigated the effect of bu-e- in hypodynamic shock in pigs. materials and methods: pigs (deutsches hausschwein, pitrain, [ ] [ ] [ ] [ ] [ ] [ ] were anesthesized with fentanyl/dormicum, ventilated (n :o = : ) and cardiovascular parameters were monitored with a complete icu-eqnipment. the hypodynamic model was established in a pilot study ( animals) to evaluate the effective concentration of bue- in healthy and endotoxin (lps)-treated animals. endotoxic shock was induced by continous infusion of ~g lps/kgkg/h ( :b , fa. difco). the hypodynamic state was defined as a decrease of cardiac output by % of steady state levels. a wedge pressure of - mmhg was kept constant by volume resucitation during the experiment. in a subsequent randomized controlled trial (rtc) groups with animals per group were studied. the groups were treated as follows: group i, lps and , % nac ; group ii, lps and bu-e- ( #g/kgkg/h); group iii, famotidine (h -blocker) pretreatment ( mg/kgkg), lps and bu-e- . results: the pilot study in healthy pigs revealed, that bu-e- had positive inotropic effects. these effects were inhibited by the h antagonist famotidin. bu-e- however had no beneficial effects in the hypodynamic phase of endotoxic shock in the rct. cardiac index (ci) and the oxygen delivery (do ) were not significantly influenced by bu-e- application (group i versus group ii). bu-e- did not ameliorate the negative inotropic effect measuring left ventricular stroke work (lvsw) in hypodynamic shock phases. on the contrary, bu-e- led to a further significant decrease of lvsw (p < , ). famotidin pretreatment did not affect the response (group iii versus group ii). conclusion: in hypodynamic shock states the h -agonism seemed to have no beneficial effect under these experimental conditions. receptor down regulation or changes of signal transduction under septic conditions may be responsible. cellular studies may help to identify these mechanisms. objectives. antithrombin iii inactivation of proccagulant proteases is so far the only inhibitory therapeutic approach to disseminated intravascutar coagulation (dic). we therefore set out to investigate whether cll substitution reduces coagulation activation in an endotoxin induced rabbit dic model. materials and methods. male rabbits chbb:hm(spf) were randomty assigned to one of the following groups. group k : naci . % (control without endotoxin, n= ). group e : endotoxin tjg kg " bolus i.v. + naci . % (control with endotoxin, n= ). group c : endotoxin pg kg - bolus i.v. + cll u kg - bolus + u kg " h "~ i,v. (treatment group, n= ). all animals were anesthetized and mechanically ventilated. blood samples were drawn prior to endotoxin administration (m ) and after (m ) and rain. (m ). thereafter, lung and liver tissue samples were taken intravitatly in a standardized fashion for h&e microscopic fibrin quantification using a triple score (fibs). from all blood samples the prothrombin time (pt), activated partial thromboplastin time (aptt), fibrin monomers (fm), and d-dimers (dd) were measured. for statistical significance of differences between the groups anovas and the wilcoxon test (fibs) were performed. results. fibs for lung/liver were significantly different (p< . ) between group e (lung , liver ) and c (lung , liver ) (group k : lung , liver ). , a synthetic serine proteinase inhibitor, has an anticoagulant activity in the absence of" antithrobim iii. gabexate has been reported to be useful in the treatment of disseminated intravascular coaguiation due to neoplastic diseases. in this study, we investigated gabexate therapy for the treatment of dic due to sepsis in the postoperative critical patients. materials and methods: from july to june , patients in the surgical intensive care unit met the criteria of dic or pre-dic. eleven were male and four were female with the mean age of . years. all these patients suffered from some complication of operations which led to the development of sepsis. foy was administered at the rate of mg/kg/hr untii the coagulation profile retumed to normal or the patient died. the coagulation parameters were monitored before and on the st, rd, th and th day. results: fourteen of these fifteen patients died despite transient improvement of the coagulation parameters in five patients. these patients suffered from sepsis resulting from surgical complications which could not be well controlled. the only survival was a case of recurrent intrahepatic duct stone with biliary tract infection complicated with sepsis and dic. after choledocholithotomy and the use of foy, the patient recovered gradually. conclusion: dic is a late manifestation of sepsis in the critical surgical patients. the most important thing is to eradicate the cause of sepsis. if the underlying septic focus cannot be controlled, dic will persist despite the use of gabexate mesilate. emergency surgery, taipei veterans general hospital, taipei, taiwan. there are main types of bradykinin (bk) receptor, namely bk~ and bk z. the bk receptor is constitutive. the bk receptor is also constitutive but in the majority of cases is inducible and involved in chronic inflammatory syndromes such as sepsis, hyperalgesia and airways hyperreactivty in animals. the mechanism(s) involved in the upregulation of the bk receptor is unclear, however a variety of agents including lps, e coil and ill are particularly efficacious in vitro and in vivo. ill and bradykinin acting at their respective receptors are believed to be involved in sirs/sepsis. we have investigated the effect of antagonists at ill (antril), bk (bradycor [cp- ]),bk~ (cp- ) and bkz/bk (cp- ) receptors on the de novo generation of bk~ receptors (reflected by hypotensive responses to a bk agonist) in the lps-treated ( ug iv) rabbit. in lps treated rabbits hypotensive responses to bk~ but not bk agonists increased with time and at time min appeared maximally induced. constant iv infusions of cp- blocked bk but not bk~ and cp- bk~ but not bk responses. cp- ,cp- +cp- and antril+cp- blocked both bk and bk~ responses. antril alone had no effect on bk or bk~ responses. within - min after stopping the infusions of antagonists the responses to bk~ and bk z agonists were the same as those in nonantagonist infused rabbits. these results indicate, at least in the lps-treated rabbit, that neither bk ,bk ~ or ill receptors alone or in combination, are involved in the de novo generation of bk receptors. in vitro studies demonstrated that beth bradycor and cp- (but not antril) were antagonists at both bk z and bk~ receptors. if both bk z and bk receptors are significantly involved in chronic inflammatory situations in man such as sirs/sepsis then the rationale for the use of compounds such as bradycor or cp- is clear. infection is a major cause of or contributor for morbidity and mortality in liver transplant recipients. effectiveness of prophylactic and therapeutic protocols is important for the success of liver transplantation ( olt ). sdd is used as prophylaxis for reduction of infection caused by gram negative or fungal microorganisms. between september and july olt's in patients were performed at our department. the actuarial -year patient survival is %. infection prophylaxis is started with sdd and ciprofloxacin once the patient is accepted as an olt candidate. perioperatively metronidazol, tobramycin and cefotaxim, postoperatively cotrimoxazol are prescribed additionally. the table shows pneumonia, peritonitis, major wound and urinary tract infection are common nosocomial infections following severe injury. in a series of severely injured patients from the university of louisville hospital, pneumonia was the most common infection followed by peritonitis, intra-abdominal abscess formation and burn wound infection. pneumonia is actually the leading cause of death from nosocomial infection. these are defined as occurring from to hours after hospital admission. this definition has important implications for antibiotic therapy because the likely pathogens and their respective sensitivities are different for community acquired pneumonia. the diagnosis of nosocomial pneumonia is difficult following major injury as many patients will have pre-existing fever, leukocytosis, tachypnea, and chest x-ray changes. reliance on sputum gram stain and culture is important and best obtained by a bronchoalveolar lavage or protected specimen brush during bronchoscopy. predisposing risk factors include severe head injury, emergent intubation and shock, and such patients have been shown to benefit by early tracheostomy. staph aureus has been the most common pathogen isolated from the sputum and the remainder gram-negative organisms with pseudomonas aeruginosa, and klebsiella pneumonia predominating. bacteria recovered by site as well as by intensive care unit is published in the six month antibiogram which also includes recent antibiotic sensitivities. this aids in empiric antibiotic selection against such nosocomial organisms. in a series of severely injured patients (iss - ), mean temp. was . f, leukocytosis was k, pan was , fin was . , and peep was . at the time of diagnosis (ards excluded). there was marked reduction in class ii histocompatibility antigen (hla-dr) density on peripheral and bal monocyte/macrophages which recovered over time with resolution of pneumonia. immune suppression occurred prior to development of pneumonia, was especially localized to the infected tissue, but recovered with clinical improvement. specific immune modulation targeted to pulmonary white cells may hasten clinical recovery and minimize pulmonary dysfunction. -clinical experience j. tnllemar amphntericin b remains the drug of choice for many systemic fungal infections. its advantages include a broad spectrum of activity and intravenous administration. the major disadvantages of amphoterlcin b is its severe side-effects, especially the nephrotoxicity. to decrease the toxic side..cffccts various liposomal amphoteficin b formulations have been produced. it was found that these liposemal formulations were as effective as amphotericin b but in contrast had a low incidence of toxicity. at present there are three ~different variations of lipid formulations under assessment: amphotericin b lipid complex (ablc), amphotericin b coloidal dispersion (abcd) or true liposomes. the ablc has a ribbon like structure. it has been shown to have a reduced toxicity and an efficacy ranging from being as effective to four times less effective that conventional amphotericin b. regarding abcd the particles have a disk-like structure with a diameter of around t am and a thickness of nm. the ami-fungal efficacy is - times less than that of conventional amphotedcin b. both ablc and abcd are presently investigated in phase ii/iii studies in the us. ambiseme is currently the only commefieally available true lipesome. ambiseme is a spherical small unilamellar lipesome with a diameter less than nm with a mutina ld of > mg/kg. it has been used in dosages up to mg/kg/day in compassionate based studies with good tolerability. the mycological efficacy range from a % response rate for invasive candida infections to % response rate for aspergillosis. ambisomc have been evaluated as anti-fungal prophylaxis in randomized trials in bone marrow (bmt) and liver transplant (ltx) recipients. it was well tolerated. in bmt recipients the incidence of proven fungal infections was % among placebo treated patients compared to % for the ambisome treated patients (ns). in ltx recipients ambisome prophylaxis was effective, significantly reducing the incidence of deep fungal infections from % to % ill placebo and ambisome treated patients respectively (p< . ). prospective randomized trials comparing these various amphotericin b preparations with conventional amphotericin b is needed to determine their future place in the therapeutical arsenal. two patlentgroups ere particularly at risk to develop serious cmv disease: cmv seronegative transplant recipients of seroposltlva donors and those patlants treated for rejection with anti t-ceil preparations, we have evaluated the value of prophylactic anti-cmv immunoglobulin (cytotect", biotest pbarma gmbh, dreieich, frg) administration in high risk heart and kidney transplant recipients, in a double blind placebo controlled study kidney transplant recipients, treated for biopsy proved re)action with rabbit atg, received globullntplacebo infusions. the preparatlons were given i,v, in a dose of mg/kg at day , , , , and after the initiation of anti = rejection therapy, passive immunization completely prevented cmv related death, although it did not reduce th~ incidence of cmv isolation, viraemia or disease, this effect was mainly observed in cmv saronegativa recipients of a serop sitive donorktdney. seroposltive recipients did not benefit from treatment and seronegatlve recipients of a seronegetlye donor were not et risk for cmv infection at e!l. in a open study the incidence of cmv infection and disease was evaluated in consecutive i~eart sllograft recipients. sixty-five patients were cmv seronagatlve and they all received passive immunlzation according to the dosage schedule used in the kidney patients, but starting on the day of transplantation, this scheme resulted in median snti-cmv igg titers of elisa units during months. cmv infection occurred in / ~eronegetlve and in / seropositive recipients (n,s,), in ssronegetive donor-recipients pairs the incidence was significantly lower ( / ] , the passively immunized seronegstive recipients of e seroposltlve donorheart showed comparable incidence of cmv infection f t ) vs the seropositive recipients. primary infection more often resulted in disease than secondary infection ( v / ), but no difference in incidence of disease ( vs / ) or severity in symptoms was noted between the immunoglobulln treated serone(]ative patients and the seropositiva recipients. apparently passive immunization induces anti-cmv immunity which crossly resembles naturally acquired resistance. abdulkadirov k.,chebotkevich v., moiseev s. the incidence of infection is still high in patients underwent bmt. this complication is the major cause of mortality if it is not recognized and treated promptly and properly. our data showed that from patients with different types of leucemia after autologous and allogenzc bmt had the episodes of fever. in the ma i ority of these episodes the bacterial etiolog$ gram negative bacflli and gram positive cocci) can be proved. on the other hand, in % of the fever cases we detected also viral respiratory (corona-, adeno-, rs-and other) infection. our previous investigations showed that even in healthy persons the viral infection has influence on antibacterial immunity, in the cases of model experimental reaction in volunteers we found the decrease of delayed hypersensitivity - days after intranasal inoculation of influenza virus a (h n - ) to bacterial (staphylococcal, streptococcal and pneumococcal) and ~iycoplasma pneumoniae antigens in the leucocyte migration inhibition test. these results showed that respiratory viruses may be the important pathogenic factor in the development of bacterial infection in posttransplanted period. we consider the constant control of latent and visual respiratory viral infection in bmt patients to be very important. ficcb the ~ter£~li of the nation~l institute of trad/~atoloqy in budapest . consecutive cases of revision hip grafting were carried out arthroplasties wlth hemoloquous bone between the years and . in the same period of time pri~ total hlp replacen~nts were performed under i entieal technical conditions. the average septic rate for the 'total hip althroplasties was less than %. in the selected i cases the septic rate was % indicating the role of bone grafting° homografts were prepared by deep freezing~ it .is recognized that the cells of the hl~grafts become destroyed by the ium~unological, response of the host~ and the patients develop ~ti-hl~, ar~tib'o~ies. the dead ~trix, however, has a bone-inducing capacity that stimulates host osteoblasts to recolonize the *i~/trix which serves as scaffolding. the sequence of events favours the infections. for this reason, beside preventive perioperative systemic ant/biotic treatment, local ~ntibioties were also applied in the form of antibiotic-//npregnated cement. the role of age and the .immune status of the patients .is discussed.. the purpose of this study is to evaluate the rate of toxemia in patients with acute panereatitis and to find this coudition to the activation of cascade systems that are encountered in the subsequent complications of the disease. we studied a series of patients with acute pancreatitis, the severeness of which was evaluated by the ranson's criteria and the apach-ii scoring system. all of them were considered to have severe acute puncreatitis. the determination of toxemia was made using the limulus test (lal test). we also determined the levels of the third (c ) and fourth (c ) complement components as weu as the coagulation factors, iibrinolysis faeters and kimns by serial measurements. the severity of the disease was serially determined by the apach-ii scoring system. it was found that complement activation ( which was also assessed using a graphically illustrated method by a aggregometer ) was followed by an increase of morbitity and mortality .we also detected that toxemia (positive lal-test) was closely correlated with complement activation and more of the ranson's criteria. a clear relation existed between the number of ranson's signs and the enmplieations' rate ( "= - . , p < . ). the documentation of toxemia and the complement activation cannot predict the kind and the severity of complications. the study of coagulation, fibrinolysis and kinms systems didn't reveal any results with statistical significance. necrotizing pancreatitis still represents a life-threatenthg disease. infectious complications dominate among the causes of death. differences in the individual immune response could possibly explain different clinical courses even in patients with comparable pancreatic morphology. to explore the inflammatory response in acute pancreatitis, the following investigation was performed. methods: peripheral-venous blood was withdrawn on admission and furthermore twice weekly in as yet patients with acute pancreatitis and tested for the parameters mentioned below. in parallel, polymorphounciear granaiocytes were isolated using density gradient centrifugation and assessed for superoxide anion and hydroxyl radical producing capacity using electron spin resonance techniques. results: total leukocyte cotmt and total lymphocyte count did neither reflect the clinical course nor predict complications. this comes tree also for serum igg, igm, iga, c , c , crp, alpha-l-antitrypsin and neopterth as well as for plasma il-la, il-ib, il- ra, il- , il- r, il- r, tnf-ct, tnf-~r (p ) and icam- . in contrast, pmn-elastase, il- and il- closely correlated to the clinical course. isolated pmn's in vitro capacity to produce oxygen radicals depended on the respective radical species and was slightly elevated (superoxide anions) or decreased (hydroxyl radicals), respectively. patients with a cd +/cd + ratio below i were seen at risk of developing septic complications. in contrast, a percentage of monocytes of % or more among total mononuclear cells indicated an uncomplicated course, in general. conclusions: the immune status of the individual patient may significantly influence the course of acute pancreatitis. the cytokine pattern in peripheral blood is very complex and most parameters are of little use for the clinician. the pmn-elastase, il- and il- , however, closely correlate to the clinical course and may prove valuable for follow-up. the cd +/cd + ratio was found the best predictor of septic complications, but it failed in non-septic patients. a percentage of % or more of monocytes among total mononuclear ceils indicated a rather mild course. the reduced ability of the pmns to produce hydroxyl radicals may help to explain the frequent development of septic complications in severe necmtizing pancreatitis. peroxidation of membrane lipids contributes to ceil injury in pancreatitis. overwhelming release of toxic metabolites by infiltrating neutrophils is regarded a major pathogenetic factor, too. as yet little is known about the mechanisms by which oxidative stress and leukocytes damage pancreatic cells. the present study examines (i) the susceptibility of pancreatic acinar cells to attacks by oxidants and leukocytes and ( ) the potential of antioxidants to prevent such damage in order to better understand the cellular mechanisms of pancreatic injury in inflammatory states. methods: freshly isolated rat pancreatic acinar ceils were exposed to a model system of oxidative stress consisting of mu/ml xanthine oxidase (xod), mm hypoxanthine (hx), mm fec and mm edta. in a second set of experiments, acinar cells were exposed to excess autologous neutrophils or neutrophils obtained from patients with acute pancreatitis. neutrophils were stimulated by zymosan a, pma, and il- . cell viability was assessed by both cellular uptake of trypan blue (tb) and by release of ldh. results: the xod/hx system caused a time-dependent acinar cell injury. this injury was effectively prevented by catalase (cat) and gfutathione peroxidase (gpx). in comrast, superoxide dismutase (sod) enhanced cell injury. addition of both sod and cat abolished the damage seen with sod alone. the non-enzymatic scavengers mannitol, dmso, dmtu and the iron chelator deferoxamine were not protective and at a higher concentration even accelerated cell decline. the newly developed antioxidants of the lazaroid type effectively prevented oxidative acinar cell damage. stimulated neutrophils, both autologous and heterologous, did not damage healthy acinar cells but had even protective effects. conclusion: pancreatic acinar ceils are very susceptible to oxidative injury. a combination of catalase and sod prevented cell damage effectively. sod when given alone may rather damage than protect aelnar cells when h is generated in concentrations overwhelming the capacity of endogenous catalase. therapeutic approaches to pancreatic disease using antioxidants should, therefore, include combinations of protective substances. the lazaroids seem to be candidates for clinical use as antioxidants in pancreatitis. the results argue against direct toxic effects of stimulated neutrophils to pancreatic acinar cells. are ch~act~z~ by the presence of a polymicrobial flora, the pmtotyi~ cffthese inf~ons is secend~,y bacterial pedtonitlw, whereby a pathololoeal process in the ~trointesfimd tract r~ful~ in tim disrup~on ofi~ inteffrlty and ¢ollseqtlent sptl]nge of inte~.i,o~.l gontents into the peritoneal c~iry. the ensuing infection invariably contains a mixtm~ of gt~m negative enteric bacilli, gram positive b~eria and anaerobe& experimental and clinical =t~ies have de~ed the eantrlbution of each of th¢~ components to ti~ ovemu virulence of these in~ons, gram negative enteri~ such as f.veher~chla coil ere endowed with a virulent l~l~x~lyse~haride ptill~ly t~sponsible for lethality, by contrast, bacteroldes sl~cles, which rarely c~se death, prornot~ abscess fonllation, a uniqm~ capsul~ polyseccluu'ide, particularly on b.j~ogiljs slrai~, oontributes to tjtis erect, several mecltanims have bccn pml~ed whereby or~ microorganism mi~t interact with its microbial ~net to augment the overall virulence of a r~xed im~edan. these include: l) provision of nutrients by one apexes which stimulates the growth of its ~opathoge& ) inhibition of host deletes by one of the migroorganisms so that the other microbes might persist and exert their virulence, ) the trant~ of vim.©n~e traits between ~renr~a.,dsms and ) the ~.mizatian d the mi~oe~vironmental con~tion$ by one d the baetez'isl pa#, so that the other might persist. exampl~ for each of these m~banisms imv~ been provided by experimental ttudies i~stigating e.co!l-b.p~flls synergistic in~ra~ons. byproducts ofg.coli metabolim l~¢ovide essential short ebath fatty acids £~ optimal b,frosili~ ga'owth. fm-ther, oxygen ¢ons~tmption by kcelt lowers oxygen tension end redox potantial to levels eomlucive to b#a#lts gro~h. coawr~ely, b,~agtlis rolea~s proteases and fatty acids wl~¢h impair pl'tsgocy~¢ ~lt rmctlon tnd permit f-..¢oli proliferation and expression of its intrinsic virulent. in summaxy, interactions among the separate microbial cemponents of mixed infections heighten the overall virttienee of these lafectiot~, this knowledge provides ~r rationale for targetting of antibiotic therapy against the knowa eantributors of these synergistic pro~¢sses, intraabdominal abscess formation and the macrophage william g. cheadle, m.d., department of surgery, university of louisville school of medicine, louisville, ky inflammation of the peritoneal cavity following bacterial contamination has been classified into primary, secondary and tertiary, the last two relating to bacteria originating from the gastrointestinal lumen. the natural history of such infection is either resolution without clinical sequelae, which is uncommon, abscess formation, or generalized peritonitis, which occurs as a result of failure of peritoneal host defenses. early clearance of microorganisms by peritoneal fluid circulation and filtration througti subdiaphragmatic lymphatics into the thoracic duct and systemic circulation occurs as well. simultaneously peritoneal macrophages and the omentum approach the area of inflammation and lead to neutrophil influx and abscess formation adjacent to the affected viscus. we have found a shift in peritoneal macrophage function from antigen presentation to proinflarnmatory cytokine production that occurs early after experimental peritonitis produced by cecal ligation and puncture. this is also reflected by reduced class ii histocompatibility antigen expression on peripheral blood mononuclear cells and peritoneal macrophages. this is accempauied by an influx of both neutrophils and macrophages into the peritoneum and subsequent abscess formation. interestingly, there is little serum endotoxin or tnf seen in this model despite tnf mrna expression in peritoneal macrophages. we believe this model is more clinically relevant than other models of endotoxemia or bacteremia in which different patterns of cytokine expression are seen. newer agents aimed at reduction of systemic manifestations of sepsis originating from intra-abdominal infection such as monoclonal antibodies against cytokines or il- receptor antagonists may need to be directed against remote organ macrophage populations while preserving peritoneal macrophage function. inflammation is a complex process involving microcirculatory changes, extravasation of fluid and a cellular influx in the affected body area. in our communication, we will only consider the regulation of the cellular infiltrate which plays a major role in the defense of the peritoneum against microbial invasion. until recently, it was thought that the influx of leukocytes in the abdomen was induced by bacterial products, local humeral factors and secretions of resident macrophages. there is now increasing evidence that this view is too simplistic. many other cell types present in the abdominal cavity or composing the peritoneal membrane (mast-cells, mesothelial cells, fibroblasts) are able to release or secrete vasoactive or chemotactic substances such as histamine, prostagtandines, or cytokines. they are most likely to play a role in the regulation of intraperitoneal inflammatory reactions. the emigration of leukocytes towards the abdominal cavity is also modulated by a previous contact with gram negative bacteria. in the rat, this intriguing phenomenon is long lasting, cannot be transferred by serum and seems independent from t lymphocytes. the clinical relevance of these various regulating mechanisms has still to be determined. kinnaert paul, h pital erasme, route de lennik , bruxelles belgium generalized response in secondary peritonitis the clinical course of an intraabdominal infection may depend on a variety of variables including the capacity of host defense mechanisms and the degree of the inflammatory response. if local defense mechanisms fail to restrict the inflammation to the abdominal cavity a generalized inflammatory reponse will result. in a first stage generalized signs of a local inflammation become detectable whereas the second stage comprises the overwhelming systemic inflammatory response. the extent of this systemic response determines the outcome. sometimes it may appear to be unrelated to the severity of the intraperitoneal findings. the activation of plasma systems and cellular elements leads to a fast release of cytokines, inflammatory mediators and other substances. these parameters precisely reflect the degree of the generalized response. inflammation of the peritoneum causes significant morbidity. objektives: to test the hypothesis that peritoneal mesothelial cells play a role in regulating inflammatory responses within the peritoneal cavity, we examined neutrophil-chemotactic activity (interleukin ) and monocyte-chemotactic cytokine (mcp) release by sytokine-etimulated mesothelial cells. confluent human peritoneal mesothelial cells were exposed to varying concentrations of phorbolmyristate-acetate (pma) and the cytokines tumorneerosis factor a (tnf a) and interleukin i~ (il-i~). the supernatant was examined for il- by elisa and for mcp by investigating the ehemotactic activity for isolated human monocytes. mesothelial cells express low levels of il and monocyte chemotactic activity when cultured. these activies were significantly increased ( -fold) after stimulation with either tnf a or il-i~. additionally macrophage inflammatory protein was detected. these observations provide a probably important mechanism whereby peritoneal mesothelial cells respond to imflammatory stimuli released during peritonitis and how leucocyte recruitment by liberation of chemotactic cytokines is regulated. the perioperative course of lps, tnfa and il- in patients with bacteriologic proven abdominal infection (intraabdominal abscess , diffuse peritonitis , pancreatic necrosis , pancreatic abscess ) was followed prospectively and evaluated for possible correlation with septic state and organ function. methods: patients were studied in a to hours period during their first surgical intervention because of intraabdominal infection. all were monitored for their cardiovascular, respiratory, hepatic and renal function. plasma samples for lps. tnfa and il- determination were drawn preoperatively, intraoperatively, and until h postoperatively in regular intervals (min /pat), results: preoperative apache ii was in median (rain , max ). patients fulfilled the criteria of sirs. of them were in septic shock.there was a significant correlation between preoperative tnfa and apache ii (p= , i, spearman coefficient). preoperative cardiovascular (systol. rr< mmhg) and respiratory (pao < mm hg) dysfunction were associated with significantly elevated tnfa (cardial: p= , i, wilcoxon; pulmonal: p= , ) and il- (cardial: p= , ; pulmonal: p= . ) overall, lps, tnfa and il- values varied considerably during the observation period. however, tnfa was markedly higher in patients with sirs and septic shock (group a: n= i , mean pg/ml) than in those who did not fulfill these criteria (group b; n= , mean pg/ml; p= , i, wilcoxon). il- was significantly higher in group a (mean pg/ml) than in group b (mean pg/ml; p= , o i wilcoxon). conclusion: perioperative tnfa and il- were shown to correlate significantly with preoperative organ function, apache ii and the severity of sepsis. these results could help to define patients that might benefit from further therapeutic strategies, e.g. antibody administration. department of surgery, university vienna, akh wien, wahringer gurtel - , wien. aim of the study: the purpose of this pilot study was to establish and to prove a standardized reproducible animal model of intraperitoneal sepsis induced by e.coli-endotoxinaemia in lew.lw-rats in order to investigate early immunoserological responses to find a mediator based evaluating system of peritonitis sepsis. materials and methods: in lew. lw-rats, diffuse peritonitis was induced by intraperitoneal injection of a mixture of e.coli (khu +) and autogenous haemoglobin solution. in the control animal group (n= ) an intraperitoneally injection of physiological saline solution was done. blood samples were obtained by heart puncture after hours. stastistieal calculations were performed on a personal computer with the spss programm vers. . (correlation with pearson's r, mann-whitney-u-test, descriptives statistics, discriminant analysis). results: in contrast to the sham treated rats, the peritonitis animals showed significant differences in the concentrations of endotoxin, interferon-gamma (wn-y), the pteridin derivate biopterin and serum pla -activities [endotoxin range from . eu/i, sd= . to . eu/ , sd- . (p < ), ifn-¥ levels, range from . pg/ml, sd- . , to pg/ml, sd= (p < . ), circulating pla -activities range from . , sd= . to . u/ , sd= . (p < . ) and biopterin range from . nmol/l sd= . to . nmol/l, sd= . (p < . )]. for the peritonitis group we found strong correlations between the degree of endotoxinaemia to elevated levels of ifn-'~ (rp = . , p < . ) and bioptefin synthesis (rv= . , p < . ). the increase of ifn-t levels was correlated to the regulatory synthesis of biopterin (r = p < . .. p • , . . ) and to the pla -actwtues (rp = . , p < . ). the biopterin synthes~s correlates slightly with the pla -actn,ities (rp= : . ; p < . ). using the para, meters of endotoxin, ifn-y levels, biopterin and the pla~ -activities only, the statistical procedure of the linear discriminant analysis makes it possible, to distinguish between non-septic animals and septic animals correctly at a rate of %. anaerobes were found in . %, anaerobes were isolated in . %. there were aerobic and anaerobic associations in . % and microflora was not found in . % of the cases. express method of anaerobes discovering let to receive information on - days early than in generally accepted nethods. intraaotal transfusion of oxygenate blood and laser irradiation of blood reduces the duration of anaerobic sow, disminishes intoxication and accelerate the patients recovery. patients with abdominal sepsis are subject to long periods of hospitalization and high associated morbidity and mortality rates. this category of patients is thus consuming extensive facilities and costs. as the age-related outcome of abdominal sepsis is not fully known, the aim of the present study was to investigate abdominal sepsis in the elderly. out of patients with abdominal sepsis treated at the surgical intensive care unit during a -year period, ( %) had an age of years or more. were women and were men, a sex distribution not differing with patients younger than years. the patients were scored according to apache ii and septic severity score (sss) upon arrival to the intensive care unit. bacterial cultures, the occurrence of organ failure, hospitalization and outcome was noted. in median two operations were performed for both "younger and elderly" patients. the median time of hospitalization in the elderly was (- ) days including in median days in the icu. figures in patients less than years of age were comparable ( (- ) days out of which in median days in the icu). apache ii and sss-scores did not significantly differ ( . vs and . vs . , respectively), between the groups. neither did the incidence of organ failure differ ( / vs / ). however, the incidence of multiple organ failure was significantly lower in elderly patients ( / vs / (p < . )). the mortality rate, however, did not differ between the groups ( / vs / ). in conclusion, severe abdominal sepsis in the elderly was not associated with an increase in mortality, incidence of organ failure or hospital stay. with the help of light transmissional scanning electron microscopy morphology of erythrosytes of peripheric blood was studied in patients with different stages of diffuse peritonitis before and after intravascu!ar irradiation of blood with heliun-neon laser. peritoneal morphology was investigated in patients who died from peritonitis, it was established that in all phases of peritonitis occured stomatocytoric and echinocytoric transformation of erythrocytes which progressed simultaneously with increase of intoxication. it combined with strongly pronounced vessels variability of microcirculatory peritoneal bed which displaied by erythrocytes aggregation, stasis and microtrombogenesis. in intravascular laser irradiation of blood number of erythrocytes which underwent to stomatocytoric and echinooytorie transformation was lower than in patients without laser irradiation. it indicated that the intravascular irradiation of blood with helium-neon laser can prevent development of severe alterations of rheological property of blood and consequently variability of microcirlatory peritoneal bed in patients with diffuse peritonitis. abdominal sepsis is still associated with high morbidity and mortality rates, frequenfly caused by multiple organ failure. it has been reported that changes in capillary permeability play a role in the pathogenesis of multiple organ failure. the present study aimed at evaluating the influence of intraabdominal sepsis induced by cekal ligation and puncture on capillary permeability in multiple organs and tissues. adult male sprague-dawley rats were subjected to laparotomy with separation of the cekum (sham operation) or induction of intraabdominal sepsis by cekal ligation and puneatre (n-- in each group). at , , , , and hours (n= /timepoint), the animals were evaluated concerning mortality and capillary permeability as determined by the passage of : i-labelled albumin from capillaries to the peritoneum, the proximal and distal small intestine, cekum, colon, spleen, kidneys, lungs. the mortality rate in rats with intraabdominal sepsis was % both at and hours. capillary permeability in the peritoneum, cekum, colon and kidneys significantly increased from hours and on in rats with intraabdominal sepsis. in septic animals, capillary permeability in the lungs and spleen increased from hours and on and in the proximal and distal small intestine from hours and on. different types of alterations in capillary permeability seem to appear: ) a temporary short increase e.g. in the proximal small intestine and spleen; ) a temporary longer increase e.g. in the colon and kidneys; ) a persisting increase e.g. in the peritoneum, cekum, distal small intestine and lungs. we conclude that experimentally induced intraabdominal sepsis induces early alterations in capillary permeability in multiple organs and tissues. such changes may contribute to explain the development of sepsis-induced multiple organ failure. despite a number of significant advances in the care of burn and non-burn traumatic injury, infection and sepsis remain major causes of morbidity and mortality. the severe immunosuppresslon often seen in patients with severe trauma or large burns may predispose these patients to life threatening infections. included among the many immune alterations are changes in the functional capabilities of neutrophlls (pmns). we have examined the expression of the p integrins (cd l a, b,c/cd ), and the fc'?r (cd , cd , and cd ), as well as several functional parameters, on pmns from thermal and non-thermal traumatic injury, pmns were obtained from patients sustaining severe trauma (initial apache ii score > ) or thermal injury (> ~ total body surface area, % full thickness), and healthy controls. the expression of cd b and c and to a lesser degree cdi a was significantly reduced on pmns. the expression of cd and cd but not cd was also significantly reduced. pmns displaying this reduction in receptor expression have a significantly reduced ability to phagocytose bacteria and undergo the oxidative metabolic burst response. thermal and traumatic injury result in global reduction in the expression of integrins and for which may lead to decreased functional capabilities, these abnormalities may in turn account at least in part for the increased rate of infection in these patlems, institute, dept. of surgery, ~ ethesda ave, cincinnalt, oh, usa, - s b, antibiotic-phagocytic cell interactions: their effect on endotoxin release. c g c-emmet , dep[baeteriolog.z, univer_sitv of glasgow, scotlan~_d increasingly it is recognised that pathogenic bacteria are capable of surviving intracellularly within phagocytic cells in addition to their capacity to produce disease whilst in the extracellular milieu. as well as providing protection from certain antibiotics which fail to penetrate the phagocyte, such intraceltular bacteria may be transported from the initial site of infection to a distant more vulnerable body site wherein they may proliferate. it is also known that some antibiotics are capable of becoming concentrated within phagocytic cells mid displaying bioactivity therein. such bioactivity might be responsible for the release of endotoxia #orn gram-negative bacteria which when liberated from the celt could ~gger the cytokine cascade. anfib,.'otic-induced damage to the ultrastructure of bacteria can also occur when the target bacteria are exposed to low (sub-mic) concentrations of certain drugs. such bacteria may present quite altered surface components m host-defense cells as well as releasing biologically active ceil wall components such as endotoxin. the nature of these interactions at the cellular level as well as the consequences for the host will be discussed. new jersey medical school: umd, newark, nj a technique of physiologic state classification has been developed based on the m~itlvariable analysis of patient derived data sets of seventeen physiologic variables. these multivariable data sets obtained from critically ill patients requiring intensive care, were aormallsed by the mean and the standard deviation of recoverin~ trauma patients who were not critically ill, the resulting normalized seventeen variable sets were then clustered. seven independent data groupings were developed. the normal stress response hyperdynamic state seen post-trauma and in compensated sepsis (a stets)/ metabolic insufficiency seen in septic decompsnsation (b stste}; early (c,) and late (e ) respiratory insufficiency associated with ards; cardlogenlc dscompensation (n state); post-trauma hyvolemla without shock (r stats). the stats closest to a new patient's values allows patient classifi atlon with regard to his previous physiologic state. classifying observations f~om patients who lived or died who fell into these physiologic states enables a probability of death (p death) to be obtalned. utilizing this criteria for the staging of severity in recent trauma patients the physiologic states accurately and significantly predicted the likelihood that the patient had an increased circulating level of the eytoklnes tnf and il- . the probability of death (p death) as well as the cytoklne levels appear to be a function of the physiologic b state with the highest levels being seen in the b state of metabolic insufficiency and the c~ state of oombined respiratory and metabolic insqffioienoy characteristic of septlc ards. the increase in the magnltude of metabolic abnormalities associated with the transition from non-sepsls to septic a, septic b, or septic c z states was associated with an increasing probability of death (p denth)(mean a state =. , mean b state = . , mean ~ state = . ). the accuraay of this estimate was prospectively analyzed in this group of m~itlple patients of whom % had sepsis and % had ssptlo ards. the survivors had a mean p death of . and the deaths had a mean p death of . . the severity of post-trauma sepsis can be quantified by probability analysis and stra~ifie~ by physiologic state. serologic tests have not been extensively tes'~ed in surgical patients but seem to be of limited value. we use nystatin as the main form of chemoprophyhxis. patients "~'ith signs of infection who do not rapidly improve with antibacterial therapy are candidates for anti-funsal therapy, amphoteradn b remains the first llne of therapy although combination therapy '~'ith flueonazole is use;l with increasing freque~;c)', the recovery of c~dida from an antra-abdominal site represents a challenging problem, anti~ngal therapy in such patients depends on the underlying disease, the nature of the infected material and overall patient risk. role of neural stimuli and pain principles and practice of anesthesiology effect of combined prednisolone, epidural analgesia and indomethacin on the systemic response after colonic surgery arginine: biochemistry, physiology and therapeutic irnplications immunosfimulatory effects of arginine in normal and injured rats arginine stimulates lymphocyte immune response in heahhy humans rote of arginine in trauma, sepsis and immunity arginine enhances wound healing in humans if labrecque t, gv campion t, and the rhll-lra phase i//sepsis syndrome study group the cleveland clinic foundation a murine-anti-human tnf-monoclonal antibody known as cb was the first anti-tnf mab which was studied in a phase ii multinational trial in the treatment of patients with severe sepsis.this was an open-label, dose-escalation trial consisting of patients who were enrolled into one of four treatment groups: ( ) . mg/kg of anti-tnf mab, ( ) . mg/kg, ( ) mg/kg or ( ) . mg/kg at study entry and the second dose hours later. the small sample size in each group (n= ) precludes detailed statistical inference in this study. nonetheless, a considerable amount of useful information was obtained from this investigation. irst, this study demonstrated the clinical feasibility of specific anticytoldne therapy in septic patients. second, the measurement systemic levels of tnf proved to be an elusive target; interleukin- may prove to be a more useful indicator of cytokine activation. third, immunologic reactions including tnf: anti-tnf mab immune complexes and human anti-routine antibodies were frequently found in these patients. despite their apparent lack of overt toxicity in this study, these immunologic reactions may complicate this form of anticytokine therapy. additionally, the potential benefits of anti-tnf mab therapy occur within the first hours of therapeutic administration in these septic patients. infecting organisms differ in their potential to induce tnf in vitro and these differences correlate with circulating tnf levels observed in septic patients. rapid methods to define those patients most likely to respond to anticytokine therapy are needed to determine the ultimate therapeutic potential of these agents in clinical medicine. wherry, j., abraham e., wunderink r., silverman h., perl t., nasraway s., levy h., bone r., wenzel r., balk r., allred r., pennington j. and the tnfa mab sepsis study group.tnfa mab (bay x ) is a murine monoclonal antibody raised against human tumor necrosis factor. tnf~ mab has been shown to reduce morbidity and mortality in animal models of septic shock and has been safely administered to septic and non septic patients.to evaluate the efficacy and safety of tnf~ mab in patients with sepsis syndrome, a prospective, multicentered, double-blind, placebo-controlled trial was conducted in hospitals in north america. patients were prospectively stratified into shock or nonshock groups and then randomized to receive a single intravenous infusion either of mg/kg tnf~ mab, . mg/kg tnf~ mab or placebo ( . % human albumin).patients received standard aggressive medical/surgical care during the day post dosing period.the three treatment arms were well balanced with respect to demographics, apache ii score and other parameters. for all infused sepsis syndrome patients, those who received tnf~ mab had slightly reduced day all cause mortality compared to placebo. among shock patients there was a more pronounced trend towards efficacy at day post dosing with lower mortality rates in both active treatment arms. among nonshock patients tn~ mab did not appear beneficial. the initial clinical experience with a chimeric anti-tnf monoclonal antibody, ca , was undertaken in septic patients. the objectives of the study were to determine the safety, pharmacokinetics and effects on cytokine levels of ca . as a single infusion or in combination with ha- a in septic patients. the study was conducted with the intent to progress to an efficacy trial based on the information collected.the trial was conducted in three stages. stage was an open label trial in which groups of patients each with the clinical diagnosis of sepsis received ascending doses of ca ( . , , , mg/kg). stage was a randomized, double blind study in which patients received a single dose of ha- a ( mg) and placebo or one of doses of ca ( , , mg/kg). stage was a randomized, double blind study in which patients received a single dose of placebo or one of doses of ca ( . , , mg/kg). in addition to usual laboratory tests, the following assays were performed: chimeric anti-tnf concentration, anti-chimeric antibody, endotoxin, tnf, il- , and il- levels.a total of patients were enrolled from clinical sites ( in stage , in stage and in stage ). primary analyses were performed on patients in stage and . there were patients who received ca exclusively and patients received placebo. administration of ca was well tolerated at doses up to mg/kg. no patient discontinued treatment due to adverse events. human anti-chimeric antibody responses were positive in % ( / ) of evaluated patients. mean cma × and auc increased proportionally with increasing doses of ca . the mean half-life was - hrs ( - hrs). a dose related decrease in tnf concentration was observed hr post infusion of ca . tnf is considered to be one of the central endogenous mediators for the inili'ation of the pathophysiological changes in patients with sepsis and septic shock. high tnf levels were demonstrated to correlate with patient outcome. blocking or neutralising tnf with specific antibodies was effective in preventing death in some animal modets of sepsis. in a placebo controlled prospective randomized study we tested the mur~ne derived antibody mak f. it is a f(ab') fragment. the fragment rather the complete antibody was selected in order to reduce the potential immunogenicity and to facilitate tissue penetration. patients with severe sepsis or septic shdck were enrolied in the study, three different doses of mak f or placebo were administered ( , ; , and i mg/kg) over a perid of hours in random order. the patients were evaluated for side effects, hemodynamics, organ dysfunction, cytokines (il , il and tnf), and outcome. at this time only an interim analysis of patients is available i indicating that mak f in all dosage groups resulted in a decrease in il . this contrasted to a further in crease of il in the placebo patients. no serious side effects have been reported so far. a more detailed analysis on all patients in the study will be presented and discussed.$ s staubach,k.h., otto, v., kooistra,a,, rosenfeid,j.a., bruch, h.p., univ. lfibeek, germany once endotoxinemia occurs in sepsis a vieieus cycle with translocation of et can be established. increasing the clearance capacity for et would therapeutically be the ulimate aim. we developed a new et on-line adsorption (ad) system in whole blood by means of polymyxin b (pb) coupled eovalently to a matrix (acrylic particles) via a atom-chain spacer. the detoxification capacity was ug[et/ml column material. the biocompatbility resulted in ~ platelet recovery. the column contained ml of admaterial and was sterilized by high steam autoclave, anticoagulation was achieved by heparine . iu/h in the inflowline after bolus injection of . iu. hp was performed on pigs at a rate of ml/min by means of a roller-pump until the animals succumbed (h). animals served as controls (c). serum et levels rose from . pg/ml to , pg/ml after hours in the c and from . pg/ml only to pg/ml in the h group after hours whieh was highly significant. survival time could be extrended from to min. results are listed in the following l. blinzler, p. zaar, m. leier, r. b( rger, d. heuser clinic of anaesthesiology , city hospital nuremberg, germany sepsis and multiple organ failure (mof) are still related with poor prognosis inspire of pharmacological and technical progress. impressed by revealing reports about blood purification the continuous veno-venous hemofiltration (cvvh) was used as supporting treatment beside the critical cam basic therapy of mof. from to consecutive patients were treated by cwh. mof was caused by hemolrhagic-traumatic noxa in °, and by septic-toxic event in %. all patients required mechanical ventilation (fio > , ) . ° showed hyperdynamic shock. % had renal and % hepatic failure. medium appache ii score amounted to , points. cvvh was performed in postdilution mode with a polyamide membrane (fh ) and high volume exchange ( l/die). anticoagulation was done with heparin. hemofiltration in mof was installed, when critical cam basic therapy including adequate respiratory and hemodynamic management, pamnteral nutrition, antibiotic treatment, etc., failed to stabilize organ functions. during consequent application of cvvh most of these patients showed improvement of their clinical course. pulmonary stabilization was seen in %, hemodynamic in % and renal in % of the cases. % of the patients survived and were discharged from hospital. of non-survivors ( %) died because of fatal mof within h after admission to icu. patients with early application of cvvh in mof showed a better survival rate.mediators of mof, i.e. products of the complement cascade measured in blood and nitrafiltrate by elisa, were partially removed by cvvh. the testing ultrafiltrate by hplc demonstrated decreasing spikes ofpolypeptides during hemofiltration. mof seems to be generated by cascade-activation of immune competent cells and plasmatic mediators (e.g. bmdykinin, eicosanoides, cytokines, anaphylatoxins, etc.). therapeutic approaches aim to inactivate or eliminate single substances. cwh with high-flux membranes in combination with high-volume exchange allows elimination of many mediators with different molecular weight and therefore may contribute to improve the prognosis of mof. other significant advantages of this teqalnique like adequate nutrition, optimized fluid balance and control of body temperature should not be negicctod. introductioni pseudomonas (p) aeruginosa has to be considered an important pathogen of nosocomial pneumonia and septic organ failure. the lung seems to be the predominant target organ for the pore-forming p. aeruginosa cytotoxin, thus inducing microvascular injury. with respect to therapeutical consequences, the potential protective effects of paf-antagonist (web ), cyelooxygenase inhibitor (diclofenac) and specific and unspecific antibodies on cytotoxin-induced pulmonary vascular reaction and mediator release were studied in the isolated perfused rabbit lung. methods: cytotoxin ( p_g/ml) was administered into the perfusion fluid in all groups, either in the absence of inhibitors (n= ), or after pretreatment with web ( xl -gm, n= ), or diclofenac ( #g/ml, n- ). furthermore, the application of specific antitoxin (mg/ml, n= ) was tested in comparison with the unspecific immunoglobulins (venimmun®, behring, . mg/ml) (n= ) and the combination of immunogiobulins, web and diclofenac (n= ). six experiments without toxin served as controls. the arterial pressure mad the weight gain as an indicator of edema formation were continuously monitored during the three hour peffusion phase. arachidonic-ucid metabolites, as well as lactate dehydrogenase (ldh) and k + concentrations were determined at rain intervals. results: cytotoxin caused a gradual increase in pulmonary arterial pressure, reaching a maximum value of . times higher than the control, starting after min and a delayed onset of edema formation resulting in a mean weight gain of g after min. this was paralleled by a significant increase in prostacyclin generation and a continuous release of k + and ldh. thromboxane synthesis exceeded about times that of controls in the toxin treated lungs. pretreatment with web or diclofenac significantly attenuated the pressure response and edema formation evoked by cytotoxin. the addition of the unspecific immunognbulin preparation alone induced a transient pressure increase within the first minutes, but mean values remained below those of the cytotoxin group in the continuing observation period. mmost complete inhibition of the pressure reaction, the edema formation and the metabolic alterations was achieved mainly by the combination of immunoglobulin, web and diclofenac and to lesser extend by the specific toxin antibody. conclusion: the current results point towards the crucial role of paf and aa-metabolites as mediators of cytotoxin induced microvascular injury. the systemic or local application of cytotoxin antibodies or even unspecific immunoglobolins in combination with paf-antagonist and diclofenac appears to be a promising therapeutic approach in the case of infection with cytotoxin-preducing strains. cytokines have long been shown to be of particular importance in the metabolic derangements occurring in lps-induced shock. recent studies strongly imply the involvement of platelet aggregating factor (paf) in the pathogenesis of gram-negative bacterial sepsis. an autocatalytic feedback network has been postulated to exist between paf and tumor necrosis factor (tnf), a key cytokine involved in septic metabolic cascade, leading to an uncontrolled amplification of inflammatory mediator release. we have previously shown that st ( -n,n,n trimethylammonium-(r)- -isovaleroyloxy-butanoic acid z- -( -chlorphtalidiliden) ethyl ester bromide) was quite effective in inhibiting the "in vitro" binding of h-paf (ki= . x - m) to rabbit platelets. the present study shows that pretreatment of c bl/ mice with st , administered by different routes, dose-dependently and significantly reduces the lethality induced by endotoxin (e.coli :b injected at mg/kg intraperitoneally). very interestingly, st administered at the same doses as above (i.e. . , . , and mg/kg body weight) results to be significantly effective in reducing the endotoxin-induced release of serum tnf. the reported dual activity of st (i.e. paf antagonism and decreased circulating tnf levels) may turn out to be greatly beneficial, in combination with current therapies, in the treatment of diseases that involve overproduction of tnf and paf such as septic shock. introduction: recently, we reported that prophylactic whole body hyperthermia ( . °c) induces heat shock protein ('asp) and increases smvival - fold in a mouse endotoxin model (am. j. physiol. in press). other investigators reported that prophylactic pharmacologic induction of hsp- by sodium arsenite improves survival in a rat sepsis model (abstract a am. rev. resp. dis. vol. , ) . the effects of heat are complex and in addition to formation of lisp- include release of cytokines, changes in cellular ph etc. thus, the protective mechanisms of heat may differ from those due to pharmacologically induced . the purpose of this study was to compare the protection of heat vs the protection of pharmacologically induced hsp- in a mouse endotoxin model to determine if different protective mechanisms were likely to be involved.. i%'lethods: both sodium arsenite ( mg/kg) and ethanol ( ~ of % ethanol) caused marked induction of hsp- in lung, gut, kidney, and liver, which was comparable to heat-induced hsp- . female nd mice weighing - gms were pretreated with arsenite or alcohol hours prior to challenge with escherichia coli endotoxin (-ld ) and survival was compared to control mice. results: survival at hrs. for arsenite treated and alcohol treated mice was % and % respectively and was statistically different from the % survival for control mice. (p< . ) (n= mice per group). however, at days post endotoxin, there were no differences in survival in the groups, i.e., ~ % survival for all groups. in contrast, the protective effect of hyperthermia remains present at days, i.e., ~ % survival vs % survival control. conclusion: the protective effect of heat is probably due to other factors such as the effect of hyperthermia to release il-lc~ and is not due solely to hsp- formation. it was the aim of the study to examine whether bacteria play a causative role in the pathogenesis of anastomotic insufficiency following gastrectomy in man.the study was carried out in form of a prospective, randemised, double-blind, multicenter trial. primary endpoints were the rate of anastomotic insufficiencies, infectious-and uncomplicated postoperative courses. all pat. received a periop, i.v. prophylaxis with cefotaxim. identical numbered vial either contained placebo or polymyxin b, tobramycin, vancomycin and amphotericin b . the vials were administered x per day from the day be ~ fore the operation until the th postop, day. insufficiencies were detected by gastrographin swallow and recorded by x-ray on day postop.. evaluation was carried out on an "intention to treat'basis. statistical analysis was done with the pearson's chi square and fisher's exact tests~ results: interim analysis was carried out in / after pat. had been recruited. along with a significant reduction of s.aureus and enterobacteria there was a reduction in the rate of anastomotic insufficiency of the esophago-jejunostomy from . % in the placebo-group to . % in the treatment group. the difference was not yet significant. the rate of nosocomial infections (e.g. respiratory tract infection and uti) were significantly reduced from . % in the placebo-group to . % in the treatment-group (p ~ . ;fisher's exact test). in march final results with more than patients will be presented for the first time. (= po < mm hg, b s-creatinin > mg%). respiratory insufficiency was the most frequent systemic complication followed by sepsis and respiratory insufficiency. etiology of pancreatitis and initial serum increase of pancreatic enzymes predicted neither complications nor outcome. only of deaths occurred during the st week, all other deaths occurred late (after - weeks), generally as the consequence of septic complications and multi-organ failure. high levels of crp were correlated with a compliacted course and a fatal outcome. although same cytokines (e.g. -- ) were found increased in severe disease, the predictive value of these markers was not better than the combination of ctinical scores (ranson, imrie, apache ii) with gt or crp. conclusions: intensive care medicine can often control the inital shock situation in severe pancreatitis. thus. only % of deaths today occur eady in the course of the disease, whereas this percentage varied between - % just years ago. nowadays, most deaths are caused by late septic complications and multi-organ failure. ranson-and ct-scores as well as serum crp predict a course with systemic complications; they are less helpful for prediction of sepsis and late mortality. it is doubtful whether measurements of cytokines will help to better predict the late outcome. as yet, only careful and continuous monitoring of patients (e.g. by apache scores) may help to early identify those who develop septic complications and multi-organ failure. the classic description of severe acute pancreatitis has hinged upon the release of large volumes of activated enzymes into the peritoneal cavity and thertce the lymphatics and blood stream. these activated enzymes escape from the pancreas due to disruption of cells with associated ischaemia and occasional infarction of tissue. for to years it has been postulated that the bocly's defence system to activated pancreatic enzymes required supplementation iu the form of anti-protease support either in the vascular space or in the peritoneal cavity. all controlled studies have shown that this is either impracftcal or unnecessary.hore recently release of a large number of cytokines from monocytes, macrophages and neutrophils have been considered to be harmful to the body and various agent~ which oppose the action of tnf alpha, paf and similar cytokines are being examined in experimental anim~is and certain clinical trials, it has clearly been shown that higher levels of cytokines are released in the patients with objectively graded severe acute pancreatitis than in those with milder disease. we now seem to be moving into an exciting phase of potentially beneficial therapy in acute pancreatitis which has had no specific effective therapy through studies utilising aprotinin, gabexate mesilate and fresh frozen plasma. inflammation cascades may play a role in the pathogenesis of acute pancreatitis. to evaluate the status of the cellular immune system we examined serum concentrations of immune activation markers in patients with acute pancreatitis ( males, females; median age: years, range: - years). concentrations of neopterin, serum soluble tumor necrosis factor receptor (stnf-r) and serum soluble intercellular adhesion molecule type (slcam- ) were determined using immunoassays (henning, bender, t cell sciences). / had increased concentrations of stnf-r compared to the th percentile obtained in healthy controls (> . ng/ml), and / patients had increased neopterin (> . nmol/i), / presented with elevated slcam- (> u/i). all patients with increased neopterin also had increased stnf-r, patients had concentrations of all three markers outside the normal range. there existed a significant correlation between neopterin and stnf-r (rs = . , p < . ). weak associations between age and stnf-r (rs= . , p=o. ) or neopterin (rs= . , p = . ) were also found. our results demonstrate activation of the cell-mediated immune system taking place in a sub-group of patients with acute pancreatitis. the finding of increased neopterin and stnf-r levels implies that activated monocytes/macrophages are involved in the pathogenesis of the disease. further data are necessary to evaluate potential associations between changes of marker concent-rations and the course of the disease. pancreatic injury after heart surgery was reported as soon as ( , ) and characterized by increased serum or urine amylase levels (in about % of patients) in the fi~t postoperafi.'ve days. this pancreatic injury, which sometimes led to acute pancreatitis, was atreaay at~buted to inappropriate perfusion of this organ. in the ffs, studies were published dealing with pancreatic suffering alter heart surgery, in large series of patients, concluding ~n~at panc~a~c injury (with a low incidence of pancreatifis) is more common than previously recognized and is a potential source of complication after camliac surgery ( , , ) . in a recent study ( ), evidence of pancreatic cellular injury was found in out of patients undergoing cardiac surgery, with out of these patients presenting abdominal signs or symptoms and developing severe pancreafitis. this injury was associated w~th preoperative renal insufficiency, valve surgery, ~..stoperalive hytxxension, calcium administered periopuratively and length of bypass. we studied patients submitted to cardiopulmunary bypass (cpb) for heart surgery and used the measurement of un:~sin, pancreatic iso-amylase and lipase in plasma for biochemical characterization of pancreatic cellular injury. blood samples were obtained before surgery, directly aller surgery (return to inte~ve care unit), hours alter surgery and in the folfowing days alter surgery (days , , , and ). computed tomography scan of pancreas was performed in patients presenting hi~ levels of amylase on day . we measured abnormal levels of trypsin and pancteatic iso-amylase in % of patients and observed simultaneous releases of these enzymes, the fi,'st one in the hours after surgery and the second more intense from day and pa~icularly on day after smgery. this second release was concomitant with abnormal levels of llpase. these biochemical observations were accompanied by radiological and clinical signs of pancreatic injury in about % of our patients : pancrealic abnormalities were revealed by scan in patients and acute pancreatitis in i patient. more pronounced pancreatic suffering was observed in patients undergoing valve replacement than in patients undergoing coronam-anrtic bypass grafm~g. analysis of trypsin and pare're, tic so-amylase are sw.cific of pancreatic cellular injury and their simultaneous ir~rease in plasma alter cpb in our padents confirms the presence of an exocrine pancreatic injury. the presence of a simultaneous peak of lipase mcaezse~ the specificity of overt pancreatic injtu diagnosis. the precise cause of th/s injury could he related to hypoperfnsion leading to ischemic injury of foe splancbnic area, pancreas being largely sensible to hypoperfnsion ( ). this hypoperfosion could he responsible for the ftmt release of pancrealac enzymes observed in our patients and would contribute to the deterioration of other organs leading to an inflammatory reaction developing in the following days and responsible for the second release of pancreatic enzymes observed in our patients. patients with necrotizing pancreatitis show a heigh rate of pulmonary, renal and septic complications, whereas the course in acute interstitial pancreatitis is generally very mild. we have prospectively analysed the value of endotoxin, interleukin- (il- ) and transferrin in compare with c-reactive protein(crp) for the early assessment of the severity of acute pancreatitis. patients aud methods: the values of endotoxin(measured by limulus-lysate-test), ii- (elisa), transferrin and crp (nephelometry) were analysed daily along the first i days of hospitalisation by patients with acute pancreatitis admitted to our hospital from / to / . it was judged whether the patients have either interstitial (aip) (n= ) or necrotizing (anp) (n=lg) pancreatitis. patients with anp have died during the course of pancreatitis (mortality= . %). results: -severity o~ pancreatitis: signifcant differences (p<o. , mann-whitney test) could be calculated between aip and anp for endotoxin, il- , transferrin and crp during the i days of monitoring. -mortality: except of il- on days , , and after ad-mission there were no significant differences between the values of analysed parameters in patients who survived and those values in patients who died. -organ failure: the patients with pulmonary, renal or multiorgan failure have significantly heigher values of endotoxin, il- and crp along the i days of monitoring in compare with those patients without an organ failure. conclusions: endotoxin, il- and transferrin, an important endotoxin carrier, are promising parameters to differentiate between the severe and mild form of pancreatitis comparable with crp. this parameters may be helpfull in the early clinical assessment of patients with acute panereatitis. the determination of cytokines may elucidate the pathophysiologic mechanisms that produce early systemic complications in acute interstitial (i) or necrotizing (n) pancreatitis (ap). the appearence of respiratory insufficiency (ri) is used to appraise the early systemic complications and is compared to the results of cytokine blood levels. in a prospective clinical trial from / - / , patients with ap were recruited and blood samples taken for cytokine determination with commercial elisa-kits. the differentiation between i (n= ) and n (n=l ) ap was made through ct-scan in all and laparotomy for drainage and necrosectomy in patients. the etiology of ap was gallstones in , alcohol abuse in , hyperlipidemia in and other or unknown causes in patients.five of patients with nap died early (n=l) or of late septic complications. none died of iap.the peak of cytnkine level in the first three days of hospitalisation was used for calculation. ri was diagnosed in the first week of hospitalisation, if oxygenmask or iutubation for at least days was nescessary to treat arterial hypoxemia. for statistical analysis u-test of wilcoxon, mann and whitney was applicated.the il- concentration in blood reached up to pg/ml in the first days depending on the severity of ap and dropped to almost zero in the next days, independently of the clinical course. the differentiation of i-versus nap, using a cutoff-line of pg/ml was correct in patients ( %, sensitivity (se): %= specificity (sp): %, (z: , ). high levels of il- over pg/ml correlated well with the prognosis ifri in the first week (accuracy: %, se: %, sp: %, c~: , ). the blood levels of il- reached a maximum of pg/ml in the first days, depending on the severity of ap, and showed a correlation to the clinical course in the following days. the peak of l,- blood levels indicated correctly the severity of ap in out of patients using a cut offtevel of pglml (accuracy: %, se: %, sp: %, ~: , ). there was no correlation between cytokine blood levels and mortality, also there seemed to be no correlation between the levels of il- and il- . in the bloodsamples of five patients with i-or nap no c~-tnf was detectable.the blood levels of il- , and to a lesser extent of [l- , can predict the severity and early systemic complications of ap in men. the excessive rise of cytokines can be explained by the stimulation of immunological cells ( macrophages, lymphocytes and endothelial cells) in the course of ap. objectives: in an opossum model, ligation of the sphincter of oddi or separate ligation of the bile and the pancreatic duct together leads to severe haemorrhagic pancreatffis while single ligation of the pancreatic duct causes only an exocdne atrophy. since bo has been discussed to be a contdbutor t¢, res dysfunction, this may depdve the organism of a potent defensive mechanism thus promoting the course of pancreatitis. the present study wa,,; carded out tc develop a new method for measuring dynamic liver res func.. tion and to test the hypothesis that bo causes a res dysfunction. material & methods: opossums were randomly placed in three groups. all received a central venous line. after midline laparotomy, a specially designe tourniquet was placed around the common hepatic duct above its junction with the pancreatic duct (group a, n= ), around the pancreatic duct (group b, n= ) or around both ducts (group c, n= ). after one week, the tourniquets were closed. using a scintillation camera, the liver uptake of nanocoll, a mtcalbumin colloid with a diameter of nm, was measured before, , and days after the obstruction. the curves were analysed by a computer and two parameters (r, s) were calculated using natural logarithmic regression. as a common measure for res function, the corrected granulopexic index (a) wa,,; determined, after day , the pancreas of each opossum was searched for necrosis by morphometdc methods. results: all animals survived till day . the opossums in groups a & had a macro-and microscopic normal pancreas, while those in group c showed a severe hemorrhagic pancreatitis. the granuinpexic index showed a significan~ change to the worse starting at day in group a & b (p<o, ) and at day it~ group c (p< ,ol). at day , res function in group c was significantly worse than that in group a & b (p< , ). the regression parameter r showed no significant change in groups a & b, but a highly significant decrease in group c starting at day (p< . ), thus showing a dysfunction of the hepatic res. conclusions: obstruction of the hepatic or pancreatic duct alone does no{ result in immediate dysfunction of the hepatic res, while obstruction of beth ducts does. because only ligation of both ducts is followed by a severe hereof.. rhagic pancreatitis, res dysfunction could be an important factor in the pathogenesis of pancreatitis and resulting organ failure.logarithmic regressinrl has proven to be a valuable tool to analyse dynamic hepatic res function. (b ) in lewis rats weighing - g. there were five groups: group a (n= ) were untreated controls; group b (n= ) were given isotonic saline intraperitoneally and observed for hours; group c (n= ) ml x e. coli intraperitoneally and observed for hours; group d (n= ) were given ml x e. coli and observed for hours; group e (n= ) ml x i e. coli and observed for hours, the rats were than killed, the spleens were removed and heparinised blood taken for immunological tests. total t-ceils, helper t-ceils, suppressor t-cells, monocytes, and the interleukin receptor were determined by monoclonal antibodies, indirect immunfluorescence, and flow cytometry (facs analyser ). statistical analysis was by the t test on rank transformed data.in group b (isotonic saline) the total t-ceils increased in the blood (p= , ) and spleen (p= , )com pared with the untreated rats (group a), in the groups with peritonitis (c, d, and e) we found highly significant rises in suppressor t-cells in the blood and spleen (both p< , ) compared with the non-infected control groups a and b. this increase was significantly higher in group d than in groups c and e. as well as other alterations, we found a uniform increase in the number of t suppressor ceils in our model of acute peritonitis that was both time and dose dependent, department of surgery, university vienna, akh wien, w~hringer gurtel - , wien. d~'na~'aics of som~ l~mnunologic indices in children with abdo;~tinal shock v.z.i~ioskalenko, s.f.vesiol$,t.v.p~bina serum (iga,:~i,g, circulating imaune co!~plexes, co:apleaentary serum activity, antimesothelialvisceral and parietal antibodies) and cellular (i-l~q~hocytes, ~s/th,b-lyaphoeytes,i nlaunoleukosis to aesothelial allergens; spontaneous blast cell transformation to phytohemaaglutinin and .nesoth<-~lial ~,togen; phagooftic neutrophil activity) im:~une indices v.'ere studied in children operated v:ith symi~to'as of abdo~ainal shock. ,'~!-~ of them had appendicnlar( - ocalized jo-diffuse, -~eneralized) and -hemoperitoni-~is (dull abdominal trau;na with injayy of 'the spleen- , the spleen and liver-q), flo patients tjere operated on for co~iplete iieus (~-intussnsc.sption, -com:lissural ileus). he follo~'~ing serum factors: cireula:;ing im~aune complexes and ~).esothelial antibodies are the aost i~portant ~ar~ers of the abdominal shock course. :yith the progress of the process the indices increase irresponsive of t.l~e cha±.aeter of pathology. in case of ileus regress and hemoperitonotis an abrupt drop in ~he titer of anti~aesothelial antibodies is noted. in bacterial peritonitis a high ~iter of anti:aesothelial parietal antibodies in great dilutions ("+++","++++"-q ; ) persisted even in the period of clinical recovery. cellular factors cham'acterize dfnaaics of the abdominal shock coulees less specifically llast cell transfor~aation and phagocyue neut~ophil activit can inderectly characterize transition of shock f~orn reversible into irreversible phases. the past so years have brought a number of major advances in burn care. the initial advance was the discovery that burn victims required large volumes of resuscitation fluid in the initial hours to maintain hsmodynamic stability, this was followed hy the dsvelopmsnt of topical antimicrohlal agents to prevent end treat infections. next, was the reporting that early excision of burn wounds, with autografting of the excised wounds was possible.finally, was the identification of the importance of aggrsssive nutritional support for the hypermstabolic burn patient.these advances have markedly increaesd survival rates for given burn mimes, s~ch that burns which would previously have been considered to be aniformly fetal, are now readily survivable.thsre remain two unsolved problems in burn care, which are limiting survivability cf burn patients.the first ie ths treatment of inhalation injury. ths sscond is how to obtain coverage of ths maaaivsly burned patient, who has limited skin graft donor sites available.this ssssion will focus on the new treatments being developed to obtain permanent coverage of burn wounds.these include the use of various tcplcal growth £aotore which can speed the rate of reepithelialization of wounds, both the beneficial and dstrimental effects of using cultured karatinooyte preparations, to obtain in excess of one squats meter of a pseudoskin from a single fifteen square centimeter biopsy of unburned skin, will be discussed, finally, the used for artificlal dermal preparations will be discussed. integumentary wound healing consists of simultaneous epithelializalion and contraction. not long ago, it was thought that healing proceeds at an optimum fixed rate and could only be delayed. the recent explosion in molecular biology and recombinant gene techniques have produced information on the biological activity of compounds previously believed to be biologically inert. many current attempts to modulate the rate of wound healing center on the topical application of various growth factors produced by integumentary cells or agents designed to modulate growth factor expression or response. epidermal growth factor (egf), plateiet derived growth factor (pdgf), transforming growth factor (tgf) and fibroblast growth factor (fgf) have been demonstrated, both in vitro and in vivo, to stimulate the proliferation and diferentiation of their designated cells types. egf and pdgf have been clearly demonstraled to increase the rate of wound closure in partial and full thickness wounds. pdgf also has demonstrated efficacy in the closure of recalcitrant pressure sores, whereas tgf increases the tensile strength of incisiona[ wounds and fgf stimulates angiogenesis. it appears that epidermal keratinocyte migration during wound healing depends on integrin-mediated interactions with compounds and ceils extracellular matrix. additionally, the activities of extracellular glycoproteins such as fibronectin, fibrinogen, laminin and thrombospondin are coordinated by multiple integrins with specific distributions and binding affinities. currently, agents which incorporate specific protein sequences essential for the interaction of the glycoproteins with the cells of the extracellular matrix are being investigated. it is has been clearly established that the rate and nature of wound healing can be influenced by the application of various agents and that sufficient quantities of these agents can be manufactured. the future challenge is to develop techniques to now the objective evaluation of the clinical efficacy of these various agents and to employ the appropriate agents in a cost-effective manner. transplantation; and ) the immune status/manipulation of the host.multiple interactive variables will determine the effect of employing allogeneic cells and tissue in wound coverage design and a batter understanding of the dynamics of the wound-graft microenvironment will facilitate the dove opment of clinicauy beneficial graft composites. cadaver allograft skin (cas) is the "gold standard" for wound coverage, but has limitations including varied availability & quality, immunogenicity leading to rejection, & potential for disease transmission. our development of a temporary living skin replacement (tlsr) addresses these issues; the tlsr consists of highly screened human neonatal fibroblasts (hf) cultured in biobrane a, a bilayer dressing consisting of nylon mesh laminated to a thin silicone membrane which controls water-vapor transmission. studies in our lab indicated that "fresw ~ tlsr grafts reproducibly close full-thickness wounds in mice & perform better than bb controls. we studied wound adherence & structural/molecular properties of fresh & cryopreserved (cryo) tlsr. methods: tlsrs were prepared by seeding /co hf in bb nylon mesh in dmem. hf quickly attached to the nylon mesh & were allowed to proliferate - wks. fibroblast mitochondrial activity was assayed by mtt assay. matrix proteins were identified by immunostaining, mrnas for specific growth factors were identified by reverse-transcription polymerase chain reaction amplification, & quantitated by gel scans. bb structure was studied by scanning electron microscopy & stretching measurements pro/post cryo. grafts were cryo'd in % dmso, quick-thawed & sutured onto x cm excised full-thickness dorsal wounds on athymic mice. "fresh" grafts were similarly placed. controls grafts were cas and aeellular bb. adherence was quantitated by a device which peeled grafts from wounds at intervals following placement, using a serve motor and a strain gauge. results: postcryo, storage and subsequent thawing tile tlsrs maintained > % cell viability by the mtt assay, indicating continued mitochondrial activity, and bb structure & stretchability were maintained. multiple matrix proteins secreted and deposited in the bb nylon mesh (types l/iii collagen, decorin, fibroneetin) were identified by specific immunostaining. growth factor mrnas in the tlsrs (afgf, bfgf, kgf, tgf~,p~,) were present in - , x higher levels in fresh/cryo tlsrs than in adult hcs. grafts adhered to wounds on mice through days of followup. histologic exams on days - showed excellent vascular ingrowth and minimal inflammation. adherence of tlsrs to wounds was >cas adherence. burn wound coverage in the massively burned patient remains a difficult problem. although cultured keratinocytes have been utilized for burn wound coverage, their impact on the patient with burns greater than % total body surface area has not been spectacular, with poor graft take and unstable epithelium.current investigations have been directed toward dermal replacement beneath either very thin split-thickness autografts (stag) or utilizing cultured keratinocytes. current products include: collagen dermal replacement with thin stag (burke, et al). collagen dermal replacement with cultured keratinocytes and fibroblasts (boyce, et ai). allograft dermis with cultured keratinocytes (cnno, et al). allograft dermis with thin stag (life cell). polyglactin acid mesh and neonatal human fibroblasts with thin stag (hansbrnngh, et al).investigations regarding culture media, use of growth factors, topical nutrients and antibiotics, and melanocytes for pigmentation as well as safety and efficacy are needed before any of the current products become viable options for coverage of the massively burned patient. the~ is a growing world-wide problem with the ujc of cadaver tissues and ocgans bae, au~ of the tren~m~s~km of dilemma such a; cmutzfeldt.jukob disease and iiiv as we ] as ready availability of urdform lis~ue~. on dec~mt~r , , the fda assumed control of as tissue bar~s in the uldtod st=tea in an attempt to bflng ~s difficult problem of dise~s~ transmission under ¢onlrol. in europe, ~om¢ of the governments are consldofll~ a c~mplcte bat) on the use of cadaverlc fissu~s such as ddn, 'this |ncroam in regulation of cadavefle ~s,quct will incmar¢ the difficulty of obtain~g and dlslflbulmg them. however, thc nc~ for these tissues contlnue~ m incrcaso, we will discuss ~'l¢ solulion to this important pmbl~n: tissue engineering. tlssu~ engineering is an in~rdisdpllnary field that applies pdnclplc~ of angin~edng and die life sclcnce~ reward the development of ~olok~¢al sub~dtute,~ ih= mslom, maintain, or improve tissue function, " ssuc ongln~cdng can provide ~ho nccassary tlssuoa for wound repair ~d ibe assuranoe fl'~t the lissuos are d.ls¢~¢ free. in addition, a ds~uo-cng~ne~n~l wound covering will bo u~lvemally acceptable and evntlublc as "off g~o shell", consis~t products, them are several approaches to restating thls function in a large wound, 'l'nosc i~elud~ tmmcdiete long term coverage, short t=nn coverage, uandtl~el coverage and compost= dssu¢ coverage, "flssuo onglncrcd wound coverings that meet those vaflous ne,.cds will he r~vlowod.cllni~:sl and experimental d~la in venous ulcer, dlabctl¢ ulcers, prossur~ ulcers and bum wounds wgj be mvlcw~, a~ welt as new approacl~s u~ csrtilag¢, bone, liver and bone marrow it~suos. c oomplon, k nadirs, w press, g wetland, j fallen iv, shrtners burns institute and massachusetts general hospital, boston, ma~schusetts, usa the clinical "take" rate o? cultured epithelial autografts (cea) has been observed to increase with transplantation to allodermls, but the reasons for the improved clinical performance have not yet been defined. the aim of this study was to determine the biological impact of normal human dermis on cea differentiation and maturation, biopsies of cea transplanted to engrafted and de-opldermlzed human homograft dermis have been compared to nopsles of cea transplanted to granulation tissue in tullthickness burn wound beds on the same patient, each patient serving as hls or her own control. paired test and control biopstes from six patients have acquired from as early as one week postgrafting to as late as years postgrafting (one patient) and analyzed histopathologlcally, ultrastructurally and immunoh[stochemloally, results demonstrate more rapid normalization of differentiation markers (e,g., involucfln, fllaggrln, cytokeratln profiles) in the cea transplanted to allodermls compared to their corresponding controls by in all patients, the proliferation rate within the basal layer ot the epidermis as determined by ki- (proliferation-associated antigen) is seen to norh~altze more quickly in the cea transplanted to allodermls in every case, persistence of allodermal matrix can be dooumented in all patients by elastic tlssue-trichrome stain, allowing visualization of the dermal elastin network. the popu;atlon densities ot intraepldarmal langerhans cells are conslstently and signlflcantly higher in cea transplanted to ,allodermls, possibly reflectlng an immunologlcal reaction to the underlying allogenlc tissue. overall, these preliminary results indicate that transplantation to a normal human dermal matrix accelerates the maturation of cea-deflved epidermis, wound closure continues to be a major problem in patients who have sustained a major thermal injury, cultured epidermal autografts (cea) have been utilized extensively since when galllco et el reported theh'use in two brothers with greater than % total body surface area burn. unfortunately, cea take rate varies widely and the resultant skin coverage is often fragile and the cosmetic results are less than optimal however the overall take rate and durability of the coverase can be markedly improved by using nn allodermls base as the recipient bed. a review of cea applications performed by physicians using cultured outologens epithelium obtained from blusurfaoe teclmology, inc. shows a marked discrepancy in the results obtained utilizing different methods of wound bed preparation. tgf-b is an important modulator coordinating complex physiological events associated with growth and development. it is assumed that tgf-b is also involved in the well-coordinated process of cutaneous wound healing by regulating proliferation, differentiation, chemotaxis and matrix deposition. the purpose of our study was to analyze the spatial and temporal pattern of tgf-b expression during granulation tissue formation in patients with accidanutl surgical trauma (monotraumata mid polytraumata) and bum wounds. after debridement (day ), the full thickness wounds were covered with epigard, a synthetic dressing until day . after this time the granulated wounds were closed by transplantation of mesh graft. biopsies of the wound center were taken from patients at the beginning of surgical treatment (day ) and after , , and days. cryosections were stained with antibodies against tgf-fi s using the apaap technique and -for standard histology -with hematoxylin-eosin. for identification of the cell type expressing tgf- , double staining immunofluorescence experiments were conducted using antibodies specific for monocytes/macrophages, polymorphoanclear neutropkils and fibroblasts. the results showed a characteristic pattern of tgf-t~ distribution during wound development. tgf-fi appearence was mainly cell-associated znd the absolute and relative number of cells that were positive increased with lime. infiltrating cells and developing blood vessels were most prominently stained; epithelial and t-cells showed no immuno-reactivity. a delay of emergence for tgf-b during the time course could be seen in one patient group. this might reflect various regulation patterns depending on the type and severity of injury.( ) pharmatec gmbh, frankfurt ( ) institut fiir immonologie and serologic, heidelberg ( immune cells extravasating specifically in skin recognize and eliminate the invading antigens (bacteria, viruses, etc.) either in situ or transport them to regional lymph nodes. they also participate in the process of skin wound healing. cells which traffic through the skin can be harvested from efferent lymph drained from a given area of skin. the type of migrating cells changes after trauma, heating and infection. we have developed a method for collection of human afferent lymph in lower limbs. the method allows obtaining immune cells from normal and injured skin and their characterization. aim of the study was to characterize skin immune cells in situ and in skin lymph with use of immunohistological methods (staining, facs). results. group , cells migrating through skin: + % t lymphocytes (cd ), + % langerhans and dendritic cells (cdla, hla dr, s ), + % cd , + % cd , no b cells (cd , ), % cd r (memory cells), + % il r. approximately % cells possessed cdlla and antigens. cd lc was expressed only on large cells. the frequency of all phenotypes was different from the blood populations. group , cells in skin: langerhans cells were found only in epidermis, cd , and , cd r , rb, ila/ cells around venules, cd (macrophages) uniformly dispersed, no il r and b cells. hla dr positive were endothelial and some dispersed mononuclear cells. group , one, three and thirty days after surgical wound (simple varicous vein extirpation): high density of epidermal langerhans cells, hla dr positive keratinocytes and all endothelial ceils, few il r cells, perivenular infiltrates of cd , r but less cd cells, high density of cdlla/ cells. classic staining of isolated and in situ located ccl!s with mgg or he did not allow to follow kinetics of changes. conclusions. this study presents the first in the literature quantitative data of immune cell traffic through normal and injured human skin. in the controlled release of biological response modifiers for soft tissue regeneration. alan s. rudolph, helmut speilberg, mariam monshipouri, and florence rollwagen, and barry j. spargo. we have employed lipid microstructures as controlled release vehicles for the delivery of growth factors in wound repair. traditional liposomes as well as novel lipid based microcylinders have been examined for their in vitro kinetics of the release of transforming growth factor beta (tgf-b). in vitro reiease has been examined by setting up models with examine the physical release of iodinated tgf-b as well as a cell based bioassay (based on the ht bioassay). the hollow lipid microcylinders ( microns in length and i micron in diameter) show an initial burst ( - ng) followed be zero order kinetics which result in the release of approximately i ng tgf/day. this release behavior can be modified by temperature based on the phase behavior of the lipid bilayer which comprises the microcylinder.we have also examined the cellular response to lipid microcylinders applied in vivo. the lipid microcylinders are mixed in agarose and implanted as a composite hydrogel block under the flank of a mouse. the blocks are removed , , and days following implant and the cells analyzed by facs sorter analysis. the observed pattern of ceil recruitment to the blocks mimics that seen in a local inflammatory response. cell surface phenotype studies included the determination of cd and cd , mac-l, and ig bearing cells. we have also begun to examine the change in cell surface phenotype and kinetics of recruitment following the inclusion of tgf-beta in the lipid microcylinders.center for biomolecular science and engineering, code , naval research laboratory, washington, dc. - . expression pattern of heat shock proteins in acute, good healing and chronic human wound tissue. abstract: wound healing is a complex biologic process that is well characterized at the histological level, but its molecular regulation is poorly understood. after clot formation, inflammatory cells are rapidly drawn into the wound, followed by migration of fibroblasts and epithelial cells that divide and repopulate the wound area. during the last decade peptide growth factors and cytokine are thought to play a key role in initiating and sustaining the phase of tissue repair. these factors which are released from different cells appear to initiate the cascade of events that lead to healing. different studys described the rapid activation of a family of proteins,named heat shock proteins (hsp) in differnt tissue that were exposed to various forms of stress (heat, toxic agents, mechanical). in this context hsp's have the ability to regulate protein folding and assembly, to transport proteins across cytoplasm and membranes, to disrupt protein complexes, to stabilize, degrade and regulate the synthesis of proteins and to take part in dna replication and repair. we now attempted to find out if hsp-gene activation is also involved in injury and wound healing, which likewise resemble a stress situation for cells. therefore we collected tissue samples during operation and single biopsies from chronic wounds (decubitus for example) and granulation tissue. after rna preparation from these samples we used rna-pcr and nothern analysis to study the expression of objectives of the study chronic, non-healing cutaneous tflcers are a challenging clinical and socioeconomic problem. several animal studies have shown that cytukines (e.g. egf, pdgf, fgf, tgfb) accelerate the healing process and tissue repair in general. results from first clinical trials indicate a promising value of cytokines in the treatment of chronic non-healing diabetic and venous ulcers. recent reports in the literature indicate that the biological activity of the solution of platlet derived wound healing formula (pdwt~) released from c~-granules (mainly pdgf & tgfi~) is greater than the activity of the recombiant single factors like e.g. pdgf-bb (robson, lancet ) . the aim of our study was to determine whether a correlation exits between the concentration of tgfi~ & pdgf and the time course of wound healing. materials and methods pdwhf was prepared from ml of auto]ogous patient blood and diluted with a special buffer to a final concentration of ng/ml g-thromboglobulin. the concentrations of pdgf and tgfg were determined by elisa-tests developed in our laboratory. patients with chronic non-healing ulcers have been evaluated alter treatment by topical application of pdwhf. pdfg and tgff~ concentrations of the topical solution were measured and two patient groups formed for analysis the time course of wound healing was regularly and meticulously documented and evaluated by photography and casting. the time from initiation of treatment instil o wound volume reduction to go of the origional size (t %) was noted• results: healing of extensive burn wounds can be accelerated by grafting cultured autologous or allogeneic keratinocytes. the stimulation of granulation tissue formation and reepithelialization is presumably based on growth factors and cytokines released by keratinocytes. we wanted to prove this hypothesis by investigating the bfgf expression during wound development, bfgf is mainly described as an angiogenic protein with mitogenic activity on various mesodermal and ectodermal cell types pointing to its stimulating potential in wound heating. in the present study we compared the pattern of human bfgf m-rna expression and the localization of bfgf protein during the first days of wound healing. biopsies were taken from juvenile human bum patients, immediately after wound debridemerit mad on day after transplantation of cultured allografts. biopsies were snap frozen and cryosected. the pattern of bfgf expression was assessed by in situ hybridization of the bfgf m-rna with a digoxigenin-labelled antisense-rna and the parallel detection of the mature protein with an anfi-bfgf monoclonal antibody. our study revealed typical patterns of bfgf-m-rna-expression and intense bfgfprotein deposition during granulation tissue formation and reepithelialjzation of healing bum wounds. 'it, is known that major thermal injuries cause early impairment of wound healing followed by decreased influx of granuiocytes st. the site of injury. the role of granuiocytes in the process of wound healing is not ~"~ "" elucidated, it is now assumed that they are not merely phagocytic cells but active participants in ~n~*' ~.,.,a+~o~: processes secreting_ a number of various cvt-;kines, in order to investigate the effect of there is accumulating evidence that neuropeptides could be involved in the pathogenesis of several inflammatory reactions. vasocactive intestinal polypeptide (vip) and substance p (sp) have been detected by immunohistochemistry in normal as well as inflammed skin mostly in perivascular and periglandular location. both vip and sp are involved in vasodilatation, mast cell degranulation and irnmunomodulation.we determined the influence of sp and vip on the proliferation of lymphocytes in patients with psoriasis and healthy individuals. peripheral blood t-lymphocytes of psoriatics and healthy controls were isolated by density gradient centrifugation and passage over nylon wool. cell enrichment was controlled by facs analysis, lx t-lymphocytes were then incubated alone or in coculture with x irradiated autologous lymphocytes in culture medium containing - mol/i sp or vip. cell proliferation was measured semiquanfitatively by tdr uptake in a betacounter. significance was tested by the wilcoxon signed-rank test.our results show that sp and vip exert only an effect on unstirnulated t-cells. in healthy individuals but not in patients with psoriasis sp increases significantly proliferation of t-cells. vip, however stimulates significantly the blastogenesis of t-lymphocytes only in psoriatics.our results confirm the psychoneuroimmunologic component in inflammatory reactions and vip and sp could be partially implicated in their pathogenetic mechanisms. moreover psoriatic lymphocytes show an altered reaction to sp and vip. this might be due to a preexisting (genetic?) or more likely to an epiphenomenal receptor defect. the adhesive interactions between endothelial cells and circulating ~enkocytes in shock and innammatory vondltions is mediated by several distinct families of ce -surface determinants. of particular importance are the leukocyte integrins cdib / cdlla-c. in this study monoclonal antibodies to two of the u chains (cdlla & cdiib) and the common [~ chain (cdib) have been used to investigate leukocyte-dependent and leukocyte-independent plasma leakage in tee skin of rabbite. plasma leakage was measured as the local accumulation of t si-hsa over a rain period, the chemotac~c peptide imlp ( . . ng) and bradykinin were used to induce cell.dependent and cell- ndependent leakage respectively, the antibodies used were . e (cdis), nri (cdlla) and antibody (cdllb). ]ntradermal in~ections of bradyklnin and ~dlp both caused a dose dependent increase in plasma extravasatien ( .~. ffi . p.l to . z b.bttl and . ,- . ~ to . z . d respectively. . e ( . - . mf,/k~ iv) caused a dose dependent inhibition of imlp-induced but not bradyldnin.inducecl plasma exudation. at . mk/kg, the plasma leakage was completely inhibited, antibody nr produced similar results, treatment with antibody did not cause inhibition o£ plasma leakage due to either tnedi~tor. in vitro, the irmnune system ex~nination in persons with bone, chest and abdominal traumatic injury (i group . patients without infectious coz~lications and group - patients with wound infections development) was carried out. to restore found immunity disorders and host defense to infection patients of the group were treated with thymalin-the biologically active peptides prepared from bovine thymus. the examination on t~e i- days after injury revealed a considerable decrease of lymphocytes, ed ",$d ~ and cd cells amo~it in the blood, cd /cd ratio and indexes of let~ocyte migration inhibition test in both groups of patients. the imm~lity disorders recovered to norm on the - days in pateents of+the i group. but stable ~eple$ion of cd and cd cells amount, lower cd /cd ratio and indexes of leukocyte migration inhibition test in patients of the group were observed~ besides that, these persons showed higher cd cells amount and ig level in the blood. after thymalin therapy valid ii~rovement of inun~e status was discovered. also good clinical effect of immunotherapy and best wo~id healing observed in % of cases. these results allow us to propose that the thymus involution and the reduction of cell-mediated immunity responsiveness with disturbances of immu_uoregulatio~ on the level of restriction of activated cd tho cells play the most important role in the pathogenesis of wound infections development in persons with traumatic injury.dept. of immunology, military-nedical academy, lebedeva str. , , st.petersburg, russia a severe impairment of neutrophil (pmn) function often occurs following severe thermal or non-thermal traumatic injury. our laboratory has previously reported that following severe burn or non-burn traumatic injury the expression of the p integrlns (cd a,b,c/cd ) and the fw receptors (cd , and cd ) were significantly decreased on pmns, in this study, the effects of gm and g-csf on the expression of the f~ r and the ~ integrln family on pmns were examined, pmns were obtained from severe trauma (initial apache ii score ;z ) or thermal injury (> ~; total body surface area, > ~ full thickness) and incubated /n v/tro with gm or g-csf. the j integrins or fcyr were detected with monoclonal antibodies and flow cytometry. gm end g-csf induced a sllght increase in the percentage of pmns expressing cd lb, cd , and cd while gm bur not c-csf induced an increase in the percentage expressing cdi a, cd lc, and cd , gm-csf and to a lesser extent g-csf induced an increase in the density ( , fold) of the ~ integrlns on pmns from normal, burn, and trauma patients, these data suggest that cytoklne modulation with csfs could have a role clinically in certain situations. institute, dept. of surgery, bethesda ave, cincinnati, oh, usa, - . funl~al infections after solid organ transplantatlon(sot) lewis flint, md and ed,~-afd e. etheredge, me) dept. of surgery tullrte univ. school of medicine new orleans. louisiana infections contribute to increased gra loss and mortaliw following sot. pr~isposing facton include diabetes, hepatitis, leukopenia, cc.¢xistem infection, and intense, especially triple drug, immunosuppression. funga] infections occur ~s isolated conditions in % and in association with bacterial infection(l %), viral infection( */.), and combined infections(it%), candida sp. is the most common fungus recovered but aspecgillus, coccidiodies, cryptococcus, histoplasma, mueor~ ghizopus, tinea, and toruiop~is s?. also are pathogens. clinical syndromes vary among orga.aizms or may be variable with a single p~tthogen, for ~ample, with aggressive immunosuppression, candlda my be localized esophagitis or cystitis or systemically iavaslve with an associated high mortality. aspergilius presents ~ a diffuse pneumonia while cryptococcus causes pulmonary and centrad nervons sy'stem infection, clinical examination, ct scanning and aggressive sampling for c'ultures a.s wall as serologic tests contribute to diagnosis. empiric the~py is ind',cated where there is a high level of suspicion. preventlon of ca.adlda izfection is ~ci~itated by early remov-a. of central }ants, ca~hetess and stents as well as by the use of oral nystatin. amphotericin ]~ remains the drug of choice for treatment of in.save fungd infection, surgical resection of infectious loci in the lung and brain is indicated in selected patients. the main problems of diagnosis in lower respirator-), tract infection are the differentation of infection from colonization or contamination, and the isolation of a reliable and true pathogen. expectorated sputum may be unreliable in pneumonia, because of contamination by oropharyngeal flora. although blood cultures may be negative, they provide a precise diagnosis and should be obtained in all pneumonias. other more invasive procedures are transtracheal needle aspiration, fibrobronchoscopic techniques including protected specimen brush and bronchoalveolar lavage with quantitative culturing and cytological analysis, transthoracic needle aspiration, thoracoscopy -guided biopsy and open lung biopsy. recently m. e -ebiary, a. torres et al, reported quantitative cultures of endotracheal aspirates for the diagnosis of ventilator-associated pneumonia offering reliable results in these patients and should be further investigated. any invasive procedure in a severely ill patient should be carefully directed weighing the risks as well as the benefits, whilst taking the underlying diseases and expected survival into consideration. -current therapeutic approach is based mainly on monotherapy with broad spectrum antibiotics. combination therapy is apparently indicated only in p. aeruginosa infections and severe s. aureus pneumonia. graft infection can lead to fulminant graft failure or rapid progressive cirrhosis. for prevention of graft infection immunoprophylaxis, i,e. administration of human polyclonal anti hbs hypedmmunoglobutin (hig), starting in the anhepatic phase during operation, has proved to be at least partially succesful when performed on a long term basis.from a total of olt in adult patients olt were performed for hbsag positive liver disease (cirrhosis n= , fulminant liver failure n= , retransplantation n= ) in pat. all pat. received . u hig in the anhepatic phase and . u/per day for the first week. a small group of pat. received hig only for i week (short term immunoprophylaxis), in all other pat. hig is administered on a long term basis to keep anti hbs serum levels above uii or until graft infection occurs (long term immunoprophylaxis);one-year survival rates are % in pat. who were transplanted for fulminant hepatitis, % in pat. with cirrhosis and long term prophylaxis, and % ir~ pat. with short term prophylaxis. all fatalities were related to hbv graft infection. the total rate of graft infection was % under short term prophylaxis and was independent from preoperative hbv dna status, under long term prophylaxis graft infection occurad in % in pat, negative for hbv dna. in hbv dna positive pat. infection rate was %, the total rate of reinfection for all pat. with long term prophylaxis was %the results of liver transplantation in hbsag positive pat. are comparable to other indications, graft infection with hepatitis b virus ist the major risk factor for these patients. under long term therapy with hig the rate of graft infection can be significantly reduced. the crucial cellular element for mods-mof: monocyi'f_./m acrophaoe ronald v. meier, m,d., f.a,c,s. the severely :injured or crldcally ill surgical patient is at high risk for immune dysfunction. a major consequence of this immune dysfunction is multiple organ dysfunction and failure leading to death, the underlying etiology is now recognized to be an uncontrolled, unfocused, disseminated activation of the host normally protective inflammatory. ,, cascades.. the resultant "mahgnant' systemic" inflan'a'natlon produces d~ffuso multiple organ bystander injury !eading to progressive organ dysfunction and failure. systemic malignant inflammation involves diffuse actlvatton of all components of the humoral and cellular inflammatory host response. of these various components, the macropha~e is the crucial central cellular element. the tissue fixed macrophage is ideally located diffusely throughout the various organs injured to orchestrate the inflammatory process. the macrophage is long-lived and highly metabolic, the macrophage regulates both the extent and the dissemination of the inflammatory processes. the macrophage is an exu'emely active c¢ capable of producing and releasing not only directly eytotoxlc agents, s irnil~, to the neutrophil, including oxidants and numerous proteases out also the multitude of other cytokines and initiators of the interacting inflammatory cascades. the macrophage is the central source for ehemotactic agents (il- , ltb , c a) for neutrophils and other inflammatory cells, production of vasoaetive arachidonie acid metabolites (tx, pgi , poe, lt's), complement components (c a, csa), thrombotic agents (pca, tx), metabolic and physiologic modulators (il, , il- or tnf), and immunosuppressivc agents (poe , il- ). these products of the macrophage are highly effective in enhancing and augmenting the inflammatory response. disseminated activation otthe macrophage is critical to the induction of the long-term diffuse activation of inflammation necessary to induce multiple organ injury and failure. our ability to elucidate the molecular mechanisms that control the macrophage will lead to our ability to conu'ol the maerophage response and prevent mods-mof.flarborview medical center, - th ave za- , seattle, wa usa key: cord- -dnsdg n authors: nan title: poster sessions date: - - journal: eur j immunol doi: . /eji. sha: doc_id: cord_uid: dnsdg n no abtract the humoral pattern recognition receptors of innate immunity include collectins, ficolins and pentraxins. ptx , the prototype of long pentraxins, plays a nonredundant role in resistance against a. fumigatus lung infection. the model proposed suggests that upon binding, ptx facilitates recognition, phagocitosis and killing of a. fumigatus conidia by alveolar macrophages, dendritic cells and neutrophils and the subsequent development of a properly th -oriented adaptive response. actually, ptx -deficient mice are highly susceptible to aspergillosis and develop th skewed responses; moreover, ptx -deficient resident macrophages and neutrophils show defective conidia phagocytosis. both in vitro and in vivo defects can be rescued by the administration of recombinant ptx , which does not show direct activity on fungal cells. finally, ptx alone or in combination with antifungal agents, induces a curative response in mice with aspergillosis, even when given prophylactically. in the present study, we investigated the mechanisms underlying the ptx -mediated opsonic activity and the involvement of complement, complement receptors and fcg receptors, by in vitro studies and genetic approaches in vivo. in vitro ptx amplified the complement-dependent effects on a. fumigatus conidia phagocytosis by human neutrophils, activated through the alternative pathway. accordingly, in the presence of ptx -opsonised conidia, cd b activation, internalization, recruitment to the phagocytic cup and cd b-dependent phagocytosis were increased. as pentraxins interact with fcgreceptors, which in turn can control cd b activation, the phagocytic assay was performed in the presence of fcgr blocking abs. data obtained strongly suggest that upon conidia opsonisation with ptx , fcgriia/cd mediates inside-out activation of cd b and consequently phagocytosis of c b-opsonised conidia. in vivo phagocytosis experiments performed with c q-and fc common gamma chain-deficient mice and complement inhibitors support in vitro data. these data confirm and extend the paradigm of cooperation among innate receptors, in particular among the humoral arm of innate immunity (complement, ptx ) and the cellular arm (fcgrs, cr ). moreover, they confirm previous studies on the interaction between pentraxins and fcgrs and support the idea that pentraxins behave as predecessors of antibodies. innate immunity is the first line of defence against pathogens and plays a key role in the initiation, activation and orientation of adaptive immunity. the humoral arm of the innate immunity includes soluble pattern-recognition receptors (prrs) such as collectins, ficolins, complement components and pentraxins. the prototypic long pentraxin ptx is rapidly produced and released by diverse cell types in response to proinflammatory signals. ptx binds selected microorganisms such as aspergillus fumigatus and restores protective immunity against this pathogen in ptx -/-mice. neonates have an immature innate immune system and are more susceptible to bacterial infection than older children or adult. a beneficial effect of breast feeding on newborn health is highly demonstrated. this protective effect is mediated by nutrients, immunomodulatory mediators (ifn-g, tnfa, or tgf-b), innate immunity factors (soluble cd , immunoglobulins, lactoferrin), and leukocytes contained in milk that can penetrate the newborn circulation. we thus hypothesized that milk may contain ptx . we found high concentration of ptx in human colostrum ( . ± . ng/ml at day post-delivery) compare to the one found in human serum ( x ng/ml). the presence of ptx in human colostrum seems to be due to the secretion of ptx by human mammary gland since we report the production of ptx by these cells. this prr is also found in human milk cells (hmc), mainly in leukocytes, and penetrate into newborn tissus after suckling. furthermore, human colostrum upregulated the ptx production by adult and neonate immunocompetent cells and we demonstrate that neonate mice present a deficit in their ptx production after lps injection. collectively, these data demonstrate that newborn have three distinct ways of ptx supplying by breast feeding: (i) soluble ptx in colostrum (ii) hmc that can secrete ptx upon stimulation in the specific tissue, (iii) an increase of ptx production by immune cells in the presence of colostrum. thus, soluble or cell-derived ptx may participate to the beneficial role of breast feeding on the newborn health. a. m. baru , j. stephani , h. wagner , t. sparwasser twincore, institute for infection immunology, hannover, germany, technical university of munich, institute for medical microbiology, immunology and hygiene, munich, germany toll-like receptors (tlrs) represent the best characterized pattern recognition receptor family in mammalian species. the family currently comprise of receptors in humans (tlr - ) and in mice (tlr - , - ). as transmembrane receptors, tlrs are expressed on the cell surface (tlr , , , , , ( ) ( ) ( ) ( ) and at endosomal membranes (tlr , , and ) . toll-like receptors recognize specific patterns of microbial components and regulate the activation of both innate and adaptive immunity. bacterial dna has been shown to possess immunomodulatory activity about a decade prior to the identification of cpg motifs. about years later to this, toll-like receptor (tlr ) was identified and shown to be the receptor for unmethylated cpg dna which is present mainly in non-vertebrate genome. studies have defined potential role of tlr as adjuvant enhancing protective immune responses against tumours and infectious diseases in murine models. although promising results are obtained from a few human clinical trials, overall efficacy and safety could not yet be translated entirely from murine studies to human trials. one explanation for these discrepancies could be the fact that expression of human-tlr (hutlr ) is restricted to b-cells and plasmacytoid dendritic cells (pdcs) whereas murine-tlr (mutlr ) is also expressed on conventional dendritic cells (cdcs). consequently, tlr ligands induce distinct cytokine profiles in mice and human thereby probably regulating immune responses in a different manner. by employing bacterial artificial chromosome (bac) technology, we generated transgenic mice with hutlr (henceforth called as hut mouse) integration in their genome under human epigenetic control. to avoid effects seen due to overlapping ligand specificities, we crossed this mouse onto mutlr knock-out background. we expect that hut -mutlr -/mice mimic the human specific expression pattern of tlr , i. e. exclusively in b-cells and pdcs, allowing us to investigate detailed in vivo functions of hutlr . by studying infection and tumour models as well as models for autoimmunity, allergy and transplantation we could then define appropriate and safe implications for employment of tlr ligands in human immunotherapy. the fractal analysis provides unique physical insights into the interactions between c q and the prp protein. if one may take the liberty to extend this to cellular surfaces, where presumably these reactions are taking place, then one has access to a possible avenue by which one may control these reactions in desired directions. if this is true, then surely, this is worth exploring further. any effort, no matter how small that assists in help providing better insights into these debilitating and neurodegenrative disorders such as alzheimers is defintely worth the effort. interleukin- is a heterodimeric cytokine consisting of the two subunits p and p . the main inducers of il- p are microbial components activating toll-like receptors with the magnitude of il- p induction depending on the specific tlr engaged. differential induction of il- p upon tlr stimulation correlated with striking differences in the kinetics of nfkb activation. cpg-dna strongly induces il- p due to its outstanding capacity (i) to induce nucleosomal remodelling in proximal il- p promoter region and (ii) to stimulate prolonged rela activity. here we were interested in further changes in chromatin structure of the il- p promoter upon tlr triggering. we did not observe a change in dna methylation, but using chormatin immunoprecipitation (chip) we were able to detect a strong increase in histone and acetylation in specific regions of the proximal promoter region. acetylation of h showed a specific distribution pattern and occured mainly in regulatory elements within the il- p promoter, whereas acetylation of h took place over all regions analyzed. tlr tolerance has been reported to be associated with specific chromatin alterations. methylation status of lysine residue on h turned out to be important for the inhibition of gene expression upon repeated stimulation. modifying the chromatin structure of gene promoter regions therefore seems to be a sensitive mechanism to modify cytokine expression to exogeneous stimuli in innate immune cells thereby allowing adaption of innate immune responses. a. d. koepruelue , w. ellmeier medical university of vienna, institute of immunology/division of immunobiology, vienna, austria macrophages are important in innate and acquired immunity. failures are associated with inflammatory and autoimmune diseases. understanding their stimulation is the basis for therapeutic targeting. members of the tec kinase family (bmx, btk, itk, rlk and tec), expressed in the haematopoietic system, constitute the second largest family of non-receptor tyrosine kinases. mutations in btk represent the source of human x-linked agammaglobulinemia (xla). a mutation in the murine btk gene accounts for a similar syndrome, x-linked immunodeficiency (xid). although the tec family members tec, btk and bmx are expressed in monocytes/macrophages, little is known about their function there. tec kinases become activated upon signaling via divers receptors including antigen receptors, receptor tyrosine kinases or tlrs. several studies in xla or xid macrophages and in monocyte/macrophage cell lines implicated roles for tec kinases in tlr signaling and as well as other macrophage effector functions like phagocytosis. inspired by these findings, we aim to determine the role of tec kinases in bone marrowderived macrophages (bmm), during macrophage activation and in other macrophage functions such as recruitment or phagocytosis. in a comprehensive functional analysis of tlr-mediated bmm activation from mice deficient for one or more of the tec family members in vitro, we reveal which of the kinases play a role in which tlr pathway. based on the results of this analysis, we set the goal to further study how tec kinases regulate the respective signaling cascades. our study will contribute insights into the role of tec kinases in this important cell population of the innate immune system. g. lunazzi , m. buxadé , j. minguillón , r. berga , j. aramburu , c. lópez-rodríguez universitat pompeu fabra, department of experimental and health sciences (dcexs), barcelona, spain nfat is a transcription factor that regulates the expression of cytokines such as tnfa and lymphotoxin b in response to osmotic stress. in addition, nfat participates in multiple processes not linked to the response to hypertonicity. in this regards, it has been recently reported that nfat is required as a novel host factor that supports hiv replication in macrophages. given the established connections between nfat , the expression of certain inflammatory cytokines, and its role in the response to specific pathogens in macrophages, we aimed at studying whether nfat could be activated by receptors for pathogens expressed in macrophages. the activation of toll-like receptors (tlrs) is central to innate immunity. upon stimulation of tlrs, cells of the immune system induce signalling pathways that lead to the activation of different transcription factors. as a result of that, cells such as macrophages and dendritic cells induce the expression of genes that participate in the response to pathogens such as those encoding proinflammatory cytokines, antimicrobial products, survival factors or mediators of cellular migration. we have analyzed whether nfat is expressed in primary macrophages through the activation of different toll-like receptors. likewise, we have explored whether the activity of nfat is induced during the response to tlrs. in addition, we have studied whether the specific inhibition of different signalling pathways positioned downstream of tlrs could interfere with the expression of nfat . our work indicates that nfat is a novel transcriptional regulator acting in response to the activation of tlrs. our work extends the knowledge about mechanisms that participate during the innate immune response to pathogens and offers a new regulatory pathway as a possible target to modulate this response. objectives: compelling evidence support a link between inflammation, cell survival, and cancer, with a central role played by nf-xb, a master switch of inflammation. recent studies implicate some tlrs in tumor development or regression, and immune escape. however, mechanisms leading to tumor growth or apoptosis induced by tlr stimulation are not fully understood. several studies strongly suggest that chronic inflammation in lungs induced by chronic bronchitis, chronic obstructive diseases, emphysema, asbestos or tobacco smoke, increases the risk of carcinogenesis. we hypothesized that some tlrs can contribute to lung inflammation and tumor development in vitro and in vivo. methods: tlr expression in lung cancer was assayed by immunohistochemistry or flow cytometry. nfxb activation was determined by western blot and nuclear translocation assay after tlr stimulation. clonogenicity of stimulated cells was analyzed by colony assay. transcriptomic analysis were performed by taqman lda technology. tumor growth in vivo was analyzed in nod/scid mice after subcutaneously engraftment of human lung tumor cell lines. we have observed that primary human lung tumors express tlr , tlr , tlr and tlr and that stimulation of these receptors in lung tumor cell lines by poly i:c, lps, loxoribine or poly u induces nfxb activation through atypical signaling pathway, with phosphorylation of ixba without its degradation and nuclear translocation of p and p nfxb subunits. interestingly, we observed that tlr stimulation induces apoptosis depending of the histological type of the tumor. on the contrary tlr , tlr and tlr stimulation induces cell survival and increases clonogenicity. this is correlated with an up-regulation of bcl- expression. moreover, despite a common atypical activation of nfxb, our transcriptomic analysis revealed major differences in gene modulation after triggering of tlr , tlr , tlr and tlr . finally, in vivo tlr stimulation of human lung tumor cells dramatically increases tumor size and metastasis. conclusions: altogether, these data emphasize that tlr , tlr or tlr triggering can directly favor tumor development whereas tlr signaling can induce tumor cell death. these data suggest that anticancer immunotherapy using tlr adjuvants should take into account the expression of these tlrs in lung tumor cells. objective: dasatinib (bms- ) is a small molecule src/abl tyrosine kinase inhibitor approved for the treatment of chronic myeloid leukaemia and philadelphia chromosome-positive acute lymphoblastic leukaemia. members of the src family of kinases are involved in normal physiological processes, and play a significant role in the induction and regulation of innate and adaptive immunity. the purpose of this study was to evaluate the inhibitory action of dasatinib on toll like receptor (tlr) signalling, natural killer (nk) cell cytotoxicity as well as antigen-specific cd + and cd + t cell function. methods: to analyse tlr signalling in vitro murine bone marrow derived (bmd) macrophages were stimulated with the tlr ligand lipopolysaccaride (lps) in the presence of dasatinib and tumour necrosis factor a (tnf-a) in the culture medium was measured. the response to tlr stimulation was also tested in vivo, dasatinib-treated mice were challenged with lps and tnf-a in the serum was quantified. in addition, the clearance of the rma-s cells, a mhc class i deficient thymoma sensitive to nk cell lysis, was analysed in mice undergoing dasatinib treatment. to investigate the inhibitory effects of dasatinib on adaptive immune responses, transgenic cd + and cd + t cells specific for ovalbumin were utilised to measure antigen specific t cell proliferation. endogenenous cd + and cd + t cell responses were determined following immunisation of dasatinib-treated mice with a nonreplicating recombinant virus. results: we show that dasatinib impairs: . innate immune response; dasatinib treatment reduced the (a) production of tnf-a following tlr stimulation of bmd macrophages in vitro, (b) production of tnf-a in vivo in response to lps and (c) ability of nk cells to eliminate mhc class i deficient cells in vivo . . adaptive immune response; dasatinib treatment inhibited (a) proliferation of antigen-specific murine transgenic t cells, (b) endogenous antigen-specific helper t cell recall-responses and (c) t cell-mediated cytotoxic effector function. conclusions: these findings suggest that dasatinib has the potential to modulate the host immune response and highlights scope for off target applications, for example therapeutic immunosuppression in the context of autoimmune pathogenesis, or in combination with other interventions for the treatment of endotoxic shock. i. zanoni , r. oatuni , m. collini , m. caccia , p. castagnoli , g. chirico , f. granucci university of milano-bicocca, biotechnology and bioscience, milan, italy, university of milano-bicocca, physics, milan, italy, singapore immunology network (sign), biomedical sciences institutes, immunos, singapore, singapore the recognition of mamps by tlrs expressed on dendritic cells (dcs) plays an essential role for the regulation of the immune responses. by recruiting different combinations of adapter proteins, individual tlrs turn on signal transduction pathways leading to the activation of different transcription factors. interleukin- (il- ) is one of the molecules produced by dcs shortly after stimulation with different tlr agonists. based on this observation and by analogy with the events following t-cell receptor (tcr) engagement leading to il- production, we hypothesized that the stimulation of tlrs on dcs might lead to activation of the ca +/ calcineurin and nfat pathway. we found that dc stimulation with lps induces extracellular ca + influxes, leading to calcineurin-dependent nfat activation. the activation of this pathway was independent of tlr engagement, depending instead exclusively on cd . we also found that lps-induced nfat activation in dcs was necessary for the efficient synthesis of cyclooxygenase- (cox- ) that, by generating prostaglandins (pgs), such as pge , regulates different dc functions including migration and polarization of t cell responses. our findings reveal novel aspects of the molecular signaling triggered by lps in dcs and define a new role for cd . given the essential involvement of cd in many diseases, including sepsis and chronic heart failure, the discovery of signal transduction pathways activated exclusively via cd represents a major step towards the development of potential treatments with modes of action involving interference with cd functions. we have examined the interaction of cd , a -kda glycosyl-phosphatidylinositol (gpi)-anchored membrane protein, with the monocyte signalling receptor, cd . human monocytes were isolated from healthy adult donor's peripheral blood. this involved labelling molecules at saturation with different coloured fluorophores and determining their positions separately by dual wavelength imaging. the cells were labelled at saturation with anti-cd antibody coupled to biotin visualised by qd- -streptavidin and anti-cd antibody coupled to allophycocyanin. the images are analysed to quantify the overlap of the particle images and hence determine the extent of co-localization of the labelled molecules. single particle fluorescence imaging (spfi) uses the high sensitivity of fluorescence to visualize individual molecules that have been selectively labelled with small fluorescent particles. the images of particles are diffraction-limited spots that are analysed by fitting with a two-dimensional gaussian function providing the basis for determining the dynamic and associated behaviour of receptors on living cells. changes in the numbers of receptors, and in the proportion of receptors showing colocalisation, indicated that lps promotes the interaction of cd and cd , suggesting a new functional role of cd as a member of a multimeric lps receptor complex. l. lundvall , r.r. schumann charité -universitätsmedizin berlin campus mitte, institute for microbiology and hygiene, berlin, germany meningitis is a life-threatening disease mainly caused by bacteria and viruses. bacterial components such as lipopolysaccharide (lps), lipoproteins or peptidoglycan breakdown products (i. e. mdp, mesodap) stimulate pattern recognition receptors (prrs), such as toll-like receptors (tlrs) and the intracellular nod-like receptors (nlrs) for an inflammatory response. we hypothesize that a synergistic effect of tlr-induced nf-xb activation and nlr-mediated caspase- induction leads to an increased release of mature il- b during bacterial meningitis in brain-derived cells. a mouse meningitis model with s. pneumoniae (d ) was established for assessing il- b induction during this disease. the murine raw . cell line, the human astroglial u- mg and the murine microglial cell-line bv- were stimulated with the tlr ligand lps, the tlr ligand pam cys, the nod ligand mdp, or the nod ligand c -ie-dap, and, as control, atp alone or in combination. we assessed il- b by elisa and caspase- and pro-il- b expression by western blot. furthermore, primary mouse astrocytes isolated from the cortices of mouse puppies were used for stimulation followed by sirna suppression of elements of the il- b induction pathway. s. pneumoniae (d ) infected mice showed a significant increase in il- b release after hours. in vitro, an increase in il- b levels after costimulation with lps or pam cys, and mdp or c -ie-dap was observed in a dose-dependent manner. a synergistic enhancement of il- b by tlr-and nlr-ligands was observed in raw cells, bv- cells, u- cells and primary astrocytes. active caspase- (p ) was induced by mdp or c -ie-dap, corresponding with high il- b responses when lps or pam cys was added. sirna experiments show that a knock-down of nod leads to a diminished il- b release after lps-and mdp-stimulation. the precursor forms of il- b and caspase- seem to be constitutively expressed in astrocytes and microglia. a synergistic enhancement between tlrs and nlrs in il- b release in brain-derived cells was observed. so a two-step stimulation seems necessary for the release of high levels of mature il- b by astrocytes and microglia. bacteria containing both, tlr-and nlr-ligands thus have the potential to induce high levels of il- b which may contribute to disease pathology and may point to novel intervention strategies. j. rosenberg toll-like receptors (tlrs), nod-like receptors (nlrs), and rig-i-like (rlrs ) are more well-characterized in their identity and expression as signaling markers which effect the ealry innate immune response and elicit adaptive immunity , . in the case of tlrs most sutides to date have delineated tlr expression and function on antigen presenting cells like dendritic cellof this research. extension of the profiling and presence of tlrs on cell characterized as adaptive immune cells such as t cells is the subject of this line of research. using a cd and cd activation model system -tlr presence on cd + cells is found in mouse t cells, human t cells and jurkat cell lines. following cd /cd activation for hours we have identified a small but distinct populationof tlr + cells. further characterization indicates these cells to be cd +cd + cells. further characterization of the expression and functional acitvity of the tlr + t cells indicates co-expression of tlr with md- indicating a functional tlr receptor. in addition lps activiation did not lead to upregulation of tlr expression in t-cells. the data indicate that tcr activation leads to tlr expression. the expression appears to be associated with cd +cd + cells and refelecting an activated t cell phenotype which will be further characterized as perhaps related to tregs or other tcell subsets. s. m. lehmann , d. kaul , c. krüger , f. zipp , r. nitsch , s. lehnardt charité-universitätsmedizin berlin, cecilie-vogt-clinic for neurology, berlin, germany, charité-universitätsmedizin berlin, institute for cell biology and neurobiology, berlin, germany the innate immune system is the first line of defense against various pathogens and requires the expression of toll-like receptors (tlrs). in macrophages, tlr plays a crucial role in immune responses elicited by gu-rich ssrna (i. e. ssrna ) as well as synthetic antiviral chemicals, including imidazoquinoline components (i. e. imiquimod) and some guanine nucleotide analogs (i. e. loxoribine). these compounds were initially described to activate mouse tlr (and human tlr ) and are potent immune response modifiers leading to important antiviral and antitumor activities. microglia serve as the major innate immune cells in the central nervous system (cns). employing various techniques including pcr, in situ hybridization, and immunocytochemistry, we demonstrate that tlr is expressed in these cells. incubation of microglia with all three of the above mentioned tlr ligands leads to activation of these cells displaying an ameboid shape and releasing inflammatory cytokines such as tnf-a and il -b in a dose-and time-dependent fashion. analysis of wild type (wt) and tlr knock out (ko) microglia by real- because neutrophil apoptosis plays a key role in resolving inflammation, identification of proteins regulating neutrophil survival should provide new strategies to modulate inflammation. using a proteomic approach, coronin- was identified as a cytosolic protein cleaved during neutrophil apoptosis. coronin- is an actinbinding protein that can associate with phagosomes and nadph oxidase but its involvement in apoptosis was currently unknown. in coronin- -transfected plb cells, coronin- overexpression did not modify the kinetics of granulocyte differentiation as assessed by cd b labeling. concerning apoptosis, increased coronin- expression in dmf-differentiated plb significantly decreased gliotoxin-induced mitochondrial depolarization as compared with controls. likewise, coronin- significantly decreased trail-induced apoptosis with less mitochondrial depolarization, caspase- and caspase- activities, but not caspase- or bid truncation suggesting that coronin- interfered with mitochondria-related events. to validate the prosurvival role of coronin- in a pathophysiological condition involving neutrophil-dominated inflammation, neutrophils from cystic fibrosis (cf) patients were studied. circulating neutrophils from cf patients had more coronin- expression assessed by immunoblotting or proteomic analysis of cytosolic proteins. this was associated with a lower apoptosis rate than those from controls evidenced by delayed phosphatidylserine externalization and mitochondria depolarization. in addition, inflammatory neutrophils from cf patients lungs showed an intense coronin- immunolabeling. we concluded that coronin- could constitute a potential target in resolving inflammation. p.-n. hsu national taiwan university, graduate institute of immunology, taipei, taiwan, republic of china human osteoclast formation from mononuclear phagocyte precursors involves interactions between tumor necrosis factor (tnf) ligand superfamily members and their receptors. many of the proinflammatory cytokines and growth factors implicated in inflammatory processes have also been demonstrated to impact osteoclast differentiation and function. recent evidence indicates that the tnf-related apoptosis-inducing ligand (trail) of the tnf ligand superfamily, which was initially thought to induce apoptosis in many transformed cell lines, can serve as an effector molecule in activated t cells. we show in this work that trail can induce osteoclast formation from human monocytes and murine raw . macrophages. we demonstrated that both cell models differentiate into osteoclast-like cells in the presence of trail in a dose-dependent manner, as evaluated in terms of tartrate-resistant acid phosphatase (trap)-positive multinucleated cells and bone resorption activity. the trail-induced osteoclast differentiation is independent of caspase activation and apoptosis induction activity. however, trail-induced osteoclastogenesis is dependent on activation of nf-xb, erk, and p map kinase. the trail-induced osteoclastogenesis was significantly inhibited by treatment with traf- sirna and traf- decoy peptide, indicating this pathway is traf- dependent. thus, our data demonstrate that trail induces osteoclast differentiation via direct engagement with the trail death receptor through a signaling pathway distinct from apoptosis. our results indicate that in addition to triggering apoptosis, trail induces osteoclast differentiation. it provides a novel role for trail in regulating osteoclast differentiation and in osteoimmunology. microglia are considered to be the local antigen presenting cells (apcs) of the central nervous system (cns) which are thought to play a crucial role in local reactivation of autoreactive t cells during cns autoimmunity e. g. in multiple sclerosis (ms) and its animal model experimental autoimmune encephalomyelitis (eae) . in this study we investigated if the anti-inflammatory nuclear transcription factor peroxisome proliferator-activated receptor gamma (pparg) that has been described to negatively regulate macrophage activation has an influence on microglia immunogenicity. sustained activation of pparg both reduced microglial signalling via mhc molecules and costimulatory molecules and concomitantly increased signalling via the coinhibitory molecules b -h and b -dc. moreover, also production of pro-inflammatory cytokines like tnf-a and il- was profoundly reduced if microglia were pre-treated with the pparg-agonist pioglitazone (pio). in contrast to this, the lack of pparg in microglia resulted in increased expression of pro-inflammatory cytokines not only following an inflammatory stimulus but also in the steady-state indicating that pparg might play a cell-intrinsic role in controlling microglia immunogenicity. importantly, if pparg was activated in microglia, the capacity to prime ovalbumin-specific t cells was impaired. t cells primed by pio-treated microglia produced reduced amounts of il- and ifn-g which could not be overcome by restimulation with acd . this indicates that t cells primed by pio-treated microglia did not undergo functional differentiation but were impaired in exhibiting effector functions. furthermore, microglia were able to induce antigen-specific differentiation of naive cd t cells into t helper (th ) cells, which have been associated with autoimmune pathogenicity during eae. however, if pparg was activated, microglia were no longer able to induce th differentiation. in conclusion, activation of pparg impairs microglial apc function leading to reduced activation of antigen-specific t cells and, in addition, inhibits the induction of th cells. therefore, activation of pparg in microglia is a promising approach to limit local activation of autoreactive t cells in the cns in cns-autoimmune deseases. bacterial lipopolysaccharide (lps) triggers monocytes and macrophages to produce several inflammatory cytokines and mediators. however, once exposed to lps, they become hyporesponsive to a subsequent endotoxin challenge. this phenomenon is defined as lps desensitization or tolerance. previous studies have identified some components of the biochemical pathways involved in negative modulation of lps responses. in particular, it has been shown that the il- receptor-related protein st could be implicated in lps tolerance. the natural ligand of st was recently identified as interleukin- (il- ), a new member of the il- family. in this study, we investigated whether il- triggering of st was able to induce lps desensitization of mouse macrophages. we found that il- actually enhances the lps response of macrophages and does not induce lps desensitization. we demonstrate that this il- enhancing effect of lps response is mediated by the st receptor since it is not found in st ko mice. the biochemical consequences of il- pretreatment of mouse macrophages were investigated. our results show that il- increases the expression of the lps receptor components myeloid differentiation protein- (md ), cd and tlr- and the myeloid differentiation factor (myd ) adaptor molecule. in addition, il- pretreatment of macrophages enhances the cytokine response to tlr- but not to tlr- ligands. thus, il- treatment preferentially affects the myd -dependent pathway activated by the tlr. c. klotz , b. lenz , r. lucius , s. hartmann humboldt-university berlin, molecular parasitology, berlin, germany chronic helminth infections are shown to be negatively associated with allergic disorders in humans and animal models and parasite cysteine protease inhibitors (cystatins) have been identified as a major class of modulators from filarial parasites. recently we showed that recombinant parasite cystatin (avcystatin), derived from the model parasite acanthocheilanema viteae, effectively abolished ova-induced allergic airway responsiveness in a mouse model of asthma (schnoeller et al., ) . the cystatin effect was blocked by the application of anti-il- receptor antibodies and by depletion of macrophages using clodronate liposomes. we hypothesize that parasite cystatin induced regulatory macrophages characterized by secretion of immune suppressive interleukin- (il- ). the aim of the present study was to elucidate the molecular mechanisms by which avcystatin induces il- in primary macrophages. in vitro experiments with peritoneal macrophages from balb/c mice confirmed specific and concentration dependent il- production after avcystatin stimulation. application of specific inhibitors revealed that the il- induction was p and erk dependent, and inhibitor titration indicated a higher sensitivity towards p . western blotting experiments confirmed the phosphorylation of p and erk in macrophages after avcystatin stimulation. in addition, by using specific inhibitor and western blotting, we showed that avcystatin induced il- is also regulated by the phosphatidylinositol- -kinase (pi k) -proteine kinase b (akt) pathway. further analysis indicated a hierarchical signalling pattern and cross regulation of the identified pathways. hence, we conclude that avcystatin renders macrophages into a regulatory state by addressing a broad range of signalling cascades that ultimately lead to the expression of il- and possibly other regulatory markers. in general, revealing fundamental knowledge about induction of regulatory macrophages by helminth immunomodulators will help to design new strategies for the treatment of inflammatory disorders. we screened approximately half the (putative) human kinome to identify novel candidates interfering with macrophage activation in response to endotoxin. this screen revealed the impact of several novel kinases as well as kinases with previously established function. one of the top candidates identified to block endotoxininduced tnf-a secretion was carkl, a gene with no previously described function. subsequent biochemical analyses unequivocally revealed that carkl is a phosphotransferase protein using sedoheptulose as a phosphate acceptor and atp as a donor. sedoheptulose is a monosaccharide consisting of seven carbon atoms and a functional ketone group. the product sedoheptulose- -phosphate (s- p) is also an intermediate metabolite of the pentose phosphate pathway (ppp) and so far was only known to be produced by condensation of ribose- p and xylulose- p via a transketolase reaction. to identify the molecular mechanism by which carkl modulates the immune response, we investigated its endogenous regulation and function in the course of macrophage activation. so far, our data favor a model where post-stimulatory downregulation, i. e. loss of carkl is essential for the activation of macrophages by various pro-inflammatory stimuli. disentangling the signaling pathways responsible for the rapid regulation of carkl unearthed nf-kb and p /jnk but not erk as driving forces. counterbalancing endotoxin induced loss of carkl by over-expression led to an impaired cytokine response and a concomitant block of free radical production. comparison of wild type and catalyticinactive forms of carkl unveiled that most of the effects of carkl on the inflammatory response were due to its phosphotransferase activity. expression profiling using gene chip analysis further supported the concept that carkl may represent a new key modulator of inflammatory processes. taken together, detailed analyses to study the molecular function of carkl should ultimately lead to a more profound understanding of cellular metabolism and especially clarify new mechanisms involved in the regulation of inflammation. in addition, connecting the ppp and its impact on the cellular redox state with inflammatory disease models might reveal new therapeutic targets. in this context, the sedoheptulose kinase carkl and its product s- p may provide a novel basis for interfering with adverse immune responses. t. bosschaerts , y. morias , p. de baetselier , a. beschin vib, cmim, vub brussels, brussels, belgium the development of classically activated monocytic cells (m ) is a prerequisite for effective elimination of parasites, including african trypanosomes. however, persistent m activation causes pathogenic damage including liver injury during infection, resulting in death of the host. we aim to identify mechanisms involved in regulation of m activity in order to dampen their pathogenicity and increase the resistance of the host to parasitic diseases. methods: we have scrutinized the phenotype and cellular origin of liver m in trypanosoma brucei infected by facs analysis and bone marrow transfer experiments. the contribution of different signaling pathways, including myd , ifng, il- , ccr and nf-kb to the development and/or recruitment of pathogenic m in the liver was investigated using knock-out mice or by delivering il- in infected mice. results: we established that cd b+ly c+cd c+ tnf and inos producing dcs (tip-dcs) represent the major m liver subpopulation. tip-dcs differentiated in an ifng/myd -dependent manner from cd b+ly c+ inflammatory monocytes in the liver of infected mice. ccr promoted the egression of inflammatory monocytes from bone marrow to blood but not their entry, differentiation and maturation to tip-dcs in the inflamed liver. as a consequence, ccr ko mice experienced reduced pathogenic symptoms. on the other hand, the absence of il- enhanced the recruitment of inflammatory monocytes as well as their differentiation and maturation to tip-dcs, resulting in exacerbated pathogenicity and early death of the host. in addition, the therapeutic liver-specific delivery of il- in t.brucei infected mice efficiently limited the differentiation and maturation to tip-dcs, hereby limiting disease-associated pathogenicity. finally, the absence of the nf-kb member p was associated with increased tissue injury associated with increased production of pathogenic tnf and no by inflammatory monocytes, but not by tip-dcs. conclusion: our data demonstrate that nf-kb p and il- play a role in preventing infection-associated pathogenicity in hosts confronted with a chronic inflammatory situation by limiting the activity of pathogenic m , in particular tip-dcs. the inflammatory activity of liver m is controlled by il- and/or p nf-kb at different levels, including recruitment of inflammatory monocytes to the liver, their differentiation to pathogenic tip-dcs, or their production of tnf and no. a. popov , j. driesen , z. abdullah , a. niño castro , t. chakraborty , m. krönke , o. utermöhlen , c. wickenhauser , j.l. schultze limes institute, laboratory for genomics and immunoregulation, university of bonn, bonn, germany, institute of molecular medicine and experimental immunology, bonn, germany, institute of medical microbiology, university of giessen, giessen, germany, institute for medical microbiology, immunology and hygiene, university of cologne, cologne, germany, institute for pathology, university clinic leipzig, leipzig, germany dendritic cells (dc) and macrophages play an important role in pathogen sensing and antimicrobial defense. here we report on a new role for the myeloid antigen presenting cells (apc) in granulomatous infections. infection of myeloid dc and macrophages with listeria monocytogenes results in a distinct regulatory phenotype characterized by expression of multiple inhibitory molecules, including indoleamine , -dioxygenase, cyclooxygenase- and cd and production of prostaglandin e (pge ) and interleukin . all these molecules are strictly dependent on autocrine tnf, released during infection, and are in concert suppressing t-cell responses; cd , expressed by regulatory myeloid cells, acts as an il- scavenger. importantly, myeloid cells with regulatory phenotype are characterized by increased resistance to infection and demonstrate significantly improved bactericidal activity against intracellular bacteria. furthermore, infected cells can transfer the regulatory phenotype to the uninfected ones in a cell-cell contact independent manner, thereby extending the pool of infection-resistant myeloid cells. induction of regulatory and protective phenotype in macrophages and dc require at least two signals provided by tnf and either pge or tlr ligands. transcriptional changes in human macrophages, infected by mycobacterium tuberculosis, resemble the ones induced in dc during infection with l.monocytogenes. in fact, granuloma in patients with tuberculosis and listeriosis are enriched for cd + ido + cox- + regulatory myeloid cells, whereas most effector cell populations, such as t cells, b cells and nk cells, are expelled from the granuloma. of note, in tuberculosis granuloma consist mostly of macrophages, whereas in listeriosis dendritic cells predominate. altogether, our studies provide strong evidence that intracellular pathogens such as m.tuberculosis and l.monocytogenes induce a specific polarization of myeloid dc and macrophages characterized by a functional preponderance of inhibitory mechanisms. we postulate that these regulatory myeloid cells play a dual role during life-threatening granulomatous infections. on one hand, they promote pathogen containment by efficiently killing intracellular bacteria; on the other hand, these myeloid cells inhibit granuloma-associated t cells and thereby might be involved in the retention of granuloma integrity protecting the host from granuloma break-down and pathogen dissemination. the interferon-gamma (ifn-g) component of the immune response plays an important and essential role in infectious and non-infectious diseases. induction of ifn-g secretion by human t and nk cells through synergistic co-stimulation with interleukin (il- ) and il- in the adaptive immune responses against pathogens is well known, whereas a similar activity by macrophages is still controversial, largely due to criticisms based on the contamination of macrophages with nk or t cells in the relevant experiments. the possible contribution of macrophages to the interferon response is, however, an important factor relevant to the pathogenesis of many diseases. to resolve this issue, we have determined the production of ifn-g at a single cell level by inmunohistochemistry and by enzyme-linked immunosorbent spot (elispot) analysis and have unequivocally demonstrated that human macrophages derived from monocytes in vitro through the combined stimulation of il- and il- or with macrophage-colony stimulating factor (m-csf) were able to produce ifn-g when further stimulated with a combination of il- and il- . in addition, naturally activated alveolar macrophages immediately secreted ifn-g upon treatment with il- and il- . therefore, human macrophages in addition to lymphoid cells contribute to the ifn-g response, providing another link between the innate and acquired immune response. a. j. denzel , m. rodriguez gomez , m. niedermeier , y. talke , n. göbel , k. schmidbauer , m. mack unversity hospital regensburg, internal medicine ii, regensburg, germany, university hospital regensburg, regensburg, germany we have shown previously that basophils recognize and react to free antigen during a memory immune response in vivo and release large amounts of il- and il- . activation of basophils is dependent on the presence of free antigen, antigen specific immunoglobulins and expression of immunoglobulin fc-receptors. we now have analysed in more detail the binding of antigen to basophils, the recruitment of basophils to lymphoid organs and the basophil dependent migration of other leukocytes during the first days of a memory immune response. following restimulation with soluble antigen only antigen specific basophils but not basophils from naïve mice migrate from bone marrow and spleen to the site of restimulation (e.g. the peritoneum) and the draining lymph nodes. peripheral blood basophils are markedly reduced during the first hours after restimulation. in the blood, spleen lymph nodes and bone marrow basophils can bind intact antigen on their surface for up to h, with basophils in the bone marrow binding the lowest amount of antigen. depletion of basophils also affects the recruitment of various other leukocyte subsets in immunized mice. our datas show that basophils are recruited to draining lymph nodes during a memory response. tnf-a is a pro-inflammatory cytokine that mediates inflammation in response to various pathogens, including mycobacterium tuberculosis. it is also a key factor in the pathogenesis of autoimmune diseases like rheumatoid arthritis. three tnf-a-blocking drugs have been approved to treat selected autoimmune diseases; two are monoclonal antibodies against tnf-a (adalimumab and infliximab); the other is a soluble tnf receptor/fc fusion protein (etanercept) . tnf-a-blockers have been shown to increase the risk of reactivation of latent tuberculosis and this risk appears to be higher in patients treated with the monoclonal antibodies. we studied the effects of tnf-a blockers on the maturation of mycobacteria-containing phagosomes in human macrophages. all three drugs had an inhibitory effect on ifn-g-induced phagosome maturation in pma-differentiated human thp- cells infected with m. bovis bcg, the avirulent m. tuberculosis h ra strain and the virulent m. tuberculosis h rv strain. adalimumab and infliximab, but not etanercept, suppressed phagosome maturation in primary human peripheral blood monocyte-derived macrophages (mdm) in the presence or absence of ifn-g. macrophages secreted tnf-a in response to infection with mycobacteria and this response was enhanced by activation of the cells with ifn-g. treatment of infected macrophages with tnf-a increased maturation of mycobacteria-containing phagosomes. these results suggest a role for tnf-a in activating phagosome maturation and highlight a novel mechanism through which tnf-a blockade can affect the host innate immune response to mycobacteria. z. g. dobreva , l.d. miteva , s.a. stanilova trakia university, faculty of medicine, molecular biology, immunology and genetics, stara zagora, bulgaria il- is a heterodimeric cytokine composed of a p subunit associated with the il- / p subunit. like il- , il- is expressed by the activated antigenpresenting cells and both cytokines induce ifn-gamma secretion by different t cell subsets. the proper balance between il- p -related cytokines controls the appearance of normal th and pathological th mediated immune responses. in this study, we examined the dynamics of inducible il- p and il- p mrna expression and protein production in purified human monocytes and how jnk and p mapks inhibitors influenced il- p and il- production. the cytokines' quantity determination was performed by elisa. quantitative real-time polymerase chain reaction (qrt-pcr) was performed for mrna transcripts detection. results were calculated in fold increase compared with gene expression in nonstimulated monocytes. il- p gene expression was higher than those observed for il- p gene at all time-points. the level of il- p mrna increased after th h and reaching a maximum level at th h ( . fold for c bgp and . fold for lps). c bgp and lps triggered il- p gene transcription were almost equal at the rd h ( . and . fold) and at th h ( . and . fold, respectively) after stimulation. the higher level of il- p gene expression was detected at th h in lps compared to c bgp stimulated monocytes ( . vs. . fold). however, il- p and il- protein production was increased in the highest level after c bgp stimulation. the inhibition of p led to the statistical significant augmentation of c bgp stimulated il- p production. the inhibition of the same map kinase enhanced lps stimulated il- p production without significant difference. the inhibition of jnk and p mapks significantly decreased c bgp stimulated il- production from human monocytic cells.in summary, the present study demonstrates the different time-course and ability of c bgp and lps to induce the expression of il- p and il- p mrnas in purified human monocytes. we showed that inhibition of p mapk down regulated il- and upregulated il- p production in stimulated monocytes. we concluded that in human monocytes p map kinase activation has an opposite effect on the il- p and il- p expression. neutrophils represent key components of the innate immune system with the ability not only to phagocytose and killing invading pathogens, but also to produce a variety of proteins, including cytokines and chemokines, with important consequences on the recruitment and activation of other immune cells, such as monocytes, dendritic cells, t and b cells. for instance, it has been shown that neutrophils can directly interact with, and induce functional maturation of, immature monocyte-derived dendritic cells (modc). indeed, upon interaction with neutrophils, modc up-regulate the expression of costimulatory molecules, such as cd , cd and cd , and secrete il- , thus acquiring the ability to induce proliferation and th polarization of naï ve t cells. in order to extend these findings, the present study was designed to address whether human neutrophils interact with peripheral blood-derived dendritic cells and the pathological consequences that such interaction could eventually produce. in human peripheral blood, dendritic cells can be divided in plasmacytoid dendritic cells (pdc) and myeloid dendritic cells (mdc), the latter further divided in three different subsets based on the expression of cd c, bdca- , and cd . by analyzing different chronic inflammatory pathologies, such as crohn's disease, psoriasis and sweet's syndrome, we found that neutrophils co-localize with a subtype of myeloid dendritic cells (mdc) with characteristics resembling the cd + subset of mdc. in order to characterize the interaction between the two cell types, autologous neutrophils, highly purified by an in-house built immunonegative selection protocol, and cd + dc were isolated from healthy donors and analyzed in a co-culture system under different stimulatory conditions. here we show that neutrophils modulate different effector functions of cd + dc, including their survival and their ability to produce il- p . besides providing the basis for a better understanding of the cellular interactions that occur in pathological conditions, our results further emphasize the importance of neutrophils in the modulation of the inflammatory response. chitin is a linear polymer of n-acetyl-d-glucosamine (glcnac) residues present in human pathogenic fungi or nematodes. chitotriosidases (cht) and acetic mammalian chitinase (amcase) have been identified as the only functional chitinases in mammalians. the expression of both chitinases appears to be strongly species dependent, indicating distinct physiological functions. amcase is considered as predominant chitinases in mice while cht is regarded as major chitinases in humans. interestingly, cht is constitutively expressed by human phagocytes at high levels while it is absent in mice phagocytes. although, amcase received increased attention as modulator of the innate immune response against chitin in mice, the physiological function of cht in humans is virtually unknown. to evaluate the physiological function of cht we have characterised the substrate specificity of human cht and the mode of substrate cleavage by analysing chtproduced fragments of chitosan, a close but water soluble derivate of chitin. degradation products of chitosan have been investigated by gel electrophoresis and maldi-tof mass spectroscopy. moreover, the application of a computer-based model of cht activity revealed the mode of substrate cleavage. we found that cht is a processive endo-cleaving chitinase resulting in the production of only small diffusible chitin/chitosan fragments. in further studies we could show that those cht-produced small chitin/chitosan fragments exhibit a strong ability to induce a pro-inflammatory response in human blood derived monocytes/macrophages as indicated by an increased release of the pro-inflammatory cytokines tnf-a, il- , il- and mcp- involving the transcription factor nfxb. moreover, these stimulated monocytes/macrophages revealed an increase of cht expression indicating an autocrine positive feed-back regulation. our data suggest that human cht is involved in the early recognition of chitin/chitosan containing human pathogens due to the generation of immuno-stimulatory chitin/chitosan fragments. m. hasenberg , s. wolke , a. brakhage , m. gunzer otto-von-guericke universität, institut für molekulare und klinische immunologie, magdeburg, germany, hans-knöll-institut, abteilung für molekulare und angewandte mikrobiologie, jena, germany since their discovery in nucleic extracellular traps (nets) released by certain cell types including neutrophil and eosinophil granulocytes were shown to play a crucial role in mediating innate immune responses towards different bacterial und fungal pathogens. recently it was found by us and others that neutrophil granulocytes release nets also upon contact to the filamentous fungus aspergillus fumigatus. in the present study we aimed to characterize this process in more detail focusing on the kinetics of net-formation as well as clarifying the responsible cell-biological mechanisms. by the use of several microscopic techniques (scanning electron microscopy, fluorescence widefield microscopy, confocal-and -photon microscopy) we initially demonstrated the generation of net like structures after coincubation of a. fumigatus germlings and freshly isolated murine or human pmn. the analysis of our time lapse video microscopy data allowed us to examine the exact time course from initial contact to the fungal surface to explosive release of nets up to hours later. moreover, we investigated the dependency of this phenomenon on the induction of an oxidative burst. therefore we added the nadph-oxidase inhibitor dpi to the cell coincubation and found clearly reduced net formation. by fluorescence staining of reactive oxygen species we could demonstrate that ros are released prior to net detection. interestingly, our data currently suggest that in contrast to other pathogens investigated so far, nets are not directly toxic to fungal elements. whether and how nets control growth of a. fumigatus currently remains open. to summarize our data, we found rapid net formation as a commonly observed immune response of neutrophil granulocytes contacting a. fumigatus. consistent with studies on different pathogens this mechanism seems to be ros-dependent, however not toxic for the fungus. thus, in the future we will have to clarify whether net-formation really occurs in vivo and how nets can control the outgrowth of a. fumigatus at sites of infection. production of type i interferons (ifn-i, mainly ifn-a and ifn-b) is a hallmark of innate immune responses to all classes of pathogens. when viral infection spreads to lymphoid organs, the majority of systemic ifn-i is produced by a specialized 'interferon-producing cell' (ipc) that has been shown to belong to the lineage of plasmacytoid dendritic cells (pdc). it is unclear whether production of systemic ifn-i is generally attributable to pdc irrespective of the nature of the infecting pathogen. we have addressed this question by studying infections of mice with the intracellular bacterium listeria monocytogenes. protective innate immunity against this pathogen is weakened by ifn-i activity. in mice infected with l. monocytogenes systemic ifn-i was amplified via ifn-b, the ifn-i receptor (ifnar) and transcription factor interferon regulatory factor (irf ), a molecular circuitry usually characterisitic of non-pdc producers. synthesis of serum ifn-i did not require tlr . in contrast, in vitro differentiated pdc infected with l. monocytogenes needed tlr to transcribe ifn-i mrna. consistent with the assumption that pdc are not the producers of systemic ifn-i, conditional ablation of the ifn-i receptor in mice showed that most systemic ifn-i is produced by myeloid cells. furthermore, results obtained with facs-purified splenic cell populations from infected mice confirmed the assumption that a cell type with surface antigens characteristic of macrophages and not of pdc is responsible for bulk ifn-i synthesis. the amount of ifn-i produced in the investigated mouse lines was inversely correlated to the resistance to lethal infection. based on these data we propose that the engagement of pdc, the mode of ifn-i mobilization, as well as the shaping of the antimicrobial innate immune response by ifn-i differ between intracellular pathogens. t. naessens , s. vander beken , p. bogaert , j. grooten ghent university, biomedical molecular biology, zwijnaarde (ghent), belgium introduction: although the effector and modulator functions of activated macrophages in innate and adaptive immunity are well documented, their exact role in the initiation and propagation of immune pathologies is still not fully understood. recent insights in monocyte and macrophage heterogeneity render the picture even more complex. in addition, it is unclear to what extend resident and elicited macrophages differ functionally and hereby differentially contribute to immune pathologies. in this study we focused on the dynamics and function of resident alveolar macrophages (ram) during and after allergic bronchial inflammation. strategy: we used an ovalbumin (ova)-alum based mouse model of allergic asthma and an ova-complete freund's adjuvant (cfa) based mouse model of hypersensitivity pneumonitis, constituting a th -driven immunological counterpart of the th -driven experimental asthma. ram were distinguished by prior in situ labelling with fluorescent polystyrene microspheres. as complementary approach, ram and elicited alveolar macrophages (eam) were distinguished using cd bone marrow chimeric mice. combined with flow cytometry and fluorescence activated cell sorting, both approaches allowed us to trace resident and elicited am populations in the course of th -and th -driven allergic airway inflammation. results: during the acute phase of the allergic response, isolated ram and eam showed distinct gene expression signatures, reflecting a possible functional heterogeneity between these two macrophage subsets. in both types of allergic inflammation, microsphere-tagged cd . + ram remained constant in cell number for the first days of chronic ova-exposure and then dropped sharply, having nearly completely disappeared from the alveoli by day of ova-exposure. as a consequence, following the clearance of inflammation, inflammation-experienced ram replaced the initial ram population. strikingly, in both types of allergic inflammation, this secondary ram population showed a markedly altered responsiveness to lps stimulation. this involved macrophage activation markers and nf-kb inducible inflammatory genes. however, especially genes induced by ifn-beta showed strongly increased expression in secondary ram as opposed to their near lack of induction in primary ram. this switch from an ifn-beta deficient to an ifn-beta adequate phenotype may increase the inflammatory sensitivity of allergic inflammationexperienced lungs as also observed in asthmatic patients, showing an increased sensitivity to bacterial infection. e. schlecker , a. stojanovic , a. cerwenka german cancer research center, innate immunity, heidelberg, germany myeloid-derived suppressor cells (mdsc) are a heterogeneous population of cells that expand during cancer, inflammation and infection. these cells play a critical role in suppressing t cell responses. the exact nature and function of mdsc remain unclear. here we show that a subpopulation of mdsc (gr- + cd b + f / + ) isolated from rma-s tumor-bearing mice did not suppress but rather activated nk cells to produce ifn-g. additionally, nk cells eliminated this subpopulation both in vitro and in vivo. in order to identify molecules and pathways that might be involved in mdsc accumulation in tumor bearing mice and their suppressive/activatory function, gene expression profiling of mdsc subpopulations was performed using whole genome microarrays. understanding the reciprocal interaction of mdsc with nk cell could improve the efficiency of cancer immunotherapy. g. solinas , f. marchesi , m. fabbri , s. schiarea , c. chiabrando , a. mantovani , , p. allavena istituto clinico humanitas, rozzano, italy, istituto mario negri, milano, italy, università di milano, milano, italy experimental and clinical evidence has highlighted that tumor-associated macrophages (tam) represent the principal component of the leukocyte infiltrate and are usually associated with tumour growth, progression and metastasis. macrophage population is generally divided into two distinct subsets: m and m . m macrophages act as a first line of defence against pathogens whereas m cells participate in wound repair and maintenance of tissue integrity. in the tumour micro-environment tam interactions with the extracellular matrix, neighboring cells, and soluble stimuli largely influence their gene expression and behavior. to investigate the role of the tumor micro-environment on macrophage differentiation, we cultured freshly isolated human monocytes with pancreatic cancer cell line supernatants, in the absence of exogenous cytokine addition.. in selected cultures, about % of the monocytes differentiated after days into macrophages. the phenotype analysis of tumor-conditioned macrophages (tc-macro) demonstrated high expression of the mannose receptor, cd , cd and low levels of mhc class ii. tc-macro produced il- , il- , tnf but not il- , even after lps stimulation. moreover, tc-macro produced a panel of chemokines including ccl , cxcl , ccl and cxcl . the transcriptional profile of tc-macro revealed that several genes in line with an m polarization are highly expressed. the nature of the tumor-derived factors inducing macrophage differentiation is currently under investigation; biochemical analysis indicated that the biological activity is excluded from exosomes and have a high molecular weight ( g . kda). il- and il- were not detectable in tumor supernatants whereas m-csf was present at low levels. by mass spectrometric techniques, we surprisingly found that the tumor-derived m-csf had peculiar migration patterns which were different from those expected for the common human homodimeric glycosilated protein, suggesting an interesting structural differences for the tumor-secreted isoforms of this primary regulator of mononuclear phagocyte. the characterization of tumor-derived factors inducing macrophage differentiation could better clarify the intricate cross-talk between tumor cells and macrophages and thus might aid in the process of devising novel anti-tumor treatments. genomic effects of glucocorticoid hormone (gc) are exerted by glucocorticoid receptor (gr)-mediated changes of gene expression. this is relatively timeconsuming process, needing hours to develop. in contrast, non-genomic effects may occur within minutes. gcs are used for a long time for the therapy of anaphylactic reactions, where mast cells play crucial role. moreover, many cells and cell lines of haemopoetic origin are sensitive to gc-induced apoptosis. recent findings indicate, that non-genomic gc effects mediated by mitochondrial gr may have important function in generating pro-apoptotic signals. we aimed the investigation of non-genomic gc effects on in vitro cultured rbl- h rat mast cell line. we demonstrate that gr nuclear translocation begins within minutes and completes after minutes in dx treated rbl- h cells. since genomic effects occur in the nucleus through gene expression changes, we considered gc effects within minutes as non-genomic. studying gc-caused apoptosis, rbl- h cells proved to be gc-resistant and no mitochondrial gr translocation neither impaired mitochondrial function could be observed upon gc treatment. in further experiments we used rbl- h cells sensitized with anti-dnp (dinitrophenyl) ige and dnp-conjugated bovine serum albumin was used for stimulation. minutes of dx treatment inhibited ca + -signaling in antigen stimulated rbl- h cells in the concentration range of nm - mm. moreover, minutes of dx treatment altered the tyrosine phosphorylation pattern of rbl- h cells. dx treatment alone caused slight increase in tyrosine phosphorylation, while dx treatment of activated cells caused also an increase in tyrosine phosphorylation compared to the solvent-treated controls. the tyrosine kinase syk plays indispensable role in regulating mast cell activation through the fc[epsilon] receptor i. our immunoprecipitation studies show, that dx treatment results in decreased syk phosphorylation in both resting and activated cells. this finding raises the possibility, that syk phosphorylation thus kinase activity may be directly or indirectly regulated by gcs via non-genomic pathway. taken together, our experiments along with the clinical experiences suggest that gcs rapidly influence mast cell activation via a non-genomic pathway, too. the elucidation of the exact signal transduction mechanisms behind rapid gc effects need further experiments. high mobility group box (hmgb ) is a non-histone nuclear protein that binds chromatin and has transcriptional and architectural functions. notably, hmgb is highly mobile in the nucleus and is passively released by necrotic cells, while it is bound firmly to apoptotic chromatin ( ) . extracellular hmgb can act as a cytokine and a chemoattractant, mediating inflammatory responses. interestingly, hmgb exerts antibacterial functions in human adenoid and testis ( ) . recent investigations have revealed that neutrophils eliminate microbes not only by intracellular phagocytosis but also by trapping them in three-dimensional structures called neutrophil extracelluar traps (nets), made of dna fibers, nuclear proteins (histones) and granule proteins. it has been shown that histones on nets have an anti-microbial activity ( ). we asked whether hmgb from neutrophils is a component of nets and whether it has a function in nets. we purified human primary neutrophils from peripheral blood of healthy volunteers on ficoll gradients. to induce net formation, we stimulated cells for or minutes with nm phorbol ester (pma), ng/ml interleukin (il- ), or ng/ml lps. the presence of nets was assessed by immunofluorescence using antibodies directed against the granule protein myeloperoxidase (mpo) and against a dna-histone h a-histone h b complex. dna was stained with hoechst. using a polyclonal antibody we found hmgb in the euchromatin of polylobulated nuclei of resting neutrophils and on the filamentous structure of nets induced by all stimuli. elisa assays revealed that hmgb is not present in the supernatants of activated neutrophils, confirming its binding to nets. in conclusion, we found that hmgb localizes on nets. we hypothesize that net-bound hmgb might exert a direct antimicrobial function, or that nets might concentrate hmgb locally to recruit macrophages to the site of infection. these receptors were present on the mast cell surface. incubation ( °c, h) of hlmc with vegf-a, vegf-b, vegf-c, vegf-d and placental growth factor- induced concentration-dependent chemotaxis that was blocked by a combination of anti-vegfr- and anti-vegfr- antibodies. these data indicate that human mast cells represent both a source and a target of vegfs and therefore may play a role in inflammatory and neoplastic angiogenesis through the expression of proangiogenic factors and their receptors. macrophages are important effector cells in immunity to intracellular pathogens and at the same time are exploited as host cells by a number of microorganisms such as mycobacterium tuberculosis. a very important mechanism of intracellular killing is delivery of invading microbes to phagolysosomes. whilst mycobacteria can block phagosome maturation in resting macrophages, and hence survive and replicate inside the host cell, the ifn-g activated macrophage utilizes a diversity of defense mechanisms to eliminate the invader. these include putative killing by antibacterial peptides/proteins and overcoming phagosome maturation block, possibly by induction of autophagy, production of reactive nitrogen or oxygen intermediates and deprivation from nutrients such as iron. mycobacteria are not eliminated even upon onset of protective immunity rather leading to persistence. we hypothesize that the very early steps of pulmonary infection directs the outcome of disease. therefore, we investigate initially infected lung cells and their role in infection in the lung with respect to their anti-microbial mechanisms against mycobacteria in vitro as well as in vivo. preliminary data show that m. tuberculosis is able to persist in the alveolar space for several weeks and bacterial numbers do barely drop even after very low dose infection, indication that bacterial killing is inefficient from the very beginning. cells harboring mycobacteria are found during early and late stages of infection. both, autophagy and nitric oxide production seems to contribute to growth restriction of mycobacteria by macrophages. neutrophils, although recruited in vast numbers to infected lungs, are not able to reduce bacterial numbers in the absence of il- . altogether, the initial response in the barrier organ lung executed by resident and immigrating cells restricted by the local environment can determine the outcome of infection. human cd molecules are dedicated to lipid presentation to t cells and are implicated in inflammatory and auto-immune responses. the cd a protein is almost exclusively expressed at the cell surface of dendritic cells and is dedicated in surveying extracellular environment. our previous studies have demonstrated that ii associated with cd a and cholesterol-dependent lipid rafts impact on cd a surface expression and cd a-restricted t cell response. bacterial infections can induce an increase in self glycolipid synthesis in dendritic cells and such activated dcs acquire the ability to stimulate cd -restricted autoreactive t cells. this mechanism of self recognition induced by bacterial infection is believed to be involved in the development of auto-immune disorders. sulfatide, which is a major component of the myelin sheath, is also the only known self-antigen presented by cd group i molecules. the functional role of these molecules has not been investigated in auto-immune diseases and we propose that regulation of glycolipid presentation by cd a molecules could impact in such pathologies. we have thus conducted a preliminary study to understand the implication of cd molecules in multiple sclerosis. we first analyzed cd expression on monocytes from ms patients and the influence of sera and plasma from these patients on dendritic cell differentiation from healthy donors. results obtained in this preliminary study demonstrate that cd a was not expressed on ms patient monocytes, while the other members of the cd family were expressed. moreover ms sera and plasma induced an earlier and more rapid dendritic cell differentiation than ab sera. these preliminary results confirm our hypothesis that cd molecule expression is modified in ms and also reveal that serum from patients with ms modifies lipid-antigen presenting cells. further studies should contribute to define precise mechanisms involved in lipid presentation by cd molecules in this context. c. ohnmacht , d. voehringer ludwig-maximilians-universität munich, institute for immunology, munich, germany basophils are effector cells of the innate immune system which are associated with allergic inflammation and infections with helminth parasites. however, their development and in vivo functions are largely unknown. here, we characterize basophil turnover, tissue localization and effector functions during infection with the gastrointestinal helminth nippostrongylus brasiliensis. for this purpose, brdu incorporation experiments and in situ fluorescence microscopy of il- reporter ( get) mice as well as in vivo depletion of basophils are used to uncover their role during type immune responses. our results demonstrate that under homeostatic conditions basophils have a lifespan of about h. n. brasiliensis induced basophilia is caused by increased de novo production of basophils in the bone marrow. basophils are found near the marginal zone in the red pulp of the spleen, in the lamina propria of the small intestine and in the lung parenchyma. activated basophils promote systemic eosinophilia, were associated with differentiation of alternatively activated macrophages in the lung and contributed to efficient worm expulsion of n. brasiliensis in the absence of th cells. these results demonstrate that basophils play a crucial role as effector cells in type immune responses which might hold great potential for the treatment of helminth infections and allergic diseases. during acute bacterial infections such as meningitis, neutrophils enter the tissue where they combat the infection before they undergo apoptosis and are taken up by macrophages. neutrophils show pro-inflammatory activity and may contribute to tissue damage. in pneumococcal meningitis, neuronal damage despite adequate chemotherapy is a frequent clinical finding. this damage may be due to excessive neutrophil activity. we here show that transgenic expression of bcl- in haematopoietic cells blocks the resolution of inflammation following antibiotic therapy in a mouse model of pneumococcal meningitis. the persistence of neutrophil brain infiltrates was accompanied by high levels of il- beta and g-csf as well as reduced levels of anti-inflammatory tgf-beta. significantly, bcl- -transgenic mice developed more severe disease that was dependent on neutrophils, characterized by pronounced vasogenic edema, vasculitis, brain haemorrhages and higher clinical scores. in vitro analysis of neutrophils demonstrated that apoptosis inhibition completely preserves neutrophil effector function and prevents internalization by macrophages. the inhibitor of cyclin-dependent kinases, roscovitine induced apoptosis in neutrophils in vitro and in vivo. in wild type mice treated with antibiotics, roscovitine significantly improved the resolution of the inflammation after pneumococcal infection and accelerated recovery. these results indicate that apoptosis is essential to turn off activated neutrophils and show that inflammatory activity and disease severity in a pyogenic infection can be modulated by targeting the apoptotic pathway in neutrophils. objectives: to investigate the existence of systemic inflammatory response to subchronic oral warfarin (wf) consumation in rats. methods: dark agouti (da) rats were treated with warfarin in drinking water ( mg and mg daily) for days. oxidative activity (cytochemical nbt reduction) and myeloperoxidase (mpo) intracellular content of peripheral blood neutrophils, plasma levels of il- and tnf-a (elisa) and superoxide dismutase (sod) activity (red blood cell lysates) were analyzed as inflammatory parameters in rats following warfarin consumation. changes in prothrombin time (pt), as basic biological warfarin activity was determined as well. results: significantly increased pt was noted at the lower wf dose, with tremendous rise after the higher dose. increase of pma-stimulated neutrophil nbt reduction capacity (neutrophil priming) was noted at both wf doses, while increase in mpo intracellular content was noted at the higher wf dose solely. warfarin consumation resulted in no changes in plasma levels of il- and tnf-a. significant decrease in the sod activity was detected in red blood cell lysates at both wf doses, suggesting systemic oxidative activity. conclusion: increased neutrophil priming as well as prooxidant activity in peripheral blood of rats following subchronic warfarin consumation imply proinflammatory effects of oral warfarin administration. absence of the rise in inflammatory cytokines in circulation, suggest low-grade inflammation in these rats. this work is funded by serbian ministry of science and technological development (grant ). objectives: although many different macrophage receptors and serum proteins have been shown to play a role in phagocytosis of apoptotic cells, the unique microenvironment of an inflammatory site will have considerable influence upon the molecular pathways which are utilized in apoptotic cell removal. we have recently reported that immune complexes (ic) are able to specifically bind to the surface of human apoptotic neutrophils which may have profound implications for their physiological clearance. in disease situations where immune complexes are present neutrophils undergoing apoptosis would be predicted to become coated with ic. here we address the consequences of ic opsonisation of apoptotic cells upon phagocytosis and cytokine response by macrophages that would be expected to be present at the earliest stages of inflammatory responses (type- macrophages, mph ), and during resolution of inflammation (type- macrophages, mph ). methods: mph / were generated by culturing cd + human monocytes for days in the presence of gm-csf or m-csf, respectively. phagocytosis by mph / of ic opsonised and unopsonised neutrophils was assessed by flow cytometry. after phagocytosis mph / were stimulated with lps and secreted il- , il- , il- , il- p and tnf were quantified by elisa. results: mph are relatively efficient phagocytes for apoptotic neutrophils whereas mph are only poorly phagocytic. opsonisation with ic leads to enhanced neutrophil uptake by both mph and mph which is specifically inhibited in the presence of a blocking mab for macrophage fcyrii. uptake of ic opsonised neutrophils causes a shift towards an anti-inflammatory cytokine profile. in both macrophage subsets il- , il- and tnf production is suppressed while il- secretion is increased. in contrast, engagement of macrophage fcyr with ic alone induces the release of pro-inflammatory cytokines. conclusion: our data demonstrate that ic opsonisation of apoptotic neutrophils increases the proportion of macrophages capable of phagocytosis and that apoptotic cell recognition interactions provide a dominant anti-inflammatory signal, suppressing macrophage responses, even in the presence of ic opsonisation. we suggest that ic present in the inflammatory milieu would opsonise apoptotic neutrophils, enhance macrophage phagocytosis and thereby facilitate the process of resolution of inflammation. excessive production of reactive oxygen species (ros) produced by neutrophils is known to be a factor accelerating ageing because of damaging effect on cells. on the other hand, intracellular heat shock proteins (hsp) are involved in protecting cells from the damaging effects, and provide cell resistance to stress. in this work, correlation analysis was applied to analyze relationship between ros production and intracellular hsp in neutrophils of elderly people. neutrophils were isolated from peripheral blood of donors of years old and older (long-livers). intracellular ros and hsp levels were registered by flow cytofluorimetry upon labeling with ', '-dichlorofluorescin diacetate (invitrogen) and anti-hsp antibody (brm- , sigma), respectively. intracellular level of hsp was also estimated in neutrophils after heat shock (hs) performed at °c for min. extracellular ros production from zymosan-activated neutrophils was detected by luminol-dependent chemiluminescence. a positive correlation was determined for intracellular ros level and zymosan-mediated extracellular ros release although the dynamics of ros release at - min time range varied within the group. the correlation was unaffected by hs of neutrophils performed for min at °c, although this short heat treatment decreased significantly ros release. there was no correlation between basal intracellular hsp (hsp basal ) and ros level, both intracellular and extracellular. at the same time increased hsp level immediately after hs (hsp ( min)) correlated negatively with intracellular ros (initial and after hs). the hsp increase value (hsp ( min) -hsp basal ) correlated negatively also with intracellular ros and extracellular ros release in response to zymosan; and the correlation with ros level became lower when hsp increase was registered in min after hs (hsp ( min) -hsp basal ). thus it was found that within this age group the alteration in hsp induced by hs in neutrophils but not basal hsp itself is the parameter associated negatively with both spontaneous ros level and ros production in response to activating action of zymosan. this work is supported by istc grant # . d. goyeneche-patiño , z. orinska , f. mirghomizadeh , s. bulfone-paus forschungszentrum borstel, borstel, germany several studies have shown different roles of mast cells (mc) in innate and adaptative immune responses. in fact, crosstalk between cd + t cells and mc has shown to induce multiple genes implicated in the signaling of specific programs such as type ifn. two novel genes, receptor transporter protein (rtp ) and virus inhibitory protein, endoplasmic reticulum-associated, interferon-inducible (viperin) are ifn inducible and were found to be over-expressed in chip analysis. the aim of this study is to characterize the expression and protein production of rtp and viperin in mast cells after tlr ligand stimulation in mice lacking of ifnra and the adapter proteins myd and trif. bone marrow derived mast cells (bmmc) from wt, ifnra -/-, mydd -/and trif -/mice, were exposed to tlr ligands (lps, pic, cpg, p(da-dt) and new castle disease virus (ndv)) during and h. mrna and protein extraction were performed for further qrt-pcr and sds page analysis for rtp and viperin. intracellular stimulation of tlr was performed transfecting cells with nucleic acids using lipofectamine . stimulation of wt cells with pic, pda-dt and ndv showed an increased expression of viperin and rtp in comparison to control cells (untreated). the same trend was observed for mc from the trif and myd knockout mice. in contrast, in the ifnra deficient mice, expression of genes and protein production was abrogated to the same levels of wt untreated cells. lipofectamine stimulation does not increase the expression/production of the genes. direct stimulation of the well recognized viral sensors tlr and , as well as, infection of mast cells with ndv (rna virus) induce the expression of rtp and viperin. the findings suggest that activation of mc with the ulterior expression of genes is type i ifn dependent. in contrast, the adaptor proteins myd and trif and the pathways that they represent are not relevant in the expression of rtp and viperin. these findings provide bases for performing further studies focused to elucidate the functions of these proteins and show an alternative role of mc in innate immune responses. in recent years, it has been suggested that the phenomenon of "myc-dependent cell competition" described in drosophila melanogaster, could be a critical step when a cell initiates nascent tumour field. we have taken a step forward and applied the phenomenon of cellular competition to the human macrophage system: inflammatory macrophages theoretically have the ability to eradicate cancer due to their tumoricidal capability and, at the same time, acting as antigen-presenting cells (apcs) to activate lymphocytes; but inflammatory macrophages do not express c-myc and, within the tumour, they encounter two powerful rivals: tumoral cells and alternative tumour-associated macrophages which express c-myc. we studied some phenomenons suggested to be myc-dependent such as the ability to feed, the ability to survive in a competitive medium, the ability to proliferate and the ability to eliminate competitors and we observed that alternative macrophages have more resources to survive in a tumoral microenvironment and could be involved in tumour growth by collaborating with tumour cells in transforming inflammatory macrophages into anergic cells which enter into apoptosis and are then phagocyted. finally, using lentiviral vectors, we over-expressed exogenous c-myc in inflammatory macrophages in an attempt to increase their chances of survival in the tumour microenvironment, in vitro and in vivo, and to determine whether it can be utilized as a potential anti-tumoral cell therapy. g. germano , e. erba , r. frapolli , m. d'incalci , a. anselmo , s. pesce , p. allavena , a. mantovani , humanitas clinical institute, rozzano, italy, mario negri institute, milan, italy, institute of general pathology, university of milan, milan, italy several lines of evidence suggest a strong association between chronic inflammation and tumor progression; therefore the use of anti-inflammatory drugs may be beneficial in anti-tumor therapies. inflammatory mediators (e. g. cytokines, chemokines) are produced at the tumor site both by tumor-associated macrophages (tam) as well as tumor cells, and are attractive target of novel anti-tumor therapies. trabectedin (et- ) is a natural product derived from the marine tunicate ectenascidia turbinate, it binds the minor groove of dna, affects transcriptional factor activity and blocks cell cycle. this novel anti-tumor agent is currently used in phase ii studies in patients with sarcoma, ovarian and breast cancer, with clinical regressions. we previously demonstrated that trabectedin is selectively cytotoxic, in vitro, to monocytes/macrophages, being active at concentrations that spared lymphocytes suggesting a possible alternative target for the anti-tumoral role of this drug. we tested the effect of trabectedin on primary cultures and liposarcoma cell lines showing that at sub-cytotoxic concentrations the production of some inflammatory mediators were down-modulated. trabectedin significantly reduces ccl , cxcl and the inflammatory protein pentraxin (ptx ) either at transcriptional and protein level, especially after tnfa/il b stimulation. down-regulation of ccl , cxcl , ptx and also of il and vegf were confirmed in primary cultures of liposarcoma. according to the previous in vitro data we now show in a mouse model , using the fibrosarcoma mnmcai , that trabectedin treatment selectively affects monocytes in the blood and bone marrow. moreover trabectedin treatment strongly reduces the number of macrophages and of cd + vessels in the tumor microenvironment, in line with its selective activity on monocytes/macrophages. overall these results suggest a possible triple role of trabectedin. besides its direct cytotoxic effect on tumor cells, trabectedin also affects tumor associated macrophages and at low dose the transcriptional activity of inflammatory genes involved in the tumor-microenvironment cross-talk. as the local expression of inflammatory mediators may play a role in tumor progression, this newly recognized effect of trabectedin makes it an attractive candidate in inflammation-associated tumors. interleukin- (il- ) is a key cytokine of the t helper cell response. il- has been found to be a major regulator of immunoglobulin class switching to ige and has important functions in the regulation of allergic diseases. here, the onset of the il- production after birth was investigated in equine neonates. the form of equine placentation does not support the transfer of cytokines or immunoglobulins in utero and maternal immunity is exclusively transferred to the neonate with the colostrum after birth. il- producing cells were measured in peripheral blood mononuclear cells (pbmc) of neonates and foals by flow cytometric analysis. at day - after birth, a small population of il- producing cells was observed in the absence of any stimuli. the il- + population was not detectable at or weeks of age. other cytokine producing cells (ifn-g, il- ) were not detected using these conditions. the stimulation of neonatal pbmc with pma and ionomycin did not alter the il- + cell population. phenotyping of the neonatal il- + cells showed that they were ige + /mhcii -/cd cells. the occurrence of cd + il- producing cells after pma stimulation increased slowly with age and did not reach adult levels by weeks after birth. magnetic cell sorting of the ige + /mhciicells identified them as basophils. previous work has shown that foals do not produce endogenous ige for at least six months of life. ige bound to the surface of neonatal basophils was found to be of maternal origin and transferred with the colostrum after birth. here, the stimulation of neonatal pbmc with anti-ige induced the secretion of il- at day after birth. neonatal pbmc collected before colostrum uptake did not produce il- in response to anti-ige. in summary, equine neonates provide a model to investigate ige mediated il- responses after birth. the transfer of maternal ige from allergic individuals could potentially provide a direct mechanism for the early induction of an allergen-specific neonatal il- response mediated by the mare's accumulated acquired immunity to allergens. s. schmechel , d. voehringer ludwig-maximilians-university munich, institute for immunology, munich, germany macrophages display broad phenotypic heterogeneity depending on their microenvironment. the initial inflammatory response to th cytokines is predominantly mediated by classically activated macrophages whereas macrophages undergo alternative activation in a stat -dependent manner when stimulated with the th cytokines il- or il- . alternatively activated macrophages (aam) are implicated in diverse disease pathologies such as host response to parasitic infection and asthma. furthermore, it has been shown that aam suppress the proliferation of t cells by a yet to be determined mechanism. currently there is still very limited information about the phenotype, migration and function of aam. we began to elucidate whether macrophage turnover and recruitment to inflammatory sites is regulated in a stat -dependent manner. to this end we generated mixed bone marrow chimeras with bone marrow from congenic wild-type and stat -deficient mice and infected these chimeras with the helminth nippostrongylus brasiliensis to determine whether lack of stat in macrophages affects their turnover and recruitment to the lung side-by-side in the same animal. the highest turnover of macrophages was found in the peritoneum, irrespective of stat expression. no major differences in tissue distribution and turnover were observed between both populations suggesting that macrophage proliferation and recruitment during parasite infection is not dependent on stat expression in macrophages. we could further confirm that in vitro generated aam from wild-type but not from stat -deficient mice have a strong inhibitory effect on t cell proliferation. we are now trying to identify the mechanism(s) by which t cell proliferation is inhibited. furthermore, we work on the cellular cross-talk between eosinophils and macrophages and try to determine the plasticity of macrophage differentiation. we have previously shown that aam can recruit eosinophils to inflammatory sites and we now try to clarify which chemotactic factor are involved in this process. to identify potential aam-derived eosinophil chemotactic factors we currently compare the gene expression profile of il- exposed macrophages from wild-type and stat -deficient mice. candidate genes will be expressed using retroviral transfections of stat -deficient macrophages and supernatants from these cells will then be used to induce eosinophil recruitment in transwell assays. macrophages are an essential component of leukocytes infiltration in the tumor. they are identified as tumor associated macrophages (tams). these cells are also present in pleural effusions which appear as a consequence of spreading of neoplasm in the pleural cavity. the aim of the study was to assess the influence of the pleural macrophages on cells from human malignant cell lines. we tested the dynamics of growth of the malignant cells, their apoptosis and expression of proteins regulating this process under the influence of conditioned media from cultures of macrophages isolated from pleural effusion. we have also attempted to interpret our results by assessing the expression of a variety of immune modulating factors, their receptors on the malignant cells surface as well as the transcription factors. in the study we used macrophages isolated from a total of pleural effusions, including malignant and nonmalignant tumors. the following human malignant cell lines were tested: a , ht , hct , sw , mcf , mda-mb , jurkat and hl . results: our results suggest that the conditioned media isolated from the cultures of pleural macrophages can up-regulate the proliferative activity of the human malignant cell lines. macrophages from pleural effusions can act as a factor promoting or inhibiting apoptosis of malignant cells. down-regulation of apoptosis may depend on modulation of expression and activity of proteins regulating this process. macrophages can affect the apoptosis regulatory proteins and their activity through the immune-modulatory molecules, e. g. cytokines, chemokines, and growth factors. the up-or down-regulation of transcription factors expression may control the expression of pro-and anti-apoptotic proteins. the results indicate that macrophages from malignant and non-malignant pleural effusions differ from each other insignificantly; however the macrophages isolated from the non-malignant tumors show a pattern comparable to m , and the tams isolated from the malignant effusions similar to m . among the alternative stimuli, glucocorticoids are the most effective stimulus up-regulating ms a a and ms a a: highest trascriptional level after h of stimulation with - m dexamethasone. ms a murine genes are differently expressed respect to the human counterpart and only the homologs of ms a a (ms a b, c and d) have a similar regulation. finally, egfp-tagged ms a a, ms a a, and ms a expressed in cho cells showed that all molecules traffic to the cell membrane. though the biological functions of these ms a proteins has not jet been defined, their membrane localization and the structural relationship with other better characterized ms a members suggest a potential involvement in signal transduction, either as components of multimeric receptor complexes or as components of ligand-gated ion channels. during inflammatory reactions endogenously produced cytokines and chemokines act in a network and interact with hormones and neurotransmittors to regulate host immune responses. these signaling circuitries are even more interfaced during infections in which microbial agonists activate toll-like (tlr), rig-like (rlr) and nod-like (nlr) receptors. on the basis of the discovery of synergy between chemokines for neutrophil attraction, we here extended this phenomenon between the chemokine monocyte chemotactic protein- (mcp- )/ccl and the gpcr ligand fmlp or the tlr agonist lipopolysaccharide (lps) on monocytes. in fact, the bacterial tripeptide fmlp, but not the cytokines il- b or ifn-g, significantly and dose-dependently synergized with ccl in monocyte chemotaxis. furthermore, lps rapidly induced the expression of interleukin- /cxcl , but not of the ccl receptor ccr in monocytic cells. in turn, the induced cxcl synergized with ccl for mononuclear cell chemotaxis and the chemotactic effect was mediated by cxcr /cxcr , because cxcl receptor antagonists or antibodies were capable of blocking the synergy, while keeping the responsiveness to ccl intact. these data recapitulate in vitro the complexity of innate immune regulation, provide a novel mechanism of enhancing monocyte chemotaxis during bacterial infections with gram-negative bacteria and demonstrate the importance of local contexts in inflammatory and infectious insults. objectives: in recent years the existence and effects of cell-derived vesicles (e. g. exosomes, microparticles) have been revealed in several physiological functions, such as antigen presentation, hemostasis or receptor transfer to innocent cells. most data were collected on endothelial cells and on thrombocytes. however, there are only few data on vesicles derived of neutrophilic granulocytes (pmn), and most of these investigations applied only pharmacological agents. our aim is to investigate pmn-derived cell-free particles and their possible role in bacterial killing methods: preparation of pmn and investigation of bacterial killing by our semi-automatic method was described by rada et al. (blood, ) . cell-free vesicles were prepared after co-incubation of human pmns with different activating agents for min at °c with gentle shaking, followed by spinning down of pmns for min, at °c and g. the supernatant was sedimented at g for min, °c, and we used the sedimented fraction for our investigations. formation of particles was followed by fluorescent and electron microscopic assays. the amount of particles was estimated with flow cytometer and by their protein content. we observed that upon co-incubation of pmns with s. aureus, opsonized by mixed human serum, pmns produce a well detectable amount of vesicles. omitting opsonization or opsonizing with heat inactivated serum caused a minimal amount of particles. production of particles could be inhibited with diphenyl-iodonium (dpi), cytochalasin b (cb) or with azide treatment. treating pmns with dnase or withdrawing glucose during co-incubation had no effect on vesicle formation. in killing assays we detected remarkable antibacterial effect, which correlated well with the protein content of the used fraction. this antibacterial activity could be inhibited by dpi, cb, azide treatment or by withdrawing glucose from the medium during the killing assay. however, treatment of the microvesicles with dnase had no effect on their antibacterial capacity. for long, cd has been used for the detection and identification of natural killer (nk) cells. recently, the presence of a minor subset of cd low cd + blood monocytes (mo) in healthy individuals and the increase in cd +cd + blood mo in patients with inflammation has been reported. the functional activity of human cd + blood mo has been studied in vitro but not tested ex vivo so far. healthy people living permanently in malaria endemic areas are exposed to plasmodium infection, and we hypothesized that blood mo of these individuals could be activated and display increased cd expression. we tested if this phenotypic expression was associated with detectable changes in the mo anti-parasitic activity. the mo phenotype of healthy malaria naï ve and malaria exposed individuals was determined by three-color flow cytometry. myeloid cell markers included cd and activation markers such as hla-dr and trem- . percentages of blood mo involved in phagocytosis activity either with or without immune sera were then identified by flowcytometry, and the potential association between a given mo phenotype and phagocytosis activity was then looked for, using spss ® and statview ® softwares. our results showed that, compared with malaria naï ve individuals, there was a . fold increase (p x . ) in the total number of circulating cd low mo present in the blood samples of healthy malaria exposed asian individuals living in thailand. according to the density of surface antigen determined by fluorescence intensity (fi), the decrease in cd and the concomitant increase in hla-dr expressions indicated that in this malaria endemic area, blood mo were mature and highly activated by comparison with surface markers of mo from malaria naï ve donors. the relative levels of cd + blood mo were associated with the percentages of membrane-bound ifn-g present at the mo surface. in conclusion, (i)-a subset of cd + blood mo expressed increased levels of cd on mo of healthy malaria exposed individuals; (ii)-blood mo with activated (hla-dr+) and mature (trem- +) phenotypes were present in these healthy individuals; (iii)-increased expression of hla-dr and cd on cd high mo was associated with a high phagocytosis activity. introduction: adipokines, initially described for their function within metabolism, have been characterized to exert a regulatory role on the immune response. for instance the appetite-regulating hormone leptin has been identified to modulate the response of the innate as well as the acquired immune system. the present work focuses on the effects of leptin on the reactivity of m -and m -polarized human macrophages. methods: monocytes were isolated from the peripheral blood by magnetic cell sorting. polarization to m and m macrophages was induced by culture in the presence of mcsf or gmcsf respectively. polarized cells were characterized by flow cytometry, stimulated with lps and response assessed by characterization of cytokine profiles via cytometric bead array (cba). results: culture of monocytes in the presence of mcsf or gmcsf induced two different phenotypes. cells cultured in the presence of gmcsf represented the m type and were cd negative but cd and mhcii positive and produced high levels of il- , tnfalpha and il- following lps stimulation. culture in the presence of mcsf resulted in induction of the m phenotype. these cells were cd positive with intermediate expression of cd and mhcii expression and produced high levels of il- , il- and il- following lps stimulation. interestingly, already baseline il- production was high in these cells. stimulation with leptin alone increased cytokine production in both cell types as compared to cells cultured in medium alone. however, if leptin was present in cultures stimulated with lps, the induction of cytokine production was significantly reduced in both, m -and m -polarized cells as compared to cells stimulated with lps alone. summary: whereas presence of leptin enhances baseline cytokine production in polarized macrophages, it reduces the cytokine production in response to stimulation with the tlr ligand lps. thus, abundant leptin levels like present in obesity or in the hypertrophied fat as present in crohn's disease patients might exert modulating effects on macrophage response to bacterial antigens. methods: hl cell line was used as a model of leukemic myeloid cell differentiation cultured in suspension or on fibronectin matrix prior to pma ( ng/ml) treatment for h. morphological evaluation was performed with conventional microscopy and electron microscopy. immunephenotype and phagocytic activity of the cells were determined by flow cytometry and immunocytochemistry. a colorimetric nitro-blue-tetrazolium reduction assay was performed to assess the production of reactive oxygen species (ros). results : besides their distinctive macrophage morphology and ultrastructure with spindle cell-like features and high granularity, the pma-treated fibronectinadherent hl cells expressed antigen receptors cd , tlr , tlr and cd , and displayed enhanced phagocytic activity and production of ros. expression of cd , cd and cd was also maintained however the cells were hla-dr and cd a negative. conclusion: we describe the enhanced ability of fibronectin-adherent hl cells to differentiate into macrophages in response to pma. hl may provide a functional model for macrophage differentiation. above all, this finding may stimulate further research on myeloid leukemia biology and potential adjuvant therapies. a. aporta , n. ferrer , a. gómez , j. gonzalo , a. arbués , a. anel , c. martín , j. pardo , apoptosis, immunity and cancer university of zaragoza, molecular and cellular biochemistry and biology, zaragoza, spain, university of zaragoza, mycobacterium genetics, zaragoza, spain mycobacterium tuberculosis is an intracellular pathogen that uses alveolar macrophages as its preferred habitat, being capable of produce both a progressive disease and an asymptomatic latent infection. it has been postulated that infected macrophage apoptosis may contribute to host defence against this intracellular infection by, firstly, eliminating supportive environment for bacterial growth and, secondly, by leading to the formation and release of apoptotic vesicles containing mycobacterial antigens. it has been proposed that m. tuberculosis inhibits host cell apoptosis thus interfering with the immune system response. however the biological relevance of this process is not clear. our group has generated so , a m. tuberculosis phop mutant strain that was shown (perez et al ) to be more attenuated than the present attenuated vaccine strain bcg and conferred protective immunity against m. tuberculosis infection in mice and guinea pigs (martin et al ) . in the present study, we compare the time course and phenotype of cell death induced by so , bcg and wild type m. tuberculosis on the murine macrophage cell line j and on bone marrow derived mouse macrophages. our results indicate that wild type m. tuberculosis induces macrophage cell death analysed by a clonogeneic assay much faster than the attenuated bacteria. of note cell death presented apoptotic features like caspase- activity and nuclear condensation. in order to analyse the consequences of this apoptotis-like cell death, it has been invetigated whether dead cells translocate phosphatydilserine to the outer part of the plasma membrane and if this traslocation is enough to promote phagocytosis by fresh macrophages. experiments are ongoing with macrophages derived from trl x deficient mice and wt animals in order to study the role and implication of those receptor on the susceptibility to infection and death induced by the virulent and attenuated phop m. tuberculosis strain. objectives: vip is a potent anti-inflammatory peptide, mainly acting as endogenous macrophage deactivating factor. type receptor for vip (vipr ) gene is highly conserved through species and, in humans, is highly polymorphic. vipr has been reported to be down-modulated in cells of the immune system after activation. an association of some snps with some autoimmune diseases has also been reported. in this study we have investigated the correlation between these snps and gene expression in monocytes exposed to lps. methods: monocytes from blood donors were separated from pbmc and stimulated with lps. total rna was reverse transcribed and the level of vipr in untreated or lps-stimulated monocytes was measured by real-time rt-pcr and protein expression. protein level was measured by western blot and densitometric analysis. the kinetic of expression of vipr after , , and h of exposure to lps was firstly analysed in monocytes from five individuals. there were two kinetics: one in which a reasonable high levels ( g %) of mrna was maintained trough time and a second one in which the decrease of mrna was pronounced. the experiments were repeated using monocytes from donors that had been typed for the relevant vipr snps. the down-regulation of vipr correlates with the presence of a t at rs mapping in the '-end of the gene (p= . ). the vipr protein level was decreased about % in monocytes of subjects typed as t/t at rs whereas subjects typed as c/c at rs maintained a high level of expression after h of lps treatment. the data show that different haplotypes of the vipr gene correlate with a different kinetics of gene expression in activated monocytes. a possible consequence of these data is that the anti-inflammatory properties of vip governed by the vipr vary in different individuals and can eventually contribute to the genetic predisposition to some autoimmune diseases. j. oujezdska , t. vavrochova , d. filipp , immunobiology institute of molecular genetics as cr, immunobiology, prague, czech republic phagocytes which appear in early mouse development (e . - . ) represent a unique embryonic macrophage lineage that differs from adult macrophages phenotypically, biochemically and by their origin. recent studies suggested that there are at least three waves of macrophages populating an early embryo: a maternallyderived one and two waves of extraembryonal, ys-derived phagocytes. in addition, the occurence of early embryonic phagocytes of undetermined origin in the anterior head mesoderm in several invertebrate and vertebrate species is well documented. this origin-related heterogeneity among early embryonic phagocyte subpopulations coupled with the lack of their specific surface markers makes it difficult to distinguish them phenotypically and study their potentially distinct physiological roles in early development. the aim of this study is to identify a set of surface markers expressed on embryonal phagocytes suitable for phenotypic distinction among embryonic phagocyte subpopulations. here we report the temporal and spatial expression of toll-like receptors (tlrs) and cd in the early mouse embryo (me). facs analysis of cell suspension prepared from . day me showed that about . - % of cells were positive for cd b. these cells exclusively were also positive for cd , tlr , and cd antigens. using qpcr and flow cytometry we show that tlrs and other tir domain-containing signaling molecules are expressed in the embryo through embryonic days , - , . reciprocal matings between wild type and mhcii-egfp knock-in mice revealed that while maternallyderived mhcii + macrophages are present in the embryo from early developmental stages (e , ), embryo-derived mhcii + macrophages start to appear in the embryo around day . multicolor facs analysis of cd b, cd , cd , f / , tlr , tlr , c-kit and mhcii surface markers revealed differential expression of tlr and c-kit on embryonal phagocyte subpopulations. moreover, the microarray analysis of cd b + tlr + cells isolated from the e , embryos has revealed significantly upregulated expression of several novel genes in comparison to their expression in murine peritoneal macrophages. these molecules are currently being tested for their use as embryonic phagocyte specific-lineage markers. these results are first to characterize the regulated expression of tlrs on early embryonal phagocytes and demonstate their potential to serve as novel markers for their detection and isolation. humans may be exposed to a variety of mycobacteria ranging from environmental or bcg vaccine to more pathogenic mycobacteria. only a minority of individuals exposed develop disease, this susceptibility may result in part from variability of host immune responses genes through simple (mendelien disease) and complex (polymorphisms with milder effect) inheritance mechanisms. interestingly, key elements of inflammatory pathways are particularly involved in this susceptibility to mycobacteria. il /il -dependent ifng pathway of macrophage activation plays a central role in inflammation and cell-mediated immune responses to mycobacteria. due to the high rate of consanguineous marriages in the north african countries, recessive genetic disorders including primary immunodeficiencies occur with a relatively high prevalence. in tunisia, among patients affected with primary immunodeficiencies presented with disseminated bcg infection (bcg-osis). among them, five have an underlying well-defined primary immunodeficiency either a severe combined immunodeficiency or a chronic granulomatous disease and have a mendelien susceptibility to mycobacterial disease. using a candidate gene strategy, we have identified in out of these patients mutations in several ifng pathway genes, other candidate genes are being investigated for the other patients. in the general population, common polymorphisms with milder effect on the risk of tuberculosis have been identified including mhc and nramp . we did focus on the study of genes which are considered as important pathogen recognition receptors of the innate immune system: tlr is the principal mediator of macrophage activation in response to mycobacteria through nfkb pro-inflammatory signaling pathway and dc-sign is the major receptor of m. tuberculosis on human dendritic cells and in contrast induces anti-inflammatory il- cytokine. using a case/household-contact cohort we did investigate polymorphisms of these genes in tunisian patients affected with active pulmonary tuberculosis and have shown specific patterns of snp and microsatellite polymorphisms associated with susceptibility/resistance to tuberculosis. host inflammatory responses play a major role in granuloma formation and control of the infection. unraveling these pathways might be crucial in order to identify new therapeutic targets and strategies including immunotherapy e. g. ifng therapy for tuberculosis, particularly in this era of emergence of multi-drug and extensively-drug resistant m. tuberculosis strains. francisella tularensis is a gram negative bacterium that is the causative agent of tularemia. research into francisella has expanded over recent years due to its designation as a potential biological warfare agent. several species of francisella exist and have varying degrees of pathogenicity. f. tularensis live vaccine strain (lvs) is an attenuated strain of the holarctica subspecies and has been shown to be an effective vaccine in humans. however, it is pathogenic in mice which can, therefore, act as a useful model of human tularemia. f. tularensis is an intracellular pathogen and is able to invade several different cell types, in particular macrophages, most commonly through phagocytosis. therefore, if phagocytosis could be disrupted via the addition of inhibitors, uptake of f. tularensis would decrease and antibiotic treatment may be more effective. a flow cytometric assay was developed to measure bacterial uptake. this method used a fitc labelled anti-f. tularensis antibody in conjunction with antibodies to cell surface markers to determine specific cell phenotypes that were positive for bacteria. a series of phagocytic inhibitors have been tested in vitro on an alveolar macrophage derived cell line (mhs) and on ex-vivo mouse lung tissue to determine whether uptake of f. tularensis lvs could be altered. the presented data shows that several inhibitors work efficiently to reduce lvs uptake by up to - % in both the in vitro and ex vivo assays. however, cytotoxicity of some of the inhibitors was high and, therefore, it was essential to concentrate on inhibitors with low cytotoxicity for further assessment. in addition, bacteriological data suggests that the combination of inhibitors with antibiotics may be a useful therapeutic against f. tularensis. it may also work against other intracellular pathogens that use phagocytic mechanisms to enter their optimal niche.ã crown copyright. dstl, . hsp are intracellular proteins but it is known that these proteins can be expressed on cell surface and contained in extracellular medium, in particular in peripheral blood serum. it is also known that extracellular hsp have pronounced immunomodulatory properties. to study the pathways of the protein modulating action on immune system we investigated effect of exogenous and cell surface hsp on reactive oxygen species (ros) release from phagocytes, namely human neutrophils, during process of phagocytosis (respiratory burst). neutrophils were isolated from human peripheral blood by using a standard protocol. respiratory burst induced by opsonized zymosan was measured by method of luminol dependent chemiluminescence. for the experiments human recombinant hsp (low endotoxin) and paraformaldehyde fixed mouse thymocytes exposed surface hsp were used. exogenous hsp was used in concentration - ug/ml, fixed thymocytes were added to neutrophil samples in quantitative ratio : and : directly before the measuring. as the control we registered amplitude of oxidative burst in samples supplemented with pbs or live mouse thymocytes having no hsp on their surface. results demonstrating effect of exogenous hsp on phagocytosis-induced ros release from human peripheral blood neutrophils have been obtained. it was demonstrated marked dose-dependent inhibiting action of exogenous hsp on amplitude of respiratory burst. the cells expressing surface hsp impacted on ros production in this model similarly. the results of chemiluminescence analysis demonstrated that zymosan induced ros production was essentially decreased under action of fixed thymocytes, and was decreased slightly in presence of live thymocytes in the neutrophil samples. the effect was more pronounced for increased amount of thymocytes added to the samples. thus, immunomodulatory effects of exogenous hsp might be caused by influence of the protein on ros release from phagocytes. we suppose that the registered effects are connected with ability of hsp to inhibit activity of nadp-oxidase -the key enzyme for ros production during respiratory burst. results: we recruited pts, with so far five complete pathological remission, five partial responses and five no responses. no substantial changes were detectable in the number of circulating monocytes. in contrast we observed a clear expansion of cd /cd and cd /cd double positive subsets. this event was transient; it abated at the later time point suggesting a causal relationship to the treatment. it correlated with sensitivity to the treatment. in fact we observed that in the responder patients the expansion of the cd / subset was clear in the first weeks of treatment and decreased there after. in contrast in non-responder patients it was already expanded before the neo-adjuvant therapy. all the patients had an initial expansion of the cd / subset. in the responder patients this population was still present at the time of surgery. the immunohistochemical study revealed a massive tumoral infiltration by macrophages that displayed clear features of alternative m polarization. conclusion: these data suggest that neo-adjuvant therapy modulates the cellular components of innate immune responses that could represent valuable predictive factors. m. dimitrijević , i. pilipović , s. stanojević , k. mitić , k. radojević , v. pešić , g. leposavić , institute of virology, vaccines and sera "torlak", immunology research centre "branislav janković", belgrade, serbia, faculty of pharmacy, university of belgrade, department of physiology, belgrade, serbia the primary aim of our current study was to ascertain whether rat resident peritoneal macrophages synthesized catecholamines and to unmask putative effects of catecholamines on nitric oxide (no) and hydrogen peroxide (h o ) production and phagocytic activity of these cells. in addition, given that chronic administration of b-adrenoceptor antagonist increases the density of b-adrenoceptors on both non-immune and immune cells and thereby affects their sensitivity to catecholamine action, we hypothesized that such treatment could also affect macrophage responsiveness. to address our proposition, we determined adrenoceptor expression on peritoneal macrophages from rats subjected to -day-long propranolol treatment and measured both no and h o production and phagocytic activity of these cells. using both immunocytochemical and flow cytometric analyses of rat peritoneal exudate cells constitutive expression of tyrosine hydroxylase and both b -and a -adrenoceptors on macrophages was revealed. furthermore, according to the characteristic assemblage of tyrosine hydroxylase and adrenoceptor subtype expression different macrophage subsets were identified. in vitro treatment of macrophages with the non-selective a,b-adrenoceptor agonist arterenol and/or the b-adrenoceptor antagonist propranolol indicated that b-adrenoceptors potentiated no production and suggested a-adrenoceptor-mediated suppression of hydrogen peroxide h o production. an increase in h o production in the presence of the a -adrenoceptor antagonist ebrantil provided support for this. chronic propranolol treatment in vivo led to increased no and h o production by peritoneal macrophages. furthermore, this treatment resulted in opposing effects on the expression of b -and a -adrenoceptors on peritoneal macrophages (a stimulatory effect on b -adrenoceptors and a suppressive effect on a -adrenoceptors). in conclusion, a subset of resident peritoneal macrophages synthesizes catecholamines, which may exert differential effects on h objectives: monocytes display great phenotypical and functional heterogeneity and are divided into two major subsets: cd ++ cd -('classical') and cd + cd + ('pro-inflammatory') monocytes. a central monocyte function is cytokine production in response to toll-like receptor (tlr) ligation. the cd + cd + monocytes display higher tlr and - expression, produce higher levels of pro-inflammatory cytokines and have increased potency for antigen presentation than the cd ++ cd monocytes, suggesting that the two subsets could play different roles in antimicrobial responses. newborns are vulnerable to infections and an immaturity of both adaptive and innate immunity has been described. studies of neonatal monocyte antimicrobial responses show contrasting results and much remains to be learned, especially regarding monocyte subpopulations. thus we aimed to compare monocytes from newborns and adults, focusing on monocyte subpopulations and responses following tlr stimulation. methods: cord blood (n= ) and peripheral-blood (n= ) mononuclear cells were stimulated in vitro for hrs with peptidoglycan and subsequently analysed for cd and cd and intracellular il- p and tnf expression. the mann-whitney u-test was used to evaluate differences between groups. results: a significantly higher percentage of neonatal monocytes were positive for il- p , both unstimulated and after peptidoglycan stimulation, as compared to adults. geomfi of il- p was low and similar between groups, although significantly higher in newborns after stimulation. in both newborns and adults, il- p (% positive cells and geomfi) was significantly higher for cd + cd + cells than for cd ++ cd cells, unstimulated and stimulated. regarding tnf, neonatal and adult monocytes did not differ in unstimulated cultures, however geomfi of tnf was significantly higher in neonatal monocytes after stimulation. whereas the tnf response following stimulation was similar between the adult monocyte subsets, in newborns the cd ++ cd cells were positive for tnf to a significantly higher extent than the cd + cd + cells. in particular the tnf response to tlr stimulation differed between newborns and adults, with neonatal monocytes having a higher per cell production of the cytokine. notably, in newborns the cd ++ cd monocytes were positive to a higher extent for tnf following stimulation pointing towards a functional immaturity of neonatal monocyte subset responses. objective: chronic granulomatous disease (cgd) is an uncommon congenital phagocyte disorder characterized by recurrent life-threatening infections. cgd generally present with recurrent suppurative infections; however, intracranial fungal abscess complicating cgd may cause a diagnostic problem to anyone who is unfamiliar with its clinical and radiological features. we report a -year-old boy who admitted with complaints of seizures during the previous months. there was a history of axillary and perianal suppurative skin infections and cavitary pneumonia. the family history was unremarkable, and the parents were unconsanguineous. physical examination was only remarkable for oral moniliasis and skin scars at axillary and perianal region. a large frontol mass with diffuse peripheral vasogenic edema was discovered on mri. subfalcine herniation was noted secondary to mass effect. cgd was suspected and the analysis with flow cytometric dihydrorhodamine assay (dhr assay), for functional analysis of neutrophils was compatible with the diagnosis of cgd and no bimodal histogram pattern spesific for x-cgd was found in the mother and sister. after the diagnosis of cgd, neurosurgical removal of the abscess cavity was performed due to peri-lesional edema and herniation risk. aspergillus fumigates grew from the culture; liposomal amphotericin b and voriconazole were started; which were found to be sensitive to the cultured species. in addition, interferon-g ( mgr/m /day, subcutaneously every other day) was started. after months, control mri showed regression of the lesion, and the anti-fungal treatment was continued for months. the screening of the other family members with dhr assay demonstrated that one of his sisters had also cgd and phenotype was autosomal recessive. mutaton analysis in "hot spot" in ncf gene concerns the well-known gt deletion in the second exon of ncf gene both at the patient and his sister. results: this was an atypical clinical presentation of cgd in an adolescent boy with cerebral aspergillosis, mimicking intra-cranial tumor. we documented a good response to the combination of ifn-g, liposomal amphotericin b and voriconazole after surgery. conclusion: cgd should be considered in the differential diagnosis for all children presenting with invasive fungal infections particularly, those involving the central nervous system. recent data suggest that ubiquitin has anti-inflammatory properties and therapeutic potential after severe trauma and brain injuries. therefore, we hypothesized that ubiquitin treatment can modulate the local inflammatory response triggered after brain injury. to test this hypothesis, a focal cortical contusion was induced using a controlled cortical impact (cci) model in sprague-dawley rats. animals (n = ) subjected to moderate brain injury were randomized, and received either . mg/kg ubiquitin or vehicle (placebo) intravenously within min after cci. levels of tnf-a, il- b, il- , il- and il- receptor antagonist were analyzed in brain tissue using real time rt-pcr at and hours after treatment. immune cell infiltration was studied by immunostaining for neutrophils and macrophages/ microglia at h and days. data were analyzed with the mann-whitney u test and a two-tailed p x . was considered significant. all cytokines were highly up-regulated hours after cci but no differences between the groups were observed at this time point. three days after trauma the levels of il- were significantly lower in the ubiquitin treated animals, whereas the levels of il- and tnf-a were higher when compared to the placebo group. interestingly, macrophages/ activated microglia were significantly increased in the pericontusional cortex after ubiquitin treatment at day . the infiltration of neutropils was not affected by ubiquitin treatment. here, we could demonstrate for the first time that a single injection of ubiquitin immediately after brain trauma is able to modulate the inflammatory response triggered after brain injury at the cellular as well as at the cytokine level. macrophage activation and oxidative metabolic changes are commonly implicated in pulmonary tuberculosis (ptb) patients. efficient plasma antioxidant activities are needed to neutralize high free radical load in pulmonary tuberculosis (ptb) patients. there is limited information about the plasma levels of neopterin (a marker of macrophage activation) and oxidative stress indices such as total plasma peroxide (tpp), total antioxidant activity (taa), malondialdehyde (mda), and oxidative stress index (osi) in ptb patients during chemotherapy with or without micronutrient supplementation. the present study was designed to assess the levels of neopterin, tpp, taa, mda, and osi during chemotherapy with (c+m) or without (c-m) micronutrient supplementation using elisa and spectrophotometric methods. thirty-eight ( ) newly diagnosed ptb patients and forty non-ptb apparently healthy subjects volunteered to participate in this study. twenty of the ptb patients were on anti-tuberculosis drugs supplemented with micronutrients (c+m) while were treated with anti-tuberculosis drug alone (c-m) for a period of four weeks. the levels of neopterin (p= . ), tpp (p= . ), osi (p = . ), mda (p = . ) were significantly raised but taa (p = . ) was significantly reduced in ptb patients compared with controls. the levels of mda (p = . ), neopterin (p= . ) and tpp (p= . ) were significantly reduced in c+m after two weeks of treatment compared with baseline values before commencement of treatment. the levels of tpp (p= . ), mda (p= . ), neopterin (p= . ), osi (p= . ) were significantly reduced while taa (p= . ) was significantly raised in c+m after weeks of treatment compared with the baseline concentrations. in c-m, only mda showed significant decreased after weeks of treatment when compared with the baseline values. plasma level of neopterin, tpp, osi and mda declined faster in c+m than c-m. therefore, micronutrient supplementation of ptb drugs with synthetic antioxidants or naturally occurring ones (fruits and vegetables) should be attempted. this will improve deranged macrophage activation and reduce oxidative stress indices in ptb patients. a. p. aguas , e.m. cunha , m.j. oliveira icbas, university of porto, anatomy, porto, portugal the acute in vivo intake of mercury (hg) microparticles ( nm in diameter) by neutrophils and macrophages was studied with the use of in situ detection of hg by scanning electron microscopy coupled with x-ray elemental microanalysis (sem-xem). the intracellular distribution of hg particles was compared, at high resolution, between macrophages and neutrophils, and between activated and non-activated phagocytes. balb/c mice were injected intraperitoneally (ip) or in a subcutaneous air-pouch with mercury chloride, and the animals were sacrificed up to minutes after the injection. in some mice, before the hg injection, peritoneal phagocytes were activacted by ip injection of bsa. pre-injections with a selenium (se) salt were also performed in order to study the putative modulatory role of se on hg intake by phagocytes. peritoneal cells were collected by washing of the peritoneal or subcutaneous cavities with pbs, they were cytospinned, fixed with formaldehyde, and processed for observation by sem-xem. five min after the hg injection more than half of the mouse phagocytes were positive for hg. a higher percentage ( %) of macrophages contained the metal particles than neutrophils ( %). phagocyte activation enhanced the number of hg particles seen inside the phagocytes. pre-injection of the peritoneal cavity of mice with se resulted in finding that more than half of the hg intracellular particles were coupled with se. subcellular topography of hg particles showed that they were presented in individual small cytoplasmic vesicles. we conclude that hg microparticles are rapidly ingested by macrophages and neutrophils, a processed that is enhanced by cell activation. hg particles are ingested by pinocytosis and sorted in the cytoplasm of macrophages and neutrophils inside individual small vesicles. this study was supported by a grant from fct, portugal. mast cells play central roles in allergic inflammatory reactions and innate immunity. swap- is a rac-interacting protein expressed in several cells types of the hematopoietic system including mast cells. in b cells and mast cells swap- regulates f-actin cytoskeletal rearrangements, cell polarisation and cell migration. (pearce et al., ; sivalenka and jessberger, ) . swap- -/-bone marrow derived mast cells (bmmc) are specifically impaired in fceri-mediated activation and degranulation and in c-kit-induced activation, migration and cell adhesion (gross et al., ; sivalenka and jessberger, ; sivalenka et al., ) . crucial regulators of these processes are members of the rho family of small gtpases such as rac and rhoa. swap- interacts with rac in vitro and preferentially binds the active gtp-bound rac . swap- supports the increase of active rac in vitro by a yet to be defined mechanism (shinohara et al., ) . in this study, in vitro pull-down assays with purified recombinant proteins were employed to characterize the interaction between swap- and rac . it was found that fulllength swap- preferentially binds to constitutively active rac (rac q l) but not to its dominant negative form (rac t n). binding assays with swap- truncated mutants showed interaction of swap- 's n-terminus with gtpgs rac or rac depleted of guanine nucleotide, whereas swap- central or c-terminal regions do not bind to any form of rac . preliminary competitive-binding assays with overlapping mer peptides, spanning the entire swap- sequence, mapped the rac binding site near the n-terminus of swap- . full-length swap- site-specific mutants will be generated to test the relevance of these interactions in mast cells in terms of adhesion, migration and activation of rho gtpases. elucidating the molecular interactions of swap- with rho gtpases and the relevance of these will shed light on the biology and biochemistry of mast cells and possibly other hematopoietic cells, which express swap- . v. c. barbosa , c. d. polli , m.c. roque-barreira , m.c. jamur , c. oliver , g. pereira-da-silva mast cells are essential cells in ige-associated immune responses. fceri crosslinking induces mast cell degranulation and release of proinflammatory mediators. we have previously shown that the lectin artinm induces mast cell activation but the mechanisms involved in this activity remain unknown. objective: the present study was undertaken to further characterize the ability of artinm to activate mast cells. methods: rbl- h cells were sensitized with ige anti-tnp and stimulated with dnp -hsa or artinm. artinm binding to rbl- h cells was assessed by flow cytometry. mast cell degranulation was determined by measurements of released b-hexosaminidase activity. microplate binding assays were utilized to assess artinm binding to ige. to investigate fceri recognition by the lectin, western blots of cell lysates were stained with biotinylated artinm and be's antibodies specific for fceri b-subunit. intracellular protein phosphorylation was detected by specific antibodies and analyzed by confocal microscopy. mcp- and tgf-b levels released by mast cells were measured by elisa. results: artinm binding to the cell surface was dependent on sugar recognition and resulted in mast cell degranulation in the presence or absence of ige. the release of b-hexosaminidase doubled when cells were sensitized by the immunoglobulin and was abrogated in the presence of d-mannose, suggesting that mast cell degranulation induced by artinm might be the result of interactions between the lectin crds and glycosylated components on the cell surface, like fceri or ige. indeed, it was observed that the lectin bound to ige in a dose-dependent manner and recognized the fceri b subunit in western blot analysis. exposure to artinm resulted also in phosphorylation of intracellular proteins, mcp- release and tgf-b production. significant increases in these activities were observed upon sensitization with ige. conclusions: these results suggest that artinm may bind to glycans of the high affinity ige receptor and/or of the ige (bound to fceri) and that such interactions would be implicated in its ability to activate and degranulate mast cells. in view of the well-established significance of mast cells in allergic inflammation, the participation of sugars as binding receptors on mast cell surface opens new ways of controlling allergic disorders. the adhesion receptor l-selectin is a key player of the innate immune response in the process of leukocyte migration from the blood stream to inflamed tissue. it is expressed on leukocytes and promotes the initial contact to the endothelium resulting in steady rolling and eventually diapedesis. a distinct feature is the exclusive presentation of l-selectin on the tip of finger-like cell membrane protrusions called microvilli which cover the entire leukocyte surface. this topography was shown to facilitate the first transient interactions of the free flowing cell to the static counterreceptor particularly in the context of high dynamic shear. other adhesion molecules such as p-selectin glycoprotein ligand (psgl- ), b and b -integrins also share this special phenotype. taken together, prominent adhesion receptor positioning reflects a widespread biological principle contributing to inflammation as well as hematogenic tumor metastasis. despite the functional relevance and frequent occurrence, however, molecular mechanisms of cell surface receptor compartmentalization remain largely unknown. in this study we identified the highly conserved transmembrane domain of l-selectin to regulate microvillus receptor positioning and adhesion under flow. taking advantage of the inverse surface expression pattern of cd (cell body) compared to l-selectin (microvilli) in a myeloid cell line, we investigated domain swapped chimeric receptors regarding their substructural surface localization and their ability to initiate rolling under flow. transmission electron microscopy showed a crucial impact of the transmembrane domain to target the chimeric receptors to a certain cell surface compartment independent of the intracellular anchorage. in turn, the receptor shift from microvilli to the cell body goes along with a substantial decrease of rolling cells in an in vitro parallel flow chamber assay. thus, contrary to the common view of single membrane spanning domains to simply act as a mechanical anchor, our results attach an important functional component as well and might point out a new general principle for targeting receptors to specific membrane compartments. objectives: macrophages are one of the principal effector cells involved in the innate immunity response. they kill microbes through phagocytosis and upon activation, secrete pro-inflammatory cytokines such as il- b, il- and tnf-a. herpes simplex virus (hsv- ) is an enveloped dna virus that infects mostly oral mucosa and sensory neurons. innate immunity responses activated by hsv infection consist of: activation of macrophages; activation of the complement cascade, and production and secretion of a variety of cytokines and chemokines. il- and tnf-a are cytokines produced by macrophages that contain known anti-hsv properties. the objective of this study was to characterise the secretome of human primary macrophages infected with hsv . methods: human monocytes were purified from the peripheral blood mononuclear cells of healthy blood donors and differentiated in vitro into macrophages. macrophages were left untreated or primed with poly(i:c) ( ug/ml), a mimetic of double-stranded rna, after which cells were left uninfected or infected with hsv- for h. after this, cell culture supernatants were collected, concentrated and proteins purified. the secreted proteins were digested into peptides, identified and quantified using itraq (isotope tagged relative and absolute quantitation) -labelling of the peptides followed by peptide fractionation by cation exchange chromatography and analysis by nanolc-ms/ms. the raw ms/ms data was analysed using proteinpilot . software. results: in the first itraq experiment over human proteins were identified in the hsv infected cell supernatants. from these proteins had at least fold increase after poly(i:c) + hsv infection compared to the uninfected cells. hsv infected cells had clearly more proteins in their cell supernatants after infection compared to the uninfected cells: itraq labelling showed a total of . fold increase in the protein amount in the poly(i:c) + hsv infected cell supernatant and a . fold increase in the hsv infected cell supernatant when compared to the uninfected cell supernatant. amongst the upregulated proteins there were known inflammatory proteins: chemokine (c-x-c motif) ligand , il- , tnf-a induced protein , complement factor b, galectin- and mxa. at present, further experiments are on-going for more detailed analysis of the hsv infected macrophage secretome. h. p. prakash , german cancer research centre, translational immunology, heidelberg, germany, max planck institute for infection biology, molecular biology, berlin, germany chlamydophila pneumoniae are the major etiological factors for worldwide pneumonia, chd and copd. chlamydia lives and multiplies inside their host epithelial cells where they confer resistance for apoptosis by inducing expression and stability of anti-apoptotic proteins called inhibitor of apoptosis proteins (iaps). the significance of cellular inhibitor of apoptosis protein- (ciap- ) and x-linked inhibitor of apoptosis proteins ( xiap) in chlamydia pneumoniae pulmonary infection and innate immune response of macrophages was investigated in ciap- and xiap knockout (ko) mice using a novel non-invasive intra-tracheal infection method. in contrast to wildtype, iap knockout mice failed to clear the infection from their lung. wildtype mice responded to infection with a strong inflammatory response in the lung. in contrast, the recruitment of monocytes and macrophages was reduced in iap ko mice compared to wildtype mice. the concentration of interferon gamma (ifn-g) was increased whereastumor necrosis factor (tnfa) was dysregulated in the lungs of infected iap ko mice compared to infected wildtype mice. ex vivo experiments on mouse peritoneal macrophages and splenocytes revealed that iaps are required for innate immune responses of these cells. our findings thus suggest a new immunoregulatory role of iaps in c.pneumonaie pulmaonry infections. methods: human monocytes were purified from venous blood of normal volunteers by ficoll density gradient centrifugation. hrgal- ( mg/ml) binding to monocytes, in the presence or absence of mm lactose or sacarose, was assessed by flow cytometry and confocal microscopy. in transwell systems, assays were performed using hrgal , laminin or fibronectin immobilized or not on the filters. these were added to wells containing soluble hrgal or rpmi and monocytes ( x ) were added into each insert. when necessary, hrgal was pre-incubated with mm lactose or sacarose. mcp- ( ng/ml) was used as positive control. we observed that hrgal- binds to the surface of human monocytes through its crd, since this interaction can be inhibited by lactose. we corroborated some data of literature that hrgal- is able to induce monocyte migration in a dose-dependent manner, resulting in a bell-shaped curve as seem with other known attractants. when we evaluated the participation of the ecm laminin and fibronectin in monocyte migration induced by hrgal- , we observed that the association between these glycoproteins and hrgal- resulted in a % increase in the number of migrating cells. both n-and c-terminal domains of hrgal- are involved in the association between laminin or fibronectin and hrgal- , since the presence of lactose resulted in % and % inhibition of monocyte migration induced by the lectin, respectively conclusions: our results showed that hrgal- induces monocyte migration by haptotaxis, through the interactions established between both n-and c-terminal domains of the lectin and ecm glycoproteins, laminin and fibronectin. in a vertebrate embryo, macrophages develop in two sites (yolk sac and liver) and constitute the primary mechanism of host defense. their phagocytic function may be required during the earliest stages of development both for survival and for organogenesis. recent studies have shown that monocyte heterogeneity is conserved in humans and mice. the different monocyte subsets seem to reflect developmental stages with distinct physiological roles but nothing is known whether the macrophage diversity arises in early ontogeny. in order to study the ontogeny of the monocyte-macrophage lineage, we developed a new culture technique using human embryonic stem cells (hesc).culturing of embryoid bodies for weeks in the presence of bmp ,vegf and a mixture of hematopoietic cytokines resulted in a generation of a significant cell population of cd +cd + cells. the sorted cd +cd + cells were further cultured for - days in the presence of m-csf and gave rise to a homogenous population of adherent mature macrophages. embryonic stem cells derived macrophages were identified by several criteria including morphology and ultrastructural features observed by microscopy and by expression of nonspecific esterase and myeloperoxidase by histochemical staining. while virtually all embryonic-derived macrophages expressed the lps-receptor cd , m-csf receptor cd and the scavenger-receptor cd , we characterized two distinct subpopulations of macrophage based on their difference in size and density and the expression of the cd and cd (fcgammariii) : the cd lowcd -and cd + cd +. trancscriptional, phenotypic and functional assays suggest the alternative (m ) polarization of cd +cd + embryonic stem cell-derived macrophages.(anti-inflammatory cytokines secretion, active phagocytosis, m -related gene expression).the exact chemokine receptor expression pattern, phenotype and transcriptional activity of their foetal counterparts are currently under investigation. collectively, our data provide insight into alternative macrophage polarization in humans and and adds further data to the growing body of evidence that establishment of macrophage heterogeneity is related to early ontogeny. b.-s. choi , p. kropf imperial college london, immunology department, london, united kingdom the balance between t helper (th) and th cell responses is a major determinant of the outcome of experimental leishmaniasis, but polarized th or th responses are not sufficient to account for healing or nonhealing. we have recently shown that arginase-induced l-arginine depletion results in local suppression of antigen-specific t cell responses in nonhealing leishmaniasis. healing, induced by chemotherapy, resulted in control of arginase activity and reversal of local immunosuppression. moreover, supplementation with l-arginine restored t cell effector functions and resulted in reduced lesions size and parasite load. however, despite the efficient production of ifn-g by cd + t cells at the site of infection and despite the reduced pathology, the mice did not heal. we hypothesised that arginase-expressing macrophages contribute to persistent disease and become refractory to ifn-g mediated signals. to test this hypothesis, we used a well-defined model of bone marrow derived macrophages and determined whether the differentiation state of parasitized arginase-expressing macrophages could be altered. in addition, we also tested whether alternatively activated macrophages can be induced to switch off arginase and upregulate inducible nitric oxide synthase (inos) to kill the intracellular parasites. vg vd t lymphocyte are activated following recognition of non-peptidic phosphorylated metabolites. the phosphoantigen isopentenyl pyrophosphate (ipp) is overproduced by tumors following hyperactivation of the mevalonate pathway of isoprenoid synthesis. previous work has shown that a molecular complex homologous to mitochondrial atp synthase (ecto-f -atpase) is expressed on many cell types and is a possible specific ligand for the vg vd tcr. the present study aims at understanding the role of f -atpase in antigen regognition. using video microscopy calcium imaging in single vg vd t lymphocytes, we can now show that the t cell response to ipp requires contact with bystander cells of variable tissue origin but that this requirement is not fulfilled by a cell line deprived of surface f -atpase. purified f -atpase immobilized on polystyrene beads can partly replace the need for cell-cell contact. ipp in soluble form is highly sensitive to terminal phosphatases and addition of these enzymes in t cell activation assays clearly shows that it is not recognized as such on tumors. however, we could detect nucleotide derivatives of phosphoantigens which are resistant to terminal phosphatases in the cell lysates of stimulatory tumors. one of these, a derivative of ipp, is barely able to stimulate vg vd cells in the absence of apcs, as opposed to the non-nucleotidic antigen ipp. however it can bind stably to f -atpase. thus the f -atpase complex acts as a presenting structure for nucleotide phosphoantigens. altogether, our data suggest that vg vd t cells are dedicated to the recognition of phosphoantigens in the form of nucleotide derivatives, on the surface of tissue cells and that antigen recognition involves multiple antigen modification steps, in including final cleavage by a nucleotide pyrophosphatase activity. surface plasmon resonance was used to analyse the molecular interaction between tcr and f -atpase. by using purified f -atpase and peptides derived from vg vd tcr sequences, interaction sites between f -atpase and tcr were identified on both ligands. based on these findings a generalized model for vg vd t cell activation is proposed. ligands for the cytotoxic lymphocyte activating receptor nkg d are highly expressed on cells stressed by numerous agents including genotoxic damage, thereby contributing to the elimination of transformed cells by nkg d(+) lymphocytes. a key question is whether this represents a primary inductive means of immune surveillance, or merely enhances responses initiated by dendritic cells and antigen-specific t cells. a second key issue is the scope and scale of events that follow nkg d activation in vivo. by transiently overexpressing the nkg d ligand rae- -beta in the skin of transgenic mice, we showed that this alone provoked rapid, coincident and reversible changes in the organization, morphology and activation state of tissue-resident vgamma vdelta gamma-delta t cells and langerhans cells (lc), that were swiftly followed by epithelial infiltration of unconventional alpha-beta t cells. these data indicate a novel primary immune surveillance pathway whereby epithelial upregulation of nkg d ligands is sufficient to provoke a series of multicomponent immunological changes. the effects on lc, which lack nkg d and presumably respond to changes initiated by local gamma-delta t cells, are particularly interesting. ongoing microarray and co-culture experiments are now providing a molecular definition of the immume surveillance response to nkg d ligands in vivo. to assess the scope of this response, ovalbumin was applied to the skin concomitant with rae induction. the primary systemic th response is increased by concomitant responses to a stress antigen. we will now resolve whether this increased response contributes to the adaptive memory pool, or whether it is a primary, regulatory response that may limit adaptive responses to auto-antigens exposed during stress. in addition, the many ligands available to the nkg d receptor suggest that different ones may play unique roles. a novel nkg d-ligand, h c, is uniquely expressed in mouse skin. when the expression of this was further increased in a novel transgenic system, there was again an overt alteration in the local immune compartment, but with features that are seemingly distinct from the action of rae- induction. such studies may help resolve a long-standing puzzle over the pleiotropy of nkg d ligands, and dissect immune surveillance of changes in gene expression levels rather than absolute levels. a.-s. invariant natural killer t (inkt) cells are a distinct lineage of t lymphocytes that co-express a highly conserved ab t cell receptor (tcr) along with typical surface receptors for natural killer (nk) cells. these lymphocytes recognize glycolipid antigens presented by the non-classical class i molecule cd d. inkt cells are characterized by their capacity to produce rapidly large amounts of both th (ifn-g, tnf) and th (il- , il- ) cytokines, which enables them to play a role in the regulation of many different types of immune responses, ranging from self-tolerance to responses against pathogens and tumors. converging studies in mouse models suggest that inkt cells can prevent the development of type diabetes. the frequency of inkt cells is lower in non-obese diabetic mice (nod mice). manipulation of inkt cells, either by increasing their frequency or by stimulating them with agonists such as a-galcer, inhibits diabetes onset in nod mice. recently, a new population of cd -nk . -inkt cells producing high levels of the pro-inflammatory cytokine il- has been identified (inkt cells). given that this cytokine has been implicated in several pathologies including autoimmune diseases, we investigated the role of inkt cells in type diabetes. interestingly, nod mice exhibit a higher frequency of inkt cells producing il- as compared to c bl/ mice. this increased frequency was observed in the thymus as well as in peripheral lymphoid tissues. as previously described in normal mice, inkt cells present in nod mice were mainly cd -nk . -, express the ror-g transcription factor and il- receptor, both molecules being usually associated with th commitment. we are currently analyzing, using co-transfer experiments, whether these inkt cells play a beneficial, a deleterious, or any role in the development of type diabetes in nod mice. j. s. dodd , r. muir , s.s. affendi , p.j. openshaw imperial college london, respiratory medicine, london, united kingdom natural killer t (nkt) cells are a heterogeneous population of innate t cells that have attracted interest because of their potential to regulate immune responses to a variety of pathogens. upon activation with their cognate glycolipid antigen presented by cd d molecules, activated nkt cells produce copious and numerous cytokines which endow these cells with potent immunoregulatory properties. consequently, nkt cells have become the focus for the development of vaccine adjuvants, cancer immunotherapeutics and modulators for autoimmune and inflammatory conditions. respiratory syncytial virus (rsv) is a common cold virus of the family paramyxoviridae. it is the most frequent viral cause of serious lower respiratory tract infection in infants and children worldwide and a significant contributor to winter deaths in the elderly. despite its global impact, there is still no safe and effective vaccine and our understanding of the immunological mechanisms that regulate protection and pathology is incomplete. it is known that cd d-deficient mice with poor nkt cell responses have inefficient induction of cd t cells and reduced clearance of rsv, perhaps because of ifn-g release by activated nkt cells. we now show that activation of lung nkt cells with intranasal agalcer during rsv infection of mice boosts th immunity (increasing il- and il- ), promoting pulmonary eosinophilia and ablating cd t cell recruitment. by contrast, intraperitonal injection of agalcer enhances nk cell recruitment and boosts pulmonary cd t cell activity (as measured by cd expression), increasing ifn-g production in the airway and lung and inhibiting viral replication. effects on illness (as measured by weight loss) were similarly distinct: intranasal agalcer induced early (d ) weight loss independent of conventional t cells, whereas intraperitonal agalcer enhanced late (d ) weight loss by a cd t cell dependent mechanism. therefore, nkt cells stimulated by agalcer administered via different routes induce distinct types of immune response to viral infection in the lung with the intraperitonal route leading to optimal viral clearance. in general, neonatal conventional t cells, especially cd + ab t cells, are regarded as immature or t h biased. vg + vd + t cells are unconventional lymphocytes: they are mhc-unrestricted and can react rapidly upon activation with pyrophosphates (e. g. (e)- -hydroxy- -methyl-but- -enyl pyrophosphate (hmb-pp)) or aminobisphosphonates (e. g. zoledronate) in adults. until now, little is known on the functional reactivity of neonatal vg + vd + t cells towards these activators. because il- is preferentially secreted by neonatal dendritic cells (dc) upon tlr stimulation, we investigated the potential costimulatory effect of this cytokine on hmb-pp and zoledronate-treated neonatal vg + vd + t cells. herein, we observed that zoledronate induced neonatal vg + vd + t cell proliferation and ifn-g production in cord blood mononuclear cells (cbmc) cultures. other t h -like cytokines like tnf-a and gm-csf were also produced upon this stimulation, but less than ifn-g, while t h -like cytokines such as il- and il- were not induced. addition of il- to zoledronate selectively costimulated ifn-g production from neonatal vg + vd + t cells. furthermore, zoledronate/il- treatment resulted in neonatal vg + vd + t cells expressing high levels of the cytotoxic mediators perforin and granzyme a. zoledronate induced the expression of the receptor for il- (il- r) and the transcription factor t-bet, which is known to be important for the production of ifn-g in gd t cells. in addition, costimulation with il- resulted in a further increase of t-bet expression in neonatal vg + vd + t cells. these changes in the expression of il- r and t-bet likely contribute to the observed selective ifn-g response towards zoledronate/il- treatment. of note, in contrast to adult peripheral blood vg + vd + t cells, hmb-pp had no or only a minor effect on the functional reactivity of neonatal vg + vd + t cells. altogether, these observations show that neonatal vg + vd + t cells are functionally active and that this t cell population might play a role in protective immune responses to infections with intracellular pathogens in early life, in particular when dc-derived il- is produced in response to microbial stimuli. the evasion of antigen presentation is a feature common to herpesviruses. one of the strategies employed to inhibit antigen presenting molecules is ubiquitination, internalisation and lysosomal breakdown by viral e ligases such as hhv encoded k , k or mhv encoded mk . these viral genes represent homologues of the march family of cellular genes whose function is the regulation of cell-surface antigen presentation and reduction of the lifetime of loaded antigen complexes. ubiquitination targets surface molecules to the lysosome via the multivesicular body (mvb), a structure which also has an important role in the budding of many viruses. we investigated the existence of alternative fates for antigen presenting molecules post-ubiquitination, and how viral e ligases manipulate them. we discovered that both the cellular march and viral e ligases ubiquitinate cd molecules. however, whereas viral molecules inhibit cd -antigen presentation, the march molecules are essential for the recirculation and function of the long-lived and lysosome-resistant cd molecules. in contrast mhc class ii was only targeted by cellular and not by viral e ligases. furthermore cd molecules could be found in viral particles as a result of ubiquitination, presumably via the mvb. thus, virally expressed and cellular e ligases have opposite effects, despite their homology. how this is achieved is a matter of active investigation. gamma delta (gd) t cells recognize stress-induced auto-antigens and contribute to immunity against infections and cancer. our previous study revealed that vd negative ( neg ) gd t lymphocytes isolated from transplant recipients infected by cytomegalovirus (cmv) killed both cmv-infected cells and ht colon cancer cells in vitro. in order to investigate the anti-tumor effects of vd neg clones in vivo, we generated hypodermal ht tumors in immunodeficient mice. concomitant injections of vd neg clones, in contrast to vd + cells, prevented the development of ht tumors. vd neg clones expressed chemokine c-c motif receptor (ccr ) and migrated in vitro in response to chemokines secreted by ht cells, among which were the ccr ligands macrophage inflammatory protein (mip)- d and monocyte chemoattractant protein (mcp)- . more importantly, a systemic intraperitoneal (i. p.) treatment with vd neg clones delayed the growth of ht subcutaneous (s. c.) tumors. the effect of in vivo gd t cell passive immunotherapy on tumor growth could be reverted by addition of a blocking anti-ccr antibody. gd t cell passive immunotherapy was dependent upon the cytotoxic activity of the gd effectors towards their targets since vd neg clones were not able to inhibit the growth of a hypodermal tumors. our findings suggest that cmv-specific vd neg cells could target in vivo cancer cells, making them an attractive candidate for anti-tumor immunotherapy. more recently, we generated ht cells expressing the luciferase and realized orthotopic injection of ht -luc cells. progressive tumor development and regression following « gd treatment » will be observed in vivo using bioluminescent imaging. intraepithelial lymphocytes (iel) compose large, oligoclonal, tissue-associated repertoires of non-mhc-restricted t cells that play key roles in immunosurveillance. it is commonly considered that the characteristic iel repertoires are positively selected by thymic epithelial molecules that are also stress-induced in specific tissues, thereby activating iel function. however, no such molecules have been identified. here we characterise skint , currently the only known determinant of a canonical iel compartment, that is selectively required for vg vd + dendritic epidermal t cell (detc) development. we show that both peripheral and thymic skint expression is essential for full detc development. its effects are highly specific since even substantial and ubiquitous over-expression neither negatively selects detc, nor affects any other t cells. unexpectedly, however, skint genes are not expressed by cell lines and are downregulated rather than activated by carcinogenesis. mouse genetic models allow powerful insight into skint function; for example, we demonstrate that the constitutive expression of wild-type skint fully restores detc development in a skint mutant mouse, but does not rescue normal detc function. thus, skint provides a novel perspective into how epithelia regulate the development and function of specific tissue-associated t cell compartments, and how normal versus dysregulated tissues may be demarcated. marginal zone (mz) b cells are strategically localized in the mz of the spleen. since most of the blood reaching the spleen is passing through this region such localization favors contact with blood born antigens and pathogens. besides being able to rapidly secrete antibodies, mz b cells may also act as professional antigen presenting cells (apcs). they are known to express high levels of cd d which is the presenting molecule for nkt cells which are also located in the mz. therefore we hypothesised that mz b cells may be efficient activators of nkt cells. to test this hypothesis, we used freshly sorted splenic mz b cells (cd + cd hi cd lo cd c -) and splenic conventional dendritic cells (cdcs) (cd c hi cd a +/-cd b +/-b -) from wt and cd d -/mice as apcs for nkt cells from va -ja transgenic or wt mice. the apcs were treated with agalactosylceramide (agalcer) or heat killed (hk) listeria monocytogenes or salmonella typhimurium. both mz b cells and cdcs proved to be highly efficient apcs for priming of nkt cells and induced robust proliferation. in contrast, other populations of b cells failed to activate nkt cells. we showed, using cd d -/mice as well as blocking antibodies to icosl, that proliferation of nkt cells depends on tcr/cd d and in case of mz b cells, also on icos/icosl interactions. importantly, apcs primed with hk bacteria were not able to induce nkt cell proliferation. interestingly, mz b cells exclusively induced production of il- by nkt cells. in contrast, cdcs mostly induced production of ifn-g and il- producing cells were scarce under these conditions. cytokine production by nkt cells proved to be independent of tcr signalling, but dependent on icos/icosl interactions when mz b cells were used as apcs, and gitr-dependent when cdcs were used. taken together, our data suggest that both mz b cells as well as cdc act as professional apcs for nkt cells. notably, the nature of apcs appears to be critical for polarization of the immune response: mz b-cell-primed nkt cells induce cytokine milieu fostering a t h response, whereas cdc-primed nkt cells rather favor a t h response. objectives: il- is an innate cytokine present in elevated levels in sera from patients suffering from autoimmunity (eg. sle and ra) and the allergic disease atopic eczema. in mice, injections of il- give rise to an early polyclonal isotype switched antibody response which is absent in inkt cell deficient (cd d -/-) mice. we set out to investigate the activated b cells in il- injected mice and how these are regulated by inkt cells. methods: mice received daily i. p. injections of il- ( mg) for days and the antibody response in serum was monitored using elisa. the b cell activation in the spleen at day was evaluated by flow cytometry and immunohistology. results: mice injected with il- developed self reactive (anti-pc and anti-dna) antibodies in the serum, in line with the autoreactive antibodies in patients with e. g. sle and atopic eczema. the antibody producing cells formed cd + cell clusters in the red pulp of the spleen, a typical feature of extrafollicular activation frequently associated with autoreactive responses. surprisingly, the antibody response induced by il- was increased in inkt cell deficient (cd d -/-) mice, in contrast to published data. an increased response to il- was also observed in ja -/mice, which lack the a-chain of the tcr used by inkt cells, and thus our data suggest that inkt cells inhibit antibody producing cells in il- induced antibody responses. further characterization of the recruitment of b cells in il- injected mice revealed a marked expansion of the marginal zone b cell (mzb) population in the spleen, suggesting an important role for mzbs in the il- induced autoreactive antibody response. mzbs are innate-type b cells that express high levels of cd d, are prone to autoantibody production and often involved in early immune responses. the il- induced antibody response in mzb deficient (cd -/-) mice was either decreased (igg) or delayed (ige), supporting the importance of mzbs in il- induced antibody responses. we conclude that the role for inkt cells in il- induced antibody responses is to inhibit the production of autoreactive antibodes from mzbs in extrafollicular foci. objectives: amoebiasis is a widespread human parasitic disease caused by the intestinal protozoan entamoeba histolytica. there are two major clinical manifestations of the disease, amoebic colitis and amoebic liver abscess (ala). interestingly, only a small proportion of e. histolytica-infected individuals develop invasive disease, whereas the majority harbors the parasite within the gut without clinical symptoms. so far, cells of the innate immune system have been described to constitute the main host defense mechanism for the control of amoebiasis, relying largely on the early production of interferon-g (ifn-g). however, information is lacking about the sources of early ifn-g production as well as the amoeba antigens involved in this activation process. methods: using a recently developed c bl/ mouse model for ala, the contribution of natural killer t (nkt) cells for protection against amoebic disease was investigated. applying nkt cells and dendritic cells as antigen-presenting cells from various ko-mice, the signaling pathways implicated in recognition of amoebic antigens and activation of cytokine-secretion by nkt cells was analysed. results: nkt cells were found to play a key role in the defense against ala. specific activation of nkt cells by a-galactosylceramide (a-galcer) induced significant protection, whereas jalpha -/-and cd d-/-mice lacking inkt as well as dnkt cells suffered from more severe abscess formation. a lipopeptidophosphoglycan, which is present in large quantities on the surfcae of e. histolytica trophozoites (ehlppg), was identified as a major amoeba antigen that activates nkt cells resulting in the production of ifn-g, but not of il- . moreover, ifn-g production required the presentation of ehlppg by cd d and signaling through the tlr receptor cascade in combination with a simultaneous secretion of il- . similar to a-galcer application, treatment of mice with purified ehlppg significantly reduced the severity of ala in amoeba-infected mice. our study provides a mechanism for the innate control of amoeba invasion that might explain why the majority of e. histolytica-infected individuals do not develop amoebic disease. a few years ago, we have observed a significant expansion of circulating effector gamma delta t cells following cytomegalovirus (cmv) infection in kidney transplant recipients (ktr). these unconventional t cells display tcr dependent cytotoxicity against both cmv-infected cells and carcinoma cells. in the present study, an extensive phenotyping of gamma-delta t cells allowed us to demonstrate an over-expression of cd in cmv-infected individuals. cd is the fcgammariiia, a natural killer cell marker usually absent on conventional t cells. we found that . ± . % of gamma-delta t cells from cmv-infected ktr expressed cd , when compared with only . ± . % in non cmv-infected ktr (p x . ). similarly, . ± . % of gamma-delta t cells from cmv-seropositive blood donors expressed cd compared to . ± . % in cmv-seronegative donors (p x . ). cd + gamma-delta t cell lines generated from cmv-infected individuals were able to produce ifn-g (a potent anti-viral cytokine) in a cd -dependent manner when activated by cmv/igg immune complexes. this production greatly increased in the presence of il- and ifn-alpha, two cytokines highly produced during cmv-infection. the supernatants of gamma-delta t cells activated with agonist anti-cd mab inhibited cmv replication in vitro and this effect was abrogated in the presence of a blocking anti-ifn-g antibody. cmv/igg immune complexes were also able to induce the expression of the cytotoxicity marker cd a on cd + gamma-delta t cell lines. cd is well-known to mediate antibody-dependant cellular cytotoxicity (adcc), especially in natural killer cells. accordingly, we demonstrated that cd + gamma delta t cell lines could make adcc against the daudi lymphoma cell line and the a skin carcinoma cell line pre-incubated either with rituximab (anti-cd ) or cetuximab (anti-egfr), respectively. in contrast, no addc could be observed against cmv-infected fibroblasts pre-incubated with polyclonal anti-cmv igg (cytogam), probably because cytogam weakly stained infected cells. these data reveal a new cd -dependent anti-cmv function of gamma-delta t cells through recognition of immune complexes and secretion of ifng. moreover, they demonstrate that these cells are able to kill through adcc lymphoma and skin carcinoma cells, two tumour types frequently encountered in ktr. dendritic epidermal t cells are a prototypic population of intraepithelial gd t cells in the mouse skin. found in the basal layer of epidermis and in close contact with langerhan's cells and keratinocytes detc facilitate vital immunological and physiological processes e. g. wound healing, homeostasis, tumor surveillance and regulation of inflammation. gd t cells respond rapidly to non-peptidic microbial and stress induced self antigens in a non-mhc restricted manner and are therefore proposed to bridge the gap between innate and adaptive immunity. by using gd t cell knock-out mice tcrd-/-, ovalbumin transgenic k mova mice and a skin grafting model we aimed to elucidate the role of gd-detc in adaptive immune responses associated with elimination of foreign antigen presented in the skin.we show that in the absence of gd t cells in the skin there is a decrease in rejection of ovalbumin expressing skin grafts compared to wildtype mice. we show that optimal regimens of antigen delivered subcutaneously in conjunction with adjuvant elicits comparable responses in wildtype and knockout mice. however frequency of primed host animals is reduced in tcrd-/-mice when antigen is delivered epidermally via skin grafting; suggesting detc enhance cross presentation of classical mhc bound antigens in the skin. considering the incapability of gd t cells to recognize peptide antigens in the context of mhc we plan to dissect the relationship between detc and professional antigen presenting cells in the skin. understanding the underlying mechanisms of this relationship will expand our knowledge of enhancing professional apc function in skin by detc and potentially other epithelia by intraepithelial gd t cells and can be useful in designing therapies to epithelial infections and malignancies. we demonstrate a rapid and hmb-pp-dependent crosstalk between gd t cells and autologous monocytes that resulted in the production of inflammatory mediators including il- , ifn-g, tnf-a, osm, ccl , cxcl , cxcl , and trail. moreover, under these co-culture conditions monocytes showed enhanced survival and differentiated overnight into inflammatory dcs with antigen-presenting functions. these cells expressed cd , cd , hla-dr, and dc-sign, and lost cd , ccr , ccr , and cxcr . addition of further microbial stimuli (lps, peptidoglycan) induced ccr and enabled these inflammatory dcs to trigger antigenspecific cd + effector ab t cells expressing ifn-g and/or il- . importantly, our in vitro model replicated the responsiveness to microbes of effluent cells from pd patients and translated directly to episodes of acute pd-associated bacterial peritonitis, where vg /vd t cell numbers and soluble inflammatory mediators were elevated in patients infected with hmb-pp-producing pathogens. conclusion: our findings suggest a direct link between invading pathogens, microbe-responsive gd t cells, and monocytes in the inflammatory infiltrate, which plays a crucial role in the early response and the generation of microbe-specific immunity. the mechanism(s) responsible for their dichotomous behaviour are poorly understood, and the outcome of nkt cell manipulation remains unpredictable. there is growing evidence that the nkt cell pool is composed of functionally distinct subsets, but such a possibility has not yet been investigated in a model of nkt cellmediated immunosuppression. we examined the differential ability of nkt cell subsets from the thymus and liver to prevent type i diabetes when transferred into prediabetic nod mice. the transfer of abtcr+dn thymocytes (a population enriched for nkt cells) has previously provided robust protection against tid development; however it has not been formally shown that nkt cells are solely responsible for the protection. our study found that while the transfer of thymic dn nkt cells can prevent tid and severe insulitis in nod mice, not all nkt cell subsets show the same tolerogenic capabilities. these findings both formally demonstrate the disease-preventing effects of nkt cell transfer in nod mice and provide further evidence that nkt cells are a functionally heterogeneous population. objective: vg /vd t cells constitute a minor t cell population in human blood that expands specifically and rapidly in response to the microbial metabolite hmb-pp. our previous microarray studies showed that vg /vd t cells stimulated with hmb-pp in the presence of il- express markers associated with a possible follicular b cell helper function. we therefore investigated in more detail whether and how hmb-pp and il- regulate expression of the b cell attracting chemokine cxcl /bca- , its receptor cxcr , and co-stimulatory molecules involved in b cell help. purified peripheral vg /vd t cells were co-cultured with autologous monocytes or b cells (as feeder cells) for up to days with and without hmb-pp, in the absence or presence of il- or il- , or in medium alone. cells were analysed by flow cytometry and immunofluorescence microscopy. results: high levels of cxcl protein were detected in co-culture supernatants only when both il- and hmb-pp were provided, implying an il- -dependent and tcr-dependent expression. vg /vd t cells were confirmed as producers of cxcl by flow cytometry and immunofluorescence. under the same conditions, activated vg /vd t cells expressed cd , cd , cd l, cd , icos and ox . in contrast, neither cxcr nor ccr changed markedly by il- stimulation of peripheral vg /vd t cells. conclusion: our findings confirm on the protein level that stimulation of vg /vd t cells with hmb-pp and il- induces markers typically associated with follicular b helper t (t fh ) cells. these data suggest that gd t cells contribute to humoral immune responses and play a role in germinal centre formation and production of high-affinity antibodies in microbial infection. ongoing analyses of gd t cells in inflamed and non-inflamed lymphoid tissues (tonsils, appendices) aim at demonstrating the physiological relevance of our findings. y. emoto , m. emoto gunma university school of health sciences, department of laboratory sciences, maebashi, japan invariant (i) natural killer (nk)t cells become undetectable after stimulation with a-galactosylceramide (a-galcer) or interleukin (il)- . although downmodulation of surface t cell receptor (tcr)/nkr-p c (nk . ) expression has been shown convincingly after a-galcer stimulation, it is unclear whether this holds true for il- stimulation. to determine whether failure to detect inkt cells after il- stimulation is caused by dissociation/internalization of tcr and/or nkr-p c or by block of de-novo synthesis of these molecules, and to examine the role of il- in disappearance of inkt cells after a-galcer stimulation, surface (s)/ cytoplasmic (c) protein expression as well as mrna expression of tcr/nkr-p c by inkt cells after stimulation with a-galcer or il- , and influence of il- neutralization on down-modulation of stcr/snkr-p c expression by inkt cells after a-galcer stimulation were examined. the s/ctcr + s/cnkr-p c + inkt cells became undetectable after in-vivo administration of a-galcer, which was partially prevented by il- neutralization. whereas s/cnkr-p c + inkt cells became undetectable after in-vivo administration of il- , s/ctcr + inkt cells were only marginally affected. mrna expression of tcr/nkr-p c remained unaffected by a-galcer or il- treatment, despite the down-modulation of ctcr and/or cnkr-p c protein expression. in contrast, ctcr + cnkr-p c + stcr -snkr-p c -inkt cells and cnkr-p c + snkr-p c -inkt cells were detectable after in-vitro stimulation with a-galcer and il- , respectively. our results indicate that tcr and nkr-p c expression by inkt cells is differentially regulated by signaling through tcr and il- r. they also suggest that il- participates, in part, in the disappearance of inkt cells after a-galcer stimulation by down-modulating not only snkr-p c but also stcr. the fetus and infant are highly susceptible to viral infections. a number of viruses, including human cytomegalovirus (cmv), cause more severe disease in early life compared to later life. it is generally accepted that this higher susceptibility to viral infections is due to the immaturity of the immune system. gd t cells are unconventional t cells that can react rapidly upon activation and show mhc-unrestricted activity. herein, we show that upon cmv infection in utero, fetal gd t cells expand and become differentiated. the response was restricted to vg -gd t cells, irrespective of their vd chain expression. differentiated gd t cells expressed high levels of ifn-g, transcription factors t-bet and eomes, natural killer receptors and cytotoxic mediators including perforin and granzymes. in addition, congenital cmv-infection induced a highly restricted complementary-determining region d (cdr d ) and cdr d repertoire, with a striking enrichment in a specific germline-encoded cdr d sequence. differentiated gd t cells and the enriched cdr d sequence were detected as early as after weeks of gestation. our results indicate that functional fetal gd t cell responses can be generated during development in utero and suggest that this t cell subset could participate in anti-viral defense in early life. results: spectratyping showed only in-frame selection for vd -jd and vgi-jg . / . rearrangements in tcrgd thymocytes and to a lesser extent in tcrgd cb cells. in contrast, clear in-frame vd -jd and vg -jg . selection was seen in pb tcrgd cells. detailed analysis of the cdr motifs revealed selection determinants in both vg -jg . (canonical length and cdr motif) and vd -jd (minimal cdr length in combination with an invariant t nucleotide) rearrangements. upon evaluation of the replication history we found a clear increase in the number of cell divisions from naïve tcrgd thymocytes (˚ ) and tcrgd cb cells ( - ) to tcrgd pb cells (˚ or more). no increase was seen between cb and pb tcrgd t cells within the first year of life, suggesting that peripheral proliferation occurs later in life. our results indicate that the human peripheral tcrgd repertoire is shaped by (antigenic) selection and proliferation processes. moreover, the ontogenetic changes in the gd repertoire between the central and peripheral immune systems are clearly influenced by proliferation. background: natural killer (nk) t cells have been implied in the regulation of disease in the non obese diabetic (nod) mouse model of type diabetes (t d). we have previously shown that transgenic expression of a cd d-restricted, va . -vb tcr in nod mice lead to an increase in cd d-restricted type ii nkt cells ( abnkt cells), and prevention of the development of t d in the transgenic mice. in this study we have investigated the requirements and underlying mechanism of disease protection by type ii nkt cells in a disease transfer model. to investigate the mode of regulation by abnkt cells, we explored a disease transfer model into nod.scid mice using transgenic diabetogenic bdc . cd + t cells, in the presence or absence of selected cells from abnkt cell transgenic mice. results: in ab transgenic mice a high frequency of activated transgenic nkt cells was found in the pancreas of the protected mice. in this organ, abnkt cells expressed a high level of cxcr and a low level of ccr and cd l, a pattern similar to that observed in t cells homing to inflammatory tissues. adoptive transfer of cd + bdc . t cells into nod.scid recipients rapidly induced onset of diabetes. using this model, we found that co-transfer of spleen cells from ab transgenic mice with bdc . cd + cells resulted in the prevention of diabetes development. the protection from disease required a minor cd + subset of ab+ nkt cells, but was independent of cd + t regulatory cells. analogs of alpha galactosylceramide (a-galcer) that may modulate the strong activation of inkt and at the same time prolong their effect upon in vivo administration are a long standing goal of research in this area due to their putative immunotherapeutical applications. a new class of non glycosidic analogues bearing an aminocyclitol ring as galactose surrogate have been synthesized and assayed in their capacity to be presented by cd d and recognized by inkt. the structural novelty of these compounds resides in the presence of a cyclohexane that substitutes the sugar moeity and the substitution of the o glycosidic linkeage with the ceramide by a n. in this basic structure, substitutions in the cyclohexane ring with oh in different conformations mimicking different sugars, differences in the length of the sphingosine lipid and differences in the orientation of the n linkeage conform a series of analogs that have been analyzed in their capacity to stimulate inkt cells. proliferation assays in bulk splenocyte cultures and cytokine secretion determinations show that inkt cells are specifically stimulated by some of the analogs tested. in particular, the active compound hs , induces in vitro inkt cell expansion and ifng and il- secretion in a similar fashion but less potently than a-galcer. dose response assays show a bias towards a th profile response after recognition by nkt cells, more similar to the response induced by och. the degree of structural similarity of the cyclitol ceramides with a-galcer parallels their cellular activities. these data open the way towards the development of a new class of a-galcer lipid analogues having charged amino substituted polar heads resistant to glycosidase degradation, thus enhancing their in vivo biodisponibility, and expands the range of potential inkt cell sphingolipid agonists that can modulate the immune response due nkt cell activation. objectives: invariant natural killer (ink) t cells represent an innate lymphocyte subset with important modulatory functions. in the presence of pathogens or tumors, inkt cells play an adjuvant function that boosts t cell immunity through cytokine secretion and dc maturation. in steady-state conditions, i.e. in the absence of pathogens, inkt cells acquire a regulatory function that promotes t cell tolerance and prevents autoimmune disease. our aim was to assess the mechanism of action of inkt cells in the steady state and, specifically, to test the hypothesis that inkt cells promote immune tolerance through modulation of dcs. methods: to assess the direct influence of regulatory inkt cells on dc maturation in resting conditions, we derived murine inkt cell lines in vitro and, after staining with agalcer-loaded cd d tetramers and magnetic purification, we tested their capacity to modulate bone marrow-derived myeloid dcs in the absence of any other maturation signals. we analyze the transcriptional profile (microarray analysis) as well as maturation, cytokine expression profile and pro-tolerogenic antigen-presenting function of inkt cell-modulated dcs (inkt-dcs). the cell-cell interaction with inkt cells provoked dramatic phenotypical changes on immature dcs that acquired the cardinal features of tolerogenic dcs such as intermediate levels of mhc class ii and co-stimulatory molecules expression and high secretion of il- with no release of pro-inflammatory cytokines. most importantly, inkt-dcs acquired tolerogenic antigen-presenting function inducing the differentiation of regulatory tr cells and immune tolerance in vivo. dcs, simultaneously stimulated with inkt cells and through toll-like receptor (lps) completely lost the pro-tolerogenic phenotype and acquired a proinflammatory cytokine profile. conclusion: it is still mysterious how inkt cells can play a dual role and either boost t cell immunity or promote immune tolerance. our results suggest that the same mechanism could underlie both inkt cell functions. in the presence of pathogen-driven maturation signals, the inkt cell-modulation of dcs favors their acquisition of a pro-inflammatory phenotype and function. on the contrary, if inkt cells are activated in the absence of pathogens, e. g. during autoimmune conditions, their interaction with immature dcs promotes their tolerogenic maturation to maintain peripheral tolerance and counter-regulate autoimmune diseases. th -type immune responses have been reported to fight extracellular bacterial infection, but as well to cause autoimmune diseases and allergy. the th immune response is characterized by the secretion of il- a and il- f. the il- locus encodes the highly conserved il- a and il- f cytokines that are syntenic in kb distance to each other. besides cd + th and nkt cells, approximately % of the il a producers are gd t-cells. like cd + th cells, il- producing gd tcells have recently been implicated to play a major role in the immune response to infections with extra-and intracellular bacteria. our findings show a difference between the il- production of gd t cells in the peripheral system and mucosal epithelia. mucosal gd t-cells generally do not produce th cytokines. in the periphery, we define novel subsets of gd t-cells that can produce either il- or ifn-g. combined with the well known classification of il- producing gd t-cells along the markers cd and cd , our data point at specialized functions of the different gd t cell subsets depending on their location and origin. functional studies are currently carried out in order to address the role of the different gd t-cell subsets for th -type immune responses in vivo. in this context, the potential redundancy of il- a and il- f may complicate the analysis. so far, most studies were carried out with il- a single-deficient or il- f single-deficient mice. to further clarify these issues, we will have to address the above mentioned findings in il- a and il- f double-deficient mice. several subsets of gd tregs have been described and intensively studied, but the potential regulatory role of innate t cells in controlling immune responses remains unclear. lymphocytes expressing gd tcr are involved in both innate and adaptive immune responses. vg vgd t cells, which represent a major peripheral blood gd t-lymphocyte subpopulation in humans, display a broad reactivity against microbial agents and tumors.here we report that tgf-b and il- differentiate in vitro a subset of gd t lymphocytes with regulatory functions (vd tregs) in the presence of specific antigen stimulation. these cells express the forkhead/winged helix transcription factor (foxp ) and, similarly to ab tregs, suppress the proliferation of anti-cd /anti-cd stimulated-pbmc. detailed knowledge about the phenotype and functionality of vd tregs will improve our understanding of the role of gd t cells in the pathogenesis and regulation of autoimmune, infectious and cancer diseases. a-galactosylceramide (a-galcer) has the potential to activate invariant (i) nkt cells, which in turn release a wide variety of cytokines that stimulate immunocompetent cells. although this rapid and vigorous cytokine release appears critical for regulation of various immune responses, it remains elusive whether protection against intracellular bacteria can be induced by a-galcer. here we show that treatment with a-galcer ameliorates murine listeriosis, and inhibits inflammation in the liver and spleen following listeria monocytogenes infection. liver infiltration of granulocytes and g/d t cells was accelerated by a-galcer treatment. granulocyte and g/d t cell depletion exacerbated listeriosis in a-galcer-treated mice, and this effect was more pronounced in granulocyte than in g/d t cell depletion. although secretion of gm-csf and il- was detected among the nkt cell population in the liver and bone marrow immediately after a-galcer treatment, infiltration of granulocytes into the liver was not prevented by neutralizing mab. yet, in parallel to the numerical increase of granulocytes expressing cd b in the liver following a-galcer treatment, numbers of cells lacking cd b diminished in the bone marrow. in addition, respiratory burst in granulocytes was enhanced by a-galcer treatment. our results indicate that a-galcer-induced antibacterial immunity is caused, in part, by accelerated infiltration of inflammatory cells, in particular granulocytes and to a lesser degree g/d t cells, into the liver. we also suggest that the infiltration of granulocytes is caused by an accelerated supply of granulocytes from the bone marrow, rather than by accelerated granulopoiesis. objectives: the aim of this work is to evaluate whether phenotypic and functional features of vgamma /vdelta t cells are influenced by the activity of mevalonate pathway in tumor cells and contribute to determine disease aggressiveness in cll. methods: eighty seven previously untreated cll patients were evaluated for in vitro vgamma /vdelta t cells expansion upon stimulation with zoledronic acid (za) and interleukin- (il- ). gammadelta t cells subset distribution and natural killer receptors profile were evaluated by multicolor flowcytometry. the mutational status of the tumor immunoglobulin heavy chain variable region (igvh) was analyzed by dna sequencing. the activity of the mev pathway was determined by ) the bioinformatic analysis of gene expression profiling data ) the quantification of mev pathway metabolites. results: proliferation of gammadelta t cells was observed in patients ( %) (responders, r), whereas patients ( %) were non-responders (nr). vgamma /vdelta t-cell subset distribution was well balanced in r patients, whereas effectors subsets [i. e., effector memory (tem), and terminally differentiated effector memory (temra)] were largely predominant in nr patients. temra of nr patients mainly expressed the inhibitory receptor ilt , whereas temra of r patients had an higher expression of the costimulatory molecule nkg d. the proliferative response of vgamma /vdelta t cells was significantly associated with igvh mutational status, which is a well known prognostic factor in cll. indeed, % of r patients were m, whereas % of um patients were nr (p x . ). given this association, we evaluated the activity of the mev pathway in tumor cells of m and um patients. the pathway was more active in tumor cells of um than m patients, suggesting that the former can more easily engage gammadelta t cells and drive their differentiation into functionally exhausted t emra . given the association between the r/nr status and the igvh mutational status we also analyzed the independent prognostic impact of r/nr status in multivariate cox analysis. nr patients had a significantly shorter time to first treatment thus pointing to r/nr status as an independent prognostic factor. conclusion: these data define a novel mechanism of immune escape which can contribute to determine disease aggressiveness in cll patients. the studies reported here were undertaken to ascertain and delineate the ability of kupffer cells to regulate the response of inkt cells to biliary obstruction. methods: c bl/ mice were not treated or rendered kupffer cell-depleted by intravenous inoculation of liposome-encapsulated dichloromethylene diphosphonate. to clarify the factors that elicit inkt cell activity, additional mice were administered anti-il- p (clone r - f ; atcc) or anti-cd- d (clone b ) monoclonal antibody (mab) prior to surgery. midline laparotomies were performed; the common bile duct was ligated twice and divided. sham-operated animals served as controls. blood and liver samples were collected at periodic intervals post-surgery. the hepatic lymphoid population was purified and characterized by flow cytometry. the nkt cell population was increased significantly in the livers of control, but not kupffer cell-depleted, mice at hours post-bdl. the response of inkt cells was diminished in mice pretreated with mab specific for il- p , a component of both il- and il- ; pretreatment with anti-cd d mab had no effect. il- rb-deficient mice also exhibited a marked increase in hepatic inkt cells following bdl suggesting that il- was not a critical factor. this suggestion is supported by the increased expression of il- p and il- p (but not il- p ) mrnas by kupffer cells purified from the livers of bdl animals. these findings imply that il- production by kupffer cells promotes the response of hepatic inkt cells to biliary obstruction. objectives: p-glycoprotein (pgp or abcb ) is a member of the abc family of transporter proteins which are characterized by their ability to pump molecules across membranes in an atp-dependent manner. although pgp was first identified for its ability to confer resistance to chemotherapeutic agents in tumor cells, it has now also been described in cells of the immune system. our work primarily focuses on gd t cells that complement and regulate the activities of ab t cells, particularly in tissues. we have recently described functional subsets of gd cells based on cd expression. gd + cells secrete interferon-g, while gd cells are capable of producing il- . this study investigates the role of pgp in gd cells with specific reference to these recently-identified cd -defined subsets. methods: pgp activity was measured based on the expulsion of rhodamine . cells were incubated with rho followed by a period in the presence or absence of the pgp inhibitor cyclosporine-a. cell populations were identified using monoclonal antibodies and flow cytometry. percentages of subpopulations were compared by anova, statistical results are shown as p values that were calculated using a newman-keuls multiple comparison post-hoc test. results: up to % of intraepithelial lymphocytes (iels) from the small intestine are tcrgd + . of these, virtually all displayed pgp activity. indeed, pgp activity was generally higher in tcrgd + than tcrab + iels. in the thymus, pgp activity was observed in only˚ % of gd + cells but not at all in gd cells. by contrast, in peripheral lymph nodes, mesenteric lymph nodes and peyer's patches, - % of gd + cells were positive for pgp activity, although their gd counterparts remained largely negative (p x . ). conclusion: this study demonstrates that subsets of gd cells display different levels of pgp activity depending on their location in the body and their expression of the newly identified functional marker cd . as pgp activity may play a role in cytokine release, cytotoxicity and protection from harmful toxins, it confirms our hypothesis that gd + and gd cells have very different roles in immune responses and provides insight into the mechanism by which gd cells cope with diverse body locations. objectives: an effective immune response orchestrates different cellular activities of both innate and adaptive immune compartments. in this context, the vgamma vdelta t cell biology presents some critical features for their ability to display a broad antimicrobial activity by directly killing infected cells and by inducing an effective adaptive immune response. the activation of vgamma vdelta t cells by aminobisphosphonate drugs such as zoledronic acid (zol) results in a massive release of cytokines and chemokines that may induce a bystander activation of other immune cells such as dendritic cells (dcs) and b lymphocytes. the aim of this work was to evaluate the ability of activated vgamma vdelta t lymphocytes to orchestrate granulocytes functions in terms of migration capability, phagocytic activity and alpha defensin release. methods: peripheral mononuclear cells (pbmc) and purified vgamma vdelta t cells from healthy donors were stimulated with different compounds (zol, ipp) for hours and supernatants from these cultures were tested for their ability to induce granulocytes activation. briefly, we analysed the migration activity, the phagocytic activity and the degranulation process by perforimg migration assays, flow cytometry and elisa tests. we showed that soluble factors released by zol-stimulated vgamma vdelta t cells activate granulocytes by inducing their chemotaxis, phagocytosis, and alpha-defensins release. proteomic analysis allowed us to identify a number of cytokines and chemokines specifically released by activated vgamma vdelta t cells. moreover, mcp- depletion by neutralizing ab revealed a critical role of this chemokine in induction of granulocyte alpha-defensins release. altogether, these data show a vgamma vdelta -mediated activation of granulocytes through a bystander mechanism, and confirm the wide ability of vgamma vdelta tlymphocytes in orchestrating the immune response. conclusion: an immune modulating strategy targeting vgamma vdelta t cells may represent a key switch to induce an effective and well-coordinated immune response, and can be proposed as a way to strengthen the immune competence during infectious diseases. objectives: the aim of this study was to analyse the activity of vg vd t lymphocytes against glioma cells and to verify the possibility to target these innate cells in new immunotherapeutic approaches. human vg vd t cells recognize and kill several cancer cells presenting a disregulation in mevalonate pathway. interestingly, drugs already in clinical use, such as zoledronic acid, are able to promptly activate vg vd t cells through an indirect mechanism involving the block of farnesyl pyrophosphate synthase of the mevalonate cycle. the vg vd t cell activation by zoledronic acid results cytokines and chemokines synthesis and cytotoxic activity. glioma are tumors arising from glia in the central nervous system. unfortunately, the majority of glioma patients die in less then of a year from diagnosis and new treatment strategies are therefore hardly needed. methods: in order to analyse the activation of vg vd t cells and their effects on the viability of glioma cells, we expanded in vitro vg vd t cells from pbmcs of healthy donors by using phosphoantigen stimulation and tested the ability of vg vd t cell lines to kill three different glioma cell lines (t , u , u ) by cytokinic/cytotoxic mechanism by flow cytometry. results: our results demonstrated that vg vd t cells lines are able to recognize glioma cells, to differentiate in effector memory cells, and to kill glioma cells by releasing perforin. moreover, we analysed whether zoledronic acid treatment could improve the susceptibility of glioma cells to vg vd t lines. we showed that zoledronic acid is able to directly induce cell death on glioma cells and to strongly enhance the cytotoxic activity of vg vd t lines. conclusions: altogether, our results suggest that the induction of a strong antitumor response in vitro of vg vd t cells by using aminobisphosphonates could represent a new interesting immunotherapeutic approach for glioma treatment. viral-induced cancers, such as cervical cancer and liver cancer, contribute to approximately % of all cancers and represent a failure of host immunity to control chronic viral infection. natural killer t (nkt) cells are a population of regulatory t lymphocytes that are pivotal to the outcome of host protection to a range of viral infections and cancers, but their role in controlling host defenses to oncogenic viruses in epithelial and cutaneous tissue is virtually unexplored. using a mouse model of chronic viral infection in the skin, in which human papillomavirus (hpv) oncoproteins are expressed as a transgene in epithelial cells, we investigated the role for nkt cells in abrogating protective immunity to viral antigens in cutaneous tissue. we show that local hpv-e protein expression in the skin attracts a large lymphocytic infiltrate, including a population of cd d-restricted nkt cells. this nkt infiltrate is required to maintain local hpv-e -induced immune suppression and results in graft survival when transplanted onto a naive, immunocompetent host. the local suppressive environment evident in e -expressing transplanted skin is dependent on interactions between populations of cd d-expressing cd c+/f + myeloid cells and nkt cells. removal of either donor-resident or host-infiltrating nkt cells is sufficient to break immune suppression and allow e graft rejection. dissecting the suppressive properties of nkt cells in this novel model of chronic viral antigen presentation in the skin will provide valuable new insight into the potential for clinical manipulation of nkt cell populations to restore chronic anti-viral and anti-tumour immunity in epithelial tissues. nkt cells were expanded from total pbmcs from healthy donors by treatment with il- and a-galcer. expression of cd a, cd d and the costimulatory molecules cd and hla-dr, was established by flow cytometry. rna was quantified by real time-pcr. functional assays were performed by analysis of nkts cytokine production (ifn-g, il- ) and cytotoxicity against treated-idcs. results: idcs stimulated with olive pollen lipids up-regulated cd d expression on the cell surface in comparison with control cells. in contrast cd a expression was decreased. cd and hla-dr slightly increased, indicating certain grade of maturation. the amount of cd d mrna was higher in treated cells than in control cells. by contrast, there was less transcription of cd a, cd b and cd c genes than in control cells. nkt cells efficiently killed treated idcs as "in vitro" cytotoxic killing assays showed. ifn-g producing cells increased slightly in response to treated idcs compared to unstimulated cells, but the number of il- producing cells was not modified. similar results were obtained using monocytes as antigen-presenting cells. conclusions: idcs treated with lipidic extracts from olive pollen up-regulate the expression of cd d on the cell surface. in addition, nkt cells are able to recognize idcs and monocytes treated with lipids from pollen, producing ifn-g and cytotoxicity. all these data suggest that nkt cells may play a role in the control of the immune response to allergens, such as the lipids present in pollen grains. outline: in humans, . - % of circulating lymphocytes express a vg vd t cell receptor, yet strikingly little is known about the function and properties of such unconventional t cells. we performed cdna microarrays to find vg -enriched genes compared to conventional mhc-restricted cd + ab t cells, and found reciprocal enrichment of nectin-like adhesion molecules igsf & crtam in gd and ab t cells respectively. because igsf binds to crtam, the data fuel a hypothesis that this may be a novel axis of communication between the two cell types. interestingly, previous studies show that activated nk, nkt and cd + ab t cells express crtam, and that engagement of igsf on epithelial cells renders the latter targets for enhanced cytolytic and cytokine responses. our data extends this to the prospect of cytolytic immunoregulatory interactions between t cells mediated by igsf /crtam. we therefore sought to answer: . what is the function of igsf /crtam on gd t cells? . how is the igsf -crtam axis regulated in t cells? results and conclusions: flow cytometry showed igsf enrichment on resting gd t cells, with expression also detected on˚ % of ab t cells. the properties of those cells are being examined. however, igsf generally correlates with markers of activation/antigen experience such as cd ro. thus, igsf cells may comprise activated-yet-resting/pseudo-memory unconventional t cells and memory-effector conventional t cells. stimulating vg + t cells in vitro led to rapid crtam induction, resulting in the majority of cells co-expressing both igsf and crtam within hours. however, engagement of igsf by crtam or vice versa is not sufficient to induce cytotoxicity, as stable cho cell transfectants expressing either molecules were not specifically lysed by pbmc in vitro, compared to efficient and parallel targeting of mica + cells. instead, our current experiments address the possibility that crtam-igsf may regulate cytotoxic interactions promoted by other receptor-ligand interactions, such as mica-nkg d. this may explain why cells can tolerate co-expression of both molecules, and would refute the hypothesis that crtam-igsf interactions are sufficient for cd t cells to kill gd t cells and/or vice versa. instead, crtam-igsf interactions may set the threshold for cytotoxic immune-surveillance responses. t cell receptor (tcr) is a multisubunit complex in which the invariant subunit cd z is a kda transmembrane protein indispensable for coupling antigen recognition by tcr to diverse signal transduction pathways. approximately - % of human peripheral blood lymphocytes express the gd tcr and the majority of these cells express the vd tcr variable segment associated with the vg segment, and recognize phosphorylated non-peptidic metabolites from microbial or self origin. these compounds trigger vg vd t cells without antigen presentation. in vitro stimulated vg vd t cells with antigens are able to produce ifn-g and tnf-a and exert a powerful cytotoxic activity against infected cells as hiv-infected cells. however, during hiv infection a marked decrease of vg vd t cells was observed and the remaining cells are unable to respond to their non-peptidic ligands. aim of the present work was to study the mechanisms of vg vd t cell anergy observed in hiv+ patients. to this aim, cd z expression and ifn-g production by vg vd t cells from hiv+ and hiv-subjects were analyzed. we show that vg vd t cells from hiv-infected patients expressed lower level of cd z compared with healthy donors. a direct correlation between cd z expression and ifn-g production capability by vg vd t cell was found. however, pkc activation by pma is able to restore cd z expression and ifn-g production. our findings may contribute to clarify the molecular mechanisms of vg vd t cell anergy found in hiv+ patients and have implication in the design of effective immune-based therapies. l. abeler-dörner , m. swamy , s.l. clarke , a. hayday king's college london, immunobiology, london, united kingdom gut intraepithelial lymphocytes (iel) constitute one of the largest t cell compartments in mice and in man. their functions and their interactions with surrounding epithelium are likely to be crucial to the fine-tuned balance between tolerance to harmless food antigens, immunity to gut-associated pathogens, and overall intestinal immune surveillance. intestinal iel comprise many unconventional t cells including tcrgd cells and tcrab cd aa or cd -cd cells, which have been assigned innate-like immune functions and key roles in surveillance of stressed tissue. unlike conventional t cells, iel might initiate an immune response rather than simply being late effector cells. it is therefore important to elucidate the "immunological information flow" in the gut. to this end, this project characterizes different subsets of iel and their interactions with epithelium in steady state and under immunostimulatory conditions in vitro and in vivo. in the past, it has been notoriously difficult to study iel ex vivo. to solve this problem, we developed a novel culture system that allows us to expand the cells ex vivo and study their responses for up to days. the cells are initially activated by plate-coated acd antibody and a cytokine cocktail and maintained further in medium containing low levels of il- . after a resting period, the cells can be restimulated in vitro. in this new system, we studied responses of different iel subsets to stimulation via tcr, nkg d and cytokine receptors, either alone or in coculture with epithelial cells. as readouts we monitored proliferation, cytokine secretion (ifng, il- ) and expression of activating and costimulatory molecules. reactivation in response to various stimuli could already be observed after hours. the in vitro data set forms the basis for analysing iel responses in vivo to stimulatory molecules ectopically expressed as transgenes in the gut. the characterization of iel responses opens new insights into the nature of gut immune responses and should provide a better understanding of the immunology of inflammatory bowel diseases which still remain a major problem in the clinic today. objective: behcet's disease (bd) is a multisystemic disorder with a possible underlying pathology of immune-mediated vasculitis. increased expression of cd in bd patients suggested that nk receptors may play a pathogenic or regulatory role in the pathogenesis. considering the regulatory functions of nkg molecules in heterodimer with cd , we screened the presence of these receptors on t cell subsets in bd. the expression of nkg a/c/d molecules on gd and cd + t cells were analyzed in active and inactive patients with bd and healthy controls. expression of nkg molecules was evaluated on cd +, gd t and cd + nk cells by using flow-cytometry. results: gd t cells were increased in patients with bd compared to controls ( . vs. . %, p= . ). in addition to the increase of gd t cells, increased expression of activating nkg c molecules was also observed on gd t cells ( % vs. %, p= . ). nkg a expression on gd t cells was found to be higher than nkg c expression in patients and controls; but nkg a expression on the t cells was not statistically different in both groups ( . vs. %). nkg d receptors were present on most of the gd t cells in both groups. however these activating molecules on cd + cells were decreased in patients with bd compared to controls ( revlimid is a therapeutic agent used to treat myelodysplastic syndrome (mds), a group of haematological disorders characterised by ineffective haematopoiesis. the mechanism of action for revlimid is poorly understood, but there has been increasing interest in the strong association reported between mds and defects within the immunoregulatory nkt cell compartment. indeed, some studies now suggest an important outcome of revlimid treatment is the restoration of normal cytokine production by nkt cell levels and an increase in their overall numbers. we have conducted the most thorough study to date of the nkt cell compartment of mds patients treated with revlimid/ancestim and can report that mds patients had normal nkt cell levels prior to treatment, and no significant increase as a result of revlimid/ancestim treatment. furthermore, nkt cells from mds patients produced high levels of th and th cytokines when stimulated with pma/ ionomycin and the proportion of nkt cells capable of cytokine production did not increase significantly after revlimid/ancestim treatment. these are highly significant findings given the recent emphasis on nkt cells as a potential therapeutic target for mds. our study provides an extensive analysis of the impact of revlimid/ ancestim treatment on the nkt cell compartment and sheds new light on the role of nkt cells in mds and the mechanism of revlimid immunomodulation. objectives: human gd t cells are potent killers of a variety of tumour cell lines, and mice lacking gd t cells suffer from high incidence of experimentally-induced tumours. however, the molecular mechanisms mediating tumour cell recognition by gd t lymphocytes remain largely unknown. we aim at identifying potential tumour antigens and co-stimulation molecules expressed in ex vivo tumours and in tumour cell lines that activate human gd t cells for tumour cytolysis. as immune evasion mechanisms that down-regulate tumour antigens may operate in vivo, we have identified candidates from human tumour cell lines of hematopoietic origin that constitute in vitro cytolysis targets for vg /vd + lymphocytes. we have screened a panel of lymphoma and leukaemia cell lines using a conventional in vitro killing assay using vg /vd + cells, and selected two susceptible ("target") cell lines (over % death in the assay) and two vg /vd + resistant ("non-target") cell lines (under % death) for cdna microarray analysis. we compared the differential expression in pairs of tumour cell lines of identical origin: the burkitt's lymphoma cell lines daudi (target) vs raji (non-target), and the pre-b cell leukemia cell lines rch-acv (target) vs (non-target), and validated the results by rt-qpcr quantification. results: we identified commonly up-regulated and commonly down-regulated genes that encode cell membrane-associated proteins in susceptible tumours. ulbp , ifitm and prame, for example, are up-regulated, whereas cd and clec d are down-regulated in target cell lines. as these encode membrane-bound proteins with relevant functions in tumour immunity, they constitute potential ligands for gd lymphocyte recognition of tumour cells. the expression of these candidate genes was studied by rt-qpcr in a broader panel of cell lines and primary biopsies. we are currently testing, in functional assays based on rna interference and overexpression, these and other candidate genes in order to determine whether they provide activating or inhibitory signals to gd t cells. the comparison between the transcriptomes of vg /vd + target versus non-target cell lines allowed the identification of candidate genes, whose individual function we are currently dissecting, that may be involved in tumour cell recognition by human gd t cells. mice and humans are the only species in which phenotype and function of inkt cells have been properly described. our aims are to directly identify this cell population and to investigate cd d, in the rat. mice and rats have very similar cd d and inkt tcr genes, with the exception of the va gene segment, which is a multimember gene family in the rat. novel monoclonal antibodies with nearly identical binding capacities to mouse and rat cd d revealed a very similar pattern of cd d distribution, and could inhibit cytokine production after agalcer stimulation of primary cells in both species. response to agalcer was studied in five different rat strains, showing big inter strain differences. notably, ifn-g and il- production was - fold lower in the best responder rat strain (f ) compared to mouse (c /bl ). since nkrp a (rat homologue of mouse nk . ) and tcr are not appropriate markers for rat inkt, cd d oligomers where tested for binding to inkt-tcr transduced cells. newly generated agalcer loaded rat cd d dimers, recognized rat inkt tcr and, although less efficiently, bound to mouse inkt tcr. however, mouse cd d agalcer dimers did not bind to rat inkt tcr. agalcer loaded rat cd d dimers were then used to stain primary intrahepatic lymphocytes. but, although mouse inkt cells were stained to some extent, the identification of a discrete population in the rat was not possible. the reasons behind could be: that the avidity of the dimers for the tcr is not high enough to stain primary cells and/or that the frequencies are so low that the detection by facs analysis is difficult. in order to clarify these issues we currently produce and test rat cd d tetramers. burkholderia pseudomallei is a highly virulent bacterium which causes the potentially fatal disease melioidosis in humans. this disease is endemic in tropical regions, especially thailand and northern australia, and has a serious outcome for many infected individuals. b. pseudomallei is an intracellular bacterium and many b. pseudomallei strains are resistant to antibiotics so antibiotic treatment is aggressive and relapse of the disease is frequent. in addition to this, no vaccine is currently available to prevent the disease. human g d t cells are involved in the immune response to infection with a number of intracellular pathogens including brucella suis and mycobacterium tuberculosis. g d t cells respond to non-peptidic phosphorylated molecules known as 'phosphoantigens' which are byproducts of essential metabolic pathways in both bacteria and mammals. phosphoantigens cause expansion and activation of g d t cells during infection with intracellular pathogens including fransicella tularensis and m. tuberculosis. analogues of natural phosphoantigens have been developed to manipulate g d t cell responses as a cancer therapeutic and are currently in clinical trials for the treatment of hepatitis c virus. we aimed to determine in vitro whether enhancing gd t cell responses in human blood using the synthetic phosphoantigen picostim could reduce growth of intracellular b. pseudomallei in the human monocytic cell line thp- . a significant (p x . ) reduction in intracellular bacterial numbers was observed (n= ) in the presence of pbmcs cultured with picostim+il- in comparison with pbmcs cultured with il- or media alone. picostim+il- caused significant expansion and activation of gd t cells following culture of pbmcs for - days. purified gd t cells stimulated with picostim were able to reduce intracellular b. pseudomallei numbers -fold. this data demonstrates that pbmcs, stimulated with the synthetic phosphoantigen picostim+il- , reduced growth of intracellular b. pseudomallei in a gd t cell-dependent manner. objectives: vgamma /vdelta (gd) t cells play a major role in innate immunity against microbes, stressed and tumor cells. they represent less than % of peripheral blood lymphocytes (pbl), but can be expanded in vitro by zoledronic acid (za)-treated monocytes or dendritic cells (dc).the purposes of this study are: ) to determine whether dc generated from multiple myeloma (mm) patients are as effective as their normal counterparts in the ability to activate gd t cells; ) to evaluate whether gd t cells can exert immunoadjuvant activity on dc generated from mm patients and primed with tumor-specific antigens (survivin-sv); ) to establish whether the same issues could be solved using a simplified protocol of dc generation. ) dc were generated from cd + cells of healthy donors/mm patients; immaturedc on day were induced to fully mature by incubation for hours with tnfa + il- b + pge in the presence or absence of mm za. after days of co-culture dc:pbl, percentages and total counts of gd t cells were determined by flow cytometry; ) idc generated from cd + cells of hla-a* + healthy donors/patients were pulsed with sv-peptide and stimulated for hours with tnfa + il- b + pge in the presence or absence of mm za; after rounds of autologous t cells stimulation by dc, the frequency of sv-specific cd + t cells was determined by svpentamers staining; ) the same experiments were performed both with dc generated following a standard protocol and a h protocol (dc fast objective: depletion of or deficiency in gd t cells aggravate colitis in different animal models. additionally, reconstitution of mice with syngeneic gd t cells ameliorated chemically-induced colitis indicating a suppressive or regulatory role for murine gd t cells in intestinal inflammation. therefore, we asked whether human gd t cells possess also suppressive or regulatory potential, which could be of therapeutical use in chronical inflammatory diseases such as ulcerative colitis or crohn's disease. hence, the proliferation, suppressive activity, and cytokine profile of human peripheral gd t cells were determined in vitro. methods: human gd t cells were isolated from whole blood of healthy donors by macs technology. the proliferation was determined by [ - h]-thymidine incorporation, while suppression of responder cell proliferation was measured by flow cytometry via cfse fluorescence intensity. the cytokine profile was determined by elisa from culture supernatants as well as by flow cytometry intracellularly. finally, the in vitro characteristics of gd t cells were compared to those of cd + cd + regulatory t cells (treg). human peripheral gd t cells show suppressive activity against responder cell proliferation, though being themselve anergic, that is, they produce negligible amounts of interleukin- on stimulation and proliferate poorly. while the proliferation of gd t cells and treg cells is comparable, the suppression of gd t cells on responder cell proliferation is even stronger than the suppression by treg cells though gd t cells being foxp negative. additionally, gd t cells are strong producers for tgf-b, particularly by the vd subset. conclusion: human peripheral gd t cells possess regulatory potential and could be of therapeutical use in treatment of chronical inflammatory diseases as they are anergic and act suppressive. their suppressive activity is even superior to treg cells and might be due to strong tgf-b secretion. for application of human gd t cells in therapy their expansion under maintenance of their regulatory properties should be elucidated. there are previous descriptions of gamma-delta t lymphocytes (gd) from behçet's disease patients (bd) but, in most of cases, they are incomplete or contradictory. it has been suggested that nkg d on gd is involved in bd lesions through interaction with mica molecules. furthermore gdcd + have been recently proposed as a new regulatory t subset (treg). objectives: to study gd phenotype in bd active (bda) (n= ) and inactive (bdna) (n= ), versus healthy controls (hc) (n= ) and patients with recurrent oral ulcerations (ru) (n= ). to determine gd cytokine profile and surface markers treg-related in bd (n= ) and hc (n= ). methods: we obtained mononuclear cells from peripheral blood (pbmc). we determined by flow cytometry: -surface expression of: gd tcr, vdelta , vdelta , cd alpha, cd beta, nkg d, nkg a and cd . -intracellular expression of ctla- , and foxp . -intracellular expression of il- , il- , ifngamma, il- and tgfbeta after pbmc polyclonal stimulation. we used two tailed test for means comparison (mann-whitney u or student's t test). -vdelta + cells were significantly increased in ru. vdelta + and gdcd + lymphocytes were significantly increased in bd versus ru and hc. -the mean fluorescence intensity of nkg d was slightly increased in gd from bda. -nkg a expression by gdcd + was not different in bd versus hc. -most of gdcd + presented cd alpha-alpha homodimers in bd and hc and were negative for cd , foxp and ctla- . gdcd + and gdcd -subsets were (in bd and hc): -high ifngamma-producers without differences. -low il- -producers: il + cells were lower in gdcd + than in gdcd -. -low il- -producers: il + cells were lower in gdcd + than in gdcd -. -low tgfbeta-producers: tgfbeta+ cells were lower in gdcd + than in gdcd --very low producers of il in most of cases. the hallmark in bd was the increase of gdcd /vdelta +. this subpopulation has recently been described as immunosuppressive in infiltrates of human tumours and its function related to nkg a in intraepithelial intestinal lymphocytes from celiac patients. we did not find a cytokine profile or a phenotype t-reg-related for gdcd +, except a lower percentage of il- + cells than in the gdcd -subset. gdcd + from bd did not show significant differences versus hc. natural killer t (nkt) cells comprise a highly heterogeneous subset of t lymphocytes that co-express a t cell receptor (tcr) and nk cells markers such as cd in humans. a subgroup, the invariant nkt cells (inkt), expresses the va vb tcr rearrangement representing a minority subset in peripheral blood and virtually absent in the newborn. objectives: to establish a method to growth cord blood-derived nkt cells (cd + cd + ), in order to evaluate their phenotypic characteristics and the tcrvb repertoire. methods: mononuclear cells were isolated from healthy umbilical cord blood samples and stimulated with ifn-g ( ng/ml), anti-cd ( ng/ml) and il- ( ui/ml). these cells were cultured for days and the expanded cd + cd + cells were isolated by immunomagnetic methods. surface markers were determined by flow cytometry. total rna was extracted from the purified cd + c + cell suspension using trizol ® reagent and mrna expression of twenty tcrvb gene families was measured by semiquantitative rt-pcr. statistical analyses were performed using mann-whitney u test and one-way anova, a p value of x , was considered significant. results: we could significantly expand cord blood cd + cd + nkt cells from , ± , % to achieve an enrichment of , ± , % (p= , ). table shows the percentage (mean±sd,n= ) of phenotypic markers in cd + cd + cells at baseline (day ) and after days of culture. expression of mrna for the vb families studied was confirmed in each individual cell culture with a significant high expression of vb and vb families (p x , ). conclusion: our results show that cord blood-derived nkt cells are mainly cd + and cd + subsets, similar to peripheral blood nkt cell with a low percent of inkt cells. additionally, we confirm a diverse tcr vb repertoire with a significant expression of the vb and vb families in these cells. l. marischen , d. wesch , p. rosenstiel , a. till , d. kabelitz institute of immunology, kiel, germany, institute of clinical molecular biology, kiel, germany gd t cells account for a minority of t cells in human blood, but represent the majority of intraepithelial t cells in the intestinal tract. due to their ability to respond rapidly and in an mhc-independent fashion to particular antigens by cytokine production, gd t cells are considered as a link between innate and adaptive immunity. in addition, the expression of distinct pattern recognition receptors such as toll-like (tlr) and nod-like receptors (nlr) are characteristic for cells of the innate immune response. recent reports have demonstrated the tlr expression in human and murine gd t cells. here we provide evidence also for a gd t cell responsiveness to muramyl dipeptide (mdp), the putative ligand of the nlr family member nod . peripheral blood mononuclear cells (pbmcs) containing gd t cells as well as freshly isolated gd t cells were stimulated via the gd t cell receptor in the absence or presence of mdp and analyzed for proliferation and ifng-production. while the proliferation of gd t cells within pbmcs was decreased, ifng-production was increased after costimulation with mdp compared to the stimulation with a non-activating dd-stereoisomer of the ligand (mdpi). the enhanced ifng production of pbmcs after costimulation was mediated mainly by gd t cells as shown by intracellular flow cytometric staining. with regard to the ifng-production after co-stimulation with mdp vs. mdpi, freshly isolated gd t cells from different healthy blood donors can be divided into responder and non-responder. responder gd t cells showed a significant increase of the ifng-production due to mdp-stimulation, whereas ifng-production was not influenced in non-responder gd t cells. in further experiments, as first approach to explain the different reactivity patterns of gd t cells, it is planned to analyze the polymorphisms of the nod gene in various donors. taken together, our preliminary data indicate that gd t cells are a major source of ifng-producing cells among pbmcs when challenged with specific antigens plus mdp, and support the role of gd t-cells as an important team player in the early immune response against bacteria. objectives & methods: an increasing of gamma-delta t cells during acute p. vivax infection and convalescent period has been reported. moreover, the activation of gamma-delta t cells leads to the inhibition of blood stage p. falciparum parasites in vitro. to determine the killing mechanisms of p. vivax parasites by gammadelta t cells comparing with what has been found in p. falciparum, the gamma-delta t cells were enriched by isopentenylpyrophosphate (ipp) from naïve pbmc. different number of gamma-delta t cells and normal pbmc were incubated with intact of p. vivax parasites and protein extract of p. vivax parasites, recombinant pvmsp and pvama proteins. gamma-delta t cells was daily determined the cytokine and granzyme intracellular releasing by flow cytometry until day culturing. results: among the enriched gamma-delta t cells, the percentage of cells expressing cd + and cd + was elevated after co-culturing with intact and the proteins of p. vivax parasites. the overall gamma-delta t cells showed proliferation at day after the co-cultivation. moreover, the gamma-delta t cells expressing ifngamma + and cd a + (lysosomal associated membrane proteins: lamp- ) elevated from the first day of pbmc collection after co-culturing with the intact and p. vivax antigens. this level was correlated with the significantly decreasing number of parasites and the increasing percentage of parasite growth inhibition. our results showed the activation of gamma-delta t cells during p. vivax infection in vitro. this suggests that gamma-delta t cells could be stimulated by p. vivax parasites and these actively activated gamma-delta t cells could kill the parasites via mechanism of granzyme and cytokines at the early stage of cell activation. this study provides more understanding in activation of the innate immunity during acute malaria infection which may lead to the selection of appropriate malaria proteins as vaccine candidates in the future. objectives: several evidence suggest that invariant nkt cells (inkt) connect innate and acquired immune system. they are able to produce both th and th cytokines after stimulation. atopic dermatitis (ad) is a chronic inflammatory skin disease. th -like and th -like cytokines have been implicated in the pathogenesis of ad, but there are controversial data on their role in ad. the frequency and absolute number of inkt cells in mononuclear cells (pbmcs) of peripheral blood of patients with atopic dermatitis (ad) (n= ) and healthy controls (n= ) were determined by flow cytometry using anti-cd and monoclonal antibody specific for the cdr loop of the invariant tcr a chain of inkt cells (clone: b ). furthermore, after pma/ionomycin stimulation for hours, intracellular ifng and il- cytokines were detected in cd +cd -, cd -cd -(dn), cd -cd + and cd +cd + subsets of inkt cells by five colour flow cytometry in patients with ad (n= ) and healthy controls (n= ). results: both frequency and absolute number of inkt cells were significantly lower in patients with ad (p x . ) compared to healthy controls. the frequency of dn subpopulation was significantly lower in ad patient (p x . ). there was a positive correlation between the frequency of dn cells and inkt cells both in ad patients (r= . and p x . ) and healthy controls (r= . and p x . ). in the intracellular ifng level there were no significant difference in any of the inkt subsets of ad patients, however the intracellular il- level was significantly higher in dn subpopulation of inkt cells of ad patients compared to healthy controls (p x . ). the frequency, the number of inkt cells and the cytokine producing capacity of the cd /cd inkt subsets are different in peripheral blood obtained from ad patients compared to healthy controls. our result suggest that the dn inkt cell subset can serve as a source of il- that promotes the th differentiation in ad patients and might play a role in the pathogenesis of this disease. introduction: intrahepatic immune cells (ihic) are known to play central roles in immunological responses mediated by the liver, and isolation and phenotypic characterization of these cells is therefore of considerable importance. aims: in the present investigation, we developed a simple procedure for the mechanical disruption of mouse liver that allows efficient isolation and phenotypic characterization of ihic. these cells are compared with the corresponding cells purified from the liver after enzymatic digestion with different concentrations of collagenase and dnase. results: the mechanical disruption yielded viable ihic in considerably greater numbers than those obtained following enzymatic digestion. the ihic isolated employing the mechanical disruption were heterogeneous in composition, consisting of both innate and adaptive immune cells, of which b, t, natural killer (nk), nk t cells, granulocytes and macrophages were the major populations (constituting . %, . %, . %, . %, . % and . % of the total number of cells recovered respectively). the ihic obtained following enzymatic digestion contained markedly lower numbers of nk t cells ( . %) . the b, t and nk t cells among ihic isolated employing mechanical disruption were found to be immunocompetent, i. e. they proliferated in vitro in response to their specific stimuli (lipopolysaccharide, concanavalin a and alpha-galactosylceramide respectively) and produced immunoglobulin m and interferon-gamma. conclusions: thus, the simple procedure for the mechanical disruption of mouse liver described here results in more efficient isolation of functionally competent ihic for various types of investigation. nature killer t cells (nkt) are a special t cell population with co-expresses nk and t cell surface markers. murine nkt cells include cd + nkt and cd -cd -nkt cells. nk . + nkt cells may release large amounts of il- , il- , ifn-g and il- after they are activated. it has been reported that a-galactorsykeramide (a-galcer), a glycolipid, may induce proliferation of nkt cells with the role of immune regulation by stimulating mouse spleen cells. this study demonstrated that superantigen staphylococcal enterotoxin b (seb) , a kind of peptide, can activate the nkt cells with the function of immune tolerance. the response ability of seb-activating effect cells to cona, lps and il- had significantly decreased compared with that of normal lymphocytes. the effect cells exerted an inhibitory effect for the response of normal lymphocytes to cona and il- . there was a significantly increase in the percent of cd + nk . + and tcrvb + nk . + nkt cells identified from the seb-activated cells. based on the cell distribution detected in the upper part of the facs picture, expression of cd molecule existed in . % of the cells from large-scale selection. the percent of cd + nk . + and tcrvb + nk . + nkt cell subsets in the giant lymphocytes were enhanced to . and . folds, respectively. under a light microscope at x magnification, the seb-activating lymphocytes in size were larger than not only the cona-activated cells but also the adherent macrophages with an increase of fold observed under a microscope. there were a few granules seen in cytoplasm. the value of cytoplasm vs nuclei was less than . and they are non-adherent cells. the differentiation pathway of the seb-activating cd + and tcrvb + nkt cells was not relative to a nk source. they were produced directly from t cell population and were considered as a subsets of t lymphocytes. our results suggest that the superantigen seb can act on the cd + nkt cell and tcrvb + nkt cells. and the two nkt cell subsets may play a critical role in seb mediated tolerance. gd t cells in the intestinal intraepithelial compartment (gd iiel) show an intrinsic activated phenotype. we hypothesised that their t cell receptor gd (tcrgd) is implicated in the activation of gd iiel. because the tcr gd ligands in mice are not well described, monoclonal antibodies (mab) directed against the gd tcr, like the clone gl which binds the d subunit of tcr gd, are important tools to specifically activate gd t cells. using cytometric indo- am measurement, we could detect calcium flux of intestinal and peripheral gd t cells from tcrd-h begfp reporter mice. stimulation with anti-gd clone gl or anti-cd clone c elicited activation of gd t cells suggesting that tcr gd and cd molecules in gd t cells are functional and signalling competent. next, using elisa and cytometric bead array, we found that iiel stimulated with plate bound gl in vitro produced ccl , ifng and tnfa. therefore, we were interested whether the ccl production of gd iiel influenced the homing of ccr cells such as lamina propria (lp) cd + foxp + cells (tregs). to test this, wt mice were i. p. treated with gl mab and lp tregs were analysed by cytometry at various time points post inoculation. we found similar frequencies of lp tregs population but a slight decrease in ccr + tregs. however, when we compared wt and tcrd -/mice, we found both lower percentages of total lp tregs and of lp ccr + tregs in tcrd -/mice compared to wt mice. in conclusion, our data suggest that intraepithelial activation of gd t cell may directly or indirectly induce changes in the iiel and lamina propria (lp) lymphocyte compartment and influence the ccr expression and the homeostasis of lp treg. the ability of nkt cells to serve a variety of different immunoregulatory functions in vivo may reflect a diversity in function of different nkt cell subsets. diversity in cytokine production by nkt cell subsets has been observed in murine and human studies, although this analysis has largely been following in vitro restimulation. here, we investigated cytokine production by murine nkt cell subsets in vivo under conditions where minimal manipulation of the cells was required. to this end, we examined il- production in g reporter strains in which dna encoding green fluorescent protein (gfp) was inserted into the first exon of the il- gene. in the absence of any manipulation gfp was expressed from the il- locus in populations of immature thymic nkt cells (predominantly cd +cd lotcrhi cells on a balb/c background, and cd +cd lonk . -on a c bl/ background) and some splenic nkt cells, with overall numbers of gfp+ cells in both tissues decreasing with age. after i. v. administration of the nkt cell ligand a-galactosylceramide, il- production was induced predominantly in cd + nkt cell subsets of the liver and spleen, and after i. n. administration, in cd + nkt cells of the airways. spontaneous and a-galcer-induced expression from the il- locus occurred in the absence of stat signalling, and did not require initial exposure to il- protein from other sources in the host. diversification in cytokine expression by nkt cells subsets therefore occurs early in ontogeny, and is also a significant feature of responses to exogenous activating stimuli. interleukin- (il- ) plays an important role in neutrophil recruitment. herein, we investigated the role of il- receptor signaling in polymicrobial sepsis induced by cecal ligation and puncture (clp). methods: adult c bl/ (wt) and il- receptor gene-deficient (il- r ko) mice were subjected to non severe (ns-clp) sepsis. intraperitoneal neutrophil migration, bacteremia, cytokine, chemokines and liver injury were evaluated hours after surgery. the ability of il- mediate the neutrophil microbiocidal activity in vitro, as well the neutrophil migration in vivo and in vitro were also evaluated. the means of different treatments were compared by analysis of variance (anova), followed by bonferroni's t test and the survival rate by the mantel-cox log rank test. results: it was observed that il- r ko mice, subjected to ns-clp sepsis, show reduced neutrophil recruitment into peritoneal cavity, spread of infection, and increased systemic inflammatory response as compared to wt. as a consequence, the mice showed an increased mortality rate. moreover, il- induced neutrophil migration in vivo and in vitro. besides, we demonstrated that neutrophils harvested from il- r ko mice already show reduced microbiocidal activity, compared with wt, suggesting a physiological role of il- receptor signaling in the microbiocidal activity of neutrophils. furthermore, wt neutrophils treated with il- showed strongly enhancement of microbiocidal activity by a mechanism dependent of nitric oxide. conclusion: during ns-clp besides the importance in recruit neutrophils to focus of infection, il- also enhances the microbiocidal activity of neutrophils. therefore, our results demonstrated that il- receptor signalization plays a critical role on host protection during polymicrobial sepsis. objectives: members of the toll-interleukin- receptor (tir) family are important for host defense, inflammation, and immune regulation. their canonical signaling pathway involves adaptor proteins and il- r associated kinases to activate nfxb and p mitogen-activated protein kinase. the il- -induced signal transduction in mast cells is poorly understood. in this work we studied the signal transduction of il- in different mast cell subsets. methods: different mast cells subsets (hmc- , human cbmcs and murine bmmcs) were stimulated with il- . the resulting signal transduction was investigated by immunoblot for activated signaling molecules (pc-kit, perk / , pakt, pnfxb, p and pjnk). additionally, we studied the signal transduction of il- in il- r transfected hek t cells. results: we found, that a tir family member, il- r, transactivates the receptor tyrosine kinase c-kit in mast cells and that il- -induced cytokine production depends on c-kit transactivation. il- r and il- r accessory protein (il- racp) form a physical complex with c-kit. thereby the complexation is dependent on the activity of c-kit. conclusion: these results show for the first time that the biological function of an il- r family member is dependent on the presence of an activated receptor tyrosine kinase. furthermore, these results reveal that certain il- -induced signaling pathways and effector functions are dependent on activated c-kit and could therefore explain the effects of il- in mast cells in absence of iger activation. ( ) . we now provide a molecular mechanism underlying this pathogenic effect by which free heme sensitizes hepatocytes to undergo tnfmediated programmed cell death. independently of newly gene transcription and/or protein synthesis, free heme cytotoxicity is mediated by the unfettered generation of free radicals in response to tnf, presumably due to the participation in the fenton reaction of the fe atom present in the protoporphyrin ix ring. once exposed in vitro to free heme, a sustained c-jun n-terminal kinase (jnk) activation was observed in hepatocytes in response to tnf, an effect that promotes further free radicals production. pharmacologic or genetic (shrna) inhibition of jnk in hepatocytes avoids free radicals accumulation and caspase- activation, also mimicked by the anti-oxidants n-acetylcystein (nac) or butylated hydroxyanisole (bha). expression of the heme catabolyzing enzyme heme oxygnease- (ho- ) in hepatocytes affords protection against heme sensitization to tnf cytotoxicity. recombinant adenovirus mediated ho- expression in the liver suppresses tnfmediated hepatocyte apoptosis and prevents the lethal outcome of plasmodium infection in mice. in conclusion our data reveals a novel signal transduction pathway via which heme sensitizes hepatocytes to undergo tnf-mediated cytotoxic effect, critically involved in the outcome plasmodium infection. the multi-step leukocyte extravasation process is governed by adhesion molecules and chemotactic factors dynamically interplaying in the presence of shear forces. responsiveness to chemotactic ligands is mediated by g protein-coupled receptors (gpcrs) which are finely regulated by a family of cytosolic proteins, betaarrestin and . recent evidence indicates that, in addition to playing a regulatory role in gpcr desensitization and internalization, beta-arrestins may contribute to gpcr signaling by functioning as scaffolds for the recruitment of signaling proteins into complexes with agonist-occupied receptors. on this basis, we investigated the physiological role of beta-arrestin in chemokine-driven dynamics associated with leukocyte extravasation, with special interest to the activation of the rap small gtpase, recently emerged as pivotal regulator of integrin function. the analysis of kc the (keratinocyte-derived chemokine) rap activation profile in rbl (rat basophilic leukemia) cells expressing mcxcr shows a bimodal kinetic, with the first peak at ''/ ' and the second at ' after stimulation. rna interference-mediated depletion of beta-arrestin specifically inhibits the occurence of the second wave of rap activation, whilst it has no effect on the early pick, thereby suggesting that beta-arrrestin is involved in rap activation and that the oscillations in the formation of rap -gtp are regulated by different molecular mechanisms. in order to elucidate the gefs and gaps involved in the gtpase regulation we are at present down-regulating the expression of c g (rap gef) and spa (rap gap): preliminary results suggest that spa- has probably a role in the early activation peak. since this oscillatory chemokine-induced rap activation is present on other myeloid cell lines (hl , d) and fresh pmn's we are also translating our research to these more appropriated cells. interestingly betaarrestins amino acid sequence and three-dimensional structure reveal a unique and evolutionary conserved proline-rich sequence in beta-arrestin , localized in a solvent exposed loop which may serve as a docking site for migration-associated transducers/adaptors. in order to find sh containing proteins that interact with beta-arrestins, we have performed an overlay screening assay of different sh domains that revealed over putative beta-arrestins putative interactors, some of which isoform specific. granulocyte-macrophage colony-stimulating factor (gm-csf), interleukin (il)- and il- stimulate proliferation, differentiation, survival and functional activation of myeloid cells. the cell surface receptors for these cytokines consist of cytokine-specific a subunits and a common b-receptor (bc), required for the activation of intracellular signaling following cytokine engagement. aberrant signalling, stimulated by these cytokines, has been implicated in the pathogenesis of many diseases, including arthritis, asthma and leukemia. as a result, we have sought to define key molecular determinants of these receptor-cytokine interactions in order to gain a greater understanding of receptor activation. here we present novel insights into the role of the ig-like domain of the gm-csfra in gm-csf binding. deletion of the ig-like domain abolished direct gm-csf binding and we identified specific residues directly involved in ligand binding by site directed mutagenesis and binding studies. the results indicate a previously unrecognized role for the ig-like domain of gm-csfra. furthermore, we address a longstanding controversy in the field of gm-csf, il- and il- receptor biology, by performing a systematic study of the role of n-glycosylation upon on the bc, and related murine b il- , in ligand-binding and receptor activation. these data demonstrate definitively that n-glycosylation does not play a role in mediating ligand-binding or receptor activation. these findings clearly establish that the determined human bc structures lacking glycosylation at asn are biologically relevant conformers of the human bc ectodomain. our results appear to suggest that the potency of receptor signalling can be influenced by the biophysical and structural properties of the extracellular receptorligand interactions and it also addresses important, poorly-understood aspects of mechanisms underlying ligand recognition and activation of the gm-csf: gm-csfra: hbc receptor complex. reference: ( ) micrornas (mirnas) are endogenous small non-coding rna molecules acting as key regulators of immune cell differentiation and innate immune responses. mirna- expression is induced by activation of the toll-like/interleukin- receptor pathway (tirpathway), where it targets essential adaptor and signaling molecules, thus serving as a regulator preventing the cells from an exacerbated pro-inflammatory response. since tnfa also up-regulates the expression of mirna- a, we decided to explore whether this mirna is involved in the regulation of apoptosis. to this end, we used the hela human epithelial cell line as a model system for tnfa signaling. following tnfa and cycloheximide (chx) treatment mirna- a transfected cells showed significantly reduced levels of the active proapoptotic caspases and (casp / ). in line with this, mirna- a conferred enhanced protection against tnfa-induced dna fragmentation and mitochondrial potential drop-down. our results demonstrate that mirna- a is a regulator of receptor-mediated apoptosis. similar to the tir-pathway, mirna- a seems to be part of a negative feedback mechanism of the tnfa signaling cascade. ongoing research focuses on the identification of the specific pro-apoptotic molecules targeted by mirna- a. furthermore, we are exploring the relevance of our observations for the mycobacterial infection of human macrophages, where the regulation of apoptosis is critical. objectives: the aim of this study was to evaluate the role of single nucleotide polymorphisms (snps) located in il- , il- r a-chain and il- r a -chain genes in hiv disease progression. methods: we studied antiretroviral treated patients (progressors) and long term non progressors (ltnp). we analyzed snps in the il- gene, snps in the il- r gene and snps in the il- r gene. in univariate analysis, we found an association between the presence of at least one mutated a allele in il- r aa and a higher possibility of being ltnp ( our study suggests that genetic polymorphisms located in il- r and il- r genes can influence the rate of disease progression in hiv+ patients, especially when a combination of aplotypes is present. mutations in the coding regions might compromise the binding of the cytokines or the intracellular signal transduction pathways, therefore leading to the alteration of cd and cd t cells homeostasis. aims: mono-adp-ribosyltransferases (arts) are gpi-anchored ectoenzymes that covalently modify cell-surface or soluble target proteins by transferring an adpribose moiety from extracellular nad+ to specific arginine residues of target proteins. in this study, we report that human tumor necrosis factor (tnf) is adpribosylated by art , and that adp-ribosylation affects both the release of tnf from cells and its cytolytic action. methods: transcription of art in human leukocytes was analyzed by rt-pcr. adp-ribosylation of tnf was detected by monitoring the incorporation of adpribose from labeled nad. release of tnf from transfected hek cells was monitored by elisa. binding of tnf to tnf receptors was analyzed by biacore. tnf cytotoxicity was monitored by flow cytometry. the adp-ribosylation site on tnf was analyzed by lc/ms mass spectrometry. results: we identified art transcripts by rt-pcr analysis in human blood leukocytes. soluble art , released from the surface of transfected cells by phosphatidylinositol-specific phospholipase c (pi-plc), adp-ribosylated recombinant human tnf in vitro. co-transfection of hek cells with art and tnf resulted in modification of tnf at at least distinct sites, i. e. one within the tnf ectodomain, and one on the stalk that remains connected with the cell membrane after cleavage by tnfa converting enzyme (tace). analysis of modified recombinant tnf by mass spectrometry provided evidence that the tnf ectodomain is adp-ribosylated at r , a site that has previously been implicated in binding to tnfr . binding assays indicated that adp-ribosylation inhibited binding of tnf to its receptors. importantly, modified tnf was less potent at inducing cell death in the human t cell lymphoma line kit than wildtype tnf. furthermore, cell surface adp-ribosylation of hek cells co-transfected with tnf and art resulted in reduced release of tnf into the supernatant. conclusions: adp-ribosylation of tnf or other cell surface proteins interferes with the biology of tnf signals by at least two distinct mechanisms. adpribosylation of tnf blocks binding to its receptors, thereby inhibiting tnf-mediated cytotoxicity. additionally, adp-ribosylation of tnf or another protein on the surface of tnf-producing cells inhibits the proteolytic release of tnf. noninflammatory chronic pelvic pain syndrome : immunological study in ejaculate g. n. drannik , t.v.poroshina institut of urology amsci of ukraine, laboratory immunology, kyiv, ukraine chronic prostatitis (cp) is a disease which likely is associated with abnormalities in local immune responses. secretions of the urinary and reproductive tract mucosa contain various protective effector molecules, produced by mucosal cells, lymphocytes, macrophages and neutrophiles. the aim of this prospective study was to observe local immunophenotypic patterns in patients with noninflammatory chronic prostatitis/chronic pelvic pain syndrome for further description and as possible surrogate markers for diagnosis and treatment. methods: patients with noninflammatory chronic prostatitis/chronic pelvic pain syndrome (cp/cpps) and control men were assessed for slpi, tnf-alpha, il- and free tgf-b in ejaculate by elisas using stat-fax plus. a to day sexual abstinence period was required from the subjects before semen collection. after liquefaction and centrifugation, seminal plasma samples were kept at - degrees c°until assayed. the materials were processed after the standard programmes for statistical analysis.the study was approved by the local ethics committee. the slpi concentration was elevated in all patients ( . ± . pg/ml, p x . ) in seminal fluid, in comparison with the healthy control subjects. the tnf-alpha concentration was elevated in all patients in seminal fluid ( . ± . pg/ml; p x . ). the il- concentration was elevated in all patients ( . ± . pg/ml; p x . ) in seminal fluid. free tgf-b was present in normal seminal plasma in high concentrations ( . ± . pg/ml), while in ejaculates of patients with noninflammatory cp/cpps tgf-b concentrations were . ± . pg/ml. conclusion: ejaculate's slpi, tnf-alpha, il- and free tgf-b are possible surrogate markers for the diagnosis and treatment of patients with noninflammatory chronic prostatitis/chronic pelvic pain syndrome. m. r. marrakchi , e. a. elgaaeid faculté des sciences de tunis, biology, tunisie, tunisia ulcerative colitis (uc) and crohn's disease (cd), collectively referred to the inflammatory bowel disease (ibd), represent a group of multifactorial autoimmune disorders of the gastrointestinal tract sharing many clinical and pathological characteristics, however, differing in histological features and cytokine profiles. the excessive production of either th or th cytokines due to perturbed regulation of immune system activation results in chronic inflammatory processes and loss of immune homeostasis that may be implicated in the genesis of ibd. studies have identified a gene that encodes the nod /card protein, which is involved in the immune system's response to bacterial infection and confirmed to influence susceptibility to cd. indead, it has been suggested that high rates of asca (saccharomyces cerevisiae ) in absence of panca (perinuclear anca: anti-neutrophil cytoplasmic) antibodies were associated with aggressive forms of cd and that the important rise of panca was more frequent at uc . in a sample of tunisian patients, we examined the contribution of nod /card gene in cd. we performed a cases /controls study upon cd patients and healthy controls. this study suggests that in northen tunisian population, insc mutation in nod /card gene is a prevalent mutation leading to the typical crohn's disease including ileal location, stricturing and penetrating clinical types and asca expression. since conflicting results were obtained on il- polymorphisms as risk factor for ibd, the aim of our study was also to explore anti-inflammatory il- cytokine genetic profile in patients with ibd. we examined the contribution of il- gene promoter polymorphisms (- and - ) to crohn's disease (cd) phenotype, and the possible genetic epistasis between these polymorphisms and card /nod gene mutations in cd presentation and location. in tunisian population, the insc insertion in nod /card gene is a marker of susceptibility to cd, while the a allele at position - in the il- promoter increases the risk of cd ileal location and severe disease presentation. a genetic epistasis between il- gene polymorphisms and card /nod gene mutation was suggested. in conclusion, genetic and serologic markers might be useful in defining patien gangliosides were shown to inhibit the il- -dependent proliferation of t-cells, implying that gangliosides interfere with one or more of the il- -driven events. it is known that the major mechanism of inhibition is the direct interaction between ganglioside and the cytokine and, as a result, the capture of il- molecule by ganglioside. but gangliosides apparently can also form complexes with il- r; such complexes influence on the signal transduction through il- r. this effect of gangliosides may lead to the failure of this pathway. unfortunately, the biological and structural aspects of this problem are poorly understood. in this study we propose possible modes of interactions between exogenous gangliosides and il- r subunits. in our work we use il- -dependent cytotoxic t-cell murine line ctll- . two different approaches for study of possible interaction types between exogenous gangliosides and il- r subunits were applied: antibody staining of il- r subunits followed by flow cytometry analysis, and photoaffinity labeling of living cells with i-dcp-gm followed by immunoprecipitation of il- r subunits. the fluorescence intensity of the antibody-labeled il- r a-subunit substantially decreases after the treatment of cells with gangliosides. it has been shown that the fluorescent labeled cell fraction decreases by . % after cells incubation with ganglioside gm , and by . % after incubation with gm . labeling of the cells with antibodies to the il- r b-subunit results in a less significant fluorescence decrease after cells incubation either with gm ( . %) or with gm ( . %). to determine the mode of this impressive masking influence of ganglioside gm , photoaffinity cross-linking has been used. in our modification this method could identify is interaction of gm with subunits of il- r occur with or without incorporation exogenous ganglioside into plasma membrane. electrophoresis followed by immunoprecipitation with appropriate antibodies resulted in appearance of the radioactive band only for b-subunit of il- r, but not for il- r a-subunit. these results demonstrate that exogenous ganglioside gm can interact with a-and bsubunits of il- r in different modes. interaction of il- r b-subunit with ganglioside gm requires incorporation of the ganglioside into plasma membrane, but it is not the case for interaction with a-subunit. a. respa , j. bukur , s. purpose: loss of interferon (ifn)-g inducibility of hla class i antigens has been found in some tumor cell lines, but the underlying molecular mechanisms of such deficiencies have not yet been elucidated in detail. this kind of tumor escape mechanism might lead to an inefficient recognition of tumor cells by the immune system. methods: phospho-specific western blot analyses were performed to verify the functionality of the different ifn-g pathway components, intra-and extracellular flow cytometry experiments were employed to determine the expression of antigen processing components and hla class i cell surface antigens, quantitative real time-pcr experiments to confirm the absence of jak and presence of pathway relevant molecules as well as, genomic pcr and chromosome typing technique to prove the deletion of jak . results: different ifn-responsive phenotypes were defined in human melanoma cell lines varying from loss to low, delayed as well as strong ifn-g inducibility. resistance to ifn-g treatment was associated in one melanoma cell line with the lack of jak expression due to a gene deletion on chromosome , whereas the expression and functionality of other ifn-g signal transduction components like stat and jak were not affected. jak blockade by two jak -specific inhibitors resulted in reduced levels of hla class i surface antigens. conclusion: structural abnormalities of jak leading to a lack of jak expression are associated with loss of ifn-g inducibility as well as reduced constitutive hla class i surface expression. in addition the jak inhibition modulates the expression of the hla class i antigen processing components. of renal cell carcinomas (rccs) are associated with the size, grade, t, n, m, stage and death of rccs patients. the genotypes were compared with those of a random sample of controls of the spanish population. methods: two il polymorphisms located on the il- promoter region, snps - a/c (rs ) and - g/c (rs ) were genotyped using taqman snp genotyping assays. the functional il- gene polymorphisms studied do not influence the susceptibility to rccs in the spanish populations (il- - p= , . il - p= , ) but may contribute to disease onset and aggressiveness: the genotype il- - cc genotype, which is associated with higher il- production, was significantly associated with higher tumor size (p= , ), grade (p= , ), t (p= , ), m (p= , ) and stage (p= , ). the influence of the il- - gg genotype was less relevant, however was correlated with higher tumor size (p= , ), grade (p= , ), t (p= , ) and stage (p= , ). the multivariate analysis with cox proportional hazard model revealed that, in this serie, nuclear grade and stage grouping were independent prognostic factors, whereas il- polimorphism can not be considered as independent prognosis factors. our results suggest that the polymorphism variants from the il- gene (il- - and - ) may be associated with an worse prognosis of rcc. high levels of il- production can play an important role in grow, invasion and metastasis of renal cancer. interleukin- (il- ) is a pleiotropic cytokine that is involved in regulation of both the innate and acquired immune response. the most prominent biologic property of il- is its ability to induce the production of ifn-gamma in presence of il- . moreover, it stimulates the expression of tnf-a and il-l, enhances the differentiation of t cells to the thl and impairs the synthesis of the anti-inflammatory cytokine il-l . then it seems that il- has a crucial role in immunity against brucella infection. since the expression of il- can be affected by polymorphisms in its gene, we decided to investigate any probable relation between six different il- gene polymorph isms and brucellosis. methods: a total of patients with brucellosis and healthy farmer who consumed contaminated row milk and dairy products from animals with brucellosis, were included in this study. all individuals were genotyped for six il- polymorphisms at positions - , - , , + , + and codon / using polymerase chain reaction-restriction fragment length polymorphism (pcr-rflp). the distribution of alleles for il- polymorphisms at position - g/+ t/+ c (correlated with high production of il- ), codon / c and - g/- c (correlated with higher production of il- ) were significantly higher in the healthy controls than the patients (p= . , p= . and p= . , respectively). discussion: as data revealed genotypes that have correlation with higher production of il- are more frequent in the controls than the patients. then it might be deduced that individuals who inherited these genotypes/alleles are able to produce higher level of il- at the onset of infection and it leads to more ifngamma production and control brucella infection before appearing brucellosis. abstract withdrawn by author objectives: a dsyregulated cytokine response has been shown to play a role in inflammatory bowel disease (ibd) pathogenesis. to dissect the influence of these cytokines, specifically interferon gamma (ifng), on the inflammatory response and colitis we have created a cell specific ifng receptor (ifngr ) mouse mutant. we have generated a mouse line carrying a conditional ifngr allele using the loxp -flp recognition target (frt) approach. a targeting vector with loxp sites flanking exons - and frt sites flanking the neomycin resistance cassette was generated. after confirmation of homologous recombination the neomycin resistance cassette was deleted by crossing with flp deleter mice. cell specific deletion is being performed by crossing conditional 'flox/flox' mice with specific cre expressing mouse lines. functional inactivation of the receptor has been demonstrated by performing western blots to detect phosphorylated and total stat following ifng stimulation. results: successful deletion of the gene and conditional mutants without the presence of the neomycin resistant cassette have been generated. functional inactivation of the receptor with no stat activation following ifng stimulation has been demonstrated in the complete knock out mice. furthermore, flox/flox mice retain full responsiveness to ifng. breeding with lysm cre and cd cre mice will been completed to create a cell specific deletion of the ifng receptor in macrophages/granulocytes and t cells respectively. the specificity of this deletion will be confirmed through cell sorting and functional assays. in order to determine the influence of ifng on a specific cell type a conditional gene targeting approach has been utilised. this has allowed the generation of conditional mice mutants with deletion of ifngr on macrophages and t cells. this has generated a very powerful tool for dissecting the role of this cytokine in numerous disease models. moreover, the ability to cross the conditional mice with additional cre lines will enable the analysis of more cell types in the future. a. gonzalez , r. carretero , p. saenz-lopez , j. cantón , j. carretero , f. ruiz-cabello , l.m. torres , cts- hospital universitario virgen de las nieves, departamento de ginecología, granada, spain, hospital universitario virgen de las nieves, departamento de análisis clí nicos, granada, spain objetives: cervical cancer is almost associated with infection by human papillomavirus (hpv). however, only a subset of infected women will ever develop the malignancy. ifng dinucleotide (ca) repeat polymorphism is responsible for genetic differences in interferon-gamma production. our objective was to investigated the relationship between ifgn polymorphism and cervical intraepithelial neoplasia (cin) methods: we have studied nineteen women with cin and normal women control. dna was extracted from blood samples, and was genotyped for functional microsatellite (ca) repeats in the first intron of ifn-gamma gene. results: heterozygosis in (ca) allele of ifgn is significant more frequent in healthy control than in cin patient (p= , ) in contrast homozygosis for (ca) allele did not show significant differences between both population. analysis of relation between this polymorphism and the cin stage showed that both heterozygosis and homozygosis is correlated with advanced stage (p= , and p= , respectively). conclusions: our study suggest that ifng (ca) allele may influence in cin risk and progression. ifng is associated with hpv clearance, but it also plays a role as inflammatory cytokine, promoting cervical malignant progression. further studies of the role of ifng and other cytokines may contribute to the understanding of cin promotions and progression. introduction: macrophages play a fundamental role in controlling of brucella infection, mainly through the secretion of cytokines such ifn-y and tnf-a. interleukin- (il- ), a th -related cytokine, triggers inflammatory cell recruitment and increases the expression of ifn-y. as the cytokine production is under the genetic control, we decided to investigate the association between il- single-nucleotide polymorphisms (snps) and susceptibility to brucellosis in iranian patients. methods: patients with brucellosis and healthy animal husband men who kept animals with brucellosis, were included in this study. all individuals were genotyped for il- (c t, g a, c a and a t) polymorph isms using pcr-rflp. results: at position , the distribution of c allele and cc genotype were significantly lower in the patients than the controls (p= . and p= . , respectively). at position , the distribution of c allele and aa genotype were significantly higher and lower in the patients than the controls, respectively (p= . , p= . ). discussion: as shown the frequency of cc genotype and c allele at position were higher in healthy controls than the patients. hence, our study provides evidence that presences of cc genotype and/or c allele are significantly associated with susceptibility to brucellosis. the frequency of aa genotype at position is lower in patients than the healthy controls and the frequency of c allele at position is higher in patients than the healthy controls. hence, our study provides evidence that presences of c allele and lack of aa genotype are significantly associated with susceptibility to brucellosis. objectives: increasing evidence is emerging regarding the ability of mammary epithelial cells to respond to various cytokines. our aim was to investigate the cytokine receptor phenotypes of two distinctive human mammary cell lines, mcf- and skbr- , as well as their ability to respond to several cytokines and to the g- gpr agonist. the mcf- and skbr- human adenocarcinoma cell lines were investigated by immunocytochemistry and flow cytometry for the expression of the following cytokine receptors: il- ra, il- rg, il- /il- /gm-csfr, il- /il- r, il- ra, il- ra, il- r (gp ), il- ra, il- r, tnfr i, tnfr ii, ifngra, cxcr , cxcr , cxcr , cxcr , cxcr . cells were incubated with il- , ifn-g, tnf-a and tnf-b (for which both cell lines displayed receptors) alone and with the gpr agonist. cytosolic ca + concentrations [ca + ] i were measured by the microfluorimetric technique. results: different cytokine receptors phenotypes emerged for the two cell lines. the less well differentiated skbr- cells were found positive for a larger number of receptors than the mcf- cells. both cell lines displayed an important heterogeneity for each of the investigated molecules, in terms of number of positive cells and expression intensity, with chemokine receptors percentages constantly higher than for the other receptors. pretreatment of mcf- cells with il- reduced the calcium response to g- , while pretreatment with ifn-g and tnf-a potentiated the calcium response to g- . tnf-b had no effect on mcf- g- stimulation. no direct effect on basal [ca + ] i stimulation could be noticed when administering the cytokines alone. in skbr- cells, pretreatment with il- or tnf-b had no effect on basal [ca + ] i and did not significantly alter the calcium response to g- , while pretreatment with ifn-g induced calcium oscillations and potentiated the calcium response to g- . pretreatment with tnf-a produced calcium oscillations and reduced the response to g- . conclusions: mcf- and skbr- cell lines express distinctive cytokine receptor phenotypes. furthermore, their ability to respond to cytokines in terms of modulating the gpr stimulation proved to be different. the susceptibility towards various soluble factors of these cell lines can offer us some insights on the complexity and individuality of each primary tumor and thus the distinctive evolution of each particular patient. results: in initial analyses of the early infection phase we identified splenic pdcs expressing ifnb/yfp. furthermore, we show for the first time in vivo that these ifnb producing pdcs are not directly infected with mcmv and that not only cdcs but also cd a -pdcs expressed gfp as a marker for mcmv infection. we observed that at early time points equal frequencies of cd a -pdcs and cdcs were infected with mcmv, whereas after h p. i. the frequency of infected cdcs wins over that of the pdcs. conclusions: with this experimental system we are able to visualize the ifnb response vs. the infection status of mcmv in vivo on a single-cell level. from our initial results we can conclude that infected cells in the spleen induce ifnb production in noninfected pdcs which initiate the antiviral immune response in this organ. recently, we have developed live-attenuated arenavirus vaccine candidates based on lymphocytic choriomeningitis viruses (lcmv) carrying the vesicular stomatitis virus (vsv) envelope gene (rlcmv/vsv-g). since interferon (ifn) signaling is known to be crucial for adaptive immune responses against wildtype (wt) lcmv and control thereof, we wanted to assess the innate and adaptive immune requirements for containing rlcmv/vsv-g infection. mice lacking both, ifn type i and ii receptors generated potent virus-specific cd t cells and neutralizing antibody responses against rlcmv/vsv-g, even exceeding the respective responses in wild type animals. in further contrast to wt lcmv infection, ifn type i and ii signaling was dispensable for rlcmv/vsv-g control. rlcmv/vsv-g infection of rag -deficient mice (lacking mature t and b cells) resulted in persistent levels of circulating viral rna that was solely detectable by qrt-pcr but not by classical measures of infectivity. overt viremia was only found in mice lacking both rag and ifn type i receptor (ar). thus, viral attenuation for vaccine use was found to considerably relax the ifn-dependence of adaptive immune responses and virus control. this redundancy of ifn and adaptive immune responses in rlcmv/vsv-g control provides a better understanding of the attenuation of this vector. it furthermore suggests that the virulence of a particular virus may influence the interferon-dependence of cd t cell and antibody responses, which may have implications for vaccine development. objectives: the class of type i interferons (ifns) consist of multiple ifnas and a single ifnb subtype. although being important for anti-viral defence they have been shown to be detrimental for the host during bacterial infections. while in general the first ifn to be produced is ifnb, the cell types responsible for its initial production remain unclear. to assess the cellular sources of ifnb and its role for the outcome in bacterial infections we use an ifnb reporter-knockin mouse model, in which yellow fluorescent protein (yfp) is expressed from a bicistronic mrna linked by an internal ribosomal entry site (ires) to the endogenous ifnb mrna. methods: to induce a type i interferon response we intravenously injected the tlr agonists poly(i:c) and cpg , respectively, or infected reporter mice or bone marrow derived macrophages (bmdms) or dendritic cells (bmdcs) with the facultative intracellular pathogen listeria monocytogenes. the spatiotemporal tracking of the ifnb response was done using facs analysis and immunofluorescent microscopy. results: after poly(i:c) injection in vivo a small subpopulation of ifnb + cd a + dendritic cells relocalize from the red pulp via the marginal zone to the t cell areas of the spleen whereas ifnb + activated pdcs were localized in the splenic white pulp at the t/b-cell interface after cpg administration. in vitro infection of bmdms, bmdcs and flt -l derived dcs with listeria monocytogenes resulted in a low frequency of ifnb + cells depending on the listeria strain used and multiplicity of infection with bmdms being the most potent producers of ifnb. in vivo mostly activated f / + macrophages were accountable for the ifnb expression. simultanous visualization of the cellular state of infection shows that ifnb + bmdms carry a higher bacterial load then ifnbcells. the cellular detection of ifnb expression reveals a remarkably low frequency of ifnb-producing cells in response to distinct pamps or the infection with intracellular bacteria. this hints at a superior role of few specialised cells for mounting a significant response against distinct stimuli or bacterial disease. additional data will be presented further resolving the timecourse of the ifnb response vs. the state of infection after bacterial challenge. the spleen is a secondary lymphoid organ that is characterized by highly specialized structures and plays a crucial role in the defense of blood-borne pathogens. we investigated the role of the spleen in the generation of antigen-specific cytotoxic cd t cell (ctl) responses in a systemic infection with recombinant adenovirus expressing ovalbumin (adova). although adenovirus mainly infects the liver, the ctl response requires the spleen, as splenectomized mice were incapable to mount an antigen-specific ctl response upon systemic challenge with adova. additionally, dendritic cells (dc), macrophages and an intact splenic structure were mandatory for the induction of an efficient ctl response. we detected adova specific ctl responses only in splenectomized mice that received splenic autotransplants but not after adoptive transfer of single cell splenocytes. furthermore, we asked how toll-like receptor (tlr) ligands influence adova-specific ctl responses. tlr ligands as "danger signals" are generally known to exert immune stimulatory effects. importantly, we observed that systemic administration of single-stranded rna (ssrna) prior to adova infection inhibited the generation of antigen-specific ctl responses in a tlr and type i interferon (ifn) dependent manner. ssrna injection induced the production of type i ifns as detected in sera and supernatants of splenocytes isolated from wild-type mice. experiments performed in ifnbeta reporter mice revealed that splenic plasmacytoid dcs represented the cellular source of type i ifns. additionally, splenic cd c+ dcs purified from ssrna pretreated mice showed a reduced capacity to cross-prime ova-specific cd t cells upon adova challenge. in vivo pre-activation of endogenous ova-specific cd t cells as well as an adoptive transfer of in vitro activated transgenic ova-specific cd t cells prevented the ssrna mediated suppression of the ctl activity. we assume that dcs preactivated by systemic ssrna were impaired in their ability to activate naive cd t cells in response to an adova infection due to an impaired cd t cell help. taken together, we show that type i ifns cannot only stimulate, but also inhibit the induction of ctl responses in the spleen. within hours after infection many viruses induce early type i interferon (ifn) responses that can confer protection against lethal infection. modified vaccinia virus ankara (mva) is a highly attenuated vaccinia virus (vacv) strain generated by more than passages in chicken embryo fibroblasts. mva stimulates systemic ifn responses, whereas vacv-induced cytokine milieus are dominated by il- . to study the impact of virus-triggered ifn on the induction of t cell responses, we used recombinant mva-gp and vacv-gp expressing the gp epitope of lymphocytic choriomeningitis virus. for adoptive transfer experiments transgenic mice expressing the p t cell receptor recognizing the gp epitope were intercrossed with mice devoid of the ifn receptor (ifnar -/-) to obtain p ifnar -/mice. upon adoptive co-transfer of p ifnar -/and p ifnar wt/wt t cells and subsequent challenge with mva-gp , a massive expansion of p ifnar wt/wt t cells was observed, whereas p ifnar -/-t cells expanded less efficiently. in contrast, challenge with vacv-gp induced a rather similar expansion of ifnar competent and ifnar deficient p t cells. in the absence of ifnar-triggering t cell expansion was associated with reduced proliferation capacity and increased apoptosis. to study the impact of ifnar-signaling on the expansion of endogenous t cells, conditional mice with a t cell-specific ifnar-ablation were infected with mva. interestingly, in those mice the virus-specific t cells showed a reduced expansion compared to t cells of wt mice. additionally, we found that ifnar-triggering of dendritic cells but not of macrophages further supported specific t cell expansion. thus, upon virus infection virus-associated properties affected the overall cytokine milieu that influenced the quality and the quantity of expansion of virus-specific t cells. to delineate h n -specific signaling patterns we used a genome-wide comparative systems biology approach analyzing gene expression in endothelial cells infected with three different human and avian influenza strains of high and low pathogenicity. blocking of specific signaling pathways revealed that h n induced an exceptionally nf-kb dependent antiviral response. irf is essential part of this interferon-response of human endothelia. furthermore, we identified hmga as a novel transcription factor specifically responsible for the overwhelming proinflammatory but not anti-viral response induced by h n . finally, nfatc was found to be a transcriptional regulator for specifically h n -induced genes. we therefore describe for the first time defined signaling patterns specifically activated by h n which, in contrast to low pathogenic influenza viruses, are responsible for an imbalance of overwhelming proinflammatory and impaired anti-viral gene programs. objectives: to study the early events of immune antagonism by influenza virus in vivo and how this process impacts the timing at which adaptive immunity is generated. methods: to dissect the contribution of the unique viral antagonist ns , delta-ns influenza virus (a recombinant influenza virus lacking the ns gene) was compared side by side with wild type influenza virus in vivo. mice infected with influenza virus were sacrificed at different time points after infection. to study the onset of inflammation during infection lung and blood samples were isolated with a selected panel of inflammatory and antiviral proteins that were measured by multiplex elisa and quantitative pcr (qpcr). to determine the bridging between innate lung inflammation and adaptive immunity, the kinetics of lung antigenbearing dendritic cell (dc) migration to the draining lymph nodes was quantitated in infected mice. further, functional in vitro and in vivo assays in infected animals with influenza-ova viruses were used to determine whether antigen-bearing migratory dcs were capable of priming transgenic t cells. to investigate the effect of ns during the onset of immunity in vivo, we systematically studied the early events occurring in the lungs and draining lymph nodes upon infection with influenza virus. strikingly, no sign of innate immunity was detected in the lungs for almost two days after infection when a sudden inflammatory burst including ifns, cytokines, and chemokines occurred. this burst preceded the robust dc migration and t cell activation in the lymph nodes. virus-loaded dcs appear in the lymph node starting days post-infection, reached its maximum at day , and triggered t cell priming in vivo. a direct comparison of delta-ns virus with wild type virus infection demonstrates that virus can only trigger rapid inflammation in vivo when it lacks the ns protein. we demonstrate that the delay in the generation of immediate lung inflammation is mediated by the influenza ns protein. thus, we propose that the virally encoded ns protein establishes a time limited "stealth phase" where replicating influenza virus remains undetected thus preventing the immediate initiation of innate and adaptive immunity. keratinocytes represent the major cell population of human epidermis which provides a first line of defense barrier for the host. in addition, keratinocytes actively participate in immune response. viral double-stranded rna (dsrna) is the most important viral structure involved in activation of innate immune response. intracellular detection of dsrna triggers secretion of soluble signaling molecules, interferons, and activates pro-inflammatory response and programmed cell death, apoptosis. here we have used subcellular proteomics to characterise dsrna-induced human keratinocytes. cells were transfected with a mimetic of dsrna, poly(i:c), after which the cells were fractionated into cytoplasmic and mitochondrial fractions. these proteomes were analysed using two-dimensional electrophoresis in combination with mass spectrometry, immunoblotting and confocal microscopy. we show that several proteins involved in apoptosis, cytoskeleton reorganization and intracellular transport are up-regulated upon dsrna stimulation. in mitochondrial proteomes the expression of structural proteins, especially fragments of cytokeratins, is highly up-regulated. we show that cytokeratin is cleaved during poly(i:c) stimulation and fragments are solely localized in mitochondria. similar degradation of cytokeratin is seen in emcv-and vsv-infected keratinocytes. cytokeratin fragmentation after dsrna stimulation is dependent on caspase activation, which indicates a role for cytokeratins in the regulation of apoptosis during viral infection. in addition, we show that - - proteins are upregulated in both mitochondrial cell fraction and cytoplasm after dsrna-stimulation and during viral infection. viral dsrna also induced transient phosphorylation of - - target proteins. thus, these results suggest that - - proteins have a regulatory role in host defence against viral infections. i. wessels , d. fleischer , l. rink , p. uciechowski institute of immunology, rwth aachen university hospital, aachen, germany objectives: the proinflammatory cytokine interleukin (il)- b mediates the expression of a variety of proteins responsible for acute inflammation and chronic inflammatory diseases. however, the molecular regulation of il- b expression is not elucidated, yet. it is known that the il- b promoter is packaged into a nontranscribed but poised architecture in monocytes, rapidly producing il- b when stimulated. b-cells which are il- b non-producers reveal an inaccessible promoter structure. in this study the chromatin structure of the il- b promoter and the impact of methylation on il- b expression were examined in a cellular monocytic differentiation model. methods: promyeloid hl- cells were differentiated into monocytic cells after dihydroxyvitamine d treatment. the monocytic phenotype was confirmed by flow cytometry. the il- b promoter was analyzed using the chromatin accessibility by real time pcr (chart) assay. to test the influence of methylation, cells were treated with -aza- -deoxycytodine (aza) and changes in il- b expression were measured by pcr, elisa and western blot analyses. results: in contrast to undifferentiated hl- cells, differentiated cells displayed upregulation of cd antigen and acquired the ability to express il- b. by comparing the accessibilities of il- b promoter we detected that the il- b promoter was not accessible in undifferentiated hl- cells but highly accessible in differentiated monocytic cells. the accessibilities of differentiated cells were comparable to that observed in primary monocytes. lps stimulation did not affect promoter accessibility in promyeloic and monocytic hl- cells, demonstrating that the chromatin remodeling of the il- b promoter depends on differentiation but is independent of the transcriptional status of the cell. demethylation via aza led to the induction of il- b expression in both undifferentiated and differentiated cells which could be increased after lps stimulation. conclusion: two independent mechanisms involved in the regulation of il- b expression were found. our data indicate that the il- b promoter is reorganized into an open conformation during monopoiesis and that the established poised structure is a privilege of mature monocytes but not of the entire myeloid lineage. as a second mechanism, il- b expression is regulated by methylation acting independent of the developmental stage of myeloid cells. a. holweg , g. wetzel , h. arnold , a. gessner microbiological institute-institute for clinical microbiology, immunology and hygiene, university hospital erlangen, erlangen, germany the p family members of interferon (ifn) inducible gtpases also known as guanylate binding proteins (gbps) are among the most abundantly induced transcripts after stimulation with ifn-g. although the stimulatory capacities of the toll like receptor ligands lps and cpg, cytokines like ifn-b and il- b and the intracellular pathogens listeria monocytogenes and toxoplasma gondii have been described to induce their expression, the function of gbps during bacterial infections is still ill defined. here we report for the first time the massive induction of murine gbps in two independent in vivo models of pneumonia (infection with streptococcus pneumoniae and pseudomonas aeruginosa). a strong and rapid induction of mgbp , , , and mrna after intratracheal infection was detected by realtime pcr analysis of bronchoalveolar lavage (bal) cells. using newly generated antibodies in western blots and fluorescence microscopy, we identified macrophages as the main producers of mgbps in bal samples. although the signaling cascade involved in the upregulation of mgbps after ifn-g stimulation has been extensively studied, the mechanisms responsible for mgbp induction upon bacterial stimuli are unclear. in this study we could show that the induction of mgbps upon infection is abrogated in ifn type i/ type ii receptor double-deficient mice and thus absolutely dependent on endogenous interferon production. in contrast, the tlr adaptor molecule myd was found to be dispensable arguing for a trif-mediated interferon production subsequentially resulting in enhanced gbp expression. based on these findings our future experiments will address the functional role of gbps in innate and adaptive immune responses against extracellular bacteria. ( )). activation of resident microglia cells and infiltrating brain macrophages appeared to play a role in modulating virus replication shortly after infection but also appeared to be responsible for the inflammation in brains of infected mice. clearance of replicating virus from the cns required mcmv specific cd + t lymphocytes whose effectors functions still remain incompletely defined (j. immunol. aug ; ( )). humoral immunity appeared to also play a role in the control of mcmv infection in developing brain. infected newborn mice treated with mcmv-specific antibodies had lower viral titers in the cns, significantly less cns inflammation and improved neuronal migration and increased cerebellar area as compared to control mice (j. virol. dec; ( ) ). conclusion: peripheral inoculation of the virus induces focal infection and inflammation in the developing mouse cns followed by strong innate and specific immune response that could also alter developmental programs required for normal development of mouse brain. passive immunization of infected newborn mice reduces mcmv-related pathology in infected brain suggesting that antiviral antibodies are an important component of immunological responses during cmv infection of developing cns. chronic inflammation is associated with the promotion and enhancement of malignancy and tumor growth. tumors enhance the accumulation of myeloid derived suppressor cells (mdsc), which contribute to tumor escape, immune tolerance and immune suppression. previously, we have shown that tumor-derived inflammatory cytokines, such as il- b in the tumor microenvironment can induce a massive accumulation of mdsc in the spleen of tumor bearing mice and induce t cell suppression. in this work, we describe a novel polymorphnuclear mdsc subpopulation -inflammatory mdsc (infmdsc) which accumulates in the bm and spleen of mice bearing inflammatory t breast cancer cells over expressing il- b ( t /il- b) tumor cells, but rarely in untransfected t cells. secretion of il- b from tumor cells is crucial for infmdsc generation and accumulation. infmdsc have the ability to suppress nk cell activity via reduction of the nk activating receptor nkg d in vivo, and in a cell-cell contact dependent manner in vitro. inflammation up-regulates il- ra expression on the cell surface, which correlates with tumor growth and induction of suppression on nk cells. our data suggest that tumor derived inflammation enhances a specific mdsc subset which has the ability induce suppression of nk cells, and perhaps can serve as a new chemotherapy target. objectives: lps constitutes a main target of innate immune recognition of gram-negative bacteria and other lps carrying pathogens. cytokine production after in vitro stimulation of whole blood with lps is used as an expression of individual lps reactivity. we assess genome-wide data to analysed the association to lps induced cytokine response, and replicated the top findings in two independent cohorts. materials and methods: we used healthy caucasian blood donors as discovery samples, and replicated snps from affymetrix -genome-wide human snp array . having p x - in two independent cohorts each consisting of blood donors using a customized chip from illumina and real-time pcr respectively. in all the three cohorts whole blood samples was stimulated for h and we measured the levels of il- , il- , il- , tnf-alfa and il -ra with r&d ® assays on luminex platform.. the association analysis was performed with wald statistical test assuming an additive model. the discovery sample statistical analysis revealed snps with p x , * - . to identify/replicate the association of cytokine production for these we reanalysed these on a cohort. a combined analysis revealed snps with p x , * - .these results are not genome wide significant. the snps showing nominal association to lps cytokine response are being analysed in a replication cohort conclusion: we find nominal associated snps between lps stimulated cytokines in blood samples of healthy caucasian blood donors. the importance of these snps are to be determined in a replication cohort. adipocytes, so far known for their lipid-storage capacity came into the focus of interest because of their immunoregulatory properties resembling those of innate immune cells. adipocyte-derived pro-inflammatory mediators contribute to the development of chronic inflammation, thereby promoting the progression of insulin-resistance/metabolic-syndrome and diabetes. the physiological signals inducing the secretion of inflammatory mediators by adipocytes are unknown. heat shock proteins, such as hsp have been identified as potent regulators of inflammatory innate immune cell-activities, whereas their influence on adipocyteactivities remained elusive. here, we investigated the regulatory effects of hsp on adipocytes. for the first time we could show a hsp -stimulated release of the proinflammatory cytokines il- , cxcl and mcp- in a time-and concentration-dependent manner from murine t -l adipocytes. analyses of hsp -signalling in these adipocytes revealed that members of the mapk-family (erk / , p ) and the transcription factor nfkb are involved in hsp -mediated induction of the mediators il- , cxcl and mcp- . binding-studies with fluorescence-labelled hsp demonstrated that the interaction of hsp with adipocytes exhibits basic features of a receptor-mediated binding. hsp -binding to adipocytes was saturable and reached its maximum at . mm. binding was inhibitable only by the unlabelled ligand ( %), but not by unrelated proteins, thereby proving the specificity of hsp -binding. further analyses to characterize hsp -receptor structures on adipocytes revealed the presence of toll-like receptor (tlr) on adipocytes. tlr has been found to be expressed on macrophages and to interact with hsp , therefore suggesting tlr as a potential receptor candidate for hsp on adipocytes. in order to identify the responsible binding-epitope of hsp we investigated the effect of specific antibodies directed against different epitopes of the hsp -molecule. incubation with antibodies directed against the n-terminus of hsp (aa - ; - mg/ml) were capable of inhibiting the hsp -binding to adipocytes ( - %) indicating that the n-terminal region of hsp is involved in receptor binding. our experiments demonstrate that hsp stimulates the release of proinflammatory adipocyte mediators. the findings implicate that the hsp -mediated induction of adipocyte mediators contributes to inflammatory processes observed in obesity-associated disorders and could serve as a target for the development of therapeutic strategies for patients suffering from diabetes or diabetes-related disorders. legionella pneumophila, a gram-negative facultative intracellular bacterium, is the causative agent of a severe pneumonia known as legionnaires' disease. classically, type i ifns (ifna/b) have been associated with antiviral immunity. ifna/b signal through the ifna/b receptor (ifnar) leading to the induction of hundreds of ifn-stimulated genes (isgs), many of which have anti-microbial activities. recently, it was demonstrated that ifna/b are also produced in host cells infected with (intracellular) bacteria or stimulated with cytosolic dna. here we show by rnai that l. pneumophila infected host cells produced ifnb dependent on irf . we observed enhanced l. pneumophila replication in mouse macrophages lacking ifnar and human cells after irf knock-down, suggesting that endogenously produced ifnb activates a cell-autonomous defence against legionella. moreover, ifnb treatment restricts legionella replication in human and murine host cells. ifna/b impacts formation of large replication vacuoles, but appears not to influence the recruitment of er markers nor fusion of the legionella-containing vacuole with the lysosome. moreover, ifna/b-mediated cellautonomous defence was independent of autophagy and pyroptosis. we thus hypothesize the crucial involvement of antibacterially acting isgs. ongoing studies focus on the role of ifn-induced immunity-related gtpases (irgs). mesenchymal stem cells (mscs) are identified by their capacity to differentiate into connective tissue cell types. mesenchymal stem cells also show a high plasticity and account for a potential therapeutic efficacy. several authors have reported the expression of alfa-smooth muscle actin (a-sm actin) by msc. this protein has been considered a marker for the myofibroblast, has the capacity to polymerize into the cytoplasm and contribute to the cell contractility. this activity may be crucial for the changes of the cell shape, for cell-cell interactions and may therefore be relevant for the physiology of msc. in this work, we study the presence of alfa-sm actin in human msc by flow cytometry and immunoflurescence. we also study the contractility of these cells under the effect of different cytokines. human bone marrow samples were obtained from bone marrow aspirates. bone marrow mononuclear cells were isolated by density gradient centrifugation and cultured in opti-mem culture medium with % of fetal calf serum at °c and % co . bone marrow nonadherent cells were removed after h, and culture medium was refreshed twice per week thereafter. cells grew adherent, with a fibroblastic morphology and expressed cd , cd , cd , cd and stro- and were negative for cd . intracellular staining was performed for alfa-sm actin. cell contractility was measured with the collagen gel contraction assay. alfa-smooth muscle actin was detected in almost % of msc. interleukin- , ifn-gamma and tnf (th cytokines) increased msc contractility, whereas il- (a th cytokine) decreased msc contractility. by immunofluorescence, we observed that il- , ifn-gamma and tnf increased the incorporation of alfa-sm actin into the stress fibers of msc, whereas il- decreased that incorporation. our results suggest that th and th cytokines regulate msc physiology by acting on their contractility. aims: thapsigargin (tg) is a sesquiterpene lactone (sl) of guaianolide type isolated from the mediterranean plant thapsia garganica l. it is widely recognized as an inhibitor of sarco-endoplasmic reticulum ca + -atpase leading to elevation of intracellular calcium. this activity is shared by trilobolide (tb), a sl from laser trilobum (l.) borkh. tg has been shown to possess prospective immunotherapeutic properties. it kills slowly proliferating and non-proliferating cells, and inhibits replication of viruses. the aim of our work was to investigate possible immunostimulatory potential of tg and tb. methods: the effects of the agents were analyzed under conditions in vitro using rat and mouse resident peritoneal cells (pecs), and human peripheral blood mononuclear cells (hpbmcs). they were cultured in density of × /ml in complete rpmi- medium. supernatant levels of ifn-g were determined by elisa. production of no by animal pecs was assayed using griess reagent. possible contamination of the samples with lps was excluded using the chromogenic limulus amoebocyte assay. results: we have found that both tg and tb at as low doses as nm and nm induce ifn-g secretion in rat pecs and hpbmcs, respectively. the concentration of ifn-g produced by the highest dose of the agents ( mm) at the -h culture interval reached the values of approximately ng/ml and ng/ml in rat pecs and hpbmcs, respectively. the increase was apparent within the interval of - h in rat pecs. the -h interval was optimal for accumulation of ifn-g in cultures of hpbmcs. only modestly enhanced secretion of ifn-g was observed in mouse pecs. production of ifn-g was found to depend on activation of nf-xb and map kinases p and erk / . it was not suppressed by the calcium chelating agents bapta-am and tmb- . the tg-and tb-induced ifn-g production was associated with activation of the high-output production of no. conclusions: sesquiterpene lactones tg and tb are potent inducers of ifn-g in animal and human cells. the effect is independent of their serca inhibitory potential. underlying mechanism(s) of the immunostimulatory effects remain to be elucidated. acknowledgements: the work was supported by the grant gacr / / . pin is a peptidil-prolil-cis-trans isomerase that specifically binds phosphorylated ser/thr-pro protein motifs and catalyzes the cis/trans isomerization of peptides. mitotic proteins, cytoskeleton, transcription factors and apoptotic proteins are pin substrates and targeting sites. recent data show that pin interacts with apo-bec g (a g). the pin /a g interaction results in a reduced a g expression and a diminished a g-mediated restriction of hiv. two single nucleotide polymorphisms (snps) in the promoter region of the pin gene (- g/c and - t/c) modulate pin expression; in particular, the - gc genotype or cc haplotype are associated with reduced protein levels (neurobiol. aging, ; - , ) . the - c/g and - t/c polymorphisms in the promoter of pin gene as well as pin protein levels were analyzed in exposed seronegative individuals (esn), heterosexual partners of hiv-infected patients; hiv-infected patients (hiv) and healthy controls (hc). the genotype and allele distributions of the - snp was skewed in esn (genotype: p= . ; allele: p= . ). in particular esn showed a significantly lower frequency of the - gg genotype compared to hiv and hc (p= . and p= . , respectively) and consequently a lower g allele frequency (p= . and p= . , respectively). no significant differences were found for the - snp. these snps are in linkage disequilibrium and combine to form haplotypes. conclusions: our findings support the role of hiv viral replication as the most important promoter of immune activation and prove the importance of art in reducing immune activation and viral replication even in a sub-saharan african environment, where patients are exposed to an abundance of other infectious agents. our data further indicates that hiv replication rather than host genetics is the key determinator of circulating cytokine levels among the studied hiv infected participants. aims: acyclic nucleoside phosphonates (anps) are synthetic analogues of natural nucleoside monophosphates. they have proved to be outstanding antivirals inhibiting replication of both dna-viruses and retroviruses. tenofovir, -(r)- [ -(phosphonomethoxy) propyl]adenine [(r)-pmpa] is broadly used for therapy of aids, adefovir, -[ -(phosphonomethoxy)ethyl]adenine (pmea) has been approved for the treatment of hepatitis b. the aim of our work was to investigate possible interactions of anps with production of cytokines and nitric oxide (no) implicated in antiviral defence mechanisms. the immunobiological effects of anps were screened in vitro using mouse resident peritoneal cells. they were cultured in density of × /ml in complete rpmi- medium. secretion of cytokines was determined after the -h culture by elisa. production of no was assayed at the interval of h using griess reagent. approximately compounds belonging to several distinct anp groups were included in the study: a) pmea derivatives, b) pmedap i. e. -[ -(phosphonomethoxy)ethlyl]- , -diaminopurine derivatives, c) -[ -hydroxy- -(phosphonomethoxy)propyl]-adenine (hpmpa), and hpmpdap derivatives, d) guanidino analogues of pmpdap, e) -heteroalkyl substituted -amino- -guanidinopurines, and f) -amino- -(purin- -yl)propanoic acid derivatives. possible presence of lps in the stock solutions of the samples was checked and excluded using the chromogenic limulus amoebocyte assay. results: approximately compounds were found to activate the secretion of anti-hiv effective chemokines rantes and mip- a and cytokines tnf-a and il- . although these anps did not stimulate biosynthesis of no on their own, they substantially augmented production of no triggered by ifn-g. no clear-cut relationship between the chemical structure and biological effects of anps was observed. however, the most effective proved to be the n -cycloalkyl derivatives of pme-dap. the effects were produced in a dose-dependent fashion, exhibiting the immunostimulatory effects at as low concentrations as to mm. the remarkably enhanced secretion of chemokines was reached within - h of the cell culture. the effects were found to depend on activation of nf-kb. conclusions: it may be suggested that anps represent a new generation of antivirotics with combined antimetabolic and therapeutically prospective immunostimulatory properties. acknowledgements. the work was supported by the grant m . host related immune factors in childhood chronic hepatitis b and change of initial profile with ifn-a treatment needs to be clarified. sixteen patients were included, and million units of ifn-a treatment three times a week for months was initiated. before and after treatment: percentages of the il- and ifn-g in cd + t cells were assessed to determine intracellular t helper cell (th ) type cytokine expression. similarly, percentages of intracellular il- and ifn-g were detected to verify cytotoxic t cell (tc ) type cytokine expression in cd + t cells. percentage of th and tc type cytokine expression, (il- and il- ) were determined in cd + and cd + t cells, respectively. six ( %) of these were evaluated as having no response and the other half with partial/complete response. all patients had higher percentages of th cells with respect to healthy controls before treatment. tc percentages, both tc and tc , were significantly different between these groups, being higher in the patient group. when values of no responder group were compared with healthy controls, il- expression was higher and the percentages of th type cells were significantly low. il- expression in th and tc cells decreased after treatment in the unresponsive group. intracellular cytokine profiles of treatment responders and normal controls were not different. this has been the first study in children comparing baseline and post treatment intracellular cytokine profiles with healthy controls. the frequency ( , %) of high concentration of igg anti-oxldl antibodies in patients with hcv infection were significantly elevated in comparison to healthy subjects ( , % according to bibliographic data). the immune response was significant but it was not assosiated with the viral load. it is probable that humoral immunity plays a critical role and contributes in an immunoinflammatory reaction of hcv-infection. abstract withdrawn by author t. schwandt , f. juengerkes , b. schumak , g. gielen , j. kalff , p. knolle , b. holzmann , a. limmer university hospital bonn, institute of molecular medicine and experimental immunology, bonn, germany, university hospital bonn, department of surgery, bonn, germany, department of surgery, tu munich, munich, germany bacterial translocation is a possible risk of abdominal surgery and could be the cause of life-threatening consequences such as organ failure and septic shock. patients surviving septic shock often suffer from opportunistic infections as well as defects in adaptive immunity. here we investigated the influence of bacteremia and bacterial translocation on systemic adaptive immune responses using murine models. to mimic abdominal surgery, mice were subjected to intestinal manipulation (im). to study septic conditions, mice underwent colon ascendens stent peritonitis (casp) or received e.coli intravenously. we monitored the distribution of gut-derived bacteria by in vivo imaging (xenogen) and additional microbiological assays and determined antigen-specific ctl responses towards subsequent infection with recombinant adenoviruses (av). we detected comparable amounts of bacteria in lung, liver and spleen of mice that underwent casp or were injected i. v. with e.coli. in addition, mice infected systemically with av lacked an antigen-specific ctl response, whereas the ctl responses of locally av infected mice were not affected. in contrast, bacteria were detected in lung and liver but not in spleen of mice that were subjected to im or received e.coli by injection into the hepatic portal vein. here, the ctl response was not impaired. depletion experiments imply that kupffer cells as well as soluble mediators such as tumor necrosis factor alpha play an important role in trapping and clearance of translocated bacteria in liver and lung. our experiments demonstrated that translocation of bacteria did not cause immune suppression as long as they did not reach the spleen in high numbers. we suggest that liver and lung fulfill a filter function to prevent systemic distribution of gut-derived bacteria. failure of or bypassing these barriers might enable bacteria to access the spleen and thus cause systemic suppression of adaptive immunity, whereas induction of local immunity is not affected. objectives: varicella-zoster virus (vzv) is one of the most frequent pathogens for which a vaccine is available. tropism of vzv for skin is the most obvious manifestation of vzv infection, producing vesicular cutaneous lesions that are associated with varicella and herpes zoster. the striking difference in the clinical outcome of infection by rush inducing circulating virulent vzv strains and asymptomatic infection by the vaccine leads to the assumption that the virus interferes with cutaneous immunity. therefore, we comparatively investigated the impact of vzv clinical isolates and the vaccine on the reciprocal crosstalk of immature dendritic cells (idcs) and epithelial gd t cells. methods: skin punch biopsies of herpes zoster patients were analyzed by dual immunofluorescence microscopy. phenotypic changes of cutaneous dcs and monocyte-derived dcs after vzv infection were investigated by flow cytometry. the cytokine profile of vzv-infected dcs and epithelial gd t cells was determined through elisa. results: we observed that innate lymphocytes recognize dcs, which infiltrate herpes zoster lesions, after infection with vzv. strikingly, only the vaccine but not vzv clinical isolates could license the bidirectional crosstalk between idcs and gd t cells resulting in release of ifn-g and il- , the signature cytokines of antiviral immune responses. this is the first demonstration that virulent virus strains disrupt dendritic cell instruction whereas the corresponding vaccine does the opposite. we describe a novel immune evasion strategy in the skin, which provides the opportunity for efficient and symptomatic virus replication. this result is also of practical importance: future vaccine design has to ensure that candidate vaccines do not impair dc instruction in order to allow stimulation of powerful antiviral immune responses. a. jafarzadeh rafsanjan university of medical sciences, rafsanjan, iran, islamic republic of objective: it has been reported that the caga + h. pylori strains induce more severe gastric mucosal inflammation. the aim of this study was to investigate the association of the h. pylori virulence factor caga with serum levels of il- and il- in h. pylori-infected duodenal (du) patients and h. pylori-infected asymptomatic (as) carriers. methods: totally, h. pylori-infected du patients ( patients were positive for anti-caga antibody and patients were negative for anti-caga antibody), h. pylori-infected as carriers ( subjects were positive for anti-caga antibody and subjects were negative for anti-caga antibody) and healthy uninfected subjects (as a control group) were enrolled to study. a blood sample was obtained from all participants and the serum levels of il- and il- was measured by elisa method. the mean serum levels of il- in total du patients ( . pg/ml ± . ) was significantly higher than those observed in total as subjects ( . pg/ml ± . , p x . ) and healthy control group ( . pg/ml ± . , p x . ). in du group, it was found that the mean serum levels of il- in subjects with positive test for anti-caga ( . pg/ml ± . ) was significantly higher than those observed in subjects with negative test for anti-caga ( . pg/ml ± . ; p x . ). the mean serum levels of il- in du ( . pg/ml ± . ) and as groups ( . pg/ml ± . ) was significantly higher than those found in uninfected control group ( . pg/ml ± . , p x . and p x . , respectively). however, no significant difference was observed for mean serum levels of il- between du and as groups. moreover, in both du and as groups the mean serum levels of il- was not significantly differ in subjects with positive test for anti-caga and those were negative for anti-caga antibody. the results of the present study showed higher serum concentrations of il- and il- in h. pylori-infected subjects as compared with control group. in du group the expression of il- influenced by the bacterial caga factor. a. aral , , a. atak gazi university faculty of medicine, department of immunology, ankara, turkey, gazi university institution of health sciences, department of immunology, ankara, turkey objective: ebv is a human herpesvirus, which infects human b lymphocytes latently and immortalizes the cells due to transformation. ebv infection is asymptomatic in childhood while it may cause a self-limiting lymphoproliferative disorder called infectious mononucleosis (im) in adolescence. in immunodefective patients, the virus may lead to malignancies like burkitt's lymphoma, nasopharyngeal carcinoma and immunoblastoma. the virus can also cause latent infections. cytokine production in response to ebv infection differs according to the phase of the infection. neopterin, ifn-g and il- levels are elevated in acute ebv infection in vitro; but in chronic phase, particularly, il- elevation could not be detected. tnf-a enhances the effects of ifn-g on neopterin synthesis while neopterin enhances the secretion of tnf-a via various stimuli. ifn-g levels are elevated particularly in the acute phase of im. since the elevation of cytokine levels changes according to the phases of disease, it's thought that the association between host defense and viral replication depends on different parameters. ifn-g is the major stimulus for neopterin synthesis, which stimulates monocytes and macrophages primarily. increased production of neopterin in body fluids can be used to monitor the activation of cell mediated immunity. method: in order to analyze the effects of neopterin release and other cytokines, mononuclear cells have been isolated from peripheral blood samples of healthy donors and transformed via ebv. neopterin, ifn-g, tnf-a and il- levels have been measured with eia kits in culture supernatants. results: neopterin levels increased dependent on time and independent of ebv transformation. in ebv-transformated cell culture tnf-a levels increased beginning from the th hour and reached to maximum levels at the st week and decreased again at the rd week; however there were no significant differences between the levels among three weeks. likely tnf, ifn-g levels also increased at the st week and started to decrease at the rd week. il- reached to its peak at the rd week. conclusion: according to these results, neopterin levels, which increased dependent on time but independent of ebv transformation, may be a helpful marker for evaluating the acute response to viral infection but not for transformation. v. jurisic , m. jurisic university of kragujevac, school of medicine, kragujevac, serbia, university of belgrade, school of dentistry, belgrade, serbia tnf-alpha is a pleiotropic cytokine that is considered as a primary modifier of inflammatory and immune reaction in response to various inflammatory diseases and tumour. we investigated tnf concentration in radicular inflamed cysts and odontogenic tumours obtained from patients undergoing surgery, under local anaesthesia, and after aspiration of cystic fluid from non-ruptured cysts. further, we estimated the role of cyst wall on production of tnf-alpha in respect of presence of inflammatory cells, degree of epithelial proliferation and degree of vascularization. the concentrations of tnf-alpha in the cystic fluids were measured by elisa, while proteins analyzed after separation by two-dimensional gel electrophoresis. the presence of pericystic inflammed cells were analyzed in thick section for routine histological examinations and by immunohistochemisty for cd , cd and cd . tnf-alpha is elevated in both cysts fluid, but higher values were found in radicular inflamed cysts in comparison to odontogenic tumours. higher concentration of tnf-a were associated with higher protein concentration, higher presence of inflammatory cells in peri cystic tissues, cysts wall thickness and higher degree of vascularisation (mann-whitney u-test, p x . ) in radicular cysts. no correlation was found, based on these parameters in odontogenic tumours, but all sumple have detectable concentrations of tnf-alpha. objectives: interactions between the neuroendocrine and immune system play an important role in maintaining and restoring homeostasis. in susceptible individuals a dysfunction of the neuroendocrine system may be one of the risk factors involved in the pathogenesis of septicemia and bacterial infection at all. we will study prolactin role in defensive reaction of immune system to bacterial infection. as a type of this bacterial infection we have chosen septic status, where we are expecting the most significant alterations of immune reaction, and specially septic statuses in blood malignancies, where we are expecting deficiency of immune system. our idea is that prolactin takes part in this defensive reaction by its cytokine effects, and it has contraregulative role against activation of adrenocortical system. the aims of this study are to extend our knowledge about the activation of peripheral prolactin expression and by this way to contribute elucidation of its role in periphery. ) drawings blood from patients and healthy donors. blood of patients and healthy persons were sampled together with past histories after getting their acquainted approval. status of patient had to meet these conditions: a) the presence of systematic inflammatory response syndrome (sirs) according to standard clinical and laboratory criteria. b) positive hemoculture or determination of septic focus with demonstration of microbiologic source. ) detection of the gene expression of prolactin and tlr- in cd + peripheral blood monocytes from patients with septic status and from healthy controls has been performed by rt-pcr at the level of mrna and flow cytometry at the level of intracellular protein results: we have found statistical significant differences (p p . ) in expression profile between patient and control groups. these differences were at both levels of expression, mrna and protein. conclusion: septic statuses change tlr- and peripheral prolactin expression in cd + monocytes. the function of interferon (ifn)-induced immunoproteasomes (i-proteasomes) is at present almost exclusively acknowledged in connection with improved processing of mhc-class i antigens. the experiments performed here challenge this existing paradigm of i-proteasome function. we demonstrate in vivo and in cellulo that the key role of i-proteasomes resides in the protection of cells against the formation of protein aggregates, which is ultimately crucial for preservation of cell viability under ifn-induced oxidative stress. ifns up-regulate the ubiquitylation machinery and enhance the formation of oxidant-damaged, nascent, poly-ubiquitylated proteins, which essentially require i-proteasomes for their efficient degradation. i-proteasome deficiency results in the formation of aggresome-like induced structures and increased sensitivity towards apoptosis. enhanced degradation of poly-ubiquitin conjugates designed to protect cells, will therefore also increase the peptide supply for antigen presentation. thus, only by executing their physiological function in the maintenance of protein homeostasis i-proteasome induction also provides a mechanism for cellular immune-adaptation. background: revived by the description of new functions, b cells are considered to be essential in the genesis of autoimmune diseases. in strong support of this interpretation, baff would play a key role in their physiology. however, the correlation between circulating baff levels and disease activity is not clearly established. conflicting results concerning levels of baff and b-cell repopulation after rituximab treatment have been reported. in fact, basal serum levels of baff reported in literature vary according to the antibodies (ab) used in the elisa and the mode of calculation. the aim of this study was to understand these variations. material and methods: different anti-baff abs were tested to verify whether they recognize glycosylated baff purified from u culture supernatant. sera from patients with autoimmune diseases were also tested. a western-blot analysis of sera was performed to evaluate anti-baff abs specificity and the best combination of anti-baff abs validated for elisa. then, different commercial baff elisa-quantification kits were compared to our "in-house" elisa. results: unexpectedly, the binding of some anti-baff ab was restricted to glycosylated baff. however, both glycosylated and non-glycosylated forms of baff were found in sera from patients with autoimmune diseases. most of the kits commercially designed to quantify baff suffer from some limitations. some sera are indeed positive with a kit and negative with another and vice versa. furthermore, there seems that rheumatoid factors (rf) interfere and correlate with the optical density of the anti-baff elisa. conflicting results concerning levels of baff and disease activity or auto-abs titers should be reconsidered in light of the glycosylation status of baff. (table- ). when tb household contacts and healthy controls were compared, cfp and esat seemed to be more useful than tst in tb contacts for displaying ltb (table- ) . although cfp spot numbers were much more than esat spots at the beginning and follow up period, statistically there was no difference in terms of median values(p: , )( table- ). both esat and cfp spots were prone to decline during the follow up period. [ is some evidence to suggest that aspects of innate immunity (e. g. triggering of cytokine production by dendritic cells) may be impaired by hcv. to gain insight into some features of the innate immune response activated in vivo in the context of acute hcv replication, we analysed cytokine and chemokine levels in serum samples from chronic hcv patients undergoing liver transplantation. luminex multiplex assays and elisas were used to quantitate serum levels of g analytes immediately prior to liver transplantation and at sequential time points up to days post-transplantation. the increase in serum hcv rna levels associated with acute viral infection of the transplanted liver was found to be associated in most patients with elevations in serum levels of cytokines and chemokines including ifn-gamma, tnf-alpha, ip- , il- and il- . notably, the pattern and magnitude of systemic analyte elevations were very similar to those accompanying the acute burst of viral replication in primary hcv infection. high-magnitude elevations in systemic type ifn levels were not observed in either setting, which may reflect an in vivo impairment of plasmacytoid dendritic cell functions by hcv similar to that observed in in vitro studies. we suggest that the liver transplant setting can be used as a model to study aspects of the innate immune response activated in acute hcv infection. to test the hypothesis that virus interactions with sialic acid receptors may play a role in innate antiviral immunity, we used recombinant viruses and differentiated cultures of human airway epithelial cells (hae). the hemagglutinin of the pandemic virus a/hong kong/ / (h n ) (hk) differs from its putative avian precursor by amino acid substitutions. we generated the complete recombinant virus rhk and its ha variants with amino acid reversions back to the ancestral avian sequence (rhk- aa-i r, n d, k n, s n, g a, human ( - ); rhk-r -l q, s g and rhk- aa-i r, n d, k n, s n, g a, l q, s g, avian ( - )). among these variants, the double mutant rhk-r and the seven mutant (rhk- aa) had a typical avian-virus-like receptor-binding specificity owing to substitutions l q and s g.we infected hae cultures with the viruses and collected samples from the apical and basolateral sides of the cultures at different times post infection. virus titers were determined in mdck cells, and concentrations of about pro-and anti-inflammatory mediators and chemokines were measured using a multiplex bead assay.concentrations of most cytokines progressively increased at the apical side of the cultures in the course of the infection. many cytokines, including t-cell-attracting chemokines such as ip- and rantes, were induced to similar levels by different viruses. however, some mediators were induced significantly stronger by avian-like viruses rhk-r and rhk- aa as compared to rhk and rhk- aa. in particular, avian-like viruses stimulated a higher release of potent chemo-attractants of innate immune cells, such as g-csf and il- , shedded adhesion molecules (cd , vcam- , icam- ), and pro-apoptotic factors (trail). remarkably, the patterns of secreted cytokines differed between the apical and basolateral sides of the cultures. whereas avian-like viruses typically induced similar or higher levels of cytokines at the apical side than did rhk and rhk- aa, the human-like viruses were stronger inducers of basolaterally secreted mediators. these data provide the first direct experimental evidence that receptor specificity of influenza viruses can significantly affect patterns of innate immune responses in human airway epithelium. further studies are warranted to determine the role of the observed effects in the host range and pathogenicity of influenza viruses in humans. a total of newborn infants were enrolled in the study. forty-nine newborn infants (group i), who met the criteria of sepsis, had a routine sepsis evaluation as well as measurement of pct, il- , and neutrophilic cd levels, procalcitonin and il- were measured by elisa technique while, neutrophilic cd by single colour flowcytometric technique. of these "infected" infants, had positive blood culture (subgroup ia: culture-positive sepsis), and infants were diagnosed to have clinical sepsis with negative blood cultures (subgroup ib: culture-negative sepsis). another newborn infants classified as control group (group ii) . results: sensitivity, specificity, positive predictive value, and negative predictive value for diagnosis of sepsis were analyzed for pct, il- , cd , and crp. il- had the highest sensitivity and specificity, % and %, respectively, using cutoff n . pg/ml. for pct, the highest sensitivity and specificity, % and %, respectively, were at a cutoff value of n . pg/ml. neutrophilic cd had maximal sensitivity and specificity of % and %, respectively, at cutoff value of . %. combinations of different markers may improve the sensitivity and specificity of biomarkers such as the tests used in this study. we found that the best combination was il- and neutrophilic cd , which together provided sensitivity and specificity of % and %, respectively, and npv %. the combination of il- and crp had high sensitivity and moderate specificity, % and %, respectively. conclusions: il- and neutrophilic cd levels determination can be used as good tests for early detection of neonatal sepsis. procalcitonin measurement might be used as an additive parameter to improve the diagnostic efficacy of the other markers in neonatal sepsis, but it is not helpful as a single test. objectives: the assembly and activation of inflammasomes are essential processes in the immune response to many inflammatory stimuli. inflammasomes are typically formed by at least one member of the cytosolic innate immune sensor family, the nod-like receptors (nlr). the nlr family members nalp , naip or ipaf and the adaptor asc are involved in caspase- activation in response to bacterial infection, triggering the processing and secretion of il- b and il- . recent studies have demonstrated that tlr-dependent inflammasome activation in macrophages is modulated by autophagy, a homeostatic mechanism for the catabolism of cytosolic constituents. autophagosome biogenesis and maturation requires activation of the class iii pi k, vps and can be inhibited with the pi k inhibitors wortmannin and methyladenine ( ma). in contrast, activation of akt, via class i pi k, results in inhibition of autophagy. the aim of this study was to determine the nature of this inflammasome and whether autophagy influences il- b secretion in dendritic cells. methods: murine bone marrow-derived dendritic cells (bmdm) were treated with tlr ligands in combination with ma, wortmannin or an akt inhibitor. supernatants were analysed for il- b by elisa. results: ma enhanced il- b secretion by bmdc treated with the tlr and tlr ligands poly(i:c) and lps, but not with any other tlr ligands tested. similar results were obtained using wortmannin. this increase in il- b secretion was greatly reduced in bmdc from nalp -/mice compared to wild type c /bl controls. treatment with the akt inhibitor had no effect on lps-induced il- b secretion by bmdc. tlr-dependent secretion of il- a was also enhanced by treatment with ma. conclusions: these data demonstrate that il- b secretion by bmdc in response to treatment with pi k inhibitors, in combination with lps or poly(i:c), is dependent on the nalp inflammasome. this response is limited to tlr and tlr agonists. inhibition of akt had no effect on lps-induced il- b production, suggesting that the effect of wortmannin and ma on inflammasome activation is not dependent on the inhibition of akt activation by class i pi k. objectives: in the last few years, several evidences have shown the modulation of toll-like receptors (tlrs) by g-protein coupled receptors, i. e. our group has recently demonstrated the attenuation of tlr signaling by the inflammatory lipid mediator sphingosine -phosphate (s p) through receptors and in human monocytes-macrophages, which could explain some of the s p anti-atherogenic effects. since adhesion of monocytes to endothelial cells is considered an important event in atherogenesis, our goal was to investigate the putative implication of tlr-s p receptors crosstalk on the expression of adhesion molecules in endothelial cells. methods: for the study, in vitro cultured endothelial cells from arterial and venous origin were challenged with a combination of tlr ligands and s p, and later analyzed by flow cytometry. a pharmacological analysis of the s p receptor subtype involved in the response was also performed. results: data from flow cytometry experiments revealed that icam- expression was increased following lps and s p concomitant stimulation in both venous and arterial cells, suggesting that tlr and s p receptors cooperate in the expression of icam- . conversely, no cooperation was observed when tlr ligands were used. in order to elucidate which s p receptor subtype was involved in the increase of icam- expression, we used a pharmacological approach with s p receptor inhibitors and pertussis toxin. results showed differences between arterial and venous cells. while in arterial cells elevated icam- after lps and s p challenge was significantly reduced by blocking s p receptor and the effect was pertussis toxin-insensitive, in venous cells the response was pertussis toxinsensitive and not blocked with inhibitors of s p receptors and , which suggest that s p receptor might be involved in the effect. conclusions: altogether these data demonstrate that tlr and s p receptors can interact to increase adhesion molecules such as icam- in human endothelial cells, and the s p receptor subtype involved in the effect differs between arterial and venous cells. with ssc without pah) and a pool of sera of healthy controls (hc) were tested. results: in dimension immunoblot, serum igg from ssc patients, patients with ipah and hc tested individually reacted with - , - and - protein bands in a human vsmc protein extract with qualitative and quantitative differences between groups, respectively. in dimension immunoblot, igg of pools of patients with ipah, igg of pools of patients with ssc with or without pah, and igg of a pool of hc recognized ± , ± , ± and protein spots respectively. twenty one protein spots were recognized by more than % of igg of pools of sera in each group of patients and not by igg of hc. twenty seven protein spots were recognized by the great majority of igg of pools of patients with a higher intensity than igg of pools of hc. identified proteins were constituents of cytoskeleton, proteins involved in oxidative stress as stress-induced phosphoprotein and peroxyredoxin and proteins involved in regulation of cell energy metabolism as triosephosphate isomerise. we have identified anti-vsmc abs in the serum of patients with idiopathic and ssc-associated pah. these abs bind to cytoskeleton, oxidative stress and cell cycle antigens. objectives: this study aimed to verify that subcutaneous lymph node transplantation inducing lymphatic regeneration is possible in healthy adult rats, as obtained in other species. methods: this rat model was used to determine the effects of lymph node fragmentation as well as sheep erythrocytes and platelet-rich plasma injection on the regeneration of the transplanted lymph nodes. results: this rat model is adequate to study the regeneration of transplanted lymph nodes. lymph node fragmentation seems to affect transplant regeneration negatively. an immune challenge by injection of sheep erythrocytes in the drainage area of the transplanted lymph nodes does not improve fragment regeneration. however, injection of syngeneic platelet-rich plasma containing several growth factors resulted in an improvement in regeneration. conclusion: lymph node fragment regeneration, although still experimental, could be relevant for lymphedema prevention. acquired lymphedema has a high prevalence in developed countries as a consequence of the removal and/or radiotherapy of tumor-draining lymph nodes in cancer patients. this disease causes lifelong disability due to chronic swelling and increased risk of infections. it currently lacks an effective treatment. methods: patients suffering from different diseases were enrolled in our study. patients were suffering from bone diseases (osteomyelitis, necrosis, tumour) whereas of them were suffering from inflammatoty diseases (soft tissue inflammation, diabetic ulceration). blood specimens were collected before hyperbaric oxygen treatment and the serum levels of icam- and vcam- were assesed by an enzyme immunoassay (elisa). results: out of the patients ( , %) had elevated levels of the intercellular adhesion molecule. out of the patients suffering from bone diseases ( , %) had raised values (mean value ng/ml) whereas patients out of the suffering from soft tissue diseases and diabetes ( , %) had raised values (mean value , ng/ml). reference value for icam- was - ng/ml. vascular cell adhesion molecule's assesment revealed no elevated levels in our patients. conclusions: our study revealed a high rate of patients ( %) having increasd levels of icam- . high icam- levels were more prevalent in patients suffering from soft tissue inflammatory diseases and diabetes ( , %) than in patients with bone diseases ( . %). mean values were found , ng/ml and ng/ml accordingly. those findings verify the positive correlation between icam- and inflammatory diseases and tissue damage but not for vcam- . colorectal cancer (crc) was the first solid tumour to be successfully targeted with anti-angiogenic therapy in the clinic. tumour angiogenesis is critical for cancer progression in that it permits expansion of the tumour mass and fosters malignant dissemination. angiogenesis is a multistep process involving endothelial cells as well as numerous stromal components within the tumour microenvironment that also represent potential therapeutic targets. inflammation dependent-angiogenesis is increasingly recognised as a central force in tumour growth and progression, while use of anti-inflammatory drugs has been found to reduce incidence of crc carcinoma potentially through repression of tumour angiogenesis. we investigated the link between inflammatory angiogenesis and colorectal cancer in archival tissues across a range of pathologies that represent diverse steps in the progression of crc: cases of ulcerative colitis (urc), adenocarcinomas developed from preexisting tubular or tubulo-villous adenomas, tubular or tubulo-villous adenomas with low grade dysplasia, and infiltrating adenocarcinomas. immunohisto- objectives: to determine the effect from the administration of preoperative pravastatina to therapeutic dose in the expression of cd in the leucocitaria adhesion to endotelio vascular in the isquémico-reperfundido miocardico weaveal endotelio vascular en el tejido miocardico isquémico-reperfundido by the circulation extracorpórea (cec). methods: they were included in way randomizada double blind patients with intervened controlled hiperlipidemia of surgery coronary low circulation extracorpórea (cec). mg of pravastatina oral hours they were administered before the procedure (group study, n= ) or placebo (group placebo, n= ), and control (group control, n= ). samples of outlying veined blood were extracted to the hours. the separation of leukocytes was made in peripheral blood, to determine the expression of cd in such. in all the samples one quantified the intensity of the expression pattern and the percentage of leucocitarias cells. results: types of patterns were distinguished: cytoplasmic, of membrane and compound. the intensity of the expression was classified in degrees: degree . without expression. degree . weak; degree . moderate; degree . intense. in the group control: most of the samples they presented/displayed a mixed pattern (cytoplasmic and of membrane) with an intensity degree - . the placebo group: mixed pattern, degree - . group study ( mg. oral pravastatina): most of the cells they presented/displayed a predominance of membrane pattern: degree - . the percentage of cells that expressed cd was greater in the group study ( mg. oral pravastatina). the preoperative oral pravastatina to therapeutic unique dose ours study produces a greater expression cd answer induced by the cec; it seems that these molecules located in the leukocytes participate in the adhesion to the activated endoteliales cells, necessary for the extrusion of the lymphocytes through endotelio towards the inflammatory center and in quimiotaxis of the leukocytes towards the inflammation sites. several surface molecules on endothelial and epithelial cells undergo regulated cleavage by the disintegrin and metalloproteinases adam and adam . we recently identified transmembrane chemokines, junctional adhesion molecule-a (jam-a), and members of the proteoglycan family as novel substrates for these proteases. here we demonstrate that cell lines and primary cells of human endothelial or epithelial origin release considerable amounts of soluble jam-a and proteoglycan ectodomains. this release is enhanced by treatment with the proinflammatory cytokines ifng and tnfa. the enhanced release was not caused by an increased gene induction but rather associated with a reduction of the surface expressed molecules. both, constitutive and induced release required the presence of adam as demonstrated by specific inhibitors, lentiviral silencing experiments as well as treatment with the recombinant catalytic domain of adam . these data suggest that the proinflammatory cytokines ifng and tnfa induce enhanced proteolytic shedding of cell surface molecules on endothelial and epithelial cells. to investigate the physiological relevance of this induced shedding, mice were treated systemically with ifng/tnfa leading to increased presence of soluble jam-a in the blood serum. both cytokines also stimulated jam-a release from excised murine aortas with was associated with enhanced adam activity in the tissue. in the presence of the adam inhibitor induction of jam-a release was suppressed. in cultured epithelial cell lines enhanced shedding of jam-a or proteoglycans was not associated with increased mrna or surface expression of adams but rather with increased activity of cellular adam as shown by means of a synthetic substrate assay. our study demonstrates that the proinflammatory cytokines ifng/ tnfa upregulate adam -mediated shedding activity rendering the protease an important modulator of endothelial and epithelial surface molecules in inflammatory settings. rium, and the haplotype vegf- / vegf+ is associated with rcc risk ( p= , ), metastases ( p= , ), nuclear grade ( p= , ), tumor stage ( p= , ), and tumor size (p= , ). on the other hand, the polymorphism vegf - a/c is not associated with rcc risk and clinical parameters. our results shed a new light to the knowledge on the association between vegf polymorphism and rcc risk and development. these data could help to improve our understanding of the rcc pathogenesis and disease progression. pten is a lipid phosphatase, whose substrate is phosphatidylinositol , , -trisphosphate. therefore, pten is one of the main antagonists of the pi -kinase, which plays a major role in many important cellular functions, such as proliferation, migration or response to inflammatory stimuli. here we investigated the role of pten in collagen induced arthritis. we show that conditional deletion of pten under the lysm promoter (lysmcrepten flox/-) leads to a significant reduction in clinical severity of collagen induced arthritis (cia). histological analysis of cia, lysmcrepten flox/mice displayed significantly reduced joint inflammation as well as erosive bone destruction. total anti-collagen antibodies, however, as well as anti-collagen iggs were identical in both groups. upon analysis of inflammatory cytokines in serum after immunisation we found a significant reduction of il- as well as il- levels. furthermore, pten deficient macrophages and dendritic cells showed reduced induction of il- as well as il- and il- mrna upon stimulation with various tlr-ligands. since these cytokines play an important role in the induction of pathogenic th- t cells, we measured th- cytokines in lymph nodes after immunisation with collagen. although dendritic cell and macrophage recruitment to the draining lymph node was comparable in both groups, there was a slight reduction of il- and a strong reduction of il- mrna in the draining lymph node of immunized lysmcrepten flox/compared to wild-type mice. one of the mechanisms through which il- exerts its anti-inflammatory effects consists in promoting the release of anti-inflammatory molecules. in this context, particularly important is the production of il- ra, whose expression is induced by lps in human neutrophils and monocytes and significantly potentiated by the presence of il- . based on our previous observation that support a direct role of il- -activated stat in the enhancement of il- ra transcription induced by lps, we plan to characterize the transcriptional activators recruited to the il- ra promoter in vivo and responsible of the increased rate of transcriptional initiation upon exposure of lps-treated cells to il- . quantitative chromatin immunoprecipitation (chip) studies were employed to examine the in vivo binding of transcriptional activators to the il- ra promoter. crosslinked nuclear lysates were immunoprecipitated and min after il- addition with different antibodies and immunoprecipitated dna was analyzed by quantitative real-time pcr for the presence of target sequence located in the il- ra promoter. chip assays showed that the pol ii recruitment to the il- ra promoter induced by lps is significantly increased by il- , further strengthening the concept that the rapid enhancement of lpsinduced il- ra gene expression by il- initially occurs by targeting transcriptional events. as expected, real-time pcr of anti-stat immunoprecipitated dna showed statistically significant levels of stat binding to the il- ra promoter only in cells stimulated with lps in the presence of il- . surprisingly, anti-p and anti-p chip assays revealed enrichment of both p and p recruitment to the il- ra promoter when il- was added to lps-stimulated cells, suggesting that il- enhances the recruitment of nf-kb to the il- ra promoter. interestingly, when nf-kb is recruited to this promoter in lps + il- -treated cells, the overall nf-kb nuclear translocation (analyzed by western blot) and dna binding activity (detected by emsa analysis) were not modified with respect to cells stimulated with lps alone. the enrichment of nf-kb at the il- ra promoter site is dependent on il- -activated stat , since it is greatly reduced when stat activation by il- is impaired. the molecular mechanism through which il- -activated stat promotes the recruitment of nf-kb to the il- ra promoter is currently under investigation. major components of mast cell secretory granules are proteases. we could recently report that intracellular stored mast cell-produced cytokines regulate mc protease activities and provided evidence that il- acts as a specific negative transcriptional regulator of mouse mast cell protease- (mmcp- ). we examined the mechanisms underlying the repression of mmcp- gene expression. our data show that the "repressor" effects of il- on mmcp- promoter activity are still operating on the mmcp- bp long minimal promoter. moreover, il- deficiency in mast cells causes a specific dysregulated expression of the transcription factors c/ebpb and yy . furthermore, chromatin immunoprecipitation revealed that il- promoted specific reciprocal recruitment of c/ebpb but not yy to the mmcp- promoter. finally, il- deficient mast cells display a predominantly non-cpg methylated pattern of the mmcp- . thus, we proposed that the expression of mmcp- and possibly other immunoregulatory genes may be regulated by il- through epigenetic modification and by balancing the content and binding of c/ ebpb and yy in mast cells. i. nagy , k. filkor , a. vörös , l. kemény , a. szász bzaka, baygen, szeged, hungary, university of szeged, department of dermatology and allergology, szeged, hungary micrornas (mirnas) are evolutionary conserved small non-coding rnas that act as key regulators of gene expression at post-transcriptional level by targeting mrnas for translational repression and/or degradation. mirnas have been shown to have unique tissue-, developmental stage-and diseases-specific expression patterns. during the last years several studies highlighted that mirnas play critical role in the differentiation and function of the adaptive as well as innate immunity. little is known however, about the differential regulation of mirnas following the activation of pattern recognition receptors. in order to tackle this issue, we treated hacat keratinocytes with staphylococcus aureus-derived peptidoglycan (pgn) once or repeatedly, the latter mimicking persistent infection. after appropriate treatments we first analyzed the expression profile of mirnas mir- , mir- a and mir- , which are known to participate in immune processes of the skin. repeated pgn-treatment significantly decreased mir- expression; in contrast, pgn re-stimulation had no further effect on mir- a and mir- expression. next, we investigated the correlation between the expression of mir- and its two known direct targets: regulatory protein p and suppressor of cytokine signalling- (socs- ). although the gene-expression profile of neither p nor socs- changed, we found that the expression of mir- reversibly correlates with both p and socs- protein expression, a phenotype that we verified by two independent protein analysis methods (western blotting and immunofluorescent labeling). importantly, transfection of hacat cells with anti-mir- prior to pgn-treatment completely abolished both p and socs- down-regulation, revealing the involvement of mir- in pgn-induced transcriptional regulation. finally, methylation-specific pcr experiments unravelled the role of dnamethylation in regulating mir- expression upon pgn-treatment. taken together, our results strongly suggest that sets of mirnas may be differentially regulated during persistent infection. results: tgfb +/-had a lower incidence and burden of benign papillomas when compared to tgfb +/+ animals. however, more scc developed in the tgfb +/-mice. after acute and chronic promotion, tgfb +/-skin showed a reduced proliferative response with no increase in epidermal tgfb or nuclear p-smad compared to tgfb +/+ mice. tpa-induced pkca activity as well as phosphorylation of specific pkc substrates in keratinocytes correlated with tgfb gene dosage. further, pharmacological inhibition of alk suppressed tpa-mediated pkca activation suggesting that physiological levels of tgfb are required for maximal activation of pkc-dependent mitogenic responses. even though the tpa-induced inflammatory response was greater in tgfb +/-skin, tgfb +/+ papillomas had more tumor infiltrating neutrophils. tpa-induced proinflammatory gene expression was sustained in tgfb +/-skin and primary keratinocytes but it was elevated in v-ras ha -transduced tgfb +/+ but not tgfb +/-keratinocytes, indicating that tgfb switches from an anti-inflammatory cytokine in the skin to a proinflammatory factor in tumors dependending on an activated ras. despite this differential proliferative and inflammatory response to tpa and enhanced papilloma formation in the tgfb +/+ mice, there was no increase in conversion to scc in this genotype. conclusions: tgfb acts to promote benign tumors enhancing cell proliferation and inflammation through its ability to regulate pkc activation in skin, yet retains a suppressive function for malignant conversion. background: proto-oncogene survivin has been recently shown as a prognostic marker distinguishing patients with destructive rheumatoid arthritis (ra). in the present study we studied the relationship between survivin and urokinase (upa), a fibrinolytic serine protease being over expressed in the inflamed joints and exhibiting arthritogenic properties. material and methods: levels of survivin and upa were measured in the paired blood and synovial fluid samples of patients with ra, using elisa and compared to controls with non-inflammatory joint diseases. the ability of upa to induce survivin and requirement of upa receptor (upar) was studied in primary synovial fibroblasts and pbmc of ra patients, human monocytic (thp- ) and fibroblast (mrc- ) cell lines employing antibodies against upar, sirna technique, and synthetic inhibitors of intracellular pathways. the ability to prevent urokinase-induced arthritis by interruption of survivin expression was evaluated in mouse model of arthritis. results: in the present material of ra patients and controls the levels of survivin correlated to urokinase (upa) (r= . ), a plasminogen activator over expressed in inflamed joints and known to exhibit potent arthritogenic properties. we found that / ra patients had high circulating levels of survivin. these patients had erosive arthritis and were characterized by high levels of upa. in vitro studies showed that upa induced survivin in leukocytes and this process was dependent on signaling through upa receptor. in turn, survivin was required for expression of upar. additionally, survivin was essential for upa production in mrc- and synovial fibroblasts. down-regulation of survivin with sirna was followed by significantly reducion of upa synthesis. finally, treatment with downregulation of survivin by sirna in vivo efficiently abrogated upa-induced arthritis in mice model. these findings indicate that survivin is an essential mediator of arthritogenic properties of upa regulating its synthesis in synovial fibroblasts and upar expression in leukocytes. close correlation between survivin and upa in patients with ra supports the improtance of these interaction for the pathogenesis of arthritis. upon cell activation, ubiquitously expressed inositol , , -trisphosphate -kinase type b (itpkb) phosphorylates inositol ( , , ) trisphosphate (ins( , , )p ), a calcium-mobilizing second messenger with pleiotropic effects. itpkb inactivation leads to severe t cell deficiency, altered thymo-independent b cell responses and neutrophil hyperactivation. we here report that itpkb-deficient (itpkb -/-) mice also display profound alterations in mast cell development and function. indeed, while mast cell number, c-kit and fcepsilonri expression were comparable in itpkb-deficient and proficient mice, itpkb -/mast cells were almost completely devoid of granules. this phenotype could be partially reversed upon treatment with sodium cromoglycate. nevertheless, fcepsilonri or c-kit activation on mast cells led to increased ca + responses and to stronger erk phosphorylations. however, itpkb -/mice displayed an attenuated sensitivity to ige-mediated passive systemic anaphylaxis, correlated to the absence of fcepsilonri-dependant histamine release and to downregulation of h and h receptor expression due to high basal histamine concentrations. production of neosynthesized mediators remained normal. finally, itpkb deficiency also severely impaired scf-induced mast cell differentiation in vitro. taken together these results demonstrate that itpkb is a key regulator of mast cell activation. itpkb antagonists might thus be of therapeutic interest for programmed and progressive depletion of histamine stores. the large percentage of immune relevant genes that are alternatively spliced and the connections between splicing and disease, strongly indicate that alternative splicing plays a central role in the regulation and fine-tuning of physiological immune responses. il- b is an important proinflammatory cytokine produced by activated macrophages and monocytes. il- b is produced as an inactive cytoplasmic precursor that is proteolytic processed by the inflammatory caspase- to generate the mature secreted active form. caspase- is also synthesized as an inactive form that requires processing by the inflammasome to become active. we have used a subset of the trc lentiviral human library to generate loss-of-function phenotypes for most of the splicing factors and splicing regulators. we were able to silence the expression of genes involved in splicing with an average -fold coverage. after the primary screen and several rounds of phenotypic validation, we have identified genes that significantly affect the production of il- b by thp- cells after a h challenge with pfa-fixed e. coli, as measured by elisa. knockdown levels were analyzed by qrt-pcr for the most significant candidates to validate the phenotypes observed. exon array analysis are being performed to identify possible targets of the most significant splicing factor candidates obtained by the shrna screening in order to dissect their mechanism of action in the regulation of the inflammasome and il- b secretion. tissue transglutaminase (tg ) has a critical role in the pathogenesis of chronic inflammatory diseases such as celiac or neurodegenerative diseases. we have previously described the key role of tg in cystic fibrosis (cf), a genetic disease characterised by chronic lung infections and inflammation. in cf, mutation on the cftr gene results in an increased tg expression and activity leading to functional sequestration of the anti-inflammatory pparg and increase of the classic parameters of inflammation. here we tested whether in vivo inhibition of tg can reverse inflammation in chronic inflammatory diseases. to assess the importance of tg not just in cf but in chronic inflammatory diseases in general, we injected cystamine, a potent tg inhibitor, in a transgenic mouse model cf and in the taz transgenic mice that spontaneously develop autoimmune thyroiditis. intraperitoneal administration of cystamine had a significant impact on the lung epithelium in the cf model, where it decreased tg expression and activity. the treatment was also able to dampen all the classic inflammatory parameters as well as restoring normal cellular levels of functional pparg. interestingly, cystamine injections could also block inflammation in the taz tcr transgenic mouse model with chronic thyroiditis, highlighting the pivotal role of tg in generating inflammation in two very different pathologies. this work underlines the critical role of tg in inflammation and provides new opportunities to develop therapeutic strategies for sufferers of chronic inflammatory diseases. angiogenesis, the growth of new blood vessels, is a process that is essential during tissue repair, foetal development, and female reproductive cycle. angiogenesis is also a relevant process associated to many pathologic conditions including autoimmune diseases and tumors. we have shown that dendritic cells activated in the simultaneous presence of pro-and anti-inflammatory signals (alternatively activated dc, a-dc) display potent angiogenic activity in vivo which is mediated by the release of biologically active vegf-a. here, we investigates the molecular mechanisms leading to vegf-a secretion in lps+pge stimulated a-dc. preliminary results indicate no accumulation of hif- alpha in a-dc, therefore suggesting that vegf-a is induced by a non-classical, hif- alpha independent pathway. in addition, we found that vegf-a secretion depends on the activation of mapk p but not erk / or jnk. inhibitor studies, nascent transcript analysis and polimerase ii recruitment on the promoter show that the induction of vegf-a is largely due to new transcription and not to changes in mrna stability. chromatin immunoprecipitation studies aimed at the characterization of the modifications of vegf-a regulatory regions in a-dc and at the identification of transcription factors bound to vegf-a promoters are being performed. this will possibly allow the description of novel transcription factors involved in vegf-a activation in a-dc. wnt proteins are secreted palmitoylated glycoproteins with multiple functions in cell proliferation, migration as well as tissue organization. they are best known for their role in embryonic development and tissue homeostasis. deregulation of wnt signaling has been shown to promote carcinogenesis. recently we identified wnt signaling to be involved in the regulation of inflammatory processes: wnt a is induced in human macrophages in response to mycobacteria and conserved bacterial structures and contributes to the regulation of the proinflammatory cytokines il- and ifn-gamma. to gain deeper insights into wnt mediated modulation of inflammatory processes we now used murine bone marrow derived macrophages and analyzed the effects of the addition of exogenous wnt homologs. we monitored wnt-mediated activation of primary macrophages by measuring the activation of signaling pathways and transcription factors, analyzed the expression of target genes by real-time pcr and measured the secretion of inflammatory cytokines by elisa. exogenous wnt a -but not wnt a -was able to induce cytokine expression in primary macrophages. in infection experiments wnt a promoted the mycobacteria-induced macrophage activation and enhanced the expression of inflammatory mediators in murine macrophages. in contrast, addition of wnt a reduced the expression of inflammatory mediators upon mycobacterial infection. these data corroborate our previous findings and further support the notion that tlr/nf-kappab and wnt signaling, both being evolutionary highly conserved pathways, are functionally interconnected infection of immunocompetent mammals with t. gondii induces a chronic infection of the brain. t. gondii cysts persist in neurons and escape elimination by the immune system. in immunodeficient individuals, the infection can be reactivated resulting in a lethal toxoplasma encephalitis (te). in te, the parasite is primarily controlled by infiltrating t and b cells. also brain resident cells may contribute to control of the disease and however, the mechanisms of brain resident cells leading to the protection of the vulnerable brain in chronic te are largely unknown. in a previous study, we could show that expression of gp on astrocytes in mice is critical for survival of te. in the present study, we analyzed the function of neuronal gp in te. after infection with t. gondii, mice lacking neuronal gp (synapsin-cre gp fl/fl ) died significantly earlier in the chronic phase of infection than control gp fl/fl mice. death of synapsin-cre cre gp fl/fl was due to a severe encephalitis with larger inflammatory lesions and higher numbers of inflammatory leukocytes. additionally, te of synapsin-cre gp fl/fl mice resulted in a substantial apoptosis of neurons both in the vicinity of inflammatory lesions and also in brain areas without inflammation. in vitro, apoptosis of gp -deficient neurons was also significantly increased upon infection with t. gondii or stimulation with tnf as compared to gp expressing neurons. interestingly, the intracerebral parasitic burden was not increased in synapsin-cre gp fl/fl mice indicating that the immunoregulatory role of neurons is more important than their anti-parasitic function. t. objectives: persistent production of tnfa in many autoimmune diseases, including intraocular inflammation (uveitis), can lead to significant tissue damage. targeting tnfa with neutralising antibodies or tnf receptor fusion proteins is often, but not always, an effective therapy. high serum concentrations of tnfa, il- b, il- and il- have been detected in a spectrum of autoimmune diseases; while in contrast, the levels of il- , il- and tgfb are reduced. this suggests, indirectly, that failure to regulate an appropriate balance of inhibitory factors contributes to the pathogenesis or propagation of tissue inflammation in autoimmunity. thus, understanding the homeostatic control of tnfa by tgfb further may generate more effective therapies. as tnfa mrna ' untranslated region (utr) contains an au-rich element (are), which targets mrnas for degradation, we wished to test whether tgfb suppresses tnfa protein production by upregulating the rnabinding protein fxr , which can bind to tnfa mrna and inhibit translation. methods: using raw . cells and mouse bone-marrow derived macrophages stimulated with lps and tgfb , we assessed mrna expression by q-pcr and tnfa protein expression by flow cytometry. the 'utr of tnfa mrna was isolated and inserted into a luciferase reporter vector on a constitutive promoter. transfected raw cells were treated with lps and tgfb and luciferase expression was quantified. cells treated with lps and tgfb were also examined for fxr expression using pcr and western blot. following fxr knockdown using sirna, the influence of tgfb and lps on tnfa protein production was examined by flow cytometry. results: we find that while tnfa mrna expression remains constant, lps induced tnfa protein expression is suppressed by tgfb . using the luciferase-tnf- 'utr vector we show that tgfb targets the 'utr of tnfa. furthermore, tgfb and il- both upregulate fxr mrna and protein; and treatment with tgfb and lps can synergistically upregulate mrna expression, more than tgfb alone. following sirna inhibition of fxr , tgfb can no longer inhibit lps-induced tnfa production. comtb up-regulated mmp- and mmp- secretion from saecs, nhbes and fibroblasts to a peak of . +/- . ng/ml, at hours. interleukin- augmented comtb-stimulated up-regulation of mmp- and mmp- secretion from saecs and fibroblasts in a synergistic manner. in contrast, interleukin- down-regulated mmp- secretion from saecs by %. interleukin- up-regulated mmp- and mmp- secretion from fibroblasts but not from saecs. timp secretion from saecs was enhanced by interleukin- but there was no effect of interleukin- . mmp up-regulation by interleukin- and comtb was inhibited by the pi kinase inhibitor ly and on western analysis akt (protein kinase b) was phosphorylated at minutes. chemical inhibition of the p d isoform of pi kinase with ic abrogated the il- and comtb driven secretion of mmp- from the small airway epithelial cells. chemical inhibition of the tumour suppressor phosphatase, pten (phosphatase and tensin analogue on chromosome ) accentuated mmp- secretion. these inhibitory effects were confirmed with sirna. mmp- up-regulation was secondary to increased gene expression with promoter activity peaking h after stimulation. in summary, interleukin- and interleukin- drive transcription dependent mmp- and mmp- secretion from airway epithelial cells and fibroblasts. interleukin- also increases timp but down-regulates mmp- gene expression and secretion. this may contribute to the matrix degrading phenotype in tuberculosis. the pi kinase pathway is central in interleukin- driven tissue destruction in the context of m. tuberculosis infection. v. delgado-maroto , l.s. moreira , e. gonzalez-rey , m. delgado institute of parasitology and biomedicine lopez-neyra , csic, granada, spain, university of seville, sevilla, spain objectives: atherosclerosis is an inflammatory chronic disease characterized by the formation in the arteries of lesions that involve inflammation, lipid accumulation, cell death and fibrosis. over time, the rupture of these atherosclerotic plaques releases prothrombotic material to the blood and causes thrombotic occlusion at the site of disruption. atherosclerosis will probably become the most common cause of death within years. one of the initial hallmarks of the disease is the uptake of oxldl particles by macrophages, which leads to intracellular cholesterol accumulation and the formation of foam cells. t cells undergo activation after interacting with foam cells, which process and present local antigens including oxldl, generating a t helper response. cholesterol metabolism is regulated by factors such as pparg (proliferator activated receptor g), srb (a class b scavenger receptor), cd or abca , that can induce cholesterol exit from the macrophage which may help to solve the lesions. expression of these factors depends on intracellular camp. adrenomedullin (am), urocortin (ucn) and vasoactive intestinal peptide (vip) are novel neuropeptides synthesized by immune cells that have various characteristics to be considered as possible therapeutic agents for atherosclerosis. they are potent anti-inflammatory agents, which downregulate a broad spectrum of pro-inflammatory mediators, and inhibit th immune response. their mechanism of action involves binding to gpcr and adenylate cyclase activation with subsequent increase of camp in the cell, which is recognized as an anti-inflammatory response. methods: we investigate am/ucn/vip effect on bone marrow-derived macrophages stimulated with oxldl. we determine the levels of pparg , srb , cd , and abca . we also analyze the cholesterol metabolism of oxldl-stimulated macrophages after neuropeptides incubation using oil red o staining of lipids drops and tritium labelled cholesterol. objective: endovascular aortic repair (evar) is considered a minimally invasive procedure, and the patients are expected to be discharged after a day or two. however up to % develop a systemic inflammatory response syndrome (sirs), resulting in prolonged convalescence. as yet there is no satisfactory explanation to this severe response. previous studies have shown a high level of il- in the mural thrombus lining the aneurysm. the thrombus is manipulated during the procedure, but whether or not it is the source of circulating il- and/or other cytokines during and after the operation is unknown. methods: quantitative analysis of the pro-inflammatory cytokines il- , tnf-a, il- b, il- and il- , and the anti-inflammatory cytokine il- , in plasma from five patients, as of yet, was carried out by means of cytometric bead array, while analysis of plasma il- was performed using the luminex platform. the cytokine levels were compared to the clinical response, in terms of sirs. results: evar induced the production of il- and il- , and in some cases, of il- . the maximal plasma levels of il- and il- were found at hours and of il- between - hours. modest plasma levels of il- were also observed, with maximal production at various time points ( - hours). by contrast, production of tnf-a, il- b and il- did not occur to a significant extent, while production of il- occurred sporadically. although our preliminary data indicate that sirs is associated with enhanced cytokine responses, the production of il- , il- , il- and il- also took place in patients who did not develop sirs. conclusion: evar is associated with the sequential production of il- , il- , il- and il- , i. e. a mixed pro-and anti-inflammatory response, even in the absence of sirs; but the production seems to be exaggerated in patients developing sirs. further studies involving patients are in progress, and will clarify this. we hypothesize that sirs is elicited by il- , activated by manipulation of the mural thrombus. to reveal whether this is the case, studies involving immunohistochemistry of the thrombus, will be performed. il- is a novel il- cytokine family member that is expressed as an intracellular precursor (pro-il- ) and is thought to be cleaved by caspase- to yield a mature bioactive form of the molecule (mat-il- ). to date however, evidence of cell-associated proteolytic processing and caspase- dependent secretion of mat-il- has not been reported. here we show that pro-il- but not mat-il- is released from uvb-irradiated keratinocytes. we demonstrate binding of pro-il- to the il- r and also il- r-dependent bioactivity of pro-il- on mast cells. we propose a previously unrecognized role for pro-il- as a pro-inflammatory mediator and suggest a direct link between uvb-mediated epithelial cell damage and cutaneous mast cell activation. we have previously shown that induction of er stress and tlr signalling synergistically enhance il- p mrna expression in myeloid cells, and markedly increase secretion of il- , but not il- , by dendritic cells. the aim of this study is to investigate the mechanism of this synergy. we examined the il- promoter for potential binding sites for er stress induced transcription factors and identified a putative site for chop . chromatin immunoprecipitation (chip) assays using anti-chop and isotype control mab were performed using nuclear lysates from u cells and il- promoter dna measured by qpcr. chop binding on the il- promoter was detected following stimulation of u cells with lps or tp alone, but this was significantly enhanced when er stress and tlr stimuli were combined. il- promoter dna was not detectable following chip with the isotype control antibody. to confirm the role of chop in il- gene transcription, u cells expressing shrna's specific for chop or non-specific gene target were tested for their ability to express il- following tlr and er stress stimulation. u expressing three independent shrna targets for chop exhibited significant reductions in il- p mrna (up to % reduction of the response to lps+tp) compared to u expressing a control shrna. chop shrna expression did not affect the expression of other lpsresponsive genes, including il- , il- , ccl and sod . to identify if er stress induction of il- mediated by chop expression plays a role in a more physiological setting, we examined the role of chop in the induction of il- p gene expression following chlamydia trachomatis (ct) infection. infection of u cells with live but not g-irradiated ct induced expression of er stress response genes, including chop . u infected with live ct exhibited increased il- p mrna expression compared to u infected with nonviable bacteria. chop silencing significantly reduced the ability of live ct to induce il- p mrna, confirming the important role of chop in this response. these data suggest that er stress induction of chop could contribute significantly to the pathogenesis of diseases in which il- plays an major role, through induction of il- and il- producing cells. the clonal deletion of thymocytes by negative selection is an important process to ensure immunologic tolerance, even though the underlying molecular mechanisms are poorly understood. here, we show that gadd b, a regulator of mitogen-activated protein kinases, is critically involved in selection processes in the thymus. gadd b expression was inducible in different in vitro and in vivo models of negative selection. strikingly, only tcr-ligating peptides resulting in negative selection induced gadd b expression, while positively selecting ligands or dexamethasone, a tcr-independent apoptosis agonist, failed to do so. expression of gadd b maintained a sustained activation of p kinase and thereby promoted tcr-mediated apoptosis. in contrast, thymocytes from gadd b-deficient mice showed only transient p activation and reduced caspase activation. interestingly, we observed a switch to positive selection in gadd b-deficient mice since a higher percentage of single positive thymocytes was found. moreover, markers of positive selection as cd and cd were elevated on gadd b-deficient thymocytes. thus, we provide evidence that gadd b and a resulting persistent activation of p constitute a novel apoptotic pathway involved in negative selection. these results also provide evidence for the novel concept that not only the on-off switch of a signaling module but also its spatiotemporal regulation may crucially determine cell fate decisions. di santo institut pasteur, paris, france, monash university, victoria, australia > the thymus represents the ''cradle'' for t cell development, with distinct thymic stroma components providing multiple soluble and cellular membrane cues that foster in a step-wise fashion developing thymocytes. although il- is recognized as an essential factor for thymopoiesis, the nature of the thymic il- niche remains poorly characterized in vivo. > using a novel bacterial artificial chromosome transgenic mice in which yellow fluorescent protein (yfp) is under control of il- promoter, we identify a subset of thymic epithelial cells (tecs) that co-express yfp and high levels of il transcripts (il- hi cells). il- hi tecs arise during early fetal thymic development, persist throughout life, and co-express homeostatic chemokines (ccl , ccl , cxcl ) and cytokines (il ) that are critical for normal thymopoiesis. in the adult thymus, il- hi cells are found in cortico-medullary regions and display traits of both cortical or medullary immature tecs. interestingly, the frequency of il- hi cells decreases with age, suggesting a mechanism for the age-related thymic involution that is associated with declining il- levels. conversely, the frequency of il- hi cells is markedly increased under severe lymphopenia imposed by genetic mutations that cause an early and profound block in t cell development. this augment indicates that thymocyte-tec crosstalk may condition il- -expression by tecs. > together, our temporal-spatial analysis of il- -expressing cells in the thymus suggests that thymic il- levels are dynamically regulated under distinct physiological conditions. this novel il- reporter mouse provides a valuable tool to further dissect the molecular and cellular mechanisms that govern thymic il- expression in vivo. two lines of evidence have recently demonstrated that the pre-b cell receptor (pre-bcr) is associated with autoimmunity, through its surrogate-light-chain (slc) components l and vpreb. it has been shown that pre-bcrs are polyreactive for several self-antigens. the polyreactivity of the pre-bcr induces pre-bcr signaling and activation. furthermore, in human a self-reactive b cell subset was identified that co-expresses immunoglobulin light chain (ig lc) and the slc components. these vpreb + lc + b cells, found in healthy individuals, are potentially harmful as they express autoreactive antibodies associated with autoimmune diseases, like sle and ra. to elucidate the contribution of pre-bcr components to the development and activation of autoreactive b cells, we have recently generated a slc transgenic (slc-tg) mouse model in which all b cells express slc proteins. slc-tg mice exhibit spontaneous igm + plasma cell development. moreover, aging slc-tg mice have elevated anti-nucleosome igm levels, accompanied by immune complex deposition in the kidney, but do not display auto-immune pathology. nevertheless, vpreb + lc + b cells may induce pathology when self-tolerance mechanisms fail. to test this hypothesis, slc-tg mice were crossed on two autoimmune-prone genetic backgrounds: (i) em-bcl -tg mice with b cell-specific overexpression of the anti-apoptotic protein bcl and (ii) fcgriib-deficient mice. both in young slc-tg;em-bcl -tg double tg mice and in slc-tg;fcgriib -/mice spontaneous germinal center (gc) formation -which is associated with autoimmunity -was significantly enhanced, when compared with control em-bcl -tg and fcgriib -/mice. in slc-tg;fcgriib -/mice, numbers of splenic igg plasma cells and serum igg levels were˚ - fold increased. importantly, serum from young slc-tg;em-bcl -tg and slc-tg;fcgriib -/mice contained high titers of igg auto-antibodies in / and / cases, respectively. these values were increased when compared with control groups: / in fcgriib -/and / in bcl -tg. finally, we found that the collagen induced arthritis (cia) was significantly enhanced in slc-tg;fcgriib -/mice, compared to fcgriib -/mice. taken together, these findings demonstrate the slc components have the capacity to induce auto-antibody formation in the mouse and and to enhance autoimmune pathology in ra. t cells are generated from progenitor cells that enter the thymus from bone marrow via blood. these progenitor cells once within the thymus have little selfrenewal capacity. differentiation from hematopoietic stem cells to early t lineage cells proceeds through a series of intermediate precursor populations. however, it is largely unknown to what extent these cell populations contribute to t cell development in the presence of other precursor populations and how the earliest intrathymic t cell progenitors are generated from extrathymic precursors. to assess the relative contribution of potential precursors to t lineage differentiation we developed a strategy based on the depletion of well-characterized precursor populations rather than their enrichment and subsequent adoptive transfer together with an equal amount of congenic non-depleted bone marrow. thus, the physiological ratio of extrathymic precursors remained largely intact and we were able to address the question whether there is only one physiological t cell precursor or many. we showed that, under such competitive conditions, t lineage progenitors are confined to the cd + cd + fraction of bone marrow cells. notably, t lineage reconstitution was not restricted to either cd hi cells, representing multipotent progenitors, or cd + cells, representing common lymphoid progenitors, both of which contributed to t lineage differentiation with different kinetics. in conclusion, our data suggest that multiple physiological extrathymic t cell precursors exist, which are able to compensate for the loss of depleted populations. thus, our findings may have implications for devising strategies for improved t lineage reconstitution after hematopoietic stem cell transplantation. background: previous results from our group have demonstrated ephb and ephb expression on both thymocytes and thymic epithelial cells (tecs). we used chimeric models to determine that those molecules govern in an autonomous and non autonomous manner thymocyte and tec development, and how they regulate interactions between both cell types. objectives: in order to better define the importance in thymus of eph-ephrin b interactions we have analyzed the effects of the lack of ephrin b and/or ephrin b , the ligands of ephb and ephb receptors. this approach is specially interesting taking into account that eph-ephrin signaling is transmitted to both the two cells participating in the interaction and that the cell responses depend on the type of signals (reverse, forward or both), their direction and intensity. methods: for this purpose we have used cre-loxp recombination systems for deleting ephrinb or ephrinb genes specifically on either thymocytes or tecs. results: animals with ephrin b deficient thymocytes showed thymic hypocellularity and alterations on t-cell development whose severity depended on the background of the analyzed mice. in these mice only a few changes occurred in the cortical tec network. on the contrary, mice with conditioned deletions in tec, especially ephrinb /b double mutants, showed a more severe phenotype that began early in the ontogeny and resulted in very small thymi exhibiting an extremely compact cortical and medullary network, decreased numbers of cd + cells in the cortex, increased proportion of k +k + cells and high presence of cysts. in addition, t-cell development was partially blocked at the dn cell stage. conclusion: these data reveal an autonomous and non-autonomous role for ephrinb and ephrinb in the development of both t cells and tecs, confirming the importance of these molecules in the establishment of a crosstalk between the main two cell types of thymus. we discuss how eph-ephrin contacts modulate cellular homotypic and heterotypic interactions that take place during thymus organogenesis and in t cell differentiation. a. rolink , d. vanhecke university of basel, developmental and molecular immunology, basel, switzerland the importance of normal t lymphocyte development in the immune system is exemplified by the occurrence of inherited and acquired human immunodeficiencies where the development or functional maturation of t cells is defective. in order to identify molecules/genes and elucidate developmental processes that are essential for human t cell development we use a novel in vitro tool, the op -dl cell culture system ( ) . using this in vitro assay we obtain large numbers of human cycd + and cd + cd + double positive thymocytes starting from umbilical cord blood (ucb) derived cd + hsc. signals and molecules that are involved in t cell development are being addressed by using blocking antibodies and/or chemical inhibitors. similar as in mice we found an essential role for il- and notch mediated signaling in the development and survival of particular developmental stages of human thymocytes. among the molecules that are rapidly induced in cd + cells upon notch signaling is cd followed by cd . t cell specification is accompanied by the induction of cd a and loss of cd on cd + cd + cells. these cd -cd a + cd + cells become dependent on continuous il and notch signaling for sustained survival and further differentiation into cd + cd ab + dp thymocytes. we found that flt l is not essential for the differentiation of cd + cd + human thymocytes but that addition of exogenous flt l in the co-cultures increases the number of cd + precursors and consequently result in higher yields of developing cd + cd ab + dp thymocytes. finally few mature tcrab + t lymphocytes develop from the cycd + cd + cd ab + dp subset in this in vitro assay suggesting that op stromal cells lack the required selecting mhc-antigen complexes and/or costimulating molecules to induce and sustain positive selection of human thymocytes. this in vitro assay will allow us now, by using rna interference, to test additional genes for their role during human lymphoid development. from a clinical standpoint, a better understanding of the mechanisms controlling human t-cell development is a fundamental step towards the development of specific therapies for the treatment of primary and acquired immunodeficiencies as well as for the treatment of malignant t-cell disorders. brain derived neurotrophic factor (bdnf) promotes various neuronal functions such as survival, regeneration and synaptic plasticity. emerging evidence also indicates an essential role for bdnf in the immune system, e.g. in the b and t cell lineages. we therefore investigated the impact of bdnf on thymocyte development using bdnf knockout (ko) mice and conditional ko mice lacking bdnf specifically in t cells. in both models, we found reduced thymocyte numbers and a significant increase in double negative thymocytes. in contrast, the percentage of naturally occurring regulatory t cells and the expression of activation markers were unaltered. moreover, the lack of bdnf did not result in enhanced thymocyte apoptosis. the increase in double negative thymocytes was due to a partial block in the transition from the dn to dn stage, where bdnf and its receptor p are expressed as revealed by real-time pcr. the observed partial block in thymic maturation results in mild peripheral lymphopenia without affecting the activation status of peripheral t cells, their homeostatic proliferation and without compromising peripheral immune responses in general. in summary, our findings point to a critical role of t cell lineage derived bdnf in thymocyte development acting in an autocrine and/or paracrine manner. r. berga , c. lópez-rodríguez pompeu fabra university, barcelona, spain nfat is a transcription factor that belongs to the rel family (nf-kb and nfatc proteins). its expression in primary cells and organs is restricted to certain proliferative tissues, like activated t lymphocytes and thymocytes, where levels of nfat are relatively high and its subcellular distribution is predominantly nuclear. recent mouse models suggest that nfat participates in thymocyte development and also indicate its involvement in t cell proliferation and survival. nfat deficient mice present a t cell immunodeficiency consistent on lymphopenia, which is more accused for cd + lymphocytes. these observations are of substantial relevance as we and others have described that, in vivo, nfat -null mice are unable to mount cd +-and cd + -immune responses. data from our laboratory indicate that nfat -null mice suffer from hyperosmolarity in plasma (hypernatremia) as a result of the incapacity to induce an osmoprotective gene expression program at a systemic level. to selectively analyze the t-cell autonomous effects derived from the lack of nfat during the development of t lymphocytes, we developed mouse models that delete nfat at early (lck-cre + /nfat flox/flox ) or late (cd -cre + /nfat flox/flox ) stages of thymocyte maturation and that present isotonic plasma. our work indicates that nfat is expressed at all stages of t cell development. in addition, analysis of mouse models that lack nfat at different points of t cell development indicate that it participates at early stages of the ontogeny of t cells. objectives: apoptosis mediated by the tumor suppressor molecule p , is regarded as a major player in tumor prevention but this may not be its only role. we have investigated this by creating a mouse (m ¿ pro) lacking residues - of the proline-rich domain of p . methods: we compared the ability of various hemaptopoietic tissues from m ¿ pro mice and wild type mice to undergo apoptosis following irradiation or treatment with pro-apoptotic drugs. apoptosis was measured by staining with annexin v in vitro and by detection of caspase activation in vitro and in vivo. we also compared their ability to undergo cell cycle arrest using brdu staining. tumor development was monitored in cohorts of m ¿ pro, p null (p -/-) and wild type (p +/+) mice, with or without prior irradiation. results: apoptotic function was lacking in m ¿ pro mice, but they were able to arrest cell-cycle progression in hematopoietic tissues. m ¿ pro developed late-onset b-cell lymphoma, but not the thymic t-cell tumors found in p -/-mice. interestingly, m ¿ pro lymphomas were comprised of incorrectly differentiated b-cells. bcell irregularities were also detected in m ¿ pro prior to tumor onset, in which aged mice showed an increased population of inappropriately differentiated b-cells in the bone marrow and spleen. we propose that the apoptotic function of p has an important role in b-cell homeostasis, which, in turn, is important for prevention of b-cell lymphomas moreover, our data suggest that the apoptotic function of p is not important for preventing thymic t-cell tumors. s. myrczek , r. pardi , a. gessner microbiological institute-institute for clinical microbiology, immunology and hygiene, university hospital erlangen, erlangen, germany, vita-salute san raffaele university school of medicine, milano, italy jab is the catalytic subunit of the highly conserved cop signalosome. this complex plays a central role in various cellular processes as proliferation and cell cycle control. jab regulates the neddylation of ubiquitin ligases and thus contributes to degradation of many proteins. furthermore jab regulates the activity of ap transciption factors. to date jab is thought to be essential for every cell type as jab knock out mice are embryonic lethal and t cell development is blocked by t cell selective absence of jab . to investigate the function of jab in b cells we established a mouse strain deficient for jab selectively in b cells. mice with floxed alleles of jab kindly provided by r. pardi were crossed with a mouse strain expressing the cre recombinase under control of the mb -locus (m. reth, freiburg). ablation of jab expression resulted in an almost complete block of b cell development at the pro b cell stage. the absence of peripheral mature b and b cells and serum immunoglobulins resulted in chronic arthritis with high pathogen burden after experimental infection with borrelia burgdorferi. the observed block in b cell development is rescued by over expression of the anti apoptotic protein bcl under the control of the m enhancer. facs analyses revealed that all b cell subtypes analyzed in the jab -deficient, bcl -transgenic mice are present albeit at reduced numbers compared to wild type animals. serum immunoglobulin titers are detectable and after borrelia infection specific antibodies are produced. we confirmed the absence of jab in sorted spleen b cells by immunoblot analysis. in summary, we show for the first time that cells are viable and functional without jab when apoptosis is prevented. t. nitta , s. murata , k. tanaka , y. takahama university of tokushima, tokushima, japan, university of tokyo, tokyo, japan, rinshoken, tokyo, japan how self-peptides are generated and displayed in the thymus to select a useful and self-protective repertoire of t cells is largely unknown, whereas the role of thymic self-peptides in eliminating self-reactive t cells and thereby preventing autoimmunity is well established. a recently identified form of proteasome, termed thymoproteasome, is specifically expressed by thymic cortical epithelial cells (ctec) and is required for the optimum generation of cd t cells. here we show that ctec display a thymoproteasome-specific spectrum of class i mhc-associated self-peptides, which is essential for positive selection of major and diverse repertoires of class i mhc-restricted t cells. indeed, cd t cells generated in the absence of thymoproteasomes display a markedly altered tcr repertoire that is defective in both allogeneic and antiviral responses. these results demonstrate that thymoproteasome-dependent self-peptides are required for positive selection of a diverse repertoire of immunocompetent cd t cells. defects in helper t cell number or function causes susceptibility to infections and in some cases autoimmunity or allergy. our understanding of the genetic control of helper t cell differentiation into specific functional subsets is still far from complete. here we present the results to date from a genome-wide enu mutation screen for mice with inherited deficits in specific helper cell subsets. these deficits were detected by multi-colour facs analysis of peripheral blood samples, and by antibody production following immunization with heat-killed b.pertussis and cgg coupled with the hapten arsonate (aba) in alum, which induce internally polarized th and th antibody responses, respectively. using this screen, a number of new mutant strains have been isolated with complete or partial loss of cd + t cells or functional deficits that selectively interfere with th or germinal centre responses. in this talk i will present data from some of the first strains that have been identified including the first strains where we have been able to identify the causative mutation. systematic genetic analysis of helper t cell differentiation in the resulting strains will illuminate how t cell help is correctly polarized for immunity and to avoid immunopathology. intrathymic t-cell development provides a unique model system to study cell fate determination because of the well-defined cellular stages and the confined microenvironment of this process. in order to highlight the differences and similarities between fetal and adult t-cell development at the molecular level we performed a microarray study. labelled rna from facs purified fetal and adult dn c-kit high (etp), dn and dn thymocyte populations was hybridised to affymetrix mouse a- . genechips. the resulting data were grouped into four distinct gene clusters: cluster i contained genes over-expressed throughout adult development and included a large proportion of transcription factors ( out of genes), illustrating a significantly different transcriptional program acting during adult differentiation. conversely, cluster ii consisted of genes that were over-expressed in fetal progenitors and included signal transducers (out of genes) such as acvr , bmpr , fzd , chemokine receptors cx cr , cxcr and integrins a , a , a , ae and av, pointing to a difference in microenvironments. genes that showed uniform down-regulation during consecutive stages of fetal and adult development were restricted to cluster iii. amongst these were transcripts governing alternative developmental choices, therefore emphasising a common mechanism of lineage restriction during thymopoiesis. on the other hand, cluster iv was limited to genes that were homogeneously up-regulated during development. these included gata- , tcf- , notch- , rag- , rag- and pre-ta, which are indispensable for t cell development. interestingly, levels of expression of these genes were elevated in fetal progenitors, especially at the etp and dn stages, suggesting that the molecular program of t-cell development is more advanced in the early stages of fetal differentiation. discriminant analysis with the use of the support vector machine arrived at the same conclusion that demonstrated a nearby clustering of all fetal stages with the adult dn population, therefore implying a more committed state of fetal progenitors. finally, transcriptional signatures of each developmental stage were defined by "recursive feature elimination" with support vector machines. this approach can now be used to classify characterised and aberrant hematopoietic progenitors and thus construct an ontological scheme of hematopoietic development based upon transcriptional signatures of populations under normal and pathological conditions. tcrgd+ cells and tcrab+cd aa+ intraepithelial lymphocytes (iels) of the gut are unconventional t cells that reside in tissues and provide innate-like immune responses to "stressed-self". as these cells share common functional properties in the periphery, we have hypothesised a common mechanism of development in the thymus; their progenitors diverging from the conventional t cell developmental pathway based on tcr signal strength at the dn stage. the pre-t-alpha chain (pta) that pairs with tcrb to generate the pre-tcr, has two isoforms; pta a and pta b . both can form a functional pre-tcr with tcrb. ligand-independent signalling by the pre-tcr is a result of spontaneous oligomerisation (followed by internalisation), that is mediated through charged residues on the pta chain. pta b lacks out of of these essential residues and therefore, we speculate results in higher surface expression and different signalling capabilities. we have hypothesised that pta a and pta b permit differential signal strength through the pre-tcr at the dn stage, facilitating the divergence of the conventional and unconventional lineages of tcrab+ t cells. preliminary semi-quantitative pcr data suggest that pta a and pta b are differentially expressed in wt thymocytes at different stages of ontogeny. retroviral transduction of pta -/-e thymocytes with either pta a or pta b alone, followed by fetal thymic organ culture, confirmed the rescue of abt cell development by both isoforms. however the two isoforms appear to differentially regulate the kinetics of thymocyte development by - days of culture; pta b expression generates a greater percentage of tcrab+ cells while pta a expression results in the accumulation of isps. these results suggest different roles for the two isoforms of pta in the thymic development of abt cells. in order to determine the mechanism by which pta a and pta b may generate qualitatively different signals, site directed mutagenesis was used to produce mutant chains of pta a and pta b that lack the "dimerisation residues" necessary for internalisation of the receptors. in addition, bac transgenic mice that express singly either pta a or pta b under the pta promoter are being generated to fully characterise their role in conventional vs. unconventional lineage commitment. erythroid, myeloid and lymphoid cells are initiated in parallel in appropriate cytokine environments so that specific number of erythrocytes, myeloid cells, natural killer cells, thymocytes and t cells, and precursor of b cells can be detected and counted at day of culture. if needed for further functional analyses, long-term proliferating lines and clones of progenitor t and b cells can be established at this point of "in vitro" development. hence the "in vitro" differentiation of es cells to different hematopoietic cell lineages and their progenitors can be quantified. it allows for testing the efficiencies for hematopoietic development of genetically or epigenetically different es or ips cells. aim: increasing evidence includes wnt proteins inside the group of master-signalling pathways which govern immune and non immune differentiation systems. although their precise functions in bone marrow and thymus are still controversial, numerous studies show that wnt signalling is able to control the proliferation of hsc and thymic progenitors and might also affect both their cell-fate decisions and subsequent maturation. in the present work we analyse the effect of transient stimulation of canonical wnt pathway in the differentiation potential of lin -cd + cd ahuman thymic progenitors, a multipotent and heterogeneous cell population which has the capacity to develop into t cells, nk cells, monocytes, conventional dendritic cells (cdc) and plasmacytoid dcs (pdcs). methods: human thymus samples from patients aged month to years undergoing corrective cardiac surgery were obtained and used according to the declaration of helsinki. transient b-catenin stabilization was triggered culturing purified thymic lin -cd + cd precursors with recombinant wnt a ( ng/ml) or with licl ( mm) for hr. active b-catenin, was detected by flow cytometry using anti-human active b-catenin mab ( e ) under conditions of phosphatase activity inhibition. wnt a or licl pre-treated precursor were assayed in chimeric human-mouse ftoc, in il- and scf-supported cultures for generation of nk cells and in co-cultures with murine bone marrow stromal st cells suplemented with il- and flt l. phenotype of recovered cells, apoptosis and cytokine receptors were analysed by flow cytometry. expression profile of transcription factors was analysed by real-time quantitative rt-pcr . our results demonstrate that giving a boost to canonical wnt signalling triggered by transient exposure of thymic progenitors to wnt a or licl, change their differentiation capacity enhancing nk cell production. on the contrary, wnt a or licl pre-treated thymic progenitors generate a significant lower number of myeloid lineage cells, monocytes and cdc, as well as reduce their capacity to differentiate into pdc lineage. as a possible mechanism for this effect we show that wnt pre-treated progenitors change their expresssion of receptors for cytokines pivotal for their expansion and differentiation, such are il- and flt l and modify the transcription factor profiles of cd + cd thymocytes mainly increasing hes- and id expression levels. human th clones and circulating th cells showed lower susceptibility to the anti-proliferative effect of tgf-beta than th and th clones or circulating th oriented t cells, respectively. accordingly, human th cells exhibited lower expression of clusterin, and higher bcl- expression and reduced apoptosis in the presence of tgf-beta, in comparison with th cells. umbilical cord blood naï ve cd (+)cd (+) t cells, which contain the precursors of human th cells, differentiated into il- a-producing cells only in response to il- beta plus il- , even in serum-free cultures. tgf-beta had no effect on constitutive rorgamma t expression by umbilical cord blood cd (+) t cells but it increased the relative proportions of cd (+) t cells differentiating into th cells in response to il- beta plus il- , whereas under the same conditions it inhibited both t-bet expression and th development. these data suggest that tgf-beta is not critical for the differentiation of human th cells, but indirectly favors their expansion because th cells are poorly susceptible to its suppressive effects. m. irla , w. reith university of medecine, pathology and immunology, geneva, switzerland objectives: medullary thymic epithelial cells (mtecs) are specialized for inducing central immunological tolerance to self-antigens. to accomplish this, mtecs must adopt a mature phenotype characterized by expression of the autoimmune regulator aire, which activates the transcription of numerous genes encoding tissue-restricted self-antigens. the mechanisms that control mature aire(+) mtec development in the postnatal thymus remain poorly understood. however, the generation of mutant mice exhibiting blocks in thymocyte differentiation at different stages, together with studies on embryonic development of the thymus, have demonstrated that reciprocal interactions between developing thymocytes and tec control not only t cell development but also the differentiation and organization of tec, a phenomenon designated as 'crosstalk'. the aim of the project outlined here is to elucidate the cellular and molecular mechanisms by which thymocytes control the numbers of mature mtec, key mediators of central tolerance. we have demonstrated by generating different transgenic mouse models, that although either cd (+) or cd (+) thymocytes are sufficient to sustain formation of a well-defined medulla, expansion of the mature mtec population requires autoantigen-specific interactions between positively selected cd (+) thymocytes bearing autoreactive t cell receptor (tcr) and mtecs displaying cognate self-peptide-mhc class ii complexes. these interactions also involve the engagement of cd on mtecs by cd l induced on the positively selected cd (+) thymocytes. conclusion: this antigen-specific tcr-mhc class ii-mediated crosstalk between cd (+) thymocytes and mtecs defines a unique checkpoint in thymic stromal development that is pivotal for generating a mature mtec population competent for ensuring central t cell tolerance. q. qiu , i. ravens , g. bernhardt hannover medical school, institute of immunology, hannover, germany cd is originally identified as human poliovirus receptor (pvr) and as rodent tage , which is overexpressed in rodent colon carcinoma. cd is also known as necl- , a particular notable nectin-like molecule belonging to immunoglobulin superfamily, owning its unique expressing frofiles. cd expression is very low in most adult organs, but is abundant in the developing or regenerating liver. in addition, cd is overexpressed in transformed cells and promotes the cell cycle. thus, cd seems to be an oncofetal protein that functions in embryonic development and cancer progression. t-cell development is characterized by the progression through several phenotypically distinct stages, defined as double negative (dn), double positive (dp) and single positive (sp) based on expression of the co-receptors cd and cd ; the dn subset is further subdivided into four stages (dn - ) by differential expression of cd and cd . thymocytes at different stages of development occupy distinct spatially restricted domains in the adult thymus, indicating that differentiation occurs concomitantly with a highly ordered migration. during their final maturation in the medulla, semi-mature sp thymocytes down-regulate activation markers and subsequently exit into periphery. while semimature cd + sp are sensitive to negative selection, it remains elusive when negative selection occurs in the cd lineage. here we show that the frequency of terminally matured cd + sp cells but not that of cd + sp present in thymus varies depending on age. in mice lacking expression of the adhesion receptor cd , a selective deficiency of mature cd + sp thymocytes was observed emerging first in adolescent animals at the age these cells start to accumulate in wild type thymus. evidence is provided that the mature cells emigrate prematurely when cd is absent thus cutting short their retention time in the medulla. moreover, in unmanipulated wild type mice semi-mature cd + sp thymocytes are subjected to negative selection as reflected by the diverging t cell receptor repertoires present on semi-mature and mature cd + t cells. in cd deficient animals, a shift in the tcr repertoire displayed by the pool of cd + sp cells was found demonstrating that cd is involved in negative selection. in the adult, steady-state, homeostatic conditions, lymphohematopoietic cell lineages display high rates of cell turnover. yet, the frequencies of simultaneously cycling cells are small, except in intermediate cellular stages of transit-amplifying precursor cell stages. the analysis of the molecular targets controlling these proliferation rates may provide relevant information to understand differentiation pathways along the ontogeny as well as mechanisms of leukemic transformation (passegué et al. j. exp. med. , , . during development, hematopoietic stem cells and their derived cell lineages need to expand to cope with continuously-increasing somatic demands. by using complementary, quantitative analyses (brdu labelings, hoescht , propidium iodide), we are dissecting the proliferation rates of hematopoietic cell lineages and their differentiation stages along the whole mouse gestation from e (e, gestational day) on, in yolk sac, splanchnopleura/agm, blood, liver, spleen and bone marrow. we have observed that around half of cd + ter + erythroid and cd + cd b + myeloid cells are simultaneously cycling (s/g /m) in the post-gastrulation mouse embryo (e - ). the peak of lymphohematopoietic cell proliferation occurs at e in a sort of wave-like pattern. these high-proliferation frequencies are present not only in immature, but also in mature cells, the latter thus displaying a different behaviour from the one present in the adult. later on, the proliferating cell subsets are restricted to fetal liver, whereas the equivalent cells become arrested in the periphery. interestingly, nucleated erythroid cells suddenly go into quiescence - hours before they enucleate, suggesting that this cell arrest is required for the enucleation process. we are also analysing the proliferation state of the first b and t lymphoid progenitors emerging at e - that give rise to perinatal lymphocytes and, in some cases, to innate-like lymphocytes displaying self-renewal in the adult. we attempt to dissect the mechanisms regulating proliferation and death in the embryo versus those of adult lymphohematopoietic precursors, which may influence the functional activities of the mature cells. objectives: the role of cd -cd interactions in t cell activation of antigen presenting cells and b cells is known, but a role for this receptor-ligand pair in hematopoiesis control has not been described. following an initial discovery that b lineage cells in the bone marrow (bm) as early as pro-b cells express cd , we hypothesised a role for cd -cd interactions in the control of b cell haematopoiesis. the objectives of this study were to investigate this hypothesis further. methods: flow cytometry was used to investigate cd expression by precursor b cells using b cell specific markers. reverse transcription of bm stromal cell rna and pcr were used to assess the presence of cd message and cell lineage specific mrna. irradiation and bone marrow transplantation (bmt) in both directions between cd -/-and wt mice was used to assess potential functional contributions of stromal or haematopoietic cd on reconstitution of b cell numbers following depletion. we show that cd is expressed by pro-b cells, and these cells proliferate in response to cd signalling in vitro. pcr identified a source of cd , negative for cd eta, in the bm of wt mice showing this cd is not provided by activated re-circulating t cells. we have shown that when cd -/-mice are recipients, but not donors of bmt, b cell recovery after irradiation is significantly delayed regardless of the donor cell source. in the in vitro experiments we found that the pta gene is expressed from the dn (cd -/cd -/cd + /cd -) to the dp (cd + /cd + ) stage, whereas no yfp expression could be observed in the b lineage. the in vivo analysis of thymocytes confirms the appearance of yfp positive cells during t cell development from the dn stage on. in the bone marrow we found yfp + /b + and yfp + /b populations. thus these pta expression analyses show closely similar pattern to those observed with hucd preta-reporter transgenic mice (gounari f. et al. , martin et al. . the bac pta reporter system can be used together with specific markers of other hematopoietic lineages and their progenitors to trace lymphopoiesis. gounari f et al., nat. immunol. , - ( ) martin c. h. et al., nat. immunol. , - ( . the individual functions and the reason for the tightly regulated expression of igm and igd during b cell development are poorly understood. our data show, that igd requires stronger stimuli than igm to induce b cell activation and that this silences autoreactive vdj recombination products when expressed as igd. in agreement with this, mhc and dhc, the respective heavy chains of igm and igd, differ dramatically in pre-bcr signaling, which represents the prototype of an autoreactive receptor. together with published data, our results reveal a novel role for igd and suggest that the differential expression of igm and igd is important to raise the activation threshold of mature b cells, thereby avoiding hypersensitivity and ensuring tolerance towards self-antigens. p. d. rymkiewicz , g. klein zmf (center for medical research), section for transplantation immunology and immunohematology, tübingen, germany thymic conduits which are exclusively found in the medullary region of the thymic lobules have been recently identified. the core of the conduits consist of fibrillar collagen bundles and is surrounded by a basement-membrane-like structure which contains the typical basement membrane components such as laminins, collagen type iv, nidogens and perlecan. a marker molecule for the conduits in the human thymus is the laminin isoform lm- which is synthesized by the medullary thymic epithelial cells (tecs) which tightly surround the conduits. functionally the conduits are too small to transport cells but they are able to transport small molecules x kda.mmp- , a secreted member of the matrix metalloproteinase superfamily, is a protease capable of digesting lm- . in the human thymus medullary, but not cortical thymic epithelial cells strongly express mmp- . by western blotting the zymogen and the activated form of mmp- can be detected in whole thymus lysates, whereas in lysates from isolated tecs mainly the activated form is present. an in situ zymographic analysis revealed an increased proteolytic activity in the medullary region of the thymus. using confocal laser scanning microscopy double immunofluorescence staining showed that lm- and mmp- can be found in close neighbourhood, but they do not exactly co-localize. why activated mmp- which can be secreted by medullary tecs does obviously not destroy the surrounding basement membrane of the conduits has not been solved so far. two natural inhibitors of mmp- , timp- and timp- , are found in the thymic medulla, but they are not expressed and secreted by tecs. whether mmp- plays a role in processing medullary chemokines which are produced by the thymic epithelial cells is presently under investigation. to study the process of t cells differentiation in more detail, we intend to establish an inducible gene expression system (tet-on system) in primary t cells. the tet-on system comprises two retroviral vectors. the response vector contains an inducible modified minimal cmv promoter which per se is unable to induce expression of the gene of interest (goi). the second vector encodes a transactivator which is constitutively expressed and undergoes conformational changes upon binding of doxycycline. in this state, the transactivator enables the minimal cmv promoter to transcribe the gene of interest. therefore, co-transduction of both vectors is required to achieve transcription of the gene of interest. to date we have tested two tet-on systems (revtet system and retro-x tet-on advanced inducible expression system) that differ in the sequence of their inducible promoters. to monitor successful transfection in retrovirus-generating phoenix cells and transduction in t cells, respectively, we have cloned the reporter gene gfp under the control of a constitutive cmv promoter, into the response vector of the revtet system. this allowed identification of transduced gfp-positive cells via facs. however, when we used a red fluorescent protein, tomato, as a surrogate goi, we detected considerable leakiness of the promoter irrespective of the presence of the transactivator or doxycycline. in contrast, we found comparably low leakiness when using the retro-x tet-on advanced inducible expression system. here, co-transfection of phoenix cells with the transactivator and supplementation of doxycycline yielded an induction of - % compared to only % basal rate. therefore, the retro-x tet-on advanced inducible expression system appears suitable for our studies. future experiments will aim at establishing this system in primary t cells. although a number of different experimental approaches has been used to elucidate impact of basal levels of adrenal gland-derived glucocorticoids (gcs) on t-cell development, and thereby t-cell-mediated immune response, their relevance for these processes is still far from being understood. the study was undertaken to explore relevance of basal levels of gcs for t-cell differentiation/maturation. eight days post-adrenalectomy in adult male rats thymocyte yield, apoptotic and proliferative rate and relationship among major thymocyte subsets defined by tcrab/cd /cd expression were examined using flow cytometry analysis. it was found that adrenal gc deprivation affects: i) thymocyte apoptosis, producing thymic hypercellularity and ii) kinetics of t-cell differentiation/maturation leading to an overrepresentation of the cd +cd + double positive (dp) tcrab low cells entering selection, and their cd +cd + dp tcrab-immediate precedents followed by underrepresentation of the selected cd +cd + dp tcrab high and the most mature cd -cd + and, particularly, cd +cd -single positive (sp) tcrab high cells. the study suggests that withdrawal of adrenal gcs produces alteration in thymocyte selection processes that may affect diversity of functional t-cell repertoire and generation of potentially self-reactive cells as indicated by the reduced proportion and number of cd -cd -double negative tcrab high cells. in addition, it indicates that gcs influencing post-selection maturation of thymocytes play a regulatory role in controlling mature cd +cd -/cd -cd + sp tcrab high cell ratio. in the thymus a specific subset of thymic stromal cells -medullary thymic epithelial cells (mtecs) -express a highly diverse set of tissue-restricted antigens (tras) representing essentially all tissues of the body, which is known as promiscuous gene expression (pge). this allows self-antigens, which otherwise are expressed in a spatially or temporally restricted manner to become continuously accessible to developing t cells. the scope of central tolerance is to a large extent dictated by the pool of promiscuously expressed genes. thus, even lack of a single tra can result in spontaneous organ-specific autoimmunity. promiscuously expressed gene which have no structural or functional commonality display two prominent features, they are highly clustered in the genome and show a preference for tras. for better understanding these features, we set out to precisely define the genomic organization of this gene pool. in particular, we probed to what extent and according to which rules predefined genomic clusters of tras are transcribed in mtecs. our analysis proceeded from the bioinformatic definition of tra clusters via gene expression analysis in mtecs using whole genome arrays to the in depth analysis of selected tra clusters by rt-pcr at the population and single cell level. patterns emerging from these studies will hopefully yield insight into evolutionary mechanisms responsible for selecting this gene pool. conceivably, positional cues in the genome and/or particular properties of self-antigens (e. g. immunogenicity) could have been driving forces during the co-evolution of pge and adaptive immunity. although catecholamines have been shown to influence thymocyte proliferation and differentiation, long-lasting b-adrenoceptor (ar) blockade failed to show any significant effects on thymic cellularity. bearing that in mind, the present study was undertaken to explore: i) a -ar expression on thymic lymphoid and nonlymphoid cells and ii) putative role of a -ar-mediated mechanisms in modulation of thymic cellularity and t-cell development. for this purpose a -ar expression on thymic cells was assessed using both immunocytochemistry and flow cytometric analyses, while their putative modulatory role in thymopoiesis was estimated by analyses of thymocyte proliferation and apoptosis, as well as expression of major thymocyte differentiation antigens (cd / cd /tcrab), in adult wistar rats subjected to -day-long treatment with a -ar blocker urapidil ( . mg/kg body weight/day s. c.). the a -ar immunoreactivity was found in both thymocytes (mainly less mature cd and cd low cells) and thymic nonlymphoid cells (thymic epithelial cells located mainly at cortico-medullary junction and cortical ed -postive cells, which comprise macrophages and dendritic cells). chronic treatment with urapidil increased thymic weight and caused the organ hypercellularity. the thymic hypercellularity reflected, at least partly, increased frequency of proliferating thymocytes, which was followed by diminished thymocyte apoptosis. in addition, in these rats changes in distribution of major thymocyte subsets delineated by cd / cd /tcrab expression were observed. these changes comprised of an increase in the percentage of cd + + tcrabthymocytes, which was accompanied by the reduction in that of cd + + tcrab low cells in urapidil-administered rats, and divergent changes in the percentage of the most mature single positive tcrab high thymocytes. compared with saline-administered controls, the percentage of cd + -tcrab high thymocytes in urapidil-administered rats was increased, while that of the cd - + tcrab high was reduced. in addition, the percentage of cd + t regulatory and cd +tcrab+ nkt cells was increased. collectively, this study clearly showed the expression of a -ar on both lymphoid and nonlymphoid thymic cells, and indicated that a -ar-mediated mechanisms may be implicated in modulation of multiple steps of t-cell development. we have recently shown that the thymus is a common target for mycobacterial infections. of notice, while bacterial growth is arrested in the spleen around weeks post infection with mycobacterium avium, it takes several weeks longer within the thymus to reach a bacterial plateau. this observation suggests that a specific immune response occurs in the thymus, although this seems to be distinct from that occurring in the spleen. since t cell differentiation occurs, to a large extent, in the thymus, and depends, among other factors, on the antigens encountered within the thymus and on the cytokine milieu of the organ, we decided to characterize the pattern of the thymic immune response against mycobacteria and investigate possible consequences on the normal function of this organ. methods: c bl/ mice infected with m. avium ( cfus, iv) were sacrificed at different time points after infection ( , and weeks). non-infected animals were used as controls. bacterial load was assessed in the spleen and thymus and cytokines (such as ifn-g, tnf, il- ) were quantified by rt-pcr in tissues (normalized for hprt expression) or by elisa in the supernatants of cultured thymocytes and splenocytes. statistical significances were determined by anova. we observe increased levels of ifn-g in infected thymi at weeks post infection. this increased expression of ifn-g is concordant with the late bacterial growth arrest within the thymus. at weeks post infection this difference is still present. throughout the course of infection no significant differences are found in the expression of tnf and il- in this organ. in the spleen, ifn-g reaches a peak of expression earlier ( weeks post infection) and this is accompanied by increased tnf expression. conclusion: our cytokine analysis of the thymus and spleen confirm that an immune response against mycobacteria is mounted within the thymus, although different in timing and pattern from the one in the spleen. since the cytokine milieu influences t cell differentiation within the thymus, our observations raise the question on the consequences of such response on the normal function of this organ. such implications should next be investigated. precise regulation of eukaryotic gene expression requires interactions between distal cis-acting regulatory sequences with the looping out of the intervening dna, but how trans-acting regulatory proteins work to establish and maintain dna loops during gene activation remains largely unexplored. lps-induced transcription of the mouse igx gene in b lymphocytes utilizes three distal enhancers and requires the transcription factor nf-xb, whose family members include rela and c-rel. using chromosome conformation capture technology in combination with chromatin immunoprecipitation, here we demonstrate that lps-induced igx gene activation creates chromosomal loops by bridging together all three pair-wise interactions between the distal enhancers and rna polymerase ii, the apparent molecular tie for the bases of these loops. rela and actin polymerization are essential for triggering these processes, which do not require new transcription, protein synthesis or c-rel. we have thus identified both essential and non-essential events that establish higher-order chromatin reorganization during igx gene activation. this investigation was supported by grants gm and ai from nih and grant i- from the robert a. welch foundation to wtg, and by grants hl and hl from nih to lst. allelic exclusion of immunoglobulin (ig) genes supports burnet's clonal selection theory. the recognition that m-chain expression is sufficient for the maintenance of the silenced allele status by a process of feedback inhibition is yet not enough to explain the earlier monoallelic activation by the rag complex. attempts to prove the probabilistic or epigenetic nature of monoallelic v(d)j recombination were insightful and favor the epigenetic hypothesis, mainly by the observation that, like autosomal imprinted or x-chromosome inactive genes, ig genes are differentially marked at the chromatin level, and replicate asynchronously in virtually any cell of the mouse organism ever since the embryonic life (mostoslavsky, singh et al. ) . we are testing this hypothesis, i. e., that an epigenetic event has previously marked, on each b cell progenitor, which of the ig alleles is going to be activated for rearrangement and expression. for this, we are generating b cell clones from b cell progenitors of (c bl/ x balb/c)f mice, because the original strains have ig heavy chain (igh) alotypes distinguishable by monoclonal antibodies. we are analyzing if individual heterozygous clones show biased expression of a particular igh allele, and we expect to map the cell stage at which the (epigenetic) allelic marks are fixed. we have already shown, for the igh gene, a clear segregation of monoallelic expressors among b cell clonal lines that were generated from the common lymphoid precursor, but no allele bias was observed among multi-potent progenitor or hematopoietic stem cell b cell clones. this result, although suggesting epigenetic silencing starting at the common lymphoid precursor stage, does not favor the prevailing epigenetic hypothesis in its original formulation. we are currently exploring this result by the analysis of the igh silenced allele rearrangement status in sorted fractions of igm+ b cell clones. we are also testing the same epigenetic hypothesis for the ig kappa light chain gene (igk), using f mice in which the igk constant region from both alleles can be distinguished by antibodies. a. giniewski , s. lang , m. stein , t. winkler friedrich-alexander university, erlangen, germany vdj recombination is considered to be regulated by lineage and stage specific changes in accessibility of the loci to rag recombinase. accessibility is expected to correlate with certain histone modifications such as acetylation (e. g. h ac) or methylation of h on lysine (h k me / ). previous studies in our lab revealed three regions in the intergenic part of the distal v h cluster (ivars), which are associated with high levels of active chromatin marks (h ac and h k me / ) only in pro b cells but not in pro t cells. it is also known that vdj h recombination is accompanied by sense and antisense transcription, however, little is known about the function and origin of the antisense transcripts. since one ivar (ivar # ) shows promoter activity in antisense orientation, it was analysed in more detail. we found three transcription start sites by 'rlm race at the ivar # element. background: t-all is a malignancy of the lymphoblast committed to the t-cell lineage with translocations between tcr genes and oncogenes as a genetic hallmark. these translocations are thought to be driven by v(d)j-recombination mechanisms. we believe that these mechanisms only partly facilitate the occurrence of tcr translocations and that the accessibility of involved genes plays an intrinsic role in promoting these events. the lmo locus, is thought to be accessible only during the double-negative (dn) and thymocyte stages based on mrna expression, implying that translocations between lmo and tcr genes can only occur within these stages. gene expression as a readout for accessibility can not elucidate the involvement of other oncogenes such as tlx (hox ), which are not expressed in thymocytes, as being accessible for translocations to occur. objectives: we aimed ) to evaluate lmo and tlx breakpoint-site accessibility during thymocyte development; ) to determine in which stage of development there is an increased chance for lmo or tlx translocation to occur based on this accessibility. methods: dna of immunologically "healthy" sorted thymocytes was isolated using faire (formaldehyde-assisted isolation of regulatory-elements). this dna was used to quantitatively assess lmo accessibility during thymocyte development at both the transcription start site (tss) and negative regulatory element (nre), and within different in t-all documented breakpoint-sites of both the lmo and tlx loci. results: quantitative analysis on the tss showed a correlation with mrna expression, with the dn and dn development stages showing the highest accessibility. the nre, showed an inverse pattern of accessibility to the tss region. analysis of breakpoint-sites revealed the highest accessibility levels within the earliest stages of development, dn , dn , dn and immature single positive (isp) stages for both lmo and tlx . conclusion: our findings show that both the lmo and tlx loci are accessible during thymic development irrespective of gene expression and that this accessibility is not restricted to dn and dn stages, suggesting that these loci are much more active than assumed, thus increasing the opportunity for translocations to occur. we addressed this issue, by building a model able to account for of v-ja gene rearrangements observed experimentally during thymus development of mice. we developed, based on experimental data, a numerical model on the whole tra/trd locus to estimate va and ja genes accessibility to rearrangements. the progressive opening of locus to v-j gene recombinations is modeled through windows of accessibility of different sizes and with different speeds of progression. furthermore, the possibility of successive secondary v-j rearrangements was introduced in the modeling. the model points out some unbalanced v-j associations resulting from a preferential access to gene rearrangements and from a non-uniform partition of the accessibility of the j genes, depending on their location in the locus. the model shows that to successive rearrangements are sufficient to explain the use of all the v and the j genes of the locus. finally, the model provides information on the kinetics of rearrangements and on the frequency of each v-j association. the model accounts for the essential features of the observed rearrangements on the tra/trd locus and may provide a reference for the repertoire of the v-j combinatorial diversity. the genetic programs of b-cell differentiation and the first dj h gene rearrangements appear in the post-gastrulation mouse embryo (e - ), shortly after the first multipotential hematopoietic progenitors do emerge. these dj h joints represent the unselected baseline of the ig repertoires. we have undergone a systematic sequencing of embryo dj h joints obtained from normal balb/c embryos and heterozygous embryos obtained from rag -/mothers mated to balb/c males (to discard any mother-derived contribution), as well as newborn and adult control groups. the embryo dj h s displayed unexpected mechanisms of diversity, including short stretches of non-templated n nucleotides in one-third of the studied sequences (in the absence of tdt expression) and frequent dj h s with large nucleotide deletions, as a consequence of ligation to joint-distal microhomology sites. because the dna polymerase m (polm), a highly-homologous tdt member of the x dna polymerase family, showed an increased expression in the embryo, we analysed the dj h s of polm -/mouse embryos. we observed that polm was mainly responsible for introducing n nucleotides at the mouse embryo dj h joints. also, and based on its dna-dependent polymerization ability, polm filled-in small sequence gaps at the coding ends, and ligated highly-processed ends by pairing to internal microhomology sites, although at the cost of germline sequence losses and the generation of "useless" gene products. we think that, more than attempting to increase diversity, polm acts as a "connector" in the embryo, subsequently participating in the repair of rag-induced double-strand breaks, to preserve genomic stability and cellular homeostasis in cells with high proliferation rates. along the end of gestation, further selective pressures acting over these first v-dj h products will contribute to establish the differential neonatal ig repertoires. although mortality from infectious diseases peaks during infancy, many vaccines are ineffective in early life. most children with infantile bronchiolitis are under months of age, and most cases are due to respiratory syncytial virus (rsv) infection, for which vaccines continue to be elusive. we now show that, compared to adults, the antibody response to rsv infection is very poor in neonatal mice and is unaffected by cd cell depletion. however, cd depletion in infancy led to a remarkable boosting of antibody responses during adult re-challenge. to test the possibility that poor antibody boosting is caused by rsv-specific cd t cells killing rsv-infected b cells, we sorted cells from the lungs of infected neonates. viral copy number was high in neonatal b cells, but viral load in surviving b cells was unaffected by cd cell depletion. in addition, fas ligand (fasl) deficient gld mice responded to rsv infection in the same way as normal mice, indicating that fasl is not required for the inhibition of antibody responses. this new mechanism of regulation of b cell responses by cd t cells has important implications for vaccine development against neonatal infections. ( ) showed that irf knockout mice had significantly reduced numbers of pre-pro-b cells in marrow and a phenotype similar to agammaglobulinemia. these results prompted us to consider icsbp/irf as a candidate gene in the pathogenesis of defective early b cell development. therefore we decide to undertake direct sequencing of the gene encoding icsbp/irf in a small cohort of patients with autosomal recessive agammaglobulinemia. methods: eight patients affected by agammaglobulinemia were included in this study. all patients were under regular ig replacement therapy. informed consent was obtained from all patients. genomic dna was extracted from whole blood and amplified with specific primers designed on the flanking regions of every exon. direct gene sequencing of the eight exons of icsbp/irf were obtained using abi prism sequencer. results: seven of the eight patients result wild type while only one patients present a synonymous snp in exon v, yet documented as rs . conclusions: although recent findings indicated that irf function is essential for early b cell development, our data in a small cohort of patients affected with autosomal recessive agammaglobulinemia did not evidence any mutations in icsbp/irf that may be responsible for this disorder. the hh/ptch signaling system is known to control the development and neoplastic transformation of several cell types. however, the role of hh/ptch for the differentiation of b and t lymphocytes from hematopoietic stem cells (hsc) has not been assessed so far. to analyze the function of hh/ptch for lymphopoiesis in vivo, we have employed a genetically engineered mouse mutant in which the ptch gene can be conditionally inactivated by virtue of the cre/loxp recombination system. we show that targeted disruption of ptch in the adult organism results in a dramatic specification and differentiation defect of the lymphoid lineage leading to rapid disappearance of newly generated b and t lymphocytes from peripheral lymphoid organs. the developmental block occurs at the level of the common lymphoid progenitor cell (clp), which defines an early branching point of hsc differentiation and lineage commitment. in contrast to the lymphoid lineage, development of cell types of the myeloid lineage from common myeloid progenitors (cmp) appears normal. our data identify hh/ptch-induced signaling as a key regulator for proper development of immunocompetent lymphocytes. hence, the progression of tumors, which are initiated upon oncogenic hh/ptch mutations, may be further promoted due to impaired tumor surveillance by a compromised immune system. l. calderon dominguez , t. boehm max-planck institute for immunbiology, developmental immunology, freiburg, germany in many organ systems of animals and plants, specialized niche microenvironments maintain and specify stem and progenitor cells. the ability to modify or artificially create such niches in vivo and in vitro has many implications for stem cell research and therapy. by analysis of several mutant mouse strains and subsequent transgenesis in the mouse, we disentangle and individually modulate niche functions responsible for collection, maintenance and specification of multipotent thymocyte progenitors. we demonstrate how an epithelial niche, rendered functionally inactive by disruption of the foxn transcription factor, can be specifically rebuilt in a modular and combinatorial fashion to only attract, or attract and maintain, or attract, maintain and specify progenitor cells into the b and t cell lineages, respectively. the strategy of engineering niche functions in a modular fashion might be applicable to other progenitor cell systems. silencing of dc-sign using lentiviral rna interference revealed its critical function for pd-l expression on dcs after m. tuberculosis infection. as a counterpart to expression of its ligand, we showed that cd and cd t cells from tuberculosis patients highly express pd- when compared to healthy uninfected individuals. in addition, analysis of pd- expression in lung biopsies from tuberculosis patients revealed that pd- is expressed on cd and cd t cells confined to lung granulomatous lesions. finally, blocking of the pd- /pd-l axis using monoclonal antibodies abrogated the down-modulation of t cell proliferation and ifn-g production induced by manlam, a mycobacterial cell wall glycolipid and ligand for dc-sign. taken together, our results suggest that the pd- /pd-l pathway is involved in the exhaustion of t cell responses to m. tuberculosis. the inflammatory canonical nfkb pathway is critically involved in virtually all aspects of inflammation in general. yet, the role of the alternative, non-canonical nfkb pathway in inflammation and adaptive immunity remains largely elusive. the alternative pathway is primarily mediated through the nfkappa-b inducing kinase (nik) which in turn leads to the phosphorylation and the cleavage of p to p . among the receptors engaging nik is the ltbr, which is also required to form the anlage for secondary lympoid tissues (slts). due to a point mutation within nik, alymphoplasia (aly) mice do not develop slts and are highly immunodeficient. however, while the immunodeficiency of aly mice is widely held to stem from their developmental malformation, it has been overlooked, that the mutation of nik itself could potentially lesion the development of immune responses. to verify this notion, we generated a series of bone marrow chimeric mice (bmc) in which the absence of slts was disconnected from the hematopoietic loss of nik function. we generated mice, which lack all slts, but are equipped with a normal systemic immune system (wt aly), and conversely, mice with normal slts, but lacking nik in all leukocytes (aly wt). surprisingly, we discovered that nik is vital for the development of autoimmune disease, while slts (ie. lns, spleen etc.) are essentially dispensable for cell-mediated immunity. we found that nik is required for the polarization of effector t cells and that th and th cells cannot be generated in the absence of nik. preliminary data implicate the involvement of nik in a discrete and novel pathway required for the formation of cell-mediated immune responses. the family of nfat (nuclear factor of activated t-cells) transcription factors is indispensable for t cells, for example playing an important role in cytokine gene regulation. in peripheral cd + t cells, nfatc and c are predominantly expressed. nfatc is synthesized in six isoforms which have partly opposing functions regarding activation and apoptosis. here we address the functional difference of the short isoform nfatc /a, which is highly induced upon t cell activation, and the long constitutively expressed isoform nfatc /c. as demonstrated by y h screen and co-ips, nfatc /c-specific c-terminus can be highly sumoylated. confocal microscopy studies revealed that upon sumoylation nfatc /c -but not the unsumoylated nfatc /a -translocates to promyelocytic leukemia-nuclear bodies (pml-nbs). this leads to interaction with hdacs followed by deacetylation of histones (co-ips), which in turn induces transcriptionally inactive chromatin (chip and confocal microscopy). as a consequence, multiple expression studies revealed sumoylation dependent suppression of the nfatc target gene interleukin- . other lymphokines like ifng and il are reversely regulated. interestingly, ntreg cells which do not express il exerted only nfatc /c, but no nfatc /a expression (qrt-pcr). these findings demonstrate that the modification by sumo converts nfatc from an activator to a site-specific transcriptional repressor, revealing a novel regulatory mechanism for nfatc function. therefore, especially ntreg cells and anergized cd + t cells might be regulated by the long sumoylatable isoform nfatc /c. lnk/sh b and aps/sh b , two members of the lnk/sh b family of adaptor proteins, play an important role as negative regulators in b cell lymphopoiesis. they possess several protein-protein interaction domains and motifs that allow their interaction with different signalling effectors. mice deficient for these proteins demonstrated that lnk inhibits expansion of pro/pre-b cells while aps controls mature b- cell population, suggesting specific roles for these adaptors during b cell development. however, the molecular mechanisms underlying their regulatory function in these cells, have not been identified. to address this question, we used primary and b cell lines at different stages of differentiation as our cellular system. analysis of lnk/aps expression pattern showed that lnk is expressed at all developmental stages, while aps is only detected in immature and mature cells. we then first examined the role of lnk in il- signalling in pre-b cells overexpression of lnk dramatically inhibits il- -dependent growth demostrating that lnk negatively regulates il- pathways. furthermore, we showed that il- stimulation induces lnk phosphorylation and its subsequent association with important signalling effectors, notably the e ubiquitin ligase cbl. we next analyzed the role of aps in mature b cells by imaging and biochemical techniques. our results showed that aps colocalizes with the bcr complex after bcr triggering. interestingly, lnk is not recruted to the bcr signalosome in these cells, suggesting that interaction of the adaptors with the receptor complex regulates their function at different development stages. moreover, we showed, for the first time, that aps can associate, upon bcr stimulation, with the signalling molecules cbl and vav . to address the functional implications of these interactions, we examined specific b cell responses, notably bcr trafficking and cytoskeleton remodelling. we demonstrated that overexpression of aps enhances ligand-induced endocytosis of bcr, possibly through interaction with cbl and affects the kinetics of bcr-induced cell spreading. our results therefore suggest a regulatory function of aps in bcr internalization and cytoskeleton dynamics. altogether, our findings demonstrate that lnk and aps display sequential specific regulatory roles during b cell development that are important for maintaining b cell homeostasis. signaling through the t-cell receptor (tcr)-cd complex is a critical event in adaptive immunity. it is still not clear how ligand binding to the tcr is communicated across the plasma membrane and leads to phosphorylation of the cytoplasmic domains of the cd complex. it is widely accepted that dimerization or multimerization of tcr is required for tcr triggering. in our model t-cell activation is initiated by recognition of monomeric mhc/peptide complexes on the surface of antigen presenting cells (apc). critical to tcr triggering is the movement of the t-cell across the apc. engagement of a mhc/peptide complex on the surface of the apc will change the mobility of the tcr leading to partitioning with lipids of lower mobility that are enriched in signaling molecules critical for t-cell activation. furthermore, the change in mobility will lead to dislocation of the itams from the plasma membrane so that they become accessible to tyrosine kinases. to test the hypothesis we established a new approach where we created a soluble bifunctional complex composed of a pmhc and a fab that recognizes an epitope tag that we express on the t-cell surface. binding of the fab to the expressed epitope tag will constrain the lateral mobility of the tcr that is engaged by the pmhc arm of the same complex. the bifunctional complexes induced activation and proliferation as well as ca influx and cytokine production in human cd + t-cell clones that displayed the epitope, but not in t-cells that did not display the epitope. activation required interaction of the fab with its epitope on the t-cell surface because no activation was observed when soluble epitope peptide, which acts as a competitor for the fab binding site, was added. these results demonstrate that a monomeric copy of a pmhc is sufficient to trigger tcr and that formation of a tcr dimer is not an obligatory step in t-cell activation. the bifunctional complex we generated may also have a great immunotherapeutic impact. exchanging the fab with a fab or cytokine directed to a surface molecule may allow an antigen specific stimulatory or inhibitory modulation of t-cell responses. adaptor proteins are crucial in signal transduction, cell cycle regulation, apoptosis and stress response. adaptor proteins containing characteristic sh or sh domains known to mediate protein-protein interactions are key players in these processes. sly (sh domain protein expressed in lymphocytes ) was identified as a putative adaptor protein containing a sh and a sam domain as well as a bipartite nls. sly belongs to a family of three molecules: sly , sly and sash .in humans, the sly gene is located on chromosome , in mice on chromosome . sly is widely expressed for example in immune tissue as well as in hematopoietic cells, brain, lung and pancreas. subcellular fractionation showed that the sly protein is located in the cytoplasm and the nucleus and to a lesser extend in the plasma membrane.to elucidate the function of sly we searched for possible interaction partners by yeast two hybrid screening with a mouse t cell lymphoma library. this approach identified sin -associated polypeptide p (sap ) as a putative interaction partner of sly . sap is a conserved member of the sin a-hdac corepressor complex that contains histone deacetylase (hdac ) and histone deacetylase (hdac ) and acts as a transcriptional repressor for a variety of genes. we confirmed this interaction by implementing coimmunoprecipitations with lysates from transiently transfected t cells. in addition, we could show a direct interaction between sly and hdac . to investigate the functional impact of this molecular interaction, we performed hdac enzymatic activity assays. we were able to show that sly increases the activity of hdac in whole cell lysates and, more precisely, in nuclear extracts of t cells. the interaction of sly with sap and hdac indicates a transcriptional function of this protein. within the sin a-hdac corepressor complex sly might act as a switch for the activity of hdac . cd -cyt and cyt are co-expressed in human t cells and undistinguishable from the cell surface. in order to determine their specific role in t cell activation, we have expressed chimeric proteins consisting of the extracellular domains of cd (b cell marker) fused to the transmembrane and intracellular domain of cd -cyt or cyt in primary t cells. we show that these two isoforms differently control human t cell function. specific cyt coengagement controlled il- secretion, while cyt coligation inhibited ifng production. moreover, our preliminary data suggest that cd -cyt inhibits the phosphorylation of several molecules known to be activated by cd stimulation. these data suggest that these two isoforms act as molecular switches for t cell activation, either promoting or turning off t cells. they demonstrate for the first time the distinct roles of cd cytoplasmic isoforms in primary human t cell activation. this also suggests that the modulation of their expression and/or activation might provide new therapeutic avenues. nck is a ubiquitously expressed adapter protein that is almost exclusively built of one sh domain and three sh domains. nck connects receptor and non-receptor tyrosine kinases to the machinery of actin reorganisation. in t cells, nck participates in different and interdependent signalling pathways linking t cell activation and effector function with actin remodelling proteins that in turn initiate changes in cell polarity and morphology. we previously showed that nck directs the death factor fasl to the cytotoxic immunological synapse when t cells encounter putative target cells. we now performed a systematic screening for interaction partners of the four individual interaction modules of nck in primary and leukemic t cells. we precipitated putative binding partners from untreated or pervanadate-treated pha blasts, jurkat and hut cells with gst fusion proteins containing full length nck, the three sh domains or the individual sh and sh domains. binding proteins were excised from gels after staining with coomassie, silver or flamingo pink and processed by tryptic in gel digestion for mass spectrometrical analysis. as expected, we observed major differences in nck binding proteins precipitated from resting versus activated t cells. we not only verified established interactions (e. g. with the tcr signalling components slp and cd epsilon, the actin-regulatory proteins wasp and wip and the nuclear protein sam ) but also identified novel nck-interacting proteins. the interaction with the actin-binding protein hip once more underscores the fundamental role of nck in tcr-mediated actinreorganization. the identification of the nuclear proteins sfpq/nono points to novel, yet unknown functions of nck that might be associated with the recently reported nuclear translocation/localization of nck. accordingly, employing laser scanning microscopy, we clearly detected nck within the nucleus also in human t cells. the present data highlight that nck serves versatile functions in t cells, which include the different interdependent pathways of tcr-induced actin reorganization but also novel, yet poorly defined protein networks that are associated with a nuclear translocation of nck. cytotoxic t lymphocytes (ctl) mediated killing is tightly regulated according to the strength of t cell receptor signal. killing is regulated by the delivery of perforin-containing lytic granules moving along microtubules towards the centrosome, which polarizes and docks at the central supramolecular activation complex (csmac) within the immunological synapse. although much has been learnt about the mechanisms controlling the strength of tcr signal and the mechanisms required for release of the lytic granules, little is known about how the strength of the tcr is able to control the degree of ctl-mediated killing so finely. here we examine how the strength of tcr signal controls polarization of the secretory apparatus leading to ctl-mediated killing using tcr transgenic ot-i ctl. decreasing the tcr signal by reducing the concentration of ova peptide or using the weak agonist peptide, g , results in a slight reduction in the number of ctl target cell conjugates formed, and the number of conjugates in which a csmac (visualized by a patch of lck-staining at the immunological synapse) was formed. tcr signals result in reduced or absent (in the case of g ) staining with psrc and perk antibodies in the immunological synapse and reduced or absent (g ) degranulation as measured by cd a assays. the centrosome docks at the csmac of the immunological synapse even with relatively weak tcr signals, but the lytic granules require a certain threshold of signaling to successfully polarize to the immunological synapse. inhibitors support a role for pi k in granule polarization. together these data demonstrate that the strength of tcr signal controls the level of ctl mediated killing at the single cell level by controlling, the number of conjugates formed, the formation of the csmac and the accumulation of psrc and perk at the synapse. the centrosome polarizes to the csmac even with relatively weak tcr signals, but granule recruitment requires a higher threshold of signaling. these findings reveal how ctl can fine tune the degree of killing in response to tcr signals at the single cell level. cytotoxic t cells play an essential role in the immune system, particularly in the elimination of tumor and virus-infected cells. cytolytic t-cell activity is mediated through the pore-forming molecule perforin allowing granzymes to enter the target cell and to initiate apoptosis. perforin and granzymes are stored in specialized secretory granules, called secretory lysosomes. they are capable of undergoing regulated secretion in response to a t cell receptor engagement which involves binding to a cognate mhc class i-peptide complex. the intracellular transport of lysosomal proteins from the golgi to the lysosomes is mediated by the cationindependent mannose- -phosphate receptor which exhibits structural and functional similarity to the vps p-receptor sortilin. sortilin was characterized predominantly in neuronal cells where its function in protein sorting was identified. in the secretory pathway, sortilin is putatively involved in trafficking of proteins in the constitutive and regulated pathway. to explore whether sortilin has a broader functional relevance, we asked if sortilin might act as an alternative receptor for the cation-independent mannose- -phosphate receptor in cytotoxic t cells. first, we demonstrate that sortilin is expressed in t cells. to examine its function during an adaptive immune response, we analysed sortilin-deficient cytotoxic t cells derived from a knockout mouse strain. in strong contrast to the results reported from neuroendocrine cells, we obtained a reverse phenotype in sortilin-deficient cytotoxic t cells. whereas the regulated release of secretory lysosomes was enhanced, the constitutive release of interferon-g was found to be decreased. the enhanced release of cytotoxic molecules from sortilin-deficient cytotoxic t cells translated into an increased cytotoxicity in vitro. thus, the deletion of sortilin imposed a specific phenotype in cytotoxic t cells which could not be compensated for by other sorting receptors. our localisation studies of sortilin in t cells were consistent with the results previously described in neuronal cells which indicated that sortilin acts as a sorting receptor during the anterograde transport of lysosomal hydrolases from the trans-golgi-network to endosomes and lysosomes. taken together, we suggest that sortilin might play a modulatory role in the regulation of the adaptive immune response through the control of the constitutive and regulated secretory pathway. there is growing interest in the soluble splice variant of ctla- (sctla- ) as an immune inhibitor secreted by t cells, because genetically determined variation in its production is associated with susceptibility to autoimmune disease. however, little is known of the biology of sctla- in immune responses. using a specific anti-human soluble ctla- monoclonal antibody, jmw- b that selectively binds the soluble isoform but not membrane bound ctla- , or cleaved fragments of it, we demonstrate that sctla- plays a vital role in regulating antigen-specific immune responses. we used antibody blockade to show that antigen-specific t cell responses are strongly enhanced upon blockade of sctla- , secreting increased amounts of cytokines including interferon-g, il- and tnf-a, but lower amounts of il- . soluble ctla- was also prepared from sera for use in experiments by antibody based affinity purification techniques. addition of sctla- induced secretion of the immunoregulatory cytokine il- by human pbmcs both in an antigen-selective and dose-dependent manner, while antibody blockade abrogated that effect. the immunosuppressive indoleamine , dioxygenase enzyme cascade was also initiated by sctla- . it is clear that the importance of this natural soluble molecule has been overlooked and like membrane-bound ctla- it is crucial to t cell inhibition. membrane-bound ctla- exists as a homo-dimer on t cells but sctla- is usually considered to be monomeric in form, implying its functional capacity is diminished because of an inability to cross-link b ligands on antigen presenting cells. a third important observation from this study is that sctla- exists both in serum and culture supernatants as a natural kda homo-dimer, and not as a monomer. this goes some way to explaining why this molecule has such potent immunoregulatory effects on antigen-specific immune responses. together, these results lead us to reappraise sctla- , concluding it to be a mediator of negative feedback, secreted as a recall regulatory t cell response to antigenic stimulus, rather than a product of resting t cells. this work also raises the possibility that where il- dependent regulation is most critical, boosting sctla- secretion by regulatory t cells could be a novel therapy for immune mediated diseases. recently, we identified a new adaptor protein, swiprosin- /efhd- , in lipid rafts of b cell lines that undergo apoptosis after b cell receptor (bcr) stimulation. swiprosin- /efhd is expressed in immature b cells of the bone marrow, in resting and activated splenic b cells, in t cells, macrophages, mast cells and some nonlymphoid tissues. ectopic expression of swiprosin- /efhd- in the immature murine b cell line wehi enhanced spontaneous and bcr-induced apoptosis. in contrast, shrna-mediated down-regulation of swiprosin- /efhd- impaired spontaneous and bcr-elicited apoptosis, but not bcr-induced g cell cycle arrest. to understand how swiprosin- /efhd enhances pro-apoptotic bcr signals, we analyzed whether swiprosin- /efhd is involved in proximal bcr signalling. in fact, ectopic expression of swiprosin- /efhd enhanced bcr-induced calcium flux in wehi cells, whereas shrna-mediated down-regulation of swiprosin- / efhd impaired bcr-elicited calcium signals. concomitantly, gst-pulldown experiments revealed that swiprosin- /efhd interacts with phospholipase cg (plcg ) and with the tyrosine kinase syk (splenic tyrosine kinase), both of which are important for bcr-induced calcium flux. the interaction of plcg and swiprosin- /efhd was further established by co-immunoprecipitation. reconstitution of bcr-elicited calcium signals through complementation of swiprosin- /efhd silenced wehi cells with swiprosin- /efhd- was inhibited by the syk inhibitor bay - . in analogy, swiprosin- /efhd regulated syk activity positively. moreover, swiprosin- /efhd re-expression accelerated tyrosine phosphorylation of several proteins, specifically tyrosine phosphorylation of plcg and of syk tyrosine residue , which is involved in syk activation. finally, reconstitution of swiprosin- /efhd knock-down cells with swiprosin- /efhd mutants revealed that the n-terminal putative sh -binding site, the first ef-hand, and to a lesser extent, the second ef-hand and the c-terminal coiled-coil domain, are important for bcr-induced calcium flux in wehi cells. interestingly, swiprosin- /efhd re-expression in swiprosin- /efhd -silenced cells induced already in unstimulated cells raft partitioning of syk, plcg and the bcr, which was reversed after min of bcr stimulation. in summary, swiprosin- /efhd is an accelerator of proximal bcr signalling and acts through syk and plcg by assembling a syk-dependent calcium initiation complex in lipid rafts. this might be relevant for memory b cell signalling or central b cell tolerance. to test the biological relevance of cbl-b e ligase activity, these mice were analyzed for t cell proliferation, susceptibility to autoimmunity, in vivo t cell tolerance responses, and tc tumor rejection. results: when stimulated, t cells from rf mutant mice hyperproliferate compared to wild type t cells, even in the absence of cd co-stimulation. preliminary data also suggest that rf mutant mice are more susceptible to autoimmunity. in addition, rf/p mice die within hours after a second challenge with p peptide, indicating a severe defect in t cell tolerance induction. more importantly, cbl-b e ligase dead mice can spontaneously reject tc tumors. conclusion: cbl-b e ligase dead mutant mice phenocopy total body cbl-b knock out mice, thus indicating that cbl-b e ligase activity is indispensable for its regulatory in vivo functions. intriguingly, our data suggest that its inactivation could be sufficient to confer anti-tumor activity. to further elucidate the cellular mechanism of cbl-b mediated tumor rejection we have now generated the conditional cbl-b e ligase dead mutant mice to for the first time study the cbl-b ubiquitination function in a tissue specific and temporal fashion. our research is also currently focused on identifying the relevant in vivo cbl-b ubiquitination substrates. interferon alpha (ifn-a) has been broadly used in the treatment of specific malignancies and chronic viral diseases. for a long time it was thought that the direct inhibitory effects on malignant or virus infected cells were the major mechanisms involved in the response to ifn-a therapy. however, recent studies in mice have revealed that ifn-a/b also exerts effects on several host immune cells. ifn-a has been shown to enhance cd t cells (ctls) responses against soluble antigens in mice. this immunostimulatory activity of ifn-a results at least partly from its direct ability to induce maturation of dendritic cells. several studies have recently demonstrated that ifn-a/b also acts directly on murine ctls, inducing clonal expansion and differentiation into effector and memory cells. to date, little is known about the effects of ifn-a on human ctls. to approach this issue, magnetically sorted untouched human cd + cd ro -t cells (mainly naï ve cells) were unstimulated or stimulated with human ifn-a and gene expression profiles were compared using an affymetrix human array. interestingly, ifn-a stimulation of highly purified human ctls without any other concomitant signals remarkably enhanced the expression of several molecules involved in death receptor signalling (trail) and chemotaxis (ip and itac). in a second genome-wide array analysis, we analyzed the effects of ifn-a on human ctls responding to antigen (signal ) and co-stimulatory signals (signal ), provided by beads coated with anti-cd /cd antibodies. gene expression patterns were compared for cells stimulated with anti-cd /cd beads alone or along with ifn-a. ifn-a regulates the expression of a number of genes that promote proliferation, activation and survival of ctls, tcr stabilization, chromatin remodelation, and, importantly, enhances the expression of genes involved in ctls effector functions (granzyme-b, ifn-g, trail, fasl) and chemotaxis (ip , itac). the enhanced expression of granzyme-b, ifn-g, trail and ip were further confirmed at the protein levels by flow cytometry analysis and/or elisa. enhancement of granzyme-b-and trail-mediated cytolitic functions was also found by functional assays using anti-cd -coated p cells and trail-sensitive caki-i cells as targets. our results show that ifn-a provides a strong signal- to human ctls leading to their differentiation into effector ctls. t cell activation is an important process of the adaptive immune system, which requires recognition of mhc-associated antigens by antigen presenting cells (apcs) via the t cell receptor (tcr). to induce a productive t cell response the interaction of t cells with apcs needs to be stabilized by adhesion molecules. junction adhesion molecules (jams) are a recently discovered group of immunoglobulin (ig) superfamily proteins, which are involved in the regulation of various inflammatory and vascular events. the third member of the jam protein family, jam-c, is highly expressed in platelets and endothelial cells, whereas expression in t cells is largely unknown. to investigate the regulation of jam-c in t lymphocytes, we determined jam-c gene expression in quiescent and activated human t cells. treatment with the polyclonal t cell activator phytohemagglutinin (pha) increased surface and total jam-c expression in t cells time-and dose-dependently, as determined by flow cytometry and immunoblot analysis. by contrast, no up-regulation of jam-a in activated t cells was detectable. the highest level of jam-c up-regulation by pha was observed in cd + foxp + and cd + cd high t cells. moreover, t cell receptor activation with combined anti-cd and anti-cd stimulation induced jam-c expression in t cells. jam-c induction occurred at the mrna level suggesting a transcriptional regulatory mechanism of jam-c expression. accordingly, we studied the regulation of the human jam-c gene promoter in transiently transfected t cells. luciferase activity of a jam-c promoter gene construct with three potential consensus sites for the transcription factor nfat was markedly induced in activated t cells. finally, pretreatment with two pharmacological inhibitors of calcineurin, cyclosporin a and fk- , but not with mapk inhibitors, blocked jam-c induction in activated t cells. in summary, the present data indicate that jam-c is induced in activated human t lymphocytes via a transcriptional mechanism and suggests a major regulatory function of jam-c for the t cell response. hiv- infection leads to immune dysfunction owing to a successive loss of the cd + t cell compartment. the molecular mechanisms underlying this depletion are not well-understood but may involve the viral nef protein. nef is a multifunctional accessory protein that is required for full hiv- virulence and the maintenance of high viral loads. nef enhances viral infectivity and replication by downregulating cell surface receptors, e. g. cd and mhc class i, and modulating signal transduction pathways. the latter is thought to raise the cellular activation level and in this way may increase the infected cell's susceptibility to apoptosis. in this study we identify a signaling complex assembling at the n-terminus of nef, which contains the kinases lck and pkcv. formation of this complex, termed nakc for nef-associated kinase complex, led to activation of lck, as assessed by in-vitro kinase assay, and recruitment of pkcv to membrane rafts, as detected by discontinuous sucrose density gradient ultracentrifugation. recruitment of pkcv to membrane rafts is a hallmark of t cell activation and has been associated with activation of the nfxb transcription factor. however, contrary to our expectations, nef-mediated nakc formation did not activate nfxb. instead, it led to a strong induction of erk / . this correlated with a nakc-mediated increase in hiv transcription that was demonstrated by luciferase reporter assays suggesting that erk / directly targets hiv transcription, possibly via induction of transcription factors. to our surprise, however, the effect of nakc on hiv transcription was found to be independent of ap- , nfat and nfxb suggesting an alternative mechanism of nakc-mediated enhancement of hiv transcription. on the basis of our previous results we propose that nef enhances hiv transcription via removal of inhibitory factors and thus derepression of the hiv promoter. how erk / is involved in this mechanism and whether nakc targets other cellular promoters, which may enhance the cellular activation level and thus sensitize the cell to apoptosis, remains to be determined. p. otahal , t. brdicka , v. horejsi institute of molecular genetics as cr, praha, czech republic aims: c-terminal src kinase (csk) and cd are key regulators of src-family kinases in leukocytes. while cd is a transmembrane phosphatase, csk is localized mostly in cytosol. however, a fraction of csk is found at the cell membrane and in lipid rafts where it inhibits signaling by phosphorylating inhibitory tyrosine of src-family kinases. currently, it is accepted that sh domain of csk binds phosphotyrosine of transmembrane adaptor protein pag and via this interaction is recruited to the cell membrane and lipid rafts. however, pag knock-out mice still have cell membrane-associated csk and do not show any apparent dysregulation of signaling which would be expected due to the low levels of membrane csk. thus, the mechanisms of membrane targeting of csk remain unclear. to analyze the role of membrane and lipid raft targeting of csk on lymphocyte signaling we targeted csk to different membrane compartments by fusing csk with transmembrane domains of lat, lax, cd and n-terminal part of src kinase. methods: csk chimeras containing n-terminal membrane targeting motif and c-terminal orange fluorescent protein were cloned into retroviral vector pmxs. jurkat t cells expressing individual constructs were subsequently prepared and analyzed for the inhibitory effect of these csk chimeras on t-cell receptor (tcr) signaling by measuring calcium flux and cd upregulation. the efficiency of inhibition depended on the membrane targeting motif, while lat-csk chimera completely inhibited tcr signaling and src-csk chimera inhibited the signaling only partially; lax-csk and cd -csk chimeras showed almost no inhibition of tcr signaling despite efficient presence at the plasma membrane. conclusions: our data demonstrate that the function of csk strongly depends on its targeting to the specific areas of plasma membrane. it also strongly supports the idea that membrane compartmentalization is critical for regulation of t-cell signaling. peripheral cd t cell tolerance can be generated outside lymphatic tissue in the liver. however, the course of events leading to tolerogenic interaction of hepatic antigen presenting cells with circulating t cells is unclear. here, we demonstrate that systemically circulating antigen was preferentially taken-up by liver sinusoidal endothelial cells (lsec) and not by other antigen presenting cells in the liver or spleen. uptake and cross-presentation of circulating antigen was followed by rapid antigen-specific naï ve cd t cell-retention in the liver but again not in other organs. using bone-marrow chimeras and tie- kb mice, we could show that antigen cross-presentation by lsec was both essential and sufficient to cause antigen-specific t cell-retention under non-inflammatory conditions, which was followed by cd t cell proliferation and expansion, but ultimately led to the development of t cell tolerance. our results show that cd t cell tolerance towards circulating systemic antigens is predominantly generated in the liver by lsec, which preferentially take-up and cross-present circulating proteins to cd t cells, leading to their rapid local antigen-specific retention and subsequent tolerisation. these insights broaden our understanding not only of physiological immune regulation towards circulating antigens but also of therapeutic manipulation of cd t cell responses. alphapix is a rho gtpase guanine nucleotide exchange factor domain-containing signaling protein that associates with other proteins involved in cytoskeletalmembrane complexes. it has been shown that pix proteins play roles in some immune cells, including neutrophils and t cells. in this study, we report the immune system phenotype of alphapix knockout mice. we extended alphapix expression experiments and found that whereas alphapix was specific to immune cells, its homolog betapix was expressed in a wider range of cells. mice lacking alphapix had reduced numbers of mature lymphocytes and defective immune responses. antigen receptor-directed proliferation of alphapix deficient t and b cells was also reduced, but basal migration was enhanced. accompanying these defects, formation of t-cell-b-cell conjugates and recruitment of pak and lfa- integrin to the immune synapse were impaired in the absence of alphapix. proximal antigen receptor signaling was largely unaffected, with the exception of reduced phosphorylation of pak and expression of git in both t cells and b cells. these results reveal specific roles for alphapix in the immune system and suggest that redundancy with betapix precludes a more severe immune phenotype. s. merluzzi , s. parusso , b. frossi , g. gri , c. pucillo university of udine, dstb, udine, italy in this study, we investigated whether primary mcs could modulate the activation and proliferation of primary b cells. we performed co-culture assays using mouse splenic b cells and bone marrow-derived mcs. naï ve and activated b cells proliferation could be induced by nonsensitized mcs while an increase in b cell proliferation was observed when mcs are activated. moreover, b cell proliferation was partially abolished when mcs and b cells were separated by the transwell membranes suggesting that cell-cell contact is important in this event. using both il- -/-mcs and anti-il- receptor antibody, we demonstrated that in co-culture of primary b cells and mcs, il- derived from activated mcs is a key cytokine implicated in the b cell proliferation. moreover, we showed that activated mcs can influence the surface expression of costimulatory molecules as cd on naï ve b cells and the interaction of cd on b cell surface and cd l on mcs is important for the further differentiation of b cells to plasmacells. indeed, we presented for the first time evidence that cytokines produced by activated mcs and interaction between cd l e cd on mc and b cells respectively can contribute to differentiate mature b cells to iga secreting cells. in conclusion, in the present report, we showed a novel role of mcs as promoter of both the survival and activation of naive b cells and of the proliferation and further differentiation of activated b cells through soluble factors production and cell-cell contact, suggesting that mcs can contribute to the regulation of specific immune response. e. fourmentraux-neves , n. bercovici , a. caignard inserm u , paris, france inhibitory killer ig-like receptors (kir dl - / ) which bind to hla-c molecules are expressed by human natural killer cells and effector memory cd + t cell subsets. these receptors suppress cd + t cell activation through recruitment of the src homology domain-containing protein tyrosine phosphatase (shp- ). to further analyse the yet largely unclear role of inhibitory kir receptors on cd +t cells, kir dl transfectants were obtained from a cd + t cell line and primary cells. the transfection of cd + t cells with kir dl dramatically increased the t cell receptor (tcr)-induced production of il- independently of ligand binding, but inhibited tcr-induced activation after ligation. kir-mediated tcr activation requires intact itim motifs, involves kir dl -itim phosphorylation, shp- recruitment, zap- and pkc-v phosphorylation. synapses leading to activation were characterized by an increase in the recruitment of p-tyr, shp- and p-pkc-v but not of shp- . in contrast, the kir dl /hla-cw interaction led to a strong synaptic accumulation of kir dl and the recruitment of shp- / , inhibiting tcr-induced il- production. kir dl may induce two opposite signaling outputs in cd + t cells, depending on whether the kir receptor is bound to its ligand. these data highlight unexpected aspects of the regulation of t cells by kir dl receptors. b cell receptor (bcr) binding by antigen initiates activating signaling cascades and facilitates the exposure of specific b cells to powerful co-stimulatory signals, such as t cell help or toll-like receptor ligands. the role of bcr binding in modulating the access to these second signals is complex and varies between stimulatory conditions. by quantitative tracking of b cell responses in vitro we can measure which signals affect b cell proliferation or differentiation, or both, and thereby establish a novel understanding of how b cells respond appropriately to different combinations of stimuli. we utilised hel-specific bcr transgenic sw hel mice to assess the effect of a specific antigen signal on b cell responses to the t-independent mitogen lipopolysaccharide (lps). the presence of antigen renders a greater proportion of cells responsive to lps stimulation and profoundly influences effector cell differentiation. antibody secreting cell formation is dramatically inhibited by hel, but we found that isotype switching to igg is strongly upregulated. both of these alterations to differentiation outcomes occur independently to the proliferative effects induced by antigen. when b cells are exposed to antigen for a limited period of time, switching to igg still occurs but some capacity to differentiate to antibody secreting cells is recovered, leading to effective secretion of igg antibody during these conditions. the observed igg switching behaviour mimics that of b cells responding to lps and il- , but is mediated by a different, stat -independent pathway. these data are indicative of the important role specific antigen signals play in regulating b cell responses in stimulatory environments. a. quintana , c. schwindling , m. pasche , c. junker , c. kummerow , u. becherer , e.c. schwarz , j. rettig , m. hoth saarland university, biophysics, homburg, germany, saarland university, physiology, homburg, germany the adaptive immune response requires the interaction between antigen-presenting cells and t cells. this cell-cell interaction, called the immunological synapse (is), facilitates the activation of t cell receptor-mediated signalling cascades including a rise of cytosolic calcium through the activation of crac/orai channels. to allow sustained activity of crac/orai channels, the calcium-dependent inactivation of the channels through local calcium microdomains has to be prevented. objectives: the purpose of the study was to analyze local and global calcium signals in t cells and to test the hypothesis that the is controls these signals through mitochondrial positioning. methods: we used different microscopy techniques including very fast wide-field microscopy with subsequent deconvolution, total-internal reflection microscopy, and confocal microscopy in combination with electrophysiological techniques in primary human t helper cells and cell lines. to test the statistical significance of our data, we used two-sided student t-tests or non-parameterized tests. results: following is formation, we found that mitochondria translocated to the is in a calcium-dependent way. the distance between mitochondria and the plasma membrane at the is was lower than nm. following accumulation at the is, mitochondria limited calcium entry to the orai channels localized right at the is by preventing their calcium-dependent inactivation. in contrast, no calcium influx was observed at sites where no mitochondria were accumulated as orai channels were inactivated at these sites. mitochondrial positioning at the is thus induced local calcium influx at the is without the necessity to enrich orai channels at the is. mitochondria took up calcium at the is distributing it further into the cytosol by releasing it at different sites, which kept the local domain at the is low enough to prevent calcium dependent orai inactivation and to prevent excessive calcium clearance by the calcium aptases in the plasma membrane, which could inhibit an efficient t cell activation. conclusion: mitochondria positioning at the is controls local calcium entry through orai channels. mitochondria prevent orai inactivation and excessive calcium clearance at the is to facilitate calcium-dependent t lymphocyte calcium signalling. we aimed to determine the functional correlates of cd + t cell tolerance and immunity in vivo. ovalbumin (ova)-specific transgenic cd + t cells were adoptively transferred into syngeneic mice immunized with soluble ova protein ± lipopolysaccharide (lps) by the i. v. route, and analyzed for a variety of immunological parameters over a period of days. under tolerogenic conditions (ova alone), cd + t cells showed substantial early activation, but their activation profile differed markedly, both in magnitude and quality (icos, - bb), from t cells activated by ova+lps. this difference in activation also translated into differing cd + t cell expansion and contraction kinetics in the early phase of the t cell response (days - ). in the late phase of the primary response (days - ), under immunizing conditions, the large majority of transgenic cd + t cells in the spleen developed into mature effectors with a prominent capacity to secrete il- , ifn-g, and il- a, and only few ova-specific foxp + regulatory t cells ( x %) were observed. germinal centers were prominent and ova-specific ig of all isotypes were generated. in contrast, under tolerizing conditions, antigen-specific cd + t cells failed to migrate into the b cell follicles, but production of ova-specific igm was nevertheless observed. in these animals, the proportion of splenic ova-specific regulatory t cells ( %) was substantially increased. on day , both groups of mice were re-challenged via the airways with ova+lps to functionally assess their immune status. in tolerized animals, the transgenic t cell population in the lung infiltrate was composed of ova-specific regulatory t cells ( %) or t cells with a reduced capacity to secrete effector cytokines. in contrast, in immunized animals, this population almost exclusively consisted of cd + effector t cells with a pronounced inflammatory cytokine profile (ifn-g, il- a). with this model we provide a comprehensive analysis of the many functional correlates of "immunity" versus "tolerance" to soluble protein antigen in vivo. we identify and characterize a number of the key players (cell surface molecules, cytokines, cell subsets) representing the decision between immunity and tolerance in the immune system. mast cells (mcs) are well-recognized as key effector cells in immunoglobulin e (ige) -associated immune responses and as prototypic regulators of innate immunity. the characteristics, importance, and molecular requirements for interactions between mast cells (mc) and cd t cells (tc) remain to be elucidated. using myelin/oligodendrocyte glycoprotein (mog), we demonstrated that mcs induce antigen-specific cd tc activation and proliferation. the antigen crosspresentation by mcs induces the secretion of interleukin- , interferon-g and macrophage inflammatory protein- by cd tc. in vivo evidence that mcs modulate t cell responses has been obtained so far in the murine experimental autoimmune encephalomyelitis (eae), the standard animal model for multiple sclerosis, in which both cd tc and mcs are now recognized as key players. one of the main central nervous system (cns) antigens recognized by autoreactive tc in eae is the myelin oligodendrocyte glycoprotein (mog). to investigate the in vivo-relevance of the identified mc-cd tc interactions, we have employed the eae as a model of organ specific autoimmune disease in wild type mice and mc-deficient w/w sh mice. wt and w/w sh mice were immunized with the mog - protein. our results provide direct evidence that mc contribute to cd -specific priming in eae and show that the tc proliferation failure is specific for cd tc from mog - -immunized w/w sh mice. the role of mc-cd tc interaction in induction of autoimmunity will be further investigated in eae. in summary, we provide the first evidence that mcs regulate antigen-specific responses of primary cd tc in vitro and in vivo. our study further supports the emerging concept that mcs, protagonists of innate immunity are also important regulators of adaptive immune responses and corresponding cd tc responses. this newly uncovered mc function might be of great biological relevance in situations where effector cd tc are critically involved, e. g. viral infections or infections with intracellular pathogens and/or autoimmune diseases such as multiple sclerosis. activation of resting t cells in vitro is triggered by combined t cell receptor (tcr) and cd engagement and can be modulated by simultaneous ligation of various other surface receptors. although the fasl is best known for its capacity to initiate cell death in fas-bearing cells, it has recently been implicated in the regulation of t cell activation. thus, a crosstalk between the tcr and fasl is likely, but far from being biochemically elucidated. we report that fasl engagement by immobilized but not soluble fasfc fusion protein and anti-fasl antibodies blocks the activation of primary human peripheral t cells even in the presence of cd costimulation at the level of an early signal initiation. inhibition is thus associated with a reduction of tyrosine phosphorylation of a number of key elements in tcr signal transduction and also with a lowered calcium response. the data presented stress the importance of the fas/fasl-system for signal initiation via the tcr/cd complex and provide further arguments for a retrograde signaling capacity of fasl or a crucial role of fas as a costimulatory molecule. golgi network (tgn). moreover, trim specifically associates with the cytoplasmic tail of ctla- , but not via any conventional motifs in this region. overexpression of trim augments ctla- surface expression, whereas down-regulation of trim expression by shrna results in disturbed ctla- localisation, mainly restricted to the tgn. ctla- vesicles and surface expression were significantly reduced but not abolished, suggesting that other factors are involved in ctla- trafficking. here, we identify additional transmembrane adapter protein (trap) family members as novel binding partners and regulators of ctla- expression. although there is some redundancy amongst traps, our results highlight the importance of this family of proteins in ctla- transport to the cell surface. it is imperative to reveal the mechanisms by which ctla- is transported to the cell surface, given that minor changes in expression can have major effects on t-cell function and in the development of autoimmunity. natural killer t (nkt) cells are found within the liver and are known to exhibit immune regulatory function. upon recognition of glycolipids presented on cd molecules, nkt cells are activated and release cytokines, including ifn-g, il- and il- . nkt cells are efficiently recruited to the liver via cxcr -dependent chemotaxis toward cxcl and constitute a large proportion of the liver-resident lymphocytes. we have previously shown, that liver sinusoidal endothelial cells (lsec) can scavenge circulating soluble antigens, and can cross-present these antigens to naive cd t cells. cross-presentation leads to initial t cell activation and expansion, but ultimately these cd t cells are rendered tolerant. as both naive t cells and nkt cells come into close contact with lsec in the hepatic sinusoids, we investigated whether nkt cells can modulate cd t cell tolerisation via interaction with lsec. to this end we analysed cd d expression on lsec and their ability to activate nkt cells by presentation of the cd d-binding glycolipid a-galactosylceramide (agalcer). we found that lsec express functional cd d, as agalcerpresenting-lsec were capable to induce tnf-a, il- , il- and ifn-g production in nkt cells in vitro. the interaction of agalcer-presenting-lsec with nkt cells led to the upregulation of cd and b -h on lsec. as naï ve cd t cell tolerisation by lsec critically depends on b -h , we hypothesise that hepatic nkt cell activity may contribute to the immunological capabilities of the liver by regulating the tolerogenic function of lsec. improved antibody responses by class-switched memory b cells require enhanced signaling from their antigen receptor (bcr). however all bcr classes on naïve and antigen-experienced b cells utilize the canonical iga/igb subunit for signaling. we identified the signal amplification mechanism of the igg-and ige-bcr. for these isotypes tyrosine-based signaling is not confined to iga/igb but extends to a conserved tyrosine residue in the cytoplasmic segments of immunoglobulin heavy chains. the phosphorylated immunoglobulin tail tyrosine recruits the adaptor grb in order to sustain protein kinase activation and generation of second messengers causing robust cellular proliferation. hence membrane-bound igg and ige not only recognize antigen but also exert bcr-intrinsic costimulation to render memory b cells less dependent on t cell help for activation. objectives: the majority of circulating human gd t cells harbor tcr containing vg , jg . , and vd gene products. they recognize nonpeptide antigens like (e)- hydroxy- -methylbut- -enyl pyrophosphate derived from pathogenic microbes and isopentenyl pyrophosphate (ipp) in malignant cells. recently, we and others found out that gd t cells express a variety of costimulatory molecules including icos, and pd- . one of the inhibitory receptors, pd- , is a member of cd /ctla- family and contains a single ig v-like domain in its extracellular region. pd- can bind to two b homologue molecules, pd-l and pd-l . it has been reported that interaction of pd- with its ligands resulted in peripheral immune regulation and tolerance in ab t cells. in this study, we show that pd- is expressed on activated human gd t cells and regulates the effctor functions of gd t cells. methods: peripheral blood mononuclear cells were resuspended in yssel's medium and stimulated with -methyl- -butenyl- -pyrophosphate plus il- to obtain gd t cells. pd-l + and pd-l -human tumor cell lines were established from cancer patients. in order to prepare anti-pd-l mabs, the pd-l extracellular domain was expressed in e.coli as inclusion bodies and refolded in the standard arginine-based buffer. mice were immunized with the refolded protein and mabs were established. to determine the function of pd- in gd t cells, we determined cytokine production and cell mediated tumor lysis by activated gd t cells in the presence of inhibitors of pd- /pd-l interaction. results: gd t cells expressed pd- upon simulation with nonpeptide antigens and many tumor cell lines expressed pd-l . we first examined whether or not the engagement of pd- receptor could modulate the cytotoxic activity of gd t cells. pd-l -expressing tumor cells tempered cytotoxic activity of pd- + gd t cells, and cytokine production such as tnf-a was down-regulated by pd- engagement. in addition, inclusion of anti-pd-l mab reversed cytotoxic activity and cytokine production when pd-l -expressing tumor cells were challenged by pd- -expressing gd t cells. conclusion: pd- delivers inhibitory signals in gd t cells upon engagement with pd-l . peripheral tolerance plays an important role in preventing t lymphocyte responses to self or harmless antigens. one of the mechanisms that contribute to this form of tolerance is anergy, which is characterized by a lack of proliferation and il- production by t cells in response to antigenic challenge. the acquisition of the anergic phenotype is an active process, with negative regulators of t cell signalling being induced. among these are the e ubiquitin-protein ligases which recognize target proteins for ubiquitination and catalyse the transfer of ubiquitin to them, directing them to the proteasome or to the endosome-lysosomal pathway, and hence downregulating their activity. the e ubiquitin-protein ligases cbl-b, itch and grail have been shown to be upregulated in anergy and to ubiquitinate and downregulate tcr signalling elements. our objectives were to determine the expression of cbl-b, itch and grail in antigen-specific cd + t cells in both the induction and maintenance phases of anergy, in vitro and in vivo, and to investigate their functional signalling role(s) in the maintenance of the tolerance phenotype. in order to accomplish these objectives we induced priming or tolerance of ovalbumin (ova - peptide)-specific t cells from do . tcr transgenic mice in vitro or, following adoptive transfer of near physiologically relevant numbers of such cells into recipients, in vivo and correlated functional outcome (via proliferation and cytokine readout assays or antibody production) with e ubiquitin-protein ligases expression and the ubiquitination status of the tcr signalling machinery. cbl-b, itch, grail and target ubiquitination status, in terms of tissue, cellular and subcellular protein expression, modification and localisation, were assessed by a combination of immunoprecipitation and western blotting studies. moreover, we have performed quantitative analysis at the single cell level by tracking such antigen-specific cells in vitro and in vivo by using laser scanning cytometry. our current work focuses on the functional consequences of adenoviral transfection of such antigen-specific t cells by mutant e ligase-, signal target-and ubiquitin-constructs. collectively, these approaches have facilitated the dissection of the potential differential roles of ubiquitin signalling in priming and tolerance of antigen-specific t cells. s. j. keppler , p. aichele immh, university freiburg, immunology, freiburg, germany interleukin (il- ) is produced by cells of the innate immune system during infection and plays an important role in controlling various pathogens. it was postulated recently that il- has a direct influence on cd + t cells in vitro, enhancing expansion and the development of effector functions as a third signal, additionally to tcr engagement (signal ) and costimulation (signal ). we analysed direct il- signaling to cd t - signaling exhibited normal degranulation activity, cytolytic functions, ifn-g and tnf-a production. however, cd t cells lacking il- signaling failed to up-regulate klrg and to down-regulate cd in the context of listeria but not viral infections. thus direct il- signaling to cd t cells determines the cell fate decision between short-lived effector cells (slecs) and memory precursor effector cells (mpecs), dependent on the pathogen-determined local cytokine milieu. cd + t lymphocytes are required for effective host defense against pathogens but also for mediating effector responses against uncontrolled proliferating self tissues. we could now reveal that individual cd + t cells are tightly controlled in their effector functions by cd (ctla- ). we demonstrate that signals induced by cd reduce the frequency of interferon-gamma (ifn-g) and granzymeb expressing cd + t cells. for this novel function cd specifically represses the transcription factor eomes, but not t-bet. a cd mediated induction of the inhibitory transcription factor ckrox has been ruled out. ectopic expression of eomes reversed cd -mediated inhibition of effector molecule production. additionally, enhanced cytotoxicity of individual cd + t cells differentiated in the absence of cd signaling could be demonstrated in vivo. the novel insights that cd -mediated signal transduction in vivo indeed alters cd + t cell cytotoxicity qualitatively at the single cell level and not only quantitatively by enhancing expansion extend the understanding how to selectively modulate immune responses of cd + t cells. objectives: atp constitutes a damage associated molecular pattern (damp) and contributes together with pathogen associated molecular patterns (pamp) to the efficient priming of the innate immune system. atp is a ubiquitous extracellular messenger, which activates plasma membrane receptors for extracellular nucleotides termed p receptors. p x - receptors open to non-selective ion channels, whereas p y , , , , - are g-protein coupled receptors, which bind preferentially adp, udp, utp or udp-glucose. as the role of p receptors in the control of b cell activation has been poorly investigated, aim of the present study is to understand better the mechanisms of intracellular atp production and release by human b cell subsets. methods: intracellular atp measurement has been performed using a bioluminescence assay while extracellular atp has been measured by hplc. storage and release of atp by b cells have been elucidated using confocal and tirf microscopy, to study vesicles distribution and dynamics near the plasma membrane. results: in both human naive and memory b cell we observed a prominent increase of atp synthesis upon tlr but not bcr stimulation. glycolytic pathway rather than oxidative phosphorilation was involved in atp synthesis. p x antagonists inhibited both proliferation and differentiation to plasma cells of human b cells thus suggesting that atp is released in the pericellular space. labelling of resting and activated human memory b cells with quinacrine, a nucleotide binding component, revealed a typical vesicular pattern of atp, confirmed with subcellular fractionation on sucrose equilibrium gradients. tirf imaging showed a fluorescently labelled vescicle underwent fusion with the plasma membrane after stimulation with anti-ig and this event was ca( +)-dependent. conclusion: these data provide evidence that atp is produced by b cell preferentially by glycolytic pathway and vesicular exocytosis is a key mediator of atp release in human b cells. atp released in the pericellular space might act as an autocrine and paracrine signalling molecule that regulates the functions of b cells. o. ballek , a. brouckova , d. filipp institute of molecular genetics as cr, laboratory of immunobiology, prague, czech republic two src family tyrosine kinases lck and fyn are critical for the proximal t-cell signaling. we have previously demonstrated that induced lck activation outside lipid rafts (lr) results in lck translocation to lr. central in this sequence of events is the rapid translocation of kinase active lck to lr, yet the mechanism underpinning this process is unknown. the main aim of this study is the characterization of molecular mechanisms and its functional elements regulating the early recruitment of signaling molecules to lr and forming immunological synapse. we have recently characterized the c-terminal yqpqp sequence as a novel cis-acting component essential for partitioning of lck to lr. here we report that the expression of the c-terminal truncate of constitutively active lck ( ¿ fqpqp) in nih t cells failed to phosphorylate several proteins detected in the presence of untruncated kinase active y flck. comparative -d gel analyses followed by ms/maldi identified rack as a candidate protein for interaction with the c-terminal tail of lck. co-expression in nih t cells of ha-tagged rack with either a wild type lck or constitutively active y flck revealed a significantly enhanced complex formation between y flck and rack compared to that of wtlck. ectopic expression of y flck with its domain-inactivating mutations showed that lck-rack interaction depends on functional sh , sh and the c-terminal tail sequence of lck. lck-rack interaction is readily detectable also in primary cd + lymph node t cells. upon their activation, only the pool of lck molecules associated with high molecular weight complexes can translocate to lipid rafts. co-purification of rack with these fractions further suggests that it plays a role in the translocation of lck to lr. in addition, lck and rack co-redistribute to both forming immunological synapse and to antibody-mediated capping clusters. moreover, the importance of interaction between activated lck and rack in the context of lck translocation to lr is further strengthen by the observation that rack is associated with elements of cytoskeleton. these results are the first to characterize rack as a candidate molecule involved in the regulation of lck translocation to lr through linking the c-terminal sequence of lck to cytoskeletal network. human b cells are currently not known to produce the pro-apoptotic protease granzyme b (grb) in physiological settings. we have discovered that b cell receptor stimulation with either viral antigens or activating antibodies in the context of the acute phase cytokine interleukin (il- ) can induce secretion of substantial amounts of grb by human b cells. grb response to viral antigens was significantly stronger in b cells from subjects recently vaccinated against the corresponding virus as compared to unvaccinated subjects. both, naï ve and memory b cells differentiated into grb-secreting cells, which featured a homogeneous cd +cd +cd -cd -igd-phenotype, improved survival and enhanced expression of co-stimulatory, antigen-presenting and cell-adhesion molecules. b cellderived grb was enzymatically active and its induction required activation of similar signaling pathways as in cytotoxic t cells. our findings suggest grb-secreting b cells play a role in early anti-viral immune responses, thereby contributing to the elevated serum grb levels found in various viral diseases. further studies will elucidate whether b cell-derived grb induces cytotoxicity towards virus-infected cells or exhibits other functions. results: transfer of otii cells augments the wild type th response to alumova inducing large early germinal centres and massive plasma cell formation with more than % of these switching to igg . the plasma cells up-regulate cxcr , but not cxcr , a chemokine receptor that attracts plasma cells to inflammatory sites. oti cells respond to alumova by producing ifng, a th -associated cytokine. when both oti and otii cells are transferred switching is diversified with plasma cells being igm (˚ %), igg a (˚ %), igg b (˚ %) or igg (˚ %). in addition to cxcr , some % of these plasma cells strongly express cxcr . the induction of cxcr in these plasma cells correlates with their increased expression of the transcription factor t-bet, which has been linked with igg a switching during th responses. this is functionally significant for oti-dependent cxcr expression, as well as induction of switching to igg a, are dependent on t-bet expression by the responding b cells, although t-bet-deficient b cells still switch to igg b. t-bet is known to be induced in b cells exposed to ifng or tlr stimulation. these two hypothetical mechanisms are currently being tested in mice injected with blocking anti-ifng antibodies or mice deficient in myd . objective: a successful immune response against malaria has to be tightly controlled. the early production of pro-inflammatory cytokines is required to control the growth of intraerythrocytic parasites but the same cytokines are also involved in the induction of severe malaria in both humans and mice. activation of t lymphocytes through tcr signalling takes place within the context of numerous other cell surface protein interactions. to prevent unnecessary activation of t cells the immune system has developed an intricate balance between positive and negative costimulatory signals. costimulatory signals determine whether antigen recognition by t lymphocytes leads to full activation or to anergy. in contrast negative costimulators expressed by t cells seem to mediate the regulation of immune responses and thus play a pivotal role in the maintenance of peripheral tolerance. recently, btla (b and t lymphocyte attenuator, cd ) was described as a novel negative costimulatory receptor. btla is predominantly expressed on t and b cells and dampens t cell activation. in this study, we analyzed the function of btla during experimental malaria infection. to study the function of btla we employed a mouse model of blood-stage malaria using p. yoelii nl infection of btla-deficient and hvem-deficient mice. p. yoelii provokes a high parasitemia in infected mice that is cleared within three weeks from time of infection. immunity in this model depends on cd + t cells and hence the role of negative costimulators that modulate t cell function can be studied using this model. results: peak parasitemia of p.yoelii-infected btla-deficient and hvem-deficient mice was much lower compared to wild type mice. the increased immunity of btla-deficient mice depends largely on cd + t cells. we found that btla::hvem interaction regulates the size and the cytokine-production of the responding t cell pool. however, in contrast to the ctla- pathway, the manipulation of btla::hvem interaction triggers no pathology during infection. the hvem::btla interaction dampens the protective immune response during experimental malaria and thus manipulation of this pathway is an attractive target for therapeutic interventions. . so far, the contribution of actin regulatory proteins to this process remained largely unknown. here we demonstrate that the actin-bundling protein l-plastin is indispensable for segregation of lfa- and cd in the psmac of untransformed human t-cells. in marked contrast, tcr/cd accumulation in the csmac is not dependent on l-plastin. the relocalization of l-plastin in the immune synapse occurs within seconds of t-cell/apc contact formation and relies on actin polymerization. importantly, binding of calmodulin to l-plastin is required for the maintenance of l-plastin in the immune synapse and inhibition of calmodulin prevents psmac formation. thus, receptor segregation in the immune synapse is a consequence of the combined activities of the actin-bundling protein l-plastin and calmodulin. protective t cell responses are based on expansion and persistence of clones with optimal affinity for antigen. presently, it is unknown which mechanisms guard the selection and expansion of the highest affinity clones from the very diverse naï ve pool. rapid cell division creates shifts in selective pressure, which is a basic biological prerequisite for elimination. therefore we hypothesized that apoptosis might play an important role during this phase of t-cell biology. here we show that the balance between the pro-apoptotic protein noxa and its antagonist mcl- regulates interclonal t cell competition during acute and chronic immune activation. we found p -independent noxa gene induction and mcl- downregulation upon t cell activation. concomitant we observed the release of death-inducing factor bim from mcl- , which was delayed in noxa -/cells. using ot- cells and altered peptide ligands we observed that the level of mcl- downregulation in activated t-cells depended on the antigen affinity of the t-cell receptor. since mcl- -/mice are embryonic lethal, noxa -/mice were used to study the functional implications of this mechanism in vivo. at a young age noxa -/mice have a normal lymphoid compartment, but accumulate effector t cells over time. upon acute influenza infection, normal levels of effector cells were generated. however, the quality of the antiviral (np ) response was impaired in these mice as many subdominant clones persisted in the effector t-cell population of noxa -/mice at the peak of infection. this increased diversity correlated with exacerbated pathology and a reduced rate of viral clearance. in a model of chronic immune activation, effector t cells rapidly accumulated in the noxa -/mice and infiltrated the peripheral organs, culminating in severe multi-organ pathology and premature death. these results establish a novel role for the noxa/mcl- axis during immune responses and suggest that the formation of a high-affinity effector population of restricted clonal diversity depends on a darwinian selection of t-cells during the expansion phase based on antigen affinity, with survival of only the fittest clones. cytotoxic t lymphocytes (ctls) are essential for immunosurveillance, a process that requires the presentation of virus-or tumor derived antigenic peptides in context of antigen presenting cells. insight into intracellular mechanisms facilitating lytic granule release and formation of the immunological synapse as a prerequisite for target cell destruction was primarily obtained from loss-of-function mutations in hereditary human diseases and gene-mutated mice. here, we refer to estrogen receptor-binding fragment-associated antigen (ebag ) as a negative regulator of secretory lysosome release. in gene deleted mice we show that loss of ebag confers ctls with enhanced cytolytic capacity, in vitro and in vivo. here, we show that ebag , which was previously identified as a snapin-interacting protein in neuronal cells, interacted with the adaptor molecule g -adaptin in t cells. both interactions suggested an involvement of ebag in endosome-lysosome related organelle biogenesis and membrane fusion. efficiency of granzyme b sorting towards secretory lysosomes was improved, which was consistent with the enhanced kinetics of cathepsin d proteolytic processing. while the formation of the immunological synapse remained unaffected in ebag -/-ctl, relative size distribution of lytic granules revealed a shift towards smaller granule diameters and volumes in ebag -deficient ctls. these data imply a role for ebag in regulating the formation of mature ctl granules and identify ebag as a tunable inhibitor of ctl-mediated adaptive immune response functions. finally, ebag defines a novel negative regulator of secretory lysosome release in ctls. thus, the elucidation of the ebag -related pathway might provide a fresh impuls on therapeutic approaches in the treatment of autoimmune disorders. the liver is known to induce tolerance, rather than immunity, through tolerogenic antigen presentation or elimination of effector t cells. recently, we could show that liver sinusoidal endothelial cells (lsec) inhibit activation of naive cd t cells by antigen-presenting dc. this regulatory effect of lsec on dc function was mhc-independent and not limited to soluble mediators, but required physical contact. interestingly, interaction with lsec led to reduced dc expression levels of cd / and il- . in addition to indirect inhibition of t cell activation by de-licensing of dc, we now detected another influence of lsec in form of direct inhibition of t cell priming. in the presence of lsec, stimulation with acd /acd or pma/ionomycin could not significantly activate cd and cd t cells. thus, lsec did not only inhibit t cell priming triggered by tcr activation but also after elicitation of ca + influx into the cytoplasm. furthermore, we found that ifn-g secreted by t cells in the early phase of activation is crucial for licensing the inhibitory function of lsec. taken together, these data indicate that the inhibitory effect of lsec is mediated by a machinery induced at the early phase of t cell activation, however, interferes with late events in the t cell activation cascade. we propose a model of "inducible inhibition", where on the one hand naive t cell priming is directly inhibited by lsec, and on the other hand tolerogenic priming by antigen-presenting lsec is still allowed. taken together, these results reveal a novel principle, operative in hepatic tolerance induction, in which lsec not only tolerize t cells themselves, but also inhibit the responsiveness to local activation stimuli. m. almena , s. carrasco , i. merida centro nacional de biotecnología csic, inmunología y oncología, madrid, spain self-tolerance acquisition is essential for the immune system to control its own response. t cells achieve self-tolerance trough thymic selection and anergy, two processes where rasgrp -ras-erk signal intensity is critical to determine the final cell outcome. rasgrp is a gef for ras that is activated in a diacylglycerol (dag)dependent manner. dag is generated by plcg after tcr stimulation and is consumed by diacylglycerol kinases (dgk). dag generation, as a result of the concerted regulation of these two enzymes, activates ras, providing a mechanism to translate the strength of the stimulus into a quantitative cell response. . analyze the impact that dag metabolism plays in t cell tolerance in vivo, using transgenic mice where dag generation is impaired. . develop a method to sense dag production and localization, in both thymocytes and peripheral t cells, and its correlation with the strength of the stimulus used. methods: we generated transgenic mice expressing a constitutively active dgk in t cell lineage. this protein was anchored to the plasma membrane, thus diminishing the lipid levels in this specific location after tcr stimulation. ot-i cd cells expressing gfp-c domains were used with peptide-pulsed apcs to study dag generation and dynamics by confocal microscopy. results: transgene expression was obtained in thymic and peripheral t cells. no major defects were observed in t cell subsets but analysis of peripheral t cells demonstrated important defects in t cell activation. we are currently studying thymic selection breeding our transgenic with h-y mice, in order to check if t cell populations are being properly selected. using t cell-apc conjugates with peptides with different tcr binding affinities we found a clear correlation between the strength of the stimulus, dag production and ras-mapk activation. conclusion: our data demonstrate that dag generation not only activates c domain containing proteins but regulates a mechanism by which t cells sense the magnitude of the stimulus received, translating it into the intensity of the generated response, a process essential in t tolerance. future experiments will help to define the exact contribution of the lipid to tcr signaling pathways and to t cell homeostasis. the inducible costimulator (icos, h , cd ), a cd -like costimulatory molecule, has an important role in the development of efficient t cell responses. early data showed that icos costimulation produced th biased responses and high production of the anti-inflammatory cytokine il- , and was essential to the development of germinal centres. however, icos can also help in the il- -dependent differentiation of inflammatory th cells. these different functions could be due to differences in the apcs bound by icos-expressing t cells and/or because of the intervention of distinct molecules binding the cytoplasmic domain of icos. icos shares with cd a yxxm cytoplasmic motif that can bind, upon tyr phosphorylation, the regulatory p subunit of class ia pi- kinases. these can complex with one of the three kda catalytic subunits (p a, p b, and p d) expressed by leukocytes that generate pip affecting cell growth, cell cycle progression, survival, intracellular traffic, cytoskeletal changes and migration. there is also evidence that the regulatory and catalytic class ia pi k isoforms fulfill specific functions in macrophages and lymphocytes. we have used proteomic and immunochemical approaches to identify molecules binding the phosphorylated or unphosphorylated cytoplasmic domain of icos, and particularly the presence of distinct pi kinase isoforms. then, the functional importance of these molecules has been analyzed by using pharmacological inhibitors specific for downstream mediators of icos activation. pull down of t cell lysates using phosphorylated or unphosphorylated synthetic peptides covering the cytoplasmic domain of icos was carried out. proteomic and immunoblot analysis of bound proteins showed that phosphorylated icos bound the different pi -k regulatory (p a, p b, p a) and catalytic (p a, p b, and p d) pi -kinase subunits expressed by leukocytes. these data were confirmed in icos immunoprecipitates from pervanadate-activated cells. icos bound regulatory and catalytic subunits in the order p a g p a g g p b and p a g p d g g p b, in agreement with quantitative rt-pcr and immunochemical estimation of subunit abundance in the t cells and t cell lines used. the use of specific pi -kinase inhibitors has confirmed the relative importance of the catalytic isoforms in icos function, including reorganization of actin cytoskeleton induced by icos ligands, or costimulation of tcr/cd -induced secretion of il- and il- . during the process of antigen recognition between t-cell and antigen-presenting cell (apc), structural and spatial changes take place at the cell-cell contact, where the molecules involved in the formation of the immune synapse (is) reorganize, displaying a segregated localization. in this context, the translocation of the microtubule-organizing center (mtoc) is an early event that occurs during the formation of the is, bringing with it the golgi apparatus, thus providing the basis for a polarized secretion. however, the molecular mechanisms involve in the localization of the mtoc at the contact area between the t cell and the apc are not completely understood yet. we have studied the possible role of scaffolding protein akap , a member of the a-kinase anchoring protein (akap) family that localizes at the centrosome and interacts with pka regulatory subunit and other signalling molecules, in mtoc polarization and immune synapse formation. either the overexpression of gfptagged c-terminal cg-nap/akap construct that acts as a dominant negative, or sirna knockdown of endogenous akap expression in t cells prevents the correct organization of cd z and pkcv to the is and mtoc reorientation towards t cell-apc contact area in antigen and superantigen-dependent human models, resulting in a disorganized is; lfa- localization was also analyzed to assess p-smac architecture and, interestingly, confocal d reconstruction revealed that lfa- ring was not clear in the akap -disrupted cells. moreover, akap was required for tcr signalling since the knock down with specific sirna and overexpression of c-terminal of akap decrease the phosphorylation of molecules such as lat, plcg and pkcv. these defective activation events as reflected in a reduction of il- production. together, our results underscore a key role for akap in the organization of the immune synapse and in the antigen-specific reorientation of the mtoc. the tcrbeta/ptalpha pre-tcr complex signals the expansion and differentiation of developing thymocytes. functional properties of the pre-tcr rely on its unique ptalpha chain, which suggests the participation of specific intracellular adaptors. in fact, we have recently identified cms, a member of the cin /cms family of adaptors, as a ptalpha-binding protein that specifically interacted with the human ptalpha cytoplasmic domain via its sh domains, and to the actin cytoskeleton via its c-terminal region. we found that cms co-localized with polymerized actin in pre-tcr clusters at the pre-tcr activation site, and also in the ptalpha endocytic compartment. since actin polymerization plays a critical role in regulating signalling through the alpha/beta tcr in mature t cells, we decided to investigate the potential function of cms as a regulator of actin polymerization and pre-tcr signalling in pre-t cells. using pre-t cells expressing a mutant pre-tcr lacking the cms-binding motif in the ptalpha tail and short hairpin irna-based gene silencing, we demonstrate that binding of cms to ptalpha contributes to cytoskeleton dynamics and pre-tcr-mediated signalling in human pre-t cells. cms-deficient cells specifically showed defects in pre-tcr-induced ca + mobilization and cell activation involving the pi k, nfat, plcg and erk signalling pathways, together with defects in actin polymerization and cell motility. cms therefore links cytoskeleton dynamics with the function of discrete pre-tcr signalling components, suggesting the functional implication of cms in human t-cell development in vivo. abstract withdrawn by author j objectives: most signaling pathways engaged after bcr activation have been described. however, several negative regulators of these pathways are unknown. the characterization of these regulators is important to understand the control of transduction pathways in adaptative immunity. carabin (tbc d c) has been recently described as a negative regulator of tcr signaling. it interacts with calcineurin and inhibits the formation of calcineurin/calmodulin complex, blocking nfat nuclear transport. moreover, carabin maintains ras protein under an inactive form, thus inhibiting ras-mapk cascade. expression of carabin is finely regulated following tcr signaling, and its knockdown (kd) enhances t cell activation. considering the important molecular similarities of antigen receptor signaling pathways in t and b cells, we studied the role of carabin in b cell. could carabin play a role of negative regulator of b cell function? methods: we studied by quantitative rt-pcr ) the expression of carabin in different purified subsets of bone marrow and splenic mouse b cells, as well as ) the kinetic of expression of carabin in bcr stimulated murine splenic mature b cells. ) we then studied the phenotype of carabin kd (shrna expressing) a b cells after bcr stimulation. ) the expression of carabin is significant in murine b cells, with an increase during b cell development, from bone marrow pro/preb to immature, to splenic t tot b cells and to follicular mature b cells. ) the kinetic of expression of carabin in bcr stimulated murine mature b cells suggests a fine regulation of carabin expression. ) bcr simulation, but not lps stimulation, of carabin kd a b cells shows an acceleration of ras target erk / phosphorylation, without any for the phosphorylation of mapk jnk, which is not targeted by ras. conclusion: carabin is expressed in murine b cells in a developmental regulated manner, with the highest expression in mature compartment. bcr stimulation leads to a fine regulation of carabin expression in wild-type mature b cells, and to a faster activation of erk / pathway in carabin kd b cells. altogether, these results strongly suggest a role of carabin as a negative regulator of b cell function toll like receptors are pattern recognition receptors, which recognize invariant pathogen associated molecular patterns. toll like receptor (tlr ) binds doublestranded rna, a nucleic acid frequently associated with viral replication. we observed that freshly isolated human cd + t cells express tlr and respond to the well characterized synthetic tlr ligand polyinosinic-polycytidylic acid [poly(i:c)]. the expression of activation markers and cytokine production by cd + t cells upon t cell receptor (tcr) stimulation is enhanced in response to co-stimulation via tlr . tlr stimulation on its own had no effect on expression of activation markers and cytokine production. to elicit the molecular basis of a potential cross-talk between tcr and poly(i:c) induced signaling, we used jurkat cells to perform luciferase assays. we observed that costimulation with poly(i:c) in comparison to tcr stimulation alone enhanced nf-kb but not nfat activation in jurkat cells. similarly to jurkat cells, tcr stimulation activated nf-kb in primary cd + t cells. this effect was further enhanced by additional poly(i:c) stimulation as shown by real-time-pcr and western blot analysis. on the other hand, we observed that poly(i:c) stimulation on its own activated the transcription factor interferon regulatory factor (irf ) as revealed by realtime-rcr analysis of ifn b and irf , whose transcription depends on the activity of irf . combined tcr and poly(i:c) stimulation further enhanced the transcription of these two genes. these results indicate that tlr signaling modulates tcr-driven responses and vice versa both in jurkat cells and in freshly isolated cd + t cells. this study was supported by dfg spp "innate immunity" (ka / - ). the initiation of protective t cell responses requires the recognition of mhc-bound peptides from pathogen or tumor antigens by the t cell receptor (tcr). how this signal is transmitted across the t cell membrane to the cytoplasmic signaling motifs is still unknown, and is the focus of this project. in textbooks, the cytoplasmic domains are depicted as flexible chains in the cytoplasm, but biochemical studies show that the cd e cytoplasmic domain (cd e cd ) binds to synthetic lipid vesicles that contain acidic phospholipids. this binding is predominantly due to electrostatic interactions between basic residues of cd e cd and acidic phospholipids. in the cell, acidic phospholipids are enriched in the inner leaflet of the plasma membrane. phosphatidylserine in particular is concentrated on the inner leaflet of the plasma membrane due to active transport mechanisms, explaining how such charge-charge interactions are generated. to study the interaction of the cd e cd with the membrane in live cells, we have developed a fluorescence resonance transfer (fret) assay which measures the proximity between a fluorescent protein (tfp) attached to the c-terminus of cd e cd and a fluorescent membrane dye (r ). with this assay, we show that the cd e cd is membrane-bound in resting cells and that binding is abrogated by introduction of mutations that disrupt lipid binding in the biochemical assay. additionally, in vitro analysis confirm functional domains for cd e cd lipid binding and conformational change. finally, nmr spectroscopy analysis reveals key features in membrane binding dynamics of cd e cd to lipid bicells. membrane binding by the cd e cd could thus be subject to dynamic regulation during the engagement of the tcr and further activation of the t cell. m. xydia , y. ge , u. quitsch , p. beckhove german cancer research center (dkfz), heidelberg, germany in peripheral tissues and the factors affecting their proliferation. cd + t cell help is believed to contribute to optimal cd + memory expansion via cd l on cd + t cells binding cd on dendritic cells. however, a few reports suggest that cd l-cd engagement may mediate direct cell-cell contacts between cd + and cd + t cells. in this study, we investigated the importance of cd -cd co-operation and cd l-cd interactions for t em proliferation. methods: we isolated human cd + and cd + t em cells from peripheral blood of healthy donors by facs or macs sorting. separated or mixed cd + and cd + populations were activated in vitro using anti-cd /cd beads. proliferation was measured by [ h]-thymidine incorporation, in some experiments after irradiation of one t em subset and/or incubation with blocking mabs against cd or cd l. furthermore, facs staining was used to assess cell-surface markers. statistical comparison was performed by student's t test. results: upon activation mixed t em populations showed a highly better proliferative response than separated cd + or cd + t em cells, demonstrating that optimal t em expansion requires direct cd -cd interactions. surprisingly, not only cd + but also cd + t em cells proliferated much more in mixed populations compared to the separated ones, indicating that optimal cd + t em proliferation depends on signals from cd + t em cells. activation induced the expression of cd on both populations and cd l on subsets of cd + and cd + t cells. blocking of cd l on cd + t em cells impaired significantly cd + t em proliferation, which confirms that the improved expansive potential of cd + t em cells in mixed populations depends on cd l co-stimulation by the cd t em subset. conclusions: our data demonstrate for the first time that activated cd + t em cells deliver help to the cd + t em subset via cd l-cd signalling and may play an important role for cd + t em expansion upon stimulation. the t cell surface glycoprotein cd , a member of the scavenger receptor cysteine-rich (srcr) family of proteins, targets to the immunological synapse upon t cell binding to antigen presenting cells (apc). however, it has not been established whether this translocation is due to the binding of a ligand expressed in the apc, or to intracellular interactions with signaling molecules or components of the cytoskeleton, that may control cd localization upon t cell:apc conjugation. we have questioned which domains of cd mediate the localization within the is, and for this we have expressed cd mutants as gfp fusion proteins in human t lymphocytes. we have also used jurkat cell lines expressing different cd mutants. t cells were incubated with superantigen-loaded raji b cells, and following the establishment of stable interactions between the cells, we analyzed the localization of cd by immunofluorescence and confocal microscopy. interestingly, our results show that the translocation of cd depends on sequences within the cytoplasmic domain, as a cd deletion mutant lacking most of the cytoplasmic tail, cd .k stop , is randomly distributed through the whole cellular surface, even in sustained t-apc interactions. the cytoplasmic domain relevant to cd translocation was mapped within amino acids glu and his since the cd .h stop mutant, just short of aa is still able to translocate to the is, whereas cd .e stop , that lacks important tyrosine residues, is no longer transported to the is upon t cell: apc interactions. although these studies do not exclude a role for the extracellular domain binding to an elusive apc-expressed ligand, they suggest that a major mechanism of regulation of cd translocation is dependent on molecular association of a short stretch of its cytoplasmic region (glu -his ) to intracellular signaling effectors. however, in hodgkin's lymphoma (hl) ebna- is missing, but lmp- is still expressed. using a hl derived cell line, we have shown that the cytokine il- can induce lmp- expression in vitro and can replace ebna- . we have investigated the molecular events for this mechanism. stat proteins bind to the palindromic ttc(n) x gaa sequence, where × is , or . a high affinity stat binding site is spaced by nucleotides. we found three potential stat binding sites in the lmp- promoter, which we named lrs, tr and edl . they were spaced by , and nucleotides, respectively. electrophoretic mobility shift (emsa) experiments were performed with nuclear extracts prepared from il- -treated or non-treated kmh -ebv cells. dna binding activity was analyzed using a double stranded oligonucleotide corresponding to the germline (gl) epsilon promoter, which is known to contain a high affinity stat binding site, or lrs-stat . a stat complex binding to the gl-epsilon promoter and lrs-stat was induced by il- . the specificity of the stat complex was shown by supershift experiments with anti-stat , but not anti-stat antibodies. when gl-epsilon or lrs-stat was used as cold competitors in a -fold excess, both unlabelled probes could compete out the labeled probe, providing evidence that the lrs-stat contains a functional stat binding site. oligonucleotides, corresponding to lrs in which the stat site had been mutated, could not compete for stat binding. interestingly, the unlabeled lrs-tr with nucleotides as spacer could also function as competitor. however, when ttc/gaa palindrom was spaced by nucleotides (lrs-edl ), it could not compete. thus, expression the transforming protein lmp- can be induced directly by the t cell derived cytokine il- in a stat dependent manner. it is likely that this mechanism operates in vivo as well and determines expression of the ebv encoded protein lmp- and thus the pathogenesis of ebv carrying hls. established knockout/ knockin mice with a fasl deletion mutant that lacks the intracellular portion (fasl ¿ intra). co-culture experiments confirmed that the truncated fasl protein is still capable of inducing apoptosis in fas-sensitive cells. preliminary immune histochemistry data suggest that, in contrast to published data, the absence of the intracellular fasl domain does not alter the intracellular fasl localization in activated t cells. we are currently investigating signalling and proliferative capacity of b-and t-cells derived from homozygous fasl ¿ intra mice. our data point to a rather inhibitory role of fasl reverse signaling during immune responses. during an immune response numerous receptor-mediated signals delivered to t cells direct their proliferation, survival and differentiation. we are using a quantitative model and in vitro methods to assess the "calculus", or decision-making algorithms t cells use to process these multiple signals. previous experiments with ot-i cd t cells revealed that tcr affinity regulated both the frequency of cells responding and the average time taken for cells to reach their first division (ji (ji . : . furthermore, affinity was the sole regulator of the rate of cell death in subsequent divisions. here we examine the same question for cd t cells. again we find that lower affinity peptides stimulate t cells to divide rapidly, however, a high proportion of cells die within each division round, revealing an important potential mechanism for affinity maturation and selection of dominant clones over time. in contrast varying the number of dendritic cells used to stimulate cd + t cells primarily affect the proportion of cd + t cells going into division rather than affecting division time or cell death in subsequent divisions. currently we are using these quantitative methods to measure the effect of cytokines and co-stimulatory molecules cd , cd and cd on parameters of cd + t cell proliferation to inform quantitative models of the immune response under different conditions. our goal is to develop quantitative models of t cell behaviour that can accommodate information at the molecular, cellular and population level. interaction between cd , a member of the tumor necrosis factor receptor superfamily constitutively expressed on antigen-presenting cell as b cells, and cd l, a member of the tumor necrosis factor family transiently expressed on activated t cells, are essential for the development of humoral adaptative immune response. various studies have shown that dual stimulation of b cell through antigen binding on bcr and cd leads to an enhancement of ig and cytokine production. the current dogma postulates that these signals are necessary and sufficient to drive naive b cell proliferation and differentiation to ig secreting plasma cells. however, recent evidence suggests that the innate immune responses could regulate humoral adaptive immune response. indeed, b cells can be activated through engagement of a variety of innate immune receptors, including toll-like receptors (tlrs). soluble cd l is unable to induce murine b cell proliferation. however, we and others have shown that recombinant mouse cd l (rmcd l) can increase proliferation induced by tlr (poly ic) and tlr (lps) agonists. by contrast, we never observed any synergy between rmcd l and tlr / (pam csk ) or tlr / (pam csk ) agonists. to go further in the study of cd l/tlr agonist synergetic effect, we have developed trimeric synthetic molecule to mimick cd l, named mini-cd ls, based on a c -symmetry core holding cd -binding motif lys-gly-tyr-tyr. in surface plasmon resonance experiments, mini-cd ls bind to immobilized human cd and compete with the binding of cd l homotrimers and diplayed effector functions that matched those of the much larger recombinant cd l homotrimers as maturation of mouse dendritic cells and activation of in vivo immune response in a mouse model of trypanosoma cruzi infection. as soluble cd l, mini-cd ls synergize tlr (lps), tlr (poly ic) and tlr / (r ) agonist-induced murine b cell proliferation but no synergy was observed between mini-cd ls and tlr / (pam csk ), tlr / (pam csk ) and tlr (odn ) agonists. synergy between cd l and tlr agonist provide the ground to use such a combination as adjuvant in vaccination strategy. however, to reach this goal, evaluation of cd l/tlr combinations on murine and human b cell activation and differentiation in antibody producing cells are under investigation. interaction of naïve cd + t cells with immature dendritic cells (idc) expressing self-peptides can result in their abortive activation (aa), which leads to the induction of cd + t cell tolerance. we have defined a phenotypic profile for cd + t cells undergoing such aa. these cells undergo limited proliferation which is associated with lack of ifn-g production, low cell surface expression of cd and cd , and high levels of expression of cd l and ly c. whereas, cd + t cells undergoing productive activation (pa), following encounter with mature dc, form effector ctl which is evidenced by extensive t cell proliferation, high levels of ifn-g, cd and cd , and loss of cd l and ly c expression. ly c is a gpi-anchored cell surface glycoprotein expressed on cells of hemopoietic origin: however, its role in peripheral tolerance induction is not understood. in this study, we show that mab-blocking of ly c in vivo and in vitro results in pa rather than aa. we hypothesize that the interaction of ly c, expressed on naïve cd + t cells, with its ligand on idcs, may be vital in controlling the induction of peripheral tolerance amongst self-reactive cd + t cells. objectives: organophosphorus compounds (opcs) are commonly used in the manufacture of insecticides and pesticides. exposure to opcs is associated with neurological toxicity but the effect on the immune system remains ill-defined. in this study, we used a subchronic exposure model to investigate the effect of the organophosphorus compound, paraoxon, on the murine immune system. methods: balb/c mice were injected i. p. daily with saline (control group) or paraoxon (experimental group) for weeks. during the treatment, animals were weighed and blood was collected weekly for determination of acetylcholinesterase activity in red blood cells. at the end of treatment, mice were sacrificed and spleen cells analyzed by flow cytometry. spleen cells were also cultured in the presence or absence of mitogens and supernatants were analyzed for cytokine content by elisa. for in vivo survival studies, mice were treated as described above and then orally infected with a virulent strain of s. typhimurium. animal survival was followed for up to days after infection. results: daily injection of paraoxon induced g % reduction in acetylcholinesterase activity by the end of the first week of treatment, a level which was thereafter maintained during the remaining weeks of treatment. mice exposed to paraoxon exhibited g % reduction in the rate of body weight gain over the treatment period in comparison with control group. at the end of treatment, ex vivo analysis of spleen cellularity and function revealed no significant differences between control and experimental groups. to analyze the status of the immune system in vivo, mice were infected with a lethal dose of a pathogenic strain of s. typhimurium and followed for survival. unexpectedly, paraoxon-treated mice exhibited a significant degree of resistance with % of mice surviving the infection compared to % in control group. protection in paraoxon-treated group was dependent on the reduced acetylcholinesterase activity as it was abrogated by coadministration of a reactivator of cholinesterase. conclusion: our data demonstrate that a reduction in the level of acetyl cholinesterase rendered mice more resistant to a virulent infection. this suggests a hitherto novel function of the neurotransmitter acetycholine in modulating the immune response to infection. t cell-dependent (td) and t cell-independent (ti) igg autoantibodies have been described in the context of the autoimmune disease systemic lupus erythematosus (sle). however, their different roles in autoimmunity are unknown. here we show that ti antigens induce anti-inflammatory igg antibodies and protect from antigen-specific immune pathology. administration of antigen-specific anti-inflammatory igg antibodies was sufficient to mediate this effect independent of the igg inhibitory receptor fcgammariib. ti but not td igg autoantibodies were further associated with inhibition of pro-inflammatory th and th cells and disease in mice deficient for fcgammariib, a spontaneous model for sle. the data suggest a novel immune regulatory function for ti immune responses through the generation of anti-inflammatory igg antibodies. objective: class i phosphoinositide -kinases (pi k) constitute a family of enzymes that generate -phosphorylated polyphosphoinositides at the cell membrane after stimulation of protein tyrosine (tyr) kinase-associated receptors or g protein-coupled receptors (gpcr). the class i pi k are divided into two types: class ia p /p heterodimers, which are activated by tyr kinases, and the class ib p g (p gamma) isoform, which is activated by gpcr. although the t cell receptor (tcr) is a tyr kinase-associated receptor, previous studies showed that p g deletion affects tcr-induced t cell stimulation. mice lacking p g show a partial defect in t cell differentiation, activation and survival. p g participates in signaling pathways that regulate pre-tcr dependent differentiation and cd +/cd + t cell lineage commitment. in the mrl/lpr mouse model of systemic lupus erythematosus, administration of a pi kg-specific inhibitor causes a reduction in the number of cd + memory t cells that mediate renal injury. similarly, pi kg deletion in p pi k transgenic mice also reduces the numbers of cd + memory t cells. there is therefore evidence that pi kg has an important function in tcr-mediated t cell activation, although the mechanism by which pi kg regulates this process is not well understood. we studied the specific role of p g in t cell activation. methods: we studied whether the tcr activates p g and the consequences of interfering with p g expression or function on t cell activation. results: we found that after tcr engagement, p g interacts with and forms a complex with ga q/ , lck and zap . tcr stimulation activates p g, which affects -phosphorylated polyphosphoinositide levels at the immunological synapse. we show that tcr-stimulated p g controls rac activity, f-actin polarization, and the interaction between t cells and antigen-presenting cells (apc). we show that p g deletion affects the activation of many pathways downstream of tcr crosslinking, as well as the interaction between t cells and apc; these findings could explain the defective activation of p g-/-t cells. our observations clarify the activation mechanism and mode of action of p g in the control of t cell activation, confirming a crucial role for p g in tcr-induced t cell activation. we investigated mechanisms controlling central location of lytic granules and kinetics of their release within immune synapses formed by cytotoxic t lymphocytes (ctl). we show that cytolytic granules in ctl can be delivered to the secretory domain via two different pathways -"short" and "long". the choice between these pathways is regulated by the kinetics of early tcr signaling which depends on the strength of tcr/pmhc/co-receptor interactions. meanwhile, the molecular hardware used to deliver the granules remains the same. we conclude that the difference in temporal and spatial coordination of the two principal events, i. e., granule movement toward microtubule organizing center (mtoc) and the mtoc polarization, accounts for two different pathways of granule delivery to the secretory domain that influence efficiency of ctl cytolytic response. our findings reveal a mechanism of well-documented flexibility in t cell responsiveness that is derived from differential use of the similar set of immune receptors, signaling proteins and intracellular effector molecules. objectives: in addition to specific immune cytokines, lymphocyte activation and immune response are modulated by universal mediators like acetylcholine. nicotine was shown to suppress both cellular and humoral immune responses. previously we found that two nicotinic acetylcholine receptor (nachr) subtypes, a b and a , expressed in mouse b lymphocytes regulate their development within the bone marrow. the aim of the present study was to evaluate the roles of these two nachrs in b lymphocyte activation. methods: b lymphocytes were magnetically separated from the spleens of c bl/ j mice. they were stained with fluorescently labeled igm-, cd -or cd specific antibodies in the presence/absence of unlabeled nachr subunit-specific antibodies to be examined by flow cytometry. b lymphocyte activation was studied by h-thymidine incorporation upon stimulation with anti-cd and nachr-specific agonists or antagonists. the antibody response of mice immunized with cytochrome c with or without a nachr antagonist methyllicaconitine (mla) was studied by elisa. results: antibodies against a or b nachr subunits inhibited binding of igm-and cd -specific antibodies but facilitated that of cd -specific antibody. in contrast, antibody against a subunit prevented binding of anti-cd but not of anti-igm or anti-cd suggesting that a nachrs are located close to cd , while a b ones are close to bcr/cd . consequently, anti-cd -induced b lymphocyte proliferation was increased by mla much stronger than by a b -specific antagonist dihydro-b-erythroidine. it was also increased when cells were incubated with the inhibitor of acetylcholine synthesis hemicholine- . in contrast, proliferation of b lymphocytes from mice consuming nicotine was significantly weaker than that of control mice. mice co-injected with cytochrome c and mla responded with igm antibodies faster than those injected with cytochrome c alone, while the secondary / igg responses were similar. the cd -mediated b lymphocyte proliferation, but not the igm-igg switch or memory b cell activation, is negatively controlled by either endogenous acetylcholine or consumed nicotine through a nachrs. therefore, acetylcholine may be regarded as an auto/paracrine regulator of lymphocyte activation.this work was supported by philip morris usa inc. and philip morris international. binding of cd + t h -lymphocytes to antigen presenting cells or of cd + cytotoxic t-lymphocytes (ctl) to their target cells lead to a tight contact between these two cells, called immunological synapse (is). formation of the is induces calcium signaling, rearrangement of the actin cytoskeleton, and the recruitment of various molecules to the is, all of which are crucial for t-cell functions such as cytokine release or target cell killing. objectives: using primary human t-lymphocytes, none of the proteins involved in either calcium influx, cytokine release, actin cytoskeleton rearrangement nor in killing of target cells can be analyzed by knock-out strategies. for testing protein functions, down-regulation by rnai technology is thus an important tool. we used short interfering rnas (sirnas) to analyze the role of proteins involved in calcium influx and proliferation (stim and trpc ), and to analyze snare proteins which were shown to accumulate at the is and are good candidates to play a role in cytotoxic granule fusion and exocytosis to kill target cells. methods: to validate down-regulation of different mrnas quantitative rt-pcr was used. down-regulation of proteins was confirmed by immunocytochemistry, western blotting and various functional assays depending on the potential role of the protein of interest (calcium imaging, proliferation, cytokine release, killing assay). results: transfection efficiency of sirnas in t-lymphocytes was about %. down-regulation of stim was confirmed by qrt-pcr and by calcium imaging, but only for early time points following activation of cd + t h -lymphocytes, probably because of stability problems. to increase stability of sirnas within t-lymphocytes we used modified sirnas published by mantei et al. (eji, ) . we show that these sirnas down-regulate various snare proteins in ctls more efficiently than non-modified sirnas. the optimal sirna concentrations for transfection in primary human t-lymphocytes was found to be - pmol, which is lower than the concentrations reported in other cell types. conclusions: following optimization, down-regulation of mrnas by sirna is a powerful tool to investigate the role of different proteins involved in the activation of t-lymphocytes in primary human cells. chemical modifications increase the lifetime and efficiency of the sirnas in primary human t-lymphocytes. stress-inducible heat shock protein (hsp ) has gained plenty of attention because of its potent adjuvant capability to induce antigen-specific cd + cytotoxic t-lymphocyte (ctl) and cd + t-helper cell (th ) responses. in this study, we investigated the behavior of t-cell subsets stimulated with endotoxin-free recombinant hsp with respect to proliferation, cytokine expression, cytotoxicity against allogeneic b-lymphoblastoid cell line (b-lcl) and k cells as well as targetindependent cytotoxicity. cd + cells exhibited a strong increase in proliferation after stimulation with hsp , with rates of up to %. in the presence of target cells, a -fold up-regulation of granzyme b mrna was observed after stimulation of cd + t-helper cells with hsp in combination with il- , - and - . the target cell-independent secretion of granzyme b by cd + cells was greatly augmented after stimulation with hsp plus il- or il- , - and - . in this study, we have shown that hsp is capable of inducing a cytotoxic response of t-helper cells in the absence of lps or any other pamps. the granzyme b secretion and the cytolytic activity of cd + t cells is induced in a target-independent way, whereas the cytotoxic activity of cd + and cd + t cells can be further enhanced in the presence of the target cells. our data provide novel insights into the role of extracellular hsp on t-cell immune response concerning the induction of target-independent t-helper cell cytotoxicity. jun n-terminal kinases (jnk) have been shown to play controversial role in regulation of cell fate. cd , which is responsible for germinal centre formation in lymph nodes, trigger jnk activation. the role of other b cell co-receptor molecules that may be involved in antigen-driven differentiation were not clarified. the aim of this study was to find out whether cd receptor contributes to jnk activation in mature human b cells. protein expression and phosphorylation were studied by western blot analysis. protein associations were evaluated by immunoprecipitation and gst-pull down assays. hpk overexpression in a model system was achieved by transfection. pjnk / expression in primary hrs cells was assessed by immunohistochemistry. ligation of cd on resting (dense) and activated (buoyant) human tonsillar b cells lead to jnk , but not jnk activation. cd ligation on primary tonsillar b cells also resulted in jnk activation. however, bcr crosslinking did not affect the level of jnk / phosphorylation. cd -mediated jnk activation was independent from sh d a/sap adaptor protein expression, and was demonstrated for all studied b-lymphoblastoid, burkitt's lymphoma and hodgkin's lymphoma (hl) cell lines of b cell origin. we were searching for serine/threonine kinase that could coprecipitate with cd and link this receptor with jnk pathway. using immunoprecipitation and gst-pull down assays we found that hematopoietic progenitor kinase (hpk ) was associated with cd in primary b cells as well as in b cell lines. cd -hpk association was independent from cd tyrosine phosphorylation and sh d a expression. overexpression of hpk in a model system significantly enhanced cd mediated jnk phosphorylation. it is known that tnf family receptors such as cd , cd , rank trigger survival signals in hrs cells. we observed the expression of pjnk / in hrs cells of primary classical hl. cd could be involved in sustained jnk activation in primary hrs cells, and this may reflect the role of cd receptor as well as other receptors in the regulation of hrs survival. overall, it was shown that jnk is activated via cd in primary b cells and in all studied cell lines of b cell origin. serine-threonine kinase hpk is involved in cd -mediated jnk activation. objectives: cd has been shown to act as a negative regulator of tcr signaling during thymocyte development. however, the molecular mechanisms involved in this process remain elusive. one potential key molecule involved in the downmodulation of tcr signaling is c-cbl, a ubiquitin ligase that physically associates with cd upon tcr crosslinking in thymocytes. the objective of this study was to determine which sequences within the cytoplasmic tail of cd are involved in c-cbl phosphorylation and association. methods: el thymoma cell line was stably transfected with wild-type human cd or hcd cytoplasmic tail mutants: cd .k stop (maintaining only a pseudo itim); cd .h stop (lacking the distal s and y in the carboxy-terminal region); cd . ¿ e -l stop (lacking the pseudo-itam, putative site for c-cbl association). phosphorylaton of y in c-cbl was analyzed, which is required for vav recruitment and c-cbl dependent degradation by the proteasome. stable clones were stimulated with anti-murine cd in combination or not with anti-human cd biotinylated antibodies and phosphorylation of c-cbl was detected by flow cytometry after intracellular staining anti-phospho c-cbl (py ) antibody. murine thymocytes were used as positive control. data was analyzed using flowjo software. unpaired two-tailed student t test was used to calculate statistical significance (p x . ). in murine thymocytes, co-crosslinking of cd with cd induces an increase in c-cbl phosphorylation compared to cd alone. analysis of the el- transfectants showed that mutants cd .k stop and cd .h stop lost the ability to costimulate cd -mediated phosphorylation of c-cbl. in contrast, cd . ¿ e -l stop mutant, was able to efficiently costimulate cd -mediated c-cbl phosphorylation, similarly to the hcd wt. our results indicate that the absence of the pseudo itam in cd does not interfere with c-cbl phosphorylation in response to cd plus cd crosslinkiing on the other hand, sequences present in the carboxy-terminal region of cd appear to be important for c-cbl phosphorylation. therefore, c-cbl phosphorylation might not require physical association with the cd cytosplasmic tail, but rather, may indirectly associate with cd through the interaction with other sh -sh domain-containing molecules, that may be recruited to cd through its carboxy-terminal region. l. kolly , s. narayan , j. tschopp , a. so , n. busso chuv, rheumatology, lausanne, switzerland, unil, biochemistry, epalinges, switzerland apoptosis-associated speck-like protein containing a caspase recruitment domain (asc) is an adaptor protein that is essential for the recruitment of pro-capase- into inflammasomes and thus plays a key role in regulating capase- -dependent il- b and il- production. despite recent evidence implicating asc in adaptive immunity against infections, hyperresponsiveness and vaccination, the cellular and molecular basis for asc involvement in adaptive immune responses remains largely unexplored. to investigate the impact of asc on t cell activation and subsequent effector function. asc +/+ and asc -/-t cells or purified cd + and cd + t cells were activated in vitro through anti-cd stimulation and their proliferative potential and cytokine profiles characterized. proliferative responses by asc -/-t cells were significantly inhibited two-fold following tcr-cd ligation when compared to asc +/+ t cells. furthermore, cytokine analysis revealed that anti-cd activated asc -/-t cells predominantly displayed a more th phenotype, producing more il- ( vs. pg/ml; asc +/+ vs. asc -/-t cells respectively; p= . ) and less ifn-g ( , vs. , pg/ml; asc +/+ vs. asc -/-t cells respectively; p = . ). when asc +/+ and asc -/-t cells were purified into cd + and cd + t cell fractions and activated individually using anti-cd , no inhibition in proliferation was observed amongst activated asc -/-cd + and cd + t cells. interestingly, the activated asc -/-cd + t cell fraction produced significantly more il- when compared to activated asc -/-cd + t cells and asc +/+ cd + and cd + t cells (asc -/-cd + t cells = pg/ml il- ; asc -/-cd + t cells = undetectable il- ; asc +/+ cd + t cells = pg/ml il- ; asc +/+ cd + t cells = undetectable il- ). cd + and cd + t cell mixing experiments revealed that asc -/-cd + t cells are able to inhibit the proliferative ability of asc -/-cd + t cells, asc +/+ cd + and cd + t cells in vitro and that this suppression appears to be mediated by a soluble factor secreted by activated asc -/-cd + t cells. collectively, these results demonstrate that the absence of asc drives cd + t cells towards a suppressor cell phenotype, suggesting that asc might play an important role in determining the fate of cd + t cells. various members of the eicosanoid family derived from arachidonic acid participate in inflammatory reactions and may act as potent regulators of the immune response. in particular, e-series prostaglandins, pge and pge suppress some t-cell functions including proliferation, activation and cytokine production. pge signals through four types of gpcrs called the ep receptors. at low concentrations, pge is believed to be necessary for t cell function, whereas at higher concentrations, pge inhibits t cell proliferation. these effects are largely governed by various cell specific stimuli and tissue microenvironment. objectives: to delineate, compare and contrast the effects of pge and ep receptor antagonists on t cell activation. methods: flow cytometry, proliferation assays, migration assays. we have observed that pge diminishes expression of early, intermediate and late t cell activation markers. in contrast, pre-treatment of cd + t cells with ep receptor antagonists was found to impair cell surface expression of cd , cd , cd and ox but not cd . suppression of t cell proliferation by pge has already been widely studied. however, blocking ep receptors in cd + t cells by the use of ep antagonists prior to activation surprisingly caused a defect in t cell proliferation. migration of cd + t cells to the chemokine sdf- b was also found to be reduced due to pre-treatment with ep antagonists. in order to study the physiological relevance of these findings we studied the trafficking of basal and activated t cells to regional lymph nodes during inflammation in the presence and absence of ep receptor antagonists. this model revealed that the use of ep antagonists causes a reduction in the amount of cd + cd + adoptively transferred t cells in the regional lymph node following the induction of a local inflammatory response. conclusions: in our study we show for the first time that ep receptors are required for expression of activation markers and activating proliferation in murine cd + t cells. our results also suggest that considering pge -mediated camp signaling in cd + t cells, it will be absolutely necessary to distinguish between transient increases, which have potentiating effects, and sustained increases, which have inhibitory effects in t cell activation. objective: our objective was to investigate how ros affect the different stages of t cell activation. because activation is initiated by changes in intracellular calcium concentration, we addressed whether and how ros affect calcium signalling. the experimental results were obtained using a combination of fluorescence microscopy, patch-clamp, t-cell activation assays and molecular biology. results: we show by direct measurement of ros that t-cells are exposed to high concentrations of oxidants when they are in close vicinity of activated phagocytes. the effect of ros on calcium signalling in jurkat t-cells as well as in primary naï ve and effector cd + human t-cells was examined. oxidation affects several ca + signalling pathways by altering the activity of ip receptors, trp channels and store operated ca + channels in a concentration dependent manner. interestingly, calcium signalling is differentially affected in naï ve and effector t cells. thiol reducing agents were able to significantly reduce the effects of oxidation implicating thiol oxidation as a major player in the regulation of ca + signalling in t-lymphocytes. cysteins are the main carrier of thiol groups in proteins and we show that orai ion channels contain reactive cysteine groups that mediate ros effects on the calcium influx pathway. conclusion: ros regulate the calcium dependent t-cell activation in a complex way, affecting all three major calcium signalling pathways. by mutational analyses of the orai proteins, we are able to pinpoint molecular targets of regulation. the activation of t cells during an immune response is a crucial but tightly regulated event. to make the grade, the t cells upregulate costimulatory but also inhibitory receptors upon antigen recognition. this enables the t cell to be stimulated for proliferation to keep pace with pathogens infection, but also to become dampened upon successful defense against the pathogens via negative feedback mechanisms. in this study we present data of the signaling mechanisms underlying the potent t cell inhibitory receptor cd (emmprin, basigin) , a member of the ig-family. previous studies reported that lymphocytes from cd knockout mouse possess enhanced mixed lymphocyte reactions and cd monoclonal antibodies can interfere with t cell activation. these observations already pointed to a negative crosstalk of cd signals with the t cell antigen receptor or co-stimulatory signals. consistent with these studies, we found that rna interference (rnai) with cd in jurkat t cells augments the secretion of the t cell growth-factor interleukin- (il- ) upon t cell activation. this up-regulation is at least partially due to an increased activity of the nuclear factor of activated t cells (nfat), which resulted in an enhanced il- promoter activity. by reconstituting the rnai-mediated knockdown with various truncated rnai-resistant forms of cd , we identified the immunomodulatory sub-domain of cd . supported by the gen-au program of the austrian federal ministry of science and research. mirnas play a critical role in the control of hematopoiesis. the goal of this project is to determine whether mirnas function also during the antigen-induced activation of mature b lymphocytes. therefore, we determined mirna profiles in primary splenic b cells before and after polyclonal activation with either lps (simulates t cell-independent activation) or a combination of anti-igm, anti-cd and il (simulates t cell-dependent activation). microarray assays identified about mirnas in unstimulated b cells. of these were downregulated and one was upregulated upon stimulation. in silico analyses with various mirna target prediction programs revealed an interesting and promising set of transcripts whose translation/stability could be controlled by mirnas during the antigen-induced activation phase of mature b cells. among these targets are bcr signalling molecules and transcription factors that control proliferation, igh class switch as well as differentiation in antibody-secreting plasma cells. one of these transcripts codes for the interferon regulatory factor- (irf- ). the graded expression of this important transcription factor has been shown to coordinate isotype switching with plasma cell differentiation. first results indicate that the expression kinetic of irf- transcripts differs from that observed for irf- protein abundance after b cell stimulation. further analysis identified the irf- transcript as a target whose expression is obviously fine-tuned by a mirna upon antigen stimulation. we are in the process to biochemically verify potential targets for each of the differentially regulated mirnas and determine the effect of ectopic and retrovirally mediated expression of mirnas on b cell differentiation. the work was in part supported by the izkf erlangen, the dfg graduiertenkolleg gk and the dfg forschergruppe for . objective: transforming growth factor-b (tgf-b) signals through type i (tgfbri) and type ii (tgfbrii) tgf-b receptors and receptor regulated smad proteins. tgf-b exerts predominantly anti-proliferative and pro-apoptotic effects which are frequently lost in cancer. the mechanisms of resistance against tgf-b have not been fully elucidated. our aim is to describe how b cell lymphoma cells respond to tgf-b compared to normal peripheral b cells, to create an overview of the different signaling pathways involved, and to characterize the mechanisms behind the loss of sensitivity to tgf-b. methods: proliferation assays were performed on different b-cell lymphoma cell lines and normal peripheral b cells to screen for tgf-b-induced effects. western immunoblotting analysis was conducted to characterize protein expression and phosphorylation related to tgf-b signaling pathways. facs analysis was used to measure tgf-b receptor surface levels. cells were treated with demethylating agents to examine changes in gene expression levels. s. manthey , f. hauck , i. berberich , f. berberich-siebelt , gk -immunomodulation institute for virology and immunobiology, university of wuerzburg, würzburg, germany, university of wuerzburg, department of molecular pathology, würzburg, germany the transcription factor ccaat/enhancer-binding protein b (c/ebpb) can not act only as a transcriptional activator but also as a transcriptional repressor. in murine cd + t lymphocytes, the transcription factor is predominantly expressed in t helper (th ) compared to t helper (th ) cells. in contrast, by binding to the c-myc promoter(s), c/ebpb represses c-myc expression thereby arresting t cells in the g phase of the cell cycle. both, transactivation and repression depend on the n-terminal transactivation domain of c/ebpb. blimp- encoded by prdm is a transcription factor necessary for terminal differentiation of b cells to plasma cells. furthermore, blimp- is expressed in differentiated effector t cells where it is higher in th than th cells. the regulation of the blimp expression is not fully understood. interestingly, we found that c/ebpb can bind to the prdm promoter and activates blimp- expression in t cells. as c/ebpb is also expressed in b cells, we hypothesize that this transcription factor might as well influence the expression of blimp- in b cells. so far, we were able to show a similar expression profile of c/ebpb and blimp- in b cells using cre recombinase. moreover we found a new putative blimp- isoform lacking exon . currently, we analyze the expression of c/ebpb and blimp- in primary b cells and b cell lines after various stimulations. to get more insights into the function of c/ebpb in b and t cells, we are generating mice carrying a b as well as a t cell-specific deletion of c/ebpb. engagement of antigen receptors on lymphocytes leads to rapid increases in intracellular free calcium concentrations via phosphorylation of phospholipase c gamma (plcy) and plays an important role in activation of cells. by screening cvid patients with a flow cytometric assay we demonstrate that calcium flux is significantly reduced in b and t cells isolated from the peripheral blood of patients in the group ia of the freiburg classification as compared to non-ia patients and healthy donors (hd). ia patients are characterized by the expansion of an unusual cd low b cell population in which calcium mobilization is strikingly lower than in other b cell subsets. common subpopulations like naï ve and mz-like b cells as well as cd + t cells but not transitional b cells or cd + t cells also revealed significantly decreased calcium peaks. the cytometric data correspond to a semiquantitative rt-pcr assay and functional data showing reduced induction of the calcium dependent macrophage inflammatory protein- a (mip- a), and abrogated activation and proliferation, respectively. preliminary data on b cell receptor (bcr) mediated phosphorylation of plcy revealed constitutively high background levels in cd low b cells of ia patients. since phosphorylation in the other b cell populations as well as calcium flux upon ionomycin were the same for patients and healthy donors, we postulate an abrogated amplification or altered inhibitory pathway targeting the signalling events downstream of plcy and upstream of internal store release, thus resulting in defective calcium signalling. the underlying mechanism yet remains to be elucidated and is part of our work in the future. c. balas , v. courtois , k. de luca , r. sodoyer sanofi pasteur, marcy l'etoile, france the presence and relative abundance of cytokines at different stages of infection is relatively well documented, but their involvement in immune status, pathogenesis or disease progression is still unclear. a potential explanation to the difficult interpretation of the results obtained might be related to the intrinsic weakness of the analytical techniques. for instance monitoring of the expression level of cytokines, such as il- , il- or il- could lead to misinterpretation if molecular isoforms are not detected by antibodies currently used to measure them. the analysis of the human transcriptome is a way to access the subset of genes involved in the immune response upon infection by various pathogens. such an analysis might be completed and enriched by the analysis of the relative expression of some cytokine splice variants. methods: genetic tools (primers and qpcr probes) capable of discriminating and quantifying alternatively spliced messenger rnas from il- , il- and il- . furthermore, the recognition by several commercial antibodies of the different cytokine isoforms (expressed as recombinant proteins) has been investigated. the genetic tools have been validated on in vitro models as well as on biological samples (please refer to the abstract no a- - - ). conclusion: implication of such kind of analysis in diagnostic application and disease progression survey will be discussed. in a different context, the same kind of analysis could be applied to the monitoring of the immune response upon vaccination or more generally for new antigens or adjuvant screening. parasitic helminths affect about one third of the world population. therefore the mechanisms, which are involved in the persistence or the expulsion of the parasite, are of special interest. from other parasitic infections it is known, that the regulatory receptor cytotoxic t lymphocyte antigen- (ctla- ) plays a crucial role during infections. here, we use the strongyloides ratti infection of mice as an experimental system to investigate the role of ctla- during nematode infections. we employed a quantitative real-time pcr (qtpcr) analysis to quantify the migrating larvae (il ) in the tissue and the released eggs and first stage larvae (l ) in the feaces. the cytokine response of lymphocytes, prepared from the spleen and the mesenteric lymphnodes (mln) upon stimulation with polyclonal a-cd and s. ratti antigen was determined. additionally the humoral response was analysed in the primary and the secondary infection. to investigate the role of ctla- during the infection, a neutralysing antibody (a-ctla- ; f ) was administered intraperitoneally ( mg) two hours before subcutaneous infection with s. ratti il . the in vivo neutralisation of ctla- -signalling by applying a-ctla- during s. ratti infection led to an altered cytokine response, compared to infected mice treated with a control antibody. we detected an increase in th cytokines, such as il- and il- and a reduction of the proinflammatory cytokines ifn-g and il- . the investigation of the humoral response showed a remarked increase of the igg -titer in the serum during secondary infection in mice that had been treated with a-ctla- during primary infection. furthermore, the blockade of ctla- resulted in a diminished worm burden as indicated by reduced release of l in the faeces. these results suggest that the blockade of ctla- during s. ratti infection induces an activation of the appropriate effectors of the immune system that are beneficial for the host defence. in particular the transition of the t cell cytokine profile towards a th response supports this hypothesis and might be the reason for the reduced worm output in the primary infection. the strong increase of igg during secondary infection also reflects the induction of a potent th response. objectives: cd is a class b scavenger receptor, which has been shown to be involved in the pathogenesis of atherosclerosis as well as in the clearance of apoptotic cells by macrophages. this clearance is important in regulating the immune system to avoid autoimmune reactions, as seen in systemic lupus erythematosus (sle). it was recently described that cd is highly expressed also on the marginal zone b cell subtype. we therefore set out to investigate the role of cd on the regulation of b cell in the setting of apoptotic cell clearance and autoimmune activation. we used a mouse model for sle where apoptotic cells were injected repeatedly in order to study the auto-reactive antibody response that follows. elisa was used to measure antibody levels and flow cytometry to study cell activation as well as cd expression. cd knock-out (ko) and wild type mice were used. results: preliminary in vivo data show a tendency for a higher antibody response towards ds-dna and the common self-antigen pc in cd ko compared to heterozygous mice. since reduced levels of cd are expressed in heterozygous mice we are currently repeating this experiment using wild type mice as controls for comparison. in support of the in vivo findings, the immunosuppressive effect of injected apoptotic cells seen in wild type mice after in vitro stimulation of splenocytes with lps is gone in cd ko mice. after one injection of apoptotic cells, cd ko b cells are activated while wild type b cells are not. after four injections a break of tolerance is seen and apoptotic cells do no longer have an immunosuppressive effect and we show that cd on b-cells are involved in setting this threshold. conclusion: our data suggest that cd is involved in the early regulation of b cell response towards apoptotic cells and production of autoreactive antibodies. it does so by being involved in regulation of the tolerance effect exerted by apoptotic cells. successful t cell immunity requires lymphocytes to be at the right time at the right place. the co-receptor cd acts as a major check-point of immune responses, but the mechanism by which cd controls peripheral t cell responses is unknown. the consequences of cd signaling on murine th cell migration were analyzed using chemotaxis assays in vitro and radioactive cell tracking in vivo. the genetic and serological inactivation of cd in th cells reduced migration towards ccl , cxcl and ccl . crosslinking of cd together with cd and cd stimulation on activated th cells increased expression of the chemokine receptors ccr and ccr , which in turn enhanced cell migration. sensitive liposome technology reveals that mature dendritic cells but not activated b cells were potent at inducing surface cd expression and cd -mediated migration-enhancing signals. importantly, migration of cd positive th lymphocytes in in vivo experiments increased, as compared to cd negative counterparts, showing that indeed cd orchestrates specific migration of selected th cells to sites of inflammation and antigenic challenge in vivo. these data show that cd signaling does not just silence cells, but selects individual ones for migration. this novel activity of cd adds to the already significant role of cd in controlling peripheral immune responses by allowing t cells to localize correctly during infection. it also suggests that interference with cd signaling provides a tool for altering the cellular composition at sites of inflammation and antigenic challenge. here we analyzed the role of cd signaling on the longevity of cd null t cells. using a sensitive staining method for cd , we show that human cd + cd null and cd + cd null t cells rapidly express surface cd . serological inactivation of cd using specific fab fragments or blockade of cd ligands using ctla ig in cd + cd null and cd + cd null t cells reduces the number of non-apoptotic cells in a fas/fasl-dependent manner. cd crosslinking on activated cd null cells prevents activation-induced cell death (aicd) as a result of reduced caspase activity. apoptosis protection conferred by cd is mediated by pi 'k dependent activation of the kinase akt resulting in enhanced phosphorylation and thereby inhibition of the pro-apoptotic molecule bad. we show that signals triggered by cd act directly on activated cd null t lymphocytes and, due to its exclusive expression as a receptor for cd /cd on cd null t cells, prevention of cd mediated signaling is likely a major target mechanism taking place during therapy with ctla ig. objectives: cd is a transmembrane protein tyrosine phosphatase (ptp) expressed in all nucleated leukocytes. it activates src family kinases (sfks) by dephosphorylating inhibitory tyrosines in their c-terminal tails. in cd -/mouse t cell signaling and development is severely impaired, while other leukocyte populations seem much less affected. at least in part, it is due to the activity of another transmembrane tyrosine phosphatase cd (ptprj, dep- ) which acts as a positive regulator of sfk in cd -/-b cells and macrophages and can compensate for cd deficiency in these cells. indeed, combined deficiency of cd and cd in mice results in defective macrophage and b cell signaling and development, a phenotype much more severe than the loss of either protein alone. naïve murine t cells do not express cd and its expression is increased only after activation. accordingly, no defects in t cell development and signaling in cd -/mice were reported so far. however, in human t cells the role of cd may be different since naive human t cells express cd at a level comparable to b cells. using cd -/-/cd -/human t cell line (jurkat-derived js- cells) we tested the ability of cd to complement cd deficiency in t cells. we used retroviral transduction to express human cd or cd in js- cells and tested their ability to reconstitute major signaling pathways. we also employed substrate trapping mutant of cd to identify direct substrates of this phosphatase. in agreement with previously published data, defective t cell receptor (tcr) signaling was observed in js- cells. expression of wild type cd or cd in js- cells resulted in more rapid calcium mobilization, enhanced tyrosine phosphorylation, and increased cd upregulation after tcr cross-linking. moreover, the carboxy-terminal tyrosine of lck, major t cell sfk, was hypophosphorylated in js- cells expressing cd when compared to control cells. finally, cd substrate trapping mutant expressed in js- cells interacted with lck in vivo suggesting that lck is a direct substrate of cd in js- cells. the results suggest a level of redundancy between cd and cd in human t cells not appreciated so far. during the past decades, great efforts have been made to get insights into the complex process of antigen-induced t cell activation and the underlying signal transduction pathways. the t cell antigen receptor signaling cascade is initiated by phosphorylation of itam-tyrosine residues through the t-cell specific src protein tyrosine kinase family member lck. during t cell activation, lck is supposed to undergo structural changes from a closed inactive to an open active conformation followed by phosphorylation of the itam-motifs. in order to resolve conformational changes of lck in living cells with high spatio-temporal resolution, we designed biochemically active conformational-sensitive förster resonance energy transfer (fret) biosensors using cyan and yellow fluorescent proteins inserted at special positions of the complete kinase backbone. for the live-fret imaging and biochemical assays we complemented lck-deficient jurkat t cells (jcam . ) with the biosensors. by introducing point mutations affecting the two major regulatory tyrosines tyr and tyr we found a dramatic decrease and increase, respectively, of intramolecular fret efficiency compared to the wild type biosensor. these results correspond to unfolding of the biosensor to its active conformation on the one hand and condensation of the kinase structure to its inactive form on the other hand. thus, our biosensor is able to detect phosphorylation modifications of key residues. however, we could not detect any overall change in fret and thus conformation of membrane-associated lck molecules during t cell activation indicating that other mechanisms, presumably reorganization of localization, underlie lck regulation. furthermore, we observed a contribution of intermolecular fret, which indicated homophilic interaction of lck. indeed, by performing single molecule analysis and native d immunoblotting we found lck dimers and higher order oligomers. together, these advanced imaging studies in the live cell context provide a novel picture of the function and regulation of this key kinase in signaling via the t cell antigen receptor. it has been reported that mitochondria accumulate under the immunological synapse (is) in response to tcr (t cell receptor) stimulation. this process seems to be required to allow proper tcr-induced calcium influx in t cells in contact with antigen presenting cells (apcs), because mitochondria can sequester calcium and thus keep crac (ca + release-activated ca +) channels open. however, antigen-induced calcium signaling is very fast, and clearly much faster than mitochondrial translocation toward the is. thus, we speculated that other signals are involved in recruiting the organelles to the contact region between t cells and apcs. we found that the adhesion molecule lfa- (leukocyte function-associated antigen ) induces localization of mitochondria at the is. this process is antigenindependent and is enhanced by the presence of chemokines in the t cell environment. however, tcr triggering stabilizes mitochondria at the synapse and it is important to sustain their recruitment in time. our data suggest that, by recruiting mitochondria to the cell-cell contact region, lfa- prepares and facilitates tcr signaling. we are performing experiments to understand the signalling pathways involved in mitochondria translocation at the is. burkitt lymphoma (bl) is a high grade b cell malignancy (non-hodgkin lymphoma (nhl)) derived from germinal center b cells, that harbours a chromosomal translocation juxtaposing the protooncogene myc next to the regulatory elements of one of the immunoglobulin loci. however, the precise contribution of myc to the pathogenesis of this tumour is poorly understood. based on the definition of a distinguishing gene expression signature for the molecular burkitt lymphoma (mbl) with myc as one hallmarking signature gene (hummel et al. ) we describe a non-viral vector based approach (vockerodt et al. ) to express myc in primary human gcb cells from pediatric tonsils. comparative whole genome gene expression profiling was performed in independent preparations. our data reveal a global change in gene expression in lymphoma precursor cells by myc giving new insight into potential changes of the gene expression program of gcb cells on the accidental way to bl in addition as a first step the function of selected signature genes in bl is accomplished. in a representative cell line with a mbl signature and with a non-mbl signature rnai directed inhibition of elements of the cd signaling cascade was conducted. after activating this particular signaling cascade (cd ) we analysed respective gene expression profiles of ikks, trafs and mapk deficient cells. based on these different rnai-mediated ge-profiles a comparison between both lymphoma types is performed. first attempts are made to reconstruct the topology of the respective signaling pathway by using the nested effects bioinformatic model, which has been described recently (markowetz et al. ). a rat thymic epithelial cell (tec) line (r-tnc. ) was established from a long-term tec culture. this line was characterized as a type of rat cortical tec with nursing activity (tnc). very little is known about molecular mechanism of the tnc/thymocyes interaction. in our previous studies we investigated molecular mechanisms involved in the binding and emperiopolesis of resting thymocytes by r-tnc. cell line in vitro. it was found that a number of adhesion molecules, such as cd , cd , cd , cd a, cd , cd , cd was involved in these processes. objectives: a main goal of this study was to define the adhesion molecules involved in the interaction between r-tnc. line and activated thymocytes. methods: experiments was performed on inbred ao rats. monoclonal antibodies (mabs)-mediated modulation of thymocyte binding and emperiopolesis was tested by adhesion and engulfment assay, respectively, using a coculture of cona and il- activated syngeneic thymocytes and unstimulated or ifn-g stimulated r-tnc. cells. we found that both the adhesion ( min and h) of activated thymoytes were partially blocked by mab to cd and cd molecules (ifn-g unstimulated and ifn-g stimulated r-tnc. cells). early adhesion was inhibited by mab to cd , abtcr, mhc class i molecule (ifn-g stimulated r-tnc. cells) and cd molecule (ifn-g unstimulated r-tnc. cells). after prolonged incubation, significant inhibition was obtained using anti-mhc class i mab (ifn-g unstimulated r-tnc. cells). almost all mabs which were inhibitory in the binding assay were inhibitory in the engulfment assay ( h), namely mab to cd , cd , cd , cd molecule (ifn-g unstimulated and ifn-g stimulated r-tnc. cells) and mhc calss i and mhc class ii molecule (ifn-g unstimulated r-tnc. cells). our results also suggest the involvement of cd a/cd dependent -cd independent pathway in adhesion and cd a/cd dependent -cd dependent pathway in emperiopolesis. the obtained results imply that adhesion, deadhesion and emperiopolesis of activated thymocytes by r-tnc. cell line are tightly regulated processes in which multiple adhesion molecules are involved. the crucial roles of cytokines in shaping t cell responses have been documented in both healthy and disease conditions. interleukin- (il- ), a recently described cytokine, has been shown to exhibit both pro-and anti-inflammatory properties. il- favours naï ve cd t cell differentiation into th cells to the detriment of th or th differentiation. the il- receptor (il- r) is a heterodimer composed of tccr, which confers ligand specificity, and gp , a signal transducing chain that is utilized by several other cytokines. il- has been demonstrated to promote cytotoxic lymphocyte functions of mouse cd t cells, but the potential impact of il- on human cd t cells has not been elucidated. our goal is to investigate the impact of il- on human cd t cell functions. we used peripheral blood mononuclear cells (pbmc) from healthy donors, either exvivo or after short term in vitro activation to perform our analyses. we first assessed whether the il- r is detectable on ex-vivo t cells using flow cytometry. we observed a greater proportion of cd than cd t cells expressing the complete surface il- r (gp +tccr). however, we detected high amounts of intracellular tccr in both, cd and cd t cells, but only polyclonal activation (anti-cd ) of cd t cells led to an actual increase of il- r surface expression. purified cd t cells from healthy donors were shortly stimulated in vitro and then analyzed using flow cytometry-based functional assays. il- activated stat and stat signalling with rapid kinetics in both cd and cd t cells, indicating the capacity of il- to signal through these molecules. addition of il- to anti-cd activated cd t cells led to a significant dose dependent increase of proliferation (as measured by cfse-based assay) and ifn-gamma and granzyme b production (determined by intracellular staining). these results demonstrate a pro-inflammatory impact of il- on human cd t cells. defects in immune regulation could result in the breakdown of immune tolerance leading to development of multiple sclerosis (ms). the pd- /pd-l pathway is associated with production of the immunoregulatory cytokine il- , the suppression of t lymphocytes proliferation by inhibition of akt phosphorylation (pakt), and the elicitation of apoptosis of antigen-specific cells; an impairment in this pathway could play a pathogenetic role in ms. we analysed by flow-cytometry the surface expression of pd-l and pd , as well as myelin basic protein (mbp)-stimulated il- production, pakt inhibition, and apoptosis (annexin v), in ms patients with relapsing-remitting disease. twenty-six patients were diagnosed as being affected by acute disease (ams); had a diagnosis of stable disease (sms). results showed that: ) pd-l -expressing cd + and cd + cells are reduced in ams compared to sms individuals (p= . ); and ) pd expression is increased in cd + t cells of sms individuals and is comparable on cd + t cells of ams and sms patients. this is associated with a significant decrease in il- production by mbp-stimulated cd + and cd + cells of ams patients (p= . ). additionally, cd + anexin v+ (av+) cells were diminished and cd + pakt+ cells were higher in ams compared to sms patients, while similar percentages of cd +av+ and cd + pakt+ were observed in both groups of individuals. data herein show that the impairments of the pd-l /pd- pathway seen in ams patients result in a reduced mbp-specific il- production by cd + and cd + cells as well as in a reduced apoptosis (annexin v) and an augmented proliferation (pakt) of mbp-specific cd + t. the pd /pdl pathway plays an important role in the pathogenesis of multiple sclerosis. monitoring of the expression of these proteins could be a novel diagnostic tool. anti- - bb in cd cells. this difference could be due to down regulation of cd by activated lymphocytes and possible preferential response of cd cells to anti- - bb costimulation. moreover, increase in ifn-g concentration in costimulated cultures also may enhance the suppressive function of mscs which again could explain the inability of costimulation in proliferation recovery. likewise, reducing tgf-ß by costimulation is not sufficient to abolish suppressive effect of mscs. in overall, these results suggest that lack of costimulation expression by mscs is not the mechanism of msc suppression and other mechanisms are involved. cytotoxic t lymphocytes (ctls) kill target cells by secretion of cytotoxic components contained in lytic granules at the contact zone between the target cell and the ctl, the immunological synapse (is). t cell receptor (tcr) enrichment at the is is one of the early and key events of is formation. objectives: soluble nsf attachment receptor (snare) proteins are required in almost all fusion events in cells. in the present study we tested if the snare protein syntaxin (stx ) is part of the is and whether it serves as a key player of is formation and/or the fusion process itself. methods: pcr-techniques, cell transfection, immunocytochemistry and different microscopic techniques like confocal microscopy and total internal reflection microscopy (tirf) were used on primary human ctls to test the function of stx . rna interference technique was also used to down regulate stx expression in primary human ctls. results: we identified stx in ctls by pcr and immunocytochemistry. stx accumulates at the is after ctl/target cell contact. when stx function was blocked by overexpression of a dominant negative stx mutant (deletion of the transmembrane region), functional studies with tirf showed a reduced accumulation and fusion of lytic granules at the is. furthermore, confocal studies showed a loss of tcr accumulation at the ctl/target contact side. conclusion: these results imply that the snare protein stx is present at the is and moreover is required for is formation in ctls. the observed block of lytic granule release is probably caused by disturbing an upstream process such as vesicle transport, recycling or sorting. objectives: despite the years history of mouse t h and t h subpopulations, relatively little is known about the differences in their signaling mechanisms and the membrane organization of critical receptors and signal transducing molecules. we have developed mouse t h hybridomas to study these differences between polarized t h cells. the in vitro established hybridomas were first characterized as t h , t h or t h phenotypes, based on their cytokine production (il- , ifng or il- ). a comparative analysis of t-bet, ifng and il- mrna levels was also done on quiescent and activated t h hybridomas. in the present study, the ca +response, membrane raft expression/organization, k + -and ca + -ion channel expression/function and sensitivity to apoptosis (aicd) were compared in these hybridomas. expressions and molecular localizations were investigated by flow cytometry and confocal microscopy, respectively. ion channnels were functionally analyzed by patch-clamp technique. apoptosis was analyzed using three markers (mitochondrial membrane potential, caspase activation, dna fragmentation) and flow cytometry. results: expression level of plasma membrane rafts/gangliosides (assessed by cholera toxin b-staining) showed the following rank: t h g t h g t h , although the membrane cholesterol level (detected with anti-cholesterol ab, ac ) was similar in the three cells. in connection, tcr displayed stronger colocalization with rafts and appeared more polarized in t h cells upon activation than in t h cells. t h cells produced a more sustained calcium response with higher amplitude than t h cells to the same tcr-mediated triggering signal. interestingly, this does not coincide with the expression of cav . and kv . ion channels, major functional determinants of the sustained calcium influx. t h cells expressed the highest levels of these two ion channels. there were also marked differences in their sensitivity to activation induced apoptosis (aicd) as assessed by three different markers of apoptosis. the results suggest that a different membrane compartmentation/organization rather than the differential expressions of certain receptors, ion channels and/or other upstream signaling molecules of these t h hybridomas may be responsible for the observed differences in their functional characteristics. objectives: bone morphogenetic proteins (bmps) belong to the tgf-b superfamily, which plays a central role in controlling cellular processes like proliferation, differentiation, apoptosis and migration. whereas tgf-b is well established as one of the most potent negative regulators of hematopoietic cells, the role of bmps in b lymphoid cells remains more elusive. in this study we investigated the effects of bmps on mature human b-cells. methods: b cells were isolated from peripheral blood of healthy donors using cd -dynabeads. cd + isolated cells were facs sorted into cd + cd naïve b or cd + cd + memory b cells. dna synthesis was measured by h-thymidine incorporation, immunoglobulin (ig) levels in cell supernatants were measured by elisa and phospho-protein levels were measured by western immunoblotting analysis. results: all bmps significantly suppressed anti-igm-induced proliferation of cd + cd naï ve b cells, of which bmp- and - were most efficient ( % suppression). similarly, all bmps suppressed cpg-induced proliferation of cd + cd + memory b cells by - %. to induce differentiation, both naï ve and memory b cells were stimulated with cd l and il- . this increased the production of igm, iga and igg - -fold compared to medium control, whereas addition of bmps inhibited the production of all ig classes. all bmps highly induced phosphorylation of smad / / in cd + b cells. the mechanisms for how bmps mediate their inhibitory effects are currently being explored in more detail. conclusion: bmps have prominent inhibitory effects on anti-igm-and cpg-induced proliferation of naive and memory human b cells, respectively. they also suppress cd l/il- -induced production of igs in mature human b cells. s. gutenberger , k. warnatz university medical centre freiburg, freiburg, germany background: signals through the b cell receptor (bcr) and co-receptors are essential for the survival, differentiation and effector function of b cells. the stimulation of the bcr initiates several independent but interrelated signaling pathways. one important pathway leads to the activation of mitogen activated protein kinases (mapk) and especially the phosphorylation of extracellular signal-regulated kinases and (erk / ). in a subgroup of patients with common variable immunodeficiency (cvid) we have previously demonstrated intrinsic defects in the activation of b cells revealed by the insufficient cd upregulation and proliferation after b cell receptor (bcr) stimulation. therefore we assessed signaling pathways downstream of the bcr in order to identify defects in the activation of b cells. methods: pbmc of hd and cvid patients were stimulated by anti-igm. different igm expressing b cell subsets were analyzed separately for erk / phosphorylation by intracellular flow-cytometry using phospho-specific antibodies to erk / . to increase the signal intracellular phosphatases were inhibited by h o . as markers of activation and initiation of proliferation, cd and ki expression were measured after days of in vitro stimulation. k. theil , p. aichele immh university freiburg, immunology, freiburg, germany type i interferons are homone-like molecules that are produced early after viral and bacterial infections. they signal via the type i interferon receptor (ifnar) and have pleiotropic effects on different cells of the immune system. their best known function is the antiviral activity. to test the direct effect of type i interferons on cd t cells in vivo we adoptively transferred lcmv glycoprotein specific tcr transgenic p cd t cells that are deficient in type i interferon receptor (ifnar-/-) into wild-type b -recipient mice and compared their expansion with wild-type (wt) p t cells after viral infection. we could demonstrate a severe impairment in the capacity of p t cells lacking type i ifnr (ifnar-/-) to expand after lcmv infection. following infection of recipient mice with recombinant vaccinia virus, recombinant vsv (vesicular stomatitis virus) or recombinant listeria monocytogenes expressing lcmv glycoprotein, p t cells expansion was considerably less dependent on type i ifnr expression. therefore direct type i ifn signalling is essential for cd t cell expansion and survival only after lcmv infection. our experiments showed that the lcmv generated cytokine milieu is responsible for the failure of expansion of ifnar-/-t cells during lcmv infection. a suitable model for elucidating the impact of the lcmv generated cytokine milieu is the transfer of p t cells into h mice. h mice ubiquitously express the lcmv immunodominant glycoprotein-epitope gp - . therefore the antigen-presentation can be uncoupled from the lcmv induced cytokine milieu when the h mice are infected with lcmv . . this is a lcmv variant that has got a point mutation in gp - and consequently cannot be recognized by the p t cells. s. frischbutter , r. baumgrass deutsches rheuma-forschungszentrum, signal transduction, berlin, germany antigen-specific stimulation of t helper cells induces activation of the main transcription factors nfat, nf-kb and ap which are important for expression of cytokines such as il- , ifng and il . it is known that the immunosuppressive drug cyclosporin a (csa) blocks the activity of the ser/thr phosphatase calcineurin and thereby the activation of the transcription factor nfat. however, we and others observed that this drug also inhibits the activation of nf-kb. to detect targets of calcineurin within the nf-kb pathway we analyzed phosphorylation and degradation levels of different nf-kb signaling proteins in the presence of csa and other calcineurin inhibitors. we found that phosphorylation of the signaling protein bcl- was prolonged in cells treated with inhibitors. our data do not indicate an enhanced bcl- phosphorylation but rather an inhibition of bcl- dephosphorylation. furthermore, calcineurin and bcl- co-precipitated with each other. interestingly, this interaction was observed only in t cell receptor-but not in tnfa-stimulated cells. in our proposed model, we hypothesize that calcineurin interacts with the carma/bcl- /malt signaling complex and dephosphorylates bcl- and, thus, promotes nf-kb activation. therefore, calcineurin is not only a hub for nfat but also for nf-kb activation. a. t. fulop , j. lamoureux , c. fortin université de sherbrooke, medicine, sherbrooke, canada objectives: aging is accompanied by a decrease in immune functions, called immunosenescence. the exact cause is still not known. changes in t cell subpopulations, thymic involution were invoked. we have demonstrated that the signal transduction is altered with aging. in the present work we studied the negative regulatory molecules in the t cell signaling to explain the altered activation of t cells with aging leading to decreased clonal expansion. methods: healthy young and elderly subjects were studied. lymphocytes were separated by fycoll-hypaque. the molecules participating in the negative control loop of lck were studied by western blot and confocal microscopy. the surface expression of ctla- has been studied by facscan. the translocation of the molecules in the membrane lipid rafts (mlr) was also studied by western blot. the activity of phosphatases was also determined. results: we found that the phosphorylation of pag was altered with aging explaining the decreased release of csk from mlr and the decreased lck activation. the activation of fynt was also altered. the phosphatase activity studies showed an increase in their activities with aging. the ctla- expression was higher after stimulation in t cells of elderly. there was differences between cd and cd t cells with aging. conclusion: these results suggest that the negative regulation is preponderant in t cells with aging on the positive activation and as such explaining the defect in t cell functions with aging. this opens new therapeutical avenues in the future. in contrast to other members of the tumour necrosis factor superfamily, fas ligand (cd l) contains a cytosolic proline-rich domain (prd) that enables interactions with sh and ww domain proteins. since fasl surface expression is regulated by adam -mediated ectodomain shedding and fasl might be subsequently released into the cytosol by regulated intramembrane proteolysis (riping) through the secretase-like enzyme sppl a, we are interested in defining interactions involving the generated intracellular fragment of fasl. employing a monoclonal antibody directed against the intracellular domain of fasl, we observed that previously described fasl-interacting proteins of the pch family selectively bind to the full length molecule but not to n-terminal fragments (ntfs). in order to identify other sh domain proteins that potentially interact with the riped fasl prd, we used a sh domain phage display library containing all sh domains expressed in humans. the screen confirmed several previously identified interactions but also revealed numerous new and interesting candidate binding proteins includig non-receptor tyrosine kinases and adaptor proteins or enzymes implicated in membrane, organelle, and actin cytoskeleton dynamics. selected interactions were verified biochemically and by laserscanning microscopy in transfected cells. it could be demonstrated that tec kinases known to be involved in immune receptor-associated signal transduction as well as members of the snx family, which are crucial regulators of endocytic and endosomal dynamics and trafficking, join the list of known fasl-interacting proteins. of note, in contrast to pch proteins, the snxs bound both ntfs and unprocessed fasl, indicating that individual interactors might influence different facets of fasl biology. in conclusion, the present data provide substantial evidence for a selective binding of individual interaction partners of fasl to the full length protein or ntfs. this more detailed glance at the fasl interactome will facilitate focussed strategies to clarify unanswered questions regarding reverse signalling and functional conse- optimal t cell activation requires the engagement of the t cell receptor (tcr) by the specific mhc/antigen complex and costimulatory signals as the interaction of b family members on antigen-presenting cells with cd on t cells. remarkably, whereas classical glucocorticoids (gcs) effectively suppress solely tcrtriggered t cell activation in vitro, additional cd co-stimulation leads to gc-resistance. in this study, we compared the non-steroidal selective glucocorticoid receptor agonist (segra), compound , with classical gcs regarding their suppressive effect on cd -costimulated t cells. human primary t cell subpopulations and jurkat cells were stimulated in vitro with plate-bound anti-cd and anti-cd , and proliferation, cytokine secretion as well as phenotypic activation parameters were determined. remarkably, a clearly improved inhibition of ifn-gamma secretion was observed in cd -costimulated human memory/effector cd + t cells by compound than by classical gcs. interestingly, apoptosis and activation antigen expression were similarly regulated. improved inhibition of lymphokine secretion by compound was also seen after pma / ionomycin stimulation of human primary t cells and jurkat cells. when investigating the in vivo effects of compound and prednisolone in acute and subacute dnfb-induced contact hypersensitivity models in mice, we observed comparable efficacy for inhibition of t cell-dependent skin inflammation when treating before hapten challenge. in contrast, however, when treating around hapten sensitization markedly stronger effects were demonstrated for compound than prednisolone. when evaluating possible mechanism for the increased activity of compound in inhibition of t cell activation we got hints for a specific inhibition of the calcineurin pathway by compound which was not prevented by the partial gc receptor antagonist, ru- , in vitro. moreover, in vivo we observed less induction of il- beta and tnf-alpha by pre-treatment with compound than with prednisolone. our data indicate that the non-steroidal segra, compound , may represent a promising drug candidate for the treatment of t cell-dependent inflammatory diseases where therapy with classical gcs is hampered by t cell resistance. influenza a infection of b mice elicits robust cd + t cell responses, with virus-specific cells showing a distinct pattern of cytokine production: tnfa+ cells always express ifng; and il- + cells are contained entirely in the ifng+tnfa+ subset. interestingly, the co-expression of ifng and tnfa varies for different epitope specificities. almost all ifng+ pa -specific cells also express tnfa, but only about half of the ifng+ np -specific cells co-express tnfa. this was originally linked to the avidity of the responding population for the specific peptide/mhc complex, with the ifng+tnfa+ phenotype representing cd + t cells with higher avidity and a more differentiated phenotype. however, the same cytokine pattern is seen in adoptively transferred cd + t cells expressing a clonal tcr, implying avidity alone cannot control development of cytokine profiles. co-expression of ifng and tnfa by adoptively transferred cfse-labelled ot-i cells following infection with influenza a virus expressing ova - peptide shows a close correlation with division in vivo. early after antigen encounter ( - divisions) the vast majority of cells express only tnfa. after - divisions cells begin to co-express ifng and tnfa. the emergence of an ifng+tnfa-phenotype increases with subsequent divisions ( - divisions), indicating cytokine profile is closely linked to cell cycling, as described previously for both b cells and cd + t cells. titration of adoptively transferred ot-i cells, which controls the level of expansion in vivo, reveals that more cd + t cells develop an ifng+tnfa-phenotype with increased expansion. thus we conclude that while tcr avidity and co-stimulation can impact the differentiation of cd + t cells, expansion plays a very important role in the regulation of cd + t cell effector function. in addition to its chemo-attractant function, sdf- a (stromal-cell derived factor- a, cxcl ) has been described to costimulate cd + t cell during tcr triggering. our objective is to clarify the mechanism regulating this costimulatory activity. tcr-driven proliferation of human cd + t cells was increased by immobilized sdf- a to a level similar to that obtained with the costimulatory molecule cd . as visualized by real time confocal microscopy, t cells entering in contact with sdf- a formed a tether and displayed an active scanning activity. since sdf- a induced a similar activity in t cells stimulated with a sub-optimal dose of anti-cd mabs, it is conceivable that the sdf- a-driven scanning may favour productive tcr engagement. to test this hypothesis, we are studying the effect of sdf- a on tcr internalization, calcium mobilization, mapk activation and actin cytoskeleton reorganization. we are also studying the role of sdf- a in the context of cd + t activation by antigen-presenting cells secreting sdf- a. this study should help us to better define how sdf- a modulates cd + t cell activation beyond its chemo-attractant function. background: propolis, an ancient herbal medicine, is well known for the management of respiratory diseases. caffeic acid phenethyl ester (cape), an active component in propolis, is known to have anti-tumor, anti-inflammatory, and antioxidant properties. in this study, the effect of cape on the functions of t cells, which play the major role in chronic airway inflammation of asthma, was evaluated. method: cd + t cells isolated from human peripheral mononuclear cells by automacs were stimulated with anti-cd and anti-cd antibodies and cape for days. cytokine levels were dertermined by elisa and lymphoproliferation was analyzed by h-thymidine incorporation method. signaling pathway of t cells was studied by western blot. result: it was found that cape significantly inhibited ifn-g and il- production and lymphoproliferation in cd + t cells stimulated by anti-cd /cd . cape could inhibit nuclear factor-kb (nf-kb) activation, but not mitogen-activated protein kinase (mapk) family phosphorylation in t cells. cape could also inhibit akt phosphorylation. conclusion: these results indicated that cape inhibits cytokine production and lymphoproliferation of t cells which might be related to the nf-kb and akt signaling pathway. this study also provided a new insight into the mechanism of cape in immunology and the rationale for propolis in the treatment of allergic disorders. objective: upon activation, cd t cells express a variety of molecules on their surface, such as mhc-class ii, cd , cd , cd , whose ligands are constitutively expressed on resting t cells. whereas these molecules are physiologically expressed on antigen presenting cells, their function on t cells is not understood. we tested the hypothesis that activated cd t cells might induce t cell proliferation and differentiation from cd resting t cells through interaction of activationinduced surface molecules and their constitutively expressed ligands. methods: cd t cells from the peripheral blood of healthy donors were co-cultured with fixed activated t cells from the same donor. after days of co-culture, the phenotype of the resulting cells was analyzed by assessing their surface molecules and production of cytokines. results: cd memory t cells but not naive t cells proliferated in response to contact with activated t cells. these cells showed a mild activated phenotype assessed by the expression of cd , cd , and cd . analysis of the cytokine profile of these cells revealed the differentiation of il- -and ifn-g-double-producing cells in response to contact with th effector cells, and il- -producing cells in response to contact with th effector cells. the levels of produced cytokines were, however, significantly lower than those produced by activated cells in response to anti-cd /cd stimulation. whereas neutralization of ifn-g or il- during culture did not diminish the frequency of the arising cytokine-producing cells, separation of the responder cell population from effector cells by a transwell system led to a significant decrease of cytokine secretion. blocking particular receptor/ligand interactions by neutralizing antibodies against hla-dr, cd , cd , and cd could not prevent cytokine production induced by t-t cell interaction. however, simultaneous addition of all antibodies significantly inhibited cytokine production to - %. conclusion: interaction of cd memory t cells with activated t cells resulted in the production of the cytokines il- , il- , and ifn-g. given the immunomodulatory capacity of il- and il- , these findings might indicate a novel potential negative feedback mechanism to control t cell-driven immunity. a. nasir , s. thompson , j.j. murphy king's college london, division of immunology infection and inflammatory disease, london, united kingdom the murine bcl leukaemia cell line can be induced to undergo plasmacytoid differentiation in vitro with cytokines il- and il- and this is characterised by a marked reduction in proliferation and production of large amounts of secreted igm. these cells were observed to express significant levels of the zinc fingercontaining protein zfp l by western blot analysis. this protein is reported to act in post-transcriptional regulation of gene expression by binding to au rich elements (ares) of mrnas of certain genes and consequently promoting mrna degradation. at a cell functional level, zfp l has been described to have roles in apoptosis, proliferation and differentiation in different cellular contexts. cytokine-induced bcl differentiation was observed to be associated with downregulation of zfp l protein. in an attempt to determine whether zfp l downregulation was directly linked to bcl differentiation, a zfp l shrna expressing lentivirus (psicor) was employed to knockdown zfp l expression. this reagent downregulated zfp l expression very effectively . shrna infected cells proliferated less well than either control virus infected cells or wild-type cells with or without cytokines. zfp l shrna infected cells also produced more secreted igm per cell than either control virus infected cells or wild-type cells in the presence or absence of cytokines. these results are consistent with a role for zfp l downregulation in promoting bcl plasmacytoid differentiation. vidual lysates of peripheral blood lymphocytes (pbl) of patients with igg multiple myeloma and healthy controls were investigated for the expression of sialic acid (sa), galactose (gal) and n-acetylglucosamine (glcnac), the sugars known to specify the glycoforms of human serum igg. the degree of glycosylation and signaling status of all isolated myeloma igg bcrs were correlated and compared with the glycosylation of the igg paraproteins isolated from sera of the same patients. it was shown that bcr igg in myeloma is more heavily sialylated when compared with normal controls, that the increased sialylation of igg bcr is associated with higher levels of tyrosine phosphorylation (signaling activity) of the igg bcr supramolecular complex and that bcr igg and serum igg paraprotein from the same patient differed in all cases in the levels of terminal sugar expression. the results suggest that the development of the malignant clone in mm from postswitch b cells expressing igg bcr at their surfaces to plasma cells secreting igg paraprotein may be followed by permanent glycosylation changes in the igg molecules. caused by thapsigargin-induced release of calcium from the endoplasmic reticulum was insensitive to tpen. conclusion: the signal with fluorescent probes for the detection of calcium ions in response to thimerosal is entirely due to zinc release, and no indication for a calcium signal was detected. in light of these observations, zinc may also contribute to calcium signals caused by mercury containing compounds other than tms, and a potential involvement of zinc release in the immunomodulatory effects of these substances should be considered. although best known for its pro-apoptotic function, it seems clear now that cd (fas, apo- ) also exerts anti-apoptotic effects associated with costimulation and the induction of proliferation. we investigated effects of fas co-ligation during tcr/cd /cd -triggered activation of freshly isolated human t-lymphocytes. to this end, tcr-triggered cells were incubated in presence or absence of different ligand concentrations of anti-apo mab, faslfc or faslstrepfc fusion proteins, or leucin zipper (lz-)cd l. interestingly for all ligands tested, we could clearly demonstrate a correlation between ligand concentration and t cell response: low doses drastically augmented proliferation in the sense of costimulation, whereas high doses completely blocked tcr-induced cell proliferation without inducing cell death. the positive costimulatory effect of fas at low concentrations is associated with elevated il- and ifng production, upregulation of activation markers, adhesion molecules and cell-cycle regulating cdks and cyclins. in addition, we observed an increased activation of important signalling molecules including mapk and caspases. using pharmacological inhibitors, we demonstrate that fas is internalized upon ligation. we also observed an increased tcr internalisation following fas co-incubation potentially resulting in the generation of larger signalling platforms that allow optimal t cell activation. in stark contrast, most fas ligands at high concentrations almost completely inhibited cell proliferation of tcr-triggered lymphocytes. in this context, crucial events associated with t cell activation, i. e. tyrosine and erk / phosphorylation, the expression of various activation markers, the il- production and caspase activation were almost completely abrogated. these findings highlight that fas-triggering accelerates or blocks t cell activation, depending on the strength of the stimulus. in addition, we provide further evidence for an anti-apoptotic function of fasl during signal initiation in human t lymphocytes. sponsored by the dfg (sfb ) and the medical faculty kiel (to oj) it has been shown that glycosylation of cell surface proteins controls critical t cell processes, including homing, thymocytes maturation, activation, and cell death. plant lectins have been long used to study changes in cell surface carbohydrate structures, to identify leukocyte cell subsets, and as surrogates for authentic t cell activation stimulus. the galb , galnac-specific lectin from amaranthus leucocarpus (all) shows a differential binding pattern to murine thymocytes and peripheral cd + and cd + t cells. in addition, mitogenic stimulus increase -fold the all binding to cd + t cells. previous studies in human pbmc showed that all binds to human cd + t cells and all-binding increased after a mitogenic stimulus using total cell cultures as murine studies. these data suggest that all detects selectively activation-related changes in cd + t cell surface carbohydrate but none study has been performed to examine the all effect on human t cell activation. to examine the effect of all on human t cell activation, we analyzed the anti-cd -dependent activation of purified cd + t cells from pbmc in presence or absent of all by measuring proliferation using cfda-se staining, expression of the surface activation marker cd and calcium influx by flow cytometry. results showed that all did not induce significantly t cell proliferation or cd expression, but enhanced the anti-cd -dependent proliferation and cd expression of purified cd + t cells. analisis of calcium influx showed that all enhanced anti-cd dependent calcium influx. our findings indicated that all alone does not affect t cell activation but suggested that all induces a costimulatory effect on human cd + t cells by up-regulating t cell activation mediated by anti-cd stimulus, as further studies have to be performed to elucidate all-induced costimulatory effect. financed in part by papiit-unam (in ) a. the adaptor protein lat (linker for activation of t cells) has a prominent role in the transduction of intracellular signals elicited by the tcr/cd complex. upon tcr engagement, lat becomes tyrosine-phosphorylated and thereby recruits to the membrane several proteins implicated in the activation of downstream signaling pathways, leading to tightly equilibrated programs of activation and survival or induced cell death. the balance between cell survival and cell death is critical for normal t cell development and activation, and is maintained by signals through lymphocyte antigen receptors and death receptors such as cd receptor. it has been previously demonstrated that cd ligation in t cells induces the proteolytic cleavage of several adaptor proteins, including gads, slp- , slap- and lat. given the dual role of lat as a transducer of activation and negative signals in t cells, we have analyzed the role of the lat cleavage in t cell functions and studied the proteases responsible for this cleavage. objective: the study is designed to explore preliminarily the need of t cells for cytokines during the culture in vitro, which are associated with the activation, proliferation and apoptosis of t cells, and by detecting the expressions of il-rs, co-stimulatory molecules and apoptotic receptors/ligands onto human peripheral blood lymphocytes (hpbls). the results may lay a theoretic and experimental basis for developing the condition media qualified especially to t cell culture. methods: pbls were isolated , and cultured in different media. both immunocytochemistry staining and cell enzyme linked immunosorbent assay (celisa) were used to detect the expressions of il- r, il- r, il- r, il- r, il- r, il- r, cd , cd , cdw ( - bb), cd (fas) and cd (fasl) on hpbls in different cultured time, i. e. d, d, d, d, d, d, d and d. using typan blue staining, the living cells, dead cells and total cells of each cultured group were counted, then their cell growth curves were drawn out. to evaluate the cellular activity, growth situation and cell cycle of t cells, both mtt and fcm analysis were also performed separately. . the expressions of several membrane immune molecules on the lymphocytes in different cultured conditions. ) the expressions of membrane immune molecules before cultured. ) expressions of the membrane molecules on hpbls during culturing. % fbs rpmi group ( group), il- group, pha group... ( ) mtt assay. ( ) proliferative times and growth curves of hpbls... . during cultured in vitro, there are expression changes of the il- rs (il- ra, il- ra, il- rg, il- r, il- r, il- r, il- r), co-stimulatory molecules (cd , cd , - bb) and apoptosis associated molecules (fas/fasl) on hpbls in different time and cultured media. the expression patterns of the most molecules checked are similar in group, il- group and pha group, but the rests are different. . our data also suggest that the hpbls cultured in cd mcab+cd mcab+il +il a group has a great proliferative potential compared with the other groups. using this condition medium, may have a practical prospect to tumor therapy. . celisa will become probably an effective test to detect the expressions of membrane receptors or molecules quantitatively on a large scale. f. beceren-braun , r. tauber zentralinstitut für laboratoriumsmedizin und pathobiochemie, berlin, germany l-selectin is a leukocyte cell surface glycoprotein involved in carbohydrate-specific ligand binding which mediates tethering of leukocytes to the endothelial surface during inflammation. apart from its role in adhesion, l-selectin functions as a signal transduction molecule. crosslinking of l-selectin with antibodies or ligand binding to the receptor have been shown to elicit a wide range of cellular responses. in addition to process signals coming from outside of the cell, the intracellular part of l-selectin (lscyto) is also able to conduct intracellular signals, e. g. activates tyrosine kinase p lck and the ras/rac signalling pathway ( ) followed by mitogen-activated protein kinases ( ) and c-jun n-terminal kinase ( ), which leads to an enhanced binding of l-selectin to soluble ligands ( ). in our previous work we described an association of lscyto with isozymes of the pkc family which phosphorylate the receptor on serine residues ( ). here we show that the protein phosphatase a inhibitor phapii is a novel direct interacting partner of the lscyto. we propose a model in which the l-selectin mediated signalling is regulated by the interaction of pkc, pp a and phapii: phapii binds to the unphosphorylated lscyto. upon l-selectin crosslinking lscyto is phosphorylated, pha-pii dissociates and inhibits the phosphatase pp a. in addition we have started structural analysis to investigate ligand binding induced conformational changes of the cytoplasmatic domain of l-selectin. v. heissmeyer , e. glasmacher helmholtz center munich, molecular immunology, munich, germany during self-antigen recognition, roquin dependent posttranscriptional downregulation of icos prevents t cell help to b cells and autoantibody production. the molecular mechanism by which roquin interferes with icos translation remained unclear. we have identified two critical regions in roquin. the amino-terminus is required for rna binding and can be functionally replaced by conserved sequences from its paralog mnab. the carboxy-terminus mediates p body localization and has specialized in roquin for efficient repression of icos in t cells. using knockout cells of dicer or ago - genes, we prove that roquin mediated repression of icos occurs in the absence of mirisc formation. instead, roquin function required intact p bodies, and was impaired after knockdown of lsm and rck or expression of dominant-negative gw . interestingly, roquin activity is blocked through induced mirisc formation implicating the mutual regulation of different mechanisms of posttranscriptional gene silencing in immune responses. s objectives: upon encountering their antigens, naï ve t cells are activated and driven to clonal expansion and differentiation into armed effector cells. according to the two-signal hypothesis, the induction of an optimal cd + t-cell immune response requires both antigen-specific and co-stimulatory signals. in contrast, stimulating naïve cd + t cells with specific antigens and costimulatory signals is insufficient to induce optimal clonal expansion and effector functions. thus, cd + t cells require additional signals for full activation and further differentiation into effector cells. methods: in this study, we adopted an in vitro approach to dissect the cellular and molecular requirements for cd + t-cell activation and differentiation. naïve cd l hi cd lo cd + t cells were sorted and stimulated by anti-cd and anti-cd antibodies. results: firstly, we show that the activation and differentiation of cd + t cells require il- provided by activated cd + t cells at the initial priming stage after stimulation. secondly, this critical il- signal is delivered through il rbg of cd + cells and is independent of il- ra. besides promoting cell proliferation, il- stimulation increases the amount of ifng and granzyme b produced by cd + t cells. conclusion: therefore, our studies demonstrate that a full cd + t-cell response is elicited by a critical temporal function of il- released from cd + t cells, providing mechanistic insights into the regulation of cd + t cell activation and differentiation. most antigenic peptides recognized by cd t lymphocytes are produced through degradation of intracellular proteins by the proteasome. however, some antigenic peptides are produced by a proteasome-independent pathway, which is poorly characterized. mage-a - is a tumor antigenic peptide presented by hla-a and widely used for vaccination of melanoma patients. we observed that proteasome and tppii inhibitors failed to block presentation of the antigen by tumor cells. however, processing of this peptide occurred in the cytosol because tap inhibition prevented its presentation. to characterize the cytosolic peptidase producing mage-a - we setup an in vitro digestion assay using a -mer precursor peptide encompassing the sequence of the antigenic peptide. we observed that only the cytosolic fraction was able to produce the antigenic peptide from this precursor. this production was abolished by treating the cytosolic fraction with o-phenanthroline, a broad-spectrum inhibitor of metallopeptidases. this inhibitor also blocked the presentation of mage-a - by tumor cells. by electroporating hla-a cells with a precursor peptide blocked at the c-and the n-terminus, we could exclude the involvement of exopeptidases in the processing of this peptide, and conclude to a major role of a cytosolic metalloendopeptidase. one such enzyme is insulin-degrading enzyme (ide). we observed that depletion of ide abolished the capacity of a cytosolic fraction to produce the antigenic peptide. furthermore, recombinant ide was able to produce the peptide in vitro from the precursor peptide. lastly, silencing of ide with sirna reduced presentation of the peptide by tumor cells. with tppii, ide is the second example of a proteasomealternative pathway in the production of class-i restricted peptides. antigen-specific t cell based tumor immunotherapy, though extensively studied, has only been of limited clinical success so far. immune escape, due to impairment of hla dependent tumor epitope presentation is believed to be one major reason for this failure. to identify novel mechanisms by which tumors can become refractory to immune elimination, human melanoma cells of different donors expressing the transmembrane mart- /melan-a tumor antigen were exposed to two or three rounds of brief co-culture with mart- /melan-a - specific cytotoxic t lymphocytes (ctls). immune selected melanoma cell clones, being resistant to lysis by mart- /melan-a - ctls due to impaired epitope processing were further investigated. our results show that in addition to previously described immune evasion mechanisms like down regulation of mhc class i and mart- expression, the ifn-gamma independent endoplasmic reticulum associated degradation (erad) pathway is crucial for mart- /melan-a - epitope generation. moreover, deregulation of several erad components is essentially responsible for the observed immune escape of the immune selected melanoma cells. in support, re-expression of down-regulated erad components in ctl-resistant melanoma cells completely restored immune recognition by mart- /melan-a - ctls. thus, our studies demonstrate for the first time that erad not only plays a central role in the production of cd + t cell epitopes from membrane proteins but also contributes to tumor escape mechanisms by cancer immunoediting. studies of t cell responses to hen egg lysozyme suggest that several conformers of peptide-mhc class ii complexes can be generated for a single peptide epitope and that distinct cd t cell repertoires known as type a and type b recognise these different conformers (lovitch and unanue, immunol rev : - , ) . type a t cells recognise peptide-mhc complexes generated from intact proteins after intracellular antigen processing under h -m (dm) control, where as type b t cells respond to synthetic peptides in the absence of dm editing, but fail to respond to processed intact protein. type b t cells escape thymic deletion in mice (petersen et al., immunity : - , ) , with implications for autoimmunity. so we studied whether type a and type b recognition patterns occur in t cell responses to autoantigens such as the rheumatoid arthritis (ra)-associated proteoglycan aggrecan, and whether naturally occurring extracellular ligands that activate type b t cells are found in inflamed joints. lymph node cells from aggrecan-immunised balb/c mice proliferated in response to intact aggrecan and to the immunodominant peptide - , whereas peptide-immunised mice responded to peptide, with low or absent responses to intact aggrecan. t cell hybridomas generated from - peptide-immunised mice either recognised peptide only (the majority) or peptide and intact aggrecan (the minority), a pattern consistent with type a and type b t cell recognition. responses to staggered and alanine-substituted peptide sets showed that type a and b t cell hybridomas recognized the - epitope in the same register, consistent with this peptide epitope binding to mhc in distinct conformers. type b t cell hybridomas recognised aggrecan fragments in supernatants from cartilage degraded by stimulation with proinflammatory cytokines that induce raassociated aggrecanases. our data suggest that inflammation generates extracellular peptides that activate type b t cells. we are also characterising human type b t cell responses as well as searching for type b t cell ligands in synovial fluid from ra patients. we propose that extracellular cartilage degradation generates ligands that induce autoreactive type b t cell responses which participate in the pathogenesis of autoimmune arthritis. a to cope with mhc i antigen presentation hcmv encodes for several post-translational strategies which have been extensively studied in transfected cells. in this study we analysed the plc in naturally hcmv-infected cells and monitored the composition of the plc throughout hcmv replication. metabolic labeling experiments revealed the absence of tapasin incorporation into the plc. in contrast, western blot analysis demonstrated only a slow decline of tapasin steady state levels in infected cells, suggesting a blocked synthesis rather than degradation. tapasin mrna levels were found to be continuously downregulated during infection, however, the tapasin transcripts were stable and long-lived. taking advantage of a novel method, in which newly transcribed rna is selectively labeled and analysed (dölken et al, ), we found, after an initial induction at hrs p. i., a strong inhibition of tapasin transcription at hrs p. i. furthermore, also reduction of tap and tap transcription was observed contrasting to the elevated levels of erp and mhc i transcripts. importantly, ectopic expression of tapasin restored the incorporation of tapasin into the plc in hcmv-infected cells. the data indicate that hcmv controls mhc i antigen presentation also on a transcriptional level and show for the first time the regulation of tapasin transcription as a viral immune evasive function. most peptides presented by mhc class i molecules are produced by the proteasome during degradation of intracellullar proteins. two main proteasome types have been described, differing in their content of catalytic subunits. the standard proteasome comprises catalytic subunits ß , ß and ß , which are replaced by their ifng-inducible counterparts ß i, ß i and ß i in the immunoproteasome. the thymoproteasome represents a third proteasome type, where catalytic subunit ß i is replaced by a thymus-specific subunit ß t. the standard proteasome is present in most tissues, the immunoproteasome in found in cells exposed to ifng and in dendritic cells, while the thymoproteasome is found exclusively in the thymus. we produced a panel of novel antibodies that recognize subunits ß i, ß i, ß i and ß in their native form. using these antibodies for successive immuodepletions performed on tumor lysates, we identified two new proteasome types that are intermediate between the standard proteasome and the immunoproteasome, i. e. they contain only one or two the three catalytic subunits of the immunoproteasome. one comprises ß ,ß and ß i (single intermediate proteasome), and the other comprises ß i, ß and ß i (double intermediate proteasome). we quantified these intermediate proteasomes in a series a tumor lines of various origins, and found that they represent - % of the total proteasome content of those tumor cells. they are also present in dendritic cells, where they represent about % of the proteasome content. we characterized the activity of these intermediate proteasomes, not only on fluorogenic substrates but also on actual antigenic peptides recognized by anti-tumor ctl. with respect to antigens known to be processed differently by the standard and the immunoproteasome, the intermediate proteasomes often behaved like the immunoproteasome. importantly, we identified two tumor antigens that are processed exclusively by either the single intermediate proteasome ( tapasin is a multi-functional protein dedicated to mhc-i biosynthesis; it serves as a structural component in the so called mhc-i peptide loading complex (plc), as a chaperone putatively acting as an active peptide editor and mhc-i quality control mechanism, as an er retention signal for immature mhc-i, and as a chaperone stabilizing tap expression and increasing tap-performance. furthermore, tapasin has been found outside the er, where it has been suggested to regulate retrograde transport of escaped immature mhc-i back to the er from the trans-golgi compartment. the role of tapasin as an active peptide-editor has been debated and we here set out to study the effect of tapasin on binding of peptides of both high-and low-affinity to a human mhc-i allele (hla-a* ) using protein interaction-and peptide-competition assays. specifically we wanted to in detail compare the binding of two peptides of the same affinity. at high concentrations all of the tested hla-a* binding peptides (tap-transported high-affinity peptide (ttp-ha), signal-peptide of high affinity (sp-ha), tap-transported mediumaffinity peptide (ttp-ma)) induced dissociation of hla-a* from tapasin, but only ttp-ha dissociated hla-a* from tapasin at lower concentrations. using peptide-competition assays against ttp-ma, a peptide of lower affinity, we could show that ttp-ha, one of the two peptides of equally high affinity was a significantly more efficient competitor than peptide sp-ha. however, analysis of mhc-i peptide loading in the tapasin-negative cell line lcl- . -a showed no competitive advantage of ttp-ha compared to sp-ha supporting a role for tapasin as a selective facilitator of mhc-i peptide binding. in conclusion, we here show that peptides of different affinities dissociate hla-a* from tapasin in a dose-dependent manner, and that tapasin facilitates ttp-ha, but not sp-ha replacement of a lower-affinity peptide (ttp-ma). together these data strongly suggest a role for tapasin as a selective facilitator of peptide binding to mhc-i. importantly, this study implies that criteria in addition to peptide-affinity determines whether tapasin will promote peptide binding to hla-a* . m. basler , , c. lauer , m. groettrup , biotechnology institute thurgau, kreuzlingen, switzerland, university of constance, division of immunology, department of biology, konstanz, germany two lmp -dependent antigens have been described that relied on the 'structural presence' of lmp in the proteasome but not on the activity of lmp . here we have investigated processing of the h- d b -restricted uty - epitope of the male minor antigen uty reported to be lmp -dependent. using splenocytes from lmp -/-, lmp -/and mecl- -/mice we found that the uty - epitope requires lmp and lmp but not mecl- . curiously, a selective lmp inhibitor did not interfere with uty - presentation. objective: we investigated why the deletion but not the inhibition of lmp interferes with uty - presentation. we hypothesized that the 'structural' requirement for lmp is based on replacement of the caspase-like activity of b in the proteasome. methods: it was determined if t a mutants of lmp and/or b can rescue the uty - epitope. we used a b -selective inhibitor to determine if the inhibition of the caspase-like activity of b preserves the epitope. finally we determined by mass spectrometry if the uty - epitope embedded within a mer precursor peptide is differentially cleaved by lmp -deficient and proficient immunoproteasomes in vitro. results: we found that t a mutants of lmp and b rescue presentation of uty [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . also inhibition of cells with a b -selective inhibitor preserves uty [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] presentation. an aspartate in position of the uty - sequence wmhhnmdli is preferentially used as a cleavage site by lmp -deficient but not half as frequently by lmp -proficient immunoproteasomes. the generation of the uty - epitope relies on the replacement of the caspase-like activity of b by lmp because the b activity destroys the uty - epitope. this is the first example for the 'structural' requirement of lmp for generation of an epitope. eliminating the activity of their constitutively expressed homologous subunits may explain the requirement for immuno-subunits of the proteasome also for the generation of other antigens. thus we have discovered a so far unrecognized mechanism how lmp and perhaps also lmp and mecl- exert their function in antigen processing. a. linnemann , a. musiol , r. lindner hannover medical school, cell biology, hannover, germany, hannover veterinary school, graduate school for biomedical sciences, hannover, germany objectives: mhc i molecules are constitutively endocytosed and recycled to the cell surface. this process is required for the turnover of aged molecules and for some forms of cross-presentation of exogenous peptides on mhc i. in fibroblasts, mhc i is known to internalize via a clathrin-independent, arf -regulated pathway that is highly sensitive towards the cholesterol-sequestering drug filipin. although this observation suggests that membrane rafts are involved in the internalization of mhc i, no evidence for an association of mhc i with membrane rafts has been found in this cell type. methods: a novel detergent extraction protocol was used to investigate the association of mhc i with membranes rafts. endocytosis of mhc i was measured with a biotinylation-based biochemical assay and with a cell biological assay employing confocal laser scanning fluorescence microscopy. for characterization of mhc i internalization pathways, dominant negative mutants of gtpases (dynamin and arf ) were overexpressed in t fibroblasts. we show that antibody-mediated oligomerization of mhc i in t fibroblasts shifted this molecule from soluble fractions to detergent-resistant membranes. this change in detergent resistance coincided with a switch to a novel internalization pathway: oligomerized mhc i internalized faster and more completely and arrived at different endocytic organelles. the two mhc i internalization pathways differed in their sensitivity towards dominant negative arf : endocytosis of oligomerized mhc i was not affected, whereas non-oligomerized mhc i endocytosed more slowly and changed its subcellular distribution. unlike transferrin receptor internalization, none of the mhc i endocytosis pathways was affected by overexpression of dominant negative dynamin suggesting internalization mechanisms independent of clathrin, caveolin and rhoa. conclusion: we propose that mhc i switches from an arf -regulated to a novel, arf -independent internalization pathway in response to a change in membrane environment induced by oligomerization of mhc i. since mhc i is one of the cellular receptors for sv virus and since sv binding triggers mhc i oligomerization, this novel pathway may be involved in sv uptake. antigen cross-presentation in dendritic cells is a complex intracellular membrane transport process, but the underlying molecular mechanisms remain to be thoroughly investigated. in this study, we tested the effect of sirna-mediated knockdown of rab gtpases, the key regulators of membrane trafficking, on antigen cross-presentation. twelve rab gtpases were identified to be associated with antigen cross-presentation, and among which rab b, c were found to be colocalized with mhc class i molecules at perinuclear tubular structure. tracing with fluorescence protein tagged beta -microglobulin demonstrated that the mhc class i molecules were internalized from plasma membrane to rab b and rab c postitive compartment. moreover, the recycling ligand transferrin was enriched in the rab b or c positive vesicles. furthermore, the rab b, c positive compartment were colocalizd with a fraction of rab a at a juxtaposition of phagosomes. together these data demonstrate that rab b and rab c positive vesicles is involved in and may constitute the recycling compartment of exogenous antigen crosspresentation. introduction: while the proteasome is thought to generate most of the hla peptidome, other proteases were also proposed to be significant for this process. both t cell based assays and proteasome inhibitors were used in the past to follow presentation of specific model hla peptides. the hla-peptidomes presented at the cell surface depend on the rate of peptide generation within the cells, their transport from the cytoplasm and loading in the er, binding stability at the cell surface and retrograde uptake of the hla molecules back into the cytoplasm. objectives: the role of the proteasome in hla peptide presentation was evaluated using proteasome inhibition, while following the turnover rates of the entire hla peptidome. the peptidomes of both the authentic membranal and a recombinant soluble form of the hla molecules were collected for analysis at different time points after the inhibition of the proteasomes. the turnover rates of the hla-peptides were followed using pulse-chase analysis with stable-isotope labeled amino acids concurrently with epoxomicin treatment. the hla molecules were immunoaffinity purified and the peptides were analyzed by capillary chromatography and orbitrap tandem mass spectrometry. both the endogenous membranal and soluble mhc molecules were studied in parallel from the same cells. peptides were identified by their ms/ms fingerprints and the turnover rates were determined by the shift from the 'light' to the 'heavy' leucine of each peptide. results: a few thousands hla-peptides were identified, and for a large portion of them, the turnover rates could be defined. proteasome inhibition did not affect the complexity of the hla peptidomes or reduced significantly the amounts of membranal hla molecules. many peptides were labeled relatively rapidly with heavy leucine, indicating that the hla peptidome contains also the products of newly synthesized and rapidly degrading proteins. the source proteins of the hla peptides seemed to have similar biological functions and cellular origins in both the inhibited and untreated cells. the centrality of proteasomal degradation in hla-peptide presentation is put into doubt and the role of the proteasome in the generation of each peptide and each cleavage site can be defined. epstein-barr virus (ebv) is a ubiquitous y-herpesvirus, infecting over % of adults worldwide. it can cause mononucleosis and several lymphomas and carcinomas, reflecting the tropism of the virus for b-lymphocytes and epithelial cells. ebv persists for life despite the presence of virus-specific adaptive immunity, indicating that it has evolved strategies to counter the host immune response. one such strategy is the persistence of the virus in the latent phase of its life cycle, where expression of viral proteins is minimized. however, for ebv replication and dissemination to occur, it must enter the lytic phase. here, over viral proteins are expressed, creating many potential antigens for presentation to cytotoxic t -lymphocytes. ebv can circumvent possible eradication by cd + t lymphocytes during the lytic phase by interference with antigen processing and presentation through hla class i in the infected cell. the viral proteins bnlf a and bglf have been shown to achieve this by impairing peptide-loading of hla class i and inducing the degradation of mrnas encoding hla molecules, respectively. a third ebv lytic phase protein, the g-protein coupled receptor (gpcr) bilf , has now been found to down-regulate cell surface hla class i expression (zuo et al, plos pathogens ). this represents a novel function for a virally-encoded gpcr. bilf is expressed early in the ebv lytic cycle and is localized predominantly at the cell surface. there it can interact with hla class i molecules, resulting in their internalization and lysosomal degradation. this has a profound effect on the ability of cytotoxic t-lymphocytes to recognize cells displaying antigens derived from ebv proteins. interestingly, bilf displays a differential effect on distinct hla class i haplotypes. furthermore, we have shown that the intracellular c-terminal tail of bilf is required for its effect on hla class i expression. however, the ability of the gpcr to activate intracellular signaling pathways is dispensable in this regard. thus, by reducing the cell surface expression of hla class i molecules, ebv bilf can hinder the recognition of virally-infected cells by cytotoxic cd + t lymphocytes, thereby facilitating the evasion of adaptive immune mechanisms. t lymphocytes mature in the thymus, generating a non-dangerous t cell repertoire. for the adquisition of tolerance, thymocytes suffer positive and negative selection processes. during t cell maturation, tcrs contact with different mhc-peptide complexes on the surface of pressenting cells, allowing tolerization against self proteins. to obtain a non-self-reactive t cell repertoire, it is of most importance that pressenting cells in the thymus express a repertoire of mhc-peptides complexes representative of the proteins that t cells will found in periphery, including tissue restricted antigens (tras)-derived peptides. in the last decade, transcription of tras in thymus has been well-reported. furthermore, the expression of many genes codifying for tras are dependent.on the expression of the autoimmune regulator (aire). aire is mainly expressed in medullar thymic epithelial eells (mtecs), which are involved in negative selection. so far no systematic study have been made to describe the peptide repertoires associated to hla molecules in the thymus. in addition, although many data of tras transcription in thymus have been reported, much less work has been performed at biochemical level, and to our knowledge, no hla ligand arising from any tra have been reported in thymus. in this report we present the results of analyzing the hla-dr-associated peptide repertoire from whole tissue samples of different human thymi by mass spectrometry. we describe natural ligands, including two peptides derived from semenogelin- , a tissue restricted antigen expressed mainly in the prostate, and present in semen. using qpcr we demonstrate that semg is transcribed in thymus from both male and female individuals. finally, we detected the semg mrna expression in a fraction enriched in stromal cells, but not in the thymocyte fraction of the thymi. the proteasome is the major protease complex for non-lysosomal protein degradation in eukaryotic cells, which generates most peptides for mhc class i antigen presentation. vertebrates express two sets of catalytic subunits, constitutive (beta , beta , beta ) and immuno-subunits (beta i, beta i, beta i). deficiency in beta i results in profound reduction of mhc class i expression, demonstrating the significance of this subunit for efficient antigen presentation. currently, this is attributed to the specific proteolytic activity of the beta i subunit, its role in the maturation of immunoproteasomes or both. however, re-expression of catalytically inactive beta i subunits is capable to rescue antigen presentation suggesting that the proteolytic activity of this subunit is not limiting in this process. here, we show that following infection with listeria monocytogenes induction of beta i expression increases the cellular proteasome content in the infected organs. our results indicate that this is due to the high chaperone activity of its propeptide which drives proteasome neosynthesis and thus enhances the overall proteasome quantity. further, mhc class i antigen presentation on beta i-deficient cells could be restored by treatment with d t, which increases the amount of proteasomes independent of beta i via induction of mixed proteasomes containing beta i, beta i and beta . consequently, not the lack of the specific proteolytic activity of beta i or immunoproteasomes, but the reduced proteasome quantity in beta i deficient cells is the major limiting factor for mhc class i cell surface expression. . we have previously shown that lc, in contrast to ddc, do not express cell surface tlr , and , which results in their inability to respond to both gram-positive and gram-negative extracellular bacteria in terms of maturation into immuno-stimulatory cells and production of inflammatory cytokines. therefore, the question remained what the role is of lc in class ii mhc-mediated activation of anti-bacterial t cells. we determined the capacity of ddc and lc to internalize and process whole bacteria and present bacterial antigens to cd + t cells. in vitro generated lcs and ddcs were cocultured with gfp-expressing bacteria and subsequently analysed by clsm and facs for their uptake capacities. furthermore we investigated their capacity to stimulate autologous bacteria-specific t cell lines as a measure for antigen presentation. results: we found that lc are principally able to internalize bacteria, but far less efficient than ddc. moreover, visualisation of bacterial uptake by em revealed different uptake mechanisms by lc and ddc. both in lc and ddc internalized bacteria were detected in the endosomal and lysosomal compartments of the mhcii processing route. nevertheless, presentation of bacterial antigens by lc on mhcii was inefficient compared to that of ddc, as indicated by a low capacity to activate autologous bacteria-specific cd + t cells. the presence of exogenous tlr and tlr ligands did not overcome the differences between lc and ddc, indicating that the impaired capacity to internalize and process bacteria and activate bacteria-specific t cells is not due to the lack of tlr signalling or insufficient expression of co-stimulatory molecules, but could be an intrinsic characteristic of lc. conclusion: we propose that the epidermis of the skin is an immune-privileged site where lc play a minor role in anti-bacterial immunity and may play a role in inducing tolerance to the bacterial skin flora by steady-state presentation of antigens from commensal skin bacteria. e. james , i. bailey , t. elliott university of southampton, cancer sciences division, southampton, united kingdom regulatory t cells (tregs) play a pivotal role in the suppression of tumour specific t cell responses. depletion of tregs in balb/c mice results in a robust immunity to the normally poorly immunogenic ct colon carcinoma. this response is long lasting and mediated by both cd and cd t cells. importantly, the treg depleted ct specific immunity is cross-protective; capable of mediating rejection of tumour lines of different histological origins (a , c , bcl , renca) implying a broader repertoire of response. we have characterised one of these cross-protective antigens, gsw , which is h -d d restricted. analysis of the generation of gsw in ct revealed that the peptide is susceptible to over-processing by the er-resident aminopeptidase eraap. inhibition of eraap in ct cells substantially increased the amount of gsw present, observed by increased t cell responses to the tumour in vitro and hplc analysis. this increase was in spite of an overall reduction of mhc class i molecules at the cell surface. to investigate whether the increase in immunogenicity following knockdown of eraap would protect mice, we generated stable eraap knockdown (kd) ct and immunised balb/c mice. greater than % of mice injected with eraap kd ct were found to reject the tumour. analysis of t cell responses revealed the presence of gsw -specific t cells, however, these responses were small ( . - %). this compared to a much larger response to ct (˚ %). preliminary results indicate that the majority of the t cell responses (non-gsw -specific) in these mice are directed toward unstable peptide/mhc complexes, possibly indicating presentation of n-terminally extended peptide antigens. this highlights manipulation of the peptide repertoire as a potent tool for the generation of t cell responses in vivo. minor histocompatibility antigens play important roles in the outcome of stem cell and organ transplantation as they are involved in the development of graftversus-host-disease and in the graft-versus-tumor reactivity in hla-identical stem cell transplantation [ ] . the di-allelic hla-a restricted minor histocompatibility antigen ha- locus codes for the highly immunogenic ha- his and the non-immunogenic ha- arg nonapeptides, differing in one amino acid. the only difference that could explain the absence of the ha- arg immunogenicity was the estimated numbers of cell surface presented copies i. e. /cell for ha- his and less than / cell for ha- arg [ ] . as ha- his/arg is hematopoietic system specific and shows additional expression on epithelial cancer cells while absent on the normal epithelial cell counterpart, the ha- his allele is currently used for boosting the graft-versus-tumor responses after hla matched ha- mismatched stem cell transplantation. to elucidate the mechanisms underlying the differential cell surface presentation of the ha- allelic peptides, we investigated the impact of the ha- his/arg polymorphism on molecular and cellular processes involved in the intracellular generation and stable cell surface presentation of hla class i-bound peptides. therefore, proteasome-mediated digestion experiments, tap translocation analyses, and hla-dissociation assays with ha- his and ha- arg peptides were performed. moreover, the crystal structures of hla-a in complex with either ha- his , ha- arg or a ha- variant with a citrulline residue at position were determined in order to obtain atomic level insights into the conformation of the hla-a /ha- peptide complexes. our results exclude a role for antigen processing in preventing ha- arg to be presented at the cell surface and both the structural and hla-dissociation data clearly show that the lack of cell surface expression essentially results from an increased instability of the ha- arg allele in the hla-a peptide binding groove [ ] . they provide a rationale for the lack of ha- arg peptide immunogenicity essential for the choice of tumor peptides for stem cell based immunotherapeutical application. proteasomes play an important role in mhc class i antigen processing. exposure of cells to proinflammatory cytokines such as tnfa or ifng leads to the expression of three facultative catalytic proteasome subunits (i. e. immunosubunits) that replace the constitutively expressed subunits in the cellular proteasome population. immunoproteasomes generate many pathogen-derived cd t cell epitopes with high efficiency and thereby shape the specificity of the pathogen-specific cd t cell response. on the other hand, immunosubunit expression is not essential for development of cd t cell-mediated protective immunity, thus the physiological relevance of these cytokine-induced proteasome subunits remains unclear. we observed that mice that lack the immunosubunits lmp (ib ) and mecl- (ib ) develop a variety of autoimmune responses, including a latent form of t d (or insulin dependent diabetes mellitus, iddm), following irradiation and bone marrow reconstitution. iddm development in these mice is characterized by inflammation of the islets of langerhans, glucose intolerance and increased water consumption, and is dependent on the presence of cd but not cd t cells. a cd t cell epitope, encoded by the islet beta cell-expressed "islet-specific glucose- -phosphatase catalytic-subunit-related protein" (igrp) mrna, was identified as an important target of the cd t cell response. this epitope, like many other known diabetes-associated epitopes, binds its presenting mhc class i molecule with low affinity. as t cells specific for low affinity binders most likely can escape central and peripheral tolerance while t cells specific for high affine binders do not, we postulate that inflammation-induced immunoproteasome expression primarily functions to replace self-peptides that are derived from tissue-associated antigens and bind mhc class i molecules with low affinity, by a higher affine peptide species towards which t cell tolerance exists. thus, the inducible proteasome subunits may play an important role in immune regulation, by removing the targets of potential auto-immune cd t cells that enter inflamed tissues. endocrine epithelial cells, targets of the autoimmune response in thyroid and other organ-specific autoimmune diseases, express hla-ii molecules with compact conformation and are therefore expected to stably bind autologous peptides. the role of these molecules is not known but they could be involved in the maintenance and regulation of the in situ autoimmune response. to study in situ t cell responses without characterizing self-reactive t cells, we have identified natural hla-dr-associated peptides from autoimmune organs that will help finding peptide-specific t cells in situ. here we report the first analysis of hla-dr natural ligands from ex-vivo graves' disease-affected thyroid tissue. using mass spectrometry, autologous peptides were identified from hla-dr-expressing cells, including thyroid follicular cells, some corresponding to predominant molecules of the thyroid colloid. most interestingly, eight of the peptides derived from a major thyroid autoantigen, thyroglobulin. cell-free in vitro binding assays were performed with the thyroglobulin peptides and some other thyroid-eluted peptides as controls, to identify to which hla alleles were these peptides associated in vivo. all but two of the thyroglobulin peptides showed low binding with the corresponding alleles. the two peptides with relatively high binding affinity were presented in the context of dr and dr . analyzing the digestion patterns used for the generation of the thyroid peptides, a preferentially cleavage after a lys and arg was observed for all of them, independent of the restricting allele. our data demonstrate that although the hla-dr-associated peptide pool in autoimmune tissue mostly belong to abundant ubiquitous proteins, peptides from autoantigens are also associated to hla-dr in vivo and therefore may well be involved in the maintenance and the regulation of the autoimmune response. the t cell response generated following herpes simplex virus type (hsv- ) infection is known to be crucial in the clearance of replicating virus and in limiting the severity of infection. despite this, the relative contributions of cd + and cd + t cells in hsv- immunity have yet to be clearly elucidated. to better understand the role of hsv- -specific cd + t cells in immune control we have identified a amino acid epitope derived from glycoprotein d of hsv- . following flank infection, gd-specific cd + t cells were first detected in the draining brachial and axillary lymph nodes (ln) -days post-infection (pi), peaking at day and declining thereafter. gd-specific cd + t cells were first recovered from the spleen, skin and dorsal root ganglia (drg) at day pi and peaked at day . while hsv-specific t cells were first observed in the draining ln at day pi, hybridoma assays showed ex vivo presentation of the gd epitope by brachial ln cells as early as days pi, with peak activity days pi before declining to background by day . however presentation of the gd epitope was much more prolonged in vivo as proliferation of transgenic gdspecific cd + t cells was observed up to days post-infection in the brachial ln. ex vivo analyses suggest that only cd c + cells were involved in gd antigen presentation at days , and post-infection. subdivision of dendritic cells (dcs) populations indicated that both skin-derived dcs and cd a + dcs can present the gd antigen to cd + t cells at day pi, whereas by day pi the skin-derived dcs were the predominant population presenting the gd epitope. together these data show that following hsv- infection, antigen presentation is initiated rapidly and persists well after clearance of replicating virus. furthermore, we present evidence that different dc populations have distinct roles in the presentation of viral antigens and that they may vary during the course of infection. complementary zippers induced complete dimer formation, whereas identical zippers impaired stable interactions of the tagged peptidases. we also verified that the zippers did not influence the substrate "preferences" of the respective erap. our results from in vitro digestions suggest that the stabilised heterodimer is significantly more efficient in the production of a model epitope than the mix of monomeric erap and erap unable to form dimers. this observation is not due to mere thermodynamic stabilisation but involves positive cooperative effects in the heterodimers. conclusion: allosteric interaction of erap /erap in heterodimeric complexes enhances the global efficiency of precursor peptide trimming in the human er. during the biogenesis of class i molecules, newly synthesized heavy chains fold and acquire disulfide bonds while interacting with the lectin-chaperone calnexin (cnx) and its associated thiol oxidoreductase erp . upon assembly of the heavy chain with b m, the class i molecule enters a peptide loading complex (plc) that consists of the tap transporter, tapasin, the calnexin homologue calreticulin (crt) plus associated erp . both crt and erp are required for efficient assembly of peptide-loaded class i molecules and their subsequent expression at the cell surface. we examined functional sites on crt and erp to gain insights into their mechanisms of action in class i biogenesis. for crt, its lectin function is thought to be crucial for its association with class i molecules. however, when crt mutants lacking lectin function were expressed in crt-deficient cells, they completely complemented all class i biosynthetic defects. thus polypeptide-based contacts either mediated through erp or directly between crt and the heavy chain are sufficient to effect the chaperone and quality control functions of crt in class i biogenesis. we also tested the notion that erp must be recruited by cnx or crt to function on class i molecules. we found that the rates of heavy chain disulfide formation were normal in cells lacking cnx, crt or both chaperones. furthermore, an erp point mutant that fails to bind to cnx or crt was just as effective as wild type erp in normalizing rates of disulfide formation. we conclude that erp does not require recruitment by cnx or crt and likely acts directly on class i heavy chains to promote disulfide formation. furthermore, in cells expressing the erp point mutant, class i heavy chains, crt and the tapasin-erp disulfide conjugate were present at normal levels in the plc, indicating that the interaction between erp and crt is not required for plc assembly. finally, we show that mutations that destroy the enzymatic function of erp have no effect on plc stability or class i surface expression, suggesting that erp plays a structural as opposed to catalytic role in plc function. autoimmune pancreatitis (aip) underlies - % of cases of chronic pancreatitis and is characterized by prominent lymphocytic infiltration. a strong association of aip with the hla-drb * /dqb * haplotype has been reported, but identification of the predisposing hla gene(s) has been precluded by strong linkage disequilibrium. here, we show that hla-dr* transgenic ab nod mice suffer from aip and additional pneumonitis after sublethal irradiation and adoptive t cell transfer from syngenic donors, leading to complete pancreatic atrophy. pancreas histology is characterized by destructive infiltration of the exocrine tissue with cd + and cd + t cells, b cells and macrophages. mice with complete pancreatic atrophy have reduced serum lipase activity, develop fat stools and loose weight on regular chow. hla-dr* transgenic mice (cd + t cell competent) develop aip even unprovoked, similar to ab nod mice (cd + t cell deficient), while hla-dr* , hla-dq or hla-dr* /dq (double-) transgenic controls all remain normal after same treatment. we conclude that hla-dr* fails to protect from aip, likely due to defects in the induction of cd + regulatory t cells. our results identify hla-dr* as a prominent risk factor for aip on the hla-drb * /dqb * haplotype. this humanized mouse model should be useful to study mechanisms that underlie the hla association of autoimmune diseases, but also immunopathogenesis, diagnostic markers and therapy of human aip. s. khan , c. britten , h. overkleeft , g. van der marel , k. melief , d. filippov , f. ossendorp leiden university medical center, section tumorimmunology, leiden, netherlands, leiden university, biosynthesis group, leiden, netherlands objective: we have targeted peptide antigens to dendritic cells by the use of synthetic peptides chemically coupled to synthetic tlr ligands to study the impact on mhc class i and class ii antigen presentation. the potency of the vaccine was addressed by monitoring antigen presentation, priming of t-cells and tumor protection. results: our data show that this type of targeting of peptides greatly improves antigen presentation and t-cell priming compared to free peptide. vaccination of mice with the tlr-ligand peptide conjugates induced high numbers of functional cd and cd t-cells that could protect mice for aggressive melanoma. this potency relies on tlr signaling since peptide coupled to a non-functional tlr ligand was unable to support induction of specific t-cells. these data indicate that simultaneous encounter of antigen and a maturation signal are crucial for optimal t-cell activation by dendritic cells, and show the potency of tlr-l peptide conjugates as a vaccine modality. y. shi , , x. hu , , a. kawana- tachikawa objectives: nef protein of human immunodeficiency virus (hiv) holds some important immunodominant ctl epitopes. two overlapping -mer and -mer epitopes (rypltfgwcf (nef - ) and rypltfgw (nef - )) were found to be presented by hla-a* and some immune escape mutants of these two epitopes have also been found in some patients, e. g. y f, y w, t c, f l, w r, f r, f y etc. or their combinations. it's important to study the molecular basis of the peptide being displayed on the cell surface, through which we can analyze the mechanism of immune escape of hiv. methods: refolding method was used to attain the soluble protein pmhc. crystals are grown using hanging drop vapor diffusion method and x-ray diffraction technology is used to determine the structure. we have determined six peptide-mhc(pmhc) structures containing nef - (wild type) and its four mostly common immune escape mutants (y f, t c, y f&t c, f l), and also nef - (wild type). we found that there was little difference between the nef - (wild type) and nef - (y f) when they were displayed in the peptide-binding groove of mhc molecule, except water molecule distribution near the anchor residue y or f . interestingly the central bulge region of the peptide was becoming very flexible for the nef - (t c) and nef - (y f&t c), which may affect the binding of peptide and the recognition of t cell receptor. for nef - (f l), the side chain of l was more flexible compared to the nef - (wild type). alignment of the nef - and nef - showed that the nef - became flat and the side chain of f was not solvent-exposed due to shortening of the length of the peptide. conclusion: as the peptide nef - was featured, while the peptide nef - was featureless, so the different topology of these two epitopes indicates that they have different tcr repertoire diversity in hiv-specific responses. different immune escape mutants of nef - was using different strategies to avoid the killing of host ctls, which indicates that the therapy strategy based on the cellular immune response should be diversity. for the in vivo or ex vivo activation of antigen-specific t cell responses long synthetic peptides are used to activate both cd + and cd + t cells. in this study we investigated the efficiency and mechanism of cross-presentation of these long synthetic peptides in mhc class i. we observed a large variation in the effectiveness of activation of specific t cells by the extended peptides corresponding to different epitopes, indicating a difference in the efficiency of processing and presentation of these peptides. for the hla-a restricted cmvpp derived nlv epitope specific t cells were most efficiently activated by n-terminally extended variants of the minimal epitope, while the use of c-terminally extended variants resulted in a - log reduction of activation efficiency. this pattern was seen for / epitopes tested in different hla restrictions. furthermore, for all epitopes tested, extending both the c-terminus and n-terminus led to - log less efficient activation of the specific t cells, compared to the minimal peptide. exchange of the c-terminal sequence of the c-terminal extended hla-b restricted cmvpp rph peptide with the c-terminal extended nlv peptide led to the enhancement of t cell activation by the exchanged nlv peptide, indicating a role of the extended peptide sequence in the efficacy of processing and presentation of the peptide. tap-deficient t cells loaded with extended nlv peptides efficiently activated nlv-specific t cells, indicating that the route of presentation was tap-independent. addition of lactacystin did not affect activation of specific t cells, illustrating that crosspre-sentation was proteasome-independent. primaquine reduced the activation of specific t cells by extended nlv peptides, but not by the minimal nlv -mer peptide, suggesting that cross-presentation was dependent on endosomal recycling. these data suggest that long synthetic peptides can be processed by peptidases in endocytic compartments and presented by recycling mhc class i molecules. not all immunogenic epitopes that have been selected in vivo for efficient processing and presentation by the classical pathway may be presented efficiently by cross-presentation. therefore, a rational design of peptides is crucial for efficient activation of cd + t cells in approaches of vaccination, adoptive transfer and immune monitoring. antigenic peptides presented by mhc class i molecules are fragments that are usually excised from intracellular proteins while these are degraded by the proteasome. recently, three antigenic peptides were found to result from the splicing of segments that are not contiguous in the parental protein. for two of these peptides, splicing was found to occur in the proteasome by a mechanism of transpeptidation resulting from the nucleophilic attack of an acyl-enzyme intermediate by a free peptide fragment. one of them is derived from melanocytic protein gp and requires excision of a four-amino acid intervening segment. the other peptide is derived from protein sp , and requires splicing in the reverse order of two segments initially separated by six amino acids. the first spliced antigenic peptide described was derived from fibroblast growth factor- (fgf- ) and was recognized by human cytotoxic t lymphocytes directed against kidney cancer cells. it is made of two spliced fragments, which are initially separated by a long segment of amino acids. the splicing mechanism of this peptide has not been worked out. the length of the intervening segment made the transpeptidation model more difficult to account for the splicing of this peptide. we therefore evaluated the role of the proteasome in the splicing of this peptide. we observed that the spliced fgf- peptide was produced in vitro after incubation of proteasomes with a -amino acid long precursor peptide. we evaluated the mechanism of the catalytic reaction by incubating proteasomes with several peptide precursors in a pair wise manner. the results confirmed the transpeptidation model of splicing. we further compared the production of the fgf- spliced peptide by cells transfected with mutant constructs encoding fgf- proteins where the intervening segment was shortened from amino acids to , or residues. we observed an increase in the production of the spliced peptide that was proportional to the reduction in length of the intervening segment, as predicted by the transpeptidation model. finally, using the spliced gp peptide model, we observed that splicing did not occur at a significant level between fragments of two distinct proteins in the cell. the polymorphic residues within the peptide binding cleft of hla class i molecules not only diversify the range of peptides presented to cytotoxic t lymphocytes but also influence the pathway of antigen presentation. in order to acquire high affinity peptides, some class i allotypes, such as hla-b* , are heavily dependent upon tapasin and other molecules comprising the peptide loading complex (plc). other class i molecules, like hla-b* , appear to largely bypass this complex but are consequently loaded suboptimally with peptide. hla-b* and b* are naturally occurring allotypes that differ by only a single amino acid, making this difference in behaviour all the more remarkable. we have previously speculated that such tapasin-independent class i molecules may have been selected in response to viral inhibitors that target the plc, such as the human cytomegalovirus us protein. to address this hypothesis, us was stably coexpressed in b lymphoblastoid cell lines expressing hla-b* or hla-b* . in the presence of us , the surface expression of hla-b* was substantially reduced whereas hla-b* expression was relatively unaffected. although us was able to form complexes with both hla class i allotypes, only hla-b* was retained intracellularly in an immature form whereas hla-b* was transported to the cell surface. accordingly, in the presence of us , hla-b* , but not hla-b* , constitutively presented a hla-b restricted alloantigen to reporter t cells, suggesting that us binds hla-b* without interfering with peptide loading. us has been reported by others to bind the plc but surprisingly we have not detected such us -plc complexes in our system. rather, in the presence of us we identified a pool of class i molecules distinct from the plc and only present in us expressing cells, implying that us may act independently of the plc. these findings demonstrate how hla class i polymorphism not only impacts upon the t cell repertoire and diversifies determinant selection, but also serves to evade the impact of viral inhibitors on antigen presentation. c. massa , b. seliger martin-luther-university halle-wittenberg, institute of medical immunology, halle, germany in the attempt to optimize vaccine dc, modifications have been proposed both in the antigen loading and in the maturation protocols. for dc loading "whole antigens" are now preferred to peptides. therefore, it is important to consider not only the costimulatory properties of the vaccine dc, but also their antigen processing abilities. this is even more important since there is the trend to stimulate dc with tlr ligands combined with ifn-y in order to induce dc not only able to correctly migrate, but also secreting the bioactive il p . since ifn-g is known to influence the expression of multiple proteases involved in antigen processing, aim of this study was to compare the various maturation cocktails for the consequences on the antigen processing capabilities of the dc in parallel to their costimulatory potential. for this purpose monocyte-derived dc were stimulated for h with the gold standard of maturation (tnfa, il b, il and pge ) or a combination of ifn-g and different tlr ligands. the dc obtained exhibit a similar expression of costimulatory and adhesion molecules together with the ability to induce proliferation of allogeneic pbmc, but differ for the pattern of proteases expression as evaluated by real time pcr. with the exception of the downregulation of the tripeptidyl peptidase ii (tppii), no dramatic differences were observed for endo-and aminopeptidase between immature and "gold standard" mature dc. in response to the "ifn-gcontaining" cocktails there was a similar tppii downregulation, but also the induction of many other enzymes. the cytosolic leucine aminopeptidase- (lap ) had a more than -fold increase in transcription levels, whereas the mrna expression of the aminopeptidases of the endoplasmic reticulum erap and erap and of the immunoproteasome subunits lmp and lmp was enhanced between and -fold under these culture conditions. with regard to the different tlr ligands used in combination with ifn-g, there was a reproducible higher mrna induction in the presence of the tlr ligand mpla in comparison to the tlr- and / ligand polyi:c and r . these data suggest that the maturation cocktail of dc may alter the peptide repertoire presented by hla class i surface antigens. it has been suggested that mast cells might serve, under certain circumstances, as antigen presenting cells for t cells. however, whether cognate interactions between mast cells and class ii restricted cd + t cells actually occur, is still an open question. we addressed this question using peritoneal cell-derived mast cells (pcmc) as an antigen presenting cell model. our results show that in vitro treatment of pcmc with ifn-g and il- induced surface expression of mature mhc class ii molecules and cd . when ifn-g/il- primed pcmc were used as antigen presenting cells for cd + t cells they induced activation of effector t cells but not of their naive counterparts as evidenced by cd up-regulation, induction of proliferation and cytokine production. confocal laser scanning microscopy showed that helper ot-ii t lymphocytes form with pcmc functional immunological synapses, characterized by pkcq enrichment and ifn-g polarized secretion towards the antigen-presenting mast cells. finally, upon cognate interaction with ot-ii t cells, mast cells lowered their threshold of activation via fceri. our results show that mast cells can establish cognate interactions with class ii restricted helper t cells, implying that they can actually serve as resident apc in inflamed tissues. h the vast majority of peptide ligands presented by mhc class i molecules is thought to be produced by cytosolic degradation of source proteins by the proteasome. although, next to cytosolic and nuclear proteins, proteins targeted to the endoplasmic reticulum (er) can also be degraded through this pathway following retrograde transport into the cytosol, antigen processing of er proteins remains little characterized. studying processing and presentation of er-targeted and cytosolic forms of proinsulin (pi), an autoantigen playing a pivotal role in triggering of cellular autoimmune responses in type -diabetes, we found that er-targeting of this model antigen has profound effects not only on how pi is degraded, but also on regulation of its synthesis. as expected, proteasome inhibition inhibited degradation of cytosolic pi as well as presentation of the epitope insulin b - to specific cd + t cells. in contrast, prior exposure of cells to proteasome inhibitors strongly reduced production of er-targeted pi (pre-pi) through induction of er stress, both in cells infected with a recombinant vaccinia virus and in cells transfected with a tetracycline-regulated expression system. experiments using conditions permissive for pre-pi expression showed that er-targeting modified proteolytic processing of pi for mhc class i presentation. these experiments suggested that two proteolytic pathways contribute to degradation of er-targeted pi, with their relative contribution depending on the stability of the protein. while degradation of unmodified pre-pi was partially dependent on the proteasome, removal of one or several disulfide bridges increased the role of the proteasome in processing of pre-pi for presentation, while introduction of a site for n-glycosylation had the opposite effect. these findings imply that er-targeting together with structural features can have profound effects both on antigen production and on the pathway of proteolytic antigen degradation and presentation. cd + t cell immune response to exogenous antigens relies on cross presentation by dendritic cells (dcs) in secondary lymphoid organs. recently, in several infectious murine models, it has been shown that in addition to dc located in tissues, de novo differentiating dc participate in the protective th immune response. the role of de novo differentiating dc in cross presentation is however poorly documented, and difficulties of human immunology prevent the accurate identification of the apc subsets patrolling for exogenous ag. a prerequisite for cross presentation is a moderate ag degradation rate in the endocytic pathway, allowing the generation of antigenic epitopes and their binding to mhc molecules. this prerequisite is of special importance considering dc precursors (such as monocytes), which are not yet dcs and may take up antigen before differentiating into dcs. the objective of our in vitro study is to evaluate whether ex vivo purified human blood monocytes are able to cross present long antigenic peptides to cd + t cells and whether they are able to sustain this cross presentation while differentiating into dcs. we have previously shown the unique property of dendritic cells to maintain for several days the capacity to stimulate cd + tumor-specific t cell clones when pulsed with long antigenic peptides (that need to be processed before presentation to cd + t cell clones, faure, , eur j immunol ( ): - ). in the present study, we address the question of the mechanisms of long peptide cross-presentation by blood monocytes along the course of their in vitro differentiation into dcs. we have shown that despite their high degradative capacity, ex vivo purified monocytes pulsed with long peptides are able to stimulate cd + t cells after their in vitro differentiation into dc, days following their antigenic pulse. the delineation of apc subsets able to sustain ag cross-presentation and t cell stimulating potential might be of clinical relevance in immunotherapy using synthetic long peptides. viral genomes contain alternative reading frames (arfs) encoding for mhc-i restricted epitopes (arf-epitope). in the siv/macaque model, ctl responses directed against arf-epitopes participate in controlling viral replication. we previously described that hiv- genome contains arfs within gag, pol and env genes encoding for a panel of hla-b* restricted epitopes. qprsdthvf (q vf/ d) is one such epitope but its parental epitope qprsnthvf (q vf/ n) has a significant higher frequency among hiv- isolates. strikingly, q vf/ d-or q vf/ n-specific ctls recognize apcs infected with hiv strains encoding for q vf/ d (e. g. hiv lai ). in contrast, hiv strains (e. g. hiv nl-ad ) encoding for q vf/ n do not activate ctl responses raising the possibility that q vf/ n epitope is not presented by infected cells. we asked whether introducing mutations within q vf might be a mean for the virus to escape ctl responses directed against this arf-encoded epitopes. we dissected the mechanism responsible for the lack of q vf/ n mhc-i presentation. we modified hiv lai to introduce a d to n mutation in q vf. introducing this single amino-acid mutation abrogated ctl recognition indicating that this asparagine (n) alters q vf mhc-i presentation. we performed in vitro proteasomal digestions of mer peptides encompassing q vf/ d or q vf/ n and cleaved polypeptides were analyzed by mass spectrometry. the asparagine (n) in q vf/ n is a preferential proteasomal cleavage site. thus suggesting that proteasome cleavages within q vf/ n might be responsible for its lack of mhc-i presentation. we then sought in hiv-infected patients for the presence of proviruses encoding for q vf/ d or q vf/ n, and ctls responses directed against these epitopes. far thus, two out of three donors tested recognized the q vf/ d peptide. we cloned and sequenced hiv- genomes from the three donors. surprisingly, out of hiv proviral genomes isolated from pbmcs of q vf/ d reactive donors, we could not find any virus bearing the q vf/ d sequence. the isolated hiv sequences either encoded for q vf/ n or had a stop codon within the epitope. in contrast, viruses encoding for q vf/ d were isolated from pbmcs of the q vf/ d nonreactive patient. altogether, our data suggest that ctls exert a selection pressure on viral arfs. hiv- seems to escape immune surveillance by introducing mutations altering processing of arf-derived epitopes. i. e. flesch , y. wang , d.c. tscharke the australian national university, biochemistry and molecular biology, canberra, australia vaccinia virus (vacv) was the live vaccine used to eradicate smallpox and some strains are now being used as vectors for recombinant vaccines. cd + t cells recognizing viral peptides in association with mhc class i molecules on infected cells play a crucial role in the defence of viruses. despite the large number of possible mhc class i-peptide combinations, cd + t cells only recognize a small number of epitopes, a phenomenon called immunodominance. using recently defined cd + t cell epitopes for vacv in mice, we have investigated how heterozygosity of mhc class i molecules influences immunodominance patterns in h- bxd f mice compared with their inbred parent strains. we find that the immunogenicity of vacv peptides defined using inbred mice is variable in f progeny, with some peptides being almost equally immunogenic in f and inbred mice, while others elicit responses that are reduced by more than % in f mice. during acute infection as well as memory responses, the dominance hierarchy in inbred mice did not predict the epitopes that would be poorly immunogenic in f mice. in line with these findings, a multiepitope construct expressed by a recombinant vacv was less immunogenic in f mice than would be predicted from its performance in parent strains. in terms of mechanism, we find evidence of altered tcr repertoires including in the case of one epitope, the loss of many diverse tcr vb clones and outgrowth of cd + t cells with a restricted vb usage in f mice. these data have implications for our interpretation of experimental vaccine work done in inbred mice and for our understanding of how mhc diversity can alter the range of epitopes that are immunogenic in outbred populations. objective: tlr ligands are being exploited as potential adjuvants, and have impact on the antigen processing and presentation by dendritic cells (dc). therefore we aimed to study the efficacy of a tlr agonist, s-[ , -bispalmitoyiloxy-( r)-propyl]-r-cysteinyl-amido-monomethoxyl polyethylene glycol (bppcysmpeg), a synthetic derivative of the mycoplasma macrophage activating lipopeptide (malp- ), as an adjuvant for cross-priming against cellular and soluble antigens. malp- has been characterized as an effective mucosal adjuvant and synthesis of bppcysmpeg further improved solubility and pharmacokinetic features of the adjuvant. methods: dc isolation, in vitro and in vivo t cell stimulation, intracellular cytokine staining, in vivo cytotoxicity assays. results: systemic administration of bppcysmpeg induced maturation of cd + and cd -dc in the spleen resulting in enhanced cross-presentation of intravenously co-administered soluble antigen in mice. in addition, administration of bppcysmpeg and cell-associated ova resulted in generation of an effective ctl response against ova in vivo in a t-helper cell-dependent manner, but independent of ifna. delivering antigenic peptides directly linked to bppcysmpeg led to superior ctl immunity as compared to giving antigens and adjuvants admixed. in contrast to other tlr ligands such as cpg, systemic activation of dc with bppcysmpeg did not result in shutdown of antigen presentation by splenic dc subsets, although cross-priming against subsequently encountered antigens was reduced. we provide evidence that bppcysmpeg stimulation of dc via tlr / results in the generation of an effective ctl response and that delivering antigenic peptides linked to bppcysmpeg is a promising strategy for vaccination. while bppcysmpeg-matured dc retain their antigen uptake and presentation capabilities, cross-priming against subsequently encountered antigens is inhibited, indicating that mechanisms beyond down-regulation of macropinocytosis and phagocytosis contribute to shut-down of cross-priming after tlr-mediated dc maturation. altogether our study promotes synthetic lipopeptides as potential adjuvant for specific applications (e. g. viral infections, cancer) for the reason that they can be chemically engineered to carry specific antigenic peptides which allows targeting of antigens and simultaneous activation. tumor immunevasion. to verify whether the loss of erap expression could confer a survival advantage on tumor cells and enhance tumor progression, we stably knocked down expression of eraap (murine erap ) in a murine t lymphoma cell line, rma. we used a method that allows an efficient and continuous expression of mirnas that directly silence eraap and obtained several eraap-deficient rma clones with different levels of eraap expression (up to % of reduction at the protein level). microsomal aminopeptidase activity and mhc class i surface expression were decreased in all clones proportionally to eraap expression. moreover, low expression of eraap affected the stability of mhc class i molecules as evaluated after acid and brefeldin a treatment. de-regulated er peptide trimming also drastically affected the tumor formation of rma cells and host survival. eraap-deficient rma clones with different levels of eraap, and % as compared to control rma cells, were injected s. c. in the flank of c bl/ syngenic mice, and analysed tumor growth. all mice injected with control rma cells developed a tumor but survived up to days after injection. all mice injected with rma clone with a % level of eraap expression developed a tumor and died within days after injection. surprisingly, any animal injected with rma clone with a % level of eraap died or showed a visible tumor. thus, knockdown of eraap expression appears differently to affect the immunogenicity of rma cells, depending on the eraap silencing level. hemophilia a is an x-chromosome-linked bleeding disorder caused by the absence or dysfunction of clotting factor viii (fviii). treatment consists of regular administration of fviii, but is complicated by the formation of inhibiting antibodies against fviii. both genetic and treatment-related factors play a role in the etiology of inhibitor development in patients with hemophilia a. the development of inhibitory antibodies in hemophilia a patients has been shown to be a cd + t-cell driven process. therefore, in order to better understand the process of inhibitor formation, we aim to identify the epitope specificity and phenotype of t cells against fviii in hemophilia a patients using mhc class ii tetramers. cd + t-cell responses of two monozygotic twins with severe hemophilia a were analyzed. one of these subjects developed a high titer inhibitor ( bu/ml) following intensive factor viii (fviii) treatment. high dose immune tolerance therapy together with anti-cd therapy resulted in eradication of the inhibitor. in contrast, his twin brother developed a low titer inhibitor ( . bu/ml) which declined rapidly after tolerance induction. fundamental differences in the twins' antibody responses were further suggested by elevated and persistent igg levels in the subject with the high titer inhibitor. in order to gain a better understanding of processes leading to inhibitor formation versus tolerance, we investigated drb * -restricted t-cell responses of the high titer inhibitor subject, using fluorescent mhc class ii tetramers loaded with -mer synthetic fviii peptides to stain epitope-specific cd + cells.cd + t-cells from the high-titre inhibitor subject recognized three peptides corresponding to the fviii a domain: fviii - , fviii - and fviii - , as well as the c domain peptide fviii - , but not any c domain peptides. the c domain peptide contains a sequence that was reported as a promiscuous t-cell epitope (jones td et al., j thromb haemost. : - , ). analysis of t cells from the lower titer inhibitor subject is expected to reveal differences in the epitope specificity and phenotypes of t cells that may underlie the discordant immune responses of these twins to infused fviii. m. forloni , s. albini , m.z. limongi , l. cifaldi , d. fruci ospedale pediatrico bambin gesù, rome, italy neuroblastoma (nb) is a pediatric tumor that derives from neural crest. the most aggressive forms are characterized by amplification of the mycn oncogene and severe reduction of hla class i expression. mycn has been claimed to hinder hla class i expression through affecting the expression of the transcription factor p nf-kb subunit. since in many human tumors the expression of hla class i molecules is positively co-ordinated with that of er aminopeptidases, erap and erap , we wondered whether in nb cell lines mycn may impair expression of these aminopeptidases. to explore this possibility, nb cell lines that differ in mycn expression were quantified for expression of mycn, erap , erap and hla class i heavy chains by western blotting and for surface hla class i expression by flow cytometry. we found that mycn negatively correlates with expression of hla class i, erap and erap . this negative correlation was confirmed in a nb cell line expressing a tetracycline repressible mycn transgene. then, by the use of tnfa (a nf-kb nuclear translocation stimulator), sulfasazine and ikba mutant (two nf-kb nuclear translocation inhibitors) and knockdown of p nf-kb subunit, we demonstrated that nf-kb is involved in erap and erap expression in nb cell lines and that mycn does not affect nf-kb expression. furthermore, we showed that mycn and nf-kb are recruited to the promoter regions of erap and erap and that mycn affects the recruitment of nf-kb binding to these promoter regions. in conclusion, the present results indicate that an enhanced mycn level, linked or not to mycn amplification, represses erap , erap and hla class i expression in nb cell lines by affecting the recruitments of nf-kb binding to their promoters. s. brosch , s. tenzer , h. schild , e. von stebut-borschitz uniklinik mainz, mainz, germany infection of inbred mouse strains with the intracellular protozoan parasite leishmania major either leads to self-healing cutaneous disease (resistant phenotype; e. g. c bl/ mice) or systemic disease (susceptible phenotype; balb/c mice) depending on the genetic background of an individual. healing of leishmania infections is based on th immunity, whereas ifng secretion of both cd + th and cd + tc cells is critically important for protection by inducing oxidative radicals in macrophages, which enables them to kill the parasite. stimulation of antigen-specific effector t cells is driven by l. major-infected dendritic cells (dc) in an il- -dependent manner. proteasome/immunoproteasome-dependent antigen processing is necessary for clearance of viral or intracellular parasitic diseases to induce effective cd + t-cell responses via the mhc class i. here, we analysed the role of the ifng inducible immunoproteasome for the priming of cd + t cells in l. major infections. using an in vivo model, we show that the functional knock-out mouse in the chymotrypsin-like catalytic domain of the immunoproteasome lpm (lmp -/-) does not exhibit an altered course of infection (lesion development, parasite loads, cytokine profiles) in intradermal, low dose infections with l. major mimiking natural transmission of the parasite as compared to wild type c bl/ mice. in addition, ex vivo co-cultures with infected dc from either lmp -/or wild type mice together with antigen-specific t cells from infected wild types showed no differences in tc cell ifng secretion and the dc restimulatory capacity of cd + t cells. furthermore, significant differences in the proliferation of antigen-specifically restimulated (with soluble leishmania antigen; sla) cd + t cells, isolated from low dose infected c bl/ wildtype or lmp -/mice, were not detected. in summary, our data indicate that despite the fact that cd responses in l. major infections are important for disease outcome, processing of antigen and thus priming of cd + t cells against l. major is independent of the lmp subunit of the immunoproteasome. studies have defined an essential requirement for autoantigen-specific b cells as antigen presenting cells in rheumatoid arthritis. however, the cellular mechanisms involved in antigen processing and presentation of joint-derived autoantigens by b cells are unknown. in this study we have developed a system to investigate how antigen-specific b cells recognise and present the proteoglycan aggrecan, a major component and candidate autoantigen of joint cartilage. we have utilised these cells to characterise the mechanisms by which aggrecan-specific b cells could induce autoimmunity. we have constructed plasmids encoding an aggrecan-specific b cell receptor and have transfected them into the b cell line a , generating b cell lines that specifically recognise and target aggrecan for presentation to t cells. in addition, we have established conditions for a panel of aggrecan-specific t cell hybridomas to recognise aggrecan pulsed b cells following fixation, to allow the kinetics and mechanisms of aggrecan processing to be studied. we used inhibitors of mhc class ii transport, endosomal ph and enzymes involved in aggrecan degradation. we found that aggrecan-specific b cell lines presented the major arthritogenic cd + t cell epitope ( - ) from the g domain of aggrecan , times more efficiently than non-specific b cells and over times more efficiently than the macrophage line j . however, despite this highly efficient aggrecan capture, processing and presentation of the - epitope took at least hours, comparable to the time required for presentation of aggrecan by j . treatment of aggrecan-specific b cells with ammonium chloride to raise endosomal ph or brefeldin-a to disrupt golgi transport inhibited presentation of the - epitope, suggesting a requirement for low endosomal ph and presentation by newly synthesised mhc class ii. interestingly, aggrecan presentation by antigen-specific b cells was also reduced by phenanthroline, an inhibitor of the aggrecan-degrading metallo-proteinases that are found in abundance in the arthritic synovium understanding the mechanisms of antigen processing and presentation by autoantigen-specific b cells may explain their role in the pathogenesis of diseases such as rheumatoid arthritis. tapasin is an mhc-dedicated chaperone that facilitates peptide loading and optimization of the peptide cargo of mhc class i molecules within the peptide loading complex (plc). class i molecules differ in their dependence on tapasin for efficient cell surface expression, dependence that is determined by the nature of amino acids at positions , and at the peptide binding groove. position also determines the strength of tapasin binding and influences peptide specificity, but its precise effect is probably context dependent. the mhc class i antigen b is strongly associated to ankylosing spondylitis (as) and other spondyloarthropathies. hla-b subtypes differ in their dependence of tapasin for cell surface expression and incorporation into the plc. tapasin also modulates b folding but not maturation and although tapasin optimizes the constitutive peptide repertoire of b* , peptide loading is relatively independent of this chaperone. we analyzed the effect of b subtype polymorphism on tapasin binding and the correlation of this feature with the affinity of the peptide repertoires, the maturation kinetics and the folding efficiency of b subtypes. the association of b heavy chain with tapasin was analyzed in c r cells transfected with hla-b subtypes and mutants by pulse-chase analysis and co-immunoprecipition with the monoclonal antibody pasta- , which recognizes human tapasin. we also analyzed the global thermostability, as a measure of the stability of the peptide cargoes, and the optimization of the b peptide repertoire with thermostability assays, by pulse-chase analysis and immunoprecipitation with the me monoclonal antibody that recognizes b properly folded b /peptide complexes. the formation of fully assembled b molecules was analyzed by pulse-chase analysis and immunoprecipitation either with the monoclonal antibody hc , which recognizes mhc class i free heavy chains (hc), or with me . maturation was analyzed by pulse-chase analysis, immunoprecipitation with me and treatment with endoglycosidase h (endo h). hla-b polymorphic positions other than , both at the a and c/f pockets modulate tapasin binding and the optimization of the peptide cargo. the stability of the peptide repertoires critically influences the folding efficiency of b subtypes. from as early as the initial phases of infection, hiv is coated with complement (c) fragments and following seroconversion, the circulating virus forms immunecomplexes with igg and complement. recent in vitro experiments revealed differences with respect to productive infection of immature dendritic cells (idcs) with differentially opsonized hiv. the opsonization pattern of hiv may additionally have profound consequences for the outcomes of the antigen-presenting capacity of dcs and their ability to mount an adequate immune response. in this context, we compared the impact of differential hiv-opsonization on the antigen-presenting capacity of dcs and found that c-opsonized hiv triggered ctl responses, while igg-coated virus did not. these in vitro generated ctls showed an enhanced ifn-g secretion and recognized the help independent ctl epitope slyntvatl. c-generated ctls also degranulated upon stimulation with specific hiv peptides and were able to elicit antiviral activity against hiv-infected cd + t cells. our results indicate that c-opsonization of hiv drives the virus towards the mhc class i pathway in dcs, thereby promoting a more efficient stimulation of naïve cd + t cells. this ctl-stimulating property of c could be exploited when searching for a novel approach against hiv. igg isolated from patients with high titers of anti-ccp antibodies showed a cross-reactivity with hcit peptides. vaccination experiments supported a triggering role of hcit for the development of arthritis in mice model. conclusions: diamination process is significantly increased in patients with ra while carbamylation is suppressed. production of specific antibodies against diaminated residues in ra patients may have a modulating role for the development of autoimmune arthritis. the classical pathway of mhc class i antigen presentation involves cytosolic degradation of viral proteins by the proteasome. peptides generated entry the endoplasmic reticulum through the transporter associated with antigen processing (tap). previous reports have shown that viral epitopes are presented to ctl independently of tap in smaller viruses. we hypothesized that presentation of vacv by mhc class i might proceed by alternative pathways. the aim of this study was to characterize these alternative pathways in tap-deficient mice. our results show that ctl derived from c bl/ mice immunized with vacv, recognized tapdeficient dendritic cells infected with the virus. approximately % of vacv global presentation in the context of h- b was independent of tap. in addition, vacv infection induced a virus-specific ctl response in mice deficient in tap. dendritic cells (dc) initiate robust ctl immunity via the presentation of antigen-derived peptides by surface major histocompatibility complex class i molecules (pmhc). two major dc subtypes have been described, cd + and cd -dc, which differ in their mhci antigen presentation capacities. cd + dc are the major dc subset responsible for cross presentation (presentation of exogenous antigen by mhci), while cd -dc display little cross presenting capacity. here, we examined the mhci antigen presentation pathway of cd + and cd -dc in more detail. first, turnover (half-life) of total mhci at the cell surface of cd + and cd -dc was determined. surprisingly, cd + dc exhibit rapid surface mhci turnover compared to cd -dc (following culture in the presence of brefeldin a). following activation of dc with cpg, mhci levels at the surface of both cd + and cd -dc were stabilized and no longer underwent rapid turnover. this suggests that cd + and cd -dc differ in their regulation of surface mhci turnover and that this is subject to regulation by antigen-associated signals. second, we examined the ability of cd + and cd -dc to generate pmhci complexes containing cross presented antigen. we utilized the model antigen ovalbumin (ova) and an antibody that can detect h- kb loaded with the ova-derived peptide, siinfekl. cd + and cd -dc isolated from ova-expressing mice (actin-ova transgenics) displayed abundant kb-siinfekl complexes at their cell surface. in contrast, in response to exogenous soluble ova protein, only the cd + dc, but not the cd -dc, displayed kb-siinfekl complexes at the cell surface. similarly, when dc were pulsed with ova-coated splenocytes, kb-siinfekl complexes were only detected on the surface of cd +, and not cd -dc. this data further validates the role for cd + dc as the major cell type responsible for cross presentation and provides insight into the mechanisms that prevent other dc subsets from accessing the important cross presentation pathway. objectives: the action radius of matrix metalloproteinases or mmps is not restricted to massive extracellular matrix (ecm) degradation but extends to the proteolysis of secreted cytokines and membrane-bound receptors and adhesion molecules. although many instances exist in which cells disintegrate, often in conjunction with induction of mmps, the intracellular mmp substrate repertoire or degradome remains relatively unexplored. the aims of the present study were to identify novel intracellular mmp targets and to answer the question whether the proteolytic modification of intracellular proteins alters the immunogenicity of released intracellular contents. methods: multidimensional degradomics technology was developed by the integration of broadly available biotechniques and applied to thp- cytosol using gelatinase b/mmp- as a model enzyme. in the first dimension, ion exchange chromatography separated the thp- proteins by their net charge and/or isoelectric point (pi) followed by cleavage of the proteins by mmp- . in the second dimension, potential substrates were separated by molecular weight on sds-page. to evaluate the effect of proteolysis by mmp- on the immunogenicity of the intracellular protein pool, mice were immunized twice with thp- cytosol in complete freund's adjuvant. lymph node t cells were isolated and stimulated with mmp- -cleaved or intact thp- cytosol. proliferation was assessed by measuring incorporated hthymidine. results: - mmp- candidate substrates were isolated, of which were identified, revealing many novel mmp- (candidate) substrates from the intracellular matrix (icm), such as actin, tubulin, stathmin,... about / of the identified substrates were described as systemic autoantigens in one or multiple autoimmune conditions. remarkably, a significantly lower t cell proliferation was observed in the presence of cleaved vs. intact cytosol. conclusion: multidimensional degradomics technology is a valuable tool for high-throughput identification of novel mmp substrates. proteolysis by mmp- decreased the immunogenicity of the intracellular contents, suggesting that mmps may contribute to the dampening of inflammation by the clearance of toxic and immunogenic burdens of intracellular (matrix) proteins released after extensive necrosis and tissue injury. a preference for hla-a versus -b molecules; e - k did not detectably associate with hla-c molecules under identical conditions. this locus specificity may provide a functional advantage to ads by inactivating t-cell receptors, while avoiding activation of nk receptors. finally, we showed that residue in hla-a and residue in e - k are highly critical for association of both proteins. this defines a putative interaction surface between e - k and class i molecules. conclusions: our studies provide novel insights into the functional relationship between e - k and the class i antigen presentation pathway. moreover, because soluble e - k can differentiate between polymorphic gene products encoded in the mhc, our results may contribute to define paradigms for how class i substrate specificity is established for er retention. overall, our studies represent an important step towards a molecular understanding of the strategy evolved by ads to establish life-long persistence in host cells. objectives: the transporter associated with antigen processing (tap) belongs to the abc transporter superfamily and is a heterodimer consisting of the two subunits tap and tap . tap transports peptides yielded by proteasomal degradation from the cytosol into the endoplasmic reticulum (er) and is thus a key element of the mhc class i antigen processing machinery (apm). methods: target-specific tap knock downs were generated by shrna technology. the resulting transfectants were subsequently analysed regarding mhc class i surface expression using flow cytometry, whereas mrna and protein expression levels of tap and tap were analysed by rt-pcr and western blots, respectively. furthermore, the protein stabilizing effect of tap on tap was investigated in the presence of two distinct proteasome inhibitors. results: previous findings obtained with rare tap mutants suggested that the lack of tap protein expression is associated with a strong reduction of tap protein levels, which could be restored by tap gene transfer, whereas no such regulation is found vice versa. to investigate this stabilizing effect of tap on tap different shrna plasmids specifically targeting tap or tap , respectively, were stably transfected into constitutively tap and tap expressing hacat keratinocytes and colo melanoma cells. in both cell types the shrna-mediated tap and tap inhibition resulted in a significant downregulation of the respective transcript and protein expression levels. the knock down of tap caused not only an almost complete loss of tap , but also a strong decrease of tap protein expression. in contrast, the tap knock down exhibited no influence on the tap expression. specific inhibition of the proteasome prevented the degradation of tap in the tap knock down variants. the results of our study emphasize that an unidirectional stabilisation of tap on tap protein expression is not restricted to rare tap mutants, but rather suggest a common regulatory mechanism for the tap complex. uv injury profoundly affects the skin immune homeostasis by promoting strong inflammation and cellular immuno-modulation. in this study, we characterized the inflammatory cell subsets that emigrate in the epidermis the days following uv exposure. therefore, the buttock skin of healthy volunteers was exposed twice to . minimal erythema dose of uv. blister roofs were then collected before and , and days after uv-exposure from un-exposed and exposed skin and the resulting epidermal cells were analysed by flow cytometry. we demonstrated that, along with the rapid activation and migration of langerhans cells (lc), uv skin radiation exposure promotes the infiltration into the epidermis of a monocytic cd + cd + and of a macrophagic cd -cd + cell subsets that emerge and days post exposition, respectively. more importantly whereas classical cd a hi cd + lc are the unique dendritic cell (dc) subset found in the epidermis of unexposed skin, we detected two new subsets of epidermal dc namely cd a low cd and cd a low cd + that emerged and days post irradiation, respectively. these two distinct populations of epidermal dc (edc) differ from classical epidermal lc by their activation/maturation profile as assessed by the strong expression of cd and hladr. finally, days post-exposure, we observed that lc represented almost the only haematopoietic cell population in the epidermis. these results suggesting that the uv-recruited edc and monocytic/macrophagic subsets participate to the progressive recovery of the epidermal immune cell network homeostasis. i. bailey , e. reeves , t. elliott , e. james university of southampton, school of medicine, cancer sciences division, southampton, united kingdom there is accumulating evidence that cd + cd + regulatory t cells (treg) play an important role in anti-tumour immunity by preventing effective t cell responses to tumour antigens. tregs have also been shown to inhibit development of organ-specific autoimmune diseases suggesting they inhibit immune responses to tissue-specific self-antigens. the depletion of tregs prior to challenge with the murine colorectal tumour, ct , stimulates a robust, protective t cell response which is also protective to challenge with other tumours of different histological origins, such as b cell lymphomas and a renal cell carcinoma. this cross protection has not been seen with other tumour cell lines. we have identified a ct -derived cross-protective antigen, gsw , which was found to be encoded within the ectotropic murine leukaemia virus (emv- ) envelope protein, gp . this protein has previously been shown to encode ct -specific cd and cd antigens, implicating it as a 'hot-spot' for ct tumour antigens. interestingly, we have identified a truncated version of gp which may be responsible for generation of gsw . expression studies have revealed increased gp expression in ct compared to other tumour cell lines, indicating the ability to cross-protect is related to the quantity of antigen (gsw ) generated. the current knowledge of hla class ii antigen presentation and peptide binding is mainly based on studies of hla-dr molecules. they contain a large hydrophobic p pocket, which can accommodate large hydrophobic amino acid residues and is the most important pocket in selecting and binding peptides, while p , p and p tune the peptide repertoire that can be presented by individual hla-dr alleles. the same rules and requirements do not necessarily exists for peptide binding to hla-dp molecules, however. the present study adresses this issue. we have expressed and affinity purified soluble recombinant hla-dp molecules from drosophila melanogaster cells and studied its binding of a number of peptides known to bind hla-dr, -dq or dp molecules. unexpectedly, the immunodominant epitope in multiple sclerosis (ms), the myelin basic protein derived peptide, mbp - , bound to hla-dp with high affinity ( - nm). binding studies of mbp - derived peptides containing single alanine substitution at each position revealed that only three of the peptides (f a, f a and k a) were affected, and only by a - fold reduction in affinity for hla-dp . the observation that none of the substitutions resulted in a complete loss of binding to hla-dp indicates that ) hla-dp binding to peptides does not depend on a large hydrophobic residue accomodated in p , or ) mbp - can bind in more than one register. we will present data addressing this issue. the hla-dp peptide binding capacity was increased at neutral as compared to acidic ph, and by the presence of n-butanol, a small organic mhc loading enhancer (mle). in summary, the hla-dp molecule binds the immunodominant epitope in ms, mbp - , possibly in more than one register. additional studies are required to resolve the hla-dp peptide binding properties, and to determine whether expression of hla-dp affects the disease course in ms patients. results: depletion of mncs for cd + cells abrogated the tg-induced cytokine production and proliferation of cd + t cells, indicating a primary role for monocytes as apcs. however, the encounter of t cell with antigens presumably occurs in b cell-rich compartments such as lymph nodes or lymphoid tissue in inflamed organs. to mimic the conditions prevailing there, we depleted pbmcs for g % of the monocytes (without significant loss of t-cells), and compared the tgelicited t-helper cell responses in the presence and absence of b cells. the tg-induced cd + t cell proliferation was significantly reduced in cd /cd -depleted mnc cultures, as compared to cultures depleted for cd + monocytes alone. the same applied to the production of il- , il- and tnf-a. production of ifn-g and il- was generally not observed. our data indicate that normal b cells are capable of inducing a pro-inflammatory cytokine response in mnc-cultures, where monocytes and monocyte-derived cells are not preponderant. studies addressing the relative contributions to this cytokine production, by b cells themselves and by t cells (following antigen-presentation by b cells), are in progress. j. kyosiimire-lugemwa , , p. pala , g. miiro , j. todd , p. kaleebu , , n. imami , f. gotch , mrc uganda, basic science, entebbe, uganda, imperial college, london, united kingdom, mrc uganda, entebbe, uganda background: hiv- -specific t-cell responses are preserved in hiv- infected individuals with non-progressing hiv- disease. "long term non progressors" (ltnps) were defined as art naï ve individuals infected with hiv- for g years, maintaining cd + t-cell counts g , and with minimal cd + decline over time. we tested the hypothesis that gag-specific t-cell responses are inversely correlated to disease progression whereas nef-specific t-cell responses are not. methods: art naï ve hiv- infected patients from the entebbe cohort in uganda were recruited and stratified by cd + t-cell count, cd + decline slopes, and time of enrolment, into groups - ltnp and rapid progressors (rp). all patients were women reflecting the patient base at the entebbe cohort. we measured plasma viral load, current cd t-cell count, and ifn-g, il- and il- elispot responses to pools of to peptides ( -mers overlapping by aa). peptides were based on consensus sequences of gag and nef from hiv- clades a , a and d. medians and inter-quartile ranges were calculated and comparisons between groups were performed using the mann-whitney u test. correlations were presented using spearmann's linear correlation coefficients. results: some gag-specific ifn-g and il- responses were significantly higher in the ltnp than in rp (p= . , ifn-g responses to gaga pool ; p= . , il- responses to gaga pool ). il- responses were low and not significantly different between ltnp and rp. there was a positive correlation between il- responses to gaga pool and cd t-cell counts (r = . , p= . ), but no correlation between either il- or ifn-g responses and viral load. cytokine responses to nef peptides were not significantly different between the ltnp and rp. conclusion: overall, gag hiv- specific responses were higher in ltnps than in rps confirming previous results. non-specific il- responses were high possibly reflecting baseline th responses to helminths a common environmental exposure in the study population. objectives: in human and murine tumors and in in vitro oncogene-transformed cells defects in the expression of components of the hla class i antigen processing machinery (apm) have been described, which were associated with a reduced antigenicity of these cells. so far, the molecular mechanisms of such defects have not been elucidated in detail. to investigate whether impaired apm component expression was due to altered transcription and associated with cell growth properties murine her /neuand her /neu + fibroblasts were employed. methods: using tapasin as a model molecule its cell cycle-dependent expression was analysed in a time kinetics upon serum starvation followed by stimulation with complete culture medium over time. cells were harvested at different cell cycle phases and expression of tapasin was analyzed by qrt-pcr and western blot. flow cytometry was employed for determination of the distinct phases using -aad for dna analysis and specific antibodies directed against the proliferation marker ki- , the m-phase specific phistone h as well as for the h- l d surface antigens. in addition, chromatin immunoprecipitation (chip) experiments with an antibody directed against rna polymerase ii were performed to investigate the transcriptional levels of tapasin in her /neuversus her /neu + cells. results: serum starvation and subsequent stimulation with complete culture medium led to the enrichment of cells at the g /g -, s-and g /m-phases of the cell cycle, which was associated with an altered tapasin transcription during the cell cycle. tapasin mrna level decreased during cell cycle progression, whereas an inverse protein expression was observed with low expression levels at the g /g -phase, which continuously raised and peaked within the s-phase. however, h- l d surface antigen expression was not altered in her /neucells during cell cycle progression. in contrast to her /neufibroblasts the her /neu + transfectants exhibit a decreased tapasin transcription, which was accompanied by an altered h- l d surface expression. this was confirmed by a reduced promoter activity and decreased accessibility of the rna polymerase ii to the tapasin promoter. conclusion: these findings lead to an improved understanding of immune escape mechanisms demonstrating a cell cycle dependent and oncogene-mediated tapasin regulation that may provide novel targets for therapeutic intervention. recent studies suggest that dendritic cells (dcs) are key players in shaping the respiratory syncytial virus (rsv) specific immune response. before, dcs within the airway epithelium were characterized as langerhans cells. in this study, in vitro counterparts of langerhans cells expressing langerin (cd ) and ccr were cultured from cd + stem cells under the influence of tgf-b (tgf-b-dcs) and compared to cd + derived dcs, which passed through a monocytic stage . after infection with rsv, both types of dcs generated viral rna and viral-proteins. although tgf-b-dcs expressed higher levels of viral proteins as revealed by flow cytometry and fluorescence microscopy, more than hundredfold more viral particles were released by il- -dcs. the increased expression of viral proteins is most likely responsible for the pronounced inhibition of t-cell functions by tgf-b-dcs. since there is evidence that langerhans cells are expressed in airway epithelium not before the age of one year, the results may indicate, that an inhibition of rsv replication is characteristic of a more mature answer against rsv. the occurrence of inhibitory antibodies against exogenous factor viii (fviii) remains the major concern of fviii replacement therapy in patients with hemophilia a. initiation of the immune response implies the endocytosis of fviii by professional antigen presenting cells (apcs): b lymphocytes, dendritic cells (dcs) and macrophages (mØ). the organ where the anti-fviii immune response is initiated and the type of apcs involved in this process have not been investigated. we hypothesized that the spleen, which is the principal filter for blood-born antigens, is the principal organ where apcs interact with fviii to initiate the anti-fviii immune response. we first administered radiolabeled fviii at therapeutic doses to fviii-deficient mice. fviii was found to preferentially accumulate in the spleen and liver of the mice. levels of fviii in the spleen remained stable for up to min following fviii administration, while they rapidly decreased in the liver. unlabelled fviii was then administered to fviii-deficient mice that had been splenectomized or sham operated and the anti-fviii humoral responses were compared. removal of the spleen resulted in significantly reduced levels of anti-fviii igg. using flow cytometry, fviii was found to preferentially accumulate with splenic mØ than dcs and b cells. elimination of apcs by treatment of the mice with clodronate-containing liposomes prior to fviii administration resulted in a drastic reduction of the anti-fviii igg response, as compared to control mice treated with pbs-containing liposomes. taken together, our results suggest that the spleen is the principal organ in the initiation of the anti-fviii immune response and that splenic mØ have an important part in this process. the interactions between antigen presenting cells (apc) and t-lymphocytes are a relevant current issue. the area of contact between an antigen presenting cell and a t-lymphocyte is termed immunological synapse (underhill et al, ) . the present work started, in experiments with leukocyte of healthy individuals from the finding that under certain experimental conditions, cell-cell association with closely contact between monocyte-derived macrophages and human autologous lymphocytes are produced when the cells are harvested from total leukocyte cell cultures. in this way, such cells selective forming rosettes with a central macrophage and adherent lymphocytes. objectives: as central hypothesis it was postulated that the phenomenon would be due to antigen presentation made (performed) by macrophages to lymphocytes and that would be t cells. methods: autologous total human leukocyte cultures, from samples of healthy blood donors were harvested at various times and centrifuged and performed as previously reported (cabral and novak, , ) . cytopreparations of each experiment were performed. statistical analysis: regression model. results: experimentally, it was found a) phagocytosis of autologous antigens by macrophages, stimulates the formation of rosettes, b) a linear relation between rosettes formation and culture-time occurs, (p x , ), anova for regression, c) the cell-cell approximation is very important and was performed by centrifugation of the cells to form pellets, d) the forming rosettes lymphocytes are t-cells, cd +, e) purified macrophages and lymphocytes produced few rosettes, however if antigens were added, the phenomenon was stimulated, f) if inhibitors of the antigen processing and antigen presentation, such as chloroquine or brefeldin a, were added, rosettes were not formed, g) monoclonal antibodies anti-human mhc ii precluded the formation of rosettes, h) gangliosides diminishes rosettes formation. conclusion: taken together, the findings suggest that the model of rosettes formation might be useful to study cell-cell interactions during antigen presentation in immunological synapses and other immunologic aspects on the cells involved, in short time assays. objective: to investigate if patients with multiple sclerosis (ms), without the typical increase of antibodies in csf, are less likely to develop neutralizing antibodies (nabs) against ifnb-treatment, and whether the absence of such an immunological response might reflect a difference in their antigen presenting ability due to a distinct genetic background. methods: overall, patients were obtained from the swedish multiple sclerosis registry and the swedish nab registry, and treatment information was available for of them. for of these patients hla-drb data was available. results: a significant correlation between lack of antibodies in csf and nab-negativity was found (p= . ). patients without csf antibodies were to a lesser extent nab-positive when treated with the ifnb- a preparations, whereas no differences were shown for ifnb- b. an association between hla-drb * and nab-negativity was detected (p= . ). the known associations between hla-drb * and csf-positive ms and hla-drb * and csf-negative ms were confirmed. conclusion: we show for the first time that patients without antibodies in csf have a different propensity to induce nabs compared to csf-positive patients, indicating an extended immunological difference between the two ms sub-groups. hla-drb potentially contributes to this, which indicates that it might have something to do with differences in antigen presentation. in csf-negative patients the reaction against ifnb- a molecules, possibly through a t-cell dependent pathway, is lower than for csf-positive patients. however, reaction against ifnb- b, which might also be activated through a t-cell independent pathway, shows no difference in seroprevalence between the groups. abstract withdrawn by author tyrosinase-derived epitope was confirmed by five independent assays: flow cytometry on multiple melanoma lines generated from patients, confocal microscopy immuno-staining of melanoma lines, frozen sections staining of authentic melanoma tissue from patients, cytotoxicity assays using tyrosinase-specific ctls, and finally mass spectrometry analysis of peptides isolated from a melanoma cell line. there was no correlation between the level of antigen presentation and mrna expression levels for the three antigens; however, our data suggest that tyrosinase protein stability may play a major role in the high level presentation of this antigen. measurement of the half lives of these proteins revealed a hierarchy in protein stability, with mart- and gp more stable than tyrosinase. by the use of the cofactor dopa, which stabilizes the tyrosinase protein, significant decrease of hla-tyr complexes presentation was achieved. in addition to the study of antigen presentation, these tcr-like antibodies can also actively participate in immunotherapy as targeting molecules, considering their high affinity and specificity. by generating a whole igg antibody, tumor cell lysis was achieved by antibody-dependent cell-mediated cytotoxicity (adcc). with the addition of point mutations in the fc fragment, which increased the affinity of the fc to the fc receptor, enhanced tumor cell lysis was achieved. g. schiavoni , s. lorenzi , f. mattei , f. spadaro , l. gabriele istitituto superiore di sanità, cell biology and neurosciences, rome, italy cross-presentation is a crucial mechanism for generating cd t cell responses against exogenous antigens (ag), such as dead cell-derived ag, and is mainly fulfilled by dendritic cells (dc), particularly cd a + dc. however, apoptotic cell death occurring in steady-state conditions is largely tolerogenic, thus hampering the onset of effector cd t cell responses. type i ifn are a family of cytokines induced upon infection and acting as danger signals by stimulating multiple arms of the immune response. in particular, type i ifn have been shown to promote the cross-priming of cd t cells against soluble or viral antigens, partly through the stimulation of dc. in this study we evaluate the role of type i ifn to affect dc capacity to capture and cross-present apoptotic cell-derived ag. by using uv-irradiated ova-expressing eg thymoma line, we show that type i ifn promote the ability of cd a + dc to capture apoptotic eg cells and to undergo phenotypic activation, both in vitro and in vivo. remarkably, ifn-treatment prolongs the survival of ag-bearing cd a + dc and the persistence of apoptotic eg -cell ag within the phagosomal dc compartment, a process that is known to facilitate the recruitment of ag into the mhc-i presentation pathway. accordingly, type i ifn-treatment increases cross-presentation of apoptotic eg -derived ova ag by dc, as revealed by higher expression levels of siinfekl peptide in association to mhc-i molecules on cell surface of phagocytic cd a + dc. as a result, eg -loaded dc become competent at inducing ot-i cd t cell proliferation and activation both in vitro and in vivo. our data indicate that type i ifn promote the cross-presentation of apoptotic cell-derived ag by cd a + dc and suggest that these cytokines may act as a switch signal for cross-presenting dc, thus skewing the immune response from tolerogenic to immunogenic. ( ). we have investigated the mechanisms of cross-presentation of soluble antigen in freshly purified splenic dc subsets. using biochemical methods, we show that only cd + dc efficiently transfer soluble antigen to their cytosol. the amount of antigen detected in the cytosol increased up to ten-fold after a short exposure to tlr ligands cpg, poly i:c or pam csk , and this correlated with enhanced cross-presentation. the increase in antigen accumulation within the cytosol was not due to increased uptake of antigen. measurement of the proteasome activity at different times after exposure to tlr ligands revealed that tlr signalling induced transient inhibition (maximum at two hours) of the proteasome in cd + dc but not cd -dc, thus promoting accumulation of exogenous antigen in the cytosol of cross-presenting dc. this correlated with formation of aggresome-like structures only in cross-presenting dc exposed to tlr ligand. by limiting the degradation of transferred proteins during early activation, when endogenous proteins are being stored in aggresome-like structures, this mechanism could favour the loading of exogenous antigen peptides over endogenous peptides, promoting cross-presentation. to our knowledge this is the first report of a direct, immediate effect of tlr activation on proteasome activity. exosomes are nano-sized membrane vesicles of endosomal origin which can exert both immune stimulatory and tolerance inducing effects depending on their cellular origin. they are currently being investigated for use in vaccination and immune therapy strategies, but their physiological role has not been elucidated. here we explore whether exosomes of different origin can selectively target different immune cells. we compare the binding of exosomes from human breast milk, monocyte derived dendritic cells and b cells to peripheral blood mononuclear cells. flow cytometry, confocal laser scanning microscopy and multispectral imaging flow cytometry (imagestream) reveal that exosomes derived from human dendritic cells and human breast milk preferably associate with monocytes, whereas exosomes from an epstein-barr virus (ebv) transformed b cell line selectively target b cells. our data suggest a highly selective association between cells and exosomes which can be a way to direct their functional effects. one of the hallmarks of cancer cells is the resistance to cell death. it has been suggested that cancer cells also have the capacity to evade the surveillance by the immune system. the proteasome and macroautophagocytosis are attractive effector mechanisms for the immune system, because they can be used to degrade foreign substances, including pathogenic protein, within cells. here, we investigated that dm , which is a saponin derivative isolated the stem bark of dracaena mannii induced autophagocytosis on a human lung cancer cell line. methods: dm induced cell cytotoxicity was measured by mtt assay and propidium iodide staining on a cells. we examined the morphological change using optical microscope. and gfp-lc punctation was measured using confocal. the protein expression was measured by western blot analysis and the mrna expression was measured using reverse transcription poly chain reaction (rt-pcr). results: dm was showed high cytotoxicity on various cancer cell line, especially a cells. dm treated a cells did not show regular dna fragmentation and caspases activation. we also analyzed protein expression of apoptotic marker, but protein level didn't change. as a result, we hypothesized that this non-apoptotic cell death is mediated autophagocytosis. we checked lc and beclin- protein and mrna expression and inhibitory effect of autophagocytosis inhibitor. the expression level of lc was increased concentration and time-dependently. beclin- also was increased. and then, we examined gfp-lc punctation on a / gfp-lc stable cells using confocal. a cells were formed gfp punctuation after dm treatment. to confirm these data, we measured cell death ratio using -methyladenine ( -ma), an autophagocytosis inhibitor. after -ma treatment, dm induced cell death was decreased comparing with dm treatment. conclusion: dm did not induce apoptotic cell death. but dm showed several characteristics of autophagic cell death. taken together, dm induced autophagocytosis on a cells. these finding indicated that therapeutic potential of dm by triggering autophagic cell death. s. b. rasmussen , s. r. paludan university of aarhus, department of medical microbiology and immunology, aarhus, denmark during viral infections, different pattern recognition receptors detect specific pathogen associated molecular patterns (pamp)s. in the case of herpes simplex virus (hsv), detection is, among others, conducted by toll like receptor (tlr) , which is a transmembrane receptor located in the endosomes where it detects unmethylated cpg rich dna of extracellular origin, e. g. viruses. upon binding to dna, tlr initiates downstream signalling cascades resulting in induction of antiviral cytokines, interferon (ifn)a and ifnb being some of the most essential ones. the exact route of hsv to the endosomes and thus tlr recognition is not clear-cut. the endocytosis pathway is believed to be a central mechanism in which viruses translocate to the endosome, but recently the role of autophagy in tlr mediated viral recognition has been drawn in to focus. we have found indications of an autophagy dependent ifn expression during hsv- infection. by use of an entry defect glycoprotein l and glycoprotein h deficient hsv- , we found that tlr dependent ifn regulatory factor activation was abrogated. in addition, inhibition by -methyladenine of phosphoinositol -kinase class iii, which is crucial in autophagosome formation, abrogates ifnb induction. these findings points to a role for autophagy in tlr dependent recognition. in the ongoing project we are examining how hsv- triggers formation of autophagosomes. especially the role of endoplasmatic reticulum stress and doublestranded rna-dependent protein kinase will receive attention. also the newly found involvement of ubiquitin and acetylation in autophagy execution and regulation will be investigated. j. zovko , i. berberich , gk -immunomodulation universität würzburg institut für virologie und immunbiologie, würzburg, germany members of the bcl- family control the integrity of mitochondria and thereby influence survival and death of cells. most bcl- family members can localize to intracellular membranes via hydrophobic sequences within their c-terminal portion. murine a and its human homologue bfl- are anti-apoptotic members of the bcl- family. a is expressed in small amounts in the bone marrow and immature b cells, but in high amounts in mature b cells. thus the protein seems to be important for b cell maturation. we analyzed the function of the c-terminus of a . unless the c-terminal ends of other bcl- proteins the tail of a does not function as a strong membrane anchor. nevertheless, the last amino acids of a are important for the protein. in fact, the c-terminus of a serves a dual function by being required for the instability and the anti-apoptotic potential of the protein. we show that a undergoes proteosomal degradation controlled by its c-terminus. interestingly, binding to the proapoptotic bcl- factor bimel results in increased stability of a . this is due to reduced ubiquitination of a after binding of bimel. we conclude that the cterminus of a /bfl- serves as a docking site for e ubiquitin ligase(s) that control the stability of a by targeting the protein to the proteasomal pathway. very recently, we have identified a putative e ubiquitin ligase that interacted with a in a yeast two-hybrid screen. currently, we are trying to confirm this interaction in mammalian cells. suppressors of cytokine signaling (socs), initially identified as negative regulators of cytokine signal transduction, have recently emerged as multi-functional proteins regulating inflammation, survival, differentiation, and apoptosis of immune cells. here we describe novel function of socs- in the suppression of rosmediated apoptosis and associated mechanisms using tnf-alpha induced t cell apoptosis as a model system. both in jurkat t cells and primary splenocytes, socs- is induced under tnf alpha-induced apoptosis conditions. the tnf-induced apoptosis was mediated by generation of ros, and the over-expression of socs- significantly suppressed tnf-induced ros levels and the subsequent apoptosis. such anti-apoptotic function of socs- was manifest not only by the suppression of jaks acting upstream of p mapk, but also protection of ptps which down-regulate the tnf alpha -induced jak / activities. we further show that tnf-alphainduced ptp inactivation can be prevented by socs which up-regulates thioredoxin levels. finally we present data that there is a molecular interaction between thioredoxin and ptp which is formed in response to ros-generating stimuli and sustained in socs- overexpressing cells. the results strongly suggest that socs- acts as an anti-oxidant and anti-apoptotic factor to sustain t cell homeostasis and survival under oxidative stress imposed upon inflammatory cytokines during infection or other immune scenarios. aim: inflammation is a cardinal host response to injury, tissue ischemia, autoimmune responses or infectious agents. the effects of inflammatory mediators on the glial function are of particular interest since astrocytes contribute to the local inflammatory responses in the cns by producing cytokines, chemokines, and maintain local homeostasis clearing released neurotransmitters. cxcl /sdf- a not only regulates cell growth and migration of hematopoietic stem cells but may also play a central role in brain development. moreover, it has been described that tnf-a mediates in cxcl effects on primary astrocytes, and it is clear that tnf-a participates in the pathogenesis of many neurological conditions. methods: we used the astrocytoma cell line u- , and sk-n-mc as neuroblastoma cells. detection of tnf-a mrna expression was carried out by rt-pcr. transcriptional activity was measured using luciferase reporter gene assays in transiently lipofectin transfected-cells. we performed cotransfection experiments of nfat promoter construct with a dominant negative version of nfat (dn-nfat). neuronal death was performed by mtt and tunel assays. nfat translocation was confirmed by western-blot. p and fas-l expression was carried out by western-blot. results: cxcl induced mrna-tnf-a and transcriptional activity of tnf-a promoter in human astrocytes. this cytokine by itself was not toxic when added directly to astrocytes, but when we investigated its effect on nb cultures, neuronal death increased in a direct and indirect way. surprisingly, tnf-a did not induce nf-kb activation in nb cells but it induced nfat activity. nfat translocation was confirmed by western-blot. neurotoxicity was absolutely reverted in the presence of ciclosporin. we discard p pathway as responsible for tnf-mediated toxicity since we did not find any alteration in p -ser levels in stimulated cells. in addition, we found increased fasl expression in neuroblastoma cells after h of tnf-a treatment. conclusions: cxcl induced-tnf-a promotes nfat activation in neuroblastoma cells and this event leads to increased apoptosis through an increase of fasl expression without alter p function/levels. s. y. demiroglu , r. dressel universitätsmedizin göttingen, zelluläre und molekulare immunologie, göttingen, germany objectives: intracellular hsp is part of the cellular stress response system and can inhibit specific apoptotic pathways. extracellular hsp on the other hand, is an immunological danger signal that can induce the antigen-specific activity of cytotoxic t lymphocytes (ctl). interestingly, hsp does not protect against cell death mediated by ctl. acute overexpression of hsp can even increase the susceptibility of target cells to ctl, which use the granule-exocytosis pathway to induce apoptosis (dressel et al. cancer res : ) . granzyme b is one of the main effector proteases of cytotoxic granules. therefore, we analyzed the effect of acute overexpression of hsp on granzyme b-induced apoptosis. methods: hsp was expressed in a human melanoma cell line under the control of a tetracycline-inducible promoter (ge-tet). the effect of hsp induction on granzyme b-mediated apoptosis was now analyzed after delivery of granzyme b into the cytosol of the target cells by the endosomolytic activity of adenovirus type . results: hsp did not protect the melanoma cells against granzyme b-mediated apoptosis when annexin v binding, loss of mitochondrial membrane potential, release of mitochondrial cytochrome c, and activation of caspase- were evaluated. instead, we observed a moderate but significant pro-apoptotic effect of hsp in ge-tet cells when late apoptosis was analyzed at the nuclear level by sub g peak measurements (p= . ). in contrast, a partial protection of ge-tet cells was observed after acute hsp overexpression when apoptosis was induced by staurosporine. conclusion: acute overexpression of hsp does not seem to protect tumor cells from granzyme b-induced apoptosis; it appears even to accelerate the progression of apoptosis to dna fragmentation and nuclear condensation. it is conceivable that this hsp effect is mediated by chaperoning pro-apoptotic molecules and improving their function as it has been reported for the caspase-activated dnase (liu et al., blood : ) . thus, granzyme b might be able to kill even those tumor cells that undergo an otherwise protective stress response. the work has been supported by the grants grk and dr / - . the authors thank prof. c. j. froelich (evanston, usa) for the granzyme b and dr. t. seidler (göttingen) for the adenoviruses. tumor necrosis factor (tnf) elicits its biological activities by stimulation of tnfr and tnfr , both belonging to the tnf receptor superfamily. tnfr -mediated signal transduction has been intensively studied and is well understood, especially with respect to activation of the classical nfkb pathway, cell death induction and map kinase signaling. in contrast tnfr -associated signal transduction is poorly defined. earlier findings demonstrated that only membrane tnf, but not soluble tnf, properly activates tnfr , resulting in depletion of traf from the cytoplasm. here we confirm that tnfr induced depletion of cytosolic traf by the use of tnfr -and tnfr -specific mutants of soluble and membrane tnf. corresponding with the known inhibitory role of traf in the alternative nfkb pathway, we show that tnfr induced activation of the alternative nfkb pathway. thus, we identified activation of the alternative nfkb pathway as a tnf signaling effect that can be specifically assigned to tnfr and membrane tnf. j. c. morales , d. ruiz-magaña , d. carranza , c. ruiz-ruiz universidad de granada, instituto de biopatologí a y medicina regenerativa. centro de investigación biomédica, armilla, spain different molecular mechanisms have been involved in the resistance of tumor cells to tumor necrosis factor-related apoptosis-inducing ligand (trail)-mediated apoptosis. epigenetic modifications commonly associated with tumor development, such as histone deacetylation, may influence the resistance of some tumor cells to trail by regulating gene transcription of trail receptors and other trail pathway related-genes. in the present study we have analyzed the effect of several histone deacetylase inhibitors (hdaci), belonging to different structural families, on trail-induced apoptosis in leukemic t cell lines. moreover, we have analyzed the activity of hdaci in normal t lymphocytes. we have found that, to a greater or lesser extent, all hdaci potentiate the induction of apoptosis in leukemic t cells by enhancing the signals triggered upon trail ligation, from the activation of the most apical caspase- . in contrast, hdaci do not sensitize primary resting or activated t lymphocytes to trail-mediated apoptosis. the analysis of the expression of several pro-and anti-apoptotic proteins involved in the trail-signalling pathway indicates that most hdaci regulate the expression of trail-r and c-flip in leukemic t cells, but not in normal cells, which may explain their selective pro-apoptotic effect on leukemic cells. zfp l is a zinc finger containing protein that is involved in post-transcriptional gene regulation. it can bind to mrnas containing adenine uridine rich (are) regions and subsequently mediate their degradation. we have previously reported a role for this protein in promotion of b cell apoptosis. one mechanism whereby zfp l may mediate cell apoptosis could be by degradation of cell survival gene mrnas. the bcl- protein is an important cell survival protein at different stages of b cell development. bcl- mrna also contains are regions that could possibly be targeted by zfp l protein. in the present study, we have tested the ability of zfp l protein to bind to a bcl- mrna are probe and to degrade bcl- mrna. recombinant bacterially expressed zfp l protein was shown to bind specifically to a bcl- are probe by rna electrophoretic shift assays (remsas). furthermore, remsas using cell lysates of ramos burkitt lymphoma b cells stimulated to express high levels of endogenous zfp l also provided evidence that endogenous zfp l in b cells could bind to the bcl- are. in order to examine whether zfp l binding to bcl- are resulted in bcl- mrna degradation, actinomycin d rna degradation assays were carried out on murine embryonic fibroblast (mefs) cells from zfp l knockout mice and wild-type mice using quantitative real-time pcr analysis. bcl- mrna was expressed in both wild-type and knockout mefs. the half-life of bcl- mrna was found to be extended in knockout mefs compared to wild-type mefs suggesting that zfp l does play a role in degradation of bcl- mrna. overall, our data are consistent with a role for zfp l in degradation of bcl- mrna which could be a mechanism for the reported role of this protein in induction of b cell apoptosis. the epstein-barr virus (ebv) is a common human herpes virus, which can predominantly infect two types of human cells: lymphoid cells and epithelial cells. its infection is associated with several human malignancies (hodgkin's lymphoma, burkitt's lymphoma, nasopharyngeal carcinoma), where it expresses limited subsets of latent proteins among which the latent membrane protein lmp . since lmp is able to transform numerous cell types, it is considered as the main oncogenic protein of ebv. the principal mechanism of lmp function is based on mimicry of activated member of the tnf receptor super family (tnfr), by its ability to bind a similar sets of adapters and to activate overlapping signalling pathways like nfkb, c-fos-jnk, pi -kinase...involved in the regulation of cellular processes. we previously generated two unique model, a monocytic (te ) and lymphocytic (nc ) immortalized by ebv and which expressing type ii latency program. here we developed original dominant negative (dn), by generating a fusion between gfp and tes or tes (transforming effectors site) derived from the c-terminal intracellular part of lmp , in inducible vectors. then, we generated cell lines conditionally expressing these dns. we showed these dns not only inhibit survival processes resulting to the impairment of nfkb and akt pathway but increase apoptosis in this cell lines. we demonstrated that this pro-apoptotic effect is due to i) the depletion of lmp 's specific adapters and ii) the recruitment of theses adapters by dns interestingly allowed generation of apoptotic complex involved tradd, fadd and caspase- . using this nc tumorigenic model in scid mice, we showed that induction of the dn lmp -tes prevent development of tumours and mouse death. these dominant negative derived from lmp could be used to develop therapeutic approaches in malignant diseases associated with epstein-barr virus, but also in inflammatory pathologies. recent studies indicate that suppressors of cytokine signaling (socs) proteins play, in addition to their action as cytokine signaling inhibitors in the immuneinflammatory response, multiple roles in cell survival, differentiation, and apoptosis in diverse cell systems. since tumor cells often exhibit aberrant expression of socs genes, which may be involved in determining resistance to anti-tumor therapies, we have investigated the role of socs isoforms during dna damageinduced apoptotic response and cell cycle changes in various tumor cell types. by using tumor cell lines transduced for over-expression or knock-down of distinct socs isoforms, it is found that socs and socs differentially affect apoptosis and cell cycle changes induced by dna damaging agents in a cell type-specific manner. in t lymphocytic leukemia cell line jurkat, socs exhibited anti-apoptotic effect in response to ionizing radiation, hydrogen peroxide, and etoposide by inducing suppression of p mapk activities, while socs promoted apoptosis with an increase in p activities. in contrast, both socs and socs display proapoptotic effect in rko colon cancer cell lines upon exposure to gamma radiation or ros-generating agents. notably, effects of socs proteins on cell cycle changes induced by dna damaging agents were rather similar in that over-expression of either socs or socs induced a slight decrease in g or s phase cells and a prominent increase in g /m cells, regardless of their distinct effects on apoptosis. the analysis of cell cycle regulator proteins, however, revealed that different mechanisms are operating to regulate cell cycle via distinct cyclins and cdk inhibitors affecting g /s transition and g /m arrest induced by socs or socs . socs promoted dna damage-induced p induction and g /m cyclin b expression, while socs induced decrease in g cyclin e expression. the results suggest that socs isoforms potentially modulate growth of tumor cells exposed to dna damage via complex network involving apoptotic response and cell cycle regulation in a cell type-specific manner. the heat shock protein (hsp ) is a highly conserved a widely expressed molecular chaperone. it is known to regulate the activity of several protein kinases or the proper folding of client proteins. hsp has also been identified as an important regulator of cellular survival. besides these intracellular functions, extracellular hsp can initiate cross presentation or immune responses. apoptotic cell death occurs permanently in multicellular organisms, without initiation of an immune response. however the mechanisms which prevent an inflammatory response to apoptotic cells are not understood to date. hsp is released during necrotic cell death and proinflammatory effects of extracellular hsp have been observed. thus, we asked whether apoptozing cells cleave hsp during apoptosis or how hsp is disposed by these cells. we induced apoptosis either in activated or resting primary human cells and analyzed the hsp protein content. we observed that hsp is degraded during apoptosis resulting in the formation of a fragment of about kda. this fragment was to be observed exclusively in activated cells, while it was not detected if resting cells were induced to undergo apoptosis. analyzing the isoforms of hsp (hsp alpha and hsp beta) we could show that the kda fragment is formed after degradation of the alpha isoform of hsp . further, we were able to show, that hsp cleavage is dependent on caspase activity and most probably mediated by calpain. analyzing the cytokine response of monocyte derived phagocytes to apoptotic cells in presence or absence of exogenous hsp and caspase inhibitors. we observed a rather proinflammatory cytokine profile, if cleavage of hsp was inhibited or if exogenous hsp was added. these results demonstrate that cleavage of hsp represents a mechanism preventing the release of proinflammatory molecules from apoptozing cells. activity. mifc integrates the advantages of flow cytometry and fluorescence microscopy in one system, the imagestream. upon induction of autophagy, cytosolic lc -i is processed to lc -ii, which then remains associated with the autophagosome until its degradation upon fusion with the lysosome. an increase in steadystate levels of autophagosomes can be due to enhanced autophagy or decreased lysosomal activity. mcf- gfp-lc cells were therefore incubated in starvation medium for hours, +/-bafilomycin, which potently inhibits lysosomal activity. classical gating strategies allowed the detection of cell populations of interest, which were further analyzed on single cell levels. we conclude that imagestream-based analysis provides an improved method in terms of objectivity, sensitivity and significance, to quantify autophagic activity. our results clearly show the need for discrimination between "steady-state" levels of autophagosomes and "current flux" of fully functional autophagy, i. e. quantification of autophagic flux. jnk seems to mediate the bcl- /beclin- control of autophagy. recently, jnk was shown to be necessary for beclin- upregulation, and jnk-mediated phosphorylation of bcl- is associated with both, starvation-and ceramide-induced autophagy. the nfkappab pathway mediates critical survival signals during starvation, which have been linked to the inhibition of autophagy. we report here the novel findings that under conditions of starvation, pharmacological inhibition of nfkappab decreased the autophagic flux in mcf- cells, while jnk inhibition shows an enhancing effect on autophagy induction. ingenol -angelate (pep ), a novel activator of protein kinase c (pkc), has been shown to induce apoptosis in acute myeloid leukemia cells. we show here, that in contrast to leukemic cells, pep provides a strong survival signal to resting and activated t cells. this anti-apoptotic effect was dependent upon the activation of pkcv, a pkc isoform restricted to t cells and myocytes. expression of pkcv in the acute myeloid leukemia cell line nb turned their response to pep from an increased to decreased rate of apoptosis. furthermore, our data show that pep inhibited t cell apoptosis through the activation of nfxb downstream of pkcv, leading to increased expression of the anti-apoptotic proteins mcl- and bcl-xl. we conclude that pkcv expression determines whether pkc activation leads to an anti-or pro-apoptotic outcome in the cell types analyzed. this finding may be of considerable importance for the development of pkc -targeting antileukemic therapies. the neuronal growth factors, neurotrophins, and their receptors are widely expressed in a variety of non-neuronal tissues including the immune system. several reports indicate that survival and activation of normal b lymphocytes are regulated by nerve growth factor (ngf) and brain-derived neurotrophic factor (bdnf) autocrine circuits. however, the production and the role of neurotrophins were not evaluated in b lymphoma cells. diffuse large b-cell lymphoma (dlbcl) is a common and often fatal malignancy. despite major advance in the treatment (r-chop protocol) which improves the clinical outcome of patients, a subset of patients does not respond or relapses after the initial treatment; the exact mechanism of such resistance is not entirely clear. we hypothesized that autocrine neurotrophin survival circuits could contribute to the chemoresistance of dlbcl tumor cells. this hypothesis was investigated with dlbcl cell lines (su-dhl). thus, we evaluated the ability of su-dhl cells to produce neurotrophins (ngf, bdnf) and to express their receptors (p , trka and trkb) in different cell culture conditions. our preliminary data show for the first time the production of neurotrophins by dlbcl tumoral cells whose level decreased in apoptotic conditions, in association with bad dephosphorylation suggesting its pro-apoptotic role. furthermore our results suggest that up-regulation of autocrine circuits (expression of trka known to be involved in survival signaling pathways) may contribute to cell survival and thus drug resistances of tumoral b cells. objectives: ataxia telangiectasia (a-t) is a rare disorder caused by mutations in the ataxia telangiectasia mutated (atm) gene. this gene encodes atm, a protein kinase which has a major role in dna double strand break response. a-t patients suffer from a variety of immune system defects including lymphopenia, immunoglobulin deficiencies and impaired class switch recombination, they also have an increased incidence of cancer especially leukaemia and lymphoma. the susceptibility to lymphoid tumours and immunodeficiency could be partly due to failure of extrinsic apoptotic processes involved in regulation of the immune system. although atm is known to have a central role in the induction of apoptosis in response to unrepaired dna double strand breaks its role in extrinsic apoptotic pathways is unclear. this study aimed to investigate if atm has a role in fas induced apoptosis. a bank of lymphoblastoid cell lines (lcls) derived from a-t and normal individuals and tumour samples with wildtype or mutant atm were used in the study. apoptosis was induced by incubating cells with the fas activating antibody ch and analysed by flow cytometry. expression of the caspase inhibitor cflip which inhibits fas induced apoptosis was detected by western blot. results: there was no significant difference in the susceptibility to fas induced apoptosis or cflip protein expression between atm mutant and control groups. however cells expressing high levels of cflip protein do show greater resistance to fas induced apoptosis than those with lower expression. whilst the lcls expressed both long and short forms of cflip, the tumour cells expressed only the long form. conclusion: atm mutations do not affect susceptibility to fas induced apoptosis or alter cflip protein expression in lcls or tumour cells. cflip protein levels and fas susceptibility vary greatly between individuals but this is independent of atm status. high expression of cflip protein correlates with reduced apoptosis in response to ch treatment but there is no clear difference in cflip expression between atm wildtype and mutant cells. labdane diterpenoids have a broad spectrum of biological activities including antibacterial, antiviral, and anti-inflammatory properties. however, little is known about their possible role in the apoptotic cell death machinery. we report that labdane diterpenoids induce apoptosis in different tumor cell lines by activating caspase in the extrinsic death receptor pathway, with subsequent participation of mitochondrial signaling. activation of caspase by diterpenoids was followed by a decrease in mitochondrial membrane potential, the release of apoptotic factors from mitochondria to the cytosol, and subsequent activation of caspases and . diterpenoids also led to time-dependent cleavage of bid. inhibition of caspase- abrogated these processes, suggesting that the death receptor pathway plays a critical role in the apoptotic events induced by labdane diterpenoids. in addition, pretreating cells with neutralizing antibodies to fas ligand, tumor necrosis factor receptor (tnf-r ), and tumor necrosis factor (tnf)-a receptor (trail) inhibited diterpenoid-induced apoptosis, revealing it to be dependent on these death receptors. diterpenoid treatment also induced a significant increase in the generation of reactive oxygen species (ros). however, increased ros production was not directly involved in diterpenoid-triggered apoptosis. these results demonstrate that labdane diterpenoids induce apoptosis through activation of the death receptor pathway. conclusion: cell proliferation and differentiation are tightly regulated networks and it is believed that in cell differentiation, even in cancer form, cells precluded from proliferation. whether these changes affect the level of differentiation or the change of survivin expression can affect the proliferation and differentiation pathways are the hypotheses that need further investigation. synthetic alkyl-lysophospholipids (alps) are a group of unnatural lipids with promising anticancer capability. a prototypic member is the ether lipid -o-octadecyl- -o-methyl-rac-glycero- -phosphocholine (et- -och ; edelfosine), which induces selective apoptosis in tumor cells through activation of fas/cd independent of its ligand fasl/cd l. fas/cd is activated by edelfosine via its translocation in lipid rafts. in this study we showed that edelfosine promotes cell death in multiple myeloma and various solid tumor cell lines in a death receptor-independent manner. edelfosine-treated cells could not be protected against cell death after inhibition of caspases by zvad-fmk while fasl-stimulated cells stayed mostly alive. furthermore cells could not be rescued by addition of zvad-fmk in combination with necrostatin- , an inhibitor of death receptor-induced necrosis. fas resistant solid tumor cells overexpressing members of the anti-apoptotic bcl -family as well as cells overexpressing the cellular regulatory protein flip went in contrast to fas stimulation to apoptosis after treatment with edelfosine. therefore we suggest that edelfosine induces a death receptor-independent cell death pathway in a wide range of tumor cells. apoptosis represents a cellular "suicide" mechanism which allows the control of cell number from tissues and elimination of cells that present dna mutations or having an abberant cell cycle, those cells being predisposed to malignant transformation. thus, elucidating the mechanisms of programmed cell death process seems to be of great importance for malignant transformation, tumour evasion and therefore for anti-cancer therapy. many anti-cancer drugs act during physiological pathways of apoptosis, leading to tumour cell destruction. the present study focused on the potential influence of oncolitical treatment ( -fluorouracyl) associated with natural compounds (curcumin, genistein, quercitin) on the dynamics of the cell cycle and levels of apoptosis in colon cancer cell lines (e. g. colo , sw , lovo, caco- , ht- ) . in addition, expression of antigens involved in tumour proliferation and apoptosis (ki- , pcna, p , bcl- ) was compared with gene expression in the presence or absence of stimuli treatment. percentages of apoptotic cells were detected by using annexin v/fitc and propidium iodide double staining, while progression through cell cycle phases was evaluated by using pi staining. correlation analyses between the individual profile of the stimuli modulated gene expression with the coded protein expression were performed by using data from rt-pcr with specific primers, and indirect immunofluorescence followed by flow cytometry, respectively. stimuli treatment of colon cancer cell lines differentially induced higher levels of apoptosis as compared to untreated tumour cells, while cell cycle distribution of dna changed. data obtained showed a various expression and functional behaviour of the markers under study associated to colon cancer cells, suggesting their possible involvement in regulating the interactions between tumour cells and host immune system. the results obtained might further lead to the establishment of an experimental pattern for the corroboration of cell and molecular mechanisms involved in the tumour progression and the treatment resistance of colon tumors using cell lines. the effect of modulatoy agents on proliferation and apoptosis could be used in clinical departments in order to elaborate new therapeutical approaches and act as useful instruments in elaboration of individualized treatment schemes. extensive tissue trauma and malnutrition results in disorders of programmed cell death influencing the patients susceptibility to infections. the purpose of our study was to assess the effect of pancreatic cancer surgery and immunonutrition on the apoptotic signaling pathways. the randomized studies were performed in patients after pancreatic cancer resection with preoperative standard or enteral immunonutrition. lymphocytes expressions of bcl- , bax, caspase , , , nfkb, parp / kda, tnfr /cd a and cd /fas were assessed by western-blot and flow cytometry. results: before and after surgery the expression of bcl- , bax, nfkb, parp was significantly lower and expression of caspases, tnfr as well as percentage of cd cells significantly higher as compared with control group. caspase expression was significantly higher as compared with nfkb, parp and tnfr . in comparison to the standard nutrition preoperative immunonutrition increased bcl- and nfkb expressions and decreased caspases and parp expressions. in addition, we found a significant down-regulation of bcl- expression after surgery, but insignificant in patients with preoperative immunonutrition. conclusion: preoperative enteral immunonutrition has an modulative effect on apoptotic signaling pathways after pancreas resection and possesses antiapoptotic properties. this modulatory effect of glutamine and omega- fatty acids has no influence on patients outcome. the capacity of medicinal herbs to modulate cellular and humoral immune response could have useful applications in some immune-mediated disorders, infections and cancers. in this study the immunomodulatory effects of salvia mirzayanii a native plant that is widely distributed to iran was investigated. s. mirzayanii is used for the treatment of infectious and inflammatory diseases and as a tonic in folk medicine. study of the effect of this plant on the activated human peripheral blood lymphocytes showed stimulatory effects at lower concentrations and inhibitory effects at higher ones (p x . ). in flow cytometry analysis, accumulation of apoptotic cells in the sub-g phase of cell cycle of the mitogen-treated lymphocytes exposed to the inhibitory doses of the extract was observed. dna fragmentation analysis of these cells showed a typical dna laddering. immunization of the extract-treated mice with the antigen decreased delayed hypersensitivity skin reaction as well as the antibody titer at higher concentrations (p x . ). these results indicated the presence of immunomodulatory compounds in the extract of s. mirzayanii and suggest that the induction of apoptosis in lymphocytes might be the mechanism responsible for the inhibitory effect of the extract observed at higher concentrations. a new randomized, double-blind, placebo-controlled clinical trial was conducted with healthy volunteers receiving either la ( cfu/day) or placebo, during days prior to uv ( × . med). blister roofs, liquid and skin biopsies were collected , and days after uv exposure from non-irradiated and irradiated skin areas and used for identification of cells involved in uv-induced immune response, quantification of inflammatory cytokines, a dna damage marker (p ). while a similar decrease of lc for both groups was observed on day after uv exposure compared to placebo, la- group presented a faster increase of a new subset of epidermal dendritic cells (dc), namely early lc precursors (cd a low cd -) associated with a minor recruitment of monocytes. concomitantly, inhibition of il- and a tendency to inhibit il- was observed in la- group compared to placebo. on day , la- group presented a greater recruitment of early lc precursors and a trend to increase cd a low cd + lc precursors compared to placebo. additionally, a faster reduction of inflammatory and immunosuppressive cytokines (il- , tnfa, il- , and il- ) was observed in la group compared to placebo. we show that la limits uv-induced immune-suppression and skin inflammation. this contributes to the recovery of the skin immune homeostasis, confirming the previously observed benefits of la supplementation for photoprotection at a lower dose ( log). the thymus is one of the primary lymphoid organs and plays a central role in the immune system. it provides the essential microenvironment for proper t cell development. in the thymus, the maturation of t cells depends on many interactions between t cells and different stromal cell types, mainly composed of epithelial cells (tecs). foxn is a winged-helix/forkhead transcription factor, which is crucially required for proper epithelial cell differentiation in the thymus. foxn appears to be expressed in all epithelial cells of the early thymic rudiment starting around e . . previously, we have used a lineage-tracing system to confirm the existence of a bi-potent epithelial progenitor cell. using the cre-loxp system, we showed that a single epithelial cell, when reverted to express foxn in a nude (foxn -deficient) background, can give rise to a functionally competent thymic microenvironment. hence, we hypothesize that the epithelial progenitor cell expresses foxn . if true, it should be possible to target this cell type by use of foxn -promoter driven transgenes. conditional targeted cell ablation is a powerful method to elucidate the physiological function of cell populations and their regenerative capabilities. currently, we are using three different strategies of conditional targeted cell ablation in order to examine functional characteristics of epithelial bi-potent progenitor cells within the thymus. intracellular accumulation of poly glutamines is known to cause neurodegenerative disorders, such as huntington's disease. considering this, we are expressing transgenic egfp variants containing either or glutamine residues under the control of foxn promoter, leading to different degrees of tec degeneration. furthermore, also under the foxn promoter, we are using the transgenic expression of human diphtheria toxin receptor and the transgenic expression of the bacterial nitroreductase enzyme that converts the pro-drug metronidatole (mtz) into a cytotoxic cross-linking agent for conditional cell ablation. preliminary results describing the phenotypes of these mice will be presented. t. kamei , , y. toriumi , k. kimura european university viadrina, frankfurt (oder), germany, shimane institute of health science, izumo, japan, shimane university faculty of medicine, department of pediatrics, izumo, japan, japan yoga niketan, yonago, japan as a method in relieving stress, yoga is popular today. many reports of physical changes describing how yoga improves respiratory, circulatory, endocrine, and metabolic functions by yogic practice have been reported until now. we examined changes of electroencephalograph (eeg) and cellular immunity before, during, and after yoga exercises, in an endeavor to detect the correlation between them. the subjects consisted of eight yoga instructors who had been practicing yoga for several years. a -minute-rest period, followed by a -minute yoga exercise called asana, a -minute respiratory exercise called pranayama (various specialized respiration methods continuously performed with the eyes closed), and a -minute meditation were performed. throughout rest and yoga, brain rhythms were continuously recorded via two disc electrodes placed on each forehead (fp ). blood samples were drawn before and after each exercise. nk activity and percentages of t-cell and b-cell subsets were measured. during the pranayama period, both a positive correlation between the change in abundance of the activated alpha waves and the ratio of changes in nk activity (r= . , p x . ), and a positive correlation between the change in abundance of the activated alpha waves and the ratio of changes in the number of t lymphocytes (r= . , p x . ) were observed. furthermore, a positive correlation was also observed between the change in amplitude of the activated alpha waves and the ratio of change in the number of cd (r= . , p= . ). these findings suggest that yoga creates a stress-free and mentally concentrative state which activates the functions of nk cells and t lymphocytes, mainly of cd , within a short period of time. we conclude from these results that yogic respiratory exercise may be able to activate cellular immunity and to help recover the mental and physical harmony of human. yoga is considered to have an effect of some re-activation of a latent ability of harmonization in which humans naturally possess. t regulatory cells play a central role in the suppression of immune responses thus serving to induce tolerance and to control persistent immune responses that can lead to autoimmunity. several recent studies suggest also that diverse populations of regulatory t cell play an important role in regulating t-helper response to allergens, maintaining functional tolerance and preventing allergy. here we demonstrate that cd + cd + t regulatory (t reg ) cells are critical in controlling the immediate hypersensitivity response of bone marrow mast cells (mcs) without affecting cytokine release. this effect is shown to require a cell-cell contact and depends on interaction between ox l expressed on mcs and the constitutive expression of ox (members of the tumor necrosis factor [tnf] and tnf receptor family, respectively) on t reg cells. this interaction does not alter the activation of plc-g, syk and lat in ige/ag stimulated mcs upon co-incubation with t reg cells, whereas it induces a decrease in the phosphorylation levels of akt. moreover, we find that upon co-incubation with t reg cells, mcs show increased levels of camp, which is known to inhibit mcs function, as a result of ox l signal. antagonism of camp in mcs reverses the inhibitory effects of t reg cells restoring normal ca + responses and degranulation. the cross-talk between t reg cells and mcs through ox -ox l interaction defines a previously unrecognized mechanism controlling mcs degranulation. loss of this interaction may contribute to the severity of allergic responses or inflammatory disease. active regulation has emerged as a very essential mechanism for both inducing and maintaining peripheral tolerance to non-pathogenic environmental antigens. a healthy immune system responds to antigens with a combination of polarized th or th effector cells and the induction of antigen specific foxp + regulatory t cells (treg). it is believed that the dominant subset determines the quality of the eventual immune response. in allergic asthma there is a clear dominance of th cell responses to non-pathogenic environmental antigens. recently it was shown that the specific transcription factors that characterize the th and treg subset, gata and foxp respectively, counter regulate each other (mantel y et al., ) . we hypothesize that children with allergic asthma will respond to allergens with low induction of foxp + tregs and high gata + th cells. in order to prove this hypothesis pbmc of children with allergic asthma and non-sensitized healthy controls are stimulated with allergens, tetanus toxoid, and lps. almost million allergic patients are sensitized to the major birch pollen allergen bet v , which cross-reacts with major allergens of fagales (e.g., alder, hazel, hornbeam, oak) pollen and plant food allergens. the epitopes of bet v recognized by allergic patients' ige antibodies belong to the conformational type and therefore have not been characterized in detail. here we used antibodies raised against peptides spanning the bet v molecule in ige competition experiments to search for sequences which are involved in ige recognition. the strongest inhibition (i.e., g %) of patients' ige binding to bet v was obtained with polyclonal and monoclonal antibodies specific for peptides comprising aa - (p ) and aa - (p ) of bet v . cross-reactive ige epitopes between bet v and related pollen allergens and plant food allergens involved primarily p . p and p are not adjacent peptides in the bet v sequence but define a surface-exposed patch on the three-dimensional structure of bet v . as determined by surface plasmon resonance, monoclonal antibody mab specific for p and mab specific for p showed high affinity, i. e., dissociation constants, k(d) = . e- m and k(d) = . e- m, respectively. interestingly, peptide-specific mabs inhibited allergic patients' ige antibodies equally well as peptide-specific polyclonal rabbit antibodies but only the latter inhibited strongly allergen-induced basophil degranulation. this finding indicates that the surface patch defined with anti-p and anti-p antibodies contains several ige epitopes. in summary, we have defined a surface-exposed patch on the bet v allergen which seems to harbor the majority of the ige epitopes and may be used for the rational design of active and passive immunotherapy strategies against birch pollen and related allergies. background: antigen-specific th cells as well as tc cells, induced by biolistic gene transfer using plasmid dna encoding the model allergen b-galactosidase (bgal) under control of the fascin promoter (pfascin-bgal), inhibited the elicitation of systemic th immune responses and suppressed ige production in an experimental mouse model. moreover, protective biolistic dna vaccination with pfascin-bgal prevented th -mediated lung pathology (eosinophilia) in sensitized mice locally challenged with bgal protein, but led to the recruitment of th /tc cells into the lung, associated with substantial neutrophilic infiltration and the induction of airway hyperresponsiveness (ahr). objective: to analyze the modalities of ahr induction in mice biolistically vaccinated with pfascin-bgal. methods: balb/c mice were immunized with pfascin-bgal using the gene gun. subsequently, mice were challenged by consecutive intranasal application of bgal protein. cd + and cd + t cells, respectively, were depleted before and during the provocation phase by intraperitoneal injection of anti-cd (gk . ) or anti-cd ( . . ) monoclonal antibodies. neutrophilic granulocytes were depleted by treatment of animals with either anti-gr- monoclonal antibody rb - c or monoclonal antibody nimp-r . one day after the last challenge airway reactivity was assessed by whole body plethysmography, bronchoalveolar lavage (bal) was performed and the frequency of ifn-g-producing cd + effector t cells in the lung was determined. results: whereas neutrophilia in the lung of immunized and challenged mice was considerably alleviated by depletion of cd + t cells, ahr was not significantly affected, implicating that the elicitation of ahr by cd + t cells is dissociated from the activity of neutrophils. this notion was verified by elimination of neutrophils during the provocation phase, likewise leading to unaltered ahr. in contrast to cd + t cells, cd + t cells induced strong neutrophilic infiltration and ahr. transfer experiments with cd + or cd + t cell, separated from the airways of vaccinated and challenged mice, will probably reveal details of the effector mechanisms of th and tc cells operative in the elicitation of airway inflammation. conclusions: robust type immune responses, although highly effective in the counter-regulation of local th -mediated pathology, might as well trigger inflammatory reactions in the lung and provoke the induction of ahr. respiratory epithelial cells function as physical barrier and have shown to be active participants within the process of defense against pathogens and recognition of allergens. upon activation they release inflammatory mediators thereby creating a micro environment in which recruited immunocompetent cells induce a local immune response. house dust mite (hdm) extract as a source of allergens has been shown to induce a broad panel of genes upon stimulation of epithelial cell line nci-h . the proteolytic activity of these hdm allergens has been proposed to be involved in the activation process. the aim of this study was to compare the influence of hdm extract on respiratory epithelial cells with grass pollen allergen-induced activation of these cells with regard to the mechanism of activation, gene expression level, and the level of induced cytokine and chemokine release. in contrast to the hdm major allergen der p , we were able to show that the major allergen of phleum pratense, phl p , although sharing molecular similarities with der p , does not display any enzymatic activity under physiological conditions. therefore, in this study respiratory epithelial cells were stimulated with grass pollen extract and purified phl p . chemokine and cytokine release was determined by multiplex enzyme-linked immunosorbent assay and mrna was used for cdna-microarray analysis. first data show that both, hdm extract and grass pollen allergens, induce the release of il- and il- from nci-h cells. furthermore, stimulation with hdm extract leads to the release of tnf-a, gm-csf and ifn-g. interestingly none of these mediators was induced after stimulation with grass pollen extract or purified phl p . in contrast to hdm extract grass pollen allergens induce the release of mcp- from respiratory epithelial cells, as well as moderate levels of il- . detailed characterization of the response on gene expression level might give new insights into the pathophysiology of grass pollen allergy and a comparison with hdm induced expression profiles will be helpful towards understanding the allergic response in general. (supported by dfg sfb tr ) results: collectively, responses to blg, but not to bsa, were observed in all groups analyzed, included healthy controls. nevertheless, distinct profiles of response were obtained: children with ige mediated cma had a significant increased level of proliferation (mean±sd of stimulation index(si): . ± . ) and of il- (mean±sd: ± pg/ml), and reduced il- (mean±sd of il- -spot forming units/ x cells (sfu): ± ), compared to healthy subjects ( . ± . si, p x . ; ± pg/ml, p x . ; ± sfu, for proliferation, il- and il- , respectively); children with non-ige mediated cma had a significant reduction of il- ( ± pg/ml), compared to ige patients (p x . ) and an increased, although not statistically significant, production of ifn-g ( . ± . sfu) compared to control ( . ± . sfu) and to ige-allergic patients ( . ± . sfu). finally, tolerant patients showed reduced il- ( ± pg/ ml, p x . ) and proliferation ( . ± . si), compared to acute ige-cma children. interestingly, the high level of il- observed in all groups might have a counter-regulatory effect, since its neutralization resulted in an increase of proliferation to blg; by contrast, il- was undetectable in all patients even blocking the il -receptor. conclusion: blg-specific, immune responses can be recalled in peripheral blood of cma patients, as well as of normal and tolerant children. a th -like response with il- and proliferation is dominant in ige-mediated cma patients; by contrast a th -skewed response with ifn-g is present in non-ige-mediated allergic and in those children who outgrew ige-allergy. y. f. tang , b. chua , f.c. lew , a. ho , k. wong , k.l. wong , d. m. kemeny national university of singapore, immunology programme, yong loo lin school of medicine, singapore, singapore allergic inflammation of the airways causes changes in the lung wall that can lead to chronic inflammatory disease such as asthma. using a mouse model, this response can be divided into an induction phase, in which cd th t cells specific for airborne allergens are produced, and an effector phase, during which they are recruited to the lung. in the lung, recruited th cells orchestrate the inflammatory response marked by eosinophilia, mucus hyper secretion and increased airway hyperresponsiveness (ahr). previously we, and others, have shown that transfer of cd t cells inhibits the induction of the th response. here we have investigated the effect of cd t cells on the effector phase of the inflammatory lung response. in vitro activated ot-i cd t cells were transferred to ovalbumin (ova)/ alum immunized mice one day before the first of airway challenges with ova. eosinophil infiltration was inhibited by transfer of cd + t cells ( . %± . % to . %± . %). when ifn-gamma -/-ot-i cd t cells were transferred, we found that the inhibitory effect on eosinophilia was reduced ( . %± . %), suggesting an important role for cd t cell ifn-gamma. cd c + cd + cd blung dcs from cd transferred mice secreted higher levels ( pg/ml) of il- p following ex-vivo stimulation as compared with animals that were not given cd t cells. these data show that, in addition to regulating the induction of the allergic immune response, cd t cells can subsequently divert the local lung environment to one that favors th immunity. the chain terminator drug abacavir triggers a serious hypersensitivity reaction in % of patients with hiv infection. this reaction is strongly associated with hla-b* and appears to be mediated by cd + t cells producing inflammatory cytokines. we show that cd +t cell responses can be primed in vitro, in normal blood donors who are hla-b* +, but not in non-b* + donors. cd t cells, but not cd t cells, are expanded by abacavir pulsed autologous apc over a -day culture, producing ifn-gamma and tnf-alpha upon re-stimulation with apc expressing hla-b* . similar responses were detected in abacavirhypersensitive hlab* + patients. responses were not detected using mutant apc deficient in tap or tapasin, or when normal apc were aldehyde fixed before loading with abacavir, indicating a reliance on the conventional mhc-i ag presentation. responses were exquisitely restricted to hla-b* since they were undetectable using apc expressing closely related hla-b or b allotypes. responses to apc expressing mutants of hla-b* demonstrated a crucial role for residue . isolation of peptide fractions from abacavir-loaded cells has led to the identification of specific fractions recognised by an abacavir-specific t cell line. our data suggests that abacavir forms a conjugate with an endogenous peptide that is presented by hla-b* triggering cd t cells. we speculate that this form of altered self is highly immunogenic, behaving like a form of allogeneic mhc-i, contributing to the responses observed in abacavir naï ve individuals. the molecular mechanisms underlying altered hla-b* may be relevant to the role of other disease-associated class i allotypes such as hla-b and b . a. jenckel , s. bulfone-paus , n. föger research center borstel, immunobiology, borstel, germany mast cells play a crucial role in acute inflammatory and allergic reactions. upon activation, mast cells secrete a vast array of preformed and newly synthesized inflammatory mediators. recent work has begun to appreciate an important role of the actin cytoskeleton in mast cell activation. the actin-associated protein coro-nin a (coro a), a coronin family protein preferentially expressed in hematopoietic cells, is critically involved in various actin-mediated cellular functions of leukocytes. recent data of our group also indicate a regulatory role of coro a in mast cell function. coronin proteins have been described to be differentially phosphorylated in vivo. however the molecular mechanisms by which coro a is regulated in response to physiological stimuli are still poorly characterized. here we investigated the modalities of coro a phosphorylation during the activation of mast cells. immunoprecipitation studies combined with phospho-specific western blotting techniques revealed a transient phosphorylation of coro a on serine residues upon antigen-specific engagement of fc-epsilon-receptors. as the phosphorylation status of coro a can influence its association with the actin cytoskeleton, we analyzed the subcellular localization of coro a during mast cell activation. cell fractionation experiments demonstrated that the association of coro a with the actin cytoskeleton significantly decreases in response to mast cell stimulation, concomitant with the increase in coro a phosphorylation. a functional correlation between coro a phosphorylation and its association with the actin cytoskeleton in mast cells was further indicated by structure function experiments employing specific phosphorylation mutants of coro a. thus, coro a is a downstream effector molecule of fc-epsilon-receptor signaling and likely is involved in the dynamic reorganization of the actin cytoskeleton during mast cell activation. allergen-specific t and b lymphocytes play an important role for the pathogenesis of asthma. t cells orchestrate the infiltration of the lung tissue with eosinophils and neutrophils and provide help for allergen-specific immunoglobulin production. recently, we have shown in a mouse model for allergic airway inflammation that b cells directly interact with t cells in the inflamed tissue and locally produce ige. to analyse t/b-interaction in the inflamed tissue in more detail, we developed a novel adoptive transfer system using ovalbumin-specific t cells and nitrophenolspecific b cells. recipient mice are then challenged intranasally with an np-ova conjugate. this system allows to track single allergen-specific t and b cells in all stages of the immune reaction using flow cytometry and immunohistology. in addition, cells can be re-isolated by flow-sorting for in-depth analysis. using this system we could define several phases of the inflammatory reaction. t and b cells first become activated in the lung-associated lymph nodes. granulocytes can be found very early in the lung tissue and also activated t cells very rapidly emigrate to the site of inflammation. however, clusters of allergen-specific t and b cells can only be found in later stages of the reaction. as another focus, we used our in vivo system to define the role of t cell costimulatory molecules for airway inflammation. costimulatory receptors are key regulators of t cell activation and differentiation and therefore promising targets for therapeutic intervention. of special interest is the t cell-specific icos molecule which is important for t/b cooperation as well as the regulation of chronic inflammatory reactions. using icos knock-out mice we were able to delineate the specific role of icos for the different stages of airway inflammation. in particular, we analysed the impact on t cell subset differentiation, cytokine production and allergen-specific immunoglobulin production. the integrity of the actin cytoskeletal network is critcal for a large variety of cellular functions. coronins constitute a family of evolutionary highly conserved wdrepeat containing proteins that have been implicated in the regulation of actin cytoskeletal dynamics. in mammalians seven coronin family members have been described. the high degree of sequence conservation amongst coronin family proteins suggests common features and functions. however, individual family members may also have developed additional selective and specific functions. our recent studies on coronin a (coro a) deficient mice have demonstrated that coro a exhibits an inhibitory function on the cellular steady-state f-actin content, is required for chemokine-mediated functions in t cells and is involved in the maintenance of t cell homeostasis. coronin b (coro b) is a closely related homolog of coro a and the two genes are co-expressed in hematopoietic cells. to address the question of functional redundancy in vivo, we have generated coro b deficient mice and crossed them with coro a deficient mice to obtain coro a/coro b double deficient mice. analysis of t lymphocytes from coro a/coro b double deficient mice revealed defective chemotactic responses and a severe peripheral t cell lymphopenia in double-deficient mice, which was significantly exacerbated as compared to the respective single knock-outs. an analysis of coronin deficient mast cells also revealed an involvement of coro a/coro b in the regulation of actin cytoskeletal dynamics and the function of mast cells. however, in contrast to the inhibitory effects of coro a/ b deficiency on t cell function, mast cell degranulation and migration was enhanced in coro a/ b double deficient mast cells. thus, depending on cell type specific requirements, coronin proteins can either exhibit positive or negative regulatory functions. additional studies will investigate molecular and regulatory mechanisms by which coronin proteins control actin cytoskeletal organization and function of immune cells. together, our studies here reinforce and expand our appreciation of the importance of actin-cytoskeleton regulatory proteins for immune cell function. initially found by serial analysis of gene expression, murine samsn (also known as hacs or sly ) is a putative adaptor and scaffold protein with a sterile-alphamotif (sam), a src homology (sh ) domain and a predicted bipartite nuclear localization signal. the samsn gene is located on mouse chromosome and encodes a well conserved protein with amino acids, which is predominantly expressed in hematopoietic tissues. initial overexpression studies suggest a contribution of samsn in b cell activation and differentiation, however its physiological function is yet unknown. to investigate samsn expression in lymphatic and myeloid cell types in greater detail we employed the sensitive method of quantitative real-time pcr. our data revealed an expression of samsn in all tested hematopoietic cell types. the highest expression level of samsn mrna was seen in mast cells compared to lower levels in macrophages, dendritic cells, cd + and cd + t cells and b cells. the other two members of the sly family of adaptor proteins -namely sly (hacs ) and sly (sash ) -were expressed only at a very low level in mast cells. the high level of samsn mrna expression in mast cells, together with minimal expression of other sly family proteins in these cells, implicates an important role of this adaptor protein for mast cells. to address the potential role of samsn in mast cell differentiation and function we are analyzing bone marrow derived mast cells from samsn deficient mice. initial in vitro experiments indicate normal proliferation and differentiation of samsn deficient mast cells. in additional studies we are now investigating the effects of samsn deficiency on mast cell activation processes, such as degranulation, cytokine production and the signal transduction cascade. analyzing the role of samsn in mast cells will help to define the biological function of this novel class of adaptor proteins. introduction: we showed previously that the ability of murine igg antibodies to mediate anaphylactic reaction is directly dependent on the amount of sialic acid residues attached to the carbohydrate chain n-linked to the antibody fc region (silva et al; j.immunol., ). then, we hypothesize that differences in the glycan composition mainly the sialylation grade observed between the anaphylactic and non-anaphylactic igg abs may be resultant of the differential expression of the glycosyltransferase, essentially sialyltransferase, coding genes during its synthesis in b cells. objective and methods: to prove this hypothesis it was analyzed the expression of st siai-v; st galnac i-iv, st gal ii -v genes quantitatively by real time-pcr in the hybridomas producer of these two types of igg abs. results: we observed that the expression of st gal i, iii and v coding genes was similar in both hybridomas, while the st gal ii and iv genes were less expressed in the hybridoma producer of non-anaphylactic igg . in addiction, the expression levels of st sia and st galnac genes in the hybridoma producer of anaphylactic igg were significantly higher when compared to those observed in the hybridoma producing of non-anaphylactic igg . conclusion: these data suggest a direct correlation between the sialylation grade observed in the carbohydrate chain attached to the igg abs and the expression of sialyltransferase enzymes in the hybridomas producer of these molecules. financial support: cnpq, capes, fapesp. basophils are innate immune cells endowed with important effector functions during allergic inflammation and parasite infection. their activation in terms of histamine and cytokine production is mediated through immunoglobulin-dependent and -independent mechanisms, raising the question whether stimulation of tolllike receptors (tlrs), which have been described in basophils, has a similar effect. we found that, in contrast to other tlr agonists tested, only the doublestranded rna poly(a:u) induced the typical t h cytokine and histamine production in vitro. this compound was also fully active when administered in vivo since it activated basophils and promoted their recruitment to the periphery. we took advantage of a murine model of allergic asthma to establish the pathophysiological relevance of this finding. using both adoptive transfer and depletion of basophils, we established not only that these cells contribute directly to the severity of asthma symptoms, but also that a mimic of viral infection can aggravate the disease through their activation. this is the first evidence for a mechanism of exacerbation of allergic asthma induced by a mimic of viral infections, mediated through basophils. ishes the airway hyperresponsiveness and airway inflammation in experiment murine asthma models. to investigate the effect of activation of nkt cells at different allergic asthma progression, we administered balb/c mice with a-galactosylceramide (a-galcer), a stimulator for nkt cell activation, before or after ova immunization and measured the airway inflammation of that mice after times of intranasal ova challenge. in our results, the total numbers of bronchoalveolar lavage (bal) cells were higher in mice administered with a-galcer before ova immunization compared to that of mice administered with a-galcer after ova immunization. moreover, significant increased percentage and cell numbers of eosinophils in bal of mice administered with a-galcer before ova immunization was noted. il- and eotaxin are the most potent cytokine/chemokines for the recruitment of eosinophils. il- and eotaxin levels in the bal fluid were higher in mice administered with a-galcer before ova immunization compared to that of mice administered with a-galcer after ova immunization. these data demonstrate that activation of nkt cells at different allergic asthma progression dictates the different outcome of asthma. in addition, the activation of nkt cells in naïve mice induces airway inflammatory responses. the potential risks of treatment with nkt cell activation on human diseases should be considered. objective: bronchial asthma is a complex disease of the lung and is characterized by a variety of symptoms such as airway hyperresponsiveness, reversible airway obstruction, high serum levels of ige and inflammation. histologically, there are infiltrations of eosinophils, degranulated mast cells and hyperplasia of airway globlet cells in addition to lymphocytes. the transcription factor irf mediates the differentiation into th cells by activating multiple genes which are independently crucial for the development of naive t cells into th cells. because irf is expressed in many different tissues, it can be considered as a master switch factor for th cell differentiation. methods: here, we tested mice deficient in irf in the murine acute asthma model to evaluate its importance in this th cell-mediated disease. the protocol setup was the following: sensitizations s. c. with ova, followed by challenges via ova inhalation and adoptively transferred wildtype cd + t cells prior to initial sensitization. in our experiments, we could demonstrate that only after priming of irf deficient mice with the help of adoptively transferred cd + t cells, asthma symptoms in these mice were more severe than in wildtype controls. as an example, eosinophil infiltration into the lung was increased by . fold. likewise, ovaspecific antibodies and numbers of goblet cells (fig. ) were also significantly higher in irf deficient mice. conclusion: interferon regulatory factor plays a role in the severity of the development of asthma. in its absence, proinflammatory parameters in the lung are increased significantly. this effect is only visible in the presence of wildtype cd + t cells. mechanistically, a potential counterregulation of asthma by th cells is not available in irf knockout mice. together with our previous report that irf represents a susceptibility gene for allergy in the human, our data highlight irf as key in regulating the severity of asthma. the sensory neuropeptide substance p (sp) acts as an important stress mediator with its own stress axis in the skin modulating mast cell as well as antigenpresenting cell (apc) activity. here we postulate that stress-dependent communication between nerve fibers and immune-competent cells can also occur in spleen and affects the course of inflammatory disease. to address this question, atopic dermatitis-like allergic dermatitis (ad) was induced in c bl/ mice by intraperitoneal sensitization and intradermal challenge using chicken egg ovalbumin. animals were additionally exposed to noise stress for hrs prior to challenge. in this model, stress lead to a relative hyperinnervation of the immune-competent areas of the spleen. at the same time, an increased number of apc could be observed in these areas and contacts between nerve fibers and apc were found. under the same conditions, we were able to show increased nk -receptor and ppt mrna levels. accordingly, sp had the capacity to raise the number of antigen presenting cells in spleen and altered the profile of cd c expressing apc substets characterized by cd and cd expression in vitro. in vivo we found a stress dependent shift of cytokine mrna levels towards a th- cytokine profile and increased levels of il- mrna. further the number of cd + t-regulatory cells was increased in vitro. additional analysis of the quality and function of neuro-immune interactions in the spleen will reveal the role of the observed stress-induced alterations in allergic inflammation. proton pump inhibitors (ppis) that are the cornerstone of gastroesophageal reflux disease therapy have been reported to improve asthma and eosinophilic esophagitis (ee) in patients with associated symptoms. the most accepted explanation for these findings is based on the belief that pathologic acidic reflux can act as a triggering factor for these diseases through proximal extent and laryngopharyngeal reflux in asthma and impairment of the epithelial barrier in ee. under these considerations, acid suppression is believed that could prevent these pathogenic mechanisms. nonetheless, a number of evidences suggested the possibility that ppis could have a direct effect in molecular pathways involved in asthma and ee: ) the inhibitory mechanism of ppis implies alkylation of cysteine residues in gastric atpase , ) asthma and ee are prototypic th diseases in which the cytokines il- and il- play a principal role through the activation of the transcription factor stat , and ) we have recently demonstrated that some chloromethyl ketones can downregulate stat by mechanisms involving cysteine alkylation. on the theoretical basis that cysteine reactivity of ppis may affect the regulation of stat , we analyzed its effect in the activation of stat by il- and il- . we found that treatment of cells with ppis inhibited the ability of il- and il- to signal stat activation in a dose-dependent manner in multiple cell types from different origin. given the important role of these mechanisms in asthma and related diseases, our findings show a novel mechanism to understand the effect of omeprazole in these diseases. in argentina more than three million people suffer from asthma, and the number is rising. asthma is defined as a disorder characterized by chronic airways inflammation that results in high mucus production and airways hyperresponsiveness. a th mediated immune response prevails in these patients. in the asthmatic exacerbation period, crisis (cr), triggered by viral infections or other factors, there is a high prevalence of bacterial overinfection. our objective is to compare immunological parameters and in vitro response of lymphocytes to bacterial antigens in the same patient, at that moment and at a time of stability between episodes (i). we studied asthmatic patients both at cr and i. we evaluated eosinophils, basophils and ige expressing b lymphocytes; as well as t regulatory cells (by expression of cd and cd high (treg)), that might inhibit the development of a th response, together with gdt cells, which function in asthma is not completely understood, but could have a role in the increased airway responsiveness. to evaluate the t cell response, mononuclear cells were cultured for hours in the presence of m. tuberculosis (m) or s. pneumoniae (spn), or absence of them (c). then the percentage of activated cells was determined (expression of cd or cd at hours). all the parameters were evaluated in peripheral blood by flow cytometry. discussion: even though the pathophysiological characteristics of asthmatic patients in periods of cr and i are different, no significant differences were observed in the parameters (cell populations and cell response to bacterial antigens) evaluated when compared for the same patient at cr and i. we might be able to detect differences if we studied cells from the lungs, the target organ. we demonstrate that murine and human lc expressed the h r. the level of intracellular ccl production in human lc was reduced after stimulation with h r agonists and basal production could be restored when h r was blocked with the specific antagonist jnj . moreover histamine and a h r specific agonist augmented the migration of lc from the epidermis as shown in ex-vivo migration experiments using human skin and in-vivo migration experiments in mice. in conclusion, the h r is expressed on murine and human lc and influences the immunomodulatory function and migration of these cells. these findings underline the relevance of the h r in allergic skin diseases and encourage further exploration of the h r as a therapeutic target in allergic skin diseases. expression data of the non-coding rna gene, prins (psoriasis susceptibility-related rna gene induced by stress) identified and characterized by our workgroup, suggests a role for prins in psoriasis susceptibility and in cellular stress response. in order to asses the function of prins, we aimed to identify genes regulated by prins and intracellular molecules interacting with this stress-induced non-coding rna. to identify prins regulated genes, we carried out a cdna microarray chip experiment on hela cells where the expression of prins was silenced. this experiment identified g p , an interferon-inducible anti-apoptotic gene that was down-regulated by prins silencing. g p was strongly expressed in proliferating keratinocytes and markedly upregulated in involved psoriatic epidermis compared to healthy epidermis. to detect prins interacting proteins we applied ribonucleoprotein (rnp) purification in hacat cells. with the help of matrix-assisted laser-desorption ionization time-of-flight (maldi-tof) method we identified nucleophosmin, a protein that physically interacts with prins rna. nucleophosmin is a ubiquitously expressed nucleolar phosphoprotein which shuttles continuously between the nucleus and the cytoplasm. immunohistochemical experiments revealed that the expression of nucleophosmin was significantly elevated in psoriatic involved epidermis, localized to the dividing cells of the basal layer. our data indicate that the non-coding prins rna forms a molecular complex with nucleophosmin that regulates stress-induced cellular processes. we suppose that the abnormal functioning of this complex may result in the altered regulation of genes among them the anti-apoptotic g p which can contribute to the pathogenesis of psoriasis. atopic dermatitis (ad) is a chronic inflammatory skin disorder based on a genetic predisposition and triggered by environmental factors characterized by eczematous skin lesions, pruritus, and typical histopathological features. rituximab is a monoclonal anti-cd antibody therapy that targets pre-b cells and mature b cells, but not plasma cells. ad is generally considered as a biphasic, with switch to initial th to chronic th -predominant disease, in which rituximab may have multiple effects. objectives: to report three patients with severe ad refractory to conventional treatments and to anti-ige monoclonal treatment (omalizumab). materials and methods: three patients with severe refractory ad with high levels of serum ige that received weekly intravenous infusions of rituximab at a dose mg/m body surface each. subsets of lymphocytes were analyzed with multiparametric-flow cytometry (facscalibur, bd) at baseline and at specific intervals after treatment. serum immunoglobulins levels were quantified by nephelometry. results: at baseline, all patients had highly elevated levels of total ige ( g , ; g , ; g , mg/dl, respectively). all patients underwent prior treatment with omalizumab for months, with only partial response. then, we started rituximab therapy, resulting in a clear and complete improvement of ad eczema area and severity of skin lesions in all patients. remission of pruritus was observed from the nd week after initiation of rituximab therapy up to year. whereas allergen-specific ige levels were not altered, we observed a large reduction in total serum ige concentrations after initiation of therapy with rituximab. in the first treated patient (follow-up year), ige levels decreased from , to , mg/dl. the other two patients are in the and -months of the follow-up period. importantly, during follow-up no other therapies were required for ad control. conclusions: treatment with an anti-cd antibody led to a dramatical improvement in our series of patients with severe refractory ad. this study support further evidence on the efficacy and safety of rituximab in severe ad. we have previously demonstrated that chronic topical exposure of mice to the contact allergen dncb or to the respiratory sensitiser trimellitic anhydride (tma) preferentially activates t helper (h) and th cells, respectively. in addition, a single application results in divergent cutaneous cytokine production and the migration of langerhans' cells (lc) with different tempos. to explore events occurring after allergen application, balb/c strain mice were exposed to a single topical dose of either %dncb, %tma or to vehicle alone for . - h. measurement of cytokine production from skin exposed to the allergens was performed by cytokine bead array. exposure to dncb provoked rapid production of il- (mean= pg/ml, n= , p x . ), il- (mean= pg/ml, n= , p x . ) and il- a ( pg/ml, n= , p x . ) in skin compared with tma-or aoo-treated mice. in subsequent experiments, mice received an intradermal injection of ng/ear of murine recombinant il- or of the known regulator of lc migration; il- b. interleukin- b induced a significant loss of epidermal lc numbers, measured as a function of reduced frequency of mhc class ii positive cells within epidermal sheets, after ( %) and h ( %) (n= , p x . ). in contrast, il- or control injections were without effect. however, il- administration caused an increase in cutaneous il- production ( pg/ml, n= , p x . ) compared with control injection and naï ve tissue ( and pg/ml, n= , ns). in addition, systemic treatment with anti-il- antibody failed to impact on lc migration provoked by dncb ( % reduction; n= , p x . ). in parallel experiments dncb-induced lc migration was blocked by treatment with anti-tumour necrosis factor (tnf)-a antibody, another cytokine known to regulate lc migration. however, dncb-induced cutaneous il- a ( pg/ml, n= , p x . ) and il- ( pg/ml, n= , p x . ) expression was reduced to baseline levels by anti-il- treatment. these data demonstrate that il- is not involved in the regulation of lc migration, unlike il- b and tnf-a. however, il- is involved in the regulation of the production of other cutaneous cytokines provoked by dncb. therefore it is hypothesised that il- may influence lc and dermal dc maturation, via the expression of il- a and il- . allergic contact dermatitis (acd) caused by nickel ions (ni) represents the most common form of human contact hypersensitivity. along with other allergies its incidence is increasing in the us and worldwide (nhanes iii survey ), but the majority of molecular events underlying this kind of t-cell mediated disease are still widely unknown. to elucidate initial molecular mechanisms (sensitization phase) taking place at the primary allergen contact site in human skin a differential proteomic approach was chosen. by applying dige technology (differential gel electrophoresis), software analysis and mass spectrometric protein identification to cell lysates of allergen stimulated human keratinocytes, seventeen proteins were identified that are specifically regulated by metal allergen ni. in the attempt to further characterize the role of a certain down regulated p -mapk-pathway related protein (p prp) in acd, we analysed its regulation, differential distribution of phosphorylated isoforms as well as its subcellular localization. our results strongly support an involvement of p mapk pathway in allergenspecific signaling responses. it is expected that identification of differentially allergen-regulated proteins and detailed analysis of acd-associated signaling events in primary keratinocytes will lead to a better biomolecular understanding of the initiation of human contact hypersensitivity. (work supported by eu-project novel testing strategies for in-vitro-assessment of allergens, lshb-ct- - , www.sens-it-iv.eu.) objectives: schnitzler's syndrome is a rare disease characterised by chronic urticaria, monoclonal gammopathy, fever, and arthralgia/arthritis with marked elevation of acute phase reactants. in the long term, % of patients develop a lymphoproliferative disorder. schnitzler's pathogenesis is unclear; immunosuppressive treatment is ineffective and high dose steroids are usually required. the recent finding that treatment with il- receptor antagonist (il- ra; kineret) is extremely effective has raised the issue of the role of the inflammatory cytokine il- b and of il- -like cytokines in the pathogenesis of the disease. methods: two patients with recently diagnosed schnitzler's syndrome were treated with kineret, obtaining rapid disappearance of fever and urticaria and normalisation of acute phase reactants in one month. blood samples were collected before and after initiation of therapy. serum cytokine levels were measured by elisa, and expression of il- -related genes by real-time pcr on mrna from blood cd + monocytes. results: compared to normal controls, schniztler's monocytes had similar expression of il- , il- bp, and caspase- , both before and after therapy with kineret. il- b expression was similar to controls before therapy, and was decreased five-fold after therapy. at the serum level, neither inflammatory (il- b, tnfa, il- ) nor anti-inflammatory cytokines (il- , tgfb) were detected. as expected, il- ra was only detectable after therapy. il- was detectable in schnitzler's sera at higher levels than in controls ( . vs. . pm) and decreased after therapy ( . pm). the circulating il- inhibitor il- bp was lower than in controls and not affected by therapy. thus, free il- levels were increased in schnitzler's patients as compared to controls ( . pm vs. . pm in controls) and decreased after therapy ( . pm). conclusions: schnitzler's syndrome is not associated to enhanced expression of il- -related cytokines (il- b, il- ), nor of the il- /il- -converting enzyme caspase- in blood monocytes. however, the high circulating levels of il- suggest an increased activity of caspase- , as in the case of autoinflammatory diseases. experiments are in progress to test this possibility. atopic dermatitis (ad) is a chronic relapsing allergic skin disease with a high and growing prevalence. currently around % of the children in industrialized countries are affected. in most cases patients exhibit increased systemic ige-levels (so-called extrinsic form) accompanied by sensitization to allergens. while ad is frequently cleared until adulthood, many patients develop allergic rhinitis and asthma. most ad-patients show topical colonization with staphylococcus aureus indicating a defective innate immune response. as a class of pattern recognition receptors, toll-like receptors (tlrs) are essential for pathogen recognition and critical for the induction of an effective adaptive immunity. all known tlrs except tlr signal via myd to induce nfxb-dependent gene transcription. tlrs are also known to be involved in the pathogenesis of autoimmune diseases. as chronic ad also has an autoimmune component, the study of myd signaling in ad might provide new insights into the function of tlrs. to investigate the role of tlrs in the immunopathology of allergic reactions and skin infections, we induced ad-like symptoms in c bl/ myd -deficient mice by repeatedly sensitizing the mice to ovalbumin (ova) after mechanical disruption of the skin barrier by tape stripping. first results show that myd -/-mice display reduced inflammation of the treated skin area compared to wildtype mice. immunostainings of skin biopsies reveal reduced acanthosis and infiltration of inflammatory cells into the dermis compared to wildtype. skin-draining lymph nodes are less enlarged in the ova-treated knockout mice compared to wildtype and differ in cellular composition. serum antibody levels determined by elisa show reduced systemic total and ova-specific igg -titers in ova-treated myd -/-mice compared to wildtype mice, although the nacl-treated myd -/-control group has higher total antibody titers than the wildtype nacl control group. total ige levels are increased in the knockout mice compared to wildtype mice under both conditions. to further investigate the role of staphylococcus aureus during ad development, we will include topical application of the superantigen staphylococcal enterotoxin b (seb) or living bacteria into our analyses. following this approach, we anticipate to obtain new insights into the role of the innate immune system in allergic reactions of the skin. introdution: the skin of vertebrates is the target for over , species of hematophagous arthropods. among these are ticks, which are long-term feeders and interact with host defenses for days to weeks. little is known about specialized strategies for eliminating ectoparasites, but ticks can induce immune responses in hosts. bovines present variable and heritable levels of resistance to the tick rhipicephalus microplus and are the only model in which distinct outcomes of infestation can be examined in the same species of host. in order to obtain some of the immune correlates of these outcomes, we examined expression of candidate genes and quantified populations of leukocytes and subpopulations of lymphocytes present in the inflammatory infiltrates elicited by tick bites in skin of genetically resistant and susceptible bovine breeds, respectively, nelore (bos taurus indicus) and holstein (b. t. taurus). methods: skin biopsies ( mm punch) were taken at the feeding sites of ticks from susceptible and resistant cattle (each phenotype n = ) or from non-infested contra-lateral sites. expression of mip- a, igf- , mcp- and ip- genes was quantified with realtime rt-pcr. sections of paraffin-embedded skin were stained with may grünwald-giemsa for differential cell counts. lymphocytes in sections of frozen skin were phenotyped with specific antibodies using immunoperoxidase technique; in infested skin, histological sections were limited to the area of the tick's cement cone. results: as expected, hosts recruit cutaneous inflammatory infiltrates around the tick's mouthparts. however the composition of infiltrates presented with significant differences that varied according to the phenotype of infestation. inflammation of nelores contained significantly more basophils, eosinophils and mononuclear cells expressing cd , cd , cd , cd , mhc class ii, and p than that of holsteins. lymphocytes expressing wc and cd antigens were significantly diminished in infested skin of holsteins when compared with control skin (p x . ). infested skin of nelores contained significantly more message for mip- a, igf- , mcp- and ip- than that of holsteins. conclusions: although ticks secrete molecules that inhibit cell adhesion and chemokines, resistance correlates with the capacity to recruit and maintain populations of leukocytes that generate effector immune responses. supported by cnpq, capes, fapesp, and icttd. in the last decade it has become clear that keratinocytes play an important role in the skin immune system. upon stimulation, keratinocytes produce high amounts of proinflammatory chemokines and cytokines and express receptors which are involved in immunoregulation. in a number of inflammatory skin diseases such as eczema or psoriasis infiltrating lymphocytes are found in close vicinity to keratinocytes, enabling interaction of these two cell types. it has been proposed (goodman et al.) that keratinocytes rather support a th response by interacting lymphocytes. we examined this hypothesis with autologous cultures of keratinocytes derived from the outer root sheet of the hair follicle co-cultured with cd + t cells from the same donor. during the coculture either seb or antigen were added. in all experimental approaches the addition of keratinocytes resulted in higher production of ifng by t cells. furthermore, we set up an experimental approach were autologous antigen-pulsed monocytes were also added. again, the induction of ifng by the presence of keratinocytes resulted in a marked and significant increase of ifng production by t cells. we were able to show that il- plays a crucial role in the induction of ifng in t cells keratinocyte interaction. in addition blocking of lfa- in the co-cultures resulted in significantly reduced ifng production by t-cells underlining that icam- /lfa- binding is also crucially important for ifng induction. we conclude from our study that keratinocytes rather support a th than a th local response pattern by virtue of il- secretion and icam- /lfa- interaction. this property of keratinocytes may account for the observed cytokine switch in allergic eczematous skin from a th like micromilieu in acute towards a th dominated milieu in chronic lesions. the genotyping of ccl l gene in patients with psoriasis could allow describing subcategories of patients based in clinical parameters and disease severity. therefore, it could be also used as a clinical diagnostic tool, potentially modulating the efficacy of new treatments, or even to be used as a therapeutical target of psoriasis. this work was supported by project grants of merck-serono and instituto de salud carlos iii (pi / ). psoriasis is an inflammatory dermatosis with % prevalence among caucasians. hla-cw allele is the gene that confers susceptibility to psoriasis and it is placed near to tnf loci with several snp in promoter region. the most common polymorphisms are two g to a transitions in - and - positions. strong association was found between polymorphisms in the - region with psoriasis. in several diseases, the association with hla and clinical manifestation is different between genders, for example in spondyloarthropathy and hla-b , and this is a question of increasing interest. the objective of this study was to identify clinical and molecular differences between male and female in brazilian psoriatic patients. sixty-nine individuals assisted at the dermatology outpatient clinic of the teaching hospital, university of campinas, with diagnosis of psoriasis of early-onset (up to years of age) were selected. hla-a -b -c -dr -dq alleles and tnf- and - snp were differentiated by pcr/ssp. analyzing the total group, patients ( . %) were male, ( . %) were female. in the male group, the mean age at disease onset was , years. severe forms were seen in this group (psoriatic arthritis in cases and erythroderma in ). seven patients ( , %) had a favorable evolution of the disease, but ( , %) developed extensive psoriasis, covering over % of body surface requiring systemic treatment. the main molecular risk factor for the disease, cw* allele was positive in cases ( , %), tnf g/a genotype was found in ( , %) and tnf g/a in ( , %). in the female group, the mean age at disease onset was , years, one case of psoriatic arthritis and one of erythroderma. twenty-nine ( , %) had a favorable evolution of the disease and ( , %) an unfavorable evolution. cw* allele was positive in cases ( , %), tnf g/a genotype was presented in ( , %) and tnf g/a in ( , %). severe disease was seen in male patients. there was no difference in frequency of cw* allele between male and female groups, but there was a tendency of significant difference in tnf g/a genotype. we found that c bl/ mice were more susceptible than balb/c and dba/ mice. higher susceptibility was reflected by higher footpad swelling and transient systemic dissemination. analysis of serum cytokine level revealed differences in production of proinflammatory cytokines, such as il- and mrp / , among different inbred strains of mice. furthermore, we identified the cells which are involved in this cytokine production. as expected, histopathological analysis showed that s. aureus infection induces an influx of monocytes and granulocytes. our study shows that not only bacteria-but also host-specific differences are associated with different courses of s. aureus skin infection. aims: to investigate the cause and to study the clinical symptoms and the laboratory findings of the anaphylactic reactions in the pediatric population of our country, considering that these are very often dangerous situations which demand direct treatment and increased alertness. methods: cases, which were studied retrospectively, included children ( boys and girls), aged - years, who had an anaphylactic reaction, out of the that were examined in total. the statistical analysis of the data was held with the spss program. the commonest causes were proved to be food ( %-particularly sea food and dried fruit), drugs ( %-usually antibiotics and non-steroidal antiinflammatory drugs), as well as insect bites ( %-mainly caused by hymenoptera). the symptoms included mainly the presence of pruritic pomphus with erythema ( %), and gastrointestinal symptoms ( %), while there were quite many cases with dyspnea, nasal congestion, but also angioedema. total ige g was found in out of the severest cases ( , %), in which the adequate control was held, while in their vast majority ( out of ) there was no previous anaphylactic reaction. on the other hand, it was proved in total, that in , % ( cases) there was a hereditary family history of atopy, while in children ( %) there was also a personal history of asthma. finally, at a great percentage ( %) eosinophilia was found, while a statistically significant seasonal distribution during spring and summer was registered. conclusions: it has, therefore, been shown that )the anaphylaxis is quite often in the pediatric population, with the commonest causes to be food and drugs, which are often thoughtlessly used. ) in particular, in many cases it is proved that there is a personal but also a family history of atopy. ) increased attention should be, thus, given in these cases -especially during spring and summer-for their early diagnosis as well as for their effective treatment (adrenaline, antihistamines and corticosteroids) particularly for the severest cases, where the hospitalization of the patient is also necessary. allergic contact dermatitis (acd) is an adaptive inflammatory response of the skin triggered upon exposures to certain chemicals or metal ions. as classical type iv delayed hypersensitivity reaction this response is mediated by t-cells. since many ingredients in consumer products might exert allergenic potency, there is a need for an appropriate screening and characterization of the chemicals used according to this toxicological endpoint. up to now the identification of potential allergens completely relies on animal testing, like buehler assay or guinea pig maximization test (gpmt). due to economical and ethical reasons, as well as driven by the enforcement of certain governmental regulations (i. e., cosmetics directive), the development of an in vitro test system for identification of potential sensitizers is mandatory. since dendritic cells (dcs) play a pivotal role in the initiation of contact dermatitis we chose dcs to characterize known sensitizers in their ability to activate these cells and subsequently examined the molecular interplay between dcs primed by allergens and t-cells in the test tube. the known allergens nickel, dinitrochlorobenzene (dncb) and cinnamic aldehyde were tested for their ability to alter the expression of several immunomodulating surface molecules on dcs derived from monocytes that display a langerhans cell (lc) type-similar phenotype. we used multicolour flow cytometry to detect differences in expression patterns of surface molecules that have been associated with maturation. in addition to the upregulation of cd we could observe dose dependent upregulation of programmed death ligand (pdl- ) and downregulation of the dendritic cell immunoreceptor (dcir). furthermore we observed enhanced t-cell proliferation in mixed leukocyte reactions (mlrs) applying lcs stimulated with allergens ex ante. since changes in the expression of only single cell molecules are unlikely of being sufficient for reliable identification of possible contact allergens, we are aimed at analyzing a wide pattern of various surface molecules by multicolour facs and propose that this might be a reasonable approach to screen for contact sensitizing properties of chemicals. our findings are of particular interest for further development of new in vitro assays, using immune cells, to detect the sensitizing potential and quantify the sensitizing potency of chemicals. we want to present the case of a year old iraqui patient with arabic ancestors who had been suffering from psoriatic arthritis since years. in march a treatment with fumaric acid esters in combination with ibuprofen was introduced. this led to the complete healing of the skin lesions. for this reason the dose could be reduced to one tablet fumaric acid esters ( mg) every second day. in april the patient presented himself in the consult with multiple livid papules with a diameter of mm in the area of the auricle. the histological examination showed an hhv- positive kaposi sarcoma. the differential blood cell count demonstrated a lymphocytopenia. the hiv-serology was negative. the staging examinations (chest x-ray, gastroscopy, coloscopy, abdominal and lymph node sonography) showed no signs of visceral involvement. after the diagnosis the treatment with fumaric acid esters was discontinued. over the course the livid papules showed a spontaneous complete regression. a spontaneous regression is known from the iatrogenic ks caused by immunosuppressive therapy when the immunosuppression is terminated. as our patient also showed a spontaneous regression of the kaposi sarcoma after stopping the treatment with fumaric acid esters we propose a causative relation. sarcoidosis is a multisystemic granulomatous disease with unknown etiology. although the immunopathogenesis of sarcoidosis remains unknown there are some supportive evidence for the significant role of th type immune response. recently, suppressor of cytokine signaling (socs) proteins have been identified as regulators of cytokine signaling pathways. in this study we aimed to evaluate the roles of socs , socs and foxp in the immünopathogenesis of sarcoidosis and their association with responsiveness to treatment. peripheral blood (pb) and broncholaveolar lavage (bal) mononuclear (m) cells from sarcoidosis patients in remission following treatment (responders, n: ), the patients who showed recurrence or progression after treatment (non-responders, n: ) and stage i/ii sarcoidosis cases which were followed up without any treatment (untreated, n: ) were evaluated for socs , socs ve foxp mrna expressions by taqman pcr, and also flow cytometric analysis was performed for lymphocyte markers including cd , cd , cd , foxp , cd + cd high , cd + foxp + . expression of socs and foxp- mrna in pbmcs and balmcs from responders were found to be significantly higher in comparison to other two groups . socs was found significantly elevated in pbmcs of responders when compared with other two groups. it was also elevated in balmcs of responders when compared with with those of untreated cases. the proportions of cd , foxp , cd +cd high , cd + foxp + cells in pbmcs and balmcs of responders were found to be increased in comparison to nonresponders and untreated cases. our data demonstrates that socs , socs and t regulatory cells may have potential roles in the control of sarcoidosis. we think that if the roles of socs and socs molecules and t regulatory cells are well characterized, new therapeutic approaches targetting cytokine signal supressors, which can strenghten the regulatory responses, may be beneficial for the sarcoidosis cases resistant to conventional therapy. the inorganic dust, containing free crystalline silicon dioxide (fcs) is critical for the development of silicosis. several studies supported the view that fibrotic responses mainly depends on the regulation of the immune response to the fcs in affected individuals. the role of fcs in induction of a local and systemic inflammation and pulmonary fibrosis are still debates.we studied the changes of neopterin, as a marker for ifn-g dependent macrophage activation and circulating immune complexes (cic), as a marker of humoral immune response, in patients with silicosis and workers exposed to dust containing fcs.we survey a group of silicosis patients, with mild ( ), moderate ( ) and severe ( ) silicosis, coal workers, exposed to inorganic dust containing fcs (exposed), and healthy workers without exposure to dust aerosol (controls).the serum quantity of neopterin and cic, containing iga(igacic), igg(iggcic) and igm(igmcic) was detected by elisa. differences between investigated groups were detected by student's t-test and a p-value less than . was considered significant.neopterin level was significantly elevated in exposed ( , ± , ng/ml) compared to controls ( , ± , ng/ml; p= , ). moreover, the neopterin level in exposed was similar to silicosis patients ( , ± , ng/ml).the levels of iggcic was significantly elevated in the exposed compared to controls ( , ± , au vs , ± , au p= , ) and to silicosis patients ( , ± , au p= , ). in contrast, igmcic was significantly elevated in silicosis than in exposed ( , ± , au vs , ± , au; p= , ).in comparison with exposed, significantly higher igmcic was found only in mild, but no in moderate and severe silicosis. in contrast, the level of iggcic in mild and moderate silicosis was significantly lower compared to the exposed (p= , and , respectively).the obtained results showed that activation of alveolar macrophages mainly depends on the presence of fcs in the respirable dust fraction and precedes the clinical data for pulmonary fibrosis. the dynamics of cic suggest the involvement of fc-receptors mediated regulation of the immune response in the progression of pulmonary fibrosis, and could be useful marker for exposure to inorganic dust containing fcs. described pathologic similarities between sarcoidosis (sa) and tuberculosis (tbc) suggest m. tuberculosis antigens as caustaive agentes. it seems that in the genetically different predisposed hosts, the same antigens may cause the development of sarcoid or tuberculous inmune response. so different hla haplotypes have been described as a predisposing factor to develop sarcoidosis (hla a* , b* , drb * (these of good prognosis) * /* /* /* ) or tbc (drb * /* /* /* and associations with dqa * /* /* ) we describe two cases of two female patients from the same geographic region with mantoux and zhiel-neelsen negative tests and high levels of tnf diagnosed of tbc and sa respectively. both of them debuted with the same clinical manifestations: fever, abdominal pain, and asthenia and shared similarities in the images from the tc study (pulmonary nodules and mesenteric adenopaties). we found the same results for the flow citometry analysis of the non-caseificant granulomes as well as the same anatomopathologic characteristics. after being treated with anti-tbc drugs, the first one presented a good clinical improvement, so she was diagnosed as tbc. the second one did not improved, so she was treated with corticosteroid, with good results. therefore, she was diagnosed as sarcoidosis. after hla analysis, we noticed that the tbc patient was hla a* , b* and drb * (sarcoidosis good prognosis haplotype) and the patient diagnosed as sarcoidosis was hla a* , drb * . as the results show, could there not be a direct relationship between the hla system and the development of sa or tbc, or in contrast, was the first patient missdiagnosed of tbc being a good prognosis sa? objectives: experimental mouse models for acute asthma are well established, yet models for chronic asthma have several shortcomings. for example, current chronic models show decreased inflammation over time and only marginal effects on airway remodelling. experimental models for chronic asthma are essential for development of new therapeutics and must include changes that closely resemble clinical conditions. ovalbumin (ova), house-dust-mite (hdm) and cockroach (cra) proteins are commonly used to trigger an asthma like response in mice and, for this reason, were used in the present study. the objectives of our work were to compare the most frequently used mouse models of chronic asthma and to develop a mouse model of chronic asthma that clearly displays pivotal features of severe human asthma. methods: for the induction of asthma, mice were initially sensitised by intraperitoneal injection of ova, hdm, cra or a combination of all three, followed by repeated challenge by intratracheal application of ova, hdm or cra. inflammation in lung was measured by analysis of cell influx into the bronchoalveolar lavage (bal) and by determination of chemokine and cytokine levels in bal and lung tissue using elisa and multiplex technology. additionally, serum levels of ige and igg antibodies were measured. airway remodelling was assessed by histological staining for mucus production, immune cell influx, smooth muscle thickening and fibrosis. results: significant differences were measured in cell influx, chemokine/cytokine and total ige levels. compared to hdm and cra, ova induced an higher cell influx in the bal, hdm showed an increase of chemokines in bal and increased ige levels in serum. using a combination of all three proteins resulted in the most severe form of asthma. conclusions: to our knowledge, this is the first study that directly compares the most commonly used mouse models in regard to their potential to display a pathology specific of severe asthma. the most sustained and severe form of asthma was induced by the combination model. this model offers particular advantages for evaluating existing and novel therapeutic agents. furthermore, this model could contribute to understanding of the mechanisms underlying chronic asthma. the present study focused on peri-smi connective tissue capsule formation, the most frequent post-operative local complication in patients receiving smi. we investigated the local immune processes via the phenotypic and functional characterization of lymphocytes within the fibrotic tissue. to this end, intracapsular lymphoid cells and peripheral blood mononuclear cells (pbmcs) from the same patients were isolated and analyzed via facs, concentrating on t-effector cells (teff) and t-regulatory cells (tregs: cd + , cd ++ , foxp + ), cytokine profiles, t-cell receptor (tcr) repertoire and reactivity against human heat shock protein (hhsp ). intracapsular tregs were visualized by immunohistochemistry and functionally tested in suppression assays. the cellular composition of intracapsular mononuclear cells showed a preponderance of cd + t-helper cells and a significant subset of tcrg/d + cells, exceeding that observed in peripheral blood. il- , il- , il- , tgf-b and ifn-g production prevailed, pointing to a th /th weighted immune response. furthermore, intracapsular t-cells displayed a restricted tcr a/b repertoire (monoclonal/oligoclonal) as well as a preferential reaction with hhsp . importantly, numbers of intracapsular tregs were inversely proportional to the degree of fibrosis and showed less suppressive capacity as compared to peripheral tregs. our results suggest that silicone triggers a specific local immune response via activated th /th cells, promoting fibrosis due to the production of profibrotic cytokines. clonal restriction of the tcr repertoire is a further indication for a specific antigen driven immune response preceding capsular fibrosis. in this context, hsp might be a prominent candidate. taking into consideration that it is ubiquitously expressed, it might be the "missing link" between local and systemic side effects of smi. the inverse correlation between the degree of capsular fibrosis and the number of intracapsular tregs suggest that tregs may initially be able to inhibit the progress of capsular fibrosis. however, as numbers of tregs, as well as their suppressive capacity decreases over time, fibrosis develops. supported by the competence center medicine tyrol (kmt) and the lore-and-udo-saldow donation. objectives: recent findings have proven that silicone induces a local inflammatory response with subsequent fibrotic reactions. the present project deals with the standardization and further development of a modified elisa test system (silisa ® ) for the identification of patients with a risk for fibrotic side effects to silicone mammary implants (smis) based on the protein signature adhering on the surfaces of such devices ( ) . the current silisa ® is a test system for the simple detection of the adhesion pattern of proteins from patients' sera to silicone. the optimization of the silisa ® comprised inter-and intra-assay standardization, robustness, specificity and sensitivity. the essay was further developed with antibodies against annotated proteins that were not yet tested in the past. all experiments were carried out in a well plate format for high-throughput analysis. statistical analysis has been performed using spss. the extended essay has been successfully established in the system with antibodies against seven already tested proteins, including c-reactive protein, collagen-i, collagen-iii, fibronectin, igg, c -complement, myeloid related protein and two new proteins, integrin-ß and fibrinogen. data from more than patients have been obtained and exploited so far. the intra-and inter-assay variability of the test was reduced to less than % and %, respectively. patients with fibrotic reactions to silicone were successfully identified using a pattern of protein deposition to silicone. conclusion: applying the silisa ® , sera from five different groups were tested: silicone patients with and without fibrotic reactions, female and male individuals without any contact to silicone and hospital's medical staff with potential silicone contact. the distribution pattern of eight proteins showed differences in patients developing strong fibrotic reactions to silicone compared to controls. muscular lesion is a frequent matter in sportive medicine and myodegenerative diseases. necrosis of the damaged tissue and activation of inflammatory response characterize the initial phase of muscle repair. this work aimed to analyze the tissue repair after induction of lesion in skeletal muscle from mouse lineages with distinct cytokine secretion patterns. it was included at least mice per group with distinct cytokine pattern: th (c bl/ , c bl/ ) and th (balb/c). muscular injury was performed by injection of bupivacaine. both th -dominant strains presented more areas with regenerating myofibers and macrophages at dpi. regional lymph nodes showed significant increase of cellularity and relative numbers of cd bupivacaine-inoculated balb/c mice compared to non-inoculated matched mice at dpi. balb/c mice showed increased collagen expression and decrease of mmp- activity associated with more mrna for tgf-b . this study shows that the immune background of the mouse may affect the remodelling processes in skeletal muscles that occur in response to bupivacaine injection promoting muscle regeneration (th cytokines) or myonecrosis and collagen deposition (th cytokines). the severe, life-threatening heart failure in some of the patients with dilated cardiomyopathy (dcm) is imputed to the stimulatory autoantibodies against the second extracellular loop (ec ii ) of the ß -adrenergic receptor (anti-ß ec ii ). to analyze their pathogenic impact as a single causal factor we used a human-analogous lewis rat model of dcm, where monthly subcutaneous immunization of the rats with the ß ec ii peptide as a glutathione-s-transferase (gst) fusion protein induced production of anti-ß ec ii and eventually dilated cardiomyopathy. in this model we isolated a ß ec ii -specific rat monoclonal antibody (clone f ), and showed by elisa that it binds to the linearized ß ec ii peptide. additionally, we confirmed with flow cytometry that f also binds the ß ec ii in its native conformation, i. e. directly labeled circular ß ec ii (dyl -ß ec ii ) peptide. moreover, we demonstrated activation of the ß -adrenoreceptor by f using a fluorescence resonance energy transfer (fret) assay system in vitro. these data further corroborate the pathogenic role of anti-ß ec ii antibodies in mediating dcm in this animal model, thus rendering them a potential therapeutic target. therefore, we investigated a novel anti-ß ec ii -specific peptide-based therapy, by intravenously applying a circular ß ec ii peptide in the dcm lewis rat model to neutralize the anti-ß ec ii antibodies. while the peptide therapy strongly reduced the anti-ß ec ii titers in the serum by up to % and consecutively lead to clinical remission, elispot assays for the detection of ß ec ii -specific antibody-secreting cells (asc) indicated no difference in the number of long-lived plasma cells in treated animals. in contrast, elispot and flow cytometrical analyses revealed a decrease in the number of ß ec ii -specific memory b cells in the treated animals, indicating that this cellular compartment is most likely also targeted by the peptide therapy. our newly developed anti-ß ec ii -specific therapy, thus, not only neutralized the pathogenic autoantibodies, but also depleted antigen-specific memory b cells involved in the generation of these autoantibodies. these results provide the rationale for further development of this therapeutic strategy for eventual application in patients with autoimmune dilated cardiomyopathy. cardiovascular diseases like myocarditis and subsequent dilated cardiomyopathy (dcm), are a frequent cause of mortality in humans with dcm being the most common reason for heart failure in young adults. infections with coxsackievirus b or cytomegalovirus can lead to an acute inflammation of the heart muscle that is followed by an autoimmune response directed autoantigens in the heart, such as the alpha isoform of cardiac myosin (myhca). immunization with the well-characterized myhca - epitope elicits autoreactive cd + t cell responses that have been shown to be the major mediators of autoimmune myocarditis in balb/c mice. it is known that professional antigen presenting cells (apcs) such as dendritic cells are crucial for initiating and maintaining t helper (th) cell responses affecting the heart muscle. however, the detailed analysis of the interaction between these cells in the context of autoimmune myocarditis has been hampered by the lack of appropriate analytical tools. we therefore generated a tcr transgenic mouse harboring t cells that specifically recognize the myhca - peptide. in a first step, hybridoma cells were generated by fusing bw tcra -cd lymphoma cells with myhca - -specific th cells. tcr expression and antigen specificity was assessed by facs analysis and elispot assay. following subcloning, the variable regions of the expressed tcr were characterized by pcr-sequencing. the rearranged v(d)j regions were subcloned into tcr cassette vectors and linearized constructs were injected into the pronuclei of fertilized oocytes. using this novel tcr tg mouse we plan to investigate in detail the activation of myhca - -specific t cells during the process of autoimmune myocarditis. furthermore, this new tool will help to generate a high resolution analysis of the contribution of different apcs in the activation and differentiation of autoreactive th cells during inflammatory heart disease. m. relle , a. schwarting , p.r. galle university medical center of the johannes gutenberg university mainz, medical clinic i, mainz, germany several mouse or rat models have been established to explore the role of proteinase (pr ), in anca-associated glomerulonephritis, vasculitis or pulmonary inflammation but these studies have demonstrated that anca alone are not sufficient to induce these diseases directly. therefore, we assessed the expression, mobilization and enzymatic activity of pr in mouse bone marrow, kidney, spleen and peripheral blood by immunohistochemistry and immunoblots, as well as the proportion of pr -positive neutrophils in the peripheral blood of frequently used mouse strains. neutrophils were mobilized from the bone marrow by an intraperitoneal injection with human il- . pr -mrna from the murine cancer cell line wehi- was amplified by race-pcr and subsequently sequenced. sequence comparisons were done with dnasis software package and the blast tool of the ncbi. promoter analyses were performed with the genomatix software matinspector. we could demonstrate, that mouse bone marrow is a reservoir for functional neutrophils, which are rapidly mobilized after injection of furthermore, we identified an alternative pr -promoter in the second intron of the mouse pr gene. this promoter is active in the bone marrow, in embyros and in cancer cell lines, indicating that its expression is not restricted to myeloid cells. fine structural analyses of this alternative promoter revealed differences not only between the rat and the mouse promoter but also between different mouse inbred strains. taken together, we have shown that the maturation processes of mouse neutrophils differ from those of human granulocytes. the identification of an alternative pr transcript and its promoter indicates that the murine pr may have additional, as yet not described, functions in hematopoiesis and cancerogenesis. objective: recent studies show that in vivo administration of och, a synthetic lipid that specifically activates natural killer t (nkt) cells, results in suppression of th mediated immune responses in autoimmune diseases. nkt cell activation depends on lipid presentation via the mhc-i like molecule cd d on antigen presenting cells such as mature dendritic cells (mdcs) and upon activation by och nkt cells rapidly produce large amounts of th cytokines. the goal of this study was to investigate the effect of och and och-primed dendritic cells on atherogenesis. methods & results: ldl receptor deficient (ldlr -/-) mice were fed a western type diet and atherosclerosis was induced via collar placement around both carotid arteries. subsequently the mice were treated i. p. with och (n= ) or pbs (n= ) twice a week for seven weeks. the injections with och did not affect atherosclerotic lesion size. to improve the presentation of och to nkt cells in vivo, bone marrow-dendritic cells were maturated via tlr activation, in the presence/ absence of och. subsequently we transferred . x mdcs (n= ) or och-primed mdcs (n= ) ( times) to ldlr -/mice. afterwards the mice were put on a western type diet to induce atherosclerosis. vaccination with och-primed dcs resulted in a . % reduction in plaque size compared to mice treated with mdcs (p x . ). during the experiment no effect on serum cholesterol levels was observed, but at the end of the experiment there was a significant . % (p x . ) reduction in cholesterol levels in the mice treated with och-primed dcs. the number of nkt cells in blood and liver was monitored and a to -fold increase in these cells was detected days after the last treatment with och-primed dcs (p x . ). additionally, the nkt cells in the liver of mice treated with och-primed dcs produced more il- . discussion: we conclude that immunotherapy using och-primed dendritic cells efficiently activates nkt cells, resulting in a th phenotype of the nkt cells and this leads to an efficient protection against atherosclerosis. these data indicate that immunotherapy based on ligand specific primed dcs may be a novel way to treat atherosclerosis. systemic lupus erythematosus (sle) is characterized by high serum titers of igg anti-nuclear antibodies secreted by plasma cells. however, the characteristics of the igg+ plasma cell antibody repertoire in sle has never been determined on a single cell level and little is known about the role of germinal center (gc) reactions for the development of sle autoantibodies. the igg inhibitory fcgriib knock-out mouse on the c bl/ background is a strain specific lupus autoimmune model that is characterized by the spontaneous development of autoantibodies to nuclear antigens such as dsdna and chromatin. to characterize the igg+ plasma cell compartment under normal circumstances and in autoimmunity we have cloned and expressed igg antibodies from single isolated gc b cells and plasma cells derived from spleen, bone marrow and lymph nodes of wild-type c bl/ and fcgriib deficient mice. igh and igl chain gene sequence analyses revealed no major differences in the ig gene usage between wild-type and autoimmune mice, but gc b cells of fcgriib were enriched for antibodies with positively charged igh cdr regions and anti-nuclear specificity. the overall frequency of autoantibodies was similiar between wild-type and fcgriib deficient mice. however, strongly autoreactive antibodies to dsdna and murine igg c were isolated only from fcgriib deficient mice, but not from c bl/ control mice and somatic mutations contributed to their generation. in summary, our data suggest that the gc reaction plays an important role for the development of self-reactive antibodies in fcgriib deficient mice. the finding that the frequency of autoreactive antibodies is higher in gc b cells than in spleen or bone marrow plasma cells may indicate that autoreactive gc b cells are partly regulated even in the absence of fcgriib. autoantibodies against double-stranded dna (dsdna) and nucleosomes (ncs) represent a hallmark of systemic lupus erythematosus (sle). however, the factors leading to the autoimmune response against these nuclear autoantigens are not fully identified. high mobility group box protein (hmgb ), a nuclear dnabinding protein and an extracellular proinflammatory mediator gets tightly bound to modified chromatin during apoptosis. it is not released, since apoptotic cells are immediately engulfed by phagocytes. conversely, in conditions of clearance deficiency, which is observed in a subset of patients with sle, non-ingested apoptotic cells, may undergo secondary necrosis, thereby releasing ncs containing the "endogenous adjuvant" hmgb . we investigated if hmgb -containing ncs contribute to the breakdown of immunological tolerance against dsdna and ncs. we found that hmgb remains associated with ncs released from late apoptotic cells in vitro. hmgb -ncs complexes were detected also in the blood of patients with sle. hmgb containing ncs from apoptotic cells induced secretion of il-b, il- , il- , and tnfa as well as expression of co-stimulatory molecules on human and murine macrophages and dendritic cells (dc), respectively. cytokine release from murine macrophages was dependent on myd and toll-like receptor . neither hmgb -free ncs from living cells nor from apoptotic hmgb -or hmgb / -deficient cells induced marked cytokine production or dc activation. specific inhibition of hmgb activity by the antagonistic a box domain significantly reduced capacity of "apoptotic "ncs to induce tnfa and il- release by macrophages. immunizations with hmgb -containing ncs from apoptotic cells induced anti-dsdna and anti-histone igg responses in non-autoimmune mice in tlr -dependent manner. in conclusion, hmgb in complex with ncs activate antigen presenting cells thereby contributing to the loss of immunological tolerance against ncs/dsdna and, hence, to the immunopathogenesis of sle. objective: apoptotic cells are considered to be a major source for autoantigens in autoimmune diseases such as systemic lupus erythematosus (sle). in agreement with this, defective clearance of apoptotic cells has been shown to increase disease susceptibility. still, little is known about how apoptotic cell-derived self-antigens activate autoreactive b cells and where this takes place. methods: injections of fluorescently labelled syngeneic apoptotic cells were traced using immunofluorescence microscopy. binding studies were performed using apoptotic cells and cho cells transfected with class a scavenger receptors (sr). repeated injections of syngeneic apoptotic cells in sr deficient and wild type mice were conducted and antibody production by autoreactive b cells was measured. autoreactivity against sr was followed in two sle prone mice strains over the development of disease and in a cohort of sle patients. an antibody against the sr was injected together with several antigens to directly evaluate the possible role of autoantibodies against the receptors. results: in this study, we find that apoptotic cells are taken up by specific scavenger receptors expressed on macrophages in the splenic marginal zone and that mice deficient in these receptors have a lower threshold for autoantibody responses. autoantibodies against sr are found before the onset of clinical symptoms in sle-prone mice, and they are also found in diagnosed sle patients. furthermore, injections of an antibody binding sr enhance the antibody production by b cells when co injected with either apoptotic cells or tnp-ficoll. conclusion: our findings describe a novel mechanism where autoantibodies toward scavenger receptors can alter the response to apoptotic cells, affect tolerance, and thus promote disease progression. because the autoantibodies can be detected before onset of disease in mice, they could have predictive value as early indicators of sle. e. glasmacher , k.p. hoefig , e. kremmer , v. heissmeyer stretches and helps in the selection of the correct splicing borders. a allele of (r h) creates a strong binding site for a splicing enhancer protein srp according to bioinformatics. our findings indicate that, the putative branch point, r h snp and the t stretch located downstream of exon two, plays a role in the alternative splicing of bank . finally, we believe that bank delta protein work as a dominant negative isoform in b cell activation and antobodies production, and may antagonize the effect of the full-length protein. these properties of the delta protein may contribute to the observed reduction in sle susceptibility. s. beermann , r. seifert , d. neumann hannover medical school, pharmacology, hannover, germany the biological function of histamine is mediated by four different receptors, namely histamine h receptor (h r), h r, h r, and h r. during an immune reaction histamine acts as a local proinflammatory mediator and contributes to the polarisation of the adaptive immune reaction by modulating the activity of dendritic cells and t cells. in these cells, histamine may modulate the synthesis of characteristic t cell cytokines such as ifng, which plays a central role in a number of autoimmune diseases. the present study was initiated to analyze the involvement of histamine on the induced production of ifng by immune cells. mouse spleen cells were stimulated in vitro by either immobilized a-cd antibodies or cpg-oligonucleotides (cpg-odn) in the presence or absence of histamine or -methylhistamine, a h r-selective agonist. ifng production was evaluated by analysis of cell culture supernatants by elisa. both, histamine and -methylhistamine concentration-dependently reduced ifng production in splenocytes obtained from control c bl/ mice induced by either a-cd antibodies or cpg-odn. this histamine effect was completely inhibited by the h r-specific antagonist famotidine, while h r-, h r-, and h /h r-selective antagonists had no or only moderate effects. interestingly, the h r-selective reagent jnj , which serves as an antagonist on human cells, did not inhibit the histamine-mediated reduction of a-cd induced ifng synthesis, but in contrast it slightly enhanced the histamine effect. thus, at the murine h r, jnj may be a partial agonist. we conclude that histamine modulates the induced production of ifng by t cells via mainly the h r and, to a much lesser extend, the h r. using this assay system, cells obtained from control c bl/ mice will be compared to those from sle-prone mrl lpr/lpr mice and the respective wild type strain mrl +/+ . i objectives: resolvins are products of omega- fatty acids and they exert potent anti-inflammatory properties. in this study we examined their effects on cytokine release in healthy subjects and autoimmune patients. to test the in vitro effects of ng/ml resolvin e (rve ) on the release of tgfb, il- and il- in the culture of peripheral mononuclear cells ( x /ml) stimulated by phorbol ester (pma) ( nm), and the combination of pma and ionomycine ( mg/ml) for hours. methods: mononuclear cells were prepared by ficoll-uromiro gradient centrifugation from healthy subjects and from patients each with sle and sjögren's syndrome (ss). level of cytokines was measured by elisa method. results: in the patients with sle (p = . ) and sjögren's (p= . ) mononuclear cell stimulation by pma resulted in a reduced release of tgf b compared with controls. rve significantly reduced tgfb release from control mononuclear cells stimulated by either pma (p= . ) or pma+ionomycin (p= . ), however rve was ineffective at reducing tgfb release in the sle and ss patients. rve caused a non-significant decrease in il- release from control mononuclear cells, but was again ineffective in sle and ss patients. the production of il- was not significantly modified by rve in any of the groups tested. the release of tgfb by ng/ml of rve can be significantly reduced in healthy control subjects but not in subjects with sle or ss. at the single dose of rve tested, il- and il- release were not significantly affected in healthy or autoimmune patients. omega- fatty acid derived rve may affect inflammation in healthy patients by reducing tgfb production but its effects on inflammation in sle and ss patients may be expected to be smaller or non-existent. in addition, the tgfb release in the pma activated mononuclear cells of sle and sjögren's patients is less than that of healthy subjects. g. e. fragoulis , a.k. tsirogianni , m. herrmann , h. m. moutsopoulos , m.n. manoussakis university of athens, dpt pathophysiology, athens, greece, university of erlangen-numberg, institute for clinical immunology, erlangen-numberg, germany objectives: altered phagocytic capacity has been shown to characterize systemic lupus erythematosus (sle) that is thought to lead to impaired clearance of apoptotic remnants. herein, we assessed comparatively the phagocytic capacity in the peripheral blood of ss and sle patients and investigated the phagocytosis of apoptotic/necrotic cells in the salivary glands of ss patients. methods: patients studied included with primary ss (american-european criteria ) and with sle (acr criteria ). age-and sex-matched healthy blood donors to the ss and sle groups ( donors each) were also studied in all assays. the phagocytosis capacity (phagocytosis index) was assessed by flow cytometry, as previously (gaipl et al, j autoimmunity, ) using heparinized whole blood from individuals studied mixed with a commercially available preparation of fluorescent microbeads (mb-phagocytosis) or a preparation of propidium iodide-stained necrotic cell-derived material obtained from heat-treated normal pbmc (snec-phagocytosis). salivary gland biopsies of patients with ss with and without malt lymphoma ( patients each) were also assessed by confocal microscopy for the presence of apoptotic/necrotic material (tunel assay) and the presence of macrophages (cd -staining). results: in agreement to previous studies, mb-phagocytosis was found significantly decreased in granulocytes and monocytes of sle patients (both for p= . ). in ss patients, defective mb-phagocytosis involved only monocytes (p x . ) and significantly correlated with the presence of extraglandular manifestations (p= . ). compared to controls, snec-phagocytosis was significantly increased in the granulocytes of sle (p x . ) and of ss (p= . ). in the salivary gland biopsies of ss patients, the lymphoepithelial lesions and germinal center-like structures manifested significantly increased infiltrations by macrophages. these lesions were also characterized by notable accumulation of apoptotic/necrotic material that resided both inside and outside the phagocytes. these phenomena were significantly more intense in the salivary gland lesions that manifested malignant in-situ b-cell lymphoma. conclusion: in a manner similar to sle, ss patients appear to manifest altered phagocytic capacity. this may be associated with the observed accumulation of apoptotic/necrotic cells in the salivary glands that in turn, may participate in the chronic autoimmune reactions and/or the lymphoma-generating processes that characterize the disorder. the autoantibodies to various enzymes are often found out in sera of systemic lupus erythematosus (sle) patients, but clinical value of such antibodies often is not understood. the purpose of work was to study the of antibodies generation to the basic enzyme of purine metabolism -adenozine deaminase (ada) in sle and to reveal the relationship of studied antibodies with clinical and laboratory features of pathological process. methods: healthy persons have been included in our study and sle patients ( women and men) with various clinical signs ( persons had st degree of disease activity, persons - nd degree of pathological process activity). women had habitual noncarrying of pregnancy (hnp) in anamnesis. antibodies of igg class to ada (anti-ada) determined by technique of indirect elisa developed by us with the use of immobilized form of ada as an antigenic matrix. b glicoprotein-i-dependent antiphospholipids (aphl) of igg classes were determined using commercial "anti-phospholipid screen igg/igm" test set (orgentec diagnostica). results: at admission an anti-ada was revealed in , %, aphl of igg class -in , % sle patients. it has been noted that igg-aphl were found out in anti-adapositive patients more often and in higher antibody titer, than in anti-ada-negative sle patients (x = , ; p x , ). development of cytopenic syndrome was noted reliable more often in sle patients with associated presence of igg-aphl and an anti-ada in comparison with patients who has not the combinations of these antibodies in blood (x = , ; p x , ). the increased levels of anti-ada were revealed in of women with hnp, and the combination of anti-ada and aphl ( / ) was found out more often than isolated anti-ada ( / , x = , ; p x , ) or isolated aphl ( / , x = , ; p x , ). conclusion: taking into account the imbalance of immunoregulatory functions in sle, the further studying of autoantibodies to ada generation seems to be very promising. presence of hnp in anamnesis is the evidence of necessity of careful biochemical monitoring of aphl and anti-ada in women for the prevention of abortus fetus and administration of adequate therapy. objectives: sjögren's syndrome (ss) is a chronic inflammatory and lymphoproliferative autoimmune disease, characterized by dryness of the mouth (xerostomia) and the eyes (keratoconjunctivitis sicca). dendritic cells (dc) are the most potent antigen-presenting cells that play a crucial role in initiating and maintaining primary immune responses. two main subsets of dc have so far been identified in human peripheral blood: myeloid dc (mdc), which can be further divided into mdc and mdc , and plasmacytoid dc (pdc), also known as ifn-a/b producing cells. the pivotal role of dc in inducing and maintaining tolerance could be critical in ss as alterations among dc populations might contribute to autoimmunity. purpose of this study was to quantify mdc , mdc and pdc in peripheral blood from primary ss patients by flow cytometry and compare the results with gender-and age-matched healthy controls. methods: blood samples from pss patients fulfilling the american european consensus group criteria (aecc) and gender-and age-matched healthy controls were collected in heparin tubes. dc populations were stained with the blood dc enumeration kit, miltenyi, according to the manufacturer's manual. cells were analyzed on a facs canto ii, bd, and data analysis was performed with flowjo software, tree star. for the statistical analysis, a two-tailed mann-whitney u test was performed using prism, graphpad. results: pss patients have significantly reduced amounts of pdc (p= , ) and mdc (p x , ) in peripheral blood. conclusion: alterations in dc populations have been considered to play a role in autoimmune diseases such as systemic lupus erythematosus (sle) or diabetes. in ss patients, up-regulation of interferon-regulated genes has been shown previously. therefore, decreased pdc numbers in peripheral blood from pss patients might explain the fact that an increased ifn signature is found in salivary glands of pss patients, but no elevated levels of ifn-a are measured in serum. recently we reported that malignant cd + b cells from patients with b chronic lymphocytic leukemia (b-cll) produce granzyme b (grb) and are rapidly undergoing apoptosis in a granzyme b-dependent manner following interleukin (il- ) stimulation. several autoimmune diseases have been linked to both elevated frequencies of cd + b cells and increased il- levels. we therefore hypothesized that il- may have similar biological effects on cd + b cells in autoimmune diseases. here we demonstrate that the amount of il- in the serum of systemic lupus erythematosus (sle) patients but not of healthy subjects highly correlated with serum levels of grb. in contrast to b cells from healthy individuals, where no baseline grb expression was found, we demonstrate that up to % of cd + b cells in sle individuals expressed grb. in-vitro experiments revealed that il- was able to induce expression of grb in b cells from individuals with sle and other autoimmune diseases including psoriasis and rheumatoid arthritis. this effect was direct and was strongly enhanced by engagement of the b cell receptor or toll-like receptor . importantly, il- significantly decreased the cd +/cd -b cell ratio in both sle peripheral blood and healthy cord blood samples, suggesting a preferential induction of cd + b cell death. these results suggest that il- -induced grb may play a regulatory role for cd + b cells similar to what we described earlier in b-cll cells. this is the first report uncovering an interrelation between il- and grb levels in sle and showing that il- reduces the cd +/ cd -b cell ratio in b cells from sle peripheral blood and healthy cord blood. endogenous il- may therefore play a disease-modifying role and may explain elevated grb serum levels in autoimmune diseases. further studies should evaluate the therapeutic potential of il- in sle and other autoimmune diseases. r. de palma , e. d'aiuto , s. vettori , g. abbate , g. valentini second university of naples, clinical & experimental medicine, napoli, italy ssc is considered an autoimmune puzzling disease whose pathogenesis is unknown. in the last years, there have been increasing evidences that an interplay between activated t cells and fibroblasts could play a pivotal role in promoting matrix accumulation in systemic sclerosis (ssc). we have previously shown that peripheral t cells from ssc patients with early diffuse disease co-cultured with autologous fibroblasts expand the same t cell clonotypes found in the affected skin. here, using the same experimental approach, we found that the t cell clonotypes expanded in co-cultures are ab positive, hla-dr positive, and promote apoptosis of autologous ssc fibroblasts. we also found that, in these co-cultures, ssc fibroblasts up-regulated fas and underwent apoptosis that paired with the expression of fas ligand (fasl) on cd + t cells. finally, when we added a blocking anti-fas antibody to the co-cultures, we observed a marked reduction of fibroblast apoptosis, suggesting that engagement of fas/fasl had a critical role in mediating apoptosis in co-cultured fibroblasts. it has to be reminded that the absence of fasmediated apoptosis in vivo could be due to several reasons, as the increase of soluble fas in sera of patients affected by ssc. moreover, in the co-culture supernatants we found tgf-beta, il- beta, il- and il- , cytokines known to have a role in promoting fibrosis in systemic sclerosis. taken together, these data suggest that t cell response in ssc may represent an attempt of the immune system to kill fibroblasts, cells that are likely to be altered and expressing (auto)antigens. indeed, fibroblasts of ssc patients have been shown to display a persistently activated phenotype characterized by excessive production of collagen and other extracellular matrix proteins. however, the overall outcome of the t cell response triggered by fibroblasts in ssc, while unable to control the activity and the growth of fibroblasts, contribute to sustain inflammatory loops leading to fibrosis. these findings may lead to change our view about the pathogenesis of this disease and other autoimmune diseases. systemic lupus erythematosus (sle) is a chronic inflammatory autoimmune disease that is associated with a major breakdown in b cell self-tolerance as reflected by elevated serum igg levels of predominantly antinuclear antibodies (anas). serum antibody titers are maintained by antibody-secreting plasmablasts and longlived plasma cells, which reside in survival niches of the bone marrow. however, the antibody repertoire of bone marrow plasma cells, which may include cells expressing autoreactive and potentially pathogenic antibodies, has not been characterized in sle. to determine the frequency, specificity and immunoglobulin gene characteristics of autoantibodies in the long-lived plasma cell compartment in sle, we cloned and expressed igg antibodies from single facs purified cd +cd +cd +cd + bone marrow plasma cells of patients with sle and tested the recombinant monoclonal antibodies for self-reactivity. our preliminary data on the ig gene repertoire and reactivity profile of human igg+ sle bone marrow plasma cells in comparison to healthy controls will be discussed. z. amirghofran , e. moazemi godarzi , e. kamali sarvestani , e. aflaki shiraz university of medical sciences, shiraz, iran, islamic republic of interleukin (il- ) has been shown to be related to the pathogenesis of systemic lupus erythematosus (sle). two polymorphisms in the promoter region of il- gene at positions - g/c and - g/c have been described that are key regulators of il- gene. in the present study the relationship between these two polymorphic sites and disease susceptibility in a group of iranian patients with sle was investigated using polymerase chain reaction-restriction fragment length polymorphism method. the genotype distribution and allele frequencies of il- gene polymorphism at - position showed no significant difference between sle patients and controls. at this position the frequency of gg genotype as well as g allele was higher than c allele in both patients and control groups. in contrary, both allelic and genotypic frequencies at the - position significantly differed in sle patients and controls. at this position gg genotype was observed in . % of patients compared to . % in the control group (p x . ). the frequency of - g allele in patients ( . %) was also higher than in controls ( . %) (p= . ). the haplotype study showed no significant difference between patients and healthy subjects. the relationship between these polymorphisms and clinical manifestations and laboratory parameters were investigated. - polymorphism was associated with the presence of antinuclear antibodies in all patients and rash and hematuria in male patients (p x . ). at - polymorphism, a significant difference with regard to photosensitivity in male patients (p= . ) was found. in conclusion, results of this study showed that - polymorphism plays an important role in susceptibility to sle and that - polymorphism could influence the presence of antinuclear antibodies in the patients. the eukaryotic constitutive proteasome is the main protease expressed in most tissues. recently we have elucidated a functional importance of the second proteasome form, inducible immunoproteasomes, in regulating nf-kb activity during the intestinal inflammation. in comparison with healthy controls and patients with ulcerative colitis (uc), there was increased expression of immunoproteasomes in the inflamed mucosa of patients with crohn's disease (cd) at both mrna and protein levels. in our very recent work we have shown that the proteasome subunit pattern might be suitable for diagnostic differentiation between cd and uc patients. since ifn-g has been shown to be the main inducer of immunoproteasomes in various murine and human cell lines and the ifn-g levels are highly elevated in inflamed intestine of cd patients, induction of immunoproteasomes in cd might be mediated by this cytokine. our data with human leukemic t cell lines and primary macrophages show a significant increase in the nf-kb controlled production of proinflammatory cytokines after the ifn-g-mediated induction of immunoproteasomes in these cells. in the dss-induced colitis model we have observed a diminished colonic inflammation in the absence of the proteasomal immunosubunits. therefore we here suppose that immunoprteasome are involved in the complex inflammatory response during the chronic intestinal inflammation by increasing nf-kb activity in the epithelial and immune cells. however, it remains to be determined whether these results have an important implication for the treatment of chronic gut inflamation in humans. objectives: inflammatory bowel diseases (ibd) including crohn's disease (cd) and ulcerative colitis (uc) are characterized by unknown etiology and chronic intestinal inflammation. noninvasive serological tests to differentiate cd from uc have been searched for a long time. testing for panca together with ascas has good predictive values to identify patients with ibd.the aim of this study was to find evidences for diversity of ascas and anti-mycobacterium paratuberculosis antibodies (anti-mpt) by elisa method. in addition, to examine whether combination of these elisas is useful for distinguishing cd from uc. methods: the study population contained patients with ibd ( with cd, with uc, with gluten sensitive enteropathy, gse) and healthy control subjects. serum asca igg, asca iga and anti-mpt antibody levels were measured by solid phase enzyme immunoassay. adsorption of asca positive sera was performed by baker's yeast suspension. results: elevated level of asca igg, iga and anti-mpt was shown in cd and gse but not in uc compared to healthy controls. serum levels of asca igg, iga showed a significant positive correlation with anti-mpt antibody levels in cd. repeated adsorptions with yeast removed asca igg and iga from sera of patients, but did not change levels of anti-mpt. these results indicate the diversity of asca igg, iga and anti-mpt (accordingly their antigens) and suggest that combination of these elisa can have a role in the differential diagnostics of ibd. it is now well recognised that the majority of lymphocytes may be located within tissues, not in blood, and yet these tissue-resident lymphocytes are relatively understudied, especially in humans. we have extracted cells from human gut biopsies (both normal and inflamed gut) in order to characterise the immune cell populations that exist therein and which molecules may be of paramount importance to their function. we show that distinct populations of t cells exist within the gut and that the ratio of these populations changes down the length of the gut, with the so-called 'unconventional' double negative t cell population (ie tcrab+ve, cd -ve cd b-ve) predominating in the healthy colon whereas these cells are overwhelmed by infiltrating cd (+) cells in inflammatory bowel disease (ibd). having previously shown in mice that gut-resident t cells express high levels of the regulator of g protein signalling- (rgs- ) protein, we have now found substantial over-expression ( - fold) of rgs- in human gut-derived t cells, particularly in this unconventional t cell population. furthermore, levels appear even higher in t cells derived from inflamed gut. transfection of rgs- decreases primary t cell responses to cxcl and ccl , strongly implying that it may regulate t cell localisation. thus, rgs- may be a novel target for modulating t cells in ibd, consistent with which snps in rgs- have been associated with both coeliac disease and type diabetes. mechanisms involved in the induction of oral tolerance (ot) or systemic immunization through the oral rout are still poorly understood. in our previous studies we have shown that when normal mice eat peanuts they become tolerant, with no gut alterations. conversely, if they are immunized with peanut proteins prior to a challenge diet (cd) containing peanuts they develop chronic inflammation of the gut. our aim is to evaluate the consequences of the introduction of a novel protein in the diet of animals presenting antigen specific gut inflammation. adult, female c bl/ j mice were divided in control (c) and experimental (e) groups. c -c received peanut protein immunization, animals of the control groups c were sham immunized, and control group c received ovalbumin (ova) immunization. the experimental group was immunized with peanut protein extract. before initial exposure to a day peanut containing cd, the experimental group was divided into groups (e -e ). ova feeding began days prior cd (e ) on day (e ), (e ), (e ) and (e ) during cd. our results show that oral exposure to a novel protein (ova) in the absence of gut inflammation (e ) leads to low levels of systemic antibody titers, comparable to tolerant animals. conversely, as off initial induction of inflammation, groups submitted to ova (ot) protocol develop increasingly higher systemic antibody (ab) titers similar to animals of the immune control group. in conclusion our protocol indicates that timing is more important than the antigenicity when a novel protein is offered, in the diet. nanoparticles of various types are increasingly used as constituents of food supplements and so called nanofood. since nanoparticles induce inflammatory reactions in the lung, there is an urgent need to also study the toxicological potential of nanoparticles in the intestine. therefore, we assessed the effect of particles on dendritic cells (dc) as key players in the manifestation of intestinal immunity.in in vitro studies we could show that ultrafine tio as well as ultrafine silica led to a mature phenotype of the cells when particles were added to cultures of immature bone-marrow-derived dc. this effect appears to result from enhanced cell death in immature dc but also from direct stimulation of the cells.to analyse the mechanisms underlying this effect we looked for apoptosis as well as for induction of the inflammasome since it has been shown that crystalline silica leads to activation of caspase and secretion of bioactive il -b.in our hands certain nanoparticles induced apoptosis of immature dc, as well as enhanced secretion of active il -b. we therefore hypothesize that particles can induce the inflammasome which leads to the activation of dc.to study the impact of nanoparticles on intestinal inflammatory processes in vivo, we induced colitis by applying % destrane sulphate sodium (dss) in the drinking water for days to wildtype mice. when ultrafine nanoparticles were administered on day and by gavage feeding, we observed an amelioration of disease symptoms when scoring the degree of epithelial disruption and inflammation. in future experiments we will also analyse the effect of different particles in the il -/model of colitis to assess the contribution of particles to the induction and pathogenesis of disease. m. schmohl , n. schneiderhan-marra , m. blum , g. stein , m. schmolz , t. joos nmi-natural and medical sciences institute at the university of tübingen, biochemistry, reutlingen, germany, edi gmbh, reutlingen, germany the human immune system represents a highly complex system that protects the organism against diseases. there is an impressive network of immunoregulatory signals within the immune system as well as between the different healthy and diseased organs. epithelial layers function as a barrier against pathogens. as the gastrointestinal tract, which is occupied with a large variety of microorganisms, represents the outside of the body, the immune system has to establish and maintain a strong presence at the mucosal boundary. the ability to discriminate between pathogens while remaining relatively unresponsive to food antigens and the commensal microflora is achieved by a plethora of largely unknown regulatory mechanisms. this ability appears to be breaking down with chronic inflammatory bowel diseases (ibd) like crohn's disease and ulcerative colitis [ ] . to date treatment options are restricted to controlling symptoms, putting and keeping the dis-eases in remission and preventing relapse. therefore, there is an urgent need for a more detailed understanding of the inflammatory events taking place during the disease. for this purpose a human organo-typic (hot) co-culture model is used, which allows analyzing the collaborative regulation between the immune system and the gut epithelial cells. the human caco- cell line, as a model for the gut-epithelium cells, are cultivated on the top side of special culture vessels, fitting as inserts into carrier wells of -well culture plates, containing whole-blood. this co-culture set up mimics the physiological barrier to perorally applied biologicals/drugs and allows measuring their effect on the immune system. as a read out miniaturized and parallelized sandwich immunoassays will be used to detect alterations in the intracellular mapk and rtk-signalling of the epithelial cells as well as in the extracellular communication via cytokines and chemokines at the interface of the two organs. this approach will provide new insight into the inter-and intracellular signalling of gut epithelium and the immune system, which will finally result in a better understanding of the etiology of inflammatory bowel diseases. inflammatory bowel disease (ibd), including crohn's disease (cd) and ulcerative colitis (uc), is characterized by an upregulation of pro-inflammatory cytokines that play an important role in pathogenesis. osteopontin (opn) is a cytokine implicated in several immunological diseases and, although expressed constitutively in normal intestine, is upregulated in intestinal mucosa and in the plasma of ibd patients. opn has been shown to be either pro-inflammatory or anti-inflammatory for experimental uc, indicating a controversy in this field, while its role in experimental cd remains unknown. in our study we investigated the role of opn in experimental colitis using two mouse models: trinitrobenzene sulphonic acid (tnbs) colitis, a t h -associated model that resembles cd, and dextran sulphate sodium (dss) colitis, a t h -like-associated model for uc. deficiency of opn (either by antibody-mediated neutralization or use of opn -/mice) resulted in suppression of disease phenotype in both colitis models, revealing that opn, and especially, the secreted isoform of opn (opn-s) is important for the initiation of acute intestinal inflammation. importantly, we discovered that opn drives il- production and t h polarization and decreases recruitment of cd + cd + foxp + t regulatory (treg) cells in mesenteric lymph nodes (mlns) of mice with colitis. also, there was an effect of opn on recruitment of cd c + dcs, which were significantly elevated in mlns of opn -/or anti-opn-treated, as compared to opn +/+ or ig-treated control mice. this finding implies that opn deficiency results in enhanced recruitment of regulatory cd c + dcs which may mediate treg induction and protect from colitis. overall, our findings indicate that opn is proinflammatory in both types of colitis, by promoting pathogenic t h and attenuating treg cell recruitment, implying also common mechanisms in the pathogenesis of cd and uc. c. shen , , g. van assche , p. rutgeerts , a. liston , j. l. ceuppens k.u. leuven, autoimmune & genetics lab, vib, leuven, belgium, k. u. leuven, experimental immunology lab, leuven, belgium, k. u. leuven, department of pathophysiology, gastroenterology section, leuven, belgium background: haptoglobin (hp) is one of the acute phase proteins synthesized during inflammation. hp- allele is associated with the disease behavior in crohn's disease but not in ulcerative colitis. however its role in inflammatory bowel disease has not been defined. aim: to determine whether hp modulates the immune responses in experimental colitis. methods: we induced types of colitis dss (th /th ), tnbs (th ) and oxazolone (th ) in hp ko mice. neutralizing anti-il- mab was injected into dss and tnbs hp ko mice. severity of colitis was evaluated by body weight, colon length and histology. th /th cells were analyzed by flow cytometry. cytokines were measured by elisa or rt-pcr. ) compared to the wt mice, hp ko mice developed much severer dss and oxa induced colitis. dss induced lethal colitis in hp ko but not in wt mice; ) in dss but not in oxa colitis mice, il- , ifn-g, tgf-b and il- were significantly increased (p x . , dss vs control) in lamina propria and mesenteric lymph nodes (mln), and this is much evident in hp ko mice compared to those in the wt (p x . , ko vs wt). in tnbs colitis, we found elevated il- and ifn-g (p x . , tnbs vs control). although not significant, il- was also somewhat upregulated; ) in dss colitis we observed that il- enhanced differentiated th cells in vitro, this effect could be abrogated by coculture with serum from wt but not hpko mice. furthermore, in vitro in the presence of tgf-b, il- and il- , more mln-t cells from hpko mice differentiated into th cells; ) anti-il- mab improved dss and tnbs colitis, and partially rescued hp ko mice from lethal dss colitis. in line with this, mice treated with anti-il- showed reduced il- , il- and ifn-g in both mln and lp (p x . , anti-il- vs control). our results reveal that hp has a protective role in the development of mucosal inflammation. in dss and tnbs colitis hp may exert its beneficial effect partially through inhibiting production of il- , supporting further pre-clinical and clinical application of hp for treatment of crohn's disease. p. engelmann , g. talabér , g. süt" o , p. németh , t. berki university of pécs, clinical center, department of immunology and biotechnology, pécs, hungary, university of pécs, clinical center, department of immunology and rheumatology, pécs, hungary objectives: inflammatory bowel disease (ibd) resembles as an autoimmune-like disease. ibd is most common in developed countries: it is calculated that . million people in europe suffer from ibd. several hypotheses are raised in the pathogenesis of inflammatory bowel disease. one of the most favored is the dysregulation of the immune response due to failure of regulatory t cells. the most well known regulatory t cells are the cd +cd hi+ t (treg) cells. furthermore, other immune-regulatory cells are known such as invariant natural killer t (inkt) cells producing both th and th cytokines rapidly upon antigen (lipid) stimulation. methods: based on this hypothesis we aimed to investigate the role of various immune-regulatory t cells in human ibd. we attempt to measure the proportions of inkt cells, treg cells in peripheral blood of patients with crohn's disease (cd) and ulcerative colitis (uc) compared to normal controls. blood samples were collected from normal controls and ibd patients; then lymphocytes were labeled for inkt and treg markers with specific monoclonal antibodies and measured with flow cytometry. results: according to our results a decline in the total inkt cells of ibd patients was observed, interestingly the proportions of cd + and double negative (dn) inkt subgroups showed a characteristic shift among the study groups. percentages of dn and cd + inkt subpopulations were assessed after gating of total inkt populations. in controls we observed high percentage of dn inkt cells ( . ± . %, mean ± sem), while cd + inkt cells ratio was moderate ( . ± . %). in uc and cd patients we found a reduced proportion of dn inkt cells (uc: . ± . %; cd: . ± . %, mean ± sem), while the percentage of cd + inkt cells was elevated (uc: . ± . %; cd: . ± . %, mean ± sem) in both disease groups. proportions of foxp + treg cells also showed a decline in ibd patients comparing to normal controls. conclusion: this study can provide useful data about the pathogenesis of ibd and can lead to identify and characterize new cellular and molecular targets with possible therapeutic use in human autoimmune disorders. objectives: the aim of this project is to explore whether exosomes from tgf-b gene modified bone marrow-derived immature dendritic cells (md-imdc) have the function of systemic immune inhibition and protective effect on the development of inflammatory bowel disease (ibd) in mice, the underlying mechanism was also investigated. methods: exosomes were isolated from supernatant of md-imdc transfected with tgf-b adenovirus (tgf-b -exo). the t cell inhibitory function of tgf-b -exo was determined by mixed lymphocyte reaction (mlr) in vitro. to evaluate the protective effect of tgf-b -exo in the development of ibd, dextran sulfate sodium(dss) induced murine ibd was established and mice were treated with tgf-b -exo. the main symptoms of ibd were observed. the inflammatory degree of colon was also evaluated by histological examination. the relative cd + foxp + treg cell numbers from spleens and mesentery lymph nodes (mlns) were analyzed by facs. results: it was demonstrated that tgf-b -exo could inhibit the proliferation of t cells in mlr in vitro. in murine ibd model, after treated with tgf-b -exo, the main symptoms of ibd such as weight loss, diarrhea and grume sanguinopurulent stool were all alleviated and the inflammatory degree of colon was also reduced. analysis of cd + foxp + regulatory t cells (treg) revealed that the relative numbers of cd + foxp + treg increased in lymphocytes from mesentery lymph nodes (mlns) of inflammatory site but not from spleens. conclusions: these results demonstrate that immunosuppressive exosomes obtained from tgf-b gene modified md-imdc can delay the development of ibd. this protective effect is mediated by the induction of cd + foxp + treg. tgf-b -exo might provide a novel strategy for the therapy of ibd. results: hcv-specific cytokine expression by cd + t-cells was similar in the four vaccinees as observed by ifng, il- production-profiles. however, the killing capacity of expanded cd + t-cells was distinct as observed by the competence to kill ns -peptide presenting transfectants in vitro. as depicted in figure , cd + t-cells cells from both vac (cleared ) and vac (chronic) produced il- and ifng after stimulation with ns -peptide . however, specific killing of the peptide loaded transfectants was only observed in vac , who was able to clear its hcv infection, and this was not observed not in any of the other chimpanzees, who became chronic carriers. [ figure ] killing of ns peptide presenting cells was restricted to the vaccinee that was able to clear hcv infection. these results suggest that controlling hcv replication as initiated by this dna-prime mva-boost vaccine-protocol was partly mediated by antigen specific cd + t-cells. hence, the effector mechanisms induced were distinct between the animals and clearance of the infection was correlated with induction of killing competent cd t-cells. objectives: infection by hepatitis c virus (hcv) is characterized by its high tendency to chronicity, which is usually associated with a low or absent t-cell response against viral antigens. immune response specific for non-structural protein ns from hcv was associated with viral clearance. we have demonstrated that fusion of an antigen to the extra domain a from fibronectin (eda) targets the antigen to tlr -expressing dendritic cells and improves its immunogenicity. thus, we tested if covalent linkage between eda and ns might constitute an alternative for vaccination against hcv infection. methods: recombinant plasmids expressing a secretable version of ns or eda-ns under the control of cmv promoter were prepared. recombinant ns and the fusion protein eda-ns were produced in e. coli. the recombinant proteins were tested in vitro on their capacity to activate maturation of bone marrow derived dendritic cells and to favour antigen presentation. hhd transgenic mice (expressing the human hla-a molecule) were immunized with the recombinant plasmids or with the recombinant proteins, in the absence or presence of poly(i:c) and anti-cd agonistic antibodies. elispot and chromium release assays were carried out to measure the immunogenicity of the different vaccination strategies. intrahepatic expression of hcv-ns rna was measured after a hydrodynamic injection with a plasmid encoding hcv ns . results: immunization of mice with the plasmids expressing eda-ns , but not ns alone, induced strong t cell responses against the main hla-a restricted cytotoxic t cell determinants from ns . the recombinant eda-ns fusion protein, but not ns , was able to activate in vitro maturation of bone marrow derived dendritic cells as well as the production of tnf-a by the thp- monocyte cell line. immunization of hhd mice with eda-ns fusion protein induced both cd + and cd + t cell responses against ns and, when immunized with poly(i:c) and anti-cd antibodies, was able to down-regulate the intrahepatic expression of hcv-ns rna. the recombinant eda-ns fusion protein may be considered for the development of prophylactic or therapeutic vaccines against hcv infection. vaccination is the most efficient strategy to prevent from microbial infections and to control epidemics but are still not available in the case of hiv infection even years after virus detection. therein we propose the intra-dermal inoculation of dna vaccine that present a plasmid vector exploiting the binding capacity of the bovine papillomavirus e protein encoding an artificial multi-component hiv antigen. this inoculation is followed by electroporation in order to increase dna uptake. we used skin as site for vaccination because, being the first line in host defence, it is populated with various cells of immune system. among them, langerhans cells (cd +cd a+), located in the epidermis, are dendritic cell subset capable to elucidate specific cd + responses. the present work emphasizes molecular and cellular biodistribution of the dna vaccine in the skin after intra-dermal vaccination in macaques, as one of the most relevant animal models in hiv studies. technical approach considers an intra-dermal injection of dna followed by topical electroporation of the injection sites. skin and draining ln biopsies were collected at different time points. these biopsies were used for ihc fluorescent staining in order to establish biodistribution dna-encoded antigens and co-localisation with different cell types. kinetic of antigen expression was studied by bioluminescence in vivo imaging. t cell responses were measured by ifn-g elispot assays up to years after dna vaccination. we show that a dna vaccine delivery method combining intra-dermal injection and electroporation dramatically increased the expression of the vaccine antigen selectively in the epidermis, increased the frequency of cd a+ cells in the draining ln in association with the antigen expression, and increased the cellular response persistence, at high levels, for more than two years after the last vaccine boost. our data suggest that electroporation after intradermal injection of dna vaccine involves langerhans cells from the epidermis that elucidate qualitative anti-hiv immune responses. this new approach that comprise new dna vaccine followed by non-invasive electroporation, induce long-lasting cellular response that could be crucial in prophylactic / therapeutic vaccine design. presenting cells was developed. murine coronavirus-based virus-like particles encoding epitopes from the lymphocytic choriomeningitis virus glycoprotein or human melan-a, in combination with the immunostimulatory cytokine gm-csf, selective targeted dcs in vitro and in vivo resulting in vector-mediated antigen expression, and efficient maturation of dcs. in mice, a single application of only low doses elicited strong and long-lasting cytotoxic t-cell responses which provided protective antiviral and antitumor immunity. furthermore, the efficient activation of human tumor-specific cd + t cells by mature dcs transduced with melan-a-recombinant human coronavirus e indicates that this novel vaccine platform mediates the delivery of antigens and immunostimulatory cytokines to those cellular components of the immune system that initiate and maintain protective immunity. as the application of gm-csf already enhanced immunogenicity, we are now trying to further modulate the coronavirus vector-induced immune response with the reverse genetic setup of recombinant coronavirus-based vectors expressing different immunostimulatory cytokines. thereby cytokines will be acting on t cell and dc level. to enhance t cell response interleukin (il ) and interleukin (il ) will be involved, and fms-like tyrosin kinase ligand (flt l) will be expressed to modulate dendritic cells. il is known to enhance early t cell expansion and limits t cell overshoot, whereaes il guarantees survival of high affinity t cells during memory phase. on the other hand flt l enhances dc proliferation and accumulation. with these approaches modulation of the immune response generated by this novel vaccine platform will be examined in viral and tumour models to get insight on the antigen specific ctl response, synergistic effects of the cytokines and protective as well as prophylactic vaccination approaches. f. demircik , ag waisman uniklinik mainz, . med, mainz, germany in murine cytomegalovirus (mcmv) infection, cytotoxic cd t cells and nk cells play a critical role. previously it was shown that mice deficient for b cells are more susceptible to mcmv-related disease, caused by virus reactivation. to better understand the role of b cells and antibodies in the response to mcmv, we made use of different mouse strains that lack b cells, secreted antibodies or il- production by the b cells. we found that for the initial t cell response to the virus b cells are important, but antibodies do not play an important role. this implicates b cells as potential important antigen presenting cells (apcs) in the activation of the virus-specific t cells. the reduced t cell response to the virus was observed whether the mice were b cell deficient from birth or if they were depleted later in life. six month after infection mice were tested for the memory cd t cell response. interestingly, we found that in mice that lack antibodies (mice that lack b cells all together and mice that have b cells but no secreted antibodies) maintain a rather high t cell response to viral peptides, in a level similar to the acute response days after infection. we conclude that antibodies probably remove residual viruses from the body and therefore prevent the continuous activation of t cells. finally, we tested the role of il- produced by b cells by conditional deletion of the il- gene in these cells. we found that b cell secreted il- has a suppressive effect on the t cell response to mcmv, as this response is elevated in these mice. we conclude that b cells are important for an efficient acute response to mcmv and that antibodies play a role in eliminating residual viral particles, thus implicating a dual role for b cells in the efficient acute and memory response to mcmv. this work is supported by the deutsche forschungsgemeinschaft grant sfb to aw. objectives: ebv infection leads to life-long viral persistence. although ebv infection can result in chronic disease and malignant transformation most carriers remain disease-free due to an effective control of the virus by t cells. ebv-specific ifng-producing t cells could be demonstrated in acute and chronic infection by many researchers. recent studies in hiv and leishmania provide, however, evidence that assessing ifng alone is insufficient to assess the quantity and quality of a memory t cell response and support the crucial role of multifunctional t cells in disease control. in this study we therefore analyzed ebv-specific t cell responses in peripheral blood (pb) and bone marrow (bm). methods: paired pb and bm samples were obtained from healthy virus carriers who underwent total hip arthroplasty. t cells were expanded for days in the presence of il- and il- with exposure to overlapping peptide pools of latent ebna- and lytic bzlf- antigens. ebv-specific immune responses were assessed exvivo and after expansion by multiparameter flow cytometry staining for live/dead discrimination marker, cd , cd , cd , ccr , cd ra, il- , tnfa, ifng and cd a. the majority of ex vivo ebv-reactive cd + t cells as well as ebna- -reactive cd + t cells were il- and tnfa-producing memory cells, the later being more frequent in bone marrow (cd +, median, ebna- : bm . %;pb . %; bzlf- : bm . %;pb . %, p= . ). after in vitro expansion a major subset of ebv-specific cd + and cd +t cells displayed a differentiated effector ifng/tnfa phenotype. a comparable number of ebv-specific cd + and cd + t cells retained, however, a tnfa single, tnfa/il- or triple producer phenotype resembling early differentiated or multifunctional memory t cells, respectively. interestingly, both cd + and cd + t cells generated from bm revealed significantly higher cytotoxic potential. sorting of ccr /cd ra differentiation subsets, revealed that ebv-specific t cells were predominantly expandable from the central memory compartment. conclusion: our data shows that multicolor assessment of ifng, tnfa and il- delineates various subsets of ebv-specific memory t cells, which reflect the profile of a protective immune response. human adenovirus (hadv) can cause serious morbidity and mortality in immunocompromised patients after allogeneic stem cell transplantation (allosct). reconstitution of hadv-specific t cells has been reported to be associated with sustained protection from hadv disease, but epitope specificity of these responses has not been further characterized. furthermore, the relative contribution of hadv-specific cd + and cd + t cells in the protection from hadv disease after allosct remains to be elucidated. in this study, we demonstrate, by sensitive measurement using intracellular cytokine staining combined with cd or peptide-mhc tetramer staining, that clearance of hadv was associated with a combined hadv hexon specific cd + and cd + t cell response in both pediatric and adult allosct recipients. based on this observation, we developed a clinical grade method for the rapid generation of t cell lines with high and defined specificity for hadv hexon epitopes for adoptive immunotherapy. activation of hadv hexon-specific cd + and cd + t cells in peripheral blood with a hexon protein-spanning pool of synthetic -mer peptides followed by ifng-based isolation allowed rapid expansion of highly specific t cell lines from healthy adults, including donors without detectable frequencies of hadv hexon-specific t cells. the frequency of hadv-specific t cells was increased to - % in the t cell lines and the absolute numbers of both hexon-specific cd + and cd + t cells were to log increased compared to the starting material. detailed analysis showed that hadv-specific t cell lines recognized multiple mhc class i and ii restricted epitopes, including known and novel epitopes, and showed specific and efficient lysis of hadv infected target cells. this strategy may be used for adoptive transfer of donor-derived hadv hexon-specific cd + and cd + t cells for treatment of disseminated hadv infection after allosct. several studies showed that hbv persistance correlates with a failure of an efficient virus-specific t-cell response. induction of hbv-specific t cells by vaccination may be an innovative approach to overcome virus persistance. dna prime-recombinant adenovirus serotype (ad ) boost strategy proved to be effective in stimulating t cell responses and control of viral infections. woodchuck hepatitis virus (whv) and its host the woodchuck are a useful peclinical model for investigating the new therapeutic approaches. the efficacy of plasmid dna and ad vaccine vectors expressing whv core protein was first examinated in c bl mice. groups of mice were immunized with a dna prime-ad boost regimen or with dna and ad alone. ad was injected i. m. or s. c. t cell response was evaluated by intracellular ifng staining of splenocytes stimulated in vitro with whc-derived peptide pools. anti-whc antibodies were detected by elisa. we detected cd + t cell responses against peptide pools and in spleens of dna and dna-ad immunized mice. however, in prime-boost group the percentage of of detected ifng+ cd + t cells was lower in comparison to dna group. in splenocytes of animals vaccinated with ad very weak cd + t cell response was observed. in dna vaccinated animals we determined high level of anti-whc already after second immunization. after boosting with ad level of antibodies did not change. those antibodies were only igg a subclass what indicates th t helper type of response. ad -immunized mice had over -fold lower level of anti-whc: both igg a and igg subtypes were detected. the weak response induced by ad may be due to the low expression of whcag. in ongoing expreriments we improved the protein expression level by insertion of an intron. we currenly investigate the new construct in mice. the new peptide construct containing four m e-peptide sequences coupled to t helper epitopes from the plasmodium falciparum cs protein and the hepatitis b virus antigen was administered together with adjuvants intranasally and subcutaneously as described (mozdzanowska et al., virology journal ) into various mouse strains. in contrast to its predecessor peptide, we found that vaccination induced much higher anti-m e serum ab titers against peptide and native m e. this correlated with a large number of m -specific ab-secreting cells in lungs and bone marrow. moreover, the serum of vaccinated mice was also crossreactive against the influenza virus subtype a/fm (h n ), which contains a variant m e-sequence different in amino acid positions. importantly, this new peptide vaccine regimen showed significant protection against viral challenge with influenza a strains x (h n ) and the highly pathogenic pr/ (h n ) with remarkably reduced viral titers in lungs and noses of mice. in conclusion, our studies show promising results towards the further development of vaccination with m e as a potential "universal" influenza vaccine. this research is supported by a nih t fellowship ca - , the nih grant ai and a grant from the commonwealth of pa. l. yu zhejiang university, zhangzhou, china interleukin- (il- ) is a cytokine produced by stimulated mononuclear macrophage system. in this report, -day-old chicken embryos were vaccined with the plasmid dna (pci-chil- ) encoding chicken interleukin- and the copy numbers of chil- in peripheral blood, spleen and bursa of fabricius at different time points post-embryonic-vaccination were detected by real-time fluorescent quantitative pcr. the polyprotein of infectious bursal disease virus (ibdv) was prepared into dna vaccine, and the dna vaccine was co-administrated with pci-chil- in -day-old chicken embryos, then boosted after two weeks, and challenged with virulent ibdv four weeks later. the results indicated that allantoic cavity vaccinated with pci-chil- could accelerate high concentrations of chil- in nonage peripheral blood, accelerate high expression of chil- in nonage spleen and bursa of fabricius and promote the body early immune response capacity. embryo vaccination with chil- could significantly enhance the nonage proliferation responses of t lymphocytes from spleen and b lymphocytes from bursa. meanwhile, it could raise the nonage neutralization antibody level and inhance the protection against virulent ibdv induced by dna vaccine. the results indicated that the nonage immune responsing to ibdv dna vaccine was highly enhanced by embryonic coadministration with chil- (p x . ). due to the unique role of the hair follicle in percutaneous penetration, drug delivery systems, which target active compounds to the hair follicle, may result in a better penetration and a higher efficiency of hair and skin therapy ("follicular targeting"). applications in immunotherapy, e. g. transcutaneous vaccination, are of particular interest, because skin antigen-presenting cells (apcs) can be found at particularly high densities in hair follicle-bearing skin, where they are concentrated around the upper portion of the hair follicles. in in vitro studies on human skin explants, we demonstrated that nanoparticles, due to their ability to aggregate in the hair follicle openings and to penetrate along the follicular duct, are promising carrier systems for transfollicular drug delivery. transcutaneously applied nanoparticles in the size range of nm, were capable of penetrating the epithelium and entered into human epidermal lcs, suggesting that such particles may be used to transcutaneously deliver active vaccine compounds, via the hair follicle. the use of the skin as target organ for vaccine has been spurred by recent implication of epithelial dendritic cells (dc) in cd cell cross-priming and suggests that vaccination via the transcutaneous (tc) route may be relevant in the induction of cellular immune responses. advanced studies in vivo using functional vaccines are, however, essential to further assess the potential of particle-based vaccines in transcutaneous vaccination. for this purpose, we developed a standard operating procedure (sop) for transcutaneous vaccine delivery on human skin based on our current knowledge on follicular penetration. in a pilot study on volunteers and a phase i study on volunteers vaccinated with an influenza vaccine, we found that this newly developed sop is safe and efficient at inducing a significant increase in cellular immune responses mostly composed of antigen-specific cd cells. induction of t cell responses has become one of the major goals in therapeutic vaccination against viral diseases and cancer. this study proposes new perspectives for the development of vaccination strategies that triggers t cell immune responses in humans. objectives: all anti-hiv- neutralizing antibodies are directed toward the viral envelope glycoproteins (gp) and the transmembrane protein gp . two sites on gp and gp are attractive targets for vaccine design: the epitope in the third hypervariable region (v ) is recognized by the human monoclonal antibody - d and the epitopes in membrane proximal external region (mper) were recognized by the human monoclonal antibodies e and f . in order to elicit anti-hiv- neutralizing antibodies we have designed virus like particles (vlps) displaying either the gp -v region or the gp -mper. the vlps are based on the acyltransferase component (e chain) of the pyruvate dehydrogenase complex of geobacillus stearothermophilus. the e chain self-assembles into a nm protein scaffold resembling a vlp and that contains copies of e . efficient display and refolding of the v and mper regions in e vlps are obtained by using engineered plasmid which allows insertion of exogenous oligonucleotides at the ' of the gene coding for e . the priming and boosting with a combination of vlps and specific hiv- envelope dna were used to immunize mice and rabbits. results: the v -e and mper-e vlps were purified as stable mers from e. coli cells after refolding in vitro from inclusion bodies followed by gel filtration chromatography. binding of - d, e and f antibodies to hiv-e monomers was confirmed by western blot. we obtained high titers of hiv- gp -specific antibodies in mice immunized with a combination of vlps plus dna (hiv- sf gp ). these antibodies generated a low ( %- %) level of neutralization. moreover immunizations were also performed in rabbits, a better model for induction of neutralizing antibodies. three doses of e vlps plus dna elicited a low titer of hiv- gp specific antibodies. additional rounds of immunizations in rabbits will be performed, in combination with gp plasmid dna, to enahance the responses to envelope and to induce neutralizing activity against these key epitopes. our results demonstrate that e vlps are able to display antigenic determinants of hiv and to induce high titers of hiv- -specific antibodies. the e vlps represent a promising tool for a vaccine design. now a day we paid for vaccination of previous generations. as a result morbidity sharply increases, but we haven't well-tried scheme of immunity renewal yet. every clinic, every center do it in there own way, while vaccination is continued, even when it's not necessary, for example, grip, nobody know strain exactly. the most unpleasantly think is that most of physicians don't know what immunity mean specifically, general they think about vitamins, that isn't fit for forming immunity because of many reasons. we offer a way of immunity according to the world scientific theory and practice. the method is based on biochemical, electrophysiology, and biology way of correction physical status. at first we normalize and activate current settings that are going to the diseased organ, vascular system, gastrointestinal tract, spleen. all of it attends indemnity necessary microelements that were extracted from wild officinal herbals. we don't concentrate only on the one or two types of immunity, fist of all we take into account structure and dynamic of immunogeneration system. in our clinic we use this method; immunity is restored very quickly and kept during long time even if organism gets any complications, which can worsen the situation. that's why when we secure new physical statement in the cns program we forming new nearest and distant men health. we tell local state mechanism of disturbances from disturbances, that develop in blood, lymphatic system, tissues and hypothalamus, when pathological process exist long time. it's completely different disturbances of physician state, which should have different therapeutic approach. the threat of an influenza pandemic has become evident in recent years, emphasizing the requirement for influenza vaccines that are broadly cross-reactive against different subtypes with pandemic potential. we have previously shown that baxter's vero cell-derived h n whole virus candidate vaccines are highly immunogenic both in animal models and in human clinical studies, and cross-protective in mice and ferrets. more recently, it was reported that cross-reactive heterosubtype immune responses against highly pathogenic h n influenza virus could also be achieved by immunizing subjects with a trivalent seasonal influenza vaccine; however the induction of cross-subtype protection could not be addressed in this study with human subjects [ ] . the study reported here evaluated whether the seasonal influenza vaccine, when used either as a monotherapy or in combination with a h n whole virus wild-type vaccine, could induce an immune response and protect mice against h n influenza virus infection. a trivalent seasonal influenza vaccine was shown to elicit anti-h n antibody and t cell responses and partially protected mice against a lethal challenge with wild-type h n virus. the protective efficacy of the trivalent vaccine derived mainly from the h n component. moreover, passively transferred serum of mice immunised with seasonal influenza vaccine protected naïve mice from infection with h n virus, suggesting that antibodies are the main contributor to protection. h n specific serum did not inhibit neuraminidase activity of h n virus suggesting that protection was not mediated by neuraminidase n -specific antibodies. next, we investigated the combination of the trivalent seasonal influenza vaccine and the h n whole virus wild-type vaccine. a prime with the seasonal influenza vaccine followed by immunisation with the h n vaccine enhanced anti-h n antibody response, cellular immunity and protection compared to a single immunization with an equivalent sub-optimal dose of the h n vaccine. hence, hetero-subtype immunity can be achieved by immunization with a trivalent seasonal influenza vaccine, which can be further boosted with a h n candidate vaccine. [ ] gioia c et al. aims: to register the compliance of the population to the old and new vaccines of the national vaccination program for the children up to years old, and to investigate the possible causes of the potential shortages, in order to approach even more successfully the further goal of this whole attempt, which undoubtedly is the future control of important generalized infections. methods: in the study we checked the vaccination history of children in the first grade of primary school in the area of central and west macedonia. there were greek and foreign children. as fully vaccinated were considered those who had already undergone at least one dose of hib, meningococcus and pneumococcus, two doses of hav, as well as four doses of dtp-sabin, while in the cases of a lack of vaccination, the causes were investigated and the adequate recommendations and information were given. in all the cases, except for the nationality, the sex, and the educational and social level of the parents were registered. results: the percentages of the compliance found, are presented in the following ) it should be underlined that, as shown in the table, the percentages of the obligatory-free of charge vaccines were close to %. ) high percentages were noted also for meningococcus, either because it is an old vaccine (it has been available for seven years), or because the bacteria is considered quite dangerous (it has been emphasized through the media). ) on the contrary, as far as the hepatitis a and the pneumococcus vaccines are concerned, low percentages were found, either because of the lack of adequate information-fact that was also shown in our study-or even because of their cost. ) finally, a statistically significant difference was found relating the response to the vaccination coverage, between greeks and foreigners, but also between the greeks themselves, in relation to their educational and socioeconomic level. objective: over the past three decades, the incidence of type diabetes has dramatically increased in europe and north america, inversely correlated to the decrease of infections. according to the hygiene hypothesis, pathogens may prevent the onset of the disease. om- , a bacterial extract of both gram positive and gram negative bacteria already used as an immunomodulatory treatment in children, has been shown to protect non obese diabetic (nod) mice from diabetes development. we aimed here at understanding the mechanism underlying this protection. methods: nod mice and nod-cd -/mice, which are devoid of natural regulatory t cells (tregs), were treated with om- . cytokine secretion, activation and proliferation of b cells and foxp + tregs were monitored. as toll-like receptors (tlr) recognise microbial molecules and trigger innate and adaptive immunological response, cells from mice deficient for tlr , tlr or the myd adaptor protein were used to further address the mechanisms driving the immunomodulatory activity of om- . two synthetic tlr agonists used as adjuvant in human (om- -dp and om- -mp-ac) were also tested for their capacity to protect nod mice from diabetes. the om- -induced protection of diabetes required natural tregs, as nod-cd -/mice were not protected. remarkably, om- activated b cells and not t cells, promoting their proliferation and il- secretion, two phenomena that were tlr -and myd -dependent. om- -dp and om- -mp-ac two synthetic murine tlr agonists effectively prevented diabetes onset in nod mice, promoted the expansion of cd + cd + foxp + t cells and the proliferation of il- secreting b cells in a dose-dependent manner. conclusion: our results argue for the involvement of tlr signaling in the protective effect of om- on development of diabetes and show that two other tlr agonists induce proliferation of b cells and their secretion of il- as well as stimulation of regulatory cd + cd + foxp + t cells. activation of the innate immunity by tlr-stimulation using those products already used in clinics, may prevent the onset of diabetes in those at risk of developing the disease. d. de wit , a. legat , s. thomas , m. van mechelen , p. hermand , m. goldman institute for medical immunology/université libre de bruxelles, gosselies, belgium, glaxosmithkline biologicals, rixensart, belgium aminoalkyl glucosaminide -phosphates (agp) are lipid a mimetics which are considered as interesting candidates for the development of synthetic vaccine adjuvants targeting toll-like receptor (tlr ). since natural lipid a from bacterial lipopolysaccharide (lps) depends on membrane-bound or soluble cd (scd ) for its tlr ligand activity, we investigated the involvement of both forms of cd in the responses elicited by crx- , a prototypical agp. first, we found that crx- efficiently induces nf-kb and irf activation in hek cells transfected with tlr and md- genes, whereas the responses to lps required co-transfection of the gene encoding membrane-bound cd . likewise, crx- efficiently induces the synthesis of nf-kb and irf- dependent cytokines in whole blood of a patient with paroxysmal nocturnal hemoglobinuria, a disease in which a defect in membrane-bound cd prevents lps responses. we then observed that monocyte-derived dendritic cells (dc) which are devoid of membrane-bound cd respond to crx- but not to lps in serum-free medium. the addition of the soluble form of cd did not modify the levels of il and tnf produced by crx- stimulated dc but increased the levels of interferon-b (ifn-b). when scd was added to hek cells expressing tlr /md- , nf-kb activity was not modified but irf activity was increased in a dose-dependent manner in response to crx- . we will further compare the responses induced by crx- in wild-type and cd deficient mice. we previously showed that the transcriptional transactivator (tat) of human immunodeficiency virus possesses the unusual ability to raise a humoral immune response in the absence of adjuvant. these observations prompted us to examine whether such a property can be used to boost the immune response raised against poorly immunogenic peptides. as we previously observed that the autoadjuvant property is controlled by a determinant located within the core-and cysteine-rich regions of the protein, we decided to investigate whether the grafting or the co-injection of a peptide partially containing this determinant (ptat) can raise a humoral immune response against two model peptides. these two peptides, which originate from diphtheria toxin (pdt) and from toxin alpha (pt), both contain an i-ad restricted t-cell epitope but are nonetheless non-immunogenic in balb/c mice of the h- d haplotype when injected with alum. the ptat, pdt, pt, ptatpt and ptatpdt constructs were prepared by chemical synthesis, purified by reverse phase hplc and characterized by mass-spectrometry. pdt+ptat, pt+ptat, ptatpt and ptatpdt were respectively injected twice at two weeks interval in balb/c mice and animals were bled and days after the second immunisation. the sera were subsequently incubated in microtiter elisa plates previously coated with pt and pdt peptides respectively in order to assess the humoral immune response. we observed a lack of antibody response for the immunizations made with the mixture of peptides (pdt+ptat and pt+ptat) but an anti-pdt and anti-pt response for the immunizations made with the two hybrid constructs (ptatpt and ptatpdt). our results indicate that a humoral immune response can be raised towards non-immunogenic peptides using a determinant involved in the autoadjuvant property of tat, that the phenomenon requires the covalent coupling to the peptide antigen and that it is therefore not related to a bystander effect. interleukin- gene polymorph isms (c t, g a, c a and a t) and susceptibility to brucellosis in iranian patients russian federation some epidemiological and observational data suggest that farm and pets exposure [ ] in early childhood may be conducive to reduced atopy. currently, there is a lack of consensus regarding underlying immunological mechanisms, especially in prenatal period. as we previously reported the decreasing of intracellular ifn-g production by cbmc statistical analysis was performed using the kruskal-wallis and mann-whitney tests. results: we revealed that newborns from rural mothers (n= ) have higher amount of both nonactivated (subtype infg+/cd -, p= . ) and activated (subtype infg+/cd +, p= . ) cbmc, producing ifn-g, as compared with newborns from urban mothers (n= ) exposure to pets and the risk of allergic symptoms during the first years of life intracellular interferon-g production by cord blood mononuclear cells as predictor of atopic dermatitis forming in infants: a one-year prospective birth cohort study pc / to what extent t-spot.tb could be used in the diagnosis of tuberculosis in children exposed to tb infection? s. a tb) in children, especially in bcg-vaccinated is difficult for diagnosis because of the low percentage of smear positivity ( - %) and clinical futures only in severe forms of disease. the purpose of the present study was to evaluate the diagnostic value of t-spot.tb (oxford immunotec, oxford, uk) compared to tuberculin skin test (tst) in children exposed to tb contact in the family. forty three children with a history for bcg vaccination/revaccination, treated in the university clinic for lung diseases in children sofia, bulgaria were enrolled in the study. the patients were divided according to age in the following groups: months - years (n= ), - years (n= ) and - (n= ) tb has the highest diagnostic value in children n years of age in early childhood the diagnostic value of t-spot.tb and tst does not differ cfp- antigen is more sensitive for detection of tb-specific t cells compared to esat- antigen. . in children with tst - mm t-spot.tb has a high diagnostic value objectives: the goal of this study is to determine the role of tlr and tlr in the development of spontaneous lupus disease by creating tlr or tlr deficient c bl/ lpr/lpr mice. methods: tlr and tlr deficient lupus prone mice have been generated by crossing c /bl -tlr -/-or c /bl -tlr -/-mice with c /bl lpr/lpr mice which develop a moderate type of lupus related to fas deficiency. we analysed the phenotype of the disease, autoantibody production and renal injury. statistical comparisons were performed using the mann-whitney u-test. results: these mice developed a less severe disease and few immunological alterations. indeed, in tlr or tlr deficient lpr mice, glomerular igg deposits and mesangial cell proliferation were dramatically decreased and anti-nuclear, anti-dsdna and anti-cardiolipin autoantibody titers were significantly reduced. however, the response against nucleosome remained unaffected, indicating a role of tlr or tlr in the production of autoantibodies directed against certain slerelated autoantigens. analysis of b cell phenotype showed a significant reduction of mz b cells, particularly in tlr deficient mice suggesting an important role of tlr in the sustained activation of these cells likely involved in autoantibody production. interestingly, the lack of tlr also affected the production of cytokines involved in the development of lupus disease. conclusion: our data show that deficiency in tlr pc / expression of full length mcl- and its splice variant in juvenile systemic lupus erythematosus (jsle) neutrophils: differential modulation by gm-csf granulocytemacrophage colony-stimulating factor (gm-csf) can prolong neutrophil survival by increasing mcl- , an anti-apoptotic protein. a splice variant of mcl- arises by removal of exon and induces cell death rather than preventing it. here we investigate the expression of both the full length mcl- (mcl- l) and its splice variant (mcl- s) in jsle neutrophils compared to controls and investigate whether the addition of gm-csf changes the expression of both isoforms of mcl- . method: neutrophils were isolated from children (diagnosed x years) with jsle (n= ) and non-inflammatory conditions (control, n= ) and incubated with control serum, jsle serum alone or with jsle serum plus pg/ml gm-csf. quantitative real time pcr was used to assess mcl- l and mcl- s mrna expression (mean ± sem) following incubation in the above conditions and immediately following neutrophil isolation the ratio of mcl- s to mcl- l was also higher in jsle patients compared to controls (p x . ). the addition of gm-csf to jsle serum was associated with an increase in mcl- l ( . ± . ) and a decrease in mcl- s ( . ± . ) mrna expression the addition of gm-csf to jsle serum can abrogate the increased neutrophil apoptosis. alternative splicing is recognised to play a significant role in the regulation of proteins involved in cell death. our results suggest that jsle neutrophils may be more apoptotic due to differential expression of mcl- compared to controls, with jsle neutrophils having greater expression of the pro-apoptotic isoform mcl- s, and less anti-apoptotic full length mcl- cyld is a tumor suppressor gene known to play an important role in the nf-kb pathway. to analyze the function of cyld in vivo we used the cyld ex / mouse strain, which is characterized by loss of the full-length transcript and overexpression of a short splice variant of the cyld gene (scyld) to further investigate the connection between scyld overexpression in t cells and colonic inflammation, we used an adoptive transfer model of colitis. therefore naive cd + cyld ex / t cells were transferred into rag -/-mice which were analysed by mini-endoscopy weekly after cell transfer. here we could demonstrate that cyld ex / cd + t cells exhibit less capacity to induce colitis compared to control cells. consequently we investigated if regulatory t cells (t regs ) of cyld ex / mice are capable to control inflammatory responses. for this purpose cd + cd + cells were co-transferred with naïve wt cd + t cells into rag -/-recipients. interestingly, rag -/-recipients of cyld ex / t regs displayed strong features of colitis compared to control recipients showing that these cells were unable to inhibit inflammatory responses. our findings demonstrate that overexpression of scyld leads to a hyperresponsive t cell phenotype and higher production of inflammatory cytokines by t cells pc / the role of hla complex in inflammatory bowel disease: crohn's disease and ulcerative colitis de investigación biomédica en red de enfermedades hepáticas y digestivas (ciberehd). university hospital virgen arrixaca the allele frequencies of hla class i in cd and uc patients were not different to those observed in controls, although we found an increased frequency of a* in cd vs uc. haplotype frequencies of hla class i and ii in cd and uc were also not different to those observed in controls. however, we found increased frequencies of drb * , * and * alleles, and a decreased allele frequency of drb * in cd vs uc patients and controls. these data are in concordance with other previous studies suggesting that, in patients with isolated colonic cd, drb * is associated with the development of severe disease and positive association of cd with drb * and drb * . indeed, drb * was negatively associated with cd. this allele appears to confer protection against all subgroups of cd, in all ethnic groups including japanese. however, hla-drb * frequency allele, associated in unselected patients with cd in other studies, was not different in our cd and uc patients, and controls. additionally, an increased frequency in hla-drb * in cd was not found in our patients in a different manner to other reported studies. on the other hand, in our uc patients, allele frequencies of drb * were strongly increased with respect to cd and controls. however, the frequency of drb * was decreased in uc with respect to cd and controls. in this sense, our data are agreed with other reports showing that hla-drb * is associated with uc in european, north american, japanese and korean populations methods: a total of children were studied, ( boys and girls), up to years of age, with symptoms suspicious for epstein-barr virus infection. the elisa method was used to look for specific antibodies against the capsid of the virus vcaigg and against the nuclear antigen ebv-igm, while taking into consideration the possible increase of the vcaigg title between two serum samples. results: totally, positive cases of children were found ( %) with active infection : boys ( x years of age, - years of age and g years of age) and girls ( x years of age, - years of age and g years of age). pharyngitis was present in children ( , %), had fever ( , %) and had lymphadenitis ( %). the lab tests revealed leukocytosis up to . leukocytes in cases ( , %) and leukocytosis g . in cases ( , %). the most frequent complication documented was streptococcal superinfection in children ( , %) and thrombocytopenia in children ( , %). a past infection (negative ebv-igm values and positive vcaigg values) was virus infection is common among children and teenagers serum negative are mainly the children of little age and ) there is no statistically important difference between the two sexes, while on the contrary there is a seasonal distribution of the infection, with winter and summer outbreaks general hospital of rethymno, rethymno, greece shows that the il- ra , previously believed to be a decoy for il- only, is able to transmit a signal via il- . our results support this and may suggest that il- / il- ra signalling causes disease in oxazolone-induced colitis. currently we are dissecting the role of single cell populations expressing il- ra to establish which cells play a role in regulating the immune response to oxazolone-induced colitis. together this data can define a role for il- or il- ra and identify specific cell populations methods: splenic apcs exposed to enteroantigen (eag) +/-probiotics were used to stimulate cultured cd + cd -t cells to which titrated numbers of tregs were added. neutralizing antibodies against il- and il- b and elisa-based cytokine analyses were used to monitor the effect of cytokines secreted in the t cell cultures. results: exposure of apcs to eag and probiotics did not influence eag-specific cd + cd -t cell proliferation. however, exposure to three of the six probiotics tested (b. bifidum bi- , l. acidophilus ncfm tm and b. bifidum bi- ) consistently reduced regulatory activity of tregs in a cell-dose dependent manner. the tregreducing activity of probiotics was analyzed using fractionated components of the b. bifidum bi- strain. data indicated that bacterial cell-wall components were responsible for reducing treg activity and not components of nucleus or cytoplasm. the probiotic-induced down-regulation of treg activity was not mediated by increased intra-culture secretion of inflammatory cytokines such as il- or il- b. conclusion: we conclude that certain probiotic strains can modify apcs to cause reduced treg activity in an eag-specific t cell proliferation assay. this effect apparently depends on a direct apc-to-treg cell contact and not secreted cytokines. the apc/probiotics-mediated inhibitory effect on tregs may oppose antiinflammatory activities desired from probiotic therapy palmieri 'la sapienza dysregulated innate and adaptive immune responses against commensal flora lead to crohn disease (cd) and ulcerative colitis (uc), two different forms of inflammatory bowel disease (ibd), a lifelong inflammatory condition of the gastrointestinal tract methods: we analyzed pediatric cd patients ( active, remission), pediatric uc patients ( active, remission), and age-matched non-ibd controls. nkg d/ligand expression was evaluated by immunostaining and multiparametric facs analysis (on pbmc subsets), and by immunohistochemistry and twocolour immunofluorescence (on intestinal biopsies). differences between groups were analyzed with non-parametric and parametric tests; a level of p x . was considered significant. results: nkg d expression is selectively upregulated on circulating "innate-like" t cell populations (g/d and cd +cd + nkt cells), in active, but not in quiescent ibd patients; receptor upregulation correlates with disease type (observed in uc, but not in cd patients). in the same patient groups, the appearance of nkg d ligands on circulating monocytes is also observed. the dramatic increase of nkg d+ lymphocytes, and the strong upregulation of nkg d ligands on both epithelial and immune components, are observed in active ibd lesions. conclusions: our observations document the dysregulated expression pattern of nkg d/ligands on selected innate immunity populations in pediatric ibd patients, both at mucosal and systemic level pc / peripheral and intestinal regulatory t cell dynamics in pediatric ibd patients is a chronic inflammatory condition of the gastrointestinal tract characterized by dysregulated innate and adaptive responses against commensal flora. regulatory t cells (t reg) represent an important mechanism to suppress uncontrolled immune responses to bacterial flora. aims: to evaluate the frequency of regulatory t cells in the peripheral blood, and in inflamed and non inflamed mucosae of pediatric ibd and non mucosal regulatory t cells were identified by immunohistochemistry; circulating regulatory t cells were analysed by immunofluorescence and facs analysis. differences were analyzed with parametric and non-parametric testsconsidered significant. results: foxp + t reg were significantly increased in the intestinal lesions of active ibd patients (cd or uc), and returned to normal levels in post-therapy remission phase. at variance, circulating cd + t reg frequency was elevated in patients affected by both forms of ibd, independently of disease activity, as it persisted in the remission phase. a selective imbalance in the frequency of t and nk subsets characterized the abundant inflammatory infiltrate present in active intestinal lesions, and the normal immunological profile was only partially restored in mucosal samples of quiescent ibd patients. conclusions: regulatory t cells dynamics are differently regulated in mucosal tissues and at the systemic level, during the distinct phases of disease; t reg dynamics in pediatric ibd patients only partially matches previous data obtained in the adults; quiescent ibd is characterized by the imbalance of selected lymphocyte subsets, both in the mucosa and systemically the increased expression of immunoproteasomes in the inflamed mucosa of ibd patients was shown to contribute to this pathology by enhancing nf-kb activation. due to the relation between nf-kb and the immunoproteasome we have investigated whether specific inhibition of immunoproteasomes is suitable for therapeutic intervention in ibd. lmp knock-out mice are deficient in the essential catalytic immunoproteasome-subunit ß i and therefore are devoid of immunoproteasomes. to test our hypothesis, we employed the dss colitis model. in contrast to wild-type mice, colitis was attenuated in lmp knock-out mice characterized by reduced weight loss and less infiltration of lymphocytes in the mucosa confirmed by histology. in addition, lmp knock-out mice had lower levels of proinflammatory cytokines and chemokines compared to wild-type mice validated by rt-pcr and elisa. especially nf-kb regulated genes show enhanced induction in wild-type mice unlike lmp knock-out mice synaptic systems gmbh, braunschweig, germany objectives: although more than million people worldwide are chronically infected with hepatitis c virus (hcv) no prophylactic or therapeutic vaccines do exist to prevent or cure hcv infections. our major objective is to develop a dendritic cell (dc)-based immunotherapy enhancing virus-specific cellular immune response for treatment of hcv infections based on this approach we aim at generating adec- antibodies conjugated with immunodominant hcv proteins to induce hcv-specific protective immunity. methods: recombinant hcv proteins are expressed using "expression-ready-clones" containing n-terminally his-tagged hcv-core (aa - ) or hcv-ns (aa - ) sequences. protein purification is performed by metal-affinity chromatography on ni-nta-agarose hcv-specific t cell responses are monitored at different time points after immunization by facs and in vitro t cell proliferation assays. results: to obtain high amounts of recombinant ns and core we successfully optimized culture and protein purification conditions. briefly, ns was purified natively using pbs-based buffers with ph-gradient. in contrast, purification of core was performed under denaturing conditions in presence of guhcl and urea and a ph-gradient elution. moreover, optimized conditions allowing conjugation of adec- to recombinant hcv proteins were established with respect to duration of conjugation and buffer requirements needed to avoid protein precipitation. efficient conjugation was verified by western blot analysis. after successful generation of adec- / hcv-protein conjugates we are currently establishing optimized vaccination conditions to induce hcv-specific immune responses pd / mva-nef vaccination induces polyfunctional cd t-cells and increases the proliferative capacity of cd t-cells in hiv- infected individuals under haart several vaccination trials have made use of the modified vaccinia virus ankara (mva) as delivery vector. in a therapeutic vaccination trial, we demonstrated that mva expressing the hiv- protein nef (mva-nef) was safe in hiv- infected individuals under haart and immunogenic in regard to the elicitation of ifn-g mediated cd t-cell responses. recent advancements in polychromatic flow-cytometry technology revealed that the sole evaluation of ifn-g provides limited information on the quality of antigen-specific t-cell responses. the evaluation of several functions is essential, as simultaneous production of multiple cytokines by t-cells is associated with superior control of viral replication. methods: in a retrospective setting, we simultaneously assessed the production of ifn-g, il- and mip- b, the expression of the activation marker cd and the differentiation marker cd ra in nef-specific cd and cd t-cell populations during the course of the vaccination trial. furthermore we applied a multi-colour cfse based proliferation assay investigating the proliferative capacity and the simultaneous expression of ifn-g, il- and mip- b. results: following mva-nef vaccination, we observed a significant increase of the total nef-specific cd t-cell response and a significant increase of polyfunctional nef-specific cd t-cells, simultaneously expressing ifn-g, il- and cd . using the standard ics no increase of nef-specific cd t-cell responses was observed. however, by the cfse based proliferation assay, we could show a clear expansion and a generally enhanced proliferative capacity of nef-specific cd t-cells following mva-nef vaccination. notebly, we observed a correlation between the increase of ifn-g, il- and cd expressing cd t-cells and the increase of proliferating cd t-cells suggesting the possibility of a causal link between the two functions. conclusions: the mva-nef vaccine is able to change the quality and quantity of the nef-specific cd t-cell immune response and has the potential to increase the proliferative capacity of nef-specific cd t-cells in hiv- infected subjects under haart this preferential binding to the complex was evident in classical immunochemistry assays, as well as in surface plasmon resonance (spr) tests. this ab inhibited hiv- mediated membrane fusion and p -detected replication. db was found to nicely recapitulate the characteristics of the unconventional, protective immune response, which is taking place in naturally resistant esn individuals. further characterization of the antibody and of its binding epitope is ongoing following intradermal vaccination with mg dna and electroporation of balb/c mice, splenocytes have been incubated with peptides representing class i and ii epitopes, and specific t cell-responses were examined by elispot-assays. the specific antibody responses have been measured by sandwich eli-sas, and neutralizing antibodies have been investigated by hi-assays. results: the vaccibody constructs have been found to be expressed and correctly folded in vitro. the in vivo experiments further demonstrate the presence of neutralizing antibodies as well as the strong induction of antigen specific cd + and cd + t cells. conclusion: antibody and cellular immune responses against influenza hemagglutinin are enhanced when targeted to apcs. methods: the hcv recombinant proteins rns ( - aa) and rns a ( - aa) were conjugated with immunomax using the heterobifunctional reagent sulfo-smcc. balb/c and dba/ j mice were immunized intraperitoneally times at a month interval with different doses ( . - mg/mouse) of the proteins without adjuvants, as conjugates with immunomax, or with complete freund's adjuvant (cfa) the other combinations were not immunogenic at given doses. it should be noted that only conjugates stimulated production of antibodies that bound not only to recombinant protein but also to peptides imitating epitopes of ns protein. immunization with rns a-immunomax conjugate and rns in cfa ( . mg/mouse) induced a similar antibody activity, but a different t-cell responses. the conjugate induced splenic accumulation of t cells specifically reacting in vitro with ns a recombinant proteins of various genotypes, with peptides and with phages by cell proliferation and/or cytokine secretion. immunization with rns a in cfa induced cells proliferating in vitro after stimulation only with peptides; none of the antigens stimulated cytokine secretion. conclusion: covalent conjugates of hcv nonstructural proteins with immunomax effectively induce humoral and cell immune responses pd / degree of cross-genotype reactivity of hcv-specific cd t cells directed against ns the existence of multiple hcv genotypes characterized by marked sequence differences is a challenge for immune control. the aim of this study was to compare the antiviral cd t cell response targeting hcv genotype (gt ) and genotype (gt ) as the most predominant genotypes in germany and to determine the extent of cross-genotype reactivity of specific t cells. we analyzed a cohort of patients with past or ongoing intravenous drug use (ivdu) hypothesizing that multiple exposures to different genotypes may occur. methods: subjects ( with gt , with gt and anti-hcv-pos/hcv-rna-neg) were analyzed. hcv-specific t cells were expanded from pbmc in the presence of peptide pools covering ns from gt or gt . individual reactive peptides and the degree of cross-reactivity between the gt and gt variants were determined by ics. complete ns is sequenced from all viremic patients pd / anti-retroviral effects of type i interferon subtypes in vivo ifna subtypes , , or suppressed fvreplication in vitro, but differed greatly in their antiviral efficacy in vivo. treatment of fv-infected mice with the ifna subtypes , or , but not led to a significant reduction in viral loads. decreased splenic viral load after ifna treatment correlated with an expansion of activated fv-specific cd + t cells and nk cells in the spleen, whereas in ifna -and ifna -treated mice it exclusively correlated with the activation of nk cells. other ifna subtypes like ifna , and are under investigation pd / elimination of immunodominant epitopes from multispecific dna-based vaccines allows induction of cd t cells that have a striking anti-viral potential immunodominance limits the tcr diversity of specific, anti-viral cd t cell responses elicited by vaccination or infection. to prime multispecific t cell responses, we constructed dna vaccines that coexpress chimeric, multidomain antigens (with cd t cell-defined epitopes of the hepatitis b virus (hbv) surface (s), core (c) and polymerase (pol) proteins, and/or the ovalbumin (ova) antigen as stress protein-capturing fusion proteins. priming of mono-or multispecific, hla-a* -or k b -restricted cd t cell responses by these dna vaccines differed. k b /ova - -and k b /s - -specific cd t cell responses did although chronic infections remain asymptomatic in most cases, immunocompromised patients can suffer from severe and life-threatening ebv-associated diseases, such as posttransplant lymphoproliferative disorders (ptld). thus, immunotherapeutic strategies using adoptively transferred ebv-specific t cells are promising. one option is the generation and expansion of cd + and cd + t lymphocytes by using ebv-specific synthetic peptides for the stimulation of pre-existing memory t cells. aim of our study was to identify a set of mhc class-ii peptides for each antigen promiscuitive peptides with high syfpeithi scores were tested for immunogenicity using an ifn-g-elispot. pbmcs of at least healthy, randomly chosen blood donors were cultured for days in the presence of each candidate peptide. functional and phenotypic analysis of t cells of several donors was performed by multicolor flow cytometry. out of tested peptides could be identified as t-cell epitopes. two of them were defined as immunodominant, as more than % of tested blood donors showed peptide-specific t cell responses. so far, eight of the tested peptides could be identified as mhc class-ii epitopes. furthermore, a highly immunodominant class ii peptide mix consisting of peptides was selected. in conclusion, we could identify several new ebv-specific mhc class-ii epitopes which can be used for united kingdom, hospital de clínicas during persistent hbv infections, patients usually develop poor or no protective immune responses against viral antigens, which not only leads to the chronicity but also the unresponsiveness to conventional treatments.in order to overcome the unresponsiveness and to generate an effective therapeutic strategy for treatment for chronic hbv infections a chimeric tcr against hbsag, which aims to increase the percentage and quality of antigen-specific cd + t cells, was developed moreover, we pre-conditioned the liver microenvironment by injection of cpg oligodeoxynucleotides (odn) to optimize the recruitment of transferred cd + t cells to the liver and to overcome the tolerogenic microenvironment of the liver. we found that the il- -exposed cd + t cells showed at least five-fold increase of survival rate in vivo than il- -exposed cd + t cells did treatment of the recipients with cpg-odn could increase the percentage and also the total amount of transferred cd + t cells mainly in the liver. by in vivo brdu incorporation, we demonstrated that the higher in vivo survival rate of il- -exposed cd + t cells and the effect of cpg-odn were due to the up-regulation of the proliferation of those cells. to sum up, the cocktail therapeutic strategy could not only increase the survival rate of transferred cells but also direct the antigen-specific cd + t cells to the liver to exhibit their effector functions. the detailed mechanisms responsible for the il- and cpg-odn effects on the regulation hyper igm (him) and wiskott-aldrich syndrome (was) than those of corresponding controls (p x . ) . there was a significant elevation of t ada and ada activities in iga deficient patients as compared to healthy individuals (p x . ) . our results hypothesized that altered ada activity may be associated with altered immunity. therefore, serum ada level could be used as an indicator along with other parameters pd / hiv- sequence evolution after dendritic cell-based immune therapy in a phase i/ii clinical trial hiv rna was extracted from plasma samples collected before the startof haart and early after vaccination when haart was terminated. rna was amplified by rt-pcr and sequenced using standard protocols. sequences of the vaccine genes tat, rev and nef as well as control genes vif, vpr, vpu and parts of env were analyzed for variation between pre-and post vaccination time points. hiv sequences spanning known and predicted epitopes of the relevant hla alleles from each participant were analyzed in detail. results and conclusion: immune therapy was well-tolerated and no severe adverse effects occurred. after haart termination, plasma viral load became detectable in all patients after - weeks. follwing the viral rebound a set point was reached, that was lower than the viral load before start of haart. using various methods we evidenced newly induced or enhanced immunity after immune therapy (see abstract b. de keersmaecker et al.). for studying sequence evolution, complete sets of both pre-haart and post-vaccination sequences were obtained in out of patients. with one exception, variation in sequences of vaccine and control genes of pre-haart samples compared to samples taken early after vaccination was limited. this indicates that there was no significant impact of the immune response on virus evolution at this stage. more focussed analysis on viral sequences spanning specific hla islamic republic of newcastle disease (nd) is regarded throughout the world as one of the most important diseases of poultry, not only due to the serious and high flock mortality, but also through the economic impacts. the purpose of this study was to be informed from the possible influence of infectious bronchitis virus on immune response of chickens to nd live vaccine. one hundred and twenty, -day-old ross broiler chickens divided randomly into groups, experimental and a group as control one. the first experimental group vaccinated by a monovalent nd live vaccine with cl/ strain, and the second experimental group vaccinated by a bivalent newcastle disease and infectious bronchitis live vaccine with cl/ and h strains, via the drinking water at days of age at the same time, and the control group received no nd vaccine. the antibody response to vaccination was assessed using the hemagglutination inhibition (hi) test by taking blood samples three times, first the day before and the next, & days post vaccination. results indicated that, although the strain of studied nd live vaccines were the same united kingdom t cell-based ifng release assays from blood are an important advance for diagnosing tuberculosis infection but do not permit reliable treatment monitoring or distinction of active tb from successfully treated disease or latent infection. t-cell cytokine profiles vary with in vivo antigen load in viral infections cd t cells from patients with active tb and patients with successfully treated tb were analysed for simultaneous expression of ifng and il at the single cell level using multi-colour flow-cytometry after hours stimulation with ppd. moreover, cells were stimulated with esat- and cfp- receiver operator characteristics analysis revealed that a percentage of ifng /il dual positive cells x % served as an accurate marker for active tb patients (specificity %, sensitivity %), while frequencies g % were observed in treated as well as active tb patients. in conclusion, quantitation of antigen-specific t cells based on the analysis of ifng only does not allow distinction of patients with active and successfully treated disease pd / necessity of postpone bcg vaccination -lesson from primary immunodeficiencies v. thon methods and results: the czech national database of primary immunodeficiencies (pid) was established in and is connected with the european database of primary immunodeficiencies (esid). the prevalence of pid in the czech republic (approximately inhabitants) is . to . among these patients there are children diagnosed with severe combined immunodeficiency (scid) and chronic granulomatous disease (cgd) too. according to the czech national database of pid, out of children with later proved scid were immunised with bcg vaccine in the first days of life. nine of them developed disseminated and generalized bcg infections. five children with scid died. moreover, reactivation of bcg was also seen in healthy children after admission of combined vaccines with hepatitis b given at the age of twelve weeks. on the other hand, this was not the case in thousands of children of hbsag positive mothers who were vaccinated against hepatitis b after delivery in the first place and later immunized with bcg vaccine. systematic vigilance against tuberculosis (tb) and vaccination significantly lower the prevalence and risks of tb. in the czech republic, the prevalence of tb is currently . to inhabitants. unfortunately, temporary interruption of bcg vaccination in three large districts in the period of to led into higher incidence of tb and appearance of new cases of aviary mycobacteriosis. these complications were not observed in vaccinated children. conclusion: we recommend a change of current practice of bcg vaccination considering new immunization schedule with hexavalent vaccine pd / novel analogues of thalidomide inhibit cd expression and production of tnf-a, il- , ifn-g, cxcl- this work describes the synthesis and characterization novel thalidomide analogues, prepared in good yields using simple methodology. our results suggest that anti-inflammatory and immunomodulatory activity of these diamine compounds is potentially applicable in treating enl and other diseases. supported by: cnpq, fapemig and capes, brazil. of the b cell follicle. cta -dd augmented gc-formations, specific antibody responses and cell-mediated immunity to the t cell-dependent antigen np-cgg, but failed to do so when used together with t cell independent antigens, such as np-ficoll or np-dextran. this effect required adp-ribosyltransferase activity, as mutant cta r k-dd failed to exert an adjuvant effect. the adjuvant function appeared to correlate with the fdc-localization and turned out to require complement and/or complement receptors (cr) chitosan formulations varying in molecular weight, counterion and structure (i. e. soluble v/s particulate) were used in assays to examine expression of maturation markers via flow cytometry and cytokine production by elisa. we found that, in contrast to alum, plg and ps particles, chitosan induced bmdc maturation on its own, as determined by the expression of cd and cd . these effects were most prevalent with soluble chitosan chloride formulations but were also notable with soluble chitosan glutamate chitosan. the effect of chitosan on cytokine production was investigated using a panel of different tlr agonists in combination with chitosan particles. results show an increase in the secretion levels of il- a and il- b, while il- levels were not affected. finally we studied the role of inflammasome activation in the enhancement of il- b production. using bmdc from nlrp -/-mice we examined il- b production in response to different tlr and chitosan combinations. results show that the ability of chitosan to enhance il- b production is dependent on nlrp . collectively our data indicate that upregulation of maturation markers and enhancement in proinflammatory cytokine secretion mediated by chitosan severe sepsis, induced in mice by cecal ligation and puncture (clp), led to ho- expression in infiltrating peritoneal leukocytes, kidney and liver. mortality rate of clp increased from % in wild type (hmox +/+ ) mice to % in ho deficient (hmox -/-) mice. hmox -/-but not hmox +/+ mice developed end-stage multiorgan failure. mortality of hmox -/-mice was associated with increased peritoneal leukocyte infiltration, but not with increased pro-inflammatory cytokine secretion or bacterial load in peritoneum, blood or organs. clp induced a significant increase in cell-free hemoglobin free heme was found to sensitize primary hepatocytes to tnf, anti-fas antibody, h o or peroxynitrite mediated apoptosis. this cell death was associated with outward nuclear translocation and extra-cellular accumulation of the late-stage pro-inflammatory cytokine hmgb . similarly, circulating and cytoplasmic hmgb was increased in hmox -/-relative to hmox +/+ mice following clp. in conclusion, these data suggest that free hemoglobin and heme, released during severe sepsis, are important factors in the organ failure and death associated with severe and b- , -linked mannose residues elicit inhibition effect. it was found that inhibition activity of oligosaccharides increases with chain length. immunization with mannan-hsa conjugate allowed for the maturation of immune response generating specific antibodies with high avidity/affinity, whereas immunization with mannan alone elicited only low-affinity antibodies. in the future, an effective antifungal subcellular vaccine would be constructed using selected mannooligosaccharidic epitope and the appropriate carrier protein as inductor of immunological memory. acknowledgements: this work was supported by the grant agency of slovak academy of sciences all subjects received dtwp vaccine at - years of age (booster vaccination), following the national vaccination schedule of iran. blood samples were collected before and - weeks after the vaccination. immunogenicity of the vaccines was assessed by elisa using commercial kits. results: the geometric mean titers (gmt) of the antibodies induced against diphtheria and tetanus by dtwp-local were . and . iu/ml and those of dtwp-pasteur were . and . iu/ml, respectively. there was no significant difference between the immunogenicity of the two vaccines against diphtheria and tetanus. the gmts of antibodies produced against pertussis were . eu/ml for dtwp-local and . eu/ml for dtwp-pasteur vaccines, respectively (p x . ). no significant differences were observed in the antibody titers against diphtheria, tetanus and pertussis between the two vaccines before vaccination. conclusion: immunogenicity against diphtheria and tetanus was similar for the two vaccines pd / united kingdom haemorrhagic septicaemia (hs) is an acute disease of cattle and buffaloes in tropical countries, caused by pasteurella multocida serotype b: , a gram-negative coccobacillus. jrmt , an aroa mutant of pasteurella multocida, constructed previously in our laboratory, attenuated for virulence in the mouse and protects mice from challenge with the virulent strain. in this work, the immune response of calves was tested after intramuscular vaccination with single dose of cfu of jrmt . a possible contributory role of cellular immunity against hs was investigated in vaccinated and in control calves after challenge. a lymphocyte stimulation assay was used to assess the effects of a cell-free extract (cfe) of p. multocida on peripheral blood mononuclear cells (pbmcs) isolated from calves at different times after challenge. the results were indicative of a possible immunosuppressive effect of challenge with p. multocida b: on calf pbmcs. the suppressive effect was further investigated by in vitro experiments. calf pbmcs obtained from normal calves were treated with cfe for h before adding concanavalin-a (cona) pd -vaccination and immunotherapy against parasitic diseases pd / evaluation of simian adenoviral vector adch expressing msp- as a candidate blood-stage malaria vaccine this successful regime incorporated a human adenovirus serotype (adhu ) prime, boosted eight weeks later with a modified vaccinia virus ankara (mva) vector. adenoviral vectors have generated great scientific interest in recent years and appear to be superior viral vectors with great potential in vaccine regimes. their potential use in humans, however, is limited by natural anti-vector immunity to human adenoviruses, but this problem could be largely circumvented by the use of simian adenoviral vaccine vectors. recent clinical trials have suggested that the simian adenoviral vector adch is a promising clinical candidate. we have developed vectors (of human and simian origin) and mva encoding a novel construct based on p. falciparum msp- and have undertaken comparative immunogenicity studies in mice. the antigen, termed 'pfm while asymptomatic per se, the heterozygous sickle cell trait confers a survival advantage against malaria, the disease caused by plasmointo carbon monoxide (co), iron and biliverdin. when infected by plasmodium hb sad mice are protected against experimental cerebral malaria (ecm), a lethal neuroinflammatory syndrome that in many aspects recapitulates human cerebral malaria. ho- expression and activity are strictly required to suppress ecm in hb sad mice, as demonstrated by functional deletion of the hmox locus or pharmacologic inhibition of its enzymatic activity. the protective effect of ho- is mediated by co, which inhibits the accumulation of protein-free heme in plasma following plasmodium infection conclusion: topical treatment of cutaneous leishmaniasis with gsno accelerated healing and reduced local parasitism in the mouse suggesting that it may be ben gp expression was confirmed by sds-page and elisa using monoclonal antibody against gp . discussion: today researchers attempt to find a suitable vaccine for leishmaniasis. although some researchers have reported proper vaccines of interest, a - recp is recognized only by sera collected from resistant bovines infested with all stages of r. microplus, but not by sera from similarly infested, susceptible hosts. furthermore, this recognition was specific since sera from resistant non-infested bovines (naï ve animals) did not react with a - recp. our results show that reverse immunogenomics can be useful for discovery of new antigens for development of an anti-tick vaccine. supported by cnpq and fapesp. for the maintenance of ab-mediated vaccine-induced protection after re-challenge with the pathogen or the vaccine antigen. memory b-cell elispot together with ab titres might therefore prove useful as independent marker for duration of protection. objective: this study focused on establishing experimental conditions and optimizing the performance of the memory b-cell elispot assay by detection of specific memory b-cells against anti-tetanus vaccine and naturally acquired toxoplasma gondii infection as a model. methodology: twelve healthy subjects who had received the tetanus vaccine at least month previously were enrolled. peripheral blood mononuclear cells (pbmcs) were isolated from each donor using cell preparation tubes (cpt). plasma was obtained after centrifugation of cpt and stored at - °c until used for elisa. specific igg-secreting b-cells were determined by elispot assay, using tetanus toxoid (tt) and t. gondii surface antigen as model antigens. results: to optimize our assay, conditions were changed and compared to the previously established protocol. we detected low frequencies of total igg memory b-cells and tt-specific memory b-cells in all donors four seropositive and seronegative donors had positive responses in elispot. no correlations were found with serum antibody titers and frequencies of memory b cells (r= . , p= . ) or with t. gondii-specific b-cells conclusions: following optimization of several assay parameters, we demonstrated that the memory b cell elispot could be reliably used to determine low numbers of antigen-specific memory b-cells in individuals naturally exposed to infection or following vaccination our previous work demonstrated that il- also affects the cells of erythroid lineage, by stimulating development of early erythroid progenitors, bfu-e, but inhibiting the growth of late stage erythroid progenitors, cfu-e, from normal murine bone marrow. we also provided in vitro evidence that at least part of its effect on cfu-e is mediated by nitric oxide (no) generation. in the present study we demonstrated that the in vivo reducing effect of il- on bone marrow cfu-e was prevented by co-treatment with the no synthase (nos) inhibitor, l-name, implying that this effect is mediated through nos activation. the data obtained in cultured bone marrow cells showed the ability of il- to upregulate the expression of mrna for both the inducible (i)nos and the constitutive, endothelial (e)nos isoform. both the nos-inducing effect of il- and il- -related inhibition of cfu-e growth were dependent on p mapk activity, since the p mapk inhibitor, sb , markedly downregulated il- -induced activation of nos and reversed the growth inhibitory effects of il- on cfu-e. the in vivo stimulating effect of il- on bfu-e colony growth in the bone marrow was not affected by co-treatment with the nos inhibitor, pointing to different mechanisms for il- effects on bfu-e and cfu-e. however, the in vivo exposure of the mice to l-name, increased the number of various hematopoietic progenitor cells in the bone marrow, indicating that no itself is important regulator of hematopoietic progenitor cell activity. overall, the data presented gave an insight into the mechanisms by which il- acts on bone marrow cells and also revealed a link between the il- , no and hematopoiesis. further studies on il- -mediated induction of both inos and enos methods: a total of blood donor samples were tested for hbsag and anti-hbc with the immunoenzymic method elisa, while simultaneously, molecular blood test (nat) was applied. the positive samples for anti-hbc were also tested for anti-hbs and anti-hbc igm. results: a total of samples ( , %) were found anti-hbc positive conclusions: it is proven, therefore, that in some cases the levels of hbsag, following an infection from the hepatitis b virus, are probable to remain low, so that it is not possible to detect them using elisa method. in these cases anti-hbc can be the only serological marker of the infection. consequently, patients with positive anti-hbc and levels of anti-hbs x iu/l are possibly not immune enough, so that they can become blood donors. that was the reason why some blood donation centers in our country, until recent years when there was no capability for nat testing of blood donors, had iu/l as a limit for anti-hbs levels. however, in present days that nat testing of blood donors is used in our country, it has offered great safety and it is possible that anti-hbc testing will not be necessary, despite the fact that many blood donor centers have preserved the safety limit of iu/l anti-hbs in all the blood units, which also goes for our study pd / neonatal allo immune thrombocytopenia and igg glycosylation patterns michaelsen , national institute of public health in milder cases it can cause petechia and in more severe cases it can cause intracranial hemorrhage and death. the reason behind the variation in clinical symptoms is not fully understood, but is probably not due to differences in immunoglobulin isotypes or antibody affinity. recently influence of glycosylation patterns of igg on the biological activity has been realized. variation in carbohydrate structures attached to asparagine can cause differences in the interaction with fc-receptors, and hence a difference in thrombocyte elimination capacity of the igg molecule. patient sera from norway and the netherlands with different levels of antibody titres and severity of symptoms have been used to affinity isolate igg antibodies against the hpa- a alloantigen and analyze the glycopeptides using mass spectrometry. the glycosylation patterns have been analyzed for a possible link between severity of symptoms and variation in the glycosylation patterns. so far patients with serious symptoms seem to have increased galactosylation and sialylation and a high level of non core-fucosylated n-glycans on their anti-hpa- a iggs we monitored children ( boys and girls) in ages from . to . years with average age of . years. in of them all was diagnosed for the first time. subject had the second relapse of all. one patient received maintenance chemotherapy, all the rest ( subjects) induction chemotherapy. methods: leukocyte count and hemiluminescent analysis of whole blood were performed for all the patients during infectious complications twice: on neutropenia background and after the recovery of neutrophil number. hemiluminescent analysis for whole blood allows to estimate the functional activity of phagocytes, namely their bactericidal power and phagocytosis completeness. we valuated spontaneous and zymosan induced hemiluminescence. we used onsonised zymosan as the inductor of "respiratory paroxysm mice with a homozygous mutation in the rc h gene, that encodes the zinc finger and ring finger containing protein roquin, develop severe autoimmune disease. the observed lupus-like phenotype involves follicular helper t cells, which express higher levels of icos. these cells provide inappropriate t cell help to b cells, leading to the production of autoantibodies (vinuesa et al. , nature , - ). it has been shown that the half-life of icos mrna is shortened when roquin is over-expressed. such repression requires the 'utr of icos, in which a bp sequence, containing a possible mir- binding site, was sufficient (yu et al. , nature, , - ). mnab is the paralogue of roquin, and has been shown to bind to nucleic acids (siess et al. , j biol chem , - ). we demonstrate that in primary mouse t cells and embryonic fibroblasts roquin, but not mnab, inhibits translation of icos. we map critical domains in the roquin protein to icos repression using deletion-and point-mutants of roquin, as well as chimaeras that swap sequences from roquin to mnab and vice versa. addressing the mechanism of roquin mediated icos repression; we demonstrate binding of roquin to icos mrna in primary mouse t cells and in cotransfection experiments. our current work dissects the requirement of cellular rnai, the stress response pathway or p-body function by testing roquin repression of icos mrna in dicer-, tia- -and ago -deficient mef cells and in knockdown approaches. acknowledments: j m m-v is a recipient of a harvard real colegio complutense (rcch) grant. work in dr tsokos' lab is supported by grant phs nih r ai . we have recently reported that -hydroxyl- -methylindole- -acetonitrile ( -hma) isolated from brassica rappa inhibit nuclear factor-kappa b (nf-xb) activity in raw . macrophages. in this report, we investigated the effect of -hma on dextran sulfate sodium (dss)-induced colitis model in mice. methods: we induced colitis with dss in mice and evaluated disease activity index (dai), including body weight, stool consistency and gross bleeding, and tissue myeloperoxidase (mpo) accumulation. through h&e staining, histological change was observed. the expression of inducible nitric oxide synthase (inos), inhibitory kappa b-a (ixba) and nf-xb were detected by western blot and immunohistochemical staining. in-vitro system, the expressions of interleukin- (il- ), monocyte chemotactic protein- (mcp- ) in ht- human colon epithelial cells were measured by rt-pcr. results: in dss colitis model, the dai score and detection of mpo accumulation brevealed -hma significantly inhibited loss of body weight, suppression of diarrhea and bleeding, and infiltration of macrophages, leukocytes. moreover, h&e staining also indicated -hma suppressed the thickness of muscle layer, edema, mucosal damages by dss. these results were related to the regulation of nf-xb activation. -hma attenuated the dss-induced phosphorylation and translocation of nf-xb subunit p . in addition, this effect was accompanied with parallel blocking degradation of ixba. moreover, pretreatment of -hma significantly reduced the mrna levels of il- and mcp- stimulated by tumor necrosis factor-a (tnf-a) in the ht- cells. pretreatment of -hma also significantly blocked the ixba degradation and nf-xb p nuclear translocation stimulated by tnf-a in the ht- cells. these results were concurred with the effect on nf-xb activation in dssinduced colitis model. conclusions: these results for the first time demonstrated that alleviation of -hma mediated by regulation of nf-xb activation and suppression of chemokines in vitro and in vivo. therefore, -hma could be new potential therapeutic agent for inflammatory bowel disease.cd serves as receptor of hiv and is a self-antigen. we have previously characterized the anti-cd igg immune response in hiv- -exposed, seronegative (esn) subjects and we know that there is a peculiar specificity of these antibodies for epitopes induced by gp -binding and that there is an epitope specificity distinct from that seen in hiv-infected patients (second cd domain preferred). to generate antibodies able to inhibit the infection of hiv virus trying to learn from what happen in nature in esn we used a particular immunization procedure. we immunized mice with autologous cells expressing gp , reacted with the external domains of soluble human cd , in the absence of the target cells expressing the co-receptor ccr . the latter is the membrane molecule, which allows the complete reshuffling of the epitopic make-up of the cd -gp complex and trigger the membrane fusion between effector (gp expressing) cells and target (ccr expressing) cells. thus, in the absence of ccr we specifically enriched our immunogens with "frozen" conformational intermediates, that are presumably transiently exposed on the cell membrane during hiv- infections. a conventional protocol for the generation of monoclonal antibodies was used. db- (igg , x), one of the anti-cd antibodies obtained, recognized preferentially cd complexed to gp , as compared to cd alone, not competed for the gp binding site on cd and was specific for the second extracellular domain of cd . g. röder , l. geironson , a. darabi , m. harndahl , c. schafer-nielsen , k. skjödt , s. buus , k. paulsson copenhagen university, institute of international health, immunology and microbiology, department of experimental immunology, copenhagen, denmark, lund university, immunology bmc d , lund, sweden, lund university, rausing laboratory, division of neurosurgery, department of clinical sciences, lund, sweden, schafer-n, copenhagen, denmark, department of immunology & microbiology, university of southern denmark, odense, denmarkcytotoxic t-lymphocytes become activated by binding to mhc-i molecules presenting antigenic peptides. the loading of peptides onto mhc-i takes place in the er and involves different chaperones and enzymes. tapasin binds mhc-i molecules, integrates them into peptide-loading complexes, and assures that only 'optimal peptides' are bound to surface exported mhc-i molecules. how tapasin exerts this quality control, and the criteria for being an optimal peptide, are still unknown. here, we have generated the first n-terminal amino acids of human tapasin, tpn , and shown that this fragment of tapasin facilitates peptide dependent folding of hla-a* . to further investigate the properties of tpn and tapasin, we generated multiple mouse monoclonal antibodies towards tpn and wildtype human tapasin. one clone, atpn - / , was found to be specific for natural human tapasin and stained cellular er localized tapasin. using peptide chip technology, the epitope of atpn / was demonstrated to be located on tapasin [ ] [ ] [ ] [ ] [ ] , which recently was shown to be a surface-exposed loop of the tapasin structure. together, these results demonstrate that, the first n-terminal amino acids of tapasin are able to facilitate peptide-binding to mhc-i, and as well, this fragment can be recombinantly expressed in e.coli and fold into a structure, which at least partially, resembles that of wild-type human tapasin. we speculate that this region of tapasin might support empty, open and receptive mhc-i peptide-binding clefts effectively allowing an otherwise inherently unstable molecule to exchange peptide; i. e. this tapasin region might be essential for enabling peptide editing. a objectives: antigen processing and presentation through hla class i molecules is critical for an effective destruction of infected or transformed cells by cd + t lymphocytes. different intracellular routes governing the processing of endogenous and exogenous antigens have been described. we show here a strategy to introduce epitopes inside the cells for a productive cross-presentation to ctls. methods: to produce genetic in-frame tat fusion proteins, dna sequence encoding for the amino acid region - of the influenza a virus nucleoprotein (np) was inserted into the expression vector ptat-ha. starting from tatnpflu recombinant protein we produce hybrid proteins, in which the hla-b* -restricted np-flu epitope (aa - ) was replaced by hla-b or hla-a -restricted epitopes of ebv and hcv, respectively. cross-presentation was evaluated according to the standard cr release assay and through the ifn-g production. results: using hla-b or hla-a restricted viral epitopes we show that the two molecules cross-present the epitopes following two different pathways of processing: the hla-b molecules follow a proteasome-independent pathway which is active in different cell types, whereas the hla-a molecules present the epitopes in a classical proteasome-dependent pathway performed by dcs. furthermore, different hla-a restricted epitopes can be inserted in tandem and presented to the specific ctls without interfering each other. the data reported here offer new insights on how a same construct containing multiple epitopes from different viral or oncogenic proteins could be designed for vaccinal strategies. these findings also enlighten hla-b as a remarkable hla-class i molecule that, differently from hla-a , can present peptides through additional, unconventional antigen presenting routes. this could concur to an imbalance of the immunological properties of the hla-b molecules leading to a more effective response towards viral as well as self -antigens. objectives: although cytotoxic t cells (ctl) in human immunodeficiency virus (hiv- )-infected individuals can potentially target multiple virus epitopes, the same few are repeatedly recognized. ctl play a key role in limiting viral replication in infections caused by e. g. epstein-barr virus, cytomegalovirus, hepatitis c virus and hiv . consistent patterns of immunodominant and subdominant ctl-responses have been found between individuals with the same hla-alleles in both acute and chronic infection. as the ctl-response frequency in a population closely correlates with its relative magnitude in an infected individual, the terms immunodominance/subdominance have been used in both contexts. however, the factors determining these ctl-response hierarchies are largely unknown. while structural differences between peptide-hla class i complexes may be important for tcr-repertoire selection and clonal expansion, it is less obvious how they impact ctl-response hierarchy formation and timing. other factors may also contribute, e. g. epitope abundance at the cell surface. methods: antigen processing efficiency of ctl epitopes from the p -gag and p region was determined in vitro. mer peptides were digested with i s and c proteasomes and the fragments identified by mass spectrometry. for epitope precursor peptides generated by the proteasome, we then determined tap affinity, trimming by eraap and hla-binding affinities and analyzed patient responses by elispot. results: we show that ctl-immunodominance in regions of hiv- p -and p -gag correlates with epitope abundance, which is influenced strongly by proteasomal digestion profiles, transporter-associated-with-antigen (tap) affinity and endoplasmatic reticulum aminopeptidase (eraap)-mediated trimming, and moderately by hla affinity. proteasomal cleavage-preferences were affected by flanking and intra-epitope ctl-escape mutations and could modulate the number and length of peptide-epitopes, thereby affecting t cell response avidity and clonality. conclusion: our analyses reveal that antigen processing plays a pivotal role in determining ctl-response hierarchies, that viral evolution may modify cleavage patterns, and suggest strategies for in vitro optimization of ctl-epitope-based vaccines. t. f. gregers , g. koster , o. landsverk , f. skjeldal , o. bakke university of oslo, molecular biosciences, oslo, norway mhc ii is synthesized and assembles in the er together with invariant chain (ii). ii facilitates mhc ii assembly followed by transport to the mhc ii loading compartment (miic) where peptide loading occurs. miic is multivesicular late endosomal compartments resembling conventional multivesicular bodies (mvbs) found in all cells. it is not known whether the biogenesis of miics is regulated by the same mechanisms as formation of mvbs. expression of ii induces the formation of enlarged endosomes and we have previously shown that ii modulates antigen processing and presentation. we have suggested that ii itself can act as a tethering factor involved in fusion of ii containing endosomes, and our main question is whether ii can regulate the formation of an endosomal pathway dedicated for antigen processing and mhc ii loading.in order to investigate this we use cell lines expressing ii controlled by an inducible promoter, thus being able to control the ii expression level and thereby the endosomal size. live imaging and high through put microscopy of ii expressing cells treated with inhibitors and/or specific sirnas have revealed that ii induced endosomal fusion is independent on type iii pi kinases and thus ptdins( )p. this is in contrast to conventional endosomal fusion and mvb formation. thus other factors might be important for miic biogenesis. by using small rnai libraries targeting proteins known to be involved in endosomal pathways and microscope based screening we aim to identify factors that are able to knock out the formation of enlarged endosomes in ii expressing cells, and thus potentially identify molecules defining an antigen presenting cell. m. bouvier , l. visvabharathy , j. fu university of illinois at chicago, microbiology and immunology, chicago, united statesobjectives: adenoviruses (ads) cause persistent infections. the e - k protein from ad targets class i mhc molecules for retention in the endoplasmic reticulum (er), thereby preventing the cell-surface presentation of viral peptides. this escape from immune surveillance allows ads to freely replicate in host cells. the molecular mechanism of e - k-mediated class i retention is mostly undefined. it is clear that further characterization of this mechanism is important to understand the susceptibility of the class i antigen presentation pathway to immunomodulatory proteins and to elucidate the molecular basis of ad pathogenicity. we used biophysical and cell-based approaches to examine interaction between ad type e - k and class i molecules.results: we showed that e - k associates with immature (peptide-deficient) and mature (peptide-filled) hla-a molecules, with the mature form being more tightly associated. we also provided evidence that e - k does not compete with the class i assembly proteins for binding onto class i molecules. importantly, immature class i molecules sequestered by e - k can still bind peptides. together, these results suggest that ads have evolved to interfere with the early and late stages of the class i antigen presentation pathway. evidence was also provided that e - k displays an allele-and locus-specificity towards class i molecules with high-density lipoprotein (hdl) reduces the risk for atherosclerotic cardiovascular disease by promotion of cholesterol efflux from macrophage foam cells and by antioxidative as well as anti-inflammatory properties. recent data indicate that qualitative changes of hdl including oxidative modifications and alterations of the protein cargo of hdl may alter its biological activity. here we analyzed the anti-inflammatory potential of hdl and compared it with hdl obtained from patients with end-stage renal disease (esrd), which are characterized by a proinflammatory state and an associated significantly increased cardiovascular mortality. we demonstrate that freshly isolated, but not oxidized hdl from healthy individuals exerts profound anti-inflammatory properties on professional antigenpresenting cells (apc) such as monocytes and dendritic cells, which are regarded as the most potent apc. production of typical proinflammatory cytokines (il- , il- , tnf-a) were significantly suppressed by hdl after stimulation of monocytes or dendritic cells with toll-receptor ligands and , but also with the t-celldependent stimulus cd l (cd ) indicating an immunomodulatory effect independent of agonist neutralization by hdl. moreover, surface expression of crucial activation and costimulatory molecules like cd , cd , and cd was inhibited by freshly isolated, but not oxidized hdl. the negative regulatory effect of hdl on cytokines and surface receptors occurred at the transcription level, while hdl did not modulate the activity of the major inflammatory transcription factor nf-kb or the map kinases p and erk- / . strikingly, hdl from esrd patients not only failed to block, but rather promoted proinflammatory cytokine production and apc activation. these data identify hdl as a novel potent anti-inflammatory regulator of professional apc, which may help to dampen excessive inflammatory responses of the innate immune system. conversely, qualitative changes of hdl leading to a loss of its anti-inflammatory function might contribute to a proinflammatory state that is linked with excessive cardiovascular mortality in esrd patients. objectives: cd + t cell abnormalities may play a role in the autoimmune pathogenesis of churg strauss syndrome (css). on one side, th (il- +) cells may sustain autoantibody formation and eosinophilia, which are hallmarks of css. on the other, th (ifn-g+) cells could participate in vessel wall damage and granuloma formation. in order to define this th / th balance and to identify potential t cell target antigens (ags), we analyzed circulating cd + t cell responses to polyclonal stimuli and to myeloperoxidase (mpo) in css and healthy subjects. methods: ifn-g and il- expression in peripheral blood cd +cd + lymphocytes were measured in ccs patients and healthy subjects (hs) upon polyclonal stimulation, both by intracellular staining and by elisa. mpo-driven il- /ifn-g production was assessed by elispot on t cells co-coltured with autologous dendritic cells, stimulated either with heat-inactivated mpo, negative control protein or hexavalent vaccine (positive control recall ags). results: upon polyclonal stimulation, higher il- and lower ifn-g intracellular expression were detected in cd + t cells from css patients, as compared to hs (il- : . ± . % vs. . ± . %, p x . ; ifn-g: . ± . % vs. . ± . %, p x . ). similar results were obtained by elisa (il- : . ± . vs. . ± . pg/ml, p x . ; ifn-g: . ± . vs. . ± . pg/ml, p x . ). elispot counts of hexavalent vaccine-stimulated cd + cells were positive for il- in / ( %) css patients and in / ( %) hs, and for ifn-g in / ( %) css patients and / ( %) hs. mpo stimulation determined significant ifn-g release in / ( %) css patients, but not in hs ( / ) no il- response to mpo in both groups was observed. conclusion: polyclonally or recall ag-stimulated cd + cells from css patients show a th -polarized cytokine profile. mpo is here first identified as a css-related ag targeted by cd + t cells, and responses towards it are instead th -polarized. these data unfold one molecular target and possible pathogenic mechanisms of cd + t cells in css. a. voigt , e. opitz , k. savvatis , k. klingel , k. stangl , u. kuckelkorn , p.-m. kloetzel charité -universitätsmedizin berlin campus mitte, berlin, germany, charite -campus benjamin franklin, berlin, germany, universität tübingen, tübingen, germanymurine models of coxsackievirus b (cvb )-induced myocarditis mimic the divergent human disease course of cardiotropic viral infection. immunoproteasomes (ip) are crucial in the modulation of adaptive immune responses, in the maintenance of protein homeostasis and in the preservation of cell viability under stress conditions. our previous work has established that ip expression in the infected myocardium is linked to a strong enhancement of viral epitope generation.here, we investigated the impact of ip function in enterovirus myocarditis. mice, which are deficient in immunosubunit lmp of the stress-induced ip, were infected with x e pfu cvb nancy strain. in concurrence to wt littermates, we observed a pronounced up-regulation of cardiac ip subunit lmp as early as day p. i. in lmp -deficient mice. however, lmp -deficiency was linked to less severe myocarditis at day p. i. (he stain of cardiac tissue sections: wt . ± . vs. lmp -deficiency . ± . (grade of myocarditis; scale - ; p x . ). whereas the cardiac output (co) was reduced in wt littermates in enterovirusmyocarditis (p x . ), there was no difference in lmp -deficient mice in comparison to sham-treated mice. maximal left ventricular pressure and dpdt max were impaired in acute myocarditis in wt littermates. in contrast, systolic function was not affected by cvb infection in lmp -deficient mice. likewise, diastolic function was preserved in lmp -deficient mice upon enterovirus infection. our findings of less severe myocarditis in lmp -deficient mice were associated with tremendously reduced viral load in the myocardium of this strain.in conclusion, this study suggests an impact of lmp -immunosubunit function in regulatory processes of viral replication. absence of lmp confers host protection in enterovirus myocarditis. h. w. liao , j. xu , j.q. huang sun yat-sen university, guangzhou, chinathe characteristic of the dengue hemorrhagic fever/dengue shock syndrome (dhf/dss) is hematologic abnormality, which results from multiple factors including thrombocytopenia, coagulopathy and vasculopathy. the pathogenesis of endothelial dysfunction associted with vascular leakage syndrome however remains unknown. in this work, we showed that dengue virus serotype (den- ) strain induced apoptosis in human umbilical vein endothelial cells (huvecs). additionally, fas expression was increased on infected huvecs. trailr and tnfr - were constantly very low whereas trailr - decreased after den- infection. fasl was expressed at similar levels on huvecs throughout den- infection. the apoptotic rates in huvecs were decreased upon addition of caspase family inhibitors and activated caspase , caspase were also observed by western blot after by den- infection. there were no significant changes of no in our study. we thus proposed that the fas/fasl pathway might be involved in apoptosis induced by dengue virus in vascular endothelial cells in vitro. dermatolymphangioadenitis is a common complication of interruption of afferent lymphatics by cancer surgery combined with partial lymphadenectomy. it seems that skin microbes normally penetrating epidermis during hand work or walking are retained in the skin and subcutis because of lack of lymph drainage and evoke host reaction. aim. to study lymph node cellular reaction to bacterial antigens before and after ligation of afferent lymphatics. materials & methods. group i. s. epidermidis was injected daily for days into wis rat paw web tissue in saline containing . x cells. group ii. s.epidermis was injected as in group after ligation of lymphatics below the popliteal lymph node. nodes were isolated on day .they were weighed, the cell number was counted and cells were stained with mabs for immunohistochemical analysis. immunohistochemical pictures were analyzed by microimage program. results. group i. skin contained some mhcii cells. the popliteal lymph nodes became enlarged on the bacteria injected side. there was an increase in lymph node weight and cell concentration per g of tissue, compared to controls by factors , and , respectively (p x . ). immunohistochemical pictures showed increase in percentage of ox (migrating dendritic cell), mhc ii, his (granulocytes), ox (stem cells) and cd (icam i) subsets in the subcapsular , follicle, paracortex and medullary areas. group ii. after ligation of afferent lymphatics the weight of nodes was not significantly increased. skin showed presence of multiple mhc ii, ed (macrophages) and ox cells. popliteal lymph nodes contained evidently less of ox , his and mhcii cells than in group i (p x . ). summary & conclusions. afferent lymphatics transport microbial cells and/or microbes phagocytized by dendritic cells and macrophages to the regional node. local skin reaction is limited, whereas lymph nodes reveal acute reaction with mobilization of granulocytes from blood perfusing nodes. interruption of lymphatics saves nodes but skin reaction is strong and long-lasting. these observations seem to explain why damage to lymphatics during mastectomy or groin dissection is followed by recurrent attacks of skin inflammation. omega- fatty acids, and in particular docosapentaenoic acid (dha) and eicosapentaenoic acid (epa) from fish origin, have recently emerged as nutrients capable of modulating the expression of genes involved in inflammation and atherosclerosis and thus reduce the risk for cardiovascular events. our presentation focuses on the role of omega- fatty acids in the prevention and treatment of cardiovascular disease. it is based on reviewing and processing data obtained by search of scientific and medical databases. search terms used were: atheroma, atherosclerosis, cardiovascular disease (cad) ,coronary disease, antiinflammatory drugs, omega- fatty acids, epa, dha. we also searched epidemiological research web sites and screened the results of numerous controlled clinical trials which monitored the effects of omega - fatty acids consumption. the results indicate that omega- fatty acids supplementation is associated with a significant cardioprotection effect on both healthy individuals and patients with an established cardiovascular disease. omega- fatty acids appear to work by decreasing endothelial responsiveness to pro-inflammatory and pro-atherogenic stimuli, affecting molecular events not targeted by other drugs thus allowing their use as complementary treatments for the already implemented pharmacological treatments in inflammatory diseases. combined therapy with omega- fatty acids and statins shows a synergistic effect. methods: on ultracentrifugation of serum at density . and , g, top % layer contained lipoproteins only and - % layer contained lipoproteins as well as immunoglobulins. the bottom layer was shown to contain immune complexes (ic) by binding to coated anti apo(a) and detection with peroxidase labelled anti human immunoglobulins.both these forms of lp(a) were western blotted and probed with jacalin-hrp, anti-gal-hrp and anti apo(a)-hrp. anti-gal was prepared by affinity chromatography on guar galactomannan and complexed with lp(a) in vitro. ic formation by lp(a) was measured in terms of reduction in response in a new elisa for lp(a) involving addition of lp(a) sample to plate-coated jacalin, followed by anti-apo(a)-hrp detection. ic formation was also shown by migration of lp(a) from free lipid layer to ic layer below in ultracentrifugation. results: anti-gal and lp(a) could be liberated from precipitated ic using specific sugar. immune complexed lp(a) in serum was found to be more o-glycosylated, larger in size and binding more anti-gal than lp(a) in free form in western blots. while ic formations within homologous free anti-gal-free lp(a) pairs were few, those within heterologous pairs were more rampant. conclusions: lp(a) is a risk factor in vascular disorders including atherosclerosis, aneurysm, stroke and peripheral vascular diseases and is a component of atherosclerotic plaques, though mechanism of its uptake remains unclear. anti-gal comprising % of serum igg is rich in igg capable of complement fixation and macrophage mobilisation. present results offer a viable mechanism of lp(a)-mediated immune injury to vessel walls leading to vascular damage. even though no receptors have been detected for lp(a), unlike for ldl, the present results may explain the internalization of lp(a) in the form of lp(a) immune complexes by macrophages since the latter can phagocytose ic. extended specificity of the a-galactoside-specific anti-gal for t-antigen in lp(a) is akin to that observed in jacalin, pea nut agglutinin and galectin- . objective: the aim of this study was to determine if the combination of two genetic alterations, one affecting cell cycle regulation, such as the e f mutation, and other affecting b cell apoptosis control, such as bcl- over-expression, can induce the development of ais. methods: mice: mice with both genetic abnormalities were generated in a non-susceptible c bl/ (b ) genetic background. e f -/-bcl- tg were obtained backcrossing e f -/-and e f +/+hbcl- tg mice. e f -/-bcl- tg, e f -/-, e f +/+hbcl- tg and control mice (e f +/+) were followed up to mo-old. serologic studies: serum samples obtained at , and month of age were test for igg and iga ana and anti-dsdna by elisa. histopathologic studies: kidney paraffin sections of , and mo-old mice were stained with hematoxylin-eosin (h&e) and masson's trichrome to identify histological changes. immunecomplex deposits were studied by direct immunofluorescence on kidney using fluoresceinated goat anti-mouse igg, igm and iga. to evaluate b cell homeostasis, absolute number of b cell in blood, primary and secondary lymph organs were assessed by flow cytometry. in vitro proliferation was measured with [h ]-thymidine and brdu was used to assess in vivo proliferation capacity of immature b cells. results: overexpression of hbcl- tg in b lymphocytes of e f -/-mice induced the production of high titres of igg and iga ana and anti-dsdna, together with the development of a glomerulonephritis characterized by a moderated mesangial proliferation, mesangial immunecomplex deposits, mainly of the iga isotype, and the presence of tubular casts and lymphoid infiltrates with the presence of glomerular deposits. e f -/-bcl- tg mice showed an altered b cell homeostasis as demonstrated in proliferation and apoptosis studies. e f -/-mice showed neither autoantibodies nor nephropathy. this study demonstrates that the isolated deficiency of e f or the overexpression of a bcl- tg in the b genetic background do not induce an ais. when combined both genetic alterations, involving deregulation of cellular proliferation and survival affect lymphocyte homeostasis, induce a mild ais with overproduction of iga autoantibodies. an alteration in the b cell compartment, but not in the t cell compartment, seems to be underlying the syndrome described in the present work. in different mouse models of the autoimmune disease systemic lupus erythematosus (sle) loss of toll-like receptor (tlr ) abolishes the generation of antinucleosome igg a and igg b autoantibodies but exacerbates lupus disease. however, the tlr -dependent tolerance mechanism is unknown. here we show that loss of tlr in b cells of lupus prone mice prevents the generation of protective t cell-dependent self-reactive igm and thereby enhances the development of th and th t cells. transfer of a synthesized monoclonal polyreactive igm to tlr deficient lupus prone mice inhibits t cell activation and abolishes development of lupus disease. thus, these results document a protective tlr -dependent tolerance mechanism in b cells that induces the generation of self-reactive igm to prevent autoimmunity. cloning and production of polyreactive or antigen-specific igm might therefore be a powerful tool to treat autoimmunity. objectives: to investigate cytokine and autoantibody levels in serum from patients with primary sjögren's syndrome (pss), and to determine possible associations with focal mononuclear cell infiltrates, lymphoid organization, and age at the time of biopsy. methods: minor salivary gland tissue was obtained from a group of patients fulfilling the revised eu-us criteria for pss (n= ) (vitali et al. ) . ninety-seven of ( %) patients had focal mononuclear cell infiltrates corresponding to focus score (fs) g (fs+), while biopsies from / ( %) patients lacked characteristic focal mononuclear cell infiltrates (fs-). germinal center (gc)-like lesions were determined in / ( %) minor salivary gland biopsies. serum samples were used for cytokine and autoantibody evaluations. the mean level of unstimulated whole saliva was significantly lower in the fs+ patients compared with the fs-patients, and in the gc+ patients compared with the gc-patients (p x . ). interleukin (il) , il- ra, il- beta, il- p , il- , macrophage inflammatory protein (mip) alpha, mip- beta, eotaxin, interferon (ifn) alpha, and il- levels were significantly increased in the gc+ patients (n= ) compared with the gc-patients (n= ). in addition, minor differences in cytokine levels were found when comparing age groups. degenerative changes such as atrophy/fibrosis and fatty cell infiltration observed in the minor salivary glands of patients with pss may represent "burned out" inflammation. no significant differences were found in autoantibody levels in either of the groups, nor when comparing cytokine levels in the fs-and fs+ subgroups. the reduced salivary flow observed in gc+ patients may be influenced by the elevated levels of il- found in these patients (gao et al. ) . increased titers of th -associated cytokines, il- , il- beta and the il- subunit il- p , may indicate a higher activity of these cells in gc+ patients (nguyen et al. ) . differences in cytokine levels may be utilized when sub-grouping the ss patients into disease phases and may consequently have implications for treatment. objectives: c-reactive protein (crp) is an acute phase protein, produced by hepatocytes in response to the pro-inflammatory cytokine il- . the rapid increase of crp during inflammation makes it an excellent inflammatory marker, but for unknown reasons, blood levels of crp typically remain low in disease flares of systemic lupus erythematosus (sle), a systemic autoimmune disease. another feature of sle is the so called 'interferon (ifn) signature' which implies high levels of ifn-alpha and/or up-regulation of ifn-alpha related genes. ifn-alpha has a wide spectrum of immunomodulatory functions but is mainly known for its antiviral and anti-tumour effects. since high levels of ifn-alpha coexist with a muted crp response in sle disease flares and in viral infections, we hypothesized that ifnalpha inhibits crp synthesis. methods: crp promoter activity was studied in a crp-promoter and luciferase reporter transfected human hepatoma cell-line, hepg . production of the acute phase protein serum amyloid a (saa) and the negative acute phase protein transferrin were analysed by elisa as reference. results: the crp-promoter activity was inhibited by all ifn-alpha subtypes. mixes of type i ifns that were induced by sle-like immune complexes or virus also inhibited the crp-promoter activity. virus-induced purified leukocyte ifn-alpha had the most prominent inhibitory effect ( g %) on crp promoter activity. saa synthesis was inhibited by ifn-alpha in a similar fashion as for crp promoter activity, whereas transferrin was unaffected. conclusion: our data indicates that ifn-alpha is an inhibitor of crp-promoter activity. we suggest that this could explain the muted crp response seen in sle disease exacerbations. further, i may contribute to differences in crp response between viral and bacterial infections. background: b cell activating factor of the tnf family (baff) is an essential b cell survival and maturation cytokine. mice overexpressing baff (baff tg mice) develop lupuslike autoimmunity, b cell hyperplasia, and lymphomas. autoimmunity in these mice involves proinflammatory autoantibodies driving nephritis and sialadenitis, and was previously found to be t cell-independent (ti) and myd -dependent. this suggested the involvement of transmembrane activator and caml interactor (taci), which is a receptor for baff that is essential for ti immune responses and is upregulated by myd -dependent tlr activation. we assessed the role of taci in baff-driven ti autoimmunity. methods: we tested the importance of taci in ti autoimmunity by generating baff tg bone marrow (bm) chimeras reconstituted with taci -/or taci +/+ bm then comparing their disease severity by flow cytometry, autoantibody elisa, immunofluorescence microscopy for ig deposition. results: as expected, baff tg chimeras reconstituted with taci +/+ bm produced high levels of circulating proinflammatory autoantibody isotypes and rheumatoid factors (rhf), and ig deposition in the kidneys and salivary glands was observed. by contrast, baff tg chimeras reconstituted with taci -/-bm had greatly ameliorated levels of circulating proinflammatory autoantibodies, rhf, and ig deposition. b cell hyperplasia was greater in taci -/- baff tg chimeras. defects in the regulation of apoptosis contribute to the pathogenesis of human systemic lupus erythematodes (sle). autoantigens not being properly removed and thus exposed to the immune systeme might lead to the emergence of autoantibodies. physiologically apoptotic cells are removed without initiation of an inflammatory immune response and myeloid dendritic cells are believed to actively tolerize t-cells after phagocytosis of apoptotic material. these processes of silent apoptotic cell clearance seem to be disturbed in sle patients. a characteristic of apoptotic cell death is the shedding of membrane coated vesicles from the cellular surface (apoptotic cell blebbing). these microparticles have been recognized as mediators of intercellular communication. therefore, we were interested whether apoptotic cell derived microparticles can influence the function of monocyte-derived dendritic cells and whether those interactions might play a role in the pathogenesis of human sle. we observed an engulfment of microparticles by monocyte-derived dendritic cells. further, apoptotic cell-derived microparticles stimulated differentiation of immature dendritic towards a mature phenotype. however, microparticles caused a remarkable downregulation of mhc class ii molecules. further, we observed only a minor release of proinflammatory cytokines from monocyte-derived dendritic cells pulsed by membrane microparticles when compared to lps stimulated dendritic cells. finally, these dendritic cells pulsed by membrane microparticles did not cause a significant t-cell expansion. interestingly, dendritic cells obtained from sle patients showed significant variations in phenotype and cytokine secretion compared to normal healthy donor cells with absence of the mhc class ii downregulation and a higher constitutive secretion of il- . objectives: increased levels of il- , an innate and inflammatory cytokine of the il- family, can be detected in serum and organs of human autoimmune pathologies, as well as in autoimmune animal models. here, expression of il- and other genes of the il- /il- r families was examined in human systemic lupus erythematosus (sle) and in mrl lpr/lpr mice, which develop a chronic progressive lupus-like syndrome. methods: serum, urine, and monocytes were collected from patients and healthy controls. lymphoid (lymph-nodes, spleen, thymus, peyer's patches) and non-lymphoid organs (kidney, lung, liver, salivary and lacrimal glands) were collected from mrl +/+ and lpr/lpr mice of different ages. il- and il- bp were measured by elisa. gene expression was assessed by real-time pcr and expressed relative to b-actin. results: in sle, serum and urine levels of total and free il- are higher than in controls. serum il- correlates with disease activity and decreases upon remission. monocyte expression of the receptor il- rb is increased and correlates with disease severity, while expression of tir /sigirr (a down-regulatory receptor of the il- r/il- r family) is reduced. in mrl lpr/lpr mice, expression of il- , caspase- and il- rb genes precedes disease onset in lymph-nodes. in other organs, changes in il- -related genes (il- and tigirr- up-regulation, tir /sigirr down-regulation) occur after disease onset. free il- levels are abnormally high in lpr/lpr lymph-nodes before disease onset, while in other organs the increase occurs with disease. conclusions: free il- levels correlate with autoimmune lupus both in mice and humans. free il- may be pathogenic in murine lymphadenopathy, while is a disease correlate in lpr/lpr and a severity correlate in sle. both in human and mouse syndromes, upregulation of il- rb is a marker of pathology, suggesting increased il- -dependent activation. both in mouse organs and human monocytes, tir /sigirr expression decreases with disease, suggesting impaired control of il- r activation. thus, il- may be involved in autoimmune lupus pathology, and il- -related molecules can be both original diagnostic markers and novel therapeutic targets in autoimmunity. in this study we compared the epitope specificity of anti-topo i autoantibodies present in sera of dcssc, lcssc and sle patients. we have constructed an antigen fragment library displayed on bacteriophage lambda and screened this library with igg purified from patients' sera. regions of topo i selected from the library were expressed as recombinant fusion proteins and were tested with elisa and western blot. we unexpectedly found that antibodies against a fragment of topo i (fragment f (amino acid (aa) - ) could be detected in sera of healthy individuals and patients with inflammatory rheumatic diseases other than ssc and sle. using sera of dcssc, lcssc and sle patients we showed that the pattern of recognized epitopes is different between these patient groups. fragment f was recognized by all patients. fragment f (aa - ) was recognized by of dcssc patients. fragment f (aa - ) was recognized by of sle patients. analysis of clinical data revealed a significant difference between the f negative and f positive groups of ssc patients in age and in the duration of the disease. according to our results the newly identified fragments f and f could represent characteristic epitopes for dcssc and sle, respectively. background: previous studies demonstrated that depletion of regulatory t cells (tregs) results in autoimmunity in mice while their adoptive transfer prevents autoimmune diseases. studies performed by us and others showed that in human connective-tissue diseases a reduced number of tregs exists and this abnormality seems to be correlated with autoantibodies production and disease activity. objectives: based on these observations and the fact that rapamycin (rapa) has the ability to expand tregs and to induce anergy, we proposed to study the possibility to restore peripheral tolerance of cd + t cells isolated from systemic lupus erythemaosus (sle) patients by ex vivo expansion of tregs. methods: pbmcs or peripheral cd + t cells from sle patients were cultured in the presence of specific stimulation with or without rapa and ril- . by facs the initial percent of tregs and after expansion protocol were determined. in order to verify the suppressive capacity of expanded tregs, cd + t cells enriched in tregs were co-cultured with activated cd + effector t cells (teff) stained with cfse, after one week teff cells proliferation was measured by facs. additionally, cytokine and igg release in cell culture media were analyzed by multiplex and elisa, respectively. expanded cd + t cells anergy was also evaluated based on cbl-b, grail and foxp mrna by realtime rt-pcr. results: in vitro expansion of tregs was more efficient when the starting cells were cd + t cells. the presence of rapa during expansion protocol significantly increased the number of tregs. sle tregs cells expanded in vitro in the presence of rapa had the capacity to suppress proliferation of both sle and hd teff cells. rapa inhibits igg secretion in the pbmcs culture, inhibition dependent on tregs level. rapa during tregs expansion protocol stimulated some type of cytokines while suppressed others. rapa had the capacity to re-establish sle cd + t cells anergy by induction of anergy genes, grail and cbl-b. conclusions: our data show that the above described protocol permits ex vivo tregs expansion and that suppressive capacity of the expanded tregs depends on the source of both tregs and teff cells. in this study, we look for a more specific approach to remove b- cells through targeting p d by shrnas strategy. methods: we used the drugs, ly and wortmannin, pan-specific inhibitors against pi ks. then we designed shrnas carried by the lentiviral system and validated that several segments of them can sufficiently knock down the expression of p d. we then introduced either pan-specific inhibitors against all pi ks or p d-targeting shrnas into an sle-prone animal model, nzb/w f mice, for therapeutic purposes. the results suggested that pi ks are not only important for the development of b- cells but also remain essential to maintain their population after birth. shrnas carried on lentiviral systems were designed to knock down the expression of p d. either pan-specific inhibitors against pi ks or p d-targeting shrnas were introduced into the sle-prone animal model, nzb/w f mice. one inhibitor, ly , and shrnas delivered by low dose of lentivirus exhibited certain potential to retard the rising of anti-dna auto-antibodies and prolonged the life span. conclusions: our findings are promising for developing treatments for sle. moreover, knowing pi ks are critical for the maintenance of b- cell populations might shed light on future treating other diseases associated with b- cells, such as certain melanoma, lymphoma, or leukemia. a. m. zaghlool , m. alarcón-riquelme , s. kozyrev institution of genetic and pathology, uppsala university, uppsala, swedenrecently, we discovered that the bank gene, which plays a role in b cells activation pathway, is associated with systemic lupus erythematosus through a nonsynonymous substitution g/a (rs , r h). we identified that bank gene expresses two alternatively spliced isoforms, a full-length, and a shorter isoform that lacks exon (delta ). the two isoforms were detected differently in susceptible lupus patients depending on the presence of a risk haplotype. to address the question of how bank is spliced and what are the signals governing the expression of each isoform, minigenes with different genetic variants were constructed and the expression of the bank isoforms were tested in vitro. qpcr analysis revealed that, another t/c snp (rs ), which is in complete ld with r h snp and located in the putative branch point, has a strong affect on the isoforms expression levels. deletion of a polypyrimidine (py) stretch downstream of the skipped exon produced a dramatic decrease in the full-length expression levels, probably due to the loss of the binding site for protein tia , which bind to t objectives: cerebral ischemia is the most common presentation of antiphospholipid syndrome (aps), but several other neuropsychiatric features, including chronic headache, dementia, cognitive dysfunction, psychosis, depression, transverse myelitis, multiple sclerosis-like disease, chorea, and seizures have been associated with the presence of antiphospholipid antibodies (apl). we report the case of a subject with atypical movement disorder related to aps successfully treated with oral anticoagulation agents. case report: a -year-old woman with a previous history of recurrent foetal losses was admitted to our hospital due to cognitive dysfunction and headache. she presented involuntary movements that were characterized as mioclonic seizures and tonic spasms lasting from few minutes to several hours, followed by bilateral arrhythmic rapid purposeless jerks of the legs. mild executive dysfunction was observed. her deep tendon reflexes were symmetric and normal. pathological reflexes were absent. biochemical analysis, renal, hepatic and thyroid functions were preserved, prothrombin time and partial thromboplastin time were all normal. the immunoglobulin g (igg) isotope of anticardiolipin antibody (acl) was elevated, whereas igm isotype and anti- gpi antibodies were undetectable. the lupus anticoagulant (la) was negative such as antinuclear antibodies (ana). no evidence of epilepsy was revealed from electroencephalogram or signs of denervation from electromyographic studies. brain magnetic resonance imaging (mri) showed multifocal encephalomalacia probably linked to previous cerebrovascular accidents. she was diagnosed as having an atypical neurologic manifestation probably linked to aps. she was thus discharged with a low-molecular-weight heparin therapy subsequently changed to mild oral anticoagulation . the therapy leads to a late, gradual improvement of symptoms that persisted at the last year follow-up evaluation. conclusions: antiphospholipid syndrome may constitute a rare but treatable cause of atypical neurologic manifestation such as myoclonic movements. due to the possibility of an effective treatment, it is important to rule out this diagnosis, moreover in women with other associated features of aps (foetal losses, livedo reticularis, thrombosis). a.-s. korganow cnrs , strasbourg, france b lymphocytes from patients with systemic lupus erythematosus are hyperactive and produce autoantibodies. several b cell phenotypic characteristics have been reported, as the expansion of activated populations, and of a newly investigated memory compartment. a few genes have been suggested to be implicated. one of the thing that makes these results difficult to interpret is the heterogeneity of the lupic disease, and sometimes the analysis all together of quiescent, paucisymptomatic and highly symptomatic patients, treated with immunosuppressors or untreated.we made the postulat that "intrinsic" abnormalities of b cells could be a common point in very quiescent patients. we choosed patients, with minor clinical and/or biological manifestations of their disease, for at least monthes. known of them received immunosuppressive drugs since this period. the mean sledai score was below . b cell surface markers expression was determined by flow cytometry. we analysed most of the already described and phenotypically distinctive b cell populations. we confirm the presence of activated b cells even in quiescent patients. we do not confirm the significant increase of a specific memory b cell compartment. above all, we described a decreased expression of the cd surface protein for all patients. this cd lower expression is associated with cd lower levels. it is not associated with an evident gene expression alteration and in vitro stimulation restores a control phenotype. these findings suggest some mechanisms in lupus genesis. objectives: tgf-beta is a pleiotropic cytokine with wide ranging effects in proliferation, differentiation, immune suppression and apoptosis. recent work from our group has shown that tgf-beta signalling in t-cells is protective in a mouse model of colitis associated cancer. smad ubiquitin regulating factors (smurf) are ubiquitin ligases that are involved in the regulation of tgf-beta signalling. the aim of this study was to determine the function of smurf expression in t-cells on the pathogenesis of experimental colitis associated colon cancer. methods: we could isolate a known splice variant of smurf lacking an exon in the c -domain. to analyse whether this form has a regulatory role in colon associated cancer we generated a transgenic mouse strain that overexpresses smurf in t-cells. smurf expression were analysed by qpcr. wild type (wt) and transgenic (tg) mice were treated once with the mutagenic agent azoxymethan (aom) followed by three cycles dextran sodiumsulfate (dss). after each cycle, the inflammation of the gut and the tumor growth and size of every mouse were monitored by colonoscopy. results: smurf expression was upregulated by tgf-beta stimulation in t-cells and smurf was markedly upregulated in tumor infiltrating cd + lymphocytes in aom/dss treated mice. whereas wt mice suffered from severe colitis resulting in colon tumors beginning at day , smurf transgenic mice had less colitis and were significantly protected from tumor development. interestingly, t-lymphocytes overexpressing smurf showed an upregulation of the tgfbrii and an activation of smad and as compared to wild-type t-lymphocytes, which were previously described as typical smurf targets for degradation. in addition the transfection of smurf and a caga-luc plasmid into cos-cells for smad -promotor studies yielded the same effect as shown by upregulation of the smad activity. conclusion: although, wt-smurf has been described as a negative regulator of the tgf-beta signalling pathway, our data show surprisingly that a smurf splice variant upregulates the tgf-beta receptor expression and increases tgf-beta signalling effects. due to immunosuppressive effects on t-cells smurf has beneficial effects on mucosal inflammation and tumor development. smurf thus emerges as an attractive target for modulation of chronic intestinal inflammation and colitis associated carcinogenesis. the transcription factor stat has important functions in cytokine signalling in a variety of tissues. however, the role of stat in the intestinal epithelium is not well understood. we demonstrate that development of colonic inflammation is associated with the induction of stat activity in intestinal epithelial cells (iec) both in humans and in mice. studies in genetically engineered mice showed that epithelial stat activation in dss colitis is dependent on il- rather than il- . il- was secreted by colonic cd c+ cells in response to toll-like receptor stimulation. conditional knockout mice with an iec specific deletion of stat activity were highly susceptible to experimental colitis, indicating that epithelial stat regulates gut homeostasis. stat iec-ko mice, upon induction of colitis, showed a striking defect of epithelial restitution. gene chip analysis indicated that stat regulates the cellular stress response, apoptosis and pathways associated with wound healing in iec. consistently, il- and epithelial stat was found to be important in wound-healing experiments both in vivo and in cell culture experiments in vitro. in summary, our data suggest that intestinal epithelial stat activation regulates immune homeostasis in the gut by promoting il- -dependent mucosal wound healing. stat seems dispensable for gut homeostasis under steady state conditions, but is activated upon challenge to drive tissue regeneration and protection in situations of increased demand, as during colitis and injury. map and ma infection induced an increase in both cd and tlr expression at day and day after infection. mycobacterial infection did not result in differential tlr expression as compared to uninfected cells. cd is involved in stimulating th pro-inflammatory responses, although map may interfere with cd signalling ( ) . tlr signalling elicits anti-inflammatory responses, which can contribute to bacterial replication ( ) .in conclusion, monocyte-derived macrophages from crohn's disease patients show an increase in cd and tlr receptor expression in response to both map and ma infection. as ma is a known human pathogen of immunocompromised hosts, this findings further support a role for map in the immunopathology of crohn's disease. objectives: for our understanding of the pathogenesis of human ibd, animal models of intestinal inflammation are indispensable. most of them are based on a compromised intestinal barrier, and a deregulated immune response against components of the flora is considered to be critically involved in the development of ibd. the occurrence of extraintestinal manifestations suggests that cross-reactions against hitherto undefined auto-antigens could be responsible for the activation of the adaptive immune system. to further dissect the pathophysiological mechanisms responsible for initiation and progression of ibd and associated extraintestinal manifestations, we established a new antigen-specific model, in which the local activation of cd t cells by exogenous antigen leads to colitis. methods: eight million naïve cd + ot-i cells, transgenic for a t-cell receptor specific for an ova-derived peptide (siinfekl) in the context of h -kb, were transferred i. v. into b mice. at day and , mice were treated intra-rectally (i. r.) with % ethanol. thirty minutes later, ovalbumin (ova) or bovine serum albumine (bsa) were applicated i. r. proliferation of cfse-labelled cells was measured at day after the injection of ot-i cells. the phenotype of effector cells was evaluated at day by measuring ifng production and by in vivo cytotoxicity assay. based on histology and immunhistochemistry for cd , the severity of colitis was scored. results: local application of the exogenous antigen ova but not of bsa led to antigen-specific activation and proliferation of adoptively transferred naïve ot-i cd + t cells. these cells differentiated into fully activated effector t cells with the capacity to secrete ifng upon re-stimulation ex vivo and possessed in vivo cytotoxicity to siinfekl-loaded target spleen cells. furthermore ova treated mice displayed an inflammatory infiltrate in the colonic lamina propria with strongly elevated numbers of cd + t cells. our study demonstrates that the local activation of antigen-specific cd t cells by exogenous antigen in the colon leads to fully activated effector t cells with the capability to promote local intestinal inflammation in non-immune-compromised b mice. aims: to determine the immune system response of the greek population against helicobacter pylori (hp), given the fact that hp infection is a frequent causal factor of gastroduodenal ulcer and gastritis, and to study the distribution by age and sex, as well as the possible correlation with anemia markers (hematocrit, hemoglobin, iron, ferritin etc). the results of express qualitative detection method for igg and iga antibodies were studied of patients, ( male and female), with age average , years of age. patients who received antibiotics and excretory medicine in the last year were excluded. anemia laboratory tests were performed (hematocrit, hemoglobin, iron, ferritin), which were followed by statistical processing, using spss, x and t-test programmes. results: in patients ( , %,with age average , years of age) no antibodies were detected. on the contrary, in the remaining , male and female, ( , %, with age average , years of age), antibodies were detected. out of them, in cases the results were strong positive ( male and female) and weak positive in cases ( male and female). the statistical analysis that followed, showed no statistically important correlation with any of the anemia markers who were determined (hematocrit, hemoglobin, iron, ferritin, mcv and rdw). conclusions: it is proven, therefore, that: ) helicobacter pylori infection is relatively common in the general population ( , %). ) there is a statistically important correlation, as far as age (increased in elderly patients) and gender is concerned (clearly greater in women). ) there seems to be no correlation with anemia. it is evident, that the method is very useful, especially in elderly patients with dyspeptic complaints, (who frequently cannot undergo invasive procedures), and should not be neglected, given the fact that there is a great risk of helicobacter pylori infection in our country. abstract withdrawn by author m. durilova , t. ulmannova , k. stechova , k. tesarova-flajsmanova , v. stavikova , j. nevoral charles university, pediatrics, prague, czech republicobjective: was to analyze composition of cytokines in breast milk of mothers whose infants were diagnosed with allergic colitis and compare it to cytokine composition in breast milk of healthy controls. methods: breast milk of mothers whose infants were diagnosed with allergic colitis and mothers of healthy infants and no history of allergic disease was analyzed for presence of cytokines. breast milk samples were collected at the time of diagnosis of allergic colitis ( - weeks, average . weeks of infant's age) or at the age of weeks in control group. concentrations of the following cytokines were analyzed using elisa method: il- , il- , il- , il- , il- , ifn-gamma, tgf-beta , egf and eotaxin. man-whitney u test was used for statistical analysis, p x . was considered statistically significant.results: il- as the only cytokine was not detected in any of the tested samples in both groups. significant difference was seen in concentration of ifn-gamma, which was higher (p x . ) in breast milk of mothers whose infants were suffering from allergic colitis (range - . pg/ml, mean . pg/ml) than in control group (range - . pg/ml, mean . pg/ml). higher concentrations of il- and lower concentration of tgf-beta were observed in breast milk received by infants with allergic colitis but the difference was not statistically significant. conclusion: immunologic factors including cytokines present in breast milk passively and actively influence the developing immune system of the newborn. although their role is not exactly known, they are important in regulation of immunologic reactions and might be responsible for protective effects of breast milk from many diseases. inter-individual differences in cytokine composition of breast milk were previously found in many studies and their presence is influenced by various factors. the results of our study indicate that there might be a risk cytokine pattern in breast milk of mothers whose infants are suffering from allergic colitis. supported by national project no. - . background: ulcerative colitis is associated with excessive neutrophil infiltration into the lamina propria and intestinal crypts leading to the formation of crypt abscesses. the chemokine il- (murine homologs kc and mip- ) and its receptor cxcr are involved in neutrophil recruitment, thus blocking this engagement offers a new therapeutic strategy for inflammatory bowel disease. this study aimed to develop and characterize a pre-clinical in vivo model to test potential therapeutics targeting neutrophil migration. methods: peritoneal exudate neutrophils from transgenic b-actin-luciferase mice were isolated h post intraperitoneal injection of thioglycollate and phenotypically (facs analysis) and functionally characterized in an in vitro chemotaxis assay. four million exudate cells were injected intravenously into recipients with dextran sulphate sodium (dss) colitis followed by bioluminescence imaging of whole body and ex vivo organs at , , and h post-transfer. anti-kc antibody or its isotype control was administered at mg/mouse one hour before transfer followed by whole body and organ imaging hours post-transfer. results: facs analysis revealed % neutrophil purity, % of which were cxcr + . in vitro, the cells migrated towards kc and this was inhibited by anti-kc. in the bioluminescent imaging model, trafficked neutrophils were evident in whole body and ex vivo organ images of dss recipients at all time points. neutrophil recruitment to the colon was detected only in dss recipients and was inhibited by anti-kc, h post cell transfer. this study describes a novel in vivo model of neutrophil trafficking that can be used for pre-clinical studies to evaluate potential inhibitors of neutrophil recruitment. the human gut contains more than bacteria (known as the commensal microbiota) that are essential for normal function of our digestive and intestinal immunologic systems. the barrier function of the mucosal epithelium is reinforced by innate defense mechanisms and by immune exclusion mediated by secretory (s)iga and sigm. sigs are generated via epithelial polymeric ig receptor (pigr)-mediated transfer of iga and igm from the lamina propria to the intestinal lumen. to assess the role of sigs in colitis development, we constructed pigr knockout (ko) mice and tested them in the dextran sodium sulfate (dss) colitis model ( . % dss in drinking water for week, followed by pure drinking water for week). pigr ko mice suffered increased morbidity and mortality compared with wild type mice, but colitis was cured by depletion of intestinal commensals suggesting that one role of sigs is to prevent pathology induced by commensal microbiota. in contrast, % dss was lethal to all commensal-depleted mice, but these mice became anemic rather than suffering from bloody diarrhea. as previously documented by medzhitov and co-workers (rakoff-nahoum et al, cell ), treatment of commensal-depleted mice with the tlr ligand lps in drinking water protected against the lethality of % dss. thus, the commensal microbiota serve two distinct roles in the dss colitis model. at dss concentration of . % they may become pathogenic and drive an intestinal inflammation. at % dss commensals protect against the toxic effect of the chemical via their tlr ligands. in mice lacking sigs, due to deleted pigr, the severity of colitis induced by . % dss was greatly enhanced suggesting that one role of sigs is to prevent commensal microbiota from becoming pathogenic. ulcerative colitis (uc) is a human inflammatory bowel disease associated with chronic inflammation of the gastrointestinal tract. although uc is associated with a type immune response, current treatment strategies use broad anti-inflammatory drugs which are aspecific for the disease. in a mouse model resembling uc, oxazolone induces il- production which is an important pathological factor. neutralizing il- or il- prevents or ameliorates disease significantly. as many aspects of the mechanisms involving these th cytokines in colitis remain undefined, we used mice deficient in il- /il- or the key receptor through which they signal, il- ra, to further dissect their role in oxazolone-induced colitis. disease was exacerbated in il- ra -/mice with increased weight loss, mortality, inflammation and immunopathological symptoms. this was in contrast to il- /il- double deficient mice which were protected from colitis. removing il- production from il- ra -/mice, by using il- ra/il- double deficient mice, reversed the susceptible phenotype to protection. together these data strongly suggest that il- mediates susceptibility in an il- ra independent manor. recent evidence pc / introduction: the activation of cd + t-cells in the lamina propria play an major role in the pathogenesis of inflammatory bowel disease (ibd). whereas cd is associated with increased production of th -like cytokines, the cytokines profile in chronic uc is characterized by the increased production of several th cytokines, such as il- ,- and il- . however, the functional role of t cell transcription factors such as nuclear factor of activated t cells (nfat) in ibd is poorly understood. the aim of this study was to further analyze the role of this signal transduction pathway and its pathogenic significance in uc. cryosesctions of uc and cd patients were analysed by immunohistochemically methods. a significantly higher expression of nfatc was found in uc and cd colonic tissue compared to control specimen. transmitted to the th -mediated oxazolone-induced colitis model, nfatc -production is significantly increased in both diseases, too. nfatc deficient mice were analyzed in colitis model and are significantly protected against the development of intestinal inflammation compared to control mice, documented by loss of weight, histological score and miniendoscopy. interestingly, cyrosections of inflamed colonic tissue displayed a higher apoptotic rate in nfatc deficient mice compared to control mice, which can be observed by tunel assays, caspase and annexin v staining, as well as in lamina propria t cells. contrary, anti-apoptotic proteins, like bcl- and bcl-xl were downregulated for induction of apoptosis. this observation was associated with a reduced production of il- , ifn-gamma, il- and il- by mucosal t lymphocytes, tested by elisa assays. further studies with the oxazoloneinduced colitis model showed that nfatc regulates il- /il- in an indirect way. last, administration of il- blocked the protective effects of the nfatc deficiency in experimental colitis, suggesting that nfatc through il- signal transduction plays a direct pathogenic role in vivo. conclusion: our data define a unique regulatory role of nfatc in colitis by controlling mucosal t cell activation in an il- dependent manner. the examination of this signal transduction pathway emerges as a potentially new therapeutic target for inflammatory bowel diseases. the pivotal role of micrornas in the regulation of gene expression, in particular genes involved in the immune response, indicates that they may play an important role in the pathogenesis of inflammatory bowel disease (ibd) as well. the study of the expression of micrornas in ibd will unravel their role in this disease. in addition, micrornas by their mechanism of action, are promising new therapeutic agents or targets. a possible therapeutic application of micrornas is the introduction of novel, artificial micrornas or microrna mimics to regulate specific genes. because ibd is a heterogeneous disease in human we decided to define microrna expression in a well defined model of experimental colitis. as a result of this study we found a number of micrornas involved in different phases of experimental colitis. to study the role of mirnas in experimental colitis in mice we have used a well defined colitis model that resembles human ibd. this colitis is mediated by cd cd rb high t cells that are injected i. p. in scid mice. in control mice in addition to the cd cd rb high t cells also regulatory cd cd rb low t cells are transferred and no colitis develops. to study mirna expression we collected colonic tissue from the mice at different time points during colitis progression. after weeks a chronic progressive colitis developed characterized by a progressing wasting disease that was terminated at weeks. microrna was isolated from colons of mice in different stages of colitis progression ( , and weeks) and control mice that do not induce colitis (n= for each timepoint). from all mice we also processed a part of the colon for immunohistochemistry to determine disease progression at the various time point after induction of colitis.the rna isolation as well as the microarray analysis has been outsourced to miltenyi biotec gmbh, bergisch galdbach, germany. we used the mirxplore tm microarrays for microrna expression profiling. from micrornas that demonstrated an induction during the development of disease we selected micrornas for in situ hybridization and for a proof of principle of the efficacy in the cd cd rb high transfer model. objective: the purpose of this clinical trial (id: nct of www.clinicaltrials.gov) is to investigate whether the expansion of the thymus in adults can restore specific immune responses by administration of growth hormone (gh). methods: successfully highly active antiretroviral therapy (haart) treated hiv infected patients that failed to elicit a humoral response to tetanus toxoid (tt), or to hepatitis a (hva) or to hepatitis b (hvb) virus have been selected for the trial. growth hormone was given for months with the hope that they will reactivate thymic input and restore their specific responses to these vaccine antigens. patients have been randomized in groups: group a (n= ) receiving haart+ gh (for months) + tt+hva/b vaccines (at month post gh adminsitration); group b (n= ) receiving haart+gh but not vaccines; and group c (hiv control group, n= ) with haart+vaccines (at month ) but without gh. all patients are followed up months further. results: preliminary results show that an increase in thymic size was observed in gh recipients and not in controls. furthernore after weeks of administaring hormone the absolute numbers of cd incresase from ± to ± cells per mm (mean and sem; p x . ). in contrast, pacients who have not received the hormone but have been vaccinated showed a significant decay of the cd absolute numbers from ± to ± cells per mm (p x . ). viral load remained undetectable in all patients. despite the increase in cd counts the percentage of recent thymic emigrants (as assessed by the expression of cd ) as well as the proportion of naï ve and memory cells remained constant throught the trial in all patients. finally, specific responses to hepatitis a virus seem to be restored in a major proportion of patients treated with gh (group a) than in the other groups. conclusions: although the clinical trial is ongoing, the preliminary results seem to indicate an increase in the thymic size and some immmune restoration in patients treated with growth hormone before vaccination. a major problem of current vaccines is the requirement for cold chains to maintain vaccine potency. in the course of the eradication of small-pox, freeze-dried vaccinia virus which proved to be extremely stable was used to overcome this limitation (dryvax ® ). before usage, dryvax has to be reconstituted before vaccination using a bifurcated needle reflecting another drawback of classical vaccination -transmission of blood-borne pathogens. an alarming report by the who has estimated that as many as one-third of immunization injections are unsafe in four of its six geographical regions. each year, an overwhelming number of infections with hiv ( , - , ), hepatitis c virus (hcv; . - . million) and hepatitis b virus (hbv; - million) are thought to originate from the reuse of needles and syringes by health-care providers. in this report, we took advantage of the stability of freeze-dried vaccinia virus mva and directly applied it into the nostrils of mice without prior reconstitution. this direct mucosal application induced systemic antibody and t cell responses comparable to those achieved by intramuscular administration. importantly, mucosal application of lyophilized mva conferred protective immunity against a lethal vaccinia virus challenge. additionally, recombinant mva expressing the model antigen ovalbumin induced long-term and protective immunity against listeria monocytogenes-ova challenge. the data clearly demonstrate the potency of a simple needle-free vaccination, combining the advantages of mucosal application with the stability and efficiency of lyophilized mva. methods and results: seventeen haart-treated asymptomatic hiv- infected patients with g cd + t-cell counts and plasma hiv-rna levels of x . log copies/ml were treated with a dc-based vaccine. the vaccine consisted of autologous mature dc electroporated seperately with either sig-tat-dc-lamp, sig-rev-dc-lamp or sig-nef-dc-lamp mrna and were each administered at a distinct anatomical site. after four monthly vaccinations haart treatment was interrupted. pbmc from timepoints, before during and after vaccination, were analysed for ifn-g production (elispot), proliferation (cfse assay) and lytic capacity (fatt-ctl) in response to the antigens used in the vaccine. pbmc were screened upon ex vivo stimulation with pools of overlapping tat, rev, nef and gag peptides and ifn-g secretion was analysed using elispot ('peptide elispot'). elispot was also performed on re-stimulated t-cells with electroporated dc ('dc elispot') in vitro. responses were considered positive when the number of spot forming units per million t-cells was g and when the responses were g times the standard deviation above the mean of replicate negative controls (mock electroporated dc). / patients were screened with both peptide and dc elispot. an increase of responses to the vaccine-antigens after vaccination was found in both assays. based on the dc elispot data, we observed immune reactivity against tat in / patients before and / patients after vaccination. for rev, / patients showed a pre-existing rev specific response and / patients responded to rev after vaccination. nef was the most immunogenic antigen used in this vaccine with already / patients responding before and / patients responding after vaccination. for our control antigen gag, we observed an anti-gag response in out of patients before vaccination and / patients after vaccination. the results of the other assays correspond to the dc elispot results be it less pronounced. conclusion: therapeutic vaccination of hiv-infected patients with dc electroporated with tat, rev and nef induces and/or enhances antigen-specific t-cell responses, especially when monitored with the dc elispot. background: enterovirus (ev ) is an etiologic agent responsible for seasonal epidemics of hand-foot-and-mouth disease and causes significant mortality among young children. no effective vaccine for ev is available yet. polysaccharide purified from ganoderma lucidum (ps-g) has been known to be a strong immunopotentiator, therefore, we studied the immune enhancing effect of ps-g as the possible adjuvant with ev inactivated virus. methods: mice were immunized intraperitoneally with mg inactivated virus /mouse with one of the following samples: pbs, cfa/ifa, and ps-g. each mouse received the same dose of boosters after , , and weeks. blood samples were collected at , , , and days. the total serum anti-ev igg level was determine by elisa, and the neutralization assay was also done. to evaluate the cellular immune responses, spleens were harvested from all mice for splenocyte proliferation assay. cytokines assay regarding ifn-g and il- from splenocytes was also measured. results: immunization with ev /ps-g showed that the anti-ev igg levels were significantly increased compared with ev alone or ev /cfa/ifa in balb/c mice. neutralization assays demonstrated an effective protection of ev /ps-g group compared to ev alone. the splenocyte proliferation test showed that production of ifn-g significantly increased in ev /ps-g-immunized mice compared to those of ev or ev /cfa/ifa-immunized mice, indicating a th cells response elicited by heat-inactivated ev vaccine with ps-g adjuvant. conclusions: vaccine design is important for the development of immune response for pathogen, and adjuvants play the very important role to enhance the effect of vaccine. the results here suggested that ps-g can be used as a novel, safe and natural vaccine adjuvant. objectives: the search for a vaccine against hepatitis c virus is hampered due to the lack of an animal model to study vaccine-efficacy other than chimpanzees. here we describe the differential modulation of cd + t-cell responses induced by a dna prime mva boost vaccine regimen in four individual chimpanzees and their association with viral clearance. methods: an ex vivo expansion protocol was used to map peptide specific cd + t-cell responses against hcv-ns , studying induction of il- , ifng and tnfa cytokine production as well as killing capacity. to assess the killing capacity and mhc restriction of the peptides, a non-radioactive killing assay was designed. peptides that induced both il- and ifng production by cd + t-cells were tested for their competence to induce killing of transfected target cells that expressed chimpanzee mhc class i molecules. introduction: high levels of hiv- -recognizing cd + cytotoxic t lymphocytes (ctl) with a widespread specificity, especially against conserved epitopes, are considered to play an important role in the control of hiv- replication, and for the prolonged survival of infected individuals. a potential immunotherapeutic strategy would be the adoptive transfer of t cells, which are reprogrammed by introduction of an hiv-specific t cell receptor (tcr). up to now, such ctl were generated by retroviral transfer of tcr-encoding genes, which harbors several challenges (i. e., activation/inactivation of genes, life-long autoimmunity). methods: therefore, we investigated the transfer of tcr-rna into cd + t cells by electroporation, and chose tcrs which were able to recognize the hla-a restricted hivpol-peptide iv , or the hivgag-peptide sl . results: t cells, reprogrammed with these receptors, released the pro-inflammatory cytokines il- , tnf, and ifng simultaneously, and showed up-regulation of the activation marker cd , after stimulation with peptide-loaded target cells or target cells (i. e. ebv-transformed b cell and cd + t cells) presenting the naturally processed epitope. furthermore, these cells maintained their ability to proliferate after stimulation. more importantly, killing assays demonstrated that the tcrreprogrammed cd + t cells were capable to specifically lyse target cells (for at least three days) loaded with the cognate peptide, or presenting the naturally processed epitope. a comparison of our reprogrammed t cells with the parental ctl showed that the transfected t cells were only one order of magnitude lower in avidity than the parental ctl. also, the parental clone's recognition pattern of mutant peptides was preserved in tcr-rna-transfected t cells. the transfection of tcr-encoding rna into cd + t cells, may represent a simple, secure, and more flexible alternative to retroviral transduction, but has the benefit that a better evaluation of the transferred tcrs is possible. background: dendritic cells (dcs) are able to capture, internalize, and process antigens leading to potent activation of antigen-specific cellular immunity. the aim of this study was to investigate the capacity of splenic dcs that ingest antigen coated magnetic beads to induce hcv cellular immune responses. methods: splenocytes of flt l pretreated balb/c mice were incubated for hrs with hcv ns -coated magnetic beads. the cells were harvested and cells that contained beads were purified by passage over a magnetic column. the isolated population contained g % dcs and was used for immunization. dc expression of the maturation markers cd , cd and cd was determined before and after ingestion of ns -coated beads, showing a significant increase of all maturation markers induced by phagocytosis. cellular immunity was assessed using a conventional ctl assay, a cfse-t-cell proliferation assay, intracellular cytokine staining and tumor challenge (with stably ns expressing sp / cells). results: in immunized animals, the ctl activity was increased -fold compared to mock immunized mice. accordingly, tumor challenge with ns expressing tumor cells showed a significant reduction in tumor growth. the number of cd + ifn-g + cells was increased g -fold and the number of cd + il- + increased g fold in the dc-ns -bead immunization group. these results paralleled the proliferative response of splenocytes to ns protein obtained from immunized animals with the most significant response in the cd + population of dc-ns -bead immunized animals. the use of ns coated beads combines three important aspects of dendritic cell based immunization in a single step: targeting of the antigen, enrichment and maturation of dendritic cells. the induction of a strong th biased t cell response makes this approach a promising new tool in therapeutic immunization in chronically hcv infected patients. the success of anti-dec- antibody as a stimulator of strong inflammatory immune responses depends on the coadministration of non-specific dendritic cell maturation factors. in their absence, anti-dec- induces antigen-specific tolerance rather than immunity. we hypothesize that regulatory t-cell epitopes contained in anti-dec- promote a tolerogenic reaction that is only overcome through the co-administration of non-specific immuno-stimulators. this hypothesis is based on our recent discovery of a set of natural regulatory t-cell epitopes derived from human immunoglobulins that induce tolerance by stimulating regulatory t cells. we have verified experimentally that these epitopes generate an expansion of regulatory t cells and suppress inflammatory immune responses. here, we embarked on a proof-of-principle demonstration that a pro-inflammatory and non-tolerogenic anti-dec- antibody can be developed. we screened the anti-dec- sequence computationally for putative hla dr -restricted, regulatory t-cell epitopes as targets for mutations that will reduce epitope binding affinity for hla. amino acid substitutions predicted to interfere with hla binding were identified and are being incorporated into an array of anti-dec- antibody variants recombinantly fused to a test antigen, hiv gag. variant antibodies that do not interfere with dendritic-cell targeting will be evaluated for reduced tolerogenicity, as well as for enhanced gag immunogenicity, in terms of cellular and humoral responses. we predict that the modification of regulatory t-cell epitopes will significantly diminish tolerogenicity, enabling the use of modified anti-dec- as a hiv antigen-delivery system that obviates the dangers associated with non-specific activation of the immune system. supported by nih r ai a live oral vaccine based on human adenovirus (ad) has proved safe and effective in us military recruits for nearly years. in these experiments, we have investigated whether replication-deficient ad can be an efficient potential vaccine carrier for oral vaccination. ad vectors were used throughout to provide a benchmark for efficacy. we generated novel ad and ad vector systems based on dna recombineering to facilitate manipulation of the vector backbone and high throughput transgene insertion (http://adz.cf.ac.uk). egfp was inserted with a hcmv ie promoter as a model transgene. preliminary in vitro studies on bloodderived human and murine cells demonstrated that primary lymphocytes are less susceptible to transduction with ad than ad . ad routinely infected and provided transgene expression in˚ % of human cd + and cd + t cells. stimulation of the hcmv ie promoter post infection increased detection of egfp to - % of cd + t cells present, showing that ad infected a surprisingly large proportion of t cells. in comparison, ad provided egfp expression in x % of either cell type, even after t cell activation. in contrast, infection rates and transgene expression in dendritic cells of both human and murine origin with ad and ad vectors were comparable. preferential infection of dcs is likely to be beneficial in the context of a vaccine. in vivo, we observed that following oral delivery both ad and ad induced restricted yet strong expression in the intestine. the vectors were rapidly taken up into the peyers patches, with optimal expression detected day after dosing, and transgene expression being reduced below detectable levels by day . interestingly, when delivered together ad and ad vectors targeted the same subset of cells. together, these data show that ad is a viable alternative to ad -based vaccines which may also avoid unwanted infection of activated t cells. aims: monoclonal antibodies (mabs) represent some of the most promising agents for anti-cancer and anti-viral immunotherapies ( recently commercialized; g in clinical trials). to date, their therapeutic antiviral efficiency has mainly been measured by their direct effects on viral spread. however, their indirect effects on long-term immune control of viral replication through their immunoregulatory properties upon interaction with other components of the immune system has hardly been assessed. as induction of long-term protective antiviral immune responses still remains a paramount challenge for treating chronic viral infections, we asked whether neutralizing mabs, in addition to blunt viral propagation, may also modulate the endogenous immune response. methods: we have developed a preclinical mouse model of fatal leukemia induced by the frcas e murine retrovirus. mice were infected with frcas e and treated, or not, with a neutralizing mab (the mab). viral propagation, survival and development of immune responses were followed up for several months. results: using this model, we have shown that -treated/infected mice develop a long-lasting protective humoral immune response as well as a strong and sustained cellular immune response with high cytolytic activity which are not observed in leukemic non-treated/infected mice. these results show that neutralizing mabs act as immunomodulatory agents capable of inducing long-term protective immunity ( g months) with both humoral and cellular contributions, despite the fact that they were administered over a short period of time (gros et al, ; gros et al, ; michaud et al. submitted) . although the initiation and maintenance of this long-term immunity is multi-factorial, we have demonstrated the crucial importance of the uptake of antibody-coated, infected cells by dendritic cells in the development of enhanced primary and memory antiviral t-cell responses. conclusions: our results show that infected-cells/antibody immune complexes play an important role in the induction and maintenance of protective antiviral immunity through enhancement of primary and memory antiviral t-cell responses. our observation might have important consequences on the design of antiviral mab-based immunotherapies. objectives: we have analyzed the potential of virus-like particles (vlps) from rabbit hemorrhagic disease virus (rhdv) as a delivery system for foreign t-cell epitopes. to accomplish this goal, we generated chimeric rhdv vlps incorporating a cd + t-cell epitope (siinfekl) derived from chicken ovalbumin (ova). the ova epitope was inserted in the capsid protein (vp ) of rhdv at two different locations: ) the n-terminus, predicted to be facing to the inner core of the vlps (rhdv-vlp- ), and ) a novel insertion site predicted to be located within an exposed loop (rhdv-vlp- ). both constructions correctly assembled into vlps and we analyzed the immunogenic potential of both chimeric rhdv vlps in vitro and in vivo. in vitro, dendritic cells (dcs) were able to process and present siinfekl peptide in the context of mhc class i from chimeric rhdv vlps for cd + specific recognition in a dose-and insert position-dependent manner. moreover, chimeric rhdv vlps activated dcs for tnf-alpha secretion.in vivo, mice immunized with the chimeric rhdv vlps without adjuvant were able to induce specific cellular responses mediated by cytotoxic (ctl) and memory t cells. although both chimeric rhdv vlps were able to induce specific ifn-g-producing cell priming, insertion of the siinfekl peptide at the amino-terminal position (rhdv-vlp- ) was more immunogenic than insertion at position for induction of ctls and anti-viral immunity.more importantly, immunization of mice with chimeric rhdv vlps at the highest dose tested was able to control an infection by a recombinant vaccinia virus expressing ova in target organs. in addition, immunization with chimeric rhdv-vlp- at the highest dose tested was able to resolve vv-ova infection. conclusion: our data demonstrated that immunization with chimeric rhdv vlps was able to protect mice from a viral challenge, suggesting the potential suitability of these constructions for new vaccine development against animal and human viral infections. objectives: a major issue pertaining to use of dna vaccines is that despite successful proof of principle results in small animal models, low efficacies have been reported in human clinical trials and large doses are required to induce protective immune responses. in this study, we describe the targeting of antigen-encoded dna directly to dendritic cells (dcs) through a pathway that results in internalisation and transfection using a cationic lipopeptide containing arginine residues and the lipid dipalmitoyl-s-glyceryl cysteine, a known tlr- ligand. methods: agarose gel electrophoresis was used to confirm the electrostatic binding of lipopeptide to dna encoding for green fluorescent protein (gfp), influenza hemagglutinin (ha) or nucleoprotein (np). transfection efficiencies of dcs using these complexes were determined by flow cytometry using specific antibodies for each encoded protein. stimulation of t cells by np-transfected dcs was also investigated by measuring their ability to induce in vitro cytokine secretion by influenza virus-specific cd + t cells. subsequently, vaccine immunogenicity was ascertained by immunisation of mice via the intra-nasal route. results: electrostatic binding of the lipopeptide to dna plasmids was confirmed by gel electrophoresis where the positively charged amino acids of the lipopeptide were able to neutralise the negative charged phosphate groups within the dna backbone and retard its ability to migrate towards the anode. high levels of gfp, ha or np were detected in murine spleen-derived cultured dcs following transfection with these complexes concomitant with the upregulation of surface mhc class ii molecules compared to when dna alone was used. the ability of transfected dcs to stimulate cd + t cells from influenza virus-infected mice was also investigated. subsequently, vaccination by lipopeptide-dna complexes resulted in the induction of higher numbers of ifn-g producing np - specific cd + t cells in the spleen and lymph nodes of mice compared to those that received dna alone. conclusion: altogether these results demonstrate that the use of a tlr- targeting lipopeptide-based system that can facilitate the delivery of dna by directly targeting and concurrently activating antigen-presenting cells, such as the dc, could prove to be advantageous in enhancing cellular responses induced by dna vaccination. the level of interferon in blood serum of non-infected mice was determined by elisa kit and by the cytopathic test in the l cell culture after aplication of ridostine and ridostine ointment. the effect of the preparation on phagocytic activity of macrophages was evaluated in the monolayer peritoneal cell culture. the statistical processing of the data was carried out by the student t-criterion. ridostine was administered to patients once or twice in the case of high temperature. the clinical signs were recorded (temperature, rhinitis, headache etc). for prophylactic and treatment purposes the ridostine ointment was intranasally applied twice per day during days. the effectiveness of the preparation was evaluated by clinical sings and the level of cd +, cd +, cd +, cd + t -lymphocytes. results: ridostine significantly decreased accumulation of the virus in lungs of infected mice at the initial stage of influenza. ridostine and ridostine ointment stimulated synthesis of interferon and phagocytic activity. ridostine in clinical practice decreased the duration of influenza, attenuated clinical signs and was more effective at an early stage of the infection. prophylactic intranasal application of ridostine ointment by healthy volunteers (nurses and doctors) resulted in a high degree of protection during the whole epidemic season and an activation of t-cell immunity. application of ointment at an early stage of disease markedly activated t-cell immunity, reduced the duration of the disease and the intoxication syndrome by , - -fold. conclusion: interferon inducer based on natural dsrna stimulates some reactions of innate immunity and resistance to influenza virus. the drugs based on dsrna show promise for treatment of influenza. objectives: the creation of gene engineering vaccines against hepatitis c virus (hcv) based on recombinant proteins is one of relevant approach. since the immunogenicity of these proteins is low as a rule, the choice of adjuvant is very important. the aim of this work was to evaluate immunogenicity of covalent conjugates between nonstructural ns and ns a hcv proteins and immunomax ® , an acid peptidoglycan of plant origin, which displays immunomodulating activity. objectives: ifn-g takes part in the development of an anti-infectious reaction of the organism, which is connected with the peculiarities of its immunomodulating action. a/h n influenza virus inhibits the ifn-g synthesis (mibayashi et al., ) and causes a decrease in cd + and cd + t-lymphocytes content in lung and lymphoid tissues associated with an impairment of this cytokine production (tumpley t. m. et al., ) . thus, ifn-g is a promising drug for prophylaxis and treatment of avian influenza under conditions of monotherapy or complex therapy. an essential defect of this cytokine is its fast degradation in blood. the goal of this work was to study immunomodulating properties of an ifn-g structural analog with increased proteolytic resistance when it was used alone or in combination with double-stranded ifn inducers. methods: a recombinant human ifn-g analog deltaferon is distinguished by a amino acid deletion at the c-end of the molecule and substitutions of amino acids in positions - (tat'kov c. i. et al., ) . deltaferon was i. p. administered to male non-inbred mice in doses of - * iu once or twice at an interval of hours, alone or in combination with double-stranded yeast rna preparation ( mg/kg). the content of ifn-a and ifn-g in mouse blood serum was determined by the immunoenzyme method, proliferative activity of splenocytes -by mtt-test (mosmann t., ) . results: when deltaferon was administered in doses of - * iu alone it did not influence the content ifn-a in blood, but caused a transient increase in the level of ifn-g. the injection of the preparation in a dose of * iu led to a an increase in both spontaneous and conconavalin a-induced proliferation of splenocytes. the two-fold administration of deltaferon in the maximal dose increased a level of ifn-g and inhibited cell proliferative activity. the combined administration of deltaferon ( * iu) and dsrna markedly increased the level of ifn-a and enhanced splenocyte proliferation. the recombinant human ifn-g analog is able to enhance ifn-g synthesis, splenocyte proliferation and to modulate the effect of ifn inducer. these data suggest that the studies of the preparation as an antiviral agent during influenza are perspective. by using a combined approach of routes of immunization and vaccine delivery systems such as poly-lactic acid (pla) biodegradable nanoparticles, we have dissected the intensity and quality of both cellular and humoral immune responses in mice. we showed that the amplitude and quality of the immune response (humoral, cellular) at systemic and mucosal sites (blood, vagina) could be largely influenced by the choice of a pertinent cutaneous route of vaccination (intradermal (id), transcutaneous (tc), subcutaneous (sc)). while id and tc route remain mostly efficient for the induction of antigen-specific cd responses (tetramer+ hiv- gag p cells), the quality of humoral responses (igg, iga) remained distinct between the two routes. in addition, sc route is less efficient than id and tc routes for the induction of cd responses after pla-p immunization. we have also shown that a lower antigenic charge of pla particles was needed when pla-p were injected using id and tc routes of immunization. currently, we are dissecting innate cellular mechanisms that gave rise to distinct quality of immune responses. this unique possibility to modulate the quality of the immune response according to the skin route of immunization paves the way for new vaccine design strategies and highlights the capacity of nanoparticles-based vaccine delivery system. b. dí az-freitas , c. prego , s. vicente , m.j. alonso , a. gonzález-fernández university of vigo. area of immunology, department of biochemistry, genetics and immunology, vigo, spain, university of santiago de compostela, nanobiofar group, department of pharmacy and pharmaceutical technology, santiago de compostela, spainobjectives: the design of effective vaccine delivery nanovehicles is opening up new possibilities for making immunization more equitable, safe and efficient. in this work, we purpose polysaccharidic-based nanocarriers as delivery structures for virus-like particle antigens, using recombinant hepatitis b surface antigen (rhbsag) as a model. our aim was to evaluate in a murine model if these nanocarriers induce an immune response after intramuscular and intranasal administration. materials and methods: loaded chitosan-based nanocarriers were prepared by cross-linking the polysaccharide chitosan, in the presence of the stabilizer poloxamer , with a counter ion, tripolyphosphate, containing the free rhbsag. the immunogenicity of these polysaccharidic-based nanocarriers with or immunizations to balb/c female mice ( weeks old) was assessed following intranasal or intramuscular immunizations. blood samples from the mouse maxillary vein were collected at different intervals (from day to post-primary immunization). specific igg antibodies levels directed against rhbsag in serum were measured by indirect elisa in miu/ml. results: chitosan-based nanoparticles with particle size in nanometric range, positive zeta potential and an important rhbsag loading were prepared. the results of the specific igg levels achieved following intramuscular administration of the antigen-loaded nanocarriers, and also of the alum-adsorbed vaccine showed the significant adjuvant effect of the nanocarriers. the response elicited was delayed respect to the alum based vaccine, and very interestingly, igg concentration was much higher using antigen-loaded nanocarriers than with the conventional vaccine. after intranasal administration, chitosan-based nanoparticles generated a lower immune response compared with the intramuscular route, but increasing over the time, showing an interesting slow release of the antigen. the igg titers elicited were enough to induce full seroprotection against the disease ( miu/ml). polysaccharidic-based nanocarriers with interesting properties for improving vaccination with complex antigens were designed and in vivo behavior of these nanocarriers suggests their potential utility as controlled release vehicles for reducing the number of intramuscular doses administered. more studies are currently underway to fully validate the potential of these nanocarrier prototypes and to optimize alternative routes of immunization such as the intranasal route. the success of immunotherapeutical approaches strongly relies on specific antigen targeting to dendritic cells (dcs) in an environment that provides optimal immunostimulatory signals. in our research group a bio-safe coronavirus-based vaccine vector platform that delivers multiple antigens to professional antigen- background: infection with human immunodeficiency virus type (hiv- ) is characterized by dysfunction of hiv- -specific t cells. to control the virus, antigenloaded dendritic cells (dcs) might be useful to boost and broaden hiv-specific t-cell responses. poly electrolyte microcapsules are potent protein delivery vehicles which can be tailored with ligands to stimulate maturation of dendritic cells. we investigated the immune stimulatory capacity of dendritic cells loaded with these microcapsules, containing both p antigen from hiv- and the tlr ligand poly i:c as a maturation factor. methods: monocyte-derived dc (mddc) from healthy subjects were cultivated with polyelectrolyte microcapsules containing, poly i:c. potential maturation of dc was evaluated by flow cytometry. mddc from hiv-infected patients under highly active anti-retroviral therapy (haart) were similarly pre-incubated with p microcapsules containing p and poly i:c. these antigen loaded mddc were used to directly stimulate autologous peripheral blood lymphocytes (pbl) in elis-pot. they were also co-cultivated for days with autologous pbl to evaluate the immunogenic capacity. potential expansion of specific t cells was measured by comparing elispot responses of pbl before and after coculture, using a pool of overlapping p peptides. intracellular staining of interferon-gamma (ifn-g), interleukin- (il- ) and cd a after p stimulation was also performed. results: mddc from hiv(-) subjects, incubated for hour microcapsules alone did not induce maturation of dc, but when poly i:c was present the dc did mature. mddc from haart treated hiv-infected individuals, cultivated with p containing microcapsules with poly i:c, were able to efficiently expand and broaden autologous effector memory t cells which contain and secrete ifn-g and il- , upon p peptide restimulation. objectives: we aimed at investigating whether and how the distance of a cytokine from the vlp surface impacts on its function and whether the relative distance towards a co-presented antigen is critical for its biological activity. methods: we inserted one, two or four ig-like domains of hcd b between our model cytokine il- and the minimal gpi-anchor acceptor sequence. subsequently, we compared particle production by western blotting for p gag, targeting of cytokines to lipid rafts and and vlp upon isopycnic membrane separation and biological activity in il- dependent proliferation assays of il- variants. results: murine il- attached to either of the four fusion partners was biologically active in vitro as shown by induction of proliferation of the il- dependent cell line ht- . we found that the membrane anchors comprising one and four ig-like domains (il- :: iggpi and il- :: iggpi) resulted in severely reduced vlp production by producer cells and despite of an increased targeting of il- :: iggpi to vlp, a reduced stimulatory capacity of producer cell crude supernatant, when compared to il- fused to the minimal gpi-anchor acceptor sequence of hcd b (il- ::gpi). il- :: iggpi, however, showed comparable particle production and biological activity in vitro when compared to il- ::gpi. furthermore, il- fused to ig::gpi showed an increased capacity to co-stimulate primary p tcr transgenic t-cells specific for lcmv-gp - in the context of h -d b . conclustions: besides the minimal gpi-anchor acceptor sequence we have identified one additional membrane anchor, which displays superior capacity to target cytokines to vlp. ig::gpi has a biological activity in vitro, which is comparable to the minimal gpi anchor. moreover, il- ::gpi displays increased co-stimulatory potential in the context of specific mhcp complexes. this work was supported by grants sfb f -b of the austrian science foundation, the austrian research promotion agency (forschungsförderungsgesellschaft) bridge grant & biomay ag, and the christian doppler laboratory for immunomodulation. a. roemhild , interdisciplinary transplant laboratory charite berlin, insitute of nephrology and medical immunology, berlin, germany immunosuppressive treatments, e. g. after transplantation are often followed by an impaired or dysfunctional immune system. missing viral immunity, particularly against ebv, is an essential key player in the development of severe infections and posttranplant lymphoproliferative disorders (ptld). ptld affects - % of solid organ transplant recipients, depending on the organ transplanted. healthy individuals control ebv infection by ebv-specific cytotoxic t lymphocytes (ctls), but some patients under immunosuppression are unable to do so. in these cases, immunotherapy is increasingly used as a new approach for re-establishing a functional immune response by retransferring in-vitro expanded autologous virusspecific t cells into the patient. currently these t cells are generated by repetitive stimulations with ebv-infected autologous lymphoblastoid cells (lcls). due to a generation time of - months, many patients suffering from missing viral immunity and subsequent severe viral disease are excluded from therapeutic benefit. therefore, shortening the generation time would be an important step to make adoptive immunotherapy available for more patients. t cell lines were generated with two different protocols. in the first protocol t cells are generated by repetitive stimulation with ebv-infected autologous lcls. the second protocol is based on stimulation with different overlapping ebv peptide-pools and immunomagnetic cell isolation. expanded t cells were analysed using multicolour flow cytometry. cells were stained for diverse surface markers and intracellular cytokine production. cytotoxic capacity and specificity was determined by a calcein release assay. our group developed a new protocol for the production of ebv specific t cells, thereby shortening the generation time from , month to days. t cell lines are composed of cd and cd cells with a mainly effector memory like phenotyp. after restimulation the cells produce more tnfa than ifng. depending on the generation protocol t cells specifically recognized and lysed autologous lcls alone or loaded with ebv-peptides. the detailed characterization of ebv-specific t cell lines should help to further improve the adoptive immunotherapy and its outcome. the novel, short time generation protocol did not affect phenotyp and cytokine production of the t cells. nevertheless their therapeutic potential in vivo has to be tested in further experiments. s. s. schmucker , m. assenmacher , a. richter miltenyi biotec gmbh, r&d cell biology, bergisch gladbach, germanyadoptive transfer of virus-specific t cells provides a promising treatment of infection in immunocompromized patients. as expansion of virus-specific t cells from antigen-experienced donors is feasible, no reliable protocols for generation of antigen-specific t cells from naive hosts exist. in this study we established a cell culture system for priming of highly rare naive cmv pp -specific cd + and cd + t cells from cmv-seronegative donors in vitro.magnetically isolated naïve (cd ro -cd -) cd + and cd + t cells from pbmc of cmv-seronegative donors were co-cultured with autologous mature monocytederived dc loaded with cmv pp peptide pool and cd -depleted autologous pbmc as feeder cells in the presence of il- , il- , and il- . already - days after primary activation pp - /a -tetramer + cd + t cells were detectable for hla-a + blood donors. to analyze cd + t cells having other specificities than for the peptide pp - as well as probably primed cd + t cells, we looked for the production of cytokines after a second stimulation. we found ifn-g secretion in up to . % of the cd + t cells and up to . % of the cd + t cells after restimulation with pp peptide pool, but not with either irrelevant ie- peptide pool or without antigen, in each of eight donors tested. for generation of t cell lines, we magnetically enriched the primed t cells according to their ifn-g secretion. subsequent cultivation for days led to a - fold expansion of pp -specific t cells, defined by their sustained capability to produce ifn-g. evaluation of the antigen-specificity of the expanded t cells also showed upregulation of the activation markers cd and cd only if restimulated with the pp peptide pool. further cytokine analysis of the cells revealed co-production of ifn-g, tnf-a, and il- , indicating the functionality of the in vitro primed and expanded t cells.in conclusion, we established a cell culture system, which enables the in vitro priming and expansion of cmv-specific cd + and cd + t cells derived from the naive compartment. this should extend the application of adoptive t cell therapy to patients for whom immune donors are not available. a. i. wolf , k. mozdzanowska , l. otvos , j. erikson the wistar institute, philadelphia, united states, temple university, philadelphia, united statesthe influenza virus a matrix protein ectodomain (m e) sequence has remained highly conserved among various human influenza a strains and is therefore a promising target for a protective vaccine. based on previous work using a synthetic m e-based multi-antigenic peptide vaccine (mozdzanowska at al., vaccine ; virology journal ), we generated a novel peptide and investigated its efficacy in inducing an anti-m e antibody (ab) response and its ability to confer protection against viral challenge.objectives: cytomegalovirus (cmv) causes significant morbidity and mortality in patients after haematopoietic stem cell transplantation (hsct). due to limitations of current antiviral therapies, alternative approaches, involving transfer of donor-derived cmv specific cd + t cells, have been considered. clinical data confirm a crucial role for antiviral cd + t cells inversely correlating with the incidence of cmv reactivation and disease. cmv specific cells have to reach protective levels in order to be effective. levels of such cells correlating with protection against cmv infection and disease have only been reported in patients expressing hla-a* and hla-b* previously. considering other frequent hla alleles cmv specific cd + t cells were monitored longitudinally in hsct patients in this study to establish the cell number thresholds at which patients are protected from cmv reactivation. methods: we have correlated the pattern of different ex vivo cmv peptide specific cd + t cell responses (frequency analysis using tetramer staining and interferon gamma elispot analysis) with the cmv viral load (dnaemia) and clinical status in patients. different response groups were compared using the mann-whitney-u test.results: our results demonstrate that the presence of different cmv specific cd + t cells inversely correlates with the ability to detect of cmv reactivation in patients at different cell number thresholds. we show that the cell number thresholds for hla-a* /pp ( - ) ( . x cells/l) and hla-b* /pp ( - ) ( . x cells/l) specific cd + t cells are significantly lower than those for hla-a* /pp ( - ) ( . x cells/l) and hla-a* /pp ( - ) ( . x cells/l) specific cd + t cells in hsct recipients post transplant. this difference is also evident in healthy cmv seropositive volunteers. conclusion: these findings suggest a differing efficiency of the responses restricted by the two sets of alleles. the data merit further studies using larger patient cohorts and are important for considerations regarding the epitope restriction and quantities of ag specific t cells to be monitored after therapeutic strategies for cmv in hsct patients. ( , - mcg) . no adverse effects were indicated during trials (up to month of observation).hiv-specific antibodies were induced by dose-dependent manner, the most prominent response was detected after th immunization with mcg of vichrepol.no differences were detected in cd + and cd + t cell counts and cd +/cd + ratio, so there was additional safety issue concerned to the possible sensitivity of vaccinees to hiv infection. the results of phase i clinical trials of vichrepol vaccine were approved by who authorized russian national control institution and transition to phase ii immunogenicity trials was recommended. objectives: to improve the vaccination efficiency of adenoviral vectors for anti-retroviral vaccination, we constructed adenoviral nanoparticles by fusion of the vaccine antigen to the adenovirus capsid protein pix. the adenoviral nanoparticle vaccine was evaluated in the friend virus (fv) mouse model and compared to conventional adenoviral vectors. methods: adenoviral nanoparticle vectors were constructed by deletion of pix from the adenoviral genome and insertion of the fusion protein encoding sequence as transgene. for vaccination against fv, that is a retrovirus complex of friend murine leukemia virus (f-mulv) and spleen focus forming virus, we constructed fusion proteins of pix and the f-mulv surface env protein gp or gag. to elucidate underlying mechanisms we produced displaying-only nanoparticles and plasmid dna encoding either pixgp or gp alone. conventional adenoviral vectors were used that express full-length f-mulv env and gag. the vaccines were tested in cb f hybrid mice that are highly susceptible to fv infection and develop viremia and splenomegaly after fv infection. results: vaccination of cb f mice with adenoviral nanoparticles expressing fusion proteins containing gp resulted in protection from viremia and splenic enlargement after fv challenge that was superior to vaccination with conventional vectors. immunological analyses showed that the adenoviral nanoparticle vaccine induced a significantly higher number of f-mulv env-specific cd + t cells and higher antibody titers than a conventional adenoviral vaccine expressing the vaccine antigen. we could show that for the beneficial effect it is necessary that the fusion protein is incorporated into the adenoviral particle and it also has to be expressed from the adenoviral vector in vivo. conclusion: adenoviral nanoparticles are a useful tool for the induction of antibody and cd + t cell responses that are superior to conventional adenoviral vectors. this new type of adenovirus-based vaccination vector combines genetic and protein vaccination and should make adenoviral vectors even more interesting for vaccination purposes. . antibody levels were monitored by elisa and hemagglutination inhibition assay, viral excretion in nasal washes was assessed by quantitative rt-pcr, and cellular production of ifn-gamma was measured via flow cytometry. results: we found that animals vaccinated with caf exhibited higher levels of serum igg and mucosal iga than the ones which received the vaccine alone, and that they excreted - % less virus. animals that received only vaxigrip were producing ifn-gamma after challenge, a sign of infection by low virulence influenza strains, whereas the animals that received also caf did not show any increase in their levels of ifn-gamma. conclusion: caf enhances the protection conferred by the commercial inactivated vaccine against strains matched by the vaccine. evaluation of the t-cell specific immune response is very important for global eradication of measles and rubella. peripheral blood lymphocytes (pbl) from children aged - years old ( boys and girls) -group , and children ( boys and girls) - years old -group were isolated on a gradient of density before vaccination ( or revaccination) with priorix, week, and months after and incubated with cfse. then million/ ml pbl were incubated in rpmi- supplemented with % fcs (the negative control), at presence of mcg/ml pha (the positive control) or at presence of the measles or rubella viruses antigens in a humidified atmosphere containing % cÎ at °c within day. intensity of a fluorescence estimated on fl by flow cytometr facscalibur (bd biosciences, usa). cytokines production was measured in the same cultures by bioplex technology (biorad, usa). in the negative control % pbl in both groups did not enter mitosis. in the positive control % of cells have passed one and more mitoses. in group measles or rubella antigens did not induced lymphocytes to enter mitosis, like in negative control, before the vaccination and in a week, however in months - % of lymphocytes demonstrated antigen-specific proliferation. in group , on the contrary, before the vaccination the most part of cells ( - %) has not entered division, but - % of cells have passed and more mitoses. in a week specific lymphocyte proliferation decreased and in months it was increased up to - %. production of the interleukin (il) , ifn-g, tnf-a, il- , il- was more informative than il- , il- , il- , il- . measles and rubella antigens induced cytokines production in pbl of immune children and did not influence on pbl of intact children. thus, it was shown, that both methods can be applied to revealing the specific cellular immune response to measles and rubella antigens. objectives: broadly neutralizing human monoclonal antibodies (mab) and patients' sera recognizing functionally conserved epitopes on hiv envelope (env), such as the gp cd -binding site (cd bs), appear to be uncommon. therefore, new approaches are needed to elicit the humoral response on these conserved epitopes. here we describe the generation of two anti-idiotype (ai) murine antibodies recognizing human anti-hiv- neutralizing polyclonal iggs directed against the cd bs. the mabs were shown to react with an anti-cd bs human neutralizing mab (b ), to elicit antibodies that recognize the gp molecule and an anti-hiv- neutralizing response in rabbits, confirming them as cd bs mimotopes. these mabs were also used as probe to detect the expression of clonally distinct anti-gp antibodies in sera of hiv-infected individuals. methods: broadly neutralizing sera were collected from long-term non-progressor patients. anti-cd bs iggs were purified and used to immunize mice for hybridoma generation. mabs reacting in elisa with the anti-cd bs igg fraction were used to immunize rabbits. rabbit sera were then tested for anti-gp titer and hiv neutralizing activity by pseudovirus-based neutralization assay. sera from hiv-infected individuals at various clinical stage of infection were studied to validate an immunoenzymatic assay able to detect the reactivity to the ais. serial dilution of b in sera from healthy hiv-negative donors were used to determine elisa sensitivity. results: two clones (p and p ) reacted in elisa only with the cd bs-directed igg fraction. the clones were also recognized in elisa by b . p and p -immunized rabbit sera showed a strong anti-gp titer. in the pseudovirus assay the ais-immunized rabbits showed a neutralization activity against virions bearing hxb strain glycoproteins. in particular, / rabbits in the p group and / in the p group showed an % hiv neutralization at dilutions ranging from : to : . the immunoenzymatic assay used, allowed to detect a p and p reactivity in hiv-positive sera and was able to detect a b concentration equal to ng/ml. conclusions: these data demonstrate that immunogens designed on the idiotype of broadly neutralizing abs are feasible and could help in the design of effective anti-hiv vaccines or diagnostic assays. yellow fever vaccines ( d and dd) are well tolerated, with a very low rate of severe adverse events (yf-sae), such as serious allergic reactions, neurotropic (yf-and) and viscerotropic (yf-avd) diseases. viral and host factors have been postulated to explain the basis of yf-sae, especially those able to modify the host immune response to the yf vaccine. however, the mechanisms underlying the occurrence of yf-sae still remain unknown. in the present investigation, we present a detailed immunological analysis of a -year-old us citizen female patient, who developed yf-and characterized by encephalitis associated with pancreatitis and myositis following d- vaccination. our findings highlighted that yf-and exhibited decreased expression of fc-g-r in monocytes (cd , cd and cd ) along with increased levels of nkt-cells (cd + cd +/-cd +/-/cd + ratio) and activated t-cells (cd + and cd + ) and b-lymphocytes. enhanced levels of plasmatic cytokines (il- , il- , il- , il- and il- ) besides exacerbated ex vivo intracytoplasmic cytokine pattern, mainly observed within nk-cells (inf-g + , tnf-a + and il- + ), cd + t-cells (il- + and il- + ) and b-lymphocytes (tnf-a + , il- + and il- + ). the analysis of cd + t-cells revealed a complex profile with increased frequency of il- + and ifn-g + and decreased percentage of tnf-a + , il- + and il- + cells. depressed cytokine synthesis was observed in monocytes (tnf-a + ) following in vitro antigenic stimuli. these results support the hypothesis that a robust magnitude of the adaptive response and abnormalities in the innate immune system may be involved in the establishment of yf-sae. this is the first case report of yf-sae investigated by members of the international laboratory network for yellow fever vaccine associated adverse events. g. mester , h.-g. rammensee , s. stevanović eberhard-karls-universität tübingen, department of immunology, tübingen, germany adenovirus (adv) is a widespread pathogen in humans and can persist in its hosts after infection. persistent virus is an important cause for severe disease in immunocompromised individuals, e. g. bmt recipients, with high rates of mortality. however, the cellular immune response against adv is poorly characterised, and very few t cell epitopes have been published up to now. thus, our aim was to detect dominantly immunogenic adenoviral cd t cell epitopes by analysing the responses of healthy blood donors who have overcome infection. we have predicted possible cd t cell epitopes for the frequent mhc class i alleles a* , a* , and a* from the proteins pii (hexon), pviii, and e a of adv strains ad and ad by using the syfpeithi software developed by our group (www.syfpeithi.de). subsequently we performed a -day recall stimulation of pbmcs from at least healthy donors with synthetic peptide followed by ifn-g elispot screenings to identify naturally occurring t cell responses and assess their frequency in the population. tetramer and intracellular cytokine stainings were also carried out to confirm the presence of specific cd t cells. we could identify new peptides eliciting ifn-g responses, several of which were confirmed as novel cd t cell epitopes. amongst others we found at least one immunodominant epitope recognised by more than half of the healthy donors for each examined hla restriction as well as, to our knowledge, the first adenoviral epitope derived from a protein other than hexon. these findings will be helpful to identify frequently immunogenic and thus promising candidate peptides for in vitro t cell priming or expansion preceding adoptive transfer, which has been proven to be a valuable therapeutic approach in the treatment of persistent viruses in immunocompromised patients. methods: sle ( ara criteria) was diagnosed in a -year-old african female patient with hiv- (clade c) infection. good initial response occurred on hydroxychloroquine and steroids followed by disease flare and drop of cd t-cell count x cells/mm . initiation of mg mmf bid was associated with biological and clinical remission of sle and cd t-cell increase. no opportunistic infections or cancers were noted during a -year follow-up and the patient remained always naive to art. hiv- -specific cd and cd t-cell responses were analyzed after months of mmf by ifn-g elispot assay and polychromatic flow cytometry assessing ifn-g, tnf-a and il- production following stimulation with a panel of hiv- -derived optimal epitopes ( / -mers) covering various hiv regions and a pool of hiv- -derived peptides ( -mers overlapping by aminoacids) encompassing the entire gag protein. all peptides are derived from hiv- consensus strain iiib. results: highly polyfunctional hiv- specific cd and cd t-cell responses against gag were detected. epitope-specific cd t-cell responses were identified: except for one response restricted by hla a* and another one by hla cw* , all the others were restricted by hla-b alleles and mostly by b* (n = ). seven out of responses were strong enough to be further analysed with regard to their functional profile and shown to be highly polyfunctional (i. e. ifn-g+, tnf-a+ and il- +) regardless of the viral region and hla restriction. conclusion: strong, broad and polyfunctional hiv- specific cd and cd t-cell responses known to be associated with nonprogressive infection were detected during mmf treatment.we therefore suggest that mmf use in the context of sle-hiv is not detrimental to the establishment or preservation of protective hiv- t-cell immunity. the rabies virus was propagated in the vero cell line. virus was titrated by focus fluorescent units. virus preparations having a titer of dl /ml were inactivated with b-propiolactone. aluminium hydroxide gel or squalene, at different concentrations were adsorbed to the inactivated rabies virus. male mice of the strain cf- of - g and no less than four weeks age, were distributed in six groups for intraperitoneal immunization, group a was immunized with virussqualene, group b with virus-aluminium hydroxide, group c with the antigen alone, group d with saline buffer-squalene, group e with saline buffer-aluminium hydroxide and group e was inoculated with mock-infected cell culture supernatant. mice were boosted at the th day. all mice were properly bled to prepare preimmune sera and hyper-immune sera. at the end of the immunization protocol the igg raised against the rabies virus was tested by an indirect elisa. results: the highest titers of neutralizing antibodies were obtained with similar concentrations of either squalene-or aluminium hydroxide-based vaccine formultaions. there was a significant difference in the neutralizing antibody titers produced by mice immunized with the antigen (inactivated rabies virus) adsorbed to the adjuvant, as compared to those obtained from mice immunized with the antigen alone, as expected, no neutralizing antibodies were detected on mice inoculated with saline buffer or mock-infected vero cell supernatant. conclusions: the use of either squalene or aluminium hydroxide as adjuvant in the canine antirabic vaccine formulation increases immunogenicity, almost to the same extent. aluminum hydroxide adsorbed to the antigen seems to be a better option, since squalene is more expensive than aluminium hydroxide. supported by: concyt- , cofaa and cgpi- . . state of vaccine-induced measles immunity was determined by means of elisa in - , - and - years since revaccination with live measles vaccine (lmv) before and after tuberculosis chemoprophylaxis. statistic data were processed with t-, w-and u-criteria. results: during the first three years since lmv revaccination igg level was middle (children with negative and long-term positive mt) and high (children with conversion and hyperergic mt). in - years since lmv revaccination uninfected and long-term infected children showed a significantly decreased (p p . ) measles immunity and antibody level much lower (p p . ) than among children with mt conversion. in - years the comparison group kept decreased (p p . ) measles immunity, the majority ( ± . %) of persons had minimal protected igg level, but the observation groups were characterized by average immunity level, which was higher (p p . ) than in the comparison group. comparing measles immunity level before and after tuberculosis chemoprophylaxis demonstrated the following: measles igg level among long-time infected children on completion of chemoprophylaxis decreased (p p . ), the majority ( . ± . %) of persons lost protected antibody level; among children with mt conversion in - years since lmv revaccination immunity state didn't change, but in further periods antibody level decreased (p p . ) to low values; among children with hyperergic mt igg level decreased (p p . ) and reached low (in - years), minimal protected (in - years) and lower than protected (in - years) values. -at the early stage of tubercular infection process measles immunity was higher compared to uninfected with mycobacterium tuberculosis persons, which fact is connected with immunomodulatory action of low-molecular peptide of bacterial cell wall -muromildipeptide.-in remote periods since lmv revaccination and on completing preventive tuberculosis treatment decreased measles immunity was observed.-in countries with high tuberculosis morbidity chemoprophylaxis level among children with latent infection is high, which can indirectly influence population measles immunity. objectives: to evaluate the balance of ifn-gamma and il- producing cells in lungs during the immunotherapy of tuberculosis with the dna vaccine encoding the heat-shock protein (dnahsp ). methods: balb/c female mice were infected by intra-tracheal route with h rv mycobacterium tuberculosis. immunotherapy with endotoxin free dnahsp genetic vaccine was done at days , , and post-infection. each dose consisted of micrograms of dna vaccine in the quadriceps. intracellular cytokine staining of cd +, cd + and gamma-delta t cells from lungs were determined and days after the end of the therapy. bacilli loads, histopathological and morphometric analysis of lungs were also evaluated. differences of p x . were considered significant (t test). results: at day after the end of the immunotherapy, dnahsp treated mice exhibit increased numbers of absolute cd + and gamma-delta t cells when compared to non-treated animals. the percentage of ifn-gamma and il- producing gamma-delta t cells were the same between treated and non-treated animals. in contrast, dnahsp treated mice showed more ifn-gamma producing cells in both cd + and cd + cell populations. at day after the end of the therapy, the main observation in mice which received dnahsp treatment was the augment of all three populations producing ifn-gamma. although non-treated animals also increased the frequency of cd + and gamma-delta t cells positive for ifn-gamma, they did not increase the numbers of ifn-gamma cd + cells, together with a more frequency of gamma-delta t cells producing il- . finally, the immunotherapeutic effects of dnahsp vaccination also included the diminution of bacilli loads in lungs, spleen and liver and the reduction of inflammation in lungs as determined by the histopathological and morphometric analysis. the results presented here indicates that cd + cells producing ifn-gamma and the reduction of the frequency of gamma-delta t cells secreting il- , are the main effects of dnahsp immunotherapy of murine tuberculosis. furthermore, these results have important implications since they indicate the importance of an appropriate balance of il- and ifn-gamma levels for the combat of the bacilli and the reduction of the immunopathologic damage in lungs. the detection of quantitative changes in mrna expression levels are currently being performed using either genome-wide (microarray) or single gene (real-time pcr) screening methods. because these techniques are technically challenging and too costly to be applied on a routine basis in resource poor settings, we have developed a reverse-transcriptase multiplex ligation-dependent probe amplification (rt-mlpa) method. rt-mlpa is a reliable, robust, low cost and user friendly technique permitting rapid mrna expression profiling of as many as loci in a single reaction. genes of interest can be selected on a tailor-made basis. the assay is highly reproducible, has an extensive dynamic range of - log depending on the genes of interest, and a pcr amplification step within the rt-mlpa ensures assay sensitivity, which is an essential prerequisite for the relative quantification of scarcely expressed genes. since this assay is relatively high throughput ( -well format), requires only ng rna per sample, and allows mrna profiling in direct ex vivo whole blood samples (from e. g. pax-gene tubes), it is an exceptionally suitable technique for performing semi-large scale gene expression analyses in human cohort studies. to illustrate this, we have been able to successfully implement this assay in different laboratories in sub-saharan africa. thus far we have applied rt-mlpa to characterize the human immune response to mycobacterium tuberculosis, with particular emphasis on the expression of genes associated with protective host cellular immunity and human disease susceptibility. a particularly useful application of the rt-mlpa is the identification and monitoring of host-biomarker profiles that predict (protection from) tuberculosis (tb) disease in latently infected household contacts or (in)adequate responsiveness to therapy in active tb patients. initial data sets already probe differences in immune reactivity in populations, yielding new candidate biomarkers associated with tb disease. these biomarkers may provide new and relevant information that can be applied in future tb studies for rapid, easy, semi-quantitative and reliable detection of host immune biomarker profiles. preclinical m. leprae infection is a major source for leprosy transmission. therefore, early detection of individuals infected with m. leprae is crucial. however, to date there are no diagnostic tests available that can identify preclinical leprosy. such tests will contribute to the prevention of leprosy disability and its further transmission by otherwise undiagnosed and untreated index cases.newly developed hla based bio-informatic tools combined with comparative genomics have created novel opportunities to help design improved tests for early detection of m. leprae infection.using this post genomic approach, we were able to identify candidate proteins and peptides unique to m. leprae containing predicted t cell epitopes restricted via several major hla-class i and ii alleles. since the selected genes were of unknown function, their expression in m. leprae bacilli was assessed.evaluation of the immunogenicity of these m. leprae proteins in pbmc from a brazilian population showed that candidate antigens induced significant ifn-g levels in m. leprae infected individuals but not in healthy controls from an endemic area.importantly, among exposed healthy controls % had no detectable igm antibodies to the m. leprae specific pgl-i, but instead responded to one or more m. leprae antigen(s). to further improve the diagnostic potential of these m. leprae sequences, synthetic peptides spanning all m. leprae proteins were analyzed similarly. determination of cumulative t cell responses towards of these peptides that activated pbmc of leprosy patients increased the sensitivity compared to single peptides to % in pb, % in rx and % in hhc, without compromising specificity.since diagnostic tools should be applicable in several populations regardless of the genetic background, these m. leprae antigens are also tested in populations on the african (ethiopia) and asian (nepal) continent.in addition, we have applied these antigens in a new user-friendly ucp-lf assay to detect different cytokines. this assay proved to be more sensitive than elisa for detection of ifn-g and can be easily applied in field sites. tuberculosis, an infectious disease caused by mycobacterium tuberculosis (mtb), affects millions of people. m. bovis bcg is the vaccine against tuberculosis but its efficiency is variable for the pulmonary form of the disease. paratuberculosis, an enzootic bacterial disease in ruminants, due to mycobacterium avium subsp. paratuberculosis (map), has a significant economic impact on livestock production, and moreover, map infection may be one of the microbial triggers of crohn's disease in humans. map vaccines can delay apparition of clinical symptoms, but they do not prevent infection and they have a confounding effect in the skin-test based bovine tuberculosis control programs. cd l, a co-stimulatory molecule preferentially expressed on activated cd + t cells, is the ligand of cd . cd -cd l interaction induces the production of il- and the initiation of a th -type immune response. several studies show that cd l is required for the activation of macrophages and the maturation of dcs. moreover, cd l enhances the capacity of cd + t cells to produce ifn-g and to lyse mtb-infected monocytes. in this study we attempt to improve existing tb and map vaccines with a recombinant bcg expressing cd l. we have constructed the recombinant bcg strain expressing cd l (rbcg ) by electroporation of bcg with pgfm /signalsequenceag b-cd lec and an another recombinant strain with empty vector pgfm (rbcg ) as a control. the expression of cd l has been evaluated by western blotting. balb/c mice were vaccinated with the recombinant bcg vaccines. bcg growth kinetics were compared by counting viable bacteria (cfu) in spleen and lungs. the immune response was evaluated by measurement of th type cytokine secretion of splenocytes after in vitro restimulation with immunodominant antigens and selected peptides. two months post vaccination, mice were challenged with mtb and map and protection was evaluated. preliminary results show normal persistence of the two recombinant bcgs. analysis of the immune response shows an effect of cd l weeks after vaccination but not at and weeks. rbcg seems to be more protective against paratuberculosis than rbcg , but not against tuberculosis. another vaccination experiment is required to confirm these results. the effects of bcg-cd l on cultured dcs in vitro will further be explored. objectives: tuberculosis is a major health problem globally and it is of critical importance to develop an effective vaccine to prevent further spread of the disease. iron is a key nutrient for both mycobacterial infection and for a successful protective immune response by the host. the regulation of iron availability within the host involves the intracellular iron-binding protein ferritin and it is proposed here that the regulation of ferritin is tightly controlled in the host immune response to tuberculosis. methods: using the guinea pig model of mycobacterium bovis bacillus calmette-guérin (bcg) vaccination, populations of immune cells were isolated and restimulated ex vivo over a time-course study using purified protein derivative (ppd) of mycobacterium tuberculosis or infected with bcg or m. tuberculosis. the expression of ferritin in co-ordination with key immuno-regulatory proteins, tnfa, ifng and il- a, was examined using real-time pcr. to determine whether immuno-regulatory proteins are involved in the regulation of ferritin, cytokine cascades were inhibited in the ppd re-stimulation studies by the addition of guinea pig specific tnfa and ifng antibodies. results: a typical pro-inflammatory immune response was observed with significant up-regulation (p x . ) of tnfa, ifng and il- a after re-stimulation with ppd and mycobacteria. of interest was a trend in ferritin down-regulation after re-stimulation with ppd and bcg and this was significant (p x . ) after restimulation with m. tuberculosis. the down-regulation of ferritin was also affected by the addition of tnfa antibody in the ppd re-stimulation study. conclusions: ferritin is important in the storage and management of intracellular iron and its regulation must be tightly controlled to restrict iron availability from invading mycobacteria from sequestering free iron. the data indicate that the regulation of ferritin is very subtle and is affected by cytokine cascades that involve tnfa. these results contribute to our understanding of the role of iron and intracellular ferritin in developing a protective immune response to mycobacteria in the guinea pig model of tuberculosis. this work is funded by health protection agency phd studentship award. methods: anti-cd mab and ag a were chemically treated with sata and sulfo-smcc respectively, in order to produce a stable crosslinker between both proteins. crosslinking was confirmed by western blotting and cd binding on cd transfected l fibroblasts. the conjugates were tested in vivo in wild type and cd + cell-depleted mice for the induction of specific anti-ag a serum antibodies. splenocytes were challenged ex vivo with ag a and were examined for their ability to produce th -related cytokines. elispot assays were performed to determine ifng production and flow cytometry was used to analyse intracellular cytokine staining for tnfa, ifng and il- . we developed a method to successfully crosslink anti-cd mab to ag a. serum antibodies against ag a were detected after immunisation with this conjugate vaccine in both wild type and cd + cell-depleted mice. t cells derived from mice immunised with conjugate vaccine, and stimulated ex vivo, showed an increase in ifng production (elispot), when compared to mice vaccinated with ag a alone. production of two other th -related cytokines, tnfa and il , was also increased in these t cells as shown by intracellular cytokine staining. conclusion: our results suggest that anti-cd conjugate vaccines could provide a new way to increase vaccine efficacy. this new conjugate vaccine may be able to by-pass the need for cd + t cell help in the production of specific antibodies, which would be a major benefit of any therapeutic vaccine to be used in immunocompromised patients. h. schäfer , r. burger robert koch-institute, cellular immunology, berlin, germany, robert koch-institute, infectious diseases, berlin, germanyobjective: immunity against mycobacterial infections is mediated by both cd -positive and cd -positive t-lymphocytes. cd -positve cells respond to peptides derived from cytosolic proteins and presented on mhc class i molecules of antigen presenting cells (apc) via the endogenous pathway. some apc however, are able to take up extracellular antigens and present peptides thereof on mhc class i molecules. this process has been termed cross-presentation and has been shown to be of importance in the immune response against intracellular bacteria. to define the contribution of cross-presentation to activation of cd + t cells in the response against mycobacterial antigens, we analyzed the secondary immune response in the guinea pig. methods: purified t lymphocytes from guinea pigs immunized with bcg or complete feund's adjuvant were labeled with the intracellular fluorescent dye cfse and incubated with ppd and/or apc for days. surface phenotype and proliferation of t-lymphocytes were analyzed by flow cytometry.results: up to % of lymph node t lymphocytes form immunized guinea pigs proliferated after in vitro restimulation with ppd-pulsed macrophages. no difference was observed between bcg-(living mycobacteria) and freund's adjuvant (heat killed mycobacteria)-immunized animals. the responses of both t cell subsets were equally strong, although the killed immunogen should primarily target the exogenous pathway of antigen presentation and therefore preferentially prime cd + t-lymphocytes in vivo. similarly, the cd -positive subpopulation should primarily respond to soluble antigens presented on mhc class ii molecules. proliferation of both the cd + and the cd + subpopulation depended on the presence of apc. stimulation oft cd + cells as a consequence of direct loading of peptides onto mhc class i molecules was ruled out by using mhc-class i-positive fibroblast cells instead of professional apc, which did not lead to proliferation of primed t-lymphocytes. conclusion: cross-presentation of soluble antigens to cd -positive t cells is a highly effective means to stimulate the response of cytotoxic t cells against mycobacterial antigens even without direct contact to infected cells. therefore cross-priming might represent an important mechanism for the induction of the cellular immune response against intracellular pathogens and should be useful for the rational design of vaccines against mycobacterial diseases. objectives: due to broad antigenic cross reactivity of purified protein derivatives (ppd) with bcg vaccine strains and environmental mycobacteria, results of currently used tuberculin skin test (tst) is not reliable to evaluate the specific anti-tuberculosis immune response. therefore, new tools are required to improve mycobacterim tuberculosis (tb) diagnosis and treatment, including enhanced ability to compare new treatment strategies. among different antigens early secretory antigenic target (esat- ) protein is highly specific for tb complex and elicit strong t-cell response in human. in order to monitor the immune response against the pathogen, iranian and afghan adults ( patients with sputum smear and culture positive tuberculosis, recovered patients during months after full course of chemical treatment and healthy individuals) were recruited to quantify the frequency of esat- and ppd specific t-cells in their peripheral blood by home made elispot ifn-gamma assay. results: considering cut off of spot forming unit ( g spots per million), we found detectable response to esat- in almost % of patients with active disease. this frequency among treated patients after disease recovery was not significantly different and % of these individuals had detectable esat- specific response even after six months completing treatment. neither of healthy individuals showed such response. t cell response against ppd was identified in %, % and % of healthy participants, active patients and healing individuals, respectively. conclusion: elispot ifn-gamma assay showed a sharp induction of th immune response, against esat- , in tb patients which persists after successful treatment and full recovery. these results may show potential application of tuberculosis-specific elispot testing as a proxy measure of tb diagnosis and treatment. bcg is the only available vaccine today to fight tuberculosis, but it has been reported to be variably efficacious in the field. both environmental and genetic host factors as well bcg strain variability and virulence of the intruding m. tuberculosis strain have been suggested to affect the efficacy of bcg vaccination. in mouse and bovine models it has been shown that pre-exposure to mycobacterium spp. negatively affected protective efficacy of bcg. we use non-human primates (nhp) for the evaluation of new tb vaccine candidates and possible identification of immune mechanisms of protection. however, in naive rhesus macaques a variable efficacy of bcg reminiscent of the clinical situation was revealed. by meta-analysis we compared immune response parameters measured after vaccination using bcg strain danish in the context of protection measured by gross pathology evaluation after experimental infection with a constant m. tuberculsosis strain erdman. although numbers of animals used are relatively low, data suggest that both breeding origin as well as the immune status of monkeys impact on the efficacy of bcg. most remarkably, while bcg induced levels of ifng secretion did not correlate with protection, kinetics of secretion monitored after in vitro stimulation of peripheral blood lymphocytes did correlate. our findings would suggest that, in accordance with mouse and bovine experimental data and epidemiologic observations, possible pre-exposure to mycobacterial antigens beyond the current sensitivity of tb diagnostics for nhp, negatively affects the protective efficacy of bcg. together, these results are relevant for evaluation and interpretation of tb vaccine tests in nhp and support further research into the identification of (mechanisms of) protective immunity in the primate host. p. s. nagpal , p.k. upadhyay national institute of immunology, pdc- , delhi, indiaobjective: study was aimed for the preparation of dry powder formulation containing live mycobacterium indicus pranii for pulmonary immunization against tuberculosis. pulmonary delivery evokes both systemic as well as mucosal immune response against the antigen. secondly, pulmonary delivery is a needle-free delivery system, long desired for vaccine delivery. dry powder aerosol one such method, in which vaccine can be directly delivered to lung without any kind of invasion and it has an edge over liquid formulation being feasibility of storage at room temperature, long term shelf stability and higher drug content per unit mass as compared to liquid one. method: sodium alginate solution with suspended mycobacterium indicus pranii was aerosolized using laboratory modified nebulizer assembly. aerosols so generated were entrapped in cacl solution with poly-vinyl alcohol (pva) as surfactant. particle so formed collected by centrifugation and lyophilized for dry powder formulation. pva and alginate concentration varies the size, surface and shape of the particles. formulation so prepared was delivered to c bl/ mice directly into lung by endotracheal intubations of mice. proliferation index (pi) of spleenocytes of immunized mice was measured after in-vitro stimulation with mycobacterium tuberculosis antigen. result: . % alginate, . % pva concentration gives particles with size of - micrometer as confirmed by particle size analyzer and scanning electron microscopy. viability of the mycobacterium indicus pranii was best achieved with % trehelose and . % pvp (poly vinyl pyrollidone). there was fold increase in proliferation index of spleenocytes and releases pico-gram of interferon gamma after week of immunization. formulation also induces the activation of dendritic cells after their in-vitro incubation as shown by % increase in cd and . % in ccr expression as compared to blank alginate formulation. bacterial exopolysaccharides (epss) are heterogeneous polymers containing a wide array of homo-or hetero-carbohydrates as well as organic and inorganic substituents. epss are produced by many bacteria and play a critical role in helping these microorganisms to cope with adverse environmental conditions. some epss contain sulphate groups as inorganic substituents. the presence of these groups contributes to the biological activity of epss, which have been shown to have anticoagulant, insulinotropic, antiviral, antitumoral and immunomodulatory properties, among others. b is a constitutively sulphated eps produced by halomonas maura, a recently discovered halophilic bacterium. in preliminary experiments we found that modification of its eps by adding sulphate groups to the native polymer (thus obtaining b s) resulted in vigorous antiproliferative activity in several haematopoietic tumour cell lines. at the same time we found that other epss produced by closely related strains had only a very limited antiproliferative effect on these same tumour cells. it was therefore of interest to determine whether the antiproliferative activity of b s was mediated by the induction of apoptosis, and if so, to dissect the pathway triggered by b s. by cell cycle analysis we determined that b s is able to induce apoptosis in up to % of jurkat and molt- t cell leukemias. the examination of a large panel of haematopoietic and nonhaematopoietic cell lines revealed that apoptosis induced by b s is restricted to cells of the haematopoietic lineage and that leukemic t cells are particularly sensitive to death induced by b s, but that untransformed cells are not. a time-course of caspases activation indicated that caspase is the first to be cleaved, followed by caspases and , thus suggesting that b s triggers apoptosis through the mitochondrial pathway. it is noteworthy that b s also induces vigorous apoptosis in primary leukemic t cells obtained from the peripheral blood of patients. therefore, b s may well provide a satisfactory therapeutic alternative to patients with acute t cell leukemias, since current antitumoral drugs are very inefficient in the treatment of these types of cancer. particulate antigen delivery tools have been shown to enhance the induction of immune responses by targeting dcs. polyelectrolyte microcapsules form a new class of microcapsules generated by the sequential adsorption of oppositely charged polyelectrolytes onto a sacrificial spherical template which is consequently dissolved, yielding a hollow microcapsule surrounded by a thin shell. this layer-by-layer approach allows an efficient incorporation of macromolecules under nondenaturing conditions. by using the biopolyelectrolytes dextran-sulphate and poly-l-arginine, biodegradable microcapsules can be obtained. in this study, we have chosen the lungs as a non-invasive route for vaccine delivery. as demonstrated by flow cytometry and confocal imaging, dextran-sulphate/poly-l-arginine microcapsules were readily taken up by local pulmonary apcs and transported to the mediastinal lymph nodes, making them excellent tools for antigen targeting towards apcs. microcapsule instillation also affected the pulmonary apc activation status, indicated by the emergence of an apc population expressing increased levels of mhcii, the co-stimulatory ligands cd , cd and cd , and of the inflammatory cytokines il- , il- and mcp- . using ovalbumin (ova) as a model antigen, we have analysed the adjuvant properties of these polyelectrolyte microcapsules. analysis of the alveolar infiltrate, cd t cell and antibody profiles revealed that polyelectrolyte microcapsules display different adjuvant properties than the standard th and th /th skewing adjuvants alum and complete freund's adjuvant (cfa). in response to ova aerosol exposure, microcapsule based vaccination resulted in an alveolar infiltrate dominated by monocytes, while alum and cfa respectively induced typical eosinophilic and neutrophilic inflammations. striking differences were also observed on the level of cd t cell responses. microcapsule based vaccination resulted in a marked induction of il- secreting th cells, without inducing strong th (cfa) or th (alum) responses. these differences were also reflected on the level of the humoral immune response, with microcapsules being the sole adjuvant producing antibodies of all isotypes tested (igg , igg c and ige).in conclusion, polyelectrolyte microcapsules allow an efficient targeting of antigens to lung apcs, and possess immune stimulating activities distinct from alum and cfa. due to their capacity to generate th responses, polyelectrolyte microcapsules may become interesting tools to combat fungal and bacterial infections. pneumolysin is an important virulence factor produced by virtually all clinical isolates of streptococcus pneumoniae. the protein binds to cholesterol in cell membranes and creates transmembrane pores, leading to cell lysis. published findings have proposed that, at sublytic concentrations, the toxin causes a range of effects including activation of host complement, activation and chemotaxis of cd + t cells and increased production of pro-inflammatory cytokines in immune cells. in this study we investigated the interaction of pneumolysin with murine dendritic cells (dc). we found that pneumolysin induced the activation of dc, reflected in the enhanced expression of the costimulatory molecules cd , cd and cd and mhc class ii molecules. the toxin alone was found to be a poor inducer of cytokine production by dc but it did enhance the secretion of tlr agonist-induced cytokines such as il- and tnf-a. previous published findings have shown that pneumolysin activates peritoneal macrophages in a tlr -dependent manner. however, we found that pneumolysin was capable of activating dc from both wildtype and tlr -defective c h/hej mice, by inducing cell maturation and synergising with tlr agonists to enhance cytokine secretion. importantly, we also found that pneumolysin is a strong inducer of il- b secretion by dc, through its effects on caspase- processing, which is also tlr -independent. the results suggest that pneumolysin is a potent stimulus for dendritic cell activation and that this does not require tlr signalling. objectives: transmission of immune competence from mothers to newborns during pregnancy and lactation is crucial for education of neonate immune system in order to develop optimal protection against early life infections. the objective of the present study was to assess whether maternal supplementation with probiotics may enhance neonatal responses to measles immunization. methods: pregnant balb/c mice were supplemented with placebo (maltodextrin) or probiotics (lactobacillus paracasei ncc (st ) or lactobacillus rhamnosus ncc (lpr), each at x cfu/day), suspended in the drinking water, throughout the gestation period and up to the weaning of pups. at weaning, pups were immunized with live attenuated measles vaccine (mv-s, aventis-pateur). weight evolution of pups was followed from week to week of life. fresh feces were collected at , and weeks of life for determination of iga levels (assessed by elisa). pups were bled and weeks after immunization for determination of measles-specific igg and igg a antibodies. analysis of microbiota composition (plating on semi-selective agar media) was performed on fresh feces collected one week after weaning. results: all newborns grew normally and no significant differences in the weight were observed between the groups all along the trial. fecal iga production increased progressively in all pups from weaning, reflecting a normal development status. nonetheless, feeding mothers during pregnancy and lactation did not significantly affect post-weaning s-iga production in pups. lpr supplementation of the mothers significantly potentiated post-weaning measles-specific antibody responses in pups in comparison to control group. interestingly, no significant effect was observed in the st -fed group. finally, a modification of the microbiota composition was observed in pups of supplemented mothers. particularly, there was a significant increase in lactobacilli in pups from the lpr group as compared to controls. conclusion: this study supports the benefit of perinatal intervention with probiotics during pregnancy and lactation on immune maturation in the offsprings. moreover, these first results seem indicate that the effects are strain specific. chitosan, ( - )- -amino- -deoxy-beta-d-glucan, is a deacetylated form of chitin, an abundant biodegradable, positively charged natural polysaccharide. chitosan (chi) is used for antigen delivery through mucosal barrier due to its ability to disrupt tight junctions. here we studied the ability of chitosan nanoparticles to form complexes with proteins of different size and charge. nanoparticles (chi-np) were prepared from kda chitosan by ionotropic gel formation. bovine serum albumin (bsa) and myoglobin, human immunoglobulin g and superoxidedismutase (sod), and chicken lysozyme were fitc labeled. chi-np were preincubated with proteins at : ration and washed times. after washing chi-np containing bound proteins were run by denaturating gel electrophoresis. all proteins were able to form complexes. most effective binding was shown for bsa, sod, and lysozyme. the stability of chi-np complexes with proteins was studied in vitro on macrophage cell line raw . by confocal microscopy. for this chi was labeled with rhodamine and nanoparticles were coincubated with fitc labeled proteins before addition to the cells. we showed co-localization of chi and fitc for all proteins studied. these results demonstrate that chi-np form stable complexes which are internalized by macrophages. the family euphorbiaceae consists of a large group of plants whose compounds have been documented to possess anti-inflammatory activities, however, their effects as modulators of innate or acquired immunity has not been described yet. in the present study, different aspects of the immunomodulatory activity of extracts from euphorbiaceaes on peripheral blood mononuclear cells (pbmc) from healthy individuals were evaluated. the pbmc were exposed to the extracts w/o phytohaemagglutinin a (pha), cycloheximide (chx) or lipopolysaccharide (lps) as agents that induce proliferation, apoptosis and cytokine production in pbmc. the lymphoproliferative activity of pbmc was evaluated by thymidine incorporation and cfse dilution assay using flow cytometry. the mitochondrial membrane depolarization (as an early apoptosis indicator) was measured using dioc /propidium iodide staining by flow cytometry and tnf-a secretion in the culture supernatans by elisa. we found that up to euphorbiaceae's extracts had the ability to modulate one or more of the immune parameters evaluated in this study. however, only the bark extract of croton spp. insoluble in hexane:diclorometane:methanol (hdm) and the latex extracts of euphorbia cotinifolia and euphorbia tirucalli induced strong proliferation, apoptosis and also tnf-a production in pbmc. these extracts were subfractioned by sephadex column chromatography obtaining three subfractions with enhanced activity in comparison to the crude extracts. additionally, we started with the characterization of the specific immune effects of these subfractions on pbmc. all three subfractions induced proliferation predominantly on cd + cells. these effect was also observed in isolated t cells indicating that accessory cells are not necessary for the subfractions'activity. the lymphoproliferative activity of these subfractions was also not inhibited by the carbohydrates d-(+) galactose or a-methyl-mannopyranoside. these results demonstrate the presence of immunomodulatory compounds in plants from the euphorbiaceae family and suggest an antigen-presenting cell-and carbohydrate-independent mechanism of the subfractions to exert their effects. we found significant increase on lymphocytes and eosinophils populations obtained from lps + p. acnes-treated group in relation to control group.on th day, we detected a significant negative correlation between eosinophils absolute number and fec. both il- and ige serum levels were increased on animals from group i when compared to control.the enhancement on th immune response pattern induced by lps and p. acnes treatment diminished drastically parasitic load. conclusion: our findings support the idea of the use of immunostimulant as a helminthiasis control strategy in sheep, which stimulate non-specific mechanisms of resistance and therefore can act against nematodes infections. vaccines based on partially purify populations from the organism or recombinant subunits proteins have been recently developed and are often not sufficiently immunogenic by themselves due to the lack of innate immune stimuli. indeed, current influenza a vaccines do not generate significant immunity against serological influenza a virus subtypes and would thus be ineffective in the face of a pandemic novel variant. hence adjuvant usually needs to be added to those types of vaccines. here we show that wittycell compounds significantly augment cellular and humoral immune responses to commercial seasonal influenza vaccines. experiments performed in mice showed induction of specific cd + ctl cells against conserved proteins that were accompanied by the induction of ifn-g producing cd +t cells, following single immunisation. in addition increased hi titres and higher levels of specific igg a and igg b antibodies were found even long periods after single vaccination with reduced doses of vaccines. consequently, protection from lethality was observed following challenge with homologous or heterogonous influenza viruses in vaccinated animals. this promising finding on the improvement of seasonal influenza vaccines by wittycell compounds in these preclinical studies strongly provides support for the careful evaluation in phase i clinical trials in humans. s. lindgren , , n. almqvist , a. lönnqvist , s. Östman , c. rask , e. telemo , a.e. wold university of göteborg, department of clinical bacteriology, göteborg, sweden, university of göteborg, department of rheumatology and inflammation research, göteborg, swedenobjective: dietary antigens normally evoke immunological tolerance. a prerequisite is their processing by intestinal epithelial cells, which leads to the appearance of a tolerogenic form in the serum of fed animals that confers antigen-specific tolerance when transferred to naï ve recipients. the gut microbiota may influence the handling of dietary antigens as atopic diseases have increased in western societies in parallel with reduced complexity of the infantile commensal microflora. we have observed that children neonatally colonized with s. aureus in the gut seem protected against development of food allergy. here we examine whether a s. aureus toxin affects tolerogenic processing by the intestinal epithelium. methods: mice were given s. aureus enterotoxin a (sea; . mg/ml) in the drinking water for days and, days later, mg ovalbumin per os. one hour postfeeding, serum was transferred to naï ve recipients, whose tolerance to ovalbumin was tested in a model of allergic airway inflammation (sensitization followed by intranasal challenge with ovalbumin). results: recipients of serum from sea pretreated ovalbumin-fed donors exhibited increased tolerance compared to recipients of serum from ovalbumin-fed donors not pretreated with sea. this was demonstrated as reduced ovalbumin-induced airway inflammation with diminished influx of eosinophils into the lungs and reduced antigen-induced production of interleukin- and interleukin- . examination of gut sections from sea treated donor mice revealed increased density of cd a + intraepithelial lymphocytes. our results show that sea promotes oral tolerance induction, possibly by facilitating tolerogenic processing of soluble antigens by the absorptive intestinal epithelium via activation of intraepithelial lymphocytes. abstract withdrawn by author to develop an efficient vaccine against cp pneumoniae we cloned chlamydial genes encoding proteins of the outer membrane like ompa, omcb, and pmp , proteins of the inclusion membrane like incc, secreted proteins like cpaf, and the heat shock protein groel. cpg-dna , a highly stimulatory oligonucleotide for apcs, was used as adjuvans. subcutaneous co-injection of ompa, omcb, pmp , or groel together with cpg-dna reduced the chlamdial burden in nasally infected mice. however, symptoms like substantial loss of body weight were not influenced. in contrast, a low dose infection with cp. pneumoniae almost completely prevented the loss of body weight upon challenge. to improve the efficacy of the vaccine we used poly-dl-lactide-co-glycolide microspheres loaded with the protein ompa or pmp together with cpg-dna . the microsphere based vaccine offers the advantage that antigen and adjuvans are delivered to the same apc. intranasal but not subcutaneous vaccination of mice with ompa or pmp microspheres efficiently lowered chlamydial burden upon challenge and prevented loss of body weight. pmp microspheres induced protective ifng-secreting cd + t-cells and raised pulmonary pmp -specific iga levels in vivo. also, pmp microspheres caused lower il- serum levels upon administration than the injection of pmp together with cpg-dna , indicating fewer side effects. objectives: staphylococcus (s.) aureus superantigens are highly potent t cell mitogens and the causative agents of toxic shock syndrome (tss) and food poisoning. most s. aureus have superantigens and patterns are highly variable. to date, the role of superantigens in bacteraemia is not well defined.to analyse whether superantigens play a role in bacteraemia, we investigated s. aureus strains and anti-superantigen antibody responses in cases of s. aureus bacteraemia in iv drug users and cases in nonaddicts. a rise in neutralising antibody titers indicates that superantigens are produced during infection. the study comprised iv drug users with positive s. aureus blood culture and an equal number of age-and sex-matched nonaddicts from the original fintrova and finlevo trials (ruotsalainen ).all s. aureus isolates were analysed by sequence-based genotyping (spa-typing), and multiplex-pcr was applied to determine the superantigen gene pattern. sera from patients were obtained at diagnosis (day ) and four weeks thereafter (day ). neutralising capacity of the sera was tested against the superantigen cocktail produced by the respective infecting strain as well as a panel of representative recombinant superantigens.results: genetic analysis confirmed our previous observation that most strains harboured superantigen genes, which were linked to staphylococcal lineages (holtfreter ) . there were no major differences in superantigen gene patterns in isolates from iv drug users and nonaddicts. interestingly, the staphylococcal lineage st (spa-type t , agr , and sea, seb, sek and seq) was much more prevalent among bacteraemia strains from iv drug users than from nonaddicts (p= . ).most iv drug users had neutralising antibodies against enterotoxins already at onset of bacteraemia, likely due to previous encounters with the infecting strain. we frequently observed a rise in antibody titers during infection. surprisingly patients with st strains did not show any elevations in neutralising antibody levels. conclusion: s. aureus bacteraemia induces an antibody formation against staphylococcal superantigens. this indicates that superantigens are produced during infection. however, the action of superantigens is frequently modulated by specific neutralising antibodies. this and the special behaviour of s. aureus st strains need further investigation. objectives: down syndrome (ds) is associated with recurrent infections, hematological malignancies and auto-immune diseases, suggesting immunological changes. to test for more severely disturbed specific antibody response we investigated the antibody response to the highly immunogenic protein antigen tetanus toxoid (tt), which is part of the dutch immunization program. methods: after booster vaccination at and years of age, quantitative (titer) and qualitative (avidity) tt responses were investigated in and ds children, respectively. samples were taken before and - weeks after vaccination. tt-specific igg and igg-subclass antibodies were measured in serum by quantitative enzyme linked immunosorbent assay (elisa), avidity of igg -anti-tt by an avidity elisa. the results were compared with reference values from the laboratory. results: at years, post-vaccination anti-tt-titers were decreased (geometric mean total igg, igg , igg and igg ). at years, ds children had lower postvaccination geometric mean igg anti-tt-titers only. post-vaccination igg -anti-tt avidity levels were decreased in / and / ds children at four years and nine years of age, respectively. the quantitative and qualitative anti-tt-responses in both ds groups are shifted downwards compared to the reference values. although the anti-ttresponse increases towards normal titers with increasing age, the avidity (qualitative response) is still abnormal at that age, showing that ds children have profound and lasting difficulties with specific anti-tt antibody formation. kda; , kda; , kda; , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] kda and , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] kda were expressed by a majority of examined strains independently of the associated diseases. we assume that these omps could be conservative proteins of h. pylori. conclusion: considering omps as potential targets in the search for disease-related biomarkers and potential vaccine antigens, the identification of h. pylori omps as well as the elucidation of their role in modifying the host immune responses seems to be very important research subjects. the increasing cases of severe diarrhoea and invasive lethal infections in children caused by salmonella typhimurium are a major public health problem in mexico. the rapid dissemination of multidrug-resistant s. typhimurium, and the lack of a licensed vaccine against non-typhoidal infections reduce the possibilities of an effective treatment. the objective of this study was to evaluate if the high incidence of non-typhoidal multidrug-resistant salmonella infections was associated with a reduced in anti-salmonella immunity. a cohort of families, from a mexican agricultural community with a high incidence of endemic salmonella infections, was followed prospectively for an -month period. sera were obtained from healthy subjects from the same community ( months to years of age). the highest incidence of salmonella-associated diarrhea, / , occurred in children under years of age. the lowest incidence, / , was observed in the population aged to . whereas serum from individuals ranging - years of age showed maximum igg , igg and iga anti-s. typhimurium titres, children less than years-old did not show detectable igg and igg titres and had weak igg , iga and igm antibody levels; only their igg levels were comparable to those detected in adults. moreover, the levels of igg and igg antibodies were lower in adults with a diarrheal-associated episode. interestingly, s. typhimurium yuhs - , a commonly isolated human strain from this endemic area, resisted the complement-fixing activity of antibodies although it was sensitive to opsonisation and to fc-mediated phagocytosis by human monocytes. these data contributes to define the protective immune response involved in anti-s. typhimurium immunity. diseases caused by the yeast of candida genus are a serious clinical and social problem. despite this fact, there is no effective prevention against these opportunistic pathogens yet. although c. albicans is the major cause of the mycoses ( %), the number of the multiresistant non-albicans isolates increases. c. dubliniensis, which was described only recently as serious human pathogen, belongs to the group of these resistant isolates. the surface mannan of candida cells is component of the cell wall mannoprotein complex and participates in an initial contact with its host and subsequently with the host defense mechanisms. because of complexicity of this homopolymer it is necessary to identify a subunit of the mannan that is most effectively recognized by the immune system and thus influences the specificity of the induced antibody response (immunodominant epitope). in our study we prepared oligosaccharides from acid-stable part of mannan c. dubliniensis by conventional acetolysis. this procedure specifically cleaves the a- , linked mannopyranose units of mannan backbone and releases the side oligomannosyl branches. obtained oligosaccharides were used in inhibition elisa and spr (surface plasmon resonance) measurements. the reason of these measurements was to quantify interactions of these oligosaccharides with anti-mannan antibodies present in rabbit serum after -fold immunization with mannan-hsa conjugate injections in week intervals as well with inactivated c. dubliniensis whole z. neščáková , s. bystrický institute of chemistry, slovak academy of sciences, bratislava, slovakiaour newest approach to sub-cellular vaccine against gram-negative bacterial pathogens exploits detoxified lipopolysacharide (detoxified lps) as the target antigen. this is achieved by conjugation of carbohydrate to a protein carrier which secures the t cell dependent immune response. the goal of the immunization with this conjugate is to generate the effective production of memory b cells. here we prepared subcellular conjugate with the detoxified lps from vibrio cholerae strain o using polymer carrier and a protein. the cell immunity induced by the vaccination with the conjugate was evaluated in mice, namely their peripheral blood and the spleen. activation and differentiation of b-cell populations in the time-dependent manner was determined by flow cytometry analysis of these samples. a single-platform approach based on flow cytometry and defined number of fluorospheres was used to count b cells. however in our hands this method, previously used in humans, had to be adapted for mouse blood samples first. the protocol allows quantifying cells simultaneously with cytometric immunophenotyping without cell loss or other cell preparation steps. like pan-b cell marker cd , expressed almost on all blood and tissue b cells, was used. here we investigate the characteristics and development of antibody (iso)types after secondary immunization with mencc or plain polysaccharide and the possible role of certain antibody responses in maintaining immunity after vaccination. methods: volunteers, age - years, were immunized with mencc or received a secondary immunization with mencc or plain menc ps. blood samples were obtained before and seven time-points after immunization. igg, iga, igm, igg , igg and avidity were assessed by a multiplex immunoassay. functional antibodies were determined by a serum bactericidal assay. results: high levels of antibodies were still present years after primary mencc immunization. secondary immunization resulted in increased igg and sba titers after to days. in primed individuals, igm was still present, and this only increased following a secondary immunization with plain ps. in addition, immunization with ps induced a higher igg response compared to mencc immunization. discussion: secondary immune responses are quiet slow. the composition of the ig (iso)type distribution is different between mencc and plain ps and might be of influence on functional titers. although this study indicates that immunological memory was previously induced by a single mencc vaccination, it highlights the importance to sustain protective antibody levels against a rapid invasive organism such as n. meningitidis. the immunological effectiveness of these two semi-synthetic immunogenic conjugates was established according to antigen-specific titers of igg, iga and igm isotypes and by phagocytic and respiratory metabolic activities of granulocytes. results: prime-boost immunization strategy resulted in enhanced production especially dlps-specific igg and igm isotype antibodies in both experimental groups (peak titers : ). igm-igg isotype switch was more pronounced with o-sp. peak values of dlps specific iga isotype were signicantly lower than igg and igm ones ( : vs. : ). flowcytometric simultaneous determination of phagocytosis and stimulated oxidative burst of granulocytes revealed conjugate induced enhancement, more evident with o-specific polysaccharide and ip final boost (stimulation index was . fold of normal control). subcutaneous immunization gave a weaker stimulation: . fold of normal control. the second de-oac conjugate exerted different pattern of stimulation, sc intervention was more effective. our results are indicative for immunological effectiveness of novel dlps derived glycoconjugates; thus promising further application in cholera subcellular vaccine. this work was supported by apvv - and vega / / grants of the slovak grant agencies. background: the yeast candida albicans is an opportunistic pathogen that causes infections in immunocompromised individuals with a high morbidity and mortality levels. a long-acting, effective and safe vaccine that protects against medically important candida species should significantly reduce the incidence of various forms of candidasis by these etiologic agents. mannan, polysaccharide component exposed at the most external layer of the fungal cell wall, contains a backbone consisting of a- , -linked d-mannopyranose units and many branches composed of a- , a- , and/or b- , -linked mannopyranose units that are connected to the backbone. investigation of oligosaccharides immunomodulatory functions could be considered as an important part of their protective immunity against fungal diseases. objective: in this study, for mice immunization, synthetically prepared oligosaccharide (heptamannoside) conjugated to protein carrier (bovine serum albumin, bsa) was used. methods: in order to study the immunogenicity of heptamannoside -bsa conjugate as inducer of hummoral and cell-mediated responses, balb/c mice were subcutaneously immunized without adjuvant ( mg oligosaccharide per one conjugate dose) two times in days intervals and then intraperitoneally or subcutaneously boosted. cell-mediated and humoral responses were analyzed on day after injections by flow cytometric immunophenotyping of peripheral blood leukocytes and by measuring the levels of mannan specific antibodies presented in serum using elisa. results: prepared conjugate was immunogenic and re-injection elicited increase of mannan specific serum antibodies levels. intraperitoneal boost elicited significantly higher igg and igm levels than subcutaneous boost. immunization also induced changes in proportions of major lymphocyte subpopulations in peripheral blood. introduction: bulgarian immunomodulator respivax enhances the natural resistance of organisms and specific immunity towards the most frequent respiratory pathogens. it is composed of killed bacterial bodies and lysates of six microbial species (streptococcus pneumoniae, branhamella catarrhalis, streptococcus pyogenes, haemophilus influenzae, staphylococcus aureus, klebsiella pneumoniae). the immune response in respiratory tract includes not only systemic immunity in lungs, but also balt as a part of common mucosal immune system. most animals develop balt after antigen stimulation and this tissue plays a central role in antigen uptake and local immune response regulation. therefore, immunostimulation of balt may contribute to more efficient mucosal immunity in respiratory tract. aim: to study balt development in different terms after oral application of respivax in guinea pigs. methods: male guinea pigs ( g- g) were treated orally with mg respivax five consecutive days. after the last application, on days , , , , and six animals on each term were sacrificed and lungs were removed. morphological changes were evaluated on mm thick serial sections, stained with hemalauneosin. the populations of cd , cd and b cells were identified on cryosections by using indirect peroxidase immunostaining. zio technique was used to detect intraepithelial dendritic cells (dc). results: balt was not identified in control animals. in the treated group on day subepithelial lymphocyte infiltrates and diffuse lymphocytes in lamina propria were found. the following two terms were presented by hyperplasia of lymph epithelium with massive complexes of intraepithelial lymphocytes. they were composed mainly of cd positive cells, which number reached maximum at the end of the second week. on day b cell lymphoid follicles with different size were found. lamina propria was presented by abundant lymphocyte infiltrates, composed of cd and cd positive cells. on days and the morphological reaction in the airways was reduced, characterized with small size lymphocyte accumulates. numerous intraepithelial dc's were detected in treated animals, comparing to controls in which only a few were identified. conclusion: oral administration of respivax in guinea pigs resulted in significant immunomorphological reaction in the airway mucosa presented by increased number of dc and balt development. g. gupta , s. majumdar bose institute, molecular medicine, kolkata, indiavisceral leishmaniasis (vl) caused by the protozoan parasite, leishmania donovani, is characterized by the loss of ability of the host to generate an effective immune response in the form of free radicals and proinflammatory cytokines. chemokines, particularly cc chemokines, have been shown to render protection against leishmania infection. there is no clear understanding about the immunoprotective role of cxc-chemokines in vl.in the present study, the comparative potential of cxc chemokines, interferon gamma inducible protein- (ip- ) and interleukin- (il- ) in restricting leishmania donovani infection via the release of nitric oxide (no) and proinflammatory cytokines was studied in an in vitro model. no, a crucial mediator for ip- mediated leishmanicidal activity, was found to be dependent on inducible nitric oxide synthase (inos ) expression and was linked to the mapk signaling pathway via antagonistic regulation of p mapk and erk / . further, ip- was also able to abrogate the survival of leishmania in an in vivo model of vl by restoration of th cytokines and no. thus this study strongly demonstrates that ip- , like cc chemokines, is involved in rendering a protective response in vl via upregulation of proinflammatory mediators. african trypanosomiaisis (at), known as sleeping sickness, is an orphan and extremely debilitating disease in human, cattle and domestic animals. at is caused by the protozoan trypanosoma brucei and at the present, there's no safe or efficient pharmacology intervention. the dna vaccines could be the answer for this disease by being able to induce production of igg antibodies and induce of th /th cytokines mediated by cd + t cells and activating cd + t helper cells. in this study, we shows that balb-c mice immunized intramuscularly with a single dose of plasmids encoding three antigenic candidate genes from trypanosoma brucei, named invariant surface glycoprotein (isg), trans-sialidase (tsa), and fosfolipase c (plc) are able to produce igg antibodies anti-trypanosoma. this immunization process was able to control the mortality level when mice were submitted to challenger assay with trypanosoma brucei brucei parasites. in mice co-infected with s. ratti and l. major (nl) neither clearance of l. major nor strongyloides infection was changed. mice co-infected with s. ratti and p. yoelii (nl) showed the same course of parasitaemia as single infected mice. these results suggest a strictly compartmentalized and successful immune response in both murine co-infection models, s. ratti and l. major or p. yoelii. if this compartmentalization is also observed in the antigen specific cytokine response of ex vivo prepared lymphocytes will be the topic of further investigations. in the present study ,we evaluated tsa -encoded dna vaccine against l.major in balb/c mice. igg and ifn-g values were markedly increased in the immunized group ,which were significantly higher than in the control groups (p x . ) following immunization and after challenge with leishmania major. il- values were increased in all groups, but there was no statistical difference between the groups(p g . ) following immunization and after challenge with leishmania major. the immunized mice with the dna vaccine presented a considerable reduction in diameter of lesion comparing to the control mice and indicated a significant difference was observed between the immunized and the control groups (p x . ) in this regard . the survival time of the immunized mice with the vaccine was significantly higher than the control groups (p x . ) after the challenge with leishmania major. the immunized mice had significantly lower parasite load comparing to the control mice(p x . ). the findings of this study indicated that the tsa -encoded dna vaccine increased the cellular response and induced protection against infection with leishmania in the mice. the tsa -encoded dna vaccine may be an excellent candidate for future vaccine developments against leishmania. there is a lot of evidence showing that bcg vaccination at mucosal site via intranasal, intragastric and intrarectal routes are effective in conferring protection against virulent mycobacterium and several non mycobacterial infectious diseases. in this study the protective effect of autoclaved leishmania major (alm) vaccine in combination of either rectal or subcutaneous bcg on susceptible balb/c mice was evaluated.one month after bcg vaccination, balb/c mice were immunized subcutaneously twice with alm+alum at week intervals. three weeks after booster injection, × stationary phase l. major promastigotes were inoculated subcutaneously in one footpad. immunological evaluation at before and post infectious challenge, showed strong proliferative responses in the spleen cells of the rectal immunized group after stimulating with parasite lysate. high level of interferon gamma was induced in the spleen and significant increase in the serum ratio of igg a/igg was observed only in rectal immunized group. rectal immunized mice showed comparable nitric oxide production and inos induction in peritoneal macrophages .the obtained results in rectal bcg vaccinated group showed no mortality but low parasite burden in the liver and spleen and suggested protective efficacy of intrarectal bcg immunization against leishmaniasis might be due to the long-lasting induction of type immunity. methods: two groups of balb/c mice were infected by l. tropica. one group was infected subcutaneously into the left footpad and the other group intradermally into the left ear dermis. mice were challenged by l. major in the right footpad after establishment of l. tropica infection. the immune response was evaluated at two intervals: one week and one month after challenge. single cell suspensions were prepared from draining lymph nodes of mice. cells were stimulated by phorbol myristate acetate (pma). cell surface markers and cytokine production were determined by intracellular cytokine assay using flow cytometry. the following parameters were assayed in the two experimental groups: lesion development, delayed type hypersensitivity (dth) to l. major challenge, production of gamma interferon (ifn-g) and interleukin (il- ), and cellular expression of cd and cd . results: infection through subcutaneous route in comparison to the intradermal route induces significantly higher levels of dth and ifn-g, lower levels of cd + lymphocytes, and higher protection against l. major challenges. conclusion: intradermal infection of l. tropica, in comparison to subcutaneous infection, induces significantly more protective immunity in balb/c mice. therefore, we propose the route of infection as an important variable in this experimental model. this factor should be considered for development of an appropriate experimental model for human l. tropica infections. objectives: many mammals exhibit a periparturient relaxation of immunity (ppri) to gastrointestinal nematode parasites culminating in increased worm burdens. it has been suggested that the extent of ppri may have a nutritional basis as this effect on host resistance is considerably augmented when protein supply is scarce. subsequent studies have shown that increased dietary protein intake can ameliorate this phenomenon. however, this effect is often confounded with increased food intake and thus increased energy levels. here, we aim to dissect the effects of protein and energy nutrition on the immune status and resistance to gastrointestinal nematodes in the periparturient host. the nippostrongylus brasiliensis lactating re-infected rat model was utilised as a well established model for mammalian ppri. lactating rats, re-infected with , infective n. brasiliensis larvae on day post parturition, were offered one of three levels of crude protein at one of two levels of metabolisable energy (me). parasite burdens were assessed by counting worms in the small intestine at day post secondary infection. histological counting of intestinal inflammatory cells, assessment of antibody levels and measurement of cytokine mrna levels in the mesenteric lymph nodes were carried out to assess the host immune status. results: increasing cp supply, but not increased me supply, reduced worm burdens. whilst feeding treatment did not affect eosinophil and goblet cell numbers, increased cp supply increased mucosal mast cell numbers and levels of n. brasiliensis specific antibody (total igg, ige, igg and igg a). this was independent of level of me supply. feeding regime did not affect levels of the type- cytokines il- and il- . conclusion: this study effectively demonstrates that increasing protein supply per se can decrease periparturient parasite burdens. this anti-parasitic effect correlates strongly with an upregulation of immune effector mechanisms, namely accumulation of mast cells and production of antibody. this data emphasises the role of immunonutrition in combating infectious disease. protein supplementation of periparturient mammals has considerable potential as a non-chemotherapeutic method of controlling gastrointestinal nematode parasites. background: gp is the major surface glycoprotein of leishmania that exhibits protease activity and has an important role in the biology of the parasite. the aim of this study was cloning and expression of gp of l.major strain mrho/ir/ /er. methods: l.major promastigotes were grown in rpmi supplemented with % fcs. l.major rna extraction and cdna synthesis were carried out. gp gene segment was amplified by specific primers and cloned into ptz r to construct ptz r/gp . the presence of gp into ptz r was confirmed by pcr. then, ptz r/gp was sent to determine the sequence of its nucleotides. after that the gp gene segment was sub-cloned into pet a (+) expression vector and transformed into e.coli bl (de ) plyss and gp protein was expressed in presence of mm iptg. objectives: the development of a vaccine against malaria caused by plasmodium falciparum is an urgent public health priority. influenza virosomes represent an innovative human-compatible antigen delivery system that has already proven its suitability for subunit vaccine design. at appropriate antigen doses, seroconversion rates of % were achieved against two synthetic malaria peptide-mimetics in malaria naï ve volunteers (genton et al., plos one, ) . the aim of this clinical trial is to proof that virosomes are a suitable delivery system for malaria peptide-mimetics in malaria semi-immune subjects. objectives include demonstration of safety and tolerability of virosome formulated malaria peptide-mimetics and determination of the humoral and cellular immune responses against these malaria peptide-mimetics. particularly, boosting of pre-existing naturally acquired anti-malaria immunity will be investigated. the study design was a single centre, randomized, controlled, double-blind, age deescalating trial including volunteers. male volunteers ( and years) for the adult group, and children of both sexes ( - years) were enrolled. subjects received virosomal formulations containing mg of ama -c (pev t), an apical membrane antigen- derived synthetic phospatidylethanolamine (pe)-peptide conjugate and ug of uk (pev t), a circumsporozoite protein derived synthetic pe-peptide conjugate. comparator groups received the influenza vaccine inflexal v. volunteers received two injections at study days , and . results: safety and tolerability defined as occurrence of local and systemic adverse events and incidence of clinically significant hematological and biochemical abnormalities are assessed. this vaccine showed a very good safety and tolerability profile in all study participants. curcumin dissolved in dmso when administered orally to p.berghei infected mice has been shown to have antimalarial activity, enabling % of the treated mice to survive till days after infection by which time all of the untreated mice had died. under such condition we found that bioavailability of curcumin was only . % of the amount fed and it remained in circulation in the blood only for minutes post feeding. we therefore prepared curcumin bound to chitosan nano particle to improve it's delivery and found that oral feeding of such particles not only increased its bioavailability to . % ( of the amount fed but it's circulation was sustained till hrs post feeding. under such conditions when mgm of curcumin bound to mgm of chitosan nano particles were fed one time daily for days post infection to plasmodium yoelii infected mice % of mice were cured and survived atleast for days without any infection and were resistant to reinfection with the same parasite. curcumin under such condition accumulated preferentially in infected erythrocytes, the quantity increasing with increase in parasitemia and fluorescence microscopy revealed that it was bound to the parasite. like chloroquine, curcumin inhibited hemozoin formation in vivo and heme polymerization in vitro in a dose dependent manner. we believe that it is one of the ways by which curcumin may be killing the parasite. among immune cells, nk and gamma-delta t cells are suspected to play a critical role in the early control of plasmodium falciparum parasitaemia and to influence malaria adaptive immunity. gamma-delta vgamma vdelta t cells, a non-conventional t cell subset specific of primates, are activated and expanded during primary p.falciparum infections in response to malaria non-peptidic phosphoantigens, and they are an important source of ifn gamma. furthermore these cells inhibit in vitro growth of p.falciparum blood stages by a granule exocytosis-dependent cytotoxic pathway and granulysin -an nk and t cell specific cytotoxic molecule has been incriminated. so far, the precise mechanism of the parasite inhibitory capacity of those cells, as well as the parasite blood stages involved remains unclear. to further investigate the anti-parasitic activity of gamma-delta t cells an rnai strategy based on a lentiviral vector approach was undertaken. we demonstrate that granulysin, but not perforin is essential for the anti-parasitic activity of gamma-delta t cells. concerning parasite blood stages, we show that both mature infected red blood cells and the free invasive form (merozoite) trigger gamma-delta degranulation and granulysin release, but noteworthy merozoites were the only stage affected by gamma-delta t cells. in addition, we also provide evidence that such a mechanism may occur in infected patients. altogether these data highlight a new mechanism by which gamma-delta t cells might directly contribute to malaria immunity opening new perspectives based on gamma-delta t cells to prevent or cure malaria. the immune system has a number of mechanisms to prevent self-destructive responses. amongst these, regulatory t cells (treg) have the ability to actively suppress effector responses. many questions surround the issue of antigen specificity of treg, since selective inhibition of only the pathogenic response, leaving the rest of the immune system intact, is the ideal therapeutic goal. the purpose of the project is to develop a model of robust, highly specific regulation operating in vivo that can be studied to understand the underlying mechanisms. such a model is provided by murine autoimmune hemolytic anemia (aiha) induced by immunisation with cross-reactive rat red blood cells (rbc). mice recover from disease due to the development of regulation with exquisite specificity, which suppresses only responses to self-epitopes whilst selectively allowing those to rat-specific determinants to be boosted. the re-establishment of tolerance is associated with the loss of t-cell proliferative responses, and emergence of il- responses, to epitopes on the dominant rbc autoantigen, anion exchanger- (ae- , or band ) protein, and protection can be transferred by injecting splenocytes from recovered mice into naï ve recipients. here we show that transfer of tolerance to naï ve recipients is dependent on ido mediated immunosuppression as mice receiving previously tolerised splenocytes under the cover of methyl tryptophan, an inhibitor of ido, were refractory to tolerance and developed hemolytic disease. induction of ido is therefore an important process in antigen-specific tolerance, and initiators of ido activity, including ctla- + regulatory t cells or soluble forms of ctla- , may also be crucial components of this regulatory pathway. consequently, this finding has important implications for our understanding of tolerance processes in autoimmune disease. objectives: it was shown alpha-fetoprotein (afp) induced immunosuppression of cell-mediated immunity in vivo. our previous work discovered afp-activated mice bone marrow hematopoietic stem cells (hscs) suppressed effector reactions of cell-mediated immunity in vitro. we investigated relationship existed between afp-induced hscs suppressor activity and immunosuppression of cell-mediated immunity during afp-produced teratocarcinoma development. methods: animal models, experimental oncology methods, immunomagnetic separation, cultural methods, cellular biology methods, flow cytometry, multiplex protein analysis, methods of molecular biology and rna interference (rnai) were used in this work. results: as a result, there was a negative correlation (r medium =- . ) between dynamics of hscs suppressor activity elevation in spleen and inhibition of nk cells, nkt cells and cd + t cells cytotoxic activities ex vivo during tumor growth. besides, the inhibition of spontaneous and induced cytokines productions such as ifng, tnf-a and tnf-b from these types of immunocompetent cells negatively correlated with increasing of suppressor factors expression such as tgf-b (r medium =- . ) and il- (r medium =- . ) in isolated splenic hscs ex vivo. analysis of effector cd + t cells in spleen showed decrease of t h cells quantity and simultaneous t h cells number increase during teratocarcinoma development. moreover, it were found elevated numbers of cd + cd + ctla- + -and cd + cd -il- + il- regulatory t cells in spleen as well as increasing suppressor activity of isolated regulatory t cells ex vivo. number boost kinetics of t h , t h and regulatory t cells were correlated (r th =- . , r th = . and r th = . and r tr = . ) with kinetics of hscs suppressor activity level. in addition, dynamics of regulatory t cells activity were linear (r th = . and r tr = . ) to hscs suppressor activity level in spleen during tumor growth. quantities of tgf-b -and il- -produced hscs in spleen were correlated (in some cases negatively but in other positively) with cell-mediated immunity effector reactions alteration during teratocarcinoma development also. however, inhibition of afp expression by rnai caused to inhibition as immunosuppression activity of hscs and their appearance in spleen as well as normalization of cell-mediated immunity effector reactions. conclusion: thus, hscs suppression activity is correlated with changes in cell-mediated immunity during endogenous afp productions by teratocarcinoma cells and may play a role in afp-mediated dysfunction of normal immunoregulation during afp-produced tumor development. syphacia obvelata, a murine pinworm gastrointestinal nematode, is common even in well-managed animal colonies. although often considered as irrelevant, pinworm infections were shown to alter hosts' immune responses and to interfere with the experimental settings. our studies showed that naturally aquired s.obvelata infection also influences the hosts' hematopoietic responses, inducing the increased production and release of the cells of granulocyte-macrophage, as well as of erythroid lineage from the bone marrow of the infected cba mice. while the enhanced myelopoiesis compensates the increased peripheral demand for a larger supply of tissue neutrophils and macrophages, the cause of stimulated erythropoiesis is less obvious, but as infection consequence clearly underscores the disturbed and altered hematopoiesis. beside cellular changes, we also evaluated the impact of the s.obvelata on mitogen-activated protein kinases (mapk) signaling in bone marrow cells and found that infection upregulated all three mapk families, p , jnk and erk. additionally, s.obvelata enhanced the expression of mrna for the inducible nitric oxide synthase (inos). to evaluate how this pinworm infection modifies hematopoietic cells' reactivity, we also examined the influence of interleukin- , t cell-derived cytokine implicated in the regulation of hematopoiesis and inflammation, on the bone marrow cells. bone marrow myeloid and erythroid progenitors from s.obvelata-infected mice displayed altered sensitivity to il- , as compared to non-infected controls. the infection also altered the effect of il- on mapk activation by preventing its stimulating effect on p mapk. moreover, in s.obvelata-infected animals il- markedly down-regulated the expression of both inos and constitutive, endothelial (e)nos, not affecting the low basal nitrite production, which was opposite to the effect previously observed in noninfected mice, i. e. il- induced no production through the activation of both inos and enos. besides highlighting the importance of working under pinwormfree conditions when using experimental murine models for immunohematopoietic investigations, the data obtained pointed to the multiple layers of modulatory ability of this pinworm parasite and confirmed that the overall orchestration of the host response to the parasites is a complex process still being unraveled at both the cellular and molecular level. .-validation of the method: in order to determine linearity, analytical range, and reproducibility, three different sera with previously identified mc were serially diluted from ⁄ to / with a normal serum pool. .-implementation as a standard method for analysis of the mc in patients with paraproteinemia. the method showed good linearity: r g . . the analytical range was from g/l to g/l. the coefficient of variation (cv) was x % for [mc] n g/dl, and x % for [mc] x g/dl. this procedure was successfully implemented to quantify the mc in serum samples between march and february, . among these samples, we have quantified light chains, heavy chains, igd and biclonal paraproteinemias. conclusions: . we have developed a simple, reproducible and low-cost method to quantify the mc using standard analyses of serum protein electrophoresis (spe), serum albumin, and densitometric quantification of mc and albumin regions. . the procedure allows monitorization of the mc in patients at diagnosis, after therapy, and evaluation of complete remission. objectives: direct influence of alpha-fetoprotein (afp) on immunosuppressor factors synthesis as well as immunosuppressor activity of bone marrow hematopoietic stem cells (hscs) was detected in our previous work in vitro. we investigated possible role of endogenous-produced afp in induction hscs immunosuppressor activity at tumor-bearing mice. methods: animal models, experimental oncology methods, immunomagnetic separation, cultural methods, cellular biology methods, flow cytometry, multiplex protein analysis, inhibition assay, colorimetric elisa, methods of molecular biology and rna interference (rnai) were used in this work. results: our results demonstrated afp endogenous synthesis adduced to elevation of immunosuppression activity of hscs in bone marrow. this afp effect becomes developed from day and reached plateau level after day of teratocarcinoma insertion. moreover, cd + cd cells showed in spleen and main lymph nodes from day and achieved plateau level after day of teratocarcinoma growth. however, immunosuppression activities of purified hscs from spleen and lymph nodes discovered at day and had a maximum pick at day of teratocarcinoma inoculation. besides, immunosuppression activity of hscs from spleen and lymph nodes was more than , times lower than in bone marrow in the same period of tumor development. isolated hscs from bone marrow, spleen or lymph nodes produced similar spectrum of suppressor factors such as tgf-b , il- , pge and no. inhibition of such suppressor factors lead to levelling of hscs immunosuppression activity ex vivo. kinetic of hscs quantity and activity had significant correlation (r cell number = . and r activity = . ) with afp level dynamics in blood serum. in addition, inhibition of afp expression by rnai caused to diminishing of hscs immunosuppression activity as well as hscs appearance in spleen and lymph nodes. conclusion: therefore, afp plays role not only specific inducer of hscs immunosuppression activity but also as a factor of activated hscs penetration into spleen and lymph nodes during afp-produced tumor development. cd -ligand molecules -that are powerful immunomodulators -are strongly expressed by activated platelets; membrane-associated cd l is cleaved to soluble (s)cd l. we sought to examine the levels of scd l in platelet concentrates (pcs) having led to an acute transfusion reactions (atr), and to test for its biological effect on b-lymphocytes. we recorded atr episodes that could lead to investigation of residual platelets in container. two fractions of aliquots from each pc (and controls) were prepared, one for assay of individual supernatant fractions and one of corresponding lysates of platelets; scd l -along with other products -were assayed by quantitative elisa. levels of il , cd p and pdgf-ab in pc supernatants and lysates from pcs associated with atr were similar to that in controls. supernatants of pcs associated with an atr contained higher scd l levels compared to controls, and -in a inversely correlative manner -corresponding platelet lysates contained lower levels of scd l . to examine if scd l was biologically active, we stimulated purified b cells recovered from healthy blood donors and exposed those normal b cells to supernatants and cell lysates of pcs implicated in atr, or control material, and we measured il- secretion. the il- concentration was consistently below - pg/ml in pc supernatants and lysates, and unstimulated b cells did not secrete detectable levels of il- . the addition of supernatant from atr-associated pc samples to purified b cells consistently resulted in sustained il- production over control (p x . ) at d after the onset of the culture, while -in a inversely correlative manner -corresponding platelet lysates contained lower levels of scd l (p x . ). pre-incubation of b cells with cd -blocking antibodies substantially abrogated il- secretion, unlike isotype-matched control. the partial blocking of cd binding on cd + b cells strongly suggests a potentially synergistic role in b cells for cytokines other than scd l (under investigation) and indicates a sustained role for pc-derived scd l. these data prompt us to investigate a larger series of events and controls to delineate on the one hand if certain factors can be responsible for an enhanced production of scd l by collected/stored cpa. objectives: there is accumulating evidence for a role of natural killer (nk) cells in the antitumor response against hematological malignances. nk cells exert their action by means of a large panel of structurally distinct activating receptors that recognize their ligands on target cells. analysis of activating receptor pathways in nk cells has revealed a dominant role for natural cytotoxic receptors (ncrs) and dnax-accessory molecule- (dnam- ) in the lysis of acute myeloid leukemia (aml) blasts. here, we investigate the expression of these activating receptors on nk cells from aml patients stratified by age. methods: we analyses by flow cytometry peripheral blood mononuclear cells (pbmcs) from aml patients before specific anti-leukemia therapy and healthy donors. all results were analyzed statistically using spss version . results: aml patients under years showed a significant reduction in the expression of dnam- , nkp and nkp compared with age-matched controls. both healthy individuals and aml patients older than years showed a reduction of these receptors compared with young donors. in contrast, we have found that nkp expression was increased in some patients of aml. on the other hand, the analysis of ligands for these activating receptors on leukemic cells showed a high variability that was not correlated with age or fab subtype. in addition, an inverse correlation in the expression of dnam- on nk cells and its ligand cd on aml blasts has been found in aml patients under years. to analyze if leukemia cell were involved in the modulation of these receptors we have performed in vitro cocultures of leukemic blast and healthy nk cells. the initial recognition of aml cells by nk cells may represent a crucial process to prevent tumor development. here we described for the first time, a decrease of dnam- expression on aml patients and confirm previous reports showing a significant decrease of nkp and nkp on aml-nk cells. altogether, these alterations of the major receptors involved in nk cell-mediated cytotoxicity of leukemic cells represent an important mechanism of immunoescape that may correlate with disease progression and patient survival. a. stelmaszczyk-emmel , e. gorska , u. demkow , m. wasik medical university of warsaw, department of laboratory diagnostics and clinical immunology of developmental age, warsaw, polandglucocorticosteroids are often used in leukemia treatment. their therapeutic use is limited due to several side effects. one of them is multidrug resistance phenomenon, which causes lack of patients response for treatment. dexamethasone is used in schedule of children's all-b treatment and the response on glucocorticosteroids therapy is very important. the aim of this study was to examine whether dexamethasone changes multidrug resistance of lymphoblasts in all-b and their tendency to begin apoptosis. the study involved children with all-b. bone marrow cells isolated by centrifugation on histopaque at the day of diagnosis were cultured for or hours with or without dexamethasone in concentration - m. analysis of: p-gp surface expression, p-gp function (rhodamine test), phi (carboxy-snarf) and apoptosis test (annexin-v and pi test) were performed with the use of flow cytometer coulter epics xl. for statistical analysis nonparametric wilcoxon test was used.the results showed that p-gp expression on lymphoblasts was , %± , . after hours of lymphoblasts incubation with or without dexamethasone any statistically significant changes were observed. average percentage of lymphoblasts with rhodamine efflux, which characterized p-gp activity, was , %± , . after hours of cells incubation with dexamethasone there was seen significantly higher percentage of cells able to eliminate rhodamine ( , %± , , p= , ). average phi in lymphoblasts was , ± , . acidification of cells incubated hours with dexamethasone was seen in - % percentage of cells ) . rest of lymphoblasts showed alkalization (phi - , ).the percentage of lymphoblasts in early stage of apoptosis after hours incubation with dexamethasone (annexin-v test) was higher than in control cells ( , % vs , %; p= , ). we concluded that dexamethasone does not influence surface p-gp expression on lymphoblasts of patients with all b but significantly increases activation of this protein. functional test should be performed to evaluate multidrug resistantace of leukemic cells, because surface expression of p-gp is not identical with its activity. moreover, dexamethasone alkalizes cytoplasm of lymphoblasts and induces early stage of their apoptosis. those effects may contribute to the treatment outcome. . however, small numbers of clonal pc can also be detected in the peripheral blood (pb) of the majority of patients and, in a minority of cases, mm is transformed into pc leukaemia (pcl). here, we describe that tumoral pc can express cd d/cd integrin with high or, sometimes, low affinity states, which is associated with their retention in or release from the bm, respectively. objectives: to evaluate the activation state of cd on malignant pc from bm and pb, as well as its regulation. methods: pc and active integrin expression on these cells were detected with anti-cd and anti-cd (clon huts ) moab, respectively, by flow cytometry. to study the integrin activation with divalent cations and pc index proliferation (brdu+ cells), we used short-term pc cultures ( hours). results: cd active form was expressed in the majority of normal and tumoral bm pc from healthy subjects ( . ± . , n= ) and mm patients in the early stages of the disease ( . ± . , n= ). in these cells, huts epitope was clearly upregulated by mn + . in contrast, circulating pc were almost all huts negative, and levels did not significantly augment when these cells were exposed to mn + . moreover, not only pb but also bm malignant cells from pcl patients were also huts negative and divalent cation refractory. it was also observed that pc from pcl patients showed an increased proliferative index ( . ± . brdu+ cells, n= ) in comparison to pc from mm patients in the first stages of disease ( . ± . brdu+ cells, n= ). these results suggest that the active form of cd must be expressed on pc to retain these cells within the bm environment. moreover, its downregulation is associated with increased numbers of circulating pc and disease progression. multipotent mesenchymal stem cells derived from (hucb) represent promising candidates for the development of future cellular therapy strategies. they are able to self-renew and they terminally differentiate into multiple lineages, including bone, cartilage, muscle, bone marrow, fat and other diverse connective tissues. in the first part of this study, we compared different protocols for the expansion of human mesenchymal stromal cells (hmsc) starting from diagnostic samples of bone marrow aspirates and the cord blood (cb). the protocols differed in the presence of either % fetal bovine serum (fbs) with and without fgf ,or % human platelet lysate (hpl), %hpl, ( % fbs + % hpl), ( % fbs + % hpl). we obtained a significantly better expansion with hpl, compared to cells with a selected batch of fbs and in fewer days.in the second part of this study, we focused on proteins that were differentially expressed during osteogenic, adipogenic and vascular muscular differentiation by western blotting. we compared the quality and the quantity of protein expression before and after differentiation (day ). two bm and two cb differentially expressed spots were observed between the two groups (before and after differentiation).we noted the low pourcentage of hmsc in cb samples: in ten samples, only two made msc colonies as in bm samples. we were also interested to the different coloration: osteogenic diffentiation was determined by alizarin red s staining, for adipogenic differentiation, the cells were stained with oil red o to visualize lipids droplets. background: inherited bone marrow failure syndromes (ibmfs) comprise a group of genetic disorders characterized by single or multiple cytopenias, as well as distinctive clinical features and varied molecular pathways. activation of p tumor suppressor pathway leads to cell cycle arrest and initiates apoptosis. we studied the presence of p dna (as a marker of cell cycle dysregulation); in bone marrow of children with fanconi anemia (fa) and those with acquired aplastic anemia (aaa). subjects and methods: this is a cross sectional study that involved: ) ten cases with fa diagnosed on the basis of dna breakage analysis, ) ten cases with aaa, and ) ten normal control cases. the presence of p dna was measured in both bone marrow and peripheral blood samples using a real-time quantitative pcr by taqman assay. results: p dna was demonstrated in bone marrow of % of children with fa, compared to % in children with aaa (p x . ), while, no p dna was seen in normal control. a positive correlation between dna breakage and presence of p dna was seen in bone marrow from fa (p x . , r . ). the presence of p tumor suppressor gene by real time pcr in bone marrow of fa may represent an early indicator of significant dna genetic alteration in those patients. key: cord- -ak pq authors: nan title: th european congress of intensive care medicine athens - greece, october – , abstracts date: journal: intensive care med doi: . /bf sha: doc_id: cord_uid: ak pq nan objectives: evaluate the levels of tnf, il- and pai-i in different moments of the ards and the possible relationships among them. methods: septic patients with ards were studied. also significant differences for: tnf, pai-i and il- in septic patients and both evaluations of ards with control gropup; pai- between septics and nd evaluation in ards, and between the ist and nd evaluation in ards; il- between septics and both evaluations in ards; and il-~ in both evaluations in ards patients in relation to mortality. conclusions: i) elevations of tnf, pai-i and il- , with clinical signs, are suggestive of infection; ) the persistent and progressive elevation of pai-i with any clinical criteria may suggest evolution to ards; ) due to its own kynetics, il- takes part later in the acute phase, its levels being related to the magnitude of the injury in the tissues. objectives: the influence of long-term volume therapy with different solutions on plasma levels of circulating adhesion molecules was studied. methods: according to a randomized sequence, patients with sepsis secondary to major surgery exclusively received either hydroxyethylstarch solution ( % hes, mean molecular weight (mw) , daltons, degree of substitution (ds) . ) or human albumin % (ha) for volume therapy for days. plasma levels of circulating (soluble) adhesion molecules (endothelial leukocyte adhesion melecule- [selam -i] , intercellular adhesion molecule- [sicam -i] , vascular cell adhesion molecule- [svcam -i] , and p-selectin ) were serially measured on the day of admission to the intensive care unit (='baseline ' value) and during the next days. results: selam-i, sicam-i, and svcam-i plasma levels were markedly higher than normal at baseline in both groups. in the hes-patients, selam-j decreased to normal range, whereas it further increased in the ha-group (from • to • during the study period, sicam-i and svcam-i plasma levels remained unchanged in the hes-patients, but further increased in the ha-group (from • to , • sgmp- increased significatly only in the ha-group ( • to • only pao /fio was significantly correlated to plasma levels of adhesion molecules. conclusions: sepsis is associated with markedly elevated plasma levels of adhesion molecules indicating endothelial activation or damage. by long-term volume therapy with hes, these levels remained unchanged or even decreased, whereas volume therapy with human albumin did not have any beneficial effects on soluble adhesion. central venous catheters are frequently used in the care of the critically ill patient. the incidence of catheter related sepsis varies in the literature. we investigated the occurrence of contamination and sepsis compared to results of the epic study as part of quality assesment in our intensive care unit. from january until august all removed central venous catheters were examined for microbiological culture. the patients who showed signs of sepsis were also registered. the results of the contaminated catheters and septic patients were compared with results from the epic study. during the month period , patients were hospitalized on our intensive care unit. central venous catheters were examined for microbiological culture. specimens appeared to be possitive ( %). patients showed clinical signs of sepsis. the incidence of sepsis due to contaminated central venous catheters was / ( %). the incidence of sepsis due to the presence of all central venous lines was / ( %). the microorganisms responsible for the sepsis syndrom were : stapylococcus aureus (n= ), escherichia colt (n= ), others (n= ). in the epic study the percentage for sepsis on the icu was . % for the netherlands and . % for europe. despite a high number of positive culture from removed intravascular lines, a low percentage of sepsis was seen compared to results of the epic study. we recommend routine bacteriological culture of all removed central venous lines and recommend to look at colonization and sepsis due to intravascular lines as a measure of quality control in the intensive care unit. objectives: prognostic assessment of simplified acute physiology score (saps) in granulocytopenie patients with septic shock (ss). methods: the medical records of admissions to an intensive care unit (icu) of granuloeytopenic patients with ss are reviewed. fiftytwo patients had haematological malignancies. seven patients had aplastie anaemia. patients were categorised as survivors (discharged from icl and non-survivors (died in the icu). saps index was calculated for patients daily during their stay in icu. all patients were severe granulocytopenic (total white cell count less than , ] ] ). results: five patients ( , %) were discharged from icu. fifty-four patients died in icu. non-survivors had saps on admission higher than survivors ( . + . and . + . , respectively, p< , , mann-whitney u test). no patient with a saps greater than survived. mortality among the patients with saps from to was , %o. the evolution of ss was rapid. the mean stay in icu among non-survivors was only hours. an analysis of the saps index on admission of non-survivors showed an inverse correlation with the duration of their stay in icu (r=- , , p= . ). all survivors recovered from granulocytopenia. they had normal white cell counts at the time of discharge from icu. there was inverse correlation in survivors between saps and white cell counts, when these parameters were evaluated daily. however, the saps index alone cannot be considered to be on individual predictor factor of mortality. patients who had failure of the malignancy to respond to chemotherapy and who had persistent granuloeytopenia died in icu despite saps index on admission and recovery from ss. conclusion: saps index greater than , failure of the malignancy to respond to chemotherapy and persistent leueopenia all point to a poor outcome of granulocytopenie patients with ss. introduction: antipyretics sometimes are used for fever control in febrile neutropenic patients with hematological malignancies(hm). we observed a dramatic fall of blood pressure(bp) and development of septic shock(ss) in some of the patients who received antipyretics. aim: to clarify can antipyretics provoke ss in neutropenic patients with infection. methods: retrospective review of medicat records of neutropenic(wbc < , / )patients with hm, admitted to the intensive care unit for ss, was performed. there was selected group of patients receiving antipyretics shortly before a fall of bp. results: there was a definite causal relationship between receiving antipyretics and fall of bp in from patients. all patients had fever due to infection and had normal level of bp before receiving antipyretics. hypotension developed within minutes up to , hours after administration of antipyretics. three patients received , g of metamisol and one , g ofparacetamol per os. in all cases we observed dramatic diaphoresis and the temperature fall to subnormal level ( . + . ~ accompanied'by hypotension. but in - hours the fever was coming back without blood pressure elevation. the fluid replacement was controlled by central venous or wedge pressures. there were required + ml colloid and cristalloid solutions for volume loading. in spite of fluid administration the hypotension persisted and all patients required inotropic therapy. only one patient survived and is alive now. conclusion: it seems to us that our data offer to state that antipyretics administration can initiate ss in febrile neutropeuic patients with infection. objectives: to assess the agreement between cardiac output (co) measured by odm t and by other methods used in icu patients. methods: we prospectively studied adu t patients requiring hemodynamic monitoring with a pulmonary artery catheter. an esophageal doppler monitor provided measurements of co (odm), stroke volume and flow time (ft) used as an indirect evaluation of patient's volume status. patient hemodynamic status was evaluated by a modified fast response pulmonary artery catheter (baxter health care corporation, santa ana, ca), allowing co measurements by thermodilution "d) and an evaluation of right ventricular ejection fraction and end diastolic volume (rvef and rv-edv). in the last six patients co was measured by transthoracic echocardiography (echo) and oxygen consumption was measured by a deltatrack ii metabolic monitor (datex) allowing co calculation according to the fick formula (fick). the agreement between methods measuring co and their reproducibility, were evaluated by bland and altman analysis. results: agreement between co measurements is expressed as bias (d) and % limits of agreement (l of a = d_+ sd . td-fick - . - . to . fick-echo . - . to . there was no correlation between ft and rv-edv. conclusions: although co measurements by odmil had the best reproducibility, the limits of agreement between the four methods tested were unacceptable for clinical purposes. further investigation is required in order to improve the accuracy of co measurement in the icu. phd, a. paltzev, v.bajbikov, b.dobryakov d.sc., a.ostanin phd, o.leplifia phd, h.chernykh phd munieip. hosp. n l, n ; inst. of clin. immunol., novosibirsk, russia objectivies: efficiency of native cytokines used in the treatment of patients with severe surgical infections has been studied. methods: for two years patients were treated with cytokine mixture (ssp) obtained by arterio-venous perfusion of swine spleen and contained the following cytokines: il- , il- , il- , tnfa, ifny, gm-csf. results: ssp intravenous infusions were shown to accompany with mortality decrease from . % to . % in patients with abscessed pneumonia and lung abscesses and from % to % if disease course was complicated with sepsis. in patients with purulent peritonitis and sepsis efficiency of ssp was decreased due to endotoxieosis. thus, we used adoptive immunotherapy with mnc activated in vitro with ssp or recombinant il- . intravenous infusions of such cells resulted in transformation of a pathologic process from destructive into productive one. moreover, clinical manifestations of sepsis were controlled in % and mortality was decreased from % to %. conclusions: the use of eytokines themselves as well as cytokine-treated lymphoeytes permits to control the disease and leads to the mortnlity decrease owing to stimulation of host defence mechanisms. background: although red blood cell transfusions (rbct) are used to increase oxygen availability in septic patients, several lines of evidence suggest that rbct may actually worsen tissue hypoxia. thus, rbct may negatively influence outcome of septic patients. objectives: to determine the association of ) rbct ; ) number of units transfused; and ) mean age of the units transfused on the first day of transfusion with mortality of critically ill septic patients. methods: we prospectively identified patients who met strict criteria for sepsis syndrome (ss) seen in the icu of st. paul's hospital from to and excluded patients who died in the first days after the onset of sepsis. we recorded clinical characteristics, multiple system organ failure score, and apache ii at onset of sepsis. then, we retrospectively recorded the total number and age of rbc units transfused during the first days after onset of sepsis. overall -day mortality was %. results: the main results are shown in the table. the mortality of patients who received rbct was nearly double the mortality of those who did not receive rbct even after adjusting for severity of illness using apache ii. objectives: gastric mucosal acidosis is frequently observed in patients with sepsis. the aim of this study was to determine whether volume infusion using pentaspan| decreases abnormal gastric mucosal pco (pico ) in patients who have sepsis syndrome (ss) who have already been resuscitated using clinical endpoints. methods: we prospectively identified patients who met strict criteria for ss, had a pulmonary artery catheter and a gastric tonometer in place, and pico > mmhg. pentaspan| ( ml) was infused in rain. measurements of hemodynamics, hemoglobin, arterial lactate, blood gas analysis, and pico were performed before and repeated miff and hr after pentaspun| infusion. we calculated the pico -arterial pco' difference (pico -paco ) and phi (using henderson-hasselbach equation). anova was used to assess statistical significance. results: all patients werereceiving adrenergie drugs. map was : : mmhg and lactate . : : . mmol/l. pentaspan| increased ci by % (p< . ) but did not change pico ( and increase m oxygen o* wery were simimny achieved in both groups. nevertheless, epinephrine was associated with a lactic acidosis and increased laetate/pyruvatemia ratio (l/p) that evoke a dysoxia rather than a metabolic effect. an higher gastric mucosal pco in the ep group compared to nor-rob suggests the hypothesis of an anaerobic production of co in favor of a splanchnic hypoxia. in both group, arterial ketone body ratio that reflects hepatic mitochondrial redox state, compared to a control group without shock was decreased but increased between and hours after restoration of arterial pressure. the association norepinephrine-dobutamine seems to be better for splanehnic circulation than epinephrine and should be used for dopamine resistant septic shock. moreover, the increase in arterial pressure with nor-dob improved gastric mueosal ph and hepatic mitochondrial redox state and argue to reconsider arterial pressure as a significant goal for resuscitation in septic shock. conclusion: significantly higher malondialdehyde and ghitathione levels and glutathione-peroxidase activity in group ns at the end of icu stay were related to mortality these findings indicate an increased generation of free oxygen radicals together with increased anfioxidant activity in this group and sapport the employment of antioxidant interventions in critically ill patients. oblecfives: to determine the role of nitric oxide (no) in the mechanism of septic shock induced by isolated limb perfuslen with recombinant tnfcr methods: we have measured tnfr~ and metebo~ites of no in patients with signs ot septic shock following treatment with isolated limb perfusion for nonresectable soft tissue tumors and melanomas of a limb. perfuslen was carried out with melphalan (burroughs wellcome) and recombinant tnfcr (boehringer). tnfc~ was determined by specific radiometric assay (medgenix diagnostics), nitrate and nitrite were measured with a modification of the guess reaction ~. results: results are shown in the table. conclusions: during isolated limb pedusion with recombinant tnf~ very high levels of tnfcr were measured in arterial blood in patients. they all showed signs of severe sepsis syndrome with shock from vasodilafion, probably due to leak of recombinant tnft~ from the peduslen circuit to the systemic circulation. tnfc~-induced vasodilation was not accompanied by a rise in serum no-metsbolites. our findings do not confirm the widely accepted theory, mainly based on animal experiments, that genera• of no is the key pathogenefic mechanism in septic vasodilafion , nor that tnfrt invariably induces forreafion of no. the precise mechanism of shock in these patients remains to be elucidated. references: . moshage h, kok b, huizenga jr, jansen plm nitrite and nitrate determinaiions in plasma: a critical evaluation. clin chem : / . . moncada s, higgs a. the l-argioine-nitrio oxide pathway. n engl j med ; : - ec is a commonly used for prolonged, stable animal anesthesia. noting that the hypotension after iv lps was attenuated by ec, we hypothesized ec also protects against lps toxicity. sprague-dawley rats received ip saline (s), thiobutabarbita mg/kg (tb), or varied doses of ec, followed hours later by bolus mg/kg iv lps. -day survival is shown below: group: s tb ec( . gmikgi ec( .sgm/kg) ec(i. gm/kg) alive (n) t ~ total (n) s s "signiflcant;y different from all other groups, p< . s / rats given lps followed hours later by ec ( . gm/kg) also died. additional rats were treated with s (n= ) or gm/kg ec (n= ) followed by mg/kg lps, then sacrificed at hours. blood glucose (bg, mg/dl),.hematocrit (hct), leukocyte count (wsc/mm~ platelet count (pltxl ~/mm ), bicarbonate (hco, mg/dl), gross bowel hemorrhage (bh, - scale) and lung myeioperoxidase activity (mpo, ~vmirvgm wet lung) are shown below ( we conclude that ec reduces the lethality and multiple organ toxit;~ty of lps. its diverse effects suggest asite of activity upstream from the cytokine cascade. these results are important for studies of lps which may use ec anesthesia and may have potential in the therapy of septic shock. [zo = hz impedance (z; {dyn.sec.cm " }); zl = first harmonic z; zc = characteristic z; z ph. = t'trst harmonic phase angle {radians}; f, #, * at least p < . between fio . and . , fio . and fio . &no - . _+ . - . _+ . # - . + . m - . + . * - . + . * - . + . * - . _+ . * in hyperoxia, compared to dogs at the same q, minipigs had a higher ppa ( + rnmhg versus + mmhg; p < . ). hypoxia increased (ppa-ppao) at all levels of q by an average of mmi-ig in minipigs and mmhg in dogs. inhaled no inhibited hypoxia-induced (ppao-ppa)/q changes in both species. conclusions: we conclude ~ that the minipig is an animal model of elevated pulmonary vascular resistance and impedance, and ~ that hypoxia-induced alterations in pvz spectrum are due to changes of resistance in small arteries. objectives: ) to determine the toxicity of ng-monomethyi-larginine (nma) administered by intravenous bolus to patients with refractory septic shock. ) to investigate the biologic activity of nitric oxide synthase inhibitors in septic shock. methods: from august to january , thirteen patients with vasopressor refractory septic shock received nma intravenously in escalating doses from to mg/kg. results: no hepatic, renal, gastrointestinal, or hematologic toxicity was observed at doses of nma as high as mg/kg. significant biological activity was observed at all dose levels consisting of increased blood pressure (systolic blood pressure from . mm hg + . to . _+ . s.e.m., p= . , systemic vascular resistance ( + to + dyne.sec/ cm s, p=. ), and a decrease in vasopressor requirements. the magnitude and duration of these effect were dose dependent. decreased cardiac output ( . _+ . to . _+ . i/min p=. ) and increased pulmonary artery pressure ( . _+ . to . _+ . mm hg; p=. ) were also observed. no significant effects on heart rate, pulmonary capillary wedge pressure, or central venous pressure were observed. four of patients survived for more than days, patients died of cancer complications (all patients had maintained blood pressure for h on nma) and patients died of complication attributable to septic shock (mods, ards, dic, refractory hypotension), and patient was unevaluable. conclusions: no adverse clinical effects have been observed in patients receiving bolus doses of nma as high as mg/kg. the increased pulmonary artery pressures observed in septic shock patients is further augmented by nma and may limit the dose which can be administered by intravenous bolus. other schedules of drug dosing may attenuate this effect. glucose-insulin-potassium (gik) solutions have been shown to improve cardiac contractility and increase oxygen availability in experimental and clinical settings of septic shock. several mechanisms have been proposed to explain these effects including a direct improvemeut of the energy balance by glucose, a direct influence of insulin on cardiac performance or an increase in intravascular volume due to the hyperosmolarity of the solution. to explore the role of hyperosmolapity, we compared the effects of gik to those of a isoosmolar hypertonic saliue solutiou in endotoxin shock in dogs. methods : the study included mongrel dogs ( • pentobarbitalanesthetized aud mechanically ventilated with air. thirty minutes after the intravenotls administration of mg/kg of e. coli endotoxin, the dogs were randomized to receive a ml/kg infusion in rain of a hypertonic ( mosm]l) solution iucludiug either a mixture of glucose % with u insulin and meq kcl/l (glk-group ) or hydroxyethyl starch . % in naci . % (hes-group ). in each dog, a . % saline infi~sion was continued to maintain the puhnonary arlery occluded pressure at baseline level. hemodynamic, blood gas aualysis and laboratory data were collecled at baseline and miu, rain, rain, and nunutes later.. results : eudotoxin administration was followed by a fall in mean arterial pressure (map) aud cardiac index (ci) and a rise in blood lactate levels. resuscitation with either gik or hes hypertoaic solutions resulted in similm increases in map, ci, oxygen delivery and left ventricular stroke index (table ) . we conclude that during resuscitation from endotoxic shock the use of gik solutions is not superior to hypertouic hes solutions. the higher blood lactate levels observed in the dogs receiving gik can be attributed to the glucose metabolism. , for group , for group ) were drawn and immediately analysed at ~ using the abl radiometer for po , pco and ph, and the osm radiometer for hbo %, hbco% and methb%. psost (i.e. the ps at ph= . , pco = mmhg and temperature at ~ c) was calculated automatically by the instruments on mixed venous blood, as was the ps "in vivo" (i.e. the ps at the patient's value of ph, pcoz and temperature), using siggaard-andersen's algorithm. the data were compared by the one-way anova test and by the t-test for paired and unpaired samples. results: the mean resulting values (in mmhg) with the statistical differences are shown in table i. in addition, the time series analysis shows the mean ps~st values as statistically below the psin vivo" in the septic patients while the opposite is shown for the cardiac patients. no differences in the time analysis are demonstrated for the second group. a possible clinical significance may be drawn from these different behaviours. objectives:toxemia degree and humoral immunity condition have been studied in patients aged from to with progressive course of sepsis and polyorganic insufficience. methods: such toxemia and humoral immunity findings as lencositlcindex of toxication (lii), level of oligopeptides of the middle molecular mass registered at the wave length of nm(mmi) & nm (mm ), distribution index (id), immunoglobulins a,m,g, concentration of circulating immunocomplexes (cici & cic ) and also some clinical and biochemical findings on the , , day after the operation serve as criteria for treatment effect. results: it was founded that in intensive therapy and detoxication, level of lii is successively decreased from . ~ . to . +. on the -th day after the operation. true decrease of the level mm from . ~. to . +. un & optimal density and increase of distribution index from . to . are argued. conclusions: in studlng the dynamics of the immunoglobulin's spectrum and the true increase of immunoglobulin g level from . +. g/i to i . +. g/i on the -th day after the operation simultaneously with the decrease of cic from . ~ to . ~ . (p . ) were founded. some stages of the investigation true increase of lymphocytes from . + . % to . + . % was noted and it appeared to be a favourable prognosis finding for disease outcome. high correlation dependence between bacillus-and segmentonuclear neutrophils and immunoglobullns g & m (r=. -. in p<. ) was discovered and it also showed positive dynamics of the course of the disease. a year old male patient was admitted to the icu with severe paraquat poisoning. treatment consisted of gastic lavage and oral administration of fullers earth. because of very high plasma levels hemodialysis together with charcoal hemoperfusion was started within one hour after admission. this treatment was further continued by continuous veno-venous hemofiltration in order to remove the circulating paraquat and also circulating cytokines. nevertheless patient s condition worsened necessitating artificial. ventilation and hemodynamic support. patient died hours after admission of acute multiple organ failure due to paraquat poisoning. serum levels of paraquat were determined by colorimetric method (table) . levels of interleukin (il ) and (il ), tumor necrosis factor (tnf-alpha), interleukin i receptor antagonist (il ra) were determined both in plasma and ultrafiltrate ( q~!ectives : evaluate in critically ill patients the effects of tow-dose dopamine on gastric mucosal blood flow (gmbf) using laser-doppler flowmetry, a continuous non invasive method of assessing microcirculation. methods : patients requiring both mechanical ventilation and pulmonary artery catheterization for multiple trauma (n= ), ards (n= ) and pancreatitis (n=l) were included. in each patient, the laser-doppler (ld) probe was inserted through a naso-gastric tube. the ld signal is proportional to the number of red blood cells moving in the measuring volume and the mean velocity of these cells. when the ld signal was satisfactory, an aspiration was created into a catheter which was fixed in parallel to the ld probe, to maintain the tip of the probe against the gastric wall at the site of measurement. data (systemic hemodynamic parameters and gmbf) were obtained at the end of a rain resting period (baseline), then min after dopamine ( mcg/kg/min) infusion, and finally rain after the end of dopamine infusion (recovery gmbf _+ (perfusion units) gmbf ~a% vs baseline) * p < . vs "baseline" and "recovery". conclusions : ) despite a slight increase in co (+ %), the dramatical increase in gmbf (+ %) with dopamine, strongly suggests a selective vasodilator effect of low-dose dopamine on gasaic mucosal perfusion. ) laser-doppler flowmetry appears a promising method to assess gastric microcircalation in critically ill patients. increasing evidence suggests that the activation of inos is the final common pathway for vasodilation in human sepsis associated with endotoxic shock. activation of the cellular immune system induces the excessive release of the pteridines neopterin (n) and , -dihydroneopterin (nh ) by human macrophages/monocytes. besides the well established diagnostic value of pteridines in several inflammatory diseases, it is speculated that these substances per se exhibit biochemical functions. thus we hypothesize that pteridines can modulate inos gene expression in vascular smooth muscle cells (vsmc) in vilro. cdtured rat aortic vsmc from female wistar kyoto rats were incubated with n ( pm), nh ( ilm), lipopolysaccharide (lps, ~g/ml), and interferone-~/(ifn-~/, u/ml) for h, respectively, inos gene expression was measured by competitive reverse transcription polymerase chain reaction. the results are summarized in the table. the present study demonstxates a neopterin induced increase in inos mrna expression at the transcriptional level in vsmc. while coincuhation of cells with n + lps resulted in an additive effect on inos gene expression, n + ifn- seem to have a more than additive effect nh did not alter inos mrna synthesis, but it suppresses the lps as well as the ifn-yinduced augmentation of inos gene expression. we speculate that this pteridine-mediated modulation of inos gene expression is involved in the regulation of the vascular tone in endotoxic septic shock. the relationship of sepsis and coagulation abnormalities is well known, mainly in severe sepsis and septic shock. still farther, the extreme expression of hemostasis abnormalities (disseminated intravascular coagulation) in sepsis, has been extensively described. we studied the changes in several coagulation and fibrinolysis markers in septic patients, trying to correlate them with the evolution of the sepsis phenomenon, with an emphasis in its early stages, where therapeutic intervention might be more drastic. in patients, with sepsis, with severe sepsis and with septic shock, as well as in healthy volunteers (control group) we measured : platelet (ptl), coagulation markers [fxii, fvii, fviii, fvw, fibrinogen (fibr) we conclude that all parts of the coagulation system are gradually changed during the evolution of sepsis phenomenon , even in the earliest stage of sepsis. the expression of an inducible nitric oxide (no) synthase (inos) plays a major role in the pathophysiology of septic shock (ss). inhibition of inos could therefore be of therapeutic value. however, such an inhibition has been shown to be detrimental, increasing tissue anoxia (and end-organ damage), possibly through the simultaneous blockade of constitutive nos (cnos). thus, selective inhibition of inos might be more suitable. we evaluated the effects of l-canavanine (can), a more potent inhibitor of inos than cnos, in an animal model of ss. method: in anesthetized rats, catheters were placed in the femoral vein and artery. rats were given an iv bolus of lipopolysaccharide (lps, mg/kg), at baseline (to). after h (t ), rats received at random an infusion of either can ( mg/kg/h; can group, n=l ) or an equivalent volume of . % naci ( cc/kg/h; nac group, n= ), giyen over h (t -t ). a third group (sham group, n= ) received . % nac in place of lps, and then was treated like the nac group. mean blood pressure (mbp), blood lactate and nitrates (no ) were measured each h. glucose, creatinine and asat were also measured in rats (n= in each group). the can _+ * + "t . + . "~ . +_ . "t + " + " *p< . can vs naci ?p< . vs sham can suppressed the hypotension, reduced the hypoglycemia and hyperlactatemia, and attenuated the biological signs of renal and hepatic dysfunction induced by endotoxemia. these effects were associated with a lesser elevation of blood no , confirming a partial inhibition of inos. conclusion: l-canavanine attenuates the hemodynamic and metabolic consequences of endotoxemia in the rat. these effects may be related to a partial inhibition of inos. they contrast with the deleterious effects described with non selective inhibitors of nos. l-canavanine could become a new tool for the treatment of septic shock. rocalc tonin :marker of sepsis, ii~flammaiiur% t~ boifi .cheval*~ jf.timsit*, m.assicot**, b.misset*,/.carlet*, c.bohuon** saint joseph heap, paris**biochemistry institut g roussy, villejuif, ce bi~)l~i~ttectives_: high serum levels of procalcitoaln (proct) have been shown to be ~ss-ocinted with bacterial infection. however, few data exist about the ability of proct to differenciate septic shock and shock from other origin in which an activation of intlmmamtory mediators has been also demonstrated. methods: thirteen patients with bacterial septic shock (ss), patients with non septic shock (nss), patients with bacterial infection without shock ( nf) and icu patients without shock and without infection (control) were compared for proct levels at dayl, , , , . patients were classified blindly and independently fi'om proct results. twelve patients were excluded because any classification was impossible due to mixed pathology. proct was measured with ebemoluminescenee (brahms diagnostica-berlin). results: dayl, proct levels are significantly different between the four groups. dayl proct levels are correlated with saps (p= . ), infection ( . +_ vs _+ ,p= . ), shock ( _+ vs +.- ,p= . ), death at day ( _+ vs _+ ,p= . ). when shock and infection are introduced in multifactor &nov& only infection remains correlated with day proct levels ( = . ) in patients with shock, dayl proct levels are correlated with saps, infection and death at day , but not with arterial lactate levels (p= . ), white blood calls (p= . ) or fever (p= . ). proct levels remain higher i~i septic shock patients at day , and ( figure) . i c edpsion: procalcitonin levels in the first three days of shock are differen[" between septic and non septic shock patients. in patients with diseases known to induce acute an inflammatory process, procaldtonin seems to be a marker o~ infection. obiectives-to evaluate the effect of endotoxic shock on the distribution of blood flow between the mucosal and the muscular layer of the intestinal wall. methods: in fasted pigs, mean aortic pressure (map, mm hg), cardiac output (co, ml/min-kg),superior mesenteric artery flow (q sma, ml/min.kg), and phi, where measured before (control) and after i.v. endotoxin ( gg/kg). the blood flow to the mucosal and the muscular layer was measured in regions (proximal jejunum (pj), mid-small intestine (mi) and terminal ileum (ti)) by colored microspheres, using adjacent samples in each region. the muscular layer was separated from the mucosa by blunt dissection, and the flow determined independently in each layer. results: endotoxin with fluid resuscitation induced the expected decrease in map ( . _+ . vs . -+ . , p< . ), and phi ( . !-_ . vs . _+ . , p< . ), with a constant co ( _+ vs _+ , p= . ) and qst, aa ( . _+ . vs . _+ . , p= . ). the results of regional pertusion are presented in the table. (flow in ml/rain g of tissue; mean _+ sem ; * p< . vs control by two-way anova) conclusions-these data indicate that the mucosal flow increased during septic shock. they suggest that a decrease in phi may be due to hypoper~usion of the muscular layer or to metabolic alterations within the mucosa, despite a % increase in flow. acute increase in wbc count (from a mean of lo.oo mm a to o /mm~), between the rd and the th day of therapy. there was a decline of the wbc count to an average of about . mm a after decreasing the daily dose of the medication to mcg there was no increase in tile absolute number of the eosinophils during the whole course of the medication. there was a slight decrease in the c complement between . to . g/i. normal values . to . g/i there was no change in c values. conclusions : an early increase in wbc count was observed ( rd day) without subsequent increase in the number of immature types from bone marrow, probably due to the mobilization of wbc from the periphery and this increase was dose dependent. there was a slight decrease in c fraction of complement, probably due to the consumption of this fraction in the process of opsonization. no adverse effects of the medication were observed, during the treatment with the above dose. these data sugest that cm csf may be a useful complement to tile main antimlcrobial treat,nent ~ of septic [cu patients. objectives: as part of a large multicentric, placebo-controlled, randomized clinical trial investigating the effects of interleukin- receptor antagonist (ii-lra) in the treatment of severe sepsis and septic shock, this substudy evaluated in dem.il the acute hemodynamic effects of ii-lra in patients who were invasively monitored. methods: in a total of evaluable patients in whom vasoactive support was little altered, hemodynamic measurements were performed at baseline (twice), and i hour, h, h, h, h, and h after the administration of mg/kg (n= ) or mg/kg (n= ) of i - ra or the corresponding placebo (n = ). / patients ( %) were treated with adrenergie agents and / ( %) with mechanical ventilation. data were analyzed by a kruskal-wallis test. results: during the study, there was no significant difference with time or between groups in arterial pressure, cardiac filling pressures, cardiac index or left ventricular stroke work (figure). burmester, "~ man and h. djonlagic medical university (internal medicine, "cardiology, *'microbiology) and "**southern city hospital, lfibeck, germany obiectives: evaluation of the incidence of bacteremia and sepsis in patients with nontyphoidal salmonella (s.) infections, specification of risk factors, need of icu treatment, clinical course, and mortality in the group of the patients who developed septic complications. methods: data of all patients with microbiologically proven s. infections hospitalized in the medical university of lobeck and in the southern city hospital of l beck from to . results: within the observation period s. was isolated from the stool cultures of patients. in patients (g m, f, median age yrs) s. could be detected in blood cultures ( s. enteritidis, s. typhimurium). in addition, in of these patients s. was also isolated from other specimens (urine, liquor, and tissue fluids derived from abscess punctures). in all patients with positive blood cultures the clinical course of s, infection was complicated: ? patients developed mof (acute renal failure, ards, hemodynamic instability, dic) and required icu treatment for at least up to days, of the patients died. the predisposing disorders in the patients with s. bacteremia were (n=): aids ( ), immunosuppressive drugs ( ), chronic alcoholism ( ), malignancies ( ), none ( ). septic complications in patients with nontyphoidal s, infections are relatively rare (in this study < % of all hospitalized patients with microbiologically proven salmonellosis) but severe (mortality of approx. %). patients at risk for a complicated clinical course are predominantly those with predisposing disorders but occasionally also patients without evidence for an underlying disease. age (yr) + + death (n) duration of shock (h) + + noradrenaline (rag/h) , _+ + temperature (~ , + , + pvr (dynxsecxcm - ) + + co (ljmin) , _+ , , + , lactate (mmol/l) + , , + interleukin- (pg/ml) _+ + interleukin- (pg/ml) , _+ , , + , tnf-alpha (pg/ml) , + , + neopterin (nmol/l) , + , + crp (rag/l) _+ +_ pro-ct (ng/ml) , + , , + there was no positive correlation between serum lactate levels, degree of shock, hypoxemia and pro-ct positivity. pts with septic shock of bacterial origin entirely developed hyperprocalcitoninemia, whereas pts with cardiogenic shock, who expired within h did not. however, in late cardiogenic shock (> h) all pts developed fever of unknown origin and consecutive hyperprocalcitoninemia. these data suggest bacterial inflammation and/or mucosal translocation of bacterial products in pts with prolonged cardiogenic shock. the use of a loading dose of quinine ( . mg/kg base in h) is recommended in previously untreated patients (pts) with sfm, particularly in multi-drug resistance areas. this protocol is difficult to validate, since the viability of microorganisms is not assessed routinely in parasitology laboratories. objectives: to examine the evolution of parasite viability during the early phase of therapy of sfm. methods: from / to / , pts with sfm (who ) treated with iv quinine for less than h were included prospectively. blood samples were collected at o, , , , , and h viability was assessed by culturing parasitized red blood cells in the presence of h-hypoxanthine, and radioactivity was determined at h by scintillation counting. viability was expressed as the percentage of radioactivity compared to the initial sample. plasma quinine was determined by liquid chromatography. tile ratio plasma quinine (pmol/ )xlo /icso for quinine (nmo]/]) was called the parasiticida/ index. results: pts were included, • saps . -+ . . the initial parasitemia was t. + . %. complications of malaria were coma ( pts), shock ( pts), renal failure ( pts) and acute lung injury ( pts). all strains were sensitive to quinine (icso -- nmol/ ). in pts who were not given a loading dose, parasite viability increased by and %, with concomitantly low quinine levels ( and #mow] at h); pt died. in pts that received a loading dose (serum quinine at h = . -- . ~mol/]) a marked decrease of parasite viability (by +_ % at h) was shown. viability was inversely correlated with plasma quinine (r=. , p-.o ) and parasiticidal index (r=. , p-.o ). conclusions: even with fully sensitive strains, the use of a loading dose of quinine seems warranted in severe falciparum malaria in order to reach rapidly adequate plasma quinine ]evels, necessary to inhibit significantly parasite viability. l nkka, e ruokonell j takala. critical care research program, department of intensive care, kuopio univ hospital, finland objective: to determine the incidence of positive blood cultures, their microbial subgroups and to evaluate the outcome of icu patients with different bacleremias. material and methods: we analysed all positive blood cultures in consecutive admission to a university hospital icu in - and the icu and hospital survival of the bacteremia patients. during these years patients had positive blood cultures that were considered as clinically relevant, excluding colonizations or contanfinations. results: patients with positive blood cultures had an icu survival of . % (vs. , % in all icu patients) and six month survival of . % (vs. . % in all icu patients). the most common bacteria were enterobacteriaceae ( , %), staphylococcus aureus ( , %) , coagulase negative staphylococci ( . %), pseudomonas ( . %) and slieptococci ( . %). obiectives: to evaluate prognostic factors and mortality in consecutive patients (pts) with hiv infection and septic shock. methods: from - to - , records of consecutivepts with septic shock (crit care med , : - ) admitted to the icu were reviewed retrospectively. results: among pts with septic shock admitted during the study period, had hiv infection- of whom had aids-(gr. i) and were hiv-negative (gr. ill. ten gr. ii pts ( %) were irnmunosuppressed because of neoplastic or immune dlsease. mechanica] ventilation was required in % gr. i and % gr. ii pts in gr . i pts ( %) a multivariate analysis demonstrated that hiv infection and sap i were independently predictive of death in pts with septic shock. ~onclusions: evidence of increased mortality, number of organ failures and higher severity scores (saps i does not take into account immunosuppression) is demonstrated in hi v-positive pts, infection with hiv appears to be an independent prognostic factor in pts with septic shock. the frequency of opportunistic infections (often responsible for delayed diagnosis and treatment) may contribute to the poor prognosis in this population. obiectives: to determine interleukin (il)-i levels in plasma of patients with sepsis and septic shock. to analyze the relationship between plasma il- and the proinflammatory mediators, tumor necrosis factor-aifa (tnf) and il- , the underlying severity of the disease and the evolution of patients with sepsis. methods: we studied critically ill patients ( men, women; - years old) in three diferents groups. group i: patients without evidence of infection, group i : patients with sepsis and with septic shock (group iii). we measured plasma il-lo, tnf and il- levels in the first hours of diagnosis. severity of illness was estimated with the acute physiology and chronic health evaluation (apache ii) scoring sytem. results: plasma levels of il- were higher in group iii (median, pg/ml; range, - pg/ml) than in group ii (median, pg/ml; range, - pg/ml; p <. ) and group i (median, pg/ml; range, - pg/ml; p <. ). median il- concentrations did not differ among patients who survived (median pg/ml; range, - pg/ml) and those who died during the overall follow-up period ( days) (median, ; range, - pg/ml); but patients who died in short-term (< hours) with catecholamine-refractory hypotension showed the highest concentrations of il-io (median, pg/ml; range, - pg/ml). in patients with bacteriemia ( %), levels of il- were higher (median, pg/ml; range, - pg/ml) than in those with negative blood culture (median, , pg/ml; range - . pg/ml; p< . ). there was a good correlation between plasma il-io concentration and levels of tnf (r= . ; p < . ) and il- (r= . ; p < . ). the correlation between levels of il- and the apache ii score was significant only in the septic shock group (r= . ; p <. ). conclusions: in septic shock, il-io and proinflammatory citokines are released in high concentrations. the significant correlation observed in patients with septic shock between il- levels and apache ii, short-term death and bacteriemia can possibly be explained by the massive inflammatory response in septic shock with fulminant course. intensive care department -calmette hospital - lille -france. in septic shock, inadequate splanchnic blood flow may play a prominent role in the pathogenesis of multiple organ failure. measurement of gastric phi has been propose to evaluate tissue oxygenation in splanchnic organs. objectives: to compare gastric phi values with hepatic icg clearance, an index of liver blood flow and function ; to determine if one of these two methods could be proposed to assess the entire splanctmic peffusion in septic shock. methods : patients (age : • years ; saps ii : • were prospectively investigated (septic shock : bone criteria). following parameters were collected during hours : systemic hemodynamic parameters (swan ganz catheter a h -ref computer -baxter lab.), calculated systemic oxygen transport (do ), oxygen consumption (vo ) by indirect calorimetry (deltatrac datex lab.), gastric intramucosal pco (pco ss) and phi (trip -ngs catheter -tonometrics lab.) and plasma disappearance rate of icg (pdr dye) (femoral artery fiberoptic/thermistor catheter , cold z computer -pulsian medizintechnik, germany). correlations were performed using a linear regression. elevated in all days with the highest value in second and third days of treatment. nonsurvivors had higher values of these parameters than survivors but differences did not reach statistical significance. another trend of changes were observed in selectin p (gmp- ) concentration. in all patients concentrations measured were elevated but in survivors after not significant decrease this parameter in second day another one had simmilar values. in patients who died we noted significant decrease in third day (p < . ) whereafter prominent increase, significant after seventh day, in comparison to third day value and value in survivors group. icam- concentrations in all patients reached high levels and in nonsurvivors after four day of treatment significant increase in comparison to survivors we found. conclusions: multiple trauma complicated with sepsis induce rapid elevation of concentrations of il- , il- and increased expressior of adhession molecules (selectin e, p, icam- ) measure of icam- and selectin p concentration determine lung injury severity and prognosis as to health and life. (clp) .pathophysiology of cip is unclear, but changes in regional bloodflow may be a ~ignificant factor. nerve blood flow (nbf)is reduced in rat models of hemorrhagic shock (g),but no information is available in sepsis. we studied the comparative effect of acute endotoxemic shock {etx)& h on perfusion of rat sciatic nerve. methods: male sprague-dawley rats were anesthetized with pentobarbital (ip), instrumented with a tracheostomy, carotid arterial & venous catheters and mechanically ventilated (fi = . ). the left sciatic nerve was surgically exposed. monitored variables included: a) mean arterial pressure (map,mmhg) ,b) nbf (ml/ o g/min) by laser doppler flow meter,c) nerve internal arterial diameter (id ~ m) by video image shearing and splitting method. after stable baseline measurements were obtained, acute hypotension was induced by randomly assigning the rats to etx ( . b , difco) in saline at mg/kg or h. both interventions produced % reduction in map within min., which recovered to baseline values spontaneously in etx group, & by reinfusion of heparinized withdrawn blood in m. data were analyzed by linear regression, two-way repeated measures analysis of variance followed by bonferroni-t method. experimental stages were:( )baseline, ( ) mid-point of map reduction; ( ) nadir of hypotension, ( )midpoint of map recovery, & ( ) after stable recovery of map. both etx & h induced shock result in similar reduction in nbf consistent with lack of autoregulation in peripheral nerve vessels independent of etiology. since cip is primarily associated with sepsis, it is not likely that acute reduction in nbf alone causes cip. direct & indirect neurotoxic effects of mediators of sepsis need to be evaluated. .':_.~::::o o:oc ., objectives : evaluate the relationship between il- , a cytokine which inhibits tnf, production and protects mice from endotoxin toxicity, and the other proinflammatory cylokines, tnf~, il and ils in severe sepsis and septic shock. methods : twenty-eight icu patients ( m, f, mean age + y) were studied as soon as they developped a severe sepsis (n = ) or a septic shock episode (n= ) as defined by a conference consensus in ( ). tnf~, il , il s and il- plasma levels were measured by immuno-radiometrie assays from medgenix (fleurus, belgium). lc mean and range. results : the comparisons between cytokine levels in severe sepsis versus septic shock were made using the logarithm of the value in order to normalize the distribution of data, and student test. il- plasma levels were higher in patients with septic shock than in patients in severe sepsis. there was a significant correlation (p < . ) between il- and tnf a (r= . ), il- and il~ (r = . ) and il- and il s (r = . ) as well as between il- and apache n score (r= . ). patients who died (n = ) had il- levels higher than patients who survived but this difference was not statistically significant ( pg/ml vs . pg/ml; p> . ). conclusions : during severe sepsis and sepsis shock, il- seems at least to follow the same evolution (increase in plasmatic level) with the severity of sepsis as the other cytokines. reference : ( ) crit care med ; : - . objectives: to evaluate the effects of steroids on hemodynamics and mortality in septic patients with konwn levels of cortisol concentration. methods: retrospectively we analyzed data ofpatients with documented septic shock who received steroids after assessment of adrenal function. in all patients hemodynamic parameters as well as the necessary vasoactive medication were assessed, before and hours after corticosteroid medication. immediately before administration of corticosteroids adrenal function was evaluated with cortisol levels before and after synthetic corticotropin ( . mg). finally we studied mortality. we defined a positive respons on corticosteroids as an elevation of map of at least mmhg and/or a decrease in the necessary vasoactive medication of at least % within hours. adrenal insufficiency was defined as a cortisol level after stimulation of less than nmol/l. results: of patients were found to respond to steroid medication, did not. mean cortisol levels before and after corticotropin were • and • nmol/l in the responder group (rg) and • and • nmol/l in the non responder group (nrg). in the rg out of ( %) were found to have an adrenal insufficiency, in the nrg out of ( %). in the rg -weeks mortality was . % (l out of ), the overall mortality % ( out of ). mortality in the nrg was % ( out of ) (p < . ) and % ( out of ) (p < . ) respectively. conclusions: in patients in septic shock there is a beneficial effect of steroids in case of adrenal insufficiency, but also in a subgroup with normal adrenal f{unction. obiectives: intercellular adhesion is a critical step in the accumulation of leukocytes. postischemic cardiac lymph has the capacity to stimulate icam-i. in the coronary microcirculation neutrophils can be trapped and in many cases obstruct capillaries, previously we found that troponin t (s-tnt) a marker for myocardial iechemia, was increased in septic patients. the aim of the study was to follow slcam- and s-tnt levels continuously starting at the beginning of sepsis. methods: patients were ingluded in this institutionally approved study after relatives had given their informed consent. all patients were included within hrs following the beginning of sepsis. blood was drawn every hrs in the first ;~ hrs, after hrs, followed once per day for days. s-tnt, icam- , elam (elisa's, boehringer mannheim inc, r&d systems ltd.) arterial and venous blood gases were determined, an ecg and a complete hemedynamir measurement including cardiac output were obtained. all patients received adequate volume and catecholamine therapy (norepinephrine, dopamine, dobutamine; median (range) . ( . - . ), . ( . - ), . ( . - . ) pg/kg/min, respectively). statistical analysis: wileoxon signed rank-sum test. . ( . - . ) . patients had s-tnt levels > . pg/l. of these died, whereas only of patients died with s-tnt values < . pg/l (p= . ). all patients that died had elevated sjcam- levels ( ilg/l:cut-off ) whereas in the survivor group only % had elevated icam- levels (p= , ). conclusions: increased slcam- and s-tnt levels were found during early sepsis in the majority of patients, a high sicam- and s-tnt value was associated with a higher mortality. the research of the noninvasive haemodynamic monitoring accelerated recently all over the world. the aim of our study was to test whether the changes of the haemodynamk parameters measured by impedance cardiography (icg) were corresponded to clinical changes in septic patients. investigations were performed on critically ill postoperative septic patients (their multiple organ failure score was - /with icg monitor. in cases the investigation~ were performed in septic shock. the measured parameters were: heart rate (hr), mean arterial pressure (map), cardiac output (co), peripherial resistance (svr),preejection period (pep), and ventricular ejection time (vet). these parameters were measured during - hours in every minutes, depending on the patients cl~tnical condition. results: at the septic patients the hr and the co ]~reased. in septic shock the co was significantly higher the svr lower than in the septic group. in the hr there was no difference between the two groups. in septic shock noradrenalin influenced more effectively the measured parameters than dobutamin. conclusion: the trend of the measured icg parameters correlated with the clinical changes of septic patient's state. the noninvasive haemodynamic monitoring by impedance cardiography helps the planning and leading the adequate intensive therapy of these critically ill septic patients. to evaluate the development of sirs, sepsis and septic shock in hospitalized patients with fever, a prospective study was performed on patients using previously defined criteria. methods: normotensive patients with fever (temperature > . ~ axillary), admitted to the department of internal medicine were evaluated for the existence of sirs during the first three days of the study and sepsis at inclusion. during a follow-up period of days the patients were daily evaluated for the development of sepsis or septic shock. results: most patients ( %) had or developed sirs within the first three days, patients ( %) did not. sepsis was present in % at inclusion. in patients with sirs, % did not progress to sepsis or septic shock, % progressed to sepsis (mean interval . • . days), and patient (< %) directly progressed from sirs to septic shock. in patients with sepsis, % progressed to septic shock (mean interval . • . days). sepsis was preceded by sirs in %. septic shock was preceded by sepsis in % and by sirs in %. conclusions: % of patients with fever in an internal medicine department develop sirs, or sepsis. furthermore, progression from sirs to sepsis or septic shock is poorly predicted by fever or sirs. nevertheless, all patients with septic shock were preceded bysirs or sepsis. taken together, this may indicate a severity hierarchy of the syndromes. however, fever, sirs and sepsis are relatively poor indicators of development of septic shock. this supports further research on additional predictors of septic shock. b. m.manuylov, v.b.skobelsky (moscow) in recent years sodium hypochlorite (sh) has been successfully used to eliminate pyo-septic complications. moreover, the mechanism of the sh effect on the immune system has not been sufficiently studied. the aim of the present investigation was to study the mechanism of sh effect in inflammatory pulmonary diseases. patients with double pneumonia were subjected to the evaluation. sh in the concentration of mg/l in the volume of - m / hours was administered by drop infusion into the central vein. to evaluate one of the defence systems the leukocytes activity by the chemoluminescence technique was studied. in all the patients baseline secondary immunodeficiency which was indicated by the decrease in the luminescence level was established. even hour after the sh administration the leukocytes activation exp-ressed by the enhancement of their chemoluminescence . - times was observed. this supports the available findings that accumulation and liberation of the oxygen active forms (ol'oh, ' , h ) are accompanied by the increased phagocytosis, i,e. the signs of "the oxydation explosion" testify to the favourable sh effect on the course of inflammation processes. the use of sh permitted to decrease the percentage of lethality in double pneumonia by % in the intensive care unit over the year. at the same time, excessive activation of free radical oxygen may be a damaging factor. therefore, precise individual control over the choice of concentration, dosage and the preparation administration rate is required. prospective, double-blind, placebo-controlled, trial of atiii substitution in sepsis r. a. balk objective: pilot study to evaluate the efficacy and safety of atiii substimtion therapy in patients with sepsis. efficacy assessed using change in mortality or organ failure/dysfunction. adult patients meeting a definition of sepsis and cared for in a tertiary care academic medical center in chicago were identified and prospectively randomied to receive either atiii (kybernin p) or placebo in a double-blind treatment protocol. all other therapy and patient management were under the direction of the patient's attending physician. all patient's were followed for days and the organ dysfunction/failure were scored using published scoring systems (jordan et al crit. care med. , goris et al arch. surg. , kuaus et al ann. surg. colldusions:wha~ we met the shomaeker objectiv% the mortality and the pro~os[s were i~ttc*. those criteria were obtained with file tradititmal t~ctor likr doht~mme, hut c.~vh ~,as ca in~aertam measure. they ac~s smxergically in the optimizatic~l of the fell vmtrictdar work index, tad fimdameatally cavh seox~s to have an impo.aat role in the better respiratory ev-altmtioa, leaving yet the possibility to coltrol the flui& r althou~l eomproved it's not aec~pt~xl file importmlce h* the diminution, of the sepsis modiat~lrs llke fnt and il- with h~wmotiltrafi(al, stopphlg the evolution to nmltiorganic failure mid de~easethe mortality. with ours clhlicals results, we could saythat cavii in multiol~atlie disfut~oa septic patieats, se~r~ to be an c xilna] supoa or troatmeat maesure. of anaesthesia and intensive therapy, medical university of prcs, p~csf hungary. objectives: since some biological effects of bacterial endotoxin require an interaction between the lps molecule and a serum factor(s), we hypothesized that lps-induced no production and cgmp accumulation in vascular smooth muscle cells (vsmc), a mechanism ~thought to underlie cardiovascular collapse associated with septic shock, is modulated by serum factor(s). methods: cultured vsmc from rat aorta were challenged with e. coli lps for - hours either in the presence or absence of fetal calf serum (fbs), and no production was monitored by radioimmunoassay determination of cgmp content of hci extracts. results: in the absence of serum, o ng/ml lps was required to increase cgmp levels, whereas the presence of % fbs shifted the lps concentration curve i times to the left. similarly to fbs, human serum also potentiated lps-induced cgmp accumulation. in contrast to lps, serum had no effect on cgmp accumulation elicited by sodium nitroprusside, a no releasing agent, suggesting that the sensitivity of vsmc to generate cgmp in response to exogenous no is not modulated by serum. heat inactivation (> ~ min) but not removal of small molecules (< , d) from the serum by dialysis, reduced the potentiation of cgmp accumulation by serum. time course studied indicated that serum is required within the first min of lps exposure to increase cgmp levels. to investigate whether the effect of serum is specific for lps, we treated the cells with increasing concentration of interleukin -~ (il-i). % fbs shifted the il-iinduced cgmp responses five times to the left. conclusions: our study suggests that lower concentrations of e. cell lps and il-i require a heat labile macromolecule in the serum in order to elicit no production. this factor is present in the human serum and it may play a potentially important role during no synthesis induction in vsmc. objective: to evaluate the factors of acquisition and the outcome of methicillin resistant staphylococcus aureus (mrsa) bacteremia in an intensive care unit (icu). methods: all patients in which bacterermia due to staphylococcus aureus developed > hours following admission to our icu, during a year period ( january through january ) were reviewed. patients (pts) were included, mean age , y (sd , ), saps , (sd , ), mac cabe ( and ) %, mortality directly due to sepsis %. pts had mrsa bacteremia and methicillin susceptible staph. aureus (mssa) . both groups were compared using the chi square (with correction of yates), fisher's exact, student's t or wilcoxon test. results: there was no statistically significant difference between mrssa and mssa regarding at age ( , + , vs , + , ) , saps ( , + , vs , + , ), use of vancomycin ( % vs %), mechanical ventilation ( % vs %), number of days (d) before the drawing of the first positive blood culture (median d, range - d vs median d, range - d). more mrsa than mssa pts had previous use of nonsteroidal anti-inflammatory drugs (nsaid) ( % vs % p< , ), central venous catheter infection due to staph.aureus ( , % vs % p< , ), but previous use of antibiotics was not significantly different ( , % vs %). the outcome of the bacteremic pts was not statistically different: saps at the first day of bacteremia ( , +_. , vs , + , ), severe sepsis and septic shock ( % vs %), persistence of the bacteremia ( % vs %), mortality directly due to bacteremia ( % vs %). conclusion: previous use of nsaid, infection of venous central catheter are more frequently associated with mrsa bacteremia. thus, similar to others studies (hershow infect control hosp epidemio ; : - ) , these results do not indicate that mrsa is associated with increased virulence. objectives: to closer definition of mosf formation mechanismes in nosocomial sepsis (ns) the complex clinicobiochemical, microbiological, immunological, functional exaroination of cases with ns had been done. methods: examination of cellular and humoral immunity, nonspecific immunologic reactivity, systemic and hepatic circulation, microbiological examination of blood,electro-and echocardiography, sonography and computer tomography of chest and abdomen organs were obligatory. autopsy findings of dead cases had been analized. results: in cases ( , %) opportunistic pathogen microscopic flora ( staphylococcus anreus,staphylococcus epidermidis, staphylococcus saprophyticus) had been found out in blood inoculations. in cases ( %) side by side with destructive process in lungs the bacterial endo-and myocarditis with blood circulation failure had been determined.in cases ( %) simultanious lesion of three organs (heart,lungs,liver) had been found. morphologic examinations of dead cases ( %) internal revealed involvement of them in mosf-syndrome.hyperplasia of adenohypophysis;sclerosis of adrenal glands cortical layer;perivascular brain oedema,paralysis of brain capillaries and plasmorrhagia, cerebral thrombosis and cerebral abscess,necrobiosis of epithelium tubules of the kidney,pletora of hepar, fatty and granular degeneration of hepatocytes had been found.atrophy of white pulp and hyperplasia of red pulp, supress of lymphoid tissue, plethora and formation of infarctious had been found in spleen. mentioned changes in spleen were indispensable in ns. conclusion: in ns spleen can not secure it functions to support and appropriate detoxication potencial of organism,elimination of microbes,toxines,antoallergenes. insolvency of immunological link of antimicrobic defence is the starting mechanism of mosf developmentin ns. %neviere, jl. chagnon, b. vallet, d. mathieu, n lebleu, f. wattel ] ept of intensive care, hop calmette, lille, france ~everal studies have described tiypoperfusion of intestine during sepsis. owever, it is unknow whether the mesenteric blood flow is associated with nucosal hypoperfusion. additionally, the effects of resuscitation on the ntestinal microcirculation remain controversial. bjectives : to describe the effects of endotoxin in a porcine model during ~hock and resuscitation. ~ethods : ten pigs ( kg) were anesthetized and instrumented for "neasurement of cardiovascular variables. gastric and gut oxygenation vere assessed by intra-mucosal ph and microvascular laser doppler lowmetry. after baseline data collection, a minute intravenous infusion )f escherichia colt (serotype h , sigma, st. louis, mo) was begun ~t a rate of pg/kg. an infusion of either saline at . ml/kg/min (group ; n= ) or saline and dobutamine at a rate of pg/kg/min (group ii; n= ) vas begun mn after the end of the endotoxin infusion. tesults : to td t ~ fl w fluid ioadin,q alone sfyras d, k perreas, e douzinas, k spanou, m pitaridis and c roussos critical care dpt, evangelismos hosp., athens univ, school of medicine. obiectives: much controversy exists concerning the beneficial effects of cvvh on sepsis. we studied the effects of cvvh application on septic patients with reference to the following parameters: i) survival rate ii) cytokines' removal and iii) timing of cwh onset. methods: patients with sepsis (criteria according to accp/sccm, ) underwent cvvh as soon as they developed renal failure or dysfunction (urinary output< ml/ h, cr> . mg/dl and bun> mgd'dl ). specimens were collected: blood samples before cvvh and therafter both blood and ultrafiltrate (uf) samples on , and hours. cytokines tnfa, i - and ii- were measured by the immunoassay method in all specimens (uf and plasma -p) and sieving coefficient ([uf]/[p]) and h solute mass transfer of tnf and i - were calculated (v h x [uf] ). the apache ii score before cvvh onset, the duration of icu stay and the timing of cwh application related to the sepsis onset in days (ta) were recorded.with respect the mortality two groups were formed, i.e. group a (survivors) and group b (non-survivors) . the morbidity period in days of those septic patients who died in the past year and were not subjected to cwh (group c) was compared to that of group b. results: group a included pts and group b pts with mean+sd age ( _+ vs _+ , ns) and apache scores( _+ vs -+ . , ns). the mean ta-+ sd was . + vs -+ , p< . . the mean_+se morbidity period of group b vs group c was _+ vs _+ . p< . . the mean values of cytokines are presented in the following figures. the sieving coefficient for tnf was . and for i - was . . the solute mass tranfer was -fold the actual plasma content at a given time. . o conclusions: i) early application of cvvh seems to favourably affect the outcome of septic patients, ii) cytokine plasma levels do not decrease although cytokine removal is substantial, iii) it seems that cwh application in sepsis of any stage helps to buy time for further treatment. the most commonly monitored variables in shock stages idclude : arterial pressure, heart rate, central venous pressure, pulmonary artery wedge pressure and cardiac index. with vigorous therapy it is possible to bring these values back into the normal range in both survivors and nonsurvivors. therapeutic goal in septic shock stages is to maximize the values of cardiac index, delivery (do ) and consumption (c ). objectives: the main purpose of this article is to determine the relationship betwee~ delivery an consumption as a sign of hypoxia. fifteen patitents with septic shock were treated with intention to maximize the value of ci,d and v . we compared the levels of these parameters between the survivors and nonsurvivors and found no significant differences after hours. high levels of do and v may not guarantee against tissue hypoxia in early stage of septic shock. zjar~iic, dj janjic, lj. gvozdenovic, a.komareevic. t.petrovic, &marjanovic, institute of surgery, novi sad, yugoslavia objectives: evaluation and mutual comparison of clinical signs, laboratory data and microbiological monitoring in the patients with burn sepsis. method: retrospective analysis of the recorded data of all burn patients treated in our department between january and december . specially attentions were given to data considering wound infection, positive haemocultures, positive urinocultures and characteristics of septic state. results: out of patient there were ( , ~) adults and ( , ( ~) children. almost two thirds of the patients ( - , ~) were males. the predominantly cause ( , ~) of children's burns was scalding b~y hot liquids and flame burns ~ , ~) in adult patients. the most frequdntly species isolated from surface swat~ were pseudomonas aeruginosa ( " in adult patients) and staphyloccocus epidermidis ( , % in children). in only five patients ( , ~ the haenmcultures were positive -pseudomonas aeruginosa was isolated in three and staphyloccocus aureus in two patients. urine infection was diagnosed in , % of all patients. the treatment protocol included use of imipenem and polyvalent pseudomonas vaccine again~ pseudomonas aeruginosa and vancomycin and aminoglycosides against staphylococcus aureus. total mortality rate in this group of burned patients was , ~, but the mortality rate caused of sepsis was low (i %) . conclusions: early detection of any signs of wound infection and symptoms of septic state is a foundation for prevention and treatment of burn sepsis. the burn sepsis could be reliable detected by continuously monitoring the patient's status and by systematic microbacteriological monitoring of the burned patients. hyperdynamic vasoplegic septic shock p.f. laterre, p. goffette, j. roeseler, j.p, fauville, a. poncelet, p. lonneux, m.s. l~eynaert. dept. of intensive care, st. luc univ. hospital, brussels, belgium. splanchnic ischemia is described as a common feature of septic shock and could determine the development of msof. therapy such as noradrenaline (na) aiming at improving blood pressure is expected to worsen splanchnic ischemia by its vasoconstrictive effect and subsequent reduction in intestinal blood flow. ob[ective: evaluate the effect of na on splanchnic blood flow. material and method : in a patient admitted for variceal bleeding, ards and sepsis with positive blood culture, a fiberoptie catheter was positionned in the portal vein after recanalisation of its portosystemic stent shunt. blood pressure (bp-mmhg) , ci, svr, do (vigilance ~ baxter), v (indirect colorimetry), arterial, mixed venous and portal vein blood gases, phi were determined before (to) and during (t ) na infusion ( , to , hcg/kg/min.) . changes in splanchnic flow were assessed by changes in portal oxygen saturation (sp ) and arterio-portal oxygen saturation gradient (sao, -spoe laterre, ,lp. pedgrim, th. dugernier, v. delrue, ph. hantson, p. mahieu, m.s. reynaert. dept. of intensive care, st. luc univ. hospital, brussels, belgium. aim of the study : prospective determination of plasma levels of in patients with ss and their correlation with the type of microorganism and outcome. material and methods : in patients (pts) with ss and severe sepsis, plasma levels of tnfti, ill-b, il and il were determined every hours for days and on day after fulfilling the criteria of ss and severe sepsis. results : in pts, sepsis was caused by a gram (-) microorganism, in pts by a gram (+) and in pts no microorganism was identified. there were survivors ( %) (s) and non-survivors ( %) (ns) . cytokines profiles and levels were not different between gram (+) and gram (-) sepsis. ill-b levels were seldom elevated whatever the group studied. tnfot and il- were significantly higher in ns than in s ( objective: to evaluate the effects on the nitric oxide synthase inhibitor l-n~ hcl ( c ) on myocardial performance in human septic shock. method: septic shock was defined as severe sepsis with either persistent hypotension (mean arterial pressure; map< mmhg) or the requirement for a noradrenaline (na) infusion >_ .i ]tg/kg/min with a map _< mmhg. cardiovascular support was limited to na _+ dobutamine (db), c was administered for up to h at a fixed dose-rate of either , . , , or mg/kg/h iv. during c infusion, na was to be reduced and if possible withdrawn, whilst maintaining map above mmhg and the cardiac index (ci) as clinically appropriate. assessments were made at baseline (t = ); at i h from the start of treatment (t = ); and at the end of treatment (t = ) with c . conclusions: c can restore systemic vascular tone in patients with septic shock enabling na therapy to be reduced and/or removed. the ci tends to fall whilst lv performance is sustained over time. c is a novel vasoacfive agent for the treatment of septic shock, which is undergoing further clinical evaluation. laterre, f. thys, e. danse, j.p. pelgrim, e. florence, z roeseler, m.s. r eynaert. dept, of intensive care, st. luc univ, hospital, brussels, belgium. therapy aiming at improving blood pressure and cardiac index in septic shock (ss) might have deleterious effects on regional blood flow. objectives : compare the influence of volume loading (vl), dobutamine (dobu) and noradrenaline (na) on sushepatic oxygen saturation (shoe) and svoe-sho, gradient in treated ss. material and methods : in patients with ss, ci (thermodilution) , doe, svo,. sho,, svoe-sho e gradient and lactate (l) were determined before (to) and after (t ); vl, dobu and na. results: in patients with treated ss, tests were performed (vl n= ; dobu n= ; na n= method: septic shock was defined as severe sepsis with either persistent hypotension (mean arterial pressure; map< mmhg) or the requirement for a noradrenaline (na) infusion ~> . ~g/kg/min with a map _< mmhg. cardiovascular support was limited to na + dobutamine (db), c was administered for up to h at a fixed dose-rate of either i, . , , or mg/kg/h iv. during c infusion, na was to be reduced and if possible withdrawn, whilst maintaining map above mmhg and the cardiac index (ci) as clinically appropriate. assessments were made at baseline (t = ); at h from the start of treatment (t = ); and at the end of treatment (t - ) with c . conclusions: c is a novel vasoactive agent that can sustain map in patients with septic shock, enabling na support to he reduced and/or removed. there is a tendency for the ci to fall during treatment, which may be reflex in response to the increase in systemic vascular tone. c is a promising new therapy for septic shock, which will now be evaluated in a randomised, placebo-controlled safety and efficacy study. k. guntupalli objective: to evaluate the acute effects of the nitric oxide synthase inhibitor l-n~ hc ( c ) on selected indices of organ function in patients with septic shock. method: septic shock was defined as severe sepsis with either persistent hypotension (mean arterial pressure; map < mmhg) or the requirement for a noradrenaline (na) infusion --> . [xg/kg/ min with a map _< mmirlg. cardiovascular support was limited to na + dobutamine. c was given for up to h at a fixed dose-rate of either , . , , or mg/kg/h iv. during c infusion, na was to be reduced and if possible withdrawn, whilst maintaining map above mmhg and the cardiac index (ci) as clinically appropriate. indices of organ function were assessed at baseline (t = ); at the end of treatment (t = ); and h after treatment (t = ) with c . results. -median values (* assessment made at h or when c discontinued). conclusions: there was no appareut dose-dependent adverse effect on these indices of organ function either during or after exposure to c . the plmelet count tended to fall whilst creadnine appeared to increase over time in all dose cohorts. this novel and promising therapy for septic shock will now be evaluated in a randomised, placebo-controlled safety and efficacy sludy. pharmacokinetics of c in patients with septic shock preliminary results z. hussein, b. jordan, c. fook-sheung, k. guntupalli objective: to evaluate the pharmacokinetics of the nitric oxide synthase inhibitor l-n~ hc ( cg ) given by continuous infusion for h in patients with septic shock. method: septic shock was defined as severe sepsis with either persistent hypotension (mean arterial pressure; map < mmhg) or the requirement for a noradrenaline (na) infusion --> . ~tg/kg/min with a map _< mmhg. cardiovascular support was limited to na • dobutamine. c was administered for up to h at a fixed dose-rate of either , . , , or mg/kg/h iv. plasma was collected from each patient over a h period and analysed for c . pharmacokinetic parameters were derived from plasma concentration-time profiles using non-compartmental pharmacokinetic analysis. results: the (cm~ -maximum plasma concentration; auc -area under curve; cl -plasma clearance; v,, s -steady state volume of distribution; t'/ -plasma elimination halflife). conclusion: the pharmacokinetics of c in patients with septic shock are dose-independent at infusion rates up to . mg/kg/h. at higher rates, clearance of c decreases without any marked change in volume of distribution. c metabolism may be partially saturable at dose-rates above . mg/kg/h. obiectives: investigate the effect of the no synthase inhibitor, l-nt-methylarginine hc ( c ) on the haemodynamics and survival rate in a conscious mouse model of endotoxin shock. methods: female cd- mice ( - g) were instrumented under gaseous anaesthesia (isofluorane, %) and connected to a swivel tether system for continuous monitoring of blood pressure and drug administration. results: after h recovery, endotoxin administration (e. col• :b , - . mgkg - i.v.) elevated the plasma concentration of nitrite/nitrate (nox) and caused a progressive fall in mean arterial pressure (map) from + to + mmhg (n= , p< . ) at h, with a survival rate at h, h and h of %, % and % respectively. c administered as a h continuous infusion ( mgkg-th -t i.v., n= ), h after endotoxin, inhibited the elevation of plasma nox and attenuated the fall in map from + to + mmhg (n= ) at h, with an improved survival rate at h, h and h of %, % and % respectively. conclusions: this study suggests that overproduction of no is involved in the hypotension and mortality characteristic of septic shock. inhibition of no synthase using c represents a novel and promising treatment for septic shock. cultures of e.coli ( , %) and candida( , %) were olso received from autopsy material of children;p.aeruginosa,unspored anaerobes,proteus sp.,s.aureus,b.pneumonia were found in the few cases. in adults the spectrum of bacterioflora was mo~ re limited speaking about the number of species and cultures. in generalized forms of bacterial pyo-septic pathology a wider specific spectrum of causative agents was revealed usua fly with associations. e.coli and k.pneumonia played the leading role in children as well as in adults. in general,k.pneumonia ( , %cultures) and common e.coli( , %)prevailed according to the date of microbiological investigations of authopsy material in pyo-septfc pathology in . objectives: .in spite of all clinical exertion sepsis is still the reason for high clinica! lethality. this study is characterizing the group of patients which survived a septi~ shock. methods: during a period of months all surgical patients on icu were registrated prospectively, more than parameters for each of them were documented'daily in a paradox file. results (see table ): of patients fulfilled the criterion of a septic shock (r. bone, ) , of them died at the lth day, while the surviving group of patients stayed almost days at icu. obiectives: to compare the effects of and % pentastarch solutions to a human albumin solution on oxygen delivery (do ) in septic patients. methods: this stud}, included septic patients with fever (t > ~ tachycardia flqr > /rain), tachypnea (rr > /min) or mechanical ventilation, leukocytosis (wbc> /mm ) or leukopcnla (wbc< ()/mm ) and a clinical source of infection, who required a fluid challenge. in each patient the pulmonary arterial occlusion pressure (paop) was < mmhg. patients were randomized to receive ml of % albunun (n:i ), hydroxyethyl starch (hes -mw /d.s. . ) % (n: ) or t % (n=i ); patients were also treated with adrenergic agents. results cardiac index (c ) increased significantly only in % lies (table) hemoglobin (hb) decreased significantly at min in the same group. there was not significant change in oxygen delivery ( do ). baseline ci alb . :: . (l'min/m ) hes % . = . hes % . polyneuropathy of the critically ill (pci ) is a well recognized complication, acquired in the course of severe illness. we undertook a prospective study, to estimate the severity, extension and time of onset of pci in a selected group of patient with established septic shock ( bone's criteria ). all patients received inotropic circulatory support and were mechanically ventilated. none received relaxants or aminoglycosides. pci was diagnose<by clinical investigation and by emg, and repeated weekly ( axonal degeneration ). after days ( day = onset of septic shock ) in % of the patients pci was demonstrable. after days % of the patients had pci. there was a strong correlation between pci and other mof and between pci and encephalopathy ( eeg ). we conclude that i. pci is a common complication after septic shock. . pci develops early in the course of illness ( % after days ). . pci can be considered as a part of mof, suggesting a common pathogenesis. objectives: we addressed two questions: ) can serial measurements of vapco and avph also reflect the onset of tissue hypoxia during endotoxemia? ) are whole body changes in vapco and avph associated with parallel changes in regional circulation? methods: in anesthetized, mechanically ventilated dogs exhaled gases were sampled for determination of oxygen consumption (vo ). a catheter was positioned into the pericardial cavity to induce cardiac tamponade. ultrasonic flow probes were placed around superior mesenteric, left renal and left femoral arteries, and the corresponding veins were cannulated. group l served as control (n= ), group (n-- ) received mg/kg of endotoxin. thirty min later, tamponade was induced to gradually reduce do . results: systemic do crit was higher ( . _+ . vs . + . ml/kg.min, p < . ) and critical oxygen extraction ratio (o ererit) was lower ( . +_ . vs . +_ . %, p< . ) in the endotoxie than in the control group. meslenteric and femoral do crit were higher in the endotoxic than in the control group ( . _+ . vs . _+ . mlll g tissue.min and . _+ . vs . _+ . ml/min, respectively, both p< . ). regional o ercrit were lower in we endotoxic than in the control group (mesenteric: . _+ . vs .i_+ . %, renal: . + . vs . -+ . % and femorah . -+ . vs . _+ . %, all p< . ). vapco and avph followed a similar pattern in both groups, increasing slightly before do crit was reached, but dramatically wh,en do fell below do crit. in the presence like in the absence of endotoxin, systemic and regional do crit calculated from vo , from vapco , or from avph were similar; do crit was also significantly higher in the mesenteric and the femoral beds of the endotoxic than the control dogs. systemic and regional do crit could be calculated from the blood lactate levels only in the control group. conclusions: during an acute reduction in blood flow i) vapco and avph can reflect hypoxic threshold in the presence like in the absence of endotoxemia. ) alterations in organ oxygen extraction capabilities induced by endotoxin can be reflected by regional changes in vapco and avph. objectives: enhanced nitric oxide (no) release has been incriminated in the vasodilation and myocardial depression characterizing septic shock, but the administration of no blockers has yielded equivocal results. the present study explored the systemic and regional effects of a no donor linsidomine (sin-i) during endotoxic shock. methods: anesthetized dogs received endotoxin ( mg/kg iv), followed min later by a saline infusion to keep cardiac filling pressures constant. oxygen uptake (vo ) was derived from the expired gas analysis. regional blood flow was measured by an ultrasonic flowmeter (transonic). results: the control endotoxie group (n= ) maintained low arterial pressure and systemic vascular resistance. the dogs receiving a continuous infusion of , , ~ug/kg.min of sin- starting min following initial fluid resuscitation (each dose for h), had a higher cardiac index ( . -+ . vs . _+ . l/min.kg, p< . ) and lower systemic and pulmonary vascular resistance than the control group ( -+ vs -+ and -+ vs -+ dyne.sec/cm , respectively, both p< . ). arterial pressure and pulmonary artery pressure were not influenced. sin- significantly increased the mesenteric blood flow from -+ to + % of baseline levels and renal blood flow from -+ to -+ % (both p< . ) but did not influence femoral blood flow. the relative blood flow was increased in the mesenteric bed (at all doses), and reduced in the femoral bed (at highest dose). systemic oxygen delivery, vo , oxygen extraction and blood lactate levels had similar course in the two groups. conclusions: in fluid-resuscitated endotoxic shock dogs, the no donor sin- increased cardiac index without deleterious effects on arterial pressure, and increased splanchnic blood fl w. objectives: sepsis syndrome is associated with the release of a variety of inflammatory mediators, such as interleukins, tumor necrosis factor and platelet-activating factor (paf). administration of paf, a naturally occuring phospolipid, to laboratory animals has been demonstrated to resullt in pathological changes that closely resemble those found in sepsis. in addition, paf antagonists of various origins have been demonstrated to attenuate cardiovascular collaps and to protect against lethality in endotoxemia. in the present study the efficacy and safety of the paf-antagonist tcv- g was investigated in sepsis syndrome patients. in addition, the putative inflammatory parameters tumor necrosis factor (tnf), interleukin il) - il- , and soluble (s) e-selectin were measured in both tcv- and placebo treated patients. metho.~a randomized, double-biind, multi-center study was undertaken to compare the safety and efficacy of intravenously administered tcv versus placebo (takeda chemical industries ltd., osaka, japan). tcv- was used as add-on to conventional therapy in the treatment of patients with severe sepsis and septic shock ( . mg/kg body weight intravenously over hrs, twice a day for one week). day mortality was registered, as well as vital signs, laboratory parameters, hemodynamic parameters, apache ii score and mof score. plasma samples for the determination of inflammatory parameters were collected twice a day for one week. the study was approved by each institutional review board and written informed consent was obtained for each patient. results: a total of patients were included in the study. one patient had to be excluded as a result of protocol violation of the remaining patients patients were randomised to receive tcv- and to be treated with placebo. there were no significant differences between the treatment group with respect to age. gender, and apache i~ score on admission. however, admission levels of il- and il- , considered to reflect severity of disease, tended to be higher in tcv treated patients: . . vs . ng/ml for il- (p. ) and . vs . ng/ml for il- (p. ) in tcv- and placebo treated patients, respectively. a remarkable difference in survival, however not significant, was observed at day i.e. the end of tcv treatment (mortality: out of in the tcv group and out of patients in the placebo group). the inc;dence ui ~-,~verse events, 'n both treatment groups were comparable. plasma il- and plasma il- levels tended to decrease more rapidly in tcv treated patients compared to controls (p=. and p=. , for il- and /l- respectively) conclusions: tcv- is safe as add-on treatment in sepsis syndrome patients. the data presented indicate that tcv- may enhance survival of sepsis syndrome which will be assessed in a phase ill study. objective: oxidation of lipids by free oxygen radicals playes an important role in sepsis. an important source of oxygen radicals is the respiratory burst of phagocytic leukocytes, especially~ polymorphonuclear leukocytes. clinically the hallmark of sepsis is the capillary leak, which is mediated in part by oxyradicals. the adult respiratory distress syndrom (ands} represents the most studied capillary leak phenomenon in sepsis. methods: in ii patients (df,~m, aged - y.) with verified septicemia had been included in this study. beginning from day oxidisable lipidautoantibodies (club), ~opterin and crp were determined in the patients serum and compared against the apache ii score during the days - . patients, f and m died @u= ring their stay at the icu, of them in less than h~urs: after admission. at the begin surviving patients showed olab values between - %, non-survivors between - %, com= pared to neopterin values of - m~ol/l in survivors and - ~mol/l in non-survivors. apache ii score didn t show any difference. during the following days olab sbowed an in= crease in survivors, while in non-survivors olab stayed equal or showed a decrease. no difference between both groups was found in neopterin, crp and apache ii score. as result we conclude, tbat lipidperoxidation playes an important role during septicemia. olab as a marker of this process are predictive for the outcome of patients. obiectives : to evaluate the influence of cardiopulmonary bypass ongastrointestinal transit. meth~xls; gastrocoecal transit time (gtt) in consecutive children on day after uncomplicated open heart surgery was compared to gtt in children after closed heart thoracic surgery and to healthy controls. gtt was measured by hydrogen breath testing, using lactulose (o, g/kg in a % aequous solution) as nonabsorbable marker. exspiratory hydrogen content was measured using a electrochemical system (stimotron). patients in both groups had continuous infusion of morphine and midazolam and were comparable in respect to catecholamine dose. children with a history of toddler's diarrhoea and children with elevated central venous pressure were excluded. results: gastrocoecal transit was delayed in all patients after cpb, no transit was achieved within minutes in / . after closed heart surgery gtt was delayed (+- ) min vs. (+- ) compared to controls, but transit was achieved in all aptients within rain. mean dopamine dose was , mcg/kgmin after cpb and , meg after closed heart surgery.. conclusions: after uncomplicated cpb gastmcoecal transit is extremely delayed. the degree of delay is not explained neither by the use of analgetic and sedative drugs neither by the use of catecholamines. factors directly related to cpb (e.g. low flow perfusion, hypothe,mia) may be responsible. these findings are of limited clinical relevance in ancomplicated cases. in critically ill patients delayed enteral feeding may contribute to infectious complications. studies of therapentic measures to accelerate gastrointestinal transit therefore are warranted. neonatal ecmo -czech experience. nekvasil r., penkova z, vasek l., plier p., bobula a novotna e., pavlicek v., hnilicka m. nicu and ecmo center brno and dept. pediatric surge ', children's hospital brno, czech republic. introduction. in spite of the fact that neonatal ecmo has been gradually considered a standard method of solving uncontrollable respiratol t failure in newborns in most countries of the western europe, the introduction of this sophisticated method in countries of the former communist block meets a lot of problem. material methods. in the course of two years, only newborns, b.w. - g, were reffered to neonatal ecmo because of uncontrollable respiratow failure. at the admission the average values of oxygenation index were + , and aado , -+ , kpa. results. newborns ( %) died shortly alter" admission or during transport without possibity to further is newborns ( , %) were treated using high-frequency ventilation (hfo or hfjv), one for conspicuous airway resistance paradoxically with low-frequency ippb. all ventilated newborns survived. ecmo was applied in newborns ( , %) and of them survived the mortality rate of group mentioned was %. conclusion. the fact that in the czech republic ( mil. population with natality rate of about . newborns/year) only small part of patients was reffered to neonatal ecmo has a number of causes. the most important are: unacquaintance of prognostic and indication criteria at forwarding workplaces and the aversion of neonatologist to all new.therapeutic interventions. last but not least, a negative role is also played by current change of health service systems when, under the influence of health-service insurance companies a newborn in serious condition given only a standard intensive care in primary and secondary nicu is literally "the golden calf", and therefore these workplaces either do not send him or send him too iate ~o an ecmo center. stndy aim: urfactant, which is being applied into the lungs for treatment of respiratory distress syndrome, can reach the circulatory blood system by absorption. it refinances local fimctiun of macrophages and lymphocytes and increases the accumulation of nentrophils into the hmgs this eunld influence the local acute phase reactiml in the hmgs after surfactant application. "ilia aim of the stud), was to investigate the influence of oatural and artificial surfactaat onto the acute phase reaction and the blood elottiog system. material :and methods: a) measurement of tnf-a, - , ltb and thromboxane b release in whole blood: each of . ml hepariaised blood of healthy adult volunteers was iucubated lbr hears with eillier ill ug/ml lps, mg/ml b&,ine smfactant (alveulhct/themae), rag/nil artificial surfactant (exosurf/wellcome), og lps + i mg/ml bovine surfactant or i ug lps + i mghal artificial sorfactant, tleparinised bhx-,d without additions was used for controls. ' e concentration of e)lokines and eicosanoids ".van measured semiquantilatively by eijsa or eia (dimmva/hamburg) after ceatrifagalion, b) measurement of platelet aggregation: each of . ml haparinised hltxxl of healthy adult vohmteers was inculmled lbr hours with eilher aghnl i,ps, i mg/ml bovine surfactant (alveafact/thomae), i mg/ml artificial snrlhctant (exosurf/wellcotne), it} ug lps + i mghnl bovine snrfactant or ug lps + i mg/ml artificial surfaetant, tlepminised blood withont additions was nsed for controls. platelet aggregation in whole blood was measured by impedance in the aggregometer (g~nstaedt). c) measurement of the dotting activity: citrated bleed of healthy adult w~lunlccrs was mixed with difl'ereul couccalrations of imvine or artificial snrfactant fad then prolhrombiu tiaras and imrlial thrmnlx~plaslia filues were ineasnrcd. for measurement of the prokoagulatioo acfivily the reagent in the qnick test was replaced by surfaclant. results: dependmlt on the dose both surfactants stimulated the release of - but not or tnf-a io wlmle blood. "illere was no suppression of the lps-indaeed eytokine-release. both surfaclants did not directly influence the extrinsic blood coagulation system, while both activated the inniasie activity dependant on the dose. platelat aggregatiun was slightly stimulated by bovine snrfactunt and strongly inhibited by by artificial surthctant. in comhinafion with . ~ both factors showed no difft.'renca to lps ahme.'fhe reltu.tse of eiconasnid~." was stimolated more by artificial surfactant then by bovine surfilctant with regard to the cyehmxygelmse (thromboxsne b ) and the lipooxygenase (lencotrieae b ) pathways. conclusion: we cooclude: ) i:lovine as well as artificial surfaelant stimulate the cellular inmnn]e response. ) bovine as well as artificial surfnctant activate the intrinsic ctmgnlation cascade. ) bovine surlilctant stimulates platelet aggregation, artificial sar-fijctant inhibits platelet aggregation.. introduction: although the use of modem noninvasive methods like measurement of tope , tcpco or san leeds to an carly infonnation about pathologic alterations of the gas exdmage of ventilated patients, these methods cannot replace invasive bloodgas analysis. a new noninvasive method to monitor the capillary gas exchange is the reflection spectrophotometry. to record reflection spectra with a ldgh sensitivity on the surface of tissue we eonstnmted a special mierolightgnide photospectronmter (empito). this spectrometer enables the investigation of local helerogenities of llbo and lb content, capillary flow rate and oxygen uptake rate. aim of the study: ) to compare the sousitivity and specifity ofthe empilo with other non-invasive method~ of monitoring the gas exchange in neonatal intensive care patients. ) to investigate the possibility of obtainiug infonnations about the local nptake and the venous as well as arterial satnration. patients and methods:we investigaled non-selected patients of oar pediatric intensive care unit. for coulinnons monitoring we conslracted a special sappert to fix file end nf the microlightguide+ during the investigation tope , tcpco and san were measured by standard methods. "fo investigate the local tisslm oxygen uptake we recorded spectres within minutes by moving the lightgnide over an described area of the skin of the patient. 'lhe reselting i(gx) l ibo vahms were sorted by a software package. on the assumption that the low values correspond to the tibo value of the veaons ends of the capillaries and the high values to those of the arterial ends we ) it is possible to gel infonnations about the local o -uptake and arterial saturation ia the peripheral tissue by photoslg'ctroscopy. ) in combination with noninvasive pl i-measorement it seems possible to reduce withdrawal of blood for invasivn bhmd gas analysis. objectives:improve ventilation,tissue oxygenation and acid base balance among extremly low birth weightand premature infants. methods: fourteen cases were s~udied,their gestational age ranged from( - )ws.continnous positive air way pressure was applied to six cases at peep level from ( - )cm h o through nasal pronge,(group i),the other cases were managed as routine,(group ii).blood gases, tcpo ,tcco ,resp.rate,depth and pattern were monitored for assessment of tissue oxygenation and ventilation, results: our rasults showed that early application of cpap improve ventilation among ( . %)of cases,while ( . %)of cases need imv.the cases of group ii need imv among ( %)of the studied cases during the second or the third day of life. conclusions:-this preliminary study showed the import-............ ance of early application of cpap before the onset of respiratory distress syndrom to improve spontaneous ve ventilation,tissue oxygenation and to gecrease the risk of intubation. comparative assessment of pediatric intensive care; a national multicenfre siudy. reinoud j.b.j. gemkc, gouke j. bonsel , the dutch picasso (pediatric intensive care assessment of outcome) study group. twilbelmina children's hospital and utrecht university, utrecht department of clinical epidemiology, academic medical center, : amsterdam, the netherlands the performance of all ( ) pediatric intensive care units (icus) in the netherlands ( tertairy and non-tertiary) was analyzed in an open prospective muiticenter study. effectiveness of care was defined as the ratio of observed to prism-score derived expected mortality aenee, standardized mortality rate). efficiency was determined by objective criteria (mortality risk > % or administration of at least icu-dependent therapy)'. consecutive admissions aged < years old were included. overall, observed and expected mortality were in good agreement (p > . ). between hospitals, crude mortality showed wide variations (mean . %, range - %). however, in each center, observed and expected mortality were similar (mean ratio . , range . - . ). in tertiary care centres, severity of illness corrected mortality in high-risk patients was less than in non-tertiary care centres; paradoxically, in low-risk patients the opposite was found. probably the large proportion of low-risk tertiary care patients suffering from severe, incurable chronic disease, explains the higher mortality in this group. this indicates that simultaneous assessment of circumstances of dying and of long term morbidity in similar future studies is imperative. the average proportion of efficient icu days was %, however large variations between units were found (range: - %). in conclusion differences in mortality rates among pediatric icus were explained by differences in severity of illness. high efficiency rates in combination with adequate effectiveness, found in several centres suggest that admission and discharge decisions might be improved by a better selection of high risk patients requiring icu-dependent therapies, especially in less efficient centres. objectives: previously published studies showed that serum lactate levels correlated with outcome of severe ill adult, 'we hypothesized that critically ill newborns are often incurred hypopeffusion manifested by elevated lactate levels. these initial blood lactate levels should be related to nicu outcome. design: prospective study with ethical comfnittee approval. setting: the -bed neonatal intensive care unit of a university hospital material and method: a total of consecutive outbem newborns admitted to nlod from , . to ., . were enrolled to the study. babies who died or were discharged from the unit within hours of treatment were excluded from the study, mean birth weight was g (+/- r), mean gestatational age was weeks (+/- . wks), mean age at the admission was h (+/- hi. multiple (~_ j organ system failure occurred jn . % of babies at the admission./~tertal lactates were measure/at the admission, among - hour and - hour of n[c'lj therapy. outcome was defined as a mortality and length of nicu stay. results" survival rate was . %, mean length of nicu stay for survivors was . days (+/- . day). we found high lactate levels at the admission in . % babies (~ . % with levels above . retool/i). the mean arterial lactate concentrations for nonsurvivors were signiftcahtly higher than for survivors durin~ consecutive da~ as follows: objectives: the purpose of our research was to analyze the frequency of bronchial asthma (b.a.) exacerbations in pregnant women and health status of infants. methods: the research was based on the epidemiological investigation and prolonged observation of pregnant women with b.a. during the gestation period. remission of b.a. before the pregnancy in excess of years was recorded in patients ( . %), patients ( . %) reported a - year remission and patients ( . %) had a remission lasting less than months before they became pregnant. results: seven patients ( . %) developed medium attacks in the second half of pregnancy, four patients ( . %) experienced light attacks of b.a. asthma attacks were most frequently caused by acute respiratory diseases and stress factors. in two cases with grave manifestation of b.a., the pregnancy ended in abortion within the first - weeks due to the frequent and heavy choking attacks. to fight b.a. attacks, five patients used adrenomimetics (salbutamol, becotid) in sprays, six women were administered theophyllinum and salbutamol in the form of tablets during - weeks. a significant portion of pregnant women with b.a. ( %) exhibited frequent complications during pregnancy (toxemia, late gestosis, threat of miscarriage). our findings prove that babies born from women with b.a. of domestic and pollen origin had a low body weight ( - gr), functional immaturity and chronic antenatal and intranatal hypoxia twice as often as the infants born from healthy women without allergic background. conclusions: preventive treatment of women with b.a. prior to pregnancy is required to maintain a stable remission of the disease, which is a key to having healthy children delivered by mothers suffering from b.a. introduction. intracerebral hemorrhage (ich) is a common event in human prematudty, affecting about % of newborns weighing below g who are born before weeks of gestation, however, little is known about the pathogenesis of ich with exception of the prematurity of the brain itself, (birth) trauma, and asphyxia. the postischemic production of oxygen free radicals (ofr) dudng reoxygenation as a cause of brain damage has been demonstrated in animal research. since almost all preventive antioxidant activity of plasma is associated with ceruloplasmin and transferdn we investigated the association of such iron-oxidizing resp. iron-binding proteins and ich. we could demonstrate significantly reduced levels of both, iron-oxidizing and iron-binding proteins, in premature asphyxiated newboms pdor to development of ich. an increase of suparoxide after hypoxia in the presence of iron ions facilitates the formation ofthe highly reactive hydroxyl radicals. our data support the theory that ich may be caused by ofr, which can damage any sensitive tissue including growing endothelial cells. the estimation of transferrin-saturation and measurement of ceruleplesmin levels might help to identify an infant at dsk before the onset of ich. with the new medos | hia-vad | cardiac assist system the missing tool in the armamentarium of cardiac surgeons is available in two pediatric sizes: i -ml and -ml pump volume. the right sided pumps are % smaller for biventricular use. between february and may we implanted this assist system in children. the indications and demographics are indicated in the following table (left ventricular assist device-lvad, right vad-rvad univentricular vad-uvad, post cardiotomy cardiac failure-pcf, dilated cardiomyopathy-cmr bland white garland syndrome-bwg, tetralogy of fallot-tof, hypoplastic left heart syndrome-hlhs). objectives: evaluate tile effeci'of inhaled nitric oxide (no) as puhnona] t vasodilating agent ill tile posloperalivc period after correclion of congenital heart defects in infant. patient n.l: kg, lnonlhs, down syndrome undenvcnl rep~fir of atrioventricular septal defect (avsd). after surgery the puhnonary arlcry pressure (pap) slowly rose to tile syslemic dcspilc tnaximal eonvcnlional fllerapy (fentanyl mcg/kg/h, hypocapnia of mmhg and metabolic alcalinization). no was delivered into tile inspiratory branch of!be breathing circuit at ppm, and the gas aoalyser for no and no (polylron dmger) were situated at the espiratory branch, a rapid dccrcasc of pap io i/ of systemic was obtained with a dramalic improvement. no was continued at ppm for six days and the baby was exlnbated if! days after surgery and discharged from the icu days after. patient n. : . kg, monlhs, onderwen! repair of avsd. the day after surgery the systemic oxygen salnralion was % wilh a pap at % of systemic. two hours of c wenlional therapy failed o improve ihc patient and no administration was slarled at ppm. so dramatically incrcased to %, but the pap dropped only to % of syslemic. nevertheless ihe clinical conditions improved and the no administration could be reduced at ppm in the following days. she was extubaled days after surgery and discharged from the icu days after. patient n. : kg, 'ears. underwen| hearl tral~splantalion for congenital heart disease with moderate hypoplasia of pulmonary arlcrics. at the end of cardiopulmonary bypass the transpnlnlonary al~erio-venoas gradient yeas higher than mnfflg and we speculaled !hat w'ls due to a degree of puhnonary vasocostrictiont. the nsnal dose of no was otilised, however no significant modilicalion of pulmonary pressure or systemic oxygen saluralion was noled, and after h no was discontinned. tile palienl was carried io the icu with maximal inotropic support, extubated after d;b's and disclmrged from the icu after days. in all patient no major adverse effect relaled to no admilfistration ",','as holed. conclusion: in our experience no ms a pulmonary vasodilaling agent is effective and easily adjustable to tile palienls requiemenls, however its use remains limited ill those palienl ill whoin tile alnonll! of fixed inlllllojliify vascular resistance is predominanl. we report the use of ecmo support in two unusual cases of severe tracheal disruption in which it had become impossible to achieve adequate ventilation. case : severe tracheal laceration due to aspiration of a share forelan bodv: a previously healthy month old toddler was referred for ecmo following aspiration of a porcelain foreign body (with razor sharp edges) which had become embedded in the right mainstem bronchus with massive extrusion of air. this was removed on veno-arteda[ ecmo support, as the patient was unventilatable prior to bronchoscopy due to ongoing airieak. ecmg was continued after bronchoscopy to permit airway healing without the presence of an endotracheal tube. unfortunately, an extensive pulmonary haemorrhage on day of ecmo necessited re-exploration of the airway. this revealed a posterior tracheal tear from the cricoid to the middle of the right lower lobe. following repair the patient was left on ecmo support together with high frequency oscillation ventilation (hfov), the latter being used to minimise potential aideak and maximise alveoli recruitment. ecmo was weaned after days ( hours) -the patient was extubated weeks later. case : tracheal wound dehiscence due to seosls -tracheal transelant on ecmo: a month old infant with a c[inically significant congenital long segment tracheal stenosis and left pulmonary artery sling underwent resection of the stenosis, followed by primary reanastomosis. this was complicated, days later, by severe mediastinitis and complete dehiscence of the anastomosis. an autologous pericardial patch was used to repair this, however, the tracheal wound again dehisced days later making mechanical ventilation impossible. in view of ongoing sepsis and a severely disrupted trachea ecmo was the only possible form of support. following resolution of the local sepsis ( days) a definitive procedure in the form of a tracheal homograft (transplant) was undertaken on ecmo. the patient was managed on ecmo and hfov for a further days, the hfov being used to optimize rapid lung inflation. unfortunately this patient died months after weaning from ecmo due to complete disintegration of the homograft, which was not deemed reparable. conclusions: ) ecmo can be used in the acute management of oxygenation when there is major airway disruption making mechanical ventilation impossible. ) hfov was a useful adjunct in aiding recruitment of lung volume on ecmo in these two patients. backoreund: persistent pulmonary hypertension of the newborn (pphn) consists of a heterogenous group of diseases ranging from transient reversibte pulmonary hypertension to fixed primary malformations of the lung (primary pulmonary dyspfasia-ppd). inhaled nitric oxide (ino), a selective pulmonary vasodilator, has been proposed as a treatment for severe pphn. obiective and methods: ino was administered to near term neonates with severe persistent pphn, oxygenation index > and echocardiogrephic evidence of pulmonary hypertension, in order to further determine the clinical role of ino in the treatment of pphn. the response to ino was also analysed retrospectively to examine whether this could be of diagnostic value in differentiating at an early stage patients with reversible from fixed causes of pphn results: twenty one of the patients studied responded to the initial trial of no ( ppm x minutes), as defined by a greater than percent improvement in pad as well as a fall in the el to < . these patients were continued on ino therapy, with patterns of response emerging: pattern babies (n= ) continued to show a sustained response to ino and were successfully weaned from it within days -all survived. pattern babies (n= ) failed to sustain their response to ino over hours, as definded by a rise in the el > . six survived, five with ecmo. pattern babies (n= ) had a sustained dependence on ino for - weeks. all three died and lung histology revealed severe primary pulmonary dysplasia (ppd). patients with ppd (pattern ) not only required ino for longer periods of time than did the sustained responders (pattern ), but also required significantly higher doses of ino we report on the air transport of paediatric intensive care patients. these transports fall into three categories: ) retrieval of critically ill neonates and paediatdc patients referred for either ecmo or inhaled nitric oxide (ino) (n = ). one patient was transferred on ind. mean transfer time . hours (se + . hrs). ) long distance international transport using chartered aircraft (n = ). the indications for these transfers included both urgent retrievals for cardiac surgery and semi-elective transfer of stable patients back to their referring unit following treatment in tertiary centres. mean transfer time . hours (se + . hrs) ) long distance international transport using commercial aircraft (n = ). indications for transfer were either semi-elective retrieval for tertiary treatment or the return of stable chronically ventilated patients to their referring hospitals. mean transfer time hours (se _+ .fhrs, longest hrs). the transport team consisted of a paediatric intensive care doctor of at least registrar grade and a registered sick chidrens nurse with intensive care experience. the administrative components of the transfer (ambulances, airlines, customs) were managed in collaboration with companies specializing in air ambulance transfers. outcome: all the patients were safely transported to their destination without mortality or morbidity. complications durino transfer ir~lv~; ) patient complications -semielective endotracheal tube change and central access needed in the only patient brought to the commercial aircraft by the referring hospital (all others retrieved directly from referral hospital), seizure in patient with known encephalopathy, severe cyanotic spells in patient with fallots tetralogy who was retrieved for urgent surgery for this indication ) mechanical compfications -ventilator failure, incubator battery failure, oxygen regulator failure -all occurred with equipment sent from referral hospital, this was unfamiliar and unchecked by our transport team -it was not the decision of the transfer team to use this equipment on this single occassion. ) administrative complications -confiscation of incubator battery by airport security police, excessive delay by custom officials ( hours) in the airport. the incidence of such problems were felt to be low and unpredictable. in conclusion: mechanically ventilated paediatric patients can be safely transported on both chartered and commercial airlines. these transports are best accomplished by trained intensive care medical and nursing staff with the backing of an air ambulance organization competent in arranging the necessary administrative details. it is essential to use your own equipment and to retrieve the patient _directly from the referrin(] hospital to minimise ootential complications. our experience with anaesthesia for paediatric electromyography _w_._pla_ti_k_a_n_o_v, r.eousseff, k.pavlova, d.marinova dpts. of anaesthesiology and int. care and clinika] neurophysiology, med. university, pleven, bulgaria ~)_b_j#~ti_v~. to t~st a " heavv sedation " regimen of anaest-es~a for the purpose of paediatric electromyography d#s~gil~ non-randomized,non-blinded human trial in the seting of an uriiversity hospetal. _m_a_t_eri_a_is_a_nd_ m_e_th_od_s_. children,asa i-if,median age years,range - who undervent eleetrcmyography required anaesthesia. they recieved low-dose ketamine + i~iazepam or midazolam via musculary route( children,age - yrs,ketamine , mg/kg, diazepam - mg total dose ) or per os ( children,ketamine - mg/kg,diazepam , mg/kg or midazclam , - , mg/kg ) _resu_l_t_s. - minutes after medication a state of heavy sedation with weak spontaneos and stimuli-provoked movements was achieved in all children, that lasted - minutes and allowed adequate needle emg and nerve conduction investigation. children recieved additional , - , vol.% halothane during the placement of the needle. non -invasive blood pressure , breath and heart sounds and hb sad by pulse oxymetry were monitored.none of the older children disclosed memories of pain when asked after they regained adequate verbal contact.no complicationes were observed. antenatal maternal steroids reduce the risk of periventricular-intraventricular hemorrhage in very premature neonates treated with natural surfactants. i.apostolidou, c.papagaroufalis, g.touloumi, m.xanthou, n.kalpoyannis a' and b" neonatal icu "ag. sophia" children" s hosp. athens, greece. dept of hygiene and epidemiology, athens university, greece. obiectives: the aim of the study was to evaluate the association of periventricular-intraventricular hemorrhage (p-ivh) in surfactanl treated premature neonates with pre-and postnatal variables. methods: the population of the study was neonates admitted during the years to , with gestational age _< weeks and severe respiratory distress syndrome (rds) (mechanical ventilation and arterialalveolar oxygen tension ratio (ajapo ) < . ), who received rescue therapy of at least two doses of natural surfactants (alveofact or curosurf) and examined with ultrasound and/or autopsy for the presence of p-ivh (papile's classification). the examined factors in each neonate were the following: gestational age, birth weight, sex, multiple pregnancy, antenatal maternal steroids (complete and incomplete course of betamethasone), a/apo before the administration of the st dose of surfeclant, delivery, apgar score at min, type of surfactant, pneumothorax and patent ductus arteriosus. the statistical methods used were x and one-way analyses of variance followed by logistic regression medels, results: the incidence ot p-ivh was . %. three factors were found to have an independent relation to p-ivh (final logistic regression model): gestalional age, a/apo before surfactant administration, and antenatal administration of maternal steroids (complete and incomplete courses). for every weeks of lower gestational age the neonates had an almost doubled associated risk of p-ivh (or: . , % c : . , . ). for every . on average decrease of a/apo before surfactant administration the risk of p-ivh in the neonates was . times higher ( % ci: . , . ). the neonates whose mothers received antenatally steroids had only one tenth of the risk of p-ivh of the neonates whose mothers had not (or: . , % ci: . , . ). conclusions: our results suggest that the antenatal administration of maternal steroids, even less than hours before delivery, reduce the risk of pqvh in very premature neonates treated with natural surfactants, whereas the small gestational age and the lung immaturity still remain the main risk factors tor the development of p-ivh. we analysed retrospectively the management of ( boys, girls) accidental ingestions of foreign bodies in children (mean age : . years, range : months- years). no child had ingested more than foreign object. the majority of the ingested foreign bodies were : coins (n : ), toy parts (n : ), jewellery (n : ), batteries (n : ), "sharp" materials such as needles and pins (n : ), "large" amounts of food (n : ). impaction of food occurs more frequently in children after oesophageal reconstruction in cases of oesophageal atresia. although according to literature "coca-cola" is reported to be effective, this was not seen in our experience. / patients had minor transient symptoms at the moment of ingestion, such as retrosternal pain. only children experienced severe manifestations (cyanosis, dysphagia). in these children, endoscopy revealed oesophageal and gastric erosions. children were seen at the emergency ward within a few hours after the accident ( mean : hours, range min. - hours). chest and/or abdominal x-ray was performed as first-line investigation ( / objects were radio-opaque), and revealed an (unexpected) oeeophageal impaction in children. in / the foreign body was in the stomach. batteries, sharp objects and objects trapped in the oesophagus were removed, either by endoscopy or by magnet-extraction whenever possible. the outcome of the patients was excellent. no complications were observed. extraction is recommended in symptomatic patients, and whenever the foreign body is trapped in the oesophagus, or if the foreign object is "sharp" or a battery. objectives: two strategies were used for management of malignant diphtheria in children aged from . to years. methods: protocol n consisted of intravenous administration of diphtheria antitoxic serum, prednisolone ( mg/kg bw/day), plasmapheresis and supportive care. protocol n included the use of antitoxic serum against the background of high-dose dexasone ( - mg/kg bw/day), hemocarioperfusion and a preventive use (before the clinical manifestation of myocardial damage) of inotropic medications, inhibitors of angiotensin-converting enzyme and pentoxyphylline. each of protocols included the monitoring of serum toxin (diphtherin) levels. results: the group of patients treated according to the protocol n consisted of children with malignant diphtheria, of them with severe malignant diphtheria (grade and ). all patients exhibited the circulation of toxin during at least three days after the start of treatment. all patients with severe grade of disease demonstrated heavy cardiovascular disturbances associated with malignant diphtheria. of the children in the group died seven. the children of the second group were treated according to the protocol n . out of total of patients of this group. patients had severe malignant diphtheria. in all children a significant reduction in serum toxin level was revealed after hemocarboperfusion. in all but one case the satisfactory control of cardiovascular function on was achieved. of children admitted to the trial survived, one child with malignant diphtheria of grade and congenital filbroelastosys of the left ventriculum died. the severity of neurological complications was similar in each of groups. conclusions: the use of hemocarboperfusion, high-dose dexasone and early prevention of heart failure as a adjunct to the standart treatment has been shown to be of benefit in the management of malignant diphtheria. t. schaible, i. reiss, j. m er, l. gortner med. university of lqbeck, children's hospital, kahlhorststr. - , l~beck, germany surfactant therapy seems a promising approach for the treatment of the biochemical and biophysical abnormalities of the pulmonary surfactant system in severe ards. patients and methods: over a months period non-neonatal pediatric ards patients (age - months) in a "pre-ecmo"-situation (oi over h) were treated with bovine surfactant (alveofact| the underlying conditions-of ards were pneumonia ( ), sepsis ( ), immunosuppression ( ), near drowning ( ), neurogenous ards ( ). a total of - mg/kg b.w. was applied in several fractions. before surfactant therapy, we first tried different ventilation (best peep-finding, inversed i/e-ratio, hfo-ventilation) while monitoring the pulmonary mechanics. for hemodynamic stabilisation both norepinephrine and epoprostenol were used to optimize pulmonary perfusion for max. hrs. if there was no improvement of the oi by at least , further treatment with surfactant was initiated. in addition to surfactant all patients received a treatment with dexamethasone of mg/kg in doses. patients with no benefit (oi remained unchanged or increased within the max. - hrs) were taken on ecmo. results: nine patients improved within hours after surfactant therapy: the oi decreased from a level of (mean, range - ) before our treatment to a level of (mean, range - ) thereafter. in patients we were able to continue the positive effects of our treatment and they could be weaned of the respirator within - days. the other patients got worse despite respiratory improvement, they suffered of multiorgan failure of more than organ systems. the last patient did not benefit from surfactant, he had to be put on ecmo, but died because of a complication (hemopericard)after days. the autopsy of the ecmo-patient showed a pulmonary fibrosis, but the other death were not due to pulmonary failure. conclusion: a different sequential ards treatment integrating surfactant therapy can reduce the number of patients requiring ecmo. but ecmo as a therapeutic tool should be available in centers involved in ards treatment. l.blindl, t.p.le, h.weinzheimer, centre for paediatrics, university of bonn, germany selective reduction of elevated pulmonary vascular resistance by inhaled prostacycliu (pgi) has been reported in adults with acute lung injury, neonates with persistent pulmonary hypertension and in one infant with idiopathic pulmonary hypertension. we report on the effect of aerosolized prostacyclin in two children with secondary pulmonary hypertension. patient : in a boy with down's syndrome an avsd had been surgically corrected at month of age. at , yr of age a catheter examination revealed a pulmonary vascular resistance of % of systemic vascular resistance in room air and at an fin of . . prostacyclin ( . mcg/ml) was administered with a jet nebulizer at an fin of . . pvr declined to . systemic vascular resistance and returned to baseline after stopping pgi-inhalation. subsequent intravenous infusion ( ng/kg rain) had to be stopped after minutes because of systemic arterial hypotension. patient : a month old male infant with bronchopulmonary dysplasia developed suprasystemic right ventricular pressure inspire of therapy with oxygen and nifedipin. while he was spontaneously breathing % oxygen via face mask pao was mmhg, arterial ph was . . systolic arterial pressure was mmhg, a rv-ra gradient of mmhg was measured by cw-doppler. while fio was maintained aerosolized prostacyclin was administered over minutes. rv-ra gradient was mmhg, systemic blood pressure mmhg, pao mmhg. two hours later nitric oxide ( ppm) was inhaled at an fio of ( , . rv-ra gradient declined from to mmhg, systemic systolic blood pressure remained stable at mlnhg. discussion: sporadic experience shows that aerosolized prostacyclin selectively reduces elevated pulmonary vascular resistance in some patients. in patient the poor response to inhaled pgi compared to inhaled nitric oxide may be explained by the fact that the action of pgi is not independent from endothelial function, limiting it's effect in severe vascular disease. during the last two years ( - ), infants weighing less than gr. admitted to our referral unit. thirty four of them ( %) survived, ( % of infants weighing - g and % of infants weighing - gr survived) for the years - - the survival of these infants was % and for the years - - , % (p< . ). we analyzed the perinatal and neonatal factors influencing the outcome of these infants. the comparison among neonatal survivors ( ) to neonatal deaths ( ) shows: gestational age: . w ( ) to . w ( ) (s). birth weight: . g ( ) to . ( ) (s). apgar score: , ( ) to . ( ) (ns). presentation and mode of delivery: breech presentation is associated with higher incidence of neonatal deaths. i.v.h. (at the age of weeks): no one of the survival infants had evidence of i.v.h. respiratory problems: intubation, at the admittance of the infants . ",,( ) to % ( ) (s) use of surfactant: % ( ) to % ( ). bpd observed in % of the babies and only one was dependent on oxygen at home. antenatal betamethasone was given in % of the mothers. in conclusion: ) a great improvement in the survival rate observed in these infants the last years in our unit. ) factors with positive effect are increasing gestational age and birth weight, the absence of i.v.h. and the use of surfactant. the breech presentation and the severe respiratory problems increase the incidence of death. animal experiments demonstrated, that brain temperature determines the amount of neuronal damage caused by hypoxia and that mild hypothermia may have a protective effect. until now there is no method described and evaluated to measure brain temperature in neonatal intensive care units. we non-invasively measured brain temperature analogues, nasopharyngeal (tnasoph) and zero-heat-flux temperature (zht) at the temple whereby under zero heat flux surface temperature represents deep head and thus brain temperature. the aim of our study was to investigate the practicability of the method, the relationship of the two brain temperature analogues to rectal temperature (trect) and their dependence on insulation, thermal environment, body activity and time course. we investigated healthy preterms less then weeks postnatal age (gestational age +_ . wks; x + sd, weight +_ g) in an incubator. tnasoph was measured by a thermistor within a feeding tube, advanced to the nasopharynx, zht temple by a thermistor and a heat flux transducers both covered by an insulating pad, and trect thermal environment was characterised by operant temperature (tair . . + twall . ). body activity was video taped. measurements were performed during the following interventions: i/ insulation increased by turning the temple with sensors onto the mattress ( rain). ii) insulation increased by a cap ( min), iii) min after its removal, iiii) increased operant temperature by . + . ~ ( min). results: seven children with ea had a gasless abdomen, the endoscopic procedure excluded ( ) or diagnosticated an upper pouch fistula ( ). in patients who suspected "h" fistula ( ) broncoscopy has strong advocated method to make diagnosis and established cervical approach. from july newborns with ea and lower pouch tef received a selective transtracheal incannulation. we were not able to proceed just in case with congenital subglottie stenosis. in these patients we provided gastric drainage by radiopaque and flexible - french catheter. the knowledge of the precise anatomic position of tef consent to adjust the tip of the endotracheal tube in order to achieve best ventilation. the presence of the catheter through the fistula helps the surgeon to identify, it quickly. no complications were correlated to the procedure and no babies had early pneumonia. alimentary continuity was achieved in all patients ( primary anastomosis, resections of tef, oesophagocoloplasty and died with gastrooesofagostomy). the late mortality . % ( ) was only directly related to the severity of associated malformations. conclusion: the advantages of this technical approach are unquestionable for the anaesthesiologist and the surgeon. in our experienc e the procedure improves perioperative management of babies and appears to be safe. relation between cytokines, prethrombotic markers and endotelial injury markers in children with septic shock objectives: to establish the relationship between cytokines (tnf, il- , il- ) prethrombotic markers (d.d., pcam) and endothelial injury markers (tm, uwf) in pediatric patients with sepsis and bacteriemia without shock, and patients with septic shock. design and methods: prospective study, children ( months- years) were admitted in our picu in with the following diagnosis: bacteriemia ( ) sepsis ( ) and septic shock ( ) according to jacob's r f criteria. measurements: il- , il- , tnf, tm, vnf, d.d. pcam and routine laboratory data on admision, , , hours and on discharge. the prism (pediatric risk of mortality score) was also recorded. results and conclusions: two patients in the septic shock group died. significant differences were found between non-shock and septic shock patients in relation to tm, dd, pcam, il- , il- and tne high levels of tnf and il- are closely associated with the severity of septic shock with purpura in children. low levels of pcam on admission were associated with severe shock. who underwent open hea~nt surgery, hypervotaemia with or without oliguria was the most frequent reason to start pd ( %). in patients pd lasted less then one week and there were no complications; in patients it lasted - days (one child had a peritonitis). instillation of dialysis fluid into the peritoneal cavity was associated with a significant increase in central venous pressure. there were no significant changes in cardiac output or arterial oxygeu saturation. in all patients pd dhnjnished fluid overload or improved the metabolic status. patients ( %) survived the postoperative course and all had complete reintegration of renal function. conclusion: pd is a useful method to treat the fluid overload and acute renal failure in paediatric patients following open heart surgery with file effects of little importance on the cardiovascular fimction. obieetives: with the marketing of computerised systems for lung function testing in newborns, there has been an increasing interest in clinical approaches. percentile curves of pulmonary parameters permit an appropriate and clinically useful interpretation. however, the manual evaluation of the results using different curves is an impractical technique. therefoi'e a computer programme was developed. methods: the percentiles ( %, %, ~ %, %) of the most important pulmonary parameters were determined non-parametrically in weight-classes. for the calculation we have taken results of our own as well as other laboratories using a meta-analysis of reference studies. in all, individual data of - healthy newborns ageing between - days were collated. using these percentiles, for every parameter in relation to the body-weight the cumulative distribution was calculated approximately using piecewise linear and exponential functions. as shown in the figure the results of computing are represented numerically as well as graphically and can be included in the patient report. conelusions: clinic~d experiences with the programme have shown that representation of all measured parameters on standardised % scales allows an easy interpretation at first sight and improves the detection of pathologic patterns in the parameters. ")supported by bmft, fp "risikoneugeborene" prism (pediatric risk of mortality) score is a well known, already validated scoring system that quantifies severity of illness based on routinely clinical and laboratory variables measuring physiological instability. once computed the score by summing up the weights corresponding to the most abnormal value recorded during the first hours, the overall risk of mortality can be predicted by using the coefficients estimated by a logistic regression where prism score is the main independent variable. (pollack mm et al, -pediatric risk of mortality (prism) score. crit. care med. ; : - . to assess the applicability and validity of prism in the italian setting we launched out a prospective data collection in a sample of pediatric icus. measures of calibration (goodness of fit statistics) and discrimination (receiver operating characteristics and area under the roc curve) are planned to be adopted in the cohort of patients recruited during year period. as the validation study started on july , data collection is still on going and validation analyses will be carried out on july . up to now centers recruited cases. at present, characteristics of the sample recruited are the following: most of the patients were male ( %); the mean age is years with % of patiens having less than days; more than half were medical cases ( %) admitted from emergency room or from hospital floor ( %); % cases were admitted with an organ failure while % to be intensively monitored. icu-mortality was l %. the paper will present final results of calibration and discrimination analyses that will be carried out in the whole sample and across subgroups known to differ in terms of clinical relevance and prognosis. if calibration and discrimination assessment will produce not satisfactoty findings, a customization of the current coefficients will be made allowing a formal comparision of previous and new parameters. jf riera-faneao, m wells, j lipman. baragwanath intensive care unit, university of the witwatarsrand, south africa. [background the prism score is designed to assess the likelihood of death in ipaediatdc icu patients, using only acute physiological disturbances, age and [operative status to predict mortality. there is no evaluation of chronic health status, [including malnutrition. this may significantly affect its ability to accurately predict outcome in a population where malnutdtion is common. aim to determine the influence of nutritional insufficiency, as indicated by a low weight-for-age on outcome prediction by prism. patients & methods we analysed prism, weight and demographic data co ected prospectively from consecutive paediatdc icu admissions over a year pedod. a proportional weight (pwt) was calculated as a percentage from the th centile of the who weight-for-age growth charts. the pwt was compared for survivors and nonsurvivors, and mortality compared for pwt categodes nho wellcome classification). multivariate statistical techniques were used to identity associations with non-survival and to develop a modified logistic regression equation including a measure of i nutdtional status. receiver operating characteristic (roc) analysis was performed including and excluding patients with low pwt for the odginal and modified equations. results non-survivors had a lower weight than survivors ( . kg and . kg medians p = ) a lower pwt ( % and % medians p = . " . the incidence of malnutdtion , in our icu population was %. the mortality of manoudshed patients was' significantly increased (p = . ), with a good correlation with the degree of malnutrition. the accuracy of prism was significantly improved when malnourished patients were excluded from the analysis (roc value increased from . to . ). ! logistic regression and discriminant analysis identified a significant association between prism, pwt and outcome; age and operative status were not significantly related to mortality. the use of a modified equation including the raw prism score, pwt category and age can significantly improve the discriminatory power (az dm/elopmental sample . , az validation sample . ). the modified formula is: legit = - . + . *prism score - . *age + . *weight category, where the probability of mortality is exp(iog/t)/ + exp(iogio. discussion although we can improve the prediction of mortality by a modified or recelibrated formula, this still does not compare with the reference prism population. the need for validation of the score itself, in the association with outcome of the acute physiological variables themselves, is thus apparent. we conclude that while the odginal prism formula can be improved significantly, a modification of the basic variables in this and other third wodd populations may be essential. a high incidence of malnutrition is an independent risk factor of mortality, and an important cause of the poor discriminatory performance of prism. in order to improve the accuracy of prism, nutritional status should be taken into account. objectives: to assess the value of inhaled no to differentiate between pulmonary vascular constriction or fixed anatomical obstruction. methods: we assessed the response to ppm inhaled no in patients( m, f, median age . months, range day to years) with signs of increased pulmonary vascular resistance, there were pre and postoperative patients. patients were divided into responders(+) or non-responders(-). a positive response was defined as a % reduction in pulmonary arterial pressure and pulmonary vascular resistance(pvr) or in the presence of a left to right shunt, a fall in pvr accompanied by increasing pulmonary blood flow. left atrioventricular valve atresia + mustard pat: pulmonary atresia vsd: ventricular septal defect asd: atrial septal defect pda: patent ductus arteriosus tapvc: total anomalous pulmonary venous connection the responders( / ) were characterised by left to right shunts or pulmonary venous hypertension( / ). patient# was weaned from ecmo with inhaled no. patient# , without congenital heart disease, underwent a lung biopsy which confirmed reversible pulmonary vascular changes. patient# had a pulmonary hypertensive crisis which responded to no. all non-responders( / ) had evidence of anatomic obstruction to pulmonary blood flow (# , , )or a low pvr(# ) on subsequent cardiac catheterisation. in patient # , lung biopsy confirmed severe obliterative vascular disease. conclusions: inhaled no appears to be an effective pulmonary vasodilator. a failed response may be evidence of either irreversible pulmonary vascular disease or a residual anatomical obstruction which may be surgically remediable in the postoperative cardiac patient. therefore, inhalation of no may be a useful diagnostic test to differentiate between fixed anatomical obstruction and reversible vasoconstriction. results: during these years, the incidence of sdra was . % of the total of admissions. the most common etiology was meningococcic septic shock. since , there is a decrease of its incidence. (from % to %) and an increase of pneumonia and immtmodeficiencies. mean age of our patients was , years ( % males, % females), total mortality by sdra was % and there is an increase up to % since mean time of stay of the dead was , days and , days those who survived. although during the late years we offer in the picu a better attendance quality to the patients with sdra and the mean stay is longer, both for those who die and for those who survive, mortality of patients with sdra have increased. the incidence of sdra secondary to the septic shock of a meningococcic etiology have decreased. on the contrary, the sdra secondary to infections by opportunistic germs in patients with congenital inmmunodeficiencies or acquired immuodeficiencies have a tendency to increase. in our series, this change of aetiology is the responsible for the increase in mortality. hospital infantil unlversitario "virgen de roclo". sevilla. espalqa aims:to assess the incidence, etiology, clinical course, sequelae and mortality of the patients admitted to a paedfiatic intensive care unit with the diagnosis of severe traumatism. material and method: cases of severe traumatism in children admitted to our icu in the period from january to june were reviewed. age of patient ranged from months to years, % were males. in our series, % of cases suffered traumatism due to a traffic collision and % had a fall from a considerable height. only in one case was traumatism due to violence to the child. we assessed the first assistance received in % of cases: where was it performed, interval of time since the accident, and steps taken. these data were also studied in relation to the latter evolution. results: % of our patients suffered cranioencephalic traumadsm (ct); in % it was an isolated picture and in % of cases was associated to other lesions. there was participation of thoracic and/or abdominal organs in % of cases. % of cases presented important maxillofacial involvement. only one case presented serious cervical medullar lesion. mortality in our series was . %. in . % important sequelae remained. all of these patients presented tepas on admission equal or lower than . % of those with traumatises had slight sequelae. . % of the total evolve towards healing. a polytraumatized child is a patient that benefits considerably of it admission in a paedriatic !cu. the rapidity in receiving first aid and its quality are essential to avoid sequelae and to make mortality decrease. after unilateral lungtransplantation % of the patients develop a lung failure with decrease of perfusion and increase of pulmonary blood pressure in the transplantated lung. the improvement of perfusion is an importent task in the postoperative period. case report: a year old girl with idiopathic pulmonary fibrosis received a left sided single lung transplantation. during the early postoperative period occured a higtter demand of oxygen and an increasment of the pulmonary vascular resistence in the left lung. the pulmonary ventilation and perfusion scintigraphy indicated in comparison with the right lung a reduced perfusion of only % in spite of a ventilation of % of the transplanted lung. to improve the perfusion of the transplant we administrated per inhalation prostacyclin in a maximal dose of ng/kg/min. the arterial blood pressure decreased but the perfusion continued nearly at the same level. during the following administration of ppm no in the respiratory air we achieved a significant reduction of the respiration pressure f~m to nun h and of the pulmonary arterial pressure. the perfusion in the transplanted lung increased to ca/of the total pulmonary perfusion. after days of administration with no we were able to withdraw the axtifical respiration without any following complications. conclusions: the perfusion of transplanted lungs is a major proble_r~ in the postoperative period. this case demonstrated the advantage of no towards the inhalativ application of prostacyclin. no showed a significant improvement of perfusion in the transplanted lung of a year old girl. results: a total of children with ards were treated with bovine surfactant (alveofact| cases were evalable. the median age was . years (range weeks to , years). in six cases ards was associated with pneumonia, in two cases with lung hemorrhage; in one case isolated ards followed hemihepatectomy. the first surfactant application was performed with a median latency of clays (range - days) after first symptoms of ards witha median doseof mg/ kg (range - mg/kg). in patients doses of surfactant were applied. during the hour before therapy, the median pao / fio -ratio was - . within min. after application of exogenous surfactant the pao / fio -ratio increased to with successive decrease over a period of hours to . accordingly, an increase in pao and oxygen saturation and (less significant) a decrease in ventilation parameters could be observed. analysis of broncho-alveolar lavage before surfactant application in children receiving repeated doses revealed in most examined cases either clear surfactant deficiency or pathological function. of treated patients survived ( of the , respectively). of the surfactant doses were applied in the surviving patients.conclusions: the application of exogenous surfactant in children with ards caused a significant increase in oxygenation, which declined over a period of - hours. the effect often could repeatedly reproduced, in one case after applications. the increase in oxygenation often allowed the reduction of fio and/or the inspiratory pressure. no side effects were observed after exogenous surfactant application.in many cases the application of surfactant wag too late after first symptoms of disease (median latency days). ards mostly due to pneumonia seemed to respond to surfactant therapy less well or not at all. permanent junctional reciprocating tachycardia (pjrt) is the most common incesant supraventricular tachycardia (svt) in children. it is usually drug resistant and its onset in early life has been associated with dilated eardiomyopathy. we report our clinical experience with patients detected antenatally and another diagnosed at months of age. method.diagnosis: negative p waves were detected in leads ii,iii and f, p'r > rp" and there was not warm-up at tachycardia onset.clinical records, ekg,x-rays, echo and holter were reviewed. ep studies were undertaken only with therapeutic purposes. results. in a year period patients under y of age fullfilled diagnostic criteria; were detected prenatally ( - weeks) and one was diagnosed at age mo. the fetuses had intermitent svt during gestation. all of them had pjrt in the first month of life at rates between and bpm. they were admitted to the icu but did not develop signs of heart failure. they were controlled with digoxine (d); d and quinidine; d and propafenone in to days. one was in sinus rhytm until age y; he then showed persistent pjrt over % of the day on repeated holters and underwent successful radiofrecuency catheter ablation (rfca).the other two patients showed initially a lowering of tachycardia rate followed by sinus rhytm for over % of the day (follow-up ran and y). the mo. old infant was admitted to the icu in severe cardiac failure. echocardiogram showed marked systolic dysfunction (shortening fraction %) treatment with digoxine, amiodarone and propafenone were unsuccessful despite lowering heart rate to ; rfca was performed at m. of age with restoration of sinus rhytm and rapid recovery of contractility. all patients were given atp at admission with transient ( to see) recovery of sinus rhytm. ff,s clinical course of pjrt is variable. atp is useful only as a diagnostic tool. initial treatment with digoxine + amiodarone or propafenone is adviced. rfca is a very useful therapeutic modality and can also be performed in young infants twelve patients ( %) died. these were meningitis, head injury, sub-arachnoid bleeds, status epileptieus, leukaemie, drowning, and multiple trauma. calculated from the a admission day p edialric risk of mortality score (prism), the probability of death (p) ranged from - %. of the deaths, i were predicted by prism analysis except for the leukaemie patient (p i%) who died from haematological complications following chemotherapy. two children predicted to die (p % & %) survived. the median length of stay was days (range - days). patlents( %) received ventilatn~ support and patienta( %) were transferred to specialist units ( neurosciences, liver, cardiac, bums). this data supports the view that many paediatric patients are being adequately treated in a dgh icu. meningitis and other neurological illness caused the majority of deaths and respiratory problems caused most admissions. most deaths ( of ) occurred within a few hours of admission. ectopic junctional tachycardia (ejt) is one of the most dangerous arrhythmias in the postoperative setting of congenital heart defects since it does not respond to antiarrhythmics or defibrilation. the object of this presentation is to report on two patients who presented f_jt in the early postoperative period and developed intense congestive heart failure which could be controlled after treatment with moderate topical hypothermia. two patients, m and y, diagnosed of atdoventficular septal defect and tetralogy of fallot developed intense heart failure in the early postoperative period. taehyeardia rate was and bpm. medical drug therapy included weaning from vasoactive drugs, iv digitalization and iv amiodarone treatment. there was not response. they were both surfaced cooled by placing plastic bags filled with cold water over the patient's chest and abdomen. temperature was monitored to obtain a central temperature of ~ there was a gradual decrease in heart rate in the following hours ( - bpm) paralel to the degree of surface cooling and clinical course estabilized.both recovered normal sinus rhytm in to hours. there were not significant arrhytmias after the procedure and postop, was uneventful. conclusions. moderate hypothermia is a very useful manuever for the treatment of drug resistant ejt. since it lacks side effects of other antiarrthymics we beleave it should be the treatment of choice for the treatment of ejt in the postoperative patient. present understanding of the pathogenesis of sepsis, based on the theory of systemic inflammatory reaction, has risen new interest in the more invasive methods of treatment, like plasmapheresis, leucapheresis and exchange transfusion (et). obiectives: evaluate the effect of et in the treatment of neonatal sepsis. material and methods: from september to december , a prospective study was carried out, where the severest cases of bacteriologically proven neonatal sepsis (n= ) were treated with et. in total newborns were treated for culture positive sepsis in the intensive care unit during this study period. diagnosis of sepsis was based on the clinical criteria of suspected neonatal sepsis, used by mc harris et al., laboratory data and positive blood culture. newborns with severe congenital malformations were excluded. et was carried out with fresh (less than hours old) adsol-conserved erythrocytes, from which buffy coat had been removed, and same donors plasma, using a slow continuous two-site technique. the mean volume of et was . ml/kg. the effect of et was assessed as a change in the score for acute neonatal physiology (snap), general treatment results were compared with a historical control group of newborns, treated for culture-positive sepsis in the same icu during the first eight months in . students ttest and chi-square test were used in statistical analysis of the data. results: with the use of el a significant decrease in mortality was achieved: death of cases during the study period, compared to deaths among the controls (p< . ). no baby, receiving et, died. the incidence of severe complications did not differ in the two groups. the snap-score showed quick improvement by the first post-transfusion day (p<o. ) and the effect persisted during the five following days. conclusions: we conclude, that the use of et in the treatment of critical cases may help to lower the mortality of neonatal sepsis. it provides a quick improvement in the clinical condition of septic neonates. the granulocyte-colony stimulating factor (g-csf) is largely employed in granulocytopenic patients. experimental studies have' demonstrated the effectiveness and safety of the therapeutic use, nevertheless the human employ is reported almost exclusively in neoplastic patients submitted to chemotherapy and in patients with fungal or pseudomona~ spp infections. the authors report the data concerning three non granulocytopenic children: in the first, o girl years old, affected by pseudomomm aerug/n~a pyeionephritis, the antibiotic therapy was not tolerated in consequence of renal and hepatic failure. the second patient, an infant months old, has been treated with salbactam-ampiciilin, ceftriaxone and chioramphenicol because of haemepldlw iafluem~ meningitis; a late neurological aggravation required the antibiotic therapy recommencement the last, a years old insulin dependent diabetic boy, was affected by cared/do spp systemic infection; the antifungnl therapy w~ not practicable in consequence of renal and hepatic involvement in all the g-csf was administred subcutaneously at the'dose of $ u for days. a significant increase of granulocytes was ever noted already after the second administration until hours after the last dose. clinical signs, fever and biochemical values are promptly influenced positively and all patients are restored to health. no side affects were noted. the granulocyte colony stimulating factor, at indicated doses, can prove of great help in critical patients, in who the antibiotic or antifangal therapies can result toxye or cannot be tolerated. the ~reptococcas p~, among the gram pmltlve~ represents the main causative organism of bacterial meningitis in children, with the majority of deaths occurring within hours of admission, the third generation cepohlosporim were indicated as antibiotic of first choice, and some literature reports have demonstrated no significant dlffereuces in dinical course of patients treated with ce~rlaxoae and of those treated with ampieibin and chlornmphenicol. the authors report the ease of on infant, male, months old, submitted to a neurosurgical interventiol after days the infant became febrile and lethargic and a bacterial meningitis by ~trepmcecctz ~ was diagnosed, in spite of he was given the eoftriaxone therapy.. an amelioration was found only when a systemic treatment with vaucomyein was added. after days an intrathecal administration of vancomyein m~day was started: the norbmlization of the spinal fluid profile was uoted two days later and the infant receve~d progressively. the follow-up after eight months doem't evidentiate motosensorini sequelae-the individuation' of ~cscc~ pmmmsm/ae strains heta-loctam antibiotics resistant requires alternative therapeutic regimens: the k~terity quead vitam and quoad valetudinem of these infections justifies an intrathecal therapy, especially failing o prompt answer to the systemic treatment objectives: to evaluate the incidence of the burnout (bo) of the staff in a picu in a university hospital. the bo is a syndrome charactedzed by many factors like emotional stress, lack of personality and reduced work ability. this syndrome represents a kind of abnormal response in the working habitat, above all, where psychological and personal characters are mostly involved. the symptoms of bo are represented by demoralisation, demotivation, incapability, boredom, isolation up to organic disturbances, like migraine, insomnia, dizziness and gastrointestinal disturbances. the major incidence of this syndrome was found in the hospital staff, especially among the nurses, working in an atmospher of high emotional dsk, just like in an intensive care unit. methods: from january ' till april ' , all the staff of the picu of the a. gemelli hospital of rome, underwent a test to evaluate the bo. the test used was taken from the book "bumout: from tedium to personal growth", by pines and aronson. twenty seven people were studied ( females, males)i out of which doctors, residents and nurses. it was considered sign!ficant as mild bo, a score between - and as severe, the score of bo >. . results: subjects ( %) resulted positive for bo, out of which were females ( %) and were males ( %). the subjects with mild bo were / : was a doctor, residents and nurses. the subjects with severe bo were / , out of which resident and nurses. conclusion: the results obtained show that bo is a condition well represented in the staff of our picu. the category most at dsk seem to be the nurses ( subjects), as well as residents ( subjects), as in literature, which shows a major incidence of the syndrome in younger subjects and having a limited partecipation of functional decision. the results obtained obliged us to start a programme of serial controls so that the subjects most exposed can have a necessary psychological support to react adequately to this condition. the term systemic inflammatory response syndrome (sirs) was adopted by the consensus conference to denote a type of systemic response to severe infection or otherinsults in critically ill patients. when sirs occurs from infection it is called sepsis. sepsis occurs more frequently in persons with perexisting illness or severe trauma. there has been tremendous advances in prophylaxis, diagnosis, and treatment of sepsis. a comprehensive model of the disease progression from sirs to mods should be developed giving priority to severity of illness scoring system and other predictive methods. some recommendations for future clinical trials include: trials should not start with humans. before proceeding to human trials, animal studies should indicate an acceptable risk/benefit ratio. appropriate patient populations must be defined and treatment protocols should be standardized. full and rapid reporting of all results should be mandatory and a central repository of published and unpublished study results could be helpful. accrual at each center should be of sufficient size, and should include the number of patients accrued, mortality rates, and patient characteristics. pivotal trial should be preceded by sufficient pilot or phase ii studies. correct drug dosage and usage should be delineated in pilot studies. large, multicenter, trials should be used to enhance the unversality of trial results. analyses should be planned a priori. definitions for the target population should be explicit, reproducible, and include illness severity scores. outcomes should be relevant reproducible and include both measures of benefit and harm. mods and its reversal should be considered as an endpoint. quality of life should also be considered as an endpoint. the estimators of overall treatment effects should be controlled for base-line prognostic factors and subgroup anaiysis should only be used for hypothesis generation and not to modify the conclusoin of the trial. economic analysis should be included as part of clinical design. evaluatin of source control should be a critical component of any study. standardized clinical mediator assays should be pursued. placebo patients in clinical trials should be studied for a better understanding of the pathogenesis and epidemiology of sirs, evidence based medicine should be used to evaluate the validity of clinical. introduction: use of inhaled nitric oxide (no) as a modulator for optimizing ventilation-perfusion or lowering pulmonary artery pressure is becoming increasingly common. no is a free radical but little toxicological research has been published. clearance of nebulized mtc-dtpa is known to be, a sensitive indicator for early function impaimaent of the alveolocapillary barrier. we investigated whether exposure to no increased clearance of ~tc-dtpa from the lung. methods: three groups of white sealand rabbits (bw . kg) were anesthetized, tracheotomized and paralyzed. groups were ventilated for six hours at pressure regulated volume control, set to deliver ml/kg with a frequency of /rain, i/e ratio = : and peep = cm hzo using a modified servo ventilator (siemens, solna, sweden) with computerized no delivery system. gas mixture per group was either / or / [no (ppm) / fioz]. after six hours of ventilation in these groups and immediately after anesthesia in group (control), ~tc-dtpa was nebulized into the inspiratory line of the breathing circuit and administered as a fine aerosol. gamma counting was measured for minutes, monoexponential curves were fitted to the data and the clearance half-time (t was calculated. the t~/ mean • sd of the different groups were: t~a (mean -sd) h"e,i witl~ arf : di.ff:erent kinds, aged .q-ore mon't.hes to [ gears o : (bodi weight .~rom ., to kg), is presen .... "ed ( i,,~u::trl:e i:ibstraclive d:lse~se... ~ .ards'- ; :~,;,,arf o~ ::entral genes:i s .- , ,~ :inc lud ing men ingeenceph it :is- ~ reye ' s ~yrtdro~e-..#~,bri~:ln pes~.re~nimatior~ disease.." ). int:lrl~]. pa-. "iiulle'i,~s ariel regymes o+ l;mv,l;i"t"v were cle'l'.ermllled by ba- 'i~ier was. about . tuber,, dopamin tiara-:. t.io; was ~.".,,'.r:~r~led. cmv,cppv d~.!"~tion raniled -~rom f to dayns.,~ < .-:in , "t -irl lo;and> davs'-in 'l~atierr~{s i'i"ai s:ltiol~ o ; patterers to imv, simv modee was per.r:)rmed, ~herl pif:' decrease.d to - ml~ar, fi ~ecreased to , . lind less with a = /,,. i:lesq.lts:{ in pat:i.ents e{ group :l, who were tre,~d.ed w&th f'f'v, teoph :i. : . l:i.r~ (is- .mg/kg/day), g lucecdr t icostei~oids ( .... :~;mg/kg/day), when r exceeded in , -.];, times normal va i tea the e aqes/,'!:l"oln ~j,, ite :i.~;::.!;, ~ml"lrj), it was possible 't'(' ce 'e~ e aad]t:..~rom ! . '.' i', to !..'; , - , mml-lg in ~}.. :~.[~ houi,!; ~d'l(:i to ru:}l",g'd!~l:i. e i::h,:~e,'~c['el';i.stil obieetives : this chapter will describe what is knovca of the psychlogical responses of infant and children to hospiuiisation and attendant procedures. the factors which may modify these responses will he discussed and important considemtiorts will be outlined for optimal anaesthetic management and postoperative period of infants and children which will minimised the rise of emotional upset. methods : in this paper the autors will discttssed the probl of: . health children (asa i, ii) facing single uncomplicated surgical elective procedures . various abnormal situations including neurotic children, children facing repeted operations, chronically ill, buaaes and tsaumatically impired ones . unfortunate young patient facing and often expoclting fatal outcome from le "ul'ukaemia, tumors, cystic fibroses or otheq" disease. : management of each child must vary greatly, ifi general the phases of emotional conditioning include home and preadmissiun preparation, admitiun preoperated and operative care and postoperative period. the authors would be happy if the child passes all stages without any trauma which could be prolonged in the future life. introduction ino is used to selectively reduce pulmonary vascular resistan(~e. we applied ino in the postoperative intensive care of patients with pulmonary hypertension and the risk of right ventricular failure after surgical correction of a congenital cardiac defect. methods - ppm no were added to the ventilatory gas mixture using a specially designed equipment (messer-griesheim, germany/austria). indications for application included pulmonary artery pressure > % systemic pressure, critically depressed right, ventricular function or an oxygenation index > . assessment of n oefficiacy consisted of on-off-on measurements according to the clinical stability of the patient including hemodynamic parameters, pulmonary gas exchange, continuous monitoring of ventitatory function and transesophageal echocardiography of the right heart. results in situations ( patients, age days- , years), ino was applied - h postoperatively. oxygenation was improved in situations from _+ to + mmhg pc ; pulmonary pressure was reduced in situations from -* % to _+ % of systemic pressure. in situations, no reduction of pulmonary pressure was present, but measurement of cardiac output or echocardiographic analysis indicated an improvement of right ventricular function (right ventricular stroke volume + -* %, cardiac output + -* %). in situations (immediately postoperativ with suprasystemic pulmonary artery pressures [n= ], multi-organ-failure [n= ]), no response to ino could be determined. conclusions for a special group of patients, the selective reduction of pulmonary vascular resistance by ino has become an important part of postoperative therapy. using this selective afterload reduction, postoperatively depressed right ventricular function can be improved. this effect of ino seems to be the most important one in the postoperative period. thus, ino appears justified to be appfleo when impaired right ventdcular function could be improved even when pulmonary artery pressure is not raised or remains unchanged. obiectives : premature infant are exposed to danger of apaea due to anaesthesia during their tirst months of life. it is yet unknown whether prematurity is corelated to any other kind of reslgratory disorder due to anaesthesia within the tirst year of life. methods : we theretbre researched retrospectively for respiratory disorders in all infants under months of life belonging to asa group . they all had been anaesthetised in . in our clinic for the following surgical reasons: ingvinal haemia, umbilical haemia, hydrocelae testis and phymosis. results : in cases we tbund: lafingospasm during induction in anaesthesia ( , %), bronchospasm during induction in anaesthesia ( , %), impaired intubation ( , ~ postanaesthetic laringospasm ( , %), supposed aspiration ( , %),postanaesthetic inspiratory stridor ( , %), postinductional inngoedema ( , %), death after months in consequative of infection pneumonie ( , %), none of these disorders was correlated the prematurity, infants suffered of post anaesthetic apnea, of them had premature medical history. concludions : prematurity does not enhance the risk of respiratory disorders due to anaesthesia within the first year of life, except the danger of postanaesthetic almea needs spetial cosideration. it could be demonstrated that aepgi lowers pulmonary vascular resistance and indirectly improves cardiac function. this effect seemed to be selective, and was comparable to ino in the doses we have examined. therefore, aepgi could represent a clinically useful alternate to inc. however, further research is necessary to work up the benefits of either therapeutic strategy. objectives: heat and moisture exchange filtem (hme) are used as artificial noses for intubated patients to prevent tracheo-bronchial or pulmonary damage resulting from dry and cold inspired gases. furthermore they are used for the prevention of bacterial contamination of the anesthetic apparatus by the patient's exspired air. so they are considered as a time-and money-saving device in anesthesia. filters are mounted directly on the tracheal tube, where they collect a large fraction of the heat and moisture of the exspired air, adding this to the subsequent inspired breath. the effective performance depends on the water-and bacteria-retention capacity of the filter. this study evaluates the efficiency of four different filters under clinical conditions. methods: four different types of filters ( dar hygrobac, gibeck humidvent, medisize hygrevent and pall bb ) were investigated dudng mechanical ventilation over a pedod of hours. minipigs with hemorrhagic shock were intubated and ventilated for days in an animal intensive care unit (icu). after hours of mechanical ventilation the filter was randomly replaced maintaining the individual ventilatory conditions. the weight of the filter was determined before use and after removal after hours. the airway pressure was monitored online to record changes during use. tracheal secretions and both sides of the filter were microbiolologically tested to see whether bacteria of the animal's respiratory system could be found on the patient's side of the filter or if they even would have penetrated the barrier. results and discussion: over a pedod of hours of types of filters showed an increase in weight of + % and airway pressure. bactedal celonisation ccured in nearly all fillers ( of ) on the patient's side, whereas only three of four types of filters showed identical bacterial colonisation on both sides. the only filter that did not show bacterial penetration, increase in weight or airway pressure was the pall-hme, a condensation humidifier without hygroscopic salts for moisture retention. with respect to our data one should use a condensation humidifier if airway conditions should remain stable dudng mechanical ventilation and desinfection of the anesthetic apparatus should be avoided after each patient. aim: to assess the clinical uses of, and experiences with, the hayek oscillator. this is a non-invasive device capable ef delivering not only continuous negative pressure (cnp) but also external oscillatory ventilation around a negative baseline (eov-nb) using an external cuirass. this type of ventilation avoids the need for intubation and intermittent positive pressure ventilation (ippv) and facilitates weaning in ventilator dependent patients. patients and methods: patients in respiratory failure, age range weeks to years in a total of patient episodes were treated using either cnp or eov-nb mode. duration of treatment varied from hours to days. indications for use ef the device were: ) to facilitate weaning from ippv ) prevent reintubation of patients following unsuccessful extubation, and ) avoid intubation and ippv altogether using the hayek oscillator as the on[y means of respiratory support. results: there was an increase in pao :fio ratio after cnp and eov-nb (p < . , and p= . respectively, wilcoxon signed rank test). patients who were in respiratory failure with hypercapnia showed a statistically significant reduction in paco both with eov-nb and cnp (p= . and p= . respectively) but the magnitude of change was individually greater in the patients who were treated with eov-nb. all patients, however, showed a fall in respiratory rate (p< . ) after the application of the cuirass in cnp mode. there was no physiological deterioration related to the application of external extrathoracic negative pressure in either cnp or eov-nb modes. conclusion: the improvement in pao :fio , the fall in paco and respiratory rate were indicators of an improvement in ventilation. the proposed mechanisms include improvement in frc, recruitment of additional alveolar units, and improvement in secretion clearance resulting in reduction in the work of breathing. meek to ~ month of the lifo,the bemodyuanicfacls were defined uitb the help of tetropolar reography method!. the excretion of !he catbocholauines fcfi] mith the urine gas detertend by taylor ll,laoorsy ~ iacg/dayl. hsaltl in the hypercuagulation stage of bic we deflorteeed the acliuutiun of the tbrubio and plasiin syaet~ mitb the increase of the inhihitnrs, in this case we registered in full uahe dot this process coabined uitb the dayl~ excreliou with lho urine epinopbr ne e], nor~pinopbr no tel and dophanine io], lbat shod the inlensificatiou of the s~nthosis prnoe-s~es and the release of ea in blood fron hissue deport the actffat on of the svnpathadrenui systen ]sfisl assisted to furl the b?perd~nanical rosins of the eircuidion and increase the ,icrocirculatinn, the klinicai sings of the insufissieutly of the circulalion have not defined,that has been associated the conpensatury character uf the ehan~es of ~ and heludy~enic status, t~e uun~u|p-lion ceugulupatby bus been donoustraled in the hypocougulatien stage ~bat man xauifosted b the exhaust of lhe confulalion nod oessel-platel heuostasis, the consuxptton of cnnpononts tbronbln ,plnstin, kallek~eiu-kinln s~slots and the forniration eat in fell canoe clot uas accoqaued bs docrea,e of fl,nfl,o, the products of the xotabolisx of c~ and the activation of xonoaninoxydasu. the decrease of the extoll'on g and the exhaust deport co indicahd about t!e ]ou fund/anal reserve of ~fl~. it was one of the lain reason of ~bo heiod~uanic disbroed iheat insnfissient]~] and the uicrncireulaflion lintestinal codeme with the low effectife periferal flow] and nul[iplay organ failure,the distrued deport of sos mitb throubocytupenin no; be one of the nechanisn the dislrood of uessej-plalol heioshasis, the correlation bolueeo changes of boiostosis c~ and circulation ore reguired aduinistration nedidns, thai reslore the love s of c~ in the blood, prevent uulliplay organ failure and hetorrnge in children with sepsis, ~b~ectives: multi-measured correlative analysis of the most number of non-invasive indices of the cardiorespiratory system function was made to determine the structure of their interrelation and the ways of their adequate and effective correction. hethods: spiremetry, capno~raphy, oxygenography, indirect fick method at recurrent respiration, plethysmography, integral rheography -in all indices were used. the received data were processed on a computer by a standard package of statistical bmdp programs. results: women with ~h-gestosis (i group) and somatically healthy pregnant women (ii group) were studied. cluster analysis has shown that the rate of the mean correlation connection between ventilation indices was % in the ist group and % in the iind group; gaseous metabolism - % and %, respectively; central hemodynamics was ~ in both groups. conclusion: cluster interpretation allowed to suggest that an increase of the rate of the mean correlation connection between the indices was characteristic of effective adaptation as the system was multi-component and well-regulated. on the contrary, the increase of the rate of strong correlation connection between the indices reveals the rigidity of the system and the tensity of adaptation mschaniams, i.e. the proximity to decompensation. it follows from this that in cases of eph-gestgsis, the reliability of regulating ventilation and gaseous metabolism decreases. seve/e hypoxemia in non intubated patients represents a major contraindicafion to fiberoptic bronehoscopy (fob) and bronehoalveolar levage (bal), but these procedures are often required for a correct diagnosis of the causative agent of pneumonia. aim of this investigation was to veaify the safety and efficacy of bronehoseopic procedures during pressure support ventilation administered through facial mask (fm-psv). five intensive care patients, all immunoeompromised, ( males and females; mean age . • were enrolled in the study. all patients presented criteria for pneumonia with pao /fio ratio ~ and were responders to fm-psv. fob and bal were performed afte~ topical anesthesia with fm-psv ( ps = em h ; peep = emh ; trigger = -lemh ) continuously admires" tered ( ' before fob fio = . ; during fob, fio = and for ' alter fob, fio = . ). pao /fio ratio as well as saturation (sat) did not show signifteative changes during the procodure (fig.l) . no complication was observed and hemodynamic conditions were stable for all patients. cmv, pnenmoeystiis ( ), legionella and mycobaetermm tuberculosis were identified from bal allowmg a prompt and targeted therapy. we concluded that mask psv can represent an excellea~ technique to pexform fob and bal in severely hypoxemic patients without deterioration of gas exchanges and avoiding endotraoheal intubation. intensive care unit, hospital general of albacete, albacet~ spain. objective: to analyze the current incidence and epidemiology of total parenteral nutrition (tpn) among critically ill patients placed on mechanical ventilation. design: prospective observational study. setting: medical intensive care unit in a tertiary hospital. patients: a total of consecutive l'ritically ill patients with non-coronary related disease needing mechanical ventilation admitted in our icu during a months period. measurements: data of sex, age, diagnosis, and outcome were recorded. severity of illness and therapeutic effort in the first hours were measured using acute physiology score and chronic health evaluation (apache ii) and therapeutic intervention scoring system (ties). r~ults: mechanically ventilated patients, male and female, were studied. only ten patients needed tpn and their main diagnoses were: five cases of multiple organ failure secondary to pneumonia ( ), ards ( ) and septic shock ( ); two eases of acute panereatitis; and one mesenteric throngmsis, one status epilepticas, and one ,prolonged cholinergic crisis b~ suicidal organophnsphate insecticide subcutaneous injection. no statistically significant differences between both tpn and non-tpn groups were found: objectives: evaluate the efficacy of prone position in ards and determine its importance in the therapeutic algorithm. methods: consecutive patients with severe ards (murray-score > , ; pao / fit < mmhg; male, female, mean age years) were conventionally ventilated (pcv, peep - mbar, i:e=i:i, ppeak < mbar). if after hours pulmonary function did not improve patients were placed in prone position. change from prone to supine position was done every hours. beside ultimate survival, parameters investigated were aado , pao /fio , and venous admixture (qs/qt). results: during the first hours in prone position of patients showed a significant decrease in qs/qt ( . % vs. . %) and aado ( vs. mmhg), and an increase in pao /fio ( vs. mmttg). changes were most pronounced in patients with high qs/qt, and in patients with an onset of ards less than hours before first application of prone position. after an average of position changes ( to ) of patients could be weaned from the ventilator. patient could leave tile hospital. i the later course letality was primarily determined by additional organ failures and by the severity of the underlying disease. negative side effects were minor, including slight cardio-vascular depression and increase in p~co , and never posed a limitation to continuation of prone position. especially in patients with septic shock skin lesions in exposed areas could not always be prevented, prone position could easily be combined with all ventilation modes and with all intensive care interventions. also immediately after major surgery and in patients with open packing prone position was possible. conclusions: in this investigation prone position proved to be an efficient and safe method in the treatment of severe ards. patients with a pronounced ventilation/ perfusion mismatch and patients in the early stages of ards appear to profit most from prone position. though the immediate effect on oxygenation is striking, still more the % of all patients die from multi organ failure and underlying diseases. a proposed therapeutic algorithm for ards is as follows: if under conservative ventilation (pcv, peep < mbar, ppeak < mbar) pulmonary function does not improve within - hours prone position should be applied. when after - position changes no lasting effect can be achieved further ventilation modes (e.g. pc-irv, aprv, no, etc.) should be used in addition to prone position. standard intensive care principles, such as fluid restriction and optimization of circulation, apply also to patients in prone position. objectives: nitric oxide reacts with superoxide to form peroxynitrite, an extremely reactive and toxic species. we quantified the presence nitrotyrosine, the stable product of the interaction ' of peroxynitrite with tyrosine residues in the lungs of pediatric patients that died with respiratory distress syndrome (rds). methods: paraffin embedded lung sections, obtained at autopsy, were incubated with a polyclonal antibody raised against nitretyrosine, followed by a secondary fluorescent antibody. alveolar structure-associated fluorescence was quantified using existing methods. results: tissue sections from patients who died with rds exhibited significant specific immunostaining which was uniformly distributed across the blood-gas barrier. in contrast only background levels of fluorescence were seen in the lungs of patients who died from non-pulmonary causes. intense staining was also seen in the lungs of rats that breathed % for h, a condition known to result in rds-type illness; no immunostaining was observed in air-breathing rats. conclusions: significant levels of peroxynitrite may be formed in the lungs of patients with acute lung injury. peroxynitrite may be contributing to the pathology of rds by damaging key components of the alveolar epithelium including the pulmonary surfactant system. mechanical ventilation time was prolonged ,g • days in patients with ardsvs , _+ l, days in control . mean staylcuwas lg _+ ,g days in the ards group vs , • , days in control group postoperative mortality rate was % in ards patients vs , % in those without respiratory failure. -ards incidence in liver transplantation is low ( , % in our sene) but it causes high mortality ( %) page, gas ventilation of the perfluorocarbon-f'dled lung, supports gas exchange and circulation in small animals (< kg) with lung disease. we hypothesized that large animals could be supported by page without adverse effects on bemodynamics. we first elucidated the determinants of gas exchange in normal sheep, and applied them to a model of adult respkatory distress syndrome (ards). methods: using the ventilator settings determined to be optimal in our pilot study (fio of . , peep of cm h , imv of bpm, it of %, and tv of ml/kg), sheep weighing . ~ . ) kg had lung injury induced by instilling ml/kg of . n hc into the trachea. ten minutes after injury, sheep with pao < ton" were randomized to continue gas ventilation (control, n= ) or to institute page (n= ). page was instituted by instilling . l of unoxygenated pefflubron into the trachea and resuming gas ventilation at the previous settings. abg's were drawn at baseline, minutes after injury, minutes after injury, and then every minutes for hours. objectives: inhaled nitric oxide (no) can improve oxygenation and decrease mean pulmonary artery pressure (papm) in hypoxemic patients with ards. in severe hypoxemic copd patients, it is not known whether inhaled no can exert a similar effect on hemodynamics and gas exchange. therefore, we investigated die response of inhaled no in hypoxemic copd patients and the results compared with those obtained in a group of ards patients. methods: ten copd patients (age _+ y;fev~ . _+ . l) and ards patients (age _+ ; lis . _+ . ) mechanically ventilated were studied. hemodynamic parameters were measured using a swan ganz catheter. arterial and mixed venous blood gas determinations, sao , svo , hb and methb were measured (abl ,osm ). mean intratracheal concentrations of no and no were continuously monitored using a chemiluminescence analyzer (nox ) . during the study the ventilatory pattern and fioz were kept constant. the protocol was for ards group: basalt, no loppm, basal~; copd group: basalz, no lo ppm, no ppm, no ppm and basal . after a steady state of rain hemodynamic and gas exchange measurements were performed. a positive noresponse was defined as a % increment in pao . results: papm was similar in both groups and decreased significantly after no (ards, basal . _+ . mmhg, no . + . mmhg, p < . ) (copd, basal . _+ . mmhg, no- . _+ . nrmhg, p< . ). all other hemodynamic variables remained unchanged after no. basal oxygenation was higher in copd group (paojfio _+ mmhg) vs ards group (paojfio _+ mmhg)(p< . ). after no- , pao increased ( _+ mmhg to _+ mmhg, p< . ) and qs/qt decreased ( + % to _+ %, p< . ) only in ards group. in both groups, significant correlations between basal papm and inhaled no-induced decrease in papm were found. inhaled no-induced increase in pao /fio was not correlated with basal paoflfio . no responders were / ( %) in ards group and / ( %) in copd group (p< . ). conclusions. in hypoxemic ards and copd patients, inhaled no decreased mean pulmonary artery pressure. however, oxygenation only ameliorated in ards group because die number of responders to inhaled no were higher in ards group and this effect seems not to be related to the basal hypoxemia. these results might be explained by the v/q abnormalities present in copd patients. grant fis / . objectives: it has been recently reported that expired con slope as a function of time is modulated by total respiratory system resistance (rrs) in critically ill patients (chest ; : - ) . in this study, we analyze the relative contribution of disease (dis), endotracheal tube resistance (rtube), airway resistance (rmin), additional resistance (~rrs), autopeep (peepi) and dylmmic/static elastance (ed/es) to the co elimination in different clinical conditions. methods: we have studied adult patients ( controls, acute respiratory failure, severe ards and copd) mechalfically ventilated (servo and c, siemens) without peep. we recorded tracheal pressure, airflow and capnograms. signals were analogic to digital converted for posterior data analysis. objectives: alveolar ejection volume (van) can be defined as the fraction of tidal volume (vt) with minimal dead space (vd) contamination. according to the classical paradigm: limvd_~ [vco /vt] =facoz, vco vs vt relationship tends asyntotically to a constant slope when approaches end-tidal volume. we have defined van as the volume that defines this relationship until a limit of % variation. methods: six subjects with normal respiratory mechanics were studied during anesthesia for minor surgery. two subjects, otherwise normals but having high values of total resistance and dynamic compliance, were also studied. capnograms were recorded in steady-state at levels of vt ( . , . and . l) and four levels of peep ( , , and cmh objectives: patients with ards presented lung abnormalities which originate an increase in airway resistance (rmin), in additional resistance (~rrs) and in static elastance (ers). application of peep further increases ~rrs. capnographic indexes reflect lung ventilation]per fusion inhomogeneities. in these conditions, the effects of peep on lung mechanics could be better understood by simultaneous measurement of capnographic indexes. methods: we studied groups of subjects. n: normal subjects scheduled for minor surgery; arf: critically ill patients with mild acute respiratory failure; ards: patients with early ards (< h). we recorded tracheal pressure, airflow and capnograms. signals were analogic to digital converted for posterior data analysis. respiratory system mechanics was assessed by constant end-inspiratory and end-expiratory occlusions technique. at equal tidal volmne ( . l) a peep level of , , and cmh was applied in all patients. we calculated ers (cmh /l), rmin, c~rrs (cmh /l/s) and autopeep. capnographic indexes were alveolar ejection volume (vae)/vt ratio and expired co slope beyond vae (sipco in contrast to synthetic surfactant natural suffactants (alveofact| are able to inhibit pmn-activation. after incubation of activated neutrophils with surfactant, l-selectin expression is decreased. these effects depends on which preparation is used. we conclude, that natural surfactant (aveofact| can perhaps influence early recruitment (,,rolling") of pmn in patients with respiratory failure like ards. with ards hormann cb, baum m, putensen c, knapp r, lingnau w, putz g . clinic for anesthesia and general lntensiv care medicine, university of lnnsbruck, anichstrabe , innsbruck objectives: in thoracic ct scans of patients with severe ards atelectasis and pleural effusion can be found in the dependent lung regions. by rotating these patients from left lateral position to right lateral position a redistribution of the ct densities, a recruitment of atelectasis and therefore an improvement of gasexchange is possible within a few days ( , ). the objective of this study was to find out the mechanism of alveolar recruitment during lateral positioning by ct scanning in left and right lateral position. methodes: after approvel by the local institutional reviewboard we investigated ventilated patients with severe ards (entry criterias: murray score > , ) in the ct scann of the university hospital. after a stabilisation period of minutes in supine position a thoracic ct scan slice cm above diaphragm was taken. then two different positions of the patients were studied in a randomized order: a) degree of left lateral position, b) degree of right lateral position. each lateral position was held for minutes. at the end of each of these periods a thoracic ct scan slice cm above diaphragm was taken. quantitative analysis of ct scan data was based on the frequency distribution of the ct numbers. to quantify the alveolar recruitment during lateral positioning by means of ct scan we defined compartments within the lungs: a) normaly inflated lung, b) poorly inflated lung, c) noninflated lung ( = atelectases) ( ). results: independant of the side of lateral positioning (l) in the non-dependent upper lung a significant increase of the normaly inflated compartment (s: %; l: %) as well as a significant decrease of the noninflated compartment (s: %, l: %) was observed in comparison to supine position (s). in the dependant lower lung the normaly inflated compartment decreased significantly (s: %, l: %) whereas the noninflated compartment increased significantly (s: %, l: %). throughout the whole studyperiode we did not observe any significant change regarding gasexchange and hemodynamic parameters. conclusions: in lateral position the non-dependent upper lung is decompressed. therefore a significant recruitment of atelectases is observed in the upper lung within minutes. on the other hand the dependent lung is compressed by the weight of the upper lung and the mediastinum. a great amount of the alveoli of the dependant lung collapse in this short time intervall. therefore the net effect of recruitment of one positioning maneuver is very small. when positioning patients one should be aware, that the patient is kept in each lateral position long enough to clean up the atelectases in the non-dependant lung and short enough to compress less lung tissue in the dependant lung. objective: to analyze effects of low-dose no inhalation ia patients with severe aeut~ respiratory distress syndrome (ards) over five days. methods: we prospectively studied patients ( men, woman) with severe ards admitted to our icu between may and may who required no inhalation with a dose of ppm for at least days. entry criteria for no injaalafioa were murray score >i . aud pat/fie < nun hg with peep >~ em i~o for at least hours. all patients were sedated, intubated and mechanicauy vantil~ed with volume assist-control ventilation, and had indwelling arterial catheters (pulmonary artery, and radial or femoral artery) to measure cardiac output (by thermodilufion) and relevant intravaseular pressures, and to calculate derived parameters. no was administered between y piece of the ventilator and endotraeheal tube and flow was adjusted to obtain ppm no in the inhaled gas. the no, no and no x concentrations were continuously measured at the distal end of the endouacheal tube by the chemiluminiscence method (nox , see-seres, france). metahemoglobinemia levels were mesured daily. no inhalation was manteined if paojfio ~ improved at least % and was stopped when the change in pao /fio ~ was below % or when the patient presented a paojf > mm hg a~er minutes without no inhalation. every day we made an on-off test to determine if no inhalation improved pao /fio ~. statistics: analysis of vmiance. data: mean + standard deviation. results: the mean age was . +_ . years and mean lung injury score was . • . . mortality was % ( / ), metahemoglobinemia . • . %, and no concentrations zero. paojf~o always improved significantly al~er ppm no inhalation (see :~ conclusions: reintubation in salf-extubated patients strongly depends on the type of meehamcal venfilatory support: the probability of needing a reintabation ff ese occurs during fult vontilatory support is higher than ff ese occurs during weaning. these data suggest that some patients may remain under weaning from mechanical ventilation for unnecessarily prolonged periods of time. objective: the aim of this study was to evaluate the acute effects on gas exehonge and hemodynamics due to positional changes from supine (sp) to prone (pp) in patients with severe acute respiratory distress syndrome (ards). methods: nine intubated, sedated, paralyzed and mechanically ventilated patients with severe ards were prospectively studied. all had a murray score > . , and a pao /f~o < with peep ~ cm h for at least h. all patients had indwelling arterial catheters in the pulmonary artery as well as in the radial or femoral artery in order to measure cardiac output (by thermodilution) mad relevont pressures, and to withdraw blood samples. arterial blood gases and hemodynamie parameters were measured first in sp, and then in pp after minutes of stabilization. vontilatoly parameters remaing unchanged during all the study. statistical analysis was done by the non parametric wdeoxon test. data are expressed as mean ~= sd. results: there were men and women with a mean age of . years ( - ) and mortality was % ( / ). main results are shown below: objective: to describe and compare a new method for obtaining p-v loops (p-vcv) by using a two-way collins valve (twv) with thosu obtained by the supersyringe method (p-vss). methodology: we prospectively studied patients who had an aeute lung injury and were intubated, sedated and paralyzed, and mechanieany ventilated. we performed the p-vev loops and p-vss loops in random order, and the static inflation pressure was limited to emh with both methods. pressure (p) was measured at the airway opening by means of a differential p transducer, and volume was obtained from flow (measured with a pneumotacograph) integration. the p-vse method has already been described (h~trf a,et al.bepr ; : - ) . the p-vev method consists in the following: the inlet of a twv is connected to the ventilator's y-piece, and both outlets are couneeted to the endotraeheal tube by means of an additional y-piece; one of this outlets has a one-way rudolph valve in order to allow inspiration but not expiration during the inflation maneuver. changing the twv tap position allows basal ventilation or progressiveinflation of the respiratory system. this maneuver is as follows: during an end-expiratory occlusion, the ventilatory settings are adjusted to deliver a ml v r with a respiratory rate of /min and i/e ratio : ; at the same time the twv tap is ehonged in order to divert flow through the one-way valve. inflation then begins alter releasing the expiratory oonlusion. pressure and flow signals were digitized and acquired by a computer for subsequent data analysis. we analyzed the following parameters: inflation compllonee ( objective: to analyze the variables which eventually may differentiate ards patients who do and do not respond to low doses of inhaled no. we prospectively studied patients ( men, woman) with severe ards admitted to our icu between may and may who were treated with no ( ppm). the onta'y criteria for no inhalation were murray score >/ . and paojfo z < mm fig and peep >/ cm i~o for at least hours. all patients were sedated, intubated and mechanically ventilated with volume assist-control ventilation. tidal volume was between and ml&g, with constant inspiratory flow, respiratory rate was - /rain, and i/e ratio between : to : . all patients had indwelling arterial catheters (pulmonary artery, and radial or femoral artery) in order to measure cardiac output (by thermodiintion) and relevant intravascular pressures, and to calculate derived parameters. no was administered between y piece of the ventilator and ondotracheal tube, and flow was adjusted to obi~a ppm no in the inhaled gas. the no, no and no x concentrations were continuously measured at the distal end of the endotracheal tube by the chemilumiinscenee method (nox , see-seres, france). metahemogtobinemia levels were measured daily. we considered a response to no inhalation when an improvement in paoz/fo above % was observed after the inhalation of ppm no (group r) . when the cha~age in paojfi z was below % it was considered a lack of response (group non-r small airways functional abnormalities have been recognized as a common feature of lung pathology. however peripheral airways contribute relatively little (~ %) resistance to flow and there disturbances can not be adequately estimated by conventional measurements of respiratory mechanics. the purpose of the study was to evaluate the relationship between raw and small airways conductance following weaning from ventilator methods. patients (age: - years; males) with no serious complications al~er mitral or multiple valves replacements and with more than hrs on mechanical ventilation have been enrolled in this study. the modified flow interrupter technique (ptg "gould" with fleish head # ; differential pressure transducer pm- -tc "statham" w amplifier "kistler ") and flow-volume recording of forced expiration (fleish head # ) have been applied before surgery and following operation on mechanical ventilation (my), after extubation (t:xtijb), on ( nay) and ( day) days. airways specific conductance (sg aw) has been calculated as a mean of - consequent measurements in each patient at each stage. the sac was estimated by max expiratory flow at and % of vc on - f-v curves (mef .~ , mef ) all the data were statistically analyzed with t-test introduction : noninvasive ventilation (niv) reduces the need for endotracheal intubation, the length of stay in icu and the mortality rate in acute exacerbation of copd. however, some patients failed to be ventilated with niv. .objectives...; to further delineate patients who failed to be ventilated with niv and to obtain predicted factors of failure. patients : a cohort of patients ( • years) presenting with acute exacerbation of copd (fevi: • ml, paco : • , ph: . • . ) and nonmvasively ventilated (pressure support through a full-face mask) between april and may twenty-seven ( %) were successfully ventilated with niv (discharged alive without the need for endotracheal intubation) while ( %) failed, requiring endotracheal intubation. .methods : patients successfully ventilated and those who failed were compared according to respiratory and nonrespiratory variables univariate analysis (wilcoxon rank-sum test and fisher-exact test) was performed to select variables included in a multivariate analysis by stepwise logistic regression. results : underlying disease assessed by the simplified acute physiologic score ( • vs • , p = . ), creatinine serum concentration ( • vs • gm/l, p = . ), blood urea nitrogen (bun : • vs mm/l, p = . ), age ( • vs • , p = . ) were higher and encephalopathy ( vs %, p = . ) more frequent in patients who failed. multivariate analysis showed that encephalopathic patients (or (odd ratio) = , p = . ) older than years (or = , p = . ) and presenting with bun >_ mmyl (or = , p = . ) failed to be ventilated with niv. variables related to the respiratory" status (i.e. paco , pao , fev ) were unable to predict tile failure of niv. conclusion : copd patients older than years, presenting with acute exacerbation, encephalopathy and bun > ram/l, should be carefully monitored because of high probability of failure with niv. methods:from february to december we studied pa_ timnts, males and females(mean age +/- ); of the se had emphysema,lo chronic bronchitis, dilatative car diomyopatia,with tracheostomy and emphysema.mean pac at admission in icu was +/- mmhg,while when weaningbegan, +/- .mean autopeep was cmh ( - ).all patients were ventilated in crpv as long as four hours to calculate st tic and dynamic cmpliance and autopeep.then the ventila tion was continued with psv+cpap(peep cmh objectives: analysis of the incidence of neurogenic pulmonary edema (npe) in a population of headtrauma patients with acute respiratory failure (arf). npe can occur after a central nervous system insult. differential diagnosis: cardiogenic pulmonary edema and other forms of non eardiogenic pulmonary edema. true incidence and pathophysiohigy remain poorly defined, however the role of catecholamines seems undeniable. early onset npe (within h after trauma) is characterised by hypoxemia, transient pulmonary hypertension and bilateral central fluffy infiltrates on chestx-ray. characteristics of cardiogenic edema or pneumonia are absent. late onset npe, (beyond hours after trauma), is more insidious. the clinical and radiographic picture has to clear within to hours. ( ) methods: all headtrauma patients admitted from january to december , in a nearotrauma icu setting were retrospectively analyzed for arf with as sole criterinm a pao -fio ratio < . results: neurotrauma patients were admitted during . patients ( %) presented with severe head injury (gcs< ), patients ( . %) with moderate (gcs - ) and patients ( . %) with minor head injury (gcs - ). overall mortulity was . % early (within h. after trauma) and delayed onset respiratory incidents were distinguished, counting for ( . %), respectively patients ( . %), patients ( . %) had early and late respiratory complications. early respiratory insufficiency was caused in patients ( . %) by aspiration, in patients ( . %) by lung contusion, in patient ( . %) by fat embolism and in patients ( %) by npe. in the late onset group patients ( . %) presented with pneumonia, ( . %) with fat embolism and ( . %) with npe. the npe group, patients, presented as follows: patients ( . %) developed early npe, and ( . %) delayed onset npe. patients ( %) died within the first days after admission, showing high mortality. gcs was less than in patients ( . %), indicating severity of head injuries. conclusions: high incidence of arf with various etiology ( , ~ was found in this population. in about % of all admitted hcadtrauma patients ( , % of arf) npe was causing attetial hypoxemia. occurrence of npe seems to be related to the severity of the brain injury and thus to outcome. these data call for extreme vigilance in respect of the insidious occurrence of npe. were included if recovering from respiratory failure and if in the opinion of the primary physician were ready for extubation. patients were excluded if undergoing compassionate withdrawal of support or had tracheostomies. the attending physicians were blinded to the measurements. included patients were placed on pressure support (ps) of em h with demand-flow continuous positive airway pressure (cpap) cm h . after a minimum of minutes on the above sehiogs: gastric intramucosai pc'o , abg, and a p . were measured. the padents were then disconnected from the ventilator for a period of one minute and the patients" respiratory rate and minute ventilation were measured using a wrights respirometer to calculate the frequency to tidal volume ratio (f/vt). patients were then extubated. extubafion failure was defined as the inability to maintain spontaneous ventilation for hours for any reason. results: twenty patients met criteria and were studied over one month period in october . six of the twenty patients ( %) failed weaning. the mean and standard deviation is outlined in failure . +/- . . +/- . . +/- . . +/- . comparison between roc areas shows phi and p . to each show a statistically significant difference from an area of . (p<o. ). the area at which the test has no discriminative value. this is not the case for either the integrative index or the f/vt ratio. the differences between areas for each curve is not statistically significant. the area's for each roc curve is shown in table #' . results. of the newborns referred to us for ecmo-therapy developed barotrauma, of the required ecmo and one of the three other patients who did not recieve ecmo-therapy died. to determine the correlation between the risk factor barotrauma and the severity of the pulmonary situation, we determined the oxygenation index of each patient referred to us for ecmo-therapy . the table demonstrates , d- giessen, frg. inhalation of nitric oxide (no) and aerosulizatiun of prostaglandin (pg) i: have been suggested to achieve selective pulmonary vasodilation and improvement of arterial oxygenation in patients with acute respiratory distress syndrome (ards). we directly compared these two modes of transbronchial vasodilator therapy in ards patients mechanically ventilated for - h (mean murray lung injury score [ ] . + . ). patients were randomized to receive either first no and then pgi (n= ), or vice versa (n= ), with an intermediate baseline period. each drug was titrated individually to find the lowest effective dose for maximum improvement of arterial oxygenation. gas exchange variables, including data from the multiple inert gas elimination technique (miget), and hemodymmaics under application of no/pgi were compared to pro-and past-challenge values. no (mean dose . + . ppm) increased the mean oxygenation index (pao /fio ) from + to + mmi-ig (p < . ) and reduced the shunt-flow from . + . to . : . % (p < . ). aernselized pgi (mean dose . + . " ng/kg rain) augmented pao /fio from + to + mmhg (p < . ), and decreased shunt from . + . to . + . % (p < . ). the miget analysis showed predominant redistribution of blood-flow from shunt areas to regions with normal ventilation-perfusion ratios in response to both agents. in patients, both no and pgi caused an increase in pao /fio by at least rnmhg. two further patients displayed a corresponding improvement of arterial oxygenation in response to either no or pgiz. the mean pulmonary artery pressure decreased from . : . to . : . mmhg in response to no, and from . : . to . : . mmhg (p < . ) in response to pgi . cardiac output, systemic arterial pressure, and right and left heart filling pressures were not affected by either agent. we r that individually titrated doses of inhaled no and aerosolized pgi~ effect selective pulmonary vasodilation and redistribute blood-flow from shunt-areas to weu-ventilated regions with nearly identical efficacy profiles. obieetives: the use of extraeorporeal bypass systems for oxygenation and co -removal is performed in patients with acute respiratory distress syndrome (ards) to reduce mortality and to improve restitution of pulmonary functions. high-dose heparin -commonly used in such patients to prevent thrombotic complications -might cause incurable bleadings as a major risk. this lead to the development of heparinized bypass systems; results of animal and first clinical studies proposed that such systems might run with low-dose heparin or even without any systemic anticoagulation. methods: since , patients with severe ards were treated with extracorporeal lung assist (ela) using heparin-coated systems (type maxima, medtronic, carmeda coating) in our icu. they received a moderate systemic heparinization. standard clotting assays (act, aptt, tt, ptt, at-ill, fbg, fsp) as well as special tests (tat, r~-tg, pf , pal-l, d-dimers, protein c, cl-inhibitor) were performed. results: covalent heparin-eoating of the ela-systems combined with lowdose heparinization could not prevent major changes in coagulation: ) in mean, platelets dropped from /nl to /nl within h after onset of ela. ) tat levels were already elevated before ela start ( ug/]) and demonstrated an additional peak h after onset of ela (up to ug/[). ) in parallel, there was a transient peak of pai-i levels (from to au/ml} h after onset of ela. ) in mean, fibrinogen levels dropped from to mg/ ml within h after onset of ela. ) in mean, cl-inhibitor levels dropped significantly from % to % after onset of ela and reached the initial levels within h. ) after membrane change, there were significant changes for c -inhibitor, fibrin-d-dimers and tt. ) global clotting tests such as aptt, ptt and tt were within normal range. conclusions: patients with severe ards develop a prethrombotic state already before they get connected to the ela bypass system. after onset of ela, they experience additional involvement of procoagulant, anticoagulant, fibrinolytic, and complement systems. in parallel to laboratory findings, clinical data suggest that the use of heparin-coated systems with accompanying systemic low-dose heparinization does not avoid thrombembolic and hemorrhagic complications in tong-term ela patients. thus, at the present, higher dosed individually adjusted heparin treatment has to be recommended. thereby, standard clotting monitoring is not sufficient. objective: poor muscle performance contributes to prolonged dependence on mechanical ventilation. longitudinal data on muscle function in icu patients is scarce. we evaluated changes in inspiratory and peripheral muscle performance during prolonged intensive care in patients with acute respiratory failure. methods: patients requiring intensive care for more than one week were studied. maximum inspiratory pressure (pimax) was measured twice a week for a maximum of three weeks. handgrip power (dynamometer) and subjective fatigue (keldet score) were obtained in survivors only. one measurement was done on the date of extubation. pimax pressure was obtained by occluding the airway for seconds or at least five inspiratory breath attempts. obiectives -measurement of peep-induced changes in lung volume helps to assess respiratory mechanics in acute lung injury (ali). we evaluated the accuracy and stability of a new respiratory inductive plethysmograph (rip), in the measurement of peep induced changes in end-expiratory lung volume (eelv) and tidal volume (vt) against volume reference, pneumotachograph (pnt), methods -two hours periods of basal ventilation and stepwise increases and reductions of peep ( - - - - cm h ) were recorded in patients with all. in addition, the new rip device (respitrace plus tm) was compared to an older rip (respigraph tm, nims, fl, usa) to determine its thermal stability using cold and warm devices and a ventilated lung model. results -the difference between the new rip and pnt in the measurement of vt was - _+ mi (x _+ se) or a - . _+ . % error. increasing peep from to cm h induced a + ml change in eelv. a difference of + ml ( . % of max. eelv change) (ns) was found between rip and pnt eelv readings. during controlled ventilation with constant settings the mean change in eelv was _+ ml/ min (fig.i) . validation of calibration showed a mean error of -+ . % after rain. the new rip had a higher drift when cold (cold + ml/ min vs warm _+ ml/ min), whereas the old rip was thermally stable (cold - + ml/ rain vs warm i + ml/ min). trauma icu, hospital traumatologia y rehabilitacidn. vall d'rebron. barcelona. spain. recent data suggest that the proportion of patients with relevant discrepancies between two jugular bulb (jb) oxygen saturation (sjo ) values is higher than suspected. objectives: determine the incidence, the significance and the pro nosis value of those differences,if they exist,in severe head injured-patients. material and methods: severe head-injured patients, coma glasgow scale (gcs) , with diffuse brain injury according to marshall criteria in the first ct scan, icp recorded, with an interval from accident-double jb monitoring < than hours, were studied. data from bilateral sjo were obtained simultaneously, every hours. discrepancies were considered significant, when differences in sjo were > %. no chan es in treatment protocol (hyperventilation, man• etc) were carried out due to this study. results: men and women were studied, aged • yrs. at arrival at hospital, gcs were < in and ) in to. the incidence of high icp() mmhg) were sz at the entry. the mean therapy index level required to control lop was ~l all patients required vasopressor therapy to maintain upp over ds mmhg. in patients a s.s f swan-ganz fiberoptic catheter was used to obtain a continuous recording of sjo . in the others , sj were intermittently controhed.the mean time of monitoring were d. • days. ten patients died within this period. a total of . blood samples were analized. at arrival, sjo discrepancies were found in patients, b %. at hours, the incidence were lower, / , . %. at th day, were h/ , z and at day , when the catheters were retired, ii[ , z showed discrepancies. the ct showed new injuries in g z of patients with differences > ~ in sd values throughout treatment period. none of those were considered for neurosurgical treatment. no correlation was found between iop and sjo values and sjo differences. conclusions: the incidence of discrepancies between sjo was higher than expected in severe head-injured patients. these situation could reflect disturbances between demands. when differences are known, and those lend to change, the ct scan, nearly always, will show new injuries. platelet-activating factor (paf) is an inflamatory mediator implicated in the pathogenesis of bronchial asthma and acute respiratory distress syndrome (ards). its inhalation in healthy subjects produces transient bronchoconstriction and mild ventilation-perfusion mismatch, together with peripheral leukopenia as a result of intrapulmonary neutrophil (pmn) sequestration. likewise our group has shown in healthy subjects and asthmatic patients that aaibutamol (s) inhibits both pulmonary and systemic effects of paf, suggesting that s may inhibit paf-induced venoconstriction in pulmonary microoirculation. the aim of the present study was to investigate if s inhalation decreases pmn by lung sequestration induced by paf. we studied healthy, non-atop• nonsmoking subjects ( m/ f, + yr), which were pre-treated with s ( ,ug) or placebo, with a randomized, double-blind, crossover, design, before paf ( ,ug) inhalation. we measured the respiratory system resistance (rrs) by forced oscillation, arterial btood gases and both total white cell and pmn count every min over a min. period. simultaneously, we recorded continuously the lung dynamics of inm-neutrophil and tc m-erythrocytes activity, with a gammacamara. after placebo, paf inhalation decreased white cells (from to x /l), and pmn(from to _+ x /l), and increased aapo (from . _+ . to . + . mmhg, p<o. ) and rrs(from . . to . . cmh .s.i q ) (p < . each). the fall in total white cell count correlated with the intrapulmonary pmn retention (r = . , p < . ). by contrast, after s, paf did not induce any change in white ceil count (from to + x /i), pmn count (from to x /i), aapo (from . to . + . mmhg), and rrs (from . . to . . cmh .s.i ). compared with placebo, after s there was a lower ~lln-pmn lung activity ( . . vs . . cts/mci/pixel, p<o. ), lower intrapulmonary pmn retention ( . . vs . . %, p=o, ), and a faster washout ( . . vs . . s ~, p= , ). our results indicate that s inhibits paf-induced intrapulmonary pmn sequestration, being consistent with the hypothesis that s may offset the venoconstriction of pulmonary microcirculadon caused by this mediator. supported inpiratory muscle fatigue with failure of force generation as def'med by a tensiontime index (tti)> . - . has been shown to occur in normal volunteers and in stable copd patients with a specific imposed breathing pattern. its role, however, in hypercapnic respiratory failure is less certain. we studied failed weaning trials in copd patients in which breathing pattern, tension-time index (tti) of inspimtory muscles, dynamic peepi, dynamic lung elastance, lung resistance, and arterial paco and ph were measured at the beginning and end of a t-piece weaning trial. in addition, the change in esophageal pressure during a mueller maneuver (apes max) was measured. a weaning trail has been prospectively defined to have failed if one of the following criteria was met: a rise in pco > mmhg from baseline accompanied by a fall in ph< . ; a respiratory frequency (f) > /min; excessive accessory inspiratory muscle recruitment; and a marked increase in dyspnea. values are expressed as mean • se. weaning failure was characterized by a more rapid, shallow breathing pattern, worsened mechanics, hypercapnia and respiratory acidemia despite an unchanged tri and pes max. we conclude that in this setting hypercapnic respiratory failure is not a consequence of inspiratory muscle fatigue. rather the adopted breathing strategy and resultant hypercapnia may represent an adaptation to forestall the onset of muscle fatigue. concerning the investigated elf-par~eters, no stadstically signhqcant differences were detected between the pgi and the control group. histopathologlcal changes occured in both groups and consisted in rare focal flaaaning f tracheal epithelium with loss of cilia and slight inflammatory cell infiltration, as well as slight swelling of alveolar typo pneumoeytes. sections of generation , and from bronchial tree were free of pathological changes. conclusion: alter h inhalation of p~ji no signs of respiratory-lract tissue damage caused by the aerosol could be detected. the minor pathological findings in the trachea are most likely due to mechanical irritation by bronchoscopy, changes of the alveolar epithelium are known for long-term mechanical ventilation . objectives: the aim of this study was to evaluate of efficiacy of ganglion stetlate blockade in patients with respiratory failure. methods: two groups of patients were investigated: group i (n = ) trauma patients with acute lung injury (ali), group if (n = ) patients with asthmatic status. in all cases continuous mandatory ventilation (cmv) was used with bennett ae. in both groups bilateral ganglion stellate blockade with antero-lateral approach was performed, using . % marcain. the following parameters were analysed: pao , sao , paco~, pip and c~t~t. results: in trauma patients with aij after bilateral ganglion stellate blockade short -lived and slight improvement of pao and sao , decrease of pacoz and pir and increase of static compliance of respiratory system were found. in second group bilateral ganglion stellate blockade interrupted the asthmatic status and significant statistical improvement of parameters of oxygenation, ventilation and respiratory system mechanics were observed. conclusions: we suggest that the bilateral ganglion stellate blockade is a very useful method in treatment of patients with obstructive respiratory insufficiency. the aim of the study was to analyse whether there exists serum and urine electrolyte disorder in patients(pts.) with acute respiratory insufficiency(ari). the study included t pts. with ari (pao : , @ , kpa. paco : , i- , kpa, ph: ~: , , hco : , :~ , mmol/ , sao : , ~- , %) who were hospitally treated due to pneumonia( pts.),emboly of the pulmonary artery( pts.) and severe attack of bronchial asthma ( pts). among tham there were ( , %) males and ( , %) females, average age , ~: , years, otherwise previously healthy. electrolyte concentracions were measured at the onset of the disease in serum and urine collected during hours (sodium-na,potassium-k, chlorine-c , calcium-ca,magnesium-mgand phosphorus-p). the measured serum and urine electrolyte concentrations were compared with respective referent values (rv). by serum electrolyte analysis, the following average velues were obtained: na:l o, the object of our investigation was a group of pts with massive pneumonias, males ( . %), females ( . %),mean age yrs.thirteen ( %) of them were smokers, ( %) nonsmokers. only pt ( . %) had pre-existing chronic respiratory disease, and ( . %) were admitted for the first lime,with no previous respiratory anamnesis. diagnose was based on anamnestic data of productive cough in pts( . %),physicaly ~onchial breathing in i~s ( . %),white cell count onder x /l in pts( . %). radiographicly, bilateral massive homogeneous shadows were found in pts ( . %), onilateral in pts( . %),pleural effusion in pts ( . %). abnormal renal function was found in pts ( . %). sputum culture was positive in pts ( %): slr.pneumoniae, str.pyogenes, pse'udomonas aerug, in , , cases respectively. all patients had remarcable hypoxernia (pao range from , to , kpa) without hypercalmea. all patients needed oxygenotherapy together with antibiotics and other .symptomatic therapy. nineteen pts had anaelioration of general condition and normalization of blood gas analyses, while pts with the lowest hypoxcmia died.in conclusion, massive pneumonias are frequently followed by respiratory insufficiency which is one of the markers of pneumonia severity. as existing hypoxemia complicates the course of the disease,prolonges the recovery, makes therapy more complexe and may be cause of death , frequent blood gas measurement is recomanded. we studied the effects of bosentan (bos), an eta and etb receptor antagonist, to examine if endogenous et mediates pulmonary hypertension in anesthetized and ventilated dogs with acute lung injury due to oleic acid (oa). the gradient between pulmonary artery pressure (ppa) and occluded ppa (ppao), and gas exchange (evaluated by arterial blood gases and sf intrapulmonary shunt) were measured at controlled flow. in dogs (treatment), data were collected at baseline, during long injury (obtained rain after intravenous administration of oa . ml/kg), and again after bos ( mg/kg intravenously). in dogs (pretreatment), data were obtained at baseline, after bos and then after oa. in treated dogs, oa increased (ppa-ppao, mmhg, table, means + sem, * p < . vs base) and deteriorated gas exchange. after oa, bos did not affect pulmonary vascular tone nor gas exchange. in pretreated dogs, bos had no effect on baseline pulmonary vascular tone but prevented the increase in (ppa-ppao) after oa. the deterioration in gas exchange after oa was not influenced by bos pretreatment. objectives: the alveolar tension is measured by the application of the alveolar air equation in which the arterial pco is used or by the simplified form of this equation in which the respiratory exchange ratio is taken at the value of . . the purpose of this study was to estimate the effective alveolar tension (pao eff) during spontaneous breathing with a new bedside technique which is simple non-invasive in normal subjects and patients with chronic bronchitis-emphysema. we also compared these values with the ideal alveolar po (pao (i)), measured from the alveolar air equation in which paco was substituted by the effective alveolar pco (paco eff) and with the alveolar po measured from the simplified alveolar air equation (pa ). this study is complemantary to previous work for the estimation of paco eff. methods: the subjects breathed quietly through the equipment assembly (mouthpiece monitoring ring, fleisch transducer head) connected to a pneumotachograph and a fast response and co analyzer. the method is a computerised calculation of the effective alveolar po quite similar to that of paco eff, obtained from the simultaneously recorded at the mouth expiratory flow, and co concentration versus time curves. results: the results showed a mean difference (pao eff-pa (i)) of - . kpa in normal subjects and - , in patients. the mean of the difference (pao eff-paq ) and (pad (i]-pao z) was much greater than . in all subjects. the limits of agreement for the difference (paozeff-pa (i))were - . to . kpa in normal subjects and - . to . in patients, while those for the differences (pao eff-pad ) and (pao (i)-pad ) were very large ( > - . to > . ) in all subjects. conclusions: the effective alveolar po is very close to the ideal one in normal subjects, tn patients pao eff may excessively deviate from pa (i) due to the observed significant difference between the alveolar/tidal volume ratio for o and that for co . the alveolar po measured from the simplified alveolar air equation (pao ) differed substantially from pao eff and pad (i) in all subjects. the essential role of glucoprotein hormone erythropoietin is to control red cell production. hypoxemia, reduced blood -carrying capacity and increased affinity of hemoglobin for are the primary stimuli for erythropoietin production. both anemia and hypoxemia induce rapidly erythropoietin secretion. kidney erythropoietin rna levels correlate inversely with hematocrit and directly with plasma erythropoietin level. similarly, hypoxemia increases kidney erythropoietin rna and plasma erythropoietin. the effect of hyperoxemia (pa >lo mmhg) on erythropoietin secretion isn't very well understood. the purpose of this study was first to evaluate the erythropoietin secretion in patients with acute respiratory failure and second to determine the effect of hyperoxemia on erythropoietin secretion in patients with and without anemia. sixteen patients with acute or acute on chronic respiratory failure needed mechanical ventilation were included in this study. these patient were divided in two groups. the patient who developed anemia were included in group i and the patients without anemia in group i . erythropoietin was estimated in venous blood in three stages. the first sample was taken during hypoxemia, the second during hyperoxemia and third during normoxemia. all the patients had high erythropoietin level during the hypoxemia period (mean value • mu/ml). during hyperoxemia etythropoietin levels were reduced in both groups ( mean value . + . mu/ml in group i, . • mu/ml in group ii). in normoxemia stage, erythropoietin increased again in anemic patients, and decreased more in the patients of group i . we conclude that hyperroxemia inhibit erythropoietin secretion in spite of anemia and tow arterial oxygen content. hyperoxemia may be a factor of the insisted anemia in with oxygen treated icu patients. the purpose of this study was to determine the relationship between clinical features of acute lung injury (all) and parameters like total proteins, total and individual phospholipids, the presence of paf, and acetylhydrolase activity in bal of mechanically ventillated patients. acetylhydrolase catalyses the cleavage of acetyl-group from the second position of the glycerylether backbone of paf, leading to its inactivation. mechanically ventillated patients were divided to three groups. group i includes patients without all; group ii, comprisespatients with moderate degree all, ( . <g-i < . ); and group iii comprises patients with severe ards (all score > . ). broncoalveolar lavage (bal) was obtained after infusion of normal saline at ~ to intubated patients and cooled immediately. cells were removed after mild centrifugation ( x g, min, oc). aliquots from the supernatant were used for total protein, phospholipid and paf analysis and determination. acetylhydrolase activity was assessed after incubation of bal with h-paf labelled on the acetyl group. released label was measured by liquid scintillation counter in the supernatant after trichloroacetic acid precipitation of the non-reacted substrate. kinetic characteristics of the enzymes were also studied. total phospholipids appear reduced in bal of patients with all, while total proteins increase. these factors appear to correlate with the severity of all. paf was not present in bal samples pretreatad with equal volume of % acetic acid to denaturate acetylhydrolase. detection limit for paf under our experimental conditions: pg paf/ml bal. instead, acetylhydrolase activity was detected in amounts increasing with the total protein content. background: intubated patients without lung injury or impaired breathing control normally display an inspiratory peak flow of below l/s. the aim of our study was to investigate the inspiratory peak flow generated by patients with acute respiratory insufficiency (ari). we had to take into account that both an inspiratory pressure support (ips) and the resistance of the endotracheal tube considerably influence the flow pattern generated by the patient. patients and methods: to investigate the non-influenced flow pattern we developed a new ventilatory mode which automatically compensates for the flow-dependent resistance of the endotracheal tube (automatic tube compensation, atc). furthermore, the mode maintains a constant tracheal pressure in inspiration and expiratio n . consequently, the measured flow pattern exactly corresponds to the flow pattern generated by the patient except that the ventilator modified for this mode (evita, driiger liibeck, germany) was not able to deliver a gas flow of more than l]s. we have investigated patients with ari arising from different reasons. results: the inspiratory peak flow measured in the atc-mode was . l/s _+ . l/s. the maximal deliverable flow of l/s was obtained in of patients. the figure shows the flow pattern under atc and ips in [~s] oi:) one of these patients. conclusions: patients with ari display a highly increased inspiratory peak flow. ventilators used for spontaneous breathing should therefore be able to deliver a gas flow of more than l/s. an overproduction of no and reactive oxygen species (ros) has been demonstratred in septic shock. ros and nitric oxide (.no) are free radicals which are known to react together leading to peroxynitrite anions that can decompose to form nitrogen dioxide (no ) and hydroxyl radical (oh~ thus, no has been reported to have a dual effect on lipid peroxidation (prooxydant via the peroxinitrite or antioxidant via the chelation of ros). in the present study we have investigated in different models the in vitro and in vivo action of no on lipid peroxidation. copper-induced ldl oxidation was used as an in vitro model of lipid peroxidation. ldl ( ~g apob/ml) was incubated with cu + ( , ~tm) in presence or absence of no donor (sodium nitroprussiate or glutathione-no) from to ~m. oxidation of ldl was monitored continuously with conjugated diene formation ( nm) and hydroxy nonenal accumulation (hne). exogenous no prevents in a dose dependent maner the progress of copperinduced oxidation. ischaemia-reperfusion injury (i/r), characterized by an overproduction of ros, is used as an in vivo model. anaesthetized rats were submitted to hour renal isehaemia following by hours of reperfusion. sham operated rats (sop) were used as control. lipid peroxidation was evaluated by measuring the hne accumulated in rat kidneys in presence or absence of l-arginine or d-arginine infusion. l-arginine, but not darginine, enhances hne accumulation in i/r but not in sop (< . nmol/g tissue in sop versus . nmol/g tissue in i/r), showing that in this experimental conditions, no produced from l-arginine, enhances the toxicity of ros. this study shows that the pro-or antioxydant effects of no are different in vivo and in vitro and could be driven by environemental conditions such as ph, relative concentration of no and ros, ferryl species...these conditions are impaired in circulatory shock. methods:" the diagnostic and therapeutic approach was standardized so that data collected over a -year period were comparable. a progressive deterioration of clinical conditions and/or pulmonary gas exchanges was considered as indication for my. variables potentially predicting the need for hv were derived from clinical and arterial gas data, extrapulmonary diseases, use of drugs, chest x-ray and ecg abnormalities. results: rv, performed with external and/or internal ventilators, was necessary in patients ( %). at the hospital admission, pac was higher and ph was lower in patients requiring rv ( pneumomediastinum, pneumothorax, ateleetasis and myocardial infarction are rarely seen in bronchial asthma. these complications occur as a result of the severe asthma.the aim of our retrospective study was to analyse the complications seen in acute asthma attacks. during the years through , patients were admitted to hospital in acute asthma episode. there were ( , %) pts with complications; mean age of yrs; females ( %). clinical history, ecg and chest radiogr~hs were analysed. the mean duration of bronchial asthma was yrs (range from months to yrs), all patients were atopics. there were four ex-smokem and one smoker. the worsening of asthma symptoms begun two days before the admission (range from to days). on ecg all patients had tschycardia. rightward shift of the qrs axis and st-t changes indicative of right ventrieutur strain were found in three pts. these were the transient fmdings that improved after curing the acute asthma attack. non-q myocardial infarction oeeured in one patlent and resulted from the hypoxaemia of asthma. hyperinfl~ion was the usual finding on the chest radiograpk pneumomediastinum and subcutaneous emphysema were apparent in five pts and required no additional treatment unilateral pneumothoraccs were present in two pts and needed eontimous intrapleural drainage; one of these patienst died in eardiorespiratory insufficiency. ateleetasis of right upper lobe was present in one patient. it oceured due to inspissated secretions and needed no additional treatment all these patients, except one who died, improved on lreaanent with oxygcr~ steroids, beta-two agonists, theophylline and antibiotics. in conclusion, complications occur in acute asthma episodes as a result of the severe asthma mediastir,*l emphysema and atelectasis are not serious complications. pneumothorax and myocardial infarction are very serious life-treatening complications and always have to i:m considered in taati~ts with sev~ asthma. acute bronchial asthmatic episodes represent one of the most common respiratory mnergendes, its maximmum expression "status asthmatiens" is one entity of low incidence, still it is a risk to the physical integrity of the patient. during a total of patients with diagnosis of status asthmabcas were hospitalized. out of these palients six had a near-fatsl asthma and they were subjected to a complex examination. near-fatal asthma was defined as either respiratory arrest or acute asttuua with paco greater than , kpa and/or an altered state of consciousness. mean age was , -d: , yrs, four male and two female sex. at presentation two patients suffered from coma, others were confused. they exh'bited severe dystmoes, diffieul~ speaking, used accessory muscles of respiration, increased whee~tg while two cases had silent chest on auscultation. cyanosis indicated a very severe asthma attack in all six patients. mean respiratory rate was ~ /min and puts rate .d: bts/imn. arterial blood gases revealed a pao of , ~ , kpa, paco of , • kpa and ph of , -+- , . area-careful evaluation they received conventional therapy (immediately continuous oxygen, impelled nebulization with high doses of betatwo agonists and ipmtropium bromide, intmvanous st~oids and theophylline). in two eases signs and symptoms of deteriorating airflow and respiratory muscle fatigue determined the need for mechanical ventilation. out of six near-fatal attacks aggressive lrealanent was suscessfull in four patients and fatal in two eases. one patient admittcxl in coma died in severe hypoxae~a upon one hour and one mechanicaly ventilated died from cardiac arrhythmia. life-threatening attacks in asthmatics in our group developed gradual worsening despite neatment which r symptoms in most other patients. one patient had "brittle asthma", other long-standing acute episodes ireated with systemic steroids. conclusions: idantitiechon of fatality prone subjects may lead to fttrther muetion of seveze episodes. respiratory affest and coma upon admission, severe dyspnoca with silent chest on ausouhation, oyanusis and use of accessory muscles of respiration constitute the basic cfinieal picture. hypoxasmia must be immediately eon'ected.the patients and physicians should be able to assess the severity of asthma, a major factor in near-fatal and fatal asthma attacks. objectives :our purpose was to asses if the evolution of patients with a adult respiratory distress syndrome (ards) ,shows any relation to the pulmonary or systemic origin of the disease and whether or not there were differences in the frequency of the syndrome in both groups. methods : randomized prospective study in multidisciplinary icu. one hundred and sixteen patients with a high risk developing ards were distributed into two groups. one was named systemic origin group(so) and the other pulmonary origth group (po).ai patients only showed one cause (pulmonary or systemic) with potential risk of ards.the patient's hemodynamic and respiratory status was evaluated every hours the first day and every hours the second and third day. at the end of hours the patients were diagnosed as ards or non-ards. measurements and main results : of the total patients, were finally included in the so group and in the po group.patients in so group and po group had comparable ages (p<. ).peep in both groups was comparable (=. ) at the mmnent of admission to the study. there were no statistically significant differences for cardiac index and systemic vascular resistances. the pulmonary vascular resistances (pvr) showed significant differences at h.(p<. ) and h. (p<. ).the oxygen comsumption (vo) in patients of the so group showed statistically significant differences at h. (p<. ) with respect to initial values.fifteen cases of ards ( . %) in the so group and twenty five cases ( . %) in the po group were identified. the time of onset of ards was _+ hours in the so group and + b hours in the po group.the final outcome was very similar th both groups : mortality of % in the so group versus % in the pc group. conclusions : the pathogenesis of ards depends on whether the lesion is originated at or outside the lung. the po group showed a sborter thne of onset of ards, a faster and more severe increase of pulmonary shunt and a higher percentage of patients developing ards compared with patients of the so group.the so group showed a higher and faster increase in puhnonary resitances tbat po group and a decrease th oxygen comsumption earlier and more severe than in the po group. these data thus seem to show that there could be two mechanisms involved in the genesis of ards depending on the cause. the fact that the ards genesis is shorter in the cases of pulmonary etiology with faster impairment of pulmonary shunt, and a slower increase in pulmonary resistances in this pulmonary group, would indicate that the underlying mechanisms responsible for the hypoxemia are different to those which thitiate the increase in pulmonary resistances. finally, the exclusive inapairinent of oxygen consumption, which appears earlier than the onset of ards in the systemic origth group, could show the generalized character of the process in this group. perfusion of prostacyclin (pgi ) to treat pulmonary hypertension in adult respiratory distress syndrome (ards) worse pulmonary gas exchange due to a marked impairement of ventilation/perfusion mismatch. recently has been shown that if prostacyclin is given by aerosol instead of intravenous the net effect is an improvement of arterial oxigenation due to a redistribution of blood flow to well ventilated areas. objectives: to asses the effects of inhaled proatacyclin on pulmonary haemodynamics and gas exchange in patients with severe ards. methods : two patients with severe ards (murray score > ) recived inhaled pgi at - ng.kg.min " using an ultrasonic nebulizer. haemodynamic measurements, arterial and mixed venous blood gas analysis were performed before and after rain of pgi inhalation. results: short-terro p~i inhalation improved pulmonary g-~ e-'~hange in both patients. arterial oxygen partial pressure (pao ) increased from to mmhg in patient and from to in patient , the ratio pao to the fraction of inspired oxygen increased from to (patient ) and from to (patient ). venous admixture decreased from % to % and from % to % in patient and respectively. mean pulmonary artery pressure decreased slightly from to mmhg in patient and from to mmhg in patient . no effects on systemic haemodynamics were observed in any patient. conclusions: pgi inhalation improves gas exchange and produces selective pulmonary vaaodilation, thus can be an alternative therapy for the treatment of pulmonary hypertension and hypexemia in patients with severe respiratory falllure. methods: we treated ards-patients (age yr ( - ) mean, range) during - . the lowest pao /fio -ratio was ( - ), the worst murray score . ( . - . ), icu-stay ( - ) days and hospital mortality %. the costs of intensive care were calculated according to intensivity of patient care as assessed by tiss-scoring (therapeutic intervention scoring system). the more intensive the care, the higher are the costs. costs per year of life saved (=life-year" in us $) were compaired by other medical treatments ( - ). it is assumed that the mean expected length of remaining life in ards-survivors after intensive care is years. treatment life-year ($) ' bone marrow transplantation (acute leukemia) lowering cholesterol using iovastatin treating hypertension using nifedipine heart transplantation intensive care of ards-patients conclusions: intensive care of patients with severe ards is highly more cost-effective as compared with many other routinely used medical treatment strategies, the usually good recovery and the reasonable quality of life in survivors justifies investments to care of these patients ( ). there is a close correlation between these two methods of measuring evlw. however there is an underestimation of . % in this kind of pulmonary edema ( oleie acid induced ) with the double dilution method. although the size of the sample is small, in normal lungs there appear not to be this underestimation. the effect of peep on evlw has been studied with contradictory results, probably as a consequence oft differences in methods of measuring evlw, variations in the type and severity of lung injury, and different timings of peep application. objective= ) to analyse the effect of different levels of peep ( , and omh ) on evlw during hpe; ) to establish whether increases in intrathoracic pressure due to high peep levels can obstruct lymphatic drainage. material and methodet hpe was provoked in groups of dogs by inflating a foley catheter in left auricular to a pressure of - r~uhg. peep levels of , i or m~hg were applied. resultst objective: to assess the effect on extravascular lung water (evlw) of the application of peep and the reduction of vt in an oleic acid pulmonary edema model in pigs, using three ventila~ary strategies. material and methods: twelve adolescent pigs (weighing over kg) were randomly divided in three gmups immediately alter infusing via a central vein . ml/kg of oleic acid to produce a permeability pulmonary edema. the ventilatory parameters for each group were as follows: group i (n= ) : vt: - ml/kg; zeep. group :(n= ) : vt: - ml/kg; peep: cm h . group :(n= ) : vt: - ml/kg; peep: emil . (resulting in permissive hypereapnla) after a four-hour period of ventilation the animals were killed and the lungs excised to calculate gravimetrically the extravascular lung water using a standardized procedure ( hemoglobin content method ). ill evlw (ml/kg) group obiective: in the postoperative period, maintenance of adeguate arterial oxygen tension is a major problem in morbidly obese patients probably because of a large reduction in functional residual capacity (frc). the aim of this study was to evaluate the effects of peep on respiratory mechamcs and gas exchange in this kind of patients. methods: in nine postoperative mechanically ventilated morbidly obese patients (bmi> kg/m ) we partitioned the total respiratory system mechanics into its lung ( ) and chest wall (w) components using the airway occlusion technique associated with the esophageal balloon, during constant flow inflation (jap ; : ) . at three different levels of peep ( , , cmh ) we measured: compliance (cst), airway (rim) and "additional" (dr) resistance, frc and gas exchange. obiectives. to describe the use of prone position in our icu we analyzed the clinical records of all patients admitted in - , selecting adult patients with arf defined as: intubation and pao /fio < mmhg plus an fio > . or peep> cm i . results. patients met the arf criteria: of them ( . %) underwent prone positioning (p+). prone position use began in the early phase of arf ( . • days from the beginning, range - , median ). out of p+ pts were treated with controlled ventilation (cppv or pcv), while were on assisted ventilation (simv+ps) and on spontaneous breathing (cpap). only pts were awake when turned prone, while pts required adjuncts of sedation to tolerate the change of position. the duration of prone positioning was variable (average lenght . • h, range . - h). only minor side effects were observed (eyelids and facial edema, chest and facial pressure bruises). we consider responders (r+) those patients presenting at least . mmhg increase in pao /fio : / patients ( . %.) were responders when first pruned. the pao /fio changes induced by prone position are reported in the figure. pao /fio increased when patients were pruned (*p< . ) and remained higher than baseline values when returning supine(*p< . ). paco remained unchanged. prone positioning was used at least twice in / ( conclusions. this retrospective analysis confirms that prone positioning improves oxtgenation in the majorib' of arf patients. altough we have no available criteria to discriminate in advance r+ from r-pts, we now routinely consider the use of prone position in the treatment of severe arf. palo a, otivei m*, galbusera c, veronesi r, sala gallini g, zanierato m, iotti g, braschi a.servizio anest. e rianim. i, *laboratorio biotecnologie e tecnologie biomediche irccs s. matteo, pavia, italy inhaled no can improve arterial oxygenation and reduce pulmonary hypertension in ards patients; little information is, however, available about the dose-response curves. methods seven ards patients (lis . +. ) submitted to mechanical ventilation randomly received inhaled no doses in increasing or decreasing sequence: . , , , , , and ppm. reference measurements were obtained before and after the entire period of no inhalation. hemodynamic parameters and blood gases were measured after min in each condition. cmv was administered under sedation and paralysis, with constant ventilation, peep (lol-_ cmh ) and fit (. +. ). the changes in vt and fit due to the no ( ppm in n ) injection in the ventilator external circuit were compensated for. results . the dose of . ppm, ineffective on papm, significantly improved oxygenation. the increase of pat and the decrease of q'va/q' and papm were nearly maximal at - ppm. no deterioration of arterial oxygenation was observed at no doses as high as ppm. co exchange was not influenced by no inhalation. systemic hemodynamic variables did not change throughout the study. these results suggest that a concentration around ppm is adequate for obtaining maximum effects on hypoxemia and pulmonary hypertension in patients with ards. low-dose inhaled nitric oxide (no) induces redistribution of pulmonary perfusion in patients with severe ards and causes improvement of oxygenation [ ] . however, addition of exogenous lowdose no in the inspiratory gas mixture might be only a replacement of missing atmospheric no ( - ppb) in hospital central-supplied medical air. [ ] we have realised nitric oxide measurements in ten healthy volunteers, ( smokers and non-smokers) breathing with a mouthpiece and occluded nostrils through a ventilator circuit, with separation of inhaled and exhaled gases by a valve. no concentration was measured with a double-chamber chemiluminometer (environnement sa, france) and with charcoal/silicate purified compressed air. there was no nitric oxide detectable in the inspirat ry limb of the ventilator. unfiltered central supply medical air contained : - ppb of no and - ppb of no , whereas central supplied oxygen was no/no free. samples were taken after equilibration periods of minutes, with increasing fit levels of . , . and . for subsequent minutes periods; paired values were recorded every s. the mean no value was . ppb (sd . ) and n o significant differences were found for different fit levels both in smokers and non-smokers. these data suggest that the no concentration of pulmonary origin in the exhaled air of' healthy volunteers is probably lower than that reported by other authors [ ] and that, previously reported, differences between smokers and non-smokers are not always striking [ ] . we suggest the use of activated charcoal/silicate filters for clinical trials in order to achieve standard conditions. [ objective: to compare efficacy and safety of two doses of salbutamol. methods: sixteen adults who had severe acute a~hma were randomly assigned to receive either rag (n= ) or rag (n= ) of nebulized sulbutamol. both groups were similar with respect to age, duration of a~hma, duration of attack before arrival at the hospital and severity of a~hma according to baseline measurements (table) . evaluation was performed , , and rain after the start of nebulization. results: compared with mg regimen, mg regimen resulted in the same improvement in peak-flow and fischl index (figure). the changes in heart rate, respiratory rate and pace did not differ significantly between both groups. the incidence of side effects, which included tremor, palpitations, cardiac arrythmlas and other symptoms, was not sj~ificanfly different in the two populations. conclusion:the results of this study suggest that nebulization of ng of salbutamol is not more effective than rag in the initial treatment of acute severe asthma in adult patients. the prognostic factors of neutropenic patients admitted to the icu remain poorly known. the aim of this study was to determine the respective weight of underlying malignancy and organ system failures on the outcome of these patients. patients and methods: the charts of neutropenic patients (wbc < /mm and/or pmn < /ram ), admitted to the icu between and , were retrospectively reviewed. the characteristics of the neoplastic disease (h~emopathy or solid tumor, tumoral evolution, duration of cancer disease and of neutropenia), the mac cabe's score, the organ system (respiratory, hemodynamic, renal, neurologic, hepatic) failures and the severity scores (saps, saps ii ,osf) were registred within the st day in the icu. when discharged from the icu, the patients were classified as alive or dead. results: fifty-seven patients ( . %) had a h~ematologic malignancy, and ( . %) a solid tumor. fifty-nine of the patients died ( . %); the mortality rate did not differ between both groups ( . and % respectively, p = . ). with univariate analysis, none of the tumoral features is linked to the prognosis; only the respiratory (p < - ) and cardiovascular (p < - ) failures, and the number of organ system failures (p < - ) are associated to the risk of death. the saps (p < - ) and saps ii scores (p < - ) were higher in patients who died. with multivariate analysis (logistic regression), only the respiratory failure is correlated to the risk of death (p = - ); neither the features of the underlying malignancy (p > . ), nor the duration of neutropenia before admission in icu (p = . ), nor the severity scores figs ii: p = . ) are linked to the outcome. conclusions: the tumoral characteristics do not modify the prognosis after admission to the icu. they should not influence the decision to admit or refuse a cancer patient in the icu. respiratory failure at icu admission has the predominent weight on the risk of death in the icu. patients with respiratory acidosis due to asthma occasionally require levels of mechanical ventilation that place them at risk for barotrauma. a few case reports have described the use of an extra-corporeal membrane oxygenator(ecmo) circuit as an alternative means of co removal. generally, this has been used for short periods of time (< h) without serious complications and with low blood flows through the extra-corporeal circuit. we report a case of refractory asthma who could not tolerate even small-volume breaths from a mechanical ventilator due to severe bilateral airleak. ecmo therapy was initiated at the referring hospital prior to helicoptor transport. high blood flows were used ( % of the patient's cardiac output), sufficient to achieve both co removal and oxygenation. satisfactory gas-exchanged was accomplished (pco = - mmhg) with nearly total lung rest for a prolonged period ( h). however, the long ecmo duration was associated with two severe complica-ti ns: ) bilateral hemothoraces due to anticoagu!ation in the extra-corporeal circuit, and ) prolonged weakness as a result of neuromuscular blockade for six days. the patient was discharged from the hospital in good condition. we present the respiratory and hemodynamic features of this case aw well as the potential complications of ecmo therapy in asthma. objectives: parameters derived from tidal expiratory flow ~e) and volume (vt) can be used to detect airflow obstruction in copd patients who might be unable to perform forced spirometry (e.g., icu). however, indices such as ave/v t and at/re are highly variable (thorax, : ; ) . methods: we investigated whether the standardized for v m effective time (teff~) of a tidal breath, which is derived by asimple mathematical procedure (teff,= j'vdt/vt ), is a more reproducible and sensitive detector of airways obstruction, we studied nine normal subjects ( male, -+ yr) and copd patients ( male, -+ yr) in the seated position, with a noseclip on. they breathed quietly, through a pneumotashograph to measure flow (v). volume was obtained by numerical integration of thellow signal. each subject had an initial - min trial run, in order to become accustomed to the apparatus and procedure. when regular breathing had been achieved, all breaths over a min time interval were recorded. the mean value of six consecutive breaths (ers criteria) for each subject was used for analysis under the condition that within session variation of tidal volume (vt) was < %. lung function tests were: in normals (mean-sd), fevl%pred = • fevl/fvc%= -+ % , and in copd patients, fev~%pred= __. and fevi/fvc%= --. %. results: values are shown as mean-..+-sd in the following a su~ve~ os literature sources p~oves that t~aditlona], i.e. medicinal medication and physiothe~apeutic methods os t~eatment often p~ove to be insufficientl~ effective both currently and in the ~emote future. the goal of this study was to investigate the efficacy os t~eatment of b~onchial asti~ma patients by means os speleo-and artificial sp~ay therapy. speleotherapy t~eatment was conducted in the conditions os mic~oclimate os salt mine in solotvino hospital. a~tis sp~ay the-~apy was conducted by means os a self-made device. ou~ method is based on the p~inci-~ le os using the majo~ facto~ of speleo-he~apy -highly dispe~sed sp~ay s sodium chloride. the obtained ~esults ~e~e analyzed in five g~adations. at the end os the speleothe~apy improvement and considerable improvement was observed in , ~ os patients; inconsiderable improvement -in , ~ os patients. having evaluated the e~s os t~eatment using a~tis sp~ay therapy the indices a~e , h and , ~ ~espectively. remote ~esults of t~eatment a~e an important index os t~eatment, the ~esult os ~hich ~e~e studied by means s a ~uestionnaive-method. patients ~ho had been t~eated by speleothe~apy mo~e f~eguently ~e-po~ted a ~elapse in disease ust afte~ the course o~ t~eatment ( , h). ho~eve~, in a ]ate~ phase the ~emission ~ould last ]on-~e~ (s months in , ~ os patients, till one yea~ in ~ ~). in , ~ os patients who passed the co~se os a~tificial sp~ay therapy a ~elapse was ~egiste~ed immediately as the co~se os t~eatment. then thei~ condition stabilized ~hile in , ~ os patients a period os ~emission lasted s ha]s a yea~. , ~ of patients dida't ~epo~t a ~elapse of the disease du~in~ one yea~. evangelismos hospital, critical care department, athens, greece method#: mechanically ventilated patients ( copd, ards, other pulmonary diseases) were studied in two phases: ) during the acute phase of respiratory failure; ) during recovery - days later. we measured mip and monitored the pattern of breathing while the patients were breathing spontaneously through the respirator (pressure support mode with - cmh ) until either the point they were unable to sustain spontaneous breathing (sb) any longer (phase ) or for two hours when they could sustain sb indefinitely (phase ). subsequently the patients were sedated, paralyzed and mechanically ventilated. then we simulated the pattern of sb at the end of the sb trial by manipulating the variables of the ventilator and assessed respiratory mechanics b y the end-inspiratory and end-expiratory occlusion technique. . during recovery, a combination of reduced inspiratory load and increased venfilatory capability makes a patient previously unable to sustain sb to breathe spontaneously. . inspiratory load is reduced during recovery, mainly because both intrinsic peep and breathing frequency are diminished. obiectives: although elevated concentrations of a few cytokines have been shown to be present in the bronchoalveolar lavage (bal) fluid (balf) of patients with the adult (acute) respiratory distress syndrome (ards), the pethogenesis of ards is largely unknown. leukemia inhibitory factor (lif), a growth factor recently recognised as a polyfunctional cytokine integrated in cytokine networks was measured in unconcentrated balf of patients from different patient groups. methods: lif was measured in balf by means of a specific and sensitive elisa (detection limit pg/ml)in balf (lavage of x ml in the right middle lobe). results: lif was not detected in the balf of healthy control patients and in only one ( pg/ml) out of patients at risk for ards (after cadiopulmonary bypass surgery) who underwent bal h after the end of the extracorporeal circulation. high and detectable levels were found in the unconcentrated balf of out of patients with full-blown ards ( + , mean + sem, range - pg/ml). there was a good correlation between the level of lif in the balf and a number of markers of inflammation: neutrophils/ml (r: . , p= . ), albumin ( r: . , p= . ) and protein level (r: . , p= . ). conclusions:the biological role of lif in these balfs is not readily explained by its currently known actions and it is unkwon whether lif contributes to or is a response to local tissue damage. our results indicate that this cytokine with lots of interesting _functions is a pert of the inflammatory cytokine cascade in ards. background and obiective : we recently demonstrated that cisapride -a new prokinetic drug -enhanced enteral feeding in a heter genoas group of ventilated icu patients by significantly accelerating their gastric clearance (crit care meal, ; : - ) . it remains unknown, however, whether certain subgroups of patients might benefit more from adding cisapfide to their enteral nutrition regimen than others. patients with chronic obstructive pulmonary disease (copd) might represent such a subgroup since their illness and its specific treatment put them at risk for gastric emptying disorders. design and setting : prospective, consecutive sample study in an adult medical intensive care unit in a university hospital. patients : mechanically ventilated and hemodynamically stable copd patients. interventions : gastric emptying was evaluated by bedside scintigraphy and expressed as the time at which % of a tcg~-labelled test meal was eliminated from the stomach (t / ). baseline data (do) were recorded after enteral nutrition reached to ml daily. scintigraphic measurements were repeated days after cisapride ( ml orally, q.i.d) had been added to this regimen (d ). patients were considered cisapride responders when gastric clearance improved by more than % from baseline. results : normal values for the test meal and for scintigraphic acquisitions obtained in the supine position were found to be + min. in healthy volunteers (crit care med, ; : - ) . five patients responded to cisapride (t / : + rain vs. + min at do and d , respectively) and five did not (t / : + min vs. _+ rain at do and d , respectively). in contrast with non-responders, all five responders had clinically significant maldigestion at baseline (excessive (> ml) gastric residues, vomiting (> times/day and abdominal distension) which disappeared in of them after the administration of cisapride. conclusion : copd patients who tolerate enteral nutrition well have basal gastric emptying times which are comparable with those of healthy volunteers and are not influenced by cisapride. however, cisapride treatment provides both scintigraphic and clinical improvement in those copd patients who exhibit clinically obvious gastric emptying disorders. cernv v., dostal p., zivny p., zabka l. dept. of anesth. and critical care, charles university, faculty hospital, i-irade~ kralove , czech republic objective: the aim of the study was to evaluate the effect of early entera nutrition started within hours of injury on the incidence of multiple orgar failure (mof) in trauma patients requiring vantilatory support. methods: after institutional approval patients were enrolled in the study enteral feeding was begun within hours of injury in trauma patients (en group) admitted to icu. nasuenteric tube was placed as soon as possible after admission into the distal duodenum under endoscopy. additional parenteral nutrition was used to meet patients energy and protein requirements. the control group (pn) consisted of patients fed during this period paretuerally. severity score apache ii, trauma score, cumulative balance of nitrogen (g), incidence of mof (three and more organs) and length of ventilatury support (days) were calculated. values are expressed as mean + sd. results: tab introduction : parenteral nutrition (pn) is an important aspect in the optimal treatment of patients on gastroenterology or intensive care. the aim of this bi-center study in patients has been to assess tolerence and efficacy of a new protein-lipid mixture for pn from a simple preparation. patients and m~hods : patients were selected in two hospitals (tenon and saint-lazare, paris) and were divided into two groups : group a (gastroenterology~ l short bowel syndrome) and group b (intensive care, surgical patients). all patients likely to require pig for a period of days (group a) or days (group b) were studied. the pn regimens administered were the following : combination with g of mct/lct fat emulsion end , g of nitrogen, in liter end glucose requirements were met by imfizsion of l liter of glucose - % via a "y " connection. lipid thus provided % of the non introgen calories. total daily calorie intake was to ] kced. this study monitored, before and at the end of infusions, the sennn albumin (alb), preaiburtun (prealb), triglycendes (tg), cholesterol (cs), and the serum ammotransferases (sgot and sgpt) end alkaline phosphatase (alp) activities. statistical significances were calculated using the wilcoxon-tost. introduction: many cu patients present a catabolic illness in response to inflammation and infection, characterized by a rapid loss in skeletal-muscle mass despite optimal nutritional support. growth hormone (gh) is responsible for a rise of lipolysis, enhancing the energetic balance, and of protein synthesis. recombinant human gh (rhgh) is nowaday available for clinical use, but its cost is very high. therefore, rhgh should only be prescribed to icu patients when its efficacy can reasonably be anticipated (ie. when the patients are catabolic or stressed, but in order to avoid overprescription for unstressed patients and for those who are overly catabolic). hence, we, as others, recently demonstrated that rhgh had no favorable effect in highly stressed icu patients. objective: to detect on a clinical basis, low (ls), mild (ms) and severe stress (ss) states in icu patients and validate this clinical judgement by objective metabolic mesurements, in order to select early those icu patients potentially able to benefit from rhgh therapy. methods: consecutive icu patients were prospectively stratified as ls, ms and ss by two experienced icu senior consultants (temperature; agitation; heart rate; arterial blood pressure; presence of an infection; respiratory rate; exogenous catecholamines). anabolic (insulin, igf- , gh) and catabolic (cortisol, ghicagon) hormones, and nitrogen balance were determined for each patient within hours after admission in the icu. metabolic and clinical data were then compared. the clinical stress states determined by icu physicians correlate with an objective metabolic assessment. therefore, the patients who will more likely benefit from adjuvant rhgh therapy can be detected simply and early. a prospective study on rhgh therapy in ms icu patients is in progress. berger mm md , chiolero r md , pannatier a phd , berger l , cayeux c , voirol p , hurni m md . surgical icu, pharmacy, and cardiac surgery, chu vaudois, ch-iotl lausanne, switzerland objective. nutrition of the compromised cardiac surgical patient is challenging. numerous factors influence the gastrointestinal (gi) absorption function, among which gut perfusion, which depends largely on the systemic hemodynamic status. patients in hemodynamic failure are prone to organ failure, and may benefit from an early jejunal feeding. the study was designed to assess the absorption function after cardiac surgery in patients with adequate and altered hemodynamic status, using paracetamol as tracer of gi absorption. methods. after cardiac surgery, patients, aged _+ years (mean_+sd) were assigned to groups (anaesthesia: fentanyl gg/kg + midazolam): group (n= ): reference group, with normal hemodynamic status, easy recovery. group ('n= ): patients in low output syndrome, cardiac index < . i/m on day (d ) after surgery, requiring prolonged intensive care, mechanical ventilation + nutritional support. paracetamol g, was given intragastrically on d + d : plasma levels measured (h.p.l.c), at administration (to), t - - - - - and rain. hemodynamic status assessed with pulmonary artery catheter. healthy subjects served as controls. results. compared to healthy controls, absorption was strongly reduced on d in all patients (no difference between groups). on d , peak paracetamol level was significantly lower in group (low cardiac output): in group the area under the curve on d and d were similar. there was a large inter-patient variability, reflecting the hemodynamic status. conclusion. gi absorption was decreased on d in all patients, and reverted to normal between d and d in case of normal cardiac function, but not in case of low output syndrome. the decrease on d can be attributed to fentanyl, known to slow down the gi transit. in patients with cardiac failure, correction of altered absorption was correlated with the hemodynamic status, suggesting that gi absorption is dependent on adequate splanchnic perfusion. the aim of the work was to define specific significance and evaluate efficiency of enteral component of infusion therapy in the intensive care of gastroenterotogic patients of surgical profile with pyo-septic complecations. there were used the methods of radial diagnostics and polyelectrography; the laboratory control on oxygen-transporting function, volumetric and hemodynamic state, changes in metabolic, hormonal and immunologic status was conducted. from january, [ till november, there was carried out the randomized study of patients with general purulent peritonitis; among them persons constituted the control group and -the main one. in the main g~oup the intestinal lavage, enterosorption, enteral introduction of nutrient solutions with gradual turn to enteral nutrition by equalized mixture "ovolaet" were started from the first hours after operation. the data obtained allowed to define the specifity of the program of artificial medical nutrition in the group of examined patients, based on necessity of individual selection of media for enteral introduction depending on the stages of intestinal insufficiency syndrome. it was shown that inclusion of enteral component into the program of infusion therapy during early periods stabilized circulation in the regime of moderate hyperdynamia, considerably decreases the deficiency of circulating blood volume, normalizes the values of oxygen transport, consumption an}d extraction, provides the optimal level of mycardial adaptive possibilities without tension of its compensatory functions and pulmonary circulation overload. due to combined application of parenteral and enteral nutrition the metabolic processes are shifted towards anabolism. this is supported by decrease to normal values in the contents of blood aggresive hormones (acth,hydrocortisone) and increase in somatotrophic hormone. the complete parenteral-andenteral nutrition influences positively on restoration of cellular and tumoral immunity, activates the factors of organism nonspecific protection and recovery from immunodepression, prevents the development of immunodeficiency. impact tm vs control. s atkinson, n maynard, r grover, e sieffert, r mason, m smithies, d bihari departments of surgery and intensive care, guy's hospital, london, u.k objectives: comparison of the effect of an immunonutrient enteral feed versus a control on the outcome of a mixed intensive care unit (icu) population. methods: admissions to this multidisciplinary adu)t icu thought likely to stay more than three days and with tube access to the gi tract ~r randomised to receive either impact tm, a feed with supplemental arginine, dietary nucleotides and omega- fatty acids, or an isocaloric and isonitrogenous control feed. study end points included mortality and icu stay. approval was obtained from the hospital ethics committee. rosults: patients were entered into the trial. the two groups were well matched for age, sex, and admission apache ii with an overall mean admission risk of death of . (std. dev. -+ . ). on an intention to treat basis, there was a no significant difference in icu mortality, icu stay or standardised mortality ratio (s.m.r.) between the two groups (see table) . similarly, there were no differences after stratification for patients receiving or more litres of feed. conclusion: there is no evidence of an effect of impact@, an enteral immunonutrient feed, on pre-determined end-points (icu mortality, icu stay or standardised mortality ratio) in a mixed intensive care unit population over that of an isocaloric, isonitrogenous control feed. objeeflves: evaluate changes of blood laatate levels according to patient medical status after cvvhd initj,~ion using dialysate solution containing lactate. method: review of medioal records of consecutive patients ~eated by cvvhd (dialysate solution hmnosol lg , hospal,uk, lactate concentration retool/l). date obtained hr before and - hrs at~er cvvhd initiation were analysed. results: all data are presented as mean + sem. in one patient, pre end post filter lactate levds were measured during standard cvvhd setting (blood flow ml/mlu, dialysate solution flow i /hr), and approximate daily lactate flux into the patient was calculated to be as high as mmol/d. lactate leveh measured after cvvhd initiation increased significenfly compared to baseline levels ( . + . axtd . + . ,respectively; p< . ,paired t-test). when patiente with increased basal lactete (~- ) were compared to paliente with normal basal values (n= ), no difference in laotete increase was fmmd (p= . , manova). patiente with severe liver dysfunction ( points in mop scomlg, n= ) had higher basal laotate levels than patiente with normal or slightly abnormal liver teste ( or point in mof scoring, n=ll), rite values being . + . and . + . , respectively (p< . , student t-test). increase in blood lactate did not differ between these two groups after cvvhd was stetted (p= . , manova). in pafiente with invasive hemedynamio mo~, no oorrelation batween changes in lactate levels and eitlm" changes in oxygen ddivery (t =o.ol; p--o. ) or oxygen consumption (reversed fie, k) (r -q).o ;p-- . ) were found after cvvhd initiation. conclusion: blood lactate increases on cvvhd with dialysate soh~on rich in lactate. this increase is predominantly caused by influx of lactate into the blood via the filter end does not seem to depend on the liver fimotion and/or oxygen metabolism changes. objectives: the study was designed in order to determine the effect on plasmatic proteins, of two types of aminoacids solutions of parenteral nutrition (pn) adapted to stress, having different concentration of branched chain aminoacids (bcaa), when applying to politraumatized critical patients. methods: a prospective study was performed using a randomized double blind design of polytraumafized patients, split in two groups of ten patients each, with mean ages of _+ an -+ years. due to their condition, all patients required p.n. for at least days. both groups were subjected to isocalorie and isonitrogenous solutions ( ci/kg/ day and . g of nitrogen/ks/day), varying only in the concentration of bcaa; solution a having a % concentration and solution b %. blood samples determinations during days , , , after the beginning of treatment with p.n. were total proteins., albumin, trandferrine, protein binding retinol; prealbumine and fibronectine. the anova test (one and two way) was used to compare the values between the two groups. results: the administration of solution a, showed statistically significant increases in the determinations of the values of protein binding retino] (p < . ) and prealbumin (p < . ). no significant increases were observed in the values of total protein, albumin, transferrine and fibronectin. solution b produced statistically significant increases only in the values of total proteins (p < . ). the remaining proteins did not changed from their control values during the whole period of pn administration. comparing both groups, no statistically significant differences were observed related to the type of diet. nevertheless, differences were found in total proteins, albumin, protein binding retinoi, fibronectin (p< . ) and prealbumin (p < . ) in relation to the time course of pn therapy. only the albumin values showed significant differences (p < . ) when considering the interaction of both the type of diet and the time course of pn. conclusions: . solutions of pn adapted to stress, can maintain the control values of slow turnover proteins and improve the values of rapid turnover proteins. . no significant differences on plasma proteins were found between the two solutions having % or % concentration of branched chain aminoaeids. &determination of rapid turnover proteins does not seems useful for discriminating different solutions of bcaa during pn. obiectives; the hormonal changes in the post-traumatic situation often leads to an elevated blood glucose and a negative nitrogen balance. to reduce the elevated glucose production by aminoacids the apprication of xylitol may be an alternative energy source. in a double-blind randomized study we investigated the effects of a xylitol/glucose solution (group a: aminoacids g/i; glucose/xylito g/ g/l) on metabolism and particularly on pancreatic and liver enzymes compared to a glucose based nutrition solution regimen (group b: aminoacids g/i; glucose g/i). methods: the clinical trial was carried out after the approval by the local ethical committee on patients with severe brain injury. there was no difference in body mass index bmi (group a: . +/- . kg/m and group b: . +/- . kg/m=), age, and sex. daily individual energy expenditure was measured by indirect calorimetry (deltetrac "~). nutrition was started - hours after trauma or surgery with carbohydrates and aminoacids. fat was added h after nutrition had started. to analyze the effects on pancreatic and liver enzymes we investigated the following parameters for days: blood gtucose, serum lipase, serum amylase, asat, alat, ~gt, ap, and serum cholinesterase (che). results: due to the daily indirect calorimetric measurements energy requirements were satisfied. there was no difference in blood glucose concentration and cumulative nitrogen balance between the two groups. neither were there any significant changes in asat, alat, ap, and che for days in both groups. serum tipase steadily rose to lull in group a and . lull in group b, respectively. conclusions: there was no measurable influence of either nutrition solution on liver enzymes. the xylitol/glucose nutrition regimen does not have any advantage over the glucose based nutrition solution concerning blood glucose level or nitrogen balance. the elevation of serum lipase to a -fold level in either group needs further investigation on trauma patients. the effects of fat emulsions in lung function, particularly in lungdamaged patients, have been attributed to alterations in pulmonary vascular tone caused by eicosanoid production modificatione. as the eicosanoid production may depend on the fatty acid profiles of the intravenous fat emulsion, haemodynamic, pulmonary gas exchange and plasma levels of prostanoids were investigated in acute respiratory distress syndrome (ards) patients, during different intravenous lipid emulsions (providing different prostanoid precursors). we studied in a randomized double-blind design groups (n= each) with ards. group i (lct) received a fat emulsion with long chain triglycerids (lct- %), group ii (mct) an emulsion containing a mixture of medium and long chain triglycerids (mct/lct / - %) and group iii placebo (control), during h ( mg/kg/min each). we measured before, at the end of h infusion, and h after the end of the infusion: lipaemia, arterial and venous blood gases, pulmonary and systemic haemodynamics, and plasmatic levels (arterial and in mixed venous sample) of eicosanoids (txb=, -keto pgf~,, and ltb ). at the end of the fat emulsion, groups (i and il) to , • to , • mmol/i), the paoz/fio z remained unchanged in the three groups; no changes in intrapulmonary shunt (qs/qt) were shown; neither in the mean pulmonary artery pressure. in contrast, only in the lct group: cardiac output and oxygen consumption increased significantly ( . % and %) (p< . ). eicosanoids were increased at baseline compared to reference values (p< , ). a decrease (p<o, l in the arterio-venous difference of the vasodilatador prostanoid ( -keto pgf~=) was shown in lct group. our results indicate that fat emulsions in ards patient do not influence pulmonary gas exchange, provided that the perfusion rate were keept at mg/kg/min. the present study was designed to evaluate the metabolic changes of a carbohydrate-based diet versus a fat-based diet on oxidation rate of substrates and energy muscle metabolism in patients with an acute exacerbation of chronic obstructive lung disease. patients were mechanically ventilated with a volume-cycled respirator. after an overnight fast, patients were randomnly treated with a continuous enteral feeding over a nasogastrie tube with a carbohydrate/fat ratio of / in group i (n= ) , and a carbohydrate/fat ratio of / in group i] (n = ) during consecutive days. indirect calorimetry was set at baseline and hours later the enteral feeding was started. muscular quadriceps biopsy was performed on baseline and on day of enteral feeding. urine was collected during hours and was used to measure h urea nitrogen production. the caloric intake was set according to the measured resting energy expenditure. respiratory gas exchange rate were measured using a computerized open-circuit indirect calorimeter. quadficeps femoral muscle samples were obtained by muscular biopsy after local anesthesia. analyses of glycogen, glucose -phosphate, lactate, phosphocreatine and adenosine triphosphate (atp) and enzymes (glycogen synthase and glycogen phosphorylase) were determined. after h of nutrition patients in group i showed an increase in carbohydrate oxidation (from . % to . %, p= . ), and in group ii a decrease in protein oxidation (from % to %, p= . ). after days of enteral feeding a tendency to increase in glycogen concentration (group i, p= . ; and group ii,p= . ) was observed. while patients in group i showed a tendency (p= . ) to decrease glycogen phosphorylase, patients in group ii maintained these enzymatic levels. the increased in glycogen was correlated in group li with degradation enzyme (p = . ). in group i, the pao /fio ratio increased after days of treatment (from . to . ; p= . ), but no changes in days of mechanically ventilation, length of stay in icu, or mortality rate was evidenced between groups. it is concluded that the oxidation rate of substrates used for energy production varies according to the caloric sources administered. with nutritional therapy muscle glycogen concentration increases in both diets, with different enzymatic influence. more studies are warranted to further assess the clinical implications of the different pathophysiologic behavior of the two diets. under certain experimental and human conditions (radiation and/or thermal injury) the gi mucosa is unable to perform this protective function and could play an important role in the pathophysiology of the syndrome of multiple organ failure (smof). the objective of the present multicenter study was to know the gi mucosal permeability in critically ill patients and the relationship with their outcome. the method used to assess the integrity of the gi mucosal barrier measured mucosal permeability to various hydrophilic compounds. specifically, we measured the urinary excretion ratio (uer) of two sugars (lactulose and mannitol) that had been previously administered through a nasogastric tube. urinary excretion rate was measured in healthy humans (control) and in patients admitted to the intensive care unit (icu) at days , and . the apache ii of the patients studied was _+ . there was an increase of uer in the critically ill patients compared with controls (o. _+ . vs . _+ . ) (p< . ). in contrast, no changes in uer between days , and were observed ( . -+ . . significant changes were also established in the different groups: major and minor surgery, general and epidural anaesthesia , transfused and non transfused patients except for iron and ferritin plasma levels which didn't change significantly in patients who had received blood. a positive significant correlation was established between crp and ferritin (r= . ) for patients under general anaesthesia, but not on epidural, regardless of the type of surgery. conclusions: iron and transferrin plasma levels fell acutely in surgical patients while ferritin and crp rose. transfusion implied an acute iron load.the established correlation between cpr and ferritin in patients on general anaesthesia suggests that ferritin behave as an acute phase reactant in that setting, while epidural anaesthesia may ameliorate the inflammatory response. objectives: to analize the effect of gastrointestinal complicacions (gic) related to the enteral nutrition (en) in the real volume of diet administered to icu adult patients. a prospective multicenter study was designed (comgine study) in wich en application, gic definition and his management were defined by collaborative consensus.patients treated with en in one month pedod were included. in each case, daily volume of diet prescribed (pv) and administered (av) was recorded and the volume ratio calculated (vr=pvxl /av). patients were divided for days of en according to the presence (group ) or absence (group ) of gic (abdominal distension, increase of gastdc residuals,vomits or regurgitation, diarrhea,constipation and bronchoaspiration). differences between groups were analized by anova and mann-whitney u test with p< . for statistical significance. (vr%) group (vr%) tolerance to the enteral nutrition (en) has been related to the severity of illness in icu patients. the aim of this study was to evaluate this relationship. methods. a prospective, multicenter, study was performed (comgine study) in wich en application and related gastrointestinal complications (gic) were defined by collaborative consensus. adult icu patients treated with en in one month pedod were included. gic were classified as: ) abdominal distension (abd), ) increase of gastdc residuals (igr), ) vomits (vom), ) diet regurgitation (reg), ) diarrhea (dia) and ) constipation (con). patients were divided in two groups: group a: patients with gic dudng their evolution. group b: patients without gic. differences between groups were analized for apache ii admission score or evolutive variables ( carbon dioxide production is a major determent of work of breathing. increase in co production during nutritional support may precipitate ventilatory failure and difficulty in weaning from mechanical ventilation. in the present study, co output was calculated while passive mechanically ventilated patient were fed with different amount of calories and different proportion of protein, carbohydrate and fat. four clinical stable and mechanically ventilated patients were studied. carbon dioxide was calculated while patients were paralyzed, sedated and mechanically ventilated. six nutritional regimens were used: a) no calories, b) t gr glucose/ h, c) calories equal to rest energy expenditure in special formula with low carbohydrates and high fat (pulmocare) d) calories equal to rest energy expenditure in standard formula (nutdson) e) calories double to rest energy expenditure in standard formula (nutrison) and f) calories equal to rest energy expenditure parenterel (tpn). carbon dioxide output was low while no calories and/or only gr/ h glucose was provided, the mean values of vco output were _+_ and !-_ ml/min respectively. there was no difference in vco output between with low carbohydrates and high fat regimen and standard regimen, mean values were t . -+ and _+ respectively.the high calories regimen and tpn resulted in higher vco output, mean values _+ and • respectively. we conclude that the special with low carbohydrates and high fat regimen did not reduce vco output, in the contrary, high calories intake as well as tpn increased vco output under tested conditions. klouche k., cristol j.p., vela c., canaud b., b raud j.j. m tabolic intensive care unit and nephrology department. lapeyronie hospital. montpeuier france. predictive factors for rhabdomyolysis (rh) -induced acute renal failure (arf) were studied in a retrospective study from january to january . patients (pts) (male: , female:lo; mean age: + y.o.) were admitted with rh defined as cpk> iu/ . etiologies were: traumatic and ischaemic , infectious , toxic , excess activity . factors studied were: simplified acute physiologic score (saps: . + . ), organ systemic failure (osf: . _-!- . ), diagnosis delay (d: +_ h), clinical parameters (sepsis, dehydration), blood chemistry data (cpk, bun, creatinine, potassium, phosphorus, calcium, proteins, hematocrit) and urinary ph. severity of rh was estimated by ward score determined according to phosphorus, albumin, potassium, cpk, dehydration and sepsis. urea appearance rate (uar) and creatinine index (ci*) were determined over a hours period. arf was observed in pts. in non-arf and arf groups respectively, saps ( . _+ . vs . + . ), deshydratation ( vs ), sepsis ( vs ), phosphorus ( . + . vs . -+ . ), calcium ( . + . vs . _+ . ), ward score ( _+ . vs . + . ) were significantly different. however, no significance was observed in uar ( -+ vs -+ ) and ci ( _+ vs _+ ). patients required hemodialysis (hd) ( : sessions) and remained dialysis free. only osf ( . _+ . vs . -+ . ), ward score ( . _-/- . vs . _+ . ) and ci ( +_ vs -+ ) appeared significantly higher in pts requiring hd. pts died from associated disease. all patients suffering from arf recovered a normal renal function. we confwmed that an elevated ward score (over ) is a good predictive index of arf. in addition we found that ci is a severity factor for arf requiring hd. thus, patients suffering for rh with elevated ward score and ci, have a fair chance of dialysis and should be treated more intensively. * ci (expressed in mg/kg) = (car + feces creatinine) / weight. where car: creatinine appearance rate; feces cr~t..= mean plasmatic creatinine x . . tr~er k., cetin t.e., tugtekin i., georgieff m., ensinger h. universit~tsklinik flir an~sthesiologie, uim, germany introduction: endogenous as well as exogenous adrenergic agonists have a profound effect on carbohydrate metabolism in human critical illness. in this study the effects of noradrenaline (nor) and dobutamine (dob) on carbohydrate metabolism during a hr infusion were investigated. methods: after approval by the local ethic committee healthy volunteers were studied. hepatic glucose production (hgp [mg/kg/min]), using , -d glucose as stable isotope tracer, as well as plasma concentrations of glucose (glc [mmol/i]) and lactate (lac [mmol/i]) were measured prior and during infusion of nor ( . pg/kg/min) and dob ( pg/kg/min). blood samples were drawn before and during the agonist infusion. results: no major changes in insulin and gtucagon plasma concentrations could be found during the study period. ::i:::: :iiiii~ ~ i ::i: ~:: : :: i:ii. mean-+sd are shown. # p< . , anova for repeated measurments. conclusions: the effect of nor on hgp and glc were smaller as compared to adrenaline (i) with a similar time course. in contrast to the effects of adrenaline and nor, dob had a different effect on carbohydrate metabolism: a decrease in hcp and glc, which is uncommon for a / -adrenoceptor agonist. since hgp is an energy consuming process that might deteriorate hepatic oxygen balance in critical illness, the differential effects of adrenergic agonists may be of importance and need further clarification. the nutritional insufficiency often accompanies post-operative hypercaloric states, inanition, serious infections and weakening chronic illnesses. that is why the early nutritional support, sufficient and appropriate for each individual base, is a fundamental component of intensive care unit as an indispensable factor for recovery. per this reason, our unit, developed a software for the implementation and nutritional control of t~e assisted patients. this software is incorporated is an expert system called ~i~su, designed and developed by the computational division of our unit. this system arrives to inferred diagnoses such as : respiratory, hepatic, renal(with and without dialysis) dysfunctions, pancreatitis, ards, decrease of consciousness, diabetes. according to these data objectives: to compare the effect of short term enteral feeding versus parenteral nutrition, when a isonitrogenous and isocaloric feeding solution is administered by either mute. methods: in a prospective controlled clinical trial patients were studied; all exhibited moderate degree of malnutrition, normal liver and kidneys, and a functi ning gastrointestinal tract. the patients were randomized to receive a free amino acid and small peptide diet ( patients) or an isonitrogenous isocaloric parenteral support (tpn) ( patients) (total energy: kcal, nitrogen: . g, carbohydrates: g, fat: g, n/non protein calories: / ) at least for days. results: there were no significant changes in anthropometric parameters within either group. nitrogen equilibrium was aqhieved by day in the tpn group and by day in the enteral group ( . % of the enterally fed patients and % of the tpn patients maintained in positive balance the day of the study). there were no significant changes in serum albumin within either group. serum level of transferrin reached a significant increase in both groups (p= . ). thyroxine-binding prealbnmin rose significantly in both groups as well (p= . and . respectively). statistically significant rises in lymphocyte counts (p= . and . respectively), in levels of c (p= . and . ) respectively), iga (p= . ), igg (p= . and . respectively) and igm (p= . ) occurred in either treatment group. there was a high incidence of negative skin tests at the start of the study in the enteral group ( . %) and the tpn group ( %). by the end of the study the incidence of negative responsiveness was . % and . % respectively. despite maintenance of similar glucose levels in both groups, tpn led to significantly higher serum insulin levels. the serum insulin increased almost linearly over the study period and eventually prevented fat mobilization and lipolysis, so that free fatty acid levels had fallen significantly. a significant elevation of the liver enzymes over the study period occurred in . % of the tpn group, but not in the enterany fed patients. conclusions: the present findings provide no evidence that enteral diets containing free amino acids and small peptides, as their nitrogen sources, are in any way inferior to isonitrogenous isoealoric regimes parenterally given. aim: the aim of this study is to describe and explore the expectations of the functions of the critical care nurse to enable the formulation of guidelines for the scope of practice for the critical care nurse with a south african context, methods: phase i was to determine the expectations of the critical care nurse, the nursing service managers and the doctors with regard to the functions of the critical care nurse. a focus group interview was held with a group of experts in the field of critical care. the results were used to compile a questionnaire. this questionnaire was sent to the critical care nurses, the nursing service managers and the doctors in south africa for completion. from these results the functions of the critical care nurse were determined. phase ii was to formulate guidelines for the scope of practice for the critical care nurse within a south african context. through usage of the date (phase i) the scope of practice was formulated. guidelines were formulated for the practise, education and research regarding the limitations of the professional-ethical authoration and the implementation of the scope of practice for the critical care nurse. objectives : high output gastric aspirates arc occasionally observed during fasting in critically ill paticnts, preventing any attempt of feeding via the enteral route. although these patients are often said to suffer from "gastroparesia", the motor correlates of this condition arc lurgcly unknown. in this stud?', wc recorded the gastrointestinal motility of critically ill patients with abundant (> ml/ hours) fasting gastric aspirates. methods : antral ( sites separated each other from . cm), duodenal ( site) and jejunal ( site) contractions were recorded simultaneously by ~eans of a multihimen tube assembly positioned trader fluoroscopic control (perfused catheter technique). tracings from prolonged recordings were obtained on a multichannel recorder ( a recorder, hewlett-packard) then anal) ,ed visually, with a special attention for the following abnormalities which are characteristic of intcstinal pseudoobstmctiou: l) absence or aberrant propagation of the migrating motor complex (mmc), ) presence of bursts (> min) of nonpropagated phasic pressure and ) presence of sustained (> min) uncnardinate pressure activity. patients with a volume of gastric aspirates of • (sd) [median ml/ hrs were investigated for - [median minutes. results : only one patient had no detectable motor abnormality. mmcs were either absent (n= ) or migrated abnormally (retrograde propagation : n= ; retrograde and stationnary : n= ) in pts. bursts of nonpropagated phasic pressure activity were present in the duodenum in pts and sustained uncoordinate pressure activity was found in pts. additional abnormalities included episodes of prominent pyloric activity. (n=l) and sustained antral pressure activity (n= }. conclusion : critically ill patients with large volume of gastric aspirates have manometric evidence of intestinal pseudoobstruction. prokinetic therapy in these patients should thus focus not only on enhancing gastric motility, but also on restoring a normal propagative contractile activity in the intestine. this prospective, open-label, randomized placebo-controlled study included patients with hypokalemia in whom rapid potassium replacement ( meq kci in h) was performed: patients received mg sulfate ( g in hours) and patients received a corresponding saline infusion. measurements were made at time , + , + and + hours results: k levels increased more in mg treated patients than in the patients who received saline infusion at time and h (p < . -students-newman-keuls). (table ). introduction. dual lumen uaso-gastrojcjunal tubes are a major ads'ance in nutritional therapy of mechanically ventilated critically ill patients since the " authorizc jejunal feeding with concurrent gastric decompression, there,, reducing the risk for aspiration. unfortunately, placcmem of these tubes in the jejunum regularly dictates to resort to endoscopy in order to facilitate pyloric intubation. recently, the remarkable gastrokinetic properties of the well known macrolide antibiotic er}lhromycin have been demonstrated in gastroparetic critically ill patients . aim. in the presem stu~,, we evaluated the feasibility of placing dual lumen naso-gastrojcjunal feeding tubes at the bedside without endoscopy, using edthromycin to help iranspy'loric migration of the tube under fluoroscopic control. methnd each patient admitted in our icu during a months period and requiring artificial ventilation and enteral nutrition for a period of at least days was included in the study.. after inserting the tube (stayput| sandoz, usa) in the gastric anmnn, e.rythromycin ( rag) was aduunistored intravenously, to help fluoroscopic positioning of the tube into the jejunum. the total duration of the procedure (from nasal intabatiun to jejunal placement), as well as the duration of ftuoroscopy were recorded in each patient. results. patients (male/female : / : mean age : . + . years; mean apacbell score : .t • . ) wore enrolled into the study.the procedure was performed within the dab,s following institution of mechanical ventilation. jejunal access was obtained in all patients without resort to enduscopy in , • . min.(total duration of the procedure). mean duration of fluoroscopy was . + . rain. conclusion. we conclude that placement of dual lmnen naso-gastrojejunal tubes can be obtained in mechanically ventilated critically ill patients without resort to endoscopy., provided that e rythromycin is used as gastrokinetic agent to help pyloric intubation. the following ad and dis parameters were considered in all patients: -mid arm circumference, triceps skinfold thickness, serum transferrin, albumine and lymphoeites and urinary creatinine/height index. patients whose results were bellow % of normal values in or more of the above criteria were considered undernourished (und).statistical analysis was performed using % analysis.statistical significance was established at p<o.o . results: a total of patients( female, male) -with a mortality rate (~) of , % -were studied, aged -+ years and a median ]enght of stay of days. -nourished (nou) at ad and at dis - ; ~ %; -nou at ad and und at dis - ; ) , % ; -und at ad and und at dis - ; ~ . %; -und at ad and nou at dis - ; ~ , %. we observed a p< . when we compared groups and (p= . ), and groups and (p= . ). variation in nutritional status and longht of stay: -nou at ad and nou at dis = > median lenght of stay days; und at ad and und at dis = > median lengbt of stay days; nutritional status and age at admission: -age > = years : nou ( ) , und ( ) -age < years: nou ( ), und ( ) nutritional status and age at discharge: -age > = years : nou ( ) , und ( ) -age < years: nou ( ), und ( ) we observed a p<o. when we compared groups and (p=o. ) and groups and (p = . ) conclusion: such study permits to conclude that most of our patients ( . %), staying in icu for more than days, were und, namely the elder. we observed that this kind ef patient had a longer median length of stay in icu, associated with higher mortality rate. in conclusion, the nutritional management is an essential element in suportive care of critical)y ill patients. ohiectives: sirs is associated with hypercataholisnl and resistance to nutritional support, despite raised circu&ting levels of growth hormone and insulin. serum igf- levels however are low; thercti~re rhigf-i administration might produce nsefnl anabolic effects. the pharmacokinetics of exogenously administration rhlge-i in such patients are likely to differ from that seen in the less seriously ill due to changes in circulating blood volume, increased capillary permeability, poor nutritional stares, and alterations in binding proteins. the ol jective of this study was to determine the clearance, apparent vohnne (if distribution (vd), time to peak concentration (tmax) and elimination half-like (tia) of a single subctkaneous il~ieetion of rhlgf-i in patients with sirs on the intensive care unit (icu). methods: icu patients with sirs were entered into the study, which had been approved by the ethics comjnittee. in each patient baseline hlood samples for circulating gf-i were obtained over a hour period, prior to il!iection of p.g/kg of rhlgf- subcutaneously. a further l samples were taken fi'om each subject over the next hours. circulating ige-i was measured in serum at each time point by radioimmtme assay, and the area under the curve tbllowing administration of rhlgf-i was derived tbr individual patients fixm~ baseline a~!justed igf-i levels. clearance values were caietdated by dividing the administered dose of rh[ge-[ by the total area under the etnve. vd and tv were calculated by regression analysis of the terminal part nf the log sertnn concentration time profiles. results: results are expressed as median (range). age ( - ) years, apache ii score ( - ), and length of icu stay ( - ) days. baseiine circulating igf- levels were low ( < - ) ng/ml reaching a peak of only ( < - ) ng/ml. of the patients had basal levels predmnhaantly below the inwel-limit of detection of the assay, and showed no appreciable rise in serum igf-[ levels tbllowing rhigf-i injection. parmacokinetic variables could not therefi)re be determined in these two patients. following rhlgf-i injection in the remaining patients tmax was at ( - ) hours, clearance was ( - ) ml/min, vd was . ( . - . ) l/kg, and tia . ( . - . ) honrs. conchlsions: there was marked patient wu-iability in response to rhlgf-i administration. vd was similar to that seen in poshq~erative maior surgery patients ( . vs . l/kg), but clearance was greater ( vs ml/min), tmax earlier ( vs hours) and t~a shorter ( . vs i . homts). if riaigp-i is to be administered to dtically ill patients the dose should he nlodified m the light of these findings. baekgronnd: acute bacterial endoearditis continues to be a condition with high morbidity and mortality. the majority of patients are treated with high dose antibiotics, but a patient group requires surgical treatment. methods and results: from january to march , patients underwent surgery for acute bacterial endocarditis: had native valve endocarditis and had prosthetic valve endocarditis. there were men and women. streptococcus viridans was the predominant infecting agent in native ~alve endocarditis and staphylococcus epidermidis in prosthetic valve endocarditis. progressive congestive failure, uncontrolled sepsis and peripheral emboli were the most common indication for surgery. although the mitral valve is more commonly involved in acute bacterial endocarditis, the aortic valve most often requires surgical inlervetion. the postoperative bleeding in the no-aprotinin group was + cc and in the aprotinin was :t: (p < , ). the mortality in icu was , %, the staphyloccocical endoearditis mortality was , % and the no-staphyloccocical endocarditis mortality was , % (p< , ). conclusions: the early surgery is indicated if endocarditis has no response with medical treatment. the mortality of staphyloceocical endoearditis involving left valves was higher than caused by other infecting agents. aprotinin treatment improved prosta#andin metabolism and preserved pletelet function during open heart surgery and could be useful in this type of interventions. urgent surgery had a high mortality ( %). selective digestive decontamination (sdd) has increasingly become the subject of critical discussion sine~ the development of multiresistant organisms has become an issue. moreover, a selection of gram-positive pathogens must also be taken into account when using the sdd regimen, which is primarily directed at the gramnegative bacterial specumn, with the methicillin-resistant staphylococcus aureus strains (mrsa) being of particular importance. the objective of our study was to determine the extent to which colonization with mrsa strains is promoted by sdd. method: patients (ventilation period > days) were randomized and allocated to the sdd group (n= ) or the control group (n= ). in their general intensive care theraw, there were no differences between the groups. the sdd regimen consisted of the four times daily administration of rag polymi~ mg tobramycin and mg amphotericin b in the nesc, mnoth and stomach. systemic prophylactic ~dmini~/rution of antibiotics was not part of the sdd regimen. smears were taken from the nose and the rectum twice wceldy and from the pharynx and trachea once wceldy, and tested for mrsa. further samples were taken as clinically reqnircr results: smears were examined in the sdd group. mrsa strains were detected in samples ( . %) from patients, and in patients they were detected for a period of up to weeks. the positive smears were districted as follows: tracheal / ( . %), nasal / ( . %), pharyngeal / ( . %) and rectal ( . %). severe mrsa-induced infections were observed in patients (infection rate . % of the colonized sdd patients). smears were examined in the control group. ivlrsa swains were r in samples ( . %) from patients, but only repeatedly over a period of up to days in patients. the po~tive snmars were distributed as follows: traclmal / ( . %), nasal / ( . %), pharyngeal / ( . %) and rectal / ( . %). there were no mrsa infections in the control group. conclusion: the data collected support the view that the use of sdd promotes a selection and persistence of mrsa strains. longer-term colonization with mrsa and sovere systemic inf~ons were only found in the sdd group. although the clinical and epidemiological impact of resistance develol~ng when sdd is applied ~maine unclear, this question should be given close scrutiny. tazobactam/piperacillin (taz/p p) is a new broad spectrum antibiotic, in which the acylaminopenicillin piperaeillin is protected by the betatactamase inhibitor tazobactam from hydrolization by bacterial enzymes. taz/pip has shown to possess a high antibacterial activity against almost all clinically relevant bacteria and is a registered drug in germany. obiectives: purpose of this investigation was to evaluate, whether faz/pip . g is suited for efficient antibacterial monotherapy of severe infections and what influence dosage frequency reveals on clinical efficacy. methods: hospitalized patients have been documented in this multicenter trial during a year period. as this investigation should reflect the usual clinical treatment, the only criteria for enrolment were the typical signs of infection as e.g. temperature > ~ leucocytosis or an isolated pathogen. exclusion criteria did not exist and the patients were treated in accordance to the severeness of infection, underlying diseases, risk factors etc. with taz/pip . g t.i.d, or b.i.d. results: patients suffered in most cases from infections of the lower respiratory tract (n= ), followed by intraabdominal (n= ) and skin and soft tissue infections (n= ). % of the lrtis wvre nosocomial acquired and in % the treatment was conducted as monotherapy. in % the lrti was treated with taz/pip b.i.d, and in % t.i.d. pseudomonas spp. (n= ) and staph..aureus (n= ) were the most isolated pathogens pretrcatment. the clinical response rates (cured/improved) after treatment with taz/pip . g b.i.d, and t.i.d, were % and % respectively. results for intraabdominal-and skin and soft tissue infections will be presented. conclusions: in hospitalized patients with severe infections successful treatment with taz/pip in monotherapy is possible. in this population a reduction of the dosage frequency to . g b.i.d, revealed equivalent clinical response rates. objectives. retrospective evaluation of cases of severe generalized tetanus (sgt), treated in our icu the last years. we review cases of sgt ( m, f), mean age . years. in eases the entry site of c.tetanus was a skin laceration, in case it proved to be the external genitalia, while in the rest no portal of entry could be determined. in the first cases incubation period was short ( - days) and so was the period of onset ( - days). all patients needed mechanical ventilation (range - days), initally through an orotracheal tube,and later through a tracheostomy, performed • days after admission. clinical manifestations of sgt included muscle rigidity and i generalized spasms, persisting for up to weeks in the most severe cases. significant autonomic nervous system dysfunction was present in cases occurring - days after the admission and following the time course of generalized spasm. besides general supportive measures, specific treatment included passive +active immunization, penicillin g, magnesium sulphate and sedation in a variety of regimens. neuromuscular blockade was required in cases. nosocomial infections occurred in eases, with sepsis and mof in one. average stay in the icu was - days. one patient died with severe septic complications and one was discharged with severe disability due to anoxaemie ancephalopathy, after a cardiac arrest on admission. ~ disinfectant in suspension test, without presence of organic load, disinfectants showed efficacy on lm. in the carrier test, in the presence of organic load, out of examined disinfectants did not exposed efficacy on lm. the results of examinations clearly showed that evaluation of disinfectant's efficacy partly depend on the used test method. antun basi , intensive care unit, kb firule split spin~ideva ! jugoslavia bacteremia and sepsis are frequent complications encouuntered in severe icu patients.microorganism identification with hemoculture presents the basis for adequate and successful antibiotic treatment.in many patients damage and vulnerability of the peripheral veins presents an obstacle for obtaining the blood culture from the central venous (cv) catheter sample could be also used. material and methods blood cultures were perfomed in lo patients on blood samples simultaneously obtained from the peripheral vein and cv catheter three times in a -hour period.criteria for the suspected bacteremia were body temperature above c and leucocytosis above ioooo leucocytes/dl. the site for venipuncture and the cv catheter stopcock port were cleansed with povidon iodine.after the initial ml of blood were discarded,lo ml were used for the blood culture.standard laboratory technique for blood cultures was used. results and discussion in ( %) patients hemocultures was negative at both sites,whereas in the remaining ( %) they were positive.for twentyone ( ~ of the positive patients the same results were obtained at both sites (peripheral vein and cv catheter),whereas in ( . %) patients the blood culture were positive only for the cv catheter samples.the cv catheters were in place for less than days in patients and for more than days in patients.from patients with positive blood culture from the cv catheter,one patient had the catheter for three days,whereas the other had the catheter from - o days. we neither found significant differences in hemodynamic dates : objectives: , to count and evaluate bacteria isolated from endotracheal (et) suctiori samples (with and without saline). . to establish the exogenous source(s) of pathogens isolated from carer's hands and the equipment involved in sampling in order to reduce the incidence of contamination and infection. method~: this prospective study included consecutive ventilated patients ( male and female, _ + yr; apache ii score -+ ) over a period of months. et aspirated samples with and without saline were taken daily from day of intubation until pathogen~ were presented in counts of _> per ml. at the same time, samples from both carer's hands were taken before and after et suction and a swab from the ventilator tube. results: the overall length of intubation varied between to days. bacterial transfer between staff and patients was noted in % of patients until day of intubation. there was no significant correlation between severity score and appearance of colonization. the incidence of pneumonia in studied patients was % with an overall mortality rate of %. acinetobacter anitratas (no ), staphylococcus aureus (no. ), klebsiella pna~moniae (no. ) and pscudomonas aeruginosa (no. ) isolates predominated in all our specimens. we noticed increased resistance to most antibiotics with the exception of imipenem for gram (-) bacteria and vancornycin for gram (+) bacteria. conclusions: i. tracheobronchial colonization appears directly in the maiority of intubated patients. . there is a close relationship between the microflora of personnel, patients and equipment. . bacteria transfer was noted both to and from patients. . strict hand disinfection policy remains an important measure for the proper care of mechanically ventilated patients to reduce respiratory infections. nnseeomial pneumonia is the most common nnsocomiai infection in the icu-settiag, reported in up to % of patients admitted to the icu following surgery. it is associated with significant mortality that ranges from ~ to %. enteric gram-negative bacilli have been implicated in % to % of ventilntor-associated pneumonias and pseudomonas aeruginosa accounts for % to % of these pneumonias. importantly, epidemics of/ - actamnse-pruducing enterobacter spp or klebsiella spp that are resistant to extended spectrum cephalosporins or penicillins, pose serious obstacles to effective antibiotic choices. carbapenems provide in ~tro activity against a wide range of enterobacteriaceaeand other gramnegative aerobic bacteria, except steaotrophomonns maltophilia. in vitro meropcnem is more active against pseudomonas spp than imipanem (especially p. aeruginosa and p. cepacia), imipenem and meropenem are effective against more than % of strains responsible for nnsocomial infections. all major pathogens associated with lrti are usually covered by the carbapenems, exceptions are pathogens involved in so-called atypical pneuomouia like mycoplasma, chlamydia and legionella. carbapenems are highly stable in the presence of most chromsomal and plasmid-mediated blactumases and usually offer a postantibiotie effect lasting for three hours against most of the enterubacteriaceae. reeent studies comparing imipenem/cilastatin with other ~-lactams and fluoroquinolones in severe lrti in icu patients resulted in favourable clinical cure rates and good tolerance, but development of resistance in p. aeruginosa and ;. aureus during treatment were of some concern. meropenem offers the advantage of greater stability against enzymatic degradation, so no concomitant administration of an enzyme inhibitor is necessary, and meropenem appears to be associated with a lower risk of seizures, particularly when used at high doses. results from studies with meropenem in lrti, especially in critically ill patients with acute exacerbations of chronic bronchitis, demonstrated excellent cure rates and better gastrointestinal tolerance of this new carbapenem. both earbapenems are effective candidates for use as empiric monotherapy in nosucominl infections of critically ill patients. qbl~ctives a favourable effect of iv immunoglobulins in septic surgical patients has been reported, but not sufficiently validated. we conducted this study on trauma patients to: i) investigate the effect of ivig on septic complications and il) quantify this effect by means of serum bactericidai activity (sba) assessment and iii) to explore the effect of temperature increase (from to ~ c) on the sba methods: twenty trauma patierits matched on admission for age, sex, inju~ severity score and glasgow coma scale, were allocated to receive either wig (ivig group; i patients) or equal volumes of human albumin % (control group; patients). wig (sandoglobulin) was administered in a total dose of g/kg divided in a four time regimen on days , , and post-admission. three blood collections were performe& before the first dose (day ) and hours after the third and the fourth dose (days and respectively). complement, lgg fractions, the sba at ~ and at o c and clinical parameters were recorded. results-similar lgg and igg] serum levels were found in groups ivig and control on day ( +_ vs • ns and + vs + , ns), whereas they were significantly higher (p< ) in the v g group on days ( _+_ vs + , p< ) and ( _+ vs +i , p< . ). the various complement-fractions increased in both groups without inter-group differences the mean (• sbas ( ~ c) at rain in ivig group vs control group were: - _+ vs - • ns for day , _+ vs - _+ p< for day and _+ vs - + p< for day . the mean (+sd) sbas ( ~ c) at rain presented a significant improvement over those of ~ c but for the control group remained negative a~d were respectively as following: -~ • vs - + , ns for day , +_ vs - _+ , p< . for day and _+ vs - _+ , p< . for day . the increase of temperature induced a -fold improvement of sba in iv g group and -fold ofcontrol-~oup positive blood cultures, and the product of the infectious episodes number multiplied by days of occurence, were significantly lower (p< ) in the ivig group than in the control ( vs , and vs , respectively). conclusions: our study shows a significantly favourable effect of ivig administration on septic complications and on sba of trauma patients. the increase of temperature results in a significant improvement of sba of patients that received ivig, which theoretically means a farther prevention of infection in the febrile state. pharmaceutical microbiology, university of bonn, meckanheimer aune , d- bonn, germany infectious diseases in intensive care patients are common in comparison to patients on other wards and out-patients. the main difference is that intensive care patients are much more sensitive even to less virulent bacteria. thus, the spectrum of infecting organisms is different. strains often regarded as pathogens with low virulence cause serious infections in these patients. strains such as serratia, however, have intrinsic resistance to most commonly used agents such as rd generation eephalosporins. furthermore, the common pathogens like staphylococci, psoudomonas aeruginosu, enterocneei and gram-negative bacteria, enterobacteriaeceae as well as the non-fermenters are less sensitive if isolated from intensive care patients. it is difficult to generalize on intensive care units as different patient groups are in different icus aud there are great changes from one hospital to another and from one country to another. if we take s. aurens strains from one study from the'overall resistance in intensive care units towards oftoxacin was %, whereas in other hospital wards the percentage of resistance was . %, in out-patients, however, only .$ %. the same trend was true for entercnecus faecnlis, coagulase-negntive staphylococci, and other bacteria as well as other drugs. one most striking difference was found with klebsialla pneumoniae and gantamycin resistance, which was $ times higher in intensive care units as compared with outpatients, whereas in the same species no difference was to be seen with the resistance towards carbapenems. however, differences between countries seem to be even more striking, as example gantamycin resistance and staph. anrens is given. the extreme difference is more than fold. thus, it is evident that there is a general trend towards higher resistance in intensive care units, but no generalizatiouis possible. therefore, surveillance studies in intensive care units are needed and the antibiotic policy has to be adapted to the specific needs of the unit. in the icu setting the most potent antimicrobial agents are required to address problem organisms including those resistant to penicillins, cephalosporins and aminoglycosides. carbapanems would appear to present a useful option in this setting. objectives of this study was the evaluation of systemic candid• in postoperative cardiac surgery patients (pts) with prolonged icu stay. methods: out of postoperative adults pts of mean age . + . years old, with a mean icu stay of . _+ . days, following an open heart surgery from july to april , pts ( %) remained in icu for more than days because of severe perioperative complications. patients were included in the protocol if they had clinical signs of infection or sepsis, and fungi isolated in blood culture or in culture from at least three different sites. the patients who developed systemic candidiasis received iv fluconazole ( mg/day) ( patients) or amphotericin-b for at least four weeks, and then they were closely monitored. results: out of postoperative pts with prolonged jcu stay, pts ( . %) developed systemic candid• usually after the th postoperative day. they were males and females of mean age +_ . years old. this group of pts had prolonged bypass and aortic cross-clamp time compared to control group ( min vs , and vs min). all these pts received inotropes per• (mean value= . ). during their icu stay, pts developed sepsis of bacterial origin, while the other two severe infection, and received antibiotic regimens for prolonged period. the patients were submitted to mechanical ventilation for a median period of days. the median icu and hospital stay was and days respectively. all pts have been improved and finally negative cultures were obtained. conclusions: . a significant percentage of patients who remained in the postoperative icu for more than days developed systemic candidiasis. . all patients who developed systemic candidiasis had received antibiotics because of sepsis or severe infection, for prolonged period. . fluconazole seems to be a very good alternative to amphotericin-b. . fluconazole is a safe antifungal agent with few side effects. botulism is the most severe and an odd food poisoning. although it is more commonly related to preserved meat derivatives, preserved fish and vegetables are also responsible for a number of cases. obiectives: to evaluate four familiar outbreaks of botulism . methods: we study the patients that were admitted in our hospital because of botulism from may to february . results: the thirteen pacients involved had a previous history of home preserved beans ingestion. after a -hours incubation period, gastrointestinal symptoms (abdominal pain, vomits, constipation) appeared and lead them to hospital consultation in the th to th day after ingestion. two patients died (acute respiratory failure before admission), seven were admitted in icu, two in ward and two of them were discharged from emergency room. clinical symptoms and the previous history of the ingestion established the diagnosis, that was emg confirmed. in all cases, symptoms were consistent with b-toxin botulism. b-toxin was isolated in serum and food proceeding from the third outbreak, and the serum was negative in the other ones. neurological symptoms were predominant: midriasis ( %), dry mouth ( %), dysfagia ( %), asthenia ( %), palpebral ptosis ( %), accomodation paralisis ( %) and urinary retention ( %). muscle weakness lead to acute respiratory failure in three patients (one of them required mechanical ventilation). four patiens developed infections (respiratory, urinary and phlebitis). both died patients and one another presented severe hypertension. all admitted patients were treated with polivalent anti-toxin. the two patients who underwent a more severe muscle weakness received also guanidine hydrochloride, with no answer in one case and provoquing a cholinergic crisis in the other one. icu length of stay was days. at hospital discharge, patients continued symptomatic, mainly with dry mouth, disfagia and impaired vision. conclusions: although botulism is a serious illness, the pronostic seems favorable if treatment and support measures are avaible. usually neurological symptoms we predominant and at discharge some of them could still persist. the arrow "hands-off" (aho) thermodilution catheter (tc) is completely shielded during balloon testing, preparation, and the insertion procedure. in order to assess the value of the aho thermodilution catheter in the prevention of systemic infections associated with pulmonary artery catheterization (siapa), we conducted a randomized prospective study over an -month period. methods : the patients (pts) were randomly assigned to two groups : group i for a standard tc customarily used in the department, versus group for the aho thermodilution catheter. the diagnosis of siapa was determined on the basis of a positive culture of tc and bacteremia with the same organism, with out any other nearby focus, in association with regression or disappearance of the clinical signs of infection after removal of the thermodilution catheter. results ( objectives: the mortality rate (mr) of tb requiring mechanical ventilation (mv) is high ( - %). the aim of the study was to evaluate mr, associated factors, and prognostic significance of mv and hemodynamic disorders from tb in icu in patients with tb. methods: clinical parameters on admission, and complications in icu were related by univariate analysis to icu, hospital, and month outcome. patients required mv; were immunocompromised (ic) including hiv. tb was pleuropulmonary in , disseminated in and meningeal in . results: mr was % in icu, % in hospital and % at month. / ( %) < . mortality was associated with a high saps score, initial shock, mv and nosocomial septicemia. the mr dramatically increased when ards occurred during illness, despite the lack of correlation between mr and initial po /fio ratio or initial murray score. the site of infection did not influence the mr. surprisingly, the mean therapy delay was shorter for non survivors. mr was not related to ic status, nor hivstatus, but was only related to previous steroid therapy. conclusion: mr of tb requiring icu is high ( % at month). need for mv increased mortality ( % vs %). general severity and respiratory dysfunction seem to be major prognostic factors in icu rather than tb per se or than therapy delay. in spite of the improvement in the prognosis of pneumococcal meningitis (pm) with third generation cephalosporins (tgc), this infection still presents a great mortality which could be increased with the appearance of antibiotic resistant streptococcus pneumoniae. objectives: to asses intensive care mortality and morbidity of pm and to define patients (pts) at risk of complicated evolution. patients and methods: a retrospective evaluation of pm cases (all diagnosed by csf culture) admitted in our icu from january tit march . in all pts we analized: demographic data, underlying disease, apache ii score, clinical symtomps, treatment, complications and outcome. statistical analysis was done using bmdp sofware package. results:a total f pts were studied, males; mean age , _+ ( - ); apache ii score , + , ; glasgow coma scale (gcs) at admission , _+ , ; ( %) pts suffer from cronic pathology; ( %) pts diabetes mellitus (dm), ( , %) pts had had a previous cranial traumatism. in cases the source of infection was otic and also in ( %) episodes of pm there were bacteriemia. in out of ( %) pts that ct was performed no radiologic abnormalities were shown, of them presented cerebral oedema and pts a cerebral abscess. twenty-eight percent presented seixures, % hemiparesia, , % respiratory failure, , % shock, i % renal failure, , % multiple organ failure (mof). as for treatment refers , % pts recieved only penicillin, , % pts only tcg, , % pts tcg followed by penicillin and , % pts tcg+vancomycin. seventy-five percelat of pts recieved corticosteroids and , % vasoaetive drugs. the mean icu stay was , : days ( - ). twelve ( , %) pts died, two of them presented pm relapse (resistant streptococcus pneumoniae) and another two pts developed neurological sequelae. factors associated statistically with bad prognosis were dm, the use of vasoactive drugs, shock, mof, the apache ii score at admission, the gcs at the and hours from admission in the icu but not the gcs at admission. didn't resulted statistiealy signifcative age, previous eronie pathology, seizures, baeteriemia, renal failure and coagulation disorders. conclusions: mortality was high and associated to apache ii score at admission, to gcs at and hours after admission, shock, vasoaetive drugs and mof. objectives:the aim of the study was to analyse some of significant immunologycai changes in surgical patients,requiring intensive health care,and to determinate the possibility for evaluation,dynamical examination and importance of immunologycal problems for treatment. methodes:the study concerns a number of patients with expanded surgical intervention or serious postoperative complications.the results has been carried out with fiowcytometryc analyses of lymphocytic suhpopulations and routins methods for investigation of humeral immunity.the"panel" for evaluation of (} immunologycal parameters has been offered:t-calls total/cd +/;t-helper/cd +/;t-supressor/cd +/ th/ts ratio;b-cells/cd +/;naturai kilier/nk/cells;skin test for cellular immune function;phagocytic and oxidative activity;serum levels of immunogiobulins-g ,a,m;protease inhibitors;c-reactive protein.all patients have been studied during suffering and after surgical procedures dynamicaly. results:there have been estimated significant changes in immunologycal parameters especially:decrease of t-cells: cd +mean= . %/ . %- . %/and cd +mean= . %/ % - . %/;inverted th/ts ratio ,mean=o. / . - , /;reduced or negative skin teste;reduced phagocytic and oxidative activity before septic complications. conclusions:dynamical examination of immunologycal parameters shows,that the prolonged t-total,t-helper lymphocytopenia with functional deficience of ceils-mediated immunity correlates with the stage of clinical condition of the patients and has prognostic importance.it's clear,that immunologycal monitoring gives a possibility for immunecorrection. patients (pts) with the human tmunodeficiency virus (hiv) infection have a decreased immune response and are particularly susceptible to infectious endocarditis (ie). the aim of our study was to analyze the prevalence of ie, its clinical and therapeutic implications in a hiv population we prospectively studied pts, . % ( / -group ie+) with ie during the clinical course of this disease. we analyzed the following parameters: age, gender, race, type of hiv, cdc classification, number of t and t type cell population and its ratio, therapeutic with azt, type and number of opportunist infections (inf, mycobacteriosis (mb), neoplasm's (nee) the echocardiographic parameters were lv internal diastolic and systolic diameters, lv percentage of fractional shortening, interventricular and posterior wall thickness, the degree of valvular regurgitations and the presence of pericardial effusion. el was located at the mv in . %, tv in . %, av in % and pv in . ~ and was multiple in . %. hiv el+ pts had larger lv diameters and more frequent significant valvular regurgitations ( % tr, pe %, mortality %). these two groups differed significantly in the following clinical parameters: the typical symptoms were watery diarrhea, high fever, tachycardia,luekocytopenia and oligouria within th postoperative days. the patients with mrsa enterocolitis had positive mrsa culture from the many materials except feces.mesa strains frequently had coagulase type ,enterotoxin a and toxic shock syndrome toxin- .eight of patients had postoperative organ failure.most of the mrsa strains in japan were similar in coagulase type to our hospital and our department.all of mesa strains were susceptible to vancomycin and arbekacin,tbough most of them showed resistant to many other antibiotics.we have employed guidelines for therapies such as oral or enteral administration of vancomycin and correction of the hemodynamics for dehydration and circulatory failure due to diarrhea from .futhermore we have placed colonized or infected patients in private room,worn gown and mask,and carefully washed our hands from . these countermeasures for prevention of nosocomial infections after significantly reduced the incidence of mrsa enterocolitis. conclusions:earlier diagnosis and treatment, and distric prophylactic measureres against mrsa infections are very important. -- cdo ivda leptespiresls affects all the organs with widespread hemorrhage that is more prominent in skin, mucosa, skeletat muscles, liver and kidneys. lung involvement is usually mild and less common. suli, it is very uncommon acute respiratory failure to be the pr sontirlg symptom. a case with leptosplrosl..,s which was presenting with acute respiratory failure is described. a year-old man admitted to icu becauso of fever, myaigla, aevere c~, hemopty~s. his blood gases showed: pao : mmhg with fio : . , pco : mmhg, ph: . , hco : mecl chest x-ray film demonstrated diffuse bilateral alveolar pattern occupying beth lung / ). trarmamlnase, bllllrubln, ~ and esr were elevated, wbc was . mm , platelet: . ram , hematesrlt: %, hemoglobin: .sgrldl=. there was no clinical or ecttlographlc evidence of left heart failure.patient fulfilled the criteria for diagnosis ards he was found to have an ~lutinatlon tlter for leptoq~lral antigens(indirect he~lutlnatlon atomy, ilia} very high ( / , negative<ill ) and iglm antibodies also very high (elisa, a= . , negative< . ) strongly suggesting leptospirosls. treatment with tetracycline and non-tnvaslve mechanical ventilation by nasal mask were started. pre~jre support mode and cpap was usod. the following days the clinical picture,the oxygenation and the chest x-ray of the patients were improved, and patient dl~herged from the icu. the control of leptosplremlc phase as well as his good cooperation during nipsv contributed to rapid recovery and excellent outcome in hiv infections, pneumonias (pnm), mainly due to pne~ mocystis carinii (pc) have an outstanding place in pu ! monary complications. the aim of this work was to compare two group> of patients admitted with pnm in our icu during the same period ( - ): group a, patients hiv+, and group b, patients hiv-. apache ii was identical in the groups (p=ns). group a required more often mechanical ventilation (p= ,o ), had a higher p(a-a)o (p= , ) and metabolic acidosis was more frequent (p= , ). regarding laboratorial parameters group a had a lower no. of linfocytes (p= , ), a higher ldh (p= , ) and a more marked hypoalbuminemia (p=o, ). mortality was higer in group a ( , %) than in group b ( , %), (p= , ). analysing the a group patients, we found no significant differences between alive and deceased patients, with exception for albuminemia, which was lower in the deceased patients (p= , ). in conclusion, the hiv+ patient's pnm have a more agres sive behavior when compared with community acquired hiv-patient's pnm. the prognosis was not influenced by the apache ii. perhaps other parameters such as p(a-a)o , metabolic acidosis, linfocytes, ldh and albumin shoud be more evaluated as possible predictive indices. some prognostic factors, usually accepted as predictive in the analysis of hiv+ patients do not seem to be worth in the late stages of aids, mainly when they reqquire intensive care. intensive care unit, onassis cardiac surgery center, athens, greece. objectives of this study was the comparison of two different antibiotic regimens as prophylaxis in cardiac surgery patients. methods: in a prospective randomised comparative study, two different forms of antibiotic regimens were investigated : a single dose of cefuroxime (zinacef, gr) (group a) given during the induction of anaesthesia, versus a four days combination of amoxiculine (amoxil, gr tid) plus netilmicin (netromycin, mg bid) (group b). a total of patients (pts) ( males and females, of mean age . + . years old) were included in the study over a period of one year; in group a and in the group b. patients were checked for the occurrence of infection during the first postoperative month. results: the total rate of infection in cardiac surgery pts was . %; . % in group a and . % in group b (p=ns). pts ( . %) developed infection following cabg, pts ( . %) following valve replacement and pts ( . %) after other cardiac surgery. they were males ( . %) and females ( . %). endocarditis has occurred . % in group a and . % in group b. severe wound infection was recorded in . % in group a and in . % in group b. one case of sepsis ( . %) in group a and in group b ( . %). respiratory infection occurred in pts of group a ( . %) and in pts of group b ( . %). two cases of urinary tract infection was in group a and one in group b. catheterrelated infection was occurred in ( . %) in group a and ( . %) pts in group b. pts ( . %) had fever of unclear aetiology in group b. conclusions: there was no statistically significant difference regarding the rate of infection in both groups. a single dose administration of cefuroxime is accordingly just as effective as a four days regimen of amoxicilline plus netiimicin. legionella pneumophila is a common bacteria of the environment, and it is an agent responsible for severe community acquired pneumonia (cap). we analyzed the patients with lpp admitted in our icu during the last years ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . they represented . % of cap. seven patients were males and female, with mean age . + . years. tiss was . + . and apache ii . + . . all, but patient, were under mechanical yen tilation (mv) during a mean period of . • (min-l, max- ) days. two pneumonias occurred beyond the season, while patients had an epidemiological history. only patient had no risk factor. in all the others tobacco smoking and alcohol abuse was quite frequent. diagnosis was based on serologic test and culture or direct fluorescent antibody staining of bronchial secretions. seven patients had a multisystemic disease with hepatic dysfunction in , renal failure in (due to rhabdomy~ lysis in ). one patient had a prosthetic valve endocarditis and another developped ards. nosocomial septicaemie occurred in patients. mortality rate was %. deceased patients had initially higher apache ii, (a-a) , and lower natriemia. comparing lpp with the other cap (n= ), both submitted to mv, mortality rate was similar ( , % versus . %). in conclusion lpp can occur all over the year. there was a high incidence of severe complications and outcome was similar to the other cap when requiring mv. prospective specimen brash (psb) with culture > cfu cfu/ml. broncho-alv~lat lavage (bal) ~= c'fu/rnl or positive blood culture. were excluded for rapture of treatment ; were analysed (shift with oral antibiotic : ; prohibited antibiotics associations : ; resistant germ : ). clinical data : age , • , ; saps • , ; mac cabe i : , % -ii : , % -iii : , . , % of the patients were intubated and under mechanical ventilation. the pneumoaiae were : primitive in ( , %), copd ( , %), aspiration pneumonia ( , %). germs were isolated (psb , bal , blood culture ) : s. pneumoniac ( , %), h. influeazae ( , %), sttep~:occns ( , %), saar ns ( , %), enterobaetdrindr ( , %), mosexella catarrhalis ( , %), othem . / ( , %) were sensitive to freatment. the ltentment was mg/kg/d of ampiclllin and mg/kg/d of sulbactam in continuous iv adminisu'ation during at least days. clinical eff~ienev : success ( %), failures ( %) with superinfeetion , worsening or relapse , dead , side effects . there was no difference between etiologies : primiti~;e~ , %, copd , %, aspiration pneamoniae , %. the bacteriological effieieacy was evaluated only for patients with eradication ( , %), eradication but super~ection ( , %) : with pseadomoaas a&ogiuosa , eater~ac~ ; beeteriological failure ( , %). in conclusion, the aasor ampicillin -sulbactam is effective for the i~eatment of severe acquired community pneumonise. objectives : to assess the efficacy of chlorhexidine (cl) gel or suspension applied in the nose and in the op for the prevention of the tmcheobronchial colonization. methods : thirty-seven patients expected to be intubated for > h were randomized to received topical application oga cl suspension ( %) qshrs, a cl gel ( %) q hrs or a placebo. in addition all vpts received a nasal and a op spray ( %) of either cl or placebo administrated according to the same schedule. semi-quantitative cultures of the anterior nares, the oropharynx (op) and the trachea were obtained on admission and once a day until extubation (just before the next application). the results were assessed according to the following criteria: success = no acquisition of gnb in the trachea ; failure = acquisition of gnb in the trachea. acquisition was defined by a follow-up culture positive for a gnb not present in the trachea on admission. results : success failure nosocomialpneumonia overall morality clsusp. placebo clgel placebo n= n= n= n= / / / * / / / / * / / / / / / / / / i *p = , byfisher'sexacttest conclusions : these results suggest that topical cl gel administered q hrs may prevent tracheal colonization by gnb. f. daumal*, m. daumal**, c. plot**, v. vurmmen ~ e.colpurt**, b. manonry** * hygiene hospitali&e, ** service de r enmmtion, * service des admissiens-urgeuces centre hospitalier g- ndral - saint-quentin -france obiectives: evaluate the nosocemial risk due to peripheral venous inserted short catheters, and the quality of care. patients-methods: the intensive tare unit (i.c.u.) is a beds unit. the prospective study includes all the patients comn~ in from / / to / / . the recruitemont uses an evaluation schedule of local clinical signs. the nurses aimed to create this evaluation data which includes the place of entry site, the duration of catheterization and the cause ot withdrawal. only patients staying longer than days in the i.c.u. are accounted for. the diagnosis of uosoenmial infection is assured by the physician taking care of the patient and by the hospital epidemiologist on the next signs: evident pus at the catheter entry site, positive culture of the strain, with or without the same pathogen in the blood sla'uam,the patient having no other distant source of infection. analyses were performed on epi/nfo. results: the occurrence of nosoeomjal inthrtions: i abcess and bacteremia during the first part of the study lent the medical staff to modify the protocol of insertion end survey of the device. so we analysed different periods: period ( / / to / / ) and period ( / / to / / ) for all .e peripheral catheters inserted in the i.c.u. period , % , % en infection due to peripheral venous device is a daily threat. the severity of some clinical situations requiring admission in icu proves it. the motivation of nurses for rigid adherence to established protocol, the daily survey of the entry site, the withdrawal of the peripheral catheter every hours aimed to reduce significantly the local signs of inflammation end infection of peripheral catheters inserted inside the i.c.u. objectives: to investigate the use of a new metabolic monitoring device for different ips levels by comparing oxygen consumption (vo ) to measurements of the mechanical work of breathing (web) and p . . methods: the study was approved by the institutiotml ethics committee. eight patients were investigated during weaning after prolonged mechanical ventilation ( - days) for various diagnoses when the clinical physician judged the patient to be ready fur weainag. ips was setto , , , mbar far rain periods each. all patients had a peep between - mbar.. respiratory frequency (f), tidal volume (tv), minute ventilation (ve) were read from the ventilator display ( ae, puritan bennett, carlsbad, usa). flow and airway pressure were measured at the endotracheal tube site. esophageal pressure was measured using an esophageal balloon catheter (fa. ruesch, frg). web was determined as the area subtended by the pleural-pressure-vohime curve. p . was determined by using standard occlusion technique and graphical analysis of the airway pressure tracing. vo and vco were measured using the pb metabolic monitor (puritan bennett, carlsbad, usa) connected to the pb ae ventilator. all data are given as mean• deviation for each ips level. comparison between the different ips levels was performed using anova for repeated measurements. significance was considered at p< . , compared to ips mbar. results: the values for breathing pattern, web, p . , vo and vco are given in the table for the different ips levels; significance is indicated by ~. objectives: fluidized beds are often used in the management of critically ill mechanically ventilated patients. critically ill patients are increasingly colonized with resistent pathogens [ie: p. aeruginosa, methicillinresistent s. aureus (mrsa), extended spectrum i~-iactamase producing enterobacteriaceae ] that can ultimately cause nosocomial infection. methods: we prospectively monitored bacterial colonization of mechanically ventilated patients and of the fluidized bed (clinitron) inwhich they were treated. multiple samples for quantitative bacterial cultures were taken from oropharynx, trachea, feces and bedsores. samples of ceramic beads from the bed were also taken both during and after patient stay (after bed operation in the absence of patient). re,~ults: episodes in consecutive patients (mean age: . years) were analyzed. all had bedsores and/or urinary catheters and fecal incontinence, patients had nosocomial pneumonia, had urinary tract infection [ with extended spectrum imactamase producing k/ebsie//a pneumoniae (ki~lse)], one had positive blood cultures with mrsa, and one patient had a ki~lse found in high concentrations ( - s cfu/ml) in occasions in feces. patients were heavily colonized: the , samples from ceramic beads showed no growth or became sterile without any sterilisation procedure (even in one case of presence of kf~lse) during the patient stay. conclusions: fluidized beds do not put patients at high risk of acquiring nosocomin pathogens, and cross-contamination between patients seems unlikely, even when multiple resistent organisms were initially present. the recommandation from some manufacturers to undergo extensive sterilization of fluidized beds after use does not seem warranted, at least with the bed used in this study. ant. koutsoukou, a, tahmitzi, p. kithreotis, m. koutonlidou, k. stavrakaki, kainis e, g. vlahogiorgos and e. eliopoulos icu-centre for respiratory failure -chest diseases hospital of athens. the cost-effectiveness issue is becoming vital in modern medicine and may lead to moral dilemmas since sometimes certain groups of patients may not have access to highly specialised modalifies. objective: our study compared the mean daily cost for antimicrobial medication in copd patients treated in icu versus all other patients in the context of relevant epidemiological, prognostic and outcome data. methods: age, sex apache ii score, length of icu stay (los) and in -icu fatality were retrieved from the files of all icu admissions over . mean daily cost for antimicrobial therapy per patient (dcat) was estimated. these variables were statistically compared between copd and non-copd patients. significance was assumed at p< . results: of the total admissions were fully evaluable. of them ( %) were copd patients. data (m---sd) results for statistical test are given in table i . copd patients were significantly older spent more time in the icu and presented with significantly higher apache ii scores. outcome and dcat were comparable in the two groups. objectives: the use of heat and moisture exchangers (hmes) during long term mechanical ventilation (mv) is increasing. in icu patients, they are routinely changed every day, according to the recommendations of the manufacturers, but the clinical basis for such a daily practice is lacking. we therefore prospectively assessed whether changing hmes (dar hygrobac, spa, mirandola, italy) every h only would affect their clinical and bacteriological efficiency. methods: two consecutive groups of patients requiring mv for > h were compared: group = hme replaced every day, n= episodes of mv in patients; group = hme changed every h, n= episodes in patients. tubings were not changed in the same patient during the whole length of ventilatory support. diagnosis of nosocomial pneumonia (np) was based on a positive quantitative culture (~ cfu/ml) of a protected specimen brush in patients with clinical signs of pneumonia. quantitative cultures of pharynx, trachea and y-cannector were performed every h. results: the groups were similar in terms of age, indication for and overall duration of mv ( +_ . vs +_ days, p= . ), and severity of illness (saps: --- . vs . +_ . , p= . ). the maximal values for peak airway pressure were identical in both groups ( . -+ . vs . • cmh , p= . ). obstruction of the tracheal tube was observed in only one instance in a group patient who had tracheal bleeding. circuit colonization was very rare, and of low grade in both groups. the level of patient colonization and the type of organisms were identical in both groups. more importantly, the incidence of np was the same ( / vs / , p= . ), as was duration of mv before the occurence of pneumonia ( • vs . +_ . , p= . ) and overall mortality rate ( vs , p= . ). conclusions: the clinical efficiency of this hme does not seem altered after days of use. indeed, replacing this hme every h only neither affect circuit and patient bacterial colonization nor the incidence of np. therefore, substantial savings could be obtained changing hmes every other day only. obiectives: to evaluate the usefulness of different paraclinical investigations for the diagnosis and prognosis of acute viral encephalitis in icu patients. methods: we reviewed patients (pts) admitted to our icu from july to december with the diagnosis of acute viral encephalitis. all were in coma and were initially treated as presumed herpes simplex virus (hsv) encephalitis. the causative agents were: hsv ( cases), herpes zoster varicellae ( ), measle ( ), rabies ( ), unidentified ( ). eleven pts survived and three presented neurologic sequelae. twelve pts were investigated by mri, and eleven also by spect and multi-modality eps. including brainstem auditory eps (baeps). these investigations were obtained as soon as possible following admission and were repeated during icu stay when possible. the clinical outcome was noted. results: six pts ( / ) had an abnormal mri. among them, pts made a complete recovery, in comparison with / pts with a normal mri. in one hsv infected patient, mri remained normal despite clinical deterioration and bad outcome. when repeated, mri became abnormal in cases (with poor outcome in one) and was improved in one. spect was found abnormal in / pts (among them, pts had thus a normal mr/). the correlation regarding the topography of brain lesions was poor between mri and spect. the findings of spect could not be correlated with a poor outcome. the baeps confmned in % of the pts the clinical diagnosis of brainstem involvement. changes in visual and somatosensory eps were mild in all the pts and were not helpful for the prognosis. eps were otherwise interesting for the follow-up of the coma in these sedated and ventilated pts. conclusions: the value of mri and eps for the diagnosis of acute viral encephalitis is of limited interest. spect seems to show early modifications, even in pts with a normal mri, but this test is poorly specific and does not correlate with mri changes when present. concerning the prognosis, larger studies should probably confmn that a normal mri could usually result in a good outcome. this serie illustrates also that hsv encephalitis could be demonstrated only in a small number of cases and that the prognosis of non hsv encephalitis is not easily assessed. objectives: to study the influence of gram (-) bacterial lung infections on liver function i~ mv icu pts. pts and methods: we studied pts, # ( , %), ( , %). hean age: , • years ( - ). mean stay in icu: , • days ( - ). they were divided in groups: a( pts) who did not suffer from pneumonia and b ( pts) who developed a gram(-) bacterial pneumonia. both groups were consisted of pts with same age, sex and disease distribution and same systemic failures. we measured sgot, sgpt, total bilirubin(tb), direct bilirubin (db), alk.phosphatase (al.ph.), v-gt and albumin (alb.) times: on days o, and of the pneumonia for group b and respectively for g~oup a. conclusions: ) in elderly intubated pts of an icu, kp is isolated more frequently than in icu pts< years (p<o, ). ) the frequency of isolation of kp in icu pts during - was - %, but now ( - ) kp becomes more and more rare ( - %) and is isolated especially in elderly pts. ) its sensibility to antibiotics remains always the same. ) the prognosis of np in our study was independent from age (p<o, ), sex, presence of bacteremia (p< , ) and type of invading microorganism. gram neg bacteria and renal failure (rf) from aminoglycoside toxicity are common and serious complications in critically ill patients. the aim of this prospective, pilot study was to compare the safety and adequancy of the endotrecheal (et) vs the intravenous (iv) administration of aminoglycosides. were measured in the plasma and the bronchial secretions at and hours after the iv (bolus) or the et (nebulized) administration of mg of gm in seven criticcally ill patients ( with established renal failure). safety was defined as peak and trough plasma gm levels _< than and ijg/ml respectively and adequancy as gm levels in bronchial secretions _> , ijg/ml. results: gentamicin was administered by the et and iv routes in and separate sessions respectively. a total of samples were assayed, in bronchial secretions (bs) and in serum. the et route resulted in higher gm levels in the bronchial secretions compared to the iv route ( , + , vs , _+ , pg/ml respectively, p = ns ). adequate bronchial gm levels were achieved in % of patients after et administration, compared to % after iv aaministretion. the blood levels of gm were significahtly lower after the et vs the iv route ( , + , vs , • , pg/ml respectively, p _< . ). the et administration resulted in toxic bronchia~ gm levels in % of the specimens. % of these samples were from patients with renal failure, however toxic blood levels were reached in only % of these. gentamicin seems to be a safe and adequate alternative route of treatment for the lrti. however, in patients with renal failure the et administration of the aminoglycosides should also be modified and continuously monitored. in order to evaluate the pathogenic role of anaerobes in nosocomial pneumonia (np), we investigated the systemic humoral response in patients who developed a np with anaerobic bacteria, especially prevotella species. methods: blood samples from groups of patients were tested. group i: patients with a np in which prevotella spp. was isolated from protected specimen brush (psb), group ih a control group of patients with a np without anaerobic bacteria, group ill: a control group of patients with dental stumps but without pulmonary infection, group iv: a control group of healthy voluntary people with prevotella spp. isolated from the dental plaque. an elisa was used to evaluate the total antibodies level against a mixture of four prevotella strains and a western-blot method was done to identify the antigenic proteins. results: data are expressed as means .+ sd. the antibody levels in patients of group i ( • was statistically higher (p=o.o ) than in the control groups (respectively: + , _+ , _+ ). using western-blot method, the intensity of the response was roughly superposable to levels obtained by elisa and the profiles were different according to the prevotella species. the occurence of a np with anaerobic bacteria (prevotella species) isolated from psb leads to an antibody response which seems specific of the prevotella species isolated. fever is common in the intensive care unit, but is not always related to an infection. we sought to define the epidemiology of febrile patients in a general medical/surgical icu. methods: we prospectively analysed the source of fever (t > . ~ c) in all adult patients admitted for >- hours in the icu during a two month period. these patients were studied for consecutive days. and werc classified in groups according to the evidence of infection (center for disease control criteria) after complete evaluation: documented infection: cdc criteria + isolation of pathogen (d); possible infectron: cdc criteria without isolation of pathogen (p); unlikely infection: patients who did nol meet the cdc criteria (u). results: of a total of patients studied, dec'eloped fever ( %). including (after complete evaluation) d, p and u palients. both the highest temperature in tile first day of fever and the maximal temperature were higher in d than in u ( . • versus . • and . -~ . ~ versus . - . , respectively p= . and p= . ). most common sources of infection in d were the lungs in patients ( %) and urina .ry tract in ( %). of these patients had positive blood cultures ( %). the overall mortality was % ( % in d, % in p and % in u. differences ns). antibiotics were given in % of d, % of p and % of u ( patients). in p there was a non significant lower mortality." in patients who received antibiotics ( / ( %) versus / ( %) patients, respectively). conclusions: in febrile icu patients both the highest first day" temperaturc and maximal temperature are significantly higher in infected than in non infected patients, but the differences are too small to be useful clinicall). mortality rate is not significantly influenced either by the presence of an infection or by the administration of antibiotics, obiective: retrospective study to determine the influence of candida infection on icu outcome. methods: patieet with a stay of more than days in inteaasive care were screened for candida infection. patients were treated with antifungal therapy due to either an increased antigen titre of -> : or clinical evidence of candida colonization. serological candida-antigens (ramco, pastorex) and antibody titres (hemagglutination, lgg-, igm-elisa) were examined routinely. seroconversion was defined as a threefold increase of antibody titre or a titre of : or higher. results: the median length of stay was (ranging from to ) days, the mean apache ii score on admission was (+_ . sd) points. of patients patients died ( . %). in the group treated with antifungnls ( patients) patients died ( . %). although of the patients only ( . %) developed a candida infection as defined above the mortality in the group that showed signs of infection was significantly higher ( . % vs. . %, p < . [chi-square-test]). in patients an antigen concentration-> : was measured. seroconversion was found in patients. the most common fungus was candida albicans ( . %). furtberm re, candida glabrata was found in . %. most of the patients were treated with x mg fluconazole ( patients). in patients therapy was changed to amphotericin b/flucytosine. in patients therapy was started with amphotericine b and flucytosine. in patients a threefold decrease of candida antigen titre was found. patients showed a decrease of candida antibody titre. conclusions: meticulous screening for eandida infection seems to be necessary since the number of patients with fatal outcome is significantly higher in the group with signs of fungal infections and thus requires immediate antifungal treatment. objective: early diagnosis of patients with ventilator-associated pneumonia (vap), and subsequent identification of causative microorganism, and selection of the appropriate therapy are critical important points that affect morbidity and mortality. the results of the quantitative bacterial cultures are not available for at least hours, while a two hours period, since the specimen are obtained is enough to know the gram stain results. the aim of this study is to determine the usefulness of gram stain in specimens obtained by bronchoaiveelar lavage (bal), through the bronchoscope. material and methods: we studied patients ( males and females, age + ) with suspected ventilator-associated pneumonia. the bal gram stain was considered positive when the specimen after a centrifugation at rpm for min revealed: i) more than leukocytes per optic field, ii) squamous epithelial cell less than percent and iii) one or more microorganisms per optic field on magnification. all patients had been receiving antibiotics, with no change during the last days, prior to bronchoscopy. results: patients had vap and patients did not. in cases the bal specimens (quantitative bacterial cultures) established the diagnosis of vap in the remaining three patients the vap diagnosis was established by other procedures (blood or pleural fluid culture, clinical outcome, autopsy). apache fl score in patients with vap was , -+ , , while in patients without vap was , + , . there was a significantly higher incidence of vap in patients who had i) coma (gcs < ) and ii) been receiving neuromuscular blockade (p< . ) . the sensitivity of the gram stain for vap diagnosis was %, the specificity , %, the positive predictive value %, and the negative predictive value , %. conclusion: our data indicate that the gram stain of bal specimens is useful for the early diagnosis of vap and the subsequent administration of the appropriate treatment. the role of anaerobes in mechanically ventilated patients with pneumonia (mvp) have been poorly investigated aim of the study : analyse the prevalence of anaerobic isolation in mvp. methods : between october and february all suspected mvp were investigated using protected specimen brush (psb) technique. brushes were rapidly transported in shaedler broth to laboratory. a special care was tooken for anaerobic isolation. results : among the psb performed for suspected mvp ( nosocomial and community-acquired pneumonia), yielded at least one micro-organism (positive psb : %). of positive psb demonstrated only aerobic bacteria and ( %) yielded with anaerobes. in out patients, anaerobes were associated with aerobic bacteria. anaerobes were mostly isolated in nosocomial pneumonia ( / positive psb). strains of anaerobes were isolated. prevotella species represent out these strains ( %) the most frequent anaerobic species were prevotella oralis ( ) p. intermedia ( ) and p. buccae ( ). comments:using adequate methods, anaerobic bacteria are frequently isolated in mvp. it could be off importance to take in account anaerobes in the choice of empirical antibiotic therapy in mvp. objectives: the majority of patients with multiple trauma are considered immunocompromised. the aim of this study was to identify risk factors of pneumonia in mechanically ventilated patients with multiple trauma or after surgery. methods: in this prospective study we studied multi-trauma patients (mean age + years, apache ii . + ), admitted to a general intensive care unit (icu). all patients were intubated and mechanically ventilated. we were considered that a patient had ventilator associated pneumonia (vap) when the specimens of bronchoalveolar lavage (bal) or protected specimen brush (psi?,), ebb'ned through the bronchoscope, had one or more microorganisms in concentrations greater than and cfu/ml respectively. all patients had been receiving antibiotics, with no change during the last days, prior to bronchoscopy. results: patients had vap, and patients didn't. in the bivariate analysis, the glasgow coma scale (gcs)< (x = . , p< . ), the administration of neuromuscular blockade (x = . , p< . ), the duration of mechanical ventilation to be greater than days (x = . , p< . ), the flail chest (x = . , p< . ), the parenteral nutrition (x = . , p< . ), the ards (x = . , p< . ), the abbreviated injury scale (ais) of more than for thorax (:,: = . , p< . ), the pneumothorax (x = . , p< . ) were statistically significant related to development of vap. in multivariate regression analysis, using the stepwise technique, three of the seventeen studied factors showed to have an indepantent association with the development of vap:the administration of neuromuscular blockade (f: . , p< . ), flail chest (f: . , p= . ), and gcs (< ) (f: . , p= . ). conclusions: in patients admitted to icu for multiple trauma or major surgery, the administration of neuromuscular blockade, the flail chest, and the gcs (< ), in the population under study, were the indepedent risk factors for vap. mof is a sereous complication of differem states: infection, sterile inflamation, extensive fissure injure, intoxication, ets. there is close correlation between extension of mof and death, developement of nasocomial infection. immunologic disfunction. in order to prgnose probability of risk of mof development among the patients with sepsis and septic shock, we achived an eqation, allowing to recive a coeficient, closely connected with this probabiliti. we have used retrospective analisis of cases of sepsis. diagnosis of sepsis was based according to bone's criterions of sepsis. mof was assessed as disfunction of or more systems according to bone's classification of mof. having used correlation analisis we have estimated factors which have had high correlation coeficient with the probability of development of mof. there were: apache-ii score points, evidenceof septic shock, endocrinopathy. with the help of multyple regression analisis we acheved next equation: y= , + , x~ + , x + , x , were x i-apache-ii score points, x -evidence of septic shock, x -endocrinopathy. the explanatory power of this quation was evidenced by roc of . , se (v - . introduction: the presence of liver dysfunction in the process of multiple organ failure is associated with an adverse outcome, particularly when it becomes progressive to liver failure. disturbances of liver function may occur early and their detection may be of significant importance for the further development of organ failure. routinely used liver function tests appear to be inconsistent indicators of hepatic damage. in this study, we used p_lasma disappearance rate (pdr) of indocyanin-green dye (icg) as an early estimate of liver function. methods: we serially evaluated pdr and routine liver function tests (serum bilirubin, sgot, sgpt), as well as acute phase and non-acute phase proteins (crp, transferrin) in patients during the first week after trauma or the onset of sepsis. patients: group : (n = ) multiple trauma iss > , group : (n = ): abdominal sepsis, acute necrotizing pancreatitis (anp) grade iii. patients were selected on the basis of clin cal estimates that these patients would require continued icu observation. pdr was determined by means of a fiberoptic catheter and a computerized system (cold z- , pulsion), which permits repeated bedside measurements. the initial values of pdr, serum bilirubin and transaminases were not significantly different in trauma, sepsis and anp. in trauma patients pdr improved during the first week. in patients with sepsis and anp pdr remained low and worsened with time. the decrease in pdr preceeded an increase in biochemical liver function tests in these patients. + . &-_ ( - ) discussion: routinely available blood tests of liver function are usually altered several days after injury. however, they are generally non-specific indicators and they are influenced by extrahepatic factors. pdr seems to be useful to evaluate impaired liver function early after the onset of sepsis and trauma. objectives: to study frequency of organ system failure (osf) and it's influence on outcome in granulocytopenic patients with hematological malignancies and septic shock(ss). materials and method: retrospective review of medical records of granulocytopenie(wbc< , xl ) patients with hematological malignancies and ss, who were admitted to the intensive care unit (icu). frequency of osf before and after ss was analysed. the patisnts were categorised on survival and non-survival. results: signs of osf were observed in . % of patients before ss and in all patients after ss. only patients presented with hypotension refractory to inotropic therapy. nevertheless there was a significant increase of frequency of acute respiratory failure (arf), acute renal failure (arenf) and liver injury (li) after ss occurred(showed on the figure). only frequency of organ failure before and after objectives: statusmetria allows to define the effective level of oxygen status and accordance to it means of carbon dioxide and elec-trolyte in critical care. the conception of syndrome int~ive care (sic) is exhausted itself and invariable outcomes of sic of multiergan system failure (mosf) confirms that. therefore, an alternative to sic should be advanced. methods: efficlenoy of treatment has been asscsaed in patients with mosf using value of metabolic rate and ability of an organism to cover it by oxygen and substrate supply. oxygen pulse (op) and index of efficacy of oxygen transport (ieto ) was monitored. ~lt~.lntenaive care is considered to be homeostasis-securing therapy (hst) if energostructure deficit is eliminated and necessary for recovery regeneration rate is .restored. op in patients with mosf was . mt-m " , and le,~ and ie'i~ w~ . units in sic. we managed to maintain op of . - . ml.m " and ieto of . - . units in hst. patients from with mosf survived in sic and patients from survived in hst. efficiency of hst appeared to be two times as much as efficiency of sic. cr of homeostasia-se-'uring therapy is advancing. the conception provides restoration of regeneration rate due to effective then in sic elimination of en=gostructure deficit. the conception may be a basis of new technology for treatment of mosf. helen f goode phd, nigel r webster phd. anaesthesia & intensive care, university of aberdeen, ab zd, uk. objectives: xanthine dehydmgenase is converted under conditions of ischemia, reperfusion and endothelial damage to xanthine oxidase, with superoxide anion as a co-product of its catalytic activity. multiorgan dysfunction syndrome is associated with splanchnic vasoconstriction resulting in significant and prolonged gut ischaemia. aggressive volume resuscitation with prompt restoration of blood flow results in reperfusion of the tissue and is likely to cause xanthine oxidase-mediated release of oxygen-derived radicals. this study investigates xanthine oxidase activation and oxygen-derived free radical-mediated damage in such patients. methods: fourteen consecutive patients on itu who met established criteria for septic shock and secondary organ dysfunction were studied. serum xanthine oxidase activity was measured using oxidation of a chromagen in a dual enzyme system and plasma malondialdehyde was measured using a specific spectrephctometdc assay. apache ii scores, blood pressure, svr, cardiac output and day survival were also recorded. biochemical data were compared with results from healthy subjects. results: xanthine oxidase activity was . + . units/i in patients (mean :t: sem) and . + . units/i in controls (p<o. , mann whitney u test), and was highest in the patients who survived (p< . ). malondialdehyde concentrations were elevated ( . • . prnol/i compared with . • . pmol/i in controls, p< . ). xanthine oxidase activity did not relate to apache score or any of the cardiovascular parameters recorded, and was not influenced by the degree of organ dysfunction. conclusions: patients with sepsis and secondary organ dysfunction have xanthine oxidase activation and evidence of free radical damage. the finding that activity was highest in those patients who survived suggests that reperfusion was incomplete in patients who died, with decreased tissue xanthine oxidase ~tash out' into the circulation. influence objective : evaluation of the feasibility of measuring the intragastrie partial pr~sure of carbon dioxide (pco ) using a chemilumin~nt optode and fibre~ptics originally developed for intrav~cular blood gas monitorlng(p ) methods the pco electrode w~ calibrated i~-vitro according to the m~ufaeturers instructions the sensor w~ then stripped of its outer c~ing and threaded down the pile tube of a g~ c enema e (gt) so ha he ens ng portion sat well within the balloon. the modified gt was inserted n~og~trically after induction of anesthesia in ten patients undergoing c~diopuimonary byp~s throughout results: according to the datas of integral rheography % of patients were revealed to have hypodynamic type of blood circulation (cardiac output was decreased on - % and general peripheral resistance increased on - %). it was effective to use complamin (xantinoli nicotinas) in the complex therapy of su~h patients. the dosage was - mg/kg during - days. as a result of such therapy was also the oliguric stage shortening (ac n ).in , % of the cases unsatisfactory datas of central hemodynamic,combined with high (more than cm h ) venous pressure,lung oedema.then nanipruss ( , - , mkg/kg/min) was succesfutly used in complex with routine therapy.in the patients with low cardiac output and low general peripheral resistance mesaton ( , - , mg/kg) and dopamine ( - mkg/kg/min) were effective.change for the worse in the hemodynamics datas, inspire of therapy during - days,was prognostically unfavourable. conclusions:we consider early diagnostic of hemodynamic disorders and adequate correction of them is one of the most important factors, determined the outcome and prognosis in arf patients. obiective: to evaluate the results of renal replacement therapy (rrt) in surgical icu patients with acute renal failure (arf). arf in icu patients is associated with high mortality rates. we investigated the relation between mortality and organ failure in different categories of surgical icu patients receiving renal replacement therapy (rrt) for arf. methods: the records of surgical icu patients with arf requiring rri-(haemodialysis or cavhd) were examined. five different patient categories were defined; .ruptured aneurysm of the abdominal aorta (raaa) n = .elective vascular surgery (evs) n = .abdominal sepsis n - .trauma n = .others ( e.g. malignancy, obstetric emergencies) n- seven organ systems (cns,circulatory,respiratory,hepatic,renal,digestive,haematologic) were scored for possible failure using the mof score. results: mortality was as follows: all patients % , group % , group % , group % , group % , group % . mortality increased with the number of failing organ systems; mortality for all patients with > failing organsysterns was % the only exception being the subgroup of trauma patients where mortality under these circumstances was o% conclusions: mortality in surgical icu patients receiving rrt for arf is high. no significant difference in mortality is found between raaa and evs. mortality increases with the number of failing organ systems. the subgroup trauma patients shows a lower mortality compared to the group as a whole, even with > failing organ systems. to look for the most accurate scoring system to measure the severity of the complications occuring in the early phase ( first day) of kidney transplantation and to asses their prognostic value. methods: in our retrospective study we applied the apache li and the goris scoring system for the kidney recipients who developed multiple organ failure (mof) as a consequence of their pulmonary and. cardiovascular complications following kidney transplantation. we evaluated the recipients the distribution of the women and men ( % ~ % ) was the same as in the kidney recipients. applying the apache ii system most of the patients had their score between and , and the function of , or organs were affected at the time of the onset of mof. the apache ii system gave adequeate information about the disturbance of the function of other organs beside the kidney failure even at the time of the transplantation. the scores and the number of the affected organs correlated with the condition of the patients in the goris scoring system but not as sensitively as in the apache ii scoring system. conclusions: both the goris and the apache ii scoring system can be applied to measure the severity of the multiple organ failure occuring during the early phase of kidney transplantation. however the apache ii system is more suitable to follow not only the stateof the patients at the time of the admission but also the changes occuring in their condition during the complication. v.v.erofeev, v.v.ivleva scientific research institute for general reanimatulogy russian amsci, moscow, russia objectives: the analysis of ssc and results of their treatment in patients following critical states showed the necessity of developing a combined antibacterial therapy. methods: according to the protocol patients ( - years old) with combined trauma and massive hemorrhagy following vast aml traumatic operations were examined. microflora's composition and resistence to up-to-date antibiotics was studied using the anaiyser iems reader by "labsisteme"(finland). general clinical, bacteriological, immunological indices, as weil as the duration of the treatment and recovering rate served as criteria of the combined antibacterial therapy effectiveness. results: it was proved expedient to administer antibiotics in staphylococcus infection in the following combinations: riphampizin with fluoroquinolones; i-ii degeneration, cephalosporins with aminoglycosides; cephalosporins with fluoroquinolones. in case of singling out the exciters of the euterobacteriaceae family, including the pseudomonas aereginosa, -fluoroquinolones combined with modern amynoglycosides; fluuroquinolones with ureidopenicillines; ureidopenicillines with amynoglycosides; amynoglycosides with the ii-iii generation cephalosporins; cephalosporins with fluoroquinolones. in severe ssc caused by combined infection (including anaerobes) clindamicin with modern amynoglycosides was prescribed. conclusion: the combined antibacterial therapy allows: ) to increase the effect on microbic agents and the efficacy of treatment in combined infections; ) to lessen the possibility of the exciters'resistence to antibiotics; ) to prevent the development of superinfection: ) to decrease the doses of medicine and its toxic effect. objectives: two methods of blood volume measurement in a group of critically ill patients were compared to investigate the practical possibilities of a new easy to use method based on carbon monoxide (co) uptake. methods: all patients had multi-organ failure and haemodynamic monitoring with a swan-ganz catheter. mean apache ii score was ( - ). when indicated, patients had blood volume measurements simultaneously based on the techniques of, i) dilution of ~cr labelled red cells, and ii) inhalation of carbon monoxide gas with measurement of the rise of carboxyhaemoglobin produced. the co was administered via a newly designed, ventilator driven, fully closed circle system ensuring co retention and co removal with automatic addition of oxygen to m}ttch patient uptake. a portable computer performed all necessary calculations. results: volumes obtained by co uptake were compared with the "gold standard" radiolabelling method. mean blood volume determined by the co method was ml ( - ml) compared with ml( - ml) with slcr labelled red cells (r= . ). regression analysis produced an intercept at ml. the slope of the regression line was . ( . - . , % confidence limits). discussion: the co method produces volumes in excess of the radiolabelling method. there appears to be a systematic error, and one possible explanation is co binding to substances other than haemoglobin. conclusion: the co method is easier to use than radiolabelling and of the lower cost, since cohb measurement only is required. aceuraey is sufficient for clinical use and our preliminary findings suggest this system will meet the requirements. objectives: this study was conducted to determine the role of nitric oxide (no) in the pathophysiologic alterations and multiple organ damage, and the possible effects of " " " (l-n -monomethyl-l-arglnlne nmma) on hemodynamics and mortality in rats caused by a prolonged hypovolemic insult. methods: a prolonged hemorrhagic shock ( - mmhg for rain) was induced in anesthetized rats followed by adequate resuscitation. l-nmma was administered intravenously at doses of . mg/kg or . mg/kg at the end of resuscitation. results: infusion of . mg/kg l-nmma diminished the fall in mean arterial pressure, significantly increased the cardiac index (ci) and stroke volume (sv), together with remarkable protection from multiple organ damage compared to the controls. the h survival rate was significantly improved from . % in the control group to . % in the treatment group (p< . ). in contrast, the high dose of . mg/kg l-nmma resulted in a strong blood pressure response but a marked reduction in ci and sv concomitant with an increased total peripheral resistance index within the observation period, and caused severe damage to various organs at h after treatment. in addition, marked elevation in both endotoxin and tnf levels were observed in animals subjected to shock insult. conclusions: these results suggest that no induced by hemorrhagic shock in rats is an important mediator for pathophysiologic alterations associating with cardiovascular abnormalities, multiple organ dysfunction, and even lethality. thus, regulation of no generation and use of no inhibitors might provide new aspects in the treatment of hemorrhage related disorders, and the use of l-nmma would be either deleterious or salutary in a dose dependent manner. (hebert, chest- ) . the purpose of this study was to assess the risk factors for hepatic dysfunction in mosf. methods: patients have been hospitalized in our icu from january to may . , ( %) with mosf. among mosf pati~ts, ( %) have had hepatic dysfunction defined according to hebert (bilirubin ~ ttmop , chest ). thirty six of these patients acquired hepatic dysfunction after admission in the icu. these patients were compared with mosf patients without hepatic dysfunction selected blindly. chrorfic diseases, severity scores, eanse of admission, clinico-biologieal and hemodyunrrfic parameters, use of vesopressors, use of hepaiotoxic drugs, use of nutritional support and mortality were compared for hepatic failare and non hepatic failure groups.twenty nine patients had postmortem hepatic histologic examination, results: univaciate analysis: only parameters with p _< . are pre~nted. including these paramet~'rs in a multivariate analysis, anly c~hosis and vascular surgery remain independent risk factors for hepatic dysfunction. in particular, pao /fio , arterial lactate, do were not different between the two groups, some de~'ee of histological abnormalities was found in all liver samples, despite a normal bilirubin level in % of the cases conclusions: in our patients, conu'ary to previous studies, hypoxic and hemody~anfic parameters were not independent risk factors for hepatic dysfantion. this might be due to the inadequacy of the usual biologic definition of hepatic dysfunction as well as to the poor sensitivity of general hamodynamic parameters. critical states of various origin are complicated with the mldtiorgan farm (moi~ oceuzr~ce. due to their and functional features the lungs become the primmy damage target in various critical.states. ard that occurs in such states is associated with pulmonary edema development because of capillary permeability increase mediated by humeral and cenular responses to amag/~ factors exposure. r nmst be emphasized that mediators and effecto~rs of this respo~e affect not only puknonary capillaries, but other organs capiu~es as wellenhancing their permeability. orsans edema is a conmm~ finding at the autopsy of patients died from mof.clinical and radiolosial findings allow to have a diagnosis of pulmonmy edema before ~mi!ar lesions in other organs occm. additionally, there are some techniques that permit quantitative assessment of pulmonary edema flv.id (evlw) volume. in conclusion, we suggest that evlw changes in .dyn~rmcs in patients with mof are considered as a critical state severity measure which reflects indirectly the edema in other organs. objectives: we compared three different dialysis membranes to find out whether or not there were differences between their clearance characteristics on substances such as inuline, creatinine, urea, and phosphate to be eliminated in acute renal failure (arf). moreover, if a loss of clearance did occur we were interested in whether this was due to heparinization and a high production of the thrombine-anti-thrombine-complex (tat). methods: we carried out a randomized controlled study on consecutive critically ill patients presenting with arf, most of them in association with multi-organ failure, to be treated by continuous pump-driven arterio-venous renal replacement therapy on continuous low-dose heparinization. three different types of high-flux filter membranes (f tm [fresenius] , ct tm [baxter] , and filtra tm [hospal]) were assessed. each filter was changed intentionally after a hours" use. together the data of filters were evaluated, each at three different times (immediately after its onset [ hi, after h, and after h). the clearances of creatinine, urea, phosphate, and inuline were measured. results: there were some significant differences in clearance characteristics of inuline, creatinine, urea and phosphate between the filters (p< , ) showing the f tm membrane excelling filtra mand ct tm the more. the loss of inuline clearance ( mi/min/m ) after h, however, was insignificant for all filter types. a continuous low-dose heparinization scheme was applied without any relevant prolongation of the aptt. even lower losses were noted for the clearances of creatinine, urea, and phosphate. we found the tat-producfion increased after h (p< , ), but it did not rise any further. conclusions: as we could demonstrate in our study the clearance data of different types of filter membranes applied during continuous renal replacement therapy do show significant differences. on the other side, no relevant loss of clearance occurs during a hours" period indicating a high efficiency over time. to consider commercial aspects as well it shows that inexpensive conventional filter membranes can successfully be applied even for a longer renal replacement period, if needed. a retrospective study was performed on patients with acute renal failure (arf). we analysed survival in continuous (cd) and intermittent dialysis (hi)). mean age of the patients was years (y), patients ( % ) were < y, patients ( %) were >= y. the incidence of dialysed arf in our mixed intensive care departement is %/admission/y. statistics: fischer's exact test, mann-whitney-u test. efioloev: the contribution sepsis, cardiac failure and aminnglycosidcs was respectively %, % and %. treatment: cavh (cd) or cvvh (cd) was used in patients ( %), hemedialysis (hd) was used in patients ( %). data: mean apache scores were the same for cd and hd ( for both groups), patients treated with continuous dialysis techniques had significantly (p<o, ) more need for ventilation ( % vs %), elevated bilirubin ( % vs %) and a higher vasopresser need ( % vs %@ than patients treated with hemedialysis. there was a trend towards more sepsis ( % vs %; p= . ) mad coaguiation disorders ( % vs %; p= . ) in cd. taere were sign/ficantly more younger patients (< y) treated with cd ( % vs %, p< .os). mean apache score was significantly lower in patiants< y than as patienta>= y ( vs ; p< . ). patients< y had significantly (i}< . ) more coagulation disorders ( % vs %) and elevated bilirabin ( % vs %). there was no significant difference in vasopressur need and ventihatio~ between age groups. outcome:. hi) had a better sr compared to cd ( % vs ~ p< . ). patiants>= y had a comparable sr vs patients< y ( ") */e vs %; p----a.s.). tha global survival rate (sr) was % ( patients). conclusions : diaiysed arf has a well known lowsurvival rate ( %): hc~raedialysed patients had a better survival rate than patients treated with continuous dialysis. this can be explained by the fact that the latter were in a worse condition considering organ failure (more vantilatian, elevated bflirubin and need for vasepressurs), apache score couldn't illustrate that. patient~ y with arf have the same survival rate as patients< y: although patients >=- y have a higher apache score they have less organ faille. the avacbe score is not a good oredictor of survival in p with organ failure. departments of surgery and intensive care, guy's hospital, london, u.g-obiectives: a randomised controlled trial of a management protocol utilising the regular measurement of gastric intramucosal ph (phim) to control the administration of dopexamine. methods: patients admitted to a multidisciplinary teaching hospital intensive care unit (icu) undergoing insertion of a pulmonary artery catheter were managed according to a resuscitation protocol. randomisation was to either the protocol alone or to insertion of a nasogastric tonometer and subsequent management guided by phim. phim < . initiated volume and inotrope resuscitation and, if unsuccessful in elevating phim, dopexamine was commenced. approval was obtained from the hospital ethics committee. results: patients were considered for analysis and the two groups were well matched for age and sex. overall, there was a high hospital mortality of . %. there was no difference in icu or hospital mortality between the two groups (see table) . objectives: to compare cardiac output (co) measurements between continuous termodilution (cco) by thermal wire on pulmonary artery catheter (cco/svo vigilance. baxter critical care), and co measurement using a trans-esophageal doppler (dco) ultrasound system (odm ii, abbott laboratories), in the immediate postoperative period of cardiac surgery. methods: patients undergoing myocardial revascularization were monitored with cco by a swan-ganz catheter and an intra-esophageal dco probe, after induction of anesthesia. exclusion criteria were: aortic valve disfunction, previous valvular surgery esophageal disease, absense of sinus cardiac rhythm, and need of ventricular or intraaortic assistance. hemodynamic parameters, co by both cco and dco, svo . sao , diuresis, pha, and hemoglobin were repeatedly registered during the first hours after surgery, as the patients were kept under sedation and mechanical ventilation. results were compared using the method described by bland and altman. results: measurements of co were obtained, ranging . objectives: a decreased tissue oxygen delivery is responsible for a higher morbi-mortality rate among surgical patients; this diminished oxygen delivery/consumption rate (dojvo ) may origin the lactic acidosis observed in the gastrointestinal tract, reported in patients undergoing hypothermic cardiopulmonary extra corporeal surgery, and can be registered by tonometry as result of the gastric mucose ph. the purpose of this study is to evaluate the reliability of the intramucosal ph (phi) measurement by a nasogastric catheter as indicator of the do /vo > its co> relation to other parameters of do /vo disturbance, and with postoperative complications and clinical course. methods: patients ( male, female) undergoing cardiac surgical procedures were included ( myocardiai revascularizations, valvular substitutions, constrictive pericarditis). mean age was + years, mean weight _+ kg. a nasogastric probe (trie tonometrics) was placed after anesthesia induction; phi values were registered in the postoperative period ( ', ', ", ' and h after surgery end). the corresponding hemodynamic parameters, venous oxygen saturation (svo ), diuresis and arterial ph (pha) were also recorded. results: phi values ranged . to . (mean . ( . ); the mean values of clinical evolution were: extubation time, _+ hr.; discharge from postoperative care unit, - hr.; and hospital total postoperative time, _+ . days. complications registered were: perioperative acute myocardial infarctions, cases of respiratory insufficiency, occlusion of coronary bypass, an ease of hyperamilasemia. all patients with severe complications needing specific treatment showed either a low phi value, or a considerable descent in comparison with the initial register. statistic correlation between low phi and presence of complications was found; the low significance (p > . ) degree may be due to the low population size. conclusions: phi measurement in cardiac surgery patients is a non invasive, uncomplicated method for prediction of doz/vo disturbances, thus reflecting risk of increased major complications, and may precede changes in other usual indicators (svo , pha, cardiac output, ...). work-in-progress with a greater population size may offer more significant results. references: ( ) gutidrrez g: lancet ; : - . ( ) landow i: acta anaesthesiol scand ; : - . the haemoglobin-level (hb) is besides the arterial oxygen saturation and the cardiac index one of the relevant parameters of oxygen supply to the tissue. in contrast to otherwise healthy patients, there is no agreement on tile so-called transfusion-trigger in critically ill patients. in i?ont of this background the question arises, whether and to what extent blood transfusion in critically ill patients improves oxygen supply io tile tissue. this study was performed in critically ill/septic patients in the postoperative period alier an inlcclive/scptie revision operation of the hip or knee joint. on cardiac/seplic reasons monitoring consisted beside other measures of a pulmonary arlery catheter and of an indwelling arterial line li~r measurering/calculating standard haem~dynamic as well as systentic oxygen parameters. the indication for blood transfusion was given by hb together with the cliuical slatus of thc patienl (asa-scorc and multiple organ dysfunction (moi))). statistical analysis w~ks performed by mann-whitney-u-test. by fisher's exact-test and by wii.coxon-test: statistical significance was set with p< . . according tu the pretransfusion value of hb and of lactate (lac) palicnts ;,,'ere divided into groups as follows: a: hb< and b: >sg/dl: i: ac< . and ii: > .smm. in either group blood transfusion results in zt significant increase in hb (a: . _+ . to . + . g/dl; b: .(~ . tt, . + . g/dl; i: . -+ . to . -+ . jdl; i : . -+ . to . + . g/dl). wlailc, however, haemodynamic parameters do not difl)r significantly from each other before and alter blood transfusion, oxygen delivery (do, -ml/min x m-') increases significantly hi either group studied (a: -+ to -+ ; b: + to + ; : -+ to -+ ; i : -+ to -+ ), in contrast oxygen consumption (vo~ -ml/min x m e) does not change significantly in either group (a: i -+ to -+ ; b: -+ to -+ ; i: -+ tu -+ ; : -+ to +_ ); oxygen exlraction ratio decreases. this study in critically ill/septic patients demonstrates, that in this group of patients studied blood transfusion at a base-line-value of > . -+ . g/dl expectedly rises do~, however, it does not improve vo=; even not in septic patients with elevated lac-values. paclitaxel in a new anticancer agent, extract from the bark of the yew tree (taxus brevifolia), employed against breast and ovarian cancers resistant to chemotherapy. it promotes the polymerization of tubuline, and disrupts the normal microtubule dynamics. hematologic toxicity, hypersensitivity reactions (bronchospasm, urticaria and hypotension), and peripheral neuropathy are the main reported toxic effects. cardiac side effects are rare: atrioventricular blocks of higher degree are reported in . % of patients; congestive cardiotoxicity was discussed only in one trial in patients treated with paclitaxel and doxorubicin. we describe the history of a -years-old worn an with a breast cancer, diagnosed in , initial staging t nim , treated with mastectomy, axillary lymphadenectomy, andchemotherapy with a cumulative dose of anthracyclines of mg/m until august . the patient complained of dyspnea and severe hypotension immediately after an intravenous infusion of mg paclitaxel, given over hour for the treatment of bilateral, malignant pleural effusion. at echocardiography die left ventricular ejection fraction was reduced to %. she died days later because of a severe cardiac low output with hepatic and renal failure; an impressive hepatic cytolysis was observed. the post mortem examination confirmed the dilatation of the cardiac cavities, especially of the right ventricle, bilateral pleural fluid, and ascites. the histology was suggestive for a cardiomyopathy secondary to anthracyclines. the electron microscopy revealed a deposition of an unusual pathological pigment in the myocytes; subsarcolemmal deposition or membranous were absent. we hypothesize that paclitaxel was the cause of a major hypersensitivity reaction with shock and severe hepatic cytolysis, worsening the myocardial damage induced by anthracyclines. the possibility that a low doge of paclitaxel could directly increase anthracyclines cardiotoxicity -as decribed in the medical literature -will be discussed. objectives: activated endothelial cells release soluble intercellular adhesion molecule- (sicam- ), vascular cell adhesion molecule- (svcam- ), and e-selectin (selam- ). sicam- , svcam- , selam- , and inflammatory cytokines were determined. methods: sicam- , svcam- , and selam- were determined by elisa. tnf-a, il- , and il- were also measured by elisa. endotoxin was measured by an endotoxin-specific endospecy test after pretreatment of new pea method. results: the sicam- and s vcam-i levels were significantly higher in the septic multiple organ failure (mof) and sepsis groups than in the non-septic mof group. the selam- level was slightly higher in the septic mof group than in the sepsis withut mof group and non-septic mof group. the increases of soluble adhesion molecules were not in agreement with changes of plasma endotoxin level. levels of soluble adhesion molecules were correlated with the levels of plasma tnf-a and il- , but the level of il- . discussion and conclusion: the slcam- and svcam- levels in septic patients closely reflected the severity of the pathophysiological conditon. it was possible that the release of sluble adhesion molecules were not stimulated by plasma endotoxin, but endotoxin in the local infectious region. tnf-c~ and il- also were suggested to be involved in the release of these soluble adhesion molecules. obiectives: cardiopulmonary bypass (cpb) surgery is associated with a systemic inflammatory response attributable to the release of various inflammatory mediators and the activation of complement or coagulofibrinolytic system. in addition, adhesion molecules, such as icam- , elam- , and vcam- , appear to be of central importance in the inflammatory process following cpb surgery. we previously reported the effects of a synthetic protease inhibitor, fut- , reduced release of inflammatory cytokines (tnf, il-lg, il- ), activation of complement (c a, c a) or coagulofibrinolytic system (tat, pic, fpa) and protected platelet function (gpib, gpiib/llla) following cpb surgery. methods: in this study, we analyzed fut- on soluble adhesion molecules following cpb surgery. patients undergoing cpb surgery were divided into two groups, group a consisted of patients who received omg of fut- in priming solution, followed by a continuous infusion at mg/kg/hr during cpb in addition to initial heparin dose of mg/kg. group b, a control group, included patients who were injected with heparin only. the plasma slcam- , selam- , and svcam- concentration was measured by elisa. results: every soluble adhesion molecules decreased during cpb in both groups, and rose after cpb. selam- and slcam- reached their peaks on hours after cpb and on pod respectively in both groups, but they remained lower in group a (selam-i: . + . vs. . • ng/ml, p< . , slcam-i: • vs. • ng/ml, p< . ), svcam- , in both groups, remained lower than preoperative levels, but did much lower in group a. conclusions: fut- reduced adhesion molecules and suggested to be the effect on postoperative organ dysfunction. in the last few :,'ears the conditions of treatment in continuous hemofiltration/hemodiafiltration were discussed controversially. a significant removal of tnf-alpha and il-i could be demonstrated in cvvhd. the aim of our study was to investigate the elimination of tnf-alpha, l- , il- , il- , s-cd- and ifn-gamma in cvvh by measurement in plasma and hemofiltrate of critically ill patients with an acute renal failure. the patients of our study were treated with a continuous veno-venous-hemofiltration (polysulfone-filter, blood flow: - ml/h, filtration rate ml/h). the samples, hemofiltrate and plasma, were taken one hour after the start of treatment. the patients suffered from septic shock ( ), the so called hepatorenal s~aldrome ( ) and a severe pancreatitis ( ). the cytokine concentrations were measured with elisa-method. in contrast to elevated concentrations in plasma for tnf-alpha ( cases), scd ( cases), il- (l case) and il- ( cases), hemofiltrates contained no activities. only il- was removed in significant amounts with even higher levels in hemofiltrate than in plasma. this phenomenon was described so far for tnf-alpha and il- and may be due to the absence of metabolic properties (possibily enz~natic) in hemofiltrate. it can be shown, that tnfalpha, il- , il- could not be eliminated in cvvh with a filtration rate to ml/h. in contrast to findings of other investigators with a higher filtration rate (> ml/h), we found no significant concentrations of tnf-alpha and il in hemofiltrate. we conclude, that for a significant removal of important cytokines higher filtration rates (> ml/h) are necessary. objectives: multiple organ dysfunction syndrome including liver and renal impairment is a fatal complication in patients with the diagnosis of sever sepsis. this study focused to the effects of removing toxic substances from inflamnatory tissue by hemodiafiltration. ~ ethods: eleven patients were admitted to the icu in emergency center and met the criteria of systemic inflammatory response syndrome in association with infection. all patients developed liver and renal dysfunction and were treated by hemodiafiltration with high flux membranes (fb-u:nipro). the hemodiafiltration were performed times using nafamostat mesilate as an anticoagulant in hours with l of substitution fluid (hf-b:fuso). the serdm levels of endotoxin, cytokines, endothelin-i (et-]), human neutrophil elastase ~ -proteinase inhibitor complex (hne-pi), fibronectin (fn), lactate, and amino acids were measured before and after the hemodiafiltration. the hemodiafiltration would be effective to renal dysfunction by reducing endothelin and beneficial to tissue metabolism represented in fisher's ratio, but might be harmful to respiratory function by activating neutropila in patients of severe sepsss. background : intermittent hd may be poorly tolerated in the early phase of arf in hemodynamically unstable patients (pts). this technic may fail to achieve steady state urea low levels in hypercatabolic pts. method : nt = consecutive pts treated with hd; n = consecutive pts treated with cvvhf. hemodynamic unstability is defined by arterial hypotension and requirement of inotropie support despite adequate filling. rate of change in urea (u), ereatinin (cr), k + , ph were computed from a linear regression .analysis of data vs time in each treatment group during the first days of application of the two technics (anova). dally worst values were recorded. results : hd-group : apach% score = _+ ; mean number of organ system failure (osf) = . -+ ; mean blood pressure (mbp) = • mmhg (first day of application of hd). cvvhf-group : apachen score : + ; osf = -+ ; mbp = + mmhg (first day of application of cwhf discussion : during the first days of application of hd/cvvhf, u and cr decreased much more rapidly in the cwhf-group. k* and ph were maintained within normal range in the two groups. initial mbp which was much lower in the cwhf-group significantly improved during the application of cvvhf while mbp remained unchanged in the hd-group. conclusion : despite higher severity of disease in cvvhf group (apachen score, osf, lower initial mbp), we obtained a better performanco with cvvhf regarding the decrease of u and cr and the improvement of mbp. in relation to the different and continuous renal replacement techniques, the continuous venovenous one is the alternative method to continuous arteriovenous for critical patients with acute renal failure (arf). we present you our experience with cvvh in patients with mof. in our intensive care unit (icu) patients with mof were treated with cvvh in the period between january in to march in . the mean (• age of our patient population was , • years, being % male and % female the whole patient population was with mof iust at the moment the technique was accomplished; % was in mechanical ventilation, % needed vasopressor support and % required both of them (mechanical ventilation and vasopressor support) apache ii score mean of the patient population was , ~: , (range - ) and ati of them were with arf oligoanudc. technique: cvvh was accomplished using a single-d~al iumen catheter, ptaced in either a temoral or subclavian vein by the stand ard seld{nger technique. pol{sultone hemofitiers were also used, and the extracerporeal circuit used standard arterial-venous blcod tubing. blood flow and hence oltrafiltration pressure, within the circuit was generated by a roller blood pump. the modulus has a roller pump, a pressure transducer connected in an arterious and venous line, such as an air-transducer which is adapted to a drip-chamber in the return way. the replacement used was a peritoneal dialysis solution. medicine , st. george's hospital medical school, london. england. hepatic sinusoidal endothelium shows a major inflammatory response in porcine sepsis that can be attenuated by the administration of dopexamine hydrochloride. dopexamine is a beta and dopaminergic receptor agonist. the specific beta adrenoceptor antagonist ici has been shown to reduce the protective effects of dopexamine. we investigated the effect of this antagonist on hepatic ultrastructure in porcine sepsis. six pigs ( - kg) divided into groups were anaesthetised and intubated. cardiac output and portal blood flow were measured using standard techniques. the groups were; placebo, (peritonitis induced); blocker, (peritonitis induced and pg/kg ici bolus infused then given hourly). caecal content was aspirated and peritonitis induced. colloid was infused to maintain pawp at - mm hg for eight hours the animals culled, hepatic tissue removed and prepared for electron microscopy. in the placebo group hepatic endothelium was swollen and the sinusoids occluded by wbc. but in the ici blocker group, much of the sinusoidal endothelium was absent and there where large extra sinusoidal spaces among the hepatocytes. an assessment of the two groups showed worse hepatic architecture in the blocker group. the b antagonist blocked any protective effect of endogenous beta adrenoceptor agonist (adrenaline) on hepatic endothelium in porcine sepsis. george's hospital medical school, london. england. dopexamine hydr chloride, a beta and dopaminergic receptor agonist reduces hepatic damage in porcine sepsis. we tested dopexamine's effect on cerebral oedema. the beta adrenoceptor antagonist ici was infused to block any protective effect of dopexamine. nine anaesthetised pigs ( - kg) were randomised into groups; placebo, (peritonitis induced); dopexamine, (peritonitis induced and ~tg/kgdar of dopexamine infused); blocker, (as in dopexamine group but in addition pg/kg ici bolus given then infused at that rate hourly). caecal peritoneum was induced and colloid infused to maintain pawp at - mmhg for eight hours when the animals were culled, cerebral tissue removed, prepared for electron microscopy and digitisation. digitisation of the area of oedema surrounding the blood vessel and expressed as a percentage of the micrograph. . _+ . , dopexamine . + . ", blocker . + . . data expressed as mean + sd. significance p< . . * dopexamine compared to placebo and blocker. in the dopexamine group the area of tissue oedema was significantly lower than either the placebo or blocker groups. there were no significant differences between the placebo or blocker groups. the antagonist completely blocked the protective effect of the drug on cerebral oedema in porcine sepsis. beta adrenoceptor stimulation is protective of cerebral oedema in porcine sepsis. objectives: the hemodynamie~ of hepatic circulation during multiple organ failure (mof) have not been suffleienly studied. we investigated liver hemodynamics in two subgroups of patients with mof, those with either liver or lungs as the main organ of involvement. methods: three groups of patients were created: i) mof-hepatic involvement (mof-hi) ( patients) with bilirubin > . mg/dl and lung injury score < . , it) mof-ards ( patients) with respective values < . and > , iii) patients with head injury with respective values < and < , served as group control. all patients were in haemodynamieally stable state with an oxygen delivery index > ml/min/m prior to measurements. two swan-ganz catheters 'were inserted, one in the hepatic veins and one in pulmonary artery and the following measurements were determined: the hepatic vein free pressure (hvfp), the hepatic vein wedge pressure (hvwp), cvp, paop and co. the gradient of hvwp-hvfp represents liver perfusion pressures. by injecting contrast media at dose of iml/lokg with the balloon inflated to achieve sinusoidai image, the hepatic blood flow (hbf) was concluded by the time in seconds of media removal after balloon deflation. results: the co, cwp and cvp were comparable to all three groups. namely, for mof-hi, mof-ards and control groups the mean (+sd) value of co was . _+ . vs . _+ . (ns) and . _+ . respectively, of the paop was . +_ . vs +: (ns) and . + . respectively and of the cvp was .+. . vs . + . (ns) and . respectively. in contrast the two mof groups were different after the cut-offinclusion criteria ie the mean (+sd) value for bilirubin was . + . vs . + . ( < . ) and . _+ . respectively and lung injury score was . objectives: oxygen delivery (do ) and oxygen consumption (vo ) are increasingly monitored parameters in the icu. there still remain controversies about an oxygen supply dependency in critical illness particularly with respect to vo determination by either indirect calorimetry (vo m) or tick calculation (vo c). the purpose of this study was to investigate the changes in vo m and vo c following do increase. methods: the relatives of critically ill patients (mean age years, mean apache ii , mean mof-score ) gave their written informed consent to participate in this institutionally approved, prospective study. do was increased by fluid loading (hydroxyethylstarch %: mean volmne ml, mean duration of infusion min) and catecholamine support (dobutamine: mean dose , ~g/kg/min). changes in vo m and v c were recorded sinmltaneously before, during and following interventions. calorimetry was obtained with the metabolic monitor integrated in the ventilator (puritan bennett, carlsbad, ca adaptive endocrine response of organism to septic shock consisting in activation of the production of adrenal hormons, renin -angiotensin -aldosterone system (raas) and other hormonal systems has an influence over microvascular changes in these states and for development of multiple organ failure (mof). in patients with peritonitis of different origins ( nonsurvivors and survivors) were followed the changes in cortisol level and raas by radioimmunological methods and many variables for evaluation of respiratory, renal, hepatic function, coagulation etc. as a signs of mof. it was observed significant increase of the level of cortisol ( +_ , nmol/ i), aldosterone ( , • , nmol/i). by factorial statistical analysis we found significantly high correlations between hormonal changes and respiratory function (for example r=- , , p < , between cortisol and pao ; r = , , p < , between cortisol and d (a-v) ; olso renin -cao r=- , , p < , , renin d ~,vl o r = , , p < , ). such significant correlations was found and for raas with respiratory, renal function, byproducts of arachidonic acid thromboxan b and p fla, soluble fibrine degradation products etc. these correlations between the degree of endocrine changes and multiple organ failure in patients with septic shock produced by peritonitis suggest that their effects upon peripheral vascular resistance and constriction of the splanchnic, splenic, renal and other organ vasculatures are not always with physiologic expediency and there are perhaps the possibilities of therapeutic influence. intredu~on : dopexamlne has previously been shown to control hyperkalaemia ia patients with acdto renal failure (arf), however effects on the subsequent course of art are undomunente~ ob_iectlv~ : to evaluate clinical progress in patients with acute renal failure (arf) in an intensive care unit (icu) with regard to biochemical control, need for -and time to -dialysis, and outcome in patients receiving dopexamine. m~ods : consecutive patients meeting standard criteria for diagnosis of arf were included in the study. full cardiovas~dar, biechemical and intervention/outcome details were recorded. dopex.~min~ was infilsed at a dose of pg/kg/min in conjunction with a regimen of inotropir support and blood volume optimization. resn]~ : following the intzoduetion of dopc',~mine ilrinr vohlmes increased slightly over the next hrs fzom + ml/ hrs to + ml/ hrs (ns). data expres,uxl as mean + sem. three patients ( %) became polyuric with urine output > ml/hr within days and did not need dialysis. in the remaining patients the time to dialysis (to correct acid-base deficits or volume overload) was . + . days. serum potassium levels were well controlled. day or immediate pre-dialysis levels were . + . mmol/l compared with pre-lreatment . + . mmol/l overall mortality in this series was / ( %). duration of acute dialysis in survivors with renal recovery was . +_ . days. patients ( %) progressed into chronic renal failure and needed continuing renal replacement therapy. no adverse cardiovascular altects were seen at this low dopoxami~ dose although its competitive inhibition to adrenergic reuptake mechanisms meant that doses of pressor agents could often be reduced. : dopcx:~minr nsed in conjunction with inotropic support and blood volume oplimitntion, can safely postpone, or even avoid, the necessity for acute haemodialysis in icu patients. no evidence of tachyphylaxis to the effect on serum potassium levels was seen over the duration of the study. hen'era m., suarez g., dagn d., varela a., ramos j., garoia jm, aragdm c, jurado l, medina a. icu. hospital regional. malaga. spain. objective: to evaluate the haemodinamic tolerance to the veno-venous continuous hemefiltration (vvchf) system in patients with systemic inflammatory response sindrome (sirs), and the possible beneficial effect of this technique on the haemodinamics in these patients. material: patient admitted to the icu, with diagnosis of sirs and monitored with a pulmonary artery catheter at the beginning of wchf. we performed a complete haemodinamic study to all these patients (cardiac output, vascular resistanoss, ph and co in arterial and mixed venous blood samples, saturation of pulmonary mixed venous blood, do and vo calculations and temperature) and determined the respiratory mechanics (compliance and pao /fie relatinship) before starting the procedure, after minutes operating with the ultraflltrate branch closed (without filtered fluid production), afler and minutes of zero fluid balance bemofiltration and after minutes of filtration with negative balanos adjusted to the patients conditions. for the statistical analisis we have performed the anova test over the mentioned variables. results: we have not detected statisticaly significant differences of the analyzed variables before the beginning after operating the pun'@ for minutes without filtered fluid production and after minutes of zero fluid balance hf. only temperature shows a meaningful decrease in time. objectives: among many organs, playing the important role in pathogenesis of multiple organ failure, the particular place is taken by the intestine. ~ethods: the study was carried out in dogs !~n"~h pi was modelled by severe operative trauma (ot). the dcm was estimated by the indices values of work time (wt), contraction frequency (cf), mean amplitude of contractions (~ac) and motility index (mi) measured by method of tensography. "sl", created on the basis of sorbit and sodium lactate ( mosm/l), was injected in the dose of .o ml/ kg into v. cephalica antebrachii after hrs of ot. the results of the present study are the evidence of "sl" stimulative action on dcm and are experimental ground for "sl" using in complex therapy of pi in clinic. with splanchnic venous blood pc p.f. laterre p. goffette, j.p. fauville, a. poncelet, p. loneux, m.s. reynaert. intensive care unit, st. luc univ. hospital, brussels, belgium. determination of gastric intramucosal ph (phi) by gastric tonometry using the henderson-hasselback equation is expected to allow the detection of splanchnic ischemia in critically ill patients. because of bicarbonate concentration and acidbase balance influences on the calculation of phi, it has been proposed to use arterio-gastric pco,_ gradient [p(gast-a)co,] to assess splanchnic perfusion. htpothesis : pcoz in the gastric mucosa is in equilibrium with intraluminal co z and with co, in the blood leaving the stomach (mesenteric and portal blood). objective: mesure pco; and ph in portal vein blood and compare its value with pco and phi obtained simultaneously by gastric tonometry. material and method : in a patient ( y.), a fiberoptic catheter (baxter r) was positionned in the portal vein after transhepatic stent shunt repermeabilisation. hemodynamic parameters, do, (vigilance n baxter), gastric co and phi (tonometrics baxter) and portal blood gas were determined at regular intervals. results : sets of data were obtained and are expressed in mean + sd. gastric pco z was , + compared to , + . mmhg for portal pco . phi was . +._ , vs . +._o, for portal ph. no correlation was found for these parameters. p (gast-a) c was . + mm hg vs + . mm hg for p (portal-a) coz (no correlation). there was a good correlation between do e and p (portal-a) co z (r = , ) [figure] but no correlation with p (gast-a) c . obiectives: desaturation is a common finding during haemodialysis (hd). pulmonary oedema might be one cause for impaired gas exchange ( ). the aim of this study was to quantitate the amount of extravascular lung water (evlw) and gasexchange in chronic renal failure patients during and after a regular hemodialysis session. methods: chronic renal failure patients without symptoms or diagnosis of cardiac or respiratory disease were studied at the start (i), at the end (ii) and two hours after (iii) a regular bicarbonate hemodialysis session. the double-indicator dilution method, with indocyanine green and the stable isotope h as tracers, was used to measure evlw ( ). arterial bloodgases and endtidal co were registered. evlw data was compared to a group of renal healthy patients ( ). dcp n evlw, ml -pao , mmhg h~o +, nmol/l control group - -- l _+ "* -+ _+ crfgroup ii -+ ~ +- ns -+ "(" iii +- t _+ ns -+ t ** p < . dcp i from dcp , t p < . dcp li or i from dcp i, :~ p < . dcp ii from dcp i the evlw at the start of dialysis was larger in the crf group than in the control group. the evlw decreased significantly to a level not different from the control group in response to the reduction in weight after hd. pao~ was normal at the start of hd and showed a nun-signficant reduction after hd. paco ( . + . kpa) and etco ( . + . kpa) were unchanged while h o+ decreased and bicarbonate increased significantly. conclusions: the elevated level of evlw at the start of hd did not impair gasexchange. the decrease in evlw did not inhibit the decrease in pao . the reduction in h + followed by a fall in alveolar vantilation is the most plausible cause for the decrease in pao in bicarbonate dialysis. . prezant lung ; : - . . wallin j appl physio ; : - . a. dona~ d. battis& l col~ r danieli, d. achill~ l viglienz;~ c. giov-anaini, p. piaropao~ oblectives: to verify if intraoperative modifications of mtramucosal gastric ph (phi) below the normal lowest value . , can be predictive for important complications, as perforation, sepsis, mof or death. methocls: we have considered patients who andenvent major abdominal surgery. all patients received the same drugs in pre-anaesthasia, the same type of anaesthesia (balanced anaesthesia) and the same treatment with h -bloekers. after the induction of anaesthesia a gastric tonometer was positioned and a catheter was positioned in the radial artery. during the operation, every minutes, the following parameters were measured at the same time: phi, arterial ph (pha), blood lactate, mean arterial pressure. in follow up we considered death and complications happened during the hospital stay, in relation to intraoperative phi falls below . . results: among the patients, had a drop of phi below . during surgery. in three of them this fall was a single episode and happened within the first hour after the begiluting of the operation. after that phi rose to nomml values until the end of the operation these patients had a normal post-operative period, without complications, the other patients had a fall of phi during the demolitive manoeuvres. two paticots of them died. the first had a lowest phi= . and the second . . the first one ~zs operated on for hepatic istiecitoma, suffered a complete del'dseenco of the surgical wound on the th day after operation and died on the th day, the second one was operated on for a hepatic carcinoma had an intraoperative haemorrhage and died ~vo hours after the end of the operation. the other patients with a fall of phi had a lowest phi= . . . . . . . respectively.the first patient,operated onfor sigmoid carcinoma, underwent on a second operation for a transmural necrosis of the colic segment on the th day; the second one, operated for carcinoma of the right colon, had a cardiac ischelnia on the th pest-operative day and a dehiscence of the surgical wound on the th day: the third one, operated on for a sigmoid carcinoma, had melena in h post~ operative da b, and finally the fonrth patient, operated on for carcinoma of the tight colon, suffered a fistula of the surgical enteral anastomosis.all these patients were discharged alive from the hospital. the other patients, who had not reductions of phi ditring the operation, had a normal pest-operative period, without complications. conclusion: phi was able to predict the arising of some complications, probably due to intraoperative ischemic events. we can say that gastric tenometry, for its low invasivi.ty, can be included among the intraoperative monitoring in patients that tmdenvent on major abdominal surgery. (ttd),t"ea~rrerj.~ of hours duraticn. all l:atients nm.'-~ms_(~lly va~ ated in eantrol wcde ard_ la':'ad a a,~m--ganz catheter, with optic fibers for contirums mmsuremmt of svo mic studies were performed, c~e before the hegir~ of hd, c~e rain after the ~, ~ne at the middle, ~ne rain before lhe erd ard one rain after the erd of hd. paired t test ~as used far slatistical eval~ti~n. results: daring i~d there was a significant'reductton (p<o.~) of: c~tr~ venc~s eres~.re ( ve)emm to . ~ ~, ) ~arn'~w capil~ ~f~e pressure (i<~p) fr~n to ~mg hg, ) i~ from to . mmg hg. ~here was also a less pmnameed bat also-slatistics/ly significant r~ucticn (p<o. ) of: i) s~o~ from to patients undergoing surgical reconstruction of the aorta due to anenrysms of its abdominal part may develop transient and sometimes sustained episodes of dysoxia despite the conventional appeoreneas of being adequately resuscitated. indirect masurement of gastric mussel ph (phi) is being widely evaluated as a minimally invasive and sensitive means of assessing the adequaey of tissue oxygenation in these circumstances. the duration end degree of gastric intramucosal acidosis are related to the risk developing injured gastrointestinal tract end its putative consequences, i.e. translceation, cytokine release, organ dysfunction end failure, sepsis end death. measurement of phi is obtained indirectly by measuring pc in the lumen of the stomach with a silicone balloon tonometer (tonometrics, inc., worcester, ma, usa) and the bicarbonate concentration in arlz,,rial blood, end substituting these two values in the henderson-hasselbalch equation. objectives: to evaluate whether perioperative phi changes are predictive of complications end outcome end how they compare to standard haemodyuamic and oxygen trensport parameters. methods: patients ( males and female) urtderwent surgical replacement of abdominal aortic anonrysm with the aortic simple or bifurcated grail. patients were operated on eleetively, had an emergency surgery for raptured enentysm. haemodynamic, arterial ph and bicarbonate concentration measurements were taken before and after induction of anaesthesia, after cross-clamping and declamping of the aorta end at admission to itu. phi calculations ware done at the sime time except before induction, as tonometers were inserted after patient had been asleep. we used pulmonary utilizing a computer billing survey as well as hospital service transfer data from a month period ( / - / ), all obstetric patients that were transferred to the med/surg icu were identified, as well as their diagnoses and procedures. twenty-eight obstetric patients were transferred to the med/surg icu, compared to , deliveries occurring during that time period for a . % rate. the following procedural indications for icu transfer occured: insertions of endotracheal tubes, arterial catheterizations, temporary tracheostumy, and venous catheter. the following medical diagnoses were found: hypertensive/eclamptic episodes, hypotensive episodes, pulmonary embolism, mitral stenosis, and coagulation deficiency. there were four episodes of respiratory failure ( %). the historical rates of respiratory failure from the medical literature seen at a university hospital ore higher than those at our community hospital despite comparable icu transfer rates for critically ill ob patients. objectives: to evaluate the usefulness of several isss used in order to predict risk of death (rd). design: prospective data collection for months in a multidisciplinary -bed icu. methods: data from consecutive patients admitted to the icu from / / to / / were studied. all the necessary informations were stored in a computer using special software for every day computation of three isss (saps, saps ii, apache ii) and of the rd predicted by saps ii, mpm , mpm and odin. data from days of hospitalisation were used for comparative evaluation of isss and rd, while sensitivity and specificity in death prediction (cut off point of rd = %) were evaluated from data collected during the day of admission (saps ii, rpm , odin) or after hrs (mpm ). agreement between rd predictions was evaluated by bland and altman analysis. results:our results were the following: objectives: a) to create and validate software specially designed for the collection of demographic data and estimation of illness severity and predicted rd and b) to provide data necessary for the evaluation of the effectiveness and the efficiency of our icu. design: prospective data collection in a multidisciplinary -bed icu. methods: prospective data on specific physiological variables, selected demographic characteristics, comorbidity and the reason for icu admission were recorded every day for consecutive patients admitted to the icu from / / to / / . data were entered into a portable computer using specially designed software allowing the calculation of usual illness severity scoring systems (isss) and the corresponding predicted rd. results: we studied men and women, with a mean age of years. we conclude that, although observed mortality was higher than predicted rd, this difference was not statistically significant. the fact that the sd of predicted rd was too large, limits the usefulness of isss predictions for a comparative evaluation of different icus. objectives :the importance of objective evaluation of patients prognosis at their admission to an emergency department is clear. the aim of this study was to appreciate the prognosis of patients with the simplified acute physiology score ii (saps ii), and correlate this score to the prognosis and orientations of patients. methods : the score which is a reduced form of saps ii was calculated from clinical variables and laboratory items for patients. sensibili}y (se), specificity (sp) and accuracy (ac) were calculated to evaluate the performances of saps i . thresholds were calculated with the youden's test (se+sp- ). results : patients were admitted in the intensive care unit (icu)and patients in medical units; patients died. the saps ii was higher in patients which died than the survivors ( , + , vs. , + , , p< , ; respectively), the best threshold was , se was de %, sp was %, the ac was / . thus for patients admitted in icu than in medical unit ( , : : , vs. , + , , p< , ; respectively); the best threshold was , se was %, the sp was %, the ac was %. conclusions : these preliminary results suggest that saps ii can be used to determine the short term prognosis and the destination of patients seen in an emergency department. design: data was collected on all admissions over a six month period. all patients had a screening hiv elisa test. confirmatory ifa tests, western blot and flow cytometry were performed on hiv positive patients. the institutional ethics committee considered the major clinical impact of the study sufficient cause to waive the patient's right to informed consent, icu staff and patients were blinded to hiv results. following discharge patients were given the option of being advised of their hiv status. setting: the study was conducted in a bedded surgical icu at a tertiary care teaching hospital. patients, participants: all patients admitted during the period in question were included. interventions: "all patients were treated according to standard icu protocols. there were no interventions. results: most patients were admitted following trauma. there were no patients with aids, hiv positive and hiv negative patients. with respect to the latter two groups, the mean age and sex distribution was similar, there was a significant difference in organ failure ( % versus %, p < . ) and septic shock ( % versus %, p , : ) but no difference in icu/hosp[tal mortality or duration of icu/hospital stay. flow cytometry changes in hiv positive patients were consistent with the quiescent phase of hiv infection. conclusion: while morbidity is higher in hiv positive patients, there is no difference in mortality. hiv status should, therefore, not be an exclusion criterion for icu admission in this patient population. , n= ) and january-march (period , n= ) were studied. interventions: a resident micu team led by a trained intensivist took over the medical care from the primary physicians when the patients were admitted to the micu. the intensivist also vetted micu admissions and decided on micu discharge. in addition, there was a resident respiratory therapist to attend to vendlatory care during office hours. after office hours, the care of the micu was delegated to the on-call team on a rotational basis among the medical departments. results: the patients in the two groups were similar with respect to age, sex, race, source of admission and apache scores. the icu and hospital mortalities decreased from . % to . % (p=ns) and . % and . % (p=ns) respectively in period . the mean icu and hospital stay was also decreased from . __+ . to . + . days (p=ns) and . __+ to . • . days (p=ns) respectively. the improved micu length of stay for survivors in period was statistically significant ( . • . days vs . • . days = . ). there was no significant differences in the vse of peritoneal dialysis ( . % vs . %) and mechanical ventilation ( . % vs . %). however, utilisation of intra-arterial lines and pulmonary artery catheters increased from % in both to . % and . % respectively in period . conclusion: the duration of critical illness was reduced in period . hence, we believe that the closed |cu which was staffed by an intensivist and had the services of a respiratory therapist was beneficial for the care of the critically ill. the intensivist, was also more likely to utilise invasive monitoring. in the absence of liver transplantation, intensive care for patients with acute hepatic decompensation arising from alcoholic liver disease (ald) is indicated only for those who are likely to benefit from conventional means of organ support. we have attempted to elucidate potential risk factors and the efficacy of organ support in relation both to hospital outcome and icu costs so as to allow the earlier identification of relatively futile intensive care in this group of patients. methods: over a period of one year, ald patients ( males; females; median age years, median apache ii score ; hospital mortality %)with severe complications ( [ %] with bleeding oesophageal varices, [ %] with hepatorenel failure, [ %] with respiratory failure, [ %] with septic shock, and [ %] others) were studied in our liver icu. organ system failure, daily therapeutic interventions and icu costs were assessed using a patient management system (riyadh intensive care program). results: whilst survivors had a significantly lower admission apache ii score compared with non survivors (medians and , p< . ), or more organ systems in failure in the first hours of icu admission was associated with a % ( ) mortality. mortality was also related to child's grading and the numbers of organs supported. of the patients who received more than one form of organ support only one survived. organs supported mortality a (n= ) % none / ; % b (n= ) . % one / ; % c (n= ) . % two or more / ; % the icu costs for the patients were only s but s ( %, patient days) was utilised by the patients who died, giving an effective cost per survivor (ecps) of s . although the cost of treating the patients with or more organ systems in failure on the day of admission was s ( % of the budget) the ecps was s . conclusion patients with ald who have or more organ systems in failure early in their icu course have a poor prognosis and in the absence of liver transplantation, traditional strategies of organ support are ineffective. this begs the question whether such costly care is appropriate as although the ecps in this sub group is comparable with other patient groups with multiple organ failure, their outcome is considerably worse. objectives: to evaluate the economic impact to the healthcare sector of using dopexamine hydrochloride infusions to deliberately increase perioperative oxygen delivery in high-risk patients. methods: effectiveness data were obtained from a controlled clinical trial in which surgical patients were randomly assigned to either a control group (n = to receive best standard optimal fluid resuscitation perioperatively, or a protocol group (n = ) to receive, in addition, an infusion of dopexamine hydrochloride to increase the perioperative oxygen delivery index to greater than ml/min per square metre. resources consumed by patients during treatment were recorded prospectively and costs of resources at / prices were obtained retrospectively. cost-effectiveness ratios were estimated by dividing the total cost for each treatment group by the total number of patients in each group and by the clinical outcome of mortality days postoperatively. results: in the protocol group, there was a % reduction in mortality days postoperatively (p = . ), a significant reduction in the number of complications (p = . ) and a reduction in length of stay in the icu and surgical ward. the average cost per protocol patient to achieve a . % survival rate days postoperatively was s , , resulting in an average cost of s , per surviving protocol patient. the average cost of a control patient was s , to achieve a . % survival rate days postoperatively, resulting in an average cost of s , per surviving control patient. the use of dopexamine to deliberately increase oxygen delivery perioperatively generated a cost saving to the nhs of s , per patient together with a % reduction in mortality at days postoperatively. hence, the cost per surviving protocol patient was s . less than the cost of a surviving control patient. conclusion: the additional cost of dopexamine in protocol patients was offset by a lower incidence of complications and a shorter stay in the icu and on the surgical ward. hence, increasing oxygen delivery perioperatively in high-risk surgical patients saves both money and lives. objective: although king's college criteria are widely used, indication and timing for liver transplantation in acute liver failure is still far from certain. long-latency sensory evoked potentials -a proven measure of metabolic brain dysfunction (grimm et al. lancet ; : - ; madl et al. lancet ; : - ) -are markedly affected in patients with acute liver failure. serial sensory evoked potential recording should provide very useful information for the management of patients with acute liver failure. methods: recordings of standard bilateral sensory evoked potentials were performed in patients with acute liver failure. findings were focused on cortical n peak -a stable negative deflection occuring • ms after median nerve stimulation in healthy subjects, which can be recorded by skull electrodes over the sensory cortex. results: nine patients recovered spontaneously, patients were referred to emergency liver transplantation, and patients died. in all patients who recovered spontaneously, n peak was detectable between and ms. all patients who were referred to liver transplantation and of patients who died, developed a loss of a prior detectable n peak during the course of acute liver failure. objective: to determine high risk arid low risk patient groups in a medical intensive care unit regarding short term and long term outcome. methods: data on all admissions of the year were collected prospectively. according to their mode of admission patients were classified as admissions by the physician staffed ambulance service (as), admissions through the emergency department (ed)i referrals from non-intensive care wards (ni), and secondary referrals from other hospitals (oth). outcome was assessed in terms of in-unit mortality, in-hospital mortality, and long time mortality. patient groups were compared using the \ -test. life table analysis were per{ormed by kaplan-meier method comparing patient groups by log-rank test. the software spss for windows . . was used for statistical calculations. results: in-unit mortality: / patients ( . %) --oth . % > as . %> ni . % > ed . %; p = . . in-hospital mortality: / patients ( . %) --oth . % > ni . % > as . % > ed . %; p = , . mean survival time in days after discharge: as < ni < oth < ed ; p = . . conclusions: despite an excess in-unit mortality of secondary referrals from other hospitals the iongtime course of this special patient group is not different to others. solsuam, j, marrugat*, g, mirs, j, nolla, a, vazqu~z-sanchez, l alvamz, ~ioio s xndioina i~siw. ir~itate l(~icipal da l~sti~isn l~di~*, ~ospits dal objective: to study the influence of modifiable variables (complications derived from therapeutic activities) on the prognosis of ~atients admitted to the icu indapemently on thn severity of illnsss. patients am methods: between january asd ]lay data from , patients over years of aqe who retained in the icu for mare than hours ~ere pr~pectively regiatered. a cohort st~ly with follo~-~ nf patients durin~ ~eir stey in the hospital was deni~.el in all patients, reasons for a~issien, principal diagnosis sad severity of illn~s moasared by the saps scare vare recorded. fastens affecting patients' outcome that my be proventsd or modified included technical :omplisafioss, heapital-acqnired infections and in~pro~riate therapeutic decisions. a logistic regression model was used to assess the relative risk (l~} for in-heapital mortality adjusted for each variable. results: ic~ mortality ~s . % and in-hospitul mortality . %. patients who died showed a higher spas score then survivors ( , ~ i ,i). after adjusting hy severity of illness, co~;licetices that statistically increased the risk of in-hospital death were septic shock secomery to hoapitul-acqdired infection ( ~ . ; % el, . to . ), pmo~othor~x related to mocasnical ventilation (@ . ; % cl, . to . ) and delay in the insertion of a fln~-quidod catheter (ii~ . ; % ic, i.i to . ). col~lusien: registration of complicaticas derived from therapeutic activities is a valuable tool far quality central in the icu. g, ~i~ , j.l mle~ma, j, ~amqat*, j..~lla, a, vazquez-saltemz, f, alvamz , servioia de nndicina l~siu. i~stitutu ~icipal de ln~sti~acidn ~ i:a*, hospital dsl objective: to dstsr~ine the incidence of self-extebatien and its effect on ~ortality. patients and ]~etheds: betveen january and april , all i~tiente in whom selfextubatien w~s registered were inclnded in a prospective study. patients were divided into @nee who needed r~intabatinn within hoers and those who did not. in all patients, dsmoqraphie and ciinical data were recorded as well as icii mortality, in-hoapital mrtality and severity of illness according to saps score. eta were analyzed usi~ the cbj-square test for cathgorical verinbls, the analysis of varianc~ (anva) for aontinuc~ ~ria~les and a leqi tic regression anal~is to estimate the relative risk (iiii) for mortality as result of celt-nxtt~ation after adjusting for severity of illness. results: a total of intnmtsd patients amre stndied. self-extu~atien occurred in ( . %) patients and . % required reintuhot~pn. when a co,arise was made between patients who did not required reint@atinn and patien~.s who did, statistically significant differences in eqe ( . v_s . years, p = .~ ), ~verity of illness ( . ~ . spas score, p = . ), dia~isstia category ( s. % v_s . % of patients with res~iratury conditiono, p = , } and mean length of stay ( , ~ , days~ p = . ) were fo~m, a~ter ad~sti~ for severity, patients with self-ext@atinn who did not reqnired reintalatien showed a . iir for mortality ( % ci, .i to . ) as co~arod with patients in when self-ext@ation did mot occur. conclnsien: self-~extamtice that does not require reint@ation is associated with a isamr in-hospital natality probably dt~ to a prolonged period of weaming. patients' admissions to ices am often delayed doe to the shortage of beds available. @ile amaltieq icu admission, these patients are treated in observation nits of @e emergency services which bare ,either tile structure nor the trained ~reomenl that are available in leb~. objective: to daterdno the effect on the patient's proqusis of a delay in tile admission to the icu when criteria for icij admission are fulfilled. ~terials and methods: between jme am l?ece~ber all patients who fulfilled criteria to be almittod to the ic who for waste~r reason retained in tile observation unit for more than hours were included in a prospective stedy. in all patients, des~raphic end clinical dabs amre recorded as well as severity of illness aencrdi~j to saps score. a cesucontrol dasi~ was eend with a total ss~ln of , patients who suffered no delay is admission to icii over a period of years. data wen analyzed using the chl.-squ~re test (to aeons the association hetwenn in-patienty mortality end categorical vari~lns) and a maltipln logistic reqression model to sstimta odds ratio for) for in-hospital mortality as result of delay in icy admission as compared with early ad~issi| after adjusting for severity of illness end use of assisted mchenical ventilation. ~ &ults: a total of patients remained in the observation nit for more than hours with a del w in igd admission of . _+ . hoers. assisted mechanical ventilation was requited in % of patients and only monitericatien in %. itsse patients were cspared with ntients from the tet~l sample ratchod by age, sp~ score and rennoss of admission. in-hospital mortality for cases warn % as compared with . % for controls (p = s). after adjamtilg fen spas, age and mobamioal ventihtien, no statistically significant differences between both ~renpa were foam, altho~b there was a tendency towards a higher mortality amen@ patients with delay in icu admission (or = . ; % ci, , to , ). conclnnien: ~se findings suggest that prognosis of critically-ill patients is no worse as a result of admission to the loll being deln~d for borers. all data appropriate for the calculation of the apache ii score (aps) together wi'th other specific cardiac details relevant to these .patients were collected daily, verified and enter~ into a computer database. results: patients were studied. six patients died and five of thee underwent cardiac surgery. the mean aps was for survivors and t for non-survivors (p < . ). the mortality ratio was . and the major markers of mortality were apache ![ score, presence of chronic ill health, mean duration of ventiiation, mean length of icu stay and need for emergen~ surgery. sixteen percent ( ) of icu bed days were occupied by % of patients (non-sarvivors) which resulted in cancellation of cardiac sot#cat sessions in momhs. conclusions: this study concludes that apache t could be used as an audit tool in a cardiac surgical icu and demonstrates the severe compromis~don of cardiac surgical throughput by a few non-survivors, organ to determine the number of organ failure free days (offd) in a cohort of survivors and non-survivors with sepsis syndrome followed over a day period. ) to determine sample size requirements for clinical trials utilizing a increase in the number of organ failure free days as the primary outcome as opposed to mortality. methods: beginning december through to april , patients who met inclusion criteria of the "cardiopulmonary effects of ibuprofen in sepsis syndrome" and who did not have hiv/aids. brain death or moribund state were prospectively identified. presence or absence of failure of organ systems (pulmonary, cvs, renal, hepatic, gi, hematologic, & cns) was recorded daily until death or until days. a score of one was assigned to each organ system free of organ failure in patients still alive, ie, maximum daily off score= , maximum day off scorn= , sample size estimations were performed for variable detectable differences in off scores (delta). alpha was set at . (two-sided), with n/group = [(z a +z b ) o conclusions: a clinically relevant increase in off days may be detected with as small a sample size as to patients per group. this represents a significantly smaller sample size than needed to detect a change in mortality from % to % ( % relative risk reduction) where the n/group= . scoring patients in this manner prevents a lethal inte~entien from providing an improved organ failure score. in addition, an intervention that prolongs survival must also provide greater organ failure free days in order to be counted by this scoring method. survival as an outcome provides no information about the quality of that survival. off days provides a measurement of burden of illness. interventions which lessens this burden may be just as valuable as those that decrease mortality by providing a measure of the quality of survival and by decreasing costs of care. they may also prove to be an accurate surrogate marker of mortality. the advantage of this approach is that the event rote is much higher and sample size requirements are subsequently smaller. this would mean that clinical trials can be completed faster and at lower cost. outcomes such as mortality could then be assessed at a later date utilizing recta-analysis. we suggest that the use of off days is a valid outcome measure that may be utilized in clihieal trials of sepsis syndrome. the icu is perceived by many as being a stressful environment for both patients and staff. stress has been defined in three ways: a stimulus producing a particular response; the physiological and psychological response to a stimulus; an interaction butwom an individual and their environment. stress is currently thought to be a dynamic system of stimulus and. response which takes into account the individual's perception of the stimulus and their ability to respond effectively. stress may, therefore, be positive and allow personal development but an individual unable to respond effectively to a stimulus will experience negative effects or strain. critical illness is an intense stimulus to which the body needs to respond effectively. physiological responses are vital and most of intensive care involves supporting these. alternatively, blocking them, for instance with atom(date, increases mortality. psyehological responses are also vital but often poorly appreciated because of communication problems. many of the problems patients experience in an icu are evidence of psychological strain. this can be exhibited in various ways, for instance, anxiety, depression, passivity and confusion. dealing with critically ill patients is perceived as stressful. we recently studied occupational stress in our icu. most aspects of intensive care were not generally perceived as stressful indicating a self-selectien of icu staff. the most stressful aspects of icu work for nursing staff were the structure of the organization and career opportunities. medical and nursing staff had different stressors and different coping strategies. support for occupational stress, therefore, should focus on the individual and concentrate on information and communication. atmosphere, and especially at intensive care units, we face up to daily decision making. in most cases these are taken on the basis of personal opinion and the processing of a very limited amount of information. rising need to optimize the results of medical attendance becomes necessary to set structured system of d@cision making in which ethical basis have a sp@dial significance in view of next considerations: -we live into a pluralist society in which the importance of values is different. -most persons consider health as the first value only in the event of illness. -medical resources available are limited, whereas medical, attendance demand from population increases in a way many people consider it unlimited. in consequence, it becomes necessary to set up priorities in patients treatment. ehtical basis that rule decision making are essentially these ones: i. beneficence: to provide the patient that is being treated the highest profit. . non maleficence: it is our first duty to avoid hurting or damaging the patient."primum non nocere" . autonomy: in every particular medical attendance, the patient has ability to decide by himself. . justice: as equity: to provide the same treatment for those who have the same pathology, ignoring another factors such as age, sex or race. severe application of these principles can cause difficulty, which resolution requires a systematization of decision making. ( - ) . the lenght of stay between survivors and non survivors didn "t show statistical significance (p = . ). the mean aiii score when considering all admissions was , ( - ) . the initial score between survivors and non survivors showed ststistical difference ( . vs . ) respectively (p < . ). univariate logistic regresion analysis demostrated a % increment in death probability for every points augmentation in the aiii score with a sensitlbity of . % and specificity of . %, the roc curve showed that the best cut off point for death prediction was points with a sensitivity of . % and specificity of . %. if a patient is classified as high risk (> ) the bayesian analysis showed a . probability of death and for one class(fed as low risk (< ) a death probability < %. conclusions: the first day aiii score in this population showed to be a good discriminator between survivors and non survivors, and the risk of death augments as the aiii does. in this population an aiii score > points is asociated with a greater risk of death. using the aiii score in conjuntion with the clinical judgement will help clinicians reducing uncertainty in the every day decision making and better predict outcome, the results from this study should been taken with caution because the data were obtained from a small sample. objective: the quality of life has been considered a "uniquely personal perception" resulting from a mixture of health related factors and social circumstances [t. m. gill, jama , : ] . the aim of this study was to evaluate two measures of pqol in intensive care unit (icu) admitted patients. patients and methods: during icu stay and six-months after hospital discharge, co-operative icu admitted patients were directly interviewed about their pqol. we administered ftrstly the uniscale (pqolu) [sage et al crit. care med. , : - ] and then a step verbal scale (pqolv): best, good, fair, poor, worst. of the studied patients, at the first interview, were able to use both scales, but ( . %) understood only the verbal one. at the second interview, patients were not able to answer, used both scales and only pqolv. statistical analysis was performed using wilcoxon signed ranks, spearman rank correlation, student's t and chi square tests. results: of all cardiac surgery pts, pts ( . %) died in icu. they were males ( . %) and females ( . %). their mean age was (+ ) years and mean ef was . (+ . ). nineteen pts ( %) had low (< . ) preoperative ef. mortality was . % in the coronary artery bypass grafting (cabg) group (n= ) and . % in the valve replacement (vr) group (n= ). in the cabg +vr group, mortality was . % (n= ), and . % in the remaining pts (n= ). cardiogenic shock was the sole cause of death in pts ( %), septic shock in pts, whereas sepsis in combination with ards in pts, sepsis and stroke in two pts. in addition, pts died from cerebrovascular accidents, one from ards and one from pulmonary embolism. the pts who died in the icu had a significantly longer bypass and aortic cross clamp time and received more blood transfusions (p< . ) than a matched control group that survived to icu discharge. the duration of mechanical ventilation and length of icu stay were greater in the pts who died in the icu than in the control group. conclusions: . although cardiogenic shock is the main cause of death ( %)in cardiac surgery pts, sepsis and cerebrovascular accident are relatively frequent causes. . patients who died in the icu had longer bypass and aortic cross clamp time and received more transfusions, compared with the control group. . although renal or hepatic failure contributed to death in some pts, they were not the primary cause of death in any patient. objectives: evaluate the acute and follow-up outcome of patients (pts) treated with primary ptca (without prior thrombolysis) in acute myocardial infarction (ami) after and up to hours after onset of typical thoracic pain ("late" primary-ptca). methods and patients characteristics: from / to / consecutive pts with ami were treated by primary ptca in the wuppertal heart center pts ( , %) were admitted to our hospital > hours and < hours after symptom onset with ongoing chest pain and typical ecg-changes.mean age was years ( - ). pts were male, four female. % had an anterior wall myocardial infarction, % suffered an inferior/postero-lateral wall myocardial infarction.two pts were in cardiogenic shock at admission. singlevessel-disease was documented in . %, multi-vessel-disease in . %. average time of onset of pain to recanalisation was min ( - ). angiography revealed timi-flow in . % of the pts, timi-flow i in . %, timi-flow ii in . %. average follow-up (fu) period was months ( - months). timi iii lv-ef ~ -day major late re-late flow p.i.* aeute/fu mortality bleeds infarction mortality . % %/ % . % . % . % % early mortality occured in the two pts, who were in cardiogenic shock at admission no pt required emergency coronary artery bypass grafting.restenosis > % was seen in % of the pts. conclusions: "late" primary ptca achieves a favourable high recanalisation rate of about % (timi ill-flow) in our study group. additionally, there seems to be a trend for lv-ef improvement in follow-up. early high mortality is influenced by the patients admitted in cardiogenic shock. there might be a trend for increased major bleeding complications. objective: to assess the validity of saps ii (new simplified acute physiology score), comparing it with the previous version, (saps), in a sample of patients recruited by giviti, a network of icu's representative of the italian icu system methods: measures of calibration (goodness-of-fit statistics) and discrimination (receiver operating characteristics curve and area under the curve) were adopted in the whole sample and across subgroups differing in relevant prognostic characteristics. of the patients recruited during one month period, a total of patients were included in this study. for the purpose of the comparison of the two scores, patients with less than years, or having cardiac surgery or staying in the icu less than hours were excluded. vital status at icu discharge in the whole sample and at hospital discharge in half cases wher adopted as outcome measure. re$ ~: saps ii fits the data equally well compared to the older version (goodness-of-fit p= . and in the new and old versions, respectively) but its performance is somewhat better in terms of capability to distinguish patients who live from patients who die (areas under the curve . and . , respectively). furthermore, saps ii is better in terms of uniformity of fit across relevant subgroups, although substantial over prediction of mortality was observed in trauma patients and in patients admitted without organ failure to be intensively monitored. saps ii performed very wet] also in the subsample where hospital mortality was the dependent variable.satisfactory measures of calibration (goodness-of-fit p-- . ) and discrimination (receiver operating characteristics area= . ) were observed. c nr saps ii, a multipurpose scoring system developed in an international study, retains its validity in this independent sample of patients recruited in a large network of italian icus. although it has shown a good performance when adopted to predict icu and hospital mortality in the entire sample, further investigations are warranted. the observed over prediction of mortality in a few subgroups indeed call for a through assessment of the impact of confounders and biases on model performance when saps ii is adopted in samples that do not reflect the "average" icu patient. objectives: ) assess the effectiveness in a group of intensive care units by means of a quality performance index (qpi); ) assess the efficiency by means of a resource use index (rui); ) evaluate the performance of individual icus with respect to both indices (clinical and economical) while controlling for severity of illness. critical from ucis in catalonia patients alearic islands have been included in the study. inhospital mortality and weighted hospital lenght-of-stay (los) have been considered the outcome variables. severity of illness has been measured with the mpm ii at admission. in each icu, expected mortality has been obtained adding the probabilities of dying for its patients. expected los has been estimated adjusting a second order polynomial to the severity of illness. performance indices have been obtained by dividing the observed by the expected outcomes. re~ult~: the overall qpi was . and it ranged from . to . in the icus. the overall rui was and it ranged l~ont . to . . there was not a trade-offpattern between clinical performance and resource use. objectives: teaching hospitals often provide [cu care across a variety of specialized services. overall, this approach appears to result in the best risk adjusted survival rates, but at the highest cost (critical care medicine ; : - ): recently, there has been increasing focus on markers of overall hospital performance. however, in large teaching institutions, such markers may fail to detect intra-institntional variation at a large tertiary care medical center. methods: first intensive care unit (icu) day, acute physiology and chronic health evaluation iii (apache iii) and active therapeutic intervention scoring system (tiss) data were collected on random admissions to specialty icus with beds (range - ) between february i and december l, . post-operative solid organ transplant recipients were excluded. units included general medical, general surgical, and trauma, neurosurgery, cardio-thoracic surgery, and coronary care units. data were analyzed for risk adjusted outcomes: icu and hospital mortality and length ef stay (los); risk of requiring active cu treatment; and icu readmissinn using apache iii risk prediction models. results: the study icus cared for a diverse group of patients. mean apache iii scores ranged from . - . ; predicted risk of hospital death ranged from . - . %. standardized mortality ratios ranged from . to . with icus performing significantly better and performing worse than predicted (p< , ). los ratios and icu readmission rates ranged from . to . (ns) and . to . % respectively. patients predicted at low risk of requiring active icu treatment ranged from , to . % conclusions: there was wide variation in the mean level of patient severity between icus. after controlling for this severity, outcomes also varied widely. no clear pattern of overall institutional performance was evident. these data suggest that efforts to assess performance, improve quality, and maximize efficiency must be focused within individual units. programmatic evaluation of outcome allows for focused review of the processes of care contributing to good outcome (best practices) and where to focus ongoing quality improvement and cost reduction activities. background and method : we compared icu mortality in different age groups presenting with the same severity of disease. we assessed severity of illness by the physiological day -apache~ (physio-aa) score (thus excluding the age related points). for each of the following physio-a n score intervals ( - ; - ; - ; - ; > ) , we compared tcu mortality within age intervals (< ; - ; - ; - ; - ; > years - , - , - ) . in these groups mortality may be twice higher in the > years patients than in the _< years. mortality does not vary with age in low (physio a n = - ) and high (physio a n = > ) risk groups. in the low risk group, mortality is low in all the age intervals because of the begninity of illness. in the high risk group, extreme severity of disease probably blunts the impact of age and leads to high mortality rates in all age intervals. introduction: to access the actual social/clinical outcome of the patients who undenvent intensive care therapy oct) is rather difficult, quality of lilr is not easih.' defined and ohserver subjectivity is a prime factor in the evaluation. mortality ratio after discharge must be established and its causes understood. obieetives: the propose of this stud)-is to look into the mortality ratio that occurred on a series of patients that undorwent ict at our unit from of the ~iew point of severity of the original illness and the diagnostic groups. material and methods: during the period of one )-ear ( ), patients were treated at the unit, of them died, and ~ere not matched in our series because os incumpletc records. thirteen patients died in hospital after their reference to other departments, twelve patients were lost after discharge. thus. at the end. only patients were evaluated on the fu. the, were classified into the follov ng three groups: acute medical, elective surge d and acute and emergency postoperative. the patients were seen at , and months after discharge. the, were evaluated in accordance to their abili~, to being self supported in their daily life and capecity to fully return and hold to their pre~ ous jobs. apache scores were evaluated for each of the three groups and correlated to the icu dead, hospital dead, and mortality after hospital discharge, spss package was used for statistical analysis. remlts/conclasions: data shows that / patients died after discharge from the hospital, of ~itch nine died in the first three months. seventy-eight per cent of the patients were fully self supported in their daily life and % showed some kind of handicap. fosty-nine per cent of the patients wore on retirement either due to age or some form of chronic disease, when admilled to our unit. thirty-two peg cent had not been able to return to work, because the" were incapacitated on discharge. only % had return to their fully jobs but the period of the stu~, is not enough for all of them to be fully physically recovered. preliminmy statistical analysis shows us significant differences among groups. the aim of the present study is to compare the prognostic performance of five general severity indices ou coronary patienta and to find out if a proper ntatistical hundling of these indices could provide better results in these patients. methods: saps ii, mpm ii (mpm ii i mpmp ii ), apach ii end gaprik were evaluated o~ patients with acute myocardial infurction admitted to intensive care units from catulunye. calibration and discrimination were calculated for each index. calibration was calculated by th bosmer-lemeshow test. discrimination was evaluated by the area under the relative operating characteristic (roc)curve. if a model did not show a good performance it was customized using multiple logistic regression. finally, tworeduced models were developed, one fro~ the mpm series (mpm ii cor) and one from the group apache-saps (sapsiicor).their performances were again evaluated. results: discrimination was high enough for all models. neverthelees, oelibration of apache ii, saps ii and mpm was not satisfactory. thus,mpm ii , saps ii and gaprik were customized for coronary patients using the logits of both models, and obtaining good calibrations. mpm ii , and apache-saps were adapted and reduced to (mpm ii cor) end to variables (sapsiicor), respectively . both models showed better oalibrutions end discriminations than the original models. conolusion| models developed for multidisciplinary patients show a good discrimination when applied on aoronar i patients, but some needed customization in order to improve calibration. the number of variables of the principal model can be reduced (even to or variables) without loosing prognostic accuracy. objective: to compare the ability of two methods to predict outcome for intensive care patients. methods: we included consecutive intensive therapy unit (itu) admissions with an itu stay> hrs in a month prospective study (exclusion criteria: burn injury and age < yrs). data were couectsd applying the criteria described by the developers [ , ] . the definition of coma (mpm ii) was modified and the best assessment within in's, rather than the admission score, was used. statistical analysis included classification tables and receiver operaung characteristics (roc) curves to assess discriminative power, and lemeshaw-hosmer statistics and calibration curves to test accuracy of prediction. results~ average abe was yrs (ranse: - ) with a male:female ratio of . : . the actual hospital mortality was . %, mean predicted death rates were . % (mpmz ii) and . % (ap hi). non-survivors had siguitlcanfly higher predicted risks than survivors applying both methods (p< . l, t-test). the total correct classification rates (tccr) for apache iii were bett~r for all decision criteria applied (tccr, decision criterion %: apache ]/i . %, mpm ii . %). the area under the roc curve was . (ap iii) and . (mpm ii) confirming the better discrimination of apache ill. accuracy of risk prediction was similar for both models (ap nl ~ - , mpm b ;( - , lemeslmw-hosmer). showing some fluctuation, calibration curves lay close to the ideal line for predicted risks -< % with increasing deviation for higher risk groups (s. figure) . apache iii underestimated the risks of hospital death for almost all risk groups (curve above diagonal), whereas considerable overestimation for predicted risks > % ceenred with mpm~ii. objective: to assess the goodness-of-fit of the apache iii model for british itu patients. methods: we prospectively studied a cohort of adult patients consecutively admitted to a medical-surgical itu over a period of months. patients with burn injury, age < yrs and itu stay < hrs were excluded. using a eomputerlsed database, we routinely recorded hrs apache ill scores. predicted risks of hospital death were computed by critical audit ltd, london. accuracy of risk prediefion was assessed by hosmer-lemeshaw chi square (;( ) statistics and calibration curves [ ]. discrimination was tested employing classification tables and receiver operating characteristics curves (roc). restths: the mean age of the male and female patients was yrs (range: - yrs). of these patients, % were medical admissions, % were admired after emergency and % after elective surgery. the observed hospital mortality was . %, the overall mean predicted death rate was . %. mean predicted risks were siguifieanfiy greater for nonsurvivors ( . %o) than for survivors ( . %, p< . l, t-test). apache iii showed good calibration (z -~ , lemeshaw-hosmer). however, the calibration curve lay above the diagonal for almost all risk groups reflecting the tendency to underestimate actual mortality (s. figure) . the best total correct classification rate (tccr) was . % (decision criterion: %). the area under the roc curve was . % confirming the good discriminative ability of the model. objectives: the aim of this study is to point out the discrepancies between needs and actual treatment of less severely ili patients admitted in italian intensive cam units (icus) requiring only intensive monitoring, and verify the substantial likelihood of data comparing those collected from a national short term study with a regional long ternl use. ~: less severely ill patients ("observed patients") were only monitored; they did not require intubation, even if for a short period (less than houm) or major cardioeiranlatory supports, and were neurologically normal. epidemiologieal national data were obtained from giviti group (gruppo italiano valutazione interventi in terapia intensiva); this cohort study, collected patients, in two months in summer in all over italy. regional data were echieved in a three years entlection ( -i ) in lombardia' icus from archidia group (arehivio diagnostieo), including patients. mortality, severity score, diagnostic category and some typical intensive procedures were analysed and compared in both studies. patients' disgunstie categories were defined as surgical, medical and trauma, according to the main diagnosis and the presence/absence of surgical procedures. rr observed patients account for . % and % of all icu's patients respectively in national and regional data. very tow mortality rate was found in national data ( . %) and extremely low mortality in regional data ( . %). in both studies mortality, s.a.p.s. and length of stay were much lowor in "observed patients" than in general icu's population (mortality: . % and . %; .a.p.s. score: . and ; iength of stay: % and ). homologous distribution of patients in the two studies was noted for what concern their diagnostic category, aside from a slight prevalence of tranmatised patients in the giviti study. in the two groups the surgical patients were respectively % vs. %, medical patients were % vs. % and traumatised were % vs. %. % of "observed patients" in national study and % in the regional did not received any intensive procedure. only a minority of these patients availed haemodynamie eonu'ol with swan-ganz or renal haemofiltration. conclusions: these results underline that about one fourth patients admitted in italian icus benefit an oversized slructure i, relation to the real needs of their pathology. in hot more than % did non received any advanced treatment and mortality and s.a.p.s. score were substantially lower respect to general population. the results obtained from these two studies are similar, suggesting an uniform distribution of the case mix in italy, even if a different recruitment period and a different gengraphieal distribution were used. some discrepancies in the two studies were found in the diagnostic categories moreover regarding the tranmatised patients ( % vs. %); this can be explained from the seasonal (summer) characteristic of the national study. mutuality, yet very low, is different in the two groups, but these data do not allow any definite explanation. finally these epidemiologieal survey suggest need of further studies settling more strict criteria of admission in icu. this study aims to evaluate patients outcome, quality of care and effectivity of therapy in our intensive care unit. the main goal was to indentify factors that the most influence that outcome. during . the authors collected data of patients outcome and predictor variables. overall mortality rate was , %. the most common causes of death were infection. the diagnosis of sistemic inflammatory response syndrome (sirs) and multiple organ dysfunction syndrome (muds) significantly correlate with death ( %). average length of stay was . days ~. % patients died in the first ten hosiptal days and only % after days. age was directly correlated with death % of dead were older then sixty years. an analysis of physiological variables showed that serum levels of gl~cose ( %) and natrium ( %) were in optimal physiological values. serum proteins ( %) and haemoglobin ( %) levels were inversely related to death. multivariate showed that alveolo-arterio difference in content was the most informative of all mortality predictors (mean value , mmhg in % patients io>mrnhg). factor that most influence the patients outcome was infection (sepsis) and muds. use of predictive indicators of outcome in critically ill patients may help to assess treatment regimens and to compare patient groups. acute physiology and chronic health evaluation (apache if) score (crit. care had. ; : - ) and the sepsis score of elebute and stoner (br. h surg. ; : - ) have been used, objectives: to compare sepsis score and apache ii score in predicting outcome of critically ill patients. methods: overall survival during the past years for patients in our icu was calculated = % (prior probability). the outcome of patients who were admitted to our icu for > hours was observed. apache ii score on admission, patient predicted risk of death (apache ii risk) and the sepsis score on the first day of antibiotic course were prospectively recorded. discriminant function analysis of the scores in relation to outcome was performed. results: apache ii and sepsis scores in the survivors were significantly lower than in those who died ( . i . v~s . • . and . • v's . • . respectively p < . ). correct prediction of outcome by each score is shown in discussion and conclusions: although both scores have been previously evaluated in predicting outcome of icu patients, studies of the sepsis score were conducted in small numbers of patients or involved additional measurements not routinely available. this study demonstrates that the sepsis score alone or in combination with apache ii score is more effective than apache ii score in predicting outcome. objective to test the hypothesis that resuscitation titrated against gastric intramucosal ph (phi) improves survival in critically ill patients as suggested by gutierrez et al~. method emergency admissions to the intensive care unit were randomized into control and intervention groups. in the control group phi was measured at , and h while in the intervention group phi measurements were made hourly for h. both groups were managed according to the same guidelines to achieve the following targets: mean arterial pressure > mmhg, systolic arterial pressure > mmhg, urine output > . /ml/kg, haemoglobin > g/dl, blood glucose < mmol/ , arterial oxygen saturation > % and correction of uncompensated respiratory acidosis. if the phi was < . after achieving these targets, or after maximal therapy to achieve the targets, patients in the intervention group were given fluid to ensure an adequate cardiac preload and then dobutamine at then mcg/kg/h, titrated against phi. this additional therapy was continued until h after entry into the study. in each year patients were subdivided in two series with random selection, so that the st series contained abeat / and the nd / of the patients. the st series of all the years constituted the devdoping data set and the nd series the validation data set. with data of the st series ( patients), we created the predictive model, using stepwise logistic regression (bmdp, usa). each patient has been evaluated in die st, th, th and th day, calculating for each lime the apache ii score (for a total of records), independent variables were, besides time and apache ii of the time ( michaloudia g,, melissaki a., alexias g., gogafi c., kolotoura a., krimpeni g., pamouktaoglou f, filias n. objectives: to determine the medical staff's attitude towards various ethical issues methods : between january and february , anonymous questionnaires were sent to intensive care units, all over greece. results : questionnaires ( , %) were replied and returned back. of them , % were answered by male and , % by female. the doctors replied in the following rate : , % aged up to , % aged between and , % aged over . questions were answered and were divided into main topics, as following: . admission criteria: limited bed availability was the main cause for refusing admission in , % of icu's. , % evaluated each case's viability and only , % used some prognostic score system. , % of icu's accepted all cases and a significant percentage ( %) gave in to pressure coming from their colleagues ( , % female and , % male). . informing the patient/relatives: only , % was willing to tell the whole truth, while , % had given selective information.. in the case of iatrogenic incident, , % withheld it, because either they feared legal implications ( , %), or lost of trust ( , %). doctors are asking consent from the patient and/or his family, in order to include him/her in research protocols, in a rate of , %, while only , % found informed consent necessary for the proposed treatment procedure. . withdrawal of therapy/dnr orders/organ donation: , % were willing to withdraw complex treatment in patients with short life expectancy, except of administi'ating intravenous fluids, feeding and analgesics. in , % such a decis~n was unanimous, while the percentage of those carrying it out was , % ( , % female, , % male). in case of brain stem death , % ( , % female, , % male) withdrew any life support. , % would like therapy withdrawal to be legally established, while only , % would perform euthanasia, if there was substantial legal cover. for these cases, relatives' consent was considered to be necessary from a percentage of only , %. , % considered organ donation to be a necessary proposal, while , % refused to ask the patients' relatives for an organ donation, either because they didn't have the psychological strength for it ( , %), or because they doubted the procedures' objectivity ( , %). note: in greece, icu beds are less than % from the total number of hospital beds available. only a percentage of - % of these admissions comes from the same hospital, with a potentially direct evaluation. usually an icu doctor has to be informed through the telephone. finally, employment conditions in greece are such that any changes of the medical and nursing staffare limited. conclusions: the mathematical model we found has been validated also in the second series and the discrimination capability increases with time. using this model we can evaluate the probability of survive at every, time. its application at different times permits a better evaluation of haemodinamically instable patient trend. introduction: the feasibility to assess pulmonary capillary pressure (pcap) offers the opportunity to determine the longitudinal distribution of pulmonary vascular resistance (pvr). the purpose of this study was to measure pcap and to calculate pvr to determine whether relevant shifts in the distribution of pvr could be expected after routine cardiac surgery. methods: the study population consisted of consecutively admitted patients after cardiac surgery. surgical procedures included coronary artery bypass graft (cabg) (n= ) and mitral valve replacement (mvr) (n=t ). pcap was estimated by analysis of the pressure decay tracing after pulmonary artery occlusion. after estimation of pcap precapillary (ra) and postcapillary resistance (rv) was calculated. a complete set of hemodynamic variables was obtained at hour and at hours after operation. results: there were no significant hemodynamic changes during the first hours after surgery. the mvr group maintained pulmonary hypertension and higher levels of pcap. ra/rv, reflecting the longitudinal distribution of resistances, remained unchanged. however, rv predominated ra during the postoperative period in both groups. objectives: evaluation of the influence of long-term continuous i.v. administration of the ace-inhibitor enalaprilat on regulators of circulatory homeostasis. methods: t trauma and sepsis patients randomly received either . mg/h (group i, n= ) or . mg/h (group , n= ) of enalaprilat i.v. or saline solution (control, n= ) as placebo for days. plasma levels of endothelin- (et), atrial natriuretic peptide (anp), renin, vasopressin, angiotensin-ii, and catecholamines were measured before injection of enalaprilat (='baseline' values) and during the next days. results: except for et, plasma levels of all vasoactive substances exceeded normal range at baseline. angiotensin-ii significantly decreased during enalaprilat infusion ( . mg/h: from . • to . • pg/ml; . mg/h: . • to . • whereas it remained significantly elevated in the untreated control patients. vasopressin increased only in the control group (p< . ) and decreased after . mg/h of enalaprilat. et remained almostunchanged in group , whereas et increased significantly in the control patients (from . • to .t• on the th day). catecholamine plasma levels (epinephrine, norepinephrine) markedly increased in the control group (p< . ), but they did not change significantly throughout the study period in both enalaprilat groups. conclusions: continuous i.v. administration of the angiotensin-converting enzyme inhibitor enalaprilat beneficially influenced systemic and local vasoactive regulators of the circulation, which are normally increased in the critically ill. thus patients at risk of (micro-) circulatory abnormalities may profit from enalaprilat infusion. objectives: to determine the time taken for hemodynamic and gas exchange variables to a reach stady-state after a change from supine to trendelenburg position (trp). methods: we prospectively studied adult patients with severe sepsis or septic shock requiring hemodynamic monitoring. usual cardiorespiratory parameters were measured at baseline, min after the patient was placed in a trp and again min after the return to a supine position. a fiberoptic pulmonary artery catheter (svo~ oximetrix, abbott) allowing continuous svo monitoring wa~used. during the protocol we also continuously measured sao~ by pulse oximetry and vco~ and vo by monitoring partial concentration of o and co ir~ inspiratory and expiratory gases (deltatrac metabolic monitor, datex). therefore, we were able to monitor cardiac output variations by dividing vo~ with arteriovenous difference according to the fick equation (co-fick). results: no significant difference in hemodynamic status was observed min after the patients were placed in trp. despite the fact that no significant change was observed in co and vo~ estimated by thermodilution, co-fick had a tendency to dedrease continuously in trp and then to return to its initial value when patients regained supine position. respiratory gas analysis showed a small but persistent continuous increase in vco without a similar trend in vo values. conclusions: we conclude that no significant hemodynamic effect was detected in our patients after min in trp. evaluation of vo from respiratory gases analysis after a change in body's position should be interpreted with caution, since the patient may not yet have reached a stady-state after rain. since vo did not change, vco~ increase was probably due to position related changes in-pulmonary gas exchange and not to a change in patient's metabolic status. objectives: to determine whether changes in svo and/or other hemodynamic parameters during weaning trials could be used to predict successful weaning. methods: we prospectively studied adult patients with a history or clinical evidence of cardiovascular dysfunction, who were unable to tolerate spontaneous breathing (sb) for hours. for all these patients right heart catheterisation was considered necessary in order to detect hemodynamic alterations during weaning. a fiberoptic pulmonary artery catheter (svo ximetrix, abbott) allowing continuous svo monitoring was sod. hemodynamic status was evaluated ~t baseline and after one hour of spontaneous breathing through a t-piece. patients were assigned to one of two groups depending on whether they tolerated sb for hours. data were analysed by analysis of variance and unpaired student's t-test we also used multiple linear regression analysis to determine which hemodynamic variables were correlated with the magnitude of svo~ change and multiple discriminant analysis to determine if asy of the above variables were associated with toleration of sb for hours and/or successful weaning (s-w). (j physiol ; ." - ) . we tested the hypothesis that the ventilatory stimulation by dead space (vd) loading and % co inhalation is accompanied by a proportionate cardiovascular change. methods: six healthy subjects, mean age, year, performed three incremental exercise tests in a randomized order: ) inspiring air without vd (air control, ac); ) inspiring air with vd of ml (avd); ) inspiring % co ; % oxygen, balance nitrogen. the ventilatory responses were examined at matched heart rate (hr) equivalent to % peak hr. results: ventilation (vi) was significantly greater (p< . ) during the avd and co tests than during the ac test at the same work rates. end-tidal co (petco ) and estimated arterial co (paco ) were significantly greater (p< . ) at w and w. oxygen saturation was significantly lower (p< . ) during the avd test than during the ac and % co exerdse. at matched hrequivalent to % peak hr, vi was significantly greater (p< . ) during the avd and % co tests than during the ac exerdse ( l, l, and /). conclusion: we conclude that the increase in xri and petco due to vd loading and % co inhalation is not associated with an acceleration in hr. sup.ported by mrc (canada). objeetlve: the production of large amounts of oxygen radicals from the onset of ~en may be responsible, st least in part, for peroxidative damage to myocardial tissue. the aim of this study was to evaluate the time dependence of plasma tbars in patients with am] receiving thrombolytie therapy (tt). patients and m~hods: filiy eight patients admitted in icu ( men and women; mean age . - . years) rec~ving systemic tt for possible am] were ~died. all patients received recorabinant haman tissue-type plasminogen activator (r-tpa). the mean time fi'om the onset of symptoms and the be~nning of tt was . - . hours. peripheral veao~s blood samples were obtained fi'om each patient before and serially after tt ( , , and hours). tbars levels woe determined by using a spectrophotometrie technique. rq~r fusion was identified by the timing of ereatine phosphate kkmse (cpk) peak (< hours). table i list the variation of plasma eoneenlrations of tbars (mean -sd) in groups (a,b, and c) as a function of time from the beginning of tr. co,arisen oftbe time cuncentzatiens reveal a difference p<o. between group ami (a and b) versus group c. the between-groups (a-c) comparison showed significant differences (p<~). ) at , and hours fi-om de begin~g of'it. comparison ofthe , and hours eonocnlratiens did not reveafl any difference becween n~ (group b) and angor (group c). condmien: we have observed a maintenance of high level of tbars in reperfused patients enly. these results suggest that the increase of tbars levels may be due to phospholipid derangament of reperfused myocytes. objective: to detemline if tbars levels may be an early index reflecting peroxidative damage in ami patients. patients and methods: by using a spectrophotometric teclmiqtm, based on a modificatien oftbe buege and aust method, tbars levels ware determined in healthy subjects (group i) , patients with several risk factors of ischaemic cardiopathy (group if) and icu patients with possible aim ( were confirmed ami (group ma) and were angor (group iiib)). peripheral blood was obtained at the time of the icu admission in the group i . electrecardiographic data and eehocardiographic wall motion were used to identify ami location (anterior, inferoposterinr and indeterminate). statistical analysis was made with a standard statistical package (rsigma, horus hardware). , h , s, , h statistically significative increase of ' ix~e/ea.ee x~* tbars levels (p< . ) only in pstients with con/tuned ami (group ilia) . no differences were found between the rest of the groups. the figure shows the relationship between the tbars values and the time from the onset of the aim's symptoms. conclusion: we have found that in am/patients the tbars levels can be used as an early index reflecting peroxidative damage occurring to ischemie tissues. perioperative risk in patients with ischemic heart disease. the protective role of diltiazem. lg. hatziantoniou, p. tassiopoulos, c. fakiolas, ! g. foussas~ m. lycourinas perioperatlve morbidity and mortality are mainly of cardiac origin, particularly among elderly patients (pts) ~ith ischemic heart disease. we performed a randomized trial, in order to investigate the possible protective activity of diltiazem (d). methods: pts (aged zo• with a history of coronary insufficiency were subjected to major urological operations. they were randomized to two groups: group i ( pts) -p:ithout d and group ii ( pts) -d ~as administered . mg/kg iv bolus hours prior to anesthesia and . mg/kg/h upon the end of operation and for at least hours post-operatively. iv was substituted by oval administration of x mg starting the next day and for the rest of hospital stay. all pts underwent daily clinical, electrocardiographic and laboratory (ck-mb) control. results: are shown in the following interactions between the heart-lung unit and a cardiac assist device are the crucial issues for successful outcome of patients under mechanical circulatory support. several factors have been indicated and we can diffrentiate between anatomical and hemodynamic interactions. especially right heart failure and pulmonary hypertension have been proven to limit left ventricutar assist device (lvad) performance. in order to study the effects of right heart failure (rhf) and pulmonary hypertension (pht) on mechanical circulatory support, we designed an experimental study in dogs and combined in six groups left heart failure (lhf) and rhf and pht, respectively. in the control group we induced lhf with subsequent occlusions of lad and cx followed by reperfusion: in the next group we supported with an lvad during minutes starting minutes after cx occlusion (group if). in the next group we added right ventricular ischemia by occluding the rca simultaneously to the cx (group iii). these both groups we repeated only with pre-existing acute pht induced by the injection of o fm glass beads (groups vi and v). a last group was similar to group v (lhf and rhf and pht), but instead of a lvad, we used bvad support. in i and v no animal survived the second perfusion period ( hours). in ii and iii (no pht), all dogs survived in stable conditions. in iv (lvf, pht), % survived, the remainder died of incomplete recovery. in vi with bvad, only % survived. in iii and v, during occlusion of the rca, the right ventricle stopped contracting and right atrial pressure and mean pulmonary pressure equalized. this lead to a significant drop in inflow on the left heart side, which still provided sufficient lvad performance in case of absent pht (iii) and lead to death due to "low lvad output" syndrome in v. this "low lvad output" snydrome could be partly overcome by bvad support. we conclude that heart-lung interactions play a major role in lvad performance. by optimizing the perioperative management, they all can be overcome, except the occurrence of alveolar leakage syndrome. obiectives : we propose a new approach of transmural pulmtmary artery occlusion pressure (paoptm), in presence ef peep, from the folluwing hypothesis : if respiratory swings of paop are compared to those of alveolar pressure (apalv = plateau pressure -total peep), an index of transmission of airway pressures to pulmonary vessels is obtained (i t = apaop/apalv). the value of the product of it by peep can be substracted from end-expiratery paop (paopee) to obtain a calculated paoptm (paoptrnc). thus paoftmc = paopee -(apaop/apalv x peep). methods : we tested this hypothesis in patients by comparing paopee, paoptmc, and paopnadir obtained within the first seconds after disconnection from the ventilator, value of reference of paoptm in absence of intrinsic peep (peepi). at zeep, peepi was absent in patients (group a) but present in others (group b). patients were studied under the peep level chosen by the attending physician-(gr a : +_ ; gr b : _+ cmh ). results : l. gr a : paopee ( _+ mmhg) differed (p < - ) from paopnadir ( • mmhg) and paoptmc ( _+ mrahg). gr b : paopee, paopnadir, paoptmc differed between themselves ( " - , + , + mmhg respectively). . good correlatkm (r - (i. ) and agreement between paopnadir and paoptm were found in gr a, while there was no agreement in gr b (bland and altman analysis). . lung compliance (v t/bpah,) correlated well with i t in both groups (r - . ), suggesting that our hypt)thesis was strtmg even in patients with peepi. conclusions : paoptm under peep ventilation can be obtained, even in patients with peepi, from parameters easily measured at the bedside. introduction: cardiopulmonary bypass (cpb) may cause ischemia in several organ systems like the heart, brain and kidneys. reactive oxygen species (ros) are formed by subsequent reperfusion and stimulation of neutrophils by cpb. ros are harmful to biological systems e.g. cellular membranes, enzymes~ dna. many natural antioxidant systems protect against the effect of ros. in patients undergoing cabg we evaluated the production of ros by total plasma antioxidant status (tas) and lipid peroxidation measured by lipofuscin flip). methods: tas (randox, uk) and concentrations ofllp (tsuchida et al.) were measured in consecutive patients undergoing cabg. all the patients had a normal serum creatinine clearance (> ml/min). serum samples were obtained a) before operation, b) after removal of the aortic crossclamp, c) at admission to the icu, d) hours after operation, e) hours after operation. results: tas was significantly decreased after removal of the aortic crosselamp ( b, c and d lower than a), followed by a subsequent significant increase of lip ( c and d higher than b). the levels of tas and lip returned to baseline hours after operation. methods: patients with preoperative lvef< % undergoing coronary artery bypass grafting were studied. after surgery, a f femoral artery catheter was inserted and connoted to a fiberoptic monitoring system (cold z- t; pulsion medizintechnik, germany); this allows, with a double-indicator dilution technique, the calculation of cardiac index (ci,l/min/m ), intrathoracic bood volume (itbv,ml/m ), pulmonary blood volume (pbv,ml/m ) and extravascular lung water (evlw,ml/kg). with a f pulmonary artery catheter, wedge (w,nunhg) and central venous pressure (cvp,mmhg) were measured, while extraction ratio (o exr,%) and oxygen delivery (do ,ml/min/m ) was calculed. peak inspiratory pressure (pawp,cmh ) and mean airway pressure (mawp,cmh ) were measured with a varflex flow transducer (bicore,sensormedics,us). the patients were studied after minutes (to) of volume controlled standard ratio ventilation (vc), and after minutes (ti) of stabilisation period of pcirv ( % inspiratory time, % pause). vt,ve and total peep were held constant in every mode of ventilation. +_ . " *'p < , versus to conclusions: these data show that pcirv : is a safe ventilatory support also in cardiac patients with impaired ventricular function, and monitoring of itbv is more reliable to measure and optimise circulatory volume status, than w and cvp. c.ledeki-,g.rldisis,s.karotzai,c.micheilidis,m.agioutantb, g.beltapaulos. objeolivee:to evaluate the influence of lvswl on the well known correlation of sr and svo . paw eight patients ( melee end females) were included in this study regerdlen of the icu ~h"niseion couse. all paints were ,'~theta~ with e fiboroptir pulmonary artery catheter connected with an oxymetfir (r)~ so /co abbot computer.for any pulmonary artery catheter insertion, two pain= of sr and svo were obtained, one dudng inserlion and one during taking the catheter out. for any pair obtained, we eleo collected the deta concemig with the pedient's hemodynamir and oxygenation end we calculated the lvswi. were significantly (p<o. ) higher in group i. conclusion: breathing room air, the less hypoxemic patients had a rather satidactory do and tissue oxygenation (pro ) because they had properly adapted their ci. the absence of this mechanism (right heart impairement ?) in the hypoxemic patients, in spite of higher blood ne and e, was responsible for the poor do and pro . objectives: acute massive pulmonary embolism (pe) increases pulmonary artery pressure (pap) and dght ventdcular aftedoad, which may lead to dght ventdcular failure. inhaled nitdc oxide (no) selectively dilates pulmonary vessels. thus, inhaled no may be of potential therapeutic value in the management of the acute phase of pe. methods: following institutional approval, ten piglets (body weight + kg) were anaesthetised (midazolam/piritramide) and ventilated using a modified servo ventilator (siemens elema, sweden; fio . ). the following variables were obtained: systolic pap (spap), mean pap (mpap), diastolic pap (dpap), mean artedal pressure (map), central venous pressure (cvp), pulmonary capillary wedge pressure (pcwp), cardiac output (co), and artedal (pao ) and mixed venous (pvo ) oxygen saturation. pulmonary vascular resistance (pvr) was calculated. to induce pe, pm microspheres (sephadex g coarse, pharmacia biotech, freiburg, germany) were injected in an amount sufficient to increase mpap to mmhg initially. rain after embolisation when haemodynamics were in a steady state (control ), inhaled no was administered. further measurements were taken min after ppm no (no ), min after ppm no (no ), and min after discontinuation of no administration (control ). the administration of inhaled no resulted in a significant decrease in spap, mpap, and pvr. the cessation of no inhalation led to a complete return to pretreatment control values (table) . co, map, cvp, pcwp, pao?, and pv~ remained unchan no administration. objectivss:evsluate the yield of recombinant tissue plasminogen activater(rt-pa, actiiyse ~ oehringer ingelheim) as a thrombolytic agent ie pulmonary embolism ie .kr~auma patients. methods: in five patients with diagnosed pulmonary embolism(blood gases, chest x-ray, echocardiography, perlesion lung scans) were given rag of actij.yse as ~ hrs infusion, followed by heparin~single j. ihjaction snd iefusion ie doses - j./kg/day).the effectiveness of rt-pg thrembo].ytic sctier~ was svaluated im . . hrs(blood gases) amd on the third dayafter t~eat-,ment(~chocardiography and lung scan). results: three hrs after actilyse was given we observed significant clinical imprevemeet,confirmed by elevation of pao and sao in dlood.echocardiegr~m on the third day showed regression of pulmonary hypertension and lung scans showed % improvement in total lung perfusien. except slight bleeding from the site of injection nc~ more thromboiytic complicatioos were observed. conclusiens: actilyse used in five hrauma patients with pulmonary embol.ism was a very effsctive and safe thrombolytic agerlt, inducing rapid and evident clinical improvement. intermittent positive pressure ventilation ( ppv) by increasing pleural pressure, decreases the pressure gradients for systemic venous return and left ventricular (lv) ejection. we have previously shown that when inspiration is synchronized with systole using synchfjv in patients with severe cardiomyopathy, that cardiac output (co) was increased relative to both ippv and non-synchronized hfjv ( ). however, it is not known if similar effects could be realized in ventilatordependent patients with lesser degrees of lv dysfunction and fluid overload. we thus compared synci-ifjv to ippv in stable patients following coronary artery bypas grafting. methods: normothermic who were stable (< % variance/h.) were studied following admission to the sicu. heart rate (hr), mean arterial (pa), pulmonary artery occlusion pressure(ppao), co by continuous thermodilutiun (vigilance, baxter, usa), mixed venous saturation (svoz) and arterial blood gases (abg) were measured after rain. of each step. ippv was simv adjusted for a rate, tidal volume and fit to insure both normal abg and a peak airway pressure < cm h,o. syncfifjv was delivered by a hfjv ventilator (acutronic) during systole with ventilator parameters adjusted to optimize abg. protocol: patients received three ippv runs (ippvi. , ) with synchfjv runs interposed (hfjvz ). statistics: anova for repeat measures and paired t-test were used to compareruns and demonstrate variability. results:no significant difference in any of the measured hemodynamic variables were seen among the ippv and synchfjv runs (table) . post hoc separation of patients by preoperative ejection fraction (ef; ef > % ; n= and < %; n= ) did not alter these results. back~ound: in man, vascular endothelium-bound ace is expressed in concentrations greater than x that in serum and is believed to be the site of synthesis of circulating angioteusin il it is unclear whether ace inlubitors interact similarly with ace in different vascular beds. coronary vessels possess all the components of the renin-angiotensin system, including ace which may be involved in normalcardiac homeostasis, as well as in the pathogenesis of various cardiomyopathies. obiecfive: to develop a method for assaying the interaction of ace inkibitors with coronary endothelium-bunnd ace in man, methods: ace a~aty was meas~ed in five patients undergoing cabg surgery, from the transeuronary hydrolysis of the synthetic ace substrate h-bpap. trace mnou~ of ~fi-bpap ( gci) were injec~d as a bolus in the root of the aorta and simultaneously blood was withdrawn from a coronary sinus catheter into a syringe containing protease inhibitors which prevented the convession of umeaet~ ai-i-bpap by blood ace. the sample was later centrifuged to separate cells from plasma and the radioactivities due to formed product (~rl-bphe) and total sh were astimated in a [b-counter. two additional such determinations of ace activity were perform~ the second in the presence of . pg/kg e (coinjected with ~-i-bpap) and the third ten minutes after e. results: all subjects were hemodynamically stable throughout the course of the there were no noticeable hemodynamic effects of e. control transcorunary metabolism of~-bpap averaged g -a: %, in agreement with previously reported data. in the presence of e, % metabolism of ~-bpap was reduced to • reflecting a • inhibition of normal ace activity. ten minutes after e, ~ri-bfap metabolism had partially recovered to :l: %, representing a -a: % inhibition of control ace activity. from this data, the dissociation constant of e for coronary ace in vivo was estimated as . x " sec "l. conclusions: we have demonstrated the feasibility of repeated, reproducible measures of coronary endothelium-bound ace activity and of its inhibition by e. this procedure is safe and can be used to study the role of ace in normal cardiac function and in card pathologies. objectives. primary pulmonary hypertension (pph) is a progressive fatal disease of unlmown origin, with median life expectancy of less than three years after diagnosis. the responsiveness of pulmonary hypertension to a variety of vasodilator agents led to the speculation that, concomitant with vascular renmdelling processes, persistent vasoconstriction is an important feature of the disease. long term use of ca-channel blockers and intravenous pgiz may improve mortality in certain populations of pph patients, but both of these treatments lack selectivity for tire lung vasculature. the aim of this study was to test the efficacy of aerosolised prostacyclin and its stable analogue, [loprost for selective pulmonary vasodilatation in pph. methods: in three patients with pph, we compared aerosolisation of prostaglandin iz (pgi ) and iloprost to a battery of vasodilatory agents (diltiazem, nifedipin, inhaled nitric oxide, intravenous pgiz). results: nebulisation of pgi and iloprost tumed out to be most favourable for achieving effective and selective pulmonary vasodilatation. pulmonary vascular resistance decreased from + to -+ dyn*s*cm (p< . ) and pulmonary artery pressure from . + . to + . mmhg (p < . ), cardiac output increased from . + . to . _+ . i/rain (p < . ), mixed venous oxygen saturation from . _+ . to . + . % (p < . ) and arterial oxygen saturation from . + . to . _+ . % (mean _+ sem of trials in patients). -month iloprost nebulisation in one patient ( gg/day in six aerosol doses) demonstrated sustained efficacy of the vasodilator r~men. conclusion: aerosolation of pgi or its stable analogue may offer as new strategy for selective pulmonary vasodilatation in pph. endothelial adhesion molecules may play an important role in the pathogenesis of myocardial cell damage, and may contribute to the progression of heart failure. we measured the plasma soluble intercellular adhesion molecule- (sicam- ), vascular cell adhesion molecule- (svcam- ), and e-selecfin (selam- ) levels in patients with acute myocardial infarction admitted within hours after onset. peripheral venous plasma-samples were collected at the time of admission, , , , , and hours after onset. plasma soluble adhesion molecule concentrations were determined by elisa. patients were divided into groups as follows: group ; killip's class (k) and without thrombolytie therapy, group ; k and with thrombolytic therapy and group ; k and . both plasma sicam- and svcam- concentrations in group and were elevated rapidly and significantly and maintained at a high level during the first days. plasma selam- level did not change in any of the groups. these results suggest that the adhesion molecules icam- and vcam- may play a role in the pathogenesis of myocardial reperfusion injury and may indicate its severity in myocardial infarction. objectives: nitric oxide (no) is known to exert cytotoxic and negative inotropic effects on cardiomyocytes. no synthase activity has been reported to be increased in infarcted area in animal model of myocardial infarction. these findings suggest that no may be an important regulator for myocardial damage and cardiac function after myocardial infarction. we measured plasma no no -(nox) levels and estimated serial changes in acute phase of myocardial infarction. methods: subjects were patients admitted within hours after onset. venous blood samples were collected at -hour intervals on the first day, -bour intervals on the nd day and -hour intervals on the rd day and th days after onset. plasma nox concentrations were determined by griess method. results: the time course of the plasma nox levels (mea~+sem) displayed a tendency to gradually increase and to make a biphasic pattern with two peaks about hours and - days after onset (basal level; . _+ . , first peak; . !-_ . , second peak; . + . ram/l). plasma nox concentration was not influenced by the thrombolytic therapy, and nox values at the time of hours after onset were significantly correlated with maximal plasma creatine kinase level (r= . , p< . ). the levels of plasma nox in the early stage of myocardial infarction (from admission to the th day after onset) did not correlate significantly with the hemodynamic parameters (left ventricular ejection fraction, pulmonary capillary wedge pressure). conclusion: the early and late increase in no production after myocardial infarction may be implicated in the deterioration of myocardial contractility and induction of myocardial damage in the early phase of myocardial infarction. range - ) fullfilling the high risk criteria of shoemaker (colectomy , gastrectomy , pancreaticoduodenectomy , others ). patients were admitted to the icu preoperatively. arterial and pulmonary artery catheters were inserted and hemodynamics and oxygen transport were measured at admission and after stabilization to predetermined physiological end points. patients were considered stable when ci > . l/min/m , pcwp > mmhg, hb > g/l, sat >. . objectives: evaluate the acute effects of , mg ipratropium bromide and , mg fenoterol (ibf) inhaled dose on pulmonary function in nonsmocers (nb:m) and smocers (s) with sever (new york heart association class ii-iii), stabile congestive heart failure(chf) and healthy subjects. methods: pulmonary function tests were performed < h postprandial. the tests consisted el arterial blood gas aspiration followed by routine spirometry and pletismography, and single-breath gas analysis. after performance of these maneuvers, the patients was administred puffs-ipratropium bromide ( , rag) and fenoterol ( , rag). for , h, spirometry was repeated. results: in resting, pulmonary abnormalities observer in the s group were more severe then abnormalities observere in the nsm group. after treatment with ibf the improvement in pulmonary function was even more marked in patients who had smoked. the mean changes by forced expiratory volume in second(eevt) was , % (p< , t) improvement and , % (p< ,ob), forced expiratory flow betwen % and % of the forced vital capacity (fef . ) was , % (p< , ) and , % (p< , ) and maxamal voluntary ventilation (mw) was , % (p< , ) and , % (p<o, ) improvement. the mean changes in normal subjects were mild (fev increased by , %,fef - by , % and mvv by , %). we observed no adverse heamodinamie conseevence and no arrthytmogenicity. conclusion." in patients with chf the airway response to ibf is highly significant. further study is needed to determine whether therapy with ibf will load to improvement quality of life in patients with chf with dearly documented venti/atory defects. compared with unilateral ima grafts, usage of bilateral ima grafts in cabg patients causes greater thoracic trauma and further reduces blood supply to the sternum and intercostal muscles. these effects may be associated with increased postoperative respiratory complications obiectives: to study the effects of bilateral ima grafts on postoperative respiratory morbidity in cabg patients. methods: two groups of patients undergoing cabg were prospectively studied: group (n= ) received both imas as grafts and group (n= ) received only the left ima. the two groups were matched for age, smoking habits, total number of grafts and preoperative ejection fraction. pao /fio ratio was measured in all patients in the icu, at , , minutes on mechanical ventilation (mv), at , , minutes after extubation (ex) and on postoperative day (pod) . in addition, radiografic scores of atelectasis and pleural effusion were assessed postoperatively. results: group had significantly longer bypass ( _+ vs + . p< , ) and aortic cross clamp time ( +- vs + , p= , ). pao /fi ratio was significantly higher in group only at minutes after extubation (table) . there was no difference in the incidence or severity of atelectasis or pleural effusion between the two groups. the duration of mv was longer in group ( +_ vs _+ , p= , ), but the length of icu stay and hospitalization were similar. there was one case of pneumothorax in group and one case of pulmonary infection in each group. after extubation m[n min min min* rain min pod ' _+ _+ + -+ - -+ _+ _+ _+ _+ -+ _+ _+ -+ mean_+sd, *p=o, o conclusions: compared with unilateral ima grafts, the usage of bilateral ima grafts in cabg patients is not associated with increased postoperative respiratory morbidity. the diagnosis of rv ischaemia is difficult in the critical care setting. the purpose of this study was to determine the ability of rv endomyocardial and interstitial ph electrodes to detect rv ischaemia in an animal model of rv infarction produced by fight coronary artery ligation. we hypothesized that changes in transmural and interstitial ph would accompany the induced tissue hypoperfusion. rv interstitial (phint) and transmural endomyocardial ph (phendo) were measured in adult anaesthetized pigs before and serially after coronary ligation. phendo and phint fell progressively and significantly following coronary occlusion. this was accompanied by ischaemic ekg changes. the absolute and relative rates of change were greater for phendo compared to phint, particularly after minutes of ischaemia, ph was unchanged in control experiments when the electrode was placed within the fight atrial or ventricular chambers, as well as when coronary ligation was not performed. these data suggest that rv myocardial ph measurements reflect coronary ligation induced rv ischaemia, ph recorded from an electrode placed against the rv endomyocardial wall via a central vein was as useful as an interstitial measurement that required a thoracotomy. this newly described technique may be helpful in clinical settings where differentiation between ischaemic and nonisehaemic causes of rv dysfunction is important. centre. depts of cardiac surgery and anesthesiology, university of bari, italy. lung injury secondary to cardiopulmonary bypass (cpb) is well recognized. nevertheless, mechanical respiratory changes after cpb have been poorly investigated. aim of the study was to evaluate the effects of cpb on respiratory mechanics. elastance of the lung (est l), chest wall (est cw), and total respiratory system (est rs) were measured in patients without previous pulmonary disease undergoing elective cardiac surgery. there were males and females; no pleurotomy was done. cpb was carried out with membrane oxygenator. mean cpb and cross clamping times were _. and • min. flow (pneumothacograph), airway opening and esophageal pressures (pressure transducers and esophageal balloon) were measured on patients sedated and paralysed. measurements were obtained before sternotomy, at the end of cpb after chest closure, four and seven hours later. est rs, est l and est cw, were measured by occluding the airways and the end of a tidal breath. before end cpb- h after h after stemotomy closed chest cpb cpb estrs (cmh /l) . + . . - . " . _+ . * . - . * est cw " . _+ . . • . + . . -+ . est l " . -- . . - . " . -_ . " . _+ . " x + sd. * p < . : anova vs "before sternotomy". our data show that cpb induces increase in est l, whereas est cw remains unchanged despite sternotomy. impairment in est l after cpb evolves within the first hours and stabilizes hours later. during the clinical course of human imunodeficiency virus infection (hiv), patients (lots) can develop congestive and dilated cardiomyopathy syndrome (dcm). the aim of our study was to analyze the prevalence of dcm among an hiv population, its clinical and echocardiographic changes under a long term follow-up and therapy with an ace-inhibitor agent (captopril). we prospectively studied pts, % ( / ) developed dcm during the follow-up study. among this group % ( / pts) were in class i and ii and % ( / pts) in class iii and iv of the nyha classification. we divided this population in a acei+ ( pts) and acei-( pts), according to the administration of captopril. we analyzed the following parameters: age, gender, race, ivda, type of hiv, cdc classification, number of t and t type cell population and its t /t ratio, therapeutic with acei, type and number of opportunist infections, mycobacteriosis and neoplasm's. we analyzed several echocardiographic parameters, which included the initial (i), final (f) and variation (a) of the lv internal diastolic (lvdd/mm) and systolic (lvsdimm) diameters, lv % of fractional shortening (lv%fs/%), ivs and posterior wall thickness and lv mass (lvm/g). the two groups differed significantly in the following parameters: left ventricular functional indices of patients (pts) with iscjhemic dilated cardiomyopathy (idcm) have a critical value in terms of clinical prognosis of this disease. three-dimensional echocardiography ( -de) is a recently developed method that gives a better evaluation of lv morphology and function. the purpose of our prospective study was to assess the clinical applicability of this new -de method in a population with ischemic dcm diagnosis and calculate its lv indices of global and regional function. we studied a group of idcm pts, mean age _+ yrs, % male gender, using a combined -de tee approach. all pts were in class ill/iv of the nyha classification. first, ekg gated images were acquired through a mechanical pullback of the tee probe, and each set of data was transferred to a conventional ibm-pc system using a dedicated -de software. computer processing and analysis of the tee images led to a -de reconstruction matrix and off-line calculation of several lv global and regional indices. in all idcm lots the lv cavity was clearly reconstructed and end-systole and diastole in several orthogonat projections, out in multiple planes and its function quantitated using -de derived indices. mean values of -de lvesv= -+ ml, lvedv= _+ ml, lvstv= _+ ml and lvef= -+ % were obtained. for all these -de parameters, good correlations were observed with -de lv measurements obtained through biplane tee analysis of the lv cavity (r>. ; p<. ) as well as regional analysis of sequential -de cut planes. conclusion: in our group of patients with the diagnosis of ischemic dilated cardiomyopathy, this new -de method could be applied. our results show that this method allows a better assessment of the lv morphology and spatial geometry, with the calculation of global and regional indices with critical clinical and prognostic value in this particular cardiovascular pathology. simultaneous left atrial (la) and left ventricle (lv) inflow analysis assessed by pulsed doppler tee illustrate the loading conditions and reflect the hemodynamics of the left heart. we performed a prospective tee pulsed doppler study with recordings of the transmitral lv filling and pulmonary venous (pv) flow drainage in a group of patients with dilated cardiomyopathy (dcm). a group of dcm patients, mean age _+ yrs, % male were studied. this population was divided according to tee severe lv dysfunction (group slvd+ % pts; group slvd- % pts) in each pt we measured the peak velocities (vel/m/sec) and time velocity integrals (vti/m) of the transmitral early (e) and late (a) filing waves, the vel and vti of the pv systolic (s), diastolic (d) and atrial contraction (c) reversal flows. -de tee evaluation of the lved, lves, lvst volumes and lvef were obtained. we calculated other parameters, such as e/a, s/d and a/c ratios and the sum of c+a vel, that refelect la systolic function and lv compliance. + -_ . simultaneous and quantitative analytical approach of the pulmonary venous and transmitral flows and ventricular volumes improve the non invasive assessment and understanding of left ventricular diastolic function and cardiac performance in dilated cardiomyopathy patients. objectives : to assess the hemodynamic effects of fluid loading (fl) in acute circulatory failure (acf) due to acute massive pulmonary embolism. methods : hemodynamic measurements (fast-response thermistor pulmonary artery catheter) were performed at baseline (baseline) and after a rapid fluid loading with (fl ) and (fl ) ml of dextl'an (rhemacrodex| in patients free of previous cardiopulmonary disease ( • yrs) with acf (ci < . l/rain/m ) due to angiographicalty proven mpe (miller score > ) . results : are expressed as mean _+ sem and compared by anova. a significant negative correlation (r = . ) was observed between baseline rvedv[ and the effects of fl on ci. such correlation was not observed between baseline rap and the fl induced increased in ci. conclusion : fusibmificantly increases ci in acf due to mpe. however, the simultaneous decrease of arterial content due to hemodilution, limits the benefits expected from improved ci on peripheral oxygenation. obiective: to examine the hemodynamic effects of external positive endexpiratory pressure (peep) on right ventricular (rv) function in acute respiratory failure (arf) patients. methods: incremental levels of peep ( - - - cmh ) were applied and rv hemodynamics were studied by a swan-ganz catheter with a fast response thermistor for right ventrieular ejection fraction (rvef) measurement in mechanically ventilated arf patients (lis = . ~- . sd). according to the response to peep , two groups of patients were defined: group a ( pts.) with unchanged or increased rv end diastolic volume index (rvedvi) and group b (h pts) with decreased rvedvi. results: in the whole sample cardiac index (ci) and stroke index (sj) decreased at all levels of peep, while rvedvi , rv end systolic volume index (rvesvi) and rvef remained anchange d. at zeep the hemodynamic parameters of the two groups did not differ. in group a, ci decreased at peep , rvef decreased at peep (~ . %)~ rvesvi increased only at peep (+ . %) and rvedv[ reded unchanged. in group b, ci and rvedvi started to decrease at peep , 'rvesvi decreased only at peep (- . %), anf rvef was unchanged. individual behaviors of the hemodynamic parameters at the levels& peep were studied. rvedvi and ci were significantly correlated in out of:l patients in group b, and in no patient of group a. on the contrary, mpap and rvesvi were significantly correlated in out of patients in group a, and in no patient of group b. the slope of the relationship between rvedvi and rv stroke work index (rvswi) expresses rv myocardial performance. this relationship was significant (no change in rv contractitity)in patients of group b and in patients of group a. in some patients of group a, increments of peep shifted the rvswi/rvedvi ratio rightward inthe plot (rv function decrease). conclusions: in arf patients peep causes more often a preload decrease with unclmnged rv conctraetility. on the contrary, the finding of increased rv volumes during the application of peep is related to a decrease in rv myocardial performance. thus, these data suggest that application of peep might be considered as a stress test to assess rv function. right introduction: after heart transplant (ht), the right ventricle can be subject to an acute pressure overload, especially in cases where there is a preexisting severe pulmonary hypertension. this provokes right ventricular failure and, occasionally, circulatory collapse in intensive care unit. desire the advances that have been made in systems for preserving the donor heart and in post-surgical management, we have failed in our attempts to totally avoid this problem. the right ventricular function, although it usually remains within tolerable limits in these patients during the post surgery period, represents a factor which limits the results achievable in clinical transplant programmes. objectives: to determine the maximum tolerance of the right ventricle (mxtrv) when faced with acute pressure overload. to study the function of both ventricles of the healthy heart (donor) when faced with different degrees of pulmonary hypertension. to detect possible interactions between the ventricles in the absence of the pericardium to approximate the experimental model to the clinical model of ht. materials and methods: the pulmonary artery is progressively constrained in an experimental model until biventricniar failure is detected. this experiment is performed in two diffferent situations: with and without pericardial integrity. results: when pericardial integrity is maintained the mxtrv faced with a pressure overload is . + . nun hg. when this pressure is exceeded there is a circulatory collapse with a sharp fall in the cardiac output and in the aortic pressure. however, when pericardectomy is performed (model similar to ht), only • . nun hg is tolerated (p < . ). conclusions: with the pericardium open, as in heart transplant, the maximum pressure that the right ventricle can support is significantly less than with the pericardium closed. the pericardium has a positive effect in protecting the systolic ventricular interaction. it is, therefore, advisable to close the pericardium after heart transplant. jb prrez-bernal, a ordrfiez, a. heroandez, jm borrego, map camacho, c cruz, mac s~nchez, j monterrubio, c garcia, e. gonz~lez. hospital uulversitario " virgen del rocio ". sevilla. espaiqa. introduction: nowadays cardiomyoplasty isused incases of cardiac insufficiency as an alternative to cardiac transplant. after surgery the patients show a noteable improvement with the aid of this "biological circulatory assistance". some researchers suspect that the improvement could also be due to the formation of new blood vessels from the muscle that wraps the heart, nourishing the ischemic myocardium. objectives: our cardiovascular research group has proposed as an objective, the detection of any possible myocardial neovascularization through the muscle used for cardiomyoplasty. in the case that there are new blood vessels to the diseased myocardium through the wide dorsal muscle in which it is wrapped and which aids it mechanically, it would be possible to confirm the worldng hypothesis that cardiomyoplasty not only improves the cardiocirculatory funcfinn mechanically but also by facilitating a better blood flow to the ischemic myocardium. materials and methods: the cardiomyoplasty technique is described using an experimental model of myocardial ischemia. the vascular cast is achieved by injecting methacrylate simulataneously into both the coronary tree and the wide dorsal muscle, in five experiments the connections between the coronary vascular system and the vascular structure of the wide dorsal muscle are demonstrated, conclusions: we have demonstrated that cardiomyoplasty, as well as improving ventricular function, favours the revascularization of the myocardium. cardiomyoplasty could be indicated for cases of ischemic cardiopathy in patients in whom it is not possible to perform direct revacularization using conventional methods. a the therapeutic cardiological manouevres necessary in cases of ischeima reperfusion have increased considerably: fibrinolysis, transluminal angioplasty, coronary revascnlarization surgery and cardiac transplant. the appearance of a specific pathology ht acute reperfusion has been related to free oxygen radicals (for) generated by oxidative damage. objectives: to evaluate the appearance of for during a conti-olled process of ischemia-reperfusion in an experimental biological model and compare it with that in clinical cases. materials and methods: transitory cardiac ischemia was performed in five rabbits by reversible surgical ligation of the descending anterior coronary artery. after minutes coronary reperfusion was performed. blood samples were taken in the basal situation, at the end of ischemia and at , and minutes after the start of reperfusion. malondialdehyde (mda) was measured to evaluate the degree of lipid peroxidation (oxidative damage to the membrane). in ten patients undergoing conventional cardiac surgery the production of for was measured after aortic clamping. results: we observed that after minutes of reperfusion there was a highly significant increase (p < . ) in the mda values (mean = . /zmols/l). these returned to basal levels after and minutes of reperfusion. conclusions: an "explosion" of oxygen free radicals was detected very quicldy, just a few minutes after post-ischemia reperfusion. thus, if antioxidant agents are to be used to reduce the toxic effects of the for, these will ordy have a therapeutic effect if they are administered in the early phases of reperfusion. introduction: aortic connterpulsation is a ventricular assistance widely used in intensive care units in patients with cardiogenic shock as a provisional ventricular assistance. paraaortic or external aortic counterpnlsation is been investigated as a definitive veutricular assistance in those cases of terminal congestive heart failure and when heart transplantation is counterindicated. aims: to assess the haemodynamic effects of an aortomyoplasty in a biological model of congestive heart failure. material and method: as specimens, we used "large white" pigs. mean weight was kg. after the administration of conventional anaesthesia, dissection of the ladssimns dorsi muscle was performed on the samples at the laboratory of experimental surgery of our hospital. then we performed a thoracotomy at the level of the fourth intercostal space to reach the thoracic aorta. the aorta is dissecated centimetres from the exit of the subclavia and it is wrapped by the dissecated muscle. a cardiomyostimulator is provided in order to allow the synchronization between the diastole and the muscle contraction. the model of heart failure was provoked using verapamil plus propanolol i.v.. results: a significant increase of the aortic diastolic pressures and a significant decrease of the left ventricle telediastolic pressures were observed. this improvement in the parameters (dpti/tti) implies an increase of the coronary perfusion in a model of heart failure. conclusions: using the external aortic counterpulsation, the aortomyoplasty improves the coronary perfnsion and the heart efficiency in patients with heart failure in whom no conventional therapeutic action is possible. the permanent character of the paraaortic counterpulsation is it main advantage. the appearance of specific pathologies as a resuk of myocardial reperfasion has been related to the oxidative damage secondary to the release of oxygen derived free radicals (ofr). during the myocardial ischemia induced during heart surgery with extraeorporeal circulation, severalsubproducts of the oxygen are produced that shall cause toxic effects after the reperfusion which could be counteracted by the physiological antioxidant systems and/or provided by the medication. aims: to asses the ofr during heart surgery. to check whether an antioxidant treatment administered in the preoperative period make decrease the levels of ofr before and after the myocardial reperfusion and to verify whether its administration have any beneficial effect on the intra and extraoperative management. material and method: the study comprehends patients studied as two groups of individuals each (a and b). all patients underwent conventional heart surgery of valvniar substitmion or myocardial revaseularization. group a patients were administered rag/ hours of vitamin e (tocopherol acetate) hours prior to the intervention as antioxidant treatment. group b patient were not administered vitamin e. we assessed the quantity of malondialdehido (mda) to assess the degree of lipidic peroxidation or oxidative damage of the membrane during the myocardial ischemia and nm after the reperfusion. conclusion: patients who underwent heart surgery and were treated with tecopherol acetate in the preoperative period presented levels of rlo significantly lower than those who were not administered the drug, both during the intraoperative period and after myocardial reperfusion. we detected in these patients a need for antiarrhythmicals and pharmacoiogical support with catecholaminas, although not significant, both in the introaperative period and the immediate postoperative period. recommendations for the treatment of pulmonary embolism (pe) in the presence of right atrial thrombus (at) are conflicting. because of a significantly higher mortality rate due to fulminam or recurrent pe, there is a necessity to treat patients (pts) with mobile type a thrombi compared to pts with adherent type b thrombi. therapeutic strategies include anticoagulation, thrombolysis (t) or surgical thrombembolectomy. combination thrombolysis (cot), predominantly used for the treatment of acute myocardial infarction proved to prevent reocclusion of the infarct related artery at a comparable rate of hemorrhagia. benefit has been related to the alteration of hemostatic proteins by non-fibrinspecific thrombolytic s. administration of cot in pe has been performed sporadically. in the present case, a -year old male with no history of prior cardiovascular disease developed acute dyspnea which was related to pe in the presence of deep vein thrombosis of the left femoral vein. therapeutic anticoagulation was installed for a couple of days until there were several bouts of deterioration. biplane transesophageal echocardiography (tee) was performed and revealed a large, wormlike, hypermobile thrombus within the right atrium. computer tomography (ct) of the chest detected a saddle embolus in the bifurcation of the pulmonary tmnk almost occluding the entire left pulmonary artery (pa) and parts of the right pat consisted of mg frontloaded rt-pa and the subsequent continuous administration of urokinase in a dosis of . u/hr for hrs followed by therapeutic anticoagulation. symptoms, blood gases and ecg improved steadily during infusion, no adverse effects, i.e. minor or major hemorragia were registered. follow-up ct promptly after termination of t showed almost complete resolution of the saddle embelus, whereas tee showed complete dissolution of the at. ' finally, the patient was switched to oral anticoagulants and had an uneventful clinical course until he was discharged. conclusion: in the present case, cot was effective for the treatment of a complicated pe without any adverse effect. introduction: nowadays we can assist hearts with problems of insufficiency by techniques other than transplant. many researchers believe that the best way of assisting insufficient heart muscle is with another muscle from the patient. this technique of ventficular assistance is known as cardiomyoplasty. we describe the surgical technique of cardiomyoplasty using a biological model. the transformed skeletal muscle is transferred to the thoracic cavity where it wraps the heart and assists it. the choice and preparation of this muscle is currently under investigation. our group has focussed on the development of protocols for electrical stimulation to transform a skeletal muscle into a muscle which resists fatigue and which is functionally similar to the myocardium. we detect the optimum time at which this muscle has been transformed, by studying the transmembrane action potentials using intracellular electrodes. when the action potential of the trained muscle behaves like cardiac muscle we consider it ready for cardiomyoplasty. conclusions: cardiomyoplasty is an alternative surgical technique to cardiac transplant, which has a great future in the treatment of patients with advanced cardiac insufficiency. we describe methodology which, by intracellular techniques, allows selection of the optimum moment of transformation of a skeletal muscle trained to perform,like cardiac muscle, without suffering fatigue. purulent pericarditis is a rare disease. its treatment associate systemic antibiotics and drainage of the pericardium. we report a ease of purulent constrictive pericarditis in which intraperieardial fibrinolysis was use. a years old patient admitted in our icu for a constrictive pericarditis as a complication of a purulent pericarditis diagnosed seventeen days before. he had also an aehalasia and the o'esogastric endoscopy had found an oesophageal neoplasm. a fistula was not seen, indeed pericardial of flora was the same that oropharyngeal. hemodynamie and echographic study had confirmed a constrictive pericarditis. because of the poor state of the patient an intraperieardial fibrinolysis was prescribed ( . ui of streptokinase on days , , , ). fluid drainage was improved and cardiac output was also improved (day : . .min "i, day : . l.min'l). no change ofhemostasis was noted. a pericardeetomy and an oesophagectomy were performed after days of evolution. eighteen months latter the patient was still alive. intraperieardial fibrinolysis seems an interesting therapeutic way if rapidly prescribed in the purulent pericarditis course. the decrease in the systolic pressure following a mechanical breath, termed ddown (delta down), has been shown to be a sensitive indicator of preload ( , ) . however, the clinical use of this method necessitates the introduction of a short apnea. we have therefore developed a respiratory systolic variation test (rsvt) which obviates the need for apnea. the test is based on the delivery of successive breaths of increasing magnitude ( , , , and ml/kg). a line of best fit is drawn between the minimal systolic values (one after each breath) and the downslope calculated as the decrease in blond pressure for each increase in airway pressure ( mmhg / cmh ). in mechanically ventilated patients the rsvt was performed during controlled mechanical ventilation under sedation. the test was repeated after the administration of ml/kg of plasma expander. the initial mean downslope of the rsvt was -. + . mmhg/cmh . following volume loading the downslope decreased to -. + . (ns). at the same time, cardiac output (co) increased by . + . l/min (p<. ), end-diastolic area (determined by tee) increased from . + . to . + . cm (ns), and paop increased from + to + mmhg ( p < . ). the preinfusion downslope value of the rsvt correlated significantly with the increase in the co (r = . ) and the eda (r = . ). methods: an expert system has been constructed running on a multimedia computer with the two objectives in mind, viz training of inexperienced staff, and protocol guidance with treatment regimes for all staff. the system is based on experience gained from two previous systems, the one for dealing with acid-base and electrolyte problems in icu patients; the second for stabilisation of patients with heart rate and blood pressure abnormalities. the training section takes the form of a stage-by-stage account of the insertion of the pac and displays of correct waveforms, coupled with indications of possible incorrect placements, and guidance when failing to achieve the perfect positioning. the treatment protocol section extends an existing protocol for correcting abnormalities in heart-rate and blood-pressure, and now takes account of all the indices as measured by the pac. the system will suggest treatment to correct such things as abnormal wedge pressures concomitant with parameter values throughout the rest of the cardiovascular system. the type of patient eg post-operative cardiothoracic or i. c. u. trauma, will be taken into account when recognising abnormal parameter values and when prescribing treatment. results: a working system which will be improved by the finetuning being carried out. the results and lessons learnt will be presented at the conference. method: septic shock was defined as severe sepsis with either persistent hypotension (mean arterial pressure; map < mmhg) or the requirement for a noradrenaline (na) infusion ~ . g/kg/ rain with a map --< mmhg. cardiovascular support was limited to na + dobutamine (db). c was given for up to h at a fixed dose-rate of either , . , , or mg/kg/h iv. during c infusion, na was to be reduced and if possible withdrawn, whilst maintaining map above mmhg and the cardiac index (ci) as clinically appropriate. assessments were made at baseline (t = ); at i h from the start of treatment (t - ); and at the end of treatment (t - ) with c . conclusions: c does not appear to increase mpap or worsen pulmonary gas exchange in patients with septic shock, when given by infusion for up to h. c is a novel vasoactive agent for the treatment of septic shock which will now he evaluated in a randomised, placebo-controlled safety and efficacy study. objectives : to compare cardiac output (q) data obtained for thermal indicators in pulmonary artery (qtpa) and aorta (qtao) and for the stable isotope hzo in aorta (q v~ o) with indocyanine green (icg) in aorta (qicg) as reference. methods : an indicator solution of ice cold h ( . ml), h ( . ml) and icg ( mg) was injected as bolus via the injection port of a swan-ganz catheter. qlco and qzmo was measured using a dual optical system (penn lab instruments, philadephia, pa, usa). qtpa and qtao was measured using a in contrast to the recoveries of thermal indicator in pa and h in aorta the :~covery of thermal indicator in aorta was significantly increased in group ii (n= boluses) over group i (n= boluses) ( . <- . vs. . +- . , p= . ). conclusions: the "overrecovery" of thermal indicator in aorta is in agreement with " biscks deconvolution study (i) and results in erroneous values for q. the most pausible explanation is the distortion of the thermal curve caused by the slow response time of the thermal detection instrument as shown by ganz ( ) objectives: to compare data obtained with the double indicator dilution method using indocyanine green (icg) and the stable isotope h for the estimation of extravascular lung water (evlw hzo) to gravimetriu lungwater data (evlwg~). methods: an indicator solution oflcg ( rag) and h ( . ml) was injected as bolus via the injection port of a swan-ganz catheter. dilution curves for icg and zh was registered in aorta with a dual optical system (penn lab instruments, philadephia, pa, usa). cardiac output and mean tranist time was measured for both tracers (qico, tlco, q n o, t o) ( ). data analysis: evlwg~av was reference for evlwzhzo calculated as q hzo times the difference in mean transit time between t nzo and rico (atm n). as reference for atzn o evlwg~,v was divided by q~cg to obtain atg~,. a reference distribution volume for h was calculated as the sum of central blood volume and evlwg=v. boluses were administrated in a group (i) of anaesthetized pulmonary healthy sheep while q was altered. another boluses were administrated in a group (ii) of anaesthetized sheep with stable oleic acid induced pulmonary oedema. evlwg~v measurement was performed postmortem. results: for boluses h parameters were not significantly different from their respective reference parameter: at vao . +_ . s vs. atg~, . + . s, evlwzh o -+ ml vs. evlwg~,~ + ml. in group i the ratio between hzo parameters and respective reference parameters (n= ) were independent of qlco from . to . l/min. obiectives: to assess the thermo dye method using indocyanine green (icg) and thermal indicator for the estimation of lung water (evlwt). methods: ice cold indicator solution of icg ( mg) in water ( ml the aim of the study was to assess left and right ventricular function in the early postoperative period after orthotopic heart transplantation to elaborate therapeutic approaches of heart function abnormalities correction. mathefial and methods. haemodynamic monitoring data of twenty one patients ( men, women ) age from to were studied. cardiac output, pulmonary artery, right atrium and pulmonary wedged pressure were measured with swan-gans catheter. central haemodynamic indices were calculated with the help of computer-based monitoring system. relations of ventricular stroke work index to it's end-diastolic pressure were used for ventficular function assessment. results. in most cases right ventricular disfunction was the main problem. isolated fight ventficular failure with high pulmonary vascular resistance (pvr) was observed in % ( pts), without high pvr-in % opts) and with left ventricular failure-in % ( pts). one of the most important reasons for fight ventricular failure was the time of heart ischemia more than min, which is of great importance in the ease of distance harvesting. the most effective treatment for cardiac failure was combination of dobutamine with i oprotherenol, atrial pacing and vasodilatators in case of right ventfieular disfunction. all cases with isolated right ventricular failure were treated sucsessfully. biventricular heart failure was a sighn of bad prognosis and the reason of death in cases. conclusion. right ventfieular disfunetion is the main problem during transplanted heart adaptation in the early postoperative period. optimal therapeutic management of cardiac disfunction includes infusion of dobutamine in combination with isoprotherenol, atrial pacing and vasodilatators. cardiology-department of clinical centre-kragujevac institution for occupational health "zastava"-kragujevac, sr yugoslavia the aim of the investigate is analisis five years survives patients with a.i.m.in dependence of locality and risk-factors. we ana~sed- ~-pat~e~ts ( males and woman), average , years. for statistic evaluation we used life-table slstem in oder to estimate prognostic determinants. patients with respkatory muscle paralysis may benefit from respiratory assistance by abdomino-diaphragmatie pneumatic belt. we used a non invasive technique, m-mode sonography, to assess the effect of this device on diaphragmatic excursion. we measured the amplitude of right diaphragm motion in seven patients with duehenne muscular dysl~ophy in supine position with various thoracic posture ( ~ ~ ~ without and during pneumatic belt respiratory assistance. without respiratory assistance, the thoracic posture had no significant consequence on the amplitude of diapttragm motion, either in quiet or deep breathing. the pneumatic belt increased the diaphragm motion amplitude from . +__ . mm to . +_ . ram (p = . ) at ~ tilt angle, and from . + . mm to . + . mm (p = . ) at " tilt angle. the tidal volume increased from + to + rut a * tilt angle, and from + to + ml at * tilt angle (p = . ). two patients could not bear the horizontal position ( ' tilt). in the five other patients, the pneumatic belt increased but not significantly the amplitude of diaphragm motion ( . + . mm to . + . ram). after an overnight respiratory assistance, pao increased from . +_. . to + . mmhg ( = . ), sao increased from . + . % to . +_. % (p = . ), and paco decreased from + . to . +_. mmhg (p = . ) according to the ventilatory pattern result, m-mode sonography allows to measure non invasively the improvement of diaphragm kinetics obtained by pneumatic belt respiratory assistance, and may be helpful for its adjustment. objective: to study the effect of flow triggering (flow sensitivity and l/min) vs pressure triggering (-lcmh ) on inspiratory effort during pressure support ventilation (psv) and assited/controlled mode (a/c) in stable copd patients non-invasively ventilated with a full face mask. methods: the patients were studied during randomized min. runs using a bird st ventilator at zero peep (zeep). trigger values for pressure (-lcmh ) and flow ( l/rain) were the lowest allowed by this ventilator. the transdiaphragmatic pressure time product per breath (ptpdi), dynamic intrinsic peep (peepi,dyn), maximal airway pressure drop during inspiration (apaw) andl ventilatory variables (ti,te,ttot,rr,vt and minute ventilation) were measured. results: no major problems due to airleaks or to auto-triggeriffg phenomena were observed in the patients, so that all of them were able to perform all the protocol runs. minute ventilation and respiratory pattern were not different using the two triggering systems. the ptpdi was significantly higher during both psv ( . + . cmh: x sec) and a/c ( . + . ) with pressure triggering, as respect to psv ( . + . , p< . ) and a/c ( . + . , p< . ) with flow triggering ( l!m). no differences were observed between and l/min flow triggers. apaw was also significantly larger during pressure triggering; peepi,dyn was reduced during flow triggering being . + . cmh (psv flow trigger) vs . + . (psv pressure trigger) and . +_ . (a/c flow trigger) vs'f~ +l (atc pressure trigger). conclusions: in stable copd patients non-invasively ventilated, flow triggering reduces the respiratory effort during both psv and aic mode as compared to pressure triggering. this may be partly due to a decrease in peepi,dyn using a flow-by system. objective. cardiac output is higher during alternating ventilation (av) (i.e. differential ventilation of the lungs with a phase shift of half a ventilatory cycle) than during synchronous ventilation (sv) of both lungs . we verified the hypothesis that the higher cardiac output depended on a lower central venous pressure and intrathoracic pressure, due to a lower mean lung volume, which we attributed to part of the expansion of the inflated lung at the expense of the expiring, opposite lung . we studied this interaction between the lungs during one-sided inflation, which we called cross-talk. method. in anaesthetized and paralyzed piglets we applied short periods ( s) of one-sided ventilation ( breaths per rain, bpm), while the other lung was open to the ambient air. the air flow into the non-ventilated lung during expiration of the ventilated lung was integrated to volume. we studied -to-r and r-to-i cross-talk at ventilatory rates of , and bpm. the amount of cross-talk was the volume displacement in the non-ventilated lung. results. during bpm the r-to-i crosstalk was _+ . % (mean +__ sd) of the tidal volume to the right lung and the -to-r crosstalk _ . % of the left tidal volume. both values increased at bpm to _ . % (p < . ) and _ . % (p < . ) respectively. the values at bpm were in between., conclusion. we concluded that the lower mean lung volume and lower thoracic expansion during av compared to sv depends on partial expansion of the inflated lung into the non-inflated lung, resulting in a lower mean intrathoracic pressure as the main reason for the higher cardiac output during av. obiective: natural surfactant given for rds in premature infants leads to a rapid improvement in oxygenation, but lung compliance did not improve in most studies. however, acute effects on lung mechanics during and immediately after surfactant administration have not been studied before. methods: a total of administrations of bovine surfactant in recommended doses was given via a small catheter into the distal endotracheal tube either as a bolus (n = ) or as a slow infusion (n = ) in infants with established rds. static compliance (c), resistance (r) and time constant (tc = cxr) of the lung were measured every minutes with a lung function cart (sensormedics ) without interrupting ventilation. infants receiving synthetic surfactant were studied as controls. results: after surfactant as a bolus or during infusion c first decreased but then increased, whereas r increased immediately with great fluctuations but did not return to baseline. this pattern was more pronounced in infusion than in bolus administration. change of c and r varied greatly in the individual case, maximum c was > %, maximum r > % of baseline value. retreatment was followed by an increase in r in all patients, but c increased only in the one who was responder. patients receiving synthetic surfactant had no change of c or r and were non-responders. ob~i ctives= acute lung injury (ali} sometimes induces severe hypoxernla which may be refractory to conventional modes of mechanical ventilation (mv). the elm of this study was to observe some cardio-pulmonary effects of an alternative method of ventilatory management of severe ali. five patients with severe ali (murray scores > ) requiring mv were studied. protocol inclusion was considered when a control-mode of mv (with a pzo~=l. and a peep level < cme=o} was not able to get either a p.ojf=o= ratio > or a s.o= > %. patients were sedated, paralyzed, and a ventilator (serve c) was used for pressuz'e-control ventilation (pcv). fio= was maintained at . and peep removed. continuous gas flow ( • ml/kg] was humidified and jet delivered through a tube ( ram id, ml capacity, . ml/cm h=o compllancel ended in a nozzle ( . mm is) attached to the endotracheal tube connector. a thermodilution flcw-dlrected catheter was inserted in pulmonary artery. following variables were recorded minutes before and after protocol started: tidal volume (vt), minute ventilation (vz), intratracheal pressures (p~w), wedge pulmonary artery pressure (wp), central venous pressure (cvp), mean arterial pressure (map), cardiac index (ci), arterial and mixed venous oxyhemoglobin saturation (sao=, svoa) , oxygen delivery (do~) , oxygen consumption (vo ) , intrapulmonary shunting (q./qt) , and oxygen extraction ratio (ero). this observation suggests that hfpv could allow to ventilate at lower fin and improve blood oxygenation during the acute phase after inhalation injury reducing toxicity risk related to high fin . further studies are necessary to confima these results and evaluate the possible implications on mortality alter smoke inhalation and for other icu pts. objectives: to design a system for volume controlled high frequency ventilation (hfv) and to estimate the dependence of the tidal volume (vt) on frequency (f) in normocapnic ventilation in rats at frequencies - hz. methods: a new system for volume controlled hfv was devised consisting of the generator of the constant flow during inspirium and the constant pressure during expirium. the ventilator allows ventilation at frequencies - hz with the relative inspiratory time (ti) . - . . the airway pressure was measured at the proximal port of tracheostomic cannula , at the same site inspiratory and expiratory flow was measured using modified lilly-type of pressure-differential flow sensor. non-linearity of flow sensor was compensated on line by derived equation based on calibration at static and dynamic conditions. flow and pressure data were evaluated on line using original software. value of the positive end expiratory pressure (peep) was serve-regulated by analogous feed-back. in animal experiments white wistar rats ( - g) narcotized with ketamine/xylazine with cannulated carotid and femoral arteries were kept at the rectal temperature ~ the arterial pressure was monitored. after traeheotomy the metal cannula ( mm [.d.) was inserted, animals were curarized and ventilated at the following condition: peep = . kpa, ti = . . the dead space of ventilator including canula was . ml. the initial frequency was hz and rain after each change of the ventitatory regimen the blood gases analysis was performed. the frequency was changed according to the following schedule : hz--> hz--> hz--> hz--> hz--> hz--~ hz--> hz. vt for each frequency was regulated to maintain normocapnie ventilation with arterial pco = + mm hg. the arterial po was always above mm hg. results: for normocapnie ventilation in rats the following tidal volumes vt [ ml/kg] were found : vt = . --+ . ml/kg for ft = hz, vt = . + . mukg for fz = hz, vt = . +_ . ml/kg forf = hz, vm = . + . ml/kg forf = hz andvmt= . + . mukg for fs = hz (presented as mean values _+ s.d., n = ). the regression analysis using the mean values resulted in the equation for normocapnic vt in rats in our experiments : vtn = . * f-e. . conclusions: the described system allowing ventilation in a wide frequency range - hz with accurate measurements of airway pressures and vt might be useful for optimisation of artificial ventilation in new-barns with different lung pathologies. supported by grants iga mz cr nr - and gacr nr . s intensive care unit. university. hospital of south manchester, uk. methods: measurements were conducted on ventilated patients (puritan bennett ac with metabolic monitor pb set to measure end tidal co ). all measurements were repeated with the patient stabilised at cm. cm and cm peep. inclusion criteria were: ) haemedynamic stab(l( .ty for hr; ) pulmonad" anon" flotation catheter in situ: ) volume control ventilation with plateau of . s: ) fio ~ > . to maintain pao~. > kpa with em peep: ) qs/ot > %; ) pao /fio ratio < . measured variab!es included: r minute volume: plateau ainvay pressure: applied and intrinsic peep: fractional end tidal co ; arterial and mixed venous blood gases and hacmod).ttamic variables. results: statistical analysis was performed using repeated measures anova. significant decreases in cardiac index (ch p< . ), compliance (p<t). ) and o,'gcn deliver, index (do , p< . ) occurred with increasing peep (table ) . no other variable showed any significant change. methods : all consecutive patients orally intubated in the micu between january to april were studied. etts were positioned according to the reference marks mentioned above (measurements from upper incisors). the position of the ett tip in relation to the carina was measured on the chest radiograph done with the patient's head in neutral position. results : a total of men and women were studied (n= ). the mean distance of ett tip to carina was . cm. using the reference markings, cases ( . %) had the ett tip < cm from the carina and cases ( . %) > cm. one case resulted in an endobronchial intubation. the mean height of all patients were cm ( - ) for males and cm ( - ) for females. of the patients with ett tip < cm from carina, the mean height was cm and cm respectively. ~ onclusion : adopting the above quoted reference marks did not result in ideal positioning of the ett in a significant proportion of cases ( . %). we postulate that [s because our asian population is generally shorter than those in previous studies. objectives: to measure the changes of pulmonary mechanics before and after tracheostomy in patients with prolonged mechanical ventilation and to determine factors that predict the outcome of liberation from mechanical ventilation. design: prospective. setting: respiratory intensive care unit (ricu) in a tertiary hospital. patients: twenty patients with chronic lung disease requiring long-term mechanical ventilation. tracheostomy is indicated for further care. intervention: tracheostomy. measurements and results: pulmonary mechanics including respiratory rate (rr), tidal volume (vt), peak inspiratory pressure (pip), intrinsic positive end ex~ piratory pressure (peepi), lung compliance (cld), mean airway resistance (rawm), work of breathing (wob), pressure time product (ptp) by bicore cp- pulmonary monitor were recorded hours before and after tracheotomy. ventilator setting parameters remained the same during surgical intervention and were also recorded for comparison. generally, the mechanics including pir wob, raw~x and ptp showed improvment after tracheostomy. but only pip was significantly reduced (pre . _+ . to post . _+ . , p < . ). changes of wobp showed significant correlation with pre-operation rr, minute volume (mv), wobp, and peep(. changes of raw m were also significantly correlated with pre-operation peep, vt, and raw m. the patients were divided into two groups according to their outcome after two week follow-up. group included eight patients who were completely weaned from ventilator; group included twelve patients who still remained ventilator-dependent or were mortality. there was no difference in age, duration of mechanical ventilation, pro, post or changes of several lung mechanics between the groups of patients. pre-tracheostomy peep i and cld showed significant difference between these two groups ( . _+ . vs . + . in peepi; . _+ . vs . _+ . in cld, p < . ). pre-tracheostomy ventilator setting in mode of assist/control also showed significant higher percentage in group ( % % in group vs . % in group ). conclusion: in prolonged mechanical ventilation patients with chronic lung disease, tracheostomy will significantly improve pip and slightly reduce wobp, raw m and ptr patients who used pressure support mode before tracheostomy had better underlying lung conditions (lower lung compliance and auto-peep) will have better chance to wean from mechanical ventilation. forty-eight infants with congenital diaphragmatic hernia presenting within the first hours of life, who underwent surgical rapair,were analysed prospectively in order to produce a reliable inde x of severity of disease that would reliably predict eventual outcome. there were survivors and deaths in this series (mortality %).using arterialpco values measured hours after surgical repairand correlating them with an index of mechanical ventilation,we have been able to clearly define two groups of diaphragmatic hernia based on their response to hyperventilation. the first group, with co retention and severe preductal shunting,was unresponsive to hyperventilation with high rates and pressures the mortality was %. the second group responded well to hyperventilation and demonstrated reversable ductal shunting only. survival in this group was %. arterial co accurately reflects the degree of lung development in this disease and separates those patients with severe pulmonary hypoplasia where the outcome is invariably fatal, from those with a well developed contralateral lung where there is excellent potential for survival. respiratory failure unit, dpt medicine, univ. thessaloniki, thessaloniki, greece the variability of arterial blood gases (po , pc ) and the ph (abg) was examined in stable icu patients, few hours before a successful weaning from the ventilator. all patients were lightly sedated and the ventgatory conti~ons were pressure support (ps) for and ps plus intermitted mantatory ventilation in ii. [n each patient, speciments of abg were measured at min intervals during a - study period. at the same time with abg the arterial blood pressure (bp), the heart rate (cf), the tidal volume (tv) and the respiratory rate (n r were measured. for all the patients, the mean coefficient of variation (c) was . percent for po , . percent for pco and . percent for hco . the average sd for ph was . , the corresponding c for systolic bp, diastolic bp, cf, tv, rf were . , . , . , . , . percent. we conclude that the spontaneous variability of arterial blood gases in icu patients is not substantial ~hen they have stable the heamodynamic and the ventilatory parameters. deptx?fa'aaesthesioiogy and reanimation, rhe sechenov medical academy, moscow, russia objective: ~he prevention and treatment of hypoxia in the critical patiems. methods: i~fusions of perphtoran -a blood substitute with gas-transporting fimclion based on perphtorhydrocarbon -in patients with acute hypovolemia, microcirculatory distnrbance~ tissue gas exchange and metabolism; pulmonary iavage in ; iongterm extrapulmonary oxigenation with tleoroearboa oxygenator in combination whb ~trafiltra!ion, hemosorption and hemodialysis -in patients. results: pe~htoran increases blood volume, co,sv, decreases svr, improves capillary blood flow, increases the blood oxygen capacity, tissue oxygen tension, del, vo by improving the rheologic properties of blood and plasma, normalizes ext., prevents and eliminates fat embolisation and ards. decreases the need for blood transfusions and infusions of plasma expanders by . - . limes. alveolar venti!ation-perfusion ratio remains unchanged with its increased effective utilization. there was no surfactant destruction during lavage. extrapulmonary oxygenation of small volumes of venous blood eliminates venous destruction and then arterial hypoxia and increases pulmonary oxygenation. the use of lluorocarbon cxygenators during hemosorption and hcmodialysis provides the atraumatic and iongterm oxygenation of arterial blood and increases elimination of co which prevents the development of hypoxic complications. conclusions: perphtoran and fluorocarb~n oxygenators are effective in the correction of hypoxia in the criticat patients. objeqtives: to determine if there are differences in oxygen consumption (vo ) during weaning from mechanical ventilation (during total ventilatory support and spontaneous ventilation with cpap), and to compare different predictive parameters of weaning in predicting success of weaning. methods; prospective study in critically ill patients treated with mechanical ventilation for at least h, who fulfilled at least of standard weaning criteria (vt> ml/kg; respiratory frecuency (f) < ; pimax > cm h ; pao /fio > ). baseline measurements: t, vt, p . , pimax, f/vt, p . *(f/vt), p . /pimax. study protocol: measurement of vo , vco (medgraphics), vt, f, ve, and arterial blood gases during total ventilatory support (cmv), and after and minutes of spontaneous ventilation with cpap cm h . the weaning trial was stopped, failure to wean diagnosed, and mv resumed it a patient presented significant tachypnea, tachycardia, bradycardia, cardiac rythm disturbances, hypertension, hypotension, hypoxemia or hypercapnia. results: four patients did not complete the weaning trial, were extubatad, and of them had to be reintubated before h, being considered also weaning failures. during cmv, vo /kg was . + . ml/kg/min, and . _+ . mlo- /kg/min after ' on cpap cm h (p < , ). of patients ( %) with standard criteria were extubated, while only of ( %) with criteria (p< , ). next objectives: compare the extent and distribution of lung injury in dogs preinjured with oleic acid (oa) and ventilated with high tpp and adequate peep in the prone and supine position. methods: lung injury was induced with oa ( . - . ml/kg) in anesthetized, paralyzed, and intubated dogs (n= ) during volume controlled ventilation: rate= /min, peep= cmh , ti/ttot= . , fio = . , vt= ml/kg. animals were rotated during the oa infusion and the following minute stabilization period to assure uniform injury. in the supine position, peep was set - cmh above the lower inflection point (as determined by the pressure-volume curve), and vt was set to obtain a tpp of cmh : animals were ventilated in either the prone (n= ) or supine (n= ) position for four hours. pulmonary artery occlusion pressure was maintained constant ( - mmhg) with saline infusion. at the end of the protocol the lungs were removed and divided by template into dependent (d) and nondependent (nd) sections for wet weight/dry weight (v~n/dw) and grading of nstologic lung injury (hli; scale - ). oseillatron | is a pneumatic device that generates high frequency, oscillation by means of a reciprocating system in the form of a membrane. it generates sinusoidai wave form at ( to ( cycles/rain. the system does not deliver gas but must be adapted to the proximal respiratory, circuit of a conventional ventilator, resulting in ci-ifo. it was developed to enhance intrapnlmona~ diffusion during mechanical ventilation and to mobilise endebronchial secretions. methods. we measured arterial blood gases and haemedynamics during a first period of conventional ventilation (cppv) followed by. two rain periods of chfo (sequences : ( and ) c/rain : group l, n = l: and c/rain : group , n = ). measurements were made at the end of each period. cardiac output was measured using thermedilution method: flu and peep were kept unchanged throughout the study. intrinsic peep was also evaluated by, means of an occlusive valve. results. pa is not significantly modified during chfo at or c/rain. paco is slightly decreased at c/rain (p = .( ). however, intrinsic peep remains unchanged. there is no sequential effect (gr. l vs gr. ). there is no more effect of chfo for patieets who are at a flu higher than . (n = ). no changes in haemodynurmcs are observed except a slight increase in central venous pressure (cvp) during ci-ifo (p < .ol). obiectives: to examine the effects of inspiratory muscles unloading on neuromuscular output at controlled levels of chemical stimuli. methods: the ventilatory response to co was examined in ten normal subjects using rebreathing method. ventilation ~) and respiratory muscle pressure output (pmus) at the same end-tidal partial pressure of co (petco~) were compared with and without combined flow and volumeproportional pressure assist in two protocols (a and b). protocol a (n = ): two levels of assist were studied; flow assist (fa) of cmh /i/sec and volume assist (va) of cmh /i (assist ), and fa of cmh /i/sec and va of cmh /i (assist ). all conditions were applied randomly. v~, tidal volume (vt) and breathing frequency (f) were measured breath by breath and plotted as a function of petco~. protocol b: in subjects, in addition to above measurements, esophageal (pes) and gastric (pg) pressures were measured and the time courses of transdiaphragmatic pressure (pdi) and pmus were calculated. one level of assist (assist ) was studied in this protocol. results: in both protocols inspiratory muscle unloading did not change the f response to c%. compared to control, with assist v t response was displaced upwards; at petco of mmhg v t was increased significantly by . + . i and . + . i in protocol a with assist end , respectively, and by . _+ . i in protocol b with assist (p< . ). ~/~ responses showed similar changes as vtresponses. in both protocols the slope of v~ response (s did not change significantly with unloading. at low petco~ ( mmhg), pdi and pmus waveforms did not differ with and without assist. with unloading, at high petco ( mmhg), pdi and pmus at the end of neural inspiration decreased by . -+ . % and . + . %, respectively, from control values. neither change was significant (p> . ). by theoretical analysis we estimated the expected changes in vt and ~/~ when the levels of assist used in both protocols were applied in the absence of : any change in neural output response to co z. the predicted response was similar to that observed, indicating that the small difference in pdi and pmus between control and unloading runs was due to intrinsic properties of respiratory muscles end respiratory system. conclusions: these results suggest that when chemical stimulus is controlled, respiratory motor output is not downregulated with unloading. the determinants of the response of the respiratory output to inspiratory flow rates (v~) were examined in awake normal subjects. subjects were connected to a volume-cycle ventilator in the assist/control mode and v~ was increased in steps from to i/min and then back to i/min. v~ pattern was square, and all breaths were subject-triggered. in six subjects the effects of breathing route (nasal or mouth) and temperature and volume of inspired gas (protocol a) and in subjects the effects of airway anesthesia (upper and lower airways, protocol b) on the response of respiratory output to varying v~ were studied. in protocol b, in order to calculate muscle pressure during inspiration (pmus), respiratory system mechanics were measured using the interrupter method at end-inspiration. independent of conditions studied breathing frequency increased . significantly and end-tidal concentration of c% decreased as v~ increased. the response was graded and reversible and not affected by breathing route, temperature and volume of inspired gas and airway anesthesia. with and without airway anesthesia (protocol ) neural inspiratory and expiratory time and neural duty cycle, estimated from pmus waveform, decreased significantly as v~ increased. at all conditions studied the rate of change in airway pressure prior to triggering the ventilator tended to increase as v~ increased. the changes in timing and drive were nearly complete within the first two breaths after transition with no evidence of adaptation during a given ~/~ period. we conclude that v~ exerts an excitatory effect on respiratory output which is independent of breathing route, temperature and volume of inspirate and airway anesthesia. the response most likely is neu~'al in origin, mediated through receptors not accessible to anesthesia such as those located in chest wall or below the airway mucosa. it has been shown, in mechanically ventilated awake normal humans, that increasing inspiratory flow rate (~/~) exerts an excitatory effect on respiratory output. it is not known if this effect persists during sleep. to test this seven normal adults were studied during wakefulness and nrem sleep. subjects were connected through a nose-mask to a volume-cycled ventilator in the assist/control mode and ~/t was increased in steps ( - breaths each) from to i/min and then back to i/min. v~ pattern was square, and all breaths were subject-triggered. forty-one trials during nrem sleep and during wakefulness were analyzed. both during sleep and wakefulness minute ventilation increased and total breath duration (ttot) decreased significantly in a graded and reversible manner as ~' increased. these changes were complete in the first breath after v{ transition. the response was significantly less during sleep than during wakefulness (p< . ); at i/min ttot, expressed as % of that at i/rain, was . +_ . % during sleep and . +_ . % during wakefulness. during wakefulness, at i/min, the rate of change in airway pressure prior to triggering the ventilator, an index of respiratory drive, was % of that at i/min (p< . ). the corresponding value during sleep, was % (p> . ). in four sleeping subjects the increase in v~ was sustained for . - min. there was no evidence for adaptation of the response; tro t, averaged over the last three breaths, did not differ from that obtained when vj was sustained for only - breaths. we conclude that ) vt exerts an excitatory effect on respiratory output, mediated by a reflex neural mechanism and ) the gain of this reflex is attenuated by sleep. chest radiographs is a common complementary technique for patients in critical care units, with a low cost and easily available. however, it has certain well-known limits in diagnosis, the most important derived from the low quality of some pictures. in this paper we make a general review of some new technical approaches developed for improving the quality of the images, and so incrensing the diagnostic value of conventional radiology. we begin deaeng with the correct positioning of the patient, trough the filtering techniques, the synchronization of radiology and ventilation, and we make reference to the new computerized systems for digital image processing. conclusions: the portable radiographic system is a device that probably with maintain for many years in critical care units as a basic non-invasive diagnostic tool. but we need an increase in the efficiency of it, applying means as simple as a correct positioning of the patient, or the use of fitlers or synchronizers. thus we should improve the general standards of portable radiography. "are circular circuits safe? quantifying undelivered tidal volume in pediatrics patients". objectives: to evaluate the overall influence of internal compliance of circular circuits on delivered tidad volume (vt). methods: we studied prospectively asa i pediatrics patients ( to yr. old) scheduled for elective general surgery. mechanical ventilation was supplied by an ohmeda excel (circular circuit). the internal compliance of the circuit (cc)-anesthesia machine plus external circuit-was determined by the supersyringe method: corrugated dar tubes of mm. id and . m. long (children < kg), and a corrugated dar set of mm. id and . m. long (children > kg) were respectively used for ccl an cc values of . and . ml/cm h . a vtof mlg/kg and respiratory frequency was adjusted for an end-tidal co (etpco ) between mmhg. tidal volumes (measured by spirometry) and airway pressure (paw) data were recorded every ten minutes. volumes and thorax-lung compliances were calculated as follows: (vt delivered = vtadjusted-vol compressible, being vol. compressible = co x ppeak (aw). apparent compliance (ca) = vt adjusted/pplateau(aw), and true compliance (ct) = =vt delivered/pplatean(aw)). comparative statistics were separately designed between calculated compliance data and tidal volumes on a paired sample ~test basis. results: calculated values for volumes and thorax-lung compliances were: conclusions: due to the elevated internal compliance of the circular circuit there is a remarkable dilference between adjusted and delivered vt: mean undelivered vt was . % and reached as high as . %. teere is also a significative error in calculating true thorax-lung compliance: its overestimation can be as high as . %. circular circuits are considered safe and cost-saving for anesthetical practice. nevertheless we conclude that anesthetists should bearin mind vt losses when using circular circuits, due to compressible volume. tracheal stenosis is one of the most serious complications of patients submitted to prolonged endotracheal intubation, in which the decrease in inner diameter of upper airway makes it very difficult to achieve a correct ventilation. objectives: compare the results of applying high frequency jet ventilation (hfjv) to some of these patients with conventional controlled ventilation (cmv). methods: we used a prototype of high frequency jet ventilator (santiago- ) developed in our university, and we developed a tracheal tube in wich we modified the distal tip (conic tip). we applied this system to two patients which were initially ventilated in the operating room with usuai controlled mecanical ventilation (cmv) following the standards of our department, and then intubated with the special endotracheal tube and ventilated with hfjv. results: we could verify a proper ventilation of both patients with cmv and hfjv. during hfjv, the airway pressures were lower than those recorded during cmv. a lower airway pressure prevents lesions due to high pressures. conclusions: hfjv is a good method of ventilation for patients with significative stenosis of the trachea, not only during surgical procedures, but also during ventilation for long periods in critically patients. the ventilatory setting is pressure support mode. the pressure level and fit were kept constant during h/d. arterial blood gas, wbc count, and mean bp was checked according to the schedule: '(immediately before h/d), ', ', ', ', ', '. respiratory drive (represented by poa), tidal volume(ti) and minute ventilation(ve) were continuously recorded by pulmonary mechanics monitor (bicore cp- ). the mean value of the breaths minutes before blood sampling were used to represent the ventilatory status of that period. anova test is used for comparison between groups. for poa, hierarchical cluster method is applied to divide the cases into two groups of similar change. conclusions: our data suggest that pl is very useful, non invasive and low-expensive emergenc e support for arf, expecially in the elderly with severe chronic pulmonary disease and relative controindications to eti. pl seems to be an effective alternative when it is not immediatly possible to perform etl. the multiple inert gas elimination technique (miget) can be used to assess the effects of any given mode of mechanical ventilation on the pulmonary and systemic factors determining arterial po and pco> however, a potential problem in mechanically ventilated patients is that the l mixing box (mb- l) placed in series in the expiratory side of the circuit of the ventilator to sample mixed expired gas may provoke substantial discrepancies between the tidal votume set in the ventilator and the effective tidal volume delivered to the patient, due to the increase in the compression volume (vc) of the circuit. the effects of the mb- l on the v c were compared with those produced by a new l mixing box (mb- l) specifically designed to produce adequate gas mixing and to prevent loss of the two most soluble gases (ether and acetone) used in the miget. at any given peak cycling pressure (p~ak, cm h~o), the v c (ml) provoked by the mb- l was substantially higher (vc= . *ppeak) than that provoked by the new mb- l (vc= . *ppeak). at a ppeak = cm h ~ the v c were ml (mb- l) and m{ (mb- l), respectively (p< . ). in a group of subjects ( m/ f, _+ years), for each of six the gases used in the miget, the regression line between the mixed expired partial pressures simultaneously obtained from mb- l and mb- l fell on the identity line. it is concluded that the new mb- l allows adequate assessment of the effect of different modalities of mechanical ventilatory support on pulmonary gas exchange, with less potential for gas compression and thus hypoventilation. objectives evaluate the influence of different pressure support ventilation (psv) levels on cardiovascular and respiratory funcion in icu polytrauma patients. metbed&we studied polytrauma icu patients , who were in weaning process , after long term mechanical ventilation for acute respiratory failure . mean age ( - ) yrs . they all were connected to servo ventilators siemens c , and all were in stable condition , without sedation , inotropes or diuretics. the hemodynamic studies were done with continuous svo , swan ganz catheter (oximetrix, abbott). they all were in spontanuous mode (spent) with cm h cpap for at least one hour. we turned them to psv with inspiratory assistance (psv cm h ) and after rain we applied psv cm h , and after min psv cm h . hemodynamlo and respiratory measurements were done before and after the application of insiratory assistance. the results were statistically analyzed with anova. resets . respiratory variables . no significant changes in minute volume (ve). tidal volume (vt) and mean airway pressure (mpaw) increased statistically significant (p< . ) . respiratory rate (rr) decreased significantly (p< . ) . blood gase showed no difference . cardiovascular variables. cardiac output (co) decreased ns , heart rate (hr) had no change , central venous pressure (cvp) , mean pulmonary artery pressure (mpap) , pulmonary capillary wedge pressure (pcwp) , increased ns , oxygen delivery (do ) decreased ns, oxygen consumption (vo ) decreased ns. conclusions. psv is a very useful respiratory mode helping patients to be weaned from long term mechanical ventilation . it has beneficial effects on respiratory function and oxygen consumption without affecting seriously the hemodynamic parameters, possibly due to a decrease of the work of breathing. a. michalopoulos, a. anthi, k. rellos, j. kriaras, s. geroulanos intensive care unit, onassis cardiac center, athens. objectives of this study was to examine the effect of different levels of peep on postoperative svo and pvo values in a group of patients, following open heart surgery. methods: upon transfer to icu, patients ( males and females) of mean age _-+ years, were randomly assigned to receive (n= ), (n= ), or cm of peep (n= ). there were no statistically significant differences in demographic data or preoperative respiratory status among the three groups. all patients were ventilated on the assist control mode with a tidal volume of ml/kg. the fraction of inspired oxygen (fio ) was adjusted to keep a pao around mmhg. mixed venous po and svo were measured at min, and hours after application of mechanical ventilation in the icu, just before extubation (be), half hour after extubation (ae), and at hours post-extubation. differences at each study time were analysed by anova. results: mean svo and pvo values among the three groups, for all study intervals, are presented in the table. conclusion: we found no differences (p=ns) in tissue oxygenation (expressed by svo and pvo ) among the three groups, at any study interval, in the early postoperative course of patients following open heart surgery. intrinsic peep (peepi), and high elastance and resistance increase inspiratory work load in copd. cpap reduces work of breathing by counterbalancing peepi. pav provides flow (fa) and volume (va) assistance proportionally to patient resistance and elastance and inspiratory effort. we studied the effects of partitioned support (cpap-fa-va) on breathing pattern and inspiratory effort in five copd patients on pav compared to spontaneous ventilation (sv) and full support (fs: cpap+fa+va). flow, volume, minute ventilation (ve) respiratory rate (rr), inspiratory swing in esophageal pressure (apes), and its integral per breath (pti/b) and per minute (pti/m) were measured. objectives: to evaluate airway pressure fluctuation (apf) during spontaneous breathing in a high compliance cpap system. methods: the cpap system consisted of two l weighted balloons in a wedge shaped holder. ventilating gas flowed from one balloon through a low resistance one way valve into a tracheal tube (ett) provided with a pycor co sensor to monitor rebreathing. the ett was connected to a piston drive mechanical lung. expired gas flowed through a low resistance valve into a second weighted balloon, from where it was exhausted through a peep valve connected in parallel with the second weighted balloon. we evaluated system performance at v r from to ml, at rr from to bpm, while closely monitoring cpap airway pressure swings. at v v of and ml the rr was limited to bpm. for comparison we explored aps of a one l balloon cpap system, the cpap mode of the puritan bennett , and siemens ventilators, when connected to a healthy adult volunteer breathing through an ett. results: the compliance (cpl.) of one l balloon system was linear over a range from . to . l, with a cpl. of . l/em h .the cpl. of the l balloon ( . l/em h ) was linear between a volume of and . l. apf of the weighted balloon system was under em h at all v r (except at a v r of ml aps was . em h ), while the apf in the l balloon was up to em h . apf witli human volunteers with the two commercially available ventilators in the cpap mode was about cm h ; while under identical conditions apf in the l balloon system was . emhzo; and in the two l balloon system was below lcm h . conelusions: cpap using the two balloon system exhibits lower airway pressure fluctuations than a single balloon system; and is substantially lower than found in the two commercially available ventilators when used in the cpap mode. objective: to perform independent lung ventilation (ilv) with individual tidal volume (vt) set at a value generating a plateau airway pressure (pplat) < crnh~o and to evaluate the usefulness of the continuous monitoring of endtidal co (etco ) as a guide to titrate individual lung vt during ilv and for the weaning from ilv. methods: in seven patients, ilv was performed with ttvo ventilators set with the same fio: and respiratory rate. each lung was ventilated with a vt that developed a pplat < cmh~o. this setting led to a lower vt on pathological lung (pl). vt was increased in pl following etco~ and paco -etco variations. ilv was discontinuated when etco~., vt and statical compliance (cst) were similar in both lungs. results: one hour after starting ilv (ti), pl mean vt was significantly lower than in normal lungs (nl) ( + ml vs + ml, p< ) two individual behaviours were observed on tl in pl: four patients presented low etco: (range - mmhg)and normal pacoz (range - mmhg), while three patients had normal etco (range - mmhg) with high pac (range - mmhg). one hour before stopping ilv (t ), vt, etc and paco were the same in each lung. the pao /fio: ratio improved in all patients from the beginning ofllv cst of pl was + % of the normal lungs' cst on ti and improved to . + % ofnl's cst on t (p< . vs conclusions: setting vt of pl to a value not overcoming a pplat threshold does not impair oxygenation and is helpful in avoiding barotraumatism. measurements of differential etco and of the differential paco -etco gradient can be used to titrate vt allocation during ilv and as a guide for the weaning from ilv. total respiratory resistance in mechanically ventilated patients exceeds values obtained in normal subjects, due to the added and highly flow dependent resistance of the endotracheal tube (rett). this can adversely effect the efficacy of pressure regulated modes of assisted ventilation, such as pressure support (psv) and proportional assist ventilation (pav). recent work demonstrates that the influence of rett during psv can be overcome by using tracheal (ptr) rather than airway opening (pao) pressure to regulate the pressure applied (intensive care med :$ , ) . the purpose of this study was to see if this approach would also be effective during pav. flow, volume, pao, ptr, and transdiaphragmatic pressure (pdi) were measured in intubated patients in which either pao or ptt were used to regulate the pressure applied during pav where volume assistance was varied from to % of respiratory elastance. representative results (mean + se) are shown below. compared to spontaneous breathing (pav %), pav increased tidal volume (vt) while reducing respiratory rate (rr) so that minute ventilation ('~e) also rose. this was associated with a reduction in inspiratory effort, as reflected by a decrease in the pressure-time integral ( [ p) of pes and pdi both per minute and per liter ~re. the effects on breathing pattern were similar for pao and ptr regulated pav. in contrast, the reduction in inspiratory effort was always greater for ptr regulated pav. in conclusion, the volume assistance provided by pav is more effective when ptr rather than pao is used to regulate the pressure applied. pav methods: retrospective data analysis of adult patients with normal pulmonary function before operation and uneventful course following coronary artery bypass graft surgery over an month period. we compared assist/controlled mandatory ventilation (s-cmv, patients), synchronized intermittent mandatory ventilation with inspiratory pressure support (s-imv/psv, patients) and biphasic positive airway pressure ventilation (bipap, patients). results: patients ventilated with bipap had a significantly shorter mean duration of intubation ( . h, p< . ) than patients treated with s-imv/-psv ( . h) and s-cmv ( . hi. with s-cmv . % of the patients required single or multiple doses of midazolam but only . % in the s-imv-/psv group and . % in the btpap group. the mean total amount of midazolam of these patients was significantly higher in the s-cmv group ( . mg) than in the s-imv/psv group ( . mg, p< . ) and in the bipap group ( . mg, p< . ). the consumption of pethidine and piritramide did not differ between s-cmv and s-imv/psv but was significantly lower during bipap (p< . ). after extubation the paco patients was highest in the s-cmv group. conclusion: ventilatory support with bipap reduces the consumption of analgesics and sedatives and duration of intubation. unrestricted spontaneous breathing as well as fully ventilatory support allow adequate adaptation to the patients requirements. bipap seems to be an alternative to s-cmv and sqmv/psv ventilation not only in patients with severe ards but also in short term ventilated patients. _objectitives: after end-inspiratory airway occlusion we examined the ensuing gradual decrease in tracheal pressure (ptr) with the following equations proposed by bates et al. and hildebrandt: pv = p'v e'~cccl~ +pst, rs (bates) [ ] where p'tr is tracheal pressure immediately after occlusion, to= is occlusion time, "r is viscoelastic time constant of respiratory system, and p t is static elastic recoil pressure of respiratory system. p~(t) = h -h log t (hildebrandt) [ ] where h~ and h are parameters depending on lung volume, and initial time is s for analytical reasons. materials & methods: we studied healthy patients intubated, anestethized with propofol, paralyzed with vecuronium, and mechanically ventilated with constant flow ( . i/s) at zeep for minor surgery. pressure was measured in the trachea. flow was measured with a pneumotachograph and volume was obtained by numerical integration. the rapid occlusions were produced by an external valve. the signals were sampled at a frequency of hz and processed on a pc. the influence of the cardiac artifacts during the occlusion time ( s) was reduced by a software low-pass filter kaiser finite duration impulse response of elevated order. results: the mean (+ sd) coefficient of correlation using eq. was , -+ . , and using eq. was . + . . the values ofz~ (eq. ), however, decreased with increasing the tidal volume (vt) according to the following equation: "~ = . - . v t, similary, the values of h~ and h increased with increasing v t according to the following functions: h~ = . + v i and h = . + . v t. conclusions: the behaviour of "% of eq. suggests that the linear viscoelastic model is not sufficient to further describe the mechanical properties of the respiratory system over the vt range ( - ml/kg) in ventilated patients. infect this model predicts that "c is constant and independent of tidal volume. on the other hand the plastoelastic model is not sufficient to further describe the mechanical properties of the respiratory system. in fact "r obtained by fitting an exponential for data of eq. , is determined by the time of endinspiratory airway occlusion. obiectives: according to the viscoelastic model, the viscoelastic pressure of the respiratory system pv=rs during lung inflation with constant flow e~ is t/ r wh t lsms ira tlmeand r given by:pv~c.~ = d~( -'e-~ )[ ] ere " ' p" tory " and "r are resistance and time constant of viscoelastic unit. in the past, the viscoaletic constants were determinated by performing a series of occlusions at different lung volumes, or a sedes of occlusions at a fixed lung volume achieved with various inflation flows. in the present study we have developed a new method for determining "c and r which requires a single constant flow inflation. our method is based on determination of pv~r, during a single breath constant flow inflation, and of z during the ensuing end-inspiratory airway occiusion. dudng the occlusion the tracheal pressure p~, declines according the following function: ptr = p'lr e " too= " z + e~t.r= [ ] where p'~r is tracheal pressure immediately after occlusion, toc c is occlusion time, p,i.rs is static elastic recoil pressure of respiratory system, and ~ is viscoelastic time constant. we first determinated "~ by analyzing the time-course of ptr according to eq and next determining r according to eq. , using the expedmental values of p,i=~, ~ and ti, as well as "~ obtained with eq. . materials & methods: we studied healthy patients intubated, anestethized with propofol, paralyzed with vecurenium, and mechanically ventilated with constant flow ( . i/s) at zeep for minor surgery. pres-sure was measured in the trachea. flow was measured with a pneumniachograph and volume was obtained by numerical integration. the rapid occlusions were produced by an external valve. the signals were sampled at a fi'equency of hz and processed on a pc. the influence of the cardiac artifacts dudng the occlusion time ( s) was reduced by a software low-pass filter kaiser finite duration impulse response of elevated order. results: the mean coefficient of correlation with eq. was . . with v t of ml/kg, the mean values (+ sd) of ': and r of the subjects amounted to . • . s and . • . cmh i "~ s. with the traditional multi breath method the corresponding values were . + . s and . _+ . cmh i " s, respectively. with the t-test the difference between new and traditional "~ was statistically significant, between new and traditional r was not significant. conclusions: with the single breath method it is possible to compute ': and r . the mean values of r with v t of nd/kg, however, was slighuy different than those obtained with the traditional multi breath method. the application of modem principles of respiratory care and mechanical ventilation in icus has resulted in increased survival of critically ill individuals with neuromuscular, skeletal and irrevers~le pulmonary diseases. in these chronically ill individunts mechanical ventilation, long term therapy (ltot) and continuous home care is considered a chronic life supporltng technique that can not be withdrawn after their discharge from an icu. the aim of this study was to present the results of a rehabilitation programme and home care that runs in our ward. twenw three patients were referred to our clinic f~om icus during - . a specific rehabilitation programme designed according to individual's needs was performed. patients that benefitted from this programme were grouped into the following disorders. ) post tb respiratow failure ( %) ) neuromuscular diseases, ( %) } undiagnosed sas { %) ) cope) ( %) ( patients had a overlap syndrom). the programme consists of : ) assessment and mechanical support ff needed of the respiratonj system with non invasive methods (nasal or via tracheostomy). ) group and individual respiratory therapy ) mobilization ) nutritional support ) educational classes for the members of the family. three from the patients passed away (during the year), are under nippv during night with or without supply, pts recieve ltot. conclusion: the development of a programme for chronically ill individuals in especially designed wards in hospitals and the overall care at home is considered necessary at least in hospitals with icus. a rehabilitation programme and home care permits the fast but safe discharge of these patients from units of acute medicine that the cost of treatment is high and besides permits beds that are invaluable. we considered that the rehabilitation prod'amine and home care in our ward is the first performed in greek chronically ill pts and even though there is no special administxative support we think that the results are quite saltsfactory. objective: we postulated that the product of the respiratory frequency (f) and the ratio of inspiratory pressure (ip) to maximal inspiratory pressure (mip) would predict the weaning outcome in deeompensated copd patients better than either variable alone or other indices previously proposed. methods: in decompensated copd patients with difficult weaning, we measured, daily, respiratory mechanics data both during mechanical ventilation and after ten minutes of spontaneous breathing. then we calculated weaning indices reported in literature and some new integrated indices. according to the results of the discriminant analysis, we considered the integrative index crop (acronym of compliance, rate, oxygenation and pressure), the rapid shallow breathing index f/vt, the load/capacity ratio ip/mip, and the following new index: f x ip/mip. we used receiver-operatingcharacteristic (roc) analysis by calculating the area under the curve considered as the overall probability of correct classification. results: main results are reported in the following objective: to evaluate the reliability of some indices of endurance in predicting the weaning outcome of decompensated copd patients. methods: in decompensated copd patients with difficult weaning from mechanical ventilation (mv) we measured, daily, blood gas analysis, ventilatory and airway pressure pattern during mv, breathing pattern (frequency (f) and tidal, volume (v~)), inspiratory pressure (ip), and maximal ip (mip) during spontaneous breathing (sb). thereafter we calculated the following weaning indices: crop (compliance * mip * (pao /pao ) / f), flvt, ip/mip. data obtained the day at which the patient was considered ready for a trial of sb on clinical grounds but weaning failed (wf) and those obtained the day of the successful weaning (ws) were compared statistically through the wilcoxon rank-sum pair analysis. in order to quantify the predictive accuracy for each index with respect to successful weaning we calculated sensitivity, specificity, and diagnostic accuracy according with the standard formulas. methods : five patients ( + yrs) suffering from ards (lung injury score > . ) for hours or less entered into the study. irv (volume controlled, decelerating flow, % inspiratory pause, lie = / ) was compared to conventional ventilation (cv) (volume controlled, constant flow, no inspiratory pause, iie= / ). these two modes were applied for hours in a randomized order, with the same levels of total peep (peept = peep + peepi), tidal volume ( . • . ml/kg), respiratory rate ( • "bpm) mad fit ( • %). measurements (respiratory mechanics, hemodynamics, arterial and mixed venous blood gases) were performed after , , and hours of application of each mode. rvsuils : are expressed as mean + sem and compared by anova. backeround and methods: periodic breathing (pb) is characterized by repetitive cyclic variation in minute ventilation. pb is considewxl to be provoked by an instability in the respiratory control. inintubated, spontaneously breathing patients conventional modes of pressure support ventilation, i.e., triggered inspiratory pressure support ps), do not allow patients to breathe with theirinherent breathing pattern. therefore, pb, if existing, will appear mainiy after extubation. since our new mode of pressure support ventilation" automatic tube compensation" (atc) continuonsly corrects for the flow-dependent tube resistance during insnmdon and expiration ("electronic" extubatim), it pemaits patients to maintain their own inherent breathing pattern. then, ff necessary, tracheal pressure can be additionally supported by volume-proportioead and/or by flow-proportional pressure support (proportional assist ventilation, pav). (~as~: we report the case of a -year-old male patient who was intubated due to acute respiratory insufficiency after acute myocardial infarction with left ventricular dysfunction. during ips of mbar the patient showed a regular breathing pattem which became periodic during atc. in addition, proportional assist ventilation of mbar/l increased periodic breathing in such a way that the typical cheyne-stokes breathing pattem occurred (see figure) . baqkground: the hering-breuer reflex (hbr) is characterized by an inhibition of inspiration during lung inflation. this response has been recognized as an important vagally mediated mechanism for regulating the rate and depth of respiration in newborn mammals. in adult man the hbr is considered to be active only at lung volumes well above functional residual capacity, i.e., at tidal volumes above ml. assessment of the hbr requires specialized methods such as single breath or multiple occlusion technique. methods; in the presence of desynchronization between ventilator and patient, which frequently occurs during triggered inspiratory pressure support ventilation (ips)(see figure) , prolongation of the interval between inspiratory efforts (indicated by negative deflection of the esophageal pressure) due to lung inflation exposes an active hbr. we examined the occurrence of hbr in intubated critically ill patients. strength of hbr was assessed by the formula: prolongation [%] = ((inspiratory interval of interest -preceding inspiratory interval)/preceding inspiratory interval) * ( . rr of patients examined showed moderate to severe desynchronization. in of these patients a (re)activation of the hbr was found. the strength of hbr amounted to + %. there was a significant correlation between tidal volume and strength of hbr. in contrast to previous reports, an active hbr was shown during lung inflation well below ml. b pck~round: triggered inspiratory pressure support ventilation (ips) is commonly used to support inspiration in intubated spontaneously breathing patients. despite its usefulness ips shows some disadvantages which can be deleterious in crificauy ill patients: -additional work of breathing to be performed by the patient due to the flow-dependent tube resistance -desynchronization between patient and ventilator due to inherent triggering failures of the ips mode suppression of the patient's inherent breathing pattern -inability to predict successful extubation in difficult-to-wean patients methods: based on the known flow-dependent tube resistance our new mode "automatic tube compensation" (atc) compensates for the pressure drop across the endotracheal tube ("electronic" extubation). then, if necessary, tracheal pressure can be supported by volume-proportional pressure support (vpps) and/or by flow-proportional pressure support (fpps). results: hitherto, we have examined patients after open-heart surgery and patients with acute respiratory insufficiency (ari) or ards using atc with/without vpps/fpps. preliminary results suggest that the new mode avoids additional work of breathing due to accurate compensation of the pressure drop across the endotracheal tube during in-/expiration prevents desynchronization between patient and ventilator allows patients to breathe with their inherent breathing pattern accurately predicts the outcome of extubation even in difficult-to-wean patients due to "electronic" extubation conclusions: the new mode atc with/without vpps/fpps allows to support ventilation in a more physiologic manner and overcomes the disadvantages of conventional modes of pressure support in intubated patients. backgound: cheyne-stokes respiration (cs) is characterized by regula]; recurring periods of hyperpnea and apnea. in normal subjects, cs may occur after hyperventilation, after arrival in high altitude, or during sleep. it has also been observed in patients with prolonged circulation time due to congestive heart failure, as well as in some neurological patients. there is no report about the influence of sedative drugs on periodic breathing (pb) and cs. methods: in intubated patients conventional modes of pressure support do not allow patients to breathe with their inherent breathing pattem. therefore, periodic breathing and cs are rarely seen. since our new mode of pressure support ventilation "automatic tube compensation" (atc) continuously corrects for the flow-dependent tube resistance during inspiration and expiration ("electronic" extubation) it permits patients to maintain their own inherent breathing pattem even if pathological, e.g., periodic. results: using this new mode of pressure support ventilation, periodic breathing was unmasked in of intubated patients, of which showed cs. in of these patients the occurrence of cs was linked to impaired left ventricular function with increased circulation time. normal left ventricular and neurologic function was found in the remaining patients. in of these patients cs disappeared after intravenous administration of the benzo-diazepine antagonist flumazenil (figure). consequently, in this patient cs was induced by benzodiazepine sedation. objecti',~s: in contrast to conventional rhodes for pressure supported spontaneous breathing, our newly developed ventilatow mode ,,automatic tube compensation" (atc) completely compensates for the flow-depandant pressure drop tlpm-r across endotracheal ttlbe (ett). in the atc mode, the ventilator supplies a flow v' in order to maintain a constant tracheal pressure p~,,~. to this end, pk,,= has to be oontinuousiy determined. since continued measurement of p,,~ by introducing a catheter via the ett is not reliable, we opted for its continuous calculation socordng to the following equation: p~ = p,,, -aperr, pw being the continuously measured airway pressure. this also requires the continual measurement .of flow v' to calculata apm-r using the non-fineer approximation: aport = kvv' + k .w. the constant tube coefficients k~ and k are mathematically determined by mesns of a least-squares-fit procadum based on laboratory investigations. tracheal secretions, however, reduca the omss-saction of the ett. consequently, ~ values of ki end k are changed rendering the p~,ch calculations inaccurate. therefore, k and ~ have to be pedodcally updated to ensure an a~urete monitoring of pn,~ and a complete tube compensation under atc at any time. background: one of the first steps in weaning patients from controlled mechanical ventilation is to stop muscle relaxation and to reduce sedation. it can take several hours, however, until the patient is able to trigger the ventilator and to breathe spontaneously. during this period, many patients display a sudden increase in peak airway pressure of up to %. patients and methods: to investigate the reason for this potentially dangerous effect, we continuously measured lung and chest wall mechanics in post-operatively ventilated patients. lung mechanics (airway resistance and lung compliance) was measured using the esophageal balloon technique as described in [ ] . chest wall mechanics (tissue resistance and chest wall compliance) was calculated from lung mechanics and total respiratory system mechanics as described in [ ] . results: we found a decrease of chest wall compliance (cw) to be the main reason for episodes of sudden airway pressure increase while lung compliance (cl) remained unchanged. the decrease of c w can be inter- gil cano a, san pedro jm ~, sandar d, herntndez . , carrizosa f, , herrero a. emergency and intensive care department, hospital of jerez, spain objective: ) to determine the incidence of hypoteasion (h) associated with emergency intabatian of mechanical ventilation, and ) to establish its relauonship with respiratory mechanics (rm) and arterial blood gases. mechanical ventilation performed in the emergency room, in a prospective eans~eative manner, were evaluated. data collected included patient demographics, diagnoses, blood pressure and arterial blood gas levels before and at~er intabatian, and p_m, including calculated pulmonary end-inspiratory volume above functional residual capacity (veic) and calculated dynamic hypetinflatien (dhc). all patients received midazolen and awaanrinm to facilitate tracheal intubatien and rm measurement. hypotension was defined as a decrease in systolic pressure higher than mmhg or an absolute decrease in systolic blood pressure below to mhg within hour of intabatian. patients were excluded because met at least one of the following exclusion criteria: preexisting shock or h ( ), cardiac arrest ( ) . there weren't any association between peepi or other airway pressures (paw) and h, but calculated pulmonary volitmes had tendency to be larger in patients with h (p < . ). high paco before lrasheal intubatian ( . - mmhg) with a quickly decrease alter starting mechanical ventilation was a usual finding (p < . ) in patients who developed h. paw. ) thexe was a good relatienship between h and high arterial paco before traqueal intahatian and its fast "washing" with mechanical ventilation. ) because cao patients had the highest incidence of h, controned mechanicel hypoventilatien driven by paco changes and pulmonary volumes monitoring instead paw, should be attempted in these patients to avoid this cemplication after tracheal intubatiert. introduction: the endotracheal tube (ett) and demand valve devices cause an added work of breathing (wobadd), which is the work necessary to overcome the resistive load of the ett and the breathing circuit ( ). application of ips has been shown to partly compensate this added work ( ). since tbe amount of wobadd is flow dependent, a fixed ips is not adequate to completly compensate the wobadd ( ). therefore, atc has been developed as a new form of assisted spontaneous breathing ( ), which provides a flow-dependent pressure support. thereby, it theoretically should compensate all the wobadd due to the tube. the purpose of this study was to evaluate the reduction of wobadd with ips and atc for different ett. methods: a mechanical lung model (ls , dr*alger, liibeck, frg) was used to generate a constant spontaneous breathing pattern. the ls was connected to an artificial trachea (at, cm long, mm id). the at was intubated with three different tubes of . , . , . mm id and connected to an evita ventilator modified to provide atc as an option (dfager, liibeck, frg). flow and airway pressure were measured between the y-piece and the ett for four different modes of ventilation: cpap, ips of and cm i and atc all with a peep of cm h . the tracheal pressure (ptrach) was measured in the at. total wobadd was calculated as the area subtended by the ptrach-volume curve below peep. results: the results for total wobadd in nd/ are shown in the figure for the three different ett: breath/mln, s=success, f=failur% *~p<. , **-p< , ns = non significant, f versus s neveltheless, in / patients, invasive ventilation was necessary in mean . _+ hours after beginning of fmpsv. there was no significant difference between the two groups (success, failure) in following parameters : sex, age, previous histoly, medical treatment, saps & , clinical signs (rr, spo , heart rate, blood pressure, glasgow score...), radiological and echocardiographic findings and standard biological parameters. only two parameters were related with failure : .a low value of pac on admission until the patients were intubated. . an increased level of cpk in relation with an acute myocardial infarction ( / cases in the failure group, vs / cases in the success group, x~(with continuity correction) : p<. ). conclusion : fmpsv is a noninvasive, safe, rapidly effective method of treatment in acpe, which may avoid tracheal intubation. further studies are necessary to precise if association of arf and low paco (< mmhg) and/er acute myocardial infarction represents an indication of immediate invasive ventilation. introduction: since the added work of breathing (wobadd) imposed by the endotracheal tube (ets and the breathing circuit is regarded as an important contribution to the total work of breathing, considerable effort has been tmdettaken to compensate for this added work. ips has been fotmd to decrease the wobadd imposed by different ventilators ( , ). because of the flow dependent pressure drop across the etf the tracheal pressure (ptr) should be measured to estimate the total imposed wobadd (wobtut) ( , ). the aim of this study was to assess the circuit imposed work (wobcirc) and wobtot (including ett) for different demand valve ventilators during cpap and/ps. methods: a mechanical lung model (ls , driiger, lfibeck, frg) generated a constant spontaneuus breathing pattern. the ls was connected to an artificial trachea (at), intubated with an . nun et]', end connected to one of four ventilators (servo c and servo , siemens,-elema, sweden; evita , driiges, liibeck, frg; pb ae, puritan bennett, carlsbad, usa). three different modes of ventilator settings were tested (cpap, ips and mbar; trigger set at maximal sensitivity, peep always mbar). flow and airway pressure (paw) were measured between the y-piece and the etr; tracheal pressure (ptr) was measured in the at. wobtot was calculated as the area under the ptr-volume curve below peep, wobcirc was calculated as the area under the paw-volume curve below peep. results: in the foti g., patroniti n., cereda m., sparacino me., giacemini m., pesenti a. inst.of anesth.and intensive care-univ.of milan -sgh monza i aim of the study was to assess cpl,rs measurement obtained by the airway occlusion method during psv. we therefore studied paralyzed cppv ventilated ali patients (lung injury score = . • that were weaned to psv. we performed end inspiratory and end expiratory airway occlusions using the hold function of the ventilator (siemens serve c), first during cppv and then within the th psv hour. airway pressure and flow signals were recorded (cpi bicore) for subsequent analysis. an airway pressure plateau was defined as a flow tracing in which airway pressure was stable for at least . sec. end inspiratory (pel,rsi) and end expiratory (pel,rse) recoil pressures were then measured as the mean airway pressure during plateaus. cpl,rs was computed as tv/ (pel,rsi-pel,rse i) cpl,rs can be adequately estimated during psv using the airway occlusion method; ) during psv inspiratory plateaus are longer than the expiratory ones; ) the length of plateaus is negatively affected by the respiratory drive. foti g., de marchi l., *tagliabue m., gilardi p., giacomini m., sparacino me., pesenti a. inst.of anesth.and intensive care,-univ.of milan *dept.of radiology-sgh monza i we retrospectively compared ct scan and gas exchange findings between a group of patients successfully weaned from vcv to psv (group s = ii patients) and a group who failed the weaning (group f = patients). we selected ali patients (lis= . • in vcv mode who had available a chest ct scan performed within days from the weaning trial. a psv trial was began as soon as the patient reached hemodynamic stability and a pao > mmhg, irrespective of fie (peep < cmh ). maximum psv level was < (pel,rs-peep) measured during vcv, where pel,rs was the respiratory system elastic recoil pressure at end inspiration. psv ventilation was considered successful if a respiratory rate < bpm, an increase in fie lower than . compared to vcv, a pace increase < % of vcv value and hemodynamic stability were maintained during the next hours of psv. if any of these conditions was not met the trial was declared a failure. interdisciplinary critical care unit, regional hospital lugano-ch *surgical critical care unit, university hospital, geneva-ch objective: to assess the degree of correlation of cardiac output measured by thoracic electrical bioimpedance and thermodilution in mechanically ventilated patients with different levels of positive end-expiratory pressure (peep). methods: prospective study with ventilated patients, after head injury and with postoperative sepsis, with normal cardiac output: simultaneous determination of cardiac output by thermodilution and thoracic electrical bioimpedance performed with different levels of peep ( - - cm h ). results: cardiac output measured by thermodilution during sequential increment of peep did not vary: . + . for peep , . + . for peep and . + . l/rain for peep . simultaneously the bioimpedance device recorded a significant increase in cardiac output from . + . for peep to . + . l/mi for peep . (p < , ). conclusion: cardiac output measured by bioimpedance cannot replace the invasive thermodilution methods of cardiac measurement output during mechanical ventilation with peep. we also isolated a subset (h) of patients who had been hypercapnic (paco > mmhg) for at least days (range to days) before the end of cv. the psv trial was started as soon as pao was > mmhg, irrespective of fie and with peep < cmh and the psv level had to be < (pplateau-peep) as measured during cv. pace , pha, base excess (be) were collected before discontinuation of cv and on the ist day of psv: ) . ) weaning is more difficult in pts with head injury(p<o, ) and iss> (p<o,ol), in elderly(p<o, ) and in pts who need fi > , (p<o, ) and peep>io cm h (p<o, ). ) pts with iss> need longer duration of mv (p<o,ol). ) the mortality rate is higher in pts with iss> (p<o,o ), with head injury (p<o,ol) and in elderly (p<o, ). ) paradoxally, compliance was found to be higher in pts> years than in pts< years (p<o, ). s. crotti, p.pelosi, d.mascheroni, m.cerisara, a.lissoni, l.gattinoni. istituto di anestesia e rianimazione -irccs, milano, italia. obiectives. we investigated by ct scan the effects of extrinsic peep (peepe) on lung inflation, tidal volume distribution and regional compliance in sedated, paralyzed chronic obstructive pulmonary disease (copd) patients with dynamic hyperinflation (peep• methods. two ct section (end expiration, end inspiration) were obtained at lung base level in patients (individual analysis for each of the eight lungs) at peepe levels: peepe = cmh (zeep) and peepe amounting to %, % and % of the peep• at zeep ( . • cmh ). tidal volume was kept constant ( • . ml/kg). each lung section was divided into zones equally spaced along the vertical axis: upper (the most ventral), middle and lower (the most dorsal) zone. for each zone we computed: gas volume at end expiration (gase) and at end inspiration (gas• tidal volume (dgas=gasi-gase) and compliance (cpl=dgas/dp;.dp=plateau pressureactual peep• we also computed the lung inflation as the entire ct section gas volume at end expiration (total gase). results. at each peep level both gase and gas• were higher in the upper than in the lower zone ( we cone!ude that in copd patients with dynamic hyperinflation only the application of peepe higher than peepi produces a further increase of lung inflation, decreasing cpl of the upper zone (the more expanded one) probably by stretching phenomena, and causing dgas redistribution from the non dependent to the dependent lung zones. i. ravagnan, p. vicardi, f. valenza, d. tubiolo. p. pelosi l. gattinoni. st . anestesia e rianimazione, universita' di milano, ospedale maggiore -irccs, italy objectives: to investigate the effects of peep and ps on respiratory. pattern and inspiratory effort during ps ventilation in patients with acute lung injury or acute exacerbation of chronic lung disease. methods: pts with acute (a) (paco = • mmhg, pao /pao = . • and pts with chronic (c)(paco = • mmhg, pao /pao = . : . ) lung disease were studied in ps ventilation during the weaning phase. measuring airway pressure, esophageal pressure and gas flow we computed respiratory rate (ril bpm), tidal volume (tv, ml) and pressure time product (ptp, cmh *s/min), which is an index of oxygen muscle consumption. measurements were collected, after rain of stabilization, at and cmh ps and at two different peep levels ( and cndd conclusions: during psv: ) breathing pattern and ptp are different between a and c; ) in both groups peep does not modify breathing pattern; ) peep reduces ptp in c but not in a; ) ps modifies breathing pattern and reduces ptp in c but not in a. to test the performance of a new heat and moisture exchanger (hme): twistair, baxter. this is a hydrophobic-hydrophilic hme without hygroscopic salts. method: we evaluated the hme performance with a previously described method (i); it consists of a "lung simulator" connected to a mechanical ventilator and a flow divider, with a dry and a wet thermal probe inserted into inspiratory and expiratory limbs. on average ps level changes were associate( with opposed changes in both p . and rr. changes in p . were remarkable, homogeneous and highly significant (p<. ), while changes in rr were less pronounced, inhomogeneous and less significant (p<. ). it seems therefore that p . is a better index than rr for estimating a change in load for the respiratory muscles. recently, the lsf method has been described as an interesitng alternative to conventional tecniques used to measure total respiratory mechanics, providing, over these latter, advantages such as no need for hold maneuvers and no need for particular flow patterns. data on the reliability of the lsf method, however, are still few. methods. sets of measurements were obtained in each of ards patients, paralyzed and ventilated in cmv with constant inspiratory flow and an end-inspiratory pause equal to % of ttot. the lsf method provided data for total compliance (ctotlsf) and total resistance (rtotlsf) by multiple linear regression analysis of airway pressure, flow and volume change, applied over the entire breath. the lsf measurements were automatic and continuous, and each value used for analysis was the average of consecutive cycles. conventional measurements of dynamic.compliance (cdyn), quasistatic compliance (cqs), minimum inspiratory resistance (rmin) and maximum inspiratory resistance (rmax) were obtained by the constant flow, end-inspiratory occlusion method, each value being the average of measuremts. ctotlsf was compared with cdyn and cqs, while rtotlsf was compared with rmin and rmax. results. ctotlsf showed a high correlation both with cdyn (cdyn=. .ctotlsf+l. , r =. ), and with cqs (cqs =. .ctotlsf + . , r =. ). the average difference between ctotlsf and cqs, yet, was much lower (+. + . ml/cmh ) than the one between ctotlsf and cdyn (+ . + . ml/cmh ). rtotlsf correlated much better with rmax (rmax=. *rtotlso+. , r =. ) than with rmin (rmin =. .rtotlsf +. . , r =. ). the average difference between rtotlsf and rmin was + . + . cmh / /s, while the one between rtotlsf and rmax was +. + . , confirming the better agreement between rtotlsf and rrnax. conclusion.the agreement between rtotlsf and rmax suggests that rtotlsf takes into account both the airway and the tissue components of resistance, unlike rmin which only expresses airway resistance. the agreement between ctotlsf and cqs indicates that ctotlsf expresses the pure elastic components of the respiratory system. in the paralyzed patient, the expiratory time constant (rce) of the unit represented by total respiratory system and ventilator can be calculated from the slope of the expiratory flow-volume curve. recently it has been suggested that an estimate of this slope is provided by the ve/v'epeak ratio, ve being the exaled tidal volume and v'epeak the peak expiratory flow.the reliability of this method is still little known. methods. sets of measurements were obtained in mechanically ventilated ards patients during paralysis and cmv. measurements of ventilator expiratory resistance (rext) and of total respiratory mechanics were performed in order to calculate these reference measurements for ve/v'epeak: rcdyn=(rmin+rext)ocdyn, rcstat=(rmax+rext)ocqs, rclsf=(rlsf+rext)~ cdyn (dynamic compliance), cqs (quasistatic compliance), rmin (minimum inspiratory resistance), rmax (maximum inspiratory resistance) were obtained by the constant flow, end-inspiratory occlusion method. clsf and rlsf respectively corresponded to measurements of total respiratory system compliance and resistance by the least squares fitting method. ve/v'epeak showed a high correlation with all reference measurements, rcdyn (ve/v'epeak= . orcdyn-. , r =. ), rcstat (ve/v'epeak = . rcstat -. , r = . ), and especially rclsf (see fig the static pressure volume curve of the respiratory system (p/vst) provides valuable information, but the extensive data analysis prevents its use in clinical routine. dynamic p/vcurves are simpler to record. the objective of this study was to develop an automated method to obtain quasi-static p/v-curves (p/vqs) during low flow inflation as well as to measure resistance. preliminary tests were done in normal subjects and subjects with varying degree of lung disease. methods: a computer/ventilator interface (cvl) was constructed for control of a servo ventilator c during an automatic sequence: a) a normal breath, b) a prolonged expiration at zero peep, c) a low flow inflation of a predetermined volume, d) analysis of ventilator pressure and flow. pressure was corrected for tube resistance. airway resistance was calculated from a) and used to derive p/vqs from c). as reference, p/vst was obtained by the occlusion method. results: in non-obstructive diseases the method performed well at volume and pressure controlled ventilation. static and quasistatic compliance were virtually identical. p/vqs clearly reflected the lower inflection point and, when present, the upper inflection point (uip). in some patients uip was more evident in p/vqs, especially at an increased inflation flow rate. in obstructive diseases, the method for subtraction of resistive pressure was inadequate. conclusions: the system for automated computer controlled studies of lung mechanics based on a standard ventilator provides comprehensive information. differences between p/vst and p/vqs imply that the latter reflects also other factors than p/vst that contribute to impedance at hyperdistension. the clinical significance of the two curves remains to be shown. obiective: to evaluate the effect of combined high frequency jet ventilation (chfjv) in patients with severe respiratory insufficiency (sri). methods: in a retrospective study of the period - we analyzed patients suffering from sri who were refractory to conventional mechanical ventilation and were treated with combined high-frequency jet ventilation (chfjv). refractory to conventional mechanical ventilation was defined as a pao /fio < , despite maximal ventilator settings: peep > cm hz , fit > . . a total of patients matched these criteria, males and females with a median age of ( - ) years. seventeen suffered from severe trauma. chfjv was started following a median period of ( - ) days of conventional mechanical ventilation. prior to chfjv ventilation parameters expressed as median were the following: fit . , pao /fio , peep cm h peak airway pressure (pap) cm h . chfjv consisted of high frequency jet ventilation with a frequency of to breaths/minute, driving pressure of . to . arm, and inspiration time of to percent, superimposed on the whole cycle of conventional mechanical ventilation with a frequency of l to breaths/minute and tidal volumes of to ml. results: following two days of chfjv of patients showed an improvement of ventilatory parameters; peep could be reduced to < cm h in patients, the pap was decreased with > cm h:o in patients, fio could be reduced to < . in patients and finally the median pao /fio ratio changed from to . during chfjv patients died, of respiratory failure and due to multiple organ failure, died within two days of chfjv. the median duration of chfjv in survivors and nonsurvivors was days in both groups. conclusions: our data show that with chfjv in the majority of patients with sri who are refractory to conventional mechanical ventilatior" the ventilatory parameters can be improved. backeround and obiectives: although ventilation with peep above the inflection point (pinf) has been shown to reduce lung injury by recruiting previously closed alveolar regions, it carries the risk of hyperinflating the lungs. in the present study we set out to develop a new strategy to recruit the lung during ventilation with small vt, while maintaining peep levels as low as possible. we hypothesized that if the lung was recruited with a sustained inflation (si) to total lung capacity, recruitment would be maintained as long as the peep level was higher than the critical closing pressure of the lung, as observed on the deflation limb of the pv curve (ajrccm ; ( ) :a ). the purpose of this study was to examine the hypothesis that a strategy using si and a peep<pinf would induce less lung injury during small tidal ventilation than ventilation at the same low peep level without using a recmitment strategy in a model of respiratory distress syndrome. methods: we used a randomized protocol with groups to ventilate nonperfused, lavaged rat lungs with small vt ( to ml/kg) for hours and studied the effect on compliance and lung injury. group : peep>ping group : peep<pin f with an si, inflation to on h over sec, to recruit volume; group : peep<pinf without si; group : control group, lungs were inflated at peep<pin f, but not ventilated. results: in group and group static compliance did not change after ventilation (table) . in group static compliance fell significantly. group showed significant changes in the pv curve, which were less severe than in group . results from morphological examination are pending but preliminary results showed less lung injury in group , compared to group . conclusions: small tidal ventilation following a recruitment manoeuvre in a model of respiratory distress syndrome allows ventilation on the deflation limb of the pv curve at a peep below pinf. this strategy ( ) minimizes lung injury as well as, or better than using peep above pint" and ( ) ensures a lower peep to minimize the detrimental consequences of high lung volume ventilation. i~peep>pin~ _objectives-this report is presenting the results of the clinical study for using eeg examination as a method of the evaluation of patients ability for weaning. methods: the study inclljqles eeg examinations with fourier spectral analysis' of patients ~vith respiratory insufficiency and prolonged control mechanical ventilation (cmv). all patients have had a-rhythm of eeg before weaning. we have followed respiratory rate, tidal volume, respiratory pa{tern, end-tidal co and blood gases during weaning. results: patients had invariable eeg activity or short -waves period (till one hour). the weaning of this patients was fast arid sucsessful. other patients have had a decreasing of a-activity, an appearence of -waves for an hour and more, a short episodes of a-and e-activity. after that this patients had gas exchange and respiratory disorders with regression of the weaning right up to cmv. conclusion: eeg could be used as a method of the evaluation of patients ability for weaning from cmv. some eeg signs shows the overstrain of compensatory systems before the change to the worse of gas exchange and respiratory pattern. s. elatrous, p. aslanian, d. touchard, d. corsi, h. lorino, l. brochard. medical intensive care unit, inserm u , hopital henri mender, cr~teil, france. in vitro comparison of flow triggering (ft) systems demonstrated advantages compared to pressure triggering (pt) systems for some ventilators (puritan bennett ) but not others (siemens serve ). we studied the two types of systems in two groups of patients mechanically assisted with pressure support ventilation ( + cmh ). in the first group (pb ) the effort of breathing, assessed by the esophageal pressure time index, was significantly lower with the ft than with the pt ( + cmh .s/min - vs + , p< . ). by contrast no significant difference appeared in the second group (serve ), as predicted by the bench study despite marked interindividual differences ( + cmh .s/min - vs + , p = . ). we conclude that ) rigorously performed bench studies can predict in vivo effects, ) mild advantages can be found for the new triggering systems on some ventilators. objectives: pressore-volume curves (pv) of the respiratory system is of interest for the determination static compliance (cs , lower (lip) and upper (uip) inflection points which indicate zones of airway recruitment and overdistension. this study aimed to compare an "automated low flow inflation" method (alfi) to the reference occlusion (oc) method. the ability of the former method to identify cst, lip and uip was tested in icu patients. me,otis: ( arf and ards) sedated paralysed patients were studied using a serve c ventilator linked to a computer which automatically forced the ventilator to insufflate at a low constant flow a velum up to - ml or a maximum paw of cm h (alfi). the quasistatic elastic pressure (pel,qs was obtained by subtraction of the resistive pressure of tubing and patient and related to volume for calculation of compliance cqst. for oc tidal volumes (v from up to - ml were followed by a s post-inspiratury pause for determination of static pal (pel,st) in relation to volume. compliance was defined from the linear part of the p/v curves. lip and uip were defined from the consistent deviation of p/v data from extrapolated the linear part. ~,~ i~: in ards, mean cst was . + . and cqst . + . ml/cm h (us), lipst . + . and lipqst . + . cm h (us), uipst . + . and uipqst . + ~ cm h (us). nosocomial pneumonias (np) are frequent and often unsuspected during ards (bell, ! ). in the present study, we evaluated prospectively the onset of np during severe ards (group b of the european study). patients and methods: the charts of patients with severe ards have been prospectively recorded. a plugged telescopic catheter (ptc) specimen has been systematically performed every hours, for quantitative bacteriological analysis. the diagnosis of np was defined by a number > colony forming units / ml. results: for the patients studied, the mean saps score (+ sd) was +_ , the initial pao /fio ratio was -&-_ , the duration of mechanical ventilation (mv) was + days. the mean delay before the onset of the first np was . + . days ( - ), and the mean pao /fio ratio was +- . respiratory symptoms (purulent aspirates, new pulmonary infiltrates, or gazometric changes) were present in % of the patients studied. alteration of gas exchange was present in of the patients ( np) . a new pulmonary infiltrate was present in only np ( %). an increase of fever was noted in patients, an increase of leukocytosis > % in patients, an increase of volume and purulence of sputum in of the patients with np. the degree ofgazometric worsening (pao /fio before np minus pao /fio during np) during the first episode of np was + mmhg. excluding the bacteriological criteria of np, the number of criterias of np present was in / patients, ( / ), ( / ) or ( / ). two patients only had a pulmonary colonization (ptc: < cfu / ml) before the first episode of np. the incidence of np is high ( %) during severe ards. the first episode occurs in average:at the th day, and is the cause of a severe hypoxemia (pao /fio ) . the onset of a np may contribute to the high mortality rate observed in our patients ( %). each worsening of hypoxemia during severe ards should induce to suspect a np. respiratory system during mechanical ventilation. the me~hod quantifies the dissipative energy consumption of the respiratory system in terms of energy loss aek, inefficiency ~k~ and respiratory dissipative resistance rk~ over a given partition of the tidal volume. the method can be applied in intensive care units with no interference to ventilatory support. it allows for monitoring the combined effects of inhomogeneities, non-linearities and visco-elastic effects, that are subject to change in the respiratory system. the method is studied on pigs~ in the presence of a log-dose response curve of methacholine (mch) induced disease. in healthy pigs~ we find a mean value of energy loss, ae, of . • j/l, a mean value of inefflency, ~ of . ~= . and a mean value of resistance, ~, of . • cm h s/ . the respiratory resistance, rk, shows a variation over the partition of tidal volume with armax ---- . • . cm h s/l. during methacholine provocation~ ae rises more than five-fold up to . • j/l~ doubles to . • and t~ increases to a maximum of • cm h s/l, with armax : . • . cm h s/ . the variation in rk becomes more pronounced with higher doses of methacholine. methods: ards patients were prospectively studied. initially they were ventilated in the amv (assist mechanical ventilation) mode with the settings prescribed by their primary physician. after stabilization, ventilatory gas exchange and hemodynamic variables were determined. patients were then ventilated in the mrv (mandatory rate ventilation) mode with breaths as the target rate. in mrv the target rate is set and the ventilator autoregulates the pressure support level delivered ~o achieve this rate. after stabilization, the measurements done on amv were repeated. finally, patients were sedated and paralyzed and ventilated in cmv (control mechanical ventilation) with the ventilatory variables they had during mrv. measurements done in amv and mrv were repeated and respiratory mechanics were assessed with the constant flow end inspiratory occlusion method. results: two groups were recognized based on their response to mrv. tn group patients responded to mrv by decreasing their v and increasing the t/t t ratio. ve, vo , and aado decreased while paco increased and tda vo ume and co remained unchanged. on the contrary, in group v, vr and ve increased; ppeak and trr t remained unchanged, paco~ decreased while vo and aado increased with constant co, the pressure support level needed to achieve the target rate was much lower in group than in group ( , -+ . vs . _+ . ). obiectives : in the newly developed mode of ventilatory support ,,automatic tube compensation" (atc) the ventilator compensates for the flow-dependent pressure drop across the endetracheat tube (ett) thus allowing ,,e]ectronic extubation". the aim of the study is to investigate whether healthy subjects perceive atc in inspiration (atc-in) and in expiration (atc-in-ex) and whether atc provides an increase in subjective comfort compared with the conventional assisted spontaneous breathing mode (asb). methods : healthy volunteers (no preceding lung disease, non-smokers, male, - years)breathed spontaneously through an uncut ett of . mm id via a mouthpiece. the ett was connected with a prototype ventilator evita modified by the manufacturer (drfiger, lebeck) for atc. flow and airway pressure were measured at the outer end of the ett. three ventilatory modes, ( ) asb ( mbarover mbar peep), ( ) atcin, ( ) atc-in-ex were selected in random order. immediately following the transition from one mode to another the volunteers answered by hand sign how they perceived the new mode compared with the preceding mode: ,,better" (+ ), ,,equal" ( ) or ,,worse" (- ). inspiration and expiration were investigated separately by presenting mode transitions (in total; including ,,placebo" transitions). results : the difference between atc and conventional asb is perceived in inspiration and in expiration. atc is positively judged; asb is nega ively judged. the diagrams show mean values _+ sd of five volunteers investigated up to now. the new mode atc is perceived as an increase in subjective comfort. our explanation is that atc preserves the natural breathing pattern better than conventional asb. objectives: to determine the role of cerebral vasoconstriction in the delayed hypoperfusion phase in comatose patients after cardiac arrest. to correlate the results with indices of cerebral oxygenation and the levels of several vasoactive hormones in the jugular bulb. methods: in comatose patients after cardiac arrest we measured the pulsatility index (pi) of the medial cerebral artery by transcranial doppler sonography. the pi is a reliable indicator of cerebral vascular resistance. we also sampled blood from the jugular bulb and measured cerebral oxygen extraction ratio and jugular bulb levels of endothelin, nitrate and cgmp. the first measurement was done within hours after cardiac arrest and repeated , , , , and hours later. results: we studied patients, females, mean age , + , years. the pi decreased s!gnificantly between th~ first and the last measurement from . _+ . to . + . (p = . ). cerebral oxygen extraction ratio decreased also from . + . to . + . (.p = . ). endothelin levels were high, but didn't change during the studied period. nitrate levels varied in a wide range, but didn't change significantly. however, cgmp levels increased significantly from very low levels in the first measurement to very high levels hours later, rasp. . pmol/ml (median; th . - th . ) and . pmol/ml (median; th . - th . ) (p = . ). eighteen and hours after the first measurement we found a strong correlation between pi and cerebral oxygen extraction ratio ( r = . , p = . and r = . , p = . ). we.also found hours after the first measurement a significant correlation between pi and cgmp levels ( r = . , p = . ). we found no correlation between pi and endothelin or nitrate levels. conclusion.~; our results show a high cerebral vascular resistance in the first few hours after cardiac arrest, gradually decreasing during the next hours. this is accompanied by an initially high cerebral oxygen extraction ratio and low cgmp levels, suggesting that the cerebral vascular resistance is induced by active vasoconstriction because of insufficient cgmp levels, leading to a decrease in cerebral blood flow and a compensatory ~ncrease in cerebral oxygen extraction. objectives: sudden cardiac arrest is a major cause of mortality in western countries accounting for over half of all cardiovascular deaths. in most cases the mechanism of death is prolonged cardio-circulatory arrest due to ver:tricular fibrillation (vf) preceding final asystole. recurrent syncopes due to idiopathic vf with good neurological prognosis have been reported in patients with and without cardiac etiology ( , ). in the past measurements of cerebral hemodynamics have been repeatedly done in humans during cpr, but until today no studies of cerebral blood flow velocity (cbfv) have been reported during controlled cardiac arrest in humans not under-going cpr. it was the purpose of our study to evaluate the acute hemodynamic effects of untreated vf on cbfv. methods: after approval by the local university ethics comittee, five male patients aged - years without evidence of cerebral disease were investigated during vf while undergoing implantation of a pacer cardioverter defibrillator system (model d; medtronic| a standard anaesthetic regimen was used (propofol, fentanyl). after implantation of the automated cardiac defibrillator vf was induced by electrical countershock to test effective sensing, pacing, and defibrillation. to measure cerebral blood flow velocities (cbfvmca) the doppler probe was placed above the zygomatic arch between the lateral margin of the orbit and the ear and directed towards the m segment of the middle cerebral artery (mca). results: a total of phases of vf were investigated. duration of vf ranged from to seconds, with cbfvmc a (mean_+sd, cm sec - ) flow pattern changing from pulsatile to laminar flow immediately after onset of vf. conclusions: the underlying mechanism of the laminar cerebral blood flow observed during vf in our patients is uncertain, but it may provide insight into the prognosis of patients with idiopathic vf. theoretically, the laminar cerebral blood flow observed in our pulseless patients may provide a substantial amount of cerebral perfusion even during clinical cardiocirculatory arrest objective: to investigate whether the intensive care nursing staff can inflate more accurately a specific air volume with the laerdal resuscitation bag when they receive feedback after each inflation about the delivered volume compared to no feedback. method: icu nurses were asked to inflate a testlung model times with a specific air volume ( ml, ,ml or ml) under three different conditions (normal, decreased compliance and increased resistance) without and with feedback. we measured the mean absolute difference from the specific airvolume after each ten inflations. results: the largest absolute difference was found when icu nurses inflated ml ( ml). the mean inflated volume for this group was ml. when the icu nurses had to inflate ml the mean absolute volume difference was ml with a mean inflated volume of ml. inflating ml produced an absolute volume difference of ml with an mean inflated volume of ml. the absolute volume difference decreased when the compliance of the testlung was decreased and even more when the resistance of the used endotracheal tube was increased. when the icu nursing staff received volume feedback after each inflation the mean absolute volume difference was reduced between the ml and ml for all specific air volumes. % of the last inflations with feedback were significantly smaller than ml from the specific air volume (p < . ). conclusion: the majority of nurses overinflated the specific air volumes. the largest over inflation occurred when ml and the smallest when inflating ml. when nurses were provided with volume feedback the performed significantly better. we concluded that icu nurses are not able to inflate a specific air volume with the laerdal resuscitation bag without receiving volume feedback. feedback is desirable in order to reduce the volume trauma. objectives: a pro_found impairment in systolic and diastolic myocardial function following successful cardiopulmonary resuscitation (cpr) has been demonstrated by using langerdorff method in rats. in the present study we have investigated post resuscitation myocardial dysfunction in a porcine model of cpr. methods: ventricular fibrillation (vf) was electrically induced by alternating current applied to the ep{cardium of the right ventricle in domestic pigs. following rain of untreated vf, precordial compression and mechanical ventilation was initiated and maintained for min. electrical defibrillation was then attempted and of animals were successfully resuscitated. results: following successful cardiac resuscitation, stroke volume index (svi) decreased from prearrest value of . ml/kg to . ml/kg (p< . ), and left ventricular stroke work index (lvswi) from . to . mmhg,ml/kg (p< . ). both svi and lvswi remained depressed for another hours. these decreases were associated with increases in heart rate from bpm to bpm (p< . ). no significant changes from baseline in mean arterial pressure, mean pulmonary pressure, right atrial pressure and pulmonary artery wedge pressure were observed. prehospital resuscitation efforts c. k ppel. g. fahron, h. lufft, a. kruger, c. th(jrk, f. bertschat, f. martens dept, of nephrology add medical intensive care, virchow-klinikum, humboldt-universit~t, d- bedin, germany obiective: the success rate of prehospital resuscitation in patients with cardiocirculatory arrest in an emergency medical system (ems) may reach - % depending on the time of calling the ems, the distance to cover by the emergency ambulance and the training of the emergency physician and his staff. in the berlin ems, which is associated with the berlin fire brigade, the time between alarm and arrival at the scene ranges from - min, mean min. resuscftation is based on the advanced cardiac life support (acls) according to the guidelines of the american heart association. if resuscitation efforts fail to restore circulation, they are terminated after - min, depending on duration of cardiocirculatory arrest, pre-existing disease, age, absence of an even transient response to cpr. however, there is a lack of practical criteria for termination of cpr in individual decision making. patients: we report cases of prehospital cpr with primary asystolia terminated after - rain of frustraneous cpr efforts including highdose epinephrine and dopamine. results: after termination of cpr, the ecg monitor remained connected and showed permanent asystolia in all patients while the emergency physician completed his records. spontaneous resumption of respiration and circulation was observed in these patients after - min and cpr efforts were immediately resumed, nevertheless, of the patients died at the scene, while could be hospitalized with stable circulation. one of them died hours after admission to the icu, the other survived for weeks in a vegetative state. spontaneous resumption of circulation and respiration is most likely due to the development of extreme hypercapnia and acidosis, which -at least in some patients -seems to be a stronger stimulant of the circulatory and respiratory brainstem centers than cpr with high-dose catecholamines, conclusion: because of the legal and ethical implications of this rare phenomenon, emergency physicians should continue ecg monitoring for at least rain. after termination of cpr efforts. pulmonary artery catheterezation is used for patient's monitoring [ ]. we reported our results on such monitoring in [f.coaobbeb,r.fe enb~-kap~monorm~, ,n ,p. - ] .however not all of the received criteria assessments meet demands that are necessary for early diagnosis of critical states. here we report the data on po ,pco (mm rg),so ,ph levels in femoral [af) and pulmonary (ap) arteries blood, as well as on summary gas pressure (sgp) calculated from pe=(po +pco ) in mm hg in ap blood. these data were derived from:i) subjects free of cardiovascular pathology according to catheterization data during their spontaneous air breathing (n group in ap blood appears to be a measure of adequacy ratio between pc and sgp in ap blood during air breathing; partly its characteristics and variations ranges are presented earlier [ j. in control group it is equal to , • mm hg. tests on sgp neither exclude nor substitute conventional (pc and pco ) tests, but rather include them as a part choosing only additive characteristic -pressure. they appear to be a part of general system of human metabolism regulation by pressure (arterial,venous,intracardiac, tissue,liquor,onco-osmotic,etc ietraabdeminal pressure produces perturbations of cardiac, pulmonary, and renal physiology. this most often occurs fonowing eeliotomy for peritonitis or intestinal obstruction; bowel edema and distention prevent wound closure without unacceptable compromise of blood pressure or pulmonary compliance. a variety of temporizing measures have been reported for managing wounds that cannot be closed: ) using towel clips to reapproximate skin only, )i sewing silastic, marlex or other prosthetic grafts to the fascia to "enlarge" the peritoneal cavity, ) using loosely tied retention sutures for partial closure, ) simply packing the wound without attempts at c~osure. these techniques either traumatize the abdominal wall (complicating definitive closure), expose the bowel to damage, or allow excessive loss of fluid and heat. since we have evolved a suturelees technique which permits the abdomen to be partially closed in a quick, safe, sterile, sealed, atraumatic fashion -while providin! decompression of unphysiologic intraabdominal pressure. methods: whenever possible omentum is interposed between bowel and the open incision. viscera are covered by a layer of sterile, non-reactive plastic, placed deep to the fascia and extending we~t beneath the edges. sump tubes are placed above the plastic and covered in turn by two layers of an adhesive plastic drape which sticks to the skin and seals the wound in all directions, the patients remain intubated and paralyzed. results: we have used this technique in a total of patients, four of whom suffered from compartment syndrome. all of the latter were males and ranged in age from to . all four showed immediate physiologic improvement. all four incisions were eventually closed without complication. one compartment syndrome patient died t days later of multiple organ failure. there were no complications related to the closure technique in any of the patients. conclusions; . selected patients with abdominal compartment syndrome will benefit from decompression using this temporary sutureless technique. the technique a) is quick, safe, sterile, sealed, and atraumatic, b) minimizes loss of fluid and heat, c) facilitates eventual definitive abdomina| closure. although m. brunner m. mitllncr objectives: to determine incidence and predisposing factors for cardiac arrest occurring during the first hours after open heart surgery. methods: the study included patients who, following open heart surgery, had adequate cardiac function and in whom cardiac arrest was not anticipated. all data were prospectively recorded and analyzed. results: from / through / , pts underwent open heart surgery at our hospital. of th~se, pts ( %) (age _+ yrs) had a cardiac arrest during the first hours after transfer to icu. they were operated on for coronary artery bypass grafting (cabg) ( pts), valve replacement (vr) ( pts), cabg and vr ( pts) and aortic aneurysm ( pt). the preoperative ejection fraction was _+ % whereas bypass and aortic cross-clamp time were + and + rain, respectively. prior to arrest, they had a cardiac index of . _+ . l/min/m and were receiving . + inotropes. arrythmias leading to cardiac arrest were ventricular tachycardia/fibrilation ( pts) and bradyarrythmia ( pts). closed-chest cpr was initially performed on all pts and was followed by open-chest cpr in pts. eighteen pts ( %) survived to icu discharge. causes of arrest included perioperative myocardial infarct (t pts, %), tamponade ( pts, %), rupture of the proximal vein gra& anastomosis ( pt, %), graft occlusion ( pts, %); no cause was found in pts ( %). conclusions: postoperative cardiac arrest in stable cardiac surgery pts is relatively infrequent (- % incidence) and is associated with a high survival rate following successful cpr. perioperative myocardial infarct is the most common predisposing factor. group ~deptof anaesthesia and intensive care, semmelweis univ. medical school, buda military hospital intensive care unit, budapest background: when a cardiac arrest occurs in-hospital, the outcome can be improved by a higher quality of basic life support provided by the witnessing health care workers until the code team arrives. this basic life ~pport (bls) should include the best available method for airway management as well. since not all medical staff are ready for carrying out endatracheal intnbation, we investigated the effieacy of the use of different airway management methods during bls. methods: we have investigated the efficacy of airway management of doctors and nurses from different hospital wards: internal medicine, department of surgery, trauma, urology and gynaecolagy. comparing the bag-valve-mask, laryngeal mask and the endotracheal intubafion, we have measured the following parameters: time needs for correct application (sec.), number of incorrect applications (out of ten trial), efficacy of artificial ventilation provided by the device. we used a computerised als trainer manikin for the evaluation of the performance. total performance score was created after the measurement between - . after the first screening we held a x hours training. doctors and nurses were trained for the endotracheal intubation (group it , t ) , doctors and nurses were trained to use the laryngeal mask (group lm , lm ) . all respondent were trained to use the bag-valve-mask device. day, month and month after the training we have carried out retention study using the same method. results: we have found that the efficacy of the artificial ventilation using the above mentioned devices were poor before the training. the average after-training performance scores of the groups are presented in the table below. (bls) should be initiated by the witnessing health care professional. the cpr study introduced a multi level code system, which means bls included sophisticated airway management, early defibrillation and early epinephrine administration provided before the code team arrives. our previous studies confirmed a poor level of cpr performance and a high demand for cpr training among health care professionals. method: we established a cpr training course centre, where doctors and nurses are being trained for in-huspital basic and advanced life support. x hours of training were held. after the theoretical introduction a step-by-step training method ws used for trainees to be familiar with all sequences of basic and advanced life support. then we synthetised all separated sequences. afterwards, a r e play of rescue groups was taken in simulated situations. we also trained the multi level alarm system fur the in-hospital resuscitations. after the training all respondents had to sit for examination. the quality of performance was scored and compared to our previous results. semi-structured interviews were carried out before and aider the training among all respondents to collect information about the course. results: we have found a remarkably high interest among doctors and nurses in our cpr training courses. it was very important to use proper equipment for the training: audio-visual training facilities, computerised als trainer manikin, manual and automatic defibrillator units. the evaluation of the examination held immediately a~er the training course showed a significant higher quality of performance than before the training. the self.-eonfidence of the trainees for initiating and carrying out resuscitation had increased. their overall feeling about the course was positive and % responded the course "very useful". . % of doctors and . % of nurses claimed fur regular training facilities with als trainers, conclusion: the cpr training for health care werkers is mandatory including the training of sophisticated airway management and use of elad~l~ills~tt~r wlaa ~en ~r a~ti~atir ~nel r rm~a'*h*nr m~thnd for training will improve the efficacy, the satisfaction of trainees, therefore their compliance for further co-operation will also increase. s objectives: the effect of reinfusion in emergency surgery and gynecology. methods: we had an experience of autologous blood transfusion in patients whom was produce t an emergency surgical or gynecological interventions in occasion with break tubal pregnancies ( . %), penetrating abdominal wounds with injuries of mesenterial vessels ( . %), injuries of the liver ( . %), blunt abdominal trauma with lien ruption ( . %). in . % patients had the previous somatic pathology. blood loss volume was - ml, & the reihfuside blood volume was - ml, consisting - % of blood loss. it was needn't to fransuse donor blood in . % in further but - ml of contanined erythrocytes were frasfused for supporting of hb concentration on the g/l ( g/dl) rate at the other patients with isovolemie hemodiluttion. results: the arterial blood pressure fast stabilisation on the perfusion level had noted after reinfusion, excluding the case, when the volume of reinfused blood had conisted just % of blood loss at the patient with massive blood loss. complications have noted in two cases. one patient with slash wound, injury of arteria gastrica dextra and total blood loss of ml, has an episode of asystoly, dic (disseminated intravascular coagulation) syndrome, acute renal failure, and acute pancreatitis that we haven't connected to reinfusion. all the complications were successfully corrected and at thirty first day patient with subcapsular wound of the lien that has happened days before complicated with external rupture of the capsull & massive intraabdominal bleeding, has the hemolytical shock, dic syndrome, acute renal failure developed after reinfusion. he was died. all another have no complications. posthemorrhagic anemia had corrected rapidly than in case when hemorrange corrected exclusively by donor blood. conclusions: we consider that simplicity, accessibility, high effectiveness, quite well further results of blood reinfusion, except the case of blood reinfusing that was for time-expired out of blood vessels (more than days in our case) will promote to the wide spreading of this method, especially in emergency surgery, in massive injuries, & in disarters, all the cases of insufficiently of time for selection of lot of donor blood. objectives: study of a reaction of the oardioreepiratory system of pregnant women to i/v microperfusion of clophelinum which is known to eliminate hemodynsmic and endocrine nociceptive reactions and can be used for treating hypertensive syndrome in pregnancy and labor. methods: the following non-invasive methods were used: capnography, spirometry, oxygenography, indirect fick principle based on the circle breathing, plethysmography and integral rheography~ functional indices of cardiorespiratory function were evaluated. results: pregnant women with ~h-gestosis were examined before and after i/v infusion of i ml of . % clophelin solution, . mg/kg/hour. before the treatment intensification of carbohydrate metabolism, hyperventilation with moderate hypooapnia and complete respiratory compensation of metabolic acidosis~ increased alveolar ventilation, decreased alveolar volume, predomination of perfusion over ventilation, hypokinetio type of circulation with dominated load by peripheral vascular resistance to the blood flow was observed in this group of patients. microperfusion of clophelin imp~-oved the ventilation/perfusion ratio, ventilatory and gaseous exchange efficiency, resulted in a decrease of congestion in the pulmonary circulation, possibly owing to a decrease of peripheral vascular resistance by %, of the heart rate by io. %, of the oardial output index by . %. conclusionm: the resulted type of circulation with a decreased load on the heart both by resistance and volume allowed to improve the cardioreepiratory system function in pregnant patients. objectives: the injury severity score is a measure of severity of anatomic injuries. iss is a sum of squares of the highest degrees of the abbreviated injury scale (ais) for each of three most severity injured regions. the purpose of the study is to establish correlation between the iss values and mortality rate in older, polytraumatized patients. methods and results: iss was determined for patients. the mean iss value was . + . while the median value was . minor injuries were present in ( %) patients with iss less than , while ( %) patients with iss more than had severe injuries. increased mortality of the older patients was noted in the range - . all patients older than died while % of patients below yrs of age survived, indicationg correlation between iss and mortality rate in polytraumatized patients above yrs of age. conclusions: this mode of evaluating severity of injuries may help in triage, determining appropriate level of care and as an indicator of future outcome of polytraumatized patients. objectives : tissue hypoxia is a non exclusive cause of hyperlactatemia. other serious medical situations induce hyperlactatemia. therefore, lactatemia could be a non specific indicator of severity in patients admitted in emergency unit. the aims of this study were to examine the correlations between lactatemia with the short term survival course prognosis and the unit of hospitalisation; intensive care unit (icu) or medicine unit, in patients admitted in our emergency department. methods -lactatemia was measured as soon as the admittance, in arterial blood sample of patients which needed arterial blond gas. sixty-one patients were included during months. to assess the statistical performances of lactatemia, sensitivity (se), specificity (sp) and accuracy (ac) were calculated for the threshold determined by the youden's test (se+sp- ). results : fifteen patients were admitted in icu and in a medical unit. fifteen patients died. a group of patients had a lactatemia up to mmol.l" . in this group of patients, had acidocetosis, had asthma, had cerebral vascular ischemia, had neoplasia, had cardiogenic shock, was epileptic, had congestive heart failure, had acute respiratory failure, had septicaemia, had hyperosmolar status finally had medicinal intoxication. lactatemia was significantly higher in non survivor than survivor ( . • vs. . + . , p<o.oool, respectively), the cut point of lactatemia was . mmoll" se was %, sp was % and the accuracy was %. on the other hand lactatemia was higher in patients admitted in icu than in medical unit ( . + . vs. . • p<o. , respectively). conclusions : this preliminary results suggest that lactatemia is a good indicator of the short term survival course in patients admitted in an emergency unit. furthermore, hyperlaotemia is present in several pathology seen in emergency. objective: to determine the pattern of injury, total peripheral white cell, neutrophil, and lymphocyte responses, and the outcome of trauma patients who are leucopaenic on admission to the icu. design: prospective, descriptive study of consecutive admissions over months. setting: a -bed surgical intensive care in a teaching hospital. patients: thirty consecutive adults admitted to the icu following trauma. leucopaenia was defined as a total peripheral white cell count of < x , neulropaenia < x , and lymphopaenia < . x ~ cells per litre. measurement and results: the total peripheral white cell count was documented daily and the neurtophil and lymphocyte counts and differential percentages on days , , and . the incidence of leucopaenia was significanfiy higher in patients with gunshot wounds (p < . ) and hollow visceral intra-abdominal injury (p < . ). eight ( %) patients died. no significant differences were found in the initial mean total white cell, neutrophil, or lymphocyte counts nor in the differential percentages between survivors and non-survivors. the total peripheral white cell count increased significantly in survivors compared to those who died (p < . ) and significant differences were found in absolute neutrophil counts (p < . ) and differential percentages (p < . ) on days and . no significant differences were found in lymphocyte counts or differential percentages. conclusions: there is a signficant association between leucopaenia, neutrepaenia, and intra-abdominal hollow visceral injury and peritoneal contamination. survival is related significantly to resolution of these white cell deficiencies. these findings suggest a possible role of granulocyte colony stimulating factor in the trauma patient with intra-abdominal infection and persistent neutropaenia. amelioration of organization emergency care team in order to improve caring for urgent cases. objectives: emergency care team (ect), as integral part of health, respectively as activity of primary health protection care all acute difficult conditions which vitaly danger patients and distressed. the aim of our investigations was to indicate on the possibilities of amelioration of organization ect in order to improve caring for urgent cases. methods: in our investigations we used epidemiologicai and statistical metods all informations and matherials from terrain ects. results: the number of traumatized in traffic accidents etc. have a epidemic character. it is well known that . % traumatized died in first two hours. ect isn't enough qualified to care a traumatized on the place of accident and during transport. because that medical personnel must have an education with solid qualities. conclusions: . besides a physician specialized (vii --+ vii -~ viii gradus) in urgent medicine, it's necessary to introduce two ( ) medical technicians instead of one as is existing now ( ~ ). .the medical technicians can drive cars and one of them always, drives the ambulance car ("b" category). .the technicians have been specially educated for urgent cases (iv ~ v ~ vi). . such a new team structure should be more capable in professional sense. .we can see an importance of team-work in ects. . such teams should also be more economic in comparison with existing one. . we think, that on the general medical studies need to include a new discipline-urgent medicine! objectives: to detoxicate an organism since we have used biological sorbents: isolated from liver and spleen of human or animal cells or tissue fragments. methods: cellular dialysis ( dialyses) in patients with an acute hepatic insufficiency poisoning by hepatotropic poisons: cc , dichloethane, mushrooms -were conducted using "artificial kidney" apparatus, by means of arteriovenous shunt, formed at forearm with disposable dialyzers. hepatocytes suspension was poured into dializing circuit of dialyzer at - mg of cells per i kg of patients weight. dialysis was performed during - hrs. patients with purulent-septic states were treated as for hemoperfusion through prepared sorptional column: . ml of hemosorbent and . ml of fragmented spleen or hepatocytes suspension with the help of roller pump with the rate of - ml/min, during - min. average volume of hemoperfusion made . . conclusions: in all cases we used both native and cryopreserved biomaterial. analysis of the dynamics of clinical, biochemical, scintigraphic and us-investigastions allows to conclude that application of cellular-sorptional method of detoxication enable to prolong life and reduce lethal outcome in some severe category of patients with endotoxicosis. after infusion of diazepam ( . mg/kg),thiopentone ( mg/ kg) and vecuronium (lmg/kg),patient was intubated and mechanical ventilation was adjusted to mantain normoca= pnia. ct scan showed left parieto-occipital hypodensity with brain swelling. after admission in neurological icu,dexamethasone ( mg/ kg/die),mannitol ( .smg/kg) and diphenylydantoin ( mg/ kg/die) were administred. two hours later neurological status of the patien~ was normal and she was extubated. the risks of stereotactie radiosnkgety are related to: hemorrhgge during the latency interval;transient radia= tion effects;permanent radiatio~einduced complications; radiation induced neoplasia. no epilepsy is reported in pts with avm and no seizure before radiosurgery. in c nclusion, epilepsy can be a posmib~e delayed eompli c~t~n, ~fe~r rgdi surgery for cerebral avm. serum potassium deficiency is a frequently reported finding in the time course of acute myocardial infarction (ami). if this is also correct for patients with ami undergoing primary percutaneous transluminal coronary angioplasty (ptca) is unknown.for that reason we analysed in patients ( m., f., + y.) serum potassium concentrations on ad ,mission and directly after ptca.the reference intervall for potassium in our clinical laboratory is , - , mmol/l. although more than % of the patients with ami undergoing ptca showed normal potassium serum concentrations on admission a measurable decrease of potassium could be found in % of patients ( ) after ptca.therefore for the majority of patients with ami potassium supplementation prior to ptca can be justified to avoid electrolyte disturbances as a possible trigger factor for cardiac arrhythmias in the setting of ami and acute reperfusion due to primary ptca. objectives: diagnostic procedures are very important in the management of abdominal injures in trauma. the aim of this paper is to present our experience with peritoneal lavage as diagnostic procedure in abdominal injury. methods: we analyzed patients to whom peritoneal lavage was applied in the surgical emergency from st of january till st of december . results; in this four years period there were abdominal injures. there were blunt injures. among them in patients peritoneal lavage was applied. results were positive in patients and negative in patients. false positive finding were in patients and false negative in two patients. in patients with positive lavage there were more than , /mm red blood cells, more than , /mm white blood cells and the level of amylase was up to u/i. the gall, urine and stool were positive as well. in patients with negative lavage there were less than , /mm red blood cells, less than /mm white blood ceils and amylase were negative. conclusion: peritoneal lavage is useful and important diagnostic procedure in the management of abdominal injures in trauma. our experience with urgent surgical management of the acute gastric and duodenal bleeding s.se en md, z.milo~evi md, *s.srdi md, k.sar ev md. clinic for abdominal and endocrine surgery, institute for surgery; *clinic for cardiology, institute for cardiovascular diseases; school for medicine novi sad, university of novi sad, yugoslavia objectives: acute gastric and duodenal bleeding are very common complication of gastric and duodenal ulcer. the aim of this paper is to present efficiency of our surgical management of these acute conditions. methods: in this paper results of surgical management of patients with acute gastric and duodenal bleeding, treated in our clinic from the st of january till st of december , are presented. results: among operated patients there were females ( . %) and males ( . %). there were patients ( . %) with gastric bleeding and patients ( . %) with duodenal bleeding. bleeding from gastric ulcer were treated with the following methods: excision of ulcer with suture in patients ( . %), ulcer suture in patients ( . %), billroth i in patients ( . %) and billroth ii in patients ( . %). heinecke-mikulicz-weinberger pyloroplastica was the most frequently used in the treatment of the duodenal ulcer bleeding. bilateral truncal vagotomy was done in patients ( %), duodenotomy with ulcer suture was done in three patients ( . %) and three patients underwent billroth ii operation. we had three complicationsduodenal dehiscence in pyloroplasty. there were no lethal complications. conclusions: based on our own experience we conclude that acute bleeding from gastric and duodenal ulcer can be safely managed with urgent and modern operative techniques. dept. of anaesthesiology. emergency center of serbia, belgrade, srj b ectives and methods: cardiac contusion was evaluated by serial determination of cpk-mb fraction and co using non-invasive doppler technique in patients with blunt trauma of the chest. results: miooardial contusion was diagnosed in ( %) patients, while in ( %) it appeared unaffected. cpk-mb of % and more in comparison to total cpkwas . + , % in patients, and , + , % in pts (p< . ).c of . + . l/min in patients with cardiac contusion, measured hrs after admission at the latest, was significantly lower than in group of patients with co of . + . l/min (p< . ). conclusions: co values were significantly lower in patients with cardiac contusion and correlated with pathological values of cpk-mb fraction. objectives: extracorporal plasmophoresis (pph) was implemented by non-stop method in patients with obstructive jaundice. methods: changes of serotonin (s) and histamine (n) in arterial (ab) and venous (vb) blood were investigated before and after one-time session of pph. s and h in blood serum were determined with fluoremetric method. results: before pph the average s contents in ab and vb did not differ. after one-time pph session s in the blood outflowing lungs was % lower than in the blood inflowing. hours after extracorporal detoxication implemented the indices kept the same level. similar dynamics after pph was observed in h. before the session h contens was equal both in the blood inflowing and in the blood outflowing lunge. after pph h contents in ab was % lower compared to the vb and did not change for hours. conclusions: dynamics of changes of biogene amines shows that under the influence of pph there takes place not only mechanical removal of their surpluses together with plasm, but there sharply grows inactivating role of lungs towards ba substances, which is confirmed by reduction of their concentration in the blood outflowing lungs. this effect is probably connected with the "deplasmation" of the lung cellular elements. simultaneously lungs' cells are freed not only from plasm but from toxic adsorbents on their surface. as a result the lung cells activate absorption of the surplus number of amines from the vessel channel. it is not excluded that s inactivation is due to growing activity of monoaminooxidase resulting in deminishing of intoxication. acute pulmonary embolism: thrombolytic therapy or pulmonary embolectomy? i.lodiena md, phd, s.shevchenko md, pphd., medical university, kharkov, ukraine objectives: pulmonary embolism (pe) remains a challenging problem for diagnosis and management for the emergency physician. the aim of this work is to identify the best treatment for massive pulmonary thromboembolism. methods: during recent years patients with symptomatic acute serious pe were reffered to our observations. selective pulmonary arteriography confirmed this diagnosis in all cases. ( . %) underwent urgent surgery, of them ( . %) with cardiogenic shock and ( . %) with massive pulmonary embolism. patients ( . %) underwent thrombolytic therapy. results: in the patients who received thrombolytic therapy successful results were achieved in cases ( . %), in ( . %) patients subsequent pulmonary embolectomy was performed. mortality rate was ( . %) in the patients who underwent pulmonary embolectomy after unsuccessful thrombolytic therapy and ( %) in the patients with cardiogenic shock and massive pe. lower extremity deep venous thrombosis caused pe in % patients. pe mortality rate is repoted to be %, but all patients with cardiogenic shock and massive pe died. this very high mortality rate related to the clinical status of the patients whose condition became worse despite intensive treatment. conclusions: the results of this study show that in most cases thrombolytic therapy is an effective rapid method of treating pe. however, surgical treatment is preferable when the patients reveal adverse effects to drug therapy, when medical therapy is unsuccessful or when the patients are seriously ill with reccurent cardiac arrest. high-quality pulmonary angiography remains the most accurate and reliable means of diagnosing pe. this prospective study was conducted to investigate the serum thyroid hormone levels in traumatized critical ill patients. serum thyroid stimulating hormene(tsh),total thyroxine(tr ),free thyroxine(ft ),total tri-iodothyronine(tr ),free tri-iodothyronine (ff ) levels were measured within hr.of intensive care unit admission(first day) in multiple tratmmtized male patients(injury severity score higher than ). in fifth day the same hormone levels were repeated. tsh,et ,ft ,tr and ft levels were determined by radioimmueoassay(ria). the levels of 'i~sh,xt ,fr ,et and ft in survivors and nonsurvivors were compared with first and fifth days (table) . in conclusion, we are convinced that there was a high correlation between low ser~n thyroid hormone levels and mortality, serum thyroid hormone levels are prognostic value in traumatized critical ill patients. objectives: glucagon improves myokardium contractility, intensifies kidney blood flow, stops bronchospasm ( ), these were the reasons of investigating its effect on hemodynamics and metabolism in hypovolemic shock expermentally and in clinic. methods : in experiments on white rats with traumatic shock glucagon (novo nordisk) was injected intraperitoneally in dose rocg/kg. patients with hypovolemic shock (trauma, hemorrage) were injected glucagon intravenously in dose mg on the background of infusion therapy. results : glucagon hightened recovery indexes of glucose in rats and patients with shock, whereas in groups without glucagon hyperglycemia developed. rats with glucagon injection had double times urea content than the norm ( , + , mmol& norm - , + , mmol/i ), whereas rats without glucagon had urea content , + , mmol/ ( p < , ). osmolarity of blood plasma in rats having glucagon injections was , + , mosm/kg h , without the drug - , + , mosm/kg h ( p < , ), ldh activity - , + . and , + , ( p < , ) mcmol cec- accordingly. lethality in the group of rats without glucagon was % /n= /, with glucagon - (n = ). in patients with hypovolemic shock in minutes after glucagon administration stroke volume increased to % (sv), miocardium contractility -to % (mc), peripheral resistance of vessels ( vr ) decreased to %. in - , hrs sv increased to %, mc-to %, pvr -decreased to %. conclusion : the study provides the evidence that glucagon has an anti-shock effect, improves hemodynamics and metabolism indexes in cases of hypovolemic shock. reference : picazo j. glucagon in acute medicine : pharmacological, clinical and therapeutic implications (norwell, mass:kluwer publishing, ), p. n, vasina (t) ,n.sebbota hph ( ). dept; of acute endotoxicosis, n.v. s~lifosovslay scientific research institute of ermergency ltealth care, boscow, lk'ussia (i}. institute for problems of cryobiology and cryouledicine of the national academy of sciences of the f/maine, kl'k r-key, [ maine ( ), objectives: the investigation of isolated hepatocytes using's efficacy in patients with acute hepatic fai lln-e. methods: native and crioconsrved allo-and xeno-!mmatocztes fixed on semi;ermeable ~mbr~s were used. results: the artificial supporting system with isolated hepatocttes as metabolic ~its was. ap-plied in patients. during tl~ treatmen~ with this cytotherapz an improvement of clinic state and biochemical rates were observed even inthe first we~s. for instance, the kncreasing of glutamin acid's level in blood up to normal value and decreasing of ammontes -concentration from i ~ mmol/l down to # mol/l were discovered. ~ae diminution of ence,%halo;athy was noted too. general lethality was' equal z, there of i z concerned acute hepatic failure (/k~). conclusions: the using of sum~rting hepatic system is able to cope the ~nenomenon of ~i~. objectives: fat embolism (ee) is an important and insufficiently studied problem of the intensive care medicine. fe is clinically diagnosed in % and morphologically in - % of the victims with severe multiple skeletal injury that is accompanied by the shock. methods: during the last years patients with the cerebropulmonary form of fe were treated in the icu. the prognosis of the probability of fe development was done using the computer system "cite-prognosis" in which the triss-method, dynamic and statistical prognosis of the injury outcome~ and the values of the most severe injury in points were realized. the treatment consisted of the surgical stabilization of the fractures with the authors' design of the rod apparatus in the acute period, long-term mechanical ventilation with peep via tracheostomy, complex fluid therapy using perfluorochemical emulsions (pe), and electrochemically active solution of na hypochlorite (snh) via the central vein. results: the treatment of patients with fe gave a good result that was manifested by the complete regression of psychoneurologic and respiratory disorders. conclusions~taking into account the prognostic data~ it is necessary to provide early complex of intensive care to the polytraumatized patients. it includes the surgical stabilization of fractures, fluid therapy using pe and snh~ early long-term mechanical ventilation. dr. alexandr e. poladko, station of medical aid. kiev ukraine. objectives: the reason of investigation is to prove the necessity of beginning of the intravenous infusion of nitroglycerin before the hospitalisation. methods: from january i to june protocols of treatment myocardial infarction patients in kiev medical aid station. results: the results of treatment are better if infusion began early or prolonged during hospitalisation. conclusions: there were patients with acute myocardial infarction treated before hospital by the cardiological groups. patients were treated with the early infusion of nitroglycerin and without. the results of treatment were: in the group with infusion: mortality in the first hours % the full disappear of pain % in the group without infusions mortality in the first hours % the full disappear of pain % that's why our opinion is that infusion of n. is necessary in the treatment of the patients with myocardial infarction from the first minutes of illness. dr. gennady d. kyrzhner, station of medical aid, kiev, ukraine. object: looking for safe medication which is good for the treatment of arterial hypertension and is simple for use. we inspected the effect of prazosin in the conditions of the cail during the year. method: we used mg prazosin tablets sub lingua and measured blood pressure ones during hours. the parameters were registrated in protocol. result." one hundred patients took part in investigation. the middle age of patients was . the reason of hypertension was not taken into consideration. in two hours after beginning of the treatment we caught the reliable lowering of blood pressure in % of patients. the general condition of patients became better in - min. it was only one accident of complication during the treatment -the orthostatic hypoter~sion, conclusion: prazosin hydrochloride in the dose mg sub lingua is safe and simple for use in the urgent situations. objectives: hypomagnesemia is frequently observed in severely burned patients during the early period after injury. increased urinary excretion is insufficient to explain the magnitude of the magnesium (mg) depletion. the possibility of exudative cutaneous mg losses has been proposed, but not yet demonstrated. the study aimed at measuring the mg content of the cutaneous exudates and urine during the first week after major burns. methods: patients, aged _+ years (mean _+ sd), and burnt _+ % of body surface, were studied from day (d ) to d . serum samples were collected at do, serum and urine from d to d , and on dio, d , d . cutaneous exudates were extracted from the textiles surrounding the patients, collected over h periods from d to d , using bidistilled acidified water. mg supplements ( . to mmol/ h) were prescribed from d to dio according to the severity of hypomg (serum mg < . mmol/i). results: mg serum levels decreased below reference ranges in patients between d and d , and normalised thereafter during supplementation. urinary mg excretion was increased in patients, mean excretion being . _+ . mmol/ h until d . mean daily mg cutaneous losses, were mmol/ h, i.e. mmo; in days. large inter-& intra-patient variability: the daily exudative losses ranged from to mmol/ h. conclusions: the mean daily mg cutaneous losses after burns were larger than the urinary losses, and equivalent to the recommended intakes for mg; the addition of the exudative and urinary losses largely explained the increased mg requirements after burns. complex immunologic alterations occur following thermal injury. the activation and consumption of the complement and dotting systems are suggested to play an important role in the development of the capillary leak syndrome, tn burned patients, a generalized inflammatory reaction as well as the impairment of the host defense are considered to be the major reasons for complications, such as sepsis and multiple organ failure. in a pig model we investigated a possible protective effect of cl-inhibitor (berinert, behring). before conducting the animal experiments the human cl-inhibitor concentrate was analyzed for its inhibitory capacity on purified pig cls and its pharmacoldnetic behaviour in pigs (t / ,id ). pigs received anesthesia and analgesia and arterial, venous and pulmonary (swan ganz) katheters were placed into the carotis and jugular veins. the pigs were scalded for seconds with ~ hot water to achieve a % tbs partial-thickness burn. blood samples were taken prior and after surgery as well as , minutes, , , and every further hours after thermal trauma. two control groups (each n= ) included animals which were either scalded or not scalded and treated only by resuscitation of ringers lactat solution. besides various parameters blood samples were analyzed for complement. the pigs (n= ) received c -inhibitor concentrate at an initial dose of u/kg body weight either immediately or hours after thermal trauma (n- ), followed by three further applications (of reduced amounts) every hours. the clinical outcome as indicated by vital parameters and as well as demonstrated by reduced edema and inflammatory tissue destruction suggested a protective effect of cl-inhibitor. complement analysis revealed a faster recovery of the alternative and classical pathway activities in cl-inhibitor treated pigs as compared to the control group but only if the regulator was given immediately after thermal trauma. from these results a protective function of c -inhibitor on thermal trauma can be assumed. objectives: the aim of study was to characterise the pattern of secretion of interleukin- (il- ) and tumor necrosis factor alpha (tnf alpha) in multiply injured or not injured critically ill patients and to relate these results to their outcome. methods: blood samples of multiply injured ( ( ) ( ) ( ) il- (nis) ( ) ( ) ( ) il- (nin) ( ) ( ) ( ) conclusions: this data suggest tnf alpha is a better prognostic marker in patients with multiple injury, than in critically ill patients without injury. il- is not significantly higher in nonsurvivors of both groups. sepsis is associated with an increased production of nitric oxide (no), as reflected by a rise in plasma levels of nitrites (no ) and nitrates (no ). severe bum injury is associated with similar symptoms of inflammation like hypotension, high cardiac output low vascular resistance and increased vascular permeability so that an increased no production may be involved. the present study included patients admitted to the intensive care unit of the burn center of brussels (age : - years; burned surfaee area - %), and control (non-septic) patients hospitalized in the neurological rehabilitation unit of the erasme hospital (age - years). the patients in both groups were fed enterally with to kcal/kg/day of a standard solution. in the burn group, daring the study period, patients were mechanically ventilated, required vasopressor (dopamine) therapy, and received antibiotics; patients were discharged alive from the intensive care unit, and no patient died during the study period. plasma was sampled for no /no determinations (griess reaction) daily from day to day (n=ll) or to discharge (day , n= ; day , n= ) in the burn group, and once the control group. in the burn group, no /no levels were elevated at day ( _+ #moles/i), reached a maximal value on day ( - p.moles/l) and progressively decreased to day (fig). these values were higher than in the control group ( . :t: . #moles/l, all differences p< . ). however, no correlation was found between the plasma no /no levels and the age of the patient or the total burned surface area; burned patients who became infected tended to have higher plasma no /no levels than those who did not ( + vs - #moles/i, ns). the present study indicates that burn noa/noa (llmol**/l) injury is associated with increasedloo] [__ ] ~ no /no plasma levels, which are ao so consistent with an enhanced no pro- o duction, ao obiectives:urapidil has been recommanded for therapy of hypotension in neurosurgical patients, because it was found not to increase intracranial pressure in patients with compromised intracranial dynamics ( ). in contrast several authors reported an increase of icp after urapidil administration in patients with head injury ( ). our study was designed to determin the influence of urapidil on mean lumbar cerebrospinal fluid pressure (csfp) in awake humans with uncompromised intracranial compliance. methods: after approval by the local university ethics comittee, six volunteers (asa , male, female) were studied in the lateral decubitusposition with the vertebral column positioned horizontally. a gauge needle was inserted into the lumbar subarachnoid space at level l /l and connected to a trancducer (mx | medex inc.) for csfp measurements. the volunteers were advised to keep respiratory rate and etco constant. after a resting period of minutes basline measurements were performed including csfp, cvp, map, hr, etco (cardiocap | datex). then the volunteers were given urapidil . mg/kg over a period of minute. minutes later measurements were repeated. results: statistics: wilcoxon signed rank test, p< . significant; values: mean_+sd, si = mmhg. - _ " _+ * _+ * - _+ " _+ csfp: cerebrospinal fluid pressure, cpp: mean cerebral perfusion pressure. conclusions: during normoventilation urapidil leads to a decrease in map parallel by an increase in csfp and therefore in icp, most likley mediated by an autoregulatory dilatation of cerebral vessels. if this cerebrovascular response is suppressed by hyperventilation, no increase in icp is found despite a uredipil induced drop in map ( ). after longterm hyperventilation cerebralbloodflow is normalized. this might be the reason why the drop in bloodpressure following urapidil administration again led to an increase in icp ( background and objectives: the intestine is one of the most sensitive tissues to ischemia-reperfusion injury. reperfusion of the intestine is often associated with increase in mucosal permeability and ulceration. d(-)-iactate is produced by indigenous bacteria found in the gastrointestinal tract and mammals do not possess the enzyme systems to rapidly metabolize it. the present study was designed to determine the kinetics of plasma d(-)-iactate alteration in rats paused by acute intestinal ischemia-reperfusion injury. methods: following the anesthesia, the superior mesentcric artery (sma) was exposed and it was occluded by a micro-bulldog clamp applied at its origin from the aorta. after min of occlusion, the arterial clamp was removed and the supply area of the sma was allowed to be reperfused ( h). plasma d(-)lactate levels were measured by an enzymatic spectrophotometric assay. the endotoxin content of plasma sample was assayed by the limulus amebocyte lysate test with a kinetic modification of the test kit procedure. results: intestinal ischemia for min resulted in a significant increase in d(-)-lactate levels within the portal vein as compared to sham-operated animals. plasma d(-)-lactate levels had a tendency to further increase after reperfusion up to h. it was showed that significant elevation of endotoxin concentrations in portal vein was alread~r detected at the end of -min ischemia, reaching peak at . h post-reperfusion and decreasing gradually throughout the study period. at the end of ischemia, marked mncosai damaged such as edema, hemorrhage and necrosis was observed. there was evidence of injury to ti, e muscuiaris propria together with diffuse mucosal damage and destruction following reperfusion, particular at h postreperfusion. in addition, penetration of bacteria was commonly found in the intestinal wall at various time points following ischemia/reperfusion injury. conclusions: these data suggest that acute intestinal ischemia is associated with permeability change of mucosal barrier resulting in increased plasma d(-)qactate, which is also remarkably influenced by subsequent reperfusion. plasma d(-)-lactate may be a useful marker of intestinal injury following both ischemia and reperfusion insult. objective: to assess the clinical usefulness of two methods of intracranial pressatre (icp) monitoring: intraparonquimatous recording (icpc) and epidural recording (icpe), versus intraventricular icp (icpv) as a gold standard. we prospectively studied patients with severe head injury (glasgow coma score < ) admitted to our icu between fobmaly and december . icpv was monitored in all pativrats by means of a multipzffolated catheter connected to a water coinnm. six of patients were additionally icpc monitored by a fiber optic transducer (comma r~), and seven of patients were icpe monitored by a couplvd fluid system (plastimedr). icpc was placed homolatetal to the focal, lesion m two cases and contralateral in three; icpe was placed homolateral to the focal lesiou in two cases and contralateral in three. all three systems were calibrated at the ear level. stali~cs: correlation coefficient and lineal regression were done between icpe and icpv, and between icpe and icpv with the hourly p~rs of values obtained dttting assistontial period. results: of the six icpc -icpv studied pa~onts, we observed a correlation coef~cieut r = . (p < . ) with a regres~on line y = . + . x. four of ~hx lcpc -icpv patients had r > . when correlaliou eoet~dent was obtained indixddually. of the seven icpe -]cpv studied patients, we observed a cortelafiau ooeffioiont r = . (p < . ) with a regression line y = . + . x. corralalmu eoetfieiont was inwer than . in all seven patients. corrdation eoelfieients for levals of icpv > man hg, > mm hg and > tuna hg with icpe showed r = . , r = . and r = . respectively; and with icpe r = . , r = . and r = . . the obtained values did not change during the study. conclusdns: in our study icpe was considered a good type of icp monitoring. /cpe signiticantly infravalorates icp values. we observed a good correlatinn between icpc and icpv values in patients with high inttacramal presanre. objective: midazolam is a benzodiazepine agonist widely used for sedation in emergency medicine. few studies in animals and humans point to a direct analgesic effect of midazolam probably mediated by spinal antinociceptive receptors and/or peripheral benzodiazepine receptors ( , ). in our experience in the berlin emergency medical system (unpublished results) with anecdotal cases of extreme chest pain due to binge drinking but no evidence of acute myocardial infarction or extreme abdominal pain due to peritonitis, acute intermittent porphyria, peutz-jeghers syndrome or testicular torsion, we found that small doses of midazolam ( - mg i.v.) were much more effective in relieving pain than repeated administration of high doses of buprenorphine or morphine, which may be associated with a considerable respiratory depressant effect. the dose of midazolam required for pain relief in these patients is non-narcotic and allowed further communication on the character and localization of' the residual pain, which might be very important for the further diagnostic procedure. patients: ten patients with abdominal pain due to acute gastrointestinal bleeding, suspected pancreatitis, suspected acute porphyria, and chest pain with no evidence of acute myocardial infarction received first-line midazolam i.v. at an initial dose of mg and were asked how it affected the intensity and character of pain. results: at the chosen dose of midazolam ( - mg), all patients were responsive to detailed questioning on basic orientation, the character, intensity and localization of the pain, and medical history. none of the patients required an additional opiate. all patients stated that the pain was tolerable after midazolam alone. conclusion: our preliminary clinical observations suggest that low-dose midazolam might be an alternative to opiates in extreme pain of presumably visceral odgin. objectives: it is known that severe head injury in elderly patients is associated with higher mortality than in younger patients. it remains however to be clarified whether the preinjury pathology which is frequent among these patients, affects the outcome. methods: in an attempt to investigate this hypothesis, patients aged over years suffering from head injury, with glasgow coma scale (gcs) of or less, were studied retrospectively. twenty-six patients ( . %) had preinjury pathology i.e. diabetes mellitus, arterial hypertension, heart failure, alcoholism, parkinson's disease etc. (group a) and fifty-three ( . %) did not (group b). the following data were recorded: mortality in the i.c.u., duration of hospitalisation, incidence of infective complications and neurologic status at discharge. results: groups were comparable in terms of mean gcs ( . vs. . ) and median age ( . vs. ). the incidence of brain pathology in the two groups was the following: epidural haematoma . % vs. . %, acute subdural! haematoma . % vs. . %, intracerebral haematoma . % vs. . %, subarachnoid haemorrhage . % vs. . %, diffuse haemorrhage . % vs. . %, contusion . % vs. . % and non-visible pathology (normal ct) . % vs. . %. unilateral pupilary dilatation was found to be . % in group a and , % in group b. the mortality during hospitalisation in the i.c.u. was almost the same: % iu group a and . % in group b patients. however, group a patients had significantly more infective complications, required longer hospitalisation and had lower gcs at discharge. conclusions: the results show that the existence of preinjury pathology does not seem to affect the short-term outcome of elderly patients with severe head injury. it has however an impact on morbidity and perhaps long-term survival of these patients. the assessment of clinical development in intensive care patients with severe head injury still remains a problem. to optimize the monitoring of intracraniel prassure (icp) we rautlr~dly implant an eplduml measuring device in our hospital. the aim of this study was to prove the correlation of the icp-values with ct findings and clinical development. during a month period ( - r the icp was monitored in p~,tients ( male, female) with severe head injury by an eplclural measuring device (epldyn~/$plegelberg| the mean age was . years ( - ). the glasgow coma scale at admission was . ( - ). in all cases the device was placed wfihln the first hours after admission. the tcp was compared with physical examination, radioidglcal or intraoperatlve findings and cunlca! outcome. the average time of measuring was . days ( - ) . the traatment depended on the !cp values recorded. rising icp-valuea ~ed to radlologlcal c ntra!s by ct-scan. in case an intracranlai hemorrhage was detected and drained. the overall survival rate was . %. showed a complete resolutl n, in other . % psychological residuals like decreased mentatlon, in . % sensomotorlc residuals like cerebral nerve dysfunction and aphasia, and . % of the injured remained in a comatous status. in % of our cases the measured values correlated with clinical course and management. in cases ( . %) we observed a displacement of the icp-pevice. there was no icp induced infecllon. istituto di anestesiologia e rianimazione, universit& ,,la sapienza", rome, italy * istituto superiore di sanit& -servizio di epidemiologia e biostatistica, rome, italy objectives: acute renal failure (arf) can be a severe complication of trauma. the current incidence of post-traumatic arf is associated with high mortality . identification of risk factors and prevention of this complication could improve the outcome of trauma patients. methods: one hundred fifty three consecutive trauma patients (age . _+ . , injury severity score . + . ) admitted to icu were studied. incidence of arf was . % ( / ). arf was defined as persisteat plasma creatinine > mg/dl with or without oligoanuria . arf was defined as early when occurring within the first hours (earf) and late when the onset was after the first four days (larf). results: earf occurred in patients while larf developed in patients. age, iss, and incidence of rhabdomyolysis and acute respiratory failure were not different in the two groups. an higher incidence of multiple organ failure (mof) and sepsis ( . % for both) were observed in larf group, when compared to earf ( % and % respectively). abdominal trauma was more frequent in earf group ( % vs %). the gs for earf and larf were respectively _+ . and _+ . while in the group who not developed arf (narf) the gs was . • conclusions: gs score difference seems suggestive and can be that an abnormal cerebral activity (hipofisary hormones?) may play a crucial role on onset of arf in these patients. moreover the frequency of acute respiratory failure in the group of arf was higher ( . versus . ) than narf group. the early ipoxia in the early phase of trauma, then, may be another crucial point for development organ failure. these are preliminary data. a more exact statistical analysis must be perform to have definitive conclusions. to compare the active compression-decompression cardiopulmonary resuscitation (acd-cpr) with the standard cardiopulmonary resuscitation (s-cpr) in out of hospital cardiac arrest patients. is a controlled, randomized study. two groups of patients with cardiac arrest out of the hospitalwere formed. group i, (acd-cpr) and group ii (s-cpr). for the acd-cpr groupweusedthecardiopumpdeviceofambulnternational. asfortherest, the erc ( ) algorithms for acls were followed. the utstein style (for out of hospitat cardiac errest) was used for listing and evaluating all cases of the study. the cpr was contucted by the crew and the doctors of our mobile intensive care units (micu). we studied consequitive patients ( in group i) and ( in .group ii). demographics pre-cpr characteristics (e.g. ecg form of cardiac arrest) and procedures (eg bystanders or second tiers crew cpr, defibrillation, drugs) were quite similar for both groups. the mean arrival time of micu was min. in group i we recorded r.o.s.c. (return of spontaneous circulation) , %, death %, continuation of cpr efforts , %. while in group ii, %, %, and , % respectively (recorded percentage until the admission to the hospital). no significant difference was found in anyofthe short term outcome parameters. no complications related to the acd-cpr technique, were noted. not any significant difference between the two methods was proven (from this small evaluated sample). the results of previous clinical studies are controversial (i) . more sophisticated studies proved the superiority, in a certain number of parameters (e.g pressures, flow, etc) of the new technique although there are many difficulties for establishing clinical results. in the pre-hospital setting that is related to many parameters (speed of the intervention, effectiveness of bystanders cpr, education ofparamedics, etc.)the evaluation is even harder. the superiority ofthe acd-cpr can be proven when it is performed in almost times increased number of studied patients as w~ll as improvement of the technique could lead us to more established results. objectives; infectious morbidity is the major cause of mortality after burn injury, and is due to multiple factors. trace elements (te), which are involved in both humeral and cellular immunity, exhibit severely altered status after burns. te supplementation has been shown to be associated with increased leukocyte counts and shortened hospital stay. the trial aimed at studying the immune responses in severely burnt patients receiving normal te supplies or early large supplements. methods: patients, aged _+ yrs (mean_+sd), with burns covering + % of body surface were studied from day (d ) to d post-injury, were randomised in groups (g): g -control receiving recommended te supplies + placebo; g -receiving in addition large supplements of cu, se and zn from d to d . enteral nutrition was started within hours of injury in all patients. immunological parameters: peripheral leukocyte counts, proliferation of mononuclear cells to mitogens, cell surface molecule expression, and neutrophil chemotaxis at d and d . infectious episodes and micro-organisms were monitored until d . results: the patients' characteristics were similar g & g . the total leukocyte counts were higher in g between d and d , due to increased neutrophils (significant from d to d ). total cd + and cdlg+ cells did not differ, whereas cd + (monocytes) were significantly increased at d . proliferation to mitogens was significantly depressed in all patients. chimiotactism was not altered. the number of infectious episodes was significantly decreased in g with a mean of . _+ . infections during the first days versus . _+ . in the control group (p < . ). conclusions: the large te supplements for days was associated with a significant decrease of the number of infectious episodes. supplementation was associated with increases in total leukocyte, monoeyte and neutrophit numbers. further studies are required to determine the precise mechanism underlying the improved immune defences. objectives: evaluate the efficiency of local adsorption (la) with the use of carbon adsorbents in case of severe burns in expertment and clinic. methods: experimental studies on la were performed on a model of % body surface area iiib-iv burn in rats. a burn eschar was excised on the rd day after burn, the wounds were dressed with the gauze bandages (control) or with adsorptive dressings (la), dressings were regularly changed. clinical investigations were carried out in the course treatment of patients with severe thermal and radiation ilia-iv burn. in the dynamics of bum disease some indices of proteometabolism and intoyacation criteria were evaluated. results: the experiments have demonstrated that the application of la after early excision of a burn eschar exerts a pronounced normalizing effect on a protein electrophoregram and the activity of proteases and their inhibitors in burned tissues preserving vitality. thus, by the th day after burn infliction the activity of cathepsin d in injm'ed muscles is times lower under an adsorptive dressing than under a gauze bandage (control) (p< , ), the activity of trypsin-like proteases is . - . times lower and the antitryptie activity does not differ significantly from the normal level. the cytotoxicity of extracts of burned tissues after the adsorptive dressing application fn vivo and adsorption in vitro is - % and - %, respectively, of the toxicity of control extracts. a similar normalizing effect of la is ok~rved for an intact muscular tissue and blood serum. the dectron-spin-resonance studies have demonstrated that la allows to normalize antitoxic activity of liver and functional activity of kidneys. the application of la in the treatment of patients with severe burns have been shown to localize a region of irreversible tissue changes, accelerate rejection of a burn eschar, attenuate an endogenous intoxication level and, as a result, shorten the time for grafting of a burn wound and accelerate wound heating. conclusions: proceeding from the obtained results, we can consider la as an effective method of localization of a region of irreversible tissue changes as well as of correction of local and general metabolism failures and overcoming burn autointoxication during burn disease. c de deyne, t vandekerckhove*, j. decruyenaere, b. vaganee, v vandewalle*, f colardyn depts of intensive care and neurosurgery*-university hospital gent-belgium. jugular bulb oximetry is the first bedside available cerebral monitoring technique providing an estimation of the adequacy of cerebral perfusion. its routine use in all patients suffering from severe head injury admitted to our ic unit enabled an extensive analysis of all very early cerebral perfusion data in order to evaluate the incidence of abnormal sjo~ data (and their possible causes) in this very eady period after traumatic insult and to search for possible implications as to the emergency management. these very early data were defined as the first hours icu data and icu admission had to occur within h of traumatic insult. over the last years, pts with severe head injury (gcs< ) were monitored by jugular bulb oximetry, starting immediately after their arrival at the icu (mean of . h after trauma, range between - h). in a total of pts (= . %), jugular bulb desaturatiens (< %) were noticed during this early h period. in pts (= %), jugular bulb saturations higher than % were observed, whereas pts (= . %) revealed no abnormal sjo data ( - %) during these first h. concerning the periods with too low jugular bulb saturations (n: ), we found the following correlation ; in pts (= . %) cerebral perfusion pressure (cpp) was below mmng, in pts (= . %) paco~ was below mmhg and finally in pts (= %) we found primary intracranial hypertension. for the high jugular saturations (n: ) we found a primary intracraniaf hypertension in f pts (= %), and a pace level above mmhg in pts (= %). in all patients we could restore jugular bulb saturation within normal range ( - %) with the correct!on of the presumed causative factor. we can conclude that ultra early jugular bulb saturation data revealed a high incidence of abnormal values, with a predominance of jugular bulb desaturations, confirming once again the high incidence of disturbed and too low cerebral perfusion within the first hours after severe head injury. these jugular bulb desaturations were especially correlated to systemic causes, as a too low cpp (caused in the vast majority by primary map insufficiency, and not by intracranial hypertension) and hyperventilation were the major causes of the desaturation periods. as jugular bulb desaturatione are known to be significantly correlated to a worse neurological outcome after severe head injury, one might improve outcome by an emergency management avoiding these possible causes of jugular desaturation. therefore, extreme attention should be paid to the maintenance of an adequate mean arterial blood pressure (above mmhg?) even duhng the few time spent at the emergency department. one should be as attentive to the maintenance of normoventilation during this very early period of admission and hyperventilation without any knowledge of icp or sjo should be abandonned. recently, indomethacine has been proposed for the treatment of therapy refractory intracranial hypertension in pts suffedng from severe head injury ( ). indomethacine, a cyclo-oxygenase inhibitor, gives rise to a significant fall in cerebral blood flow by inducing cerebral vasoconstriction. therefore, its use could result in a drastic lowering of the intraeranial pressure (;cp) in pts suffering from intracranial hypertension secondary to cerebral hyperaemia and in whom the use of other cerebral vasoconstrictive drugs (barbiturates or hyperventilation) appears insufficient to control icp. for the last months, we included the use of indomethacine in our therapeutic flow chart for severe head injury management. pts revealing intracranial hypertension (icp> mmhg) and cerebral hyperaemia (sjo~> %) and in whom icp was not efficiently controlled by the combined use of hyperventilation and barbiturates were given indomethacine in a trial to control icp. a total of head injured pts received treatment for intracranial hypertension over the last months. six of them met the criteria set for the administration of indomethacine. in pts, no decrease in icp or in sjo was observed and both pts died due to therapy refractory intracranial hypertension. in the other pts, a significant fall in icp and in sjo was observed shortly after indomethacine administration. in pts we observed a catastrophic fall of sjo= even below %, indicating an extreme cerebral vasoconstriction with the possible risk of inducing cerebral ischaemia. in one of the pts, icp remained under control without further administration of indomethadne, but he died days later in multiple organ failure. the other pts, needed multiple indomethacine administrations (for pt even during consecutive days) to finally control icp. in all pts, icp was finally controlled, but only pt survived. both other pts died from systemic causes (multiple organ failure in pt, massive gut infarction in the other tat, possibly due to the systemic vasoconsttictive effects of the indomethacine administration). in conclusion, indornethacine might have a role in the treatment of intraoranial hypertension, especially when caused by cerebral hyperaemia. we observed however a poor final outcome and a threatening high incidence of systemic events (multiple organ failure, gut infarction) in those pts receiving indomethacine for icp control. therefore, indomethacine in the treatment of intracranial hypertension should be reevaluated in controlled study settings, before its routine use can be considered. untill recently, intracranial hypertension (ich) in pts suffering from severe head injury was managed in a staircase approach, with csf drainage as first therapeutic step, mannitol as second step, hyperventilation as third step, and finally, barbiturates as the last rescue step for therapy refractory ich. this staircase approach for the treatment of tch was only guided by the intracraniat pressure, and not by other parameters such as e.g. the actual state of cerebral perfusion of the concerned pt. jugular bulb oximetry provides us with the first, bedside and continuous available, estimation of cerebral perfueion. its implementation in a rigourous flow chart, based on as well icp-as jugular bulb oximetry-data might result in an altered strategy for ich management. we adopted a '~ugular bulb saturation (sjo~)-guided approach" for ich management in consecutive pts, suffering from severe head injury (gcs< ). we maintained csf drainage as first therapeutic step, but the decision for the second step was guided by sjo information. pts revealing ich and sjo=values above %, were treated with hyperventilation, and did not receive mannitol. if ich persisted, barbiturates were added as a third step. on the other hand, pts with ich and sjo= vales less than %, received mannitol administration as second step. hyperventilation and/or barbiturates were only added if ich persisted and if no cerebral hypoperfusion was discerned (sjo=> %). our objectives were to prospectively analyze this new therapeuticstrategy, as compared to the formerly used staircase approach of ich. we managed pts with ich, with an overall mortality of . % due to therapy refractory ich. all pts received standard primary care with head elevation, full sedation and normovenfilation. fer pts, csf drainage alone was sufficient to control ice of the remaining pts, pts received mannitol and pts were hyperventilated as second approach. in the third line, pts were managed with barbiturates, with mannitol and pts with hyperventilation. finally, barbiturates were used as the final rescue in pts. these results reveal a less frequent use of mannitol as only pts received mannitol, compared to the pts that would have received mannitol using the former staircase approach. hyperventilalien was used much earlier in the treatment course, as lots were already hyperventilated in the second line approach, were this was formerly exclusively reserved for the third line approach. finally, also barbiturates were used much eadier ( pts received barbiturates as third approach). we may therefore conclude to a important change in the management of ich, induced by a sjo -guided flowchart. however, future studies will have to elucidate if this new strategy for the intensive care management of severe head injury will also result in an improved outcome. obsectives: in a first series of experimental brain injury we investigated the course of brain po , icp and cerebral blood flow after traumatic brain injury (tbi), whilst accordingly there are very few data available and the mechanisms leading to secondary brain damage are poorly understood. methods: in piglets ( days old, , - kg) of either sex we produced a moderate brain injury ( , arm., msec.) using a lateral fluid percussion {fp) device. complete measurements were made before and min. after brain trauma and after , and hours including blood gases, cardiac output (htermodilution), heart rate, eeg, laser doppler flow probe (ldf} and icp values (camino), brain temp., po by a clake type oxygen electrode (licox) and coloured microspheres for regional blood flow. results: immediately after the trauma a typical "cushing"response to the icp peak up to mm hg being highly significant (before mean i mm hg, range - mm hg) could be observed: mean arterial blood pressure rose from appr. mm hg to ii mm hg for - min. in two animals this was followed by an ischemic period lasting min. accordingly icp values gradually returned to starting measures within hours; in the ischemic animals they remained at a level of about mm hg.-no secondary increase of icp could be observed, once icp dropped to starting values within hours. cerebral blood flow (ldf) fell from mean values being i before trauma to appr. zero and recovered to around . brain po started at mean values of mm hg (range - mm hg) and fell to around zero depending upon the severity of the ischemic reaction. on average values of mm hg were reached over the time course. conclusions: with our fp trauma model we can reproduce the well known "cushing"-response after brain injury; secondary icp elevations cannot be achieved, although local edema is observed. direct brain po measurement seems to be a very sensitive variable for detection of cerebral ischemia and anticipates eventually following icp elevations by far. pulmonary aspiration s,traoaras. v. sgountzos, p. agouridakis, m eforakopoulou, e. ioannidou. intensive care unit (tcu) of "kat" hospital, athens, greece ob!e=ives: the reported mortality rate after pulmonary aspiration is variable in several series. the purpose of this study was to find out the influence of preexisting disease or situation on morbidity and mortality of intensive care unit (icu) patients with pulmonary aspiration. methods: patients who were treated in icu and had pulmonary aspiration, were studied, entrance's criteria in the study, all of them obliged, were: ) suction of gastric contents from trachea during intubation, ) presense of a predisposing factor, e.g. coma. ) recent hypoxaemia or new infiltrates in xray. preexisting disease was recorded and correlated with complications and outcome. patients with glasgow coma scale , because of cerebral injury, and patients who died within days from cause other than aspiration, were excluded from the study. method of statistical analysis: chi-square test, results: one hundred forty five patients were studied. the trauma patients were and the non trauma patients . from the trauma patients, had cerebral injury and were polytreumatized without cerebral damage. from the non trauma patients, had malignant neoplasms, neurological diseases in terminal stage, old age, drug overdose, and several diseases. eighty seven from trauma patients ( %) and from non trauma patients ( %) manifested several complications (pneumonia, ards, etc), so there was no statistical difference in complications' frequency between the groups (p> , ). the severity of complications was also proportional in the groups. eighteen deaths were recorded in the trauma patients (mortality %). only deaths correlated directly or indirectly with the aspiration ( %). in non trauma patients, deaths were recorded ( %). twelve deaths were recorded in patients with neoplasms, deaths in patients with neurological diseases, deaths in aged patients, death in drug overdose patients, and death in patients with several diseases, the mortality difference in trauma and non trauma patients was statistically significant (p< , ). in patients with drug overdose the mortality was significantly lower from the other non trauma patients and the difference was statistically significant (p< , ). conclusion: the preexisting disease or situation plays a major role in the outcome of the patients with pulmonary aspiration. the mortality of patients with aspiration seems to be caused by severe preexisting situations rather, that lead to death, than from the pulmonary aspiration per se, which may be a final happening in a predetermined course. obiectives; the purpose of this study was to compare fluconazole and amfotericin-b in the treatment of fungal infections in severe trauma patients. methods: thirty five severe trauma patients who were treated in intensive care unit (icu), were studied prospectively. they all developed fungal infections, prooved with blood positive cultures and at least one of the following: fever, positive urine or bronchial secretions cultures, infiltrates in xrays. the patients were separated randomly in groups. the patients of group a ( patients) received fluconazole rag/day for days. and the patients of group ( patients) amfotericin-b rag/day for also days. compaiison's criteria were the clinical responce to treatment (fever etc), the fungal elimination (blood and other cultures), the relapses of the disease, the side effects of drug, and the outcome of the patients. as method of statistical analysis was used the chi-square test. results: nine patients from of the group a ( %), and from of the group b ( %), presented remission of fever (patients of group b had better clinical responce than patients of group a, and the difference was statistically significant, p< , ). all the patients before treatment had positive for fungi blood cultures. after days of treatment, patients of group a and none of group b had positive cultures. eight patients (from who had positive cultures of bronchial secretions before treatment) of group a. and (from ) of group . had positive cuttures of bronchial secretions after days of treatment, so positive bronchial secretions were fewer in group b than in group a, but this difference wasn't statistically significant, (p< , and p> , ): ten patients (from ) of group a and patients (from ) of group b had positive urine cultures, after days of treatment (positive urine cultures were fewer in group b than in group a and this difference was statistically significant. (p< , ). two patients of group a and none of group b had a relapse of fungal disease. in group a, no side effects were obsepced, while in group b were observed only minor side effects (small increase of serum creatinine in patients, chills and fever during infusion in patients, and hypokalemia in patients). three patients of group a and patient of group b died, because of sepsis. conclusion: amfotericin-b (even i~ short regimen of days), is superior to fluconazole in the clinical and laboratory responce and also in the relapse of fungal disease, fluconazole is superior to amfotericin-b as it has no side effects. ob!ectives: flail chest after thoracic trauma is a serious injury. it is controversial if flail chest by itself orthe concomitant intrathoracic injuries e.g. pulmonary contusion, is the cause of the reported significant morbidity and mortality. in this study we searched the influence of concomitant thoracic injuries in the course and outcome of patients with flail chest. methods: eighty five patients with flail chest after isolated chest injuries were studied, for the purpose of analysis, we separated the patients into groups, patients with isolated flail chest were included in group a, patients with flail chest and hemo-pneumothorax in group b, patients with flail chest and pulmonary contusion in group c, and patients with flail chest and hemo-pneumothorax and pulmonary contusion in group d. complications from the chest, duration of mechanical ventilation and mortality were compared in the groups. statistical comparison of results belween groups was made using chi-square and t-studend tests. results: the patients were . all patients received mechanical ventilation, twenty eight patients were ihcluded in group a, in group b, in group c. and in group d. seventy three patients manifested complications from the chest, especially pulmonary infections. there was no statistical difference among the groups as to number of complications ( twenty four patients had chest complications in group a, in group b, in group c, and in group d. p> , }. the duration of mechanical ventilation was not statistically different among the groups (the mean duration was , days in group a, , in group b, , in group c, and , in group d, p> , ). there was also no statistical difference in mortality among the groups (six patients died in group a. in group b, in group c, and in group d, p> , ). conclusion: flail chest by itself is a serious thoracic damage with many complications, regardless of the presense of other thoracic injuries, which don't contribute to greater morbidity and mortality. the present study investigated the correlation between blood lactate mortality and organ failure in trauma patients admitting between december , and july , in the icu. road traffic accidents were the most common cause of trauma in this studded population. brain damage was the main cause of mortality .nevertheless, of patients died from sepsis and multiple organ failure without significant brain damage and these deaths were potentially preventable. respiratory failure was the most common complication and was developed in ( %) of survivors and in ( %) of non survivors .we noted low fncidence of renal failure may be do to the early and aggressive ittv'asive hemodynamic monitoring and cardiopulmonary support. as part of our routine case protocol serial blood lactate levels were measured in each patient at least times a day until the valses returned within the normal range or until death. we analysed the blood lactate levels on admission, the highest value and the number of days until the first normal value (<l.smeq/i). initial lactate and highest lactate levels were significantly higher in patients with than without organ failure.( . vs . , . vs . meq/l, p< . )the duration of hyperlactemia also was higher in the patients with organ complications. ( . vs . days, p< . ). both initial lactate and highest lactate levels were higher in the non survivors than in the survivors.( . vs . , . vs . meq/l, p< . ) the present study demonstrated that not only the degree but also the duration of hyperlactemia can be related to the development of posttraumatic complications. serial blood lactate measurements are reliable indicators of morbidity and mortality following trauma. s current evidence suggest that peep only affects intracrenisl pressure (zcp)when it interferes with systemic arterial pressure, although the effects of peep over cerebral oxygenation remains unknown. objective: determine the effects of peep over icp and cerebral metabolism measured by sj , kjdo and ceo in severe head injured patients. meterial ~ methgds: patients with glasgow coma scale l at arrival, with difusse brain injury in the first gt, according to marshall criteria,lop and jo monitored, under analgesia and sedation, normoventilated with zeep, without mannitol treatment in the previous hours, normal x ray chest and within the first hours from injury, were considered candidates. peep value where increased fro~ zero to cmh , in three different steps, each one with min of duration. all date were analized on line, pmax p'p ..... p.._, from the ventilator, systemlc haemodynem]c data, cerebra perfuslon pressure (cpp), icp and sjo , in the case of hemodynamic deterioration, during the study, ppc ommhg, extra-volume were administered. if persistence, dopamine were begun or increased dosage. if no good response were obtained, patients were retired from the study. results. patients were candidates, of them were exc;uded due to haemodynamic instability, k total of completed all steps and were e~amined, men and women, age ! . yrs. at hospite; arrival, glasgow were < in patients, and > in the rest . patients mmhg at the beginning. zeep ob/ectives. critically ill patients are transpoded to an intensive care unit(icu), under conditions, which have not been systematically evaluated. therefore, we set suite investigate transportation and admission condition of these patients to our department. methods. we studied patients( females), aged (mean-..+-sd) . _ . yrs, which were consecutively (from august to march ) admitted to the icu, through the greek national emergency transporta~on service. apache ii severity score upon admission was . -+ . (range - ). the following data were evaluated: ) number of medical departments, where health care was provided until final admission to the icu, ) ambulance transportation conditions, ) catheters and tubes inserted before admission, ) vital signs upon admission ) information provided by referring physician (scored on a to scale: history, electrocardiogram, chest x-ray, laboratory data, drug therapy already administered), ) comparison of the state of the patient described by referring physicians, to the actual state u pen admission. resu/ts. one to four medical departments had provided health care before the palient was admitted the icu ( : . %, : . %, : . %, : %). thirty/ ( . %) patients were escorted by a physician. twenty-six/ ( . %) were transported on oxyge n, fio (mean__.sd): -+ %, pao : . -+ . mmhg. five of the remaining , for whom no oxygen was provided, had pao : . -+ mmhg. twelve/ ( . %) were intubated and ventilated during transportation. thirtyfour/ had a peripheral venous line, / had an arterial line, / had a nasogastdc tube, / had a urinary catheter. eleven/ were sedated and / were paralysed. three/ were on inotropes. vital signs upon admission were: arterial blood pressure, systolic . -+ mmhg, diastolic -+ mmhg, heart rate -+ bpm, temperature . -+ cc. patient information score was --. . . the actual state upon admission was found substantially different, as compared to the description of the referring physician, in / ( . %) patients. conclusions. we conclude that several aspects of the greek national emergency transportation service to an icu should be reevaluated and further improved, i. e. ventilatory support, adequacy of information provided and accuracy of prior description of the patient's state. a new perspective must be applied for critically ill patients transportation since . % of the patients were evaluated and treated in more than one, medical departments, mostly primary care, before they were finally admitted to our icu. dclhb is a human derived hemoglobin molecule that has been cross-linked to stabilize and permit heat pasteurization to remove residual proteins and inactivate viruses. dclhb is mixed with a lactated electrolyte solution to yield a total hemoglobin concentration of log/dl objective: to present an overview of four recently completed clinical safety studies of dclhb in the u.s. and europe, and to discuss the properties, actions and potential indications for dclhb. method: patient populations in the four studies included males and females ranging in age from to years. dosing ranged from mglkg to mg/kg. the controlled randomized safety studies were conducted in chronic renal failure patients, surgical patients undergoing total hip replacement or abdominal aorta repair and in hemorrhagic hypovolemic shock patients. these very diverse patient populations allowed safety evaluation of the product in patients who were generally elderly, often hypertensive with some degree of cardiovascular disease, and receiving medications for treatment of other conditions. results: over patients received dclhb in the four:studies. no product related sarious adverse events occurred during the clinical trials. conclusion: results from phase itll safety studies of dclhb in patients undergoing chronic renal dialysis, abdominal aorta repair, or total hip replacement and in patients in hemorrhagic hypovolemic shock, indicate that the product was well tolerated in these distinct populations. although these studies were designed to evaluate safety, the data suggest clinical benefit. follow-up efficacy trials are indicated. prehospital emergency services represent the extension of emergency care into the community and constitutes the manpower, communications, transportations and facilities used to provide care for patients outside hospital. one of the main points of the system is how to decide the hospitalization of patients and what kind of facilities to provide : emergency medical service, fire brigade, locat general praclitionner or ambulance officers. objectives : to realize guidelines for using the prehospital emergency medical service in case of patient'calls outside hospital. methods : from st june to july , all the calls for emergency care were analysed using a questionnaire of items (origin of the call, responses to the questions of an emergency practitionner, kind of emergency service provided and the issue of the patient). after taking account of the appropriatness of the decision, statistical method used was a logistic regression. results : calls were analysed. the criteria, for prehospital emergency medical service using, given by the logistic regression were as following : existence of a call for emergency, thoracic pain, dyspnea, seizures, cyanosis, drug intoxication, fall of the patient, fracture, age, the state of consciousness and the neurologic reactivity. the minimal and maximal predictive values of the model given by the logistic regression are respectively % and %. the performance of the model is %. conclusion : it seems possible to help medical decision of emergency medicine by using only some easy criteria and a predictive model. (italy) objective: to evaluate the incidence of blunt carotideal injury (bci) in patients admitted to our icu after head injury. methods: we reviewed the medical records of all patients diagnosed to have a bci. at admission, the severity of trauma was assessed either with glasgow coma scale (gcs) and with ct scan. bci was demostrated by doppler ultrasography (us) and by angiography (ang). results:since may to april , patients were admitted to our icu with bci ( m, f, age + ). a history of direct trauma was present in patients. admission gcs was in all patients, and was associated with hemiparesis in of them; the last became paretic hours thereafter. two patients had concomitant injuries (a homoiateral clavicular and a controlateral zygomatic fracture, respectively). the initial ct scan was negative in every patient, and showed signs of ischemia after a variable timespan ( - days) after the onset of the symptoms. the bci was diagnosed with us and ang, which demonstrated a thrombosis of the internal carotid artery (ic). in two patients, an intimai dissection was also present. three patients were treated with heparin associated with antiaggregating agents and were discharged alive. the last patient was referred to our icu after the development of a massive hemispheric infarction, and died three days after the admission. at necropsy, the ic thrombosis was associated to an extensive homolateral extra and intracranial venous thrombosis. conclusions:the presence of focal neurological signs despite a negative ct scan should address the diagnosis toward a bci, thus implementing the diagnostic workup with us and/or ang. tab i: distribution of l~tients (%) in the groups the outcome were monitorett results were sabmitted to statistical analysis using a continence table x in z test. res.cl~s: of patients were submitted to thrombolysts and died. the higher incidence of bracb, ar~lhmias (ii degree gg p t e and av block. i degree av block. avsb . <ii. sinus arrest) that requited the insertion of avsc . <cl temporar~ pace-maker was recorded in group a b vs c . ns in % of the cas ~. the rapid recognition of life tab li:;~ test. threatening conditions suggests the monitoring of v r-lead r b' in the course of inferior ami. the authors report their own expirience in application of ringer lact.(rl)and , %nacl/ %dextran . methods:in polytraumatized patients with developed hae morrhagic shock,injures of the chest(qg)and abdomen(q ) prevalid.replacement of the volume loss egan in institutions of general medicine,frequently by rl and dextran or haemaccel.after receiving necessary medical aid,polytraumatized patients wer transported to mma by helicopter,continuing replacing of circulation volume by the same solution. results:average quantities of the solution dministered ~o the injured wer~ - ml.a group of the inoured which,after the admission to mma was resuscitated by adml nistration of the , %nacl/ %dextran @(groupii)( -sml/kg observed to the final surgical management,had statistically higher map(increase of % in relation to admission), cvp(average increase of , cmh~o),diuresis(averagely omi after min.),better gas ~nalyses and acid-base status in relation to the group which was administered only rl(groupi)(increase of the map of q %,average increase c~ of cvp , cmh and average diuresis oml after omin). total quantity of the administered solutions was significantly smaller in the groupii( ml: ml).the only ob served complication was hyperhatr~f~a::and~moderat~hyper osmolerity of plasma which disappeared within hours. the quantity of replaced blodd was considerably smaller in the groupii(qooofnl: ooml).in the group i interstitial pulmonary oedema was observed in two patients(qo%),while: in the group ii no complication was observed.coagulation i disorder and hypotension were not recorded because of the fast infusion of hypertonic/hyperoncotic solution in the groupil as the infusion of , %nacl/ %dextran lasted minutes. w.scherzer(l~,k.lechner( ),r.simon( ). ll)dept.anaesthesiology,trauma hospital,vienna-meidlin !( )neurotraum.rehabilitation center,vienna-meidling both austrian workers'compensation board,vienna,austria what we usually call"unconcious" means only that the patient is not able to interact in any kind of way.experiences in intensive care medicine (robinson, ) ,in general anaesthesia (bennet, ) and with evoked potentials show,that unconcious patients are not automatically unhble to process and recall information.this implicates a challenge to start the efforts to restore the traumatica-!ly desintegrated brain function as early as possible in the acute phase ia (zieger, ) .we present a method to ~repare the patient for the rehabilitation goals of the postacute phase ib,the phase of stabilisation ii and of rehabilitation iii within one concept. to achieve sensory stimulation in phase la we use: attempt at some kind of dialogue by speaking to and tou-ching the patient,to make him experience his body limits, ~acio-oral therapy by ice application,tast and smell pro vocations and trials of feeding,early adaption to circadian rhythm and guiding the patient in every-day-life-ac-tivities e.g.:in the brushing of teeth,washing etc. to achieve motor stimulation and orientation in terms of ~-ace,time and gravity in phase la we use: avoidance of perceptual impairment as produced by air-:sack-beds or habituation to supine position,using method~ according to affolter( ) ,basal stimulation (bienstein, ) , bobath-therapy( ) and facilitation of a physiological moving pattern(pnf)according to knott( ) . conclusions: by integrating all these measures in the ~herapy and nursing even in the acute phase la one prei ares the comatose patient for multisensory stimulation nd motor training and all other rehabilitation activities in the following phases of therapy. s objectives." measurement of cardiac output (co) and stroke volume (sv) requires insertion of a central venous catheter and is associated with a considerable risk for complications. non-invasive methods for determination of hemodynamic parameters have been introduced recently. one of these methods is the aortic modelflow program, which provides co and sv continously using a three-element windkessel model. the aim of the study was to evaluate the accuracy of the aortic modelflow program in critically ill patients. methods: patients (mean age: ) admitted to the emergency department after cardiopuimonary resuscitation (n=ll) and for myocardial infaction (n= ), acute congestive heart failure (n= ) and anaphylactic shock (n=l) were included to the study protocol. after stabilization all patients received a swan-ganz catheter (baxter~; edwards swan-g-anz) via a central vein. co and sv were measured by the use of a cardiac output catheter and injection of rrd ice-cold glucose % non-invasive cardiac output measurement was done by using the continuous cardiac output software package modelflow (tno; portapres| results: overall, paired measurements were used for the evaluation of the accuracy of non-invasive obstained sv and co. mean values, standard deviation and range of co and sv evaluated by invasive and non-invasive methods are summarized in the aortic modelflow provides reliable hemodynamic data in critically ill patients. it may be a useful alternative due to its non-invasive approach and continous measurement. objectives: to determine the feasability and accuracy of a rapid urine screening test (triage tm, merck-diagnostika) in an emergency department. methods: prospective analysis of the triage tm testkit (tt) in patients admitted because of suspected drug abuse during an observational period of months. the tt is a eompetitve immunoassay which gives qualitative results within i minutes for methadone, benzodiazepines, cocaine, amphetamine, opiates, barbiturates and cannabis. urine samples were analysed with the tt and compared to the results of a enzyme-lihked tmmuno-assay (el.a) in our labratory (gold standard). results: during the study patients were enrolled, in of these patients substance abuse was proven by tt and verified by eia. we recorded no false positive or false negative results for benzodiazepines, barbiturates, opiates, cocaine and cannabis. two results were false positive and one false negative for methadone; one result was false negative for amphetamines. the triage-testldt had an overall sensitivity of . and specificity of . . conclusions: the triage tm testkit seems to be a a useful rapid test device in addition to quantitative tests for drugs of abuse in an emergency department. objectives: the purpose of this study was to evaluate the influence of several factors of the renin-angiotensin-aldosterone system on blood pressure response in patients with hypertensive crises. methods: patients with systolic blood pressure > rorohg and diastolic blood pressure > nunllg were included into the study. prior to treatment blood samples for determination of plasma renin activity (pra), angiotensin converting enzyme (ace), angiotensin ii (ang ii) and aldosterone (aldo) were collected. all patients received rog enalaprilat intravenously. success of treatroent was defined as a reduction of systolic blood pressure below mmi-ig and diastolic blood pressure below mmi-ig within minutes after start of treatment. results: patients were included in our study, ( %) patients responded successfully to treatment. mean arterial pressure decreased in responders by . mmhg and in non-respenders by . mmhg (p< . ). responders and non-respenders differed signii'icantly concerning pra (p= . ), ace (p= . ) and ang ii (p= . ). . . the extent of blood pressure reduction correlated positively with the pretreatment pra and ang ii concentrations (correlation coefficient for pra: r= . ; ang ii: r= . ). conclusion: our data confirm that in patients with hypertensive crises blood pressure response to ace inhibition is mainly determined by circulatory pra, ace and ang ii. as the extent of blood pressure reduction correlates with pra, ace-inhibitors in patients with suspected high renin status cannot be recommended, as excessive blood pressure reduction, which carries a considerable risk for further organ damage, may occur. f. staikowsky, n. grillon, f.pevirieri, c.jedrecy, c. zanker, f. michard, a. haft medical emergency department. hospital bichat, paris epidemiology of acute intentional self medications-poisoning (smp) in france is especially known by data of poison control centei,s and intensive care units (icu). the purpose of this study is pro~,ided characteristics of this problem in a med for adults. method: july to june , files of patients consulting to the ed for smp have been retrospectively analyzed. results: patients, women and men, . + years old (range - ) have been admitted for episodes of smp ( % of all consultations) whose relapses during the period of study. psychiatric disorders, drug addiction or hiv patients was found for respectively . %, . % and , % of patients. the interval of time between the ingestion and emergency consultation was noted for % of smp ( + min, ranges - ). the involved products name was known in totality in % of cases with an average number by episode of . + drugs (ranges - ). the most often, ( %) or ( %) different products were interfered. the nonbarbiturate psychotropic drugs accounted for . % of the products (benzodiazepines %, antidepressants . %, neuroleptics %, carbamates . %, imidazopyridines . %, cyclqpyrrol nes . %). analgesics and nonsteroidal antiinflammatories represented . % of all drugs, anticonvulsants . %, cardiovascular drugs %, antiinfective agents . %, drugs against cough . %, muscle relaxants . % and antihistamines h . %. the benzodiaz pines were present in episodes, alone in episodes. in . % of cases, there was a simultaneous intoxication with alcohol. the processing consisted of gastric lavage in . % of cases, activated charcoal in . % of cases, flumazenil in . % of cases, naloxone and acetylcysteine in . % of cases; orotracheal intubation was performed in patients. admission in hospital was effective for patients, in medical ward (n = ), psychiatry (n = ) or icu (n = ); no fatal case was recorded. conelusion: smp to ed are often benign. the benzodiaz pines are the most often incriminated but the new anxiolytics and hypnotics (imidazopyridines and cyclopyrrolones) take a growing place. the latsion burn center of athens. its planning constructive and functional refinements j. ioannovich, a. petalas-vourekus, d~ serbetis, h. carsin a bed burns unit is under construction following a donation to the general hospital of athens. the plan of the unit, covering a surface of approximately . m is based on the principle of three identical bed satelites which may function totally independent from each other. in the center of the unit the common facilities are installed, like operation theatres, storage rooms etc. this new modification in the plan of a burn unit is presented in this paper. the advantages from the fucntional, administrative and medical point of view are discussed. tiffs anisotropic conduodon could favour the ocenrence of a circular movement of the impulse that leads to tachyeardias by reentry. purposes of this work were to study, with the help of epicardial mapping, the influence of a trieyclie antidepressant, clomipramine (c), on the conduction velocity longitudinal (vl) and transverse (vt) to myocardial fiber orientation and on anisotropy (a = ratio vl/vt), and their modificutions by the sodium bicarbonate ( ). method: a plaque of electrodes, positioned on the left anterior ventricular wall of anesthetized dogs, allowed to deliver, thanks to central electrodes, programmed electrical stimulations inducing vcuttienlar complexes, and to collect them. each entailed unipolar dectrogram was processed by a computer system that drew the isochrones and a map of activation allowing the calculation of v. the c was infused ( . mg/kg/min iv) during rain; at t , dogs received the b until the retuni of qrs to its initial value fro). a lengthening of qrs of at least % of its value at to was demanded before the administration of b. results: dog was excluded because of an.~nsufficient prolongation of qrs before the administration of b. all values (map : mean arterial pressure, i-ir : heart rate, qrs andqt intervals, v) differed significatively ( < . ) compared to values control fro)except qrs at t . the b ( + ml/kg; ranges . and . ml/kg) modified no studied dements outside of the ( }rs. to ti t t t t t a , + , , + , , + , , + , , + , , + , , +- ,~ conclusion : the c slowed v l and v t without modify the anisotropy. the b did not modify the v of~conduction while the qrs prolongation was corrected. the c acts as a class i antiarrythmie drug on the inward sodium current during the phase of action potential; the gap junctions have shown to be important in the conduction and an action on the gap junctions such as a modulation of the junctional resistivity, can not be rule out. is the doctor a heroe ? p. t.schies~.he, t. bauer, m. seyr dept. of anaesthesiology and intensive care, aokh krems, austria objectives: helicopter emergency services (hes) are getting popular more and more. the results concerning outcome are encouraging. however, some recent accidents with dead or badly wounded hescrew-members have shown the relatively high risk for the crews. therefore we were interested to eval ate the motivation of physicians to participate in a hes. this survey was designed to investigate current concerns about safety and motivation of doctors on emergency call. methods: a questionnaire was sent to doctors of the austrian emergency system. the survey consisted of multiple choice questions and subjective scoring tables from (--full agreement) to (=disagreement). overall, "/. of the active emergency physicians participated in the survey. results: . % of the doctors assume the system is basically safe, experienced doctors tended to have less trust in safety. only % would not hesitate to go into action by dark. . % stdctly refuse night flights to accidents outdoors. although defibrillations are assumed to be safe dudng flight, only % would do it. . % of the doctors would rather stop flying. the most common reasons for ,uitting were wish of family and fear of an accident. . % conclusioq: short transportation times help to avoid trauma related stress, pain and shock-induced organ complications. therefore the physiologic and economic advantages of hes are undebatable. however, the survey data indicate a considerable concern about safety of the medical personal in a hes. crash landings within less than years with deadcases and badly wounded crew members in a small country like austda make desire for safe flying conditions understandable. obiectives: to evaluate the clinical usefulness of trachlight. methods: trachlight is a new device facilitating endotracheal intubation. a stylet with a lightprobe is inserted into the endotracheal tube. intubation is guided by the light glowing through the neck tissues, thus rendering direct laryngoscopy unnecessary. intubation using trachlight was studied in patients (age - years). the indication for intubation was elective surgery in patients (asa i-ii) and emergency intubation in patients. in the elective patients, anaesthesia was induced with thiopentone supplemented with fentanyl, and intubation was facilitated with vecuronium. the cause for intubation in the emergency patients was dyspnea in , cardiac arrest in , trauma in, and unconsciousness due to drug overdose or seizures in patients. intubation was facilitated with medication in patients. results: of the elective patients, ( %) were successfully intubated. six patients ( %) needed two attempts before successful intubation. the duration of intubation exceeded seconds in patients ( %). of the emergency patients, ( %) were successfully intubated. six patients ( %) needed two attempts, and the duration of intubation was more than seconds in patients ( %). in % of all patients, intubation was assessed as easy. no or insufficient glow, prolonging intubation or necessitating two attempts, was noted in patients ( %). oesophageal intubation occurred in patients. conclusions: trachlight may be a valuable adjunct for intubation in varoius settings provided that adequate training is provided. a learning curve was found to exist. objectives: to compare enoxaparin and standard heparin in cavhd and calculate the value of laboratory controls in the treaanent. patients and methods: twenty patients needing dialysis for acute renal failure participated in the study. the main exclusion criteria were massive bleeding or a thrombocyte level < x e /i. in each treatment the same type (av- , fresenius ag, germany) of a polysulfone capillary haemofilter was used. the study scheme consisted of two consecutive four-day cavhd treatments, one course for each type of heparin. the order of heparin administration was counterbalanced between patients. the standard heparin was given as a continuous infusion aiming at an activated coagulation time between and s. the initial enoxaparin dose was rag every :th hour intravenously, but was modified by any signs of coagulation in the dialysis blood lines or bleeding complications. results: the dialysis treatment was adequate in both treatment modes, with mean blood urea levels . and . mmol/l respectively (ns). the bleeding complications were moderate and similar in both treatment modes. the mean life-span of haemofilter using enoxaparin as an anticoagulant was some longer than using heparin ( . + . h versus . + h, ns). the mean aptt-levcl during heparin treatment was s and during enoxaparin treatment s (ref. - s). the mean daily dose of heparin was nag, that of enoxaparin lg mg. the mean anti-xa activities were . u/mi and . u/mi, respectively, reflecting a better bioavallability of enoxaparin. conclusions: both anticoagniation modes were equally effective and well tolerated. the amount of enoxaparin needed for a proper anticoagulation was, however, less than half of that of standard heparin. the changes in aptt level were too slight to make its use possible in controliing the dose of enoxaparin. the use of enoxaparin seems to be rather safe in cavhd even without laboratory controls. the adv~ucea in the management of computerized data of an intensive care unit have been petalled to the clinical advauces and the increasing sophistication of methods of diagnosis fop the clinical application an therapy. this has led our unit to design and develop a computational system called timbu which is used to help physicians assist patients. among its various uses, this system has a software for the hemodynsmic control of a critic patient. this program was carried out to get as fast as possible the hemodynamic data of the patients in an intensive care unit. as an example, we can mention that when we load data obtained through direct measurement from the monitors and the lab, the program calculates parameters that guide, intelligently, to the diagnosis and therapeutic behaviour of the hemodynamic problem through screen messages. the validation of this program in the unit of intensive care has demonstrated that its use allows a more efficient handling of the patient with serious hemodynamics and respiratory disorders. ohieetlve: traema is a heterogeneotm 'disease' that ecatr~ a~"o~s all age ~oupe with v~ying degrees of severity. this imerogeneity has made the di~e, trmma, diflkaflt to r the ehn of this stady wa~ to assr the fitaen of saps in ibis popeleties. methode: in order to compute the ~ probability, a model derived from logistic regression w~ developed. meam'e of calibration (goodaess-of-fit stetislj.r and di~'riminafion (roc ou~e) were adopted in developmm~ and validetlon set randomly taken from a database of pts eeeseemivety admitted in icu (arohidia). ~ witho= salm, p~ yom~ am is yam, with los ~horter thma hotam wore exr fa'om thi~ mmly~ir thi~ model v~s then evahmed on the ~per ~mbgro~ (i.e., trmma pts). if'it did t~t fit the data well ~, new model wm developed rer the logit only on trm=~apm. reims: data were availabte for pts during aperiod of three .y~m , treama pts were . %), teats of calibration iadioaled probability model did mot provide m adequate refle~on of the mortality ezperieace in pm with ireutae, being the observed mortality lower flma the expected (figm'o). a aew model was then variable. this oastomized model fit~ the de~t of trmara pts very well (g =- a p> . ; roc = , ). the di:lferencea between the two modele were evident. conclusion: this ltudy shows that mortality in iramna pts is over wcfe~d when ~se~ed by menm of saps. however the r mode! meets high standmcd in terms of calibration mid dil~'iminat'~o~ ']"he advaatage of ~imd models meaas the colleotion of the ~ set of variables for all pm admitted in icu e~einat the ase of diasma specific ~oring syatex~. ("sl"): effects on cardiovascular and hemostasis systems (cvs, hss) a.oborin~ph, ~.~yndiuk~ph, b.kondratsky ~pt. of'""su~gery and transfusiology, research institute of hematology, lvov, ukraine objectives: great interest has been shown recently in the use of hoss for the initial resuscitation of hypovolemic shock. methods: the study was carried out in dogs -~h hs was induced by jet momentary hemorrhage (h) from a. femoralls (the bloodloss volume made . + . ml/kg). the treatment was begun after .u+o. hrs of h. "sl", created on the basis of-sorblt and natrium lactate ( mosm/l) was injected into v. femofalls at the dose of io. ml/kg. results: it is established that before treatmen-~rterial blood and central venous pressures (abp, cvp) diminished to . mm hg and - . + . cm h (p .o ), while heart rate (hr)-increased to . + . per min (p<.o ). by this the indices of ~latelet counts (pic) and plasma fibrinogen (pf) lowered by . % (p<.i) and . % (p~. ), while fibrin degradation products (fdp) enlarged by . % (p~ . ). after - min of treatment termination abp and cvp increased to . + . mmhg and . +o. cm h (p<.o ), and ~[r diminished to t . + . per min (p>. ). at the same time the indtces of pic and pf enlarged by . % and . % (p>.i), while fdp diminished by . % (p>.i). one of dogs survived. life duration of the other dogs was . + . hrs. conclusions: the obtained data are ~he evidence of normalizing influence of "sl" on cvs and hss, and allow to recommend it as a mean of initial resuscitation of hs in clinic. oblectives: we prospectively studied icu patients with severe head injury (hi), which cerebral lesions monitorized with sjo through opljcal fiber and the cerebral flux with tcd. methods: since january until june , we collected ht admitted to the icu, and of them monitorized with optical fiber in the right jugular bulb and tcd. all patients needed mechanical ventilation related to gcs <__ , with ct in admission (classifing lesions according to marshall and al.) . we related the final results to the evolution of sjo and tcd, with other monitorizing methods like gcs, ct and icp. ~sults: conclusions: in patients with gcs _< , sjo is useful to evaluate the evolution towards vegetative state, still more in cases with ct type ii in admission and higher apache ill. elevation of icp implies an evolutive nsk to brain death and data of tcd is a good indicator of brain death, the complete monitorization of these patients can improve the therapeutic control of this neurologic problem, , ( m, f) , (m. age: + years), divided in two groups (a and b) under specific criteria(tremor and/or fever during admission in i.c.u., or not). the injury severity score was > in all studied patients. tbe group a ( m, ") had no tremor and/or fever on admisskm, while em group b (tin, the above criteria were ix)sitive. bhx~d samplings were taken - hours after accident and - rain. after admisskm in i.c.u. micro-eli~ method was used for measuring cytokinc-levcls. statistic analysis was performed by studcnt-t test. as control group, healthy people were examined. _resu!_ts-il-lct, il-ii~, il- and tnf-tt levels were similar to control group levels in both groups a and b. i!,- and g-csf levels were found increased in both groups (p< jxjl), while il- levels were statistically significant comparing to group a. in con_tin_skin, during immediate post raumatic period,proinflamatory cylokines il-i~, il-i~ and tnf.-ct, produced in an earlier stage than ,. , cannot be detected,whereas .- was increased significantly, especially in group b. g-csf was fimnd in increawal levels in both gr(mps, without statistically significant difference between gnmps a and i|. objectives-l~valantc proteolitic activity, disorders in" eariy, period after combined trauma and p(~.ssibilit, i' of their correction by injection of proteo[ysis inhibitors contrycal and s-fto~:nracil in combination with driving an isotonic snlu~ion of sodlum chloride and polig[ucine. methods: biochemicai studies of proteolitic activity in dogs with limited deep burn and acute bloodloss, . result:s: in case of deep % burn, cornplicated by bloodshed the of blood grows at - times. it; is the restdt of the pancreas glandischemi demage, caused by the centralised circulation of blood and intensifies the deviations of haemodiaamics and albumin exchange. the degree of endogene intoxication by mean mofecular peptides which are the products of albumin decay reses to %, and % in hours. in hours after the trauma the-process is accompanied b ! , % lower inhibitory activity of blood, where as at the peak of the trauma it was , ~ higher. that proves the nnfavuurahle process of the shock in case a combined trauma. conclusion: the vein injection of 'proteolysis inhihitotz cnntrycal and -fforuraei[ in cumbination with driving an isotonic solution of sodium chloride and p.dligh]cine to refill lhe loss of blood helps to lower at times the profeolitic activity of blood. but it still remains above the initial level. the degree of endogene intoxication lowers at times; [ emodinamics aml albumin exchange stahilised. objectives: nimodipine, a known calcium antagonist, has been shown to dispose a beneficial effect on patients with subarachnoid hemorrhage, but its efficacy on traumatic or spontaneous intracerebral hematoma has not been justified. therefore, we studied the effect of nimodipine on the histopathological changes following an experimental intracerebral haematoma in rabbits. methods: twenty-three new zealand albin rabbits of both sexes, weighing - , kgr and at age of - months were anesthetized and a small burr hold in the left parietal aerea was carried out under aseptic conditions. the dura was opened and . ml (this volume assuring a normal incranial pressure after kaufman ) of autologous blood was injected into a depth of mm via a needle of . mm bore. the wound was closed and the animals were left to recover. nimodipine, of , mg/kgr of by weight per day was given via a nasogastric tube to fifteen animals for a period of time of fifteen days (group b). six rabbits were given water and served as control (group a). both groups of animals weie sacrified on the fifteenth day, their brains were removed and immersed into % formalin solution. tissue sections of ~ were embedded into paraphin and stained with haematoxyline and eosin, mason and gfap stain for gliac cells. results: two animals died after the surgical procedure, because they developed large intracerebral bematoma. no animal developed neurological deficit except one of group a which manifested a right side hemiparesis. the results of the bistopathological changes are the following: i) the mean -+ sd diameter of the lesions in the group a was --. ~t while that of group b was + ~t (p< , ) ii) secondary ischaemic neural tissue changes, characterized by the extravasatlon of red cells, the presence of haemosiderin-containing macrophages and signs of low grade inflammation zpredominated in the specimens of group a and were totaly absent from those of group b. iii) a ring of gliac hyperplasia and a low grade local fibrosis was found, encircling the lesions in the specimens of group a in contrast to those of group b. conclusions: nimodipine when administered in rabbits following the development of a non increasing the icp experimental intracerebral haematoma, prevents the extention and the severity of the lesion. objectives: to study the efficacy and side effects of adding intramuscular clonidine (clophelinum) to analgesic regimen in early management of patients with serious burn injury. methods: pts with - % bsa second to third degree flame burns (respiratory tact injury excluded) to yrs of age were randomised to study (n= ) and control (n= ) groups. burn shock was treated with hypertonic saline -bicarbonate solutions ( mmol/l na +) ml/kg/%bsa for the first hours and ml/kg/%bsa for second day. analgesia in control group for the first hours was provided by regular hourly intramuscular administration of mg of morphine sulphate and mg of analgesic -antipyretic analgin with mg of diphenhydramine (dimedrol). from the rd day regular administration of morphine was finished. in the study group ixg of clonidine was added -hourly for hours and dose of morphine halved. vas, verbal rating scale for sedation (vrs, - ), sleeping time, spo , hr, bp, diuresis, vomiting and other complications were comparatively evaluated during patients' stay in icu. results: addition of ~g of intramuscular clonidine daily allowed to achieve better analgesia and sedation with halved consumption of morphine. mean vrs in study group for the first days was . - . vs . - . in control group with twice longer sleeping time. there was significantly less tachycardia in study group; dynamics of bp for the first hours did not differ considerably; later, there, was tendency for hypotension in study group without adverse effects on diuresis or other indices of tissue perfusion. because of high incidence of chronic ethanol abuse among study population pts of control group suffered from psychomotor agitation or delirium, probably as a sign of alcohol withdrawal syndrome (aws). this made regular evaluation of vas impossible. in the study group only pt showed sign of aws. mean vas score was in . - . range for first postburn days. pts appeared excessively drowsy due to clonidine, but it had no adverse effect on their overall clinical course. mean spo values in study group were in - % range, among controls - %; vomiting was absent in. cionidine group vs cases among controls conclusions: clonidine could be a valuable addition to analgesic -sedative regimen in burns, especially for prevention of aws and deserves further study in this regard. hemodialysis -hemoflltration modifications and/or intratracheal gas insuflation have been recently used for blood gas exchange in several models of respiratory failure. objectives: evaluate the combination of cavh-m and igi for respiratory support in experimental acute lung injury. methods: five mongrel dogs ( -+ kgr) were mechanically ventilated inroom air, paralysed, heparinized, connected with a cavh-m system (diafilter- polysulphone membrane) and remained stable for one hour (pao~= . • peco = -+ mmhg, ph= . -+ . , bp= -+ mmhg and pap= -+ mmhg). all was induced two hours after oleic acid infusion ( . ml/kgr) into the pulmonary artery (poo~= . _+ -p< . , paco~- . _+ -p< . , ph= . -+ . -p< . , bp= -+ -p=ns, and pap= _+ -p< . ). fio % for the next minutes did not significantly altered the b ood gas abnormalities. afterwards, pure oxygen applied simultaneously a) through the inlet of the filtrate's compartment of the hemofilter ( l/min) while filtrate and gas were removed from the outlet port (bypass flow ml/min) b) through a thin intratracheal catheter positioned cm above the carina ( l/min). the fio given through the ventilator readjusted to %. results replacement fluids/filtrate during the next four hours were not exceed . l/hour, whilst the blood gases and pressures were improved as follow: cavh-inlet:pao.= . objective. to compare the changes in humoral immunity in trauma patients following massive transfusion of autologous and homologous blood. methods. we studied randomised clinical groups of patients each containing patients with trauma and operation of large arterial vessels. the amount of autologous or homologous blood transfused to the patients was exceeding ml, while the patients in the control group did not recieve blood or blood products. results. we recorded most pronounced and characteristic changes on the -st and on the -th day in the group of patients recieving homologous blood transfusion, i.e. decreased amount of igg,iga,igm,c and c fractions of the complement system, haptoglobin and significant and sustained rise of circulating immune complexes up to the end of the study period. in the control group of patients the decrease was weaker and lasted only during the -st post-operative day; the dynamics of the circulating immune complexes level were almost the same as in the first group of patients. in the group of patients recieving autologous blood transfusion, the parameter values did not change significantly from preexisting levels after the -st day, while on the -th and on the -th day showed a tendency towards aslight rise. conclusions. autologous blood has a favourable effect upon humoral immunity and should be the transfusion medium of choice in cases where autologous blood reinfusion is technically possible. ivan petkov, m.d., rumen farashev, m.d. and dimitar terziiski, m. d. medicine, military medical academy, g. sofiiski str., sofia, bulgaria objective. the amount of blood lost during trauma and operation could hardly be forseen and donor blood supplies are not always available in sufficient amounts. rare blood group types and/or unexpected haemorrhage pose a great challenge to the transfusion therapy and the methods of intraoperative autologous blood transfusion. methods. we report a case of a -year old male patient with extremely massive intraabdominal haemorrhage ( m( blood loss ) during an abdominal aorta reconstruction following a traumatic injury of the abdominal aorta. we achieved a successful reinfusion of ml of autologous blood using an original autotransfusion system developed by us ( pat. no / . . ) . results and conclusions. the autotogous blood in the case reported here was the only and the most suitable transfusion medium for the rapid intraoperative compensation of the acute haemorrhage and the favourable outcome of the patient. the post-operative period was smooth and no significant disorders in the clinical course as well as in the laboratory tests ( morphological,biochemical,coagulation and immunological) were recorded. there were no complications during the postoperative period despite the fact that the amount of blood reinfused to the patient was slightly exceeding his own volume of circulating blood. objective. the haemoglobin concentration and the perfusion pressure value could not be the only criteria for the early signs of tissue and organ dysfunction. because of this, we employed the extensive monitoring of oxygen transport during severe trauma in order to. achieve dynamic evaluation of physiologic compensatory mechanisms and to assess the efficacy of intensive care management. methods. we conducted a prospective controlled trial on the blood oxygenation, oxygen transport and tissue perfusion during the first days after the trauma in patients with polytrauma. we used a swan -ganz pulmonary artery catheter (beckton -dickinson, u.s.a.), deseret cardiac output computer (medical inc., u.s.a.) and hewlett -packard monitor (hewlett -packard, germany) to measure and calculate all the parameter values. the severity of the injury was assessed using the apache ii score system. all the patients had scores over . results. the results show a significant decrease in the arterial blood oxygen content and in the arterio-venous difference, as well as an increase in alveolo-arterial oxygen difference and in the transpulmonary right-to-left shunt. the tissue oxygen supply and the tissue oxygen consumption reveal a tendency towards a decrease below the physiologic minimum of adeqate values. the erythrocyte current velocity and the ratio between oxygen transport and erythrocyte current velocity also decrease inspite of the optimal blood rheology. conclusions. the dynamics in the parameters values are most pronounced between the -nd and the -th hr after trauma, which predisposes patients to the risk of developing stable hypoxemia and characterizes this period as the most critical for tissue metabolism and organ dysfunction. posttraumatic changes in immune mechanisms in lung compartment in trauma were analyzed in ao and da inbred strains of rats which differ in their immunological reactivity: the former being low responder and lat-~er hiperresponsive. methods: the levels of tnf-alpha activity in the supernatants of cultured lung lobes and dynamics of cells migration from tissue explants in h lung cultures were assessed in ao and da rats subject ted to severe burn trauma. results: increased levels of tnf activity ( + pg/ml compared to + . pg/ml in control) were found od day following trauma in lung sups of ao rats while no changes in the levels of activity of this cytokine were found in lung-sups od da rats more pronounced extent and dynamics of cell emigration were noted in da rats, while almost unchanged in ao rats sharp rise in pmn percentages h following trauma ( - % compared to rare pmns in control), followed by increase in lymphocyte numbers at later time points among lung cell emigrants was detected in ao rats. slower but persistent increase ( %, h following trauma and % and % on days and after trauma infliction, respectively) in pmn numbers among da lung cell emigrants was detected, which appeared to be activated, as judged by their nbt reduction capacity. increased percentages of peripheral blood pmns and increased state of leukocyte aggregation/adhesion were detected in both strains, but different levels of plasma tnf: increased levels in ao rats on days and following trauma, and initially but persistently high levels of plasma tnf alpha in da rats ( - fold higher compared to initial levels in ao rats). conclusions:different patterns of local (lung) and systemic changes in cell numbers and cytokine levels implicate differential posttraumatic migratory capacity of pmns vs. lymphocytes in lungs in ao and da rats. early diagnosis of acute intestinal ischemia by color doppler sonography e. danse, b.van beers, p.goffette, f.hammer,aav.dardenne, f.thys, p-f.laterre, m,s. reynaert, .lpringot dept of radiology (profb.maldague) and dept of intensive care ( prof m,s.reynaert), st.luc univ.hospital, brussels, belgium ob emergeny medical squad service is the most important segment in the process of saving the people, in the cases of mass accidents, like industrial accidents caused by the: explosion, fire, chemical poisoning, traffic accident, elemental catastrophes and the war. because of that, each emergency medical squad service needs to have in its motor-pool vehicle for the mass accidents/ for provoding at least people, wounded as well as the people became ill/. objectives: presentation of such special vehicle, produced by "zastava-kamioni" and it's medical-technical equipment. methods: descriptive and comparative analysis of the medical and technical characteristics, based on the actual norms/din, , iso , yus.../ results: on the base of doctrinaired requirements of the emergency medical squad in the case of mass accidents, our researches resulted in the following medical and technical characteristics -the vehicles for mass accidents are gvw/with a payload off cca - t, with the fixed, closed body, type: universal van, -technical equipment aggregates, stretches, anti-fire device, equipment for pitching the tent and for maintaing technical conditions of the work -medical equipment: linen bags with complete sets of bandage material, means for the reanimation and immobilization, for the infusion, medical instruments and remedies as well as the tent for lodging at least wounded and sik people. in federal republic yugoslavia, it was proposed such vehicles for the emergency medical squad needs. conclusion: we suggest to introduce this vehicle in the production range of the ambulance vehicles for saving, especially in the circles where can occur serious accidents. introduction : carbon monoxide (co) poisoning commonly generates central nervous system abnormalities though an important cardiac morbidity and mortality must be considered. long-term exposure to co with cohb levels < % may be more dangerous than short-term levels of - %. we report a case of an adolescent who after prolonged exposure to co developed a severe reversible cardiac dysfunction with low levels of bloed cohe c a.ase history : a year old boy was found comatose at home. his mother in the neighbouring bathroom died severn hours earlier of what was later proven to be a co intoxication. on arrival the gcs was / and the patient was breathing spontaneously. a postictal status with eventual postanoxic encephalopathy was suspected. a coh'b level of % was objectivated. the cardiorespiratory situation quickly deteriorated requiring mechanical ventilation. chest x-ray showed diffuse bilateral patchy infiltrates. ecg revealed signs of ischemia. severe left ventricular dysfunction was evidenced by pulmonary artery catheterisation and echecardiography and later by isotopic angiography (lvef %). treatment was intensified with inotropic support, intta-aortic balloon counterpulsation and oxygen therapy. the clinical course was further complicated by a crush syndrome and renal failure. the patient's condition gradually improved and he fully recovered without any residual lesions (lwf %) conclusion : even after prolonged exposure cohb levels can be misleadingly low. high tissue levels of accumulated co can be associated with coma and fulminant cardiorespiratory failure requiring advanced life support facilities. introduction : both neuroleptics (nlp) and tricyclic antidepressive agents (tca) can induce arrhythmias, prolongation of the qt segment and the pr interval and hypotension. we report a case illustrating that combined overdose of these agents increases the toxicity of each compound and the risk for adverse cardiac events. .c, gse history : a year old male ingested mg doxepin (sinequanr), a tca and mg prothipendyl (dominalr), a potent nlp in an attempted suicide. upon arrival in the emergency department the patient was unconscious (gcs / ), breathing superficially, and presenting signs of recent vomiting. physical examination revealed a taehycardia of b.p.m., an arterial blood pressure of / mmh g. ecg showed a brood qrs complex tachycardia. a chest x-ray revealed the presence of an aspiration pneumonia. laboratory investigation demonstrated increased levels of crcatine phosphokinase, lactate dehydrogenase and aspartate transaminase ; hyperglycemia and leucocytosis were present. the plasma concentrations of doxepin and prothipendyl were respectively gg/l (toxic level #g/l) and i.tg/l (no reference). treatment consisted of mechanical ventilation, gaslric lavage and administration of activated charcoal and iv fluids and antibiotics. a hemodynamically well tolerated veatricular tachycardia developed / h later. nahco ( meq/ h) was administrated inducing an ectopic atrial tachycardia with a normal qrs complex and prolonged qt. h after admission a normal sinus rhythm was present; the prolongation of the qt segment persisted for days. the patient fully recovered. conclusion : the treatment with nahco~, alkalizing the blood and thus increasing the protein binding of the tricyclic antidepressant molecule, can readily correct the potentially life-threatening cardiac arrhythmias and therefore should be part of the routine treatment of combined tca-nlp overdose. ob/ectives: the development of diabetes insipidus (di) in patients with brain injury is a known negative prognostic sign. the aim of this study was to investigate whether this is also a reliable early prognostic sign of brain death. methods: this is a retrospective study of patients treated" during a two year period ( - - to - - ) in our i.c.u who meeted the following criteria: ( ) coma score _< gcs within the first hours, ( ) positive brain ct scan on admission classified according to marshall's diagnostic classification (classes - ), ( ) normal renal function during the entire icu stay. for the definition of di were used the usual di criteria plus hypematriaemia (serum na" >_ meq/l). survival was defined up to the th postadmission day. conclusions: according to the findings of this study, the development of diabetes insipidus in brain injured patients seems to be a highly specific index for brain death (positive predictive value = . ). however, further prospective studies are needed for the definitive evaluation of these findings in such patients. emergency care in italy, despite all efforts, is still lacking a nationwide organized prehospital care system and, until today, there are only different regional solutions. the majority of these realities imply rather simple ambulance first-aid services without attending emergency physicians and without resuscitation equipment. the emergency medical service (ems) system in falconara m., italy, was implemented in august by a collaboration between the school of anesthesiology and intensive care of the university of ancona and the, already existing, volunteer rescuer organisation "yellow cross". according to the guidelines pubblished in [ ] the pre-existing equipment of the volunteers was completed with type a ambulances and special equiped motorcar (patient monitor, defibrillator) for ambulance indipendent physician transpur[. a special data collecting schedule was created to memorise every emergency intervention in a computerised data-base. the intraining members of the school of anesthesiology and intensive care provide hour ready intervention. in this report the authors describe their experience concerning primary firstaid medical interventions. for a preliminary evaluation we considered, retrospectively, consecutive emergency interventions in the time period from novembre , to april , . the emergency physicians treated male ( %) and female ( %) patients, patients died before hospital admission and patients ( %) were treated at home by the ambulance indipendent physician and did not need any further medical treatment. in the same time period year earlier (november to april ) without attending physician the volunteer rescuers transferred all first-aid interventions to near-by hospitals. we conclude that the presence of an attending, iudipendently motorised physician in emergency interventions is essential for the establishment of precise priorities and may be helpful to reduce hospital admissions by ambulance intervention, though reducing primary" health care costs. we have developed the method of liquor filtration which allows to purify the cerebrospinal liquor from blood and its decay products in the subarachnoid bloodstroke. the hemipermeable dialysis membrane was used as a filter, which lets only in water, electrolytes and substances with small molecular weight. the liquor filtration was used for the treatment of patients with the subarachnoid bloodstrokes of different etiology. the perfusion of liquor was performed at the rate ml/min in the recirculatory mode. its duration was - min depending on the bloodstroke intensity. the filtration makes possible the most completely purifying of the hemorragic liquor, the reducing of the content of blood ceils and its decay products - times as less. the monitoring of the patient's state during the perfusion didn't revealed the departure from the norm of the main vital part. the liquor filtration technique compares favo-~ rsbly with the routine method of cleaning by the absence of toxical effect of heterogenous solutions on the central nervous system. the filtrstion of the cerebrospinal liquor in the subarachnoid bloodstroke sllows to provide the the early cleaning of liqour, the regression of meningeal syndrome and to improve the patient's state of health. e tabli~mczr bd ~ of rei~idnal medical first-aid zhoulittoing, ed., tan zi, m.d. dept. of sargery, the first teaching t[ospitat, yejin-l)a-l)ao, wuhan fltlna objectives: the medical first-aid is the most important task of the public hc atth department. in general, single hospital model couldn't fatty, effective ly rescue mony severe patients who need mergant treatment in the scene. bub establishing the medical first-aid network, the severe patients can be given the most timely und the most scientific emergent treatment. so that, the suc cessfut rate of the saving wilt be greatly increased. methods..; our hospital is a general big hospital. through developing and cons tructlng for more than ten years, the medical first-aid network distributed art over the area under our jurisdiction has been set up. it consists of thr ee units: the medical first-aid unib center comartd and mnagment unit, co m~nlcation and tiaison unit. the principle of the network operation is with oat having to far to mergoncy, specialized emergency and the best merge acy. results: the results of the network operation were notable. cmpari~ the to tat successful rate of the saving ( . ~), the successful rate of saving tra ma ( .~), the suscessfut rate of saving shock ( .~) and the successful rate of cardioputmonary resuscitation ( . ~) daring the three years after t he network operated with these before ( . ~), ( ]. ~), ( . ~) and ( ft. ~), the successful rates after operating were remrk~iy higher ( p<i).o ). conctusions~ the above results showed estabiishmnt and mena emont of region at medical first-aid network had very si~nificont action during mergentty s avin the severe patients. moreover, the regi~at medical first-aid network atso take an important part in prevention and health protection of the mass. objectives: se related mortality is due to the occurrence of both rv and lv dysfunction evoking cardiogenic shock and pulmonary edema (f. abroug, intensive care med ; s. nouira. chest ) . prospective evaluation of scorpion antivenon (sav), the only available specific treatment, is lacking. the aim of this study is to assess the effects of sav on the basis of a case-control study. methods:among consecutive victims of se presenting to the emergency department, were managed using sav (group sav+). these patients were matched to envenomated patients who did not receive sav (group sav- chest injuries are the cause of death in % of trauma fatalities and a major contributing factor in an additional %. pneumothorax is a major complication of chest injuries and can be initially overlooked bringing to additional morbidity and mortality especially in patients who require mechanical ventilation. the real incidence of pneumothorax in severe blunt chest trauma hasn't yet been established, as many pneumothorax can be missed on the initial chest x-ray (cxr) and computed tomography (ct) has not been routinely used. to evaluate the incidence of pneumothorax in patients with severe blunt chest trauma. methods: a prospective study. from january to december , all trauma patients with one of the followings conditions: fractures of four or more ribs, signs of lung contusion on the initial cxr or presenting a severe hypoxemia on admission (pao /fio < ) in the absence of pre-existing pathologies, were evaluated by initial cxr and subsequently submitted routinely to a ct. results: severe trauma patients were considered. of them, presenting a severe hypoxemia on admission, had no evidence of major chest trauma (ais< ). hypoxemia was the consequence of severe trauma in other districts; these patients were therefore excluded. patients with severe thoracic trauma (ais>= ) were admitted into the study. the mean iss was . ( - ). thirty-six patients required artificial ventilation for at least hours during the icu slay. three of them, who had a tension pneumothorax, were submitted to an emergency thoracic decompression on the field by the emergency helicopter team. in cases pneumothorax was diagnosed an the initial cxr more patients had a pnx which was identified only on the ct. in cases a large pnx with lung collapse was missed on the cxr. in our group of severe blunt trauma patients, % ( / ) presented a pnx that required the insertion of a thoracic drainage. only one third ( / ) of the pneumothorax could be recognised on the initial cxr, while other were decompressed before performing the cxr. as many as % of the cases of clinically significant pnx were missed on the cxr, and a ct performed soon after admission allowed an early diagnosis bringing to changes in the treatment. (as the patients were mechanically ventilated a chest tube was inserted in all these cases). in cases, the initial cxr overlooked a huge tended pnx which was the cause of hemodynamie instability. conclusion: in patients with severe blunt chest trauma even large pnx can be missed on the initial cxr. moreover due to the non compliant compressible lung, a % pneumothorax which can be recegnised only on a ct, can bring to high intrapleural pressure altering eardiopulmonary function. n. andoeli , .~osid, m.zesevid, m.risovid, d.stepi , d.djokid b~rga~yc~qterclinicalcaqterafserbia, belgrade cb~ctives:~lis study ~ the use of ~rq]ofol earbired with k~t~ine (aq a~sjgh~ic s@~qt widn inirjrsic armlgesic pro~mities) or with fsqtmtyl,with psrtial azgmsis an hgenxlyn-a~ic ~ durirg ~ ~ re:~ver~ f~m ~ in hxh ~ of ~ti~. ~: yali~mial and ~bod: a~it p~tie~ts a~ i-ii were included in ibis shxly. patients were rsrd]nly dieided in two ~ns. all d~tie~ts ~me given - prcpofol bolus doses (o, ~gkg) for ird~iqn of ~. ~ia ~s m~sjn~ with an infusion ~ ~ropafol. as sdflitianal were given fan-i~l (o, n]g) ~tely before ~ anj trad~e~ irfojoation followad by feasted bolus of o,i mg in ~ro o l.patients in gr~ o received i~ (an initial bolus dose of rg slowly intcavax~ rd mg as infusion over ~ rain) .infusions of pro~fol or imcpofol with kg~mine ~ stopfsj - rain ]:~o~ extuhation.arterial blood ~ (sistolic arterial blood preassu-re~zap,mean ~rterial blood pr~,d~lic arterial preassure-[zp a~ h~art rate-~) ~ m~ before induction of a~ io, snd rain aftem ~ intutation. results: arterial blood preasstre ~s decreases duri~ irn~ction of sn~wd~sia in hy~ ~n~s,tnt mare in th~ ~ who r~eived fsqtanyl.~ere w~s statisticslly sifnific~ntly difemerme dmir~ m~ of an~ia. arterial blood r~easatre and heart rate were stable in the t-..e~min -~a ~. all th~,fl-e keta'nire grcqo hsd e~rly :~e~y time. ctrmlusi~s: ~e ombiretion of protxfol wilh keta/ne for irduorion a~d ~ of sn~sd~esis w~s yell accept~ by p~tierfcs anj coald he ~ as an alterrstive ~o ccnva~icrsl a~es -d~sia. objectives : assess the relation between cytokine or endotoxin release and indices of splanchnic malperfasion after hemorragic shock in multiple trauma patients. ]~r study was approved by the local ethical committee. trauma patients admitted to the emergency room who met the entrance criteria of more than hour map < mmhg or use of vasoactive agents or blood lactates > mmol/ were selected for study. a nasogastric tonometer (tonometrics, inc, plastimed, france) and a swan ganz catheter were placed on admission. phi, lactates, hemodynamics, plasma cytokine and endotoxin concentrations were measured on admission and at . , , , hrs. an immunoradiometric assay was used to determine plasma concentrations of il (n< . ng/ml) and tnfc~ (n< pg/ml). plasma endotoxin concentrations were measured using a chromogenic limulus assay (n< . eu/ml)( endotoxine unit= pg). results : severe multiple trauma patients (age = _+ yrs, iss = -!-_ , saps = +'~, mean-+sd) were studied. they received + packed red cells during the first h. mean duration of collapsus before inclusion was . _+ . hrs. death occm'red in ~tients. ~ pglml, *: ng/ml, etox : endotoxin(eu/ml), lact: lactate (retool/l) a significant correlation between initial il level and saps was observed. in the early post-injury period phi, sao , svo , vo were significantly associated with ;il release (p< . at ho, h , h ). later a significant correlation existed between lactates and ii (h , h ). a peak of tnf was detected at and hrs. it was associated with low phi and low arterial ph of the early post-injury period (p< . iat ho, h , h ,h , h ) and with high lactate levels of later period (_>h ). only the late release of endotoxins (i{ ) was correlated significantly with initial !oxygea-delivered parameters. iconclusion : there was a marked increase in il in the early phase of trauma . i and tnf release after major trauma iwith hemorragic shock is associated with splanchnic malperfusion, as assess by the ivery low values of phi. lactates seem to be a later indice. toxic effects are a well-known complication of an overdosage of prescription theophylline. what is less known is that over-the-counter (otc) asthma medications contain theophylline, and that in some cases this might cause toxic effects. a case seen by us involved toxic effects from theophylline in an otc medication and to date is the only published case in the english literaturet the rationale for this study was to delineate the otc products containing theophylline from whatever data sources available. hyperthermia frequently occurs in intensive care treated patients and intentional application of whole body hyperthermia together with chemotherapy is a therapeutical access to treatment of malignant disorders. anaesthetic support is required in either condition. due to the marked decrease in systemic vascular resistance seen in hyperthermia an additional vasodilatory effect of the anaesthetic is unwanted. the vascular effects of anaesthetics in hypertherm organisms is not known in detail. therefore, we performed an experimental study to detect the effects of inhalational anaesthetics in whole body hyperthermia. in sprague-dawley-rats katheters were inserted into trachea, jugular vein, and carotid artery. for continuous monitoring of cardiac output a flow probe was placed around the aortic arch. the rats were mechanically ventilated with different concentrations of inhalational agents in oxygen. we compared the effects of enflurane, isoflurane, and halothane in stepwise increased body temperature by submerging in a temperature controlled water bath. results: isoflurane lowers arterial pressure more than halothane or enflurane. the inhalational anaesthetics lower the cardiac output similarily and independently of temperature. isoflurane decreases systemic vascular resistance independently of core temperature and the decreasing effect of halothane on the resistance is completely abolished in hyperthermia. conclusions: the influence of hyperthermia on the systemic vascular resistance is dangerous. this allows no additional effect of the anaesthetic management. in spite of the vasodilating effect of inhalational agents in normotherm subjects, this effect is abolished in hypertherms using halothane. the condition of management of analgosedation in hyperthermia is different from normothermia. objectives: to evaluate a bedside computer processed cerebral function monitor for assessment of brain wave activity when clinical/visual clues are not present. methods: ten icu patients undergoing neuromuscular blockade monitored with the aspect brain wave monitor from january to june , . results: time to onset and depth of sedation were readily apparent to icu physicians not specifically trained in eeg reading. objectives: to determine whether non-depolarising neuromuscular blockade reduces oxygen consumption (vo ) in sedated, apnoeic patients. methods: haemedynamic. metabolic and oxygen transport variables were determined in sedated, apnoeic patients with severe acute lung injury. all patients were ventilated using a puritan-bennett ae ventilator with integrated metabolic monitor. inclusion criteria were; ) stable cardiorespirator s" status; ) systemic and pulmonary artery catheters already in situ; ) inspired oxygen < %. patients were sedated with midazolam or propofol to abolish response to verbal stimuli, and sufficient morphine or alfentanil to abolish all spontaneous respiratory efforts. following baseline measurements, neuromuscular blockade was induced with intravenous vecuronium, ug/kg, followed by an infusion of ug/kg/h to maintain the train-of-four ratio at . a further four sets of measured and calculated variables were obtained at min intervals. results: statistical analysis was by repeated measures anova. there were no significant changes in any variable over time. the changes in calculated oxygen consumption (vo fick) , and measured oxygen consumption (vo gas), and in energy expenditure (ee), are shown in the table. objetive: to study the effects on coronary hemodyrtamics and myocardiai metabolism of administering propofol during postoperation sedation of patients with normal coronary circulation and good ventricular function undergoing cardiac surgery. patients and methods: patients ( women and men) undergoing aortic and/or mi~-a/ valvular cardiac surgery were selected, with an ejection fraction greater than . and normal coronary circulation. for postoperation sedation propofol was administered in . mg/kg i.v. bolus, followed by a . mg/kgth perfusion. all data were registered before administering propofol and after minutes, the patients being hemodynamically stable and a rectal temperature of _+ . -~ systemic and pulmonary hemodynamics, and global, as well as regional myocardial blood flow, and metabofic variables were measured. results: the patients studied were about years old, and the average period of aortic cross-clamp was . min. the adminstering of propofol caused a decrease in the coronary blood flow (- %), great curonary vein flow (- %), myocardial oxygen consumption (- %), regional myocardial oxygen constanption (- %), myocardial oxygen extraction (- %), regional myocardial ooxygen extraction (- %), while coronary vascular resistances and global coronary vascular resistances did not change. oxygen saturation increased in the coronary sinus (+ %) as well as in the great cardiac vein (+ %). in no patient were significant changes suggestive of myocardial ischemia objectified. there was also found a decrease in systolic (- %), diastolic (- %) and mean (- %) arterial pressure, systemic vascular resistance (- %), and cardiac output (- %). conclusions: in accordance with the clinical conditions of this study, the administering of propofol is not likely to cause changes in coronary autoregulation, oxygenation and myocardial metabolism. obietive: analyse the effects of . % "end tidal" isoflurane (sedative dosage) on the metabolism and coronary hemodynamics during the postoperation period of patients undergoing cardiac surgery. patients and methods: patients ( women and men) undergoing aortic and/or mitral valvular cardiac surgery, with an ejection fraction greater than . and normal coronary anatomy, were selected. after the surgical operation, . "end tidal" isoflurane was administered for postoperadon sedation. the determination of variables to be studied was carried out before and minutes after administering isoflurane, die patients being hemodynamically stable and a rectal temperature of _+ . -+c. systemic and pulmonary hemodynamics, and global, as well as regional myocardial blood flow, and metabolic variables were measured. results: the average age of the patients studied was -+ . years. during surgical operation the period of aortic cross-clamp was . _+ . rain. the administering of isoflurane was followed by a statistically significant drop in coronary perfusion pressure (- %), coronary vascular resistance (- %), regional coronary vascular resistance (- %), regional myocardial oxygen consumption (- %), regional myocardial oxygen extraction (- %) and accompanied by a significant rise in oxygen saturation in the coronary sinus (+ %) and in the great cardiac vein (+ %). myocardial oxygen consumption, myocardial exu'action of lactate and regional myocardial lactate extraction did not change. in no patient were enzyme or electrocardiograph changes objectified. systolic (- %), diastolic (- %), mean (- % ) arterial pressure, and systemic vascular resistances (- %) decreased, while cardiac output did not. discussion: the administering of . % "end ddal" isoflurane, in the clinical conditions of this study, produced a decrease in systemic arterial pressure due to a reduction of systemic vascular resistance without deteriorate cardiac output. at coronary circulation level, has and effect on coronary autoregulation but had no effect on oxygenation and myocardial metabolism. the idea of tiva implies the realisation of major anesthesia components (los of consciousness, neurovegetative inhibition, analgesia, myorelaxatiou, providing the adequate gas-exchange) through i.v. introduction of drugs exclasively. aim: providing for the main tiva components with minimal side effects of the drugs used, taking into consideration the patients characteristics and the surgery specific character. methods: anaesthesias have been conducted in patients aged years ( females, males), undergoing planned and urgent operations with the pathology of lower, extremities, perinaeum, small pelvis, hypogastrium and with reserved spontaneus respiration against a background of % insnffladon through mask. operations lasted from . - . h. anaesthesia adequacy was assested by constant monitoring: "cardiocap" (nibr hr, rr, sao , t), through glykhaemia level and mimicry reactions. standart premedicatioo of m-cholinolytics ( . mg/kg) and h -blockers ( . mg/kg) on the operational table was sumplemented by administration of . - . mg/kg of lidocaine, . . mkg/kg of clonidine, . - . mg/kg of pentamidine by the tachifilaxia method. the premedication adequacy was assessed through haemodynamics characteristics. sedation: . - . mg/kg of droperidoi, .l- . mglkg of diazepam and analgesia: - mkg/kg of phentanyl, . -- . mg/kg of ketamine were introduced fractionally according to indications. infusion rate of ringer-lactat solution was - ml/kg/h and depended on the intraoperational blood loss volume and on the patients preoperational condition. the duration of postoperative analgesia was registered. results: clinical assessment of analgesia according to this techniques allowed to decrease the anaigetics dosage to the subauaesthetic levels. smooth stabilisation of haemodynamics (bp) at proper age norms in patients with the initial hypertension by the -th min. of anaesthesia as well as the absence of its increase in response to the additional introduction of anaesthetic have been achieved. (hr) had no abrupt changes and remained in the range of - per rain. adequate external breathing: decrease (rr) by - per rain., with sao increase from % to - %. hypoventilation was avoided by respirate ventilator. according to unauthentic data the glykhaemia level had been lowered by -t % to the end of the operation with the initial moderate hyperglykhaemia of up to mmol/l the cutaneous covering grew warm and got pink colouring. no mimicry reactions. in the postoperative period patients were in the superficial sleep state ( - ) and analgesia lasted - b. there were no complications due to anaesthesia. conclusion: combined using of bz, opiates, neuroleptics potentiate the i.v. anaesthetics effects allowing lowering of each tiva component dosage and, as a consequence avoiding their negative influence on respiratory and heart vascular systems. complex application of adrenergetics (therapeutic doses of cionidine and pentamini with using of taehfilaxy effects) permitted to provide for analgetic and neurovegetative components of general anaesthesia under subanacsthetic doses of tiva main components, and manifestation of hyperdynamic reactions of haemodynamics decreased while using of lidocaine -the economicai activity of heart-vascular system. good level of muscle relaxation was achieved allowing for widening of surgical intervention extent without respirator ventilators and inhalation anaesthetics application. anaesthesia is easily controlled due to fractional introduction of drugs with quick recovery of cns functions after anaesthesia. postanaesthetic analgesia is increased while concurrent opiates doses are decreased. absence of marced haemodynamic, endocrine and metabolic reactions during the operation and after it resulted in shortening the period of patients staying in hospital. a yo white man was admitted to hospital for dyspnea and a productive cough. he had cabg in past, but no recent cardiac ischemia. physical exam: decreased breath sounds over right lung. chest xray: consolidation of right lung. admission medications included diltiazem, furosemide (both were continued) and trazodone (which was discontinued). admission ecg: sinus rhythm, qt . /qtc . sec, with st and t wave abnormalities similar to prior tracings. he required intubation and mechanical ventilation for progressive hypoventilation and hypoxemia. between icu days and he received haloperidol, - mg/d (cumulative dose rag) for agitation and delirium. icu day : qt . /qtc . sec. icu day : for better control of delirium, trazodone " mg q hs was added. icu day : he developed frequent nonsustained ventdcular ectopy. icu day : qt . /qtc . sec, pha . , paco mm hg, pao mm hg, k . meq/l, mg . meq/l. later in icu day the patient had brief episodes of torsades de pointes, each responding to precordial thump, and finally rhythm stabilized with i.v. lidocaine and magnesium. haloperidor and trazodone were discontinued. ecg was unchanged and myocardial infarction was ruled out. next day, icu day : qt . /qtc . sec. torsades de pointes, a form of ventricular tachycardia characterized by a twisting qrs axis, is commonly associated with qt prolongation. haloperidol is used frequently in icu for control of agitation and delirium, with reported doses up to mg/day. over past decade, cases of torsades de pointes with prolonged qt related to haloperidol have been reported. trazodone may also prolong qt and cause ventricular arrhythmias, especially in patients with pre-existing cardiac disease. in this patient, trazodone likely exacerbated qt prolongation from halopeddol leading to torsades de pointes. critical care physicians must be aware of this interaction. it is imperative to follow the qt interval for patients receiving halopeddol, especially when another drug also known to prolong qt is added. one must consider discontinuing the drug when qt/qtc becomes prolonged. objectives: analgesics and intravenous anesthetic drugs are routinely used in critically fll patients, who often suffer from a secondary impairment of the immune system. previous in vitro studies have demonstrated inhibitory effects of these drugs on polymorpho nuclear cells (pmn). the potentially important role of endothelial cells (ec), however, was not investigated, since suitable test systems were not available until recently. therefore a physiologically more relevant in vitro migration assay through cultured human endothelial cell monolayers (ecm) we established. using this assay system, the comparative effects of fenlanyl, sufentanil, propofol and the known pmn inhibitor thiopontal were tested. methods: human umbilical vein endothelial cells (huvec) were isolated and cultured on microporous membranes (cyclopererm) until an ecm was grown. pmn from male and female volunteers were separated by standard procedures. ecm and pmn were preincubated with clinically relevant concentratious of thiopental ( m), propofol ( p_g/ml), the solvent of propoful (intralipid), fentanyl ( ng/ml) and sufentanil (sng/ml). after preincubatiun (ecm minutes, pmn minutes) with the reslx~tive drug, leukocyte migration towards the chemoatfractant fmlp ( o - m) was measured in a two chamber well system for hours. the migration rate of untreated (untr.) and treated (treat.) pmn through untreated and treated ecm were determined. as a control untreated pmn and untreated ecm were used. results are given as means from independent duplicate determinations and expressed as a percentage of control (table) . statistical analysis was done with student's t-test. results: clinical concentrations of fentanyl, sufentanil and prupofol showed similar inhibitor~ effects as the known pivin inhibitor thit e ). % conclusions: for the first time we could show that analgesics and anesthetics exert their inhibitory effects not only on pmn, but mainly on the interaction of pmn with endothelial cells. moreover, we could shmv a significant suppressive effect of the opinids fentanyl and sufentanil on both ec and pmn. the known inhibitory effect of thiopental obtained in ec-free test systems were also confirmed in our physiologically more relevant assay system. objectives: to investigate when and how sedation is used in a consecutive cohort of patients admitted in a large sample of italian intensive care units (icus), gathered in a network named giviti, representative of the italian icus system. methods; the study called for a recruitment period of one month, from january to february , , data collection included age and other demographic variables, acute diagnostic broad profiles, severity of illness scores, treatments, lenght of stay and vital status at icu discharge. as concerned sedation, each patient was observed until discharge or for a maximum period of seven days. information on all the drugs used for analgesia/sedation, the route and modalities of administration, the timing, dosages and purpose of the administration have been recorded. results: the study involved the cooperation of icus, of which enrolled at least one case. the total sample included patients. overall, . % of patients analyzed (t / ) received at least one prescription of sedative during their stay. globally, at least one sedative drug was prescribed to these patients in days in icu. although over drugs were reported to be used, pharmacological principles accounted alone for % of all prescriptions. opioids were actually used in % of prescriptions; propofol in % and benzodiazepine in . %. as regards the way of administration, intravenous administration was applied in % of cases and, followed by intramuscular in . %. moreover, non-steroidal anti-inflammatory drugs (nsald) were used in % of patients and neuromuscular blockade agents (nmba) in %. detailed analysis on certain subgroups (surgical, trauma, ventilated patients etc.) have been also carried out in order to describe the practice of sedation in these peculiar subgroups. findings will be widely discussed during the presentation. conclusions: these results should be interpreted keeping in mind how peculiar is the intensive care setting compared to many other less complex settings of hospital care. in conclusion we thought it was important to present the data currently available in the most neutral form, to start moving in a direction which will enable us -by means of more specific and detailed studies, and with the cooperation and involvement of all those participating in the project -to shed light on one of the many aspects of medical practice in the field of intensive care which deserve closer attention. introduction: the aged run perilously high risks in cardiac surgery: among others, of haemodynamic fluctuations, respiratory depresskm and organ failure. response to anaesthetics is a crucial determinant for post<)perative complications, none the less being reintubation due to mechanical ventilation difficulties which increase morbidity, mortality and intensive cdre unit (icu) stay. objective: we wanted to assess our a,aesthesia window (selection, and a view of the induction -extubation period) for predicting safe and swift awaking, thus: icu dismissal for the aged. methods: in , selected patients (pts) (> y, f) followed a regular elective cardiac surgery protocol (propofol given at precisely designated time intervals). upon cu arrival, they were subjected to an admission protocol. our predictive criteria for early extubation at h included: a) alertness and ready response to commands; b) adequate gag reflex and sufficient protection for respirak)ry tract; c) pao > mmhg with flu < . ; d) stable ph> . with spontaneous respiration; d) stable haemodynamics without dysrhythmias; e) adequate perfusion and diuresis (> .(i ml/kg/h); f) mediastinal bfeeding< ml/h for at least h; g) normothermia (core temp> ~ and no shivering). subsequent reintubation was for: ) rr> /min; ) spontancx)us ventilation for rain with paco > mmhg; ) pao < mmhg with fio > . ; ) ph> . ; ) heart rate>] bm; and/or ) non mental alertness; and ) other medical disorders, after which adequate weaning therapy was necessary. then, successful weaning after h was considered: ) spontaneous breathing without any forrn of mechanical assistance; ) stability in haemodynamics; and ) elimination of fever threat. results: pts ( %) were extubated at h without complication; other pts ( %) at h but had to be reintubated because they were hypoxic and began weaning therapy; finally, they were all re-extubated by h. only pts ( %) proved problematic. conclusion: a,aesthesia wimhlw options (selectkm, extubation, reintubation and weaning) predicted quick (times propofol administration) and safe (rigid criteria) extubation ( %= h and %= h), exempting pts with developed post-operative complications ( %=extubation< h) unrelated to al~aesthesia window or icu protocol. dismissal and recovery then became an abbreviated question of time. fifisetll p, domeneg~i ~, sforzini i., veronesi i~, maconi a.g. *, breg~ massone p.p h [] ic+pca request conclusions:using e~aprenorphine, a synthetic,long-acting, ago-antagemist opinid drug as analgesic, in the major surgery we obtained the best clinic results with association of conttheus infusion of haft dose drug with bohts of pca in the first - hours and just pca in the secmad day after surgery when the patient is less sleepy. in this way we dent have a great sav~g of suppled drug but the major well-belng of patient without ~erious side-effects and quick mobilization; the dosage used don't compromise a good awake of patient: all patients are sleepy but ready for answer, no allueinatian, bradipnea but not less than b/m without ipoxia. also the patient proffered this kind of truit meut than the traditional at demand. the ward staff feel it useful] and rehabl~ the negative feed-back technology of the electronic infuser system makes possible to use it safe in the ward with high drug's concentration too. the infusion rate of low dose of drug assure a continuative analgesic covering ~n the first postoperative periad; the pca mode involves the patient him-self in the managemenl of therapy and enables him to choose the best way to confront the dll~icuity of postoperative period without call medical stall using pca-device we have had no probicm~ no accident. analgesia during extracorporeal shook wave lithot ripsy a .levit, b.grinbezg regional hospital, ekaterinbu~g, russia b~ectives: our task was to compare ~he analgetic effect of norphin and tramel. methods: study was made of two groups of uro-li~patients aged - . group a ( patients) received baprenorphine hydrochloride (norphin) at dosages of #. • mg/kg. group b ( patients) received tramadel hydrochloride (t~aasl) st dosages of . z . mg/kg. before the procedure diazepam was administrated i.v. ( . ! . mg/kg). blood saturation (spoz), hemodynamics incides (bp, hr,sv,co,sap,svr) were examined and the patients' subjective assessments of snsesthesis quality were analyzed. the hospital ethics committee approved the investigation. results: when using norphin hr increased by . % on the onset of the procedure while sap and sv decreased by .%% and . %, respectively (p< . ). however, there were no reliable co chsnges. spoz ~educed by @. % (p< . ) and remained lower than the initial one after the procedure was oyez. when administrating tramsl min. after ste~ting the procedure sap and svr increased by ~ . % and . % respectively. sv and co decreased insignificantly. nine patients in group b saffeting some dlscomfo~t needed additional tm~msl in~ection. in the course of the whole p~oced~e spo, was constant and was highez than that in ~he case of nozphin (p<o. ). conclusion: respiratory suppression by no~phin can limit its use in case of lithotzip@y while the advantages of tremsl a~e: lack of dyspnoe and good contact with the patti-b_ the role of uncommon agents of antinociception, placed epidurally, in the control of pain transmission in the spinal cord is well documented. somatostatin as an inhibitory agent is found in the dorsal horn of the gray matter of spinal cord. the effects of somatostatin is similar to the opiates if injected into the epidural space (ref. i, , , ) . the aim of the present study is to present the clinical effect of above mentioned palliative therapy in the case of cancer and non cancer pain. informed consent had been obtained from every patient before treatment was initiated under the legalisation of the local ethics committee. using the permanent epidural catheter and continuous infusion pump, we administer somatostatin in the dose of up to microgram per hour for one up to three days. the patients paln feeling was evaluated using a vas (visual analogue scale). we found that the opiate resistant pain level decreased by %. the rest pain could be controlled by pereral opiate analgetics or by combination of somatostatin and an opiate plus a local anaesthetic agent epidurally. theoretically, it seems that epidural administration of somatostatin and local anaesthetic agent is useful in the control of acute pancreatic pain and systemic effect of somatostatin is beneficial in the treatment of acute pancreatitis. few studies have been performed in the critically ill (ci) and because of the complex pharmacodynamic and pharmacokinetic changes that occur, it is difficult to predict accurately drug responses and interactions in individual patients. midazolam (m) is a watersoluble benzodiazepine with a rapid onset and short duration of action in normal individuals; however its metabolism is decreased in the ci. critical care physicians every day experience suggest that among the ci there is a different pattern of response to m; here it is hypothized that such a response could be due to different metabolic behaviours of ci. m (i.v.infusion rate - rag/h) and antipyrine ( rag p.o.) were infused to ci with ( patients -group ) and without ( patientsgroup ) endotracheal intubation. blood samples were collected at fixed times during and after m infusion. total urine nitrogen and antipyrine elimination half-life demonstrated different metabolic characteristics in group chypermetabolizer") as compared to group chypometabolizer"). results follow. the results of our study are preliminary and more kinetic data in critically ill patients are needed to statistically confirm our approach of "hypometabolizer" and "hypermetabolizer". objectives: some clinieai and experimental studies suggest that propofol decreases myocardial contractility in coronary artery bypass grafting (cabg) patients. the intrinsic negative inotropic action of the drug may be independent from changes of preload and afterload, whereas the myocardial depression induced by propofol may depend on changes in ca ++ ious availability. aim of this study was to verify the hemodynamic effects of propofoi in a group of cabg patients with uneompromised contractile function and to minimize myocardial depression, during induction of anesthesia, choosing to associate calcium chloride (cac ) in an other group and to define its role in producing this effect. methods: fourty patients, randomly divided in two groups ( pts a and pts b), undergone elective cabg, were enrolled in this study. anesthesia was induced by senior anesthetist in both groups by means of fentanyl mg• kg < in ", pancuronium . mg x kg - and propofol mg • kg - in ". a blind investigator administered via another venous way at same speed ( ") saline in patients of group a and mg x kg - cac in patients of group b. hemodynamic data (heart rate hr, mean arterial pressure map, mean pulmonary artery pressure pap, pulmonary capillary wedge pressure pcwp, central venous pressure cvp, cardiac index ci, stroke volume index svi, systemic and pulmonary vascular resistances indexed svri and pvri) were obtained at baseline (to), ' after anesthesia induction (t ) and " after tracheal intubation (t ). results: hr decreased moderately in both groups (p = ns). map decreased as well, more markeatly in group a at ti and t . this trend was statistical significant (p < . ) in both groups. pap, pcwp and cvp unchanged following induction in any of two groups. ci decreased in group a (p < . ) dramatically dropping at t , while in group b it showed a negligible decrease at t (p = ns) and value similar to baseline at t . svri and pvri did not exhibit statistically significant changes. conclusions: the use of propofol as a starter of anesthesia in cabg patients allows to reduce total dose of fentanyi, dramatically reducing post-operative intubation time. propofol-fentanyl association prevented the hyperdynamic response to stress (i. e. laryngoscopy, intubation) but severely depressed ci and svi. cac simoultaneous administration effectively minimized the negative inotropic effect of propofol. moreover no unwanted side-effects due to cac were observed. diseoordinated labor activity (dla) is one of most serious in obstetric pathology.medication sleep effect to a woman in birth is of limitation of pathologic impulsation from the central nervous system, but it does not influence processes resulting in and supporting dla on the organ level. constant discoordinated uterine contractions during medication sleep (ms) imerease disturbances of uterine blood flow, organ hepoxemia connected with hyperergia of vascular wall and myometrium to catecholamines, that reduces efficiency of anesthesiologic protection. we applied hexoprenalin in complex with ms to primaparas. efficiency control was being accomlished through hysterograms, grade scale of analgetic effect where hemodynamics reaction to pain, significance of emotional reactions and their vegetative manifestations before and during ms were registered, and also through hydrocortisone level in plasma before and during ms. the examined women's average duration of ms was hours more than in control and made hours, though their average labor duration and in control was the same and made hours. % of the examined women experienced independant labor, as for the control group -only %. supplementary methods of anesthesia were required in % in control,that increased medicamentous depression of the fetus, but for the examined group -only in %. the examinees's newborns experienced favourable terminations, in control newborns were in state of light rate asphixia. analgetic effect was complete in control just in i %, and of examinees -in %. on the women's hysterogrnms before ms discoordinated contractions were registered on the background of increased tonus. during ms in control similar uterine contractions were constantly registered, but for the examinees they were not marked at all or had the proper character. the examinees' hydrocortisone level was reduced for % and in control only for %. the study has allowed to make a conclusion that hexoprenalin applications in complex with ms in case of dla make anesthesiologic care safer and more effective, promote simultaneous removal of patholgic processes in the central nervous system and uterus, which lead to dla development. objectives: the aim of report is to present a clinical study results for the evaluation of anesthesia for a patients in unconscious states by eeg examination: methods: the study was conducted on patients after a severe abdominal operations on stomach and intestine and on one patient after traumaticat exarticulation of upper extremity. all patients have different unconscious state levels from somnolence to coma ]. also thay hav~ had respiratory insufficiency and control mandatory ventilation have been used. we were used intrave,~ous injections of morphine hydrochloride from initial dose of mg. than we were increased the dose to mg i.v. by drops with an accompanying eeg monitoring and fourier spectral analysis. initially all patients have had -rhythm with slow and a-activity. an c~-activity index have been lower than %. results: after anesthesia the o-and a-activity have disappeared. the or-activity index have increased to %. also we have noted decreasing of the unconscious state levels to a possibility of a verbal contacts ( - points gcs). conclusion: the quality of anesthesia could be evaluated by eeg monitoring. a regaining consciousness afte~ the injections of high doses of morphine, probably, may be a result of an opiate deficiency (full or partial). anesthesia with the following drugs: clonidine, l-arginine, l-n-arginine-methyl-ester (l-name), and their combinations. tail-flick latency (tfl) was determined at time zero and min after each treatment. clonidine (i- ~g/mouse) prolonged tfl in a dose-dependent manner. at a clonidine concentration of gg/mouse, tfl increased . -fold compared to time zero. l-arginine increased tfl at a high concentration (i gg/mouse). l-name suppressed tfl . -fold compared to time zero at a concentration of gg/mouse. conclusion: we have found that no is involved in clonidine spinal analgesia. studies are currently being undertaken to assess the effect of combination treatment of the above drugs on clonidine supraspinal analgesia. objectives: hemodynamic changes ussualy accompany laryngoscopy and tracheal intubation.the aim of this prospective study vas tu present the effectivness sufentanil in preventing reflex circulatory respone of laryngoscopy and tracheal intubation and provides stable hemodynamio condition for induction of balanced anaesthesia. methods: adult asa i-ii patients sheduled for elective surgery.they were allocated randomly and divided into two groups:fifteen patients received single bolus of placebo prior to laryngoscopy.and tracheal intubation. anaesthesia induction was then performed with thiopental (smg/kg) and atracurium ( , mg/kg), followed by neurolept anaesthesia. mean arterial preassure (map) and heart rate (hr) were measured before, during and min after intubation. althought ecg was recorded at the same time intervals. results: the groups were compared with regard to the changes in arterial blood pressure,heart rate and electrocardiogram. mean arterial preassure and heart rate were incresed significantly during and after laryngoscopy and tracheal intubation in control group but remained unohange the baselin values in the group who received sufentanil. control group ecg suggested more changes than sufentanil group. conclusions: the results indicated that , mg/kg sufentanil for anaesthesia induction reducing the pressor response to laryngoscopy and tracheal intubation. obiective: the aim of this experimental study was to evaluate morphologically the influence of anesthesia in liver as well as in isolated hepatocytes (hc). methods: a total of female swine ( - kg) were used as liver donors and were randomly assigned to one of two groups regarding anesthesia protocol: group a (n= ): induction with intravenous sodium thiopental in a dose of mg/kg, group b (n= ): propophol in a dose of mg/kg for induction'and mg/kg/h for maintainance of general anesthesia. both groups were under the same preanesthetic regimen (diazepam, ketalar and atropin) and received standarised doses of fentanyl and pancuronium during anesthesia. in beth groups total hepatectomy was performed and the liver was perfused with - it of cold ( oc) university of wisconsin solution for a period of hours before hepatocyte isolation. liver tissue samples were fixed in formalin for the morphological study and stained with hematoxylin-eosin and pas. for hc isolation collagenase solution (type v, sigma, c- , . mg/ml) was infused via portal vein and the liver was incubated at oc for rain. cells were filtered and then washed in cold hanks' solution. isolated hc were fixed and prepared for staining or simply cryopreserved (- oc). results: histology was completed in / experiments ( in group a and in group b). mononuclear infiltration (halothane type injury) was found in all specimens ( / ). focal zonal necrosis and acidophilic bodies were found in specimens from group a ( / and / respectively) while portal inflammation with piece meal necrosis was found in one specimen from group b ( / ). smears of hepatocytes -beth formalin fixed and cryopreserved at - oc -were stained with hematoxylin-eosin and showed morphologically intact hepatocytes. conclusion: pre[imineq~' study of our material reveals mote frequent livet injury in the thiopental group, but this finding has yet to be established by increasing the number of observations. objectives: in this paper we present our experience and point out the importance of sedation of those patients who suffered severe head injuries/contusic cerebri with signs of decelebration/and who were put on ventilatory machine to obtain better mechanical ventilation in neurosurgical intensive care unit of emergency center in belgrade. methods: patients were treated from jan. till dec. results: in patients we successed to reduce agitation and decelebration. conclusions: the major demand of sedation in neurosurgical patients are that the patient should be calm, comfortable and painless. references we compared three analogous groups of patients in each, with orthopedic operations on lower extremities. in each group we applied different kind of analgesia: first group rece-ned local anesthetic ( ml . ~ bupivacaine) when analgesia was first demanded; second group received ml fentanyl via peridurai catheter when analgesia was first demanded, and third group received m] fentanyi intravenously on demand for analgesia. we used visual analog scale (vas) for estimation of pain intensity and success of applied therapy. statistical data analysis was performed using student's t-test. tha best vas had group in which local anesthetic was applied periduraly. second was the group with periduraly applied fentanyl, and third was the group with intravenously applied fentanyl the longest duration of the effect of analgesia ~/as in the second group. there were no adverse effects and complications, apart from mild sedation in the third group. for orthopedic operations on lower extremities, application of peridural catheter for anesthesia and successful postoperative analgesia with local anesthetic, or opioid analgesics is acceptable. background and obiective : tympanic temperature measurement (ttm) is a relatively new procedure which provides accurate estimates of core temperature in stable postoperative patients and in patients admitted to the emergency ward. however, experience with this technique in hemodynamically unstable adult icu patients is limited. design : prospective study with patients serving as their own control. se:ttijlg : adult medico-surgical icu in a tertiary care hospital. patients : consecutively enrolled patients with thermodilution catheters in place and without contraindieation for rectal temperature measurement and without hypothermia (core temperature < ~ interventions : tympanic temperature, measured by a commercialized tympanic thermometer (genius a first temp) was compared with pulmonary artery (pat) and rectal temperature (rt). within minutes, core temperature was assessed in each patient by the same trained individual using the three techniques in random order. measurements and main results : mean bias (ix~ and its variability (sdr of method comparison pairs were calculated using the methods comparison analysis as described by bland and alunan (lancet, ; i : - objective: to measure the prevalence of pressure sores on the intensive care unit and the effect of risk calculation on pressure score prevalence and pressure score severity. method:in during a period of months a series of prevalence studies was carried out on the sicu to investigate how many patient days with pressure sores were taken care for by the icu nursing staff, what kind of preventive interventions were done and what the patient risk was on developing a pressure score. results: on average there was a prevalence of pressure sores on the buttocks of . % on the short stay unit and . % on the long stay unit; preventive interventions increased when the pressure score was visible for the nurse and the majority of patients had a high risk or extra high risk for developing pressure sores according the pressure score risk calculator. as a result of these findings the nursing management decided in that nurses had to complete daily on every patient the pressure score risk assessment in order to be able to take adequate preventive interventions. when the results of the two studies were compared, the most important results were that: ) there is no indication of a reduction in patient days with pressure sores (short stay unit: %, long stay unit: %); ) there is a reduction in severity of pressure sores (grade iv decreased from . % to . %) because adequate preventive measurements are initiated by icu nurses in an earlier stage. possible explanations for the increase of patient days with pressure sores on the long stay unit are that the average pressure score risk score was increased from . to . and the mean frequency of nursing patients on alternate sides decreased from . to . times each day. conclusion: daily assessment of the individual patient risk on developing pressure sores does not decrease development but reduces the severity of the developed pressure sores. method: the conventional woundcare method of patients with an open abdomen is to change frequently the saline soaked ribbon gauze pads ( - times each day). this method is labour intensive for the itu nursing staff and rather uncomfortable for the patient. problems that often occur are: insufficient hygiene, frequent nursing interventions, a progressive risk for deterioration of the skin and underlying tissues, difficult to measure abdominal output and in case of bowel fistulas enteral feeding is hardly possible. in using the new method, stomahesive is applied on the skin surrounding the wound. a large size of surgical film dressing, opsite | is applied over the abdomen. two or more foley ~ catheters ch are inserted through an opening in the surgical filmdressing. the application of several lateral openings in the catheters is advised. the drains are held firmly in place between two layers of sterile opsite | "flexigrid ~'' x with the so called bookend method and are connected to a low vacuum suction system ( - kph) results: we studied the frequency of dressings changes on patients in the itu. in total they had days with an open abdomen. during this period there were dressing changes. this means one dressing change every days. further advantages of the new method are; more hygiene in patient care, no maceration and irritation of the skin, nursing on alternate sides is possible, output can be measured, in case of bowl fistula, enteral feeding is possible and an abdominal lavage is possible on the sicu by opening the opsite | conclusion: this new method of woundcare management is more patient friendly, prevents several nursing complications and decreases the work load for the nursing staff. objective: to investigate the interaction between the ventilator and the closed suction system related to possible induced negative pressure by this sytem. method: we studied in a testlung model what the effect was of the different ventilators (evita: dr~iger; servo c: siemens), ventilation modes ppv, simv) and ventilator settings (tv, trigger level, frequency, peep level) on the induced negative pressure by the closed suction system during suctioning. results: when using the evita the magnitude of the negative pressure increased when the ventilation frequency and tv decreased.. the largest negative pressure (- cm h ) was produced with a tv of ml and a frequency of /min in the ippv mode with trigger level on - cm h and a peep level of + cm h . the servo c showed a complete different pattern. the largest negative pressures were measured when no trigger level was set ( cm h ), the induced negative pressure was not depended on the tv or ventilation frequency but more depending on the ventilator mode. the simv mode produced the smallest negative pressures ( - cm h ) and the ippv mode the largest ( - cm h ). conclusion: the effectiveness of the closed suction system is very much depending on the kind of ventilator, ventilator mode and ventilator setting that is used. if the interaction with the used ventilator is not known by the icu nurse this piece of equipment can cause damage to the patient by inducing large negative pressure in the comprimised lung and there fore creating the possibility of alveolar collaps and atelectasis or oedema. introduction : ethical problems are daily and disturbing preoccupations for the icu staff. objectives : improving ethical management in icu by creation of an intelprofessional group of ethical reflexion ( iger ). methods : existing was evaluated by a preliminary formulated series of questions sended to the whole staff (q , n = , items) after two training sessions (laws, deontology, professional values, culture, norms of quality) followed by persons ( %) and directed by an external chairman, satisfaction and needs of the staff were evaluated by a second series of questions (q , n = , items). at the issue, iger was created. results : q : responders ( %). law's misinformation : to %, disagreement for limitation of intensive cares : %, hope to improve collective decisions and creation of iger : % q : responders ( %) : satisfied by the training sessions : %, non satisfied : %, want to continue the project : %, refuse : %. iger was constitute by physicians, nurses, secretary, dietetic (n = ). the first working theme was {< therapeutic's withholding and withdrawing decisions in icu ~>. four subgroups of iger's members (having access to an ethical library) worked independautly and submitted their reflexions in a tdmestrial plenary session of iger in the presence of an external chairman, allowing a synthesis. at the issue a report was writted to be used as a reference for bedside and individual decisions. conclusions : constitution of iger seems to improve ethical management in icu. the first result of iger is that it is now possible to began collectively a reflexion concerning therapeutic's withholding and withdrawing in icu. the work is going on and further subjects will be studied. objectives: ) to compare the value of heat-moisture exchangers with bacterial filters (hmef) and without bacterial filters (hme) in the prevention of colonization of ventilator tubing and ventilator-associated respiratory infections. ) to asses the temperature and relative humidity of inspired all using both types of heat-moisture exchangers. methods: mechanically ventilated patients were randomized, to either hmef or hme. endotraeheal aspirates, pharyngeal swabs and samples from tubing were collected for bacterial cultures on the st, nd day mechanically ventilation and weekly thereafter. temperature and relative humidity were measured in patients ( hmef and hme) h and h after placing the hme or the hmef. results: both groups were comparable as regards age, mechanical ventilation period, severity score (saps ii), leukocyte count, and number of patients with prior antibiotic treatment. from the hmef group, ( %) ventilator tubing yielded microorganisms in, at least, one sample as compared to ( %) of the hme group; p=ns. the incidence of respiratory infection was similar in both groups ( % vs %, p:ns, for hmef and hme respectively). among the bacterial species isolated from ventilator tubing in the hmef group, ( %) were not isolated from pharyngeal swabs. a similar ratio was shown in the hme group ( / , %). both heat-moisture exchangers were efficacious in keeping a good relative humidity of inspired air ( % • vs % • .%; p=ns, for hmef and hme respectively). relative humidity was significantly higher after h of mechanical ventilation in the hme group as compared to hme group ( . % • vs . % • %; p= . ). conclusions: both types of heat-moisture exchangers have the same effect on the prevention of colonization of ventilator tubing. similar relative humidities are achieved when using either type of heat-moisture exchanger. results: tumor and nontumer enhrgements of the thyroidea were present in ~ of the operated, surgicel adrenal disease in io!, hyperplssle or persthyroid gland tumor in ~ end endocrine pancreatic tumors in %. in the intensive oere unit, these patients wore screened by noninwsive monitoring in ~ of cases: and invasive monitoring was applied in % of ceses.the basic noninvesive methods included: electrocardiogram with standard end precerdial leeds, percutaneous eutomotlc measurement of systolic, diastolic and mean arterial pressure, measurement of hourly diuresis and body temperature, frequency, hearing capacity and rhythm of one s own breathbng bs well as pulse oxymetry. a special plece in monitoring and control of vital parameters in postoperative period belonged to the nurse, thoroughly trained for enelysis end interpretation of the observed parameters which would be discussed in the paper. it has been believed that the leader sits at the pinnacle of power. over the years, this has proven to produce frustruation and anguish instead of the expected results. leaders have not been able to produce the changes they know are essential to their organization's survival with this command-and-control paradigm. through literature reviews and evaluating leadership styles, one can clearly see the most effective form is that of empowering people to a new level of performance -not ordering it. changing the leadership paradigm to a manner/style that has been shown to be effective and one of people empowerment shifts the focus to personal responsibility for performance. removing obstae}es~ stimulating self-directed actions, and determining focus and direction are just a few elements used to create the successful environment of empowerment. with increasing pressure in the health care arena, it becomes critical that a leader's job is to get the people to be responsible for their own performance. developing ownership, creating an environment where people want to be responsible, being a mentor or coach, and learning faster while encouraging others to do so demonstrates the commitment to effective leadership. this presentation will illustrate the critical components that are achieved when every person in the institution is empowered to perform at a level that is directed toward positive, effective results. herrera m. (md) . icu. hospital regional. malaga. spain. the systems of veno-vanous continuous haemofiltration (wchf) have a high cost and a limited life span. in an attempt of lengthening their mean life it has been proposed to accomplish programmed washes of the ~-stems. this practice supposes an increase in nursing workload. in order to evaluate the real efficiency of this practice we have accomplished this study. material: prospective randomized study of all the filters of vvchf used during the last year in our icu. we have determined two groups of filters, in the first (group a) we accomplished washed in a programmed way, and in the other (group b) only when the alarms of the system suggested a clotting of the filter. for the statistical analysis we used the kaplan-meier test for survival analysis. results: we have studied a total of patient submitted to wchf during the last year. we used a total of filters with this results. objectives. sounding out the nurses about the need to inform patients" relatives and the rigth kind of such information, like a preliminary approach to an information cuality assessment, methods: we inquired all the nurses of the intensive care unit of an regional hospital by an semiestructurated questionary which included personal data: age, sex, contractual relation, professional experience.., and opinion data: do you think to inform relatives is a nurse task?. which of the next informafions do you think is more important?, please, write others topics about information you think are relevant. we process the data on epi-info estatistical program and use x test to compare the results. results" from nurses of staff refused to flu the quetionary, and were not available. of the remaining, %were v~men and % men. the mean age were . % had an svable contract and ( eventual, the mean professional experience were of years and % worked in the unit since more than years. the % answered that offer information to relatives is part of the nurse activities. we did not find differences with nurses who answered negatively comparing by sex, age, contractual relation or proffesional experience. the three information topics found out like more important were: ) to inform about patient mood. ) to inform about happenings from the last visit. ) to inform about dressing instrument required by the patient, nurses who answered negatively think that to inform is a doctors task or that nurses are not competent. conclusion~ intensive care unit teams (nurses, doctors and auxiliar personnel) should get accord on who and how to inform relatives, we consider the nurses' role on information as unquestionable. objective: investigate the respiratory and cardiovascular response after discontinuing oxygen therapy durir~ intr~/]o~pital transport. desiqn: fifty-one patients ( male and female, aged + , and , , years respectively, ~+sym) being on therapy were studied prospectively in two consecutive intrahospital transports. oxygen therapy was continued in the first transport while the second one was performed as usually, i,e, without . during transport each patient was monitored by pulse oxymeter and holter whereas arterlal blood gases were tested just before a~xl aft~-trar~portation. results: compared to daseline, pa and sa were signif~canthy decreased in the case of oxygen discontinuation (p< , i). paco was significantly inur~ds~i only in the subgroup of patients with obstructive lun[ disease (p< , ) . heart rate increased in all phases of the transport when administratlon was discontinued. blood pressure remained stable in either case. the percentage of supraventricu!ar extrasysto!es, ectopic v~r[hicui~r contractions and st-s ~ment depression was progressively increasing and became very high at the end of transport in the case of therapy discontinuation. other arrhythmias did not change significantly. conclusion: discontinuation of oxygen therapy during intrahospital transport causes severe drop of pao and sa , increases the heart rate and contributes to the appearance of arrhythmias which were not present before. methods:for evaluation of the functional state of brain the complex of methods was used,whieh included electro encephalngraphy ( brain mapping ), rheoencephalography, tetrapolar transtorax rheography. for the estimation of humoral status the level of histamine and serotonine, products of free-radical oxidation,enzimatic markers of ishemic damage of brain and of endogenous intoxication was investigated. results: patients with encephalopathies after resuscitation were observed.asystolia was as a result of:shock, trauma, asphyxia,poisonings,appiication of drugs, eclamp sia,injury of the heart,diseases of fhe cardiac vessels. all patients with postasystolic syndrome entranced in comafose condition.in the group (reconvalescents) the depth of coma by glasgo~ pittsburg"s scale was , +- , . the duration of coma was from rain. to hour,average , +- ,sh.ln the group (the deads) the depth of come was , +- , .the artificial lung ventilation was used in all patients:in the group , +- , days,in the ~ , +- , days.apallish syndrome developed in cases,in patients diagnozed <<brain death~. the th degree encephalopathy (coma) was found to be associated with disorganized eeg-pattern with predomi nant alpha-( group) and slow ( group) activity. the relative power of delta-and theta-ranges was about - per cent.the patients with apallich syndrome demonstrated greatly elevated theta-range power, that of beta -range was essentially decreased. the degree of disturbances of circulation in the both groups was different.cardiac index (ci) and stroke index (si) in the group decreased on , % and , z, in the -on , % and (; , %.general peripheral resistance (gpr) increased in the group on , %,in the -on (; , %.in the group brain blood flow was increased, in the -decreased on , n.in the group there was the normotonic type of reg-curve,in the -atonic or hypotonic type (without reaction on pharmacologic influ ence).disturbances of permeability of cellular membranes (enhanced free-radical oxidation with an increase in di enic conjugates on and n) and vascular ones (an increase of serotonin concentration on and %, hi stamine content on and %) resulted in hyperfermentia (an increase of concentration of creaune phosphoki nase in the group on %,in the - %.the level of middle molecules (mm) increased in the group on , %, in the -on , %,of polyamines (p) (spermidin,spermin,putrescin).coefficient of correlation between mm and p was , . immediate correction of neurocyte metabolism is impossible due to marked brain oedemaswelling,severe dis ordes of cerebral circulation, disturbances of membrane permeability.thus,the following treatment algorythm might be advisable: . protective inhibition of brain and reduction of its energy requirements. . recovery of cell and vascular membrane fuoctions. .recovery of blood circulatiom .oxygenation of nervous tissue and drainage of brain disintegration products.in patientshbo carried out. .active methods of detoxication (homo-and enterosorption). .correction of cerebral metabolism. . dehydrative therapy must be very cautious. conclusion: the activation of energetic metabolism in neurocytes must be carred out only under neurophysiolngical control and after definite preparation. ( ). the clinical estimation of acute cerebral failure carried out by olasgo-pittsburg"s classification of coma. we studied such groups of patients: poisoning co ( ), asystolia ( ), eclampsia ( ), acute renal failure ( ), control group ( ). methods: electroeneephalngraphy (brain mapping) was fulfilled by means of encephalograph <,medicor ~ in monopolar channels. the estimation of eeg patterns by zhirmunskaya method ( ) was carried out. there were analyzed and changes of eegb by results of rapid transformation furie after rhythmical photostimulation(phts) , , , hz. we studied also the figures of absolute and relative power in such diapason : delta ( - hz), theta ( - hz), alpha ( - hz), betal( - hz), beta ( hz and more). results: all eeo changes were divided into following types: -organized ( , groups); iii -asynehronic ( group); iv -disorganized with prevalence of alpha-waves ( , , groups); v -disorganized with prevalence of delta-and theta activity ( , , ; , groups) akkording to our model of hormonal-metabolic disbalance in pregnancy , one of the main reason of destosis is hypoergos , reesults in endotoxemia and neuro-endokrine disregulation. so, stady of central nervous system function give us the information about adaptation processes. the aim of our work is to study the degree of neurologikal deficit in destosis when olifen and lipin were used. there were studied pregnants with different severe forms of gestosis. the degree of neurologikal deficit was valued by datas of neurologikal status . eeg with ,~brain mappinng~, , electrikal resistance of the skin. the komplex inteensive therapy with applikation of olifen and lipin allowed to improve the patient,s condition , decrease the degree of neurolodikal deficit and mortality . kostenko v.phd,kabanko t.,phd,galalu s.md,beliavtseva n. ,shramenko k. phd intensive care department,donetsk medical university, donetsk, ukraine plasmapheresis (pph),as other extracorporal methods of detoxicatin,has a great importance in contemporary medicine. the role of pph is special,because of its simplicity, effectiveness, atraumatic.we incalcated pph in obstetrical clinic. there are rough changes in agregate condition of blood in more of pregnants.these changes lead to disturbances of central,organ and peripheral hemodynamic.the therapeutic effect of pph is due to influence on agregate condition of blood. we considered that application of pph is very effective in pregnants with:bronchial asthma, severe forms of diabet, psorias, gestosis, allergic diseases. we used the membrane pph in pregnants: apparatus-,,hemos-pf>,, plasmofllter pmf- ,with effective area- cm,the volume of extracorporal contour- ml.such pph has no the ~ agressive effect,,, as in cases of application another extracorporal methods. this method was incalcated in our practice recently, so results will be reported in further publications. ( ). post-operative cerebral neoplasm ( ), post-operative subdural hematoma ( ). icp was monitored via a catheter inserted in the lateral ventricle and values were continuously digitally recorded by means of a bedside computer data acquisition system (maclab). the fiberoptic tracheobroucosenpe, which guided the procedure, was passed between the nasotracheal tube and the trachea in order to avoid hypoventilalion. the patients had stable baseline hemodynaimcs. propofol infusion and fentanyl boli were administered to mantain stable mean arterial pressure values. peak (mean(sd)) icp duping the minutes pre-ciaglia procedure (baseline values) were compared with values during ciaglia procedure, and the minutes p st-ciaglia procedure. data were compared with repeated measures anova. results: ciaglia procedure duration was (mean(sd)) ( ) objectives: transient global amnesia (tga) is a syndrome caracterized by impairment of short-term memory, inability to form new memories, retrograde amnesia and repetitive queries, without other neurological signs and symptoms. the pathophysiology of tga is unknown; thromboembolic, epileptic, migrainous and metabolic mechanisms have been suggested. to address some of these issues, we undertook a study of cases of tga in whom we examined clinical, laboratory data, electroencephalogram, ct of the head, ultrasonography ecodoppler. methods: patients were included in this study: men and women. the mean age was years. all cases underwent a standard clinical examination, electrocardiogram, routinary humoral tests and x-ray, electroencephalogram (eeg), ct scan of the head, ultrasonography ecodoppler. results': the mean duration of amnesia was h. m. +/- h. m. hypertension was found in patients ( %), ischemic heart disease in patients ( %), hypercholesterolemia in patients ( %), hypertrigliceridemia in patients ( %), smoking in patients ( %), atrial fibrillation in patient ( %), history of epilepsy in patient ( %), migraine history was not recorded. ct scans of the head showed multiple small deep infarcts in patients ( %), a single hypodense lesion in patients ( %). in patients electroencephalogram was normal ( %), in patients there were widespread nonspecific electrical changes ( %), in patients there were focal nonspecific eeg abnormalities ( %). conclusion: in our study tga was more common in women ( %). we showed a prevalence of hypertension, hypercholesterolemia and cerebral infarcts compared to normal controls. we have demonstrated a higher incidence of nonspecific electrical changes in tga of lower length, while ischemic lesions in ct of the head were more frequent in tga of greater length. these data seem to be in agreement with the hypothesis that tga is a heterogeneous clinical syndrome, consisting of pure, epileptic, and ischemic types. however we did not find any correlation useful in discriminating pure from associated tga forms. from our study it is tempting to speculate that pure tga is a rare event, underlying still unknown mechanisms wich differ from ischemic, epileptic, migraineous causes. objectives: aneurysmal subarachnoid haemorrhage (sah) is special condition increasing intracranial pressure (icp) in various ways. at the other hand cerebral vasospasm and related delayed ischaemic deficit (did) could answer for the poor outcome. triple h therapy seems today a basic option to prevent did, but it may increase the icp worsening the altered intracranial pressure condition and thereby the cerebral perfusion pressure (cpp). is there any way to individualise the triple h therapy when it is necessary? methods: between sept. march thirty-seven patients with intracranial aneurysms were operated on within hours following sah. five patients were in hunt-hess iv at admission. all patients received triple h therapy in a preventive fashion following surgery and were monitored by daily transcranial doppler ultrasonography (tcd). icp and cpp was measured in twenty-four cases. twenty-two of them received lumbar liquor drainage (lld) and nineteen were administered induced hypertension. the other group was treated by basic triple h therapy. results: in group with monitored icp the outcome was twenty-one excellent, one poor, two died (one of them died from extracranial decease). in the other group four had excellent, six moderate, two poor outcome, and one died. conclusion: according to our recent observation the patients can be divided into two groups of therapy. in group i, the patients with elevated tcd values and either low or high icp reacted to lld. we are concerned that haemodilution and slight hypervolaemia should dominate in the triple h therapy. in group ii patients having high icp with tcd and/or symptomatic vasospasm should be managed by the induced hypertensionhypervolaemia dominated therapy focusing on cpp (icp) and focal neurological signs. air emboli were detected in lo% (n= ) of natients undergoing coronary srtery bypass craftin~ (cabg). central nervous system ~ysfunction occured in ~$ of the nstients with air embnli and in none of those ~ithhout air embo!i. hvtothermia is the classic form of oro-tect~on used dur~nc ~"~" " ~ ~ ca~.,~modu] :r, on~_,_. bj/oass. the surf~eon sho,;,ed thorough!~: evecnnte air from the heart, but the onesthesio!o[[ist can signifieamt!y influence the outcome by emt!oyin ~ methods to detect and treat air emboli. the changes in head rate are primarily due to alterations of autonomic tone. the heart rate variability (hrv), that express the degree of heart rate fluctuation around the mean heart rate, reflects somehow the condition of central nervous system. hrv may be measured by a number of techniques. short-term time-domain variables of hrv are reflect generally the vegal activity. in this study the changes in hrv variables of patients with brain damage, and in addition the changes in hrv measurements in comparison with the clinical evolution were evaluated. eight patient with brain damage and six normal individuals as control group were studied. a elecrocardiographer with availability of computation the sequence of beat-to-beat intervals for one minute was used. the following variables of hrv were measured: ) standard deviation (sd) of beat to beat r-r interval differences that reflects the respiratory control, )the maximum/minimum (max/rain) interval that reflect variability related to baroreflex and thermoregulation and ) the coel~cient of variation (cv), the results are shown in the in the patients with brain death and in vegetate state there were virtually no hrv. increased hrv pattern was found with clinical improvement, the changes of hrv precede of the changes of gcs, we conclude that time-domain hrv could reflects the degree of brain damage, it is good prognostic index of the brain damage and may change earlier than the gcs. objectives: cerebral co vasoreactivity is an important determinant of cerebral blood flow (cbf) and has been shown to be of prognostic value in head trauma (acta anaesthesiol. scand. ; : - ) . we wondered whether co vasoreactivity could be selectively altered in one hemisphere in comatose patients. methods: patients ( m/ f, age - yrs, glasgow - ) in coma due an acute brain lesion (trauma, hemorrhage, or infection) were studied. cbf was measured bilaterally using jugular thermodilution at paco , , , and mmhg by increasing pico with mechanical ventilation kept constant. normal co vasoreactivity was defined as an increase in cbf of at least i ml/min. g per mmhg paco . results: patients had normal co vasoreactivity bilaterally, patients had altered co vasoreactivity at both sides, and patients had a normal response at one side (left or right) with an altered response on the other side (dght or left). for the patients left cbf was in mean ! ml/min. g lower than right cbf (figure methods: following institutional approval piglets (body weight :tl . ) were anaesthetized by % fluothane. a catheter was placed in the right femoral artery for blood pressure monitoring and a fiberoptic catheter (oxymetncs- abbott) was advanced via the right internal jugular vein to the jugular bulb for sjo determinations. another catheter with a balloon on the tip was advanced in the right atrium via the right femoral vein. a mean arterial pressure (bp) at mmhg was achieved by appropriate balloon inflation for rain and two groups were cleated: i) the hypoxemic group by respirator disconnection (*) and it) the hyperoxemic group by fio =l on respirator (o). samples were obtained at time ( ), ' min at hypoperfusion ( ) arid at reperfijsion at ' ( ), ' ( ) and ' ( ). pao , pjo and oxidative brain stress evaluation was performed from jugular bulb blood. the latter included: i) no synthase (nos) and xanthine oxidase (xo) activities by a method based on the oxidation of scopoletin detected fluorometrically, it) no levels estimated as onoo-by luminol enhanced chemiluminescence in the presence of ~tm hydrogen peroxide (h ). resul'~s: the mean pao was mmt-ig for group i and methods: we retrospectively reviewed all upper gi-endoscopies, performed in the period january -july in patients ( men and women) admitted at the icu's of our hospital. results: it concerned surgical, medical, eardiological and neurological patients with a mean age of . yrs (range: - ). in %, the endoscopy was performed at the icu and in % at the endoscopy department. in % of the cases, the endoscopy was primarily diagnostic, of which % was performed for localization of upper gi blood loss. in % the endoscopy was primarily thempentic, of which % was performed for placement of a duodenal feeding canula. location of the upper gi bleeding was: variees ( %), duodenal ulcer ( %), oesophagitis ( %), gastric ulcer ( %), others ( %) and none ( %). as coincidental findings were noted: cesophagitis ( %), gastritis ( %), gastric deer ( %), duodenal ulcer ( %), duodenitis ( %), oesophageal ulcer ( %) and others ( %). conclusions: there were marked differences in indications and findings of endoscopy at the different icu's. these differences reflect an admission bias and differences in populations and treatment preferences. compared with cardiological and neurological icu's, substantially more endoscopies were performed at surgical and medical icu's. in a considerable number of cases, no source of upper gi blood loss could be found endoscopicaiiy. when upper gi blood loss was the icu admission diagnosis, the main cause was needing varices, which could be controlled endoscopically in the vast majority of cases. when upper gi blood loss was ndt the icu admission diagnosis, peigie ulcer and oesophagifis were the main causes of bleeding. because of the considerable number of coincidental almom~adities found at endoscopy, there is still room for debate whether antacid medication and/or motility stimulating agents should be given prophylactically at icu's. many studies have shown that blood lactate levels in survivors and nonsmvivors of traumatic and septic shock are significantly different. the degree of multiple organ failure is related to the duration of lactic acidosis ( ). the aim of this study was to evaluate blood lactate level as a prognostic marker of high risk postoperative patients who may benefit from invasive hemodynamic monitoring and aggressive fluids administration and early inotropic support based on oxygen transport parameters. methods: patients undergoing elective long term vascular and abdominal surgery (asa i-bi) were studied. blood lactate levels were measured after icu admission. in the case of blood lactate level above mmoltl, measurement was repeated every hours for hours or until normaiisation (blood lactate level less than mmol/ ). type of surgery, length of surgery, amount of fluids delivered intraoperatively and postoperatively, hemoglobin levels, hemodynamic variables, diuresis, postoperative complications, length of icu stay and clinical outcome were recorded. because no attempts were made to randomisr therapy or change our standard therapy protocol institutional approval was not required. rebuts: the frequency of postoperative complications was , % and mortafity was , % in a group of patients with blood lactate level less than , mmol/l (n = ). frequency of complications ( , %) was significantly increased in a group of patients with blood lactate levels , - mmol/l (n = ), mortality was , %. mortality ( %) and frequency of complications ( %) were significantly increased in a group of patients with blood lactate levels above mmol/l (n = ). conclusion: blood lactate levels can serve as early marker of high risk postoperalivr patients and may predict increased risk of postoperative complications mad ~e death. objective.~: investigated practicability and clinical value of the routine measurement of hepatic venous oxygen saturation (shvo ) after major liver surgery, as shvo is considered an indirect parameter for splanchthc and hepatic blood flow. methods: consecutive patients were included in this study after liver resections for primary or secondary liver tumors. patients suffered from liver cirrhosis (childs a). immediately after post-operative admission on the icu a pa-catheter ,was inserted under fluoroscopy via the right jugular internal vein into the hepatic vein contralateral to the resection area. hepatic venous and arterial blood samples were drawn every two hours. shvo was correlated to the clinical course, macro hemedynamics, abgs aug other established lab parameters. results: in out of attempts the catheter could be placed correctly. in four cases after right hemihepatectomy the left hepatic vein could not be intubated due to a dorso-lateral tilting of the left liver. this is also reflected in a significantly longer time of fluoroscopy for catheterization of the left hepatic vein ( . _+ % rain vs. . + . rain; p < . ). the procedure requires a total of between and minutes. relevant clinical complications were not observed except for short term supraventricular arrhythmias during passage of the catheter through the right atrium. hemodynamics and pulmonary function could be considered normal in all individuals at time of measurement. shvo showed a span from . % to . % with a mean of . % -+ . %. the following statistically significant findings could be obtained: (a) patients with liver cirrhosis showed a significantly lower shvq than patients without ( . % • . % vs. . % • . %; p < . ). (b) a negative correlation between shvo immediately after operation and the duration of intraoperative hepatic vascular occlusion could be observed (r = - . ; p < . ). this correlation could also be seen for the first post-operative hours (r = - . ; p < . ). (c) a negative correlation between shvo and the difference between arterial and hepatic venous lactate levels was found (r = - . ; p < . ). conclusions: the routine measurement of shvo appears to be a promising extension of post-operative monitoring after major liver surgery. it is a safe method easily feasible on any major surgical icu though relatively time consuming. a further validation of this method is necessary in larger studies. therapeutic recommendations on the basis of shvo findings cannot be given yet. methods: in cases after major liver resection, in which abnormally low readings of shvo suggested an impaired hepatic blood flow, pgi was applied at a dose rate of ng/kg/min. as shvo can be considered an indirect parameter for hepatic blood flow, the effect of pgi infusion on shvo was measured. moreover, the changes of macro hemodynamics and pulmonary function were monitored. results: before the application of pgi z mean shvo for all patients .was . % ( - - - ). in three cases without major structural alteration of the remaining liver tissue the continuous intravenous administration of pgi lead to a sustained increase of shvo z to an average of . % ( . - , ). the postoperative course in these three cases was uneventful. in two cases with compensated liver cirrhosis after hepatitis c no change in shvoz under pgi infusion could be observed. both patients died and days respectively after operation in protracted liver failure. side effects of pgi included a slight decrease of systemic and pulmonary vascular resistances. consequently map decreased by up to % as did intrapuimonary right-left shunt increase. in none of the observed patients did these side effects posed a limitation of continuous application of pgi z. conclusions: in patients without structural alteration of the liver the systemic application of prostacyclin at a dose rate of ng/kg/min could significantly increase an abnormally low hepatic venous oxygen saturation after major liver resections, tn two cases of severe liver cirrhosis a similar increase could not be observed. after first clinical investigations and with the results of recent studies in animal further controlled clinical studies of prostacyclin in the postoperative management after liver surgery appear justified. any delay in gastric emptying can promote micro-aspiration and give rise to ventilator associated nosoarnnial pneumonia. h -receptor antagonists have been suspected of promoting pneumonia by changing the gastric ph. in a few tri',ds on humans ranitidine was noted to delay gastric emptying. the aim of this prospective, randomised, blinded study was to evaluate in a ventilated icu population if there was a difference between cimetidine (c) and ranitidine (r) on the gastric filling index (gfi conclusion: in this population there was no difference in gfi between c and r; however the age and creatinine were significantly different and could have favoured the c group. also the very long t/ could have hidden smaller differences between c and r as has been described in volunteers. between april , and april , , patients with severe acute pancreatitis were admitted to participating hospitals. patients were entered into the study if severe acute pancreatitis was indicated, on admission, by multiple laboratory criteria (imrie score >_ ) and/or computed tomography criteria (balthazar grade d or e). patients were randomly assigned to receive standard treatment (control group) or standard treatment plus selective decontamination (norfloxacin, colistin, amphotericin; selective decontamination group). all patients received furl supportive treatment, and surveillance cultures were taken in both groups. results: fifty patients were assigned to the selective decontamination group and were assigned to the control group. there were deaths in the control group ( %), compared with deaths ( %) in the selective decontamination group. (adjusted for imrie score and balthazar grade: p = . ). this difference was mainly caused by a reduction of late mortality (> weeks) due to significant reduction of gram-negative panreatic infection (p = . ). the average number of laparotomies per patient was reduced in patients treated with selective decontamination (p < . ). failure of selective decontamination to prevent secondary gram-negative pancreatic infection with subsequent death was seen in only three patients ( %) and transient gramnegative pancreatic infection was seen in one ( %). in both groups of patients, all gram-negative aerobic pancreatic infection was preceded by colonization of the digestive tract by the same bacteria. reduction of gram-negative colonization of the digestive tract, preventing subsequent pancreatic infection by means of selective decontamination, significantly reduces morbidity and mortality in patients with severe acute necrotizing pancreatitis. ieco by sodium hypochlorite (nacio) infusion is considered to be a model of microsomal oxidation in liver on cytochrome p- . active c provides oxidation of toxic metabolic products in the blood and exfused during plasmapheresis plasma, and also hydrophobic to hydrofilic transformation of substanses. sterile nacio in necessery concentrations was obtained by electrolysis of saline ( , - , % naci solution) in electrochemical set e~io- (russin,moscow). methods: . the nacio in concentration ragfl ( - ml/ h ) was administred into central veins in patients with extensive peritonitis and endotoxicosis - /t. erytrocytes resistance to nacio, circulating blood volume glycemia and hemostasis were initially estimated. . after plasmapheresis exfused toxic plasma was mixed with nacio conccantration of i mg/t in : ratio in sterile "hemacons".the effectiveness of plasma detoxication and possibility of its reinfusion were evaluated by determination of albumin effective concentration (eca g/l), the concanlration of medium molecular oligopeptides (mm , ) and other biochemical tests (bilimbin, creatinine, carbomide and so on). results: . the intravenous administration of nac excels detoxicative effect of hemosortion by - % provides effictive presentation of protein components and blood cells and improves the transport function of albumin by %. . the return of exfused plasma after its purification ieco was - %. only the remaning - % of deficient plasma were compensated by fresh cryoplasma and albumin solutions. ischemic hepatitis (ih) is a severe complication in critically ill patients. acute circulatory failure of multiple etiology can lead to splachnic hypoperfusion and cause acute and reversible anoxic damage. over a period of mos pts, m and f, mean age + . yrs developed liver disease compatible with ih. eight pts had a documented hypotensive episode (six pts with septic shock and two hypovolemic shock), while cardiogenic pulmonary edema in the absence of hypotension was responsible for ih in the remaining four pts. all the pts had a rapid striking elevation of ast, &lt and ldh with equally rapid resolution of these parameters to near normal wimin days (mean . ). the mean peak level of ast, alt and ldh was iu/l (range to ), iu/l (range to ) and iu/l (range to ) respectively. serum total bilirubin levels rose transiently with a moan t:eak level of . mg/dl (range . to . ), while altered coagulation paran-,ete's (pt> . times normal) was observed in four pts and clinically significant coagulopathy with fibrin degradation products occurred in one pt ( . %). renal impairment (cr> . mg/dl) was manifest in all pts; six pts developed non-oliguric renal failure ( %) while two pts required hemodialysis. ten lots required vasoconstrictor inotropes [dobutamine (range - pg/kg/min) and dopamine (range - pg/kg/min), while replacement of circulatory blood volume was performed in two pts with hypovolemic shock. eight lots expired ( . %), but none died as a direct result of hepatic damage. the mortality rate was higher among pts with concurrent renal failure ( %). it is concluded that: ) ih is not uncommon complication in the icu with the prognosis depending on the underlying disease. ) clinically significant coagulopathy is uncommon complication of ih. ) titration of inotropes is required to obtain optimal cardiac output support and subsequently liver blood flow. it is difficult to ascertain the perfusion of free flaps such as jejunal loops after surgery. objectives: to assess ischaemia as evidenced by intramural ph of jejunal free flaps used for reconstructive surgery following total pharyngolaryngectomy. methods: the sigmoid ph tonometer ( tonometrics inc.,usa ) was used to monitor intramural ph of the jejunal free microvascular flaps ( phig ) in patients who underwent total pharyngolaryngectomy. a standard general anaesthetic was given and all patients were admitted to the icu for controlled ventilation and monitoring. all had similar postoperative care. phig was measured pre, post-revascularization of the flap and on icu admission, , and hours postrevascularization. objectives: to classificate the wide spectrum of itc of anp into distinct pathophysiological patterns according to presentation and course. patients (pts) and methods: pts, ~( , %), ( , %) were admitted in the icu because of anp and acute respiratory failure(arf), ilean age: , • years. hean stay in icu: , • days. pts were operated, of them twice. hean value of ranson's scale: , • ( - ). we analyzed hemodynamic measurements,arterial blood gases(abg), x-ray findings(xrf), ct-scans and operative records. results: patterns of pleuropulmonary complications were identified: a)early hypoxia without xrf - pts. b)early ards with typical xrf - pts( died), c)early arf with xrf(atelectasis,infiltrates)- pts( died). d)late ards with typical xrf- pts( died), e)pleural effusions in various combinations with the above patterns - pts. overall mortality rate: / = , %. conclusions: l)frequent x-rays and abg are important for the classification of itc of anp. )even though patterns of classification in anp are not clearly distinguishable,they facilitate an anticipatory management. )deterioration of abg and xrf indicates that preventive measures for arf must be intensified and agressive surgical therapy is required. )delay of surgical therapy is related to worse prognosis(p<o, ) and prolonged hospitalisation time(p<o,ol). in the contrary, the early timing of the operation before infection and extrapancreatic necrosis is essential for a better prognosis (p<o, ). objectives: the development of cholestasis during intensive care treatment is allied with an inferior prognosis. this study investigates the sensivity, specification and also the positive and negative forecast of patients survival -exemplary for bilirubine. methods: during a period of months all surgical patients on icu were registrated prospectively (n = ). for documentation of disease development a standard foldcrform based on a computer-data-system was used. the results of this kind of documentation showed aapprax. , m!ll. of single patient informatioas. results: of patients didn't suffer from liver disease or have any operation on liver or bile tract. of them have had a normal scale of bilimbine in the postoperative period, a higher scale of bilirabine was regisu'atcd by patients. at a ,,cut-off" of rag% (total-bilirubine) the sensitivity was , , specivity , , positive predicive level , , negative predictive level , for survival criteria of a patient. the max. reached level of bilirubine, also the daily increase turned out almost the same results as they were found for other parameters nf cholcstasis like ap or gamma-gt. objective: the aim of this experimental protocol was to evaluate the morphological and functional parameters of isolated pig liver grafts, perfused in an extracorporeal liver support system. methods: the system consists of the graft liver, a membrane oxygenator (oxygenation surface . cm z, flow= itlmin), a heater, a centrifuge pump and a fluid reservoir. twenty pig livers, mean weight gr (min , max gr) were obtained and after a mean cold ischemia time of min were perfused fo} a mean of . h (min . , max h). perfusion solution consisted of r/l and hemacell. inflow to the graft liver was performed through the portal vein at a mean pressure of ( - ) mmhg at ~ while outflow was secured through the suprahepatic inferior vena cava. during perfusion the following parameters were evaluated through pre-and posthepatic hourly (to-t ) sampling: po , " + + " pco , ph, hco -, na , k , ca , osmolality, glucose, lactate, ast, alt, alp, u bilirubin and coagulation factors i, v and viii. biopsies were obtained periodicallty. b_p_~: results were as follows: mean ph fell from . at t o to . at t while mean output po fell from at t o to at t . mean output ast values increased from at t o to > at t while mean output alp values increased from . at t o to at t . mean output k + values increased from . at t o to > at t . histology revealed lesions of ischemic necrosis, more prominent after t . conclusion: results show that the isolated liver graft presents satisfactory function and morphology at least for a five hour perfusion period in the described extracorporeal circuit. correction of ph contributed to an increase in bile flow. between and the practice of transplantation has changed drasticaily in switzerland -besides kidneys also hearts, heart and lung, lung, iiver and pancreas transplantation has started in several centers. major information efforts have been made, organ exchange rules were set up and a national coordination center was initiated. the aim of this retrospective single center study was to assess the influence of transplantation on organ donation. in the past eleven years organs were donated from potential donors i single, multi organ donations) analysis of refusal was evaluated categorized into medical and/or familiar reasons. the number of potential donors increased from ( ) ,to ( ) with a concomitant drastic reduction of donations from % in to % in ; amounting to a net unchanged number of donations over the last years ( = ; = ) . the import and export of donor organs was balanced since the introduction of the national coordination center. in contrast multi organ donation increased from % in to % in despite of the more stringeant selection criteria, in conc]usion the introduction of a full range of transplantation procedures at several new university programs and the increase of multi organ donation has not had the forecasted impact on organ donation despite a sustained informative and promotional campaign, objective: monitoring hepatic venous oxygen saturation (svho ) provides online information about hepatic-splanchnic oxygen supply-demand ratio [ ]. previously, x~ reported hepatic venous catheterization in patients undergoing orthotopic liver traru~lantation (olt) [ ] . in the present study, we assessed the effects of nitroglycerin (ng), a vasudilator that affects the venous capacitance vessels more than arterial vessels and prostaeyclin (pgi , flolan r~, wellcome, uk), an arterial and splanchnic vasodilator on hemodynamies and hepatic venous oxygen saturation (svho ) in human liver transplantation. methods: with institutional approval and informed consent, consecutive patients, mean age - -_ years, were studied following olt. postoperatively, fiberoptic pulmonary artery catheter was inserted into the right hepatic vein. timed infusions of ng at a rate of . gg/kg/min and pgi at ng/kg/min were initiated for a rain period. each sequence was followed by baseline therapy for rain. results are expressed as mean=tsd. statistical analysis was performed using friedman's-two-way-anova-test, significance was accepted at p< , . results: ng at . gg/kg/min induced a decrease of mean arterial pressure (map) ( _ [baseline] vs. + mmhg) and pulmonary artery wedge pressure (pcwp) ( j: [baseline] vs. : mmhg). cardiac index (ci) ( - vs. + l/rain/m ), oxygen delivery index (do i) ( -+ vs. + mgnfin) and svho ( _~ vs. -l-_ %) were decreased (p< . ). pgi at ng/kg/min induced a reduction in map ( • nm~. _g) and pcwp ( + mmhg). ci ( _+ l/rain/m ), do i ( : ml/min) and svhoz ( + %) were increased (!o< . ). vasedilatation induced by ng decreased systemic oxygen supply and impaired splanclmie oxygenation. pgi increased systemic oxygen delivery in parallel with svho , suggesting a corresponding improvement of hepatic-splanchnic okygenation. thus, if vasedilator therapy is indicated in th orient receiving liver grafting, pgi appears to be advantageous. however, due to its platelct aggregation inhibiting properties, the usefulness and safety of pgi in olt patients has still to be determined. objectives: to analyze the effect of steroid treatment given to donor on the early function of transplanted kidney. methods: from january, until now donors were involved into this prospective study. every other donor was treated with mg/kg solu-medrol one hour before organ retrieval. according to the steroid treatment of the donor the recipients were divided into two groups: group -steroid pretreatment goup (y~= ), and group -control group (n= ). the donors and the recipients were treated using the same kidney transplantation protocol onl~r the adults, and the first cadaver kidney transplanted patients were involved into the study. the daily routine parameters were analyzed pre-and intraoperafive, and on the - th, th and th postoperative days. results: we could not show any clinically important differences between the two groups in respect of donor parameters. preoperative, the patients in group had slightly lower ereatinin level ( -+ g.,non vs. -+ gmol/ ) which persisted into the early postoperative phase. the values of the other examined pre-and intmoperativc parameters were almost the same. during the first postoperative days the patients in group i needed less diuretics (furosemide and renal dose of dopamine) and their sodium excretion was closer to the physiological range than in group . the other parameters did not differ significantly. the less furosemide need in group ! pe~isted to the end of the first month. conclusions: according to our data the steroid treatment of the donors improves the early function of the transplanted kidney in some respects. to prove the real benefit of the donor steroid treatment needs more data and further analysis. objectives: severe infections may compromize the outcome of liver transplantation..determination of new parameters may increase the knowledge of pathophysiologic mechanisms and may lead to changes in postoperative therapeutic management of patients at risk. methods: between august and september , patients with transplants were monitored for cytokines and extracellular matrix pammeters on a daily basis. serious infections (n= ) included microbiologic evidence and more than secondary organ failures. patients with cholangitis (n=ll) or uneventful postoperative course (n= ) referred as control groups. results: -year patient survival was . % ( / ): patients died due to serious infections, while died for other reasons. mean bilimbin, stnf-rii-, ifn- -, il- -, il- -, il- -, laminin-and neopterin levels were significantly elevated in patients with serious infections compared with patients experiencing mild cholangitis or with an uneventful postoperative course. a further increase of all parameters was observed in patients who subsequently died; tnf-ri/: _+ pg/ml vs • pg/ml; ifn- : _+ pg/ml vs . -+ . pg/ml; il- : -+ pg/ml vs -+ pg/ml; il- : -+ pg/ml vs _+ pg/ml; il- : _+ pg/ml vs • pg/ml; laminin: -+ ng/ml vs -+ ng/ml; neopterin: _+ nmol/ vs _+ nmolb for non surviving vs-surviving patients. a significant decrease of sialic acid yeas observed in patients with serious infections; and a further decrease occurred in patients who subsequently died: -+ mg/l vs • mg/ . conclusions: the increase or decrease of various cytokines and extracellular matrix parameters may be indicative for severity of infectiolx routine monitoring of these parameters may improve current diagnostic tools and poss~ly lead to changes in therapeutic management of patients at ~k. objectives: evaluation of the cytokine network after liver transplantation may give some insight in pathophysiologic mechanisms of rejection and may lead to detection of patients at high risk. methods: patients with transplants were monitored for various cytokines on a daily basis between august and september . rejection was assessed by histology in combination with clinical signs of rejection and laboratory investigations. results: during the first postoperative month, patients ( . %) developed rejection; patients were successfully treated with methylprednisolone (steroid-sensible rejection), while further patients required additional treatment with fk or okt (steroid-resistant rejection). patients subsequently developed chronic rejection. mean levels of various cytokines and extracellular matrix parameters including tnf-rii, ifn- , il-ib, il- r, il- , il- , il- , hyaluronic acid and neopterin were significantly higher in patients with steroid-resistant than in patients with steroid-sensible rejection. a further increase of some parameters was observed in patients who subsequently developed chronic rejection; bilirubin: . -+ . mg/dl vs . -+ . rag/all; tnf-rii: -+ pg/ml vs _+ pg/ml; il- : +- pg/ml vs -+ pg/ml; neopterin _+ nmol/ vs -+ nmol/ ; hyaluronic acid: _+ ~tg/l vs _+ ~tg/l for patients with chronic versus patients with acute steroid-resistant ~ejection. sialic acid levels decreased in patients with acute steroidresistant rejection; and a further decrease was observed in patients who tieveloped chronic rejection: _+ mg/l vs _+ mg/ . ~onclusions: various cytokines and extraeeuular matrix parameters were indicative of severity of rejction. the extensive increase of bilirubin, tnf-ii, il- , hyaluronic acid and neopterin may indicate subsequent chronic ection. monitoring of these parameters may, therefore, lead to changes in immunologic management after liver transplantation. background : combined kidney and pancreatic transplantation is being performed with increasing frequency in patients with diabetes mellitus and renal failure, as it offers more chances of success and better results than kidney transplantation alone. mycotic arterial aneurysm constitutes a devastating complication following pancreatic transplantation. all cases of mycotic arterial aneurysms have been however reported with exocrine pancreatic drainage into the gastrointestinal tract. intervention : we describe a series of consecutive whole kidney-pancreas transplantation performed at the university of geneva hospitals ( beds) between december and may . exocrine pancreatic drainage into the bladder (epdb) was performed to improve early detection of rejection episodes. epdb was hypothesized to reduce the risk of contamination from the gastrointestinal tract and the subsequent possible occurrence of potentially fatal infectious complication. in all patients the dual transplantation was performed through a median incision according to the procedure described by nghiem. results : two out of the patients who received kidney-pancreatic transplant developed arterial mycotic aneurysms and days following surgery. aneurysms developed at the site of the arterial anastomosis used to rearterialize the homograft. both patients had peritonitis caused by candida albicans requiring surgical drainage and intravenous antifungal therapy. rupture with hemorragic shock occured in both patients leading to graft removal in one patient, and three episodes of lffetreateniug hemorragic shock followed by graft failure and removal days after transplantation in the other. conclusion : arterial mycotic aneurysm constitutes an early, lifetreatening complication of kidney-pancreatic transplantation; it mandates graft removal. although exocrine pancreatic drainage into the bladder consitutes a definitive advantage for caller diagnosis of graft rejection, it does not eliminate the risk for retrograde colonization and subsequent severe infection in our experience. s. bocharov, i. teterina, regional clinical hospital, irkutsk, russia acute profound loss of blood can result from the very different injuries and hepato-pancreato-duodenai operations enter such a rank. ill-timed and inadeguate correction of operation hemorrage is one of the reasons for postoperation complications, including polyorganic insufficiency. the pathogenesis seems to be very complex. in early stages of bleeding the liquid enters the vessel bed, followed by hypoproteinosis and hematocrit fall. however, as decompensation develops, the fluid leaves the vessel system in the result of increasing postcapillary resistance and lowering col-ioidnooncotic blood pressure (cop). the resulting hypovolemia causes primarily acute disturbance of central hemodynamics and then of microcirculations and transcapillary exchange. central hemodynamic failure after acute loss of blood manifests itself through cardiac output lowering and capillary blood flow deceleration. taking into consideration, that % is critical value for cpv loss and for cev it is %, we consider arising the level of cop to the immediate task. cop raising allows to normalize transcapillary exchange, which we assess through cop and mcp (mean capilary pressure) gradient. the next task is to make up for globular volume till homeostasis providing level. considerable attention is given to catabolism inhibition and maximum possible enegry provision. control over high proteolitic activity of blood and callicreinkinin system activity implies direct proteases inhibitors. reologic, membrane stabilizing, antihypoxanthine and anticoagulant therapies are obligatory. virehow clinic, dept. of surgery, humboldt university berlin, germany regarding a high mortality up to % of fulminant hepatic failure orthotopic liver transplantation seems to be the only promising therapeutic approach in many cases. this study shows experiences from a transplantation center. between june and april patients suffering fulminant hepatic failure were admitted to our surgical intensive care unit all patients showed severe liver dysfunction with grade ii to iv encephalopathy. after a period of diagnostics and conservative treatment ranging from few hours to days (mean . days) we reported of these patients as possible organ recipients to eurotransplant. all of these patients were transplanted within hours, ( %) of them even within hours. the principal aetiologies were hepatitis b ( ), hepatitis c ( ), nanb hepatitis ( ), mushroom poisoning (amanita phalloides ). after transplantation patients suffered from initial-non-function and underwent re-transplantation. the one-year-survival rate was %, patients died within months after transplantation due to various reasons. patients were not referred for liver transplantation. of them never met transplantation criteria, improved by conventional therapy and could finally be discharged from hospital. the known reasons for liver failure in this group were mushroom poisoning ( ), paracetamol intoxication ( ) and fulminant hepatitis a ( ). patients suffering from fulminant hepatitis ( ) or intoxication ( ) were excluded from emergency liver transplantation for various contraindications. of these patients ( %) died despite conventional intensive care. we don't know if some of the patients in the transplantation group would have survived without transplantation, because whenever we decided on transplantation we could perform the operation within hours. but the good survival rate in the transplantation group ( %) the % recovery rate in the group, where there was no transplant-indication in our opinion and the fatal outcome ( % mortality) in patients with contraindications are an encouraging proof of a successful therapeutic strategy in acute liver failure. these results are based on a close cooperation between experienced transplant surgeons, hepatologists and intensive care doctors, using sophisticated laboratory and imaging techniques in a specialized center. introduction: during brain death patients suffer from multiple endocrinologic disturbances. one of the most important are those related with thyroidal axis. it is well described the euthyroid sick syndrome whose more frequent pattern consist of decreased triiodothyronine (t ), increased reverse t (rt ) with normal levels of tetraiodothyronine ( " ) and tsh, this lacking in " " levels lead to a change from aerobic to anaerobic metabolism which results in tissular damage. objective: .to study thyroidal pattern in brain death patients potential organ donors. .to avoid organ impairment by administration of t . .to study the hemodynamic and hormonal changes after the administration of t in these patients. material and methods:population: brain death patients of any etiology potential organ donors admitted to the intensive care unit. patients were classified in hemodynamically stable (group ) and unstable (group ). group received a bolus of . p.gr/kg. and a perfusion at a dose of - . p.gr]h of t . hormonal assays: total t (tt ), total " (tt ), tsh. fxee t (ft ), free " (ft ) and rt were determine at the moment of clinical brain death ( hrs) and in group two these assays were repeted at hours , and . results: patients ( male) with a mean age of years (range to yrs.) were studied. the clinical brain death was confirm later with other explorations (eeg, doppler). there were patients in group ( , %) and patients in group ( , %). hormonal pattern: at the moment of brain death tt was normal in cases ( , %) and decreased in i ( , %); tt was normal in patients ( , %) and decreased in ( , %); ft was normal in cases (i , %), decreased in ( , %); fl' was normal in patients ( , %) , decreased in ( , %) .rt was normal in cases ( , %) and increased in cases ( , %). there were no statistically significant differences in hormonal pattern between the two groups. only t levels at hours , and were significant in group . in the cases with ft decreased, the tt was normal in ( %) and decreased in ( %), tt was decreased in ( , %) and normal in ( , %), tsh was decreased in i ( , %), normal in ( , %) and increased in i( , %) and ft decreased in ( , %) and normal in ( , %) and rt was normal in ( , %) and increased in ( , %). there were no statistically significant differences in cardiac index, vascular resistances and pulmonary shunt before and after the administration ef t . conclusions: . the hormonal pattern most often find in brain death patients was: normal tt , decreased tt , normal tsh, decreased ft , normal fr and normal rt . . there were discrepancies in the values of ft and tt . there were no statistically significant differences in hemodynamic and pulmonary parameters. objectives: magnetic resonance angiographie (mra), a non-invasive procedure, provides flow-related information additionly to the anatomy of the vascular system. measurement of signal intensity and edge detection of vessel structures permits to calculate blood flow velocity and vascular diameters. we examined whether cerebral hemodynamic changes by altering the arterial pressure of carbon dioxid (pace ) could be detected by mra. methods: following institutional approval and informed consent, mechanically ventilated patients without elevated intracraltial pressure underwent mra with defined periods of hyper-, hypo-and normoventilation (pace : , , mmhg; arterial blood gas probes; avl). mra was performed with a . tesla magnetom (vision, siemens). two different mra techniques were used: a conventional time-of-flight- d-angiography (tr: ms; te: ms; fl: deg; slab: mm) for vessel diameter detection and a flash- d-gradient-echo-sequence (tr: ms; te: ms; fl: dog) for measurements of blood flow velocity. an axial view parallel to the ac-pc-iine (anteriorposterior-commissur-line) was used for repeated imaging of identical regions of interest toi) of the proximal part of the internal carotid (ica) and middle cerebral artery (mca) as well as of peripheral branches of the mca and the posterior cerebral artery (pca). results: changes of pace correlated with changing signal intensities, whereby under hyperventilation a decrease of , % (p . ) and under hypoventilation an increase of . % (p . ) was observed compared with normoventilation. blood pressures were stable throughout the whole study period, pace dependent changes in vessel diameters were more pronounced in peripheral branches of mca and pca. a change from normo-to hyperventilation produced a decrease in proximal vessel diameter of - . % (p _< . ) and in peripheral diameter of - . % (p _< , ). a change from normo-to hypoventilation produced an increase in proximal diameter of + . % (p < . ) and of + . % (p -< . ) in peripheral diameter. conclusions: pace related changes of cerebral vessel diameter can be easily detected by mra without injecting a contrast agent. the results confirm that co -reactivity is more pronounced in peripheral cerebral vessels, which are subjected to greater changes in diameter than major basal arteries. hyperventilation leads to a decrease and hypoventilation to an increase in signal intensity thus reflecting the corresponding changes in blood flow velocity, intensive care unit (icu) of "kat" hospital, athens, greece, ob!ective$; the value of bronchoscopy in pulmonary atelectasis of icu patients is under question the presence of an air bronchogram sign in xrays, which is considered as evidence of central bronchus patency, is referred in several studies as a negative criterion for bronchoscopy, whereas its absence as a positive one. it is also referred that air bronchogram sign correlates with delayed resolution of atelectasis, probably because of obstruction of many periferal airways (not central). the purpose of this prospective study was the evaluation of the air bronchogram sign on frontal chest film as a negative criterion for bronchoscopy and as criterion of delayed resolution of atetectasis, methods: icu patients with atelectasis were studied prospectively. they underwent bronchoscopy, bronchoscopic findings, presense of air bronchogram sign, and outcome of atelectasis were recorded, correlations were made, between: ) bronchoscopic potency of airways and air bronchogram sign } resolution time of atelectasis and broncoscopic potency of airways. ) resolution time'of atelectasis and air bronchogram sign, methods of statistical analysis were the t-student test and the chi square test, results:the patients were , men women , seventeen patients had atelectasis of whole lung, of upper lobe, and of lower lobe. ten patients had atelectasis in right and in left lung. eight from patients had air bronchogram sign in x-ray, there was no statistical correlation between air bronchogram sign and bronchoscopic potency of airways [ from patients with air bronchogram sign ( %) and from without air bronchogram sign ( %), had bronchoscopic potency of airways, p> . ], resolution time of atelectasis didn't correlate statistically with bronchoscopic potency of airways (mean resolution time in patients with bronchoscopic potency , days and in bronchoscopically closed bronchi , days, p> , ). there was also not a statistical correlation between resolution time of atelectasis and air bronchogram sign (mean resolution time in patients with air bronchogram sign , days, and without air bronchogram sign , days. p> ). conclusion~i; the presense of an air bronchogram sign in x-ray of icu patients with atelectasis, does not coexist obligatorily with bronchoscopic patency of airways and cannot be used as a negative criterion for bronchoscopy, neither as a criterion of delayed resolution of atelectasis. th. wertgen chest sonography (cs) is routinely used in our department to examine icu patients with clinical symptoms of pulmonary embolism, pneumonia, pleural effusion or unclear chest pain. we perform cs with a sector transducer ( . mhz) and a linear transducer ( . mhz) using acuson xp/ c. the sonographic signs of pulmonary embolism and infarction are most well demarcated, mainly wedge shaped and triangular pleural based lesions, more roughly structured, observed with a hyperechoic reflex in the center corresponding to the bronchitic (fig. ) . pneumonia is characterized by homogenously hypoechoic, wedge shaped parenchymal lesions, containing air or fluid bronchograms; they move with respiration (fig. ) . pleural effusions are spaces of various echogenicities, from anechoic to homogeneously echogenic, which may contain floating strands or complex septa, located between visceral and parietal pleuras (fig. ) . from march to april we did examinations by cs in icu patients ( male, female; age from - ). patients examinations pulmonary embolism pneumonia pleural effusion us-guided thoracic punctions were performed in patients. in two patients we found pneumonia or pleural effusion caused by a lung carcinoma. another two patients showed a normal cs (diagnosis: inflammation of the gall bladder, inflammation of the myocardium). conclusion: cs is a very useful method for icu patients with chest diseases. it takes less time and is less expensive than ctand sometimes of a higher diagnostic value than x-ray. last but not least cs is invaluable for the icu patient, because the examination is done save and quickly at bed side and the results of cs are very helpful in diagnoses and treatment. results : inter-observer reliability was evaluated as an % concordance. results of the tee classification were : class : n = ( %) ; class : n = ( %) ; class : n = ( %) ; class : n = ( %) class : n = ( %). therapeutic implications of tee in class patients were : cardiac surgery in patients (two cases of acute mitral regurgitation, two valvular abscesses and one hematoma compressing the left atrium), discontinuation of peep in one ventilated patient with an atrial septal defect, weaning of mechanical ventilation in one patient with an atrial septal defect, prescription of antimicrobial therapy in patients with endocarditis and prescription of anticoagulant therapy in patients with left atrial thrombus. the only noteworthy complication was a case of spontaneously resolving supraventrieular tachycardia. conclusion : tee is safe and well tolerated, and is useful in the management of icu patients with shock, unexplained and severe hypoxemia or suspected endecarditis. the aim of this study was to determine whether ultrasound guidance can help interns to improve the results of jugular vein access in icu. methods : in a prospective and randomized study, we compared, in patients admitted to the icu, an ultrasound-guided method (ultrasound group : patients) with an external landmark guided technique (control group : patients). all jugular vein accesses were performed by young interns with an experience of < procedures. results : internal jugular cannulatian vein was aci~ieved in all patients in the ultrasound group and in patients ( p.cent) in the control group (p < . ). average access time was longer in the control group ( • sec. vs • see. ; p = . ) and puncture of the carotid artery occurred in patients in each group (p = . ). patients ( p.cent) in the ultrasound group and patients ( p.cent) ia the control group (p < . ) were cannulated in rain. or less. the cannula was therefore unabie to be inserted within minutes in patients in the control group, with failure of eannulation in of these patients ( p.cent). failure was due to thrombosis (n = ), small calibre of the internal jugular vein (< ram) (n = ), abnormal vascular relations (n = ) or cervical irridation (n = ). among the primary failures of cannulation, an internal jugular vein catheter was able to be inserted in cases by an experienced physician on the side initially selected and with ultrasound guidance in cases. the catheter was inserted into the contralateral internal jugular vein under ultrasound guidance in the remaining cases. jugular cannulation was obtained at the first attempt in p.cent in the control group and p.cent in the ultrasound group. conclusion : ultrasound guidance improved the success rate of jugular vein cannulation by inexperienced operators in icu patients. when the internal jugular vein has not been successfully eannulated within minutes by the external landmark guided technique, the authors recommend the use of the ultrasound guidance. in the majority of cases right atrial or ventricular thrombi represent pulmonary emboli in transit. these may be fatal in patients (pts) treated conservatively with anticoagulation only. in literature the incidence of right heart thrombi in pts with proven pulmonary embolism (pe) is said to be in the range of - %. extremely mobile, long, worm-shaped masses in the right heart cavities carry an especially high early thrombus-related mortality rate which ranges from - %. current therapeutic strategies favour fibrinolytic therapy with consecutive anticoagulation. we report five cases ( male, i female, - years) of right heart and pulmonary thromboembolism. in these pts diagnosis and regression of thromboemboli following systemic intravenous lysis therapy with recombinant tissue-type plasminogen activator (rt-pa) was documented by transesophageal echocardiography (tee). a submassive pe occured in pts, a massive pe in pts. one patient (pt) had a cardiac arrest. in all cases tee clearly identified the extensive thrombns formation in the right-sided cavities of the heart and in the central pulmonary artery in cases. all pts were treated with mg rt-pa, pts in a front-loaded regimen over minutes, pt over minutes, and, due to the life threatening situation, in one case a bolus injection as ultima ratio was performed with no intracerebral bleeding complication. regression of thromboembolic masses after fibrinolytic therapy was demonstrated by transthoracic and transesophageal echocardingraphy after to hours. all pts survived and were put on coumadine, pt developed an intracerebral bleeding with persistent hemiplegia. conclusions: the use of thrombolytic therapy is highly efficacious for the therapy of pts with pe and concomitant right or ventricular thrombus formation. transthoracic and especially transesophageal echocardiography are powerful bed-side diagnostic tools for the immediate diagnosis and follow-up of successful treatment in this life-threatening condition. although widely used, catheterisation of the femoral vein in the groin using "landmark" technique is frequently complicated by accidental arterial puncture. suboptimal hygiene and patient discomfort are also associated with this technique. with regard to these last two factors cannulation of the femoral vein - cm below the inguinal ligament would seem an attractive alternative. as "landmark" technique is not possible for the cannulation of the femoral vein in this part of the thigh, ultrasound was used to locate the vessel and the results of this technique were evaluated. methods: a portable compact ultrasound device (site rite,dymax corp.) featuring a . mhz transducer (ultrasound depth - cm) fitted with a needle guide and a cm screen was used by residents with no previous experience in ultrasound guided cannulation. patients consisted of a surgical icu population. results: in patients catheters were introduced.in cases more than one ( - ) attempt was made and in patients the procedure was unsuccesfull due to the fact that the vessel was situated out of reach of the ultrasound (vessel depth > - cm), during the procedures one accidental arterial punction was registered. the catheters remained in situ for a mean of days (range - ) and were used for volume suppletion, medication, parenteral nutrition and haemodialysis.co-ionisation rates compared to those of subclavian catheters in our icu. in the first patients cases of asymptomatic thrombosis of the femoral vein were seer on ct-scans performed for other indications, in the following patients duplex scanning performed after removal of the catheter yielded another cases of asymptomatic femoral vein thrombosis. conclusions: ultrasound guided femoral vein catheterisation - cm below the inguinal ligament is a safe and simple technique that can easily be performed by residents without prior experience. the incidence and impact of thrombo-embolic complications associated with this technique are still subject to further investigation. objectives: to estimate the cost of antibiotherapy (ab-cost) in a multidisciplinary -bed greek icu and to correlate ab-cost with total cost of drugs and consumables and with patient's outcome, severity of illness and type of admission. methods: prospective data from consecutive patients admitted to the icu from / / to / / were studied. a tick chart was designed to record all drugs, materials and consumables regularly used for icu patients, but did not include low price drugs and consumables, which are provided from hospital's pharmacy as stock and were included in a fixed icu cost calculated for a month period. the chart also contained demographic details and data necessary for the calculation of several illness severity scoring systems. obiectives: over years evaluate the necessary efforts and expenses to implement a cis in the routine of a -bed stcu. methods: in june a commercially available, unix-based cis was installed on a -bed surgical icu. the goal was a paperless documentation at the bedside. after more than years clinical experience two aspects were investigated: what effort is necessary to install and support a cis, and what is the benefit for patients and personnel on the icu? results: the installation and support of a full-fledged cis requires a considerable effort: (a) the conceptual framework for the cis has to be defined. this includes the definition of documentation standards, as well as nursing and therapeutic standards, which is the essential basis for the configuration of any cis. (b) configuring a cis, i.e. "fine-tuning" it to the user's specific needs, is always a laborious task. moreover, constant maintenance is necessary. these tasks require the following personnel: experienced health care professionals for defining the conceptual framework, - trained health care professionals for configuration, system administrator. on a single icu ( - beds) these are not considered full-time jobs. (c) training is best done employing the "train-the-trainers" approach. (d) beside the necessary amount of man power and money to install and purchase a cis, administrative and mis support is needed, especially when interfaces to the hospital and laboratory information systems have to be set up. in general, a cis needs the commitment of all people involved. without a really professional approach with a longterm goal any major cis can turn into an unnecessary but inevitable night mare. after years clinical use and a thorough implementation of a cis on a major sicu it can be said that full-fledged cis offers an opportunity to dramatically improve the working environment on an icu. moreover, it adds to patient safety, quality of care and cost efficiency in one of the most advanced and expensive areas of medicine. conclusion: a major investment in man power and money is necessary to install and maintain a full-fledged cis. a sincere professional commitment to the goals of a cis is necessary. in exchange, a well configured and well maintained cis dramatically improves the quality of therapy and care on the icu. even return of investment and financial profitability of a cis seem feasible todayl from the clinical perspective it appears that the users themselves are the central determinant whether a cis makes a dream come tree or turns into a night mare. objectives: to establish a relationship between the activities of the staff and the occurrence of auditory alarms on the i. c.u. ard to evaluate confusion between auditory alarms. methods: laboratory based studies which investigated aspects of confusion between alarms in current use on the i. c. u. the observational studies were conducted over an month period and examined the frequency and duration of alarms together with the concurrent activites being undertaken by staff on the unit. the laboratory based studies showed that there were enduring confusions between the alarms on various items of medical equipment, for example a ventilator alarm and an e. c. g. monitor alarm. the results of the observation studies demonstrated that alarms are activated when specific activities are being undertaken by staff. sounds could be used in future recommendations for alarms on medical equipment. suggestions are also discussed for improving and rationalising auditory warnings in the i. c. u. obiectives: we investigated inferior petrosal sinus (ips), the lowest affluent to jugular bulb (jb), as a possible source of contamination of samples in jb for monitoring oxyhemogiobin saturation (sjbo ). pulling back the catheter the oxyhemoglobin saturation usually rises indicating extracerebral contamination (jakobs en met al: j cereb blood flow metab ; : ). methods: the study was carried out on patients undergoing ips sampling to differentiate cushing disease from ectopic acth syndrome and to lateralize any resulting pituitary lesion. we studied the value of oxyhemogiobkn saturation high in jb (sjbo ), at ips (sipso ) and at mid jugular vein ( th cervical vertebra) (smj ) bilaterally. results: we found significant differences between right sjbo and both right sipso (p= . ) and right smjo ( p= , ) and between left sjbo and both left sipso (p= . ) and left smjo (p= . ) we did not fred any difference bilaterally. objectives: we studied various methods of receiving and editing of clinical datas in critically ill patients (different ethiology). patients were investigated in regional intensive care center. methods : the following datas were studied : anamnesis, status praesens objectivus ( organs and systems ) ,. clinical and biochemical markers of critical condition , datas of eeg ,rheography . the medical information complex contained : channel electroencephalograph, -channel roencephalograph, ad-converter ( analog inputs, bit resolution, k hz), ibm dx , software includes set of routines for spectral eeg analysis, eeg-mapping, correlative analysis, and brain bloodstream reg-monitoring (written in turbo pascal . ), expert programs for estimation objective and humoral patient status (written in clipper . ) and statistics. there were used following programme-language instruments : borland c++ . , nantucket clipper . , ca-clipper tools ii. as the methods of statistical processing of dates were used: t-students criterion , fisher criterion, methods of correlation analisis, calculation of the regression levels, dispersion analysis, results : there was created the optimal structure of hard and sofware complex of search steady objective regularity in dynamic of critically ill patients condition. conclusion : the created system allowed to value effectiveness of intensive care and give us new opportunities in study pathogenesis of systems disorders in critical condition . over a five year period a patient data management system has been installed which allows individualised patient data to be accurately collected. using this data a costing system has been developed which ascribes costs thus: . direct costs -drugs, fluids, consumables, interventions. these are ascribed to individual patients, according to data collected from the pdms. . indirect costs -energy, depreciation, admm costs, maintenance etc. these are summed for the year and ascribed as an overhead per patient day. n.b staffcusts contain art element of both cost types the aim is to make as many costs as possibie 'direct', hence 'activity costs' have been calculated winch comprise staff time, drugs and consumables -these are direct costs. these costs of patient care are then searnlessly integrated into the financial and budget management of the icu environment. it was found that by calculating costs in this manner % of the total cost of icu are captured within the 'direct' element, and so are able to be ascribed to individual patients. this is much more accurate than simply dividing the total costs of ~cu by the number of patient days. temporal costs (variations during patient stay) and cross sectional costs (cost differences between admitting specialities) were also noted with interest. results of the initial analysis of data captured by the system will be presented. little is known about the resource costs (not simply cash costs) of icu. even less is known about individual patient costs, with previous estimates of these costs varying widely. however, if cost effectiveness studies are to be undertaken accurate calculation of individual, group and total icu cost is an essential, prerequisite, which, via this system of costing, is now achievable. information about intensive care of cancer patients is limited in the literature, despite the increasing use of such facilities in oncology over the two last decades. in order to determine if and how critical care facilities can be used specifically for these patients, we performed a world-wide inquiry in anticancer centers selecting the hospitals by using the international directory of cancer institutes and organizations. we mailed a questionnaire to centers and we received responses ( . %). there was at least one uncological (i.e. with > % of cancer patients) icu in (% % an -year old woman with graves disease presents with sore throat, vomiting, diarrhea, sinus tachycardia at /minute and a temperature of ~ several weeks before, treatment with propylthiouraeil had been stopped (rash and fever) and replaced by methimazole and ledide prior to a minor surgery. however, both drugs were discontinued by the patient two weeks before admission. shortly after arrival in hospital, patient's condition progressed to respiratory failure (upper airway edema), delirium and shock requiring icu admission, intubation and resuscitation with fluids and vasopressors. white blood count was /mm ~ with neutrophils. patient's hemodynamic data showed initial hyperdynamic profile followed by low output state with decreased sv ( %) (n - %) and cardiac index ( , ) (n , - ). echocardiogram confirmed cardiac chambers dilation as previously described in thyroid storm. lithium carbonate, corticosteroids, antibiotics and beta-blocker perfusion were given. plasmapheresis was started. free t& (n= , - pmo/l) went from , to , after the first two pheresis. after a remarkable clinical recovery, sub-total thyroideetomy was done i days after admission. in life-threatening thyroid storm, plasmapheresis is a very effective therapy when anti-thyroid drugs are counterindicated. purpose: to compare the reliability of prognostic indexes in crhically iu patients admitted in an intesive care unit (icu) who had acute renal failure (arfi and were treated with different dialytic techniques. material and methods: patients were included in a prospective study from june to november . patients presented arf defined by creatinin serum leve(s greater than pmol/l and previous normal levels. patients were divided in three groups. group i (control) : patients with arf who did not receive substitutive techniques. group ih patients under intermittent hemodialysis (hd) or peritoneal dialysis (pd). group ii : patients under continuous hemodiafiltrstion (hf). the statistical analysis was chi-square test and analysis of variance. results: the table shows the results we obtained, we did not find any significant difference betwen the two groups of patients undergoing dialysis. d(fferences were observed only between group i and the other groups as shown below. we did not find any significant association between the theoretical mortality predicted and the observed mortality according to saps in the three groups. due to exposure to a wide variety of unpleasant stimuli, for example, tracheal suctioning, venipuneture and physiotherapy, most pataents admitted to the icu will require some form of sedation. this review will describe the suggested properties of an ideal sedative agent for use in the icu and review the current limitations of some of the available agents from this perspactive. methods used to quantify the level of sedation, such as the ramsay score, glasgow coma score, newcastle sedation score and visual analogue scores, and their deficiencies will be examined. consideration will be given to defining the optimal level of sedation and the circumstances under which sedation might be varied over the icu course will be discussed. preliminary results from an ongoing study examining the role of light versus heavy sedation and ischaemia in a cardiac surgical icu population will be presented. the pharmacceconomics of icu sedation will be briefly addressed. finally, the role that sedation may play in increasing morbidity, pastieuiarly nosocomial pneumonia, in the icu will be discussed. objectives : therapy cost(tc) in icu patients is a substantial component of total hospital care cost. estimation of tc during this year, partitioning to various groups of drugs used and attempt to minimise it, were considered practically useful. methods : in collaboration with the hospital pharmacy we were able to have a complete report of au drugs used for icu patients (including enteral and parenteral nutrition). mean apache ii severity score upon admission was . and mean length of tcu stay was . days. price per drug unit and cost per group of drugs were also available drugs were divided into two groups: antibiotics ( ) cardiovascular drugs ( ), gastrointestinal system drugs ( ), enteral and parenteral nutrition ( ), respiratory system drugs ( ), sedative, analgesics and paralysing agents ( ), parenteral solutions with electrolytes, vitamins and trace elements ( ), anti-inflammatory agents ( ), protein substitutes and immunomodulation agents ( ), anticoagulative agents ( ). antibiotics were further subdivided into those "freely" prescribed (a) and those whose prescription and administration requires filling of a relevant form (b). results : !) tc for icu patients/day was . drs ($ ). total tc/patient was . drs ($ . . ). ii) partitioning total tc per group of drugs reveals : ( ) %, ( ) . %, ( ) . %, ( ) . %, ( ) . %, ( ) . %, ( ) . %, ( ) . %, ( ) . %, ( ) . %. t ) concerning antibiotics which consist the major cost component, group a and group b contributed by . % and . % to the total icu tc respectively. group b were administered to . % of all icu patients. conclusions : i) for the above studied patient population antibiotics consist almost half of total tc followed by protein substitutes and immunomodulation agents. ii) if tc control could be attempted in the icu, prescription of beth groups must be reviewed. appropriate treatment should be prescribed and readily provided to any patient. clinical significance of routine protein substitution, currently controversial, should be re-evaluated. new antibiotics (third & fourth generation cephalosporins, quinolones, carbaponems) should be prescribed on the basis of strict diagnostic procedures using modern technology available. rationalisetion of antibiotic therapy will lead to cost control, redistribution of icu expenses and substantial contribution to infection policy in our country. objectives: i -to investigate the clinic efficiency of the monitoring of the rso cerebral, in relationship to the stroke prevention, in patient undergoing carotid surgery. -to determinate the variations of the rso during the different surgical and anesthetic procedures in these patients methods: ten patients undergoing carotid endarterectomy. precise neurological exploration previously to the surgery and in the immediate postoperative period. angiography evaluation to the extend of carotid artery disease. invasive blood pressure, ecg, pulse-oximetry ( pso ) and rso were collected previousty to the induction of anesthesia. the premedication was administered intravenously -midazolam ( mcgr/kg) and fentanyl (i rncgr/kg) -. thiopental ( mg/kg),fentanyl ( mcgr/kg) and atracnrium ( , mg/kg) have been used for induction of anesthesia. co te is monitoring al~er the orotraqueal intubation ! the anesthetic maintenance is accomplished with lsofluorane ( , - , %) and bolus of atracurium and fentanyh the surgical procedure is standard (without arterial shunt during the carotid cross-clamping). we register each minutes: blood pressure, cardiac frequency, pso , co te and rso . the rso cerebral variate in relation with: the anesthetic induction, blood ~ressure, co te, cross-ulampping carotid and with the modifications of the head position. the maximum decrease of rso cerebral was in relation with the :ross-clampping carotid ( minimal value: ). no patient had neurologic complications and postoperative stroke after carotid endarterectomy were not observed. objectives: there are more than anesthesia in chelyabinsk emergency hospital every year. to % patients of it emergency anesthesia is applied. more than patients have ishemie heart disease (ihd), hypertansion (hp) and previos miocardial infarction (pmi). more than % of all patients are old patients (op). the resalts deep noninvasive bioimpedance monitoring (nbm) in surgical patients have been studied by us. methods: our nbm system "kentavr" includes parameters of cardiac and vessels function. it is realised by monitors in operation theatres and computer network. moreover we are able to examine surgery patients before anesthesia and perioperatively by using special computers system for cardiovascular reflex control by fast fourie transform (fft) of parameters simultaneously. results: pathients extremly needed peryoperative monitoring of hemodinamics. from these patients more % had stroke volume (sv) less than ml, n -co less than . /mim/m , % -ejection fraction (ef) less than n and % -puls bioimpedans microvessels (pbm) less than morn. patient had intensive care in special department. out of died. comparing with survived with these patients before operation hr was larger, sv, co,ef, pbm and puls bioimpedance aortha was smaller. much more of these patients were with ihd, pmi, hd, op. even with survived patients these parameters decreased the towards the end of operation. surgery patients had different variability of basic hemodinamical parameters with common tendency to increase power amplitude in low frequency by fft. conclusions: using of bioimpedanee noninvasive parameters allows to have criteria for corrections (infusies, vasodilatators, inotrops and others) and then us the final goal, to have more sucssesful surgery. with survived patients was perioperatively and postoperatively care more intensive. obiectives: the aim of the study was to compare the phi with the hemodynamically derived tissue oxygenation indexes as: oxygen delivery (do ), oxygen consumption (vo ), cardiac index (el), and arteriovenous difference in oxygen [(a-v)do ]. methods: patients ( males and females) with major trauma or major abdominal surgery were studied. on admission, a nasogastric tube allowing phi measurement was introduced and a pulmonary artery catheter was inserted for optimal hemodynamic management. each phi measurement was accompanied with a complete hemodynamic study comprising systemic and pulmonary artery pressures, blood gases, and cardiac output measurements with the thermodilution method. derived parameters vo , do , ci, (a-v)do were measured according to the standard formula. hemodynamic parameters were opt• as soon as possible with fluids, inotrepes, and vasopressors according to repetitive hemodynamic measurements. all patients were under mechanical ventilation. after hemodynamic stabilisation phi and hemodynamic measurements were repeated every eight hours, during a -hour study period. a total number of measurements were obtained and compared. statistics: results are presented as means + sd, correlations were performed between phi and the hemodynamically derived oxygenation parameters. a p< . value was considered as significant. results: mean values were phi= . + . , do = + , vo = + , c. = . + . , (a-v)do = . + . . no correlation was found between phi and do , phi and vo , phi and c.i, phi and (a-v)do . on the contrary in patients phi remained below . for more than hours despite adequate hemodynamically derived tissue oxygenation parameters. mortality in this group of patients was very high ( %). conclusion: no correlation was found between phi and the hemodynamically derived tissue oxygenation parameters our data suggest that phi is a better oxygenation indicator than the hemodynamically derived tissue oxygenation parameters, because it is closely related to the patient's outcome. objectives: the pathogenesis of septic shock and multiorgan failure is believed to be related to tissue hypoxia of the gastrointestinal tract. therefore new monitoring techniques, preferably organ specific, are required to establish the adequacy of tissue oxygenation. peep is used to reduce pulmonary shunt volume and improve blood oxygenation, but is accused to impair splanchnic perfusion. we studied mucosal oxygenation and perfusion on the capillary level in the stomach and the duodenum. methods: we used the erlangen microlightguide spectrophotometer (empho ll) together with a specifically designed fibre probe (bodenseewerk ger~tetechnik, berlingen) in combination with a standard gastroscope. measurements were performed on ventilated, traumatized patients (ages - years), with no evidence of shock or severe infection, after informed consent was obtained from the relatives. all patients were hemodynamically stable without inotropic support. an area of cm was analysed in the gastric corpus, the antrum and in the duodenum. in three patients we simultaneously measured the muc sal blood flow using a laser doppler flowmeter ( objectives: to investigate the influence of hb-o affinity in the monitoring of svo~ during improvement of cardiac index (ci) in cardiogenic shock. design: to state whether changes in svo: were associated in changes in actual pso (p~ ) and standard p~ (ps st) consecutive measurements of artero-venous bga, before an.d after therapy-induced changes in ci, were evaluated in patients (mean age -* y) suffering from cardiogenie shock, all under mechanical ventilation in psv modality. methods: together the hemodynamic measures, m~xed venous samples were analysed at ~ c using the abl radiometer for po , pco: and ph, and the osm radiometer for hbo %, hbco% and methb%. psost (i.e. the p~ at ph= . , pco:= mmhg and temperature at ~ c) was calculated automatically by the instruments on mixed venous blood as was the ps "in vivo" (i.e. the pso at the patient's value of ph, pco and temperature), using siggaard-andersen's computerizated algorithm. mean time between paired measurements was . -* . houm. the data were compared by anova test for linear regression and t-test for paired samples. results: a dose linear relationship was found between svo and oxygen extraction ratio (oer), r= . ,p= . . the improvement of ci ( . -* . to . + . l/min/m , p< . ) induced a significant increase in svo~ ( . -* . to . • . %, p<. ). a significant decrease in p ( . • . to . • . mmhg, p< . ) without any significant change in p~ st ( . • . to . • . mmhg, p=ns) was also found. these data show that either oer or the shift to the left of the oxygen dissociation curve account for increase in svo occurring with restoration of systemic blood flow. the program is intended to help the intensive care unit interne providing him with a practical tool when making decisions concerning patients in a critical condition. in his daily practice in intensive care unit, in this case the interne of the unit, uses this program for each patient as follows: on the first stage of data collection he should complete the following modules: ( )personal data ( )patient's pathology ( ) laboratory and~ monitor lug data ( )drugs prescribed or toxic elements ingested. in this way, the system allows optionally the consult with a computerized data base about the drugs prescribed, standardized parameters and techinques performed by the central laboratory. ( )reference to an antibiotics guide regarding becterian sensitivety in our unit, whitch ee checked every six month ( ) access to de questionnaired apache ii to load up new data. ( ) statistcs about patient's admission and discharge. results: once all data collection is finished the system performs the followin duties: ( )detailed drugs interactions, including toxic elements ( )diagnosis starting from the clinical, laboratory and monitoring data. in some cases, it also establishes therapeutic strategies, e.g. a coagulopathy ( ) give the l~narmacological incompatibilities between the drugs p~escribed and %he diagnosis established, and ( )perform dosage adjustments based upon the personal and pathological data. objeatve: to assess the power of diseri~,~ion ofa multiperpose severity score (sai~) when applied to subgroups ofpatieals (pta) according to their lemg~ of ~ay (los) in icu. design: in order to compute the saps probability, a model derived fi~m legible regression was developed. meaumree of calibration (goodmem..of.fit statistics) end discrimination (roc cm've and relative area under the cm've) were adopted in develotammtul asd validation set. the whole databue was ~ati~ed in five gronps reeked on los as follows: los = days, los = - days, los = - da~, los = - days, los > day~. area under the carve (auc) was ud~ninted for each ro~. s~ing: imlimlcus. patents: of ~ pts comec~ively admired ~ a period of three yeet~ ( ) ( ) ( ) , a total of was i~leded in this study. pts without saps, p~ yolmger them yearn, p~ with los shorter ~ hom'~ were excluded from this maly~is. iaterventinns: nose mema'onm~ end result: the logistic model developed gave good remits in terns of calibration md discrimin~on, both in developmental set (do.s g : . , p > . ; auc = . i- . ) and in validation ~t (g.o.g g : . , p > . ; auc = . ..+ . ). auc of each grottp showed a loss in di~zimination (i.e., prediaton) closely related with los, being . i- . in pts with los = days el . ~. ia tm with los > da~ (figure). following the present guidelines of integral management, in order to achieve optimization of sanitary resources and better use of facilities, we feel that the setting up of objetives is a key factor in the continuous process of improvement of quality care. postsurgical intensive care services maintain an interdepent relationship with other hospital services. within the general plan of the hospital it's of the utmost importance to delegate autonomy to the various depertments and service units in determining and achieving objetives. it's also necessary to establish mechanism for coordination of the activities in order to assure the succes of the program. the objetives cannot be improvised, they must be carried out in a specific manner in the following stages: .-analysis of the present situation (starting point). where are we?. defining objetives and making explicit the activities and methods to achieve them is to anticipate the future; it is of the utmost importance to comunicate said plans to all whom affect by encouraging them to attain the desired results. in the present paper we intend to show the guidelines to follow in carrying out a course of objetives. introduction:we presents results related to the quality of life (qol)of critical patients, from paeec project data. material and methods: the paeec project is a multicentre study define the type of patients cared for in spanish icus, and the therapeutic activity provided. ninety-five icus from spain are taking part. this study analyzes the qol of critical patients prior to their icu admission.for the evaluation of qol a questionnaire designed by our team for critical patients was used, with items grouped in sub-scales: physiological functions ( items); functional capacity ( items) and subjective aspects ( items). qol is classified in levels: normality ( points); slight deterioration ( - points);moderate deterioration ( - points); significant deterioration (>i points). the we present results related to therapeutic activity in critical patients and their age, from the paeec project. material and methods: the paeec project is a multicentre study to define the type of patients in spanish icus, and the therapeutic activity provided. ninetyfive icus from spain are participating. this study analyzes therapeutic activity in the first hours as evaluated by tiss, and related factors. results: the sample was , patients, sge . ~ . years. severity by apache ii system was . • points. the tiss score was . • points, distributed as follows: i (<i points): %; ii (i - points): %; iii ( - points): %; iv (> points): %.there is a positive correlation between the level of therapeutic activity and severity by apache ii (r = . , p < . ), and a very weak but negative correlation between tiss and age (r = - . , p < . ), so that an increase in age corresponds to a lower level of therapeutic activity.patients the multivariate analysis of the relationship between tiss and age took into account: severity, existence of previous history, need for mechanical ventilation, size of hospital, diagnosis and mortality. it indicated that there continued to be a relationship between therapeutic activity and age, so that as age increased, therapeutic activity diminished. conclusions: therapeutic activity performed on critical patients is less in the oldest patients, in whom excessively aggressive procedures are limited. a relational data base management system in the icu. c. kotsavassiloglou*, d.matamis, g. dadoudis, j. kioumis, d. riggos. icu dep., g. papanicolaou gen. hosp., exohl, thessaloniki, and * a' neurological clinic of aristotelian university, thessaloniki, greece. objectives: the introduction of the information technology in the i. c. u seems to be unavoidable because of the large amount of produced data and the need for their systematic analysis. such an information system should be a) easy to use, b) friendly to the user, c) powerful and d) modular. on that basis, we created a patient data management system (pdms) according to the expectations of the medical staff of an eighteen bed multidisciplinary icu. methods: we selected paradox for windows v . for the implementation of a relational data base because this program meets the above mentioned criteria. informations regarding the patients include a) demographic data, b) previous medical history, c)diseases upon admission, d)complications during hospitalization and e) outcome data. the diseases' registration consists of items classified in categories upon the principal system affected. specific informations about the need and duration of mechanical ventilation, nutrition, renal replacement, right heart catheterization and icp monitoring are also available. an extension was added concerning icu infections and related informations about antibiotic-resistant pathogens. all icu pathogens can be matched to their resistance or sensitivity and cost of antibiotics. the program can perform queries and various statistical analyses based on complex criteria. new modules can be added later according to the future needs and remarks of the users. results: the program was well accepted by the medical staff and patients were registered as a test. the first analysis of the data related a) observed mortality versus the apache ii predicted mortality, b) mortality according to the age, gender, pathology aud duration of icu stay and c) pathology upon admission and icu related complications. conclusions: the long term use of this pdms can be an efficacious research tool. it can be used in retrospective or prospective studies by addition of necessary modules. the first data analysis revealed the iack of an international diseases' classification system. the development of a worldwide common classification system is essential for the compatibility of the data analysis among various icus. this will allow the realization of multicenter trials on a large scale. s. nanas= n. sphiris, a. precates, a. lymberis, m. pirounaki, and ch. roussos dept. of critical care, university of athens, athens, greece the complexity of the cases submitted to an icu, the variety of underline disease, tbe severity, as well as the large number of substances administered to each patient constitute obvious the need of support with an easy available dss. this system will assure the safety of the administered treatment will help to adjust the dose according to the situation of each patient and it will screen for possible interaction and incompatibilities between the administered drugs. the goal of the present effort is the design and development of a software system acting as a decision support tool to physicians of icu. the application is organised around a relation database management system (rdbms) that consist of: a) all available substances ( . ), b) all generic names of medications available in our country for each substance, c) incompatibilities ( . cases) and d) interactions with other substances ( . cases). the following figure shows the structure of the rdbms. y ta~ortato~ [ c~rs using the stored parameters for each patient the dose and the rate of administration of selected substances will be possible to calculate. the continuous monitoring of the treatment for each patient supports the medical staff to make the necessary changes of the prescriptions. the application is currently developing in wireless pen based computer systems which place patients at the centre of "islands of information" located throughout icu. in conclusion this dss is a powerful and useful tool for icu staff because it provides without additionai work to the routine of daily practice, the currently available information for each order concerning drug interaction and incompatibilities as well as treatment monitoring is to obsea~ among critically ill pfdieats, stdjdivided following the diagn~s at the adn~ssio~ the diffmeax:es in the ~ and oxyplx~efic l~mmems bawe~ strvwors [s] and non sumvors ins] and to test the pc~'bih'ty to have soar survival criteria, as earliest as tx~able. method~ :we made a ~ study on consexa~e ~ilically ill paliffas, subdivided in series following the diastases at the admission: medical pafiea~ ( s and ns), surgical patients ( s and ns), a~d poliwauntas ( s and ns). follow up was done at d,.ays from the admission in ice. all the patienls were ramitored with a ~ c~eter and laeno:lymmi. "c and o .x.xyphorefic txuamaers va:~e couected at fin~es (t): at fiae ~draission (t ), at x~ars from t (t ), at (f ), (y ), (t ), % (t ) and horus from t cf ). in~,h ~ies, for ~y ~ a all the lin'~ n~an and sandaid d~viation was ~ tx~h for s and for ns. th~ betw~ s and ns tl~ roeaas of ~h porarneter ~e ccmpared tt~ng t-lest and p < . w~ considered ska~ significant in each series in the t wheae the mast significative diffemx:as ~goeamd bet~en s and ns, we made a txedictive criterion, asamting as predictive indices for stnvival the i:r values, higher or lower than flae treans of the ~rar~ers of au flae patients, axx)rdhlg to those ones t~iatistically diff~'e~ betw~m s and ns. fhmlly xse co:weatxt onaong the series the nrametees of the st~rs with the analysis of variance, to daserve the lxjsable differealt irea~ of sty hflices, following the diagn~s of admission: :nedkal, angical patient or poll~tam results: we c~ld not find ~ predictive criterion for politraonaas, perhaps ixx:ause of the few ntanber of l~fients. for high ri~ saw~cal patieras the following criterion at t has a sensitivi .ly of ~ ,and a ~ecificity of . %: sv > . nffmin/n~, map> mmhg, pmap< nmalqg cvp<i nunhg will nmah& lvswi> g m/m , sxo > ~ do > mlhnin/m , o er< %. for lx~dical l~tienls at t the following criteric~a has a ser~tivi.ty of % and a ~zificity of . ~ cvp< . mn~g, sao > %, s,g) > ~ vo i< ml/nfin/m , o er< %, shunt< % survlvops' data of the series ~ signitic~atly differenl~ both for the t~mody~nic a~ for fl~e ox rphomfic lxlmn~s; moreover we ~ that the vatt~ of hemodynamic mad ox.~ho~tic indices were higher in politrautms. conclus'ions: acx~ording to the fftffe~mt patho!o~es, the ~ rnelabo~c needs are diffeten~ so that it is juslified to mash ~ the~alceutic goals, following the type oflmthology. hen~ we foru~d for high ~k mrgical pmka~ and for medical patier~s assme, ff mllslied, a good prognosis while, if n [ ntljsfled~ the plinsliclioil ofdl~tth is no[ g(ioct finally, ab~ high iis~ supgical palieaats, according to what other atmhors say, txatws sh ~'n~ers ' therapeutic goalsvvould seem inadeqt~te, bec~jse they need a gear physiologic and themtx~ic elth~ in rdation to the rretabolic needs. figure ) . thus, the smaller european nations had a greater participation than ~e larger ones, with the exception of norway. a similar result was evidenced for contributions to intensive care medicine (figure ). these findings can be explained by different submission policies and language banners. however, there was no significant correlation with the gross national product of each country. conclusion: we conclude that the smaller european countries generally contribute more to international intensive care journals than the larger ones. objectives: to evaluate the agreement between a new and three old methods measuring ctp and to assess their reproducibility. methods: we studied patients ventilated with a siemens c respirator. we measured ctp by dividing the tidal volume with the increase in airway pressure (paw), either with the respirator setting used (ca) or with a fixed setting (cf). by modifing the inspiratory time (ti) without changing inspiratory flow, we were able to deliver two series of inflations ( , ,... ml) before and after curarisation of the patient. the same volumes were also inflated in paralysed patients with a super syringe. at the end of each inflation a plateau of sec was performed and paw was recorded. the above three sets of pressure-volume (pv) points were used to reconstruct the corresponding pv-curves (( , c , c the new method for ctp measurement without a super-syringe had the best reproducibility in paralysed patients and gave similar results without curarisation in the majority of them. however, agreement between the methods tested was unacceptable for clinical purposes. further investigation is required in order to improve the accuracy of ctp measurement in icu patients. m kunert, r.sorgenicht, l.scheuble, k.emmerich, h.g ker med.clinic b (dept.of cardiology) i heart center of wuppertal/university witten-herdecke,germany objective to determine the accuracy of activated partial thromboplastin time (apl-l) and activated clotting time (act) studies when samples are drawn through heparinized central venous catheters (cvc). methods a total sample of paired act/p't-/" values was analysed in patients ( m., f., + y.) for monitoring heparin therapy.all patients had a cvc (certofix trio,braun,frg) in the internal jugular vein receiving a continous infusion of . u heparin via the central catheter.act (hr-act, hemotec,usa) and ap'i-f (neothromtin, behring,frg) samples were drawn from the cvc using the double syringe technique (removing and discarding ml blood before drawing the sample). these blood samples were compared to act/ap'cf blood samples obtained by venipuncture (v.fem.) at the same time, act values were analysed directly in the intensive care unit (icu),api-i samples were measured in the hospital laboratory within minutes. results ac-i -~ pi-f~ cact/~pi r = , ) cvc samples + + . v.femoralis samples " + + p-value n.s. n.s. conclusion there is no difference in heparin anticoagulation studies drawn from heparinized central venous catheters compared to those obtained by femoral venipuncture,withdrawing ml blood prior to obtaining the blood specimen is a safe way for eliminating heparin contamination.not only the aptt test but also the act test is a useful method for heparin anticoagulation assessment in the icu. objectives: evaluation of the delicate balance between filter-coagulation and patient-hemorrhage using heparin as anticoagulant in continuous renal replacement procedures. methods: from january through august , we studied filter surviva[ and hemorrhagic complications during filter periods in critically d[ patients, treated with continuous arterio-venous hemo(dia)filtration, with special emphasis on the heparin dose, concurrent use of coumarins, systemic activated partial thromboplastin tirne(aptr), platelet count, mean arterial bloodpressure and the type of filter used. results: filters ( %) were disconnected because of coagulation. mean survival of multiflow an filters was twofold shorter compared to survival of fh gambm filters. a total of hemorrhagic complications occurred of which three patients died at aptt values of respectively , and seconds. after adjustment for mean arterial bloodpressure, platelet count and the type of the filter, the risk for filter-coagulation decreased % (relative risk . , %c . - . ) for each ten seconds increase in aptt. the risk for patient-hemorrhage increased % (relative risk . , %ci . - . ) at an aptt-increase of ten seconds. the occurrence of filter-coagulation and patienthemorrhage was not correlated with the administered dose of heparin. concurrent use of cournarines had a positive effect on filter-survival, without increasing the overall incidence rate of patient-hemorrhage. conclusions: the systemic apt]" is a good predictor of the risk for filtercoagulation and patient-hemorrhage. heparine therapy seems optimal at an aptt between and seconds, although one should realize that fatal hemorrhagic complications still can occur. objectives: the alterations in vascular tone which are primarily regulated by adreno-sympathetic tone(ast) are compensatory responses in hemorrhagic patients. this study was designed to evaluate the correlation between vascular tone and ast in patients with hemorrhage, methods: the vascular tone was expressed by volume elastic modulus (ev) that is defined as; ev = ap/(av/v) (ap; the arterial pulse pressure, av/v; the volume change ratio). ev was measured using a non-invasive transmittance infrared photoelectric plethysmography (tipp) and a volume oscillometric sphygmomanometer . we prospectively studied patients with hemorrhage. the initial ev measurement was performed on arrival and repeated for a hours duration. as a parameters of ast, serum concentrations of adrenalin (ad), noradrenalin (nor), plasma renin activity(pra) were measured simultaneously. we analyzed the correlation of ev and conventional parameters to ast by multivariate statistical analysis. results: ev values at transmural pressure mmhg on admission and hours later were respectively . + . mmhg, . +_ . mmhg (mean + sd). systolic pressure(pas) and serum hormones on arrival and hours later were respectively, pas; . _+ . , + . mmhg, ad; . _+ . , . _+ . ng/ml, nor; . _+ . , . + . ng/ml, pra; . _+ . , . _+ . ng/ml/hr. the ev values correlated significantly with ad (r= . , p= . , n= ), nor (r= . , p= . , n= ), pra (r= . , p= . , n= ). by multivariate statistical analysis, ev correlated more significantly with ad and nor and pra (p= . ) than the conventional parameters such as pas, heart rate and pulse pressure. conclusions: the alterations of ev correlates closely with ast. the compensatory mechanism in hemorrhagic patients can be detected noninvasively by ev monitoring. obiectives and method: autologous oxygenator blood was processed at the end of cardiopulmonary bypass (cpb) by either hemofiltration (hf , , m , fresenius) or by cell washing with a onntinous autologous transfusion system (cats, fresenius). prospectively the blood of patients for each group was processed and then retransfused intravenously to the patient. besides, volume and time requirements, standard hematologic chemistry, coagulation and complement activation were measured. results (mean values for oxygenator blood at the end of cpb, and results of concentrate after processing by filtration or washing): both processing techniques show excellent hemoconcentration of the diluted cpb blood with a good transfusion effect for the patient. filtration retains all plasma proteins and large molecular weight plasma bound waste products. in contrast, cell washing with cats significantly depletes plasma proteins and waste products. the newely developped cats machine gives eonsisinnt laboratory result in a fully automatic continuous processing mode. in conclusion, both filtration and washing are effective for processing cpb blood. filtra tion yields a highly concentrated whole blood, whereas cats washing produces a high quality autologous erythrocyte concentrate. soluble fibrin has during the last years gained interest as a marker for the activation of the coagulation in connection with various clinical conditions, e.g. disseminated intravascular coagulation, deep venous thrombosis and myocardial infarction. elevated levels of soluble fibrin in plasma can be detected by the chromogenic assay coaset fibrin monomer, relying on the ability of fibrin to enhance the tpa-catalyzed conversion of plasminogen to ,plasmin. using this test, it has been shown that the level of soluble fibrin can be correlated to severeness of illness in critically ill intensive care unit patients. a revision of the coaset fibrin monomer kit has now been made and the new product, coatest soluble fibrin, is considerably more convenient to handle and gives higher resolution at low fibrin levels. the test is performed by the addition of a buffer dilution of the plasma sample to a microstrip well containing the colyophilized mixture of tpa, plasminogen and the plasmin specific cbromogenic substrate s- . the reaction is allowed to proceed at,. room temperature for minutes before discontinuation. the absorbance at nm, measured in a microplate reader, is proportional to the content of soluble fibrin in the sample. the assay is carefully standardized and calibration curves are provided in the kit. the convenient and rapid assay procedure makes the coatest soluble fibrin test well suited for single test analysis in acute situations. objectives : blood coagulation abnormalities have been reported in the systemic blood of patients with cerebral lesions. the physiopathology of such events is not yet completely understood. we compare the coagulation profile of blood from the right jugular bulb with systemic blood of patients with head injury. methods: we studied patients, who were admitted to our neurosurgical intensive care unit between january and march with head injury and no other associated pathology (age - yrs), a glasgow coma score <= g, no abnormality in baseline coagulation profile and no history of coagulopaties. the patients did not undergo angiography. a one-way gauge certofix catheter was inserted through the right internal jugular vein up to the jugular bulb. an identical catheter was inserted through a subclavian vein. blood was sampled from either catheter (a=atrial; j=jugular) - hours after trauma (t ) and t hours later (t the inddence dpontolx'rative thmmhi~e and haumord~gic complieatiom were assessed in padents treated with indobefen, heparin calcine caeca), low mollecolar weight heparin (lmwh) (f.nosheparin) and undergoing hemodiludun, blood predeposhing, intra mad postoperative blood saving. ]'he indolmfon tempota~.norks platelet aggregation through ,,elective inhibition of the cyclatygenasis and thus atacbldonicadd( ).tbe n'mimum effect occurs after hours from the fast administration and is still present after hours. ~- patients, mean age --- yrs., weight --- kg were studied. ( . %) were male and ( . %) female. onderwent hip prosthesis ( previously plate and screw removal) hip revim'un ( stem, cop and stem + cop), tutal knee prosthesis, in the st anaesthesidogy depl from - to - - . as for antithromboembolic ptephylam, apart from hemodihitiun pts were with treated indobufen ndo), with heparin ealdum caeca) and with low mo!lecular weight hepam (lwr, ). as the slightest clinical and/or imtmmental suspidon of deep vein thrombosis (dv'i') or polmonary umbolism(pe), a phlebogram or sdndgram were respectively carried out. -the inddence of homologom transhisiom was significandy lower (p= . l) in the padeats treated with indobufen ( . ) compared .'ith heca ( . %). the con~gency table shows statistical signifleance for the use of heca in patients with vein deficiency in the lower limbs, past dvr and/or pe, coronary heart disease (cdh'), while there is no correlation for renal, cardiac or liver defidency, obesity, systemic hypertemion, atrhythmy, diabetes, chronic bronchitis and rheumatoid arthritis. by comparing the postoperative cumplications with the risk factors, there ks a highly significant correlation (p= . l) between cdh and thrombotic and humord~agic complieatiom (pe, death, he~atoma, die use of hum_ologous blood). thee data show that hep~in, preferred in patients with c'dh, roost likely for leagal-tuedical reasons, did not have the de~'ed effect. conclusions -the stastisfical aar~ais shows ~nifieanfly different efflea~ (pro . ) between the therapies (see table) : it can be seen that in patients undergoing autotramfusiun and hemedihidon, indobufen produo~ a lower incidence of haemotrhagic complieatiens compared to heca and lmwh and is more effective in the prevention d ~c complications at clinical e~idence. the duration of i~toperadve hospital stay is signi~cantlylonger for patients transfused with homologous red ceils and treated with hec, .a ( . -+ . days) and lmwh ( . +- a days) compared with indo(ll. _+ a days). one of the main causes for postoperative complications in major orthopaedic surgery is postopemtive bleeding with local effects in the operation site (hematomata, pain and delayed mobilization) and/or systemic and subsequent cardiodrculamry repercussions that are sometimes severe. the aim of this study is to assess the possibility to apply a new system of monitoring, control and saving postopemtive blood loss from the drainage. the bt recovery dideco (marandola, modena-italy) ~ used since it is the only apparatus capable of doing this. the apparatus consists of a pressure transducer, adjustable from - a + mmhg, which activates a peristaltic pump connected m drainage robes. the bt recovery display shows hourly bleeding in the first hours, total bleeding, time passed since the start of monito~g and subsequent salvage and the aspimtioo pressure on the drainage robes; the latter is inserted at - mmhg and then modified according to bleeding/minute, g bt recovery also has an alarm that sounds automatically if.' blood loss is more than ml/hour; air is in the circuit; the batteries are running low. materials and methods: pts were studied ( m and ~), aged . -+ .lyears, basal hemoglobin . -+ (range . - . )g/all, treated from st january, to mst december, in the st service of anesthesia and intensive care unit of our hospital. the patients underwent the following surgical treatment: total hip revision ( pts), cup revision (~ipts), stem revision ( pts), total knee revision ( pts). the average dumtion of the operations was -+ min. intranpemtive monitoring and blood salvage was applied to all patients. genera! anesthesia was used on pts. and integrated (epidural analgesia + light general) on the remaining t . anttthromboembolic prophylaxis consisted of external pressure bandage, isovolemic hemodilution with iodobufen in ( . %)pts., calalc heparin in ( . %)pts., low molecular weight heparin in ( . %)pts.; pt did not give a predepoalt of blood, gave unit, pts units, pts units, pts units. the data obtained was statistically analysed using contingency tables and anova. results: average intmop salvage was -+ ml, average postop salvage was -+ mi the average intra+postop +- ml. average postop loss was -+ ml. the global incidence of postop complications was: h~natomata . %, dvt . %, pulmonary thromboembolism , , myocardiac ischemia . %, acute myocardic infarction . %, respiratory deflciecy . %, arrhythmia %, cystitis . % there were nn complications in . % of pts. postop bleeding over ml in under minutes (with bleeding alarm activation) occurred in pts ( . %). this sta~tically correlates only with the type of operation performed (more frequently in total hip revision p= . ) and with a significant decrease (p~ . ) in the pruthrombic activity detected about hours after the operation. this bleeding, also made the alarm sound, calling the attention of staff who could act accordingly, by making the drainage pressure positive and incre~sthg the tension of the external pressure bandage. conclusions postop monitoring, control and blood loss salvage combined with predepoalting and intmop salvage has enabled allogenic transfusions in % of cases to be avoided in operations with high postop blood loss like hip or knee revision. the usefulness of the system can be seen by the fact that in the patients with so much bleeding to set off the alarm, there was no significant difference in the incidence of allotransfusions and complications. references )borghi b., bassi a., de simone n., laguardia am., fonnaro g. an injury of the brain may result in various disorders of hemostasis caused by the release of • into the circulation through a damaged blood-brain bar tier. disseminated intravascular coagulation(dic) is one of these disorders. it is a freguent but relatively rare ly diagnosed complication of subaraohnoidal haemorrhage. the aim of this study was to evaluate some parameters of both blood coagulation and fibrynolisis in patients with sah.in addition one wanted to find out wh~ther potential changes correlated with the pa• condition in the acute phase of sah and whether they influenced the course of this disease. patients with sah were studied. in of them sah was due to closed eraniocerebral injury and in the rema ining resulted from vascular malformation. the following parameters were evaluated:the prothrombine time,the activated partial thromboplastin time, the thrombine time,level of factor v,fibrinogen degrada tion products and fibrin monomers. the results let us show the presence of oic in patients with closed craniocerebral injury and in with vas. cular malformation despite the lack of clinical symptoms the tests in posttraumatic patients and in patients from second group showed incomplete dic.on admission patients with such changes in measured parameters were in poor condition.the course of the disease and the effe cts of treatment were also worse in these patients. the results showed ihal in patients with sah complex disorders of both coagulation and fibrynolisis occur, and they depend on clinical condition of the patient. they also influence the course of the disease. methods : charts of all patients admitted with d.i.c. over a ten year period ( - ) were reviewed. diagnosis of dic was based on the association of fibrinogen < g/ -platelets < / -fpd > ~tg/ml in the hours of the admission. results : patients -mean age + y -saps +_ -gestanional age _+ weeks -the two first conditions associated with d.i.c. were placental abruption ( %) and preeclampsia or eclampsia ( , %). bleeding episode was present in pts ( %) and surgical treatment has always been necessary. pts ( %) were given packed red ceils ( + u) and fresh frozen plasma ( + u). patients were given platelets packs. heparin was never administered. pts required mechanical ventilation and two patients hemodialysis. all the patients survived. correction of prothrombin time (p.t.) and fibrinogen (f) was quick (p.t. at t h ~ % -f at t h , + , g/i). but platelets count remained low (plat. at t h + / ) -no difference was observed in patients who received platelets. conclusion : prognosis of critically ill o.p. is good. blood loss is the main complication. correction of hypovolemia and anemia with concomitant surgical treatment are essential. the administration of coagulation factors or platelets is still under discussion. objectives: to evaluate the effects of antithrombin iii i at-iii) and a protease inhibitor, gabexate mesilate foy), on the coagulation and fibrinolysis in disseminated intravascular coagulation (dic). methods: after the approval of our institution and consent from patient's family, patients with a dic score ( , japan) more than points (dic or having a risk for dic) entered this study. they were randomly divided into two groups, foy (i- mg/kg/h for days or more) treated group and no foy group, each of patients. platelet count (plt), fibrinogen (fen), at-iii fibrin degradation product (fdp), d-dimer (do), fibrin monomer (fm), thrombin-antithrombin complex (tat), plasmin-plasmin inhibitor complex (pic), and prothrombin time ratio (ptr) were measured before the start of treatment (at admission) and i, , and days after the admission. at-iii at units for days was administered if the at-iii at admission was less than %. finally the patients were divided into four groups: group a, foy (+) and the at-iii ~ %; group b, foy (+) and the at-iii < %" group c, foy (-) and the at-iii %; group d, foy (~) anffthe at-iii < %, each of patients, to match the patients for backsrounds. all parameters, dic score and survival rate in a month following treatment were compared among the four groups. results: the at-iii and plt from day to were significantly higher in groups a and c than in groups b and d. the fdp, dd, tat, and pic after treatment decreased significantly from the baselines in groups a and c but not in groups b and d. the fgn and fm were not significantly different among the four groups. the ptr decreased in groups c and d but increased in group b. the dic score decreased significantly in groups a and c than in groups b and d. survival rates were %, %, % and % in groups a, b, c and d, respectively, although not significantly different. conclusions: in patients with dic or a risk for dic, foy had no expected effects but at-iii had suppressive effects on the coagulation and fibrinolysis mechanisms. a prognostic factor ? carbon monoxyde intoxication is a classical complication of inhalation injury. carbon monoxyda is also physiologically produced during the heme metabolism: heme is conversed to bi]irubin by the hemeoxygenase which is an intracellular stress protein. icu patients (pts) were studied prospectively for apache ii score and carboxyhemnglobin (hbco) arterial level to assess if hbco level could be correlated with the severity of the pts. objective: to evaluate a new technique of non-surgical tracheotomy. patients: adults, mean age years and children, mean age months ( me.- yrs). method: through a needle inserted in the trachea, a guide wire is retmgradely pushed out of the mouth and attached to a special device formed by a flexible plastic cone with pointed metal tip joined to an armoured tracheal cannula. this device is then pulled back through the oral cavity, larynx and trachea, and outwards across the neck wall by applying traction on the wire with one hand and counterpressure on the neck wall with the fingers of the operator's other hand. when the cone and / of the eannula have emerged, the cannula is cut off from the cone, straightened perpendicular to the skin, rotated and advanced caudally to its final position. results: endoscopic control facilitates and improves the safety of all manoeuvres. the pointed cone easily pierces the tissues, and the cannula is extracted without difficulty since it has the same outer diameter as the cone. tissue adherence around the cannula is absolute thus preventing local inflammation. the time in apnea required for dilation and cannula placement does not exceed see., and it is well tolerated because within safety limits in patients hyperventilated with oxygen. only one case of bleeding occured in a patient on dialysis with severe coagulopathy. autoptic findings in subjects who died due to progression of primary disease showed a very regular stoma with an almost complete lack of hematic and flogistie infiltration in recent tracheotomies. .conclusions: translaryngeal tracheotomy (tlt), by virtue of its greater inherent safety and lower tissue trauma than percutaneous techniques, can also be carded out in infants and children, a severe test bench for any tracbeotomy technique. further specific indications are recently stemotomized patients, since tlt is associated with a low rate of infection, and short term tracheotomies after laryngeal surgery, to prevent obstructive complications. references: fantoni a., translaryngeal tracheotomy, apice, ed. gullo, trieste, , . background: inhalation of no has been shown to reverse hypoxic pulmonary vasoconstriction , to reduce pulmonary pressure in pulmonary hypertension of different origin and to improve gas exchange. in putmoflary embolism, pulmonary hypertension is caused by mechanical vascutar obstruction and by reactive vasoconstriction. the effects of inhaled no in putmonary embofism has been partiatly studied' the purpose of this study was to investigate and determine the effects of no inhalation on pulmonary hemodinamica and gas exchange in a hypoxic canine model of pulmonary embolism. methods: two groups of adult mongrel dogs were studied: group (control} dogs and group (no inhaled) dogs. both groups were anestesized with tiopental, mechanically normoventilated with an hypoxjc mixture of and n~ (f[q , ) and instrumented (swang-ganz catheter, femoral artery catheter) pulmonary embolism (pe) was induced by fisher's method s. no inhalation ( ppm) in group was started rain. pdor to pe and kept constant throughout the experiment. no inhaled concentration was analyzecf by chemiluminiscence technique. pulmonary artery pressure (pap), central venous pressure and sistemic arterial pressure were continuosly recorded. cardiac output, artedat po~ (pan ) and mixed venous po~ were measured in both groups under hypo)dr conditions, before pe and , , and rain. after pe. pulmonary vascular resistance (pvr) and gas exchange (pao fio:~ ratio), were calculate using standard formulas. data were process and analyzed with non pararnetdc test, and reported as mean -so and statistical significance was considered if p < , . : no produced an increase in arterial oxigenation (pao /fio~ ratio) and reduced pap before pe induction in group . after pe we found no significant difference with .respect to the time eour.se of pap, pvr and gas exchange between beth groups throughout the experiment. probably, the severe mechanical obstruction produced in pulmonary embolism masked the small effects of no inhaled. obiectives: blood volume measurement would be useful in critically ill patient management if it were easy to perform. this is not the ease and current methods are based on radiolabelled red cell dilution. inhalation and uptake of a known mass of carbon monoxide (co) gas and measurement of earboxyhaemoglobin increase can give results accurate enough for clinical use. this requires a rebreathing system providing oxygenation and carbon dioxide removal, yet complete retention of all carbon monoxide administer&l, and so most authors hand ventilate with a bag and waters soda-lime canister, adding oxygen as necessary. we aim to popularise this method by; i)design of an automatic co administration system driven by the itu ventilator and ii)writing of software for a portable computer to perform all necessary calculations method: we show the computer is use estimating the co dose required and later estimating the blood volume. we also show the new gas administration system. this is a fully closed circle attached to a "bag in bottle", driven by the ventilator. the novel feature is the mechanism by winch driving gas (set to % ) spills automatically into the circle, balancing o uptake by the patient, yet allowing no co loss. conclusions: this equipment is easy to use, reduces human error and allows optimum ventilator settings to remain. the operator merely administers the volume of co determined by the computer and takes blood on two occasions. carboxyhaemoglobin measurement is easy to perform, thus there is a cost saving also. with our modifications use of this technique may potentially become more widespread, the video demonstrates the method in use in our itu. - ( %) underwent conventional surgical therapeutics. " ( %) with resection of tracheal stenosis with end-to-end anastomosis(rts). i ( %) with broncoscopic dilatation. one patient died and the others still have stable patency(sp) without continued treatment. - ( , %) have received endoscopic laser ablation with or without calibration tubes. of them ( , %) are receiving continued endotracheal treatment until now. ( , %) have sp wihout continued treatment. -i ( , %) endoscopic laser therapeutic case turned to rts and is having sp. conclusion: conventional surgical aproach has been progressively replaced in our hospital by endoscopic laser ablation and silicone calibration tubes. this study suggests that these technics are effective and could be the elective treatment for iatrogenic stenosis. obiectives: hemorrhagic disorders due to thrombocytopenia and thrombocyiopathia remain one of the most serious complications during long-term extracorporeal membrane oxygenation (ecmo) in patients with severe acute respiratory distress ~drome (ards). in the presented study, nitric oxide (no), kwown as a potent endogenous platelet antiadhesive, disaggregating and antiaggregating compound, was evaluated for its possible antagonistic effect on platelet trapping when added to the gas compartment of membrane oxygenators (mo). meti~ods: two parallel separated extracorporeal circuits, consisting of heparin bonded hollow fiber oxygenators (minimax, medtronic, carmeda eioactive surface), tubing systems, low pressure reservoirs, and roller pumps were prepared. for each measurement, a pair of circuits was simultaneously filled blood from the same volunteer. low-heparinized fresh warm blood was obtained from four healthy volunteers, who had no drugs for at least two weeks. the gas inlets of both oxygenators received dry gas ( % oxxygen, % carbon dioxide, % nitrogen); gaseous no ( ppm) was added to the gas of one of the oxygenators (no-mo), whereas the other one (mo) was used as control. after minutes no gas was switched off, so that the no-mo received no more no, and no was added to the gas inlet of the membrane, which had no no before_ to assure iutracircnit volume stability, drawn blood for measurements was replaced with saline, and platelet counts were corrected for dilution by hemoglobin values. the mean of four platelet counts (coulter counter) of each timepoint (start, , , , , , , , and minutes) was used for statistical analysis (paired sample t-test). results: in the no-mo platelets remained at + , % (percentage of baseline value, mean -+ sd) until min. in contrast, platelets of the mo continuously decreased after start and were significantly lower after minutes ( , + , % vs _+ , %(p< . ); min. , -+ , %vs , _+ , %(p< . ); min. , _+ , % ( p < . ). after switching of no gas to the mo, further decrease of plateleta was stopped and platelets remained at , +_ , % until termination of circulation. platelets of the former no-mo decreased slightly after cessation of no gas to , _+ , %. conclusions: these data indicate that gaseous no significantly attenuates platelet trapping in hollow fiber oxygenators, when added to the gas compartment. this might be a new therapeutical approach for membrane oxygenator induced thrombocytopenia during long-term ecmd. objectives: nitric oxide (no) plays a pivotal role in regulation of vascular hemostasis. several studies elucidated the antiadhesive, antiaggregating, and disaggregating properties of endothelially synthesized no to platelets. additionally, agonist-induced no production in platelets by the l-arginine-no pathway was found as a negative feedback mechanism after platelet activation. although noplatelet interactions were intensively studied by several investigators, no data exist, about changes in platelet surface molecule expression in no-modulated platelets measured by flow cytometry using monoclonal antibodies (moabs). methods: p-selectin (alpha-granule-membrane protein, gmp- , cd p) and glycoproteiu (gp , lysosomal protein, cd ) are expressed only after platelet activation and degranulation. activation was quantified in thrombin ( . u/ml) and adp ( . ram) stimulated platelet rich plasma samples (prp). blood was obtained from healthy volunteers (n= ), who had no drugs for at least days. for evahiation of no-modulated activation, the spontaneously noreleasing compound sin-i ( . mm) ( -morpholino-syndonimin-hydrochlorid) was added in parallel prepared samples prior to the addition of agonist. platelet surface molecule expression was evaluated with moabs directed against cd a (gpilbliia, fibrinogen-receptor, phycoerythrin(pe)-conjugated), cd p (fitcconjugated), and cd (fitc). only cd a-positive signals were gated in sideangled light scatter, and assayed for activation marker expression (defined as percent of gated population). results: basal p-selectin expression was . + . %, and increased to . _+ . % after thrembin-activation, and to . + . % in adp-stimulated samples. addition of sin- attenuated p-selectin expression to . - - % in thrombin (p<. , two-tailed paired t-test), and . + . % (p<. ) in adpactivated platelets. basal gp expression was . _+ . % and increased to . + . % in thrombin, and to . _+ . % in adp-stimulated samples. with sin-l, gp expression decreased to _+ . % (p<. ) in thrombin, and . : . (p . ) in adp-stimulated samples. conclusions: these data implicate, that no leads to a significantly reduced activation of surface molecule expression in thrombin and adp-stimulated platelets. in addition, flow cytometry might be a useful tool for studying modulation of platelet activation by no or no-releasing compounds. introduction: acute cadmium poisoning is very rare. on initial presentation may mimic metal-fume fever, but acute inhalation cadmium toxicity may produce fatal chemical pneumonitis. case report: we present a case of acute fatal respiratory failure secondary to cadmium-fume irthalation. a year old patient was trasferred from another hospital with acute respiratory failure presumably due to pneumonia. the last days before he had had commom cold symptoms. he had been cutting with a welder during one hour without any respiratory protective measure. three hours after exposure he developed progressive dispnea and was admitted to hospital. with presumtive diagnosis of respiratory infection, antibiotics were begun, however be failed to improve. all microbiological studies were negative. chest x-ray showed bilateral diffuse infiltrates. on seventh day he needed intubation and mechanical ventilation and on th he was admitted to our icu. antibiotics were stopped and new microbiological studies were performed including brochoalveolar lavage and virologic studies. all results were negative. he developed progressive hipoxemia and hipercapmia and finally, multiorganic disfunction syndrome. he died days after exposure. the metal he had been working with was a % cadmium alleation. blood cadmilam concentration days after exposure was . mcg cd/g cr, and urine cadmium concentration was . mcg/l. on postmortem examination, tissue cadmium concentrations were: blood ng/ml, liver ng/g, kidney ng/g and lung ng/g. these values confirm that cadmium was the cause of the fatal respiratory illness in this patient. conclusion: this case evidences the considerable hazard of acute poisoning after inhalation of eadmium-fume and stresses the need of appropiated safety measures against metal-fume poisoning. aim : lactic acidosis is considered the hallmark of cyanide poisonirig. however, the relationship between plasma lactate and blood cyanide levels has not been determined. the aim of this study was to determine the significance of plasma lactate concentration (plc) during the course of cyanide poisonings. methods : the patients were included according to the clinical suspicion of pure cyanide poisoning at the time of presentation. fire victims were excluded. serial blood samples were collected before and after intravenous hydroxocobalamin (hoco). blood cyanide concentration (bcc) was measured colorimetrically. plc was measured enzymatically. results : patients were studied. on admission, plc ranged from . to mmol/l, and bcc from . to gmol/l. mean systolic blood pressure was • mm hg, mean arterial ph . • . , mean anion gap was . + . mmol/l and mean pao . • . kpa. three patients died. before antidotal treatment, there was a significant correlation between plc and arterial ph (p = . ), anion gap (p = . ) and bcc (p = . ) but not with heart rate, pao , paco and blood glucose, or blood pressure. during the whole course of the poisoning, a plc _> retool/ was a sensitive and specific indicator of a blood cyanide concentration > ~tmol/ . sustained catecholamine administration reduces the correlation coefficient. conclusion : baseline measurement of plc allows assessment of severity of acute cyanide poisoning. thereafter, plc may be used to assess the adequacy of antidotal treatment, more especially in patients not requiring sustained infusion of catecholamines. aim: the aim of this case report was [o study the correlation between the plasma lactate levels and several clinical, biological, and toxicological parameters serially measured during the course of a cyanide poisoning treated with a high dose of hydroxocobalamin. a -year-old male ingested potassium cyanide leading to cardiac arrest. cpr was performed prior to hospital arrival where the patient received g hydroxocobalamin. sbp rapidly returned to normal allowing withdrawal of epinephrine. the patient remained comatose and died from brain injury days after the ingestion. methods plasma lactate and blood cyanide levels were measured serially. blood cyanide levels were measured using a colorimetric method.~ plasma lactate levels were measured using an enzymatic method. for correlation spearman rank correlation test was used. results. initial plasma lactate and blood cyanide levels were mmol/l and gmol/l, respectively. there was no overall correlation between sbp and either blood cyanide or plasma lactate levels. similarly, there was no overall correlation between arterialvenous oxygen saturation difference with either blood cyanide or plasma lactate levels. in contrast there was a strong correlation between blood cyanide and plasma lactate levels (r= . , p< . ). the time-course of the blood cyanide concentrations was described by a mono-exponentiai decay (r = . ) with a blood half-life of . h. similarly, the time-course of plasma lactate levels was described by a mono-exponential decay (r = . ) with a blood half-life of . h. discussion. in this case of acute human poisoning, sbp was a much poorer indicator of continuing cyanide effect both before and after antidotal treatment, than was lactate production. this suggests a potential clinical role for following serial plasma lactate levels as a marker of the evolution of cyanide toxicity. aim : cyanide (cn) poisoning in fire victims is frequent and rapidly fatal. in a prospective study we tried to assess the clinical tolerance of a high dose of hydroxocobalamin (hoco) administered at the scene of the fire in fire victims suspected of cn poisoning. methods : inclusion criteria : soot in mouth or sputum ~ any degree of neurological impairment. exclusion criteria : children, pregnant women, burns of total surface body area > %, multiple trauma. protocol desigrl following examination and the collection of a blood sample in dry heparin, a g dose of hoco ( g in case of cardiovascular collapse) was administered intravenously over min. the systolic blood pressure was monitored before and after the administration of hoco, and one hour later. results : there were females and males. the mean blood cn concentration was • pmol/ . the mean blood carbon monoxide was . • . mmol/ . nineteen fire victims eventually died. among the non-cn-intoxicated patients (blood cn < ~mol/ ), there was no significant change in arterial blood pressure. in the cn-intoxicated patients (blood cn > gmol/ ) a significant increase in blood pressure was observed both immediately (p < . ) and hour later (p < . ) after the admistration of hoco. no allergic reactions were observed. conclusions : in fire victims with cyanide poisoning, the administration of a high dose of hydroxocobalamin was associated with an improvement in systolic blood pressure. hydroxocobalamin is well tolerated in fire victims without cn poisoning. objectives: tricyclic antidepressant (tca) overdose can lead to serious complications including cardiac arrhythmias [ ] . because of the known risk of early deterioration and the implication for management, emergent evaluation is essential. we determined the diagnostic usefulness of the electrocardiogram (ecg) in tca poisoning. methods: retrospective study of all patients with tca intoxication (pos. ,toxicology screening in urine and/or pos. history) in a -beduniversity hospital from through . the severity was graded with mild= no symptoms or agitation; medium= disorientation, somnolence, tachycardia, or convulsions; and sever~ coma, significant arrhythmias or death. we analysed the first ecg after admission with a special emphasis on qrs-and qtc-intervals and the terminal ms frontal plane qrs-vector (tqrs), which, was reported to lie typically between + and * + + • the best correlation with severity grade was found with qrs-and qtc-duration (p= . ), the tca-dose (p= . ) and hf (p= . ); tqrs did not correlate. patients died ( . %). conclusion: qrs-and qtc-prolongation in the admission ecg, and the reported dose of ingested drugs are useful predictors for severity of poisoning due to tricyclic antidepressants. we did not find additional benefit in determining the terminal ms frontal plane qrs-vector. objectives: since treatment of amphetamine poisoning is usually symptomatic and often associated with a fatal outcome, a search for specific drugs to help the amphetamine-intoxicated victim is sorely needed. methods: we report a case of a suicidal ingestion of large amounts of the amphetamine-derivative , -methylenedioxy-ethamphetamine (mdea) and heroin (diacetylmorphine) and present the hypothesis that the two drugs produce opposing clinical effects. results: a year old caucasian male was admitted to the emergency ward because of acute-onset confusion. at presentation, he was agitated and showed increased muscular rigidity. he had taken tablets of "eve" (mdea, approx. g) and g of "smack" (heroin) by oral route approximately h before admission. because of rapidly progressive tachypnea and exhaustion, the patient was intubated and ventilated. the serum concentration of "eve" on admission was ng/ml (lethal range - ng/ml). trace amounts of cocaine and substantial amounts of heroin ( ngtml; mean value in heroin-related deaths: ng/ml) were also found in the serum. the patient was successfully weaned from the ventilator by day and recovered without persistent neurobehavioral disturbance. despite high serum levels of both drugs, the patient did not present with the classic signs and symptoms normally seen during intoxication with these drugs. amphetamines in general, and mdea in particular, have opposite clinical effects to heroin or diacetylmorphine. none of these were however present in the case presented despite the high ingested doses and the serum levels in the lethal range. conclusions: the fascinating fact that, apart from the respiratory depression, none of the clinical signs reported after massive overdose with these two drugs were present, might be attributed to the opposite pharmacological effects of mdea and heroin. we believe that the patient unwittingly saved his own life by the oral coingestion of both mdea and heroin. our clinical data raise an interesting point about the pharmacological treatment of acute poisoning with amphetaminederivatives. introduction: the acute attack of aip still carries a significant risk of mortality of around %. a succesful outcome depends on early diagnosis, removal of pricipitating factors and provision of intensive supportive therapy. objectives: twenty one patients ( females, male) with documented aip were seen over a -year period in the university hospital. patient was in clinical remission and were with the acute attack of aip, among them with respiratory paralysis were required artificial lung ventilation and -assistant ventilation with peee pathologic treatment during the attack was normosany, adenil, androgenes, glueosa, riboxin parenteral and enteral nutrition via nasogastric tube. symtomatic treatment -pethidine, propranoton, antibiotics, bronchoscopia. methods: intermittent phasmapheresis was performed on patients. the following measurements were peformed: level of porphobilinogen (pbg) in the wire and delta-aminolevulinic acid in the blood. hematological and routine chemical evaluations, hepatic, hemodynamic and respiratory function. results: after plasmapheresis the median pbg excretion (normal range - mkg per/ kgr creatinine) fill from mkg on admission . mkg, then on - day raise to mkg and then during treatment with normosong and prasmapheresis lowest level was . mgk. fatalities occured in two females during attacks with proforma cerebral involvement and patients attained clinical remission. conclusion: after therapy with plasmapheresis normosong we found that there was consistently reduce the urinary excretion of pbg and shortening the duration of the acute attack. objectives: pigs has been reported to present with a higher pulmonary arterial pressure (ppa) and stronger pulmonary vascular reactivity than many other species, including man. aim of the present study was to compare pulmonary vascular impedance (pvz) before and after embolisation in weight-matched adult dogs and minipigs. methods: we investigated pvz spectra in anaesthetized and ventilated (fio . ) minipigs and dogs. after baseline measurements the animals were embolised with autologous blood clots to reach a ppa above mmhg. results: flow ( and ppa matched pvz data (mean-+sem) are shown in the table. [zo = hz impedance (z; {dyn.sec_em- }); zl = first harmonic z; zc = characteristic z; z phase = first harmonic phase a@e {radians}; fmin = frequency of pvz the first m{n~mam; *, f p at least < . between dog and minipig, and before v~. after embolisation respectively]. before case report: a -yr-o]d woman affected by legs recurrent thmmbophlebitis, was admired in medmine department for tach.~pnea, chest pain, tachycardia and cyanosis. before starting two-dimensional transesophageal echocardiography (tee) to confirm the suspicion of pulmonary embolism, she suddenly had ventricular fibrillation. resuscitation and defibrillation were readily performed. when sinus rhythm was reinstituted she was in superficial coma with preserved corneal and light reflexes: right hemiplegia, poor perfusion and h~posphygrma of the left arm. tee showed dilation of rigth ventricle (rv), incomplete occlusion of pulmonary arter~ (pal at it~ hifurcation, severe tigth-to-left shunt through a patent foramen ovate, paradoxical embolism with incomplete occlusion of left subclavian artery mechanically ventilated with vt= ml, rr= /mm, fio =l, the patient had ph= . , pao = mmhg and paco = . systemic bp was / mmhg and hr= b/min with low dose epinephrine ( . g/kg/min) a thrombolytic infusion (rtpa: mg/ h) through a peripheral vein was started tee imaging and clinical status hours later were unmodified. a new rtpa infusion was performed through the pulmonary hole of a swan-ganz catheter with the tip close to the embolus. one hour later pa pressure decreased from / mmhg to / mmhg, etco increased from to mmhg and sao improved from % to % three days later the parietal, spontaneously breathing and with normalized tee scans of rv and pa, was transferred to rehabilitation service to perform physical therapy. conclusions: massive pulmonary embolism in a patient with patent foremen ovale, paradoxical embolism and refractory hypoxaemia was unaffected by systemic rtpa infusion, while intrapulmonary rtpa administration dramatically improved gas-exchange, hemodinamics and the general conditions of the patient. the presence of a large rigth-to-left _atrial shunt and the rapid rtpa metabolism could likely explain the effectiveness of its intrapulmonary administration in front of failure of systemic thrombolysis. introduction. cardiogenic shock during massive pulmonary embolism (blpe) is due to an acute increase of right ventricle (rv) afterload and possibly rv ischemia causing a failure of rv pump function. the rec~;mmended therapeutic strategies are: xoiume augmentation ~n ~rder m }ncrease rv pre-h~ad, adrenergic drugs to increase t'ontractillly and maybe coronary perfusion, fibrinolytic drugs to delermine clot lysis. there have been several reports of noradrenaline (na) as a useful drug in this setting for its sluing ~z, but also ~, properties. case report.an obese },ears old woman was transferred to our icu for tetanus. she was given the usual antibiotic and immunoglobuline therapy. l'wo thoracic epidural catheters were put in place at different levels and replenished with marcaine qid. a continous infusion of sedation (diazepam § was started together with mechanical ventilation. curarization ~,as given occasionally. fraxiparine . /die was used for prophylaxis of thrombotic disease, on day th at . a.m. she started to be hypoxic (sa %), tach ,tardic l l(i b/rain.), her blood pressure(rp) dropped frum norma~ values to r mm/hg, the central venous pressure (cvp) raised [rom lb to mm/hg and the end tidal co was mm/hg lower than one hour before. the physical examination of the chest revealed a clear bilateral ventilation and the chest x-ray was normal apart from an elevation of the :tiaphragm as compared to the previous. an e.c.g. showed sinus tachycardia, right bundle branch block and a possible inferior necrosis (which was already present on admission). a trans-thoracic echozardiography was performed which showed "an acute overload of the right centricle wilh remarkable dilatation. tricuspidal regurgitation ++. paradoxical movement of septum. small left ventricle with normal wall kinetics". the cardiac enzymes were later shown to be normal. an acute massive pulmonary embolization was assumed m be present.. a bolus of streptokinase x i(i u. was given fonowed by a continous infusion . two liters of colloids were also given in a sh~rt time, two hours later the patient was still deeply hypotensive, hypoxemic and anurir(bp / mm/hg, cvs mm/hg, spo %) despite a cominnus infusion of dobutamine fag/kg/min and adrenaline . ~tg/kg/min. at this stage a bolus of aoradrenaline ,g was given followed by a cnntinous infusion of . !*g/kg/min. an immediate improvement of the hemodynamics was noticed and one hour later the bp was / mmhg, the cvp mm/hg, the sao % and a brisk diuresis started. the hemodynamics kept stable and weaning from vasoactive drugs was achieved within two days. one month iater the patient was discharged home in good conditions.. con c i u sio n.ne administration may help to restore rv coronary flow and ;~ump function during mpe. aeute putmonary t~omboembo~sm [ffe) cou be mamfeslated with either respiratory or cardiovascular syndromes or both. the arm of the study was to establish leading respn'atory symptoms, frequency and form of the roendganographic (rig) changes as well as blood gas disturbance degree in acute pte with dommam respiratory disease appearance. the study includes retrospeotive analysis of i pte patients (pts), males (average age , yrs) and .q females (average age , yrs). they were admitted at university, olinie" with suspection ofpleuropnlmonary disease, including pte. final diagnosis of pte was based o~ evident risk factors in , % of the eases (deep venous thrombosis, surgery, trauma, imobilisation, malignancy ere), acceptable clinical, rtg, sdntigraphic and laboratory findings, as well as deep veins examination by dopple~-sonographie and radioisotopic -~enogmphy. respiratory symptoms appeared in all cases: sudden pleural pain ( %), dyspnea ( %), hemoptysis ( %), cough ( %) with association of two or more symptoms in %. chest xrays findings were abnormal in % with diaphragmal elevation ( , ~ lung opaeilies ( , %), atelectasis ( , %), plemal effusion ( , %), main pulmonary brancah asimetry ( , ~ oligemia ( %), heart shadow changes ( , %) and pulmonary arteries "cut off' ( , %). the association of two or more abnormalities was found in , % while normal chest x-rot was found in ~ of the cases. hypoxemia with pao < , kpa was found in , % followed with hypocapnia and respiratory alealosis in , % in , % of the gas exchage analysis were within normal limits. among cardiovascular symptoms short syn~cpa appeared in i , %, ecg changes-st q t type in "~ , %. results show high frequency of positive ~g findings in pte pts that is opposite to oppinion that chest x-ray in acute fie is the most ofran normal. leading symptoms are pleural pain and dyspnea, while hemoptysis were found in a half of the study group. blood gas changes were present in two thirds of the cases. kakkar, in his classic work ,clearly demonstrated the efficiency of low doses of heparin in prevention of deep vein thrombosis (lancet : , ) .after this first study the application of heparin prophylaxis became more and more diffused until to be considered a routine in many surgical departement.actually application of blood saving technique induces postoperative hemodilution effect. in that condition prophylaxis routinely applied seems a nonsense and can be at risk for postoperative hemorrhage. methods: to analize this problem we compared patients arrived in our intensive care unit (i.c.u.) in. : (group a) with arrived in : (group b) .every patient was operated for major abdominal surgery.in each one we considered the hemoglobin (hb) value,hematocrit(hct), and coagulation pattern (c.p.) at the arrive in i.c.u. and hours later. the patients was also divided in those receiving heparin prophylaxis (i) from not treated patients (ii) results:the application of blood saving technique clearly appears from the hb and hct level wich have a mean value of , +/- , (hb) and +/- (hct) in group a while in group b mean value are , -/- , (hb) and +/- (hct).patients of group a (ii) are the only one where a pathologycal c.p. with statistical significance has been demonstrated.in this goup we got four cases of evidence of venous thrombosis and one of pulmonary embolism.in patients of group b(i) we encontered the incidence of two cases of severe hemorrhage despite the absence of statistical significance in c.p.modifications. oxygen desaturation during broncho-alveolar lavage: role of oxygen saturation monitoring in prevention of acute respiratory insufficiency g. galluccio, b. valeri, s.batzella, m. di lazzaro*, servizio di endoscopia toracica, ospedale forlanini, rome, italy * servizio die anestesia a rianimazione, osp. forlanini the broncho-alveolar iavage is a diagnostic procedure employed in interstitial diseases of the lung. it requests the introduction through the working channel of a fiberoptic bronchoscope, after occlusion of a segmentary bronchus, of aliquots of saline solution at c, subsequently gently reaspired, in order to remove cells and proteins from elf (endoalveolar lining fluid), which is related to interstitial medium. bronchoalveolar lavage induces deep effects on pulmonary function: -lowering of the alveolar surface of exchange; -shunt effect, depending on the perfusion of non-ventilated districts; -increased pulmonary arterial pressure, due to hypoxic vasoconstriction; -decrease of lung compliance. in this report the authors present the result of oxygen saturation monitoring in a group of patients with interstitial lung disease, who underwent diagnostic broncho-alveolar lavage. in most patients with severe interstitial involvement, the lavage performed without supplement of oxygen induced a severe fall in the oxygen saturation during the late phase of the procedure. if supplementary oxygen was delivered during bronchoscopy, since its beginning, only slight modifications of the curve were detected. in patients without thickening of interstitium, in whom the lavage was performed in order to obtain material for bacterial or cytologic examination, no modification of oxygen saturation was observed in standard procedure. as conclusion the authors strongly reccomend monitoring oxygen saturation in patients with radiologic evidence of interstitial involvement also in patients with no evidence of dyspnoea. g. galluccio, b.valeri, s.batzella, m. di lazzaro*, servizio di endoscopia toracica, ospedale forlanini, rome, italy * servizio die anestesia a rianimazione, osp. forlanini the treatment of choice in patients with alveolar proteinosis consists of pulmonary lavage. this procedure requests the introduction, through the working channel of a fiberoptic bronchoscope, segment by segment, of aliquots of saline solution at c, subsequently gently reaspired, in order to remove the proteins deposited in the alveolar spaces. the method is very similar to that used in bronchoalveolar iavage, a diagnostic procedure used to obtain cells and substances from elf (endoalveolar lining fluid), which is related to interstitial medium. as known, bronchoalveolar lavage induces oxygen desaturation, because of shunt effect. understandably, one lung lavage has remarkably more deep effects on pulmonary function than bronchoalveolar lavage, for the amount of fluid introduced, the length of the procedure and the conditions of controlaterai lung. in this report the authors present the result of oxygen saturation monitoring in a patient who underwent pulmonary lavage for alveolar proteinosis. in the lavage performed without supplement of oxygen a severe fall in the oxygen saturation was observed during the late phase of the procedure. if supplementary oxygen was delivered during bronchoscopy, since its beginning, only slight modifications of the curve were detected. as conclusion the authors strongly reccomend the subministration of supplementary oxygen in pulmonary lavages, also in patients with excellent respiratory conditions. a. b. dublisky prof., m. r. isaakjan ass., v. a. zasukha, s. m. vinichuk prof., v. p. tserty ass. prof., chair of anaesthesiology, resuccitation and medicine of catastrophes, neurology of ukrainian state medical university, kiev, ukraine. objectives: detection of plasmophoresis's influence of results in treatment of ishemic insult. methods: we ve investigate patients with ishemic insult, treated with reverse plasmopheresis in complex treatment. after primary infusive therapy we took ml of patients' blood and separated it within min with rotation frequensy of /rain. after separation of erythrocytes from plasma, the latter has been returned to patients. we made - procedures during - days. hemoglobin, hematokrit, time of blood coagulation were determinated. the brain blood flow in internal carotid arteries, regional volum brain blood flow and total brain biood flow were evaluated with tetrapotar chest rheography and tetrapolar rheoencephalography. obtained date were comparised with control group after traditional treatment. results: it was found that after reverse plasmopheresis the hemoglobin and hematokrit levels decreased significantly in studied patients' plasma (from + . g/l to _+ . g/ and from + . % to _+ . % respectively). the time of blood coagulation by lee-white has increased by - . times (up to - rain). the level of brain blood flow has been increased significantly after reverse plasmopheresis in comparison with control group. the following tests of brain blood flow have been increased: a) the total volume brain blood flow from . + . ml/min to . _+ . ml/min (p < . ); b) the regional brain blood flow from . _+ . ml/min to . + . ml/min (p < . ); c) the brain blood flow in internal carotid arteries from . _+ . ml/min to . + . ml/min (p < . ). conclusions: the use of reverse plasmopheresis in complex treatment of patients with ishemic insult aiiows to improve rheological blood patterns, helps to increase volume brain blood flow. it results in quicer reparation of neurological functions. objectives: a prospective evaluation of the efficacy of continuous infusion of verapamil in reducing the incidence of postoperative atrial fibrillation after pulmonary surgery. methods: a total of consecutive patients, on verapamil, on placebo was included after lobectomy or pneumouectomy. a loading bolus of verapamil ( mg over minutes) was followed by a rapid loading infusion ( . mg/min) for minutes and finally a maintenance infusion ( . rag/rain) for hours. results: a mean plasma level of verapamil of ng/ml was obtained only after more than hours. atrial fibrillation occurred in five out of patients who tolerated the verapamil infusion, and in out of patients on placebo (p = . ). verapamil infusion was not tolerated in patients because of hypotension or a heart rate of less than /min, within hours of the start of the therapy. when atrial fibrillation occurred, the ventricular response, mean _+ sd, was not significantly slower during verapamil infusion ( + ) compared to placebo ( + ). conclusions: because of its frequent side effects and the only modest efficacy verapamil should not be considered for prophylactic therapy of atrial fibrillation after pulmonary surgery, and is probably not a good first choice for slowing the heart rate in case of rapid ventricular response once atrial fibrillation has occurred in these patients. results: study of haemostasis in these patients has showed deep disturbances of blood coagulation. fibrogen level has reduced to . + . g/l, fibrinogen and/or fibrine degradation products concentration have enhanced to . _+ . g/l, monofibrin soluble complex concentration to . -+ . g/l, blood plasmin level was enhanced to . + . mmol/ , plasminogen proactivator level was also enhanced to . + . ram, plateletes aggregation has decreased to %. after plasmopheresis aggregation was decreased in . times. it has been connected with decrease of fibrin and/or fibrinogen degradation products level and level plasmin in . times, and plasminogtnt activator level in . times. at the same time we have observed increase in total antifibrinalitic activity of blood in . times. activity of activators plasmine and plasminogene proactivators has decreased in . times and in the same time activity of activation inhibitors and antiplasmines has increased in times. conclusions: plasmapheresis leads to considerable improvement of a general condition and reduction of the haemorrhagic syndrom's sings (controlling of gastrointestinal haemorrage, reduction of intensity of subcutaneons haematoma). evaluation of continuous cardiac output (cc ) monitoring based on thermodilution technique in critically ill patients. methods: cardiac output (co) was monitored continuously using a modified pulmonary artery (pa) catheter, on which a heating filament is located and by which energy is transmitted to the circulating blood. a microprocessor calculated co by a new algorithm. standard bolus thermodilution technique ( ml of ice-cold saline solution) was used to compare cc with intermittent bolus cardiac output (ic ) measurements. the following subgroups were prospectively studied: i. heart rate (hr) > beats/min, . cardiac output > i/min . cardiac output < . i/min, . rectal temperature > . ~ and . pa catheter was inserted for more than days. results: a total of pairs of ic and cc measurements were obtained from the patients. bias (ico measurement minus cc measurement) of all measurements were . • i/min and the % confidence limits (mean difference• were - . / . i/min. also in the subgroups, cc measurement agreed closely with ico measurement (c > i/min: bias= . • i/min; co < . i/min: bias=- . • i/mln). elevated temperature and prolonged lay-days of the pa catheter did influence agreement of cc measurement with ic measurement neither (> ~ bias= . • i/min). conclusions: monitoring of cc using a modified pulmonary artery catheter with a heated filament has proven to be accurate and precise also in the critically ill when compared with "standard" intermittent bolus thermodilution technique. this method enhances our armamentarium for more intensive monitoring of these patients under various circumstances. background: the number of patients who need coronary artery surgery was) grows every year. most of these surgical operations are with extrar eircuiation (ecc). since january , this surgery is made without ecc in selected patients in our hospital. this technique is exceptional in spain. this type of surgery has proved useful in patients requiring revascularization of the left anterior descending, eireunflex or right coronary artery (not for grafting the pos~tefio~r descending branch}. blethods and results: since , patients aged to years (mean years) underwent cas without ecc. the mortality in programmed surgery was %. no patient was reexplored for hemorrhage. the mean values of some clinics parameters v~ere: a) blood requeriments: units per patient, b) need of mechanical ~entilation: i , hours, c) postoperative bleeding: cc, d) days at icui , . we used the student % t test or fisber~s exact test to compare these results with the mean values of surgery with ecc: a) blood requeriments per patient (p< , ), b) need of mechanical ventilation: hours (p< , ), c) postoperative bleeding: cc (p< , ), d) days at icu: (p< , ), e) programmed surgery mortality: % (p< , ). conclusion: our limited experience shows that this surgery is an alternative in the treatment of coronary disease, especially for aged patients with associated pathology and in jehova's witness. the need of mechanical ventilation, days at icu, blood requeriments and morbi-mortality were fewer than surgery with ecc. to study the hemodynamic and antiarrhythmic influence of ace-inhibitor enalapril in acute myocardial infarction (mi). methods: holter ecg monitoring, heart rate variability analysis, echocardiography ( and l days after beginning of the treatment), stress-echocardiography and stress ecg ( - -th day after the onset of mi). enalapril was included into the treatment of pts with mi (study group), with normal or increased blood pressure, from the -st day of the disease. the data were compared with pts treated without enalapril (control group). results: silent ischemia during stress-test was registered in pts of the study group and of control group, the arrhythmia episodes during stress test -in and pts and episodes of silent nocturnal isehemia -in and pts correspondingly. enalapril importantly attenuated the hypertensi~re re~aetioh % stress test. in pts of the study group the number of perifocal hypokinesis zones decreased; in the control group it didn't change. the quantity of ventricular extrasystoles in the patients of the study group decreased by %; the heart rate variability indices improved as well; in the control group the character of ventrieulir arrhythmias, heart rate and its va]~i~bili%y didn't change significantly. conclusions: the inclusion of enalapril into the treatment of mi is a useful t ol to improve hemodynamie parameters and decrease the incidence of ventricular arrhythmias. objectives: to study left ventricular (lv) systolic function in the patients with acute myocardial infarction (ami) before and after peroral captopril test. methods: the original echocardiographic parameter of lv contractility, "coefficient of effective systolic function" (cesf), was proposed in the study. cesf is calculated from lv stroke volume (sv), obtained from doppler aortic flow in lv outflow tract and lv end-diastolic diameter (edd): cesf =sv/edd. the study included patients with ami, who had local lv dyskinesia and global lv systolic dysfunction (ef< %). besides cesf, the ejection fraction was calculated before and after administration of mg eaptopril (on the fifth day of ami) by methods of bullet and simpson. results: the dynamics of these parameters, as well as heart rate (hr) and mean blood pressure (bp), is shown in the tabte. before cal~topril ef (bullet) . • . ef (simpson) . introduction: the cold system is a monitoring system for measurement of right (copa) and left (coart) ventricular cardiac output, cardiac function index (cfi), fight ventricular ejection fraction crvef), fight ventricular cnddiastolic volume (rvedv), intrathoracic blood volume (!tbv), global enddiastolic volume (gedv), lung water (etv) and excretory liver function (pdr). patients and methods: pts have been monitored by the cold system. above mentioned parameters are measured by thermal dye dilution and a fiheroptic femoral artery catheter. copa, rvef and rvedv measurements additionally were compared to measurements by the baxter explorer. :::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: ;;;k;;;;i cov (%) explorer ! ! [ gedv, itbv and pdr showed a significant decrease dufing the first - h after the operation, cfi and rvef si~canfly improved after k wheras etv showed a i~ in the early postoperative phase and fell to normal ranges at h. comparison of cold/explorer m~ements sb wed good correlations. discussion: concerning m ~toring of ri,ght ventric~ar function cold and explorer can he seen as equal. rvef gives an ar report about the performance of the right ventricle without use o f echocardiography. measuring itbv and gedv ~ improve ~gement and con~ol of th.e volume status, monitoring etv helps preventing lung edema. pdr shows good corre|ati n to liver blood chemistry and is bedside avai|ab|e. thus the cold system offers additional parameters for comprehensive m~nitofing of pts. ~e~ ~c surgery. obiectives: to evaluate the influence of an a!'~ered cardiac function on the cardiovascular response to the increase in oxygen demand induced by an increase in core temperature. methods: this preliminary study included adult critica!ly ill patients monitored by arterial and pulmonary artery catheters in whom thermodilution cardiac index {ci) and arteria! and mixed-vef)ous blood gases measurements could be obtained before and after an acute change in core temperature of at least . ~ (max rain apartl the patients were separated in two groups according to their cardiac function: patients had an impaired cardiac function as defined by a history of cardiac disease and an ejection fraction below % and patients had normal cardiac function. results: individual data are shown in the figure. in contrast to the control group (continuous line) in which c! increased without changes in oxygen extraction ( er), the q er in patients with impaired cardiac function (dottled line) increased without changes in ci. conclusions: the increase in oxygen demand associated with changes in temperature is met by an increase in c! in patients with unaltered cardiac function and in an increase in o er in patients with altered cardiac function. temperature should be taken into account in the assessment of the adequacy of cardiac output in patients with impaired cardiac function. objectives: to define the hemedynamic and metabolic response to physical therapy(pt) in relation to the type/level of sedation and the cardiac status in icu patients. methods: we studied mechanically ventilated icu patients ( • years) in stable hemodynamic status (no change in vasoactive treatment for at least hours), separated in groups: group = deep sedation, cardiac dysfunction required dobutamine (n= )r group = deep sedation (barbiturates), unaltered cardiac function (h=lo), group = moderate sedation, altered cardiac function (h= ) and group = moderate sedation, unaltered cardiac function (n= ). complete hemodynamic data, arterial and mixed venous blood gases, respiratory gas analysis (metabolic cart ccm, medgraphics) were obtained at baseline ( x) and twice (q. min) during leg mobilization. data were analyzed by anova. calcium channel blockers were used in complex preoperative preparation of hypertensive surgical patients. patients were allotted to groups based on their hemodynamic profile: hypokinetic: ejection fraction (ef)< . , patients; eukinetic (ef> . ),i patients and hyperkinetic (ef> . ),i patients. the most noticable change in hemodynamics was in the hypokinetic group: ef and cardiac output (co) were significantly decreased (p< . ) while systolic arterial pressure (sap) (p< . ) and peripheral resistance (pr) (p< . ) were elevated. the results showed that in hypokinetic patients on nifedipine ef (p< . t) stroke volume (sv) (p< . l) and co (p< . ) were increased while pr(p< . t), sap(p< . ) and diastolic arterial pressure(p< . ) were decreased. eukinetic type patients also showed an increase in ef,albiet to a lesser extent,than in the hypokinetic group. increased sv and co(p< . ) were observed in eukinetic patients though this was to a lesser extent than in the hyperkinetic group. in the hyperkinetic group of patients nifedipine had no effect on the aforementioned parameters except for a decrease in sap(p< . i). nifedipine increased ef in all hypokinetic patients. comparative results show that isoptin was less effective than nifedipine in decreasing peripl~eral vascular resistance and had a depressive effect on the myocardium. it can be concluded that the action of calcium channel blockers normalizing the circulation in the hypertensive surgical patient depends on: the condition of myocardium, the patients hemodynamic profile and their pharmacological properties. they were most effective in the hypokinetic group. zalo/nthinos e., daniil z. zakynthinos s., armaganidis a., kotanidou a., nikolaou ch..,roussos ch. critical care department, university of.athens, evangelismos hospital, athens, greece. introduction : surgical is the optimal treatrnent for ioculated effusions and the preferable procedure when multiple bands are seen in the pericardial sac by echo. patients : palients, post cardiac surgery, uremic ( men, women) with large pericardial effusion and clinical or echocardiographic findings of tamponade or both. these particular patients displayed numerous linear echo-dense bands and s~'ands crossing the pericardial space (in one of them a ioculated effusion compressed the left ventricule). one had aptt increased, four were mechanically ventilated. technklue : a fr polyurethane catheter with end and multiple side holes over ga needle was echo-guided to the ideal site (fluid abundant and closest to the transducer). the catheter was attached to a close system with a heimlich valve for continuous drainage (pneumothorax kit). subcostal entry was selected in one patient and chest wall in five. the patient's position was changed every hour at least. (we believe that the small changes in the position of the catheter and the mechanical breaking of the bands in relation with the movement of the heart assist the pericardial fluid to remove). results : in all cases only a small quantity of fluid was withdrawn in the first minutes( - ml) with some clinical and echo-findings improvement. the fluid was bloody or serosanuginous with high protein content (ht= % ,protein , gr/dl) in all cases. in first hours the mean volume of fluid removed was ml ( to ml). in that period echo showed no residual fluid. the catheter remained within the pericardium to days .. no complications are mentioned. conclusion : cardiac tamponade due to hemorrhagic high protein pericardial effusion in uremic and postcardiac surgery patients,, as it is revealed by echo dense bands, can be faced by -d echo guided perieardiocentesis. a -fr polyurethane catheter with multiple side holes, attached to a heimlich valve was effective to evacuate the pericardial fluid. no catheter was occluded though heparin infusions were not used. multiple changes of the patient's position may be fundamental. this -d echo guided pericardiocentesis performed in in~nsive care unit seems to be useful , safe and quick technique. determining the best inotropic drug represents a very serious problems. the use of more selective and potential inotropic and vasodilatative drugs does not always lead to improvement of hemodynamic parameters in patients with low cardiac output syndrome. this paper presents patients with acbp who need an inotropie support after extracorporeal circulation in first hours. the patients were divided into dobutamin et dopamine groups. the heart rate (hr). mean sistemic arterial pressure [map), central venous pressure (cvp). and termodilution cardiac index (ci) were measured. the measurements were without using inotropic drugs, and then using them after rain, min, and finally with one hour rate, within first hours. the statistical analysis shows that both drugs lead to an increase in hr in the first hour of the application. the final effect of dobutamine is no change in hr, whereas the effect of dopanime is very significant increase in hr. thus. an absence of taehyeardie response selects the dobutamine as a better choice. backeround: pulmonary vascular eadothelium possesses major metabolic functions, which when altered contribute to the development of serious pathologies such as ards. one such function is the conversion of angiotensin i to angiotensin ii, catalyzed by angiotensin converting enzyme (ace), located on the luminal surface of the endothelial cells. ace activity has been extensively studied in animals in vivo, by means of indicator-dilution techniques, providing: i) under toxic conditions, an early index of lung injury, and it) under normal conditions, estimations of dynamically perfused capillary surface area (pcsa). objectives: to validate the use of these techniques in matt: i) for pulmonary endothelial function assessment, and it) for pcsa estimation. methods: ace activity was estimated in ten adult haman volunteers, with no pulmonary medical history and normal pulmonary artery pressures, undergoing cardiac catheterization for coronary artery disease assessment. single-pass traspulmonary hydrolysis of the specific ace substrate hbenzoyl-phe-ala-pro (bpap; p.ci) was measured by means of indicatordilution techniques, and expressed as %metabolism (%m) and v=-hi( -m). bpap was injected as a bolus i) into a main pulmonary artery, and it) inside the right atrium, to assess ace activity in one and both lungs. we also calculated a,~,/i~, an index of pcsa. pulmonary plasma flow (fv) was determined by thermodilution. fp in one lung was estimated as . xf v. results: similar values of %m ( . + . vs . • and v ( . • vs . • were observed in both and one lung respectively. a~k~ decreased from • ml/min (both ltmgs) to :~ (one lung). conclusions: i) pulmonary endothelial ace activity and thus pulmonary endothelial function may be assessed in humans by means of indicator-dilution techniques, it) our data denote homogeneous pulmonary capillary ace coneentratious and capillary transit times in both haman lungs, iii) the % reduction of a=~/k~ in one lung suggests that this procedure can be used to quantify pcsa in man. (supported by the fonds de la recherche en saute du quebec and the national health system of greece). objective: verify whether antioxidant activity is higher in reperfused than in no-reflow myocardium after i.v. thrombolysis for acute myocardial infarction (ami). methods: patients with ami were included. blood for estimation of catalase (cat), glutathione peroxidase (gpx) and mn-superoxide dismutase (sod) was drawn before initiation of i. the mechanism of myocardial cell defence against free radicals is probably identical in both reperfusion and no-reflow phenomena. therefore, antioxidants cannot be used as reperfusion markers. objectives_ to evaluate the precipitating factors of hypothermic phrenic nerve injury following cabg with lima. methods: fifty two consecutive patients ( females), with a mean age of + (mean +sd) years were studied. during the ischemic arrest time topical hypothermia was obtained in al~ patients wffh ice slush and no cardiac insulation pad was used. all patients received a lima graft, with or whithout additional vein grafts. supramaximai, bilateral phrenic nerve stimulation was performed percutaneously preoperatively and whithin hours postoperatively. square wave stimuli of . msec duration were applied at the posterior border of the sternomastoid muscle. the compound muscle action potential of the diaphragm was recorded, using surface electrodes on the anterior chest wall. the time interval from the application of stimulus to the onset of diaphragmatic activity, phrenic nerve conduction time (pnct), was measured. values exceeding . msec were considered as abnormal. besults: preoperatively, all patients had normal (mean+sd) pnct, . • msec for the left nerve and . • mseo for the right nerve. on the first postoperative day, right pnct was normal in atl patients ( . • msec) , whereas left pnct was normal in patients ( . • msec) and abnormal in patients (incidence . %). in patients the left phrenic nerve was inexcitable and in patient left pnct was prolonged ( . msec). comparing patients with normal and abnormal pnct there was no difference in age, gender, number of grafts used, aortic cross-clamp and bypass time. however, patients with abnormal pnct had a lower preoperative ejection fraction ( • vs • p= . ). moreover, in all of them lima was dissected from its origin ligating all upper arterial branches, which provide the blood supply to the left phrenic nerve, whereas in those with normal pnct the small vessels originating from the upper to cm of lima were preserved (p= . ). conclusiojel~ a hypoperfused left phrenic nerve seems to be more susceptible to hypothermic injury during cabg with a lima conduit. objectives: to test if necessary interventions on systemic vascular resistance (svr) along with preset pump flew (q) during cpb could adversely affect autoregulatory response and cause vo shifts. methods: we studied males ( - yrs) who underwent cpb for cardiac surgery. at o oesophageal temperature - c we set pump flow at . i.m~ .min - . when map was higher than mmhg we calculated vo by using fick equation. then we infused sodium nitropruaside (sn) to control map at - mmhg for min and we calculated vq . without changing the sn infusion rate we set q at . i.m' .min " . ten min later we measured vo . we took vo changes into consideration if greater than %. statistical analysis using students-t-test for paired data and analysis of variance was used as appropriate. results: depending on the biphasic vo response to sn infusion during low and high q we classified pts in four groups (table). i. vo increases with sn and increases further during high q unmasking hypoperfusion and supply dependency. ii. vo increases with sn but the addition of high q results in systemic shunt. iii. vo increase during high q proves that vasodilatation can turn flow insufficient. iv. vo does not change with any intervention. the small number of pts and the wide standard deviation did not allow any statistical significance. conclusions: cpb is an interesting model for the behavior of microcirculation. intervention on svr and q can improve or impair effective regional oxygen delivery, resulting in either better perfusion or systemic shunt. vo monitoring seems necessary during cpb. preoperative cardiovascular optimization (opt) to ci > . l/min/m , _< paop < mm hg,and svri __< mmhg/ll/min/m decreases cardiac events (events) and mortality (mort) in peripheral vascular surgery patients (pvs). objectives: to determine if opt to the same endpeints decreases events in patients undergoing abdominal aortic aneurysm repair (aaar) and to study the r predictive value in pvs patients. methods: aaar patients and pvs patients were admitted to the s cu monitored with e pa and arterial catheters and treated to achieve opt. patients underwent surgery independent of success of opt data included demograph cs, incremental risk factors, laboratory and hemodynamic data pre, intra, a~nd postoperatively events, and mort. events included arrhythmias requiring treatment or prolonging the sicu stay > hours, a st depression > !mm or t wave inversion, an acute mr defined by a new q wave > . sec or cpk-mb > %. results are presented as means _ -. sd. opt was achieved in of ( %) and in of ( %) in the pvs and aaar group, respectively. events did nat differ between groups of ( , %) and of ( , %) in the pvs and aaar group, respectively (p>o. ). mort was of ( %) and of ( . %) in the pvs and aaar group, respectively (p > . ), while there was no difference in endpoints of opt between patients with and with.out events in the aaar group, there was a significant difference in ci between patients with and without events in the pvs group. of note, of ( %) patients who developed events in the pvs group had a ci < . in contrast to of ( %)in the aaar group. the positive and negative predictive value were % and % in the pvs and % and % in the aaar group. conciusione: f. the endpoints of opt used for pvs patients cannot be ~sed to reduce events in aaar patients; . pvs patients who have net achieved opt are at extraordinary risk of perioperative events; . preoperative card ovascu ar opt in aaar patients makes no difference in cardiac related events, background : comparison of the right and left filling pressures (cvp/pcwp ratio) is considered as a useful diagnostic clue : the normal ratio is _< . ; ratio >_ . may suggest right ventricul~ infarction while equalization of the cvp and pewp is a classic sign of tamponade ( ). however after cardiac surgery, many conditions (diastolic dysfunction, pulmonary hypertension, positive pressure ventilation) are susceptible to modify the '*normal" cvp/pcwp ratio. material and method : we determined cvp/pewp ratio in consecutive patients (pts) after uncomplicated cardiac surgery ( coronary artery bypass grafts; valvular replacements) measurements were made before and after tracheal axtubation. results :cardiac index : . _+ . /minlm~; laotate: + rag/i; cvp range : - rnmhg; pewp range : - mmhg. mean cvp/pcwp ratio before extubation is . ( % confidence imerval : . - . ) and after extubation, . ( % confidence interval : . -. . ), (ns, paired t-test). in % of the pts, cvp was higher than pewp. there are no correlation between the cvp/pcwp ratio and c! before (r = - . ) and after extubation (r = - . ) nor between the cvp/pcwp ratio and mean pulmonary arterial pressure (mpap), before (r = . ) and after extubation (r = - . ), discussion : cardiac performance is adequate according to ci and lactate. however the cvp/pcwp ratio is markedly higher than the "normal" (_< . ) ratio. this difference is not related to mechanical ventilation because the ratio is similar before and after extubation, nor to pulmonary hypetaension because of absence of any correlation with mpap, post-cpb diastolic dysfunction of the right ventricle could be an alternative explanation. in this group of pts, increased cvp/pewp is not associated with any impairment of cardiac performance (absence of correlation with ci), conclusions : cvp/pcwp ratio as high as within a large range of cvp ( - mmhg) and pcwp ( - mmhg) may still be considered as normal after cardiac surgery. this emphasizes the limitations of the hemodynamic monitoring after cardiac surgery (in comparison with echographic technics). careful analysis of the morphology of the cvp and right ventricular pressure curves (x descent, y descent, dip-plateau) is mandatory rather than relying on the quantitative assessment alone. reference : ( ) ntensive care.-university hospital -m~laga (spaink introduction. fibrinolitic treatment (ft) permits the treatment of acute myocardial infarction (ami) addressing the etiology, thereby eading to mproved ventncular function and a marked reduction m mortality. the main clinical oroblem is the reduced time of application. delay in hospitalization, which can be from to minutes, is potentially the most avoidable delay. method. to reduce delays in hospitalization, the following was carried out in two chases. audit: analysis of the time lapse from onset of symptoms to start of ft. showed that during "(he period june to december , patients with chest paros were treated within a eriod varying from minutes to hours from onset of symtoms. ages ranged from to (average , ), oelng males and females. they were glved initial ecgs to determine st mcreases suggesting ami. median t~me for this orocedure was l m.. potentia ami patients were then admitted to the coronary unit, [)atients, under age with no contraindications received ft the median time apse from admission to corona-y care and administration of ft was minutes ( . ), -he total median delay was minutes ~ -i h. min,~ delays n start of this procedure are grouped as follows: extra-hosdita delays (from onset of symtoms to arrival at hospital) diagnostic delays (from hospital arrival to ecg). treatment delays (from diagnosis to ft). objectives: protocol of procedure to implement a fast-track method. a protoco was drawn up with the object of reducing diagnostic delays to -i minutes and treatment delays to less than i minutes results. following rmplementatlon of this protocol in january , fts were glven, with an over all average delay of minutes. this fast-track method did not reveal any inappropnate ft or any increase m complications, conclusions: detailed study of the various times taken for diagnosis ane treatment of ami patients, showed up weaknesses in the system and improvements througn the protocol based on performence orocedures which led to a % reduction in the start of ft background: the importance of the early use of thrombo!ytic agents in acute myocardial infarction (ami) is based in the better remaining ventrictjlar function and smaller mortality rate because of the greater reperfusion and sma!ler infarction size, therefore, it is very impodant to apply this treatment to the maximum number of patients without thrombolytic contraindicati n, and within the minimun period of time. the "thrombolytic fast track" implementation allows to optimize the time to administrate thrombelytic agents avoiding multiple delays~ methodology: we anal!ze the application of thromboly c agents to patients with suspect of ami from the begin!ng of september until the end of february . in this time there are two different periods, during the first months thrombolytic agent were admin!strated at intensive care unit (icu), and during the second period we carried out a protocol of quick detection and thrombolysis therapy in susceptible patients at the emergency room in order to reduce the time to treatment. ma!n results are shown in the faffewins de ay h=hours m=minutes the implementation of the fast track does not need supplementary personal or equipment but a protocelized approach and training of the personal involved the main problem detected was the usual attendance overload of the emergency department that makes difficult to follow many structurated actions. conclusions: pratocqlized changes in the management of ami can significantly reduce the detay in the administration ef thrombolytic agents. it is not necessary to eomplet the procedure iq the emergency department, as the use of bolus schedules allows to begin the treatment in this area and to transfer the patient to icu afterwards. elective cardiac surgery. b calvet, f ryckwaert, p trinh duc, p colson. anesthesia -reanimation, hopital arnaud de villeneuve, montpellier, france. obhectives: the study was aimed at analysing the incidence of renal dysfunction following cardiac surgery and its prognosis (acute renal failure, post-operative morbidity and mortality). methods: two hundred and thirty seven patients (aged from to ) were consecutively operated on for elective cardiac surgery and retrospectively included in the study. patients with preoperative infections and operated on in emergency were excluded. each patient had preoperative invasive cardiac investigation with angiography and calculated ejection fraction (ef). anaesthesia, cardiopulmonary bypass (cpb) and cardiac arrest management were similar in all patients. general body temperature was reduced to - ~ c. renal dysfunction was defined as a % increase from baseline of serum creatinine. demographic data, asa, treatments, pre-operative creaunine level, cpb and clamping (axc) times, intra and postoperative use of inotrope, serum lactate level before surgery, at the end of cpb, at the time of admission in intensive care unit (icu) and on post operative day one and apache score were compared in patients with or without renal dysfunction using anova test for repeated mesures and x when appropriate. data are expressed as mean +__sd. p value less than . was considered statistically significant. results: thirtytwo patients ( , %) suffered from renal dysfunction. age, serum lactate level at the end of cpb, at admission in icu, at pod and apache level at admission in icu, intra-operative use of inotropes were statistically different in patients with or without renal dysfunction (p< , ). mortality rate was statistically different in patients with or without renal dysfunction(~, , % and %, respectively, p= , ). incidence of acute renal failure following renal dysfunction was , % ( patients required hemodialysis). conclusions: although our cdteria for defining renal dysfunction were very sensitive, the incidence of renal dysfunction following elective cardiac surgery was lower than communly accepted in the litterature ( ). however renal dysfunction appeared significantly associated with a poor prognosis. reference: -settergren g, ohqvist g current opinion in anaesthesiology , : - r ; , tzelepis, g. , , late complications were observed in % of cannulations: local infection in (i, %), catheter displacement by the patient in cases ( , %), catheter displacement during nursing care in ( , %) and malfunction in cases ( , %). conclusions: central venous catheterizations are followed by immediate and late complications in almost the same percentage acute poisoning with amphetamines (mdea) and heroin: antagonistic effects between the two drugs methods: after institutional approval and informed consent, selected patients ( _+ years) undergoing peripheral vascular surgery (n= ) or carotid endarterectomy (n= ) were investigated. patients included had either documented cad (n= ) or two or more (n= ) dsk factors (age > years, smoking, diabetes meltitus, hypertension, hypercholesterolaemia > mg/dl). -lead ecg recordings were carded out preoperatively, on ardval in the postanaesthetic care unit, and h, h, h, and h postoperatively. ecg recordings were analysed by an independent blinded cardiologist for signs of pmi (new st segment depression > . mv and/or new t inversion). in addition results: of the patients investigated developed ecg-documented pmi, % occurdng in the immediate postoperative phase. troponin i levels > . ng/ml were found in of these patients thus, comparing a cardiac troponin i cut-off level of ng/ml with intermittent -lead ecg recordings, we found a sensitivity of % and a specificity of % methods: demographic, clinical and ecg data were analyzed. . % of patients were male; . % female. cad was the most common underlying cardiac disease ( . %) and . % underwent open heart surgery. % received proeainamide for supraventricular and % for ven~cular arrhythmias. % received a loading dose. maintenance was provided by iv route in . % and by po in . % ( . %sr end . % ir). . % of patients were obese right ventricular function following cardiopulmonary bypass: is important the mode of myocardial protection we underwent this study in order to examine its safety and usefulness in pts with trustable coronary conditions (unstable angina ua the mean age for group a was • years, for group b • years, and for group c • years. a history of previous myocardial infarction was present in pts of group a, in of group b and in of group c. three pts in group a, in group b and in group c had previous coronary artery bypass grafting. the median time between the onset of symptoms and a was days ( - ) for group a we used a continuous fixed intravenous a infusion at a dose of the sn was % in groups a and b, % in c, and sp % for group a, (fixed defects included) and % for groups b and c. there was no difference of side effects among groups: chest pain (i pt -group a, pts -group b, and pts -group c), transient hypotension ( pt -group c), headache ( pts, group c), dyspnea ( pt -group a), while st depression was seen in pts of group b and in pts in group c. the rate of a infusion was decreased to /kgr/min in one group b pt due to development of chest pain s five year follow up of humoral immunity in paced patients athens polyclinic hospital, department of cardiology athens, greece author index a abiad ch bertschat, e betbes blanch, l del nogal saez e -meneza nolla, j. nolla-salas pilz~ u puig de la bellacasa e scarpa, n. van de wetering objectives: only % of patients suffering from acute guillain-barr@ syndrome (gbs) respond promptly to established therapies like plasma exchange or intravenous immunoglobulines. in contrast to serum, cerebrospinal fluid (csf) of gbs and ctdp patients contains enriched portions of antiexcitatory factors(i) and cytokines ( ) able to induce pronounced conduction block ( ). to reduce or remove such pathologic factors we introduced a technique with direct access to the subarachnoid space. methods: with informed consent we lumbally inserted g catheters in gbs-and cidp -patients under sterile conditions. some of them had not responded very well to established therapies. - ml of csf were withdrawn and retransfused by a bidirectional pump (flofors) after passing newly developed filters (pall). daily filtrations with several cycles were performed ( - ml) over one week. results: the gbs patients improved after days (median) for one grade (according to the gbs-scale from the gbs study group) . the ventilator dependent patients were weaned after days (median). patients not at all treated before ( / ) responded better than patients that had been pretreated ( / ) with plasmaexchange or intravenous immunoglobulines. / cidp patients drew benefit from treatment, stabilized iongterm. conclusions: csf-filtration is a relatively save and well tolerated additional procedure. the costs are considerably lower ( / ) than those for plasmaexchange or intravenous immunoglobulines. references:( )wsrz aet al: csf and serum from patients with inflammatory polyradiculopathy have opposite effects on sodium channels. muscle nerve ( ) . ( ) clinical observations were made in patients admitted to the clinic. they were in coma associated with acute alcohol intoxication.standard evaluations (ecg-monitoring, electrocardiography, neuromonitoring, studies of acid-alkali condition, biochemical and toxicologic investigation of blood and urine) prior to and following the treatment conducted were undertaken in all the patients.to correct irreversible impairement of functions twofold laser blood irradiation by means of alok- apparatus, the exposure within minutes, was carried out.the data obtained confirm more rapid coma withdrawal of the patients, reconstruction of the heart and central nervous system electrophysiologic indeces, reliable reduction in complications compared with the control group. objective: to know the actual incidence of the critical illness polyneuropathy(cip). setting: fourteen intensive/critical care unit beds, in bed university hospital, covering . inhabitants (majority rural area). the icu patients are medical, surgical and coronary, excluded the neurotrauma and neurosurgical. design: a conseculive and prospective study. all the patients admitted during three months, from january lth to march th , were eligible (patients with admittance diagnosis of polyneuropathy were excluded ). methods: patients with apache ii score > , at the admission and six days after admissions were included into the study protocol. diagnosis of sepsis, mof, and all the drugs administered days before were recorded. a complete neurological exam, by a neurologist, in absence of ssdatives and muscles reliant ( th, ~ and th days after icu admittance) was made. we evaluated the nerve and muscles function with and electromyography study in all patients, at same days. in some paeents with cip we performed a nerve biopsy. results: from patients ( apache ii score: . ) admitted in the icu, ( . %) enter the study protocol. seven ( , %) had an axonal polyneuropathy(cip), three very severe. only four of the patients with cip had pathologic clinical exam. apache ii score: cip vs non-cip was . vs . . the incidence of cip by diagnosis (cip/diagnosis) was: sepsis, / and mof, / . conclusions: . -we think that it is necessary to define the "critically ill" for some score, before designing a study to know the incidence of this syndrome. . -we think that the incidence of the cip is lower that the latest papers say. objectives:acute pancreatitis(ap)is becoming a more important problem among the elderly as the population ages. the increasing presence of gallstone disease,as well as the use of certain drugs,may also contribute to the occurrence of pancreatitis. methods:all patients(> years)admitted to our medical department over an eight year period were included.pancreatitis was confirmed by biochemical tests and imaging techniques.scores were developed using ranson's criteria and a multiple organ system failure(mosf)index . overall, patients were evaluated; ( %)had pancreatitis of unknown etiology . results:( )patients with pancreatitis of ~nlqnown etiology were sicker and had greater morbidity( % vs %),mortality( % vs %),and longer hospital stays than p~tierf~ with pancreatitis of known cause.( )the best predicto~of severity and outcome was the mosf index and not ranson's criteria;the higher the score,the greater the associated disease,the worse the outcome.( )curlously,no difference existed in associated medical conditions between patierts withknown and ur ~own causes of pancreatitis. conclusions:greater organ dysfunction exists in patients with pancreatitis of unknown etiology, even though age and associated medical conditions do not differ . the application of the total enteral nutrition in the burns disease has minimized the complication rate and consequently increased the survival rate of children and adults. time of initiation, composition, duration and way of administration are very important in obtaining the optimum beneficial effect from the treatment and diminishing the complication rate and side effects. the above features will be discussed in view of our experience in cases. ta buckle?,, ra freebalm, c gomersall g joynt, r young. tg short. department of anaesthesia and intensive cm+e, prince of wales hospital. the chinese university of hong kong, shatin, hong kong introduction: gastric mucosal ph (phi) monitoring has been proposed as a relatively noninvasive index of the adequacy of aerobic metabolism in the gut. to examine the accuracy of gastric intramucosal pit measurements as a function of time and as a function of the catheter itself to determine whether the measurement error between catheters is clinically acceptable. patients with a gastric tonometer (trip tm, tonometrics, worcester. ma) insitu for > days were studied. following informed consent two new tonometers were inserted equidistantly & correct position was confirmed radiographically. measurements of intramucosal gastric ph were then performed over a hr period. eight -ten measurements were made in each of ten critically ill patients.percent differences between the two new catheters were . % ie at ph . _+ . ( % limits) and between old & new catheters were . %, ie ph j _+ . ( % limits). conclusions: the results suggest that the function of the tonometer deteriorates over time and that the absolute values of phi m~ not ~ufficiently accurate. however as a trend monitor phi may be useful in the clinical setting. despite a continuous decline both in li'equency and severity of gastro-intestinal stress-lesion/-bleeding (gisb) due to both improvement in preclinical support and in intensive care medicine, patients with cerebral lesion are still considered at high risk for developing gis . therefore the question arises, whether m> specific (}lsb-prophylaxis besides general and neurological intensive care, specific pharlnaeothcrapy or even the combination of two specific drugs reveals any protective efli~ct on frequency and severity of gisb.this pntspcclive randomized study has been perfornted in patients snfrering t'rttna head-injury/cerebral lesion and with a glasgow-coma-scale on admission (gcs:,)of < . according to randomization the patients have been grouped as tbllows: h analgesia/sedation (n= ); ih analgesiajsedation plus pirenzepine mg/day (n= ); .[ih anatgcsia/sedalkm plus sncraltate x [ g/day (n= ); iv: analgesidsedatkm plus pirenzcpine mghlay plus sucralfate x e/day (n= ). slalislical analysis has been performed by chl:*tt~sl. rank correlatinn and unpaired t-test; statistical significance has been set with p < . . / patients ( . %) developed gisb. although the mean gcs~-value (x -+ sd) did not reach significance between patients with and without gisb ( . + . vs . -+ . ). a significant inverse correlation between gcs:, and the incidence of gtsb (rs~ = . ) has been shown. the frequency of gisb among the groups is as follows: h . %; lh . %; llh . %; iv: . % (ch -~ = . ; not signilicant). no gisb-induced blood translusion or mortality, respectively, could be demonstrated. survival rate between the groups did not differ significantly (chi-" = . ; p= . ) and reached an overall-value of . %.drug-specific glsb-prophylaxis -administered either as monotherapy (pirenzepine, sueralfate) or in combination of these two specific-drugs -reveals no additional significant influence on the incidence of gisb in patients with cerebral lesion compared to no specific prophylaxis besides the general trauma-/disease-specific intensive care measures. critical care dpt, evangelismos hospital, athens university scho~" of medicine objectives: the correlation of longterm presence of nasogastric tube (ngt) to gastroesophageal reflux (ger) is still in question. in case of positive correlation, peg should represent an alternative to tube feeding in patients unable to be fed orally. therefore, we investigated: i) the correlation between ng and ger and ii) the effect of peg on ger. methods: a -h esophageal ph-metry was performed in patients in recumbent position at ~ who had a ngt for more than days and were on sucralfate for gastric mucosal protection. the tip of the ph-probe was lied cm over the esophagogasttie junction, confirmed by x-rays. patients who presented a percentage of ger-total (i.e. with a ph less or more than ) (ger-t) more than %, underwent ~t peg. the presence of a creseent-notch on the esophagogastric junction persisting on inspiration and the grade os endoseopic and histologic esophagitis (scale= - ) was noted. two ph-metrles repeated on h and on days post-peg were compared to the pre-peg one, with the followin~ parameters taken in consideration: i) % ger-t, ii) number of ger-total per hour (no/h ger-t) and iii) the duration that ph was less than (tph< ). in case ot ger persistence at the ph-metry on ?th day post-peg (group ii) another endoscopy was performed, while patients with reduced ger (group i) were considered as esophagifis-free.results: out of patients presented a ger-t> %. eleven out of group i group (n= ) i ( objectives: the aim of the present study was to compare the performance of a specially modified version of a photo-and magnetoacoustic (pa/ma) gas analyzer (br~)el & kjaer, denmark) with a conventional quadrupole mass spectrometer (ms) (innovision, denmark) in inert gas rebreathing (rb) tests such as determination of functional residual capacity (frc), pulmonary capillary blood flow (pcbf) and lung tissue volume (vtc). methods : from simultaneous readings of inert gas concentrations with the ms and the pa/ma analyzer during rb experiments a comparison was made of the pcbf, vtc and frc values. the rb tests were performed during rest and exercise ( , and w) in ten healthy subjects. results: the differences (mean +/-sd) between simultaneous estimates of rebreathing parameters were the following (pa/ma -ms) for pooled data, pcbf: . +/- . i/min, vtc: - +/- ml and frc: . +/- . liters. conclusions: smell but significant differences were found between the estimates of pcbf, vtc and frc using the ms and pa/ma, respectively. reference: p. clemensen, p. christensen, p. norsk, and j. gr~nlund. a modified photo-and magnetoacoustic multigas analyzer aplied in gas exchange measurements. j appl physiol ; : - . objectives: because transcranial doppler (tcd) has been proposed to explore cerebral co vasoreactivity in brain injury (stroke ; : - ), we compared this technique with the kety-schmidt reference method to assess cerebral vasoreactivity in comatose patients. methods: mechanically ventilated patients (age - yrs, glasgow - ) in coma due to acute brain injury were investigated during stepwise changes in paco ( , , , and mmhg) by increasing inspired pco . middle cerebral artery velocity (vm) was measured by tcd. after insertion of a catheter in the ipsilateral jugular bulb, cerebral blood flow (cbf) was determined by the kety-schmidt method, using the inhalation of % n through the inspiratory line of the ventilator. for each patient a cerebral co~ vasoreactivity index was calculated as the slope of linear relationship between vm or cbf and paco . objectives: after cardiac surgery the fluid shill, between interstitial and intravasal space may be marked. this is due either to the intraoperative volume loading by the extracorporeal circulation or the increased postoperative diuresis. therefore, infusion of a large amount &fluids is necessary during the first postoperative hours. it still remains unclear which of the substances at disposal is the best for this purpose. aim of the present study was to compare the different fluids with special regard to postoperative bleeding and rheological behaviour. methods: patients undergoing cabg-surgery were investigated and randomizedly distributed to three different groups of postoperative volume replacement to stabilize the mean arterial pressure at mm hg. . ringer's solution, . . % gelatine solution, . % hydroxyaethylstarch (mean m.w. . ). we evaluated the following parameters within intervals of min: arterial and central venous pressure, heart rate, postoperative bleeding, urinary output, volume replacement. results: there was no statistically significant difference between the groups with regard to urinary output and bleeding. in spite of larger amounts of fluids necessary in the ringer treated group patients of this group showed symptoms of hypovolemia. hematocrit was increased in the ringer patients. this was statistically significant. introduction: pulmonary wedge pressure (pcwp) and central venous pressure (cvp) are frequently used as parameters for cardiac preload, although it is known that both are poorly correlated to the cardiac index (ci). it has been claimed that intrathoracic blood volume (itbv) measured with the thermal dye dilution method reflects cardiac preload better than pcwp and cvp. we studied the correlation between itbv and ci in a mixed population of critically ill patients. methods: in consecutive patients ( sepsis/sirs, acute heart failure, ards, transjugular intrahepatic portosystemic shunt) monitored with a pulmonary artery catheter, itbv was measured on regular intervals using the pulsion cold z- system (pulsion, munich, germany). ci, pcwp, and cvp were recorded simultaneously. results: a total of ol measurements was made. pcwp and cvp did not correlate to ci, nor did apcwp or acvp correlate to aci. itbv was correlated to ci in a non-linear fashion (f - , df = , p < . , (figure) ). aitbv was correlated to ac in a linear fashion (r = . , f = , df = , p < .o ). a rapid and efficient circulatory support system may save a patient in cardiogenic shock. left heart bypass with percutaneous and transseptal placement of the aspiration canuia simplifies the circuit and avoids the need for an oxygenator. we assessed this preclinical set-up in anaesthetized pigs using a centrifugal pump with a f arterial catheter and a f left atrial aspiration line. animals were supported for two hours at a mean flow of . liter ( ' rpm), a mean hematocrit of % and low heparinisetion (act double baseline). hemodynamic and laboratory samples were taken at baseline (a), minutes (b), one hour ( pulmonary hypertension (ph) usually involves obliteration and loss of functional pulmonary microvasculature. the microvaseular endothelium normally acts as a major metabolic organ, converting angiotensin i to angiotensin ii via the angiotensin-converting ectoenzyme (ace). it is unknown whether the loss of functional vasculature and altered pulmonary blood flow seen in ph will affect lung ace metabolic activity. we therefore estimated pulmonary vascular ace activity in patients with ph of various causes: primary; post atrial septal defect closure (asd); chronic thromboembolic (te); anorexigen; iv drugs; collagen disease. single-pass transpulmonary hydrolysis of the specific ace substrate h-benzoyl-pbe-ala-pro (bpap) was measured and expressed as % metabolism (%me . we also calculated an index of peffused functional capillary surface area (amax/km). all patients with ph had an abnormality of %met or amax/km, or both. as compared to control humans (mean %met = . % _+ . % s.d.), the mean %met in ph patients was . % _+ %. the %met in ph patients correlated inversely with cardiac output (r= . ), possibly reflecting more complete bpap hydrolysis with longer pulmonary transit times. amax/km was markedly decreased in ph ( + ml/min) as compared to controls ( _+ ml]min), consistent with a significant loss of functional capillary surface area. patients with collagen disease, asd and anorexigen-induced ph had the most marked abnormalities. in conclusion, patients with pulmonary hypertension have decreased pulmonary endothelial angiotensin converting enzyme activity, likely due to a loss of functional or perfused pulmonary microvaseulature. supported by the funds de la recherche en same du quebec and the national health system of greece. objective: to investigate adrenocortical function in patients with ruptured aneurysm of the abdominal aorta (raaa). studies investigating adrenocortical insufficiency in critically ill patients report an incidence ranging from % to less than %. this may in part be explained by difference in methods used (single cortisol measurement vs short acth stimulation test) and populations studied (heterogenous groups of patients with great individual variation in underlying disease as well as duration and severity of illness). methods: we investigated the adrenocortical function in patients with (raaa).a short acth stimulation test (synacthen test; ug - acth iv) was performed at hrs within hrs of admission. plasma cortisol was measured before (cort basal) and after stimulation (cort stim). a plasma cortisol level > . umol\l before or after stimulation was considered normal, severity of illness was assessed using apache ii. results: of the patients investigated died and survived. mean cort basal in nonsurvivors was significantly (p< .o ) higher than in survivors; . (range . - . ) vs . (range . - , ). this difference between nonsurvivors and survivors was also present for cort stim but lacked significance; . (range . - . ) vs . (range . - . ). while patients showed a cort basal < . , no cort stim < . was found. there was no significant difference in mean age or apache ii score between survivors and nonsurvivors; vs and vs . conclusions: single plasma cortisol levels were inadequate to assess the adrenocortical function in the patients studied, judged by a short acth stimulation test, our investigation in patients with raaa showed no adrenocortical insufficiency. mortality in raaa is associated with elevated plasma cortisol levels. obiectives: mortality in acute myocardial infarction (ami) prinicipally depends on hemedynamic impairment. thus, patients (pts) with elevated pulmonary wedge pressure (pwp) present high in-hospital mortality. however, the complete right heart catheterization is laborious, so the central venous pressure (cvp) alone is frequently used to assess the severity of ami. the accuracy of cvp in estimating pts with ami was tested in this retrospective study. methods: pts. aged + years, admitted to our ccu from to with their first ami, were inctuded in this study. all had undergone right heart catheterization because of overt or suspected heart failure. swan-ganz catheters ( f, cm, abbott, il, usa) had been used, every treatment had been temporarily interrupted l h before the calheferization. based on ecg findings the pts were retrospectively divided into groups. in group a we included pts with anterior ami, in group b, pts with inferior ami, and in group c, pts with inferior and right ventricular ami. the initial values of cvp and pwp were considered for the linear regression of the pwp variable on cvp and p< . was accepted as statistically significant.results: in g~oup a, the cvp and pwp vaiues were + mmhg and _+ mmhg respectively. despite the signifanf correlation (p< . ) between the two variables, it was not possible fo predict the exact value of pwp based on cvp value, pts ( %) presented cvp> mrnhg and of these ( %) had pwp_> mmhg. in group , the cvp was _+ mmhg and the pwp, _+ mmhg. significant correlation (p< . ) between the two variables also existed, however it was impossible to predict the pwp value. pts ( %) had cvp> mmhg but only of these ( %) had pwp> mmhg, similar was the relation between cvp and pwp in group c (p< . ). cvp averaged + mmhg, and pwp, _+ mmhg. pts ( %) had cvp> mmhg and from these ( %) presented pwp> mmhg,conclusions: a single measurement of cvp in ami does not ensure an accurate assessment of pwp. because every pt with ami needs optimal values of pwp in order to prevent pulmonary congestion or manifestations of low preload, the significance of complete right heart catheterization becomes apparent. in patients (pts) with advanced hf the need and the prognosis for heart transplantation (ht) can be predicted from vo= max. indirect measure of functional capacity with the six-minute walk test can also predict smvival in moderate hf. to predict vos max from indirect astinmtions of functional capadty such as - ~q~/, pulmonary and heart function tests, and to assess the prediddve value of the above parameters in hf pts survival. we evaluated pts (age + yeats nyha class: ii, hi, iv) with hf for pit. they underwent a pmgmmive exercise test on cycle ergometer for vo max determination, a -mw, a right heart catheterization and a spirometry and dlco estimation. introduction: brain death causes myocardial impairment by mechanisms that are not well understood yet. the aim of this work was to assess the echocardiographic features found in these patients from the clinical onset of brain death to somatic death, methods: seven brain dead patients were studied (patients" relatives refused to allow them to be used as donors). mean age was . ( - ) years old. four of the patients were female, none of the patients had any history of cardiac disease. transthoracic echocardiogram (echo) and electrocardiogram (ecg) were obtained at the onset of clinical brain death and were repeated every hours until somatic death. we we detected severe diffuse hypokinesia (ef< %) in patients and mild hypokinesia in others (ef - %). systolic function was strictly normal in only patients. corrected qt interval (qtc) in ecg was . _+ . msec (normal range - msec) just before somatic death (b). conclusion: in patients with brain death we observed a significant increase of left ventricular mass due mainly to ivs "hypertrophy" without any important change in the dimensions of the left ventricle. to our knowledge, this finding has never been reported before and its importantance in heart transplantations may be of particular interest. predict right ventricular outcome. l. jacquet, r. dion, p. noirhomme. m. van dijck. m. goenen cardiothoracic intensive care unit, st-luc univ. hospital(ucl) we have registred: heart rate (hr), blood pressure (bp), pulmonary artery pressures (pap), central venous pressure (cvp), pulmonary capillary wedge pressure (pcwp), pulmonary and systemic vascular resistances (pvr, svr), right ventricle end-diastolic end end-systolic volume (redv, resv), right ejection fraction (ref), right sistolyc ventricular work (rsvw) and cardiac output (co) using a thermodilution thechnique and a microprocessor (model ref- ; baxter-edwards laboratory); duration of cpb and aortic clamping, and the requirements of haemodynamic support after cpb.results: in the c group an increase post-cpb of the fc ( + . + . , p < . ) was produced without significantly changes in the redv, resv, ref, rsvw neither co. in the w group, hr increased from . + . to . + . (p < . ); redv was reduced from . -+ to . _+ . (p < . ); resv was reduced from • . to + . (p < . ). there were not changes in the other haemodynamyc parameters. there was a trend (no significantly) to an increase of ref in the w group ( . + . |• . ) compared with the c"group ( • . ($ . • . ) post-cpb. the need for haemodynamic support was similar in both groups.conclusions: the warm, continuous, anterograde-retrogade myocardial protection has obtained a decrease of preload, hr, and a trend to an increase in the ref, making an improvement in the right ventricular global performance when is compared with the classic form of cold myocardial protection. objective: to evaluate the effect of dobutamine on gastric mucosal ph (phi) after coronaly artery bypass surgery. design: prospective study in a university hospital intensive care unit (icu). subjects: elective cardiac surgery patients. interventions: dobutamine was infused at ug/kg/min for hours immediately after admission to the icu. hemodynamics were measured every minute periods until hours and again hours after stopping dobutamine. results: there were no significant differences in mean gastric phi between the groups but mean phi decreased in both groups during the study period. oxygen delivery and consumption both increased during dobutamine infusion but decreased to the control group level after stopping the dobutamine infusion. lactate levels did not change. baseline objectives: the aim of the study was to evaluate the usefulness of a low dobutamine dose in conjunction with intraaortic balloon pumping and mechanical ventilation in cardiogenic shock. we studied patients . -+ t . years of age suffered of post infarction cardiogenic shock characterized by a systolic arterial pressure< mmhg, urine output< ml/h and mental confusion or purpueral signs of low output, non responded to dobutamine infusion up to pg/kg/min. all patients underwent mechanical assistance by the intra-aortic balloon pump (iabp). five patients were additionally placed on mechanical ventilation due to blood gases disturbances. the end points in our study were: reversion of cardiogenic shock, improvement of patients survival or both on the th post infarction day and months later. results: three patients refused iabp treatment and / survived on the th day. on the th day / supported by the iabp and / that underwent mechanical ventilation plus iabp were alive (p < . ). on the th month / supported by the iabp and / that underwent mechanical ventilation plus iabp were alive (p< . ). conclusions: in conclusion, the combined use of mechanical ventilation and iabp assistance in severe cardiogenic shock might improve survival. obiectives: the study was aimed at analysing predictive factors of swan ganz pulmonary catheter (pc) requiremen t during elective cardiac surgery according to the need of sustained inotropic support after surgery. methods: three hundred patients (aged from to ; females and males)were consecutively operated on for elective coronary artery bypass surgery (cabg, n= ), valvular replacement (vr, n= ), combination of both (vr-cabg, n= ), or others (n= ) and retrospectively included in the study. each patient had preoperative invasive cardiac investigation with calculated ejection fraction (ee). anaesthesia, cardiopulmonary bypass (cpb) and cardiac arrest managements were similar in all patients. pc requirement was estimated from the need of either dobutamine, adrenaline, dopamine or enoximone use during the first hours after cardiac surgery. demographic data, asa and nyha classifications, preoperative ef and treatments, type of surgery, cpb and aortic cross clamping (axc) times, and postoperative incidence of complications were compared in patients with or without inotropic support using either student's t test or x with continuity correction when appropriate. results: seventy hree patients ( . %) required inotropic support after surgery. axc .and cpb times, mean stay in icu were significantly longer in patients with inotropie support (p< . ). type of surgery, preoperative ef, and nyha classification are the first significant factors related to inotropic support (p< . ). most patients operated on for double-vr or vr=cabg required inotropic support ( and %, respectively). postoperative mortality was higher in patients receiving inotropic support ( , % vs , % 'overall mortality, p= . ). conclusions: since pc insertion is most.often justified because inotropes are required, these results suggest that elective rather than routine systemic pc insertion could be helped by considering several but selected preoperative factors. background: cardiovascular depression due to anaesthesia, old age and major gastrointestinal surgery is becoming an increasingly frequent challenge .to the anaesthesia-surgory team. deliberate preoperative manipulation of haemodynamics and oxygen transport parametres towards prede~t~mined optimal values may prove to be effective "in reducing morbidity ~nd mortality in high risk surgical patients,. a new concept of using conlimaous perioperative measurement of cardiac'output to obtain and maintain supranormal oxygen delivery (do i) is presented. methods: continuous measurement of cardiac output is a relatively new form of on-line monitoring, in which trains of impulses are emitted from a thermal filament mounted on a pulmonary artery catheter. computer software recognizes patterns generated by minute changes in blood temperature and ealoalates cardiac output every - seconds. cardiac output and mixed venous blood oxygen saturation are displayed graphically on line. in tins tm study cardiac output was measured continuously by vigilance cardiac outpu t compl/ter (baxter). preoperative haemodynamic optimization was performed with the goal of increa- sing do i to at least ml/min/m accordfing to shoemaker's algorithm . this was.done by infusing colloids (albumin or hydroxy ethyl starch (haes-steril| until the desired do was reached. infusion was stopped if cardiac output ceased to increase with infusion, if there were signs of pulmonary oedema or if wedge pressure reached mmhg. vasoactive or inotropic drugs were infused if the desired do was not reached by infusion alone. anaesthetic technique included continuous thoracic epidural and isoflourane anaesthesia. expected mol:bidity and mortality rates were calculated by the "possum" score aasing preoperative clinical and paradinical estimates of organ function as well as surgery characteristics . materials: asa group ill-iv patients with a mean age of years (range - ) and a mean weight of kg (range - )) scheduled for major abdominal surgery were included. results: patients were excluded because do i could not be raised at all. mean do i was increased from ml/min/m (range - ) to ml/min/m (range - ). mean volume of preoperativdy infused colloid was ml (range - ). during surgery ml (range ) of colloid was infused. mean length of surgery was minutes (range - ). mean blood loss was ml (range ). expected mortality and morbidity rates ("possum") were % and %, respectively, whereas patient follow up upon discharge or at death revealed mortality and morbidity rates of % and %, respectively. conclusion: based on experience from the present study, continuous measurement of cardiac output has proved to be a valuable tool for perioperative optimization of do in asa group ili and iv patients during major surgery. however further studies including a greater number of patients are necessary to confirm the promising preliminary findings. we studied the hemodyn~c effects of three different combinations of positiv inotropic .agents, vasodilators, diuretics and av-filtration (av) in patients (pts) with severe left heart faille (left veutrieul x filling pressure (lvfp) > mmhg) due to acute myocardial infarction. hemodynamic measurements (intravascular pressures (lvfp), thermodilution (cardiac index (ci)) were made before (control) and after each therapy. in furosemide (f) + d butamin (d) + nitroglycerin (ni) reduced lvfp and a small increase of ci occurred. in of these pts :(group a) nitroprusside (hip) instead of ni increased ci significantly, in the other pts adding of amrinone (a) resulted in a pronounced increase of ci. group c (n= ): the combination of ni and av reduced lvfp but did not increase ci which was achieved by av+d+ni. in order to optimize the treatment of acute heart failure a combination of inotropic agents, vasodilators, diuretics and av-filtration should he used guided by hemodynamic monitoring. arias jr, miragaya d, sandard, san pedro dm ~, herndndez d, valenzuela . objectives: to evaluate the variation in nomdrenaline (na) plasma concentrations in patients with acute myocardial infarction (am ) after thrombolytic therapy with noniltvasive reperfusion criteria (clinical, electrocardiographic and enzymatic), in relation to infarct size and location.methods: consecutive patiens with ami, from october , to february , , admitted within hours alter onset of symptoms, undergone successfull systemic thrombolysis. of them were anterior (group a) and inferior (group b) . noradrenaline plasma levels at (na ), (na ) and (na ) minutes after admission were compared with ck-peak plasma levels by linear regression. differences were tested for significance by student-t-test for paired and unpaired values. na plasma concentration was measured by high-presssure liquid chromatography. p< ns . ns means -sem (normal limit for our laboratory: na < / pg/ml; ck < u/i ) conclusions: . the na plasma levels at admission (nai) are more increased in anterior than inferior amis, probably in relation to infarct size. . the decrease in na is more evidence in amis with anterior location. . this decrease is probably due to the major efficacy of thrombolytic therapy in amis with anterior location. arias jd, miragaya (group b) , probably due to certain degree of t~cg'rfueion. . there is not significant variation in na in conventional treated ami (group c). v.suchanov, a.levit, p.trofimov, icu, regional hospital, ekaterinburg, russiaobjectives: our task was to improve the technique of preservation of platelet rich plasma. methods: patients scheduled for multiple cardiac valve replacement in were divided into two groups: group i ( patients) -without pp; group ii ( patients) -pp was performed preoperatively. the first pp was made ten days and the second - days before the operation. prp was preserved by cryoconservation. our technique of cryoconservation is distinguished by the speed of freezing ( - ~ and absence of dmso. this made it possible to preserve % functionally active platelets during days. the prp was transfused back after heparin neutralization. the hospital ethics committee approved the investigation.results: the blood loss through the st p. o. d. was significantly greatest in the group i ( _+ ml) and all the patients required transfusion of the donor blood ( + ml) whereas the blood loss in group ii was +_ ml and olny patients required the donor blood. the number of platelets on the st p.o.d, was _+ . /l (group i) and + . /l (group ii), p < . .conclusions: our technique of prp cryoconservation makes it possible to avoid the crystallization phase during freezing of prr thus the infusion of prp may improve hemostasis after open heart surgery and limit the use of the donor blood. in-hospital outcome of women suffering an ami is generally considered worse than that of men, but it is still debated whether female sex is per sea negative prognostic factor or is merely associated with other negative determinants of prognosis. the purpose of the present study is to evaluate the independence of the association between female sex and mortality (in the patients of the swiss centers) and in the patients randomized in the isis- trail mortality rate in women was . % ( / ) compared to . % ( / ) in men; in switzerland: in-hospital mortality for women was . % ( / ), for men . % ( / ).the table shows the results of isis- in terms of odds ratios and their % confidence intervals either after unadjusted analysis or after adjustment for age, known to be the major confounding variable when prognosis of women after myocardial infarction is considered, and for all the available clinical and epidemiological characteristics collected at trial entry: these observations suggest that there is a small but independent effect of female sex on short-term mortality after acute myocardial infarction. ( ) and bubble ( ) oxygenators a, ere used. anaesthesia was balanced and pts were extubated to hrs after cpb. pts were monitored with swan-ganz catheters (sgc) for hrs after cpb. at that time qs/qt was calculate( according to )be standard shunt equation. after the sgc had been removed, an estimated shunt was calculated. measurements of qs/qt were performed: before induction of anaesthesia ( ), after induction of anaesthesia (i[), mins after cpb (iii) (iv) and (v) hrs afiter cpb, rains after extubation (vi), hrs after cpb (v[ ) and on the nd, rd, th, th and tb postoperative day (pd) (viii, x, x, xi, xi , respectively). analysis of data was performed by two-way analysis of variance, p < . being regard as significant.results: the figure shows the values for qs/qt expressed as means + sd. there was a significant increase in qs/qt above b~setine throughoul the whole investigated period except on the th pd. qs/qt reached maximum at rains after extubation (vi). objectives: many stndies have shown advantages of membrane oxygenalors over ubbie type oxygenators. the aim of this study was to evaluate the influence of x 'genator type on pulmonary shunt (as/at) after coronary surgery. methods: patients (pts) gave their informed consent to the study which was approved by the university ttuman research committee. pts were divided into two groups: a (n = ) with a membrane o~genator and a (n = ) with a bubble oxygenalor used during cardiopulmonary bypass (cpb). ths were monitored with swan-ganz catheters (sgc) for hrs after cpb. at that tfme os/ot was calculated according to the standard shunt equation. alter the sgc had been removed, an estimated shunt was calculated..measurements of os/qt were performed: betore induction of anaesthesia (i), mins after extubation ( ), hrs alter cpb ( ) and on the nd, rd, th, th and th postoperative day (iv, v, vi, vii> viii, respectively). analysis of data was performed by one-way analysis of variance, p < . being regarded as significant.results: the figure shows the values for qs/qt expressed as means _+ sd. os/qt was significantly greater at rains after extubation (ii) in a group. the difl'ereuce between the two groups was no more significant from hrs after cpb (iii) to the end of the investigated period. ! i * p < a. s betw~n ~o~ conclusions: membrane ox 'genation during cpb is accomplished by reduction in blood cellular destruction and less alteration in blood. the results of our study show the influence of oxygenator type on value of qs/ot only after extubation ( to hrs after cpb). the difference in qs/qt disappeared his after cpb and since that time the oxygenator type had no influence on qs/qt. it may be of particular importance in patients with severe forms of cardiopulmonary disease who are at risk of higher postoperative morbidity and mortality. objectives: hypomagnesemia has been reported with a variable prevalence ( to % ) in icu patients. magnesium deficiency can induce a number of climcal symptoms (primarily cardiovascular and neuropsychiatric) but can also be clinically silent ( - % are asymptomadc), methods: we measured whole blood ionized magnesium (lmg++) in patients on admission to the icu, using a nova electrolyte analyzer (nova biomedical), containing an img++ electrode. blood was collected in syringes with dry heparin (radiometer qs ). normal range of img++ was found between . - . mmot/l (healthy volunteers). results: for the entire population, we found a % prevalence ( / ) of hypomagnesemia (figure ) . among the surgical patients, the prevalence was highest after cardiac surgery ( %) and after thoracic surgery ( %) and was lowest after neurosurgery ( %). hypomagnesemia was also common in patients after liver transplantation (lvtx) or with hepatic failure ( % for both groups). conclusion: our findings confirm that hypomagnesemia is common in acutely ill patients, especially in those after cardiothoracic surgery or those with liver disease. nevertheless. it is difficult to define the associated factors with sufficient specificity, so that measurements of img++ are warranted to diagnose hypomagnesemia. hepariu influences platelet function and may lead to thrombocytopenia called heparin-associated thrombocytopenia (hat) regardless of the dose and route of administration. additinnal venous and/or arterial thrombosis may lead to life-threatening complications. the incidence of so-calied heparin-associated thrombocytopenia and thrombosis (hatt) ranges between i- %. hatt is confirmed by a heparin induced platelet activation assay (hipa). results: from / to / consecutive patients of our icu were reviewed retrospectively. all patients were treated with heparim the incidence of hatt was % ( ). in all cases diagnosis was proven by a positive hipa. / patients died. in / hatt could be confirmed before severe thromboembolic complications occured. / patients developed a deep vein thrombosis (dvt), / dvt and pulmonary embolism (pe), / dvt, pe and arterial thrombosis (at) and / a dvt, pe~ at and a sinus thrombosis. conclusion: the incidence of hatt in a r series of pts. is %. presence of thrombocytopenia and thrombosis of the great 'vessels is associated with a significant mortality ( / ). computed tom graphy (ct) and transthoracic/transesophageal echocardiography (tte/tee) are important tools in diagnosing and monitoring the extent of cenlrai venous and arterial thrombosis. a. cabral md, m. shahla md c. meneses-oliveira md and jl vincenl md.phd. department of intensive care. erasme university hospital, brussels, belgium objective: to determine extreme hemodynanuc patterns in cardiogenic shock. although ~.~xdiogenic shock is characterized by a low cardiac index (ci), high systemic w~,scular resistance index (svri), and high cardiac filling pressures, some patients may develop art atypical pattern. we reviewed the hemodyuamic pattern of patients with cardiogenic shock, as defined by an initial ct below . l/rain/m: in the presence of myocardial dysfimction attributed to ischemic heart disease (n= ), heart failure (n= ), valvulopathy (n= ) or recent cardiac surgery (n= ). after exclusion of patients with concurrently suspected/documented infection, this study included patients, of whom ( . %) survived. treatment of shock included dopamine (n= ), dobutamine (n= ), norepinephrine (n= ) and epinephrine (n= ). patients with arterial hypertension (ah) and initially law plasnla renin activity (pra) had been studied. in all patient changes of arterial pressure (ap) after single administration of enap was studied. nypotensive reaction wiht deereasin e of average ap about - mm hg ayter single drug administration observed only in patients. ezap monotherapy accomplished during one week with mg daily dose. hypotensive effect observed in patients including ones which were susceptible to single enap administration. after that first stage of therapy all patints began to combinate enap with hypothyazid in dose of mg per day~ after week of treatment such drugs combination lead to veritable ap lowering in addition patients. in the remaining resistant to such drug combination patients was add corinfar in daily dose of mg. this new drug combination permits to lower ap in patients. subsequent discontinuation of enap administration to such patients aid not connected with increasing of again.therefore the most of the patients with ah and law pra( , %)did not susceptible to enap therapy and enap and hypothyazid combination. on the contrary-combination of corinfar with hipothyazid was effective in % patients with ah and low pra. methods: in patients with cardiogenic shock due to ischemic heart disease (n= ), heart failure (n= ) and valvulopathy (n= ), hemod aamic data including measures of intravascular pressures, cardiac output and mixed venous gases were collected at regular times intervals, at least times a da?. all measurements were obtamed in a relative steady state and in the absence of severe anemia or hypoxemia. treatment of shock included dobutamine (n= ), dopamine (n= ), norepinephrine (n=i ) and epinephrine (n= objective: based on our previous studies of the function of isolated liver grafts, this experimental protocol aims at developing a novel extracorporeal liver support circuit, with an incorporated pig liver. methods:the graft liver was obtained from pigs weighing - kg. under general anesthesia the aqimals underwent total hepatectomy,following cannulation of the portal vein, the infrarenal aorta and the infrahapatic vena cava and peffusion wit h it of heparinised r/l solution at ~ the circuit consisted of the graft liver connected to a fluid reservoir and a centrifuge pump. ten healthy pigs weighing - kgr were connected to the circuit as follows: the rt carotid artery was connected to the portal vein of the graft and the rt jugular vein was connected to the fluid reservoir, through the centrifuge pump. the fluid reservoir collected the outflow from the graft's suprahepatic inferior vena cava. the cystic duct of the graft was ligated and the bile.duct cannulated for bile collection and measurement. bridges were adapted to the circuit to bypass the graft liver when necessary, in cases of by pass blood perfusing the graft was oxygenated through a bubble oxygenator. mean total priming volume of the circuit was ml. temperature was maintained at ~ and portal vein pressure at ( - ) mmhg. the flow was . - . ml/gr of graft liver mass per minute. observation period was hours (t ). results: results of the hemadynamic and metabolic monitoring of the recipients [map (t = mmhg , t = mmhg), hr (t = , t = ), rap (t = mmhg , t = mmhg), pap (t = mmhg, t = mmhg), pcwp (t = mmhg, t = ~mhg), svr (t = dyn'sec/cm ' , t = dyn'seclcm~ pvr (t = dyn.sec/cm o, t = dyn.sec/cm ,'~), co (t = . t/min, t = . t/min), do (t = ml/min, t = . ml/min), vo (t = ml/min, t = ml/min), o er (t = . %, t = . % ), ph (to= . , t = . ), po (t = mmhg, t = mmhg), pco (t = mmhg, t = mmhg), pvo (t = mmhg, t = mmhg), svo (t = %, t = %), be, na, k, ca ++, lactate, osmolality, ast, alt, pt, aptt, revealed hemodynamic and metabolic stability of the animal. consumption, co production and tissue oxygenation of the graft were also studied. conclusion; the described circuit proved to be safe and well tolerated by healthy animals but its value for temporary liver support is currently being estimated, in a surgically induced experimental fulminant hepatic failure modal. introduction: prosthetic materials like silikone, dacron, teflon e.tc. produce auto immune responses and may even trigger clinical syndromes like scleroderma, sjogren, sle el.c. in our study we followed the evolution of humorial immunity parametrs for up to five years in a cohort of paced pts with implanted metallic and silicone materials. method: paced pts (mean age +- yrs) without clinical or laboratory findings of malignancy or immune disorders were included. we measured the immunoglobulins, the complement, the auto antibodies and the proteins involved in inflammatory reactions every months. the initial and final mean values are shown in the obiectives: hsp, a systemic leucocytoclastic vasculitis and anaphylactoid purpura can be accompanied by abdominal pain and life-threatening intestinal bleeding. recently we could disclose, that these patients develop severe fxiii-deficiency and immense haemorrhagic oedema of the intestinal wall. by the following case report we will demonstrate and discuss the importance of fxiiideficiency for pathogenesis, therapy and outcome in hsp. case report: a year old man developed typical skin manifestations of hsp following an episode of severe (biliary ?) pancreatitis and percutaneous draining of a pancreatic pseudocyst. two days later he had a paralytic "ileus with immense hemorrhagic wall-oedema and massive dilatation of the small bowel. he got fever up to . ~ and developed severe gastrointestinal haemorrhage (blood transfusions necessary). the coagulation data disclosed a severe fxhi-deficiency (activity %), whereas quickvalues, platelet count and atiii-level were found to be within the normal range. elastase was markedly elevated. substitution of fxiii to normal levels leeds to the cessation of bleeding symptoms and abdominal pain, later resulting in a restitutio ad integrum. conclusions: hsp with intestinal involvement is a life-threatening vasculitis, in which careful and frequent examinations of the coagulation system, especially of fxiii are necessary. detailed analysis of the coagulation data suggest, that the severe fxiiideficiency is due to a specific degradation by proteolytic enzymes (like elastase) as well as consumption within the immense haemorrhagic oedema of the intestinal wall. knowing these facts, even most severe cases of hsp with intestinal involvement can be successfully treated by substitution of fxih. a -year-old woman presented a year history of occasional self-limited episodes of weakness, generalized edema and o!!~aria. the immunologic testing showed no~nnai levels of complements, clq inhibitor, and serum chemistry values, between or during a attack, she was not treated. she was a~mitted to the hospital with symptoms including nausea, vomiting, weakness and ol!guria. on examination, the patient presented facial and g~neralized edema. the systolic blood pressure was mm hg, pulse beats/mir~ute, hematocrit . , seln~n protein /i, and se~um albumin q/l. an leg-kappa pa[apfotein was demostrated ( . g/l) and urine was neaative for puotein. c~'stalloid and colloid don't increased the blaod pressure but resulted in anasarca, with a total of ii lit[as of in~ravenous fluids. therapy wink flozen plasma, . units of clq inhibitor, cortlcosteroids, annihistwnines and antifibrinolytic agents was uns~iccessfull. the a~minist~ation of dopamine, norepineph~ne and epinephrine was inefective. the patient died at the bores, only a few cases have been reported, all had igg paraprotein, the pathophysio!o~] is urd~no~n% but is possible that the paraprotein may be zesponsib!e for the increased capillary pe~leabilityo despite efforts to res~scinate the patients during an acute attack, the syndrome is often fatal. the variable course of systemic uapiliary leak syndrome and the unpredictability and self-limited nature of attacks cloud assessment of therapeutic inte~-vention. the purpose of the present work is to provide some information about the nursing care and results from our experience in continous arteriovenus hemofiltration (cavh).cavh is an extracorporeal technique, especially applicable in the critically ill patients, for disturbances, and for the control of azotemia.we used this method in critically ill patients men and women ages from - who had sepsis -arf congestive heart failure postoperative multiple organ failure and polytrauma .this method was applied to these patients from to hours. % of the patients recovered completely their kidney function, % improved their kidney function and % died.we concluded therefore that this method was very effective for the critically ill patients to whom it was applied, but it requires excellent and continuous nursing care; under the above mentioned circumstances the method works effectivelly. an animal model with rats undergoing a dialysis procedure was designed to test the hypothesis that recovery from ischemic acute renal failure (airf) may be affected by the type of membrane used in hemodialysis. male sprague dawley rats were allocated to groups: in group i, (n= ) airf was inducted by bilateral renal artery clamping for rain. group h (n= ) rats underwent a sham procedure. in each group, rats were dialyzed twice ( th and th day) with either a cuprophan (cupro), a hemophan (hemo) or a pan (an ) minidialyscr or stayed nondialyzed (no hi)). renal function was monitored daily by measuring urea and creatinine values and by two single shot inulin clearances on the days following dialysis. additionally hemolytical activity of complement was determined. inulin clearance on day was reduced significantly but there was no difference in the degree of decrement in glomular filtration rate (gfr) between dialyzed and undialyzed rats, nor between the dialyzed animals with different membranes (gfr: no hi): . _+ . ; cupro: . _+ . ; hemo: . _+ . ; an : . _+ . ). the evaluation of renal function by day nine revealed significant recovery for all airf-groups compared to day (p< . ), irrespective of wether they underwent dialysis or not, or the type of dialysis membrane. complement activation could be detected in all dialyzed groups but no statistical differences between the animal groups dialyzed with different membranes were noticed. our findings refute the hypothesis that in airf exposure to complement-activating cellulosic membranes impairs the recovery of renal function in rats. changes patients: patients who underwent first cadaver kidney transplantation in our unit between january and december in were involved. the recipients were divided into groups: group i." non functioning graft (n= ); group ii: delayed graft function (n= ), group ili: good graft function (n= ). the grouping criteria were: a/haemodialysis in the fii~t postoperative days, b/diuresis in the i st postoperative day, c,' scram crcatininc difference between the st postoperative day and the preoperative level. all of the parameters were involved into the exarainatio, which we measllre in our every, day practice. results: the preoperative haematocrit level differed significantly between group i. ( . ) and croup ii. and iii. ( . and . , p< . ). intmo! emtive significant differences were found between the different groups in systolic blood pressure (group i. hgrmn, group ii. hgnnn, group iii. hgmm, p< . ), mean arterial pressure (group i. hgmm, vs. group ii. hgnun p< . , vs. group iii. hgmm p< . ), and pulse-amplitude and rate-pressure product too. the second warm ishaemic time in group iii. was significantly shorter than in the other two groups (group iii. inin. vs. group ii. rain. p< . , vs. group i. rain. p< . !). the rejection rate was higher in the first days in the patients with non-functioning grafts (group i. % and group ii. % vs. group iii. %) . the other examined parameters have not differed significantly. conclusion: according to our results the success of the kidney transplantation is mnitifactorial. the most important factors of this relationship are: the perioperative fluid-balance, the maintenance of adequate perfusion blood pressure during the operation, good surgical technique and immunological problems. key: cord- -uj fe y authors: nan title: scientific abstracts date: - - journal: reprod sci doi: . / sha: doc_id: cord_uid: uj fe y nan by reduced placental oxygenation, hypoxia-induced oxidative stress is a predominant mechanism. we investigated the effect of hypoxic pregnancy, with and without antioxidant treatment, on placental and maternal circulatory indices of oxidative stress in rats. methods: on pregnancy day , wistar rats were randomised into: normoxia ( % o litters), hypoxia ( % o litters) and hypoxia + vit c ( % o + mg. ml - vit c in water, litters). on day , dams were anaesthetised, maternal blood taken, pups measured and weighed, placentae weighed and frozen. only placentae from two male pups from any one litter were investigated. blood was processed for ascorbate, urate, l-cysteine and glutathione (gsh) measurement. placental protein was analysed for heat shock protein (hsp ; western). results: hypoxia + vit c did not affect maternal food or water intake. vit c elevated maternal ascorbate by % of baseline; a similar increment to human trials (poston et al. ) . hypoxia elevated placental hsp and maternal plasma urate and l-cysteine, but decreased gsh. vit c in hypoxic pregnancies prevented all stimulated effects, but the reduction in gsh persisted. hypoxic pups had a reduced ponderal index and elevated head diameter: body weight ratio; effects also prevented by vit c. fetal oxygen uptake in normal and gdm pregnancies. emanuela taricco, tatjana radaelli, veronica cozzi, gabriele rossi, danila puglia, giorgio pardi, irene cetin. department of obstetrics gynecology "l. mangiagalli", irccs policlinico, mangiagalli e regina elena, milan, italy. background. diabetes in pregnancy has been associated with alterations of fetal growth probably due to increased nutrient availability and placental transport. fetal hypoxia and acidemia have been reported in pregestational diabetic pregnancies with poor glicemic control but this is still uncertain in well controlled patients. the role of placental function and the relationship between maternal and fetal circulation is crucial for efficient exchanges of oxygen and nutrients. since umbilical blood flow can be obtained by us in utero, we studied normal and gdm pregnancies in order to evaluate fetal oxygen uptake. methods. normal (n) and gdm pregnancies were studied at term, at the time of elective caesarean section. umbilical vein volume flow (qumb) was measured by us before caesarean section and blood samples from umbilical vein (uv) and artery (ua) were obtained. blood gases and acid-base balance were evaluated. results. average fetal weights were similar in both groups (n= ± ; gdm= ± g) while placental weights were significantly different (n= ± ; gdm= ± g). n and gdm pregnancies showed similar values of qumb and qumb/kg of fetal weight. (qumb: . ± . in n and . ± . ml/min in gdm; qumb/kg: . ± . in n and . ± . ml/min/kg in gdm). in fetuses from gdm pregnancies a significant reduction in o sat, o cont and po and a significant increased lactate conc was found in both uv and ua compared to n (table ) . o umb uptake (n= . ± . ; gdm= . ± . mmo/l/min) and o umb uptake/kg (n= . ± . ; gdm= . ± . mmo/l/ min/kg) were significantly lower in gdm compared to n fetuses. conclusions. our data indicate that fetuses from gdm pregnancies show a significant reduction in oxygen supply despite a normal blood flow/kg of fetal weight. these data may suggest that a good maternal metabolic control is not sufficient to ensure normal placental oxygen supply and/or utilization by the gdm fetus. background: synthetic glucocorticoids (sgc) are given to mothers at risk of preterm delivery to promote fetal lung maturation. evidence is emerging indicating long-term effects of such treatment on endocrine function and behavior in offspring. however, virtually nothing is known concerning potential transgenerational influences on growth, endocrine function and behaviour. we hypothesize that repeated treatment of grandmothers (f ) with sgc will alter hypothalamic-pituitary-adrenal (hpa) function in f offspring with no manipulation of the f pregnancy. methods: pregnant guinea pigs (f ) were subcutaneously injected with betamethasone (beta; mg/kg) or vehicle (veh) on gestational days / , / / . adult f female offspring from each group were mated with control males. hpa function was assessed in adult f offspring by non-invasive measurement of salivary cortisol concentrations: ) under basal conditions, ) during and following exposure to psychological stress (high frequency strobe light) or psychological/physical stress (forced swim) and, ) following dexamethasone suppression. results: there was no effect of beta (f ) on bodyweight from birth to adulthood in f offspring. basal salivary cortisol in the betaf females was lower than in the vehf group in the morning but not the afternoon; there were no differences in male f offspring. in contrast, both male and female betaf failed to mount adrenocortical responses to psychological stress compared to vehf offspring that mounted robust responses (p< . ). swim stress induced robust adrenocortical responses in all groups (p< . ), however, the response was consistently lower in betaf offspring. beta exposure also led to a significant difference (p< . ) in the cortisol response to dexamethasone suppression in female (f ) offspring, with a similar trend in males. conclusion: prenatal exposure to sgc (f ) causes transgenerational programming of adrenocortical function in adult f offspring. grand maternal exposure to beta results reduced basal adrenocortical activity in betaf female offspring, and caused stress hypo-responsiveness in both males and females. dexamethasone suppression tests indicate altered central glucocorticoid feedback. these findings have important ramifications for the management of human preterm labor. support: canadian institutes of health research. in the uterine and umbilical vasculatures, we hypothesized that their remodeling would also be blunted in pregnant enos -/-mice, leading to an elevated vascular resistance and decreased blood flow to the placenta contributing to fetal growth restriction. methods: utero-and umbilical-placental blood velocity waveforms and umbilical arterial diameters were measured using mhz ultrasound biomicroscopy in control (c bl/ j) and enos -/-mice at . days of pregnancy (n= mothers). spiral artery and fetal capillary morphologies, and uterine arterial diameters were evaluated from vascular corrosion casts. tissues were collected for hydroxyprobe- and actin immunohistochemistry to identify hypoxic and smooth muscle regions. we calculated resistance index ((s-d)/s) from systolic (s) and diastolic (d) velocities, and blood flow from mean velocity and vessel area. results: calculated uterine blood flow normalized to the weight of the uterus and its contents was % lower (p< . ) in pregnant enos -/-mothers due to large reductions in uterine artery diameter (- %, p< . ) and mean velocity (- %, p< . ). uterine arterial resistance index was % higher (p< . ), and the spiral arteries were less coiled and contained more smooth muscle actin in enos -/-mice than controls. more intense hypoxic immunoreactivity was detected in the spongiotrophoblast and trophoblast giant cell layers of the junctional zone of enos -/-placentas, whereas fainter staining was only detected in the spongiotrophoblast cell layer in controls. in the umbilical circulation, flow normalized to fetal weight was not significantly changed although the resistance index was slightly elevated ( %, p< . ) and capillary lobule length was reduced by (- %, p< . ). fetal organs showed increased hypoxic immunoreactivity suggesting reduced organ oxygen delivery in enos -/-fetuses. conclusions: results suggest that enos plays an important role in uterine and spiral artery remodeling and in augmenting utero-placental blood flow during pregnancy. it also appears to enhance oxygen delivery to the placenta and fetus. enos may contribute to normal fetal growth by these mechanisms. integrin methods: hpmcs isolated from term placentas were assessed for their phenotype markers, mutilineage capacity, and the expression of integrin molecules. the hpmcs were induced to endothelial cell differentiation in the presence of endothelial cell growth medium with % of fcs and ng/ml vegf for to days. the angiogenesis ability of these cells was demonstrated by using an in vitro angiogenesis kit and in vivo chick chorioallantoic membrane assay from ten-day-old embryos. blocking antibodies specific to integrin , associated with gdm. this study was adequately powered to detect association in the caucasian and asian group. the absence of association suggests that gdm and t dm may have more divergent molecular pathophysiology than previously suspected. background: uterine endometrium has a unique cycle of physiological angiogenesis. in mice, endothelial progenitor cells (epc) contribute to endometrial angiogenesis being incorporated after oestradiol administration. objective: to determine whether circulating © epc number and function vary through the menstrual cycle in response to changes in circulating sex steroid concentrations. methods: ten healthy, nulliparous, pre-menopausal, non-smoking women (mean age years) with regular menses ( - days) were studied. venous blood was collected during menstrual, follicular, periovulatory and luteal phases of a single cycle (days - , - , - , - ) . cepcs, serum oestradiol (e) and progesterone (p) were measured at each phase. cepcs were quantified by phenotype using flow cytometry (leukocytes co-expressing cd , kdr and cd ) and function by the epc-colony forming unit (cfu) assay. epc-cfus were stained for endothelial markers including uptake of acetylated low-density lipoprotein, binding of lectin (ulex europaeus) and endoglin. results: luteal p was > nmol/l in all women. cepc numbers increased in the menstrual and follicular phases being -fold higher in the follicular compared to periovulatory phase (p< . ). there was no significant variation in cepc function over the menstrual cycle. there was no correlation between serum e or p levels and cepc number or function. conclusion: cepcs number but not function (epc-cfu assay) vary during the menstrual cycle with numbers increasing during the menstrual and follicular phase and falling in the periovulatory and luteal phase of the menstrual cycle. this may represent mobilisation of cepcs from the bone marrow and subsequent incorporation into the endometrium. neither function nor cepc number correlate with serum p or e. our previous studies demonstrated that tissue factor (tf), which binds factor vii to act as a potent pro-coagulant and angiogenic factor, is over-expressed in endometriotic lesions. thus, we determined whether tf could serve as a target for the elimination of pre-established ectopic human endometrial growth in a mouse model. icon is composed of a mutated factor vii (fvii) domain targeting tf and an igg fc (fvii/igg fc) effector domain that activates antibody-dependent immune responses against tf bearing endothelial cells. methods: athymic, ovariectomized and estrogen-treated mice received intraperitoneal (i.p.) injections of . mg of proliferative phase human endometrial tissue derived from normal (disease-free) women. twelve days after inoculation to establish lesions, icon protein ( ug) was delivered i.p. once a week for weeks. after sacrifice, animals were subject to gross inspection. residual endometriotic tissue was formalin fixed, paraffin embedded and immunostained for von willebrand's factor (vwf) and tf. results: compared to control mice, treatment with ug of icon abolished all lesions in of mice and reduced both size ( . to . mm) and number of lesions ( . to per diseased mouse) in the remaining mice. moreover, residual lesions from icon treated mice were atrophic and displayed significant reductions in vessel areas of % +/- % (p= . , mean +/-sem, n= ) as determined by vwf immunostaining. no hemorrhagic or thrombotic sequelae were observed in icon treated mice. conclusions: unlike other treatments that target developing angiogenesis, icon can target both developing and established human endometriotic lesions in athymic mice. the gross and microscopic vessel analysis suggests that icon directly or indirectly destroys endometriotic vessels. thus, icon presents a novel, non-toxic therapy for endometriosis. compared to the miscarriage and top groups. ihc for crisp demonstrated increased secretion of crisp in the glandular epithelium and expression in the leucocytes of the tubal uterine decidua. conclusions: there are differences in decidual gene expression in tubal compared to iu pregnancies. we believe that potential biomarkers of tubal pregnancy can be discovered by focusing on secreted proteins associated with uterine decidualization. one of these proteins, crisp , is significantly increased in decidua of tubal ectopic pregnancies and we are currently investigating its expression pattern in sera from women with tubal compared to iu pregnancies. progesterone and hoxa regulate gaba-a pi receptor expression, membrane translocation and activation. homayoun sadeghi, hugh s taylor. obstetrics, gynecology and reproductive sciences, yale school of medicine, new haven, ct, usa. objective the expression of the gaba-a pi receptor has been previously described in the human endometrium in both luminal epithelium and stroma. it is upregulated during stromal decidualization in the rat and in the implantation window of human endometrium. the gaba receptor is modulated by progesterone metabolites, with the resultant opening of the receptor ion channels which allow the water flux necessary for trophoblast attachment. here we identified regulators of pi subunit receptor gene expression and activity. the well-differentiated human endometrial adenocarcinoma cell line (ishikawa) and human endometrial stromal cells (hesc) were transfected with hoxa , treated with progesterone, or treated with vehicle or empty plasmid controls. gaba-a pi receptor mrna upregulation was evaluated by real time rt-pcr. protein expression was evaluated using immunohistochemistry. results gaba-a pi receptor mrna expression was increased with either progesterone treatment ( %, p= . ) or hoxa transfection ( %, p= . ). coadministration of progesterone along with increased hoxa transfection had no additive effect on the expression of gaba-a pi receptor mrna (p= . ). gaba-a pi receptor protein expression was similarly increased by each treatment. either hoxa or progesterone independently caused translocation of the gaba receptor from the cytoplasm to the cell membrane in ishikawa cells. conclusion gaba-a pi receptor expression is increased in the human luminal epithelium and stroma in the window of implantation. activation of the pi subunit leads to opening of ion channels, likely allowing flux of water into the epithelial cells and out of the uterine lumen. progesterone and hoxa each increase both pi subunit receptor expression and membrane translocation. the lack of additive effect suggests progesterone induced pi subunit receptor expression is likely mediated indirectly through progesterone's regulation of hoxa expression. finally, after receptor expression and translocation, progesterone mediated gaba receptor ion channel activation mediates water resorption necessary for implantation. cancer. suzy davies, donghai dai, kimberly k leslie. obstetrics and gynecology, university of new mexico, albuquerque, nm, usa. objectives: endometrial cancer is the most frequent gynecologic cancer in women. it affects an estimated , women in the us every year, and long term outcomes for patients with advanced stage or recurrent disease are poor. targeted molecular therapy against the vascular endothelial growth factor (vegf) and its receptors constitute a new therapeutic option for patients that is now under study by the gynecologic oncology group in a phase trial, gog e (now in second stage accrual). the goal of our work was to assess the potential effectiveness of vegf/vegfr blockade in preclinical endometrial cancer models. methods: these studies employed two agents, bevacizumab (avastin, a vegfa blocking antibody) and vandetanib (zactima, a tyrosine kinase inhibitor of egfr and vegfr ). ic experiments were performed on endometrial cancer cells in culture using four established cell line models. xenografted athymic mice were also employed to test the ability of compounds to inhibit tumor growth in vivo. tumors were isolated from controls and treated animals, mrna was extracted, and affymetrix gene expression arrays were performed to determine the genes consistently modulated by treatment. results: compared to vandetanib with an ic of . m, bevacizumab showed little activity on cell proliferation in vitro, and cell numbers were not reduced by % using concentrations up to . m. however, bevacizumab demonstrated robust activity in the athymic mouse model, resulting in a significant decrease in tumor formation and growth compared to vehicle treated animals when dosed bi-weekly in a concentration of . mg/mouse ip. tumors from this model demonstrated that eighteen genes were consistently up or down regulated in the presence of bevacizumab. among the regulated transcripts was microrna , which was significantly down-regulated. microrna is an anti-apoptotic factor, and its inhibition by bevacizumab predicts for increased expression of the tumor suppressor pten, decreased cell proliferation, and a reduced capacity for metastasis. conclusions: these studies confirm that the vegf pathway is a good target for new therapies against endometrial cancer. blocking this pathway not only inhibits angiogenesis, but also results in changes in gene expression that enhance apoptosis and reduce cellular proliferation and tumor invasion. we collected fibroid and adjacent normal tissues following hysterectomy with patient consent and institutional irbs. to date, data points for each gene from arrays have been collected from patients. the fluorescence readings were log-transformed and normalized with the robust quartile normalization method. quality control of the normalized data was performed to remove arrays that deviated from twice the inter-quartile range calculated from the array signals. results: the custom microarray profiling identified genes that were differentially expressed between normal and fibroid tissues (p < . ; fdr< . ). among them, genes encode receptor tks, genes encode tk ligands, and genes encode cell cycle and apoptosis proteins. clustering analysis of the mean log ratios of these genes has led to the division of the patients into two major groups. thirty four ( %) belong to a group characterized by the significant downregulation of the cyr (ccn ) gene in fibroid tissues. the cyr -down group can be further divided into two sub-groups based on the expression of another tk ligand, efn a. other receptor differentially expressed tk did not segregate with the three defined sub-groups. finally, there was no sub-group segregation based on age of the patient, menstrual phase, or weight of the fibroid. conclusion: our results have demonstrated that tyrosine kinases and their ligands are uniquely differentially expressed between normal and fibroid tissues. unique tyrosine kinase ligands in our population were cyr and efn a, and these markers created three molecular classification groups based on their differential expression. these results support the hypothesis that tyrosine kinase ligands are involved in fibroid growth, and may offer targets for a strategy to avoid hysterectomy. microvascular perfusion sonographic imaging to detect early stage ovarian cancer. joanie mayer hope, arthur c fleischer, brian day, stephanie v blank, bhavana pothuri, robert wallach, john p curtin, david a fishman. gynecologic oncology, new york university school of medicine, new york, ny, usa; radiology, vanderbilt university medical center, nashville, tn, usa. objective: epithelial ovarian cancer (eoc) is the th leading cause of death in us women due to the inability to detect early stage disease. recent sonographic developments involving harmonics, pulse inversion, and the use of contrast agents justify the hope that depiction of aberrant tumor microvascularity associated with early disease can occur. this study utilizes these new techniques to assess the unique microvascularity associated with early stage eoc. methods: we used pulse inversion harmonic microvascular imaging (mvi) technology to depict differences between benign and malignant ovarian lesions. contrast enhanced harmonic transvaginal (tv) sonography was performed using the philips iu scanner after intravenous injection of g of definity (bristol-myer-squibb). split screen real-time images were acquired displaying conventional sonographic views adjacent to harmonic, low mechanical index images. morphologic features (thickened wall, papillary excrescence, calcifications) and aberrant vascularity were noted. q-lab quantification of wash-in, peak enhancement, and wash-out times as well as area-under-thecurve (corresponding to microvessel perfusion) were compared using student's t-tests. results: to date, contrast enhancement patterns of ovaries have been analyzed, benign and malignant. of the malignancies ( fallopian tube, ovarian: stage i, stage iii), / women were correctly identified using conventional tv imaging and others were identified using mvi / . all benign lesions were correctly identified by mvi / while tv detected normals ( false negatives). the lesions detected as malignant by mvi were -stage i fallopian tube and -stage i eoc. contrast enhancement kinetics of malignant lesions demonstrated similar wash-in ( . ± . vs . ± . , p = . ), greater peak enhancement ( . ± . vs . ± . , p < . ), longer wash-out ( . ± . vs . ± . (p < . ), and greater perfusion ( . ± . vs . ± , p < . ) when compared to benign lesions. conclusion: contrast enhancement patterns are significantly different in benign vs. malignant ovarian masses. this technique has clear potential in differentiating benign from malignant lesions and for detecting occult stage i disease. identification and characterization of mir- a as regulator of ikk expression and its function in ovarian cancer cells. rui chen, ayesha b alvero, thomas rutherford, gil mor. department of obstetrics and gynecology, yale university school of medicine, new haven, ct, usa. introduction: the proinflammatory environment associated with tumor growth and chemoresistance is produced by both immune cells and cancer cells. the nf-b pathway plays a critical role mediating the capacity of cancer cells to produce pro-inflammatory cytokines. recently, we described a group of epithelial ovarian cancer (eoc) cells characterized by ikk expression as the main factor promoting nf-b activation and cytokine production. in this study we evaluated the regulation of ikk in eoc cells. we describe the identification and characterization of mir- a as a regulator of ikk expression, thus indirectly of nf-b activity and function. materials and methods: human eoc cell lines were established from malignant ovarian cancer ascites. protein expression were determined by western blotting. ikk mrna was measured by rt-pcr. cytokines were profiled by the luminex system. ikk transfection was done with roche fugene transfection reagent. mirna microarray was done with invitrogen ncode multi-species mirna microarray kit. mir- a qrt-pcr was performed with invitrogen ncode sybr greener mirna qrt-pcr analysis kit. mirna transfection was done with ambion siport neofx agent. the ikk '-utr luciferase reporter plasmid was established based on ambion pmir-report mirna expression reporter vector. results: ikk expression was associated with nf-b cyclic activity and the ability of type i eoc cells (but not type ii) to produce inflammatory cytokines. transfection of ikk into type ii eoc cells reversed their phenotype. mirna microarray identified mirnas differentially expressed in type i versus type ii cells, one of which, mir- a, had putative binding sites in the '-utr of ikk mrna. mir- a introduction into type i cells inhibited ikk expression, and direct inhibition through ikk 's '-utr was confirmed by luciferase assay. conclusion: we describe for the first time the identification of ikk as a potential key switch between chemo-resistant and chemo-sensitive phenotypes, by regulating nf-b activity in eoc cells. furthermore, we identified mir- a as a direct regulator of ikk expression. ikk expression may represent an adaptational stage in tumor progression allowing cancer cells to create their own inflammatory environment. these findings may provide novel molecular targets and potential markers for individual therapy selection. regulation of cd in the rat uterus by nitric oxide and the involvement of p kinase pathway. uma yallampalli, rebakah elkins, pawel goluszko, chandra yallampalli. obstetrics gynecology, university of texas medical branch, galveston, tx, usa. objective. cd is expressed in many cell types including uterine cells and has been shown to play an important role in protecting against compliment attack. we have previously shown in endometrial cell lines that cd levels were down regulated by nitric oxide (no) . in this study we extend our previous observations to determine if no down regulates cd in rat uterine tissues and assess its mechanisms of action. methods. non pregnant rats ( g; b wt) were bilaterally ovariectomized under ketamine anesthesia. groups of ovariectomized rats were implanted with alzet mini pumps to deliver nitro-l-arginine melthylester (l-name) at or /mg/rat/day in saline or saline alone. uteri were obtained from these rats at or hours after infusion. in another set of experiments uteri were obtained from ovariectomized rats and cut in to small pieces and incubated in vitro with either l-name ( mm), diethylenetriamine-no mm) or worthmanin ( . m) in mem without phenol red for hours. uterine tissues were homogenized in trizol and mrna levels for cd were measured using rt-pcr and expressed as a ratio to s. results. results show that in vivo treatment with l-name to ovariectomized rats caused elevations in uterine cd mrna levels in a time and dose dependent manner with maximal responses seen with mg l-name and at hours. in vitro studies show that deta-no suppressed cd mrna levels in the rat uterus. both l-name and pi kinase inhibitor, worthmanin caused increases in cd levels in these tissues and the effects of worthmanin are reversed by deta-no. these results suggest that cd levels in the rat uterus are down regulated by no and are upregulated when no synthesis is inhibited by l-name. further, pi kinase appears to be involved in cd regulation in the rat uterus and no donor appears to modulate this response. lung. lakeitha r foster, daniel b hardy, carole r mendelson. dept of pediatrics; depts of biochemistry and ob/gyn, ut southwestern medical center, dallas, tx, usa. during % of human pregnancy, the maternal uterus is maintained in a state of almost complete quiescence by elevated circulating levels of progesterone (p ). we previously observed that p acting through the progesterone receptor (pr) serves an anti-inflammatory role and inhibits uterine contractility by antagonizing nuclear factor b activation and cyclooxygenase- (cox- ) expression (hardy et al., ) . our previous findings also suggest that the fetus provides an important signal for the initiation of labor near term through augmented secretion of the major lung surfactant protein, sp-a, into amniotic fluid (condon et al., ) . secreted sp-a, in turn, activates fetal macrophages which migrate to the maternal uterus where they release cytokines and promote an inflammatory response, leading to labor. in the present study, we tested the hypothesis that maternal p also maintains uterine quiescence by inhibiting sp-a production by the fetal lung. age matched icr mice were injected s.c. once daily either with sesame oil (control) or p ( mg/ml) from to days post-coitum (dpc). as expected, treatment with p delayed parturition by - h. this also was associated with a decrease in uterine cox- mrna expression, as compared to the vehicle-injected controls. interestingly, maternal p treatment caused a marked decrease in the levels of immunoreactive sp-a protein secreted by the fetal lungs into in amniotic fluid at dpc. furthermore, sp-a protein and mrna levels were reduced in the fetal lungs of p -injected mothers, as compared to controls. this was associated with an inhibitory effect of p treatment on cox- protein and mrna levels in the fetal lungs. these findings were of interest, since cox- expression is markedly upregulated during differentiation of human fetal lung (hfl) explants in culture (hardy et al., ) and endogenous and exogenous prostaglandins increase sp-a expression in hfl (acarregui et al., ) . collectively, these findings suggest that maternal p treatment prevents increased uterine contractility, in part, by inhibiting inflammatory response pathways within the fetal lung. this, in turn, blocks the developmental induction of sp-a expression and its secretion into amniotic fluid. in this manner, maternal p inhibits an important fetal signal leading to labor. supported by nih p hd ; nih r hl . recent evidence suggests that leukocytes infiltrate uterine tissues at the time of parturition implicating inflammation as a key mechanism of human labor. ccl- is a pro-inflammatory cytokine that may contribute to the development of inflammatory reaction in the myometrium. previously we showed upregulation of rat ccl- gene expression in myometrium during term and ru -induced preterm labor. also ccl- was elevated specifically in the gravid horn of unilaterally pregnant rats suggesting that mechanical strain imposed by the growing fetus controls its expression in the myometrium. the objective of this study was to investigate the role of mechanical stretch as a possible regulator of myometrial leukocyte infiltration and ccl- as a mediator of this stretch response. we also studied the effect of progesterone (p ) on the myometrial secretion of ccl- . methods. we used primary culture of rat myometrium smooth muscle cells (smcs) to study in vitro ccl- gene and protein induction by static mechanical stretch. ccl- gene expression analysis was performed by real-time rt-pcr and immunoreactive (ir) protein content was measured by elisa assay. we used primary rat monocytes to access whether stretch-induced ccl- production by myometrial smcs resulted in enhanced monocyte chemotactic activity. results. myometrial cells were stretched for - hours and the supernatants collected. analysis of media conditioned by primary myometrial smcs revealed that static mechanical stretch ( % elongation for hours) caused a significant accumulation in ir ccl- which was repressed by pretreatment with p ( um). the rise in ccl- protein levels was preceded by a transient increase on ccl- mrna. the migration of primary rat monocytes in response to conditioned medium from stretched myometrial smcs was much greater than that of conditioned medium from control non-stretched cells. co-incubation with a neutralizing antibody to ccl- significantly reduced the chemotaxis of monocytes in response to the stretch-conditioned medium. conclusion: uterine smcs play an active role in uterine inflammation by producing chemokines and promoting the chemotaxis of immune cells into the myometrium. the blockade of this effect by p offers a potential explanation for the therapeutic actions of this hormone in the prevention of preterm birth. membranes during human labor. nardhy gomez-lopez, , guadalupe estrada-gutierrez, lourdes vadillo-perez, felipe vadillo-ortega. direction of research, instituto nacional de perinatologia, mexico city, df, mexico; escuela nacional de ciencias biologicas, ipn, mexico city, df, mexico. introduction. leukocytes arriving to the choriodecidua (chd) during labor are capable to secrete cytokines and matrix metalloproteinases that may play a role in the fetal membranes (fm) extracellular matrix degradation. objective. the aim of this work was to identify changes in the leukocyte subpopulations in the chd and fm during human labor. methods: fm were obtained from two groups of women: ) term without labor (n= ) and ) term with spontaneous labor (n= ). chd cells were isolated and analyzed by flow cytometry. explants of fm were embedded in paraffin and analyzed by confocal microscopy. in both techniques, cd , cd , cd , cd and cd subpopulations of leukocytes were identified. intracellular mmp- was also identified in these cells. results: major changes in leukocytes subpopulations during labor involved a higher amount of cd +, cd+ and cd + cells, both in the chd and inside the fm. mmp- was associated to cd + cells. cd + exhibited a more widespread localization in the fm and cd + cells were localized in the contact with the trophoblast layer during labor. conclusions: leukocyte populations changes both in the chd and fm during labor and are characterized by arrival and infiltration of specific subpopulation of lymphocytes and monocytes. nk cells are enriched in mmp- , which may be related to a role in extracellular matrix degradation leading to the rupture of fetal membranes. women with a history of a pregnancy complicated by preeclampsia or intrauterine growth restriction (iugr) have an increased risk of future cardiovascular disease. excessive weight, particularly abdominal fat mass, is associated with cardiovascular morbidity and mortality. objectives: the aim of this study was to investigate differences in body composition and fat distribution between women with a history of preeclampsia or iugr and uncomplicated pregnancies. methods: from a genetically isolated population in the southwest of the netherlands, non-pregnant women with a history of preeclampsia (n= ), iugr (n= ) and uncomplicated pregnancies (n= ) were recruited at a mean follow up time of . years after pregnancy. body composition and fat distribution were assessed by dual energy-x-ray absorptiometry (dxa) and anthropometric measurements. results: women with a history of preeclampsia compared to controls had higher mean total-, fat-and lean mass (p < . ) as well as higher mean indices of body mass, fat mass and lean mass (p< . ). no significant differences were found for these variables between women with a history of iugr and controls. women with a history of preeclampsia had higher waist circumferences and waistto-hip ratios (p< . ) as well as excess of android fat mass and increased android-to-fat ratios (p< . ). women after pregnancies complicated by iugr had higher waist-to-hip ratios (p < . ). after controlling for body mass index, both women with a history of preeclampsia or iugr had higher waist circumferences (p < . ) and waist-to-hip ratios (p < . ) as well as smaller hip circumferences (p < . ). conclusion: despite differences in body mass index, both women with a history of preeclampsia and women after pregnancies complicated by iugr have a metabolically adverse fat distribution, marked by an excess of fat deposition in the abdominal region relatively to the hip region. these findings may explain, at least partly, their increased cardiovascular risk. women with a history of preeclampsia. marc ea spaanderman, timo h ekhart, robert aardenburg, louis lh peeters. obstetrics and gynecology, radboud university medical center, nijmegen, netherlands; obstetrics and gynecology, university medical center maastricht, maastricht, netherlands. background: a history of preeclampsia is associated with persistent short-term alterations in circulatory function and remote cardiovascular disease. in this study we tested the hypothesis at least years after preeclamptic pregnancy renal and central hemodynamic function is impaired as compared to women with uncomplicated pregnancy. methods: in formerly preeclamptic women (pe) and healthy parous controls (control) who were normotensive at weeks post-partum follow up, we assessed at least years after delivery blood pressure (mmhg), cardiac output (co, doppler ultrasonography, l/min) and effective renal plasma flow (erpf, pah clearance, ml/min/ . m ) after which we calculated total peripheral vascular resistance (. dyne.s/cm ) and renal vascular resistance (. dyne.s/cm ). data were analyzed parametrically (p< . ). results: age and bmi were comparable between groups. blood pressure was higher in formerly pe. moreover, % of formerly pe women and % of control were hypertensive (p< . ). although cardiac out was comparable between groups, total peripheral and renal vascular resistance were about % higher and erpf % lower in formerly pe women as compared to control. conclusion: at least years after gestational hypertensive disease, women who were normotensive at direct follow up have impaired renal and central hemodynamic function and developed more often chronic hypertension. long-term follow up may also be warranted in apparently healthy formerly pe women who are normotensive at post-partum follow up. circulatory function in formerly preeclamptic women and healthy parous controls co erpf tpvr rvr formerly pe . ± . ± * ± * ± * control . ± . ± ± ± * = p< . pregnancy increases blood-brain barrier permeability coefficient (l p ) to lucifer yellow: role of estrogen. marchien j wiegman, , marilyn j cipolla. neurology, ob/gyn and pharmacology, university of vermont, burlington, vt, usa; ob/gyn, university medical center groningen, groningen, netherlands. background: eclampsia is similar to posterior reversible encephalopathy syndrome in which an acute rise in blood pressure causes breakthrough of autoregulation, blood-brain barrier (bbb) disruption, and cerebral edema formation. we previously showed that late-pregnant (lp) animals developed cerebral edema during breakthrough, a response that was absent in nonpregnant (np) animals. in the current study we hypothesized that pregnancy predisposes the brain to edema during acute hypertension by enhanced bbb permeability. we further hypothesized that the underlying effect of pregnancy on the bbb permeability is due to elevated estrogen levels. methods: permeability coefficients (l p ) to lucifer yellow (ly), a polar compound that does not pass through tight junctions, were compared in posterior cerebral arteries (pca) from groups of sprague dawley rats: np (n= ), lp (d ; n= ), ovariectomized and implanted with -estradiol ( . mg, -day release) and estriol ( . mg, -day release) pellets for days (ovx+e; n= ), and ovariectomized and implanted with placebo pellets for days (ovx; n= ). pcas were isolated, pressurized in an arteriograph, and perfused with . mg/ml ly in saline. concentration changes of ly outside the vessel wall were determined at pressures from - mmhg. the slope of the pressure vs. permeability curve is the rate of flux, or l p for ly. results: l p for ly was significantly increased in pcas from lp and ovx animals vs. np (p< . ; figure ). estrogen was protective of the bbb only in ovariectomized animals, decreasing l p % in ovx+e vs. ovx. however, pregnancy did not afford protection and had a l p that was % greater than np. conclusions: pregnancy significantly increases bbb permeability to ly, an effect that may predispose the brain to edema formation during acute hypertension. these data also show that estrogen modulates l p in ovariectomized animals differentially than pregnancy, suggesting that the increased bbb permeability in pregnancy is caused by a mechanism other than elevated estrogen levels. in both pre-and early pregnancy, the sympathoinhibitory response to volume expansion is blunted in formerly preeclamptic women with low plasma volume. ineke krabbendam, marc ea spaanderman, dorette a courtar, robert aardenburg, ben j janssen, fred k lotgering, louis lh peeters. obstetrics and gynecology, radboud university nijmegen medical centre, nijmegen, netherlands; obstetrics and gynecology, university hospital maastricht, maastricht, netherlands; pharmacology and toxicology, university of maastricht, maastricht, netherlands. background: the circulation of formerly preeclamptic women with a low plasma volume (lpv) is characterized by sympathetic dominance. these women respond to a new pregnancy with an aberrant rise in atrial natriuretic peptide (anp) and a times higher chance to develop recurrent gestational hypertensive disease compared to their counterparts with normal plasma volume (npv). anp has sympathicomimetic capacity. we postulate that the sympathetic overdrive in lpv-women is associated with a reduced venous capacitance. to this end, we compared the response to volume expansion (ve) in women with lpv and npv, both before and in pregnancy. method: in non-pregnant normotensive formerly preeclamptic women, we measured pv (hsa i indicator dilution method) at least months post partum. we intravenously infused ml of iso-oncotic fluid over minutes. during the infusion, we recorded changes in heart rate (hr, bpm), blood pressure (bp, mmhg), cardiac output (co, l/min), sympathetic activity (lfsys, mmhg , low frequency component of spontaneous fluctuations in systolic bp, portapress) and anp (nmol/l). eight women became pregnant within year and were evaluated at weeks gestation. changes in circulatory and autonomic function between and within groups were analyzed non-parametrically (p< . ). results: before pregnancy, ve leads to comparable changes in hr, bp and co in women with lpv ( / ) and npv ( / ). in npv, lfsys decreased %, but only % in lpv (p< . ). anp remained unaltered in npv, but increased in lpv. in the pregnant group, women had lpv and had npv. in both groups, pregnancy did not alter the response to ve. conclusion: irrespective of pregnancy, the sympathoinhibitory response to ve is diminished in lpv. these data suggest that in these women ve leads to venous overfill, giving rise to anp-release and consequently sympathetic activation, flattening the normal baroreceptor-mediated sympathoinhibitory response. we speculate that this mechanism contribute to circulatory maladaptation to pregnancy, sympathetic dominance and subsequent gestational hypertensive disease. in both pe and ht, serum sflt- was increased, and plgf reduced at all gestations (p< . ). seng levels were also increased in pe. after weeks (but not before) antihypertensive treatment was associated with a significant fall in serum sflt- and seng, in pe only. the concentrations of both sflt- and seng were significantly higher in the placentas of women with pe, but not ht, compared with controls (p= . ). only sflt- was significantly reduced in the placenta in women who received antihypertensive therapy. conclusion in pe, antihypertensive therapy after weeks' gestation is associated with a significant fall in serum sflt- and seng, and in placental sflt- . these findings raise the possibility that these drugs may have an effect on the pathophysiology of pe other than their known antihypertensive action. the synergistic effect of soluble vegf receptor pre-treatment and small doses of tnf-on endothelial cells. tereza cindrova-davies, debbie a sanders, olivera spasic-boskovic, graham j burton, d stephen charnock-jones. dept of pdn, university of cambridge, united kingdom; dept of obstetrics and gynaecology, university of cambridge, united kingdom. introduction: preeclampsia is marked by an enhanced endothelial inflammatory response manifested by maternal endothelial activation. soluble fms-like tyrosine kinase- (sflt- , svegf-r ), a naturally occurring circulating antagonist of vegf-a and plgf, is one of the secreted factors implicated in the pathogenesis of preeclampsia. in women who develop preeclampsia, sflt rises sharply, preceding the onset of the clinical disease. the aim of this study was to examine the effect of a combined treatment with recombinant sflt- and tnf-on the activation of human umbilical cord endothelial cells (huvec) by examining leukocyte adhesion, and the expression of icam, vcam, endothelin and vwf. methods: huvec were seeded, grown overnight and pre-treated with recombinant sflt ( - ng/ml) in a basic % fbs-dmem medium for hr. on day , low doses of tnf-( . ng/ml) were applied to pre-treated cells for hr. antagonism of the vegf-a action was mimicked at the protein level by pre-incubating huvec with anti-flt antibody ( - g/ml), anti-kdr antibody ( g/ml), anti-vegf antibody ( g/ml), or vegf receptor inhibitor su ( m). at the rna level, the effect of sflt was mimicked by sirna transfection of huvec with siflt or sikdr. at the end of each experiment cells were either harvested for western blotting, fixed in % pfa for immunofluorescence or incubated with labelled hl leukocyte cells, followed by fluorescent detection of adhesion. results: pre-incubation of huvec with sflt and subsequent treatment with low doses of tnf-increased the adhesion of hl leukocytes and increased icam- , vcam- , endothelin and vwf, compared to tnf-treatment alone. similar results were obtained when cells were pre-treated with su , anti-flt, anti-kdr or anti-vegf. transfection knock-down of flt or kdr gene also significantly increased leukocyte adhesion when small doses of tnfwere added. conclusions: pre-incubation with recombinant sflt, anti-flt, anti-kdr, anti-vegf, su or knocking-down flt or kdr transcripts all antagonised the autocrine actions of vegf and/or plgf. this predisposes huvecs to be more sensitive to the effect of tnf-. our study shows that sflt and tnfcombine to induce an enhanced synergistic effect, activating endothelial cells. maternal obesity is associated with increased production of inflammatory cytokines and risk of poor perinatal outcome. inflammatory cytokines can stimulate production of reactive oxygen (ros) and nitrogen (rns) species, which can covalently modify protein function. placental oxidative and nitrative stress are increased in pathological pregnancies and associated with altered placental function. objectives: determine the effect of increasing body mass index (bmi) on placental nitrative stress, measured by the expression and localization of nitrated (nitrotyrosine) and oxidized proteins. methods: placental tissue was collected at term ( - wks) from lean kg/m ), overweight ( - . kg/m ) and obese ( - kg/m ) patients (n= or /group). tissue was sectioned for immunostaining with nitrotyrosine antibody. protein samples were either dot blotted onto nitrocellulose membrane, probed with nitrotyrosine ab, and nitrated proteins detected using ecl, or derivatized using , -dinitrophenylhydrazine (dnph) (oxyblot® kit), separated on sds-page, probed with anti-dnph ab ( : ) and oxidized proteins detected using ecl. protein band intensity was measured by densitometry. oxidized proteins were selected for maldi mass spectrometry analysis. results: nitrotyrosine residues were immunolocalized primarily in the fetal capillary endothelial cells and the villous stroma, but were almost absent in the syncytiotrophoblast. by dot blot, nitrotyrosine expression differed across the three groups (p< . , anova) with expression in tissue from obese women being significantly increased compared to lean (p< . ) and overweight (p< . , tukey test). several oxidized proteins were detected with significantly greater expression seen in the lean versus overweight groups (p< . , mann-whitney u). one oxidized protein was identified by maldi-ms with four peptide matches and . % coverage as -beta hydroxysteroid dehydrogenase ( bhsd). discussion: with increasing bmi, an increase in nitrative stress appears to occur in parallel with a decrease in oxidative stress. oxidative stress is apparently reduced as ros are consumed by the interaction with rns to give nitrative stress. bhsd is involved in the biosynthesis of steroid hormones and glucocorticoids. its activity and hence steroid metabolism in the placenta may be regulated by oxidation with implications for fetal development. background teenagers are susceptible to delivering small-for-gestationalage (sga) infants. previous studies implicate continued maternal growth as a causal factor . growing adolescent sheep have reduced fetal birthweight due to impaired placental development and nutrient transfer . we hypothesized that placental function is impaired in human teenage pregnancy if there is maternal growth. methods placentas were collected from teenagers ( - years) and adults. activity of the amino acid transporter, system a, was quantified by the sodium-dependent uptake of c-methylaminoisobutyric acid into placental fragments. teenagers were defined as growing (> mm increase in kneeheight per days) or non-growing. system a activity was analysed in relation to individualised birthweight centile and maternal growth. results placental system a activity was significantly lower in teenage compared to adult pregnancies (p< . ). this was unrelated to birthweight; teenagers who delivered infants appropriate-for-gestational age (aga) had significantly lower placental system a activity compared to aga infants delivered to adults (p< . ). growing teenagers did not deliver lower birthweight infants than non-growing teenagers (median birthweight g and g respectively). furthermore, system a activity in placentas from growing teenagers was significantly elevated compared to that in non-growing teenagers (p< . ), and was similar to placental activity in adults. conclusions system a activity was reduced in placentas from teenagers compared to adults. this suggests that inherently lower placental function predisposes teenagers to sga, but that other factors (e.g. adequate nutrition) can compensate in those teenagers delivering aga infants. in contrast to our hypothesis, placental system a was elevated in growing teenagers and mimicked that of adults. this may be related to a hormonal-milieu in growing teenagers that is conducive to fetal growth, in part through stimulating placental transport. homocysteine inhibition of system a amino acid transport activity in human placenta. eleni tsitsiou, susan l greenwood, colin p sibley, stephen w d'souza, jocelyn d glazier. maternal and fetal health research group, st. mary's hospital, university of manchester, manchester, united kingdom. background: elevated plasma levels of the amino acid homocysteine (hcy) during pregnancy are associated with vascular-related complications and adverse neonatal outcomes including a reduced birthweight. fetal and maternal plasma hcy concentrations are positively correlated suggesting placental transport of hcy may be an important determinant of fetal plasma hcy. the mechanisms involved in placental hcy transport are uncharacterised. evidence that the system a amino acid transporter, which transports neutral amino acids in a na + -dependent manner, is important in promoting fetal growth and that a reduced system a activity is associated with intrauterine growth restriction (iugr), led to our hypothesis that system a provides one mechanism for placental hcy transport. this hypothesis was tested by measuring the ability of hcy to inhibit system a activity in isolated microvillous plasma membrane (mvm) vesicles and placental fragments. materials and methods: mvm vesicles and placental fragments were isolated from placentas of normal pregnancies at term. system a activity was measured at initial rate ( s and min respectively) as na + -dependent cmethylaminoisobutyric acid (meaib) uptake into mvm vesicles ( . mm) or fragments ( . mm) in the absence (control) or presence of l-hcy and dl-hcy or model substrates. results: mm l-hcy (custom-synthesised) and dl-hcy (commercial source) significantly (p< . ) inhibited na + -dependent c-meaib uptake into mvm vesicles compared to control; comparable in magnitude to other model substrates (meaib, l-ala, l-ser, l-met; n= , kruskal-wallis with dunn's multiple comparison test). l-hcy, l-met and meaib ( . - mm) caused a dose-dependent inhibition of na + -dependent c-meaib uptake into mvm with ec values (in mm; mean ± se) of . ± . , . ± . , and . ± . respectively, n= ). na + -dependent c-meaib uptake into fragments was reduced substantially in the presence of mm l-hcy or dl-hcy causing a ± and ± % reduction (mean ± sd, n= - ) of control respectively. conclusion: these observations suggest that hcy is a relatively high affinity substrate for system a in the human placenta. we speculate that inhibition of placental amino acid uptake by hcy could impact on fetal growth and development. supported by the mrc and action research endowment fund. glucose regulates placental mtor activity and glucose deprivation down-regulates placental system l activity in an mtor-dependent manner. sara roos, theresa l powell, thomas jansson. department of physiology, institute of neuroscience and physiology, gothenburg, sweden; department of obstetrics and gynecology, university of cincinnati, cincinnati, oh, usa. placental amino acid transporters are down-regulated in intrauterine growth restriction (iugr). we have previously shown that mammalian target of rapamycin (mtor) regulates placental system l transporter activity and that placental mtor activity is decreased in iugr. however, the upstream regulators of placental mtor are unknown. in iugr, fetal hypoglycemia and reduced maternal glucose levels are common and the placenta may therefore be exposed to low glucose levels. hypothesis: we hypothesized that glucose availability regulates placental amino acid transporter activity mediated by changes in mtor signaling. methods: cytotrophoblast cells were isolated and cultured until syncytialization at hours. cells were cultured for an additional hours in culture media containing . mm, . mm, or mm glucose (control), which corresponds to standard culture media. at hrs, the activity of the mtor signaling pathway was assessed by measuring the protein expression of s k phosphorylated at thr- , the primary site of mtor phosphorylation. in another set of cells, system l activity was assessed by measuring the bchinhibitable uptake of h-leucine. results: as compared to control, phospho-thr- -s k expression was reduced by % in cells incubated in . mm results: fgr pregnancies were delivered earlier ( ± . v ± . w; p< . ), weighed less ( . ± . v . ± . kg; p< . ) and had higher s/d ratios ( . ± . v . ± . ; p< . ). eaa concentrations were similar between groups, but there were differences (non-parametric testing; p< . ) in transport rates between groups with his crossing considerably faster and ile crossing slower in fgr . the table shows fetal vein/maternal (fv/m) ratios standardized to the leu fv/m ratio for all eaa for both groups. amino acids fell into groups for fgr pregnancies: ) his (ratio . ), ) leu, phe, met (ratios ), and ) val, thr, ile, trp, lys with intermediate ratios ( . ns conclusion: this is the st study to compare the relative rates of in vivo placental transport for all eaa between normal and fgr human pregnancies showing striking differences in the transport rates of two eaa. in the absence of eaa concentration differences between groups, the higher his transport rate in fgr suggests higher utilization by the placenta. vasculature of women who received exogenous estrogen compared to those who received placebo. the purpose of this investigation was to identify the gene expression in response to the estrogen, equilin, as the major component of conjugated equine estrogen and genistein, a phytoestrogen in human coronary artery endothelial cells. human coronary artery endothelial cells from a -year old female were used. cells were treated with estradiol [e ], equilin [eq] ( . nm) or genistein [gen] ( micromolar) for hours. focused oligomicroarrays for cardiovascular disease and endothelial cell function were used to study gene expression. rt-pcr was used to confirm the transcription of genes analyzed in oligoarrays. vascular adhesion molecules and genes involved in the inflammatory response were most affected with the three estrogen sources. significant reduction of integrin alphae was seen with all three . , . and . for e , eq and gen respectively. similarly nfkb was also reduced . , . and . fold compared to controls. significant reduction of ccl was demonstrated with eq ( . fold) and gen ( . fold). tnfreceptor a and b were only significantly decreased by gen. vcam- which is regulated by proatherogenic factors and upregulated mainly at atherosclerosis -prone sites, was significantly reduced ( . -fold) with genistein, and a slight reduction was seen with e and no change was observed with eq. our data support the clinical findings that estrogen has favorable effects on the genes involved in atherosclerotic disease. while e has been shown to be slightly more effective than equilin in the modulation of gene expression, genistein was significantly more effective in the favorable expression of genes involved in particularly the inflammatory response. olga n lekontseva, sandra t davidge. physiology and obstetrics/gynecology, university of alberta, edmonton, ab, canada. introduction: the prevalence of cardiovascular disease in women dramatically rises in the postmenopausal period. although deficiency of estrogen has been implicated in the pathophysiology of systemic vascular dysfunction, the effects of estrogen on vasculature are complex and not completely understood. we have previously shown that estrogen exerts a beneficial effect on the aging vascular system by reducing circulating levels of the inflammatory cytokine tnf . tnf is a known regulator of matrix metalloproteinases (mmps), proteolytic enzymes that may modulate vascular tone through cleavage of vasoactive peptides such as big endothelin- (et- ). the role of estrogen in this pathway is unknown. we tested the hypothesis that in aging/estrogen deficiency, tnf -induced mmp activity mediates greater vasoconstriction, in part, through the et- pathway. we further hypothesized that estrogen replacement reduces vascular sensitivity to the constriction by preventing mmp activation. methods: aged ( month old) female sprague dawley rats were ovariectomized and treated with either placebo [ovx] , -estradiol [ovx+e ], or the tnf inhibitor etanercept [ovx+etan] . after four weeks, resistance mesenteric arteries were isolated and studied on the pressure arteriograph. concentrationresponse to exogenous big in the absence or presence of mmp inhibitor (gm , μm) was assessed in the vessels. results: treatment of "menopausal" [ovx] rats with either estrogen or the tnf inhibitor reduced sensitivity of arteries to big et- (ec = . ± . μm in ovx versus . ± . μm in ovx+e or . ± . μm in ovx+etan groups; p< . ). although mmp inhibition attenuated maximal constriction in all of the arteries, there was a significantly greater (p< . ) role of mmps in big et- -induced vasoconstriction in the ovx+e group (reduction in max constriction= . ± . %) compared to ovx ( . ± . %) and ovx+etan groups ( . ± . %). conclusions: both estrogen and tnf inhibition reduced big et- vasoconstriction. however, contrary to our hypothesis, tnf is not contributing to mmp modulation of et- vasoconstriction. interestingly, our study demonstrates a novel role for estrogen to increase mmp contribution to big et- vasoactivity with the net effect being less vasoconstrictive. understanding this unique pathway of regulation by estrogen in the aged vasculature will allow for development of new therapeutic options for women. mo, usa. introduction: the etiology of pelvic organ prolapse is multifactorial, with both inherited and acquired components. the molecular mechanisms of prolapse have not been established yet. we have previously shown that lysyl oxidase (lox) expression is suppressed in uterosacral ligaments of women with pelvic organ prolapse. it has also been shown that lox is a tumor suppressor gene inactivated by methylation in human gastric cancers. hypothesis: the aim of this study was to analyze the dna sequence of the promoter region of the lysyl oxidase gene in tissues from women with pelvic organ prolapse and identify whether methylation is present. our hypothesis is that the promoters of the lox gene in women with pelvic organ prolapse have significantly more methylation sites than women without prolapse. materials and methods: genomic dna was isolated from the uterosacral ligaments of eight women with pelvic organ prolapse and women without prolapse (controls). genomic dna samples were treated with ez dna methylation kit (zemo research, orange, ca). the lox gene promoter region of - to + was amplified by pcr and then cloned into pcr . -topo (invitrogen, carlsbad, ca) and transformed into an e. coli dh a strain. amplified plasmid dna samples containing the lox gene promoter region from each woman were sequenced and methylated cpg islands were identified by sequence comparison. results: a total of methylated cpg sites were found in the patient group with pelvic organ prolapse while only methylated cpg site was found in the non-prolapse control group. conclusion: these findings suggest that methylation in the promoter region suppresses lox gene expression in women with pelvic organ prolapse. background: reports from case-control and cohort studies have suggested an inverse association between lactation and breast cancer risk, but findings have been inconsistent. methods: we conducted a prospective observational cohort study of , parous women participating in the nurses' health study ii from to . our primary outcome was incident premenopausal breast cancer. results: during the study period, cases of premenopausal breast cancer were diagnosed during , person-years of follow-up. women who had ever breastfed had a % lower incidence of premenopausal breast cancer ( % confidence interval [ci] - %) compared with women who never breastfed, adjusting for parity, age at first birth, year of first birth, height, body mass index (bmi), bmi at age , family history, personal history of benign breast disease, participant birth weight, preterm birth, age at menarche, oral contraceptive use, physical activity, and alcohol consumption. no trend was observed with duration of lactation (p= . ). the association between ever-breastfeeding and premenopausal breast cancer was modified by use of medication to suppress lactation (p= . ); in analyses restricted to women who had never used suppressive medication, ever-breastfeeding was associated with a % ( %ci - %) reduction in incident disease. the association between lactation and premenopausal breast cancer was further modified by family history of breast cancer (p= . ). among women who had never used suppressive medication and reported a family history, those who had breastfed had a % lower covariate-adjusted risk of premenopausal breast cancer ( % ci - %) than women who had never breastfed. among women without a family history, ever-breastfeeding was not associated with breast cancer incidence (hazard ratio . , % ci . - . ). among women who had ever breastfed, we observed no association between breast cancer risk and duration of lactation amenorrhea or exclusive breastfeeding. conclusion: in a large, prospective cohort study, ever-breastfeeding was inversely associated with risk for premenopausal breast cancer. at the durations observed in our cohort, we observed no trend with duration of breastfeeding or lactation amenorrhea. the inverse association with ever-breastfeeding was stronger among women with a family history of breast cancer. regulatory and nkt cells at the maternal-fetal interface. thomas f mcelrath, rachael a clark. brigham women's hospital, boston, ma, usa; harvard skin disease research center, brigham women's hospital, boston, ma, usa. introduction: pregnancy represents an immunologically challenging event requiring maternal tolerance of the fetal semi-allograft. an increase in decidual cd + cd + t cells has been documented but it is unclear if these represent foxp + t cells (tregs) . the possibility also exists that other cd + lineages with potential regulatory function exist within the decidua parientalis. we examined if cd + cd + cells are true foxp + tregs and evaluated the frequency of other t cell subsets with possible regulatory potential. methods: we extracted t cells from term deciduas of planned cesarean deliveries. cells were stained with directly conjugated monoclonal antibodies and were analyzed on a six color flow cytometer. results: we found that only a subset (median %) of cd + t cells were true foxp + regulatory t cells. from donors, foxp + tregs accounted for a median of . % of all cd + cells. these cells were memory cd ro + t cells lacking ccr , and l-selectin but expressing cd , ctla- , gitr, and hla-dr. additionally we found that a median % of cd + t cells expressed the nkt marker cd . these cells were a mixture of cd and cd -cd -t cells, with variable numbers of cd t cells, suggesting they do not represent merely recently activated t cells. there was enrichment for t cells with nkt markers after culture on hela cells expressing cd d, suggesting that these cell represent true nkt cells. nkt cells were of the non-classical type, with a diverse t cell repertoire ( . % iv jq) and also expressed cd , hla class ii, cd ro but were ccr and l-selectin low. comments: we find that only a minority of cd -expressing t cell in the decidua are true foxp + tregs. because much of the work on treg in pregnancy has used cd as a treg marker, this suggests additional studies are needed to confirm the role of these cells in pregnancy. we find that a novel population of non-classical nkt cells exists in the human decidua. nkt cells can either promote or antagonize tolerance, depending on the immunologic context. the large number of these cells in the decidua suggests they may play a role equal or exceeding that of regulatory t cells. prolapse. marsha k guess, kathleen a connell, richard bercik, lloyd g cantley. obstetrics, gynecology reproductive sciences, yale university school of medicine, new haven, ct, usa; internal medicine/neprhology, yale university school of medicine, new haven, ct, usa. objectives: thy- is a cell surface glycoprotien expressed in human fibroblasts, neurons, hematopoetic stems cells and endothelial cells. thy- expression affects fibroblast proliferation and migration, cell-cell, as well as, cell-matrix interactions. moreover, thy- expression has been shown to play a critical role in fibroblast dedifferentiation into myofibroblasts, as well as in extracellular matrix (ecm) production and fibrosis. women with pelvic organ prolapse (pop) have alterations in vaginal ecm protein expression and metabolism, as well as decreases in smooth muscle fractional content. in the current study, we evaluated thy- as well as the smooth muscle markers alpha-smooth muscle actin (asma) and desmin expression in women with pelvic organ prolapse compared to women with normal pelvic support (controls). methods: anterior apical vaginal wall specimens from women with pop and controls were collected at the time of hysterectomy. messenger rna and protein expression of thy- , asma and desmin were evaluated using semiquantitative rt-pcr, real-time pcr and western blot analysis. gapdh and beta actin were used as internal controls. results: rt-pcr demonstrated the presence of thy- , asma and desmin in vaginal tissue from women with pop and controls. further, thy- mrna expression was downregulated % in women with pop compared to controls (p = . ). a parallel decrease in thy- protein was seen in women with pop compared to controls (p= . ). although a % and a % decease were seen in asma and desmin mrna, these differences were not statistically significant. similarly, no differences were seen in asma and desmin protein expression. conclusion: we demonstrate that there is significantly less thy- expression in vaginal tissue from women with pop compared to controls. differential expression of thy- in prolapsed vaginal tissues suggests that thy- may have a functional role in mediating ecm metabolism in the female genitourinary tract. hur expression is altered in ectopic endometrium. s karipcin, t altun, ua kayisli, e seli. ob gyn, yale u., new haven, ct, usa. introduction: cytokines and growth factors contribute to cyclic turnover of the normal endomterium and to the pathogenesis of endometriosis. cytokine and growth factor messenger rnas (mrnas) undergo rapid turnover that is primarily mediated by au-rich elements (are) that consist of multiple stretches of adenylate and uridylate residues located in the ' untranslated region ( '-utr) of their mrnas. hur is a ubiquitously expressed rna-binding protein that stabilizes are containing mrnas and prolongs their expression. we hypothesized that hur may play a role in the regulation of cytokine expression during normal menstrual cycle and in endometriosis. methods: tissue sections obtained from normal (n= ) and ectopic (n= ) endometrium were immunostained for hur. staining intensity was evaluated by hscore and grouped according to menstrual cycle phase. statistical analysis was done with one-way anova. cultured stromal cells isolated from normal endometrium were treated with vehicle, estradiol (e ; - m), or progesterone (p, - m) for and h, and hur expression was determined using western analysis and normalized to ß-actin. results: hur immunostaining was nuclear in endometrial cells. hur immunoreactivity was significantly lower in the early proliferative and late secretory phases ( . ± . and . ± . , respectively), compared to the mid-late proliferative ( . ± . ) and early-mid secretory phases ( . ± . )(p< . ). moreover, hur expression was significantly lower in ectopic endometrial cells when compared to normal endometrium in midlate proliferative and early and mid-secretory phases (p< . ). progesterone suppressed hur levels significantly in cultured endometrial stromal cells at both and h compared to control (p< . ) while estrogen did not cause a significant change. discussion: decreased hur levels in the late secretory and early proliferative phases are likely to contribute to degradation of cytokines and result in lower cytokine levels observed mid-cycle. late secretory decrease in hur levels may be mediated by progesterone as suggested by in vitro findings. in ectopic endometrium, persistent low expression of hur compared to normal endometrium most probably results from elevated cytokine levels associated with endometriosis. the effect of lower hur expression in ectopic endometrium on other are-containing transcripts, and on the pathogenesis of endometriosis remains to be elucidated. tissue. hong zhao, joy innes, scott reierstad, mehmet bertan yilmaz, serdar e bulun. department of obstetrics and gynecology, northwestern university, chicago, il, usa. background: aromatase is the key enzyme for estrogen biosythesis, and is encoded by the cyp a gene. thus far, only three unique untranslated first exons associated with distinct promoters in the mouse cyp a gene were described (brain-, ovary and testis-specific exon i). however, it remains unknown whether aromatase is expressed in other mouse tissues via previously unknown tissue-specific promoters activating new exon is. methods: real-time pcr was used to examine the aromatase expression levels in various c bl mouse tissues. '-rapid amplification of cdna ends ( '-race) was used to determine the transcriptional start sites of cyp a transcripts. promoter activity was measured using serial deletion mutants of dna fused to the luciferase reporter gene. results: real-time pcr results showed that aromatase was expressed in male gonadal fat and the expression level is lower than that in testis. the adipose tissue-specific untranslated exon i of cyp a transcript was isolated using '-race and this novel gonadal fat-specific exon i of cyp a mrna did not show sequence similarities to previously reported ones. this new adiposespecific exon i was mapped to kb upstream of the translation start site in the coding exon ii. the genomic region upstream of the adipose-specific exon i was cloned into luciferase plasmids. transfection of murine t -l cells with these plasmids showed that promoter activity was conferred by the sequence located at - to - bp upstream of the transcriptional start site. dexamethasone significantly induced activity of the adipose-specific promoter region. conclusion: taken together, our results suggest that a novel cyp a transcript is regulated by a tissue-specific promoter in male murine gonadal fat. these sacrificed; reproductive and selected other organs were removed, weighed and evaluated histologically by an observer blinded to treatment. results: the table below shows that tcc augmented effects of tp on weights of accessory sex organs. histological assessment revealed that tcc induced greater glandular distention with more secretions compared to the effects of tp alone; furthermore, mitotic figures were seen only in prostates from rats exposed to tp+tcc. conclusions: tcc is a newly identified endocrine disruptor with unique and novel actions resulting in potentiation of androgen effects on sex organs. these observations underscore possible environmental risks related to exposure to tcc. background: blastocyst implantation is dependent on the differentiation of human endometrial stromal cells (hesc) into decidual cells. transforming growth factor family members have well defined roles in cell differentiation and proliferation. activin a, a tgf superfamily member, enhances hesc decidualization and localizes to decidualized cells in human endometrium. other tgf superfamily members, including bmp , bmp , bmp , gdf , gdf (myostatin), gdf and nodal, may also be present in decidual cells and therefore may also play a role during this important process. this study aimed to determine whether activin is the major family member driving decidualization or whether other family members contribute to the process. methods: broad ranging activin inhibitors (activin-m a and sb ) that effect receptor-ligand interactions of other tgf superfamily members were used in hesc decidualization. protein localization was examined in secretory phase endometrium and first trimester decidua by immunohistochemistry and mrna expression was examined in an ex vivo model. the secretion of candidate proteins was measured during hesc decidualization and certain recombinant proteins added during decidualization to examine their effect. results: m a ( nm) and sb ( m) significantly reduced decidualization ( % and % respectively) demonstrating that activin and possibly other tgf family members are involved in decidualization in vitro. bmp , gdf and tgf protein were detected in decidual cells of mid-late secretory endometrium and first trimester decidua whilst all ligands except nodal, were expressed by hesc. both bmp and tgf secretion increased during hesc decidualization and administration of both these proteins significantly enhanced decidualization in vitro. conclusions: these data support a role for activin a, bmp and tgf in hesc decidualization. this is important as the elucidation of factors involved during decidualization will aid in better understanding implantation and fertility. abnormal chromatin remodeling in diabetic murine oocytes. laura lawrence, ann ratchford, cybill esguerra, qiang wang, kelle moley. ob/ gyn, washington university, st. louis, mo, usa. background: diabetic women experience increased miscarriages and adverse pregnancy outcomes. previous studies suggest adverse diabetic outcomes may occur earlier than the preimplantation period, particularly during oogenesis. we hypothesize that diabetes affects chromatin remodeling and chromosomal condensation in murine oocytes. methods: mii oocytes from diabetic and control mice were fixed with % pfa, permeabilized with . % triton x- for min, and immunostained against a-tubulin and the nucleus for hr at rt. images were obtained with laser confocal scanning microscope. protein expression levels of chromatin with dimethyl h k modifications were measured in nondiabetic and diabetic denuded murine oocytes at hours post pmsg ( hour) and at six hours post hcg ( hour) via western immunoblots. mature nondiabetic denuded oocytes were fixed in %pfa and permeabilized with . % triton x- . they were stained via immunohistochemistry against histone protein h at lysine (h k me ) and heterochromatin. results: immunohistochemistry reveals that diabetic mii oocytes have aberrant spindle formations and metaphase chromosome alignment. approximately / ( %) diabetic oocytes examined had abnormal spindles and metaphase alignments compared with only about / normal oocytes ( . %). western blot demonstrated times higher expression of dimethylated chromatin in diabetic oocytes at time compared with nondiabetic oocytes. at time hours, diabetic oocytes had significantly fewer h k modifications than the controls. when staining mature murine nondiabetic oocytes for dimethylation of h k me by immunohistochemistry, we demonstrated h k me expression in a condensed heterochromatin ring surrounding the nucleolus, consistent with transcriptional silencing. conclusions: diabetic mii oocytes have a significant increase in abnormal spindle formation and metaphase chromosome alignment. they also have increased dimethylation compared with normal oocytes at a time point when they should be transcriptionally active, storing maternal mrna in preparation for the silencing period. in addition, after hcg injection to trigger maturation and gene silencing, the diabetic oocytes had decreased dimethylated chromatin changes. our findings suggest that diabetic oocytes may be exiting the transcriptionally active period prematurely and may ultimately experience decreased, partial, and incomplete gene silencing. and xenopus epab genes and proteins were performed. expression of human epab and pabpc mrna was tested in ten different somatic tissues, testes, and ovaries by rt-pcr. amplification with actin primers provided a positive control and allowed semi-quantitative analysis. epab and pabpc expression in human prophase i (pi) and metaphase ii (mii) oocytes, -cell embryos and blastocysts was evaluated using quantitative real time pcr. results: human epab is a aa protein with % identity and % similarity to mouse epab and contains rna recognition motifs and a pabp domain. human epab mrna is detected in ovaries and to a lesser extent in testes and several somatic tissues including kidney, liver, and muscle. similar to its mouse orthologue, human epab mrna is expressed in pi and mii oocytes, but not in -cell embryos or blastocysts. pabpc mrna is ubiquitously present in all tissues as well as -cell, and blastocyst stage embryos. however, its levels are significantly lower than that of epab in oocytes. conclusions: in this study we report the identification of human epab. similar to that observed in xenopus and mouse, human epab is the predominant poly(a) binding protein in oocytes and it is replaced by pabpc following zga, which occurs at -to -cell stage in human. our findings suggest that the unique translational regulatory pathways that control gene expression during oogenesis and early embryo development may be common between model organisms and humans. objective: c-jun nh -terminal kinase (jnk), a member of mitogen-activated protein (map) kinase family, is involved in cell proliferation, differentiation, and survival. fsh is required for granulosa cell proliferation and antral follicle growth but its mechanism of action in preantral stages is not well defined. we previously showed that pharmacological inhibition of jnk pathway halts invitro growth of murine preantral follicles in serum free media supplemented with fsh ( miu/ml). sigcs (spontaneously immortalized rat granulosa cell line) have characteristics similar to preantral granulosa cells and hence they were used in this study to determine whether the jnk pathway plays a key role in preantral granulosa cell proliferation. our specific aims were to determine whether: a) fsh activates jnk pathway in granulosa cells; b) the inhibition of jnk pathway blocks cell cycle progression. material and methods: sigcs were treated with miu/ml recombinant fsh in serum free media two days after serum starvation. activation of jnk pathway was analyzed with if and wb using phospho-jnk and phospho-cjun expression, respectively. fsh receptor expression (fshr) was analyzed with if. the inhibition of jnk pathway on cell cycle progression was analyzed by facs using a jnk inhibitor sp . results: fshr protein was expressed in sigc indicating that they can respond to fsh (fig a) . likewise by if, phospho-jnk expression was significantly increased in sigc hour post fsh exposure (fig- a) . similarly on wb, phospho-cjun expression increased as early as min after fsh exposure and peaked at hrs. cjun phosphorylation was abolished hr after treatment with sp ( mm) (fig b) . facs analysis showed that the inhibition of jnk by sp resulted in cell cycle arrest at g /m transition in a dose dependent fashion (fig c) . conclusion: these results strongly suggest that the proliferative effect of fsh on immature granulosa cells is mediated through the activation of jnk pathway. this is the first experimental observation implicating jnk signaling in granulosa cell cycle control. (nichd - ). the induction of {alpha}-hydroxylase (cyp ) expression in granulosa cells. satin s patel, victor e beshay, william e rainey, bruce r carr. reproductive endocrinology and infertility, university of texas at southwestern medical center at dallas, dallas, tx, usa; physiology, medical college of georgia, augusta, ga, usa. according to the traditional two-cell two-gonadotropin hypothesis of the ovary, androgen production arises exclusively from theca cells. the granulosa cells, in turn, utilize androstenedione, which is aromatized eventually to estradiol. studies involving immunohistochemical analysis of normal ovaries have shown that granulosa cells express significantly higher levels of the activator protein- (ap- ) transcription factor, cfos compared to theca cells, where cfos expression is virtually absent. we hypothesize that cfos functions to inhibit the expression of cyp in granulosa cells, thereby suppressing androgen production. hence, the inhibition of cfos activity might result in cyp expression in the granulosa cell. our objective was to define the role of cfos, in the regulation of cyp expression in granulosa cells. transformed human luteinized granulosa (hgl ) cells were utilized for all experiments. hgl cells were cultured in monolayer for h. cells were treated for h with and without pd (pd), a mapkk inhibitor, which also blocks cfos expression. rna was isolated and real time rt-pcr was performed for cyp . cfos rna interference experiments were carried out using rnaifect, cfos smartpool sirna and scrambled sirna for h. rna was isolated and rt-pcr was also performed for cyp . immunochistochemical studies were performed on normal ovaries, staining for cfos and cyp . treatment of hgl cells with the mapkk inhibitor pd for h, resulted in a -fold increase in cyp mrna expression compared to basal conditions. in cfos gene silenced cells, cyp mrna expression also increased by -fold compared to control sirna conditions. immunohistochemical staining for cfos and cyp showed significant staining of cfos in the granulosa cell layer, but absent staining for cyp . conversely, the theca cell layer did not stain for cfos, but staining was evident for cyp . these results suggest that the ap- transcription factor, cfos, may play a role in the inhibition of cyp expression in granulosa cells. this may provide an explanation for the lack of cyp expression in granulosa cells. the g /m stages of the granulosa cell cycle. as clip- has been identified as an mtor substrate, we hypothesized that its function at the mitotic spindle would be positively regulated by mtor during the late g and m phases of the cell cycle in granulosa cells. during periods of stress (e.g., mtor inhibition), mtor would fail to phosphorylate clip- , leading to spindle checkpoint failure and follicle undergrowth. objectives: the expression of clip- and mtor were evaluated. computational analysis of potential clip- phosphorylation sites and comparison with residues on known mtor targets were performed. clip- threonine and serine were chosen and evaluated as bona fide mtor phosphorylation sites. a preliminary assessment of the effects of mtor inhibition upon clip- function was performed. methods: for protein expression analyses, western blots, immunostaining of tissues and primary granulosa cells in culture were performed. computational analysis of potential clip- phosphorylation sites was followed by in vitro assessment of mtor kinase activity upon clip- and a peptide substrate. results: clip- was expressed in the ovarian stroma, blood vessels (including the endothelial cells of both arteries and veins), granulosa cells, and in the oocytes of primordial and growing follicles. overlapping expression was found between clip- , mtor, and the mtor cofactors raptor and rictor in granulosa cells. this expression was conserved between the mouse and the human. evaluation of clip- phosphorylation supported thr as a bona fide mtor target. conclusions: clip- was supported as an mtor substrate protein during granulosa cell mitosis. the mechanism of mtor action during granulosa cell growth and survival is likely to include the phosphorylation of clip- and subsequent positive regulation of mitotic spindle function. the effect of a selective oxytocin antagonist (barusiban) in threatened preterm labour: a randomised, double-blind, placebo-controlled trial. steven thornton, thomas m goodwin, gorm greisen, morten hedegaard, joan-carles arce. warwick medical school, university of warwick, coventry, united kingdom; maternal-fetal medicine, university of southern california, los angeles, usa; dept of neonatology, rigshospitalet, copenhagen, denmark; dept of obstetrics, rigshospitalet, copenhagen, denmark; clinical research development, ferring pharmaceuticals, copenhagen, denmark. objective: a mixed oxytocin/vasopressin v a antagonist, atosiban, has been shown to reduce uterine contractions in placebo-controlled clinical trials and is useful in the management of preterm labour. the objective of this study was to determine the effect of a selective oxytocin antagonist, barusiban, in delaying delivery and reducing uterine contractions in women with threatened preterm labour at a late gestational age and relatively high risk of delivery. methods: this was a randomised, double-blind, placebo-controlled multicentre study in countries. a total of women between + and + weeks gestation, and with uterine contractions of sec duration during min, cervical length mm, and cervical dilatation > and < cm were randomised to receive a single intravenous bolus dose of either barusiban . mg, mg, mg, mg or placebo. rescue tocolytics were prohibited. the primary end-point was percentage of women who did not deliver within h. uterine contractions were monitored by cardiotocography. obstetrical and neonatal outcomes were determined. results: there were no significant differences between the placebo and any barusiban group in percentage of women who did not deliver within h ( % in the placebo group and % to % in the barusiban groups). there was no dose-effect relationship nor an effect at or h. none of the barusiban groups were associated with a significant reduction in number of uterine contractions compared to placebo at any time point up to h post-dosing. postpartum blood loss and time to established lactation were not significantly increased with barusiban. barusiban was well tolerated and was not associated with safety concerns for the women, fetus or neonates. conclusion: a single intravenous bolus of a selective oxytocin antagonist, barusiban (dose range . - mg), did not delay delivery or reduce uterine contractions compared to placebo in women with preterm labour at late gestational age and with short cervical length. the results contrast those of the mixed oxytocin/vasopressin v a antagonist, atosiban. prolonged delivery intervals in triplet gestations. tracy a manuck, heather l mertz, leah passmore, david c merrill. obstetrics and gynecology, wake forest university health sciences, winston-salem, nc, usa. objective: delayed interval delivery is one management strategy for previable preterm labor affecting multiple gestations. prior reports of asynchronous deliveries have examined twins and higher-order multiples as a group. this study was conducted to analyze the unique situation of asynchronous triplet deliveries. study design: cases of asynchronous triplet deliveries resulting in an ongoing twin gestation were ascertained through medline. data were abstracted and combined with two similar previously unpublished cases. patients were grouped by management with and without rescue cerclage. variables compared included use of tocolytics, antibiotic administration, gestational age at delivery of each fetus, interdelivery interval, delivery mode, birthweights, and short and long term outcomes. chi-square or t-test analyses were used where appropriate. results: fifty-one cases of asynchronous triplet deliveries met inclusion criteria and were analyzed. twenty-three patients ( . %) underwent placement of a rescue cerclage following delivery of the first infant. these patients delivered the first fetus at a significantly earlier gestational age as compared to those patients without a cerclage ( . +/- . weeks vs. . +/- . weeks, p= . ). patients with a rescue cerclage had a significantly longer prolongation of the remaining twin gestation ( . +/- . days vs. . +/- . days, p= . ). no significant differences in use of tocolytics or antibiotics, gestational age at delivery of triplets "b" and "c," mode of delivery, short term outcome (alive at hours), or long term outcome (alive at discharge) were noted, despite delivery of triplet "a" at a significantly younger gestational age. conclusion: rescue cerclage, particularly when placed following previable delivery of a first triplet, may significantly prolong the delivery interval for the remaining twin gestation. mean pregnancy prolongation (days). objective the aim of our study was to evaluate the role of amnioinfusion in pregnancies complicated by pprom. we studied singleton pregnancies with pprom at < weeks gestation. all patients were managed conservatively with bed-rest, prophilactic antibiotics, tocolytics and steroids. only patients without vaginal bleeding and/or contractions were included: patients showed an amniotic fluid pocket (afp) persistently cm and did not undergo amnioinfusion (group b) whereas had a maximum afp < cm and were offered amnioinfusion to restore an adequate amount of amniotic fluid (group a). in patients of group a amnioinfusion was successful (afp cm for hours following the procedure: group a ) whilst in it was unsuccessful (afp< for hours: group a ) and repeated. results were analized with the student t test for unpaired samples and with the when appropriate. p values < . were considered significant. results the group where amnioinfusion was not successful (group a ) showed the worst outcome (see table) . there were intrauterine deaths, all in this group. pulmonary hypoplasia was present in / ( . %) newborns (both survived and deceased) newborns, / in group a . no maternal complications were recorded. conclusions our data confirm that a conservative-active management with amnioinfusion can be considered a reasonable option in women with pprom. in our series it was effective in preventing both neonatal death and pulmonary hypoplasia. group a (infusion successful) background. synthetic progestogens are effective in reducing the risk of spontaneous preterm birth in high risk singleton, but not multiple, pregnancy. we hypothesized that myometrial stretch may inhibit the response of human myometrium to progestogens. methods. myometrial strips obtained with written consent at the time of term planned cesarean section were studied using a modification of the method of young and zhang (jsgi ; : - ) . strips were maintained in individual tubes in tissue culture media in an incubator for a period of three days. the effect of prolonged stretch was assessed by comparing strips connected to a . g weight with those connected to a . g weight. the effect of prolonged exposure to progestogen was studied by adding medroxyprogesterone acetate (mpa, nm or nm). following the day incubation, myometrial strips were transferred to an organ bath containing kreb's solution. all were placed under g tension and responses obtained to mm potassium then oxytocin. contractility was expressed as the ratio of the maximum response to potassium or oxytocin to the wet weight of the tissue (units = g.tension per gram), summarized as the mean (sem) and compared using student's paired t test. prolonged stretch increased the maximum response to both potassium ( . g weight = . [ . ]; . g weight = . [ . ], n= , p= . ) and oxytocin ( . g weight = . [ . ]; . g weight = . [ . ], n= , p= . ). in strips with a . g weight, incubation in mpa for three days reduced the maximum response to potassium (vehicle = . [ . ]; mpa = . [ . ], n= , p= . ) and there was a trend towards a reduced maximum response to oxytocin (vehicle = . [ . ]; mpa = . [ . ], n= , p= . ). in strips with a . g weight, incubation in mpa for three days had no effect on either the maximum response to potassium . prolonged stretch increases human myometrial contractility in vitro. . prolonged exposure to a progestogen inhibits the contractility of human myometrium but this effect is blocked by prolonged stretch. these properties of human myometrium may explain the failure of oh progesterone caproate to reduce the incidence of spontaneous preterm birth in multiple gestations. sangeeta jain, william l maner, janet l brandon, gary dv hankins, robert e garfield. obstetrics and gynecology, the university of texas medical branch, galveston, tx, usa. objective: to determine if transabdominal uterine electromyography (emg) correlates with parturition factors such as measurement-to-delivery time (mtdt), cervical dilation (cd), cervical effacement (ce), and station (s) for preterm labor patients with and without tocolysis. materials and methods: pregnant preterm labor women were included. uterine electromyography (emg) was measured for minutes. cd, ce, and s were assessed at or near the time of uterine emg measurement. the power density spectrum peak frequency (pdspf) was calculated on emg. patients were grouped (g : tocolysis, n= ; g : no tocolysis, n= ). pearson-product-moment test was used for correlation. significant differences were sought between groups using student-t test. p< . significant. results: there was a significantly higher uterine emg activity (pdspf: . ± . vs. . ± . ), but no difference in cd ( . ± . vs. . ± . ), for patients delivering within days of emg recording compared to those who delivered later, regardless of tocolysis. there was no apparent difference in uterine emg in tocolytic vs. non-tocolytic patients, regardless of mtdt (table ) . conclusions: uterine emg activity is significantly greater in patients in preterm labor who delivered within days of measurement, making it a viable alternative diagnostic parameter for assessing the state of parturition. tocolytics may not affect uterine emg, but this should be further verified with larger studies. supported by grant nih r -hd . objective: calcium sensitizers are a novel class of drugs with unique molecular and phsiological actions. levosimendan, the best characterized of these compounds and is used in the treatment of acute and chronic heart failure. levosimendan can exert an inotropic effect via sensitization of myofilaments to calcium. it also exerts a relaxant effect on vascular smooth muscle through the opening of atp-dependent potassium channels and has been shown to be a potent inhibitor of human uterine contractions in vitro. for these reasons we investigated the effects of levosimendan on uterine contractions, both spontaneous and agonist induced, in the presence of glibenclamide, a k-atp channel blocker. method: biopsies of human myometrium were obtained at elective caesarean section (n= ). dissected myometrial strips suspended under isometric conditions, undergoing spontaneous and oxytocin-induced contractions, were exposed to glibenclmide ( mmol) followed by cumulative additions of levosimendan in the concentration range of nmol/l to mmol/l. control experiments were performed simultaneously. results: levosimendan exerted an inhibitory effect on spontaneous and oxytocin induced contractions in human m yometrium in vitro, in comparison to control experiments. the effect of levosimendan was significantly antagonized by glibenclamide with the mean maximal inhibition seen due to levosimendan greatly reduced (n= , p< . ). conclusion: the calcium sensitizer levosimendan exerted a potent relaxant effect on human uterine contractility in vitro. this action was antagonized by glibanclamide and this study demonstrates that the effect of levosimendan on uterine smooth muscle is mediated at least in part through the k-atp channel. introduction: the determination of the beginning and ending points for "bursts" of electrical activity occurring during uterine contractions is sometimes difficult. if bursts cannot be discerned, the preferred burst-by-burst analysis cannot be performed. one solution to is to analyze any given electrical recording in its entirety. but this approach has often lead to meaningless results when traditional analytic methods are applied. chaos analysis, using lyapunov exponents, may provide an answer. materials and methods: term patients were included in the analysis: were in labor (group ), while were non-labor (group ). minute recordings were analyzed using "lyapunov exponent." for each pair of subsequent trajectories in phase space, only the most positive lyapunov exponent was calculated. the mean largest exponent was found by averaging over all of the trajectories in the recording. the lyapunov exponent is given in units of bits per data sample. thus a value of + means that the separation of nearby orbits doubles on the average in the time interval between data samples. the mean largest exponent was found for each patient recording. these values were compared using t-test (p < . considered significant). results: the mean and sd of the lyapunov exponent for all the patients was . ± . . moreover, the lyapunov exponent calculated for each patient was positive. comparing lyapunov exponents of the two groups showed a statistically low value (low chaos) for the laboring group, compared to the non-laboring group (figure) . conclusions: there is a chaotic component associated with uterine emg traces, since small but non-negative lyapunov exponents were found in all the traces observed. the lyapunov exponent indicated significantly lower chaotic behavior in the whole emg traces of patients who were in labor than found in those who were not in labor, implying that this measure could be a good diagnostic parameter for labor, possibly eliminating the need for tedious burst-by-burst analysis. supported by grant nih r -hd . comparing uterine emg to tocodynamometer for monitoring contractions. robert e garfield, lynette b mackay, sangeeta jain, william l maner. obstetrics and gynecology, the university of texas medical branch, galveston, tx, usa. objective: to determine whether uterine electromyography (emg) plots contractions similarly to tocodynamometer (toco). study design: pregnant term labor patients were recorded using both uterine emg and toco simultaneously. uterine emg signals were sampled at hz and band-pass filtered in the . to . hz range. root-mean-square (rms) function was calculated from the uterine emg signals in order to produce a "toco-like" trace from the original emg trace. emg-generated rms contraction plots were then compared to toco contraction plots using the following criteria: contractions were assigned a marker value of " ." in-between contraction periods were assigned a " ." from these marker values, contraction rates were established. correlation was found between the contraction rates of rms and toco. temporal overlap of contractions plotted by the two methods was used to find overall percent agreement (opa), positive percent agreement (ppa), and negative percent agreement (npa). these parameters were corrected for within-patient variation using a bootstrap method. results: uterine rms contraction plots were seen to correspond with toco contraction plots (fig. ) . corrected opa, ppa, and npa were high at . %, . %, and . %, respectively. there was a large, statistically significant correlation between uterine emg and toco contraction frequency (fig. ) . conclusions: the similarity between toco and uterine emg contraction plots (specifically, using rms to convert) will allow emg to be used interchangeably with toco in the clinic. supported by grant nih r -hd . introduction -this is a secondary analysis of women participating in a center randomized placebo controlled trial (rct) evaluating the impact of -ohpc in preterm birth (ptb) prevention among women with twins. objective -to evaluate the relationship between plasma -ohpc concentrations and gestational age (ga) at delivery in women with twins receiving weekly injections of -ohpc. methods -women with twins were randomized between - weeks to receive weekly im injections of either -ohpc ( mg) or placebo until weeks. after a minimum of five consecutive injections had been administered to assure steady state concentrations a plasma sample was collected between - weeks. the sample drawn just prior to the next scheduled injection was analyzed for -ohpc by hplc-ms in a blinded manner. the lower limit of quantification of -ohpc was ng/ml. we conducted univariate analyses to assess the association of -ohpc concentration and ga at delivery. we also conducted a proportional hazards model to evaluate the time from sample draw to spontaneous delivery (censoring indicated preterm deliveries), and a logistic regression to evaluate ptb< weeks; in both analyses we adjusted for bmi, race and ga at sample draw. results - women assigned to -ohpc were included; all received all of their scheduled injections. the concentration of -ohpc was significantly higher in women delivering < weeks compared with those women delivering > weeks (p= . , table) . concentration of -ohpc was significantly correlated with ga at delivery (r = - . , p= . ). each unit increase of -ohpc was associated with a % increased odds of delivering < weeks (odds ratio . , % ci, . - . , p= . ) and a % increase in hazard of spontaneous delivery (hazard ratio . , % ci, . - . , p= . ) after adjusting for confounders. gestational age at delivery mean (sd) -ng/ml < weeks (n= ) . ( . ) > weeks (n= ) . ( . ) conclusion plasma -ohpc concentrations after weekly injections were inversely related to ga at delivery in women with twins. since -ohpc induces its own metabolism it is possible that higher concentrations during initial treatment are associated with lower plasma concentrations and reduced efficacy in later pregnancy . clearly more studies are needed. objective: in many non-human species, maternal circulating progesterone levels fall prior to delivery, leading to the theory that in humans progesterone withdrawal occurs on a local and/or functional level. our objective was to characterize maternal and fetal progesterone in human preterm and term labor. methods: women between . and . weeks' gestation (cases) or term controls ( - weeks) with either labor with intact membranes or premature rupture of the membranes prior to labor (prom) were enrolled in a prospective case-control study. progesterone was measured by immulite assay in maternal serum collected upon enrollment and again within minutes after delivery and in umbilical cord serum obtained at delivery. maternal progesterone treatment was not used in any subjects. results: cases and controls were studied (see table for comparisons). controls p value ga at enrollment, weeks . ± . . ± . < . interval to delivery, days (median, range) ( - ) ( - . ) < . maternal progesterone at enrollment, ng/ml ± ± < . maternal progesterone after delivery, ng/ml ± ± . cord progesterone, ng/ml ± ± . among cases, fetal but not maternal progesterone was significantly lower in preterm labor with intact membranes ( ± ng/ml, n = ), as compared to prom ( ± , n = ), p< . . this difference increased further when cases of clinical chorioamnionitis were excluded. conclusions: serum progesterone in laboring patients prior to delivery is higher at term than in the preterm period, which may be attributable to increased placental mass in late pregnancy. this disparity disappears shortly after delivery of the fetus and placenta. fetal progesterone levels are several-fold higher than peripartum maternal levels. preterm labor with intact membranes is associated with diminished fetal progesterone, a phenomenon unrelated to clinical infection. these findings suggest the possibility of fetally regulated progesterone withdrawal as a mechanism underlying preterm labor with intact membranes. [snps] and ptb. however, many of these studies are inconclusive and non reproducible. the challenge of identifying robust associations between genetic variation and either susceptibility or protection from ptb is enormous. a systematic review of literature followed by metaanalysis was performed to understand true associations between snps and ptb. methods: for systematic review, articles were chosen based on medline and embase searches ( ( -april and relevant articles were chosen based on stringent inclusion criteria. primarily, studies reporting genetic associations between snps in maternal dna in singleton pregnancies and spontaneous ptb were included. other criteria included, but not limited to, provided genetic data in a complete enough format so that it could be evaluated in meta-analysis and defined the clinical outcome clearly. meta-analysis was performed wherever > replication data sets were available results: a total of abstracts were reviewed and were selected for full text review. data were extracted from articles. over associations were reported between snps on various candidate genes and ptb; however only had replication dataset. meta-analysis documented significant association between pon a g (odds ratio [or]= . ( %ci . - . ), pon (rs# )(or= . ; ci- . - . ), tnfrsf - a/g (fas) (or= . ; ci- . - . ) and ptb. two snps pon s c (or= . ; ci- . - . ) and ifn gamma (rs ; or- . ; ci- . - . ) documented protective effect. conclusions: systematic review concludes significant heterogeneities leading to lack of reproducible data in genetic association studies of ptb. heterogeneities are contributed predominantly by lack of adequate power, poor phenotype selection, and population admixture. the functional relevance of the risk and protective alleles needs to be verified. jignesh parvadia, mounira habli, jeff livingstone, william polzin, foong lim, timothy crombleholme. pediatric and thoracic surgery, cincinnati children's hospital medical center, cincinnati, oh, usa; obstetric and gynecology, university of cincinnati, cincinnati, oh, usa; obstetric and gynecology, good samaritan hospital, cincinnati, oh, usa. objective little is known about the response of ttts to treatment either by amnioreduction or selective fetospopic laser in triplet pregnancy, particularly the survival of the bystander fetuses. in order to define the response of triplet pregnancies to treatment for ttts we reviewed our experience with higher order multifetal gestations complicated by ttts. study design retrospective chart review of patients diagnosed with in high order gestation from - was performed. results among cases of ttts / ( . %) patients with high order gestations were identified (n= ) with a mean ga at diagnosis of . ± . weeks. pregnancies ( . %) were dichorionic triamniotic and ( . %) were monochorionic triamniotic. cincinnati modification of quinterro staging was utilized to characterize recipient cardiomyopathy as mild (stage iiia, n= ), moderate (stage iiib, n= ) and severe (stage iiic or iv, n= ) categories. / ( . %) were treated with amnioreduction alone (ar), / ( . %) with selective fetoscopic laser photocoagulation (sflp) alone, / ( . %) with ar followed by sflp and / ( . %) with ar followed by intrafetal radio frequency ablation (rfa). / ( . %) patient had a cervical cerclage. / ( . %) patients were treated but remain undelivered. mean ga at delivery was . ± . weeks. overall survival was / ( . %) with bystander survival was / ( %), donor survival / ( . %), recipient survival was / ( . %). conclusion triplet pregnancies treated for ttts have % survival rate for bystander fetuses and have . % survival rates for donor and recipients comparable to twins treated for ttts. ga at diagnosis . ± . weeks cincinnati modification of quinterro (i), (ii), (iii), (iiia), (iiib), (iv) ga at delivery . ± . weeks live birth -donor / ( . %)* # -recipient / ( . %)*# -bystander / ( %) # birth weight -donor to assess the effect of breast feeding (bf) on perinatal outcome in relation to maternal antenatal methadone dose. study design a retrospective chart review study of methadone dependent mother and infant pairs. patients were categorized into groups based on maternal dose at time of delivery: group : dose mg, group : dose - mg, group : dose > mg. the finnegan's scoring system was used to monitor neonatal abstinence syndrome(nas). treatment for nas was initiated if there were scores of . neonatal outcome data included:% nicu admission, % of babies discharged(d/c) at time of maternal d/c, % nas, % treated for nas and total hospital stay. data were analyzed by t-test and fisher's exact test. maternal characteristics between the groups were similar. regardless of maternal methadone dose, bf infants have shorter hospital stays and higher rates of d/c at time of maternal d/c, lower incidence of nas and fewer nicu admission (table) . in all three groups, breast feeding did not impact the severity of nas as reflected in finnegan's score(fs). (table) conclusion regardless of maternal methadone dose, breast feeding improves perinatal objective: to evaluate the effects of preventive collagen plugging of the fetoscopic access port at the time of balloon removal on pregnancy outcome in fetoscopic endoluminal tracheal occlusion pregnancies. study design: fifty-one pregnancies involving fetuses with severe congenital diaphragmatic hernia (cdh) were studied. all patients underwent feto between - weeks gestational age (ga) and fetoscopic balloon removal around weeks ga. at the time of balloon removal, a purified dried collagen plug was inserted through the fetoscopic access port in consecutive pregnancies but not in the first pregnancies considered as controls. all patients underwent post-plugging ultrasound and magnetic resonance imaging studies to evaluate for membrane dehiscence, amniotic fluid volume and fetal well being. ga at delivery, incidence of premature rupture of the membranes (pprom), bleeding at port retrieval and adverse fetal effects were compared in both groups. results: mean (sd) ga at feto [ . ( . ) vs. . ( . ) weeks; p= ns] and balloon removal [ . ( . ) vs. . ( . ) weeks; p= ns] was similar in the treatment and in the control group. incidence of pprom following the second fetoscopy was / in the study group compared to / in the control group (p< . ). mean (sd) ga at delivery was . ( . ) weeks in the study group, compared to . ( . ) in the control group (p= . ). bleeding from the trocar insertion site occurred in cases in both groups, but clinically significant bleeding occurred only in one of the controls. membrane dehiscence was noted in patient in the treatment group compared to in the control group (p=ns). conclusion: preventive collagen plugging of the fetal membrane defect created by the fetoscopic access resulted in a significant reduction in pprom rates and a trend towards increased ga at birth without adverse fetal effects in feto pregnancies. wider application of this technique should be considered, but needs evaluation in larger, randomized trials. hydrops fetalis is an uncommon fetal condition characterised by the abnormal accumulation of fluid in two or more body cavities, traditionally associated with a poor prognosis. the relative rarity of this presentation has meant that published case series have consisted of small numbers. a retrospective review of case notes of all cases managed at kemh between and was performed. in western australia, kemh is the only tertiary maternity hospital incorporating a maternal-fetal medicine unit. cases were obtained from the mfm database. in the period to there was a total of pregnancies affected by hydrops (incidence . per births). the average maternal age was years. in cases a fetal abnormality had occurred in a previous pregnancy. the median gestational age at diagnosis was weeks (range - weeks). in just over half ( %) of cases, the diagnosis was confirmed prior to delivery. a post-mortem was performed on all but of the babies not born alive. edema was present in at least cavities in over half of cases (n= ). chromosomal anomalies included trisomy , trisomy and turners syndrome. in all cases of infection, parvovirus b was implicated. cardiac arrythmias included svt and atrial flutter. cases classified as other included alpha thalassemia and syndromic disorders. in cases an interruption of pregnancy was performed at a mean gestational age of weeks. of those who did not elect to terminate the pregnancy, there were fetal deaths in utero, live borns with neonatal death. for the live borns, the median gestational at delivery was . weeks (range to . weeks). the causes of hydrops in live birth cases included cardiac arrythmia (n= ), infection (n= ), chromosomal abnormality (n= ), unknown (n= ) and other (n= (dmv) have been noted to play a role in the development of hemorrhagic and periventricular leukomalacic lesions in premature babies. since deep vein drainage system is relatively more prominent in the developmental brain than adult brain, we investigated if dmv anomalies could be associated with clastic lesions in-utero. methods two senior neuroradiologists reviewed fetal brain exam performed between and , seeking for unequivocal anomalies in dmv, such as periventricular venular engorgement. all mr scanning is performed at . tesla, using surface abdominal coils and single-shot fast spin-echo t -weighted - mm thick sections, with . - . mm in planar spatial resolution. we found cases with dmv anomalies (tab. ). most of the dmv engorgement is located at frontal lobes level. from this limited preliminary series it appears that dmv involvement plays a role in the development of periventricular leukomalacia and periventricular hemorrhagic necrosis. the observation that these lesions are mostly located at frontal level may suggest that some of the term neonates carrying sequelae of atypically located leukomalacia (i.e. deep frontal lobe) might have developed these lesions in-utero. it is of interest to notice that most of our cases were related to heart failure. therefore, central venous hypertension affecting immature deep cerebral venous system has to be taken into account. in our center, patients with an estimated risk for chromosomal abnormalities at term greater that in after st trimester combined screening test (ft) are offered non-directive genetic counseling. the aim of this study was to evaluate the responses of women younger than attending this genetic counseling session. material and methods: data from patients referred for a positive ft from september , to july , was retrieved from our database. information concerning women younger than years of age at the estimated date of delivery was extracted and tabulated. results: during the study period women had genetic counseling for positive ft. thirteen patients were excluded from further analysis ( had incomplete clinical documentation and had spontaneous miscarriages prior to weeks gestation). four hundred and twenty-five patients were older than and were younger than at the estimated date of delivery. table depicts summary statistics for studied variables in this younger group of women. conclusions: overall this data suggests that approximately % of this younger group of women opted for chorionic villous sampling (cvs), % for amniocentesis and more than % declined prenatal genetic testing. moreover, this data also suggests that: ) these women opted for cvs when the ft risk (mean = in ) almost doubled the cvs procedure related risk quoted at % and, ) when the ft risk is between in and in almost half ( out of ) declined not only the st trimester cvs but also the nd trimester amniocentesis. we believe that understanding our patient population is important to optimize both the efficiency and efficacy of the alternative prenatal screening programs. acceptance for prenatal genetic testing after a positive first trimester combined screening test in women of less than to determine the optimal diagnostic test using prenatal ultrasonography and mri for predicting pulmonary hypoplasia in fetuses with congenital diaphragmatic hernia. methods: relevant papers were identified by searching medline ( medline ( - , embase ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) and the cochrane library ( issue ). in addition, the specialist literature on the topic and reference lists were hand searched for relevant articles. studies were selected if they examined diagnostic tests for the prenatal prediction of pulmonary hypoplasia in fetuses with congenital diaphragmatic hernia. the primary outcome measure was perinatal survival. study selection, quality assessment and data abstraction were performed independently and in duplicate by separate observers. results: of a total number of articles (published studies), there were eighteen studies that fulfilled the entry criteria. six examined entirely unique heterogeneous parameters. of the remainder, all examined the ultrasound measurement of lung to head ratios (lhr) at a 'cut-off of greater than or less than the thresholds of . , . , . , . . in addition, the presence of liver in the fetal thorax was included (if present) as a co-variable (liver "up"). a lhr > . compared to < . provided the strongest association with perinatal survival (peto or . , % ci . - . ). the finding of "liver up"in the fetal thorax had a negative association with survival (peto or . , % ci . - . ). only three studies provided data for lhr in conjunction with the presence of liver in the fetal thorax (peto or survival for lhr > . compared to < . was or . , % ci . - . ). data was also available for liver up and lhr > . which had a peto or of . ( % ci . - . ). discussion: the data supports the view that lhr may be a useful prognostic indicator of perinatal survival in fetuses with congenital diaphragmatic hernia. however, heterogeneity still exists regarding the timing of ultrasound measurement and the use of mri. the majority of studies have small sample sizes. objective: to estimate, in fetal anaemia, the diagnostic value of fetal ultrasonography and doppler blood flow in the evaluation of fetal anaemia methods: literature from to was identified using medline and embase, the cochrane library and relevant specialist register of the cochrane collaboration, and by checking reference lists of known primary studies and review articles. studies were selected if the accuracy of the fetal ultrasound parameters or doppler studies of blood flow in the fetal vessels was estimated compared with a reference standard. diagnostic tests evaluated were ultrasound measurement of the fetal spleen and liver length and doppler studies from the umbilical vein and middle cerebral artery. data from the selected studies were abstracted as x tables comparing the diagnostic test result with the reference standard. results were pooled where appropriate. diagnostic accuracy was expressed as sensitivity and specificity. twenty-nine primary studies were identified containing suitable data. twentyone of these gave data on middle cerebral artery doppler peak systolic velocity (mca-psv) and could be pooled in the meta-analysis giving a sensitivity of . ( . - . ) and a specificity of . ( . - . ) for cases in detecting severe anaemia. four studies gave data on spleen perimeter and it was possible to pool three of these giving a sensitivity of . ( . - . ) and a specificity of . ( . - . ) for cases. three studies had data for liver length measurements and two were pooled. the sensitivity was . ( . - . ) and the specificity was . ( . - . ) but only cases were used in the analysis. there were three studies on umbilical vein maximum velocity doppler studies and all were suitable for meta-analysis giving a sensitivity of . ( . - . ) and a specificity of . ( . - . ) with cases analysed. two studies gave data on middle cerebral artery time-averaged mean velocity score giving cases. the sensitivity was . ( . - . ) and specificity was . ( . - . ). discussion: middle cerebral artery peak systolic velocity dopplers remain the gold standard for non-invasive screening of fetal anaemia. middle cerebral artery time-averaged mean velocity scores require further investigation. other tests perform poorly when diagnostic accuracy is assessed. cochrane review of treatment in twin to twin transfusion syndrome. twin-twin transfusion syndrome is a condition affecting monochorionic twin pregnancies associated with a high risk of perinatal mortality and morbidity. the objective of this review was to evaluate the impact (maternal,fetal and pediatric)of treatment modalities in twin-twin transfusion syndrome. register and cochrane controlled trials register. we also searched conference proceedings and made personal contact with experts active in the area of the review. randomised and quasi-randomised studies of amnioreduction versus laser coagulation, septostomy versus laser coagulation or septostomy versus amnioreduction. eligibility was assessed by one reviewer. study authors were contacted for additional information. two studies were included. this review shows that laser coagulation of anastomotic vessels results in less fetal (rr . ; % ci . , . ) and neonatal deaths (rr . ; % ci . , . ) than amnioreduction ( figure ). there is no difference in perinatal outcome between amnioreduction and septostomy. more babies in the laser arm are alive without neurological abnormality at six months of age (developmental delay at < years rr . , % ci . , . )( figure ). conclusions: endoscopic laser coagulation of anastomotic vessels should be considered in the treatment of all stages of twin twin transfusion syndrome to improve perinatal outcome. further research on the effect of treatment on milder forms of twin twin transfusion syndrome (quintero stage and ) are required. (produced in collaration with the cochrane collaboration, uk). (cvs) concluded that although the risks of pregnancy loss are relatively low, lack of adequate controls tends to underestimate the true added risk of prenatal invasive procedures (obstet gynecol. ; ( ): - ). the west midlands is a large region within the uk containing approximately % of the total uk population. the congenital anomaly register for this region is able to monitor pregnancy outcomes with accurate deminator data. results: there were first trimester cvs performed, by ten operators, in the west midlands (uk) in . this equates to . procedures per births. significantly higher rates were noted in areas of high socioeconomic status. the median number of procedures performed per operator was (range - ). all operators were performing other invasive tests such as amniocentesis or cordocentesis,etc. the most common indication for cvs was: i) fetal anomaly on dating scan ( . %) ii) abnormal (> in ) risk on combined first trimester screening ( . %) iii) molecular genetic diagnosis ( . %) and iv) maternal request ( . %). using a combination of first trimester scanning and cvs, % had abnormal karyotype/structural anomaly.the corrected loss rate (background and procedure-related) following cvs in normally formed, singleton pregnancies was . % ( thci . - . %) up to days postnatal (perinatal loss) and . % ( thci . - . %) with days of procedure. the proportion of cvs in which an adequate sample was not obtained was . % ( th ci . - . %). conclusions: this epidemiological study using accurate demoninator data provide interesting statistics relating to the uptake and prenatal risks of first trimester cvs. with the increasing prevalence of obesity in the last two decades, we have seen a tremendous increase in bariatric procedures in reproductive aged women. malnourishment and vitamin deficiency are common complications after gastric bypass which may impact on fetal development. we present the case of a yo who underwent a duodenal switch procedure in . she was discovered to be pregnant during evaluation for persistent malnutrition in . multiple prenatal ultrasounds were performed; the first at weeks gestation was unremarkable. the week sono revealed a male fetus with shortened femurs and humeri bilaterally, nasal bridge hypoplasia, macroglossia, poorly defined hands, and possible clubbed feet. amniocentesis revealed a normal karyotype. d ultrasound redemonstrated the abnormal facial findings. a fetal echocardiogram was normal. the lagging long bone measurements continued to worsen, ultimately with femurs weeks behind. the fetal thoracic circumference was two standard deviations below the mean, giving rise to concern for pulmonary sequelae. growth restriction was noted at the wk sonogram. delivery by cesarean section was at / weeks secondary to nonreasuring fetal status. the birthweight was gm; apgars were , , and . postnatal radiographs confirmed antenatal ultrasonographic findings and demonstrated evidence of epiphyseal stippling. the infant remained intubated until weeks of life, after which it died secondary to respiratory complications associated with pulmonary hypoplasia. gene mapping studies have not found any point mutations on the recessive gene as an etiology of this disorder. rhizomelic chondrodysplasia punctata refers to a heterogeneous group of bone dysplasias with a familiar radiographic phenotype involving punctate calcifications and epiphyseal stippling. the etiology of this may be secondary to chromosomal abnormalities, mendelian gene disorders, or teratogens, notably warfarin. this case may be explained by vitamin k deficiency of the embryo due to maternal malabsorption after bariatric surgery. the maternal vit k level was <. ng/dl at time of delivery(normal > . ). the teratogenic effects of vitamin k deficiency in this instance highlight the need for strict counseling and screening for vitamin deficiency in those women undergoing bariatric surgery since previous obesity-related anovulation is reversed as patients lose weight, resulting in unexpected pregnancies and potentially preventable fetal abnormalities. term preeclampsia: any risk for the neonate? sindhu k srinivas, jamie bastek, christina m andrela, emmanuelle pare, michal a elovitz. obstetrics and gynecology; crrwh, university of pennsylvania, philadelphia, pa, usa. introduction: preeclampsia continues to be a major contributor to maternal morbidity and mortality worldwide. preeclampsia at term is not associated with the same risk of neonatal morbidty and mortality as preterm preeclampsia . however, neonatal outcomes in term women with preeclampsia have not been adequately studied. we sought to compare short term neonatal outcomes in term infants born to women with and without preeclampsia. methods: this study was part of a large case control study. women with preeclampsia (n= ) and term controls (n= ) were prospectively identified. infants with congenital anomalies were excluded. hospital length of stay (los), admission or transfer to the nicu, and use of mechanical ventilation or cpap within first week of life were assessed. associations between neonatal outcomes and preeclampsia were evaluated using chi-square analysis and wilcoxon rank sum test. significant confounders were controlled for using multivariable logistic regression. results: discharge day of life was significantly different between neonates born to women with preeclampsia (median = ; mean = . ) versus those born to women with uncomplicated term deliveries (median = ; mean = . , p< . ). this difference persisted even when neonates with iugr and those born to diabetic mothers were excluded (p< . ). term infants born to women with preeclampsia have a higher odds of being admitted or transferred to the nicu (aor= . [ . - . ], p= . ) after controlling for iugr, delivery mode, race, and gestational age at delivery. these infants also have a higher odds of mechanical ventilation (aor= [ . - . ], p= . ) and cpap use (aor= . [ . - . ], p= . ) after controlling for the same confounders. there was no difference in ivh or nec between the two groups. conclusion: neonates born to women with preeclampsia have differences in short-term morbidity when compared to neonates born to women without preeclampsia, despite being born at term. whether this increase in neonatal morbidity is attributable to medications used in preeclampsia, such as magnesium sulfate, is unclear. these findings may have implications for patient counseling as well as hospital resource allocation. further investigation correlating these findings with long-term morbidity is warranted. could confined placental mosaicism account for adverse perinatal outcomes in ivf pregnancies? benoit c jacod, gh schuring-blom, kd lichtenbelt, jse laven, d van opstal, mjc eijkemans, nick s macklon. reproductive medicine gynaecology, university medical centre, utrecht, netherlands; clinical genetics, university medical centre, utrecht, netherlands; obstetrics gynaecology, erasmus medical centre, rotterdam, netherlands; clinical genetics, erasmus medical centre, rotterdam, netherlands. background: ivf singletons have poorer perinatal outcomes than singletons from spontaneous conceptions. this may be due to the influence of ovarian stimulation on the chromosomal constitution of the embryos which could be translated into localized chromosomal anomalies in the placenta. aim: to compare the incidence of confined placental mosaicism (cpm) in ivf/icsi pregnancies and spontaneous conceptions. methods: multicentre retrospective analysis of karyotype results obtained by chorionic villus sampling (cvs) performed because of advanced maternal age ( years at weeks of gestation) in the netherlands between and . results: from a total of pregnancies, cvs results were analysed: in the ivf/icsi group and in the control group. the mean age of women in both groups was . years (mean difference - . , % ci - . - . ). foetal karyotype was missing in cases of possible cpm, all in the control group. when taking into account missing data, the incidence of cpm was lower in the ivf-icsi group than in the control group, . % vs. . % (odds ratio . , % ci . - . ) whereas the incidence of foetal chromosomal anomalies was increased . % vs. . % (odds ratio . , % ci . - . ) although both differences are not significant. conclusions: the incidence of confined placental mosaicism is not increased in ivf/icsi pregnancies compared to spontaneous conceptions. cpm probably does not account for the adverse perinatal outcomes following ivf/icsi. fetal rh d, cc and ee genotyping using fetal dna from maternal blood is not impaired by the presence of maternal alloimmunization. chad a grotegut, stacey l jeronis, john p gaughan, enrique hernandez, ossie geifman-holtzman. obstetrics and gynecology, duke university, durham, nc, usa; obstetrics, gynecology and reproductive sciences, temple university, philadelphia, pa, usa; biostatistics, temple university, philadelphia, pa, usa. this study was conducted to assess the impact of maternal alloimmunization on the accuracy of fetal rh d, cc and ee genotyping from maternal blood. we performed a literature search of english-written articles describing fetal rh d, cc and ee determination from maternal blood (am j obstet gynecol ; : - ) . using this database, we determined the accuracy of rh d, cc and ee genotyping in the presence of maternal alloimmunization. for each subgroup, % confidence intervals for a proportion were calculated and compared between groups. we found english-written publications reporting non-invasive rhd genotyping and reporting non-invasive rh ce genotyping from maternal blood. fourteen ( . %) of the rh d articles and three ( %) of the rh ce articles provided accuracy results in the setting of alloimmunization. the accuracy results are reported as follows: . % of the samples were determined correctly in the presence of alloimmunization.* this accuracy was significantly lower than the accuracy reported for all rh cc samples. when only studies utilizing free fetal dna for rh cc genotyping were used (vs fetal cells), fetal cc genotype was determined correctly in / ( %, % ci . , ), which was similar to the overall success rate for rh cc determination. overall, there were no differences in the success of rh d, cc or ee determination in the setting of alloimmunization compared to the overall accuracy seen when free fetal dna was used. the presence of maternal alloimmunization does not reduce the accuracy of fetal rh d, cc or ee non-invasive genotyping from maternal blood utilizing free fetal dna. further research into structure and rearrangements of the rh d, cc and ee genes may further improve diagnostic accuracy of free fetal dna from maternal blood. fetal dna, most likely of trophoblast origin, is present in both the plasma of pregnant women and provides a potential basis for non-invasive fetal diagnostic tests. however, fetal dna in maternal plasma is highly contaminated with maternal dna, and this contamination is the main technical challenge in trying to accomplish non-invasive detection of fetal chromosome abnormalities. existing methods for the selective amplification of fetal dna have generally relied on specific sequence differences between the mother and fetus. as an alternative, we have developed a method for selective amplification of fetal dna that makes use of observation that trophoblast dna is globally hypomethylated in comparison with dna from other sources. in this method, a dna mixture is first digested with a methylation sensitive restriction enzyme and then amplified by linker-mediated pcr. after an initial amplification, a second isothermal rolling-circle amplification is performed. this procedure results in the differential amplification of short, relatively hypomethylated fragments. after amplification, the resulting "representations" are comparatively hybridized to a microarray consisting of oligonucleotides that correspond to restriction fragments generated by the initial digest. copy number differences are then detected through statistical analysis of array addresses that show significant amplification. to test the feasibility of this method for detecting aneuploidy, we have prepared mixtures of peripheral blood dna and first trimester trophoblast dna from either normal or from samples with trisomy and trisomy . we present data showing that aneuploidy can be detected even when % of the starting dna sample was derived from a euploid source and only % was from an aneuploid trophoblast sample. two different approaches to data analysis are presented. one relies on prior analyses of trophoblast methylation and the second is independent of any prior knowledge or analysis. both methods provide similar ability to detect aneuploidy. future work will focus on testing whether this approach can be used for non-invasive prenatal diagnosis. chromatin immunoprecipitation and emsa analysis of nf-b and c/ ebp synergism on the otr promoter. shirin khanjani, yun s lee, vasso terzidou, mark r johnson, phillip r bennett. institute of reproductive developmental biology, imperial college, london, united kingdom. we have shown, the transient transfections of the transcription factors nf-bp and c/ebp leads to a synergistic increase in otr promoter activity in human myocytes. this effect is mediated through a bp region of the promoter between - to - from tss. we now report that this sequence binds both nf-bp and c/ebp in vitro and chip studies show binding of both transcription factors to be increased by il- in vivo. materials and methods: emsa studies were performed using a bp oligonocleotide sequence (- to - ) , containing the bp region responsible for the synergistic activation of the otr promoter and nuclear extracts from primary human myocytes treated with il- ng/ml for hours. for chip analysis, dna protein complexes were crosslinked and antibodies recognizing p , c/ebp and h (positive control) and igg (negative control) were used for immunoprecipitation. primers were designed to amplify the region - to - , which includes the bp response sequence. to further confirm the interaction between nf-bp with c/ebp a xnf-b consensus/luc reporter construct was cotransfected with an expression vector for either nf-bp or c/ebp . results: specific nf-bp and c/ebp binding was seen in the emsa study. preincubation with antibodies to nf-bp and c/ebp led, not to supershift, but to elimination of dna binding for both nf-kb p and c/ebp . chip analysis confirmed increased in vivo binding of nf-b p and c/ebp to this region of the otr promoter following stimulation with il- . transfection of the nf-b/luc reporter construct with an expression vector for c/ebp caused a significant reduction in the basal promoter activity suggesting that c/ebp is binding to nf-kb. this interaction was further confirmed using a tf-tf interaction array. conclusion: these data support the role of nf-b and c/ebp in regulation of otr. the bp region contains a c/ebp but not a nf-b dna binding site suggesting that c/ebp primarily binds to this part of the otr promoter but also interacts with nf-b. the emsa data shows that the bp region binds both nf-b and c/ebp . the loss of the supershift observed in previous emsa studies suggests that the antibodies inhibit the interaction between c/ebp and nf-b, therefore inhibiting dna binding. chip analysis supports the concept that il- leads to binding of nf-b and c/ebp to the bp region. regulation of pro-labour genes by c/ebp, nf-b and ap- in human uterine myocytes. suren r sooranna, shirin khanjani, yun s lee, phillip r bennett, mark r johnson. imperial college parturition research group, imperial college, london, united kingdom; irdb, imperial college, london. introduction: the transcription factors c/ebp (lap), nf-b (p ) and ap- (c-fos and c-jun) are implicated in inflammatory processes such as parturition. the promoter regions of the pro-labour genes il- , pghs- and otr contain putative transcription factor-binding sites for these transcription factors. our aim was to determine the effect of transfecting these transcription factors either alone or in combination into uterine myocytes and to determine their effects on the expression of pro-labour genes including il- , pghs- , otr, connexin- and fp. methods: primary cultures of human uterine myocytes (n= ) were grown from myometrial samples obtained at the time of elective lscs. cells were cultured in well plates to % confluence at which point expression constructs for c/ ebp (lap), nf-b(p ), ap- (c-fos and c-jun) were transfected either alone or in different combinations. the empty expression vector psg was used as a filler construct. cells were cultured for and h after which culture medium was collected for elisa and cells frozen at - °c prior to rna extraction. copy numbers of il- , pghs- , otr, fp, connexin- and gapdh were measured by qpcr using a rotor-genetm (corbett research). results: h post transfection with c/ebp (lap), nf-b (p ), c-fos and c-jun alone or in combination showed no significant changes in pghs - , otr and connexin- expression. over expression of p alone or together with either c-fos or c-jun increased fp expression by , and % respectively (p= . ). nf-bp consistently increased il- expression either alone (by %; p= . ) or in combination with lap, c-fos or c-jun (by , and % respectively; n= ; p= , ). rel a, lap, c-fos and c-jun together also increased il- expression (by %; p= . ) and a small but significant increase was seen with a combination c-fos and c-jun (by %; p= . ). the changes observed in il- expression were reflected in the medium il- concentration at h post transfection. in the presence of rela and c-fos il- increased from . ± . to . ± . pg/ml of culture medium (mean ± sem; n= ). conclusions: nf-b (p ) consistently increased fp and il- expression in human myometrium. the data suggest that pghs- activation has greater dependence upon other transcription factor(s) in addition to p . true identity of myometrial pr-c: fact or fiction? yun s lee, suren r soorana, mark r johnson, jan brosens, phillip r bennett. imperial college parturition research group, hammersmith campus, london, united kingdom. progesterone is thought to be central to maintenance of pregnancy. multiple progesterone receptor (pr) isoforms underlie complex and diverse biological action of progestins. previously two human pr isoforms have been identified: pr-b and pr-a. pr-a is n-terminally truncated form of pr-b (initiation site methionine ). in some cells pr-a inhibits pr-b. it has been proposed that increased expression of pr-a in myometrium underlies a 'functional progesterone withdrawal'. the breast cancer cell t d contains a kda progestin-specific binding protein that is not found in pr negative cells. it was proposed that there is a downstream methionine (met ) which serves as a translation initiation site for the generation of a pr isoform of kda. based on such findings condon et al (mol endo ) have focused on the possible role of kda pr-c in human parturition. they found that expression of "pr-c" using pr antibody (sc- santa cruz, sc) is increased in upper segment myometrium with labour and that overexpression of this protein inhibits pr-b function. we cloned the same human pr cdna into psg expression vector. in vivo translation produced a protein of only kda. furthermore overexpression of pr-c did not significantly decrease pr-b activity in human myocytes. we examined other downstream initiation sites, which may produce a kda protein. we constructed potential pr isoforms in psg vector with initiation sites at met and . in vivo translation produced proteins of approximately and kda respectively. to determine the effect of pr isoforms on pr-b function, myocytes were co-transfected with pr-a, pr-c , pr-c and pr-c with constant amounts of pr-b. the progesterone dependent pre/luc was used as reporter. unlike pr-c both pr-c and pr-c significantly inhibited ligand dependent pr-b mediated transcriptional activity. we found in western analysis that the antibodies pgr- (nc) and the sc- (sc) detected both endogenous and overexpressed pr-a and pr-b but none of the pr-c isoforms. the sc- antibody detected only pr-a and pr-b very poorly. our data suggests that the sc- antibody would not detect any pr-c protein and that none of the commercially available antibodies in the uk do so. great care needs to be taken when over-expressing pr isoforms to ensure that proteins are of the expected size. if pr-c does exist in vivo then the kda but not the kda isoform might inhibit pr-b function. tnf receptor antibody (tnf ri ab), nf-b inhibitor (nf-b activation inhibitor) and erk inhibitor (u ) (p< . ), but not by tnf receptor antibody (tnf rii ab), p mapk inhibitor (sb ) and jnk inhibitor (sp ). by western blot analysis, we found that the protein level of tnf receptor associate factor (traf ) was higher than that of tnf receptor associate factor (traf ) (traf >traf ) in untreated ct cells. however, after tnf treatment for h to h, traf protein level was increased, but traf protein level was reduced (traf >traf ). the increase of traf and decrease of traf were blocked by tnf ri ab, but not by tnf rii ab. we also found that tnf rapidly (within - min) and significantly increased phosphorylation of ikk , erk / and jnk / / and the phosphorylation of these protein kinases by tnf was reduced significantly by tnf ri ab, but not by tnf rii ab. moreover, we found that the changes of increased traf and decreased traf in ct cells (traf >traf ) resulted in a dramatic deficiency in phosphorylation of the above protein kinases induced by tnf compared with the normal ct cells (traf >traf ). nuclear localization of nf-b p in tnf treated cells was increased compared to untreated controls. conclusion: we have demonstrated for the first time that tnf stimulates mmp- production in ct cells through tnf ri-trafs-ikk /erk-nf-b signaling pathways, but not through the jnk/p -ap- pathway. these studies reveal steps within this pathway as possible therapeutic targets to inhibit mmp- expression potentially attenuating tnf -induced degradation of extracellular matrix and pre-term rupture of the fetal membranes. objective: women with preterm birth are at elevated risk of cardiovascular disease, but mechanisms that might relate these conditions are not understood. we hypothesized that women with spontaneous preterm vs. term births may have early gestation evidence of activation of the fibrinolytic cascade, as measured by the thrombin-antithrombin iii (tat) complex. we also tested if this relation may be associated with inflammation. methods: tat was measured in plasma collected < weeks gestation (mean . weeks, sd . ) among women without chronic medical conditions, preeclampsia or growth restriction who delivered singleton liveborn infants. inflammation was assessed by c-reactive protein (crp) measured in serum from the same samples. women with spontaneous preterm birth (sptb) < weeks (n= ) and -< weeks (n= ) were compared to women with term births >= weeks (n= ). high tat was defined as > . ng/ml (highest quartile among women with term births) and high crp was defined as >= ug/ml. multinomial logistic regression was utilized to relate elevated tat and inflammation to risk of sptb subtypes. results: women with sptb were more likely to have tat concentrations in the highest quartile compared to women with term births (< weeks, . %; -< weeks, . %; >= weeks . %, p< . ). women with high tat concentrations had a . -fold ( % ci: . - . ) increased risk for sptb < weeks, after adjustment for body mass index, race, age and gestational age at sampling. there was no relation between high tat and sptb -< weeks (or . , % ci . - . ). additional adjustment for elevated crp (>= ug/ ml) did not effect the estimates associated with tat, and elevated crp was independently related to risk for both sptb subtypes (< weeks, or . [ . - . ]; -< weeks, or . [ . - . ]). thus, women with high tat and elevated crp appeared to be at particularly elevated risk of sptb < weeks (or . , % ci . - . ). conclusions: the thrombin-antithrombin iii complex was elevated early in gestation among women with sptb < weeks, perhaps secondary to microvascular injury. this effect was independent of inflammation, suggesting that the elevated fibrinolytic cascade may function independently from inflammation among women with sptb. plasma cortico-releasing hormone and cortisol concentrations and psychological stress among pregnant women. katherine p himes, hyagriv n simhan. obstetrics and gynecology, university of pittsburgh medical center, magee womens hospital, pittsburgh, pa, usa. objective: many studies have found an association between psychological stress and preterm birth. we sought to determine if women with greater psychological stress during pregnancy had higher concentrations of plasma cortico-releasing hormone (crh) or cortisol. study design: this is a secondary analysis of a multicenter case-control study, nested within an observational cohort. of , participants, plasma crh and cortisol concentrations at and weeks gestation were available in controls who delivered after weeks and cases who delivered before weeks. the abbreviated scale for the assessment of psychosocial status in pregnancy (asaps) was available for all women. concentrations of crh and cortisol were compared between women above and below the lowest quartile score on the asaps among cases and controls. the same analysis was done for the portion of the scale related to psychological stress. concentrations of crh and cortisol and psychological stress were also compared between black and non-black cases and controls. univariate analysis was performed with kruskal wallis or chi-square. results: there was no difference in crh or cortisol concentrations at or weeks among women above or below the lowest quartile on the asaps (controls:p= . - . cases:p= . - . ). greater psychological stress was not associated with higher concentrations of crh or cortisol at or weeks(controls:p= . - . cases:p= . - . ). crh concentrations were not different between blacks and non-blacks. among both cases and controls, cortisol concentrations at and weeks were lower in black women than non-black women (controls:p< . cases:p< . ). the median cortisol concentration among control black women was . g/ml at weeks compared to . g/ml among non-black women and . g/ml compared to . g/ml at weeks. black women reported less psychological stress than non-black women (p= . ) conclusion: we found no relationship between psychological stress and plasma crh or cortisol. furthermore, while stress is hypothesized to play a role in the racial disparity of preterm birth, black women reported less psychological stress and had lower cortisol concentrations than non-black women. improved assessments of psychological stress and additional biomarkers involved in the stress response may broaden our understanding of how stress contributes to preterm birth. expression, tissular traffic and activation of mmp- in human fetal membranes during labor. rodrigo vega-sanchez, arturo flores, marisol castillo, nardhy gomez, felipe vadillo-ortega. direction of research, instituto nacional de perinatologia, mexico city, df, mexico. introduction. rupture of the fetal membranes (fm) during human labor occurs as a consequence of extracellular matrix degradation. this process is controlled by increased secretion and activity of matrix metalloproteinases, particularly mmp- .several evidences suggest that mmp- is mainly produced by infiltrating choriodecidual leukocytes that could arrive from placental circulation. characterization of the synthesis, transport and activation of mmp- within the fm is critical to understand the process of membrane rupture during human labor. objectives. expression and secretion of mmp- in placental leukocytes, trafficking of the enzyme through the fm and one possible mechanism for its activation were analyzed. methods. leukocytes were isolated from placental blood of women after active labor. maternal leukocytes were used as controls. cells were cultured for h. relative expression of mmp- by rt-pcr and enzyme secretion by elisa and zymography were followed at different times. to analyze the traffic of mmp- through the fm, fluorescein-conjugated prommp- was added to the choriodecidual side of the fm in an in vitro system that allows the separation of amnion and chorion. labeled mmp- was localized at distinct times by confocal microscopy. the protease responsible for the activation of mmp- was identified using neutralizing antibodies and specific inhibitors. results. no difference in the relative expression of mmp- in leukocytes throughout the culture was found. however, secretion of the enzyme significantly increased since h (p< . ). experiments using labeled mmp- , repeatedly showed that after h in culture, the enzyme was mainly localized within the amniotic epithelium. specific inhibition of mmp- significantly decreased the activation of pro-mmp- . conclusions. our results demonstrate that the increased secretion of mmp- by placental leukocytes is not associated to increased gene expression, suggesting that homing of a specific leukocyte subpopulation to the choriodecidua is occurring during labor. mmp- can be trafficked from the choriodecidua to the amnion, suggesting a transmembranal pathway that may regulate the tissular localization of the enzyme to the area of the fm with high content of connective tissue. once secreted by the placental leukocytes, activation of mmp- depends mainly on mmp- , which seems to be derived from the same leukocytes. objective: preterm labour is a major problem in terms of perinatal morbidity and mortality. the histone-deacetylase inhibitor (hdaci), trichostatin a (tsa) has been shown to have an inhibitory effect on myometrial contractility. the aim of this study was to evaluate the effect of the hdaci's suberic bishydroxymate (sbha) and valproic acid (vpa) on human uterine contractions and hence their potential role as tocolytic agents. methods: biopsies of human myometrium were obtained at elective caesarean section (n= ). dissected myometrial strips suspended under isometric conditions, undergoing spontaneous and oxytocin-induced contractions, were exposed to cumulative additions of sbha in the concentration range of nmol/l to mmol/l and vpa ( nmol/l- mmol/l). control experiments were run simultaneously. results: sbha and vpa exerted a potent and cumulative inhibitory effect on spontaneous and oxytocin-induced contractions, compared to control strips. the mean maximal inhibition (mmi) values for sbha were . % for spontaneous contractions (n= ; p< . ), and . % for oxytocin-induced contractions (n= ; p< . ). the mmi values for vpa were . % for spontaneous contractions (n= ; p< . ), and . % for oxytocin-induced contractions (n= ; p< . ). conclusion: these results raise the possibility that hdaci's may have tocolytic potential, in addition to their current clinical indications. the inhibitory effect observed may be linked to the ability of hdac inhibitors to induce the expression of genes involved in the maintenance of myometrial quiescence via epigenetic mechanisms but may potentially also involve non-epigenetic pathways. progestin suppresses thrombin-enhanced interleukin- expression in term decidual cells: implications for abruption-induced preterm delivery. edward kuczynski, lynn f buchwalder, frederick schatz, charles j lockwood. obstetrics/gynecology reprod. sciences, yale university school of medicine, new haven, ct, usa. background and objective: decidual hemorrhage (abruption) promotes binding of factor vii to decidual cell (dc)-expressed tissue factor to generate thrombin. thrombin in turn induces several biological effects leading to preterm delivery via activation of cell surface protease-activated receptors (pars). interleukin- (il- ) is a pleiotropic proinflammatory cytokine induced by pars. this study assessed the separate and interactive effects of thrombin and medroxyprogesterone acetate (mpa) on il- expression in term dcs. methods: term decidua from stripped fetal membranes were isolated and the dcs were purified on a percoll gradient, grown to confluence and passaged until leukocyte-free. confluent dcs were primed in - m estradiol (e ) of e + - m mpa for days, then incubated in a defined medium (dm) with corresponding steroids ± thrombin. after hours, il- levels in conditioned dm were measured by elisa and western blotting. in parallel -hour incubations, il- mrna levels were assessed by quantitative rt-pcr and normalized to -actin mrna. results: secreted il- levels were similar in cultures maintained in e alone ( . ± . ) and e + mpa ( . ± . pg/ml/ug protein; mean ± sem; n = ). the addition of thrombin ( . u/ml) enhanced secreted il- levels by . ± . fold (p< . ) in incubations with e and by . ± . -fold (p< . ) in incubations with e + mpa. the inhibitory effect of mpa was statistically significant (p< . ). in confluent dcs incubated with e + mpa, exogenous thrombin ( . - . u/ml) elicited a concentration-dependent increase in secreted il- levels. hirudin acted as a pure thrombin antagonist, exerting no agonist effects alone, but counteracting thrombin-enhanced il- secretion. western blotting confirmed the elisa results. quantitative rt-pcr confirmed that il- m-rna levels corresponded to protein changes. conclusion: thrombin enhances il- mrna and protein expression in term dcs and progestin blunts these effects. since thrombin-generating abruption is closely associated with preterm delivery, anti-inflammatory effects induced by progestin inhibition of dc-derived il- may contribute to the protection against ptd, and may explain the reported protective effects of administration of -oh-progesterone in recent clinical trials. introduction: catechol-o-methyltransferase (comt) catalyzes the methylation of the phenolic hydroxyl groups in a variety of catechols. during estrogen metabolism, this enzyme converts the catechol estrogen, -hydroxyestrogen ( ohe ), to -ethoxyestrogen ( meohe ). the comt substrate, -ohe , can exhibit an anti-estrogenic effect in multiple biologic assays while the methoxyestrogen ( -meoe ) can exhibit an estrogenic effect. the biologic activities of these estrogen metabolites ( ohe meoe) depend upon their concentrations and tissue type. since comt activity ultimately controls levels of these metabolites, it appears to be a key factor in regulating the cellular estrogenic milieu. we have recently reported that amnion layers of human fetal membranes from laboring women exhibited folds higher comt mrna expression when compared to non-laboring women (wentz et. al. sgi ) . objective: to investigate the impact of comt inhibition on prostaglandin e (pge ) production by human amniotic membrane explants. study design: explants consisting of -cm circular sections of the amnion layer (obtained from term pregnant women who underwent elective repeat cesarean section) were prepared and placed in tissue culture explants media at ºc. after a -hour incubation, explants were treated with the selective comt inhibitor ro - , at and m concentrations. the incubation media was harvested after and -hour intervals. the levels of prostaglandin e (pge ) in the media were measured by sensitive elisa and were normalized against total protein concentration. results: ro - comt inhibitor induced major reductions in pge production in media collected from amnion explants of human fetal membranes. in the group treated with m of ro - for hours there was %± % reduction of pge after hours compared to untreated control (p< . ). in the amnion explants treated with m of ro - , there was %± % after hours and %± % after hours of treatment compared to untreated control (p< . ). conclusions: this finding indicates that comt activity in the amnion layer of human fetal membranes affects pge production. by facilitating a pro-estrogenic milieu in human fetal membranes in late gestation, increased comt activity may indirectly increases production of factors associated with labor such as pge . hypothesis: an extensive remodeling of the human cervical connective tissue takes place throughout pregnancy with a decrease in the total concentration of collagen and proteoglycans due to an altered higher metabolic turnover. we hypothesize that the profound changes in proteoglycan production in the human pregnant cervix can be seen in corresponding cervical fibroblasts as well. we also hypothesize that proteoglycan production in cervical fibroblasts from preterm partal women are simmilar to the production in fibroblasts from partal women. method: cervical biopsies were obtained from non-pregnant women, women during elective cesarean section, woman after spontaneous parturition and after a preterm vaginal delivery. by explant technique fibroblasts were cultured from the biopsies. produced proteoglycans were metabolically labeled with s during hours and then purified by ion-exchange chromatography and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. results: the total proteoglycan production decreases with approximately % in partal and preterm partal cell cultures. the reduction of proteoglycans in preterm partal and partal cell cultures is significant compared to non pregnant fibroblast cultures. the distribution of biglycan and perlecan are similar in partal and preterm partal cells. biglycan are significantly reduced by around % and perlecan are significantly increased by around % compared to non pregnant cultures. preterm partal cervical fibroblasts secreates significantly more heparan sulfate proteoglycans compared to non pregnant cultures. the changes in total proteoglycan production in the human pregnant cervix can be seen in corresponding cervical fibroblasts as well. both partal and preterm partal cell cultures differ in their proteoglycan production compared to their non pregnant counterpart, suggesting a role for proteoglycans in cervical ripening. the role of the oxytocin/oxytocin receptor system is not well defined in human amnion. previous studies in rabbit amnion have demonstrated an up-regulation of oxytocin receptors in the end of pregnancy and have shown that there is a large increase in the ability of ot to stimulate pge production. we and others have previously shown a role for nf-b in otr regulation. in whole genome array analysis of human amnion we found that otr was the gene with the second highest increase associated with activation of nf-b (the highest being cox- ). the present work was directed towards further understanding of otr expression and function in human amnion at term. we have shown that pge release by pre-labour primary human amnion cells is significantly increased after oxytocin treatment for hrs (ot: - m; n= ; triplicate samples; fold increase p< . ). the expression of otr in labour (+) and labour (-) primary amnion cells was measured with real time rt-pcr. we found a significantly higher level of expression in the labour (+) cells (n= ; duplicate samples; p< . ). western blot analysis confirmed the upregulation of otr in labouring amnion. treatment with il- b resulted in a significant upregulation of otr which peaked with a fold induction after hours (p< . ). il- caused a fold increase in otr mrna levels in labour (-) cells, bringing the expression level up to that found in labour (+) cells. our findings provide further evidence for a role of otr in human amnion. expression of otr in human amnion is significantly increased after the onset of labour. term non laboured amnion can be stimulated with il- to increase otr expression to levels of laboured amnion. one of the functions of otr in amnion cells is stimulation of prostaglandin synthesis. expression of antioxidant defence proteins in human myometrium before and after the onset of labour. vasso terzidou, mandeep s kandola, shirin khanjani, jan j brosens, phillip r bennett. parturition reserach group, irdb, imperial college, london, united kingdom. background oxidative stress is a result of an imbalance between the production of reactive oxygen species (ros) and the antioxidant defence mechanisms present in biological systems. parturition and infection-induced preterm labour resemble inflammatory processes that are linked to the production of ros including super-oxide (o -), hydrogen peroxide (h o ) and peroxynitrite. low concentrations of ros can act as second messengers in the regulation of several cellular functions. in an attempt to maintain redox homeostasis cells are equipped with machineries to both produce and scavenge ros. enzymatic scavengers include superoxide dismutase (sod), glutathione peroxidise and catalase. sod is the first enzymatic step in the defence system against oxidative stress. nuclear factor-kappa b (nf-b), a transcription factor family classically associated with inflammation, is activated in response to infection and proinflammatory cytokines, such as those prevalent during labour. labour is associated with an increase in nf-b activity in human myometrium and in fetal membranes. nf-b is known to regulate a range of genes associated with the onset of labour. in a study using affymetrix whole genome arrays analysis nf-b overexpression was associated with increased expression of sod- ( . fold). to determine changes in ros scavenging potential upon onset of labour, lower segment myometrial biopsies were taken at term before and after the onset of labour (n= ; each group). cdna was extracted from the tissues and real time rt-pcr was performed for sod- , serum/glucocorticoidinduced protein kinase- (sgk- ) and the dna repair enzyme gadd a. we found that labour onset is associated with a two fold increase in the levels of expression of each. oxidative damage at the fetomaternal interphase has been extensively studied in the placenta as part of investigations for first trimester pregnancy losses, iugr and pre-eclampsia. the role of ros is less well defined in human decidua and the underlying myometrium. our results suggest a role for oxidative stress and redox homeostasis in the maintenance of myometrial quiescene during pregnancy and onset of labour. plasma anandamide levels increase during labour induction and appear to delay labour progression. vijianitha nallendran, anthony h taylor, patricia mw lam, stephen c bell, david j taylor, justin c konje. cancer studies and molecular medicine, university of leicester, leicester, united kingdom. background: the evidence for a role of the endocannabinoid, anandamide (aea) in labour is conflicting. we previously showed elevated aea plasma levels in labouring women whilst another group showed that activation of functional receptors for anandamide actually inhibited uterotonin-induced contractions through an adenylate cyclase pathway . the aim of this study was to explore further the relationship between labour and plasma aea levels. methods: plasma aea levels in women undergoing induction of labour for various indications at term, were measured by a sensitive isotope dilution method using hplc-ms/ms. each volunteer had an assessment of her cervix prior to the start of the induction when the first sample for aea was collected. once active labour was established (cervix cm dilated; contractions every - minutes), a second sample was collected. results: seventeen ( %) of the subjects were multigravida. the inductions were for postdates(n= ); decreased fetal movements(n= ), fetal growth restriction(n= ), symphysis pubis dysfunction(n= ), diabetes mellitus(n= ), pre-eclampsia(n= ), fetal cardiac complication(n= ), spontaneous rupture of newborn offspring with persistent pulmonary hypertension, despite enhanced fetal membranes(n= ). plasma aea levels increased significantly once labour was established (figure) and demonstrated in ( %) of the cases. the median (interquartile range) plasma aea level increased from . nm ( . - . ) at induction to . nm ( . - . ); *p= . ; mann-whitney u-test) at active labour. there was a positive correlation between plasma anandamide levels taken before induction and the time taken for the women to enter into active labour (r = . , p= . pearson correlation). plasma aea levels were higher in labouring women compared to non-labouring women after the induction of labour confirming our previous observations and suggesting a direct role for this endocannabinoid in labour. further studies are required to elucidate this role. nuclear factor-kappa b (nf-b) is a transcription factor family classically associated with inflammation, activated in response to infection and proinflammatory cytokines, such as those prevalent during labour. as a cytokineinducible transcription factor it plays a key role in the expression of a variety of genes involved in inflammatory responses and cell survival. nf-b dna binding and transcriptional activity plays an important role in labour associated gene expression in myometrium. in this study we examined the range of genes regulated by nf-b in myometrium using transient transfections and wholegenome array analysis. myometrial cells were extracted from myometrial biopsies taken at the time of elective caesarean sections at term. transient transfections of primary myocytes were performed using expression vectors for nf-b p .the amount of dna was constant and psg was used as a control construct. cdna was made from myocytes transfected with either nf-b or psg . affymetrix genechip u microarray was performed (n= , each group). we found that genes were significantly differentially expressed between control and overexpressed nf-b samples. twenty eight of these genes were upregulated with nf-b and were down regulated. several chemokines and cytokines were identified in the upregulated group. interleukin demonstrated the highest, fold, induction in the upregulated group, followed by tumor necrosis factor, a-induced protein (tnfaip ), chemokine ligand (ccl ), chemokine ligand (ccl ), pentaxin related gene (ptx ), interleukin (il ) and superoxide dismutase (sod- ). nine genes were present in the nf-b group that were absent in the control group. these included chemokine ligand (ccl ), chemokine ligand (ccl ), chemokine ligand (cxcl ) and il . ingenuity pathway analysis demonstrated that immune response, inflammatory, cell growth and proliferation and cell death were the main pathways involved. standardisation experiments have been performed, and the microarray results were confirmed with real time rt-pcr in several candidate genes. our results provide further support in the role of nf-b in human labour and suggest its direct link in upregulation of inflammatory genes, cytokines and chemokines, consistent with the imflammatory nature of the biochemistry of labour. inflammation is widely accepted to be a key feature of human labor. secretory leukocyte protease inhibitor (slpi), an innate immune molecule, has been shown to be an antimicrobial and anti-inflammatory protein. the aims of this study were to verify its expression and localization in human myometrium methods: specimens were obtained at time of cesarean delivery with or without labor. expression and localization of slpi was detected using immunohistochemistry. slpi expression pattern relative to nf-b p subunit was compared between not in labor and in labor subjects, between different tissue sections as well as in in vitro model systems including myometrial explants, uterine smooth muscle cells (usmc) and ishikawa endometrial adenocarcinoma cells. slpi was predominantly localized to the nuclei of myocytes. the observed nuclear immunoreactivity of myocytes was increased during the labor relative to not in labor, paralleled with p nuclear translocation. the nuclear pattern of slpi is specific to myometrium since slpi immunostaining was present exclusively in the cytoplasm of all other tissues examined, including amnion, chorion, decidua and endometrium. slpi staining was also positive in macrophages, indicated by co-localization of slpi with cd positive cells. treatment with il- or tnf-induced nuclear translocation of p in myometrium explants and usmc, but not in ishikawa cells. in human myometrium, slpi is predominantly localized in the nuclei of myocytes and in macrophages. the nuclear expression pattern of slpi is myometrium-specific and increased following the onset of labor and correlated with nf-b activation. further understanding of its physiological significance may suggest new strategies aimed at preventing preterm birth. application of a new proteomic technology on amniotic fluid in prom. sara consonni, niccolo bosso, marianna andreani, agnese pizzardi, fulvio magni, anna locatelli. obstetrics and gynaecology, university of milano-bicocca, monza, italy; experimental medicine, university of milano-bicocca, monza, italy. objective: mass spectrometry (ms) is the obligatory tool for proteomics studies. biological samples must be purified before ms analysis due to the matrix complexity. a recent approach combines active surface prepurification with maldi-tof (matrix assisted laser desorption) analysis. clinprot technology provides the prepurification of the sample through the use of magnetic beads (mb) with activated surface. this technique can be carried out by robot in an automated way on a large number of samples. a unique example of the use of mb before maldi-tof analysis to determine proteomic profiles of amniotic fluid (af) is reported for rapid detection of fetal aneuploidies (wang ) . the objective of our study was to verify the applicability of clinprot prepurification before maldi-tof analysis on amniotic fluid collected noninvasively in premature rupture of membranes (prom). methods: we sampled af from vaginal posterior fornix of women with preterm prom (group , n= ) and term prom (group , n= ). samples were prepared with mb and analyzed with ms maldi-tof in order to generate proteomic profiles. results: it was possible to generate average proteomic profiles in the two study groups and the observed profiles were different. samples of af non invasively collected in prom can be analyzed by ms maldi-tof after preparation with mb. this technique allows to retain part of the eluted sample for characterization of protein peaks of interest. due to the less laborious characteristics of this method in comparison with techniques based on bidimensional electrophoresis its application can be useful in clinical proteomics. progestins accentuate the maternal but not fetal inflammatory response of women with intra-amniotic inflammation. sonya abdel-razeq, irina a buhimschi, michael cackovic, guoyang luo, antonette dulay, victor rosenberg, mert bahtiyar, errol norwitz, edmund funai, catalin buhimschi. ob./gyn. reprod.sci., yale university, new haven, ct, usa. introduction: data from animal research suggests that progestins have a marked pro-inflammatory capacity. recent studies support the administration of -hydroxyprogesterone caproate in women at risk for preterm birth. we sought to determine the impact of progestins during gestation on the extent of maternal and fetal inflammatory responses in pregnant women with intraamniotic inflammation. methods: amniotic fluid, placenta and cord blood were obtained from women who delivered preterm (median[range], ga: [ - ] wks). an amniocentesis was done to rule out infection. women exposed to progestins (n= ) within one week prior to amniocentesis were matched to controls (crl) by age, parity, history of preterm birth ga, membrane status and interval to delivery. proteomic profiling of amniotic fluid [mass restricted (mr) score] identified the presence or absence of intra-amniotic inflammation. an mr score of or confirmed intra-amniotic inflammation. amniotic fluid and umbilical cord interleukin- (il- ) levels were measured by elisa. histological chorioamnionitis was graded based on recognized criteria. results: overall, women and their fetuses exposed to progestin did not exhibit an increased inflammatory response (table) . however, sub-analysis restricted to women with mr or (n= ) showed that in the context of intraamniotic inflammation, progestins were associated with significantly elevated amniotic fluid il- levels compared to unexposed women (progestins (n= ): vs. crl (n= ): [ . - ] ng/ml, p= . ). these relationships were maintained after correction for steroid and antibiotic exposure. such significance was not found for amniotic fluid glucose, ldh, wbc count or cord blood il- . conclusion: our results suggest that progestins may amplify the maternal, but not the fetal inflammatory response of women with intraamniotic inflammation. objective: recently progestins have been shown to reduce the incidence of recurrent preterm labor in women. progestins have long been known to inhibit preterm labor in some species including rats and are also known to delay term labor. nitric oxide (no) donors, including ng, inhibit uterine contractility and have been used as tocolytics. the aim of this study was to examine the inhibitory effects of r on preterm labor in rats induced with a progesterone antagonist (onapristone, zk) with and without ng. materials and methods: charles river s-d timed pregnant rats (n= /group) were treated with zk ( mg/rat, s.c.) alone or vehicle (controls) on day of gestation. other groups of rats were treated with zk in combination with various doses of r ( . , or mg/rat s.c) with and without ng ( mg s.c. pellet) from days to of gestation. all rats were sacrificed on day of gestation and the number of fetuses and implantation sites were counted to determine the preterm delivery rate. one way anova was used for statistical analysis. p< . was considered significantly different. results: rats treated with zk alone delivered all their fetuses prematurely compared with controls (p< . ) treated with vehicle only (ca. % fetuses delivered). ng treatment alone did not affect the delivery rate (p> . ) compared to controls. similarly zk + ng did not reduce the preterm delivery rate compared to zk alone (p> . ). however r dose dependently reduced (p< . at all doses) the number of fetuses delivered prematurely in response to zk and the premature delivery rate was further reduced when treatment included the combination of r plus ng (p< . ). conclusions: zk effectively induces premature delivery. premature delivery produced by zk can be effectively reduced with a r , a progestin known to bind with high affinity to nuclear progesterone receptors. ng by itself, at the dosage used, does not reduce the prematurity rate caused by zk. however, r and ng act synergistically to reduce the preterm delivery rate. this study indicates that combinations of a progestin with an no donor may be an effective treatment for preterm labor and delivery. monkeys. pl grigsby, jp rasanen, , dw sadowsky, m bertolino, m carbonatto, e gillio tos, s canali, j lacy, a chollet, mj novy. reprod sci, oregon primate res ctr, beaverton, or; ob/gyn, oregon health sci univ, or, usa; rbm, merck serono, italy; merck serono, switzerland. objective: to investigate the pharmacokinetics (pk) and pharmacodynamics (pd) of as , a novel orally active non-peptide oxytocin (ot) antagonist in rhesus monkeys. study design: dose finding and pk/pd studies were done in chronically instrumented non-pregnant (n= ) and pregnant ( - dga, term d; n= ) monkeys. maternal and amniotic fluid compartments were serially sampled during dosing and washout. treatment phases included: ot infusion control, ot infusion + as , and ot rescue therapy. ot infusions ( - mu/kg/hr, iv) were given to determine the lowest dose required to produce stable, sub-maximal uterine contractions in each animal. as was administered p.o. at , , mg/kg and the effects on inhibition of uterine activity (hca, mmhg.sec/hr) were compared. to verify antagonist reversibility, objectives: infection such as chorioamnionitis is thought to be the cause of premature rupture of the membrane and induce uterine contractions leading to preterm delivery. however, the mechanism for enhancement of uterine contractility is not well understood. we have reported that non-selective cation channels (nsccs) regulate pacemaker potentials to generate rhythmical contractions and should be targets of magnesium ions used for tocolysis. the purpose of this study is to investigate the changes of the expression of nsccs during normal pregnancy and the effect of inflammation in preterm. methods; atp receptors (p x) and transient receptor potential canonical (trpc) channels were examined as uterine nsccs. the mrna was extracted from the rat myometrium of non-pregnant and pregnant rats at days , , and . the expression of each subtype of p x and trpc channels was measured by real time rt-pcr (abi ) with taqman probes (abi). as an inflammatory model lipopolysaccharide (lps; . mg/kg) was injected into intraperitoneal cavity at day and the tissue was sampled after six hours. results; p x and p x were determined to be dominant subtypes of p x channel. the expression of p x was increased by % at day , compared with day and that of p x was enhanced by %. on the other hands, trpc and trpc were detected dominantly. the expression of trpc was increased three times in the late stages of gestation. however, trpc was suppressed by %. in the lps treated rat myometrium cox- mrna expression was measured to be fold higher than that of the control rat, showing inflammatory effects in the myometrium. in this model the expressions of p x , p x and trpc were enhanced by . , . and . times, but trpc was not changed. the mrna expression of p x , p x and trpc channels in rat myometrium was increased in the late stages of pregnancy. these channels are suggested to be concerned with onset of labor. in the inflammatory model the expression of these channels was accelerated dramatically and these values were much higher than those in normal pregnancy. this finding supposed inflammation may enhance some types of nsccs to accelerate uterine contractility and induce preterm delivery. anandamide ( to determine the mechanism of rho protein activation during oxytocin and carbachol induced contraction, freshly prepared myometrial strips in krebs henseleit buffer were treated with oxytocin ( nm) and carbachol ( μm) under isometric and tension free conditions. control strips were exposed to buffer only. treated myometrial strips were solubilised and separated into membrane and cytosolic extracts and equal aliquots were immunoblotted with rhoa and rhob antibodies. rhoa translocated to the membrane after oxytocin and carbachol stimulation under both isometric and tension free conditions (p< . ). there were no significant changes in rhob membrane to cytosol ratios relative to control. agonist induced contraction in human myometrium is associated with rhoa but not rhob membrane translocation. during pregnancy components of the intracellular camp signalling pathway show increased gene expression resulting in the maintenance of myometrial quiescence until term where a substantial decrease in expression of these genes is observed. protein kinase a regulatory subunit riia (rii ) is upregulated at both the mrna and protein levels in the human myometrium during pregnancy. this particular subunit is membrane-bound and by directing phosphorylation to myometrial cytoskeletal proteins may affect contractile machinery thus playing a role in maintaining uterine relaxation. acetylation of histones promotes a favourable chromatin environment for transcriptional activity of many genes. this process is largely inhibited by histone deacetylases (hdacs), whereby its activity leads to transcriptional repression. hdacs can be recruited to the promoter region of a gene by other transcription factors such as sp proteins. since the rii promoter contains three sp - consensus binding sequences in its proximal part, we investigated whether this gene is a target for transcriptional regulation by hdacs. using dna precipitation assays we found that sp , and as well as hdac and form complexes with biotin-labelled fragments relating to sp - elements in the promoter region of rii . additionally, treatment of myometrial primary cell cultures with hdac inhibitor trichostatin a (tsa), or with the methyltransferase inhibitor -azac resulted in increased mrna and proteins levels. further studies with full and truncated luciferase constructs of the promoter region of the rii gene in transiently transfected myometrial cells confirmed that all three sp - elements are involved in the transcriptional regulation of the gene. this process involves hdacs, as h treatment with tsa significantly increased the luciferase signal. changes in the binding of sp proteins and hdacs to the promoter after tsa and azac treatment were investigated employing chromatin immunoprecipitation assays. alterations in the methylation status of the promoter after treatment were examined by bisulfite modification and dna sequencing. together, this study highlights the importance of chromatin modifications in the maintenance of uterine quiescence during pregnancy as well as identifying a potential mechanistic target for drugs that may reduce the incidence of preterm labour. objective: progesterone maintains pregnancy by promoting myometrial quiescence. typically progesterone effects are thought to be mediated through the classic genomic pathway. there is evidence, however, that progesterone also acts via a non-genomic pathway by interacting with specific membrane progesterone receptors (mprs) and in particular progesterone receptor membrane component - (pgrmc- ). the role of non-genomic progesterone actions in human pregnancy and parturition is not clearly understood. the goal of this study was to measure the extent of mpr expression in biopsy specimens of human myometrium obtained at cesarean delivery, and to determine whether expression changes with advancing gestation or the onset of labor. study design: lower uterine segment myometrial biopsies were obtained at the time of delivery from consenting women who were at term and not in labor (n= ), preterm and not in labor (n= ) and term and in labor (n= ). protein extracts were prepared and subjected to polyacrylamide gel electrophoresis and immunoblot analyses for pgrmc- and gapdh. abundance of pgrmc- protein relative to gapdh was determined by digital densitometry. we also performed immunohistochemistry (ihc) to determine the cellular localization of pgrmc- in the human pregnancy myometrium. results: pgrmc- protein was identified in each biopsy specimen. there was a -fold increase in pgrmc- protein in term compared with preterm biopsies (relative to gapdh p< . ). the relative level of pgrmc- protein was not different between biopsy specimens from laboring and non-laboring women at term (compared to gapdh, p= . ). pgrmc- immunoreactivity was localized to granular cytoplasmic staining. conclusion: this is the first description of the presence of pgrmc- protein in the human myometrium during pregnancy. its presence suggests that progesterone may influence contractility non-genomically via these receptors. the functional significance of the gestational age associated two fold increase in pgrmc- is unclear. changes in expression of this receptor during pregnancy may be important for the hormonal control of parturition and can be the focus of future studies. (ruddock et. al., abstract smfm, ) . the aim was to examine the mechanism of p inhibition. methods: uterine tissues from women (n= ) at term with cesarean section, were suspended in organ chambers and exposed to various agents or solvents. contractility was registered, stored, analyzed and compared before and after addition of agents or kcl. tissues were treated with p alone ( - to - m) or p bound to bovine serum albumin (bsa/p , - to - m p ), a progestin with low affinity to mpr (r , - - - m), or a non-sex steroid (cholesterol, - to - m). other tissues were pretreated with selective inhibitors of adenylate cyclase (sq , - m), guanylate cylase (odq, - m), phosphodiesterase (rolipram, - m), nitric oxide (no) synthases (l-name, - m) or a nuclear p receptor antagonist (mifepristone, mif, - m), followed by p . data were analyzed by anova for statistical differences (p< . ). results: p rapidly (< hour), effectively ( %) and dose-dependently inhibits spontaneous and kclinduced contractility (ed of < - m incubation of strips with zd- , at concentrations up to m for one hour prior to the experiment, led to no significant reduction in the total work done. however, incubation of strips with l- with a concentration of nm for one hour prior to the experiment, led to a statistically significant reduction in the total work done after one and a half hours. furthermore, when increasing concentrations of pge ( - to - ) were added to l- ( nm) pre-treated strips, total work done per contraction was comparable to that of non-treated controls. similar effects were not observed in zd- ( nm) pre-treated strips. since the reduction in contractility caused by l- was greater than that caused by zd- , it is likely that the stimulating effect of pge acts predominantly via ep receptors. taken together with our previous data showing an increase in ep at labour, this data shows that targetting an ep receptor may be a useful strategy in managaing pre-term labour. recently a truncated kda isoform of the er-has been described in human endothelial and testicular cells. we describe the presence of this kda erisoform in pregnant and immortalized non-pregnant human myometrial cells. methods: myometrial tissue obtained from non-laboring pregnant women undergoing cesarean section at term (n= ) was dissected prior to being finely minced. a portion of the tissue was placed in pbs and sonicated, while the remainder of the tissue was dissociated with collagenase prior to filtration and placement on culture plates. cells were then cultured in mem w/ % fetal bovine serum (fbs) until confluence. cultured cells were then dissociated with . % typsin/edta, subsequently re-plated and grown to confluence. immunohistochemistry directed toward smooth muscle protein was used to verify myometrial phenotype.) protein was extracted and quantified. western blot was performed with mouse monoclonal anti-er-receptor antibody (santa cruz biotechnology) to the f- domain of the human er-receptor. an immortalized non-pregnant human myometrial cell line provided by ann word, htert, was cultured and isolated as described above. results: htert cells expressed both the truncated ( kda) and the full length ( kda) er-isoforms. fresh and cultured myometrial cells from non-labored term pregnant patients also expressed both isoforms of er-. subsequent subcultures of myometrial tissue continued to express the kda erisoform, yet the expression of the kda isoform was lost. a representative blot is below. conclusions: we demonstrate, for the first time, the presence of a kda erisoform in cultured and non-cultured pregnant and cultured nonpregnant human myometrial tissue. discovery of this isoform of er-in myometrial tissue could provide insight into the molecular mechanisms involved in parturition. the action of prostaglandin f (pgf ), a potent uterotonic stimulant that is associated with labour at term and preterm, is mediated by its receptor, ptgfr. myometrial ptgfr mrna levels fall during pregnancy and this likely plays a role in uterine quiescence. however, the mechanisms by which this occurs are poorly understood. we previously reported that pgf downregulates fp mrna expression in cultured human myometrial ultr cells in a protein kinase c (pkc) dependent manner. in addition to the downregulation of mrna levels, receptor desensitization may also represent another mechanism of decreasing ptgfr activity because ligand binding to g protein coupled receptors often results in receptor internalization. we therefore hypothesized that pgf treatment of ultr cells also results in pkc dependent ptgfr internalization. methods: near confluent cultured human myometrial ultr cells were treated +/- - m or - m pgf for , , or hr. cells were fixed with formaldehyde and visualized for localization of ptgfr by immunofluorescence. to examine the potential involvement of pkc in the process, ultr cells were treated +/- - m pgf and +/- m myristoylated pkc inhibitor ( - ) and examined for ptgfr cellular localization as described above. results: pgf treatment resulted in a dose dependent decrease in ptgfr membrane signal at , , and h. this decrease was dependent on pkc as cotreatment with the myristoylated pkc inhibitor ( - ) prevented the pgf induced decrease in membrane ptgfr at h treatment. there was no visible effect of the pkc inhibitor on ptgfr membrane signal on its own. conclusion: we conclude that pgf decreases membrane levels of ptgfr protein in human myometrial ultr cells in a pkc dependent manner. these results suggest that pkc may be required for both the pgf induced internalization and desensitization of ptgfr protein and downregulation of ptgfr mrna in human myometrial ultr cells. therefore pkc may play a crucial role in downregulating ptgfr expression and activity and maintaining uterine quiescence during pregnancy. rhoa is a small gtpase that acts as a molecular switch to control a variety of signalling pathways in smooth muscle, including contractility. it is thought that increases in rhoa-gtp levels facilitates phosphorylation of target proteins such as cpi- , promoting contractility at pre-term and term labour in humans. however, in situations of acute or chronic hypoxia in the uterus, it is important that myometrial contractility and subsequent labour is not facilitated prematurely. given the importance of rhoa to a cell's response to hypoxia in other cell types, it was hypothesised that rhoa plays a central role in the mechanism controlling smooth muscle contraction in the uterus too. following acute hypoxia ( . % o ) for one to six hours, rhoa mrna, total protein and activation (rhoa-gtp) levels were analysed, using semi-quantitative pcrs and western blot, and compared to normoxic non-pregnant human uterine smooth muscle control cells. next, we investigated whether reduced oxygen conditions affected oxytocin induced activation of rhoa, following a two hour treatment of nm oxytocin. firstly, our results demonstrate that the rhoa itself is significantly activated under low oxygen conditions, resulting in phosphorylation of myosin phosphatase, myosin light chain and cofilin, three proteins known to be central in contraction and actin filament organisation. secondly, hypoxia significantly reduced the coupling of oxytocin to rhoa activation under the conditions examined. we observed a significantly reduced level of rhoa expression and activation which correlated with an increase in the level of another rhogtpase protein, rhoe. we propose that rhoa inactivation occurs through a rhoe-mediated mechanism, suggesting a balance in the activity of these two antagonistic rhogtpases in oxytocin-induced hypoxic human uterine smooth muscle cells. these results provide a possible explanation for the reduced coupling of oxytocin as a stimulant of myometrial contractions during slowly progressing labours. oxytocin-induced release of calcium ions (ca + ) from sarcoplasmic reticulum (sr) and sensitisation of contractile proteins to ca + have been suggested to mediate the oxytocin-induced potentiation of myometrial contractions. objective: we investigated the effects of oxytocin in the presence of nifedipine, a known inhibitor of the l-type calcium channel (ltcc). method: samples of myometrium were obtained from women undergoing term caesarean section with the approval of the local ethics committee. a standard organ bath system (ad instruments, uk) was employed to analyse contractile activity. stable spontaneous contractions were recorded for - minutes before addition of nifedipine. results: in agreement with our previous findings, application of oxytocin to spontaneously active strips produced a two-component effect: a transient tetanus-like contraction, followed by prolonged augmentation of phasic contractions. nifedipine ( um) rapidly abolishes spontaneous contractions, subsequent addition of nm oxytocin produced an initial, transient rise in force, approxiamately % compared to oxytocin alone, followed by high frequency oscillations in > % of strips. calcium-free solutions were used to confirm that oscillations were due to ca + entry. disabling the sr store using thapsigargin ( um) had no effect on oscillations, confirming the sr not to be involved. the t-type calcium channel blocker, mibefradil ( um ) showed no inhibition of oscillations. an ip receptor and store-operated calcium channel inhibitor, -aminoethyldiphenylborate ( -apb) um also had no effect on oxytocin-induced oscillations. the store-operated calcium channel inhibitor skf- ( um) showed partial inhibition of oscillations. conclusions: based on these results, we propose that the most likely mechanism of ca + entry producing oxytocin-induced oscillations in the presence of nifedipine is the transient receptor channel-c (trpc) channel, known to be present in the human myometrium. further work needs to be completed to clarify this further. identification of stim and orai in human myometrium. a new paradigm in store-operated calcium signaling. evonne c chin-smith, mark r johnson, rachel m tribe. division of reproduction and endocrinology, king's college london, london, united kingdom; department of maternal fetal medicine, imperial college school of medicine, chelsea and wesminster hospital, london, united kingdom. background: recent reports have suggested that two novel proteins, stim and orai, are involved in the regulation of store-operated calcium entry. we have previously reported that members of the trpc family, putative basal and store operated calcium entry channels, are present in human myometrium and regulated by labour associated stimuli il- beta and mechanical stretch. although stim and orai isoforms have been reported in other smooth muscle cell types, there are no published reports of stim and orai expression in human myometrium. the aim of this study was to identify mrna expression of stim , stim , orai and orai in human myometrium. methods: human myometrial biopsies were obtained from women undergoing elective caesarean section at term (prior to labour) with informed written consent and institutional ethics committee approval. whole myometrial tissue was either snap frozen and stored at - o c or used for cell culture. rna was extracted from whole tissue (n= ), primary cultured myometrial cells (n= ) and passaged (p ) myometrial cells (n= ) and stim , stim , orai and orai mrna expression was assesed by quantitative real-time pcr. results: all four genes were expressed in whole myometrial tissue and cells. stim and stim mrna expression in cultured myometrial smooth muscle cells (primary and passaged) was significantly reduced compared to myometrial tissue expression (p< . ). however, there was no significant difference in either orai or orai expression in whole tissue versus cultured myometrial smooth muscle cells. conclusion: to our knowledge this is the first report of stim / and orai / mrna expression in human myometrium. these genes may contribute to the regulation of calcium signalling in human myometrium, but the functional significance of their expression remains to be determined. funded by the bbsrc. obstetrics and gynecology, national university of ireland, galway, galway, ireland. objective: the ability of uterine smooth muscle cells to stimulate collagen contraction has been well established as an in vitro model of myometrial contractility. devost and zingg ( ) reported that the contractility of human myometrial cell lines layered onto collagen matrices was increased by oxytocin while another group described the stimulation of human uterine smooth muscle cell contractility, cultured within collagen lattices, with endothelin- (dallot et al., ) . our study investigated the response of human primary uterine smooth muscle cells cultured within collagen lattices, to various compounds, including the non-specific depolarizing agent potassium chloride (kcl), the inflammatory cytokine tnf , the rock- inhibitor y- , oxytocin, and oxytocin plus its clinically used antagonist, atosiban. methods: human primary uterine smooth muscle cells (utsmc) (lonza) were maintained in dmem high glucose media. cells ( , per well) passage - , were embedded in . mg/ml collagen, in . ml aliquots, in dmem-f media on well plates (invitrogen) (dallot et al., ) . effects on contraction were studied by monitoring changes in gel area (alpha innotech imager, image j software (nih)). statistical significance was determined by the student t test. results: the utsmcs displayed basal contraction of the collagen gels while the non-contractile cell line hek cells, did not. kcl ( nm) stimulated an % increase in contractility (n= , p= . ) while nm tnf resulted in an . % increase (n= , p= . ), in comparison to unstimulated cells embedded in gel. the rock inhibitor y- ( m) inhibited contractility, with a . % decrease in collagen gel contractility (n= , p= . ). a % increase (n= , p= . ) in utsmc embedded collagen contractility was observed with nm oxytocin, which was antagonized by atosiban ( m) (n= ). conclusion: this study highlights the importance of the development and optimisation of a reproducible human in vitro myometrial contractility model, to evaluate the effect of various known labor-associated, and also unknown compounds. this should aid in our understanding of the many complex biochemical pathways involved in myometrial contractility at labor, and ultimately contribute to the prevention of preterm labor. changes in intra-mural myometrial blood flow during spontaneous labour using d power doppler angiography (pda). nw jones, , nj raine-fenning, h mousa, mj taggart, k jayaprakasan, gj bugg. nottingham university hospitals nhs trust, united kingdom; nottingham university, united kingdom; university of newcastle upon tyne, united kingdom. methods d pda was used to measure the percentage (%) change in the vascularization index (vi), the flow index (fi) and the vascularization flow index (vfi) in a volume of myometrium at the uterine fundus of nulliparous women at term, in the first stage of uncomplicated spontaneous labour. d data sets were obtained during a single cycle of uterine relaxation (r ), contraction and subsequent relaxation (r ). measurements were made independantly by two authors (nwj and gjb) using vocal® (ge kretz) and the mean value was used for analysis. the results from each woman are presented as a % change of r . data is presented as medians [interquartile range (iqr)] and analysed using non-parametric tests. the median volume (cm ) of interest for r was . cm ( . - . cm ), for the contraction was . (fig. ) . the % change for all three indices between r and r was not significantly different. however, there was a significant difference between the contraction and r (p< . ). the mean intra-class correlation coefficient and % confidence interval (ci) of vi, fi and vfi for the authors (nwj and gjb) were . ( . - . ), . ( . - . ) and . ( . - . ), indicative of good inter-observer reliability. conclusion d pda is a useful and reliable tool in the assessment of changes in intramural myometrial blood flow, which was found to reduce significantly during a contraction but increase again during the following uterine relaxation. . raine-fenning nj, et al. the inter-observer reliability of d pda acquisition within the female pelvis. ultrasound obstet gynecol. ; : - . the role of pgf on myometrial contractility; studied with a selective fp antagonist. shankari arulkumaran, andre chollet, phillip r bennet. imperial college parturition research group, institute of reproductive and developmental biology, hammersmith hospital campus, london, united kingdom; merck serono, geneva, switzerland. objectives: human myometrial strips established in culture will usually begin contracting after one hour. we have previously shown that stretch upregulates prostaglandin synthesis and have therefore hypothesized that spontaneous contractions occur because of stretch-related prostaglandin synthesis. methods: experiments were performed using x mm human pre-labour, lower uterine segment myometrial strips in a dmt myograph ms in oxygenated kreb's solution, with adi powerlab software. spontaneous contractions required stretch force and initial experiments determined that the maximum number of strips attaining spontaneous contractions was greatest at a force of - g. a novel antagonist to pgf (fpa) was studied in this model. the fpa has a ki of nm for fp and is - fold selective for fp compared with other prostanoid receptors. results: addition of pge and pgf after the commencement of spontaneous contractions caused a statistically significant increase in the total work done by the strips. the effect of pge was being greater than that of pgf . pre-incubation of the baths with a novel and selective fp antagonist (fpa) with concentrations up to m ( - ) did not affect the total work done by spontaneous contractions compared to non-treated controls. however, increasing concentrations of the fpa ( - to - ) decreased the total work done by -fold on strips treated with pgf beforehand in comparison the pgf -treated strips alone. conclusion: these data suggest that stretch and synthesis of prostaglandins is essential for spontaneous contractility in human myometrial strips. since fpa is able to block pgf induced but not spontaneous contractions, it is likely that pgf does not play a role in spontaneous myometrial contractility in vitro, although it may do so in vivo. because other factors may combine to increase contractility, the combination of an fp antagonist with other inhibitors may therefore, be a more effective strategy in reducing pre-term deliveries. objectives: brain natriuretic peptide (bnp) is synthesized in fetal membranes and inhibits oxytocin-induced contraction of preterm human myometrium. we hypothesized that bnp may be a paracrine mediator of human myometrial quiescence. we showed bnp content is higher in membranes from preterm pregnancies absent labor and significantly decreased with idiopathic preterm labor. while bnp activates natriuretic peptides receptors a (npr-a), b (npr-b), and its clearance receptor (npr-c), we have shown bnp does not inhibit myometrial contraction via npr-a or npr-b. herein, we test in part the hypothesis that bnp inhibits myometrial contractions by activating npr-c by quantitating npr-c in human myometrium at different gestations and labor status. methods: myometrial samples were obtained at the time of cesarean section after informed consent from groups of patients: preterm not in labor (pt-nl), preterm in labor (pt-l), term not in labor (t-nl) and term in labor (t-l). myometrial samples were obtained from women - weeks' gestation, and term between and weeks. mrna for npr-a, b and c were semiquantitated by realtime pcr (normalized by s mrna), and npr-c protein by western blot. results: natriuretic peptide receptors a, b and c mrnas were identified in all groups. while there were no differences in mrna levels among groups, npr-c protein was increased in samples from term laboring women. conclusion: since bnp inhibits the contraction of human preterm but not term myometrium independent of npr-a and npr-b, we have previously speculated that bnp activates another "unknown" receptor. herein we find that myometrial npr-c protein but not mrna increases during human labor at term. the increase in npr-c at term could compete with the unknown quiescent receptor to functionally reduce the availability of bnp and thus permit or promote myometrial activation/contraction. (supported by a grant from chilean government fondecyt ). objective: bnp is synthesized within human chorion and amnion and inhibits oxytocin-induced contraction of human myometrium. bnp activates guanylate cyclase (gc) natriuretic peptides receptors a (npr-a) and b (npr-b). bnp also stimulates the clearance receptor (npr-c) whose action is not mediated by gc. the intracellular pathway of bnp/npr-c inhibition is not known. we determined that bnp does not inhibit myometrial contraction via npr-a or -b. we hypothesized that bnp inhibits myometrium by npr-c activation. we test aspects of our hypothesis by determining whether bnp/npr-c pathway inhibits myometrial contractions via myometrial nos pathway. methods: bnp and canp (specific npr-c agonist) were added to primary human myometrial cell cultures and enos/inos activity/expression determined. cell cultures (n= ) were prepared from myometrial samples of nonlaboring, term pregnant women. after confluence ( d), the cells were incubated ( h) with nm bnp and/or canp. nos activity was measured ( l-citruline assay) and transcription/translation semi quantitated (realtime pcr and western blotting). results: bnp had no effect on either nos expression or activity. canp significantly reduced inos but not enos mrna level. canp did not alter the protein level of nos but significantly reduced nos activity. co-incubation with bnp and canp reduced both mrna levels (p< . ) and significantly decreased enos, but not inos, protein without change in overall nos activity. within the female pelvis. ultrasound obstet gynecol. ; : - . conclusion: the activation of npr-c by canp reduces nos activity and expression. the same effect was not observed by bnp. the difference may be explained because bnp (but not canp) activates all natriuretic peptide receptors, and they may have opposite actions on nos pathway. we conclude unlikely bnp inhibits human myometrial contraction by npr-c activation via the nos pathway. ( introduction: ultr is a retroviral immortalized human uterine smooth muscle cell line which we use as a model for uterine hypertrophy studies. however, this cell line has limited usage due to a reduced rate of cell division and eventually replicative senescence in culture. one of the mechanisms of human somatic cellular senescence is un-compensated shortening of telomeres, the specialized dna structures located at the ends of eukaryotic chromosomes. introduction of human telomere reverse transcriptase (htert) has been shown to induce telomerase activity and telomere elongation, and extend life-span of normal human cells. objective: to recover ultr cell division without altering the phenotypic characteristics of smooth muscle cells by introducing htert, therefore improving ultr cell line. methods: ultr cells were transfected with a modified htert expression vector at a relatively early stage (passage ) in the presence of selective antibiotic. ultr cell growth rate, with (ultr-ht) or without transfection, was determined by plating cells in multiple plates at a fixed density and counting cell numbers after days. single cell clones of stably transfected cells were further isolated by plating in well plates. expression of htert, smooth muscle specific genes (smc-sgs), and target genes was identified by rt-pcr of total rna extracted from ultr and ultr-ht cells. results: rt-pcr demonstrated successful introduction of htert into ultr cells. the growth rate of ultr-ht cells was increased and cell morphology was improved (free of the typical aneupoid appearance). at passage , ultr-ht cells grew . fold (p< . ) and . fold (p< . ) faster than ultr cells at passages and , respectively. currently single cell clones have been selected. ultr-ht cells express a set of smc-sgs, including -actin, caldesmon, calponin, myosin heavy chain, sm , and smoothelin, confirming that the smooth muscle phenotype is preserved. a panel of genes involved in angiotensin ii signalling, including angiotensin ii receptors (at / ), nox family (nox , , and duox ), nox-associated genes (p phox, p phox, p phox, and rac / ), was also preserved. conclusion: this is the first report of rescuing uterine smooth muscle cell replicative senescence via activation of telomerase. we have established a human uterine smooth muscle cell line which will provide an improved in vitro model for studying human myometrium. at term, myometrium is characterized by spontaneous contractions which vary in the amplitude, frequency and duration depending on specie and hormonal status. repetitive depolarization of plasma membrane followed by transient elevation in [ca + ] i is thought to underlie the contractions. however, the detailed pathways which regulate the spontaneous contractility remain unclear. objective: this study was designed to elucidate the effect of protein kinase c (pkc) on the spontaneous contractions and [ca + ] i transients in the myometria from term pregnant mice and women. methods: the human samples were obtained from women undergoing cesarean section with the approval from the institutional review board committee while mice myometria were dissected from the days pregnant mice sacrificed according to the animal study protocol. the isometric force and [ca + ] i were measured simultaneously in fura- loaded myometrial strips using spectrofluorometer equipped with force transducer. results: the pkc activator phorbol , -dibutyrate (pdbu) applied at - m first stimulated the amplitude of spontaneous contractions in mice and human myometria followed by their inhibition. under the pdbu treatment the frequency of the contractions was first increased and then decreased in both species. at the same time, pdbu didn't initially increase the amplitude of [ca + ] i transients but attenuated it over time. however, the frequency of the transients was first increased and later decreased upon the pdbu exposure. in addition, pkc activation with pdbu resulted in the elevation of the uterine basal tone without corresponding changes in the basal level of [ca + ] i in human myometrium. in mice myometrium, on the contrary, pkc activation didn't result in an increase of the muscle tone and the basal level of [ca + ] i . conclusions: we propose that pkc causes bi-phasic effect on uterine spontaneous contraction in mice and human first to potentiate the amplitudes and the frequencies and later decreases them. the amplitude of [ca + ] i was not initially potentiated by pdbu suggestive of the dissociation of the contractile and [ca + ] i response. the stimulatory effect of pdbu on the basal muscle tone in human myometrium and the absence of the effect in mouse suggest different mode of pkc action on uterine contraction in human and mice. introduction: mechanical stretch of uterine myocytes is detected through integrins on the cell surface which form part of the focal adhesion complex, this signals through mapk, ultimately leading to the up-regulation of various pro-labour genes including pghs- and il- . on integrin activation fak is recruited to the focal adhesion complex and activated by autophosphorylation at tyr- , creating a src binding site. this promotes further fak phosphorylation at tyr- , and , enhancing fak catalytic activity. however fak can also act as a scaffold protein and its exact role in the expression of pro-labour genes is uncertain. in this study we used inhibitors for the kinase activity of fak and mek / in order to test the affect of fak kinase activity on expression on pghs- mrna. methods: primary human myometrial cell cultures were grown from myometrial biopsies taken from women undergoing elective caesarean section. cells were plated onto -well flexible bottom plates coated in type i collagen. cells were subjected to % static stretch for up to minutes. cells were also incubated with either the rho kinase inhibitor y or the mek / inhibitor u prior to being stretched. western blots were performed using antibodies to fak phospho- , fak phospho- and erk / phospho- / , -actin was used as a loading control. rna was also extracted and levels of pghs- measured using q-pcr. results: stretch increased levels of phosphorylation at both tyr- and tyr- with the greatest increases occurring at and minutes. however the increase at tyr- appeared to be greater than at tyr- . incubation with the rho kinase inhibitor reduced phosphorylation of fak- , however this did not affect phosphorylation of erk / or stretch induced up regulation of pghs- mrna. in contrast incubation with the mek / inhibitor reduced erk / phosphorylation and expression of pghs- mrna, whilst also reducing fak phosphorylation (n= ; p= . ). conclusions: fak has previously been shown to be important in activation of the stretch induced mapk cascade and pghs- expression. however these data suggest that while stretch causes fak phosphorylation fak kinase activity is not essential to pghs- expression. this suggests fak may be acting as a protein scaffold. . our aim was to examine the effects of both natural progesterone and hp on spontaneous myometrial contractions. myometrial biopsies were taken with informed consent and ethics approval from non-labouring women at elective caesarean section weeks gestation. strips of myometrium mm long , mm wide were cut and suspended under a resting tension of mn in organ baths of krebs gassed with % o / % co within hours of collection. progesterone or hp were added in a cumulative manner at -minute intervals. changes in amplitude were recorded. results were compared using anova. following equilibration for hours, myometrial strips contracted in a rhythmic manner (amplitude . ± . mn, n= pairs). progesterone ( nm- m) produced a concentration dependent inhibitory effect on myometrial contractions (fig ), which was greater than that of vehicle (p< . ). maximum inhibition measured . ± . % and . ± . % for progesterone and vehicle, respectively. hp exerted an inhibitory effect, this was not significantly different from the vehicle (p> . ). progesterone exerts an inhibitory effect on myometrial contractility in vitro. this is apparent within minutes suggesting a nongenomic action. our data are in agreement with some reports in the literature but conflict with others[ ]. this acute inhibition of myometrial contractility may contribute to the mechanism by which progesterone prevents preterm birth. in contrast, we were unable to demonstrate an inhibitory effect of hp on contractility despite its demonstrated ability to reduce the incidence of preterm delivery [ ] . our data suggest that natural progesterone may be a more effective tocolytic agent in the acute setting than hp. obstetrics and gynecology, ramathibodi hospital, mahidol university, bangkok, thailand; pathology, ramathibodi hospital, mahidol university, bangkok, thailand; obstetrics and gynecology, samuel lunenfeld research institute, toronto, canada. objective: chemokines has been shown to play an important role in regulating uterine function. recent evidence demonstrates that monocyte chemotactic protein (mcp) level in amniotic fluid increases during spontaneous labor. the aim of this study was to examine the expression of mcp in human myometrium. methods: myometrial biopsies were taken from nonpregnant women undergoing hysterectomy and term pregnant women undergoing cesarean section followed written consent and local ethics committee approval. elective cesarean section was performed before the onset of labor while emegency section was done after the onset of labor. immunolocalization (n = each) was performed on paraffin sections by avidin biotin complex (abc) technique using monoclonal antibody specific to human mcp . reverse transcriptionpolymerase chain reaction (n = each) using gene specific primer against mcp and mcp receptor was performed to identify mcp messenger(m) rna in human myometrium . results: immunohistochemical findings demonstrated mcp in human myometrial cells from nonpregnant, term pregnant women before and after the onset of labor. mcp was labelled on plasma membrane and cytoplasm of myocytes from these three groups of women. similarly, mcp and mcp receptor mrna were found in nonpregnant and term pregnant women before and after the onset of labor. in this prospective study a group of fetuses was consecutively enrolled. one ultrasound examination was performed to each patient within days from delivery. we considered fetal macrosomia a birthweight g and big babies a birthweight g. cut-off points for identifying the best value of fetal abdominal circumference for fetal macrosomia prediction were chosen by receiving operator characteristics' curve (roc) analysis. using the best cut-off indicated by roc analysis, specificity and sensitivity were calculated. mean gestational age at delivery was + weeks ( + sd). neonates weighted less than g, had a weight range between and g and weighted more than g. the fetal ac measurement was the selected criterium to evaluate the risk of fetal macrosomia. to identify macrosomic fetuses the roc curve analysis identified a cut off of ac > mm which allowed to select fetuses. analysing this population we found true negative cases, false negatives, false positives and true positives, with a sensitivity of . % and a specificity of %. to detect big babies the roc curve analysis identified a cut off of ac > mm (n. fetuses), reaching a sensitivity of % and a specificity of %, without false negative cases and with false positives ( true positives, true negatives). the cases that weighted between and g, were included in the cases considered false positives by this cut-off. conclusions. these results clearly indicated that ultrasounds alone can not be used to manage a pregnancy suspected for fetal macrosomia or big babies. in fact, to avoid shoulder dystocia and all other complications strictly related to macrosomic fetuses, clinicians should perform a large number of useless elective cesarean sections. . pathway analysis of differentially expressed genes (pa; z-score . ) showed up-regulation of axon guidance, folate biosynthesis, nitrogen metabolism and down-regulation of steroid biosynthesis, insulin signaling, oxidative phosphorylation, tgf-beta signaling, and ubiquinone biosynthesis pathways in cm vs cf. comparison of mnr vs c in f showed genes up-and down-regulated (n= , ; p< . ). pa showed up-regulation steroid biosynthesis, fatty acid metabolism, glycolysis/gluconeogenesis, phosphatidylinositol signaling, ketone body metabolism, and ubiquinone biosynthesis pathways and down-regulation of bile acid biosynthesis, cell adhesion molecules, dna polymerase, notch signaling and ti diabetes mellitus pathways in mnr vs c. comparison of mnr vs c in m showed genes up-and down-regulated (n= , ; p< . ). pa showed up-regulation of jak-stat signaling, autophagy, renin-angiotensin system (ras), and ubiquinone biosynthesis pathways and down-regulation of apoptosis, basal transcription, folate biosynthesis, nitrogen metabolism, protein export, and snare interaction pathways in mnr vs c. only the ubiquinone biosynthesis pathway up-regulation of mnr vs. c was common to both m and f. conclusions: ta and pa demonstrate sex-specific transcriptome expression in fetal kidneys at . g. in addition, these results show sexspecificity in response to mnr. we have seen similar effects of mnr on gene expression for autophagy, apoptosis, ras, ubiquinone and cell adhesion pathways at . g, suggesting these pathways may contribute to persistent affects of mnr. finally, we postulate that stress in utero may contribute to sex differences in risk of hypertension in adult life. leptin and neuropeptied y protein expression paradoxally increased in gestationall food restricted dams. louiza belkacemi, chun-hung chen, andrea jelks, michael g ross, mina desai. dept. of ob/gyn, harbor-ucla med. ctr., torrance, ca, usa. objective: placental insufficiency is associated with marked increase in placental leptin production. this results in a rise in maternal leptin levels that serves as an early index of placental dysfunction. further increased placental leptin and suppressed neuropeptide y (npy) are associated with preeclampsia. leptin, an anorexigenic hormone and npy, an orexigenic peptide regulate food intake. importantly, leptin also serves as a placental and fetal growth factor. we have shown that maternal food restriction (mfr) results in intrauterine growth ... sf ()to:!)) df(,..)l) ...._ bf(jtolo) ... -m bf(,..lj) =- ( . ) <(u.)) ( . ) )( . )"" ' ~. ) !( . )' d.'cbom< ( ) f( ))""" s( l) background teenagers are more likely to deliver small-for-gestational age (sga) infants than adults, even after adjustment for socioeconomic factors , . previous studies of mostly black and hispanic subjects in the usa have suggested that maternal growth may contribute to reduced infant birthweight, due to preferential nutrient partitioning to the mother . the impact of maternal growth on birthweight and nutrient partitioning in pregnant teenagers in the uk has not been examined. methods skeletal growth (change in knee-height from st to rd trimester), weight gain and skinfold thicknesses were measured in pregnant teenagers (n= , % non-white) in london and manchester. key mediators of nutrient partitioning and metabolism: insulin-like growth factor(igf)- , igf binding protein(bp)- and leptin, were measured in maternal plasma ( weeks gestation). results maternal growth (defined as increase in knee-height > mm/ days) was detected in % of pregnant teenagers. this growth was not associated with sga birth; in fact these mothers were more likely to deliver large-forgestational age (lga) infants (p< . ). maternal weight gain and fat accrual at peripheral and central sites were greater in growers (p< . ). these parameters correlated positively with maternal igf- and leptin but negatively with the igf inhibitor, igfbp- (p< . for all). subjects delivering sga infants gained significantly less weight (p< . ) and had lower igf- levels (p< . ) than those delivering non-sga infants. conclusion maternal growth in teenage pregnancy was not associated with reduced birthweight. indeed the increased weight gain and fat accrual observed in growing teenagers may protect against sga birth and promote fetal growth. igf- and leptin promote fetal growth, primarily through effects on maternal metabolism and nutrient partitioning to the fetoplacental unit. these data suggest that higher maternal igf- and leptin in growing teenagers may provide an anabolic drive for both maternal and fetal growth. background. umbilical oxygen uptake (o umb uptake) has been estimated in human pregnancies only in acute experiments at the time of caesarean section. the recently developed possibility to measure umbilical blood flow by ultrasound in utero, prompted us to study normal and iugr pregnancies in order to evaluate fetal oxygen uptake utilizing the fick principle, i.e. uptake equals umbilical blood flow times (a-v) differences. methods. thirty-six iugr pregnancies were studied at the time of elective caesarean section and compared to twenty-one controls (c) (gestational age: c= . ± . and iugr= . ± . wks). an ultrasound examination was performed within hours from the caesarean section in all the recruited patients. umbilical vein absolute volume flow (qumb) was measured as the result between umbilical vein area and the time-averaged peak velocity * . . blood samples from umbilical vein (uv) and artery (ua) were obtained after the delivery and blood gases and acid-base balance were evaluated. umbilical oxygen uptake was calculated as o umb uptake = qumb*(uv-ua) o content. results. as expected, average fetal and placental weights were significantly different in the studied groups ( ± and ± g in iugr vs ± and ± g in n) . iugr pregnancies showed a significant reduction in qumb ( . ± . vs . ± . ml/min; p< . ) but no differences in the qumb/kg of fetal weight. iugr fetuses showed a significant reduction in o sat, o cont and po in both uv and ua compared to n. (uv-ua)o content ( . ± . vs . ± . mmol/l; p< . ) and o umb uptake/kg ( . ± . vs . ± . mmol/min/kg; p< . ) were significantly reduced in iugr. conclusions. we here report an evaluation of fetal oxygen uptake that proved surprisingly similar to the values reported in chronically catheterized animals. however, iugr fetuses showed a significant reduction in both blood and oxygen supply: this latter was reduced more that % on a per kg basis. iugr fetuses therefore utilize less oxygen than normally grown fetuses. circulating levels of vitamin d and il- in pregnancies with iugr. calvin j hobel, chander p arora, adegoke adeniji, priya arora, susan e jackman, olga miadel, baldjyan lilit. ob-gyn, cedars-sinai medical center, burns ans allen research institute, los angeles, ca, usa; university of california los angeles, los angeles, ca, usa. background: any condition resulting in under exposure to sunlight, including the use of sun block or poor nutrition may result in insufficiency ( . - nmol/l) or even deficiency of vitamin d (< . nmol/l).vitamin d regulates placental development and function. vitamin d deficiency has been linked to increased risk of serious chronic and inflammatory diseases. objective: to determine if the circulating levels of vitamin d and interleukin - (il- ) in maternal plasma correlates to pregnancies resulting in fetus with intrauterine growth restriction (iugr). hypothesis: the metabolism of vitamin d initiates the biochemical cascade of events leading to the expression of il- and the inflammatory response in iugr births. study design: in a behavior in pregnancy study, plasma samples at all three time points were analyzed in a cohort of women for (oh)d using elisa. the samples were also analyzed for il- at three stages of pregnancy: t ( - weeks), t ( - weeks) and t ( - weeks). iugr was defined as birth weight below the tenth percentile for gestational age. none of the iugr cases had spontaneous preterm birth. results: iugr was diagnosed in of women with available samples from a behavior in pregnancy study (bips) . out of these subjects, were selected as matched case controls. circulating levels of vitamin d ( (oh)d) were significantly lower in iugr cases at each visit (p<. ). the levels indicated deficient ( . ± . nmol/l) vitamin d in iugr group at t but sufficient vitamin d levels ( . ± . nmol/l) in controls. subsequent visits also showed lower levels in the iugr cases compared to the control group (t : ± . nmol/l vs ± . nmol /l; t : ± . nmol/l vs ± . nmol /l). at all three time intervals, significantly (p<. ) higher levels of il- were associated with the iugr cases ( pg/ml, pg/ml and pg/ml respectively) as compared to the controls ( pg/ml, pg/ml and pg/ml respectively). conclusions: vitamin d deficiency or even insufficiency may be an unrecognized cause of iugr. it is possible that a primary non-infectious inflammatory process is activated by vitamin d deficiency. combined assessment of vitamin d deficiency and il- expression during different stages of pregnancy may facilitate the resognition of the risk of developing iugr. response to global % maternal nutrient restriction (mnr). nathan drever, thomas j mcdonald, peter w nathanielsz, cun li. obstetrics and gynecology, university of texas health science center at san antonio, san antonio, tx, usa. background: igf-ii is a major growth factor in the developing pancreas and studies in the human fetus demonstrate that the peptide localizes to b cells of the islets (j endocrinology : ). rodent studies show decreased fetal pancreatic growth and igf-ii and ins abundance and increased apoptosis with mnr (j endocrinology : ). we previously demonstrated a fall in most components of the placental and fetal baboon liver igf systems with mnr. here we have evaluated fetal pancreatic igf-ii and ins changes in response to mnr. methods: pregnant baboons were fed ad lib (ctr, n= ) or % of wt adjusted ctr diet (mnr, n= ) from . gestation (g) and fetuses were recovered at c-section under general anesthesia at . (n= ; ctr and mnr) and . g (n= ; ctr and mnr). igf-ii and ins expression were determined by immunohistochemistry (ihc) and quantified by image analysis for fraction (area immunostained/area of the field x %) and density. data are expressed as mean + sem; ctr data are expressed] first; comparison made with two tailed t-test. results: at . g there was no difference in igf-ii or insulin fraction or density between groups. at . g, igf-ii fraction ( . ± . vs . ± . ,p< . ) and density ( . x ± . x vs . x ± . x , p< . ) and insulin fraction ( . ± . vs . ± . , p= . ) were reduced. conclusion: moderate mnr decreases abundance of fetal pancreatic igf-ii and ins at . g. in the fetal baboon and supports the extant evidence for impaired pancreatic development with mnr seen in rodents. maintenance of liver growth in the hypoxic growth restricted fetal sheep: a role for intrahepatic glut ? sheridan gentili, janna l morrison, i caroline mcmillen. sansom institute, unisa, adelaide, south australia, australia. objective: we have previously demonstrated that there is a differential tissue response to chronic placental and fetal growth restriction. growth of fetal tissues such as the brain and the adrenal are consistently spared in the face of chronic substrate restriction, whilst we have demonstrated that the growth of the fetal liver may be either maintained or reduced. it is unclear whether the growth response of the fetal liver to hypoxia and hypoglycemia are determined by intrahepatic metabolic adaptations. hypothesis: we hypothesize that there will be a differential profile of hepatic expression of glut , hsd , the gluconeogenic and glycolytic enzymes pepck and g pdh and the transcription factors pgc and ppar in animals in which liver growth is maintained or reduced. methods: carunclectomy was performed in non-pregnant ewes to induce placental restriction (pr). vascular catheters were inserted in pr and control (c) fetuses at - d and arterial blood samples were collected for blood gas analysis. mean gestation po < mmhg was defined as hypoxic (h; normoxia, n). post mortem was performed at - d. hepatic mrna expression of glut , hsd , pepck, g pdh, pgc and ppar was determined using qrt-pcr. results: four experimental groups were defined by fetal po and liver growth (c-n, pr-n, pr-h and pr-h-reduced liver growth). fetal weight correlated with mean gestational po (r = . , y= . x+ . , p< . ). liver weight was significantly lower in a cohort of pr-h fetuses (c, . ± . ; pr-n . ± . ; pr-h . ± . ; pr-h-rlg . ± . g:kg; p< . ). glut expression was highest in those pr-h fetuses in which liver growth was maintained, whilst the expression of hsd , pgc , ppar and pepck was highest in the pr-h fetuses in which liver growth was reduced (p< . ). conclusions: in the pr-h fetuses in which liver growth was reduced, the increase in hsd expression may be associated with an increase in hepatic exposure to cortisol and an associated increase in pepck, pgc and ppar . interestingly the compensatory increase in hepatic glut expression did not occur in fetuses in which liver growth was reduced. predicting the trajectory of fetal growth. racine n edwards-silva, jeffrey gornbein, calvin j hobel. obstetrics gynecology, los angeles, ca, usa; biomathematics, david geffen school of medicine at university of california, los angeles, ca, usa; obstetrics gynecology, david geffen school of medicine at university of california, los angeles, ca, usa. objective: to evaluate twelve potential predictors of fetal growth trajectory defined as the rate of fetal weight change over time. study design: a longitudinal prospective study of singleton fetal growth trajectory. the twelve potential predictors considered were: fetal gender, gestational age, parity, race, bmi, age, weight at st clinical exam, cumulative weight gain at each exam, smoking, and alcohol use. estimated fetal weight was computed using the hadlock formula and sonographic fetal biometric parameters at - weeks, - weeks, and - weeks. the rate of change in fetal weight was defined as x (fetal wt at exam j -fetal wt at exam i )(j > i)/ (gestational age at exam j -gestational age at exam i ). bivariate statistical analysis included the non-parametric spearman rank correlation and wilcoxon rank sum test. all factors were assessed multivariately using multiple linear regression. results: there were multi-ethnic women included in the study, after were excluded. they underwent a total of , exams. fetal gender (p= . ), maternal weight at st exam (p= . ), and cumulative maternal weight gain at exam (p < . ) were significant predictors of fetal growth trajectory. in this model, male fetuses had an average rate of fetal weight change of . grams per days higher than females. the rate of fetal weight change increased by an average of . grams per days for each lb increase in maternal weight at the st exam. the fetal growth rate increased . grams per days for each lb of cumulative weight gain at the rd exam. conclusions: in this study, maternal weight at the st exam and cumulative maternal weight gain were the significant determinants of fetal growth trajectory. adequate initial maternal weight and cumulative gestational weight gains probably ensure sufficient nourishment for normal placental growth, uteroplacental blood flow, and fetal nutrient uptake. this supports the emphasis on periconceptual nutritional counseling for all pregnant women. the implication of this study is that similar to fetal programming of adult diseases, there is nutritional programming of fetal growth trajectory. high risk patients. other predictors include the known risk factors of age < and > , single, tobacco/alcohol use, and african american race. interestingly, hispanic and native american patients have a lower lbw rate compared to other groups. introduction: catecholamines released by the sympathetic nervous system and adrenal medulla act via b-ars to regulate glucose and insulin function in liver, pancreas, adipose and muscle tissue. b-ar knock out mice show increased fat mass and glucose intolerance (asenslo et al.,diabetes, ) . mnr animal models have increased sympathetic activity. we, therefore, evaluated effects of mnr on baboon fetal liver b -ar. methods: baboons were fed as ad lib controls (ctr) or % of wt adjusted ctr diet (mnr) from . gestation(g) with fetuses retrieved at c-section under general anesthesia at . or . g. protein expression determined by immunohistochemistry for b -ar in the central liver lobule was quantified by image anaysis and expressed as fraction = area immuno-stained/area of the field x %. all data are expressed as mean + sem with ctr data presented first. liver glycogen expression was determined by the periodic accid schiiff (pas) method. comparisions were made with student's t-test with alpha level set at . . results: fetal body and liver wts were not changed by mnr at either age. pas stained liver glycogen at . g = . ± . vs. . ± . %, p< . . b -ar fraction was lower following mnr at . g and at . g (p< . ; fig ) conclusions: mnr decreased b -ar over % at . g and % at . g. decreased fetal liver b -ar in mnr alters glucose metabolisam and may result in reduced lipolysis predisposing to fatty liver. infection with noncytopathic bovine viral diarrhea virus (ncpbvdv) during early bovine pregnancy (< d gestation) results in fetal immunotolerance, persistent infection (pi) and intrauterine growth restriction (iugr). in contrast, infection after the development of adaptive immune competence (> d gestation or postnatal) results in a transient infection (ti). we have previously reported an iugr in pi fetuses presenting as decreased body weight and ponderal index. a growth defect can be the result of many factors, including placental insufficiency and nutrient restriction, however it was hypothesized that the iugr seen in bvdv pi fetuses may be an immunopathological effect caused by the persisting virus. our two part experimental design examined the relationship between bvdv and its pi host. in experiment (exp) , blood cell mrna was collected from pi steers (n= ; confirmed by virus isolation), or uninfected control steers (n= ) and used to identify differentially expressed genes using microarray (affymetrix) and qtrt-pcr approaches. in exp , bvdv naïve pregnant heifers (n= per group) were not infected (control) or infected with ncpbvdv on d. or d. of pregnancy creating pi and ti fetuses, respectively. fetuses were collected by c-section on d. ; infection was confirmed by elisa and qtrt-pcr. histology of placental tissue revealed no placentitis or pathology, and glucose and lactate levels in fetal serum were normal. microarray analysis revealed genes that were differentially regulated in pi vs. controls (p< . , > . fold). qtrt-pcr of steer and fetal blood revealed a significant upregulation of activators and products of the antiviral type-i interferon (ifn-i) pathway. as ifn-i can act as a growthsuppressive cytokine, a long-term upregulation may contribute to the iugr seen in persistent bvdv infection and in other viral infections observed during pregnancy. nricg - from the csrees. patients with a short cervix are at an increased risk for spontaneous preterm delivery. therefore, it is possible that women with a short cervix during pregnancy are also at risk to deliver an sga neonate. this study was conducted to address this question. study design: patients > weeks of gestation were prospectively enrolled into an observational study ( / to / ). transvaginal sonographic examinations were performed every weeks until delivery. the shortest cervical length between - weeks was used for analysis. sga was defined as less than the tenth percentile of birth weight. results: asymptomatic patients were studied. . % of patients delivered an sga neonate ( / ). the median cervical length was mm ( to mm); patients had a cervical length < mm. of these, % ( / ) delivered an sga neonate. similarly, % ( / ) patients with a cervical length < mm had an sga neonate. no relationship was found between a short cervix and sga (short cervix was defined as either < mm and < mm). the frequency of sga was not significantly different between women with a short cervix and those with a long cervix [ % ( / ) vs. % ( / ); p= . ]. newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced background folate is an essential micronutrient for cellular growth. recommendations on periconceptional folic acid use are mainly focussed on prevention of neural tube defects, despite growing evidence that folic acid use may have positive effects on birth weight. objective to examine associations between folic acid use, intrauterine fetal growth and birth weight. design the study was embedded in the generation r study in rotterdam, the netherlands, a population-based prospective cohort study from early pregnancy onwards. methods information on folic acid use was obtained by questionnaires and categorized into three groups: ) preconception start of folic acid use; ) start of folic acid use in first ten weeks of gestation; ) no folic acid use at all. fetal growth measurements included head circumference, abdominal circumference and femur length measured in mid-and late pregnancy, i.e., gestational age - and > weeks, respectively, and birth weight. fetal weight in mid-and late pregnancy was estimated using the haddock method. results data from , pregnant women were available. overall, folic acid use was positively associated with fetal growth. preconceptional folic acid use resulted in an increased growth of grams ( %ci . - . , p< . ) per week from late pregnancy to birth, compared to no folic acid use. similarly, start of folic acid use in the first ten weeks of gestation resulted in an increased growth of grams ( %ci . - . , p= . ) per week from late pregnancy to birth. both preconceptional folic acid use and folic acid use started in the first ten weeks of gestation resulted in higher birth weights of grams ( %ci - ) and grams ( %ci - ), respectively, compared to no folic acid use. a tendency was found for an increased risk of birth weight less than grams when folic acid was not started preconceptionally (or . , %ci . - . ). conclusion periconceptional folic acid use is significantly associated with increased fetal growth resulting in a higher birth weight. ductus venosus isovolumetric relaxation in severely premature growth-restricted fetuses. jason l picconi, katherine drennan, farhan hanif, michael kruger, giancarlo mari. obstetrics and gynecology, wayne state university/dmc, detroit, mi, usa. objective: ductus venosus (dv) doppler waveforms are characterized by two periods in which blood velocity decreases. the first represents the isovolumetric relaxation (ir) at the end of ventricular systole and the second represents atrial contraction at the end of ventricular diastole (a). ductus venosus reversed flow (dvrf) occurring at the time of the a-wave is considered a risk factor for intrauterine fetal demise (iufd). we have previously reported that absent or reversed a-wave flow can be present for weeks before iufd occurs or delivery is performed for non-reassuring fetal testing. the guiding hypothesis for this study is that decreased flow at the time of ir in combination with absent or reversed a-wave flow allows a more accurate prediction of fetal outcome than a-wave absent or reversed flow alone. material and methods: ductus venosus doppler was serially studied in severely premature iugr fetuses (estimated fetal weight < th percentile and umbilical artery pulsatility index > th percentile) from diagnosis until demise or delivery. ductus venosus waveforms were assessed quantitatively for peak systolic velocity (psv), isovolumetric relaxation velocity (irv), and end diastolic velocity (edv). the psv/irv + edv were compared to fetal and neonatal outcome. a kruskal-wallis one way anova, mann whitney u post-hoc test, and a roc, were used for statistical analysis. a p < . was considered statistically significant. results: all fetuses were delivered at < weeks. six cases resulted in iufd, five cases resulted in neonatal demise (nd), and six cases resulted in neonatal survival (ns) at the time of discharge from the hospital. the psv/irv+edv correlated better than a-wave reversal of flow with perinatal outcome. a psv/irv+edv score less than - . resulted in iufd, whereas a score greater than - . resulted in live birth. live births segregated based on estimated gestational age, where those fetuses at less than weeks resulted in nd and those fetuses at or greater than weeks resulted in ns. all results were statistically significant. conclusions: the isovolumetric relaxation velocity is a novel doppler parameter in the assessment of severely premature iugr fetuses. these data indicate that assessment of irv should be considered part of the evaluation of severely iugr fetuses. background: fetal growth restriction has been linked to an increased incidence of chronic hypertension, which may be the result of extracellular matrix changes (ecm) within the vascular tree. in previous studies, ecm changes were observed in the umbilical arteries of preterm growth restricted infants. objective: to determine if there are alterations in collagen subtypes within the umbilical cords from growth restricted fetal rat pups after a period of maternal nutrient restriction. methods: timed pregnant sprague-dawley rats were fed either a % food restricted diet (mfr; n= ) or were fed ad libidum (control; n= ) from d until d of gestation. litter size, fetal weights and placental weights were then noted and umbilical cords from randomly selected pups in each litter were snap frozen. gene expression for collagens i, iii, xiv and decorin was evaluated by real-time rt-pcr with normalization to the gadph housekeeping gene. data were analyzed from fitting general linear regression models with estimation based on the quasi-likelihood estimation (generalized estimating equations) to account for clustering of responses within litters. data are shown as cycles to amplification (ct), which is inversely proportional to mrna levels. results: no difference in median litter size was detected. however, fetal and placental weights in the mfr group were significantly less than those from control dams. no significant differences in umbilical cord gene expression for collagens i, iii, xiv or decorin were detected between mfr and control pups. conclusions: maternal food restriction does not result in any detectable alterations in collagen or decorin expression within the intact umbilical cord, despite causing significant reductions in fetal growth. control (n= ) p-value litter size (median, range) ( , ) ( , ) . fetal weight (mean ± sd) g . ± . . ± . < . placental weight (mean ± sd) g . ± . . ± . . collagen i (mean ± sem) ct . ± . . we have previously demonstrated that the restriction of placental and fetal growth results in fetal hypoxia and fetal brain sparing suggesting a redistribution of cardiac output. furthermore, placentally restricted (pr) hypoxic fetuses are more dependent on their sympathetic nervous system for the maintenance of blood pressure during late gestation. nerve growth factor (ngf) plays a significant role in sympathetic innervation. hypothesis: we hypothesize that the expression of ngf will be higher in the aorta and femoral artery of the pr hypoxic compared to the control fetus. method: carunclectomy was performed in non-pregnant ewes to induce pr. vascular catheters were inserted in pr and control (c) fetuses at - d and arterial blood samples were collected for blood gas analysis. all pr fetuses introduction elevated sflt- levels have been shown to be a feature of pre-eclampsia and is considered to play a significant role in the pathogenesis of the condition. the role of angiogenic factors in fetal growth restriction has not been as well established. this study evaluated the levels of circulating vascular endothelial growth factor (vegf) and its soluble receptor sflt- , in normal pregnancies and isolated placental vascular disease without evidence of pre-eclampsia. method maternal peripheral venous samples were collected antenatally from two groups of pregnant women between - weeks of gestation. group a: uncomplicated normal pregnancies (n = ) and group b: pregnancies complicated by isolated placental vascular disease( n = ) as defined by birth weight less than th centile for gestation and umbilical artery doppler s:d ratio above the th centile for gestation, with no evidence of maternal preeclampsia or pre-existing hypertension. plasma vegf and sflt- levels were measured using standard eliza techniques. comparison between groups were performed by using one way analysis of variance. the maternal plasma sflt- levels (figure ) in group a: normal pregnancies increased with gestation (p < . ). the sflt- levels in group b: isolated placental vascular disease were significantly higher throughout all gestations (p = . ) and did not show a significant variation with gestation ( p = . ). the plasma vegf levels were below the detectable levels of the assay in all samples except normal pregnancies. the significantly increased maternal plasma sflt- levels in established isolated placental vascular disease without evidence of pre-eclampsia suggest a disease of placental origin. the dysregulation of angiogenic factors may be part of a repair and regeneration process in the placenta. objectives: production of -reduced neurosteroids is critical for reducing fetal vulnerability to stressors in pregnancy and inhibition of -reductases ( r) increase acute hypoxia-induced apoptosis in the fetal brain (yawno et al neurosci : . we have developed a model of placental insufficiency that results in fetal growth restriction (gr) in the guinea pig (palliser et al repro sci # ). the aim of the present study was to determine the effects of suppression of -reduced steroid synthesis on apoptosis in vulnerable regions of the fetal brain and on neurosteroid synthetic enzymes in pregnancies compromised by chronic placental insufficiency. methods: placental insufficiency was induced in guinea pig dams by surgical ablation of uterine artery branches at mid gestation (term d). sham operated or gr dams received finasteride (a r inhibitor; mg/kg/day) during late gestation ( d until term). activated caspase- , a marker of apoptotic cell death, was measured by immunohistochemistry and steroidogenic enzymes, r and cytochrome p side chain cleavage (p scc) were measured by real time pcr and western blotting in fetuses ( d) and neonates h after birth. results: placental insufficiency significantly reduced fetal body and organ weight by % whilst sparing brain weight. the number of activated caspase- positive cells was significantly increased in the fetal hippocampus of gr fetuses and further increased in the cortex of gr fetuses receiving finasteride. the neonatal brain also exhibited changes in caspase- activation following gr and finasteride treatment. the fetal adrenal and the placenta responded to the compromise with increased expression of p scc mrna and r protein, respectively. conclusion: the combination of placental insufficiency and suppressed neurosteroid system leads to markedly increased apoptotic cell death in the fetal brain which continues to affect the neonatal brain. the fetus responds to these conditions by increasing steroid synthetic enzyme expression in the placenta and adrenal glands suggestive of a possible neuroprotective feedback process. to study the effect of smoking by mothers on the fetal and neonatal brain using non-invasive magnetoencephalography technique (meg). materials and methods: using fetal magnetoencephalography, cortical auditory evoked responses (aer) were measured from fetuses ranging from to weeks gestational age for a total of recordings. measurements were taken from mothers with a history of smoking (sm) and from mothers with no smoking history (ns). after delivery, five sm and five ns newborns had meg aer measurements twice for a total of recordings. aer was quantified by cross-correlation analysis and its significance was assessed by boot-strap technique. results: aers were detectable in out of fetal recordings in the ns group and of in the sm group. the neonatal response rate was % for each group. the latencies were divided into three components: c ( - ms), c ( - ms) and c ( - ms). in both fetuses and neonates as well, there was a statistically significant difference (p< . ) between the two groups in the c -component and the sm group showed faster aer compared to the lr group. conclusion: meg technique provides a non-invasive approach to study the effects of smoking on developing fetal and neonatal brain. the observed decrease in the latency of fetuses and neonates of the smoking mothers could indicate a hypersensitive cortical response to auditory tone. adenosine (ado) modulates metabolism in adult mammals through multiple mechanisms that involve ado a and a a receptors. objective: this study was designed to test the hypothesis that ado a and a a receptors participate in fetal metabolic homeostasis. methods: experiments were performed in chronically catheterized fetal sheep (> . term). intravascular infusion for h of dpcpx (a receptor antagonist) or zm (zm, a a receptor antagonist) was performed alone or in concert with ado administration. the highly selective ado receptor antagonists were also infused in fetuses in which hypoxia was induced for h by having the ewe breathe a hypoxic gas mixture (fio = . ). data were analyzed by two-way repeated measures of anova. results: blockade of ado a receptors (n= ) increased significantly (p < . ) fetal concentrations of glucose [control (c): . ± . (se)]; experiment (e): . ± . mg/dl] and lactate (c: . ± . ; e: . ± mg/dl) without significantly altering insulin levels and arterial blood gases or ph. antagonism of ado a a receptors (n= ) did not affect plasma levels of glucose, lactate, or insulin. intravenous infusion of ado (n= ), which did not alter pao or paco , increased concentrations of glucose (c: . ± . ; e: . ± . mg/dl) and lactate (c: . ± . ; e: . ± . ). zm (n= ), but not dpcpx (n= ), abolished ado-induced rise in glucose and lactate concentrations. isocapnic hypoxia (pao torr), which increases ( - fold) fetal plasma ado levels to those similar to ado infusion, decreased arterial ph (c: . ± . ; e: . ± . ), and increased fetal levels of glucose (c: . ± . ; e: . ± . mg/dl) and lactate (c: . ± . ; e: . ± . mg/dl) without altering changing insulin concentrations. these effects of hypoxia were not altered by dpcpx or zm. conclusions: ) a receptors modulate plasma levels of glucose and lactate in normoxic fetuses; ) a a receptors mediate the adoinduced rise in plasma glucose and lactate; and ) a and a a receptors are not significant modulators of hypoxia-induced changes in plasma levels of glucose and lactate. supported by usphs hd- . objective: to investigate the dynamic changes of the relationship between leptin, adiponectin and resistin in maternal and fetal circulation during pregnancy and in the early post-natal period. materials and methods: thirty pregnant women with uncomplicated singleton pregnancy delivered at term. maternal and fetal/neonatal venous blood samples were obtained at delivery and at hours from birth. leptin, adiponectin and resistin were measured by specific elisa assays. neonatal anthropometric measurements, glucose metabolism and lipid profile, blood pressure information were obtained. statistical analysis was performed by anova followed by student t test or duncan's test whenever appropriate. correlations were calculated by using the pearson coefficient. results: adipokines concentration at birth and at h from birth was showed in table. multivariate regression analysis showed that fetal leptin levels were positively associated with female gender and adiponectin levels, but not with anthropometric characteristics. fetal leptin and adiponectin levels were not correlated with maternal concentration, whereas fetal and maternal resistin levels were. fetal and maternal resistin concentration was positively associated with gestational age and birth weight and fetal resistin levels correlated negatively with lipid profile. after birth leptin concentration in maternal and neonatal circulation decreased dramatically and the correlation between leptin and adiponectin levels was lost. a positive correlation between resistin and leptin concentration in neonatal circulation was found at h from birth. conclusions: in contrast to adult life a positive correlation between leptin and adiponectin is present in fetal life. placental secretion of leptin, but not of adiponectin and resistin, contributes significantly to maternal and fetal circulating levels. significant changes in the relationship among adipokines occurred immediately after birth and may affect growth and development in early post-natal period. adipokines concentration in maternal and fetal circulation ) antagonism has been shown to normalize placental perfusion and fetal growth in several rat models of fetal growth restriction. however, direct administration of et a antagonists to newborn rats within hours of delivery has been consistently associated with neonatal demise due to failure of the ductus arteriosus to close, raising concerns about the safety of their use late in pregnancy. perinatal exposure to et a antagonists (maternal administration in late gestation) and its impact on rat pup survival and oxygen saturation has not been investigated. objective: to determine the impact of a maternally administered et a antagonist on oxygen saturation in newborn and -day-old rat pups. methods: timed pregnant sprague-dawley rats were treated with fr ( mg/kg/day; et a antagonist) or . % nahco vehicle, by subcutaneous osmotic pump connected to an intravenous catheter, from gestational day (term= days) through parturition. all five pregnant rats in each group delivered spontaneously and nursed their pups through postpartum day . oxygen saturation of each rat pup was measured by pulse oximeter on postpartum days and . results are presented as means ± se. newborn -day neonate vehicle . ± . . ± . eta antagonist . ± . . ± . there were no statistically significant differences between the treatment groups. maternal administration of an et a antagonist from gestational day through parturition, at a dose sufficient to ameliorate fetal growth restriction, has no adverse impact on oxygen saturation in neonatal rat pups. helen l torrance, jan b derks, martijn a oudijk, avnesh s thakor, tereza cindrova-davies, frank van bel, gerard ha visser, graham j burton, dino a giussani. perinatal center, university medical center utrecht, netherlands; department of physiology, development neuroscience, university of cambridge, united kingdom. introduction: the management of perinatal asphyxia remains a major concerns in obstetrics. umbilical cord compressions (ucc) induce fetal asphyxia and ischaemia-reperfusion (i/r). i/r increases reactive oxygen species, for instance via activation of the xanthine oxidase (xo) pathway, which may promote oxidative stress in the fetal circulation. while treatment with allopurinol of asphyxic human neonates reduced free radicals and improved cardiovascular status, treatment started postnatally was deemed too late to prevent oxidative damage (benders et al. arch dis child : , ) . consequently, in complicated pregnancy, recommendations to treat the fetus via the mother, rather than the neonate, with allopurinol are being entertained today. we investigated the effects of maternal allopurinol treatment on indices of oxidative stress in the fetal heart following repeated ucc in sheep. methods: at . of gestation, surgically instrumented sheep fetuses were submitted to i/r ( x min repeated ucc) under maternal allopurinol (n= ) or saline vehicle (n= ) infusion. fetal hearts were collected h after i/r and snap frozen for measurement of (anti)oxidant proteins by western blot. hearts from uninstrumented fetal sheep at . gestation served as controls. statistical comparisons were made using one-way anova. results: i/r episodes led to increased expression of cox- , enos, hsp and decreased expression of sod and gluthatione peroxidase (gpx) in the fetal heart, findings consistent with cardiac oxidative stress. maternal treatment with allopurinol ameliorated these effects (fig. objectives: hypoxic-ischemic fetal brain injury (hie) is a major cause of neonatal death and morbidity. evidence suggests that the brain cell injury associated with chronic hpx (in contrast to an acute ischemic reperfusion injury) is a complex process reflecting a series of adaptive intracellular events that are duration and gestational age dependent. we applied advanced proteomic tools as a next step toward ascertaining a more complete understanding of the impact of hpx on the fetal brain proteome. methods: time-mated guinea pigs were housed in a chamber beginning on day for d, breathing either room air (nmx), or . % or % o ( % o hpx or . % o hpx). on day (term), the fetal brains were removed and preserved for study. total protein was extracted, and the proteome first characterized by d gel electrophoresis. the density of the resulting protein spots were acquired and analyzed using the gs densitometer and pdquest software. identified spots of interest were trypsin digested and subject to maldi mass spectrometry using the proteomics analyzer mass spectrometer for peptide mapping or sequencing. the hpx-induced protein spots were identified based on a minimum of a x change from nmx. results: hpx had a clear effect on the fetal brain proteome. superoxide dismutase (sod), heat shock protein (hsp ), actin (actg ), interleukin- (il- ), and glutamine synthetase (gs) were each up regulated, while cofilin- , brain-type creatine kinase (bb-ck), and - gtp binding protein were each down regulated. all protein changes were proportional to the hpx ( % o hpx vs nmx, % o hpx vs . % o hpx, p< . ). conclusions: this initial application of proteomic techniques confirms that fetal brain damage secondary to chronic hpx (in contrast to acute hpx) is a complex processes characterized by fetal adaptations mediated by multiple protein activations and inactivations. while sod, il- , and gs have each been previously investigated, the potential roles of actg , bb-ck, beta- gtp binding protein, and cofilin- in the fetal response to hpx and the associated brain damage are unclear and represent strong candidates for future investigation. acknowlegement: this study was supported by grants from the phs (r hl - , cpw) and cdc grant (u dp - , cpw). intermittent umbilical cord occlusion occurs in % of human pregnancies.given that elastogenesis within the vascular wall is in part mediated by hemodynamic conditions during development, the blood pressure response to acute hypoxic insults such as cord occlusion may alter arterial composition. elastin content of a central and peripheral artery and blood pressure responses in fetal sheep exposed to varying degrees of cord occlusion were determined. methods: over a day period, near term fetal sheep received total umbilical cord occlusion (uco) lasting min/ hour (mild group; n= ), min/hour (moderate group; n= ), min/hour (severe group; n = ) or no occlusion (control group; n= ). fetal arterial blood samples were drawn min prior to and at the end of cord occlusions. mean arterial pressure (map) was monitored continuously. the carotid and superior mesenteric arteries were excised and a colorimetric assay (biocolor) performed for determination of elastin content. results are presented as mean ± sem. results:umbilical cord occlusions produced decreases in fetal arterial oxygen pressure and oxygen saturation that were progressively more pronounced across mild, moderate and severe uco groups (p < . ). lactate concentration rose during occlusions in the moderate and severe groups, but not the mild group (p < . ).elastin content of the superior mesenteric artery did not differ between the experimental groups and the control group. elastin content of the carotid artery and map response elastin content (μg/mg tissue) max ∆ in map duration of rise in map (min) control . ± . . ± . -mild . ± . . ± . ** . ± . moderate . ± . . ± . ** † . ± . † severe . ± . * . ± . ** † † . ± . † † * p < . ; ** p < . : experimental groups compared to control † p < . ; † † p < . : compared to mild group the max in map was positively correlated with elastin content (r = . , p < . ). conclusions:the transient rise in blood pressure and preferential blood flow to the brain that occur in response to acute hypoxemia during severe intermittent umbilical cord occlusion induce an increase in elastin synthesis in the carotid artery.this may give rise to adaptive programming of postnatal central arterial compliance. electrocortical activity during repetitive umbilical cord occlusions (uco) with worsening acidemia in the ovine fetus near term. martin g frasch, roy mansano, michael g ross, robert gagnon, bryan s richardson. obgyn, chri, univ of western ontario, london, canada; obgyn, harbor-ucla med ctr, torrance, ca. objective: uterine contractions during labour can restrict umbilical blood flow compromising fetal oxygenation and leading to adverse neonatal outcome including newborn encephalopathy/subsequent cerebral palsy. while electronic fetal heart rate (fhr) monitoring is widely used for assessment during labour, abnormal fhr patterns as used clinically have a poor positive predictive value for concerning/significant acidosis at birth. there is limited study of fetal electrocortical activity (ecog) in animal models with induced hypoxia/ acidemia as might be seen during labour. thus, we aimed to induce repetitive ucos in fetal sheep leading to worsening acidemia to determine the predictive value of ecog activity for fetal compromise. methods: near-term fetal sheep (n= ) underwent chronic preparation with artery catheters, ecg/ecog electrodes and placement of an inflatable umbilical cord occluder. following a baseline recording period, fetuses underwent a series of mild ( min every min), moderate ( min every min) and severe ( min every min) ucos with each series lasting h or until fetal arterial ph decreased to < . . fetal arterial blood samples for blood gases and ph were taken at selected time points during the baseline, ucos, and recovery periods. arterial blood pressure (abp) and fhr were continuously monitored and spectral edge frequency (sef) was calculated from ecog. correlation of sef with fhr change was calculated for time intervals using sef maxima and fhr minima during uco series. data are presented as means±sem. results: repetitive ucos led to development of a marked acidosis (ph . ± . to . ± . , p< . ). ± min prior to fetal ph drop < . , the sef of ecog began to increase abruptly during each fhr deceleration from ± hz up to ± hz (p< . ) and was correlated to both fhr change and a decrease in fetal abp during each fhr deceleration at this time (p< . ). conclusion: our findings suggest that in the animal model studied ( ) fetal ecog activity is impaired with progressive acidemia accompanied by fhr decelerations and pathological abp decreases; ( ) there is a consistent temporal relationship between the occurrence of ecog alterations and the subsequent critical drop of ph < . . these findings could contribute to improvement in the clinical ability to predict fetal compromise during labour using ecog/fhr monitoring. background: the ductus arteriosus (da) plays a pivotal role in fetal development and circulation. during gestation, patency of the fetal da is maintained by nitric oxide (no) and prostaglandins (pg). no and pgs are downstream effectors of estrogen (e ) and progesterone (p ). however, the roles of e and p in da regulation have not been studied. objective: we hypothesized that: e and p have opposite effects in the da; that rising e and falling p levels help trigger da closure via specific hormone receptors; and that no and/or pgs mediate da responses to e and p . methods: expression of er , er , pra, prb, and the putative membrane receptors mpr-, -, -, pgrmc- , - , and serbp- was examined by rt-pcr and qpcr on days , , (term), and p . the effects of e and p ( - - - m) on the d fetal da were examined in a cannulated microvessel myography system. e and p effects were also studied in the presence of receptor antagonists (ici , ru ) and no and pg inhibitors (l-name, indomethacin). results: er and pr receptors were expressed at low levels; pgrmc- expression was stronger than other membrane prs, and increased with advancing gestation. under fetal o conditions, e induced rapid, concentrationdependent dilation at - - - m (n= ); p induced progressive vasodilation at all doses (n= ). e and p -induced dilation occurred within - seconds of exposure. while e -dilated das did not respond to ici, ici-pretreated das failed to dilate to the same dose of e (n= ) suggesting antagonism of e effects. the vasodilatory effects of e were partially inhibited by pretreatment with l-name. p -dilated das did not respond to ru , whereas ru pretreated das showed a small, significant response to p (n= ), suggesting partial antagonism or signaling via alternative, ru -independent pathways. pre-treatment with indocin did not block the vasodilatory effects of p . conclusions: contrary to our expectation, both e and p have dilating effects on the fetal da, via no, pgs and non-no, non-pg pathways. expression studies and the rapid response of ex vivo das are consistent with non-genomic actions via membrane receptors. hormone shifts in parturition may have longterm effects on da preparation for postnatal closure, but strategies to maintain fetal da patency or treat newborns with pda will require better understanding of this process. background. for the fetus, arterial blood gases are critical in the regulation of cerebral blood flow (cbf) and cerebral oxygenation. however, the relation of cbf, cortical tissue po (tpo ), and electrocorticographic (ecog) activity to arterial o tension (pao ) are not well defined. in an effort to elucidate these interrelations, we tested the null hypothesis that in the near-term fetus, acute hypoxic-associated cerebral oxygenation and related variables are not closely associated with ecog state. methods. by use of a laser doppler flowmeter with a fluorescent tissue o probe, and with fluorescent-labeled microspheres, and with ecog electrodes, in near-term fetal sheep (n = ) we measured laser doppler cbf (ld-cbf), tpo , ecog (root mean square (rms) voltage with high voltage low frequency, hvlf versus low voltage high frequency, lvhf) and spectral edge frequency- % (sef ) in response to min moderate isocapnic hypoxia. results. ld-cbf, cerebral o delivery, tpo , and several other variables correlated highly with ecog state. in the normoxic control fetus, in association with a shift from hvlf to lvhf ecog activity, tpo decreased briefly to ± from a control value of ± torr; however, as ld-cbf increased ± %, and sef increased to ± from ± %, tpo returned to near normal value. with acute hypoxia (pao = ± torr) when in the lvhf state ld-cbf increased only ± %, as opposed to a ± % increase when in hvlf ecog state. with this degree of hypoxia, tpo decreased to ± torr, sef remained at ± %, and cerebral metabolic rate for o (cmro ) decreased ± % (p< . ). conclusions. for the near-term fetus, normoxia with changes in ecog state was associated with brief periods of decrease in tpo , which were restored quickly by increased ld-cbf. in contrast, acute hypoxia was associated with a significant depression of cortical tpo , cmro , and ecog state, with increased ld-cbf failing to restore cortical tpo . thus, we reject the null hypothesis that in such fetuses, hypoxia demonstrates no compromise in cerebral oxygenation. (supported by usphs hd- ). releasing hormone and arginine vasopressin in the ovine fetus. charles a ducsay, kanchan m kaushal, malgorzata mlynarczyk, kimberly hyatt, dean a myers. ctr. for perinatal biol., loma linda univ., loma linda, ca; ob/gyn, univ. oklahoma hlth. sci. ctr., oklahoma city, ok. background: long term hypoxia (lth) profoundly affects the hypothalamicpituitary-adrenal axis of the fetal sheep. we previously showed that lth causes augmented corticotrope function. the present study was designed to test the hypothesis that lth enhances sensitivity to the acth secretagogues; cortiocotrophic releasing hormone (crh) and arginine vasopressin (avp) resulting in increased anterior pituitary corticotrope secretion of acth. methods: pregnant ewes were maintained at high altitude ( , m) from day to - of gestation (dg), when they were returned to the lab and a maternal tracheal catheter was implanted. maternal po was maintained at a level comparable to that observed at altitude ( mmhg) by nitrogen infusion. on dg, lth (n = ) and age-matched, normoxic control (n = ) fetuses were implantated with vascular catheters. each fetus received a min infusion of either saline vehicle, ng/kg of ovine crh or ng/kg of avp (estimated body weight/min) in a randomized order over consecutive days ( - dg). blood samples were collected at min (baseline prior to infusion), , , and min following the start of the infusion and analyzed for acth, as well as the acth precursors pro-opiomelanocortin and the major processing intermediate kda proacth. results: vehicle had no effect on any of the measured parameters. with crh infusion, acth (pg/ml) increased in both groups over the course of the study. however, peak concentrations (at min) were significantly higher in the lth group compared to control ( ± vs. ± , respectively; p< . ). acth precursor secretion (pm) was greater in lth fetuses compared to controls during the experiment (p< . ). in response to avp, peak acth concentrations were also higher in the lth fetuses compared to control ( ± vs. ± respectively; p< . ), however peak levels were reached at between and min after start of infusion with levels in both groups returning to pre-infusion values. a similar pattern was observed with precursor levels ( . ± . vs. . ± . , p< . , lth vs. control). conclusions: lth significantly increases pituitary sensitivity to both crh and avp. this enhanced sensitivity may be mechanism of our previously observed enhanced corticotrope function. (supported my nih grants hd and hd ). martin g frasch, roy mansano, michael g ross, robert gagnon, bryan s richardson. ob/gyn, chri, university of western ontario, london, canada; ob/gyn, harbor-ucla med. ctr., torrance, ca. objective: repetitive uco leading to worsening fetal acidosis with fetal heart rate (fhr) decelerations (fhr dec ) are accompanied by pathological decreases of fetal arterial blood pressure (bp). we hypothesized this bp change may be caused by the bjr, a vagally mediated reflex with bradycardia and hypotension to reduce cardiac workload, via stimulation of cardiac chemoreceptors during systemic acidemia. methods: ten near-term fetal sheep ( ± dga) underwent chronic preparation with brachial artery catheters and placement of an inflatable umbilical cord occluder. after a control period, fetuses underwent a series of mild ( min every min), moderate ( min every min) and severe ( min every min) uco each lasting h or until ph decreased to < . . fetal arterial blood samples were taken at selected time points during the control and uco periods. bp and fhr were continuously monitored. individual fhr nadir during each fhr dec and accompanying bp change ( bp = [bp at the time of a fhr nadir] -[bp at baseline preceding a uco series]) were determined during all uco. data are presented as means±sem. results: control period ph ( . ± . ), fhr ( ± bpm) and bp ( ± mmhg) were within the physiological range. average depth of fhr dec for all uco was ± bpm and increased with higher lactate concentrations (r = . , p < . ) and lower ph (r = . , p = . ). bp during all uco demonstrated an initial hypertensive response to fhr dec , which decreased with lower ph (r = . , p < . ). bp increased ± mmhg (p < . ) during mild uco (ph to . ± . ), ± mmhg during moderate uco (ph to . ± . , p < . ) and ± mmhg during severe uco (ph to . ± . , p < . ). conclusion: these results suggest that bjr, a short-acting, ph dependent depressor reflex, blunts the physiologic hypertensive response to cord occlusion insults leading to worsening acidemia as might be seen in the human fetus during labour with repetitive variable fhr dec . the failure to increase bp may prevent optimal blood flow distribution responses necessary for preservation of vital organs. role of nos and pde in placental dysfunction following fetal bypass. mitali basu, r scott baker, christopher t lam, kenneth e clark, pirooz eghtesady. cardiothoracic surgery, cincinnati children's hospital, cincinnati, oh, usa; ob/gyn, university of cincinnati, cincinnati, oh, usa. introduction rising placental vascular resistance following fetal cardiopulmonary bypass (bypass) remains the achilles' heel of fetal cardiac surgery. we have previously shown in real-time that nitric oxide (no) production rises during bypass and falls post-bypass, while cgmp levels rise throughout. using immunohistochemical and western analysis of placenta, we examined the involvement no pathway components in this placental vascular pathophysiology. ovine fetuses at - days gestation were placed on bypass for minutes and followed post-bypass for hours. placental samples were collected immediately prior to bypass and at and min post-bypass (n= ) and compared to a group of similarly instrumented controls, (n= ). placental enos, inos and pde protein expression was measured using standard methods and relative expression normalized to beta-actin. statistical analysis utilized students t-test, and anova for trend and group-wise analysis, (significance at p= . ). pre-bypass protein expression did not differ between groups. pde protein levels and phosphorylated pde- expression were both elevated min post bypass, and reduced min post bypass compared to sham, (p= . ). enos levels in the bypass group increased linearly from pre-bypass to min postbypass (p< . ), and were also elevated compared with shams (p< . ), while shams had declined significantly by min post bypass, (p< . ). similarly, phosphorylated enos expression in the bypass group increased linearly from pre-bypass to min post-bypass, (p= . ), while shams trended towards decline by min post-bypass. simultaneously, placental inos expression remained stable within groups, but was lower in the bypass group at and min post-bypass, (p< . ). the preceding data correlated with observed immunohistological changes in the same placental cotyledons. fetal bypass leads to significant increases in placental protein levels of pde , phosphorylated pde- , enos and ostensibly phosphorylated enos. increased pde expression may be a response to increased no and the generated cgmp. this data suggests a compensatory upregulation of pde and enos that eventually fails, leading to increasing placental vascular resistance and subsequent lethal placental dysfunction. angiotensin receptors (rat-ii) in neuronal nuclei of the brainstem have been implicated in integration of baroreceptor responses. in near term fetal sheep, we have previously shown that days of mild chronic hypoxemia increases heart rate (hr) and blood pressure (bp). the aim of the present study was to investigate the effects of chronic mild hypoxemia on the expression of rat-ii in the medulla oblongata and on baroreflex control of the circulation. methods: at days of gestational age, pregnant sheep were submitted to days of hypoxemia ( % of fetal arterial po ; at day fetus ± vs ± . ; mother ± vs ± mmhg). hr and arterial bp were continuously recorded from mother and fetus in control (n= ) and hypoxemic (n= ) animals. baroreflex sensitivity (brs) as well as hr and diastolic bp variability were analyzed (nevrokard software). brainstem was collected and rat-ii expression in the nucleus tractus solitarius (nts) and dorsal motor nucleus of the vagus (dmnx) was measured by autoradiography using i-sarthran labeling. data are shown as mean±sem and were analyzed by t-test. results: hypoxemia induced a greater total binding of i-sarthran in nts and dmnx resulting from an increased expression of at receptors. in the time-domain analysis of brs hypoxemia induces lower baroreflex sensitivity in the fetus (brs in ms/mmhg; . ± . vs. . ± . , p< . ). in the frequency domain, hypoxemia changes the relative power of low frequencies (lf: . - . hz) and high frequencies (hf: . - . hz) of rri and dbp with an increased value of the lf/hf ratio (rri lf/hf . ± . vs ± . , p< . ; dbp lf/hf . ± . vs . ± . , p< . ). also the alpha index for baroreflex sensitivity is decreased in hypoxemic animals (alpha lf . ± vs . ± . , p< . ; alpha hf . ± . vs . ± . , p< . ). no differences were observed in maternal variables. conclusions: our results suggest a link between a prenatal insult, alterations in cns receptors and functional alterations of baroreflex responses. a higher sympathetic outflow, suggested by a greater lf/hf ratio, and impaired reflex gain, both possibly mediated by increases in nts rat , may have potential long-term consequences for the development of hypertension. hd ;hd ;hl . objective : we evaluated velocity profiles, relative wall distension rate (rwdr), and wall shear rate (wsr) of fetal descending aorta (fda) in uncomplicated singleton gestations. study design: ninety seven uncomplicated singleton fetuses were studied throughout gestation using multigate spectral doppler analysis (msda) working with gasp software. this consists of a personal computer (pc) add-on board including a single high-speed digital signal processor. the analysis of echo-signals backscattered from range cells located along the axis of the interrogating ultrasound (us) beam. post-processing was accomplished using gasp software. statistical analysis consisted of spearman correlation and chi-square test. results: velocity profiles, wall distension, wall shear rate were obtained from fetal descending aorta throughout gestation establishing gestational age specific norms. wdr[%] is highly correlated with gestational age in appropriate growth fetuses ( . ± . , rs= . p< . ) with linear regression with standardized coefficient of . (p< . ). in contrast, wsr ( ± ) is unchanged during the first , second and third trimesters (p= . ).conclusion: we speculate that the relative wdr changes observed during gestation, in normally grown fetuses, may be secondary to adaptive vascular and autonomic responses and the evolving composition of the vessel wall, particularly with respect to elastin. conversely, the mean wsr for the study group was independent and constant throughout the gestation. these findings suggest that there is an increase in the diameter of the fetal aorta, which provides adaptation to the progressive flow demands, while preserving other key hemodynamic parameters. in this study, we have established normative values of rwdr and wsr, which are new rheological parameters that may be useful in distinguishing normal and pathologic hemodynamic states. objective: placental dysfunction is a key barrier to successful fetal cardiopulmonary bypass (cpb) for repair of congenital heart defects in utero. endothelial cells regulate vascular tone during fetal cpb through interactions of vasodilation by nitric oxide (no) and endothelin- (et- )-mediated vasoconstriction. the objective was to determine the time during fetal cpb when endothelial cell-mediated changes occur. methods: human umbilical vein endothelial cells (huvec) were cultured in media containing % serum collected from ovine fetuses (n= ) that underwent min of cpb, then were maintained for min. serum was collected before cpb, from pump prime before initiation of cpb, min on cpb, or and min after fetal cpb. control cells were cultured in normal fetal serum. cells were harvested and hr after addition of fetal serum. no production was measured in real time with an electrochemical detection system (inno-t, harvard apparatus). et- was measured in the culture media by elisa. results: no production by huvec after and hr was stimulated above control levels by fetal serum collected during and up to min after fetal cpb (p< . ). serum collected from fetuses that were surgically instrumented, but not yet subjected to cpb, decreased no levels below controls (p<. ). stimulation of et- after and hr of huvec culture peaked with serum collected at min after fetal cpb (p<. compared with control), but was elevated above control levels at each collection time point (p<. , table ). conclusions: fetal cpb releases serum proteins that elevate endothelial cell no and et- production during and for at least min after cpb. although the specific regulatory proteins remain to be identified, the no and et- pathways share circulating mediators and participate in a feedback loop to modulate vascular tone. to test the hypothesis that hypoxia-induced upregulation of no is linked to cardiac inos expression, a selective inos inhibitor, l-nil (l-n -( -iminoethyl)-lysine), was administered in vivo to fetal guinea pigs and no levels measured in fetal hearts. methods: pregnant guinea pigs were exposed to either normal room air (normoxia; %o ) or . %o (hypoxia) in a hypoxic chamber for days prior to term (term= d). l-nil was administered to pregnant normoxic and hypoxic guinea pigs via their drinking water at a dose of - mg/kg/d for days. at d gestation, pregnant sows were anesthetized and near-term fetuses removed via hysterotomy. the fetal hearts were excised, weighed, and normalized to their respective fetal body weights. left cardiac ventricles were obtained and frozen in liquid n and stored at - o c until ready for analysis. the effect of total no product (no and no -, nox) of left ventricles of fetuses exposed to normoxia (n= ), hypoxia (n= ) and hypoxia plus l-nil (n= ) was quantified by a commercial fluorometric no assay kit. results: intrauterine hypoxia significantly reduced fetal body weight by % and increased placenta/fetal body wt by % as expected for hypoxic stress. hypoxia induced a slight increase in heart/fetal body wt by %. fetal cardiac nox levels (pmoles/mg) were increased by hypoxia ( . + . ) by . fold compared to normoxic controls ( . + . ). l-nil significantly decreased (p< . ) nox levels in hypoxic hearts by % ( . + . vs . + . ; hypoxic vs hypoxic+l-nil, respectively). conclusion: l-nil inhibits inos-derived no generation in the hypoxic fetal guinea pig heart. since previous study in our lab showed a significant increase in inos expression but a decrease in enos and no change in nnos expression, we hypothesize that hypoxia upregulates cardiac no generation via the inos pathway. further study is needed to identify the important role of cardiac inos in the adaptive response of fetal hearts to chronic intrauterine hypoxia. objective: fetal asphyxia-mediated metabolic acidosis results in a ph decrease and an increase of lactate and base deficit in blood (bd blood ) and extracellular fluid (bd ecf ). it is not clear whether bd blood and bd ecf are similarly altered with worsening fetal acidosis. in the present study we sought to study the dynamic relations of bd blood and bd ecf to lactate in the ovine fetus subjected to repetitive uco with worsening acidemia. methods: ten near-term fetal sheep ( ± dga) underwent chronic preparation with brachial artery catheters and placement of an inflatable umbilical cord occluder. following a baseline recording period, fetuses underwent a series of mild ( min every min), moderate ( min every min) and severe ( min every min) uco with each series lasting h or until fetal arterial ph decreased to < . . fetal arterial blood was sampled at baseline, immediately before and during the first mild, moderate and severe uco and at min intervals during the moderate and severe uco. bd gap (bd blood -bd ecf ) was studied in relation to lactate. presented as means±sem. results: lactate correlated to bd blood (r= . , p< . ). bd ecf correlated strongly with bd blood (r= , p< . ). bd ecf increased by . ± . mmol/l more than bd blood from baseline to ph nadir, with bd gap therefore decreasing with increasing lactic acidemia ( fig. ) . lactate, bd blood , bd ecf and bd gap correlated to ph (r= . , r= . , r= . and r= , respectively, all p< . ) and ph could therefore be predicted with any of the four acid-base parameters. conclusion: the increases of bd blood and bd ecf and the decrease of bd gap with increasing lactic acidemia suggest a relatively more rapid accumulation of [h+] in ecf during uco of increasing severity. this may be because the metabolic build-up of acidosis occurs primarily in the tissues and the endothelial permeability for [h+] is impeded during increasing acidosis with atp depletion thus decreasing [h+] movement from ecf to plasma. previous studies have shown that intraperitoneal transplantation of human umbilical cord blood (hucb)-derived mononuclear cells led to the specific 'homing' of these cells to a hypoxic-ischemic brain lesion in perinatal rats. motor deficits resulting from the lesion were alleviated upon transplantation. thus, the presence of hucb cells at the lesion site seems to be a major prerequisite for their potential beneficial effect. however, the mechanisms of cell 'homing' are still unclear. in this study, we focused on elucidating mechanisms underlying the specific migration of hucb-derived mononuclear cells to the brain lesion. one possibility to induce cell 'homing' are chemotactic signals present at the lesion site. the cxc chemokine stromal derived factor- (sdf- ), which was previously shown to be a potent chemoattractant for directed migration of other stem and progenitor cells, is a putative candidate in our lesion paradigm. therefore we investigated the spatial and temporal expression of sdf- in brain hemispheres with or without hypoxic-ischemic lesion. sdf- expression was substantially increased at the lesion site during the investigated period of fourteen days after the insult. furthermore, hla-positive hucb cells were mainly detected in sdf- expressing brain regions and we were able to show that these cells express the sdf- receptor cxcr on their surface. the functional implication of sdf- in directing hucb cell migration was determined by application of neutralizing sdf- antibodies in vivo, resulting in a reduced number of hucb-derived mononuclear cells at the lesion site. with these functional effects, together with the observed timing and location of its expression, the involvement of the chemokine sdf- in hucb cell 'homing' seems conceivable. novel pathways in inflammation-induced fetal brain injury. michal a elovitz, jinghua chai. obstetrics and gynecology; crrwh, university of pennsylvania, philadelphia, pa, usa. intro: while survival of extremely preterm infants continues to improve, the number of children with cognitive impairment and/or cerebral palsy is increasing. if preterm birth (ptb) cannot be prevented, then strategies to identify and treat fetal brain injury in the setting of a ptb must be investigated. these studies were performed to explore novel pathways involved in fetal brain injury in the setting of inflammation-induced ptb. methods: cd- mice on e receive intrauterine injection of lipopolysaccharide (lps) or saline. hours after injection, fetal brains from the left upper horn were harvested. fetal brains were removed from each dam with dams per treatment group. separate rna samples were prepared and used for microarray (ma) analysis. all protocols were conducted as described in the affymetrix genechip expression analysis technical manual in the ma core facility using the moe av chip. data analysis was performed using significance analysis for microarray (sam). the brain samples from the same dam were considered as dependent samples. pathway analysis and functional annotation clustering tools were used with david. validations studies using qpcr with fetal brain samples (n= - per group) were performed. results: while there was significant differences in gene expression between lps and saline exposed fetal brains, variability existed even between pups from the same dam. genes were significantly differentially expressed between lps and saline brains (p< . ). with a p value of < . , genes were differentially expressed. pathway analysis revealed significant involvement of ) the cadeherin, cadherin-like, cell fraction and calcium binding (enrichment score . ) and ) ribosomal processing, rna metabolism, pyrimidine metabolism (enrichment score . ). specifically, genes involved in neurogenesis, synaptic function, and neuronal and glial metabolism were most differentially regulated. qpcr confirmed observed fold changes in / genes analyzed. inflammatory pathways were not differentially regulated. conclusions: current theories regarding fetal brain injury in ptb focus on activation of inflammatory processes as essential events. this data suggests that long-term neurological injury in a ptb may be secondary to altered neuronal function, metabolism and/or communication. disruptions in these pathways should be explored as key mechanisms to adverse neurological outcomes in preterm neonates and should be targets for future investigations. regulation of microglial activation in the developing murine brain. mariya hristova, virginia zbarsky, daniel cuthill, adam wallace, donald peebles, gennadij raivich. institute for women's health, university college london, london, united kingdom. introduction: the aim of this study was to assess the process of microglial differentiation in developing white matter, an area of the brain that is particularly vulnerable to damage pre-myelination (approx weeks gestation). microglia form a distinctive non-neuronal component. although related to peripheral macrophages they undergo highly specific processes of regional maturation and differentiation inside the brain with a slimming of the cell body, development of very elaborate crenulated arborised branches and downregulation of most macrophage activation markers. this process is relatively rapid in most grey matter brain regions, but is retarded in and around the subcortical white matter (swm) giving rise to the phagocytic fountains of microglia (fom). methods:we examined the process of deactivation and morphological differentiation in the cortex and swm of mice - days after birth (p -p ) using confocal microscopy for monoclonal antibodies against alpha and beta integrin subunits and the costimulatory factor b . , colocalised with standard microglial marker iba . results: strikingly, only the fom macrophages, but not cortical microglia, strongly expressed typical activation markers alpha- , alpha- , alpha-m, alpha-x, beta- and b . . the data for alpha-x are shown as an example in the figure; cortical microglia are shown in the grey bars and swm in black. fom activation was maximal at p , decreased linearly over p and p and disappeared at p . this process followed the presence of ingested phagocytic material but correlated only moderately with ramification, demonstrated by non-ramified but inactive p cortical microglia and formation of stubby processes in p fom. conclusion: these data describe strong and selective biochemical activation of fom phagocytes in p -p swm, roughly equivalent to early rd trimester human foetal development. this presence of highly active phagocytes in the neighbourhood of vulnerable wm could play an important role in the genesis of axonal damage in the foetus and premature neonate. , pp. - ) . mbp is expressed in mature oligodendrocytes (jakovcevski and zecevic, glia ) . although myelination in the fetal sheep brain, a model extensively used to evaluate fetal development, has been described using conventional staining techniques (barlow, j comp neurol ) the use of specific mbp staining allows a more precise determination of the onset of myelination (antonow-schlorke et al., reprod sci . , , a) . aim: to use mbp expression to determine the trajectory of development of different white matter tracts of fetal sheep brain. methods: the ontogenetic profile of mbp expression was estimated in healthy fetuses at . (n= ), . (n= ), . (n= ), . (n= ), . (n= ), . (n= ) and . (n= ) of gestation (term days). after brain fixation and embedding in wax, sections at the level of the optic chiasm were stained with a monoclonal anti-mbp antibody using the abc-technique. immunohistochemical distribution of mbp was morphometrically assessed in the dorsal internal capsule and cortical white matter tracts, i.e. the lateral centrum semiovale, superficial white matter and median corpus callosum using an image analysis system (scion image . , nih, usa). results: mbp expression of various fetal white matter structures started at different time points; initially in the internal capsule at . of gestation followed by the cortical white matter structures at . of gestation. cortical myelination advanced from the cortical deep white matter via superficial white matter to the corpus callosum reflecting different rates of progression. conclusions: the onset of mbp expression estimated here explicitly antedates previous observations in sheep (barlow, ) the way the embryonic heart functions before cardiac morphogenesis is completed is still subject of many studies. to gain more insight into the early cardiac structure-function relationship, doppler blood flow velocity waveforms at four different locations in the embryonic chicken heart during cardiovascular development were assessed. we collected waveforms using high frequency ultrasound biomicroscopy with a -mhz transducer at hh stages , and which are comparable to humans at to weeks of gestation. waveforms were obtained at the inflow tract, the primitive left ventricle, the primitive right ventricle, and at the outflow tract in ten different embryos per stage. by exploring the time relation between the waveforms, cardiac cycle events were outlined. our results demonstrate that stage and location dependent, intracardiac blood flow velocity waveforms can be obtained in the chicken embryo which reflect stage dependent pumping mechanisms. the blood flow profiles assessed at the four locations in the embryonic heart demonstrated a developmental related increase in velocity. in the primitive ventricle the passive filling wave decreased, whereas the active filling wave increased resulting in a decreased p to a ratio in the course of time. high frequency derived cardiac blood flow velocity characteristics support previous findings that the embryonic heart functions like a suction pump at early development and transforms towards another pumping mechanism at later developmental stages. these findings are of importance for the interpretation of human first trimester cardiac velocimetry studies. figure a . changes in the perimeter of the thymus with gestational age by fetal gender are depicted in figure b . background. for the fetus, the roles of arterial blood gases are recognized to be critical in the regulation of cerebral blood flow (cbf) and cerebral metabolic rate for oxygen (cmro ), cortical tissue po (tpo ), and electrocorticographic (ecog) activity. in addition, metabolites such as adenosine (ado) are important in this regard. nonetheless, the relation of adenosine and its metabolites to these indices of cerebral oxygenation are not well defined. in an effort to elucidate these interrelations, we tested the null hypothesis that acute hypoxic-associated cerebral oxygenation and related variables are not closely associated with adenosine. the most common cause of perinatal brain injury is chronic hypoxia/ischemia. the mechanisms are complex and poorly understood. applying proteomics tools, we identified a set of hypoxia-related proteins in the fetal guinea pig (gp) brain (companion abstract). one protein, cofilin- , which regulates the rapid cycling of actin assembly and disassembly, was dramatically altered by chronic brain hpx. herein, we test the hypothesis that confilin- modifications during hpx contributes to brain damage via apoptosis and is an example of an adaptive response that becomes maladaptive. methods: time-mated gps were housed in a chamber from d to d (term) with either room air (nmx) or . % or % o . fetal brains were removed and sections prepared for double staining specific to bax and dephosporylated cofilin- . total protein was isolated and equal amounts loaded on a sds gel, separated by electrophoresis, and transfered to pvdf membranes probed with primary antibodies to dephosphorylated and phosphorylated cofilin- , bax and bcl-xl, and incubated with second antibody. protein signals were detected, quantified by densitometry, and normalized to -actin. total rna was isolated, reverse-transcribed, and quantified by real-time pcr using sybr green i labeling. expression was calculated by the ct method using s for control. melt analysis was performed to confirm specificity of the amplification, and efficiency determined by the slope of the standard curve. the mitogen activated protein kinase (mapk) signalling pathway is upregulated in perinatal hypoxic-ischaemic brain. the role of the extracellular signal-regulated kinase (erk) cascade, a pivotal component of mapk signalling, remains unclear with reported actions ranging from mitogenic and trophic effects to a neuronal death-promoting role. the aim of this study was to assess the role of neuronal erk activity using selective neuronal inhibition in a perinatal model of hypoxic-ischemic brain injury. methods: we explored the effects of selective neuronal inhibition of erk activation using transgenic mice expressing dominant-negative (dn) mek , the upstream activator of erk / , which was under the control of the pan-neuronal talpha alpha-tubulin promoter. p-erk immunoreactivity was quantified following a hypoxic inschemic (hi) insult in p c bl/ mice, caused by unilateral occlusion of the carotid artery, followed by hypoxia with % oxygen for mins at °c. the volume of surviving brain on the affected hemisphere was expressed as a % relative to the control side. results: expression of the mek dn was associated with a reduction in erk / activation following hypoxia ischaemia, as assessed by p-erk immunoreativity. compared with the wild type, littermate controls, mek dn animals exhibited significantly decreased volume of forebrain damage brain following unilateral, min hi insult (*p< . , **p< . , anova). similar protective effects of mek dn were observed in cerebral cortex, hippocampus, thalamus and striatum when compared to wild type littermate controls and correlated with a significant reduction in microglial activation in all brain areas. conclusion: overall, these results suggest that neuronal mek and its downstream signals have an important death-inducing role in this model of perinatal brain injury and could serve as potential targets for therapeutic intervention. oup, ) . however, whether melatonin protects placento-fetal development during hypoxic or undernourished pregnancy is unknown. we investigated in rats the effects on placental and fetal growth of maternal treatment with melatonin in hypoxic and undernourished pregnancy. methods: on pregnancy day , wistar rats were divided: control ( % o ) + melatonin ( g.ml - drinking water), hypoxia ( % o ) + melatonin, and undernutrition ( % reduction in food intake) + melatonin (n= per group). on day , dams were anaesthetised, the pups, placentae and fetal brain were weighed. results: relative to controls (fetal weight: . ± . g; brain/body weight ratio: . ± . mg/g; n= ), both hypoxic ( . ± . g; . ± . mg/g, n= ) and undernourished pregnancy ( . ± . g; . ± . mg/g, n= ) promoted asymmetric iugr (p< . ), without affecting placental weight. melatonin treatment had no effect on iugr, but it decreased placental weight in normoxic ( . + . , n= ), hypoxic ( . ± . , n= ) and undernourished ( . ± . , n= ) pregnancies relative to untreated controls ( . ± . , n= ; p< . ). when fetal body weight was expressed relative to placental weight, a measure of placental efficiency, melatonin prevented the fall in the ratio in undernourished, but not hypoxic pregnancies (fig. objective: midkine (mk) is a -kda heparin-binding growth factor with various functions including cell proliferation, migration, differentiation and angiogenesis. mk expression is strictly regulated in temporal sequence; it is highly expressed during midgestation. we recently studied mk expression in the midgestation human fetus, and showed that the highest mk expression were observed in the adrenal gland, brain, lung and kidney. in the present study, we investigated the profile of mk in human amniotic fluid (af) and amnion (am). methods: amniotic fluid and amniotic membranes were collected at diagnostic amniocentesis, preterm no labor, and term no labor. mk protein levels were analysed by western blot. expression of transcripts encoding mk and putative mk receptors were examined by rt-pcr and real-time quantitative rt-pcr (qpcr). results: ) western blot analysis demonstrated abundant mk protein in the human am; mk levels were higher at midgestation than at term ( -fold: wks vs. wks). ) tissue transglutaminase known to polymerize mk was abundant in af. ) qpcr revealed that mk mrna was not expressed in am whereas it was highly expressed in the fetal adrenal, kidney and lung (positive controls). ) among the receptors implicated in mk signaling, lowdensity lipoprotein receptor-related protein and syndecan- were expressed in am while protein-tyrosine phosphatase, anaplastic lymphoma kinase, and syndecan- were not. conclusions: abundant mk protein in the midgestation af is likely to be derived from the fetus. mk in af may play a role in feto-amniotic communications and/or development of fetal organs exposed to af. short term hfix expression. we hypothesized that long term hfix expression could be achieved in fetal sheep using an aav vector, without stimulating an immune response to the transgenic hfix protein. we injected aav hfix vector ( - x p/kg) into the peritoneal cavity of fetal sheep under ultrasound guidance in early (n = ) or late (n = ) gestation. fetal blood was retrieved by ultrasound guided sampling from the intra-hepatic umbilical vein. fetal and lamb blood was tested for hfix expression using elisa, antibody responses using functional assays, and for liver damage up to a year after birth. vector spread was detected in maternal and fetal tissues by quantitative pcr analysis. results: the highest level hfix was detected days after late ip injection ( % and % normal human levels). early gestation ip injection gave . % and . % at and days after injection. hfix levels dropped rapidly correlating with the increase in size of the fetal liver and lamb. however, hfix was detectable at a low levels ( . %) year after birth in early and months after birth in late gestation injected lambs. up to year after birth, liver function tests and bile acid levels were normal, showing no evidence of liver pathology. no functional antibodies to the hfix protein or aav vector were detectable. high vector levels were detected in the fetal liver, and other peritoneal organs; no vector was present in fetal gonadal tissue. conclusion: hfix expression is detectable up to year after delivery of aav vector to the fetal sheep using a clinically applicable method. this is the first study to show long term hfix expression after fetal gene therapy in a large animal. further work will include testing for immune tolerance to exogenous hfix protein in these animals. objective: placental estrogens play a pivotal role in the endocrine control of pregnancy and may be involved in the key changes occuring during parturition. it has been established that crh interacting with crh receptor has a positive effect on estrogen production throughout pregnancy. urocortin (ucn ), a novel peptide of the crh family binding exclusively crh receptor , is expressed by human placenta and the aim of the present study was to evaluate the influence of ucn on estrogen biosynthesis in cultured trophoblast cells. methods: cultured term placental cells were treated with various concentrations of ucn in the presence of the estrogens precursors dehydroepiandrosterone sulphate (dhea-s), androstenedione or testosterone. estradiol secretion was measured in the culture medium using a specific elisa, p arom mrna and protein expression were evaluated by real time pcr and western blot analysis respectively. results: the addition of ucn , in presence of dheas significantly increased e levels at , and hours treatment, while in presence of androstenedione and testosterone an increase in e secretion was detected only at , and hours (at different ucn doses). both p arom mrna and protein were up-regulated in presence of each estrogen precursor and the addition of ucn caused a synergistic increase. anti-sauvagine (a crh type receptor antagonist) resulted in a significantly attenuated ucn effects on e secretion and on p arom mrna and protein expression. conclusions: the present study supports a possible role of ucn on placental e biosynthesis: e secretion and p arom transcript and protein expression were significantly increased after ucn treatment. in conclusion, the crf family may play a major role on placental steroidogenesis, stimulating dheas secretion in fetal adrenals by crh and controlling placental estrogen biosynthesis through ucn . a possible influence on the mechanisms of parturition may be hypothesized. increased expression of kiss- gene in preterm placenta. letizia galleri, michela torricelli, chiara voltolini, fernando m reis, felice petraglia. department of pediatrics, obstetrics and reproductive medicine, university of siena, siena, italy. introduction placenta expresses a large number of peptides involved in delivery. neuropeptides, cytokines, are expressed and secreted by human placenta at the time of preterm delivery. human placenta is a major source of kiss- , a -amino-acid peptide encoded by a putative metastasis suppressor gene. kiss- acts on its placental g protein-coupled receptors (kiss- r); this peptide stimulates release of oxytocin in rats, the most potent known uterine stimulant, suggesting a possible role of kiss- in the mechanisms of labor. aim of study the aim of this study was to evaluate placental expression of kiss- at preterm labor. material and methods placental tissue and plasma samples (both maternal and fetal) were collected at term in the absence of labor (tnl), at term spontaneous vaginal delivery (tl), and at preterm labor (ptl). changes in placental mrna expression were determined by real-time quantitative rt-pcr analysis. kiss- protein levels were measured by specific immunoenzymatic assay (elisa). results placental kiss- mrna expression was significantly higher (p< . ) in ptl than in tnl and in tl. however, maternal and fetal plasma kiss- levels did not differ among tnl, tl, and ptl samples. conclusion the present study showed that placental kiss- mrna expression is increased in preterm delivery. further studies are needed to better understand the role of kiss- on cascade leading term and preterm labor. placental angiogenesis is essential for placental development, which occurs under a hypoxic environment ( - % o ) during normal pregnancy. it has been reported that in transformed human dermal microvascular endothelial cells, hypoxia activates erk / , which stabilizes hypoxia-inducible transcription factor- (hif- ). however, signaling mechanisms governing placental angiogenesis under hypoxia is largely unknown. herein, we tested whether hypoxia affected fgf -and vegf-stimulated cell proliferation partly via activating erk / and stabilizing hif- protein levels in human placental artery endothelial (hpae) and transformed human placental microvascular endothelial (hpme) cells. methods: cells cultured under normoxia ( % o ) or hypoxia ( % o ) for days were treated with fgf and vegf. after days, the number of cells was determined. activation of erk / and levels of hif- protein were determined by western analysis. results: under normoxia, fgf and vegf stimulated (p < . ) cell proliferation and induced ( min, p < . ) erk / phosphorylation in hpae and hpme cells. hypoxia promoted (p < . ) fgf -and vegf-stimulated cell proliferation in hpae cells, whereas hypoxia blocked (p < . ) such actions in hpme cells. hypoxia enhanced fgf -, but not vegf-induced erk / phosphorylation in hpae cells. in contrast, hypoxia promoted (p < . ) vegf-, but not fgf -induced erk / phosphorylation in hpme cells. hypoxia increased (p < . ) hif- levels in the hpae cells: first detected at . hr and maintained up to hr. hpme cells had high basal levels of hif- ( folds; p < . ) compared with hpae cells. hypoxia did not alter hif- levels up to hr and decreased (p < . ) hif- levels at hr in hpme cells, conclusions: in hpae cells, hypoxia enhances fgf -and vegf-stimulated cell proliferation possibly partially via promoting activation of different protein kinases. this stimulatory effect is associated with increased hif- protein levels. however, inhibition by hypoxia on fgf -and vegf-stimulated hpme cell proliferation is associated with relatively high hif- levels in the first hr and decreased hif- levels at hr. thus, these data suggest that different signaling mechanisms are involved in hypoxia-modulated growth factor-induced placental angiogenesis in which an increase in hif- levels plays a critical role. objective: corticotrophin releasing factor (crf) is a well established neurohormone involved in the initiation of the stress response. we recently documented that rat placenta is analogous to human placenta in the expression of crf-mrna and protein, and that placental crf release may contribute to in utero meconium passage. time-of-day variation in crf expression is a highly recognized phenomenon at the brain cellular sites of crf synthesis. we sought to determine whether crf expression in rat placenta is subject to time-of-day variation. method: time-dated pregnant rats were obtained on day of gestation (term= days), housed in under h- h light-dark conditions (lights on ). lab chow and water were continuously available. on day , pregnant rats were quickly anesthetized by exposure to isoflurane, abdomen opened and fetuses and placenta exteriorized either at - hr (zeitgeiber time, zt ) or - hr (zt ). individual placentas were processed either for rna extraction or immunohistochemical investigation. the levels of crf-mrna were assessed by pcr using rat specific pcr primers. pcr bands were subsequently cloned and sequenced. bouin's solution fixed paraffin sections of placenta were subjected to crf immunohistochemistry with antibodies specific to rat species and intensity of immunostaining was analyzed using image pro-plus software and expressed in arbitrary units (au). results: one specific pcr band of bp size was consistently identifiable in placenta harvested at zt but not at zt . nucleotide sequence of the pcr band confirmed its identity as crf-mrna. intensity of crf-immunostaining was significantly greater at zt in giant trophoblast (gt) cells (gt-crf: zt = . ± . au, zt = . ± . au, p= . ) but not in spongiotrophoblast cells (stc) (stc-crf: zt = . ± . au, zt = . ± . au, p=ns) or labyrinth cells (lbc) (lbc-crf: zt = . ± . , zt = . ± . au, p=ns) . conclusion: similar to adult brain, rat placenta expresses crf mrna and protein. time-of-day variation of crf expression originally seen in central nervous system neurons is also identifiable in giant trophoblasts cells at zt . these findings suggest that stress-mediated placental crf release, and potentially fetal meconium passage, may be dependent upon time-of-day. objectve: transplacental water flow is essential for the provision and maintenance of fetal body water and amniotic fluid. water flow across membranes is regulated by aquaporin (aqp) water channels in many tissues. thus aqp , expressed in the placenta, is a candidate to regulate maternal to fetal fluid exchange. maternal beta-mimetics have been hypothesized to augment fetal growth by increasing the availability of nutrients and perhaps water. as camp acts as a second messenger to increase expression of selected aqps in other tissues, we sought to determine if betamimetics acting through camp could modulate placental aqp , and potentially influence placental water transfer. methods: trophoblastic cell cultures were established in first trimester-derived extravillous htr- /svneo cells and term placenta-like trophoblast carcinoma cell line jeg- . cultures were treated with sp-camp, a membrane-permeable and phosphodiesterase resistant camp, and forskolin, an adenylate cyclase stimulator, in doses of . , and m for hrs (aqp mrna expression) and hrs (aqp protein expression). for time course experiments, cells were incubated with μm of either sp-camp or forskolin for , and hrs at °c, % co . after cell harvest, mrna and protein expression were assayed using real time pcr and western blotting. results: sp-camp and forskolin increased aqp mrna expression in both cell lines after hrs (p< . ) in a dose-dependent manner. protein expression paralleled the increase seen in the mrna. m of sp-camp and forskolin stimulated aqp mrna expression after hrs in htr- /svneo cells and after hrs in jeg- cells (p< . ). m of either sp-camp or forskolin stimulated aqp protein expression in both cell lines after hrs (p< . ) and the expression remained high at hrs. conclusion: aqp gene expression in trophoblast cells is up-regulated by camp agonists. these results suggest that maternal beta-adrenergic agonists or antagonists may modulate maternal-fetal water flux via modulation of aqp water channels. rat placenta expresses corticotrophin releasing factor-binding protein and mrna. jayaraman lakshmanan, thomas r magee, bindu cherian, sharon k sugano, hanalise huff, michael g ross. dept. of ob/gyn, harbor-ucla med. ctr., torrance, ca, usa. objective: corticotrophin releasing factor-binding protein (crf-bp), a kda secreted glycoprotein, was originally isolated from human plasma. it binds crf and urocortin i with an equal or greater affinity than does the crf receptor. crf-bp expression has been reported in target tissues such as brain and placenta. based on its ability to inhibit crf actions in vitro, it is speculated to function as a "gate keeper" for crf-initiated stress responses. we recently documented expression of crf and urocortin- in rat placenta. in the present study we sought to establish the expression of crf-bp in rat placenta by immunohistochemical and pcr-analyses. methods: placental tissues (n= ) collected from sprague-dawley pregnant rats on day of gestation (term= ) were either fixed in % paraformaldehyde solution (ph . ) and paraffin embedded or processed for total rna extraction. paraffin sections of five micron thickness were subjected to immunohistochemical analysis with goat polyclonal antibodies to human crf-bp precursor (sc- santa cruz biotechnology, ca) by avidinbiotin-peroxidase complex technique. the structure of cloned human crf-bp precursor exhibit significant amino acid sequence homology among all species studied. immunoreactivities on the sections were quantified using image pro . software and staining intensity (od/area) expressed as arbitrary units (au). all values are expressed as mean±sem. for pcr, cdna was synthesized and pcr amplified with a one step rt-pcr kit. fragments were gel purified, clone into plasmid vector and dna sequenced. results: the crf-bp antibody elicited strong positive staining in decidua, giant trophoblasts, spongiotrophoblasts and labyrinth cells. the results of image analyses revealed (au): decidua: . ± . , giant trophoblast cells: . ± . , spongiotrophoblasts: . ± . , fetal membranes: . ± . . pcr analyses identified a single bp band, consistent with crf-bp. conclusion: our study establishes for the first time that rat placenta is analogous to humans in that both express crf-bp mrna and protein. immunohistochemical findings reveal that crf-bp protein expression occurs at multiple sites within the placenta. expression of per clock protein in rat placenta: an internal timer for placental functions. jayaraman lakshmanan, reuben lakshmanan, sharon k sugano, michael g ross. dept. of ob/gyn, harbor-ucla med. ctr., torrance, ca, usa. objective: corticotrophin releasing factor (crf) expression time-of-day variation occurs in giant trophoblast cells on the maternal side of the rat placenta. this temporal change in crf expression pattern is very similar to that of central nervous system neurons, suggesting that placental cells may use a similar mechanism to read time of the day. the self-sustaining rhythm generating capacity of the suprachiasmatic nuclei is believed to be derived from cell-autonomous, transcriptional feed-back loops dependent on a number of canonical clock genes. in the present study we sought to determine whether rat placenta expresses period gene (per ), one of the clock/cycle related genes. methods: time-dated pregnant sprague-dawley rats were received on day of gestation and housed in a controlled environment ( - h lights on) with free assess to food and water. placentas collected on days and of gestation (term= days) between and . am were fixed in % paraformaldehyde and paraffin embedded. for each gestational ages a total of placentas from different pregnant rats were used. paraffin sections ( sections per placenta) were subjected to immunohistochemical analysis with antibody to per ( : , santa cruz biotechnology, ca) by abc technique and , 'diaminobenzidine as a chromagen. sections were examined under microscope, imunostaining quantified by image pro . software and expressed in arbitrary units (au). data are presented as mean ± sem. statistical significance was analyzed by anova with a p value < . as significant. conclusion: the present results indicate that rat placental cells, devoid of any neural innervations, express per clock protein, similar to central nervous system neurons. based on the differences in the relative intensities between trophoblasts and labyrinth cells on day of gestation, we conclude that fetalmaternal interactions in per regulation disappear at term (or at the time of birth.). our findings imply that per may function as internal physiological modulator in placenta. to determine the mechanism(s) underlying placental time-of-day crf variation, we examined the effect of maternal administration of betamethasone (a synthetic glucocorticoid) on nuclear gc receptor expression in placental cells. method: time-dated pregnant rats (n= ) on day of gestation were given a single subcutaneous injection (at : am) of betamethasone ( μg/kg body weight) while control pregnant rats (n= ) received saline. dams were exposed to isoflurane anesthesia at : am, and placentas harvested, fixed in bouin's solution and paraffin embedded. sections were subjected to immunohistochemical analysis with gc-receptor antibody (sc- , santa cruz biotechnology), with immunoreactive material identified by abc technique using , ' diaminobenzidine as a chromagen. percentages of gc-nuclear receptor (gc-nr) positive cells and their intensities (od/area) were quantified using image pro . plus software. all values are expressed as mean ± sem. statistical analysis was by anova with p< . considered significant. results: in placentas of saline exposed pregnant rats, isolated labyrinth (lb) cells (< %) were positive to gc-nr. no positive staining for gc-nrs was seen either in giant trophoblasts (gt) or in spongiotrophoblasts (st). betamethasone administration was associated with a significant and marked increase in gc-nr staining in all three placental cell types (p< . ). among the cells, gt ( ± %) demonstrated a greater percentage of cells expressing gc-nr than did st ( ± %) or (lb= ± %) (p< . ), though there was similar immunoreactive intensities (gt: . ± . , st: . ± . , lb: ± . au; p=ns) conclusion: our findings indicate that all placental cells respond to gc with upregulation of gc-nr with an enhanced response among gt cells. these results suggest that endogenous or exogenous maternal stress-induced gc exposure may influence signaling responses within both maternal and fetal placental compartments. ovine placenta at very-preterm gestation expresses corticotrophin releasing factor (crf), crf-binding protein (crf-bp) and glucocorticoid receptors (gr). jayaraman lakshmanan, john d richard, guo l liu, sharon k sugano, michael g ross. dept. of ob/gyn, harbor-ucla med. ctr., torrance, ca, usa. objective: in humans, the placenta is the major source of maternal and fetal plasma crf. based on its critical functions, crf is considered as a "placental clock" of parturition. we recently reported that ovine fetal as well as maternal plasma contain measurable amounts of crf at near-term but not at very preterm gestation. we interpret the absence of crf in plasma at very preterm gestation is either due to lack of placental crf expression and/or placental crf release. here, we examined the expression status of crf, crf-bp (a known specific binding protein for crf and a known regulator of crf functions in human placenta) and gr (a known positive regulator of crf expression in human placenta) in ovine placenta collected at very-preterm gestation. method: placenta harvested from time-dated pregnant ewes on ± days gestation were fixed in bouins's solution and processed for paraffin embedding. paraffin sections were subjected to immunohistochemical analysis with polyclonal antibodies specific to ovine crf( : - : , phoenix pharmaceuticals, ca,) crf binding protein ( : - : , sc ) and gr ( : - : , sc: , santacruz biotechnology, ca). immunoreactive material on the sections were identified as brown staining by abc technique using diaminobenzidine as chormagen. immunoreactive material was quantified by image analysis using image pro . plus software and the immuoreactive intensity (od/area) expressed as arbitrary units (au). results: strong positive staining for crf ( . ± . au), crf-bp ( . ± . au) and nuclear-gr ( . ± . au) were noticed in syncytiotrophoblast cells in all placental sections obtained from pregnant ewes at days gestation. control sections exhibited no positive staining. conclusion: our findings indicated that ovine placenta at very-preterm gestation expresses crf, crf-bp and gr. these results suggest that absence of measurable crf in maternal and fetal plasma at very-preterm gestation is not due to lack of placental crf expression but rather due to the absence of regulated crf secretory mechanisms. rat placenta expresses urocortin i protein and mrna. thomas r magee, michael g ross, john d richard, sharon k sugano, jayaraman lakshmanan. dept. of ob/gyn, harbor-ucla med. ctr., torrance, ca, usa. objective: urocortin i (ucn- ), a amino acid neuropeptide belongs to corticotrophin releasing factor (crf) stress hormone family. in humans, the placenta and several gestational tissues have been reported to express ucn- protein and ucn- mrna. a number of published studies indicate that ucn- is similar to crf in expression pattern and biological functions. we recently reported that rat placenta is a site of crf protein and crf mrna expression. in the present study we sought to determine whether rat placenta expresses ucn- protein and ucn- mrna. methods: placenta (n= ) collected from pregnant rats at day gestation were either fixed in bouin's solution and processed for paraffin embedding or placed in rna later preservative and frozen for rna extraction. for immunohistochemical localization, five micron thickness paraffin sections were cut and immunostained with rabbit polyclonal antibodies to ucn- ( : to : , sigma) by standard abc technique. control sections were incubated with omission of ucn- antibody. immunoreactive materials on the sections were identified using , ' daminobenzidine as chromagen. immunostaining intensity (od/area) was quantified by image-pro plus software and expressed in arbitrary units (au). for pcr, cdna was synthesized and pcr amplified with a one step rt-pcr kit. fragments were gel purified, cloned into a plasmid vector and dna sequenced. all values are given as mean ± sem. results: ucn- polyclonal antibody elicited strong immunostaining in placental trophoblast cells with variable intensity. the pattern of immunostaining is as follows: giant trophoblast cells: . ± . au, spongiotrophoblast cells: . ± . au and labyrinth cells: . ± . au. the relative intensity in labyrinth cells was significantly lower than the other two cell types (p< . ). pcr analyses revealed the presence of a single band of bp, consistent with ucn- mrna. conclusion: similar to human placenta, rat placenta expresses ucn- protein and ucn- mrna, with most expression localized to the maternal side trophoblast cells. these results support our hypothesis that rat placenta can be used as a model to understand the role of this peptide in feto-maternal stress. the role of the nuclear hormone receptors fxr, pxr and car in placental bile acid homeostasis in normal and pathological pregnancy. victoria geenes, peter dixon, selina raguz, jenny chambers, kishore bhakoo, catherine williamson. imperial college, london, united kingdom. background: obstetric cholestasis (oc) is a pregnancy specific liver disorder characterised by raised maternal serum bile acid levels and associated with adverse fetal outcome. the aetiology of oc is complex and not fully understood, but the fetal complications are likely to result from an accumulation of bile acids in the fetal circulation. bile acids are the toxic end products of hepatic cholesterol metabolism and are synthesised from weeks gestation. in common with other waste products, accumulation in the fetal compartment is prevented by excretion across the placenta into the maternal compartment. however, studies of maternal and cord serum from normal and oc pregnancies have suggested that bidirectional transfer of bile acids is possible. hepatic bile acid transport and metabolism is regulated by members of the nuclear hormone receptor family, namely fxr, pxr and car, but the mechanisms for regulating placental transfer are unknown. objectives: this study used placenta from normal and cholestatic pregnancies to investigate the expression of genes involved in hepatic bile acid homeostasis. methods: villous trophoblast samples from oc and normal pregnancies (np), and human livers were collected and preserved in rnalater. explant cultures were prepared from a further np placentas and cultured for days at % oxygen. on day they were treated with chenodeoxycholic acid, lithocholic acid or vehicle for hours prior to fixing in rnalater. total rna was extracted using trizol, and reverse transcribed to cdna. quantitative real-time pcr was performed using sybr green. target gene mrna abundance was calculated from a standard curve and normalised to l . results: the expression of fxr, pxr and car, the nuclear hormone receptors responsible for hepatic bile acid homeostasis and several fxr target genes (shp, mdr and bsep) was found to be very low in np, and unaffected by the presence of maternal cholestasis. furthermore, the expression of these genes could not be induced by bile acid treatment in an in vitro model. here we have shown that the nuclear hormone receptors (fxr, pxr and car) involved in hepatic bile acid homeostasis are expressed at very low levels in normal and pathological pregnancy and are thus likely to be of limited importance in placental bile acid transfer. a placental phenotype for obstetric cholestasis. victoria geenes, jenny chambers, jo wyatt-ashmead, kishore bhakoo, catherine williamson. imperial college, london, united kingdom; hammersmith hospital, london, united kingdom. background: obstetric cholestasis (oc) is a pregnancy specific liver disorder that affects . % of pregnant women in the uk. biochemically, oc is characterised by liver dysfunction with elevated maternal serum bile acids (sba), and clinically by an increased risk of fetal complications including fetal distress, meconium staining of the amniotic fluid, preterm labour and sudden intrauterine death. the aetiology of oc is complex and not fully understood, but the fetal complications are likely to result from the toxic effects of bile acids, which can cause vasoconstriction in the placenta and fetal cardiac dysrrhythmias. furthermore, in a rodent model of oc, bile acids cause oxidative stress in the placenta and a reduction in litter size. these changes are absent in animals treated with ursodeoxycholic acid (udca), a drug used to treat oc. however human data are lacking. objectives: this study used whole placenta and explant cultures to investigate the effects of bile acids on human placental architecture and to establish whether udca protects the placenta against bile acid induced damage. methods: villous trophoblast samples were collected from oc and normal pregnancies (np) and fixed in formalin. explant cultures were prepared from a further np placentas, and cultured for days at % oxygen. on day a subset were treated with udca overnight, and on day the explants were treated with taurocholic acid, taurochenodeoxycholic acid or vehicle for hours prior to fixing. m sections were stained with haematoxyllin and eosin. slides were reviewed by a perinatal pathologist and syncytial knots (sk) counted. results: oc tissues showed marked alterations in morphology, including congestion of the terminal villi, and loosening of the stroma and fibrotic change of the membranes of the stem villi. there were significantly more sk the oc samples (mann-whitney u test p= . ). furthermore, sk were increased in explants treated with bile acids, but not those treated with udca prior to the addition of bile acids. conclusions: here we describe several morphological abnormalities of the placenta associated with maternal cholestasis. these changes were confirmed using a placental explant model, which also showed that udca protects the placenta against bile acid induced damage. in summary, this study indicates that udca is likely to protect the fetus in oc. the placenta has complex metabolic and endocrine activities and is essential for the growth and survival of the fetus in utero. ultrasound is the most sensitive and less invasive method to evaluate placental size, morphology and function. the three-dimensional approach allows to calculate placental volume in the i and ii trimester of pregnancy. pregnancies from assisted reproduction technologies (art) show an increased rate of pathologies potentially related to placental insufficiency such as intrauterine growth restriction and preterm delivery. aim. to determine placental volume in art pregnancies in a cross sectional study in the first trimester of gestation. design. using three-dimensional ultrasound machine (ge ) placental volume measurement from art pregnancies (n= ; - wks) were compared with data from normal controls (n= ) matched for gestational age. data were analysed with software stata . results. mean placental volume was . ml (sd± . ) in art pregnancies and . ml (sd± . ) in normal controls. mean difference resulted in . ml (- - . ) and was statistically different (p= . ). multiple linear regression analysis showed a statistically significant interaction (p= . ) between gestational age and case status, i.e. differences in placental volume increase significantly with advancing gestational age between cases and normal controls. mean gestational age at birth was not essentially different between the two subsets ( . weeks ± . ) however a statistically significantly lower mean fetal birthweight was found in art pregnancies: g (sd± ) vs .(sd± . ) (p= . ). conclusions. placental volume is slightly decreased in art pregnancies. measurements of placenta volume by d ultrasound may play a role in identifying the degree of placental growth early in gestation in a risk population. in vitro effects of adenovirus-mediated gene transfer of insulin like growth factor- (ad-igf- ) in trophoblast cells. mounira habli, datis alae, foong yen lim, jignesh parvadia, timothy crombleholme. obstetrics and gynecology; pediatric surgery, university of cincinnati/children's hospital, usa. objective:recently, ad-igf- corrected both fetal and placental weights in rat model of fetal growth restriction (fgr) . to elucidate the mechanism of action of ad-igf- ;we examined the effect of ad-igf- on trophoblast proliferation, differentiation and invasion. methods: rcho- trophoblast cell line derived from rat choricarcinoma was used. ad-igf- and galactosidase transgene (ad-lacz) were given at moi of : and : in all the experiments.transduction efficiency was assessed hr after infection with ad-lacz by galactosidase enzyme activity. the invasiveness of rcho- was measured by using bdmatrigel invasion chamber kit. rcho- proliferation cell density was assessed by crystal violet assay. morphologic analysis of rcho- differentiation in response to treatment (differentiated cells are multinucleated giant cells) was assessed byimunocytochemistry staining using placental lactogen- antibodies(specific hormone produced by giant cells). results: % efficiency of gene transfer of ad-lacz to rcho- cells was observed at both moi's.there was no significant difference in proliferation between treated control group regardless of the dose at and hrs. ad-igf- induced morphologic differentiation of trophoblast cells compared to control (fig ) at hrs.ad-igf- induced higher rate invasion in a dose dependent fashion as compared to control(ad-lacz) group( % at moi vs . % in control p< . )(fig ). conclusion: ad-igf- gene transfer induces differentiation and invasiveness of trophoblast cells. these may be the mechansim of correction of fgr in rat model of placental insufficiency. placental gene transfer may be an effective treatment strategy for placental insufficiency. types of human placenta. martina dieber-rotheneder, ursula hiden, gernot desoye, mila cervar-zivkovic. department of obstetrics and gynaecology, medical university graz, graz, austria. background and hypothesis: endothelin- is a polypeptide with a wide range of functions. in the placenta it acts as a potent regulator of vasotonus on endothelial and smooth muscle cells, whereas on the trophoblast it regulates cell proliferation, invasion and apoptosis. endothelin- action is differently signaled through two endothelin receptor (etr) subtypes, etr-a and etr-b. several alternative splice variants of etr were identified. here we hypothesize that the etr-a splicing varies with gestational age and is different in trophoblast vs endothelial cells of the human term placenta. methods: mrna of placental tissue from first trimester (pregnancy terminations, missed abortions) and term of gestation, first trimester and term trophoblasts, arterial and venous placental endothelial cells as well as cell lines representing first trimester trophoblast (ach p) and term placental endothelial cells (hpec) were analyzed for full length etr-a and known as well as unknown splice variants by sqrt-pcr using primers spanning the exons - . the predominant dna bands in agarose gel were excised and sequenced. results: all tissues and cells expressed full length etr-a. in all tissues an additional -deletion variant was identified which was not found in the isolated cells. trophoblasts expressed another yet unidentified splice variant. the endothelial cells expressed a -deletion variant and a novel splice variant, which was identified as partial -partial deletion. this novel splice variant was found regardless of the arterial or venous origin of the cells as well as in the cell line (sv -transformed). in tissues und trophoblast no difference in splicing was found between first trimester and term. conclusion: gestational age does not alter splicing of endothelin receptor-a. splicing is different between trophoblasts and endothelial cells. the endothelial cells contain a novel splice variant. the differential splicing may allow maternal and fetal endothelin- to induce different effects on the two major cell types of the placenta. (supported by grant , jubilee fund, austrian national bank, vienna). adrenomedullin production and secretion by human trophoblast cells are regulated by glucocorticoids and hypoxia. francesca ciardo, katia pacioni, emanuela marinoni, giovanna corona, massimo moscarini, alfredo patella, romolo di iorio. department of gynecology, perinatology and child health, university "la sapienza", rome, italy; department of obstetetrics and gynecology, university of ferrara, ferrara, italy. objectives: adrenomedullin (am) is produced by intrauterine tissues and is involved in the regulation of implantation, placental hemodynamics and endocrine function. circulating am is increased in pregnancy complications such as preeclampsia and intrauterine growth retardation. we investigated whether am output by human trophoblast cells is regulated by hypoxia and/or glucocorticoids. study design: trophoblast cells obtained by human placentas at term (n= ) were cultured in presence or absence of hypoxia ( % o ) and treated with or without betamethasone at the dose of - , - , - m. media and cells were collected at h and at , and h from syncytiotrophoblast cultures. am was measured in cultured media by specific ria kit. protein expression in trophoblast cells was evaluated with immunohistochemistry and western blot. results: hypoxia stimulated am output and protein expression by cytotrophoblast and syncytiotrophoblast cells. betamethasone induced an increase in am production and secretion in a time-and dose-dependent manner (figure). effects of hypoxia were partially reversed by betamethasone in a dose and time-dependent fashion. conclusions: am production in trophoblast cells is up-regulated by hypoxia and glucocorticoids, independently. increased am levels in pregnancy complications characterized by placental insufficiency might derived by an increase in am placental secretion stimulated directly by hypoxia or indirectly by an increase in fetal cortisol levels. preeclampsia complicates - % of pregnancies, and while associated with significant maternal and fetal morbidity and mortality, the mechanisms responsible for preeclampsia are not completely understood. recent advances suggest there are imbalances of pro-and antiangiogenic factors, present from the time of implantation affecting vascular responsiveness of the fetal placental unit and maternal vasculature. in this study, we investigate vasomotor responsiveness of placental arteries from normal and preeclamptic (pet) pregnancies using an in vitro muscle bath contractility assay. transverse rings of placental arteries were cut and equilibrated in the muscle bath containing a bicarbonate buffer aerated with % o / % co at °c. viability was demonstrated by contraction to mm kcl prior to all experiments. cumulative logarithmic dose dependent responses to four different contractile agents: pgf , serotonin, carbochol, and norepinephrine were compared. placental arteries precontracted with pgf at half maximal concentration were relaxed with three vasorelaxants: sodium nitroprusside (snp), forskolin (fsk) and lactate (lct). agonist studies revealed contraction to both pgf and serotonin but not to carbochol or norepinephrine. there were no statistically significant differences between the responses of normal and pet arteries. both normal and pet arteries demonstrated heightened responsiveness to sodium nitroprusside compared with forskolin and lactate. this study reveals that the placental vessels are more sensitive to sodium nitroprusside, that mediates relaxation through nitric oxide -cyclic gmp signal transduction pathway, than forskolin that induces relaxation through the cyclic amp pathway. cancer complicates approximately one in , pregnancies. depending on the type of cancer and trimester, patients can terminate the pregnancy or choose treatment such as chemotherapy. since there is limited knowledge on the safety of chemotherapy on fetal tissues in utero, clinicians cannot efficiently analyze the risks of particular anticancer agents when deciding on a course of therapy. since carboplatin is among the most common anticancer agents used for treatment of cancer during pregnancy the primary objective of this study was to evaluate if carboplatin will readily cross to placental barrier and determine total potential platinum fetal exposure. this project utilizes an ex vivo placenta perfusion model to determine the concentration of carboplatin that crosses the human placental barrier. placentas are obtained within minutes of spontaneous delivery and re-perfused. two carboplatin concentrations were selected of ng/ml and ng/ml to represent clinically relevant maternal plasma concentrations. antipyrine was used as the internal control. serial samples were collected every minutes for total minutes in open-open model then experiment repeated with closed circuit model. antipyrine concentrations were determined by hplc methods. platinum concentrations in media samples were determined with a validated atomic absorption assay. a cell culture-based approach will be used to determine whether cell cycle or apoptotic proteins are altered (p , bax, bcl- , aif, and pro-caspase- ) using western blotting as a detection method. a total of two placentas have been completed at the low carboplatin concentration and one at the high concentration. the mean carboplatin clearance was . +/- . ml/min and . +/- . ml/min at the low and high carboplatin concentrations, respectively. an estimated % increase in the transport fraction was observed in the high concentration experiments compared to the low concentrations model. fetal carboplatin exposure ranged from to ng/ml. the placental perfusion experiments conducted at carboplatin and ng/ml indicate that carboplatin crosses the placenta through simple diffusion. the toxicology assays are ongoing to determine potential effect on fetal tissues. preterm delivery represents one of the predominant causes of perinatal mortality and morbidity, and is one of the most unpredictable gestational disturbances. urocortin is a peptide expressed by trophoblast and gestational tissues (amnion and chorion), whose maternal levels correlate with gestational length, since they are increased at preterm delivery and decreased in post-term pregnancy, when compared to term pregnancy. the addiction of urocortin enhances contractility of human myometrial strips, suggesting a possible role on uterine contractility. high maternal urocortin levels in threatened preterm delivery correlates with the timing of delivery, suggesting a possible role in the predictivity pf preterm delivery. in the present study urocortin concentrations in amniotic fluid collected at mid gestation for amniocentesis were correlated with gestational age at delivery. a case-control study with amniotic fluid obtained from healthy women undergoing amniocentesis for genetic indications was performed; urocortin concentrations were measured by a specific elisa. amniotic fluid urocortin concentrations resulted significantly lower (p= . ) in women delivering preterm (n= ; ucn= . ± . pg/ml) (m ± se) than in those delivering at term (n= ; ucn= . ± . pg/ml). in conclusion, the present preliminary data showed that amniotic fluid urocortin concentrations at mid gestation may represent predictive marker of preterm delivery. human placentas throughout pregnancy. annunziata mastrogiacomo, elisabetta federico, francesca caprio, maria teresa schettino, gabriele coppola, antonio de luca, luigi cobellis. department of obstetrics and gynecology, second university of naples, naples, italy; department of medicine and public health, section of anatomy, second university of naples, nales, italy. hypothesis apoptosis is intimately involved in placental homeostasis, growth and remodeling and the apoptotic rates increase progressively during normal pregnancy as part of normal placental development. moreover, apoptosis increases in pregnancies complicated by some pathologies such as preeclampsia, fetal growth restriction, diabetes. in the present study, we describe differences in the expression of pro-apoptotic protein bax, in first trimester voluntary termination of pregnancy, first trimester abortion (reserved abortion), caesarean birth, spontaneous birth, preeclampsia and diabetes. material and methods human placental samples were obtained with informed consent from patients undergoing surgery such as first trimester voluntary termination of pregnancy (n= ), first trimester abortion (miscarriage) (n= ), delivery section (n= ), spontaneous birth (n= ), preeclampsia (n= ) and diabetes (n= ). the gestation period ranged from to weeks. the specimens were immediately fixed in formalin for immunohistochemistry. we first observed a strong increase of bax expression in the cytotrophoblast, stroma, endothelial cells and decidua of placentas of the first trimester abortion compared to the low/moderate bax immunopositivity in all the placental compartments during the first trimester voluntary termination of pregnancy. secondly, we showed a more intense immunopositivity for bax in the third trimester spontaneous birth respect to the third trimester caesarean birth. thirdly, we observed an increase of bax expression in preeclamptic placentas compared to the normal full-term placentas. on the contrary, we observed a moderate bax expression in diabetic placentas only slightly decreased compared to the normal full-term placentas. our results seem to suggest that deregulation of apoptotic turnover may lead to placental dysfunction and pathologies. objectives: maternal endothelial activation in preeclampsia (pe) is attributed to the release of unknown factors from a hypoperfused placenta. the application of proteomic technologies such as mass spectrometry promises the reward of identifying and characterising these factors. using a placental explantconditioned culture media model system we have developed a proteomics workflow to obtain relative quantification of proteins released into placental explant culture media. methods: term villous explants were cultured in serum-free conditions and exposed to differing oxygen concentrations ( % %) to mimic physiological and non-physiological intervillous o tensions. the d media was concentrated, immunodepleted to remove fibrinogen (using beckman igy- columns) and proteins labelled using an itraq (applied biosystems) kit following the manufacturer's protocol. labelled peptides were fractionated by strong cation exchange and then analysed using lc-maldi on a maldi-tof/tof (applied biosystems) mass spectrometer. data was analysed using protein pilot . (applied biosystems). results: when matched pooled (n= ) media samples were subjected to our proteomics workflow over proteins were identified with a calculated false positive identification rate of < %. of these proteins a total of display a statistically significant difference in protein levels between the % % o samples (p=< . ). from a list of proteins of interest we selected proteins for validation using elisa/western blotting to measure their relative abundance in both pooled and individual explant media samples. interleukin is an example of a low-abundance differentially expressed protein identified in our proteomic workflow and subsequently validated by elisa; il- (pg/ml) in % o : . , range . to . ; % o : . , range . to . ; values are median with interquartile range. *** p< . , mann-whitney test (n= ) conclusions: we demonstrate a successful reduction to practice of a relative quantitative proteomics strategy applied to placental explant-conditioned culture media. having optimised and validated this proteomic workflow we will apply the same methods to compare proteins released by normal and preeclamptic placentas. a proteomics screen of human placental microvillous syncytiotrophoblast (stb) revealed the expression of dysferlin (dysf), a membrane repair protein associated with certain muscular dystrophies (vandré et al., ) . a second ferlin protein, myoferlin (myof), was also discovered which has not yet been characterized in the placenta. in human c c myoblasts, dysf and myof are reciprocally expressed as a function of differentiation into multinucleate syncytial myotubes, with dysf predominating in differentiated cells. we hypothesized that dysf and myof would show a similar reciprocal expression pattern in human trophoblasts as a function of syncytialization. methods term placentas from uncomplicated pregnancies were obtained with informed consent (n= ) and either fixed for immunofluorescence (if) labeling or flash frozen for subsequent immunoblotting (ib). human trophoblastic (bewo, jar, and jeg- ) and other cell lines (mrc- , hl- , huvec) were cultured in the absence or presence of μm forskolin or solvent control for - days. dysf and myof expression was examined by rt-pcr, ib, and if. results ib validated proteomics data showing myof expression in term placenta. by if, myof labeling was predominant in apical and basal stb plasma membranes. myof was expressed in trophoblastic cell lines (bewo, jar, and jeg- ), cultured endothelium (huvec), and a fibroblast cell line (mrc- ), but was not detected in a leukemic cell line (hl- ). dysf was constitutively expressed in jar, but minimally expressed in unstimulated bewo. following forskolin-induced syncytialization, we observed a time-dependent increase in dysf expression in bewo concomitant with increasing syncytin- levels. by if, dysf expression was restricted to bewo cells in syncytial structures. in contrast, myof expression was robust in mononuclear bewo cells and not down-regulated over the course of days of differentiation. conclusions two ferlin-family genes are expressed in trophoblasts. fusion-competent bewo cells behave similarly to cultured myoblasts and ctbs with regard to dysf expression, which is restricted to syncytializing cells. myof, in contrast, is expressed constitutively in bewo and other models. while both proteins are likely to function in stb plasma membrane repair (e.g., following syncytial sprouting), the relative contributions of each to this process awaits clarification. features of chronic placental insufficiency are significantly more common in small for gestational age placentas from infants with intrauterine growth retardation. emily king, violetta kolesnikova, carri tillotson, jean-marie guise, terry morgan. pathology; center for biostatistics; obstetrics and gynecology, ohsu, portland, or, usa. background: there is a strong association between intrauterine growth restriction (iugr) and small for gestational age (sga) placentas (heinonen et al. placenta ( ), : - ) . the cause is likely multifactorial, but we hypothesize that chronic uteroplacental insufficiency may play a role. our objective was to test whether sga placentas from human neonates with iugr show significantly more pathologic features of placental insufficiency compared to controls. design: we performed a retrospective review of consecutive singleton placentas submitted to ohsu pathology ( - ), excluding elective terminations and spontaneous abortions before weeks gestation. clinical records were reviewed and pregnancy outcomes recorded, including: ) neonatal sex; ) weight (iugr calculated by routine methods), ) trimmed weight of the placenta (sga calculated by routine methods), ) gross placental infarctions, ) gestational age at delivery, and ) maternal features (e.g. race, gravida). controls were defined as cases within the series without sga or iugr (e.g. submitted for meconium, infection, etc). histologic sections were scored by two pathologists while blinded to clinical diagnoses as positive or negative for features of placental insufficiency, including accelerated villous maturation (avm), chorangiosis, and microscopic infarctions. significant associations were tested by analysis and logistic regression for multiple variables. results: similar to prior reports, we observed a strong association between iugr and sga placentas ( . ; p < . ). this relationship was independent of maternal race, fetal sex, and parity, although it was more common in primigravidas. avm and placental infarctions were significantly more frequent in sga placentas with superimposed iugr. controls ( introduction: messenger rna expression peripheral blood cells (pbc) has been recently used as biomarkers of environmental exposures (ionizing radiation or tobacco), physiological conditions (stress) and diseases (hypertension, neurological disorders). pbc evaluation is a useful diagnostic tool in an era of individualized medicine. since there is an urgent need for non-invasive methods for determination of fetal (f) and placental (p) function, this study was designed to evaluate the genes differently and commonly expressed in p tissue and leukocytes in maternal (m) and f circulation.material and methods. p (n= ), f (n= ) and m blood (n= ) were obtained during cesarean section in pregnant baboons at term. total rna from a buffy coat pellet was isolated using a modified procedure of the qiagen rneasy mini kit (qiagen). anti-sense rna (arna) was synthesized and purified using the illumina rna amplification kit (ambion, usa). hybridization of arna to illumina sentrix human whole genome (wg- )beadchips and subsequent washing, blocking and detection was performed using illumina's beadchip ´ protocol. samples were scanned on the illumina beadarrayer gx reader using illumina beadscan image data acquisition software (ver. . . . ). differential gene expression data analysis was performed using illumina beadstudio software (ver. . . . ). results. the detection level of gene transcripts using illumina methodology with control human rna was - at p< . . gene transcripts were detected in f blood and in maternal blood. transcripts were uniquely expressed in fetal blood. transcripts were found in m, but not in f leukocytes. the number of gene transcripts expressed in p tissue was and of these genes were not expressed in m leukocytes. conclusion. despite white blood cells trafficking through the p barrier there is a set of unique genes expressed only in p or in the m or f circulation. the application of these genes as the biomarkers of p barrier function still need to be evaluated. objective: to evaluate if a relationship exists between duration of placental exposure to meconium in vivo and histologic evidence of severity and extent of meconium uptake by macrophages. study design: from a cohort of / ( %) consecutive singleton liveborn infants delivered at term with thick meconium-stained fluid, ( %) had placental histologic examination performed, and in / the timing of meconium appearance after membrane rupture was documented. placental histologic examination quantitatively evaluated the intensity of meconium uptake by resident macrophages based on the number of macrophages/field, and the extent of uptake based on histologic location, graded in a score to . results: mean interval between meconium appearance and delivery was . ± . min (range - ). after exclusion of cases in which severe placental inflammation interfered with analysis, meconium uptake by macrophages was documented in / cases at the level of amniochorionic membranes, in / cases at the placenta, and in / cases at the umbilical cord. there was no correlation between the interval meconium appearance-to-delivery in relation to presence of meconium in the membranes (p= . ), in the placenta (p= . ), in the cord (p= . ), or score of severity of meconium uptake (p= . ). the results did not change after correcting for gestational age, oligohydramnios, presence of placental acute inflammatory or vascular lesions. conclusion: there is no relationship between duration of placental exposure to meconium and the extent and intensity of its uptake by macrophages in cases with exposure up to . hours. transfer of bisphenol a across the human placenta. biju balakrishnan, , kimiora henare, , eric thorstensen, , murray d mitchell. , the liggins institute, university of auckland; national research centre for growth and development, auckland, new zealand. introduction: there are growing concerns over the effects of developmental exposure to the xenoestrogen bisphenol a (bpa). animal studies have shown that bpa is transferred through the placenta and can cause deleterious effects to the fetus. the presence of bpa in feto-placental tissues in humans has also been reported. however, a detailed study of the time-course of bpa transfer across the human placenta has not been performed. the aim of this research was to study the transfer of bpa in ex-vivo perfused human placental tissues. methods: a dual recirculating single cotyledon perfusion was used to monitor the placental transfer of bpa. bpa ( ng/ml), antipyrine (ap) ( μg/ml), and fitc dextran (fitc-dx) ( . μg/ml) were added to the maternal perfusate, and perfusion was continued for hours. perfusate samples were collected from both reservoirs at timed intervals and analysed. bpa, ap, and fitc-dx were determined by hplc with fluorescent detection, hplc with uv detection, and spectrofluorometry respectively. the viability and metabolic activity of the placentae were assessed by measuring -hcg (elisa), glucose utilization, and lactate production (autoanalyzer). fetal pressure, ph, flow rates and fluid shifts were monitored continuously. results: the biochemical validation parameters (glucose consumption, lactate production and -hcg secretion) indicated that the placental tissue was metabolically active and viable throughout the -hour perfusion period. the physical parameters observed (fetal pressure < mmhg, ph range . - . ) were in concordance with other published works for placental perfusion. membrane integrity was confirmed by fluid shifts from either circuit of < ml/hr, and by < % materno-fetal transfer of fitc-dx. an observed ap transfer of - % further validated our model. bpa first appeared in the fetal compartment within minutes of perfusion and reached a peak of about - % of maternal concentration within hours of perfusion. this figure is likely to be an underestimate since it does not include conjugated bpas. conclusion: this first study of bpa transfer in ex-vivo perfused human placental tissue shows that our model can serve as a useful tool to study the transfer kinetics and metabolism of bpa in human term placentae. we found that bpa rapidly crosses the human placenta at environmentally-relevant doses, with potentially harmful effects on the human fetus. angiogenic growth factor secretion by uterine natural killer cells in co-culture with extravillous trophoblast. gendie e lash, katsu naruse, , barbara a innes, stephen c robson, judith n bulmer. institue of cellular medicine, newcastle university, newcastle upon tyne, tyne and wear, united kingdom; obstetrics and gynaecology, nara medical university, nara, japan. objectives. uterine natural killer (unk) and extravillous trophoblast (evt) cells have been proposed to play roles in remodeling of uterine spiral arteries through secretion of various angiogenic growth factors. we have previously demonstrated that unk cells are a significant source of angiogenic growth factors within the decidua, many of which alter with gestational age. however whether the secretion of these proteins is altered by interactions of unk cells with evt is unclear. hypothesis. unk cell angiogenic growth factor secretion is regulated by evt in early pregnant decidua. methods. placental and decidual samples were collected from women undergoing termination of pregnancy with written informed consent ( - and - weeks gestation, n= each group). . × cd + unk cells were positively selected from decidua and co-cultured with evt ( . × ) or cytotrophoblast (ctb; . × ) purified from the same placenta for h in direct or indirect contact (n= each group). angiogenin, ang , pdgf-bb, fgf-basic, timp , icam and vegf-a were measured by fast quant ® angiogenesis multiplex assay system, and vegf-c, ang and plgf by elisa. results of unk co-culture with evt or ctb (negative control) at each gestational age were analyzed with wilcoxon rank test. in addition, the effect of direct and indirect co-culture of unk cells with evt at each gestational age was compared with mann whitney u test. results. at - weeks gestation unk secretion of ang (p= . ), icam (p= . ) and plgf (p= . ) was increased in the presence of evt compared with ctb. no differences were observed at - weeks gestation. in addition, at - weeks gestation unk secretion of ang (p= . ), vegf-c (p= . ) and ang (p= . ) was increased in direct co-culture with evt compared with indirect co-culture. at - weeks gestation unk secretion of timp- (p= . ) was reduced in direct co-culture with evt compared with indirect co-culture. conclusions. unk cell secretion of several key angiogenic growth factors was altered by direct culture with evt. these data suggest that a membrane bound molecule (such as hla-g) mediates this modulation of unk cell activity. abnormalities in placentation and impaired placental circulation can lead to fetal growth restriction. -d ultrasound can be used to evaluate this through the quantification of the power doppler signal that may be expressed as a percentage of colour voxels within a user-defined volume (vi: vascularisation index). we aimed to test the hypothesis that increased fetoplacental blood flow correlates with an increased vi, using the in vitro dual perfusion model of the human placental lobule. three term lobules were dually perfused through both circulations with earles bicarbonate buffer (ebb), and supplemented on the fetal-side only with adult erythrocytes, prepared to a % haematocrit. following initial equilibration perfusion at normal flow values, fetal-side flow was varied between and ml/min, whilst maternal-side flow was held at ml/min. images were obtained with a 'voluson i' ultrasound machine and a neonatal transducer (pulse repetition frequency = . hz, wall motion filter = low , and gain = . ). three -d datasets were acquired at each flow rate from each placental lobule and these were measured in triplicate using vocal. a sphere was centred on a visibly perfused cotyledon along the chorionic-decidual axis, with a diameter corresponding to placental thickness. linear regression analysis was used to assess the relationship between the total fetal-side flow and mean vi. the mean vi showed a high degree of correlation with total fetal-side flow for each lobule ( figure ) suggesting increased vascular perfusion and the inclusion of perfused vessels that cross the detection threshold with increased flow. this data provides qualifying information for translation to a clinical application, where early gestational fetoplacental blood flow will be assessed to predict the onset of fetal growth restriction. anthony n imudia, brian a kilburn, anelia petkova, samuel s edwin, roberto romero, d randall armant. objective survival of first trimester cytotrophoblast cells depends on their upregulation of heparin-binding egf-like growth factor (hbegf), which is downregulated in placental tissues diagnosed with preeclampsia. we have examined the expression and cytoprotective activity of hbegf in term villous explants subjected to hypoxic stress in vitro. non-pathological placentas were collected by cesarean section at term (n= ). chorionic villous explants were prepared and cultured at either % or % o and treated with the hbegf antagonist crm or recombinant hbegf. paraffin sections were assayed for trophoblast cell death by the tunel assay, proliferation by immunohistochemical labeling of nuclear ki and hbegf expression by semi-quantitative immunohistochemistry. data were compared using anova and the student-newman-keuls posthoc test. results crm ( mg/ml) increased trophoblast cell death after culturing villous explants h at % o (p< . ), but only slightly affected proliferative capacity. culture at % o increased trophoblast cell death % above explants incubated at % o (p< . ). trophoblast cell proliferation decreased after h in explants cultured at either % or % o (p< . ). exogenous hbegf ( nm) prevented the elevation of cell death during hypoxia (p< . ) and maintained nuclear ki expression at %, but not %, o . contrary to first trimester trophoblast, hbegf was not upregulated by hypoxia in term trophoblast. the failure of term trophoblast to elevate hbegf expression in response to hypoxia could contribute to their decreased survival at low o compared to early gestation. endogenous hbegf signaling appears to facilitate survival of term trophoblast during villous explants culture. exogenous hbegf supplementation prevented cell death due to hypoxia and maintained trophoblast proliferation rates under in vitro conditions. therefore, hbegf, which is downregulated in preeclampsia, could have significant impact on trophoblast survival during late gestation. supported by the intramural research program of nichd. conspicuous meconium-laden macrophages in the chorionic plate are an independent predictor of clinically significant fetal distress. violetta kolesnikova, emily king, carrie tillotson, jean-marie guise, terry morgan. pathology; center for biostatistics; obstetrics and gynecology, ohsu, portland, or, usa. background: meconium is a common indication for placental examination, present in approximately % of placentas routinely submitted to pathologists (beebe et al. obstet gynecol ; : - ) . prolonged meconium exposure leads to accumulation of meconium-laden macrophages in the chorionic plate (estimated to require at least hours). our objective was to test whether prolonged meconium exposure is associated with clinically significant fetal distress. design: retrospective review of consecutive singleton placentas submitted to ohsu pathology ( - ) was performed, excluding abortions before weeks gestation, and placentas without representative sections of the chorionic plate. clinical records were reviewed and pregnancy outcomes recorded, including: ) evidence of fetal distress prompting c-section (non-reassuring fetal heart rate), ) gestational age, ) maternal diagnosis, ) apgar scores, and ) neonatal length of hospital stay. routine histologic sections were independently scored by two pathologists while blinded to clinical diagnoses and outcomes. cases were scored as positive or negative for: ) diffusely conspicuous meconium-laden macrophages in the chorionic plate (at least / hpf), ) chorioamnionitis, and ) features of placental insufficiency. significant associations were tested by the mann-whitney u test for paired comparisons, x analysis, and logistic regression for multiple variables. results: conspicuous meconium staining of the chorionic plate was common ( / , %) and was significantly more frequent in c-sections performed for fetal distress ( / cases, %) (x . ; p-value < . ). logistic regression modeling showed that this association was independent of chorioamnionitis and features of placental insufficiency. there was no association between meconium and neonatal outcome, including no difference in apgar scores or length of hospital stay. conclusions: our data support the hypothesis that meconium-laden macrophages in the chorionic plate are associated with fetal distress prompting c-section. whether meconium is the cause or consequence of this distress is uncertain. however, given the acute time frame between clinical diagnosis and c-section, we suspect prolonged meconium exposure may be a significant cause of non-reassuring fetal heart rate changes. apoptotic index in normal and intrauterine growth-restricted rat placentas. elissa scotland, tri nguyen, s chiang, radmila runic. dept. of ob/gyn, harbor-ucla med. ctr., torrance, ca, usa. objective: intra-uterine stress caused by maternal food-restriction may have adverse fetal and placental effects. we sought to assess the effect of maternal food restriction during pregnancy on placental apoptosis. methods: rat placentas were analyzed from control dams fed ad libitum and dams % food-restricted from day of gestation. placentas were harvested at embryonic days and (n= ). placentas were fixed in % pfa and two methods of analysis were utilized to determine the proportion of apoptotic to non-apoptotic cells within maternal food restricted and control rat placentas: immunohistochemistry (ih) using fas-ligand and in situ tunel (terminal deoxynucleotidyl transferase biotin-dutp nick end labeling). tunel: after rehydration, slides were subjected to tdt enzyme. dab was used to visualize brown apoptotic nuclei. dna-se treated slide was used as a positive control. ih: primary rabbit polyclonal fas-ligand antibody was used at : dilution, after which secondary antibody linked to peroxidase and dab staining was performed. results: food-restricted placentas demonstrated . % relative apoptotic index when compared to . % in the control group. the iugr iod (integrated optical density) per unit area in the placental membrane was higher than in the control group. fasl demonstrates an iod of . + . in food restricted placentas as compared to . + . per μm surface area in controls. apoptosis was seen in the amnion as well as trophoblast (syncytialtrophoblasts and some cytotrophoblasts). conclusion: the increased apoptotic index in maternal food restricted placentas suggests that the accompanying iugr may be a result of both maternal/fetal nutrient restriction and increased fetal stress. this study found that maternal food restriction during pregnancy affects placental apoptosis. there is more apoptosis in the iugr placental bed at e than at e , with the reverse being true for the placental membrane. more research is required to statistically validate the data. the suggested next step is to evaluate fas and fas-l and to address possible mechanisms of placental apoptosis. cord. jayaraman lakshmanan, avish arora, lilit baldjyan, sharon k sugano, olga miadel, adegoke adeniji, michael g ross, calvin j hobel. ob-gyn, harbor-ucla medical center, torrance, ca, usa; ob-gyn, cedars-sinai medical center, los angeles, ca, usa. background: crf-bp is a kda protein, that specifically binds corticotrophin releasing factor (crf). the structure of cloned crf cdnas in all species examined predicts that the precursor is larger than the kda in size and contains one n-glycosylation site. marked reductions in plasma crf-bp levels seen in pregnant women prior to both preterm and term delivery led to the notion that crf-bp is a "gatekeeper" of crf responses. recently we identified crf-bp expression in term human umbilical cord (uc) by immunohistochemical analyses. objective: to characterized the expression of crf-bp by immunohistochemical and biochemical analyses in human uc. methods: freshly obtained human preterm ( to < weeks, n= ) umbilical cord ( pieces of mm thickness taken at - cm intervals close to placenta) were fixed in bouin's solution and paraffin embedded. also, pieces of ucs were dissected, arteries and vein separated, weighed and frozen at - c. for immunohistochemical localization, uc sections were subjected to immunostaining with polyclonal antibodies to human crf-bp precursors. for western blot analyses, whole uc as well as isolated arteries and vein were homogenized in a buffer containing detergents and protease inhibitors. the homogenate supernatant proteins were subjected to western analyses by standard protocol. immunoreactive protein bands were identified by chemiluminescent reagent. results: both goat and rabbit crf-bp polyclonal antibodies elicited weak to moderate positive staining in uc epithelial layers, vascular musculature and barely endothelial cells. they identified a strong kda band in whole uc as well as in isolated artery and venous preparations. in addition minor immunoreactive protein bands of , , kda in size were noticed in all three preparations. conclusion: we conclude that preterm uc expresses crf-bp. we postulate that the kda major band is either glycosylated crf-bp precursor or glycosylated kda mature protein. the low molecular weight immunoreactive proteins likely represent proteolytically processed, glycosylated crf-bp precursor or a proetolytically processed mature da crf-bp. uc obtained at delivery could be useful as a tool to understand the critical functions of crf-bp in feto-placental unit. objective: adenosine, known to be released from inflammatory sites and tisse ischemia, has many important biologic roles. four specific adenosine receptors have been cloned to date, termed a , a a, a b, and a . recently our study has shown that increased a receptor in the trophoblast of preeclamptic pregnancy was noted and non-vascular and trophoblast-mediated a receptor may play an important role in the pathogenesis of preeclampsia. there are evidences of impaired trophoblast invasion related to matrix metalloproteinase (mmp) in preeclampsia and the relationship between adenosine receptor and mmp in other fields. the objective of this study is to evaluate the effect of mmp expression by adenosine a receptor in preeclamptic villous explants at different oxygen conditions. methods: placental villous explants from normal (n= ) and preeclamptic (n= ) pregnancies were cultured at high ( %) and low ( %) oxygen levels for days. explants were analyzed for mmp- /- and timp- /- by rt-pcr and western blot. preeclamptic villous explants in hypoxic culture condition were treated with a receptor agonist, cl-ib-meca and a receptor antagonist, mre. mmp- / - expression was determined in a time-and dose-dependent manner by rt-pcr, western blot. also mmp- /- activity was evaluated by zymogram assay. results: there were significantly increased a receptor intensity and reduced mmp- /- and timp- /- expression at low oxygen level in normal and preeclamptic villous explants. interestingly, in preeclamptic villous explants, after high oxygen culture mmp- /- and timp- /- expression were recovered to almost same level compared to those in normal villous explants. treatment of preeclamptic villous explants with cl-ib-meca in low oxygen level resulted in a time-and dose-dependent enhanced expression of mmp- /- . this cl-ib-meca-induced expression of mmp- /- was inhibited by pretreatment with mre. conclusion: to our knowledge, this study is the first to evaluate modulation of mmp secretion by adenosine a receptor in preeclamptic villous explant. our results provide evidence for the existence of functional adenosine a receptors in the trophoblast and suggest that adenosine a receptor will be investigated as a therapeutic target in preeclampsia. murray d mitchell, timothy a sato, anderson wang, jeffrey a keelan, anna p ponnampalam, michelle glass. liggins institute; department of pharmacology and clinical pharmacology, university of auckland, auckland, new zealand. introduction: it is well established that prostaglandins play critical roles in multiple aspects of pregnancy and that the fetal membranes are an important site of intrauterine prostaglandin production. endocannabinoids have been implicated in the maintenance of pregnancy and parturition in women and are a source of arachidonic acid which is a substrate for the production of prostaglandins. the aim of the present study was to determine the effects of endocannabinoids on the production of prostaglandins in extraplacental membranes -amnion, chorion and decidua. methods: explants of term amnion and choriodecidua were established and treated with endogenous endocannabinoids -arachidonoyl glycerol ( ag) and anandamide (aea) and with the synthetic cannabinoid cp , , to determine the ability of these substances to modulate prostaglandin e (pge ) production. pge was measured by radioimmunoassay. the explants were also treated with cp , in the presence of either sr a (a selective antagonist of the cannabinoid receptor cb ) or ns (a cox- inhibitor), to determine whether any observed stimulation of pge production was mediated through cox- activity and/or the cb receptor. cox- , cox- , cpla and pgdh protein levels were measured by western blotting. results: all three cannabinoids caused a significant increase in pge production in amnion but not in choriodecidua. however, separated fetal (chorion) explants responded to cannabinoid treatment in a similar manner to amnion, whereas maternal (decidual) explants did not. the enhanced pge production caused by cp , was abrogated by co-treatment with either sr a or ns , illustrating that the cannabinoid action on prostaglandin production in fetal membranes is mediated by cb agonism and cox- . preliminary data from western blotting show that cannabinoid treatment results in the up-regulation of cox- expression. however, there was no change in cox- expression and no evidence either for up-regulation of cpla or for down-regulation of pgdh expression. conclusion: this study demonstrates a potential role for endocannabinoids in the modulation of prostaglandin production in late human pregnancy, with potentially important implications for the timing and progression of term and preterm labour and membrane rupture. inactivation of vegf receptor- , but not vegfr- or vegfr- , during the peri-implantation period prevents normal pregnancy development in the rodent through disruption of uterine angiogenesis. nataki c douglas, hongyan tang, raul gomez, bronislaw pytowski, daniel j hicklin, jan kitajewski, mark v sauer, ralf c zimmermann. division of reproductive endocrinology and infertility, columbia university, new york, ny, usa; imclone systems, inc., new york. objective: vegf is involved in the regulation of uterine angiogenesis and implantation in both rodents and non-human primates. vegfr- , r- , and r- are expressed in the uterine decidua and are involved in the regulation of vessel formation in many systems. to determine if these receptors have a functional role in the regulation of post-implantation angiogenesis and pregnancy development, we examined the effects of blocking vegfr- , r- , and r- function. design: prospective animal laboratory material and methods: to avoid effects of vegf receptor neutralization on ovarian function, we utilized a progesterone replaced, ovariectomized mouse model. vegf receptor blocking antibodies were administered on ed . , prior to embryonic expression of these receptors. embryonic development was evaluated on ed . , blood vessel density and apoptosis on ed . , and cellular proliferation on ed . (n= per time point in each group). anova with bonferroni correction was used to compare sample means. results: see tables. conclusions: neutralization of vegfr- and vegfr- , but not vegfr- resulted in a significant reduction in cellular proliferation and decidual angiogenesis. vegfr- mediates decidual angiogenesis, but is not required for normal pregnancy development. in contrast, an intact vegf/vegfr- pathway is required for the decidual angiogenesis that mediates early pregnancy development. effect of vegfr neutralization on ed . control anti r- anti r- anti r- no. of implantation sites . +/- . +/- . . +/- . . +/- . hoxa encodes a transcription factor required for endometrial receptivity and embryo implantation. our objective was to identify and to characterize those molecular markers regulated by hoxa in multiple cellular model-systems. using microarray technologies, we identified putative hoxa target genes involved in early implantation. liposome-mediated transfection delivering either empty vector or the same plasmid constitutively expressing hoxa was introduced into newly impregnated mice during laparotomy or layered onto cultured human endometrial stromal-cells (hescs). rna products from these in vivo and in vitro transfections were used to identify targets and to validate the microarray screen employing semi-quantitative real-time pcr (qrt-pcr). we identified statistically-significant genes regulated by hoxa overexpression of which genes were down-regulated greater than -fold when compared to controls. cellular ontogenies of differentially-expressed genes include: cell adhesion molecules, signal transduction factors as well as metabolic regulators. furthermore, we identified the -phosphoglycerate dehydrogenase gene, (pgdh) whose products are regulated by hoxa during implantation in both murine model systems in and cell culture. this genes codes for an enzyme critical to de novo l-serine biosynthesis via a phosphorylation-dependent pathway. microarray analysis demonstrated a fold expression decrease when hoxa is overexpressed. this diminution in pgdh expression was noted in the validation experiments using qrt-pcr and corroborated in hesc cells where the mrna levels decreased to % when compared to controls. the repression of pgdh during the implantation window may represent a conservation of activity as secretory-phase protein synthesis may be suppressed in order to promote cellular differentiation and resultant implantation. these regulatory relationships identified in mouse implantation likely function to enhance uterine receptivity and may have a role in human implantation. objective: hoxa is expressed in endometrium, where it is regulated by sex steroids and is necessary for endometrial receptivity and implantation. hoxa is also expressed in leukocytes. here we hypothesized that hoxa would be regulated by sex steroids in both cell types. we further hypothesized a correlation between expression of hoxa in peripheral blood cell (pbcs) and endometrium in both mice and in humans. methods: real-time pcr was used to determine differential expression of hoxa mrna in u cells, a monomyelocytic cell line, in response to increasing concentrations of estradiol. to determine if hoxa is expressed in leukocytes in vivo, peripheral leukocyte hoxa mrna expression was measured over sequential estrus cycles in mature cd nulliparous mice and correlated to vaginal smear-cytology. additionally, peripheral leukocytes were isolated either from pregnant or normally-cycling women to assess hoxa mrna expression. results: there was a direct, dose-responsive correlation between exposure to increasing estradiol and hoxa mrna expression levels in u cells. in a murine model, we demonstrated that hoxa mrna expression-levels varies throughout the estrus cycle with a marked increase in expression following vaginal plug detection. the nadir of hoxa expression is prior to proestrus and increased up to -fold during the receptive phase. this increased expression continues throughout gestation. the heightened expression in murine leukocytederived hoxa mrna also is demonstrated across species. our preliminary data suggests that the greatest fold-increase of expression occurs during the window of implantation of the secretory phase in normal, cycling women. this level was sustained in pregnancy. there appears to be a trend with the highest levels of expression associated with viable gestations. women with attenuated expression profiles had non-viable gestations. conclusions: hoxa expression is regulated by sex steroids in both leukocytes and endometrium. the temporal pattern of peripheral hoxa transcript expression demonstrated in mice and humans mimics the differential rna expression documented within the uterus. leukocyte hoxa expression during the reproductive cycle in mice and humans is a marker of endometrial receptivity. peripheral leukocyte expression of hoxa mrna may correlate with implantation success. carolien m boomsma, annemieke kavelaars, marinus jc eijkemans, gijs teklenburg, bart cjm fauser, cobi heijnen, nick s macklon. reproductive medicine and gynaecology and laboratory of psychoneuroimmunology, university medical center, utrecht, netherlands. the purpose of this study is to assess the association between the intra-uterine cytokine expression profile at the time of embryo transfer and successful embryo implantation following ivf/ icsi treatment. materials and methods women undergoing ivf/ icsi underwent endometrial secretion aspiration prior to embryo transfer. known soluble mediators of implantation were measured using a multiplex immunoassay, namely il ß, il , il , il , il , il , il , il , tnf , vegf, ifn , eotaxin, mcp- , ip- , dkk- and hbegf. mif was determined using an elisa. the total protein concentration was measured for normalization purposes. data were log transformed to obtain normal distribution. multivariable logistic regression analysis with a backward elimination procedure (p< . ) was used, potential confounders (age, blood contamination, embryo quality) were included in a forward stepwise model. ten mediators of the analysed were detectable in - % of the samples. il was detectable in % of samples, dkk- %, il- %, il- %, il- % and hbegf was detectable in % of samples. ifn-was not detectable in any of the samples. multivariable logistic regression showed only logmif concentrations to have a significant correlation with achieving clinical pregnancy (p = . ). higher mif concentrations were correlated with a higher chance of conceiving. endometrial secretion analysis represents a novel means of assessing the intra-uterine milieu encountered by the embryo and offers new perspectives in the study of endometrial receptivity. in this large prospective study assessing an array of cytokines, mif was found to be significantly correlated with pregnancy. mif, macrophage migration inhibitory factor, is a cytokine with numerous proinflammatory, immunomodulatory, angiogenic and tissue remodelling properties. mif induces the synthesis and secretion of matrix metalloproteinases by endometrial cells, which may contribute to embryo invasion. its expression is particularly increased during the secretory phase, suggesting a role in reproductive processes. analysis of aspirated endometrial secretions offers a direct clinical test of endometrial receptivity which can be applied during treatment cycles without disrupting implantation. endometrial receptivity and secretory differentiation require progesterone (p). it has been hypothesized that low p levels result in delayed endometrial differentiation and infertility due to reduced receptivity. objective: test the effects of low luteal p on histologic and molecular markers of differentiation and function. methods: normal cycling women (n= ) were treated with daily leuprolide ( . mg/d) beginning in the midluteal phase and continuing through the protocol. after menses, subjects received transdermal estradiol (e, . mg/d) for days. after day of e, subjects also received daily i.m. injections of p, randomized to mg/d (sub-physiological) or mg/d (physiological). endometrial biopsy was performed after days of combined e and p treatment. additional untreated women had biopsies performed days after spontaneous lh surge. endometrial histologic dating was performed by two individuals according to the criteria of noyes et al. mrna levels were assessed using real-time rt-pcr. results: mean(s.d.) of peak and trough p serum concentrations in the mg/d p group were . ( . ) and . ( . ) ng/dl, respectively, while those in the mg/d group were . ( . ) and . ( . ) ng/dl. there were no differences between treatment groups for histologic dating; the mean(s.d.) histologic date was . ( . ), . ( . ), and . ( . ) for the mg, mg, and spontaneous cycle groups, respectively. there also were no differences among the three groups in mrna levels of ten functional markers (er , pr, integrin subunit, osteopontin, cyr , egr- , fkb , c-fos, and cd ), although variability of gene expression was greater in those who received mg/d p than in those who received mg/d p. there was also no correlation between serum progesterone level and gene expression or histologic date. conclusions: sub-physiological levels of progesterone, in the range seen in ovulatory women, do not induce detectable changes in expression of marker genes or histological dating, although low p levels were associated with greater variability of gene expression. these data suggest that abnormalities in endometrial histologic development and function likely result from intrinsic abnormalities rather than from low levels of p secretion. (supported by unc nova carta fund and nih u hd- ). endometrial ip attracts trophectoderm through cxcr interaction. simcha yagel, caryn greenfield, hen sela, jacob hanna, irit manaster, orna singer, ronit haimov-kochman, shira natanson-yaron, diana prus, benjamin reubinoff, debra s goldman-wohl, ofer mandelboim. obstetrics and gynecology, hadassah-hebrew university medical centers, jerusalem, israel; lautenberg center for immunology, jerusalem, israel; human embryonic stem cell research center, jerusalem, israel. introduction: implantation is initiated in part by attraction of the blastocyst to the endometrium lining the uterus. we hypothesized that this process is partly accomplished by chemokines expressed by the endometrium interacting with chemokine receptors on the blastocyst, suggesting that they play an important role in implantation. materials and methods: chemokine receptors were characterized in jeg cells, placental villi, primary trophoblast cell culture, trophectoderm cells derived from human es cells, blastocyst trophectoderm, and st trimester placental tissue sections. expression of chemokines was tested in decidua, endometrium, and ishikawa and hec- cell lines. immunohistochemistry, intracellular staining, elisa, facs analysis, and rt-pcr were employed to characterize chemokine receptor and ligand expression. functional testing was performed using transwell migration assays and in a nude mouse model using a matrigel gel plug cell attraction assay followed by facs analysis. results: trophoblasts demonstrated expression of various chemokine receptors, most prominently cxcr and cxcr . immunohistochemistry of trophoblast from placental villi plated on matrigel expressed cxcr and cxcr as well as hla-g. noteworthy is that trophectoderm cells derived from hes cells treated with bmp- and jeg cells, and blastocyst trophectoderm expressed principally cxcr . elisa and immunohistochemistry showed that decidua and endometrium expressed chemokines ip and il . migration assays demonstrated that ip significantly attracted various trophoblast and trophectoderm cells in vitro, and in the mouse model in vivo. taken together these results demonstrate the interaction between trophoblasts and endometrial cells is mediated by cxcr and cxcr , and il and ip . these interactions are important in the attraction of trophoblasts at the feto-maternal interface. however, ip -cxcr is the most relevant to early implantation as only cxcr is expressed consistently by trophectoderm. the endometrial proteome: changes from proliferative to secretory phase. lois a salamonsen, jenny i-c chen, xian mak, peter j stanton, david m robertson, andrew n stephens. prince henry's institute of medical research, melbourne, vic, australia. global gene analyses have demonstrated major changes across the menstrual cycle, but which of these are reflected in the proteome is not known. this study aimed to globally assess proteins differentially expressed in the endometrium between the proliferative and. secretory phases. d page analysis with dige minimal dye labelling was conducted across the pi range - on endometrial tissue from either the mid-proliferative or midsecretory phases (n= /group). profiles were assessed using samespots software. differentially expressed proteins were identified using maldi-tof ms and the interrelationship of proteins examined using ingenuity software. a total of spots were detected: were differentially expressed (p < . ) with spots having an overall false discovery rate < % (q < . ). hierarchical clustering analysis revealed that these proteins lay within three main branches in the protein dendrogram. one cluster had proteins upregulated in the proliferative phase and two contained proteins up-regulated in the secretory phase. the unique protein profiles were also revealed using principle component analysis (pca): proteins clustered into two main groups, according to cycle phase. pca thus indicated similar unique protein signatures as suggested by hierarchical clustering. thirty one of the differentially expressed proteins were identified using maldi-tof ms. these proteins could be grouped into seven categories, which included structural ( ), transport ( ), regulatory ( ), membrane ( ), enzyme ( ), motor ( ) and others ( ). proteins involved in matrix assembly and those needed for subsequent establishment of secretory endometrium were up-regulated in proliferative endometrium. proteins important for cellular organisation and communication as well as products responding to environmental stress and the immune system were highly up-regulated during the secretory phase. biological pathways were constructed based on the proteins identified. the top network for secretory endometrium clustered around tgf-b. others related to inhibition of cell death / cell viability and leukocyte extravasation. these studies provide a global approach to the cyclic changes of the endometrium and highlight the complex dynamics of protein expression in human endometrium. hormonal regulation of prokineticins in the human fallopian tube: potential regulators of embryo transport. aw horne, hn jabbour, p lourenco, s wright, s battersby, arw williams, hod critchley. reproductive and developmental sciences, university of edinburgh, edinburgh, united kingdom; mrc human reproductive sciences unit, queen's medical research institute, edinburgh, united kingdom. background: understanding the factors regulating embryo transport in the fallopian tube (ft) has important clinical implications. embryo retention in the tube due to ft dysfunction is thought to lead to tubal pregnancy, a considerable cause of morbidity and occasional mortality. transport of the embryo through the ft is, for the greater part, accomplished by smooth muscle contraction. a group of multi-functional proteins and their receptors, called prokineticins, have been shown to affect smooth muscle function in other tissues, such as the intestine. the expression pattern of prokineticins, and their receptors, was examined in normal human ft obtained throughout the menstrual cycle, and the effect of the sex steroids on prokineticin expression was examined in an in-vitro model of the ft. methods: ft biopsies (n= ) and sera (for measurement of oestradiol and progesterone for endocrine staging) were collected from women undergoing gynaecological procedures for benign conditions. using a combination of quantitative taqman rt-pcr and immunohistochemistry, the mrna and protein expression pattern of prokineticins, and their receptors, were examined in the ft throughout the menstrual cycle. tubal explant culture was established using surgical tissue from the biopsies and exposed to varying concentrations, and time courses, of oestrogen and progesterone. results: prokineticin (pk ) and prokineticin receptor (pkr ) mrna are up-regulated in the progesterone-dominant mid-secretory phase. pk and pkr protein are expressed in the epithelium, smooth muscle and around the blood vessels of the ft. stimulation of tubal explant cultures with a physiological concentration of progesterone showed an up-regulation of pk and pkr . conclusions: prokineticins show temporal variation in expression in human ft and appear to be regulated by progesterone. their role in embryo transport needs to be investigated to further understanding of pregnancy complications, such as tubal pregnancy. translating mouse to human: a dynamic model of xenografted human endometrium. alex j polotsky, liyin zhu, nanette santoro, jeffrey w pollard. albert einstein college of medicine, bronx, ny, usa. in the mouse endometrium, the hormonal environment controls cellular proliferation and cell cycle activity. estradiol (e ) inhibits glycogen synthase kinase beta (gsk- ), resulting in nuclear accumulation of cyclin d and progression of the cell cycle, as well as dna replication licensing. in utero administration of the gsk- inhibitor, licl, results in epithelial cell proliferation in the absence of e (zhu, pollard. pnas. ; : ) . in this study, we derived a functional model of xenografted human endometrium to perform mechanistic studies of human endometrial proliferation. methods: human endometrial samples were obtained from volunteers aged - . immuno-compromised mice were transplanted with disaggregated/ recombined human epithelial glands and stroma under the kidney capsule. after weeks of out-growth, mice were ovariectomized, and replaced with e or licl. xenografts were harvested and processed for immunohistochemistry (ihc) and glandular labeling index (li). t test was used to compare group means. results: - % of engraftments were successful, resulting in a vascularized endometrium with characteristic architecture (a, b). hoechst staining confirmed that xenografts were made up of human cells ©. e (e) induced significantly greater proliferation compared to control (d) as assessed by ihc for ki , with li of . ± . and . ± . , respectively, p = . ). minichromosome maintenance- , a protein involved in dna replication licensing, was more sensitive for e -treated cells synthesizing dna than was ki staining (i). estrogen (g) and progesterone receptors (h) were expressed in xenotransplant tissue, the latter being up-regulated by e . licl (f) induced proliferation similar to e and greater than control (li = . ± . , p= . for e vs. licl). conclusion: xenografted human endometrium provides a dynamic model of endometrial proliferation that is well suited for translational studies. administration of licl in the absence of e induced glandular proliferation, supporting the notion that similar mechanisms are operative in human proliferation as in the mouse. gnrh analogs have been extensively used in assisted reproduction. although the main effects of gnrh analogs are via gnrh receptors on the pituitary gonadotrope, gnrh and gnrh receptors have been identified in many reproductive tissues including human endometrium, suggesting their potential action at endometrial level. in the present study, we examined the potential regulatory action of gnrha, la on sex steroid mediated gene expression in human endometrium. human endometrial surface epithelial (hes) cells and isolated endometrial stromal (esc) cells were used as in vitro models. all experiments lasted for h. the cells were treated with estradiol (e , nm, h), progesterone (p , nm, h), gnrha (la μm, h), e ( h) followed by p (last h) and gnrha plus e plus p where, gnrha added either first, second or last in order at h intervals. total rna was extracted, reversed-transcribed and subjected to real-time pcr simultaneously, measuring the expressions of il- , il- , il- , il- , il- , il- , il- , il- p , il- p , il- , ifn-and tnf . both hes and esc expressed majority of these cytokines with the exception of low to undetectable levels of il- , il- , il- and ifn-. treatment with e and p , either alone, or in combination significantly upregulated the expression of many of these cytokines at varying extend as compared to controls. however, gnrha either alone or in combination with e and p significantly diminished e , p or e plus p induced mrna expression of cytokines. the suppressive effects of gnrha on some of the cytokines varied significantly by the order which gnrha was introduced into the culture medium. we conclude that gnrha by acting directly on the endometrial cells effectively suppresses the mrna expression of several key proinflammatory cytokines upregulated by ovarian steroids. our results imply that gnrha therapy during assisted reproduction may modify endometrial receptivity via downregulation of proinflammatory cytokines induced by estradiol and progesterone. supported by nih grant hd . introduction: endometrial angiogenesis is characterized by a rapid increase during the early proliferative phase that peaks midcycle, followed by a gradual decrease in the secretory phase, while menses involves generalized endometrial inflammation, necrosis, and vascular thrombosis. vascular endothelial growth factor (vegf), whose expression is regulated by sex steroids, is a mediator of endometrial angiogenesis. recently, myoferlin, a kd transmembrane protein, was identified in endothelial cells where it mediates vegf-dependent endothelial cell proliferation, migration, and nitric oxide synthesis by affecting vegf receptor- function and stability. myoferlin also appears to play a role in vesicle trafficking and membrane repair. objective: to characterize myoferlin protein expression in endometrium. methods: western analysis of cultured human endometrial stromal cells (hesc) and human endometrial endothelial cells (heec) treated with physiologic concentrations of estradiol and progesterone was performed using a polyclonal rabbit antibody against myoferlin. subsequently, immunohistochemistry (ihc) was performed on human endometrium samples obtained from various stages of the menstrual cycle. results: myoferlin protein was expressed in hescs and heecs, but expression was not affected by sex steroids. ihc staining for myoferlin was specific, intense, and localized to the apical membrane of glandular epithelial cells and endothelial cells, with less intense staining in the stroma. h-score quantification showed that in endometrial endothelial cells, myoferlin protein expression was highest during the early proliferative and early secretory phases, while glandular and stromal myoferlin expression peaked during the late proliferative/early secretory phase. conclusion: myoferlin expression in human endometrium correlates with periods of greatest endometrial angiogenesis, and expression is not limited to endothelial cells, but also includes glandular and stromal cells. given the involvement of myoferlin in vegf signaling as well as membrane repair, understanding its role in human endometrium may further elucidate an understanding of endometrial development both under physiologic and pathologic conditions. the human embryo is the primary regulator of embryo-endometrial molecular cross talk during early implantation. gijs teklenburg, , cobi heijnen, esther baart, karima amarouchi, carolien boomsma, janet carver, helen mardon, annemieke kavelaars, nick macklon. reproductive medicine and gynaecology, and laboratory of psychoneuroimmunology, university medical center, utrecht, netherlands; nuffield department of obstetrics and gynaecology, university of oxford, women's centre, john radcliffe hospital, oxford, united kingdom. introduction: uterine receptivity and implantation are controlled by locally acting trophic factors and cytokines. in humans, the regulation of the embryo-endometrial dialogue beyond the early blastocyst stage is poorly understood. we hypothesized that the interaction between a healthy conceptus and the receptive endometrium is associated with a distinct local regulation of cytokine production favouring implantation. methods: human embryos, cryopreserved at day after fertilization and donated for research, were thawed and cultured under standard conditions until day five. forty-two embryos from donors developed to the blastocyst stage. following removal of the zona pellucida, they were placed in individual coculture on a confluent monolayer of endometrial stromal cells. on day , the developmental potential of each embryo was assessed as early arrested, late arrested or developing. culture supernatants were analysed for concentrations of il- , il- , il- , il- , il- , il- , il- , il- , tnf-, mcp- , ip- , eotaxin and hb-egf using a multiplex immunoassay. day supernatants from culture systems in which no embryo had been placed were also analysed as controls. results: out of ( %) embryos continued to develop and were able to attach and invade into the stromal cell compartment of the co-culture environment. twelve late arrested embryos showed signs of degradation on day and the totally disintegrated embryos were assigned to the early arrested group. supernatants from both early and late arrested embryo cultures contained significantly lower levels of a number of cytokines and growth factors in comparison to developing embryo cultures. moreover, the levels of these mediators in co-cultures were significantly lower than those in non-embryo control stromal cell culture supernatants. conclusion: these data suggest a pivotal role of the embryo in embryoendometrial cross talk. whether reduced mediator expression in co-cultures reflects a selective down regulation of stromal cell cytokine and growth factor production is now being investigated. epithelial cell dynamics during human implantation. hiroshi uchida, tetsuo maruyama, toru arase, masanori ono, takashi kajitani, maki kagami, hideyuku oda, sayaka nishikawa, yasunori yoshimura. department of obstetrics and gynecology, keio university school of medicine, shinjuku, tokyo, japan. epithelio-mesenchymal transition (emt) is thought to play a role in functional differentiation of endometrial epithelial cells during human implantation. molecular mechanism of epithelial sheet remodeling caused by embryo invasion remains elusive. to address this, we investigated cellular dynamics of n-cadherin and vimentin, the two representative major markers of emt, during implantation. in in vitro implantation assay using a human endometrial epithelial cell line, ishikawa, and a human choriocarcinoma cell line, jar (uchida et al., hum reprod ), we pre-treated human ishikawa cells with or without ovarian steroid hormones ( -estradiol + progesterone; ep), fa- (n-cadherin blocking ab), or suberoylanilide hydroxamic acid (saha), one of histone deacetylase inhibitors, which has a potential to improve in vitro implantation (ibid). implantation or treatment with or without ep or saha enhanced the expression of n-cadherin and vimentin but down-regulated ecadherin. furthermore, treatment with ep or saha accelerated ishikawa cell motility and increased the number and spreading area of jar spheroids. in vitro implantation assay, the most prominent staining intensity of n-cadherin was observed just around the adhered spheroid from which its intensity decreased away. functional blockade of n-cadherin by fa- resulted in the complete suppression of ishikawa cell motility, the unique distribution of n-cadherin around jar spheroids, and the spreading area of jar spheroids, while it did not affect the number of the adhered spheroids. human implantation consists of the multiple steps, including apposition, adhesion and penetration. thus, these results collectively indicate that emt may take place after the apposition and that n-cadherin may be required for the remodeling and emt of the epithelial sheet during embryo invasion. n-cadherin may enhance the recruitment of spheroid-neighboring cells, suggesting its role in the covering-up of the invading embryo through acceleration of epithelial cell motility. endocannabinoid regulation in human endometrium. jessica g scotchie, marc a fritz, steven l young. obstetrics and gynecology, university of north carolina, chapel hill, nc, usa. background: research in mouse models demonstrates two cyclically regulated endocannabinoids produced in murine endometrium, anandamide and -arachidonoyl glycerol ( -ag); both play critical roles in murine embryonic implantation. no studies of endocannabinoids in human endometrium have been performed. objectives: determine menstrual cycle expression and localization of synthetic and degradative enzymes for anandamide and ag in human endometrium. methods: human endometrium was collected from volunteers across the menstrual cycle (n= ). quantitative rt-pcr was performed analyzing the expression of: n-acylphosphatidylethanolamine (nape) and fatty acid amide hydrolase (faah), the synthetic and degradative enzymes for anandamide, respectively; sn- -diacylglycerol lipase-a (dagla), and b (daglb), the synthesis enzymes for ag; and monoacylglycerol lipase (magl) and cyclooxygenase- (cox ), the degradative enzymes for ag. the constitutive gene ppia was used for comparison. immunohistochemical localization of nape protein was performed using nape-pld polyclonal antibody (cayman chemical, # ). anova and student's t-test analysis performed on samples grouped by proliferative (pro), early, mid-, and late secretory (es, ms, ls) phases. results: mrna expression of all enzymes responsible for synthesis and degradation of anandamide and ag was detected throughout the cycle. no significant cyclic change in nape, faah, or daglb gene expression was seen. a decrease in dagla gene expression in the ms and ls phases compared to the pro and es phases (p= . ) was seen. magl gene expression was higher in the secretory phase than the pro phase (p= . ). cox gene expression was detected at low levels in the pro, es and ms phases, with marked increase in the ls phase (p< . for all comparisons). protein localization of nape showed a cytoplasmic epithelial location, with increased staining on pro and es samples compared to ms and ls samples. immunolocalization of remaining proteins is ongoing. conclusion: this is the first report documenting the presence of endocannabinoid synthetic and degradative enzymes in human endometrium. genes controlling anandamide expression do not fluctuate significantly across the cycle. however, ag's degradative enzymes increase in the secretory phase, suggesting that lower ag levels may be advantageous for embryo implantation. our findings suggest that human endometrial endocannabinoid regulation differs from murine regulation. interleukin- beta (il- ) regulates il- signaling in decidua-implication in the pathophysiology of preeclampsia (pe). sj huang, cf yen, cp chen, f schatz, cj lockwood. obstetrics, gynecology and reproductive sciences, yale university, new haven, ct, usa; ob/gyn, chang gung memorial hospital, tao-yuan, taiwan; ob/gyn, mackay memorial hospital, taipei, taiwan. objective: previously, we found much higher cytoplasmic immunoreactive il- levels in the preeclamptic decidual cells than in adjacent interstitial trophoblasts. such decidual cell-derived il- contributes to the systemic endothelial cell dysfunction that elicits the proteinuria and hypertension of the maternal syndrome. il- promotes the transition from innate to adaptive immunity. moreover, by skewing monocyte differentiation from a dendritic to a macrophage phenotype, decidual il- may promote the macrophage excess observed in the preeclamptic decidua. macrophages impair trophoblast decidual invasion to foster incomplete spiral artery remodeling that elicits placental ischemia and hypoxia. the current study: ) localized il- mrna levels in preeclamptic versus normal decidual sections; ) evaluated mechanisms regulating il- synthesis by targeting intracellular signaling pathways with specific inhibitors; ) identified potential il- targets by immunolocalizing the il- receptor (il- r) to specific cell types in placental bed biopsies. methods: in situ hybridization localized il- mrna in normal versus preeclamptic decidua. il- r was immunolocalized in placental bed biopsies. leukocyte-free first trimester decidual cells were incubated with e and mpa ± il- ( ng/ml) ± an inhibitor of p mapk (sb ) or protein kinase c (calphostin c) or nf b (activation inhibitor iii) for hrs. an elisa measured secreted il- levels. results: il- mrna was present primarily in decidual cells with increased il- mrna levels observed in pe. preferential expression of the il- r was observed on decidual cells in placental bed biopsies. compared with basal il- levels ( . ± . pg/ml/ g cell protein) by decidual cells, il- enhanced il- output by decidual cells ( . ± . pg/ml/ g cell protein). only the p mapk inhibitor significantly reduced this output to . ± . pg/ml/ g cell protein (n= , p< . ). our results indicate that inflammatory cytokine enhances il- synthesis in decidual cells of the preeclamptic decidua by a mechanism involving p mapk. such il- is likely to act as an autocrine/paracrine effector via decidual cell-expressed il- r to contribute to the macrophage excess observed in the preeclamptic decidua. heparanase is up-regulated by estrogen and during the secretory phase of the human endometrium. ronit haimov-kochman, shira natanson-yaron, caryn greenfield, achinoam lev-sagie, lichtenstein michal, haya lorberboum-galsky, israel vlodavsky, simcha yagel, arye hurwitz. ob/ gyn, jerusalem, israel; cellular biochemistry human genetics, hadassah hebrew university medical centers, jerusalem, israel; cancer and vascular biology research center, technion school of medicine, haifa, israel. introduction: heparanase is an endoglycosidase that cleaves heparan sulfate (hs) proteoglycan of the extracellular matrix. the full-length proheparanase is activated by cleavage into an active isoenzyme, resulting in the release of hsbound cell-differentiation factors, such as hb-egf. the cycling endometrium involves remarkable steroid hormone-induced tissue remodeling. in vivo, increasing exposure to unopposed estrogen may lead to endometrial malignant transformation. aim: to investigate heparanase expression and regulation in the cycling endometrium. materials and methods: heparanase mrna levels were measured by quatitative rt-pcr in naturally menstruating women and in hec a, estrogen receptor (er)-negative and ishikawa, er-positive endometrial carcinoma cell lines exposed to increasing doses of estradiol. heparanase isoenzymes were localized by immunohistochemistry using specific anitbodies in murine endometrium and human normal, hyperplastic and malignant endometrium. results: heparanase mrna level increased fold in secretory phase (d ) compared to proliferative phase (d ) endometrium. heparanase transcript levels increased fold during hr culture in er positive adenocarcinoma cell line exposed to increasing doses of estradiol, but not in hec a, er negative cell line. both heparanase isoforms were localized to murine glandular endometrium. human glandular endometrium at both proliferative and secretory phases was immunoreactive with the active isoform of heparanase. proheparanase was detected in basal membrane of endometrial glands and endometrial stroma during secretory phase. along with malignant transformation of the endometrium the presence of proheparanase increased dramatically from none in stroma of normal and hyperplastic endometrium to abundance in malignant tumors. conclusions: heparanase gene expression is higher during the window of implantation and up-regulated with estrogen in endometrial cells via er in vitro and vivo. heparanase is differentially localized in the secretory phase of the endometrium compared to the proliferative phase, suggesting a role for this molecule during the window of implantation in man. interleukin- (il- ) system mrna and protein expression in the human fallopian tube with ectopic implantation. hong-yuan huang, , tien-hung huang, chin-jung li, chyi-long lee, , hsin-shih wang, , yung-kuei soong. , obstetrics and gynecology, chang gung memorial hospital, kwei-shan, tao-yuan, taiwan; obstetrics and gynecology, chang gung university and school of medicine, kwei-shan, tao-yuan, taiwan. objective: ectopic pregnancy, an abnormal implantation of a fertilized ovum outside the uterine cavity, has been increasing in number at a staggering pace of all pregnancies. il- system is one of the major cytokines involved in human endometrium during embryo implantation and might perform a defensive role against maternal immune response. very little information is available regarding the expression and synthesis of cytokines in the pathogenesis of fallopian tube with ectopic gestation. the purpose of this study is to investigate il- system expression in human fallopian tubes with ectopic pregnancy. methods: paired segments of human fallopian tubes with ectopic implantation site and side portion close to ectopic gestation (n= ) were collected from women undergoing laparoscopic salpingectomy after informed consent and irb approval. segments of fallopian tubes from women undergoing tubal ligation (n= ) were used as control groups. total extracted rna was reverse transcribed and amplified by pcr using specific primers for gapdh ( bp), il- ( bp), il- bp ( bp) and il- r ( bp). quantitative il- and il- bp mrna expression in human fallopian tube was determined by real-time pcr. to determine the presence of il- system proteins, tissues were fixed and processed for immunohistochemical study. data analysis was done with anova and pearson's correlation. results: il- and il- bp as well as il- r mrna were all expressed in tubal ectopic implantation and normal tubes. according to real-time pcr with c t value quantification and -ct method, a significantly higher il- expression in tubal ectopic implantation and lower ratio of il- antagonist to agonist in portion close to ectopic implantation is demonstrated in comparison to normal tubes (p< . ). immunoreactive il- system at the protein levels was also present in human fallopian tubes with ectopic implantation and normal tubes. conclusions: these results suggest that fallopian tube il- system expression may play a crucial role during the process of early embryonic implantation. the expression and ratio of antagonist to agonist in fallopian tubes may indicate an earlier "dialogue " in human fallopian tubal gestation prior to uterine implantation. replacement. marcia c ferreira, ines kd cavallo, fernando m reis. gynecology, ufmg, belo horizonte, mg, brazil. activin a is a growth factor expressed in the endometrium, where it modulates tissue remodelling and enhances decidualization. the effects of activin a are counteracted by two binding proteins, namely follistatin and follistatin-like (fstl ). while the endometrial expression of activin a increases during the secretory phase of menstrual cycle, the effects of ovarian steroids on these proteins and their mrnas has not been assessed yet in postmenopausal women or in ovariectomized animals. we have evaluated the effects of estrogen alone or estrogen plus progestin on the endometrial expression of activin beta-a subunit, follistatin and fstl in ovariectomized rats. adult female wistar rats (n= ) were ovariectomized and received one week later a single dose of estradiol benzoate ( . mg/kg body weight, i.m. injection), either alone (n= ) or associated with depot medroxyprogesterone acetate ( . mg/kg body weight, i.m. injection, n= ), or oil vehicle (control group, n= ). one week after the hormone or placebo treatment, the animals were sacrificed and their uteri were removed and processed by immunohistochemistry and real-time pcr. data were normalized to the expression of ribosomal phosphoprotein p (rpp ) and analyzed with the delta-delta ct method, anova and newman-keuls test. activin beta-a subunit mrna levels increased significantly in the uteri of rats treated with estradiol alone ( . fold increase over controls, p< . ) and to the same extent in rats receiving estradiol plus medroxyprogesterone ( . fold increase over controls, p< . ). this was accompanied by increase of beta-a subunit immunostaining in estradiol and estroprogestin-treated rats, which was noted only in the surface endometrial epithelium. follistatin mrna expression, conversely, showed a significant decrease in the groups treated with estrogen alone ( . fold compared to controls, p< . ) and estrogen plus progestin ( . fold compared to controls, p< . ), while follistatin immunostaining in the glandular epithelium was weaker in estradiol and estroprotestin-treated rats compared to controls. fstl expression was similar in the groups. in conclusion, the expression of activin beta-a subunit increases and that of follistatin decreases following estrogen replacement in the endometrium of ovariectomized rats, and these effects are not further altered by the addition of progestin. endometrial nk cells are a unique inert nk subset until pregnancy. simcha yagel, irit manaster, jacob hanna, ronit haimov-kochman, miri godin, yuval bdolach, caryn greenfield, shira natanson-yaron, arye hurwitz, debra s goldman-wohl, ofer mandelboim. obstetrics and gynecology, hadassah-hebrew university medical center, jerusalem, israel; lautenberg center for tumor immunology, hadassah-hebrew university medical center, jerusalem, israel. introduction: we recently demonstrated that nk (natural killer) cells play a critical role in trophoblast migration and angiogenesis at the fetal maternal interface. nk cells populate the endometrium at the secretory phase of the menstrual cycle, the time of anticipated blastocyst implantation. peripheral blood (pb) nk cells and decidual nk (dnk) cells express a variety of activating receptors, including nkp , nkp and nkp , collectively known as natural cytotoxicity receptors (ncrs), and nkg d which regulate nk cell killing and growth factor production. to compare endometrial nk cell (enk) activating receptor expression and function to pbnk and dnk cells and endometrial ligand expression, with a focus on their roles in blastocyst implantation. patients and methods: subjects were ivf patients undergoing natural menstrual cycles. endometrium samples were collected on treatment days and . a lymphocyte profile of the endometrial cells and pb was performed. facs analysis was performed on isolated endometrial nk cells, pbnk cells and dnk cells for cd , cd , nkp , nkp , nkp and nkg d. ncr ligand expression was characterized on adherent endometrial cells using ncr-ig fusion proteins and nkgd -ig and nkg d specific ligands as well as control ccmi-ig. redirected killing assays and cytokine secretion assays of ifn , vegf, plgf, and il- with and without il- were performed. results: endometrial lymphocytes of day and in these women are mostly cd bright cd -nk cells, with a significant amount of t cells, similar to pbnk cells and in marked contrast to dnk cells. unlike pbnk and dnk cells, enk receptors do not express nkp , nkp . nkp and nkg d are the only activating enk receptor expressed. like decidual cells, adherent stromal endometrial cells expressed the ligands for nkp , nkp and nkg d, suggesting that these nk cells have potential for activation. finally enk cells could not kill or secrete cytokines. conclusions: these findings of a unique activating receptor profile on endometrial nk cells, unlike that of dnk and pbnk, suggest that enk cells are a special local population of nk cells that change dramatically and are activated at the onset of pregnancy. variation in platelet activation throughout the menstrual cycle. fiona c denison, amy o robb, imogen b smith, nicholas l mills, hilary od critchley, david e newby. centre for reproductive biology; centre for cardiovascular sciences, the university of edinburgh, united kingdom. background: platelet-monocyte aggregation (pma) is a sensitive and novel measure of platelet activation with important proinflammatory consequences including release of cytokines and chemokines. previous studies using less sensitive techniques suggest that platelet activation alters during the menstrual cycle in response to circulating concentrations of sex steroids. the effect of sex steroids on circulating (c) pmas during a single menstrual cycle is not known. objective: to determine whether cpmas, platelet surface (ps) p-selectin and plasma (p) p-selectin vary through the menstrual cycle in response to changes in circulating sex steroid concentrations. methods: healthy, nulliparous, pre-menopausal, non-smoking women (mean age years), with regular menses ( - days) were studied. subjects gave written informed consent and the study had ethical approval. serial venous blood samples were taken at menstrual, follicular, periovulatory and luteal phases of a single cycle (days - , - , - and - ). cpmas (monocytes positive for the platelet marker cd a) were measured by flow-cytometry. psp-selectin expression was calculated on cd a positive cells. isotype-matched controls were used. serum oestradiol (e) and progesterone (p), plasma and pp-selectin were measured by elisas. data were analysed by one-way anova with repeated measures and bonferroni's post-tests for multiple comparisons. results: luteal phase p was > nmol/l in all women. numbers of cpmas and expression of psp-selectin were both significantly higher during menstrual compared with periovulatory phase of the menstrual cycle ( . ± . vs. . ± . %, p= . and . ± . vs . ± . %, p< . , respectively). there was no significant difference in pp-selectin concentration during the menstrual cycle (p= . ). there was no correlation between levels of serum e or p and numbers of cpmas, expression of psp-selectin or pp-selectin concentration. conclusions: numbers of cpmas and expression of pspselectin are maximal at menstruation with neither numbers of cpmas nor expression of psp-selectin correlating with serum e or p levels. this study suggests that activated platelets may potentially contribute to the inflammatory response at menstruation by releasing inflammatory mediators. by angiogenic factors and peri-cellular proteases in decidual secretory endometrium (dse), decidua parietalis (dp), and basalis (db) of miscarriage patients and matched controls. comparison of these parameters between the two groups enabled hypothesizing about their correlation with the occurrence of miscarriages. methods: decidua was obtained during st trimester termination of pregnancy (control group) and vacuum aspiration of missed abortions (case group). vascularization was studied by cd -immunohistochemistry. the expression of vascular endothelial growth factor-a, placental growth factor, flt- , kdr, angiopoietin- , angiopoietin- , tie- and the membrane-type matrix metalloproteinases mt -, mt -, mt -and mt -mmp were determined at mrna and antigen level and cd -positive unk cells, cd -positive macrophages, proliferation (ki ) and apoptosis (activated caspase- ) were evaluated by immunohistochemistry in consecutive serial sections. results: the decidual vascularization pattern showed differences between cases and controls: i.e. fewer vessels with larger circumference in cases, and this correlated with the differential expression of various angiogenic factors and proteases at mrna and antigen level. moreover, the endothelial protein expression of flt , kdr, mt -and mt -mmp was increased at the implantation site of cases. ki and active caspase- showed similar levels in the two groups and also the immune cells, both unk cells and macrophages, showed no differences at the implantation site between both groups. conclusion: differences between cases and controls appeared not to be based on altered proliferation, apoptosis, and/or inflammation. the differences in vascularization pattern and in the expression of angiogenic factors and proteases between both study groups suggest a correlation between decidual vascularization and the occurrence of miscarriages. respond to adrenomedullin. yaun-lin dong, hong y wen, janice endsley, alison hogg, hui-qun wang, manubai nagamani, chandra yallampalli. background: natural killer (nk) cells are the predominant lymphocytes present in human implantation site. decidual nk cells express perforin, an essential molecule required for lysis. formation of the placenta involves cooperation between maternal nk cells and fetal trophoblast cells that remodels the blood supply; however, the interaction between trophoblasts and decidual nk cells is largely unknown. adrenomedullin (adm) has been implicated in regulating early placental function and fetal growth. objective: to determine the role of multifunctional peptide adm in the decidual nk cells and fetal trophoblast cells interactions. methods: decidual and placental tissues were obtained from normal firsttrimester pregnancies terminated for social reasons. ethical approval to use these tissues was obtained from the irb of university of texas medical branch. cell preparations containing all decidual mononuclear cells were isolated by collagenase enzymatic disaggregation. cd decidual nk cells were purified by magnetic bead isolation. results: ) immunohistochemical analysis showed that adm is expressed primarily in decidual cells and trophoblast cells at the human implantation site; ) confocal imaging analysis demonstrated that decidual nk cells, which were identified by anti-cd staining, express adm receptor components crlr/ramp /ramp and their mrna expressions were futher confirmed by rt-pcr; ) k target cell killing assay indicates that adm inhibits cytokine il- /il- -induced decidual nk cell cytotoxicity; and ) immunofluorescent labeling and flow cytometric analysis revealed that adm suppresses perforin expression by decidual nk cells. conclusion: trophoblast-derived adm inhibits decidual nk cell cytotoxicity via suppressing perforin expression, thus, our results provide evidence for a new paradigm of embryonic-maternal communication involving a adm mediated interaction between decidual nk cells and fetal trophoblasts. leandro g oliveira, gendie e lash, judith n bulmer, barbara a innes, roger f searle, stephen c robson. institute of cellular medicine, newcastle university, newcastle upon tyne, tyne and wear, united kingdom. background: we have previously demonstrated that co-culture with extravillous trophoblast cells (evt) (expressing hla-g) alters cytokine secretion by uterine natural killer (unk) cells, particularly at - weeks gestation. we have also reported that unk cells can stimulate evt invasion, but only at - weeks gestation (not at - weeks gestation). in addition, unk cell cytokine profiles alter with increasing gestational age. other reports have suggested that evt or hla-g expressing cells may alter the expression of cytokines and angiogenic growth factors by unk cells. hypothesis: hla-g expressing cells alters unk secretion of cytokines. methods: cd + unk cells were isolated from early pregnancy decidua ( - and - weeks gestation, n= each group) using enzyme digestion and positive immunomagnetic bead separation. the human b lymphoblastoid . transfected with either hla-g ( g) or a mock cdna ( cdna) were obtained as a kind gift from mr r apps (university of cambridge, uk). isolated unk cells were cultured in the presence or absence of the two cell lines in either direct or indirect contact (n= each group and each gestational age) for hours. cell supernatants were analysed for cytokines using a fastquant® th /th multiplex protein assay (il- , il- , il- , il- , il- , tnf-, il- , il- , ifn-) or by standard elisa (tgf- ). the effect of direct co-culture of unk cells with g compared with co-culture with cdna at each gestational age was tested using mann whitney u test. the effect of co-culture of unk cells with g in both direct and indirect contact was also tested using mann whitney u test. results: there was no difference in the level of cytokines secreted by the g or cdna cells. cytokine secretion by unk cells was not altered after direct co-culture with either g or cdna cells at either gestational age. in addition, direct or indirect co-culture of g or cdna with unk did not alter cytokine secretion at either gestional age. conclusions: hla-g does not alter the secretion of cytokines by unk cells from either - or - weeks gestation. other evt or decidua derived factors (including hla-e) may be responsible for the alteration in secretion of cytokines by unks with increasing gestational age. introduction: natural killer (nk) lymphocytes are central to innate immunity and contribute to tissue homeostasis by eliminating altered cells. their nkg d receptor pathway plays a fundamental role in target elimination through binding nkg d ligands on the cell surface. reduction in the nkg d ligand, ulbp , expression is associated with immune resistance in neoplastic processes. we have previously shown that fibroblasts from adhesion tissue (at) are characterized by increased extracellular matrix molecules and inflammatory cytokines compared with normal peritoneal (np) fibroblasts. objective: to determine if there is a difference in nk lymphocyte-mediated elimination between np and at fibroblasts and to investigate potential role of nkg d pathway in this process. material and methods: expression of nkg d ligands; ulbp , ulbp , mica, and micb was evaluated by flow cytometry and western blot in primary cell cultures of fibroblasts from np and at, established from two patients. peripheral blood nk lymphocytes (cd +cd -) from three healthy volunteers were isolated using macs system with purity greater than % and kept in interleukin overnight. fibroblast elimination with and without ulbp blocking was investigated following -hour co-incubation with allogeneic nk lymphocytes using our established flow cytometric cell mediated cytotoxicity assay. paired t test was used in statistical analysis. results: the flow cytometry studies showed that nkg d ligands (ulbp , ulbp , mica and micb) were lower in at compared to np fibroblasts, reaching a statistical significance in ulbp expression (p = . ). western blot analysis also revealed a lower ulbp protein level in at than np fibroblasts. furthermore, nk lymphocyte-mediated elimination was % lower in at in comparison with np fibroblasts. blocking ulbp expression resulted in decreased nk lymphocyte-mediated np fibroblast elimination by %, supporting the role of nkg d receptor pathway in the process. conclusions: our results demonstrate that nkg d pathway is operational in at fibroblast resistance to immune elimination, and extends our prior observations of the potential role of immunological mechanisms in the pathogenesis of adhesion development. objective: galectin- is an anti-inflammatory lectin that has pleiotropic regulatory functions at the crossroad of innate and adaptive immunity. human galectin- is expressed in the placenta and immune privileged sites and it has been implicated in establishing immune tolerance. the aim of this study was to examine the evolution and placental expression of the lgals gene in primates. methods: seven primate nucleotide sequences were generated, aligned to vertebrate orthologs from all classes and subjected to phylogenetic analysis. deduced amino acid sequences were analyzed for functionally important substitutions. placental galectin- expression was studied by immunohistochemistry and western blot. results: ) the lgals gene had high sequence identity among all investigated species. ) phylogenetic analysis revealed that intense purifying selection had been acting on the lgals gene in placental mammals (dn/ds= . ); ) residues responsible for sugar binding or molecule stabilizing were highly conserved in primates. ) immunostaining showed a uniformly abundant and ubiquitous galectin- expression pattern in human, old and new world monkey and prosimian placentas, regardless the type of placentation. the lgals gene has conserved sequence and placental expression pattern in primates that may suggest its important function in maternal-fetal immune interactions. these results support the view that immune interactions at the maternal-fetal interface evolved in concert with invasive placentation and that these interactions have been maintained regardless of the degree of placental invasion in primates and other mammals. expression of interleukin- in human endometrium throughout the menstrual cycle and early pregnancy. yesim h uz, , william murk, umit a kayisli, aydin arici. department of obstetrics, gynecology and reproductive sciences, yale university school of medicine, new haven, ct, usa; department of histology and embryology, trakya university school of medicine, edirne, turkey. background: interleukin- (il- ) is a recently discovered heterodimeric cytokine, comprised by a novel p subunit and a p subunit shared by il- . it has biological activities that are similar to but distinct from il- , and is known to be involved in th /th cell class switching and the regulation of cytokines such as ifn-gamma, il- , tnf-alpha, and il- . early pregnancy is associated with alterations in the maternal immune response, such as changes in cytokine expression, and leukocyte recruitment and subtype switching. we hypothesized that expression of il- in the human endometrium is menstrual cycle-and pregnancy-dependent. materials and methods: endometrial samples from women (n= ) undergoing surgery for benign gynecologic conditions, and decidual tissues from women (n= ) with clinically normal pregnancies terminated voluntarily in the first trimester, were obtained after receiving informed consent. endometrial samples were grouped according to menstrual phase. paraffin sections were stained with il- p antibodies and evaluated semi-quantitatively with hscore. statistical analysis of the data was done using anova, with p< . considered significant. results: il- immunoreactivity was predominantly located in the cytoplasm of both endometrial stromal (esc) and glandular (egc) cells. escs showed mild il- immunoreactivity without significant changes in intensity throughout the menstrual cycle. on the other hand, first trimester decidual cells showed significantly stronger il- staining compared to escs from non-pregnant endometrium (p< . ). il- immunoreactivity in egcs was high in the late proliferative phase, as compared to other cycle phases and first trimester tissues (p< . ). moreover, egcs from the early secretory phase (p< . ) and first trimester tissues (p< . ) showed higher il- immunoreactivity compared to the early proliferative and late secretory phases. conclusions: this is the first study describing il- expression in the human endometrium and decidua. these results suggest that il- has a cycledependent expression in endometrial cells and may be involved in regulating cytokine expression and immune cell modulation during the menstrual cycle and early pregnancy. gercel-taylor, douglas d taylor. obstetrics, gynecology, and women's health, university of louisville, louisville, ky, usa. objective: estrogen appears appear to be a critical regulator of the immune system. since hypoestrogenism is present in the postmenopausal woman, our objective was to determine whether t cell activation and function, defined as il- production and signaling molecule expressions at the transcriptional and translational levels, were affected by a low estrogen environment. design: prospective study in a university research laboratory. materials and methods: jurkat . t cells, initially grown in estrogen free media, were incubated in pm (representing postmenopausal levels) or pm (premenopausal levels) of estradiol (e ) for hours. cells were either resting or activated with a phorbol ester, -phorbol -myristate -acetate (pma), and ionomycin. enzyme-linked immunosorbent spot assay (elispot) was performed to analyze production of il- . expression of signaling protein components, cd and jak, were determined by western immunoblotting. real time-polymerase chain reaction was performed to quantify cd , jak , and jak gene expression. a p value of < . was considered significant. results: jurkat cells exposed to pm e and activated exhibited significantly diminished numbers of il- producing colonies compared to t cells exposed to pm ( . ± . vs. . ± . colonies, p< . ). analysis of cellular cd and jak protein demonstrated that jurkat cells incubated in pm e expressed a . -fold decrease in cd and . -fold decrease in jak compared with cells incubated in pm e (p< . ). these diminished protein levels appeared to be the consequence of suppressed transcription, as the mrna levels of cd , jak and jak were significantly decreased in jurkat cells incubated in low levels of estrogen ( . , . , and . fold, respectively, compared to pm). conclusions: jurkat cells exposed to low postmenopausal estrogen levels produce significantly less il- following activation, which was associated with a significant decrease in signaling proteins. the diminished levels of signaling proteins appear to result from decreased cd , jak and jak gene expression in the presence of low estrogen. these findings support the observation of decreased cellular immune response in postmenopausal women and may provide a basis for the increased risk of infections and cancer proliferation associated with aging. support: dept. of ob/gyn research seed fund. the expression pattern of novel cytokines (il- and ) in human fetal membranes. judith eckardt, , stephen j fortunato, holger maul, ramkumar menon. the perinatal research center, nashville, tn, usa; womens' hospital, university of heidelberg, heidelberg, baden-wuerttemberg, germany. objective: interleukin (il) and are novel cytokines produced by various immunological cells in response to microbial antigens. the functions of these cytokines in reproductive system is unknown. this study examines the expression pattern of il- and il- in human fetal membranes from preterm and term births and in in vitro in normal term membranes in response to bacterial endotoxin (lipopolysaccharide-lps). methods: fetal membranes collected (n= ) from cesareans at term (normal, not in labor) were placed in an organ explant system for hours and were stimulated with lps for an additional hrs. fetal membranes were also collected (n= ) either at preterm or term after vaginal deliveries. in a case -control study (preterm birth vs. normal term deliveries) amniotic fluids (af) (n= ) were collected to document the role of il- and il- in ptb. tissue expressions of il- and il- were studied by rt-pcr using specific primers. elisa documented culture media and af cytokine concentrations. statistical analysis was performed using non-parametric mann-whitney u test. results: both il- and il - expressions were seen in fetal membranes in culture (in vitro) regardless of stimulation. in vivo in membranes from preterm and term deliveries and membranes at term not in labor also documented the expression of these cytokines. culture media analysis documented higher concentration of il- after lps stimulation (lps- . vs. . pg/ml; p= . ) whereas no difference was noticed in il- concentration (lps- . control- . pg/ml; p= . ) between the two groups. af analysis, regardless of the status, did not document detectable concentrations of either of the cytokines (lower limit . pg/ml for both). conclusion: this is the first study to document the expression of two novel cytokines in laboring and non laboring human fetal membranes and also in membranes from preterm deliveries. il- production was stimulated by lps whereas il- was not affected. these cytokines are not physiological components of af and their role in fetal membranes is unclear. higher il- concentration in response to lps but lack of its presence in term or preterm af is suggestive of an autocrine immune response during pregnancy in response to a microbial antigen. evidence for a selective migration of fetus specific cd + cd bright regulatory t cells from the peripheral blood to the decidua in human pregnancy. tamara tilburg, , dave l roelen, barbara j van der mast, godelieve m de groot, carin kleijburg, sicco a scherjon, frans hj claas. department of obstetrics, leiden university medical centre, leiden, netherlands; department of immunohematologie and bloodbank, leiden university medical centre, leiden, netherlands. during pregnancy the maternal immune system has to tolerate the persistence of fetal alloantigens. many mechanisms contribute to the prevention of a destructive immune response mediated by maternal alloreactive lymphocytes directed against the allogeneic fetus. murine studies suggest that cd + cd + t cells provide mechanisms of specific immune tolerance to fetal alloantigens during pregnancy. previous studies by our group demonstrate that a significantly higher percentage of activated t cells and cd + cd bright t cells are present in decidual tissue in comparison with maternal peripheral blood in human pregnancy ( ) . in this study we examined the phenotypic and functional properties of cd + cd bright t cells derived from maternal peripheral blood and decidual tissue. depletion of cd + cd bright t cells from maternal peripheral blood demonstrates regulation to a rd party umbilical cord blood cells comparable to non-pregnant controls, whereas the suppression capacity to umbilical cord blood cells of her own child is absent. furthermore, maternal peripheral blood shows a reduced percentage of cd + cd bright foxp + and cd + cd bright hla-dr + cells compared to peripheral blood of non-pregnant controls. in contrast, decidual lymphocyte isolates contain high percentages of cd + cd bright t cells with a regulatory phenotype that are able to down regulate fetus-specific and non-specific immune responses. these data suggest a preferential recruitment of fetus-specific regulatory t cells from maternal peripheral blood to the fetal-maternal interface where they may contribute to the local regulation of fetus specific responses. ( ) tilburgs t, roelen dl, van der mast bj, van schip jj, kleijburg c, de groot-swings gm, kanhai hh, claas fh, scherjon sa. differential distribution of cd (+) cd (bright) and cd (+) cd (-) t-cells in decidua and maternal blood during human pregnancy. placenta. apr; suppl a:s - . alicia del toro-arreola, lourdes nunez-atahualpa, juan velazquez-rodriguez, laura gonzalez-lopez, jorge i gamez-nava, adrian daneri-navarro. there is evidence that the maternal immune system is influence by changes in the hormonal levels during the menstrual cycle (mc). so far, the information related to the levels of t, treg, nk cells and receptors of activation and inhibitors is scarce. aim: to analyze the populations of t, treg, nk cells and their receptors of peripheral blood of healthy women and their correlation with hormones during mc. material and methods: we studied to women not using hormonal contraceptives in the day th of the follicular phase and st of the luteal phase of the mc. pbmc subsets and their receptors were determined by flow cytometry and hormone levels by chemiluminescence method. we found that the progesterone and prolactin were positive correlated (rho= . , p< . and rho= . , p< . , respectively) with cd /nkg in t cells and negative correlated (rho= - . , p< . and rho= - . , p< . , respectively) with nk cells. meanwhile cortisol was positive correlated (rho= . , p< . ) with the receptor nkg d expressed in nk cells. the results observed in this study in the luteal phase of mc on the expression of cd /nkg inhibitor receptor and nkg d activator receptor were related to a particular hormone (progesterone, prolactin and cortisol) might contribute to understanding the physiological role of the neuroendocrine axis on the expression of some receptors of the immune system in order to keep the homeostasis milieu of the mc. objective: sp-d, a key component of the innate immune system, is detected in amniotic fluid (af) and believed to originate in the fetal lung. however, sp-d is produced by other cells and therefore extra-pulmonary sources must be considered. the objective of this study was to determine the maternal and fetal plasma and af concentrations of sp-d to gain insight into the behavior of this natural antimicrobial peptide in pregnancy. moreover, we studied sp-d expression in maternal and fetal peripheral leukocytes. methods: maternal and fetal plasma and af samples were obtained from patients in the following groups: ) term not in labor (tnl; n= ); ) term in labor (til; n= ); ) preterm labor without histologic chorioamnionitis (ptl; n= ) and ) preterm labor with histologic chorioamnionitis (ptl-hc; n= ). sp-d concentration was measured by elisa. sp-d mrna expression in maternal and fetal leukocytes was evaluated by real-time qrt-pcr. flow cytometry and confocal microscopy were used to study the localization of sp-d in leukocytes. results: ) the af sp-d concentration increased as a function of gestational age (mean, til: , . ng/ml vs. ptl: , . ng/ml; p< . ); ) in contrast, the maternal and fetal plasma sp-d concentrations decreased with advancing gestational age (mean, til: . ng/ml vs. ptl: . ng/ml; p< . , and til: . ng/ml vs. ptl: . ng/ml; p< . , respectively); ) the maternal plasma sp-d concentration was lower than that of fetal plasma (mean: . ng/ml vs. . ng/ml; p< . ); ) however, sp-d mrna expression in maternal leukocytes was . fold higher than that of fetal leukocytes (p< . ); ) neutrophils (both maternal and fetal) expressed sp-d as demonstrated by flow cytometry and confocal microscopy. conclusion: ) the concentrations of sp-d in the maternal and fetal circulation decreased with gestational age while the af concentration increased; ) the expression of sp-d mrna is higher in maternal leukocytes than in fetal leukocytes; ) we report for the first time that maternal and fetal neutrophils are a source of sp-d and propose that this molecule plays a role in host defense against infection and in the modulation of the maternal and fetal immune response. introduction intrauterine insemination (iui) is a fertility technique that allows for couples to have intercourse after the procedure is performed. it has been postulated that intercourse after iui may increase the pregnancy rate by either endometrial stimulation or because it may represent a second spermatic flow in the periovulatory period. in the present study we evaluate the effect of intercourse on the pregnancy rate of patients undergoing iui. material and methods: from to patients were enrolled in the study. every couple undergoing iui was instructed at the moment of insemination to decide whether to have or not intercourse on the same day of the procedure. all couples were abstinent three to seven days before iui. the information regarding intercourse was recorded the day after treatment. ovulatory, insulin resistant, cervical, male, tubal and endometrial factors as well as parity and time of infertility were compared between the two groups. all these factors were analyzed based on number of follicles that ovulated in each group. our principal outcome was to determine the pregnancy rate. intercourse was practiced by . % of the couples. the global pregnancy rate was . %. the pregnancy rate for the couples who had intercourse was . % and . % for those who did not have intercourse (p< . ). even though age, parity, time of infertility and stimulation protocols were similarly distributed in both groups, the proportion of tubal and endometrial factors were higher among those who had intercourse (p< . ). when subjects with tubal and endometrial factors were excluded, the pregnancy rate between both groups (n= ) was similar ( . % vs . % for positive and negative intercourse, respectively). the average number of ovulatory follicles was . + . for the group that had intercourse and . + . for those who did not. according to our results, intercourse after iui does not improve pregnancy rate after this procedure is performed. furthermore our study indicates that iui does not interfere with sexual intimacy since almost % of the couples decided to have intercourse on the same day of the procedure. . we sought to investigate the effects of gravidity on mmc and fmc in healthy, parous women. methods: mc was assayed in dna extracted from peripheral blood mononuclear cells (pbmc). hla-genotyping was first conducted and mc quantified employing a q-pcr assay targeting a non-shared maternal-or fetalspecific hla polymorphism. gravidity was dichotomized as a history of one pregnancy compared with two or more, and the prevalence of mc was analyzed using logistic regression. possible confounders were included as appropriate, including subject age and time since last pregnancy. adjustment for possible correlation between values was also made when there were repeated measures for the same subject. results: for the mmc analysis, there were subjects with observations. for the fmc analysis, there were subjects with observations. table provides a summary of mmc and fmc by gravidity. mmc was significantly decreased with higher gravidity. fmc was not affected by gravidity. gravida gravida or more adjusted* or ( %ci) mmc / ( %) / ( %) . ( . - . ) fmc / ( %) / ( %) . ( . - . ) *adjusted for possible correlation between values within a subject, subject age, and time from last pregnancy, as appropriate. increasing gravidity is significantly associated with a decreased prevalence of mmc. despite additional sources of fmc, there does not appear to be an increase in fmc prevalence with increasing gravidity. the biology of mc is incompletely understood, and the nature of mmc and fmc are likely to be different given that acquisition of the former, but not the latter, occurs within a nascent immune system. these data raise interesting questions when considered as interactions of acquired grafts within a host, including whether emergence and persistence of one dominant source of mc may be most advantageous for an individual. anti-igd antibody treatment as a novel immunosuppressive agent for autoimmune diseases and its effects on th /th gene expression. tommie g nguyen, eileen d gallery, , jonathan m morris. elevated t-helper cell type- (th ) and type- (th ) cytokine expression have a role in autoimmune diseases, allograft rejection and pregnancy-related complications. thus, molecules that can shift the immunity away from th /th responses toward a th response represent a novel therapeutic treatment for these conditions. we have previously demonstrated that pregnancy is associated with a suppression of t-bet in peripheral t cells. in this study, we examined a novel effect of anti-igd antibody on t-bet expression, th /th gene expression in human peripheral blood mononuclear cells (pbmc) and its therapeutic effects in an animal model of collagen-induced arthritis. methods: human pbmc were isolated and then cultured in the presence of anti-igd antibody at various time points followed by stimulation with pma/ionomycin (p/i) for hrs. gene expression was examined by rt-pcr, western blotting and elisa. for in vivo study, arthritis-prone dba/j mice were induced to undergo joint inflammation by intradermal injections with bovine type-ii collagen. these mice were then given daily doses of mg/kg of intravenous injection with anti-igd antibodies as preventive or therapeutic treatments (n = per group). results: treatment with anti-igd antibodies significantly suppressed p/iinduced expression of t-bet (a master regulator of th immunity), tnf-(a classical pro-inflammatory th cytokine), and il- (a critical proinflammatory th cytokine) in human pbmc. this suppression is highly specific to these genes because anti-igd antibodies have no effects on the expression of ifn-g and il- (two classical th cytokines). in vivo experiment showed that anti-igd antibody treatment markedly reduced clinical severity of joint inflammation when comparing the clinical score of control mice group ( . ± . , mean ± s.e.m), preventive group ( . ± . ) and therapeutic group ( . ± . ) . conclusions: our study has demonstrated that suppression of t-bet by anti-igd antibodies, similar to the changes seen in human pregnancy is a novel in vivo anti-inflammatory effect. given the essential role of t-bet, tnf-and il- in the pathogenesis of human autoimmune diseases, anti-igd antibodies may represent a novel immunosuppressive treatment that needs further studies and evaluation. objective: women with circulating anti-phospholipid antibodies (apl) are at risk for recurrent miscarriage, preeclampsia and preterm labor. apl antibodies directly target the placenta by binding to phospholipids or phospholipid-binding proteins expressed on the surface of viable trophoblasts. the objective of this study was to determine the effects of apl antibodies on first trimester trophoblast cells. methods: two mouse igg anti-human beta -glycoprotein i monoclonal antibodies (mabs), designated id and iic , were used in these studies. the first trimester trophoblast cell line, htr , was incubated with either medium, a mouse igg control, id or iic ( - g/ml), in the presence or absence of unfractionated heparin ( ng/ml). trophoblast cell death and apoptosis was determined using a viability assay, hoechst staining and a caspase activity assay. cytokine production was evaluated by multiplex analysis. results: following a hour incubation, significant trophoblast cell death was induced by iic ( . ± . %) and id ( . ± . %) at the high dose of g/ml, when compared to the medium and mouse igg controls (p< . ). hoechst staining showed that id -and iic -induced trophoblast cell death was a result of apoptosis. moreover, id and iic induced a significant increase in caspase- , - and - activity (p< . ). treatment of trophoblasts with heparin significantly inhibited the effects of iic and id on cell death by . ± . % and . ± . % %, respectively (p< . ). following a hour incubation at lower concentrations ( g/ml), treatment of trophoblast cells with id or iic resulted in a significant upregulation of il- , mcp- , gro production (p< . ), and a significant reduction in il- secretion (p< . ). conclusion: this study demonstrates that at low levels apl antibodies can modulate trophoblast cytokine production, while at higher levels, the same antibodies induce trophoblast apoptosis in a caspase-dependent manner. these findings shed new light on the mechanisms by which apl antibodies may impact placental survival and function. antigenic targets for the diagnosis of premature ovarian failure. hc bohler, c gercel-taylor, lt ku, st nakajima, dd taylor. obstetrics, gynecology, women's health, university of louisville, louisville, ky, usa. objective: premature ovarian failure (pof) is a premature depletion of ovarian follicles before the age of , affecting approximately % of women < years. the involvement of autoimmune mechanisms in pof has been suggested and similar mechanisms have been postulated for other ovarian pathologies, including idiopathic infertility, polycystic ovary syndrome (pcos), or endometriosis. while the association of autoantibodies has been demonstrated for these ovarian pathologies, variation in specificity and frequency of false positivity have limited the diagnostic use of autoantibodies. the objective of this study was to develop an antigen array to differentiate antibody recognition patterns of pof from other infertility pathologies. design: prospective study in a university research laboratory. materials and methods: patients diagnosed with infertility were included in this pilot study: endometriosis (n= ), pcos (n= ) and pof (n= ). autoantibodies were assayed by dot immunoblotting using an antigen array derived from endometrial and ovarian cells. for the cellular antigen preparations, solubilized total proteins were separated by reverse phase-hplcquid chromatography and the individual proteins were blotted onto nitrocellulose membranes and reactivity visualized by peroxidase-labeled antihuman igg. results: patients with pof, endometriosis, and pcos all exhibited autoantibodies reactive with these cellular proteins. while some antigenic reactivities were shared by all infertility patients, the pattern of antigen recognition was distinct for patients with pof. patients with pof all recognized a common antigenic proteins (row , antigens a,b,d-g). conclusions: alterations in autoreactivity are observed in patients with the diagnosis of infertility; however, distinct patterns of autoantibody recognition can be demonstrated for patients with different pathologies. while this study needs to be expanded to reliably establish the specificity, sensitivity and positive and negative predictive values, patients with pof clearly exhibit a shared recognition pattern that may be useful a diagnostic marker. cicek gercel-taylor. ob/gyn, university of louisville, louisville, ky, usa. objective: americans' consumption of nutraceuticals is one of the most rapidly expanding health markets, growing at a rate of % annually. multiple nutraceuticals containing phytoestrogens have been marketed as "immune boosters" despite suboptimal evidence-based medicine to support such statements. as immunomodulatory therapies should affect downstream cytokine expression, the relative effects of estradiol and genistein in regulating expression of cd -and jak were tested. these markers were chosen since they are central to t cell signaling. cd -is a critical transducer of tcr activation and regulates t cell proliferation and cytokine production. jak upregulation is a specific marker for hematopoietic cell stimulation. methods: to test the immunomodulatory effects of phytoestrogens and estrogen, jurkat . (t cell leukemia) cells were grown in estrogen-free, phenol red-free media for hours. these cells were then exposed for hours to pm, pm (postmenopausal), or pm (premenopausual) of estradiol in the presence of increasing concentrations of genistein ( , . , . , . , , and m). cells were then solubilized and cellular protein quantitated. protein concentrations were standardized and western blots for each set of culturing conditions were run in triplicate. cd -and jak expression were quantitated following visualization with chemiluminescence by digital pixel quantification. results: our findings show that in the absence of estradiol and at postmenopausal levels of estradiol, genistein induced a dose dependent increase in cd -reaching a maximum of fold. although cultivation of t cells in pm of estradiol significantly increased the levels of cd -and jak relative to hypoestrogenic conditions, the genistein mediated dose response was not observed. conclusions: these in vitro results indicate that genistein can at least partially reverse suppression of signaling molecules observed in postmenopausal estrogen environments. clinically, this suggests that phytoestrogens may have greater immunomodulatory properties for postmenopausal females than those that are premenopausal. maternal serum il- as a biomarker of acute immunologic rejection of pregnancy. joaquin santolaya-forgas, juan deleon-luis, isabel galan. obstetrics and gynecology, brigham and women's hospital, boston, ma, usa; amarillo women's health research institute, texas tech university health science center, amarillo, tx, usa. objective: markers of acute rejection of pregnancy are very scarce. in this study we aimed at determining if rapid changes in maternal serum concetration of a variety of biomarkers could be used for this purpose. we used an established baboon model for in utero stem cell therapy to introduce at - days from conception and via ultrasound-guided celocentesis, human hematopoietic stem cells with different proportions of natural killer t-cells (nk). maternal blood was collected before and hours after celocentesis for quantification of hormones and il- using solid phase, enzyme labeled, chemiluminescent sequential immunometric assays. pearson correlation analysis was used for determination of significant changes from baseline (p< . ). results: all animals survived their pregnancies. seven animals receiving < % concentration of nk delivered at term ( days gestation) while animal receiving more than % concentration of nk had dead fetuses on ultrasound evaluation hours after celocentesis. table depicts mean maternal serum concentration of the biomarkers investigated (all n.s.). figure shows mean il- changes from baseline in continuing (n.s.) and rejected pregnancies (p< . ). conclusions: we have described a model in which in utero graft vs host disease can be studied. these preliminary results suggest that of all the biomarkers investigated, il- might be the most sensitive for detection of an acute rejection of pregnancy. biomarkers of acute immunologic rejection of pregnancy biomarker unit pre-celocentesis ( ) the activity of cytotoxic cd + t cells during pregnancy protects the mother and fetus from infection. however, pregnancy's effect on the proliferation and apoptosis of cd + t cells has not been clearly defined. objective: to determine if normal pregnancy changes the number of proliferating or apoptotic splenic cd +t cells. methods: female c bl/ mice were used unmated (um) or underwent timed mating. one day prior to harvest, mice were i.p. injected with bromodeoxyuridine (brdu), which is incorporated into replicating dna. harvested spleens were homogenized, enumerated, and stained for cell surface expression of cd and t cell receptor beta chain (tcr ). apoptotic cells were detected by treatment with terminal transferase and fitc-dutp (tunel). the numbers of cd +tcr + cells that were brdu+ or tunel+ were calculated from the percentage of positive cells obtained by flow cytometry and the absolute number of cells counted. for each experiment, the ratio of the number of positive cells in pregnant to um mice was compared by anova with dunnett's post-test. results: at day of pregnancy (n= ), the number of brdu positive cd + t cells was two fold higher than that found in um (n= , p< . ). the number of proliferating cd + t cells continued to be non-significantly elevated at day ( . x, n= ), day ( . x, n= ), and day of pregnancy ( . x, n= ) compared to um. by day of pregnancy (n= ) the number of proliferating cd + t cells returned to the um level, however by this time the total number of splenic cd + t cells was . fold higher than um (n= p< . ). on gestational day , the number of proliferating cd + t cells declined further ( . x, n= ), and the number of splenic cd +t cells returned to the um level (n= , p> . ). compared to um mice, there was no significant difference in the number of cd + t cells undergoing apoptosis at any gestational day examined ( . - . x, p> . ). in normal murine pregnancy, the number of cd + t cells is increased in late gestation, and then returns to baseline at the end of pregnancy. this is due to an early increase then gradual decline in cd + t cell proliferation, accompanied by a steady rate of apoptosis. this argues that the maternal immune system undergoes dynamic homeostatic changes, and is not globally suppressed. supported by nihro hd - niht ai . arturo cerbulo-vazquez, cun li, , gene hubbard, natalia e schlabritz-loutsevitch, , peter w nathanielsz. objectives: early thymocyte (t) maturation occurs in the cortex while later stages occur in the medulla. thymic epithelial cells (tec) synthesize gc and t express gr. tec may influence t cell maturation by regulating apoptosisinduced gc-gr interactions. igf- (also synthesized by tec) may support thymocyte prolifetarion. human fetuses of mothers in premature labor are exposed to gc. gc administered to pregnant baboons at . , . , and . gestation (g) alters fetal lymphocyte populations at . (g) (j repro immunol, , : ) . we determined if fetal gc exposure alters thymic ) structure; ) gr and igf-i protein. methods: pregnant baboons received saline (control ctr; n= ) or betamethasone i.m. ( μg/kg daily for two days at . , . and . g; gc group; n= ), c-sectioned at at . g under general anesthesia and thymic gr and igf-i evaluated by immunohistochemistry. results: gr localized to medulla and a few cortical cells. igf-i localized to cortex with little medullary expression. medullary necrosis was greater in ctr than gc fetuses. t gr was located in cytoplasm. no gross differences were observed between ctr or gc fetuses for either igf-i or gr. conclusions: a) early thymocyte maturation may be supported by igf- , b) later differentiation involves gr, and c) after exposure to gc doses equivalent to human therapy, no gross effects were detected on gr or igf, but d) natural thymic necrosis was inhibited. lindsay s christensen, peyman bizargity, elizabeth a bonney. ob/gyn, university of vermont, burlington, vt, usa. background: the exact mechanism by which bacterial products trigger preterm delivery and the immune cellular circuits involved remain unclear. our recent data in normal c bl/ (b ) or recombinase deficient c bl/ mice (rag-ko) indicates that t and b cells are not critical for lps-induced preterm delivery and stresses the importance of related innate mechanisms. rag-ko mice are more susceptible to lps, suggesting that t or b cells may control the innate response. macrophages are vital to innate immunity and produce proinflammatory cytokines that can activate prostaglandin synthesis and myometrial contraction. we questioned whether differences in susceptibility between the strains are due to differences in the uterine macrophage response to lps and thus examined macrophages at the maternal-fetal interface early after injection. objective: to compare uterine macrophages levels at and hours after lps injection in pregnant b and rag-ko mice. methods: b and rag-ko mice were mated and on gestation day , females were injected intraperitoneally with μg lps in l saline (pbs) or l pbs alone. euthanasia and uterine harvest occurred or hours after injection. frozen uterine sections were stained with the macrophage marker f / or an isotype-matched control followed by an alexafluor -conjugated secondary and a nuclear stain (dapi). sections were visualized by fluorescence microscopy. for each mouse, f / + dapi+ and total dapi + cells were counted in areas of representative section and the percentage of f / + dapi+ cells was calculated. the mean percentage for at least representative areas per experimental group was analyzed by anova. results: two hours post-injection, macrophages levels were similar in b and rag-ko mice injected with pbs (b , n= , . ± . ; rag-ko, n= , . ± . % + per area). lps injection increased macrophages (p< . ) in both strains (b , n= , . ± . ; rag-ko, n= , . ± . ,); no difference was evident between strains. the percentage of f / + cells was similar hours post-injection (b , n= , . ± . v. rag-ko, n= , . ± . ), and not elevated relative to the hour time point. objective. decay-accelerating factor (cd ), is expressed in the plasma membranes and protects mammalian cells against the lytic action of serum complement. phosphoinositide -kinases (pi ks) are involved in the regulation of cell functions by synthesizing a second messenger molecule ptdins ( , , ) p . akt, a serine-threonine kinase acts downstream of pi k and regulates cell survival, growth and proliferation. the pi k-akt activity is controlled by tumor suppressor gene pten. in this study we assessed whether the pi k-akt activity affects the expression of cd in human endometrial and cervical cells. methods. endometrial and cervical cell lines which differ in the constitutive pi k activity were used in this study. ishikawa and rl - endometrial cell lines harbor pten mutation and have high levels of phosphorylated akt (p-akt). hec- -a and kle endometrial cell lines and hela cells express wild-type pten and have minimal or no demonstrable levels of p-akt. the expression of cd was evaluated by rt-pcr, immunoblotting and flow cytometry. the pi k activity was assessed by immunoblotting with anti-p-akt antibodies. the effect of inhibition of pi k-akt pathway on cd expression was evaluated in cells treated with wortmannin ( nm), ly ( mm), or with akt inhibitor sh ( mm). results. immunoblotting densitometry and measurements of mean fluorescence intensities showed that the level of cd expression correlates with the status of pi k-akt pathway. the cd expression was lowest in hec- -a, ishikawa and rl - cells which constitutively express p-akt. higher cd expression was found in hela cells and kle cells which express wild-type pten product and has no detectable phospho-akt. mean fluorescence intensities were . -fold higher for kle cells and -fold higher for hela cells compared to hec- -a cells. treatment of cells with akt inhibitor led to . - . -fold increase in cd expression. the . - . -fold increase following treatment with pi k inhibitor wortmannin was found in ishikawa cells, rl - and kle cells. conclusions. human endometrial cell lines with elevated pi k-akt activity express lower level of cd compared to cell lines with intact pten gene function. these findings may indicate that structural alteration at the dna level and resultant overexpression of pi k-akt pathway are involved in the downregulation of cd . endometrial and cervical cells. pawel goluszko, chandra yallampalli. obstetrics gynecology, university of texas medical branch, galveston, tx, usa. objective. cell shape is determined by the cytoskeleton, which provides the mechanical support and is involved in cellular signaling. apoptotic cells undergo major morphological changes such as rounding and contraction, a process regulated by caspases, the cysteine proteases responsible for events controlling the cell disassembly. the motifs in certain cytokeratins make them substrates for caspase degradation. anti-apoptotic serine/threonine kinase akt provides a survival signal by phosphorylating downstream effector molecules including caspase- . while studying the akt distribution in human endometrial cell line we found that akt shows filamentous pattern of staining resembling cytoskeleton organization. in this study we evaluated whether akt staining correlates with the microfilaments (mf), microtubules (mt) or intermediate filaments. endometrial ishikawa, rl - , hec- -a, kle and cervical hela cell lines were used in the study. incubation with cytochalasin d, ( ug/ml) or nocodazole ( mg/ml) and labeling with bodipy fl phallacidin, anti -tubulin or anti-akt antibody were used to assess whether cytoskeleton disruptors affect akt distribution and mf and mt organization. for colocalization, cells were stained with anti-cytokeratin mouse antibody followed by anti-mouse alexa conjugate, and then stained with anti-akt rabbit antibody and anti-rabbit alexa conjugate. the scans collected with laser scanning confocal microscope from channels filtered for alexa and alexa were combined digitally and evaluated with imaris colocalization analysis software. filamentous pattern of akt staining was most pronounced in ishikawa and less obvious in hec- -a cells. treatment with cytochalasin d or nocodazole resulted in disruption of mf and mt but had no effect on cytokeratin organization and akt distribution. double staining with anti-cytokeratin- and anti-akt antibody showed overlapping staining for cytokeratin and akt. analysis of digitally acquired images showed highest correlation for colocalized channels in rl - cells ( . ) followed by ishikawa ( . ) and hela cells ( . ). lowest correlation was found for kle ( . ) and hec- -a cells ( . ) conclusions. this study indicates a strong colocalization pattern of serine/ threonine kinase akt with cytokeratins, and suggests a mechanism by which cytokeratins might be protected against cleavage by caspase- and caspase- in the early apoptotic stages. background: cd + cd + t regulatory cells (t-reg), express foxp ,suppress antigen-specific immune responses and are important for allograft tolerance. during pregnancy the mother tolerates an allograft expressing paternal antigens (the fetus), requiring substantial changes in immune regulation over a programmed period of time. the presence of t-reg cells (cd + cd highfoxp +) was assessed in the peripheral venous blood of non-pregnant, pregnant and seven postnatal healthy women by flow cytometry. human decidua was obtained by surgical termination of pregnancy in the first (n= ), second (n= ) and third (n= ) trimester of human pregnancy. paraffin sections were immunostained for foxp and cd . foxp +cells were quantified in x fields and results compared between first, second and third trimester samples and according to the presence of extravillous trophoblast. results: fluorometric studies of blood samples indicate an increase % of circulating cd + cd highfoxp t-cells in pregnant ( . % [range . - . %]) vs. non-pregnant controls ( . % [range . - . %]; p< . ). a progression from st, nd and rd trimesters indicated the % of cd + cd highfoxp t-cells was . %, . % and . %, respectively. low numbers of foxp + cells were detected in all decidua samples and their distribution mirrored that of cd + cells. in st trimester samples, foxp + cells were often detected in lymphoid aggregates adjacent to endometrial glands. increased numbers of foxp + cells were detected in st ( . ± . ) compared with nd trimester decidua ( . ± . ; p< . ) but there was no difference between st and rd trimester ( . ± . ), nor between nd and rd trimester decidua. in st trimester decidua, numbers of foxp + cells were increased in areas without extravillous trophoblast. conclusion: normal human pregnancy is associated with an increase in the number of circulating cd + cd highfoxp t-cells. the presence of foxp + cells in early gestation human decidua may be important in the initiation of materno-fetal tolerance at an autocrine level. (supported by mrc). aims: -defensins are small cationic peptides with antibiotic and antimicotic activity. hyaluronan and its degradation products have been described as endogenous ligands for tlr and tlr , whose involvement in -defensin expression has been reported in different epithelial tissues and cell lines. we aim to investigate weather low molecular weight hyaluronic acid induces -defensin release by keratinocytes, via tlr and tlr . methods: the induction of -defensin production following in vitro treatment of human keratinocytes with a low molecular weight hyaluronic acid solution was evaluated by pcr-analysis and elisa techniques. studies on the involvement of tlr and tlr in -defensin production have been performed using specific blocking antibodies. results: pcr and elisa revealed an intense -defensin production following hyaluronic acid treatment in human keratinocytes. the -defensin production induced by hyaluronan was abolished following block of tlr and tlr by specific antibodies, demonstrating the involvement of these receptors. the same hyaluronic acid treatment did not induce activation of inflammatory genes, such as il- , tnf-, il- and il- . conclusion: our data show that hyaluronic acid is an efficient inducer ofdefensin production in keratinocytes, via tlr and tlr . this observation might be important to open new perspectives in the development of possible topical products containing hyaluronic acid, to improve the release ofdefensins by keratinocytes, ameliorating the self-defence of the skin in case of skin infections. therefore, one of the possible applications for this kind of topical products might be the treatment of infective vulvitis, one of the most distressing gynaecological diseases for adult women. pregnancy outcome? kiera von besser, serena wu, mary d stephenson. , obstetrics and gynecology, university of chicago, chicago, il, usa; obstetrics and gynaecology, university of british columbia, vancouver, bc, canada. objective: to investigate whether gender of an ongoing pregnancy impacts the probability of a successful outcome, and, to ascertain whether the gender of prior live birth(s) impacts subsequent pregnancy outcome, among women with rm/aps. materials and method: cohort-control study. rm subjects were evaluated by mds between - (stephenson, . couples who met aps criteria (wilson et al, ) , restricted to rm only, were followed prospectively. cohort data was compared to live birth data from the vital statistics agency of british columbia from - . secondary sex ratios (ssrs) among successful pregnancy outcomes were calculated by dividing the number of male live births by female. sex ratios were calculated for all pregnancies weeks, regardless if they ended in success or demise. pearsons test with yates continuity correction was applied. results: subjects were identified. subjects had prior live births of known gender ( male/ female), giving a ssr of . . there were also prior fetal demises wks of known gender ( / ) giving a sex ratio for all prior pregnancies at wks of . . subjects delivered subsequent live births of known gender ( / ), giving a ssr of . . there were also subsequent fetal demises ( / ) giving a sex ratio for all subsequent pregnancies of . . subjects delivered both prior and subsequent live births. the ssr was . ( / ) among their prior and . ( / ) among their subsequent live births. including fetal demises, subjects had ongoing prior and subsequent pregnancies. the sex ratio was . among their prior pregnancies and . among their subsequent. as the control, a ssr of . ( , / , ) was calculated from vital statistics data. when the prior and subsequent ssrs of the cohort were compared to each other, as well as to the control, there were no statistically significant differences. conclusions: our findings, from the largest study of its kind to date, suggest that, in patients with rm/aps, the gender of an ongoing pregnancy does not significantly affect the probability of a successful outcome, to any greater degree than it does in the general population. also, the gender of a prior ongoing pregnancy does not significantly impact the likelihood of developing rm/aps. oocyte maturation. jk friend, fb bezirci, e seli. ob gyn, yale u., new haven, ct, usa. introduction: oocyte maturation is associated with repression of transcription. during oocyte maturation, fertilization, and early embryo development until the onset of zygotic gene expression, proteins are synthesized from maternallyderived mrnas. the regulation of protein expression from these maternal mrnas is post-transcriptional, and occurs mainly via poly(a)-tail elongation. the embryonic poly(a)-binding protein (epab) is the predominant poly(a)binding protein before the activation of the zygotic genome, and plays a critical role in the activation of certain maternal mrnas, those bound by cpeb and probably pumilio. we are characterizing additional functions of epab during the process of oocyte maturation. methods and results: our model system is the xenopus laevis oocyte where oocyte maturation is induced by the addition of progesterone. our preliminary findings indicate that epab is phosphorylated, and that levels of phosphorylated epab increase upon progesterone-induced oocyte maturation. moreover, glycerol gradient centrifugation revealed that nonphosphorylated and phosphorylated epab are contained in distinct complexes that change mobility upon oocyte maturation. furthermore, oligo-dt selection for poly(a)-containing mrnas strongly suggests that these mrnas are bound exclusively by phosphorylated epab. using affinity purification, we have determined that nonphosphorylated epab exists primarily in a large protein complex prior to oocyte maturation that is later disassembled after the addition of progesterone. conclusions: based on these preliminary findings, we conclude that prior to oocyte maturation, the bulk of epab is nonphosphorylated and is found in a protein-rich complex separate from poly(a)-containing mrnas. upon oocyte maturation (when certain maternally-derived mrnas are activated for translation), the majority of epab becomes phosphorylated, and this phosphorylated form of epab is likely bound to translationally-active mrnas. we are currently investigating what kinase phosphorylates epab and whether this phosphorylation plays a role in translational up-regulation of epab-bound mrnas. introduction: reactive oxygen species (ros) play important roles in all aspects of cellular fate. nadph oxidase isoforms, a family of genetically preserved enzyme complexes, have been shown to be the main sources of ros in various cell types. however, the role of nadph oxidase isoforms in human myometrium proliferation and differentiation has not been defined. in the myometrium, different smooth muscle phenotypes maybe associated with specific physiologic functions. we have shown that angiotensin ii (angii) stimulates hypertrophy but not cell proliferation in ultr cells, an in vitro model of human myometrium. ultr cells at greater than passages display a replicative senescent phenotype. by introducing human telomerase reverse transcriptase (htert) gene, we have obtained a stable cell line (ultr-ht) which has a significantly increased division rate and distinct cellular morphology than the original ultr cells. objective: to determine the relationship of expression of nadph oxidase to ultr cellular fate. methods: early and late passages (p - ) of ultr and ultr-ht (p - ) cells were grown on either plastic or collagen iv (cn )-coated surfaces. ultr-ht cells were further stimulated with angii ( . um) for hrs. expression of nadph oxidase core (nox - and duox / ) and associated subunits (p phox, p phox, noxo , p phox, noxa , p phox, and rac / ), and angii receptors at / was identified by rt-pcr from cellular total rna. fluorescent immunohistochemistry (ihc) was employed to determine protein expression and localization. results: the mrna level of house keeping gene -actin was unchanged by any cellular manipulation. the senescent phenotype of ultr cells was accompanied by an apparent down-regulation of nox , p phox, and noxa genes, and an up-regulation of at / . overexpression of htert did not reverse nox , p phox and noxa expression while cell division rate was increased. however, there was a down-regulation of nox , at / and rac . plating ultr-ht cells on cn induced nox / down-regulation and up-regulation of duox / , with no apparent change of at / . however, exposure to cn re-directed cellular response to angii such that only nox was induced by angii stimulation. conclusion: expression of nadph oxidase isoforms nox , , , and duox / are correlated with ultr cell differentiation and cell fate control. data also suggests that ang ii-induced myometrial hypertrophy involves nox mediated ros generation. fluids from reproductive women -the influence of aging. eriko y fujii, masahiro nakayama. women's health, national center for child health and development, tokyo, japan; aska reproductive clinic, nara, japan. [introduction] receptor for advanced glycation end products (rage) is a multiligand type glycoprotein, and is characterized based on its ability to bind ages, adducts formed by non-enzymatic glycation and oxidation of protein and lipids. this process occurs during normal course of aging. ages/rage interaction regulates various physiological function, such as inflammation, angiogenesis through vegf inducement. a soluble form of rage (srage) works as a decoy in the body and inhibits intracellular signaling. [objectives] the balance of these factors may contribute to reproductive dysfunction by aging. we aimed to measure the ages, srage and vegf concentrations in plasma and follicular fluids from reproductive women, and examined the differences of those factors between young group and old group. [material and methods] patients' plasma and follicular fluid were collected with consent based on regulations of the ethical committee, and we measured ages (pentosidine, cml), srage and vegf in duplicate using commercially available elisa kits (fushimi co, r d and cyelex). concentrations were calculated from each standard curve, and compared between the young group under years old, and the old group over y.o. data were evaluated for the difference in two groups by student's t test , and the significance was determined by p< . . [results] ) srage in plasma, ± pg/ml (mean±sd), n= in the young group was significantly higher than ± pg/ml, n= in the old group. there was no significant difference in plasma vegf. ) vegf in follicular fluid was ± pg /mg protein, n= in the young, and ± pg /mg protein, n= in the old was increased significantly. ) we could not see statistical difference of pentosidine nor cml concentrations between two groups in plasma and follicular fluid samples. [conclusions] it has been reported that higher concentration of vegf in follicular fluid may relate to worse pregnancy rate in art. there was a significant decrease of plasma srage in older group in our result, and because of this decrease of 'decoy', focal ages-rage-vegf signaling might be activated in older women. our results showed the possibility that ages/rage and vegf regulation may contribute to the reproductive dysfunction by aging. and leiomyosarcoma cells. qun pan, xiaoping luo, nasser chegini. ob/ gyn, university of florida, gainesville, fl, usa. as a part of a novel endogenous rna silencing machinery, a noncoding short rna strand referred to as "microrna" (mirna) has been identified to regulate the stability of the target gene expression through mrna degradation and repression. we have identified the expression of many of these mirnas in leiomyoma, myometrium, their isolated smooth muscle cells (lsmc and msmc), transformed lsmc (t-lsmc) and sklm (leiomyosarcoma cell line), including mir- which is predicted to target the expression of many genes, including tgf-b and tgf-b type ii receptor (tgf-brii). however, the biological significance of these mirnas in various cellular processes remains to be established. as such in the present study we examined the expression, regulation and function of mir- in lsmc, msmc, t-lsmc and sklm. we found that mir- is expressed and regulated by b-estridiol and medroxyprogesterone acetate ( - m) in these cells (p< . ). we further assessed the regulatory function of gain of and loss of function of mir- on the expression of tgf-brii. transfection of lsmc, msmc, t-lsmc and sklm with pre-mir-and anti-mir- oligonueclotides resulted in a significant increase and/or inhibition of mir- expression in these cells, respectively as determined by realtime pcr (p< . ). over-expression of mir- resulted in a significant reduction, while transfection with antimir- increased the expression of tgfbrii mrna and protein in these cells as compared to controls (p< . ). we concluded that mir- is expressed in leiomyoma and myometrial cells, its expression is regulated by the ovarian steroids and it functions by targeting the expression of tgfbrii and possibly other genes with key regulatory action on cell growth, angiogenesis, transcription regulation, ecm turnover and apoptosis that results in leiomyomas growth and regression. (supported by nih grant hd ). objective: placenta and a number of gestational tissues are well recognized to express corticotrophin releasing factor (crf), urocortin (ucn ), ucn , ucn and crf -r and crf-r receptor subtypes together with crfbinding proteins locally. ucn and ucn are implicated in the reversal of stress responses initiated by crf. in the present investigation, we evaluated functions of crf and ucns by quantifying their contents in venous smooth muscle layers using image pro . in human umbilical cords collected at preterm and term gestation methods: umbilical cord specimens ( - mm thickness, pieces per umbilical cord) collected at preterm and term (n= each) at delivery were fixed in bouin's solution and paraffin embedded. sections were subjected to immunohistochemical analyses with polyclonal antibodies to crf ( : ), ucn ( : ), ucn ( : ) and ucn ( : ) (peninsula laboratory, pa and sigma aldridge, ms) by standard abc technique. immunoreactive materials on the sections were identified using , '-diaminobenzidine as a chromagen. immunostaining intensities (od/area) on uc-sections were quantified by image pro . software and expressed as arbitrary units (au). all values were expressed as mean ± sem. differences between groups were evaluated by anova, followed by the post-hoc tukey test for multiple comparison. results: antibodies to crf and ucns elicited positive immunostaining of variable intensity in venous smooth muscle layers in uc-sections of preterm and term gestations. immunostaining intensity (au) of venous smooth muscle layers at preterm (pt) and term (t) are as follows: crf-pt= . ± . vs crf-t= . ± . (p< . ); ucn -pt= . ± . vs ucn -t= . ± . (p< . ); ucn -pt= . ± . vs ucn -t= . ± . (p=ns); ucn -pt= . ± . vs . ± . (p< . ). conclusion: crf content in venous smooth muscle layer markedly increased at term, while ucn and ucn contents significantly decreased and no significant change occurred in ucn content. based on the opposing changes in crf vs ucn and ucn immunostaining, we speculate that crf, but not ucn or ucn , is the major key player of vasodilation and function locally at the level of venous smooth muscle cells at term. ucn and ucn may perform cytoprotective functions at preterm. physiol. ; : - , am j physiol. ; : - ) . these studies showed that when ad libitum (al) feeding was resumed in cr females, fertility was sustained well beyond the age at which al-fed females became infertile. however, much of what is known on the effects of cr on fertility derives from models in which cr was initiated at weaning. further, there is large variation in how the experiments were conducted. objective: herein we tested if adult-onset cr could delay age-related infertility in females. methods: cr ( %, nia protocol as described in j geront. a:b -b ) was initiated in c bl/ female mice at months and continued until . months of age, at which time al feeding was resumed. matings were initiated at months of age. for mating during cr, a male mouse was housed overnight in a cage with a female and removed the next morning, so that the female mice could be fed their dietary food ration. al-fed and cr females followed the same mating regimen. the number of offspring born and that survived to weaning (day ) were recorded. results: fertility was lost in of al-fed females by months of age and continued to decline through . months of age. age-matched females maintained on cr during the same period exhibited poor fertility, with a total of pregnancies achieved out of females. although cr females showed poor fertility while on cr, their fertility improved dramatically after the reinitiation of al feeding at . months of age. while only out of , al-fed females achieved a pregnancy between . - . months of age, out of age-matched cr-then-al-fed females achieved a total of pregnancies in this -month time. notably, % of the pups born to cr-then-al-fed females between . - . months of age survived and developed to weaning without complications. conclusions: adult-onset cr allows maintenance of female fertility into advanced maternal age after the reinitiation of al feeding. how long fertility can be maintained and the minimum time needed for the beneficial effects of cr to be realized remain to be investigated. nonetheless, these observations suggest that there may be ways to safely extend fertility in females at ages when reproductive function is suboptimal (nih r -ag ). bile acids in human ovarian follicular fluid. laura p smith, , kaila deiorio-haggar, jason reindollar, alan s penzias, , anny usheva-simidjiyska. ob/gyn reproductive endocrinology infertility, bidmc, boston, ma, usa; boston ivf, boston, ma, usa; endocrinology, bidmc, boston, ma, usa. introduction: bile acids are known to serve important functions in the hepatobiliary and gastrointestinal systems. the presence of bile acids in the human ovary and relation with fertility potential have never been previously evaluated. methods: human follicular fluid (ff) from large follicles and small follicles was obtained at vaginal oocyte retrieval. human serum was obtained before and hours after human chorionic gonadotropin (h-cg). follicular fluid and serum samples were analyzed for total bile acids by spectrophotometry. bile acid concentrations were correlated with age, number of retrieved and mature oocytes, number of fertilized oocytes, and pregnancy. results: bile acid concentrations were analyzed and compared to the normal human serum bile acid concentration which ranges from to micromoles/l. bile acids are present in follicular fluid with a mean concentration of . micromoles/l in large follicles and . micromoles/l in small follicles (p= . ). pre and post h-cg serum bile acid concentrations differed significantly ( . micromoles/l vs. . micromoles/l, p= . ). there was a trend toward higher bile acid concentration in large follicles of young patients < years old compared to older patients years old ( . ± . vs. . ± . , p= . ). there was also a trend toward higher pre h-cg serum bile acid concentrations in older patients ( . ± . vs. . ± . , p= . ). there was no correlation between serum and follicular fluid bile acid concentrations and number of oocytes retrieved or number of mature oocytes, but there did appear to be a positive correlation between pre h-cg serum bile acid concentration and number of fertilized oocytes (spearman's correlation coefficient . , p= . ). conclusions: this is the first demonstration of the presence of bile acids within human ovarian follicular fluid. there may be a relationship between bile acid concentration and fertility potential. the precise function of bile acids in human ovarian follicular fluid is under investigation. objective to investigate the impact of ovarian hyperstimulation treatment on the biomarkers of the homocysteine pathway in blood and follicular fluid, and their association with the follicle diameter as a measure of follicular maturation. methods in women undergoing ivf/icsi treatment blood samples were collected on cycle day and the day of hcg administration. during oocyte retrieval in each woman the diameter of two follicles was measured and the corresponding follicular fluids were collected. in blood and follicular fluid total homocysteine (thcy), folate, cobalamin and pyridoxal' 'phosphate (plp) concentrations were determined. women with a serum folate . nmol/l were classified as folic acid supplemented. results ovarian hyperstimulation significantly decreased thcy and cobalamin blood levels (both p . ). the blood and follicular fluid concentrations of thcy, folate, cobalamin and plp were significantly correlated (all p . ). in the total group, a two-fold increase of thcy in follicular fluid resulted in a . mm decrease of the follicular diameter (p . ). in non-supplemented women this decrease was . mm (p . ). in supplemented women a twofold increase of follicular fluid folate resulted in a . mm decrease of the follicular diameter (p . ). conclusions ovarian hyperstimulation reduces thcy blood levels independent of folic acid supplementation. however, high follicular fluid thcy and folic acid supplementation may have detrimental effects on the maturation of the follicle. ), post-fixed for rain in % w/v osmium tetroxide in the same buffer, quickly dehydrated in a series of ethanol solutions, and embedded in epon. thin sections were stained with uranyl acetate followed by lead citrate and were observed at electron microscope. results: ten blastocysts from each group were collected and analyzed. compared to the in vivo embryos, blastocysts generated in vitro exhibited significant differences. the surface of their trophectoderm (te) layer had a reduced number of microvilli, the number of the apoptotic cells in the inner cell mass (icm) was higher and the presence of non functional mitochondria was elevated. in this study we have, for the first time, compared the ultrastructure of the in vivo and in vitro conceived blastocysts. taken together, these results suggest that both, the higher rate of apoptosis and the morphological alterations in the mitochondrial structure in ivf embryos, are associated with stress during in vitro embryo culture. therefore, these parameters can be used, in the future, as markers for the assessment of the embryo wellbeing in the ivf settings. deteriorating oocyte quality is a critical hurdle in the management of infertility, especially one associated with advancing age.here, we explore a newly discovered role of nitric oxide (no) in the sustenance of oocyte quality. methods: sibling oocytes from superovulated mice were subjected to intracytoplasmic sperm injection (icsi) with cauda-epididymal spermatozoa following exposure to either the no donor, s-nitroso n-acetyl penicillamine (snap, . m/min); a soluble guanylyl cyclase inhibitor, h-[ , , ] oxadiazolo [ , -a] quinoxalin- -one (odq, m) or an no synthase inhibitor, n w -nitro-l-arginine methyl ester (l-name, mm). their sibling oocytes were subjected to icsi either before (young) or after culture for the corresponding period (old). outcomes of fertilization, cleavage and development to the morula and blastocyst stages were compared. some embryos from each subgroup were also subjected to tunel assay for apoptosis. results: oocytes deteriorated in their ability to undergo normal fertilization and development to morulae/blastocysts after aging in culture, as compared to their sibling cohorts subjected to icsi immediately after ovulation (p< . ). this deterioration was prevented after oocyte exposure to snap. while, exposure to l-name or odq resulted in a significant compromise in fertilization and development to the morulae/blastocysts (p< . ) with detection of apoptosis, which was also noted in embryos derived from aged oocytes but not in those from young or snap exposed oocytes. conclusions: no is essential to sustain oocyte fertilizability and developmental ability, and to prevent blastomere apoptosis. objective to investigate the effects of the levels of the biomarkers of the homocysteine pathway on ivf outcome. methods from women undergoing an ivf or icsi procedures, two blood samples and two mono follicular fluid samples were collected for determination of folate, cobalamin, and total homocysteine (thcy). total protein was determined in follicular fluid to adjust the biomarker concentrations for follicular maturation. primary endpoint of the study was oocyte quality measured by fertilization and embryo quality (range - ; with being best quality). secondary endpoint was the occurrence of pregnancy. results % of the included women used a folic acid supplement (serum folate . nmol/l). in non-supplemented women higher cobalamin levels in follicular fluid correlated with a better embryo quality (estimate - . ; p . ) and higher thcy levels (median . mol/l, range . - . ) correlated with a worse embryo quality (estimate . ; p . ). in supplemented women higher follicular fluid thcy (median . mol/l, range . - . ) correlated with better embryo quality (estimate - . ; p . ). the follicular fluid folate level of oocytes that did not fertilize was . -fold higher than in the fluids of a fertilized oocyte ( % ci . - . ; p . ). a two-fold increase of follicular folate corresponded with a . higher chance to achieve pregnancy ( % ci . - . ). conclusions cobalamin levels in follicular fluid are correlated with embryo quality. folic acid supplementation modifies the thcy and folate levels in follicular fluid and thereby affects oocyte quality. the level of folate in follicular fluid is important in the fertilization process. we recently reported that increasing relative oocyte immaturity is associated with worsening outcome, and that cycles with many immature oocytes are more common in younger women. (moore et al, asrm annual meeting, ) to further investigate this trend, we conducted a case/control analysis of patients with repeated cycles of high-level oocyte immaturity (hloi). methods: oocyte maturity data was collected on all icsi cycles starting in . we defined a cycle with hloi as having > % immature oocytes (> sd's above the median). a case was defined as a patient with hloi on more than one cycle. control subjects were age-matched and defined as having </= % ( sd above median) immature oocytes on all cycles. results: from subjects, we identified cases of recurrent hloi (comprising icsi cycles) and control subjects. at baseline, cases had more oocytes retrieved per cycle than controls ( . vs. . , p<. ). all other baseline characteristics were similar. all case subjects were younger than . outcomes are shown in table . discussion: clinical pregnancy and live births were reduced in the case group, but more than expected based on % immaturity. during cycles, there were no live births among the case group. patients with recurrent hloi represent a previously undescribed group of young patients with a very poor prognosis during icsi treatment. this study may suggest that the problem underlying the immaturity may relate to overproduction of oocytes during stimulation, a factor which may be modifiable. however, the additional trend (though not significant) that even the mature oocytes from the case patients tend to have poor outcomes suggests the possibility of a maturation defect and warrants further consideration. data on response to gonadotropins, fert rates, and implantation rates will be presented. amongst those, were performed for an initial diagnosis of premature ovarian aging (poa), for the diagnosis of premature ovarian failure, and for repeated pregnancy loss. in women under age , b-fsh levels over miu/ml and up to < miu/ml denoted a diagnosis of poa, while levels of miu/ml were considered diagnostic for pof. all patients signed consent permitting review of medical records for clinical research purposes. results: day fsh levels ranged from . to . miu/ml (mean . ± . miu/ml). amh levels ranged from < . to . ng/ml (mean . ± . ) and allele repeats ranged from to , (mean for allele- , . ± . ; allele- , . ± . ). mixed model analysis of variance for all patients, adjusted for age, race, and allele- revealed a statistically significant correlation between b-fsh levels and number of repeats in allele- ( figure ; p < . ). we did not observe a significant linear relationship between serum amh levels and cgg repeat counts, however cgg repeat counts above were significantly associated with serum amh levels less than ng/ml (chi square = . ). conclusions: triple repeats above , a level currently considered well within normal, are associated with evidence of premature decline in ovarian function. the evaluation of triple repeats in frm gene offers a first objective assessment of ovarian function and should be predictive of autologous reproductive life span in most women. objective: the levels of mis in the serum and in the ovary have been demonstrated to decrease in response to increasing doses of cisplatin. we hypothesize that ovarian stimulation also will reveal dose related decreases in ovarian and serum mis levels after cisplatin exposure, further demonstrating the follicular damage from chemotherapy. methods: adult female sprague-dawley rats were treated with saline, . mg/kg cisplatin or . mg/kg cisplatin as two weekly injections. five days following the last cisplatin injection, the rats were injected with pregnant mare serum gonadotropin (pmsg) and euthanized hours following the pmsg injection. serum was collected and both ovaries were obtained for protein analysis. serum and ovarian levels of mis were determined using an elisa kit. western blot analysis, immunohistochemical analysis, and tunel analysis were also performed on the ovarian proteins. results: stimulated mis levels declined in a linear fashion in response to increasing cisplatin doses (p< . ). ovarian protein levels measured by elisa and western blot both indicated that stimulated ovarian mis levels also decrease in a linear fashion (p= . and p< . respectively.) immunohistochemical analysis revealed that while the total number of follicles remains the same, both the total number of mis positive follicles, and the percentage of mis positive follicles declines in a linear fashion (p= . for both). tunel analysis reveals that there is no change in the incidence of apoptosis in the stimulated ovaries from any treatment group. conclusions: the administration of pmsg after treatment with cisplatin causes a dose dependent decrease in the levels of mis in both the serum and in the ovary. serum mis levels following ovarian stimulation may be useful as a serum biomarker in this animal model to assess ovarian damage following the administration of cisplatin. correlations of micro-rnas a, b, - pp, and - p with inflammatory and steroidogenic factors in human granulosa/cumulus cells. tannaz toloubeydokhti, orhan bukulmez, kenneth c drury, r stan williams, nasser chegini. obstetrics and gynecology, university of florida college of medicine, gaineville, fl, usa. as part of the novel endogenous rna silencing machinery, noncoding short rna strands, referred to as micrornas (mirnas), have been identified to regulate the stability of the target gene expression through degradation and repression. the expression of many of these mirnas has been identified in human tissues however, their biological significance in various cellular processes remains to be elucidated. in this cross-sectional study of human granulosa cells, we aimed to study the potential correlations between five selected mirnas, mir- a, mir- b, mir- - p, mir- and mir- - p, and mrna expressions of some of their predicted target genes namely, cyclooxygenase (cox)- , il- , steroidogenic acute regulatory protein (star), aromatase and estrogen receptor (er) . granulosa/cumulus cells were obtained from women (age range - ; mean age . ± . ) undergoing oocyte retrieval for assisted reproduction. total rna was extracted from these cells and reversed-transcribed and real-time pcr was performed to measure steady-state levels of mirnas and mrna transcripts. we observed that the expression of mir- a, mir- - p, mir- and mir- - p displayed a significant and positive correlation with the expression of cox- . moreover, mir- b significantly correlated with star mrna expression, but not with other genes tested (p< . ). with advancing age, the expression of mir- - p and cox- was significantly decreased, with cox- expression displaying a positive correlation with aromatase, star and er mrna expressions (p< . ). none of the factors tested showed any significant correlations with peak estradiol levels and number of oocytes retrieved. receiver operating characteristic curve analysis revealed that female age cut-off value of . y best predicted whether mir- - p was below its mean arbitrary value. in conclusion, given the importance of mirnas' regulatory function in gene expression, our results suggest that mir- b may play an important role in gene expression related to granulosa cell luteinization, while variation in mir- - p expression with female age may suggest its potential role as a predictor of oocyte quality and granulosa cell function. given the importance of mirnas in gene regulation, the role of mirnas in ovarian physiology and aging requires further investigation. support: nih grant . introduction: a symbiotic relationship between ovarian granulosa cells (gc) and the enclosed developing oocyte is critical to reproductive efficiency. genetic modulations in gc's can lead to reproductive insufficiency, highlighting the critical role of gc's in reproductive competence. defects in the oocyte-gc repertoire in women with dor include lower e levels, luteal deficiency, poor fertilization rates, higher incidence of aneuploidy, lower pregnancy and higher miscarriage rates. utilizing global gene expression analyses in cumulus gc's, we attempt to enhance our understanding of biological mechanisms that may contribute to poor reproductive capacity in young women with dor. methods: infertile women (< years old) undergoing ivf were prospectively enrolled into two groups based on ovarian reserve (normal reserve, fsh< dor, fsh > miu/ml). at egg retrieval, cumulus gc's were isolated, rna extracted transcribed into cdna. microarray targets were generated cdna hybridized to affymetrix human genome u plus . genechips. microarray data were analyzed (array assist) and normalized (robust multichip analysis). a difference in gene expression of > fold was considered biologically relevant. results: of the , genes identified to be differentially expressed in young women with dor compared to normal reserve, genes demonstrated consistency of expression across five different normalization schema; were down regulated and up-regulated. expression of gremlin, a member of the dan family of genes known for its highly regulated expression pattern during folliculogenesis, was noted to be down-regulated -fold over two probe sets (- . ) in women with dor versus normal reserve; this down-regulation was confirmed by real-time pcr (- . ) . conclusions: this is the first demonstration linking differential expression of gremlin with etiology of infertility in women. gremlin is a downstream effector of oocyte-derived gdf- which facilitates cumulus cell expansion, a critical event in reproductive physiology. our finding of a significant downregulation of gremlin expression in cumulus gc's associated with dor may partly explain the physiology of poor reproductive performance. nih k cd . nih k rr . ferring pharmaceuticals. the objective of the present study was to investigate the effects of the gnrh antagonist ca on expression of mis and aromatase (cyp ) using luteinized human granulosa cells obtained during in-vitro fertilization cycles and an immortalized human granulosa cell line (hgl ). the granulosa cells were cultured +/-dibutyryl camp ( mm) and incubated +/-the gnrh antagonist, ca, for - h. rna was isolated, reversed-transcribed and real-time pcr was performed to measure mrna transcripts for ovary-specific cypiia and mis. we observed that camp markedly induced aromatase mrna in both the primary and immortalized human granulosa cells. interestingly, camp treatment of these cells also caused an upregulation of mis mrna. objectives: bisphenol a (bpa), a known endocrine disruptor, is a chemical used as a plasticizer in the manufacture of polycarbonate plastics and epoxy resins and is present in multitude products, including the interior coating of food cans, milk containers, and baby formula bottles. bpa can leach into foods during heat processing and is known to exert a variety of endocrine-like effects on different cell types acting as an estrogen because it contains two hydroxyl groups in its diphenyl structure. in this study we focused on the effects of bpa on aromatase expression and estradiol production in the human granulosa kgn cell line. we also evaluated its effects on several transcription factors crucial in cyp expression. materials and methods: kgn cells were cultured in f- dmem and were starved for h before experiments. subsequently they were treated for h with vehicle (control), fsh ( ng/ml), and/or bpa ( , , , , um) . messenger rna expression was quantified by real time pcr and estradiol secrection was measured in supernatants by elisa. results: fsh induced a -fold increase in aromatase expression. bpa induced a dose-dependent decrease in cyp production, with the greatest effect at um (p< . ), resulting in +/- % (mean+/-sem) inhibition, compared to aromatase expression induced by fsh alone. bpa also reduced levels of estradiol secretion in a dose-dependent manner, with the greatest inhibition at um (p< . ) resulting in +/- % decrease. we also evaluated expression of transcription factors known to be involved in regulating the activity of the ovary-specific aromatase proximal pii promoter. interestingly factors known for induction of aromatase such us steroidogenic factor- and gata- , mimic the pattern of cyp expression after bpa treatment, whereas, other receptors previously reported to act as aromatase inhibitor, such as ppar gamma were up-regulated by the addition of bpa. moreover, expression of creb remained virtually intact, suggesting that most likely mechanisms governing endocrine disruption by bpa are highly selective. conclusions: bpa inhibited fsh stimulated aromatase expression and downstream estradiol secretion. we propose that constant exposure to this chemical may result in endocrine disruption which may contribute to reduced fertility and early ovarian senescence. oocyte maturation occurs during folliculogenesis as a result of complex cell-tocell communications between the granulosa cells and the oocyte. maintaining the granulosa cells' spherical structure and network of gap junctions surrounding the oocyte is critical. this project tests the ability of a novel substrate-free threedimensional culture system to form viable granulosa cell spheroids. methods: after irb approval, freshly obtained follicular fluid from in vitro fertilization was obtained and granulosa cells were purified by percol gradient. nonadhesive agarose hydrogels, containing cylindrical round bottom recesses m in diameter, were cast from micro-molds designed using computer-assisted rapid prototyping. granulosa cells seeded at a density of , cells per gel were incubated for up to days. cellular viability was assessed with live:dead assay. results: after three days in culture, granulosa cells formed spheroids of densely packed cells that were difficult to disperse with multiple pipettings. the cells remained viable for at least days. conclusions: granulosa cells can be cultured in a novel substrate-free threedimensional culture system. the cells form tightly adherent spheroids that remain viable for extended culture. the cohesiveness of the cells suggests the formation of gap junctions. this is under investigation with immunohistochemistry and electron microscopy. these experiments suggest that a substrate-free threedimensional hydrogel culture system may be ideal to reassemble follicular structure important for future in vitro evidence testing and oocyte maturation. known to function as responders to pathogens, inflammation, and tissue injury. previous studies in our laboratory demonstrated that saa was produced in mouse granulosa and production was regulated by cytokines. ovulation has long been considered an inflammatory reaction and patients with chronic inflammatory conditions often experience infertility. the present study was undertaken to explore the role of saa in human ovarian function. methods: ovarian granulosa and luteal cells were obtained from surgically removed specimens and mural and cumulus granulosa-luteal cells were obtained from ivf aspirates. rna was extracted from fresh or cultured cells. some cells were treated in vitro with tnf or other cytokines for h. expression of saa was assessed by quantitative rt-pcr. in addition, serum levels of saa were determined using a commercial elisa in women undergoing ovulation induction (oi) and art. saa levels were measured at baseline, during oi, on the day of hcg administration and at the time of the pregnancy test. results: saa mrna was expressed in theca, granulosa, and granulosa-luteal cells. in granulosa-luteal cells both saa and saa mrnas were expressed at higher levels in cumulus compared to mural granulosa. expression of saa and saa in theca was increased following treatment with tnf in vitro. serum levels indicated that patients with ovulatory dysfunction had increased levels of saa at the time of hcg injection while patients without ovulatory dysfunction had lower saa levels as compared to the baseline level. in addition, patients undergoing oi who achieved pregnancy exhibited increased levels of saa at the time of the pregnancy test compared to baseline levels, whereas patients who did not become pregnant had lower post-cycle levels of saa. interestingly, saa levels did not change in art patients that became pregnant without undergoing oi (donor egg or frozen embryo transfer) conclusions: human ovarian cells express saa mrnas which can be altered in vitro. serum levels of saa may correlate with the responsiveness of the ovary to gonadotropins as evidenced by altered levels in women with ovulatory dysfunction, and by an increase in pregnant patients following ovarian stimulation. yeh, beom su kim, felicia hercules, jennifer peresie, armando arroyo. gynecology-obstetrics, university at buffalo, buffalo, ny, usa. objective: cisplatin is a common chemotherapeutic agent given to women for treatment for a wide variety of cancers. we hypothesize that one mechanism by which cisplatin may cause damage to ovarian structures is by decreasing the amount of anti-oxidant activity in the ovary. we examined super-oxide dismutase (sod ), a critical anti-oxidant enzyme that has been shown to be affected by cisplatin in other tissues, in the ovaries of cisplatin treated animals. methods: adult female sprague dawley rats were injected with saline, cisplatin . mg/kg or cisplatin . mg/kg as weekly doses. five days following the last injection, the rats were euthanized and both ovaries were excised. one ovary was processed for immunohistochemistry and the other was processed for protein analysis using western blot techniques for sod . the anti-sod antibody was purchased from santa cruz. the immunohistochemical sections were scored using a semiquantitative h scoring method. results: immunohistochemistry analysis of the expression pattern of sod following cisplatin administration revealed that there was a significant linear decrease in a dose response pattern in the expression of sod in antral follicles and in corpora lutea (p< . for both). no change was found in the h score of sod in other ovarian structures. western blot analysis of sod in the ovaries following increasing doses of cisplatin revealed no changes in the overall protein levels of sod in the ovary. conclusions: this is the first report that administration of cisplatin causes changes in the expression pattern of sod in antral follicles and in copora lutea. cisplatin decreases the amount of sod available in these structures, possibly leading to increased oxidative stress and free radical damage, thereby leading to ovarian damage found after cisplatin administration. is there evidence for aromatase activity in the stroma of postmenopausal ovaries? mf landay, rh fogle, rb allen, s patel, fz stanczyk, rj paulson. ob/gyn, usc, keck school of medicine, los angeles, ca, usa. background: following menopause, the ovaries continue to secrete androgens and estrogens. we have recently confirmed the production of androstenedione, testosterone and estradiol (e ) up to ten years after menopause by measuring gradients from ovarian venous effluent and peripheral blood. anti-müllerian hormone (amh) and inhibin b have been shown to be markers of follicular activity. peripheral levels of these hormones have previously been found to be undetectable in menopause, suggesting the absence of follicular activity in the postmenopausal ovary. objective: to investigate if the postmenopausal ovary continues to demonstrate evidence of follicular activity as the source of steroid production. design: observational study materials and methods: eight subjects aged ± . yr (range - ) were enrolled. postmenopausal status was confirmed by preoperative fsh levels of more than u/l and/or amenorrhea greater than months. serum was collected from the ovarian veins during total abdominal hysterectomy and bilateral oophorectomy. peripheral blood was also collected pre-operatively, intraoperatively and postoperatively. all samples were analyzed for amh and inhibin b using elisas with sensitivities of . ng/ml and pg/ml, respectively. androgen and estrogen levels in these samples have previously been reported, and documented a gradient between ovarian venous effluent and peripheral serum in all cases. results: ) six patients had no detectable follicular activity by amh and inhibin b levels. ) one patient demonstrated detectable inhibin b levels with an -fold gradient between ovarian venous effluent ( pg/ml) and peripheral blood ( pg/ml), however no amh was detected. ) in one patient, aged and months postmenopause, both amh and inhibin b were detected. peripheral inhibin b levels were high at pg/ml. amh was detectable at levels of . ng/ml. conclusions: ) in the majority of patients, continued e and androgen production in the ovary occurs in the absence of follicular activity as detected by amh and inhibin b production. ) some patients have evidence of follicular function up to ten years after menopause. ) since e is produced in post-menopausal ovaries in the absence of follicular activity, these data provide evidence for aromatase activity in the stroma of post-menopausal ovaries. to elucidate the process by which prostaglandin f (pgf ) mediates luteal regression, this study examined the role that the transcription factor yin yang (yy ) and histone deacetylase (hdac ) play in altering luteal cholesterol uptake by the scavenger receptor class b type i (sr-bi), intracellular cholesterol transport by steroidogenic acute regulatory protein (star), and cholesterol processing by p side chain cleavage enzyme (p scc) expression. rat luteal cells ( days post-ovulation) were treated with pgf ( hr) and trichostatin a (tsa; hr), a potent hdac inhibitor. protein expression was measured post-treatment by western blot and cholesterol was localized via filipin staining. star and sr-bi promoter activation was also assessed in hek t cells treated with yy , myy , a deletion mutant lacking an essential region required for transcriptional repression, and tsa. pgf caused a significant -fold decline in star (p< . ), and smaller declines in sr-bi and p scc which occurred concomitantly with an increase in yy ( -fold, p< . ) and intracellular lipid staining ( % increase). in contrast, nm tsa treatment caused a dose dependent increase in sr-bi, star, and p scc protein levels, . -fold (p< . ), . -fold (p< . ), . -fold (p< . ), respectively, and a . -fold decline (p< . ) in intracellular lipid levels. tsa prevented the pgf -induced decline in sr-bi, star, and p scc expression. promoter analysis demonstrated that wildtype yy , but not myy , repressed sr-bi and star activation while the addition of tsa overcame the repressive effects. this study shows the critical role that yy plays in pgf induced luteal regression by recruiting a histone deacetylase to block three essential processes in steroid production. in luteal cells yy -mediated global repressive action prevents cholesterol metabolism by inhibiting cholesterol uptake, intracellular transport, and processing thus leading to functional and structural luteal demise. supported by nih hd and nih hl . regulation of intermedin expression in cycling rat ovary. madhu chauhan, rebekah elkins, chandra yallampalli. obstetrics gynecology, university of texas medical branch, galveston, tx, usa. background: intermedin (imd) is a ct/cgrp family peptide involved in a variety of physiological functions, including vasodilatation and fetoplacental growth. this peptide is expressed in a variety of tissues such as stomach, placenta, uterus, pituitary and ovary suggesting its different functions including in reproduction. imd gene has an estrogen response element and we have shown that the plasma concentration of immuno-reactive imd is elevated in rats with pregnancy. however, expression of imd in the ovary and its regulation during estrous cycle is not known. we hypothesize that imd is expressed in the ovary and its expression is hormonally regulated throughout the estrous cycle in rat. objective: ) to assess expression of imd mrna and its receptors components calcitonin receptor like receptor (crlr), and receptor activity modifying proteins, ramp and ramp mrna in rat ovary; on diestrus, proestrus and estrus stages of rat estrus cycle and ; ) to demonstrate immunohistolocalization of imd, crlr, ramp , ramp and ramp in rat ovary. methods: groups of sprague-dawley non-pregnant and pregnant rats on day of gestation were used in this study. non-pregnant rats were sacrificed on diestrus, proestrus and estrus stage. ovaries were collected and total rna was extracted using trizol method. rna was treated with dnase before performing the reverse transcriptase polymerase chain reaction (rt-pcr). immunohistolocalization of imd, ramp , ramp and ramp proteins were assessed in tissue sections of ovaries from pregnant rats sacrificed on day . results: ) imd mrna is regulated in rat estrus cycle and its expression is significantly downregulated in estrus stage compared to the diestrus and proestrus; )expression of imd receptor crlr is highest in the diestrus stage, followed by a decline in proestrus which further declined during estrus; ) expression ramp mrna is higher in proestrus compared to diestrus and estrus (p< . ) but there is no significant change in the ramp expression during the estrus cycle and ; ) imd, ramp , ramp and ramp are immunolocalized in rat ovary in granulose cells of all follicles and granulosa cells of the corpus luteum. conclusion: imd mrna and protein are expressed in rat ovary suggesting a possible role for imd in ovarian function. defining and defying deterioration in oocyte quality with advancing chronological age: role of nitric oxide. pt goud, ap goud, mp diamond, b gonik, hm abu-soud. div reprod endocrinology, dept ob/gyn, wayne state university, detroit, mi, usa; div maternal and fetal medicine, sinai grace hospital, wayne state university, detroit, mi, usa. nitric oxide (no) is a ubiquitous free radical essential for oocyte maturation, function and sustenance of oocyte quality post-ovulation. the current study investigates the role of no insufficiency in the causation of oocyte quality deterioration with advancing chronological age. methods: in set , oocytes were retrieved from the following superovulated b d f mice: (a) young breeders (yb, n= ); (b) retired breeders (rb, n= ), and © old animals (oa, n= ), aged - , - , and - weeks respectively; and studied for zona pellucida dissolution time (zpdt), spindle ( -tubulin fluorescence immunocytochemistry, sigma) and chromosome morphology (dapi, vector), ooplasmic microtubule (mt) dynamics (omd) in response to taxol [ ], and cortical granule (cg) status (rhodamine conjugated lectin, vector). in set (n= ), oocytes from retired breeders were studied after exposure in vitro to an no-donor, s-nitroso acetyl penicillamine (snap in m- , . m no/min , n= , h, °c, % co ), while their sibling control oocytes were cultured for corresponding period under identical conditions without snap. [ ]. zpdt, spindle and chromosome morphology, omd and cg status were assessed with a confocal microscope and compared between the subgroups using anova, chi square and/or fisher's exacts test and appropriate post-hoc tests. results: in set , a significantly fewer oocytes were retrieved per animal (mean) in rb ( ) and oa ( ) compared to yb ( . , p< . ). advancing age was also associated with a significant increase in zpdt, omd and cg loss in rb compared to yb (p< . ). furthermore, significantly fewer oocytes from rb than yb had normal spindle and chromosome morphology (p< . ). oocytes from oa had significant spindle and chromosome disarray, a near total cg loss and significantly harder zp (p< . ). these oocytes also exhibited diminished omd in response to taxol although, metaphases were exquisitely sensitive to disruption with taxol. in set , exposure to snap in oocytes from rb resulted in a significantly lowerzpdt, omd and cg loss, and significantly higher incidence of normal spindles ( the gamma-aminobutyric acid (gaba), its biosnthetic enzyme gad and gaba-a receptors have been found in the oviduct and ovary. objective: this study examined the expression of gaba-a receptor subunit genes and gad and whether the gaba-a receptor could alter cytosolic ca + in gc. methods: for qrt-pcr, both human cumulus and mural gc were obtained at the time of oocyte retrieval for ivf and cultured separately. total rna was isolated separately from each gc type and from human brain ( positive control). the total rna from patients was pooled for each gc type and all rna was treated with dnase i. two-step qrt -pcr was performed using gene-specific lux™ primers for all gaba-a receptor subunits, gad and gad plus gapdh. single and specific qrt-pcr products were verified by melting curve analysis, gel electrophoresis, and dna sequencing. for ca + study, gc were cultured on coverslips. gc were loaded with fura- -am and changes in ca + concentration of gc were studied using a dynamic digital ca + imaging system. gaba-a agonist, muscimol, was used to study any dose-dependent effects on gc. gaba-a antagonist, m bicuculline, were perfused min. prior to and during application of muscimol. the responses were represented as changes in the / nm fluorescence ratio over time. m atp was used as positive control. results: the qrt-pcr results indicated that all gaba-a receptor subunits were expressed to various degrees in both types of gc, with the expressed highest in both cell types. gaba-a receptor subunits showing the next highest expression in both cell types were , and . gad isoenzyme was more abundantly expressed in cumulus and mural gc than gad . ca + imaging showed that muscimol, had the ability to increase ca ++ in gc, about % gc (n= ) cells responded to muscimol. muscimol increased intracellular ca + in a dose-dependent manner. the muscimol responsive cells was reduced by bicuculline, from % to % (n= , p< . ). conclusion: qrt-pcr indicates that gaba-a receptor subunit gene and gad expression occurs in both cumulus and mural gc. the ability of bicuculline to inhibit the ca + response to muscimol suggests the activation of gaba-a receptor. the current study confirms the presence of functional gaba-a in gc for the first time, and suggests that gaba may exert trophic effects in the ovary via gaba-a receptor. the present study test the hypothesis that administration of l-nitro-l-arginine methyl ester (l-name), a nos inhibitor, prior to hypoxia prevents the hypoxiainduced activation of p mapk, erk and jnk in the cerebral cortex of the guinea pig fetus during gestation. to test this hypothesis guinea pig fetuses at and days gestation were assigned to normoxic (nx, n= ), hypoxic (hx, n= ) and hypoxic pretreated with nos inhibitor (hx+l-name, mg/kg i.p., n= ) groups. hypoxia in the fetuses was induced by exposing the pregnant guinea pigs at both gestational ages to an fio of . for min. cerebral tissue hypoxia was documented biochemically by determining the tissue levels of atp and phosphocreatine (pcr). neuronal nuclei were isolated, purified and proteins separated by sds-page, and then probed with specific phosporylated erk, jnk and p antibodies. in the days gestation group: expression of p-p was . ± . (nx), . ± . (hx) . ± . (hx+l-name). p-erk expression was . ± . (nx), . ± . (hx), . ± . (hx+l-name). p-jnk expression was . ± . (nx), . ± . (hx), . ± . (hx+l-name). in the days gestation group: expression of p-p was . ± . (nx), . ± . (hx), . ± . (hx+l-name). p-erk expression was . ± . (nx), . ± . (hx), . ± . (hx+l-name). p-jnk expression was . ± . (nx), . ± . (hx) . ± . (hx+l-name). the data show that administration of l-name prior to hypoxia decreased the expression of phosporylated p , erk and jnk at both gestation ages however expression of phosporylation was higher at term as compared to preterm. since a nos inhibitor prevented the hypoxia-induced phosphorylation of p , erk and jnk in both gestational ages, we conclude that the hypoxia-induced activation of p , erk and jnk in the cerebral cortical nuclei of preterm and term guinea pig fetus is no-mediated. we speculate that no-mediated modification of cysteine residue leading to inhibition of map kinase phosphatases results in increased activation of p , erk and jnk in the guinea pig fetus. (nih-hd , nih-hd and st. christophers foundation for children). the the endocannabinoid, anandamide (aea), is involved in the hormone-cytokine network that regulates implantation and early pregnancy maintenance with levels at the endometrial level considered a major checkpoint . high levels ( nm) in culture are associated with embryo death while plasma levels (> nm) at weeks in women undergoing ivf-et are associated with failed pregnancy . what is uncertain is whether systemic aea levels after spontaneous conception in women presenting with threatened miscarriage are predictive of pregnancy outcome. our aim was therefore to measure plasma aea levels in women presenting with threatened miscarriage and to relate these to outcomes. methods: plasma aea levels were measured using a sensitive and previously validated hplc-ms method at - weeks gestation in women (nonsmokers, bmi < kg/m ) presenting with threatened miscarriage and in whom a viable pregnancy was confirmed by ultrasound scan. results: nine of the women subsequently had a miscarriage. the plasma aea levels in those women who had live births was . -fold lower than that in those who subsequently miscarried ( . ± . nm versus . ± . nm, mean ± sem; respectively; p< . unpaired student's t-test). the roc analysis revealed an area under the curve of . ± . with a sensitivity of % ( %ci of . % to . %) and a specificity of . % ( % ci of . % to . %). using an aea level of . nm as the optimal cut-off point, a single plasma aea measurement provided a sensitivity of % and a specificity of % with a negative predictive value of % and a positive predictive value of % for subsequent miscarriage. conclusion: these findings suggest a possible predictive role for plasma aea in women presenting with threatened miscarriage. the data also indicate that systemic aea levels may reflect local endometrial levels and therefore the local hormonal milieu in the early stages of normal pregnancies and in those complicated by threatened miscarriage. introduction: follicle stimulating hormone (fsh) mediates cyclic follicle growth and development and is widely used for controlled ovarian stimulation. the ovarian response of different women to fsh is variable, ranging from poor response to ovarian hyperstimulation and has been partly attributed to two common variants of the fsh receptor (fshr). we have previously identified four abnormal fshr splicing products ( exon deletions and intron insertion) in women with low and high response to fsh. two of the splice variants, deletion of exon and deletion of exon , showed a correlation with low and high response to fsh, respectively. in the current study, we evaluated the functional competence of the mutant fshrs in vitro. methods: we established stable hek cell lines expressing wild-type (wt) and splice-variant (del) fshr under the control of the inducible tet on/off system. the cells were transfected with a camp-responsive luciferase reporter plasmid and stimulated with fsh. results: the subcellular distribution of all splicing variants was the same as the controls and the protein localized mainly on the cell surface. all four splicing variants showed markedly decreased camp activation compared to controls when stimulated with fsh. however, all variants were able to form functional heterodimers with the wt receptor when co-expressed. interestingly, the heterodimer containing the form of fshr lacking exon , found in patients with decreased response to fsh, resulted in lower intracellular camp compared with the wild-type homodimer. conclusion: our findings suggest that fshr variants can contribute to abnormal response to stimulation in certain women undergoing ivf treatment. the variants require the presence of wild-type receptor in order to initiate signaling in response to fsh. further analysis of this signaling cascade in granulosa cells is underway to estimate the final production of estrogen from these heterodimeric receptors. objective: to compare the proteome of human endometrium in the prereceptive versus the receptive phases of the menstrual cycle. m m: endometrial biopsies were collected and days after urinary lh surge in the same menstrual cycle from three fertile women (n= ). proteins were extracted using sample grinding kit (ge healthcare) and interfering substances removed using -d clean-up kit (ge healthcare). labelling was performed with cydye dige fluors (ge healthcare) and proteins were separated using difference gel electrophoresis (dige). for the isoelectric focusing, cm ipg-strips in the nonlinear range of ph - were used. the second dimension was carried out using sds-page. differentially expressed proteins were detected by image analysis using decyder v . and the statistical module eda (ge healthcare). the spots of interest were subject to protein identification based on in-gel digestion, maldi-tof/tof mass spectrometry and database searching. western blot analysis were performed in the same biopsies in order to validate some candidate proteins. results: table displays the differentially protein abundance between days lh+ and lh+ (> -fold change). of these proteins, were increased at lh+ in comparison with lh+ , whereas only proteins were decreased. stathmin was found more than -fold decrease in lh+ compared to lh+ in the three patients studied in the validation studies performed by western blot. conclusions: this study shows that the human endometrium has a differential protein repertoire during the window of implantation compared to the prereceptive phase. the role of these proteins in the molecular events directed to embryo implantation is under research. differential protein abundance in human endometrium ( the extracellular signal-regulated kinases and (erk / ) are a mitogen-activated protein kinase (mapk) subfamily that act as key links in eukaryotic intracellular signaling transduction. activated by phosphorylation in response to specific stimuli, erk / is known to play a role in the regulation of cellular proliferation and survival. the human myometrium is a tissue known to undergo cycle-dependent proliferative and apoptotic changes in response to sex steroids. we hypothesized that erk / activity is involved in mediating menstrual cycle-dependent changes in the myometrium. materials and methods: immunostaining for phospho-erk and total-erk was performed on myometrial tissues obtained from normal women (n= ) at different phases of the menstrual cycle. staining intensities were evaluated by hscore. myometrial smooth muscle cells were isolated and cultured from normal women and treated with vehicle, estrogen ( - m), and progesterone ( - m) for and minutes, and then subjected to western blot analysis for p-and t-erk. statistical analysis was performed using one-way anova. results: tissue staining revealed that p-erk was mostly nuclear in all tissues, while t-erk was cytoplasmic and nuclear. p-erk staining was significantly stronger in the secretory phase and strongest at the early secretory phase, compared to other phases (p< . ). t-erk staining intensity was moderatehigh without variation across the menstrual cycle. in cultured myometrial cells, progesterone significantly increased p-erk levels within and minutes (p< . ) when compared to control. our results suggest that erk activity in the human myometrium is regulated in a menstrual cycle-dependent manner. the increased phosphorylation in the secretory phase suggests the involvement of progesterone in erk activation, as supported by our in vitro results. this increased erk activity may play a role in regulating myometrial proliferation. measures of insulin resistance (ir) including: fasting serum insulin, gir and homa. measures of plasma levels of endothelin- (et- ), soluble intercellular adhesion molecule- (sicam- ), soluble vascular cell adhesion molecule- (svcam- ) and high sensitivity c-reactive proteins (hscrp). results: women with pcos exhibited significantly higher levels of et- (p < . ), sicamp- (p < . ), svcam- (p < . ) and hscrp (p < . ) as compared with age-matched controls, respectively. positive correlations were evident between et- and fai (r = . ; p < . ) but et- negatively correlated with shbg (r = - . ; p < . ). svcam- positively correlated with total t (r = . ; . ), hscrp correlated with: bmi (r = . ; p < . ), and homa (r = - . ; p < . ), respectively. conclusions: women with pcos exhibited abnormal levels of endothelial and inflammatory markers, which appear to be inter-related to hyperandrogenaemia. ( ), and a higher expression of fatty acid amide hydrolase (faah) in peripheral lymphocytes post-ovulation ( ), suggest an involvement of the endocannabinoid system in menstrual cycle control. our aims were to investigate changes in plasma aea levels and in endometrial faah expression throughout the menstrual cycle. methods: plasma aea levels were measured using a hplc ms/ms method from women, median age yrs (range - ) and bmi kg/m (range - ), with regular menstrual cycles, with no medical problem and not on any medication for the preceding months. the menstrual cycle was divided to early follicular d - (n= ), late follicular d - (n= ), ovulatory d - (n= ), early luteal d - (n- ) and late luteal phases d - (n= ). uterine biopsies were taken from hysterectomy specimens taken for benign conditions and subjected to immunohistochemistry for faah with polyclonal antibodies. results: aea levels were significantly higher around ovulation in comparison to the pre-ovulatory or post-ovulatory phases as shown in the figure. faah expression in the endometrial stroma was unchanged throughout the follicular phase but increased during the mid to late luteal phase reaching a maximum in the late luteal phase. a high aea level in the early follicular phase and during ovulation suggests a role for aea in ovulation. the lower levels of aea in the luteal phase, essential for successful implantation, may be regulated by increased faah expression at the uterine level. the modulation of plasma aea levels during the menstrual cycle strongly suggests that it is regulated by gonadal steroid hormones. ( , , , , , , and , as well as at pregnancy. binding activities of igfbp and their protein levels (igfbp , , , , , ) were assessed by western ligand blot (wlb) and western blot (wb), respectively. the glyscosylation and phosphorylation status of igfbps were examined by deglycosylation treatment. our results showed that both glycosylated and unglycosylated igfbp elevate in fetal and newborn rat and graduately decline to a lower level at day and kept lower constant level since then. interestingly, biding activity of glycosylated igfbp was not detected by wlb assay. the igfbp- cleaved products were observed after rat day age day , which associated with a decrease in full length of igfbp- , suggesting endoproteolytic processing may involved in decreasing igfbp content. igfbp- , with heterogeneous glycosylation, were appeared after age day and disappeared during pregnancy, recurrence again postpartum. glycosylation of igfbp has no effect on its binding activity. unglycosylated and glycosylated igfbp were constant in life time. physiologically constant igfbp were detected by wb in protein, but not by wlb in binging activity. conclusion: highly elevated circulation igfbp suggest physiological role in new born and early postnatal development. its binding activity are well regulated by its posttranslation modification, such as glycosylation of igfbp in inactivating binding activity and possible involvement of active endoproteolytic processing in maintaining binding active igfbp- at low background: cigarette smoking affects hormone biosynthesis, storage, release, binding, transport and clearance, resulting in changes in circulating hormone levels. we used metabolomics to analyze the effects of cigarette smoking and second hand smoke (shs) on changes in hormone levels in women of childbearing age. methods: this is a three arm study; women aged - years who are active smokers, exposed to shs (passive smokers), or non-exposed were recruited from the washington d.c. area. all women completed a detailed staffadministered questionnaire probing their medical history, occupational, lifestyle factors and diet. blood and urine samples were collected at the follicular phase. hormone profiles were determined using metabolomics for serum thyroxine and triiodothyronine and steroid hormones, as well as for cotinine using isotopedilution tandem mass spectrometry (lc/ms/ms-api ). in addition, all samples were analyzed for serum lh, fsh, tsh and creatinine. results: the relationships of cigarette smoking, age, relative weight, and dietary intake to serum thyroxine, triiodothyronine, estradiol (e ), estrone (e ), progesterone (p), -hydroxyprogesterone ( ohp),dehydroepiandrosterone (dhea), dehydroepiandrosterone sulfate (dheas), androstenedione, cortisol, -deoxycortisol, corticosterone, testosterone, aldosterone and vitamin d were analyzed using lc/ms/ms. mean tsh in non-exposed, shs-exposed and smokers: . , . , and . miu/ml respectively. similarly, mean t . , . ( % increase) (p= . ), . μg/dl (- %) in active smokers; (active vs. exposed p= . ). shs increased dhea levels ( % higher, p = . ), dheas ( % higher, p = . ), cortisol ( % lower, p = . ), aldosterone ( % lower, p = . ) and androstenedione ( % higher, p = . ). these data suggest that active smoking and shs can have a profound effect on serum t , adrenal steroid and sex hormone concentrations in women of childbearing age. objective: to determine the prevalence and predictors of the metabolic syndrome (mbs) among in saudi women with polycystic ovary syndrome (pcos) in comparison to women without pcos and to assess the role of androgens and insulin resistance (ir) in mbs development. design: a prospective case control study. setting: tertiary referral university hospital. subjects: six hundreds saudi women living in the jeddah area were classified as follows: with pcos and age-matched women without pcos. interventions: blood samples were collected from all women with or without pcos between : - : , after an overnight fast. main outcome measures: measures of abdominal obesity, blood pressure and that of serum levels of lh, fsh, tsh, ft , -ohp, -a, dheas, total t, free t, shbg, insulin, hdl-c, triglycerides and plasma levels of glucose. measures ir including: fasting serum insulin, gir and homa. results: age-adjusted prevalence of mbs was higher in women with pcos ( . %, % ci: . - . %) as compared with women without pcos ( . %, % ci: . - . %) (p< . ). in the same age group, the risk of mbs in women with pcos was greater than that for women without pcos (p< . ). markers of ir were significantly abnormal in women with both pcos and mbs in comparison to those without mbs (p< . ). the most common abnormal components of mbs in women with both pcos and mbs (after adjustment for age) were: decreased hdl-c ( . ± . %); increased triglycerides ( . ± . %); and increased bmi ( . ± . %), respectively. the prevalence of mbs from lowest to highest tertile of free t level was . , . and . %, respectively; in women with both pcos and mbs. in women with pcos, % exhibited all components of mbs; . % had components, and . % had components. conclusions: women with pcos exhibited significantly higher prevalence of mbs ( . -fold) as compared with age-matched control without pcos. ir is a possible common pathogenetic factor for both mbs and the pcos. it is suggested that more intensive screening and/or therapy monitoring of mbs among women with pcos should be part of the therapeutic modalities of the condition. case of sisters with complete androgen insensitivity syndrome and discordant mullerian remnants. jennifer l nichols, jennifer j gell, eric j bieber. reproductive endocrinology and infertility, geisinger medical center, danville, pa, usa. complete androgen insensitivity is an x-linked recessive disorder resulting in the abnormal expression of the androgen receptor. affected individuals are most commonly phenotypically female but genotypically male. the prevalence of this disorder is in , live male births. we present a case of complete androgen insensitivity in members of the same family with differing residual mullerian tissue. sister a presented at age for evaluation of primary amenorrhea. a karyotype revealed ,xy. an mri of the pelvis showed a hypoplastic uterus but no ovaries. this patient underwent laparoscopic bilateral gonadectomy and hemihysterectomy. on examination under anesthesia, she was noted to have a normal vagina with no cervix noted. at laparoscopic evaluation, she was noted to have bilateral elongated gonads and what appeared to be a remnant of uterine tissue. pathology revealed portions of immature testicles and fragments of smooth muscle in what grossly appeared to be the uterine remnant. the patient's total testosterone following surgery was noted to be elevated at . ng/ml. other laboratory evaluation showed fsh . miu/ml, lh . miu/ml, free testosterone . pg/ml, and estradiol . pg/ml. approximately two years later sister b presented at age for evaluation of primary amenorrhea. no uterus or ovaries were visualized on pelvic ultrasound. again a karyotype revealed ,xy. laboratory evaluation demonstrated fsh . miu/ml, lh . miu/ml, estradiol . pg/ml, total testosterone . ng/ml, and free testosterone . pg/ml. she underwent a laparoscopic bilateral gonadectomy. no uterus, cervix or pelvic masses were identified on exam under anesthesia. at laparoscopy, both gonads were visualized and removed without difficulty but no uterus was visualized. pathology reported two testicular and epididymal-like structures. this case demonstrates the presentation and laparoscopic results of complete androgen insensitivity syndrome discovered in two siblings. although both girls are genotypically male, they differ in the presence of vestigial mullerian tissue. the case shows with complete androgen insensitivity, as an x-linked defect, one should consider apparent sisters of affected individuals, as well as offspring of unaffected individuals with a family member diagnosed. background: anandamide (n-arachidonoylethanolamine, aea) is an endocannabinoid that binds to and activates the cannabinoid receptors, cb and cb and may have important roles in the regulation of human reproduction. the traditional lipid extraction methods used for aea are cumbersome, slow and of low efficiency. the aim of this study was to determine whether the use of a solid phase (spe) method of aea extraction from human plasma would offer any advantages over the traditional liquid phase (lpe) method. methods: pooled human plasma was obtained from the local blood transfusion unit and aliquots stored at - °c prior to extraction. an internal standard of . pmol of deuterated aea (aea-d ) was added to each plasma sample and aea extracted from aliquots on each occasion over three days using the lpe and spe methods. spe was performed with waters oasis hlb cc/ mg cartridges on a waters vacuum manifold. cartridges were activated with methanol and water, the samples applied and washed with % methanol at ml/min. aea was eluted with ml acetonitrile and the eluents dried under a stream of nitrogen before reconstitution in ml acetonitrile. aea levels were measured using uplc-ms/ms against authentic standards. results: these are shown in the table. conclusion: the spe method was -fold more efficient at extracting aea compared to the traditional lpe method. the intra-day and inter-day assay variability were similar for both techniques, although the spe method was quicker, cheaper and required less plasma to generate data similar to that from the traditional lpe method, suggesting that the spe method may be more efficient than the lpe method, and thus making it more suitable for routine analysis of multiple plasma aea samples. background: the precise role of the endocannabinoid, anandamide (narachidonoylethanolamine, aea) in reproduction has been hampered by difficulties in its accurate measurement. aea levels have previously been measured by tlc, gc-ms and hplc-ms but these are laborious. our aim was to improve the analysis of aea using uplc and tandem ms/ms with a standard isotope-dilution method , . methods: authentic non-labelled and deuterium-labelled aea (aea-d ) diluted in acetonitrile were maintained at °c before analysis and separation by uplc on a c ( . x mm) column maintained at °c using a gradient of % a, . min: % a, . min: % a, . min: % a, . min: % a with a flow rate of . ml/min. the mobile phases were a ( mm ammonium acetate, . % formic acid) and b (acetonitrile, . % formic acid). the analytes were quantified using multiple-reaction monitoring in positive ion mode with a quattro premier mass spectrometer. entry, collision and exit energies were - , and - ev, respectively. calibration curves were performed in triplicate with . pmol aea-d as the internal standard. transitions employed were . > . and . > . for aea and aea-d , respectively. results: calibration curves ( . to fmol aea on column; n= ) were linear, producing a mean (±sd) gradient of y = . ± . x, crossing the y-axis very close to the origin ( . ± . units). variability was limited, with an r = . . measurements were precise, fmol aea produced a cv of only . %, and the retention time was consistent at . ± . min (n= ). the limit of quantification (signal/noise> ) was . fmol on column and the limit of detection (lod) was . fmol on column (signal/noise= ). accuracy for . fmol, . fmol and fmol aea was . ± . %, . ± . % and . ± . %, respectively. conclusions: the method described is an improvement over other lc-ms/ms methods , with a lower lod [ . fmol v. fmol or fmol ] and shorter run time [ min v. min or . min ]. thus, an improvement in terms of speed, increased sensitivity and better reproducibility will allow for a more accurate assessment of aea concentrations in a number of biological samples. objectives: the gata family of transcription factors consists of six zinc-finger proteins with a critical role in tissue-specific and developmentally-regulated gene expression. gata factors exert their effects alone and through interactions with cofactors, such as friend of gata (fog), as well as with nuclear hormone receptors, including steroidogenic factor- (sf- ), a well-described activator of gonadotropin gene expression. the objective of these studies was to define the role of gata family members in the gonadotrope. methods: ) total rna was extracted from the gonadotrope cell line, l t , and analyzed by standard rt-pcr analysis. ) the cv- fibroblast cell line was transiently transfected by the calcium phosphate precipitation method with a rat - /+ lh promoterreporter vector as well as cmv-driven expression vectors for gata- , gata- , dngata- , fog- and/or sf- . results: gonadotrope l t cells were found to express transcripts encoding gata- , gata- , and gata- as well as fog- and fog- . gata- and gata- stimulated lh gene promoter activity by approximately -fold (p< . ) and synergistically increased the sf- response ( -fold versus -fold for sf- alone; p< . ). the gata-mediated increase in lh gene expression was nearly eliminated with co-transfection of fog- . similarly, co-transfection with a gata- dominant negative construct blunted wild-type gata- effects in a dose-dependent fashion. conclusions: these data demonstrate expression of both gata and the functionally related fog proteins in a well-characterized gonadotrope cell line. furthermore, they demonstrate a functional role for these factors in regulation of gonadotrope function, specifically expression of the lh gene. low-dose dexamethasone (dex) therapy early in pregnancy is used in fetuses with suspected risk of congenital adrenal hyperplasia. several adverse neurological events in prenatally treated children have been reported and the fetal hypothalamic-pituitary-adrenal (hpa) axis may be involved. aim: to investigate the immediate and long-term effects of early maternal dex administration on fetal growth and pituitary-adrenal activity in sheep. method: pregnant ewes carrying singleton fetuses (total n= ) were randomized to control ( ml saline/ewe) or dex treatment ( . mg/kg ewe weight) consisting of four intramuscular injections at -hourly intervals over hours on - days of gestation (dg). at , , , and dg fetal weights were recorded. i -ria, qrt-pcr and in-situ hybridisation were used to measure fetal plasma cortisol and acth levels and to analyse adrenal and pituitary mrna expression. results: dex-exposed fetuses were lighter than control animals at dg*, but not at other times; in general fetal organ weights were similar between treatment groups. fetal plasma acth was unaffected by dex and did not differ between genders. similarly, pomc and pc- mrna in pars distalis were unaltered after dex. however, fetal plasma cortisol was reduced after dex in both male and females at dg*, was similar at and dg, then elevated at dg*. plasma cortisol in female fetuses in control and after dex was significantly higher than in males. the increases in cortisol after dex at dg* were associated with increased fetal adrenal expression of p c and bhsd mrna in females, reduced expression of mc r in males, but no difference in star mrna. conclusion: we conclude that in sheep, early dex programs the developmental trajectory of the fetal hpa with increased activation directly of the adrenal, but not pars distalis function. in females this effect may be attributed to increased fetal adrenal steroidogenic activity. the effect of dex in increasing cortisol in males, albeit at a significantly lower level than in females, appears to be independent of the enzymes that we have measured.*p< . . synaptophysin and gonadotropin-releasing hormone (gnrh) are colocalized in rat hypothalamus. armando arroyo, beom su kim, amanda biehl, blenna cl bett, , john yeh. gynecology-obstetrics, university of buffalo, buffalo, ny, usa; physiology and biophysics, university at buffalo, buffalo, ny, usa. the cellular and molecular mechanisms that control gonadotropin-releasing hormone (gnrh) release are not completely understood. gnrh is stored in synaptic vesicles and released by exocytosis at gnrh nerve terminals. there are currently nine families of synaptic vesicle proteins that are involved in neurotransmitter release by exocytosis. synaptophysin is one of the most common synaptic vesicle proteins present in synaptic vesicles in neurons. the hypothesis of this study is that synaptophysin is expressed in gnrh neurons. we obtained sections from the hypothalamus of female sprague dawley rats. double-label fluorescence immunohistochemistry was performed on free-floating sections. sections were incubated with a mixture of mouse monoclonal antibody against gnrh ( : -chemicon international) and with a rabbit polyclonal antibody against synaptophysin ( : -santa cruz biotechnology) for h. after incubation the sections were washed and incubated with a mixture of alexa conjugated goat antimouse and alexa conjugated goat antirabbit ( : ; molecular probes) for h. slices were visualized with confocal microscopy (zeiss lsm- ). fifteen out of a total of fifteen gnrh cell bodies in the medial preoptic area and most gnrh neuron terminals in the median eminence showed intense immunostaining for synaptophysin. this is the first study to demonstrate that synaptophysin is expressed in rat gnrh neuron terminals. this suggests that synaptophysin is present in gnrh neuron vesicles. thus, gnrh release by exocytosis may be mediated by synaptic vesicle proteins. objective: our aim was to identify the effects of early gestation gc exposure on fetal and postnatal hpa axis development and function in postnatal life. method: pregnant ewes carrying singleton fetuses were randomized to control ( ml saline/ewe) or dex groups ( . mg/kg), consisting of four at h intervals on days - of pregnancy. at months postnatal age, catheters were implanted; a bolus injection of crh ( . mg/body weight) and avp ( . mg/body weight) were administered and arterial blood samples were taken at - , - , , , , , , , and min. levels of hepatic cbg mrna were determined by qpcr, expressed relative to s rrna. plasma cortisol and cbg levels were measured by radioimmunoassay. results: both total and free cortisol levels in the dex females (n= ) were lower than in dex males (n= ) from to min (p . ) and lower than in control females (n= ) at minutes (p . ), also in the dex-m group were higher than in control males (n= ) at min (p . ). plasma cbg levels and cbg mrna expression were not altered by dexamethasone exposure or sex. conclusions: these findings suggest that prenatal glucocorticoid exposure alters the development of the hpa axis differentially according to the sex of the exposed fetus. to determine maternal injections of synthetic glucocorticoids in early gestation can alter expression of hippocampal corticosteroid receptors at months postnatal age. method: pregnant ewes carrying singleton fetuses were randomized to control ( ml saline/ewe) or dexamethasone treatment ( . mg/kg) consisting of four injections at h intervals on days - . hippocampal was collected from the offspring at months postnatal age. levels of mrna of gr and mr were determined using qpcr and levels relatived to s rrna. results: dexamethasone-treated male animals (n= ) had significantly higher levels of mr gene expression than both control males (n= ; p= . ) and females (n= ; p= . ). gr gene expression levels were higher in treated vs. control males (p= . ), but in females levels in treated and control animals were similar. total body, brain and hippocampus weights were similar. conclusions: maternal dexamethasone administration in early pregnancy resulted in gender-dependent changes in hippocampal gene expression when measured in the offspring seven months after birth. objective: diminished ovarian reserve (dor) affects many younger women. we analyzed superovulation with intrauterine insemination (so) cycles in vitro fertilization (ivf) cycles of women with dor to determine if women < with dor differ from their older counterparts. method: irb approved retrospective review of so ivf cycles from / - / with follicle stimulating hormone (fsh) levels based on age < or . results: so cycles were performed in women. no differences were noted in clinical pregnancy. women < were more likely to have inseminates with a lower mean tms/ins, median tms/ins was . among pregnancy cycles compared to for unsuccessful cycles. for ivf, mean total gonadotropin dosage was significantly lower in women < , mean number of follicles mm peak e were significantly higher in women < . so ivf measures are in tables , respectively. conclusions: similar clinical pregnancy outcomes were seen despite age. in ivf, women < required less gonadotropins and generated more follicles but with no significant difference in number of mature oocytes or clinical pregnancies. of note, pregnancy rates for so in women < with dor are substantially lower than expected in our clinical practice. as ivf yielded a substantially higher chance of pregnancy, consideration should be made to expedite progression to ivf. to assess the effects of intraovarian injection of ad-fshr on the reproductive system of fshr (-/-) mice. methods: about l containing x pfu of ad-hfshr were injected directly into each ovary of treated group and same amount of ad-lacz were injected into the ovaries of control animals. vaginal smears were collected and body weight was measured daily. four weeks after the injection animals were sacrificed and all organs were weighted and evaluated by h e. fsh, e and p measured before and after treatment. results: ad-hfshrtreated mice showed obvious estrogenic changes in vaginal smear while in control animals vaginal smear remained at diestrus stage. significant increase in total body weight and estrogen dependent organs weight (uterus, ovary, vagina) was observed in treated animals compare to control group (p< . ). no significant weight changes were observed in other organs. h e evaluation of the ovaries showed significant increases in both the total number of follicles and the collective diameter of the follicles in treated animals compare to controls. on average follicles/ovary were observed in ad-hfshr-treated group of which follicles were at the antral stage while only follicles observed in ad-lacz control group, with zero follicles at antral stage. a . to folds increase in e and about % decrease of fsh observed in treated animals compared to control mice. there was no significant change in serum progesterone level between treated and control groups. conclusion: intraovarian injection of an adenovirus expressing human fshr gene is able to restore folliculogenesis and resume estrogen hormone production in female forko mice. objective: the sentinel issue surrounding multiple gestations following ivf is the inability to precisely estimate the reproductive potential of individual embryos with the currently used embryo grading systems based on embryo cleavage rate and morphology. recently, metabolomic profiling of spent culture media using raman and near-infrared spectroscopy have been reported to predict reproductive potential of embryos. in this study we applied proton nuclear magnetic resonance (¹h nmr) spectroscopy to analyze metabolomic profile of embryo culture media and to identify components of the media that correlate with reproductive potential. methods: eighteen spent media samples from embryos that failed to implant, and samples from embryos that resulted in pregnancy and delivery were individually collected after embryo transfer on day , and evaluated using ¹h nmr. the spectra obtained were analyzed using a selective genetic algorithm (ga) to determine regions predictive of pregnancy outcome as determined by logistic regression. to avoid random correlations, a leave-one out crossvalidation was used. sensitivity and specificity of predicting pregnancy (described as implantation and delivery) were calculated. results: using the ga, two areas in the ¹h nmr spectral region were identified as most discriminatory between the two groups. viability indices calculated by ¹h nmr using these regions were significantly higher in culture media of embryos with proven reproductive potential ( . ± . ) compared to those that failed to implant ( . ± . ) (p< . ). compiled outcomes from the leave-one-out cross-validation of the logistic regression using the ¹h nmr measurements resulted in a sensitivity of % and a specificity of . %. quantification by integration showed significantly decreased glutamate levels (p= . ) and a trend toward an increase in pyruvate levels (p= . ) in culture media of embryos that did not cause pregnancy. conclusion: metabolomic profile of spent embryo culture media using ¹h nmr correlates with the reproductive potential of embryos. the lower glutamate levels detected in culture media of embryos that failed to implant could potentially be due to the toxicity associated with increased embryonic glutamate uptake. additional studies using complementary approaches are needed to further delineate molecular components associated with reproductive potential. objective: beta-carotene and other carotenoids are known antioxidants previously identified in human follicular fluid (ff). in addition to their inherent antioxidant properties, carotenoids have been identified as precursors of the antioxidant retinol in bovine ff. high retinol levels in bovine ff are associated with non-atretic follicles. this would suggest a possible role for retinol and its carotenoid precursors in follicular heath and the general oxidative state of the follicle. we sought to measure carotenoids and retinol in the ff of women undergoing ivf and correlate these levels with normal fertilization as a marker of follicular/oocyte health. design: prospective cohort study materials and methods: ff from a single - mm follicle was obtained from women (age - ) undergoing ivf and intracytoplasmic sperm injection (icsi). serum was also obtained at the time of oocyte retrieval. retinol, vitamin e ( , , and tocopherol) and carotenoids ( -carotene,cryptoxanthin, lycopene and lutein/zeaxanthin) were measured using hplc. we correlated ff carotenoid and retinol levels with oocyte fertilization status following icsi. results: as previously reported, retinol, vitamin e and carotenoids were all identified in the ff. each fat-soluble vitamin level was significantly lower in ff compared to serum (p< . for all analytes) and the levels were strongly correlated (r > . , p< . for all analytes). mean levels of ff -carotene were significantly higher in those follicles that resulted in a fertilized oocyte ( . +/- . g/ml vs. . +/- . g/ml, p= . ). other carotenoids, retinol, and vitamin e levels did not correlate with fertilization outcomes. conclusions: our finding of a strong association between ff -carotene concentration and subsequent normal fertilization of the oocytes suggests an important antioxidant role for -carotene in the health of the human ovarian follicle/oocyte. the lack of correlation with other carotenoids, retinol and vitamin e suggests that the antioxidant properties of -carotene act by preventing singlet oxygen and scavenging the peroxyl radical and may directly influence oocyte competence. mature oocytes obtained during egg retrieval have been shown to undergo spindle depolymerization as a result of cooling. for this reason, ivf programs strive to maintain constant temperature during follicle aspiration. we sought to elucidate if there was a difference in temperature of fluid aspirated into a hand held (hh) or heating block (hb) encased collection tube. the experiment was performed in an ambient temperature of °c. thermistors, pinhead sized sensors with an accuracy of %, were used to monitor temperature differences between control fluid (cf) (sterile water ºc) and aspirated fluid. data was recorded using an -channel data acquisition system from dataq instruments. one thermistor was placed into the cf, the other was placed into an empty collection tube set in the heating block or held in a gloved hand. a cm, gauge, single lumen needle connected to cm of tubing was used to aspirate into the empty collection tube. temperature was recorded x/second, each experiment was repeated times. baseline empty collection tube temperature was significantly cooler in the hh vs the hb group ( . ± . °c vs . ±. °c, p=. ). two seconds after aspiration, the lowest aspirate temperature was observed, (hh . ±. °c, hb . ±. °c, p>. ) no difference between groups. in both groups, temperature quickly increased as aspiration progressed (hh . ±. °c, hb . ±. °c, p>. ). the hb group took an average of . min to return to baseline ( °c), the hh group never returned to baseline. substantial cooling of aspirated fluid occurs during oocyte retrieval, with a mean temperature decrease of . ±. °c corresponding to . °c. considering this dramatic decrease, the difference between temperatures in the hh vs. the hb group is negligible. current aspiration systems poorly regulate temperature, thus the choice of aspirating into a test tube warmed by hand or by heating block is inconsequential. clinical management of twin gestations with recurrent preterm labor symptoms. lesley de la torre, luis roca, niki b istwan, debbie j rhea, gary j stanziano, victor hugo gonzalez-quintero. obstetrics and gynecology, university of miami medical center, miami, fl, usa; clinical research, matria healthcare, marietta, ga, usa. objective to examine pregnancy outcomes in women with twin pregnancies receiving nifedipine tocolysis (nt) who experienced recurrent preterm labor symptoms (rptlsx). study design twin pregnancies enrolled for outpatient preterm labor (ptl) surveillance services prescribed nt following an initial episode of ptl were identified from a database (n= ). eligible for inclusion were patients later hospitalized with acute rptlsx (n= ). included were those < weeks' gestational age (ga), with intact membranes, remaining undelivered for > hours after rptlsx . pregnancy outcomes of women resuming nt (rnt group, n= ) following hospitalization were compared to those having an alteration in treatment (alttx group, n= ) to continuous subcutaneous terbutaline. per normality assumptions, either independent student´s t or mann-whitney u test statistics were used for continuous variables; pearson´s chi-square for categoric. all p-values presented as two-sided, significant at < . . results overall, ( . %) of twin pregnancies prescribed nt experienced rptlsx; ( . %) were not eligible for continued tocolysis. pregnancy outcomes are presented in table. conclusion rptlsx are common. in twin pregnancies receiving nt, alteration of tocolytic treatment following rptlsx had a positive impact on pregnancy prolongation and neonatal outcomes. routine cervical dilatation during elective cesarean section -is it really necessary? arie koifman, avi harlev, eyal sheiner, fernanda press, arnon wiznitzer. obstetrics and gynecology, soroka university medical center, ben-gurion university of the negev, beer-sheva, israel. objective: the purpose of this study was to examine the necessity of routine cervical dilatation during elective cesarean section. material and methods a retrospective cohort study was performed, including all cases of elective cesarean sections performed at a tertiary medical center during . stratified analysis, using the mantel-haenszel technique, was done to control for confounders. results out of a total of elective cesarean deliveries (cd), underwent routine cervical dilatation. the overall rate of febrile morbidity was . %. no significant differences in postpartum febrile morbidity were noted between the groups ( . and . %; p= . ). in addition, hospitalization duration did not differ between the groups ( . ± . and . ± . days p= . ). about % of all elective operations were repeated cd. there was no difference in febrile morbidity between the groups even in that subgroup of the elective cd. likewise, there was no difference in anemia rate between the two groups (hemoglobin . ± . mg and . ± . mg p= . ). controlling for a previous vaginal delivery, using the mantel-haenszel technique, no significant association was noted between cervical dilatation and fever (weighted or= . ; % ci . - . ; p= . ). nevertheless, in a subgroup of patients following a previous vaginal delivery, cervical dilatation was significantly associated with post-operative fever (or= . the majority of women with an unfavorable cervix undergoing iol at term deliver vaginally, even with a prolonged first stage of labor. this is important information to discuss with women prior to iol when establishing labor expectations. providers should consider the ongoing success of labor induction when contemplating a diagnosis of "failed induction". objective: this study was performed to investigate and compare changes of the lipid peroxide levels and the protein carbonyls formation in the maternal venous plasma of preterm premature rupture of membrane (pprom) during antibiotics administration. materials and methods: thirty-six pregnant women with pprom between and weeks of gestation were chosen for this study. eighteen patients (group ) were treated with amoxicillin and erythromycin for day period while the other patients (group ) were treated with rd generation cephalosporin and erythromycin for the same period. samples of maternal blood were obtained from the two groups at before the antibiotics administration, day , and day after the antibiotics administration. lipid peroxide levels were measured by thiobarbituric acid reaction and protein carbonyl contents were determined by the , -dinitrophenylhydrazine method. results: . the lipid peroxide levels and protein carbonyls formation in the maternal venous plasma of pprom was significantly higher than that of normal pregnancy ( . ± . vs . ± . nmol/mg protein, p< . ), ( . ± . vs . ± . nmol/mg protein, p< . ). . there were no significant differences in the lipid peroxide levels and protein carbonyls formation of the maternal venous plasma with pprom mixed and incubated by amoxicillin, cefodizime, and erythromycin (in vitro). . there were no significant differences in the lipid peroxide levels and protein carbonyls formation of the venous plasma of group among before the antibiotics administration, day , and day after the antibiotics administration. . the protein carbonyls formation in the venous plasma of group was significantly decreased at day and day after the antibiotics administration than that of before the antibiotics administration ( . ± . , . ± . , . ± . nmol/mg protein, p< . ). conclusion: in the maternal venous plasma of pprom, the lipid peroxide levels and protein carbonyls formation were increased. the results suggest that reactive oxygen species formation by inflammatory reaction is suppressed by combined treatment of rd generation cephalosporin and erythromycin. objective: the aim of the present prospective cohort study was to evaluate the correlation between blood flow pulsatility index in fetal middle cerebral artery (mca) and the onset of spontaneous labor. study design: doppler evaluation of fetal mca were performed between and weeks gestation in consecutive pregnant women with a singleton pregnancy and known gestational age. the study population was divided according to the mca pi (< . or >/= . mom) and, using survival analysis and cox regression, the two subgroups obtained were compared. in these analyses, the event was delivery (following spontaneous labor) and the time variable was time elapsed since doppler exam. results: the median time elapsed between doppler evaluation and spontaneous labor was significantly shorter in the women with mca pi lower than . mom ( . days, interquartile range - ) in comparison with the women with mca pi higher or equal than . mom ( . days, interquartile - . days (p< . , mann-whitney test). after correction for birth weight and umbilical artery pi, survival analysis and cox regression confirmed that mca pi was independently associated with the number of days elapsed from doppler to spontaneous labor and delivery (p< . , exp(b) . , ci % . - . ). conclusions: the present data show that, at term of pregnancy, fetal cerebral resistance reduction anticipates the onset of spontaneous labor. novel calcium sensitizers and human uterine contractility. mark p hehir, audrey t moynihan, terry j smith, john j morrison. department of obstetrics and gynaecology, national university of ireland, galway, ireland. objective: the factors regulating contractility of uterine smooth muscle are central to the occurrence of preterm labour and delivery. calcium sensitizers are a novel class of drugs with unique molecular actions. levosimendan, the best characterized of these compounds, is used in the treatment of acute and chronic heart failure and is a compound which exerts a number of effects on smooth muscle. it can exert an inotropic effect via sensitization of myofilaments to calcium and also exerts a relaxant effect by opening atp-dependent potassium channels. for these reasons we hypothesized that levosimendan may have an effect on myometrial contractility and investigated its action on both spontaneous and agonist induced contractions. method: biopsies of human myometrium were obtained at elective caesarean section (n= ). dissected myometrial strips suspended under isometric conditions, undergoing spontaneous and oxytocin-induced contractions, were exposed to cumulative additions of levosimendan in the concentration range of nmol/l to mmol/l. two sets of control experiments were performed simultaneously as follows: . strips exposed to either physiological salt solution (pss) only, for spontaneous contractions, or . nmol/l oxytocin; . strips exposed to pss/oxytocin and vehicle for levosimendan. results: levosimendan exerted an inhibitory effect on spontaneous and agonist induced contractions, compared to control strips. the mean maximal inhibition values were as follows: . ± . % for spontaneous contractions (n= ; p< . ) and . ± . % for oxytocin-induced contractions (n= ; p< . ). no significant difference was found between control and control for both spontaneous and oxytocin induced contractions. conclusion: the calcium sensitizer levosimendan exerted a potent relaxant effect on uterine contractility in vitro. this action was seen in both spontaneous and agonist induced contractions. the results from this study raise the possibility of calcium sensitizers holding potential tocolytic properties in vivo and further studies are required to investigate the potential benefits of this novel class of drugs. to determine to what degree extensive patient movement affects uterine emg signals and toco signals. study design: pregnant term labor patients were recorded using both uterine emg and toco simultaneously. baseline recordings were obtained when patients were still, and these were used as control records. test recordings for both devices were obtained from all patients by asking the patients to perform movements. area under the rectified-voltage amplitude curve of the uterine emg signals and area under the curve for the toco signals were found. mean %-increase was calculated for the uterine emg and toco devices (each device %-increase values were averaged over all patients). mann-whitney rank-sum test was used to look for any statistical differences in %-increase in area for uterine emg vs. toco methods (p < . considered significant). results: there was a large increase in activity (artifact) on both devices' signals during patient movement (figure ) . both devices' traces eventually returned to baseline after the patient movement stopped. toco movement artifact was significantly higher than emg movement artifact ( table ) . conclusions: both uterine emg and toco signals experience artifact when patients move the uterine emg electrodes and toco pressure transducer, respectively. toco seems to be more adversely affected by such movements. uterine emg may be a preferred method for monitoring contractions of laboring patients in the clinic. supported by grant nih r -hd . results: % of obstetricians completed the questionnaire. none would counsel patients against labour unless there were contraindications. the majority would recommend labour for all indications for previous caesarean section investigated although in all instances, personal preferences were lower (p< . ); after a failed instrumental delivery, % obstetricians would recommend labour but only % would choose that option for themselves (p< . ). overall, female obstetricians would contemplate and recommend labour more readily than male obstetricians. labour augmentation and induction were recommended to patients more frequently ( % and % respectively) than chosen for personal care ( % and %). reluctance for labour augmentation and induction was greatest among younger consultants. conclusion: consultants have responded to consumerism and aim to meet the requests of their patients. they more readily recommend labour than they would choose for personal care, and a majority would recommend labour induction when necessary. informed patient choice is paramount rather than attaining a target figure for women attempting to labour, and the views of those requesting delivery by caesarean section should be respected. (table) . cd increased in nullipara singleton, cephalic, term pregnancy in spontaneous labor, mostly due to dystocia; nullipara singleton, cephalic term pregnancy with induced labor, mostly due to non-reassuring fetal status and dystocia; singleton, cephalic term pregnancy with previous cd, mostly due to elective cd; singleton cephalic preterm. no changes in neonatal outcome were observed. conclusion: clinical audit was useful to keep under control cd rate in our institution in comparison with italian reality, but it was not sufficient to maintain stable cd rate suggesting the need of other, multifaceted strategies. elective delivery at weeks' gestation is a common obstetric intervention. the purpose of this study is to estimate the maternal mortality rates (mmr) associated with this acog -approved intervention. there are no reliable us data describing mmr by mode of delivery. therefore, we base our estimates on british data indicating a procedure related mmr of . / , , . / , and . / , for vaginal, elective cesarean section (c/s) and emergency-unplanned c/s deliveries, respectively. a decision tree model was constructed assuming that all eligible patients (approx. , , /annually in the u.s.) would be delivered at weeks by either an elective c/s or an induction of labor. we further assumed that % of inductions would result in a vaginal delivery. our estimates show that the annual, delivery-related maternal mortality associated with an elective delivery of all patients at weeks would be and for elective c/s and induction of labor, respectively. because vaginal delivery results in the lowest mmr, we performed a oneway sensitivity analysis to identify the impact of changing the success rate of induction on the estimated mmr. the results indicate that once the success rate exceeds %, this intervention would be associated with a lower mmr as compared to elective c/s. our estimates indicate that although the overall mmr associated with elective delivery at weeks is relatively low (approximately . / , deliveries), it is certainly not negligible. in addition, the mmr is highly dependent on the likelihood of a successful vaginal delivery following induction of labor. when the success rate for induction of labor falls below %, an elective c/s appears to be the safer delivery method. background: the first obstetric visit is an opportunity to provide the pregnant patient information regarding substances that can cause potential harm to the pregnancy. little is known about how obstetric care providers handle these topics. objective: to examine patient-provider communication about substance use during the first prenatal visit. methods: we audiotaped first prenatal visits and qualitatively analyzed those tapes in which patients disclosed substance use. we invited patient participants to return for a semi structured interview during which they reviewed their audiotaped conversations and described their reactions to the providers' communication styles. results: providers ( residents, midwives, nurse practitioners) and patients participated. providers asked about smoking, alcohol and drug use in all ( %) visits. patients reported being smokers, reported alcohol use, and reported drug use. provider responses to smoking disclosures included brief discussions of smoking effects on pregnancy, encouragement to quit/cut down, and referral to smoking cessation programs. provider responses to alcohol or drug disclosures included only general statements regarding effects on pregnancy (e.g., "we find that this is bad for babies.") and referral to ultrasound/genetics for reassurance. few alcohol or drug discussions assessed whether the patient had intentions or concerns regarding continued use during the pregnancy. few discussions addressed strategies for behavioral change. none included assessment for motivations, readiness, or barriers to change. in follow up interviews, patient participants said they expected to be asked about substance use but advised providers to ask non-judgmentally. those who used alcohol/drugs wanted more information on potential effects of these substances on the pregnancy/fetus and appreciated the reassurance from referrals to ultrasound/genetics. discussion: counseling for risky behaviors in the first obstetric visits contained only limited discussion of the effects of the risky behaviors and primarily focused on referral-which may be a proxy for avoiding a difficult and time consuming conversations. background. there are conflicting results regarding the association of maternal psychiatric disorders or psychological distress due to stressful life events with pre-term birth and low birth weight. aims. to investigate the association between maternal psychiatric disorders and/or stressful life events and intrauterine growth abnormality, low birth weight or preterm birth. method. three mutually exclusive and homogeneous groups of pregnant women ( with actual psychiatric disorder, with stressful life events, and healthy comparisons) underwent serial fetal ultrasound examinations and uterine and umbilical artery doppler velocimetry at (+ ), (+ ) and (+ ) weeks of gestational age. subjects were recruited from all consecutive women attending two antenatal clinics. the presence of any maternal medical illness, drug treatments, fetal chromosomal and/or structural malformations, were exclusion criteria. all women recruited underwent a structured interview at - weeks for the psychiatric diagnosis (mini international neuropsychiatric interview -mini) (sheehan et al, ) ; moreover, the -item hamilton rating scale for depression and the hamilton rating scale for anxiety were included in the assessment. the person who obtained obstetrical clinical data was blind to the results of psychological evaluations. results. the three groups were comparable for: age, parity, socioeconomic status, smoking, alcohol consumption, body mass index. gestational age at birth was not different in the three groups. infants of women with psychiatric disorders had significantly lower birthweight ( + g) and higher percentage of birth weight below the th centile for gestational age ( %) than infants of healthy mothers ( + g and %, respectively). there was also a trend towards lower mean birth weight ( + g) and higher incidence of birth weight below the th centile ( %) in the stressful life event group. there was no significant difference among groups in the percentage of abnormal uterine or umbilical doppler results. conclusions. maternal psychiatric disorders are associated with a lower birth-weight, but the effect is unlikely to be due to abnormal utero-placental or feto-placental vascularisation. objective: the purpose of this study was to evaluate antenatal ultrasound as a tool for the detection of intrauterine growth restriction (iugr) and small for gestational age (sga) infants among subjects with elevated human chorionic gonadotropin (hcg) levels on second trimester serum screening. although iugr has been linked to elevated hcg levels, the optimal screening regimen for antenatal sonographic surveillance has not been previously established. methods: a retrospective cohort study was performed at saddleback memorial medical center where serial ultrasounds from weeks-delivery are generally recommended for patients with hcg levels > . mom. all pregnancies were dated by second trimester ultrasound ± last menstrual period. subjects with an hcg > . mom, who had at least one antenatal ultrasound evaluation for iugr, were identified from an electronic ultrasound database used for clinical report generation. ultrasound data were then linked to an obstetrical birth outcomes database for relevant demographic/delivery information using unique identifiers. iugr was defined as a sonographic estimated fetal weight (efw) < %ile for the estimated gestational age (ega). sga was defined as an actual birthweight < %ile for the ega at the time of delivery. results: from - , there were subjects with elevated hcg levels who underwent antenatal ultrasound surveillance for iugr and who had known delivery information. a total of ultrasound examinations were performed. the median number of examinations per subject was with a range from - examinations per subject. the incidence of iugr and of sga were . % (n= / ) and . % (n= / ), respectively. no fetus with iugr demonstrated absent or reverse end diastolic umbilical artery doppler flow. antenatal ultrasound examinations only identified . % (n= / ) of sga infants. however, the sensitivity for the detection of sga was % when an efw cut-off of % was used. conclusions: although the majority of sga infants did not demonstrate growth restriction on antenatal ultrasound, a sonographic efw > %ile appears to be a safe cut-off to rule out fetuses at risk for sga. neonatal dry lung syndrome after pprom: reason to change intrauterine referral practice in the netherlands? ellen s hoogakker, christiaan v hulzebos, gerda g zeeman. obstetrics and gynecology, university medical center groningen, groningen, netherlands; pediatrics, division of neonatology, university medical center groningen, groningen, netherlands. background: neonatal dry lung syndrome (dls) is a distinct clinical entity following pprom, mimicking pulmonary hypoplasia but with dramatic respiratory improvement during the first - h . its pathogenesis implies complete collapse of small airways to a degree that capillary forces impede distension by ordinary ventilatory pressures. in the netherlands, women with pprom remain admitted at a level iii nicu perinatal center until they reach wks. when they are referred back to their community hospital, unless they live in the neighborhood of the tertiary center. we question this referral pattern because we still see severe respiratory problems occur > wks. we sought to determine the prevalence of such morbidity, in particular dls, in women with pprom who deliver > wks. methods: retrospective descriptive study of neonatal outcome data of singleton pregnancies complicated by pprom between - wks, who deliver > wks (latency > h) , during the -yr period of - at a single academic center. data were extracted from medical records and electronic department databases. results: pprom pregnancies were identified. all but received at least full course of steroids. ( %) delivered > wks. newborns born at the community hospital needed emergency transportation to a level iii nicu for respiratory morbidity. neonatal outcome data (means ± sd) are listed in the table. there were no cases of late onset sepsis, nec or perinatal mortality. conclusions: respiratory morbidity still occurs after pprom with delivery > wks. further investigation of pregnancy-related characteristics, such as the presence of anhydramnios and the latency period, with regards to dls is needed. modification of current referral practice depends upon complete data derived from all dutch level iii perinatal care centers (n = ). tertiary care center (n = ) to determine if an association exists between macrosomia (birthweight > g) and perinatal outcomes in women with and without gestational diabetes mellitus (gdm). study design: this is a retrospective cohort study of , singleton pregnancies. the study cohort was stratified by the diagnosis of gdm, with the presence or absence of macrosomia as the dependent variable. perinatal outcomes examined included neonatal hyperbilirubinemia, hypoglycemia, respiratory distress syndrome (rds), shoulder dystocia and erb's palsy. chisquare tests were performed as well as multivariable analyses controlling for confounders, using p< . to indicate statistical significance. results: in women diagnosed with gdm, macrosomia is associated with a higher frequency of hypoglycemia, respiratory distress syndrome, shoulder dystocia and erb's palsy. though the prevalence of these outcomes is relatively decreased in patients without gdm, they are still more prevalent in macrosomic patients. macrosomia is associated with a higher prevalence of adverse perinatal outcomes in women with and without gdm. therefore, it is important to evaluate neonates with birthweights greater than grams for hypoglycemia and unrecognized erb's palsy. conclusion a significant proportion of our obstetrical patients are obese and many do not perceive themselves to be obese. while our finding of an inverse correlation between level of education and bmi may be confounded by socioeconomic status, our results suggest that in order to address the problems of obesity in our population an important first step will be improving the education of our reproductive age women regarding normal weight gain and nutrition in pregnancy. this may have a significant impact on improving pregnancy outcomes of today's obstetrical population. objective: the aim of the study was to evaluate the impact of a "flat" oral gct upon the outcome of pregnancy. the gct was considered flat when the difference between the basal value and the after load value was %. methods: we prospectively analyzed the outcomes of pregnancies who delivered at our department. inclusion criteria were singleton pregnancy; bmi < kg/m , absence of major risk factors for diabetes and of pregestational diabetes. g -hour oral gct was performed at - weeks of gestation. women were subdivided into groups according to the result of the gct: group = negative (load glucose > % and < mg/dl) women ( . %); group =flat (load glucose % than basal and < mg/dl) women ( . %); group =positive gct/negative ogtt, women ( . %). data are mean ± sd. differences were calculated with the student t test for unpaired samples and test. regression analysis were performed by the least squared method. p-values were considered significant at p< . . results: the characteristics and obstetric outcomes in the three groups are presented in the table . in all patients there was a significant linear relationship between the load and basal glucose values (load value= . + . basal value; r= . ; p< . ) and between birthweight and load values (birthweight= . + . load value; r= . ; p< . ). the relationships were significant also in group and separately but not in group . in this preliminary study we found no major outcome differences in women with flat gct compared to women with normal and gct positive/ ogtt negative results. it seems important, however, to futher investigate the meaning of a flat curve in a bigger population and/or by means of metabolic studies with the use of stable isotopes. results characteristic of the study population and obstetric outcomes are presented in table . the probability to be primiparous and to deliver babies < ° centile decreased significantly with bmi whereas the risk of cesarean section, of post partum hemorrhage at vaginal delivery and to deliver babies > ° centile increased significantly. also the risk to develop preeclampsia and gestational diabetes was increased, although not significantly, with bmi. compared to lean and normal, obese were more likely to be hypertensive and diabetic before pregnancy (p< . ) and to start pregnancy without any medical and obstetrical risk but obesity ( . % vs . and . %; p< . ). african women exhibited the highest bmi and the highest rate of obesity (table ). conclusions we confirm that obese women have an increased risk of prepregnancy and pregnancy complications: less than % have an uncomplicated pregnancy. at delivery there is an increased risk of cesarean section and post partum haemorrhage. objective: overweight and obese women often give birth to larger babies, which is associated with traumatic birth injuries and an increased risk to develop obesity, diabetes and hypertension in childhood and later in life. the mechanisms underlying fetal overgrowth in obese pregnant women are largely unknown. the aim of this study was to establish a mouse model of obesity/high fat diet in pregnancy. we hypothesized that a moderately high fat diet prior to and during pregnancy would result in increased maternal adiposity, fetal overgrowth and a metabolic profile similar to that of obese newborn offspring with persistent pulmonary hypertension, despite enhanced pregnant women. method: c bl/ j female mice were fed control (c, % of energy from fat) or isocaloric high fat (hf, % of energy from fat) diets ad libitum for weeks prior to mating and during gestation. at gestational day . maternal blood samples were obtained to measure adiponectin, leptin and cytokine levels and maternal fat pads were isolated and weighed. in a sub set of animals measurements of transplacental transport of neutral amino acids and glucose were performed in vivo under ketamine anesthesia using h-meaib, and c-glucose. results: no significant differences were observed in maternal pre-pregnancy bodyweight, total caloric intake, weight of the dam at e . or litter size between treatment groups. fetal weight was increased in the hf group by % (p< . ). maternal adiponectin levels were significantly (p< . , n= ) decreased (hf ± , c ± g/ml) and leptin levels increased by % in animals fed a high fat diet, but this difference did not reach statistical significance (n= ). adiposity (fat pad weight) was increased by % (p< . , n= in the dams fed high fat diet, however no difference was observed in maternal il- levels and neither group had measurable levels of tnf-. maternal red blood cell lipid profiles were altered in high fat animals with an increase in stearic and linoleic acids but decreased oleic acid levels. preliminary data showed that placental uptake and transfer to the fetus of glucose and meaib were increased by at least % in dams fed high fat diet. conclusion: this murine high fat diet model has several features consistent with human obesity in pregnancy and the maternal metabolic environment is similar to that seen in the human. the increase in placental uptake and transfer of nutrients constitute a key mechanism underlying fetal overgrowth in overweight/obesity in pregnancy. objective: epidemiological studies have demonstrated a positive relationship between maternal overnutrition and the development of the metabolic syndrome in the offspring. we previously reported that lambs of well fed ewes have increased plasma glucose levels in early life, this may be a consequence of altered hepatic glucose production. increased hsd expression is associated with an increase in intracellular cortisol, promotion of hepatic insulin resistance and a consequential increase in gluconeogenic activity. hypothesis: we hypothesised that maternal overnutrition in late gestation in the sheep results in increased hepatic expression of hsd in the lamb before and after birth. methods: ewes were provided with either % (control,c) or % (well fed, wf) of maintenance energy requirements from d gestation until delivery. post-mortem was performed on either ± d gestation (c= ,wf= ) or and postnatal day (c= ,wf= ). plasma glucose and leptin concentrations in the fetuses and lambs were determined. the relative hepatic mrna expression of hsd and reference gene rpp were determined by qrt-pcr. results: relative liver weight was significantly higher in lambs of wf ewes compared to c at d (p< . ). the expression of hsd mrna was significantly higher in the postnatal compared to the fetal lamb (p< . ) independent of maternal nutritional treatment. however, there was no effect of maternal overnutrition on the hepatic expression of hsd mrna before or after birth. there was no effect of prenatal nutrition on fetal or postnatal plasma cortisol concentrations. the expression of hsd in the liver of lambs of wf, but not c ewes, was inversely related to plasma glucose concentrations in the first hrs after birth (r =- . , p= . ). we have therefore demonstrated that exposure to prenatal overnutrition results in an inverse relationship between hsd mrna expression in the liver at d and plasma glucose concentrations on the first day of life. this suggests that exposure to high glucose levels before and immediately after birth results in a reduced expression of hepatic hsd . we would expect a reduction, rather than promotion of intra-hepatic cortisol production, and therefore it is unlikely to explain the hyperglycemia present in lambs of wf ewes in early life. ninety-seven ( %) of mothers were classified as obese (bmi> ) based on their first pregnancy weight. glycemic control at weeks was superior in the non-obese group (table ) . there were no differences in glycemic control during the last week of pregnancy. obesity was significantly associated with increased maternal weight gain during pregnancy. mean birth weights, ponderal indices and rates of macrosomia were significantly higher in infants born to obese women when compared to non-obese (table ) . primary cesarean deliveries rates were comparable. the rate of neonatal hypoglycemia, hyperbilirubinemia, phototherapy and neonatal icu admissions did not differ between obese and non-obese diabetic women (table ) . although not statistically significant, there was a trend towards an increased rate of birth injuries in the obese group. a similar comparison between obese and non-obese women treated with medication (glyburide or insulin) demonstrated a higher mean birth weight ( g+ vs. g+ , p=. ) and higher rate of macrosomia ( vs. %, p= . ). there were no differences in glycemic control, cesarean delivery rates and other neonatal outcomes between the obese and non-obese treated with medication. conclusion: obese women with type ii and gdm give birth to larger infants than their non-obese counterparts and have a higher incidence of fetal macrosomia. there was a trend towards increased rate of birth injuries in the obese group. despite these differences other maternal and neonatal outcomes were similar which may be a reflection of glycemic control. introduction: tlrs are key components of the innate immune system which recognize conserved sequences on the surface of pathogens and trigger effector cell functions. the placenta, and more specifically the trophoblast, may play an important role in the response to infection. previously, we described the expression of tlr- by human trophoblast and their ability to respond to polyinosinic-polycytidylic acid (polyi:c), a synthetic double strand rna which mimics viral rna. in the present study we evaluate the effect of polyi:c in mouse pregnancy and characterize the local and systemic response. material and methods: human first trimester trophoblast cell line, htr , was treated with polyi:c. c b/ wild type and tlr- knock out mice were injected intraperitoneally with polyi:c at . gestation day. cytokine and chemokine level were determined in supernatant and lysates using the bio-rad multiplex assay and analysis was done using the bio-plex is. results: polyi:c induced cytokine (il , il and il ) and chemokine (il , rantes, gro , mcp and mip ) secretion and production by human cultured trophoblast in a time ( - h) and a dose ( - g/ml) dependent manner. injection of polyi:c to c b/ wild type mice induced preterm delivery within h at a dose of mg/kg body weight. no effect was observed in tlr- knock out mice. a robust systemic (spleen and serum) and local (placenta and amniotic fluid) inflammatory response was observed and h following polyi:c treatment. trophoblast cell cultures from tlr- ko mice confirmed that the response to polyi:c is tlr- dependent. conclusion: we demonstrate that viral infection may trigger an immune response leading to preterm labor. furthermore we show that the trophoblast is able to recognize and respond to viruses through the expression of tlr- . our findings provide a novel mechanism of pathogenesis of preterm labor associated with tlr- mediated inflammatory response. introduction: adverse neurological outcome is a major cause of neonatal morbidity after a preterm birth (ptb). a growing body of evidence demonstrates the involvement of inflammatory pathways in ptb and implicates these pathways as a causative factor in fetal brain injury. however, activations of cytokine pathways in normal labor (whether iatrogenic preterm or at term) has been observed. what remains understudied is the effect of labor on the preterm fetal brain and whether an inflammatory stimulus is essential for fetal brain injury. methods: mouse models were utilized: ( ) a model of intrauterine inflammation (lps into uterine horn) (n= ); controls received intrauterine saline (n= ) and ( ) autism is a neurodevelopmental disorder with a strong genetic component and several known environmental risk factors, such as infection. in addition, its onset of etiology is likely to occur during prenatal development. we propose that subjecting fetal sheep via amniocentesis to the bacterial endotoxin lipopolysaccharide (lps) injected to the amniotic fluid at gestational day (gd) will result in morphological alterations in the offsprings' cerebellum resembling alterations found in the cerebellum of patients with autism. using high precision design-based stereology, we investigated mean total-and layer-specific volume and mean total granule and purkinje cell (pc) number in the cerebellum of lps infected animals and controls. the results of the present study showed preserved volumes of the total cerebellum as well as of the molecular layer, outer and inner granular cell layers and white matter. interestingly, compared to controls, the lps infected brains showed a statistically significant increase (+ . %) in the mean total number of granule cells, whereas the pcs did not show any difference between the groups. these seemingly paradoxical results might be explained by ( ) the so-called time of origin of these neurons, i.e. the pcs develop prenatally whereas the granule cells develop postnatally or ( ) the direct correlation between pcs and granule cell number in the cerebellum. these results might contribute, as an animal model, to our understanding of the biological basis for interindividual differences in morphological alterations found in the brains of patients with autism. the previous studies have shown that there is a higher incidence of spontaneous preterm birth and poorer neonatal outcome in pregnancies with a male fetus. in vitro studies also report a sex dependent pattern in different placental enzymatic systems. we have shown previously that lactobacillus rhamnosus gr- supernatant is able to antagonize the actions of lps on cytokines and ptgs in placental trophoblasts. we hypothesize that fetal sex will influence the production of cytokines and prostaglandin regulating enzymes in lps and lactobacilli treated placental trophoblast cells. methods: term placentae were collected from women undergoing elective caesarean section. placental trophoblasts were isolated using established primary culture protocols. cells were pretreated with lactobacilli supernatant and subsequently treated with lps. pgdh and ptgs expression levels were measured by western blot analysis and tnf-, and il- concentrations measured by elisa. results: lps stimulation caused a marked increase in production of tnf-( . pg/ml to . pg/ml, n= , p< . ), an effect that was greater in placentae of the male fetuses ( . pg/ml, n= ) compared to female fetuses ( . pg/ml, n= ). lactobacilli supernatant abolished this response in both sexes. lps-activated trophoblasts from placentae of the male fetuses showed an increase in il- production (n= , p< . ) and ptgs expression (n= , p< . ). however, there was no response to lps in placentae of the female fetuses. lactobacilli supernatant up-regulated pgdh (n= ) by %, and this effect was greater in placentae of the female fetuses (n= ). conclusion: we conclude that human placentae from pregnancies carrying male fetuses are more responsive to lps by producing more pro-and anti-inflammatory cytokines, as well as ptgs . conversely, placentae of the female fetuses upregulate pgdh with lactobacilli treatment. these findings may explain the underlying mechanism for the higher incidence of preterm birth and adverse pregnancy outcomes seen with male fetuses in the clinical setting. , ) . this study investigates whether iai is associated also with altered expression of neuropilin- . methods: (i) immunohistochemistry (ihc) was performed on tissue sections of term decidua with or without clinical / histologic evidence of iai (n= for each). neuropilin- expression was scored by an investigator blinded to the identity of the samples. (ii) cultured term dscs were retrieved from elective cesarean (n= ), purified, and depleted of leukocytes. after treatment with - m estradiol (e ), - m medroxyprogesterone acetate (mpa), both, or vehicle for days, dscs were stimulated with il- b ( - ng/ml), tnfa ( ng/ ml), or thrombin ( . iu/ml) for h. since no elisa exists for neuropilin- , protein expression was determined by immunocytochemistry (icc). (iii) total rna was extracted and the effect of il- b on neuropilin- mrna expression measured by real-time rt-qpcr and corrected for b-actin mrna. results: neuropilin- expression in term decidua was increased in tissues with iai vs controls (p< . ), and localized primarily to dscs. using icc, an increase in neuropilin- was noted after stimulation with il- b and tnfa, but not thrombin. il- b increased neuropilin- mrna expression in dscs by . ± . -fold (from . ± . to . ± . neuropilin- mrna/b-actin mrna; p= . ). conclusions: iai is associated with increased expression of the vegf receptor, neuropilin- , in term decidual tissues. il- b and tnfa (but not thrombin) stimulated neuropilin- expression in term dscs, and this effect appears to be mediated at the level of gene transcription. since aberrant vegf function alters vascular permeability, these data provide a mechanism by which iai can promote 'decidual activation' and preterm labor. inflammation, university of glasgow, glasgow, united kingdom. objective: asthma is associated with inappropriate activation of airway smooth muscle, chemokine expression and accumulation of mast cells which drive smooth muscle reactivity. labour is similarly associated with smooth muscle activation and expression of cxcr and cxcr ligands. the role of mast cells in human parturition is unknown; however, mast cell products can stimulate myometrial contractions and preterm labour in animal models. we have quantified mast cells in association with human labour and determined whether they express cxcr and cxcr . methods: lower segment myometrial and cervical biopsies were taken at term caesarean section from women not in labour (nil) (myometrium n= ; cervix n= ) and in labour (il) (myometrium n= ; cervix n= ). mast cells were localised in myometrial and cervical sections by icc with a primary antibody against c-kit. the number of cell transects in randomly selected high-powered fields ( x) was quantified blindly by two observers for each specimen, with median density and interquartile range (iqr) calculated. backto-back icc was performed to determine whether c-kit co-localised with the chemokine receptors cxcr and cxcr . results: mast cells were in close association with myometrial smooth muscle in non-labouring lower segment myometrium. labour was associated with a significant influx and increase in mast cells numbers (nil median . , iqr . - . ; il median . , iqr . - . , p= . ). in contrast no significant increase in mast cells was observed in cervical tissue in association with labour (nil median . , iqr . - . ; il median . iqr . - . , p= . ). analysis of chemokine receptor expression demonstrated co-localisation of cxcr to c-kit positive cells present within the myometrium. conclusions: human labour at term is associated with an increase in mast cells within the myometrium, with close approximation to smooth muscle bundles. these mast cells express the chemokine receptor cxcr , the ligands of which we have previously shown to be up-regulated in labouring myometrium. mast cells are not accumulated in cervix in association with labour suggesting a less critical role in cervical ripening. further analysis of the role of mast cells in modulating myometrial smooth muscle physiology is warranted. progestins and the glucocorticoid receptor in human myometrial and amnion-derived wish cells. alison j tyson-capper, stephen c robson. reproductive sciences, newcastle university, newcastle upon tyne, united kingdom. background and objectives: progesterone (p) can reduce the risk of preterm birth in some high risk women. in this context there is accumulating evidence that this, at least in part, may be due to anti-inflammatory and immunoregulatory properties of p. target tissue responsiveness to p is considered to be determined by the progesterone receptors (pr) and nuclear co-factors that directly interact with pr. pr and glucocorticoid receptors (gr) share several structural and functional characteristics, including similarities in dna sequence recognition by binding to the same hormone response elements. pr and gr interact with similar chaperones in the absence of ligand and with a similar group of co-activators in the presence of hormones; both can display comparable anti-inflammatory activities under specific physiological conditions. in this study we aimed to investigate whether the anti-inflammatory properties of progestins may be mediated by pr and gr signalling. methods: primary cultures of non-pregnant and term pregnant human myometrial (passage - ) and wish cells were serum starved for hrs and treated with -hydroxyprogesterone ( -hp), progesterone (p), dexamethasone (dex) and immunofluorescent staining and immunoblotting analyses performed. in some experiments cells were pre-treated with ru (a pr/gr antagonist) or org (a pure pr antagonist). results: in the absence of hormone gr appeared to be predominantly cytoplasmic, whereas, upon treatment with -hp and p ( and m) and dex ( nm) gr was abundant within the nuclei of myometrial cells. immunoblotting analyses demonstrated that levels of gr progressively increased within the nuclear fractions of both pregnant myometrial and wish cells in response to increasing concentrations of p ( nm to m), and decreased sequentially within cytoplasmic fractions. in the presence of org gr protein levels remained constant within the cytoplasm. there also appeared to be a slight increase in gr expression, though not statistically significant (p> . ) within both cells types in response to p. conclusion: in this study we show that gr is activated by -hp and p and translocates to the nuclei of human myometrial and amnion-derived cells. in addition, levels of gr increase in response to p. whether p and -hp act as agonists or antagonists for gr in the regulation of hormone response genes associated with the onset of term and preterm labour remains to be elucidated. objective: using a mouse model of inflammation-induced preterm birth (ptb), we have demonstrated dramatic cytokine elevations in the uterus and placenta with concomitant, though less dramatic, cytokine elevations in the fetal liver and brain, associated with neuronal injury. because precise mechanisms of fetal injury in ptb remain unclear, we sought to examine inflammatory cell trafficking, and target organ damage by histopathologic assessment of the placenta, fetus, and fetal brain. study design: hours after intrauterine infusion of saline or lps into the right lower uterine horn of cd- mice, the left upper horn, with the gestational sacs(gs) in situ, was removed en bloc(n= per group) each with - gs with fetuses/treatment group. specimens were fixed, bisected and processed for histology and ihc. inflammatory and hematopoietic cells were quantified using pas, gata- (erythroid precursors), cd , and bm (macrophage-mp) within the placenta, liver, extremity mesenchyme, brain and leptomeninges. the presence of hemorrhage, necrosis, and apoptosis (h ax stain) was assessed. erythopoietin (epo) levels were measured in brain and liver by elisa. results: more neutrophils were present in maternal decidual vessels in lps compared to saline (p= . ). in lps-exposed, fetal mp were increased in the placenta (p= . ), fetal extremity mesenchyme (p= . ), fetal liver (p= . ) and leptomeninges (p= . ) but not in the brain or spinal cord compared to saline. no necrosis, hemorrhage or increased apoptosis was noted in the fetal brains. % of lps-exposed fetuses and % of saline-exposed had liver hemorrhages (p< . ). increases in nucleated erythrocytes and erythroid precursors were found in fetal vasculature of the placenta in lps-exposed (p= . ). epo levels were not elevated in either group. conclusion: intrauterine lps infusion induces acute inflammation predominantly in the maternal circulation of the placenta. in the fetus, there is widespread mp activation, liver hemorrhage and increased erythroid precursors seen in the fetal circulation of the placenta. although histologic evidence of cns damage was not evident, the increased mps present in the leptomeninges may play an important role in inflammatory-mediated cns damage. non-toll-like innate immune proteins: do they change during pregnancy? juan m gonzalez, hua xu, ella ofori, michal a elovitz. obgyn; crrwh, university of pennsylvania, philadelphia, pa, usa. introduction: trem- (trigger receptors expressed on myeloid cells) is an important regulator of innate immunity. the natural ligand for trem has not been identified. activation of trem- in the presence of toll-like receptors results in substantial amplification of the host inflammatory response (klesney-tait et al nature immunology ). since inflammatory pathways are implicated in adverse pregnancy outcomes, this novel mediator of inflammation may play a critical role in preterm birth (ptb). therefore, we sought to determine trem- expression in the uterus, cervix, and placenta across gestation and to determine if trem- levels are altered by intrauterine inflammation. methods: in cd- mice, trem- was investigated in non-pregnant (np) and throughout gestation e , e (n= - per group). uterine, cervical, and placental tissues were harvested. using an established mouse model of inflammation-induced ptb, uterine tissue was collected hours after intrauterine infusion of saline (n= ) or lipopolysaccharide (lps) (n= ). for a non-pregnant model, using cd- mice, lps (n= ) or saline (n= ) was injected into the uterine horn following same procedures as with pregnant mice. uteri were harvested hrs later. quantikine ® mouse trem- immunoassay was utilized for these studies. statistical analysis was performed using oneway anova followed by pair-wise comparison if statistical significance was reached (p< . ) results: trem levels are significantly different between np and pregnant uterine tissues (p= . ). e and e trem expression is significantly increased . and . -fold compared to np (p= . and . respectively). trem- levels in the placenta and cervix were not significantly different between e and e . trem levels increased about -fold in the uterus after intrauterine infusion of saline or lps compared to e controls. in nonpregnant, trem levels were significantly different (p= . ) with a -fold increase in trem expression in uteri exposed to lps or saline compared to controls. conclusions: non-toll-like innate immune proteins are differentially regulated during pregnancy compared to the non-pregnant state. the role of trem- in inflammation-induced ptb requires further study. research is warranted to determine if uterine up-regulation of trem in gestation is associated with an increased likelihood of responding to pathogens or severe as a protective mechanism. reduced plasma levels of vitamin d in caucasian women at term are associated with increased rate of infection. chander p arora, adegoke adeniji, susan e jackman, babak forooghi, isaac mostadim, phillip yadegari, calvin j hobel. og-gyn, cedars-siani medical center, los angeles, ca, usa; university of california los angeles, los angeles, ca, usa. background: vitamin d plays an important role in human pregnancy by acting as a regulator of immunity at the fetal-maternal interface. inflammatory changes associated with pro-inflammatory cytokines were reduced by vitamin d while anti-inflammatory cytokines were increased in t lymphocytes. vitamin d status has been defined as deficiency (< . nmol/l), insufficiency ( . to nmol/l) and sufficiency (> nmol/l) . objective: to assess the involvement of vitamin d in the occurrence of maternal infection during pregnacy in women with term deliveries. hypothesis: vitamin d metabolism could affect the rate of infection during pregnancy. study design plasma levels of (oh)d were determined by elisa and the rate of infection was recorded in a behavior in pregnancy study. in this study, ethnically diverse women were evaluated at - weeks (t ), - weeks (t ) and - weeks (t ). maternal infections were documented at each stage as well as at baseline visit with history of infection in current pregnancy. of these subjects who delivered at term two groups ( with no infection during pregnancy, with infection or history of infection) were matched further for non-smoking status, non-diabetics, ethnicity and maternal age. plasma from these were assayed for (oh)d and analyzed for the rate of maternal infection using fisher´s exact test or chi-square test. results although the women delivered at term, the levels of (oh)d in caucasians were significantly lower in the subjects with infection than the ones without (p<. ). women with vitamin d insufficiecy in the first trimester were more likely to develop infection during pregnancy ( . nmol/l ± . at t , . nmol/l ± . at t and . nmol/l ± . at t ; all p<. ) but not subjects with sufficient vitamin d at t ( . nmol/l ± . at t , . nmol/l ± . at t and . nmol/l ± . at t ; all p<. ). conclusion the results reveal a positive association between (oh) d concentrations and greater risk of infection. vitamin d deficiency or even insufficiency may, therefore be involved in the pathogenesis of maternal infection during pregnancy. it is probable that vitamin d deficiency or even insufficiency could modulate the maternal susceptibility to infection during pregnancy by a proinflammatory mechanism. in vitro and in vivo observational data suggest that infection leads to caspase activation and apoptosis in the placenta and membranes, however currently there are no data on the role of apoptosis in the pathogenesis of infection associated preterm delivery. here we used group b streptococcus (gbs) as a model pathogen and examined the role of caspase dependent and independent apoptosis in preterm delivery. methods: we injected ( . x ) heat killed group b streptococcus organisms (hk-gbs) intraperitoneally (i.p.) in . day pregnant c b/l mice. the mice were euthanized at hr (n= ) and hr (n= ), the placentas and membranes were removed and assessed for apoptosis by tunel staining. caspase activation and expression were determined by immuno-histochemistry (ih) and western blotting. the effect of apoptosis on preterm delivery was assessed by i.p. treating the pregnant mice with pbs (n= ), dmso (n= ) or pancaspase inhibitor z-vad-fmk (n= ) prior to hk-gbs and observing the animal for delivery for hrs. results: there was a time dependent, gbs-induced increase in apoptosis by tunel assay and caspase activation in the placenta and membranes. in addition hk-gbs-induced the expression of caspase and caspase independent m-calpain in the placenta. z-vad-fmk ( mg/kg), at the maximum concentration that did not induce maternal illness, did not prevent hk-gbsinduced preterm delivery. conclusions: caspase dependent and independent mechanisms are activated in the placenta upon exposure to gbs. systemic adminstration of a pan-caspase inhibitor did not impact upon the occurance or timing of bacterially induced preterm delivery. further studies are needed to assess the role of apoptosis in the pathogenesis of infection associated preterm delivery. early and ( . %) had a late sptb. the mean + sd gestational age at blood draw was + weeks. the median level of bb was higher in women with early as compared with late sptb or term births (p= . for trend). women with bb in the top quartile were . times more likely to have an early sptb as compared with women who had lower levels of bb ( % ci . to , p = . ). there was no association between bb and late sptb (rr= . , % ci = . to ). the adjusted or of an elevated bb for early sptb was . ( % ci = . to , p= . ). when the analysis was restricted to the women with sptb the rr of an elevated bb for early sptb was . ( % ci . to . , p = . ). conclusions: a significant relationship was found between an elevated bb in early pregnancy and early sptb suggesting inflammatory events in early pregnancy, perhaps infection-related, are part of the pathogenic mechanisms. objective: genital tract infection and/or inflammation appears to contribute to the majority of ptds preceding weeks of gestation. ptd in humans has been associated with colonization and/or infection with a variety of different organisms including gram positive and negative bacteria, mycoplasma, ureaplasma, trichomonads, malaria parasites and viruses. the innate immune response to these pathogens is produced by a family of pattern-recognition cell membrane receptors known as the toll-like receptors (tlrs). these studies sought to characterize the tlr isoforms expressed in the preterm mouse uterus, and their modulation during lipopolysaccharide (lps)-induced ptd. methods: using sterile surgical technique, day- pregnant cd- mice underwent intrauterine injection of g lps. subsequently, the mice were euthanized at , , , , and hours after lps injection. uterine tissue was harvested and placed in rnalater; subsequently total rna was isolated using the trizol reagent and genomic dna was removed using turbo dnafree. cdna was made using iscript cdna synthesis kit. pcr primers were designed using published mouse tlr gene sequences. real-time quantitative rt-pcr was performed using the power sybr green master mix and an abi prism multicycler. to confirm tlr amplicon sizes, the rt-pcr products were visualized on a % agarose/tbe gel stained with gelred. results: these studies have confirmed mrna expression of all of the reported mouse tlr isoforms. these tlr amplicons range in size from to bp as expected; amplicon sequences are pending. quantitative rt-pcr studies performed using uterine tissues from five mice at each time point demonstrated that at hours after lps injection, tlr increased -fold and tlr increased -fold (both p< . ). in contrast, the expression of tlr (the ligand for lps) remained stable during the hours after lps; the expression of tlr , , , , , and also remained stable. tlr expression trended upward and tlr trended downward, although neither was statistically significant. conclusions: these studies have confirmed expression of all tlrs within the preterm pregnant mouse uterus, along with characterization of their modulation during lps-induced ptd. these observations are important because they contribute to our understanding of the immunologic signaling events leading to ptd. (funded by nih hd ). udp-glucose and its receptor p y as a new innate immune system in the female reproductive tract. toru arase, tetsuo maruyama, hiroshi uchida, takashi kajitani, masanori ono, maki kagami, hironori asada, yasunori yoshimura. obstetrics and gynecology, keio university, shinjukuku, tokyo, japan. objective: innate immune system involving toll-like receptors has recently emerged in the female reproductive tract (frt). we hypothesize that there may exist new other mucosal immunity in frt. recently, it has been reported that p y , a g protein-coupled p y receptor for udp-glucose (udp-g), is involved in the lung epithelial immune system. the aim of this study is to investigate whether udp-g and p y have a potential as the defense immune system in frt, in particular endometrium. materials and methods: we obtained human endometrial tissues from consenting reproductive-aged patients. the spatiotemporal expression of p y in human and mouse endometrial tissues was analyzed using rt-pcr and ihc. isolated human endometrial cells and a human endometrial epithelial cell line, ishikawa, were cultured, treated with udp-g, and subjected to rt-pcr analysis for il- mrna expression. we also measured the il- secretion using elisa. small interfering rna was used to knock down p y expression. the chemotactic activity of udp-g on neutrophils was tested using transwell assay with ishikawa cells. lastly, mouse uterine tissues were incubated with udp-g and subjected to rt-pcr analysis for mrna expressions of kc and mip- , the il- homologues in mice. results: p y was exclusively expressed in the glandular and luminal epithelium both in human and mouse uteri. treatment with udp-g induced the secretion of il- in ishikawa and human endometrial glandular cells, but not stromal cells, in a dose-and a time-dependent manner. p y knockdown abrogated udp-g-induced il- production. treatment with udp-g also significantly increased the chemotaxis of neutrophils, which was attenuated by co-addition of anti-human il- neutralizing antibody. in the mouse uterus stimulation of udp-g significantly up-regulated the expressions of kc and mip- mrna. conclusions: udp-g is an endogenous molecule and released into the extracellular environment in a lytic manner after cell damage. taken together, our results collectively substantiate a model in which udp-g released from endometrial cells damaged by infection stimulates il- production via p y in endometrial glandular epithelium, which, in turn, recruits neutrophils thereby preventing the progression of infection. thus, udp-g and its receptor p y may be involved in the trans-species mucosal immune system in frt. (jsgi ; , (suppl) , abst # ) but in utero effects on fetal lung remain to be established. we have examined the relationship between duration of iai and subsequent azi treatment on the severity of fetal lung histopathology. we hypothesized that early treatment would prevent the development of advanced lesions, while late treatment may reduce the severity of lung damage. study design. thirteen chronically instrumented rhesus monkeys received intraamniotic inoculation of u. parvum (serovar ; - x cfu) at ± . days gest. age (dga, mean ± sem, term= d). six of these animals received maternal i.v. azi ( . mg/kg q h or q h for d) either alone (n= ) or in combination (n= ) with dexamethasone (dex; mg/kg/d i.v. for d) and indomethacin (indo; mg/d p.o for d). tissues were obtained at cesarean section for histopathologic assessment. leukocytic infiltration of aveolar spaces and septal walls, type ii pneumocyte hyperplasia and peribronchiolar lymphocytic aggregates were scored as absent ( ), minimal ( ), moderate ( ) and severe ( ). results. inoculation-to-delivery interval was - d for combined treatment groups and was similar to long duration infection without treatment ( - d). treatment effects were tabulated as mean scores and compared as follows: control (n= ), score ; short duration infection ( - d; n= ), score ; long duration infection ( - d; n= ), score ; short duration infection + treatment (n= ), score ; long duration infection + treatment (n= ), score . conclusions. histopathologic findings of fetal pneumonia progressively worsen with duration of u. parvum iai. early maternal azi treatment prevents development of advanced lung lesions, while later treatment may reduce the severity of fetal lung damage. our results suggest that in utero treatment of iai may lower the risk of neonatal bronchopulmonary dysplasia. support: nih hd , rr . brain associated with adverse neurodevelopmental outcome. lps triggers proinflammatory responses through toll-like receptor- (tlr ). mitogen activated protein kinases including c-jun-n-terminal kinase (jnk) have been reported to be implicated in tlr signalling pathways and play important role in both onset of labor and brain injury. in the present study, we used a mouse model of intrauterine infection-associated preterm labor to determine whether the administration of specific inhibitor of jnk signaling, d-jnk inhibitory peptide (d-jnki) can (i) inhibit jnk activity in vivo, (ii) delay lps-induced preterm delivery, and (iii) improve neonatal outcome in the presence of intrauterine inflammation. intrauterine administration of tlr- specific lps to cd pregnant mice at day of pregnancy caused preterm delivery after to h with % pup mortality. in vitro kinase assay demonstrated the activation of jnk in response to lps in the maternal uterus and fetal brain. furthermore, the brain specific jnk was found to be the major jnk isoform activated by lps in the fetal brain. co-administration of d-jnki with lps to pregnant mice delayed significantly (p< . ) lps-induced preterm delivery and reduced pup mortality up to %. this was associated with inhibition of jnk activity in both maternal uterus and fetal brain. in addition, we have found that treatment with lps significantly up-regulated cox- , cxcl (il- equivalent) and ccl in myometrium and this is significantly suppressed after co-administration of d-jnki. we conclude that specific inhibition of jnk signaling may have a potential of controlling preterm labor and preventing fetal brain damage as a result of infection/inflammation. the prostaglandin -deoxy- , -prostaglandin j delays lps-induced preterm delivery and reduces mortality in the mouse. intrauterine infection is a common trigger for preterm birth, and is also a risk factor for the development of neurodevelopmental abnormalities in the neonate. bacterial lipopolysaccharide (lps) binds to toll-like receptor- (tlr ) to activate pro-inflammatory signaling pathways. the transcription factor nuclear factor kappa b (nf-kb) is a key player in the orchestration of the inflammatory response and has a central role in parturition. we have previously shown that exposure to the anti-inflammatory cyclopentenone prostaglandin -deoxy- , -prostaglandin j ( d-pgj ) inhibits il- b-induced nf-kb activity and cox- expression in human myometrial and amnion epithelial cells in vitro. in the present study, we used a mouse model of intrauterine infection-associated preterm labor to determine whether the administration of d-pgj can (i) inhibit nf-kb in vivo, (ii) delay lps-induced preterm delivery, and (iii) improve neonatal outcome in the presence of intrauterine inflammation. intrauterine administration of tlr specific lps to cd pregnant mice at day of pregnancy caused preterm delivery after to h with % pup mortality. co-administration of d-pgj with lps to pregnant mice delayed significantly (p< . ) lps-induced preterm delivery and conferred protection from lps-induced fetal mortality up to %. we have looked at the expression profile of several labor associated genes in myometrium hours after lps administration. (otr, connexin and , cox- , cox- , cxcl (il- equivalent) and ccl ). we have found that treatment with lps significantly up-regulated cox- , cxcl and ccl and this is significantly suppressed after with co-administration of d-pgj . western analysis for ser -phosphorylated p and ikkb in-vitro kinase assay has demonstrated that lps induced activation of nf-kb at both h and h. co-administration of d-pgj was associated with inhibition of nf-kb activation in both the maternal uterus and the fetal brain. conclusion d-pgj may have potential as a therapeutic agent in the management of preterm labor and, by targeting the player nf-kb, may have the added advantage of preventing detrimental effects to the fetus that may result from infection/inflammation. synergistic macrophage response to co-activation of tlr- and tlr- : mechanisms and implications for bacterial/viral co-infection. vladimir ilievski, emmet hirsch. , department of ob/gyn, evanston northwestern healthcare, evanston, il; department of ob/gyn, feinberg school of medicine, northwestern university, chicago, il. background: toll-like receptors (tlrs) recognize structural components of pathogens and initiate host defenses. tlr- responds to gram-positive organisms and peptidoglycan (pgn), a gram-positive cell wall constituent. tlr- is activated by viral infection in response to double-stranded rna. polyinosinic-cytidylic acid (poly(i:c)) is a tlr- ligand. we have shown that pgn and poly(i:c) have a synergistic effect on the expression of downstream genes for both tlr- and tlr- . here we identify mechanisms underlying this synergy. methods: mouse peritoneal macrophages or a mouse macrophage cell line (raw . ) were treated in tissue culture with either pgn ( g/ml), poly(i: c) ( g/ml) or both pgn and poly(i:c) either simultaneously or sequentially for - hours. total rna was extracted and duplex rt-pcr was performed for inducible nitric oxide synthase (inos), interleukin (il- ), tumor necrosis factor (tnf), the chemokine rantes and tlr- , normalized to the housekeeping gene gapdh. results: compared to stimulation with either pgn or poly(i:c) alone, costimulation of raw . cells with both pgn and poly(i:c) resulted in synergistic expression of inos, il- , tnf and rantes (p< . for all) at and hours . sequential stimulation with either pgn or poly(i:c) for h followed by incubation for an additional h with the alternate ligand also induced synergistic expression of the same rnas, albeit at lower levels than were elicited by simultaneous stimulation. in contrast, incubation with either pgn or poly(i:c) for h followed by medium for h induced minimal to no gene expression. both pgn and poly(i:c) induced tlr- mrna after h but not h. tlr- mrna was not detectable by rt-pcr. in primary peritoneal macrophages, similar synergy due to pgn and poly(i:c) was seen. conclusions: simultaneous or sequential exposure to pgn and poly(i:c) exerts a synergistic effect on the expression of inflammatory mediators in macrophages. interestingly, either one of these two tlr pathways can prime cells for activation of the other pathway. a possible mechanism for this effect may be induction of tlr- by either tlr- or tlr- activation. these observations have implications for bacterial/viral co-infection. this is a secondary analysis of a prospective cohort study. after irb approval, daily blood samples were obtained from pprom subjects and analyzed for il- by elisa. paired maternal serum il- levels from subjects were divided into groups: il- levels obtained - hours prior to completion of antibiotics and those obtained - hours after completion of antibiotics. the wilcoxon signed rank test was used to perform the data comparison on the analyze-it statistical software program. statistical significance was defined as p< . . of the pprom subjects, the maternal age was . yrs; gestational age at admission was . weeks; latency was . days; gestational age at delivery was weeks; and infant birth weight was grams. median maternal serum il- levels obtained off antibiotics were significantly higher when compared to those on antibiotics ( . vs. . pg/ml, p< . ). the results of this investigation suggest that maternal serum il- levels rise after discontinuation of antibiotics. the optimal duration of antibiotics administration in the setting of pprom is unknown. this data suggests a role for continuation of antibiotics in women with pprom in order to prolong the latency period and potentially decrease neonatal morbidity. to identify clinical and pathologic factors associated with fetal inflammatory responses in the placenta from term parturients. methods: retrospective cohort study of consecutive term parturients with submitted placentas in . placentas with histologic chorioamnionitis were divided into two cohorts: group -maternal inflammatory responses only, and group -maternal and fetal inflammatory responses. maternal and fetal inflammatory responses in the placenta were classified according to guidelines established by the amniotic fluid infection nosology committee of the perinatal section of the society of pediatric pathologists. selected demographic, intrapartum, newborn and placental characteristics were analyzed with t-tests and chi-square tests as appropriate. multiple logistic regression was used to identify independent predictors of fetal inflammatory responses in the placenta. results: of consecutively submitted placentas, had histologic chorioamnionitis: with maternal inflammatory responses only (group ) and with both maternal and fetal inflammatory responses (group ). fetal inflammatory responses observed in group were associated with higher stages of maternal inflammatory responses (p<. ). % of fetal inflammatory responses were stage i (acute chorionic vasculitis or phlebitis).group patients were more likely to have had amnioinfusion ( % v %, p=. ) and less likely induction of labor ( % v %, p=. ). group was more likely to have had intrapartum fever ( % v %, p=. ) and maternal tachycardia ( % v %, p=. ). newborns from group were more likely to have been observed for sepsis ( % v %, p <. ) and have an apgar score at minutes ( % v %, p=. ). a logistic regression model showed that stage ii or greater maternal inflammatory responses (or . ) and amnioinfusion (or . ) were independent predictors of fetal inflammatory responses. conclusion: higher stages of maternal inflammatory responses in the placenta and amnioinfusion were independent predictors of fetal inflammatory responses in term parturients with histologic chorioamnionitis. objective: previous studies have shown that sulfasalazaine (sasp) inhibits lipopolysaccharide (lps)-induced nf-b activation and decreases lpsstimulated interleukin- (il- ) production by cultured explants of placenta, amnion and choriodecidua with no effect on cell viability. bacteria-induced il- production in the cervix is a potential mechanism for premature cervical ripening that may lead to preterm birth. it is unclear, however, whether sasp can inhibit il- production by endocervical cells. therefore, we performed a series of in vitro studies to determine if sasp inhibits il- production by endocervical epithelial cells stimulated with bacterial pathogens associated with preterm birth. methods: human endocervical epithelial cells were incubated with - . mm sasp overnight and then stimulated with ccu/ml ureaplasma parvum, cfu/ml escherichia coli, or x cfu/ml gardnerella vaginalis for another overnight incubation in -well cultures. conditioned medium was then harvested and production of il- was quantified by elisa. viability of the cells was ascertained at the end of the experiment with the mtt-assay. each treatment was applied in quadruplicate wells and experiments were repeated times using different batches of cells for each pathogen. results were evaluated using the general linear models procedure of sas for a randomized block design. results: sasp had no detectible effect on il- production by endocervical cells not treated with bacteria. at the highest concentration tested ( . mm), sasp significantly inhibited il- production by cultures stimulated with e. coli (p< . ), u. parvum (p< . ), and g. vaginalis (p< . ). viability of the cells, however, was significantly reduced by sasp at . and . mm in the both the presence and absence of bacteria for all experiments. conclusion: although high concentrations of sasp inhibit il- production by cultures of endocervical cells stimulated with pathogens associated with preterm birth, this effect may be due to toxicity of the antibiotic on the cells. the effect of -hydroxyprogesterone caproate on lipopolysaccharide stimulated peripheral blood mononuclear cells from pregnant women. richard h lee, aimin li, frank z stanczyk, jinwen i lin, t murphy goodwin. obstetrics and gynecology, university of southern california, los angeles, ca, usa. introduction: -hydroxyprogesterone caproate ( -ohpc) reduces the rate of recurrent preterm birth in high-risk women. however, there are lines of evidence to suggest that -ohpc alters inflammatory response in the setting of gram-negative infection. in a mouse model, -ohpc decreased the rate of preterm birth but was associated with significantly increased maternal morbidity when mice were exposed to lipopolysaccharide (lps). in non-pregnant women, -ohpc pre-treatment of whole blood exposed to lps significantly increased the production of tnf-. objective: to determine if -ohpc has an effect on the production of proinflammatory cytokines from peripheral blood mononuclear cells (pbmc) in pregnant women. methods: pbmc were isolated from fresh whole blood samples of pregnant women between and weeks. pregnant women had no prior history of preterm birth. cells were treated with -ohpc ( m) and escherichia coli lipopolysaccharide (lps, g/ml) alone or in combination. after and hours of culture, supernatants were collected and tested for tnf-and il- levels by enzyme-linked immunosorbent assay (elisa). statistical analysis was performed using non-parametric tests. p < . was considered significant. results: pbmc exposed to lps significantly increased tnf-and il- secretion compared to untreated controls (p= . and p= . ). tnf-concentrations were not significantly different between lps and lps+ -ohpc treated cells at both and hours (p= . and p= . ). il- production was significantly increased after -hour treatment with lps+ -ohpc compared to lps alone ( , [ , - , ] background. recent clinical trials indicate that progestins reduce the incidence of preterm birth in some high risk pregnancies. it has been proposed that progesterone promotes uterine quiescence, in part, via its antiinflammatory properties with inhibition of pro-inflammatory gene expression. it is intriguing that progestins are clinically effective given the considerably increased background circulating levels of the hormone during pregnancy. we hypothesised that non-classical progesterone signalling pathways contribute towards mediation of the anti inflammatory effects of progestins. to the endotoxin lipopolysaccharide (lps) by activation of inflammatory pathways. myometrial cell cultures were treated with lps ( g/ml)+/progestin ( nm). two progestin compounds were investigated. natural progesterone (p) is known to have a strong affinity with progesterone receptor (pr) analogues in contrast to -hydroxyprogesterone ( -hp) which, in the absence of esterification with caproate or acetate, has been reported to have no progestogenic activity at pr. the effect of p and -hp on the activation of two inflammatory genes known to be associated with labour (cycloxygenase [cox- ], toll-like receptor [tlr- ]) was evaluated. cox- and tlr- were detected at the protein and mrna levels using sds-page and rt-pcr. results. lps-induced expression of cox- and tlr- proteins were significantly inhibited by both p (p< . and p< . , respectively) and -hp (p< . and p< . , respectively). furthermore, preliminary results indicate that co-incubation with the anti-progesterone mifepristone, fail to abrogate the anti inflammatory effect associated with progestin treatment. conclusion. non-classical progesterone signalling pathways have a role in mediating the anti-inflammatory properties of progestins. further elucidation of the molecular actions of progestins may allow novel approaches to ameliorate the inflammatory response associated with preterm labour. detection and transcriptional expression of tlr- , tlr- and tlr- at the maternal-fetal interface. jacqueline p tilak, karen zakharian, alexandra tungol, gabriel o schubiner, david m svinarich. patient care research, providence hospital, southfield, mi, usa. preterm labor and delivery remains a leading cause of neonatal morbidity and mortality and bacterial infection is considered to be the most common etiology. toll-like receptors (tlr's) and the associated components of the innate immune system may represent a first line of defense against these pathogens. tlr's belong to a family of pattern-recognition receptors that bind to highly conserved pathogen-associated molecular patterns (pamps) including lipopolysaccharide (lps), lipotechoic acid (lta) and cpg dna, and are a key component of the innate immune system. this study was undertaken to characterize the transcriptional expression and regulation of tlr- , tlr- and tlr- within gynecologic and gestational tissues. human first trimester trophoblasts, endometrial mesoderm, ectocervix, choriocarcinoma and neonatal epithelium, were cultured in media alone or in the presence of either lps ( mg/ml) or lta ( mg/ml) for , , , , and hours. total rna was isolated and semi-quantitative rt-pcr was performed using intron-spanning amplimers to tlr- , tlr- , tlr- and gapdh. following gel electrophoresis, the integrated optical densities were determined for the corresponding pcr products and normalized with respect to gapdh levels. inducible transcription of tlr- with lta was observed in choriocarcinoma cells ( -fold, h), and endometrial mesoderm cells ( -fold, h). tlr- induction with lps was observed in ectocervical cells ( -fold, h), choriocarcinoma cells ( -fold, h) and endometrial mesoderm cells ( -fold, h). tlr- induction with lps was observed in choriocarcinoma cells ( fold, h) and neonatal epithelial cells ( -fold, h). all cell lines showed at least constitutive levels of transcription for tlr- , tlr- and tlr- . these data demonstrate that tlr- , tlr- and tlr- are transcriptionally expressed either constitutively or inducibly in both gynecologic (endometrial mesoderm, ectocervix) and gestational (chorion, trophoblast), tissues. translation of these receptors suggests that the innate immune system may function at the maternal-fetal interface to help protect the fetus against infection and prevent or diminish the likelihood of prematurity. objective: further to our development of a sheep model of intrauterine ureaplasma spp infection, we aimed to examine the capability of ureaplasma colonization in the amniotic fluid to infect the fetus and alter lung and brain development. methods: at days of gestation (d, term= d) ewes bearing single fetuses were given a single ultrasound-guided intra-amniotic injection of (a) x colony forming units (cfu) of u parvum (serovar , n= ; serovar , n= ), (b) x cfu of u parvum (serovar , n= ; serovar , n= ) or (c) media control (n= ). at d, fetuses were delivered by cesarean section. amniotic fluid and umbilical arterial blood samples were collected. fetal body weight was recorded, fetal cerebrospinal fluid (csf) collected and a descending pressurevolume curve constructed after inflation of the lungs to cmh o. fetal membranes were immersion fixed and the fetal brain was perfusion fixed with % paraformaldehyde. the fetal brain and membranes were blocked, paraffin embedded, stained and viewed under the light microscope. results: chronic intra-amniotic colonisation with u parvum serovar or (ureaplasmas) did not result in fetal abortion or death. amniotic fluid ureaplasma titers at delivery were not dose-dependent. chronic ureaplasma exposure did not affect fetal body or brain weights, or result in a fetal systemic inflammatory response. umbilical arterial blood gases at delivery were similar between ureaplasma-and media-exposed fetuses. chronic intra-amniotic exposure to ureaplasmas resulted in higher inflammatory cell scores in the fetal membranes compared to media controls (p< . ). lung compliance was increased in ureaplasma-exposed fetuses compared to controls (p< . ). there were no gross anatomical changes observed in the white or grey matter in the cerebral hemispheres of fetuses that had been exposed to ureaplasmas; even in animals (n= ) that had csf ureaplasma colonisation. conclusion: chronic ureaplasma colonisation enhances fetal lung compliance without gross anatomical changes in the fetal brain. background: an important source of vitamin d is its synthesis by skin from uv solar radiations. the skin pigment melanin absorbs uv photons thus reducing the vitamin d synthesis by more than % making african americans at high risk for vitamin d deficiency. low maternal vitamin d status during pregnancy has been linked to molecular pathways for adverse outcomes. objective: the nf b transcription factor regulates genes involved in inflammation and immune processes, and is proposed to play a major role in the successful outcome of pregnancy and labor. prior immunohistochemical (ihc) and biochemical studies have been conflicting regarding nf b activation in human intrauterine tissues. the aim of this study was to quantify nuclear localization of p , the major tranactivating nf b subunit, in full-thickness fetal membranes (fm) and myometrium using ihc. methods: a retrospective analysis of paired fm, decidua, and myometrial samples was conducted using tissues collected from women in cohorts: preterm no labor (pnl, n= ), preterm labor (ptl, n= ), spontaneous term labor (stl, n= ), and term no labor (tnl, n= ). subjects were delivered between gestational ages - weeks (preterm) and - weeks (term) by cesarean. paraffin sections were stained with polyclonal (rabbit) anti-human p and microscopically examined. myometrial samples were categorized in a blinded fashion to groups of staining: no nuclear p (-), rare nuclear p (+), and > % nuclear p (++). a p nuclear labeling index (nli; % nuclear p /total cells) was calculated for each histological layer in full-thickness fm specimens using a blinded targeted randomization scheme consisting of non-overlapping low-magnification fields. results: nuclear p labeling was rare in amnion and inconsistently present in chorion. in decidua, p nuclear labeling was observed in all cases; however, decidual nli did not vary significantly across cohorts [pnl- % ( - %); ptl- % ( - %); tnl- % ( - %); stl- % ( - %); all values are median (iqr)]. in myometrium, ++ p nuclear labeling was significantly associated with labor, but present only in a portion of cases (ptl- %; stl- %). despite a trend, decidual nli was not significantly correlated with myometrial nuclear p labeling: myometrial specimens classified as -, +, and ++ corresponded with decidual nli of % ( - %) [median (iqr)], % ( - %), and % ( - %), respectively. conclusions: in a comprehensive ihc analysis, significant nuclear p immunolabeling was observed in myometrial cells following labor. nuclear p labeling in decidua was present in all cases, and did not vary significantly among the cohorts. the inflammatory cytokines interleukin- and tnf-increase g-csf expression in term decidual cells. objectives: chorioamnionitis (cam) elicits premature rupture of the membranes and pre-term delivery. previously, we found that the decidua from cam patients contains much higher neutrophil numbers than control decidua, but macrophage numbers are similar in both groups. granulocyte colony-stimulating factor (g-csf) enhances granulocyte colony formation chemoattraction and activation. the amniotic fluid during cam contains elevated tnf-and il- levels. to account for the marked influx of neutrophils infiltration in cam-complicated decidua, tnf-and il-effects were assessed on g-csf expression in term decidual cell (dc) monolayers. methods: confluent leukocyte-free term dcs from normal term deliveries were primed with - m estradiol (e ) + - m medroxyprogesterone acetate (mpa) for days, then switched to serum-free defined medium (dm) with steroids ± tnf-or il- . after h, aliquots of conditioned dm supernatants, cell lysates and extracted rna from h parallel incubations were frozen. secreted g-csf was measured by elisa in conditioned dm and normalized to cell protein and mrna was assessed by quantitative real time rt-pcr and normalized to -actin mrna. results: in cultured first trimester dcs, basal g-csf levels in conditioned dm were . ± . pg/ml/ug cell protein [mean ± sem, n= ] and did not differ from e +mpa basal levels. the addition of ng/ml of tnf-or il- significantly elevated g-csf output to . ± . and ± respectively (p< . ), representing more than a -fold and , -fold change; respectively. western blotting confirmed the elisa results. quantitative rt-pcr demonstrated corresponding changes in g-csf mrna levels as found for the elisa measurements. concentration-dependent effects for both tnfand il- from . to . ng/ml were observed. conclusions: when extrapolated to the placental milieu of cam, the marked increase in g-csf elicited in term dcs by tnf-and il- may provide a mechanism to account for the selective increase in decidual neutrophils versus macrophages. background. the immunological mechanisms of the female reproductive tract are unclear. toll-like receptors (tlrs), innate immune receptors that combat microbial infections and establish adaptive immunity, may play a role. infection in pregnancy has been associated with preterm birth and tlrs may modulate the host response to such infections. we hypothesised that the distribution of tlr and tlr in cervical epithelial cells may differ with pregnancy, and that oestrogen may contribute to the modulation of these receptors. aims and objectives. . to compare tlr and tlr protein expression, using immunocytochemistry, in the cervical epithelium of pre-menopausal non-pregnant women with pregnant women at term. methods. fresh non-pregnant (n= ) and pregnant (n= ) human cervical cells were obtained by smear and tlr and tlr expression investigated by immunocytochemistry. cervical epithelial cells from nonpregnant women obtained by smears were then coincubated with varying concentrations of estradiol, and tlr and tlr protein expression quantified by immunocytochemistry. results. using pixel-based image analysis software, we demonstrated a reduction in tlr expression in late pregnant compared with non-pregnant cervical epithelium (p= . ), whilst tlr did not appear to differ. there was an apparent up-regulation of tlr protein expression in response to beta-oestradiol (n= ) objective: to evaluate in vitro antimicrobial activity of cranberry-exposed urine against common urinary pathogens. subjects were pregnant women enrolled in a clinical trial evaluating the effect of daily cranberry juice cocktail or matching placebo on asymptomatic bacteriuria and other urinary tract infections. methods: -hour urine samples from pregnant women who were randomized to cranberry or placebo in three treatment arms: a. cranberry two times daily with meals (c, c; n= ), b: cranberry in the am, then placebo at dinner (c, p; n= ), c: placebo two times daily with meals (p, p; n= ). we identified non-pregnant, reproductive-aged controls, randomized them to the same treatment groups and collected -hour urine specimens from them. the ph of all urine specimens was adjusted to ph= and filtered. aliquots of each were independently inoculated with overnight culture of - cell/ml each of single strains of e. coli with fimbriae type i and type ii, k. pneumoniae, and c. albicans. after hours of incubation for e. coli and k. pneumoniae, and hours for c. albicans cfu/ml of each specimen were enumerated by subculture with quantitative plate counts in duplicate. results: there were no differences for any of the antimicrobial activities between pregnant and non-pregnant groups, or based on treatment allocation. we were able to perform antimicrobial assays on the urine of women exposed to cranberry juice or placebo, but unable to demonstrate differences based on treatment allocation or pregnancy. this may be due to beta-error. further studies are planned. supported by r dk - ; clinical trials registration nct . ...,.. e. coli . x .:!: . x . x .:!: . x group a (c, c) group b ( c, p) . x + . x . x + . x . . . group c (p, p) . x o"::!>s x o" . x ~ .ox: o• k. ~neumoniae group a ( c, c) . x + . x . x + . x . . . group b (c, p) . x + . x . x + . x group c (p, pl . x ~ . x . x ~ . x c. al bicans group a (c, c) . x '+ . x . x + . x group b (c, p) . x o•+ . x . x + . x . . . group c (p, p) . x ~ . x . x ~ . x • objective: to measure compliance, we sought to evaluate the use of a bioassay for cranberry in the urine of women enrolled in a clinical trial designed to determine the effect of daily dosing of cranberry juice cocktail or matching placebo on the incidence of asymptomatic bacteriuria (asb) and other urinary tract infections (uti) during pregnancy. methods: we collected -hour urine specimens from pregnant women who were randomized to ingest cranberry or placebo in three treatment arms: a: cranberry two times daily with meals (n= ), b: cranberry in the am, then placebo at dinner (n= ), c. placebo two times daily with meals (n= ). we identified non-pregnant, reproductive-aged controls, randomized them to the same treatment regimens (group a: n= , group b: n= , group c: n= ), and collected -hour urine specimens from them. bacterial anti-adhesion effects of the cranberry-exposed urine were evaluated utilizing a human red blood cell hemagglutination assay specific for uropathogenic p-fimbriated e. coli. activity was quantified as , , and %. results: when combining all subjects and dosing regimens, the sensitivity of the assay was %, range % in the pregnant group assigned single daily dose of cranberry to % in the non-pregnant group assigned to multiple daily doses. the specificity ranged from % to %. conclusions: these data indicate that the bioassay can be applied to the pregnant patient population, although the sensitivity of the assay is variable. higher daily dosing appears to confer greater sensitivity. further studies are needed to evaluate the utility of this bioassay for compliance. supported by r dk - . clinical trials registration nct . objective: increasing evidence suggests that inflammation plays a crucial role in initiation of labour both at term and preterm. we have previously shown upregulation of pro-inflammatory cytokines in myometrium, cervix and membranes at term labour. we have also shown that myometrium and cervix is invaded by leukocytes at the time of labour, and these leukocytes express pro-inflammatory cytokines. in this study, we aimed to test the hypothesis that pro-inflammatory cytokines and toll-like receptor and (tlr and ) mrna expression are up-regulated in circulating leukocytes during term and preterm labour. methods: peripheral blood samples were taken from pregnant women: - weeks gestation (w) preterm not in labour (ptnl) n= ; - w preterm in labour (ptl), n= ; - w term not in labour (tnl) n= ; and - w term in labour (tl) n= . leukocytes were isolated using dextran sedimentation. total rna was isolated and reverse transcribed using high capacity cdna archive kit, and mrna expression determined by real-time pcr (abi, taqman). student's t-test was used to compare outcomes between groups. results: messenger rna expression of il- , icam- , mcp- , tlr and tlr was significantly increased in tl compared to gestation matched tnl. the expression levels of il- b, il- and tlr- were significantly greater in ptl compared with gestation matched ptnl. (table i) . conclusions: we show up-regulation of pro-inflammatory cytokine production in circulating leukocytes in both term and preterm labour. thus, the pathophysiology of labour seems to involve the upregulation of proinflammatory cytokine production in peripheral blood leukocytes, followed by their invasion into the myometrium and cervix. this work further contributes to our understanding of the pathophysiology of parturition, and suggests that peripheral blood leukocytes may be potential targets for therapeutic agents aimed at modifying the time course of parturition. introduction. the mechanisms of innate immunity and tolerance in the female reproductive tract (frt) are ill-understood but involve the pattern recognition toll-like receptors (tlrs). we have previously demonstrated high expression levels of tlr gene and protein in fresh human cervical epithelium. aims. in order to gain insight into the immunological role of cervical epithelial cells (cecs), we sought to determine cec responses to the following tlr- agonists: macrophage-activating lipopeptide (malp- ), pam csk , and peptidoglycan. methods. cecs were isolated by smears and compared between pregnant ( rd trimester) and nonpregnant women. following cell preparation, flow cytometry was performed using a direct staining procedure with mouse anti-human tlr primary antibody and its igg , isotype control, to determine tlr- protein expression. a further nonpregnant smear samples were each prepared, and seeded at a concentration of , cervical epithelial cells/ml into -well cell plates. cells were incubated at °c in % co overnight with the tlr agonists malp- and pam csk (at concentrations of and ng/ml), peptidoglycan( ng/ml), il- ( ng/ml, positive control) and rpmi + -glutamine media only (vehicle). following centrifugation, all resulting supernatants were stored at - °c until the concentration of il- was determined by elisa and an array of cytokines by bead assays. results. both pregnant and nonpregnant cecs expressed tlr (specific mean fluorescence . vs . respectively) but did not differ (p = . ). unstimulated cells incubated with buffer alone demonstrated high il- levels ( pg/ml), which did not differ following stimulation with malp- ( pg/ml), pam csk ( pg/ml) or peptidoglycan ( pg/ml). results of an array of pro-and anti-inflammatory cytokines following incubation of cells stimulated with tlr agonists are pending. conclusion. high basal il- levels suggest constitutive expression by cecs but the physiological relevance of this intriguing observation is unclear. that cec stimulation with tlr- agonists did not lead to further release of il- may epitomise the resistance of these cells to antigenic challenge by the vaginal commensal flora. cecs may play a pivotal role in modulating the immunological tolerance necessary to minimise inappropriate inflammation in the cervix. there are several epidemiological and clinical studies that omega- fatty acids and related fish oils can decrease the premature labor and birth. however, the scientific mechanisms are not well elucidated. this study was carried out to investigate the effects of omega- fatty acids on producing pge and il- , in human umbilical vascular endothelial cell(huvec) media with artificial inflammation as an infection-induced premature labor tissue model. also, if there are some significant effects with omega- fatty acids, we want to investigate the possible mechanisms. materials and methods; human umbilical vascular vascular cell primary culture was done. in the adequate cell confluence in each cell dish, pretreatment with various concentrations of epa, dha and troglitazone (another ppar-ligand) and incubation were done for hours in °c, co incubator. after that, g/ml conc. of lipopolysaccharide(lps) was treated to the each cell dishes and incubated for hours. the cell media were collected, and pge and il- concentration were checked by elisa. the each cell lysates were collected and western blot anaysis for cyclooxygenase(cox)- were done. on the other hand, nuclear factor kappa b (nf b) luciferase vector was transfected and then did the same pretreatment with epa, dha and troglitazone and lps treatment to each cell dishes. after that, nf b luciferase activity was checked with luminometer. statistical analysis was done by student t-test. results: epa, dha and troglitazone decreased the pge (p< . ) and il- (p< . ) significantly in elisa. cox- expression revealed the significant reduction with pretreatment of epa, dha and troglitazone in higher concentration ( , m) in the western blotting (p< . ). nf b luciferase activity were also significantly decresed with pretreatment of epa, dha and troglitazone in higher concentration (p< . ). conclusion; this study offers some scientific mechanisms, by which omega- fatty acids (epa, dha) and troglitazone may be one kind of the preventive medicine for infection-induced preterm labor and delivery. also, these effects may come from the common mechanism of epa, dha and troglitazone, ppar-ligands. the next study would be how the cox- , nf b and pparare related. ( mg/ml). cytokine expression was measured in medium using the bio-plex suspension array system (bio-rad). mean expression was compared between the two groups using t test. results: -aminopurine significantly inhibited lps stimulated cytokine production by human myometrium. in contrast, there were no significant differences in expression after mpa treatment, although a trend towards inhibition of proinflammatory cytokine and an increase in il- production was noted. introduction: intrauterine infection is now recognised as a major antecedent of white matter injury (wmi) in the preterm infant brain, which can manifest later as cerebral palsy or as varying degrees of cognitive dysfunction. wmi in these infants is characterized by damage to immature oligodendrocytes, and has been linked both to microglial activation and to elevated levels of tnfa, il- b and il- . we have developed a fetal sheep model for diffuse and focal wmi, based on repeated administration of e. coli lipopolysaccharide (lps) as the stimulus for an inflammatory response. methods: surgery to implant fetal vascular catheters was performed on pregnant ewes carrying single fetuses at d - of gestation. fetuses received repeated iv injections of lps ( ng/kg estimated fetal weight/day) between d and d . plasma levels of pro-inflammatory cytokines were determined in fetal arterial blood samples taken between d and d . at d ewes and fetuses were euthanized and fetal brain tissue samples collected for histological analysis. results: five days after final administration of lps to fetuses we observed a pattern of wmi similar to that seen clinically, ranging from focal to diffuse injury within the periventricular region, impairment of white matter (cnpase; marker for immature/mature oligodendrocytes) tracts, and thinning of the corpus callosum, characterised by oligodendrocyte disruption and microglial proliferation in the surrounding and sub-cortical white matter. we also found a progressive rise in fetal plasma tnf levels between days and (day two of treatment to third day following final dose of lps). background: plasma fatty acid (fa) levels are associated with altered host inflammatory responses, blood pressure regulation, and insulin resistance, characteristic features of pregnancy-induced hypertension (pih). most studies compare the n- and n- polyunsaturated fatty acids (pufas). in addition, recent data demonstrate that saturated and trans-fas promote inflammation. based on the immunomodulatory activity of various fas, we explored their effects on placenta inflammatory responses ex vivo. methods: placenta cells were isolated from fresh human (anonymous), term placentas and treated with/without lipopolysaccharide (lps) with various fas, including saturated fas, trans-fas, and n- and n- pufas (at physiological concentrations). after an overnight treatment, tnf-alpha (tnf) production was determined. data were analyzed by analysis of variance (anova) followed by the dunnett's test. results: oleic acid ( : n- ), a cis-monosaturated fa common in the mediterranean diet, did not induce significant placenta tnf production (fig. a) . by contrast, elaidic acid ( : n- ), a trans-monosaturated fa, induced tnf production by -fold compared to vehicle (fig. a) . likewise, palmitic acid ( : ), a saturated fa, induced placenta tnf production by -fold (fig. a) . to mimic inflammation, placenta cells were treated with lps ex vivo in the presence/absence of fas. docosahexanoic acid ( : n- , dha) reduced placenta tnf production by up to % following stimulation (fig. b) . similarly, treatment of placenta cells with linoleic acid ( : n- , la) or arachidonic acid (n : n- , aa) suppressed tnf production by up to % and %, respectively (fig. b) . conclusions: both saturated fas and trans-fas, which positively correlate with hypertension, induce placental tnf production. the n- fa precursors to prostaglandins, aa and linoleic acid, reduce placental tnf production following stimulation. likewise, dha, a product of n- fa metabolism commonly consumed in fish oil and associated with improved blood pressure, suppresses tnf production by stimulated placenta cells. vegf and flk mediated glomerulogenesis in offspring exposed to maternal hypernatremia. roy z mansano, mina desai, ahmed abdel-hakeem, thomas r magee, tazmia q henry, cynthia nast, john s torday, michael g ross. dept. of ob/gyn, harbor-ucla med. ctr., torrance, ca, usa. objective: growth restricted human and animal offspring, resulting from maternal nutrient restriction or uteroplacental insufficiency, consistently demonstrate reduced glomerular number and a predisposition to adult systemic hypertension and renal compromise. in contrast, maternal water restriction (wr) produces newborns with a unique programmed phenotype of increased glomerular number. glomerulogenesis is dependent, in part, on appropriately timed and quantified vascular development. as vegf and its receptor flk have crucial stimulatory effects on renal vasculogenesis, we hypothesized that maternal wr-induced morphologic renal changes are secondary to vegfmediated vasculogenic effects. methods: from day to term gestation, pregnant rats received either ad libitum food and water (control, n= ), or wr to produce an increment of meq/l in plasma sodium (n= ). following delivery, all dams received ad libitum food and water. at d after birth, offspring kidneys were extracted for mrna. vegf and its receptor, flk , mrna levels were determined using real-time rt-pcr (presented as fold difference normalized to s). at d after birth, offspring glomerular number were determined in formalin fixed m sections using histomorphometric analysis. values are means±se. results: wr offspring (day ) had higher glomerular number than control (wr ± , control ± per mm , p< . ). flk mrna expression was increased in wr offspring kidneys (wr . ± . , control . ± . , p< . ) as compared to controls. in contrast, vegf mrna expression in wr offspring kidney was comparable to control (wr . ± . , control . ± . , p= . ). conclusion:prenatally wr offspring demonstrate significantly increased glomerular number. as vegf recruits angioblasts to the developing glomerulus via its receptor flk , increased receptors (flk ) with the same level of the ligand (vegf) suggest that enhanced vasculogenesis may represent a putative mechanism for increased nephrogenesis in wr offspring. modulation of newborn vasculogenesis via vegf and flk expression may represent a therapeutic option for growth restricted offspring with decreased glomerular number. objective: maternal food restriction (mfr) during gestation (embryonic day to birth) reduces rat kidney glomerular number by % at weeks of age. undernutrition during human gestation leads to similar impaired nephrogenesis and increased hypertension in adults. renal development may be delineated into stages including ureteric bud branching, mesenchymal to epithelial transformation and glomerulogenesis. previously, we detected dysregulation of genes controlling nephrogenesis at gestational ages, e -e , suggesting that mfr has significant effects on ureteric bud branching. in the present study we evaluated the effect of this dysregulation on early nephron development in e fetal kidney explants from dams with mfr. methods: e fetal kidneys were collected from % mfr pregnant rats and ad libitum control rats (n= per group and time point), incubated in dmem/f k medium for , , , , and days, and fixed with % paraformaldehyde. kidneys were stained with fluorescein-labeled dolichos biflorus agglutinin (dba), images captured and terminal ureteric buds quantitated. all values are presented as mean ± sem. differences were considered significant at p< . . results: mfr ex-vivo kidneys at day demonstrated an initial moderate reduction in terminal branches versus control (c) samples (mfr: ± vs c: ± terminal branch ends per kidney). both mfr and control (c) kidneys demonstrated significant (p< . ) increases in branching in explant culture to maximum values at days (mfr: ± vs c: ± ). with increasing culture days, the percent reduction in mfr branching increased with terminal branch point decreases of % at day , % at day , % at day , % at day , and % at day (p< . , mfr vs c at day ). conclusion: kidney explant cultures from mfr treated fetuses display basal and culture-based decreased branching compared to controls. this decrease in terminal ureteric buds, in combination with our previous findings of dysregulation of branching-associated kidney transcription and growth factors, suggests mfr induces early (e ) dysregulation of branching as a major mechanism of the associated nephropenia. these results indicate that early programming events in kidney development induce nephropenia and renal disease in adults. intrauterine growth restriction (iugr) has important implications for the neonate not only at the time of birth, but also as an adult. in humans and animals with iugr, the elevated risk of developing hypertension is thought to involve an upregulation of the renin-angiotensin system (ras). however, the link between the intrauterine insult and enhanced ras is not known. a novel mechanism leading to hypertension has emerged recently involving two orphan g-protein coupled receptors (gcr and gcr ) and their endogenous ligands, succinate and -ketoglutarate. infusion of succinate into adult mice induces hypertension, an effect which was eliminated in gcr knockout mice or by pretreatment with ace inhibitors. interestingly, succinate levels have been shown to increase in the circulation under conditions of oxidative stress, one of the hypothesized mediators of developmental programming of hypertension. thus, we hypothesized that gcr and gcr are upregulated in a rat model of iugr and may contribute to in utero programming of hypertension. timedpregnant rats were fed either control (c, % casein) or low protein (lp, % casein) diet throughout gestation. kidneys were collected on embryonic day (e ), post-natal day (d ) or (d ), n . real-time pcr was used to compare gcr , gcr and renin expression. data were standardized to a housekeeping gene (s ) and are expressed as fold increase/ decrease compared to control. a student's t-test was used to determine significance between groups (p< . ). offspring from lp dams were significantly smaller at birth and did not display catch-up growth. in kidneys from lp fetuses, both gcr and gcr were significantly elevated compared with controls ( . fold and . fold respectively; p= . ), whereas renin was . fold lower. on post-natal d , there was a trend towards increased expression of both gcr ( . fold; p= . ) and renin ( . fold; p= . ) in offspring from lp dams. at d , there were no significant differences between groups. in conclusion, these preliminary data suggest that in iugr offspring from lp rats, the gcr / pathway may be enhanced, indicating a novel mechanism for the programming of hypertension. objective: in addition to excess adipose tissue, obesity is accompanied by increased fat storage in organs such as the liver. peroxisome proliferatoractivated receptors (ppars) are ligand-activated transcription factors involved in the regulation of lipid metabolism, and lipid-associated inflammatory response. obesity represents a state of chronic low-level inflammation, with ppar and ppar implicated in this process. we have previously shown that nutrient restriction in pregnancy results in intrauterine growth restricted (iugr) newborns which develop adult obesity with elevated c-reactive protein (crp) levels. as crp is derived from the liver, we hypothesized that iugr-induced obesity inhibition of hepatic ppar and ppar is associated with an increased inflammatory response. methods: control dams received ad libitum food, whereas study dams were % food-restricted from pregnancy day to to produce iugr newborns. all pups were nursed by control dams and weaned at weeks to ad libitum feed. at day and months of age, male offspring were analyzed for hepatic ppar , ppar and crp mrna (real time rt-pcr) and protein (western blot) expression. data was normalized to -actin and presented as fold change for protein levels. at months, hepatic triglyceride content was determined enzymatically. results: at d of age, iugr pups showed significant downregulation of ppar ( . -fold) and ppar ( . -fold) expression, though crp expression was significantly upregulated ( -fold). findings persisted at months of age, with continued downregulation of ppar and ppar ( . -fold) and upregulation of crp expression ( -fold). furthermore, iugr adults had significantly increased hepatic triglyceride content ( ± vs. ± mg/g liver, p< . ). conclusions: reduced expression of hepatic ppar and ppar in iugr offspring may contribute to elevated hepatic crp levels and triglyceride content. thus, developmental hepatic dysregulation leads to programmed obesity-induced inflammation in iugr offspring. offspring. mina desai, ederlen casillas, guang han, darran n tosh, , michael g ross. dept. of ob/gyn, labiomed at harbor-ucla med. ctr., torrance, ca, usa; dept. of physiology, univ. of adelaide, australia. objective: leptin and insulin mediate central anorexigenic signaling responses via different receptor molecules: leptin binds to the obrb receptor activating jak-stat and pi k pathways, whereas insulin activates pi k pathway by binding to its receptor (ir) and substrate (irs ). maternal food restriction in pregnancy results in iugr newborns that develop hyperphagia and adult obesity. the iugr newborns have significantly decreased plasma leptin levels with increased hypothalamic expression of basal obrb, stat and decreased expression of ir and irs , suggesting altered anorexigenic pathways. we studied the response of hypothalamic leptin/insulin signal molecules to peripheral leptin in iugr newborns. methods: control dams received ad libitum food, whereas study dams were % food-restricted from pregnancy day to . day old male offspring were given saline or leptin ( g/g, i.p). hypothalamus was dissected at , and minutes and protein expression of total stat , phosphorylated stat (p-stat ), inhibitor of leptin signal (socs ), ir, irs , total akt and phosphorylated akt was determined by western blot (normalized to -actin). data is compared between leptin and saline treatments in iugr and controls. results: in response to peripheral leptin, iugr newborns showed marked dysfunction in stimulated hypothalamic leptin and insulin signaling responses. ( ) jak-stat : leptin-treated controls show progressively increased pstat ( -fold) with initial suppression of socs ( . -fold) as compared to salinetreated controls. conversely in leptin-treated iugr, the pattern is reversed such that there is sustained decline in pstat expression ( . -fold) with failure to downregulate socs . ( ) pi k pathway: leptin-treated controls showed a significant reduction in irs ( . -fold) and pakt ( . -fold) as compared to saline-treated controls. however, leptin-treated iugr newborns exhibited a paradoxical increase expression of irs ( -fold) and pakt ( -fold). conclusion:the iugr offspring demonstrate persistent upregulation of leptin receptor, a reduced phosphorylated stat (p-stat ) response in conjunction with an enhanced socs- response. the persistent increase in insulin responses indicates a dysfunction in dynamic signaling, leading to altered anorexigenic response and development of programmed obesity. we have previously demonstrated that maternal food restriction (mfr) in rats induces a marked increase in the expression of vegf protein in the aorta and mesenteric arterioles, accompanied by an increase in tgf-and collagen in both vessel types in adult rat offspring (am j physiol, ) . the aim of this study was to determine if this in vivo finding could be reproduced in an in vitro preparation. methods: two types of preparations were used in this study. we isolated endothelial cells from week old male control rat aortas. these cells were used after the third passage. staining with von willerbrand factor demonstrated that these cells were pure endothelial cells. the second type of preparation was aortic explants from week old male control rats. endothelial cells and aortic explants were transfected with a vegf adenovirus ( - viral infective particles) or a -galactosidase-adenovirus as a control. after hours of culture protein was isolated from cells and explants for western blot analysis using rat specific antibodies. culture media was assayed for vegf by elisa. results: transfection of vegf adenovirus induced a dose-dependant increase in the expression of vegf protein in primary endothelial cells and aortic explants. the transfection of vegf into the endothelial cells showed a bell shaped curve, and was accompanied by an increase in media levels of vegf protein. maximal secretion of vegf was found with viral infective particles. vegf adenovirus transfection induced a dose-dependant increase in c-reactive protein (crp) (inflammatory marker), and tgf-protein in both aortic explants and primary endothelial cells. these results indicate that upregulation of vegf in blood vessels induces inflammation and tgf-expression which in turn can induce collagen synthesis. thus the increased collagen expression and reduced compliance previously reported by us in vessels of mfr offspring can be explained by the over-expression of vegf which we reported. therapeutic intervention aimed at prevention of the increase in vascular vegf expression in mfr offspring could potentially prevent programmed hypertension. maternal regulation of high fat nourishment during lactation period reduce a hypertension of male offspring. hidenori takahashi, toshiaki okawa, keiya fujimori, akira sato. obgyn, fukushima med.univ., fukushima, fukushima, japan. objective: exposure to undernutrition or high fat nourishment during fetal life has been proposed as an underlying cause of adult hypertension, but the effect of maternal feeding regulation during lactation period on blood pressure of offspring is unclear. our objective was to investigate the effects of either high-fat diet (hfd) during gestation to lactation period or restrictive fed a hfd during lactation period on blood pressure in male rat offspring. we use types pregnant wistar rats as fed with normal nutrition (group a), with a high fat diet (hfd) during gestation to lactation period (group b) and with hfd nutritionally restricted by feeding with % of the normal lactationmatched dietary intake from the day of delivery to the end of lactation period (group c). the male offspring was measured blood pressure at , and weeks by using indirect tail-cuff method. statically analysis was performed using oneway annova. results: body weight was significantly reduced in c offspring compared to a and b in male offspring at day after delivery (p< . ). at weeks old, the body weight of c offspring was no difference to catch up compared to a and b offspring. systolic and diastolic blood pressures were significantly elevated at all , and weeks in offspring of b > c >a. (p< . , vs. a) conclusions: under high-fat nutrition during gestation to lactation period induced hypertension in male rat offspring. maternal high fat environment make a hypertensive offspring, but regulation of fat feeding during lactation period may reduce adulthood hypertension. background: uterine artery (uta) doppler velocimetry has been validated in populations of heterogeneous parity in the second trimester for prediction of obstetric outcomes requiring preterm delivery to include: fetal growth restriction, fetal demise, hypertensive disorders of pregnancy, abruption, and indicated preterm delivery. understanding that parity may affect uta doppler indices in subsequent pregnancies, we sought to validate these predictive values in the first trimester in a homogeneous population of multiparous women. study design: multiparous women undergoing first trimester screening of singleton pregnancies were enrolled and followed prospectively until delivery (n= ). these women were divided into controls, ri < . (n= ) and cases, ri . (n= ), based on prior studies. demographic, clinical, and sonographic data (including uta indices and assessment of notching) were obtained. statistical analysis included student's t test and chi square. results: cases were not significantly different from controls in terms of maternal age, ethnicity, bmi, or medical history. uta doppler indices were significantly different between the two cohorts in terms of the presence of unilateral or bilateral notching ( % vs, % p< . and % vs. %, p< . , respectively). in contrast to that observed in patients of heterogenous parity previously, ri . was not associated with adverse pregnancy outcomes despite an average ri of . , significantly above this threshold. conclusions: in this multiparous cohort ri was not predictive of adverse obstetrical outcome, in contrast to that observed in cohorts including nulliparous patient. parity may affect uta vasculature and obscure the ability of doppler velocimetry to predict adverse obstetric outcome in multiparous women. presence of uta doppler notching in the first trimester remained a robust predictor of adverse obstetric outcomes in multiparous patients. objective: epidemiological studies have shown that offspring exposed to preeclampsia during fetal development are more susceptible to airway disease later in life. we have shown previously that gender, but not sflt- over-expression during pregnancy determines higher reactivity in the offspring airways at months of age. the objective of this study was to examine the effect of preeclampsia on the trachea from female and male offspring in our model of sflt- induced preeclampsia at year of age and compare responses between the two age groups. methods: cd- mice at day of gestation were injected via the tail vein with adenovirus carrying sflt (adsflt , pfu/ l) or mfc (admfc, pfu/ l). mice were allowed to deliver. tracheas were isolated from female and male offspring at months and year of age, and rings were mounted in organ chambers for isometric tension recording. responses to potassium chloride (kcl, mm), the mast cell degranulating agent compound / ( / , g/ml), and concentration-responses curve to acetylcholine ( - - - m) were obtained. results: there was no significant difference in responses to acetylcholine, kcl, or compound / between year old offspring born to the sflt and mfc groups. when comparing offspring within the same pregnancy exposure groups, responses to acetylcholine in adsflt -treated group were significantly higher in year old females than males. comparison between age groups by pregnancy exposure revealed that in the mfc group, year old male offspring had higher responses to compound / and acetylcholine than months old males. responses to kcl were significantly higher in months old males than year olds independent of maternal treatment during pregnancy. in females, the only difference between age groups was observed in the mfc group, where months old offspring demonstrated significantly higher responses to acetylcholine compared to year old offspring. conclusions: our findings did not show that airways of year old offspring born to mice with a preeclampsia-like syndrome induced by sflt- over-expression have airway hyperreactivity. however, sex and age differences in airway responses dependent on maternal exposure during pregnancy were observed, and needs to be explored further to elucidate underlying mechanisms. objective: maternal food restriction (fr) results in iugr newborns that when normally nursed exhibit rapid catch-up growth and adult obesity. continued fr during nursing delays catch-up growth and prevents adult obesity. igf- , which modulates growth and is secreted by the liver, may contribute to these morbidities. igf- is epigenetically regulated involving two promoters, alternative exon splicing and multiple transcription termination sites. we determined if hepatic igf- mrna levels correlate with obesity, and whether these changes are due to programmed epigenetic modification. methods: control pregnant rats received ad libitum food from gestation day to and lactation, whereas study dams were % fr. fr pups were nursed by either control (fr/adlib) or fr dams (fr/fr) and weaned to ad libitum feed. at day and months, male livers were analyzed for igf- mrna variant levels (real time rt-pcr). chromatin immunoprecipitation (chip) was performed using the antibody for h k trimethyl, and associated levels of each igf- species were measured by pcr. results: at months, obese fr/adlib males showed increased mrna levels of igf- a, igf- b, igf- exon , and igf- exon as compared to controls ( ± , ± , ± , and ± %). comparing fr/adlib month to newborn offspring, h /k was increased at igf -promoter , promoter , exon , utr# and utr# ( ± , ± , ± , ± , and ± %), though there was no differences between control month and newborns. in contrast, month fr/fr males had comparable mrna levels to the controls except for igf- b (% of control: ± ). further, fr/fr month h /k was only different from newborns at utr# (% of newborn: ± ). conclusion: iugr newborns with rapid catch-up growth and adult obesity have increased postnatal hepatic igf- mrna levels, likely a result of igf- histone and chromatin structure modifications to h k trimethylation. conversely, iugr with delayed catch-up growth and absence of adult obesity have levels similar to that of controls. thus, modulation of the rate of iugr newborn catch-up growth may protect against igf- epigenetic modifications. introduction: igf-ii is synthesized as a pro-hormone (proigf-ii; -amino acid peptide) which is then processed into its active forms: "big" igf-ii ( - ) and mature igf-ii ( - ). these active forms are essential for placental and fetal development and have also been shown to persist into postnatal life. since maternal smoking is known to adversely affect feto-placental growth and postnatal development, we postulated that these effects might be mediated through nicotine-induced alterations in igf-ii processing. methods: in the present study, nulliparous female wistar rats ( - g) were given nicotine ( mg/kg/day) or saline for days prior to mating, during pregnancy, and throughout lactation. at gestational day , and , dams were euthanized and we collected serum (fetal and maternal), amniotic fluid and recorded fetal body weight. a subset of dams were allowed to deliver at term. following parturition, serum samples from the offspring were collected at birth (pnd ) and weaning (pnd ). body weight was recorded weekly from birth to weaning. pro, "big" and mature igf-ii levels were determined by western blot analysis. results: maternal nicotine exposure during pregnancy resulted in a significant reduction in fetal body weight by gestational day . however, there was no effect of nicotine on fetal serum or amniotic fluid igf-ii levels at any gestational age examined. in maternal serum, mature igf-ii in control animals decreased with advancing gestational age such that igf-ii levels were lowest at gestational day . nicotine administration prevented this decline, which resulted in significantly higher mature igf-ii levels in nicotine-exposed mothers at gestational day . in postnatal life, nicotine exposed offspring had significantly lower levels of "big" igf-ii expression at weaning (pnd ). conclusions: these data demonstrate that nicotine can alter the amount of the active forms of igf-ii in the mother and the newborn. dysregulation of maternal igf-ii occurs concomitantly with suboptimal fetal growth. results from this study suggest a mechanism by which maternal smoking causes impaired fetal growth and adverse postnatal health outcomes. objective: maternal food restriction in pregnancy results in iugr newborns which develop adult metabolic syndrome. programming of both increased appetite-mediated hyperphagia and enhanced adipogenesis contribute to the development of obesity. transcription factors, peroxisome-proliferatoractivated-receptor (ppar ), ccaat/enhancer binding-protein (c/ebp ), and sterol regulatory element binding-protein (srebp c) regulate adipogenesis and lipogenesis. although iugr offspring exhibit acute upregulation of the adipogenesis signaling cascade prior to the development of obesity, we determined if this increased adipogenic potential was an intrinsic cellular response, and thus maintained in cell culture. we further examined the responses to adipocyte stimulators (ppar activator-ligand rosiglitazone) and inhibitors (ppar repressor-ligand badge). methods: control dams received ad libitum food, whereas study dams were % food-restricted from pregnancy day to term. adipocytes from day old iugr and controls were isolated and cell proliferation rate was determined (mtt). primary adipocyte cell cultures were established and following % confluence, iugr and control adipocytes were treated to two doses ( and m) of either rosiglitazone or badge for h. mrna and protein was extracted for expression of ppar , c/ebp , srebp c. data was normalized to -actin and compared to the respective untreated cells. results: iugr adipocytes had significantly increased protein expression of ppar ( . -fold) and c/ebp ( -fold) as compared to control adipocytes, though srebp c levels were unchanged. mrna levels showed similar changes in iugr newborns. importantly, iugr adipocytes exhibited increased cell proliferation ( % of control, p< . ) and showed greater response to rosiglitazone ( . -fold), though similar response to badge, as the control adipocytes conclusion: iugr primary adipocytes cell culture exhibit basal phenotypic characteristic of programmed upregulation of adipogenic transcription factors which promote adipose cell proliferation. the enhanced response to the adipogenic stimulant is further evidence of the predisposition to obesity. in contrast, the normal suppressive response to the inhibitor suggests that iugr adipocytes may respond to pharmacologic approaches to prevent obesity during this period. objective: maternal nutrient restriction results in intrauterine growth restricted (iugr) newborns which develop programmed obesity despite a normal post-weaning diet. the epidemic of obesity has been attributed in part to programmed "thrifty phenotype" and exposure to "western" diets. hepatic igf- is epigenetically regulated involving two promoters, alternative exon splicing, and multiple transcription termination sites. iugr offspring with normal post-weaning diet have increased postnatal hepatic igf- mrna levels, likely a result of igf- histone and chromatin structure modifications to h k trimethylation. we hypothesized that iugr newborns that develop programmed obesity would demonstrate discernable hepatic igf- changes which are distinct from diet-induced obesity. we determined igf- hepatic mrna levels and epigenetic characteristics in programmed (iugr) and dietinduced (dio) offspring. methods:: control pregnant rats received ad libitum food whereas study dams were % maternal food restricted from day to . all pups were nursed on ad libitum fed dams. controls were weaned to high-fat (fat, %) diet whereas iugr were weaned to normal ad libitum diet (fat, %) to produce diet-induced (dio) and programmed obese groups, respectively. at months, male hepatic igf- were analyzed for igf- mrna variant levels (real time rt-pcr). chromatin immunoprecipitation (chip) was performed using the antibody for h k dimethyl and h k trimethyl, and associated levels of each igf- species were measured by pcr. result: relative to dio control males, iugr had increased mrna of igf- a, exon and exon ( ± , ± , ± %). chip with h k dimethyl showed increased igf- exon ( ± %) and with h k trimethyl, increased igf- promoter and promoter ( ± , ± %) as compared to dio controls. conclusion: adult obese iugr males exposed to normal postweaning diet have increased hepatic igf- a mrna and h k dimethylation and trimethylation of igf- than dio controls. changes in igf- in adulthood from a prenatal insult thus suggest that igf- is programmed during the fetal period and may be associated with programmed adult obesity. rebekah elkins, pandu gangula, chandra yallampalli. obstetrics gynecology, university of texas medical branch, galveston, tx, usa; internal medicine, university of texas medical branch, galveston, tx, usa. objectives: we previously reported that the offspring of rats fed % protein during gestation develop hypertension at two to four months and that the hypertension is exacerbated in males. this study is to evaluate: ) changes in estrogen receptor (er) angiotensin ii subtype receptor (at -r) and endothelial nitric oxide synthase (enos) in the mesenteric artery and aorta of offspring and assess if these changes, if any, are gender specific. methods: pregnant sprague dawley rats were fed either % protein (ctrl) or % protein (lpd) from day of gestation. the offspring were evaluated for hypertension by means of systolic blood pressure measurements. at four months for the males and nine months for the females, mesenteric artery and aorta were collected in rnalater. expression of estrogen receptor a (er-a) and b (er-b), at -r, and enos were analyzed by western immunoblotting and rt-pcr and expressed relative to b-actin or s. results: mesenteric artery shows no differences between ctrl and lpd female offspring in at -r, enos, er-a or er-b. similarly mesenteric artery shows no diet exposure related changes in at -r, er-a or er-b in male offspring. however, enos expression was lower in mesenteric artery of lpd male offspring. on the other hand, in the aorta both er-a and er-b levels are lower in lpd female offspring while there were no changes in at -r or enos. no changes in at -r, er-a or er-b were observed in male offspring aorta of ctrl and lpd rats. conclusion: the in utero exposure to lpd results in adult hypertension in both male and female offspring. some mechanisms for hypertension include the decrease in er-a and er-b but not at -r or enos in females, and the decrease in enos but not at -r or er in males indicating gender related differences. offspring. hidenori takahashi, toshiaki okawa, keiya fujimori, akira sato. obgyn, fukushima med. univ., fukushima, fukushima, japan. our objective was to investigate the effects of either severe undernutrition during late gestation or lactation period on blood pressure and the development of vascular function in male rat offspring. we use normal pregnant wistar rats (group a), nutritionally restricted by feeding with % of the normal gestation-matched dietary intake from day of gestation to delivery (group b) and % restricted after delivery to the end of lactation period (group c). the offspring was measured blood pressure at and weeks by using indirect tail-cuff method. rings of thoracic aorta with intact endothelium from the male offspring of a and b at weeks, were equilibrated at g passive tension in organ chambers filled with krebs-henseleit solution continuously bubbled with %co in air ( °, ph . ) for isometric tension recording. concentration-response relationships to norepinephrine (ne) and angiotensinii(atii) were obtained in the absence or presence of n(omega)-nitro-l-arginine methyl ester (l-name) or a selective atii type- receptor blocker (valsartan). responses to cumulative concentrations of sodium nitroprusside (snp) and to - m oxyhemoglobin (hb, nitric oxide scavenger) were also determined. contractions were expressed as a percent of the reference contraction induced by potassium chloride ( mm). statically analysis was performed using one-way annova. results: body weight was significantly reduced in b offspring compared to a and c in male offspring at day (p< . ). at weeks the body weight of offspring of b was no difference to catch up compared to a and c offspring. systolic and diastolic blood pressures were significantly elevated at both and weeks in offspring of b > c >a. ne concentration-dependently stimulated tension of aortic rings from in a and b offspring, which was not significantly (n= ). maximal contractions to ne were significantly stimulated by l-name in a (p< . ), but not b offspring. valsartan significantly inhibited aortic contractions by ne in r (p< . ), but not a offspring. there was no significant difference on responses of aortic rings by atii, snp and hb in a and b offspring. conclusions: severe under nutrition during not only late gestation but also lactation period induced hypertension in male rat offspring in adulthood. fetal origin of adult hypertension might be vascular endothelial dysfunction. fujimori, akira sato. obgyn, fukushima med. univ., fukushima, fukushima, japan. objective: exposure to undernutrition during fetal life has been proposed as an underlying cause of adult hypertension, but the effect of either high fat nourishment or undernutrition during lactation period on blood pressure is unclear. our objective was to investigate the most effective maternal nourishment and feeding period for offspring induced adulthood hypertension in using high-fat diet (hfd). study design: we use types pregnant wistar rats as fed with normal nutrition (group a), nutritionally restricted by feeding with % of the normal gestation-matched dietary intake from day of gestation to delivery (group b), % restricted after delivery to the end of lactation period (group c), with a high fat diet (hfd) during gestation to lactation period (group d) and with hfd nutritionally % restricted from the day of delivery to the end of lactation period (group e). the offspring was measured body weight (bw) and measured blood pressure at , and weeks by using indirect tail-cuff method. statically analysis was performed using one-way annova. results: bw was significantly reduced in b offspring compared to another (a, c, d, e) male offspring at day (p< . ). at day after delivery, bw was significantly reduced in c, e offspring compared to a, d in male offspring (p< . ). at weeks old, bw of all type offspring was no difference. systolic and diastolic blood pressures were significantly elevated at weeks in offspring of d> b > e > c >a. (p< . , vs. a). at weeks, hypertensive offspring as b>d>e>c>a (p< . , vs. a). at weeks, d> e > b > c >a (p< . , vs. a). conclusions: maternal high fat environment make a hypertensive offspring, but regulation of fat feeding during lactation period may reduce adulthood hypertension. in case with normal food, restrictive feeding during late gestation is more effective than lactation period for inducing hypertensive male offspring. regulation of maternal feeding not only during late gestation but also lactation period may control adulthood hypertension. the strongest epigenetical factor of maternal nutrition is high fat feeding during pregnancy to lactation period for f blood pressure, respectively. or residence at high altitude, impacts fetal growth and development. in a preliminary study, we observed a significant decrease in birth weight, subsequent compensatory postnatal growth, and an increase in relative right ventricular (rv) weight at postnatal (pn) day in female offspring of rats exposed to hypoxia ( , ft; . % po ) from days thru of gestation (dga). thus, our objective was to further elucidate the impact of prenatal hypoxia on fetal growth and postnatal development. methods: pregnant dams (hx, n= ) were hypoxic from - dga with additional control dams either fed ad libitum (al, n= ), pair-fed with the hx dams throughout gestation (pg, n= ), or only pair-fed during the window of hypoxia (ph, n= ). female offspring from hx, pg, and ph dams were cross-fostered onto additional al dams (n= /litter) by h after birth. results: at birth, there was no difference in litter size; however, body weight (bw) of the hx, pg, and ph pups was lower (p< . ) than that of al pups, and hx pups were lighter (p< . ) than ph pups. weight of hx offspring remained lower (p< . ) than al pups until the termination of the study at pn , while the pg and ph pups reached weights comparable to the al offspring by pn . relative to bw, heart weight and left ventricular/septal (lvs) weight was not different among groups; however, right ventricular weight (rv/bw) was greater (p< . ) in the hx offspring at pn as was rv/lvs (p< . ). cardiac function was evaluated by echocardiography at pn . rv wall thickness was % greater (p< . ) in hx pups as compared to al pups, confirming the significantly higher relative rv weight observed at necropsy. pep, pep/at, and pep/et were %, %, and % higher respectively in the hx offspring relative to the al offspring. lv end diastolic and end systolic diameters were smaller (p< . ) in hx and ph offspring relative to the al group. myosin heavy chain (mhc) and mrna concentrations in the rv were evaluated by qrt-pcr, and the mhc / mrna ratio was greater (p< . ) in the hx pups. conclusion: prenatal hypoxia from - dga impacted both fetal and postnatal growth, altered postnatal heart development and function, with the primary impact being on the rv. supported by nih hd . reproductive sciences; pharmacology experimental therapeutics, university of maryland, baltimore, md, usa. background: exposure to nicotine (nic) is a significant risk to normal fetal development. fetal nic, which readily crosses the placenta, can be acquired from pregnant mothers by smoking or nicotine replacement therapy. the impact of nic on fetal organs may be mediated directly and/or via intrauterine hypoxia (hpx) via constriction of the uterine circulation. in adult hearts, both nic and hypoxia stimulate gene expression of matrix metalloproteinases (mmp), although the study of nic and hypoxia on gene expression in fetal organs remains incomplete. because mmps are involved in the regulation of extracellular matrix turnover and cardiac remodeling, we tested the hypothesis that prenatal nic and intrauterine hpx upregulate protein expression and activity of mmp in the fetal guinea pig heart. methods: pregnant guinea pigs were placed in either normoxia (nmx) or hpx ( . %o in chamber) for d prior to term ( d). in two separate groups, nic was also added to the drinking water ( mg/kg/d) for d at a dose that generates fetal nic levels ( ng/ml cotinine) equivalent to a moderate smoker. anesthetized near-term fetuses ( d) were excised and weighed. left ventricles of hearts were obtained and frozen at - c for storage. mmp protein levels and enzymatic activity were measured by western analysis and gel zymography, respectively. results: nic alone (nmx+nic) decreased (p< . ) fetal body wt by %, increased (p< . ) the relative fetal brain wt (brain wt/fetal body wt ratio) by . % and had no effect on relative placental or fetal heart wts. hpx alone decreased (p< . ) fetal body wt by . %, increased the relative fetal brain wt by % but, in contrast to nic alone, increased relative placental wt by . %. both mmp protein levels (mmp /a-actin density values) and activity (clear band density) were increased (p< . ) by nic alone (by . and . fold, respectively) and hpx alone (by . and . fold, respectively). in addition, both protein and activity levels of hpx hearts were further increased by nic (by . and . fold) compared to hpx alone. conclusion: prenatal nic upregulates mmp expression in nmx fetal hearts and is potentiated by hpx. this suggests that under conditions of intrauterine stress cardiac remodeling by mmp activation may be an important mechanism by which nic and hpx affect fetal heart function. objective: in addition to peripheral hypoglycemic effects, insulin induces central anorexigenic responses via stimulation of the phosphoinositide- kinase (pi k) pathway and cellular growth by mitogen activated protein kinase (mapk) pathway. the pi k signaling cascade is activated by insulin binding to its receptor (ir), recruiting ir substrate (irs- ), and phosphorylating pi k. activated pi k in turn causes phosphorylation of protein kinase b/akt which subsequently modulates hypothalamic anorexigenic responses. in contrast, the mapk (erk /erk ) signaling pathway likely involves irs- . further, insulin signaling is inhibited by the lipid phosphatase pten. we have previously shown that maternal food restriction (mfr) during pregnancy results in iugr newborns that develop hyperphagia, obesity and insulin resistance as adults. we sought to determine if altered hypothalamic basal insulin signaling expression of pi k and mapk pathways contribute to reduced satiety responses and thus enhanced growth in iugr newborns methods: pregnant control dams received ad libitum (n= ) food, whereas study dams were % mfr (n= ) from pregnancy day to to produce iugr newborns. at day , hypothalamic region was dissected and analyzed for mrna levels (real time rt-pcr) of insulin signaling components via pi k (ir, irs , pi k and akt) and mapk (irs , erk , erk ) pathways, and pten. data is presented as fold difference normalized to beta- -microglobulin. results: at d of age, iugr pups exhibited downregulation of the entire pi k pathway with significantly decreased (p< . ) mrna levels of ir ( . -fold), irs- ( . -fold), pi k ( . -fold) and akt ( . -fold). further, iugr pups showed similar decreased mrna expression of erk ( . -fold) and erk ( . -fold). however pten expression was similar to the controls. conclusion: reduced insulin-mediated pi k signaling likely contributes to the suppressed anorexigenic responses and development of obesity in iugr offspring. reduction of central mapk signaling suggests a potential maldevelopment of additional neuronal pathways in iugr offspring. objectives: in the rat, uteroplacental insufficiency restricts fetal growth and impairs mammary development further compromising postnatal growth. both male and female growth-restricted offspring have a reduced nephron endowment but only males develop hypertension with glomerular hypertrophy, which can be reversed by improving the lactational environment. this study used cross-fostering to assess the influence of the prenatal and postnatal environments on renal development and nephrogenesis. methods: bilateral uterine vessel ligation (restricted, r) or sham surgery (control, c) was performed on day of gestation in wky rats. control and restricted pups were cross-fostered onto c or r mothers on postnatal day (pn ). post mortem was carried out on pn (c and r) and pn (c-on-c, c-on-r, r-on-c, r-on-r). results: body and kidney weights were decreased in r and r-on-r pups on pn and pn (p< . ). there was some evidence of accelerated pup growth for r-on-c relative to r-on-r on pn . male, but not female, relative bmp mrna expression on pn was higher in r than c (p< . ) while gdnf, tgf and at receptor were not different. on pn , wnt (but not at r, vegf-a) mrna expression (males only) was relatively higher in r-on-r (p< . ) when compared to c-on-c (p< . ). this and the histological analyses suggests an up-regulation of nephrogenic activity with more immature nephrons (males and females) in r-on-r (p< . ) when compared to c-on-c, while r-on-c remained intermediate. intrauterine growth-restricted pups were born lighter and with smaller kidneys. this was partially rescued by improving lactational nutrition (r-on-c) at pn . higher bmp mrna expression indicates impaired branching morphogenesis in pn r male, but not female kidneys, suggesting the timing and/or molecular mechanisms underlying the nephron deficit may be sex specific. at pn there was evidence of extended and increased nephrogenic activity in r-on-r, however, this was unable to restore the later nephron deficit. improved lactation for r-on-c, which prevented the adult nephron deficit and hypertension, increased and extended nephrogenesis to a lesser degree than r-on-r suggesting that the restoration of nephron endowment was likely to have occurred prior to pn . amy m tetrault, sarah b lieber, marya shanabrough, tamas l horvath, hugh s taylor. obstetrics, gynecology and reproductive sciences, yale university school of medicine, new haven, ct, usa. objective: classically recognized for its role in energy balance, body weight and appetite, ghrelin has also been implicated in reproduction. ghrelin (-/-) mice are infertile while administration of ghrelin to wt mice results in decreased litter size and constrained embryonic growth. here we investigate the effect of maternal ghrelin deficiency on in utero developmental programming of the female reproductive tract. hox genes determine developmental identity of the paramesonephric duct. we determined that hoxa is regulated by ghrelin in vitro and that in utero ghrelin deficency alters f hoxa gene expression and reproductive success. methods: wild-type females mice parented by ghrelin +/-b d f (ghrellin deficient) mice were analyzed for litter size, oocyte, and corpus luteum number. rna was extracted from the uterus of mice exposed to ghrelin deficiency in utero. ishikawa cells were treated with ghrelin with/without receptor (ghsr) antagonist, or saline and rna extracted. in both hoxa expression was analysed by real time rt-pcr normalized to -actin and also determined by ihc. experiments were repeated in triplicate and mrna expression compared by student's t-test. results: wild-type female offspring of ghrelin deficient dams had smaller litter sizes than controls (n= , . ± . pups; n= , . ± . pups, respectively; p< . ). no differences were seen in oocyte or corpus luteum number suggesting a uterine defect. hoxa mrna and protein expression were decreased in the uterus of the f females. ghsr was expressed in uterine endometrium. treatment of ishikawa cells with nm to nm ghrelin resulted in a to % increase (p< . ) in hoxa expression. treatment with ghrelin and ghsr antagonist resulted in similar increases in hoxa expression indicating a non-receptor mediated mechanism. conclusion: ghrelin contributes to reproductive tract developmental programming; in utero ghrelin deficiency compromises reproduction in female offspring. the developmental effects of ghrelin were mediated by alteration in hox gene expression and not through the classic ghsr receptor. obesity and decreased ghrelin may lead to defects in developmental programming of the reproductive tract. these findings demonstrate the importance of nutrition, energy utilization and appropriate ghrelin levels on normal uterine development. we have previously studied the deleterious effects of lack of the endothelial nitric oxid synthase (nos ) in mouse dams and their offspring. our laboratory demonstrated that adaptive responses in subsequent pregnancies may offset the harmful effects of the genetic deficiency of nos . in this study we aimed to determine hepatic and renal histopathologic damage in nos deficient pregnant mice comparing animals carrying their first versus their second pregnancy. study design: gravid nos +/+wt and nos -/-ko mice during their first (p ) or second (p ) pregnancy were sacrificed at day of gestation. livers and kidneys were stained and analyzed for the presence and extent of histopathologic lesions. results: nos +/+wt dams displayed a low incidence of significant renal or hepatic lesions in either the first or the second pregnancy. in nos -/-ko mice the incidence of liver necrosis and inflammation during the first pregnancy was % and %, respectively. in nos -/-ko dams sacrificed during the second gestation the incidence rates for the same lesions were % and %, respectively (p< . ). this correlation persisted when we analyzed the relative severity of hepatic lesions between p and p animals. although a similar trend was observed, the difference between p and p animals with regards to kidney lesions did not reach the level of statistical significance in our study. conclusions: a second pregnancy in this animal model of hypertension was associated with a significantly improved hepatic histopathology compared with the first pregnancy. this observation is consistent with our previous studies showing a decrease in systemic vascular resistance in p versus p nos -/-ko mice. the beneficial effects of a prior pregnancy may partially underlie the phenomenon of a decreased risk of preeclampsia in multiparous versus nulliparous women. further studies are required to delineate the counterregulatory mechanisms leading to improved cardiovascular function in subsequent pregnancies in these genetically modified animals. maternal hypomethylation is associated with congenital heart defects in down syndrome. lmjw van driel, , r de jonge, wa helbing, bd van zelst, j lindemans, eap steegers, rpm steegers-theunissen. , , , obstetrics gynecology; pediatrics; clinical chemistry; epidemiolog y biostatistics; clinical genetics; erasmus university mc, rotterdam, netherlands. background: maternal age and hyperhomocysteinemia are risk factors for having a child with down syndrome (ds) and congenital heart defects (chds), respectively. evidence is rising that ageing is associated with a state of hypomethylation. objectives: to investigate whether the risk of a child with ds and chd is associated with maternal hypomethylation. methods: we conducted a case-control triad study at months after the index-pregnancy. case-children (n= ) were included if they had ds and chd. children (n= ) without a major congenital malformation served as controls. the concentrations of s-adenosyl methionine (sam), s-adenosyl homocysteine (sah), sam/sah ratio, and homocysteine in maternal blood were measured as biomarkers for methylation. the data were analyzed using the mann-whitney u test and a logistic regression model. results: maternal age was included in the model as potential confounder. the levels and the crude and adjusted or( %ci) of the biomarkers are shown in table . an increase of the sam/sah ratio with unit decreases the risk of a child with ds and chd with percent. moreover, every increase of mol/l of homocysteine . fold increases this risk. conclusions: maternal hypomethylation is significantly associated with an increased risk of having a child with ds and chd. since, the effects are confounded by maternal age, hypomethylation can be considered as feature of ageing. the developmental origins hypothesis postulates that during critical ontogenetic periods, transient environmental stimuli perturb developmental pathways and induce permanent changes in gene expression, metabolism, and chronic disease susceptibility. one likely mechanism is via early nutritional influences on epigenetic gene modification consisting of the presence of a methyl group on the carbon of a cytosine residue. this modification is responsible for an important form of gene regulation in eukaryotes. in the present study, we have tested the hypothesis that maternal low-protein diet altered epigenetic regulation of specific gene of the offspring. c bl/ female mice were mated and on the day the plug was detected, these females were then randomly allocated to be fed isocaloric diets consisting % protein or % protein. at delivery, offspring were killed and the livers were removed immediately, frozen in liquid nitrogen and stored at - c. genomic sequencing after bisulfite modification is used to study site-specific dna methylation. dna methylation status of oct- and sphk- gene upstream regions in the mouse liver was analyzed. hepatic oct- or sphk- promoter methylation was not significantly different between both groups. however, dna methylation pattern of the genomic dna is specific in low-protein diet group. aberrant oct- and sphk- gene expression may cause perturbations in cell differentiation. we suggest that the epigenetic mechanism consisting of dna methylation underlies the fetal programming theory. primary human cytomegalovirus (hcmv) infection during pregnancy can have devastating consequences for both the mother and fetus. hcmv infection has been implicated in the development of pre-eclampsia and intrauterine growth retardation (iugr), as well as congenital cmv syndrome in newborns exposed in utero. previously, we have shown that hcmv infection of placental cytotrophoblasts inhibits their normal invasion, proliferation, and migration. however, the mechanisms occurring during early establishment of placental infection are largely unknown. we assessed the impact of hcmv infection on cytotrophoblasts by performing immuno-based assays for various cytokines and cellular growth factors. we detected significant cytokine dysregulation at both and hours after in vitro hcmv infection of cytotrophoblast cells. soluble cytokines involved in recruitment of monocytes and macrophages (gro-a, mcp- ) were downregulated at both and hours after infection. sdf- , which is chemotactic for lymphocytes during early inflammation, was also decreased. these results suggest that recruitment of cells involved in the anti-viral immune response is being interrupted early in the course of infection by hcmv. additionally, a large decrease in the amount of soluble hgf was seen. hgf normally induces migration of cytotrophoblasts along the invasive pathway, and downregulation of this factor could severely affect these processes. finally, we saw increased amounts of soluble icam- , contrasted by decreased amounts of vcam- , indicating dysregulation of adhesion molecules that are necessary for successful placental invasion to occur. all together, these data indicate significant alterations in cytokine profiles as early as hours after hcmv infection, which could provide important clues to the pathogenesis of hcmv in placental invasion and inflammation. additional studies will further elucidate this dysregulation, and determine whether these effects are due to alterations in pre-existing cellular factors or if transcriptional alterations are involved. method: studies were performed in pregnant ewes (n= in each group) with twin fetuses at - days (very preterm) and - days (near-term). neither mother nor fetuses were instrumented prior to the time of blood collection. maternal blood was collected from the jugular vein before sedation. anesthesia was then induced with isofluorane, the abdomen was opened, fetuses exteriorized and blood collected from umbilical cords. blood samples were transferred to plastic tubes containing ethylene-diamino tetraacetic acid and reduced glutathione, plasma separated and stored at - c. commercial radioimmunoassay kits for ovine-crf (phoenix pharmaceuticals, b : . ± . pg/ml) and cortisol (diagnostic laboratory, b : . ± . ng/ml) were used according to the manufacturer's instructions. results: in very-preterm gestations, maternal and fetal plasma crf levels were undetectable. maternal, but not fetal, plasma cortisol levels were measurable ( ± ng/ml). in near-term gestations, both cortisol and crf were measurable in maternal (crf: ± pg/ml; cortisol: ± ng/m) and fetal plasma (crf: ± pg/ml; cortisol: ± ng/ml). plasma crf levels were higher in nearterm fetuses than in their maternal ewes (p < . ). conclusion: ovine plasma crf levels are measurable in maternal and fetal plasma in near-term but not very-preterm gestations. the absence of crf in preterm plasma, perhaps due to reduced placental expression and/or placental crf release, may contribute to the rarity of in utero meconium passage in preterm gestations. interleukin - objectives: interleukin- (il- ) is a pro-inflammatory cytokine produced in adipose cells. recent studies suggest il- may be a marker of maternal obesity and in utero fetal programming. our hypotheses were ) il- correlates with maternal obesity and ) il- mediates the effect of maternal obesity on infant birth weight. methods: the parity, inflammation, and diabetes (pid) study is a longitudinal study of adipokine levels in a diverse sample of pregnant women. we present a cross-sectional analysis of first trimester il- levels from non-diabetic women who underwent a live birth. the independent variable was il- (pg/ml), measured with monoclonal antibody elisa assays. the dependent variable was infant birth weight (gms). maternal bmi categories were: normal/underweight (< kg/m ); overweight ( - kg/m ); and obese (> kg/m ). data on demographic and clinical factors, nutrition and physical activity were collected at baseline. average il- levels were compared across bmi categories using anova. the association of il- levels with infant birth weight was estimated using multiple linear regression, adjusting for covariates. results: average il- levels were significantly higher in obese women ( . ± . ) compared to overweight ( . ± . ) and normal/underweight women ( . ± . ) [p< . ]. after adjustment, il- levels was positively correlated with pre-pregnancy bmi [regression coefficient (rc) . ; % ci: ( . , . )]. as shown in the table below, elevated levels of il- were statistically significantly associated with a . gm higher infant birth weight after full adjustment. each unit increase in pre-pregnancy bmi was associated with a gm higher infant birth weight. table . association of il- with infant birth weight characteristic regression coefficient % confidence interval interleukin- . . , . pre-pregnancy bmi . , gestational weight gain - - , bmi = body mass index; coefficients adjusted for demographics, clinical factors, pre-pregnancy bmi, gestational weight gain, non-fasting first trimester glucose levels, gestational age, nutritional intake and physical activity conclusion: il- and pre-pregnancy bmi were associated with infant birth weight after adjustment for covariates. our findings suggest that the effect of maternal obesity on infant birth weight may be mediated through il- or an alternative independent pathway. we have demonstrated that -tetrahydrocannabinol (thc), in physiologically relevant concentrations, inhibits the growth and tight transcriptional control of the bewo trophoblast cell line . the mechanism involved the decreased expression of the transcriptional regulator histone deacetylase (hdac ). in these experiments we sought to answer the question, 'does anandamide (aea) work in the same manner as thc?' methods: the first trimester human trophoblast cell, bewo were plated at or x cells /well to -well and -well plates for growth and rna experiments, respectively. after growing to - % confluence, cultures were treated with varying concentrations of aea up to a maximum of m for hr. cell numbers were determined using the xtt apoptosis/proliferation assay. total cellular rna was prepared and the relative levels of hdac and gapdh determined by end-point rt-pcr. aea exhibited an inhibitory effect on the bewo cell cultures, but only at concentrations in excess of m where confluency was significantly reduced from % at m to - % at m and m (*p< . ; one-way anova with tukey's hsd test; n= ). cultures treated with m and m aea did not exhibit increased cell death or failure to attach to the substratum, as evidenced by the lack of increase in the shedding of cells into the spent medium. bewo cells treated with aea showed a dose-dependent decrease in hdac mrna expression with a significant effect at . m (*p< . ; oneway anova with tukey's hsd test; n= ). at this dose, the effect of aea had reached an effective maximum decrease in hdac mrna levels ( %) because hdac mrna levels were not decreased further by either m aea ( %) or m aea ( %). the alteration of hdac gene expression by aea in bewo cells with its associated decrease in cell number suggest that the trophoblast cell may be an important target for circulating endocannabinoids during the st trimester of pregnancy. the data indicates that although exocannabinoids and endocannabinoids both inhibit bewo cell growth, they do so using different transcriptional mechanisms. further understanding of the mechanism(s) by which aea alters placental physiology may lead to new strategies for the prevention of pregnancy complications such as st trimester miscarriages. ( ) taylor background: anandamide (aea) exerts its effects by acting on two cannabinoid receptors, cb and cb with the main regulator for aea levels being the metabolising enzyme, fatty acid amide hydrolase (faah). aea, cb , cb and faah constitute the endocannabinoid system and previous studies have shown that faah and cb are expressed in term human placenta( ) suggesting that the endocannabinoid system might be present earlier in gestation. this study aimed to document changes in faah, cb and cb expression in the placenta during the first trimester of pregnancy. methods: first trimester samples ( to weeks gestation) were fixed in % neutral-buffered formalin for days before embedding into paraffin wax or frozen in liquid nitrogen for rna analysis. for immunohistochemistry, faah polyclonal antibodies were used at an optimal dilution of : in pbs, cb at : and cb at : . transcripts were measured using q-pcr with gene-specific primers. results: immunoreactive faah, cb and cb were detected in all samples. faah immunoreactivity in the syncytiotrophoblast increased between the th and th gestational week and by week faah was barely detectable within large parts of the placenta. simultaneously, faah immunoreactivity increased in the mesenchymal core of the developing villi. immunoreactive cb and cb localised to the syncytiotrophoblast, cytotrophoblast and mesenchymal core with cb immunoreactivity showing diminished intensity after week , although this did not reach significance at the transcript level. cb immunoreactivity was absent from fetal blood cells and infiltrating maternal plasma cells, whereas cb and cb immunoreactivity was detected in endothelial cells but not in the vascular smooth muscle cells of blood vessels. the intensity of cb immunoreactivity in the syncytiotrophoblast differed from that of cb and faah in that it remained constant throughout. conclusion: the data suggest that placental faah and cb levels do not alter significantly during the first trimester, but alter their cellular distribution from the syncytiotrophoblast to the mesenchymal core. the significant loss of cb expression from the syncytiotrophoblast after the th week of gestation, a point of critical alteration in the developing placenta, suggests that its retention may be detrimental to normal placental development. reference: park, b. et al., ( ) hankins, chandra yallampalli. obstetrics gynecology, university of texas medical branch, galveston, tx, usa. background: numerous angiogenic proteins synthesized in the placenta are thought to be involved in placental vascularization and development; however, the molecular mechanism modeling the angiogenic process in early pregnancy remains elusive. calatonin gene-related peptide (cgrp) is a multifunctional peptide expressed at the human implantation site; but its influence on in vitro angiogenesis by human micro vascular endothelial cells is not known. objective: the present study was designed to determine the influence of cgrp on angiogenesis by human dermal microvascular endothelial cell (hdmvec) in vitro. methods: hdmvecs (vec technologies) were cultured in mcdb- complete solution containing cgrp ( - m), cgrp plus its antagonist cgrp - ( - m), in well plates with x cells per well. the existence of cgrp receptor components calcitonin receptor-like receptor (crlr) and receptor activity modifying protein (ramp ) was determined using immunofluorescent staining. cell proliferation was examined using methylthiazoltetrazolium (mtt) assay. the pro-angiogenic bioactivity of cgrp was evaluated using cell migration and capillary like tube formation on the matrigel. results: ) immunofluorescent staining showed that cgrp receptor components crlr and ramp are abundantly expressed by hdmvecs. replacement of the primary antibodies with preimmune serum resulted in a negative staining; ) cgrp dose-dependently ( - to - m) stimulated hdmvec proliferation, and this effect was totally blocked by cgrp antagonist, cgrp - ; ) quantitative analysis for cell migration revealed that cgrp increases hdmvec migration in a dose and time-dependant manner; and ) cgrp promotes hdmvec capillary like tube formation, and the length of capillary tube induced by cgrp ( - m) was significantly increased over that of the untreated controls. this increase was observed at hours of treatment and further increase was noted at hours of culture. conclusion: cgrp induces in vitro angiogenesis by promoting microvascular endothelial cell proliferation, migration and capillary like tube formation. therefore, trophoblast derived cgrp at the implantation site may play a role in placental angiogenesis and fetal growth. over-expression of socs- gene promotes il- production by jeg- trophoblast cells. qin dong, , ruping fan, yang gu, david f lewis, yuping wang. biochemistry, harbin medical university, harbin, heilongjiang, china; obstetrics and gynecology, lsuhsc-shreveport, shreveport, la, usa. objective: suppressor of cytokine signaling- (socs- ) plays an important role in negative regulation of inflammatory response in biological cells. evidence has shown anti-inflammatory cytokine il- expression was significantly reduced in trophoblasts of preeclamptic (pe) placentas. we sought to determine if over-expression of socs- in placental trophoblasts could promote il- production. methods: full-length socs- open reading frame (socs- cdna) was generated by rt-pcr from total rna samples isolated from human leukocytes and cloned into a pzsgreen -n vector, which encodes a green fluorescent protein zsgreen . successful socs- cloning was confirmed by sequencing. for transfection, jeg- cells were placed into well/cluster plates at a concentration of . x /well. the tranfection was carried out with . g of socs- /zsgreen plasmid (psocs- /zsgreen ) for hours when cell reached - % confluence. siport lipid were used. jeg- cells transfected with zsgreen plasmid (pzsgreen ) only was used as control. after approximately hours of transfection, cells were treated with il- at and ng/ml. medium was then collected and measured for il- by elisa. il- production was calculated as the percentage of increase by psocs- /zsgreen transfected cells compared to the cells transfected with pzsgreen only. result: il- production was increased by psocs- /zsgreen transfected jeg- cells compared to the cells transfected with pzsgreen only when stimulated by il- , control: . % increase; ng/ml il- : . % increase and ng/ml il- : % increase, p< . , respectively. data are means from three independent experiments. conclusion: over-expression of socs- gene could promote il- production by placental trophoblast cells. reduced sil- r release and increased ratio of sgp /sil- r production by placental tissues from women with preeclampsia. shuang zhao, ruping fan, jingxia sun, yang gu, david f lewis, yuping wang. obstetrics and gynecology, lsuhsc-shreveport, shreveport, la, usa; obstetrics and gynecology, first hospital, harbin medical university, harbin, heilongjiang, china. objective: placental tissue/trophoblasts release more inflammatory cytokines (il- , il- and tnf ) in preeclampsia (pe) than in normal pregnancies. however, the reason for increased inflammatory cytokines released by pe placentas is not clear. soluble il- receptor (sil- r) and membrane receptor il- /gp complex play an important role in the negative regulation of cytokine signaling in suppressor of cytokine signaling (socs) pathway. in contrast, soluble gp (sgp ) is an antagonist for il- /il- r trans-signaling. this study was undertaken to determine sil- r and sgp production by villous tissues from normal and pe placentas. methods: placentas delivered by normal and pe pregnant women were used in this study. placental explants were incubated with dmem for h. the culture medium was collected. placental villous tissue productions of sil- r and sgp were measured by elisa. all samples were assayed in duplicate. data are expressed as mean ± se and analyzed by mann whitney test. a p level < . was considered statistically different. results: placental tissues from pe produced significantly less sil- r than tissues from normal pregnancies, . ± . vs. . ± . pg/mg of wet tissue, p< . . soluble gp production was relatively compatible between pe and normal placental tissues: . ± . vs. . ± . pg/mg of wet tissue. the ratio of sgp /sil- r release was significantly higher in pe than in normal placentas, . ± . vs. . ± . , p< . . conclusion: reduced sil- r production and/or increased ratio of sgp /sil- r production by pe placentas suggest less cytokine inhibitory activity in pe placentas, which may contribute to the increased toxic cytokine production in placentas from pe. using chemiluminescence immunoassays. peter s uzelac, jing dai, frank z stanczyk, daniel r mishell, jr. obstetrics and gynecology, university of louisville, louisville, ky, usa; obstetrics and gynecology, university of southern california, los angeles, ca, usa. objective: characterizing human chorionic gonadotropin (hcg) levels throughout normal pregnancy is critical to its use as a bio-marker for abnormal gestations. there is a paucity of data describing hcg trends during pregnancy and most of the relevant studies use older, less specific assays. our first objective was to characterize hcg levels throughout normal gestation using two different contemporary chemiluminescence immunoassays. our second objective was to compare hcg patterns in healthy gestations with pregnancies affected by diabetes, a common obstetrical complication. methods: a single blood sample was collected from healthy pregnant women and diabetic pregnancies. gestational age was confirmed by ultrasound. serum hcg levels were quantified by chemiluminescence immunoassays using the acs- and immulite systems. data was grouped in -week intervals until weeks of gestation and -week intervals thereafter, with to samples in each interval for healthy women and to samples in each interval for diabetic women. paired t-test and wilcoxon rank sum test were used for statistical analysis. results: using the acs- system, mean hcg levels (miu/ml) for healthy pregnant women were , at - weeks, , at - weeks, , at - weeks, , at - weeks, , at - weeks, and , at - weeks. mean hcg levels obtained from the immulite system were similar. for diabetic pregnancies, mean hcg levels (miu/ml) were , , , , , , , , , and , , respectively. between - weeks of gestation, the hcg levels were significantly lower in diabetic pregnancies (p= . ) compared to healthy controls. ) compared to healthy pregnancies, diabetic gestations have significantly lower peak hcg levels. the cause of this difference is an area deserving further investigation. background: mouse and human placentae share several cellular and molecular features, including a haemochorial interface, allowing the murine labyrinth to be compared with the human fetal placenta. there is an autonomous embryonic ras from at least the time of implantation. ace converts angiotensin i to the active angiotensin ii (angii). angii's actions as a pro-inflammatory agent, in promoting cell migration, angiogenesis and cellular growth and apoptosis, strongly suggest a rôle in placentation. hypothesis: there would be counterregulation of the placental ras if the effect of ace were removed. this study investigated for the first time whether the knockout of somatic ace affected the expression and localisation of various components of the ras in the placentae of wild-type (wt/wt), heterozygous (wt/ ) and ace knockout ( / ) mice. methods: immunohistochemistry (dako envision plus) was used to localise and semiquantify ang type receptor (at r), ang type receptor (at r), ace, and ace- in the three genotypes (n= /group). placental sections were blinded to genotype; a score range of - was used. the test was used (spss version . ) to analyse the difference in staining score by genotype. results: immunoreactivity of all antigens increased in the placental labyrinth of / mice compared to wt/wt and wt/ mice (at r p< . ; at r p<< . ; ace p<< . ; ace- p<< . ). at r and ace- displayed increased staining in the fetal vascular endothelium of / mice (at r p<< . ; ace- p= . ), and in the cells lining the maternal central artery (at r p<< . ; ace- p<< . ). ace- expression was very high in cytotrophoblast lining the maternal blood space in all genotypes. no gross structural differences were seen. comment: the antibody used did not differentiate between ace and the membrane-bound "testicular"-ace (tace). immunohistochemically-identified ace expression was upregulated in / placentae despite the loss of somatic ace, suggesting that t-ace can be expressed placentally. we believe this is the first demonstration of such expression. ace and t-ace are catalyticallysimilar in converting angi to angii. furthermore, angii acting via the at rs is vasodilatory and ace- catalyses production of the vasodilatory ang ( - ); placental blood flow was presumably well-maintained and pregnancy outcome is normal in / mice. it is generally accepted that prostaglandin production plays a crucial role in both term and preterm parturition, and recently the administration of alpha hydroxyprogesterone acetate has been shown useful in preventing preterm labor. what is not clear is the biochemical pathway of prostaglandin production during labor and what, if any, effect alpha hydroxyprogesterone acetate has on this pathway. in order to address this question, we used immunohistochemical staining techniques to evaluate the effect of treating human placental amnion and chorion decidua with alpha hydroxyprogesterone acetate. membranes from unlabored patients were obtained at cesarean section and immediately seperated into control and drug treated specimens. controls were from the same placenta and were subjected to all experimental procedures except for the addition of alpha hydroxyprogesterone acetate to the culture media. specimens were compared at zero, six, and twenty four hour intervals. at the appropriate time, each specimen was formalin fixed and then paraffin blocked. tissue sections were then mounted on slides which were immunohistochemically stained using appropriate primary and secondary antibodies and standard techniques. the slides were then analyzed via light microscopy for changes in staining of three enzymes involved in prostaglandin production--cyclooxygenase (cox- ), cyclooxygenase (cox- ), and -hydroxy prostaglandin dehydrogenase (pgdh). compared to control, the slides treated with alpha hydroxyprogesterone acetate had differing amounts of enzyme expression. cox- was relatively unchanged and pgdh was only slightly increased, but cox- was noticeably decreased in the treated slides. these results were time dependent. this data suggests that alpha hydroxyprogesterone acetate decreases prostaglandin production in fetal membranes primarily by downregulation of the cyclooxygenase enzyme. objectives: in the human placenta, proliferation, differentiation and fusion of cytotrophoblasts (ct) are essential events in the formation of the multinucleated syncytiotrophoblast, however the regulation of these processes is poorly understood. using an explant model of human first trimester placenta we have established that both igf-i and -ii enhance ct proliferation, differentiation and survival mediated via igf r signalling. we have also shown that non-specific inhibition of protein tyrosine phosphatases inhibits igf-mediated signalling in trophoblast; therefore, we have now used sirna-mediated knockdown to investigate the role of the tyrosine phosphatase shp- in this pathway. methods: amaxa nucleofector technology was used to deliver shp- or scrambled sirna ( nm) to bewo cells or first trimester villous tissue fragments. knockdown was confirmed by q-pcr and western blotting. transfected cells and tissue were maintained in culture for hours, then treated with igf-i or igf-ii ( nm) for a further hours before immunohistochemical (ihc) analysis for cell proliferation (ki , brdu) or apoptosis (m ). results: sirna-mediated knockdown of shp- in bewo cells ( % reduction on western blot) demonstrated that igf-induced proliferation was reduced from . ± . % to . ± . % (p< . , n= ). ihc analysis of tissue demonstrated that shp- is localised to ct. following knockdown ( % decrease by q-pcr), igf-i-and igf-ii-induced ct proliferation was decreased by . ± . % and . ± . % respectively (p< . , n= ). furthermore, the ability of igf-i-and igf-ii to prevent ct apoptosis (m staining) was reduced by . ± . % and . ± . % respectively (p< . , n= ) after shp- knockdown. conclusions: igf stimulation of cytotrophoblast proliferation is mediated by shp- . exogenous igf rescues cytotrophoblast from apoptosis, and this pathway is also shp- -dependent. villous endothelial cells. emily j su, zhi-hong lin, ping yin, scott reierstad, joy innes, serdar e bulun. obstetrics and gynecology, northwestern university feinberg school of medicine, chicago, il, usa. background: within the human vascular system, estrogens have been shown to enhance vasodilatation in both normal and abnormal endothelium. estrogenic function occurs by activation of one or both of two estrogen receptors, estrogen receptor-alpha (esr ) and estrogen receptor-beta (esr ). these estrogen receptors are expressed in a wide variety of tissues. within the vasculature, estrogen receptors regulate the expression of multiple vasodilator and vasoconstrictor proteins. specifically, esr has been shown to be critical in maintaining normal vascular physiology in a murine model, where esr knock-out mice demonstrate significant systolic and diastolic hypertension. we hypothesize that within placental endothelium, estrogen plays an important role in maintaining normal vascular function that is critical for normal fetal growth and development. methods: term placentas from uncomplicated pregnancies were obtained, and the decidua was removed. an iv cannula was inserted into the umbilical vein, which was perfused with a collagenase/dispase solution. the perfusate was collected and subjected to further purification. these cells were cultured in complete medium, and after the initial passage of these cells, purity was confirmed via immunofluorescence and flow cytometric studies. estrogen receptor expression was determined in these cells via western blotting. additionally, these endothelial cells underwent treatment with varying doses of estradiol ( - m to - m), and quantitative real-time pcr was performed thereafter for mrna levels of various genes important in prostanoid production. results: western blotting demonstrated that esr is the only estrogen receptor expressed within villous placental endothelial cells. estradiol induced cyclooxygenase- (cox- ) mrna levels -to -fold, as quantified by real-time pcr (p< . ). conversely, there was no effect of estradiol on cyclooxgenase- (cox- ). conclusion: these results suggest that estradiol and esr are important in mediating the balance of prostanoid production that is essential in maintaining placental vascular health. future studies will further delineate estrogenic effects on prostanoid production within placental endothelial cells in health and disease. supported by the smfm/aaogf scholarship award and the nih grant u -hd . previously we established that proteins secreted by the decidua promote the differentiation of extravillous trophoblasts (evt) from a proliferative phenotype (characterized by cx , her- and alpha integrin protein expression) to an invasive phenotype (characterized by her- and alpha protein expression). the ability of decidua-conditioned media (dcm) to induce trophoblast differentiation was inhibited in the presence of the her- receptor antagonist, ag . furthermore, dcm-induced jar cell migration was also attenuated in the presence of ag . thus, the purpose of this study is to define the role of her signaling in evt differentiation and invasion. methods: evt differentiation was assessed in placental villous explant outgrowths and jar cells using antibodies against markers of the proliferative and invasive phenotypes. trophoblast migration was assessed using jar cells in transwell migration assays. results: treatment of placental villous explants with egf, a her- ligand, resulted in the downregulation of her- and an upregulation of her- expression, as well as an induction of alpha integrin expression. pre-treatment of placental villous explants with ag blocked this effect. in the jar cell line, egf treatment mimicked the differentiation-promoting effects of dcm by downregulating her- and upregulating her- expression, effects that were both blocked when jar cells were pre-incubated with ag . in contrast to dcm however, egf stimulation did not induce jar migration. stimulation of jar cells with hb-egf, a her- /her- heterodimer ligand, induced jar migration in a dose-dependent manner. analysis of dcm using antibody arrays confirmed the presence of many members of the egf family including hb-egf. immunohistochemical assessments of placental villous explants verified the expression of her- in evt outgrowths and in jar cells; her- expression was not affected by stimulation with either egf or hb-egf. conclusions: her signaling is an important and necessary component of the invasive evt differentiation cascade. our data supports a role for her- signaling in the induction of the invasive evt phenotype. chandrasekhar thota, chandra yallampalli. obstetrics gynecology, university of texas medical branch, galveston, tx, usa. background: parathyroid hormone related peptide (pthrp) is expressed in trophoblast cells and may play a role in placental growth and function. studies conducted in pregnant rats using pthrp antagonist showed decreases in fetoplacental growth during mid gestation. objective: to assess the effects of pthrp silencing on the expression of growth factors in immortalized first trimester trophoblast cells (htr- /svneo cells). methods: htr- /svneo cells cultured at º c and %co in rpmi- medium supplemented with % fbs were transiently transfected with nm of three different sirna sequences of pthrp, si , auaccuaacucaggaaacuuu; s i , g a g c u g u g u c u g a a c a u c a u u ; a n d s i , caagauuuacggcgacgauuu. for control, a scrambled sirna sequence was used. at % confluency, cells from each well were split and transfected again in triplicates with respective sirna sequences. total rna was isolated using trizol reagent h after transfection, and protein was extracted using lysis buffer h after transfection. the isolated rna and protein were subjected to reverse transcription and polymerase chain reaction (rt-pcr) and western analysis, respectively, using primers and antibodies specific for pthrp, plgf, vegf and lif. the results are expressed relative to either s for changes in mrna expression or -actin for changes in protein expression. results: rt-pcr of total rna obtained from htr cells subjected to double transfection with sirnas for pthrp showed a significant decrease in pthrp expression. expression of growth factors plgf, vegf and lif showed decreases with all the three sirna sequences used compared to the scrambled sequence. western analysis of cell lysates obtained from htr cells subjected to transfection with sirnas for pthrp showed a significant decrease in protein expression of pthrp and vegf. however the protein expression for plgf, and lif decreased in cells transfected with only si sequence of pthrp. conclusions: our studies showed that transient transfection of htr cells with sirna for pthrp caused decreases in both mrna and protein expression of pthrp. our results further suggests that decrease in pthrp peptide in transfected cells resulted in a decrease in vegf, plgf and lif suggesting that pthrp may play role in regulating trophoblast cell functions. objective. maternal obesity poses an increased risk to the fetus during pregnancy, and has long term consequences for the progeny. we tested the hypothesis that maternal caloric excess effects growth-related gene expression changes in the murine placenta. methods. female c bl/ mice were fed a hypercaloric diet ( % fat, % sugar) or standard chow for six weeks prior to mating and throughout pregnancy. near-term (day gestation) the dams ( controls, overfed) were sacrificed. following placental rna extraction, we used the affymetrix mouse a_ . array to measure gene expression changes. we performed pathway analysis on regulated genes. results. maternal overfeeding was associated with a two-fold increase in body fat mass. probe sets, corresponding to genes showed differential expression (p < . ); twenty-seven of which were up-regulated, and ten down-regulated, as compared to the placenta of control fed dams. of note, several genes related to obesity, diabetes, dna methylation, and the tgf beta pathway were differentially expressed. conclusions. diet-induced obesity in mice was associated with altered placental gene expression, including genes involved in tgf beta signaling and dna methylation pathways. these findings may have important implications for placental growth and epigenetic regulation. (supported by usphs hd- , earnest eu framework , tommy's the baby charity, uk). objective. maternal dietary protein restriction has been shown to have deleterious effects on placental development, and has long-term consequences for the progeny. to comprehend more completely stress responses to maternal protein restriction, we measured gene expression changes in the mouse placenta. methods. pregnant fvb/nj mice were fed an isocaloric diet containing % less protein than normal chow ( % vs. % protein content) from embryonic day . (e . ) to e . . following placental rna extraction, we used the affymetrix mouse a_ . array to measure gene expression changes. we performed pathway analysis on the regulated genes, and used both qrt-pcr and immunohistochemistry to verify the results. results. the weights of the e . pups were decreased % (p< . ). probe sets, corresponding to genes, were regulated by protein restriction (p< . ); ninety-one being up-regulated and down-regulated. of particular note, several genes related to the p pathway were up-regulated. along with p itself, positive regulators of p (zmiz , jmy, hipk ) and genes activated by p (inpp d, cebpa) were induced. for selected genes we confirmed these results using qrt-pcr and immunohistochemistry. conclusions. by microarray analysis, we have described the genetic response to maternal protein deprivation in the mouse placenta. we observed that pups were growth restricted, and genes related to the p pathway were regulated. we propose a model through which intrauterine growth restriction is triggered, in part, by activation of the p pathway. (supported by usphs hd- and the sgi medical student grant to cpg). purpose: human placental villous tissue cultures have been underused in the study of placental drug disposition. thus we assessed the utility of this model by studying the effect of time in culture on the viability and integrity of the tissue and the, expression and function of proteins involved in the formation and efflux of -chloro- , -dinitrobenzene (cdnb) conjugate , -dinitrophenyl-s-glutathione (dnp-sg) as a model system for phase ii metabolism and cellular efflux. methods: placental tissue samples were obtained within minutes of cesarean deliveries following normal pregnancies in three patients. villous tissue was cultured in m medium to hr. at , , , , , and hr post culture, villous tissue was preincubated without or with atpase inhibitor sodium orthovanadate, exposed to μm cdnb, rinsed and incubated in buffer at °c to determine formation and efflux of dnp-sg, which was assayed by hplc. changes in expression of gstp - , abc transporter isoforms b , c and g (abcb , abcc , and abcg , resp.) were assessed by immunoblotting. lactate dehydrogenase (ldh) release, methyl tetrazolium thiazolyl blue (mtt) incorporation, and total tissue glutathione content were monitored up to hr. villous tissue morphology was assessed by immunohistochemistry. results: villous tissue structure and protein expression of glutathione-stransferase isoform p - (gstp - ) and abcg remained unchanged over hr in culture. expression of abcb and abcc , and total tissue glutathione decreased with culture time. ldh release was unchanged up to hr and increased at hr, while mtt incorporation remained constant to hr and decreased at and hr suggesting a decline in tissue integrity and viability at hr. however, dnp-sg formation, dnp-sg buffer/tissue ratio, and the extent of inhibition of dnp-sg efflux by sodium orthovanadate remained unchanged through hr. sodium orthovanadate decreased the dnp-sg buffer/tissue ratio by . ± . % (p< . ), consistent with inhibition of apical abc transporters. conclusions: these results support the use of this model to study the coordinated function of metabolizing enzyme gstp - and apical abc transporters in the formation and efflux of the model substrate dnp-sg. the model may be useful to study metabolism and transport of other compounds. syncytiotrophoblast. shauna f williams, ewa fik-rymarkiewicz, stacy zamudio, nicholas p illsley. obstetrics, gynecology and women's health, umd-new jersey medical school, newark, nj, usa. introduction: multiple inputs influence placental protein synthesis. nutritional, endocrine and metabolic factors have been implicated but its regulation has not been investigated. one of the factors shown to be associated with the inhibition of protein synthesis is hypoxia. the goal of this study was to determine the effects of hypoxia on a marker of placental protein synthesis. eukaryotic initiation factor (eif ) is a subunit of eif which is required for initiation of translation however when phosphorylated, eif is unable to participate in the assembly of the initiation complex. hypoxia has been shown previously to cause increased phosphorylation of eif . we hypothesized that hypoxia would increase the levels of phosphorylated eif in term syncytiotrophoblast, thus inhibiting protein synthesis. methods: primary syncytiotrophoblast cultured from term cytotrophoblast were incubated for hr in atmospheres of , , or % o or in the presence of the hypoxia-mimetic, dimethyloxalylglycine (dmog, . - . mm) in % o . cell extracts were analyzed by western blotting to determine the degree of eif phosphorylation. results: incubation in , , or % o did not increase eif phosphorylation relative to the % o control (n= , separate placental preparations). incubation in dmog concentrations up to . mm did not affect eif phosphorylation however incubation in . mm dmog increased eif phosphorylation by ± % (p < . , n= ). conclusions: contrary to our expectations, inhibition of protein synthesis via the eif regulatory pathway was not apparent except when induced by the highest concentration of dmog, consistent with severe hypoxia. thus while eif phosphorylation does occur, we did not observe changes at dissolved oxygen levels of %. these data suggest that a reduction in syncytial protein synthesis via the eif pathway takes place only under severe hypoxic stress. (supported by nih hd ). the increasing prevalence of overweight and obese women of childbearing age is a growing public health concern. the impact of maternal obesity on placental aa transport, which is essential for normal fetal development, remains poorly defined. there are three sub-types of the placental na-dependent system a transporter, snat , and which mediate neutral aa transport. snat is ubiquitously expressed in mammalian tissues and is likely responsible for the majority of placental system a activity. objective: to examine the impact of maternal obesity and over-nutrition on the fetal: maternal (f:m) aa ratio and placental protein abundance for snat . methods: nonpregnant ewes were randomly assigned to a control (c, % of nrc recommendations) or obesogenic (ob, % of nrc) diet from - to days of gestation (dg). under isofluorane anesthesia, maternal and fetal blood samples were collected for aa analysis by hplc from five twin bearing ewes in each dietary group. after euthanasia, placental cotyledonary (cot) tissue was separated from caruncular tissue, frozen in liquid nitrogen and stored at - c for western blot analysis. results: fetuses from ob ewes were % heavier (p< . ) than those from c ewes at dg ( ± vs. ± g). blood concentrations of asn, thr, cit, arg, tau, tyr, trp, val, phe, leu and orn were higher (p< . ), or tended to be higher (met and lys, p< . ) in ob than c ewes. in contrast, f:m ratios, for asn, ser, gln, his, gly, thr, cit, arg, b-ala, tau, ala, tyr, trp, met, val, phe, leu, orn and lys were reduced (p< . ) in ob compared to c ewes. snat content in cot tissue was reduced in ob when compared to c ewes ( . ± . vs. . ± . arbitrary units; p< . ). conclusions: maternal obesity in pregnancy reduced expression of placental snat protein and efficiency of placental aa transport in ewes, providing a mechanism whereby fetuses may mitigate excessive delivery of aa under conditions of maternal obesity and over-nutrition. decreased aa transport to the fetus may play a role in altered cellular structure and function. nih inbre p rr . early gestation utero-placental hemodynamics in an ovine model of fetal growth restriction. lucia dohnal, james s barry, henry l galan, randall b wilkening, russell v anthony. perinatal research center, university of colorado health sciences center, aurora, co. objective: fetal growth restricted (fgr) pregnancies, during late gestation, exhibit altered placental hemodynamics, and reduced capacity for o and nutrient transfer. it was our objective to examine utero-placental hemodynamics and o uptake during early gestation in an ovine model of fgr. methods: singleton-bearing ewes were instrumented with uterine artery flow probes, uterine venous and femoral artery catheters before being placed into a highambient temperature (fgr; n= ) or normothermic (con; n= ) environment at days of gestation (dga). maternal arterial and venous blood, uterine artery flow, heart rate, arterial pressure and respiration rate was collected until dga, at which time umbilical venous blood, fetal weight, placental weight and tissue were harvested. data reported here were analyzed by students t-test. results: maternal respiration rate ( . ± . vs . ± . breaths/min) and arterial po ( . ± . vs . ± . mmhg) were increased (p . ), whereas maternal heart rate ( . ± . vs . ± . beats/min), blood pressure ( . ± . vs . ± . mmhg) and arterial pco ( . ± . vs . ± . mmhg) were reduced (p . ) in fgr pregnancies. at dga, fetal weight was not different (p . ), but placental (total placentome) weight ( . ± . vs . ± . g) was reduced (p . ) in fgr pregnancies. while uterine artery (pregnant horn) flow ( . ± . vs . ± . ml/min) tended (p= . ) to be reduced in fgr pregnancies, relative uterine artery flow ( . ± . vs . ± . ml/min/g fetus; . ± . vs . ± . ml/min/ g placenta) was not different (p . ). uterine o uptake (mmol/min), relative uterine o uptake (ml/min/g fetus or ml/min/ g placenta) and uterine o extraction (%) were not different (p . ) between fgr and con pregnancies. at dga, umbilical vein po (mmhg), o content (mm) and o capacity (mm) were also not different between fgr and con pregnancies. conclusions: reduction in absolute uterine artery flow (ml/min) did not impact utero-placental o uptake or transfer to the umbilical vein, and may have resulted from reductions in maternal cardiac output. relative uterine artery flow was not reduced, suggesting that uterine blood flow and delivery of o to the conceptus does not mediate the ongoing placental growth restriction initiated during early gestation. supported by nih hd . barton c staat, anna maria marconi, cinzia paolini, alex cheung, henry l galan, frederick c battaglia. obstetrics gynecology and pediatrics, univ of colorado at denver health sciences center, aurora, co, usa; dept of obstetrics and gynecology, san paolo institute of biomedical sciences, university of milano, milano, italy. objective: to determine relative contributions of transplacental flux vs fetal production for myo-inositol and mannose in normal term pregnancies using stable isotopic methodolgy. background: myo-inositol and mannose are important in biologic functions. an external supply of mannose may be required for glycoprotein synthesis. low maternal myo-inositol is associated with spina bifida. mannose concentrations are known to be higher in the mother than the fetus. in contrast, myo-inositol concentrations are higher in the fetus than the mother. what remains unknown is whether fetal levels of these polyols are a result of direct maternal transport or from conversion of glucose. design: four term uncomplicated pregnancies undergoing an elective ceasaran section were infused with c labeled isotopes of glucose, myo-inositol and mannose over hours prior to delivery. maternal samples were obtained prior to infusate being administered, and at hour (h), . h and h. fetal concentrations were measured from umbilical artery and vein plasma. the concentrations of labeled and unlabeled glucose, mannose and myo-inositol were measured using high pressure anion exchange chromatogrpahy permitting detection of polyols and sugars at concentrations in the μm range. the feto-maternal molar percent enrichment (mpe) ratio was calculated for each glucose, mannose, and myo-inositol as the ratio between fetal plasma enrichment and the maternal plasma enrichment at steady state. steady state was calculated as the mean of the three maternal samples taken during infusion. results: the feto-maternal mpe ratios of mannose ( . ± . , p= . ) and glucose ( . ± . , p= . ) were not significantly different from . , consistent with transplacental supply. the feto-maternal ratio for myo-inositol ( . ± . , p= . ) indicates little transplacental flux ( % of fetal inositol derived from maternal plasma). conclusion: in normal term pregnancies, fetal mannose and glucose concentrations are dependent upon maternal transplacental supply. in contrast, fetal myo-inositol concentration is not dependent upon transplacental supply, but fetal demands are met by placental conversion, likely from glucose. christian wadsack, manuela augsten, christian guelly, ursula hiden, ingrid lang, manfred moertl, uwe lang, gernot desoye. clinic ob/gyn; center of med res; inst cell biol, histol embryol, med univ graz, austria. background placenta and fetus need lipids for growth and synthesis functions. part of the lipids is supplied from maternal sources by transplacental transfer. recently, we identified in human placenta the high density lipoprotein (hdl) receptor scavenger receptor class b type i (sr-bi). among other functions it mediates hdl-induced ser -phosphorylation of endothelial nitric oxide synthase (enos) resulting in enos activation. this mechanism allows hdl to contribute to regulation of vasotonus in arteries. we hypothesized that term placental endothelial cells (ec) express sr-bi at levels different between arteries and veins. methods sr-bi was localized by ihc and quantified by qrt-pcr in rna isolated from arterial and venous vessels. arterial (eca) and venous (ecv) placental ec were rigorously characterized. sr-bi levels were measured by qrt-pcr and immunoblotting. hdl binding and uptake was measured in eca and ecv with i-labelled hdl. hdl from human donors was used to stimulate ser enos phosphorylation. epigenetic regulation was studied by methylationspecific pcrs for cpg-rich promoter regions of sr-bi. pdzk a key adaptor for sr-bi mediated enos activation was measured by sqrt-pcr. in situ analyses (ihc, qrt-pcr) showed more sr-bi in arteries than in the vein. the differential expression persisted in vitro in isolated eca and ecv even after passages and culture under same conditions suggesting epigenetic mechanisms regulating sr-bi. however, no methylation was found in eca or ecv. sr-bi was functional since hdl cell association was -fold higher in eca than in ecv ( ± . vs ± . ng hdl/mg prot). hdl did not induce ser enos phosporylation in eca or ecv, which was stimulated by ionomycin about -fold in both cell types. pdzk was undetectable in eca and ecv, whereas it was expressed in placental tissue. conclusion more sr-bi is expressed in ec from arteries than from veins in situ and in vitro. this is not the result of different methylation of sr-bi promoter and, hence, unlikely an epigenetic phenomenon. mechanism of differential expression and its functional consequences for vasotonus regulation is yet unknown. the lack of pdzk may account for the failure of hdl to activate enos. (grants , , oenb). cells. juan a arroyo, brad ziebell, mi-hye park, henry l galan. obstetrics and gynecology, university of colorado andgealth sciences center, aurora, co, usa. introduction: mtor is a protein that regulates cell growth in response to nutrients and growth factors. downstream effectors of the mtor pathway include the p and the ebp proteins. activation by phosphorylation of these proteins increases protein synthesis. given that various signaling pathways are regulated by hypoxia in human trophoblast and that mtor is expressed in human trophoblast, our objective was to determine the effects of hypoxia in the activation of mtor, p and ebp in cultured human trophoblast. study designs: trophoblast cell were isolated from term uncomplicated placentas using a trypsin, dnase and disapase solution. cytokeratin immunocytochemistry confirmed trophoblast cells culture purity. trophoblast cells were treated with hypoxia ( % o ) or normoxia ( % o ) for and hours. western blot for p-mtor, mtor, p-p , p , p- ebp , and ebp were done for each time studied. results: trophoblast cells demonstrated: ) positive staining for cytokeratin, ) non significant differences for mtor at either ( . -fold; p= . ) or hours ( . -fold, p= . ), ) no differences in p protein at ( . fold; p= . ) or hours ( . -fold, p= . . ), ) no differences for ebp at either or hour. conclusion: we conclude that the mtor pathway is not regulated under hypoxic conditions in cultured trophoblast, which suggests that hypoxia does not affect protein synthesis in cultured human trophoblast. however, this may not reflect what happens in vivo in iugr. (supported by nih grant r hl - a ). increased expression of phospho-mtor, phospho-p , phospho-akt and phospho-erk in an ovine model of fetal growth restriction. juan a arroyo, brad ziebell, henry l galan. obstetrics and gynecology, university of colorado and health sciences center, aurora, co, usa. objective: both phosphorylated (p) mtor and p are known to be involved in protein synthesis and are regulated by physiological conditions such as fetal growth restriction (fgr). in a hyperthermic (ht) ovine model of fgr we hypothesize that mtor, p , ebp , erk and akt will be phosphorylated (activated) in the placentae of age (dga) animals. study design: ewes were exposed to ht conditions for days to induce iugr and were placed in ambient conditions. at necropsy ( dga), placentomes were separated into the maternal (caruncle) and fetal (cotyledon) components and frozen for western blot analysis with antibodies against (p) mtor, mtor, (p) p , p , (p) ebp , ebp , (p) erk, erk, (p)akt and akt. results: compared to control animals, fgr animals had smaller fetuses ( ± g v. ± g; p= . ) and smaller placentae ( ± g v. ± g; p= . ) at dga. fgr cotyledon showed an increase in p-mtor ( . -fold; p= . ), p-p ( . -fold; p< . ), p-erk ( . -fold; p< . ) and p-akt ( . -fold; p< . ). in contrast, caruncle (maternal) did not show any changes for the mtor pathway. conclusion: in fgr ovine pregnancies, the fetal placental tissues (cotyledons) showed upregulation of the mtor pathway for protein synthesis via phosphorylation of the p but not ebp while this was not seen in the maternal (caruncle) tissues. in addition neither the cotyledon or caruncle tissues at mid-gestation ( dga) showed changes in these endpoints, which is prior to the exponential fetal growth that starts at mid-gestation. (supported by nih grant r hl - a ). hyperuricemia has long been recognized as a common clinical finding in preeclamptic (pe) women. to date, elevated uric acid concentrations in these women have been considered a marker of disease severity. however preeclamptic pregnancies with hyperuricemia, are associated with an increased frequency of preterm birth and fetal growth restriction. over the past decade several pathogenic roles for uric acid have become evident, raising the possibility of a role(s) for uric acid in the altered vascular and placental functions associated with pe. objective: examine the effects of syncytial uric acid uptake on system a amino acid transport across the human placenta using a primary placental villous explant model. methods: placental villous explants from placentae of healthy, term pregnancies were incubated for hours with uric acid ( . mg/dl), corresponding to concentrations of uric acid observed in pe women. these experiments were conducted in the presence or absence of probenecid ( m), a uric acid cellular uptake inhibitor. system a amino acid transport was subsequently assayed using a radiochemical assay in which na+-dependant uptake of radio-labeled system a substrate, [ c] methyl-amino-isobutyric acid, was measured over minutes. data were analyzed using a paired student's t-test and presented as mean ± sem. results: uric acid attenuated system a amino acid placental transport by % (± . %, p< . ). this inhibitory effect of uric acid on system a activity was prevented by probenecid. conclusions: uric acid reduces placental amino acid transport at concentrations observed in pe women. this inhibitory effect of uric acid is dependant upon syncytial uptake of uric acid, being inhibited by the uric acid transporter inhibitor probenecid. these results may be relevant to the increased frequency of fetal growth restriction observed in hyperuricemic pe. additionally the results of this study, indicating a detrimental effect of hyperuricemia on placental function, also suggest a role for uric acid in the pathophysiology of pe. hyperuricemia, a well-documented clinical finding in preeclamptic women, is associated with pre-term birth and intrauterine growth restriction. uric acid is higher in women destined to develop preeclampsia as early as weeks of gestation at a time when cytotrophoblast are invading decidua and myometrium and remodeling uterine spiral arterioles. we propose that elevated concentrations of uric acid may have detrimental effects on placental development in part through inhibition of trophoblast invasion through the decidua. objective: examine the effects of increasing concentrations of uric acid on trophoblast invasion through a reconstituted extracellular matrix. methods: using the in-vitro matrigel invasion assay, the effects of increasing concentrations of dissolved uric acid ( . mg/dl, . mg/dl and . mg/dl) on the ability of immortalized first trimester extravillous trophoblast cells (htr -svneo) to invade through a reconstituted extracellular membrane were assessed. the concentrations of uric acid used were comparable to those measured in healthy pregnant women and preeclamptic women with an increase in uric acid of two or four standard deviations above normal. cells that successfully invaded through the matrigel membrane within hours were fixed with methanol, stained with hematoxylin and counted. data were analyzed using a one-way analysis of variance with fisher's post-hoc analysis. results: uric acid attenuated trophoblast invasion in a dose-dependent fashion (p< . ), with decreases of % (± . %), % (± . %) and % (± . %) respectively compared to untreated controls. conclusions: exogenous uric acid, at physiological and pathological concentrations, is capable of attenuating trophoblast invasion through a reconstituted extracellular membrane in a dose dependent fashion. these results suggest uric acid is a potential contributor to the pathophysiology of altered placental perfusion in preeclamptic pregnancies. previous studies have shown that transforming growth factor (tgf)-is a key inhibitory factors in the invasion of early trophoblast cells, suggesting therefore that overcoming tgf-beta signaling may be necessary for successful implantation. smad ubiquitin regulatory factor (smurf ), a hect type e ubiquitin ligase, is a key regulator of tgf-signaling pathway, targeting tgf-receptors and various smads for proteasome-mediated degradation. in this context, smurf has been shown to play important roles in embryonic development, cell senescence and tumor formation. as a key regulator of tgfbeta signaling, we wished to determine whether smurf has a physiological role during embryo implantation, especially in trophoblast invasion. we have examined the spatio-temporal expression of smurf in human placental villi during pregnancy. we have also investigated the possible function of smurf in trophoblast cell migration and invasion in a model system involving a human extravillous trophoblast cell line, htr /svneo. our results showed that expression of smurf in placental villi was the highest during the first trimester and the expression decreased in the nd trimester. expression of smurf was lowest in placental villi at parturition. overexpression of smurf in htr /svneo cells reduced tgf beta type i receptor levels and attenuated the inhibitory effect of tgf-on cell migration and invasion. conversely, rnai-mediated down-regulation of smurf resulted in significant increase of tgf-type i receptor protein levels. in contrast, the levels of smad , another potential target of smurf , was unchanged. in conclusion, the present study suggests that smurf participates in trophoblast cell migration and invasion by down-regulating the expression of tgf-type i receptor. our previous data demonstrated the extensive expression of mmp- in various kinds of trophoblast cells in human placenta at the early pregnancy. however, the modulation of the enzyme in trophoblasts is largely unclear. in the present study, the effects of the two types of gonadotropin releasing hormone (gnrh) on mmp- expression were examined in an immortalized human cytotrophoblast cell line, b tert- that has been established in this lab. real-time quantitative pcr and western blot analysis revealed that both types of gnrh (gnrh i and gnrh ii) could increase mmp- mrna and protein levels in b tert- cells in time-dependent manners. in particular, regulatory effect of gnrh i on mmp- expression was concentration-independent, whereas that of gnrh ii was dose-dependent. moreover, both gnrh i and gnrh ii could evidently activate jnk kinase, and sp , an inhibitor of a jnk kinase, reversed the up-regulation of mmp- induced by either gnrh i or gnrh ii. on the other hand, it is not likely that erk / pathway participates in the signaling of gnrh i or gnrh ii. collectively, our observations suggest that gnrh i and gnrh ii elicit their modulation effects in human trophoblastic cells through jnk pathway leading to up-regulation of mmp- . during the first trimester of pregnancy, the oxygen tension of the developing trophoblast cells is less than %. however, the majority of studies on primary trophoblast cell development have been performed at % oxygen. primary third-trimester trophoblast cells are believed to be nonproliferative syncytiotrophoblast cells. we have previously demonstrated that low oxygen tension dramatically affects the differentiation pathway of these cells. we now hypothesize that cell culture in low oxygen tension will improve cell growth and restore proliferation. methods: primary trophoblast cells were purified from third-trimester placenta by enzymatic dispersion and cd- negative selection and cultured at %, % or . % oxygen tension for up to days. the number of cells in culture was assessed by cell counting and by measuring genomic dna. live:dead and mtt assays were used to determine viability. proliferation was assayed with brdu and immunohistochemistry for proliferating cell nuclear antigen. to assess cellular activity, radioactivity of protein precipitated from cells cultured in the presence of tritiated leucine was measured. results: there were no obvious morphologic changes in the cells cultured in different oxygen tensions. the amount of cell loss was directly proportional to oxygen tension: at % oxygen % of the cells remain in culture; at . % oxygen tension % of the cells remained. the cells at . % oxygen tension were proliferating and had a five-fold increased metabolic activity. conclusions: it was previously believed that third-trimester trophoblast cells are non-proliferative. we have demonstrated that low oxygen tension increases the survival of primary third-trimester trophoblast cells. this may reflect the change in the differentiation pathway of these cells. however, the cells also begin to proliferate and increase their metabolic activity. trophoblast cells in vivo form a three dimensional structure which promotes critical complex cell-to-cell interactions that cannot be studied with traditional monolayer cell culture. we developed a substrate-free three-dimensional trophoblast culture system capable of studying cellular interactions without a confounding artificial matrix. methods: nonadhesive agarose hydrogels containing cylindrical recesses m in diameter were cast from molds designed using computer-assisted prototyping. tcl trophoblast cells were seeded into the gels ( , cells per) for up to days. viability and cellular stress were assessed and the threedimensional structures of the spheroids were analyzed. results: tcl trophoblast cells formed uniform spheroids within three days of seeding. the spheroids remained intact after being removed from the mold. when placed in traditional cell culture dishes the cells adhered to the plate within one hour and rapidly proliferated into a monolayer. repetitive reseeding allowed easy transition between monolayer and spheroid without affecting cellular morphology. serial sectioning on days , and revealed central vacuolization forming a trophoblast vesicle with an outer rim . m (+/- m) thick. this rim size remained constant for at least days. live:dead assay demonstrated that the outer cells remained viable and staining against proliferating cell nuclear antigen demonstrated that the cells were proliferating. the inner cells undergo apoptosis as demonstrated by caspase- staining. there is an abundance of vegf staining in the cells remaining in the on the inside of the sphere suggesting a gradient of nutrient or oxidative stress. the formation of a vesicle has been confirmed with confocal imaging. em imaging revealed the structure of the rim. conclusions: trophoblast cells cultured in a novel substrate-free three dimensional system form trophoblast vesicles within days of seeding. these vesicles remain viable after long-term culture and can be repeatedly reformed with repetitive seeding. this new cell culture technique allows us to better study placental cell-cell interactions with the potential of forming microtissues. the transcription factor glial cell missing- (gcm ) mediates cell cycle arrest and differentiation of human trophoblast progenitors into villous syncytiotrophoblast and invasive extravillous cytotrophoblast (evt). micro-array analysis of total rna extracted from cultured bewo cells, in which gcm mrna and protein were repressed using sirna, identified tissue inhibitor of metalloproteinase- (timp- ) in the highest ( -fold) upregulated group of genes. confirmatory rtpcr demonstrated a -fold mrna induction. in placental villi, gcm acts as a transcription factor promoting expression of the fusogenic protein syncytin that mediates syncytial fusion into the overlying syncytiotrophoblast. by contrast, syncytial fusion is uncommon in evt. rather these cells invade several millimeters into the distal myometrium where they transform spiral arterioles. to investigate the role of timp and gcm in the trophoblast we assessed its mrna by qrt-pcr and protein by western blot in cellular extracts from both bewo cells grown under standard cultivation conditions (synchronized by prior thymidine exposure) and floating cultured first trimester villous explants cultured in % oxygen with prior exposure to either gcm sirna or anti-sense oligo-nucleotides to gcm . gcm inhibition in the bewo system was associated with a - % increase in timp- protein expression and alteration of cell proliferation and differentiation in both models. we are presently utilizing the explant model of evt invasion (explant tips cultured on matrigel in % oxygen) to test the hypothesis that gcm mediates metallo-proteinase expression and evt invasion via timp- . presently we conclude that gcm -mediated evt differentiation involves more than an arrest of mitosis and may include promotion of invasion via repression of timp- . funding: cihr. scott h purcell, jeremy d cantlon, virginia d winn, russell v anthony. , colorado state university, fort collins, co; university of colorado health sciences center, aurora, co. background: periattachment factor (pf) is a nuclear protein first described in the bovine conceptus. our research in sheep has shown pf mrna concentration peaks when the conceptus is undergoing elongation and initial apposition to the endometrium, and that pf is a nuclear protein localized to the trophoblast. in silico analysis identified a human homolog, hprr . objective: the objective of this experiment was to determine if pf was expressed in the human placenta, and to develop short-hairpin (sh) rnas for hpf to begin investigating its function. materials and methods: immunohistochemistry was performed on paraffin embedded first and second trimester human placental samples. placental sections were immuno-stained using rabbit polyclonal antiovine pf or anti-human cytokeratin- . cytotrophoblasts from first trimester pregnancies (n= ) were subjected to an in vitro invasion assay and rna was harvested following , , and h. quantitative rt-pcr was performed on these samples with intron-spanning primers for hpf, and normalized on hs mrna concentrations. based on the human pf sequence, four putative shrna constructs were generated and cloned into a lentiviral expression vector. bewo human choriocarcinoma cells were treated with one of four shrna contructs or an empty vector for h and then rna was harvested from cells for analysis by quantitative rt-pcr. results: periattachment factor was present in the nuclei of both first and second trimester cytotrophoblasts. hpf mrna concentration increased as invasion occurred from , , to h in all samples; while hypoxia decreased expression at h of invasion compared to h under normoxic conditions. the four lentiviral vectors expressing shrna against hpf resulted in hpf mrna concentrations at , , and % of hpf mrna concentration with the control vector. conclusion: the presence of pf in the human placenta and the increase in pf mrna during cytotrophoblast invasion may indicate this gene plays a role during implantation. we have developed shrnas against pf that result in greater than % mrna knockdown and will be using these to begin to elucidate the function of pf in the human placenta, specifically during the invasion process. recently we demonstrated that infusion of imd antagonist (imd - ) in rat caused distorted labyrinth indicative of a deficient vasculature in placenta. we hypothesize that imd has a role in migration of first trimester trophoblast cell (htr- /svneo) via regulating human leukocyte antigen (hla-g) and stimulating mek / / erk / phosphorylation. objectives: ) to asses the effect of imd on migrating capacity of htr- sv/neo cells using scratch assay in presence or absence of mek and ras/raf inhibitor, u and manumycin a, respectively ; ) to assess the effect of imd peptide on phosphorylation of mek / and erk / in first trimester htr cells ) to analyze the effects of imd on the expression of human leukocyte antigen, hla-g, a critical factor involved in the invasion and vascular remodeling of spiral uterine arteries and subsequent pregnancy in human. methods: htr- sv/neo cells were used to assess the effect of imd ( - m) on the expression of hla-g mrna and phosphorylation of erk / and mek / protein by reverse transcriptase polymeration chain reaction (rt-pcr) and western blot analysis respectively. scratch wound assay was used to determine the migration capacity of htr cells. total rna was isolated from cells using trizol reagent and processed for rt-pcr and results are expressed relative to s mrna. trichloroacetic acid was used for the extraction of total protein for western blot analyses. results: our data demonstrates that, ) imd enhances the migrating capacity of htr cells (compared to the untreated cells) and these effects are inhibited by mek and ras/raf inhibitors, u and manumycin a, respectively; ) imd ( - m ) stimulates phosphorylation of erk / and mek / proteins in htr cells, ) imd increases the expression of hla-g mrna in htr cells. conclusion: imd promotes migration of first trimester htr cells through mek/ erk signaling pathway and modulates the expression of immunoregulatory molecule, hla-g in these cells. chymotrypsin-like protease promotes the placenta tissue release of sflt- . yang gu, shuang zhao, david f lewis, yuping wang. obstetrics and gynecology, lsuhsc-shreveport, shreveport, la, usa. objective: the placenta is a major source of soluble vegf receptor- (sflt- ) in the maternal circulation during pregnancy. increased placental release of sflt- is believed to play an important role in the pathophysiology and pathogenesis in pe. however, the mechanism of increased placental sflt- release in pe is unknown. we recently reported increased chymotrypsin-like protease (clp) activity and expression in placental trophoblasts from pe. in this study, we tested if proteolytic effects of chymotrypsin may play a role in promoting sflt- release by placental trophoblasts. methods: placentas delivered by normal pregnant women (n = ) were used. we tested if chymotrypsin could promote sflt- release by placental tissue, in which villous explants were cultured with dmem containing chymotrypsin at . , . , and . g/ml for hours. the culture medium was then collected for measuring sflt- . we then determined the specificity of chymotrypsin induced sflt- release. villous tissues were cultured with or without chymotrypsin inhibitor (ci) in culture and then the medium was collected and measured for sflt- . soluble flt- was measured by elisa. all samples were assayed in duplicate. data are presented as mean ± se and analyzed by anova. a p level < . is considered as statistically different. results: ) sflt- concentrations in the medium were increased when chymotrypsin was present in culture and the increased sflt- release induced by chymotrypsin was in a concentration-dependent manner: control: . ± . ; . g/ml: . ± . ; . g/ml: . ± . ; and . g/ml: . ± . (p< . ) pg/mg tissue/hour. ) ci could attenuate sflt- release. this inhibitory effect was also revealed in a concentration-dependent manner: control: . ± . ; ci . g/ml: . ± . ; and ci . g/ml: . ± . (p< . ) pg/mg tissue/hour. conclusions: increased placental sflt- release stimulated by chymotrypsin and decreased placental sflt- release inhibited by chymotrypsin inhibitor suggest that the proteolytic effect of clp may play a role in sflt- generation. therefore, increased clp activity in placental trophoblasts may contribute to the increased placental sflt- production in pe. (supported nih grants hl and hd ). the change of autophagy-related proteins, lc and beclin- , by tnfa stimulation in cultured primary trophoblasts. soo-young oh, kyung hee kim, suk-joo choi, jong-hwa kim, cheong-rae roh. department of obstetrics and gynecology, samsung medical center, sungkyunkwan university school of medicine, seoul, korea. objective: our previous work have demonstrated that the expression of lc , but not beclin- , was increased in placentas from pregnancies complicated by severe preeclampsia (sgi abstract # ) . to understand the regulatory mechanism of these autophagy-related proteins in trophoblast cells, we investigated the changes in these proteins in response to cytokine or hypoxic stimulation in cultured primary trophoblast. material and methods: primary human cytotrophoblasts obtained from normal term placenta were cultured with stimulation of tnf-a or cocl for a given time and the changes of beclin- and lc were assessed using immunoblot analysis. paired t test was used for statistic analysis. results: tnf-a stimulation induced a significant increase of the expression of lc -ii in cultured primary trophoblasts while decreasing the expression of beclin- (p< . for each). however, cocl stimulation did not induce a significant change of both lc -ii and beclin- . conclusions: our data suggests that tnf-a stimulation in cultured primary trophoblasts is associated with increased autophagic activity. background: thyroid hormones play vital roles in the development of the fetal brain. mutations in mct , recently recognised as a specific thyroid hormone transporter, define a novel syndrome of severe x-linked psychomotor retardation accompanied by elevated serum t . we previously reported that mct expression in n-tera- (nt ) cells (a human embryonal cell line with characteristics of cns precursors), as well as mct -null jeg- choriocarcinoma cells, resulted in markedly reduced cell proliferation. further, the s x mct mutation, as reported in males affected by severe psychomotor impairment, resulted in a similar repression of proliferation to wild type, whereas the l p mutant failed to influence cell turnover compared with control. methods: we now examine the effect of "knocking down" mct via sirna and evaluate the effects of cell proliferation (mtt and [ h]-thymidine assays) and tri-iodothyronine (t ) uptake. results: repression of endogenous mct expression in nt cells by % caused a significant increase in proliferation compared to matched-dose nonspecific sirna treatment, independent of t concentration ( . %, . % and . % induction at , and nm t , n= , p< . ). we also sought to examine the role of mct in t uptake. in jeg- cells, wild type mct induced a . -fold increase in the uptake of i-labelled t . by contrast, mutants s x and l p failed to significantly augment t uptake, though r h caused a mild but significant . fold induction in uptake, hence retaining approximately % of wt activity. in parallel experiments, co-transfection of mu-crystallin, a t binding protein, resulted in a similar increase in t uptake compared with control ( . -fold; n= ; p< . ), implying that mct plays only a minor role in thyroid hormone efflux in jeg- cells. mutants s x, l p and r h showed analogous responses to those in the absence of mu-crystallin. conclusion: these results further extend the evidence of a potential role for mct in the modulation of cell proliferation, independent of t transport. to determine predictors of failure for labor induction in women with preeclampsia. we conducted a retrospective cohort study to examine cesarean delivery rates in all the preeclamptic women at a single institution undergoing labor induction between - with a singleton pregnancy >= weeks gestational age (ga). bivariate analyses informed the creation of multivariable logistic models to predict the risk of cesarean delivery using multiple predictors (maternal age, race/ethnicity, unfavorable cervix, gestational diabetes, diabetes, and gestational age). analyses were stratified by parity. our study population included , preeclamptic women undergoing labor induction. in the bivariate analyses, the risk of cesarean delivery ranged from as low as . % (p= . ) among multiparous women - weeks ga to as high as . % (p< . ) among nulliparous women with diabetes. a total of , women had adequate data to be included in the multivariable analyses. odds ratios of the predictors are presented in the objective: preeclampsia and cardiovascular disease share many risk factors, and women with preeclampsia are at increased risk of cardiovascular mortality later in life. we investigated whether risk factors associated with cardiovascular disease and preeclampsia remain elevated months postpartum. methods: we measured plasma sflt , endoglin, plgf, cellular fibronectin (cfn), uric acid, homocysteine, and asymmetric dimethylarginine (adma) in women with uncomplicated normotensive pregnancies compared to women with preeclampsia in samples collected at pre-delivery and again several months postpartum (average . ± . months). data are mean±sd or median (interquartile range). statistical analysis was by wilcoxin rank-sum or students unpaired t-tests with statistical significance accepted at p< . . results: the mean concentration of sflt , endoglin, plgf, homocysteine, adma, cfn, and uric acid were all significantly different in samples collected pre-delivery in subjects with preeclampsia compared to controls (table). adma, cfn and uric acid remained significantly higher postpartum in subjects with previous preeclampsia compared to postpartum controls (table) . conclusions: biological markers associated with altered vascular function or cardiovascular risk are elevated in women with preeclampsia, and some remain significantly higher in postpartum preeclamptic women. these data suggest that vascular dysfunction persists in women with previous preeclampsia, and may contribute to the increased risk of future cardiovascular disease. funded in part by national institutes of health nih- mo -rr and nih- po -hd . (ajp, ) . as leptin is a known potent angiogenic factor we hypothesized that leptin deficiency and/or resistance to leptin-induced vegf expression might be a mechanism for reduced angiogenesis in mfr offspring. methods: pregnant sprague-dawley rats had % mfr from day of gestation until delivery. mfr and control offspring were sacrificed on day of life (p ). some tissues were used to determine the expression of leptin by western blot analysis. for culture experiments, thoracic aortas were dissected, cut into - mm explants and incubated with leptin ( - ng/ml) in dmem ( % fbs). after hours of culture, rna was extracted from the tissues and subjected to real time rt-pcr using specific rat primers for vegf, vegfr and r , and ob-ra, stat and socs ( s mrna as control). culture media was analyzed for vegf protein by elisa. results: expression of leptin mrna and protein in p mfr aortas was significantly reduced. in culture, leptin significantly increased expression of vegf, vegfr and vegfr mrna in explants of aortas obtained from the control but not mfr tissues. as expected, control but not mfr aortic explants secreted significantly more vegf in vitro. to determine the mechanism for resistance to leptin-induced vegf in mfr offspring, we assessed expression of leptin receptor (ob-ra) in explants treated with leptin. leptin was found to induce the expression of ob-ra in aortas from both dietary groups. this upregulation of leptin receptor was accompanied by significant upregulation of stat and socs mrna in the control tissues. in contrast, in mfr explants only the ng/ml concentration of leptin induced an increase in stat mrna, and the magnitude of socs mrna increase by both concentrations of leptin was significantly less in the mfr explants. conclusion: these results indicate that reduced angiogenesis in mfr vessels is in part due to reduced leptin expression and ability of leptin to stimulate vegf expression. although in vitro leptin induced the expression of its receptor in both groups, it was only in the mfr group in which leptin up-regulated vegf and its receptors. our results suggest that this defect in leptin receptor function in mfr vessels is due, in part, to defects in jak/stat signaling. objective: women with a history of early-onset preeclampsia are at increased risk of developing major cardiovascular disease (cvd) related events, that have a detrimental effect on their long-term health and life expectancy. in this follow-up study, we measured established risk factors predictive of first cvd events after early-onset preeclampsia. study design: over a -year interval, primiparous women with a history of early-onset preeclampsia (delivery < weeks gestation) were included and tested for major cardiovascular risk factors at least six months after delivery, in addition to a population-based control group of healthy non-pregnant women. women with chronic hypertension were excluded. results: mean age was . years for cases compared to . years for controls (p<. ). after adjustment for age, we observed significantly increased mean values for weight (p=. ), body-mass index (p<. ), systolic blood pressure (p<. ), diastolic blood pressure (p<. ), total cholesterol (p=. ), ldl cholesterol (p<. ), triglycerides (p=. ), fasting blood glucose (p<. ), and lower hdl cholesterol (p<. ) in women with previous early-onset preeclampsia. no difference was found for height, smoking, diabetes, and ethnicity. estimated -year risk of first cvd events by framingham risk scores remained < % for all women (low-risk). nonetheless, at mean (sd) . ( . ) years after early-onset preeclampsia, % of women met the criteria for metabolic syndrome, % of women exhibited >= , % of women >= and % of women >= major cvd risk factors. conclusion: the majority of women with a history of early-onset preeclampsia exhibit at least one modifiable risk factor for future cvd. although most of these women are classified as low-risk according to the current aha guidelines, this is mainly due to their young age masking other, mostly modifiable, major risk factors. our data thus support life-style intervention programs aimed at primary prevention of cvd in women with a history of early-onset preeclampsia. it has recently been shown that antihypertensive drugs can stimulate cytokine release in normal and hypertensive pregnancy. there is evidence that these cytokines alter the secretion of inhibin a. inhibin a and activin a levels are increased in pre-eclampsia (pe), but it is not known if antihypertensive therapy can affect their secretion. patients and methods we recruited women with hypertensive disorders in pregnancy ( pe and non-proteinuric hypertension [ht]) and matched normotensive controls. inhibin a and activin a levels, before and - hours after initiating antihypertensives, were measured in serum and urine, using an elisa. the same markers were measured using validated assays in placentas delivered at cesarean section at similar gestational age ( pe, ht and controls). analysis the three study groups were compared using anova multiple comparisons with bonferroni post hoc testing. the data were normally distributed after logarithmic transformation. marker levels before and after antihypertensive therapy were compared using paired t-test. we compared placental concentrations between the group which received antihypertensive therapy and the group which did not, using an independent t-test. data were analysed using spss®. in pe, both serum and urine levels of inibin a and activin a were increased at all gestations (p< . ). activin a (but not inhibin a) level was also increased at all gestations in ht (p< . ). after weeks' gestation (but not before), antihypertensive treatment was associated with a significant fall in both inhibin a and activin a serum levels, and urinary inhibin a, in both pe and ht. the placental concentration of inhibin a, but not activin a, was significantly higher in women with pe compared with controls (p< . ). there was no significant difference in either marker between controls and women with ht. the fall in serum levels of inhibin a and activin a following antihypertensive treatment after weeks' gestation may indicate that these drugs have an effect on the pathophysiology of pe other than their known antihypertensive action. pre-eclampsia (pe) is a placental disease of unknown etiology. anti-angiogenic factors, such as soluble fms-like tyrosine kinase (sflt- ) and soluble endoglin (seng), and pro-angiogenic factors, such as vascular endothelial growth factor (vegf) and placental growth factor (plgf), are believed to play an important role in its pathophysiology. maternal plasma concentrations of these markers are altered in pe, even weeks before the clinical manifestations. the aim of this study was to compare the concentration of these markers in placental extracts of normotensive pregnant women, and women with pe and non-proteinuric hypertension (ht). patients and methods placental samples were collected at cesarean section from women with pe (n = ), ht (n = ) and normotensive pregnancies of similar gestational age (n = ). these samples were stored at - ºc. the frozen tissue samples were homogenised and these four markers measured by specific, validated enzymelinked immunosorbent assays. analysis the three study groups were compared using anova multiple comparisons with bonferroni post hoc testing. the data were normally distributed after logarithmic transformation. data were analysed using spss®. the concentrations of both sflt- and seng were significantly higher in the placentas of women with pe, but not ht, compared with controls (p= . ). there was no significant difference in plgf concentration between controls and women with pe or ht. placental vegf concentrations in both pe and ht were higher than in controls (p< . ); there was no significant difference between the levels in pe and ht (p= . ). the fact that placental concentrations of sflt- and seng mirrored the known rise in serum levels in pe suggests that the placenta is the main source of these circulating factors. although sflt- was significantly raised in pe, plgf was not reduced. this suggests that the lower levels of free plgf found in the serum of women with pe are not the result of impaired placental production or secretion, but are due to increased binding by (the increased levels of) sflt- in the serum. objective. the purpose of this study was to evaluate whether systematic screening with uterine artery doppler (utad) and serum biochemical markers of oxidative stress, endothelial dysfunction and vasculogenesis performed during the first trimester predict efficiently pre-eclampsia (pet), specifically early-onset pet, in an unselected chilean population. methods. this nested case-control study involved asymptomatic pregnancies scanned at + - + week of gestation. the subjects for biochemical testing were women who were delivered due to pet (n= ) and normotensive controls (n= ) that were enrolled during the first trimester scan. mean pulsatility index (pi) of the utad was calculated. blood samples were obtained and stored at - o c until biochemical analysis of oxidative stress, endothelial dysfunction and vasculogenesis were performed. normally distributed data were analysed by the unpaired t test, and non-normally distributed data by the mann-whitney rank sum test. chi-square tests were used for the comparison of categorical variables. a probability level of p< . was considered significant. multiple logistic regressions were used to develop a combined predictive index. results. there was % and % significantly increased of the mean pi utad in women who later developed pet or early-onset pet compared to control pregnancies during the first trimester scan. although oxidative stress and endothelial dysfunction biochemical markers were not different between all pet pregnancies and control groups, plasma levels of sflt ( . ± . vs. . ± . pg/ml, p< . ) and placenta growth factor ( . ± . vs. . ± . pg/ml, p< . ) were significantly higher in women who subsequently developed early-onset pet compared to controls. multivariate logistic regression showed that a combination between abnormal utad and both biochemical markers of abnormal vasculogenesis were the best predictor test for early-onset pet, being its detection rate % with % false positive rate. conclusion. this study has shown early and selective changes in markers of impaired placentation and angiogenic state in women who later developed earlyonset pet, without alteration in oxidative stress and endothelial dysfunction. supported by fondecyt . currently it is unknown whether maternal inflammatory changes are specific to pregnancy or reflect an innate susceptibility to inflammation. c-reactive protein (crp) and interleukin- (il- ) are markers of the acute-phase inflammatory response and predictive of future cardiovascular events. we compared crp and il- levels after influenza vaccination, as an in vivo model for lowgrade inflammation, in non-pregnant women with a history of early-onset preeclampsia and controls with only uneventful pregnancies. methods: forty-four women with a history of early-onset preeclampsia (delivery < weeks' gestation) and twenty-nine controls with at least one uneventful pregnancy received an influenza vaccination. we then compared plasma levels of crp and il- at baseline, . days and . days after vaccination. results: median baseline crp and il- levels of women with a history of early-onset preeclampsia were comparable to controls ( . versus . mg/l; p= . and . versus . pg/l; p= . , respectively). however, high crp and il- responses to vaccination were more common in cases (ors for response > th, > th, > th, > th and > th percentile based on the distribution of control values of . , . , . , . and for crp [p for trend . ] and of . , . , . , . and . for il- [p for trend . ], respectively). the relationship between high il- responses and early-onset preeclampsia persisted after adjustment for body-mass index (p for trend . ). conclusion: women with a history of early-onset preeclampsia more frequently exhibit an innate pro-inflammatory phenotype not specific to pregnancy. background altered maternal inflammatory responses play a role in the development of preeclampsia and the hemolysis, elevated liver enzymes and low platelets (hellp) syndrome. we examined whether allelic variants of the innate immune receptors toll-like receptor (tlr ) and nucleotide-binding oligomerization domain (nod ), that impair the inflammatory response to endotoxin, are related to preeclampsia and hellp syndrome. we determined five common mutations in tlr (d g and t i) and nod (r w, g r and l fs) in primiparous women with a history of early-onset preeclampsia, of whom women developed hellp syndrome and in women with a history of only uneventful pregnancies as controls. in addition, we assessed plasma levels of pro-inflammatory biomarkers c-rp, il- , sicam- , fibrinogen and von willebrand factor in a subset of women included at least six months after delivery. after adjustment for maternal age and chronic hypertension, attenuating allelic variants of tlr were more common in women with a history of early-onset preeclampsia than in controls (or . [ % ci . - . ]). highest frequencies for tlr variants were observed in women who developed hellp syndrome (adjusted or . [ % ci . - . ]). in addition, high levels of il- and fibrinogen were associated with a history of early-onset preeclampsia. combined positivity for any of the tlr and nod allelic variants and high levels of il- was . -fold more common in women with a history of early-onset preeclampsia ( % ci . - . ) compared to controls. we observed an association of common tlr and nod gene variants, and pro-inflammatory phenotype with a history of early-onset preeclampsia and hellp syndrome, that suggests involvement of the maternal innate immune system in severe hypertensive disorders of pregnancy. thus, we sought changes in pbef in the serum of patients with mild and severe pe, compared with matched controls. immunodistribution of pbef in fetal membranes and placentas from similar patients was also studied. methods: serum samples ( ) were collected with clinical data including; gestational age, medications, ethnicity and recognized complications. patients in labor or infection were excluded. the standard bp and proteinuria criteria was utilized to classify cases for pe grouping; no pe (n= ), mild pe (n= bp; / - / , proteinuria; trace to + or - mg/ hr urine) and severe pe (n= bp; > / , proteinuria; > + or > g/ hr, or other associated symptoms). pbef concentration was determined by eia (phoenix pharmaceuticals) in accordance with the manufacturers instructions. fetal membranes and placentas of additional patients, no pe (n= ) and with pe (n= ) were fixed and embedded in paraffin. sections ( um) were immunostained with pbef antibody / (pheonix) and treated with abc reagent (vector labs) followed by dab ( . % mg/ml), washed, counterstained, mounted and viewed under brightfield microscopy. results: the concentrations of pbef in serum were between - ng/ml and were significantly higher in those patients with mild pe (p= . ) and further significantly elevated in those with severe pe (p= . ) compared with the matched controls. pbef was detected by immunocytochemistry in the placental syncytiotrophoblast and in the amnion and choriodecidua of the fetal membranes. conclusions: pbef was elevated in the serum of patients according to the degree of pe severity and may be derived from the placenta and/or fetal membranes. (supported by nih #u rr - to the pacific research center for early human development, university of hawaii). background: platelet-monocyte aggregation (pma) is a novel sensitive measure of platelet activation and indicates a proinflammatory state (cytokine release). less sensitive techniques demonstrate platelet activation during pregnancy and pre-eclampsia (pe) but platelet activation has not been assessed by pma.objective: longitudinal study of pma in normal pregnancy and pe. methods: healthy, non-smoking primigravida with an uncomplicated pregnancy and primigravida women with pe were studied. pe was defined by standard definitions. informed consent was obtained and the study had ethical approval. serial venous blood collected at , , , wks in controls, at time of diagnosis in pe cases and wks post-natal (pn) in all. pma, platelet surface p-selectin (psp-sel) and monocyte cd expression (mcd ) were analysed by flow-cytometry and plasma (p) p-sel by elisa. results: groups were matched for mean age and bmi. in controls, pmas, psp-sel and mcd expression and pp-sel increased with gestation and decreased post-natally (table ) . for pe analysis, data was divided into pre-term (sampled at mean wks), and term (mean wks). pp-sel was lower in pre-term pe than control (normal pregnancy wks; p= . ) there was no significant difference in other measures between pe and control ( objective: much effort has been put into the evaluation of novel markers to identify pregnant women at risk for the development of pre-eclampsia. soluble endoglin (seng) and soluble fms-like tyrosine kinase (sflt ), two antiangiogenic agents, appear to be involved in the pathogenesis of pre-eclampsia. despite several studies describing higher midtrimester serum concentrations of these markers in women with subsequent pre-eclampsia, information on first trimester serum levels is scarce. the aim of this study was to assess seng and sflt as first trimester serum markers for the prediction of pre-eclampsia. methods: sera were obtained between + and + weeks of gestation from women who later developed late-onset pre-eclampsia and from controls matched for gestational age, maternal age, maternal pre-pregnancy weight, and storage. using commercially available microplate enzyme immunoanalytical methods, seng and sflt were determined and the results analyzed using non-parametric statistical tests. results: the serum concentration of seng was found to be increased in women with subsequent pre-eclampsia when compared to controls (mean ± sd, . ± . ng/ml versus . ± . ng/ml, p = . , unpaired mann-whitney test). similarly, the serum levels of sflt were higher in women later developing late-onset pre-eclampsia ( ± ng/ml) when compared to controls ( ± ng/ml, p = . ). sensitivities and specificities for predicting pre-eclampsia were % and % for seng and % and % for sflt- , respectively. the combination of the two markers by multiplication yielded a sensitivity of % and a specificity of %. conclusion: seng and sflt , showing increased first trimester serum levels in women with subsequent pre-eclampsia, might both fulfill the characteristics of first trimester markers to predict pre-eclampsia. the combination of the two, however, did not improve the sensitivity nor the specificity compared to their individual determinations. moderate sensitivities and specifities, however, limit the clinical use of these molecules as single markers. pregnancy is associated with increased monocyte/platelet aggregate formation in whole blood. beth a bouchard, adrienne schonberg, gary j badger, ira m bernstein. biochemistry; obstetrics and gynecology; medical biostatistics, univ of vt, burlington, vt, usa. background preeclampsia is associated with increased rates of platelet clearance, changes in platelet function and platelet activation. the goal of the current study was to examine basal levels of platelet activation through pregnancy beginning prior to conception, and to examine the association of platelet activation with the development of hypertensive complications during pregnancy. methods two indices of platelet activation, platelet cd expression (%cd ) and monocyte/platelet aggregate (%mp) formation, were measured in whole blood by flow cytometry using specific, fluorescentlylabeled monoclonal antibodies in healthy, nonsmoking women during the follicular phase of their menstrual cycle (pp, cycle day . + . ). all women subsequently conceived singleton pregnancies and were re-examined in early (ep, - wks) and late pregnancy (lp, - wks). five of these women were diagnosed with hypertensive complications ( hypertension, preeclampsia) at term although hypertension was not observed at any study time point. data are expressed as mean±sem. p< . was accepted for significance. results subjects were . + . years old with a bmi of . + . kg/m at the time of prepregnancy studies. a significant increase in the %mp formed over time of pregnancy was observed (p= . ). there was little change in the %mp formed between pp and ep (pp, . ± . % and ep, . ± . %, p= . ). however, the %mp increased significantly in lp ( . ± . %) as compared to pp (p= . ) and ep (p= . ). this increase occurred independent of the development of hypertensive complications (p= . ) and independent of pp platelet activation status. although statistically significant increases in cd expression were not observed, the change in cd expression over pregnancy correlated with the change in %mp over pregnancy (r= . , p= . ) and cd expression correlated with %mp in lp (r= . , p= . ). conclusion these combined observations suggest that pregnancy is associated with increases in levels of unstimulated platelet activation and that these increases occur in the presence or absence of subsequent hypertensive complications. furthermore, we observed a correlation between the changes in two distinct platelet activation events, %mp and cd expression, during pregnancy. supported by nih hl . backgound sildenafil citrate (sc) has been proposed as a therapy to improve uterine perfusion in pregnancies complicated by iugr or preeclampsia. we sought to determine the effects of sc on uterine vascular resistance, uterine blood flow and cardiac output in young healthy nulliparous women. methods eleven young healthy nulligravid women were studied during the luteal phase (cycle day + ) of the menstrual cycle. women were randomized in a double-blind fashion to receive placebo (pl), or sc at a dose of or mg. uterine artery vascular resistance, uterine artery volumetric flow, brachial artery volumetric flow and cardiac output were measured at baseline and at and hours post dosing employing color doppler ultrasound. comparisons were made by anova between those randomized to pl (n= ) versus sc (n= ). p< . was accepted for significance. data are expressed as mean + s.e.m. results there were no significant differences in subject age, cycle day, body mass index, uterine blood flow, brachial blood flow or cardiac output at baseline comparing the two groups. there was a tendency towards increased uterine blood flow in subjects randomized to receive sc ( % increase) compared to pl (no change), changes in uterine blood flow, brachial blood flow and cardiac output are outlined in the objective reduced maternal plasma volume in the third trimester has been associated with both fetal growth restriction and preeclampisa. we sought to determine the degree to which third trimester plasma volume is dependent on plasma volume prior to pregnancy. methods sixteen young ( . + . years) healthy nulligravid women had their plasma volume measured during the follicular phase (cycle day . + . ) of the menstrual cycle and subsequently conceived. subjects were predominantly caucasian ( / ) with a mean prepregnancy bmi of . + . kg/m . plasma volume was re-estimated at - weeks gestation. all patients were placed on sodium and total calorie balanced diets for days prior to each plasma volume determination. plasma volume was determined employing evans blue dilution with multiple post injection sampling time points. data are expressed as mean + s.d. results baseline prepregnant plasma volume was , + ml or + ml/unit bmi. third trimester plasma volume was , + ml representing a % increase (p< . ). the range of plasma volume expansion was - % dependent upon prepregnant plasma volume. plasma volume in the third trimester of pregnancy was strongly correlated to prepregnant plasma volume r= . (p= . ). plasma volume expansion was consistent across the range of prepregnancy plasma volume. conclusions pre-pregnancy plasma volume contributes approximately % of the variance in third trimester plasma volume. the observed increase is plasma volume is independent of prepregnancy volume resulting in a greater percentage increase for those starting at the lower end of the plasma volume range. as third trimester plasma volume is strongly associated with pregnancy outcome the correlation of prepregnancy plasma volume to third trimester plasma volume suggests that prepregnancy status contributes to these adverse reproductive events.this work was supported by nih hl . background: hydatidiform mole is a rare disorder of pregnancy and may predispose the mother to severe morbidity. molar pregnancies are known to be associated with high risk for the development of early onset preeclampsia. in recent years, the expression of sflt- (soluble vegfr- ) was found to be increased in preeclampsia, and contributes to the pathogenesis of the maternal systemic disease. the objective of the present study was to examine the expression of sflt- in placentae from molar pregnancies. methods: placental samples from unique cases of twin pregnancies with complete molar pregnancy in one sac and developing fetus in the other sac were prospectively collected (n= ). the first set delivered at weeks due to excessive bleeding. the second set delivered at weeks due to severe iugr and elevated blood pressure. mrna level of sflt- was measured by quantitative real-time pcr using specific taqman primers and probe. protein expression of sflt- in placental tissue lysates were measured by western blot analysis using a polyclonal antibody against flt- . immunohistochemistry of paraffin embedded samples was performed using specific antibody for sftl- . results: mrna level of sflt- was increased by . fold in the molar placentae compared to matched controls. the placentae of the developing fetuses which were growth restricted exhibited . fold increase compared to controls. sflt- protein expression in the molar placentae was increased by . fold compared to controls, while the co-twin placentae exhibited a . fold increase compared to controls. immunohistochemistry revealed strong positive immunoreactivity for sflt- in the trophoblast layer of both molar pregnancies and iugr co-twin relative to controls. conclusion: our data suggest that sflt- expression is increased in placentae from molar pregnancies and thus may explain the increased risk for developing early onset preeclampsia. the expression of sflt- in the growth restricted twin placenta is also increased compared to controls and support our previous observation (supported by cihr and owh/igh). (n= ); ) neonates of patients with pe (n= ); and ) sga neonates (n= ). cord blood was collected immediately after delivery and pz plasma concentrations were measured by elisa. pz deficiency was defined as a cord plasma concentration < th percentile of the normal pregnancy group. non-parametric statistics were used for analysis. results: ) cord plasma pz concentration differed significantly among the study groups (kruskal wallis, p< . ); ) neonates of patients with pe and sga neonates had a significantly lower median cord plasma pz concentration than those delivered after normal pregnancy (pe: median . g/ml, range . - . , p< . ; sga: median . g/ml, range . - . , p= . ; normal pregnancy: median . g/ml, range . - . ); ) there were no differences in the rate of pz deficiency among the groups; and ) there was no relationship between placental histologic findings and median cord plasma pz concentrations between and among the sga and pe groups. conclusions: ) at the time of delivery, the median cord plasma pz concentration was lower in sga neonates and those born to women with pe than in neonates born to normal pregnancies; ) there was no difference in the rate of pz deficiency among the study groups, suggesting that the lower median pz cord blood concentrations in pe and sga groups may result from activation of the coagulation cascade rather than an inherited pz deficiency. objective: periconceptional multivitamin (mv) use may be related preeclampsia risk. we examined the relation between timing and frequency of periconceptional multivitamin use and the risk of preeclampsia. methods: women in the danish national birth cohort who delivered singleton liveborn infants (n= , ) reported upon enrollment at . weeks (sd . ) the number of weeks of regular multivitamin use during a week periconceptional period (lmp- to lmp+ ). preeclampsia cases were identified using icd- codes (n= , . %). logistic regression was used to estimate the effect of frequency (number of weeks of use) and timing of use to lmp+ ] and post-conception [lmp+ to lmp+ ]). results were stratified a priori by overweight status. results: overall, , women ( %) reported mv use in the periconceptional period. after adjustment for bmi, smoking, parity and chronic hypertension, infrequent mv use (< weeks of use) had no relation to risk (or . ; % ci . , . ) but regular use (>= weeks) was associated with modestly reduced risk ( . ( . , . ). similarly, when mv use was modeled as a continuous variable, each additional week of use was related to reduced risk for preeclampsia (or . , % ci . - . ). this potential dose effect of periconceptional mv use appeared to be limited to normal weight women (bmi < kg/m², or . ; % ci . - . ), with no apparent effect among overweight women (bmi kg/m², or . ; % ci . - . ). a total of , women reported regular mv use in both the preconception and post-conception periods, and , women reported regular use only in the post-conception period. among normal weight women, regular use in the preconception period had no effect on preeclampsia risk (or . , % ci . - . ). in contrast, use in the post conception period was associated with reduced risk for preeclampsia (or . , % ci . - . ). conclusions: regular periconceptional mv use was associated with a modestly reduced risk for preeclampsia among normal weight women. if causal, mv use immediately after conception appeared to be the critical exposure window. background: pre-eclampsia (pe), a disorder of pregnancy characterized by maternal inflammation, results in immune, cardiovascular and metabolic dysfunction. in non-pregnant persons, inflammatory disorders are treated with and prevented by pharmaceuticals and lifestyle methods such as physical activity (pa). while most pharmaceuticals are contraindicated for pregnant women, pa during pregnancy has been found safe, healthy and beneficial for both mother and baby. clinical evidence has found pa can beneficially affect pregnancy outcome, decrease excessive inflammation and decrease the risk of pe. epidemiological studies indicate that pa may be useful in preventing pe. unfortunately, previous studies have quantified pa based on recall of postpartum women and have not controlled for differences in women's interpretations of amount, type or intensity of pa. however, investigating pa utilizing a laboratory-based exercise intervention to control these variables inflicts difficulties translating the intervention into a community-based program that attracts and retains pregnant women in order to enhance public health. method: a retrospective study was performed to determine the rates of pe among the , women who gave birth at yale-new haven hospital (ynhh) during and , and a person subset of this group who performed prenatal pa in a community-based program that is evidence-based and standardized, thereby controlling for type and intensity of pa. additionally, the program is established in the community, has been offered to the public for years and is internationally known. results: during during - , the pe rate for the general population at ynhh was . %. for the pa group, the rate was . %. two women in the pa group were diagnosed with pe in the last month of pregnancy and delivered normal infants at term. no pe was observed in this group (pa group) during the second or early third trimesters nor was there any prematurity in this group. significance: these findings support the hypothesis that adequate physical activity provided in a standardized community-based group setting may provide a non-pharmacological approach for preventing pe. growth restriction. margreet plaisier, esther streefland, pieter koolwijk, frans m helmerhorst, jan jaap hm erwich. department of gynecology and reproductive medicine, leiden university medical center, leiden, netherlands; department of obstetrics and gynecology, university medical center groningen, groningen, netherlands; department of physiology, vu univerity medical center, amsterdam, netherlands. objective: disturbances in decidual and placental vascular development may play a role in the pathogenesis of pregnancy complications, like pre-eclampsia (pe) or fetal growth restriction (fgr). whether the regulation of decidual vascular adaptation to implantation is altered in these illnesses, is not elucidated yet. the present study focused on the role of first-trimester angiogenic factors in the pathogenesis of pe and or fgr. methods: first-trimester decidua samples were obtained during routine chorionic villous sampling. the expression of vascular endothelial growth factor (vegf-a), placental growth factor (plgf), flt- , kdr, angiopoietin- (ang- ), angiopoietin- (ang- ) and tie- mrna was determined by rt-pcr. the expression of the angiogenic factors was related to the pregnancy outcome, i.e. uncomplicated, pe or fgr. results: the first-trimester decidual tissues expressed all angiogenic factors. mrna levels of vegf-a, plgf, kdr, ang- , ang- and tie- appeared increased in fgr cases compared to matched controls. in addition, plgf, ang- and tie- mrna appeared increased in pe cases compared to matched controls. the differential expression of angiogenic factors was more pronounced in cases with fgr than pe. the large inter-individual variation disallowed a significant outcome. conclusions: various angiogenic factors showed differential mrna expression in st trimester decidua of patients developing pe or fgr in later pregnancy compared to their matched controls. the first-trimester decidual samples provided a unique opportunity to obtain information regarding the onset of pe and fgr. early st trimester changes in angiogenic factor expression may well occur as a compensatory mechanism. in turn, this may set the stage for increased non-branching angiogenesis and altered decidual and placental vascular adaptation, which may be part of the pathogenesis of pe and/or fgr. complications. beth a bouchard, adrienne schonberg, gary j badger, ira m bernstein. biochemistry; obstetrics and gynecology; medical biostatistics, univ of vt, burlington, vt, usa. background preeclampsia is characterized by endothelial dysfunction. the goal of the current study was to prospectively measure plasma levels of the soluble endothelial cell adhesion molecules, sicam- and svcam- , beginning prior to pregnancy and determine if subjects destined to develop hypertension complicating pregnancy had differences in the concentrations of these molecules. methods serum levels of sicam- and svcam- were measured in healthy, nonsmoking women (cycle day . + . , prepregnancy) by elisa. all women subsequently conceived singleton pregnancies and were re-examined in early (ep, - weeks) and late pregnancy (lp, - weeks). five of these women developed hypertensive complications ( gestational hypertension, pre-eclampsia) near term. all subjects were normotensive at all study time points. data are expressed as mean ± sem. p< . was accepted as significant. results subjects were . + . years old with a mean bmi of . + . kg/m at the time of prepregnancy studies. significant differences in sicam- levels as a function of pregnancy were observed (p= . ) and are outlined in table p= . differences were dependent upon the stage of pregnancy in those women who were not diagnosed with hypertensive complications with a decrease in sicam- levels in ep (p= . ) followed by an increase in sicam- levels in lp (p= . ). in women with hypertension in pregnancy, these differences in sicam- levels were not evident (p= . ). there were no differences in sicam- levels comparing women with or women without hypertensive complications prior to pregnancy (p= . ). in contrast to sicam- , we observed no significant differences in svcam- levels over pregnancy or between those with and without hypertension. conclusions these combined observations suggest that levels of the soluble adhesion molecule sicam- change significantly over time in normal pregnancies. subjects destined to develop hypertension did not demonstrate the early pregnancy reduction in sicam- . supported by nih hl . yuval bdolah, uriel elchalal, shira natanson-yaron, hadas caspi, tali bdolah-abram, angelika bord, caryn greenfield, debra goldman-wohl, ariel milwidsky, franklin h epstein, s ananth karumanchi, simcha yagel, drorith hochner-celnikier. ob/ gyn, jerusalem, israel; ob/gyn medicine, beth israel deaconess medical center, harvard medical school, boston, ma, usa. objectives: nulliparity is a risk factor for preeclampsia (pe) with a reported incidence of up to - times higher than multiparous pregnancies. soluble fms-like tyrosine kinase- (sflt ), a circulating anti-angiogenic molecule of placental origin plays a pivotal role in pe by antagonizing placental growth factor (plgf). increased sflt and sflt /plgf have been shown to antedate clinical signs in pe. we therefore hypothesized, that the higher risk of pe in nulliparous pregnancies is associated with high sflt (or sflt /plgf). methods: maternal serum samples from nulliparous (n= ) and multiparous (n= ) term singleton pregnancies without pe, at the time of admission to delivery room, were used. serum samples were analyzed for levels of sflt and plgf by elisa. statistical analysis was performed applying t-test and the kruskall-wallis test and using spss software. results: for nulliparous and multiparous pregnancies, the mean serum sflt levels were , ± and , ± , (p= . ), the mean serum plgf levels were ± and ± (p= . ), and the mean ratios of sflt- /plgf were ± and ± (p= . ), accordingly. in a subgroup of multigravidous nulliparous pregnancies, sflt levels were , ± . correcting for maternal age did not alter the results. moreover, results did not differ between multiparous pregnancies with a - years interpregnancy interval compared with a - years interval. conclusions: in nulliparous pregnancies, circulating sflt levels and sflt / plgf ratios are significantly higher than in multiparous pregnancies. these findings suggest that the increased risk of pe in nulliparous pregnancies may involve anti-angiogenic imbalance. nulliparity may be more substantial than primigravity, as a risk factor for pe, suggesting that first semester abortions in primigravidas may not protect from pe in a subsequent term pregnancy. nevertheless, even - years intervals from the previous gestation do not increase the risk for pe. different normograms of angiogenesis should be used, when assessing the risk for pe in multiparous versus nulliparous pregnancies. objective: we determined whether maternal serum levels of angiogenic proteins namely soluble fms like tyrosine kinase (sflt- ), soluble endoglin (seng), and placental growth factor (plgf) -measured during the first trimester are associated with the subsequent development of placental abruption. methods: we performed a prospective, nested case-control study of women enrolled in the massachusetts general hospital obstetric maternal study (moms). first trimester serum samples from placental abruption cases and normal pregnancies were measured for angiogenic factors. cases and controls were matched by body mass index and age. placenta abruption was diagnosed by standard clinical findings and pathological examination of the placenta. women with confirmed preeclampsia or chronic hypertension were excluded. results: compared to controls, cases had more pregnancies, delivered infants at an earlier gestational age and with lower birth weight. first trimester levels of seng were significantly increased in cases compared to controls: . ± . ng/ml vs. . ± . ng/ml, p < . . there were no significant difference in serum levels of plgf, . ± . ng/ml versus . ± . ng/ml, p=ns, although sflt levels were lower in cases: . ± . ng/ml vs. . ± . ng/ml, p=ns. in logistic regression analysis adjusted for age, race, smoking, number of pregnancies, gestational age at delivery, gestational age of blood sampling, and blood pressure at first prenatal, seng levels remained independently associated with subsequent risk (odds ratio . , % ci . - . ) of placental abruption. examining this relationship by tertiles of seng, in the unadjusted model, women in the second (or . , % ci . - . ) and third (or . , % ci . - . ) tertiles were at increased risk of developing placental abruption compared with women in the lowest tertile. after adjusting for known risk factors of placental abruption, women in the second (or . , % ci . - . ) and third (or . , % ci . . ) tertiles remained at increased risk for placental abruption. conclusion: increased first trimester maternal serum levels of seng are associated with increased risk of subsequent placental abruption. ob/gyn, univ of vermont. shear stress is the most potent physiologic stimulus for elevating endothelial no production for flow-mediated vasodilatation. we measured in vivo shear stress during the proliferative and secretory phases and at and weeks of gestation hypothesizing that uterine blood flow (ubf) elevations in turn increase shear stress in early and late gestation. methods: during proliferative, secretory phases, and at and - weeks of pregnancy ua blood velocity and internal radius were measured bilaterally using color doppler ultrasound. blood viscosity was measured at shear rates in excess of /sec. results: compared to the proliferative phase viscosity was decreased at weeks and more so at weeks gestation (p< . ). . ± . . ± . nsd . ± . *** . ± . *** ua velocity (cm/sec) . ± . . ± . *** . ± . *** . ± . *** ubf (ml/min) . ± . ± *** . ± . *** . ± . *** shear stress (dynes/cm ) . ± . . ± . *** . ± . *** . ± . *** internal ua radius was not altered by the menstrual cycle, and was greater at weeks (+ %) and weeks (+ %). compared to proliferative, secretory phase showed significant rises in unilateral ubf and velocity that rose progressively during gestation. in contrast, shear stress increased in secretory ( %) and did not rise further in early pregnancy but by weeks shear stresses was further elevated %. conclusions: equivalent rises in shear stress during the secretory phase and weeks gestation demonstrate increases in radius and profound remodeling of uas that reflect the physiologic process of "normalization of shear". by late gestation, continued but modest rises in radius illustrate that further increases in shear stress occur almost solely due to rises in ubf via falls in down stream impedance. continued rises in shear stress into late gestation provide progressive stimuli for no production by ua endothelium. nih hl , hd , hl background: hypertension during pregnancy is associated with altered uterine vascular reactivity and blood flow, although its effects on arterial myogenic behavior have not been explored. the purpose of this study was to evaluate the effects of hypertension and no inhibition on myogenic tone in pregnancy, as the ability of a vessel to constrict and dilate in response to pressure plays a key role in regulating blood flow to the uterus. methods: three groups of sprague dawley late pregnant (day ) rats were used: control (n= ), hypertensive ( . g/l l-name in the drinking water, n= ), and treated with l-name and hydralazine (also in the drinking water, . g/l, n= ) to prevent the blood pressure increase, yet maintain no inhibition. resistance-sized radial arteries (< m) were mounted in a pressure arteriograph and equilibrated at mmhg (in pss containing l-nna and indomethacin) to induce a myogenic response. vessels were then subjected to pressure steps from to mmhg. tone (%) was calculated by comparing the vessel diameter at each pressure with the passive diameter at the same pressure (determined by incubation with . mm papaverine and m diltiazem). results: myogenic tone developed in controls ( ± % maximal), and was maintained over a broad range of transmural pressures . arteries from the l-name group did not develop tone at any pressure. co-treatment with hydralazine reinstated tone ( ± % maximal) over the same range of pressures as in the control group. the reduction in average placental weights in the l-name group ( vs. mg, p< . ) was restored by hydralazine ( mg, p< . vs. l-name). average fetal weights were also reduced in the l-name group ( . vs. . g, p< . ), but only partially restored by hydralazine co-treatment ( . g, p< . vs. control and l-name groups). conclusions: surprisingly, radial uterine arteries from the l-name group did not develop tone over any range of pressures, despite the fact that matched arteries from late-pregnant controls developed significant myogenic tone. this abolishment of tone was reversed by hydralazine, which also had beneficial effects on fetal and placental growth. these results implicate hypertension rather than no inhibition as the key factor in the suppression of myogenicitiy and dysregulation of uterine blood flow. charles r rosenfeld, timothy roy. division of neonatal-perinatal medicine, ut southwestern medical center at dallas, dallas, tx, usa. background: upbf b rises -fold in ovine pregnancy; but the mechanisms responsible for the rise and maintenance are unclear. we (jsgi ) reported that uterine vascular smooth muscle bk ca k + channels contribute to uterine vasodilation and upbf b maintenance; but up-stream activators are unclear. uterine vascular prostacyclin synthesis increases in pregnancy, but cyclooxygenase inhibition does not alter upbf b (ajp ) . vascular nitric oxide synthase (nos) also increases; but acute inhibition with l-name decreases upbf b only % (jci ) . it is unclear if l-name doses were insufficient or if prolonged nos inhibition has a greater effect. objective: to determine if local nos inhibition with l-name dose-dependently decreases upbf b and if prolonged inhibition exerts a greater effect on upbf b . methods: pregnant ewes were studied at - d gestation age (ga). had doseresponse studies with uterine artery l-name infusions to achieve . - . mg/ml over min. had h arterial l-name infusions to achieve and maintain levels of . mg/ml at (n= ), (n= ) and d (n= ) ga, while measuring arterial pressure (map), heart rate (hr) and upbf b before, during ( , , , , , , and h) and after ( , h) infusions. uterine arterial and venous cgmp were measured. data were analyzed by anova. results: acute nos inhibition decreased upbf b - %, but was not doserelated. h arterial l-name infusions decreased upbf b by - h at each ga (p . , anova) and values returned to baseline by h postinfusion. sensitivity did not differ between ga (p= . , anova), upbf b falling - % at each ga. contralateral upbf b was unaffected at all ga. map rose and hr fell during infusions at and d ga, p . ; but were unaffected at d ga. venous-arterial cgmp concentration differences were seen at d and absent at h of l-name infusion, p= . . conclusions: uterine vascular nos increases in ovine pregnancy, but its inhibition decreases upbf b %, suggesting study doses were insufficient to fully inhibit vascular nos activity. alternatively, nos contributes to the maintenance of upbf b , but other mediators, not yet identified, are more important in activating bk ca and regulating upbf b . notably, l-name reached the systemic circulation, and although further diluted, map rose - %, suggesting the systemic vasculature may be more sensitive to nos inhibition than upbf b . charles r rosenfeld, xiao-tie liu. division of neonatal-perinatal medicine, university of texas southwestern medical school, dallas, tx, usa. background: uteroplacental blood flow (upbf) rises -fold in ovine pregnancy, reflecting increases during pre-implantation, placentation and finally, in the last third of gestation. we reported that bk ca density in uterine vascular smooth muscle (uvsm) increases in ovine pregnancy (sgi ) and inhibition with tetraethylammonium ( . mm) dose-dependently decreases basal upbf % in the last third of pregnancy (jsgi ) . it is unclear how bk ca subunit expression changes and activity is regulated in pregnancy; since uterine vascular nitric oxide is increased, signaling via cgmp-dependent protein kinase g (pkg) is one potential pathway. objective: to determine simultaneous changes in uvsm bk ca density and regulatory -subunit expression and the cgmp signaling pathway for activation in ovine pregnancy. methods: endothelium-denuded segments of nd generation uterine arteries obtained from nonpregnant (n= ), pregnant (n= , - d gestation; term d) and postpartum (n= , - d) sheep were used to measure expression of bk ca -and regulatory -subunits and signaling proteins in uvsm by immunoblot analysis and immunohistochemistry. results: uvsm bk ca density, reflected as a change in the kda -subunit, rose % with placentation (p< . ) and was unchanged thereafter. expression of the kda regulatory -subunit paralleled the rise in bk ca density during placentation, increasing % (p< . ), but increased another -fold and exponentially in the last third of pregnancy (p< . , r = . , n= ). changes in subunit immunostaining in uvsm paralleled increases in protein. although uvsm soluble guanylyl cyclase was unchanged in pregnancy (p= . , r= . , n= ), pkg expression rose -fold (p= . , r= . , n= ) and gradually returned to nonpregnant levels by d postpartum (p= . , r= . , n= ). uvsm cgmp is being measured. conclusions: these are the st data suggesting that increases in ovine upbf during placentation involve vascular growth and bk camediated vasodilation. the rise in upbf in the last third of pregnancy reflects bk ca -mediated vasodilation due to enhanced channel activation via increases in uvsm pkg, bk ca phosphorylation and changes in subunit stoichiometry due to an exponential rise in the regulatory -subunit. it is unclear what initiates and directs these changes in bk ca expression and sensitivity. relaxin (rlx) is a hormone traditionally associated with changes in the female reproductive tract during pregnacy. recent evidence suggests that rlx may play a pivotal role in regulating cardiovascular function during gestation. analogous to pregnancy, administration of recombinant human relaxin (rhrlx) to nonpregnant female rats reduces systemic vascular resistance, as well as increases global arterial compliance. additionally, we demonstrated that, concurrent with rlx´s influence on overall cardiovascular function, small renal arteries (sra) from rhrlx-treated mice and rats are characterized by reduced passive stiffness and increased arterial wall area whereas external iliac arteries (eia) are not. we hypothesized that rlx regulates arterial passive mechanical properties by altering the cellular and biochemical composition of the arterial wall. nonpregnant female mice were administered rhrlx for days after which sra and eia were isolated. we measured arterial collagen, elastin, and total protein using the sircol collagen assay, the fastin elastin assay, and the pierce bca protein assay, respectively. additionally, we quantified arterial smooth muscle cell (smc) density using immunofluorescent techniques. sra isolated from rhrlx-treated mice were characterized by a significant reduction in collagen to total protein ratio ( . ± . vs . ± . μg collagen/μg protein; mean±sem; p< . ) as well as a significant increase in smc density ( . ± . vs . ± . cells/ μm ; p< . ) compared to control mice with no significant change in elastin content ( ± vs ± μg elastin/mg dry weight). in contrast, there were no significant changes in collagen to total protein ratio ( . ± . vs . ± . μg collagen/μg protein), smc density ( . ± . vs . ± . cells/ μm ) or elastin content ( ± vs ± μg elastin/mg dry weight) in eia from the rhrlx-treated mice compared to control mice. of note, comparable results were observed for rlx knock-out (rlx -/-) and wild-type mice with rlx -/mice exhibiting increased arterial collagen and decreased smc density. we conclude that the rlx-induced decrease in passive stiffness of sra that we previously reported is, at least in part, due to rlx-induced alterations in arterial wall cellular and biochemical composition. further, our findings suggest that these vessel wall remodeling effects of rlx are artery-type specific. relaxin is a peptide hormone that emanates from the corpus luteum of the ovary and circulates during pregnancy. this hormone plays an important role in renal vasodilation and hyperfiltration, two fundamental maternal adaptations in pregnancy. chronic administration of recombinant human relaxin (rhrlx) to both virgin rats and mice inhibits myogenic reactivity and increases compliance of small renal arteries, thus further mimicking pregnancy. we hypothesize that these arterial responses to rhrlx are mediated by the lgr , and not the lgr receptor. both lgr and lgr receptor-deficient, and wild-type virgin mice were investigated. the mice were chronically infused with rhrlx or vehicle (veh) for days. small renal arteries were isolated and mounted in a pressure arteriograph and myogenic reactivity was assessed (% change in diameter over baseline in response to a mmhg step increase in intraluminal pressure). small renal arteries from rhrlx-infused lgr wild-type mice showed inhibited myogenic reactivity with a . ± . % increase in diameter whereas the arteries from rhrlx-infused lgr knock-out mice exhibited robust myogenic reactivity with only a . ± . % change in diameter (p= . ). in contrast, myogenic reactivity of small renal arteries was inhibited in both the rhrlx-infused lgr knock-out and wild-type mice. the veh-treated lgr and lgr mice, regardless of genotype, exhibited robust myogenic reactivity. arterial compliance was also assessed for each genotype/treatment group. rhrlx infusion increased arterial compliance of small renal arteries from lgr wild-type, but not from lgr knock-out mice (p= . ). in contrast, the arteries from rhrlx-infused lgr wild-type and knock-out mice showed increased compliance relative to veh-infused animals. conclusion: relaxin-induced inhibition of myogenic reacitivity and increase in compliance of small renal arteries is mediated by the lgr , and not the lgr receptor. smoking is associated with adverse pregnancy outcomes including fetal growth restriction. pathologic effects of smoking on maternal vasculature is a potential mechanism leading to fetal growth restriction. the objective of this study was to determine whether cigarette smoke exposure during pregnancy affects the functional properties of uterine and peripheral arteries using a gravid murine model. study design: pregnant and virgin c bl/cj mice were exposed to whole body side stream smoke using an inhalational chamber for hours/day. smoke exposure was increased from day of gestation until late pregnancy (day - ) with mean total suspended particle levels of mg/m , representative of moderate to heavy smoking in humans. control animals were exposed to ambient room air. late pregnant and virgin mice were sacrificed and uterine, mesenteric, and renal arteries were isolated and studied in a pressure arteriograph system (n= - in each group). plasma cotinine was measured by elisa. means were compared using t-test or analysis of variance. results: fetal weights were significantly reduced in mice exposed to smoke compared to control fetuses ( . ± . g vs . ± . g, p= . ), while litter sizes were not different. cotinine levels in smoking mice were significantly elevated compared to control mice ( . ± . vs . ± . ng/ml for virgin mice and . ± vs . ± . ng/ml for pregnant mice). there was no significant difference in phenylephrine responses between groups. endothelial mediated relaxation responses to methacholine were significantly impaired in both the uterine and mesenteric vasculature of pregnant mice exposed to cigarette smoke during gestation. no difference in endothelial-mediated relaxation was seen in isolated renal arteries in pregnant mice exposed to cigarette smoke, however relaxation was significantly reduced in renal arteries from smokeexposed virgin mice. conclusions: passive cigarette smoke exposure is associated with impaired vascular relaxation of uterine and mesenteric arteries in a gravid murine model. functional vascular perturbations during pregnancy, specifically reduced uterine blood flow and impaired peripheral vasodilation, may be a mechanism by which smoking results in fetal growth restriction. human chorionic gonadotropin (hcg) is essential during early human gestation for "rescue" of the corpus luteum. however, its potential contribution to the widespread maternal vasodilation of pregnancy that occurs at this stage of pregnancy remains uncertain. our objective was to use the renal circulation of conscious rats as an experimental model in which to test the vasodilatory potential of hcg. in addition, we investigated both myogenic reactivity and relaxation responses in small renal and mesenteric arteries isolated from rats, as well as in small human subcutaneous arteries using a pressure arteriograph. we chronically instrumented rats for measurement of renal function. five were ovariectomized, and sham-ovariectomized. after days of surgical recovery, baseline glomerular filtration (gfr) and effective renal plasma flow (erpf) were measured on two separate days and the values averaged. then, an osmotic minipump containing hcg ( iu/min) was implanted s.c. and renal function was again assessed and days thereafter. gfr and erpf significantly increased and calculated effective renal vascular resistance decreased from baseline in the intact (p< . vs. baseline), but not ovariectomized (p=ns) rats on both days and of hcg administration. in the intact, but not ovariectomized rats, plasma osmolality declined and progesterone increased (both p< . vs baseline). plasma hcg concentrations were , miu/ml and comparable in both groups of rats. incubation of small renal arteries from rats with recombinant human relaxin (rhrlx, - ng/ml), but not hcg ( , - , miu/ml) in vitro inhibited myogenic reactivity and relaxed phenylephrine (pe)-constricted arteries. in contrast, both rhrlx and hcg inhibited myogenic reactivity and relaxed pe-constricted small mesenteric arteries from rats and small human subcutaneously arteries. in conclusion, consistent with our earlier work showing a virtually exclusive role for relaxin in mediating the renal circulatory changes of pregnancy, hcg is likely to play little or no role. in contrast, hcg and relaxin are both likely to contribute to the vasodilation of other organ circulations during pregnancy. pierre-andre scott, , michele brochu, jean st-louis. , research centre, chu ste-justine, montréal, qc, canada; pharmacology, université de montréal, montréal, qc, canada. uterine vasculature undergoes major structural and functional changes during pregnancy. estrogens have been shown to induce increased uterine blood flow in this circulation. we have reported that estradiol ( -e ) induced direct vasorelaxant response on smooth muscle of the uterine arteries that, for it major part, is not mediated through tissue nitric oxide. to investigate the cellular effectors mediating this vasorelaxant effect of -e , we set-up uterine arteries of non-pregnant rats in wire myograph systems for microvessels. -e and -e were equipotent in relaxing phe ( μmol/l)-preconstricted uterine arteries, although the later produced significant smaller relaxations. genistein, a phytoestrogen presumed to inhibit phosphatases, also produced uterine artery relaxation with significant lower potency. to try interfere with the vasorelaxant effect of -e , tissues were preincubated with different substances. cycloheximide (protein biosysthesis inhibitor), ici , (estrogen receptor modulator), and kt (pka inhibitor) did not significantly influenced the response to -e . rp- -pcpt-cgmps (pkg inhibitor) slightly displaced the concentration-relaxation curve to -e to the right. inhibitors of potassium channels, penitrem a (bkca) and glybenclamide (katp), showed opposite effects for -e concentration-response curve; the former producing a right shift and the latter a small not significant left shift. these data indicated that the direct acute effect of -e in uterine artery is the result of complex interactions within smooth muscle cells, involving potassium channels, and protein kinases and phosphatases. the renin-angiotensin-aldosterone system is paradoxically activated during pregnancy, since blood pressure decreased. earlier data showed that the high levels of aldosterone present during pregnancy may be involved in cardiovascular adaptation to pregnancy. in order to delineate this effect of aldosterone on vascular tone, potassium canrenoate, an antagonist of mineralocorticoid receptors (mr), was administered ( mg/kg/day) to pregnant rats from the day to of gestation. rats were sacrificed at day , and of gestation together with untreated and day pregnant rats. the thoracic aorta was quickly removed and set up in tissue baths as ring ( - mm) preparation. as observed previously, canrenoate enhanced responsiveness to phe for all time of treatment tested, but only at day ( days treatment) for kcl responsiveness. aortic contractile responses to tea (tetraethylammonium) gradually decreased during pregnancy to almost disappear at the end of gestation. in the present results, canrenoate treatment made the response statistically different by day of pregnancy. finally, the activity of na/k-atpase was measured by the relaxant effect of kcl added to physiological solution without potassium. the activity of the pump was decreased when approaching parturition compare to day of pregnancy. canrenoate treatment abolished this effect. the present data show that vascular changes that occurred during pregnancy are markedly modified by treatment of rats with an antagonist of mineralocorticoid receptors, suggesting that aldosterone may be involved in vascular adaptation to pregnancy. background impaired endothelium-dependent vasodilatation has previously been demonstrated in small myometrial arteries from women with gestational diabetes. this impairment may play a role in mediating the complications observed in diabetic pregnancies. it is not known which mechanisms of endothelium-dependent vasodilatation are affected in myometrial arteries by gestational diabetes. aim to investigate mechanisms of endothelium-dependent vasodilatation in uterine arteries using a mouse model of pregnancy complicated by diabetes. methods diabetes was induced in female c bl /j mice (streptozotocin; mg/kg) prior to mating. mice were culled at day of pregnancy (term) and uterine arteries dissected, mounted on a wire myograph, normalised to mmhg and equilibrated ( °c; %co /air). arteries were constricted with phenylephrine ( m) and a concentration-response curve to the endotheliumdependent vasodilator acetylcholine (ach; . nm- m) constructed in the presence and absence of a nitric oxide synthase inhibitor (l-nna; m), a cyclooxygenase inhibitor (indomethacin; m) or a combination of the two to determine the contribution of nitric oxide (no), prostacyclin and endothelium derived hyperpolarising factor (edhf) to vasodilatation. results sensitivity to ach was comparable between diabetic and vehicle treated mice (ec . ± . nm vs . ± . nm). the contribution of individual endothelium-dependent vasodilators was significantly altered in arteries from diabetic mice. at m ach, edhf-mediated relaxation was significantly reduced (p= . , one-way anova) compared with controls ( . ± . vs . ± ). in comparison, no-mediated relaxation was significantly increased (p= . , one-way anova) compared with controls ( . ± . vs . ± . %). conclusions endothelium-dependent relaxation was not reduced in uterine arteries of diabetic mice compared with controls. however, there was a profound change in the contribution of endothelium-dependent vasodilators in arteries of diabetic mice. this may alter compensatory capacity as disease progresses. supported by mfn training grant (cihr). raf- serine/threonine protein kinase has been extensively studied as the upstream kinase linking ras activation to the mek/erk module. mek/erk has been shown to play a role in the modulation of vascular contraction. however, the role of raf kinase in vascular contraction and its possible involvement in alteration of maternal vascular function during pregnancy is not known. objectives: to determine ( ) if raf kinase contributes to phenylephrine (pe)induced contractile response, ( ) the role of raf kinase inhibitor (gw ) in regulating vascular tone during pregnancy, and ( ) mechanism by which gw produces vasodilatation in rat mesenteric arteries. methods and results: conscious non pregnant (np) and pregnant (p) sprague dawley rats received increasing doses of pe in the absence or presence of gw . pe induced a dose-dependent increase in map in both np and p rats but responses were substantially depressed in pregnancy. gw shifted the pe-induced dose response to the right in both np and p rats. gw itself did not affect basal blood pressure. isometric tension studies in mesenteric arteries showed that gw did not change the kcl-evoked contraction but significantly inhibited the contraction to pe in both np and p arteries. interestingly, at a given concentration, gw produced greater inhibition of pe-induced contractile response in p than in np arteries. also, in p arteries the inhibitory effects of gw were greater as the pregnancy progressed from day to day . raf kinase expression and activity was significantly decreased in arteries of p compared to np rats. in mesenteric vascular smooth muscle cells (vsmcs), pe stimulated activation of raf kinase as indicated by phosphorylation of its immediate target, mek / in a time-and dose-dependent manner. measurement of [ca + ] i with fura- showed that gw -mediated inhibition of pe-induced contraction was not associated with decrease in [ca + ] i . vsmcs treated with pe exhibited higher levels of the contractile proteins, p-mypt and p-mlc which was inhibited by gw . conclusion: to our knowledge, for the first time we show that raf kinase plays an important role in the regulation of vascular contractility. decreased expression and/or activity of raf kinase during pregnancy may explain in part, for decreased systemic vascular reactivity during late gestation. the mechanism(s) that contribute to reduced vascular sensitivity to phenyleperine (pe) during pregnancy is not well understood. pe, in addition to activating the classical contractile pathways, also stimulates growth factor pathways that results in activation of ras/mitogen-activated protein kinases. it is not clear whether these pathways play a role in the modulation of vascular contraction. hence, the present study was designed to determine ) if ras is involved in mediating the pressor response to pe in non pregnant (np) and pregnant (p) rats, ) if so, the mechanism by which ras protein contributes to pe-induced vascular contraction, and ) any differential expression and/or activity of ras during pregnancy that might contribute to altered vascular response. methods: ) mean arterial pressure (map) was measured in conscious np and p rats. ) isometric tension was measured in mesenteric arteries of np and p rats. ) expression of contractile proteins, p-mlc and p-mypt were studied in cultured vascular smooth muscle cells (vsmcs). ) expression and activity of ras in mesenteric arteries of np and p rats were measured by western blot analysis. results: ( ) intravenous administration of pe resulted in a dose-dependent increase in map but responses were substantially depressed in pregnancy. inhibiting ras activation with manumycin, decreased pe-induced increase in map in both np and p rats. manumycin by itself had no effects on basal map. ( ) isometric contraction studies in myograph with mesenteric arteries showed that manumycin shifted pe-induced dose response curve to the right with a decrease in e max in np and p rats. higher concentration of manumycin was required to inhibit pe-induced contraction in np ( μm) compared to p ( - μm) rats. ( ) pretreatment with manumycin inhibited pe-induced increase in the extent of phosphorylation of both mlc and mypt in mvsmcs. ( ) compared to p rats, mesenteric arteries of np rats revealed increased basal and pe-induced expression and activity of ras. reduced expression of this contractile ras pathway might contribute to decreased vascular reactivity during pregnancy. conclusion: ras protein plays a role in regulation of vascular contraction. the decreased amount and activity of vascular ras may be a novel mechanism that explains the reduced vascular resistance during pregnancy. overweight affects the relaxin-induced response of mesenteric arteries. joris van drongelen, arianne van koppen, marc ea spaanderman, paul abm smits, frederik k lotgering. obstetrics and gynecology, radboud university nijmegen medical centre, nijmegen, netherlands; pharmacology and toxicology, radboud university nijmegen medical centre, nijmegen, netherlands. objective: overweight affects pregnancy-induced vascular adaptation and predisposes to gestational hypertensive disease. relaxin plays a key role in normal gestational vascular adaptation by increasing endotheliumdependent vasodilation and compliance, and reducing myogenic reactivity. we hypothesized that overweight blunts the vascular response to relaxin. the vascular responses to flow and pressure of mesenteric arteries after pre-treatment to human recombinant relaxin (rlx) or placebo (plac) were examined in overweight (ow) and normal weight (nw) rats in a pressure-perfusion myograph. overweight was established by a high-fat diet. the endothelium-dependent vasodilatation was measured in response to flow: e (flow inducing % dilatation) and e max (maximal dilative effect). active contractile (myogenic reactivity) and passive dilative (compliance) vascular responses to pressure were determined. all vascular responses were calculated as the proportional change in diameter to % precontraction with u . results: in nw rats rlx decreased e and increased e max to flow and attenuated myogenic reactivity without affecting vascular compliance. in ow plac-treated rats, compared to nw rats, an upregulated vasodilative state was present (decreased e and increased e max , and lower myogenic reactivity). in ow rats rlx increased e to flow and unaffected e max . rlx increased myogenic reactivity mildly in absence of changes in vascular compliance. values are presented as mean±sem; * p< . between nw/ow, # p< . within nw/ow. whereas rlx stimulates endothelium-dependent vasodilation to flow and myogenic reactivity in nw rats, ow overturns the flow-induced response and decreases myogenic reactivity to a lesser extent than present in nw rats. we speculate that these overweight-induced adverse effects of rlx prelude to vascular maladaptation and gestational hypertensive disease. compared to the luteal (lut) phase, uterine blood flow is increased in vivo during the follicular (fol) phase and more so during pregnancy (preg). both of these are physiologic states of high estrogen and shear stress. endothelial cells express enos and produce greater amounts of nitric oxide (no) in response to elevations of shear stress. phosphorylation of enos is a signaling marker of activation. we have recently validated lut, fol and preg uaec culture models for evaluating enos phosphorylation responses to shear stress and vascular mediators. we hypothesized that uaecs derived from fol and preg sheep will show greater enos phosphorylation than lut phase uaecs, and with more robust responses in the presence of estrogen (e ). methods: uaecs were cultured until % confluence, and then subjected to (static control), or dynes/cm for hrs in the absence or presence of e ( nm). western analysis was used to compare optical densities of ser -penos (penos) normalized to total-enos (mean + sem). results: in lut uaec penos was equally increased two fold by and dynes/cm (from . + . to . + . and . + . , respectively). compared to lut uaecs, the static control fol uaecs appeared to have higher penos levels ( . + . ) and this was further increased . - . fold with dynes/cm ( . + . ) and dynes/cm ( . + . ). as seen with fol uaecs, preg uaec static penos ( . + . ) appeared higher than lut uaec, but unexpectedly, neither nor dynes/cm significantly raised these levels of penos ( . + . and . + . ). regardless of shear stress level, e replacement in the culture media only increased the penos levels in the nonpregnant, but not the pregnant derived uaecs. conclusions: increasing amounts of shear stress have a corresponding increase in the ratio of enos that is phosphorylated at serine in lut and fol uaecs. pregnant uaecs do not appear to increase the constitutive ratio of serine phosphorylation of enos at either or dynes/cm . in contrast to our hypothesis, chronic treatment with e for hrs did not augment the ratio of enos phosphorylation in pregnancy. nih hl , hd , hl . cells. honghai zhang, wu xiang liao, dong-bao chen. reproductive medicine, university of california san diego, la jolla, ca, usa. s-nitrosylation (sno) is a rapid, reversible, and nitric oxide (no) dependent post-translational protein modification critical for signal transduction. estrogen stimulates endothelial cell (ec) no production but yet to be determined is if this leads to increased formation of sno-proteins in ec. hypothesis: we hypothesize that estrogen stimulates the formation of sno via a receptor and endogenous no dependent pathway in huvec or ovine uterine artery ec. methods: cells on glass coverslips were treated with mm of no donor gsno or dea nonoate, estradiol- ( nm) in the presence of l-name ( mm) for min. the cells were fixed with methanol. in situ sno-proteins were detected by a rapid biotin derivatization method by blocking thiols by methylmethanethiosulfonate (mmts), followed by ascorbate reduction and labeling with texas red-labeled ethylmethanethiosulfonate (mtsea) and examined by fluorescence microscopy. cells ( x /group) were lysed in hen buffer, and then similarly prepared but labeled with biotin-mtsea. a portion of the samples were separated on sds-page and blotted with anti-biotin antibody for total sno-protein patterns. the biotin-labeled sno-proteins were captured by avidin, followed by immunoblotting with specific antibodies. results: basal sno-protein labeling was apparent in both ec types. exogenous no donated by gsno or dea nonoate significantly increased red fluorescence labeling of sno-proteins in the cytosol of both ec types. treatment with estradiol- also increased total sno-protein labeling in both ec types. pretreatment with l-name significantly reduced sno-protein labeling. sds-page and immunoblotting with anti-biotin antibody analyses identified multiple bands of sno-proteins. in avidin captured biotinylated samples, multiple proteins were indentified. conclusion: exogenous no donated by gsno or dea nonoate and endogenous no upon estrogen stimulation increased s-nitrosylated proteins in ec (supported by nih ro hl and ). background and objective: the renin-angiotensin system (ras) functions both systemically and locally as a primary regulator of blood pressure and fluid volume and is therefore involved in the etiopathogenesis of essential hypertension as well as preeclampsia, a pregnancy-induced hypertensive disorder in pregnant women. while much progress has been made in the development of strategies to block the systemic ras and thus treat essential hypertension, relatively less advance has been achieved in the development of effective local ras inhibitors to treat preeclampsia, primarily due to the fact the conventional systemic ras inhibitors generate potential teratogenic risks to pregnant women during critical stages of their pregnancies. ginger has been widely and effectively used in pregnant women to treat pregnancyinduced nausea and vomiting with minimal adverse effects. the present study was designed to investigate whether ginger could down-regulate renin secretion by human decidual cells (the major source of renin in the tissue-based uteroplacental ras), as an initial step toward a long-term goal to develop potential safe and effective ras inhibitors for use in obstetric patients. methods: full-term normal human placentas were obtained within one hour of vaginal deliveries or cesarean sections. decidual cells were isolated from the decidua parietalis. after an initial culture for days in a serum-containing medium, the decidual cells were treated with ginger juice at various doses in a serum-free medium for hours. the culture supernatants were then harvested and subject to western blot analyses of renin protein contents. the ginger juice used was freshly prepared from the ginger roots purchased from a local grocery store and different batches of juice preparations were standardized based on their optical density values at a wavelength of μm on a spectrophotometer. results: a dominant band of renin at approximately kd was detected in all samples. when compared with the control (i.e. %), ginger juice decreased the renin protein contents in the culture supernatants in a dose-dependent manner. no significant difference in the cell morphology was observed between the control and treatment groups. conclusion: ginger root juice down-regulates renin secretion in human decidua, showing a great potential of a novel ras inhibitor, particularly, in obstetric patients. noninvasive quantification of the autonomic nervous system throughout pregnancy. dietmar schlembach, karoline pickel, daniela ulrich, philipp klaritsch, isa alkan, uwe lang, manfred g moertl. obstetrics and gynecology, medical university of graz, graz, styria, austria; cnsystems medizintechnik ag, graz, styria, austria. introduction: the analysis of heart rate variability (hrv), blood pressure variability (bpv), and baroreflex sensitivity (brs) has become a powerful tool for the assessment of autonomic control. although hrv analysis was initially developed for risk stratification in cardiology, the field of clinical application has broadened in recent years. the aim of our study was to investigate the adaptation of autonomic control during pregnancy based on analysis of heart rate variability and baroreceptor sensitivity. methods: patients with uncomplicated pregnancy were measured with the taskforce® monitor i made by cnsystems. all measurements were performed in supine position under standardized resting conditions. autonomic parameters were recorded at rest and within the "deep breathing method" as a "cardiovascular challenge test". results: throughout pregnancy a shift to higher sympathetic activity respectively a lower parasympathetic activity was observed. , and . ± . [> wks]) (figure ). during the deep breathing maneuver we could show an increase of the sympathetic / parasympathetic ratio (figure ). the noninvasive determination of autonomic parameters throughout pregnancy is possible. these results can be used as basic parameters for classifying and assessing autonomic changes in pathological conditions in pregnancy such as hypertensive disorders. how far the characterization and challenge of these autonomic functions can be utilized for diagnosis and prediction of preeclampsia has to be shown in future studies. hemodynamic parameters measured noninvasively throughout pregnancy. daniela ulrich, manfred g moertl, karoline pickel, philipp klaritsch, isa alkan, uwe lang, dietmar schlembach. obstetrics and gynecology, medical university of graz, graz, styria, austria; cnsystems medizintechnik ag, graz, styria, austria. background: hemodynamic changes throughout pregnancy have been measured predominantly by invasive techniques. the discussion on the valence und the low acceptance of these invasive procedures by pregnant women demands a noninvasive method for evaluating and distinguishing cardiovascular adaptative mechanisms throughout normal and especially complicated pregnancy. method: healthy patients with uncomplicated pregnancy were measured with the taskforce® monitor i (cnsystems, austria) at different time points throughout pregnancy. cardiovascular parameters were recorded in supine and left lateral position under standardized conditions. results: throughout pregnancy an increase of global parameters such as heart rate (hr), blood pressure (bp), total peripheral resistance (tpr) and total peripheral resistance index (tpri) were observed. stroke volume (sv), stroke index (si), cardiac output (co), contractility index (ci), acceleration index (aci), and left ventricular ejection time (lvet) decreased throughout pregnancy (table). discussion: the noninvasive determination of cardiovascular parameters throughout pregnancy is possible and the results of this pilot study can serve as basic parameters for classifying and assessing cardiovascular changes in pathological conditions in pregnancy such as hypertensive disorders. assigning pregnant hypertensive women into hyper-and hypodynamic groups may aid in planning individual therapeutic strategies. objective: both gnrh i and gnrh ii are expressed in humans. gnrh i is produced by the hypothalamus under the regulation of gonadal steroids and stimulates pituitary gonadotropins. during pregnancy gnrh i is also produced by the placenta and affects hcg. gnrh ii, the ancient isoform, is produced by numerous human tissues including immune and reproductive tissues and circulates in blood in quantifiable levels. we have demonstrated that gnrh ii analogs directly affect fertilization and uterine function, and propose that it acts via immune regulation. during pregnancy it is known to regulate numerous hormone productions, yet the levels of gnrh ii has not been reported. in these studies we have determined the circulating levels of gnrh ii throughout pregnancy and in early pregnancy loss. study design: thirty-three women having normal pregnancies were followed prospectively. plasma samples were drawn at , , , , , , weeks gestation and during labor. plasma was also collected from patients have spontaneous abortions (n= ). circulating gnrh ii and crh and gnrh i were measured by specific radioimmunoassays. results: circulating gnrh ii increased from non-pregnant levels ( +/- pg/ ml, mean+/-sem) to +/- pg/ml by -week lmp. gnrh ii concentrations continued to increase through weeks gestation over the -week levels, although a significant increase was only attained by weeks. gnrh ii continued to increase in late term, attaining levels of +/- pg/ml which was significantly higher than that of early gestation. during labor and delivery gnrh ii in maternal plasma was further increased to +/- pg/ml, i.e., . times that of -week gestation ( +/- pg/ml). circulating gnrh ii did not parallel gnrh i in early pregnancy but did in late gestation. gnrh ii did correlate with crh in early pregnancy but not in late gestation. patients having spontaneous abortion had increased circulating gnrh ii at -weeks lmp ( +/- pg/ml) as compared to normal pregnancies ( +/- pg/ml). conclusion: gnrh ii increased throughout pregnancy attaining highest concentrations during labor and delivery. patient with early pregnancy loss had increased gnrh ii expression. the function of gnrh ii or factors affecting this increased gnrh ii in normal or abnormal human pregnancy should be investigated. introduction: plasma levels of the endocannabinoid, anandamide (aea) decrease during the luteal phase of the menstrual cycle and early pregnancy and increase during parturition. high plasma aea levels at weeks in women undergoing ivf-et was associated with a failure to achieve an ongoing pregnancy . what is uncertain is what happens to aea levels when the pregnancy has already failed. we aimed to quantify aea levels in women presenting in early pregnancy with a diagnosed non-viable pregnancy and to compare them to those of a viable pregnancy. methods: plasma aea was measured by a sensitive isotope dilution hplc-ms/ms method from women in early pregnancy ( - weeks) of whom had a viable pregnancy and had a non-viable pregnancy. serum hcg and progesterone were measured in blood samples by standard elisa methods. results: the ages and bmi of both groups were similar ( ± . versus ± . years; mean ± sd; p= . ; student's unpaired t-test and ± . versus ± . kg/m ; p= . ) respectively. plasma aea levels in women with non-viable pregnancies were significantly higher than those in women with viable pregnancies ( . ± . versus . ± . nm; p= . ). there was no correlation found between aea levels and hcg or progesterone levels p= . and r= . ; p= . , respectively) , despite progesterone being significantly lower in the non-viable group ( . ± . versus . ± . ng/ml; p= . ). conclusion: higher plasma aea levels were associated with early pregnancy failure, and this association appeared to be independent of serum hcg or progesterone concentrations. these data suggest that plasma aea levels are linked with pregnancy failure through a mechanism that does not involve hcg or progesterone production. the precise involvement of anandamide in early pregnancy needs to be investigated further. n- ) and arachidonic (aa, : n- ) acids, the major brain long-chain polyunsaturated fatty acids (lc-pufa) are generated by elongation-desaturation of dietary essential fatty acids (efa). the maternal liver is principally responsible for efa elongation-desaturation. objetive. we determined effects of protein restriction in pregnancy on expression of d, d and elongases and (elov and ) in the maternal liver rat. methods. pregnant rats were fed control ( % casein; c) or restricted ( % casein; r) isocaloric diets. at day gestation maternal blood and livers were collected. serum triglycerides, cholesterol and glucose were determinate with the synchron cx autoanalyser and leptin and insulin by ria. liver fat determination was performed by the soxhlet method. liver ara and dha were calculated by gas chromatography based upon retention times from methyl ester standards. liver d, d, elov and mrna were measured by rt-pcr and northern blot. data are m ± sem; analysis by t-test. results. liver weights did not differ in c and r. total liver fat ( ± vs ± mg) serum leptin ( ± . vs ± . ng/ml) and insulin ( . ± . vs . ± . ng/ml) differed in c and r respectively (p< . ). serum triglycerides, cholesterol, and glucose did not differ. desaturase and elongase mrna and % of maternal liver aa and dha were lower in r (fig ) . conclusion. low liver fat content and desaturase and elongase mrna in r indicate impaired lc-pufa synthesis which may adversely impact fetal development, especially the brain. objective: to test the hypothesis that obstetrical dic results from an excessive leak of fetal material into the maternal circulation. a secondary objective was to assess maternal morbidity. methods: all cesarean hysterectomy cases for hemorrhage at our hospital from to were included. intravascular presence of fetal material was determined by two pathologists, blinded to each other and any clinical information. the percentage of those with any fetal material in the maternal circulation was calculated for each diagnosis for hemorrhage. for a given diagnosis, the percentage of intravascular fetal material in those with the given diagnosis was compared to those without that diagnosis using fisher's exact test. most patients had multiple hemorrhage diagnoses. a two sample t-test was used to evaluate the difference in mean blood loss between those with and without intravascular fetal material. results: primary outcome* % of patients received blood products and % received clotting agents. the average blood loss was cc. there were no maternal deaths and injuries to adjacent organs, all to the bladder. conclusions: there was no association between the presence of intravascular fetal material and any specific hemorrhage diagnosis or amount of blood loss. although the power to detect a relationship was low, the excessive leakage of fetal material as a specific and exclusive mechanism of obstetrical dic, as postulated, seems unlikely. while % of the population was transfused, there were few intraoperative complications and no maternal deaths. hypertension, tel-aviv university, sheba medical center, ramat-gan, israel. objective: the joint national committee on high blood pressure (jnc ) determined blood pressure of - / - in adults to be prehypertension that requires health promoting life style modifications to prevent cardiovascular disease. similarly, we hypothesize that gestational prehypertension in pregnancy is associated with increased risk of pregnancy complications and its management would improve pregnancy outcome. methods: prospective and retrospective recruitment resulted in a study group consisting of patients who were diagnosed in the first and early second trimester (< weeks) with blood pressure of - / , and a control group consisting of patients with blood pressure < / at similar gestational age. comparisons between the two groups were accomplished for demographic characteristics and outcome measures using the chi-square test statistic and two-sample t-tests for categorical and continuous variables, respectively. results: all outcome measures analyzed resulted in poorer results being associated with the study group compared with control as follows: % versus % of the study and control group pregnancies, respectively, experienced preeclampsia (p= . ); and % versus % of the study and control group, respectively, were associated with gestational hypertension (p= . ). % of the control group deliveries were associated with admission to the nicu, compared to only % in the control group (p= . ); % versus % of the study and control group deliveries, respectively, were preterm (p= . ); twenty-nine percent of the study group were cesarean deliveries, compared to % in the control group (p= . ). on admission mean sbp and dbp was and mmhg, respectively, in the study group, compared to and mmhg in the control group (p= . for sbp and p= . for dbp). conclusions: "gestational pre-hypertension" may be a real pregnancy condition that when is recognized, physician attention, patient close monitoring during prenatal care and delivery and early intervention in patients at risk, may all promote improved pregnancy outcome. larger scale study is in progress to evaluate and confirm these findings in the larger pregnant population. are contractions at - weeks gestation less painful than those at full term? kristy a ruis, karin blakemore, abimbola aina-mumuney, valerie jones. gynecology and obstetrics, johns hopkins hospital, baltimore, md, usa. objective to determine if gestational age plays a role in severity of pain associated with uterine contractions. study design self-reported pain from women in labor, on a scale of - , is assessed and recorded in an obstetrical database by nurses at regular intervals at two hospitals within our medical system and was retrospectively reviewed from - . pain at various dilatations ( - cm) of women who were laboring and then delivered at - weeks' gestation was compared to women in labor at term. maternal demographics of age, race, education level, bmi, presence of support person, request for analgesia at any point in labor, and epidural placement were abstracted from maternal records. categorical data were analyzed using chi square or fisher exact test; continuous variables using student t-test. results laboring patients between - weeks gestation and at term were identified. term and preterm patients did not differ with respect to maternal demographics. pain reported by preterm patients was significantly less at - cm dilatation ( . vs. . ‚ p= . ) and significantly greater at - cm ( . vs. . ‚ p= . ). pain reported at - cm and - cm was not statistically different between the groups. of note, fewer of the preterm patients received an epidural despite the request for analgesia. conclusion preterm laboring patients between - weeks gestation may perceive less pain at - cm dilatation compared to term laboring patients; however, their pain perception at advanced dilatation was comparable to those at term despite the fact that they were less likely to have an epidural in place at the time of pain evaluation. in light of these findings, the widely accepted etiology of labor pain as the result of cervical dilatation may need to be re-examined. also, factoring in the patient's discomfort level into the evaluation for onset of early active labor may not be valid in the preterm patient. background and objective: asthma is a risk factor for adverse pregnancy outcome. this has been attributed to the effect of allergy in pregnancy. mast cell mediators can induce uterine contractility. the objective of the study was to test the hypothesis that pregnant women with asthma have different uterine electrical signals from those generated by normal pregnant women. materials and methods: uterine electromyography (emg) recordings from gestational age matched pregnant women at term were analyzed. the cases consisted of patients with asthma and the controls, those women without asthma. emg was recorded for approximately minutes from surface electrodes placed upon the maternal abdomen, with the electrical signals filtered in the uterine-specific range of . to . hz to remove noise components. the recordings were analyzed in their entirety first by power spectrum analysis and then by lyapunov analysis. the power-spectrum-largest-peak frequency and the largest lyapunov exponent were calculated for each patient, and then the mean and standard deviation of these parameters was compared using student's t-test. results: patients with asthma had significant lower power spectrum frequency and higher lyapunov exponent than women without asthma. see ) the power spectrum frequency and the lyapunov exponent used to analyze uterine emg signals were different in women with and without asthma; ) the results suggest that uterine electrical activity is altered in women affected by asthma; ) these findings may explain the observed increased frequency of preterm birth in women with asthma. further studies are required to dissect the mechanisms. fetal microchimeric cells trafficked into the maternal circulation persist in blood and tissues for years after pregnancy. increasing data suggest that microchimerism occurs after every pregnancy, but the biological role of fetal microchimerism or the cell types involved is unclear. while persistent fetal cells were initially implicated in autoimmune disease, animal studies suggest these fetal cells play a broader role in response to tissue injury. methods: appendix specimens were acquired from women undergoing appendicectomy during pregnancy. detailed reproductive histories were obtained. fluorescence in situ hybridisation (fish) with two different probes allowed investigation of the presence of male presumed-fetal cells and nested pcr amplification of sry gene confirmed male dna in the appendix. immunostaining was used to determine the fetal cell phenotype. results: male cells were identified in appendix tissues from women with known male pregnancies (n= ) and also from a woman with no sons and a previous miscarriage of undetermined gender (n= ). no male cells were observed in the control (n= ), a woman with daughters. male cells of presumed fetal origin were evenly distributed in the muscle, mucosa and submucosa layers of the appendix. morphology and co-localisation analysis suggested the identified male cells had differentiated primarily into muscle cells or lymphocytes. combined immunostaining and y-fish demonstrated male desmin+ muscle cells and cd + and cd + lymphocytes. finally, the presence of male dna in the appendix specimens was confirmed by nested pcr. male cells of presumed fetal origin were identified in the appendix of pregnant women. microchimerism frequency varied according to the reproductive history and the degree of inflammation. microchimerism rates were higher in the appendix from women with current male pregnancies than in those with previous male pregnancies and were higher in those with histological acute inflammation, when compared to milder cases. male microchimeric cells identified were of haematopoietic and mesenchymal origin. this study suggests that fetal cells are present in sites of tissue injury and may participate in tissue repair during pregnancy. collagen i and collagen-binding integrins mrna expression in rat cervix during gestation. huiling ji, tanya l dailey, vit long, edward ks chien. obstetrics and gynecology, maternal fetal medicine, women and infants' hospital of rhode island, providence, ri, usa. objective cervical remodeling is associated with cell proliferation and extracellular matrix (ecm) remodeling. integrins are a family of multifunctional cell adhesion receptors which mediate ecm-cell interactions including binding collagen. of the integrin (i) heterodimers, , , and are primary receptors for collagen. the predominant cervical collagen is collagen type . the purpose of this study is to investigate collagen and integrin expression in the pregnant rat cervix. the cervix was harvested from timed pregnant sprague-dawley rats. non pregnant (np)and timed pregnant day , , , , and animals were euthanized using a protocol approved by the iacuc. four animals were sacrificed on each day of gestation. quantitative rt-pcr was used to evaluate mrna expression and normalized to -actin. a standard curve was generated from a single sample. data was analyzed using anova and multiple comparisons testing. collagen i mrna expression increased through mid-gestation but decreased to np levels on day . a similar pattern was seen with the integrin beta subunit. the pattern of integrin alpha subunit expression was different for each subunit during pregnancy. the changes in collagen, integrin alpha , , and integrin beta were found to be statistically significant. (figure) . the pattern of collagen binding integrin alpha subunit expression during the rat gestation appears to vary independently of each other. the expression of the subunit paralleled col expression. collagen binding integrins may play independent roles in signaling and biomechanics during gestation. funding: women infants' hospital research fund; phs nih-ncrr p rr p rr . background. the effects of chemotherapy on human fetal development are poorly studied, mainly due to the lack of adequate animal models in which placentation and embryological development resembles humans and pharmacokinetic studies, prenatal sonography as well as histological studies can be performed. aim of the study. to test the baboon as model for studying pharmacokinetics and fetal effects of chemotherapy administered during pregnancy. methods. experiments were performed in pregnant baboons at a mean gestational age of (+/- ) days. detailed ultrasound examination (biometry, doppler, screening) was performed days before, at and day after the drug administration. the administration of different schemes of chemotherapy and the fetal samplings occurred under endotracheal anaesthesia. maternal blood samplings, as well as percutaneous ultrasound guided fetal blood ( ml) and amniotic fluid ( ml) samplings were performed at least once immediately after drug administration. in case of fetal demise, the fetus underwent detailed macro-and microscopic examination. in the other animals mother and fetus were euthanized h after the experiments, with collection of blood, amniotic fluid and tissues for further analysis. results. all mothers survived the experiments, one mother developed paralitic ileus. none of the fetuses died during the acute phase of the experiment but / ( %) fetuses died within h following the experiment. none of the animals showed significant clinical or histological signs of infection or anaemia. a total of ultrasound examinations were performed in fetuses, allowing the creation of sonographic growth charts in this model. at the time of drug administration the mean maternal weight was . kg (range . - . ) and the estimated fetal weight was gr (range - ). sixteen out of cordocenteses ( %) and all amniocenteses ( %) were successful in obtaining the required samples for analysis. conclusion. the pregnant baboon can be used as a model for studies on pharmacokinetics and fetal effects of chemotherapy administered during gestation. prenatal ultrasound is comparable to the human situation, and amniocentesis and cordocentesis can be performed with a high success rate and short term survival. therapy to prevent preterm birth is limited". moreover, -hydroxyprogesterone ( ohp) caproate is a category d drug according to the fda (evidence of fetal harm). p is produced in the adrenal glands, gonads and brain. after weeks gestation p is secreted from the placenta independently to the mother and the fetus. p is the precursor of aldosterone and after conversion to ohp, of cortisol and androstenedione. baboons have similar reproductive system and development to humans and are used to study mechanisms of labor and prevention of preterm labor. currently, there are no normative data on the concentration of p, ohp, cortisol ©, estradiol (e ) or inhibin (i) throughout their pregnancy. therefore, a doseresponse or the teratogenic effects of p or ohp caproate cannot be studied in a controlled manner in this animal model. the aim of this study was to generate reference values for these hormones during baboon gestation and early postpartum life. material and methods: hormonal quantitative measurements were performed (immulite analyzer) results: the mean concentrations of these hormones are depicted in figure (maternal serum, from conception to term days) and figure (newborn serum) conclusion: this study provides reference values for hormones quantified during the baboon gestation and early postpartum life. this may be useful for future research concerning the effects of p and ohp administration during pregnancy. ( )non-pregnant women eligible for ivf treatment and ( )pregnant women receiving prenatal care. the pre- / samples were drawn in the year prior to / / ; the post- / samples were drawn within months after. the pre- / and post- / normal pregnant samples were drawn at + weeks' gestation. the non-pregnant samples were from ivf candidates with serum e levels< newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced pg/ml and fsh serum levels< . miu/ml. the samples were assayed for acth and cortisol by a commercially available immulite™ system. hsp and hsp were measured by commercially available elisa. these results were compared in the patient populations before and after / . t-tests were used for analysis. statistical significance was set at p< . . results: in the pregnant subjects, there were no statistical differences in the mean levels of acth, cortisol, and hsp pre-and post- / (table ) . only serum hsp levels were significantly increased in the pregnant women post- / .* mean serum acth, cortisol, hsp , and hsp levels were all significantly different in non-pregnant subjects pre-and post- / . conclusions: except for hsp , serum markers for stress were unchanged in pregnant subjects after the stress of / / in comparison to non-pregnant women whose serum hsp , hsp , cortisol, and acth levels were significantly different. the lack of change in serum markers of stress in pregnant women suggests that the hormonal milieu of pregnancy may buffer against acute environmental stressors. objective: in human pregnancy, maternal body composition provides an indicator of maternal nutritional status and metabolic capacity. previously, we reported that ß hsd- activity was significantly reduced in term placentas from thin women and women with a smaller mid-upper arm circumference before conception. this suggests that these fetuses may have been exposed to inappropriate levels of maternal cortisol, which is a mediator of gestation length. in this study, we hypothesize that the duration of gestation will be shorter in women who tend to be thin and have a lower mid-upper arm circumference prior to conception. methods: within the longitudinal, population-based southampton women's survey (sws), analyses were performed on women whose estimated date of conception was set using an algorithm that combined menstrual and early ultrasound scan data and who had a spontaneous onset of labour and delivered after weeks of gestation. linear regression was used to examine the relationships between maternal body composition and the duration of gestation. results: within the women surveyed, lower maternal body mass index, mid-upper arm circumference and arm muscle area before conception were all associated with shorter duration of gestation at delivery (r= . , p= . ; r= . , p= . ; and r= . , p= . , respectively). a lower subscapular skinfold thickness, sum of skinfolds and height were also associated with shorter duration of gestation (r= . , p= . ; r= . , p= . and r= . , p= . , respectively). mother's age, own birthweight and ratio of subscapular/triceps skinfold thickness were not related to the duration of gestation. conclusions: in this study, we found that thinner women with a lower midupper arm circumference and arm muscle area tended to have a shorter duration of gestation. our findings of shorter gestation length in thinner women are in keeping with our previous observation of lower ß hsd- activity in term placentas from thinner women. we conclude that metabolic capacity prior to conception could influence duration of gestation through mechanisms that include alteration in placental metabolism and fetal cortisol exposure. fecal incontinence (fi) is a debilitating condition affecting - % of the us. our prior studies found that % of women report new onset fi after childbirth. the goal of our study was to examine the impact of fi on postpartum quality of life (qol). women reporting fi on a statewide survey who agreed to participate in a -year study of qol were included in the analysis. women were considered to have fi based upon the nih definition of fi. the quality of life survey was based upon the uebersax incontinence impact questionnaire and was administered every months for years. qol in women with fi was examined using chi square, and impact of severe fi (stool incontinence) was determined by multivariate logistic regression. results women with fi were surveyed and of those, ( %) returned at least survey during the study period, completed all survey questions, and were included in the final analysis. among women with fi, % felt frustrated due to fi, % reported fi impacted their emotional health, . % reported fi impacted child-caring abilities, and . % reported a negative impact on social activities. qol was similar across survey periods. one out of three women with fi reported severe symptoms (incontinence of stool). women with severe symptoms were - times more likely to report negative impacts on qol compared to milder (e.g. flatus) fi after adjusting for age, parity and urinary incontinence. . % felt their stool was stored elsewhere before bowel movements, . % reported using digital defecation and more than half ( %) reported symptoms of urinary leakage. despite the substantial impact on postpartum quality of life, few women sought medical help with only % of women at months, . % at year, and . % at years ever reporting their symptoms to a medical provider. postpartum women report that fecal incontinence has a substantial negative impact on their qol after delivery including their emotional health and ability to care for their newborn. despite this profound impact, few women will discuss fi with medical providers. these data suggests there may be a benefit for providers to inquire about fi at postpartum visits. objective: the objective of this study was to determine what characteristics contribute to racial/ethnic differences in breastfeeding rates. study design: a retrospective cohort study of all women who delivered a viable infant (n= , ) was conducted. the primary outcome was breastfeeding upon discharge of the hospital. we first examined the association between race/ ethnicity and breastfeeding. next, we conducted stratified analyses examining a variety of predictors of breastfeeding within the racial/ethnic groups including maternal demographics, obstetric interventions, and perinatal complications. results: we found that both asians (or . , % ci . - . ) and blacks ( . , % ci . - . ) had statistically significant lower rates of breastfeeding, while latinas (or . , % ci . - . ) showed a trend towards higher rates of breastfeeding. in latinas, we found that rd and th degree lacerations were significantly associated with lower rates of breastfeeding, but were not predictive of breastfeeding in the other racial/ethnic groups. epidural use was predictive of lower rates of breastfeeding in caucasians and blacks, but was not predictive in latinos and asians. some of the other predictors which differed between the racial/ethnic groups were obesity and induction of labor (table ) . conclusion: race/ethnicity is significantly associated with breastfeeding, with blacks and asians having the lowest rates of breastfeeding at discharge. a variety of other factors are associated with breastfeeding and interestingly, their effect appears to differ between the racial/ethnic groups. future studies might elucidate the sociocultural and biomedical reasons that explain the differences. these results help focus our efforts during the peripartum period to advocate for mothers who have factors that might impede breast feeding. epidemiology, erasmus medical centre, rotterdam, netherlands; pediatrics, erasmus medical centre, rotterdam, netherlands; public health, erasmus medical centre, rotterdam, netherlands; clinical genetics, erasmus medical centre, rotterdam, netherlands. background recommendations on folic acid use to prevent neural tube defects are launched in several countries. however, the adequate use of folic acid supplements during the periconception period seems to be low. objective to assess the prevalence of adequate folic acid use, defined as the preconception start of supplements, and to identify its determinants in a multi-ethnic population. design the study was embedded in the generation r study in rotterdam, the netherlands, a population-based prospective cohort study from early pregnancy onwards. methods from all women in the cohort who delivered between april and january information on folic acid use and potential determinants was obtained by questionnaires and physical examination. logistic regression models were used to identify determinants of periconception folic acid use. results. data from , pregnant women were available. of all women % adequately used folic acid supplements. the most important risk factors for inadequate use were unplanned pregnancy (or . , p< . ), nonwestern ethnicity (or . , ci . - . , p < . ) and a low educational level (or . , ci . - . , p< . ). other risk factors were single marital status, smoking, multiparity (all p< . ) and alcohol use (p< . ). prior spontaneous abortion was associated with increased adequate folic acid use (p< . ). conclusion adequate periconceptional folic acid use is low. improved preconception care and public health education programs are necessary to improve the uptake of folic acid. although methamphetamine (ma) use has been front and center of the united states drug control policy since , little attention has been given to ma use in pregnancy, despite the fact that pregnant admissions to drug treatment for ma have been rising sharply. to determine trends in the prevalence of ma treatment admissions during pregnancy, we undertook a secondary analysis of the treatment episode data set (teds), an administrative data set that captures at least % of all known treatment admissions in the us. in particular, we investigated risk factors for ma use and how these characteristics have changed over time. demographic, geographic and substance use data were collected for the , pregnant admissions captured between and . logistic regression models were constructed by year. confounding was assessed via backwards elimination with a change-in-estimate criteria of . considered substantial. trend results were reported as adjusted proportions and represented graphically. overall ma prevalence, reported as the primary drug of use upon admission, rose from . % in to . % in . although white women had times the odds of using ma in (adjusted or ( %ci) . [ . , . ]), this had dropped to . [ . , . ] by , mostly due to an increase in latina ma use in pregnancy. pregnant women admitted for ma had fewer prior treatment admissions, were more likely to be unemployed, and less likely to use alcohol or marijuana. we found great geographic variability in use. ma was more common in the pacific region and least common in new england. there was little change in regional variation over the study time period. less is known about the perinatal effects of ma compared with other substances, yet it is the primary drug of choice for pregnant women admitted into treatment. as pregnant women occupy a unique place in drug treatment, analysis of national trend data is essential to guide both policy and research. background: epidemiologists have grouped the multiple disorders that lead to extremely preterm delivery in a variety of ways. we sought to identify characteristics that would support the combining or dividing of the disorders that lead to preterm delivery. methods: we enrolled , women who delivered a live born singleton infant between and completed weeks gestation at tertiary centers in the united states. each delivery was classified according to the complication that prompted presentation: preterm labor, preterm premature rupture of newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced fetal membranes (pprom), preeclampsia, placental abruption, cervical incompetence, and fetal indication/intrauterine growth restriction (iugr). we compared these entities on the frequency of characteristics identified by standardized interview, chart review, histological examination of the placenta, and culture of placenta parenchyma. results: the percents of women who presented with each antenatal complication were: preterm labor ( ), pprom ( ), preeclampsia ( ), placental abruption ( ), cervical insufficiency ( ), and fetal indication/iugr ( ). after considering antecedents and correlates of the processes leading to preterm delivery, we observed two overarching epidemiologic patterns. the first pattern, characterized by recovery of organisms from the placenta and by histologic chorioamnionitis, tended to be associated with preterm labor, pprom, placental abruption, and cervical insufficiency. the second pattern, characterized by a paucity of organisms and inflammation and the presence of histologic features of dysfunctional placentation, tended to be associated with preeclampsia and fetal indications/iugr. conclusions: disorders leading to preterm delivery can be categorized broadly into two groups: those associated with intrauterine inflammation and those with aberrations of placentation. predictors of compliance with tuberculosis screening in pregnancy. nadav schwartz, sarah wagner, sean keeler, may tam, julian mierlak, , aaron caughey. obstetrics and gynecology, nyu school of medicine, new york, ny; obstetrics and gynecology, ucsf, san francisco, ca; obstetrics and gynecology, gouverneur health care services, new york, ny. objective: poor compliance with tuberculosis screening and treatment is a major obstacle to the containment of this disease. we sought to identify predictors of ppd and cxr compliance during pregnancy. methods: a retrospective cohort study at a single institution which serves a largely immigrant population in nyc, from november , through june , . data on maternal age, ethnicity, country of origin, level of education, ppd and cxr status were collected. results: of the , pregnancies, asian and hispanic race/ethnicities accounted for . % and . % of the population, respectively, with . % being us-born. the mean age was . years and the overall ppd+ rate was . %. there was % non-compliance with ppd testing, and % of ppd+ patients were non-compliant with their chest x-rays. asian women were more likely to be ppd-compliant than hispanic or caucasian women. ppd+ asian women were also more likely to be compliant with cxr. us-born women were significantly less likely to be compliant with their ppd or with their cxr. women > years old were less likely to be compliant with their cxr, while women with an elementary school education or less were more likely to be compliant. (see table for odds ratios and %ci) conclusions: age, education, immigrant status and other cultural and ethnic factors appear to play a role in compliance with tuberculosis screening. further elucidation of these effects may help clinicians target at-risk subpopulations and improve overall compliance, working towards better control of this disease. background: in the us, differential outcomes in cervical cancer among medically underserved women are linked to multiple barriers impacting loss to follow-up and failure or delay in diagnostic resolution. prior studies have found risk factors for acquisition of hpv and for inability to obtain a pap smear. no study has explored health literacy and physician communication as a key factor. methods: this research represents the formative phase of a randomized controlled trial of african american (aa) and hispanic women aimed to improve communication and abnormal pap smear follow-up in chicago. semi-structured interviews and focus groups were conducted face to face in spanish or english. each interview lasted minutes, and each focus group lasted - minutes. we recruited patients ( hispanic, aa) and providers from a purposive sample representative of two large clinics that serve low-income women. results: all interviews were transcribed by two investigators. each interview was coded by two investigators separately and then a third investigator reviewed the transcripts and coding to achieve triangulation. codes for these themes were developed and the responses were tabulated using the coding scheme. atlas ti was utilized to analyze all qualitative data. from these data, we uncovered provider-patient challenges in cancer communication including: the providers' trade off between medical accuracy and/or literacy when communicating with their patients. for the patients, the word cancer was important to hear since they wanted the truth and needed to hear this word in order to encourage them to respond more quickly. however, many providers believed that the word cancer was too "scary" and to "extreme" of a word that may communicate too much exaggerated information. both the hispanic and aa patients did not seem to differ in their responses. conclusion: results exemplify not only the importance of health literacy and patient provider communication, but demonstrate the wealth of information gained from qualitative research-a method of data collection seldom utilized in ob/gyn investigations. funding: women's reproductive health research award: hd - ; r ca (spring). population. kristine beckers, isabelle guelinckx, greet vansant, roland devlieger. obstetrics and gynaecology, university hospital gasthuisberg, leuven, belgium; clinical nutrition, katholic university leuven, leuven, belgium. objective: to generate reference charts for weight gain during pregnancy for the different bmi-categories (underweight, normal weight, overweight, obesity), based on recent data in a homogeneous caucasian population. methods: in a retrospective study at the department of obstetrics of the leuven university hospital (belgium), weight gain and pre-pregnancy bmi were determined in belgian pregnant women with accurately dateable, uncomplicated singleton pregnancies. centile curves for the different bmicategories were constructed using of the linear mixed model, one set of charts based on the absolute weight gain, another set based on the relative (expressed as percentage) weight gain. the effect of parity on weight gain was examined. results: overall mean weight gain was . ± . kg ( . ± . lbs). mean weight gain was . ± . kg ( . ± . lbs) in the underweight population, . ± . kg ( . ± . lbs) in the population with normal weight, . ± . kg ( . ± . lbs) in the overweight population and . ± . kg ( . ± . lbs) in the obese population. weight gain (pattern and amount) of the underweight and normal weight patients differed significantly of the overweight and obese patients. parity had a statistical, but no clinical significant influence on amount and evolution of weight gain. conclusion: by using strict inclusion criteria, bmi-category-specific reference charts were generated representing the optimal gestational weight gain, rather than the mean weight gain. this enables the weight charts to be used as a clinical tool during the counselling of pregnant women. further studies are required to assess the effectiveness of this clinical tool. female fshr(-/-) mice are sterile, because of a block in folliculogenesis at the primary follicle stage, show decrease in e and elevated fsh. this animal model is an appropriate model for studying hypergonadotropic ovarian dysgenesis and infertility, caused by c t mutation in fshr gene. objective: to investigate the effects of bmt on serum hormonal levels, follicular maturation and fertility of fshr(-/-) mice. methods: fshr (-/-) mice at - weeks of age were randomized into treated versus control groups.bm from syngenic female donor was injected into the tails of recipients. control group received vehicle alone. vaginal smears were collected, body weight was measured daily. sample animals were sacrificed at , , , , and weeks post bmt. all organs were weighted and examined by h e. fsh, e , and p were measured before and after treatment. for donor cell tracking, dna was extracted from various organs. specific primer sets were designed for normal and mutant hfshr gene. pcr amplification was done and pcr products were analyzed in % agarose gel. results: total body weight significantly increased in treated animals(p< . ). significant increase in both the total number of follicles, and the collective diameter of the follicles in treated animals observed(p< . and p< . ). six out of treated animals showed estrogenic changes in daily vaginal smear. e level increased . - . times and fsh level dropped to % in treated animals. normal (donor allele) fshr gene was amplifiable in out of recipient mice, and was detected only in the ovaries and uterus but not in any other tested organs.control group did not show any changes in vaginal smear, hormonal level, and normal fshr allele. conclusion: intravenously injected syngeneic bone marrow cells were able to home to the ovaries of female fshr (-/-) mice and restore folliculogenesis and resume steroid hormone production. potential mechanisms for these observations will be discussed. conclusion: neural stem cells (nscs) give rise to progenitors, which expand by rapid proliferation until cell cycle arrest followed by differentiation along one of cns cell lineages: neurons, astrocytes and oligodendrocytes. increased differentiation of neuronal cells with ins and even more with leptin suggests that iugr-associated reduction of these growth factors may contribute to impaired hypothalamic pathway development. objective: to develop a technology of modulating hucb in order to achieve neuronal cells for future potential replacement of damaged neuronal tissue. design: we developed a two-dimensional ( d) tissue culture technology for isolation and differentiation of collagen-adherent hucb neural progenitors (hucbnps). we further used the extracellular matrix protein collagen, organized in a three-dimensional ( d) gel, supplemented with neuronal conditioning medium and nerve growth factor (ngf), to facilitate ex-vivo long term neuronal differentiation of hucbnps. we developed a stable green fluorescence protein (gfp)-pc cell model, to be used as a positive control for monitoring neuronal outgrowth for proper evaluation of the hucbnps growth in the d scaffold, mimicking a neural tissue organization. results: our experimental data indicate that d collagen environment is neuronal biocompatible, supporting attachment, long-term survival, proliferation and differentiation of both hucbnps and gfp-pc cells. conclusions: improvement of the d technology with cultures of hucbnps that is in progress in our laboratory might be the first step in validating the concept for the feasibility of generating a neuronal d tissue construct for future potential treatment of neurodegenerative disorders. piane, justin tsai, gnanaratnam giritharan, emin maltepe, paolo rinaudo. reproductive sciences, university of california san francisco, san francisco, ca, usa. objective: ivf embryos are often used to generate stem cells. however, it is unknown if stem cell lines originated from in vitro generated embryos are different from stem cell originated from in vivo embryos. trophoblastic cells, due to their external position within the embryo, are most susceptible to environmental factors encountered in vitro. furthermore, our previous data showed that trophoblast transport functions may be impaired after culture in vitro and that the number of trophoblastic cells is reduced in the embryo after ivf. in this study, we assess the differentiation characteristics of ts cell lines obtained from in vivo and in vitro fertilized embryos. methods: oocytes were isolated from superovulated c bl/ j female mice and in vitro fertilized with sperm from male c bl/ j mice. the resulting late-cavitating blastocysts were harvested. in vivo controls were obtained by flushing the blastocysts from the uteri of superovulated pregnant mice days post hcg. ts cells from the two groups were allowed to develop and maintained in vitro in the presence of fgf and heparin, using a feeder layer of human placental fibroblasts. ts cells were then allowed to differentiate without fgf and heparin, fixed on day , stained with -tubulin and zo- antibodies and then observed under fluorescence microscopy. three ts cell lines per group were analyzed and their differentiative capacity was evaluated using morphological criteria. results: in vivo and ivf derived ts exhibit a similar differentiation pattern. in particular, the number and timing of trophoblast giant cells and spongiotrophoblasts derivation is similar in the two groups. there are no obvious abnormalities in the immunologic staining morphology of the different cell lines at different time points. conclusion: there are no apparent morphological alterations in ts cells lines derived from ivf embryos as compared to in vivo embryos. this finding in an animal model increases our confidence in the reliability of human stem cells derived from ivf. as a further investigation, markers of trophoblast giant cells, spongiotrophoblast, syncytiotrophoblast and chorionic trophoblast cells will be examined and compared in the two different groups by northern blot hybridization. genomewide high density snp-cgh reveals several new deletion copy number variants on the x chromosome in pof patients. erik ah knauff, cisca wijmenga, ruben van 't slot, lude franke, bart cjm fauser. reproduction gynecology, university medical centre, utrecht, netherlands; complex genetics group, university medical centre, utrecht, netherlands. introduction: around % of women have a post-menopausal hormonal profile before age , referred to as premature ovarian failure (pof). pof could act as a genetic model for accelerated follicle loss and may render useful information about the polygenetic background of major individual differences in menopausal age. macrodeletions on the x chromosome are associated with pof but karyotyping has a maximal resolution of mb. when using genotyping whole genome arrays it is now possible to detect submicroscopic deletions and duplications (copy number variants (cnv)) up to kb effective resolution. we have screened for microdeletions on the x chromosome in a well-phenotyped cohort of pof patients. methods: our study included caucasian, ,xx patients with spontaneous secondary amenorrhea before age , fsh > iu/l and absence of (low) x/ xx mosaicism and fmr premutations. dna analysis was performed using illumina k cnv arrays containing , probes. , probes were located on the x chromosome ( probe for every kb), of which , probes were specifically designed to detect known cnvs. ten samples with call rates < % were removed from the analysis and one sample gave inconsistent x probe intensities. we screened for deletions ( kb) using an algorithm detecting at least five consecutive probes with intensities (logr > - . ) below the mean probe intensity. we identified a total of x chromosomal microdeletions, divided in loci and varying between - kb in size. of the samples showed at least one deletion on the x chromosome. % of the identified deletions had already been recorded in the database of genomic variants. in the newly identified newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced loci, we found coding regions containing genes. eight of these are established or potential pof candidate genes, including five that are clustered on the terminal xq critical pof region. conclusions: we observed abundant variation in the cnv regions, a proof-of-principle for this specially designed cnv-chip. snp comparative genomic hybridization revealed possible new deletions specific to pof on the x chromosome. data on duplications, validation and whole genome cnv analysis, compared to a control cohort, will become available soon and will be presented at the sgi annual scientific meeting. unexplained intrauterine fetal death (iufd) is associated with long qt syndrome. irene cetin, patrizio antonazzo, sabrina cozzolino, stefania calabrese, lia crotti, francesca ferrari, roberto insolia, fabio facchinetti, peter j schwartz. dept. of obst/gynecol, univ. of milano, milano, italy; molec cardiol lab, policlinico san matteo, pavia, italy; mother-infant dept., univ. of modena/reggio emilia, modena, italy. introduction: in developed countries, nearly in every pregnancies ends in late fetal loss. many iufds can be attributed to maternal disorders, fetal pathology, placental pathology and fewer to complications of labor and delivery. however, - % of cases remain unexplained. we recently demonstrated that % of sudden infant death syndrome (sids) cases carry functionally relevant genetic variants in long qt syndrome (lqts) genes. aim of the study was to analyze whether lqts genes are associated with iufd. materials and methods: patients with iufd were enrolled in two years, as part of an italian multicentre study. iufd was defined as fetal death at weeks or more of gestation according to the definition of late fetal death of the who. out of cases were classified as "unexplained stillbirths" according to the wigglesworth and aberdeen classifications. at birth placental and/cord samples were collected and dna extracted. the main lqts genes kcnq , kcnh , scn a, kcne and kcne were screened through dhplc and sequence analysis. any amino-acid substitution identified in the samples was checked for in a control population of caucasian women with uneventful pregnancies. preliminary data on the first cases (gestational age of death: - weeks) are reported. results: a total of missense mutations were identified in of stillbirths ( %), two on scn a and one on kcnh . the two mutations on scn a (v l; p a) were observed in two iufd occurred at term; they had been previously associated to sids and shown to increase the late sodium current. the mutation on kcnh is a novel genetic variant absent in reference alleles, never described in any control populations; this mutations was present in a case of iufd diagnosed at weeks of gestation. we are currently performing the electrophysiological cellular studies to define its functional effect. conclusions: these preliminary data indicate that a potentially significant number of currently unexplained iufd might be caused by ion channel diseases such as lqts. the potential prevention of sids or iufd recurrence and the identification of other affected family members could have important implications for the affected families. alternative splicing of epab is regulated by exonic splicing enhancers. e seli, a yaba, o guzeloglu-kayisli, md lalioti. ob gyn, yale u., new haven, ct, usa. introduction: alternative splicing is an important mechanism by which the genome gives rise to the observed diversity of proteins. embryonic poly(a) binding protein (epab), expressed exclusively in oocytes and early embryos, mediates translation of maternal mrnas. we identified an alternatively spliced form of epab lacking exon (cex del), and investigated its regulation as a model for alternative splicing in early development. specifically, we evaluated: imprinting (expression from maternal or paternal allele only); rna editing (post-transcriptional single nucleotide substitution); and exonic splicing enhancers (eses, exonic sites that bind splicing proteins). methods: a single nucleotide polymorphism (snp) detected in exon (c a/g) served as a marker for the parental origin of the spliced form. snp genotyping was performed by pcr amplification of exon followed by restriction enzyme digestion. to evaluate imprinting, we characterized heterozygous mice (a/g) that inherited the snp from either the mother or the father. to test for rna-editing and exonic enhancer contribution we tested mice homozygous for the exon snp (a/a or g/g). efficiency of alternative splicing in different genetic backgrounds was tested using real-time pcr normalized to actin expression. results: in mice heterozygous (a/g) for the exon snp, cex del was only expressed from the (a) allele. however, this was independent of the parental origin of the allele, ruling out imprinting. in mice homozygous (g/g) for the exon snp, the cex del variant also contained (g). therefore, rna editing did not occur. further sequence analysis led to the identification of an additional snp in exon (c g/c) that co-segregated with the exon snp. presence of c g led to the formation of an ese that binds splicing regulatory protein srp , leading to efficient exclusion of exon . real time pcr revealed a five-fold increase in the expression of the cex del alternative splicing variant in animals carrying the enhancer (homozygous g/g) for the exon snp compared to those that did not (homozygous c/c) at the same locus (p < . ). conclusions: in this study, we found that eses mediate the alternative splicing of oocyte-specific transcripts. our findings suggest that single nucleotide polymorphisms may alter the ratio between alternative splicing variants of oocyte-specific proteins. the role that these subtle differences play in determining individual reproductive outcome remains to be identified. epigenetics and chromosomal abnormalities in human oocytes. ilse van den berg, , joop se laven, robert jan galjaard, j hikke van doorninck. , obstetrics gynaecology; clinical genetics, erasmus medical center, rotterdam, netherlands. humans have a low fertility rate compared to other mammalian species. moreover their fertility declines with increasing age. both phenomena are largely due to an increasing number of chromosomal abnormalities in human oocytes during life. identifying factors that cause aneuploidy in oocytes may offer possibilities to diminish these abnormalities in vitro or in vivo. chromosomal segregation errors can result from aberrant recombination but little is known about epigenetic factors that may cause aneuploidy. epigenetic modifications such as histone acetylation and subsequent de-acetylation in oocytes are necessary for a correct progress through meiosis. if disturbed it may lead to aberrant segregation of chromosomes or chromatids due to decreased kinetochore function and imperfect spindle figures resulting in aneuploidy. mice oocyte data have shown a correlation of abnormal histone de-acetylation and aneuploidy and a correlation between age, remaining histone acetylation and aneuploidy (akiyama et al., ) . our research focused on human oocytes and investigated whether a similar relationship between histone acetylation, age and aneuploidy is present. human oocytes were surplus from standard ivf/icsi treatments (ic+). human oocytes showed immunostaining for histone , lysine acetylation (h k ) at the germinal vesicle stage and complete deacetylation at the mii stage in % of the oocytes, while % keep high levels of h k acetylation. treatment of germinal vesicle oocytes with a histon deacetylase inhibitor (tsa) during in vitro maturation until mii stage resulted in high levels of acetylation. remaining acetylation in tsa treated oocytes was correlated significantly with abnormal spindle figures (tubulin staining), a hallmark of developing aneuploidy. similarly, % of oocytes with naturally remaining h k acetylation showed abnormal spindle figures. in contrast % of the normal de-acetylated oocytes showed normal spindles (p= . ). this suggests that defective de-acetylation of h k in human oocytes leads to abnormal spindle figures and subsequent aneuploidy. advanced maternal age is associated with a reduction in de-acetylation during in vitro maturation and an increase in spindle abnormalities. these results may stimulate the development of assays for histone modifications as biomarkers to follow oocyte quality in in vitro maturation studies or in optimizing general ivf treatments. objective: racial disparity in preterm birth (ptb) is unexplained and genetic risk factors are suspected as a major cause. this large scale candidate gene study examines differences association of single nucleotide polymorphisms [snps] in caucasians (c) and african americans (aa) to help elucidate racial disparity in preterm birth (ptb) methods: in this case (preterm birth < weeks) control study (term birth > weeks) maternal and fetal dna from ( cases and controls) c and ( ptb and term) aa were collected. a high-throughput candidate gene association study was performed examining snps in genes of selected from hypothesized ptb pathways. single locus association analyses were performed separately on maternal and fetal samples. results: snps in genes associated with ptb (p< . ) were common between races with both maternal and fetal dna analyses. however, snps in genes in c and in aa in both maternal and fetal dna differed in its association with ptb. in c maternal dna, the single strongest association between ptb and snp was in plasminogen activator tissue (plat) gene (c- t; rs ) at both allelic (p = . x - ) and genotypic (p= . x - ) level with an odds ratio (or) of . ]. the single strongest effect in c fetal dna was observed in a snp in the interleukin- receptor antagonist gene (a g; rs ) for both allele (p= . ) and genotype (p= . x - ), or . ). in aa, the strongest associations were in interluekin- (c t-rs , allele p= . x - , genotype p= . x - ) in maternal dna with an or= . [ci . - . ] and in fetal dna interleukin- receptor b (a g; rs , allele p= . x - , genotype p= . x - ) with an or . ]. conclusion: large scale high throughput analyses of snps in candidate genes of ptb pathway reveals significant differences between races at both maternal and fetal levels. we found that the strongest single locus associations differed in the two races in both maternal and fetal dna samples. these findings support the hypothesis that underlying genetic predispositions may differ between these populations, perhaps partly explaining racial disparity in preterm birth. candidate gene association study indicates differential etiologies in gdm and in t dm. johann urschitz, tarik sultan, kenneth ward. obgyn, jabsom, university of hawaii, honolulu, hi, usa. objective: recently several genome-wide association studies have associated multiple single nucleotide polymorphisms (snps) in multiple genes with increased risk of type diabetes. as risk factors are similar between t dm and gestational diabetes (gdm), we investigated a possible association of those snps with gdm. study design: blood was collected from caucasian ( cases, controls) women who met coustan-carpenter criteria for gdm and non diabetic controls. dna was extracted and a candidate gene association study was performed (taqman, abi). results: chi contingency tests were used to analyze genotype and allele frequencies in controls and gdm affected pregnancies (see table below). none of the snps showed a significant association after bonferroni correction. conclusion: several polymorphisms which displayed highly significant associations with type diabetes are not associated with gestational diabetes. these results suggest that gdm is not a simple unmasking of a t dm predisposition by the metabolic demands of pregnancy; rather it appears that different biological mechanisms are responsible for the respective diseases. introduction: histone acetylation/deacetylation plays an important role in the regulation of gene expression. histone deacetylase inhibitors (hdaci), in general, maintain gene expression, although in some cases they cause repression of specific genes. in this context we have previously shown that the hdaci tsa suppresses activation of the pro-contractile gene cox- in human myometrial and amnion-derived cells [reproductive sciences, vol , no (supplement, # )]. we have also shown that expression profiles for hdacs ( - , ) differ significantly within upper and lower regions of the myometrium during pregnancy and in labour. objectives: since changes in both acetylation and phosphoryation status can influence the activity of individual hdacs we aimed to define whether there are any changes in the levels of acetylation and phosphoryation of myometrial hdac , and during pregnancy and labour. methods: protein homogenates prepared from non-pregnant, term and labouring myometrium were treated +/-shrimp alkaline phosphatase and subjected to sds-page and western immunoblotting (wb) using antibodies to hdac , and . the acetylation status of the hdacs was assessed by a co-immunoprecipitation assay using anti-hdac and anti-acetylated lysine antibodies. results: distinct patterns of acetylation were observed for the individual hdacs; hdac and appeared to be more acetylated in non-pregnant and labouring lower tissues when compared to pregnant myometrium. in contrast, hdac appeared to be slightly more acetylated in lower uterine samples from pregnant and labouring myometrium. in experiments to evaluate changes in phosphorylation status we observed that ) myometrial hdac appeared to less phosphorylated in both upper and lower labouring samples when compared to non-pregnant and pregnant samples; ) hdac appeared to be more phosphorylated in labouring samples than non-pregnant and pregnant myometrium; ) no changes in phosphorylation levels were observed for hdac using this test system. conclusions: the difference in hdac acetylation and phosphorylation levels in the human myometrium may indicate differential regulation of the activity of the hdacs within the distinct myometrial regions, perhaps leading to the alteration of their epigenetic effect on genes related to myometrial quiescence and contraction and the subsequent onset of both term and preterm labour. objective: native hawaiians and pacific islanders experience a higher perinatal morbidity secondary to the increased incidence and earlier onset of heart failure. this increased incidence is likely multi-factorial and includes co-morbidities and genetic factors. mutations in the human adrenergic receptor gene may play a role in the determination of heart function. mutations in the b -adrenergic receptor (adrb ) located on chromosome have been identified as a cause progressive cardiomyocyte loss leading to a dilated cardiomyopathy. the minor allele frequencies for most of these polymorphisms have been determined for caucasian, japanese, african-american and chinese as part of the hapmap project. the frequencies have not been determined in the pacific islander groups. this study helped determine the hawaiian allele frequency and determine the allele frequency in the presence of cardiac or other vascular co-morbidities such as hypertension, preeclampsia, and gestational diabetes. study design: real time pcr technology (taqman genotyping assays, applied biosystems) was used to screen maternal dna (n= ) from the phenotyping sample core of the pacific center for early human development (prcehd) for the rs single nucleotide polymorphisms (snps). genotype frequencies were also determined in % complete ethnic controls as determined by a four grandparent descent. results: chi square was used to analyze allele and genotype frequencies differences between controls and affected pregnancies. the results are summarized in the following tables. conclusion: the rs snp was not significantly associated with hypertension, preeclampsia or gdm in our overall hawaiian population. the genotype frequency in the % pacific islander group was significantly different from the caucasians (p= . ) and asians (p= . ) in our overall hawaiian population. angiotensin-converting enzyme and -adducin polymorphisms in preeclamptic mothers and fetuses. c mando, p antonazzo, s tabano, f colleoni, a martinelli, s calabrese, c benedetto, l marozio, f facchinetti, m miozzo, i cetin. inst. obstetrics and gynecology, fondaz. irccs policlinico mangiagalli regina elena, univ. milan, milan, italy; dept. biology and genetics, univ. milan, italy; dept.obstetrics and gynecology, univ. torino, italy; univ. modena and reggio emilia, modena, italy. background the genes encoding angiotensin-converting enzyme (ace) and -adducin (add ) share the potential of influencing blood pressure. previous studies demonstrated in humans the association of hypertension with the combined effect of both ace insertion/deletion (i/d) polymorphism, which leads to a different activity of the enzyme, and add g w non-sense single nucleotide polymorphism (snp). ace i-i genotype has been associated with low serum ace activity. controversial studies concerning the association of ace polymorphism with preeclampsia (pe) were reported and the possible combined effect of both ace i/d and add g w polymorphisms yet remains to be investigated. population association study we genotyped polymorphisms (ace i/d and add g w) in women: with pe ( / with severe pe) and controls. moreover, we investigated a subset of their fetuses: from severe pe, from mild pe and controls, in order to identify specific maternal and/or fetal genotypes conferring a higher risk to develop pe. we both evaluated the single and the combined effects of ace and add genotypes on mother-fetus couples and singularly on mothers and fetuses. ace i/d genotype was analyzed using a pcr method; an allelic discrimination approach was performed to detect the add snp. in mother-fetus genotype couples, neither ace nor add polymorphisms are associated with pe, nor are the combination of their genotypes separately in mothers and in fetuses. nevertheless in mothers with mild pe ace i-i genotype is significantly less frequent ( % vs %; p< . ) and ace i-d is significantly more frequent ( % vs %; p< . ) compared to controls. ace i allele frequency is not significantly different in mild pe compared to controls ( % vs %). in women with mild pe the i allele seems to move from i-i to i-d genotype. it leads to the increase in mild pe women of those genotypes with a higher ace activity conferring a higher susceptibility to develop pe. this association with mild pe and not with severe pe could be due to the contribution in the latter of many other genetic and environmental factors. introduction: maternal mrnas stored in the oocyte are critical for early development as transcription ceases upon oocyte maturation, and gene expression until zygotic genome activation (zga) is mediated by translation of maternal transcripts. cytoplasmic polyadenylation element binding protein (cpeb) plays a central role in this process by stabilizing maternal mrnas and regulating their timely activation. this function has been well characterized in xenpous, and a similar role in mammals is suspected as the cpeb knockout mouse displays infertility as a result of arrested oogenesis. in order to investigate if translational regulation of maternal transcripts by cpeb is maintained in mammals, we characterized the spatial and temporal expression of cpeb- in the mouse and human. methods: ten different somatic tissues, testes, and ovaries were tested by rt-pcr for the expression of cpeb mrna in mouse and human. cpeb mrna expression in mouse was also tested in prophase i (pi) and metaphase ii (mii) oocytes, -cell, -cell, -cell, -cell embryos and blastocysts. in human, pi and mii oocytes, -cell embryos and blastocysts were evaluated. sequencing of the pcr products was performed to confirm specific amplification of cpeb. amplification with actin primers provided a positive control and allowed semi-quantitative analysis. results and discussion: highest level of cpeb mrna expression was detected in ovaries and testis of both mouse and human. in addition, differential expression of cpeb in somatic tissues was also observed. among these, prominent expression was present in brain, where a role for cpeb in facilitating gene expression through cytoplasmic polyadenylation has been proposed. these data would suggest that translational control of mrna by cpeb may be a mechanism utilized by multiple somatic tissues to regulate gene expression. in the mouse, cpeb was expressed in pi and mii oocytes and -cell and -cell embryos, and became undetectable in -cell or more advanced embryos. human cpeb was expressed in pi and mii oocytes, but not in -cell embryos or blastocysts. zga occurs at late -cell stage and -to- -cell stage, in mouse and human, respectively. the tightly controlled temporal expression of cpeb prior to zga in both mouse and human is consistent with a conserved role for cpeb in the regulation of maternal mrna expression during early development in mammals. background embryonic stem cells (esc) express specific transcription factors that are indicative of their ability to maintain pluripotency. these factors are activated during preimplantation embryo development. the interplay between the transcription factors pou f (oct / ) and caudal-homeobox domain (cdx ) is thought to regulate inner cell mass (icm) and trophectoderm (te) differentiation; however, the mechanism of cell fate determination in mammalian embryos is poorly understood. genetic ablation of oct / in mice prevents icm development, while cdx knockout embryos fail to implant. esc deficient in nanog, a second key regulator of pluripotency, fail to maintain pluripotency and undergo differentiation. expression of nanog in primate embryos has not been investigated, therefore the localization of oct / , nanog and cdx was determined in rhesus macaque blastocysts. methods oocytes were collected from rhesus macaque females, fertilized and cultured in vitro to the blastocyst stage. embryos were collected hours post insemination, then fixed prior to incubation with primary antibodies directed against oct / , nanog and cdx . following incubation with cy conjugated secondary antibodies, nuclei were counterstained with dapi and embryos visualized using an olympus bx fluorescence microscope. results nanog protein was restricted to the icm of monkey blastocysts. unlike the mouse embryo, oct / protein was detected in both the icm and te, based on two different antibodies. the expression of cdx was localized specifically to the te. conclusions the ubiquitous pattern of oct / expression is consistent with observations in human, cow and pig embryos. significantly, lack of restricted oct / protein, and icm localization of nanog in primate blastocysts, suggests that nanog more specifically determines cell fate in primate embryos. these results contrast markedly with current mechanistic hypotheses, although other factors may lie upstream of nanog. importantly, this difference may underlie observations that regulatory mechanisms in esc differ between mice and primates. further investigations will focus on determining the onset of marker expression, and the upstream regulators of nanog activation. supported by nih grants r hd r rr , and the intramural research program of nichd. in vitro-conceived mice tend to be smaller at birth and throughout their life. luisa delle piane, annemarie donjacour, francesca di sebastiano, gnanaratnam giritharan, paolo rinaudo. obstetrics, gynecology and reproductive sciences, university of california san francisco, san francisco, ca, usa. objective: epidemiological evidence indicates that ivf is associated with an increased incidence of low birth weight. this phenomenon has not been studied in a mouse model; in addition, it is unknown if the resulting offspring show different growth pattern later in life. we therefore created an ivf mouse model to follow growth patterns till adulthood. methods: we generated experimental groups of b mice: one cohort of in vivo generated mice (in vivo group), one cohort of in vitro generated animals (ivf group) transferred to cd foster mothers and one cohort of animals fertilized in vivo and transferred to cd foster mothers (flushed blastocysts group, fb). the fb group was generated because our preliminary results showed that embryo transfer to b foster mothers was not successful. all pups were delivered at term, measured and weighed at birth and then weekly up to weeks. parametric tests (anova with bonferroni correction) were used as appropriate. a mixed model was used to compare growth curves. results: average litter size was . (in vivo), . (fb) and . (ivf). birth weight of male mice both ivf ( . mg) and fb ( . mg) was larger than male in vivo mice ( . mg) (p< . ). ivf mice tended to be smaller than fb mice in both sexes (p= . ). the bmi (body mass index) was not different among all the groups. male fb growth curves were different from in vivo mice (p< . ) and more importantly from ivf growth curves (p= . ) (fig. ) . conclusion: the method of conception and the maternal environment play a significant role in determining birth weight in this mouse model, emphasizing that the fb group is the best control for the effects of ivf in this model. these preliminary results confirmed for the first time an ivf effect on growth and interestingly the ivf males did not show a catch-up growth. the finding of smaller ivf mice when compared to fb is both noteworthy, because it confirms human data, and worrisome, because lower birth weight is associated with long term sequelae according to the barker hypothesis. the effect of betamethasone exposure in mid-gestation on renal sodium excretion in male sheep. lijun tang, luke carrey, nancy valego, philip deibel, james perrott, jorge figueroa, mark chappell, james c rose. obestetrics and gynecology, wake forest university, medical school, nc, usa; hypertension center, wake forest university, medical school, nc, usa. objective: whereas prenatal exposure of ovine fetuses to clinically relevant doses of glucocorticoids during the time of peak nephrogenesis results in a reduction in nephron number in adulthood, there is little information about its effect on sodium excretion. in the present study, we evaluated the effect of exposure to betamethasone on renal sodium excretion in adult male sheep. methods: we studied nineteen conscious adult rams at . years of age which were exposed to either vehicle or betamethasone at - days gestation. we implanted vascular and bladder catheters and then allowed the animals a - days recovery period prior to study. inulin and para-aminohippuric acid (pah) clearances were performed for estimating glomerular filtration rate (gfr) and renal plasma flow (rpf) respectively. following determination under basal conditions, an acute hypertonic sodium load was administrated intravenously by a continuous infusion of nacl ( . meq/kg/min at . ml/min) for minutes. urine was continuously collected for determination of na + excretion. results: basal gfr was decreased in steroid exposed adults ( . ± . ml/min/kg) compared with the vehicle animals ( . ± . ml/min/kg) (p= . ). rpf was similar in the vehicle and steroid exposed group ( . ± . ml/min/kg vs . ± . ml/min/kg). at basal conditions, na + excretion (u na v) was similar in vehicle and steroid exposed group ( . ± . μmol/h vs . ± . μmol/h). this similarity was also present after normalization by body weight ( . ± . μmol/h/kg vs . ± . μmol/h/kg). the vehicle group excreted . ± . % of the dose of na during the experiment while the betamethasone exposed group excreted only . ± . % (p< . ). gfr and prpf did not change during the experiments. these results suggest that prenatal exposure to glucocorticoids affects renal function in adult male sheep which results in a decreased basal gfr and an attenuated ability to excrete on acute sodium load. the elevated blood pressure previously observed associated with this prenatal steroid treatment may be related to the alteration in renal function. the study is supported by nih grants hd , hl and hd . characterisation of human fetal cardiac stem cells. marah alfakir, nicholas dawe, annette meeson, stephen c robson. school of surgical reproductive sciences, newcastle university, newcastle upon tyne, united kingdom. objectives: for many patients with severe cardiac disease treatment is limited to surgical intervention and/or heart transplantation. the heart has a limited regenerative capacity but it is insufficient to repair extensive damage. cellular therapies might provide an alternative treatment. we have identified stem cells (sp cells) in the adult mouse and human heart and have shown that the mouse cardiac sp cells can differentiate along a cardiac lineage. sp cells can be identified using facs combined with hoechst dye efflux. this ability to efflux hoechst dye is due to expression of abcg (an abc transporter). we have hypothesized that the fetal heart would contain more cardiac stem cells, relative to the adult, and the aim of this project was to determine the spatial and temporal expression of known stem cells markers in the embryonic/fetal heart. methods: human fetal hearts were collected aged between - wk. we used rt-pcr, using primers for several stem cell (abcg , cd , cd ) and cardiac specific (mlc- v, mhc) markers, and ihc on frozen and paraffin sections, using a panel of antibodies (abcg , cd , cd , islet ). cdna was generated from the following regions of the heart at different developmental stages: right and left atrium, right and left ventricle, and outflow tract. results: abcg mrna was highly expressed in all regions of the fetal heart between - wk. expression was down regulated at - wk especially in the la and lv. a similar pattern of expression was observed for cd . cd showed low/moderate expression in all heart regions examined; however, this expression was absent in the lv at - wk. mrna and protein analysis showed similar results. whilst protein expression of abcg was robust at wk (approximately - %), it declined with increasing gestational age. cd was also strongly expressed at weeks of age. there was no co-localisation between abcg and islet . conclusion: abcg and cd are both expressed between - wk of age. based on this analysis, further studies are underway to isolate and characterise both the abcg and cd expressing fetal cardiac cells which might be a potential source of cardiac stem/progenitor cells. xanthine oxidase plays a significant role in the fetal cardiovascular defence to hypoxia. ja hansell, a kane, e herrera, da giussani. physiology, cambridge university, united kingdom. prenatal hypoxia remains a major concern in obstetrics. the fetal defence to hypoxia includes redistribution of the cardiac output, away from peripheral and towards essential ciculations, such as those perfusing the brain (cohn et al . ajog : , ) . the physiology underlying this response is well characterised and involves chemoreflex and endocrine responses (giussani et al. fet mat med rev : , ) . more recently, local factors such as nitric oxide (no) and reactive oxygen species (ros), and the interaction between them, have been shown to play a role. antioxidants, such as vitamin c, scavenge hypoxia-induced ros, maintain no high and thereby depress peripheral constriction (thakor et al., sgi ) . in this study, we address the source of hypoxia-induced ros production and investigated the effects on the in vivo fetal femoral constrictor response to hypoxia of maternal treatment with the xanthine oxidase inhibitor allopurinol in sheep. methods: under anaesthesia, sheep at . gestation, were instrumented with maternal and fetal catheters and a fetal femoral transonic probe. five days later, all animals were subjected to h normoxia, . h hypoxia (mat fio to reduce fetal pao to ca. mmhg) and h recovery, either following maternal i.v. treatment with vehicle or allopurinol in low ( mg.kg - over min) or high ( mg.kg - over min ) doses. treatments finished min prior to hypoxia. the low allopurinol dose was adopted from human studies (benders et al. arch dis child : , ) . the timing of the hypoxic challenge coincided with peak concentrations in fetal sheep plasma of oxypurinol (active metabolite). we evaluated the effect of bpa exposure on uterine gene expression in a nonhuman primate model. method: a total of nine vervet monkeys (cercopithecus aethiops sabaeus) were ovariectomized. three monkeys were treated with bpa via alzet pumps ( microgram/kg/day), two with estradiol benzoate and three received combined treatment with bpa and estradiol. the remaining monkey was untreated and served as a control. following days of treatment the monkeys were hysterectomized and immunohistochemistry performed to examine endometrial gene expression. we evaluated hoxa , proliferating cell nuclear antigen (pcna), and caspase iii gene expression as markers of differentiation, proliferation, and apoptosis respectively. differential expression was evaluated by h-score. results: exposure to a combination of estradiol and bpa resulted in increased expression of hoxa and pcna compared to control (p< . ). lower, but increased, levels of hoxa and pcna gene expression were seen in the specimens exposed solely to estradiol (p< . ). those specimens exposed to bpa alone demonstrated a small induction in hoxa expression (p< . ) with no change in caspase iii or pcna expression when compared to the control. conclusion: bpa induced the expression of hoxa in the uteri of a non-human primate. exposure in the presence of endogenous estrogen further augmented estrogenic stimulation confirming that bpa is a xenoestrogen. bpa's effect of developmental gene expression may alter uterine differentiation. bpa acts as an endocrine disruptor by altering estrogen regulation on a gene required for fertility. contractility and meconium passage. jayaraman lakshmanan, john d richard, guo l liu, sharon k sugano, ahmet karadag, michael g ross. dept. of ob/gyn, harbor-ucla med. ctr., torrance, ca, usa; dept. of pediatrics, harbor-ucla med. ctr., torrance, ca, usa. objective: endogenous glucocorticoids (gcs) increase with advancing fetal age suggesting that gcs function as a hormonal modulator of organ maturation. although fetal colonic motility and meconium passage primarily occur near term, the role of gc in colonic maturation is poorly understood. we sought to examine gc-nuclear receptor (gc-nr) expression in ovine fetal distal colon smooth muscle from very-preterm to term gestation to define the cascade of gc initiated biochemical changes as a prerequisite for maturation-associated meconium passage. methods: ovine fetal distal colonic segments removed at very-preterm (vpt: - d gestation), preterm (pt: - d), near term (nt: - d) and term (t: - d) (n= fetuses in each group) were fixed in bouin's solution and paraffin embedded. sections were subjected to immunohistochmical analysis with gc-receptor antibody ( : , sc- , santacruz biotechnology, ca) using standard abc regimen. immunoreactive material identified by , 'diaminobenzidine as chromogen. the percentage of gc-nr staining in smooth muscle-enteric unit was analyzed by counting dark brown immunostained nuclei at different fields at x magnification. all values are expressed as mean ± sem. results: the gc-receptor antibody immunostained nuclei both in smooth muscle and enteric units and the observed pattern (expressed as percentage of total nuclei) are as follows: nuclear gc expression varies with gestational age with maximal expression occurring near-term in smooth muscle and enteric neurons, in a synchronized manner. a near total disappearance of gc-nr expression occurs at term. these results suggest that peak gastrointestinal gc-nr mediated effects may occur prior to term with secondary signaling effects resulting in distal colonic motility maturation and meconium passage. the rna-binding protein hud co-localizes with choline acetyl mechanisms of in utero meconium passage. jayaraman lakshmanan, john d richard, guo l liu, sharon k sugano, octavio balbuena, michael g ross. dept. of ob/gyn, harbor-ucla med. ctr., torrance, ca, usa. objective: we have previously speculated that crf, acting through its receptor crf-r , increases gastrointestinal (gi) motility and potentiates in utero meconium passage. patients with paraneoplastic syndrome with auto-antibodies to hud, a neuronal rna binding protein, develop severe gi dysmotility, indicating a role for hud in gi motility. hud binds mrna for actylcholinesterase implying a role in the cholinergic neurotransmitter system. based on these known functions, we hypothesized that hud may regulate post-transcriptional activity in fetal colonic cholinergic neurons expressing crf-r . method: bouin's solution-fixed paraffin-embedded sections of distal colonic segments were prepared from very preterm (vpt: - days), preterm (pt: - days), near term (nt: - days) and term ( - days) ovine fetuses (n= at each age). sections were immunostained with anti-human neuronal protein huc/hud antibodies ( : to : ) with abc reagents and examined at x. neurons positive for hud staining were quantified. double immunofluorescence and laser confocal analyses evaluated co-expression of hud in neurons expressing peripheral choline acetyl transferase (pchat) (a marker for peripheral cholinergic neurons) and crf-r receptor. results: hud immunostaining was seen in either entire cytoplasm (c) or cytoplasm and nuclear regions (c+n) in both submucosal and myenteric neurons. a greater percentage of submucosal (vpt: ± , pt: ± , nt: ± , and t: ± %) than myenteric neurons (vpt: ± , pt: ± , nt: ± , t: ± %) exhibited positive staining at all ages. the percentages of neurons with c+n staining in submucosal neurons were significantly lower at very-preterm gestation. confocal studies co-localized the hud staining with pchat and crf-r receptor immunoreactivity both in submucosal and myenteric neurons. conclusion: in the fetal enteric nervous system hud may function both as a rna-binding protein and as a nuclear cytoplasmic shuttling protein. colocalizaion studies suggest that both pchat-mrna and crf-r mrna species are target transcripts for hud in myenteric neurons. we speculate upregulation of hud contributes to in utero meconium passage. meconium passage during mild stressors. jayaraman lakshmanan, guo l liu, john d richard, sharon k sugano, raina khan, octavio balbuena, kimberly chap, virender rehan, michael g ross. dept. of ob/gyn, harbor-ucla med. ctr., torrance, ca, usa; dept. of pediatrics, harbor-ucla med. ctr., torrance, ca, usa. objective: mr exhibit a greater affinity to glucocorticoids (gc) than do glucocorticoid receptors (gr). thus low circulating gc levels may preferentially bind mr during mild-stress periods, while the high gc levels may bind gc-receptors. mr antagonism increases gc-mediated responses, suggesting that mrs have a regulatory impact on gc-mediated stress responses. we recently documented that fetal in utero meconium passage is a neurovisceral stress response. to test our hypothesis that mrs play a primary role in mediating suppressive gc effects, we examined the expression patterns of mr in ovine fetal distal colon. methods: bouin's solution fixed paraffin sections of distal colonic segments collected from ovine fetuses (n= for each gestational ages) at very preterm (vpt: - days gestation), preterm (pt: - days), near term (nt: - days) and term (t: - days) were subjected to immunohistochemistry with polyclonal antibodies to mr. digital photos ( field per colonic ring, rings each gestational age) taken at x were used to count the number of intensely stained neurons, referred to as "mr-capped neurons". differences over time were determined with anova. results: mr antibody elicited a punctate staining pattern in all layers of ovine fetal distal colon. significant immunoreactive intensity was observed in submucosal and myenteric ganglia at all ages: submucosal ganglia: vpt= . ± . , pt= . ± . , nt= . ± . , t= . ± . ; a subpopulation of enteric neurons exhibited dense staining ("mr-capped"). advancing gestation was associated with a significant decreased in the percentage of mrcapped myenteric (vpt = ± , pt = ± , nt = ± , t = ± %; p< . ), though not submucosal ganglia neurons. results indicate a significant decrease in the percentage of mr capped myentric neurons with advancing gestation. in view of the potential inhibitory effect of mr on gc-mediated stress responses, these results suggest that the decrease in mr expression may contribute to near term and term in utero meconium passage. the ambiguity of myometrial progesterone receptor expression in pregnancy and labour. alison j tyson-capper, elizabeth a shiells, stephen c robson. surgical reproductive sciences, institute of cellular medicine, newcastle university, newcastle upon tyne, united kingdom. background and aims: in contrast to many other species, human parturition is not due to a reduction in circulating levels of progesterone (p) but appears to be related to changes in expression, ratios or signalling events of p receptors (pr-a and pr-b). the literature on pr expression in human myometrium in pregnancy and labour remains conflicting. we hypothesise this is due, at least in part, to differing specificities of the various pr antibodies employed. we carried out a comprehensive analysis of pr expression using ten 'pr-specific' antibodies that recognise different amino acid epitopes within the pr proteins. two phosphorylation-specific antibodies for pr were also included to define whether phosphorylation status of myometrial pr changes in pregnancy and in labour. methods: western immunoblotting and semi quantitative rt-pcr were undertaken using protein lysates and rna prepared from myometrial tissue from: -first (n= ) and second (n= ) trimesters, paired upper and lower segment myometrium from preterm ( - wks, n = ), term not in labour (n = ), term spontaneous labour (n = ) and non pregnant (n = ). results: using the same set of myometrial lysates for each antibody, we found that the specificity of individual pr antibodies, in particular those raised against internal and c-terminal epitopes of the pr proteins, varied considerably resulting in different patterns of expression. there also appeared to be temporal and spatial differences in levels of myometrial pr proteins ( - kda/ - kda) in pregnancy and in labour with use of the phosphorylation-specific pr antibodies, however, it remains to be resolved which pr isoforms these may be. in contrast, data using four antibodies all of which react with the amino terminal domain of pr consistently indicated that expression of pr, in particular pr-b, decreased significantly at term (p < . ) and in labour (p < . ) when compared to non-pregnant levels. a decrease in levels of pr-b protein was also observed when comparing preterm levels to non-pregnant levels. rt-pcr using primers specific to pr-b consistently indicated that levels of pr-b mrna decreased at term and in labour. conclusion: interpretation of gestation-related changes in myometrial pr expression and phosphorylation status must take into account the pr antibodies used. further studies are now underway to validate the specificity of the pr antibodies. objective to test the hypothesis that expression of fshr in mural gl cells from ivf follicles varies with the infertility diagnosis and correlates with the outcome of ovulation induction (oi). materials and methods women undergoing ivf were classified as: . "no ovarian factor", (tubal or male factor and egg donors, nof;n= ); . endometriosis (endo; n= ); . poor responders (pr; n= ); . polycystic ovary syndrome (pcos; n= ). ovulation induction was carried out using a long or microflare or antagonist protocol based on clinical parameters and gonadotrophin doses were selected based on ovarian reserve (day fsh and e and basal antral follicle count) and adjusted to the individual response. after ultrasound guided egg retrieval, mural gl cells were isolated from pooled follicular fluids from each patient using a percoll gradient and anti-cd immunobeads to eliminate wbcs, viability was assessed by trypan blue. fshr was measured by rt-pcr as relative expression compared to beta actin. statistical analysis was performed with the spss using pearson´s correlation, one way anova and student´s t-test. results fshr expression in nof ( ± . ) was statistically significantly higher than in pr ( ± . ) and lower than in pcos ( ± . ). both endo ( ± . ) and pr levels of fshr were statistically significantly lower than in pcos. analysis of all cycles showed that fshr expression correlates positively with the number of total (r= , ; p< , ) and mii (r= , ; p< , ) oocytes and negatively with the units of fsh (r=- , ; p< , ) and lh (r=- , ; p< , ) administered for oi. separate analysis of nof showed a positive correlation with the number of total and mii oocytes retrieved and with estradiol levels on day of hcg but not with the total dose of gonadotropins received during oi. fshr expression correlates negatively with day fsh levels in all patients except in pcos (r=- , ; p< , ). conclusions these results suggest that expression of fshr in the hyperstimulated ovarian follicle is "average" in normoresponders, high in high responders (pcos) and low in poor responders (pr and endo). knowledge of the level of expression of fshr might be useful to individualize gonadotrophin doses and oi protocols thus improving pregnancy rates. comparison of intracellular atp levels between non-vitrified and surviving post-vitrification/thawed human oocytes. somjate manipalviratn, zhi-bin tong, eric widra, , alan h decherney. reproductive biology and medicine branch, nichd/nih, bethesda, md, usa; department of obstetrics and gynecology, georgetown university hospital, washington, dc, usa; shady grove reproductive science center, washington, dc, usa. objective: to compare intracellular atp level in non-vitrified and surviving post-vitrification/thawed human oocytes using an atp bioanalysis assay. design: prospective match-controlled laboratory study material and methods: all oocytes were obtained from women undergoing controlled ovarian stimulation for ivf/icsi. oocytes were discarded due to nuclear immaturity at the time of planned icsi. all immature oocytes (gv and mi) were incubated overnight. oocytes from patients with or more discarded eggs which matured to mii stage were used in this study. oocytes from each women were randomly divided into groups. in group , the oocytes were placed in l of ultrapure water and kept at - o c for further atp analysis. in group , the oocytes were vitrified using % ethylene glycol, % dimethyl sulphoxide and . m sucrose as cryoprotectant. these oocytes were kept in liquid nitrogen for - days before thawing with a rapid thawing method. oocyte survival was determined by morphological assessment after minutes of incubation then oocytes were placed in l of ultrapure water and kept at - o c for further atp analysis. intracellular atp level was determined using luciferin-luciferase bioluminescent assay. result: ninety-one discarded human oocytes were obtained from women. oocytes were vitrified/thawed before measuring for atp content. the other oocytes were measured for their atp content without undergoing vitrification/thawing process. oocyte survival rate after vitrification/thawing is . % ( / ). mean oocyte atp levels in non-vitrified oocytes is significantly higher than surviving post-vitrification/thawed oocytes (table ). coefficient of variation of luciferin-luciferase bioluminescent assay is less than %. conclusion: oocyte atp level in surviving post-vitrification/thawed human oocytes is significantly lower than non-vitrified oocytes. objective to study the expression of genes involved in cell proliferation and steroidogenesis in the human follicle (kl , kl , fshr, papp, p ) and its relationship with ivf outcome. materials and methods patients with different infertility diagnosis underwent ovulation induction with either a long or microflare or antagonist protocol based on clinical parameters; the dose of gonadotrophin used for ovulation induction was selected based on the ovarian reserve (day fsh and e and basal antral follicle count) and adjusted to the individual patient response. ultrasound guided egg retrieval was performed hours after administration of . iu of hcg. mural granulosa-lutein cells (gl cells) were isolated from pooled follicular fluids (ff) from each patient using a percoll gradient and anti-cd immunbeads to eliminate wbcs, viability was assessed by trypan blue. the genes under study were measured by rt-pcr as relative expression compared to actin. statistical analysis was performed with the spss statistical software using pearson´s correlation and mann-whitney u. results kl expression correlates positively with kl levels (r= , ; p< , ) and with genes implicated in granulosa cell function such as fshr (r= , ; p< , ), papp (r= , ; p< , ) and p (r= , ; p< , ). the number of mii oocytes obtained is positively correlated with both fshr (r= , ; p< , ) and papp (r= , ; p< , ) expression, but not with the other genes. kl expression in women who became pregnant during the ivf cycle studied was higher ( , ± ) than in non pregnant women ( , ± , ).(n= , mann-whitney u p< , ). conclusions patients who become pregnant have increased expression of kl , which is associated with optimal granulosa cell proliferation and maturation. this is in accordance with the presence of lower apoptosis in granulosa cells in the same patients. syndrome during assisted reproduction treatment. k jayaprakasan, r jayaprakasan, h al-hasie, js clewes, bk campbell, ir johnson, nj raine-fenning. school of human development, university of nottingham, nottingham, united kingdom. objective: to test the hypothesis that an increased pre-treatment ovarian blood flow is associated with the development of ovarian hyperstimulation syndrome (ohss) and to evaluate ovarian vascularity as a predictor of ohss during invitro fertilization (ivf). methods: subjects undergoing first cycle of ivf had d transvaginal ultrasound in the early follicular phase of the menstrual cycle preceding ivf. of them developed ovarian hyper-response, defined as retrieval of oocytes and ohss. subjects had normal ovarian response with retrieval of to oocytes in the absence of ohss. antral follicle count (afc), ovarian volume (ov), and ovarian vascularity (vascularisation index, vi; flow index, fi and vascularisation flow index, vfi) were measured and an unpaired t-test was used to compare these parameters between the ohss and the control groups. multiple logistic regression analysis was used to assess the predictive value of these variables against age, bmi and basal fsh for the development of ohss. results: the ovarian vi ( . ± . vs. . ± . ), fi ( . ± . vs. . ± . ) and vfi ( . ± . vs. . ± . ) were similar in both the groups. afc and ov were significantly higher (p< . ) in the ohss group ( . ± . and . ± . cm respectively) than in the control group ( . ± . and . ± . cm respectively). afc was the only significant (p= . ) predictor of ohss on multiple regression analysis (table ) . conclusion: women developing ohss during ivf do not demonstrate an increased pre-treatment ovarian blood flow as measured by d ultrasound but do have a significantly higher afc, which is the only significant predictor of ohss. compared to other species, little is known about the steroid biosynthetic capacity and gene expression profiles of human cumulus cells. objective: to examine the ability of human cumulus cells in primary and long-term culture to synthesize steroids and respond to gonadotropin or camp-dependent stimulation. methods: human cumulus cells were isolated from cumulusoocyte complexes during assisted reproductive technology (art) and placed in primary culture or propagated to third passage. at subconfluence, cells were transferred into serum-free conditions in the presence of vehicle, forskolin, fsh, or hcg. at h, media was collected and progesterone (p ) and estradiol (e ) levels were determined by eia. aromatase activity was measured by the tritiated water assay. aromatase (cyp ) and cholesterol side-chain cleavage (cyp a ) mrna abundance was determined by quantitative real-time pcr (qrt-pcr). transcriptional regulation of the cyp and cyp a promoters was investigated by transient transfection of cumulus cells with promoter luciferase constructs. results: substantial amounts of p and e were synthesized by cumulus cells in culture, which were further elevated by forskolin, hcg, or fsh treatment. the presence of cyp and cyp a mrna in cumulus cells was confirmed by qrt-pcr, and the relative mrna abundance of both transcripts was induced by forskolin, hcg, or fsh treatment. changes in cyp and cyp a gene expression in response to camp and gonadotropin stimulation were associated with increased transcriptional regulation of both the cyp and cyp a gene promoters. our studies demonstrate that human cumulus cells are sites of significant p and e biosynthesis and respond to camp-and gonadotropin-stimulation in vitro. the ability to examine human cumulus cells in primary and long-term culture provides a unique model system to investigate cumulus cell function, and the paracrine role of cumulus cells in oocyte development, maturation, and subsequent fertilization. background: there is increasing evidence suggesting that events occurring during fetal development may result in increased predisposition to adult conditions, such as diabetes. in vitro fertilization (ivf) is a new environmental stressor and the long-term effects of these manipulations are currently unknown. there is some evidence that lower number of insulin-secreting cells at birth signals predisposition to diabetes later in life. in this study, we compare areas of pancreatic insulin-secreting cells in newborn mice conceived in vivo versus in vitro. methods: oocytes were collected from super ovulated cf- mice and fertilized in vitro with cauda epididymal sperm from b d f /j mice. fertilized eggs were cultured in whitten medium under % co in humidified air at °c for h. blastocysts were transferred to the uteri of pseudo-pregnant recipients. control mice were allowed to conceive in vivo. the newborn animals were sacrificed within hours of birth. pancreases were sectioned, immunostained with anti-insulin, and total areas and stained areas were compared using t-test with two-tailed distribution. p value of < . was considered significant. results: there were total of newborn animals: females ( ivf, controls) and males ( ivf, controls). percentage of total pancreatic area occupied by insulin-secreting cells was lower in female ivf ( . mm , . %) animals compared to female controls ( . mm , . %) and higher in male ivf ( . mm , . %) animals compared to controls ( . mm , . %). the average of males and females was slightly lower for ivf ( . mm , . %) than controls ( . mm , . %). none of these differences were statistically significant. there was a trend in newborn female mice towards lower amount of insulin-secreting cells in the ivf offspring, suggesting possible predisposition to diabetes later in life. this effect was not observed in newborn males. while our study failed to demonstrate consistent and significant differences in the areas of insulin-secreting cells between newborn mice conceived in vitro and in vivo, the number of animals in the study may have been too small to show significant results. larger studies are needed to further investigate this question. peter s uzelac, phyllis risch, kassi shelton, steven t nakajima. obstetrics and gynecology, university of louisville, louisville, ky, usa. objective: several fertility preservation methods currently available may not be viable options to certain patients due to their high cost and limited number of centers offering these services. for women undergoing bilateral oophorectomy, in vitro maturation (ivm) of oocytes retrieved from unstimulated whole ovary specimens may represent a more practical solution. here we describe out initial experience in offering ivm as a fertility-preserving measure to two patients undergoing total abdominal hysterectomy and bilateral salpingo-oophoectomy (tah-bso). case one was a year old nulligravid with chronic pelvic inflammatory disease unresponsive to medical management. case two was a year old nulligravid with stage ia endometrial carcinoma. surgery was planned for the mid-follicular phase in order to collect prophase i oocytes before the onset of late-follicular phase atresia. upon surgical removal, suction aspiration of all identifiable follicles was performed. ovaries were then serially sectioned and all tissue was examined for the presence of prophase i oocytes. results: total number of prophase i oocytes retrieved was and , maturation rate after hours was % ( / ) and % ( / ), fertilization rate after hours was % ( / ) and % ( / ), maturation rate after hours was % ( / ) and % ( / ) and fertilization rates after hours % ( / ) and % ( / ), for case and case respectively. ivm of oocytes retrieved from unstimulated whole ovary specimens may be a simple and inexpensive approach to fertility preservation in women undergoing bilateral oophorectomy. by requiring minimal adjustments to in vitro fertilization protocols, this treatment could easily be implemented in centers which currently have no fertility preservation program. patient age at time of retrieval may be predictive of developmental potential. continued patient enrollment and follow-up studies on the developmental potential of embryos derived from this technique are necessary to fully evaluate its potential for a role in fertility preservation. the routine use of progesterone as luteal phase support in women with a diagnosis of polycystic ovary syndrome (pcos) undergoing ovulation induction cycles with oral agents has not been fully elucidated. we hypothesize that women with pcos utilizing either clomiphene citrate or letrozole, an aromatase inhibitor, should administer intravaginal progesterone in the luteal phase to increase pregnancy rates. design: retrospective chart review. materials and methods: cycle data from women with pcos undergoing ovulation induction with clomiphene citrate or letrozole at the university of cincinnati medical center from to were evaluated. diagnosis of pcos was based on rotterdam criteria and all other infertility diagnoses were excluded. clinical pregnancy rates (presence of fetal cardiac activity on ultrasound at - weeks gestation) in women who received intravaginal micronized progesterone ( mg bid) following ovulation induction (with and without intrauterine insemination) were compared to those who did not receive progesterone. results: no significant differences were noted in demographic parameters, including patient age or bmi. a total of cycles were evaluated in women treated with clomiphene citrate. clinical pregnancies were documented in . % ( / ) of cycles in the progesterone group compared to . % ( / ) of the non-progesterone group (p < . ). forty-three cycles were evaluated in patients treated with letrozole. clinical pregnancies were documented in . % ( / ) of cycles in the progesterone group compared to none ( / ) in the non-progesterone group (p < . ). conclusions: patients with pcos who used letrozole for ovulation induction had superior clinical pregnancy rates when using intravaginal micronized progesterone compared to women who did not receive luteal phase support. there was a trend toward increased clinical pregnancy rates in pcos patients utilizing luteal support following clomiphene citrate for ovulation induction. therefore, in women with pcos undergoing ovulation induction with oral agents, luteal supplementation with progesterone should be strongly considered, especially in those using letrozole. were measured and an unpaired t-test was used to compare these parameters between poor and normal responders. multiple logistic regression analysis was used to assess the predictive value of these variables against age and basal fsh for poor ovarian response. results: the ovarian vi ( . ± . vs. . ± . ), fi ( . ± . vs. . ± . ) and vfi ( . ± . vs. . ± . ) were similar in both poor and normal responders. afc and ov were significantly lower (p< . ) in poor responders ( ± . and . ± . cm respectively) than in normal responders ( . ± . and . ± . cm respectively). afc was the best (p< . ) predictor of poor ovarian response on multiple regression analysis ( objective: gay male couples increasingly seek parenthood through in vitro fertilization using an oocyte donor and a gestational carrier, but no studies describe this unique experience. the purpose of this study was to determine medical and psychosocial issues unique to gay men using art. design: qualitative analysis of semi-structured interviews with gay male couples seeking parenthood through art. materials and methods: sixteen gay males (eight couples) entering an art program were assessed through the use of a semi-structured interview. characteristics evaluated included age, relationship status, duration of their relationship, psychological health and stability, how the decision was made concerning who would donate the sperm, the decision to use an anonymous or known oocyte donor, and whether their gestational carrier was someone previously known to them or not. results: the average age of the men in this study was years. all eight couples were in a committed relationship and had been together for an average of . years. five of the couples ( %) had been joined in a civil union which is legal in the state of connecticut. all subjects were psychologically stable and in good health. six couples ( %) were very clear about which partner would inseminate the oocytes. of those couples, two felt that the older partner should donate; two felt that the partner who cared more about a genetic connection to the child should donate; and two felt that the partner with "better genes" should donate. the remaining two couples chose to inseminate equal numbers of oocytes in order to transfer an embryo from each partner. all couples chose an anonymous oocyte donor, two couples chose relatives as their gestational carriers, while the others chose carriers recruited through an agency. conclusions: participants in this study were determined to become parents through assisted reproduction. they had given thoughtful consideration to the medical and psychosocial issues unique to this process including which partner would be the genetic parent, and why. clomiphene superovulation. rb allen, az steiner, rh fogle, mj kalan, rj paulson. ob/gyn, usc keck school of medicine, la, ca, usa; ob/gyn, unc, chapel hill, nc, usa. intro: micro-dose hcg has been demonstrated to increase ovulation and pregnancy rates following clomiphene administration in women resistant to clomiphene. since micro-dose hcg stimulates growth in follicles expressing the lh receptor, we hypothesized that its use following clomiphene in women with unexplained infertility would increase the number of follicles that ovulate and subsequently, pregnancy rates. m m: irb approval was obtained for this prospective pilot study of women with unexplained infertility. on day # a baseline ultrasound was performed and serum fsh and e levels were measured. subjects were given mg of clomiphene daily from days - and then returned for serial ultrasounds on day # . when at least follicles mm were present, iu of hcg im daily was initiated. cycles were monitored by ultrasound every days until a +lh surge occurred. all subjects had previously undergone a cycle using clomiphene alone for superovulation followed by iui and these cycles were used for comparison. results: subjects, aged . ± . yrs (mean±sem) and bmi . ± . kg/m with . ± . yrs of infertility were enrolled. the mean fsh and e levels were . ± . miu/ml and . ± . pg/ml, respectively. there was an average of . ± . days from starting clomiphene to the attainment of follicles mm. out of the subjects had follicles mm by day # . a mean of . ± . days of treatment were required until ovulation occurred. there were twice as many follicles in the micro-dose hcg cycles, although due to the small sample size this did not reach statistical significance ( . ± . follicles, measuring . ± . mm in the micro-dose hcg cycles, compared to . ± . (p= . ), measuring . ± . mm (p= . ) in the control cycles). there was no difference in the endometrial thickness at the time of ovulation between the micro-dose hcg cycles and the control cycles: . ± . mm vs. . ± . mm (p= . ), respectively. all subjects had at least ovulatory sized follicles in the study cycles. % of subjects had an iui performed, however there were no pregnancies resulting from this study. conclusions: ) the addition of micro-dose hcg to clomiphene may allow more follicles to reach an ovulatory size, which should theoretically increase pregnancy rates ) this pilot study demonstrates that adding micro-dose hcg may increase the effectiveness of clomiphene when given in a superovulatory protocol. jessica salas, donald maier, john nulsen, claudio benadiva, lawrence engmann. obstetrics gynecology, university of connecticut, farmington, ct, usa. objective: to evaluate the antepartum, intrapartum, and neonatal complications in patients undergoing ivf using a gnrh-antagonist protocol where a gnrhagonist was used to induce final oocyte maturation. materials and methods: a retrospective review of data from high responders undergoing their st or nd ivf cycle using a gnrh-antagonist protocol who were triggered with leuprolide acetate (study group) or hcg (control) and achieved at least a singleton pregnancy reaching the third trimester. patients younger than years old were included. both groups received luteal phase support with intramuscular progesterone. the study group received estrogen patch supplementation. outcomes measured were antenatal and intrapartum complications, order of gestation, gestational age at delivery, birth weight, neonatal adverse outcomes, and congenital anomalies. pearson chi square, fisher's exact test, or independent t-test were used as appropriate. results: the baseline characteristics were different (table ) . maternal antenatal and intrapartum complications were similar in both groups ( . % vs . %, p= . ). there were more singletons in the study group ( . % vs . %, p< . ). a subgroup analysis of gestational age at delivery, birth weight, neonatal complications and congenital anomalies in singletons showed no difference (table ) . conclusions: this is the first study reporting the maternal and perinatal outcomes after gnrh-agonist trigger. differences in response to ovarian stimulation may dictate use of gnrh-agonist triggering for prevention of ohss, which may explain the differences in baseline characteristics. maternal and neonatal complications remain unaffected. table lupron (n= ) objective: moderate to severe ovarian hyperstimulation syndrome (ohss) occurs in - % of assisted technology cycles and carries the potential for severe complications. traditional management consists of hospitalization with intravenous fluids (ivf), bedrest, and close monitoring. early and aggressive paracentesis is an alternative method of treatment for ohss in an outpatient setting, but the economic consequences of the two treatment regimens have not been compared. design: cost-effectiveness analysis materials and methods: two scenarios, outpatient management with transvaginal paracentesis and conservative therapy with hospitalization, were compared. potential initial outcomes were analyzed for the conservative group to include hospitalization either in a ward hospital bed or the intensive care unit (icu) for an average of seven days. costs included ivf administration, ultrasound, and daily bloodwork. initial outcomes for the outpatient management group included no further therapy beyond the initial transvaginal paracentesis, bloodwork, ivfs, and ultrasound versus admission for an average of three days to a ward bed with similar management. the probability of the negative outcome for the conservative group was set at % (icu admission) and % for the outpatient group (regular hospitalization). results: the cost of conservative therapy including first tier complications ranged from a low of $ , to a high of $ , . the cost of outpatient management with aggressive paracentesis and its first tier complications ranged from a low of $ to a high of $ , . this resulted in an estimated cost burden of $ , to $ , for conservative management with hospitalization. the main improvement factor was that patients in the outpatient management group had a much lower likelihood for prolonged hospitalization than the conservatively managed group. conclusions: aggressive outpatient treatment of moderate to severe ohss with early paracentesis appears to be a cost-effective strategy. induction. zeynep alpay, michael p diamond, michael l kruger, elizabeth e puscheck. obstetrics and gynecology, division of reproductive endocrinology and infertility, hutzel hospital, wayne state university, detroit, mi, usa. introduction: aromatase inhibitors (ai) are a new method of ovulation induction and is proposed to replace clomiphene citrate (cc) due to their reported advantages. their superior effect is thought to be due to up-regulation of the estrogen receptors and increase in the sensitivity of the endometrium, resulting in better proliferation despite low estrogen levels in the circulation. objective: to compare the effect of different ovulation induction methods, including ai, cc and gonadotropins (gt) on the endometrium in infertility patients. methods: we reviewed ovulation induction cycles performed in our institution in the last one-year period retrospectively. there were gt, cc and ai cycles. age, gravida, day (d et) and midcycle (mcet) endometrial thickness, endometrial pattern (ep) on day of hcg injection, endometrial growth during the induction cycle (d-et), which was calculated by the difference between mcet and d et, and the pregnancy rates (pr) were compared. the ep was categorized as type a (homogenous and hyperechogenic), type b (intermediate isoechogenic pattern and a poorly defined central echogenic line), and type c (multilayered triple-line). chi-square, anova, t test and pearson correlation were used for statistical analysis. results: in all cycles, ep was closely related to the d-et (p < . ). this correlation appeared to be more pronounced when observed ep was compared with d-et (r = . ; p < . ) rather than with mcet (r = . ; p = . ). there was a reverse relationship between the age of the patients and ep (r = - . ; p < . ). a negative correlation was also found between the gravida and ep (r = - . ; p < . ). pregnancy rate was significantly correlated with ep (r = . ; p < . ). pregnancy rate was % in type a ep, . % in b, % in c when all cycles were analyzed. aromatase inhibitors and gt treatments each resulted in % type c pattern in contrast to % with cc (p = . for ai vs. cc; p = . for gt vs. cc). the d-et was greater in gt cycles compared with ai or cc (p < . ). conclusion: these results show that ep has a strong correlation with pr in ovulation induction cycles. additionally, ai and gt treatments have similar effects on ep. both ep and the growth of the endometrium during the induction (d-et) are good prognostic variables for the successful pregnancy initiation. ovarian response in patients undergoing ovarian stimulation after myomectomy. hyacinth browne, , desiree mccarthy-keith, , barbara stegmann, , alicia armstrong. , reproductive biology and medicine branch, nichd, nih, bethesda, md, usa; reproductive endocrinology and infertility, walter reed army medical center, washington, dc, usa. objective: the impact of myomectomy on ovarian function has not been wellstudied. other surgical treatments of fibroids, such as hysterectomy and uterine artery embolization, have shown an increase of fsh into the peri-menopausal range. the objective of this study is to examine ovarian response in infertile women undergoing ovarian stimulation after abdominal myomectomy. design: retrospective analysis. materials and methods: a retrospective analysis of all infertile women with known fibroids who had a failed art cycle, from january to , followed by an abdominal myomectomy and a subsequent art cycle was performed. women served as their own controls. ovarian function pre and post-myomectomy was assessed by age, day and fsh levels, days of stimulation, total gonadotropins used, peak estradiol level, number of oocytes retrieved, embryos obtained, and high-grade embryos, and pregnancy outcome. quantitative results are presented as mean + sd. results: four women had a failed art cycle and underwent an abdominal myomectomy prior to a subsequent art cycle. the mean age was and pre-and post-myomectomy, respectively. all subjects had uterine factor infertility. two of these women also had tubal factor infertility, and one had endometriosis and male factor infertility. we found no difference in ovarian response pre and post-myomectomy. pre-myomectomy post-myomectomy age (years) + . + . day fsh (u/l) . + . . + . day fsh (u/l) . + . . + . objective: anti-mullerian hormone ( amh) is produced by developing primordial follicles. amh levels are stable through the menstrual cycle and therefore can be drawn randomly. the objective of this study was to assess the association of anti-mullerian hormone ( amh) and oocytes obtained at ivf retrieval. design: cross-sectional cohort study. materials and methods: the study cohort is comprised of thirty women in cycles of in vitro fertiization (ivf). serum levels antimullerian hormone (amh) were drawn before starting an ovulation induction cycle for ivf. standard ovulation induction was performed with leuprolide flare and miu/ml per day of follicle stimulating hormone. we performed linear regression of log convert d oocyte number against the log converted amh level. serum amh was measured using an enzymatically amplified two-site immunoassay dsl- - active mis/amh elisa. statistical analysis was performed using spss version . . continuous values are presented as mean std error. results: the log number of oocytes retrieved was directly related to the log value of amh (p = . ). conclusions: our data demonstrate an association between serum amh and oocytes produced in response to ovulation induction. using amh levels in addition to fsh levels in evaluating for ovarian reserve and pregnancy outcome may help improve predictions of reproductive performance. support: foundation for reproductive medicine. context: prediction of outcome after in vitro fertilization (ivf) can be difficult due multiple factors. human chorionic gonadotropin (hcg) levels correlate with pregnancy outcome, but data that can be used to easily counsel patients on their possible outcome is lacking. objective: to investigate the use of hcg levels along with other significant factors to predict the likelihood of an ivf pregnancy progressing to the point of detection of cardiac activity by ultrasound design: retrospective data analysis of ivf cycles performed from january to july resulting in pregnancies. multiple logistic regression analysis modeling was performed to determine the factors most predictive of an ongoing early pregnancy and to assess possible confounding variables. setting: an academic fertility center. patients: patients undergoing in vitro fertilization using autologous fresh embryos. intervention: none main outcome measure: pregnancy continuation to the documentation of cardiac activity by ultrasound. results: maternal age, day (post-oocyte aspiration) hcg level, and day hcg levels were significant in predicting pregnancy outcome. day and day hcg levels were highly correlated and can be considered proxies for each other. the most accurate predictive model used only a single day hcg level and maternal age. the type of fertilization method used, the cycle number for that patient, and the number of embryos transferred were not found to be significantly different. ongoing pregnancy rates were directly proportional to day hcg level, and inversely proportional to maternal age. the incidence of multiple pregnancies also increased proportionally to the initial hcg level. % of ongoing pregnancies had hcg level > miu/ml. conclusions: a single day post-oocyte aspiration hcg level and maternal age are the most predictive of an ongoing ivf pregnancy. there was no difference in outcome between fertilization methods or number of embryos transferred. there is no benefit to obtaining serial hcg levels after the initial one. erk activation with u ( m)) reduced fsh stimulated cyclin d mrna expression by %. fsh has also been shown to stimulate mtor, a regulator of growth and proliferation of many cell types. inhibiting mtor activation with nm rapamycin for min significantly reduced fsh-mediated cyclin d mrna expression. dht exposure for hours also inhibited fsh stimulated mtor signaling, as shown by a % reduction in the phosphorylation of its down stream target p s kinase. furthermore, pretreatment with dht resulted in significantly reduced fsh-mediated tsc- phosphorylation, which is an upstream regulator of mtor pathway. further studies revealed that fsh mediated tsc- phosphorylation and mtor signaling is regulated by erk, but independent of akt. based on these results we conclude that elevated reduced metabolites of androgens inhibit fsh-stimulated pka-erk pathway resulting in the inhibition of multiple mitogenic signaling pathways leading to defective follicle maturation culminating in anovulation. thus, rescuing the erk activation might serve as a potential therapeutic target to restore normal granulosa cell proliferation in hyperandrogenic states. (supported by nih grant hd- ). searching pcos-genes: results of a genome screen for pcos in a dutch founder population. olivier valkenburg, annemarie g mulders, aida bertoli-avella, ben a oostra, joop se laven. department of gynecology and obstetrics, erasmusmc, rotterdam, netherlands; department of internal medicine, erasmusmc, rotterdam, netherlands. context: although the etiology of pcos is not yet fully understood, evidence has accumulated for a complex genetic background i.e. a combination of multiple genetic and environmental factors. to reduce genetic heterogeneity, this study was conducted in a genetically isolated population in the netherlands . objective: in order to identify genomic loci that are associated with pcos, a whole genome screen using highly polymorphic microsatellite markers was performed in a founder population. subjects and methods: pcos patients ( rotterdam criteria) were identified in the founder population (rucphen, the netherlands) of ± . inhabitants. patients underwent a standardized screening procedure that included clinical, ultrasound and endocrine evaluation. all patients plus unaffected first-degree family members were genotyped using polymorphic markers on (microsatellites) from the abi prism ® linkage mapping set md- (average spacing cm). association-analysis was performed with the transmission disequilibrium test (tdt, genehunter software). linkage analysis was performed in clusters of or more closely related patients (simwalk ). results: there was an average number of . alleles per polymorphic marker. the tdt identified two non-adjacent markers on chrome (d s and d s ) that showed significant association with pcos (p , ). other markers that showed significant association were positioned on chromosomes (d s ), (d s ), (d s ) , (d s ), (d s ) and seventeen (d s ) (all p values . ). linkage analysis in sub-pedigrees revealed no significant linkage for these, or other, loci with pcos. moreover the results of a prior report of linkage of a marker on chromosome (d s ) could not be confirmed. conclusions: the lack of consistent results suggests the absence of a consistent genetic background in this select group of pcos patients. this supports the hypothesis of a complex genetic background for pcos that allows relatively small contributions of multiple risk-genes to be involved in the pathogenesis of this syndrome. in this founder-population the genetic heterogeneity was not sufficiently reduced to find risk-loci in a genome wide screen using highly polymorphic markers with an average spacing of cm. objectives: to objectively quantify uterine and endometrial blood flow in women with polycystic ovarian syndrome (pcos) and to examine if this was different in women with different phenotypic expressions of the disease. methods: transvaginal d and d ultrasound was performed in women with pcos, as defined by the rotterdam criteria, and sub-group analysis conducted based on the subjects' body mass index (bmi), ovulation status, and hirsutism score. anova was used to compare the mean values between the groups. results: pcos women with clinical hyperandrogenaemia had significantly lower endometrial and subendometrial blood flow than their anovulatory normoandrogenic counterparts (table ). there were no differences between lean and obese women or between anovulatory and ovulatory women with pcos. the pulsed wave doppler parameters were similar in all three phenotypic groups. conclusions: hirsute women with pcos have impaired endometrial perfusion compared to their normoandrogenic counterparts which is only evident with d ultrasound and not conventional pulsed wave doppler. nusayba a bagegni, jill blaine, anuja dokras. obstetrics gynecology, university of iowa hospitals clinics, iowa city, ia, usa; obstetrics gynecology, university of pennsylvania, philadelphia, pa, usa. background: there is conflicting evidence on the association between pcos and early and late obstetric complications. it is unclear if the reported risks are independent of bmi, preexisting hypertension and diabetes. we examined the risk of early and late obstetrical complications in a large group of women with pcos compared to controls. methods: we reviewed pregnancy records of women with pcos (rotterdam criteria, n= ) and controls (tubal infertility, n= ) after in vitro fertilization at university of iowa from - . the wilcoxon rank sum test and fisher's exact test were used to evaluate differences between variables and logistic regression analysis was used to determine the independent risk of pcos. results: subject demographics and medical history are shown in table. the first trimester miscarriage rate was % in women with pcos and % in controls. after logistic regression analysis pcos was not associated with miscarriage (p= . ). the prevalence of gestational dm (gdm) was similar in both groups % pcos vs % controls. pcos was not associated with gdm after adjusting for age and bmi (p= . ). however, bmi was significantly associated with gdm after adjusting for age and pcos (p= . ). risk of both pre-eclampsia and pih was % in pcos and % in controls, but not statistically significant after adjusting for age, bmi and twin gestation. preexisting htn showed a significant association with preeclampsia (p< . ). there was no significant difference in preterm delivery, cesarean section, twin gestation, intrauterine fetal death and intrauterine growth restriction in the groups. conclusion: despite adequate power, our study did not detect an increased risk of miscarriage in women with pcos. obesity was a significant contributor to late obstetric complications, namely gdm. these findings may warrant aggressive counseling of women with pcos on the potential benefits of weight loss prior to pregnancy. objective: to assess the prevalence of polycystic appearing ovaries (pcao) as defined by the rotterdam criteria; and to determine whether metabolic parameters differ between regularly cycling women with pcao and those with normal ovaries. studies have demonstrated a population frequency of pcao of - %, with - % among women with regular cycles. most were performed in a young reproductive age population; none examined the impact of age. all were performed prior to adoption of the rotterdam criteria. recent studies have shown an increased prevalence of metabolic syndrome among women diagnosed with polycystic ovarian syndrome (pcos). however studies focused on women in their s found no increase in bmi or fasting insulin with pcao but regular menses. participants: women aged - with regular cycles (every - days) enrolled in the ova study, a population-based study of ovarian aging. each subject underwent a transvaginal ultrasound for assessment of ovarian volume and antral follicle count (afc). outcomes collected included waist measurements and hdl, ldl, total cholesterol, triglycerides, fasting glucose and insulin. pcao were determined by the rotterdam criteria (afc of on one ovary or ovarian volume > cc). student's t-test and chi-square tests were used to assess differences between those with and without pcao for continuous and categorical variables, respectively. the prevalence of pcao decreased with increasing age (table ) . of the women with pcao, % met the afc criterion only, while % met the volume criterion only, and % met both. women with and without pcao did not differ in waist measurements, or in fasting lipids, insulin, or glucose. the rotterdam criteria, while less subjective than those described by adams in , have led to an increased prevalence of pcao among women with regular menses, over / of women in their s. women with pcao do not differ from those with normal ovaries in metabolic parameters associated with pcos. consideration should be given to adopting an age-adjusted criterion for afc, or a combination of afc and ovarian volume, for diagnosing pcos. background: pcos, especially accompanied by obesity, has been reported to be associated with a characteristic dyslipidemia comprising elevated triglycerides (tgs) and depressed hdl, especially the hdl- fraction. this is an atherogenic profile; in fact, studies suggest low hdl- may correlate most strongly with cardiovascular disease risk. weight loss is a mainstay of treatment and improves all manifestations of the disease, but the optimal diet to recommend remains undetermined. preliminary studies show a high protein diet may improve total hdl levels and insulin responses. however, the effect of weight loss and dietary composition on hdl- levels in pcos has not been investigated. objective: to evaluate the fasting lipid profile in newly diagnosed obese pcos patients and to determine the effects of a high-protein diet with or without metformin on weight loss, hdl- and other lipoproteins, and menstrual cyclicity. methods: in this pilot retrospective observational study, the fasting lipid profile of obese women newly diagnosed with pcos was determined. they were then placed on a high protein ( - g/day), low carbohydrate ( - g/day) diet with or without metformin ( and %, respectively) and followed monthly for an average of months (range - ). results: at diagnosis, % had low hdl and % had low hdl- ; only % had elevated tgs. on the diet, the patients demonstrated an average weight loss of . lbs ( . to . , p< . ) and decreased bmi of . kg/m ( . to . , p< . ). hdl levels increased significantly ( % increase from . to . , p< . ), especially the hdl- fraction ( % increase from . to . , p< . ). triglycerides decreased as well ( . to . , p< . ). ldl decreased but did not reach statistical significance. resumed menstrual cycles, were started on oral contraceptives, and had a hysterectomy. pregnancies occurred. no difference was seen with metformin use. conclusion: a majority demonstrated decreased hdl and hdl- at diagnosis. the high protein diet resulted in significant weight loss and improvement in hdl- levels, as well as improvements in total hdl, tgs and menstrual cyclicity. metformin produced no added benefit. prospective trials based on these data will help determine the optimal diet to reduce the significant short-and long-term morbidity in the large population of women with pcos. resistin is an adipokine that has been associated with obesity and insulin resistance in animal models. studies on the role of resistin on insulin resistance in humans have been controversial. recently resistin has been shown to exert atherosclerotic effects and elevated resistin levels have been observed in women with coronary heart disease (chd). women with polycystic ovary syndrome (pcos) are at high risk for chd. our present study investigates potential association of resistin and markers of inflammation, c-reactive protein (crp) and insulin resistance in women with pcos. methods: thirty two women with pcos participated in the study. all were hirsute and had irregular cycles. nineteen women with normal ovulatory cycles who matched the pcos patients in bmi served as controls. fasting glucose, insulin, resistin and crp levels were measured in all women. after a high carbohydrate diet for days, a standard oral glucose tolerance test (ogtt) was performed. blood samples were collected for glucose and insulin before and , and hrs after g oral glucose. the area under the curve (auc) for insulin and glucose was calculated. women with overt diabetes were excluded from the study. results: fasting insulin levels ( . + . μu [±se] /ml) and insulin response to oral glucose (auc) ( . + . μu/ml) were higher (p < . ) in women with pcos compared to controls. all were insulin resistant with homa-ir value > . mol x μu / l . resistin levels in women with pcos ( . ± . ng/ml) was significantly (p < . ) higher compared to control women ( . ± . ng/ml). crp levels in women with pcos ( . ± ng/ml) was also significantly (p < . ) higher than the controls ( . ± ng/ml). there was a significant positive correlation between resistin and crp levels (r = . , p < . ). there was no correlation between resistin levels and fasting insulin levels or insulin auc. there was also no correlation between resistin levels and fasting glucose or glucose auc. conclusions: our results indicate that ( )women with pcos and insulin resistance have increased resistin levels, ( ) there is a strong association between resistin and crp ( ) elevated resistin and crp levels may predict women who are at increased risk for chd ( ) ) it is unlikely that resistin plays a major role on insulin resistance in women with pcos. vuk p jovanovic, enrico carmina, prati vardhana, michel ferin, rogerio a lobo. department of obstetrics and gynecology, columbia university, new york, ny, usa; department of internal medicine, university of palermo, palermo, italy. current diagnostic criteria for pcos includes both ovulatory (ov) and anovulatory or "classic" (c) phenotypes. in an effort to further characterize differences and /or similarities between these phenotypes, we studied hyperandrogenic women with pcos (age . ± . , bmi . ± . ) and age matched controls (age . ± . , bmi . ± . ). women with pcos were divided into weight matched (ov) n= and (c) n= groups. fat and weight distribution were assessed by dexa (total fat, r fat, trunk fat) as well as fasting levels of lh, e , mis/amh, kisspeptin and testosterone, the adipocytokines (leptin, adiponectin, visfatin and retinol-binding-protein- rbp ) and serum glucose, insulin and crp. the hyperandrogenic pcos groups had characteristically altered hormone profiles compared to matched controls. although total fat mass was comparable, women with c-pcos had a significantly larger waist circumference ( . . vs. cm, p< . ) trunk fat, r fat and %trunk and %r fat compared to ov-pcos (p< . ). leptin, rbp and visfatin did not significantly differ among the pcos subgroups although adiponectin was lower in the c-pcos group (p< . ). quicki was significantly lower in c-pcos ( . ± . vs. . ± . , p< . ) and insulin was higher ( . ± . vs. . ± . , p< . ). serum lh was also higher ( . ± . vs. . ± . , p< . ) but kisspeptin, testosterone and estradiol were similar. mis/amh ( . ± . vs. . ± . ng/ml, not significant) and crp ( . ± . vs. . ± . , p< . ) were higher in c-pcos. significant correlations (p< . ) were noted among kisspeptin/rbp (r . ), mis/testosterone (r . ), insulin/ %trunk fat (r . , p< , ), total fat/leptin (r . , p< . ), %trunk fat/adiponectin (r - . ). in conclusion, women with c-pcos when compared to similarly hyperandrogenic women with ov-pcos with similar bmi, have increased abdominal fat, and appear to have differences in serum lh, crp and increased insulin resistance. the latter, as well as subtle differences in the adipocytokines may explain these differences in anthropometric findings. problems of normal oogenesis and folliculogenesis but also disturbed oogenesis and folliculogenesis in polycystic ovaries are not fully understood. oocyte specific genes play an essential role in oogenesis and folliculogenesis. there are suggestions about possible role of some oocyte specific genes in etiopathophysiology of pcos. zona pellucida gene (zp ) is recently identified gene, which belongs to zona pellucida genes such as zp , zp and zp . the role of zp , contrary to above-mentioned other zp genes is not well described. the aim of this study was to analyze zp coding sequence and expression in patients with polycystic ovary syndrome. material included blood received from patients (mean age , +/- , years; mean bmi , +/- , kg/m ) with polycystic ovary syndrome. all patients with pcos were diagnosed with the use of eshre/asrm criteria from . dna was isolated from blood cells( after separation of blood cells from serum) using a dna isolation kit (qiagen ). genomic dna was used for in vitro amplification by pcr with a specific set of primers complementary to the coding sequence of the zp gene. products from each pcr reaction were examined by sscp method. samples with changes detected by sscp in comparison to control probes were cloned into plasmid vector and then automatically sequenced from a total of patient samples with pcos, we identified nucleotide changes in the zp coding seguence : silent nucleotide changes in exons , , , , and nucleotide change in the exon ( position , t>g). the mutation in exon ( t>g ) results in substitution of cystein for glycin of amino acid in position of zp protein. in summary, our data demonstrate that zp nucleotide changes account for % of patients with pcos. relationship between serum mullerian inhibiting substance levels and insulin in women with polycystic ovary syndrome. rebecca a chilvers, shilla chakrabarthy, summer james, xin ma, manubai nagamani. ob/gyn, university of texas medical branch, galveston, tx, usa. müllerian-inhibiting substance (mis) is a member of the transforming growth factor-superfamily of growth factors. it is expressed exclusively in granulosa cells and is believed to play a role in the regulation of follicle selection and maturation. women with pcos have anovulation and most of them have hyperinsulinemia. the purpose of our study is to investigate possible association between insulin and mis in the dysregulation of folliculogenesis in pcos. methods: twenty one women with pcos who had anovulatory cycles and hyperandrogenism were recruited for the study. sixteen women with ovulatory cycles and matched the pcos patients in age and bmi served as controls. an oral glucose tolerance test (ogtt) was performed in all women. blood samples were obtained at fasting and , and hours after glucose ingestion for measurement of glucose and insulin. to investigate the effect of hyperinsulinemia on mis secretion, mis levels were measured in ten patients during the ogtt. fasting mis, testosterone, dheas, fsh, and lh levels were measured in all patients. results: fasting insulin levels ( . + . μu/ml) vs . + μ u/ml [+ se]) (p < . ) and area under the curve (auc) of insulin ( . + . vs . + . μu/ml) (p < . ) were significantly increased in women with pcos compared to control women, while the glucose levels were normal indicating insulin resistance. mis levels were significantly (p < . ) increased in women with pcos ( . + . ng/ml) compared to controls ( . + . ng/ml). there was no correlation between age and mis levels in pcos patients while there was a highly significant negative correlation between the age and mis levels in the control women ( r = - . , p< . ). there was significant negative correlation between mis and fasting insulin levels (r = - . , p < . ) and insulin auc during the ogtt (r = - . , p < . ). there were no changes in mis levels during ogtt. conclusions: results of our study indicate that in women with pcos, ( ) there is an increase in secretion of mis, ( ) higher insulin levels are associated with lower mis levels, ( ) acute increase in insulin levels has no effect on mis levels, ( ) lower mis levels in women with severe hyperinsulinemia could be due to associated increased follicular atresia and a decrease in the ovarian reserve. further studies are needed to investigate the role of insulin on mis secretion. hiroyuki asakura, noriko tanaka, kyoko nishio. ohgimachi ladies' clinic, osaka, japan. objective: elevation of serum anti-müllerian hormone (amh) levels among polycystic ovary syndrome (pcos) patients has been reported. however, the regulatory factors of amh in pcos remain unknown. we examined correlations between amh values and various indices of insulin resistance in pcos women. methods: infertile women compatible with rotterdam criteria for pcos were recruited under informed consent. the subjects underwent g oral glucose tolerance test ( ggtt, sampling at , , minutes), and -step elisa for amh (sensitivity . pmol/l, inter intra-assay c.v. : . %, . %, respectively). p< . was considered as statistical significance. results: average amh level of the subjects (age: . + . years, bmi: . + . kg/m , mean+s.d.) was . + . pmol/l, which was higher than normo-ovulatory women ( . + . pmol/l, n= ). amh levels had significant positive correlation with total ovarian volume by ultrasound ( . + . cc), but not with bmi, waist-hip ratio ( . + . ), and levels of serum lh ( . + . iu/l), total testosterone ( . + . ng/dl). although fasting glucose levels ( . + . mg/dl) were positively correlated with total ovarian volume (r= . ), amh levels had no significant correlation with fasting insulin ( . + . iu/l), fasting glucose/insulin ration ( . + . ), homa-ir ( . + . ), and the sum of insulin levels ( . + . iu/l)during ggtt. conclusions: increased serum amh level of pcos and its positive correlation with total ovarian volume implies that determination of serum amh level would aid in confirming diagnosis of the ovulatory disorder. absence of relationship between amh and various clinical indices of insulin resistance suggests alternative regulatory factors of amh gene expression in the follicular compartment. reproductive tissues. amisra a nikrodhanond, keeley l mui, helen h kim. ob/gyn, university of chicago, chicago, il, usa. background: although primarily a neuroendocrine hormone, gonadotropinreleasing hormone (gnrh) is also produced in reproductive tissues, where it acts locally. the study of gnrh regulation in these tissues is limited by low levels of expression. objective: to elucidate mechanisms that regulate gnrh expression in male tissues, our objective was to identify regions of the gnrh gene that target expression in vivo. methods: transgenic mice were generated with different fragments of the mouse gnrh gene promoter (- /+ and - /+ bps), fused to the luciferase reporter gene. in these mice, luciferase activity (detected as light) reflects gnrh promoter activity. using bioluminescent imaging, gnrh promoter activity was assayed in live gnrh-luc mice and in their reproductive tissues ex vivo. to confirm imaging results, luciferase activity was also measured as relative light units (rlu) in tissue homogenates. for each dna construct, male mice were examined. with whole-body imaging, bioluminescence was detected in the genital region of the gnrh-luc transgenic mice (fig.a) . in mice that incorporated the - luc transgene, examination of reproductive tissues ex vivo revealed bioluminescence in the prostate and penis, but not in the seminal vesicles, epididymis, or testes (fig.b) . in contrast, in the - luc mice, bioluminescence was detected in the testes, but not in other tissues (fig.c) . examination of luciferase activity in tissue homogenates from - luc mice confirmed gnrh promoter activity in the prostate ( ± rlu) and penis ( ± rlu) and absence in the testes ( ± rlu). in the - luc mice, however, luciferase activity was detected only in the testes ( ± rlu) and not in the prostate ( ± rlu) or penis ( ± rlu). conclusions: our studies demonstrate that gnrh is present in male reproductive tissues, but may be differentially regulated in the testes vs. prostate/penis. promoter elements, contained within the proximal - bp of the mouse gnrh gene, are sufficient to mediate testicular gnrh expression while - bp of the gene promoter are necessary to direct expression to the prostate and penis. characterization of mouse ringo/speedy homologues. z walton, s uckac, o guzeloglu-kayisli, md lalioti, d sakkas, e seli. ob gyn, yale u., new haven, ct, usa. introduction: ringo/speedy (ringo/spy) is a recently discovered cyclindependent kinase (cdk) activator that functions similar to cyclins in controlling the cell cycle. ringo/spy plays a crucial role during meiotic maturation in xenopus by inducing meiotic g /m progression and germinal vesicle breakdown (gvbd). more recently, padmanabhan and richter demonstrated that ringo/spy mrna is repressed in the xenopus oocyte cytoplasm by pumilio . upon meiotic reactivation, pumilio loses its interactions with ringo/spy permitting its translation. ringo/spy is then expressed, leading to activation of cpeb by phosphorylation, which in turn elicits polyadenylation and translation activation of the mrna for a critical oocyte maturation factor, the mos kinase. in this study, we investigated the expression of ringo/spy in mouse. methods: ten different somatic tissues, testes, and ovaries were tested by reverse transcription-polymerase chain reaction (rt-pcr) for the expression of ringo/spy homologues and their alternative splicing variants. ringo/spy mrna expression was also tested in prophase i (pi) and metaphase ii (mii) oocytes, -cell, -cell, -cell, -cell embryos and blastocysts. amplification with actin primers provided a positive control and allowed semi-quantitative analysis. results: we analyzed the two previously identified homologues of ringo/spy (a and b). the two alternative splicing variants of ringo/spy a (a and a ) were separately studied for their expression profile. we also identified an additional alternative splicing variant of ringo/spy b and evaluated similarly. ringo/spy a ( and ) were expressed in testes, ovaries, and certain somatic tissues including brain, and spleen. ringo/spy b (both variants) were only expressed in testis. ringo/spy a or b were not present in mouse oocytes or early embryos. conclusions: our findings indicate that the previopusly described ringo/spy homologues a and b are not expressed in mouse oocytes or early embryos suggesting that either an alternative cdk-activator or a yet to be identified homologue of ringo/spy may be mediating meiotic g /m progression in mouse. testis specific expression of ringo/spy b, and previously described role of ringo/spy in meiosis suggests a role for this protein in male gametogenesis in mouse. treated with glyburide. jennifer aguayo, gladys a ramos, alethea hanley, carri r warshak, thomas r moore. reproductive medicine, university of california, san diego, san diego, ca, usa. objective: to determine the risk factors that may predict inadequate response to first-line glyburide monotherapy in pregnant women with type diabetes mellitus (dm) or gdm and to assess if non-responders are at increased risk of adverse pregnancy and neonatal outcomes. study design: this was a retrospective cohort of women diagnosed with type ii or gdm initially treated with glyburide at a single institution from - . maternal characteristics, adequate glycemic control defined as more than % of fasting glucose < mg/dl and one-hour post-prandials < mg/dl, and neonatal outcomes were assessed. non-responders were defined as failure to achieve adequate glycemic control on maximum daily doses of glyburide or intolerance due to side effects, necessitating switch to insulin therapy. statistical methods included bivariate analyses. results: of the women initially treated with glyburide, ( %) failed to achieve adequate glycemic control or did not tolerate glyburide. reasons for glyburide non-response were maximum dose ( mg/d) reached ( %), maternal hypoglycemia ( %) and other side effects ( %). there were no statistically significant differences between non-responders and responders with respect to family history of diabetes ( % vs. %, p= . ), prior history of gdm ( % vs. %, p= . ) and macrosomia (> g) ( % vs. %, p= . ) or hour glucose challenge test ( + vs. + mg/dl, p= . ). non-responders had a higher rate of obesity (bmi> ) ( % vs. %, p= . ) and earlier gestational age at initiation of therapy ( + . vs. + . wks, p= . ). there were no differences between the groups in the mean -week fasting ( + vs. + mg/dl, p= . ) and post-prandial glucose values ( + vs. + mg/dl, p= . ). no significant differences were observed in incidence of pre-eclampsia, primary cesarean delivery or birth weight. neonatal outcomes including ponderal index, neonatal hypoglycemia, nicu admission, and birth injuries also did not differ between the groups. conclusion: women who are obese and who require earlier initiation of glyburide therapy are at increased risk of non-response to glyburide monotherapy. however, despite non-response, these women can be managed with subsequent insulin therapy to achieve similar glycemic control and pregnancy and neonatal outcomes. a different diagnostic strategy using the gram, -hour glucose tolerance test for the diagnosis of gestational diabetes mellitus. yvonne w cheng, ingrid block-kurbisch, jennifer lydell, aaron b caughey. obstetrics, gynecology and reproductive sciences, university of california, san francisco, san francisco, ca, usa. objective: to examine whether a simplified strategy using the gm, -hour glucose tolerance test (gtt) may be useful for the diagnosis of gestational diabetes mellitus (gdm). methods: this is a retrospective cohort study of women with singleton pregnancy who received the -gm, -hour glucose challenging test (gct) for initial screening and the gm, -hour gtt as confirmatory tests for the diagnosis of gdm between and . various combinations of the gm, -hour gtt results were examined and compared to the diagnostic criteria for gdm established by the carpenter and coustan criteria using the receiver-operator characteristic (roc) curves. perinatal outcomes of women who would have had a false-positive or false-negative test results were compared to those who did not have gdm using the carpenter and coustan criteria. potential confounding factors were controlled for using multivariable regression models. results: , women had gm, -hour gtt results available for analysis during the study period. using gdm diagnosed by the carpenter and coustan criteria as reference, various diagnostic strategies was compared using roc curves (figure ) . summation of the -hour and -hour gtt results with a diagnostic threshold of mg/dl yielded the most optimal balance between sensitivity ( . %) and specificity ( . %). when compared to women without gdm, women who were diagnosed with gdm by c c criteria but not by summation of -hour and -hour gtts had higher odds of operative vaginal delivery (aor= . , % confidence interval [ci] . - . ) and neonatal birthweight > gm (aor= . , % ci . - . ). conclusion: using only the summation of only the -hour and -hour gtt results of the gm -hour gtt offers an alternative test strategy which may be more convenient and less costly for the diagnosis of gdm. however, women who would have gdm by the carpenter and coustan criteria but not by the summation method have higher odds of having an operative vaginal delivery and neonatal birthweight > gm. outcome of induction of labor indicated for term prom among women with or without a uterine scar. yifat ochshorn, avital skornick rapaport, adi reches, joseph b lessing, ariel many. obstetrics gynecology, lis maternity hospital, tel aviv sourasky medical center, tel aviv, israel. objective: we aimed at comparing the outcome of induced term deliveries presenting with prom with or without a previous uterine scar. methods: the computerized files of women delivered following induction of labor due to term prom, were reviewed. perinatal outcome parameters such as the mode of delivery, indication for cesarean section, rate of low ' apgar scores and nicu admissions were compared between women with and without a previous cesarean. results: during the study period women delivered in our institution following prom and induction of labor , of them had a previous uterine scar. parturients of both groups (with and without a scar) were similar with regard to age, gestational age at delivery and parity. cesarean section rate was higher for the previous scar group ( % vs %, p< . ). the most common indication for cesarean was arrest of dilatation and/or descent among women with previous scar accounting for % and % (p< . ) for women with and with no uterine scar, respectively. no differences were noted in neonatal outcome parameters such as rate of low ' apgar scores and nicu admission rate between the two groups. conclusion: induction of labor due to prom culminates in higher cesarean rate in women with one previous scar compared with parturients with no scar. perinatal outcome is similar between the two groups. acid base balance and immediate perinatal outcome of vertex compared with breech presentation in elective cesarean section. avital skornick rapaport, yifat ochshorn, joseph b lessing, yuval yaron, michael kupferminc, ariel many. obstetrics gynecology, lis maternity hospital, tel aviv sourasky medical center, tel aviv, israel. backround: apgar scores, umbilical blood ph and bicarbonate are generally lower and pco levels are higher in vaginally delivered breech neonates compared to cephalic deliveries. although cesarean delivery improved apgar scores there is a debate wether it improved the acid base status. this retroprospective study compared umbilical cord blood acid-base values and perinatal outcome of elective cesarean breech-delivery with those of elective cephalic cesarean delivery and to determine whether a different metabolic status and perinatal outcome should be expected in neonates in breech presentation. study design: the study group included singleton pregnancies delivered by elective cesarean section at term between january and march . computerized files of singleton breech presentation elective cesarean sections were compared to those of singleton vertex neonates delivered by elective cesarean section. demographic data included: maternal age, pregnancy week at delivery and parity. perinatal outcome measures checked were: birth weight, apgar scores at 'and ', umbilical cord venous and arterial ph and base excess. results: during the period between january and march there were singleton elective cesarean sections, of them were breech and vertex. the mean age, gravida and parity were significantly different between groups ( . vs. . , . vs. . . and . vs. . respectively, p< . ). the birth weight was significantly different - . gram for the vertex and . gram in the breech deliveries (p< . ) there were no differences in either apgar scores or umbilical ph between the breech and vertex neonates delivered by cesarean section at term ( - + w). venous and arterial po and pco levels were significantly different, though the differences were very small and we doubt if these differences have any clinical importance. conclusion: although vaginal breech deliveries are associated with increased risk of asphyxia during delivery, elective cesarean breech deliveries are not at increased risk for lower ph levels or apgar scores. the clinical significance of bleeding during the second trimester of pregnancy. arie koifman, amalia levi, yaron zaulan, avi harlev, eyal sheiner. obstetrics gynecology, soroka university medical center, ben gurion university of the negev, beer-sheva, israel; epidemiology and health services evaluation, faculty of health sciences, ben gurion university of the negev, beer-sheva, israel. objective: this study aimed at investigating clinical importance and pregnancy outcome in women suffering from bleeding during the second half of their pregnancies. methods: a population based study including all deliveries which took place in the soroka university medical center between the years - were examined. comparison was performed between patients with and without second trimester bleeding pregnancies terminated before weeks, multiple gestations and women lacking prenatal care were excluded . stratified analysis, using the mantel-haenszel technique, and a multiple logistic regression model were performed . results: during the study period, , singleton deliveries occurred in our institute. of these, ( . %) were complicated with bleeding upon admission during the second half of pregnancy. the cases were attributed to placental abruption ( . %; n= ) and placenta previa ( . %; n= ). independent risk factors associated with bleeding, were oligohydramnios, polyhydramnions, (odds ratio [or]= . ; % confidence interval [ci] . - . ; p= and . ; . - . ;p< . respectively ), suspected intra uterine growth restriction (iugr, . ; . - . ; p<. ), gestational age, previos abortions and maternal age. these patients subsequently were more likely to deliver by cesarean section (cs, . % vs. . %, or= . ; %ci . - . ; p< . ). perinatal mortality among patients admitted due to second half bleeding was significantly higher as compared to patients without bleeding (p<. ). conclusion: bleeding upon admission during the second half of pregnancy is an independent risk factor for perinatal mortality. careful surveillance, including fetal monitoring, is suggested in these cases in order to reduce the adverse perinatal outcome. crude and adjusted odds ratios for perinatal mortality among patients with vaginal bleeding. characteristics or % ci p crude or for perinatal mortality . . - . < . or adjusted for: < . iugr . . - . < . oligohydramnios . . - . < . premature rupture of membranes . . - . < . the predictors included neonatal pi, bmi, or birthweight while the outcomes included hypoglycemia, shoulder dystocia, acidemia and hyperbilirubenemia. roc curve analyses were utilized to evaluate the relationship between sensitivity and specificity for each of the predictors and outcomes, with an area under the curve (auc) significantly greater than . indicating a screening test that is better than chance. results: neonatal pi, bmi and birthweight are poor predictors of nicu admission, acidemia and hypoglycemia with all auc values not statistically significantly different than chance alone. they are a reasonable predictor of shoulder dystocia, with birthweight functioning the best (p< . ). in general, neonatal anthropometric measurements do not appear to be good predictors of short term neonatal outcomes. although they are a reasonable predictor of shoulder dystocia, they are not useful clinically for this outcome since they cannot be calculated until after birth. in future studies, new models of neonatal anthropometrics should be created to better predict adverse neonatal outcomes. neonatal anthropometric measurements and their relationship to perinatal outcomes expressed as auc and % confidence intervals history of at least one spontaneous preterm delivery were offered protocolbased prenatal care including first-or second trimester bacterial vaginosis screening, repeated ultrasound cervical length assessment, and supportive care by specialized nurses. women with a positive test for bacterial vaginosis were treated with oral metronidazol therapy, and women with a cervical length below . cm (before weeks of gestation) underwent a vaginal cerclage. progesterone administration was not provided. data on obstetrical and medical history, pregnancy, delivery and neonatal outcome were prospectively collected and evaluated. results: in total, pregnant women were prospectively followedup. eighty-seven women had experienced a preterm delivery in their last pregnancy, of whom had delivered before weeks. three women were diagnosed previously with a bicornual or septal uterus. twelve women received metronidazol therapy and women underwent a vaginal cerclage. eighty-four women ( . %) delivered at weeks or beyond. one-hundred and three women ( . %) delivered no earlier than weeks, and only one woman delivered at weeks. two women gave birth to a twin (at and weeks, respectively). two neonatal deaths due to pulmonary hypoplasia were recorded. conclusion: women at high risk of preterm delivery who were offered protocol-based prenatal care not including progesterone therapy had a better pregnancy outcome than would be expected from previous studies. our findings may question the reported beneficial effects of prenatal progesterone administration. premature rupture of membranes. tracy a manuck, alexandra g eller, m sean esplin, robert m silver. obstetrics gynecology, university of utah health sciences, salt lake city, ut, usa. objective: historically, outcomes following expectant management of midtrimester preterm premature rupture of membranes (pprom) have been uniformly poor. thus, many patients elected pregnancy termination. however, outcomes may be improved with recent advances in neonatal medicine. our purpose was to assess outcomes in expectantly managed early pprom in an era of improved maternal and neonatal care. study design: this is a retrospective cohort of patients from tertiary healthcare systems from - experiencing pprom . weeks gestation. patients electing immediate termination of pregnancy, carrying fetus(es) with lethal anomalies, or delivering within hours of pprom were excluded. survival without major morbidity was the primary outcome. data were analyzed using student t-test and chi-square as appropriate. results: a total of women carrying fetuses ( singleton, twin, triplet, quadruplet) met inclusion criteria. only the fetus in the "ruptured sac" was studied. pprom occurred at a mean of . (+/- . , range . - ) weeks. the average latency period was . (+/- . , range . - ) days, with a mean delivery gestational age of . (+/- . , range . - ) weeks. this communication provides the first report of fundal uterine necrosis following placement of multiple uterine compression sutures. only two previous published cases report occurrence of uterine necrosis following application of a uterine compression suture --both identified as the b-lynch technique --and neither of these cases report necrosis confined to the fundus. in our case, uterine atony was refractory to pharmacologic therapy and manual compression of the uterus appeared to decrease the amount of blood loss. therefore, we applied a traditional b-lynch which effectively contracted the majority of the uterus, while the fundus remained atonic. a second horizontal, square suture was then placed between the cornua and carried over the fundus (see figure one: red reflects b-lynch suture; blue represents second fundal, square stitch). the patient subsequently experienced significant, persistent fevers despite antibiotic therapy. a repeat laparotomy was performed, at which time we found uterine necrosis confined to the uterine fundus (see figure : photograph taken at the time of hysterectomy). the placenta showed no histologic evidence of chorioamniotis --hence, the patient's main risk factor for fundal necrosis was the compression sutures. physicians should be aware of the risk of uterine necrosis, especially after placement of multiple compression sutures and, more specifically, after placement of a compression suture confined to the uterine fundus. background: migraines are far more common in women than in men. the incidence of migraines increases in girls after puberty, reaching an incidence of % in women who experience migraine at least once a year around middle age. migraine has been postulated as one of the major risk factors for stroke during pregnancy and the puerperium.triptans are a class of serotonin receptor agonists used in the treatment of migraine headaches. triptans administered in combination with other drugs have been known to precipitate serotonin syndrome, a rare but potentially life-threatening condition clinically manifested by a triad consisting of mental-status changes, autonomic hyperactivity, and neuromuscular abnormalities. objective: to determine whether triptan monotherapy is associated with serotonin syndrome methods: using data mining techniques we analyzed the entire food and drug administration's (fda) adverse event reporting system (aers) database. results: after excluding reports of concomitant serotonergic medication or other potentially confounding medication, eleven reports remained of serotonin syndrome associated with triptan use without concomitant serotonergic medication. the mean age for these eleven cases was . years; nine cases occurred in females and two occurred in males. there were no apparent instances of overdose among these eleven cases. conclusions: serotonin syndrome is a rare but serious occurrence with the use of triptan monotherapy. such cases were seen with eletriptan, rizatriptan, sumatriptan, and zolmitriptan. because of the spontaneous nature of voluntary reporting to aers, the actual number of occurrences of serotonin syndrome in patients using triptans is probably higher and cannot be assessed from aers data. users of triptan in combination with an ssri or snri should be warned of this rare but serious adverse effect. during periods of drug initiation, dose escalation, or the addition of another serotonergic agent, patients should be particularly vigilant for symptoms of concern and seek urgent medical attention if any occur. the offending agents should be withdrawn and the patients closely monitored and treated with supportive measures as required. rising maternal mortality in the midst of modern technology. oormila p kovilam, jane khoury, padmini c sekar, ralph c buncher. obstetrics gynecology, university of cincinnati, cincinnati, oh, usa; center for epidemiology and biostatistics, cincinnati children's hospital medical center, cincinnati, oh, usa; environmental health, university of cincinnatic, cincinnati, oh, usa. introduction: maternal mortality rate (mmr) is an index of overall wellbeing of the community and safe motherhood should be given utmost priority in obstetric care. as per , who estimates % of maternal mortality occurs in developed countries . this rate has established without much decline in recent years. the mmr for ohio for to was reported by cdc to be . per , live births with % confidence interval . to . . objective: our objective was to audit the trend in maternal mortality in the state of ohio for the last years. methods: information from the death certificates completed by attending physicians, medical examiners, coroners, funeral directors filed with ohio state registration offices were analyzed. we only used women who were residents of the state of ohio at the time of death, and cause of death was coded as a "complication of pregnancy, childbirth and the puerperium", icd codes to and icd codes o to o . five year maternal mortality pattern and long term trend was assessed. results: the number of maternal deaths recorded as due to pregnancy complications varied over the years from a high of in to a low of in and . in general the five-year mortality rate was decreasing over time until the last four years to , when we may be seeing an upward trend compared to to (p= . ). the rates and associated % confidence intervals (ci) are shown in the table below. years mmr % ci ci - ci . . - . ci - . . - . - . . - . - . . - . - . . - . the rate of maternal mortality is on the rise after a period of downward trend. we should develop a multidisciplinary approach to analyze and target the root causes of maternal death. introduction. women with thrombophilia are at higher risk of vte during pregnancy due to the acquired hypercoagulable state. it is likely that not only maternal veins but also placental vessels are more prone to the development of thrombosis. our objective was to compare maternal and fetal placental circulation disorders in women with intra-uterine fetal death (iufd) and thrombophilia and women without thrombophilia. methods. in a dutch multi-centre study on iufd, during the period - we studied singleton deaths > weeks of gestation for which the diagnosis of iufd was determined before labour. factor v leiden and prothrombin g a were tested at induction of labour. panel classification of cause according to the tulip classification was performed by assessors after individual investigation of structured patient information. we studied the cause of death group "maternal and fetal placental circulation disorders": placenta bed pathology with abruption or infarction as origin of mechanism and placental parenchyma pathology with fetal thrombotic vasculopathy and massive perivillous fibrin deposition as origin of mechanism. results. of the women tested for factor v leiden, ( . %) were carriers. of the deaths caused by "maternal and fetal placental circulation disorders" ( . %) mothers were carriers of factor v leiden. of the deaths with another cause of death ( . %) mothers were carriers of factor v leiden (p= . ). of the women tested for prothrombin g a, ( . %) were carriers. of the deaths caused by "maternal and fetal placental circulation disorders" ( . %) mothers were carriers of prothrombin g a mutation. of the deaths with another cause of death ( . %) mothers were carrier of prothrombin g a mutation (p= . ). in women with iufd and factor v leiden or prothrombin g a mutation "maternal and fetal placental circulation disorders" did not seem to cause iufd more often than in non-carriers. , university medical center groningen, groningen, netherlands; pathology, university medical center groningen, groningen, netherlands; trial coordinating center, dept of epidemiology, university medical center groningen, groningen, netherlands; hematology, university medical center groningen, groningen, netherlands. introduction. growing evidence suggests that women with thrombophilic defects may be at higher risk of fetal loss. although some paternal components to the predisposition of preeclampsia have been demonstrated it is not known whether paternal components contribute to intra-uterine fetal death (iufd). our objective was to investigate the relation between paternal thrombophilic defects and iufd. methods. in a dutch multi-centre study on iufd, from - we studied singleton deaths > weeks of gestation for which the diagnosis of iufd was determined before labour. we tested male partners of women with iufd for antithrombin (at), protein c, protein s type i and iii, factor v leiden, prothrombin g a (factor ii) and factor viii: ag. standard tests were performed in one laboratory. normal ranges were determined in healthy male blood donors. we compared prevalence of thrombophilic defects to reference values from the literature. . of the men tested for factor v leiden, ( . %) were carrier versus ( . %) non-carriers. prevalence of factor v leiden in the normal population is % (p= . ). men were tested for prothrombin g a, ( . %) were carriers and ( . %) non-carriers. all were heterozygous. prevalence of prothrombin g a mutation in the normal population is % (p= . ). decreased levels of antithrombin, protein c, protein s type i and iii and increased levels of factor viii: ag were observed significantly more often in male partners of women with iufd compared to the normal population. conclusion. in our iufd group the prevalence of male factor v leiden carriers was comparable to the normal population, prevalence of prothrombin g a mutation was lower. the difference in other factors imply an as yet unexplained association of male thrombophilia with fetal death in their partner. objective fetal macrosomia, the most important complication of gdm, increases the risk of shoulder dystocia, erb's palsy and intrauterine hypoxia. we compared frequencies of fetal macrosomia and erb's palsy in two gdm cohorts with different severity of glycemic disturbance and in healthy controls. methods we studied consecutive gdm women with singleton childbirth and or abnormal values in -h ogtt with g of glucose. abnormal plasma glucose values of the ogtt were: fasting . , -h . and -h . mmol/l. insulin treatment was started when values were . preprandially or . mmol/l postprandially in the -h glucose profile done within days of diagnosis. if the same woman had more than childbirth during the study period, only the last pregnancy was included. the control group consisted of women from a nearby town with singleton childbirth in the same hospital. women with diabetes were excluded from controls. macrosomia was defined as birth weight (bw) > . sd above the mean of a standard population. erb's palsy was diagnosed by pediatric surgeons. results gdm women were older, more obese, had more childbirths and more often a previous child with bw > g than controls. insulin was started in gdm women ( . %). c/s rate was . % in controls, . % in diettreated and . % in insulin-treated gdm women. macrosomia rate was . % in controls, . % in diet-treated and . % in insulin-treated gdm women (p< . compared with controls or diet-treated gdm women). the frequency of macrosomia did not differ between the diet-treated gdm and control women. erb's palsy occurred in . % of controls, in . % of diet-treated and in . % of insulin-treated gdm women. the frequency of erb's palsy was significantly higher in both gdm groups than in controls (p= . ). by regression analysis, previous child with bw > g (p< . ), previous c/s (p= . ), mother's age (p= . ), bmi (p= . ), insulin treatment (p= . ) and fasting plasma glucose of the -h profile (p= . ) were significant independent predictors of fetal macrosomia. conclusions the -h glucose profile done shortly after the diagnosis of gdm clearly distinguishes between low-risk (diet-treated) and high-risk (insulintreated) gdm pregnancies for fetal macrosomia. unfavourable fetal body composition of diet-treated gdm women is the likely explanation for the high rate of erb's palsy in this group. . '-:·:tpartq,-, '-:·:tpartq,-, '-:·:tpartq,-, % p= . ). first trimester uta doppler indices were similar in the two cohorts in terms of the resistance and pulsatility indices (ri . vs. . , p= . ; pi . vs. . , p= . ) . however, bilateral notching was much more common in the cohort of prior adverse outcomes ( % vs. % p= . ) as well as in patients destined to have a subsequent poor outcome (p= . ). conclusions: not surprisingly, prior poor obstetric outcome was strongly associated with recurrent adverse obstetric outcome. uta notching was robustly associated with prior poor obstetric history as well as a recurrent poor outcome. this clinical history had no discernable influence on ri or pi. reassurance may not be offered based on first trimester uta ri and pi. anti-retroviral therapy is associated with increased blood loss and uterine atony for patients undergoing primary cesarean section. carey eppes, alice cootauco, melissa russo, jessica bienstock. gynecology and obstetrics, johns hopkins university school of medicine, baltimore, md, usa. objective: anti-retroviral therapy has been associated with gastrointestinal smooth muscle dysfunction. it has also been hypothesized that myopathies can occur secondary to anti-retroviral therapy due to mitchondrial alterations. in our experience, we have noticed an increased incidence of uterine atony in our hiv patients on anti-retroviral therapy. we sought to examine the incidence of postpartum hemorrhage and uterine atony in patients currently on anti-retroviral therapy. study design: a retrospective case-controlled study was conducted on all hiv positive pregnant women on anti-retroviral therapy undergoing a primary low segment transverse cesarean section from through . these patients were obtained from an irb approved database containing hiv positive patients within our institution. patients' medical records were abstracted for demographic data, use of uterotonics, preoperative and postoperative hematocrits, and incidence of blood transfusions. controls were matched for age, parity, gestational age and surgical indication. data was analyzed using the t-test, fischer's exact test and chi square. results: there were no differences in demographics, incidence of chorioamnionitis or magnesium sulfate use between groups. patients on anti-retroviral therapy had a statistically greater decrease in hematocrit and estimated blood loss compared to controls. they also had an increased need for uterotonics and blood transfusions. conclusion: anti-retroviral therapy may impact uterine smooth muscle contractility in pregnancy, as evident by the increased incidence of uterine atony and change in hematocrit. additional research is needed to elucidate the mechanism. clinicians should be aware of the potential for uterine atony and excessive blood loss in patients on anti-retroviral therapy. background: adolescent pregnancy is frequently associated with adverse outcomes, especially small-for-gestational age (sga) deliveries. some studies have implicated maternal nutritional status in these poor outcomes. methods: in a prospective longitudinal study, ethnically-diverse, pregnant adolescents were studied from booking to parturition, with collection of anthropometric and nutritional variables. blood samples ( - weeks' gestation; n= subjects) were assayed for a spectrum of nutritional biomarkers. logistic regression was used to determine significant associations between studied variables and pregnancy outcomes. results: median age at recruitment was . years (iqr: . - . ). outcome data was available for subjects. ( . %) had uncomplicated pregnancies. median birthweight was , g (iqr: , - , g) and median birthweight centile was . % (iqr: . - . %). ( . %) infants were born sga and ( . %) were preterm. spontaneous vaginal deliveries occurred in . % of cases. there were ( . %) cases of pre-eclampsia and ( . %) admissions to neonatal care. . % of subjects reported smoking at booking. iron deficiency anaemia was prevalent in . % of subjects by - weeks and was strongly associated with higher infant birthweight centile (p< . ). . % had serum -hydroxy vitamin d concentrations < ng/ml, although this was not associated with any outcomes. low folate status, as indicated by low red cell folate (p= . ), low serum folate (p= . ) and high serum homocysteine (p= . ) concentrations, was associated with higher rates of sga birth. conclusion: adolescent pregnancy in inner-city populations is associated with a high risk of sga and preterm birth, increasing the likelihood of health problems in later life and perpetuating social disparities in health. the association between anaemia and higher birthweight is unexplained. impaired maternal folate status may contribute to impaired fetal growth. we suggest that sga birth in this population could be reduced by the use of antenatal supplements or the mandatory fortification of flour with folic acid. amount of cigarette use pre-pregnancy and during pregnancy were self-reported and subgrouped into nonsmokers, quitting during pregnancy, and continued smoking throughout pregnancy. categorical outcomes were compared using chi-square test. multivariable logistic regression analyses were used to control for potential confounders (continued smoking compared to quitting during pregnancy). results: women who quit smoking during pregnancy had higher rates of pregnancy-associated hypertension and cesarean delivery but lower rates of preterm delivery and neonatal birthweight < gm compared to non-smokers. compared to women who quit smoking during pregnancy, those who continue to smoke have lower odds of pregnancy-associated hypertension and cesarean delivery, but higher odds of preterm delivery, neonatal birthweight < gm, and apgar score < at minutes (see table) conclusion: quitting cigarette smoking during pregnancy appears to reduce undesirable neonatal outcomes, though an increase in pregnancy associated hypertension. these findings should be emphasized to women who are smoking during pregnancy. to achieve effective tamponade the balloon was inflated initially up to - ml with incremental increases of volume by ml until bleeding stops. to measure intrauterine balloon pressures we used a standard pressure transducer (edwards lifesciences) connected to the balloon port and to a pressure monitor (ge dash ). the pressure transducer was mounted on the bedrail at uterine level, primed with sterile saline and then connected to the balloon port of the catheter. the system was zeroed to atmospheric pressure, the stopcock of the intrauterine balloon port was opened and the pressure was recorded (p : the total pressure on the fluid). to verify the accuracy of measurements we used another balloon catheter (reference), inflated with the same amount of saline, connected to the pressure transducer and zeroed to atmospheric pressure. the reference balloon measured p (the intrinsic balloon pressure caused by distension at that volume). the system was then zeroed to the reference balloon. the pressure transducer was then reconnected with the intrauterine balloon and the actual intrauterine pressure was recorded (p ). we calculated the correlation coefficient between p and systolic, diastolic and mean blood pressure using a linear regression (statsdirect statistical software there is a growing body of evidence to suggest that peripartum assessment of fetal or neonatal lactate levels are as good as or better than standard blood gas analysis in the prediction of neonatal outcome. in this study we have evaluated the ability of umbilical cord blood gases and lactate levels in the prediction of neonatal hypoxic-ischaemic encephalopathy (hie). department of neonatal paediatrics, king edward memorial hospital, perth, western australia, australia; women and infants research foundation, king edward memorial hospital, perth, western australia, australia. objective: current evidence suggests that umbilical cord ph at delivery provides the most sensitive reflection of birth asphyxia. paired umbilical artery/vein blood gases have been routinely collected at king edward memorial hospital over the last years. the objective of this study was to determine: local reference ranges; accuracy of sampling; and the rates of metabolic acidosis. of the , births ( ) ( ) ( ) ( ) , accurate paired results were available on % of births. over the study period there was a progressive improvements in accuracy rates of paired sampling (p< . ). the median ( . th , . th centile) values for cord arterial blood gases were: ph . ( . , . ); po . mmhg ( . , . ); pco . mmhg ( . , . ); base excess - . (- . , . ); and lactate . mmol/l ( . , . ). there was a progressive improvement in all blood gas measures over the years of this study (all p< . ). moreover, there were significant reductions in all measures of metabolic acidosis (see table) . the progressive improvement in the measures of metabolic acidosis remained significant after multivariate analysis including obstetric, fetal, and demographic factors associated with metabolic acidosis. the introduction of universal umbilical cord blood gas analysis to all births is associated with significant improvements in all markers of metabolic acidosis. together with other compelling evidence in the literature, these data support the routine use of cord arterial and venous gases at all births; however, improved accuracy rates on paired sampling requires an ongoing education program umbilical artery indicators of acidosis, percentage of total ph < . ph < th centile* objective: intrapartum pcn prophylaxis aims to prevent early-onset gbs sepsis by interrupting vertical transmission from colonized mothers to their newborns. however, despite its wide clinical use, systematic pharmacokinetic evidence in support of the current pcn dosage regimen is lacking. current cdc guidelines recommend intensified surveillance and testing of infants exposed to < h of prophylaxis. our goal was to examine the relationship between maternal time of exposure to pcn and fetal serum pcn levels among maternal-fetal dyads exposed to short durations of pcn prophylaxis (< h) compared to those exposed to longer durations. study design: ninety-eight laboring gbs positive women were administered million units (mu) of intravenous pcn to be followed by . mu every hours until delivery (cdc ) . subjects with renal disease, multiple gestation, and preterm delivery (< wks) were excluded. umbilical cord blood samples were collected at delivery and pcn levels measured by high-performance liquid chromatography. intra and inter-assay coefficients of variation were < %. results: the pcn concentrations (mean±sd) by duration of prophylaxis were: < h, . ± . g/ml (n= ); - h, . ± . g/ml (n= ); - h, . ± . g/ ml (n= ); - h, . ± . g/ml (n= ); - h, . ± . g/ml (n= );> h . ± . g/ml (n= ); and for those without a second dose after h, . ± . g/ml (n= ). fetuses exposed to short duration (< h) had higher levels of pcn than those exposed to > h (p= . ). in multivariable linear regression analysis, fetal pcn levels were determined by total duration of exposure, time since last dose, dosage, and number of doses, but not maternal bmi. pcn levels in cord serum increased linearly until hour; thereafter, they decreased rapidly, but all groups were significantly above the minimum inhibitory concentration (mic) for gbs ( . g/ml)(p< . ). furthermore, every sample individually remained - fold above the mic. conclusion: in this study, even short durations of prophylaxis achieved levels above the mic, suggesting a benefit to prophylaxis even in precipitous labors. the data also suggests that the current cdc designation of infants exposed to < h of pcn prophylaxis as particularly at risk for gbs sepsis may be inaccurate from a pharmacokinetic standpoint. objective: -oh aa is a metabolite of tryptophan with pro-oxidant and proapoptotic properties and has been shown to increase in umbilical cord blood in pregnancies with intra-uterine infection. since labour-related events may also activate inflammatory pathways, we sought to determine the placental release of -oh aa into the umbilical circulation in labouring vs non-labouring patients at term. methods: twenty-six patients were studied (term labour n= , and term elective cesarean section n= ) with blood sampling from a clamped segment of umbilical cord after delivery of the fetus and from the cord at its insertion into the placenta after delivery of the placenta, with subsequent measurement of blood gases/ph and -oh aa (isocratic hplc using fluorometric detection with assay sensitivity at pmol). results: -oh aa measurements from respective umbilical and placental cord vessels were all variably higher in the labouring group vs the elective cesarean group patients (table ) . for labouring group patients, the -oh aa levels from the umbilical vein were significantly higher than those from the umbilical artery, indicating net release from the placenta into the fetal circulation. placental vein levels were also significantly higher than those from the umbilical vein, indicating continued placental release of -oh aa into the cord blood after delivery of the fetus. conclusion: labour at term is associated with changes in the placental metabolism of tryptophan resulting in the increased release of -oh aa into the fetal circulation with the potential for pro-oxidative and apoptotic effects in many tissues, including the brain. obstetrics, gynecology, and reproductive sciences, new brunswick, nj, usa; department of obstetrics and gynecology, mineola, ny, usa. objective: ischemic placental disease (preeclampsia, small for gestational age, sga and placental abruption) is a major contributor to pregnancy-related morbidity. although the placenta is considered a fetal organ, it is accepted that ischemic placental disease (ipd) can present clinically with either fetal or maternal manifestations. we hypothesized that the pattern of diagnosis (maternal versus fetal) varies by gestational age and would provide insights in to origins of indicated and spontaneous preterm birth. methods: this was a retrospective cohort study utilizing the maternallylinked reproductive history data for missouri residents , restricted to singleton live births. women who experienced spontaneous onset of labor and subsequently delivered preterm were classified as spontaneous preterm birth. medically indicated preterm birth included women who delivered preterm through a labor induction or (prelabor) cesarean delivery. ipd was classified as maternal (preeclampsia only), fetal (sga only) or both (preeclampsia with sga or abruption, and all conditions). results: among term births with ipd, . % presented as maternal disease only, . % as fetal disease, and the remainder ( . %) as both. among spontaneous preterm births with ipd, a greater proportion were of fetal presentation ( bethesda, md, usa; dept ob/gyn, wayne state univ, detroit, mi, usa; dept pathology, wayne state univ, detroit, mi, usa; dept ob/gyn, soroka univ medical center, israel. objective: hemoglobin (hg) and its catabolic products have been observed in cases of amniotic fluid (af) discoloration, which is a risk factor for intraamniotic infection/inflammation (iai). the study aimed to determine the association between af fetal hg concentration and gestational age, term and preterm labor and iai. study design: this cross-sectional study included: ) mid-trimester (n= ); ) term not in labor (tnl) (n= ); ) term in labor (tin) (n= ); ) preterm labor (ptl) who delivered at term (n= ); ) ptl without iai (n= ); ) ptl with iai (n= ); ) preterm prelabor rupture of membranes (pprom) with (n= ) and without iai (n= ). af fetal hg concentrations were determined by elisa. results: ) fetal hg was detected in . % of all af and . % of mid-trimester samples; ) women at tnl had a higher median af fetal hg concentration than patients at mid-trimester ( . ng/ml, iqr - vs . ng/ml, iqr . - . , p= . ); ) no differences were found in median af fetal hg concentration among patients with and without labor at term (til: . ng/ml, iqr - . ; p= . ); ) median af fetal hg concentration was not significantly different among the ptl subgroups [ptl with iai: . ng/ml, ptl who delivered preterm: . ng/ml, ptl without iai who delivered at term: . ng/ml, p= . (kruskal wallis) ]; ) in pprom, there were no differences among patients with and without iai ( . ng/ml, respectively; p= . ) ; ) median af fetal hg concentrations were significantly higher in ptl or pprom, with or without iai than in pregnant women at term, with and without labor (p< . for all comparisons). conclusions: ) immunoreactive af fetal hg increases with gestational age; ) among women with ptl or pprom, the median af fetal hg concentration is not associated with iai; ) the median af fetal hg concentration is higher in pregnancies complicated with ptl or pprom than in term pregnancies. the impact of latency time to delivery after preterm premature rupture of membranes (pprom) on neonatal outcome. dan nayot, deborah penava, barbra de vrijer, orlando da silva, bryan s richardson. obstetrics and gynaecology; pediatrics, university of western ontario, london, on, canada. objective: there continues to be controversy as to the management of pprom with conservative management to advance gestational age (ga) versus aggressive management with early induction to avoid chorioamnionitis. we have therefore used the perinatal and neonatal databases of a large regional patient population to determine the association of pregnancy variables with latency time to delivery after pprom and the impact of latency duration on adverse neonatal outcomes. methods: the perinatal/neonatal database of st. joseph's health care, london, ontario was used to obtain demographic and neonatal outcome information for all patients with pprom > and < weeks gestation, singleton and no major anomalies, delivering between january , and december , . patients were grouped according to ga at pprom stratified for latency time < hrs vs > hrs with incidences for those pregnancy related variables and neonatal outcomes available from the database then compared with the use of logistic regression analysis. results: there were patients who met the inclusion criteria of whom %, %, and % had pprom at - wks , at - wks, or at - wks, respectively, and with these pprom groupings showing a stepwise decrease in the percentage of patients with latency to delivery > hrs, at %, %, and %, respectively. pregnancy related variables and neonatal outcomes for these patient groupings are as shown in table . conclusion: despite a to fold increase in the incidence of chorioamnionitis with latency to delivery > hrs, a policy of conservative management to advance ga after pprom will result in decreased severe infant morbidity until weeks, and moderate infant morbidity until weeks. hospital. outcomes studied were prolongation of latency period with intact membranes and prom using magnesium sulfate (mgso ), nifedipine, terbutaline and tocolytic agents. secondary outcomes examined were maternal complications. statistical analysis performed were t-test and . comparisons were expressed as odds ratios ( % ci). results: in a -year period, twin gestations were admitted in ptl and administered tocolytic agent(s) with mean gestational age of . weeks. when tocolytic agents were used, maternal complications were: ( . %) with intact membranes and ( . %) with prom had pulmonary edema (or= . , ci . - . ); ( . %) with intact membranes and ( . %) with prom had chorioamnionitis (or= . , ci . - . ); ( . %) with intact membranes and ( . %) with prom had postpartum hemorrhage (or= . , ci . - . ). conclusion: there is no difference in prolongation of latency period achieved in twin gestations in ptl with intact membranes or prom using tocolytic agents. similarly, there is no difference between the groups with latency period hrs. there is an increased likelihood of pulmonary edema and chorioamnionitis with use of tocolytic agents. however, results are not significant due to type ii error. mean latency period and latency ≥ hrs using single or multiple tocolytic agent ( introduction: high sensitivity crp (hscrp) is a serum marker of inflammation and has proven clinical utlility in predicting cardiovascular disease (cvd). considering the hypothesized association between preeclampsia (pre) and inflammation and cvd, it is plausible that hscrp may have utility in predicting pre. prior to widespread utilization of this marker, the affect of labor on levels of crp needs to be clarified. we assessed the association between labor and hscrp levels in term deliveries and between elevated hscrp and adverse perinatal outcomes. a secondary analysis comparing hscrp in women with preeclampsia (pre) to those without was performed. methods: women presenting for term delivery or pre were prospectively identified as part of a case-control study. clinical data and serum were collected for all subjects. a standard immunoturbidimetric assay was used to measure hscrp levels. women presenting for induction of labor or planned cesarean delivery (non-labor) were compared to women presenting in labor. a secondary analysis comparing non labor women with and without pre was performed. serum was collected prior to labor induction in the non-labor group. nonparametric comparisons were made using wilcoxon rank sum tests. mvlr was used to evaluate dichotomous outcomes and control for confounders. results: women were included (non-labor group (n= ), labor group (n= ), non-labor pre (n= )). the median and mean hscrp levels were and and and in the non-labor and labor groups respectively (p= . ). elevated levels of hscrp in these term deliveries were not associated with chorioamnionitis (p= . ), maternal postpartum complications (endometritis, hemorrhage, transfusion) (p= . ), mode of delivery (p= . ), or admission to the nicu ( . ). levels of hscrp were significantly greater in the non-labor pre group compared to non labor without pre (mean . vs. . , median vs. , p< . ). conclusion: use of hscrp as a biomarker may improve clinical prediction of obstetrical complications such as pre. levels of hscrp are affected by labor and this should be taken into account when studying the utility of this biomarker. further, crp levels are elevated in women with pre even after excluding patients in labor. further investigations to determine if crp elevation in term labor is associated with adverse outcomes may be warranted. the role of hscrp as a valid and discriminating biomarker in pre should be assessed. review of the electronic labor record facilitated collection of maternal demographic, intrapartum, and newborn data. data analyzed with t-tests, chi-square tests, and calculated odds ratios with % cis as appropriate. a forward stepwise logistic regression analysis was used to identify predictors of histologic chorioamnionitis. results: of submitted placentas, had histologic chorioamnionitis (cases) and did not (controls). the groups were similar with respect to age, race, gbs status, and mode of delivery. gestational age, birthweight, duration of labor and ruptured membranes, and number of vaginal exams were greater in the cases (p . ). the cases were more likely to have had epidural anesthesia (or . ), internal monitoring (or . ), fever (or . ), maternal tachycardia (or . ) and fetal tachycardia (or . ) and less likely to have had induction of labor (or . ). newborns in the histologic chorioamnionitis group were more likely to have been observed for sepsis (or . severe sepsis defined as sepsis associated with acute respiratory distress syndrome (ards) or cardiovascular dysfunction(cvd) or with or more other organ dysfunction.all patients were resuscitated with fluids and treated with broad spectrum antibiotics and supportive care as needed. outcome data were: etiology, management, maternal complications, duration of icu stay and perinatal survival. results patients were young (mean age= . ± . years) with a mean gestational age at delivery . ± . weeks. etiologies were pyelonephritis(n= ), septic abortion(n= ), endomyometritis (n= ), chorioamnionitis (n= ), ruptured appendix(n= ), pneumonia( n= ) and one unknown. eighteen ( %) were diagnosed during antepartum and ( %) postpartum period.there were ( %) maternal deaths and high rate of major morbidities (table) . among the antepartum patients,there were, abortions, iufd, neonatal death for a perinatal survival rate of only %. conclusion pregnancies complicated with severe sepsis/septic shock are associated with substantial maternal and perinatal morbidities. the low maternal mortality rate in our study as compared to previous reports is attributed to early diagnosis and aggressive management of maternal complications. fetal loss rate, however continues to be high when septic shock develops antepartum. and tnfa levels are elevated. we developed a dynamic computer (in silico) model of pregnancy (uterine myometrial environment -infection/inflammation and endocrine crosstalk). mathematical differential equations were used to describe the interactions between molecules. infection was represented as increased levels of activated, nuclear transcription factor nf-kb. in the model ru inhibited both the glucocorticoid receptor and progesterone receptors. simulations were run adding different concentrations of ru ( . um= dose used in patients, . um, . um) at different time points during infection (before, at the time of or after nf-kb activation). ru degradation kinetics was also included. the effect of ru on nf-kb induced il- and tnfa levels was assessed. results: infection induced nf-kb activation led to increased il- and tnfa levels. there was a subsequent increase in cortisol that led to dampening of nf-kb activation, il- and tnfa levels. in the presence of ru , il- and tnfa levels continued to rise. the effect of ru on nf-kb induced il- and tnfa was dose dependent and was more prominent in slow metabolizers who had ru in the system for a longer time. addition of ru after the onset of nf-kb activation led to increased il- and tnfa levels above those observed without ru . the addition of misoprostol (prostaglandin e analogue), at the concentrations used together with ru for medical abortion, did not add to the effect of ru on nf-kb induced il- or tnf. conclusions: ru has dose dependent effects on infection induced immune activation and may contribute to the pathogenesis of c sordelii induced sepsis syndrome. the increased susceptibility of neonates to infection remains a major clinical problem. we previously found that after intraperitoneal (ip) listeria monocytogenes infection, neonatal mice have an ld that is orders of magnitude lower than adults. we also found that the inflammatory response of neonatal mouse macrophages, but not neutrophils, in the peritoneal fluid was deficient, and that this correlated with low levels of macrophage chemokines mcp- and rantes. given that the liver and spleen are important organs in listeria infection, we sought to characterize the innate immune response in these organs. a sublethal dose of listeria was injected intraperitoneally into balb/c - week old adult mice and - day old neonatal mice. liver and spleen was collected at , , and hours, sectioned serially for staining with hematoxylin-eosin and primary antibodies (rabbit anti-listeria monocytogenes igg; mhc class ii rat anti-mouse igg, a marker for activated macrophages; f / rat antimouse igg, a marker for macrophages) and secondary antibodies (cy- goat anti-rabbit igg; af goat anti-rat igg) were applied. negative controls used only secondary antibody. real time pcr was used to compare the levels of the chemokines mcp- and rantes in adult and neonatal liver. after ip infection, both adults and neonates showed similar influx of neutrophils to the sites of infection within the liver and spleen. at hours post-infection with listeria, adult liver and spleen showed increased staining for f / and class ii, which increased further and became confluent surrounding microabscesses at and hours. in contrast, the listeria infected neonatal mouse showed some increase in f / around microabscesses but no apparent increase in staining for mhc class ii. the neonates showed greater staining for listeria at each time point. mcp- and rantes levels were higher in infected neonatal liver compared to adults. in the neonatal mouse, the innate immune response in the liver and spleen was characterized by a deficiency of activated macrophages. this deficiency was not correlated with hepatic expression of macrophage chemokines. is there a seasonal pattern in the incidence of post-cesarean endometritis? tamula m patterson, alan tn tita, william w andrews. obstetrics and gynecology, the university of alabama at birmingham, birmingham, al, usa. objective: several theories, including one suggesting a peak in july coincident with resident turnover, postulate seasonality in post-cesarean infections. we assessed whether there is seasonal variation in endometritis. a retrospective cohort study of post-cesarean endometritis at our university-based institution using our obstetric computerized database to compare annual variation in monthly incidence patterns from to . prior to establishing an average aggregate seasonal pattern for all years, years were assessed separately for a recurrent pattern of peaks and nadirs in incidence. peak incidence (or nadir) was defined as any monthly incidence that differed from the mean incidence for the year by over %. results: a total of . % ( , ) of , deliveries from to were by cesarean. annual cesarean rates increased by an absolute rate of over %; while, post-cesarean endometritis rates decreased from % to %. monthly incidence of post-cesarean endometritis did not reveal a consistent recurrent pattern of peaks (figure ) or nadirs. the month of july accounted for only out of a total of peaks for all years. the adjacent months of june and august accounted for only each. the month with the highest number of peaks was april with only . these findings contraindicated the establishment of an average aggregate monthly pattern for all years. the incidence of post-cesarean endometritis did not follow a seasonal pattern. chi-square analyses were used to compare associations between race (black (bl) vs non-black (nbl)) and dichotomous characteristics. student´s t-test was used to compare continuous variables. results , patients were evaluated ( cases and controls). % and % of cases and % and % of the controls were bl and nbl respectively. the baseline prevalence of chtn in bl and nbl controls was . % and . % (p= . ). when comparing bl and nbl cases, bl women had a higher mean systolic blood pressure and screening bmi. bl women also had a trend toward being discharged on post partum blood pressure medicine when compared to nbl women. there was no difference in chtn, diabetes, severity of disease, iugr, or delivery < wks between the two groups (table) . to determine the leading causes of death in a case series of stillborn infants examined in a large hospital autopsy service, and to describe the most common post-mortem observations. study design: one hundred and sixty one stillborn infants were examined. gross pathology observations were recorded at autopsy and during the placental exam, and tissue sections were collected and examined microscopically. immediate and underlying cause of death (cod) were recorded, along with contributory cod, concomitant/significant cod, and incidental findings. statistical analysis was conducted using the software spss v. . . results: the immediate anatomic cod could be determined in . % of all infants examined. in over % of these cases, cod was attributable to placental or umbilical cord findings affecting the maternal-fetal blood supply. the most prevalent among these findings were placental lesions (maternal floor infarction, placental abruption, fetal thrombotic vasculopathy), umbilical cord lesions (entanglement, true knot, compression, excessive length/twisting), and infectious/inflammatory processes (chorioamnionitis, chronic villitis objective: advanced maternal age (ama) is associated with increased risk of intrauterine fetal demise (iufd). antenatal testing (at) is widely used in clinical practice to prevent iufd due to uteroplacental insufficiency and has been suggested to reduce the risk of iufd in ama women. we sought to assess the impact of at on obstetrical interventions and compliance with new practice recommendations. methods: retrospective cohort of ama women ( and older at their due date) who delivered at or after weeks. non-exposed women delivered from july to december (when at for ama was not routinely recommended); exposed women delivered from july to december (after at for ama was introduced at our institution). subjects were identified through the perinatal database; records were abstracted for demographics, medical history, and labor/delivery variables. outcomes included rates of at and induction of labor (iol) and mode of delivery. associations between at for ama and outcomes were tested using t test and chi square. assuming a baseline rate of iol of %, we had % power to detect an increase to % after the introduction of at. results: women met the inclusion criteria: delivered before the introduction of at (non-exposed=before at) and delivered after the introduction of at (exposed=after at). baseline clinical characteristics were similar in both groups. as anticipated, at was more common in the after at group than in the before at group ( % vs %; p< . ). women were not eligible for or declined trial of labor, thus not "at risk" for iol and not included in all analyses. ob intervention rates were increased after at compared to before at (table ) . the corrected iufd rates were similar in both groups ( / vs / ). conclusion: at an academic center, compliance with new practice recommendations was excellent. introducing at testing for a new indication seemed to increase iol and cs rates. these findings should be considered when assessing the risks:benefits ratio of antenatal testing. . we stratified indications for cesarean into the following: failed induction (cervix < cm dilated), arrest of dilation, arrest of descent, failed operative delivery, fetal intolerance of labor (fil), and "other" reasons (e.g., pre-eclampsia, abruption, chorioamnionitis, malpresentation). both fil and "other" reasons were more likely to occur among the medically indicated group (rr . , . physicians in solo practice had higher rates of elective inductions (p< . ), but there was no association between cesarean and practice type. conclusions: inductions accounted for . % of cesareans. these results suggest an increased risk for cs for patients undergoing medically indicated inductions at our institution. there was no association between cesarean and type of practice, whether solo or group, suggesting institutional clinical policies may be more important than practice type in determining delivery outcome after induction. further research is needed to understand how age, race/ethnicity, or other unmeasured patient factors may impact these findings. given rising rates of both cesarean delivery and inductions, this information may be pertinent to women considering elective induction prior to weeks. objective: fetal demise can lead to a consumptive coagulophathy ("fetal death syndrome") traditionally attributed to the release of "tissue thromboplastin", now known as "tissue factor" (tf). tf is the most potent activator of coagulation. despite the appeal and acceptance of this proposed pathophysiology, there is no evidence supporting this view. this study was undertaken to determine if fetal death prior to development of fetal death syndrome is associated with changes in maternal plasma concentration of cd l (a marker of platelet activation), tf and its soluble inhibitor (tfpi). methods: a cross-sectional study included the following groups: ) women with normal pregnancy (n= ) and ) patients with fetal demise without disseminated intravascular coagulation (n= ). plasma concentrations of scd l, tf and tfpi were measured by elisa. standard coagulation tests were performed. non-parametric statistics were used for analysis. results: ) patients with fetal demise had a higher median maternal plasma scd l concentration than women with normal pregnancy (median . pg/ml, range - vs. median . pg/ml, range . - . , p< . ); ) there was no significant difference between the groups in the median maternal plasma tf concentration and ) in contrast, the median maternal plasma tfpi concentration was significantly lower in patients with fetal demise than in women with normal pregnancy (median . ng/ml, range . - . vs. median . ng/ml, range . - . , p< . ). conclusions: ) a change in the plasma concentration of tf was not demonstrated; ) a change in the ratio of tf/tf inhibitor pathway may predispose to thrombin generation and activation of the coagulation cascade; ) however, maternal platelet activation is present in patients with a fetal demise without fetal death syndrome and ) the role of tf in fetal death syndrome remains to be proven. lipoic acid inhibits matrix metalloproteinase production, activity and prostaglandin e secretion by cultured amnion epithelial and mesenchymal cells. r moore, j novak, d kumar, j moore. case western reserve university, cleveland, oh, usa. introduction: cytokines, free radicals, matrix metalloproteinases (mmp) and prostaglandins (pg) have been implicated in processes of fetal membrane rupture and labor. dietary anti-oxidant supplementation has been suggested as a possible therapy for high risk patients, however, clincal evidence supporting the efficacy of agents such as vitamin c or n-acetylcysteine remains controversial. in fact, we have previously shown that vitamin c increases matrix metalloproteinase (mmp) activity in isolated fetal membrane fragments and fails to inhibit tumor necrosis factor (tnf) induced fetal membrane weakening in vitro. in this study, we examine the effect of the naturally occurring anti-oxidant, -lipoic acid, on tnf induced mmp activity/protein and pge secretion in isolated amnion epithelial and mesenchymal cells. methods: amnion epithelial and mesenchymal cells were pre-treated with increasing doses of -lipoic acid ( - mm/ h), then with increasing doses of tnf ( - ng/ml/ h). medium and cells were analyzed by gelatin zymography/western blotting for mmp /mmp activities/protein. pge output was determined by immunoassay. results: tnf induced a dose dependent increase in mmp production, secretion and activity in amnion epithelial cells. tnf ( ng/ml) induced an fold increase in cellular active mmp production and fold increase in secreted mmp enzyme activity by amnion epithelial cells. these increases were reduced - % following h pre-treatment with . - . mm -lipoic acid. mmp protein/activity and pge secretion by amnion epithelial cells were barely detectable and unaffected by tnf and/or -lipoic acid treatment. in striking contrast, mesenchymal cells exhibited little basal or tnf induced mmp protein/activity. mmp protein/activity in mesenchymal cells were unaffected by either tnf and/or -lipoic acid. however, tnf treated mesemchymal cells exhibited a dose dependent increase in pge production ( fold increase/ ng/ml tnf/ h) that was inhibited by %- % following . objective: nearly fifty years after the discovery of microphthalmia-associated transcription factor (mitf), its gene was identified as a specialized transcription factor that dictates cell-specific differentiation. unique mitf isoforms are generated from alternative promoter usage. an isoform of mitf (mitf-cx) is down-regulated in cervical stromal cells of the ripened cervix. further, mitf-cx inhibits il- gene expression and thereby suppresses signaling of the final pathway in cervical ripening. since mitf binds to canonical eboxes (canntg) in promoter regions of target genes, we sought to determine if mitf regulated its own promoter through ebox motifs. methods: the kb genomic dna sequence upstream of the mitf-cx transcription start site was cloned into pgl luciferase reporter vectors which were co-transfected with wild type or mutmitf-cx (impaired dna binding) into cervical stromal cells or hek cells. at h, promoter activity was determined and normalized for transfection efficiency. results: gel-shift assays conducted with oligonucleotides corresponding to eboxes in the mitf-cx promoter revealed two strong binding sites (eboxes - to - and - to - ). specific binding was established using oligonucleotides with or without mutated ebox, antibody supershift experiments, competition with cold probe, and absence of binding to mutmitf-cx. binding specificities were confirmed in nuclear extracts from cells that overexpressed mitf-cx, but not control or mutmitf-cx. reporter gene studies indicated that mitf-cx, but not mitf-m, increased mitf-cx promoter activity -to -fold. whereas mutations in ebox or resulted in significant decreases in mitf-stimulated promoter activity, mitfinduced increases in promoter activity were abolished by mutations in both eboxes. conclusions: collectively these experiments indicate that mitf-cx is a vital regulator of its own promoter activity and acts in a positive feedforward loop through two specific binding sites in its promoter. moreover, isoform-specific amino acids are important to mediate mitf-induced mitf-cx promoter activity. decreasing mitf protein or mutating its promoter would interrupt this loop resulting in rapid reduction of mitf synthesis. since mitf-cx suppresses il- gene expression in cervical stromal cells, we suggest that preservation of mitf-cx-induced mitf gene expression is an important mechanism to maintain the "brake" on cervical ripening and ensure cervical competency during pregnancy. the role of cd in cervical remodeling. denisse sanchez, brenda timmons, mala mahendroo. obstetrics and gynecology, ut southwestern medical center, dallas, tx, usa. objective: prior to the onset of parturition, the uterine cervix undergoes a remodeling process from a closed, rigid structure, to one that is soft and dilatable. many changes occur, including increases in hyaluronan (ha), a glycosaminoglycan that facilitates loosening of the collagen matrix. in the postpartum period, the concentration of ha is reduced to that of the nonpregnant state. cd , a transmembrane glycoprotein expressed in hematopoietic and epithelial cells, is a receptor for ha. cd expression by immune cells is important in extravasation of leukocytes into tissue. cd may also be required for ha catabolism through the action of hyaluronidases and . in the cervix, cd is expressed in the endo-cervical epithelia as well as in immune cells localized in the stromal matrix. to study the importance of cd in cervical remodeling, mice with a null mutation for cd (cd -/-) were evaluated during pregnancy, parturition and postpartum. methods: changes in ha amount and size distribution were assessed using ha molecular weight gels and fluorophore assisted carbohydrate electrophoresis. to identify defects in cervical remodeling in the cd -/mice, differences in expression for genes regulated in the cervix were studied by quantitative real time pcr . to determine whether cd plays a role in the recruitment of immune cells during cervical ripening and postpartum repair, immunohistochemistry with antibodies against leukocytes was done. in the postpartum period, there is a higher ratio of high molecular weight ha relative to low molecular weight ha in the cd -/mice. this suggests that postpartum breakdown of ha in cd -/cervices is delayed. as compared to wt cervix, there was a significant increase in hyaluronidase (hyal- ) mrna in the postpartum period. the distribution and relative numbers of immune cells in the cd -/cervix was similar to wt. conclusion: these studies provide evidence that cd may play a role in remodeling of the postpartum cervix back to the nonpregnant state. our current data suggests that the catabolism of ha after birth is delayed in the mutant mice and upregulation of hyal may compensate to allow ha removal. furthermore, the activity of hyal- may be dependent on cd . little difference in the recruitment of immune cells between cd and wt animals suggest that cd expression is not required for this process. these experiments provide a greater understanding for the role of cd and ha in cervical remodeling. in background: during in vitro experiments we have shown that separation of amnion from choriodecidua occurs as an integral part of the process of fetal membrane (fm) rupture. although spontaneous amnion and choriodecidual separation is seen in fm after both svd and elective c/s deliveries, its etiology is uncertain. biochemical degradation at the amnion-choriodecidua interface may be a key contributing factor. our previous biomechanical studies have demonstrated that separated fm require less physical work to rupture than intact membranes. the purpose of this study was to determine whether fm separation was associated with clinical differences in the birth process. hypothesis: during term, normal labor, spontaneous separation of fm is associated with differences in the clinical parameters of labor and delivery. study design: fm from consecutive term deliveries were cut off the placental disk. separated areas of fm were cut from the intact areas. both were weighed and their weight ratios determined. maternal medical, pregnancy, and delivery data were collected and analyzed. results: term fm had the following characteristics: maternal age ± . yr, gravida . ± . , gestation ± . wks, elec. cs . %, duration of rom ± min, duration of contractions ± min, african american %. % of the fm had < % separation; % had more than % separation. srom fm with > % separation (vs. < %) had significantly shorter duration of rom (p= . ) and admission to birth (p= . ) times. srom fm with > % separation (vs. < %) had even shorter rom (p= . ), duration of contractions (p= . ) and admission to birth (p= . ). the > % group (vs. < %) was further along, gestationally (p= . ). srom fm (vs. arom) had shorter admission to birth (p= . ), but longer rom to birth (p< . ) times. absence of epidural (p= . ), srom mode of rupture (p= . ), svd (vs. elec. c/s) (p= . ), and the presence of meconium (p= . ), were all associated with increased fm separation. conclusion: spontaneous separation of fetal membranes is nearly universal and is associated with increased gestation, spontaneous rupture of membranes, shorter duration of contractions, and svd. speculation: we speculate that programmed biochemical changes initiate fm separation which then facilitates rom and childbirth. whole genome array and si-rna investigation of the function of nfkb in human amnion epithelial cells. sheri e lim, shirin khanjani, yun s lee, tg teoh, , philip r bennett. institute of reproductive and developmental biology, imperial college, london, united kingdom; maternal fetal medicine, st. mary's hospital, london, united kingdom. introduction: labour is associated with activation of nfkappab in the amnion. nfkappab increases prostaglandin synthesis through the upregulation of cyclooxygenase- (cox- ), which is essential to the labour process. cox- mrna expression increases with gestation in the amnion. primary amnion epithelial cells cultivated from tissue collected prior to the onset of labour display a spectrum of nfkappab activation, similar to the spectrum of cox- expression presumably relating to the nearness of labour. our aim was to investigate the full range of genes under nfkappab control in amnion epithelial cells by using whole genome arrays. methods: amnion from women undergoing elective caesarean section was collected and primary cell cultures established. total rna and protein were extracted from each culture. nuclear localization of nfkappab is required for its activation. western analysis of nuclear p was therefore performed to identify the samples displaying the lowest and highest nfkappab activity. the corresponding rna samples displaying the three lowest and three highest nuclear p protein concentrations were used for whole genome analysis using affymetrix u arrays. results and conclusions: we identified significantly regulated genes. the gene with the highest fold change was cox- (x . ) followed by oxytocin receptor (x . ), ch orf (x . ), integrina (x . ), and connective tissue growth factor (x . ). other significant genes included interleukin- (il- ) (x . ). pathway analysis revealed the majority of other nfkappab associated genes were involved in cell signaling, turnover and proliferation. we used real-time pcr (rtq-pcr) to validate cox- and il- expression. to prove that cox- is directly regulated by nfkappab, primary amnion epithelial cells were then transiently transfected with nfkappab p sigenome smart pool and sicontrol non-targeting sirna pool. western blot analysis confirmed knockdown of nfkappab p associated with inhibition of cox- demonstrating that nfkappab is essential for cox- expression. objective: premature birth is a major public problem accounting for over , deaths and , surviving infants with life-long morbidity yearly. in order to develop a rational basis for treatment and prevention of premature fetal membrane (fm) failure, we first need to understand the sub-failure fm structural and mechanical behavior at near full term. methods: we utilized planar biaxial mechanical testing, which approximates the physiologic loading state, for mechanical evaluation of the fm, and a structural constitutive model approach was used to offer insight into the structure-strength of the fm by integrating information on tissue composition and structure. small angle light scattering (sals) was used to nondestructively quantify the collagen fiber architecture of both intact and separated fm layers. results: in the stress free state, the gross collagen fiber architecture of the fm and separated layers were not homogenously align but exhibited small regions of fiber alignment. the amnion layer displayed the greatest alignment. the model fit the equi-biaxial strain data well (r = . ) and indicated that fm collagen fibers were rapidly recruited and straightened well below failure stress levels. collagen fibers were gradually recruited followed by a drastic increase in fiber recruitment. conclusion: this study provided the first data on the effective collagen fiber stiffness in the intact fm under physiologic biaxial loading, which was related to quantitative collagen fiber architectural measures. modeling results indicated that the collagen fibers became fully loaded and straighten well below physiological loading levels. failure did not occur during physiological loading, indicating that fibers do not begin to fail until all collagen fibers are fully straightened and bearing load. this result suggested modest structural reserve in the fm collagen architecture, and may be an important aspect of its failure properties. previously, we demonstrated that a physically "weak zone" exists overlying the cervix in the fm, evident of collagen remodeling and cellular apoptosis. we are currently extending the present study to include the "weak zone" tissues, allowing us to elucidate the micro-mechanical mechanisms that facilitate failure in this newly identified fm zone. supported by nih . the extracellular matrix of the cervix undergoes extensive remodeling during parturition. hyaluronan (ha) is a major constituent of the extracellular matrix of the term pregnant cervix. the onset of labor is preceded by an increase in ha and after delivery, the concentration of cervical ha gradually decreases to that of the non-pregnant state. these dramatic changes suggest that ha plays an important role during parturition. hyaluronan synthase (has ) is one of three known ha synthases and the most abundant isoform in the pregnant cervix. transcripts for has are regulated by two alternative promoters, one upstream of the first coding exon (proximal promoter) and another upstream on an untranslated exon (distal promoter). the focus of the current study is to further our understanding of the transcriptional regulation of has during cervical ripening. methods: rna blotting was carried out using transcript specific probes corresponding to the distal and proximal promoter of the mouse has gene. the regulation of has was evaluated in a cervical epithelial cancer cell line (caski cells) which we have previously shown to express endogenous has . regulation of has expression by epidermal growth factor was assessed in the caski cells by western blotting and quantitative real time pcr assessment of transcripts. results: has mrnas in the nonpregnant (np) and pregnant cervix are transcribed from the distal promoter upstream of exon . two transcripts of approximately . kb and . kb were detected that arise from use of polyadenylation sequences. as compared to np, the expression of has is increased , , and fold on gestation days , and shortly postpartum respectively. has mrna expression is increased upon treatment of caski cells with epidermal growth factor ( ng/ml) and is suppressed in cells treated with ag ( m), an inhibitor of egf receptor phosphorylation. maximal stimulation was observed at and hours of treatment. conclusion: has is the major ha synthase expressed during cervical ripening and the majority of transcripts are driven by the distal promoter in the has gene. in vitro studies using caski cells suggest has is regulated in part by the egf signaling pathway resulting in a several fold increase in has expression. these results provide an understanding of has gene regulation at the time of cervical ripening which will ultimately enhance our understanding of the molecular mechanisms important to cervical ripening. chorion and decidua cells. chad a grotegut, bernard j canzoneri, liping feng, phil heine, amy p murtha. obstetrics and gynecology, duke university, durham, nc, usa. objective: preterm premature rupture of the fetal membranes accounts for approximately % of all preterm deliveries. cigarette smoking independently carries a fourfold increase risk for pprom. our laboratory has previously demonstrated that the chorion layer undergoes apoptosis in women with pprom. this study was conducted to determine if extract of cigarette smoke causes cell death in specific cells of the fetal membrane. fetal membranes were collected at the time of elective cesarean section from women without labor and at term. the chorion and decidua layers were separated and purified on a gradient spin column and then plated near confluence. cigarette smoke extract (cse) was collected in cell media and used to treat chorion and decidua cells in culture at concentrations ranging from % to % in -well plates. cell viability was determined at , and hours following treatment with a non-radioactive cell viability assay. data were analyzed using paired t test (analyse-it, leeds, uk). chorion and decidua cells underwent cell death when exposed to cse in a dose dependent fashion. increasing concentrations of cse resulted in increased cell death at hours in both cell types (figure ). at hours, chorion exhibited greater percent cell death compared to decidua at concentrations of , and % cse (p= . , . , and . , respectively). for any given concentration of cse, the degree of cell death increased with increasing length of exposure ( , and hours) for each cell type. chorion cells routinely exhibited greater percentage of cell death following treatment with cse at concentrations ranging from - % compared to decidua cells. human chorion and decidua cells in primary cell culture exhibit a dose-response and time dependent cell death in the presence of cse. human chorion cells show greater sensitivity to cell death when compared to decidua cells. further studies are needed to determine the mechanisms through which these cell types undergo cell death and the implications for the differential sensitivity to cigarette smoke. yoon ha kim, tae-bok song, cheol hong kim, jong woon kim, moon kyoung cho, sung yeul yang, bong whan ahn. obstetrics gynecology, chonnam national university medical school, gwangju, korea; biochemistry, chonnam national university medical school, gwangju, korea. objective: to investigate the lipid peroxide levels and protein carbonyls levels in the amniotic fluid of pregnant women with preterm premature rupture of membranes (pprom). the lipid peroxide levels in the amniotic fluid of normal pregnancy (n= ) and pregnant women with pprom (n= ) were newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced measured by thiobarbituric acid reaction. the protein carbonyl contents in the amniotic fluid of normal pregnancy (n= ) and pregnant women with pprom (n= ) were determined by the , -dinitrophenylhydrazine method. after amniotic fluid of them were mixed and incubated up to hours with . ml of mm moxalactam, cefodizime, amoxacillin, erythromycin, the lipid peroxide levels and protein carbonyl contents in them were measured. results: . the lipid peroxide levels in the amniotic fluid of pregnant women with pprom was significantly higher than that of normal pregnancy ( . ± . vs. . ± . nmol/mg protein, p< . ). . the protein carbonyl levels in the amniotic fluid of pregnant women with pprom was significantly higher than that of normal pregnancy ( . ± . vs. . ± . nmol/mg protein p< . ). . the lipid peroxide levels and protein carbonyls formation by moxalactam in the amniotic fluid of pregnant women with pprom was significantly higher than basal level ( . ± . vs. . ± . nmol/mg protein, . ± . vs. . ± . nmol/mg protein, p< . ). . the lipid peroxide levels and protein carbonyls formation by cefodizime in the amniotic fluid of pregnant women with pprom was significantly lower than basal level ( . ± . vs. . ± . nmol/mg protein, . ± . vs. . ± . nmol/mg protein, p< . ). . there were no significant differences in the levels of lipid peroxide and protein carbonyls by amoxacillin and erythromycin in the amniotic fluid of pregnant women with pprom between antibiotics-induced and basal levels. background: chorioamnionitis (cam) is a major antecedent of preterm delivery (ptd) associated with elevated amniotic fluid tnf and il . we hypothesized that these cytokines enhance the term decidual cell (dc) expression of the matrix metalloproteinases (mmp) and , which can then promote ptd by degrading the extracellular matrix of the decidua, fetal membranes, and cervix. methods: immunostaining for mmp- , mmp- , and vimentin (a dc marker) was performed on cam-complicated (n= ) and gestational age-matched control decidua (n= ), and staining intensities were evaluated by hscore. confluent, leukocyte-free term dcs were primed with - m estradiol (e ) or e + - m medroxyprogesterone acetate (mpa), and then switched to a defined medium with e +/-mpa with or without ng/ml of il or tnf . secreted mmp- and mmp- levels were measured by elisa (n= ), and quantitative rt-pcr assessed mmp- and mmp- mrna levels (n= ). results: tissue staining revealed that mmp- and mmp- levels in cam-complicated decidua (hscore mean±sem: ± and ± , respectively) were significantly higher than in control decidua ( ± , and ± respectively; p< . ). in cultured term dcs incubated with e , tnf and il significantly increased secreted levels of mmp- compared to e alone (pg/ml/ g protein: . ± . and . ± . , respectively, vs. . ± . ; p< . ). in parallel incubations with e +mpa, basal mmp- output was lowered by %, and tnf -and il -elicited mmp levels were blunted by % and %, respectively. rt-pcr confirmed that tnf and il increased mmp mrna levels (p< . ), although mrna levels in e +mpa incubations were not different from those of e alone. mmp levels in all treatments were similar. conclusions: mmp- and mmp- are elevated in cam decidua compared to controls. our in vitro results suggest that mmp- expression is enhanced by the high levels of il and tnf associated with cam, and that mpa may be able to blunt this effect. we have previously found a similar regulatory mechanism of mmp- and mmp- and their over-expression in cam-complicated tissues. synergy among these mmps may represent a potent pathogenic mechanism of cam that can be targeted through the therapeutic use of progestins in preventing cam-induced ptd. domerudee preechapornprasert, patama promsonthi, wasun chantratita, mana rochanawutanon, patcharee karnsombut, chutatip srichunrusami, stephen j lye, boonsri chanrachakul. obstetrics and gynecology, ramathibodi hospital, mahidol university, bangkok, thailand; pathology, ramathibodi hospital, mahidol university, bangkok, thailand; obstetrics and gynecology, samuel lunenfeld research institute, toronto, canada. objective: cervical ripening is an inflammatory process involving chemokines, cytokines and various mediators. recent study has shown that the level of monocyte chemotactic protein (mcp) , a chemokine, increases in amniotic fluid during spontaneous labor. the aim of this study was to examine the localization and expression of mcp in human cervix before pregnancy, during pregnancy and after the onset of labor. methods: this study was approved by the local ethics committee and written informed consent was obtained from each participant. cervical biopsies were taken from groups of women; nonpregnant women, first trimester pregnant women, term pregnant women with and without labor. tissue samples were fixed in % formal saline for paraffin section. immunohistochemistry (n = each) was performed by avidin biotin complex (abc) technique using monoclonal antibody specific to human mcp . the mcp messenger(m) rna was identified by reverse transcription-polymerase chain reaction using gene specific primer against mcp and mcp receptor (n = each). results: immunohistochemistry demonstrated mcp in cervical tissues from all four groups of women. mcp was localized on plasma membrane and cytoplasm of both squamous epithelial and columnar cell lining of endocervical gland. mcp and mcp receptor mrna were identified in nonpregnant, first trimester and term with and without labor human cervix. conclusion: mcp and mcp receptor were located in cervical tissues of nonpregnant and pregnant women at different gestation both before and after the onset of labor.ongoing studies are investigating the role of this chemokine during pregnancy and labor. whether preterm cervical ripening is just an aberrant regulation in timing or whether divergent mechanisms and pathways are involved in preterm versus term cervical ripening remains to be elucidated. methods: cervical tissue was collected from groups of cd- mice. group : mouse model of ptb that utilizes intrauterine infusion of lipopolysaccharide (lps). group : e dams representing preterm controls. group : e . - dams selected from a timed pregnant batch where half of the dams had delivered representing term cervical ripening. n= dams/treatment group. separate rna samples were used for microarray analysis (ma). significance analysis for ma and partek software was used for biostatistical analysis. pathway analysis was performed using david. quantitative pcr was performed to confirm the most differentially regulated genes. results: using a cut-off of -fold change with p value of < . , genes in the cervix were differently regulated between the groups. principal component analysis revealed three distinct groups (see graph). functional annotation clustering demonstrated the following pathways: ) in preterm cervical ripening (e lps vs e ): immune and inflammation response, defense response ) in term cervical ripening compared to preterm controls: negative regulation of cellular process and biological process, ecm, cell-cell communication. qprc confirmed the highly significant differences found in ma (see table) . conclusions: the molecular mechanisms and pathways governing preterm and term cervical ripening are distinctly different. elucidating these unique pathways can lead to improved therapeutics for prevention of ptb as well as for postdate pregnancies. cervical ripening at term involves activation of apoptotic enzymes. maria kb sennstrom, valentina ciani, gunvor e ekman. obstetrics and gynecology, women and child health, karolinska institute, stockholm, sweden; obstetric and gynecology, university of siena, siena, italy. aim: to investigate if the human cervical ripening at term involves programmed cell death. during the final cervical ripening the extracellular matrix dominated cervix undergoes an extensive remodelling of the tissue. inflammatory mediators such as the cytokines increase in ripening cervical tissue at term. programmed cell death, apoptosis, has been suggested as important in this process. apoptosis can be induced by inflammatory mediators such as cytokines. we also looked upon the distribution of inflammatory cells in cervical tissue. materials and methods: cervical biopsies from pregnant women at term and post partum women with fully ripened cervix were studied. biopsies from non-pregnant women served as controls. immunohistochemical analysis of the apoptotic enzyme caspase- and the inflammatory cell marker cd was performed on paraffin embedded sections of cervical tissue. double staining was performed. mann-whitney u-test was used for statistical analysis. results: there was a significantly higher frequency of caspase- staining in the post partal sections from ripened cervical tissue compared to tissue from term pregnant (p= . ) with unripe cervix and from non-pregnant patients (p= . ). the inflammatory cells staining for cd increased in post partal and term pregnant sections compared to non-pregnant (p= . ). there was a higher frequency of caspase- positive cells in the post partal tissue than of cd positive cells (p= . ). the localization of cd- positive staining was highest in the epithelia and basal lamina while caspase- staining was most pronounced in stromal tissue and around vessels. conclusion: our data show apoptotic activity in stromal tissue in fully ripened human cervix at term of pregnancy, suggesting that apoptotic mechanisms are involved in the extracellular matrix remodelling at term. the apoptotic activity is not co-localized with inflammatory cells suggesting non-infectous inflammation with apoptosis as important for cervical ripening at term. objective: cervical biomechanical responses are important for accommodating the increased stress induced by an enlarging uterus. a mechanical testing system was modified to evaluate the stress relaxation response in the pregnant cervix. methods: tissue harvested from the non pregnant and timed pregnant (days , , , , and ) sprague-dawley rats underwent tensile testing using an instron material testing system. the testing regimen consisted of tissue extension to near maximal strain over seconds followed by a period of constant strain for minutes, then return to rest over seconds. this cycle was repeated additional times with a minute rest period between cycles. strain and force measurements were recorded at second intervals. - animals were used for each time point. in addition, stress and strain at the yield point was also determined for each gestational time point. results: the pregnant and non pregnant samples exhibit marked differences in response in both stress and strain. the timed pregnant tissue demonstrated progressively increased compliance and lower nominal stress compared to non pregnant tissue. peak nominal stress declined with each successive cycle. this was demonstrated in the peak nominal stress and strain values at the yield point. conclusion: the cervix becomes more distensible (compliant) but less resistant to force with increasing gestational age. ...,.. e. coli . x .:!: . x . x .:!: . x group a (c, c) group b ( c, p) . x + . x . x + . x . . . group c (p, p) . x o"::!>s x o" . x ~ .ox: o• k. ~neumoniae group a ( c, c) . x + . x . x + . x . . . group b (c, p) . x + . x . x + . x group c (p, pl . x ~ . x . x ~ . x c. al bicans group a (c, c) . x '+ . x . x + . x group b (c, p) . x o•+ . x . x + . x . . . group c (p, p) . x ~ . x . x ~ . x • introduction: a growing body of evidence supports that inflammatory processes are implicated in spontaneous preterm birth (ptb). using an inflammatory and non-inflammatory mouse model of ptb, we sought to determine if activation of these inflammatory pathways are essential for ptb and/or cervical ripening to occur. methods: timed pregnant cd- mice were used in these two models of ptb: ) a model of intrauterine inflammation where lipopolysaccharide (lps) is injected into the uterine horn (n= ); controls for this model received intrauterine saline (n= ) and ) a non-infectious model of ptb using ru sq ( ugrams/dam) (n= ); controls for this model received no intervention (n= ) were used for these studies. for both models, hours later uterine and cervical tissues were harvested. the tissues were processed for protein and rna studies. elisas were performed to assess il- and tnf-alpha in the uterine tissue. mrna expression of ifn , il- , il- , il- , tnf-alpha were assessed in cervical tissue from both models by quantitative pcr. results: in uterine tissues, both il- and tnf were significantly elevated in the lps-induced ptb when compared to control, saline, and non-infectiousinduced preterm birth (p< . ) (figure ). in cervical tissue, an increase in il- , il- beta and tnf mrnawas observed in both models, while ifn-gamma and il- were only increased in the lps model. (figure ). conclusions: up-regulation of pro-inflammatory cytokines in the uterus do not appear to be essential for ptb. cytokine expression in the cervix is greater in an inflammatory model of ptb but is also present in a non-infectious model. these studies suggest that targeting a cytokine response in the cervix may hold the most promise in prevention of ptb. the initiation of labor at term and preterm is associated with an inflammatory response, with increased interleukins in amniotic fluid (af) and infiltration of the myometrium by neutrophils and macrophages (m ). whereas, in preterm labor, intra-amniotic infection may provide the stimulus for increased af interleukins and inflammatory cell migration, the stimulus for these events at term has remained uncertain. in studies using pregnant mice, we observed that the m that invade the maternal uterus near term arise from the fetus. furthermore, we obtained compelling evidence that surfactant protein-a (sp-a), a developmentally regulated c-type lectin secreted by the fetal lung into af near term, activates af m , which migrate to the uterus where they promote an inflammatory response culminating in labor. we propose that interactions of m surface receptors with sp-a, at term, or bacterial lipopolysaccharide at preterm, initiate changes in m phenotypic properties, resulting in the enhanced expression of genes that promote their migration to the uterus. the objectives of the present study were to analyze the numbers and phenotypic properties of mouse af m during late gestation and to identify their putative tissue source(s) of origin. to assess changes in the number of m in af during late gestation, af cells were isolated and stained for the m marker f / . the density of adherent f / + cells greatly increased in equivalent volumes of af between . and . days postcoitum (dpc) ( . dpc = term). interestingly, the f / + cells at . dpc were highly similar in morphology to those present in . dpc fetal lung, but distinctly different from those in fetal liver, suggesting their pulmonary origin. the af m were foam cell-like, suggesting the presence of lipid inclusions, a property shared by adult alveolar m . to further analyze gestational changes in the m population(s) in mouse af, we used flow cytometric analysis. in our initial studies, cells isolated from af were stained for f / and for cd , a pan-leukocyte marker. we observed that af from . and . dpc mice contained two sub-populations of cd + f / + cells. studies are in progress to analyze these af m populations for expression of cell surface antigens indicative of their maturity, activation state and chemotactic properties in association with the developmental induction of sp-a synthesis and secretion by the fetal lung. april bleich, patrick keller, r ann word. ob-gyn, ut southwestern, dallas, tx, usa. the role of prs, proinflammatory cytokines, cell adhesion molecules, cox- , and toll-like receptors in mediating cervical ripening prior to labor is not clear. the objective of this study was to quantify pr isoforms and determine the relative expression of certain inflammation-related genes in cervical stroma from nonpregnant and pregnant women in early gestation (eg), term before and after cervical ripening, and during labor. methods: standard curves of pr-b, -a, pra+b and qpcr were used to quantify total and pr-b in cervical stroma from nonpregnant (proliferative, n = ; progestin treatment, n = ) and pregnant women undergoing hysterectomy (eg, n = ; term before ripening, n = ; after ripening, n = ; in labor, n = ). cervical status was determined by modified bishop scoring. results: total pr expression was maximal in nonpregnant women in the proliferative phase ( . ± . pg/ug cdna) and decreased % by progestins ( . ± . pg/ug cdna). this level was maintained in stroma from pregnant women before labor ( . ± . , eg.; . ± . before ripening; . ± . after ripening pg/ug cdna). in contrast, total pr was decreased significantly in the dilated cervix ( . ± . pg/ug, p < . ). interestingly, whereas pr-b was ± % that of total pr in the nonpregnant cervix, pr-b mrna levels were ± % to ± % in cervical tissues from all pregnant women and did not vary with labor status. using immunoblot analysis and pr-specific antibodies (pgr ), pr-b immunoreactivity was % that of total pr in all samples from pregnant women, and both pr-a and -b were downregulated significantly in the dilated cervix (from ± to ± units/atub). decreased expression of pr in the dilated cervix was accompanied by significant increases in il- , mcp- , tlr- , cd l, cox- , and s a mrna (all p < . , anova) but not cd b or tlr- . pgdh mrna was decreased significantly in the dilated cervix. with the exception of cox- , expression of these genes was similar before labor regardless of cervical ripening. conclusions: both pr and pr-b are decreased proportionately in the dilated cervix, but not during cervical ripening. further, a number of inflammatory gene products are increased dramatically in the cervix during labor, but not before. taken together, the results suggest that cervical ripening is distinct from cervical dilation and involves upregulation of cox- but not il- , mcp- , or toll-like receptors. ifn-y il- il- , il- tnf- "p value < . lps/saline ru /controls . "" . " . " . * " " . * . . " association between interleukin- (il- ) and il- to examine association of amniotic fluid interleukin (il- ) concentration with il- and its receptor il -r haplotypes in term and preterm caucasians (c) and african americans (aa) samples. methods: in this study case (preterm birth -ptb [< weeks]) and control (term [> weeks]) amniotic fluid (af) il- concentrations were analyzed for association with haplotypes of the il- and il r genes in aa and c separately. in il- , eight, and in il- r, single nucleotide polymorphisms (snps) were examined. aa and c maternal and fetal genotypes were assessed (aa: maternal:cases- controls- fetal: cases- fetal controls-; c: maternal cases- , controls- , fetal:cases- ; controls= ). haplotype associations were performed by using a sliding window with outcome il- concentration. analyses were performed separately on maternal and fetal dna. results: the strongest haplotype associations were observed in il- r rather than in il- . in c fetal dna il- r haplotypes defined by markers - bp from the transcription start site associated most strongly with af il- concentrations (global p= . x - ) and in aa maternal il- r haplotype markers at - - - (global p= . x - associated with il- concentrations. in the c fetal cases the - haplotype with the highest concentration was a-g (log(cytokine) = . pg/ml). in aa maternal samples the highest concentration was observed for haplotype t-t-g-c at - - - .this was seen in both cases and controls at (case log (cytokine) = . ; control log(cytokine) = . pg/ml). significant associations from haplotype analyses converged on three regions of the il- r in both races. no strong differences were observed between the haplotypes of cases with and without microbial invasion of the amniotic cavity (miac), with the exception of aa fetal samples that showed two overlapping haplotypes in il- that associated in cases with miac but not in cases without miac (- and - ; - and - )(both with p < x - ) conclusion: differences in the af il- concentration in ptb do not result from single snp effect on il- but are a result of complex relationships between il- and il- r haplotypes. these associations exhibit racial disparity. the role of tnf-in parturition. helen alexander, amanda tattersall, mark tattersall, suren sooranna, peta grigsby, leslie myatt, mark johnson. obstetrics gynaecology, imperial college, london, united kingdom; obstetrics gynaecology, university of cincinnati college of medcine, cincinnati, oh, usa. introduction: tumour necrosis factor-alpha (tnf-) is thought to play a role in inflammation-induced preterm labour since the decidua produces tnfin response to bacterial products and amniotic fluid tnf-concentrations are increased in the presence of intra-amniotic infection. the aims of this study were to (i) investigate the expression of myometrial tnf-and its receptors in relation to the onset of preterm and term labour; (ii) to identify which intracellular pathways are activated by tnf-; and to investigate the effect of tnf-alone and in combination with il- or il- on gene expression in uterine myocytes. methods: biopsies of human myometrium were taken at caesarean section from women before and after the onset of preterm and term labour and analysed for tnf-and its receptor mrna expression. a further samples were obtained before the onset of labour (n= ) from which myocytes were isolated and cultured in -well plates. when cells were - % confluent either tnf-at a concentration of ng/ml was added to the cells for , , , and min and the cells analysed by western blotting or tnf-at concentrations of , . and ng/ml was added either alone or in combination with similar concentrations of il- or il- to cells for hours and mrna was extracted and converted to cdna to determine il- and gapdh gene expression by qpcr. results: there was no change in tnf-mrna expression in relation to the onset of labour, but the expression of tnfr and tnfr mrna levels were significantly increased with gestation and further increased with the onset of labour. incubation of uterine myocytes with tnf-( ng/ml) activated all three mapk substypes: erk, jnk, and p , activation peaked between - minutes. preliminary data suggest that tnf-induces il- mrna expression but exposure to il- or il- itself did not enhance this response. conclusions: although there is no significant increase in tnf-concentration from baseline during labour we have shown tnf receptor mrna levels do increase with labour at term. exposure of isolated uterine myocytes to tnfcauses activation of all mapk subtypes and an increase in il- mrna expression. this enhanced mapk-dependent il- expression at term may be mediated via increased myometrial sensitivity to tnf-through increased tnf receptor expression. remodeling. brenda c timmons, anna-marie fairhurst, mala s mahendroo. obstetrics and gynecology, ut southwestern medical center, dallas, tx, usa; immunology, ut southwestern medical center, dallas, tx, usa. objective: the molecular mechanisms involved in cervical ripening are not well understood. immunohistochemical studies from our lab report a recruitment of inflammatory cells to the cervical stroma one day before birth in the mouse using a neutrophil/monocyte marker (neutrophil / ). in this study, we sought to identify and quantitate inflammatory cells migrating into the mouse cervix and to determine if this recruitment was affected by changes in progesterone levels. peripheral blood was also evaluated to see if changes in the cervix was paralleled in blood. methods: flow cytometric analysis was performed using cervical cells and peripheral blood obtained before and during cervical ripening along with - h postpartum. dispersion of cervical cells was optimized. these cells were stained with a panel of fluorescent conjugated antibodies directed against leukocyte antigens and analyzed on an lsrii flow cytometer. cells were also sorted and stained to visualize cell morphologies. to determine the effect of progesterone on the migration of leukocytes, gestation d mice were treated for h with a progesterone receptor (pr) antagonist prior to tissue collection. results: neutrophils do not appear to increase in the cervix until after birth. monocyte (mo) numbers do increase during cervical ripening (late day , d . ) and remain high through postpartum (pp). macrophages (mØ) are present prior to cervical ripening and steady state levels are maintained during labor and pp. pr antagonist treatment on d resulted in a premature increase in mo but not neutrophils or mØ. in contrast to the cervix, mo and neutrophil numbers do not significantly increase in the peripheral blood until pp. results from lymphocyte studies suggest a low level of b and t cells in the cervix. in the peripheral blood, b cells remain consistent through parturition and the t cells decrease by d . and continue to decrease pp. conclusion: tissue mo are increased in the cervix during ripening. this recruitment is dependant on loss of pr function. in contrast, neutrophils are increased in the pp cervix while mØ numbers appear constant. timing of changes in mo and neutrophil numbers in the peripheral blood differed from that observed in the cervix suggesting quantitation of these cell types in blood is not reflective of what is occurring in the cervix during ripening, dilation and pp repair. novel interactions between nf-b and other labour-associated transcription factors identified by a tf-tf array. shirin khanjani, yun s lee, suren r sooranna, mark r johnson, phillip r bennett. irdb, imperial college, london, united kingdom. introduction: external stimuli lead to changes in cellular gene expression through activation of inducible transcription factors. nf-b is a ubiquitous transcription factor classically associated with inflammation, which is activated in response to infection and proinflammatory cytokines such as those prevalent during the labour. the tf-tf interaction array uses a novel technology for detecting interactions between transcription factors based on binding of tfs to their own consensus dna binding sequence. materials and methods: primary myometrial cells were grown until - % confluent and stimulated with ng/ml il- prior to nuclear protein extraction. the nuclear extracts were incubated with the provided set of biotin-labeled, double-stranded oligonucleotide probes, which represent a known library of cis-elements. during the incubation step, these tf probes bind to their specific tfs in the nuclear extract. next, immunoprecipitation was performed using an antibody against nf-bp , which pulled out nf-bp and any tfs bound to it, bound to corresponding cis-elements. normal igg was used in a parallel experiment to represent a negative control. free cis-elements and nonspecific binding proteins were washed away and the cis-elements were finally eluted and hybridized to the array membrane, which is spotted with different tf consensus sequences. results: table shows the different tfs interacting with nf-bp based on the degree of binding stimulated by il- compared to no-il- control. conclusion: these data show that il- stimulation causes nf-b to bind to a wide variety of other treansciption factors. of particular interest in the area of parturition is binding to the other pro-inlammatory tfs such as c/ebp and ap- , which are known to regulate labour-associated genes in synergy with nf-b. the lack of association between nf-b and pr without il- stimulation supports the concept that with the onset of labour inflammation leads to functional progesterone withdrawal, rather than pr acting to inhibit inflammation. table high ap- , c/ebp, cbf, creb , c-myb, e f- , ets, ets- /pea ,fast- , gas/isre, hse, mef- , mef- , myc-max, nf- , nfatc, nf-e , nf-e , pax- , objective: pre-partum cervical ripening involves remodeling of collagen structure and inflammatory immune cell activity (jsgi : , ; reprod biol endo : , ) . although parturition is associated with systemic or local progesterone withdrawal (ajog : , ) , effects of a decline in progesterone on cervical ripening is not known. the present study tested the hypothesis that progesterone withdrawal promotes collagen degradation, innervation, and immune cell trafficking in the cervix of nonpregnant mice. methods: adult virgin female c bl mice received capsules (sc) with oil vehicle (v) or estradiol (e) and progesterone (p) to simulate concentrations in pregnancy (hum reprod : , ) . after days, mice in the v and e+p groups were euthanized. the p capsule was removed from some mice on day (e-p) and groups killed on days and . cervix sections were stained for collagen, nerve fibers, macrophages, or neutrophils (n= /group/day; sections/ cervix; biol reprod : , ; jsgi : , ) . stained macrophages and neutrophils were counted (image pro-plus , media cybernetics). results: e+p treatment for days promoted hypertrophy of the cervix compared to v controls, i.e., collagen content and structure diminished, cell nuclei density declined, and nerve fibers increased. removal of p did not affect these endpoints. for immune cells, e+p for , , or days decreased immune cell numbers. by contrast, p removal increased macrophages and neutrophils in the cervix on days and (p< . , e-p vs e+p groups, respectively). the census of resident immune cells in e-p groups at and h after p removal equaled that in the v group. conclusions: mimicking gonadal steroid concentrations in circulation during pregnancy promotes hypertrophy and suppresses immigration of immune cells in the cervix. in this non-pregnant murine model for parturition, progesterone withdrawal recruits immune cells, but fails to promote further remodeling or hyperplasia of nerve fibers in the cervix. the findings raise the possibility that ripening of the cervix requires not only recruitment, but also activation of immune cells. whether proinflammatory activities by specific immune cells affect nerve fiber hypertrophy or neural activity, as part of the mechanism for ripening of the cervix, remains to be determined. we have previously identified and characterized a truncated pr (pr-m), that localizes to the mitochondrion by multiple experimental techniques, including confocal imaging of a recombinant gfp fusion protein, western blot analysis after cellular fractionation of nuclear pr negative t d-y breast cancer cells and western blot analysis of purified human heart mitochondrial proteins. initial studies with nuclear pr negative mcf- a breast epithelial cells shown to express pr-m demonstrated an increase in mitochondrial membrane potential (mmp) with progesterone/progestin treatment. these studies led to the hypothesis that progesterone modulates cellular respiration via the mitochondrial receptor, pr-m. objectives: the present studies sought to further localize pr-m to the outer, inner or matrix portion of the mitochondrion and to correlate the increase in mmp with total cellular atp production. additionally, the potency of progesterone and synthetic progestins on the change in mmp was evaluated. methods: the location of pr-m was determined by western blot analysis after fractionation of human heart mitochondria with digitonin treatment and differential centrifugation. mmp was determined in mcf- a breast epithelial cells and a rhabdomyosarcoma cells by the change in fluorescent emission of jc- . total atp was determined by a bioluminescent assay. results: western blot analysis after mitochondrial fractionation showed pr-m localization exclusively in the outer membrane. a dose-dependent increase in mmp was seen in cell lines with - min treatment with progesterone and r which were inhibited by a specific pr antagonist, rti- - b, and not affected by the translational inhibitor, cycloheximide. similar changes in mmp were seen with the same concentration of progesterone, mpa and r . progesterone/progestin treatment for min led to an increase in total cellular atp without a change in cell number. conclusions: progesterone/progestin treatment results in an increase in cellular respiration in cells expressing an outer mitochondrial membrane pr and known to lack nuclear pr expression. this may represent a mechanism whereby progesterone enhances cellular energy production to meet the demands of pregnancy. introduction pge is a major product of the fetal membranes, decidua and myometrium and plays an important role in cervical ripening and myometrial contractions. there are four pge receptors, ep- and ep- mediate contractions whilst ep- and ep- mediate quiescence. ep- contains multiple consensus sequences for transcription factors known to be of importance in labour, in particular nfkappab. we therefore performed experiments to determine the effect of activation and inhibition of nfkappab upon ep- expression. myocytes plated in well plates were treated with il- ( ng/ml), to activate nfkappab. myometrial cells were also transiently transfected with nfkappab p sigenome smart pool and sicontrol non-targeting sirna to knock down nfkappab p . rna was extracted for amplifying ep- using quantitative rt-pcr with amplification of l as a control to normalise data. il- caused an increase in expression of ep- . knock down of nfkappab using si-rna resulted in a further increase in ep- . this data suggests that although il- stimulates expression of ep- it does so through an nfkappab independent mechanism. the upregulation of ep- with sirna knock down of p suggests that activation of nfkappab would inhibit ep- expression consistent with the concept that activation of nfkappab at term causes the myometrium to adopt a more contractile phenotype. introduction: a role for the pro-inflammatory cytokine il- is suggested in preterm and term birth, independent of the presence of infection. we previously showed that mice with a null mutation in the il- gene (ko) delivered one day later than wild type (wt) mice due at least in part to altered timing of uterine expression of prostaglandin (pg) f receptor, ptgfr, mrna. we also observed differences in mrna expression of other uterine activation proteins (uaps), pg h synthase (pghs)- , oxytocin receptor (otr) and connexin- (cx- ), suggesting multiple physiological effects for il- in term delivery. objective: to examine the effect of il- deficiency on the peri-partal uterine mrna expression of the pge receptors (ep) , and ; the post-partum changes of all uaps; and the relationship of these to serum progesterone (p ) concentrations. methods: gestational length was observed in pregnant c bl/ wt (n= ) and ko (n= ) mice. uap mrna levels were measured by real time rt-pcr in wt and ko dams sacrificed from d through delivery and up to h postdelivery. serum p was determined by ria. data were analyzed by one-way and two-way anova using the holm-sidak test to differentiate treatment effects at p< . . results: birth was delayed in the il- ko mice ( . ± . d vs. . ± . d), and this affected the timing of peri-partal changes for all uaps similarly. both ep and ep (relaxatory receptors) were elevated at d in ko dams, but returned to low levels before delivery and were elevated at delivery (ep ) or afterwards (ep ). ep levels did not change. otr increased several hours before delivery in all dams. cx- increased at delivery, then fell, while pghs- increased at delivery and remained elevated afterwards. ptgfr mrna increased - -fold at delivery and a further - -fold after delivery, suggesting loss of pgf permitted enhanced ptgfr expression. p serum concentrations fell pre-partum in both groups. conclusions: il- ko alters the expression pattern of several pregnancy and parturition-related genes and may delay the pre-partum p fall, suggesting a potential ovarian effect. a uterine role for il- in regulating the timing of normal term parturition cannot be ruled out. objective: recent studies have highlighted the prevalence of vitamin d deficiency in pregnant women, particularly in those from ethnic groups with darker skin who require higher levels of uv light to make parental vitamin d. as the active form of vitamin d, , -dihydroxyvitamin d ( , (oh) d ) is a potent immunomodulator, we postulated that vitamin d deficiency may lead to dysregulated placental immunity. both trophoblast and decidua express the enzyme -hydroxylase (cyp b ) which catalyzes synthesis of , (oh) d from the inactive pro-hormone -hydroxyvitamin d ( ohd ). in view of the role of trophoblast as a barrier site protecting the fetus against infection, we investigated the impact of cyp b , ohd and , (oh) d on innate immune responses in trophoblastic cells. methods: a trophoblast cell line was used to assess the effect of vitamin d on innate immune responses. the cells were treated for hrs with various concentrations of , (oh) d ( - nm) and antimicrobial cathelicidin expression was assessed by real time pcr. to assess whether expression of cyp b was affected by pathogenic stimuli, a cells were treated with ligands for toll-like receptor (tlr) - , cyp b expression (real time pcr) and ohd utilization were assessed. results: a trophoblast cell line; which shows temperature-sensitive differentiation, revealed expression of cyp b with higher levels of enzyme activity under conditions of syncytiotrophoblast development. cells treated for hrs with , (oh) d showed dose-dependent induction of the antimicrobial defensin cathelicidin expression ( . - fold induction), whilst cells treated pro-hormone ohd ( nm) showed . -fold induction of cathelicidin expression. activation of tlr (poly i:c) and tlr (lipopolysaccharide) enhanced the expression of cyp b ( -and . -fold) and increased the sensitivity to ohd as a consequence. conclusion: these data show that autocrine synthesis of , (oh) d from ohd can stimulate trophoblast immune responses in a similar fashion to macrophages. as ohd is the major circulating form of vitamin d, we hypothesize that trophoblast innate immunity may be significantly compromised under conditions of vitamin d deficiency. introduction: secretory leukocyte protease inhibitor (slpi) is a potent -kda protein inhibitor of neutrophil elastase and it is a mediator of mucosal immunity and an inhibitor of nfkb regulated inflammatory responses. however, its source, function and regulation within the uterus during pregnancy and at parturition are not well defined. it has previously been shown to be present in fetal membranes and cervical mucus and in amniotic fluid, where its levels is increased from second trimester to term and with a further increase at parturition. slpi has also been shown to be responsive to progesterone in human epithelial cells. our aim was to determine the effects of il- on slpi gene expression in human myometrium. methods: primary human uterine myocytes were isolated from non labouring myometrium and cultured in well plates and when cells were - % confluent they were serum starved overnight and incubated with m methyl- -hydroxy-progesterone acetate for h and with or without ng/ml il- for a further hours. at the end of incubations rna was extracted and converted to cdna. paired upper and lower segment myometrial tissue was collected at caesarean section either before or after the onset of term or pre-term labour and frozen for extraction of rna (n= for ptnl, ptl, tnl and l). copy numbers of slpi, gapdh and beta-actin were measured by qpcr. results: h incubation of uterine myocytes with ng/ml il- caused a marked increase in slpi by % (n= ; p< . ). incubation of uterine myocytes with m progesterone for h also increased slpi by % (n= ; p< . ). incubation with il- for h in the presence of h with progesterone increased slpi: gapdh mrna ratio from . ± . to . ± . (mean ± sem; p< . ). slpi expression was similar in the upper and lower segment myometrium in preterm patients. in term myometrium the slpi: beta-actin mrna ratio was increased by -and -fold in term labour versus term non labour samples in the lower and upper segment respectively (mean ± sem; p< . ). these data show slpi is present in human myometrium and that it is increased by il- . progesterone also increases its expression. its expression is increased in term labour where its anti-bacterial, anti-fungal and anti-viral properties could allow it to act as an endogenous block to infections. objective: pre-b cell colony-enhancing factor (pbef) downstream mapk signaling and transcription factor activation. introduction: pbef is expressed in all layers of the human fetal membranes and the myometrium and is upregulated by labor, infection, nf-kb, ap- and stretching. pbef has the ability to protect a variety of cells from apoptosis, however it also appears to act as an insulin mimetic via the insulin receptor (fukuhara et al. science : ; - ) . its levels are elevated in obese patients, those with type diabetes and in gestational diabetes. because pbef has poorly understood biological activities an investigation of mapk signaling and transcription factor activation induced by pbef has been undertaken in order to gain insights into its mechanisms of action. methods: primary aec were isolated from fetal membranes and treated with rhpbef ( ng/ml) for h, lysed and the resultant proteins labeled with cy or cy (amersham). there were used on a protein microarray containing constituents of the mapk signaling pathways (sigma). isolated aec were transfected with luciferase constructs for nf-kb, ap- , cre, hse and gre response elements using the exgen reagent. the cells were treated with rhpbef ( . , . , , ng/ml) hrs, lysed and ul of sample was analyzed for luciferase activity with dual-luciferase reporter assay system (promega). co-transfection with gfp and sv luciferase constructs to control for transfection efficiency and cell number (respectively) was also performed for each experiment. results: pbef significantly upregulated mapk signaling components including functioning as part of the erk pathways and belonging to p signaling. it also activated nf-kb and camp response elements. however, it significantly down regulated signaling molecules belonging to the jnk pathway that resulted in decreased c-jun phosphorylation. conclusions: pbef signaling in aec is consistent with its anti-apoptotic ability and its upregulation of the pro-inflammatory cytokines. although some mapk components responsive to pbef could be associated with insulin receptor signaling, some may not. therefore, it appears likely that pbef interacts with another potentially unique, but currently unidentified receptor. was the first effector to be characterised, but camp is now known to have other effectors, including camp receptor protein, cyclic nucleotide-gated channels and camp-guanine nucleotide exchange factor/exchange protein directly activated by camp (camp-gef/epac). two epac's have been identified and they consist of functional domains: camp-binding domains, a dep domain, a ras exchange motif and a gef domain. our aim was to determine the presence of epac's in human myometrium and to study the effect of camp responses on prolabour genes such as il- , pghs- , otr and fp. methods: primary human uterine myocytes were isolated from non labouring myometrium and cultured in well plates until - % confluent. cells were serum starved overnight and incubated with μm methyl progesterone, . mm sodium -bromo-camp, . mm forskolin, μm kt , ng/ml brefeldin a and . mm -pmeopt- 'o-me-camp either alone or in combination for h. rna was extracted and converted to cdna. paired upper and lower segment myometrial tissue was collected at caesarean section either before or after the onset of term or pre-term labour and frozen for extraction of rna (n= for ptnl, ptl, tnl and l). copy numbers of epac , epac , il- , pghs- , otr, fp, gapdh and -actin were measured by qpcr. results: epac and epac were present in the upper and lower segment of myometrium with epac levels being some -fold higher than those of epac . there was no change with the onset of labour at or before term. treatment with il- for h significantly increased epac (n= ; p< . ) but had no effect on epac . when conditions mimicked pregnancy, (the presence of forskolin and progesterone), brefeldin a, an epac antagonist, increased basal pghs- and fp mrna expression (n= ; p< . ), but had no effect on il- and tended to reduce otr. the il- -induced increase in pghs- and il- , but not fp, was greater with the epac antagonist. conclusions: these data show epac's are present in human myometrium and that camp acts via epacs to reduce pghs- and fp expression during pregnancy. background: inflammatory events have been implicated in the process of labour. glucocorticoids mediate strong anti-inflammatory effects through binding to the glucocorticoid receptor (gr), which on activation translocates to the nucleus and either increases or decreases the expression of responsive genes thereby suppressing inflammation. objective: to characterise the expression profile for gr protein and mrna in human myometrium during fetal maturation and parturition. methods: western immunoblotting (wb) was employed to characterise gr protein expression in first (n= ) and second trimester myometrium (n= ), and in paired upper and lower segment pregnant (non-labouring, p, n= ) and labouring (l, n= ) myometrium; as compared to non-pregnant (np, n= ) control samples. immunofluorescence staining with confocal microscopy and rt-pcr were also undertaken. results: detection of gr protein by wb revealed two bands at - kda, representing the alternatively spliced isoforms, gr-and gr-. densitometric analysis showed that gr levels decreased significantly during pregnancy and remained at very low levels at term and in labour when compared with np samples (p< . ); this decrease was seen for gr-and gr-. no significant temporal variations were observed in gr protein levels at term or during labour. gr protein was localised by immunofluorescence staining to the nuclei and cytoplasm of cells. less intense staining was apparent in p compared to np tissues; consistent with wb data. rt-pcr showed a consistent predominance of gr-to gr-mrna in all the tissues used. the observed decrease in protein expression was also mirrored at the mrna level: gr-mrna levels appeared to gradually decrease throughout gestation (p< . ). differences between the upper and lower myometrial regions were only observed in the labouring samples, where gr-mrna levels were significantly decreased in the lower segment (p< . ). nested pcr was additionally used to amplify gr-, but no consistent pattern of mrna expression was obtained. conclusions: these data are the first to characterise gr expression in human myometrium. spatial and temporal variations have been found with expression evolving through the three trimesters of pregnancy and in labour. further studies are now underway to evaluate whether gr contributes to the sequence of inflammatory events implicated in triggering term and preterm labour. these studies sought to determine the mechanism by which pas modulate the immune response. methods: ) an in vitro co-culture model mixed with human cervical epithelial hela cells and pma-induced human macrophage u cells at epithelial/macrophage cell ratio of : was employed. ) the co-culture was pre-treated with medroxyprogesterone acetate (mpa), progesterone (prog) and dexamethasone (dex) at concentrations of , , and nm for hrs followed by day stimulation of g/ml lipopolysaccharide (lps). these experiments were repeated using hela cells transfected with sirna for glucocorticoid receptor (gr). the production of cytokines il- , il- and il- , il- and tnf were determined using elisas. ) the co-culture was pre-treated with nm of each pas for hours prior to lps stimulation at g/ml for . , . , , , and hours. the phosphorylation of p mapk and gr and the expression of mapk phosphatase (mkp ) were determined by western-blotting. results: il- , il- and il- , il- and tnf were elevated by . fold, . fold, fold, . fold and . fold in the co-culture model in response to lps(p< . for all). pretreatment of mpa and dex, but not prog, inhibited the lps-induced cytokine production. the anti-inflammatory effect of mpa occurs in a dose-dependent manner. in the absence of gr, the inhibitory effect of mpa and dex on il- , but not on il- , was lost. mpa and dex, but not prog, induced the phosphorylation of gr and expression of mkp . p mapk was activated by lps for up to hrs and pretreatment of mpa and dex attenuated this response. the ability of mpa and dex to suppress the inflammatory response in cervical tissues appears to be mediated through a gr-dependent pathway, specifically though p mapk. our studies suggest that prog is not a significant immunomodulator in these tissues. background: progesterone (p ) has been shown to play a critical role in maintaining pregnancy and preventing preterm birth. these effects are likely related to its immunomodulatory properties. we investigated whether camp plays a role in the immunosuppressive action of progesterone on fetal mononuclear cells. methods: umbilical cord blood mononuclear cells were isolated using density gradient centrifugation. to establish a p effect and optimize concentrations, cells were pretreated with p ( - m- - m), dexamethasone ( um) or vehicle for hour prior to overnight lps ( ng/ml) stimulation. supernatants were assayed for tnf-using elisa. ldh assays confirmed the absence of a cytotoxic effect. next, cells were pre-incubated with forskolin (adenylate cyclase activator) or db-camp (camp agonist) prior to lps to determine the effects of camp on tnf production. finally, cells were pretreated with rp-camp (camp antagonist) prior to p incubation and lps stimulation to determine whether the p effect was reversed by a camp antagonist. results: p significantly inhibited lps-induced tnf production in a dose dependent manner, with maximum suppression observed at - m. both forskolin and db-camp suppressed lps-induced tnf production in a doserelated manner (fig ) . finally, a dose-dependent partial reversal of tnf production was observed when cells were pretreated with rp-camp. (fig ) conclusions: progesterone suppresses the inflammatory response in fetal mononuclear cells as measured by tnf expression. this effect is mimicked by adenylate cyclase activators and camp agonists and partially reversed by camp antagonists. this implicates the camp pathway in mediating the immunomodulatory actions of p . objectives estrogen improves endothelial function after vascular injury via largely unknown mechanisms. endothelial progenitor cells (epcs) are known to be implicated in various vascular events requiring endothelialization. we hypothesized that estrogen and progesterone could influence the function and the change of epcs in menstrual cycle. material and methods peripheral blood mononuclear cells (pbmcs) were isolated from peripheral blood of healthy young women in each menstrual period by density gradient centrifugation with ficoll separating solution. pbmcs were seeded in endothelial basal medium with or without estrogen or progesterone. on the th day in culture, nonadherent cells were removed. on the th day in culture, adherent cells were incubated with di-ldl, fixed with paraformaldehyde, and stained with fluorescein isothiocyanate-labeled lectin. the number of ldl-and lectin-positive cells was measured as epcs using flowcytometry. the expression of estrogen and progesterone receptor mrna in epcs were measured by real time pcr in menstrual and luteal period. the number of epcs was significantly increased in the menstrual and luteal period compared with the follicular phase. estrogen and progesterone significantly increased the number of adherent epcs dose dependently in menstrual period, but not in luteal period. the expression of estrogen-alpha receptor in menstrual period was higher than luteal period. also, estrogen-beta receptor in luteal period was strongly expressed compared with menstrual period. these results suggest the expression of estrogen receptor and progesterone receptor play important roles to regulate epcs' proliferation during menstrual cycle. objective: elevated levels of fetal plasma avp are associated with the development of oligohydramnios, in part a result of avp-mediated reduction in fetal urine production. as avp urinary concentration effects are mediated via upregulation of renal tubular aqp water channels, we propose that avp modulates placental aqp channels, influencing bidirectional maternal-fetal water flow. we sought to study the effect of avp on trophoblast aqp gene expression using the first trimester-derived extravillous htr- /svneo cells and the term placenta-like trophoblast carcinoma cells jeg- . methods: cultures of both cell lines were treated with a physiological concentration of avp ( . nm) to determine aqp mrna and protein expression. negative controls consisted of cells incubated in medium supplemented with % fbs without avp. to determine whether avp regulation of aqp occurs by a camp signaling pathway, jeg- cells were preincubated with μm -(tetrahydro- '-furyl) adenine (sq ), a cell-permeable camp inhibitor, before being treated with avp ( . nm). cells were incubated for hrs for aqp mrna expression and for hrs for protein extraction at °c. after harvest, real time pcr and western blotting analysis were used to detect the aqp mrna and protein expression levels, respectively. results: avp increased aqp mrna expression in both cell lines by . and . fold after hrs. aqp protein expression paralleled the increase seen in the mrna (p< . ). pretreatment of jeg- cells with sq inhibitor completely blocked the stimulatory effect of avp. conclusion: aqp gene expression is up-regulated by avp in first trimester and term trophoblast cells, with a higher induction in the later. avp activation of aqp gene expression occurs via a camp mediated pathway, as the adenyl cyclase inhibitor blocked avp effects on aqp gene expression. these results suggest that increased fetal plasma avp may contribute to oligohydramnios by an increase in aqp-mediated fetal to maternal water flow. objective: changes in expression, ratio and activity of progesterone receptors within human myometrium have been proposed as potential contributory mechanisms for the functional progesterone withdrawal effect which precedes labour. progesterone receptor c (pr-c) is an n terminally truncated isoform of the full length progesterone receptor; pr-c has been shown to be abundant in fetal membranes, placenta and upper segment of laboring myometrium ( , ). the function and regulation of pr-c is at present unclear. in this study we aimed to investigate the regulation of pr-c in cultured human myometrial and amnion-derived wish cells. methods: myometrial primary cell cultures were prepared from non pregnant and term pregnant uteri. both myometrial and wish cell cultures were treated with natural progesterone ( , nm, mm, mm, mm) for , and hrs. western immunoblotting was employed using nuclear and cytoplasmic extracts prepared from treated cells to observe the effect of progesterone on a) expression and b) subcellular localisation of pr-c within both cell types. immunofluorescent staining and confocal microscopy were also employed. experiments were also undertaken whereby nuclear and cytoplasmic extracts were subjected to shrimp alkaline phosphatase treatment to evaluate the phosphorylation status of pr-c in response to hormone. results: data indicates that a kda cytoplasmic protein, representing pr-c is abundant in myometrial and amnion cell cultures. we show that expression of pr-c increases in both cytoplasmic and nuclear fractions within both cell types in response to progesterone. evidence also suggests that pr-c is phosphorylated in a progesterone-independent manner. concurrent treatment with progesterone and the pr-antagonists ru and organon in amnion-derived cells still led to an increased abundance of pr-c but seemed to alter the phosphorylation status of pr-c. conclusion: pr-c expression and subcellular localisation within myometrial and wish cells alters in response to progesterone. speculation is generated about potential role of pr-c in reproductive tissues and its contribution to functional progesterone withdrawal. . taylor smooth muscle responses have been studied, data on regional blood flow (bf)distribution are scarce. we have studied the effect of a single dex course on relative bf distribution within the brain, skeletal muscle, heart, omental fat, placenta and adrenal in fetal sheep at . g, the equivalent of weeks. methods: fetal sheep received amniotic, jugular, carotid and femoral artery and vein catheters at days g (dg). experiments started at dg in dex ( mg) and saline treated controls (ctr), each receiving injections h apart. fetal blood pressure was recorded continuously (windaq pro+). microspheres ( . x total; sterispheres, biopal) were injected at : am into fetal jugular and femoral veins before maternal i.m. injection (t ) and h after the rd injection (t ) in both groups. tissues were collected hours after the th injection for assessment of bf (biopal). results: fetal bp increased above baseline after dex ( . + . mmhg; p= . ) with no change in ctr (- . + . mmhg). regional bf distribution is presented as a percent of the total counts per g tissue (table ) and after normalization within each animal to placentomal flow, shown previously to be unchanged by dex, in table . no significant differences in flow were found. discussion: although dex altered bp, regional distribution of bf among key organs was unaffected. whether gc does not affect regional bf, or the rise in bp is the result of a non-specific, system-wide vasoconstriction, or the impact of gc on bf is similar in all organs, will be subject of further investigation that will include determination of regional vascular resistance and absolute flow. stimulator while crf-r is an inhibitor of gi motility at times of stress. we recently hypothesized that stress-induced in utero meconium passage in fetuses is analogous to stress-induced defecation in adult rats and likely mediated by crf pathway. in support, we demonstrated hypoxia-induced meconium passage in conjunction with marked increases in fetal plasma crf levels. as the mouse is a common experimental model, with known genome, we sought to determine the presence of crf-r and crf-r receptors in mouse fetuses. methods: time-dated cd- pregnant mouse were anaesthetized and fetuses (n= ) were collected at e (term = e ). whole gi tracts were dissected, fixed in bouin's solution, paraffin embedded and sectioned at five micron thickness. sections were immunostained with rabbit polyclonal antibodies to crf-r and crf-r by abc technique with vector abc reagents. immunoreactivity was identified as brown staining using ', diaminobenzamidine as chromagen. slides were counter stained with mayer's hematoxylin and examined under the microscope. immunoreactive signals in the smooth muscle layers of gi tracts were quantified by image analysis using image pro . plus software. results: immunostaining for both crf-r and crf-r was seen in the smooth muscle layers of all lumens examined ( lumens per section). the immunostaining intensity expressed as intensity over density (iod) in arbitrary units for crf-r (maximal iod: . ± . au and minimal iod: . ± . au) and for crf-r (maximal iod: . ± . au and minimal iod: . ± . au) greatly varied between lumens. the mouse fetal gi tract expressed both crf-r and crf-r receptors, suggesting that the mouse is an appropriate model for fetal-stress induced neurovisceral motor responses. the non-uniform distribution of crf receptors in the fetal gi tract suggests the existence of regional differences in the expression patterns of crf-receptors of both types. we speculate that mouse devoid of crf and crf-r or r receptors will be useful to confirm the participation of crf pathway in stress-induced in utero meconium passage. antenatal exposure to glucocorticoids (gc) is associated with a reduction in nephron number and hypertension in adult life. one of the mechanisms for the development of hypertension is thought to be the long term effect of single nephron hyperfiltration. however, in most studies there is only a - % reduction in nephron number with no change in gfr. thus, although a reduction in nephron number may be a contributing factor it is unlikely the only variable. the aim of the present study was to evaluate nephron mass, renal function and blood pressure in a cohort of adult sheep exposed antenatally to betamethasone. methods: pregnant sheep were treated with two im doses of betamethasone (bm, . mg/kg) or vehicle (ctr) -hs apart at days gestational age and allowed to deliver at term. at . yr of age in female (f) and male (m) offspring, glomerular filtration rate (gfr) was measured as inulin clearance and effective renal plasma flow (erpf) as pah clearance. blood pressure was measured though an indwelling catheter in the femoral artery over a -hour period. nephron number was measured by the acid maceration technique. data mean±sem were analyzed by anova and/or two sample t test. results: antenatal bm was associated with an elevation in arterial blood pressure and a reduction in nephron number in both f and m offspring. in contrast, a reduction in gfr and erpf was present only in males. plasma and urinary electrolytes as well as urinary protein excretion were not different from ctr. conclusion: our data show that prenatal exposure to a single course of gc at . gestation has long-term effects on blood pressure regulation. interestingly, while the decrease in nephron number was of similar magnitude in m and f, evidence of alterations in renal function was present only in males. these suggest that the decrease in renal function observed in males is not solely a consequence of the decrease in nephron number. furthermore, it is possible that different mechanisms are responsible for the elevation in arterial blood pressure observed in m and f. hl ; hd p hd . clr l>t-x to evaluate the effects on blood pressure, urine output, sodium excretion and gfr of the intrarenal infusion of angiotensin - in the male sheep with or without prenatal exposure to b. we studied male sheep which had received either b (n= ) or vehicle (n= ) at - days gestation and were born at term. catheters were placed in the femoral artery, femoral vein, left renal artery and the bladder. the right kidney was removed. after days recovery the sheep received an infusion of ang - ( ng/kg/min) with or without its antagonist, [d-ala ]-ang-( - )(d-ala, ng/kg/min), into the renal artery for hours. blood pressure, gfr, urine output and sodium excretion were measured before and after the infusion. two-way analysis of variance (anova) was used to test mean values between the groups. with or without d-ala, map and urine output didn't change significantly during ang - infusion. however, the infusion of ang - resulted in a decrease (change from baseline) (p= . ) in na + excretion of . ± . meq/kg/ h in the vehicle animals and . ± . meq/kg/ h in the b animals. there was no significant change in gfr during ang - infusion. gfr was lower in the b group during the combined ang - and d-ala infusion ( . ± . ml/min vs . ± in vehicle group, p= . ). intrarenal infusion of ang - at ng/kg/min is followed by a significant decrease in sodium excretion but no significant change in urine output and gfr. the decrease tended to be greater in the vehicle animals and was not markedly attenuated by the antagonist d-ala. this suggests the effect of ang - may be mediated by a receptor other than the mas receptor for ang - and may be of lesser magnitude in animals exposed to b before birth. and norepinephrine ( . - . g/kg/min) and to an l-name ( mg/kg) bolus were studied. vascular reactivity was analyzed by curve fitting of either the blood pressure or hr responses to obtain the maximal response. data are expressed as mean±sem of change from baseline. statistical significance was established using t test. results: bm-exposed animals displayed a higher blood pressure response to at-ii (atii max ± vs ± % of baseline, p< . , figure left panel). no differences in blood pressure or heart rate responses to norepinephrine infusion were detected in bm when compared to control animals (figure right panel) . maximal response to l-name was also elevated in bm-exposed animals but did not reach statistical significance with the current sample size. conclusion: we have shown that antenatal gc treatment significantly increases blood pressure in sheep. here we show an enhanced in vivo response to at-ii. this response may contribute to the increased blood pressure observed. the greater response to l-name most likely represent an increased no production as a compensatory mechanism to the higher blood pressure observed. hl . women at risk of delivering preterm are frequently treated with glucocorticoids to facilitate fetal lung maturation. animal studies have revealed that prenatal exposure to glucocorticoids may alter aspects of hypothalamic-pituitary adrenal functioning in later life. in this study we examined the effects of clinically relevant prenatal betamethasone treatment on the responsiveness of pituitary cells (in terms of adrenocorticotropin, acth, secretion) isolated from adult sheep. time-dated pregnant sheep were injected with betamethasone ( . mg/kg) or vehicle on days and of gestation. thereafter pregnancy was allowed to continue unimpeded and offspring were born. pituitaries were isolated from adult sheep aged between and months and cells were dispersed and plated at a density × cells per well in well plates. after h, cells were stimulated with normal medium, nm arginine vasopressin (avp), nm avp, nm corticotropin releasing factor (crf) or nm crf for h. medium was collected and analyzed for acth using a commercially available kit. acth secretion (given as % increase from untreated cells) was similar between the control and betamethasone groups following nm avp ( . ± . vs. . ± . ) and nm crf ( . ± . vs. . ± . ). at higher stimulatory concentrations of both avp and crf, acth secretion remained similar in control cells ( . ± . and . ± . respectively), but was significantly decreased in betamethasone cells ( . ± . and . ± . respectively). these findings suggest that prenatal exposure to clinically relevant doses of betamethasone can alter pituitary responsiveness to both avp and crf in adulthood. this research was supported by nih grants hd and hd . lcc is supported by an rjr-leon golberg fellowship in pharmacology and toxicology. objective: newborn meconium (mec) passage normally occurs within the first - h after birth. in contrast, more than half a million infants born annually in the us pass mec in utero. neither the physiological mechanism(s) nor causes for in utero mec passage are well understood, though both fetal maturation and stress are associated. we recently reported in utero mec passage and marked increases in plasma crf levels in term fetal rats exposed to maternal hypoxic stress. we hypothesize that stress-induced in utero mec passage is mediated by the crf pathway in a manner analogous to stress-induced defecation in adult rats. in the present investigation we examined placental crf content prior to and following maternal hypoxia, to determine the source of increased plasma crf. methods: time-dated pregnant rats (n= ) were exposed to a paradigm of graded, stepwise hypoxia on day (term= ) (pediatr. res. : - , ) . control pregnant rats were exposed to % oxygen for a similar duration as experimental animals. at the end of the study, placentas were harvested, fixed in % paraformaldehyde and paraffin embedded. sections (n= per placenta) were subjected to immunohistochemical analyses with rat/human crf antibody (peninsula laboratory) by abc technique. immunoreactivities on placental sections were quantified using the image pro . software and intensity expressed as arbitrary unit (au). all values are mean± sem. results: in control maternal rats exposed to normoxia, positive staining for crf was seen in decidua ( . ± . au) and in all three major placental cells types (giant trophoblast cells: . ± . au, spongiotrophoblast cells: . ± . au and labyrinth cells: . ± . au.) in contrast, in animals exposed to hypoxia, there was a total absence of crf staining in the placental cells, with only positive crf staining seen only in the decidua ( . ± . au). conclusion: the total absence of crf staining in both the basal and labyrinth zones in placentas of pregnant rats subjected to hypoxic stress suggests that placental cells rapidly release crf into the maternal and fetal circulation in response to hypoxic-stress. our findings support our hypothesis that the peripheral crf pathway mediates in utero mec passage. offspring. angela g massmann, jie zhang, jorge p figueroa. department of obstetrics and gynecology, wake forest university school of medicine, winston-salem, nc, usa. objective: in rats and sheep, exposure to glucocorticoids (gc) in the perinatal period induces hypertension in adult life. recent evidence suggests that endothelin b (etb) receptor plays an important role in the regulation of sodium balance and blood pressure. renal medulla is an important site of expression and action of etb receptor. furthermore, in kidney it is the medulla (km) where the highest concentrations of immunoreactive et- and etb receptor are found. the aim of this study was to measure eta and etb expression in adult sheep kidney exposed antenatally to gc. methods: pregnant sheep were treated with two im doses of betamethasone (bm, . mg/kg) or vehicle (v) hours apart at days of gestational age and allowed to deliver at term. at . yr of age, male and female offspring exposed to either vehicle or bm were euthanized and the kidneys harvested. kidney medulla (km) was obtained by sharp dissection. eta and etb protein and mrna expression were evaluated by western blot and rnaase protection assay. data are expressed as mean±sem and were analyzed by two way anova. results: a significant decrease in etb (f= . , p= . ) but not eta protein was observed in km of bm exposed male and female adult sheep. at the mrna level, bm exposure also affected etb, but not, eta expression. however, bm treated animals had higher mrna levels than control sheep (f= . ; p= . ). conclusion: our data show that prenatal exposure to a single course of gc at . gestation has long-term effects. the activation of etb receptor by et- inhibits sodium transport function in the collecting duct and the selective gene deletion of the etb in medulla results in hypertension.therefore, our finding of a significant reduction in etb protein suggests that there may be an impairment in the kidney's ability to regulate sodium reabsorption. the functional relevance of the alterations in etb protein expression as well as the discrepancies in mrna regulation need to be established. hl ; hd p hd . in the placenta, -hydroxysteroid dehydrogenase type ( hsd ) regulates the transfer of cortisol. maternal glucocorticoid (gc) administration has been shown to affect the ovine fetal growth trajectory and regulate hsd expression in mid and late gestation. however, little is know about the effects of gc administration in early gestation. we hypothesized that maternally administered low-dose dexamethasone (dex) in early gestation would affect hsd expression, which will subsequently alter fetal birth weight. pregnant ewes were randomized and received intramuscular injections consisting of either saline ( ml saline/ewe) or dex ( . mg/kg) given hours apart starting on days of gestation (dg). the animals were sacrificed at , , , and dg and fetal weights were recorded and placental tissue was collected. qrt-pcr was used to measure hsd placental mrna expression. statistical analysis was done by anova with student's t-test posthoc. overall, there was no significant effect of dex treatment on placental hsd mrna expression across gestation. however at dg, there was a significant increase in hsd expression after dex (p= . ). there was an increase in placental hsd mrna progressively from dg (mean = . ) to dg (mean = . ), independent of treatment. overall, a positive correlation between hsd and fetal weight (r = . ) was apparent at dg and at dg (p= . ) in both control (p= . ) and dex (p= . ). significant positive correlations between placental hsd and fetal weight (r = . ) were found in females (p= . ) and a similar trend was found in male fetuses (p= . ). we conclude that in the sheep placenta, hsd expression increases throughout gestation and may respond, at least transiently in early gestation, to elevations in maternal gc. the positive correlation between hsd and fetal weight is consistent with the thesis that the fetal growth trajectory may be influenced by maternal cortisol levels and that placental hsd is an important mediator of these effects. antenatal gc exposure induces hypertension in the young adult rat (neuroendocrinol; ; : ) and increases femoral vascular resistance in the lamb (jphysiol; ; : ) . unpublished own examinations in preparation of this study have shown age dependency of the vascular reactivity. vascular contractility of the middle cerebral artery (mca) decreased in response to k + and noradrenaline (na) between mo and , y of age (p< . ). vascular relaxation to acetylcholine (ach) but not to pge decreased during the same time (p< . ). vascular contractility of the renal artery (ra) increased in response to k + (p< . ). aims: to examine if antenatal gc alter vascular reactivity in the aged individual when cardiovascular diseases are predominant. we studied the ra because its vascular tone is involved in modulation of arterial pressure control (amjphysiol; ; :r ) and the mca because depressive disorders that are associated with dysregulation of the hpa axis (jcem; ; : ) increase stroke mortality for unknown reasons (amjpsychiatry; ; : ) . to be clinically relevant, we administered dexamethasone at the dose used to enhance lung maturation in babies threaten premature labor. methods: pregnant dams received saline (n= ) or μg/kg dexamethasone (n= ) i.p. at day e / equivalent to x mg dexamethasone administered to a kg pregnant woman. at . years of age, vascular response of the ra and mca to endothelium-dependent and independent mediators was measured using wire myography. vessels were inspected histologically for intact endothelium. results: basal vasoconstrictory and dilatory responses to all mediators were less pronounced in the mca than in the ra probably reflecting autoregulatory properties of cerebral vessels (p< . ). after prenatal dexamethasone exposure, contractility but not sensitivity to k + and na was enhanced in the mca and even more pronounced in the ra (p< . ). relaxation to ach and pge was similarly diminished in both vessels after precontraction with na (p< . ). conclusions: prenatal dexamethasone exposure at the dose used clinically increases renal and cerebral vascular tone in the aged rat by endotheliumdependent and independent mechanisms. this effect may be a potential mechanism of fetal programming of cardiovascular diseases in later life. betamethasone (bmz) therapy during pregnancy to improve fetal lung maturity may result in long term side effects, including hypertension, during adulthood. the kidney is an important organ which regulates systemic blood pressure via the local renin angiotensin system (ras). the major enzymes of the intrarenal ras include angiotensin converting enzyme (ace), angiotensin converting enzyme (ace ), and neprilysin. the hypothesis of this study is that prenatal bmz exposure will result in alterations of the enzymes regulating the intrarenal ras. specifically, sheep that are treated with bmz in utero will have higher amounts of ace activity in kidney cortex compared to control animals. pregnant sheep of known mating date were either treated with vehicle (n= ) or with two doses of bmz (n= ), . mg/kg, hours apart at days of gestation. renal cortex from the male offspring was harvested at - months of age. cortical membranes were then solubilized and incubated at °c with either i-ang i or i-ang ii in the presence or absence of lisinopril to inhibit ace activity, mln to inhibit ace activity, or sch to inhibit neprilysin activity. the metabolic products were separated by reversephase high performance liquid chromatography (rp-hplc). the rate of enzyme activity was quantified by calculating the area under the curve for each product and converted to fmol of product per mg protein per minute of incubation. statistical analysis was performed using student's t-test. p< . was considered significant. results: bmz treatment resulted in a significant increase in ace activity compared to control animals (p= . ). ace and neprilysin levels were not significantly different between treatment groups. when expressed as a ratio of ace/ace activity, bmz treated animals exhibited a . fold greater proportion of ace activity versus control animals (p= . ). this study demonstrates that bmz exposure during fetal life results in a significant increase in renal ace activity during adult life. this increase in enzyme activity would be expected to be associated with increased levels of ang ii in the kidney and na + retention, possibly underlying the development of hypertension. hd , hd . we recently hypothesized that stress-induced in utero meconium passage in term fetuses utilizes peripheral and/or central crf pathways in a manner analogous to stress-induced defecation in adult rats. as participation of central crf pathway requires the crf circuitry system in brain-gut axis, we exploited immunohistochemical techniques to address whether term fetal colon is wired by crf-nerve fibers. methods: fetal distal colon segments (n= ) were dissected from term ovine fetuses ( - d gestation). in addition, lumbosacral spinal cord with spinal roots and dorsal root ganglia (drg) (n= ) were dissected from ovine fetuses, and vagal nerve trunk with nodose ganglia (n= ) were dissected from mouse fetuses. paraffin sections fixed either in bouin's solution or zamboni's were subjected to immunohistochemistry with rabbit polyclonal antibodies specific to ovine-crf (ocrf). immunoreactivity on the sections was identified by standard abc technique, immunostaining quantified by image-pro plus software and expressed (intensity over area) as arbitrary units (au). results: ocrf antibody elicited strong positive staining on the serosal surface (port of entry for extrinsic nerve fibers) in distal colonic segments objective: we hypothesize that stress-induced in utero meconium passage is mediated by the crf pathway. ucn-i, similar to crf, is a potent contractility inhibitor of preterm ovine fetal distal colon, which has abundant crf-r receptors. both ucn- and crf thus may inhibit colonic motility and prevent preterm in utero meconium passage via crf-r receptors. however, ucn-i is reported to stimulate contractility of adult rat colonic smooth muscle strips more strongly than crf. we sought to study the pattern of ucn-i innervation in distal colon of ovine fetuses to delineate whether ucn- functions as a facilitator or inhibitor of colonic propulsive motility. methods: bouin's solution fixed, paraffin sections of very preterm (vpt: - days gestation), preterm (pt: - days), near term (nt: - ) and term (t: - days) ovine fetal distal colon rings were subjected to immunohistochemistry with polyclonal antibodies to human ucn- ( : sigma) by abc system. intensity of ucn- immunostaining in smooth muscle layers and myenteric neurons were quantified by image-pro plus software and expressed (intensity/area) in arbitrary units (au). differences over time were assessed with anova. in all groups very preterm was significantly greater than term (p< . ). conclusion: ucn- immunoreactivity in all three muscle layers, as well as myenteric and submucosal neurons, is highest at very preterm as compared to more mature gestations. these results suggest that the reduction of ucn- inhibitory function may facilitate stress-induced meconium passage at term. to evaluate the effect on blood pressure (bp), urinary output (uop), sodium excretion (nae) and gfr of the intrarenal infusion of ang ii in the male sheep after prenatal exposure to b. methods: we studied male sheep which had received either b (n= ) or vehicle (n= ) at - days gestation. catheters were placed in the femoral artery and vein, left renal artery and in the bladder. a right side nephrectomy was performed. after days of recovery, the sheep were infused with ang ii ( ng/kg/min) with or without candesartan ( ng/kg/day) or pd (pd g loading dose and ng/kg/min infusion) into the renal artery over hours. bp, gfr, uop and nae were measured before and after the infusion. two-way analysis of variance was used to test mean values between the groups. results: bp and uop did not change with ang ii infusion with or without candesartan. the infusion of pd did not affect uop but did increase bp in the b sheep (p= . ). ang ii decreased the nae in both groups(p= . ). candesartan increased nae among controls(controls . and b . meq/kg/h, p= . ). pd infusion decreased nae among controls but this was not significant. baseline gfr was higher among vehicle compared to b animals (p= . ). during ang ii infusion there was a decrease in gfr among control compared to b sheep (- . vs. - . ml/min, p= . ). this difference was not seen during candesartan or pd infusion. conclusion: ang ii infusion led to a decrease in nae in both groups and gfr among controls but not b animals. infusion of ang ii with candesartan increased nae while pd decreased nae among controls but not b animals. this suggests that prenatal steroid exposure can alter at receptor mediated response in renal function by decreasing the effect of ang ii on renal sodium excretion. the differences in gfr suggest that this may be due to an effect on tubular sodium reabsorption rather than glomerular perfusion. antalarmin antagonism of acth-induced cortisol secretion by ovine adrenal cells probably not at acth receptor. nancy k valego, james c rose. center of research for ob/gyn, wake forest u. school of medicine, winston-salem, nc, usa. in addition to regulating the hpa axis, crh and its type receptor (crhr- ) occur peripherally and in the female reproductive system. late in human pregnancy, a surge in placental crh probably stimulates adrenal secretion of cortisol and dheas requisite to parturition. we previously reported that, in dispersed fetal or adult ovine adrenal cortical cells, hour incubation with the specific crh-r antagonist, antalarmin (ant), significantly reduced both cyclicamp and cortisol responses to acth, suggesting an interaction with acth-r (like crh-r , a g-protein-coupled membrane receptor). however, forskolin (fsk; direct stimulant of adenylyl cyclase)-stimulated cortisol secretion was also attenuated by hour co-incubation with ant. our objective was to clarify the effect of ant on binding of acth to its receptor after a short ( . hours) treatment. method: acth binding: the adrenals from adult sheep were obtained at necropsy and the cortex cells dispersed and plated @ cells/well. after hours in culture, wells were rinsed x and refilled with serum-free dmem/ f containing vehicle (dmso) with or without ant. after . hours, wells were washed very gently and the binding assay (adapted from rainey et al; j biol chem, : , ) completed. triplicate vehicle or ant wells were treated with i -tyrosine human acth ( - ) with or without - m acth (for non-specific binding). after hour, wells were chilled, washed, and the cells lysed and counted on a gamma counter. fsk treatment: cells were treated as above except that fsk ( - m) was added to the wells. after . hours, medium was removed and stored @ - ºc for camp eia and cortisol ria. results (mean±se) of . hour incubation on acth binding and fskinduced secretion. vehicle antalarmin acth binding (net cpm) n= ± ± cortisol (ng/ml/ . hr) n= ± ± * (p=. ) camp (pmol/ml/ . hr) n= . ± . . ± . * (p=. ) * indicates significant difference from vehicle alone. conclusion: the crh-r antagonist, antalarmin, attenuates acthstimulated cortisol secretion but not by preventing acth receptor binding. however, its effect is early in the secretory process and is associated with a reduced camp response. objective glucocorticoids are often administered to pregnant women to prevent neonatal respiratory distress syndrome. prenatal steroid treatment increases blood pressure in adult sheep. exposure to excess corticosteroids before birth is hypothesized to be a key mechanism underlying the fetal origins of adult disease hypothesis and effects on the renin-angiotension system (ras) may modulate the steroid-induced increases in blood pressure. we therefore sought to determine if renin processing and secretion were altered in adult female sheep exposed antenatally to betamethasone ( ) and to compare them with data from males studied previously. methods pregnant sheep were randomized to receive doses of . mg/kg of or vehicle, at and days of gestation; the offspring were studied at and months of age. in female offspring, active renin concentration (arc) and total renin concentration in plasma were measured by ria of angiotensin i generated by incubation with excess substrate. prorenin concentration (prc) is the difference between total and active renin. nine or control exposed female animals born at term ( - days of gestation) were brought from the farm at - months of age, and had vascular and bladder catheters placed. five days after surgery, a sodium load of hypertonic nacl ( . meq/kg/min at . ml/min) was given for min. blood samples were obtained. data are expressed as mean sem and were analyzed by t test. the prc was significantly higher in the females than in the males ( . ± . vs . ± . p< . ) but there was no effect of prenatal steroid treatment. arc was similar in both genders. however, arc was a significantly greater percent of the total plasma renin concentration in the males ( . ± . vs . ± . p< . ) during the na infusion experiment, the exposed females had lower arc than did the control females ( . ± . vs . ± . p< . ). conclusion the data suggest that prenatal exposure to didn't alter the processing and secretion of renin in adult female sheep. prenatal steroid treatment does not appear to alter the effect of gender on plasma renin levels in adult sheep.it seems unlikely that the elevated blood pressure seen in adult ewes after prenatal exposure is the result of increased secretion or processing of renin. supported by hd and hd . background: mrap is a recently discovered protein with alpha and beta isoforms, a common amino terminal region and divergent c-terminal sequences generated by alternative splicing. in human and mouse mrap plays an essential role in the generation of a functional, g-protein coupled acth receptor (melanocortin- receptor; mc r) but has not been described for ruminants. we previously reported that lth fetal sheep exhibited elevated basal acth - yet decreased expression of key steroidogenic enzymes and the mc r in the adrenal cortex while basal cortisol levels were not different from control. we hypothesized that mrap could play a key role in mediating the effect of lth as well as developmental regulation of fetal adrenal cortisol synthesis in fetal sheep. the goals of the present study were to determine the ontogenic pattern of mrap expression in the sheep fetus and to determine if lth alters mrap expression. methods: we searched the bovine genomic sequence database (www.ncbi.nlm. nih.gov/genome/guide/cow/) and found a sequence with > % homology to the human mrap n-terminal residues, with partial homology to the beta isoform in the carboxyl region ( %). using primers based on the bovine sequence, we confirmed the presence of mrap in the ovine fetal adrenal cortex. adrenal cortical tissue was collected from sheep fetuses from - (n= ) and near term ( - ; n= ) days of gestation (dg) as well as from lth (n= ) fetuses (exposed to high altitude hypoxia from to - dg) and age-matched normoxic controls (n= ). cyclophilin was used as a housekeeping mrna. data are expressed in fg mrna/ ng total rna. results: the expression of mrap, based on quantitative rt-pcr, was low between - dg ( . ± . ) and increased (p< . ) approx. -fold near term ( - dg; . ± . ). levels of mrna for mrap were highly correlated to cyp mrna in individual samples. mrap expression in control ( . ± . ) and lth ( . ± . ) did not differ between groups. gender differences in hypertension are well described and there is growing evidence that the regulation of the renin angiotensin system (ras) is influenced by sex hormones. the major enzymes of the ras include angiotensin converting enzyme (ace), angiotensin converting enzyme (ace ), and neprilysin. the peptide products of these enzymes have opposing actions. angiotensin ii (ang ii), the product of ace, is a potent vasoconstrictor. on the other hand, the peptide products of ace and neprilysin, ang ( - ) and ang ( - ), exhibit vasodilatory properties. thus, modifications in the relative proportions of these enzymes and their peptide products can result in alterations of systemic blood pressure. the purpose of this study was to describe the gender differences in angiotensin converting enzyme (ace), angiotensin converting enzyme (ace ), and neprilysin (nep) enzyme activities in adult sheep kidney cortex. renal cortex from male (n= ) and female (n= ) sheep were harvested at - months of age. the tissue membranes were solubilized and incubated at °c with either i-ang i or i-ang ii in the presence or absence of lisinopril to inhibit ace activity, mln to inhibit ace activity, or sch to inhibit neprilysin activity. the metabolic products were then separated by reverse-phase high performance liquid chromatography (rp-hplc). the rate of enzyme activity was then quantified by calculating the area under the curve for each product and converted to fmol of product per mg protein per minute of incubation. statistical analysis was performed using student's t-test. p< . was considered significant. results ace activity was over times greater ( ± . vs. . ± . fmol/mg/min, p< . ), ace activity was . times greater ( ± . vs. . ± . fmol/mg/min, p= . ) and nep activity was nearly times greater ( . ± . vs. . ± . fmol/mg/min, p= . ) in female kidney cortex. in adult sheep, key enzymes of the intrarenal ras have signficantly greater activity in female kidney cortex. these findings suggest that there are fundamental, gender specific, differences in the regulation of the enzymes of the intrarenal ras. the physiologic significance of these findings remain to be elucidated. hd , hd . in rats and sheep exposure to glucocorticoids (gc) in the perinatal period is associated with a reduction in nephron number and hypertension in adult life. furthermore, antenatal exposure to gc alters glucose tolerance in animals and in people. the aim of the present study was to determine ) if insulin resistance is a contributing factor for the development of hypertension in adult sheep exposed antenatally to gc and ) if diet-induced obesity has a more pronounced effect in sheep exposed antenatally to gc. methods: pregnant sheep were treated with two im doses of betamethasone (bm, . mg/kg) or vehicle (ctr) -hours apart at days gestational age and allowed to deliver at term. at mo of age, female sheep were randomly allocated to be fed at either % of recommended nutritional allowance or ad libitum for three months. sheep were chronically instrumented under general anesthesia to place intravascular catheters. insulin sensitivity was evaluated both by iv glucose tolerance test (ivgtt) and euglycemic clamp (hec)techniques. for the ivgtt a . g/kg glucose bolus was used and for the hec mu/kg human insulin was used. data mean±sem were analyzed by anova and/or two sample t test. results: ad lib fed sheep gain > % of the original weight. antenatal bm was associated with an elevation in basal and ivgtt plasma insulin values. as shown on the figure, diet-induced obesity significantly increased insulin plasma levels during ivgtt in bm-exposed adult female sheep (f= . ;p< . ). insulin sensitivity derived from hec was significantly decreased by obesity in bm-exposed adult female sheep. conclusion: our data show that prenatal exposure to a single course of gc at . gestation has long-term effects on glucose metabolism regulation. bm-exposed sheep exhibit alterations in glucose tolerance, hyperinsulinemia and insulin resistance. these abnormalities are exagertated by diet-induced obesity. hl . syngergistic induction of -hydroxysteroid deyhdrogenase type expression by cortisol and interleukin- in human fetal lung fibroblasts. z yang, p zhu, cm guo, l myatt, k sun. school of life sciences, fudan university, shanghai, china; obstetrics and gynecology, university of cincinnati, cincinnati, oh, usa. objectives: glucocorticoids acting via glucocorticoid receptor (gr), serve as crucial hormones in fetal lung maturation. glucocorticoids and proinflammatory cytokines to induce b-hydroxysteroid dehydrogenase type ( b-hsd ) which converts inactive cortisone to active cortisol, but their effect on b-hsd expression has not been addressed in human fetal lung. we examined the interactions and mechanism of cortisol and interleukin- b (il- b) effect on b-hsd in human fetal lung fibroblasts (hfl- cells). methods: the expression of b-hsd in hfl- was examined with immunocytochemistry and pcr. b-hsd , prostaglandin h synthase- (pghs- ) and cytosolic phospholipase a a (cpla ) mrna levels in cultured human fetal lung fibroblasts treated with cortisol and il- b were measured with real time pcr. the roles of gr and c/ebps in the effect of cortisol and il- b were studied using a gr antagonist (ru ) and transfection of plasmid carrying c/ebp-specific dominant-negative gene (cmv -a/cebp). results: hfl- cells expressed a high level of b-hsd . both cortisol ( - - - m) and il- b ( . - ng/ml) induced b-hsd mrna expression in a concentration-dependent manner, an effect blocked by the mrna transcription inhibitor , -dichlorobenzimidazole riboside ( μm). ru ( - m) blocked the induction of b-hsd by cortisol. induction of b-hsd mrna expression by cortisol ( - m) was synergistically increased by co-treatment with il- b ( . - ng/ml) in a concentration-dependent manner. in contrast, the induction of cpla and pghs- expression by il- b was inhibited by cortisol, suggesting a different mechanism of interaction. transfection of the cells with c/ebp-specific dominant-negative plasmid attenuated induction of b-hsd mrna expression by either cortisol or il- b. these data suggest induction of b-hsd expression by cortisol is a gr dependent process involving c/ebps, which also mediate induction of b-hsd expression by il- b. conclusions: cortisol and il- b synergistically induce b-hsd expression in human fetal lung fibroblasts. this would lead to greater local cortisol production, perhaps providing either a self-attenuating mechanism for control of inflammation or a mechanism for enhancing fetal lung maturation when the fetus is exposed to cytokines e.g. with infection-induced preterm labor. do alterations in placental -hydroxysteroid dehydrogenase ( hsd) activities explain differences in fetal hypothalamic pituitary adrenal function following periconceptional undernutrition or twinning in sheep? kl connor, pl van zijl, cw rumball, , al jaquiery, , je harding, , mh oliver, , fh bloomfield, , jrg challis. medicine, university of toronto. periconceptional undernutrition (pcun) leads to activation of fetal hypothalamic pituitary adrenal (hpa) function, whereas twinning results in delayed fetal hpa activation. we hypothesized that these differences in fetal hpa activity were the result of altered patterns of expression of placental of hsd isozymes and hence of the maternal glucocorticoid (gc) effect on the fetus. we developed a mass spectrometric assay for the measurement of hsd- and - activities and validated this method against a widely used thin layer chromatography method. sheep were randomly assigned to ad libitum (n control) concentrates throughout gestation or were undernourished (un) from days before until days after mating to reduce maternal body weight by %, with ad libitum feeding thereafter. placentomes were collected on days , , , and of gestation (term, d) and hsd- and - activities were determined by measuring the rate of interconversion between cortisone to cortisol. with un, there was a trend towards lower hsd- activity at d (p= . ), a significant reduction at d (p< . ), but no difference at d or d . hsd- activity was not different between n and un animals at any time. there was no effect of twinning on hsd- or - at d . however, with twins both hsd- (p= . ) and - (p< . ) activities increased at d . hsd- activity was reduced in twins (p= . ) at d but was higher than any singleton pregnancies at d (p= . ). hsd- was not different between singletons and twins at either d or d . overall, hsd- was lower in placentae of male compared to female fetuses in late gestation; hsd- was higher in male than female fetuses. there was no interaction with un in either sex. we conclude that pcun and twinning result in alterations of placental hsd activities in sheep. modifications in these enzymes during critical periods of fetal development may affect transplacental transfer or placental generation of gcs reaching the fetus potentially influencing the timing of activation of the fetal hpa axis, fetal maturation and later life development. methods: pregnant sprague-dawley rats had % mfr from day of gestation until delivery. control animals had ad libitum food. offspring were sacrificed on day of life (p ) and at months (n= - per group). adrenals were dissected and snap frozen in liquid nitrogen for later extraction of rna. real time rt-pcr using specific rat primers was used to quantify mrna levels ( s as control). we evaluated the expression of -beta hydroxylase (cyp b ), aldosterone synthase (cyp b ), acth receptor (mcr ), p side chain cleavage enzyme (cyp a ), star protein, -hydroxysteroid dehydrogenase type (hsd ) and type (hsd ), glucocorticoid receptor (gr), and mineralocorticoid receptor (nr c ). fold changes in mrna expression in controls and mfr offspring was compared by student's t-test. results: there was a marked downregulation in expression of cyp b (p=. ), cyp b (p=. ), hsd (p=. ), p (p=. ), acth receptor (p=. ), star (p=. ) and nr c (p=. ) mrna in p mfr offspring, with no changes in hsd and gr. gender specific differences were found in the adult mfr offspring. in the male mfr offspring the expression of hsd and gr were significantly upregulated with a trend towards an increase in acth receptor (p=. ) whereas in the female mfr the expression of acth receptor (p=. ) was increased and nr c (p=. ) and cyp b were decreased (p=. ). in combined data for adult male and female offspring the expression of acth receptor was significantly (p=. ) increased. conclusion: these results indicate that mfr has a suppressive effect on steroidogenic enzymes of the newborn offspring regardless of gender. this may be an adaptive mechanism in the fetus/newborn to offset the high circulating maternal glucocorticoids in response to undernutrition. in adult male mfr offspring, increased hsd indicates an increase in gc synthesis whereas in the females there were no changes in gc synthesizing enzymes with a suppression of mc synthesizing pathway. the common finding of an increased acth receptor expression in both genders would suggest an increased sensitivity of adult mfr offspring adrenals to the effects of stress. objective: during gestation the fetal adrenal undergoes phases of active growth ( - d), quiescence ( - d) followed by reactivation (> d) before birth. insulin like growth factors play an important role in stimulating adrenal growth throughout late gestation. interestingly the prepartum activation of the adrenal is delayed in the female compared with the male fetus, but the mechanisms underlying this delay are unknown. hypothesis: we hypothesize that there are gender specific differences in the gestational profile of adrenal igf mrna expression between male and female fetuses. methods: a total of twin fetuses were used in this study. post mortem was performed at either - d, - d or - d gestation. adrenal mrna expression of igf , igf , igf r, igf r and cyp was determined by qrt-pcr. results: fetal weight was not different between males and females, and increased (p< . ) with increasing gestational age. the relative adrenal weight was lower however after d when compared to the earlier gestation age group (p< . ). adrenal igf mrna expression was lower (p< . ) at - d when compared to the earlier gestation age groups. interestingly, igf r expression was highest at - d (p< . ). there was an interaction between the effects of age and gender on adrenal igf and igf r mrna expression such that the expression was higher in males compared to females at - d (p< . ), but was not different to either the earlier or later gestational ages. there was no effect of either gender or gestational age on adrenal cyp mrna expression. conclusions: it has been speculated that a delay in prepartum activation of the adrenal in female fetuses may be due to gender specific differences in the intra-adrenal bioavailability of igf . in this study we have demonstrated there is an increase in both igf and igf r mrna expression in males compared to female fetuses. this may be evidence that adrenal igf expression plays a role in the earlier activation of the adrenal gland in male fetuses. we have previously shown that in response to a secondary stressor, in vivo cortisol secretion is elevated in long term hypoxic (lth) ovine fetus despite lower acth receptor mrna expression, and no differences in plasma adrenocorticotropic hormone (acth) levels when compared to normoxic controls. the present study was designed to determine the potential mechanism(s) of this enhanced cortisol secretion. specifically we tested the hypothesis that post receptor signaling events including camp production and expression of steroidogenic acute regulatory protein (star) are enhanced following lth. methods: for the lth group, pregnant sheep were maintained at high altitude ( , m) from day to near term. on days - (term = days), fetal adrenal glands were collected from lth (n= ) and age-matched, normoxic signal transduction pathways that control embryogenesis, cell differentiation, cell proliferation and cell death. the roles of extracellular signal-regulated kinase / (erk / ) and p map kinase in the differentiation and invasion of human trophoblasts have been studied. however, the in vivo expression and activation of erk / and p at the placental bed has not been elucidated. the study group consisted of placental bed biopsy tissues obtained from the pregnancies without preeclampsia (n= ) and with preeclampsia (n= ) between and weeks of gestation. we evaluated the expressions and phosphorylations of erk / and p map kinase in the invasive trophoblasts in the placental bed tissues using immunohistochemistry. results: p and phospho-p map kinase were not detected in invasive trophoblasts in cases or controls. erk / and phospho-erk / were positive in invasive trophoblasts albeit with variable staining. phosphorylation of erk / was significantly less frequent in invasive trophoblasts in placental bed biopsies from women with preeclampsia compared with normotensive controls. conclusion: these findings suggest that preeclampsia is associated with decreased activation of erk / in invasive trophoblasts in vivo. objective: placentae from preeclamptic pregnancies are characterized by an excess of immature hyperproliferative trophoblast cells, however the molecular mechanisms regulating cell cycle progression in this pathology are unclear. our aim was to examine the expression of g phase cell cycle regulators in normal and preeclamptic placentae and to establish whether the hyperproliferative state of trophoblast cells in preeclampsia may result from a developmental delay. methods: human placental samples were collected from normal pregnancies throughout gestation (n= ) and from severe early onset preeclamptic (n= ), and age-matched control placentae (n= ). protein expression of cyclin e , cyclin d , cyclin d and cell cycle inhibitors, p , p , p , and p was assessed by western blot analysis. spatial and temporal localization of cyclins e , d , d , p and p was determined by fluorescence immunohistochemistry. expression of cyclin e and cyclin d mrna was evaluated by qpcr analysis. results: immunohistochemical analysis showed cyclin e and cyclin d to be localized to cytotrophoblast (ct) cells of the chorionic villi; additionally cyclin e was also expressed in the extravillous trophoblast cells of the anchoring columns. in contrast, cyclin d expression was predominantly restricted to the villous stroma. cell cycle inhibitor p was expressed in both ct and syncytiotrophoblast (st) cells whereas p was restricted to the st. during normal placentation, levels of both cyclin e and cyclin d were high in the first trimester and decreased with advancing gestation. the expression of cyclin d , p and p showed an inverse correlation to cyclins e and d whereby their expression increased towards term. levels of p and p remained constant throughout pregnancy. preeclamptic placentae showed a significant increase in both cyclin e and p and a decrease cyclin d and p expression, as compared to age-matched control tissues. interestingly, preeclampsia was associated with an increased number of cyclin e positive progenitor ct cells in the floating villi and an increased expression of p in the endothelial cells lining the villous vessels. conclusion: preeclampsia displays an altered expression of g phase cell cycle regulatory molecules portraying an expression profile that closely resembles that of first trimester placentae. (supported by cihr, owh/igh). we have also demonstrated elevated, hif- -mediated placental and circulating sflt- at high altitude. we sought to correlate circulating levels of plgf, free vegf and sflt- with maternal and fetal oxygen tensions. we hypothesized that circulating sflt- and free vegf would be increased at m, and that plgf, known to be up-regulated by higher oxygen tension, would be decreased. methods: we collected both serum and plasma samples, the latter treated with inhibitors of platelet activation. maternal and umbilical samples were obtained from and healthy mother-infant pairs living at m and m respectively. plgf, free vegf and sflt- were measured in both serum and plasma using commercially available elisa kits (r d). data were log-transformed and analyzed by unpaired student's t test or anova as appropriate. results: plgf did not differ between altitudes. free vegf ( ± pg/ml vs. ± pg/ml, p<. ) and sflt- ( . ± . vs. . ± . ng/ml) were increased at m. however concentrations of all the angiogenic growth factors were reduced when platelet activation was prevented (- ± % plgf; - ± % free vegf; - ± % sflt- , p<. ). cord blood free vegf was -fold greater than in the mothers, but inhibition of peripheral cell activation abolished detectable levels in % of the babies. similar results were obtained for sflt- . thus, while free and total maternal circulating vegf and sflt- are elevated at high altitude, these increases are due to activation of peripheral blood cells. moreover, free vegf does not exist in detectable amounts in human pregnancy. there was no relationship between variation in the angiogenic growth factors and maternal or fetal oxygen tensions. the objective of this study is to identify candidate genes responsible for the variance between severe preeclampsia (spe) and hellp syndrome (hs). placental biopsies from spe (n= ) and hs (n= ) were collected. diagnosis of spe was confirmed by blood pressure and protein criteria. hs was diagnosed in preeclamptic patients who developed characteristic laboratory abnormalities. placental tissues were embedded in oct for sectioning, h e staining and rna isolation (invitrogen, carlsbad). gene expression data was obtained by hybridizing fluorescently labeled reverse transcription products to spotted cdna microarrays. the microarrays were produced by the laboratory of microarray technology at the van andel institute (grand rapids, mi) using a custom microarrayer. microarrays were scanned using an agilent g b scanner. images were analyzed using genepix . (axon). limmagui was used to generate lists of discriminating genes for these data, while david provided functional annotations. there were differentially expressed genes between spe and hs (p<. ). among these candidate genes, were up-regulated and were downregulated. the most up-regulated genes are ( )-deoxyribonucleotidase (dnt- ), superoxide dismutase , hydroxy-delta- -steroid dehydrogenase, caspase , and general transcription factor iih. further analysis of functional groups revealed the most enriched gene categories are related to cellular energy (mitochondria), cell cycle regulation, and protein metabolism. the underlying role of the placenta in the development of hs is currently unknown. this study shows that there is a relatively mdest number of genes that are differentially expressed between hs versus spe placentals. thes data provide the molecular context for placental changes seen in this variant of preeclampsia and provides an opportunity to study the role of the effected genes in disease variance. ( ), severe preeclampsia ( ), hellp syndrome ( ) and eclampsia ( ) and control group ( patients) uncomplicated pregnancies. total rna was isolated and reversely transcribed to c-dna. the mrna level of tert was detected with a probe-specific real-time quantitative pcr assay using ß-actin as the reference gene. crossing point (cp) values were obtained during the pcr amplification and the relative expression level of htert equals to (cp htert -cp ß-actin) . statistical analysis was performed using the student's t test. immunohistochemistry (ihc) staining was employed to localize htert protein on placenta tissue sections using abc method, incubation with rabbit anti-htert antibody followed by application of a goat anti-rabbit antibody with results evaluation using microscope. objective tgf s are involved in the regulation of trophoblast differentiation and invasion, and we have previously reported that tgf- is over expressed in pre-eclamptic placentae. tgf s signal via a receptor complex composed of type ii (t r-ii) and type i (t r-i) receptor. to date, seven type i receptors, designated as activin receptor-like kinase (alk - ) have been identified. our aim was to investigate the expression pattern of alk and alk receptors, known to exert tgf signaling, in preeclamptic and iugr placentae. methods human placental tissue throughout gestation was used in order to determine the development profile of the receptors. in addition, placental tissue from preeclamptic and iugr pregnancies and from age matched controls was collected. all iugr pregnancies were characterized by absence of end diastolic velocity in the umbilical artery and had no evidence of preeclampsia. expression of alk and alk mrna was measured by real-time pcr analysis, and protein by western blot analysis using alk and alk antibodies. immunoblot analysis demonstrated a unique developmental profile whereby alk expression increased with advancing gestation. in preeclamptic placentae alk expression was significantly increased compared to preterm and term controls, whereas alk expression was significantly decreased. preeclamptic placentae also exhibited decreased phosphorylation of smad , a tgf signaling molecule, which is activated by alk and increased phosphorylation of smad , which is triggered by alk . the expression of alk and alk in iugr placentae differed from that of preeclampsia as both alk and alk mrna levels were significantly increased in iugr compared to preterm and term controls. however, only alk expression was significantly increased in iugr placentae at the protein level, while no differences in alk protein levels were noted between iugr and controls. conclusions imbalance between alk and alk signaling pathways might play a role in the pathogenesis of preeclampsia and iugr. as tgf signaling via either alk or alk has been found to differentially regulate vasculogenesis, changes in these signaling pathways may contribute to the altered vasculogenesis found in these pregnancy-related disorders. (supported by cihr and owh/igh). ( ), severe preeclampsia ( ), hellp syndrome ( ) and eclampsia ( ) and patients, the control group who had uncomplicated pregnancies. total protein was isolated from placental tissue. gadd a and its downstream signal proteins--phospo-p , phospho-mkk /mkk were assessed by western blot. immunohistochemistry (ihc) staining was employed to localize the expression of gadd a and sflt- proteins in placenta tissue sections using abc method. huvec cells were cultured to - % confluence and were divided into groups: control, stress induction (sorbitol, . m, h), p inhibition (sb- , ug/ml, ug/ml and ug/ml, hour) + sorbitol ( . m, h). total protein was isolated from cells and the supernatant of huvec was collected. western blot was processed to detect the induction of gadd a and phospho-p . supernatant sflt- was measured with an eia kit and the results were read at nm wavelength. results: gadd a protein was elevated in the preeclamptic placentas with its downstream proteins (mkk and p ) activation compared with control. overexpression of gadd a and sflt- in preeclamptic placentas was observed with ihc staining. in huvec cells, gadd a was induced by sorbitol, triggering the activation of the downstream p- pathway and the accumulation of sflt- in the supernatant. the up-regulation of sflt- secretion by inducing gadd a was depleted when treated with p- inhibitor. conclusions: our study reveals that gadd a and its down stream p- pathway were activated in preeclamptic placentas and this stress inducible signal pathway regulates the secretion of sflt- , which is a key player in preeclampsia. it provides novel evidence that links placental stress to sflt- secretion via the gadd . trophoblast adipose triglyceride lipase (atgl) expression is upregulated in preeclampsia. beth a plunkett, jennifer a doll, emily j su, serdar e bulun, mona cornwell, susan e crawford. obstetrics and gynecology, northwestern university, chicago, il, usa; pathology, northwestern university, chicago, il, usa. objective: preeclampsia is characterized by placental endothelial cell dysfunction and elevated maternal triglyceridemia (tg). tg traverse the placenta in a process of uptake followed by lipolysis with subsequent release of fatty acids to the fetus. although three placental lipases (hormone sensitive lipase, endothelial lipase and lipoprotein lipase) have been identified, they do not account for all lipolytic activity. here, we introduce a new lipase, adipose triglyceride lipase (atgl), which is responsible for the hydrolysis of triglycerides to diglycerides in adipocytes and was recently identified as a receptor for endothelial cell modulator pigment epithelium-derived factor. the purpose of this study is to determine if expression of atgl, a potential modulator of both lipid metabolism and vasculature, is upregulated in preeclampsia. methods: immunohistochemical studies were performed on placental tissues from normal pregnancies (n= ) and those complicated by severe preeclampsia (n= ) with anti-atgl antibodies. the degree of positivity in the trophoblasts and endothelial cell was scored ( =none, =spotty, light, =consistent, dark). to determine if atgl is a product of placental endothelial cells (plec), microvascular cells were isolated from normal placental tissue. purity of the sample was confirmed using flowcytometry (> / positivity for factor antigen). atgl was detected immunohistochemically and via western blot using anti-atgl antibodies. results: mean atgl expression in preeclamptic trophoblast was significantly higher than normal placentas ( . + standard deviation versus . + . , p = . ). endothelial expression was not significantly different in preeclamptic ( . + . ) versus normal placentas ( . + . , p> . ). atgl stained intensely and demonstrated a beaded pattern in the endothelial cells, suggestive of a lipid droplet pattern. immunohistochemistry of plec and western blot analysis of cell lysates revealed strong immunopositivity for atgl, although at a smaller size than anticipated. conclusion: these findings demonstrate that a novel lipase, atgl, is produced by trophoblasts and is upregulated in severe preeclampsia. plec express high levels of atgl, suggesting that atgl could prove to be important in the vascular dysfunction and lipid abnormalities characteristic of preeclampsia. increased endothelial chymotrypsin-like protease (chymase) expression is responsible for endothelial activation in preeclampsia. yuping wang, yang gu, yanping zhang, david f lewis. obstetrics and gynecology, lsuhsc-shreveport, shreveport, la, usa. objective: endothelial (ec) activation is an important component of inflammatory phenotypic changes in preeclampsia (pe). our previous study showed enhanced chymotrypsin-like protease (clp)/chymase expression in the maternal vessel endothelium in women with pe. in this study, specific effect of placental-derived clp on ec activation was examined. methods: human uterine microvascular endothelial cells (utmvecs) were used. placental conditioned medium (cm) was prepared by culturing villous explants from normal and pe placentas. confluent utmvecs were treated with placental cm with or without depletion of chymotrypsin. ec adhesion molecule expressions for icam, vcam, p-selectin and e-selectin were determined by a colorimetric assay at od nm. depletion of chymotrypsin from cm was performed by immunoprecipitation. to further determine if activation of endogenous clp/chymase in ecs is responsible for up-regulation of p-selectin and e-selectin expression, chymase sirna was applied to ec culture before the cells were treated with normal or pe cm and then ec adhesion molecule expressions were examined. data was expressed as mean ± se and analyzed by anova. a p level < . was set for statistically different. results: ) expressions of vcam, p-selectin and e-selectin, but not icam, were significantly increased in pe-cm treated utmvecs compared to those of normal-cm treated cells and untreated controls, p< . ; ) there was no difference for adhesion molecule expression in utmvecs between normal-cm treated with untreated controls; ) utmvecs transfected with chymase sirna significantly reduced p-selectin and e-selectin expressions when exposed to pe-cm, p< . . conclusion: placental-derived clp/chymase is responsible for activating ecs and inducing ec adhesion molecule expression. activation of ec chymase may be directly related to the inflammatory phenotypic changes that occur in ecs in pe. (supported nih grants hd and hl ). corin is a transmembrane serine protease that is important in processing natriuretic peptides (nps) and maintaining normal blood pressure. genetically modified mice without corin function develop a syndrome during pregnancy similar to preeclampsia. corin is present in the pregnant uterus and a deficiency in the enzyme may lead to hypertension and preeclampsia. we tested the hypothesis that corin expression was increased in human myometrium from women with preeclampsia. methods: myometrium was obtained from groups of women at the time of primary cesarean section: (i) preterm no labor with preeclampsia (n= , ptspe, . weeks); (ii) preterm labor (n= , ptl, . weeks); (iv) term no labor (n= , tnl, . weeks); (v) term labor (n= , tl, . weeks). microarray gene profiling was performed using affymetrix human genome u plus . arrays. women who were not in active labor at the time of delivery (tnl) served as the control group. conventional and real time pcr was performed to verify corin expression in the arrays was directionally accurate. corin protein expression was examined by western blot. results: compared to tnl control, corin levels decreased nearly -fold in myometrium from women in term labor (tl). there was a small increase in corin gene expression of preeclamptic women (ptspe) and little-to-no change in myometrium from women in preterm labor (ptl). these results were confirmed by pcr analysis. conclusion: blood volume surges during pregnancy, increasing the potential for hypertension and preeclampsia in the mother. the corin-np control system could contribute to this condition. however, there are no reports on corin message or protein levels in women with preeclampsia. our preliminary results suggest that preeclampsia is not a consequence of a corin deficiency (decreased corin preeclampsia). acknowlegement: supported by grants from the phs (r hl - , cpw) and cdc grant (u dp - , cpw). the expression of gp at implantation site: implication for the pathogenesis of preeclampsia. objective: gp is a common shard signal transducing subunit of the il- cytokine family which is critical for implantation, and viewed as marker of endometrial blastocyst receptivity. preeclampsia (pe) is highly related to the restricted trophoblast invasion, which leads to impaired spiral artery remodeling. our hypothesis was that the poor placentation of pe is associated with the altered expression of gp at the implantation site. methods: human decidua from patients with pe and uncomplicated term deliveries (n = , respectively) were immunostained for gp . the intensity and distribution of immunostaining on decidual cells and extravillous traphoblasts were evaluated with hscore. statistical analysis of the data was performed using student's t-test and kruskal-wallis one way anova on ranks followed by post hoc test. results: immunostaining of gp was significantly higher in decidual cells of patients with pe compared with normal specimens, with hscore (median, [interquartile range]) value [ . - . ] and , respectively (p< . ). in pe specimen, the hscore of decidual cells was also significantly higher than that of trophoblast ( [ - ]; p< . ). there were no difference in the comparison of hscore of trophoblasts between the pe and normal specimens, and in normal specimens between decidal cells and trophoblasts. conclusions: the increase of gp expression in the decidual cells of preeclamptic placenta may be implied to the pathogenesis of poor placentation in preeclampsia. we have previously demonstrated that decidual cells are the major source of renin at the human uteroplacental interface, but little is known regarding the human decidual renin expression in hypoxic conditions. therefore, the present study was undertaken to determine the effect of hypoxia on renin secretion by human decidual cells in vitro. methods: full-term normal human placentas were obtained within one hour of vaginal deliveries or cesarean sections. decidual cells were isolated from the decidua parietalis. after an initial culture for days in a serum-containing medium, the decidual cells were exposed to normoxia or hypoxia ( % or % oxygen) in a serum-free medium for hours. the culture supernatants were then harvested and subject to western blot analyses of renin protein contents. a dominant band of renin at approximately kd was detected in all samples. when compared with the cells cultured in the normoxic condition, the cells cultured in both hypoxic conditions (i.e. % and % oxygen) had significantly lower renin protein contents in their culture supernatants. conclusion: our data for the first time show that hypoxia down-regulates renin secretion in human decidua, suggesting a link between the uteroplacental ras and oxygen tension during human gestation. the effect of nucleated fetal red blood cells derived from preeclamptic patients on endothelial progenitor cell proliferation. keiichi matsubara, emiko abe, yuko matsubara, shinji hyodo, masaharu ito. obstetrics and gynecology, ehime university school of medicine, toon, ehime, japan. objectives inadequate uteroplacental circulation results in placental ischemia and the development of preeclampsia (pe). endothelial progenitor cells (epcs) are thought to be a key player in the fetal angiogenesis. vascular endothelial growth factor (vegf), which is up regulated in pe, is involved in epcs proliferation. recently, it was reported that fetal nucleated red blood cells (nrbcs) have the capability to generate vegf. we hypothesized that nrbcs could influence epcs proliferation in the placenta and may be involved in the pathogenesis of pe. material and methods mononuclear cells (mncs) were isolated from the umbilical venous blood of normal pregnant women and preeclamptic patients by density gradient centrifugation. mncs were incubated with anti-cd antibody conjugated with microbeads. nrbcs were collected using a mini macs separator. nrbcs were incubated for hours with or without angiotensin ii (ang ii) and erythropoietin (epo). vegf and placental growth factor (plgf) concentrations in the supernatant were measured using elisa. also, pbmcs without nrbcs were seeded in endothelial basal medium with or without nrbcs using a boyden chamber. these samples were incubated for days with or without ang ii and epo. the adherent cells were incubated with di-ldl, fixed with paraformaldehyde, and stained with fluorescein isothiocyanate-labeled lectin. di-ldl and lectin positive cells was considered to represent epcs and the number was measured using flowcytometry. the number of nrbcs derived from umbilical venous blood was significantly increased in pe. both ang ii and epo significantly increased vegf concentration in the supernatant of nrbcs derived from normal pregnant women. however, ang ii and epo did not influence the nrbcs' vegf production in pe patients. plgf was not detectable in the supernatant. the number of epcs in the umbilical venous blood was significantly decreased in pe and the number was not changed by nrbcs. on the other hand, the number of epcs was significantly decreased in the culture with nrbcs. epo significantly increased the number of epcs in pe at the lower concentration of epo compared with normal pregnant women. conclusions it appears that fetal nrbcs may inhibit fetal epcs proliferation in pe. since epo reduced the inhibitory reaction of nrbcs without vegf production epo may affect epcs proliferation independently of nrbcs. relationship between the hypoxia-inducible factor- (hif- ) and the receptor svegf-r /sflt- : implication for phatophysiology of preeclampsia. julio e valdivia-silva, , juan c gonzalez-altamirano, keisy lopez- the trophoblast invasion is critical for the establishment of the uteroplacental circulation. at early phases of this process local oxygen pressure in the placenta is lower, that pathologically in preeclampsia remain constant. because of this, is important to understand the response of placental cells against these stimuli. in the present work, we use primary cultures of trophoblast cells, fibroblasts of the villous, and human umbilical endothelial cells, isolated of preterm and term placentas (with and without preeclampsia), to explore the effect of the oxygen pressure in the expression and synthesis of vegf, svegfr- /sflt- , hif- and- . our results show that the low pressure of oxygen resulted in a significant increase of the mrna and the protein of the receptor svegf-r selectively in the cts. the vegf's expression and synthesis was raised in three cellular types, but the free protein (not bounded to svegf-r ) of the cts was diminished. on the other hand, the expression of the arnm of hif- or - in cells was comparable in all the types of placentas, nevertheless, the protein hif- was more increased in the cts of preeclamptic placentas. to evaluate the relation of hif- and the increase of the receptor svegfr- , we used sirna-hif . in response to the inhibition, the expression of the receptor svegf-r diminished dramatically. the blockade of hif- did not alter vegf's expression. our data are the first that propose that the protein of the factor of transcription hif- is one of the molecules involved in the selective expression of the receptor svegf-r in trophoblast cells during hypoxia. figure: reduction of the expression to soluble receptor flt- /svegfr- after transfection with sirna-hif in trophoblast cells. sirna= interference rna, lu=luciferase. luc-sirna was used as control. effects of estradiol on synthesis, secretion, and activation of von willebrand factor in endometrial endothelial cell. shumei zhao, chainarong choksuchat, michael s scholfield, todd d deutch, thomas d kimble, david f archer. obstetrics and gynecology, eastern virginia medical school, norfolk, va, usa. objectives: heavy menstrual bleeding (hmb) is a serious clinical condition affecting % of women. due to inefficient and ineffective medical interventions, women with hmb often elect endometrial ablation or hysterectomy to eliminate hmb symptoms. morbidity and loss of fertility linked to these surgical treatments support the search for more effective medical remedies. von willebrand factor (vwf), a principle initiator of blood clotting produced by endothelial cells. women with von willebrand disease (vwd), have a high incidence of hmb indicating poor clotting. these findings suggest vwf heavily impacts the amount of blood loss during menstruation. estrogen stops/reduces hmb and has been used to treat hmb in women with vwd, although the mechanism(s) is unknown. this proposal addresses the hypothesis that estradiol (e ) increases the synthesis and activation of vwf, promoting clotting. materials and methods: immortalized human endometrial endothelia cells (heecs) were used for the studies. to determine if e increases synthesis of vwf, we treated heecs with e at . μm, . μm and . μm for hours. vwf protein and mrna levels were determined by western blotting and real time pcr, respectively. to establish if e can convert vwf from an inactive to an active conformation, the release of activated vwf by cells into culture medium will be assessed by elisa. decreased adamts (a disintegrin and metalloproteinase with thrombospondin type motif) activity results in increased amounts of active vwf. rrelease of activated adamts will be determined by fret. to ascertain if e regulated vwf secretion is by genomic pathway, heecs will be exposed to the estrogen receptor antagonist ici , . elisa and fret will assess the release of active vwf and adamts , respectively. results: western blotting demonstrated e increases vwf mrna and protein levels in a dose-dependent manner in heecs.conclusion: the project will determine if e acts at the heecs to increase synthesis, secretion and activation of vwf. preliminary results show e increases intracellular vwf protein and mrna levels in heecs, supporting our hypothesis that e increases synthesis of vwf in heecs in vitro. if e increases activated vwf, it could reduce/stop hmb by increasing activated vwf, providing justification for a clinical trial of e to treat hmb. over-expression of vegf-d does not induce lymphangiogenesis in the mouse endometrium. peter a rogers, jacqui f donoghue, marc g achen, steven a stacker, jane e girling. obstetrics gynaecology, monash university, melbourne, victoria, australia; ludwig institute for cancer research, melbourne, victoria, australia. background: the human endometrial functionalis has reduced lymphatics compared to the basalis and myometrium . this study examines the distribution of lymphatics in mouse uterus and investigates if over-expression of the lymphangiogenic growth factor vascular endothelial growth factor-d (vegf-d) stimulates growth of new endometrial lymphatic vessels. methods: the distribution of uterine lymphatics was examined in c bl/ jxcba mice collected during the oestrus cycle, early pregnancy and following oestrogen and progesterone treatment. human ebna cells with/without stable transfection of vegf-d were injected into the uterine horn of nod/scid mice. uteri were collected after weeks. serial sections were immunostained with lyve- and/or vegfr (lymphatic endothelial cell markers), cd (blood endothelial cell marker), mab (human vegf-d), mab (human mitochondria) and pcna (proliferative cell nuclear antigen). results: lymphatic vessel profiles were mostly found in the connective tissue between the longitudinal and circular muscle layers of the myometrium. they were rare in the endometrium and only observed in % of the sections. when present in endometrium, lymphatic vessel profiles were usually situated adjacent to the endometrial/myometrial border. ebna tumours formed inside and outside the uterine horn of both the control (n= of ) and vegf-d group (n= of ). localization of ebna cells within the mouse uterus was confirmed by anti-human mitochondrial expression. vegf-d immunostaining confirmed that transfected ebna cells expressed vegf-d in vivo. over-expression of vegf-d did not stimulate endometrial lymphangiogenesis, although there was an increase in vessel diameter of lymphatics in the myometrium adjacent to tumours. initial analysis shows no significant effect of vegf-d ebna cells on endometrial blood vascular density or endothelial cell proliferation. conclusions: minimal lymphatics are present in the mouse endometrium, as is the case for lymphatic vessels in the human endometrial functionalis. the lack of endometrial lymphangiogenesis in response to vegf-d suggests the presence of an inhibitory factor limiting lymphatic growth in this tissue. in early pregnancy, decidual-derived vegf mediates angiogenesis and is required for implantation and placentation. the role of decidual vegf in later pregnancy is poorly understood. decidual hemorrhage (placental abruption) generates excess thrombin and is a major risk factor for pprom and preterm birth. this study compares immunohistochemical (ihc) localization of vegf in decidual tissue sections from term pregnancies complicated by abruption and gestational age-matched controls, and investigates the effect of thrombin on vegf expression by cultured human term decidual stromal cells (dscs). study design: ihc was performed on serial sections of term placental tissues (no labor) with (n= ) and without (n= ) abruption. purified term dscs were passaged until > % free of cd + cells by facs. confluent dscs were primed with - m estradiol (e ), - m medroxyprogesterone acetate (mpa), both, or vehicle for days. after h incubation in defined medium with corresponding steroids ± thrombin ( . - . iu/ml), conditioned supernatants were analyzed for vegf by elisa. extracted total rna was used to assess vegf mrna levels by quantitative rt-pcr using established primers. results: vegf expression was localized by ihc primarily to dscs in placental tissue sections, and was increased in tissues from placental abruption vs controls. in term dscs, thrombin increased vegf secretion in a dosedependent fashion irrespective of the hormonal milieu (eg, . -fold stimulation by . iu/ml thrombin from . ± . to . ± . pg/ml per mcg protein for e +mpa; p= . ). this effect was abrogated by the thrombin inactivator, hirudin. vegf mrna were similarly increased by thrombin with or without steroid hormones (eg, . -fold for e +mpa; p< . ). conclusions: placental abruption is associated with increased vegf expression in term decidual tissues in vivo with thrombin enhancing vegf mrna and protein expression in term dscs in vitro. excess thrombinmediated vegf expression in term decidua aberrantly increases endothelial cell permeability to further generate thrombin by continuous exposure of tissue factor-expressing decidual cells to circulating factor vii. thrombin-enhanced matrix metalloproteinase expression in term dscs would degrade decidual and fetal membrane extracellular matrix to induce pprom and preterm birth. basal directional release of angiotensin ii by endothelial cells stimulated by chymotrypsin-like protease (clp)/chymase. yuping wang, david f lewis, yang gu. obstetrics and gynecology, lsuhsc-shreveport, shreveport, la, usa. objective: chymotrypsin-like protease (clp)/chymase is a serine protease which plays a major role in angiotensin ii (ang ii) generation in the human heart. our previous study showed a higher clp activity in the maternal plasma in women with pe than in normal pregnancies. we also found enhanced chymase expression in the maternal vessel endothelium in women with pe. in this study, we determined if clp could promote endothelial cell (ec) generation of ang ii. methods: we specifically examined basal directional release of ang ii by cultured ecs. ecs were grown on cell culture insert ( well/plate, micron pore size). when ecs reached confluence, chymotrypsin (chy) at concentrations of . , . , . , . , and . g/ml were added to the upper chamber of the cell insert. after hours of culture, medium in the lower chamber was collected. medium concentrations of ang ii were measured by enzymelinked immunoassay (eia). all samples were measured in duplicate. data are expressed as mean ± se and analyzed by anova. a p level < . was considered statistically different. results: chymotrypsin produced a concentration-dependent increase in basal directional release of ang ii by cultured ecs, control: . ± . g/ml; chy . : . ± . g/ml; chy . : . ± . g/ml; chy . : . ± . g/ml; chy . : . ± . g/ml (p< . ); chy . : . ± . g/ml (p< . ), respectively. data are means from independent experiments. conclusion: apical exposure of ecs with chymotrypsin-like protease could promote basal directional release of ang ii. our result implicates that in pe, elevated clp levels in the maternal circulation are very likely to affect ec generation of ang ii. basal directional released ang ii may bind to its receptor on underlying vascular smooth muscle cells and contribute to the increased vasoconstriction in pe. (supported nih grants hd and hl ). vegf stimulates angiogenesis and vasodilation critical for dramatic rises in materno-feto interface blood flows directly linked to fetal growth/survival. extracellular signal-regulated kinase (erk / ) pathway mediates partially vegf-induced angiogenic and vasodilatory responses in placental endothelial cells (ec). it is, however, unknown how this vegf-induced signaling is organized in placental ec. objectives: ovine fetoplacental artery ec (ofpaec) and its transformed counterpart, sv -of to test whether: ) vegf-activated erk / signaling is compartmentalized in the caveolae and disruption of caveolae interferes vegf-induced erk / activation and; ) caveolin- , the structure protein of caveolae, regulates vegf-stimulated erk / phosphorylation. methods: ofpaec or sv -of cells were cultured in mcdb- / % fbs/antibiotics. serum-starved subconfluent ( %) cells were treated with rhvegf ( to ng/ml) for various times. caveolae were disrupted by -cyclodextrin ( -cd, mm, min) or caveolin- scaffolding domain (cav-sd, m, hr). sv -of cells were used for fractionation of caveolae membranes by discontinuous sucrose gradient ( %/ %/ %) ultracentrifugation. activation of erk / signaling pathway were analyzed by western-blotting with specific antibodies. results: in total cell extracts, vegf stimulates erk / phosphorylation in a time-and dose-dependent manner. erk / phosphorylation maximized by vegf ( ng/ml) at - min, which was abrogated by -cd or cav-sd. all the molecules for compromising the erk / signaling module, plc , pkc , src, ras, raf- , mek / and erk / , were detectable in purified caveolae membranes positive for various markers including caveolin- , enos, flotillin- , and -adaptin. in caveolae, vegf dramatically increased phosphorylated erk / without altering total erk / in a time-dependent manner similar to that in total cell extracts, which also maximized at - min. pretreatment with -cd or cav-sd blocked vegf stimulation of erk / phosphorylation in caveolae. conclusion: vegf activates the erk / signaling pathway in caveolae and caveolae integrity is essential for vegf-activated erk / signaling pathway. we conclude that caveolae/caveolin- serves as a platform for compartmentalizing the vegf-induced erk / signaling pathway in placental ec (hl and hl ). hypoxia upregulates gcm in human placenta. david mccaig, fiona lyall. institute of medical genetics, university of glasgow, glasgow, united kingdom. introduction: studies in transgenic mice have shown that a variety of genes regulate the differentiation of trophoblast cells. these genes include gcm . gcm is also expressed in the human placenta. placental gcm- protein has been reported to be reduced in pre-eclampsia, in view of the close link between hypoxia, hypoxia-reoxygneation, pre-eclampsia, placental development and the reported reduction in gcm we hypothesised that gcm expression would be affected by hypoxia. aim: the aim of this study was to determine the effects of hypoxia on gcm expression in the human placenta. two model systems were used; ( ) free floating villous explants and ( ) cultured primary cytotrophoblast and syncytiotrophoblast cells as described previously*. methods: explants or cell cultures were exposed to either hypoxia or hypoxia followed by re-oxygenation. western blot analysis was used to assess gcm protein levels. bands on the gels were quantified using scanning densitometry. statistical differences (n= experiments for both models used) were calulated by anova and turkey's post-hoc test. results: gcm protein was detectable at a low level in villous explants maintained for h in % o . a striking increase in gcm protein was observed when villous explants were incubated for h in % o (p< . ). incubation of villous explants for h in % o followed by re-oxygenation for h in % o resulted in a marked decline in gcm protein (p< . ). expression of gcm was also analysed in primary cytotrophoblast and syncytiotrophoblast cultured in % o or reduced oxygen ( % o ) conditions. gcm protein was not detected in any of the experimental conditions used. discussion: the present study has shown that acute hypoxia increases gcm- protein in villous explants. the experiments with purified trophoblast do not support a role for hypoxia increasing gcm- in these cells under the experimental conditions used. the present findings are in keeping with the complex effects of oxygen depending on the conditions used. the observed hypoxic effects on gcm warrant further investigation. the effect of acute alcohol exposure on histone -lys modification in the mid-gestation embryonic lung. xiangyuan wang, debra wolgemuth, , , laxmi baxi. ob/gyn; genetics development; human nutrition, columbia university medical center, new york, ny, usa. objective maternal alcohol abuse during pregnancy produces an array of birth defects comprising fetal alcohol syndrome. lung development depends on a balance between cell proliferation and apoptosis. we have previously shown that the acute alcohol exposure in the mid-gestation embryo can delay lung development and induce apoptosis. acute exposure to ethanol of selected tissues in mouse embryos has been reported by others to initiate apoptosis within hours after exposure and result in histone modifications. specifically, histone acetylation and deacetylation are involved in transcriptional activation and repression, respectively, but can also involve apoptosis. in the present study, we have investigated the effect of alcohol on acetylation of histone at lysine (ach lys ) in the mid-gestation embryonic lung. pregnant c bl/ j mice at day . of gestation (e . ) were injected intraperitoneally with doses of % ethanol ( . g/kg ), hr apart (alcoholexposed: ae) or with ringers solution (controls: c). ae and five c fetuses were retrieved and hr later and the lungs were fixed and processed for morphological evaluation and staining with rabbit polyclonal anti-ach ly antibody. the entire lung tissue field was evaluated for the levels of ach lys staining and scored as (-) to (++++). three areas were selected randomly from each sample and the total number of cells and staining positive cells were counted in the bronchial epithelium and in the mesenchyme. twelve hr after alcohol exposure at e . , the morphology of ae embryonic lungs was normal. however, high levels of ach lys were detected in % of the bronchial epithelial cells and % of the mesenchymal cells. the expression level in both lineages decreased hr after alcohol exposure. in the controls, the expression of ach lys was virtually undetectable in both the bronchial epithelium and the mesenchymal cells. our previous study showed that ae e . lungs significantly increased apoptotic cell in both bronchial epithelium and mesenchyme hours after alcohol treatment. we now observe that elevated expression of ach lys in the embryonic lung preceded the observation of apoptosis, suggesting that alteration in the acetylation of h could be one of the molecular mechanisms involved in the induction of apoptosis following acute alcohol exposure. objective: our purpose was to evaluate the effect of vitamin c and e supplementation on lipid peroxide levels, total antioxidant ability, and antioxidant levels in the umbilical venous plasma. materials and methods: women at risk for preeclampsia (nullipara, previous preeclampsia, chronic hypertension) were recruited at to weeksgestation and randomly assigned to receive either mg of vitamin c and iu of vitamin e (study group, n= ) or placebo (control group, n= ) daily until delivery. umbilical venous blood were collected after full term delivery. lipid peroxide levels, oxygen-radical absorbance capacity (orac) values, antioxidant levels were measured by each method (thiobarbituric acid reaction, cao's method, and high performance liquid chromatography). results: . the lipid peroxide levels in the umbilical venous plasma of study group were significantly lower than that of control group ( . ± . vs. . ± . nmol/mg protein, p< . ). . the orac values in the umbilical venous plasma of study group were significantly higher than that of control group ( . ± . vs. . ± . u/ml, p< . ). . the -tocopherol levels in the umbilical venous plasma of study group were significantly higher than that of control group ( . ± . vs. . ± . nmol/ml, p< . ). . there were no significant differences in the ascorbic acid, uric acid, -carotene, retinol, and -tocopherol levels in the umbilical venous plasma between control and study group. conclusion: this suggested that maternal vitamin c and e supplementation may affect the oxidant-antioxidants balance of the utero-placenta unit and fetus. placental tnf-related apoptosis-inducing ligand (trail) in normal pregnancy and pre-eclampsia. xilian bai, jenny e myers, philip n baker, john d aplin, ian p crocker. maternal and fetal health research group, the university of manchester. introduction: enhanced placental trophoblast apoptosis is well known to occur in pre-eclampsia. however, the potential role of membrane associated or soluble trail, an apoptosis-inducing ligand, and its death receptor, dr , is not known. we tested the hypotheses that the trail/trail-receptor system is compromised in pre-eclampsia and that soluble trail is a circulating factor which triggers the vascular complications of pre-eclampsia. method: this study was conducted on placental samples and plasma (edta) from women with uncomplicated pregnancies (n= ) and with pre-eclampsia (n= ) at - weeks gestation. protein expression levels and tissue localisation of trail and dr were defined in villous tissue by western blotting and immunohistochemistry. soluble trail (strail) was measured in maternal plasma from both groups using a commercial elisa (diaclone). results: placental villous trail and dr protein was unaltered in preeclampsia compared to normal pregnancy. whilst there was differential distribution of trail and dr within the component cells, this also was unaffected in pre-eclampsia. within the villi, trail was mainly confined to the cytoplasm and perinuclear regions of cytotrophoblast, syncytiotrophoblast, stromal cells and the fetal capillary endothelium. conversely, dr was restricted to trophoblast only, distributed evenly between cytoplasm, plasma membranes and nucleus. strail was present in plasma from non-pregnant women of childbearing age [ . ( . - . ) , median (interquartile range), pg/ml, n= ]. however, there was no significant increase either in pregnancy [ . ( . - . ) pg/ml, n= ] or pre-eclampsia [ . ( . - . ) pg/ ml, n= ] (kruskal-wallis test, p> . ). conclusions: these results suggest that placental villous trail is not adversely regulated in pre-eclampsia. the absence of dr in stromal and endothelial cells may increase resistance to apoptotic stimuli in cells of the villous core. the co-localisation of trail and dr in trophoblast suggests a role in autocrine regulation of cell turnover in this cell type. introduction: preeclampsia is associated with apoptosis of the syncytial layer of placenta and the release of particulate and soluble factors which are deleterious to maternal endothelial function. the goal of the current study was to determine whether laser capture microdissection (lcmd) and western blotting could be used to assess levels of syncytial fas ligand (fasl), a key protein in the apoptotic cascade. methods: frozen sections of term placenta delivered from uncomplicated pregnancies at term were used for study (n= ). following staining with mayer's hematoxylin (n= ), lcmd (leica instruments) of an intact terminal villus was carried out using a focused laser pulse directed to the area of interest using a microscope. in the first round of microdissection the placental villus core consisting of fibroblast, macrophages, fetal vessels, and connective tissue, were removed. in the second round of lcmd, the syncytial layer of that same villus was removed and collected in lysis buffer containing detergent and protease inhibitors, and the number of nuclei per sample was recorded. this procedure was repeated until syncytial tissue from - villi were collected. electrophoretic separation of lysate proteins was then carried out, and western blotting and immunodetection using an anti-fasl antibody was performed. results: we observed that fasl was detected in microdissected syncytial specimens at a molecular weight of approximately kda ( figure) , consistent with our previous reports. it is of note, that western blotting of samples containing approximately (lane ), (lane ), (lane ), and (lane ) nuclei revealed that fasl could be reliably detected in specimens containing as few as nuclei. conclusions: since fasl is a cytokine of low to moderate abundance in placenta, this suggests that lcmd coupled with western blotting will be a valuable methodology to elucidate pathways of syncytial apoptosis and pathophysiology in pregnancies complicated by preeclampsia. supported in part by nih grant hd . the placenta of preeclamptic pregnancies shows oxidative and nitrative stress. we have shown low protein abundance but paradoxically high activity of inducible nitric oxide synthase (inos) in the placenta with severe preeclampsia compared with normal pregnancies. protein nitration and phosphorylation are post-translational modifications possibly involved in nos activity. objectives: examine inos localization and tyrosine phosphorylation in the preeclamptic placenta and the effect of a peroxynitrite generator (sin- ) on inos expression in primary human placental microvascular endothelial cell (hpmec) cultures. methods: placental lysates from normal (n= ), mild (n= ) and severe (n= ) preeclamptic pregnancies were western blotted for inos and what are the roles played by peroxynitrite in preeclamptic women? yoshikatsu suzuki, tamao yamamoto, yoshimasa watanabe, takeo itoh, hidetaka izumi. obstetrics and gynecology, nagoya city university, nagoya, japan; pharmacology, nagoya city university, nagoya, japan; obstetrics and gynecology, izumi women's hospital, fukuoka, japan. aim preeclampsia is characterized by hypertension plus proteinuria. it is hypothesized that the endothelial cell function might be activated by placental faculty in early pregnancy and the activation might cause the vascular disease in preeclampsia. peroxynitrite, which is produced by combination with superoxide (o -) and nitric oxide (no), is strong oxidant as well as o -.we investigated whether or not the localization of peroxynitrite in both placenta and resistance artery might play important roles of developing preeclampsia. omental arteries or placentas were obtained from severe preeclamptic and term-normotensive pregnant women at cesarean section. they were stained by ant-nitrotyrosine (nt, a marker of peroxynitrite) antibody. furthermore, the concentration of nt was measured in omental arteries. in formed consent was obtained from all patients in written. preeclampsia was diagnosed according to the criteria of japan society of obstetrics and gynecology. the localization of nt was seen in placentas obtained from sever preeclamptic women ( / ), although it was not seen from normotensive pregnant women ( / ). the localization was also seen in placentas from of severe preeclamptic women with intrauterine fetal restriction. in omental arteries from both groups, the localization of nt was seen to same degree, and the concentration of nt was similar ( . ± . for sever preeclampsia and . ± . for normotensive pregnant women). from these results, it was suggested that an increase in o production, a decrease in no as well as peroxynitrite production in the placentas might cause the vascular dysfunction and subsequently damage uteroplacental blood circulation in preeclampsia. however, the role played by peroxynitrite in resistance artery might be different and more complicated. background: preeclampsia (pe) is associated with shallow cytotrophoblast (ct) invasion of the decidua, leading to impaired vascular transformation and poor uteroplacental perfusion. ct invasion requires the selective proteolysis of the peri-decidual cell (dc) extracellular matrix. we hypothesized that il and tnf , cytokines that have been linked to pe, may induce aberrant expression of the matrix metalloproteinases (mmp) and in dcs, thereby preferentially degrading the decidual ecm and interfering with sequential ct invasion. methods: immunostaining for mmp- , mmp- , and vimentin (a dc marker) was performed on decidua from normal (n= ) and preeclamptic (n= ) women, and staining intensities were evaluated by hscore. confluent, leukocyte-free first trimester dcs were primed with - m estradiol (e ) or e + - m medroxyprogesterone acetate (mpa), and then switched to a defined medium with e +/-mpa with or without ng/ml of il or tnf . secreted mmp- and mmp- levels were measured by elisa (n= ) and confirmed by western blotting. quantitative rt-pcr assessed mmp- and mmp- mrna levels (n= ). results: tissue staining revealed that mmp- and mmp- levels in preeclamptic decidua (hscore mean±sem: ± and ± , respectively) were significantly higher than in normal decidua ( ± and ± , respectively; p< . ). in cultured first trimester dcs incubated with e , tnf increased secreted mmp- and mmp- levels by ± and ± -fold, respectively, while il increased them by ± and ± fold, respectively (p< . ). in parallel incubations with e +mpa, basal mmp- and mmp- output were lowered by approximately % and %, respectively, while tnf -and il elicited mmp- and mmp- levels were blunted by - %. western blotting confirmed the elisa results, and mrna levels corresponded to changes in mmp- and mmp- secreted protein levels. conclusions: over-expression of mmp- and mmp- in decidual cells may promote pe by disrupting decidual ecm and impairing normal ct invasion. the high levels of il and tnf associated with pe may be contributing to this over-expression. our in vitro observations that mpa blunts tnf -and il -elicited mmp expression suggests that exogenous progestin may offer a novel therapeutic approach in preventing pe. to produce -methoxyestradiol ( -me), a compound with diverse biological activities including inhibition of hif- , a transcription factor that mediates cellular response to hypoxia. circulating levels of -me and placental comt activity are significantly reduced in preeclampsia, raising the possibility that reduced production of -me contributes to the pathophysiology of preeclampsia by altering placental response to hypoxia. genetic variation in the comt gene is linked to comt activity and has been associated with intrauterine fetal growth restriction. we determined if a snp in exon of the comt gene (rs ), which does not change amino acid sequence ( leu ), but reduces comt mrna translation, was associated with preeclampsia. we analyzed comt genotypes in paired dna samples extracted from maternal and cord blood from normal pregnancies and pregnancies complicated by preeclampsia by allele discrimination. the study population was predominantly (> %) african-american. the frequency of the minor rs "g" allele, which is associated with low comt activity, was similar in maternal cases and controls (cases: %; controls: %, p= . ), but was significantly greater in fetal dna from pregnancies complicated by preeclampsia compared to control pregnancies (g allele frequency cases: %; control: %; p< . ). likewise, fetal carriage of the rs "g" allele conferred a significantly greater risk of preeclampsia (odds ratio: . ; % c.i.: . , . , p< . ). there was also a significant discordance between paired maternal and fetal rs genotypes with significantly greater discordance for the "g" allele in fetuses hosted in preeclamptic pregnancies (odds ratio: . ; % c.i.: . , . ). we conclude that genetic variation in the comt gene is associated with risk of preeclampsia, possibly through a mechanism involving reduced production of -me. supported by nih p md . smoking is associated with elevated adma in preeclampsia. michael p frank, robert w powers. , magee-womens research institute, pittsburgh, pa, usa; obstetrics gynecology, university of pittsburgh, pittsburgh, pa, usa. objective: cigarette smoke exposure paradoxically reduces the risk of preeclampsia. asymmetric dimethylarginine (adma) is an endogenous competitive inhibitor of nitric oxide synthase (nos), an independent risk factor for cardiovascular mortality, adma is elevated in women who develop preeclampsia, and adma has been reported to be both higher and lower in smokers. the objective of this study was to investigate the concentration of adma in pregnant smokers and nonsmokers with and without preeclampsia. study design: case-control study of women with uncomplicated pregnancy (controls), and women with preeclampsia matched for gestational age at sample collection. adma was measured by hplc. cigarette smoke exposure was determined by questionnaire and confirmed by plasma cotinine. data are mean±sd. analysis was by two factor anova with fishers post-hoc testing, significance accepted at p< . results: as previously reported, maternal plasma adma concentrations were higher in women with preeclampsia compared to controls (p< . ). in addition, the concentration of adma was significantly higher in preeclampsia smokers compared to controls and preeclampsia nonsmokers (p< . ). in contrast, there was no difference in adma concentration between control smokers and nonsmokers. conclusion: these data may suggest a differential effect of cigarette smoke exposure on circulating adma concentrations between women who do and do not develop preeclampsia. previous data has suggested that cigarette smoking is associated with lower adma in low risk elderly patients, and higher adma in high-risk subjects with diabetes. therefore, the data in preeclamptic and non-preeclamptic subjects may be consistent with these studies, however, the underlying biological explanation for this differential effect has yet to be determined. objective: eclampsia is similar to hypertensive encephalopathy in which an acute elevation in blood pressure causes autoregulatory breakthrough, hyperperfusion and edema formation. we previously reported that the pressure of breakthrough was similar between nonpregnant (np) and late-pregnant (lp) rats, but only lp animals developed edema. this study tested the hypothesis that lp animals have decreased in cerebrovascular resistance (cvr) and hyperperfusion in response to breakthrough vs. np. we further hypothesized that acute hypertension would cause greater blood-brain barrier (bbb) permeability in lp rats due to elevated hydrostatic pressure. methods: in vivo models of bbb permeability and cerebral blood flow (cbf) were used in np (n= ) and lp (d - ; n= ) rats that were either normotensive or hypertensive (np-htn, lp-htn) by infusion of phenylephrine to raise mean arterial pressure. permeability was determined in anterior and posterior brain regions by calculating the flux of kd dextran into the brain tissue, measured by a fluorescent spectrophotometer after flushing the vasculature with saline. cbf and cvr were measured by infusion of m fluorescent microspheres and determined based on the flow rate and fluorescence intensity of a reference sample for each animal. animals were ventilated to maintain blood gases within normal ranges (po > mmhg, pco = - mmhg). results: although the pressure change was similar between np and lp ( and mm hg), lp animals responded to acute hypertension with hyperperfusion. cbf increased from ± to ± ml/ g/min in np ( %) and ± to ± ml/ g/min in lp ( %; p< . vs. np). hyperperfusion in lp animals was associate with decreased cvr vs. np ( . ± . vs. . ± . mm hg/(ml/ g/min); p< . ). bbb permeability was significantly increased in lp animals at breakthrough vs. np in both anterior and posterior brain regions. the flux of dextran in anterior and posterior brain regions for np vs. lp animals was: ± vs. ± for anterior (p< . ) and ± vs. ± for posterior (p< . ). conclusions: these data demonstrate that pregnancy decreases cvr and causes hyperperfusion of the brain during acute hypertension. because increases in cvr is a protective function in the brain, impairment of these mechanisms during pregnancy may predispose the brain to edema when blood pressure is elevated, as in eclampsia. introduction: this study used the in vitro dually perfused human placental lobule to test the hypothesese that placental release of vegf and the fetoplacental vasodilatory response to exogenous vegf- are altered by tissue oxygenations that mimic healthy and preeclamptic pregancies. methods: lobules were dually perfused for six hours under one of two oxygenation conditions, representing 'normoxia' and 'hypoxia' (n = each): delivering maternal side inflow perfusate at oxygen concentrations of . % and %, respectively, distributed via cannulae; and fetal side inflow perfusate oxygen concentration of - % in both systems. venous perfusates were sampled and assayed, appropriate to side of release, for erythropoietin (epo), macrophage inflammatory protein- alpha (mip- alpha) (reference oxygen sensitive hormones), free vegf, svegfr- and plgf. in separate perfusions, fetoplacental vasodilation in response to pm vegf was investigated, following preconstriction of the fetoplacental vasculature to steady state fetal-side inflow hydrostatic pressure (fihp) with the thromboxane mimetic, u , (n = each). results: maternal-side mip- alpha release was higher in the 'hypoxic' than the 'normoxic' system ( ± and ± pg/ml, respectively, at hours, mean ± se; -way anova: p< . ). maternal-side epo and fetal-side soluble free vegf and svegfr- release were not different between groups. fetal-side release of plgf was higher in the 'hypoxic' group than the 'normoxic' group ( . ± . and . ± . pm, respectively, at hours, mean ± se; -way anova: p < . ). there was no difference in the vasodilatory response to vegf- in the fetoplacental vasculature between the groups ( . ± . and . ± . % change in fihp, mean ± se). discussion: differences in mip- alpha and plgf release provide evidence for metabolic separation of the adapted systems, caused by a changed oxygen environment. our failure to observe differences in epo, vegf and svegfr- release may be explained by longer lag-times for up regulation of their gene expression. vegf associated endothelial signalling appears to be unaffected by 'hypoxia' in the placenta over the time course studied here. background: there is a placental renin-angiotensin system (ras) from very early pregnancy. ang iv mediates various effects by binding to its specific receptor, the at r, the active site of which is an insulin-regulated aminopeptidase (irap). there is at r expression in both endothelial and smooth muscle cells. ang iv at low concentrations is vasodilatory, increasing blood flow via the at rs; it also stimulates nf-kappa beta and modulates glut- . to date at r expression has not been investigated in the placenta. we propose that at r plays a part in the placental vascular development necessary for successful pregnancy, and that reduced at r expression may be associated with inadequate vascular adaptation contributing to pre-eclampsia (pe). aim: to identify and locate at r expression in both np and pe placentae. methods: the study had hospital ethical approval; written informed consent was obtained from all women. placental samples were obtained from np and pe at delivery (gestational ages: and weeks respectively). samples were taken from areas, near cord, middle and outer edge of the placenta. paraffin embedded sections were immunostained for irap reactivity using a rabbit polyclonal antibody (gift from professor david james, garvan institute, australia). immunoreactivity of trophoblast and uterine cell populations was assessed using a semi-quantitative grading system. grade = no positive labelling, = - %, = - %, = - % and = - % of cells positively labelled. median (max, min) are shown. results: ) at r immunostaining was prominent in the syncytiotrophoblast and hofbauer cells of all placental villi examined, with no differences in expression between sampling sites. ) at r positivity was reduced in near cord pe samples ( . ( . , . )) compared to np ( . ( . , . ) p= . ). conclusion: we have shown for the first time, dense at rs in syncytiotrophoblast and hofbauer cells in np placentae, and their down-regulation in pe. reduced ang iv/at r binding may contribute to increased placental vasoconstriction resulting in increased ischemia/reperfusion. this in turn may stimulate xanthine oxidase, which is itself stimulated by angii, leading to increased superoxide production. further work is needed to clearly define the role of this newly identified component of the renin-angiotensin system in normal pregnancy and pe. pre-eclampsia is associated with lower percentages of regulatory t cells in maternal blood. jr prins, hm boelens, jj hm erwich, j heimweg, s van der heide, ajm van oosterhout, aej dubois, jg aarnoudse. obstetrics, university medical center groningen, groningen, netherlands; laboratory of allergology and pulmonary disease, university medical center groningen; pediatric allergology, beatrix children's clinic, university medical center groningen. pre-eclampsia is a serious disease of human pregnancy and immunological mechanisms play a role in its pathophysiology. normal pregnancy is associated with an increase in regulatory t (treg) cells and with a predominant th immune profile. treg cells are a subpopulation of cd + lymphocytes and are specifically characterized by the lineage specific transcription factor foxp . treg cells seem to induce immunological changes that have a protective role in maintaining normal pregnancy. we hypothesised that percentages of treg cells are decreased in pregnancies complicated by pre-eclampsia. methods in total, women with pregnancies complicated by pre-eclampsia and healthy pregnant controls were enrolled. to obtain control umbilical cord blood as well, control group i consisted of eighteen healthy pregnant women at term. in addition, since women with pre-eclampsia delivered preterm, control group ii (peripheral blood only) consisted of women during normal pregnancy with a gestational age matched for the preterm pre-eclamptic group. treg cells were measured from whole blood using four-color flow-cytometry. women with a pregnancy complicated by pre-eclampsia had a significantly lower percentage of cd + foxp + treg cells ( . vs . %; p< . ). in the pre-term group the pregnancies complicated by pre-eclampsia showed a significantly lower percentage of cd + foxp + cells in the peripheral blood as compared to the healthy pregnant controls. at term this percentage was also lower but not significantly so. between pre-term and term pregnancies both complicated by pre-eclampsia no significant difference was found in the percentage of cd + foxp + treg cells. no difference was found in umbilical cord blood ( . vs . %). conclusions our data suggest that pre-eclampsia is associated with a diminished percentage of treg cells in peripheral blood. we conclude that a deficiency of regulatory t cells may play a role in the pathophysiology of pre-eclampsia. background: preeclampsia and eclampsia are significant causes of maternal and fetal death. however, the pathophysiology of these conditions is unclear. we and others have reported that inhibition of endogenous nitric oxide (no) synthesis produces symptoms similar to preeclampsia in pregnant rats. several studies demonstrate that fetoplacental weights are altered in pregnancies of spontaneous hypertensive (shr) rats. in addition, impaired synthesis of tetrahydrobiopterin (bh ), a major co-factor for endothelial nitric oxide synthase (enos) activity and enhanced expression of enos has been observed in the pathogenesis of hypertension. in the current study, we examined whether supplementation of bh and sepiapterin (sep, a precursor for bh biosynthesis in the salvage pathway) reduces increased blood pressure and improves fetoplacental weights in shr pregnant rats. methods: groups ( - ) of shr pregnant rats were either treated with bh , sep ( mg/k.g body weight/day/rat, oral tablets) or vehicle (normal diet) beginning from day of pregnancy until day of gestation. animals were sacrificed on day of gestation and fetoplacental weights were recorded immediately. western blot analysis was performed to determine vascular enos expression (enos/gapdh). results: significant (p< . ) elevations in blood pressure (bp, mmhg) were observed in shr (shr, ± . vs. wky, ± . mmhg) compared to wistar-kyoto (wky) group. supplementation of either bh or sep ( ± . ), significantly (p< . ) reduced elevated bp beginning from day of pregnancy. fetal but not placental weights were significantly (p< . ) reduced in shr ( . ± . grams) compared to wky ( . ± . ) rats. bh ( . ± . ) treatment partially (p< . ) increased fetal weights compared to the shr group. vascular enos expression is significantly (p< . ) elevated in shr ( . ± . ) compared to wky rats ( . ± . ). further, treatment with bh ( . ± . ) but not sep ( . ± . ) significantly (p< . ) reduced elevated enos protein expression in shr rats. conclusions: bh may be beneficial treatment of preeclampsia to reduce blood pressure and improve fetal perfusion to increase fetal weights. genetic risk factor for severe preeclampsia: significance of endothelial nitric oxide synthase gene t- ->c and missense glu asp variants. toshihiro yoshimura, michihiro yoshimura, masafumi nakayama. obstetrics and gynecology, kumamoto university school of medicine, kumamoto, japan; cardiovascular medicine, jikei university school of medicine, tokyo, japan; cardiovascular medicine, kumamoto university school of medicine, kumamoto, japan. introduction: we recently identified two endothelial nitric oxide synthase (enos) gene polymorphisms, a glu asp missense variant in exon and a t- -->c variant in the '-franking region, which are associated with coronary spasm and myocardial infarction in japanese population. and we also identified a missense glu asp variant is associated with severe preeclampsia and placental abruption. our objective was to analyze the association between the t- -->c and severe preeclampsia. materials and methods: the study participants included patients with histories of severe preeclampsia. this is a preliminary study, therefore, the comparisons were made with the general normal population. results: the analyses revealed that the frequency of the missense glu asp variant (n= / , %) was significantly higher than the general population (n= / , %), as we previously published. however, the frequency of the t- -->c variant (n= / , %) was not different from the general population (n= / , %). interestingly, only one patient had both t- -->c and missense glu asp variants, and she developed placental abruption. conclusion: although our sample size is small, it is very unlikely that the t- -->c variant is associated with severe preeclampsia. the t- -->c variant may not be a genetic susceptibility factor to severe preeclampsia. the t- -->c may have some reproductive significance in combination with missense glu asp variant, however huge number of patients would be needed to analyze such rare ( . %) combination of the variance. the association between the development of preeclampsia and methylenetetrahydrofolate reductase, angiotensinogen, vascular endothelial growth factor single nucleotide polymorphism genotype combinations. hyun soo park, jong kwan jun, chan-wook park, joong shin park, bo hyun yoon, hee chul syn. obstetrics and gynecology, dongguk university international hospital, goyang-si, gyeonggi-do, republic of korea; obstetrics and gynecology, seoul national university college of medicine, seoul, republic of korea. objective this study was conducted to investigate if there exists any genotype combination of multiple single nucleotide polymorphism (snp)s which is frequently found in preeclampsia patients. study design one hundred sixty two preeclampsia patients and normotensive pregnant women were included in this study between jan and jul . diagnosis of preeclampsia and assignment of severity were made according to the criteria by national high blood pressure education working group and american college of obstetricians and gynecologists. the patients were reclassified as early ( weeks or before) and late-onset ( weeks or beyond) disease. genotypes were measured with pcr-rflp for methylenetetrahydrofolate reductase (mthfr) c t, angiotensinogen (agt) m t, vascular endothelial growth factor (vegf) c t with the dna extracted from maternal blood. case-control study for each snp was done and the frequencies of genotype combination were compared. anova, t-test, chi-square test, fisher's exact test and logistic regression analysis were used for statistical analysis. a p value of < . was considered statistically significant. results genotypes of mthfr polymorphism showed significant difference between late onset preeclampsia and control (cc+ct/tt, or: . , p< . ) but agt and vegf polymorphism did not show statistical difference between any case-control combination. only out of possible genotype combinations were found and there was no statistical difference in the frequencies of genotype combination between case and control group. conclusion mthfr polymorphism might be associated with the development of preeclampsia, but there was no combination of mthfr, agt and vegf polymorphisms which is associated with the development of preeclampsia. diffuse staining and vascular smooth muscle (vsm) staining. resistance-sized vessels ( - μm) were evaluated. results: for mpo, the intensity of staining (fig) and the % vessels with neutrophil, diffuse and vsm staining was significantly greater for obese than for overweight or normal weight patients: % diffuse staining ( . ± . vs. . ± . vs. . ± . , p< . ); % vsm staining ( . ± . vs. . ± . vs. . ± . , p< . ). for mmp , obese and overweight patients had a greater (p< . ) % vessels with neutrophil, diffuse and vsm staining than normal weight patients: % diffuse staining ( . ± . vs. . ± . vs. . ± . ); % vsm staining ( . ± . vs. . ± . vs. . ± . ). conclusions: neutrophils infiltrate the systemic vasculature of obese women and release mpo and mmp . speculation: obesity may put women at risk for pe because their vasculature may already be dysfunctional due to neutrophil infiltration and release of mpo and mmp . hl . collagen is an important protein that maintains the structural integrity of tissues. disruption of vascular smooth muscle collagen could result in vascular dysfunction in women with preeclampsia. recently, neutrophil infiltration of the systemic vasculature was demonstrated in preeclamptic women. neutrophils produce inflammatory mediators, such as reactive oxygen species (ros) and tnf-. we hypothesized that neutrophils, ros and tnf-would alter expression of collagen regulating genes. methods: primary cultures of human vascular smooth muscle cells (vsmc) were seeded into t- flasks ( , cells/flask) and grown for days to % confluence. the cells were treated for hours with medium control, ros (hx, . mm + xo . u/ml), tnf-( ng/ml); and neutrophils ( , ) activated with arachidonic acid, μm, ( : ratio of neutrophils to vsmc). rna was extracted from cell homogenates and analyzed for gene expression with an rt profiler pcr array system for human extracellular matrix genes (superarray). to determine the fold-change of gene expression, the results were first normalized to a housekeeping gene and then ct was calculated across two rt-pcr arrays where group was the control and group was the experimental treatment. results: table . conclusions: neutrophils, ros and tnf increased mmp expression. interestingly, genes involved in collagen synthesis (col a ) or inhibition of mmp- activity (timp ) were either not affected or down-regulated. these data suggest that neutrophil infiltration in preeclamptic women could cause vascular dysfunction by creating an imbalance between collagen synthesis and collagen breakdown favoring breakdown. hl , fogarty d tw , p md . objective: hypoxia increases membrane attack complex (mac) binding to cultured human trophoblasts, and mac enhances apoptosis in trophoblasts exposed to low compared to normal fio . trophoblast microparticles and cellular fragments released into the maternal circulation in vivo may contribute to the systemic pathophysiology of preeclampsia. we tested the hypothesis that hypoxia induced mac deposition on cultured human trophoblasts yields microparticles and fragments coated with mac. study design: primary cytotrophoblasts from term human placentas (n= ) were cultured h in % and % oxygen in dmem with % human serum with active mac or heat inactivated serum (control). media were centrifuged to obtain pellets of microparticles and cell debris which were immunostained for mac or exposed - h to confluent, phorbol myristate acetate differentiated u macrophages. the percentage of macrophages that ingested trophoblast debris was quantified by counting the number of macrophages with immunofluorescence for trophoblast cytokeratin filament staining, as assessed by confocal microscopy. results: cultures exposed to normal human serum, but not heat inactivated control serum, showed mac immunofluorescence on microparticles and fragments in medium, with the highest level of mac in cultures exposed to extreme hypoxia. the maximal percentage of macrophages that ingested the trophoblast debris coated with mac from cultures with % oxygen was . %, not different from the . % from cultures exposed a % fio and control serum. conclusion: trophoblasts exposed to hypoxia and active complement release microparticles and cellular fragments coated with mac into the extracellular environment. mac coating does not influence phagocytic removal of the debris by macrophages suggesting that placental derived, membrane bound mac could circulate to yield systemic affects on maternal endothelium. supported by nih hd and hd . is there a role for fatty acids in the pathogenesis of pre-eclampsia? nicola j robinson, laura j minchell, jenny e myers, philip n baker, carl a hubel, ian p crocker. maternal and fetal health research group, the university of manchester; obstetrics, gynecology and reproductive sciences, university of pittsburgh. objectives: women with pre-eclampsia (pe) display altered lipid metabolism as characterized by elevated circulating triglycerides and non-esterified fatty acids (nefa) and these changes are evident before the disease is clinically apparent. we have tested the hypothesis that the increased circulating levels of nefa contribute to endothelial dysfunction in pe. methods: human umbilical vein endothelial cells were incubated for h with pooled plasma ( %) from normal or pe pregnancies, or with palmitic, oleic and linoleic acid in culture media at the concentrations and molar ratios to albumin identified in normal ( , , m, ratio . ) and pe pregnancies ( , , m, ratio . ) , . lipid droplet accumulation was determined using an oil red o absorbance assay. endothelial metabolism was measured using the mtt test and mitochondrial membrane potential determined by jc- assay as a marker of early apoptosis. results: plasma from pe pregnancies increased endothelial cell lipid droplet accumulation compared to normal plasma (p< . , wilcoxon signed ranks, n= ). this change was replicated following exposure to nefa at the combined concentrations found in pe compared to normal pregnant controls (p< . , n= ). plasma from women with pe caused a significant decrease in mitochondrial dehydrogenase activity (mtt test; p< . , n= ) and a reduction in jc- fluorescence (p< . , n= ), compared to normal plasma, suggestive of mitochondrial membrane depolarization and increased cellular apoptosis. again these effects were replicated using nefa in culture medium at the levels found in pe compared to normal pregnancies (mtt test: p< . , n= ; jc- assay: p< . , n= ). conclusions: in endothelial cell cultures, plasma from women with pe caused increased lipid droplet accumulation, decreased cellular metabolism and increased apoptosis. these changes to cellular function were mirrored using nefa in culture medium at the concentrations and molar ratios to albumin previously reported in pe. these findings provide evidence that the changes in endothelial cell function induced by plasma from women with pe may be due to the increased nefa circulating levels and that increased palmitic, oleic and linoleic acid, in combination, could play a role in pathogenesis of pe. lorentzen ( ) bjog; endresen ( ) ajog. background: skewing of the maternal endothelial phenotype in pre-eclampsia (pe) is attributed to the release of unknown factors from a hypoperfused placenta. we hypothesise that factors secreted from pe placental tissue will impair endothelial cell function. we have tested the effect of soluble factors by menopausal status, there was a significant increase in bmi in pre-but not in postmenopausal women. in postmenopausal women there was insufficient power to note a statistically significant change in bmi. results are summarized with the follow-up for each group represented by "n" in the graph below. premenopausal patients were divided into hysterectomy with oophorectomy (pbso) versus hysterectomy alone (ph). the ph group showed an increase in bmi that plateaus at time . in the pbso group the bmi continued to increase over time. subgroup analysis comparing ph to pbso demonstrates initial weight loss in pbso but a significant increase in bmi from baseline at time compared to ph. conclusions: hysterectomy appears to be associated with an increase in bmi over time. subgroup analysis suggests that, in premenopausal women, oophorectomy is more strongly associated with continuing weight gain than hysterectomy alone. purpose: obesity is implicated as a key risk factor in chronic disease, but no studies have associated central obesity to the presence of chronic abdominal and/or pelvic pain. we set out to identify the prevalence of chronic abdominal/ pelvic pain in an underserved, primarily latina population by a cross-sectional study in the olive view-ucla outpatient gynecology clinic. we sought to identify an association between the presence and severity of abdominal/pelvic pain and central obesity. methods: nonpregnant women presenting to the gynecology clinic were prospectively evaluated and grouped according to the presence of abdominal/ pelvic pain ('none-mild' or 'moderate-severe' pain). body mass index (bmi) and abdominal circumference (ac) were measured. patients with 'moderate-severe' pain completed standardized questionnaires for pelvic pain and global health scores. results: / ( %) of patients has 'none-mild' pain, and ( %) had 'moderate-severe' pain. pain prevalence was not significantly associated with bmi (mean: 'none-mild' . + . kg/m ; 'moderate-severe' . + . kg/m , p= . ), nor was pain severity (p= . ). pain prevalence was significantly associated with ac (mean: 'none-mild' . + . inches; 'moderate-severe' . + . , p= . ). a borderline positive association exists between ac and pain severity (p= . ). conclusions: we demonstrate an association between both the presence and severity of chronic abdominal/pelvic pain and central obesity, independent of bmi. ac appears to be a more relevant factor than other traditional measures of habitus in patients with this chronic malady. to improve preventative care in women's health management, further evaluation of the role of central obesity in the pathogenesis of chronic pain is necessary. aims: vulvitis is one of the most frequently diagnosed gynaecological infections. we aim to assess the efficacy against infective vulvitis of a new topical medical device containing low molecular weight hyaluronic acid (lmw-ha). the ability of this molecule to stimulate -defensin release in keratinocytes has been recently shown. methods: we report preliminary data regarding women suffering from infective vulvitis, as assessed by a gyneacologist: patients were randomly selected to receive sphg ( patients), a cream containing low-molecular weigh hyaluronic acid, or vehicle ( patients). patients were asked to apply the cream to the vulva twice-daily for days. at the end of treatment, evaluation of efficacy, tolerability and acceptability of the cream was assessed by a specific questionnaire. results: preliminary results show that patients receiving sphg report a significative improvement of vulvitis symptoms, in terms of itch, redness of the skin and burning, in comparison to vehicle. sphg also showed a good tolerability, cosmetic acceptability and symptomatic relief perceived by patients. conclusion: sphg seems to be efficacious in ameliorating the symptoms of infective vulvitis. this activity may be probably related to the lmw-ha presents in this formulation: in fact, low molecular weight hyaluronic acid has been recently shown to induce -defensin production by human keratinocytes. since this peptide exerts antimicrobial and antimicotic activity, the improvement of symptoms assessed in patients receiving sphg might be linked to a reduced infective charge, attributable to the activity of -defensin . background: allogeneic hematopoietic stem cell transplant (hsct) is a treatment used for many malignant and nonmalignant diseases of the bone marrow and immune system. hsct may be complicated by chronic graft-versus-hostdisease (cgvhd) in to % from matched unrelated donors. genital cgvhd complicates about % of hsct and may uncommonly result in labial fusion. case: year old woman with a history of ewing's sarcoma and acute myelogenous leukemia, had received chemotherapy and total body irradiation (tbi) followed by a matched unrelated donor hsct. menarche occurred at years of age after normal pubertal development. she menstruated regularly until cancer diagnosis. premature ovarian failure resulted after chemotherapy and tbi and oral contraceptive pills were used for hormone replacement. after transplant, she developed chronic gvhd involving the skin, eyes, mouth and joints, and concomitantly complained of vulvar pruritus. she was presumed to have a yeast infection which was treated with fluconazole without a pelvic exam. she was evaluated by a gynecologist when she was unable to insert a tampon. pelvic exam revealed dense labia minora adhesions from the clitoris to urethral meatus and posteriorly leaving a cm opening at the urethra. pelvic mri revealed a normal uterus and ovaries. after weeks of topical estrogen cream, the adhesions remained dense and were lysed under general anesthesia. vaginal examination revealed pale, minimally rugated, normal mucosa. cervical cytology was normal. post-operatively she used daily topical estrogen and hydrocortisone creams. at months after surgery, her urinary stream was stronger. on pelvic exam, the labial opening was cm, but a small posterior forchette adhesion elicited severe pain. after using dilators coated with topical steroids and estrogen, she was able to insert a tampon. conclusion: genital gvhd should be considered in women with genital tract complaints after hsct. labial fusion secondary to chronic gvhd may be treated successfully with surgery and medical therapy. support: rbmb/nichd/nih. dana r ambler, mazen e abdallah, rahi victory, michael p diamond, elizabeth e puscheck, jay m berman. obstetrics and gynecology, wayne state university/detroit medical center, detroit, mi, usa. objective: to determine which factors are predictive of a ruptured ectopic pregnancy, and whether endometrial stripe thickness can be used as an alternative to such criteria in the diagnosis of an ectopic pregnancy. design: retrospectively collected ectopic pregnancy database. setting: detroit medical center, detroit, michigan. patients or participants: women with a diagnosis of an ectopic pregnancy were studied, with surgically confirmed tubal rupture cases. interventions: abstracted data included hcg(iu), gestational age (days), presenting symptoms of pain and/or bleeding, hemoglobin (hgb), hematocrit (hct), historical risk factors, ultrasound-determined ectopic size, endometrial stripe thickness (mm), amount of cul de sac fluid (cds), tubal rupture at time of surgery, and estimated blood loss (ebl)(ml). covariates included demographics. results were significant when p< . . results: chi square analysis revealed that there is a relationship between endometrial stripe thickness and hcg levels. logistic regression models demonstrated that endometrial stripe thickness was not predictive of ectopic rupture, (or . , p= . ) . however, logistic regression, both forced and forward stepwise analysis, demonstrated that hcg (or= . , p< . ), a large volume of cul-de-sac fluid (or= . , p< . ), and increasing pain (or= . , p= . ) were associated with increased risks of rupture. gestational age, and ectopic related risk factors were also not predictive of rupture. conclusions: endometrial stripe thickness is not a useful predictor for the diagnosis of a ruptured ectopic pregnancy. serum hcg measurement, cds fluid volume and the presence of pain are much stronger diagnostic indicators of ectopic pregnancies. clinical profile of migraineurs in a university hospital gynecology department in japan. [objectives] the changes in hormonal milieu associated with menarche, pregnancy, menopause, and post-menopause are frequently accompanied by changes in the patterns and frequency of migraine. little is known on the relationship of women's issues of migraine though the balance between estrogen and progesterone is critical in the elimination of migraine. our aim was to investigate the relations among the prevalence of migraine, the reproductive stage, and gynecologic diseases. [materials and methods] female patients (average age: . years old) who consulted a physician and agreed to answer about the questionnaires during september, -june, . migraineurs were diagnosed with the migraine screener by the japanese headache medical treatment promotion committee in . and the patients answered questionnaires that screened about menopausal disorder and abnormal menstruation at once. they were conducted a survey in the form of a questionnaire and sometimes were taken blood samples. [results] in patients had migraine ( . % average age: . years old). prevalence were . % in one's twenties, higher than another ages. most patients with migraine was complicated by menstruation disorders (premenstrual syndrome %, dysmenorrhea . %), sterility ( . %), and severe menopausal disorder ( %). when trh and the lh-rh test were examined for the sterility patient, migraineurs had higher prolactin basal level and lh level after lord but lower fsh level before and after lord than nonmigraineurs. on the other hand, the prevalence of migraine for postmenopausal women and women who had gynecology cancer treatment was low, and there was no relation between migraine and pregnancy history. [conclusions] this study provides migraine headache is influenced by reproductive stage and that women with migraine are frequently complicated by menstruation disorder. it is thought that migraine account for abnormality of hormone milieu including abnormality of the hypothalamus-pituitary system. objective: this study evaluated the efficacy of doses of estradiol (e ) gel . % (divigel ® ), a novel formulation of e consisting of mg e per g transdermal gel, to reduce frequency and severity of vasomotor symptoms and signs of vulvar and vaginal atrophy (vva) associated with menopause. design: postmenopausal women were evaluated in a -week study comparing placebo to e gel . % at doses of . g/day, . g/day, and . g/day with estimated nominal daily deliveries of . mg, . mg, and . mg of e respectively. endpoints included mean change from baseline in daily frequency and severity of moderate to severe hot flashes (msvs). vaginal ph and % superficial cells were collected at baseline and end of study. results: e gel . % showed statistically significant improvements from placebo gel as early as week (table) that were maintained throughout treatment. signs of vva (vaginal ph and % of superficial cells) showed statistically significant improvements from baseline with all doses of e gel . % compared to placebo. conclusion: e gel . % significantly decreased the frequency and severity of msvs at all doses evaluated in this trial. e gel . % offers multiple dosing options to individualize patient therapy, including the lowest effective dose that was studied ( . mg e , delivering . mg e /day), to treat vasomotor symptoms associated with menopause. estradiol (e ) gel . % (divigel ® ) is a novel formulation of e consisting of mg e per g transdermal gel for the treatment of vasomotor symptoms associated with menopause. safety and tolerability of e gel . % were evaluated in a large placebo-controlled trial. design: postmenopausal women participated in a -week study comparing placebo to . g/day, . g/day, and . g/day of e gel . %. circulating e and estrone (e ) concentrations were measured. safety analyses included the incidence of adverse events (aes) and clinical laboratory evaluations, including plasma levels of sex hormone binding globulin (shbg). application site tolerability was assessed using the draize scale. results: all doses of e gel . % produced physiologic e :e ratios similar to those seen in premenopausal women. e :e increased from a baseline mean of . to . , . , and . with, respectively, the . , . , and . g/day doses of e gel . %. the most frequently reported aes were breast tenderness and postmenopausal bleeding that appeared to be dose-related and would be expected with increased circulating estrogen concentrations. there were no remarkable changes in hematology, blood chemistry, urinalysis, lipid, coagulation, and carbohydrate values following treatment with e gel . %. shbg levels remained unchanged after weeks of treatment at all doses. the vast majority of patients had no evidence of skin irritation throughout the treatment period. mean draise scale scores after , , and weeks of treatment were . for all treatment groups except for a mean value of . ± . for the . g dose group after weeks of treatment. conclusion: e gel . % is a safe and well-tolerated therapy for the treatment of menopausal symptoms. design: pharmacokinetic parameters, dosing, efficacy, and safety information for divigel ® , elestrin ™ , evamist ™ , estrogel ® , and estrasorb ® were obtained from current prescribing information (obtained from manufacturer websites) and the data were compared. results: together, these new transdermal therapies offer multiple dosing/ delivery options and contain a wide range of e ( . mg to . mg), with the lowest systemic daily delivery of e attained by the divigel ® . g dose. following weeks of treatment, across the dosing options, each treatment significantly reduced both the frequency and severity of moderate to severe vasomotor symptoms (msvms) compared to placebo. change in frequency of msvms ranged from - . (divigel ® . g) to - . (estrasorb ® ); change in severity scores for msvms ranged from - . (divigel ® . g) to - . (divigel ® . g). all treatments are safe and well tolerated. breast tenderness was the only adverse event reported in % of subjects, occurring with all therapies. conclusions: current treatment guidelines recommend using the lowest effective dose of estrogen for the treatment of menopausal symptoms. this side-by-side comparison of the recently available e non-patch transdermal options to the standard transdermal therapies is meant to assist physicians in individualizing treatment for their patients. introduction: cdb- (cdb) is a relatively new progesterone receptor modulator being clinically evaluated for contraception and treatment of fibromas. its use in an intrauterine device/system (ius) has not been reported. in this study we prepared cdb-filled intrauterine devices (cdb-ius) and evaluated their effects on endometrial growth and bleeding patterns in rhesus macaques. methods: short ( . - . cm) lengths of silastic tubing (od . mm), either empty (n= ),or filled with silicone rubber matrix containing % of micronized cdb (n= ), were inserted into the uterine lumens of ovariectomized rhesus macaques. animals were induced to cycle by sequential treatment with systemic estradiol and progesterone (p) as reported (brenner et al, ann ny acad, ) . after . cycles, at the end of the follicular phase, the uterus was removed and processed. results: when systemic p treatment was withdrawn at the end of each cycle, animals with empty ius menstruated normally, while animals with cdb-ius bled little or not at all. over the whole . cycles, animals bearing a blank ius bled for an average of . ± . days while cdb treated animals bled for an average of only ± . days. at the end of treatment, animals exposed to blank ius had mean endometrial wet weights of ± mg while the cdb-treated endometria weighed only ± mg. the proliferation markers ki- and phospho-h were substantially lower in the cdb-ius treated than in the blank treated animals. histologically, the cdb exposed endometria were atrophied with evidence of glandular degeneration while the blank controls were proliferative and normal. summary: in cycling rhesus macaques, a cdb-ius prevented progestational development, blocked menstruation after p withdrawal, and suppressed endometrial proliferation. if these effects are confirmed in women, the cdb-ius could provide estrogen-free and bleed-free contraception, and could help control heavy menstrual bleeding. supported by rr , hd and the population council. introduction: irregular uterine bleeding is a major side effect and cause for discontinuation of ltpoc use. while endometria of ltpoc-exposed women display abnormally enlarged, fragile blood vessels (bv), decreased blood flow and evidence of oxidative stress, the mechanisms by which structural and vasomotor endometrial dysfunction occurs remains unknown, in part by the difficulty of manipulating hormone levels in women. the aims of this study were ) to validate the guinea pig (gp) as a model to study uterine effects of ltpoc and ) to investigate ltpoc-effects on endometrial histology and oxidative stress markers. methods: oophorectomized gps were implanted s.c. with time release pellets containing either placebo (crl,n= ); estradiol (e ,n= ); medroxyprogesterone acetate (mpa,n= ) or e +mpa (n= ). after days, uterine horns were weighed and frozen or paraffin embedded. angiogenesis was assessed by quantitative image analysis of vonwillebrand factor staining and included bv density and size. oxidative stress was detected by isoprostane and -oh-deoxyguanosine ( oxog). apoptosis was investigated by the tunel method. statistical analysis was by -way and -way anova. results: gp uteri were enlarged by both e (p< . ) and mpa (p= . ). effects of mpa on uterine weight differed significantly depending on e levels (p< . ), where mpa opposed the e effect in combined treatments. angiogenesis parameters were similarly impacted upon. thus, mpa alone increased bv density (p= . ) and bv average area (p= . ). the presence of e significantly decreased these parameters (bv density mean± sem: crl: . ± . %, e : . ± . %, mpa: . ± . %, e +mpa: . ± . %, p= . ). these changes were associated with highly elevated -isoprostane content in e +mpa-treated uteri compared to all other groups (p< . ). abnormalities in the e +mpa group were consistent with chromatin redistribution, nuclear pyknosis, karyolysis and increased nuclear oxog staining and a marked increase in tunel labeling. conclusions: ltpoc exposure alters endometrial vascular and tissue morphology consistent with oxidative stress and apoptosis in a complex interplay with endogenous estrogens. the gp is an excellent model for the study of ltpoc effects on the uterus. physiologic and psychological symptoms associated with injectable and oral contraceptive use. abbey b berenson, susan odom, carmen r breitkopf, mahbubur rahman. ob/gyn, utmb, galveston, tx, usa. objective: to compare physiologic and psychological symptoms over mo among users of depomedroxyprogesterone acetate (dmpa), an oral contraceptive with micrograms ethinyl estradiol (oc), and non-hormonal (nh) contraception. methods: a total of women reported the presence of symptoms prior to initiating contraception ( dmpa, oc, nh) and every mo thereafter for mo. longitudinal relationships between symptoms and contraceptives (reference: nh), as well as race/ethnicity (non-hispanic black, non-hispanic white, and hispanic) were assessed by gee-analysis after adjusting for age, visits and baseline status of symptoms. persistence, resolution, and new development of symptoms were noted by method in mo increments for mo and compared with that of nh controls. the gee analyses showed that oc was protective against mastalgia (or= . ), cramping (or= . ), hair loss (or= . ), acne (or= . ), nervousness (or= . ) and mood swings (or= . ). when race was considered, oc was protective for all women against acne, for whites against mastalgia and cramping, and for non-whites against nervousness. for whites, it was a risk factor for bleeding between menses. oc use resulted in resolution of acne within mo in nearly % of those with it at baseline, but no significant resolution after mo. also mastalgia ( % at baseline) was less likely to persist at and mo. bleeding between menses was reported for the first time at mo by % of oc users. dmpa users of all races had an increased risk of missed periods (or= . ), bleeding between menses (or= . ), bleeding > d (or= . ) and loss of libido (or= . ) relative to nh users. it reduced cramping (after mo) and bloating (all mo). racial differences were observed with dmpa protective against mastalgia and mood swings in whites, cramping in whites and hispanics, and bloating in non-whites. it was a risk factor for loss of energy in whites only. neither method affected depressive symptoms. conclusion: side effects of these two methods are mostly related to abnormal bleeding. very low dose pills can be protective against symptoms (mastalgia, cramping, hair loss, acne, nervousness and mood swings) commonly associated with pills while dmpa protects against cramping and bloating. knowledge about racial differences will allow physicians to individualize therapy. counseling should include that some symptoms can develop after mo while resolution often occurs within mo. methods: twenty-four women were enrolled in irb approved prospective, randomized, cross-over trial comparing months of oc (ortho cyclen) vs. tc ortho evra). the daily oc administers micrograms of ee; the weekly tc contains . milligrams of ee. each treatment was followed by months of washout and months of the alternative contraceptive. blood was drawn at baseline and final week of treatment for each arm of the study. ee was quantified by ria, with preceding organic solvent extraction and celite column partition chromatography. data were analyzed by t test with boferroni's correction, p < . . results: after two months of treatment the mean (+/-sem) ee levels for tc = . pg/ml (+/- . ) and oc = . pg/ml (+/- . ). the ee level is not significant different for the two medication (p = . ). conclusions: there is no difference in ee levels with the use of these oral and transdermal contraceptives. this suggests the transdermal contraceptive, despite the lack of the hepatic first pass effect, has similar levels of ethinyl estradiol compared to oc. the continuous elevation in ee seen with the tc route of administration, versus the episodic increases seen with the oc route, raises concerns of constant exposure to ee. this may explain the increased risk of estrogen-induced thrombotic events with this tc route of administration. impact of paracervical block, in combination with general anesthesia, on post-abortion pain. gweneth b lazenby, tod aeby. obstetrics and gynecology, university of hawaii, honolulu, hi, usa. objective to evaluate the impact of paracervical block with a long-acting local anesthetic, in conjunction with general anesthesia, on post-operative pain. methods a power analysis determined patients per arm were needed to demonstrate a significant difference of in mean pain scores. seventy-two patients were allocated to one of two arms using urn randomization. all patients received standardized anesthesia; intravenous sedation for gestational age under weeks and general anesthesia for over weeks. thirty-nine patients were randomized to receive a paracervical block with . % bupivacaine and thirtythree were randomized to no local anesthesia prior to surgical abortion. patients completed visual analog scales for pain and anxiety prior to the procedure, upon awaking, and minutes post-operatively, and prior to discharge. data were analyzed using an anova and students t-test the experimental and control groups were equivelent in age, ethnicity, gravidity, parity, prior abortions, prior vaginal deliveries, prior c-sections, gestational age, number of laminaria, pre-operative and intra-operative dilation, operative time, estimated blood loss, and reported complications. pain and anxiety were not significantly affected by placement of a paracervical block. these data do not support the hypothesized benefit of local anesthesia, prior to surgical abortion under general anesthesia, to reduce post-operative pain. we do not recommend the routine use of a paracervical block to decrease post-operative pain. age, parity, history of abortion and contraceptive choices affect the risk of repeated abortion. oskari heikinheimo, mika gissler, satu suhonen. ob gyn, university of helsinki, helsinki, finland; national research and development centre for welfare and health, helsinki, finland. objective the rate of repeat abortion varies from to % in northern europe. however, risk factors for repeat abortion are poorly understood. we characterized risk factors (demographic, as well as those related to abortion and postabortal contraception) of repeat abortion. design a prospective cohort study of women undergoing medical abortion between august and december . the subjects were followed by means of finnish registry on induced abortions until december ; the follow-up time (mean ± sd) was . ± . months. results altogether ( . %) of the subjects requested repeat abortion within the follow-up time. in univariate analysis previous abortion, parity, young age, smoking and failure to attend the follow-up visit were associated with increased risk of repeat abortion. immediate -in contrast to postponed -initiation of any contraceptive method was linked to lower risk of repeat abortion. in comparison to combined oral contraceptives, use of intrauterine contraception was most efficacious in reducing the risk of another pregnancy termination. in multivariate analysis the effects of young age, parity, previous abortion and type of contraception on the risk of another abortion persisted. conclusions increased focus on young, parous and those with the history of an abortion may be efficacious in decreasing repeat abortion. contraceptive choices made at the time of abortion have an important effect on the rate of reabortion. postabortal use of intrauterine contraception, specifically that of lng-ius, might decrease the rate repeat abortion. objective. to determine the rate of failure and to analyze factors associated with failure for the essure permanent birth control device at the detroit medical center (dmc). methods. a chart review was conducted on patients who underwent essure placement at the dmc from january through june . patient demographics, past medical and surgical history, anesthesia type, procedure time, intraoperative complications, and procedure failures were noted. data were analyzed for statistical significance using spss. results. there were essure procedures attempted at the dmc from january through june . of the attempted procedures, there were failures ( . %). of the failures were attributed to difficulty visualizing the ostia ( %). other causes of failure included expulsion of the device ( ), tubal spasm ( ), uterine perforation ( ), and tubal ostia too large for the device ( ). there were cases of failed placement for undocumented reasons, one case requiring a laparoscopic tubal ligation secondary to postprocedure tubal patency, and post-procedure pregnancies. age, race, body mass index, gavidity, parity, history of sexually transmitted infections, medical history, history of cesarean section, tobacco or illicit drug use, anesthesia type, and physician experience with the procedure were not significantly associated with placement failure or difficulty visualizing the ostia. a longer procedure time was significantly associated with failure ( . vs . min, p = . ), and history of ectopic pregnancy was significantly associated with difficulty visualizing the ostia ( . % vs . %, p = . ). conclusion. the failure rate for placement of the essure permanent birth control device at the dmc is . % with a pregnancy rate of . %. the majority of failures may be attributed to difficulty visualizing the ostia. a history of ectopic gestation was significantly associated with difficulty visualizing the ostia; thus, it may be reasonable to advise these women that success in essure placement may be reduced. introduction. the essure permanent birth control device is a relatively new form of minimally invasive sterilization for women. under hysteroscopic guidance, a dynamically expanding micro-insert is introduced into the proximal portion of the fallopian tube. the micro-insert induces local fibrosis and ultimately occlusion of the tubal lumen. a hysterosalpingogram (hsg) is performed three months after the procedure to confirm bilateral tubal occlusion. objective. to determine the follow-up rate for the post-essure hsg for a clinic population. methods. a retrospective chart review was conducted on university health center (uhc) patients who underwent placement of the essure permanent birth control device at the detroit medical center from january through june . follow-up for the post-essure hsg as well as the result of the hsg were noted for each patient. results. placement of the essure permanent birth control device was attempted in uhc patients of which were successfully completed. of the patients, ten underwent a post-essure hsg ( . %). the hsg was performed three to six months after placement of the essure permanent birth control device. bilateral tubal occlusion was documented in all ten patients. conclusion. despite counseling patients prior to their procedure that a hsg is needed and providing an information sheet, the follow-up rate for the post-essure hsg for this clinic population is only . %. for those in whom a hsg was performed three to six months after essure placement, bilateral tubal occlusion was confirmed in all. steps and or approaches to improve compliance with post-procedure confirmation of tubal occlusion should be employed to increase follow-up in the future. towards fibroids gene therapy: adenovirus mediated delivery of herpes simplex virus thymidine kinase gene/ganciclovir shrinks uterine leiomyoma in the eker rat model. memy hassan, dong zhang, salama salama, cheryl walker, hala el-mazar, ayman al-hendy. ob/gyn, utmb; md anderson; ob/gyn, meharry medical college, nashville, usa. aim: assessment of the efficacy of gene therapy of uterine leiomyoma in the immune-competent eker rat model using adenovirus mediated delivery of herpes simplex- -thymidine kinase gene followed by ganciclivir treatment (ad-tk/gcv). method: female eker rats with mri-confirmed uterine fibroid lesions were randomized to a single treatment with direct intratumor injection of ad-tk/gcv, ad-lacz/gcv, or medium.the tumor volume was evaluated by serial mri scanning and confirmed with caliper measurement at time of euthanasia. sample rats were selected randomly and killed at the following time points; , and days post treatment. samples were collected from tumors, other body organs and blood to assess the safety and efficacy of the treatment. results: ad-tk/gcv treatment produced dramatic shrinkage of the total uterine fibroid volume by % ± , % ± and %± of pretreatment volume at days , and respectively. the tumor size in negative control animals receiving ad-lacz/gcv continued to grow by + %± , + %± , + % ± while receiving media continue to grow by + % ± , + % ± and + % ± at same time points. ad-tk/gcv induced significant increase in caspase activity, bax expression, decrease in bcl and parp proteins expression and increased tunnel apoptosis index. additionally ad-tk/gcv treatment decreased cyclin d and pcna expressions. ad-tk/gcv did not produce any significant change in liver function tests or relative uterine horns weight to total body weight. the adenovirus transfection did not disseminated significantly to other distal organs except to liver and myometrium in limited number of animals. h e staining of non targeted organs did not revealed any sign of tissue damage. ad-transfection increased local cd and cd expressions as well as serum anti-ad antibodies. conclusion: ad-mediated delivery of hsv tk gene by direct intra-tumor inoculation followed by sc treatment of gcv for ten days effectively shrinks uterine leiomyoma lesions in eker rats. this effect is mediated via induction of apoptosis and decreasing the proliferation. the treatment regimen is well tolerated. these studies provide essential preclinical data for the development of gene therapy as an alternative non-surgical treatment option for women with symptomatic uterine fibroids. inhibitors of catechol o-methyl transferase shrinks uterine fibroids in the eker rat model. memy hassan, dong zhang, hala el-mazar, cheryl walker, ayman al-hendy. ob/gyn, utmb; md anderson; ob/gyn, meharry medical college, nashville, usa. background :the sex hormone dependent pattern of uterine leiomyomas and their high content of catechole -o-methy transferase (comt) raise the possibility for the development of novel treatment option using comt inhibitors.aim : to assess the potential therapeutic utility of a synthetic comt inhibitor (ro - ) in the eker rat model of uterine leiomyoma. methods female eker rat were evaluated by mri to confirm the prescence uterine fibroid lesion, then randomized for sc treatment with ro - mg /kg ,twice/ day for days versus vehicle injection.fibroid tumor burden was evaluated by serial mri measurement and confirmed by direct caliper measurement at time of euthanasia. sample animals were euthanized at and weeks. at that time tumor tissue, blood and most of animal organs including long bones were collected and subjected for further evaluations. hours urine samples were collected for evaluation of estrogen (e ) metabolites and bone resorption marker. results:animals treated with ro - exhibited significantly lower uterine fibroid tumor burden ( % and %) of pretreatment volume at and weeks post treatment respectively. conversely, the tumor size in control animals continued to grow and reached %, and % of pretreatment size at the same time points. ro - treatment resulted in an increase in urinary / hydroxy e metabolite ratio. in addition ro - increased bax expression and decreased parp , pcna and cyclind experssions . all ro - treated animals tolerated the treatment protocol with no signs of toxicity. h e staining of different body organs did not reveal any signs of tissue damage. furthermore, there was no significant change in both liver function tests (alt, ast, billirubin) and bone resorption marker , deoxypyridinoline croslinks, between treated and control rats. conclusion ro - , a synthetic selective comt inhibitor, caused immediate arrest of the growth of eker rat uterine leiomyoma. this effect might be in part due to modulation of various estrogen dependent genes regulating leiomyoma apoptosis (parp, bax) and proliferation (pcna, cyclin d ). this anti-estrogenic effect is due to the accumulation of the the antiestrogenic metabolite hydroxy estrogen secondary to comt inhibition.comt inhibitors might present an alternative non-surgical option for the treatment of women with symptomatic uterine fibroids. background development of uterine leiomyomas (fibroids) is the most common pathological feature in the female reproductive tract. they negatively impact patients of virtually every gynecologist. despite such morbidity, leiomyoma development is poorly understood. we have recently demonstrated that leiomyomas have a genomic expression pattern that limits retinoic acid (ra) exposure. our group and others have demonstrated that tgf-beta regulation is altered as well. these two pathways likely play central roles in leiomyoma development. the central feature of uterine leiomyomas, the extracellular matrix (ecm), is regulated by both all-trans retinoic acid and tgf- , focusing on versican as a critical ecm component. human uterine leiomyoma and patient-matched myometrium were obtained from surgical specimens under an irb-approved protocol. these tissues were immortalized and treated with either all-trans retinoic acid, tgf- , or anti-tgf- antibody. rna was isolated for qrt-pcr. human immortalized leiomyoma cells demonstrated the same increased template expression of tgf- ( . ± . fold), retinoic acid metabolizing protein (cyp a ; . ± . ), and versican variant v ( . ± . fold) as was found in the progenitor tissue. when treated with all-trans ra, expression of versican variant v decreased to levels found in myometrial cells ( . ± . fold). conversely, when treated with tgf- , expression of versican variant v increased . ± . fold. to confirm that tgf- was central to the overexpression of v , we treated leiomyoma cells with anti-tgf- antibody, and found that baseline over-expression of v template was decreased to expression levels similar to untreated myometrial cells. finally, we elucidated a link between the ra and tgf-pathways by assessing the impact of ra treatment of tgf- expression, demonstrating that tgf- template decreased to levels comparable to myometrial cell expression ( . ± . fold). the disrupted leiomyoma ecm, of which versican is a central component, defines the leiomyoma phenotype. in this study, the leiomyoma fibrotic phenotype regressed when treated with ra and increased when treated with tgf- , providing the basis for novel therapeutic interventions directed at cell differentiation and ecm formation. objectives: uterine fibroids are the leading cause for hysterectomies in the us. the lack of an appropriate in vitro cell model for the initiation of fibroid growth has hindered advancement in understanding the cellular and molecular basis for the development and progression of uterine fibroids. fibrosis is the underlying mechanism of uterine fibroid formation and myofibroblasts cells are the principal fibrogenic cell type in the uterus. we sought to develop a myofibroblast in vitro cell model for analyzing the initiating molecular events of uterine fibrosis. methods: smooth muscle cells (smcs) were enzymatically isolated from the myometrium of non-pregnant women and cultured in the presence of % serum until % confluent. for the next h cells were cultured in serum-free media followed by replacement with serum containing media. cells were fixed at , , , , m later. cell fine structure and cytoskeletal organization were evaluated by transmission electron microscopy. smooth muscle specific alpha-actin ( -sma) and progesterone receptors (pr) were detected by western blot. results: we observed smc differentiation into myofibroblasts, marked by the presence of notched nuclei ( figure) and the increased expression of -sma m after serum replacement. pr-a and pr-b were detectable at , and m. conclusions: the development of myofibroblasts is important in wound healing and fibrosis. we show for the first time that uterine myofibroblasts can be derived in culture from myometrial smcs. thus, these cells will be utilized as a model for developing "in vitro fibroids". this model will enable the study of myofibroblast activation, cytokine signaling, intracellular regulation of uterine fibrogenesis, production of extracellular matrix proteins and development of antifibrotic drugs. the presence of prs in our model enables us to evaluate pr mediated events in fibroid pathogenesis and treatment. this model will be more useful in determining the molecular biology of fibroid initiation than cell models derived from established fibroids that are already well past their initial stages of development. novel approach to genome-wide expression profiling analysis. liping feng, morgan walls, insuk sohn, millie behera, sin-ho jung, phyllis leppert. obgyn; biostatistics and bioinformatics, duke university medical center, durham, nc, usa. background: microarray studies have examined the differential gene expression between uterine fibroid and normal myometrium. all previous studies considered the fibroid as a whole and analyzed only fold changes. we have developed a novel statistical approach to genome-wide expression analysis comparing two fibroid tissue sites to myometrium. methods: using affymetrix tm u a genechip, we have compared the gene expression between c and e and matched adjacent m. data has been analyzed by considering the specimens per subject and subjects as individuals. we used a block one-way anova method to test if each gene was differentially expressed among the three sites. the p-value is calculated using a permutation method accounting for possible dependency among three lesions. the multiple testing issue was addressed by controlling the false discovery rate. expression values were calculated using the robust multichip average (rma) method. rma estimates are based upon a robust average of background corrected perfect/mismatch (pm) intensities. normalization was done using quantile normalization. expression values were then transformed by taking logarithm base . confirmatory rt-pcr was performed. results: we applied a hierarchical clustering analysis to all raw data sets and then displayed a dendrogram, where the height of each branch point indicates the similarity level at each generated cluster. identical gene expression among sites clusters together. due to a strong site effect, m tissues clustered separately from e and c combined. genes were differentially expressed when we used a . q-value cut off. expression data revealed concordant changes in genes regulating cholesterol biosynthesis, gene transcription, estrogen and extracellular matrix formation when both e and c were examined. cyp was detected and we report for the first time that scc- (a cell cycle-regulated molecule) folliculin and l-selectin are differentially expressed suggesting that they may be involved in the regulation of cell growth and proliferation of uterine fibroids. conclusions: our novel robust analysis of gene expression provides new clues to the relevant pathways of fibroid development. this new statistical approach that can be used in clinical and/or translational studies to identify differentially expressed genes comparing treatment regimens, cells or tissues. objectives: thrombospondin- (tsp- ) is a large matricellular glycoprotein secreted by many cell types. matricellular proteins modulate interactions between cells and their environment, regulate cell adhesion and are expressed during tissue formative processes. they are especially important in fibrosis. tsp- plays an important role in angiogenesis and is an activator of tgf - . in a previous study, we found that differential expression of tsp- in uterine fibroids may contribute to an altered healing process leading to fibrosis. this alteration in tissue response to injury initiates the development of abundant, nonaligned collagen fibrils and changes in other components of the ecm. methods: we measureed the pattern of mrna and protein expression of tsp- by rt-pcr and western blot in an in vitro serum-deprived differentiated myometrial cell (myofibroblast) model. specifically, smooth muscle cells (smcs) were enzymatically isolated from the myometrium of non-pregnant women and grown in primary culture to % confluence. then smc were serum deprived for h and treated back with % serum for , , , and m. cells were collected for rna and protein, and tsp- expression was evaluated. in addition, cells were stained using the combination of anti-cd and anti-smooth muscle -actin or combination of anti-cd d and anti-smooth muscle -actin as well as the appropriate single and double negative controls. stained sections were analyzed using zeiss axio imager widefield fluorescence confocal microscopy. results: tsp- mrna and protein was present in cells in this serumstarvation model after the addition of serum and the expression level remained elevated for m following the addition of serum. fluorescence staining analysis indicated that these cells were positive for human smooth muscle -actin, but negative for leukocyte antigen cd and platelet marker cd d suggesting that the myofibroblasts cells themselves were the source of the tsp- . conclusions: unlike skin wounds, where tsp- is derived from the blood macrophages, monocytes and platelets, differentiated myometrial cells appear to produce tsp- . elucidating the roles of tsp- in myometrium physiology and pathobiology will increase our understanding of the etiology of uterine fibroids and may lead to improved therapies. further studies utilizing this cell model to determine the role of tsp- in the activation of tgf - are indicated. phospholipid s p via gi, rac and rho pathways. yoel smicun, armando wu pimentel, jennifer gilman, david a fishman. obstetrics gynecology, new york university school of medicine, new york, ny, usa. objectives: sphingosine- -phosphate (s p) levels are elevated in serum and ascites of ovarian cancer patients. we have demonstrated that low concentration s p enhances while high dose s p inhibits invasion of epithelial ovarian carcinoma (eoc) cells in a dose and attachment mode dependent manner. we sought to further dissect the pathways by which s p affects invasion, using specific inhibitors. methods: dov eoc cells were pretreated for -hrs with vehicle, . m or m s p and with inhibitors for gi, p -mapk, rac and rock, thereafter cells were detached and tested for invasion towards m lpa chemoattractant in matrigel-coated chambers. conditioned media from pretreated cells and invading cells were quantified for upa activity using colorimetric assays. the significance of results was calculated by student's t-test. results: inhibition of gi mildly increased invasion of both control ( p= . ) and m s p treated cells ( p= . ). inhibition of both p -mapk and rac did not affect m s p treated cells, in contrast invasion of control cells was mildly increased (p= . , . ). inhibition of rock, a protein effector downstream of rho, highly elevated invasion of both control and m s p treated cells ( fold, p= . , fold, p= . ). both upa and gelatinase activities were higher in conditioned media of invading cells than of attached cells. gelatinase activity was enhanced by both concentrations of s p (p= . , . ). ptx fully inhibited gelatinase activity of control and . m s p treated cells, and partially of the m s p treated cells (p< . ). . m s p significantly increased upa activity of attached (p= . ) but not of invading cells. this increase was sensitive to ptx and rac inhibitor. m s p inhibited upa in both attached and invading cells (p= . ), this inhibition was rock dependent. conclusions: these findings suggest a strong inhibition of invasion and upa by the rho pathway, of both control and m s p treated cells. this inhibition is induced partially by upstream gi protein. increased invasion by . m s p is associated with elevation of gelatinase activity through gi, rac and rock pathways. this suggests that attached cells and invading cells affect upa activity through different pathways. objectives: sphingosine- -phosphate (s p) levels are elevated in serum and ascites of epithelial ovarian cancer (eoc) patients. we have demonstrated that invasion of attached eoc cells differentially react to s p as compared to invading cells. we examined the impact of the inhibitors for gi and rac on attached and invading eoc. methods: dov eoc cells were pretreated for -hrs with vehicle, . m or m s p and with inhibitors for gi (pertussis toxin (ptx)), and rac (nsc , (rac-i)), and cells were detached and assayed for invasion towards m lpa in matrigel-coated chambers. to distinguish the response of attached from invading cells, inhibition of cells pretreated with inhibitors was either continued or not in the invasion chambers. conditioned media (cm) of invading cells were quantified for upa and gelatinase activity by fluorometric and colorimetric assays. significance of results was calculated by student's t-test. results: the significant (p= . ) increase of invasion by . m s p was inhibited by both ptx and rac-i, either by pretreatment alone or by continuous treatment (p= . - . ). however, the invasion was higher in cells inhibited continuously than cells inhibited only in dishes. both inhibitors did not affect cells treated with m s p. control cells invasion was increased ( . fold, p= . ) by continuous rac-i treatment. . m s p increased gelatinolysis in cm of invading cells. this and control cells activity was inhibited by ptx pretreatment. continuous treatment with ptx or rac-i elevated and fold gelatinase activity of control and m s p treated cells. in contrast upa activity was inhibited by both . m and m s p. activity of control cells was inhibited by both pretreatment and continuous treatment with both ptx and raci. upa activity of cells treated with . m s p was increased only by pretreatment with ptx and raci. in contrast, in cells treated with m s p upa was inhibited only by continuous inhibition with ptx and raci. conclusions: invasion of s p induced eoc cells correlated with the gelatinase and upa activities in their microenvironment. ptx and rac-i affect attached and invading cells in different manner, inhibition of invasion and gelatinolysis of attached and increased invasion of invading cells. this suggests a dual effect of the gi-rac pathway, inhibiting attached and stimulating invasion via gelatinolysis of invading cells. lysophosphatidic acid (lpa) levels are elevated in the ascites and plasma of early-and late-stage ovarian cancer patients, underscoring the unique role this bioactive lipid plays in the pathobiology of epithelial ovarian cancer (eoc). lpa binding to its cognate g-protein-coupled receptor can transactivate the receptor tyrosine kinase, egfr, which is often overexpressed in ovarian tumors. in the current study, we investigated the role that lpa activation of egfr plays in the processing of the metalloproteinase, mmp- , and eoc dissemination. lpa stimulates tyrosine phosphorylation of egfr in ovarian cancer cells, and egfr kinase activity is required for optimal lpa induction of pro-mmp- activation. lpa-dependent pro-mmp- activation does not require the egfr ligands, amphiregulin, b-cellulin, egf, hb-egf, and tgf-a. we previously reported that when cells are cultured at high density, lpa represses rhoa activity to induce loss of stress fibers, and these changes in actin microfilament organization contribute to pro-mmp- activation. in the current study, inhibition of rho-kinase/rock with y- reversed the repression of lpa-stimulated pro-mmp- processing observed with treatment of the egfr kinase inhibitor, ag . correspondingly, lpa induced the loss of stress fibers, while inhibition of egfr kinase restored stress fiber formation in lpa-treated cells. this suggests that lpa acts through egfr to modulate microfilament organization and pro-mmp- processing. finally, lpa-induced cellular haptotactic migration and invasion are abrogated when egfr kinase activity is blocked. taken together, these results suggest that egfr signaling plays a critical role in lpa regulation of metastatic pathways by mediating changes in the cytoskeleton which impact protease activity. objectives: membrane-derived vesicles are active modulators of tumor dissemination; they promote and contribute to extracellular matrix degradation and tumor cell invasion. we have isolated vesicles from ascites of ovarian cancer patients and from ovarian cancer cells in vitro. here we analyzed the functional consequences of exposure of cancer cells to vesicles derived from a different type of malignancy in order to evaluate their proinvasive properties. methods: ovarian cancer (dov ), breast cancer (mda mb ) and mesothelioma (hp- ) cells were grown in media supplemented with % fbs. after serum deprivation, % vesicle-free fbs was added for hr to induce vesicle release. vesicles were isolated from media by differential centrifugation and quantified with a bradford assay. fusion to cells was followed by fluorescence of the lipophilic tracer dii. each cell line was stimulated with and g/ml of each type of vesicles and tested in a matrigel invasion assay. changes in proliferation were evaluated in an mts assay. gelatin zymography was used to assess matrix metalloproteinase activity of vesicles. results: zymographic analysis showed that vesicles contained mmp- and . fusion experiments showed that all vesicles fused to all cell types. all vesicles induced the invasion of their respective cell types in a dose-dependent manner. when vesicles were used at g/ml they induced significantly high levels of invasion in all cell types tested. at g/ml they significantly inhibited invasion in different cell types. both concentrations of vesicles stimulated cell proliferation in all cell types tested. conclusions: membrane derived vesicles are potent mediators of invasion for mesothelioma, breast and ovarian cancer cells in vitro. this effect occurs in a dose-dependent manner when vesicles induce invasion of the same cell type from which they were isolated. when vesicles are used to induce a different cell type, low concentrations induce invasion while high concentrations inhibit invasion, this effect was independent of cellular proliferation. these results suggest that a novel mechanism may be at play where activation of recognition of self and non-self specific pathways may determine the invasive potential of the tumor cell upon fusion to microvesicles. the identification of signaling pathways responsible for this heterotypic signaling is a current effort. vegfr- as a potential target to inhibit lpa-induced epithelial ovarian cancer (eoc) invasion. sonia dutta, fengqiang wang, elaine barfield, david a fishman. obstetrics and gynecology, new york university school of medicine, new york, ny, usa. objective: vegf and vegf receptors (vegfrs) play important roles in ovarian cancer metastasis. in this study, we examined the expression profiles of vegf and vegfrs (vegfr , vegfr , co-receptor nrp and nrp ) in established eoc cells lines (dov , r , ovca , skov ) and an immortalized normal ovarian epithelium (iose- ). the effect of lysophosphatidic acid (lpa) on vegf and vegfrs expression and the effect of vegfr- silencing by rnai on lpa-induced invasion were also evaluated. methods: vegf and vegfrs expression in ovarian cancer cell lines and normal ovarian epithelium was quantified by real time pcr. cell invasion differentiate into either a pro-tumor or anti-tumor phenotype depending on the specific tumor microenvironment. previously, we described a subgroup of epithelial ovarian cancer (eoc) cells with a functional tlr- -myd -nf-b pathway (type i eoc cells). these cells have constitutive nf-b activity and constitutively secrete il- , il- , mcp- , and gro . we hypothesize that type i eoc cells, but not type ii, can promote macrophage differentiation into a tumor-supporting immune cell. methods: eoc cell conditioned media (cm) was prepared by incubating eoc cells in log-phase growth in optimem for h. migration assay was done using an in vitro cell culture insert with m-size pore and pkh red fluorescentlabeled thp- . cytokine profile of thp- cells co-cultured with eoc cell cm was determined using xmap technology. modulation of response to apoptotic bodies was determined by "pre-educating" thp- cells with eoc cell cm for h prior to exposure to apoptotic bodies ( : ratio with thp- cells). results: monocytes migrate toward eoc cells. however, migration is significantly higher towards type i eoc cells. type i, but not type ii, eoc cells alter monocytes' cytokine profile by inducing the secretion of high levels of progrowth and angiogenic cytokines il- , il- , mcp- , and gro . furthermore, type i eoc cells modify monocytes response to apoptotic bodies by inducing a significant increase in the secretion of il- , il- , mip- , mip- , and gro ( -fold, . -fold, -fold, -fold, and -fold respectively). conclusion: we demonstrate for the first time a differential interaction between two subtypes of eoc cells and monocytes. we showed that the microenvironment created by type i eoc cells is able to modify the function and differentiation of immune cells towards a tumor supporting phenotype. understanding the molecular mechanisms mediating this tumor-immune interaction will help to design appropriate immune therapies that will take into consideration the tumor microenvironment. objectives: the standard of treatment for patients with ovarian cancer (oc) is intravenous combination chemotherapy (ct) after primary cytoreductive surgery. although initial response is above %, most of these patients experience recurrence. the only approach for these patients is salvage ct which may prolong their lives for months. early detection of patients who are not responding to current therapy or are at risk of experiencing an early relapse of disease might improve response rates and survival if alternative therapy is possible. no single predictive marker has yet been proven sufficiently sensitive or specific to find a place in the daily clinic. in the present study we use a newly described biomarker test for the detection of oc (visintin et al clinical cancer research) and evaluated the ability of the test to monitor ct response methods: patients with oc, figo stage i-iv, were included in this study. all patients recieved postsurgery first line combination ct (paclitaxel/carboplatin). samples were evaluated at ) baseline (mean days after surgery), prior to the first cycle of ct, and ) after cycles of ct. μl serum samples were analyzed by multiplex assay (beadlyte® cancer biomarker panel kit) for six markers. changes during ct and differences in markers between patients with short time to progression (ttp) and patients with long ttp were determined. results: positive test for oc was observed in % of the patients evaluated at baseline. all patients had residual disease after surgery. from patients with long ttp, / patients (specificity %) had a negative test after cycles. from the patients with short ttp, / had a positive test (sensitivity %) p= . ( ²). the risk of experiencing a ttp shorter than months when having a positive test after cycles of ct was %. conclusion: we describe for the first time the use of a panel of biomarkers for oc that might have an application for monitoring ct response and risk of relapse. the test detects the presence of residual disease following debulking surgery, and differentiates between long term and short term progression. a longitudinal study is performed to determine how early during ct the test can identify responder versus non responder. ksp inhibitor (arry- ) as an alternative for paclitaxel in myd -positive epithelial ovarian cancer cells. ki h kim, ayesha b alvero, yanhua xie, david trollinger, gil mor. obstetrics, gynecology, and reproductive sciences, yale university school of medicine, new haven, ct, usa; array biopharma inc., boulder, co, usa. background: we previously described that epithelial ovarian cancer (eoc) cells ubiquitously express tlr- , but not the adaptor protein myd . when treated with paclitaxel, a known tlr- ligand, myd -positive eoc cells exhibited nf-b activation, increased secretion of il- , il- , mcp- , and gro , and increased p-erk levels . since majority of eoc patients are given paclitaxel in combination with a platinum drug, it is not only important to distinguish those patients that should not be given paclitaxel; it is also important to identify alternative chemotherapy agents that would benefit this sub-group of patients. the objective of this study is to determine if the ksp inhibitor, arry- , can be an alternative for paclitaxel in myd -positive ovarian cancer patients. methods: myd -positive and myd -negative eoc cell lines isolated from either ascites or tumor tissue were treated with increasing concentrations of arry- ( to nm) or paclitaxel ( . to m) for , , and hours and cell viability was determined using the celltiter aqueous one solution cell proliferation assay. cytokine profiling was performed from supernatants using xmap technology. nf-b activity was determined using a luciferase reporter system. p-erk levels were measured by western blot analysis. results: arry- and paclitaxel exhibited the same cytotoxic effect on myd -negative and positive eoc cells. the ic at h for myd -negative eoc cells was . m and . um for arry- and paclitaxel, respectively. for myd -positive eoc cells, the ic at h was m and m for arry- and paclitaxel, respectively. however, unlike paclitaxel, arry- did not induce nf-b activation or enhance the secretion of il- , il- , mcp- , and gro , and did not induce erk phosphorylation on myd positive cells. conclusions: administration of paclitaxel to patients with a myd -positive tumor could have detrimental effects due to the paclitaxel-induced enhancement of cytokine production which promotes chemoresistance and tumor growth. arry- has similar anti-tumor activity in eoc cells as that of paclitaxel. however, unlike paclitaxel, it does not induce cytokine production in myd positive eoc cells, and therefore, the ksp inhibitor arry- may represent an alternative to paclitaxel in this subgroup of eoc patients. introduction: nf-b activation has been associated with cell proliferation, angiogenesis, metastasis and suppression of apoptosis in ovarian cancer. in addition, nf-b activity induces the production of pro-inflammatory cytokines which may contribute to chemoresistance. inhibition of nf-b may represent a new approach to prevent tumor growth and reverse chemoresistance. in the present study we described the characterization of a novel nf-b inhibitor, eriocalyxin b (erib) that is able to re-establish the apoptotic cascade in chemoresistant ovarian cancer cells by suppressing pro-inflammatory cytokines and antiapoptotic proteins. materials and methods: eoc cell lines were isolated from malignant ovarian cancer ascites. caspase activity was determined by caspase-glotm assay. cytokine production and secretion were determined using multiplex assay. erib effect on cancer cells was evaluated in a time and dose dependent manner using celltiter cell proliferation assay. protein expression was determined by western blot analysis. combination studies were done with paclitaxel and carboplatin in addition to erib treatment. nf-b activity was determined by monitoring the expression of a nf-b luciferase reporter. results: erib decreased cell viability of ovarian cancer cells with an ic of . - μm in hours and was associated to increasing levels of caspases , and activity. intracellular changes induced by erib included: ) inhibition of nf-b activity; ) decrease in cytokine production; ) down regulation of anti-apoptotic proteins xiap and flip, and reversal of resistance to tnf-and fasl-mediated cell death; and ) chemosensitization to carboplatin and paclitaxel. at present, evidence is accumulating regarding the existence of unique populations of specialized tumor-initiating, stem-like cells within various tumor types of distinct origins. these cancer stem cells (csc), with characteristics reminiscent of normal stem cells, are thought to be responsible for driving tumor growth. we propose that ovarian cancers arise from csc, and are using microarray-based technology to identify specific genes/cell surface markers associated with ovarian csc that will permit distinction of these rare cells from the remaining tumor bulk. to identify unique gene signatures associated with an ovarian tumor-initiating cell population, we have utilized an in vivo serial transplantation model. this model selects for primary human ovarian tumor cells with increased tumorigenic capacity, given that time to tumor formation decreases with successive serial transplant despite fewer cells injected. our initial studies used cells derived from a human ovarian clear cell carcinoma, serially transplanted for three passages in nod/scid mice. rna derived from these transplanted tumors was analyzed on human genome microarrays. from these analyses, several differentially expressed genes were identified. the differential expression noted for potential genes of interest is currently being validated by rt-pcr. of particular interest, expression of the transmembrane glycoprotein muc was found to increase both at the rna and protein level with successive transplant of this clear cell carcinoma. further studies are ongoing to determine the functional significance of muc and other identified differentially expressed genes in ovarian clear cell cancer. in addition, we are carrying out parallel microarray analyses in other ovarian tumor subtypes. background recent observations suggest a decreased incidence of neoplastic lesions in hiv infected individuals treated with highly active anti-retroviral therapy comprised of protease inhibitors, such as ritonavir. the polycomb group protein ezh is associated with aggressive human malignancies via transcriptional suppression of dna repair proteins. objective objective of the present study was to assess the antineoplastic impact of ritonavir on epithelial ovarian cancer (eoc) cell lines. methods eoc cell lines (mdah and skov- ) were treated with serial dilutions of ritonavir ( - mm) dissolved in dmso. normal diploid human fibroblasts served as controls. growth curves, apoptosis and cell cycle analysis were performed with cell counting kit- , annexin v and flow cytometry. signal transduction was studied with western blotting. dna double strand breaks (dsb) were induced with . mm etoposide treatment for hours. homologous recombination (hr) repair of dna damage was measured by assessment of rad foci formation in the nucleus of the cells as visualized by fluorescence microscopy according to previously published criteria. untreated eoc cells expressed higher levels of ezh and lower levels of rad and xrcc as compared to controls and in turn, had lower prevalence of rad repair foci formation in response to dsb. serial treatments of eoc cells with ritonavir resulted in a decrease in expression of ezh and an increase in expression of rad and xrcc when compared to untreated eoc cells. after induction of dsb, the rad repair foci formation was significantly more prevalent in eoc cells treated with ritonavir as compared to untreated eoc cells. in addition, ritonavir induced apoptosis in ovarian cancer cell lines by down-regulation of akt pathway and caused g cell cycle arrest mediated by down modulating levels of prb phosphorylation and depleting the cyclindependent kinase and . ritonavir effectively induces apoptosis, cell cycle arrest and improves repair of dna damage by hr in ovarian cancer cell lines. as impaired hr is a key event in causation and progression of neoplastic lesions, ritonavir may have a role in chemoprophylaxis and treatment of human malignancies. a rare case of ovarian cystic lymphangioma. tomer singer, tamer seckin, noa feldman, susan jormark, michael divon. department of obstetrics and gynecology, lenox hill hospital, n.y., ny, usa; department of pathology, lenox hill hospital, n.y., ny, usa. cystic lymphangioma (cl) is a rare, benign malformation of the lymphatic system whose exact etiology remains uncertain. cl may arise in different sites: the spleen, the mediastinum, the axillary region, the retroperitoneum, and the mesentery. retroperitoneal cl is extremely rare and its true incidence is unknown. the majority of cases are symptomatic during childhood. clinical presentation of adult cl is variable and may be misleading. typically, this is a slow-growing tumor and it remains asymptomatic for a long period of time. it is most often found incidentally during abdominal or pelvic imaging studies, surgeries or autopsies. total surgical removal of the lesions with microscopically clear margins is the best approach when it is possible. we report, for the first time, a case of cystic lymphangioma arising from the ovary in a post-menopausal woman and present the feasibility and the advantages of laparoscopic surgery, allowing accurate diagnosis, optimal treatment and minimal risk for the patient. lgsc) is a chemoresistant ovarian neoplasm that has been molecularly linked to low malignant potential tumor (lmp), which often behaves in a non-invasive fashion. micropapillary features within lmp (lmp-mp) are associated with increased invasive behavior. the aim of this study was to determine the differential gene expression of lmp, lmp-mp, and lgsc to identify genes involved in malignant transformation and carcinogenesis. methods: laser capture microdissection was used to isolate epithelial cells from snap-frozen lmp (n= ), lmp-mp (n= ) and lgsc (n= ). rna was extracted, amplified, reverse transcribed to cdna and hybridized to affymetrix u plus arrays. data was analyzed by significance analysis methods: medical records were reviewed for patients who underwent hysterectomy for all indications at wsu hospitals from / / - / / . pathology reports were reviewed to identify the incidence of adenomyosis. data obtained from medial records included: age, race, insurance, bmi, social history, medical history, and presenting symptoms. the correlation between adenomyosis and all the above factors was tested using the appropriate statistical methods. results: patients were included. adenomyosis was confirmed by pathology in patients, an incidence of . %. incidence was not significantly different among races after controlling for parity. . % in african americans, . % in caucasians, and . % in others. incidence was not statistically different between uninsured( . %), privately insured ( . %), and medicaid ( . %) patients. p=. . incidence of adenomyosis was not different between smokers ( . %) and nonsmokers ( . %). p= . . table i shows the correlation between adenomyosis and different risk factors. conclusion: adenomyosis was diagnosed in . % of hysterectomy specimens. race, socioeconomic status or social habits didn't affect its incidence. diabetes and endometrial cancer were negative risk factors for adenomyosis, whereas htn, hypothyroid, breast cancer, fibroids, polyps and endometriosis didn't affect its incidence. menorrhagia, dysmenorrhea, dyspareunia, and chronic pelvic pain but not metrorrhagia had a positive correlation with adenomyosis. there is a protective adipocytokine profile. we hypothesize that this finding may precede some ill-defined threshold of fat mass and/or insulin resistance after which adiponectin decreases. the objective: to correlate reproductive hormone production with menstrual bleeding patterns among women in the menopause transition. methods: a sub-cohort of the swan study consisting of women age - was studied. each woman collected daily first void urine samples for one complete menstrual cycle or days (whichever came first) once a year for years. urine was assayed for excreted levels of fsh, lh, estrogen metabolites and progesterone metabolites which were normalized for creatinine concentration. ovulation was detected by a validated algorithm. menstrual bleeding parameters were derived from daily calendars. correlations between bleeding characteristics, hormone concentrations, and other potential clinical predictors were analyzed using multivariate logistic regression models. results: the cohort was ethnically diverse with a median age of and with % in the early perimenopause at the start of the study. % of all cycles were anovulatory. short cycle intervals (< days) were associated with the early perimenopause (or . , ci . , . ) and with anovulation (or . , ci . , . ). long cycle intervals ( + days) were associated with late perimenopause (or . , ci . , . )and with anovulatory cycles (or . , ci . , . ). both short ( - days) and long ( + days) duration of menstrual bleeding were significantly associated with anovulation (or . and . , respectively). women with anovulatory cycles were less likely to report heavy menstrual bleeding than women with ovulatory cycles. menorrhagia was not associated with steroid hormone concentrations but was associated with obesity (or . , ci . , . ) and with the self-reported presence of fibroids (or . , ci . , . ). conclusions: among women in the menopause transition, abnormalities in timing of menstrual bleeding (cycle intervals or bleeding duration) have a hormonal basis and are frequently associated with anovulation. in contrast, abnormally heavy periods do not have a hormonal basis and are less likely following anovulatory cycles. heavy periods are associated with obesity and fibroids. to undetectable levels. the use is vaginal e is contraindicated, although these women have even higher rates of av. topical testosterone (tt) has successfully treated vulvar atrophy; testosterone receptors are also present in the vaginal epithelium. objective: assess the effect of tt on vaginal maturation index (mi) and relief of av symptoms. methods: postmenopausal women on aromatase inhibitor with symptomatic atrophic vaginitis were enrolled in prospective, irb approved study. estradiol (e) and testosterone (t) levels, av questionnaires (score - ; none to most severe symptoms of dryness, irritation, and dyspareunia), gynecologic exam, and vaginal smears (for ph and vaginal maturation index, mi, by meisels criteria) were performed at baseline and after month of daily treatment with mcg of tt. data was assessed by t test and fischer's exact test; significant p < . . results: e levels were undetectable at baseline and following treatment. t levels increased (mean +/-sem) from baseline ( +/- . ) to treatment ( +/- . ); the difference was not significant; p = . , although one patient had an appreciable rise in serum t level. two av symptoms improved significantly with tt use; comparing baseline to treatment scores: vaginal dryness ( . vs. . ; p = . ) and dyspareunia ( . vs. ; p = . saliva analysis is a convenient, non-invasive and rapid method for assessing estradiol (e ) levels. however, particularly in postmenopausal women, the low salivary e levels are often near or below the sensitivity of available assays, compromising both accuracy and precision. we present results using an extraction step prior to e assay, which concentrates the sample to increase sensitivity and removes potentially interfering substances. morning saliva samples were obtained from premenopausal (mid-luteal phase, n= , ) and postmenopausal women (n= , ) not taking hormones, and from postmenopausal women receiving oral conjugated equine estrogens (cenestin, n= ; premarin, n= ), oral micronized e (estrace, n= ; compounded e , n= ), transdermal e patches (climara, n= ; vivelle, n= ); or topical e cream (compounded e , n= ). e levels were determined by an automated enzyme immunoassay (eia) after solid phase extraction. the functional sensitivity of the assay was determined to be . pg/ml, compared with > pg/ml without extraction. . . . . . oral conjugated equine estrogens; oral e ; e patch; topical cream salivary e levels corresponded with the hormone dosage, suggesting a reliable assessment of unbound e levels with each formulation, dosage and type of estrogen therapy. extraction prior to eia in an automated assay dramatically increased precision and accuracy at low concentrations. omitting the extraction step may have contributed to poor serum versus saliva correlations in other studies. this method may therefore allow reliable monitoring of estrogen therapy without the need for expensive and inconvenient blood tests. the role of fibulin- in pelvic organ prolapse. david d rahn, jesus f acevedo, patrick w keller, lihua marmorstein, r ann word. ob-gyn, ut southwestern, dallas, tx, usa; visual sciences, univ arizona, tuscon, az, usa. objectives: fibulin- (fib- ) is crucial for normal elastic fibers in the protein showed a statistically significant reduction in its expression (p= . ). lox, loxl , and proteins were detected by immunohistochemistry in all three layers of vaginal skin biopsies: ( ) stratified squamous epithelium; ( ) the lamina propria and ( ) the muscularis layer from both patients with pop and controls. significantly, in both groups we detected a numerous macrophages scattered throughout the vaginal thickness which displayed a very strong immunostaining to loxl . patients with severe pop showed reduced expression of proteins regulating collagen and elastin biogenesis. our finding raises the possibility that failure of ecm homeostasis could underlie the etiology of pop in women. fibroblast proliferation is regulated by hoxa : molecular implications for pelvic organ prolapse. kathleen a connell, marsha k guess, richard bercik, hugh s taylor. obstetrics, gynecology reproductive sciences, yale university school of medicine, new haven, ct, usa. objectives: the integrity of the extracellular matrix (ecm) is maintained by a delicate balance between collagen synthesis and degradation. previously, we have demonstrated that hoxa is essential for the development of the uterosacral ligament in mice and regulates the expression of collagen type iii and mmp . we have also shown that hoxa is deficient in the uterosacral ligament of women with pelvic organ prolapse (pop) compared to women with normal support. the exact mechanisms by which hoxa regulates pelvic organ integrity and repair remains to be elucidated. the aim of this study was to determine the effect of hoxa expression on fibroblast proliferation, and its potential role in pop. methods: nih t cells, a murine fibroblast cell line, were seeded onto a six well plate ( x cells/well) and transfected with either a vector carrying a hoxa cdna or empty vector alone as a control. immunohistochemistry using bromodeoxyuridine (brdu) was performed to evaluate cell proliferation. cell division in the uterosacral ligament (usl) was also compared in women with and without pop. usl specimens were obtained at the time of hysterectomy for benign disease. immunohistochemistry was performed on paraffin embedded sections of the usl to evaluate expression of two mitotic markers, cyclin b and phospho-histone . results: constitutive expression of hoxa in murine fibroblasts resulted in significantly higher proliferation. cells transfected with hoxa had a mean brdu incorporation of . + . cells/ cells compared with . + . cells/ cells in controls (p= . ). in the usl obtained from women with and without pop, cell proliferation as determined by cyclin b and phospho-histone expression was not significantly different. cyclin b and phosphor-histone were expressed in comparable numbers of cells in both groups. conclusion: hoxa is necessary for usl development and promotes proliferation of adult fibroblasts. hoxa may have a similar function in vivo in usl, and may regulate fibroblast proliferation during growth and the acute phase response following trauma when fibroblasts are activated to proliferate and remodel the ecm. it is likely that hoxa mediated proliferation of usl fibroblasts contributes to the tensile strength and resilience of these structures and thereby prevents pop. supported by nichd wrhr: k hd - . connective tissue composition, in term of collagen i, iii and proteoglycans content, in support and nonsupport structures of women with uterine prolapse. elisabetta trabucco, gennaro acone, sara d'avino, marco torella, gennaro trezza, annamaria marenna, nicola colacurci, luigi cobellis. department of obstetrics and gynecology, second university of naples, naples, italy; loreto mare hospital, naples, italy. objective. connective tissue consists mainly of collagen and elastic fibers, glycoproteins and proteoglycans (pgs) and is considered an important factor of the support and nonsupport structures of the genitourinary region. it has been already demonstrated altered morphologic features in the pelvic support connective tissue in women with genital prolapse. however, analysis of nonsupport tissue may provide a more accurate reflection of body collagen. the objective of our study was compare the expression of collagen i, iii and four pgs (decorin, fibromodulin, lumican, biglycan), essential for synthesis and regular assembly and diameter of the collagen fibrils, in uterosacral ligaments and in a nonsupport tissue, the uterine cervix, between premenopausal women with uterine prolapse respect to age matched controls. we characterized uterosacral ligaments and uterine cervix of premenopausal women with uterine prolapse and controls. this immunohistochemical study was performed on paraffin-embedded sections. results. uterosacral ligaments of the prolapsed uteri are characterized by a higher expression of collagen iii, decorin, fibromodulin and byglican and a lower quantity of collagen i. no differences in the immunoreactivity of lumican between the two patients groups. the abnormalities of support connective tissue composition are not observed in the uterine cervix of patients with prolapse. conclusions. our results suggest an altered remodeling of connective tissue in the ligaments of premenopausal patients with prolapse, with a significant decrease in collagen i content and an increase in collagen iii and pgs expression . in the prolapse patients this abnormal collagen metabolism and organization, mainly related to the observed change in pgs expression, might affect significantly the tensile strength of the connective tissue and consequently the support that is provided by the suspensory apparatus to the uterus. the alterd remodeling of support tissues, not detectable in nonsupport tissues, may suggest a predominant role of local biomechanical stresses (childbirth, chronic straining) in the pathogenesis of prolapse respect to systemic connective tissue deficiency. objective: to achieve vaginal delivery with minimal maternal injury, vaginal smooth muscle (sm) contractility likely decreases. the objective of this study was to characterize changes in rat vaginal sm contractility in pregnancy that affords circumferential vaginal distension. methods: a tubular segment of the vagina of virgin, late pregnant , immediate and late post vaginal delivery rats were mounted in an organ bath between a force transducer and an adjustable support block. dose response curves to norepinephrine (ne) and potassium (k+) were used to assess contractility. relaxation capacity was determined in ne preconstricted vagina, using cumulative doses of sodium nitroprusside (snp). differences in active vs. passive length-tension curves were used to measure sm contractility relative to the vaginal wall forces. sm -actin levels were measured using quantitative confocal immunofluorescence. results: cumulative doses of ne induced a maximum constriction force of . ± mn/g in virgin vagina while no measurable force was generated in response to ne in late pregnant or postpartum vaginas. virgin vagina relaxed with cumulative doses of snp; no measurable relaxation response was observed from pregnant or postpartum animals. in the presence of the high dose k+ ( mm), virgin vagina generated the greatest contractile force ( . ± mn/g) vs. late pregnant vagina ( . ± . mn/g, p< . ) or immediate post partum vagina ( . ± mn/g, p= . ). four week postpartum k+ induced forces were similar to virgin levels ( . ± mn/g). sm force generation (difference in active vs. passive length tension curves at mm of displacement) was decreased in late pregnancy ( . ± mn/g) compared with virgin ( ± mn/g, p < . ) and week postpartum vagina ( ± mn/g, p < . ). the expression of sm -actin was lowest in late pregnancy ( ± . , p < . ) and immediate postpartum vagina ( ± . , p < . ) relative to virgin ( ± ) with a return to virgin levels week postpartum ( ± ). conclusions: vaginal sm contractility diminishes in pregnancy. the altered contractility is mirrored by a decrease in sm -actin protein expression. near complete recovery to pre-pregnancy levels occurs by weeks post partum. these functional and biochemical changes likely represent maternal adaptations designed to minimize trauma during passage of the fetus(es). background: diagnosis or exclusion of endometriosis usually requires laparoscopy and peritoneal biopsy. we have described a novel and consistent observation of small nerve fibers in the functional layer of eutopic endometrium in women with endometriosis (tokushige et al, ) . these nerve fibers are not present in women without endometriosis. this finding allows the possibility endometriosis in the nude-mouse and inhibited mmp- expression in human endometrial stroma. the present findings may lead to the development of novel treatments of endometriosis involving statins. supported by nih u hd . k-ras actived immunocompetent retrograde menstruation model of endometriosis. ching-wen cheng, , di licence, , cristin g print, , d stephen charnock-jones. , pathology, university of cambridge, cambridge, united kingdom; obstetric gynaecology, university of cambridge, cambridge; department of molecular medicine pathology, university of auckland, auckland, new zealand. objective: to establish a new murine model of endometriosis that mimics the retrograde menstruation theory using immuno-competent mice. method: ovariectomised donor female k-ras v +/-/cre +/+ /rosa r-lacz +/+ transgenic mice were treated sequentially with steroid hormones. to induce decidualization and activation and k-ras transgene, mg/kg b-nf dissolved in maize oil was injected into the uterine lumen. tissue degeneration mimicking menstruation was induced by hormone withdrawal. menstruating endometrial tissue was collected from donor mice hours after last hormone treatment, re-suspended in matrigel, and implanted subcutaneously in female c bl/ recipients. immunohistochemical and morphometric methods were used to characterize the endometriosis-like lesions. microarray analysis (illumina, n= in each group), was used to study the molecular changes in "menstruating" uteri following k-ras activation. result: simple transplantation of decidualised endometrial tissue into immunocompetent animals does not lead to endometriotic lesion development but using tissue with the genetic modification described here overcomes this. viable endometriosis-like lesions are visible days after implantation. these lesions are histologically similar to those seen in man with intact glands, functional blood vessels, leukocyte infiltration and collagen deposition. statistical analysis revealed that transcripts were differentially regulated in the ras activated and control tissue. gene ontology (go) analysis indicated that transcripts associated with epithelial cell function and differentiation were over represented within this gene set (chloride transport and epidermis development) as were those associated with the acute inflammatory response and neutrophil chemotaxis. we developed a new model of endometriosis using immunocompetent mice to mimic retrograde menstruation induced endometriosis. this permits for the first time, the ready use of transgenic and knock-out tools to investigate the cellular and molecular mechanisms underlying endometriosis. since the animals have an intact immune system it also allows the testing of therapeutic agents that modify the inflammatory response. stra is expressed and induced by progesterone in the endometrium and is absent in endometriosis. mary ellen pavone, serdar e bulun, scott s reierstad, joy innes, magdy p milad, you-hong cheng. obstetrics and gynecology, northwestern univeristy, chicago, il, usa. objective: retinoic acid (ra) plays important roles in development, growth and differentiation by regulating the expression of target genes. in the mouse endometrium, ra deficiency leads to hyperkeratinization, while high concentrations of retinoids promote secretory characteristics. this leads many to believe that ra mediates important actions of progesterone in the endometrium and may account for progesterone resistance in endometriosis. the mechanism for regulation of ra production by progesterone is unknown. moreover, the conversion from retinol to retinoic acid is not different between normal endometrium and endometriotic tissue. we hypothesize that retinol intake into cells rather than conversion of retinol to ra is the critical step that determines ra activity in the endometrium and endometriosis. stra , a widely expressed multitransmembrane domain protein and member of a group of "stimulated by retinoic acid" genes, has been identified as the retinol binding protein receptor. it is strongly expressed in adult mice in several tissues including the female genital tract. we hypothesize that ra activity in the endometrium may be regulated by stra . here we investigate the differential expression of stra in the endometrium and endometriosis. design: we studied primary stromal cells isolated from the eutopic endometrium of disease free women and walls of ovarian endometriomas from women with endometriosis with respect to stra expression and regulation by progesterone. materials and methods primary culture of eutopic endometrial and endometriotic stromal cells were treated with - m estradiol (e ), - m r , a progesterone agonist, or vehicle for hours. real-time pcr was employed to quantify stra mrna levels. we treated cells for hours and quantified protein levels by immunoblotting. results basal mrna of stra levels in endometrial cells (n= ), were > , fold higher than those in vehicle-treated endometriotic cells (n= , p<. ). in endometrial stromal cells (n= ), r stimulated stra mrna levels by . - fold. r induced stra protein levels in endometrial stromal cells (n= ). conclusions stra is highly expressed and regulated by progesterone in endometrial stromal cells, whereas it is practically undetectable in endometriotic cells. stra may mediate important actions of progesterone in endometrium. upregulation of aromatase expression in endometrial cells disseminated into the peritoneal cavity (pc) may influence their survival and persistence via local estrogen synthesis. the inducing factors for the upregulation of aromatase in endometriosis are not well defined, but increased expression of sf- has been suggested to play an important role. given that the aromatase substrate androstenedione (a ) is the predominant steroid in peritoneal fluid, we aimed to determine whether a has a role in the regulation of aromatase expression in human endometrium. we found that culture of primary endometrial stromal cells and explants with a ( - nm) for h significantly upregulated expression of aromatase mrna transcripts containing exon iia at their '-ends. moreover, in human endometrial surface epithelial cells (hes), dose-response studies with a ( - nm) revealed that nm a maximally upregulated expression of both aromatase and sf- . when tissue samples were evaluated from women with endometriosis and control endometrium, expression of aromatase mrna mirrored sf- . treatment of hes cells ( h) with tritiated a demonstrated its metabolism to estradiol (e ), testosterone (t), dihydrotestosterone (dht) and androstanediol (a-diol). although equimolar concentrations of a , t and e upregulated aromatase and sf- mrna expression in hes cells, the nonaromatizable androgens, dht and a-diol, had no effect. a positive feedback role of estrogen in aromatase upregulation was suggested by the finding that the estrogen receptor antagonist, ici , , markedly diminished aromatase and sf- mrna expression induced by a . finally, chromatin immunoprecipitation revealed that a significantly enhanced recruitment of sf- to its response element (- bp) upstream of cyp exon iia. collectively, our findings strongly suggest that exposure of endometrial cells within the pc to c -steroids may cause an acute upregulation of cyp gene expression through their aromatization to estrogens. thus, estrogen may play a critical positive feedback role in the pathogenesis of endometriosis. supported by nih r -dk . the hpa axis and depression/anxiety scores in chronic pelvic pain patients. b stegmann, b frank, j gemmill, g chrousos, m ballweg, p stratton. rbmb, nichd/nih, bethesda, md; som, wake forest university, winston-salem, nc; endometriosis association, milwaukee, wi. stress, pain, anxiety and depression adversely affect the hypothalamic-pituitaryadrenal (hpa) axis. we examined the influence of depression and anxiety on the hpa axis response to corticotropin-releasing hormone (crh) testing in women with and without chronic pelvic pain (cpp). methods: healthy women, aged - , with without cpp, with regular menses and off hormonal contraception were studied. none had pelvic infections, untreated depression, manic depression, fibromyalgia, or chronic fatigue syndrome. after ovine crh injection ( mcg/kg), serial blood samples (- , , + , + , + minutes) were obtained for acth and cortisol measurements. depression and anxiety were scored using the duke quality of life questionnaire. hpa response was abnormal if peak acth levels were > pg/ml without a rise in cortisol levels. subject groups were: no pain and normal acth response (npnr), pain and normal acth response (pnr), no pain with an abnormal acth response (npar) and pain with an abnormal acth response (par). student t-test was used for comparisons. results: women ( cpp, controls) had a mean age of . ± . years (range: - ). women responded normally ( pnr, npnr) and women responded abnormally ( par, npar). peak and absolute rise in acth were significantly higher in abnormal (a) vs normal (n) responders; (peak: . a vs . pg/ml n, p< . ; absolute: . a vs . pg/ml n, p= . ). there was a significant difference within groups: peak acth: . par vs . pg/ml pnr, p= . ; . npar vs . pg/ml npnr, p< . ; absolute acth: . par vs . pg/ml pnr, p= . , . npar vs . pg/ml npnr, p< . ). however, total cortisol level was not different between or within the groups ( . a vs . mcg/dl n, p= . ). mean depression score did not differ among cpp patients ( . par vs . pnr, p= . ), but differed among controls ( npar vs . npnr, p= . ). anxiety scores did not differ between or within groups ( . npar vs . npnr p = . ; par vs . pnr, p= . ). conclusions: chronic pelvic pain is associated with an abnormal hpa response, regardless of anxiety or depression symptoms. abnormal hpa responses in control women, however, appear to be influenced by depression. the mechanism and clinical significance of these findings should be explored. support: rbmb/nichd/nih and endometriosis association. objective: the eutopic endometrium in women with endometriosis demonstrates altered endometrial receptivity and altered gene expression. it is unknow if the endometrium is defective giving rise to a predisposition toward endometriosis or alternativly if the endometriosis causes the altered endometrial receptivity. here we created experimental endometriosis in a mouse model through allotransplantation of the uterus to the peritoneal cavity in immunocompetent mice and examined the expression of several markers of endometrial receptivity in the eutopic endometrium. materials and methods: the uterus of -week-old cd female mice was transected at cervicovaginal junction and each horn divided and transplantated into the abdomidnal cavity of eight cd mice. seven controls received sham surgery only. after weeks the uterus was removed, snap-frozen in trizol. total rna was extracted and cdna generated. quantitative real time rt-pcr using sybrgreen was performed and normalized to -actin. fold changes in normalized hoxa , hoxa , bteb , emx , igfbp , integrin - , total progesterone receptor (pr-ab) and progesterone receptor-b (pr-b) were assessed. all experiments were conducted in duplicate, repeated at least three times and compared using mann-whitney rank sum test. results: hoxa , hoxa , bteb , emx , and igfbp mrna expression showed . -fold (p= . ), . -fold (p< . ), . -fold (p= . ), . fold (p= . ), and . -fold (p< . ) decrease in the uterus of mice with experimental endometriosis compared with controls. pr-ab and pr-b mrna showed . -fold (p= . ) and . -fold (p= . ) increase in endometriosis group compared to controls, respectively, however the ratio of pr-b to pr-ab (b/ab) showed no significant change in both groups ( . vs. . , endometriosis vs. control, p> . ). there was no significant change in integrin - mrna expression ( . -fold, p> . ). conclusion: we demonstrate significant changes in multiple markers of endometrial receptivity in eutopic endometrium after induction of endometriosis. these findings suggest that origionally normal endometrium can develop defects with the creation of endometriosis; an abnormal endometrium is not a prerequisite for the development of endometriosis or associated abnormalities. this data also suggest the existance of a signal conduction pathway from endometriosis that alteres endometrial gene expression. endometrial expression patterns of relaxin and relaxin receptor mrna suggest involvement of relaxin in endometriosis. sara s morelli, felice petraglia, gerson weiss, stefano luisi, pasquale florio, jeff gardner, andrea wojtczuk, laura t goldsmith. obstetrics, gynecology and women's health, new jersey medical school, newark, nj, usa; pediatrics, obstetrics and reproductive medicine, university of siena, siena, italy. objective: in normal endometrium, relaxin is a potent inhibitor of matrix metalloproteinases, which have been implicated in the invasive process of endometriosis. we tested the hypothesis that relaxin plays a role in endometriosis by comparing expression of relaxin and its lgr receptor in normal human endometrium to levels in samples from patients with endometriosis. materials and methods: total rnas, extracted from ectopic (n= ) and eutopic (n= ) endometrium of patients with endometriosis and from endometrium of normal controls (n= ), were reverse transcribed into cdnas. real-time pcr was performed using primers previously shown to identify human lgr relaxin receptor mrna, h relaxin mrna, and beta-actin mrna, with sybr-green detection of double stranded dna products. the comparative c t method ( -ct ) determined relative lgr and relaxin mrna expression (normalized to beta-actin mrna expression). relaxin mrna was detectable in normal endometrium from of ( %) control patients. in contrast, relaxin mrna was detectable in a lower proportion of samples [ of ( . %)] from patients with endometriosis, among whom relaxin mrna was detectable in a lower proportion of ectopic samples [ of ( %)] than in eutopic samples [ of ( . %)]. lgr relaxin receptor mrna was detectable in all samples, with lower expression in endometriosis samples than in endometrium from normal controls. relaxin receptor lgr mrna levels vary with cycle phase, with greater expression in the secretory phase (sp) than in proliferative phase (pp): in normal controls . -fold higher levels in the sp than pp; and in endometriosis patients . -fold higher levels in the sp than pp. in both phases, lgr mrna levels were lower in ectopic samples than in either eutopic samples ( . -fold lower in pp and . -fold lower in sp) or endometrium from normal controls ( . -fold lower in pp and . -fold lower in sp). eutopic endometrium had similar lgr mrna expression to controls throughout the cycle. decreased local expression of relaxin and relaxin receptor mrna in ectopic endometrium from patients with endometriosis throughout the menstrual cycle suggests that relaxin may be protective against endometriosis. ovarian reserve after laparoscopic cystectomy of endometriotic ovarian cysts. shinichi hayasaka, takashi murakami. obsterics and gynecology, tohoku university, sendai, japan. objective laparoscopic ovarian cystectomy is generally recommended for endometriotic ovarian cysts because it has been associated with a higher pregnancy rate and a lower recurrent rate. however, residual ovarian function after laparoscopic cystectomy of endometriotic ovarian cysts has been questioned. in this study, we retrospectively evaluated ovarian response to hyperstimulation in women selected for ivf and icsi cycles who previously underwent laparoscopic cystectomy of a monolateral endometriotic ovarian cyst. methods a retrospective review of the patients between january and september was performed. the operated ovary and contralateral intact ovary were compared in terms of number of oocytes retrieved, number of follicles with a mean diameter > mm at the time of hcg administration. the patients who had recurrent endometriotic ovarian cysts were excluded. in total, ten patients were identified. the mean(±sd)number of oocytes retrieved was . ± . in the control ovary and . ± . in the previously operated ovary(p= . ). the mean(±sd)number of follicles with a mean diameter > mm was . ± . in the control ovary and . ± . in the previously operated ovary(p= . ). the age of patients and diameter of the operated ovary had little influence on the difference between the response of the control ovary and one of the previously operated ovary. laparoscopic cystectomy of endometriotic ovarian cysts is associated with a significant reduction in ovarian reserve. background pre-eclampsia (pe) is associated with systemic maternal endothelial dysfunction, which is thought to result from the presence of circulating factors released following placental damage. it is hypothesised that this occurs as a result of reduced oxygen (o ) delivery. some features of placental pathology seen in pe, such as increased apoptosis, can be reproduced by culture of placental explants in % o . metabolomics operates to study 'all' metabolites within a biological system. this strategy has previously identified differences in maternal plasma between normal pregnancies and those complicated by pe. in these experiments we used metabolomics to investigate differences in the metabolic profile of explants cultured in different o tensions. hypothesis the metabolic profile of placental villous explants is altered by culture in different o tensions. methods placental villous explants were cultured with either % serum (n= ) or in serum-free conditions (n= ) for h in %, % and % o . after h, conditioned culture medium and tissue were collected and snap frozen. tissue was homogenised in % ice cold methanol prior to analysis. samples were analysed using gas chromatography-mass spectroscopy (gc-ms). samples cultured at %, % and % o were compared to identify concentration differences in metabolites in conditioned cultured medium and tissue lysate. kruskal-wallis test was used for statistical analysis. due to the large number of metabolites identified, a p value of . was considered significant. results the mean intra-assay variability was . %. there were no differences in the metabolic profile of conditioned culture medium from % and % o . several metabolites differed in culture medium from % compared to % and % o including: deoxyribose, glycerol and threionic acid. these changes were present in both serum and serum-free conditions. using these metabolites alone, culture in % o could be completely discriminated from % and % o . deoxyribose was also elevated in tissue lysate from % o compared to % o . conclusion this metabolomic strategy can identify differences in metabolic profile in placental tissue cultured in % o . these novel compounds may provide further insight into pathophysiology of pe. objective a strategy of metabolic footprinting (the study of extracellular metabolites, which are related to intracellular metabolism) was used to detect a wide array of low molecular weight metabolites. metabolic profiles were employed to differentiate between placental explants cultured at different oxygen tensions. two separate studies were combined to validate initial observations. methods metabolic footprints of placental villous explants, obtained from uncomplicated pregnancies, (n= ) were cultured for h in %, % and % oxygen. conditioned culture medium was then collected and frozen, prior to analysis by uplc/ltq-orbitrap mass spectrometry in both negative and positive ion mode. samples cultured at %, % and % were compared to identify differences in the metabolic profiles. this procedure was repeated for different explants and the results compared across the studies in order to validate initial findings. approximately unique peaks were detected in both sample sets in negative ion mode and peaks in positive ion mode. a number of metabolites were identified as being significantly different using kruskal-wallis (pval< . ) under different oxygen tensions. some differences were seen between the results obtained from both runs due to separate batch preparation of the medium but good reproducibility was obtained for many metabolites between batches. metabolites of biological interest included amongst others -deoxyribose, valine, tyrosine and aspartic acid. the largest differences were those seen between o % and o %. comprehensive metabolic profiles were detected and employed to identify differences in the metabolic footprints of explants cultured under differing oxygen conditions. a number of biologically interesting metabolites were characterized. objective: brain weight and dna content were reduced in leptin deficient (lep ob /lep ob ) mice postnatally. leptin is detected in the sera and its receptors are expressed in the brain of the mouse fetus. we examined the role of leptin in the proliferation and differentiation of neural lineage cells in the mouse fetus. methods: the number of total cells in the cerebral cortex was compared between (lep ob /lep ob ) and wild type fetuses from embryonic day (e) to . the number of brdu positive cells was also compared on e and e . brdu uptake, the ratio of viable colony number to plated cell number, the proportion of multipotent, neuronal or glial progenitor colonies, and the expression levels of hes mrna were compared between leptin-and non-treated neurosphere cells. moreover, we examined stat phosphorylation by leptin stimulation with elisa to investigate whether or not jak/stat pathway transduces the leptin signal in the prenatal period as in the adult. results: lep ob /lep ob fetuses had reduced total cell numbers in the ventricular zone (vz) on e and e , and in the cortical plate on e . the number of brdu-positive cells was reduced in vz of e and e lep ob /lep ob fetuses. brdu uptake and the number of viable colonies were increased by days-leptin treatment in the neurosphere culture. the proportions of glial-restricted and multipotent precursor colonies were increased by leptin, whereas that of oligodendrocyte precursor colonies were decreased. hes mrna expression was enhanced in neurosphere cells by leptin. neither the amount of phosphorylated stat nor that of stat was increased in neurosphere cells by leptin stimulation. conclusion: our study suggests that leptin maintains the neural stem and glial-restricted precursor cells through upregulation of hes expression and increases the number of cerebrocortical cells in the mouse fetus, however, jak/stat pathway does not mediate the leptin signal in undifferentiated neural lineage cells. a vaginal wall, and > % of fib- knockout (ko) mice develop pelvic organ prolapse by weeks of age. in contrast, fib- and - deficiencies do not result in prolapse, and fib- kos die shortly after birth. herein, we evaluated the role of fib- in pelvic organ support. methods: two observers serially measured the degree of vaginal, perineal, and anal prolapse in fib- ko (fib -/-) and in fib -/-mice severity of prolapse was significantly related to age, but not parity, and the average age at diagnosis was wks. fib -/-animals with advanced prolapse had attenuated uterosacral ligaments and descent of the bladder and uterus caudal to the symphysis. pro-mmp ( -fold, p= . ), active mmp ( -fold, p= . ), and prommp ( -fold, p= . ) were increased in vaginal tissues from mice with gross prolapse compared with age-, strain-, and cycle-matched controls. this increase in protease activity, however, was also accompanied by increased expression of fib- and tropoelastin ( . -fold, p< . ). regardless of prolapse maternal morbidtties tf ards-n(%) ( ) acute ren~m f lure-n (%) ( ) car~tovascular ~ysfunrtion-n ("/o) ( ) hepatc fa..lure-n (%) dt sst'dlm atl:d inlfavascul..-coagul tipalhy-n(%) ( ) durallon of !cu suy (days. mean+sd) :t . hospital my (days.. me +sd) . condon, j. c., hardy, d. b., kovaric, k., and mendelson, c. r. ( ) mol endocrinol ( ), - . objective: innervation of the uterine cervix is important for the process of parturition (physiol beh : , ; j histochem cytochem : , jsgi : , ) . the hypogastric nerve is a major pathway that innervates the uterine cervix, yet its contribution to processes associated with cervical ripening and parturition is not known. the objective of this study was to determine the effect of hypogastric nerve transection on processes associated with cervical remodeling and parturition. methods: time-dated pregnant rats were sham-operated or the hypogastric nerve bilaterally transected just anterior to the inferior mesenteric ganglion on day post-breeding. live pups were born spontaneously by all rats at term on days - post-breeding. on the day of birth, the cervix was excised, postfixed overnight, sectioned, and processed to evaluate collagen content and structure (nih image j). sections were also processed by immunohistochemistry to assess cell nuclei density, the census of resident macrophages, and area of tissue that contained nerve fibers. results: hypogastric neurectomy did not affect cell nuclei density, the number of macrophages, or density of nerve fibers in the cervix. the failure of hypogastric nerve transection to affect indices of cervical remodeling is consistent with the finding that duration of pregnancy and timing of birth were not different in sham-operated and hypogastrectomized rats. conclusions: nerve fibers in the hypogastric nerve that innervate the lower uterus and cervix, including sympathetic and neuropeptidergic projections, are thus not essential for birth. these novel findings provide support for the contention that innervation of the uterine cervix other than through the hypogastric nerve contributes to processes associated with cervical ripening and parturition. receptor system in prostaglandin synthesis in pregnancy. eugenie r lumbers, , della m yates, carolyn m mitchell, jonathan j hirst, , tamas zakar. , school of health sciences, university of newcastle, newcastle, nsw, australia; mothers and babies research centre, hunter medical research institute, newcastle, nsw, australia; obstetrics and gynaecology, john hunter hospital, school of medical practice and public health, university of newcastle, newcastle, nsw, australia. the fetal membranes and decidua contain prorenin, which requires proteolytic activation. ngyuen (j clin invest. : ) described a renin/prorenin receptor that conformationally activates prorenin, so that it can form ang i from aogen. in addition, receptor-bound prorenin can stimulate intracellular pathways directly. prostaglandins (pgs) participate in the control of term and preterm labour, and renin can stimulate pg production by amnion and decidua when angiotensin's action is blocked mitchell placenta : ) . the prorenin receptor may mediate these effects. our aim was to find out if the prorenin receptor gene is expressed and colocalised with prorenin and prostaglandin h synthase- (pghs- ) in human placenta, amnion, chorion and decidua. we have also determined if there is any change in the expression of the prorenin and prorenin receptor genes with labor. placentae with attached membranes were collected after term birth either by elective caesarian section or by spontaneous labor, and prorenin, prorenin receptor and pghs- mrna levels were quantitated relative to beta-actin mrna by real-time rt-pcr in amnion, chorion, decidua and placenta. before labor, mean prorenin mrna levels were . times higher in decidua and . times higher in chorion and placenta than in amnion (p< . ). there were no significant changes with labor. proenin receptor mrna was expressed in all tissues. proenin receptor mrna levels in prelabor amnion, chorion and placenta were similar, while levels in decidua were % of those in amnion (p= . ). this low level of prorenin receptor mrna in decidua persisted after labor (p< . ). pghs- mrna expression was highest in amnion; in decidua and placenta it was only and % of amnion. similar differences were present after labor. we conclude that the decidua is the principal source of prorenin within the pregnant uterus, and all gestational tissues are targets for prorenin. decidual prorenin may affect pghs- expression in amnion through the prorenin receptor forming a maternal-fetal paracrine system that stimulates pg production at labor. the immuno-suppressive effects of progesterone on umbilical cord blood mononuclear cells and the effect of culture media estradiol content. nadav schwartz, xiangying xue, christine n metz. obstetrics and gynecology, nyu school of medicine, new york, ny, usa; feinstein institute for medical research, manhasset, ny, usa. objective: progesterone (p ) is known to supress the maternal immune response and similar effects have been seen with fetal cells. we sought to ascertain whether p action was modulated by the phenol red and/or estrogen content of the culture media. methods: umbilical cord blood mononuclear cells were isolated using a density gradient centrifugation. the cells were incubated with p ( - m) hour prior to overnight stimulation with lps. supernatants were assayed for tnf-using elisa. the following culture media were used: a)'red' media: rpmi+ % fbs, b)'yellow' media: phenol red-free rpmi+ % fbs, and c)'clear' media: phenol-free rpmi+ % dextran/charcoal treated fbs. finally, the 'clear' media experiments were repeated with estradiol add-back. results: p suppressed tnf production in all medias. tnf levels were suppressed to . % (p< . ) of controls using 'red' media and to . % (p< . ) in 'yellow' media. the degree of suppression was not as substantial using 'clear' media where tnf levels were . % (p< . ) of control. (fig ) adding estradiol to the 'clear' media had little or no effect on the degree of tnf suppression. (fig ) conclusions: progesterone exerts an immuno-suppressive effect on lpsstimulated fetal mononuclear cells which is more pronounced when using media containing untreated fetal bovine serum. the phenol-red/estradiol content of the culture media did not modulated the p effect. dextran/charcoal treatment of fbs appears to deplete the media of factors other than estradiol that are necessary for p to exert its full effects. culture conditions should be optimized when studying progesterone's effects on immune response. ovine fetal regional blood flow following prenatal dexamethasone (dex) at . gestation (g). antonine d van heesewijk, jan g nijhuis, susan l jenkins, peter w nathanielsz, mark j nijland. ob/gyn, academisch ziekenhuis maastricht, maastricht, netherlands; ob/gyn, cpnr, uthscsa, san antonio, tx, usa. background: pregnant women at risk for premature labor receive antenatal glucocorticoids (gc) to reduce neonatal morbidity and mortality. while fetal blood pressure (bp), heart rate and vascular endothelial and control fetuses (n= ). dispersed adrenal cortical cells ( . x cells/tube; in duplicate) were challenged with - m acth. samples were collected at time (baseline), , , , and min after acth treatment, and tissue and media samples were frozen for determination of cortisol, camp, and star. results: cortisol output (ng/ . x cells) was higher in the lth group compared to the control, (p< . ) at min ( . ± . vs. . ± . ), min ( . ± . vs. . ± . ), and min ( . ± . vs. . ± . ). peak camp levels (fmol/ . x cells) were observed at min but did not differ between control ( . ± . ) and lth ( . ± . ) adrenal cells. western analysis demonstrated that star protein expression was higher in the lth adrenal cortex compared to control (p< . ) at min ( . ± . vs. . ± . ), and at min ( . ± . vs. . ± . ), relative optical density units. conclusions: results from the present study taken together with those of previous in vivo studies suggest that the enhanced cortisol output in lth fetuses is the result of increased adrenal acth sensitivity. this enhanced sensitivity is not due to differences in camp generation. star, which is a key element involved in cholesterol transfer at the level of the mitochondrial membrane appears to play a key role in the enhanced cortisol response to acth in the lth group. (nih grants hd , hd and llu-nih imsd r gm - ). expression. hiroaki itoh, makoto kawamura, shigeo yura, haruta mogami, tsuyoshhi fujii, norimasa sagawa. obstetrics and gynecology, national hospital organization osaka national hospital, osaka, japan; gynecology and obstetrics, kyoto university graduate school of medicine, kyoto, japan; obstetrics and gynecology, mie university school of medicine, tsu, japan. objective: epidemiological evidences suggested that undernutrition in utero develops risk factors for adult cardiovascular disorders, such as blood pressure increase. the present study was designed to prove the hypothesis that maternal iso-caloric high protein diet alleviates blood pressure increase in the adult offspring by affecting placental beta-hydroxysteroid dehydrogenase type ( beta-hsd ) expression. methods: the % calorie restriction was applied to pregnant mice by using either standard protein diet (spd; % casein protein; spd-un) or high protein diet (hpd; % casein protein; spd-un). in some groups, these pregnant mice were sacrificed at . d.p.c.; then maternal and fetal plasma as well as placental tissues were corrected. while in other groups, systolic blood pressure was measured in the offspring at wks. fetal and maternal corticosterone levels were measured by elisa. the placental beta-hsd gene expression was measured by quantitative taqman pcr. results: systolic blood pressure in the spd-un offspring at weeks ( mmhg) was higher than that in spd-nn offspring ( . mmhg). by contrast, systolic blood pressure in the hpd-un offspring ( . mmhg) was similar to that in spd-nn offspring. maternal calorie restriction with spd and hpd caused similar elevation of maternal plasma corticosterone concentrations. by contrast, fetal plasma corticosterone levels in hpd-un offspring ( ng/ml) was lower than those in spd-un offspring ( ng/ml), indicating the amelioration of fetal exposure to high corticosterone by maternal hpd. the placental beta-hsd gene expression in the hpd-un was % higher than that in spd-un, suggesting that maternal hpd augmented placental beta-hsd gene expression in the placenta and probably facilitated the inactivation of maternal cortocsterone in the process of placental transfer to fetal circulation. conclusion: the preset study suggests that maternal high protein ingestion may elevate placental beta-hsd gene expression and ameliorate blood pressure increase in the adult offspring via modification of fetal exposure to maternal corticosterone. elevated cortisol levels at birth exert critical maturational effects through action at glucocorticoid receptors, gr. in contrast, the low cortisol concentrations in the preterm fetus, before the time of increased fetal cortisol secretion, are near to the kd for cortisol at the higher affinity mineralocorticoid receptor, mr. we hypothesized that endogenous cortisol in the fetus exerts physiologic actions at mr in tissues without appreciable cortisol-inactivating enzyme, hsd , including the fetal lung and brain. we therefore tested the effect of infusion of a mr-antagonist, ru (mra, . mg/h over h) into - day fetal sheep. we tested for effects on lung liquid production rate, lung liquid and plasma electrolytes, plasma acth and cortisol, and hematocrit at - h after start of the infusion. although there was no significant difference in the liquid production rate in fetuses infused with ru , the ratio of na + to k + in lung liquid was significantly greater in mra-treated fetuses than in the control fetuses ( . ± . vs . ± . ). plasma acth was significantly greater in the mra-treated fetuses than in control fetuses at h ( ± vs ± pg/ml); in the mra-treated fetuses, acth concentrations at h were greater than in the same fetuses at h ( ± pg/ml), wheras there was no increase in plasma acth in the control fetuses ( h: ± pg/ml). similarly, plasma cortisol concentrations at and h were greater in the mra infused fetuses than in control fetuses ( h: . ± . vs . ± . ng/ml), and cortisol increased significantly over time in the fetuses infused with mra, but not in control fetuses ( h, mra: . ± . , control: . ± . ng/ml). infusion of mra did not alter fetal plasma electrolyte, fetal blood pressure or fetal heart rate. however, fetal packed cell volume was significantly increased at - h of infusion of mra ( h: ± vs ± %) and fetal pco was significantly greater at h of infusion of mra as compared to control fetuses ( . ± . vs . ± . ). these results suggest that endogenous cortisol concentrations in the preterm fetus exert negative feedback control of acth via action at mr in the fetal brain, and play a role in regulation of fetal lung liquid composition and in fetal fluid balance. preparturient increases in fetal acth secretion are cyclooxygenase- (cox- ) dependent. charles e wood. dept. physiology and functional genomics, university of florida college of medicine, gainesville, fl, usa. maturation of the fetal hypothalamus-pituitary-adrenal axis is critical for the timely somatic development of the fetus and readiness for birth. in sheep, increased preparturient activity of the fetal hpa axis appears to be responsible for triggering parturition itself. this study was designed to test the hypothesis that prostaglandin generation, mediated by cox- in the fetal brain, is critically important for stimulation of the preparturient increase in fetal acth secretion. singleton fetal sheep were chronically catheterized with vascular, amniotic, and lateral cerebral ventricular catheters. nimesulide, a selective cox- inhibitor, was infused ( mg/day, n= ), and vehicle ( % dimethylsulfoxide, n= ) was infused. arterial blood samples were drawn from fetuses at hour intervals for measurement of plasma hormone concentrations and arterial blood gases. in the vehicle-treated fetuses, fetal plasma acth, pomc, and cortisol concentrations increased exponentially (p< . by anova) before spontaneous parturition at ± . days. in the nimesulide-treated fetuses, fetal plasma acth and pomc were constant and did not increase prior to parturition (p=ns by anova). in contrast, fetal plasma cortisol increased independent of acth (p< . by anova) and the fetuses were born spontaneously on ± . days gestation. the slight delay in spontaneous parturition in the nimesulide-treated fetuses was statistically significant (p< . by mantel-cox test). intracerebroventricular nimesulide infusion did not decrease fetal plasma concentrations of prostaglandin e , demonstrating that the action of the nimesulide was restricted to the fetal brain. fetal blood gases were normal in all of the fetuses, and there were no differences in blood gases between groups. we conclude that the spontaneous increase in fetal acth and pomc prior to parturition is cox- dependent. however, we also conclude that the increase in fetal plasma cortisol concentration can occur independent of increases in fetal acth, suggesting that the increase in cortisol can result from increases in adrenal sensitivity to acth. the results of this study demonstrate that the timing of parturition in the sheep is not dependent upon increased acth secretion, and the results suggest that parturition is regulated primarily by changes in adrenal sensitivity. expression of extracellular signal-regulated kinase / and p mitogen-antiphosphotyrosine. immunolocalization of inos was performed in the same tissues. hpmec were used to determine effect of sin- ( mm) on inos protein expression ( min, and h). results: inos protein was detected in microvascular endothelium, smooth muscle and syncytiotrophoblast of the placenta. inducible nos levels in placentas from severe preeclampsia were significantly decreased compared to normal (p< . ) but were not different from mild preeclampsia. aligning western blots of anti-phosphotyrosine and inos revealed a phosphorylated band corresponding to the molecular weight of inos. the proportion of tyrosine phosphorylated inos was reduced by % in severe preeclampsia compared with normotensive. preliminary data with ip for phosphotyrosine and crossblotting with inos confirmed this finding. sin- treatment decreased inos protein abundance at and h in hpmec. conclusions: our results demonstrate decreased tyrosine phosphorylation of inos in preeclamptic placenta. phosphorylation of tyrosine in inos has been reported to negatively regulate its activity. we therefore postulate that decreased phosphorylation of inos may be responsible for the increased catalytic activity of the enzyme that we have previously observed. the decreased levels of inos observed on sin- treatment may be due to nitration, an effect analogous to preeclampsia, where the presence of peroxynitrite has been well established. it remains to be seen if protein nitration has an effect on enzyme stability. objective: -isoprostane ( -iso), a prostaglandin-like compound formed in vivo, is a reliable measure of oxidative stress which occurs in response to many different stimuli, including infection. periodontal disease is a chronic oral infection associated with fetal growth restriction and preeclampsia. our objective was to determine if maternal periodontal disease is associated with oxidative stress as measured by -iso. methods: a secondary analysis was conducted using prospective data from the oral conditions and pregnancy study. a cohort of healthy women enrolled < weeks underwent oral health examination and serum sampling. maternal periodontal disease was categorized as healthy, mild, or moderate-severe by clinical criteria. maternal serum was analyzed for -iso by ultra-sensitive elisa. elevated -iso was defined by a value th %tile. maternal factors associated with elevated -iso were determined using chi-square or t-tests as appropriate. a logistic regression model was created to determine adjusted odds ratios ( th %ci) for elevated -iso. results: women had complete data for analysis. the median -iso level (iqr) was , ( - , pg/dl). using bivariate analysis, moderate-severe periodontal disease, non-white race, use of wic/food stamps, unmarried status, obesity, and lack of insurance were associated with elevated -iso. the logistic regression model for elevated -iso is shown below. adjusted or ( th ci)* mild periodontal disease . maternal periodontal disease and utilization of public assistance are associated with oxidative stress in pregnancy. the relationship between periodontal disease, social hardship, oxidative stress, and adverse pregnancy outcome remains to be determined. antioxidant therapy could represent novel therapy for the prevention of adverse pregnancy outcome. severe transient immunological thrombocytopenia in a preeclampsia patient whose bone marrow production of platelets had been restricted. toshihiro yoshimura. obstetrics and gynecology, ntt-west kyushu general hospital, kumamoto-city, kumamoto, japan. introduction: idiopathic myelofibrosis is a chronic myeloproliferative disorder in which clonal haemopoetic stem cell proliferation is accompanied by reactive fibrosis. case report: a -year-old primiparous woman was referred to our hospital for prenatal care after weeks of gestation. her medical history was significant for idiopathic myelofibrosis, and congenital agenesis of the spleen. the laboratory workup showed the patient's white blood cell count to be , / l; hemoglobin, . gm/dl; and platelet count, , / l. her bleeding time was normal ( min). until the th week, the patient's pregnancy was uneventful. during the th week, however, the patient's platelet count declined to , / l, when proteinuria became evident. her bleeding time was prolonged to min. the coagulation system was normal. by the th week, the patient's blood pressure was elevated to / mmhg, and her urinary protein excretion exceeded g/day. our initial diagnosis was thrombocytopenia due to bone marrow suppression. the patient had no history of platelet transfusion, but we expected it would increase the platelet count, particularly since in the absence of destruction by the spleen, the lifespan of the platelets would be prolonged. this was not the case, however. elective induction of labor was carried out after weeks. at the same time, the patient was transfused with units of platelets, but her platelet count remained unchanged ( , / l). it was then that we realized that immunological thrombocytopenia may be involved, and massive doses ( mg/kg) of gamma globulin were given intravenously for one day. thereafter, platelets ( units) were again transfused, this time raising the platelet count to , / l. cesarean section was promptly carried out under general anesthesia. we later found that the patient's serum platelet associated immunoglobulin (paigg) level was positive, though antiplatelet and antinuclear antibodies were negative. the postoperative course was uneventful. the maternal platelet count declined to the pretransfusion level within days after delivery, but gradually increased to the prepregnancy level by weeks. comment: this idiopathic myelofibrosis patient in whom bone marrow production of platelets had been severely restricted demonstrates that immunological destruction may play a major role in the development of thrombocytopenia in preeclampsia. early l-arginine therapy improves notably the fetal growth in preeclamptic women. a randomized controlled trial. keisy lopez-molina, juan c gonzalez-altamirano, julio e valdivia-silva. , oncoinmunología y biología vascular, facultad de medicina, universidad nacional san agustín, arequipa, peru; cardiología y cirugía de tórax, hospital nacional case essalud, arequipa, peru; inmunología, instituto de investigaciones biomédicas, méxico, distrito federal, mexico. objective: to assess the benefit of early administration to l-arginine, the precursor to nitric oxide, on fetal growth. methods: one hundred women with preeclampsia were randomized to receive either l-arginine or placebo until day postpartum. live singleton infants were born after preeclamptic pregnancies and compared those with other control infants. birth size was expressed as the ratio between observed and expected birth weights, and infants smaller than two standard deviations from expected birth weights were classified as small for gestational age (sga). all the women had neither previous precedents of the preeclampsia nor other factors that they cause sga. results: no significant differences existed between the groups with preeclampsia before randomization. preeclampsia was associated with a % ( % confidence interval [ci] %, %) reduction in birth weight. women with hellp syndrome had to leave the study. the risk of sga was three times higher (relative risk [rr] = . ; % ci . , . ) in infants born after preeclampsia without l-arginine therapy than in control pregnancies, and two times higher (relative risk [rr] = . ; % ci . , . ). in infants born after preeclampsia with l-arginine therapy. conclusion: fetal growth improve markedly with l-arginine therapy in women with preeclampsia. introduction: although the pathogenesis of severe preeclampsia (pe) and intrauterine growth restriction (iugr) is poorly defined, inadequate remodeling of uterine spiral arties may promote reperfusion injury leading to focal infarction and reduced nutrient flow between mother and fetus. our goal was to determine whether an intravillous inflammatory cytokine cascade was associated with pe and iugr. methods: immunohistochemistry (ihc) examined il- and il- expression in preterm placentas with severe pe+iugr, and idiopathic preterm controls (ptc) with no evidence of clinical or histological chorioamnionitis. cultures of fibroblasts (fibs) and syncytiotrophoblasts (scts) from term placentas (n= ) were treated with il- and cytokine levels in conditioned media were determined using elisa or multiplex array. results were analyzed by student's t test or anova. results: the gestational age at delivery was not significantly different in pe+iugr and ptc groups ( . ± . vs . ± . wks). ihc of pe+iugr samples revealed intense villus core staining of il- adjacent to infarcts. villi distal to infarcts stained with a lower intensity. in contrast, staining was virtually undetectable in ptc. the pattern of il- staining was nearly identical in pe+iugr and ptc groups, was localized to the syncytium, and did not differ with respect to distance from infarct. to identify cellular targets of il- action, primary cultures of fibs and scts were incubated for h in serum-free medium ± ng/ml il- . by elisa we observed that il- treatment of fibs increased il- levels from ± to ± pg/ g protein (p< . ). similarly, multiplex array revealed that levels of il- , il- , and il- were induced -, -, and -fold, respectively in fibs. the presence of ng/ml il- receptor antagonist reduced the il- -mediated increase in il- levels ± % (p< . ), indicating that this response was mediated through il- receptor. in marked contrast, il- treatment did not significantly affect levels of il- , il- , il- or il- in scts, indicating that this response was cell type-specific. conclusions: these results indicate that increased expression of il- in the placental villus core in pe and iugr may promote an inflammatory cascade in fibs at this site leading to focal infarction and reduced flow of nutrients to the fetus. introduction: in human pregnancy, decreased responsiveness to angiotensin ii (angii) starts in week , promoting an expanded vascular bed. at the same time levels of the vasodilatory hemopexin increases . during pre-eclampsia the vascular bed is contracted and the responsiveness upon angii persists. this may be due to the increased levels of extra cellular atp, a natural inhibitor of hemopexin . in the present study we tested whether extra cellular atp is toxic in the pregnant condition. methods: pregnant rats were infused with either atp ( g/kg bw; n= ) or saline (n= ) on day of pregnancy (permanent jugular vein cannula/ hr infusion). non-pregnant rats (n= ) were infused with atp identically. one day before and , , days after infusion, hr urine and blood samples (wbc count and hemopexin activity) were collected. seven days after the infusion, rats were sacrificed and kidney tissue processed for immunohistology. results: urinary albumin excretion was increased in pregnant atp infused rats only (fig ) . wbc were also increased only in pregnant rats infused with atp (days and vs pre-infusion value). in pregnant atp infused rats intraglomerular influx of monocytes was increased, which correlated with urinary albumin excretion on the same day (r = . ). staining of glomeruli for the angii receptor (at- r) showed decreased at- r expression in control pregnant rats as compared to non-pregnant rats, while at- r expression in atp infused pregnant rats was increased as compared to control pregnant rats. hemopexin activity was increased on days and in control pregnant rats as compared to all other groups. discussion: these data support the notion that atp is toxic exclusively in the pregnant condition. it may be suggested that atp induced an inflammatory response, although the exact mechanism by which atp induced its effects needs further investigation. background: hepatocyte growth factor (hgf) is a multifunctional cytokine that is known to promote division, motility, invasion and morphogenesis of a wide range of cell types and to inhibit apoptosis. the effects of hgf are mediated through its interaction with the tyrosine kinase receptor c-met. in pregnancy hgf/met system is involved in the physiologic growth and development of the feto-placental unit. hgf/met effects are counteracted by transforming growth factor- (tgf- ), that is known to promote progressive fibrosis in human tissues. moreover tgf- plays a major role in trophoblast growth and differentiation. tgf- inhibits hgf expression as well as hgf inhibits tgf- expression. an understanding of the mechanisms regulating placental balance of these growth factors may provide insights into the processes that occur in complications of pregnancy, such as preeclampsia (pe) and fetal growth restriction (fgr). objective: to verify the hypothesis that hgf, c-met and tgf- are differently expressed in tissues of normal and pe placentas. methods: we studied placentas from pregnancies complicated by pe ( with normal fetal growth and with fgr) and placentas from normal pregnancies. from each placenta random samples were excised and rna was extracted; quantitative real-time rt-pcr analysis was used to investigate mrna expression of hgf, c-met and tgf- in pe (with or without fgr) and in normal placentas. results: gestational age, neonatal weight and placental weight were, as expected, lower in pe groups. the mrna expressions of the three molecules are higher in pe than in normal placentas, but the difference is statistically significant only for c-met. the higher values are mainly due to the increase in the pe group without fgr for c-met and tgf- and the increase in the pe-fgr group for hgf. conclusion: increased levels of expression of hgf/met system in pathological placentas could be explained as an attempt to repair placental damages; nevertheless placental regeneration could be inhibited by the increase of tgf- , that promote fibrosis. placental expression of hgf, c-met and tgf- is increased in all pe placentas, but particularly in placentas of pe with normal fetal growth, where placental tissue is probably less compromised. obesity is a risk factor for preeclampsia (pe), but the reason for this risk is unknown. previously, we found that neutrophils infiltrated into the vasculature of pe women released myeloperoxidase (mpo) and matrix metalloproteinase (mmp ), products that can cause oxidative stress and vascular dysfunction. if neutrophils infiltrate the vasculature of obese women and release mpo and/or mmp , this may help explain why they are at risk of developing pe. hypothesis: systemic vascular tissue of obese women will have a significant presence of mpo and mmp as a result of neutrophil infiltration. methods: subcutaneous fat, which is highly vascularized, was obtained at abdominal surgery from normal weight, overweight and obese women. formalin fixed, paraffin embedded μm sections of fat biopsies were stained using immunohistochemistry with specific antibodies for mpo and mmp . data were evaluated for intensity of vessel staining by visual score ( - ), density of staining using image analysis software, and % vessels with neutrophil staining, liberated from serum-free placental villous explant cultures from normal and pe pregnancies on human endothelial cells, and identified candidate mediators of their differential effects on metabolism and behaviour. methods: term placental villous tissue from normal (n= ) or pe (n= ) pregnancies were explanted for days at % oxygen. conditioned h medium (day - ) was applied to human umbilical vein endothelial cells (huvecs). an angiogenesis assay was conducted in which tubule length and number were measured by morphometric imaging following seeding on % matrigel. the effect of explant conditioned medium on endothelial cell metabolism was determined by mtt and bioluminescent atp assay. the release of vasoactive metabolites (nitrite, endothelin- , prostacyclin) from huvecs exposed to this medium was also measured. finally, a luminex bead array was used to screen the explant media for a panel of chemokines/cytokines. results: in the angiogenesis assay, tubule length (p< . , mann-whitney u test) and number (p< . ) were significantly decreased in pe compared to normal pregnancy medium. there was no change in mtt reduction, but endothelial cellular atp levels were also significantly reduced following exposure to pe explant medium (p< . ). both normal and pe-derived medium stimulated huvecs to produce vasoactive metabolites. the following cytokines were detectable in the explant media: interleukin (il)- , il- , gro-, monocyte chemotactic protein- and macrophage inflammatory protein- (mip- ). higher levels of both il- and gro-were present in the normal medium compared to pe (both p< . ). mip- was present in pe conditioned media but undetectable in media generated from normal placental explants. conclusions: these results suggest that pre-eclampsia stimulates the release of soluble factors from the placenta which have adverse effects on endothelial cell angiogenic potential and metabolism. altered levels of several cytokines were detected in the explant medium, and the effects of these on endothelial cell function are currently being addressed. yang gu, davis f lewis, yuping wang. obstetrics and gynecology, lsuhsc-shreveport, shreveport, la, usa. objective: placentas from women with preeclampsia (pe) release more chymotrypsin-like protease (clp). the purpose of this study was to determine if placenta-derived clp was responsible for altering endothelial (ec) barrier function in pe. approaches: endothelial junctional protein complex ( -catenin/ve-cadherin/ p ) expression and junctional protein ve-cadherin distribution were examined in ecs treated with pe placental conditioned medium. methods: ) confluent ecs were treated with pe placental conditioned medium (cm). the association of ec junctional protein complex ve-cadherin/catenin/p was examined by a combined immuno-precipitation (ip) and immuno-blotting (ib) assay, in which total cellular protein was immunoprecipitated with monoclonal antibody against -catenin and then immunoblotted with antibodies against ve-cadherin or p- . ) confluent ecs grown on cover slips were exposed to pe placental cm with or without depletion of chymotrypsin. ec junction protein ve-cadherin distribution was examined by fluorescent microscopy. results: ) ve-cadherin and p are expressed in control ecs but not in ecs exposed to pe cm, which indicate that the junctional protein complex vecadherin/ -catenin/ p is lost in ecs exposed to pe cm. ) ecs exposed to pe placental cm showed a discontinuous distribution and reduced expression for ve-cadherin at cell contact areas. the zipper like structure was lost and cleft was formed at cell-cell contacts. these observations indicate that the homotypic cell-cell adhesion and junction protein intracellular partner complex are disrupted. these disruptive phenomena in cells treated with pe conditioned medium were not present in control cells and in cells treated with cm after depletion of chymotrypsin. conclusion: clp released by the placenta could be a candidate agent that is responsible for disrupting ec integrity and inducing endothelial permeability in pe. (supported by nih grants hl and hd ). introduction: chronic pelvic pain (cpp) syndromes such as endometriosis, irritable bowel syndrome, and interstitial cystitis are associated with visceral hyperalgesia, and often coexist in the same patient. one possible explanation for this phenomenon is viscero-visceral cross-sensitization in which increased nociceptive input from an inflamed pelvic organ sensitizes neurons that receive convergent input from an unaffected organ to the same dorsal root ganglion (drg). nociception induces upregulation of perk and substance p. the purpose of this study was to determine, in a rodent model, whether uterine inflammation increased the number of perk-and sp-positive neurons in sensory ganglia innervating both uterus and colon. methods: colonic and uterine drgs were retrogradely labeled with fluorescent tracer dyes micro-injected into the colon and uterus. ganglia were harvested, cryoprotected and cut for fluorescent microscopy. results:approximately % neurons were colon-specific and % were uterus-specific. among these uterus-or colon-specific neurons, up to % of labeled drg neurons in the l -s levels innervated both visceral organs. uterine inflammation increased the number of perk and sp-immunoreactive neurons in drg neurons innervating colon, uterus, and those innervating both organs. furthermore, this effect was specific as non-retrograde labeled drg neurons did not manifest a significant increase in perk or sp immunoreactive cells. conclusions: localized uterine inflammation leads to increased expression of sp and perk in uterine afferents as well as dichotomizing afferents innervating both uterus and colon, suggesting that viscero-visceral convergence is present at the level of drg primary afferent cell bodies. this visceral sensory integration may underlie the co-morbidity of female pelvic pain disorders and may provide basic information regarding the etiology of cpp syndromes. purpose: success rates of medical and surgical modalities for the treatment of severe fecal incontinence due to obstetric trauma are modest. we tested whether the intrasphincteric injection of autologous myoblasts is clinically safe and feasible for postobstetric fecal incontinence. methods: using ultrasound guidance a suspension of autologous myoblasts was injected in the anal sphincter muscle complex in three women with severe postobstetric fecal incontinence. main outcome measures were safety, feasibility and the wexner grading score. secondary outcome measures were incontinence episodes, rockwood fecal incontinenece quality of life scale, external anal sphincter morphology and anal pressure values. results: all procurement procedures and injections were performed without complications; there were no clinically or laboratory signs of infection or inflammation. three months post myoblast injection, wexner continence grading scores and rockwood incontinence scales were markedly improved and incontinence episodes reduced in all three patients. no change could be observed in sonographic anal sphincter morphology. mean anal squeeze pressure improved. conclusion: autologous myoblast injection for fecal incontinence is clinically feasible and safe; further studies will evaluate the efficacy of this approach. objective: to evaluate whether hysterectomy is associated with a change in postoperative bmi. methods: a retrospective cohort study was conducted of hysterectomies performed for benign indications from june to june . institutional review board approval was obtained. the data were collected by a medical student blinded to the hypothesis. basic demographic data were recorded. body mass index (bmi) was determined at time points: time (immediately postoperatively), time ( weeks to months postoperatively), time ( months to year postoperatively) and time ( to . years postoperatively). one-way anova was performed using ncss statistical software. a bonferroni multiple comparison test (p< . ) was used to compare median changes in bmi from baseline. the starting bmi served as the control group. results: there was a statistically significant increase in bmi at time compared to baseline in all women ( . to . ). when sorted was measured by matrigel invasion assay. small interference rna targeting vegfr- were predesigned by ambion inc. and cells were transfected using ambion siport neofx according to the optimized protocol. results: of the four receptors (vegfr- , vegfr- , nrp- and nrp- ), vegfr- was the predominant receptor that expressed in the more invasive cell lines (dov , skov and r ). lpa, at - m, significantly induced vegfr- expression in dov and skov cells (p< . ), without significantly affecting vegfr- expression. lpa ( - m) also significantly induced the expression of vegf and vegf (p< . ) in dov and skov cells. by small interference rna (sirna) transfection, we demonstrated that inhibition of vegfr- expression could significantly decrease dov cells' invasiveness (p< . ) and moderately decrease skov cells' invasiveness. moreover, silencing of vegfr- by sirna significantly suppressed lpa-induced dov and skov invasion. conclusion: these results suggest that knocking down vegfr- expression by rnai may be an effective strategy to inhibit lpa-induced ovarian cancer metastasis. cancer. fengqiang wang, david fishman. obstetrics and gynecology, new york university school of medicine, new york, ny, usa. objectives: expression of matrix metalloproteinases, such as mmp- and mmp- , has implicated in epithelial ovarian cancer (eoc) invasion and metastasis. osteopontin (opn) was also expressed in various human cancers and associated with tumor progression, invasion and metastasis. in the present study, we examined the correlation of mmp- , mmp- and osteopontin expression with tumor stage in ovarian tumor tissues and normal ovaries using a commercial tissue scan array. methods: complimentary dna (cdna) from normal ovaries (n = ) and ovarian tumors (n = ) of different stages (stage i, n = ; stage ii, n = ; stage iii, n = ; stage iv, n = ) was amplified by real time pcr using gene specific primer pairs for mmp- , mmp- , and osteopontin. gapdh was also amplified as a reference control. the expression level of mmp- , mmp- and osteopontin was calculated as relative expression normalized to that of gapdh in each tissue sample, and the expression in sample that has the lowest target gene expression was arbitrarily set as . the average expression of mmp- in ovarian tumor tissues ( ± ) is fold of that in normal ovaries ( ± , p = . ). using an arbitrarily set standard, of normal ovaries ( . %) has elevated mmp- expression; in ovarian tumor tissues, the percentage is . % ( of ), significantly higher than in normal tissues (p = . ). in early stage tumors (stage i/ii, n = ), of them have elevated mmp- expression ( . %, p = . vs. normal ovaries), whereh the average expression of mmp- is fold of that in normal ovaries (p = . ) and . fold of that in late stage tumors (stage iii/iv, n = ). the mmp- expression in ovarian tumors (n = , ± ) is fold of that in normal ovaries (n = ), however, the difference is not significant (p > . ) due to the extremely high expression of mmp- in two tumor tissues. osteopontin expression in ovarian tumor tissues is . fold of that in normal ovarian tissues ( ± . , n= vs. . ± . , n = , p< . ). in early stage (i/ii) ovarian tumors, osteopontin expression is . fold of that in normal ovaries, while in late stage (iii/iv) ovarian tumors, its expression is . fold of that in normal ovaries (p< . ), suggesting an increase trend associated with disease stages. conclusions: our results suggest that mmp- , mmp- and osteopontin alone or combined may have clinical value for ovarian cancer detection. objectives: -tubulin acetylation has been proposed to control the dynamic nature of microtubule stability. acetylated -tubulin plays a role in regulating microtubule functions in mitosis and cell migration. here, we sought to identify the relationship between post-translational -tubulin acetylation and the expression of epithelial cell adhesion molecules (ep-cam) after exposure to microtubule interacting agents in ovarian cancer cells. methods: epithelial ovarian cancer cell line, hey was treated with microtubule stabilizing agents (taxol, epothilone b and discodermolide), microtubule-destabilizing agent (vinblastine), hdac inhibitor trichostatin a (tsa), anti-metabolite fluorouracil ( fu), or alkalyting agents (cisplatin and carboplatin). cells were separately treated with either ic or -fold ic of each agent, and incubated at °c for h, h and h. acetylation oftubulin and pan--tubulin were evaluated by western blot analysis followed by protein quantification. cell surface ep-cam expression was examined by flow cytometry. results: increased acetylation of -tubulin was seen with taxol, epothilone b, discodermolide, vinblastine and tsa. -tubulin acetylation was time and dose dependent. the highest level of -tubulin acetylation ( . -fold) was observed with vinblastine at -fold ic after h. exposure to microtubule interacting agents and tsa resulted in increased cell surface expression of ep-cam in a time and dose dependent manner. the highest level of cell surface ep-cam expression ( . fold) was observed with -fold ic of vinblastine at h. the increase in acetylated -tubulin and ep-cam expression was clearly detectable after h treatment. this data reveals a similar dose and time dependent increases between the acetylation of -tubulin and the increase of ep-cam expression. conclusions: these data demonstrate the promotion of -tubulin acetylation and cell surface ep-cam expression by a microtubule destabilizing agent and by microtubule stabilizing agents. interestingly, vinblastine induces the highest -tubulin acetylation and ep-cam expression. acetylation of alpha-tubulin may be associated with redistribution of cell surface antigens in ovarian cancer cells. objective: epothilone b (epob) is similar to taxol in its ability to enhance tubulin polymerization and to stabilize microtubules. epob is currently being evaluated as an antitumor agent and is in phase iii clinical trials. the tumor suppressor gene, p plays an important role in the induction of apoptosis by a variety of anticancer drugs. p mitogen-activated protein kinase is activated by a wide array of stress stimuli including chemotherapeutic agents and promotes apoptosis. since both p and p activation induces apoptosis, we hoped to evaluate the relationship between p , p-p and parp cleavage, an early indicator of apoptosis, in ovarian cancer cells after treatment with epob. methods: hey cells were treated with epob ( to nm) for h. parp cleavage product p as well as p-p , p , phospho-p (ser- ), p and survivin were determined by western blot analysis. a wild type ovarian cancer cell line hey, was treated with or without m sb , a specific inhibitor of p-p , followed by treatment with epob ( or nm) for h. lysates were prepared and western blot analysis was performed with polyclonal phospho-p and anti-phospho-p antibodies. results: epob induces p activation and apoptosis demonstrated by increased parp cleavage product. time course studies indicated that phosphorylation of p precedes phosphorylation of p in hey cells. the expression of p targeted gene, p (survivin) were differentially expressed depending on the dose of epob and the duration of drug exposure. pretreatment with specific inhibitor of p markedly inhibited the p phosphorylation at serine . conclusions: nm epob (a concentration that triggered mitotic arrest) causes a decrease in p expression and an increase in survivin expression. epob induces parp cleavage. p inhibitor sb inhibits epo-b -induced p phosphorylation. these results suggest that phosphorylation of p may lead to p activation and these signaling events may be related to epo-b induced cell death in ovarian cells. conclusions: nf-b has been shown to control the expression and function of anti-apoptotic proteins and pro-inflammatory cytokines. in the present study we demonstrated that specific inhibition of nf-b by erib reversed the anti-apoptotic state of chemoresistant ovarian cancer cells, therefore may provide a new potential venue for the treatment of ovarian cancer patients. expression in cervical cancer. madhu chauhan, chandra yallampalli. gynecology, university of texas medical branch, galveston, tx, usa. background: intermedin (imd)/adrenomedullin is a ct/cgrp family peptide. imd is expressed in a variety of tissues such as pituitary, stomach, placenta, uterus, and ovary .we have shown that imd plays a role in a variety of physiological functions, including vasodilatation and fetoplacental growth regulation. further we have data indicating an angiogenic activity of imd in first trimester trophoblast cells. we hypothesize that imd is expressed in cervix and may have a role in the pathology of cervical cancer objective: ) to analyze expression of imd mrna in human cervix, rat cervix from non-pregnant and pregnant rats; ) to assess the differences in the expression of imd in normal human cervix and cervical carcinoma tissues and ) to analyze the effect of imd on the expression of tnf-alpha and il- beta in human epithelial cervical carcinoma cells (hela). methods: groups of sprague-dawley, non-pregnant and pregnant rats on day of gestation were used in this study. cervical tissues were collected from the non-pregnant and pregnant rats, women undergoing hysterectomy and from women diagnosed with cervical carcinoma. total rna was extracted using trizol method. hela cells were grown to % confluency in rpmi medium supplemented with % fbs. the cells were starved in %fbs for hrs followed by treatment with imd ( - m) in presence or absence of imd antagonist, imda ( - m). cells were further incubated for hrs and total rna was extracted using trizol reagent. rna was treated with dnase before performing the reverse transcriptase polymerase chain reaction (rt-pcr). the rt-pcr data was normalized to that of s. results: ) imd mrna is expressed in rat and human cervix and in hela cells. ) expression of imd is elevated in pregnant rat cervix as compared to the non-pregnant. ) imd has no effect on tnf-alpha expression in hela cells treated with imd but caused a decline in the expression of il- beta mrna and, ) expression of imd mrna is significantly elevated (p< . ) in cervical carcinoma as compared to the normal cervix. conclusion: imd is expressed in both rat and human cervix and is elevated in pregnant rat suggesting that it may have a role in cervical function during pregnancy in rat. in addition we demonstrate that imd is involved in the pathology of cervical carcinoma and thus may have a clinical significance in the pathology of cervical cancer. objective: there is minimal information about the impact of treatment delays or dose reductions on chemotherapeutic treatment responses and overall outcomes in ovarian cancer patients. the primary objective of this study was to quantify the impact of treatment delays and dose reductions in an in vivo xenograft ovarian cancer model and to evaluate if the growth factors could improve overall survival. methods: heya- and skov -ip xenograft mouse models to determine the effect of treatment delays or dose reductions on tumor response. results: the results indicated that full dose of paclitaxel ( mg/kg) and carboplatin ( mg/kg) delivered on timeevery daysachieved better tumor response in both aggressive (heya- ) and metastatic (skov -ip ) human ovarian tumors compared to the non-treatment and vehicle groups. in heya- mice, paclitaxel/carboplatin alone and paclitaxel/carboplatin followed by growth factor support agents including pegfilgrastim ( μg/kg) and darbepoetin ( μg/kg) improved overall survival rate. growth factors also improved the tolerability in heya- model. for skov -ip mice, the treatment delays resulted in a significant reduce in overall survival time compared to full dose, on time treatment (p< . ). for the dose reduction group, there was a significant different survival comparing to full dose, on time treatment (p< . ). conclusion: treatment delays had a negative impact on tumor response and overall survival compare with treatment controls. in addition, use of growth factor agents also improved treatment response and tolerability of chemotherapy and ultimately overall survival. - ) . median age at recurrence was ( - ), and median interval to recurrence . months ( - ). ( %) patients died of recurrent disease and ( %) of other causes. site of recurrence and was local in , groin in and distant in . of the local recurrences were managed with wide local excision (wle) radical surgery ( radical excisions with skin flaps, posterior exenterations, anterior exenteration, total exenteration), wle and radiotherapy (rt)/chemort, chemo/chemort/rt, and palliation. groin recurrences were managed surgically ( adjuvant rt), rt alone, chemotherapy alone, and palliation. patients with distant metastases were managed surgically, with rt/chemo/chemort, and palliation. overall median survival after recurrence was months. median survival (months) by site of recurrence was: local , groin , distant , groin or distant . there was a significant difference in survival in local vs groin node recurrence (p< . ), local vs groin and distant (p< . ), and groin node vs distant (p . ). and years after recurrence survival rates were: local % and %, groin % and % distant , . conclusion patients with vscc remain at risk of recurrent disease therefore long term follow up these patients is essential. patients with local recurrence often have a good prognosis. outcomes for groin and distant recurrences are poor, but a proportion of those with groin recurrences can be salvaged. therefore early identification of local and groin recurrences is essential for improving outcomes in recurrent vscc particularly in cases with more conservative management of primary disease. introduction: endocrine disrupting chemicals (edcs) are environmental agents possessing hormone-like activity. exposure to edcs during differentiation can interfere with hormonal signaling, resulting in predisposition to some cancers. it has been suggested that some of these effects may be epigenetic, mediated by changes in dna and histone methylation. we hypothesized that edcs, diethylstilbestrol (des), and bisphenol-a (bpa), may act by altering the expression of dna and histone methyltransferases (dnmts and hmts). methods: ishikawa (endometrial) and mcf (breast) cells were cultured in steroid-free, phenol-free media with bpa ( m), des ( x - m) or vehicle for hours. pregnant cd- mice were treated with intraperitoneal injections of des ( mg/kg) or vehicle on days - of gestation. weeks after birth, offspring were sacrificed and tissue obtained. mrna was extracted, cdna was generated and quantitative real time rt-pcr was performed and normalized to -actin. all experiments were conducted in triplicate, repeated three times and compared using anova. results: dnmts (dnmt , a, and b) and hmts (mll and ezh ) were screened after in vitro exposure to edcs (table ) . ezh mrna expression was significantly increased in ishikawa cells after treatment with des or bpa. a similar response in ezh expression was seen in mcf cells treated with des or bpa. due to the consistent induction of ezh in all cells with either treatment, we examined the effect of des exposure on ezh expression in vivo. in adult mice exposed to des in utero, ezh expression was persistently increased ( . ± . fold, p< . ). conclusions: breast and uterine cell lines show increased expression of ezh in response to bpa or des exposure. this differential expression persists in the adult offspring of mice exposed to des in utero. ezh has been identified as a risk for neoplastic progression in the breast and for increased proliferation in uterine cancers. des induced ezh expression may be a mechanism for the increased incidence of breast and reproductive tract cancers seen in des exposed women. defects in cellular programs that control apoptosis lead to an imbalance of cell proliferation and cell death in ovarian cancer. recent evidence suggests that the use of some anti-inflammatory drugs decreases risk of ovarian cancer. natural curcumin-based anti-inflammatory therapies were shown to be beneficial in preclinical models of ovarian cancer (lin et al., ) . in this study, we examined the effects of plant derived curcumin on cell proliferation, apoptosis, and vegf expression in cultivated ovarian cancer cells. ovarian cancer igrov cells were cultured under standard conditions to study the effects of curcumin on cell kinetics and on vegf expression. cell proliferation was measured by srb and mtt assays. apoptosis was determined by measuring cytoplasmic histone-associated-dna-fragments (cell death detection elisa, roche, germany). vegf gene promoter-reporter activation and real-time quantitative reverse transcription polymerase chain reaction were used. conditioned media concentrations of vegf were measured with a commercially available enzyme-linked immunosorbent assay (quantikine, r d systems, minneapolis, mn). we observed that curcumin dose-dependently suppressed cell growth in igrov cancer cells (ic = um). treated cells showed a - fold increase in dna fragmentation compared to controls. curcumin also resulted in a significant decrease of vegfmrna expression and vegf protein secretion into the conditioned media in a dose-dependent manner. in this study, we have demonstrated that curcumin induces apoptosis, suppresses growth, and inhibits vegf gene and protein expression in an ovarian cancer cell line. experiments are underway to identify specific mechanism of curcumin action. curcumin may act as a chemosensitizing drug by potentiating the antitumor effects of standard treatments including taxols and platins in ovarian cancer. supported by the caldwell family foundation and the vesa w. and william j. hardman, jr. charitable foundation inc. daniel r christie, faheem m shaikh, john a lucas iii, susan l bellis. obsterics and gynecology, university of alabama at birmingham, birmingham, al, usa; physiology and biophysics, university of alabama at birmingham, birmingham, al, usa. introduction: previous work has shown that an upregulation of the enzyme st gal i, which is responsible for the , sialylation of integrins, confers a more tumorigenic/metastatic phenotype to colon carcinoma cell lines. it is not known, however, if this is unique to colon cancer, or if it is more broadly applicable to other forms of cancers. quantitatively increased expression of st gal i has been reported in ovarian cancers versus controls, but no biochemical or functional assays have been described to date. objective: because integrins are involved in cell adhesion and migration, and because metastasis of epithelial ovarian cancers is largely an intraperitoneal dissemination, we hypothesized that upregulation in the expression of st gal i in an ovarian cancer cell line would enhance binding with the extracellular matrix, increase invasiveness, and alter migration. methods: in the present study we forced a stable transduction of st gal i into the ov ovarian tumor cell line, which we found to be lacking the st gal i enzyme, establishing a parental (par), an empty lentiviral vector (ev), and an st gal i expressing (st ) cell line. a collagen i cell adhesion assay was performed and quantified by staining adherent cells and measuring absorbance. a haptotactic collagen i cell migration assay was performed by seeding cells in boyden chambers lined with collagen i concentration gradient, and quantified with cell staining and absorbance measurement. cell invasion through a reconstituted basement membrane (matrigel) was quantified as previously described for the cell migration assay. results: we were able to demonstrate successful creation of the ov -st gal i cell line by western blot analysis. functional assays demonstrated increased adhesion to collagen i (p < . ), increased haptotactic collagen i cell migration (p < . ), and increased invasiveness (p < . ) in the st cell line as compared to par and ev when analyzed by one-way anova. conclusion: this initial study into st gal i in ovarian cancer may have future therapeutic implications, and, in addition, lend greater insight into the intraperitoneal dissemination of disease.of microarrays (sam), arrayassist and binary tree structured vector quantization. differentially expressed genes were integrated with a database of known predicted protein-protein interactions (ophid) and key genes are being validated with real-time pcr and immunohistochemistry. results: unsupervised hierarchical clustering revealed collective clustering of all tumors, irrespective of their pathological classification. sam analysis has highlighted probe sets as differentially expressed between lmp and lmp-mp, probes between lmp and lgsc, and probes between lmp and lmp-mp+lgsc. no differential gene expression was detected between lmp-mp and lgsc. ophid analysis demonstrated gene members of the egfr and mapk / pathways to be differentially regulated between the non-invasive and the invasive tumors. to date, we have successfully validated members of the mapk / pathway-poly adp ribose polymerase (ppol) and traf family associated nf kappa b activator (tank)-using real-time pcr. conclusion: our data demonstrate that although the tumors have related genetic profiles, lmp-mp and lgsc are similar to each other and different from lmp, in keeping with their clinical behavior. members of the mapk / and egfr pathways appear to play a key role in low grade serous cancer. identification of novel genes associated with malignant transformation, may lead to development of more effective targeted therapy for lgsc. background: approximately % of pap smears with the ambiguous diagnosis of atypical squamous cells, cannot exclude high grade (asc-h), are negative for dysplasia in follow up colposcopic examination and biopsy. although hpv testing provides excellent negative predictive value (npv) ( %), the prevalence of high risk hpv infection is high in young women and the positive predictive value (ppv) in asc-h pap smears is no better than cytologic diagnosis alone ( %). recent studies have shown that immunostaining for p ink a , or proex tm c, supports a diagnosis of dysplasia in surgical biopsies. our objective was to determine whether staining for p or proexc provides sufficient predictive value to reliably distinguish high grade dysplasia in asc-h pap smears. design: we retrospectively collected samples from liquid based pap smears diagnosed as asc-h at either ohsu or portland oregon kaiser permanente ( - ). known hsil and negative pap cases were included as immunostaining controls. asc-h cases with followup cervical biopsies (n= ) were included for subsequent immunostaining according to manufacture's instructions. samples were also sequestered for hpv testing (pending). immunostained slides were scored as positive or negative by two independent cytopathologists (as and tm) while blinded to pap diagnoses and surgical biopsy outcomes. results: we observed excellent agreement between pathologists' scores (pairwise kappa statistic . ). the correlation between p and proexc scores was moderate (kappa . ). chi-square analysis comparing staining to biopsy outcome revealed a significant association between proex c positivity and cervical dysplasia (*** p< . jing lin, zhenmin lei, ch v rao. ob/gyn women health, university of louisville health sciences center, louisville, ky, usa. introduction: matrix metalloproteinases (mmps) are a family of highly homologous zinc-dependent endopeptidases, which degrade all kinds of extracellular matrix proteins. the degradation process is required for tissue growth and remodelling. these enzymes are probably involved in uterine growth and devolopment and its differentiated functions. ovarian steroid hormones, estradiol (e ) and progesterone (p ), are known to regulate some of the uterine mmps. since it is now known that lh/hcg can directly regulate uterine growth and devolopment and its functions, we questioned whether these gonadotropins could also regulate mmps in the uterus. methods: sixty day old wild type (wt) and lh receptor knockout (lhrko) mice and -day e and p treated -day old animals were used. in addition, primary cultures of uterine epithelial cells from wt animals were treated for hrs either with no hormone or with a single or a combination of pg/ml of e or p , ng/ml of hcg. then mmp- , - and - mrna levels were quantified by the semiquantitative rt-pcr. results: while the uterine mmp- mrna levels were unaffected, mmp- mrna levels significantly decreased and mmp- mrna levels significantly increased in lhrko animals as compared with wt siblings. e and p treatment reversed mmp- and mmp- changes in lhrko animals. treatment of wt type primary endometrial epithelial cell cultures with hcg had no effect on mmp- mrna levels, but it did increase mmp- mrna levels. this increase was synergistic with both e or p . conclusion: while lh/hcg do not regulate uterine mmp- and mmp- , they seem to co-regulate mmp- with ovarian steroid hormones. cancer. radhika gogoi, christine ardalan, dorota popiolek, leslie gold, john curtin, stephanie v blank, bhavana pothuri. new york university, new york, ny, usa. objective: megesterol, a synthetic progestin with strong androgenic properties, is used in the medical management of patients (pts) with atypical endometrial hyperplasia (aeh) or endometrial carcinoma (emca). our hypothesis was that androgen receptor (ar) expression in the endometrium of aeh and emca pts would correlate with degree of response to treatment. methods: pre-and post-treatment endometrial biopsy specimens were obtained from pts treated with megesterol for aeh or emca. ar expression was investigated by immunohistochemical (ihc) analysis with appropriate positive and negative controls. ihc staining was scored for intensity ( - ) and percentage of positive cells ( - ) in the glandular and stromal compartments by a pathologist, blinded to clinical response data. a composite score utilizing both intensity and percentage of positive cells was calculated. we evaluated pre-and post-treatment ar expression in responders and nonresponders as well as ar expression in emca, aeh and normal endometrium. the mann whitney u and the wilxocon signed rank tests were utilized for statistical analysis. results: eight pts' pre-and post-treatment samples were obtained; with emca and with aeh. three pts had no response, , a partial response and , complete responses. ar expression in emca samples when compared to the normal endometrium was significantly lower in both the glands (mean . vs. ; p< . ) and the stroma (mean . vs. ; p< . ). although there was no statistically significant difference in glandular ar expression in the pretreatment biopsies of responders compared to nonresponders (mean . vs. . ; p= . ), there was a significantly higher level of glandular ar expression in the post treatment biopsies of responders compared to non-responders (mean . vs. ; p< . ). furthermore, we noted a trend towards higher levels of glandular ar expression in the post-treatment versus the pre-treatment biopsies in the responders (mean . vs. . ; p= . ). we noted a significant decrease in ar expression in emca compared to the normal endometrium. increased ar expression after treatment in responders suggests an important role of the ar in pts treated conservatively with progestational therapy, and needs further prospective validation as a means to predict treatment response in these pts. objectives: study the correlation between adenomyosis and some potential non-surgical risk factors. objective: o -methylguanine-dna methyltransferase (mgmt) acts to repair dna damaged by alkylation of guanine residues. mgmt promoter methylation and gene silencing is seen in a variety of cancers and pre-cancerous changes. the loss of mgmt activity is associated with increased sensitivity to alkylating agents and is a favorable prognostic indicator in gliomas. we sought to determine if mgmt promoter methylation plays a role in endometrial cancer. methods: one hundred and twenty primary endometrial cancers were analyzed for mgmt promoter methylation by combined bisulfite restriction analysis (cobra). the cohort included endometrioid endometrial cancers, endometrial tumors of adverse histologic type, and endometrial cancer cell lines. twenty one endometrioid and mixed endometrioid ovarian cancers were also analyzed. a subset of the primary tumors was analyzed for mgmt expression by immunohistochemistry. results: no mgmt promoter methylation was seen in the endometrial cancers evaluated or the endometrial cancer cell lines. one of the ovarian cancers showed methylation. immunohistochemistry for mgmt expression is ongoing. conclusion: mgmt promoter methylation is an infrequent event in endometrial cancer. mgmt expression in tumor cells and repair of alkylguanine residues could explain in part the limited response of endometrial tumors to alkylating chemotherapy. objective: uterine sarcomas ( % of gynecological malignancies) originate from uterine mesenchyma. patients with high-risk disease (i.e. high grade) or at advanced stages have poor -years overall survival (< - %). pelvic radiation and/or chemotherapy have not demonstrated to improve survival. identification of new therapies for this malignancy is a major goal. few in vitro models have been established to test therapeutic agents for uterine sarcoma. here we sought to establish a new human uterine sarcoma cell line and to test the effects of chemotherapeutic drugs: tnf-related apoptosis inducing ligand (trail) used alone or in combination. methods: tissue sample was obtained from a woman with uterine sarcoma undergoing hysterectomy. sarcoma cell lines were established using a published protocol for endometrial cancer. phenotypic characterization was made through the different passages ( to ) by western blot. levels of estrogen (er), progesterone (pr) and trail receptors were also studied (rt-pcr, w-b). cells were incubated with chemotherapy agents (cisplatin, paclitaxel and doxorubicin and trail) and cytotoxicity (mts assays) and apoptosis (flow cytometry, parp cleavage by w-b, and dna laddering) measured. results: human uterine sarcoma cell line was established from a highgrade uterine sarcoma. through the different passages the cell line remains expressing cytokeratin, vimentin, tissue factor, caveolin- and -actin. this cell line expresses low er and pr levels. trail receptors (r and r ) were also detectable (rt-pcr, w-b). cisplatin, paclitaxel and doxorubicin ( um for h) produced low cell cytotoxicity (< %). trail ( ng/ml for h) induced about % cell cytotoxicity. apoptosis was confirmed by parp cleavage. doxorubicin significantly enhances trail mediated cytotoxicity (up to %), this was demonstrated by a significant increase in the sub g /g region in the dna histogram. conclusions: we establish a human uterine sarcoma cell lines using protocols for endometrial cancer. more importantly, we demonstrated that doxorubicin enhances trail effect on this uterine sarcomas cell line. thus, this combination might be considered as a treatment for high-risk uterine sarcomas. (financial support fondecyt ). defining the tumor initiating capacity of cd + human endometrial cancer cells. anne m friel, petra a sergent, christine l cummings, rosemary foster, current data suggest rare subpopulations of cells with tumor initiating capabilities are a common feature of solid tumors. several investigators have recently identified cd , a cell surface marker expressed on primitive cells of neural, hematopoietic, endothelial and epithelial lineages, as a potential tumor initiating cell (tic) marker in solid tumors of the brain, colon and pancreas. in our efforts to investigate such a population in human endometrial cancers (enca), we developed an in vivo model that is based on serial transplants of tics from endometrial tumor explants. serial transplant experiments were initiated in nod/scid mice that were injected with primary human enca cells. a subset of cells derived from the generated tumor was subsequently transplanted into new recipients. tumors formed following serial transplantation retained the original histological phenotype of the primary enca, and the number of cells required to initiate tumor formation was reduced at each successive transplant stage suggesting an increase in the ratio of tics to non-tics. we exploited this model in our initial efforts to investigate the tumor initiating potential of cd -expressing enca cells. to evaluate the tumorigenic potential of cd + cells, the tumor initiation capacity of xenograft derived cd + and cd cells were compared following subcutaneous injection of each population into nod/scid mice. only the cd + fraction was tumorigenic consistent with the hypothesis that tumors are generated and maintained by a subpopulation of cells with phenotypically distinct profiles. we are further investigating the functional significance and characteristics of this fraction in vitro and in vivo. objectives. central issues in tumor biology are the understanding of factors that control tumor cell proliferation and the identification of extracellular matrix cues controlling the signaling transducing repertoire that make cancer cells proliferate and invade the host tissues. among these factors, small leucine-rich proteoglycans (srlps):decorin, fibromodulin, lumican, biglycan, are emerging as powerful modulators of angiogenesis and cell growth, by affecting several key elements including matrix assembly, growth factor binding, and receptor tyrosine kinase activity. recently has been demonstrated that srlps can act as a pan-erbb ligand and, in doing so, down-regulate the activity of one of the most potent oncogenic proteins, erbb , whose overexpression is linked to poor prognosis and increased cancer mortality in breast, ovary, and prostate. since srlps have not been previously investigated in the endometrial cancer biology, we investigated their role in the benign and malignant endometrial neoplasia. method. the sampling has been obtained in women (n= ; mean age ± . ) with endometrial hyperplasia (n= ), polyps(n= ), and cancer(n= ), during therapeutic and diagnostic procedures (hysterectomy, colpohysterectomy, hysteroscopy). physiological endometrial samples (n= ) were obtained from menopausal women (n= ; mean age ± . ), during procedures for others gynaecologic indications. immunohistochemestry was the biology technique used for the detection of srlps.results. in the physiological endometrial samples immunoreactivity for decorin, fibromodulin and biglycan was intense (+++), while it was weak in polyps and hyperplasia (+) and absent (-) in cancer. no significant difference in the staining of lumican between the physiological and pathological samples. conclusions. these results could provide a mechanism by which naturally occurring proteins normally synthesized by fibroblasts and smooth muscle cells, the two key components of the tumor stroma, may play a protective role in the genesis and progression of endometrial neoplasia counteracting the growth of neoplastic cells and suppressing tumor cell-mediated angiogenesis. although further studies are necessary to understand mechanisms whereby srlps might influence endometrial cell growth and survival, these molecules may represent potential target for pharmacological cancer therapy. myostatin is a member of the tgf-beta superfamily of protein and a wellknown inhibitor of skeletal muscle proliferation. the muscular component of the uterus is the myometrium, a tissue that regulates its mass in response to different physiologic conditions under the influence of steroids. we determined the expression of myostatin mrna in immortalized pregnant human myometrial (phm ) cells and we verified its biological activity. functional assays showed myostatin induced phosphorylation of smad- and reduction of proliferation of phm cells in a time and dose-dependent manner. to investigate the physiological relevance of our in vitro findings, the expression of myostatin in rat uterus was examined at various phases of the estrous cycle. for the first time we report that myostatin is expressed in rat uterus and that levels of myostatin mrna change during distinct phases of the estrus cycle. uterine levels of myostatin peaked during late estrous and were the lowest at pro-estrous. to further examine the role of steroids in myostatin regulation, we examined the effects of gonadal steroid administration in ovariectomized (ovx) rats. ovaryectomy increased myostatin expression compared to normal cycling controls. estrogen treatment strong decreased myostatin levels while progesterone produced less robust effects on myostatin expression. these findings taken together suggest an important role for myostatin in the regulation of myometrial functionality. her neu over-expression and pi kinase/akt pathway activation in paget's disease of the vulva. amy stenson, bita behjatnia, jaime shamonki, jianyu rao, amer karam, jonathan berek, oliver dorigo. ob/gyn and pathology, ucla, los angeles, ca, usa; ob/gyn, stanford university, palo alto, ca, usa. background: paget's disease of the vulva is rare with high recurrence rates. treatment of recurrent disease is challenging due to its extent and location. non-surgical approaches have limited clinical efficacy, obviating the need for novel therapies. in contrast to paget's of the breast with well-described overexpression of her neu, molecular features of vulvar paget's are poorly characterized. our objective was to study therapeutic targets in vulvar paget's, including the her neu protein and the phosphorylated oncoprotein akt (pakt). in addition, detailed clinical characteristics were correlated with molecular expression. methods: specimens with vulvar paget's disease were retrieved from ucla's department of pathology. protein expression was evaluated by immunohistochemistry on slides from paraffin embedded tissue using the hercep test (dako) for her neu expression and a standard protocol to assess expression of activated pakt. slides were scored by two independent pathologists based on a nominal scale of (negative) to (strongly positive). clinical data was retrieved via chart review. results: between and , patients with vulvar paget's were identified. median age was yrs ( - yrs). a family history of cancer was found in / ( %), / ( %) were smokers and / ( %) had a history of hormone use. intraepithelial lesions accounted for the majority ( / , %), while / ( %) demonstrated invasion and / ( %) were associated with underlying gi malignancy. / ( %) had at least one recurrence, with median time to recurrence months. so far, specimens were stained for her neu and pakt. overexpression of her neu was found in / ( %.) positive staining for pakt was evident in / ( %.) statistical analysis suggested a correlation between her neu and pakt expression. conclusions: our study demonstrates that overexpression of her neu and activation of the pi kinase/pakt pathway are important features of vulvar paget's disease. to the best of our knowledge, this is the largest series evaluating these molecular pathways in vulvar paget's. our data suggest that her neu and pakt may be important molecular targets for novel therapies using for example the monoclonal antibody trastuzumab directed against her neu, or a pi kinase pathway inhibitor like rapamycin. introduction: uterine leiomyomas are the most frequent tumor of the female reproductive tract and are the primary indication for hysterectomy in women worldwide. these tumors occur in up to % of adult women, and their prevalence is especially high in africa-american women. currently there is no effective and safe medical treatment for uterine fibroids and surgery is the main stay. epigallocatechin gallate (egcg) constitutes the main solid extract of green tea and is believed to contributes most of the antioxidant and antiinflammation capacity of green tea. egcg has been shown to have beneficial effects on prostate cancer and breast cancer by inducing apoptosis and inhibiting proliferation of cancer cells. in this study, we investigated the ability of egcg to inhibit proliferation of human leiomyoma cells (hlm) in vitro. methods: human immortalized leiomyoma cells were cultured at ºc in a humidified atmosphere of % co - % air in smbm medium supplied with %fbs, . % insulin, . % hfgf-b, . % ga- and . % hegf(lonza). the cells were plated at density of × cells/well in -well plates and grown under the same conditions above. the monolayer cultures at approximately % confluence were treated with various concentrations ( , . , . , , , and μm) of egcg (sigma) for days. a fluorometric assay using hoechst dye (sigma) for dna quantitation was conducted at the following designed time points, day , , , and post egcg treatment. the cells were lysed and dna content was determined using hoechst dye solution ( μg/ml). fluorescence was measured after excitation at nm and emission at nm. results: egcg exhibited marked anti-proliferation effect on the growth of hlm cells. compared with untreated control, the inhibitory effect of egcg on hlm cells was observed at μm and peaked at μm concentration. the difference was statistically significant (p = . ). evaluation of the mechanism of action of egcg is cuurently under investigation in the lab.conclusion: egcg significantly inhibited the proliferation of human leiomyoma cells in a dose-depended pattern. egcg may present a potential effective and safe medicinal treatment for uterine fibroids. objective: choriocarcinoma is an aggressive form of germ cell tumor that exhibits rapid growth with early metastases. choriocarcinomas autonomously secrete hcg which acts as an autocrine/paracrine growth factor in these cancers. we hypothesize that a novel hcg antagonist (hcg-ant) can limit tumor expansion by blocking hcg-induced expression of pro-invasive genes. we investigated if hcg-ant could alter rna expression of matrix metalloproteinases (mmp- and mmp- ), which facilitate basement membrane degradation and hence invasion, and metastin (kisspeptin), a strong suppressant of metastasis, in the choriocarcinoma cell line jeg- . design: in vitro experiments using the jeg- cell line. materials and methods: after plating jeg- cells in a well tray overnight in rpmi media containing % fbs, cells were washed twice with pbs and then cultured in ul of serum-free rpmi media containing one of four treatments: ) saline; ) hcg ( iu); ) hcg ( iu) plus hcg-ant ( iu); or ) hcg-ant ( iu). rna was extracted from each well using trizol and reverse transcribed using sensiscript (qiagen). the relative expression of mmp- , mmp- , and metastin mrna was quantified using sybr-green based real time pcr. the expression of the housekeeping gene hprt was used to normalize expression data. results: treatment of jeg- cells with hcg-ant vs. hcg alone reduced expression of mmp- ( . ± . vs. . ± . ) and . hcg-ant reduced mmp- and mmp- expression by % and %, respectively (p< . ). treatment with hcg-ant vs. hcg increased metastin expression ( . ± . vs. . ± . ). metastin expression was increased by % following hcg-ant treatment. conclusion: treatment of the jeg- choriocarcinoma cell line with hcg-ant reversed the increased expression of mmp- and mmp- following treatment with hcg. metastin expression was increased by hcg-ant. this data suggests that hcg antagonism is capable of altering gene expression thereby inhibiting invasion in a choriocarcinoma cell line. the role of hcg-ant as an adjuvant therapy in hcg sensitive tumors offers promise. retinoids, but not progesterone, directly induce differentiation and apoptosis of endometrial cancer cells. you-hong cheng, serdar e bulun. ob/gyn, northwestern university feinberg school of medicine, chicago, il, usa. objectives: the opposing actions of estrogen and progesterone during the menstrual cycle regulate endometrial proliferation, differentiation and secretion. the unopposed action of estrogen contributes to the development of type i endometrial cancer. however, the mechanisms for progesterone protection of estrogen-induced carcinogenesis in endometrium remain unclear. methods and results: in the current study, we demonstrated that retinoids ( -cis retinoic acid (ra) or all-trans ra (atra)) significantly inhibited basal and hormone-stimulated ishikawa cell proliferation by over % using mtt assay. the same experiment indicated that estrogen had no significant effect, whereas progesterone slightly induced, on cell proliferation. cell cycle analysis indicated that atra significantly increased the g /g cell population by % and decreased s phase cells by %, suggesting that ra induces cell cycle arrest at the s phase. knock-down of rar alone or both rar and rxr significantly increased proliferating cell nuclear antigen (pcna) levels in epithelial ishikawa cells, suggesting that ra signaling via rar/rxr activation is critical for normal endometrial growth and differentiation. to determine whether retinoids are naturally secreted by the endometrial stromal cells, we cultured primary stromal cells and analyzed the culture media using hplc. we found that retinol is the predominant retinoid form secreted by stromal cells. the average concentration of retinol in the cultured media of eutopic endometrial stromal cells was approximately to ng/ml/ cells (n= ). although there was less than . ng/ml/ cells of all-trans retinal or atra in the cultured media, we did find a small peak for all-trans retinal and atra in the media using hplc analysis. furthermore, progesterone significantly increased secreted retinol levels from eutopic endometrial stromal cells, but decreased retinol levels secreted from endometriotic stromal cells. retinol is a precursor for ra that is converted to retinal and then to ra. conclusions: we demonstrated that progesterone signaling via pr induces endometrial stromal cells to secrete paracrine retionids that in turn control the phenotype of adjacent epithelial cells. conversely, this interaction is disrupted in diseased endometrial tissues, such as endometrial cancer and endometriosis. the effects of hormonal contraceptives on antimullerian hormone by obesity status. anne z steiner, frank z stanczyk, stan patel, alison edelman. ob/gyn, university of north carolina, chapel hill, nc, usa; ob/gyn, usc keck school of medicine, los angeles, ca, usa; ob/gyn, oregon health and sciences university, portland, or, usa. background: antimullerian hormone (amh) is emerging as a predictor of reproductive potential. serum levels of fsh, a commonly used measure of ovarian reserve, are suppressed with the use of oral contraceptives (ocs) thereby limiting its use. the impact of ocs and on serum amh levels in normal and obese women is unknown. objective: to examine the impact of ocs on serum amh levels by obesity status in reproductive-age women. materials and methods: ovulatory women, ages - years, of normal (< kg/m ; n = ) and obese (> kg/m ; n = ) body mass index (bmi) received a low dose oc ( mcg ethinyl estradiol/ mcg levonorgestrel) for two cycles. serum was obtained at three time points: after days of active pills (cycle , day ), at the end of the -day hormone-free interval (cycle , day ), and during the first week of active pills in cycle (cycle , day , , or ). amh levels were quantified by elisa; fsh and lh levels were determined by chemiluminescent immunoassay. amh at the three time points was compared using repeated measures anova. models were used to assess the relationship between amh and cycle day by obesity status. amh and gonadotropin levels were compared using spearman's correlation. results: amh levels did not differ by oc cycle day (p anova = . ) or by active versus placebo pill use ( . ng/ml ± . vs. . ng/ml ± . , p= . ) among normal bmi women. among obese women, amh levels differed by oc cycle day (p anova = . ), but not by use of active or placebo pill ( . ng/ml ± . vs. . ng/ml ± . , p = . ). across the cycle, cv(standard deviation/mean) averaged . %± . in the obese and . %± . in the normal bmi women ( p= . ). modeling to determine differences in amh throughout the cycles based on obesity status showed a significant interaction (p = . ) and lower amh levels in the obese group (p< . ). among both bmi groups, serum amh and fsh levels did not correlate during active pills or after days of placebo pills. conclusions: in young, normal bmi women serum amh levels do not appear to fluctuate during oc use. among obese women, amh levels are lower and fluctuate significantly. these fluctuations do not appear to mirror changes in gonadotropins and may complicate clinical interpretation of amh. background: menopausal hormone therapy (ht) is a confusing topic for many clinicians and patients. objective: to assess comprehension of basic clinical trial features among prospective participants for the keeps trial, designed to study the effects of ht initiated within years of menopause on chd markers. methods: screening materials were provided giving an overview of study purpose, duration, medications, likelihood of receiving drug vs. placebo, ht related risks and side effects. at a subsequent interview, a -item questionnaire assessed the participant's level of comprehension. a score of % was adequate to complete the informed consent process. those scoring < % were re-counseled and retested. demographic variables determining the likelihood of an initial score > % were evaluated by univariate and multivariable analyses. results: % ( / ) scored > % on initial testing. all women scored > % after re-counseling and retesting. likelihood of an initial response > % correct was unrelated to age or time since menopause. ability to correctly respond was influenced by highest educational level attained. none of women whose furthest educational level was high school scored > % on initial testing, significantly less than those with a college education (p= . ). a higher proportion of college graduates ( / ) scored > % compared to those attaining further education ( / ) (p= . ). comprehension was greatest for study purpose and duration ( / and / correct responses respectively) and least for questions related to results of the whi hormone trial breast cancer and chd. that e alone was not associated with an increased risk of chd ( %) or breast cancer ( %) was poorly understood. conclusion: comprehension of the risks and benefits of ht by potential research subjects is low despite provision of reading materials prior to the informed consent process. (supported by the montefiore medical center/albert einstein college of medicine site for the kronos longevity research institute and k -hd to ns). variation in menopausal symptoms within a sample of hispanic women: swan, the study of women's health across the nation. robin r green, alex j polotsky, aileen p mcginn, carol a derby, rachel p wildman, lhasa ray, kavitha t ram, gerson weiss, nanette f santoro. albert einstein coll of med, bronx, ny; univ of med and dent of new jersey, newark, nj. background: menopausal symptoms are experienced by over % of women. purpose: to describe symptom frequency in a sample of midlife hispanic women from different countries of origin. methods: the study of women's health across the nation (swan) recruited women at baseline who self-identified as hispanic. their baseline responses to validated questionnaires regarding common menopausal symptomatology were examined. symptoms were reported over the previous two weeks and scored on a frequency scale ranging from (not at all) to (every day). for all analyses, symptoms were dichotomized into "absent" vs. "present" variables. responses were correlated with acculturation ( -item scale: marin, hisp j behav sci : , ) and analyzed by sub-ethnicities: central/south american (c/s am), puerto rican (pr), dominican (dr), and cuban (cu). associations between symptoms and sub-ethnicity were tested by chi-square. logistic regression was used to test for associations with acculturation and being us-born. there was significant variation in several menopausal symptoms. while puerto-rican women had the highest likelihood of reporting trouble sleeping (or= . , %ci: . - . ) and headaches (or= . , %ci: . - . ), dominican women were most likely to report vaginal dryness (or= . , %ci: . - . ) acculturation and being us-born did not explain the variation between subethnicities in any of the models tested conclusion: there appear to be significant differences among hispanic women with respect to menopausal symptomatology. these differences were not readily explained by measures of acculturation. (supported by the study of women's health across the nation (swan) has grant support national institutes of health, dhhs, through the national institute on aging, from the national institute of nursing research and the nih office of research on women's health (grants nr ; ag , ag , ag , ag , ag , ag , ag , ag and cd ). purpose: to compare the relative effects of conjugated equine estrogen, raloxifene, and tamoxifen therapies on cognition among women aged years or older. participants and methods: annual modified mini mental state ( ms) examinations were used to assess global cognitive function among the , women enrolled in the two randomized placebo-controlled clinical trials of the women s health initiative memory study (whims) and women enrolled in co-star, the cognitive substudy of the nsabp's study of tamoxifen and raloxifen (star) trial who had baseline ms testing. associations between baseline cognitive risk factors common to both trials and baseline ms scores were assessed and interactions used to examine whether risk factor relationships differed between cohorts. factors for which relationships were similar were used as covariates in analyses comparing ontrial ms scores. factors for which relationships did not appear to be similar were used to stratify analyses. results: compared to placebo, each of the active therapies was associated with a small mean relative deficit in ms scores of . units or less, which was fairly consistent between women with and without prior hysterectomy. overall, relative deficits appeared to be most marked for tamoxifen (unadjusted p= . ) but were also evident for raloxifene (p= . ) and cee (p= . ). conclusions: while unmeasured differences between trials may have confounded our analysis, these findings suggest that both tamoxifen and raloxifene may adversely affect cognitive function in older women. weight gain and increased abdominal fat have been found in women after menopause and is associated with higher levels of leptin, and decreased levels of the cardioprotective adipocytokine adiponectin. at the same time, bmd characteristically decreases. in an effort to determine the evolution and correlates of these changes, we studied postmenopausal women (pm) within years of menopause (age . ± yrs) of normal weight (bmi . ± ) and compared them to weight matched (bmi . ± ) premenopausal (pre) controls (age . ± yrs.) all subjects had bmd and body composition studies by dexa and measurements of leptin, adiponectin, visfatin and retinol-binding protein (rbp .) while total fat was similar in the groups, pm had more trunk and abdominal fat ( ± ; ± g) compared to pre ( ± g p< . ; ± g p< . ) pm also had greater %trunk fat and %central abdominal fat compared to pre, p< . .serum leptin ( . ± . vs ± pg/ml) and visfatin ( . ± vs. . ± . ng/ml) were similar but adiponectin ( ± vs. . ± g/ml) and rbp ( . ± vs. . ± ng/ml) were higher, p< . in pm. while in pre, abdominal fat correlated negatively with adiponectin (p< . ) in pm only leptin correlated with various parameters of fat mass, p< . , and adiponectin did not correlate but correlated positively with age (r . , p< . .) as expected, pm had reduced bmd at the lumbar spine and hip ( . ± . vs. . ± . g/cm ; . ± . vs. . ± . g/cm , p< . ) but there was a correlation between total and trunk fat in pm and lumbar bmd (r . , . , p< . ) but not with hip bmd; or any correlations in pre. there was a correlation between leptin and lumbar bmd in pm (r . , p< . ) but not in pre. in summary, in normal weight early pm, abdominal fat is increased, but only adiponectin and rbp are altered with an increase in the former correlating with age. lumbar bmd correlated with fat mass in pm and is partially explained by increases in leptin. it is suggested that in spite of increasing abdominal fat in normal weight early pm, (which correlates with a higher lumbar bmd) david d rahn, jesus f acevedo, r ann word. ob-gyn, ut southwestern, dallas, tx, usa. objectives: matrix metalloprotease (mmp) activity is increased in the postpartum vagina of wild type (wt) animals, and this activity is accompanied by a burst in elastic fiber synthesis. the mechanisms that precipitate these changes are unclear. the goals of this study were to determine how vaginal distention (such as in parturition) affects elastic fiber homeostasis in the vaginal wall and the potential significance of these changes in the pathogenesis of pelvic organ prolapse. methods: nonpregnant ( ) and pregnant (d ) wt mice underwent either vaginal distention with a μl balloon x min (to simulate parturition) or sham procedure. tissues were obtained at and h for immunoblot analysis, zymography, and histology. distention was also performed in young fbln -/-, fbln +/-, and wts, and prolapse was quantified for weeks. results: distention resulted in marked increases in mmp activity in nonpregnant animals (prommp , . -fold; active mmp , . -fold; prommp , . -fold; active mmp , . -fold; all p < . compared with sham) which was accompanied by fragmented and disrupted elastic fibers in the vaginal wall. abundant amounts of tropoelastin and fibulin- in the nonpregnant vagina were not increased further by distention. in pregnant animals (which normally have suppressed vaginal wall fibulin- and tropoelastin), however, distention resulted in -fold upregulation of both proteins (p< . ). distention also induced increased mmps in pregnant animals (mmp- , . -fold; prommp- , . -fold; active mmp- , . -fold; all p < . ). thirteen young fbln -/-( - wks prior to age of prolapse development), het siblings, and age-matched wts underwent serial exams after ballooning. distention induced rapid increases in perineal bulge and vaginal diameter (within d) in fbln -/mice which never recovered. conclusions: in pregnant mice, vaginal distention results in increased protease activity in the vaginal wall but also increased synthesis of proteins important for elastic fiber assembly. distention may thereby contribute to the burst of elastic fiber synthesis in the postpartum vagina. the finding that distention results in accelerated pelvic organ prolapse in fbln -/animals, but not wt, indicates that distention results in loss of pelvic organ support if elastic fiber synthesis is compromised. further, the data suggest that elastic fiber synthesis is crucial for recovery of the vaginal wall from parturition/distention-induced increases in vaginal protease activity. the molecular etiology of pelvic organ prolapse (pop) is complex and not yet well understood. defects in the connective tissue, such as a decrease in extracellular matrix (ecm) protein content may predispose women to pop. our objective was to study the expression and the enzymatic activity of matrix metalloproteinases (mmps) and their tissue inhibitor (timps) in vaginal tissue of patients with advanced pop and controls. after informed consent, pre-menopausal caucasian patients affected by pop ( grade by pop-q), and control patients (no pop) matched for age and bmi, undergoing vaginal and abdominal hysterectomy respectively were recruited. full thickness anterior vaginal epithelial tissue was obtained from the surgical cuff of patients and controls in the proliferative phase of the menstrual cycle. total protein was extracted using ripa lyses buffer. western immunoblot analysis was performed (patients: n= , controls: n= ) to study the protein expression of mmp- , - , timp- , - , - . gelatin zymography was used to quantify the activity of mmp- and - . immunohistochemical analysis for timp- and - was performed on pfa-fixed vaginal biopsy tissue (n= ) in each group. both patient and control vaginal biopsy samples expressed latent and active forms of mmp- , and mmp- . the protein expression of the kda active form of mmp- was significantly increased in patients with pop compared to controls (p= . ). zymography detected the enzymatic activity of the proform and active form of both mmp- and mmp- . we found a significant increase in gelatinolytic activity of both kda pro-form and kda active forms of mmp- (p= . and p< . respectively) as well as kda active form of mmp- (p< . ) in patients with pop compared to controls. timp- protein expression in vaginal tissue showed a significant reduction in pop patients compared to controls (p= . ). timp- and - immunostaining were mainly localized in the smooth muscle cells at the muscularis layer of the vaginal biopsies. in vaginal tissue of patients with pop, we have shown a decrease in timp protein expression paralleled by an increase in mmp protein expression and activity. these findings reflect an active ecm remodelling that may compromise the structural integrity of the pelvic floor leading to pop. aberrant elastin and collagen synthesis may play a role in the pathogenesis of pelvic organ prolapse (pop). the lysyl oxidase (lox) family of enzymes direct cross linking of collagen and elastin polymers, however to-date no information is available on the expression and localization of these proteins in human vaginal tissue. our objectives were to study the expression and in situ localization of lox, loxl , loxl and loxl proteins in the vaginal tissue of patients with advanced pop and asymptomatic controls. pre-menopausal caucasian patients affected by pop ( grade by pop-q) and control patients (no pop) matched for age and bmi, undergoing vaginal and abdominal hysterectomy respectively were recruited. full thickness anterior vaginal epithelial tissue was obtained from the surgical cuff of patients and controls in the proliferative phase of the menstrual cycle. total protein was extracted using ripa lyses buffer and western immunoblot analysis was performed (patients: n= , controls: n= ). pfa-fixed vaginal biopsy tissues (n= for each group) were used for immunohistochemical study. vaginal biopsy samples from both patient and control groups expressed all four members of lox family proteins: lox ( kda pro-form and kda active form), loxl ( kda pro-form and kda active form), loxl ( kda) and loxl ( kda). the expression of all lox family proteins was reduced in patients with pop compared to controls; however only the pro-form of lox of making the diagnosis of endometriosis from an endometrial biopsy. objective: to evaluate the diagnostic value of examining endometrial biopsy specimens for small nerve fibers in women with pelvic pain or infertility in a double-blinded prospective comparison with laparoscopy. methodology: we undertook to compare the detection of endometrial nerve fibers with laparoscopy and peritoneal biopsy for the diagnosis of endometriosis in women (aged . years; range - years) who presented with chronic pelvic pain or infertility. small nerve fibers were detected in the functional layer of endometrium using immuno-histochemical staining with the highly specific pan-neuronal marker pgp . , using a carefully validated technique and blinded assessment of nerve fiber density. laparoscopic assessment of the presence of endometriosis and peritoneal biopsies was undertaken by three experienced gynecologic endoscopic surgeons. data from these assessments were recorded independently of endometrial findings and maintained under blinded coding until the codes were broken. results: small nerve fibers were detected in all of the women in whom endometriosis was surgically diagnosed and in none of the women in whom endometriosis was excluded at laparoscopy, giving the specificity and sensitivity of %. the density of nerve fibers in the endometriosis cases was . per mm²± . (mean ± sd). conclusions: endometrial biopsy provided a reliability of detection or exclusion of endometriosis which was equal to that of diagnostic laparoscopy carried out by experienced gynecologic laparoscopists. background: erk / are mapk intracellular signaling proteins involved in cell survival and differentiation. endometriosis requires angiogenesis for ectopic implant growth. we hypothesized that the endometriotic peritoneal environment, known to be high in estrogen (e ), vegf, and cytokines such as il- and mcp- , stimulates angiogenesis in human endometrial endothelial cells (heec) through erk signaling. methods: serial sections from normal (n= ) and ectopic (n= ) endometrium were immunostained for total-(t-) and phospho-(p-) erk / and cd (an endothelial progenitor cell marker); results were quantified by computer densitometry and grouped by menstrual phase. cultured normal heec were treated with control, e ( - m), il- , mcp- , and vegf (all ng/ml) for min, and western blotted for p-/t-erk (n= ). heec were treated with peritoneal fluid (pf; diluted : in basal media) from normal (n= ) and endometriotic (n= ) women, with or without pd erk / inhibitor ( m) for h, and cell viability was analyzed by mtt assay. statistical analysis was done with one-way anova. results: tissue staining revealed that ectopic cd + endothelial progenitor cells undergoing angiogenesis (vessel sprouting and/or angiogenic cell cord formation) exhibited the strongest levels of p-erk. heec of ectopic foci showed moderate-high p-erk staining, while surrounding mesothelial capillaries were weak for p-erk. in normal endometrium, p-erk was cycledependent, with low levels in the late secretory phase vs. other phases (p< . ). p-erk of ectopic heec showed no cycle dependence, with moderate-high levels in all phases. t-erk in all tissues was high and invariable. in cultured heec, treatment with pf from endometriotic women significantly increased heec viability after h compared to normal pf (p< . ). this effect was abrogated by erk / inhibitor. among factors known to be high in endometriotic pf, vegf increased p-erk in cultured heec in and min (p< . ), while e , il- , and mcp- had no effect. conclusions: high p-erk found specifically in sprouting endothelial progenitor cells and focal ectopic capillaries suggests that erk / is involved in endometriotic angiogenesis. the peritoneal microenvironment of endometriosis may be persistently stimulating erk-mediated endometrial angiogenesis through vegf. further investigation into erk / inhibitors may offer a novel treatment of endometriosis. the events leading to the development of post operative adhesions are unknown, though recent observations emphasize the crucial role played by the superoxide ion generated by hypoxia. exposure of normal peritoneal fibroblasts to hypoxia caused the development of the adhesion phenotype, which is characterized by increased extracellular matrix molecules and inflammatory cytokines as well as high protein nitration and low nitric oxide. superoxide dismutases (sod) are a family of metalloenzymes that eliminates superoxide by converting it to hydrogen peroxide, protecting mammalian organisms against superoxide. objective: to determine whether sod is differentially expressed between normal peritoneal and adhesion fibroblasts and whether its expression is modulated by hypoxia. methods: fibroblasts from normal peritoneal and adhesion tissues were cultured under normal ( % o ) and hypoxia ( % o ) conditions for and hours. real time rt/pcr was utilized to measure the absolute mrna levels for sod in both cell lines before and after hypoxia exposure. results: normal peritoneal fibroblasts exhibited significantly higher basal mrna levels for sod ( . pg/ grna, p< . ) as compared to adhesion fibroblasts ( . pg/ grna, p< . ). short exposure to hypoxia resulted in a significant increase in sod mrna levels to . and pg/ grna in normal and adhesion fibroblasts respectively, p< . . in contrast, long exposure to hypoxia resulted in a significant decrease in sod mrna levels to . and . pg/ grna in normal peritoneal and adhesion fibroblasts respectively, p< . . conclusion: sod mrna levels are lower in adhesion as compared to normal fibroblasts, both basally and following short and longer exposure to hypoxia. reduced sod expression creates an milieu with greater free radical levels, which leads to the development of the adhesion phenotype. enhancement of sod levels and/or function are likely to be of benefit for reduction of postoperative adhesion development. objectives: development of endometriosis requires ectopic attachment and proliferation of endometrial tissue. the invasive process required to establish endometriosis may involve matrix metalloproteinases (mmps), including mmp- . recently, we have demonstrated that statins inhibit proliferation of endometrial stroma. this study evaluated the effects of simvastatin on a nude mouse model of endometriosis and on modulation of mmp- . methods: proliferative phase human endometrial biopsies were established as organ cultures or utilized for stromal cell isolation. to establish endometriosis in the nude mouse, endometrial tissues were maintained in nm estradiol (e) for hrs and subsequently injected intraperitoneally into ovariectomized nude mice. mice were treated with e ( μg, silastic capsule implants) and simvastatin ( - mg/kg/day) by gavage for days beginning day after tissue injection. control mice received e+vehicle. subsequently, animals were sacrificed and endometrial implants were evaluated. studies of endometrial stroma involved plating the cells in the presence of e ( nm) or e+medroxyprogesterone acetate (mpa; pm) with and without simvastatin ( - μm). some cultures additionally received interleukin- (il- , ng/ml). conditioned media was subjected to western analysis for expression of mmp- . results: in vivo studies demonstrated a dose-dependent inhibitory effect of simvastatin on number and volume of endometrial implants in mice. at the highest dose of simvastatin ( mg/kg/day), the number of endometrial implants was decreased by % and the volume by % in comparison to the control group. isolated stromal cells expressed abundant levels of mmp- following treatment with e, but minimal levels in cultures additionally receiving mpa or simvastatin. although il- induced a dramatic increase in mmp- secretion from cells pretreated with e alone, treatment with either mpa or simvastatin prevented this induction. cultures receiving e+mpa+simvastatin were the most resistant to mmp- induction by il- . objective: to increase awareness of potential presentations of adenomyosis and the differential of a uterine mass. material and method: in a tertiary medical center, a patient was being evaluated for a uterine cystic mass and cyclic dysmenorrhea. the patient is a year old nulliparous woman who has been complaining of cyclic dysmenorrhea for several years. the pain starts with the onset of menses and lasts around weeks. the patient did not improve on contraceptives. the patient had prior laparoscopy and imaging studies which misdiagnosed the mass as either a leiomyoma or an adnexal hemorrhagic cyst. the patient underwent exploratory laparotomy and resection of the mass. results: on ultrasound, the mass appeared as an echodense cyst within the uterus. intraoperatively, a cm thick-walled well circumscribed uterine chocolate cyst was identified. the resection of the cyst was performed in similarly to an ovarian cystectomy. tissue examination confirmed the diagnosis of cystic adenomyosis. following surgical intervention, the patient reported significant improvement of symptoms. conclusion: this case highlights an unusual presentation and treatment of adenomyosis and cyclic dysmenorrhea. cystic adenomyosis should be on the differential of uterine cystic mass, particularly in young women with cyclic dysmenorrhea and menorrhagia. the earlier misdiagnosis probably resulted from the unfamiliarity of physicians with this condition. similar clinical presentations may occur with congenital uterine anomalies (with an obstructed uterine segment) and cystic degeneration of a leiomyoma. the incidence and pathogenesis of cystic adenomyosis are unknown. oral contraceptives may be helpful as a first line therapy. however, resection of the adenomyotic cyst appears to be more effective, particularly in women seeking fertility. objective: it is hypothesized that abnormalities within the eutopic endometrium of females with endometriosis can cause the ectopic growth of the endometrial tissue at extrauterine sites. previous studies have shown that gene expression in eutopic endometrium of women with endometriosis is markedly different from disease-free females. inflammatory cytokines and receptor-dependent kinase pathways are widely recognized as therapeutic targets for immune disorders, which is believed to be the underlying pathogenesis of endometriosis. in this study we asked whether responses of primary eutopic endometrial cell cultures are dysregulated between females with or without endometriosis. methods: a total of biopsies of endometriotic eutopic endometrium (eee) and disease-free endometrium (dfe) were obtained from proliferative phase females. the primary cell cultures established from these biopsies were treated with tnfa to induce expression of inflammatory mediators. parallel cultures were also treated with kinase inhibitors of p , mek, pi k and ikk. after a period of or hours, concentrations of il- , mcp- , and gm-csf in cell culture supernatants were analyzed by elisa. results: in eee, basal concentrations of mcp- and gm-csf were - times higher, while il- was times higher compared to dfe. as expected, tnfa treatment stimulated higher levels of cytokine secretion in dfe mimicking disease state, however, the same treatment had almost no effect on eee. kinase inhibitors were very effective in lowering the cytokine levels of dfe, however, their effect on eee were less dramatic. conclusion: eee expresses higher levels of inflammatory cytokines under basal conditions, which is in concert with the current literature. our results validate that high il- levels in endometrium are diagnostic for females with disease. the increase of gm-csf, il- and mcp- following tnfa treatment was expected in dfe however; tnfa's effect was blunted in eee, which implies that eee are already highly activated. the effect of kinase inhibitors on cytokine production from eee was unaltered relative to dfe, which implies that tnfa stimulated kinase pathways are modified even in eee. endometrial cancer is the fourth leading cause of cancer in women, and hyperplasia and adenocarcinoma are more commonly seen in women with polycystic ovary syndrome (pcos). mig- , a negative inhibitor of egf signaling, is expressed in normal secretory phase endometrium and associated with hyperplasia in mig- knockout mice. objective: we examined and compared endometrium from normal and pcos women for mig- expression and characterized its regulation using in vivo and in vitro models. methods: immunohistochemistry (ihc) and real-time pcr were performed in endometrium from normal (n= ) women and pcos (n= ) women. regulation of mig- was studied in ishikawa and ecc- endometrial cell lines, treated with sex steroids or egf. endometrial xenografts from normal and pcos women were implanted in ovxd rag -(c) immunocompromised mice, treated with e or e + p pellets to mimic a natural cycle. results: mig- protein expression was low in the functionalis of the proliferative phase and high in the secretory phase; this pattern was reversed in the basalis layer. pcos secretory phase endometrium had significantly less mig- protein and mrna than normal endometrium (p < . ). xenografts using pcos samples had paradoxically elevated expression of mig- compared to normal, and appeared unresponsive to steroid treatments. mig- expression in endometrial cell lines was regulated by egf but not by ovarian steroids, e or e + p. conclusions: mig- expression is low in proliferating endometrium and regulated by egf. risk of hyperplasia or cancer in pcos women can be explained by altered expression of mig- . reduced expression mig- provides insight into endometrial function and may lead to better treatment options for disorders of endometrial proliferation including endometriosis, adenomyosis, endometrial hyperplasia and cancer. supported in part by nih-u -hd (sly), u hd (lcg) and u hd (fjd). could androgens influence human luteal cells function? anna tropea, mariateresa orlando, maria francesca gangale, federica romani, isamaria loiudice, federica tiberi, stefania catino, antonio lanzone, rosanna apa. cattedrà di fisiopatologia della riproduzione, università cattolica del s. cuore (ucsc), roma, italy; istituto scientifico internazionale (isi) "paolo vi", ucsc, roma, italy; istituto di ricerca "associazione oasi maria ss onlus", troina (en), italy. in pcos patients reproductive dysfunctions are frequently observed even if ovulation occurs. an impaired luteal function could partially explain this subfertility, since luteal steroidogenesis deficiency and premature luteal degeneration have been described in pcos patients. based on the frequent observation of high plasmatic levels of androgens in pcos, we investigated whether hyperandrogenism could negatively affect luteal function.to this aim, we tested the in vitro effects of androgens on progesterone (p) and on vascular endothelial growth factor (vegf) production by human luteal cells. indeed, vegf is essential for normal luteal development and function being an important regulator of angiogenesis and vascular permeability. since prostaglandins (pgs) play a central role in modulating luteal function, the influence of androgens on pge and pgf a secretion was also investigated. in order to investigate whether testosterone and androstenedione act directly or after local aromatisation, we tested the in vitro effects of estriol, estrone and --estradiol on p, vegf, pge and pgf secretion by human luteal cells. moreover, we tested the effects of testosterone and androstenedione in presence of exemestane -an aromatase inactivator. highly purified human luteal cells were cultured for h with medium alone (control) or in presence of increasing concentrations of testosterone or dihydrotestosterone or androstenedione (from - to - m) or in presence of increasing concentrations of estriol, estrone and --estradiol (from . to ng/ml). moreover, testosterone and androstenedione - m were tested in presence of exemestane (from to nm). in the culture medium vegf, p, pge and pgf secretion was assayed by commercially available elisa kits. in order to evaluate the influence of androgens and estrogens on vegf mrna expression on luteal cells real-time rt-pcr has been performed. our results demonstrated that testosterone, androstenedione and dihydrotestosterone were all able to significantly reduce vegf secretion in human luteal cells, while no effect was seen on vegf mrna expression. androgens were also able to significantly decrease p and pgf secretion, while an increase was observed on pge production. moreover our preliminary results demonstrated that in human isolated luteal cells estriol, estrone and -estradiol at all tested doses are able to mimic androgens effects on p and pge production. on the contrary estrogens were able to increased vegf release. estrogens seemed to have no effect on pgf  released. data regarding exemestane inhibition of testosterone and androstenedione are still in progress. increased levels of androgens were able to decrease luteal vegf, p and pgf release and might be involved in pcos-luteal phase defect. nevertheless, the observed effects could probably be attributed -at least in part -to estrogens locally produced via the aromatase enzyme, rather than be directly mediated by testosterone and androstenedione. the in vitro effect of proghrelin-derived peptides on purified human luteal cells. anna tropea, mariateresa orlando, maria francesca gangale, federica romani, isamaria loiudice, federica tiberi, stefania catino, antonio lanzone, rosanna apa. cattedrà di fisiopatologia della riproduzione, università cattolica del s. cuore (ucsc), roma, italy; istituto scientifico internazionale (isi) "paolo vi", ucsc, roma, italy; istituto di ricerca "associazione oasi maria ss onlus", troina (en), italy. ghrelin, well known mediator of neuroendocrine effects, has been demonstrated to have a role as signal for energy insufficiency. several evidences suggested that ghrelin might also operate as regulator of reproductive function. indeed, we recently demonstrated that both p and vegf released were significantly decreased by ghrelin in isolated human luteal cells. moreover ghrelin was also able to reduce prostaglandin (pg) e and increase pgf luteal cells release. in the present work we investigated whether two ghrelin-related peptides derived by the same ghrelin precursor (unacylated ghrelin and obestatin) might affect human isolated luteal cells function. conventionally regarded as an inert form of ghrelin, unacylated ghrelin has been recently proven as biologically active in specific cellular contexts. obestatin has been suggested to directly control porcine ovarian cell functions. in these assumptions, we investigated whether unacylated ghrelin and obestatin could directly affect luteal progesterone (p) release. moreover, since vascular endothelial growth factor (vegf) is known to play a critical role in luteal development and function, the influence of unacylated ghrelin and obestatin on luteal vegf, pgf and pge production was also analysed. highly purified human luteal cells were incubated for h with medium alone (control) or with hcg ( ng/ml) or with cocl ( μm) or in presence of increasing concentrations of unacylated ghrelin ( - nm) and obestatin ( - nm). in the culture medium vegf, pgf , pge and p secretion was assayed by commercially available elisa kits. moreover real-time rt-pcr has been performed in order to evaluate whether unacylated ghrelin and obestatin could affect vegf mrna in human luteal cells. our preliminary results demonstrated that either unacylated ghrelin or obestatin are able to negatively affect luteal steroidogenesis. moreover, both peptides seemed to increase the release of two luteotropic factors -vegf and pge and to reduce pgf release -a luteolytic prostanoid -from isolated human luteal cells. finally data regarding the expression of vegf mrna are still in progress. our results suggest that unacylated ghrelin and obestatin -two ghrelin-related peptides -could play a role in regulating luteal function affecting both luteal steroidogenesis and luteotropic/luteolytic imbalance. the mechanisms responsible for labor progression have yet to be fully elucidated. in a previous study, over-expression of small conductance calcium-activated k + channel isoform (sk ) in transgenic mice compromised parturition, suggesting a role for sk channels in this process. based on these findings, we hypothesized that sk channel expression is reduced late in pregnancy to enable the uterus to produce the forceful contractions required for parturition. we investigated the effects of sk channel expression on gestation and parturition by comparing transgenic mice over-expressing mice sk (sk t/t ) mice to wild-type (wt). in wt mice, sk transcript and protein are significantly reduced during pregnancy. the force produced by uterine strips from sk t/t mice on the day of delivery was significantly less than wt or sk knockout control (sk dox ), and the contractile force reached wt levels by application of the sk channel inhibitor, apamin. moreover, lipopolysaccharide and ru , which induce pre-term labor in wt mice, failed to result in completion of delivery in sk t/t mice. thus, stimuli that initiate parturition under normal circumstances are insufficient to coordinate the uterine contractions needed for the completion of delivery when sk channel activity is in excess. the mechanism(s) down-regulating this channel in the uterus during pregnancy is unknown. the sk gene promoter region contains two specificity protein (sp) binding sites; ) sp , is a transcription factor known to enhance the transcription of genes in response to estrogen, and ) sp , which competes for the same binding motif as sp , reduces gene expression. sp protein expression in mice uteri dramatically decreases in late pregnancy, while sp protein level remains consistent. our data indicate that sk channels must be downregulated for the gravid uterus to generate labor contractions sufficient for delivery in both term and preterm mice. the changes in sk channel expression may be transcriptionally regulated by sp and sp .